OA20573A - 6,7-dihydro-5H-pyrido[2,3-c] pyridazine derivatives and related compounds as Bcl-xL protein inhibitors and pro-apoptotic agents for treating cancer. - Google Patents

6,7-dihydro-5H-pyrido[2,3-c] pyridazine derivatives and related compounds as Bcl-xL protein inhibitors and pro-apoptotic agents for treating cancer. Download PDF

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OA20573A
OA20573A OA1202200028 OA20573A OA 20573 A OA20573 A OA 20573A OA 1202200028 OA1202200028 OA 1202200028 OA 20573 A OA20573 A OA 20573A
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methyl
amino
mmol
benzothiazol
pyrido
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OA1202200028
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Maïa CHANRION
James Edward Paul Davidson
James Brooke MURRAY
Attila Paczal
Ana Leticia MARAGNO
Jérôme-Benoît STARCK
Frédéric COLLAND
Tibor Novak
Simon Bedford
Mark Philip DODSWORTH
Andras Herner
Paul Webb
Andras Kotschy
Emma Sanders
Matyas Pal TIMÀRI
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Les Laboratoires Servier
Vernalis (R&D) Limited
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Abstract

The present invention discloses 6,7-dihydro-5Hpyrido[2,3-c]pyridazine, 1,2,3,4-tetrahydroquinoline, 1H-indole, 3,4- dihydro-2H-l,4benzoxazine, lH-pyrrolo[2,3-b]pyridin-1-yl, 7Hpyrrolo[2,3-c]pyridazine, 5H,6H,7H,8H,9Hpyridazino[3,4-b]azepine derivatives and related compounds of formula (I) as Bcl-xL protein inhibitors for use as pro-apoptotic agents for treating cancer, autoimmune diseases or immune system diseases. Formula (1). The description discloses the preparation of exemplary compounds (e.g. pages 113 to 354 examples 1 to 221) as well as pharmacological studies with relevant data (e.g. pages 355 to 367; examples A to E; tables 1 to 5). Exemplary compounds are e.g. 2-{6-[(1,3-benzothiazol-2-yl) amino]-1,2,3,4tetrahydroquinolin-1-yl}-1,3-thiazole-4-carboxylic acid (example 1) or e.g. 3-{1-[(adamantan-1yl)methyl]-5- methyl-1H-pyrazol-4-yl}-6-{3-[(1,3benzothiazol-2-yl)amino]-4- methyl-5H,6H,7H,8Hpyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid (example 24).

Description

6,7-DIHYDRO-5H-PYRIDO[2,3-C]PYRIDAZINE DERIVATIVES AND RELATED COMPOUNDS AS BCL-XL PROTEIN INHIBITORS AND PRO-APOPTOTIC AGENTS FOR TREATING CANCER
FIELD OF THE INVENTION
The présent invention relates to 6,7-dihydro-5//-pyrido[2,3-c]pyridazin-8-yl dérivatives, to pharmaceutical compositions containing them and their uses as pro-apoptotic agents. The compounds of the présent invention inhibit the activity of the Bcl-xL protein and may be of interest in the treatment of cancer, immune and autoimmune diseases.
BACKGROUND OF THE INVENTION
Apoptosîs (programmed cell death) is an evolutionarily conserved pathway essential for tissue homeostasis, development and removai of damaged cells. Deregulation of apoptosîs contributes to human diseases, including malignancies, neurodegenerative disorders, diseases of the immune System and autoimmune diseases (Hanahan and Weinberg, Cell. 2011 Mar 15 4;144(5):646-74; Marsden and Strasser, Anna Rev Immunol. 2003;21:71-105; Vaux and
Flavell, Cttrr Opln Immunol. 2000 Dec; 12(6):719-24). Evasion of apoptosîs is recognized as a hallmark of cancer, participaling in the development as well as the sustained expansion of tumors and the résistance to anti-cancer treatments (Hanahan and Weinberg, Cell. 2000 J an 7; 100(1):57-70).
The Bcl-2 protein family comprises key regulators of cell survival which can suppress (e.g., Bel-2, Bcl-xL, Mcl-1) or promote (e.g., Bad, Bax) apoptosîs (Gross et al., Genes Dev. 1999 Aug 1;13(15):1899-911, Youle and Strasser, Nat. Rev. Mol. Cell Biol. 2008 Jan;9(l):47-59).
In the face of stress stimuli, whether a cell survives or undergoes apoptosîs is dépendent on the extent of pairing between the Bcl-2 famîly members that promote cell death with family 25 members that promote cell survival. For the most part, these interactions involve the docking of the Bcl-2 homology 3 (BH3) domain of proapoptotîc family members into a groove on the surface of pro-survival members. The presence of Bcl-2 homology (BH) domain defines the membership of the Bcl-2 family, which is divided into three main groups depending upon the particular BH domains présent within the protein. The prosurvival members such as Bcl-2, 30 Bcl-xL, and Mcl-f contain BH domains 1-4, whereas Bax and Bak, the proapoptotîc effectors of mitochondrial outer membrane permeabîlîzation during apoptosîs, contain BH domains 1 φ 3 (Youle and Strasser, Nat. Rev. Mol. Cell Biol. 2008 Jan;9(l ):47-59).
Overexpression of the prosurvival members of the Bcl-2 family is a hallmark of cancer and it has been shown that these proteins play an important rôle in tumor development, maintenance and résistance to anticancer therapy (Czabotar et al., Nat. Rev. Mol. Cell Biol. 2014 5 Jan; 15(1):49-63). Bcl-xL (also named BCL2L1, from BCL2-like 1) îs frequently amplified in cancer (Beroukhim et al., Nature 2010 Feb 18;463(7283):899-905) and it has been shown that its expression inversely correiates with sensitivity to more than 120 anti-cancer therapeutic molécules în a représentative panel of cancer cell lines (NCI-60) (Amundson et al., Cancer Res. 2000 Nov l;60(21):6101-10).
In addition, several studies using transgenic knockout mouse models and transgenic overexpression of Bcl-2 family members highlighted the importance of these proteins in the diseases of the immune System and autoimmune diseases (for a review, see Merîno et al., Apoptosis 2009 Apr;14(4):570-83. doi: 10.1007/s10495-008-0308-4. PMID: 19172396). Transgenic overexpression of Bcl-xL within the T-cell compartment resulted in résistance to 15 apoptosis induced by glucocorticoid, g-radiation and CD3 crosslinking, suggesting that transgenic Bcl-xL overexpression can reduce apoptosis in resting and actîvated T-cells (Droin et al., Biochim Biophys Acta 2004 Mar l;1644(2-3): 179-88. doi: 10.1016/j.bbamcr.2003.10.011.PMID: 14996502 ). In patient samples, persistent or hîgh expression of antiapoptotic Bcl-2 family proteins has been observed (Pope et al., Nat Rev 20 Immunol. 2002 Jul;2(7):527-35. doi: 10.1038/nri846.PMID: 12094227). In particular, T-ceils isolated from the joints of rheumatoid arthritis patients exhibited increased Bcl-xL expression and were résistant to spontaneous apoptosis (Salmon et al., J Clin Invest. 1997 Feb l;99(3):439-46. doi: 10.U72/JCI119178.PMID; 9022077). The use of BH3 mimetics has also shown benefit in pre-clinical models of diseases of the immune System and autoimmune 25 diseases. Treatment with ABT-737 (Bd-2, Bcl-xL, and Bcl-w inhibitor) resulted in potent inhibition of lymphocyte prolifération in vitro. Importantly, mice treated with ABT- 737 in animal models of arthritis and lupus showed a significant decrease in disease severity (Bardwell et al., J Clin Invest. 1997 Feb 1;99(3) :439-46. doi:
10.1172/JCI119178.PMID: 9022077). In addition, it has been shown that ABT-737 prevented 30 allogeneic T-cell activation, prolifération, and cytotoxicity in vitro and inhibited allogeneic Tand B-cell responses after skîn transplantation with hîgh selectivity for lymphoid cells (Cippa et al., .Transpl Int. 2011 Jul;24(7):722-32. doi: 10.1111/j. 1432-2277.2011.01272.x. Epub 2011 May 25.PMID: 21615547).
φ The findings indicated above motivated the discovery and development of a new class of drugs named BH3 mimetics. These molécules are able to disrupt the interaction between the proapoptotîc and antîapoptolic members of the Bcl-2 family and are potent inducers of apoptosis. This new class of drugs includes inhibitors of Bcl-2, Bcl-xL, Bcl-w and Mcl-1. The 5 first BH3 mimetics described were ABT-737 and ABT-263, targeting Bcl-2, Bcl-xL and Bclw (Park et al., J. Med. Chem. 2008 Nov 13;51(21):6902-15; Roberts et al., J. Clin. Oncol. 2012 Feb 10;30(5):488-96). After that, sélective inhibitors of Bcl-2 (ABT-199 and S55746 Souers et al., Nat Med. 2013 Feb;19(2):202-8; Casara et al., Oncotarget 2018 Apr 13;9(28):20075-20088), Bcl-xL (A-1155463 and A-1331852 - Tao et al., ACS Med Chem
Lett. 2014 Aug 26;5(10):1088-93; Leverson et al., Sci Transi Med. 2015 Mar 18;7(279):279ra40) and Mcl-1 (A-1210477, S63845, S64315, AMG-176 and AZD-5991 Leverson et al., CellDeath Dis. 2015 Jan 15;6:el590.; Kotschy et al., Nature 2016, 538, 477482; Maragno et al., AACR 2019, Poster #4482; Kotschy et al., WO 2015/097123; Caenepeel et al., Cancer Discov. 2018 Dec;8(l2): 1582-1597; Tron et al., Nat. Commun. 2018
Dec 17;9(1):5341) were also dîscovered. The sélective Bcl-2 inhibitor ABT-199 is now approved for the treatment of patients with CLL and AML in combination therapy, while the other inhibitors are stili under pre-clinical or clinical development. In pre-clinical models, ABT-263 has shown activity în several hematological malignancies and solid tumors (Shoemaker et al., Clin. Cancer Res. 2008 Jun 1; 14(11);3268-77; Ackler et al., Cancer
Chemother. Pharmacol. 2010 Oct;66(5):869-80; Chen et al., Mol. Cancer Ther. 2011 Dec; 10(12):2340-9). In clinical studies, ABT-263 exhibited objective antitumor activity in lymphoid malignancies (Wilson et al., Lancet Oncol. 2010 Dec;ll(12):1149-59; Roberts et al., J. Clin. Oncol. 2012 Feb 10;30(5):488-96) and its activity is being investigated in combination with several thérapies in solid tumors. The sélective Bcl-xL inhibitors, A25 1155463 or A-1331852, exhibited in vivo activity in pre-clinical models of T-ALL (T-cell
Acute Lymphoblastic Leukemia) and different types of solid tumors (Tao et al., ACSMed. Chem. Lett. 2014 Aug 26;5(10):1088-93; Leverson et al., Sci. Transi. Med. 2015 Mar 18;7(279):279ra40). The Mcl-1 sélective inhibitors hâve shown promising in vivo activity in several types of hematological cell malignancies in preclinical models and three of them, 30 S64315, AMG176 and AZD5991, are currently being investigated in clinical trials (Yang et al., Eur. J. Med. Chem. 2019 May 8;177:63-75). Therefore, BH3 mimetics represent a highly attractive approach for the development of novel thérapies in oncology and in the field of immune and autoimmune diseases. In particular, the need exists for small molécules that inhibit selectively the Bcl-xL protein. The présent invention fulfills this need.
SUMMARY OF THE INVENTION
The présent invention provides potent sélective Bcl-xL inhibitors of formula (I) as defined below. We hâve shown that these compounds are able to induce apoptosis of cancer cells in vivo, triggerîng tumor régression in mice. Based on their pro-apoptotîc propertïes, the compounds of the invention could be of interest for the treatment of pathologies involving a de régulation in apoptosis, such as, for example, cancer, auto-immune diseases and diseases of the immune system. In addition, these compounds were well toierated in mice, with no clinically relevant body weight loss upon treatment with efficacious doses, indicating a possible therapeutic margin for the use of these Bcl-xL-targeting small molécules in cancer treatment. In agreement with the previously described rôle of Bcl-xL in the régulation of platelets life-span (Zhang et al., Cell Death Differ. 2007 May; 14(5):943-51; Mason et al., Cell. 2007 Mar 23;128(6):1173-86), we observed a réduction în the number of circulating platelets after treatment of mice with these inhibitors, with recovery after treatment discontinuation. Considering this effect in plalelel survival, the Bcl-xL inhibitors of the présent invention could also be used for treating diseases or conditions characterized by an excess or a deregulated activity of platelets, such as, for exampie, pro-thrombotic conditions.
DETAILED DESCRIPTION OFTHE INVENTION
In a first embodiment (El), the présent invention provides compounds of formula (I):
wherein:
♦ the Het moiety represents a fused aromatic or non-aromatic ring composed of from 5 to 7 ring members, which may contain, in addition to the nitrogen, one additional heteroatom or group selected from oxygen, sulphur and C=O,
A4 and As independentiy of one another represent a carbon or a nitrogen atom, preferably A4 and As represent each a nitrogen atom, ♦ Zi represents a bond, -N(R)-, or -O-, wherein R represents a hydrogen or a linear or branched Ci-Côalkyl, ♦ Ri represents a group selected from: hydrogen; linear or branched Cj-C(,alkyl optionally substituted by a hydroxyl or a Ci-Cbalkoxy group; Cs-Côcycloalkyl; trifluoromethyi; linear or branched Ci-Csalkylene-heterocycIoalkyl wherein the heterocycloalkyl group is optionally substituted by a a linear or branched Ci-Cealkyl group;
♦ R2 represents a hydrogen or a methyl;
♦ R3 represents a group selected from: hydrogen; linear or branched Ci-C+alkyl; -XiNRaRb; -Xi-\ RJM<; -Xi-O-Rc; -Xj-COORc; -Xi-PO(OH)2; -Xi-SO2(OH); -X1-N3 and :
—ΧΊ-=C H i
♦ Ra and Rb independentiy of one another represent a group selected from: hydrogen; heterocycloalkyl; -SCh-phenyl wherein the phenyl may be substituted by a linear or branched Ci-Côalkyl; linear or branched Ci-Côalkyl optionally substituted by one or two hydroxyl groups; Ci-Cûalkylene-SOzOH; Ci-C6alkylene-SO2O/ Ci-C&alkyleneCOOH; Ci-Côalkylene-PO(OH)2; Ci-C6alkylene-NRdRe; Ci-C6alkylene-N+RdReRf; QCôalkylene-phenyl wherein the phenyl may be substituted by a Ci-Cbalkoxy group;
the group:
or Ra and Rh form with the nitrogen atom carrying them a cycle Bi;
or Ra, Rb and Rc form with the nitrogen atom carrying them a bridged Cs-Csheterocycloalkyl, ♦ Rc, Rd, Re, Rt, independentiy of one another represents a hydrogen or a linear or branched Cj-Côalkyi group, or Rd and Re form with the nitrogen atom carrying them a
a cycle El·, or Rd, Re and Rf form with the nitrogen atom carrying them a bridged Ca-Csheterocycloalkyl, ♦ Heti represents a group selected from:
♦ Het2 represents a group selected from:
♦ A! is -NH-, -N(Ci-C3alkyi), O, S or Se, φ ♦ Α2 is N, CH or C(R5), ♦ G is selected from the group consisting of:
-C(0)0RG3, -C(0)NRgiRg2, -C(0)Rg2, -NRgiC(0)Rü2, -NRG!C(O)NRgiRg2,
-OC(O)NRGiRG2, -NRgjC(O)ORG3, -C(=NORgi)NRgiRg2,
-NRoiC(=NCN)NRgiRg2, -NRGiS(O)2NRGiRg2, -S(O)2RG3, -S(O)2NRgiRG2,
-NRGiS(O)2Rg25 -NRgiC(=NRG2)NRgiRg2, -C(=S)NRgiRg2, -C(=NRG|)NRgiRg2, halogen, -NO2, and -CN, in which:
- Rgi and Rg2 at each occurrence are each independently selected from the group consisting of hydrogen, Cj-Côalkyl optionally substituted by 1 to 3 halogen atoms, 10 C2-Cfialkenyl, Cs-Cealkynyl, Ca-Côcycloalkyl, phenyl and -(CH2)i-4-phenyl;
- Rgi is selected from the group consisting of Ci-Cûalkyl optionally substituted by 1 to 3 halogen atoms, Cz-Côalkenyl, C2-C6aikynyl, Cs-Côcycloalkyl, phenyl and -(CH2)i-4phenyl; or
Rgi and Rg2, together with the atom to which each is attached are combined to form a
Ca-Cgheterocycloalkyl ; or in the alternative, G is selected from the group consisting of:
wherein RG4 is selected from Ci-Csalkyl optionally substituted by 1 to 3 halogen atoms, C2-Cealkenyl, Cz-Cealkynyl and Ca-Cficydoalkyl,
A ♦ R4 represents a hydrogen, fluorine, chlorine or bromine atom, a methyl, a hydroxy 1 or a methoxy group, ♦ R? represents a group selected from: Ci-Côalkyl optionally substituted by 1 to 3 halogen atoms; Cs-Ccalkenyl; Cz-Cealkynyl; halogen or-CN, ♦ Rt, represents a group selected from:
hydrogen;
-C2-Côalkenyl;
-X2-O-R7;
-X2-NSO2-R7;
-C=C(R9)-Yi-O-R7;
Cî-Côcycloalkyl;
Ci-Cbheterocycloalkyl optionally substituted by a hydroxyl group;
C3-CôCycloalkylene-Y2-R7 ;
Cs-Côlieterocycloalkylene-Yi-R? group, an heteroarylene-R? group optionally substituted by a linear or branched Ci-Côalkyl group, ♦ R7 represents a group selected from: linear or branched Ci-Côalkyl group; (Ca-Cûjcycloalkylene-Rs; or:
wherein Cy represents a C3-Cscycloalkyl, ♦ Rs represents a group selected front: hydrogen; linear or branched Ci-Cbalkyl, NR’aR’b; -NR’a-CO-OR’c; -NRVCO-IC; -N-ICJUJC.; -O-R’c; -NH-X’2N+R’aR’bR’c; -O-X’2-NR’aR’b, -X’2-NR’aR’b, -NR’C-X’2-N3 and :
-NRV—X'?^=CH
♦ R9 represents a group selected from linear or branched Ci-Cealkyl, trifluoromethyl, hydroxyl, halogen, Ci-Cbalkoxy, ♦ Rio represents a group selected from hydrogen, fluorine, chlorine, bromine, -CF3 and methyl, ♦ Ru represents a group selected from hydrogen, halogen, CuCsalkylene-Rg, -O-CiCaalkylene-Rs, -CO-NRhRi and ~CH=CH-Ci-C4alkylene-NRhRi, -CH=CH-CHO, CCscycloalkylene-CH2-Rs, Ca-Csheterocycloalkylene-CHz-Rs, ♦ Ri2 and R13, independently of one another, represent a hydrogen atom or a methyl group, ♦ Rj4 and R15, independently of one another, represent a hydrogen or a methyl group, or Ri4 and R15 form with the carbon atom carrying them a a cyclohexyl, ♦ Rh and Ri, independently of one another, represent a hydrogen or a linear or branched Ci-Côalkyl group, ♦ X] represents a linear or branched
Ci-C4alkylene group optionally substituted by one or two groups selected from trifluoromethyl, hydroxyl, halogen, Ci-Ccalkoxy, * X2 represents a linear or branched
Ci-Csalkylene group optionally substituted by one or two groups selected from trifluoromethyl, hydroxyl, halogen, Ci-Cbalkoxy,
♦ X’2 represents a linear or branched Ci-Cealkylene, ♦ R a and R’b independently of one another, represent a group selected from: hydrogen; heterocycloalkyl; -SÛ2-phenyl wherein the phenyl may be substituted by a linear or branched Ci-Csalkyl; linear or branched Ci-C^alkyl optionally substituted by one or two hydroxyl or Ci-Cealkoxy groups; Ci-Côalkylene-SChOH; Ci-Côalkylene-SOsO-; Ci-Côalkylene-COOH; Ci-C6alkylene-PO(OH)2; Ci-CôalkyIene-NR’dR’e; Ci-Cûalkylene-bPR’dR’eR’f; Ci-Câalkylene-O-Ci-Cûalkylene-OH; Ci-Cûalkylenephenyl wherein the phenyl may be substituted by a hydroxyl or a Ci-Cûalkoxy group; the group;
or R’a and R’b form with the nitrogen atom carrying them a cycle B3, or R’a, R’b and R’c form with the nitrogen atom carrying them a bridged CvCsheterocycloalkyl, ♦ R’c, R’<i, R’e, R’f, independently of one another, represents a hydrogen or a linear or branched Ci-Cealkyl group,
or R’d and R’e form with the nitrogen atom carrying them a
cycle B4,
or R’d, R’e and R’f form with the nitrogen atom carrying them a
bridged Cs-Csheterocycloalkyl, ♦ Yi represents a linear or branched Cj-C4alkylene, ♦ Y2 represents a bond, -O-, -O-CH2-, -O-CO-, -O-SO2-, -CH2-, -CH2-O, -CH2-CO-, < , O -CO-, -CO-O-, -CO-CH2-, -CO-NH-CH2-, -SO2-, -SO2-CH2-,
- NH-CO-, -NH-SO2-, ♦ m=0, 1 or 2, ♦ p=l, 2, 3 or 4, ♦ Bi, B2, B3 and B4, independently of one another, represents a Cs-Csheterocycloalkyl group, which group can: (i) be a mono- or bi-cyclic group, wherein bicyclic group includes fused, briged or spiro ring System, (ii) can contain, in addition to the nitrogen atom, one or two hetero atoms selected independently from oxygen, sulphur and nitrogen, (ni) be substituted by one or two groups selected from: fluorine, bromine, chlorine, Iinear or branched Cj-Cc,alkyl, hydroxyl, -NH2, oxo or piperidinyl, it also being understood that:
aryl means a phenyl, naphthyl, biphenyl or îndenyl group, heteroaryl means any mono- or bi-cyclic group composed of from 5 to lü ring members, having at least one aromatic moiety and containing from 1 to 4 hetero atoms selected from oxygen, sulphur and nitrogen (including quaternary nitrogens), cycloalkyl means any mono- or bi-cyclic non-aromatic carbocyclic group containing from 3 to 10 ring members, which may include fused, bridged or spiro ring Systems, “heterocycloalkyl” means any mono- or bi-cyclic non-aromatic carbocyclic group, composed of from 3 to 10 ring members, and containing from one to 3 hetero atoms selected from oxygen, sulphur, SO, SO2 and nitrogen, it being understood that bicyclic group may be fused or spiro type, heteroarylene, cycloalkylene, heterocycloalkylene mean a divalent heteroaryl, cycloalkyl and heterocycloalkyl, its enantiomers and diastereoisomers, and addition salts thereof with a pharmaceuticaliy acceptable acid or base.
Among the pharmaceuticaliy acceptable acids there may be mentioned, without implying any limitation, hydrochloric acid, hydrobromic acid, sulphuric acid, phosphonic acid, acetic acid, trifluoroacetic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, tartaric acid, maleic acid, citric acid, ascorbic acid, oxalic acid, methanesulphonic acid and camphoric acid.
Among the pharmaceuticaliy acceptable bases there may be mentioned, without implying any limitation, sodium hydroxide, potassium hydroxide, triethylamine and tert-buty lamine.
Further enumerated embodiments (E) of the invention are described herein. It will be recognized that features specified in each embodiment may be combined with other specified features to provide further embodiments of the présent invention.
The compound according to El, which is a compound of formula (IA):
Ei The compound according to El or E2 wherein Zi represents -NH- or -O-, £4. The compound according to any of El to E3 wherein R3 represents -Xi-NRaRb, 5 preferably the group -C2H5-NH-CH3.
The compound according to El or E2, which is selected from:
E6, The compound according to E5, which is a compound of formula (IB):
Het2
E7, The compound according to E6 wherein Zi represents a bond and R3 represents a hydrogen atom.
E8, The compound according to El, which is a compound of formula (IC):
wherein A.i represents an oxygen or a sulphur atom.
E9. The compound according to any one of El to E8 wherein Ri represents a hydrogen atom, a methyl or a cyclopropyl group, preferably a methyl.
EIO, The compound according to any one of El to E9 wherein Heti represents:
Eli, The compound according to any one of El to E10 wherein Het2 represents:
o
COOH r6
Eli The compound according to any one of El to E10 wherein Het2 represents:
E13. The compound according to Eli wherein Rô represents a -X2-O-R7 group wherein X2 is a propylene group.
ELL The compound according to E13 wherein R7 represents the following group;
El5. The compound according to El3 wherein R7 represents the following group:
El 6· The compound according to E13 wherein R7 represents the following group:
Eli The compound according to any of E14 to E16 wherein Rg represents NR’aR’b.
El8. The compound according to any of E14 to E16 wherein Rg represents a group selected from: dimethylamino, diethylamino, diisopropylamino, diisobutylamino, methylamino, ethylamino, ethyl(methyl)amino, 4-methyl-piperazin-l-yl, piperazin-l-yl, pyrrolidin-l-yl, azetidin-l-yl, 1-piperidyl, 4-morpholinyl, 4,4-difluoropiperidin-l-yl, 3,3-difluoropiperidin-lyl, 3-hydroxy-l-pipendyl, (lS,5R)-3-azabicyclo[3.1.0]hexan-3-yl, 4-(l-piperidyl)-l-pipeiidyl, φ 3-oxo-2,8-diazaspiro[4.5]decan-8-yl, (lS,5/?)-6,6-difluoro-3-azabicyclo[3.1.0]hexan-3-yl, 2(dimethylamino)ethylamino, 3-piperazin-l-yI, (3R,5S)-3,5-dimethylpiperazin-l-yI, (but-3-ynl-yl)amino, (but-3-yn-l-yl)(methyl)amino, (3-azidopropyl)amîno, (3azidopropyl)(methyl)amino (3-aminopropyl)amino, (pent-4-yn-l-yl)amino, methyl(pent-45 yn-l-yl)amino, (prop-2-yn-l-yi)amino, (hex-5-yn-l-yI)amino, 3-[(hex-5-yn-lyl)(methyl)amino, (4-azidobutyl)amino, (4-azidobutyl)(methyl)amino, [2-(2hydroxyethoxy)ethylj(methyl)amino, and:
m The compound according to any of E14 Lo El6 wherein Rs represents a group selected from: bis[(35')-3,4-dihydroxybutyl]amino, amino, [(3.5)-3,4-dihydroxybutyl]amino, [(3R)-3,4dihydroxybutyl]amino, acetyl(methyl)amino, 3-hydroxypropylamino.
E20, The compound according to E13 wherein R? represents:
wherein Ru is selected from 3-(dimethylamino)propyl, 3-(methyIamino)propyl, aminomethyl, 2-(dimethylamino)ethyl, 4-(dimethylamino)butyl, 2-(methylamino)ethyl, 4(methylamino)butyl, 3-(azetidin-l-yl)propyl, 3-(4-melhylpiperazin-l-yI)propyl, 3-pyrrolidin1-ylpropyl, 3-morpholinopropyl, 3-(l-piperidyl)propyl, 3-[(lR,5S)-6,6-difIuoro-3azabicyclo[3.1.0]hexan-3-yl and 3-(3-oxo-2,8-diazaspiro[4.5]decan-8-yl)propyl.
E21. The compound according lo E13 wherein R? represents a group selected from:
E22. The compound according to E12 wherein R(, represents:
E23. The compound according to E22 wherein R? represents a group selected from:
wherein R» represents -0-X’2-NR’aR’b or -Xù-NR’aR’b.
E24. The compound according to E22 wherein R? represents a group selected from :
wherein Rs represents a group selected from: hydrogen, 2-(methylamino)ethoxy, 2(dimethylamino)ethoxy,
2-[(2-sulfoethyl)amino]ethoxy,
2-[methyl(2sulfoethyl)amino]ethoxy, 4-methylpiperazin-l-yl and:
E25. The compound according to E22 wherein R? represents a group selected from :
wherein Rs represents a group selected from: 2-pyrrolidin-l-ylethoxy, 2-(4-methylpiperazin5 l-yl)ethoxy, 2-[[(37ï)-3,4-dihydroxybutyl]-methyl-amino]ethoxy, 2-(4hydroxybutylamino)ethoxy, 2-[[3-hydroxy-2-(hydroxymethyl)propyl]amino]ethoxy, 2-[bis(2hydroxyethyl)amino]ethoxy, 2-[[2-hydroxy-l-(hydroxymethyl)ethyl]amino]ethoxy, 2-(2-(2hydiOxyethoxy)ethylamino]ethoxy, 2-[bis(3-hydroxypropyl)amino]ethoxy, 2-(3hydroxypropylamino)ethoxy, 2-[bis(4-hydroxybutyl)amino]ethoxy, 2-morphoIinoethoxy, 210 (l-piperidyl)ethoxy, 2-piperazin-l-ylethoxy, 2-(azepan-l-yl)ethoxy, 2-(4-isopropylpiperazinl-yl)ethoxy, 2-[(4-hydroxyphenyl)methylamino]elhoxy, 2-(2hydroxyethyl(methyl)amino]ethoxy, 2-[3-methoxypropyl(methyl)amino]ethoxy, 2-[4hydroxybutyl(methyl)amino]ethoxy, 3-pyrrolidin-l-ylpropyl, 3-(dimethylamino)propyl, 3-(4methylpiperazin-l-yl)propyl, 3-morpholinopropyl, 3-(3-hydroxypropylamino)propyl, 3-(415 hydroxybutylamino)propyl, 3-[ [(35)-3,4-dihydroxybutyl]amino]propyl, 3-hydroxy-2(hydroxymethyl)propyl]amino]propyl, 3-[4-hydroxybutyl(methyl)amino]propyl, 3-[3hydroxypropyl(methyl)amino]propyl, 3-[3-[bis(3-hydroxypropyl)amino]propyl, 3-piperazin1-ylpropyl.
E26. The compound according to any of El, E2 and E6 wherein Ri represents -Xi20 PO(OH)2, -Xi-SO2(OH), -Xi-NRaRb; -Xi-N*RaRbRc, wherein R;1 or Rb, or both of them, represent a group selected from Ci-Côalkylene-SChOH, Ci-C6alkylene-SO2O- and Ci φ C+iIkylcncPOjOH)?.
Ε27, The compound according to any one of El, E2 and E6 wherein Rg represents NR’aR’b; -N+R’aR’bR’c; -NH-X’2-N+R’aR’bR’(;, w'herein R’a and R’b, or both of them, represent a group selected from Cj-Côalkylene-SOzOH and Ci-C6alkylene-PO(OH)2.
E28. A compound according to El selected in the following group:
2-(3-(1,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido[2,3-c]pyridazin8-yI]-5-[3-[4-[3-(dimethylaminû)prop-l-ynyl]-2-fluoro-phenoxy]propyl]thiazole-4carboxylic acid,
2-{3-((1,3 -Benzothiazol-2-yl)amino] -4 -methy 1-5//,6//, 7//,8//-pyridof 2,3-c]pyridazin10 8-yl}-5-(3-{2-fluoro-4-[3-(methylamino)prop-l-yn-l-yl]phenoxy}propyl)-l,3thiazole-4-carboxylic acid,
- 2-{3-[(l,3-Benzothiazol-2-yl)amino]-4-methyl-5//,6//,7//,8//-pyrido[2,3-c]pyridazin8-yl}-5-(3-{4-[3-(dimethyIamino)propyl]-2-fluorophenoxy}propyl)-l,3-thiazole-4carboxylic acid,
- 2-(3-( l,3-Benzothîazol-2-ylarmno)-4-methyI-6,7-dihydro-5//-pyrido[2,3-c]pyridazin8-ylJ-5-(3-[2-fluoro-4-[3-(4-methylpiperazin-l-yl)but-lynyl]phenoxy]propyl]thiazole-4-carboxylic acid,
2-(3-(1,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5Z/-pyrido[2,3-c]pyridazin8-yl]-5-[3-[2-fluoro-4-(3-pyrrolidin-l-ylprop-l-ynyl)phenoxy]propyi]thiazole-420 carboxylic acid,
- 5-(3-{4-[3-(Azetidin-l-yl)prop-l-yn-l-yl]-2-fluorophenoxy}propyl)-2-{3-((l,3benzothiazol-2-yI)amino]-4-methyl-5//,6//,7//,8//-pyrido[2,3-c]pyridazin-8-yl}-l,3thiazole-4-carboxyIic acid,
- 2-(3-(1,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido[2,3-c]pyridazin25 8-yl]-5-(3-[2-fluoro-4-[3-(4-methylpiperazin-l-yl)prop-l-ynyl]phenoxy]propyl] thiazole-4-carboxylic acid, . 2-{3-[(l,3-Benzothiazol-2-yl)amino]-4-methy 1-5//,6//,7//,8//-pyrîdo(2,3-c]pyridazin8-yi}-5-(3-{4-[3-(4,4-difluoropiperidin-l-yl)prop-l-yn-l-yl]-2fluorophenoxy}propyl)-l,3-thiazole-4-carboxyIic acid,
- 2-{3-((l,3-Benzothiazol-2-yl)amino]-4-methyl-5//,6//,7//,8Z/-pyrido[2,3-c|pyridazin8-yl}-5-(3-{4-[3-(3,3-difluoropiperidin-l-yl)prop-l-yn-l-yl]-2fluorophenoxy}propyl)-l,3-thiazole-4-carboxylic acid,
- 2-(3-(1,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/f-pyrido[2,3-c]pyridazin8-yl]-5-[3-(2-fluoro-4-[3-(3-oxo-2,8-diazaspiro[4.5]decan-8-yI)prop-l-ynyl] phenoxy ] propy I] lhiazole-4-carboxy 1 ic acid, 2-(3-(1,3-Benzothiazol-2-y lamino)-4-methyl-6,7-dihydro-52/-pyrido(2,3-c]pyridazin5 8-yl]-5-[3-[4-[3-[(15,5R)-6,6-difluoro-3-azabicyclo[3.1.0]hexan-3-yl]prop-l-ynyl]-2fluoro-phenoxy]propyl]thiazole-4-carboxylîc acid, 2-(3-(1,3-Benzothiazol-2-ylamino)-4-melhyl-6,7-dihydro-5E/-pyrido[2,3-c]pyridazin8-yl]-5-[3-[2-fluoro-4-(3-piperazin-l-ylprop-l-ynyl)phenoxy]propyl]thiazole-4carboxylic acid,
- 2-(3-(1,3-Benzothiazol-2-ylamino)-4-methy 1-6,7-dihydro-57f-pyrido[2,3-c]pyridazin8-yl]-5-[3-[4-[3-[(3/?,55)-3,5-dimelhylpiperazin-l-yl]prop-l-ynyl]-2-fluorophenoxy]propyl]thiazoIe-4-carboxylic acid,
- 2-(3-(1,3-Benzothiazoi-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin8-yl]-5-[3-[4-[3-(diethylamino)prop-l-ynyl]-2-fluoro-phenoxy]propyl]thiazole-415 carboxylic acid,
- 2-(3-(1,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/f-pyrido[2,3-c]pyridazin8-yl]-5-[3-[4-[3-(diisopropylamino)prop-l-ynyl]-2-fluoro-phenoxy]propyl]thiazole-4carboxylic acid,
- 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/f-pyrido[2,3-c]pyridazin20 8-yl]-5-[3-[4-[3-[2-(dimethylamino)ethylamino]prop-l-ynyl]-2-fluorophenoxy]propyl]thiazoIe-4-carboxylic acid,
- 2-{3-[(l,3-Benzolhiazoi-2-yl)amino]-4-methyl-6-[2-(methylamino)ethoxy]5H,6/f,7//,8//-pyrido[2,3-cJpyridazin-8-yl}-5-(3-{4-[3-(dimethylamino)prop-l-yn-lyl]-2-fluorophenoxy}propyI)-l,3-thiazole-4-carboxylic acid,
- 2-(3-(1,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5£Lpyrido[2,3-c]pyridazin8-yl]-5-[3-[4-[l-[(dimethylamino)methyl]-3-bicyclo(l.l.l]pentanyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylic acid,
- 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido[2J3-c]pyridazin8-yl]-5-[3-[2-fluoro-4-[3-inethyl-3-(methylamino)but-l30 ynyl]phenoxy ]propyI]thiazole-4-carboxylic acid,
- 2-(3-(1,3-benzothiazol-2-yiamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin8-yl]-5-[3-[2-fluoro-4-[3-(prop-2-ynyIamino)prop-l-ynyl]phenoxy]piOpyl]thiazole-4carboxylic acid, 6-{3-((l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido[2,3-c]pyridazin
8(5//)-yl}-3-[l-({3-[2-(dimethylamino)ethoxy]-5,7-dimethyladamantan-l-yl}methyl)5-methyl-l//-pyrazol-4-yl]pyridine-2-carboxyIic acid.
6-{3-[(l,3-benzothiazol-2-yl)ammo]-4-methyl-6,7-dihydropyrido[2,3-c|pyridazin8(5//)-yl}-3-[l-({3,5-diniethyl-7-[2-(methylamino)eihoxy]adamantan-l-y]}methyl)-5methyl-l//-pyrazol-4-yl]pyridine-2-carboxylic acid, 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido[2f3-c]pyridazin8(5H)-yl}-5-(3-{4-[3-(ethyIamnio)-3-methylbut-l-yn-l-yl]-2-fluorophenoxy}propyl)l,3-thiazole-4-carboxylic acid, 3-{l-[(Adamantan-l-yl)methyi]-5-methyl-U/-pyrazol-4-yl}-6-{3-[(l,3-benzothiazol2-yI)amino]-4-methyl-5Ef, 8H-pyrido[2,3-c]pyridazin-8-yl}pyridme-2carboxylic acid, its enantiomers and diastereoisomers, and addition salis thereof with a pharmaceutically acceptable acid or base.
E29. A compound according to El selected in the following group:
- 6-{3-[(l,3-benzothiazol-2-yI)ammo]-4-methyl-6,7-dihydropyrido[2,3-c]pyridazin8(5H)-yl}-3-[l-({3-[2-(dimethylammo)ethoxy]-5,7-dimethyladamantan-l-yl}methyI)5-methyl-lH-pyrazol-4-yl]pyridme-2-carboxyIic acid,
- 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5ELpyrido[2,3-c]pyridazm8-yl]-3-[l-[[3,5-dimethyl-7-(2-pyrrolidin-l-ylethoxy)-l-adamantyl]methyl]-5-methyl· pyrazol-4-yl]pyridine-2-carboxylic acid,
- 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido[2,3-c]pyridazin8-yl]-3-[l-[[3,5-dimethyl-7-[2-(4-methylpiperazin-l-yI)ethoxy]-l-adamantyl]methyl]5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid,
- 6-[3-(l,3-benzolhiazol-2-ylammo)-4-methyl-6,7-dihydro-5/7-pyrido[2,3-c|pyridazm8-yl]-3-[l-[[3-[2-(3-hydroxypiOpylamino)ethoxy]-5,7-dimelhyl-l-adamantyl]methyl]5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid,
- 6-(3-(1,3-benzothiazol-2-ylamino )-4-methyl-6,7-dihydro-5//-pyrido[2,3-c]pyridazm-
8-yl]-3-[l-[[3-[2-(4-hydroxybutylamino)ethoxy]-5î7-dimethyl-l-adamantyl]methyl]5-methyl-pyrazol-4-yI]pyridine-2-carboxyIic acid, 6-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido[2,3-c|pyridazin8(57/)-yI}-3-(l-{[3-(2-{ [(35)-3,4-dihydroxybutyl]amino}ethoxy)-5,7dimethyIadamantan-l-yl]methyI}-5-methyl-lH-pyrazol-4-yl)pyridine-2-carboxyiic acid.
- 6-{3-[(l,3-benzothiazol-2-yI)amino]-4-methyl-6,7-dihydropyrido[2,3-c]pyridazin8(5H)-yl}-3-(l-{ [3-(2-{ [(3/i)-3,4-dihydroxybutyl]ammo}ethoxy)-5,7diinethyladamantan-l-yl]methyl}-5-methyl-l//-pyrazol-4-yl)pyridine-2-carboxylic acid,
- 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido[2,3-c]pyridazin8-yl]-3-[l-([3-[2-[2-hydroxyethyl(methyl)amino]ethoxy]-5,7-dimethyl-ladamantyI]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid, 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido(2,3-c]pyridazin8-yl]-3-(l-[[3-[2-[4-hydroxybutyl(methyl)amino]ethoxy]-5,7-dinielhyl-l10 adamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid,
6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido[2,3-c]pyridazin8-yl]-3-[l-[[3-[2-(((3JÏ)-3,4-dihydroxybutyl]-methyl-amino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid, 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido[2,3-c]pyridazin15 8-yl]-3-(l-[[3,5-dimethyl-7-(2-piperazin-l-ylethoxy)-l-adamantyl]methyl]-5-methylpyrazol-4-yl]pyridine-2-carboxylic acid, 6-{3-((1,3-benzothiazol-2-yl)amino]-4-methy 1-6,7-dihydropyrido[2,3-c]pyridazin8(5f/)-yl}-3-[l-({3,5-dÎniethyI-7-[2-(methylamino)ethoxy]adamantan-l-yl}methyl)-5methyl-l/7-pyrazol-4-yl]pyridine-2-carboxylic acid,
- 6-(3-(1,3-benzothiazo]-2-ylamino)-4-methyl-6,7-dihydro-5/7-pyrido(2,3-c]pyridazin8-yl]-3-[l-[[3s5-dimethyl-7-[2-(l-piperidyl)ethoxy]-l-adamantyl]inetbyl]-5-methylpyrazol-4-yl]pyridine-2-carboxylic acid,
- 3-[l-[[3-[2-(azepan-l-yl)ethoxy]-5,7-dimethyl-l-adamantyl]methyl]-5-methylpyrazol-4-yI] -6-(3-( 1,3-be nzothiazol-2-y lamîno)-4-methy 1-6,7-dihydro-5//25 pyrido[2,3-c]pyridazin-8-yl]pyndine-2-carboxylic acid,
6-(3-(1,3-benzothiazol-2-ylamino)-4-metby 1-6,7-dihydro-5H-pyrido[2,3-c]pyrîdazin8-yl]-3-[l-[[3-(2-(4-isopropylpiperazin-I-yl)ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyi-pyrazol-4-yl]pyridine-2-carboxylic acid,
- 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido[2,3-c]pyridazin-
8-yl]-3-[ 1-((3,5-dimethyl-7-(2-morpholinoethoxy)-l-adamantyl]methyl]-5-methylpyrazol-4-yl]pyridine-2-carboxylic acid,
- 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/Apyrido[2,3-c]pyridazin-
8-yl]-3-[l-[[3-(2-[3-methoxypropyl(methyl)amino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid,
- 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyI-6,7-dihydro-5/:/-pyrido[2,3-c]pyridazin8-yl]-3-[l-[[3-[2-[2-(2-hydiOxyethoxy)ethylamino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyI-pyrazol-4-yl]pyridine-2-carboxylic acid, 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-
8-yl]-3-[l-[[3-[2-[[2-hydroxy-l-(hydroxyniethyl)ethyl]amino]ethoxy]-5,7-diniethyl-ladamantyl]inethyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid, 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin8-yl]-3-[l-[[3-[2-[[3-hydiOxy-2-(hydroxymethyl)propyl]amino]ethoxy]-5,7-dimethyll-adamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid,
- 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/7-pyrido[2,3-c]pyridazin8-yl]-3-[l-[[3-[2-[bis(2-hydroxyethyl)amino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyI-pyrazol-4-yl]pyridine-2-carboxylic acid,
- 6-(3-(1,3~benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/f-pyndo[2,3-c]pyridazin-
8-yl]-3-(l-[(3-[2-(bis(3-hydroxypropyl)amino]eihoxy]-5,7“dimelhyl-l15 adamantyl]methyl]-5-methyI-pyrazol-4-yl]pyridine~2-carboxylic acid,
- 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5i/-pyrido[2,3-c]pyridazin-
8-yl]-3-[l-([3-[2-[bis(4-hydroxybutyl)amino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazoI-4-yl]pyridine-2-carboxylic acid,
- 6-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido[2,3-c]pyridazin20 8(5H)-yl}--3-{ 1-((3,5-dÎmethy]-7-{2-[(2-sulfoethyi)amino]ethoxy}adamantan-lyl)methyl]-5-methyl-l/f-pyrazol-4-yl}pyridine-2-carboxylic acid,
- 6-(3-(1,3-benzothiazol-2-ylamino)-4-methy 1-6,7-dÎhydro-57f-pyrido[2,3-c]pyridazin8-yl]-3-[l-[[3-(2-((4-hydroxyphenyl)methylaniino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yi]pyridine-2-carboxylic acid,
- 2-(3-(1,3-Benzothiazoi-2-ylamino)-4-methy 1-6,7-dihy dro-5//-pyrido[2,3-c]pyridazin8-yl]-5-[3-[4-[3-(dimethylamino)prop-l-ynyl]-2-fluoro-phenoxy]propyl]thiazole-4carboxylic acid,
- 2-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido[2,3-c]pyridazin8-ylj-5-[3-[4-[3-[((35')-3,4-dihydroxybutyl]amino]prop-l-ynyl]-2-fluoro- phenoxy]propyl]thiazole-4-carboxylic acid,
- 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-
8-yl]-5-(3-[2-fluoro-4-[3-(3-liydroxypropylamino)prop-lynyl]phenoxy]propyl]thiazole-4-carboxylic acid,
its enantiomers and diastereoîsomers, and addition salts thereof with a pharmaceuticaliy acceptable acid or base.
E30· Process for the préparation of a compound of formula (I) according to E6, which process is characterized in that there is used as starting material the compound of formula (II):
which compound of formula (II) is subjected to a leaving group incorporation (using iodination preferably) to yield the compounds of formula (III):
wherein L.G represents a leaving group (preferably a halogen atom, more preferably iodine), whîch compound of formula (III) is further subjected to a coupling reaction, in an aqueous or organic medium (preferably acetone), in the presence of a base (preferably césium carbonate), with a compound of formula (IV):
G1
(IV) wherein G1 represents a Ci-C^alkyl group or a (4-methoxyphenyl)methyl group and (P.G represents a protecting group (preferably a reri-butoxycarbonyl group), to yield the compound of formula (V):
which amino group of the compound of formula (V) îs deprotected (using preferably 1,1,1,3,3,3-hexafluoroîsopropanol) to yield the compound of formula (VI):
which compound of formula (VI) is subjected to a Suzuki coupling reaction, in an aqueous or organic medium, in the presence of a phosphine palladium complex (preferably Pd(AtaPhos)2Cl2), of a base (preferably CS2CO3) and of a compound of formula (VII):
wherein R7 is as defined in formula (I), to yield the compound of formula (VIII):
which compound of formula (VIII) is further subjected to an intramolecular Buchwald coupling reaction, în an aqueous or organic medium, in the presence of a phosphine palladium compiex (preferably PdfAtaPhosjzCk) and at least one base (preferably CS2CO3 and DI PEA) to yield the compound of formula (IX):
(IX) which compound of formula (IX) is subjected to a Buchwald reaction, in an aqueous or 5 organic medium, in the presence of a palladium catalyst (preferably Pd2(dba)3), of a base (preferably DIPEA), of a phosphine (preferably Xantphos) and of the compound of formula (X):
(X) wherein R4 and m are as defined in formula (I), to yield the compound of formula (XI):
(XI) the ester function of which compound of formula (XI) is hydrolysed (using preferably LiOHxHbO or TFA) to yield the compound of formula (I), which compound of formula (I) may be purified according to a conventional séparation technique, which may be converted into its addition salts with a pharmaceuticaliy acceptable 5 acid or base and which is optionally separated into its isomers according to a conventional séparation technique, il being understood that, at any time considered appropriate in the course of the abovedescribed process, hydroxy, amino, carboxylic and phosphono groups of the reagents or intermediates of synthesis may be protected and then deprotected according to the 10 requirements of synthesis.
OL Process according to E30 wherein the group R? is selected from:
wherein R,s, Riz and R13 are as defined in formula (1).
E32. Process for the préparation of a compound of formula (I) according to E6, which process is characterized in that there is used as starting material the compound of formula (II):
(Π) which compound of formula (II) is subjected to a Mitsunobu reaction in the presence of triphenylphosphine in toluene, an appropriate coupling reagent (preferably di-tert20 butylazodicarboxylate) and of the compound of formula (ΧΙΙ-a) or (Xll-b):
wherein Ai, A2 and Rô are as defîned in formula (I), G1 represents a Ci-Cfialkyl group or a (4methoxyphenyl)methyl group and ( P.G ) represents a protecting group (preferably a tertbutoxycarbonyl group), to yield the compound of formula (ΧΠΙ-a) or (XIII-b):
(XlU-b) which amino group of the compound of formula (ΧΙΙΙ-a) or (ΧΙΙΙ-b) is further deprotected to yield the compound of formula (XlV-a) or (XlV-b):
(XlV-b) (i) which compound of formula (XlV-a) is further subjected to an intramolecular coupling reaction, in an aqueous or organic medium, in the presence of a base (preferably CS2CO3) to yield the compound of formula (XV-a), or (ii) which compound of formula (XlV-b) is further subjected to an intramolecular Buchwald 15 coupling reaction, in an aqueous or organic medium, in the presence of a phosphine palladium compiex (preferably Pd(AtaPhos)2Ch) and at least one base (preferably C52CO3 and DIPEA) to yield the compound of formula (XV-b),
(XV-a) or
(XV-b) which compound of formula (XV-a) or (XV-b) is subjected to a Buchwald réaction, in an aqueous or organic medium, in the presence of a palladium catalyst (preferably Pd2(dba)3), of a base (preferably DIPEA), of a phosphine (preferably Xantphos) and of the compound of formula (X):
(X) to yield the compound of formula (XVI-a) or (XVI-b):
G1
R6
H N m
(XVI-a) or
the ester function of which compound of formula (XVI-a) or (XVI-b) is hydrolysed (using preferably LiOHxH2O or with TFA) to yield the compound of formula (I), which compound of formula (1) may be purified according to a conventional séparation technique, which may be converted into its addition salts with a pharmaceutically acceptable acid or base and which is optionally separated into its isomers according to a conventional séparation technique, it being understood that, at any time considered appropriate in the course of the abovedescribed process, hydroxy, amino, carboxylic and phosphono groups of the reagents or intermediates of synthesis may be protected and then deprotected according to the requirements of synthesis.
E33. Synthesis intermediate according to E30 or E31 selected in the following group:
wherein R? is as defined in formula (I) and G1 represents a Cj-Côalkyl group, preferably a methyl group, or a (4-methoxyphenyl)methyl group.
E34. Synthesis intermediate according to E32 selected in the following group:
wherein R6 is as defined in formula (I) and G1 represents a Ci-C6alkyl group, preferably a methyl group, or a (4-methoxyphenyl)methyl group.
E35, The compound according to El wherein R4 represents a hydrogen, fluorine, chlorine 5 or bromine atom, a methyl or a methoxy group.
E36. The compound according to El wherein Rs represents a group selected from; hydrogen; linear or branched Ci-Côalkyl, -NR’aR’b; -NR’a-CO-OR’c; -N+R’aR’bR’c; -O-R’c; NH-X’z-bER’aR’bR’c; -O-X’2-NR’aR’b, -NR’C-X’2-N3 and :
-NR'C— X,2^=CH m The compound according to El wherein R’a and R’b independently of one another, represent a group selected from: hydrogen; heterocycloalkyl; -SOz-phenyl wherein the phenyl may be substîtuted by a linear or branched Ci-Cbalkyl; linear or branched Ci-Céalkyl optionally substîtuted by one or two hydroxyl groups; Ci-Cbalkylene-SChOH; Ci-Côalkylene-
SOO: Ci-Côalkylene-COOH; Ci-Csalkylene-POfOH),; Ci-Csalkylene-NR'aR’o;
Ci-Cfiaikylene-N+R’dR’eR’f; Ci-Cbalkylene-O-Q-Cbalkylene-OH; Ci-Côalkylene-phenyl wherein the phenyl may be substituted by a Ci-Cealkoxy group;
the group:
or R’a and R’b form with the nitrogen atom carrying them a cycle B3, or R’a, R’b and R’c form with the nitrogen atom carrying them a bridged C3-Csheterocycloalkyl.
E3S. Compound according to any of El to E27 wherein m=l.
Pharmacological study of the compounds of the invention has shown that they hâve proapoptotic properties. The abilîty to reactivate the apoptotic process in cells is of major therapeutic interest in the treatment of cancers and of immune and auto-immune diseases. In particular, the compounds according to the invention will be useful in the treatment of chemo15 or radio-résistant cancers.
In another embodiment, the compounds of the invention could be used for treating diseases or conditions characterized by an excess or a deregulated activity of platelets, especially prothrombotic conditions.
As used herein, the term “treat”, “treating or treatment of any disease or disorder refers in 20 one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treat”, treating or treatment refers to alleviating or ameliorating at least one physical parameler including those which may not be discernible by the patient. In yet another embodiment, “treat”, treating or treatment refers to modulating the disease or 25 disorder, eîther physîcally, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.
Among the cancer treatments envisagea there may be mentioned, without implying any limitation, the treatment of haematological malignancies and solid tumors. Haematological malignancies include myeloma, especially multiple myeloma, lymphoma, especially NonHodgkin Lymphoma (NHL) and more especially Diffuse Large B-cell Lymphoma (DLBCL), and leukemia, especially Chronic Lymphocytic Leukemia (CLL), T-cell Acute Lymphoblastic Leukemia (T-ALL), B-ceil Acute Lymphoblastic Leukemia (B-ALL) and Acute Myelogenous Leukemia (AML). Solid tumors include the bladder, brain, breast, utérus, œsophagus and liver cancers, colorectal cancer, rénal cancer, melanoma, ovarian cancer, prostate cancer, pancreatic cancer and lung cancer, especially non-small-cell lung cancer and small-cell lung cancer.
In particular, T-ALL results from the leukemic transformation of thymie cell precursors and their arrest at spécifie stages of différentiation. Despite recent and extensive insights into the molecular and cellular mechanisms responsible for T-ALL onset and progression, this knowledge has not been translated into efficient targeted thérapies. Current clinical treatments include chemotherapy associated or not with hematopoietic stem cell transplantation with survivai rates remaining around 50 and 70% in adult and pédiatrie cases, respectively. Both in pédiatrie and adult cases, relapses show very poor prognosis, reinforcing the need of the discovery of novel therapeutic options (Passaro et al., Immunol. Rev. 2016 May;271(l):15672). It has been shown that dual Bcl-2/Bcl-xL inhibitors, like ABT-263 and ABT-737, hâve promising activity în T-ALL patient derived xenograft models (Van Delft et al. Cancer Cell 2006;10:389-99; Suryani et al., Clin. Cancer Res. 2014,20:4520-31). Other studies hâve reported a differential requirement for Bcl-xL or Bcl-2 for survivai of mature versus very immature (ETP subgroup) T-ALL (Chonghaile et al., Cancer Dtscov. 2014;4:1074-87). The sélective Bcl-xL inhibitor A-1331852 described previously hâve also shown to hâve in vitro and in vivo activity in the mature T-ALL cell line xenograft model Molt-4 (Leverson et al., Sci. Transi. Med. 2015 Mar 18;7(279):279ra40). In a particular embodiment, tumor growth inhibition was also observed in MOLT-4 xenograft model upon treatment with the Bcl-xL inhibitors of the invention. These data support the use of the présent compounds in the treatment of T-ALL.
Among the treatments of autoimmune diseases envisaged there may be mentioned, without implying any limitation, the treatment of rheumatoid arthntis (RA) and systemic lupus erythematosus (SLE).
The present invention relates also to pharmaceutical compositions comprising at least one compound of formula (I), as the active ingrédient, in combination with one or more pharmaceutically acceptable excipients. In particular, these pharmaceutical compositions are interesling for use as pro-apoptotic and/or anti-proliférative agents, particularly, in the treatment of cancers and of auto-immune and immune System diseases.
Suitable excipients according to the invention include diluents, lubricants, binders, disintegration agents, stabilisers, preservatives, absorbents, colorants, sweeteners and flavourings.
By way of non-limiting example there may be mentioned:
* as diluents·. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycerol, ♦ as lubricants; silica, talc, stearîc acid and its magnésium and calcium salts, polyethylene glycol, ♦ as binders: magnésium aluminium silicate, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidone, ♦ as disintegrants; agar, alginic acid and its sodium sait, effervescent mixtures.
Among the pharmaceutical compositions according to the invention there may be mentioned more especially those that are suitable for oral, parentéral, nasal, per- or trans-cutaneous, rectal, perlingual, ocular or respiratory administration, especially tablets, dragées, sublingual tablets, capsules, glossettes, capsules, lozenges, injectable or drinkable préparations, aérosols, eye or nose drops, suppositories, creams, ointments, dermal gels.
Actual dosage levels of the active ingrédients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingrédient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will dépend upon a variety of factors including the activity of the particular compound of the présent invention employed, the route of administration, the time of administration, the rate of excrétion or melabolism of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with 5 the particular compound employed, the âge, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A suitable daily dose of a compound of the invention will dépend upon the factors described above and may range from 0.01 mg to 2.5 g per day in one or more administration(s).
In another aspect, the présent invention relates also to the combination of a compound of 10 formula (I) with an anticancer agent selected from genotoxic agents, mitotic poisons, antimetabolites, protéasome inhibitors, kinase inhibitors and antibodies, and also to pharmaceutical compositions comprising that type of combination and their use in the manufacture of médicaments for use in the treatment of cancer.
In another aspect, the compounds of the invention can be used in combination with 15 radiotherapy in the treatment of cancer.
Alternatively, the compounds of the invention may be linked to monoclonal antibodies. Antibody Drug Conjugales (ADCs) represent a new class of therapeutics that is formed by chemically linking a cytotoxic drug to a monoclonal antibody through a linker. The 20 monoclonal antibody of an ADC selectively binds to a target antigen of a cell (e.g. cancer cell) and releases the drug into the cell. ADCs hâve therapeutic potential because they combine the specificity of the antibody and the cytotoxic potential of the drug. Nonetheless, developing ADCs as therapeutic agents has thus far met with limited success owing to a variety of factors such as unfavorable toxicîty profiles, low effîcacies and poor 25 pharm acological parameters. Accordingly, there is still a need for new ADCs that overcome these problems and can selectively deliver Bcl-xL to target cancer cells.
In another aspect, the compounds of the invention may be linked to fragments of monoclonal antibodies or linked to scaffold proteins that can be related or not to monoclonal antibodies. Antibody fragments must be understood as fragments of Fv, scFv, Fab, F(ab')2, F(ab'), scFv20573
Fc type or dîabodies, which generally hâve lhe same specificity of binding as the antibody from which they are descended. Accordîng to the présent invention, antibody fragments of the invention can be obtained starting from antibodies by methods such as digestion by enzymes, such as pepsin or papain, and/or by cleavage of the disulfide bridges by Chemical réduction. 5 In another manner, the antibody fragments comprised in the présent invention can be obtained by techniques of genetic recombination likewise well known to the person skilled in the art or else by peptide synthesis by means of, for example, automatic peptide synthesizers such as those supplied by the company Applied Biosystems, etc.
Scaffold proteins that can be related or not to monocional antibodies are understood to mean a 10 protein that contains or not an immunoglobulin fold and that yields a binding capacity similar to a monocional antibody. The man skilled in the art knows how to select the protein scaffold. More particularly, it îs known that, to be selected, such a scaffold should display several features as follow (Skerra, J. Mol. Recogn. 2000, 13, 167-187): phylogenetically good conservation, robust architecture with a well-known three-dimensional molecular organîzatîon (such as, for example, crystallography or NMR), small size, no or only a low degree of post-translational modifications, easy to produce, express and purify. Such a protein scaffold can be, but without limitation, a structure selected from tbe group consisting in fibronectîn and preferentiaily the tenth fibronectin type III domain (FNfnlO), lipocalin, antîcalîn (Skerra, J. Biotechnol. 2001, 74, 257-75), the protein Z dérivative from the domain
B of staphylococcal protein A, thioredoxin A or any protein with a repeated domain such as an “ankyrin repeat” (Kohl et al. PNAS 2003, 100, 1700-1705), “armadillo repeat”, “leucinerich repeat” or “tetratricopeptide repeat”. There couid also be mentioned a scaffold dérivative from toxins (such as, for example, scorpion, insect, plant or mollusc toxins) or protein inhibitors of neuronal nitric oxide synthase (PIN).
The following Examples illustrate the invention but do not limit it in any way. Ail intermediates for preparing Examples are either commercially available or can be obtained by the person skilled in the art using conventionai Chemical reactions described in the iîterature.
GENERAL PROCEDURE
Ali reagents obtained from commercial sources were used without further purification. Anhydrous solvents were obtained from commercial sources and used without further drying.
Column Chromatography
Automated flash column chromatography was performed on ISCO CombiFlash® Rf 200 or CombiFlash® Rf+ Lumen™ using RediSep® Rf Normal-phase Silica Flash Columns (3570pm, 60 A), RediSep Rf Gold® Normal-phase Silica High Performance Columns (20-40pm, 60 Â), RediSep® Rf Reversed-phase C18 Columns (40-63 pm, 60 Â), or RediSep Rf Gold® Reversed-phase C18 High Performance Columns (20-40 pm, 100 Â).
TLC
Thin layer chromatography was conducted with 5 x 10 cm plates coated with Merck Type 60 F254 silica-gel.
Microwave Reactions
Microwave heating was performed with a CEM Discover® SP, or with an Anton Paar 15 Monowave Microwave Reactor.
NMR
1H-NMR measurements were performed on a Bruker Avance ΠΙ 500 MHz spectrometer, a Bruker Avance III 400 MHz spectrometer, or a Bruker DPX-400 spectrometer using DMSOde or CDCI3 as solvent. IH NMR data is in the form of delta values, given in part per million 20 (ppm), using the residual peak of the solvent (2.50 ppm for DMSO-t/ό and 7.26 ppm for
CDCI3) as internai standard. Splitting patterns are designated as: s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), sept (septet), m (multiplet), br s (broad singlet), dd (doublet of doublets), td (triplet of doublets), dt (doublet of triplets), ddd (doublet of doublet of doublets).
Analytical LC-MS
Certain compounds of the présent invention were characterized by high performance liquid chromatography-mass spectroscopy (HPLC-MS) on Agilenl HP1200 with Agilent 6140 quadrupole LC/MS, operating in positive or négative ion electrospray ionisation mode. Molecular weight scan range is 100 to 1350. Parallel UV détection was done al 210 nm and φ 254 nm. Samples were supplied as a I mM solution in ACN, or in THF/H20 (1:1) with 5 pL loop injection. LCMS analyses were performed on two instruments, one of which was operated with basic, and the other with acidic eluents.
Basic LCMS: Gemini-NX, 3 pm, C18, 50 mm x 3.00 mm i.d. column at 23 °C, at a flow rate 5 of 1 mL min-1 using 5 mM ammonium bicarbonate (Solvent A) and acetonitrile (Solvent B) with a gradient starting from 100% Solvent A and finishing at 100% Solvent B over various/certain duration of time.
Acidic LCMS: K1NATEX XB-C18-100A, 2.6 pm, 50 ιηπι*2.1 mm column al 40 °C, at a flow rate of 1 mL min-1 using 0.02% v/v aqueous fonnic acid (Solvent A) and 0.02% v/v 10 fonnic acid in acetonitrile (Solvent B) with a gradient starting from 100% Solvent A and finishing at 100% Solvent B over various/certain duration of time.
Certain other compounds of the présent invention were characterized HPLC-MS under spécifie named methods as follows. For ail of these methods UV détection was by diode array detector at 230, 254, and 270 nm. Sample injection volume was 1 pL. Gradient elutions were 15 run by defîning flow rates and percentage mixtures of the following mobile phases, using
HP LC-grade solvents:
Solvent A: 10 mM aqueous ammonium formate + 0.04% (v/v) formic acid
Solvent B: Acetonitrile + 5.3% (v/v) Solvent A + 0.04% (v/v) formic acid.
Rétention times (RT) for lhese named methods are reported în minutes. Ionisation is recorded 20 în positive mode, négative mode, or positive-negative switching mode. Spécifie details for individual methods follow.
LCMS-V-B methods
Using an Agilent 1200 SL sériés instrument linked to an Agilent MSD 6140 single quadrupole with an ESI-APCI multimode source (Methods LCMS-V-B! and LCMS-V-B2) or 25 using an Agilent 1290 Infinity II sériés instrument connected to an Agilent TOF 6230 with an
ESI-jet stream source (Method LCMS-V-B1); column: Thermo Accucore 2.6 pm, Cl8, 50 mm x 2.1 mm at 55 °C. Gradient details for methods LCMS-V-B 1 and LCMS-V-B2:
Time (min) LCMS-V-B1 LCMS-V-B2 Flow (mL/min)
Solvent A (%) Solvent B (%) Solvent A(%) Solvent B (%)
0 95 5 60 40 1.1
0.12 95 5 60 40 1.3
1.30 5 95 2 98 1.3
1.35 5 95 2 98 1.6
1.85 5 95 2 98 1.6
1.90 5 95 2 98 1.3
1.95 95 5 95 5 1.3
LCMS-V-C method
Using an Agilent 1200 SL sériés instrument linked to an Agilent MSD 6140 single quadrupole with an ESI-APCI multimode source; column: Agilent Zorbax Eclipse plus 3.5 pm, Cl8(2), 30 mm x 2.1 mm at 35 °C. Gradient details for method LCMS-V-C:
Time (min) Solvent A (%) Solvent B (%) Flow (mL/min)
0 95 5 1
0.25 95 5 1
2.50 95 5 1
2.55 5 95 1.7
3.60 5 95 1.7
3.65 5 95 1
3.70 95 5 1
3.75 95 5 1
Préparative HPLC
Certain compounds of the présent invention were purified by high performance liquid chromatography (HPLC) on an Armen Spot Liquid Chromatography or Teledyne EZ system with a Gemini-NX® 10 μΜ C18, 250 mm x 50 mm i.d. column running at a flow rate of 118 5 mL min-1 with UV diode array détection (210 - 400 nm) using 25 mM aqueous NH4HCO3 solution and MeCN or 0.1% TFA in water and MeCN as eluents.
Certain other compounds of the présent invention were purified by HPLC under spécifie named methods as follows:
HPLC-V-A methods
These were performed on a Waters FractionLynx MS autopurification system, with a Gemini® 5 μηι C18(2), 100 mm x 20 mm i.d. column from Phenomenex, running at a flow rate of 20 cm3min1 with UV diode array détection (210^400 nm) and mass-directed collection. The mass spectrometer was a Waters Micromass ZQ2000 spectrometer, operating ïn positive or négative ion electrospray ionisation modes, with a molecular weight scan range 15 of 150 to 1000.
Method HPLC-V-A1 (pH 4):
Solvent A: 10 mM aqueous ammonium acetate + 0.08% (v/v) formic acid; Solvent B: acetonitrile + 5% (v/v) Solvent A + 0.08% (v/v) formic acid
Method HPLC-V-A2 (pH 9):
Solvent A; 10 mM aqueous ammonium acetate + 0.08% (v/v) conc. ammonia; Solvent B: acetonitrile + 5% (v/v) Solvent A + 0.08% (v/v) conc. ammonia
HPLC-V-B methods
Performed on an AccQPrep HP125 (Teledyne ISCO) system, with a Gemini® NX 5 μηι C18(2), 150 mm x 21.2 mm i.d. column from Phenomenex, running at a flow rate of 20 25 cnPmin 1 with UV (214 and 254 nm) and ELS détection.
Method HPLC-V-B1 (pH 4):
Solvent A: water + 0.08% (v/v) formic acid; solvent B: acetonitrile + 0.08% (v/v) formic acid.
Method HPLC-V-B2 (pH 9):
Solvent A: water + 0.08% (v/v) conc. ammonia; solvent B: acetonitrile + 0.08% (v/v) conc. 30 ammonia.
Method HPLC-V-B3 fneutral):
Solvent A: water; Solvent B: acetonitrile.
Analytical GC-MS
Combination gas chromatography and low resolution mass spectrometry (GC-MS) was 5 performed on Agilent 6850 gas chromatograph and Agilent 5975C mass spectrometer using 15 m x 0.25 mm column with 0.25 pm HP-5MS coating and hélium as carrier gas. Ion source: EI+, 70 eV, 230°C, quadrupole: 150°C, interface: 300°C.
High-resolution MS
High-resolution mass spectra were acquired on an Agilent 6230 time-of-flight mass 10 spectrometer equipped with a Jet Stream electrospray ion source in positive ion mode.
Injections of 0.5μ1 were directed to the mass spectrometer at a flow rate 1.5 mi/min (5mM ammonium-formate in water and acetonitrile gradient program), using an Agilent 1290 Infinity HPLC System. Jet Stream parameters: drying gas (N2) flow and température: 8.0 1/min and 325 °C, respectively; nebulizer gas (N2) pressure: 30 psi; capillary voltage: 3000 V;
sheath gas flow and température; 325 °C and 10.0 1/min; TOFMS parameters: fragmenter voltage: 100 V; skimmer potential: 60 V; OCT 1 RF Vpp:750 V. Full-scan mass spectra were acquired over the m/z range 105-1700 at an acquisition rate of 995.6 ms/spectrum and processed by Agilent MassHunter B.04.00 software.
Chemical naming
IGPAC-preferred names were generated using ChemAxon’s ‘Structure to Name’ (s2n) functionality within MarvinSketch oïJChem for Excel (JChem versions 16.6.13 — 18.22.3), or with the Chemical naming functionality provided by Biovia® Draw 4.2.
Abbreviations
Ahx 6-hexanoic acid monomer
AgOTf s il ver tri fluoro nie thanesulfonate
’BuOH tm-butanol
5 cc. concentrated
CyOH cyclohexanol
dba (l£,4£)-l,5-diphenylpenta-l,4-dien-3-one, dibenzylideneacetone
DCM dichloromethane
DIPA TV-isopropyIpropan-2-amine, diisopropylamine
10 DIPEA A-ethyl-X-isopropyl-propan-T-amine, diisopropylethy lamine
DMAP 4-dimethylaminopyridine
ee. enatiomeric excess
eq. équivalent
EtOAc ethyl acetate
15 HFxPyr Hydrogen fluoride pyridine
hs homo sapiens
LDA lithium diîsopropylamide
MeCN acetonitrile
MeOH methanol
20 NMP A-methyl-2'pyrroIidone
Pd(AtaPhos)2Ch bis(di-tert-butyl(4- dimethylaminophenyl)phosphine)dichloropalladium(II)
rt room température
RT rétention time (în minutes)
25 on overnight
TBAF tetrabutylammonium fluoride
TBAOH tetrabutylammonium hydroxide
TBDPS-CI tert-butyl-chloro-diphenyl-silane
TBSC1 tert-butyl-chloro-dimethyl-silane
TEA A/W-diethylethanamine
5 TFA 2,2,2-trifluoroacetic acid
pTSA 4-methylbenzenesulfonic acid
THF tetrahydrofuran
TMP-MgCl 2,2,6,6-tetramethylpiperidinylmagnesium chlorîde lithium chloride complex solution
10 DIAD diisopropylazodicarboxylate
Xantphos 4,5-Bis(diphenyiphosphino)-9,9-dimethylxanthene
BrettPhos 2-(Dicyclohexylphosphino)-3,6-dimethoxy-2',4',6'-triisopropyl-l)l'biphenyl
15 JosiPhos (2R)-l-[(UÏ)-l-(Dicyclohexylphosphino)ethyl]-2(diphenylphosphino)ferrocene
JosiPhos Pd G3 {(R)-l-[(5p)-2-(Dicyclohexylphosphino)ferrocenyljethyldi-tertbutylphosphine}[2-(2'-amino-l,r-biphenyl)]palladium(II) methanesulfonate
20 Xantphos Pd G3 [(4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene)-2-(2'-amino-l,l'biphenyl)]paliadium(II) methanesulfonate
BINAP 2.2'-Bis(diphenylphosphino)-l,l,-binaphthyl
rac-BINAP Pd G3 [(2,2'-Bis(diphenylphosphino)-1,1 '-binaphthyl)-2-(2'-amino- 1,1'bipheny!)]palladium(II) methanesulfonate
Pd(dppf)Ch.CH2C12 [1,1'-Bis(diphenylphosphino)ferrocene]dichloropaliadium(II)
25 Pd2(dba)3 T ris(dibenzy lideneacetone)dipallad ium(0)
Named General Procedures
The following are représentative experimental procedures that are referred to by name in subséquent Préparations.
Sonouashira General Procedure
The mixture of 1 eq. of aryl halogenide, 2 eq. of acetylene, 0.05 eq. of Pd(PPh3)2C12, 0.05 eq. of Cul, and DIPA (1 mL/mmol) în THF (5 mL/mmol) was kept at 60°C. After reachîng an appropriate conversion the volatiles were removed under reduced pressure, the crude intermediate was purified via flash chromatography using heptane / EtOAc as eluents.
Deprotection with HFIP General Procedure
Substrate in HFIP (10 mL/mmol) was kept at 100-120°C in a pressure bottle. After reachîng an appropriate conversion the volatiles were removed under reduced pressure, the crude intermediate was purified via flash chromatography using heptane i EtOAc as eluents.
Deprotection and Hydrolysis General Procedure
The mixture of 1 eq. of substrate and 100 eq. of HFxPyr in MeCN (15 mL/mmol) was stirred 15 at 60°C. After reachîng an appropriate conversion, the volatiles were removed under reduced pressure, the residue was suspended in a 1:1 mixture of THF - water (30 mL/mmol), 150 eq. of LiOH x H2O was added, and the mixture was stirred at rt. After reachîng an appropriate conversion, the volatiles were removed under reduced pressure; the crude product was purified via flash chromatography using DCM and MeOH (containing 1.2% NH3) as eluents.
Alkylation General Procedure
The mixture of 1 eq. of phenol/carbamate, 1-2 eq. of alkyl iodide/bromide, and 2-3 eq. of CS2CO3 in acelone (5 mL/mmol) was stirred at rt for phénols and at 55 °C for carbamates. After reachîng an appropriate conversion the volatiles were removed under reduced pressure, the crude intermediate was purified via flash chromatography using heptane / EtOAc as 25 eluents.
Alkylation with tosvlate General Procedure
An oven-dried vial was equipped with a PTFF-coated magnetic stirring bar, and was charged with 1 eq. tosylate and 5 eq. as the appropriate amine were suspended in MeCN (5 mL/mmol). The reaction mixture was then warmed up to 50°C and stirred at that température untii no further conversion was observed. The réaction mixture was diluted with DCM then it was injected onto a DCM preconditioned silica gel column. Then it was purified via flash chromatography using DCM and MeOH (1.2% NH3) as eluents.
Alkylation of Silyl-Protected Phénols General Procedure
The mixture of 1 eq. of silyi-protected phénol, 1 eq. of alkyl iodide, and 1.15 eq. of TBAF (1 M in THF) in THF (2 mL/mmol) was stirred at rt. After reaching an appropriate conversion the volatiles were removed under reduced pressure, the crude intermediate was purified via flash chromatography using heptane / EtOAc as eluents.
Buchwald General Procedure I
The mixture of 1 eq. of chloro-substrate, 2 eq. of l,3-benzothiazol-2-amine, 0.1 eq. of Pd2(dba)3, 0.2 eq. of XantPhos, and 3 eq. of DI PEA in CyOH (5 mL/mmol) was kept at 140°C. After reaching an appropriate conversion, the reaction mixture was diluted with DCM (10 mL/mmol), injected onto a preconditioned silica gel column and was purified via flash chromatography using heptane / EtOAc as eluents.
Buchwald General Procedure II
The mixture of chloro compound, 2 eq. of l,3-benzothiazol-2-amine, 10 mol% of JosîPhos Pd (G3) and 3 eq. of DIPE suspended in 1,4-dioxane (5 mL/mmol) were stirred at reflux untii no further conversion was observed. Celite was added to the reaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography on 120 20 g silica gel column using heptane-EtOAc or DCM-MeOH (1.2% NH3) as eluents.
Mitsunobu General Procedure
To the mixture of 1 eq. of aliphatic alcohol, 1 eq. of carbamate/phenol, and 1 eq. triphenylphosphine in toluene (5 mL/mmol) was added 1 eq. of di-tert-butyl azodicarboxylate. The mixture was stirred at 50°C for the carbamate and at rt for the phénol. 25 After reaching an appropriate conversion the volatiles were removed under reduced pressure, the crude intermediate was purified via flash chromatography using heptane ! EtOAc as eluents.
Finkelstein General Procedure
The mixture of 1 eq. of alkyl chloride and 2 eq. of Nal in acetone (5 mL/mmol) was kept at 30 reflux. After reaching an appropriate conversion the volatiles were removed under reduced pressure, the crude intermediate was purified via flash chromatography using heptane /
EtOAc as eluenls.
Ouaternary sait formation General Procedure
An oven-dried vial was equipped with a PTFE-coated magnetic stirrîng bar, and was charged with 1 eq. tosylate and 20 eq. as the appropriate amine were suspended in CyOH (5 mL/mmol). The reaction mixture was then warmed up to 140°C and stirred at that température until no further conversion was observed. The reaction mixture was diluted with DCM then it was injected onto a DCM preconditioned silica gel column. Then it was purified via flash chromatography using DCM and MeOH (1.2% NH3) as eluents.
Ouaternary sait deprotection General Procedure
To a THF (5 mL/mmol) solution of the appropriate qualernary sait 3 eq. TB AF was added, and then it was stirred at rt until no further conversion was observed. The reaction mixture was the evaporated to dry under reduced pressure. To a suspension of 1 eq. desalilated quaternary sait in dry MeCN (15 mL/mmol), 100 eq. of HF x Pyr added, and then was stirred at 60°C. After reaching an appropriate conversion, the volatiles were removed under reduced pressure, the residue was suspended in a 1:1 mixture of THF - water (30 mL/mmol), 150 eq. of LiOH x H2O was added, and the mixture was stirred at rt. After reaching an appropriate conversion, the volatiles were removed under reduced pressure. The crude product was purified via flash chromatography using DCM and MeOH (containing 1.2% NH3) as eluents.
Propargylic amine préparation General Procedure
An oven-dried vial was equipped with a PTFE-coated magnetic stirrîng bar, it was charged with 2 eq. PPh3 and 2 eq. imidazole then DCM (5 mL/mmol) was added. To the resulting mixture 2 eq. iodine was added portionwise then stirred for 15 min at rat. To the resulting mixture 1 eq. of the appropriate alcohol was added dissolved in DCM and stirred at rt until no further conversion was observed. To the generated iodo compound 20 eq. of the appropriate amine was added and then stirred for 30 min at rt, while fuil conversion was observed. Celite was added to the reaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography using DCM and MeOH (1.2% NHs) eluents.
Silver catalvzed propargylic amine préparation General Procedure
A 24 ml vial was equipped with a stirrîng bar, and charged with 1 eq. of 2-(3-(1,3benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3-c]pyrÎdazin-8-yl]-5-(3-(420573 ethynyl-2-fluoro-phenoxy)propyl]thiazole-4-carboxylic acid.
eq.
paraformaldehyde/acetone and 20 eq. of the appropriate amine were stirred in dry éthanol (5 ml/mmol) in presence of 20 mol% silver tosyJate at 80°C until no further conversion was observed. Celite was added to the réaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography using DCM and MeOH (1.2% NHa) as eluents.
Hydrolysis General Procedure
The appropriate methyl ester was suspended in a 1:1 mixture of THF - water (5 mL/mmoI) and 10 eq. of LiOH x H2O was added, and the mixture was stirred at 50°C. After reaching an appropriate conversion, the volatiles were removed under reduced pressure; the crude product was purified via flash chromatography using DCM and MeOH (containing 1.2% NH3) as eluents.
Amine substitution and Hydrolysis General procedure
To the product from any of the Préparations 12, 13 and 14 in a 1:1 mixture of acetonitrile and Ar-methyl-2-pyrrolidone (10 ml/mmol), was added the appropriate amine (3-10 eq), and the réaction mixture was stirred at 50 °C for 2-24 h. After the purification of the substitution product by column chromatography (silica gel, using DCM and MeOH as eluents), the product was dissolved in THF (10 ml/mmol), and water (2 ml/mmol) and LiOHxH2O (3-5 equ) was added. Then, the reaction mixture was stirred at 20-40 °C for 1-4 h. The hydrolysed product was purified by préparative HPLC (using acetonitrile and 5mM aqueous NH4HCO3 solution as eluents) to give the desired product.
Préparations
The following experimental details describe the préparation of synthetic intermediates.
Préparation la: Methyl 2-(fort-butoxycarbonylamino)-5-[3-(2-fluoro*4-iodophenoxy)propyl]thiazole-4-carboxylate
Step A: methyl 2-(tert-butoxycarbonylamino)-5-iodo-thiazole-4-carboxylate
50.00 g methyl 2-(terr-butoxycarbonylamino)thiazole-4-carboxylate (193.55 mmol, 1 equiv) was suspended în 600 mL dry MeCN. 52.25 gN-iodo succinimide (232.30 mmol, ) was added and the resulting mixture was stirred overnight at room température.
The reaction mixture was diluted with saturated brine, then it was extracted with EtOAc. The combined organic layers were extracted with 1 M NasSsOs, then with brine again. Then dried over Na2SO4, filtered and the filtrate was concentrated under reduced pressure. The crude product was purified via flash chromatography using heptane as eluent to obtain 60 g of the desired product (156 mmol, 80% Yield).
Ή NMR (400 MHz, DMSO-d6): S ppm 12.03/11.06 (br s), 3.78 (s, 3H), 1.47 (s, 9H); 13C NMR (100 MHz, DMSO-d6) δ ppm 153.8, 82.5, 77.7, 52.3, 28.3; HRMS-ESI (m/z): [M+H]+ calcd for C1ÜH14IN2O4S; 384.9713; found 384.9708.
Step B: methyl 2-(tert-butoxycarbonylamino)-5-(3-hydroxyprop-I -ynyl)thiazole-4carboxylate
A 500 mL oven-dried, one-necked, round-bottom flask was equipped with a PTFE-coated magnetic stirring bar and fitted with a reflux condenser. It was charged with 9.6 g of the product from Step A (25 mmol, 1 equiv), 2.80 g prop-2-yn-l-ol (2.91 mL, 50 mmol, 2 equiv) and 36.10 g DIPA (50 mL, 356.8 mmol, 14.27 equiv) then 125 mL dry THF was added and the system was flushed with argon. After 5 minutes stirring under inert atmosphère 549 mg Pd(PPh3)2Cl2 (L25 mmol, 0.05 equiv) and 238 mg Cul (1.25 mmol, 0.05 equiv) was added. The resulting mixture was then warmed up to 60°C and stirred at that température until no further conversion was observed. Celite was added to the reaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography using heptane and EtOAc as eluents to give 7.30 g of the desired product (23 mmol, 93% Yield) as a yellow solid.
‘H NMR (400 MHz, DMSO-d6): δ ppm 12.1 (br s, 1H), 5.45 (t, 1H), 4.36 (d, 2H), 3.79 (s, φ 3Η), 1.48 (s, 9H); 13C NMR (100 MHz, DMSO-d6) S ppm 12.1 (br s, 1H), 5.45 (t, 1H), 4.36 (d, 2H), 3.79 (s, 3H), 1.48 (s, 9H); HRMS-ESI (m/z): [M+H]+ calcd for CnHnNzOsS: 313.0852, found 313.0866.
Step C: methyl 2-(tert-butoxycarbonylamino)-5-(3-hydroxypropyl)thiazole~4^carboxylate
An 1 L oven-dried pressure bottie equîpped with a ETEE-coated magnetic stir bar was charged with 44.75 g of the product from Step B (143.3 mmol, 1 equiv), 7.62 Pd/C ( 7.17 mmol, 0.05 equiv) in 340 mL éthanol, and then placed under a nitrogen atmosphère using hydrogénation system. After that, it was filled with 4 bar H2 gas and stirred at rt ovemight. Fuli conversion was observed, but only the olefin product was formed. After filtration of the catalysts through a pad of Celite, the whole procedure was repeated with 5 mol% new catalysts. The resulting mixtures were stirred overnîght to get full conversion. Celite was added to the reaction mixtures and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography column using heptane and EtOAc as eluents to give 31.9 g of the desired product (101 mmol, 70.4% Yield) as light-yellow crystals
Ή NMR (500 MHz, DMSO-dô): δ ppm 11.61 (br s, 1H), 4.54 (t, 1H), 3.76 (s, 3H), 3.43 (m, 2H), 3.09 (t, 2H), 1.74 (m, 2H), 1.46 (s, 9H); I3C NMR (125 MHz, DMSO-d6) δ ppm 162.8, 143.1, 135.4, 60.3, 51.9, 34.5, 28.3, 23.4; HRMS-ESI (m/z): [M+H]+ calcd for C^iNzOsS: 317.1165, found 317.1164 (M+H).
Step D: methyl 2-(tert-butoxycarbonylamino) -5-[3-(2-ftuoro-4-iodo- phenoxy)propyl]thiazole-4-carboxylate
A 250 mL oven-dried, one-necked, round-bottomed flask equîpped with a ETEE-coated magnetic stir bar, was charged with 3.40 g 2-fluoro-4-iodo-phenol (14 mmol, 1 equiv), 5.00 g of the product from Step C (16 mmol, 1.1 equiv) and 4.10 g PPh3 (16 mmol, 1.1 equiv) dissolved in 71 mL dry toluene. After 5 min stirring under nitrogen atmosphère, 3.10 mL 25 DIAD (3.20 g, 16 mmol, 1.1 equiv) was added in one portion while the réaction mixture warmed up. Then the reaction mixture was heated up to 50°C and stirred at that température for 30 mîn, when the reaction reached complété conversion.
The reaction mixture was directly injected onto a preconditioned silica gel column, and then it was purified via flash chromatography using heptane and EtOAc as eluents. The crude 30 product was crystalized from MeOH to give 4.64 g of the desired product (9.24 mmol, 66% Yield).
Ή NMR (500 MHz, DMSO-d6) δ ppm 11.64 (br s, 1H), 7.59 (dd, 1H), 7.45 (dd, 1H), 6.98 (t, φ IH), 4.06 (t, 2H), 3.73 (s, 3H), 3.22 (t, 2H), 2.06 (m, 2H), 1.46 (s, 9H); 13C NMR (125 MHz, DMSO-dô) δ ppm 134, 124.9, 117.6, 68.2, 51.9, 30.5, 28.3, 23.2; HRMS-ESI (m/z): [M+H]+ calcd for C19H23N2O5FSI: 537.0350; found 537.0348.
Préparation 1b: Methyl 2-(tert-butoxycarbony lamino)-5-13-[4-[3- [iert5 butoxycarbonyl(methyl)amiïio]prop-l-ynyl]-2-fluoro-phenoxy]propyl]thiazole-4carboxylate
A 500 mL oven-dried, one-necked, round-bottom flask was equipped with a PTFE-co-àted magnetic stirrîng bar and fitted with a reflux condenser. It was charged with 13.41 g Préparation la (25 mmol, 1 equiv), 8.46 g tert-butyl A-methyl-TV-prop-Z-ynyLcarbamate (50 10 mmol, 2 equiv) and 50 mL DIPA (36.10 g, 50 mL, 356.8 mmol, 14.27 equiv) then 125 mL dry THF was added and the System was flushed with argon. After 5 minutes stirrîng under inert atmosphère 549 mg PdfPPhsjzCh (1.25 mmol, 0.05 equiv) and 238 mg Cul (1.25 mmol, 0.05 equiv) were added. The resulting mixture was then warmed up to 60°C and stirred at that température until no further conversion was observed. Celite was added to the reaction 15 mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography using heptane and EtOAc as eluents to give 10.5 g of the desired product (18.2 mmol, 72.7% Yield).
Hl NMR (500 MHz, DMSO-d6) Ô ppm 11.65 (br s, IH), 7.31 (br d, IH), 7.21 (br d, IH), 7.14 (t, IH), 4.23 (s, 2H), 4.1 (t, 2H), 3.73 (s, 3H), 3.23 (t, 2H), 2.86 (s, 3H), 2.07 (m, 2H), 20 1.46/1.41 (s, 18H); 13C NMR (125 MHz, DMSO-dû) δ ppm 129.1, 119.2, 115.4, 68.1, 5 L9,
38.6, 33.8, 30.5, 23.2; HRMS-ESI (m/z): [M+H]+ calcd for C28H37FN3O7S: 578.2330; found 578.2331.
Préparation le: Methyl 2-(tert-butoxycarbonylamino)-5-[3-[4-[3(dimethylamino)prop-l-ynyl]-2-fluoro-phenoxy]propyI]thiazole-4-carboxylate
A 250 mL oven-dried, one-necked, round-bottom flask was equipped with a PTFE-coated magnetic stirrîng bar and fitted with a reflux condenser. It was charged with 5.36 g Préparation la (10 mmol, 1 equiv), 1.66 g AyV-dimethyIprop-2-yn-l-amine (20 mmol, 2 equiv) and 20 mL DIPA (142.7 mmol, 14.27 equiv) then 50 mL dry THF was added and the system was flushed with argon. After 5 minutes stirrîng under inert atmosphère 220 mg
Pd(PPh3)2Ch (0.5 mmol, 0.05 equiv) and 95 Cul (0.5 mmol, 0.05 equiv) were added. The φ resulting mixture was then warmed up to 60°C and stirred at that température until no further conversion was observed. Celite was added to the reaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography using DCM and MeOH (1.2% NH3) as eluents to give 4.5 g of the desired product (7.8 mmol, 78% Yield).
Ή NMR (500 MHz, DMSO-d6) δ ppm 11.66 (s, 1H), 7.29 (dd, 1H), 7.19 (m, 1H), 7.12 (t, 1H), 4.09 (t, 2H), 3.73 (s, 3H), 3.44 (s, 2H), 3.23 (t, 2H), 2.24 (s, 6H), 2.07 (m, 2H), 1.45 (s, 9H); I3C NMR (125 MHz, DMSO-d6) 5 ppm 162.8, 147.3, 129, 119.2, 115.4, 84.3, 68, 51.9, 48.1, 44.2, 30.6, 28.3, 23.2; HRMS-ESI (m/z); [M+H]+ calcd for C24H3iFN3O5S: 492.1962; found 492.1956 (M+H).
Préparation Id: Methyl 2-{[frert-butoxy)carbonyl]amîno}-5-(3-iodopropyl)-l,3thiazole-4-carboxylate
To a solution of the product from Préparation la, Step C (5 g, 15.8 mmol, 1 eq) in diethyl ether (175 mL) and acetonitrile (35 mL) was added imidazole (1.57 mL, 23.71 mmol, 1.5 eq) followed by triphenylphosphine (3.73 g, 14.22 mmol, 1.5 eq) and iodine (6.02 g, 23.71 mmol, 15 1.5 eq). The mixture was stirred at ambient température for 1 h. The réaction was partitioned between ethyl acetate (150 mL) and 10% aqueous sodium thiosulfate (250 mL), and the organic phase was successively washed with water (200 mL) and brine (150 mL), dried (magnésium sulfate) and concentrated in vacuo. The residue was dissolved in diethyl ether and left to âge at frîdge température overnight. The résultant crystals were removed by 20 filtration and the filtrate was concentrated in vacuo. Purification by automated flash chromatography (Combiflash Rf, Silica 80g RediSep column) eluting with a gradient of 0 50% ethyl acetate in Ao-heptane afforded the desired product (5.77 g, 13.53 mmol, 85%) as a whîte solid.
LC/MS (C13H19IN2O4S) 427 [M+H]+; RT 0.88 (LCMS-V-B2)
NMR (400 MHz, DMSO-d6) δ 1 L67 (s, 1 H), 3.79 (s, 3H), 3.29 (t, J = 6.8 Hz, 2H), 3.203.12 (m, 2H), 2.09 (dq, J = 8.7, 6.8 Hz, 2H), 1.48 (s, 9H).
Préparation 2a: 3-(3,6Dichloro-5-methyl-pyridazin-4-yl)propan-l-ol
Step A: [(pent-4-yn-l-yloxy)methyl]benzene
To an oven-dried flask was added 4-pentyn-l-ol (11.1 mL, 119 mmol, 1 eq) in THF (100 mL) and the solution was cooled to 0 °C. Sodium hydride (60% dispersion; 7.13 g, 178 mmol, 1.5 eq) was added portionwise and the mixture was allowed to stir for 30 min at 0 °C before the dropwise addition of benzyl bromide (15.6 mL, 131 mmol, 1.1 eq). The mixture was allowed to warm to ainbient température and was stirred for 16 h, then cooled to 0 °C, quenched with saturated aqueous ammonium chloride (30 mL) and diluted with water (30 mL). The mixture was extracted with ethyl acetate (2 x 150 mL), and the combined organic extracts were washed successively with dilute aqueous ammonium hydroxide ammonium hydroxide (150 mL) and brine (100 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 330 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% ethyl acetate in ûo-heptane afforded the desired product as a yellow liquid (19.5 g, 112 mmol, 94%).
LC/MS (C12H14O) 175 [M+H]+; RT 1.28 (LCMS-V-B1) *H NMR (400 MHz, Chloroform-d) Ô 7.37 - 7.32 (m, 4H), 7.31 - 7.27 (m, IH), 4.52 (s, 2H), 3.58 (t, J = 6.1 Hz, 2H), 2.32 (td, J = 7.1, 2.6 Hz, 2H), 1.95 (t, J = 2.7 Hz, IH), 1.83 (tt, J = 7.1,6.2 Hz, 2H).
Step B: [(hex-4-yn-l -yloxy)methyl]benzene
To an oven-dried flask was added the product from Step A (19.5 g, 112 mmol, 1 eq) and tetrahydrofuran (200 mL) and the solution was cooled to -78 °C. n-Butyllithium (66.9 mL, 135 mmol, 1.2 eq) was added dropwise over 30 min and the reaction was stirred for 1 h then iodomethane (10.5 mL, 168 mmol, 1.5 eq) was added dropwise and the mixture was allowed to warm to 0 °C over 1 h. The reaction was quenched by the addition of saturated aqueous ammonium chloride (40 mL), diluted with water (40 mL), extracted with ethyl acetate (3 x 100 mL), and the combined organic extracts were successively washed with 2M aqueous sodium thiosulfate (200 mL) and brine (200 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 330 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% ethyl acetate in zso-heptane afforded the desired product as a yellow liquid (19.2 g, 0.1 mol, 91%).
LC/MS (CbHiôO) 189 [M+H]+; RT 1.34 (LCMS-V-B1) φ 'H NMR ¢400 MHz, DMSO-d6) δ 7.41 - 7.23 (m, 5H), 4.46 (s, 2Η), 3.48 (t, J - 6.3 Hz, 2H), 2.23 - 2.14 (m, 2H), 1.72 (s, 3H), 1.70-1.65 (m, 2H).
Step C: 4-[3-(benzyloxy)propyl]-3,6-dichloro-5-methylpyridazine
A solution of 3,6-dichloro-l,2,4,5-tetrazine (5 g, 33.1 mmol, 1 eq) and the product from Step 5 B (7.48 g, 39.8 mmol, 1.2 eq) in tetrahydrofuran (30 mL) was heated at 160 °C for 19 h in a sealed flask. The reaction was allowed to cool to ambient température then concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 220 g RediSep™ silica cartridge) eluting with a gradient of 0 - 30% ethyl acetate in rso-heptane afforded the desired product as an orange oil (7.32 g, 23.5 mmol, 71%).
LC/MS (C)5Hi6CkN2O) 311 [M+H]+; RT 1.35 (LCMS-V-B1) *H NMR (400 MHz, DMSO-d6) δ 7.45 - 7.18 (m, 5H), 4.48 (s, 2H), 3.53 (t, J - 5.9 Hz, 2H), 2.96 - 2.83 (m, 2H), 2.42 (s, 3H), 1.88 - 1.69 (m, 2H).
Step D: 3-(3,6-dichloro-5-methylpyrîdazin-4-yl)propan-I -ol
To a cooled solution of the product from Step C (7.32 g, 23.5 mmol, 1 eq) in dichloromethane 15 (100 mL) was added boron trîchloride solution (1 M in dichloromethane; 58.8 mL, 58.8 mmol, 2.5 eq) dropwise and the mixture was allowed to stir at ambient température for 1 h. The reaction was quenched by the addition of methanol and concentrated in vacuo. The residue was partitioned between dichloromethane (100 mL) and saturated aqueous sodium bicarbonate (150 mL), and the organic phase was washed with brine (150 mL), dried 20 (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 80 g RediSep™ silica cartridge) eluting with a gradient of 0 - 80% ethyl acetate in iso-heptane afforded the desired product as a yellow oil (4.19 g, 19 mmol, 81%).
LC/MS (C8H10CI2N2O) 221 [M+H]+; RT 0.84 (LCMS-V-B1) *H NMR (400 MHz, DMSO-d6) 5 4.67 (t, J = 5.1 Hz, 1H), 3.49 (td, J = 6.0, 5.1 Hz, 2H), 2.91 - 2.80 (m, 2H), 2.43 (s, 3H), 1.72 - 1.59 (m, 2H).
Préparation 2b: 2-[iert-butyl(diphenyl)silyl]oxy-3-(3,6-dÎchloro-5-methyl-pyridazin4-yl)propan-l-ol (enantiopure, from Enantiomer 2 ofStepA)
Step A .· ethyl 3-(3,6-dichloro-5-methyl-pyridazin-4-yl) -2-hydroxy-propanoate
Μ Το 3,6-dichloro-4,5-dimethyl-pyridazîne (26.5 g, 150 mmol) în dry THF (375 mL) was added dropwise TMP-MgCl x LiCl (165 mL, 165 mmol, 1.1 eq.) at -78°C, then the resulting mixture was stirred for 2 h at 0 °C. The generated Mg sait was transferred to a solution of ethyl 2oxoacetate (45.9 g, 225 mmol, 1.5 eq.) în dry THF (375 mL) at 0 °C, then it was stirred for 30 5 min at 0 °C. After quenching the reaction with saturated aqueous NH4CI solution and extraction with EtOAc, the combined organic layers were dried, filtered, concentrated, and purified via flash chromatography on silica gel using heptane and EtOAc as eluents to give 11 g (26.3%) of the desired compound,
Ή NMR (500 MHz, DMSO-dfi) δ ppm 5.85 (d, IH), 4.33 (m, IH), 4.12 (q, 2H), 3.19 (d, 2H), 10 2.45 (s, 3H), 1.17 (i, 3H); 13C NMR (125 MHz, DMSO-d6) δ ppm 172.8,157.6, 157.2, 141.4,
139.3, 68.8, 61.2, 35.2, 17.3, 14.4. HRMS-ESI (m/z): [M+H]+ calcd for C10H13CI2N2O3: 279.0303, found 279.0301.
Enantiomers of lhe desired product was separated on a AS-V chiral column (100*500 mm, 20 pm) using 10:90 EtOH-heptane as eluents to give the Enantiomer 1 (eluded first) of 99.6% ee 15 and Enantiomer 2 (eluded last) of 99.1% ee.
Step B: ethyl 2-[tert-butyl(diphenyl)silyl]oxy-3-(3,6-dichloro-5-methyl-pyridazin-4ybpropanoate
To the Enantiomer 2 of Step A (4500 mg, 16 mmol, imîdazole (2200 mg, 2.0 eq.) in THF (81 mL) was added dropwise TBDPS-CI (8900 mg, 2.0 eq.), then it was stirred at rt for 18 h. The 20 product was purified via flash chromatography using heptane and EtOAc as eluents to give the desired product (6200 mg, 74%).
’H NMR (400 MHz, DMSO-dc,) δ ppm 7.52-7.27 (m, 10H), 4.46 (dd, IH), 3.83 (m, 2H), 3.35 (dd, IH), 3.19 (dd, IH), 2.34 (s, 3H), 0.93 (t, 3H), 0.87 (s, 9H).
Step C: 2-[tert-butyl(diphenyl)silyl]oxy-3-(3,6-dichloro-5-methyl-pyridazin-4-yl)propan-I-ol 25 To lhe enantiopure product of Step B (3600 mg, 6.95 mmol) in MeOH (35 mL) was added portionwise NaBH4 (2.63 g, 10 eq.) at 0 °C over a period of 5 min and stirred at thaï température for 30 min. After quenching lhe reaction with the addition of saturated aqueous solution of NH4CI, it was extracted twice with EtOAc. The combined organic layers were dried, filtered, concentrated, and purified via flash chromatography on silica gel using heptane 30 and EtOAc as eluents to give lhe desired product (1.6 g, 48%).
NMR (500 MHz, DMSO-d6) δ ppm 7.57-7.3 (m, 10H), 4.9 (brs, IH), 4.05 (m, IH), 3.38/3.32 (dd+dd, 2H), 3.13/3.11 (dd+dd, 2H), 2.3 (s, 3H), 0.8 (s, 9H); 13C NMR (125 MHz, φ DMSO-dô) ppm 72.7, 65.5, 35.5, 26.9, 17.2; HRMS-ESI (m/z): [M+H]+ calcd for C24H2ç>Cl2N2O2Si: 475.1369, found 475.1362.
Préparation 2c: 2(3,6-DÎcliloro-5-methyl-pyrÎdazin-4-yI)ethanol
Step A : 3-(3,6-dichloro-5-methyl-pyridazin-4-yl)propane-I,2-diol
To 700 mg (2.5 mmol) of the product from Préparation 2b, Step A in 3 mL of methanol was added 285 mg (3 eq.) of NaBH4 at 0 °C and the mixture was stirred at 0 °C for 0.5 h. After quenching the reaction with a saturated solution of NH4CI, the crude product was purified via flash chromatography on silica gel using DCM and MeOH (1.2% NH3) as eluents to give 500 mg (84%) of the dcsîred compound.
ÀH NMR (400 MHz, DMSO-d6) δ ppm 4.90 (bd, 1H), 4.83 (bs, 1H), 3.75 (m, 1H), 3.47 (dd, 1H), 3.38 (m, 1H), 3.00 (dd, 1H), 2.87 (dd, 1H), 2.45 (s, 3H).
Step B: 2-(3,6-dichloro~5~methyl-pyridazin-4-yl)acetaldehyde
To a solution of 237 mg of the product from Step A (1 mmol.) in a 5 mL acetone/H2O (4:1) were cooled to 0 °C, then 427 mg sodium perîodate (2 mmol, 2 eq.) was added portionwise. 15 After 2 h stirring at rt, the mixture was purified by flash chromatography using heptaneEtOAc as eluents to give 200 mg of the dcsîred product (97%).
*H NMR (400 MHz, DMSO-d6) δ ppm 9.71 (s, 1H), 4.27 (s, 1H), 2.35 (s, 3H).
Step C: 2-(3,6-dichloro~5-methyl-pyridazin-4-yl)ethanol
To a solution of 200 mg of the product from Step B (0.97 mmol) in 3 mL of methanol was 20 added in small portions 110 mg (2.92 mmol, 3 eq.) of sodium borohydride at 0 °C. After 15 min stirring, the reaction mixture was diluted with saturated aqueous NH4C1 solution and extracted with EtOAc. The combined organic layers were dried, filtered, concentrated, and purified by flash chromatography using heptane-EtOAc as eluents to give 180 mg (89%) of the desired product.
JH NMR (500 MHz, DMSO-dû) δ ppm 4.9 (t, 1 H), 3.65 (m, 2H), 3 (t, 2H), 2.45 (s, 3H); i3C NMR (125 MHz, DMSO-d6) δ ppm 157.5, 157.2, 140.9, 140.7, 59.1, 34, 17.1; HRMS-ESI (m/z): [M+H]+ calcd for C7H9CI2N2O: 207.0086, found 207.0083.
Préparation 2e: 3-(3,6-l)idiloi'o-5-methvlpviidazin-4-vl)propanal
To an oven-drîed flask was added dimethyl sulfoxide (3.08 mL, 43.4 mmol, 2.4 eq) and dichloromethane (100 mL) and the solution was cooled to -78 °C. Oxalyl chloride (2M in dichloromethane; 13.6 mL, 27.1 mmol, 1.5 eq) was added dropwise and the reaction was 5 allowed to stir for 1 h. A solution of the product from Préparation 2a (4 g, 18.1 mmol, 1 eq) in dichloromethane (20 mL) was then added dropwise and the mixture was allowed to stir for 1 h. Triethylamine (15.1 mL, 109 mmol, 6 eq) was added and the reaction was allowed to warm to 0 °C over 1 h. The reaction was quenched with water (50 mL), then partitioned between saturated sodium bicarbonate (50 mL) and dichloromethane (200 mL), the aqueous phase was extracted with dichloromethane (200 mL), and the combined organic extracts were washed with brine (100 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 50% ethyl acetate in iso-heptane afforded the desired product as an off-white solid (2.27 g, 10.4 mmol, 57%).
LC/MS (C8H8ChN2O) 219 [M+H]+; RT 0.87 (LCMS-V-B1)
Ή NMR (400 MHz, DMSO-d6) δ 9.71 (s, 1H), 3.03 (dd, J = 8.7, 7.0 Hz, 2H), 2.86 - 2.69 (m, 2H), 2.44 (s, 3H).
Préparation 3a: Methyl 2-(3-chloro-4-methyl-6,7-dihydro-5H-pyridoi2,3c]pyridazÎn-8-yl)-5-[3-(2-fluoro-4-iodo-phenoxy)propyl]thiazole-4-carboxylate
Step A: methyl 2-{[(tert-butoxy)carbonyl][3-(3,6-dichloro-5-methylpyridazin-4yl)propylJamino}'5-[3-(2-fluoro-4-iodophenoxy)propyl]-lf3-thiazole-4~carboxylate
Using Mitsunobu General Procedure starting from 4.85 g Préparation la (9.04 mmol, 1 equiv) as the appropriate carbamate and 2 g Préparation 2a (9.04 mmol, 1 equiv) as the appropriate alcohol, 4.6 g of the desired product (69% Yield) was obtained.
41 NMR (500 MHz, DMSO-d6) δ ppm 7.56 (dd, 1H), 7.44 (dm, 1H), 7.08 (m, 2H), 6.96 (t,
1H), 4.05 (t, 2H), 3.75 (s, 3H), 3.21 (t, 2H), 2.82 (m, 2H), 2.4 (s, 3H), 2.06 (m, 2H), 1.88 (m, 2H), 1.48 (s, 9H); I3C NMR (125 MHz, DMSO-d6) Ô ppm 162.7, 157.6, 156.7, 156.5/153.2, 152.2, 147, 142.1, 139.8, 134, 124.9, 117.6, 84, 82.4, 68.1, 52.1, 46.1, 30.4, 28.1, 27.5, 25.8, 23.1, 16.4; HRMS-ES1 (m/z): [M+H]+ calcd for C27H31CI2FIN4O5S: 739.0415, found 30 739.0395.
A Step B: methyl 2’[3-(3,6-didil<>r<>-5-niethyl-pyri(laziii~4-yl)piopylcniiiiioj-5-l3-(2-jli(ot<>-4iodo-phenoxy)propyl]thiazole-4-carboxylate
Using Deprotection with HFIPA General Procedure starting from the product from Step A as the appropriate carbamate, 3.70 g the desired product (97% Yield) was obtained.
Ή NMR (500 MHz, DMSO-d6) δ ppm 7.71 (t, 1 H), 7.59 (dd, 1 H), 7.44 (dm, 1 H), 6.96 (l, 1 H), 4.03 (t, 2 H), 3.7 (s, 3 H), 3.29 (m, 2 H), 3.11 (t, 2 H), 2.84 (m, 2 H), 2.39 (s, 3 H), 2 (m, 2 H), 1.76 (m, 2 H); 13C NMR (125 MHz, DMSO-df,) δ ppm 164.6, 163, 152.3, 147.1, 134.1, 124.8, 117.6, 82.4, 68.1, 51.9, 44, 30.7, 28, 26.9, 23.3, 16.4; HRMS-ESI (m/z): [M+H]+ calcd for C22H23CI2FIN4O3S: 638.9891, found 638.9888.
Step C: methyl 2‘(3-€hloro-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl)-5-13-(2fluoro-4-iodo-phenoxy)propyl]thiazole-4-carboxylate
A suspension of 3 g of the product from Step B (4.69 mmol, 1 eq) and 1.81 g césium carbonate (9.3853 mmol, 2 eq.) were stirred at 80°C for 3 h in 25 mL dry 1,4-dioxane to reach complété conversion. Reaction mixture directly was evaporated to Celite, and then purified by 15 flash chromatography on using DCM-MeOH as eluents to obtain 2.67 g of the title compound (94% Yield).
NMR (500 MHz, DMSO-dô) δ ppm 7.57 (dd, 1H), 7.43 (dm, 1H), 6.97 (t, 1H), 4.23 (t, 2 H), 4.08 (t, 2 H), 3.77 (s, 3 H), 3.22 (t, 2 H), 2.86 (t, 2 H), 2.29 (s, 3 H), 2.08 (m, 2 H), 2.03 (m, 2 H);
13C NMR (125 MHz, DMSO-d6) ô ppm 163.1, 155.4, 152.2, 151.6, 151.2, 147, 142.5, 136,
134.8, 134, 128.9, 124.9, 117.6, 82.3, 68.4, 51.9, 46.3, 30.7, 24.2, 23, 19.7, 15.7; HRMS-ESI (m/z): [M+H]+ calcd for C22H22C1FIN4O3S: 603.0124, found 603.0108.
Préparation 3b: Methyl 5-(3-hydroxypropyl)-2-[4-methyl-3-[(Z)-(3-(2trimethylsilylethoxymethyl)-l,3-benzothiazol-2-ylidene]amino]-6,7-dihydro-5//25 pyrido[2,3-c]pyridazin-8-yl]thiazole-4-carboxylate
Step A: methyl 2-(tert-butoxycarbonylamino)-5-[3-[tert-butyl(dimethyl)silyl]oxyprop~lynylJthiazole-4-carboxylate
AIL oven-dried, one-necked, round-bottomed flask equipped with a PTFE-coated magnetic stir bar was charged with 20 g Préparation la, Step A (52.05 mmol, 1.0 eq.), 17.73 g tert30 butyl-dimethyl-prop-2-ynoxy-silane (21 mL, 104.1 mmol, 2.0 eq.) dissolved in 250 mL dry
A THF/25 mL DIPA and then placed under a nitrogen atmosphère through a gas inlet. Then this solution was charged with 572 mg Pd(PPh3)2Cb (1.30 mmol, 0.025 eq.) and 247 mg Cul (1.30 mmol, 0.025 eq.). The reaction mixture was then warmed up to reflux and stirred at that température untii no further conversion was observed. Celite was added to the reaction mixture and the volatiles were removed under reduced pressure. Then it was purified in two parts via flash chromatography using heptane and EtOAc as eluents to obtain 18.00 g of the desired product (81% Yield).
*H NMR (400 MHz, DMSO-î/6) δ ppm 12.13 (br., 1H), 4.62 (s, 2H), 3.79 (s, 3H), 1.48 (s, 9H), 0.89 (s, 9H), 0.13 (s, 6H); I3C NMR (100 MHz, DMSO-d6) δ ppm 161.2, 52.4, 52.4, 10 28.3, 26.2, -4.6; HRMS-ESI (m/z): [M+H]+ calcd for C19H31N2O5SSK 427.1717, found
427.1711.
Step B: methyl 2-(tert-butoxycarbonylamino)-5-[3-[tertbutyl(dimethyl)silyl]oxypropyl]thiazole-4‘Carboxylate g of the product from Step A (30.42 mmol, 1.0 eq.) was dissolved in 150 mL EtOH and 15 charged with 3.23 g Pd/C (3.04 mmol, 0.1 eq.). A 250 mL oven-dried autoclave equipped with a FTFE-coated magnetic stir bar was charged with the solution, and then placed under a nitrogen atmosphère usîng the hydrogénation system. After that it was filled with 10 bar H2 gas. After 2 hours stirring at rt the reaction reached complété conversion. Celite was added to the reaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography heptane and EtOAc as eluents to obtain 9.95 g of the desired product (78% Yield).
*H NMR (500 MHz, DMSO-dô) δ ppm 11.62 (br., 1H), 3.76 (s, 3H), 3.62 (t, 2H), 3.12 (t, 2H), 1.78 (quint., 2H), 1.46 (s, 9H), 0.86 (s, 9H), 0.03 (s, 6H); 13C NMR (125 MHz, DMSOd6) δ ppm 162.8, 62, 51.9, 34.3, 28.3, 26.3, 23.3, -4.9; HRMS-ESI (m/z): [M+H]+ calcd for 25 C19H35N2O5SS1: 431.2030, found 431.2025.
Step C: methyl 2-[tert-butoxycarbonyl-[3-(3,6-dichloro-5-methyl-pyridazin -4yl)propyl]amino]-5-[3-[tert-butyl(dimethyl)silyl]oxypropyllthiazole-4~carboxylate
Using Mitsunobu General Procedure starting from 9.91 g of the product from Step B (23.0 mmol, 1 eq.) as the appropriate carbamate and 5.1g Préparation 2a (23.0 mmol, 1 equiv) as 30 the appropriate alcohol, 13.02 g of the desired product (89% Yield) was obtained.
Ή NMR (500 MHz, DMSO-tfc) δ ppm 4.09 (t, 2H), 3.77 (s, 3H), 3.61 (t, 2H), 3.12 (t, 2H), 2.82 (t, 2H), 2.41 (s, 3H), 1.88 (qn, 2H), 1.79 (qn, 2H), 1.39 (s, 9H), 0.85 (s, 9H), 0.02 (s, φ 6H); 13C NMR (125 MHz, DMSO-d6) Ô ppm 162.8, 157.7, 156.3, 156.1, 152.8, 144.5, 142.1, 139.9, 135.3, 79.4, 62.1, 52.1, 46.1, 34.1, 28.6, 27.5, 26.3, 25.9, 23.2, 18.4, 16.4, -4.9; HRMS-ESI (m/z): [M+H]+ calcd for CsvHwChNiOsSSi; 633.2095, found 633.2091.
Step D: methyl 5-[3-[tert-butyl(dimethyl)silyl]oxypropyl]-2~[3-(3,6-dichloro-5-methyl- pyridazin M-yl) propylaminoJthiazole-4-carboxylate
Using Deprotection with HFIPA General Procedure starting from the product from Step C as the approprïate carbamate, 10.4 g of the desired product (95% Yield) was obtained.
*H NMR (500 MHz, DMSO-iZe) δ ppm 7.69 (t, 1H), 3.71 (s, 3H), 3.60 (t, 2H), 3.30 (q, 2H), 3.01 (t, 2H), 2.85 (t, 2H), 2.41 (s, 3H), 1.78 (qn, 2H), 1.71 (qn, 2H), 0.86 (s, 9H), 0.02 (s, 6H);
I3C NMR (125 MHz, DMSO-d6) δ ppm 164.3, 163.1, 157.7, 156.9, 142.5, 140.0, 137.6, 136.5, 62.0, 51.7, 44.1, 34.4, 28.0, 26.9, 26.3, 23.4, 18.5, 16.5, -4.9; HRMS-ESI (m/z): [M+H]+ calcd for CszHssCk^ChSSi: 533.1570, found 533.1566.
Step E: methyl S-[3-[tert-butyl(dimethyl)silyl]oxypropyl]-2-(3-chloro-4-methyl-6,7-dihydro5H-pyrido[2,3-c]pyridazin-8-yl)thiazole-4-carboxylate
A 250 mL oven-dried one-necked, round-bottom flask equîpped with a PTFE-coated magnetic stir bar was charged with 10.4 g of the product from Step D (19.57 mmol, 1.0 eq.), 12.75 g CS2CO3 (39.13 mmol, 2.0 eq.) and 100 mL dry 1,4-dioxane. The reaction mixture was then warmed up to reflux température and stirred at that température for 8 h. Celite was added to the réaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography using heptane and EtOAc as eluents to obtain 6.40 g of the desired product (66% Yield).
*H NMR (500 MHz, DMSO-rfô) δ ppm 4.26 (t, 2H), 3.79 (s, 3H), 3.65 (t, 2H), 3.14 (t, 2H), 2.89 (t, 2H), 2.32 (s, 3H), 2.04 (m, 2H), 1.82 (m, 2H), 0.87 (s, 9H), 0.04 (s, 6H); 13C NMR (125 MHz, DMSO-dô) δ ppm 163.1, 155.3, 151.8, 151.3, 143.4, 136.1, 134.6, 129.0, 62.1, 25 52.0, 46.3, 34.4, 26.3, 24.2, 23.1, 19.7, 15.7, -4.8; HRMS-ESI (m/z): [M+H]+ calcd for
C22H34CIN4O3SSÎ: 497.1804, found 497.1796.
Step F: methyl 2f3-(l,3-benzothiazol-2-ylamino)-4-methyl-6r 7-dihydro-5H-pyrido[2,3c!pyridazin -8-ylj~5-[3-[tert'butyl(dimethyl)silyl]oxypropyl] thiazole-4-carboxylate
A 250 mL oven-dried, one-necked, round-bottom flask was equîpped with a PTFE-COated magnetic stirring bar and fitted with a reflux condenser. It was charged with 6.43 g of the product from StepE (12.94 mmol, 1.0 eq.), 3.88 g l,3-benzothiazol-2-amine (25.87 mmol, 2.0 equiv) and 6.75 mL DIPEA (38.81 mmol, 3.0 eq.) then 65 mL CyOI-I was added. And then the System was flushed with argon. After 5 minutes stirrîng under inert atmosphère 1.18 g Pd2(dba)j (1.29 mmol, 0.1 eq.) and 1.49 g XantPhos (2.587 mmol, 0.2 eq.) were added. The resulting mixture was then warmed up to 140°C and stirred at that température for 1 hour to 5 reach complété conversion. The reaction mixture was diluted with DCM, and directly injected onto a preconditioned silica gel column, and then il was purified via flash chromatography using heptane and EtOAc as eluents to obtain 6.85 g of the desired product (87% Yield).
1H NMR (500 MHz, DMSO-e/6) δ ppm 7.82 (br., IH), 7.52 (br., IH), 7.37 (t, IH), 7.19 (t, IH), 4.25 (t, 2H), 3.80 (s, 3H), 3.66 (t, 2H), 3.16 (t, 2H), 2.87 (t, 2H), 2.33 (s, 3H), 2.04 (m, 10 2H), 1.84 (m, 2H), 0.92 (s, 9H), 0.07 (s, 6H); 13C NMR (125 MHz, DMSO-d6) δ ppm 163.2,
155.6, 148.8, 148.6, 142.3, 134.5, 127.6, 126.5, 122.5, 122, 62.0, 51.9, 46.3, 34.4, 26.4, 23.9,
22.9, 20.3, 12.8, -4.8; HRMS-ES1 (m/z): [M+H]+ calcd for QwHyjNsOsSzSi: 611.2288, found 611.2284.
Step G: methyl 5-[3-[tert-butyl(diniethyl)silyl]oxypropyl]-2-[4-m^ 3-(215 trimethylsilylethoxymethyl)-l,3-benzothiazol-2-ylidene]amino]-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]thiazole-4-carboxylate
5.00 g of the product from Step F (8.18 mmol, 1.0 eq.) was dissolved in 50 mL dry DCM and 50 mg DMAP (0.41 mmol, 0.05 eq.) and 2.85 mL DIPEA (16.37 mmol, 2.0 eq.) was added at 0°C. Then 2.24 mL 2-(chloromethoxy)ethyl-trimethyl-silane (12.69 mmol, 1.5 eq.) was added 20 over 5 minutes period of time at 0°C, and the resulting mixture was put in the frîdge for a night, while complété conversion was observed. Celite was added to the reaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography using heptane and EtOAc as eluents to obtain 3.85 g of the desired product (63% Yield).
‘H NMR (500 MHz, DMSO-dô) δ ppm 7.6-7.15 (m, 4H), 5.83 (s, 2H), 4.42 (t, 2H), 3.92 (s, 3H), 3.74 (t, 2H), 3.73 (t, 2H), 3.24 (t, 2H), 2.86 (t, 2H), 2.37 (s, 3H), 2.12 (m, 2H), 1.97 (m, 2H), 0.96 (t, 2H), 0.95 (s, 9H), 0.1 (s, 6H), -0.07 (s, 9H); Ï3C NMR (125 MHz, DMSO-d6) δ ppm 163.6, 157,7, 156.4, 154.7, 148.5, 143.7, 137.6, 134.1, 132.6, 126.1, 125.6, 73.2, 66.9,
62.5, 51.9, 46, 34.3, 26.1, 24.2, 23.4, 20.6, 18.0, 12.9, -1.4, -5.2; HRMS-ESI (m/z): [M+H]+ 30 calcd for C35H53N6O4S2S12: 741.3102, found 741.3098.
Step H: methyl S-(3-hydroxypropyl)-2-[4-methyl-3-[(Z)-[3-(2-trlmethylsilylethoxymethyl)l,3-benzothiazol-2-ylidene]amino]-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl]thiazole-420573 carboxylate
3.85 g of the product from Step G (5.19 mmol, 1.0 eq.) and 362 mg camphor sulfonic acid (1.56 mmol, 0.3 eq.) were dissolved in 40 mL DCM/MeOH (2:1). The reaction mixture was then warmed up to 50 °C and stirred at that température overnight. The réaction reached 5 complété conversion, The reaction mixture was cooled to room température and quenched by the addition of saturated aqueous NaHCCh solution and then then il was extracted with EtOAc for two times. Celite was added to the combined organic layers and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography using heptane and EtOAc as eluents to give 2.50 g title compound (76% Yield).
Ή NMR (500 MHz, DMSO-ί/σ) δ ppm 7.83 (dm, 1H), 7.44 (dm, 1H), 7.42 (m, 1H), 7.23 (m, 1H), 5.84 (s, 2H), 4.57 (brs, 1H), 4.26 (t, 2H), 3.80 (s, 3H), 3.72 (m, 2H), 3.48 (t, 2H), 3.14 (m, 2H), 2.86 (t, 2H), 2.36 (s, 3H), 2.04 (m, 2H), 1.81 (m, 2H), 0.91 (m, 2H), -0.11 (s, 9H); i3C NMR (125 MHz, DMSO-d6) Ô ppm 127.1, 123.3, 123.2, 111.9, 72.9, 66.7, 60.6, 51.9, 46.4, 35.0, 23.8, 23.2, 20.4, 17.8, 13, -1.0; HRMS-ESI (m/z): [M+H]+ calcd for 15 CzsHmNôO+SzSî: 627.2237, found 627.2236.
Préparation 3c: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/7pyrido[2,3-c]pyridazin-8-yl]-5-[3-(4-ethynyl-2-nuoro-phenoxy)propyl]thiazole-4carboxylic acid
Step A: methyl2-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3-cJpyridazin-8-yl)-5-[3-[220 fluoro-4-(2-trimethylsilylethynyl)phenoxy] propyl}thiazole-4-carboxylate
A 250 mL oven-dried, one-necked, round-bottom flask was equipped with a PTFE-coated magnetic stirring bar and fitted with a reflux condenser. It was charged with 5 g Préparation 3a (8.29 mmol, 1 eq.), 2.34 mL ethynyl(trimethyl)silane (16.58 mmol, 2 eq.) and 10 mL DIPEA, then 40 mL dry THF was added and the system was flushed with argon. After 5 25 minutes stirring under inert atmosphère 182 mg Pd(PPh3)2Cb (0.41 mmol, 0.05 eq.) and 79 mg (0.41 mmol, 0.05 eq.) were added. The resulting mixture was then warmed up to 60°C and stirred at that température for 2 hours to reach complété conversion. Celite was added to the reaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography using Heptane-EtOAc as eluents to give 4.26 g of the desired 30 product (89% Yield).
ΣΗ NMR (500 MHz, DMSO-îZû) δ ppm 7.31 (dd, 1H), 7.23 (dn, 1H), 7.13 (t, 1H), 4.25 (t, φ 2Η), 4.12 (t, 2H), 3.77 (s, 3H), 3.24 (t, 2H), 2.87 (t, 2H), 2.31 (s, 3H), 2.1 (m, 2H), 2.03 (m, 2H), 0.21 (s, 9H); 13C NMR (125 MHz, DMSO-dô) δ ppm 163.0, 155.3, 151.7, 151.3, 136.1, 129.4, 129.0, 119.4, 115.3, 104.6, 93.7, 68.2, 51.9, 46.3, 30.7, 24.1, 23.0, 19.7, 15.7, 0.4; HRMS-ESI (m/z): [M]+ calcd for C27H30ClFN4O3SSi: 572.1481, found 572.1480.
Step B: methyl 2~[3-(l,3-benzothiazol~2-ylamino)-^methyl-ô,7-dihydro-5H-pyrido/2,3c]pyridazin~8-yl]-5-[3-[2-fluoro-4-(2-trimethylsilylethynyl) phenoxy]propyl]thiazole-4carboxylate
A 100 mL oven-dried, one-necked, round-bottom flask with a PTFE-coated magnetic stirring bar was charged with 4.25 g of the product from Step A {7 A mmol, 1.0 eq.), 2.23 g 1,310 benzothiazol-2-amine (14.8 mmol, 2.0 eq.) and 3.87 mL DIPEA (2.87 mg, 22.2 mmol, 3.0 eq.) then 40 mL cyclohexanol was added and the System was flushed with argon. After 5 minutes stirring under inert atmosphère 679 mg Pd2(dba)3 (0.74 mmol, 0.10 eq.) and 858 mg XantPhos (1.48 mmol, 0.20 eq.) were added. The resulting mixture was then warmed up to 140°C and stirred at that température for 30 min to reach complété conversion. The reaction 15 mixture was diluted with DCM and directly injected onto a preconditioned silica gel column, and then it was purified via flash chromatography using heptane and EtOAc as eluents. The pure fractions were combined and concentrated under reduced pressure to give 3.90 g of the desired product (77% Yield).
*H NMR (500 MHz, DMSO-d6) δ ppm 12.27/10.91 (brs, 1 H), 8.1-7.1 (brin, 4H), 7.34 (dd, 20 IH), 7.24 (dm, IH), 7.16 (t, IH), 4.25 (t, 2H), 4.15 (t, 2H), 3.78 (s, 3H), 3.28 (t, 2H), 2.87 (t, 2H), 2.34 (s, 3H), 2.13 (m, 2H), 2.04 (m, 2H), 0.19 (s, 9H); HRMS-ESI (m/z): [M+H]+ calcd for C34H36FN6O3S2Si: 687.2038, found 687.2020.
Step C: 2f3-(l,3-henzothiazol~2-ylamino)-4-methyl-6,7-dihydro-5H-pyridol2,3-c]pyridazin8-yl]~5-[3-(4-ethynyl-2-fluoro-phenoxy)propyl]thiazole~4-carboxylic acid
A 10 mL oven-dried, one-necked, round-bottom flask was equipped with a PTFE-coated magnetic stirring bar and fitted with a reflux condenser. It was charged with 343 mg of the product from Step B (0.5 mmol, 1.0 eq.) dîssolved in 2.5 mL THF/H2O (4:1). Then 105 mg LiOH x H2O (2.50 mmol, 5.0 eq.) was added and the resulting mixture was heated to 60°C and stirred for 4 h at this temp. The reaction reached complété conversion. Celite gel was 30 added to the reaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography using DCM and MeOH (1.2% NH3) as eluents to give 200 mg title compound (66% Yield).
φ Ή NMR (500 MHz, DMSO-î/6) δ ppm 7.88 (d, IH), 7.49 (br., IH), 7.37 (t, IH), 7.36 (dd, IH), 7.25 (dm, IH), 7.19 (t, IH), 7.16 (t, IH), 4.27 (t, 2H), 4.15 (t, 2H), 4.11 (s, IH), 3.27 (t, 2H), 2.87 (t, 2H), 2.33 (s, 3H), 2.14 (m, 2H), 2.04 (m, 2H); 13C NMR (125 MHz, DMSO-d6) Ô ppm 164.2, 151.5, 147.9, 129.4, 126.5, 122.5, 122.3, 119.5, 115.5, 114.5, 82.9, 80.5, 68.5, 5 46.2, 31.0, 23.9, 23.1, 20.3, 12.9; HRMS-ESI (m/z): [M+H]+ calcd for C30H26FN6O3S2:
601.1486, found 601.1498.
Préparation 3d: Methyl 2-[3-(1,3-benzothiazol-2-y lammo)-4-methy 1-6,7-dihydro5/7-pyrido[2,3-c]pyndazin-8-yl]-5-[3-[2-fluoro-4-(3-hydroxyprop-lynyl)phenoxy]propyl]thiazole-4-carboxylate
Step A: methyl 5-/3-/4-13-/tert-butyl(dimethyl)silyl]oxyprop-l-ynyl]-2-fiuorophenoxy]propylJ-2-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3-cJpyridazin -8-yl)thiazole4-carboxylate
Using Sonogashira General Procedure starting from 4.00 g of Préparation 3a (6.63 mmol, 1.0 eq.) and 2.26 g tert-bittyl-dimethyl·prop-2-ynoxy-silane (13.27 mmol, 2 eq.) as the 15 appropriate acetylene, 2.80 g of the desired product (65% Yield) was obtained.
NMR (500 MHz, DMSO-dâ) δ ppm 7.27 (dd, IH), 7.19 (dd, IH), 7.14 (t, IH), 4.51 (s, IH), 4.25 (m, 2H), 4.12 (t, 2H), 3.77 (s, 3H), 3.24 (t, 2H), 2.87 (t, 2H), 2.3 (s, 3H), 2.1 (quint., 2H), 2.03 (m, 2H), 0.88 (s, 9H), 0.12 (s, 6H); I3C NMR (125 MHz, DMSO-d6) δ ppm 163.0, 128.9, 119.1, 115.5, 68.3, 52.1, 51.9, 46.3, 30.7, 26.2, 24.2, 23.0, 19.7, 15.7, -4.6; HRMS20 ESI (m/z): [M+H]+ calcd for Csi^ClFNÆriSSi: 645.2128, found 645.2120.
Step li: methyl 2-[3-(I,3-benzothiazol-2-ylatnino)-4-methyl-6,7-dihydro-5H‘pyrido[2,3cIpyridazin -8-yl]-5-[3-[4-[3ftert-butyl(dimethyl) silylIoxyprop-l-ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylate
Using Buchwald General Procedure II starting from 2.8 g of the product from Step A (4.34 25 mmol, 1.0 eq.) and 1.30 g l,3-benzothiazol-2-amine (8.67 mmol, 2.0 eq.), 2.1 g of the desired product (64% Yield) was obtained.
*H NMR (500 MHz, DMSO-d6) δ ppm 12.25/10.91 (brs IH), 7.88 (br, IH), 7.51 (br, 1H), 7.37 (t, IH), 7.29 (dd, IH), 7.2 (t, IH), 7.2 (dd, IH), 7.17 (t, IH), 4.49 (s, 2H), 4.25 (t, 2H), 4.14 (t, 2H), 3.77 (s, 3H), 3.27 (t, 2H), 2.86 (t, 2H), 2.32 (s, 3H), 2.13 (qn, 2H), 2.04 (qn, 2H), 30 0.87 (s, 9H), 0.1 (s, 6H); 13C NMR (125 MHz, DMSO-d6) δ ppm 163.2, 155.7, 151.6, 148.5, φ 147.6, 141.5, 128.9, 127.6, 126.5, 122.5, 122.3, 119.1, 116.9, 115.5, 114.8, 88.2, 84, 68.4, 52.1, 51.9, 46.4, 31, 26.2, 24, 23.1, 20.4, 12.9, -4.6; HRMS-ESI (m/z): [M+H]+ calcd for C38H44FN6O4S2Si: 759.2613, found 759.2609.
Step C: methyl 2~[3~(l,3-benzothiazol-2-ylamino) -4-methyl-6,7-dihydro-5H-pyrido[2,35 c]pyridazin-8‘yl]dF[3f2fluoro-4f3-hydroxyprop~l-ynyl)phenoxy]propyl] thiazole-4carboxylate
A 100 mL oven-dried, one-necked, round-bottom flask was equipped with a ETEE-coated magnetic stirring bar and fitted with a reflux condenser. It was charged with 2.10 g of the product from Step B (2.76 mmol, 1.0 eq.) dissolved in 15 mL THF. Then 3.32 mL TB AF 10 (3.32 mmol, 1.2 eq., 1 M in THF) was added dropwise via syringe over a period of 2 minutes, and stirred at that température for 30 min. The reaction mixture was quenched with saturated NH4C1, then directly evaporated to Celite and it was purified via flash chromatography using heptane- EtOAc as eluents to give 1.6 g of the desired product (90% Yield).
*H NMR (500 MHz, DMSO-d6) δ ppm 11.14 (brs, 1H), 7.83 (brd, 1H), 7.49 (brs, 1H), 7.36 15 (m, 1H), 7.24 (dd, 1H), 7.19 (m, 1H), 7.18 (dm, 1H), 7.15 (t, 1H), 5.08 (t, 1H), 4.28 (m, 2H),
4.27 (d, 2H), 4.17 (t, 2H), 3.8 (s, 3H), 3.29 (m, 2H), 2.89 (m, 2H), 2.35 (s, 3H), 2.15 (m, 2H), 2.07 (m, 2H); HRMS-ESI (m/z): [M+H]+ calcd for C32H30FN6O4S2: 645.1748, found 645.1738.
Préparation 3e; Methyl 2-[3-(l,3-benzothiazol-2-ylammo)-4-methyl-6,7-dihydro20 5if-pyrido[2,3-clpyridazin-8-yl]-5-[3-[2-fluoro-4-(3hydroxypropyl)phenoxy]propyl]thiazole-4-carboxylate
Step A: methyl 2-(tert~butoxycarbonylamino)-5-[3~[4-[3-[tert-butyl(dimethyl)silyl]oxypropl-ynyl]^2‘fluoro-phenoxy]propyl]thiazole-4-carboxylate
Using Sonogashira General Procedure starting from 4.00 g of Préparation la (7.45 mmol, 25 1.0 eq.) and 2.54 g tert-butyl-dimethyl-prop-2-ynoxy-silane (14.90 mmol, 2.0 eq.) as the appropriate acetylene, 1.70 g of the desired product (39% Yield) was obtained.
Ή NMR (500 MHz, DMSO-ds) δ ppm 11.64 (s, 1H), 7.27 (dd, IH), 7.19 (dm, 1H), 7.14 (t, 1H), 4.51 (s, 2H), 4.1 (t, 2H), 3.73 (s, 3H), 3.23 (t, 2H), 2.07 (m, 2H), 1.46 (s, 9H), 0.89 (s, 9H), 0.12 (s, 6H); 13C NMR (125 MHz, DMSO-d6) δ ppm 88.2, 83.8.
Step B: methyl 2-(tert-butoxycarbonylamino)-5-l3-[4-(3-[tertbutyl(dimethyl)silyl]oxypropyl]-2‘fluoro-phenoxy]propyl]thiazole-4-carboxylate
A 50 mL oven-dried autoclave was equipped with a PTFE-coated magnetic stirring bar. It was charged with 1.70 g of the product from Step A (2.9 mmol, 1.0 eq.), 310 mg Pd/C (0.29 5 mmol, 0.10 eq.) and 15 mL éthanol, and then inertized using vacuum and nitrogen, finally fi lied with 10 bar pressure hydrogen gas. Then the mixture was stirred at rt température for 3 hours to reach complété conversion. Celite was added to the reaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography using heplane and EtOAc as eluents to give 1.2 g of the desired product (70% Yield).
lH NMR (500 MHz, DMSO-ifc) δ ppm 11.64 (br„ 1H), 7.02 (t, 1H), 7.01 (d, 1H), 6.89 (d, 1H), 4.02 (t, 2H), 3.74 (s, 3H), 3.54 (t, 2H), 3.22 (t, 2H), 2.54 (t, 2H), 2.04 (quint., 2H), 1.70 (quint., 2H), 1.45 (s, 9H), 0.85 (s, 9H), 0 (s, 6H); 13C NMR (125 MHz, DMSO-d&) δ ppm 162.8, 156.2/153.5, 152.0, 144.7, 141.9, 135.8, 135.5, 124.6, 116.2, 115.5, 68.1, 62.0, 51.9, 34.3, 30.8, 30.8, 28.3, 26.2, 23.2, -4.9.
Step C: methyl 2-[tert-butoxycarbonyl-[3-(3,6-dichloro-5-methyl-pyridazin-4yl)prppyl]amino]-5-[3-[4-[3-[tert-butyl(dimethyl)silyl]oxypropyl]-2-fluorophenoxyJpropyl]thiazole-4-carboxylate
Using Mitsunobu General Procedure starting from 1.16 g of the product from Step B (2.0 mmol, 1.0 eq.) as the appropriate carbamate and 484 mg of Préparation 2a (2.2 mmol, 1.1 20 eq.) as the appropriate alcohol, 1.2 g of the desired product (77% Yield) was obtained.
*H NMR (500 MHz, DMSO-ife) δ ppm 7.02 (m, 1H), 6.99 (d, 1H), 6.89 (m, 1H), 4.08 (t, 2H), 4.02 (t, 2H), 3.75 (s, 3H), 3.54 (t, 2H), 3.22 (t, 2H), 2.81 (t, 2H), 2.53 (t, 2H), 2.40 (s, 3H), 2.05 (quint., 2H), 1.87 (m, 2H), 1.70 (quint., 2H), 1.48 (s, 9H), 0.85 (s, 9H), 0.00 (s, 6H); 13C NMR (125 MHz, DMSO-dô) δ ppm 162.7, 156.4/153, 152.0, 144.7, 143.6, 142/139.8, 141.9, 25 135.5, 124.6,116.2, 115.4, 68.1, 62.0, 52.0, 46.1, 34.2, 30.8, 30.7, 28.0, 27.5, 26.2, 25.8, 23.2,
16.4, -4.9;
Step D; methyl 5-[3-[4-[3-[tert-butyl(dimethyl)silyl]oxypropyl]-2-fluoro-phenoxy]propyl]-2[3-(3,6-dichloro-5-methyl-pyridazin-4-yl)propylamino]thiazole-4-carboxylate
Using Deprotection with HFIPA General Procedure starting from 1.2 g of the product 30 from Step C as the appropriate carbamate, 790 mg of the desired product (75% Yield) was obtained.
B Step E: methyl 5-[3-[4-[3-[tert-butyl(dimethyl) silyljoxypropyl]-2-fluoro-phenoxy]propyl]-2(3-chlorO'4-methyl-6,7-dihydro-5H-pyrido[2,3-cJpyridazinS-yl) thiazole-4-carboxylate
A 25 mL oven-dried pressure bottle equipped with a PTFF-coated magnetic stir bar was charged with 1.2 g of the product from Step D (1.75 mmol, 1.0 equiv) and 680 mg cesiunt 5 carbonate (3.50 mmol, 2.0 equiv) suspended in 10 mL 1,4-dioxane. The reaction mixture was then warmed up to 80°C and stirred at that température for 3 h, when the reaction reached complété conversion. Celite was added to the reaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography DCM and MeOH (containing 1.2% NH3) as eluents to give 1.0 g of the desired product (88% Yield).
Steo F: methyl 2-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-l3-{4-[3-[tert-butyl(dimethyl)silyl]oxypropyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylate
Using Buchwald General Procedure II starting from 630 mg of the product from Step E (0.97 mmol, 1.0 eq.) and 291 mg l,3-benzothiazol-2-amine (1.94 mmol, 2.0 eq.), 600 mg of 15 the desired product (81%) was obtained.
Step G: methyl 2-[3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-[3-[2-fluoro-4-(3-hydroxypropyl)phenoxyjpropyl]thiazole-4-carboxylate
A 250 mL oven-dried, round-bottom flask was equipped with a PTFF-coated magnetic stirring bar. It was charged with 600 mg of the product from Step F (0.78 mmol, 1.0 eq.) 20 dissolved in 10 mL THF, and then 936 uL TBAF (0.963 mmol, 1.2 eq.) was added dropwise.
After 1 hour stirring full conversion was observed. Then reaction mixture was quenched with saturated aqueous NH4C1 solution, Celite was added to the reaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography using heptane and EtOAc and MeOH (1,2% NH3) as eluents to give 450 mg of the desired product 25 (89% Yield).
NMR (500 MHz, DMSO-dû) δ ppm 7.87 (br, IH), 7.49 (br, IH), 7.37 (t, IH), 7.19 (t, IH), 7.06 (m, IH), 7.05 (d, IH), 6.92 (dd, IH), 4.44 (br, IH), 4.25 (t, 2H), 4.08 (t, 2H), 3.78 (s, 3H), 3.36 (l, 2H), 3.27 (t, 2H), 2.85 (t, 2H), 2.52 (t, 2H), 2.32 (s, 3H), 2.1 (qn, 2H), 2.04 (qn, 2H), 1.65 (qn, 2H); 13C NMR (500 MHz, dmso-d6) δ ppm 163.2, 155.6, 152.0, 148.5, 144.7, 30 141.7, 135.9, 134.8, 127.6, 126.5, 124.7, 122.5, 122.3, 116.3, 116.0, 115.6, 68.6, 60.4, 52.0,
46.4, 34.6, 31.2, 31.0, 23.9, 23.2, 20.4, 12.9.
B Préparation 3f: Ethyl 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-niethyl-5Ff,6Ff,7/7,8/fpyrido[2,3-i?]pyridazin-8-yl}-l,3-thiazole-4-carboxylate
Step A: ethyl 2-[(hex-4-yn-l-yl)amino]-l,3-thiazole-4-carboxylate
To a solution of ethyl 2-bromo-l,3-thiazole-4-carboxylate (1.17 g, 4.97 mmol, 1 eq) in 5 acetonitrile (16 mL) was added hex-4-yn-l-amine (725 mg, 7.46 mmol, 1.5 eq) and triethylamine (1.04 mL, 7.46 mmol, 1.5 eq) and the mixture was heated at 150 °C for 4 h under microwave irradiation. The reaction was partitioned between ethyl acetate and brine, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) 10 eluting with a gradient of 0 - 60% ethyl acetate in iso-heptane afforded the desired product as a beige solid (741 mg, 2.94 mmol, 59%).
LC/MS (Ci2Hi6N2O2S) 253 [M+H]+; RT 2.32 (LCMS-V-C)
Step B: ethyl 2f3-chloro-4-methyl-5H,6H,7H,8H-pyrido[2,3-cjpyridazin-8-yl}-l,3-thiazole4-carboxylate
To a solution of 3,6-dichloro-l,2,4,5-tetrazine (443 mg, 2.94 mmol, 1 eq) in tetrahydrofuran (15 mL) was added the product from Step A (741 mg, 2.94 mmol, 1 eq) and the mixture was heated in a sealed tube at 110 °C overnight. The reaction was concentrated in vacuo and the residue was triturated with methanol, filtered and dried under vacuum to afford the desired product as a beige solid (607 mg, 1.79 mmol, 61%).
LC/MS (C14H15CIN4O2S) 339 [M+H]+; RT 2.41 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) Ô 8.06 (s, 1H), 4.38 - 4.25 (m, 4H), 2.92 (t, J = 6.3 Hz, 2H), 2.34 (s, 3H), 2.14 - 2.01(m, 2H), 1.31 (t, J = 7.1 Hz, 3H).
Step C: ethyl 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H, 7H,8H-pyrido[2,3cJpyridazin -8-yl}-l,3-thiazole-4-carboxylate
To an oven-dried microwave vial was added the product from Step B (607 mg, 1.79 mmol, 1 eq), 2-aminobenzothiazole (404 mg, 2.69 mmol, 1.5 eq) ), XantPhos (207 mg, 0.36 mmol, 0.2 eq), césium carbonate (1.17 g, 3.58 mmol, 2 eq) and 1,4-dioxane (36 mL) and the vessel was evacuated and flushed with nitrogen then tris(dibenzylideneacetone)dipalladiun)(0) (164 mg, 0.18 mmol, 0.1 eq) was added and the mixture was sparged with nitrogen (10 mins) then 30 heated at 150 °C for 4 hours under microwave irradiation. The reaction was diluted with ethyl acetate and filtered through celite, then washed with brine, dried (magnésium sulfate) and
B concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in isoheptane afforded a solid that was triturated with diethyl ether, filtered and dried under vacuum to afford the desired product as a yellow solid (329 mg, 0.73 mmol, 41%).
LC/MS (C21H20N6O2S2) 453 [M+H]+; RT 2.73 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 7.99 (br s + s, 2H), 7.65 (br s, 1H), 7.43 - 7.31 (m, 1H), 7.28 - 7.15 (m, 1H), 4.35 - 4.25 (m, 4H), 2.96 - 2.85 (m, 2H), 2.36 (s, 3H), 2.15 - 2.00 (m, 2H), 1.32 (t, J = 7.1 Hz, 3H).
Préparation 3g: Ethyl 5-(3-hydroxypropyl)-2-(4-methyl-3-{[(2Z)-3-{[210 (trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3-benzothÎazol-2-ylidene]amino}5/f,6H,7Ar,8H-pyrido[2,3-c]pyrÎdazin-8-yl)-l,3-thiazole-4-carboxylate
Step A: ethyl 2-(4-methyl-3-{[(2Z)-3-{[2-(trimethylsilyl) ethoxy]methyl}-2,3-dihydro-l ,3benzothiazol-2-ylidene]amino}-5HfiH, 7H,8H -pyrido[2,3-c]pyridazin -8-yl) -1,3-thiazole-4carboxylate
To a solution of the product from Préparation 3f (11.7 g, 25.8 mmol, 1 eq) in dimethylformamide (700 mL) was added A^V-diisopropylethylamine (13.5 mL, 77.4 mmol, 3 eq). After 5 min the mixture was cooled to 0 °C and 4-(dimethylamino)pyridine (630 mg, 5.16 mmol, 0.2 eq) and 2-(trimethylsilyl)ethoxymethyl chloride (13.6 mL, 77.4 mmol, 3 eq) were added and the mixture was stirred at ambient température overnight. The reaction was 20 concentrated in vacuo, then partitioned between dichloromethane and brine, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 330 g RediSep™ silica cartridge) eluting with a gradient of 0 - 40% ethyl acetate in iso-heptane afforded the desired product as a yellow solid (9.61 g, 16.5 mmol, 64%).
LC/MS (C27H34N6O3SiS2) 583 [M+H]+; RT 2.90 (LCMS-V-C) ‘H NMR (400 MHz, DMSO-d6) δ 7.99 (s, 1H), 7.82 (dd, J = 7.7, 1.1 Hz, 1H), 7.49 - 7.38 (m, 2H), 7.28 - 7.19 (m, 1H), 5.86 (s, 2H), 4.38 - 4.23 (m, 4H), 3.77 - 3.67 (m, 2H), 2.89 (t, J = 6.2 Hz, 2H), 2.38 (s, 3H), 2.13 - 2.01 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H), 0.91 (dd, J = 8.5, 7.4 Hz, 2H), -0.11 (s, 9H).
Steo B: ethyl 5-bromo-2-(4-methyl-3-{l(2Z)-3-{[2-(trimethylsilyl)ethoxy]methyl}'2,3dihydro-l,3-benzothiazol-2-ylidene]amino}-5Hf6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl)-l,3thiazole-4-carboxylate
To a solution of the product of Step A(9.61 g, 16.5 mmol, 1 eq) in dichloromethane (400 5 mL) was added 7V-bromosuccînimide (3.52 g, 19.8 mmol, 1.2 eq) and the mixture was stirred at ambient température overnight. The réaction was partitioned between dichloromethane and water, and the organic phase was washed with brine, dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 220 g RediSep™ silica cartridge) eluting with a gradient of 0 - 40% ethyl acetate in iso10 heptane afforded the desired product as a yellow solid (9.66 g, 14.6 mmol, 89%).
LC/MS (C27H33BrN6OsSiS2) 663 [M+H]+; RT 3.13 (LCMS-V-C)
NMR (400 MHz, DMSO-d6) δ 7.84 (dd, J = 7.5, 1.1 Hz, 1H), 7.59 - 7.38 (m, 2H), 7.24 (ddd, J = 8.3, 6.7, 1.7 Hz, 1H), 5.85 (s, 2H), 4.37 - 4.23 (m, 4H), 3.72 (dd, J = 8.5, 7.4 Hz, 2H), 2.87 (t, J = 6.2 Hz, 2H), 2.38 (s, 3H), 2.13 - 2.00 (m, 2H), 1.32 (t, 3H), 0.95 - 0.81 (m, 15 2H), -0.12 (s, 9H).
Step C: ethyl S-[(lE)-3-[(tert-butyldimethylsilyl)oxyJprop-l-en-l-ylj-2-(4-methyl-3-{[(2Z)-3{[2-(trimethylsilyl)ethoxyjmethyl}-2,3-dihydro-l,3-benzothiazol‘2-ylidene]amino}5H,6H, 7H,8H-pyridol2,3~c]pyridazin -8-yl) ~1,3-thiazole-4-carboxylate
To an oven-dried sealed flask was added the product from Step B (9.66 g, 14.6 mmol, 1 eq), 20 (£)-3-(ierZ-butyldimethylsilyloxy)propene-l-yl:boronic acid pinacol ester (5.74 mL, 17.5 mmol, 1.2 eq), potassium carbonate (6.05 g, 43.8 mmol, 3 eq), [Ι,Γbis(diphenylphosphino)ferrocene]dichloropalladium(n) (1.19 g, 1.46 mmol, 0.1 eq), tetrahydrofuran (360 mL) and water (120 mL), and the mixture was sparged with nitrogen (10 min) then heated at 120 °C for 2 h. The reaction was partitioned between ethyl acetate and 25 water, and the organic layer was washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 220 g RediSep™ silica cartridge) eluting with a gradient of 0 - 30% ethyl acetate in isoheptane afforded the desired product as a yellow solid (6.46 g, 8.58 mmol, 59%).
LC/MS (C3i,H52N6O4Si2S2) 753 [M+Hf; RT 1.62 (LCMS-V-B2)
Ή NMR (400 MHz, DMSO-d6) δ 7.80 (dd, J = 7.6, 1.0 Hz, 1H), 7.51 - 7.38 (m, 3H), 7.24 (ddd, J = 8.3, 6.8, 1.8 Hz, 1H), 6.28 (dt, J = 16.0, 4.3 Hz, 1H), 5.85 (s, 2H), 4.37 (dd, J = 4.4, 2.1 Hz, 2H), 4.35 - 4.25 (m, 4H), 3.72 (dd, J = 8.5, 7.4 Hz, 2H), 2.88 (t, J = 6.3 Hz, 2H), 2.37
φ. (s, 3Η), 2.09 - 1.99 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H), 0.93 (s, 9H), 0.92 - 0.83 (m, 2H), 0.11 ( (s, 6H), -0.11 (s, 9H).
Step D: ethyl 5-{3-[(tert-butyldimethylsÎlyl)oxy]propyl}-2-(4-inethyl~3~{[ (2Z)~3~{/2(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3-benzothiazol‘2-ylideneJamino}5 5H,6H, 7H,8H-pyrido[2,3-c]pyridazin -8-yl) ~l,3-thiazole-4-carboxylate
To a solution of the product from Step C (6.46 g, 8.58 mmol, 1 eq) in ethyl acetate (300 mL) was added platinum (IV) oxide (390 mg, 1.72 mmol, 0.2 eq) under a nitrogen atmosphère. The vessel was evacuated and backfilled with nitrogen (x3), then evacuated, placed under an atmosphère of hydrogen, and shaken for 3 days at ambient température. The 10 reaction was fîltered through celite, eluted with ethyl acetate and concentrated in vacuo to afford the desired product as a brown gum (6.72 g, 8.9 mmol, >100%).
LC/MS (C36H54N6O4Si2S2) 755 [M+H]+; RT 1.67 (LCMS-V-B2) ‘H NMR (400 MHz, DMSO-d6) δ 7.76 (d, 1H), 7.48 - 7.35 (m, 2H), 7.24 (ddd, J = 8.2, 6.5, 1.9 Hz, 1H), 5.84 (s, 2H), 4.33 - 4.22 (m, 4H), 3.76 - 3.62 (m, 4H), 3.15 (t, J = 7.5 Hz, 2H), 15 2.87 (t, J = 6.4 Hz, 2H), 2.37 (s, 3H), 2.10-1.98 (m, 3H), 1.91 - 1.79 (m, 2H), 1.31 (t, J - 7.1
Hz, 3H), 0.95 - 0.85 (m, 11H), 0.06 (s, 6H), -0.12 (s, 9H).
Step E: ethyl 5-(3-hydroxypropyl)-2-(4-methyl-3-{[(2Z)-3-{[2ftrimethylsilyl)ethoxyjmethyl}2,3-dihydro-l ,3~benzothiazol~2-ylidene]amino}-5H,6H, 7H,8H-pyrido[2,3-c]pyridazin ~8-yl) 1,3-th iazole-4-carboxylate
To a solution of the product from Step D (6.72 g, 8.9 mmol, 1 eq) in 1,4-dioxane (400 mL) was added hydrochloric acid (4M in dioxane; 67 mL, 267 mmol, 30 eq) and the mixture was stirred al ambient température for 1 h. The reaction cooled to 0 °C and neutralised with IN aqueous sodium hydroxide (300 mL), then partitioned between ethyl acetate and water, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification 25 by automated flash column chromatography (CombiFlash Rf, 120 g RediSep™ silica cartridge) eluting with a gradient of 0 - 80% ethyl acetate in Zso-heptane gave a solid that was triturated wilh diethyl ether, fîltered and dried under vacuum to afford the desired product as a white solid (3.87 g, 6.04 mmol, 68%).
LC/MS (C3oH4oN604SiS2) 641 [M+H]+; RT 2.80 (LCMS-V-C) ’H NMR (400 MHz, DMSO-d6) δ 7.83 (dd, J = 7.6, 1.1 Hz, 1H), 7.48 - 7.37 (m, 2H), 7.23 (ddd, J = 8.3, 6.7, 1.8 Hz, 1H), 5.85 (s, 2H), 4.56 (t, J = 5.1 Hz, 1H), 4.33 - 4.22 (m, 4H), 3.72 φ (dd, J = 8.6, 7.3 Hz, 2H), 3.48 (td, J = 6.3, 5.1 Hz, 2H), 3.17 - 3.08 (m, 2H), 2.88 (t, J = 6.4 Hz, 2H), 2.38 (s, 3H), 2.11 - 1.99 (m, 2H), 1.87 - 1.75 (m, 2H), 1.31 (t, J = 7.1 Hz, 314), 0.96 -0.86 (m, 2H), -0.11 (s, 9H).
Préparation 4a: 4-[l-[(Dimethylamino)methyl]-3-bicyclo[l.l.l]pentanyl]-2-fluoro5 phénol
Step A: tricyclo[1 .l.l.Odlpentane
AIL 3-neck flask equipped with a stirrer bar was assembled with a still-head attached to a condenser and 250 mL collection flask with schlenk tap, a 250 mL dropping tunnel, and a thermometer [ali glassware was assembled hot, then connected to the Schlenk line and 10 allowed to cool under a stream of nitrogen]. A solution of l,l-dibromo-2,2bis(chloromethyl)cyclopropane (59.4 g, 200 mmol, 1 eq) in diethyl ether (200 mL) was cooled to -45 °C and phenyllithium (1.9 M in n-butyl ether; 211 mL, 400 mmol, 2 eq) was added over 25 min by dropping funnel. After complété addition the mixture was allowed to wann to 0 °C and stirred for 2 h. After this time the receiving flask was cooled to -78 °C, and 15 the connection to the manifold was briefly closcd and replaced with a vacuum pump attachaient (with pressure-equalising inlet connected to the nitrogen manifold). Before switching on the pump the dropping funnel and thermometer were replaced with pre-greascd glass stoppcrs. The pump was brought to a pressure of 200 mbar and then the connection was opened. Over 3 mins the pressure was gradually reduced to 120 mbar and then the reaction 20 vessel was allowed to warm to ambient température. The pressure was then cautiously reduced to 45 mbar and this pressure was maintained for 45 mins. After this time the vacuum was released with nitrogen and the résultant clear and colourless distillate was stored at -20 °C. The concentration of the desired product was determined to be 0.45 M by *H NMR.
NMR (400 MHz, Chloroform-d) δ 2.04 (s, 6H).
Step B: bromo(3-fluoro-4‘methoxyphenyl)magnesium
To a 3-neck 50 mL flask equipped with a stirrer bar and condenser was added magnésium (681 mg, 28 mmol, 1.4 eq) and the apparatus was heated strongly (~ 500 °C) with a heat gun for 5 mins with vigorous stirring and then allowed to cool to ambient température under nitrogen. Diethyl ether (5 mL) was added followed by 1,2-dîbromoethane (172 pL, 2 mmol, 30 0.1 eq). The mixture was heated to reflux 4-5 times over 5 mins and then left to stand for 10 mins after which lime a gentle reflux was observed. The mixture was brought to a steady reflux with hand-heat, and then slow stirrîng was initiated. At this point a solution of 4bromo-2-fluoroanisole (4.1 g, 20 mmol, 1 eq) in diethyl ether (10 mL) was added at such a rate as to maintain steady reflux and stirrîng speed was increased (300 rpm). Addition was complété after 15 mins. The mixture was allowed to stir at ambient température for 0.5 h after which time a clear biphasic System had resulted. The lower dark straw-coloured layer (10.15 mL) was transferred to a dry Schlenk flask via syringe through a 0.2 um PTFE filter. The concentration of the solution was calculated to be 1.38 M by titration against a solution of iodine in dry tetrahydrofuran. The product solution was used directly in the next step without further characterisation.
Step C: ethyl 3-(3-fluoro-4-methoxyphenyl)bieyclo[ 1.1.1 ]pentane-i -carboxylate
To an oven-dried 50 mL ACE pressure vessel equipped with a stirrer bar was added the product from Step B (1.38M in diethyl ether; 4.83 mL, 6.67 mmol, 1 eq) followed by the product from Step A (0.45M in diethyl ether, 14.8 mL, 6.67 mmol, 1 eq) and the vessel was sealed with a tefion screw-top fitted with a front O-ring, and placed in a pre-heated heater block behind a blast shield at 105 °C for 3 h. The mixture was allowed to cool at ambient température for 20 mins, and then in i ce-water for 10 mins. The tefion screw top was replaced with a subaseal attached to the nitrogen line, and the reaction was cooled to -78 °C. Ethyl chloroformate (5.1 mL, 53.3 mmol, 4 eq) was added and the mixture was allowed to warm to ambient température for 1.5 h. The reaction was partitioned between saturated aqueous ammonium chloride and diethyl ether, and the aqueous phase was extracted with ether. The combined organic extracts were washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 80 g RediSep™ silica cartridge) ehitîng with a gradient of 0 — 10% ethyl acetate in isoheptanc afforded the desired product as a colourless liquid that was a mixture of desired product and byproduct. This material was further purified by automated flash column chromatography (CombiFlash Torrent, 200 g RediSep™ silica cartridge) eluting with a gradient of 0 - 80% dichloromethane in heptane afforded the desired product (640 mg, 3.78 mmol, 56%).
‘H NMR (500 MHz, DMSO-d6) Ô ppm 7.09 (t, 1 H), 7.09 (dd, 1 H), 6.98 (dm, 1 H), 4.08 (q, 2 H), 3.8 (s, 3 H), 2.22 (s, 6 H), 1.2 (t, 3 H). 13C NMR (500 MHz, dmso-d6) δ ppm 169.8, 151.8,146.6, 133.0, 122.8, 114.2, 114.2, 60.6, 56.5, 53.2, 41.0, 36.9, 14.6.
HRMS-EI (m/z): M+ calcd for C15 H17 F 03: 264.1162, found 264.1156.
Step D: 1 -(3-fluoro-4-methoxy-phenyl)bicyclo[l.l.l]pentane-3-carboxylic acid
200 mg of the product from Step C (0.76 mmol, 1 eq.) and 159 mg of LiOHxHzO (3.78 mmol, 5 eq.) were mixed in 1,4-dioxane (2 mL/mmol) and water (2 mL/mmol) then stirred at rt for 1 h when full conversion was observed. Reaction mixture was made basic with 1:1 HCl 5 solution then the précipitation was filtered and washed with water then dried in vacuum for o.n. 170 mg (95%) of the desired product was isolated as a white solid.
NMR (500 MHz, DMSO-dQ δ ppm 12.41 (s, 1H), 7.09 (m, 1H), 7.09 (m, 1H), 6.97 (dm, 1H), 3.80 (s, 3H), 2.18 (s, 6H); 13C NMR (125 MHz, DMSO-dQ δ ppm 171.7, 151.8, 146.6, 133.3, 122.7, 114.2, 114.1, 56.5, 53.1, 40.8, 37.0; GC-MS-EI (m/z): [M]+ calcd for 10 CisHjîFOî: 236.0849, found 236.0840.
Step E: 1-(3-fluoro-4-methoxy-phenyi)-N,N-dimethyl-bicyclo[l.l.l]pentane-3-carboxamide
164 mg of the product from Step D (1.04 mmol, 1 eq.) and 278 mg of N,N-diethylethanamine (1.39 mmol, 2 eq.) were mixed in EtOAc (3 mL/mmol) then 663 mg of 2,4,6-tripropyl7,3,5,2)4(5),4)4{5),6λ^(5)-trioxatriphosphinane 2,4,6-trioxide (50w% in EtOAc, 1.04 mmol, 15 1.5 eq.) was added in one portion then stirred at rl for 40 min. After the reaction time 0.52 mL oîN-methylmethanamme (2 M in MeOH, 1.04 mmol, 1.5 eq.) was added and stirred at rt unlil full conversion was observed (60 min). Reaction mixture was diluted with DCM then washed with cc. NaHCO? then the organic phase was washed with cc. NaCl, dried over MgSO4, filtered, concentrated, dried in vacuo to give 187 mg (quant.) of the desired product as a solid 20 with peach color.
*H NMR (500 MHz, DMSO-dQ δ ppm 7.14 (m, 2H), 6.86 (m, 2H), 3.72 (s, 3H), 3.08 (s, 3H), 2.81 (s, 3 H), 2.26 (s, 6H); 13C NMR (125 MHz, DMSO-de) δ ppm 168.9, 158.6, 132.5, 127.6, 114.1, 55.5, 54.2, 42.0, 39.0, 37.4, 35.9; HRMS-ESI (m/z): [M+H]+ calcd for C15H19FNO2: 264.1394, found 264.1389.
Step F: 1 -(3-(3-fluoro-4-methoxy-phenyl) -1 -bicyclof1.1 .l]pentanyl]-NJS-dimethylmethanamine
182 mg of the product from Step E (0.69 mmol, 1 eq.) was dissolved in THF (5 mL/mmol) then 1.38 mL of L1AIH4 (1 M in THF, 1.38 mmol, 2 eq.) was added under nitrogen atmosphère at ambient température then stirred until full conversion was achieved (ca. 1 h). 30 The mixture cooled lo 0°C then quenched with cc. NH4CL After quenching ~5 mL water and ~10 mL EtOAc were added and shaked well. 2 M HCl was added and the (acidic) water phase was separated then the organic phase was extracted with further 2 M HCL The combined water phases were made basic with 2 M NaOH and extracted with DCM. The combined organic phases was washed with brme, dried over MgSOi and concentrated, dried in vacuo. 119 mg (69%) of the desired product was obtained as viscous oil.
Ή NMR (500 MHz, DMSO-î/û) δ ppm 7.07 (t, 1H), 7.01 (dd, 1H), 6.93 (dm, 1H), 3.79 (s, 3H), 2.35 (s, 2H), 2.16 (s, 6H), 1.90 (s, 6H); *3C NMR (125 MHz, DMSO4) δ ppm 151.8, 146.2, 134.5, 122.5, 114.1, 114.0, 60.7, 56.5, 52.9, 46.6, 41.7, 38.0; HRMS-ESI (m/z): [M+H]+ calcd for C15H21FNO: 250.1602, found 250.1596.
Step G: 4-]1 -[(dimethylamino) methyl]-3-bicy cio] 1.1.1 ]pentanyl]-2-fluoro-phenol
113 mg of the product from Step F (0.45 mmol, 1 eq.) was dissolved in DCM (5 mL/mmol) then 1.36 mL of BBr3 (1 M in DCM, 1.36 mmol, 3 eq.) was added under nitrogen atmosphère al Ü°C then stirred for 15 min at 0°C and at rt until full conversion was achieved (ca. 45 min). DCM was added then poured into NaHCO? solution, stirred for a few minutes then made il neutral with cc. NH4CI. Separated and washed with brine, dried over MgSÛ4 and concentrated, dried in vacuo. 47 mg (quant.) of the crude desired product was obtained as viscous oil.
*H NMR (400 MHz, CDCI3) δ ppm 6.95 (t, 1H), 6.90 (dd, 1H), 6.85 (dm, 1H), 3.84 (s, 2H), 3.17 (s, 6H), 2.24 (s, 6H); 13C NMR (100 MHz, CDCh) δ ppm 122.4, 117.4, 113.4, 59.5, 54.8, 46.0, 43.8, 34.8; HRMS-ESI (m/z): [M+H]+ calcd for C14H19FNO: 236.1445, 236.1445.
Préparation 4b: 4-[3-(Dimethylamino)prop-l-ynyl]-2-fluoro-phenol
Using Sonogashira General Procedure starting from 10.00 g of 2-fluoro-4-iodo-phenol (42.0 mmol, 1 eq.) as the appropriate phénol and 5.24 g of M-Y-dimethyIprop-2-yn-l-amine (63 mmol, 1.5 eq.) as the alkyne, 7.30 g (90%) of the desired product was obtained.
Ή NMR (500 MHz, DMSO-d6) δ ppm 7.20 (dd, 1H), 7.07 (dm, 1H), 6.91 (m, 1H), 3.39 (m, 2H), 2.21 (m, 3H); 13C NMR (125 MHz, DMSO-d6) δ ppm 150.9, 146.2, 128.9, 119.5, 118.4, 113.6, 84.5, 84.2, 48.2, 44.3; HRMS-ESI (m/z): [M+H]+ calcd for C11H13FNO: 194.0976, found 194.0981.
• Préparation 4c: zert-Butyl Ar-[3-(3-fluoro-4-hydroxy-phenyl)prop-2-ynyl]-Ar-methylcarbamate
Using Sonogashira General Procedure starting from 10.00 g of 2-fIuoro-4-iodo-phenol (42.0 mmol, 1 eq.) as the appropriate phénol and 10.67 g of ieri-butyl N-methylW-prop-25 ynyl-carbamate (63.1 mmol, 1.5 eq.) as the alkyne, 10.8 g (92%) of the desired product was obtained.
*H NMR (500 MHz, DMSO-d6) δ ppm 10.32 (s, 1 H), 7.22 (brd, 1H), 7.08 (dm, 1H), 6.92 (dd, 1H), 4.21 (s, 2H), 2.85 (s, 3H), 1.41 (s, 9H); 13C NMR (125 MHz, DMSO-df,) δ ppm 150.8, 146.4, 129.0, 119.6, 118.4, 113.2, 84.4, 82.7, 38.5, 33.8, 28.5; HRMS-ESI (m/z): [M10 C4H8+H]+ calcd for CnHiiFNCh: 224.0717, found 224.0720.
Préparation 4d: 4-[3-(Dimethylamino)propyl]-2-fluorophenol
To a solution of the product from Préparation 4b (1.5 g, 7.76 mmol, 1 eq) in ethyl acetate (54 mL) and éthanol (18 mL) under nitrogen was added platinum(IV) oxide hydrate (353 mg, 1.55 mmol, 0.2 eq). The vessel was evacuated and backfilled with nitrogen (x3), then 15 evacuated, subjected to an atmosphère of hydrogen, and shaken at ambient température overnight. The reaction was filtered through celite, eluted with ethyl acetate and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% IN methanolic ammonia in dichloromethane afforded the desired product (652 mg, 3.31 mmol, 42%) as an off-white 20 solid.
LC/MS (CiiHiôFNO) 198 [M+H]+; RT 0.44 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 9,52 (s, 1H), 6.96 (dd, J = 12.5, 1.9 Hz, 1H), 6.88 - 6.76 (m, 2H), 2.47 (dd, J = 8.5, 6.8 Hz, 2H), 2.20 - 2.13 (m, 2H), 2.11 (s, 6H), 1.69 - 1.57 (m, 2H).
Préparation 4e: 4-|2-(Dimethylamino)ethoxy]phenol
Step A: 4-(methoxymethoxy)phénol
To a solution of hydroquinone (0.76 mL, 9.08 mmol, 1 eq) in acetone (30 mL) was added potassium carbonate (2.51 g, 18.2 mmol, 2 eq) and chloromethyl methyl ether (0.69 mL, 9.08 mmol, 1 eq) and the mixture was stirred at ambient température overnight. The reaction was partitioned between ethyl acetate and water, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by au tomate d flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 — 20% ethyl acetate in wo-heptane afforded the desired product as a brown oil (601 mg, 3.9 mmol, 43%).
NMR (400 MHz, DMSO-d6) δ 9.03 (s, 1H), 6.91 - 6.80 (m, 2H), 6.72 - 6.62 (m, 2H), 5.05 (s, 2H), 3.36 (s, 3H).
Step B: {2-[4-(methoxymethoxy)phenoxy]ethyl}dimethylamine
To a solution of the product from Step A (400 mg, 2.59 mmol, 1 eq) in tetrahydrofuran (20 mL)was added MA-dimethylethanolamine (526 gL, 5.19 mmol, 2 eq), di-tert-butyl 10 azodîcarboxylate (1.19 g, 5.19 mmol, 2 eq) and triphenylphosphîne (1.36 g, 5.19 mmol, 2 eq) and the mixture was heated at 50 °C for 3 h. The réaction was concentrated in vacuo, partitioned between dichloromethane and saturated aqueous sodium bicarbonate, and the organic phase was separated (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) 15 eluting with a gradient of 0 - 7% methanol in dichloromethane afforded the desired product as a brown oil (383 mg, 1.7 mmol, 66%).
LC/MS (C12H19NO3) 226 [M+H]+; RT 0.88 (LCMS-V-C)
Mi NMR (400 MHz, DMSO-d6) δ 6.99 - 6.90 (m, 2H), 6.94 - 6.82 (m, 2H), 5.10 (s, 2H), 3.98 (t, J = 5.8 Hz, 2H), 3.36 (s, 3H), 2.59 (t, J = 5.9 Hz, 2H), 2.20 (s, 6H).
Step C: 4-[2-(dimethylamino)ethoxy]phenol
A solution of the product from Step B (383 mg, 1.7 mmol, 1 eq) in hydrochloric acid (4M in 1,4-dioxane; 5 mL, 20 mmol, 11.7 eq) was stirred at ambient température for 1 h. The reaction was concentrated in vacuo, then dissolved in methanol, loaded onto a methanol-wet SCX cartridge (10 g), washed with methanol, and eluted with 1.75N methanolic ammonia and 25 concentrated in vacuo to afford the desired product as a brown solid (249 mg, 1.37 mmol, 812%).
LC/MS (C10H15NO2) 182 [M+H]+; RT 0.24 (LCMS-V-C)
NMR (400 MHz, DMSO-d6) δ 8.91 (s, 1H), 6.79 - 6.70 (m, 2H), 6.70 - 6.62 (m, 2H), 3.92 (t, J = 5.9 Hz, 2H), 2.57 (l, J = 5.9 Hz, 2H), 2.20 (s, 6H).
Préparation 4f: 4-[2-(Pyrrolidin-1-yl)ethoxy]phénol
Step A; 1-(2-(4-(methoxymethoxy)phenoxy]ethyl}pyrrolidine
To a solution of the product from Préparation 4e, Step A (525 mg, 3.41 mmol, 1 eq) in tetrahydrofuran (20 mL) was added l-(2-hydroxyethyl)pyrrolidine (0.8 mL, 6.81 mmol, 2 eq), di-tert-butyl azodicarboxylale (1.57 g, 6.81 mmol, 2 eq) and triphenylphosphine (1.79 g, 6.81 mmol, 2 eq) and the mixture was heated at 50 °C overnight. The reaction was concentrated in vacuo and partîtîoned between dichloromethane and saturated aqueous sodium bicarbonate, and the organic phase was separated (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 7% methanol in dichloromethane afforded the desired product as a brown oil (556 mg, 2.21 mmol, 65%).
LC/MS (C14H21NO3) 252 [M+H]+; RT 1.09 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) Ô 6.98 - 6.91 (m, 2H), 6.91 - 6.82 (m, 2H), 5.10 (s, 2H), 4.00 (t, J = 5.9 Hz, 2H), 3.36 (d, J = 6.0 Hz, 3H), 2.75 (t, J = 6.0 Hz, 2H), 2.50 - 2.42 (m, 4H), 1.74 -1.61 (m, 4H).
Step B: 4~[2~(pyrrolidin-l-yl)ethoxy]phenol
A solution of the product from Step A (556 mg, 2.21 mmol, 1 eq) in hydrochloric acid (4M in 1,4-dioxane; 7 mL, 28 mmol, 12.7 eq) was stirred at ambient température for 30 min. The reaction was concentrated in vacuo, then dissolved in methanol, loaded onto a methanol-wet SCX cartridge (10 g), washed with methanol, eluted with 1.75N methanolic ammonia and concentrated in vacuo to afford the desired product as a brown solid (453 mg, 2.19 mmol, 99%).
LC/MS (C12H17NO2) 208 [M+H]+; RT 0.28 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) 5 8.90 (s, 1H), 6.79 - 6.70 (m, 2H), 6.70 - 6.62 (m, 2H), 3.94 (t, J = 6.0 Hz, 2H), 3.17 (d, J = 4.3 Hz, 2H), 2.73 (t, J = 6.0 Hz, 2H), 2.49 (dt, J = 4.1, 1.4 Hz, 2H), 1.74-1.60 (m, 4H).
Préparation 4g: 4-[2-(Dimethylamino)ethyl]-2-fluorophenol
Step A: 2-fluoro-l-methoxy-4-[(E)-2-nitroethenyl]benzene
To a solution of 3-fluoro-4-methoxybenzaldehyde (400 mg, 2.6 mmol, 1 eq) and nitromethane (339 pL, 6.23 mmol, 2.4 eq) in methanol (50 mL), cooled to 0 °C, was added IM aqueous sodium hydroxide (20 mL, 20 mmol, 7.71 eq) dropwise and the résultant mixture was stirred at 0 °C for 1 h. The mixture was added portionwise to 8M aqueous hydrochloric acid (12 mL, 96 mmol, 37 eq), cooled to 0 °C, and the résultant suspension was allowed to warm to ambient température and stir for 30 min. The precipitate was collected by filtration, washed with water and dried under vacuum to afford the desired product (393 mg, 1.99 mmol, 76%) as a yellow solid.
Ή NMR (400 MHz, DMSOd6) δ 8.20 (d, J = 13.6 Hz, 1H), 8.10 (dd, J = 13.5, 1.0 Hz, 1H), 7.88 (dd, J = 12.6, 2.1 Hz, 1H), 7.70 (dt, J = 8.6, 1.5 Hz, 1H), 7.29 (t, J = 8.8 Hz, 1H), 3.92 (s, 3H).
Step B: 2-(3-fluoro-4-methoxyphenyl)ethan-l-amine
To a solution of the product from Step A (393 mg, 1.99 mmol, 1 eq) in tetrahydrofuran (12 mL) was added lithium aluminium hydride (IM in tetrahydrofuran; 5.98 mL, 5.98 mmol, 3 eq) and the mixture was heated at 40 °C overnight. The reaction was quenched with water (1.2 mL) and concentrated in vacuo. The residue was dissolved in 2N aqueous hydrochloric acid (20 mL) and washed with ethyl acetate (x2). Tartaric acid (2.1 g) was added to the aqueous phase and the pH was adjusted to pH 11 with concentrated ammonium hydroxide. The mixture was extracted wîth dichloromethane (x3) and the combined organic extracts were separated (PTFE phase separator) and concentrated in vacuo to afford the desired product as a yellow oil (252 mg, 1.49 mmol, 75%).
LC/MS (C9H12FNO) 170 [M+H]+; RT 0.14 (LCMS-V-B1)
Ή NMR (400 MHz, DMSO-d6) δ 7.16 - 7.03 (m, 3H), 3.80 (s, 3H), 3.38 - 3.30 (m, 2H), 2.79 - 2.69 (m, 2H), 2.62 - 2.54 (m, 2H).
Step C: [2-(3-fluoro-4-methoxyphenyl) ethyl]dimethylamine
To a solution of the product from Step B (252 mg, 1.49 mmol, 1 eq) in methanol (5 mL) was added formaldéhyde (13.4M in water; 123 pL, 4.47 mmol, 3 eq) followed by sodium triacetoxyboro hydride (947 mg, 4.47 mmol, 3 eq) and glacial ace tic acid (0.05 mL) and the mixture was stirred al ambient température for 1 h. The reaction was partitioned between ethyl acetate and saturated aqueous sodium bicarbonate, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 -10% methanoi în dichloromethane afforded the desired product as a yellow oil (92 mg, 0.47 mmol, 31%).
LC/MS (CnHifiFNO) 198 [M+H]+; RT 0.82 (LCMS-V-C) 1H NMR (400 MHz, DMSO-d6) δ 7.16 - 6.94 (m, 3H), 3.80 (s, 3H), 2.64 (dd, J = 8.5, 6.7 Hz, 2H), 2.41 (dd, J = 8.5, 6.7 Hz, 2H), 2.16 (s, 6H).
Step D: 4-[2-(dimethylamino) ethyl]-2-fluorophenol
To a solution of the product from Step C (92 mg, 0.47 mmol, 1 eq) in dichloromethane (3.5 mL), cooled to 0 °C, was added boron tribromide (IM in dichloromethane, 1.4 mL, 1.4 mmol, 3 eq) and the mixture was stirred at ambient température overnight. The reaction was cooled to 0 °C and quenched with methanoi, then concentrated in vacuo. The residue was dissolved in methanoi, loaded onto a methanol-wet SCX cartridge (5 g), washed with methanoi, eluted with 1.75N methanoiic ammonia, and concentrated in vacuo to afford the desired product as a brown oil (41 mg, 0.22 mmol, 48%).
LC/MS (CkjHhFNO) 184 [M+H]+; RT0.36 (LCMS-V-C) *Η NMR (400 MHz, DMSO-d6) δ 9.55 (s, IH), 7.03 - 6.95 (m, IH), 6.88 - 6.78 (m, 2H), 2.59 (dd, J = 8.7, 6.7 Hz, 2H), 2.38 (dd, J = 8.6, 6.7 Hz, 2H), 2.15 (s, 6H).
Préparation 4h: 3-[(Diniethylamino)methyl]-5-tluoro-l-methyl-LW-indol-6-ol
Step A: 6-(benzyloxy)-5-fluoro-lH-indole-2~carboxylic acid
To a stirred solution of 6-benzyloxy-5-fluoro-lH-indole-2-carboxylic acid methyl ester (2.5 g, 8.35 mmol, 1 eq) in a mixture of tetrahydrofuran (25 mL) and methanoi (25 mL) was added a solution of sodium hydroxîde (4 g, 100 mmol, 12 eq) in water (30 mL) and the mixture was stirred for 2.5 h. The réaction was cooled în ice-water and acidified with stirrîng by slow addition of 2N aqueous hydrochloric acid (60 mL) resulting in précipitation. Water (80 mL) was added and the mixture was stirred for 45 min, then the solids were collected by filtration, washed with water and dried under vacuum to afford the desired product (2.25 g, 7.89 mmol, 94%) as an off-white solid.
φ LC/MS (C16H12FNO3) 284 [Μ-H]’; RT 1.16 (LCMS-V-B1) *H NMR (400 MHz, DMSO-d6) δ 12.85 (s, 1H), 11.71 (d, J = 2.3 Hz, 1H), 7.54 - 7.39 (m, 5H), 7.39 - 7.32 (m, 1H), 7.10 (dd, J - 7.4, 0.8 Hz, 1H), 7.01 (dd, J = 2.2, 0.8 Hz, 1H), 5.19 (s, 2H).
Step fi: 6-(benzyloxy)-5-fluoro-l H-indole
A mixture of the product from Step A (1.25 g, 4.38 mmol, 1 eq) and diphenyl ether (60 mL) was heated at 290 °C (external) for 45 min. The reaction was allowed to cool to ambient température then diluted with heptane (180 mL) and loaded onto a hexane-wet pre-packed silica column (80 g) under vacuum. Purification by automated flash chromatography 10 (CombiFlash Rf, Silica 80g RediSep column) eluting with a gradient of 0 - 80% ethyl acetate in hexane afforded the desired product (372 mg, 1.54 mmol, 35%) as a beige solid.
LC/MS (C15H12FNO) 242 [M+H]+; RT 1.26 (LCMS-V-B1)
NMR (400 MHz, DMSO-d6) δ J1.00 (s, 1H), 7.52 - 7.45 (m, 2H), 7.49 - 7.37 (m, 2H), 7.41 - 7.30 (m, 2H), 7.25 (t, J = 2.8 Hz, 1H), 7.13 (dd, J = 7.3, 0.8 Hz, 1H), 6.34 (ddd, J = 3.0, 15 2.0, 0.8 Hz, 1H), 5.18 (s, 2H).
Step C: 6-(benzyloxy)-5-fluoro-l-methy l-l H-indole
To a stirred solution of the product from Step B (365 mg, 1.51 mmol, 1 eq) in dimethylformamide (10 mL), cooled in an ice-water bath, was added sodium hydride (60% dispersion; 72.6 mg, 3.03 mmol, 2 eq) and the mixture was stirred for 15 min. lodomethane 20 (0.11 mL, 1.82 mmol, 1.2 eq) was added, then the mixture was allowed to warm to ambient température and stir for 1 h. The réaction was cooled in ice, then quenched by dropwise addition of saturated aqueous ammonium chloride and slowly poured onto stirring ice-water (40 mL) resulting in précipitation. Further ice was added (20 mL) and after 30 min stirring the solids were coliccted by filtration, washed successively with ice-cold water (2 x 30 mL) and 25 hexane (2 x 10 mL) and dried under vacuum to afford the desired product (259 mg, LOI mmol, 67%) as a beige solid.
LC/MS (Ci6H14FNO) 256 [M+H]+; RT 1.36 (LCMS-V-B1)
Ή NMR (400 MHz, DMSO-d6) δ 7.55 - 7.48 (m, 2H), 7.47 - 7.38 (m, 2H), 7.40 - 7.29 (m, 3H), 7.25 (d, J = 3.1 Hz, 1H), 6.33 (dd, J = 3.1, 0.8 Hz, 1H), 5.21 (s, 2H), 3.76 (s, 3H).
Step D: {[ô-(benzyloxy) -5-fluoro-I -methyl-lH-indol-3-yl]methyl}dimethylaniine
To a stirred mixture of 1,4-dioxane (5 mL) and glacial acetic acid (5 mL) was added aqueous formaldéhyde (37 wt%; 1.18 mL, 14.54 mmol, 14.6 eq) foliowed by aqueous dimethy lamine (40 wt%; 1.42 mL, 12.6 mmol, 12.6 eq). This solution (1.6 mL) was added to a stirred 5 solution of the product of Step C (255 mg, 1 mmol, 1 eq) in 1,4-dioxane (1 mL) and the mixture was stirred at ambient température for 4 h. The reaction was concentrated in vacuo, then 2N aqueous sodium hydroxide (4 mL) was added and the résultant thick suspension was diluted with water (10 mL), stirred and cooled in ice-water for 15 min, then filtered, washed with water (x3) and dried under vacuum to afford the desired product (290 mg, 0.93 mmol, 10 93%) as a cream solid.
LC/MS (C19H21FN2O) 268 [M+H-NHMe2]+; RT 0.99 (LCMS-V-B1)
NMR (400 MHz, DMSO-d6) δ 7.55 - 7.47 (m, 2H), 7.47 - 7.38 (m, 2H), 7.40 - 7.31 (m, 2H), 7.28 (d, J = 7.3 Hz, IH), 7.13 (s, IH), 5.20 (s, 2H), 3.71 (s, 3H), 3.44 (s, 2H), 2.12 (s, 6H).
Step E: 3-[(dimethylamino)methylj-5-fluoro-l-methyl~lH-indol-6-ol
A flask was charged with 10% Pd/C (50 mg, 0.05 eq), then evacuated and flushed with nitrogen (x 2). A solution of the product from Step D (285 mg, 0.91 mmol, 1 eq) in éthanol (20 mL)was added and the flask was evacuated and flushed with nitrogen (x 3), then evacuated and flushed with hydrogen (x 3), then subjected to an atmosphère of hydrogen and 20 shaken for 4 h at ambient température. The reaction was filtered through an HM-N cartridge, eluted with éthanol, and concentrated in vacuo. Purification by reverse phase automated flash chromatography (CombiFlash Rf, C18 50g Gold RediSep column) eluting with a gradient of 10 - 100% acetonitrile in water afforded the desired product (69.8 mg, 0.27 mmol, 29%) as an off-white solid (hydrochloride sait).
LC/MS (Ci2Hi5FN2O) 178 [M+H-NHMe2]+; RT Ü.36 (LCMS-V-B1)
NMR (400 MHz, DMSO-d6) δ 10.08 (s, IH), 9.66 (s, IH), 7.60 (d, J = 11.8 Hz, IH), 7.40 (s, IH), 6.96 (d, J = 7.5 Hz, IH), 4.29 (s, 2H), 3.71 (s, 3H), 2.66 (s, 6H), 1.23 (d, J = 6.5 Hz, IH).
Préparation 4i; 4-[4-(Diniethylamino)butyl]-2-fluorophenoI
SleR.A: [3-(l,3-dioxo-2,3-dihydro-lH-isoindol-2-yl)propyl]triphenylphosphanium bromide AE(3-bromopropyI)phthalimide (2.75 g, 10.26 mmol, 1 eq) and triphenylphosphine (2.69 g, 10.3 mmol, 1 eq) were stirred in toluene (25 mL) and heated at reflux overnight. The reaction was allowed to cool to ambient température and the solids were collected by filtration and dried under vacuum to afford the desired product as a white solid (2.55 g, 4.81 mmol, 47%). ‘H NMR (400 MHz, DMSO-d6) δ 7.95 - 7.85 (m, 7H), 7.82 - 7.71 (m, 12H), 3.80 - 3.64 (in, 4H), 1.99 - 1.90 (m, 2H).
Step B: 2-[(3E)-4-(3-fluoro-4~methoxyph enyl) but-3-en-l -yl]-2,3-dihydro-lH-isoindole-l,3dione
To a solution of the product from Step A (2.55 g, 4.81 mmol, 1 eq) in toluene (25 mL) was added 3-Fluoro-4-methoxybenzaJdehyde (741 mg, 4.81 mmol, 1 eq), followed by 18-crown-6 (108 pL, 0.48 mmol, 0.1 eq) and the mixture was stirred at ambient température overnight. The reaction was partitioned between ethyl acetate and water, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 30% ethyl acetate in /so-heptane afforded the desired product as a white solid (1.48 g, 4.55 mmol, 95%).
LC/MS (C19H16FNO3) 302 [OTHER]; RT 2.25 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 7.91 - 7.79 (m, 4H), 7.13 - 6.98 (m, 3H), 6.43 - 6.33 (m, IH), 5.61 (dt, J = 11.7, 7.4 Hz, IH), 3.82 (s, 3H), 3.70 (t, 2H), 2.64 (qd, J = 7.2,1.8 Hz, 2H).
Step C: (3E)~4-(3-fhioro-4-methoxyphenyl)but-3-en-l-amine
To a solution of the product from Step B (1.48 g, 4.55 mmol, 1 eq) in éthanol (60 mL) was added methylamine (2M in methanol; 24 mL, 665 mmol, 146 eq) and the mixture was heated at reflux overnight. The reaction was allowed to cool to ambient température and was concentrated in vacuo. The residue was trituraled with diethyl ether, filtered and dried under vacuum. The crude solid was dissolved in ethyl acetate and extracted with IN aqueous hydrochloric acid (3 x 100 mL). The combined aqueous extracts were basified with 4M aqueous potassium hydroxide, extracted with ethyl acetate (x2), and the combined organic extracts were dried (magnésium sulfate) and concentrated in vacuo to afford the desired product as a pink gum (395 mg, 2.02 mmol, 45%).
φ LC/MS (ChHkFNO) 196 [M+H]4; RT 1.25 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 7.24 - 7.04 (m, 4H), 6.44 - 6.30 (m, 1H), 5.63 (dt, J = 11.6, 7.2 Hz, 1H), 2.65 (t, 2H), 2.37 (qd, J = 7.1, 2.0 Hz, 2H).
Step D: 4-(3-fluoro-4-methoxyphenyl)butan-l-amine
To a solution of the product from Step C (395 mg, 2.02 mmol, 1 eq) in methanol (10 mL) was added platinum(IV) oxide (45.9 mg, 0.2 mmol, 0.1 eq) under a nitrogen atmosphère. The vessel was evacuated and backfilled with nitrogen (x3), evacuated, then placed under an atmosphère of hydrogen and shaken at ambient température ovemight. The reaction was filtered through celite, eluted with methanol and concentrated in vacuo. The residue was 10 dissolved in methanol, loaded onto a methanol-wet SCX cartridge (5 g), washed with methanol, eluted with 1.75N methanolic ammonia and concentrated in vacuo to afford the desired product as a peach gum (219 mg, 1.11 mmol, 55%).
LC/MS (CuHiôFNO) 198 [M+H]+; RT 1.18 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.10 - 7.01 (m, 2H), 6.99 - 6.90 (m, 1H), 3.80 (s, 3H), 15 2.57 - 2.44 (m, 2H), 1.61 -1.42 (m, 4H), 1.39 - 1.26 (m, 2H).
Step E: [4-(3-fluoro-4-methoxyphenyl)butyl]dipiethylamine
To a solution of the product from Step D (219 mg, 1.11 mmol, 1 eq) in methanol (5 mL) was added aqueous formaldéhyde (37 wt%; 91.8 pL, 13.4 M, 3.33 mmol, 3 eq), sodium triacetoxyborohydride (706 mg, 3.33 mmol, 3 eq) and glacial acetic acid (6.36 pL, 0.11 20 mmol, 0.1 eq) and the mixture was stirred at ambient température ovemight. The reaction was concentrated in vacuo, then partitioned between ethyl acetate and saturated aqueous sodium bicarbonate, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 — 10% methanol in dichloromethane afforded the 25 desired product as a clear oil (163 mg, 0.72 mmol, 65%).
LC/MS (C13H20FNO) 226 [M+H]+; RT 1.30 (LCMS-V-C) XH NMR (400 MHz, DMSO-d6) δ 7.10 - 7.00 (m, 2H), 6.98 - 6.90 (m, 1H), 3.80 (s, 3H), 2.56 - 2.47 (m, 2H), 2.23 - 2.15 (m, 2H), 2.09 (s, 6H), 1.59 - 1.47 (m, 2H), 1.43 - 1.31 (m, 2H).
Step F; 4-[4-(dimethylamino)butyl]-2-fluorophenol
To a solution of the product of Step E (163 mg, 0.72 mmol, 1 eq) in dichloromethane (5 mL), cooled to 0 °C, was added boron tribromide (IM în dichloromethane; 2.17 mL, 2.17 mmol, 3 eq) and the mixture was stirred at ambient température overnight. The reaction was cooled to 5 0 °C, quenched with methanol and concentrated in vacuo. The residue was dissolved in methanol, loaded onto a methanol-wet SCX cartrîdge (5 g), washed with methanol, eluted with 1.75N methanolic ammonia and concentrated in vacuo to afford the desired product as a yellow oil (110 mg, 0.52 mmol, 72%).
LC/MS (Ci2Hi8FNO) 212 [M+H]+; RT 0.96 (LCMS-V-C)
Ή NMR(400 MHz, DMSO-d6) δ 9.54 (s, IH), 6.94 (dd, J = 12.5, 2.0 Hz, IH), 6.88 - 6.75 (m, 2H), 2.47 (t, J = 7.6 Hz, 2H), 2.21 - 2.13 (m, 2H), 2.08 (s, 6H), 1.56 - 1.44 (m, 2H), 1.42 1.30 (m,2H).
Préparation 4j: tert-butyl N-[2-(3-fluoro-4-hydroxyphenyl)ethyl]-Nmethylcarbamate
StepA: ethyl N-[2-(3-fluoro-4-methoxyphenyl) ethyl] carbamate
To a solution of 2-(3-fluoro-4-methoxyphenyl)ethan-l-amine (263 mg, 1.55 mmol, 1 eq) in dichloromethane (10 mL) was added triethylamine (315 mg, 3.Tl mmol, 2 eq) and the mixture was cooled lo 0 °C before the addition of ethyl chloroformate (149 pL, 1.55 mmol, 1 eq) and the mixture was stirred at ambient température overnight. The reaction was 20 partitioned between dichloromethane and saturated aqueous sodium bicarbonate, and the organic phase was separated (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartrîdge) eluting with a gradient of 0-1% methanol in dichloromethane afforded the desired product as a white wax (245 mg, 1,02 mmol, 65%).
LC/MS (C12H16FNO3) 242 [M+H]+; RT 1.81 (LCMS-V-C)
NMR (400 MHz, DMSO-d6) δ 7.13 (t, IH), 7.11 - 7.01 (m, 2H), 6.98 - 6.92 (m, IH), 3.96 (q, J = 7.1 Hz, 2H), 3.80 (s, 3H), 3.16 (td, J = 7,3, 5.8 Hz, 2H), 2.64 (t, J = 7.3 Hz, 2H), 1.13 (t, J = 7.1 Hz, 3H).
S4
Ste# B: [2-(3-fluoro-4-methoxyphenyl)ethyl](methyl)amine
A solution of the product from Step A (245 mg, 1.02 mmol, 1 eq) in tetrahydrofuran (3 mL) was cooled to 0 °C. Lithium aluminium hydride (IM in tetrahydrofuran, 2.54 mL, 2.54 mmol, 2.5 eq) was added and the mixture was heated at reflux overnight. The reaction was 5 cooled to 0 °C and water (96 gL) was added, followed by 15% aqueous sodium hydroxide (96 gL), then water (288 gL). Further tetrahydrofuran was added to aid stirring and the mixture was stirred at ambient température for 30 min. Magnésium sulfate was added, followed by ethyl acetate, the mixture was stirred for 15 min, then fîltered through celite and eluted with ethyl acetate. Solvents were removed in vacuo and purification by automated flash column 10 chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% methanol in dichloromethane afforded the desired product as a clear oil (89 mg, 0.49 mmol, 48%).
LC/MS (CwHuFNO) 184 [M+H]+; RT 0.75 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 7.15 - 7.01 (m, 2H), 6.96 (ddd, J = 8.3, 2.0, 1.0 Hz, 1H), 15 3.80 (s, 3H), 2.69 - 2.57 (m, 4H), 2.27 (s, 3H).
Step C: 2-fluoro-4-[2-(methylamino) ethyl]phénol
To a solution of the product of Step B (89 mg, 0.49 mmol, 1 eq) in dichloromethane (4 mL), cooled to 0 °C, was added boron tribromide (IM in dichloromethane, 1.46 mL, 1.46 mmol, 3 eq) and the mixture was stirred at ambient température for 4 h. The reaction was cooled to 0 20 °C and quenched with methanol, then concentrated in vacuo. The residue was dissolved in methanol, loaded onto a methanol-wet SCX cartridge (5 g), washed with methanol, eluted with 1.75N methanolic ammonia and concentrated in vacuo to afford the desired product as a brown gum (62 mg, 0.37 mmol, 75%).
LC/MS (C9H12FNO) 170 [M+H]+; RT 0.24 (LCMS-V-C) ‘H NMR (400 MHz, DMSO-d6) δ 7.02 - 6.93 (m, 1H), 6.88 - 6.76 (m, 2H), 2.67 - 2.53 (m, 4H), 2.27 (s, 3H).
Step D: tert-butyl\~l2-(3-fluoro-4-hydroxyphenyl)ethyl]-N-methylcarbamate
To a solution of the product from Step C (62 mg, 0.37 mmol, 1 eq) in dichloromethane (5 mL) was added triethylamine (153 gL, 1.1 mmol, 3 eq) and 4-(dimcthylamino)pyridinc (4.48 30 mg, 0.04 mmol, 0.1 eq), followed by di-ieri-butyl dicarbonate (0.09 mL, 0.44 mmol, 1.2 eq) and the mixture was stirred at ambient température for 3 h. The réaction was partitioned between dîchloromethane and saturated aqueous sodium bicarbonate, and the organic phase was dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 85% ethyl acetate în iso-heptane afforded the desired product as a clear oil (48 mg, 0.18 mmol, 49%).
LC/MS (C14H20FNO3) 170 [M-Boc+H]+; RT 2.54 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 9.60 (s, IH), 6.95 (d, J = 12.3 FIz, IH), 6.89 - 6.72 (m, 2H), 3.34 - 3.27 (m, 2H), 2.73 (s, 3H), 2.64 (t, J = 7.1 Hz, 2H), 1.28 (s, 9H).
Préparation 4k: Ce//-Butyi V-[4-(3-lluoro-4-hydroxyphenyl)butyl]-Amethylcarbamate
Sien A : ethyl N-[(3E)-4-(3-fluoro-4-methoxyphenyl)but-3-en-l-yl]carbamate
To a solution of the product from Préparation 4i, Step C (397 mg, 2.03 mmol, 1 eq) in dîchloromethane (20 mL) was added trîethylamine (0.57 mL, 4.07 mmol, 2 eq) and the mixture was cooled to 0 °C. Ethyl chloroformate (194 pL, 2.03 mmol, 1 eq) was added and the mixture was ailowed to warm to ambient température and stir overnight. The reaction was partitioned between dîchloromethane and saturated aqueous sodium bicarbonate, and the organic phase was separated (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 25% ethyl acetate in iso-heplane afforded the desired product as a clear oil (310 mg, 1.16 mmol, 57%).
LC/MS (C14H18FNO3) 268 [M+H]+; RT 2.06 (LCMS-V-C) ‘H NMR (400 MHz, DMSO-d6) δ 7.24 - 7.05 (m, 3H), 6.42 - 6.33 (m, IH), 5.57 (dt, J = 11.7, 7.2 Hz, IH), 4.00 (dq, J = 26.6, 7.1 Hz, 2H), 3.84 (s, 3H), 3.08 (q, J = 6.8 Hz, 2H), 2.42 (qd, J = 7.1, 1.9 Hz, 2H), 1.16(1, 3H).
Step B: ethylN~[4’(3-fluoro-4-methoxyphenyl)butyl]carbamate
To a solution of the product from Step A (310 mg, 1.16 mmol, 1 eq) in methanol (12 mL) was added platinum(IV) oxide (26.3 mg, 0.12 mmol, 0.1 eq) under a nitrogen atmosphère. The vessel was evacuated and backfilled with nitrogen (x3), evacuated, placed under an atmosphère of hydrogen and shaken at ambient température overnight. The reaction was filtered through celite, eiuted with methanol and concentrated in vacuo to afford the desired product as a clear oil (275 mg, 1.02 mmol, 88%).
LC/MS (C14H20FNO3) 270 [M+H]+; RT 2.07 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.12 - 7.00 (m, 3H), 6.99 - 6.91 (m, 1H), 3.96 (q, J = 7.1 Hz, 2H), 3.80 (s, 3H), 2.97 (q, J = 6.7 Hz, 2H), 2.52 - 2.45 (m, 2H), 1.51 (p, J = 7.8, 7.4 Hz, 2H), 1.37 (p, J = 7.2 Hz, 2H), 1.14 (t, J = 7.1 Hz, 3H).
Step C: [4-(3-fluoro-4-methoxyphenyl)butyl](methyl)amine
To a solution of the product from Step B (417 mg, 1.55 mmol, 1 eq) in tetrahydrofuran (5 mL), cooled to 0 ÜC, was added lithium aluminium hydride (IM in tetrahydrofuran; 3.87 mL, 3.87 mmol, 2.5 eq) and the mixture was heated at reflux overnight. The reaction was cooled to 0 CC, water (150 pL) was added, followed by 15% aqueous sodium hydroxide (150 pL) and water (450 pL). The mixture was diluted with tetrahydrofuran and stirred for 30 min. Magnésium sulfate and ethyl acetate were added, and the mixture was filtered through celite and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RedîSep™ silica cartridge) eluting with a gradient of 0 ~ 15% methanol in dichloromethane afforded the desired product as a clear oil (222 mg, 1.05 mmol, 68%).
LC/MS (C12H18FNO) 212 [M+H]+; RT 1.28 (LCMS-V-C) ‘H NMR (400 MHz, DMSO-d6) δ 7.09 - 6.99 (m, 2H), 6.94 (dd, 1H), 3.80 (s, 3H), 2.51 2.48 (m, 2H), 2.44 (t, 2H), 2.24 (s, 3H), 1.61 - 1.47 (m, 2H), 1.47 -1.31 (m, 2H).
Step D: 2-fluoro-4-[4-(methylamino)butyl]phenol
To a solution of the product of Step C (222 mg, 1.05 mmol, 1 eq) in dichloromethane (10 mL), cooled at 0 °C, was added boron tribromîde (IM in dichloromethane, 3.15 mL, 3.15 mmol, 3 eq) and the mixture was stirred at ambient température for 3 h. The reaction was cooled to 0 °C, quenched with methanol and concentrated in vacuo. The residue was dissolved in methanol, loaded onto a methanol-wet SCX cartridge (5 g), washed with methanol, eiuted with 1.4N methanolic ammonia and concentrated in vacuo to afford the desired product as a brown gum (63 mg, 0.32 mmol, 30%).
LC/MS (ChHiôFNO) 198 [M+H]+; RT 1.01 (LCMS-V-C)
4l NMR (400 MHz, DMSO-d6) δ 7.00 - 6.91 (m, 1H), 6.88 - 6.75 (m, 2H), 2.49 - 2.40 (m, 4H), 2.24 (s, 3H), 1.58 - 1.45 (m, 2H), 1.43 - 1.33 (m, 2H).
Step E: tert-butyl N-[4-(3-fluoro-4-hydroxyphenyl)butyl]-N-methylcarbamate
To a solution of the product of Step D (63 mg, 0.32 mmol, 1 eq) in dichloromethane (5 mL) was added triethylamine (133 pL, 0.96 mmol, 3 eq) and 4-(dimethylamino)pyridine (3.9 mg, 0.03 mmol, 0.1 eq) and the mixture was cooled to 0 °C and di-tert-butyl dicarbonate (66 5 pL, 0.29 mmol, 0.9 eq) was added and the mixture was stirred at ambient température for 1 h. The reaction was partitioned between dichloromethane and saturated aqueous sodium bicarbonate, and the organic phase was separated (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 35% ethyl acetate in iso-heptane 10 afforded the desired product (33 mg, 0.11 mmol, 35%).
LC/MS (Ci6H24FNO3) 198 [M-Boc+H|4; RT 2.18 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 9.54 (s, 1H), 6.95 (dd, J = 12.4, 2.0 Hz, 1H), 6.88 - 6.74 (m, 2H), 3.21-3.11 (m, 2H), 2.74 (s, 3H), 2.49 - 2.41 (m, 2H), 1.53 - 1.40 (m, 4H), 1.37 (s, 9H).
Préparation 5a: l-(l-AdamantylmethyI)-5-methyl-4-(4,4,5,5-tetraniethyl-l,3,2dioxaborolan-2-yl)pyrazole
Step A: 1 -(l-adamantylmethyl)-4-iodo-pyrazole
The mixture of 35.9 g of 1-adamantylmethanol (216 mmol), 73.48 g of triphenylphosphine (280 mmol, 1.3 eq.), 54.25 g of 4-iodo-l//-pyrazole (280 mmol, 1.3 eq.) and 64.4 g of tert20 butyl V-(ter/-butoxycarbonyliminomethyIene)carbamate (266 mmol. 1.3 eq.) in 1078 mL of THF was stirred at rt for 48 h. After the addition of extra 10.94 g of 4-iodo-l/f-pyrazole (56 mmol, 0.26 eq.), 12.81 g of Zert-butyl W-(tert-butoxycarbonyliminomethylene)carbamate (53 mmol, 0.26 eq.) and 14.69 g of triphenylphosphine (56 mmol, 0.26 eq.), the reaction was stirred at rt for 24 h then concentrated, purified via flash column chromatography using DCM 25 as eluent, triturated in cold MeOH, and filtered off to give 53.6 g (73%) of the desired product as while powder.
Step B: I-(l-adamantylmethyl)-4-iodo-5-methyl-pyrazole
To 9.8 mL of diisopropylamine (69.5 mmol, 1.1 eq.) in 180 mL of THF was added dropwise 33.4 mL of a 2.5 M solution of butyl lithium (84 mmol, 1.3 eq.) at -78 °C and the mixture was 30 stirred at -78 °C for 0.5 h, treated with 22.0 g of the product from Step A (64.28 mmol, 1 eq.) φ in 90 mL of THF, stirred at -78 °C for 1 h, treated with 4.67 mL of methyliodide (73.3 mmol, 1.14 eq.), and stirred at -78 °C for 18 h. After quenching with cc. NH4CI, the reaction was extracted with EtOAc and the combined organic phases were washed with brine, dried, concentrated, triturated in MeOH, and filtered off to give 21 g (92%) of the desired product.
Step C: 1 -(1-adamantylmethyl) -5-tnethyl-4-(4,4,5,5-tetramethyl-If3,2-dioxaborolan-2yl)pyrazole
To 21 g of the product from Step B (58.95 mmol, 1 eq.) in 300 mL of THF was added 28.3 mL of a 2.5 M solution of butyllithium (70.8 mmol, 1.2 eq) at -78 °C and the mixture was stirred at -78 °C for 0.5 h, treated with 16.4 g of 2-isopropoxy-4,4,5,5-te tramethy 1-1,3,210 dioxaborolane (88.1 mmol, 1.5 eq.) (addition in portions over 40 min), and kept at -78 °C for 24 h. After quenching with cc. NH4CI at rt, the reaction was extracted with EtOAc and the combined organic phases were washed wîth brine, dried, concentrated, triturated in MeOH, and filtered off to give 19.7 g (94%) of the desired product as off-white crystals.
*H NMR (500 MHz, DMSO-r/6) δ ppm 7.45 (s, 1H), 3.69 (s, 2H), 2.36 (s, 3H), 1.91 (m, 1 H), 15 1.64/1.54 (m, 6H), 1.50 (m, 6H), 1.24 (s, 12H); 13C NMR (500 MHz, , DMSO-do) δ ppm
146.9, 144.1, 104.6, 59.7, 40.6, 36.S, 35.4, 28.1, 25.1, 12.1; HRMS-ESI (m/z): [M+H]+ calcd for C21H34BN2O2; 357.2713, found 357.2704.
Préparation 5b: l-{[l-(3-Methoxypropyl)cycloocty!Jmethyl}-5-methyl-4-(4,4,5,5tetramethyl-l,3,2-dioxaborolan-2-yl)-Lff-pyrazole
Step A: methyl l-(3-methoxypropyl)cyclooctanecarboxylate
To 4.74 g (1.14 eq.) of diisopropyiamine in 90 mL of tetrahydrofuran was added 18.8 mL (1.14 eq.) of a 2.5 M solution of butyl lithium at -78 °C and after 0.5 h al -78 °C, 7.0 g (41.1 mmol) of methyl cyclooctanecarboxylate in 40 mL of tetrahydrofuran was added over 1 h. After 1 h at -78 °C, 7.2 g (1.14 eq.) of l-bromo-3-methoxy-propane was added and the 25 mixture was stirred for 18 h. After quenching the reaction with the addition of saturated NH4CI solution, the mixture was extracted with EtOAc and the organic phases were dried over MgSÛ4 and concentrated to give 8.0 g (80%) of the desired product.
Ή NMR (400 MHz, CDCli) δ ppm 3.66 (s, 3H), 3.33 (t, 2H), 3.31 (s, 3H), 2.03-1.94 (m, 2H), 1.64-1.38 (m, 16H).
Step B: [l-(3-methoxypropyl)cyclooctyl]methanol
To 9.0 g (37.13 mmol) of the product from Step A in 93 mL of diethyl ether was added 1.76 g (1.25 eq.) of lithium aiuminum hydride portion wise at 0 °C. After stirring at rt for 2 h, the reaction was quenched by the addition of icy water and EtOAc and a 10% solution of NaOH 5 were added. The mixture was extracted with EtOAc, dried, and concentrated to give 7.4 g (93%) of the desired product.
Ή NMR (400 MHz, CDCb) δ ppm 3.37 (t, 2H), 3.34 (s, 3H), 3.30 (s, 2H), 1.61-1.23 (m, 18H).
Step C: 4-iodo~l-[[l-(3-methoxypropyl)cyclooctyl]methyl]-lH-pyrazole
To 1.39 g (6.5 mmol) of the product from Step B and 1.64 g (1.3 eq.) of 4-iodo-lff-pyrazole in 33 mL of tetrahydrofuran was added 2.22 g (1.3 eq.) of triphenylphosphine and 1.95 g (L3 eq.) of di-ieri-butyl azodicarboxylate and the mixture was stirred at rt for 67 h. To the mixture was added 278 mg of 4-iodo-lff-pyrazole, 444 mg of triphenylphosphine, and 390 mg of diterr-butyi azodicarboxylate and was stirred at rt for 24 h. After the addition of reagents and 15 stirring at rt for 24 h was repeated (115 h stirring in total), the mixture was concentrated and purified via flash column chromatography (silica gel) using heptane and EtOAc as eluents to give 1.24 g (49%) of the desired product.
*H NMR (400 MHz, CDCh) Ô ppm 7.47 (s, 1H), 7.42 (s, 1H), 3.93 (s, 2H), 3.37 (t, 2H), 3.36 (s, 3H), 1.68-1.18 (m, 18H).
Step D: 4-iodo-I-[[l-(3-methoxypropyl)cyclooctyl]methyl]-5-methyl-lH-pyrazole
To 1.2 g (3.07 mmol) of the product from Step C in 5 mL of tetrahydrofuran was added 3.7 mL (1.2 eq.) of a 1 M solution of LDA at -78 °C. After 0.6 h at -78 °C, 0.5 mL (1.14 eq.) of methyl iodide was added dropwise to the mixture and it was let to warm up to rt over 20 h. Reaction was quenched with a saturated solution of NH4CI and extracted with EtOAc. The 25 combined organic phases were dried, concentrated, and purified via flash column chromatography (silica gel) using heptane and EtOAc as eluents to give 0.79 g (64%) of the desired product.
Ή NMR (400 MHz, CDCI3) δ ppm 7.43 (s, 1H), 3.85 (s, 2H), 3.38 (t, 2H), 3.35 (s, 3H), 2.29 (s, 3H), 1.69-1.24 (m, 18H).
φ StepE: 1 -[[l-(3-methoxypropyl)cydooayl!methyl]-5-methy 1-4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan -2-yl)-lH-pyrazole
To the solution of 0.81 g (2 mmol) of the product from Step D in 15 mL of tetrahydrofuran was added 0.96 mL (1.2 eq.) of a 2.5 M solution of butyl lithium dropwise at -78 °C. After 0.5 5 h, 0.5 mL (1.2 eq.) of 2-isopropoxy-4,4,5,5-tetramethyl-l,3,2-dioxaboroIane was added over 20 min and the mixture was kept at -78 °C for 6 h and at rt for 6 h. After quenching the reaction with saturated solution of NH4CI and extractîng with EtOAc, the combined organic phases were washed with brine, dried, and purified via flash column chromatography (silica gel) using heptane and EtOAc as eluents to give 0.33 g (34%) of the desired product.
‘H NMR (500 MHz, dmso-d6) δ ppm 7.46 (s, 1H), 3.75 (s, 2H), 3.27 (t, 2H), 3.21 (s, 3H), 2.36 (s, 3H), 1.66-1.1 (m, 14H), 1.57 (m, 2H), 1.24 (s, 12H), 1.24 (m, 2H). 13C NMR (500 MHz, dmso-d6) δ ppm 147.3, 144.5, 104.5, 73.2, 58.2, 54.4, 40.5, 33.2, 25.1,23.6, 11.8. IR: 2922, 1556, 1246, 1144, 1055. HRMS-ESI (m/z): [M+H]+ calcd for C23H42N2O3B: 405.3289, found 405.3329.
Préparation 5c: l-{[l-(3-Methoxypropyl)cydohexyl]methyl}-5-methyl-4-(4,4,5,5tetramethyl-l,3,2-dioxaborolan-2-yl)-l//-pyrazole
Step A: methyl l-(3-methoxypropyl)cyclohexanecarboxylate
To 6.84g (1.09 eq.) of diisopropylamine in 130 mL of tetrahydrofuran was added 27 mL (1.09 eq.) of a 2.5 M solution of butyl lithium at -78 °C and after 0.5 h at -78 °C, 8.8 g of methyl 20 cyclohexanecarboxylate in 50 mL of tetrahydrofuran was added over 1 h. After 1 h at -78 °C, 10.7 g (1.13 eq.) of l-bromo-3-methoxy-propane was added and the mixture was stirred for 18 h. After quenching the reaction with the addition of saturated NH4C1 solution, the mixture was extracted with EtOAc and the organic phases were dried over MgSO4 and concentrated to give 12 g (92%) of the desired product.
lH NMR (400 MHz, CDCh) Ô ppm 3.67 (s, 3H), 3.35 (d, IH), 3.32 (d, 1H), 3.31 (s, 3H), 2.11-2.03 (m, 2H), 1.60-1.16 (m, 12H).
Step B: /1 -(3-methoxypropyl)cydohexyl] methanol
To 12 g (56.41 mmol) of the product from Step A in 140 mL of diethyl ether was added 2.68 g (1.25 eq.) of lithium aluminum hydride portion wise at 0 °C. After stirring at rt for 2 h, the 30 reaction was quenched by the addition of icy water and EtOAc and a 10% solution of NaOH
P were added. The mixture was extracted with EtOAc, dried, and concentrated to give 9.37 g (89%) of the desired product.
Ή NMR (400 MHz, CDCh) δ ppm 3.41 (s, 2H), 3.38 (t, 2H), 3.35 (s, 3H), 1.56-1.27 (m, 14H).
Step C: 4-iodo-l [[l-(3-methoxypropyl) cyclohexyl]methyl]pyrazole
To 1.21g (6.5 mmol) of the product from Step B and 2.58 g (2.05 eq.) of 4-iodo-l H-pyrazole in 33 mL of tetrahydrofuran was added 3.5 g (2.05 eq.) of triphenylphosphine and 3.07 g (2.05 eq.) of di-/trr-butyl azodîcarboxylate and the mixture was stirred at rt for 2 h. To the mixture was added 140 mg of 4-iodo-l//-pyrazole, 230 mg of triphenylphosphine, and 200 10 mg of di-fôrt-butyl azodîcarboxylate and was stirred at rt for 24 h. After the addition of reagents and stirring at rt for 24 h was repeated twice (96 h stirring in total), the mixture was concentrated and purified via flash column chromatography (silica gel) using heptane and EtOAc as eiuents to give 1.4 g (59.5%) of the desired product.
* H NMR (400 MHz, CDCh) δ ppm 7.47 (s, 1H), 7.41 (s, 1H), 4.00 (s, 2H), 3.36 (t, 2H), 3.35 15 (s, 3H), 1.62-1.21 (m, 14H).
Step D: 4-iodo-l-[[l-(3-methoxypropyl)cyclohexyl]methyl]-5-methyl-pyrazole
To 3.7 g (10.21 mmol) of the product from Step C in 15 mL of tetrahydrofuran was added 12.3 mL (1.2 eq.) of a 1 M solution of LDA in tetrahydrofuran at -78 °C. After 0.6 h at -78 °C, 0.73 mL (1.14 eq.) of methyl iodide was added dropwise to the mixture and it was let to 20 warm up to rt over 20 h. Reaction was quenched with a saturated solution of NH4CI and extracted with EtOAc. The combined organic phases were dried, concentrated, and purified via flash column chromatography (silica gel) using heptane and EtOAc as eiuents to give 2.85 g (74%) of the desired product.
‘ 11 NMR (400 MHz, CDCh) δ ppm 7.44 (s, 1H), 3.92 (s, 2H), 3.38 (t, 2H), 3.35 (s, 3H), 2.29 25 (s, 3H), 1.58-1.13 (m, 14H).
Step E: 1-[[1 -(3-methoxypropyl)cyclohexyl]methyl]-5-methy 1-4-(4,4,5,5-tetramethyl-l,3,2dioxaborolan-2-yl)pyrazole
To the solution of 5.0 g (13.3 mmol) of the product from Step D in 71 mL of tetrahydrofuran was added 6.38 mL (1.2 eq.) of a 2.5 M solution of butyl lithium dropwise at -78 °C. After 0.5 30 h, 4.1 mL (1.5 eq.) of 2-îsopropoxy-4,4,5,5-tetramethyl-l,3,2-dioxaborolane was added over 40 min and the mixture was kept at -78 °C for 6 h and at rt for 6 h. After quenching the ) reaction with saturated solution of NH4CI and extracting with EtOAc, the combined organic phases were washed with brine, dried, and purified via flash column chromatography (silica gel) using heptane and EtOAc as eluents to give 2.3 g (46%) of the desired product.
NMR (500 MHz, dmso-d6) δ ppm 7.47 (s, 1H), 3.84 (s, 2H), 3.27 (t, 2H), 3.2 (s, 3H), 2.37 5 (s, 3H), 1.54-1.07 (m, 10H), 1.46 (m, 2H), 1.32 (m, 2H), 1.24 (s, 12H). 13C NMR (500 MHz, dmso-d6) δ ppm 147.3, 144.4, 104.6, 73.1, 58.2, 55.7, 37.9, 30.6, 25.1, 23.1, 12.0. IR: 2927, 1556, 1257, 1144, 1053. HRMS-ESI (m/z): [M+H]+ calcd for C21H38N2O3B: 376.2897, found 376.3019.
Préparation 6a: Ethyl 5-bromo-2-(4-methyl-3-{[(2Z)-3-{[210 (trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3-benzothiazoI-2-ylidene]amino}5//,6//, 7//-pyrrolo[2,3-c]pyridazin-7-yl)-l,3-thiazole-4-carboxylate
Step A : ethyl 2-/(pent-3-yn-l-yl)amino]-l,3-thiazole-4-carboxylate
To a solution of ethyl 2-bromo-l,3-thiazole-4-carboxylate (538 mg, 2.28 mmol, 1 eq) in acetonitrile (10 mL)was added pent-3-yn-l-amine hydrochloride (300 mg, 2.51 mmol, 1.1 15 eq) and triethylamîne (0.7 mL, 5.02 mmol, 2.2 eq) and the mixture was heated at 150 °C for 3 h under microwave irradiation. The reaction was partitioned between ethyl acetate and brine, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 — 40% ethyl acetate in rso-heptane afforded the desired product as 20 a beige solid (221 mg, 0.93 mmol, 41%).
LC/MS (C11H14N2O2S) 239 [M+Hf ; RT 2.22 (LCMS-V-C) * H NMR (400 MHz, DMSO-d6) δ 7.93 (t, J = 5.7 Hz, 1H), 7.52 (s, 1H), 4.22 (q, J = 7.1 Hz, 2H), 3.38 - 3.28 (m, 2H), 2.44 - 2.33 (m, 2H), 1.75 (l, J = 2.5 Hz, 3H), 1.27 (t, J = 7.1 Hz, 3H).
Step B: ethyl 2-{3-chloro-4~methyl~5H,6H,7H-pyrrolo[2,3-c]pyridazin-7-yl}-l,3-thiazole-4carboxylate
To a solution of 3,6-dichloro-l,2,4,5-tetrazine (140 mg, 0.93 mmol, 1 eq) in tetrahydrofuran was added the product from Step A (221 mg, 0.93 mmol, 1 eq) and the mixture was heated at reflux overnight. The reaction was concentrated in vacuo and the residue was triturated with dichloromethane, filtered, and dried under vacuum to afford the desired product as an offwhite solid (148 mg, 0.46 mmol, 49%).
LC/MS (C13HBCIN4O2S) 325 [M+H]+; RT 2.32 (LCMS-V-C) * H NMR (400 MHz, DMSO-d6) δ 8.11 (s, IH), 4.41 (dd, J = 8.8, 7.7 Hz, 2H), 4.30 (q, J = 7.1 Hz, 2H), 3.34 - 3.24 (m, 2H), 2.29 (d, .1 = 1.1 Hz, 3H), 1.31 (t, J = 7.1 Hz, 3H).
Step C: ethyl 2~{3-[(l ,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H, 7H-pyrrolo[2,3c]pyridazin-7-yl}-l ,3-thiazole-4-carboxylate
To an-oven dried microwave vial was added the product from Step B (148 mg, 0.46 mmol, 1 eq), 2-aminobenzothiazole (103 mg, 0.68 mmol, 1.5 eq), XantPhos (52.7 mg, 0.09 mmol, 0.2 eq), césium carbonate (297 mg, 0.91 mmol, 2 eq), and 1,4-dioxane (20 mL) and the vessel was evacuated and flushed with nitrogen then tris(dibenzylideneacetone)dipalladium(0) (41.7 mg, 0.05 mmol, 0.1 eq) was added and the mixture was sparged with nitrogen (10 min) then heated at 150 °C for 2 h under microwave irradiation. The reaction was diluted with ethyl acetate, filtered through celite, washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% methanoi in dichloromethane afforded the desired product as a yellow solid (103 mg, 0.23 mmol, 52%).
LC/MS (C20H18NÈO2S2) 439 [M+H]+; RT 2.67 (LCMS-V-C)
NMR (400 MHz, DMSO-d6) δ 10.94 (br s, IH), 8.06 (s, IH), 7.95 (br s, IH), 7.66 (br s, IH), 7.44 - 7.33 (m, IH), 7.28 - 7.15 (m, IH), 4.42 - 4.34 (m, 2H), 4.30 (q, 2H), 3.32 - 3.28 (m, 2H), 2.34 (s, 3H), 1.32 (t, J - 7.1, 2.4 Hz, 3H).
Step D: ethyl 2-(4-methyl-3-{[(2Z)-3f[2-(trimethylsilyl)ethoxyJmethyl}-2,3-dihydro-l,3benzothiazol-2-ylidene]amino}-5H,6H, 7H-pyrrolo[2,3-cjpyridazin - 7-yl)-l ,3-thiazole-4carboxylate
To a cooled solution of the product of Step C (103 mg, 0.23 mmol, 1 eq) in tetrahydrofuran (15 mL) and dimethylformamide (5 mL) was added ΛζΝ-dnsopropylethy lamine (81.8 pL, 0.47 mmol, 2 eq). After 5 min, 4-dimethylaminopyridine (5.74 mg, 0.05 mmol, 0.2 eq) and [2-(chloromethoxy)elhyl]trimethylsilane (103 pL, 0.59 mmol, 2.5 eq) were added and the mixture was stirred at ambient température overnight. The reaction was partitioned between ethyl acetate and water, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash
Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 70% ethyl acetate in isoheptane afforded the desired product as an off white solid (98 mg, 0.17 mmol, 73%).
LC/MS (C26H32N6O3S1S2) no ionisation; RT 3.08 (LCMS-V-C) * H NMR (400 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.91 (d, J = 7.6 Hz, 1H), 7.60 - 7.51 (m, 2H), 5 7.39 - 7.30 (m, 1H), 5.96 (s, 2H), 4.48 (t, J = 8.1 Hz, 2H), 4.40 (q, J = 7.1 Hz, 2H), 3.87 - 3.78 (m, 2H), 3.48 - 3.36 (m, 2H), 2.44 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H), 1.07 - 0.98 (m, 2H), 0.00 (s, 9H).
Step E: ethyl 5-bromo-2-(4~methyl-3-{[(2Z)-3-{[2-(trimethylsilyl)ethoxy]methyl}-2,3dihydro-1,3-benzothiazol-2-ylidene]amino}-5H,6H, 7H-pyrrolo[2,3-c]pyridazin -7 -y 1)-1,310 thiazole-4-carboxylate
To a solution of the product of Step D (98 mg, 0.17 mmol, 1 eq) in dichloromethane (15 mL) was added A'-bromosuccinimide (39.9 mg, 0.22 mmol, 1.3 eq) and the mixture was stirred at ambient température for 3 h. Purification by automated flash column chromatography (CombiFlash Rf, 12g RediSep™ silica cartridge) eluting with a gradient of 0 15 - 50% ethyl acetate in Lw-heptane afforded the desired product as an off-white solid (98 mg,
0.15 mmol, 88%).
LC/MS (C26H3iBrN6O3SiS2) no ionisation; RT 3.22 (LCMS-V-C) * H NMR (400 MHz, DMSO-d6) δ 7.81 (dd, J = 7.6, 1.1 Hz, 1H), 7.50 - 7.39 (m, 2H), 7.28 7.21 (ni, 1H), 5.85 (s, 2H), 4.40 - 4.24 (m, 4H), 3.68 - 3.58 (m, 2H), 3.27 (t, J = 8.0 Hz, 2H), 20 2.32 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H), 1.02 (dd, J = 8.5, 7.4 Hz, 2H), -0.12 (s, 9H).
Préparation 7: ieri-butyl-diphenyl-[2-[[3,5-diniethyl-7-[[5-methyl-4-(4,4,5,5-tetramethyll,3,2-dioxaborolan-2-yl)pyrazol-l-yl]methyl]-l-adamantyl]oxy]ethoxy]silane
Step A : 3-bromo-5,7-dimeth yladamantane-1 -carboxylic acid
After stirring iron (6.7 g, 120 mmol) in bromine (30.7 mL, 600 mmol, 5 eq) at 0 °C for 1 h, 25 3,5-dimethyladamantane-1 -carboxylic acid (25 g, 1 eq) was added and the reaction mixture was stirred at rt for 2 days. After the addition of EtOAc, the reaction mixture was treated carefully with a saturated solution of sodium-thiosulfate at 0 °C and stirred for 15 min. After filtration through a pad of Celite and rinsing with EtOAc, the organic phase was separated, washed with a saturated solution of sodium-thiosulfate and brîne, dried, concentrated to give 30 the desired product (34.28 g, 74.6%), which was used without further purification.
‘H NMR (400 MHz, DMSO-d6): δ ppm 12.33 (br., IH), 2.21 (s, 2H), 1.96/1.91 (d+d, 4H), 1.50/1.43 (d+d, 4H), 1.21/1.14 (dm+dm, 2H), 0.86 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ ppm 176.8, 66.8, 54.0, 48.7, 48.5, 45.7, 43.3, 35.5, 29.4; HRMS-ESI (m/z): [M-H]- calcd for CUH.-BrO,: 285.0496; found 285.0498.
U 10 Z 7
Step B; 3-bromo-5,7-dimethyl-l-adamantyl-methanol
To the product from Step A (34.3 g, 119 mmol) in THF (77.6 mL) was added slowly a 1 M solution of BH3-THF in THF (358 mL, 3 eq) and the réaction mixture was stirred for 18 h. After the addition of methanol and stirring for 30 min, purificationby column chromatography (silica gel, heptane and MTBE as eluents) afforded the desired product (16.19 g, 49.6%).
*H NMR (400 MHz, DMSO-d6): δ ppm 4.51 (t, IH), 3.Ü5 (d, 2H), 1.91 (s, 2H), 1.91 (s, 4H), 1.19/1.09 (d+d, 2H), 1.19/1.05 (d+d, 4H), 0.85 (s, 6H) 13C NMR (100 MHz, DMSO-dù) δ ppm 70.4, 68.9, 54.9, 49.8, 49.3, 43.8, 41.4, 35.7, 29.7; HRMS-ESI (m/z): [M-Br]- calcd for C13H2]O: 193.1598 found: 193.1589.
Step Cz 1 -[3-bromo-5,7-dimethyl-l-adamantyl]methyl]pyrazole
To the product from Step B (16.19 g, 59.26 mmol) and l/Tpyrazole (4.841 g, 1.2 eq) in toluene (178 mL) was added cyanomethylenetributylphosphorane (18.64 mL, 1.2 eq) in one portion and the reaction mixture was stirred at 90 °C for 2 h. Purificationby column chromatography (silica gel, heptane and MTBE as eluents) afforded the desired product (17.88 g, 93%).
‘H NMR (400 MHz, DMSO-d6): δ ppm 7.63 (d, IH), 7.43 (d, IH), 6.23 (t, IH), 3.90 (s, 2H), 1.92-1.02 (m, 12H), 0.83 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ ppm 139.0, 131.8, 105.2, 67.7, 61.4, 54.4/48.8/44.6, 50.4, 35.7, 29.6; HRMS-ESI (m/z): [M]+ calcd for C]6H23BrN2: 322.1045 found: 322.1014.
Stej)Dz 5~methyl-l-[[-3-bromo-5,7-dimethyl-l-adamantyl]methyl]pyrazole
To the solution of the product from Step C (17.88 g, 55.3 mmol) in THF (277 mL) was added butyllithium (2.5 M in THF, 66 mL, 3 eq) at -78 °C, then after 1 h, iodomethane (17.2 mL, 5 eq) was added. After 10 min, the reaction mixture was quenched with a saturated solution of NH4CI, extracted with EtOAc and the combined organic layers were dried and concentrated to give the desired product (18.7 g, 100%), which was used in the next step without further 30 purification.
*H NMR (400 MHz, DMSO-d6): δ ppm 7.31 (d, 1H), 6.00 (d, 1H), 3.79 (s, 2H), 2.23 (s, 3H), 2.01 (s, 2H), 1.89/1.85 (d+d, 4H), 1.23/1.15 (d+d, 4H), 1.16/1.05 (d+d, 2H), 0.83 (s, 6H); I3C NMR (100 MHz, DMSO-d6) δ ppm 139.2, 138.0, 105.2, 67.8, 57.8, 54.4, 50.6, 48.8, 44.8, 41.5, 35.7, 29.6, 11.8; HRMS-ESI (m/z): [M+H]+ calcd for C]7H26BrN2 : 337.1279 found: 5 337.1289.
Step E: 2-[f-3,5-dimethyl-7-[(5-methylpyraz.ol-l-yl)methyl]-!-adamantyl]oxy] éthanol
The mixture of the product from Step D (18.7 g, 55.3 mmol), ethylene glycol (123 mL, 40 eq), and DIPEA (48.2 mL, 5 eq) was stirred at 120 °C for 6 h. After the reaction mixture was diluted with water and extracted with EtOAc, the combined organic layers were dried and 10 concentrated to give the desired product (18.5 g, 105%), which was used in the next step without further purification.
Ή NMR (400 MHz, DMSO-dù): δ ppm 7.29 (d, 1H), 5.99 (d, 1H), 4.45 (t, 1H), 3.78 (s, 2H), 3.39 (q, 2H), 3.32 (t, 2H), 2.23 (s, 3H), 1.34 (s, 2H), 1.27/1.21 (d+d, 4H), 1.13/1.07 (d+d, 4H), 1.04/0.97 (d+d, 2H), 0.84 (s, 6H); i3C NMR (100 MHz, DMSO-d6) δ ppm 139.0, 137.8, 15 105.1, 74.0, 62.1, 61.5, 58.5, 50.1, 47.0, 46.1, 43.3, 39.7, 33.5, 30.2, 11.9; HRMS-ESI (m/z):
[M+H]+ calcd for C19H31N2O2 : 319.2386 found: 319.2387.
Step F: tert-butyl-diphenyl-[2-[[-3,5-dimethyl-7-[(5-methylpyrazol-l~yl)methyl]-ladamantyl]oxy]ethoxy]silane
To the mixture of the product from Step E (17.6 g, 55.3 mmol) and imidazole (5.65 g, 1.5 eq) 20 in DCM (150 ml) was added fôri-butyLchloro-diphenyl-silane (18.6 g, 1.2 eq) and the reaction mixture was stirred for 1 h. Purificationby column chromatography (silica gel, heptane and MTBE as eluents) afforded the desired product (27.0 g, 87.8%).
’H NMR (400 MHz, DMSO-ds): δ ppm 7.72-7.34 (m, 10H), 7.29 (d, 1H), 5.99 (br., 1H), 3.78 (s, 2H), 3.67 (t, 2H), 3.44 (t, 2H), 2.21 (s, 3H), 1.33 (s, 2H), 1.26/1.18 (d+d, 4H), 1.12/1.06 25 (d+d, 4H), 1.03/0.96 (d+d, 2H), 0.98 (s, 9H), 0.82 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ ppm 139.0, 137.8, 105.1, 74.2, 64.4, 61.7, 58.5, 50.0, 46.9, 46.0, 43.4, 39.6, 33.5, 30.1, 27.1, 19.3, 11.9; HRMS-ESI (m/z): [M+H]+ calcd for C35H49N2O2Si : 557.3563 found: 557.3564.
Step G: tert-butyl-diphenyl-[2-[[3-[(4-iodo~5-methyl-pyrazol-l-yl)methyl]-5,7-dimethyl-ladamantyl]oxy]ethoxy]sUane φ Το the solution of the product from Step F (27.0 g, 48.56 mmol) in DMF (243 mL) was added /V-iodosuccinimide (13.6 g, 1.25 eq) and the reaction mixture was stirred for 2 h. After the dilution with water, the mixture was extracted with DCM. The combined organic layers were washed with saturated solution of sodîum-thiosulphate and brine, dried, and concentrated to 5 afford the desired product (30.1g, 90%).
Ή NMR (400 MHz, DMSO-d6); δ ppm 7.68-7.37 (m, 10H), 7.45 (s, 1H), 3.89 (s, 2H), 3.67 (t, 2H), 3.44 (t, 2H), 2.23 (s, 3H), 1.30 (s, 2H), 1.26/1.17 (d+d, 4H), 1.12/1.05 (d+d, 4H), 1.00/0.96 (d+d, 2H), 0.98 (s, 9H), 0.82 (s, 6H); 13C NMR (100 MHz, DMSO-dô) δ ppm 142.5, 140.8, 133.7, 64.4, 61.7, 60.3, 59.9, 49.9, 46.8, 45.9, 43.2, 39.7, 33.5, 30.1, 27.1, 19.3, 10 12.2; HRMS-ESI (m/z): [M+H]+ calcd for C35H48IN2O2Si: 683.2530 found: 683.2533.
Step H: tert-biityl-diphenyl-[2-[[3,5-dimethyl-7-[[5-methyl~4-(4,4,5,5-tetramethyl-l,3,2dioxaborolan-2-yl)pyrazol-l-yl]methyl]~l-adamantyl]oxy]ethoxy]silane
To the product from Step G (17.5 g, 25.6 mmol) in THF (128 mL) was added chloro(isopropyl)magnesium-LiCl (1.3 M in THF, 24 mL, 1.2 eq) at 0 DC, stirred for 40 min, 15 treated with 2-isopropoxy-4,4,5,5-tetramethyl-l,3,2-dioxaborolane (15.7 mL, 3 eq), and the reaction mixture was stirred for 10 min. After dilution with a saturated solution NH4CI and extraction with EtOAc, the combined organic phases were concentrated and was purified by column chromatography (siiica gel, heptane and MTBE as eluents) to give the desired product (15.2g, 86.9%).
Ή NMR (400 MHz, DMSO-dù): δ ppm 7.65 (dm, 4H), 7.47 (s, 1H), 7.45 (tm, 2H), 7.40 (tm, 4H), 3.80 (s, 2H), 3.66 (t, 2 H), 3.44 (t, 2H), 2.35 (s, 3H), 1.35-0.94 (m, 12H), 1.24 (s, 12H), 0.97 (s, 9H), 0.83 (s, 6H); 13C NMR (100 MHz, DMSO-ds) δ ppm 146.9, 144.3, 135.6, 130.2, 128.2, 104.7, 83.0, 74.2, 64.4, 61.7, 58.4, 30.1, 27.1, 25.2, 19.3, 12.0; HRMS-ESI (m/z): ΓΜ+Η1+ calcd for C..H...BN.O.SÎ: 683.4415 found: 683.4423.
L J 41 60 2 4
Préparation 8: ieri-butyl- [3-[3,5-dimethyl-7-[I5-methyl·4-(4,4,5,5-tetramethyJ-l,3,2dioxaborolan-2-yl)pyrazoLI-yl]methyl]-l-adamantyl]propoxy]-diphenyl-silane
Step A: 1 ‘[[3-allyl-S, 7-dùnethyl-l -adamantyl]methyl]-5-methyl-pyrazole
To the product of Step D of Préparation 7 (15.66 g, 46.43 mmol) and AgOTf (597 ni g, 0.Ü5 eq) in THF (232 mL) was added a 2 M solution of allyl-Mg-Cl in THF (46.4 mL, 2 eq) and 30 the reaction mixture was stirred for 0.5 h. After quenching with a saturated solution of NH4CI and extracting with EtOAc, the combined organic phases were concentrated and purified by column chromatography (silica gel, heptane and MTBE as eluents) to give the desired product (11.32 g, 81.7%).
Ή NMR (400 MHz, DMSO-d6): δ ppm 7.27 (d, 1H), 5.98 (m, 1H), 5.76 (m, 1H), 5.01/4.96 (dm+dm, 2H), 3.73 (s, 2H), 2.22 (s, 3H), 1.83 (d, 2H), 1.15-0.93 (m, 12H), 0.78 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ ppm 139.0, 137.7, 135.0, 117.7, 105.0, 59.0, 47.8, 44.2, 35.0, 31.8, 30.6, 11.9; HRMS-ESI (m/z): [M+H]+ calcd for C20H31Nr 299.2487 found; 299.2485.
Step B : 343,5-diinethvl- 7-f(5-methylpyrazol-l -vl) methyll-1 -adamantvl]propan-l -ol
To the product of Step A (10.2 g, 34.17 mmol), în THF (85 mL) was added a 1 M solution of BH3-THF in THF (85.4 mL, 2 eq) and the reaction mixture was stirred for 1 h. After treatment with a 10 M solution of NaOH (24 mL, 7 eq) and a 33 % solution of hydrogen peroxide (73 mL, 25 eq) at 0 °C, the reaction wasstirred at rt for 1 h. Then, it was quenched with aqueous HCl solution, extracted with EtOAc, and purified by column chromatography (silica gel, heptane and MTBE as eluents) to give the desired product (9.75 g, 90%).
>H NMR (400 MHz, DMSO-dô): δ ppm 7.28 (d, 1H), 5.98 (m, 1H), 4.33 (t, 1H), 3.73 (s, 2H), 3.32 (m, 2H), 2.22 (brs, 3H), 1.32 (m, 2H), 1.12-0.92 (m, 12H), 1.06 (m, 2H), 0.78 (s, 6H); 13C NMR (100 MHz, DMSO-dô) δ ppm 137.7, 105.0, 62.1, 59.1, 39.7, 30.7, 26.5, 11.9, HRMS-ESI (m/z): [M+H]+ calcd for C20H33N2O: 317.2593 found: 317.2590
Step C: tert-butyl-[3-[3,5-dimethyl-7-[(5-methylpyrazol-l-yl)methyl]-l-adamaiityljpropoxy]diphenyl-silane
To the product of Step B (9.75 g, 30.8 mmol) and imidazole (3.1 g, 1.5 eq) in DCM (92 ml) was added h?ri-/ni(y/-chloro-diphenyl-silane (9.45 mL, 1.2 eq) and the reaction mixture was stirred for 1 h. Purificationby column chromatography (silica gel, heptane and MTBE as eluents) afforded the desired product (12.5 g, 73%).
*H NMR (400 MHz, DMSO-dâ): δ ppm 7.63-7.39 (m, 10H), 7.27 (d, 1H), 5.98 (d, 1H), 3.72 (s, 2H), 3.59 (t, 2H), 2.21 (s, 3H), 1.42 (m, 2H), 1.1-0.92 (br„ 12H), 1.09 (m, 2H), 0.98 (s, 9H), 0.77 (s, 6H); 13C NMR (100 MHz, DMSO-dû) δ ppm 137.7, 105.0, 64.8, 59.1, 39.3, 38.0, 34.2, 31.8, 30.6, 27.2, 26.1, 19.2, 11.9; HRMS-ESI (m/z): [M+H]+ calcd for C,.Hs.NQOSi: 555.3771 found: 555.3770.
Step D: tert-butyl-[3-[3-[(4-iodo-5-methyl~pyrazol-l-yl)methyl]-5,7-dimethyl-l-adamantyl] propoxyj-diphenyl-silane
To the product of Step C (12.5 g, 22.54 mmol) in DMF (112 mL) was added Λ7iodosuccinimide (6.34 g, 1.25 eq) and the reaction mixture was stirred for 2 h. After quenching with a saturated solution of sodium thiosulfate and extraction with DCM, the combined organic phases were washed with saturated sodium thiosulphate and brine, dried, 5 and evaporated to afford the desired product (16.3 g, 105%). LC/MS (CîôHsoINzOSî) 681 [M+H]+.
Step E: tert-butyl-[3-[3,5-dimethyl-7-[[5-methyl-4-(4,4,St5-tetramethyl-l,3,2-dioxaborolan2-yl)pyrazol-l-yl]melhyl]-l-adamantyl]propoxy]-diphenyl-silane
To the product of Step D (16.25 g, 23.9 mmol) in THF (119 mL) was added 10 chloro(isopropyl)magnesium-LiCl (1.3 M in THF, 22 mL, 1.2 eq.) at 0 °C, the mixture was stirred for 40 min, treated with 2-isopropoxy-4,4,5,5-tetramethyl-l,3,2-dioxaborolane (14.6 mL, 3 eq), and stirred for 10 min. After dilution with a saturated solution NH4CI and extraction with EtOAc, the combined organic phases were concentrated and was purified by column chromatography (silica gel, heptane and MTBE as eluents) to give the desired product 15 (11.4 g, 70%).
Ή NMR (400 MHz, DMSO-d6): δ ppm 7.59 (d, 4H), 7.46 (s, 1H), 7.45 (t, 2H), 7.43 (t, 4H), 3.74 (s, 2H), 3.59 (t, 2H), 2.35 (s, 3H), 1.41 (qn, 2H), 1.24 (s, 12H), 1.09 (m, 2H), 1.08 (s, 4H), 1.05 (s, 2H), 0.98 (s, 9H), 0.98 (s, 2H), 0.94 (s, 4H), 0.78 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ ppm 146.9,144.2, 135.5, 133.8,130.3, 128.3, 104.6, 83.0, 64.7, 64.7, 59.0, 50.6, 20 48.2, 46.5, 44.1, 39.2, 37.9, 31.8, 30.7, 27.2, 26.1, 25.2, 19.2, 12.0; HRMS-ESI (m/z):
[M+Hf calcd for C42H62BN2O3Si: 681.4623 found: 681.4631.
Préparation 9: fer^butvl-[2-ί[3-ίί5-methyl-4-(4,4,5,5-tetramethyl·l,3,2-dioxaborolan-2yl)pyrazol·l-yl]nlethyl]-l-adamantyl]oxy]ethoxy]-dipheπyl-silane
StepA: (3-bromo-l-adamantyl) methanol
To 3-bromoadamantane-l-carboxylic acid (10.0 g, 38.6 mmol) in THF (25 mL) was added slowly a 1 M solution of BH3-THF in THF (115 mL, 3 eq) and the mixture was stirred for 48 h. After the addition of methanol and stirring for 30 min, purificationby column chromatography (silica gel, heptane and MTBE as eluents) afforded the desired product (8.37 g, 88%).
100
Φ *Η NMR (400 MHz, DMSO-d6): δ ppm 4.50 (t, 1H), 3.02 (d, 2H), 2.28/2.21 (dm+dm, 4H), 2.11 (m, 2H), 2.07 (s, 2H), 1.66/1.56 (dm+dm, 2H), 1.48/1.39 (dm+dm, 4H); ,3C NMR (100 MHz, DMSO-dô) δ ppm 70.9, 69.3, 51.3, 49.0, 40.6, 37.3, 35.1, 32.3.
Step B: !-[(3-bromo-l-adamantyl)methyl]pyrazole
To the product from Step A (8.37 g, 34.1 mmol), lH-pyrazole (2.79 g, 1.2 eq) in toluene (100 mL) was added (cyanomethylene)tributylphosphorane (10.7 mL, 1.2 eq) and the reaction mixture was stirred at 90 °C for 2 h. Purificationby column chromatography (silica gel, heptane and MTBE as eluents) afforded the desired product (8.50 g, 84%).
NMR (400 MHz, DMSO-d0): δ ppm 7.63 (dd, 1H), 7.43 (dd, 1H), 6.23 (t, 1H), 3.87 (s, 10 2H), 2.24/2.13 (m+m, 4H), 2.1 (m, 2H), 2.07 (s, 2H), 1.63/1.50 (m+m, 2H), 1.47/1.43 (m+m,
4H); 13C NMR (100 MHz, DMSO-d6) δ ppm 138.9, 131.7, 105.1, 68.0, 61.8, 51.8, 48.5, 39.8, 38.3, 34.6, 32.1; HRMS-ES1 (m/z): [M+H]+ calcd for C14H20BrN2: 295.0810 found: 295.0804.
Step C: l-[(3-bromo-l-adamantyl)methyl]-5-methyl-pyrazole
To the product from Step B (1.70 g, 5.76 mmol) in THF (30 mL) was added butyllithium (2.5 M in THF, 12 mL, 5 eq) at -78 DC. After 1 h, iodomethane (7.2 mL, 5 eq) was added to the mixture. After 10 min, the reaction mixture was quenched with a saturated solution of NHiCl, extracted with EtOAc and the combined organic layers were dried and concentrated to give the desired product (2.0 g, 112%), which was used in the next step without further 20 purification.
NMR (400 MHz, DMSO-d6): δ ppm 7.31 (d, 1H), 6.01 (d, 1H), 3.76 (s, 2H), 2.25/2.15 (d+d, 4H), 2.24 (s, 3H), 2.16 (s, 2H), 2.10 (m, 2H), 1.63/1.52 (d+d, 2H), 1.52/1.49 (d+d, 4H); 13C NMR (100 MHz, DMSO-d6) δ ppm 139.2, 138.0, 105.2, 68.2, 58.3, 52.1, 48.5, 40.5, 38.4, 34.5, 32.2, 11.8; HRMS-ESI (m/z): [M+H]+ calcd for C15H22BrN2: 309.0966 found: 25 309.0962.
Step D: 2-[[3-[(5-methylpyrazol-l -yl)methyl]-! -adamantyl]oxy] éthanol
The mixture of the product from Step C (2.00 g, 6.47 mmol), ethylene glycol (14.4 mL, 40 eq), and DI PEA (5.6 mL, 5 eq) was stirred at 120 °C for 6 h. After diluting with water and 30 extracting with EtOAc, the combined organic phases were purified by column
101
A chromatography (silica gel, heptane and MTBE as eluents) to give the desired product (1.62 g, 86.6%).
Ή NMR (400 MHz, DMSO-d6): δ ppm 7.28 (d, 1H), 5.99 (m, 1H), 4.46 (t, 1H), 3.75 (s, 2H), 3.40 (m, 2H), 3.32 (m, 2H), 2.23 (brs, 3H), 2.13 (m, 2H), 1.61/1.52 (m+m, 4H), 1.47/1.43 5 (m+m, 2H), 1.45 (s, 2H), 1.44-1.35 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ ppm 137.8,
105.1, 61.8, 61.5, 59.0, 44.6, 40.8, 39.6, 35.7, 30.0, 11.9; HRMS-ESI (m/z): [M+H]+ calcd for C17H27N2O2: 291.2073 found: 291.2069.
Step E: tert-butyl-[2-[[3-/(5-methylpyrazol-1-yl)methyl]-l-adamantyl]oxy]ethoxy]-diphenylsilane
To the product from Step D (6.52 g, 22.5 mmol) and imidazole (2.29 g, 1.5 eq) în DCM (67 ml) was added rert-butyi-chloro-diphenyl-silane (6.9 mL, 1.2 eq) and the reaction mixture was stirred for 1 h. Purification by column chromatography (silica gel, heptane and MTBE as eluents) afforded the desired product (11.0 g, 92.7%). LC/MS (C33H45N2O2S1) 529 [M+H]+.
Step F: tert-butyl-[2-[[3-[(4-iodo-5-methyl-pyrazol-l -yl)methylj-1 -adamantyl]oxy]ethoxy]· 15 diphenyl-silane
To the product from Step E (11.0 g, 20.8 mmol) in DMF (105 mL) was added Niodosuccinimide (5.85 g, 1.25 eq.) and the reaction mixture was stirred for 3 h. After the reaction mixture was diluted with water and extracted with DCM, the combined organic phases were washed with saturated sodium thiosulphate and brine, dried, and evaporated to 20 gel the desired product (11.0 g, 81%).
*H NMR (400 MHz, DMSO-d6): δ ppm 7.70-7.36 (m, 10H), 7.44 (s, 1H), 3.86 (s, 2H), 3.67 (t, 2H), 3.45 (t, 2H), 2.24 (s, 3H), 2.12 (m, 2H), 1.66-1.32 (m, 12H), 0.98 (s, 9H) 13C NMR (100 MHz, DMSO-dû) δ ppm 142.4, 140.9, 64.4, 61.4, 60.4, 60.3, 30.0, 27.1, 12.2; HRMSESI (m/z): [M+H]+ calcd for C33H44IN2O2Si: 655.2217 found: 655.2217.
Step G: tert-butyl-[2-[[3-[[5-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)pyrazoll -yl]methyl]-l -adamantyl]oxy]ethoxy]-diphenyl-silane
To the product from Step F (11.0 g, 16.8 mmol) in THF (84 mL) was added chloro(isopropyl)magnesium-LiCl (1.3 M in THF, 17 mL, 1.2 eq) at 0 °C, and the reaction mixture was stirred for 40 min, treated with 2-isopropoxy-4,4,5,5-tetramethyl-l,3,230 dioxaborolane (10.3 mL, 3 eq), and stirred for 10 min. After dilution with a saturated solution NH4CI and extraction with EtOAc, the combined organic phases were concentrated
102 andpurified by column chromatography (silica gel, heptane and MTBE as eluents) to give the desired product (9.0 g, 82%).
NMR (400 MHz, DMSO-d6): δ ppm 7.66 (d, 4H), 7.47 (s, IH), 7.45 (t, 2H), 7.40 (t, 4H), 3.77 (s, 2H), 3.67 (t, 2H), 3.44 (t, 2H), 2.36 (s, 3H), 2.11 (br, 2H), 1.60/1.48 (d+d, 4H), 1.44 (d, 2H), 1.44 (s, 2H), 1.40 (d, 4H), 1.23 (s, 12H), 0.97 (s, 9H); 13C NMR (100 MHz, DMSOd6) δ ppm 146.9, 144.2, 133.8, 130.2, 128.3, 125.7, 104.6, 83.0, 72.5, 64.4, 61.4, 58.9, 44.6, 40.7, 39.6, 38.7, 35.6, 30.0, 27.1, 25.2, 19.3, 12.1; HRMS-ESI (m/z): [M+H]+ calcd for CJKBN.A.Si: 655.4102 found: 655.4108.
Préparation 10: methyl 3-bromo-6-[3-(3,6-dichloro-5-methyI-pyridazin-4l)propylamino]pyridine-2-carboxylate
Step A: methyl 6-[bis(tert-butoxycarbonyl)aminoJ-3-bromo-pyridine-2-carboxylate
To methyl 6-amino-3-bromo-pyridine-2-carboxylate (25.0 g, 108.2 mmol) and DMAP (1.3 g, 0.1 eq) in DCM (541 mL) was added Boc2O (59.0 g, 2.5 eq) at 0 °C and the reaction mixture was stirred for 2.5 h. After the addition of a saturated solution of NaHCO? and extraction with DCM, the combined organic phases were dried and concentrated to afford the desired product (45.0 g, 72.3%).
LC/MS (Ci7H23BrN2Û6Na) 453 [M+Na]+.
Step B*. methyl 3-bromo-6-(tert-butoxycarbonylamino)pyridine-2-carboxylate
To the product from Step A (42.7 g, 74.34 mmol) in DCM (370 mL) was added TFA (17.1 mL, 3 eq) at 0 °C and the reaction mixture was stirred for 18 h. After washing with a saturated solution of NaHCO3 and brine, the combined organic phases were dried, concentrated, and purified by column chromatography (silica gel, heptane and EtOAc as eluents) to give the desired product (28.3 g, 115.2%).
NMR (400 MHz, DMSO-d6): δ ppm 10.29 (s, IH), 8.11 (d, IH), 7.88 (d, IH), 3.87 (s, 3H), 1.46 (s, 9H) 13C NMR (100 MHz, DMSO-d6) δ ppm 165.6, 153.1, 151.8/148.3, 143.5, 116.3, 109.2, 53.2, 28.4. LC/MS (CizHisBrNzOiNa) 353 [M+Na]+.
Step C: methyl 3-bromo-6-[tert-butoxycarbonyl-[3-(3t6-dichloro-5-methyl-pyridazin-4yl)propyl/aminû]pyridine-2-carboxylate
103
To the product from Step B (10.0 g, 30.1967 mmol) in acetone (150 mL), were added CS2CO3 (29.5 g, 3 eq) and 3,6-dichloro-4-(3-iodopropyl)-5-methyl-pyridazine (9.9 g, 1 eq) and the reaction mixture was stirred for 18 h. After dilution with water and extraction with EtOAc, the combined organic phases were washed with brine, dried and concentrated to give the desired product (17.5 g, 108%).
NMR (400 MHz, DMSO-d6): Ô ppm 8.13 (d, IH), 7.78 (d, IH), 3.91 (t, 2H), 3.89 (s, 3H), 2.79 (m, 2H), 2.38 (s, 3H), 1.82 (m, 2H), 1.46 (s, 9H); 13C NMR (100 MHz, DMSO-d(1) δ ppm 165.3, 157.6, 156.6, 153.2, 152.9, 147.2, 143.1, 142.2, 139.7, 122.6, 111.8, 82.2, 53.3, 46.4, 28.1, 27.7, 26.5, 16.3; HRMS-ESI (m/z); [M+Na]+ calcd for C20H23BrCl2N4NaO4: 555.0177 found; 555.0172.
Step D: methyl 3-bromo^6-[3-(3,6-dichlQrQ-5-nιethyl·pyridazin-4-l)propylamino]pyridiι^e-2carboxylate
The product from Step C (17.5 g, 32.7 mmol) in 1,1,1,3,3,3-hexafluoroisopropanoI (330 mL) was stirred at 110 °C for 18 h. Purificationby column chromatography (silica gel, heplane and EtOAc as eluents) afforded the desired product (9.9 g, 70%).
>H NMR (400 MHz, DMSO-d6): δ ppm 7.63 (d, IH), 7.22 (t, IH), 6.57 (d, IH), 3.83 (s, 3H), 3.30 (m, 2H), 2.83 (m, 2H), 2.37 (s, 3H), 1.74 (m, 2H) 13C NMR (100 MHz, DMSO-d6) δ ppm 166.5, 141.5,112.6, 52.9, 40.9, 28.0, 27.0, 16.4.
Préparation 11: (4-inethoxyphenyl)methyl 3-bromo-6-[3-(3,6-dichloro-5-methylpyridazin-4-yl)propylamino]pyridine-2-carboxylate
Step A: S-bromo-ô-fô-fSfi-dichloro-S-methyl-pyridazin-d-ytypropylaminofyyridine-icarboxylic acid
The mixture of the product from Préparation 10 (35.39 g, 81.52 mmol) and LiOHxHzO (13.68 g, 4 eq) in 1,4-dioxane (408 mL) and water (82 mL) was stirred ai 60 °C for 1 h. After quenching with a 1 M solution of HCl and extraction with EtOAc, the combined organic phases were dried, concentrated, and purified by flash chromatography (silica gel, using DCM and MeOH as eluents) to give the desired product (27.74 g, 81%).
LC/MS (CnHuBrChN+O?) 421 [M+H]+.
Step B: (4-methoxyphenyl) methyl 3~bromo-6-[3-(3,6-dichloro-5-methyl-pyridazin-4yl)propylaminoJpyriditie-2-carboxylate
104
To the product of Step A (27,7 g, 65.9 mmol), (4-methoxyphenyl)methanol (16.4 mL, 2 eq), and PPh3 (34.6 g, 2 eq) m toluene (660 mL) and THF (20 ml) was added dropwise diisopropyl azodîcarboxylate (26 mL, 2 eq) and the reaction mixture was stirred at 50 °C for 1 h. Purifîcationby flash chromatography (silica gel, using heptane and EtOAc as eluents) afforded the desired product (23.65 g, 66.4%).
Ή NMR (500 MHz, dmso-d6) δ ppm 7.62 (d, IH), 7.37 (dn, 2H), 7.21 (t, IH), 6.91 (dm, 2H), 6.56 (d, IH), 5.25 (s, 2H), 3.74 (s, 3H), 3.30 (q, 2H), 2.81 (m, 2H), 2.33 (s, 3H), 1.73 (m, 2H); 13C NMR (500 MHz, dmso-d6) 8 ppm 165.9, 159.7, 157.6, 157.5, 156.8, 148.0, 142.7, 141.5, 139.7, 130.6,127.8, 114.3, 112.6, 101.6, 67.0, 55.6, 40.9, 28.0, 27.1, 16.4; HRMS-ES1 (m/z): [M+H]+ calcd for C22H22BrC12N4O3: 539.0252, found: 539.0246.
Préparation 12: methyl 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//pyrido[2,3-c]pyridazm-8-yl]-3-[l-[[3,5-dimethyl-7-[2-(p-tolylsulfonyloxy)ethoxy]-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylate
Step A: methyl 6-[3-(3$-dichloro-5-methyl-pyridazin-4-yl)propylamino/-3-15-methyl-l-[[3[2-[tert-butyl(diphenyl)silyl]oxyethoxy]-5,7-dimethyl-l-adamantyl]methyl]pyrazol-4yl]pyridine-2-carboxylate
The mixture of the product from Préparation 10 (15.0 g, 34.55 mmol), the product from Préparation 7 (30.7 g, 1.3 eq), CS2CO3 (33.8 g, 3.0 eq), and Pd(AtaPhos)2Ch (1.53 g, 0.1 eq) in 1,4-dioxane (207 mL) and H2O (34.5 mL) was stirred at 80 °C for 1.5 h. Purification by column chromatography (silica gel, heptane and EtOAc as eluents) afforded the desired product (18.5 g, 58%).
‘H NMR (400 MHz, DMSO-d6): δ ppm 7.69-7.37 (m, 10H), 7.32 (d, IH), 7.23 (s, IH), 6.98 (t, IH), 6.63 (d, IH), 3.82 (s, 2H), 3.67 (t, 2H), 3.58 (s, 3H), 3.46 (t, 2H), 3.35 (m, 2H), 2.86 (m, 2H), 2.40 (s, 3H), 2.06 (s, 3H), 1.78 (m, 2H), 1.35 (s, 2H), 1.27/1.2 (m+m, 4H), 1.15/1.09 (m+m, 4H), 1.05/0.97 (m+m, 2H), 0.97 (s, 9H), 0.84 (s, 6H); HRMS-ES1 (m/z): [M+H]+ calcd for C-ALXLNO.Si; 909.4057 found: 909.4053.
711/ OD ώ U
Step B‘. methyl 6-(3~ch loro-4-methyl-6,7-dihydro-5H -pyrido[2,3-cjpyridazin -8-yl) -3 -[5methyl-1 -[[[2 -[tcrt-butyKdiphenyl)silyl]oxyethoxy]-5,7-dimethyl-l adamautyl]methyl]pyrazol-4-yl]pyridine-2-carboxylate
105
The mixture of the product from Step A (18.5 g, 20.3 mmol), Cs2CO3 (13.2 g, 2 eq), DIPEA (7.1 mL, 2 eq), and Pd(Ataphos)2Cl2 (900 mg, 0.1 eq) in 1,4-dioxane (102 mL) was stirred at 110 °C for 18 h. After filtration and concentration, the residue was taken up with DCM, washed with water, and purified by column chromatography (silica gel, DCM and EtOAc as eluents) to give the desired product (12.6 g, 71%).
‘H NMR (400 MHz, DMSO-d6): δ ppm 7.85 (d, 1H), 7.69 (d, 1H), 7.66 (dm, 4H), 7.47-7.36 (m, 6H), 7.38 (s, 1H), 3.97 (t, 2H), 3.87 (s, 2H), 3.68 (t, 2H), 3.66 (s, 3H), 3.47 (t, 2H), 2.87 (t, 2H), 2.30 (s, 3H), 2.14 (s, 3H), 1.99 (br., 2H), 1.38 (s, 2H), 1.32-0.96 (br„ 10H), 0.98 (s, 9H), 0.85 (s, 6H); l3C NMR (100 MHz, DMSO-d6) δ ppm 139.9, 137.6, 120.5, 64.4, 61.7, 58.9, 52.3, 46.0, 43.4, 30.2, 27.1, 24.6, 21.0, 15.5, 10.9; HRMS-ESI (m/z): [M+H]+ calcd for C50H62ClN6O4Si: 873.4290 found: 873.4291.
Step Ci methyl 6-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl)~3-[l-[[3(2-hydroxyethoxy) -5,7-dimethyl-l-adamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2carboxylate
To the product from Step B (8.46 g, 9.68 mmol) in THF (95 mL) was added a 1 M solution of TB AF in THF (10.6 mL, 1.1 eq) at ü °C and the reaction mixture was stirred for 2 h. After quenching with a saturated solution of NH4CI and extraction with EtOAc, the combined organic phases were washed with brine, dried, and purified by column chromatography (silica gel, DCM and MeOH as eluents) to give the desired product (5.38g, 88%).
>H NMR (400 MHz, DMSO-d6): δ ppm 7.86 (d, 1H), 7.71 (d, 1H), 7.38 (s, 1H), 4.46 (t, 1H), 3.97 (t, 2H), 3.87 (s, 2H), 3.70 (s, 3H), 3.40 (m, 2H), 3.35 (t, 2H), 2.87 (t, 2H), 2.30 (s, 3H), 2.15 (s, 3H), 1.99 (m, 2H), 1.42-0.95 (ni, 12H), 0.87 (s, 6H); HRMS-ESI (m/z): [M+H]+ calcd for C34H44C1N6O4: 635.3113 found: 635.3112.
Step Di methyl 6-[3-(l ,3-benzothiazol-2-ylamino) -4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-3-[1 -[[3-(2-hydroxyethoxy) -5,7-dimethyl-1 -adamantyl]methyl]-5-methylpyrazol-4-yljpyridine-2-carboxylate
Using Buchwald General Procedure I at 130 °C for 1 h, starting from 3.7 g of the product from Step C (5.78 mmol) and 1.74 g of l,3-benzothiazol-2-amine (2 eq), 3.1 g of the desired product (72% Yield) were obtained.
’H NMR (400 MHz, DMSO-d&): δ ppm 7.96 (d, 1H), 7.82 (br., 1H), 7.70 (d, 1H), 7.50 (br., 1H), 7.38 (s, 1H), 7.35 (t, 1H), 7.17 (t, 1H), 4.46 (br., 1H), 4.00 (t, 2H), 3.88 (s, 2H), 3.70 (s,
106 φ 3Η), 3.40 (brt„ 2Η), 3.35 (t, 2H), 2.86 (t, 2H), 2.32 (s, 3H), 2.16 (s, 3H), 2.03-1.94 (m, 2H), 1.42-0.96 (m, 12H), 0.87 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ ppm 139.8, 137.5, 126.4, 122.4, 122.1, 119.0, 62.1, 61.5, 59.0, 52.6, 45.4, 30.2, 24.3, 21.7, 12.6, 10.9; HRMSESI (m/z); [M+H]+ calcd for C4]H49NgO4S: 749.3597 found: 749.3595.
Step E·. methyl 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6i7-dihydro-5H-pyrido[2,3c]pyridaziti-8-yl]-3-[l-[J3,5-dimethyl-7-[2-(p-tolylsulfonyloxy)ethoxy]-l-adamantyl]methyl]5-methyl-pyrazol-4-ylJpyridine~2-carboxylate
To the product from Step D (3.85 g, 5.14 mmol) and trie thy lamine (2.15 mL, 3 eq) in DCM (50 mL) was added p-tolylsulfonyl 4-methylbenzenesulfonate (2.51 g, 1.5 eq) and the re action 10 mixture was stirred for 1 h. Purification by column chromatography (silica gel, heptane and EtOAc as eluents) afforded the desired product (3.2 g, 69%).
Al NMR (400 MHz, DMSO-d6): δ ppm 7.96 (d, 1H), 7.81 (br., 1H), 7.77 (d, 2H), 7.70 (d, 1H), 7.50 (br., 1H), 7.46 (d, 2H), 7.39 (s, 1H), 7.35 (t, 1H), 7.17 (t, 1H), 4.06 (t, 2H), 4.00 (t, 2H), 3.85 (s, 2H), 3.69 (s, 3H), 3.49 (t, 2H), 2.86 (t, 2H), 2.40 (s, 3H), 2.32 (s, 3H), 2.15 (s, 15 3H), 1.99 (m, 2H), 1.32-0.93 (m, 12H), 0.84 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ ppm
139.8, 137.6, 130.6, 128.1, 126.4, 122.4, 122.1, 119, 71.5, 58.8, 58.4, 52.6, 45.4, 30.1, 24.3, 21.7, 21.6, 12.6, 10.9; HRMS-ESI (m/z): [M+H]+ calcd for C48H55N8O6S2: 903.3686 found: 903.3685.
Préparation 13: (4-methoxy phenyl) methyl 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl20 6,7-dihydro-5//-pyrido[2,3-c]pyridazin-8-yl]-3-[l-[[3,5-dimethyl·7-[3-(ptolylsulfonyloxy)propyl]-l-adamantyl]methyl]-5-methyl-pyrazol-4-yi]pyridine-2carboxylate
Step A: (4-methoxyphenyl) methyl 3-[l -[[3-[3-[tert-butyl(diphenyl)silyl]oxypropyl]-5,7dimethyl-l-adamantyl]methyl]-5-methyl-pyrazol-4-yl]-6-[3-(3,6-dichloro-5-methyl25 pyrldazin -4-yl)propylamino]pyridine~2-carboxylate
The mixture of the product from Préparation 11 (3.67 g, 6.79 mmol), the product from Préparation 8 (5.09 g, 1.1 eq), Pd(AtaPhos)2Cl2 (301 mg, 0.1 eq), and CS2CO3 (6.64 g, 3 eq) in 1,4-dioxane (41 mL) and H2O (6.8 mL) was stirred at 80 °C for 18 h. Purificationby column chromatography (silica gel, heptane and EtOAc as eluents) afforded the desired 30 product (4.43 g, 64%).
107 φ Ή NMR (400 MHz, DMSO-d6): δ ppm 7.62-7.38 (ni, 10H), 7.32 (d, 1H), 7.26 (s, 1H), 7.10 (m, 2H), 6.98 (t, 1H), 6.83 (m, 2H), 6.63 (d, 1H), 4.98 (s, 2H), 3.74 (s, 2H), 3.70 (s, 3H), 3.58 (t, 2H), 3.35 (m, 2H), 2.84 (m, 2H), 2.34 (s, 3H), 2.02 (s, 3H), 1.77 (m, 2H), 1.43 (m, 2H), 1.18-0.85 (m, 12H), 1.09 (t, 2H), 0.97 (s, 9H), 0.77 (s, 6H); HRMS-ESI (m/z): [M+H]+ calcd 5 for C.RH7.CLN,O.Si: 1013.4683 found: 1013.4683;
Step B‘. (4-methoxyphenyl)methyl 3-[l-[[3-[3-[tert-butyl(diphenyl)silyl]oxypropyl]-5,7dimethyl-l-adamantyl]methyl]-5-methyl-pyrazol-4'yl]-6-(3-chloro-4-methyl-6f7-dihydro5H-pyrido[2,3‘C]pyridazin-8-yl)pyriditie-2-carboxylate
The mixture of the product from Step A (4.43 g, 4.37 mmol), CS2CO3 (2.84 g, 2 eq), DI PEA 10 (1.5 mL, 2 eq) and Pd(Ataphos)2CI2 (193 mg, 0.1 eq) in 1,4-dioxane (22 mL) was stirred at
110 °C for 18 h. After quenching wîth water and extracting wîth EtOAc, the combined organic phases were dried, concentrated, and purified by column chromatography (silica gel, DCM and EtOAc as eluents) to give the desired product (2.83 g, 66%).
NMR (400 MHz, DMSO-d6): δ ppm 7.84 (d, 1H), 7.68 (d, 1H), 7.59 (d, 4H), 7.44 (t, 2H), 15 7.42 (t, 4H), 7.38 (s, 1H), 7.14 (d, 2H), 6.87 (d, 2H), 5.07 (s, 2H), 3.96 (t, 2H), 3.78 (s, 2H),
3.71 (s, 3H), 3.59 (t, 2H), 2.86 (t, 2H), 2.29 (s, 3H), 2.08 (s, 3H), 1.97 (qn, 2H), 1.43 (qn, 2H), 1.12 (s, 4H), 1.10 (s, 2H), 1.09 (l, 2H), 0.97 (s, 9H), 0.95 (s, 2H), 0.94/0.91 (d+d, 4H), 0.78 (s, 6H); I3C NMR (100 MHz, DMSO-d6) δ ppm 166.9, 159.6, 156.3, 153.6, 150.8, 147.7, 140.1, 137.5, 137.3, 136.0, 135.5, 133.8, 130.3, 130.1, 129.1, 128.3, 127.6, 123.1, 120.5, 115.5, 20 114.3, 66.8, 64.8, 64.8, 59.6, 55.6, 50.5, 48.1, 46.4, 46.0, 44.2, 39.3, 38.1, 31.7, 30.6, 27.2,
26.1, 24.6, 21.0, 19.3, 15.5, 10.9; HRMS-ESI (m/z): [M+H]+ calcd for C58H70ClN6O4Si; 977.4916 found: 977.4915.
Step C: (4-methoxyph enyl) methyl 6-(3-chloro-4-methyl-6,7‘dihydro-5H-pyrido[2,3‘ cIpyridazin -8-yl) -3-[1 -[[3-(3-hydroxypropyl) -5,7-dimethyl-l -adamantyl]methyl]-5-methyl25 pyrazol-4-yl]pyridine-2-carboxylate
To the product from Step B (2.83 g, 2.89 mmol) in THF (95 mL) was added a 1 M solution ot TB AF in THF (3.2 mL, 1.1 eq) at 0 °C and the reaction mixture was stirred for 2 h. After quenching with a saturated solution of NH4CI and extracted with EtOAc, the combined organic phases were washed with brine, dried, concentrated, and purified b y column 30 chromatography (silica gel, DCM and MeOH as eluents) to give the desired product (2.21 g, 103%).
108 1H NMR (400 MHz, DMSO-dû): Ô ppm 7.85 (d, 1H), 7.70 (d, 1 H), 7.39 (s, 1H), 7.17 (d, 2H),
6.90 (d, 2H), 5.09 (s, 2H), 4.34 (t, 1H), 3.96 (t, 2H), 3.79 (s, 2H), 3.74 (s, 3H), 3.32 (q, 2H), 2.86 (t, 2H), 2.29 (s, 3H), 2.09 (s, 3H), 1.98 (qn, 2H), 1.34 (qn, 2H), 1.13 (s, 2H), 1.13 (s, 4H), 1.06 (t, 2H), 0.99/0.95 (d+d, 4H), 0.97 (s, 2H), 0.78 (s, 6H); 1JC NMR (100 MHz, DMSO-de) δ ppm 166.9, 159.7, 156.4, 153.6, 150.8, 147.7, 140.2, 137.5, 137.3, 136.0, 130.2, 129.1, 127.6, 123.1, 120.4, 115.5, 114.3, 66.8, 66.8, 62.1, 59.7, 55.6, 50.6, 48.2, 46.5, 46.0, 44.3, 39.7, 38.1, 31.8, 30.6, 26.5, 24.6, 21.0, 15.5, 10.9; HRMS-ESI (m/z); [M+H]+ calcd for C^H„C1N.O,: 739.3739 found: 739.3739.
Step D·. (4-methoxyphenyl) methyl 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro5H-pyrido[2,3-c]pyridazin-8-yl]-3-[1 -[(3-(3-hydroxypropyl) -5,7-dimethyl-l adamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylate
The mixture of the product from Step C (1.71 g, 2.31 mmol), l,3-benzothiazol-2-amine (695 mg, 2 eq), Pd2dba3 (212 mg, 0.1 eq), XantPhos (268 mg, 0.2 eq), and DIPEA (1.2 mL, 3 eq) in cyclohexanol (14 mL) was stirred at 130 °C for 1 h. Purification by column chromalography (silica gel, heptane, DCM and MeCN as eluents) afforded the desired product (1.25g, 63%).
‘H NMR (400 MHz, DMSO-d6): δ ppm 12.08/10.87 (brs/brs, 1H), 7.95 (d, 1H), 7.81 (br, 1H), 7.68 (d, 1H), 7.50 (br, 1H), 7.39 (s, 1H), 7.35 (t, 1H), 7.18 (d, 2H), 7.17 (t, 1H), 6.90 (d, 2H), 5.10 (s, 2H), 4.34 (l, 1H), 3.99 (t, 2H), 3.79 (s, 2H), 3.74 (s, 3H), 3.33 (q, 2H), 2.85 (t, 2H), 2.32 (s, 3H), 2.11 (s, 3H), 1.98 (qn, 2H), 1.34 (qn, 2H), 1.14 (s, 4H), 1.14 (s, 2H), 1.07 (t, 2H), 1.00/0.95 (d+d, 2H), 0.99/0.95 (d+d, 4H), 0.79 (s, 6H); 13C NMR (100 MHz, DMSOdô) Ô ppm 140.0, 137.6, 130.2, 126.4, 122.4, 122.0, 119.0, 114.3, 66.7, 62.1, 59.6, 55.6, 50.6, 48.2, 46.5, 45.4, 44.3, 39.7, 30.6, 26.5, 24.3, 21.7, 12.6, 11.0; HRMS-ESI (m/z): [M+H]+ calcd for C49H57N8O4S: 853.4223 found: 853.4229.
Step E: (4-methoxyphenyl) methyl 6-(3-(1,3-benzothiazol-2-ylamino) -4-methyl-6,7-dihydro5H-pyrido(2,3-c]pyridazîn-8-ylJ-3-(l-([3,5-dimethyl-7-(3-(p-tolylsulfonyloxy)propyl]-l· adamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridùie-2-carboxylate
To the product from Step D (1.25 g, 1.47 mmol) and triethylamine (0.61 mL, 3 eq) in DCM (15 mL) was added p-tolylsulfonyl 4-methylbenzenesulfonate (717 mg, 1.5 eq) and the reaction mixture was stirred for 1 h. Purification by column chromalography (silica gel, heptane and EtOAc as eluents) afforded 800 mg (54%) of the desired product.
109 • *H NMR (400 MHz, DMSO-dü): δ ppm 7.95 (d, 1H), 7.88 (brs, 1H), 7.77 (m, 2H), 7.68 (d, 1H), 7.62 (brs, 1H), 7.47 (m, 2H), 7.39 (s, 1H), 7.35 (brs, 1H), 7.17 (brs, 1H), 7.10 (ni, 2H), 6.90 (m, 2H), 5.09 (s, 2H), 4.00 (ni, 2H), 3.98 (t, 2H), 3.77 (s, 2H), 3.74 (s, 3H), 2.85 (t, 2H), 2.40 (s, 3H), 2.32 (s, 3H), 2.09 (s, 3H), 1.98 (m, 2H), 1.45 (m, 2H), 1.17-0.8 (m, 12H), 0.98 5 (m, 2H), 0.77 (s, 6H); HRMS-ESI (m/z): [M+H]+ calcd for C„H„NR0,S,: 1007.4312 found:
J JO t),) O U ώ
1007.4318.
Préparation 14: (4-methoxyphenyl)methyl 6-[3-(l,3-benzothîazol-2-ylamino)-4-methyl6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl]-3-[5-methyM-[[3-[2-(ptolylsulfonyloxy)ethoxyJ-l-adamantyl]methyl]pyrazol-4-yl]pyridine-2-carboxylate
Step A : (4-methoxyphenyl)methyl 3-[l-[[3-[2-[tert-butyl(diphenyl)silyl]oxyethoxy]-ladamantyl]methyl]-5~niethyl-pyrazol-4~yl]-6-[3-(3,6-dichloro-5-methyl-pyridazifi-4yl)propylatnino]pyridine-2-carboxylate
The mixture of the product from Préparation 11 (3.67 g, 6.79 mmol), the product from Préparation 9 ( 4.89 g, 1.1 eq), Pd(AtaPhos)2Ch (301 mg, 0.1 eq), and CS2CO3 (6.64 g, 3 eq) 15 in 1,4-dioxane (41 mL) and H2O (6.8 mL) was stirred at 80 °C for 12 h. Purification by column chromatography (silica gel, heptane and EtOAc as eiuents) afforded the desired product (3.0 g, 45%).
*H NMR (400 MHz, DMSO-d6): δ ppm 7.69-7.37 (m, 10H), 7.31 (d, 1H), 7.24 (s, 1H), 7.12 (m, 2H), 6.98 (t, 1H), 6.83 (m, 2H), 6.62 (d, 1H), 4.99 (s, 2H), 3.76 (s, 2H), 3.70 (s, 3H), 3.66 20 (t, 2H), 3.45 (t, 2H), 3.35 (m, 2H), 2.85 (m, 2H), 2.34 (s, 3H), 2.12 (m, 2H), 2.Ü2 (s, 3H), 1.77 (m, 2H), 1.65-1.33 (m, 12H), 0.97 (s, 9H); HRMS-ESI (m/z): [M+H]+ calcd for C„H„CLN,OsSi: 987.4163 found: 987.4158.
Step B: (4-methoxyphenyl) methyl 3-[l-[[3-[2~[tert-butyl(diphenyl)silyl]oxyethoxy]-l adamantyl]methyl]-5-methyl-pyrazol-4-yl]-6-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3‘
c]pyridazin-S-yl)pyridine-2-carboxylate
The mixture of the product from Step A (3.0Ü g, 3.00 mmol), CS2CO3 (1.95 g, 2 eq), DIPEA (1.0 mL, 2 eq), and Pd(Ataphos)2Cb (212 mg, 0.1 eq) in 1,4-dioxane (15 mL) was stirred at 110 °C for 18 h. Purification by column chromatography (silica gel, DCM and MeOH as eiuents) afforded the desired product (1.74 g, 60%).
110
Ή NMR (400 MHz, DMSO-dâ): δ ppm 7.84 (d, IH), 7.68 (d, IH), 7.68-7.37 (m, 10H), 7.36 (s, IH), 7.16 (m, 2H), 6.87 (m, 2H), 5.08 (s, 2H), 3.96 (m, 2H), 3.81 (s, 2H), 3.72 (s, 3H), 3.67 (t, 2H), 3.46 (t, 2H), 2.87 (t, 2H), 2.29 (s, 3H), 2.13 (m, 2H), 2.09 (s, 3H), 1.98 (m, 2H), 1.65-1.37 (m, 12H), 0.97 (s, 9H); HRMS-ESI (m/z): [M+H]+ calcd for C55H64ClN6O5Si: 951.4396 found: 951.4397.
Step C: (4-methaxyphenyl) methyl 6-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl)-3-[1 -[[3-(2-hydroxyethoxy) -1 -adamantyl]methyl]-5~methyl-pyrazol-4yl]pyridine-2-carboxylate
To the product from Step B (1.73 g, 1.82 mmol) in THF (20 mL) was added a 1 M solution of TBAF in THF (2.0 mL, 1.1 eq) at 0 °C and the reaction mixture was stirred for 2 h. Purification by column chromatography (silica gel, DCM and MeOH as eluents) afforded the desired product (1.06 g, 82%).
*H NMR (400 MHz, DMSO-dû): δ ppm 7.85 (d, IH), 7.71 (d, IH), 7.36 (s, IH), 7.19 (m, 2H), 6.90 (m, 2H), 5.10 (s, 2H), 4.47 (t, IH), 3.96 (m, 2H), 3.81 (s, 2H), 3.75 (s, 3H), 3.40 (m, 2H), 3.34 (t, 2H), 2.87 (t, 2H), 2.29 (s, 3H), 2.14 (m, 2H), 2.10 (s, 3H), 1.98 (m, 2H), 1.67-1.36 (m, 12H); HRMS-ESI (m/z): [M+H]+ calcd for C^H^CIN^: 713.3218 found: 713.3217.
Step D; (4-methoxyphenyl) methyl 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro5H-pyrido[2,3-c]pyridazin -8-yl] -3-[l -[[3-(2-hydroxyethoxy) -1 -adamantyl]methyl]-5-methyl· pyrazol~4-yl]pyridine-2-carboxylate
The mixture of the product from Step C (1.00 g, 1.40 mmol), l,3-benzothiazol-2-amine (421 mg, 2 eq), Pd2dba2 (128 mg, 0.1 eq), XantPhos (162 mg, 0.2 eq), and DTPEA (0.72 mL, 3 eq) in cyclohexanol (10 mL) was stirred at 130 °C for 1 h. Purification by column chromatography (silica gel, heptane, then DCM and MeOH as eluents) afforded the desired product (600 mg, 53%).
*H NMR (400 MHz, DMSO-de): δ ppm 12.18/10.84 (brs/brs, IH), 7.94 (d, IH), 7.83 (br, IH), 7.69 (d, IH), 7.57 (br, IH), 7.36 (s, IH), 7.35 (brt, IH), 7.20 (d, 2H), 7.17 (brt, IH), 6.91 (d, 2H), 5.Tl (s, 2H), 4.47 (brt, IH), 4.00 (t, 2H), 3.81 (s, 2H), 3.75 (s, 3H), 3.41 (brq, 2H), 3.35 (t, 2H), 2.85 (t, 2H), 2.32 (s, 3H), 2.14 (m, 2H), 2.12 (s, 3H), 1.99 (qn, 2H), 1.62/1.53 (d+d, 4H), 1.53 (s, 2H), 1.49/1.44 (d+d, 2H), 1.44 (s, 4H); 13C NMR (100 MHz, DMSO-d6) δ ppm 139.9, 137.6, 130.1, 126.4, 122.4, 122.0, 118.9, 114.2, 66.7, 61.9, 61.5, 59.5, 55.6, 45.4,
111 φ 44.7, 40.8, 39.5, 35.6, 30.1, 24.3, 21.7, 12.6, 10.8; HRMS-ESI (m/z): [M+H]+ calcd for C.,Hs1N„0,S: 827.3703 found: 827.3709.
Step Et (4-methoxyphenyl) methyl 6-(3-(1,3-benzotkiazol-2-ylamino) -4-methyl-6,7-dihydro5H-pyrido[2,3-c]pyridazin -8-yl]-3-(5-methyl-l -((3-(2~(p~tolylsulfonyloxy) ethoxy]-! - adamantyl]methylJpyrazol~4-yl]pyridine-2-carboxylate
To the product from Step D (600 mg, 0.726 mmol) and NW-diethylethanamine (0.31 mL, 3 eq) în dichloromethane (7 mL) was added p-tolylsulfonyl 4-methylbenzenesuIfonate (357 mg, 1.5 eq) and the reaction mixture was stirred for 18 h. Purification by flash chromatography (silica gel, using DCM and MeOH as eluents) afforded 354 mg (50%) of the desired product.
’H NMR (500 MHz, dmso-d6) δ ppm 12.22/10.85 (brs/brs, 1H), 7.94 (d, 1H), 7.81 (br, 1H), 7.77 (d, 2H), 7.70 (d, 1H), 7.52 (br, 1H), 7.45 (d, 2H), 7.37 (s, 1H), 7.35 (t, 1H), 7.19 (d, 2H), 7.17 (t, 1H), 6.89 (d, 2H), 5.10 (s, 2H), 4.05 (t, 2 H), 4.00 (l, 2H), 3.79 (s, 2H), 3.74 (s, 3H), 3.49 (t, 2H), 2.86 (t, 2H), 2.40 (s, 3H), 2.32 (s, 3H), 2.11 (m, 2H), 2.11 (s, 3H), 1.99 (qn, 2H), 1.55-1.36 (m, 12H); 13C NMR (500 MHz, dmso-d6) δ ppm 139.9, 137.6, 130.5, 130.3, 128.1, 15 126.4, 122.4, 122.0, 118.9, 114.2, 71.4, 66.8, 59.4, 58.2, 55.6, 45.4, 30.0, 24.2, 21.6, 21.6,
12.6, 10.9; HRMS-ESI (m/z): [M+H]+ calcd for C53H„N8O7S2; 981.3792 found: 981.3795.
Préparation 15: ethyl 2-(3-chloro-4-methyl-6,7-dihydro-5/7-pyrido[2,3-c]pyridazin-8-yl)5-[3-(2-fluoro-4-iodo-phenoxy)propyl]thiazole-4-carboxylate
Step A: 2-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido(2,3-cJpyridazin-8-yl)-5-(3-(2-fluoro-420 iodo-phenoxy)propyl]thiazole-4-carboxylic acid
The mixture of the product from Préparation 3a (35.39 g, 81.52 mmol) and LiOHxHzO (4 eq) in 1,4-dioxane (408 mL) and water (82 mL) was stirred at 60 °C for 1 h. After quenching with a 1 M solution of HCl and extraction with EtOAc, the combined organic phases were dried, concentrated, and purified by flash chromatography (silica gel, using DCM and MeOH 25 as eluents) to give the desired product (27.7 g, 81%).
NMR (500 MHz, dmso-d6) δ ppm 7.56 (dd, 1H), 7.43 (brd., 1H), 6.96 (t, 1H), 4.18 (t, 2H), 4.05 (t, 2H), 3.28 (t, 2H), 2.84 (t, 2H), 2.29 (s, 3H), 2.07 (m, 2H), 1.97 (m, 2H); 13C NMR (500 MHz, dmso-d6) δ ppm 166.4, 154.8, 152.1, 151.8, 151.1, 147.1, 143.9, 135.7, 134.0, 133.8, 129.0, 124.9, 117.6, 82.3, 68.8, 46.3, 31.0, 24.0, 22.5, 19.8, 15.7; HRMS-ESI 30 (m/z): [M+HJ+ calcd for C21H20CIFIN4O3S: 588.9973 found: 588.9969.
112 φ SteP Bi ethyl 2-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl)-5-[3-(2fluoro-4-iodo-phenoxy)propyljthiazole-4-carboxylate
To the mixture of the product of Step A (27.7 g, 65.9 mmol), éthanol (2 eq) and PPh3 (2 eq) in toluene (660 mL) and THF (20 ml) was added dropwîse diisopropyl azodicarboxylate (2 eq) 5 and the reaction was stirred at 50 °C 1 h. Purification by flash chromatography (silica gel, using heptane and EtOAc as eluents) afforded the desired product (23.65 g, 66.4%).
XH NMR (500 MHz, dmso-d6) δ ppm 7.59 (dd, 1H), 7.44 (dm, 1H), 6.98 (t, 1H), 4.29 (m, 2H), 4.25 (q, 2H), 4.08 (t, 2H), 3.24 (t, 2H), 2.89 (t, 2H), 2.32 (s, 3H), 2.09 (m, 2H), 2.04 (m, 2H), 1.28 (t, 3H); 13C NMR (500 MHz, dmso-d6) δ ppm 162.6, 155.4, 152.2, 151.7, 151.3, 10 147.0, 134.0, 124.9, 117.6, 82.4, 68.3, 60.7, 46.3, 30.8, 24.1, 23.1, 19.7, 15.7, 14.6; HRMSESI (m/z): [M+Hf calcd for C23H24CIFIN4O3S: 617.0286, found: 617.0282.
113
Example 1: 2-{6-[(l,3-Benzothiazol-2-yl)amino]-l,2,3,4-tetrahydroquinolin-l-yl}-l,3thïazole-4-carboxylic acid
StepA: ethyl2-(6-bromo-l,2,3,4-tetrahydroquinolin-l-yl)-l ,3-thiazole-4-carboxylate
To a solution of benzoyl isothiocyanate (380 pL, 2.83 mmol, 1.2 eq) in acetone (10 mL) was added 6-bromo-l,2,3,4-tetrahydroquinoline (500 mg, 2.36 mmol, 1 eq) and the mixture was heated at reflux for 1 h. The mixture was poured onto ice water and the precipitate filtered, washed with water and dried to give a pale yellow solid. The solid was added to IN aqueous sodium hydroxide (10 mL) and the suspension was heated at 80 °C for 30 min, cooled to ambient température and poured onto cold IN aqueous hydrochloric acid. The pH was adjusted to pH 8 with saturated aqueous sodium carbonate and the solids were collecled by filtration and washed with water to afford a yellow solid. A suspension of the solid and ethyl bromopyruvate (296 pL, 2.36 mmol, 1 eq) in éthanol (10 mL) was heated at reflux for 2 h. The mixture was allowed to cool to ambient température, then partitioned between ethyl acetate and water. The aqueous phase was extracted with ethyl acetate (3 x 50 mL) and the combined organic extracts were washed with brine (50 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% ethyl acetate in isoheplane afforded the desired product as a yellow gum (232 mg, 0.63 mmol, 27%).
LC/MS (CisHisBrNzChS) 367 [M+H]+; RT 1.42 (LCMS-V-B1)
NMR (400 MHz, DMSO-d6) Ô 7.99 (d, 1H), 7.87 (s, 1H), 7.53 - 7.48 (m, 1H), 7.44 - 7.38 (m, 1H), 4.27 (q, 2H), 3.83 (t, 2H), 2.83 - 2.73 (m, 2H), 2.00 - 1.90 (m, 2H), 1.29 (t, 3H).
Step B: ethyl 2-{6-[(1,3-benzothiazob2-yl)amino]-l,2,3,4-tetrahydroquinolin-l-yl}-l,3thiazole-4-carboxylate
To an oven-dried microwave vial was added the product from Step A (93.8 mg, 0.26 mmol, 1 eq), 2-aminobenzothiazole (46.0 mg, 0.31 mmol, 1.2 eq), césium carbonate (166 mg, 0.51 mmol, 2 eq) and 1,4-dioxane (4 mL), and the mixture was sparged with nitrogen (10 mins)
114 • before adding BrettPhos (13.7 mg, 0.03 mmol, 0.1 eq) and tris(dibenzylideneacetone)dipalladium(0) (23.4 mg, 0.03 mmol, 0.1 eq), and heating at 120 °C for 2 h under microwave irradiation. The reaction was partitioned between ethyl acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 50 mL), and the combined 5 organic extracts were washed with brine (50 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 20% ethyl acetate in iso-heptane afforded the desired product as a yellow gum (85.1 mg, 0.19 mmol, 76%).
LC/MS (C22H20N4O2S2) 437 [M+H]+; RT 1.40 (LCMS-V-B1) ‘H NMR (400 MHz, DMSO-d6) δ 7.87 (d, 1H), 7.83 - 7.78 (m, 1H), 7.69 - 7.65 (m, 1H), 7.63 - 7.57 (m, 2H), 7.36 - 7.29 (m, 1H), 7.18 - 7.13 (m, 1H), 7.03 - 6.98 (m, 1H), 4.28 (q, J = 7.08 Hz, 2H), 3.92 - 3.84 (m, 2H), 2.80 (t, J = 6.30 Hz, 2H), 2.00 - 1.89 (m, 2H), 1.30 (t, J = 7.09 Hz, 211).
Step C: 2-{6-[(1,3-benzothiazol-2-yl)amino]-l,2,3,4-tetrahydroquinolin-l-yl}-l ,3-thiazole-415 carboxylic acid
To a solution of the product from Step B (85.1 mg, 0.19 mmol, 1 eq) în tetrahydrofuran (2 mL) and methanol (1 mL), was added a IN aqueous sodium hydroxide (0.39 mL, 0.39 mmol, 2 eq) and the mixture was heated at 50 °C for 3 h. The reaction was partitioned between ethyl acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 30 mL), and the 20 combined organîc extracts were washed with brine (50 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 6% methanol in dichloromethane afforded material that was further purified by préparative HPLC (HPLC-VA2) to afford the desired product as a cream solid (1.2 mg, 1.5%).
HRMS-ESI (m/z) [M+H]+ calcd for C20H17N4O2S2: 409.0793, found 409.0830
Example 2: 2-{5-[(l,3-Benzothiazol-2-yl)amÎno]-l/7-indol-l-yl}-l,3-thiazole-4carboxylic acid
115
StepA: methyl2-(S-bromo-lH~indol-l-yl)-1,3-thiazole-4-carboxylate
To a cooled solution of 5-bromoindole (150 mg, 0.77 mmol, 1 eq) in dimethylformamide (2 mL) was added sodium hydride (60% dispersion; 36.7 mg, 1.53 mmol, 2 eq) portionwise and 5 the mixture was stirred at 0 °C for 30 min, before the addition of methyl 2-chloro-4thiazolecarboxylate (272 mg, 1,53 mmol, 2 eq), then allowing to warm to ambient température and stir overnight. The reaction was partitioned between ethyl acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 30 mL), and the combined organic extracts were washed with brine (3 x 30 mL), dried (magnésium sulfate) and concentrated in 10 vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g
RediSep™ silica cartridge) eluting with a gradient of 0 — 20% ethyl acetate în iso-heptane afforded the desired product as a yellow solid (72.8 mg, 0.22 mmol, 28%).
LC/MS (Ci3H9BrN2O2S) 339 [M+H]+; RT 1.35 (LCMS-V-B1) ‘H NMR (400 MHz, DMSO-d6) δ 8.41 - 8.38 (m, 1H), 8.37 (s, 1H), 8.02 (d, J = 3.5 Hz, 1H), 15 7.93 (d, J = 2.0 Hz, 1H), 7.57 (dd, J = 8.8, 2.0 Hz, 1H), 6.86 (dd, J = 3.5, 0.8 Hz, 1H), 3.89 (s,
3H).
Step B: methyl 2-{5-[(l,3-benzothiazol-2-yl)amino]-lH-indol-l-yl}-l,3-thiazole-4carboxylate
To an oven-dried microwave vial was added the product from Step A (72.8 mg, 0.22 mmol, 1 20 eq), 2-aminobenzothiazole (38.9 mg, 0.26 mmol, 1.2 eq), césium carbonate (141 mg, 0.43 mmol, 2 eq) and 1,4-dioxane (2 mL) and the mixture was sparged with nitrogen (10 mins) before adding BrettPhos (11.6 mg, 0.02 mmol, 0.1 eq) and tris(dibenzylideneacetone)dipalladium(0) (19.8 mg, 0.02 mmol, 0.1 eq), then heating at 120 °C for 2 h under microwave irradiation. The reaction was partitioned between ethyl acetate 25 and water, the aqueous phase was extracted with ethyl acetate (3 x 30 mL), and the combined organic extracts were washed with brine (30 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g
116 φ RediSep™ siiica cartridge) eluting with a gradient of 0 - 20% ethyl acetate in Lw-heptane afforded the desired product as a pale yellow solid (10.5 mg, 0.03 mmol, 12%).
LC/MS (C20H14N4O2S2) 407 [M+H]+; RT 1.35 (LCMS-V-B1) *H NMR (400 MHz, DMSO-d6) δ 8.39 (d, J = 8.9 Hz, 1H), 8.34 (d, J = 4.4 Hz, 1H), 7.96 (d, 5 J = 3.6 Hz, 1H), 7.84 - 7.78 (m, 1H), 7.67 - 7.57 (m, 2H), 7.39 - 7.30 (m, 2H), 7.20 - 7.12 (m, 1H), 6.92 (d, J = 3.7 Hz, 1H), 3.90 (s, 3H).
Step C: 2-{5-[(l,3-benzothiazol-2-yl)amino]-lH~indol-l-yl}-l,3-thiazole-4-carboxylie acid
To a solution of the product from Step B (10.5 mg, 0 mol, 1 eq) in tetrahydrofuran (2 mL) and methanol (1 mL) was added IN aqueous sodium hydroxide (0.05 mL, 0.Ü5 mmol, 2 eq) and 10 the mixture was heated at 50 °C for 2 h. The reaction was partitioned between ethyl acetate and water, the aqueous phase was extracted with ethyl (3 x 30 mL), and the combined organic extracts were washed with brine (50 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by préparative HPLC (HPLC-V-A2) afforded the desired product as a cream solid (3.5 mg, 0.01 mmol, 35%).
HRMS-ESI (m/z) [M+H]+ calcd for C19H13N4O2S2: 393.0480, found 393.0503
Example 3: 2-{5-[(l,3-Benzothiazol-2-yl)amino]-2,3-diliydro-l/Z-indol-l-yl}-l,3thiazole-4-carboxylîc acid
Step A: ethylZ-fS-bronio^fl-dihydro-IH-indol-l-yty-lJ-thiazole-d-carboxylate
To a solution of benzoyl isothiocyanate (0.33 mL, 2.42 mmol, 1.2 eq) in acetone (10 mL) was added 5-bromoindoline (400 mg, 2.02 mmol, 1 eq) and the mixture was heated at reflux for 1 h. The reaction was poured onto ice water and the precipitate filtered, washed with water and dried to give a pale yellow solid. The solid was added to IN aqueous sodium hydroxide (10 mL) and the suspension was heated at 80 C for 30 min, aliowed to cool to ambient température and poured onto cold IN aqueous hydrochloric acid. The pH was adjusted to pH
117 with saturated aqueous sodium carbonate, and the solids were collected by filtration and washed with water to afford a yellow solid. A suspension of the solid and ethyl bromopyruvate (253 uL, 2.02 mmol, 1 eq) in éthanol (10 mL) was heated at reflux for 2 h then allowed to cool to ambient température. The reaction was partitioned between ethyl 5 acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 50 mL), and the combined organic extracts were washed with brine (50 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 20% ethyl acetate in isoheptane afforded the desired product as a yellow solid (377 mg, 1.07 mmol, 53%).
LC/MS (Ci4HbBiN2O2S) 353 [M+H]+; RT 1.39 (LCMS-V-B1)
NMR (400 MHz, DMSO-d6) δ 8.00 (d, J = 8.4 Hz, 1H), 7.92 (s, 1H), 7.57 - 7.53 (m, 1H), 7.47 - 7.44 (m, 1H), 4.30 (q, J = 7.1 Hz, 2H), 4.08 (t, 2H), 3.34 - 3.26 (m, 2H), 1.31 (t, 3H).
Step B: ethyl 2-{5-[(l,3-benzothiazol-2-yl)amino]~2,3-dihydro-lH-indol-l-yl}-l,3-thiazole4-carboxylate
To an oven-dried microwave vial was added the product from Step A (100 mg, 0.28 mmol, 1 eq), 2-aminobenzothiazole (51.0 mg, 0.34 mmol, 1.2 eq), césium carbonate (129 mg, 0.4 mmol, 2 eq), and 1,4-dioxane (2 mL) and the mixture was sparged with nitrogen (10 mîns) before adding BrettPhos (10.6 mg, 0.02 mmol, 0.1 eq) and tris(dibenzylideneacetone)dipalladium(0) (18.2 mg, 0.02 mmol, 0.1 eq), then heatîng at 120 20 °C for 1 h under microwave irradiation. The reaction was partitioned between ethyl acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 50 mL), and the combined organic extracts were washed with brine (50 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient 0 - 40% ethyl acetate in iso-h eptane 25 afforded the desired product as a brown gum (29.8 mg, 0.07 mmol, 36%).
LC/MS (C2iHi8N4O2S2) 423 [M+H]+; RT 1.38 (LCMS-V-B1) ‘H NMR (400 MHz, DMSO-d6) δ 8.01 (d, 1H), 7.87 (s, 1H), 7.83 - 7.78 (m, 1H), 7.61 - 7.57 (m, 1H), 7.45 (s, 1H), 7.33 - 7.28 (m, 1H), 7.18 - 7.10 (m, 1H), 7.00 (td, J = 1.24, 7.55 Hz, 1H), 4.31 (q, J = 7.12 Hz, 2H), 4.13 - 4.06 (m, 2H), 3.43 - 3.34 (m, 2H), 1.33 (t, J = 7.12 Hz, 30 3H).
118
Step C: 2-{5-[(l,3-benzothiazol-2~yl)amino]-2,3-dihydro-lH-indol-l-yl}-l,3-thiazole-4carboxylic acid
To a solution of the product from Step B (29.8 mg, 0.07 mmol, 1 eq) in tetrahydrofuran (2 mL) and methanol (1 mL), was added IN aqueous sodium hydroxide (0.14 mL, 0.14 mmol, 2 eq) and the mixture was heated at 50 °C for 1 h. The reaction was partitioned between ethyl acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 30 mL), and the combined organic extracts were washed with brine (50 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 15% methanol in dichloromethane afforded material that was further purified by préparative H P LC (HPLC-VA2) to afford the desired product as a cream solid (3.7 mg, 0.01 mmol, 13%).
HRMS-ESI (m/z) [M+H]+ calcd for C19H15N4O2S2: 395.0636, found 395.0659
Exampie 4: 2-{7-[(l,3-Benzothiazol-2-yl)amino]-3,4-dihydro-2i/-l,4*benzoxazin-4-yl}l,3-thiazole-4-carboxylic acid
Step A: ethyl2-(7-bromo-3,4-dihydro-2H-l,4-benzoxazin-4-yl)-1,3-thiazole-4-carboxylate
To a solution of benzoyl isotbiocyanate (301 pL, 2.24 mmol, 1.2 eq) in acetone (10 mL) was added 7-bromo-3,4-dihydro-2/f-benzo[b][l,4]oxazine (400 mg, 1.87 mmol, 1 eq) and the mixture was heated at reflux for 1 h. The reaction was poured onto ice water and the precipitate filtered, washed with water and dried to give a pale yellow solid. The solid was added to IN sodium hydroxide (10 mL) and the suspension was heated at 80 °C for 30 min, cooled to ambient température and poured onto cold IN aqueous hydrochloric acid. The pH was adjusted to pH 8 with saturated aqueous sodium carbonate, the solids were collected by filtration and washed with water to afford a yellow solid. A suspension of the solid and ethyl bromopyruvate (235 pL, 1.87 mmol, 1 eq) in éthanol (10 mL) was heated at reflux for 2 h then allowed to cool to ambient température. The reaction was partitioned between ethyl
119 acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 50 mL), and lhe combined organic extracts were washed with brine (50 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient 0 - 20% ethyl acetate in isoheptane afforded the desired product as a yellow solid (264 mg, 0.71 mmol, 38%).
LC/MS (CuHisBrNzOsS) 369 [M+H]+; RT 1.36 (LCMS-V-B1) ‘H NMR (400 MHz, DMSO-d6) δ 8.14 (d, J = 8.76 Hz, IH), 7.94 (s, IH), 7.26 - 7.11 (m, 2H), 4.37 - 4.21 (m, 4H), 4.07 - 3.99 (m, 2H), 1.29 (t, 3H).
Step B: ethyl 2-{7-[(1,3-benzothiazol-2-yl)aminoj-3t4-dihydro-2H-1,4-benzoxazin-4-yl}'-1,3thiazole-4-carboxylate
To an oven-dried microwave vial was added the product from Step A (173 mg, 0.47 mmol, 1 eq), 2-aminobenzothiazole (105 mg, 0.7 mmol, 1.5 eq), potassium Zerr-butoxide (105 mg, 0.94 mmol, 2 eq) and 1,4-dîoxane (5 mL), and the mixture was sparged with nitrogen (10 mins) before adding BrettPhos (37.7 mg, 0.07 mmol, 0.15 eq) and tris(dibenzylideneacetone)dipalladium(0) (42.9 mg, 0.05 mmol, 0.1 eq), then heating at 140 °C for 1 h under microwave irradiation. The reaction was partitîoned between ethyl acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 50 mL), and the combined organic extracts were washed with brine (50 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 30% ethyl acetate în iso-heptane afforded the desired product as a pale yellow solid (91.4 mg, 0.21 mmol, 45%) that was used directly in the next step without further purification.
LC/MS (C21H18N4O3S2) 439 [M+H]+; RT 1.35 (LCMS-V-B1)
Step C: 2-{7-[(l J'benzothiazol-Z-ytyaminoj-S^-dihydro^H-l ,4-benzoxazin-4-yl}-l ,3thiazole-4-carboxylic acid
To a solution of the product from Step B (91.4 mg, 0.21 mmol, 1 eq) in tetrahydrofuran (3 mL) and methanol (1 mL), was added IN aqueous sodium hydroxide (0.42 mL, 0.42 mmol, 2 eq) and the mixture was heated at 50 °C for 2 h. The reaction was partitîoned between ethyl acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 30 mL), and the combined organic extracts were washed with brine (50 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by préparative HPLC (HPLC-V-A1) afforded the desired product as a créant solid (1.5 mg, 2%).
120
P LC/MS (C19H14N4O3S2) 411 [M+H]+; RT 1.18 (LCMS-V-B1) *H NMR (400 MHz, DMSO-d6) δ 10.51 (s, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.81 (dd, J = 7.8, 1.2 Hz, 1H), 7.69 - 7.59 (m, 3H), 7.33 (td, J = 7.7, 1.3 Hz, 1H), 7.24 (dd, J - 8.9, 2.5 Hz, 1H), 7.16 (td, J = 7.6, 1.2 Hz, 1H), 4.30 (t, J = 4.4 Hz, 2H), 4.03 (t, J = 4.5 Hz, 2H).
HRMS-ESI (m/z) [M+H]+ calcd for Ci5H15N4O3S2: 411.0586, found 411.0610
Example 5: 6-{5-[(l,3-Benzothiazül-2-yl)animo]-LH-indol-l-yl}pyridine-2-carboxylic acid
Step A: 6-(5-iodo-lH-indol-l-yl)pyridine-2~carboxylic acid
To a stirred solution of 5-iodoindole (170 mg, 0.7 mmol, 1 eq) in 1,4-dioxane (5 mL) / dimethylformamide (1 mL) was added sodium hydride (60% dispersion; 20.1 mg, 0.84 mmol, 1.2 eq) portionwise over 20 minutes, then the mixture was stirred for 30 min before the addition of ethyl 6-chloropicolinate (143 mg, 0.77 mmol, 1.1 eq) and stirring at 70 °C overnight. The reaction was partitioned between ethyl acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 30 mL), and the combined organic extracts were washed with brine (3 x 30 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 30% ethyl acetate in iso-heptane afforded the desired product as a yellow solid (38.3 mg, 0.11 mmol, 15%).
LC/MS (C14H9IN2O2) 365 [M+H]+; RT 1.22 (LCMS-V-B1) ’H NMR (400 MHz, DMSO-d6) δ 13.47 (s, 1H), 8.64 (d, J = 8.8 Hz, 1H), 8.20 - 8.17 (m, 1H), 8.16 - 8.13 (m, 1H), 8.08 - 8.02 (m, 2H), 7.93 (dd, J = 7.5, 0.8 Hz, 1H), 7.59 (dd, J = 1.8 Hz, 1H), 6.78 (dd, J = 3.6, 0.7 Hz, 1H).
121 φ Step B: 6-{5-[(1,3-benzothiawl-2-yl)amino]~lH-indol-1 ~yl}pyridine-2-carboxylic acid
To an oven-dried microwave vial was added the product from Step A (38.3 mg, 0.11 mmol, 1 eq), 2-aminobenzothiazole (19 mg, 0.13 mmol, 1.2 eq), sodium tert-butoxide (20.2 mg, 0.21 mmol, 2 eq) and 1,4-dioxane (2 mL) and the mixture was sparged with nitrogen (10 mins) 5 before adding BrettPhos (5.65 mg, 0.01 mmol, 0.1 eq) and tris(dibenzylideneacetone)dipalladium(0) (9.63 mg, 0.01 mmol, 0.1 eq), then heating at 140 °C for 4 h under microwave irradiation. The réaction was partitioned between ethyl acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 50 mL), and the combined organic extracts were washed with brîne (50 mL), dried (magnésium sulfate) and concentrated 10 in vacuo. Purification by préparative HPLC (HPLC-V-A2) afforded the desired product as a cream solid (0.8 mg, 2%).
HRMS-ESI (m/z) [M+H]+ calcd for C21H15N4O2S: 387.0916, found 387.0943
Example 6: 2-{5-[(l,3-Benzothiazol-2-yl)aniîno]-l/f-pyrrolo[2,3-/>]pyridin-l-yI}-1,3thiazole-4-carboxylic acid
Step A: ethyl 2-{5-bromo-lH-pyrrolo[2,3-b]pyridin-1 -yl}-l,3-thiazole-4-carboxylate
To a solution of 5-bromo-lW-pyrrolo[2,3-b]pyridine (250 mg, 1.27 mmol, 1 eq) in 1,4dioxane (5 mL) and dimethylformamide (2 mL) was added sodium hydride (60% dispersion; 36.5 mg, 1.52 mmol, 1.2 eq) portionwise over 20 mins and the mixture was stirred at ambient 20 température for 30 min before the addition of ethyl 2-bromo-l,3-thiazole-4-carboxyIate (449 mg, 1.9 mmol, 1.5 eq). The mixture was heated at reflux for 2 h then allowed to cool to ambient température and the résultant precipitate was collected by filtration and dried under vacuum to afford the desired product as a cream solid (300 mg, 0.85 mmol, 67%).
LC/MS (CisHwBrNjChS) 352 [M+H]+; RT 1.41 (LCMS-V-B1)
122 φ *Η NMR (400 MHz, DMSO-d6) δ 8.61 (d, J = 2.2 Hz, 1H), 8.48 (d, J = 2.2 Hz, 1H), 8.38 (s, 1H), 8.33 (d, J = 3.9 Hz, 1H), 6.88 (d, J = 3.8 Hz, 1H), 4.34 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H).
Step B; ethyl2-(5-/(I,3-benzothiazol-2~yl)amüio]-lH-pyrrolo[2,3-b]pyridin-l-yl}-l,35 thiazole-4-carboxylate
To an oven-dried microwave vial was added the product from Step A (200 mg, 0.57 mmol, 1 eq), 2-amînobenzothiazole (128 mg, 0.85 mmol, 1.5 eq), césium carbonate (370 mg, 1.14 mmol, 2 eq) and 1,4-dioxane (3 mL) and the mixture was sparged with nitrogen (10 mins) before the addition of tris(dibenzylideneacetone)dipalladium(0) (52 mg, 0.06 mmol, 0.1 eq) 10 and Xantphos (64.8 mg, 0.12 mmol, 0.2 eq) then heating at 120 °C for 6 h under micro wave irradiation. The reaction was partitioned between ethyl acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 30 mL), and the combined organic extracts were washed with brine (30 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFIash Rf, 12 g RediSep™ silica cartridge) 15 eluting with a gradient of 0 — 70% ethyl acetate in Ao-heptane afforded the crude desired product as a yellow gum that was used directly in the subséquent step without further purification.
LC/MS (C20H15N5O2S2) 422 [M+H]+; RT 1.37 (LCMS-V-B1)
Step C: 2-{5-[(l,3-benzothiazol-2-yl)amino]-lH~pyrrolo[2,3-b]pyridin-1 -yl}-l,3-thiazole-420 carboxylic acid
To a solution of the product from Step B (61.2 mg, 0.15 mmol, 1 eq) in tetrahydrofuran (3 mL) and methanol (1 mL), was added IN aqueous sodium hydroxide (0.29 mL, 0.29 mmol, 2 eq) and the mixture was heated at 50 °C for 2 h. The réaction was concentrated in vacuo and the residue suspended in water and acidified to pH 6 with IN aqueous hydrochloric acid. The 25 mixture was partitioned between ethyl acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 30 mL), and the combined organic extracts were washed with brine (50 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by reverse phase automated flash chromatography (CombiFIash Rf, C18 5.5g RediSep column) eluting with a gradient of 5 - 95% acetonitrile in water afforded the desired product as a white solid (2.5 30 mg, 0.01 mmol, 4%).
HRMS-ESI (m/z) [M+H]+ calcd for CJ8H12N5O2S2: 394.0432, found 394.0459
123
Φ Example 7: 2-{5-[(l,3-Benzothiazol-2-yl)amino]-lJyf-pyrrolol2,3-dpyridin-l-yl}-l;3thiazole-4-carboxylic acid
Step A: ethyl 2-{5-chloro-lH-pyrrolo[2,3-c]pyridin-l-yl}-l,3-thiawle-4-carboxylate
To a solution of 5-chloro-l/f-pyrrolo[2,3-c]pyridîne (300 mg, 1.97 mmol, 1 eq) in 1,4dioxane (5 mL) and dimethylformamide (2 mL) was added sodium hydride (60% dispersion; 56.6 mg, 2.36 mmol, 1.2 eq) portionwise over 20 mins and the mixture was stirred at ambient température for 30 min, before the addition of ethyl 2-bromo-l,3-thiazole-4-carboxylate (696 mg, 2.95 mmol, 1.5 eq) and heating at reflux for 2 h. The reaction was allowed to cool to 10 ambient température and the résultant precipitate was collected by filtration and drying under vacuum to afford the desired product as a pale brown solid (518 mg, 1.68 mmol, 86%).
LC/MS (C!3HioC1N302S) 308 [M+H]+; RT 1.19 (LCMS-V-B1)
NMR (400 MHz, DMSO-d6) δ 9.48 (s, 1H), 8.39 (s, 1H), 8.33 (d, J - 3.5 Hz, 1H), 7.85 (d, J = 0.9 Hz, 1H), 6.93 (dd, J = 3.5, 0.8 Hz, 1H), 4.37 (q, J = 7.1 Hz, 2H), 1.36 (t, J = 7.1 Hz, 15 3H).
Step B: ethyl 2-{5-[(l,3-benzothiazol-2-yl)aminoj-lH-pyrrolo[2,3-c]pyridin-l-yl}-l,3thiazole-4-carboxylate
To an oven-dried microwave vial was added the product from Step A (300 mg, 0.97 mmol, 1 eq), 2-aminobenzothiazole (220 mg, 1.46 mmol, 1.5 eq), césium carbonate (635 mg, 1.95 20 mmol, 2 eq), and 1,4-dioxane (7 mL) and the mixture was sparged with nitrogen (10 mins) before the addition of tris(dibenzylideneacetone)dipalladium(0) (89.3 mg, 0.1 mmol, 0.1 eq) and Xantphos (113 mg, 0.19 mmol, 0.2 eq) then heating at 130 °C for 8 h under microwave irradiation. The reaction was partitioned between ethyl acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 50 mL), and the combined organic extracts were washed 25 with brine (30 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge)
124 eluting with a gradient of 0 - 70% ethyl acetate in iso-heptane afforded the desired product as a yellow solid (17.3 mg, 0.04 mmol, 4%).
LC/MS (C2ûH15N5O2S2) 422 [M+H]+; RT 1.34 (LCMS-V-B1)
Ή NMR (400 MHz, DMSOM6) δ 11.52 (s, IH), 9.53 (t, J = 0.9 Hz, IH), 8.34 (s, IH), 8.22 (d, J = 3.5 Hz, IH), 7.87 (d, IH), 7.61 (d, J = 8.1 Hz, IH), 7.54 - 7.49 (m, IH), 7.40 - 7.27 (m, IH), 7.21 - 7.14 (m, IH), 6.94 (dd, J = 3.5, 0.7 Hz, IH), 4.40 (q, J = 7.1 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H).
Step C: 2-{5-[(l,3-benzothiazol-2-yl)amino]-lH-pyrrolo[2,3-c]pyridin-l-yl}-l,3-thiazole-4carboxylic acid
To a solution of the product from Step B (17.3 mg, 0.04 mmol, 1 eq) in tetrahydrofuran (3 mL) and methanol (1 mL), was added IN aqueous sodium hydroxide (0.08 mL, 0.08 mmol, 2 eq) and the mixture was heated at 50 °C for 2 h. The reaction was concentrated in vacuo and the residue was suspended in water and acidified to pH 7 with IN aqueous hydrochloric acid. The solîds were colîected by filtration, washed with methanol, then diethyl ether, and dried under vacuum to afford the desired product as a cream solid (9.1 mg, 0.02 mmol, 56%).
HRMS-ESI (m/z) [M+H]+ calcd for C18H12N5O2S2: 394.0432, found 394.0452
Example 8: 2-{3-[(l,3-Benzothiazol-2-yl)amino]-7f/-pyrrolo[2,3-i:]pyridazîn-7-yl}-l,3thiazole-4-carboxylic acid
Step A: ethyl 2f3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-l,3-thiazole-4-carboxylate
To a solution of 3-chloro-7/LpynOlo[2,3-c]pyridazine (285 mg, 1.86 mmol, 1 eq) in 1,4dioxane (5 mL) and dimethylformamide (2 mL) was added sodium hydride (60% dispersion;
53.4 mg, 2.23 mmol, 1.2 eq) portionwise over 20 mins, then the mixture was stirred at ambient température for 30 min, before the addition of ethyl 2-chlorothiazole-4-carboxylate 25 (533 mg, 2.78 mmol, 1.5 eq) and heating at reflux for 2 h. The reaction was partitioned
125 between ethyl acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 40 mL), and the combined organic extracts were washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 — 40% ethyl acetate in Ao-heptane afforded the desired product as a peach solid (388 mg, 1.26 mmol, 68%).
LC/MS (Ci2H9C1N4O2S) 309 [M+H]+; RT 1.14 (LCMS-V-B1) *H NMR (400 MHz, DMSO-d6) δ 8.69 (d, J = 3.8 Hz, 1H), 8.46 (s, 1H), 8.30 (s, 1H), 6.97 (d, J = 3.8 Hz, 1H), 4.36 (q, J = 7.1 Hz, 2FI), 1.35 (t, J = 7.1 Hz, 3H).
Step B: ethyl 2-{3-[(1,3-benzoihiazoE2-yl)amiiiol-7i l-pyiroi()l2,3-c]pyrid(t-in-7-yl}-1,3thiazole-4-carboxylate
To an oven-dried microwave vial was added the product from Step A (388 mg, 1.26 mmol, 1 eq), 2-aminobenzothiazole (283 mg, 1.89 mmol, 1.5 eq), césium carbonate (819 mg, 2.51 mmol, 2 eq), and 1,4-dioxane (10 mL) and the mixture was sparged with nitrogen (10 mins) before the addition of trîs(dibenzylideneacetone)dipalladium(0) (115 mg, 0.13 mmol, 0.1 eq) and Xantphos (145 mg, 0.25 mmol, 0.2 eq), then heating at 130 °C for 6 h under microwave irradiation. The reaction was partitioned between ethyl acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 50 mL), and the combined organic extracts were washed with brine (30 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in Ao-heptane afforded the desired product as a brown solid (112 mg, 0.27 mmol, 21%).
LC/MS (Ci9Hi4NûO2S2) 423 [M+H]+; RT 1.29 (LCMS-V-B1)
Al NMR (400 MHz, DMSO-d6) δ 11.84 (s, III), 8.58 (d, J = 3.9 Hz, 1H), 8.41 (s, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.91 (s, 1H), 7.71 - 7.63 (m, 1H), 7.40 (ddd, J = 8.2, 7.2, 1.3 Hz, 1H), 7.24 (td, J = 7.6, 1.1 Hz, 1H), 6.99 (d, J = 3.9 Hz, 1H), 4.36 (q, J = 7.1 Hz, 3H), 1.35 (t, J = 7.1 Hz, 3H).
Step C: 2f3-/(1,3-benzothiazol-2-yl) amino]- 7H-pyrrolo[2,3-cJpyridazin - 7-yl}-l ,3-thiazole4-carboxylic acid
To a solution of the product from Step B (112 mg, 0.27 mmol, 1 eq) in tetrahydrofuran (5 mL) and methanol (1.5 mL) was added IN aqueous sodium hydroxide (0.53 mL, 0.53 mmol.
126
Φ 2 eq) and the mixture was heated at 50 °C for 2 h. The reaction was concentrated in vacuo, the residue was suspended in water, and the solids were collected by filtration. Purification by reverse phase automaled flash chromatography (CombiFlash Rf, C18 5.5g RediSep column) eluting with a gradient of 5 - 95% acetonitrile in water afforded the desired product as a pale 5 yellow solid (4.9 mg, 0.01 mmol, 5%).
HRMS-ESI (m/z) [M+H]+ calcd for Ci7HnN6O2S2: 395.0385, found 395.0406
Exampie 9: 2-{3-[(l,3-Benzothiazol-2-yl)amino]-7//-pyrrolo[2,3-c]pyridazin-7-yl}-5-[3(2-fluorophenoxy)propyl]-l,34hiazoIe-4-carboxylic acid
Step A: ethyl5‘bromo-2-acetamido-l,3-thiazole-4-carboxylate
To a solution of ethyl 2-amino-5-bromothiazole-4-carboxylate (4 g, 15.9 mmol, 1 eq) in dichloromethane (70 mL) was added ace tic anhydride (1.65 mL, 17.5 mmol, 1.1 eq) and 4dimethylaminopyridine (2.24 g, 18.3 mmol, 1.15 eq) and the mixture was stirred at ambient température overnight. The reaction was allowed to cool to ambient température, then washed with water followed by brine, dried (magnésium sulfate) and concentrated in vacuo. The résultant solid was triturated with diethyl ether, filtered, and dried under vacuum to afford the desired product as an off-white solid (4.15 g, 14.15 mmol, 89%).
LC/MS (C8H9BrN2O3S) 294 [M+H]1; RT 0.82 (LCMS-V-B1) rH NMR (400 MHz, DMSO-J6) δ 12.80 (s, 1H), 4.28 (q, J = 7.1 Hz, 2H), 2.15 (s, 3H), 1.30 20 (t, J = 7.1 Hz, 3H).
Step B: ethyl 2-acetamido-5-(3-hydroxyprop-l-yn-1 -yl)·! ,34hiazole-4-carboxylate
Tetrakis(triphenylphosphine)palladium(0) (813 mg, 0.7 mmol, 0.05 eq) was added to a solution of the product from Step A (4.13 g, 14.1 mmol, 1 eq), propargyl alcohol (1.64 mL, 28.2 mmol, 2 eq), triethylamine (5.87 mL, 42.2 mmol, 3 eq) and copper (I) iodide (0.27 g, 25 1.41 mmol, 0.1 eq) in dimethylformamide (60 mL) under a nitrogen atmosphère, and the
127 mixture was heated at 100 °C for 3 h. The reaction was concentrated in vacuo and purification by automated flash column chromatography (CombiFlash Rf, 120 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% methanol in dichloromethane afforded the desired product as a cream solid (3 g, 11.2 mmol, 79%).
LC/MS (C11H12N2O4S) 269 [M+H]+; RT 0.63 (LCMS-V-B1)
Ή NMR (400 MHz, DMSO-d6) δ 12.80 (s, 1H), 5.45 (t, J = 6.0 Hz, 1H), 4.37 (d, J = 6.0 Hz, 2H), 4.27 (q, J = 7.1 Hz, 2H), 2.16 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H).
Step C: ethyl 2-acetanüdo-5-(3-hydroxypropyl)-l,3-thiazole-4~carboxylate
Ethyl acetate (30 mL) and methanol (30 mL) were added to a flask containing the product 10 from Step B (3 g, 11.2 mmol, 1 eq) and platinum(IV) oxide hydrate (508 mg, 2.23 mmol, 0.2 eq) under a nitrogen atmosphère. The vessel was evacuated and back-filled with nitrogen (x3), then evacuated and placed under an atmosphère of hydrogen and shaken at ambient température for 24 h. The reaction was fîltered through celite (10 g), eluted with methanol, and the solvent removed in vacuo. Purification by automated flash column chromatography 15 (CombiFlash Rf, 80 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% methanol in dichloromethane afforded the desired product as a brown solid (1.89 g, 6.94 mmol, 62%).
LC/MS (C11H1ÛN2O4S) 273 [M+H]+; RT 0.61 (LCMS-V-B1) XH NMR (400 MHz, DMSO-d6) δ 12.38 (s, 1H), 4.54 (t, J = 5.1 Hz, 1H), 4.25 (q, J = 7.1 Hz, 2H), 3.44 (q, J = 6.1 Hz, 2H), 3.20 - 3.08 (m, 2H), 2.12 (s, 3H), 1.82 - 1.68 (m, 2H), 1.29 (t, J 20 - 7.1 Hz, 3H).
Step D: ethyl 2-amino-5-(3-hydroxypropyl) -I J-thiazole-d-carboxylate
To a solution of the product from Step C (500 mg, 1.84 mmol, 1 eq) in éthanol (15 mL) was added hydrochloric acid (4M in 1,4-dioxane; 4.59 mL, 4 M, 18.4 mmol, 10 eq) and the mixture was heated at 60 °C overnight. The reaction was allowed to cool to ambient 25 température and then concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% methanol in dichloromethane afforded the desired product as a beige solid (422 mg, 1.83 mmol, 100%).
LC/MS (C9H14N2O3S) 231 [M+H]+; RT 0.50 (LCMS-V-B1) *H NMR (400 MHz, DMSO-d6) Ô 8.04 (br s, 2H), 4.26 (q, J = 7.1 Hz, 2H), 3.44 (t, J = 6.3 Hz, 2H), 3.05 - 2.96 (m, 2H), 1.76 - 1.64 (m, 2H), 1.29 (t, J = 7.1 Hz, 3H).
128
Step E: ethyl 2-bromo-5-(3-hydroxypropyl)-l,3-thiazole-4‘carboxylate reri-Butyl nitrate (0.26 mL, 2.2 mmol, 1.2 eq) was added dropwise to a stirred solution of copper (II) bromide (491 mg, 2.2 mmol, 1.2 eq) în acetonitrile (6 æL) and the mixture was heated to 60 °C then a suspension of the product from Step D (422 mg, 1.83 mmol, 1 eq) in 5 acetonitrile (8 mL) was added slowly. The mixture was maintained at 60 °C for 2 h then allowed to cool to ambient température and quenched by the addition of 2N aqueous sodium hydroxide, then extracted with ethyl acetate. The organic extract was washed with water, brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a 10 gradient of 0 — 50% ethyl acetate în tso-heptane afforded the desired product as a colourless oil (271 mg, 0.92 mmol, 50%).
LC/MS (C9H12BrNO3S) 296 [M+H]+; RT 0.76 (LCMS-V-B1) ‘H NMR (400 MHz, DMSO-d6) 5 4.59 (t, J = 5.1 Hz, 1H), 4.29 (q, J = 7.1 Hz, 2H), 3.44 (td, J - 6.3, 5.1 Hz, 2H), 3.24 - 3.15 (m, 2H), 1.81- 1.69 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H).
Step F: ethyl 2-bromo-5-[3-(2-fluorophenoxy)propyl]'l,3~thiazole~4-carhoxylate
A solution of the product from Step E (271 mg, 0.92 mmol, 1 eq), 2-fluorophenoI (0.12 mL, 1.38 mmol, 1.5 eq) and triphenylphosphine (362 mg, 1.38 mmol, 1.5 eq) in tetrahydrofuran (10 mL)was cooled in an ice-bath then diisopropyl azodicarboxylate (0.27 mL, 1.38 mmol, 1.5 eq) was added slowly and the mixture was stirred at 0 °C for 30 min then at ambient 20 température for 3 h. The reaction was partitioned between ethyl acetate and water, and the organic phase was washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 60% ethyl acetate in /so-heptane afforded the desired product as an orange oil (302 mg, 0.78 mmol, 85%).
LC/MS (Ci5Hi5BrFNO3S) 390 [M+H]+; RT 1.23 (LCMS-V-B1) *H NMR (400 MHz, DMSO-d6) δ 7.26 - 7.07 (m, 3H), 7.01 - 6.88 (m, 1H), 4.27 (q, J = 7.1 Hz, 2H), 4.09 (t, J - 6.1 Hz, 2H), 3.39 - 3.29 (m, 2H), 2.16 - 2.03 (m, 2H), 1.28 (t, J = 7.1 Hz, 3H).
129
Step G: ethyl 2-{3-chloro-7H-pyrrolo[2,3-c]pyridazin-7-yl}-5-[3-(2-fluoropheiioxy)propyl]l,3-thiazole~4-carboxylate
To a stirred solution of 3-chloro-7/f-pyrrolo[2,3-c]pyridazine (179 mg, 1.17 mmol, 1.5 eq) in 1,4-dioxane (10 mL) and dimethylformamide (3 mL) was added sodium hydride (60% 5 dispersion; 22.4 mg, 0.93 mmol, 1.2 eq) portionwise over 20 minutes and the mixture was stirred for 30 mins before the addition of the product from Step F (302 mg, 0.78 mmol, 1 eq) and stirring at ambient température for 2 h and at reflux overnight. The reaction was partitioned between ethyl acetate and water, the aqueous phase was extracted with ethyl acetate (3 x 30 mL), and the combined organic extracts were washed with brine, dried 10 (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 50% ethyl acetate in fso-heptane afforded the desired product as a paie yellow solid (122 mg, 0.27 mmol, 34%).
LC/MS (C21H18CIFN4O3S) 461 [M+H]+; RT 1.41 (LCMS-V-B1) JH NMR (400 MHz, DMSO-d6) δ 8.63 (d, J = 3.8 Hz, IH), 8.28 (s, IH), 7.26 - 7.07 (m, 3H), 6.99 - 6.87 (m, 2H), 4.32 (q, J = 7.1 Hz, 2H), 4.15 (t, J = 6.1 Hz, 2H), 3.48 - 3.37 (m, 2H), 2.28 - 2.14 (m, 2H), 1.32 (t, J = 7.1 Hz, 3H).
Step H: ethyl 2-{3-[(l,3-benzothiazol-2-yl)amino]-7H-pyrrolo[2,3-c]pyridazin-7-yl}-5-[3-(2fluoropheiioxy)propyl]-l,3-thiazole-4-curboxylate
To a micro wave vial was added the product from Step G (122 mg, 0.27 mmol, 1 eq), 2aminobenzothiazole (59.7 mg, 0.4 mmol, 1.5 eq), césium carbonate (173 mg, 0.53 mmol, 2 eq), tris(dibenzylideneacelone)dipaIladium(0) (24.3 mg, 0.03 mmol, 0.1 eq), Xantphos (15.3 mg, 0.03 mmol, 0.1 eq) and 1,4-dioxane (7.5 mL), and the mixture was heated at 120 °C for 6 h under microwave irradiation. The mixture was partitioned between ethyl acetate and water, 25 and the aqueous phase was extracted with ethyl acetate (3 x 40 mL), washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 50% ethyl acetate in /so-heptane afforded the desired product as a yellow solid (42.8 mg, 0.07 mmol, 28%).
LC/MS (C28H23FN6O3S2) 575 [M+H]+; RT 1.47 (LCMS-V-B1) XH NMR (400 MHz, DMSO-d6) δ 11.80 (br s, IH), 8.53 (d, J = 3.8 Hz, IH), 7.95 (d, J = 7.7 Hz, IH), 7.88 (s, IH), 7.69 - 7.63 (m, IH), 7.40 (ddd, J = 8.2, 7.3, 1.3 Hz, IH), 7.26 - 7.16
130 φ (m, 3H), 7.15 - 7.11 (m, 1H), 6.98 - 6.91 (m, 2H), 4.32 (q, J = 7.1 Hz, 2H), 4,17 (t, J = 6.1 Hz, 2H), 3.48 - 3.39 (m, 2H), 2.28 - 2.17 (m, 2H), 1.33 (t, J = 7.1 Hz, 3H).
Step I: 2-{3-[(l,3-benzothiazol-2-yl)amino]-7H-pyrrolo[2,3-cJpyridazin-7-yl}-5-[3-(2· fluorophenoxy)propyl]-l ,3-thiazole-4-carboxylic acid
To a solution of the product from Step H (42.8 mg, 0.07 mmol, 1 eq) in 1,4-dioxane (2 mL) was added 1.25M aqueous lithium hydroxide (0.12 mL, 0.15 mmol, 2 eq) and the mixture was heated at reflux for 2 h. The reaction was concentrated in vacuo and purification by préparative HPLC (HPLC-V-A2) afforded the desired product as a yellow solid (2.3 mg, 6%).
HRMS-ESI (m/z) [M+H]+ calcd for C2ûH2oFNû03S2: 547.1022, found 547.1010.
Example 10: 2-{3-[(l.,3-Benzothiazol-2-yl)aiîiiiio]-5fL6/f,7Zf-pyrrolo[2,3-c]pyridazin-7yl}-l,3-thiazole-4-carboxylic acid
Step A: tert-butyl 3-chloro-5H,6H,7H-pyrrolo[2,3-c]pyridazine-7-carboxylate
To a solution of V-(but-3-yn-l-yl)-6-chloro-l,2,4,5-tetrazin-3-amine (381 mg, 2.08 mmol, 1 eq) in tetrahydrofuran (15 mL) was added di-iert-buty 1 dicarbonate (1.36 g, 6.23 mmol, 3 eq) and 4-dimethylaminopyridine (12.7 mg, 0.1 mmol, 0.05 eq) and the mixture was stirred at ambient température overnight. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 70% ethyl acetate in iso-heptane afforded the desired product as a red solid (89 mg, 0.35 mmol, 17%).
LC/MS (C11H14CIN3O2) 256 [M+H]+; RT 2.06 (LCMS-V-C) ’H NMR (400 MHz, DMSO-d6) δ 7.62 (t, J - 1.6 Hz, 1H), 3.97 (dd, J = 8.9, 7.9 Hz, 2H), 3.10 (ddd, J = 9.4, 7.8, 1.6 Hz, 2H), 1.51 (s, 9H).
131
Step B: 3-chloro-5H,6H,7H-pyrrolo[2,3-c]pyridazine
To a solution of the product from Step A (89 mg, 0.35 mmol, 1 eq) in dichloromethane (3 mL) was added trifluoroacetic acid (1.5 mL) and the mixture was stirred at ambient 5 température for 1 h. The reaction was concentrated in vacuo then loaded onto a methanol washed SCX cartridge (5 g), washed with methanol, then eluted with 1.4N methanolic ammonia to afford the desired product as a beige solid (51 mg, 0.33 mmol, 94%).
LC/MS (CûH6C1N3) 156 [M+H]+; RT 0.37 (LCMS-V-C) ’H NMR (400 MHz, DMSO-d6) δ 7.27 (br s, 1H), 7.24 - 7.20 (m, 1H), 3.55 (td, J = 8.2,1.1 10 Hz, 2H), 3.06 (ddd, J = 9.7, 7.8, 1.7 Hz, 1H).
Step C: ethyl 2-{3-ehloro-5H,6H,7H~pyrrolo[2,3~cJpyridazin-7-yl}-l,3-thiazole-4carboxylate
To an oven-dried microwave vial was added the product from Step B (51 mg, 0.33 mmol, 1 eq), ethyl 2-bromo-l,3-thiazole-4-carboxylate (92.9 mg, 0.39 mmol, 1.2 eq), trans-Ν,Ν'15 dimethylcyclohexane-l,2-diamine (10.3 gL, 0.07 mmol, 0.2 eq), copper(I) iodide (6.24 mg, 0.03 mmol, 0.1 eq)and potassium phosphate tribasîc (139 mg, 0.66 mmol, 2 eq), and 1,4dioxane (3 mL) and the vessel was evacuated and flushed with nitrogen then heated at 150 °C for 1 hour under microwave irradiation. The reaction was diluted with ethyl acetate, fîltered through celite, then washed with brine, dried (magnésium sulfate) and concentrated in vacuo. 20 Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 3% methanol in dichloromethane afforded the crude desired product as a beige solid (19 mg, 0.06 mmol, 19%) that was used directly in the next step without further purification.
LC/MS (C12H11CIN4O2S) 311 [M+H]+; RT 2.24 (LCMS-V-C)
Step D: ethyl 2-{3-[(l^-benzothiazol^-yllaminoJ-SHfiH,7H-pyrrolo[2,3-cjpyridazin 7-yl}-
J-thiazole-d-carboxylate
To an oven-dried microwave vial was added the product from Step C (19 mg, 0.06 mmol, 1 eq), 2-aminobenzothiazole (13.8 mg, 0.09 mmol, 1.5 eq), Xantphos (7.08 mg, 0.01 mmol, 0.2 eq), césium carbonate (39.8 mg, 0.12 mmol, 2 eq), and 1,4-dioxane (3 mL) and the vessel was 30 evacuated and flushed with nitrogen then tris(dîbenzylideneacetone)dipalladium(0) (5.6 mg, 0.01 mmol, 0.1 eq) was added and the mixture was sparged with nitrogen (10 mins) then
132 heated at 150 °C for 1 h under microwave irradiation. The reaction was diluted with ethyl acetate and filtered through celite, washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% methanol in dîchioromethane afforded the desired product as a brown solid (9 mg, 0.02 mmol, 35%).
LC/MS (C19H16N6O2S2) 425 [M+H]+; RT 2.53 (LCMS-V-C) ‘H NMR (400 MHz, DMSO-d6) Ô 7.95 - 7.89 (m, IH), 7.69 - 7.58 (m, 2H), 7.41 - 7.35 (m, IH), 7.33 - 7.29 (m, IH), 7.25 - 7.15 (m, IH), 4.40 - 4.26 (m, 4H), 1.33 (t, 3H).
Step E: 2-{3-[(l,3-benzothiazol-2-yl)amino]-5H,6H,7H-pyrrolo[2,3-c]pyridazin-7-yl}-l,3thiazole-4-carboxylic acid
To a solution of the product from Step D (9 mg, 0.02 mmol, 1 eq) in 1,4-dioxane (2 mL) was added lithium hydroxide monohydrate (3.56 mg, 0.08 mmol, 4 eq) and the mixture was heated at reflux for 6 h. The reaction was concentrated in vacuo, then dîssolved in methanol and loaded onto a methanol-washed PE-ΑΧ cartridge (5 g), washed with methanol, eluted with 10:1 dîchioromethane / formic acid, and concentrated in vacuo. The crude material was triturated with dîchioromethane, filtered, and dried under vacuum to afford the desired product as a beige solid (2.42 mg, 0.01 mmol, 29%), as a formic acid sait.
HRMS-ESI (m/z) [M+H]+ calcd for CnHuN^Sz: 397.0541, found 397.0529.
Example 11: 2-{3-[(l,3-Benzothiazol-2-yi)amino]-4-methyl-51/,6/f,7//-pyrrolo[2,3c]pyridazin-7-yl}-l,3-thiazole-4-carboxylic acid
-¼ .N
HN N S^N
Step A: S-chloro-d-methylSHfiH,7H-pyrrolo[2,3-c]pyridazine
To a solution 3,6-dichloro-l,2,4,5-tetrazine (600 mg, 3.97 mmol, 1 eq) in tetrahydrofuran (16 mL)was added pent-3-yn-l-amine hydrochloride (475 mg, 3.97 mmol, 1 eq) and triethylamine (553 gL, 3.97 mmol, 1 eq) and the mixture was heated at 110 °C in a sealed
133 φ tube for 8 hours. The reaction was diluted with methanol, filtered through a pad of celite, and the filtrate was partitîoned between dichioromethane and saturated aqueous sodium bicarbonate, the aqueous phase was extracted with dichioromethane, and the combined organic extracts were dried (magnésium sulfate) and concentrated in vacuo. Purification by 5 automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ siiica cartridge) eluting with a gradient of 0 - 10% methanol in dichioromethane afforded the desired product as a beige solid (96 mg, 0.57 mmol, 14%).
LC/MS (C7H8CIN3) 170 [M+H]+; RT 0.54 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 7.12 (s, 1H), 3.56 (td, J = 8.4, 1.2 Hz, 2H), 3.04 (ddd, J = 10 9.2, 7.9, 1.3 Hz, 2H), 2.13 (d, J = 1.3 Hz, 3H).
Step B: ethyl 2-{3-chloro-4-methyl-5H.6ll,7H-pyrrolol2,3-clpyrida~.iii-7-yl)-l,3~thiazole-4· carboxylate
To an oven-dried microwave vîal was added the product from Step A (96 mg, 0.57 mmol, 1 eq), ethyl 2-bromo-l,3-thiazole-4-carboxylate (187 mg, 0.79 mmol, 1.4 eq), trans-N,N'15 dimethylcyclohexane-l,2-diamine (17.9 pL, 0.11 mmol, 0.2 eq), copper (I) iodide (10.8 mg, 0.06 mmol, 0.1 eq), potassium phosphate tribasic (240 mg, 1.13 mmol, 2 eq), and 1,4-dioxane (8 mL) and the vessel was evacuated and flushed with nitrogen then heated at 150 °C for 1 h under microwave irradiation. The reaction was diluted with ethyl acetate, filtered through celite, washed with brine, dried (magnésium sulfate) and concentrated în vacuo. Purification 20 by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ siiica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in i'so-heptane afforded the desired product as a beige solid (18 mg, 0.06 mmol, 10%).
LC/MS (C13H13CIN4O2S) 325 [M+H]+; RT 2.32 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 8.11 (s, 1H), 4.41 (dd, J = 8.8, 7.6 Hz, 2H), 4.30 (q, J = 7.1 25 Hz, 2H), 3.34 - 3.27 (m, 2H), 2.29 (d, J = 1.2 Hz, 3H), 1.31 (t, J = 7.1 Hz, 3H).
Step C: ethyl 2-{3-[Π ,3-benz(Hhiazol-2-yl)(iminol-4-methyl-511,611,7H-pyrrolo[2,3c]pyridazin-7-yl}-l ,3-thiazole-4~carboxylate
To an oven-dried microwave vial was added the product from Step B (27 mg, 0.08 mmol, 1 eq), 2-aminobenzothiazoIe (18.7 mg, 0.12 mmol, 1.5 eq), Xantphos (9.62 mg, 0.02 mmol, 0.2 30 eq), césium carbonate (54.2 mg, 0.17 mmol, 2 eq) and 1,4-dioxane (4 mL), and the vessel was evacuated and flushed with nitrogen then tris(dibenzylideneacetone)dipalIadium(0) (7.61 mg,
134 φ 0.01 mmol, 0.1 eq) was added and the mixture was sparged with nitrogen (10 mins) then heated at 150 °C for 1 h under microwave irradiation. The reaction was diiuted with ethyl acetate and filtered through celite, then washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash 5 Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in isoheptane afforded the desired product as a yellow solid (15 mg, 0.03 mmol, 41%).
LC/MS (C20H18N6O2S2) 439 [M+H]+; RT 2.67 (LCMS-V-C) ‘H NMR (400 MHz, DMSO-d6) δ 8.06 (s, 1H), 7.88 (s, 1H), 7.53 (br s, 1H), 7.38 (t, J = 7.5
Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H), 4.38 (t, J = 8.0 Hz, 2H), 4.31 (q, J = 7.1 Hz, 2H), 3.32 10 3.21 (m, 2H), 2.33 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H).
Step D: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H-pyrrolo[2,3-c]pyridaziu-7yl}-l,3-thiazole-4-carboxylic acid
To a solution of the product from Step C (15 mg, 0.03 mmol, 1 eq) in 1,4-dioxane (2 mL) was added lithium hydroxide monohydrate (5.74 mg, 0.14 mmol, 4 eq) and the mixture was heated 15 at reflux for 3 h. The reaction was concentrated in vacuo, dissolved in methanol, loaded onto a methanol-washed PE-ΑΧ cartridge (5 g), washed with methanol, eluted with 9:1 dichloromethane / formic acid and concentrated in vacuo. The residue was triturated with dichloromethane, filtered and dried under vacuum to afford the desired product as a cream solid (9.03 mg, 0.02 mmol, 64%).
HRMS-ESI (m/z) [M+H]+ calcd for CisHisNûOzSz: 411.0698, found 411.0701.
Example 12: 2-{3-[( 1,3-Benzothiazol-2-yl ) ami no }-5Η,6Η,ΊΗ, 8W-pyrido|2,3-dpyridazin8-yl}-l,3-thiazole-4-carboxylic acid
135
Step A: pent-4-yn-l-yl methanesulfonate
To a solution of 4-pentyn-l-ol (3.32 mL, 35.7 mmol, 1 eq) in dichloromethane (60 mL) was added triethylamine (6.45 mL, 46.4 mmol, 1.3 eq) and the mixture was cooled to 0 °C before the dropwise addition of methanesulfonyl chloride (3.31 mL, 42.8 mmol, 1.2 eq) and stirring at ambient température overnîght. The reaction was partitioned between dichloromethane and water, and the organic phase was washed successively with saturated sodium bicarbonate and brine, dried (magnésium sulfate) and concentrated in vacuo to afford the desired product as an amber oil (5.8 g, 35.8 mmol, 100%).
Ή NMR (400 MHz, DMSO-d6) δ 4.26 (t, J = 6.2 Hz, 2H), 3.19 (s, 3H), 2.88 (t, J = 2.7 Hz, 1H), 2.29 (td, J = 7.1, 2.7 Hz, 2H), 1.91-1.80 (m, 2H).
Step B: 5-azidopent-l-yne
To a solution of the product from Step A (5.8 g, 35.8 mmol, 1 eq) in dimethylformamide (30 mL) was added sodium azide (5.81 g, 89.4 mmol, 2.5 eq) and the mixture was heated at 70 °C for 3 h. The reaction was diluted with water, the aqueous phase was extracted with diethyl ether (x3), and the combined organics were dried (magnésium sulfate) and concentrated in vacuo to afford the desired product as a yellow oil (5.65 g, 51.8 mmol, >100%).
JH NMR (400 MHz, DMSO-dô) δ 3.42 (t, J = 6.7 Hz, 2H), 2.85 (t, J = 2.7 Hz, 1H), 2.25 (td, J = 7.0, 2.7 Hz, 2H), 1.75 - 1.64 (m, 2H).
Step C: pent-4-yn-l-amine
A solution of the product from Step B (3.9 g, 35.7 mmol, 1 eq) in diethyl ether (40 mL) was cooled to 0 °C, triphenylphosphine (14.1 g, 53.6 mmol, 1,5 eq) was added and the reaction stirred at 0 °C for 6 h. The reaction was quenched by the addition of water (5 mL) and stirred at ambient température overnîght. The mixture was poured onto 4Ν aqueous hydrochloric acid (300 mL) and extracted with diethyl ether (x3). The aqueous phase was basified with portionwîse addition of sodium hydroxide and further extracted with diethyl ether (x2). The combined organic extracts were dried (magnésium sulfate) and concentrated in vacuo to afford the desired product as a yellow oil (1.51 g, 18.16 mmol, 51%).
JH NMR (400 MHz, DMSO-d6) δ 2.73 (t, 1H), 2.58 (t, J = 6.7 Hz, 2H), 2.19 (td, J = 7.2, 2.7 Hz, 2H), 1.55 - 1.44 (m, 2H).
136
Step D: ethyl 2-[(pent-4-yn~l •yl)amino]-l,3-thiazole-4~carboxylate
To a solution of ethyl 2-bromo-l,3-thiazole-4-carboxylate (750 mg, 3.18 mmol, 1 eq) in acetonitrile (15 niL) was added the product from Step C (396 mg, 4.77 mmol, 1.5 eq) and triethylamine (0.66 mL, 4.77 mmol, 1.5 eq) and the mixture was heated at 150 °C for 10 h under microwave irradiation. The réaction was partitioned between ethyl acetate and brine, and the organic phase was dried (MgSO4) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFIash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 — 50% ethyl acetate in Ao-heptane afforded the desired product as a colourless solid (263 mg, 1.1 mmol, 35%).
LC/MS (CijHi4N2O2S) 239 [M+H]+; RT 2.20 (LCMS-V-C)
NMR (400 MHz, DMSO-d6) δ 7.84 (t, J = 5.4 Hz, 1H), 7.51 (s, 1H), 4.22 (q, J = 7.1 Hz, 2FI), 3.28 (td, J = 6.9, 5.4 Hz, 2H), 2.82 (t, J - 2.6 Hz, 1H), 2.25 (td, J = 7.1, 2.7 Hz, 2H), 1.73 (p, J = 7.0 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H).
Step E: ethyl 2-{3-chloro-5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl}-l,3-thiazole-4carboxylate
To a solution of 3,6-dichloro-l,2,4,5-tetrazine (103 mg, 0.68 mmol, 1 eq) in tetrahydrofuran (12 mL) was added the product from Step D (163 mg, 0.68 mmol, 1 eq) and the mixture was heated at 90 C overnight. Purification by automated flash column chromatography (CombiFIash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 70% ethyl acetate in fso-heptane afforded the desired product as an off whîte solid (141 mg, 0.43 mmol, 64%).
LC/MS (Ci2Hi3ClN4O2S) 325 [M+H]+; RT 2.42 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 8.08 (s, 1H), 7.77 - 7.71 (m, 1H), 4.40 - 4.33 (m, 2H), 4.30 (q, 2H), 2.94 (t, J = 6.1 Hz, 2H), 2.11 - 2.00 (m, 2H), 1.32 (t, J = 7.1 Hz, 3H).
Step F: ethyl 2-{3-[(l,3-benzothiazol-2-yl)amino]-5Hi6H,7H,8H-pyrido[2,3-c]pyridaz.in-8yl}-l ,3-thiazole-4-carboxylate
To an oven-dried micro wave vial was added the product from Step E (141 mg, 0.43 mmol, 1 eq), 2-aminobenzothiazole (97.8 mg, 0.65 mmol, 1.5 eq), Xantphos (50.2 mg, 0.09 mmol, 0.2 eq), césium carbonate (283 mg, 0.87 mmol, 2 eq) andl,4-dioxane (15 mL) and the vessel was evacuated and flushed with nitrogen then tris(dibenzyiideneacetone)dipalladium(0) (39.8 mg, 0.04 mmol, 0.1 eq) was added and the mixture was sparged with nitrogen (10 mins) then
137 heated at 150 °C for 2 h under microwave irradiation. The réaction was diluted with ethyl acetate and filtered through celite, washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in iso5 heptane afforded the desired product as a yellow solid (29 mg, 0.07 mmol, 15%).
LC/MS (C2oHi8N602S2) 439 [M+H]+; RT 2.64 (LCMS-V-C) XH NMR (400 MHz, DMSO-d6) δ 11.67 (s, 1H), 8.02 (s, 1H), 7.98 (d, 1H), 7.66 (d, J = 7.9 Hz, 1H), 7.42 (dt, J = 15.0, 7.2 Hz, 1H), 7.35 (s, 1H), 7.23 (t, J = 7.5 Hz, 1H), 4.41 - 4.24 (m, 4H), 2.96 (t, 2H), 2.12 - 2.02 (m, 2H), 1.32 (t, J = 7.1 Hz, 3H).
Step G: 2-{3-[(l,3-benzothiazol-2-yl)amino]-5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl}-l,3thiazole-4-carboxylic acid
To a solution of the product from Step F (29 mg, 0.07 mmol, 1 eq) in 1,4-dioxane (6 mL) was added lithium hydroxide monohydrate (13.9 mg, 0.33 mmol, 5 eq) and the mixture was heated at reflux for 5 h. The reaction was concentrated in vacuo, dissolved in methanol, then loaded 15 onto a methanol-washed PE-ΑΧ cartridge (5 g), washed with methanol, eluted with 9:1 dîchloromethane / formic acid, and concentrated in vacuo. The residue was trilurated dichloromethane, filtered and dried under vacuum to afford the desired product as a cream solid (9.86 mg, 0.02 mmol, 36%), as a formic acid sait.
HRMS-ESI (m/z) [M+H]+ calcd for C18H15N6O2S2: 411.0698, found 411.0722
Example 13: 5-{l-[(Adamantan-l-yl)methyl]-5-methyl-lH-pyrazol-4-yl}-2-{3-[(l,3benzothiazol-2-yI)aminoJ-4-methyl-5HJ677J7//-pyrrolo[2,3-c]pyridazin-7-yl}-l,3-thiazole4-carboxylic acid
138
Step A: ethyl5-{!-[(adamantan-1 -yl) methyl]-5-methy l-l H-pyrazol-4-yl}-2-(4-methy 1-3{[(2Z)-3-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3-henzothiazol-2-ylidene]afnino}5H,6H,7H-pyrrolo]2,3-c]pyridazin-7-yl)-l,3-thiazole-4-carboxylate
To an oven-dried microwave vial was added the product from Préparation 6a (98 mg, 0.15 mmol, 1 eq), the product from Préparation 5a (64.7 mg, 0.18 mmol, 1.2 eq), potassium carbonate (62.7 mg, 0.45 mmol, 3 eq), [1,1'bis(diphenylphosphino)ferroccne]dichloropalladium(II) (11.1 mg, 0.02 mmol, 0.1 eq), tetrahydrofuran (3 mL) and water (1 mL) and the mixture was sparged with nitrogen (10 min) then heated at 120 °C for 1 h under microwave irradiation. The reaction was parti tîoned between ethyl acetate and water, and the organic layer was washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 -50% ethyl acetate in iso-heptane afforded the desired product as a cream solid (57 mg, 0.07 mmol, 47%).
Ή NMR (400 MHz, DMSO-d6) δ 7.78 (d, 1H), 7.56 (s, 1H), 7.48 - 7.38 (ni, 2H), 7.27 - 7.2Ü (m, 1H), 5.85 (s, 2H), 4.37 (t, J = 8.1 Hz, 2H), 4.17 (q, J = 7.1 Hz, 2H), 3.79 (s, 2H), 3.76 3.67 (m, 2H), 3.45 - 3.36 (m, 2H), 2.34 (s, 3H), 2.23 (s, 3H), 2.02 - 1.90 (m, 3H), 1.73 - 1.52 (m, 12H), 1.16 (ί, 3H), 0.96-0.87 (m, 2H), -0.11 (s, 9H).
Step B: ethyl 5-{l-[(adamantan-l-yl)methyl]-5-methyl-lH-pyrazol-4-yl}-2-{3-](l,3benzothiazol-2-yl)amino]-4-methyl-SH,6H, 7H-pyrrolo]2,3-c]pyridazin-7-yl}-l ,3-thiazole-4carboxylate
To a cooled solution of the product from Step A (57 mg, 0.07 mmol, 1 eq) in dichloromethane (6 mL) was added trifluoroacetic acid (0.6 mL) and after 10 min the mixture was allowed to warm to ambient température and stir overnight. The reaction was partitioned between dichloromethane and saturated aqueous sodium bicarbonate, dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in iso-heptane afforded the desired product as a yellow solid (17 mg, 0.03 mmol, 36%).
LC/MS (C35H38N8O2S2) 667 [M+H]+; RT 1.55 (LCMS-V-B2) 3H NMR (400 MHz, DMSO-d6) δ 7.85 (d, J = 7.6 Hz, 1H), 7.62 - 7.44 (m, 2H), 7.42 - 7.31 (m, 1H), 7.20 (t, J = 7.6 Hz, 1H), 4.37 (t, J = 8.1 Hz, 2H), 4.18 (q, J = 7.1 Hz, 2H), 3.80 (s,
139 φ 2Η), 3.34 - 3.24 (m, 2Η), 2.34 (d, J = 3.4 Hz, 3H), 2.24 (s, 3H), 2.02 - 1.93 (m, 3H), 1.74 1.51 (m, 12H), 1.18 (t, 3H).
Step C: S-{l-[(adamantan-l-yl)methyl]-5-methyl-lH-pyrazol-4-yl}-2-{3-[(l,3-benzothiazol2-yl) amino]-4-tnethyl-5H,6H, 7H~pyrrolo[2,3-cJpyridazin -7 -yl}-l ,3-thiazole-4-carboxylic 5 acid
To a solution of the product from Step B (17 mg, 0.03 mmol, 1 eq) in 1,4-dioxane (6 mL) was added lithium hydroxide monohydrate (10.7 mg, 0.25 mmol, 10 eq) and the mixture was heated at reflux for 5 h. The reaction was concentrated in vacuo, dissolved in methanol, loaded onto a methanol-washed PE-ΑΧ cartridge (5 g), washed with methanol, eluted with 10 9:1 dichloromethane l formic acid, and concentrated in vacuo. The residue was triturated with diethyl ether and acetonitrile, filtered and dried under vacuum to afford the desired product as a beige solid (2.4 mg, 3.7 pmol, 15%).
HRMS-ESI (m/z) [M+H]+ calcd for C33H35N8O2S2: 639.2324, found 639.2310
Example 14: 2-{3-[(1,3-Benzothiazol-2-yl)amino]-4-methy7/7-pyrrolo[2,315 c]pyridazin-7-yl}-5-(l-{[l-(3-methoxypropyl)cyclooctyl]methyl}-5-methyl-lH-pyrazol-4yl)-l,3-thiazole-4-carboxylic acid
Step A: ethyl 5-(l-{[l-(3-methoxypropyl)cyclooctyl]methyl}-5-methyl-lH-pyrazol-4-yl)-2-(4methyl-3-{[ (2Z) -3 -{[2-(trimethylsilyl) ethoxyJmethyl}-2,3-dihydro-l,3-benzothiazol-220 ylidene]amino}-5H.6 H, 7H-pyrrolo[2,3-cJpyridazin - 7-yl) -1,3-thiazole~4-carboxylate
To an oven dried microwave vial was added the product from Préparation 6a (34 mg, 0.05 mmol, 1 eq), the product from Préparation 5b (25.5 mg, 0.06 mmol, 1.2 eq), potassium carbonate (21.8 mg, 0.16 mmol, 3 eq), [1,1‘bis(diphenylphosphino)fenOcene]dichloropaHadium(n) (3.84 mg, 0.01 mmol, 0.1 eq), 25 tetrahydrofuran (3 mL) and water (1 mL) and the mixture was sparged with nitrogen (10 min)
140 then heated at 120 °C for 1 h under microwave irradiation. The reaction was partitîoned between ethyl acetate and water, and the organic layer was washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 50% ethyl acetate in iso-heplane afforded the desired product as a white solid (29 mg, 0.03 mmol, 65%).
LC/MS (C43H60N8O4SiS2) 845 [M+H]+; RT 1.79 (LCMS-V-B2)
Ή NMR (400 MHz, DMSO-d6) δ 7.77 (d,l H), 7.58 (s, IH), 7.49 - 7.38 (m, 2H), 7.27 - 7.19 (m, IH), 5.85 (s, 2H), 4.37 (t, J = 8.2 Hz, 2H), 4.17 (q, J = 7.1 Hz, 2H), 3.85 (s, 2H), 3.77 3.66 (m, 2H), 3.45 - 3.34 (m, 2H), 3.31 - 3.26 (m, 4H), 3.23 (s, 3H), 2.33 (s, 3H), 2.22 (s, 3H), 1.74 - 1.48 (m, 8H), 1.47 - 1.20 (m, 8H), 1.18 (t, 3H), 0.96 - 0.87 (m, 2H), -0.11 (s, 9H).
Step B: ethyl 2-{3-[(l,3~benzothiazol-2-yl)amino]-4-methyl-5H,6H, 7H-pyrrolo[2,3c]pyridazin-7-yl}-5-(l -{[ 1 -(3-meihoxypropyl) cyclooctyl]methyl} -5-methyl-lH-pyrazol-4-yl) 1,3-thiazole-4-carboxylate
To a cooled solution of the product from Step A (29 mg, 0.03 mmol, 1 eq) în dichloromethane (5 mL) was added trifluoroacetic acid (0.9 mL) and after 10 min the mixture was allowed to warm to ambient température and stirred overnight. The reaction was partitîoned between dichloromethane and saturated aqueous sodium bicarbonate, dried (PTFE phase separator) and concentrated in vacuo afforded the desired product as a yellow solid (13 mg, 0.02 mmol, 54%).
LC/MS (C37H46N8O3S2) 715 [M+H]+; RT 1.59 (LCMS-V-B2) ’H NMR (400 MHz, DMSO-d6) δ 7.86 (s, IH), 7.59 (br s + s, 2H), 7.37 (t, IH), 7.20 (t, J = 7.6 Hz, IH), 4.37 (t, J = 8.1 Hz, 2H), 4.19 (q, J = 7.0 Hz, 2H), 3.87 (s, 2H), 3.34 - 3.26 (m, 6H), 3.25 (s, 3H), 2.34 (s, 3H), 2.23 (s, 3H), 1.73 - 1.49 (m, 8H), 1.48 - 1.21 (m, 8H), 1.18 (t, 3H).
Step C: 2-{3-[(1,3-benzothiazol-2-yl) amino}-4-methyl-5H,6H, 7H-pyrrolo[2,3-c]pyridazin -7yl}-5-(l -{[1 -(3~methoxypropyl)cyclooctyl]methyl}-5-methyl-lH‘pyrazol~4-yl) -1,3-th tazole-4carboxylic acid
To a solution of the product from Step B (13 mg, 0.02 mmol, 1 eq) in 1,4-dioxane (3 mL) was added lithium hydroxide mono hydrate (11.5 mg, 0.27 mmol, 15 eq) and the mixture was
141 heated at reflux for 5 h. The reaction was concentrated in vacuo, and the residue was triturated with water, filtered and dried under vacuum to afford the desired product as a yellow solid (5.64 mg, 0.01 mmol, 45%), as a lithium sali.
HRMS-ESI (m/z) [M+H]+ calcd for C35H43N8O3S2: 687.2900, found 687.2932
Exampie 15: 2-{3-[(1.3-Benzothiazol-2-yl)amino|-4-inethvl-5//,6//,7//,8//-pyrido[2,3c]pyridazin-8-yl}-l,3-thiazole-4-carboxylic acid
StepA: 2-{3-[(1,3-benzothiazol-2-yl)amino]-4~methyl-5H,6H,7H,8H-pyrîdo[2,3c]pyridazin-8-yl}-l,3-thiazole-4-carboxylic acid
To a solution of the product from Préparation 3f (24 mg, 0.05 mmol, 1 eq) in 1,4-dioxane (6 mL) was added lithium hydroxide monohydrate (33.4 mg, 0.8 mmol, 15 eq) and the mixture was heated at reflux for 7 h. The reaction was concentrated in vacuo, then dissolved in methanol, loaded onto a methanol-wet PE-ΑΧ cartridge (5 g), washed with methanol, eluted with 9:1 dichloromethane / formic acid and concentrated in vacuo. The residue was triturated 15 with dichloromethane, filtered and dried under vacuum to afford the desired product as a beige solid (13.5 mg, 0.03 mmol, 60%), as a formic acid sait.
HRMS-ESI (m/z) [M+H]+ calcd for C19H17N6O2S2: 425.0854, found 425.0845.
Example 16: 2-{3-[(l,3-Benzothiazol-2-yI)amino]-6-methyl-5H,6f/,7L/-pyrrolo[2,3c]pyridazin-7-yl}-l,3-thiazole-4-carboxylic acid
142
Step A: ethyl 2-[(pent-4-yn-2-yl)amino]~l ,3~thiazole-4-carboxylate
To a solution of ethyl 2-bromo-l,3-thiazole-4-carboxylate (1.87 g, 7.93 mmol, 1 eq) in acetonitrile (18 mL) was added pent-4-yn-2-amine (989 mg, 11.9 mmol, 1.5 eq) and 5 triethylamîne (1.66 mL, 11.9 mmol, 1.5 eq) and the mixture was heated at 170 °C in a sealed tube overnight. The reaction was partitîoned between ethyl acetate and brine, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 50% ethyl acetate in iso-heptane afforded the desired product as a yellow oil 10 (555 mg, 2.33 mmol, 29%).
LC/MS (C11H14N2O2S) 239 [M+H]+; RT 2.21 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.85 (d, J = 7.4 Hz, IH), 7.51 (s, IH), 4.27 (q, 2H), 3.91 3.79 (m, IH), 2.89 (t, J = 2.6 Hz, IH), 2.51 - 2.45 (m, IH), 2.44 - 2.41 (m, IH), 1.30 - 1.21 (m, 6H).
Step B: ethyl 2f3‘Chloro-6-methyl-5H,6H,7H-pyrrolo[2,3-c]pyridazin-7-yl}-I,3-thiazole-4carboxylate
To a solution of 3,6-dichIoro-l,2,4,5-tetrazine (352 mg, 2.33 mmol, 1 eq) in tetrahydrofuran (15 mL) was added the product from Step A (555 mg, 2.33 mmol, 1 eq) and the mixture was heated at reflux overnight. The reaction was concentrated in vacuo and purification by 20 automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in Ao-heptane afforded the desired product as a red solid (124 mg, 0.38 mmol, 16%).
LC/MS (C13H13CIN4O2S) 325 [M+H]+; RT 2.39 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 8.12 (s, IH), 7.71 (t, J = 1.6 Hz, IH), 5.11 - 4.97 (m, IH), 25 4.31 (q, J = 7.1, 1.4 Hz, 2H), 3.65 - 3.53 (m, IH), 3.00 - 2.88 (m, IH), 1.50 (d, J = 6.3 Hz,
3H), 1.31 (t, 3H).
143
Step C: ethyl 2-{3-[(l,3-benzothiazol-2-yl)arnino]-6-methyl-5H,6H,7H-pyrrolo[2,3cjpyridazin - 7-yl}-I,3-thiazole-4~carboxylate
To an oven-drîed microwave vial was added the product from Step B (124 mg, 0.38 mmol, 1 eq), 2-aminobenzothiazole (86 mg, 0.57 mmol, 1.5 eq), Xantphos (44.2 mg, 0.08 mmol, 0.2 eq), césium carbonate (249 mg, 0.76 mmol, 2 eq), 1,4-dioxane (4 mL) and tris(dibenzylideneacetone)dipalladium(0) (35 mg, 0.04 mmol, 0.1 eq) and the mixture was sparged with nitrogen (10 mins) then heated at 150 °C for 2 h under microwave irradiation. The reaction was diluted with ethyl acetate and filtered through celite, then washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 — 100% ethyl acetate în iso-heptane afforded a solid that was triturated with diethyl ether, filtered and dried under vacuum to afford the desired product as a beige solid (37 mg, 0.08 mmol, 22%).
LC/MS (C20H18N6O2S2) 439 [M+H]+; RT 2.62 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-dô) δ 11.69 (s, 1H), 8.07 (s, 1H), 7.95 (d, J = 7.9 Hz, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.44 - 7.37 (m, 1H), 7.36 (s, 1H), 7.23 (td, J = 7.6, 1.1 Hz, 1H), 5.07 4.95 (m, 1H), 4.31 (q, 2H), 3.65 - 3.52 (m, 1H), 3.03 - 2.93 (m, 1H), 1.49 (d, J = 6.3 Hz, 3H), 1.32 (t, J =7.1 Hz, 3H).
Step D: 2-{3-[(l,3-benzothiazjol-2~yl)amino]-6-methyl-SH,6H,7H-pyrrolo[2,3-c]pyridazin-720 yl}-J,3-thiazole-4-carboxyUc acid
To a solution of the product from Step C (37 mg, 0.08 mmol, 1 eq) in 1,4-dioxane (8 mL) was added lithium hydroxide monohydrate (53.1 mg, 1.27 mmol, 15 eq) and the mixture was heated at reflux for 7 h. The reaction was concentrated in vacuo, dissolved in methanol, then loaded onto a methanol-wet PE-ΑΧ cartridge (10 g), washed with methanol, eluted with 9:1 dichloromethane / formic acid and concentrated in vacuo. The residue was triturated with dichloromethane, filtered and dried under vacuum to afford the desired product as a beige solid (24.8 mg, 0.06 mmol, 72%), as a formic acid sait.
HRMS-ESI (m/z) [M+H]+ calcd for C18H15N6O2S2: 411.0698, found 411.0695.
144
Example 17: 2-{3-1(1,3-Benzothiazol-2-yl)amino]-4-methy 1-5/7,6//,7W-pyrrolo[2,3c[pyndazin-7-yl}-5-(l-{[l-(3-methoxypropyl)cyclohexyl]methyI}-5-methyl-l//-pyrazol-4yl)-l,3-thiazole-4-carboxylic acid
Step A: ethyl 5-(I-{[I-(3-methoxypropyl)cyclohexyl]methyl}-5-methyl-lH-pyrazol-4-yl)-2(4-methyl-3-{[ (2Z)-3-{[2-(trimethylsilyl) ethoxy /methyl}-2,3-dihydrO'i ,3-benzothiazol-2ylidene]amino}-5H,6H,7H-pyrrolo[2,3-c]pyridazin‘7-yl)-l,3-thiazole-4-carboxylate
To an oven-dried micro wave vial was added the product from Préparation 6a (37 mg, 0.06 mmol, 1 eq), the product from Préparation 5c (25.8 mg, 0.07 mmol, 1.2 eq), potassium carbonate (23.7 mg, 0.17 mmol, 3 eq), [l,r-bîs(diphenyiphosphino)ferrocene] dichloropalladium(II) (4.18 mg, 0.01 mmol, 0.1 eq), tetrahydrofuran (3 mL) and water (1 mL) and the mixture was sparged with nitrogen (10 min) then heated at 120 °C for 1 h under microwave irradiation. The réaction was partitioned between ethyl acetate and water, and the organic phase was washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartrîdge) eluting with a gradient of 0 - 60% ethyl acetate in «o-heptane afforded the desired product as a white solid (22 mg, 0.03 mmol, 47%).
LC/MS (CiiHsôNsCUSiSz) 818 [M+H]+; RT 3.45 (LCMS-V-C) *Η NMR (400 MHz, DMSO-d6) δ 7.79 - 7.74 (m, IH), 7.58 (s, IH), 7.48 - 7.38 (m, 2H), 7.27 - 7.20 (m, IH), 5.85 (s, 2H), 4.37 (t, J = 8.1 Hz, 2H), 4.17 (q, J = 7.1 Hz, 2H), 3.93 (s, 2H), 3.76 - 3.66 (m, 2H), 3.44 - 3.36 (m, 2H), 3.34 - 3.25 (m, 2H), 3.23 (s, 3H), 2.33 (s, 3H), 2.23 (s, 3H), 1.60- 1.27 (m, 14H), 1.18 (t, J = 7.1 Hz, 3H), 0.98 - 0.85 (m, 2H), -0.11 (s, 9H).
Step B: ethyl 2-{3-[(1,3-beuzothiazol-2-yl)(iiiiÎ!iol-4-inc’lhyl-5ll,6H,7H-pyri<)lo}2,3c]pyridazin-7-yl} -5-(l-{[l-(3-methoxypropyl) cycloh exyl]methyl}-5-methyl-lH-pyrazol-4-yl) 1,3-thiazole-4-carboxylate
To a cooled solution of the product from Step A (22 mg, 0 mol, 1 eq) în dichloromethane (5 mL) was added trifluoroacetic acid (1.5 mL) and after 10 min the mixture was allowed to
145 wann to ambient température and stir overnight. The réaction was partitioned between dichloromethane and saturated aqueous sodium bicarbonate, dried (PTFE phase separalor) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 100% ethyl 5 acetate in Eo-heptane afforded the desired product as a yellow solid (10 mg, 0.01 mmol, 54%).
LC/MS (C35H42N8O3S2) 688 [M+H]+; RT 3.02 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 7.91 - 7.79 (m, 1H), 7.59 (br s + s, 2H), 7.38 (t, J - 7.7 Hz, 1H), 7.25 - 7.13 (m, 1H), 4.38 (t, J = 8.2 Hz, 2H), 4.19 (q, J = 7.0 Hz, 2H), 3.95 (s, 2H), 10 3.34 - 3.27 (m, 4H), 3.25 (s, 3H), 2.34 (s, 3H), 2.24 (s, 3H), 1.60 - 1.45 (m, 6H), 1.44 - 1.29 (m, 6H), 1.28 - 1.22 (m, 2H), 1.18 (t, 3H).
Step C: 2-{3-[(l,3-benzothiawl-2-yl)amino]-4-methyl-5H,6H,7H-pyrrolo[2,3-c]pyridazin-7yl}-5-(l-{[i-(3-methoxypropyl)cyclohexyl]methyl}-5-methyl-lH-pyrazrol-4-yl)-l,3-thiazole‘4· carboxylic acid
To a solution of the product from Step B (10 mg, 0.01 mmol, 1 eq) in 1,4-dioxane (3 mL) was added lithium hydroxide monohydrate (12.2 mg, 0.29 mmol, 20 eq) and the mixture was heated at reflux for 5 h. The reaction was concentrated in vacuo, triturated with water, filtered and dried under vacuum to afford the desired product as a yellow solid (5.71 mg, 0.01 mmol, 60%).
HRMS-ESI (m/z) [M+H]+ calcd for C33H39N8O3S2: 659.2587, found 659.2577.
Example 18: 2-{4-Methyl-3-[(l,3-thiazol-2-yl)amino]-5//,6/Z,7/7-pyrrolo[2,3c]pyridazin-7-yl}-l,3-thiazole-4-carboxylic acid
146
Step A: ethyl 2-{4-methyl-3-[(l,3-thiazol-2-yl)amino]-5H,6H,7H-pyrrolo[2t3-c]pyridazin-7yl}-1,3-thiazole-4-carboxylate
To an oven-dried microwave vial was added the product from Préparation 6a, Step B (100 mg, 0.31 mmol, 1 eq), 2-aminothiazoie (46.3 mg, 0.46 mmol, 1.5 eq), Xantphos (35.6 mg, 0.06 mmol, 0.2 eq), césium carbonate (201 mg, 0.62 mmol, 2 eq), 1,4-dioxane (4 mL) and tris(dibenzylideneacetone)dipailadium(0) (28.2 mg, 0.03 mmol, 0.1 eq) and the mixture was sparged with nitrogen (10 min) then heated at 150 °C for 1 h under microwave irradiation. The reaction was diluted with ethyl acetate, filtered through celite, washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% methanol in dichloromethane gave a solid that was trîturated with acetonitrile, filtered and dried under vacuum to afford the desired product as a yellow solid (23 mg, 0.06 mmol, 19%).
LC/MS (CiôHiôNcOzSz) 389 [M+H]+; RT 2.29 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 10.57 (br s, 1H), 8.04 (s, 1H), 7.44 (br s, 1H), 7.06 (br s, JH), 4.40 - 4.32 (m, 2H), 4.29 (q, 2H), 3.30 - 3.22 (m, 2H), 2.31 (s, 3H), 1.32 (t, 3H).
Step B : 2-{4-meth yl-3-f (1,3~thiazol-2-vl)amino]-5H,6H, 7H-pyrrolo[2,3-c]pyridazin · 7-yl}1,3-thiazole-4-carboxylic acid
To a solution of the product from Step A (23 mg, 0.06 mmol, 1 eq) in 1,4-dioxane (6 mL) was added lithium hydroxide monohydrate (37.3 mg, 0.89 mmol, 15 eq) and the mixture was heated at reflux for 5 h. The reaction was concentrated in vacuo, dissolved in methanol, loaded onto a methanol-wet PE-ΑΧ cartridge (5 g), washed with methanol, eiuted with 9:1 dichloromethane / formic acid and concentrated in vacuo. The residue was trîturated with dichloromethane and methanol, filtered and dried under vacuum to afford the desired product as a beige solid (8.84 mg, 0.02 mmol, 41%).
HRMS-ESI (m/z) [M+H]+ calcd for C14H13N6O2S2: 361.0541, found 361.0531.
Example 19: 2-{3-[(4,5-Dimethyl-l,3-thiazol-2-y])amino]-4-methyl-5Æ,6/f,7Efpyrrolo[2,3-c]pyridazin-7-yl}-l,3-thiazole-4-carboxylic acid
147
Step A: ethyl2-{3-[(4,5-dimethyl-l,3-thiazol-2-yl)amino]-4-methyl-5H,6H,7H-pyrrolo[2,3c]pyridaz,in-7-yl}-l,3-thiazole-4-carboxylate
To an oven-drîed microwave vial was added the product from Préparation 6a, Step B (100 5 mg, 0.31 mmol, 1 eq), 4,5-dimethyl-l,3-thiazol-2-amine (59.2 mg, 0.46 mmol, 1.5 eq), Xantphos (35.6 mg, 0.06 mmol, 0.2 eq), césium carbonate (201 mg, 0.62 mmol, 2 eq), 1,4-dioxane (3 mL) then tris(dibenzylideneacetone)dipalladium(0) (28.2 mg, 0.03 mmol, 0.1 eq) and the mixture was sparged with nitrogen (10 mins) then heated at 150 °C for 1 h under microwave irradiation. The reaction was diluted with ethyl acetate and filtered through celite, 10 then washed with brine, dried (magnésium sulfate) and concentrated in vacuo. The residue was triturated with methanol, filtered and dried under vacuum to afford the desired product as a yellow solid (64 mg, 0.15 mmol, 50%).
LC/MS (Ci8H2oN602S2) 417 [M+H]+; RT 2.42 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 8.03 (s, 1H), 4.39 - 4.21 (m, 4H), 3.25 (t, J = 8.0 Hz, 2H), 15 2.27 (s, 3H), 2.23 (s, 3H), 2.16 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H).
Step B: 2-{3-[(4,5-dimethyl~l,3-thiazol-2'yl)amino]-4-methyl-5H,6H!7H-pyrrolo[2,3· c]pyndazin-7-yl}‘l,3~thiazole‘4-carboxylic acid
To a solution of the product from Step A (64 mg, 0.15 mmol, 1 eq) in 1,4-dioxane (15 mL) was added lithium hydroxide monohydrate (96.7 mg, 2.3 mmol, 15 eq) and the mixture 20 was heated at reflux for 5 h. The reaction was concentrated in vacuo, dissolved in methanol, then loaded onto a methanol-wet PE-ΑΧ cartridge (10 g), washed with methanol, eluted with 9:1 dichloromethane / formic acid and concentrated in vacuo. The residue was triturated with methanol, filtered and dried under vacuum to afford the desired product as a beige solid (17.9 mg, 0.05 mmol, 30%).
HRMS-ESI (m/z) [M+H]+ calcd for Ci6Hi7N6O2S2: 389.0854, found 389.0847.
148
Example 20: 6-{3-[(l,3-Benzothiazol-2-yl)aminol-4-methyl-5//,6F/,7//-pyrrolo[2,3c]pyridazin-7-yl}pyricline-2-carbûxylic acid
Step A: tert-butyl 6-[(pent~3-yn-l-yl)amino]pyridine-2-carboxylate
To a solution of teri-butyl 6-fluoropyridine-2-carboxylate (219 mg, 1.11 mmol, 1 eq) in dimethylacetamide (5 mL) was added pent-3-yn-l-amine hydrochloride (133 mg, 1.11 mmol, 1 eq) and A,A-diisopropyleihylamine (0.39 mL, 2.22 mmol, 2 eq) and the mixture was heated at 120 °C overnight in a sealed tube. The reaction was partitioned between ethyl acetate and water, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo.
Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 30% ethyl acetate in wo-heptane afforded the desired product as a clear oil (48 mg, 0.18 mmol, 17%).
LC/MS (C15H20N2O2) 261 [M+H]+; RT 2.42 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.49 (dd, J = 8.4, 7.2 Hz, 1H), 7.11 (dd, J = 7.3, 0.8 Hz, 15 1H), 6.89 (t, J = 5.7 Hz, 1H), 6.66 (dd, J = 8.5, 0.8 Hz, 1H), 3.45 - 3.29 (m, 2H), 2.44 - 2.35 (m, 2H), 1.75 (t, J = 2.6 Hz, 3H), 1.53 (s, 9H).
Step B: tert-butyl 6-{3‘Chloro-4-methyl-5H,6H,7H-pyrrolo[2,3-c]pyridazin-7-yl}pyridine-2carboxylate
To a solution of 3,6-dichloro-l,2,4,5-tetrazine (27.8 mg, 0.18 mmol, 1 eq) in tetrahydrofuran 20 (3 mL) was added the product from Step A (48 mg, 0.18 mmol, 1 eq) and the mixture was heated at 110 °C for 1 h under microwave irradiation. The reaction was concentrated in vacuo and purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 30% ethyl acetate in iso-heptane afforded the desired product as a pink solid (12 mg, 0.03 mmol, 19%).
LC/MS (C17H19CIN4O2) 347 [M+H]+; RT 2.67 (LCMS-V-C)
149 • lH NMR (400 MHz, DMSO-d6) δ 8.84 (dd, J = 8.6, 0.8 Hz, 1H), 8.01 (dd, J = 8.6, 7.4 Hz, 1H), 7.65 (dd, J = 7.4, 0.8 Hz, 1H), 4.36 (dd, J = 8.9, 7.8 Hz, 2H), 3.22 (t, J = 8.3 Hz, 2H), 2.27 (s, 3H), 1.57 (s, 9H).
Step C: tert-butyl 6-{3-[(l,3-benzothiazol-2-yl)amino]-4-niethyl-5H,6H,7H-pyrrolo[2,35 cjpyridazin - 7-yl}pyridine-2-carboxylate
To an oven-drîed microwave vial was added the product from Step B (26 mg, 0.07 mmol, 1 eq), 2-aminothiazole (16.9 mg, 0.11 mmol, 1.5 eq), Xantphos (8.68 mg, 0.01 mmol, 0.2 eq), césium carbonate (48.9 mg, 0.15 mmol, 2 eq), 1,4-dioxane (4 mL) then tris(dibenzylîdeneacelone)dipalladium(0) (6.87 mg, 0.01 mmol, 0.1 eq) and the mixture was 10 sparged with nitrogen (10 mins) then heated at 150 °C for 1 h under microwave irradiation.
The reaction was diluted with ethyl acetate, filtered through celite, washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 40% ethyl acetate in rso-heptane gave a solid that was triturated with diethyl ether, filtered 15 and dried under vacuum to afford the desired product as a yellow solid (7 mg, 0.02 mmol, 20%).
LC/MS (C24H24N6O2S) 461 [M+H]+; RT 2.69 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 8.98 (d, J = 8.7 Hz, 1H), 8.01 (t, J = 8.0 Hz, 1H), 7.88 (s, 1H), 7.68 - 7.57 (m, 2H), 7.38 (t, J = 7.5 Hz, 1H), 7.28 - 7.14 (m, 1H), 4.32 (t, J = 8.2 Hz, 20 2H), 3.20 (t, J = 8.1 Hz, 2H), 2.33 (s, 3H), 1.58 (s, 8H).
Step D: 6-{3-[(1,3-benzothiazol-2-yl)amino]-4-methyl~5H,6H,7H-pyrrolo[2,3-c]pyridazin-7yl}pyridine-2-carboxylic acid
To a solution of the product from Step C (7 mg, 0.02 mmol, 1 eq) in 1,4-dioxane (3 mL) was added lithium hydroxide monohydrate (9.57 mg, 0.23 mmol, 15 eq) and the mixture was 25 heated at reflux for 6 h. The reaction was concentrated in vacuo, then dissolved in methanol, loaded onto a methanol-wet PE-ΑΧ cartridge (5 g), washed with methanol, eluted with 9:1 dichloromethane / formic acid and concentrated in vacuo. The residue was triturated with dichloromethane and methanol, filtered and dried under vacuum to afford the desired product as a yellow solid (2.0 mg, 32%).
HRMS-ESI (m/z) [M+H]+ calcd for C2oHi7N602S: 405.1134, found 405.1122.
150 φ Exampie 21: 6-{3-[(1,3-Benzothiazol-2-yl)amino]-4-methyl-5/Z,6//,ΊΗ,8/f-pyrido[2,3c]pyridazin-8-yl}pyridine-2-carboxylic acid
StepA: ethyl6-{[3-(3,6-di€hloro-5-methylpyridazin-4-yl)propyl]amino}pyridine-25 carboxylate
To a solution of the product from Préparation 2e (500 mg, 2.28 mmol, 1 eq) and ethyl 6aminopicolinate (455 mg, 2.74 mmol, 1.2 eq) in methanol (18 mL) and acetic acid (6 mL) was added sodium triacetoxyborohydride (968 mg, 4.56 mmol, 2 eq) and the mixture was stirred at ambient température for 16 h. The reaction was quenched by the addition of IN aqueous 10 sodium hydroxide (50 mL), extracted with ethyl acetate (3 x 50 mL), and the combined organic extracts were successîvely washed with saturated aqueous sodium bicarbonate and brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 60% ethyl acetate in Δο-heptane afforded the desired product as a white solid 15 (400 mg, L08 mmol, 47%).
LC/MS (C16H18CI2N4O2) 369 [M+H]+; RT 1.19 (LCMS-V-B1)
Ή NMR (400 MHz, DMSO-d6) δ 7.52 (dd, J = 8.4, 7.2 Hz, IH), 7.18 (dd, IH), 6.99 (t, J = 5.7 Hz, IH), 6.69 (dd, J = 8.5, 0.8 Hz, IH), 4.27 (q, J = 7.1 Hz, 2H), 3.42 - 3.35 (m, 2H), 2.92 - 2.82 (m, 2H), 2.41 (s, 3H), 1.87 - 1.74 (m, 2H), 1.29 (t, J = 7.1 Hz, 3H).
Step B: methyl 6-{3-chloro-4-methyl-5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl}pyridine-2carboxylate
To a solution of the product from Step A (170 mg, 0.46 mmol, 1 eq) in alpha,alpha,alphatrifluorotoluene (4 mL) was added césium carbonate (300 mg, 0.92 mmol, 2 eq) and the mixture was heated in a sealed tube al 160 °C for 5 days. The reaction was ailowed to cool to 25 ambient température and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0
151
- 10% methanol in dichloromethane afforded the desired product as a cream solid (74 mg,
0.23 mmol, 50%).
LC/MS (C15H15CIN4O2) 319 [M+H]+; RT 1.10 (LCMS-V-B1)
Ή NMR (400 MHz, DMSO-d6) δ 7.97 - 7.84 (m, 2H), 7.75 (dd, J = 7.1, 1.2 Hz, 1H), 4.05 3.96 (m, 2H), 3.89 (s, 3H), 2.92 - 2.81 (m, 2H), 2.31 (s, 3H), 2.05 - 1.92 (m, 2H).
Step C: methyl 6-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,3cjpyridazin-8-yl}pyridine~2-carboxylate
To a solution of the product from Step B (74 mg, 0.23 mmol, 1 eq), 2-aminobenzothiazole, (52.3 mg, 0.35 mmol, 1.5 eq) and Al/V-diisopropyiethylamine (0.12 mL, 0.7 mmol, 3 eq) in 1,4-dioxane (5 mL) was added Xantphos (13.4 mg, 0.02 mmol, 0.1 eq) and tris(dibenzylideneacetone)dipalladium(O) (10.6 mg, 0.01 mmol, 0.05 eq) and the mixture was heated in a sealed tube at 150 °C for 20 h. The reaction was allowed to cool to ambient température, then partitioned between ethyl acetate (20 mL) and brine (25 mL), and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in tso-heptane afforded the desired product as a yellow solid (20 mg, 0.05 mmol, 20%).
LC/MS (C22H20N6O2S) 433 [M+H]+; RT 1.15 (LCMS-V-B1) *H NMR (400 MHz, DMSO-d6) δ 8.03 (dd, J = 8.5, 0.9 Hz, 1H), 7.87 (dd, J = 8.5, 7.3 Hz, 1H), 7.83 (br s, 1H), 7.68 (d, J = 7.3 Hz, 1H), 7.53 (br s, J H), 7.36 (t, J = 7.6 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 4.11 - 3.99 (m, 2H), 3.90 (s, 3H), 2.86 (t, J = 6.5 Hz, 2H), 2.33 (s, 3H), 2.05 -1.94 (m,2H).
Step D: 6-(3-/(1,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,3c]pyridazin-8-yl}pyridine-2-carboxylic acid
To a solution of the product from Step C (15 mg, 0.03 mmol, 1 eq) în 1,4-dioxane (2 mL) was added lithium hydroxide monohydrate (2.91 mg, 0.07 mmol, 2 eq) and the mixture was heated at reflux for 1 h. The reaction was allowed to cool to ambient température and concentrated in vacuo. Purification by reverse phase automated flash chromatography (CombiFlash Rf, Cl 8 4.3g RediSep column) eluting with a gradient of 5 - 95% acetonitrile in water afforded the desired product as a cream solid (10 mg, 0.02 mmol, 69%).
HRMS-ESI (m/z) [M+H]+ calcd for C21H19N6O2S: 419.1290, found 419.1287.
152
Example 22: 2-{3-[(1,3-Benzothiazol-2-yl)amino]-4-methyl-5H,6H,7/Z-pyrrolo[2,3c]pyridazin-7-yl}-5-(3-{4-[3-(dimethylamino)prop-l-yn-l-yl]-2-flnorophenoxy}propyl)l,3-thiazole~4-carboxylic acid
Step A: ethyl5-[(lE)-3-[(tert-butyldimethylsilyl)oxy]prop-l-en-l-yl]-2-(4-methyl-3-{[(2Z)-3{[2-(trimethylsilyl)ethoxy]methyl}~2,3-dihydro~l,3-benzothiazol-2-ylidene]amino}5H,6H, 7H-pyrrolo[2,3-c]pyridazin -7-yl) -1,3-thiazole-4-carboxylate
To an oven-dried sealed flask was added the product from Préparation 6a (3 g, 4.63 mmol, 1 eq), (£)-3-(ierr-butyldimethyIsilyloxy)propene-l-yl-boronic acid pinacol ester (1.82 mL, 5.56 mmol, 1.2 eq), potassium carbonate (1.92 g, 13.9 mmol, 3 eq), [Ι,Γbis(diphenylphosphino)ferrocene]dichloropalladium(n) (339 mg, 0.46 mmol, 0.1 eq), tetrahydrofuran (150 mL) and water (50 mL) and the mixture was sparged with nitrogen (10 min) then heated at 120 °C for 1.5 h. The reaction was allowed to cool to ambient température then partitioned between ethyl acetate and water, and the organic phase washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 80 g RediSep™ silica cartridge) eluting with a gradient of 0 - 70% ethyl acetate în iso-heptane afforded the desired product as a cream solid (1.86 g, 2.52 mmol, 54%).
LC/MS (C35H5oN604Si2S2) 739 [M+H]+; RT 3.69 (Shortneg2) XH NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 7.6 Hz, 1H), 7.55 - 7.38 (m, 3H), 7.30 - 7.20 (m, 1H), 6.30 (dt, J = 15.9, 4.3 Hz, 1H), 5.85 (s, 2H), 4.41 - 4.26 (m, 4H), 3.77 - 3.67 (m, 2H), 3.45 - 3.20 (m, 4H), 2.32 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H), 0.93 (s, 9H), 0.92 - 0.86 (m, 2H), 0.11 (s, 6H), -0.11 (s, 9H).
153
Step B: ethyl 5-{3-[(tert-butyldiinethylsilyl)oxy]propyl}-2-(4-methyl-3-{[(2Z)-3-{[2(trünethylsilyl)ethoxy]methyl}-2,3-dihydro-I,3-benzothiazol-2-ylidene]ainino}~5H,6H,7Hpyrrolo[2,3-c]pyridaziu-7-yl)-l,3-thiazole-4-earboxylate
To a solution of the product from Step A (900 mg, 1.22 mmol, 1 eq) in ethyl acetate (600 5 mL) was added a catalytic amount of platinum (IV) oxide under a nitrogen atmosphère. The mixture was evacuated and backfilled with nitrogen (x3), then evacuated and backfilled with hydrogen and shaken for 3 days at ambient température under an atmosphère of hydrogen. The reaction was filtered through celite, eluted with ethyl acetate and evaporated under reduced pressure to afford the desired product as a beige solid (950 mg, 1.28 mmol, 10 >100%%).
LC/MS (C35H52N6O4S12S2) 741 [M+H]+; RT 1.88 (LCMS-V-B2) *H NMR (400 MHz, DMSO-d6) δ 7.77 (d, J = 7.6 Hz, 1H), 7.50 - 7.39 (m, 2H), 7.28 - 7.18 (m, 1H), 5.85 (s, 2H), 4.37 - 4.22 (m, 4H), 3.77 - 3.62 (m, 4H), 3.31 - 3.14 (m, 4H), 2.32 (s, 3H), 1.93 - 1.80 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H), 0.96-0.80 (m, 11H), 0.06 (s, 6H), -0.11 (s, 15 9H).
Step C: ethyl 5-(3-hydroxypropyl)-2-(4-methyl-3-{[(2Z)-3f[2-(trimethylsilyl)ethoxylmethyl}2,3-dihydro-l,3-benzothiazol-2-ylidene]amino}-5H,6H,7H-pyrrolo[2,3-c]pyridazin-7-yl)1,3-thiazole-4-carboxylate
To a solution of the product from Step B (950 mg, 1.28 mmol, 1 eq) in 1,4-dioxane (150 20 mL) was added hydrochloric acid (4M in dioxane; 50 mL, 200 mmol, 156 eq) and the mixture was stirred at ambient température for 1 h. The reaction was partitioned between ethyl acetate and saturated aqueous sodium bicarbonate, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFIash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 80% ethyl acetate in Ao-heplane 25 afforded the desired product as an off-white solid (577 mg, 0.92 mmol, 72%).
LC/MS (CzoHssNôChSiSz) 627 [M+H]+; RT 2.68 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 7.5 Hz, 1H), 7.49 - 7.38 (m, 2H), 7.28 - 7.19 (m, 1H), 5.85 (s, 2H), 4.57 (t, J = 5.2 Hz, 1H), 4.37 - 4.22 (m, 4H), 3.76 - 3.67 (m, 2H), 3.53 3.44 (m, 2H), 3.30 - 3.13 (m, 4H), 2.32 (s, 3H), 1.86 - 1.77 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H), 30 0.96 - 0.86 (m, 2H), -0.11 (s, 9H).
154
Step D: ethyl 5-(3-chloropropyl)-2-(4-methyl-3-{[(2Z)-3-{[2-(trimethylsilyl)ethoxy]methyl}2,3-dihydro-l ,3-benzothiazol-2-ylidene]amino)-5H,6H,7H-pyrrolof2,3-c]pyrîdazin-7-yl)l,3-thiazole-4-carboxylate
The product from Step C (577 mg, 0.92 mmol, 1 eq) was dîssolved in thionyl chloride (30 mL) and stirred at ambient température for 5 h. The reaction was concentrated in vacuo, then partitioned between dîchioromethane and brine, and the organic phase was dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 50% ethyl acetate in iso-heptane afforded the desired product as a beige solid (341 mg, 0.53 mmol, 57%).
LC/MS (C29H37ClN6O3SiS2) 645 [M+H]+; RT 2.91 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 7.80 (d, IH), 7.50 - 7.39 (m, 2H), 7.29 - 7.18 (m, IH), 5.85 (s, 2H), 4.38 - 4.22 (m, 4H), 3.78 - 3.67 (m, 4H), 3.30 - 3.21 (m, 2H), 2.32 (s, 3H), 2.20 - 2.06 (m, 2H), 1.31 (t, J = 7.1 Hz, 2H), 0.97 - 0.86 (m, 2H), -0.11 (s, 9H).
Step E: ethyl 5-(3-{4-[3-(dimethylamino)prop-l-y n-l-yl]-2-fluorophenoxy}propyl)-2-(4methyl-3-{[(2Z)-3-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3-benzothiazol-2ylidene]amino}-5H,6H, 7H-pyrrolo[2,3-c]pyridazin-7-yl)-l,3-thiazole-4-carboxylate
To an oven dried sealed flask was added the product from Préparation 4b (133 mg, 0.69 mmol, 1.3 eq) in dnnethylformamide (70 mL). Sodium hydrîde (60% dispersion; 52.8 mg, 1.32 mmol, 2.5 eq) was added to the solution and the mixture stirred for 2 min. A solution of the product from Step D (341 mg, 0.53 mmol, 1 eq) in dimethylformamide (30 mL) was added and the mixture was sparged with nitrogen (10 min) and heated at 100 °C for 1 h. The reaction was partitioned between ethyl acetate and water, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 7% methanol in dîchioromethane afforded the desired product as a beige solid (203 mg, 0.25 mmol, 48%).
LC/MS (C40H48FN7O4S1S2) 802 [M+H]+; RT 2.59 (LCMS-V-C) rH NMR (400 MHz, DMSO-d6) δ 7.79 (d, IH), 7.47 - 7.38 (m, 2H), 7.31 (dd, IH), 7.27 7.11 (m, 3H), 5.84 (s, 2H), 4.36 - 4.20 (m, 4H), 4.13 (t, 2H), 3.75 - 3.66 (m, 2H), 3.39 (s, 2H), 3.31 - 3.19 (m, 4H), 2.30 (s, 3H), 2.19 (s, 6H), 2.18 -2.09 (m, 2H), 1.28 (t, 3H), 0.95 0.84 (m, 2H), -0.11 (s, 9H).
155 φ Step F: ethyl 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H, 7H~pyrrolo[2,3cjpyrldazin - 7-yl}-5-(3-{4-[3fdimethylamino)prop-l -yn -1 -yl]-2-fluorophenoxy}propyl) -1,3thiazole-4-carboxylate
To a solution of the product from Step E (203 mg, 0.25 mmol, 1 eq) in dichioromethane (10 5 mL) was added trifluoroacetic acid (5.0 mL, 65.8 mmol, 260 eq) and the mixture was stirred at rt for 6 h. The reaction was diluted with dichioromethane, cooled to 0 °C and neutralised by the addition of 2M aqueous sodium hydroxide. The organic phase was dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ siiica cartridge) eluting with a gradient of 0 10 - 5% methanol in dichioromethane afforded the desired product as a yellow solid (114 mg,
0.17 mmol, 67%).
LC/MS (C34H34FN7O3S2) 672 [M+H]+; RT 2.04 (LCMS-V-C)
NMR (400 MHz, DMSO-d6) δ 11.36 (br s, 1H), 7.89 (d, J = 7.9 Hz, 1H), 7.58 - 7.47 (m, 1H), 7.43 - 7.28 (m, 2H), 7.26 - 7.12 (m, 3H), 4.33 (t, 2H), 4.27 (q, 2H), 4.15 (t, J = 6.1 Hz, 15 2H), 3.40 (s, 2H), 3.34 - 3.22 (m, 4H), 2.33 (s, 3H), 2.21 (s, 6H), 2.16 (t, 2H), 1.29 (t, J = 7.1
Hz, 3H).
Step G: 2--/3-/(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H-pyrrolo[2,3-c]pyridazin-7yl}-5-(3-{4-[3-(dimethylamino)prop-l -yn-1 -yl]-2-fluoroph enoxy}propyl)-1,3-thiazole-4carboxylic acid
To a solution of the product from Step F (114 mg, 0.17 mmol, 1 eq) in 1,4-dioxane (15 mL) was added lithium hydroxide mono hydrate (71.2 mg, 1.7 mmol, 10 eq) and the mixture was heated at reflux for 7 h. The reaction was concentrated in vacuo, and the residue was triturated with water and acetonitrile, filtered and dried under vacuum to afford the desired product as a yellow solid (64.7 mg, 0.1 mmol, 59%), as a lithium sait.
HRMS-ESI (m/z) [M+H]+ calcd for C32H31FN7O3S2: 644.1914, found 644.1908.
Example 23: 3-{l-[(Adamantan-l-yl)methyl]-5-methyl-l/f-pyrazol-4-yl}-6-{3-[(l,3bcnzothiazol-2-yDamino|-4-mcthyl-7//-pyriOlo|2,3-c|pyridazin-7-yl}pyridine-2carboxylic acid
156
Step A: tert-butyl N-(pent-3-yn-l -yljcarbamate
To a solution of pent-3-yn-I-amine hydrochloride (5 g, 41.8 mmol, 1 eq) in tetrahydrofuran (130 mL) and water (130 mL) was added sodium bicarbonate (10.5 g, 125 mmol, 3 eq), 5 followed by di-teri-butyl dicarbonate (9.12 g, 41.8 mmol, 1 eq) and the mixture was stirred at ambient température overnight. The reaction was diiuted with ethyl acetate, successively washed with saturated aqueous sodium bicarbonate and brine, dried (magnésium sulfate) and concentrated in vacuo to afford the desired product as a yellow oil (8.2 g, 44.8 mmol, >100%).
Ή NMR (400 MHz, DMSO-d6) δ 6.89 (t, J = 5.9 Hz, 1H), 2.99 (td, J = 7.3, 5.9 Hz, 2H), 2.25 - 2.15 (m, 2H), 1.73 (t, J = 2.6 Hz, 3H), 1.38 (s, 9H).
Step B: ethyl 3-bromo-6-{[(tert-butoxy)carbonyl](pent-3-yn-l-yl)amino}pyridine-2carboxylate
To an oven-dried sealed flask was added the product from Step A (8.2 g, 44.8 mmol, 1 15 eq), ethyl 3,6-dibromopicolmate (13.8 g, 44.8 mmol, 1 eq), Xantphos (2.59 g, 4.47 mmol, 0.1 eq), césium carbonate (29.2 g, 89.5 mmol, 2 eq) and 1,4-dioxane (180 mL) . The vessel was evacuated and flushed with nitrogen then tris(dibenzylideneacetone)dipalladium(0) (2.05 g, 2.24 mmol, 0.05 eq) was added and the mixture was sparged with nitrogen (10 mins) then heated at 130 °C for 1 h. The reaction was diiuted with ethyl acetate and filtered through 20 celite, then successively washed with water and brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromalography (CombiFlash Rf, 330 g RediSep™ silica cartridge) eluting with a gradient of 0 - 8% ethyl acelate in isoheplane afforded the desired product as a yellow oil (9.95 g, 24.2 mmol, 54%).
LC/MS (Ci8H23BrN2O4) 357 [Μ-Έυ]+; RT 2.59 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 8.14 (d, J = 8.9 Hz, 1H), 7.71 (d, J = 8.9 Hz, 1H), 4.38 (q, J = 7.1 Hz, 2H), 3.96 - 3.87 (m, 2H), 2.49 - 2.38 (m, 2H), 1.66 (t, J = 2.5 Hz, 3H), 1.48 (s, 9H), 1.33 (t, J = 7.1 Hz, 3H).
157
Step C: ethyl 3-bromo-6-[(pent-3-yn-l -yl)amino]pyridine-2-carboxylate
To a solution of the product from Step B (9.95 g, 24.2 mmol, 1 eq) in dichloromethane (120 mL) was added trifluoroacetic acid (19.9 mL, 260 mmol, 10.8 eq) and the mixture was stirred at ambient overnight. The réaction was diluted with dichloromethane, cooled to 0 °C and 5 neutralised by the addition of 4M aqueous sodium hydroxîde. The organic phase was dried (phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 220 g RediSep™ silica cartridge) eluting with a gradient of 0 - 30% ethyl acetate in zso-heptane afforded the desired product as a yellow oil (6.68 g, 21.5 mmol, 89%).
LC/MS (Ci3Hi5BrN2O2) 313 [M+H]+; RT 2.12 (LCMS-V-C)
Ή NMR (400 MHz, DMSOM6) δ 7.63 (d, J = 9.0 Hz, 1H), 7.2Û (t, J = 5.8 Hz, 1H), 6.58 (d, J = 9.0 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 3.29 (td, J - 7.1, 5.7 Hz, 2H), 2.41 - 2.29 (m, 2H), 1.74 (t, J = 2.6 Hz, 3H), 1.30 (t, J = 7.1 Hz, 3H).
Step D: ethyl 3~bromo-6-{3‘chloro-4-methyl-7H-pyrrolo[2,3-c]pyridazin-7-yl}pyridine-215 carboxylate
To a solution of the product from Step C (6.68 g, 21.5 mmol, 1 eq) in 1,4-dioxane (220 mL) was added 3,6-dichloro-l,2,4,5-tetrazine (6.48 g, 42.9 mmol, 2 eq) and the mixture was heated in a sealed flask at 120 °C for 72 h. The reaction was diluted with methanol, filtered through a phase separator and concentrated in vacuo. Purification by automated flash column 20 chromatography (CombiFlash Rf, 120 g RediSep™ silica cartridge) eluting with a gradient of 0 - 40% ethyl acetate in Tso-heptane afforded the desired product as a red solid (1.49 g, 3.77 mmol, 18%).
LC/MS (Ci5H12BrClN4O2) 397 [M+HJ+; RT 2.36 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 8.93 (d, J = 8.9 Hz, 1H), 8.63 (d, J = 3.9 Hz, IH), 8.53 (d, 25 J = 8.9 Hz, 1H), 7.11 (d, J = 3.9 Hz, 1H), 4.45 (q, J = 7.1 Hz, 2H), 2.64 (s, 3H), 1.37 (t, 3H).
Step E: ethyl 3-{l-[(adamantan-l-yl)methyl]-5-methyl-lH-pyrazol-4-yl}-6~{3-chloro-4methyl-7H-pyrrolo[2,3-c]pyridazin-7-yl}pyridine-2-carboxylate
To a solution of the product from Step D (1.49 g, 3.77 mmol, 1 eq) in tetrahydrofuran (5 mL) and water (15 mL) was added the product from Préparation 5a (1.48 g, 4.14 mmol, 1.1 30 eq) and potassium carbonate (1.56 g, 11.3 mmol, 3 eq). The vessel was evacuated and flushed with nitrogen then Pd(dppf)Cl2.CH2Ch (308 mg, 0.38 mmol, 0.1 eq) was added and the
158 φ mixture was sparged with nitrogen (10 mins) then heated at 90 °C overnight in a sealed flask. The reaction was partitîoned between ethyl acetate and water, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 80 g RediSep™ silica cartridge) eluting with a gradient of 0 5 - 40% ethyl acetate in iso-heptane afforded the desired product as a white solid (1.08 g, 1.98 mmol, 53%).
LC/MS (C30H33CIN6O2) 545 [M+H]+; RT 2.72 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 8.97 (d, J = 8.6 Hz, IH), 8.69 (d, J = 3.9 Hz, IH), 8.17 (d, J = 8.6 Hz, IH), 7.43 (s, IH), 7.10 (d, J = 3.9 Hz, IH), 4.22 (q, J = 7.1 Hz, 2H), 3.80 (s, 2H), 10 2.65 (s, 3H), 2.23 (s, 3H), 2.03 - 1.89 (m, 3H), 1.73 - 1.50 (ni, 12H), 1.16 (t, 3H).
Step F: ethyl 3-{i-[(adamantan-1-yl)methyl]-5-methyl-lH-pyrazob4-yl}-6-{3-[(1,3benzothiazob2-yl)amino]-4-methyl-7H-pyrrolo[2,3-c]pyridazin-7-yl}pyridine-2-carboxylate
To an oven-dried sealed flask was added the product from Step E (1.08 mg, 1.98 mmol, 1 eq), 2-aminothiazole (594 mg, 3.96 mmol, 2 eq), ΛζΝ-diisopropylethy lamine (1.03 mL, 5.93 15 mmol, 3 eq) and 1,4-dioxane (80 mL). The vessel was evacuated and flushed with nitrogen then JosiPhos (183 mg, 0.2 mmol, 0.1 eq) was added and the mixture was sparged with nitrogen (10 mins) then heated at 150 °C for 2 days. The reaction was diluted with ethyl acetate, successively washed with water and brine, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column 20 chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 70% ethyl acetate in iso-heptane afforded the desired product as an orange solid (510 mg, 0.77 mmol, 39%).
LC/MS (C37H38N8O2S) 659 [M+H]+; RT 2.9 (LCMS-V-C) ’H NMR (400 MHz, DMSO-d6) δ 9.13 (d, J = 8.6 Hz, IH), 8.61 (d, J = 3.9 Hz, IH), 8.16 (d, 25 J = 8.6 Hz, IH), 7.91 (br s, IH), 7.62 (br s, IH), 7.44 (s, IH), 7.41 - 7.32 (m, IH), 7.27 - 7.11 (m, IH), 6.99 (d, J = 3.9 Hz, IH), 4.22 (q, J = 7.1 Hz, 2H), 3.80 (s, 2H), 2.65 (s, 3H), 2.23 (s, 3H), 2.03 - 1.89 (m, 3H), 1.76 - 1.52 (m, 12H), 1.16 (t, 3H).
Step G: 3-{l-[(adamantan-l-yl)methyl]-5-methyblH-pyrazol-4-yl}-6-{3-l(l,3-benzothiazob 2-yl)amino]-4-methyl-7H-pyrrolo[2,3-c]pyridazin~7-yl}pyridine-2-carboxylic acid
To a solution of the product from Step F (400 mg, 0.61 mmol, 1 eq) in 1,4-dioxane (15 mL) was added lithium hydroxîde monobydrate (255 mg, 6.07 mmol, 10 eq) and the mixture
159 was heated at reflux overnight. The reaction was concentrated in vacuo, and the residue was triturated in water, filtered and dried under vacuum. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 — 14% methanol in dichloromethane afforded a pale yellow solid that was triturated with 5 methanol, filtered and dried under vacuum to afford the desired product as a yellow solid (154 mg, 0.24 mmol, 40%).
HRMS-ESI (m/z) [M+H]+ calcd for C35H35N8O2S: 631.2604, found 631.2600.
Example 24: 3-{M(Adamantan-l-yl)niethyl]-5-methyl-l//-pyrazoL4-yl}-6-{3-[(l,3benzothiazol-2-yl)aminoJ-4-methyl-5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl}pyridine10 2-carboxyIic acid
Step A: ethyl 3-{I-[(adamantan-l-yl)methyl]-S-methyl-lH-pyrazol-4-yl}-6-aminopyridine-2carboxylate
A biphasic solution of ethyl 6-amino-3-bromopicolinate (5.14 g, 21 mmol, 1 eq), the product 15 from Préparation 5a (7.47 g, 21 mmol, 1 eq) and potassium carbonate (8.7 g, 62.9 mmol, 3 eq) in tetrahydrofuran (100 mL) and water (20 mL) was mixed vigorously while sparging with nitrogen (10 min). Pd(dppf)Cl2.CH2C12 (2.57 g, 3.15 mmol, 0.15 eq) was added and the mixture was heated at reflux for 16 h. The reaction was allowed to cool to ambient température and was filtered through celite. The fîltrate was diluted with ethyl acetate (200 20 mL), washed with water (100 mL), and the organic phase was washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 220 g RediSep™ silica cartridge) eluting with a gradient of 0 - 90% ethyl acetate in Ao-hcptane afforded the desired product as a cream solid (5.28 g, 13.4 mmol, 64%).
LC/MS (C23H30N4O2) 395 [M+H]+; RT 1.32 (LCMS-V-B1)
160
Φ *Η NMR (400 MHz, DMSO-dô) δ 7032 (d, IH), 7.21 (s, IH), 6.58 (d, IH), 6.24 (s, 2H), 4.05 (q, 2H), 3.72 (s, 2H), 2.10 (s, 3H), 1.98 - 1.88 (m, 3H), 1.71 - 1.47 (m, 12H), 1.10 (t, J = 7.1 Hz, 3H).
Step B: ethyl 3-{l-[(adainantan-l-yl)inethyl]-5-methyl-lH-pyrazol-4-yl}-6-{[3-(3,6-dichloro5 5-methylpyridazin-4-yl)propyljamino}pyri.dine-2-carhoxylate
To a solution of the product from Préparation 2e (1.92 g, 8.76 mmol, 1 eq) and the product from Step A (3.8 g, 9.64 mmol, 1.1 eq) in methanol (40 mL) was added acetic acid (15 mL) and sodium cyanoborohydride (2.75 g, 43.8 mmol, 5 eq) portionwise and the mixture was heated at reflux for 1 h. The reaction was allowed to cool to ambient température then 10 poured onto IN aqueous sodium hydroxide (50 mL) and extracted with ethyl acetate (3 x 50 mL). The organic phase was successively washed with saturated aqueous sodium bicarbonate (100 mL) and brine (100 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 — 70% ethyl acetate in iso-heptane 15 afforded the desired product as a white solid (3.99 g, 6.68 mmol, 76%).
LC/MS (C31H38CI2N6O2) 597 [M+H]+; RT 1.53 (I.CMS VB1 ) XH NMR (400 MHz, DMSO-d6) Ô 7.34 (d, J = 8.6 Hz, IH), 7.22 (s, IH), 6.95 (t, J = 5.7 Hz, IH), 6.63 (d, J = 8.6 Hz, IH), 4.06 (q, J = 7.1 Hz, 2H), 3.72 (s, 2FI), 3.41 - 3.31 (m, 2FI), 2.92 - 2.83 (m, 2H), 2.42 (s, 3H), 2.10 (s, 3H), 1.98 - 1.89 (m, 3H), 1.86 - 1.74 (m, 2H), 1.71 20 1.48 (m, 12H), 1.08 (t, J = 7.1 Hz, 3H).
Step C: ethyl 3-{l~[(adamantan~l-yl)methyl]-5-methyl-lH-pyrazol-4-yl}-6-{3-chloro-4methyl-5H,6H,7H,8H-pyrido[2,3-cJpyridazin-8-yl}pyridine-2-carboxylate
To a solution of the product from Step B (3.99 g, 6.68 mmol, 1 eq) in alpha,alpha,alphatrifluorotoluene (150 mL) was added césium carbonate (4.35 g, 13.4 mmol, 2 eq) and the 25 mixture was heated in a sealed tube at 160 °C for 3 days. The reaction was allowed to cool to ambient température and concentrated în vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 120 g RediSep™ silica cartridge) eluting with a gradient of 0 - 70% ethyl acetate in iso-heptane afforded the desired product as a white solid (0.95 g, 1.69 mmol, 25%).
LC/MS (C31H37CIN6O2) 561 [M+H]+; RT 1.55 (LCMS-V-B1)
161
Φ ‘Η NMR (400 MHz, DMSO-d6) δ 7.85 (d, J = 8.6 Hz, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.35 (s, 1H), 4.15 (q, J = 7.1 Hz, 2H), 4.01 - 3.92 (m, 2H), 3.77 (s, 2H), 2.88 (t, J = 6.6 Hz, 2H), 2.31 (s, 3H), 2.19 (s, 3H), 2.04 - 1.88 (m, 5H), 1.73 - 1.50 (ni, 12H), 1.13 (t, J = 7.1 Hz, 3H).
Step D: ethyl 3-{l-[(adamantan -1 -yl)methyl]-5-methyl-lH-pyrazol-4-yl} -6-{3-[(1,35 benzothiaz.ol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl}pyridine-2carboxylate
To a solution of the product from Step C (946 mg, 1.69 mmol, 1 eq), 2-aminobenzothiazole, (380 mg, 2.53 mmol, 1.5 eq) and JV,Abdiisopropylethylamine (0.88 mL, 5.06 mmol, 3 eq) in 1,4-dioxane (30 mL) was added Xantphos (97.6 mg, 0.17 mmol, 0.1 eq) and 10 tris(dibenzylideneaœtone)dipalladium(0) (77.2 mg, 0.08 mmol, 0.05 eq) and the mixture was heated in a sealed flask at 160 °C for 60 h. The reaction was allowed to cool to ambient température and was dîluted with ethyl acetate (20 mL), washed with brine (25 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 80 g RediSep™ silica cartridge) eluting with a gradient of 0 15 - 75% ethyl acetate in iso-heptane afforded the desired product as an orange solid (606 mg,
0.9 mmol, 53%).
LC/MS (C38H42N8O2S) 675 [M+H]+; RT 1.61 (LCMS-V-B1)
Ή NMR (400 MHz, DMSO-d6) Ô 7.95 (d, J = 9.6 Hz, 1H), 7.83 (br s, 1H), 7.69 (d, 1H), 7.57 (br s, 1H), 7.40 - 7.31 (m, 2H), 7.18 (t, J = 7.5 Hz, 1H), 4.15 (q, J = 7.1 Hz, 2H), 4.05 - 3.97 20 (m, 2H), 3.77 (s, 2H), 2.86 (t, J = 6.5 Hz, 2H), 2.33 (s, 3H), 2.20 (s, 3H), 2.04 - 1.89 (m, 5H),
1.72 - 1.50 (m, 12H), 1.13 (t, 3H).
Step E: 3-{l-[ (adamantan-1 -yl)methyl]-5-methyl-lH-pyrazol-4-yl}-6-{3-[(l,3-benzothiazol2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl}pyridine-2-carboxylic acid
To a solution of the product from Step D (600 mg, 0.89 mmol, 1 eq) in 1,4-dioxane (10 25 mL) was added lithium hydroxide monohydrate (74.6 mg, 1,78 mmol, 2 eq) and the mixture was heated at reflux for 1 h. The reaction was allowed to cool to ambient température and concentrated in vacuo. The residue was taken up în water, acidified to pH 4 and the solids were collected by filtration, washed with water and dried. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a 30 gradient of 0 - 10% methanol in dichloromethane afforded the desired product as a yellow solid (93 mg, 0.14 mmol, 16%).
HRMS-ESI (m/z) [M+H]+ calcd for C36H39N8O2S: 647.2917, found 647.2913.
162
Example 25: 2-{3-[(l,3-Benzothiazol-2-yl)amino]-4-methyl-5H,6/f,7H,8H,9/fpyridazino[3,4-fr]azepin-9-yl}-l,3-thiazole-4-carboxylic acid
Step A: ethyl2-{[(tert-butoxy)carbonyl](hept-5-yn~l-yl)amino}-l,3-thiazole-4-carboxylate
To ethyl 2-{[(rm-butoxy)carbonyl]amino}-l,3-thiazole-4-carboxylate (1.62 g, 5.94 mmol, 1 eq) in tetrahydrofuran (50 mL) was added hept-5-yn-l-ol (1 g, 8.92 mmol, 1.5 eq) and triphenylphosphine (2.34 g, 8.92 mmol, 1.5 eq), followed by dropwîse addition of diethyl azodicarboxylate (1.62 mL, 8.92 mmol, 1.5 eq) and the mixture was stirred at ambient température for 16 h. The reaction was partitioned between dichloromethane and brine, and the organic phase was dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 — 15% ethyl acetate în zxo-heptane afforded the desired product as a yellow oil (1.92 g, 5.24 mmol, 88%).
LC/MS (C18H26N2O4S) 367 [M+H]+; RT 2.53 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 8.07 (s, 1H), 4.27 (q, J = 7.1 Hz, 2H), 4.11 - 4.00 (m, 2H), 2.22 - 2.13 (m, 2H), 1.79 - 1.68 (ni, 5H), 1.55 (s, 9H), 1.48 - 1.34 (m, 2H), 1.30 (t, J = 7.1 Hz, 2H).
Step B: ethyl 2-1(hept-5-yn-l-yl)amino]-l J-thiazole^-carboxylate
To a solution of the product from Step A (1.97 g, 5.38 mmol, 1 eq) in dichloromethane (50 mL) was added trîfluoroacetic acid (4.94 mL, 64.5 mmol, 12 eq) and the mixture was stirred at ambient température overnight. The reaction was cooled to 0 °C and diluted with dichloromethane, basified with 2N aqueous sodium hydroxide, and the organic phase was dried (PTFE phase separator) and concentrated in vacuo to afford the desired product as a yellow oil (1.46 g, 5.48 mmol, >100%).
LC/MS (C13H18N2O2S) 267 [M+HT; RT 1.91 (LCMS-V-C)
163 • Ή NMR (400 MHz, DMSOM6) δ 7.81 (t, J = 5.4 Hz, IH), 7.49 (s, IH), 5.77 (s, IH), 4.21 (q, J = 7.1 Hz, 2H), 3.21 (td, J = 6.9, 5.3 Hz, 2H), 2.19 - 2.07 (m, 2H), 1.73 (t, J = 2.6 Hz, 3H), 1.67 - 1.55 (m, 2H), 1.54 - 1.41 (m, 2H), 1.26 (t, J = 7.1 Hz, 3H).
Stw C: ethyl 2-{3-chloro-4-methyl-SH,6H,7H,8H,9H-pyridazino[3,4-bJazepin-9-yl}-l,3- thiazole-4-carboxylate
To a solution of 3,6-dichloro-l,2,4,5-tetrazine (827 mg, 5.48 mmol, 1 eq) in tetrahydrofuran (20 mL) was added the product from Step B (1.46 g, 5.48 mmol, 1 eq) and the mixture was heated at 120 °C in a sealed flask for 48 h. The reaction was concentrated in vacuo. And purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ 10 silica cartridge) eluting with a gradient of 0 - 60% ethyl acetate in iso-heptane afforded the desired product as a pink solid (0.92 g, 2.61 mmol, 48%).
LC/MS (C15H17CIN4O2S) 353 [M+H]*; RT 2.00 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 7.85 (s, 1 H), 4.28 (q, J = 7.1 Hz, 3FT), 4.22 - 4.13 (m, 2H), 2.95 - 2.88 (m, 2H), 2.40 (s, 3H), 2.02 - 1.89 (m, 2H), 1.84 - 1.75 (m, 2H), 1.30 (t, J = 7.1 Hz, 15 3H).
Step D: ethyl 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H)8Hi9Hpyridazino[3,4-b]azepin-9-yl}-l ,3-thiazole-4-carboxylate
To an oven-dried micro wave vial was added the product from Step C (920 mg, 2.61 mmol, 1 eq), 2-aminothiazole (470 mg, 3.13 mmol, 1.2 eq), Xantphos (151 mg, 0.26 mmol, 0.1 eq), 20 A^-diisopropylethylamine (1.36 mL, 7.82 mmol, 3 eq) and 1,4-dioxane (15 mL) . The vessel was evacuated and flushed with nitrogen then tris(dibenzylideneacetone)dipalladium(0) (119 mg, 0.13 mmol, 0.05 eq) was added and the mixture was sparged with nitrogen (10 min) then heated at 150 °C for 8 h under microwave irradiation. The réaction was partitioned between ethyl acetate and brine, and the organic phase was dried (magnésium sulfate) and 25 concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash
Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in isoheptane gave a solid that was triturated with acetomtrile, filtered and dried under vacuum to afford the desired product as a yellow solid (0.81 g, 1.74 mmol, 67%).
LC/MS (C22H22N6O2S2) 467 [M+H]+; RT 2.20 (LCMS-V-C)
164
Φ m NMR (400 MHz, DMSO-d6) δ 7.88 (br s, 1H), 7.75 (s, 1H), 7.49 (br s, 1H), 7.44 - 7.34 (m, 1H), 7.27 - 7.16 (m, 1H), 4.28 (q, J = 7.1 Hz, 2H), 4.14 - 4.01 (s, 2H), 2.90 - 2.78 (m, 2H), 2.42 (s, 3H), 1.96 - 1.83 (m, 2H), 1.82 - 1.70 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H).
Step E: 2-{3-[(I,3-benzothiazol-2-yl)amino]-4-tnethyl-5H,6H,7H,8H,9H-pyridazino[3,45 b]azepin-9-yl}-l,3-thiazole-4-carboxylic acid
To a solution of the product from Step D (30 mg, 0.06 mmol, 1 eq) in 1,4-dioxane (6 mL) was added lithium hydroxide monohydrate (27 mg, 0.64 mmol, 10 eq) and the mixture was heated at reflux for 6 h. The reaction was concentrated in vacuo, then dissolved in MeOH, loaded onto a methanol-wet PE-ΑΧ cartridge (5 g), washed with methanol, eluted with 9:1 10 dichloromethane / formic acid and concentrated in vacuo. The residue was triturated with dichloromethane and methanol, filtered and dried under vacuum to afford the desired product as an off-white solid (22.4 mg, 0.05 mmol, 79%).
HRMS-ESI (m/z) [M+H]+ calcd for C20H19N6O2S2: 439.1011, found 439.1003.
Exampie 26: 3-{3-[(1,3-Benzothiazol-2-yl) amino]-4-methy 1-5//,6//,7//,8//-pyrido [2,315 c]pyri(lazin-8-yl}benzoic acid
Step A: ethyl 3-{[3-(3,6-dichloro-5-inethylpyridazin-4-yl)propyl]amino}benzoate
To a solution of the product from Préparation 2e (100 mg, 0.46 mmol, 1 eq) and ethyl 3aminobenzoate (79.2 mg, 0.48 mmol, 1.05 eq) in methanol (6 mL) was added acetic acid (2 20 mL) and sodium cyanoborohydridc (57.4 mg, 0.91 mmol, 2 eq) portionwise and the mixture was stirred overnight. The reaction was quenched by the addition of IN aqueous sodium hydroxide and extracted with ethyl aeetate (3 x 50 mL), and the combined organic extracts were washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge)
165 eluting with a gradient of 0 - 40% ethyl acetate in iso-heptane afforded the desired product as a colourless oil (88 mg, 0.24 mmol, 52%).
LC/MS (C17H19CI2N3O2) 332 [M-HC1+H]+; RT 1.34 (LCMS-V-B1) ΣΗ NMR (400 MHz, Chloroform-J) δ 7.40 (dt, 7 = 7.6, 1.3 Hz, 1H), 7.28 (dd,J = 2.6, 1.5 Hz, 5 1H), 7.23 (t, J = 7.9 Hz, 1H), 6.78 (ddd, J = 8.1, 2.6, 1.0 Hz, 1H), 4.36 (q, J = 7.2 Hz, 2H),
3.32 (t, J = 6.7 Hz, 2H), 2.98 - 2.86 (m, 2H), 2.40 (s, 3H), 1.96 - 1.83 (m, 2H), 1.38 (t, J = 7.1 Hz, 3H).
Step B: ethyl 3-{3-ehloro~4~methyl-5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl}benzoate
To a solution of the product from Step A (88 mg, 0.24 mmol, 1 eq) in dichloromethane (3 10 mL) was added trifluoroacetic acid (0.1 mL) and the mixture was stirred at ambient température overnight. The reaction was neutralised by addition of IN aqueous sodium hydroxide and the mixture was extracted with dichloromethane (2 x 20 mL). The combined organic extracts were washed with brine, dried (magnésium sulfate) and concentrated in vacuo to afford the desired product as a yellow solid (48 mg, 0.14 mmol, 61%).
LC/MS (C17H18CIN3O2) 332 [M+H]+; RT 1.27 (LCMS-V-B1) *H NMR (400 MHz, DMSO-d6) δ 7.90 (t, J = 1.9 Hz, 1H), 7.80 (dt, J = 7.6, 1.5 Hz, 1H), 7.66 - 7.60 (m, 1H), 7.56 (t, J = 7.7 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 3.76 - 3.70 (m, 2H), 2.87 (t, J = 6.5 Hz, 2H), 2.25 (s, 3H), 2.09 - 1.97 (m, 2H), 1.32 (t, J = 7.1 Hz, 3H).
Step C: ethyl 3-{3jU,3-benzothiazol-2-yl)amino]-4-methyl-5Ht6H,7H,8H-pyrido[2,320 c]pyridazin-8-yl}benzoate
To a solution of the product from Step B (80 mg, 0.24 mmol, 1 eq), 2-aminobenzothiazole, (43.5 mg, 0.29 mmol, 1.2 eq), A,A-diisopropylethylamine (0.13 mL, 0.72 mmol, 3 eq) and 1,4-dioxane (5 mL) was added Xantphos (14 mg, 0.02 mmol, 0.1 eq) and tris(dibenzylideneacetone)dipaliadium(O) (11 mg, 0.01 mmol, 0.05 eq) and the mixture was 25 heated în a sealed tube at 160 °C for 24 h. The reaction was allowed to cool to ambient température, then partitioned between ethyl acetate (20 mL) and brine (25 mL) and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by reverse phase automated flash chromatography (CombiFlash Rf, C18 4.3g RediSep column) eluting with a gradient of 5 - 95% acetonitrile in water afforded the crude desired product as a 30 yellow glass (18 mg, 0.04 mmol, 17%) that was used directly in the subséquent step without further purification.
166 φ LC/MS (C24H23N5O2S) 446 [Μ+Η]+; RT 1.31 (LCMS-V-B1)
Step D; 3-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5tI,6H,7H,8H-pyrido[2,3cjpyridazin -8-yl}benzoic acid
To a solution of the product from Step C (18 mg, 0.04 mmol, 1 eq) in 1,4-dioxane (5 mL) was 5 added lithium hydroxide monohydrate (3.39 mg, 0.08 mmol, 2 eq) and the mixture was heated at reflux for 1 h. The reaction was allowed to cool to ambient température and concentrated in vacuo. The residue was taken up in water, acidified with IN aqueous hydrochloric acid and concentrated in vacuo. Purification by préparative HPLC (HPLC-V-A1) afforded the desired product as a yellow solid (10.6 mg, 0.03 mmol, 63%).
HRMS-ESI (m/z) [M+H]+ calcd for C22H20N5O2S: 418.1338, found 418.1334.
Example 27: 2-[3-(l,3-Benzothiazol-2-ylamin<>)-4-methyl-6,7-dihydro-5//-pyrido[2,3cJpyridazin-8-yl]-5-[3-[4-[3-(dimethylamino)prop-l-ynyl]-2-ttuorophenoxy]propyl]thiazole-4-carboxylic acid
Using Propargylic amine préparation General Procedure starting from Préparation 3d and dimethylamine as the appropriate amine. Then Hydrolysis General Procedure starting from the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C34H35FN7O3S2: 672.2221, found 672.2205.
Example 28: 2-{3-[(l,3-BenzothÎazol-2-yl)amînol-4-cyclopropyl-5Jf,6//,7F/-pyrrolo[2,320 c]pyridazin-7-yl}-l,3-thiazole-4-carboxylic acid
167
HN N N^S
Step A: ethyl 2-{[(tert-butoxy)carbonyl](4-iodobut-3-yn-l-yl)amino}-l,3-thiazole-4carboxylate
To ethyl 2-{[(terEbutoxy)carbonyl]amino}-l,3-thiazole-4-carboxylate (3.16 g, 11.6 mmol, 1 5 eq) in tetrahydrofuran (150 mL) was added 4-iodobut-3-yn-l-ol (3.41 g, 17.4 mmol, 1.5 eq) and triphenylphosphine (4.56 g, 17.4 mmol, 1.5 eq), foilowed by dropwise addition of diethyl azodicarboxylate (2.74 mL, 17.4 mmol, 1.5 eq) and the mixture was stirred at ambient température for 16 h. The reaction was partitioned between ethyl acetate and brine, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification 10 by automated flash column chromatography (CombiFlash Rf, 120 g RediSep™ silica cartridge) eluting with a gradient of 0 - 15% ethyl acetate in zso-heptane afforded the desired product as a white solid (3.75 g, 8.33 mmol, 72%).
LC/MS (C15H19IN2O4S) 451 [M+H]+; RT 2.45 (LCMS-V-C) ‘H NMR (400 MHz, DMSO-d6) δ 8.09 (s, 1H), 4.28 (q, J = 7.1 Hz, 2H), 4.16 (t, J = 6.7 Hz, 15 2H), 2.77 (t, J = 6.7 Hz, 2H), 1.56 (s, 9H), 1.30 (t, J = 7.1 Hz, 3H).
Step B: ethyl 2-/(4-iodobut-3-yn-l-yl)amino]-l,3-thiazole-4-carboxylate
To a solution of the product from Step A (3.75 g, 8.33 mmol, 1 eq) in dichloromethane (50 mL) was added trifluoroacetic acid (15.3 mL, 200 mmol, 24 eq) and the mixture was stirred at ambient température for 1 h. The reaction was cooled to 0 C, diluted with dichloromethane, 20 basified with 2N aqueous sodium hydroxide, and the organic phase was dried (PTFE phase separator) and concentrated in vacuo to afford the desired product as a white solid (2.74 g, 7.82 mmol, 94%).
LC/MS (C10H11IN2O2S) 351 [M+H]+; RT 1.84 (LCMS-V-C)
Hl NMR (400 MHz, DMSO-d6) δ 7.98 (t, J = 5.7 Hz, 1H), 7.53 (s, 1H), 4.22 (q, J = 7.1 Hz, 25 2H), 3.40 - 3.30 (m, 2H), 2.61 (t, J = 6.8 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H).
168
Step C: ethyl 2-{3-chloro-4-iodo-5Hf6H,7H-pyrrolo[2,3-c]pyridazin-7-yl}-l,3-thiazole-4carboxylate
To a solution of 3,6-dichIoro-1,2,4,5-tetrazine (1.18 g, 7.82 mmol, 1 eq) in tetrahydrofuran (8Ü mL) was added the product from Step B (2.74 g, 7.82 mmol, 1 eq) and the mixture was 5 heated at reflux overnight. The reaction was allowed to cool to ambient température and the precipitate was collected by filtration, washed with tetrahydrofuran and dried under vacuum to afford the desired product as an off-white solid (1.06 g, 2.43 mmol, 31%).
LC/MS (Ci2HioClIN402S) 437 [M+H]+; RT 1.99 (LCMS-V-C) XH NMR (400 MHz, DMSO-d6) δ 8.17 (s, 1H), 4.45 (t, J = 8.1 Hz, 2H), 4.31 (q, J = 7.1 Hz, 10 2H), 3.46 - 3.33 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H).
Step D: ethyl 2-{3-chloro-4-eyclopropyl-5H,6H,7H-pyrrolo[2,3-c]pyridazin-7-yl}-l>3thiazole-4-carboxylate
To a sealed tube was added the product from Step C (120 mg, 0.27 mmol, 1 eq), potassium cyclopropyitrifluoroborate (102 mg, 0.69 mmol, 2.5 eq), potassium carbonate (114 mg, 0.82 15 mmol, 3 eq), tetrahydrofuran (16 mL) and water (4 mL). The vessel was evacuated and flushed with nitrogen then Pd(dppf)Cl2.CH2Ch (44.9 mg, 0.05 mmol, 0.2 eq) was added and the mixture was sparged with nitrogen (10 min) then heated at 150 °C for 40 h under microwave irradiation. The reaction was partitioned between ethyl acetate and brine and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by 20 automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 90% ethyl acetate in rso-heptane afforded the desired product as a white solid (40 mg, 0.11 mmol, 42%).
LC/MS (Ci5Hl5ClN4O2S) 351 [M+H]+; RT 2.00 (LCMS-V-C) ’H NMR (400 MHz, DMSO-d6) δ 8.12 (d, J = 9.9 Hz, 1H), 4.43 - 4.23 (m, 4H), 3.42 - 3.29 25 (m, 2H), 2.03 - 1.94 (m, 1H), 1.30 (t, 3H), 1.14-1.04 (m, 2H), 0.98 - 0.84 (m, 2H).
Step E: ethyl 2-{3-[(l,3-benzothiazol-2~yl)amifio]-4-cyclopropyl-5H,6H,7H-pyrrolo[2,3c]pyridazin-7-yl}-l:f3-thiazole-4-carboxylate
To an oven-dried microwave vial was added the product from Step D (40 mg, 0.11 mmol, 1 eq), 2-aminothiazoIe (25.7 mg, 0.17 mmol, 1.5 eq), V,V-diisopropylethy lamine (59.6 pL, 0.34 30 mmol, 3 eq) and 1,4-dioxane (3 mL). The vessel was evacuated and flushed with nitrogen then JosîPhos (10.5 mg, 0.01 mmol, 0.1 cq) was added and the mixture was sparged with
169 nitrogen (10 min) then heated at 150 °C for 1 h under microwave irradiation. The reaction was partitioned between ethyl acetate and brine, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in iso-heptane afforded the desired product as a yellow solid (18 mg, 0.04 mmol, 34%).
LC/MS (C22H20N6O2S2) 464 [M+H]+; RT 2.31 (LCMS-V-C) ΣΗ NMR (400 MHz, DMSO-d6) δ 10.58 (br s, 1H), 8.06 (s, 1H), 8.02 - 7.89 (m, 1H), 7.76 7.62 (m, 1H), 7.47 - 7.32 (m, 1H), 7.30 - 7.13 (m, 1H), 4.42 - 4.25 (m, 4H), 3.46 - 3.37 (m, 2H), 2.20-2.01 (m, 1H), 1.32 (t, J = 7.1 Hz, 3H), 1.21 - 1.06 (m, 2H), 0.93 -0.74 (m, 2H).
Step F: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-cyclopropyl-5H,6H, 7H-pyrrolo[2,3c]pyridazin-7-yl}-l,3-thiazole-4-carboxylic acid
To a solution of the product from Step E (18 mg, 0.04 mmol, 1 eq) in 1,4-dioxane (6 mL) was added lithium hydroxide monohydrate (16.3 mg, 0.39 mmol, 10 eq) and the mixture was heated at reflux for 8 h. The reaction was concentrated in vacuo, dissolved in methanol, then loaded onto a methanol-wed PE-ΑΧ cartridge (10 g), washed with methanol, eiuted with 9:1 dichloromethane / formic acid and concentrated în vacuo. The residue was successively trîturated with dichloromethane and water, filtered and dried under vacuum to afford the desired product as a beige solid (2.44 mg, 0.01 mmol, 14.43%), as a formic acid sait.
HRMS-ES1 (m/z) [M+H]+ calcd for CzoHnNôOiSi: 437.0854, found 437.0853.
Example 29: 3-{l-[(Adamantan-l-yl)methyl]-5-methyl-LH-pyrazol-4-yl}-6-{3-[(l,3benzothiazol-2-yl) amino] -4-methy9/7-pyridazino [3,4-à]azepin-9yl}pyridine-2-carboxylic acid
170
Step A: [(hex-5-yn-J-yloxy)methyl]benzene
To a stirred solution of 5-hexyn-l-ol (5.36 g, 54.6 mmol, 1 eq) in tetrahydrofuran (35 mL), cooled to 0 °C, was added sodium hydride (60% dispersion; 3.28 g, 81.9 mmol, 1.5 eq) portionwise and the mixture was allowed to stir for 30 min. Benzyl bromide (6.49 mL, 54.6 mmol, 1 eq) was added dropwise and the mixture was allowed to warm to ambient température and stir for 90 h. The reaction was cooled to 0 °C and quenched by the addition of saturated aqueous ammonium chloride (30 mL) then diluted with water (30 mL). The mixture was extracted with ethyl acetate (2 x 150 mL), and the combined organic ex tracts were washed with brine (100 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 120 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% ethyl acetate in Ao-heptane afforded the desired product as a yellow oil (10.2 g, 54.2 mmol, 99%).
LC/MS (CbHiôO) 189 [M+H]+; RT 2.21 (LCMS-V-C) ‘H NMR (400 MHz, Chloroform-d) δ 7.40 - 7.24 (m, 5H), 4.45 (s, 2H), 3.44 (t, J = 6.3 Hz, 2H), 2.77 (t, J = 2.7 Hz, 1H), 2.17 (td, J = 7.0, 2.6 Hz, 2H), 1.70 - 1.57 (m, 2H), 1.56 - 1.37 (m, 2H).
Step B: [(hept-5-yn-l-yloxy)methyl]benzene
A solution of the product from Step A (10.2 g, 54.2 mmol, 1 eq) in tetrahydrofuran (90 mL) was cooled to -78 °C and u-butyllithium (2.5M în hexanes; 26 mL, 65 mmol, 1.2 eq) was added dropwise over 30 min. After stirring for 1 h, iodomethane (4.05 mL, 65 mmol, 1.2 eq) was added dropwise and the mixture was allowed to warm to 0 ”C over 1 h. The reaction was quenched with aqueous saturated ammonium chloride (40 mL), diluted with water (40 mL), and extracted with ethyl acetate (3 x 100 mL). The combined organic extracts were successively washed with 2N aqueous sodium thiosulfate (200 mL) and brine (200 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 220 g RediSep™ silica cartridge) eluting with a gradient of 0 - 6% ethyl acetate in Ao-heptane afforded the desired product as a clear oil (10.4 g, 51.3 mmol, 95%).
LC/MS (C)4H18O) 203 [M+H]+; RT 2.37 (LCMS-V-C)
NMR (400 MHz, DMSO-d6) δ 7.40 - 7.24 (m, 5H), 4.45 (s, 2H), 3.43 (t, J = 6.4 Hz, 2H), 2.17 - 2.08 (m, 2H), 1.73 (t, J = 2.6 Hz, 3H), 1.67 - 1.55 (m, 2H), 1.54 - 1.41 (m, 2H).
171
Step C: 4-[4-(benzyloxy)butyl]-3,6-dichloro~5-methylpyridazine
A solution of 3,6-dichloro-l,2,4,5-tetrazine (3.23 g, 21.4 mmol, 1 eq) and the product from Step B (5.2 g, 25.7 mmol, 1.2 eq) in toluene (40 mL) was heated at 130 °C overnight in a sealed flask. The reaction was allowed to cool to ambient température and was concentrated 5 in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 120 g RediSep™ silica cartridge) eluting with a gradient of 0 — 30% ethyl acetate in Zso-heptane afforded the desired product as a red oil (3.27 g, 10.1 mmol, 47%).
LC/MS (CisHisChlSkO) 325 [M+H]+; RT 2.32 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 7.40 - 7.24 (m, 5H), 4.46 (s, 2H), 3.49 (t, J = 6.1 Hz, 2H), 10 2.87 - 2.78 (m, 2H), 2.41 (s, 3H), 1.74 - 1.62 (m, 2H), 1.61 -1.51 (m, 2H).
Step D: 4-(3,6-dichloro-5-methylpyridazin~4’yl)butan-l-ol
To a solution of the product from Step C (3.27 g, 10.1 mmol, 1 eq) in dîchioromethane (50 mL), cooled in an ice-water bath, was added boron trichloride (IM in dîchioromethane; 50.3 mL, 50.3 mmol, 5 eq) dropwise and the mixture was allowed to warm to ambient température 15 and stir for 1 h. The reaction was cooled to 0 C, quenched by the addition of methanol and concentrated in vacuo. The residue was partitioned between dîchioromethane (100 mL) and saturated aqueous sodium bicarbonate (150 mL), and the organic phase was washed with brine (150 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) 20 eluting with a gradient of 0 - 80% ethyl acetate in tso-heplane afforded the desired product as a yellow oil (2.21 g, 9.4 mmol, 94%).
LC/MS (C9H12CI2N2O) 235 [M+H]+; RT 1.36 (LCMS-V-C) ’H NMR (40Ü MHz, DMSO-d6) δ 4.44 (t, J = 5.1 Hz, IH), 3.45 (dd, J = 6.0, 5.0 Hz, 2H), 2.87 - 2.76 (m, 2H), 2.43 (s, 3H), 1.62 -1.48 (m, 4H).
Step E: 4-(3,6-dichloro-5-methylpyridazin-4-yl)butanal
An oven-dried flask was charged with dimethyl sulfoxide (1.6 mL, 22.6 mmol, 2.4 eq) and dîchioromethane (60 mL) and the mixture was cooled to -78 °C. Oxalyl chloride (2M in dîchioromethane; 7.05 mL, 14.1 mmol, 1.5 eq) was added dropwise and the mixture was stirred for 1 h. A solution of the product from Step D (2.21 g, 9.4 mmol, 1 eq) in 30 dîchioromethane (20 mL) was added dropwise and the mixture was stirred for 1
h. Triethylamine (7.84 mL, 56.4 mmol, 6 eq) was added and the mixture was allowed to warm
172
A to 0 °C over 1 h. The reaction was quenched with water (50 mL), diluted with saturated aqueous sodium bicarbonate (50 mL) and extracted with dichloromethane (2 x 200 mL). The combined organic extracts were washed with brine (100 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash 5 Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 70% ethyl acetate in isoheptane afforded the desired product as a yellow oîl (6.58 g, 6.78 mmol, 72%).
LC/MS (C9H10CI2N2O) 233 [M+H]+; RT 1.51 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 9.70 (t, J = 1.1 Hz, 1H), 2.86 - 2.76 (m, 2H), 2.63 (td, J = 7.0, 1.1 Hz, 2H), 2.45 (s, 3H), 1.81 - 1.68 (m, 2H).
Step F: ethyl 3-{l-[(adamantan-1 -yl)methyl]-5-methyl-lH-pyrazol-4-yl}-6-{[4-(3,6-dichloro5-inethylpyridazin-4-yl)butyl]amino}pyridine-2-carboxylate
To a solution of the product from Step E (1.04 g, 4.45 mmol, 1 eq) and the product from Example 24, Step A (1.93 g, 4.89 mmol, 1.1 eq) in methanol (30 mL) and ace tic acid (10 mL) was added sodium cyanoborohydride (559 mg, 8.89 mmol, 2 eq) and the mixture was 15 stirred at ambient température overnight. The reaction was quenched with IN aqueous sodium hydroxide (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were successively washed with saturated aqueous sodium bicarbonate and brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 20 - 70% ethyl acetate in tso-heptane afforded the desired product as a white gum (1.71 g, 2.8 mmol, 63%).
LC/MS (C32H40CI2N6O2) 611 [M+H]+; RT 2.65 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 7.32 (d, J = 8.6 Hz, 1H), 7.21 (s, 1H), 6.81 (t, J = 5.4 Hz, 1H), 6.60 (d, J = 8.6 Hz, 1H), 4.05 (q, J = 7.1 Hz, 2H), 3.72 (s, 2H), 3.32 - 3.23 (m, 2H), 2.90 25 - 2.81 (m, 2H), 2.43 (s, 3H), 2.10 (s, 3H), 1.94 (s, 3H), 1.74 - 1.48 (m, 16H), 1.07 (t, J = 7.1
Hz, 3H).
Step G: ethyl 3-{1-[(adamantan-1 -yl)methyl]-5-methyl-lH-pyrazol-4-yl}-6-{3-chloro-4methyl-5H,6H, 7H,8H,9H-pyridazino[3,4-b]azepin -9-yl}pyridin e-2-carboxylate
To a solution of the product from Step F (646 mg, 1.06 mmol, 1 eq) in alpha,alpha,alpha30 trifluorotoluene (6 mL) was added césium carbonate (1.03 g, 3.17 mmol, 3 eq) and XantPhos Pd G3 (50.1 mg, 0.05 mmol, 0.05 eq) and the mixture was sparged with nitrogen (10 min)
173 then heated at 120 °C overnight. The reaction was partitioned between ethyl acetate and water, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ siiica cartridge) eluting with a gradient of 0 - 60% ethyl acetate in Zso-heptane afforded the desired product as an off-white solid (119 mg, 0.21 mmol, 20%).
LC/MS (C32H39CIN6O2) 575 [M+H]+; RT 2.66 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) 5 7.47 (d, J = 8.8 Hz, 1H), 7.29 (s, 1H), 6.80 (d, J = 8.7 Hz, 1H), 4.22 - 4.16 (m, 2H), 4.12 (q, 2H), 3.73 (s, 2H), 2.89 - 2.81 (m, 2H), 2.45 (s, 3H), 2.14 (s, 3H), 2.00 - 1.89 (m, 3H), 1.80 - 1.49 (m, 16H), 1.10 (t, 3H).
Step H: ethyl 3-{l-[(adamantan-l-yl)methyl]-5-methyl-lH-pyrazol-4-yl}~6~{3-[(l,3benzothiazol-2-yl)amino]~4-methyl-5H,6H,7H,8H,9H-pyridazino[3,4-b]azepin-9yl}pyridine-2-carboxylate
To a solution of the product from Step G (119 mg, 0.21 mmol, 1 eq), 2-aminobenzothiazole, (62.2 mg, 0.41 mmol, 2 eq) and N,V-diisopropylelhylamine (108 pL, 0.62 mmol, 3 eq) in 1,4dioxane (8 mL) was added JosiPhos (19.2 mg, 0.02 mmol, 0.1 eq) and the mixture was heated in a sealed tube at 150 °C for 72 h. The reaction was aliowed to cool to ambient température, then diluted with ethyl acetate (20 mL), washed with brine (25 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ siiica cartridge) eluting with a gradient of 0 - 50% ethyl acetate in iw-heptane afforded the crude desired product as a yellow gum (49 mg, 0.07 mmol, 34%) that was used directly in the subséquent step without further purification.
LC/MS (C39H44N8O2S) 690 [M+H]+; RT 2.81 (LCMS-V-C)
Step I: 3-{l -[(adamantan -1 -yl)methyl]-5-methyl-lH-pyrazol-4-yl}-6-{3-[(I,3-benzothiazol-2yl)amino]~4~methyl-5H,6H,7H,8H,9H-pyridazino[3,4-b]azepin-9-yl}pyridine-2~carboxylic acid
To a solution of the product from Step H (49 mg, 0.07 mmol, 1 eq) in 1,4-dioxane (15 mL) was added lithium hydroxide monohydrate (44.8 mg, 1.07 mmol, 15 eq) and the mixture was heated at reflux for 1 h. The reaction was cooled to ambient température and concentrated in vacuo. Purification by reverse phase automated flash chromatography (CombiFlash Rf, C18 4.3g RediSep column) eluting with a gradient of 5 - 95% acetonitrile in water afforded the desired product as a yellow solid (6.24 mg, 0.01 mmol, 13%).
174 φ HRMS-ESI (m/z) [M+H]+ calcd for C37H41N8O2S: 661.3073, found 661.3097
Example 30: 2-{3-[(l,3-BenzothiazoI-2-yl)amino]-4-cyclopropyl-5H,6J7,7/7,8//pyritlo[2,3-c]pyridazin-8-yl}-5-(3-{4-[3-(dimethylamino)prop-l-yn-l-yl]-2fluorophenoxy}propyl)-l,3-thiazole-4-carboxylic acid
S
StepA: tert-butyl[(5-cyclopropylpent-4-yn-l-yl)oxy]dimethylsilane
To a solution of cyclopropylacetylene (8 mL, 94.5 mmol, 1.1 eq) in tetrahydrofuran (200 mL), cooled to -78 °C, was added n-butyllithium (2.0M in hexanes; 47.3 mL, 94.5 mmol, 1.1 eq) and the mixture was stirred at this température for 2.5 h. l,3-dimethyl-3,4,5,6-tetrahydro10 2(lH)-pyrimidinone (12 mL, 98.8 mmol, 1.15 eq)was added and after 15 min (3bromopropoxy)-tert-butyldimethylsilane (15 mL, 85.9 mmol, 1 eq) was added dropwise and the mixture was allowed to warm to ambient température and stir overnight. The reaction was partitioned between ethyl acetate and saturated aqueous ammonium chloride, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 330 g RediSep™ silica cartrîdge) eiuting with a gradient of 0 - 6% ethyl acetate in Zsoheptane afforded the crude desired product as a ciear oil (8.62 g, 36.2 mmol, 42%) that was used directly in the subséquent step without further purification.
Step B: 5-cyclopropylpent-4-yn-l-ol
To a solution of the product from Step A (8.62 g, 36.2 mmol, 1 eq) in tetrahydrofuran (150 mL) was added tetrabutylammonium fluoride (IM in tetrahydrofuran; 39.8 mL, 39.8 mmol, 1.1 eq) and the mixture was stirred at ambient température for 1 h. The reaction was concentrated in vacuo, partitioned between ethyl acetate and water, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 120 g RediSep™ silica cartrîdge) eiuting with a gradient of
175 — 50% ethyl acetate in ώο-heptane afforded the desired product as a clear oil (2.14 g, 17.2 mmol, 48%).
*H NMR (400 MHz, DMSO-d6) δ 4.43 (t, J = 5.2 Hz, 1H), 3.46 - 3.38 (m, 2H), 2.12 (td, J = 7.1, 2.0 Hz, 2H), 1.58 - 1.47 (m, 2H), 1.29 - 1.16 (m, 1H), 0.74 - 0.65 (m, 2H), 0.54 - 0.46 (m, 5 2H).
Step C: ethyl 2-{[(tert-butoxy)carbonyl](5-cyclopropylpent-4-yn-l-yl)amino}-l ,3-thiazole-4carboxylate
To a solution of ethyl 2-[(rm-butoxycarbonyI)amino]-l,3-thiazole-4-carboxylate (3.13 g, 11.5 mmol, 1 eq) and the product from Step B (2.14 g, 17.2 mmol, 1.5 eq) in tetrahydrofuran (80 10 mL) was added polymer-supported triphenylphosphine (4.52 g, 17.23 mmol, 1.5 eq) and the mixture was cooled to 0 °C and diethyl azodicarboxylate (2.73 mL, 17.2 mmol, 1.5 eq)was added dropwise then the mixture was allowed to warm to ambient température and stir overnight. The reaction was partitioned between dichloromethane and water, separated (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column 15 chromatography (CombiFlash Rf, 80 g RediSep™ silica cartridge) eluting with a gradient of 0 - 15% ethyl acetate in iso-heptane afforded the desired product as a colourless solid (3.86 g, 10.2 mmol, 89%).
LC/MS (C19H26N2O4S) 379 [M+H]+; RT 2.60 (LCMS-V-C)
NMR (400 MHz, DMSO-d6) δ 8.07 (s, 1H), 4.28 (q, J = 7.1 Hz, 2H), 4.11 (t, J = 7.Û Hz, 20 2H), 2.21 - 2.13 (m, 2H), 1.84 - 1.75 (m, 2H), 1.58 (s, 9H), 1.55 (s, 4H), 1.29 (t, 3H), 1.23 -
1.14 (m, 1H), 0.73 - 0.61 (m, 2H), 0.56 - 0.45 (m, 2H).
Step D: ethyl 2-{3-chloro-4-cyclopropyl-5H,6H,7H,8H-pyridol2,3-c]pyridazin-8-yl}-l ,3thiazole-4-carboxylate
To a solution of the product of Step C (3.86 g, 10.2 mmol, 1 eq) in toluene (120 mL) was 25 added 3,6-dichloro-l,2,4,5-tetrazine (1.54 g, 10.2 mmol, 1 eq) and the mixture was heated in a sealed flask at 130 °C for 24 h. The réaction was concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 80 g RediSep™ silica cartridge) eluting with a gradient of 0 - 60% ethyl acetate in iso-heptane afforded the desired product (677 mg, 1.86 mmol, 18%).
LC/MS (Ci6Hi7ClN4O2S) 365 [M+Hf ; RT 2.18 (LCMS-V-C)
176 φ ‘Η NMR (400 MHz, TFA added / DMSO-d6) δ 8.04 (s, IH), 4.36 - 4.22 (m, 4H), 3.07 (t, J = 6.2 Hz, 2H), 2.11 - 2.00 (m, 2H), 1.92 - 1.81 (m, IH), 1.31 (t, J = 7.1 Hz, 3H), 1.20 - 1.06 (m, 2H), 0.76 - 0.67 (m, 2H).
Step E: ethyl 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-cyclopropyl-5H,6H,7H,8H-pyrido[2,3-
c]pyridazin-8-yl}-I,3-thiazole-4-carboxylate
To an oven-dried sealed tube was added the product from Step D (627 mg, 1.72 mmol, 1 eq), 2-aminobenzothiazole (387 mg, 2.58 mmol, 1.5 eq), JV,N-diisopropylethy lamine (0.9 mL, 5.16 mmol, 3 eq) and 1,4-dioxane (22 mL) and the mixture was sparged with nitrogen (10 min) then Josiphos Pd G3 (162 mg, 0.17 mmol, 0.1 eq) was added and the mixture was heated 10 al 150 °C for 20 h. The reaction was partitioned between ethyl acetate and brine, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in iso-heptane afforded the desired product as a beige solid (261 mg, 0.55 mmol, 32%).
LC/MS (C23H22Nf,O2S2) 479 [M+H]+; RT 2.44 (LCMS-V-C) *H NMR (400 MHz, TFA added / DMSO-d6) δ 10.45 (s, IH), 8.07 - 7.92 (m, 2H), 7.75 7.60 (m, IH), 7.40 (t, IH), 7.30 - 7.16 (m, IH), 4.38 - 4.22 (m, 4H), 3.08 (t, J = 6.1 Hz, 2H), 2.15 - 2.01 (m, 2H), 1.98 - 1.85 (m, IH), 1.32 (t, J = 7.1 Hz, 3H), 1.27 - 1.12 (m, 2H), 0.74 0.53 (m, 2H).
Step F: ethyl 2-{3-[(l,3-benzothiazol-2-yl) ({[2-(trimethylsilyl) ethoxy]methyl})amino]-4cyclopropyl-SH,6H, 7H,8H-pyrido[2,3-c]pyridazin -8-yl}-I,3-thiazole-4-earboxylate
To a solution of the product of Step E (1.47 g, 3.07 mmol, 1 eq) in dimethylfonnamide (240 mL) was added N,N-diisopropylethylamme (1.61 mL, 9.21 mmol, 3 eq) and after 5 min the mixture was cooled to 0 °C and 4-(dimethylamino)pyridine (75.1 mg, 0.61 mmol, 0.2 eq) and 25 2-(trimethylsilyl)ethoxymethyl chloride (1.62 mL, 9.21 mmol, 3 eq) were added and the mixture was allowed to warm to ambient température and stir overnight. The reaction was concentrated in vacuo, then partitioned between dichloromethane and brine, separated (PTFE phase separator) and the organic phase was concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with 30 a gradient of 0 - 50% ethyl acetate in iso-heptane afforded the desired product as a yellow gum (f .36 g, 2.23 mmol, 73%).
LC/MS (C29H36N6O3SiS2) 609 [M+H]+; RT 2.96 (LCMS-V-C)
177 φ *Η NMR (400 MHz, DMSO-d6) δ 7.97 (s, 1H), 7.80 (d, 1H), 7.48 - 7.37 (m, 2H), 7.22 (ddd, J = 8.3, 7.0, 1.5 Hz, 1H), 5.82 (s, 2H), 4.36 - 4.21 (m, 4H), 3.76 - 3.66 (m, 2H), 3.06 (t, J = 6.2 Hz, 2H), 2.13 - 2.02 (m, 2H), 1.95 - 1.84 (m, 1H), 1.31 (t, J = 7.1 Hz, 3H), 1.23 -1.15 (m, 2H), 1.13 - 1.05 (m, 2H), 0.93 - 0.84 (m, 2H), -0.10 (s, 9H).
Step G: ethyl 2-{3-[(1,3-benzothiazol-2-yl)({[2-(trimethy Isilyljethoxy]methyl}) amino]-4cyclopropyl-SH,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl}-5-bromo-l ,3‘thiazole-4-carboxylate
To a solution of the product of Step F (1.36 g, 2.23 mmol, 1 eq) in dichloromethane (40 mL) was added A-bromosuccinimide (596 mg, 3.35 mmol, 1.5 eq) and the mixture was stirred at ambient température overnight. The reaction was partitioned between dichloromethane and 10 brine, and the organic phase was dried (PTFE phase separator) and concentrated in vacuo.
Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 40% ethyl acetate in fw-heptane afforded the desired product as a yellow solid (1.43 g, 2.08 mmol, 93%).
LC/MS (C29H35BrN6O3SiS2) 689 [M+H]+; RT 3.17 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.86 - 7.81 (m, 1H), 7.50 - 7.39 (m, 2H), 7.27 - 7.20 (m, 1H), 5.82 (s, 2H), 4.32 (q, 2H), 4.29 - 4.21 (m, 2H), 3.75 - 3.66 (m, 2H), 2.13 - 2.01 (m, 2H), 1.98- 1.86 (m, 1H), 1.32 (t, 3H), 1.28-1.16 (m, 2H), 1.15 - 1.05 (m, 2H), 0.94 -0.83 (m, 2H), -0.10 (s, 9H).
Step H: ethyl 5-[(1E) -3-[(tert-biityldimethylsilyl) oxy]prop-l ~en-l-yl]-2-(4-cyclopropyl-320 {](2Z)-3-{]2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3-benzothiazol-2-ylidene]amino}5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl)-l,3-thiazole-4-carboxylate
To an oven-dried sealed llask was added the product from Step G (1.43 g, 2.08 mmol, 1 eq), (£)-3-(ter/-butyldîmethylsilyloxy)propene-l-yl-boronic acid pinacol ester (0.82 mL, 2.5 mmol, 1.2 eq), potassium carbonate (862 mg, 6.24 mmol, 3 eq), [Ι,Γ25 bis(dîphenylphosphmû)ferrocene]dichloropalladium(ll) (170 mg, 0.21 mmol, 0.1 eq), tetrahydrofuran (60 mL) and water (20 mL) and the mixture was sparged with nitrogen (10 min) then heated at 120 °C for 1.5 h. The reaction was partitioned between ethyl acetate and water, and the organic phase was washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash 30 Rf, 80 g RediSep™ silica cartridge) eluting with a gradient of 0 — 30% ethyl acetate in isoheptane afforded the desired product as a yellow solid (1.05 g, 1.35 mmol, 65%).
LC/MS (C38H54N6O4Si2S2) 779 [M+H]+; RT 1.66 (LCMS-V-B2)
178
Φ JH NMR (400 MHz, DMSO-d6) δ 7.79 (d, IH), 7.51 - 7.37 (m, 3H), 7.27 - 7.18 (m, IH), 6.27 (dt, J = 16.0, 4.3 Hz, IH), 5.82 (s, 2H), 4.40 - 4.34 (ni, 2H), 4.34 - 4.23 (m, 4H), 3.75 3.66 (m, 2H), 3.06 (t, J = 6.1 Hz, 2H), 2.13 - 2.01 (m, 2H), 1.96 - 1.84 (m, IH), 1.31 (t, J = 7.1 Hz, 3H), 1.22 - 1.14 (ni, 2H), 1.13 - 1.04 (m, 2H), 0.93 (s, 9H), 0.89 - 0.82 (m, 2H), 0.11 5 (s, 6H),-0.10(s,9H).
Step I: ethyl 5-{3-[(tert-butyldiniethylsilyl)oxy]propyl}-2-(4-cyclopropyl-3-{f(2Z)-3-{[2(tritnethylsilyl)ethoxy]methyl}-2,3-dÎhydro-l,3-benzothiazol-2-ylidene]amino}5H,6H, 7H,8H-pyrido[2,3-c]pyridazin -8-yl) -1,3-thiazole-4-carboxylate
To a solution of the product from Step H (1.05 g, 1.35 mmol, 1 eq) in ethyl acetate (60 10 mL)was added platinum (IV) oxide (91.8 mg, 0.4 mmol, 0.3 eq) under a nitrogen atmosphère. The vessel was evacuated and back-filled with nitrogen (x3), then evacuated, placed under an atmosphère of hydrogen, and shaken at ambient température for 2 days. The réaction was filtered through celite, eluted with ethyl acetate and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ 15 silica cartridge) eluting with a gradient of 0 - 40% ethyl acetate in zso-heptane afforded the desired product as a clear gum (913 mg, 1.17 mmol, 87%).
LC/MS (C38H5ûN6O4Si2S2) 781 [M+H]+; RT 1.71 (LCMS-V-B2)
Ul NMR (400 MHz, DMSO-d6) δ 7.74 (d, IH), 7.49 - 7.36 (m, 2H), 7.27 - 7.20 (m, IH), 5.82 (s, 2H), 4.34 - 4.21 (m, 4H), 3.75 - 3.62 (m, 4H), 3.15 (l, J = 7.5 Hz, 2H), 3.05 (t, J = 6.3 20 Hz, 2H), 2.12 - 2.00 (m, 2H), 1.96 - 1.78 (m, 3H), 1.31 (t, 3H), 1.21 - 1.16 (m, 2H), 1.13 1.05 (m, 2H), 0.91 (s, 9H), 0.87 - 0.81 (m, 2H), 0.06 (s, 6H), -0.10 (m, 9H).
Step J: ethyl 2-(4-cyclopropyl-3-{[(2Z) -3-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3benzothiazol-2-ylidene]amino}-5H,6H, 7H,8H -pyrido[2,3-c]pyridazin -8-yl) -5-(3hydroxypropyl) -1,3-thiazole-4-carboxylate
To a solution of the product from Step I (819 mg, 1.05 mmol, 1 eq) in 1,4-dioxane (18 mL)was added te trabutyl ammonium fluoride (IM in tetrahydrofuran; 1.15 mL, 1.15 mmol, 1.1 eq) and the mixture was stirred at ambient température for 2 h. The reaction was partitîoned between ethyl acetate and water, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography 30 (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 50% ethyl acetate in dichloromethane afforded the desired product as a yellow gum (650 mg, 0.97 mmol, 93%).
179
Φ LC/MS (C32H42N6O4SiS2) 667 [M+H]+; RT 2.85 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-dô) δ 7.81 (dd, J = 7.5, 1.0 Hz, 1H), 7.48 - 7.36 (m, 21-1), 7.27 7.17 (m, 1H), 5.81 (s, 2H), 4.56 (t, J = 5.1 Hz, 1H), 4.34 - 4.21 (m, 4H), 4.12 (q, J = 7.1 Hz, 3H), 3.75 - 3.66 (m, 2H), 3.48 (td, J = 6.3, 5.1 Hz, 2H), 3.17 - 3.08 (ni, 2H), 3.05 (t, J = 6.3 5 Hz, 2H), 1.96 - 1.84 (m, 1H), 1.83 - 1.74 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H), 1.20 - 1.15 (m, 2H), 1.13 -1.04 (m, 2H), 0.93-0.85 (m, 2H), -0.10 (s, 9H).
Step K: ethyl 5-(3-chloropropyl)-2-(4-cyclopropyl-3-{[(2Z)-3-{[2(trimethylsilyl) ethoxy]methyl} -2,3-dihydro-l ,3-benzothiazol-2-ylidene]amino}5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl)-l,3-thiazole-4-carboxylate
The product from Step J (291 mg, 0.44 mmol, 1 eq) was dissolved in thîonyl chloride (10 mL) and stirred at ambient température for 8 h. The reaction was concentrated in vacuo, then partitioned between dichloromethane and brine, and the organic phase was dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 15 - 40% ethyl acetate in fso-heptane afforded the desired product as an orange gum (147 mg,
0.21 mmol, 49%).
LC/MS (C32H4iClN&O3SiS2) 685 [M+H]+; RT 3.15 (LCMS-V-C)
NMR (400 MHz, DMSO-d6) δ 7.80 (dd, J = 7.4, 1.0 Hz, 1H), 7.48 - 7.36 (m, 2H), 7.27 7.18 (m, 1H), 5.81 (s, 2H), 4.34 - 4.21 (m, 4H), 3.78 - 3.64 (m, 4H), 3.27 - 3.18 (m, 2H), 3.05 20 (t, J = 6.2 Hz, 2H), 2.16 - 1.96 (m, 4H), 1.98 - 1.81 (m, 1H), 1.32 (t, J = 7.1 Hz, 3H), 1.22 -
1.14 (m, 2H), 1.13 - 1.01 (m, 2H), 0.95-0.82 (m, 2H), -0.11 (s, 9H).
Step L: ethyl 2-(4-cyclopropyl-3-{[(2’Z)-3-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3benzothiazol-2-ylidene]atnÎno}-5Hf6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl)-5-(3-{4-[3(dimethylamino)prop-l -yn-1 -yl]-2-fluorophenoxy}propyl)-l,3-thiazole-4-carboxylate
To a solution of the product from Préparation 4b (53.9 mg, 0.28 mmol, 1.3 eq) in dimethylfonnamide (30 mL) was added sodium hydride (60% dispersion; 21.5 mg, 0.54 mmol, 2.5 eq) and the mixture stirred for 2 min. A solution of the product from Step K (147 mg, 0.21 mmol, 1 eq) in dimethylformamide (10 mL) was added and the mixture was heated at 100 °C for 1.5 h. The reaction was partitioned between ethyl acetate and water, and the 30 organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge)
ISO eluting with a gradient of 0 — 5% methanol in dichloromethane afforded the desired product as a yellow gum (145 mg, 0.17 mmol, 80%).
LC/MS (C43H52FN7O4S1S2) 842 [M+H]+; RT 2.76 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 7.79 (dd, J = 7.5, 1.1 Hz, 1H), 7.48 - 7.36 (m, 2H), 7.34 7.27 (m, 1H), 7.26 - 7.12 (τη, 3H), 5.81 (s, 2H), 4.30 - 4.20 (m, 4H), 4.14 (t, J = 6.1 Hz, 2H), 3.75 - 3.66 (m, 2H), 3.38 (s, 2H), 3.27 (t, J = 6.3 Hz, 2H), 3.05 (t, 2H), 2.19 (s, 6H), 2.17 2.Ü9 (m, 2H), 2.08 - 1.99 (m, 2H), 1.94 - 1.83 (m, 1H), 1.29 (t, 3H), 1.21 - 1.14 (m, 2H), 1.13 - 1.05 (m, 2H), 0.94 - 0.82 (m, 2H), -010 (s, 9H).
Step M: ethyl 2-{3-[(1,3-benzothiazol-2-yl)amino]-4-cyclopropyl-5H,6H, 7H,8H-pyrido[2,3cJpyridazin -8-yl}-5-(3f4-[3-(dimethylamino)prop-l -yn -1 -yl]-2-fluorophenoxy}propyl) -1,3thiazole-4-carboxylate
To a solution of the product from Step L (175 mg, 0.21 mmol, 1 eq) in dichloromethane (6 mL) was added trifluoroacetic acid (6 mL) and the mixture was stirred at ambient température for 7.5 h. The reaction was diluted with dichloromethane, cooled to 0 °C and neutralised with 2N aqueous sodium hydroxide. The organic phase was dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 7% methanol in dichloromethane afforded the desired product as a yellow gum (16 mg, 0.02 mmol, 11%).
LC/MS (C37H38FN7O3S2) 712 [M+H]+; RT 2.18 (LCMS-V-C)
Hl NMR (400 MHz, DMSO-d6) δ 7.94 (d, J = 7.7 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.43 7.34 (m, 1H), 7.30 (dd, J -11.9, 2.0 Hz, 1H), 7.26 - 7.10 (m, 3H), 4.30 - 4.20 (m, 4H), 4.14 (t, J = 6.1 Hz, 2H), 3.36 (s, 2H), 3.30 - 3.23 (m, 2H), 3.10 - 3.00 (m, 2H), 2.19 (s, 6H), 2.16 2.09 (m, 2H), 2.08 - 1.99 (m, 2H), 1.95 - 1.86 (m, 1H), 1.28 (t, J = 7.1 Hz, 3H), 1.25 - 1.21 (m, 2H), 1.21-1.13 (m, 2H).
Step N: 2-{3-[(1,3-benzothiazol-2-yl)amino]-4-cyclopropyl-5H,6H, 7H,8H-pyrido[2,3cJpyridazin -8-yl}-5-(3-{4-[3-(dimethylamino)prop-l -yn-1 -yl]-2-fluorophenoxy}propyl)-1,3thiazole-4-carboxylic acid
To a solution of the product from Step M (16 mg, 0.02 mmol, 1 eq) in 1,4-dioxane (5 mL) was added lithium hydroxide monohydrate (9.43 mg, 0.22 mmol, 10 eq) and the mixture was heated at reflux overnight. The reaction was concentrated in vacuo, and the residue was
181 triturated in water then diethyl ether, filtered and dried under vacuum to afford the desired product as a yellow solid (10.4 mg, 0.02 mmol, 68%), as a lithium sait.
HRMS-ESI (m/z) [M+H]+ calcd for C35H35FN7O3S2: 684.2227, found 684.2223.
Example 31: 2«{3>[(l^>Benzothiazol-2-yI)amîno]-4-methyl-5fî,<îH',7/I',8Ht9H>
pyridazino[3,4-è]azepin-9-yl}-5-(3-{4-[3-(dimethylamino)prop-l-yn-l-yl]-2Ι1ροι·ορΗεηοχγ}ρΓοργ1)-1,3-ίΗΪ3ζοΐ6-4-υ3Γ0οχ}'Ιΐυ acid
Step A: ethyl2-(4-methyl-3-{[(2Z)-3-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3benzothiazol-2-ylideneJamino}-5H,6H, 7H,8H,9H-pyridazino[3,4-b]azepin-9-yl) -1,310 thiazole-4-carboxylate
To a solution of Example 25 (4.97 g, 10.7 mmol, 1 eq) in dimethylformamide (180 mL) was added N,A-diisopropyle thylamine (5.57 mL, 32 mmol, 3 eq). After 5 min the mixture was cooled to 0 °C and 4-(dimethylamino)pyridine (260 mg, 2.13 mmol, 0.2 eq) and 2(trimethylsilyl)ethoxy methyl chloride (5.61 mL, 32 mmol, 3 eq) were added and the mixture 15 was stirred at ambient température overnight. The réaction was concentrated in vacuo, partitioned between ethyl acetate and water, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFIash Rf, 120 g RediSep™ silica cartridge) eluting with a gradient of 0 - 40% ethyl acetate in iso-heptane afforded the desired product as a yellow gum (5.21 g, 8.73 mmol, 20 82%).
LC/MS (CmHsûNgOîSîSs) 597 [M+H]+; RT 2.87 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.82 (dd, J = 7.7, 1.1 Hz, 1 H), 7.74 (s, 1 H), 7.52 - 7.39 (m, 2H), 7.25 (td, J = 7.5, 1.4 Hz, 1H), 5.88 (s, 2H), 4.27 (q, J = 7.1 Hz, 2H), 4.15 - 4.01 (m, 2H),
182 φ 3.72 (dd, J = 8.4, 7.4 Hz, 2H), 2.89 - 2.81 (m, 2H), 2.43 (s, 3H), 1.95 - 1.83 (m, 2H), 1.82 1.69 (m, 2H), 1.29 (t, J = 7.1 Hz, 3H), 0.91 (dd, J = 8.5, 7.4 Hz, 2H), -0.11 (s, 9H).
Step B: ethyl 5-bromo-2-(4-methyl-3-{[(2Z)-3-{[2-(trimethylsilyl)ethoxy]methyl}-2,3dihydro-l,3-benzothiazol~2-ylidene]amino}-5H,6H, 7H,8H,9H-pyridazino[3,4-b]azepin -95 yl)-l ,3-thiazole-4-carboxylate
To a solution of the product of Step A (5.21 g, 8.73 mmol, 1 eq) in dichloromethane (100 mL) was added V-bromosuccinimide (1.71 g, 9.6 mmol, 1.1 eq) and the mixture was stirred at ambient température overnight. The reaction was partitioned between dichloromethane and 10% aqueous sodium thiosulfate, and the organic phase was washed with brine, dried (PTFE 10 phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 120 g RediSep™ silica cartridge) eluting with a gradient of 0 - 30% ethyl acetate in iso-heptane afforded the desired product as a yellow gum (5.23 g, 7.74 mmol, 89%).
LC/MS (C28H35BrN6O3SiS2) 677 [M+H]4; RT 3.08 (LCMS-V-C) ‘H NMR (400 MHz, DMSO-d6) δ 7.84 (dd, J = 7.9, 1.1 Hz, 1H), 7.52 - 7.38 (m, 2H), 7.31 7.20 (m, 1H), 5.88 (s, 2H), 4.30 (q, J = 7.1 Hz, 2H), 4.18 (dd, J = 6.8, 4.6 Hz, 2H), 3.78 - 3.66 (m, 2H), 2.95 - 2.85 (m, 2H), 2.44 (s, 3H), 2.01 - 1.88 (m, 2H), 1.86 - 1.72 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H), 0.96 - 0.87 (m, 2H), -0.10 (s, 9H).
Step C: ethyl 5-[(lE)-3-[(tert-butyldimethylsilyl)oxy]prop-l-en-l-yl]-2-(4-methyl-3-{[(2Z)-3~ 20 {[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3-benzothiazol-2-ylidene]amino}5H,6H, 7H,8H,9H-pyridazino[3,4-b]azepin-9-yl)-l,3-thiazole-4-carboxylate
To an oven-drîed sealed flask was added the product from Step B (5.23 g, 7.74 mmol, 1 eq), (£)-3-(teri-butyldimethylsilyloxy)propene-l-yl-boronic acid pinacol ester (3.04 mL, 9.29 mmol, 1.2 eq), potassium carbonate (3.21 g, 23.2 mmol, 3 eq), [1,1'25 bis(diphenylphosphino)ferrocene]dichloropalladium(II) (632 mg, 0.77 mmol, 0.1 eq), tetrahydrofuran (150 mL) and water (50 mL) and the mixture was sparged with nitrogen (10 min) then heated at 120 °C for 1 h. The réaction was partitioned between ethyl acetate and water, and the organic phase was washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash 30 Rf, 120 g RediSep™ silica cartridge) eluting with a gradient of 0 - 40% ethyl acetate in isoheptane afforded the desired product as an orange oil (5.17 g, 6.74 mmol, 87%).
LC/MS (C37H54N6O4Si2S2) 767 [M+H]+; RT 1.60 (LCMS-V-B2)
183
Ή NMR (400 MHz, DMSO-d6) Ô 7.82 (dd, J = 7.8, 1.2 Hz, 1H), 7.53 - 7.35 (m, 3H), 7.26 (ddd, J = 8.3, 7.2, 1.3 Hz, 1H), 6.01 (dt, J = 15.9, 4.3 Hz, 1H), 5.88 (s, 2H), 4.36 - 4.235 (m, 4H), 4.12 - 3.99 (m, 2H), 3.78 - 3.67 (m, 2H), 2.89 - 2.78 (m, 2H), 2.43 (s, 3H), 1.95 - 1.82 (m, 2H), 1.81 - 1.68 (m, 2H), 1.30 (t, J = 7.1 Hz, 3H), 0.97 - 0.84 (m, UH), 0.07 (s, 6H), 5 0.12 (s, 9H).
Step D: ethyl 5-{3-[(tert-butyldiniethylsilyl)oxy]propyl}-2-(4-methyl-3-{[(2Z)-3-{[2(triniethylsilyl)ethoxy]inethyl}-2,3-dihydro-l,3-benzothiazol-2-ylidene]amino}5H,6H, 7H,8H,9H-pyridazlno[3,4-b]azepin -9-yl)-l ,3-thiazole-4-carboxylate
To a solution of the product from Step C (5.17 g, 6.74 mmol, 1 eq) in ethyl acetate (120 mL)was added platinum (IV) oxide (459 mg, 2.02 mmol, 0.3 eq) under a nitrogen atmosphère. The vessel was evacuated and backfilled with nitrogen (x3), then evacuated, placed under an atmosphère of hydrogen and shaken at ambient température for 2 days. The reaction was fîltered through celite, eluted with ethyl acetate and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 120 g RediSep™ 15 silica cartridge) eluting with a gradient of 0 - 40% ethyl acetate in tw-heptane afforded the desired product as an orange oil (4.46 g, 5.8 mmol, 86%).
LC/MS (C37H56N6Û4SÎ2S2) 769 [M+H]+; RT 1.62 (LCMS-V-B2)
Ή NMR (400 MHz, DMSO-d6) δ 7.79 (dd, J = 7.9, 1.2 Hz, 1H), 7.53 - 7.40 (m, 2H), 7.31 7.21 (m, 1H), 5.87 (s, 2H), 4.26 (q, J = 7.1 Hz, 2H), 4.10 - 3.97 (m, 2H), 3.77 - 3.66 (m, 2H), 20 3.61 (t, J = 6.1 Hz, 2H), 3.60 (t, J = 7.6 Hz, 2H), 3.11 - 2.99 (m, 2H), 2.88 - 2.76 (m, 2H), .44 (s, 3H), 1.96 - 1.81 (m, 2H), 1.81 - 1.67 (m, 4H), 1.29 (t, 3H), 0.91 - 0.81 (m, 11 H), 0.01 (s, 6H), -0.11 (s, 9H).
Step E: ethyl 5-(3-hydroxypropyl)-2-(4-methyl-3-{[(2Z)-3-{[2-(trimethylsilyl)ethoxyjmethyl}2,3-dihydro-l .3-benzothiazol-2-ylidene]amino}-5H,6H,7H,8H,9H-pyridazino[3,4-b]azepin25 9-yl)-l,3-thiazole-4~carboxylate
To a solution of the product from Step D (4.46 g, 5.8 mmol, 1 eq) in tetrahydrofuran (150 mL) was added tetrabutylammonium fluoride (IM in tetrahydrofuran, 8.7 mL, 8.7 mmol, 1.5 eq) and the mixture was stirred al rt for 1.5 h. The reaction was partitioned between ethyl acetate and water, and the organic phase was dried (magnésium sulfate) and concentrated in 30 vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 80 g RediSep™ silica cartridge) eluting with a gradient of 0 — 50% ethyl acetate in dichloromethane afforded the desired product as a beige gum (2.63 g, 4.02 mmol, 69%).
184 φ LC/MS (C31H42N6O4S1S2) 655 [Μ+Η]+; RT 2.77 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.83 (dd, J = 7.9, 1.2 Hz, 1H), 7.52 - 7.39 (m, 2H), 7.25 (ddd, J = 8.3, 7.1, 1.3 Hz, 1H), 5.88 (s, 2H), 4.50 (t, J = 5.1 Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H), 4.07 - 3.98 (m, 2H), 3.72 (t, J = 7.9 Hz, 2H), 3.42 (td, J = 6.3, 5.1 Hz, 2H), 3.04 (dd, J = 8.9, 5 6.4 Hz, 2H), 2.90 - 2.77 (m, 2H), 2.43 (s, 3H), 1.95 - 1.82 (m, 2H), 1.81 - 1.65 (m, 4H), 1.29 (t, J = 7.1 Hz, 3H), 0.91 (dd, J = 8.4, 7.4 Hz, 2H), -0.12 (s, 9H).
Step F: ethyl 5-(3-chloropropyl)-2-(4-methyl-3-{[(2Z)-3-{[2-(trimethylsilyl)ethoxy]methyl}2,3-dihydro-l,3-benzothiazob2‘ylidene]amino}-5H,6H,7H,8H,9H-pyrida~ino[3,4-b]azepin9-yl)-l,3-thiazole-4-carboxylate
The product from Step E (1.35 g, 2.06 mmol, 1 eq)was dissolved in thionyl chloride (20 mL) and stirred at ambient température for 5 h. The reaction was concentrated in vacuo, then partitioned between dichloromethane and brine, and the organic phase was dried (PTFE phase separator) and concentrated in vacuo. Purification by automatcd flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting wîth a gradient of 0 15 - 50% ethyl aeetate în iso-heptane afforded the desired product as a yellow gum (989 mg,
1.47 mmol, 71%).
LC/MS (C3iH4iClN6O3SiS2) 673 [M+H]+; RT 3.02 (LCMS-V-C)
NMR (400 MHz, DMSO-d6) δ 7.82 (dd, J = 7.8, 1.2 Hz, 1H), 7.52 - 7.39 (m, 2H), 7.30 7.21 (m, 1H), 5.87 (s, 2H), 4.27 (q, J = 7.1 Hz, 2H), 4.11 - 3.98 (m, 2H), 3.72 (t, 2H), 3.67 (t, 20 2H), 3.19 - 3.09 (m, 2H), 2.88 - 2.80 (m, 2H), 2.44 (s, 3H), 2.07 - 1.96 (m, 2H), 1.94 - 1.83 (m, 2H), 1.82 - 1.69 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H), 0.91 (dd, J = 8.4, 7.4 Hz, 2H), -0.11 (s, 9H).
Step G: ethyl 5-(3-{4-[3-(dimethylamino)prop-l -yn-1 -yl]-2-fluorophenoxy}propyl) -2-(4methyl-3-{[(2ZJ-3-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3-benzothiazol-225 ylidene]amino}-5H,6H, 7H,8H,9H-pyridazino[3,4-b]azepin-9-yl)-l,3-thiazole-4-carboxylate
To a solution of the product from Préparation 4b (369 mg, 1.91 mmol, 1.3 eq) în dimelhylforniamide (150 mL) was added sodium hydride (147 mg, 3.67 mmol, 2.5 eq) and the mixture stirred for 2 min. A solution of the product from Step F (989 mg, 1.47 mmol, 1 eq) in dimethylformamide (50 mL) was added and the mixture was heated at 100 °C for 1.5 h. 30 The reaction was partitioned between ethyl aeetate and water, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0
185 — 5% methanol in dîchioromethane afforded the desired product as a yellow oil (728 mg, 0.88 mmol, 60%).
LC/MS (C42H52FN7O4SiS2) 830 [M+H]+; RT 2.61 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.82 (dd, J = 7.7, 1.1 Hz, IH), 7.52 - 7.39 (m, 2H), 7.31 7.22 (m, 2H), 7.21 - 7.15 (m, IH), 7.12 (t, J = 8.7 Hz, IH), 5.88 (s, 2H), 4.24 (q, J = 7.1 Hz, 2H), 4.10 (t, 2H), 4.08 - 3.99 (m, 2H), 3.77 - 3.68 (m, 2H), 3.38 (s, 2H), 3.18 (t, J = 7.6 Hz, 2H), 2.86 - 2.77 (m, 2H), 2.43 (s, 3H), 2.20 (s, 6H), 2.11 - 1.98 (m, 2H), 1.94 - 1.83 (m, 2H), 1.82 - 1.69 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H), 0.91 (dd, J = 8.4, 7.4 Hz, 2H), -0.12 (s, 9H).
Step H: ethyl 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H ,8H,9Hpyridazino[3,4-b]azepin-9-yl}-5-(3-{4-[3-(dimethylamino)prop-l-yn-l-yl]~2fluorophenoxy}propyl)-l,3-thiazole-4-carboxylate
To a solution of the product from Step G (728 mg, 0.88 mmol, 1 eq) in tetrahydrofuran (18 mL) was added ethylènediamine (176 pL, 2.63 mmol, 3 eq) and tetrabutylammonium fluoride (IM in tetrahydrofuran; 2.64 mL, 2.63 mmol, 3 eq) and the mixture was heated at 60 °C for 24 h. The reaction was partitioned between ethyl acetate and water and the organic phase was washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 7% methanol in dîchioromethane afforded the desired product as a yellow gum (224 mg, 0.32 mmol, 37%).
LC/MS (C36H38FN7O3S2) 700 [M+H]+; RT 2.05 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 11.67 (br s, IH), 7.87 (d, IH), 7.51 (d, J = 8.0 Hz, IH), 7.38 (ddd, J = 8.2, 7.3, 1.3 Hz, IH), 7.26 (dd, J = 12.0, 2.0 Hz, IH), 7.23 - 7.16 (m, 2H), 7.12 (t, J = 8.7 Hz, IH), 4.24 (q, J = 7.1 Hz, 2H), 4.09 (t, 2H), 4.05 - 3.97 (m, 2H), 3.38 (s, 2H), 3.17 (t, J = 7.6 Hz, 2H), 2.86 - 2.77 (m, 2H), 2.40 (s, 3H), 2.20 (s, 6H), 2.11 - 1.96 (m, 2H), 1.92-1.82 (m, 2H), 1.80- 1.69 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H).
Step J: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H,9H-pyridazino[3,4bJazepin -9-yl}-5-(3-{4-[3-(dimethylamino)prop-l -yn-i-yl]-2-fluorophenoxy}propyl) -1,3thiazole-4-carboxylic acid
To a solution of the product from Step H (224 mg, 0.32 mmol, 1 eq) in 1,4-dioxane (15 mL) was added lithium hydroxide monohydrate (134 mg, 3.2 mmol, 10 eq) and the mixture was heated at reflux overnight. The reaction was concentrated in vacuo, and the residue was
186 triturated with water, fîltered and dried under vacuum to afford the desired product as a yellow solid (202 mg, 0.3 mmol, 94%), as a lithium sait.
HRMS-ESI (m/z) [M+H]+ calcd for C34H35FN7O3S2; 672.2227, found 672.225.
Example 32: 2-{3-[(l,3-Benzothïazol-2-yl)amino]-4-methyl-5//,6H,7Z/,8//-pyrido[2,3c]pyridazin-8-yl}-5-(3-{2-iluoro-4-[3-(methylamino)prop-l-yn-l-yl]phenoxy}propyl)-l,3thiazole-4-carboxylic acid .N
Step A: ethyl5-{3-[4-(3-{[(tert-butoxy)carbonyl](methyl)amino}prop-l-yn-l-yl)-2fluorophenoxy]propyl}‘2-(4-methyl-3-{[(2Z)-3-{[2-(trimethylsÎlyl)ethoxy]methyl}~2,3dihydro-1,3-benzothiazol-2~ylidene]amino}-5H,6H, 7H,8H-pyrido[2,3-c]pyridazin -8-yl)-l,3thiazole-4-carboxylate
To a solution of the product from Préparation 3g (500 mg, 0.78 mmol, 1 eq) in toluene (15 mL) was added the product from Préparation 4c (327 mg, 1.17 mmol, 1.5 eq), followed by triphenylphosphine (307 mg, 1.17 mmol, 1.5 eq) and diisopropyl azodicarboxylate (230 gL, 1.17 mmol, 1.5 eq) and he mixture was heated at reflux overnight. The reaction was partitioned between dichloromethane and water, and the organic phase was dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 50% ethyl acetate în iso-heptane afforded the desired product as an off-white foam (715 mg, 0.79 mmol, >100%).
LC/MS (C45H56FN7O6SiS2) 902 [M+H]+; RT 1.46 (LCMS-V-B2) XH NMR (400 MHz, DMSO-d6) Ô 7.82 (dt, J = 7.6, 0.9 Hz, 1H), 7.48 - 7.37 (m, 2H), 7.33 (d, J = 11.6 Hz, 1H), 7.28 - 7.13 (m, 3H), 5.84 (s, 2H), 4.32 - 4.17 (m, 6H), 4.15 (t, J = 6.1 Hz, 2H), 3.72 (dd, J = 8.5, 7.4 Hz, 2H), 3.27 (d, J = 15.4 Hz, 2H), 2.93 - 2.75 (m, 5H), 2.36 (s,
187 φ 3H), 2.19 - 2.10 (m, 2H), 2.10-1.98 (m, 2H), 1.40 (s, 9H), 1.28 (t, 3H), 0.96 - 0.89 (m, 2H),
-0.11 (s, 9H).
Step B: ethyl 2-{3-[(IJ-benzothiazotâ-ytyaminol-d-methylSH^H, 7H,8H-pyrido[2,3c]pyridazin-8-yl}-5~(3-{2-fluoro-4-[3-(methyla}nino)prop-l‘yn-l-yl]phenoxy}propyl)-l>35 thiazole-4-carboxylate
To a solution of the product from Step A (1.67 g, 1.85 mmol, 1 eq) in acetonitrile (17 mL) was added hydrogen fluoride-pyridine (3.22 mL, 37 mmol, 20 eq) and the mixture was heated at 60 °C for 2 h. The reaction was partitioned between 3:1 dîchloromethane / isopropanol and 2N aqueous sodium hydroxîde, and the organic phase was washed with brine, dried (PTFE 10 phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 80 g RediSep™ silica cartridge) eluting with a gradient of 0 - 7% methanol in dîchloromethane afforded the desired product as a yellow solid (1.02 g, 1.52 mmol, 82%).
LC/MS (C34H34FN7O3S2) 672 [M+Hf; RT 2.06 (LCMS-V-C) ’H NMR (400 MHz, DMSO-d6) Ô 7.89 (dd, J = 7.8, 1.2 Hz, IH), 7.50 (d, J = 8.1 Hz, IH), 7.38 (ddd, J = 8.2, 7.3, 1.2 Hz, IH), 7.32 - 7.25 (m, IH), 7.23 - 7.12 (m, 3H), 4.32 - 4.21 (m, 4H), 4.15 (t, J = 6.1 Hz, 2H), 3.45 (s, 2H), 3.32 - 3.23 (m, 2H), 2.89 (t, J = 6.4 Hz, 2H), 2.35 (s, 3H), 2.31 (s, 3H), 2.20 - 2.10 (m, 2H), 2.09 - 1.97 (m, 2H), 1.30 (t, J = 7.1 Hz, 3H).
Step C: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H>6H,7H,8H-pyrido[2,320 c/i)yri(lazifi-<S-yl}-5-(3-l2-Jliioro-4-l3-(!nethy!iiinino)pi(>p-J-yn-l-ylli)heiioxy}i)ropyl)-l .3thiazole-4-carboxylic acid
To a solution of the product from Step B (L02 g, 1.52 mmol, 1 eq) in 1,4-dioxane (50 mL) was lithium hydroxîde monohydrate (637 mg, 15.2 mmol, 10 eq) and the mixture was heated at 110 °C overnight. Purification by automated flash column chromatography 25 (CombiFlash Rf, 80 g RediSep™ silica cartridge) eluting with a gradient of 0 - 70% 0.7N methanolic ammonia in dîchloromethane gave a solid that was triturated with acetonitrile, filtered and dried under vacuum to afford the desired product as a yellow solid (657 mg, 1.02 mmol, 67%).
HRMS-ESI (m/z) [M+H]+ calcd for C32H31FN7O3S2: 644.1914, found 644.1930.
188
Exampie 33: 2-[(6/ï)-3-[(l,3-Benzothiazol-2-yl)amino]-6-hydroxy-4-mediyI5H,6H,7H,8ZZ-pyrido[2,3-c]pyridazin-8-yl]-1,3-thiazole-4-ca rboxylie acid
Step A: (4S)-3-[2-(benzyloxy)acetyl]-4-(propan-2-yl)-1,3-oxazolidin-2-one
To a solution of (S)-4-isopropyl-2-oxazolidinone (10 g, 77.4 mmol, 1 eq) in tetrahydrofuran (200 mL) was slowly added sodium hydride (60% dispersion; 3.72 g, 92.9 mmol, 1.2 eq) at 0 °C. After 1 h, benzyloxyacetyl chloride (12.8 mL, 81.3 mmol, 1.05 eq) was added dropwise and the mixture was stirred for 1 h. The reaction was quenched by the dropwise addition of saturated aqueous ammonium chloride (20 mL) at 0 °C, and extracted with ethyl acetate (250 mL). The organic extract was washed successively with water (250 mL) and brine (2 x 250 mL), dried (magnésium sulfate) and concentrated in vacuo. The solids were suspended in heptane (250 mL) and stirred vigorously for 1 h, then filtered, washed with heptane (2 x 100 mL) and dried under vacuum to afford the desired product as a white powder (19.9 g, 71.7 mmol, 93%).
LC/MS (C15H19NO4) 278 [M+H]+; RT 1.19 (LCMS-V-B 1) XH NMR (400 MHz, DMSO-d6) δ 7.42 - 7.35 (m, 4H), 7.34 - 7.28 (m, 1H), 4.64 (d, 2H), 4.57 (d, J = 0.8 Hz, 2H), 4.42 - 4.29 (m, 3H), 2.26 - 2.15 (m, 1H), 0.85 (dd, J = 20.8, 6.9 Hz, 6H).
Step B: (4S)-3-[(2R)-2-(benzyloxy)hex-4-ynoyl]-4-(propan-2-yl)-1,3-oxazolidîn-2-one
To a solution of the product from Step A (8.7 g, 31.4 mmol, 1 eq) in tetrahydrofuran (350 mL), cooled to - 78 °C, was added sodium bis(trimetbylsiiyl)amide (IM in tetrahydrofuran; 47.1 mL, 47.1 mmol, 1.5 eq) dropwise and the mixture was stirred for 1 h at this température. A solution of l-iodobut-2-yne (16.9 g, 94.1 mmol, 3 eq) in tetrahydrofuran (30 mL) was added dropwise and the mixture was allowed to warm to -40 °C and stir for 3 h. The reaction was quenched with saturate aqueous ammonium chloride (200 mL), partitioned between ethyl acetate and water, and the organic phase was successively washed with water and brine, dried
189 (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 120 g RediSep™ silica cartridge) eluting with a gradient of 0-25% ethyl acetate in Zso-heptane afforded the desired product as a yellow gum (4.17 g, 12.7 mmol, 40%).
LC/MS (C19H23NO4) 330 [M+H]+; RT 1.31 (LCMS-V-BIPsNeg)
NMR (400 MHz, DMSO-d6) δ 7.41 - 7.34 (m, 4H), 7.34 - 7.27 (m, 1H), 5.13 (t, J = 5.7 Hz, 1H), 4.57 (d, J = 11.9 Hz, 1H), 4.55 - 4.40 (m, 2H), 4.39 - 4.26 (m, 2H), 2.68 - 2.56 (m, 2H), 2.20 - 2.07 (m, 1H), 1.71 (t, J = 2.6 Hz, 3H), 0.84 (dd, 6H).
Step C: (2R)-2-(benzyloxy)hex-4-yn-l-ol
To a cooled solution of the product from Step B (4.17 g, 12.7 mmol, 1 eq) in tetrahydrofuran (45 mL)was added a solution of sodium borohydride (623 mg, 16.5 mmol, 1.3 eq) in water (12 mL) and the mixture was stirred at ambient température for 3 h. The reaction was quenched with saturated aqueous ammonium chloride (100 mL) and partitioned between ethyl acetate and water. The organic phase was successiveiy washed with water and brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 30% ethyl acetate in zso-heptane afforded the desired product as a colourless oil (2.01 g, 9.84 mmol, 78%).
lH NMR (400 MHz, DMSO-d6) δ 7.41 - 7.31 (m, 4H), 7.31 - 7.23 (m, 1H), 4.70 (t, J = 5.5 Hz, 1H), 4.59 (d, J = 1.9 Hz, 2H), 3.57 - 3.40 (m, 3H), 2.46 - 2.27 (m, 2H), 1.75 (t, J = 2.6 Hz, 3H).
Step D: {[(2R)-2-(benzyloxy)hex-4-yu-l-yl]oxy}(tert-butyl)diphenylsilane
To a cooled solution of the product from Step C (2.01 g, 9.84 mmol, 1 eq) in dichloromethane (50 mL) was added imidazole (1.34 g, 19.7 mmol, 2 eq) and tert-butyl(chloro)diphenylsilane (5.12 mL, 19.7 mmol, 2 eq) dropwise and the mixture was allowed to warm to ambient température and stir for 4 h. The reaction was quenched with 2M aqueous ammonium chloride and partitioned between dichloromethane and water. The organic phase was successiveiy washed with water and brine, dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 80 g RediSep™ silica cartridge) eluting with a gradient of 0 — 8% ethyl acetate in rso-heptane afforded the desired product as a colourless oil (3.66 g, 8.27 mmol, 84%).
190
LC/MS (C29H34O2S1) weak ionisation; RT 1.73 (LCMS-V-BIPOsNeg) *H NMR (400 MHz, DMSO-d6) Ô 7.68 - 7.61 (m, 4H), 7.51 - 7.38 (m, 6H), 7.37 - 7.32 (ni, 4H), 7.32 - 7.25 (m, 1H), 4.65 - 4.52 (m, 2H), 3.83 - 3.68 (m, 2H), 3.67 - 3.56 (m, 1H), 2.49 - 2.41 (m, 2H), 1.71 (t, J = 2.5 Hz, 3H), 1.00 (s, 9H).
Step E: 4-[(2R)-2-(benzyloxy)-3-[(tert-butyldiphenylsilyl)oxy]propyl]-3,6-dichloro-5’ methylpyridazine
A solution of 3,6-dichloro-l,2,4,5-tetrazine (4.99 g, 33.1 mmol, 4 eq) and the product from Step D (3.66 g, 8.27 mmol, 1 eq) in toluene (50 mL) was heated in a sealed flask at 150 C overnight. The reaction was concentrated in vacuo and purification by automated flash 10 column chromatography (CombiFlash Rf, 80 g RediSep™ silica cartridge) eluting with a gradient of 0 - 15% ethyl acetate in iso-heptane afforded the desired product as a bright orange oîl (3.76 g, 6.65 mmol, 80%).
LC/MS (C3iH34ChN2O2Si) 566 [M+H]+; RT 1.72 (LCMS-V-B1) *H NMR (400 MHz, DMSO-d6) δ 7.72 - 7.62 (m, 4H), 7.56 - 7.40 (m, 6H), 7.27 - 7.11 (m, 15 3H), 6.93 - 6.84 (m, 2H), 4.44 (d, J = 12.1 Hz, 1H), 4.10 (d, J = 12.1 Hz, 1H), 3.91 - 3.78 (m,
2H), 3.76 - 3.66 (m, 1H), 3.15 (dd, J = 13.8, 10.0 Hz, 1H), 3.00 (dd, J = 13.8, 3.6 Hz, 1H), 2.27 (s, 3H), 1.04 (s, 9H).
Step F: (2R)-2-(benzyloxy)-3-(3,6-dichloro-5-methylpyridazin~4-yl)propan-l-oÎ
To a solution of the product from Step E (3.76 g, 6.65 mmol, 1 eq) in letrahydrofuran (25 20 mL)was added tetrabutylammonium fluoride (IM in tetrahydrofuran; 7.31 mL, 7.31 mmol, 1.1 eq) and the mixture was stirred at ambient température for 1 h. The réaction was diluted with ethyl acetate (100 mL), successively washed with water (150 mL), saturated aqueous sodium bicarbonate (150 mL) and brine (2 x 100 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash 25 Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 80% ethyl acetate in isoheptane afforded the desired product as an orange solid (1.68 g, 5.14 mmol, 77%).
LC/MS (C15H16CI2N2O2) 327 [M+H]+; RT 1.06 (LCMS-V-B1)
Ή NMR (400 MHz, DMSO-d6) δ 7.25 - 7.12 (m, 3H), 7.01 - 6.93 (m, 2H), 4.99 (t, J = 5.5 Hz, 1H), 4.53 (d, J = 12.1 Hz, 1H), 4.22 (d, J = 12.0 Hz, 1H), 3.69 - 3.51 (m, 3H), 3.08 - 2.91 30 (m, 2H), 2.29 (s, 3H).
191
Step G: ethyl 2-{[(2R)-2-(benzyloxy)-3-(3,6-dichloro-5‘methylpyridazin-4-yl)propyl]amino}-
1,3- thiazole-4-carboxylate
To a solution of the product from Step F (1.68 g, 5.14 mmol, 1 eq) in tetrahydrofuran (55 mL) was added triphenylphosphine (2.7 g, 10.3 mmol, 2 eq) and ethyl 2-[(teributoxycarbonyl)amino]-l,3-thiazole-4-carboxylate (1.68 g, 6.16 mmol, 1.2 eq), followed by diîsopropyl azodicarboxylate (2.02 mL, 10.3 mmol, 2 eq) and the mixture was stirred at ambient température for 2 h. The reaction was partitioned between ethyl acetate and water and the organic phase was successively washed with water and brine (2 x 50 mL), dried (magnésium sulfate) and concentrated in vacuo. The material was dissolved in dichloromethane (30 mL) and trifluoroacetic acid (7.87 mL, 103 mmol, 20 eq) was added and the mixture was stirred at ambient température overnight. The reaction was diluted and neutralised with saturated aqueous sodium bicarbonate, the layers were separated and the aqueous phase was extracted with dichloromethane (3 x 100 mL). The combined organic extracts were washed with brine (150 mL), dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 80% ethyl acetate in iso-heptane afforded the desired product as a yellow solid (1.94 g, 4.02 mmol, 78%).
LC/MS (C21H22CI2N4O3S) 482 [M+H]+; RT 1.28 (LCMS-V-B1) *Η NMR (400 MHz, DMSO-d6) δ 8.14 (t, J = 5.9 Hz, 1H), 7.57 (s, 1H), 7.24 - 7.06 (m, 3H), 7.01 - 6.93 (m, 2H), 4.54 (d, J = 11.9 Hz, 1H), 4.31 - 4.15 (m, 3H), 3.97 - 3.86 (m, 1H), 3.70 - 3.59 (m, 1H), 3.52 - 3.41 (m, 1H), 3.06 (dd, J = 13.9,40.2 Hz, 1H), 2.92 (dd, J = 13.9, 3.2 Hz, 1H), 2.24 (s, 3H), 1.27 (t, J = 7.1 Hz, 3H).
Step H: ethyl 2-[(6R)-6-(benzyloxy)-3-chloro-4-methyl~5H,6H,7H,8H-pyrido[2,3c]pyridazin -8-yl]-l ,3-thiazole-4-carboxylate
To a solution of the product from Step G (2.09 g, 4.34 mmol, J eq) in acetonitrile (130 mL) was added potassium carbonate (1.2 g, 8.68 mmol, 2 eq) and copper (I) iodide (827 mg, 4.34 mmol, 1 eq) and the mixture was heated at reflux for 10 h. The reaction was diluted with water and extracted with ethyl acetate (3 x 60 mL). The combined organic extracts were washed with brine (2 x 50 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by reverse phase automated flash chromatography (CombiFlash Rf, C18 130g RediSep column) eluting with a gradient of 5 - 95% acetonitrile in water afforded the desired product as a pale brown solid (0.7 g, 1.58 mmol, 36%).
192
Φ LC/MS (C21H21CIN4O3S) 446 [Μ+Η]+; RT 1.36 (LCMS-V-B1)
Ή NMR (400 MHz, DMSO-d6) δ 8.05 (s, 1H), 7.32 - 7.17 (m, 5H), 4.99 - 4.86 (m, 1H), 4.59 (s, 2H), 4.39 - 4.23 (m, 3H), 4.07 (dd, J = 13.6, 2.1 Hz, 1H), 3.29 - 3.02 (ni, 2H), 2.32 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H).
Step I: ethyl 2-[(6R)‘3-[(l,3-benzothiazol-2-yl)amino]-6-(benzyloxy)-4-methyl5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl]-l,3-thiazole-4-carboxylate
To an oven-dried sealed flask was added the product from Step H (700 mg, 1.57 mmol, 1 eq), 2-aminobenzothiazoIe (473 mg, 3.15 mmol, 2 eq), A^V-diisopropylethylamine (0.82 mL, 4.72 mmol, 3 eq), JosiPhos Pd G3 (291 mg, 0.31 mmol, 0.2 eq) and 1,4-dioxane (17.5 mL) and the 10 mixture was sparged with nitrogen (10 min) then heated at 100 °C for 3 days. The réaction was partitioned between saturated aqueous sodium bicarbonate and ethyl acetate, the aqueous phase was extracted with ethyl acetate (3 x 80 mL), and the combined organic extracts were washed with brine (70 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ 15 silica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in /so-heptane afforded the desired product as a yellow solid (548 mg, 0.98 mmol, 62%).
LC/MS (C28H2ûN6O3S2) 559 [M+H]+; RT 1.03 (LCMS-V-B2) XH NMR (400 MHz, DMSO-d6) δ 8.00 (s, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.50 (br s, 1H), 7.38 (td, J = 7.7, 1.2 Hz, 1H), 7.33 - 7.16 (m, 6H), 4.96 - 4.85 (m, 1H), 4.61 (s, 2H), 4.38 - 4.25 20 (m, 3H), 4.15 - 4.05 (m, 1H), 3.25 - 3.14 (m, 1H), 3.13-3.02 (m, 1H), 2.35 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H).
Step J: ethyl 2-[(6R)-3-[(l,3-benzothiazol-2-yl)amino]-6-hydroxy-4-methyb5H,6H,7H,8Hpyridol2,3-cJpyridazin -8-yl]-l }3-thiazole~4-carboxylate
To a cooled solution of the product from Step I (548 mg, 0.98 mmol, 1 eq) in 25 dichloromethane (30 mL) was added boron trichloride (IM în dichloromethane; 4.9 mL, 4.9 mmol, 5 eq) dropwise and the mixture was stirred at ambient température for 2 h. The reaction was cooled to 0-5 °C and quenched with methanol (5 mL). Further methanol (5 mL) was added and the mixture was heated at reflux for 1.5 h. The reaction was concentrated in vacuo and purification by reverse phase automated flash chromatography (CombiFlash Rf, 30 C18 43g RediSep column) eluting with a gradient of 5 - 95% acetonitrile in water afforded the desired product as a yellow solid (328 mg, 0.7 mmol, 71%).
193
Φ LC/MS (C21H20N6O3S2) 469 [Μ+Η]+; RT 0.71 (LCMS-V-B2) £Η NMR (400 MHz, DMSO-d6) δ 8.01 (d, J = 9.3 Hz, IH), 7.89 (br s, IH), 7.51 (br s, IH), 7.38 (t, J = 7.5 Hz, IH), 7.20 (t, J = 7.4 Hz, IH), 5.38 (d, J = 3.2 Hz, IH), 4.47 (dd, J = 12.8, 4.8 Hz, IH), 4.41 - 4.34 (m, IH), 4.30 (q, J = 7.1 Hz, 2H), 4.19 (dd, J = 12.9, 2.5 Hz, IH), 5 3.08 - 2.88 (m, 2H), 2.35 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H).
Step K: 2-[(6R)-3-[(l,3-benzothiazol-2-yI)amino]-6-hydroxy-4-methyl-5H,6H,7H,8Hpyrido[2,3-c]pyridazin-8-yl]-l,3-thiazole-4-carboxylic acid
To a solution of the product from Step J (60 mg, 0.13 mmol, 1 eq) in 1,4-dioxane (10 mL) was added a solution of lithium hydroxîde monohydrate (10.8 mg, 0.26 mmol, 2 eq) în 10 water (2 mL) and the mixture was heated at reflux for 2 h. Purification by reverse phase automated flash chromatography (CombiFlash Rf, Cl 8 13g RediSep column) eluting with a gradient of 5 - 95% acetonitrîle in water afforded a yellow solid. The solid was dissolved in methanoi, then loaded onto a methanol-wet PE-ΑΧ cartridge (10 g), washed successively with methanoi and dichloromethane, eluted with 10% formic acid in dichloromethane, and 15 concentrated in vacuo to afford the desired product as a yellow solid (11.4 mg, 0.03 mmol, 20%), as a formic acid sait.
HRMS-ESI (m/z) [M+H]+ calcd for C19H17N6O3S2: 441.0804, found 441.0827.
Example 34: 2-{3-| (1,3-Benzothiazol-2-yl)amino]-4-methy 1-57/,8H-pyrido[2,3c]pyrÎdazin-8-yl}-5-(3-{2-fluoro-4-[3-(inethylamino)propyl]phenoxy}propyl)-l,320 thiazole-4-carboxylic acid
Step A: ethyl5-{3-[4-(3-{[(tert-butoxy)carbonyl](niethyl)amino}propyl)-2fluorophenoxy]propyl}-2-(4-methyl-3f[(2Z)-3-{[2-(trimethylsilyl)ethoxy]methyl}-2,3dïhydro-1,3-benzothiazol-2-ylidene]amino}-5H,6H,7H,8H-pyrido/2y3-c/pyrida7,iii-8-\l)-i ,325 thiazole-4-carboxylate
194
To a solution of the Example 32, Step A (447 mg, 0.5 mmol, I eq) in 1,4-dioxane (11 mL) was added platinum (IV) oxide (22.5 mg, 0.1 mmol, 0.2 eq) under a nitrogen atmosphère. The vessel was evacuated and back-filied with nitrogen (x3) then evacuated, placed under a hydrogen atmosphère and shaken at ambient température for 24 h. The reaction was filtered through celite, eluted with ethyl acetate and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of iso-heptane to 50% ethyl acetate in iso-heptane afforded the desired product as a yellow solid (424 mg, 0.47 mmol, 94%).
LC/MS (QsHûûF^OûSîSz) 906 [M+H]+; RT 1.45 (LCMS-V-B2)
Ή NMR (400 MHz, DMSO-d6) δ 7.82 (d, 1H), 7.48 - 7.38 (m, 2H), 7.28 - 7.20 (m, 1H), 7.12 - 7.02 (m, 2H), 6.98 - 6.91 (m, 1H), 5.84 (s, 2H), 4.33 - 4.20 (m, 4H), 4.08 (l, J = 6.2 Hz, 2H), 3.77 - 3.67 (m, 2H), 3.27 (t, J = 7.6 Hz, 2H), 3.18 - 3.04 (m, 2H), 2.88 (t, J = 6.3 Hz, 2H), 2.72 (s, 3H), 2.45 (t, J = 7.3 Hz, 2H), 2.37 (s, 3H), 2.17 - 1.99 (m, 4H), 1.78 - 1.62 (m, 2H), 1.29 (t, 3H), 1.24 (s, 9H), -0.11 (s, 9H).
Step B: ethyl 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,3c]pyridazin-8~yl}-5-(3f2-fluoro-4‘[3-(methylamiiio)propyl]phenoxy}propyl)-l,3-thiazole-4carboxylate
To a solution of the product from Step A (424 mg, 0.47 mmol, 1 eq) in acetonitrile (6 mL) was added hydrogen fluoride-pyridine (0.81 mL, 9.36 mmol, 20 eq) and the mixture was heated at 60 ÛC for 3 h. The reaction was partitioned between dichloromethane and 2N aqueous sodium hydroxide, and the organic phase was washed with brine, dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12g RediSep™ silica cartridge) eluting with a gradient of 0 - 15% methanol in dichloromethane afforded the desired product as a yellow solid (147 mg, 0.22 mmol, 47%).
LC/MS (C34H38FN7O3S2) 676 [M+Hf; RT 2.06 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 7.87 (dd, J - 7.9, 1.1 Hz, 1H), 7.53 - 7.45 (m, 1H), 7.41 7.32 (m, 1H), 7.18 (td, J = 7.6, 1.2 Hz, 1H), 7.12 - 7.01 (m, 2H), 6.97 - 6.89 (m, 1H), 4.34 4.19 (m, 4H), 4.09 (t, J = 6.2 Hz, 2H), 3.32 - 3.21 (m, 4H), 2.88 (t, J - 6.4 Hz, 2H), 2.41 (t, 2H), 2.34 (s, 3H), 2.24 (s, 3H), 2.17 - 1.99 (m, 4H), 1.69 - 1.57 (m, 2H), 1.30 (t, J = 7.1 Hz, 3H).
195
Step C: 2-{3-[(l ,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H, 7H,8H-pyrido[2,3c]pyt‘tdazin-8-yl}~5-(3-{2-fluoro-4-[3-(methylamino)propyl]pheiioxy}propyl)-l>3-thiazole-4carboxylic acid
To a solution of the product from Step B (147 mg, 0.22 mmol, 1 cq) in 1,4-dioxane (15 mL) was added lithium hydroxide monohydrate (91.3 mg, 2.18 mmol, 10 eq) and the mixture was heated at reflux overnight. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 25% 7N methanolic ammonia in dichloromethane gave a solid that was triturated with acetonitrile, filtered and dried under vacuum to afford the desired product as a yellow solid (83.8 mg, 0.13 mmol, 60%).
HRMS-ESI (m/z) [M+H]+ calcd for C32H35FN7O3S2: 648.2227, found 648.2246.
Exampie 35: 5-{3-[4-(Aminomethyl)-2-fluorophenoxy]propyl}-2-{3-[(l,3-benzothiazol2-y I)amino] -4-methy 1-5/7, βΗ,ΊΗ, 8/Z-pyrido [2,3-c]pyridazi n-8-yl}-1,3-thiazole-4carboxylic acid
Step A: ethyl5-[3-(4-cyano-2-fluorophenoxy)propyl]-2-(4-methyl-3-{[(2Z)-3-{2-[2(trimethylsilyl)ethoxy]ethyl}-2,3-dihydro-l,3-benzothiazol-2-ylidenejamino}-5H,6H,7H,8Hpyrido[2,3-c]pyridazin -8-yl)-l,3-thiazole-4-carboxylate
To a solution of the product from Préparation 3g (200 mg, 0.31 mmol, 1 eq) in toluene (6 mL) was added 3-fluoro-4-hydroxybenzonilrîle (64.2 mg, 0.47 mmol, 1.5 eq), followed by triphenylphosphine (123 mg, 0.47 mmol, 1.5 eq) and diisopropyl azodicarboxylate (92.2 pL, 0.47 mmol, 1.5 eq) and the mixture was heated at reflux overnight. The reaction was partitioned between dichloromethane and water, and the organic phase was dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0
196
- 50% ethyl acetate in iso-heptane afforded the desired product as a beige solid (207 mg, 0.27 mmol, 87%).
LC/MS (C37H42FN7O4S1S2) 760 (M+H]+; RT 3.00 (LCMS-V-C) lH NMR (400 MHz, DMSO-d6) 5 7.89 - 7.78 (m, 2H), 7.70 - 7.63 (m, 1H), 7.49 - 7.38 (m, 2H), 7.36 (t, J = 8.6 Hz, 1H), 7.28 - 7.19 (m, 1H), 5.85 (s, 2H), 4.33 - 4.18 (m, 6H), 3.77 3.68 (m, 2H), 3.32 - 3.23 (m, 2H), 2.88 (t, J - 6.2 Hz, 2H), 2.37 (s, 3H), 2.22 - 2.11 (m, 2H), 2.10 - 1.99 (m, 2H), 1.28 (t, J = 7.1 Hz, 3H), 0.96 - 0.86 (m, 2H), -0.11 (s, 9H).
Step B: ethyl 5-{3-[4-(aminomethyl)-2-fluorophenoxy]propyl}-2-(4-methyl-3-{[(2Z)-3-{2-[2(trimethylsilyl) ethoxy] ethyl} -2,3-dihydro-lf3-benzothiazol‘2-ylidene]aniino}-5H,6Ht 7H,8Hpyrido[2,3-c]pyridazin-8-yl)-l,3‘thiazole-4-carboxylate
A solution of the product from Step A (336 mg, 0.44 mmol, 1 eq) in ethyl acetate (60 mL) was hydrogenated using an H-Cube® Pro (ThalesNano) (110 °C, 85 bar). The mixture was concentrated in vacuo and purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 8% methanol in dichloromethane afforded the desired product as a yellow gum (282 mg, 0.37 mmol, 84%).
LC/MS (C37H4ÛFN7O4S1S2) 764 [M+H]+; RT 1.31 (LCMS-V-B1) *H NMR (400 MHz, DMSO-d6) δ 7.84 - 7.78 (m, 1H), 7.48 - 7.37 (m, 2H), 7.28 - 7.13 (m, 2H), 7.12 - 7.00 (m, 2H), 5.84 (s, 2H), 5.87 (s, 2H), 4.33 - 4.20 (m, 4H), 4.09 (td, J = 6.2, 1.9 Hz, 2H), 3.78 - 3.66 (m, 2H), 3.31 - 3.21 (m, 4H), 2.86 (t, J = 6.3 Hz, 2H), 2.36 (s, 3H), 2.19 1.97 (m, 4H), 1.30 (t, J = 7.1 Hz, 3H), 0.95 - 0.84 (m, 2H), -0.12 (s, 9H).
Step C: ethyl 5-{3-[4-(aminomethyl)-2-fluorophenoxy]propyl}-2-{3-[(l,3-benzothia.zob-2yl)amino]-4-methyl-SH,6H7H,8H-pyrido[2,3-c]pyridazin-8-yl}~l,3-thiazole-4-carboxylate
To a solution of the product from Step B (94 mg, 0.12 mmol, 1 eq) in tetrahydrofuran (5 mL) was added tetrabutylammonium fluoride (IM în tetrahydrofuran, 0.74 mL, 0.74 mmol, 6 eq) and ethylenedi amine (49.3 pL, 0.74 mmol, 6 eq) and the mixture was heated at 100 °C for 1 h under microwave irradiation. The reaction was partitioned between ethyl acetate and water, and the organic phase was washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 — 10% methanol in dichloromethane afforded the desired product as a yellow solid (29 mg, 0.05 mmol, 37%).
LC/MS (C31H32FN7O3S2) 634 [M+H]*; RT 1.96 (LCMS-V-C)
197 φ *Η NMR (400 MHz, DMSO-d6) δ 7.84 (d, J = 7.6 Hz, 1H), 7.46 (d, J = 7.9 Hz, 1H), 7.38 7.30 (m, 1H), 7.26 - 7.00 (m, 4H), 4.32 - 4.21 (m, 4H), 4.09 (t, J = 6.2 Hz, 2H), 3.32 - 3.21 (m, 4H), 2.87 (t, J = 6.5 Hz, 2H), 2.33 (s, 3H), 2.17 - 1.98 (m, 4H), 1.29 (t, J = 7.3 Hz, 3H).
Step D: 5-{3-[4-(aminomethyl) -2-fluorophenoxy]propyl}-2-{3-[(l,3~benzothiazol-25 yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,3~c]pyridazin-8-yl}-l,3-thiazole-4-carboxylic acid
To a solution of the product from Step C (29 mg, Û.05 mmol, 1 eq) in 1,4-dioxane (5 mL) was added lithium hydroxide monohydrate (19.2 mg, 0.46 mmol, 10 eq) and the mixture was heated at reflux overnight. Purification by automated flash column chromatography 10 (CombiFlash Rf, 4g RediSep™ siiica cartridge) eluting with a gradient of 0 - 25% 7N methanolic ammonia in dichioromethane gave a solid that was triturated with diethyl ether, filtered and dried under vacuum to afford the desired product as a yellow solid (3.0 mg, 4.89 μιηοΐ, 28%).
HRMS-ESI (m/z) [M+H]+ calcd for C29H29FN7O3S2: 606.1757, found 606.1782.
Example 36: 2-{3-[(l,3-Benzothiazol-2-yl)amino]-4-niethyl-5H,6H,7H,8H-pyrido[2,3c]pyridazin-8-yl}-5-(3-{4-[3-(dimethylamino)propyl]-2-fluorophenoxy}propyl)-l,3thiazole-4-carboxyIic acid
Step A: ethyl5-(3-{4-[3-(dimethylamino)prop-l-ynd•yl]-2-fluorophenoxy}propyl)-2-(4~ 20 methyl-3-{[(2Z) -3-{[2-(trimethylsilyl) ethoxy]methyl}-2,3-dihydro-l ,3-benzothiazol-2ylidene]amino}-5H,6H,7H,8H-pyrido[2,3-c]pyndazin-8-yl)-l,3-thiazole-4-carboxylate
To a solution of the product from Préparation 3g (1 g, 1.56 mmol, 1 eq) in toluene (30 mL) was added the product from Préparation 4b (452 mg, 2.34 mmol, 1.5 eq), followcd by triphenylphosphine (614 mg, 2.34 mmol, 1.5 eq) and diisopropyl azodicarboxylate (461 pL, 25 2.34 mmol, 1.5 eq) and the mixture was heated at reflux overnight. The reaction was
198 partitîoned between dichloromethane and water, and the organic phase was dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in i'so-heptane afforded the desired product as a brown gum (1.16 g, 1.42 mmol, 91%).
LC/MS (C4iH5oFN7O4SiS2) 816 [M+H]+; RT 2.70 (LCMS-V-C) lH NMR (400 MHz, DMSO-d6) δ 7.84 - 7.77 (m, IH), 7.47 - 7.37 (m, 2H), 7.31 (dd, J = 12.0, 1.9 Hz, IH), 7.27 - 7.12 (m, 3H), 5.83 (s, 2H), 4.32 - 4.20 (m, 4H), 4.14 (t, J - 6.1 Hz, 2H), 3.76 - 3.67 (m, 2H), 3.37 (s, 2H), 3.31 - 3.21 (m, 2H), 2.86 (t, J - 6.3 Hz, 2H), 2.36 (s, 3H), 2.19 (s, 6H), 2.17 - 2.09 (m, 2H), 2.07 - 2.00 (m, 2H), 1.28 (t, J = 7.1 Hz, 3H), 0.90 (dd, J = 8.5, 7.4 Hz, 2H), -0.11 (s, 9H).
Step B: ethyl 2f3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,3c]pyridazin-8-yl}-5-(3-{4-[3-(dimethylamino)prop-l-yn-l-yl]-2-fluorophenoxy}propyl)-l,3thiazole-4-carboxylate
To a solution of the product from Step A (262 mg, 0.32 mmol, 1 eq) in 1,4-dioxane (10 mL) was added ethylenediamine (21.4 pL, 0.32 mmol, 1 eq) and tetrabutylammonium fluoride (IM in tetrahydrofuran, 0.96 mL, 0.96 mmol, 3 eq) and the mixture was heated at 70 C overnight. The reaction was partitîoned between ethyl acetate and water, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 6% methanol in dichloromethane afforded the desired product as a yellow gum (114 mg, 0.17 mmol, 52%).
LC/MS (C35H36FN7O3S2) 686 [M+H]+; RT 2.08 (LCMS-V-C) 1H NMR (400 MHz, DMSO-d6) δ 11.43 (br s, IH), 7.88 (d, J = 7.6 Hz, IH), 7.50 (d, J = 8.0 Hz, IH), 7.42 - 7.35 (m, IH), 7.31 (dd, IH), 7.26 - 7.12 (m, 3H), 4.32 - 4.22 (m, 4H), 4.15 (t, J = 6.1 Hz, 2H), 3.40 (s, 2H), 3.28 (dd, J = 8.6, 6.7 Hz, 2H), 2.88 (t, J = 6.3 Hz, 2H), 2.35 (s, 3H), 2.21 (s, 6H), 2.20 - 2.10 (m, 2H), 2.07 - 2.02 (m, 2H), 1.30 (t, J = 7.1 Hz, 3H).
Step C: ethyl 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,3c]pyridazin-8-yl}-5-(3-{4-[3-(dimethylamino)propyl]-2-fluorophenoxy}propyl)-l,3-thiazole4-carhoxylate
199 φ Το a solution of the product from Step B (119 mg, 0.17 mmol, 1 eq) in ethyl acetate (40 mL) was added platinum (IV) oxide (7.88 mg, 0.03 mmol, 0.2 eq) under a nitrogen atmosphère and the vessel was evacuated and backfilled with nitrogen (x3), then evacuated, placed under a hydrogen atmosphère, and shaken at ambient température for 3 days. The 5 reaction was filtered through celite, eluted with ethyl acetate and concentrated in vacuo.
Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% methanol in dichloromethane afforded the desired product as a yellow gum (55 mg, 0.08 mmol, 46%).
LC/MS (C35H4OFN7O3S2) 690 [M+Hf ; RT 2.10 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 11.44 (br s, 1H), 7.88 (dd, J = 7.5, 1.1 Hz, 1H), 7.50 (d, J = 8.1 Hz, 1H), 7.38 (td, J = 7.7, 1.3 Hz, 1H), 7.20 (td, J = 7.5, 1.1 Hz, 1H), 7.12 - 7.02 (m, 2H), 6.97 - 6.89 (m, 1H), 4.32 - 4.21 (m, 4H), 4.09 (t, 2H), 3.33 - 3.22 (m, 4H), 2.89 (t, J = 6.3 Hz, 2H), 2.35 (s, 3H), 2.19 - 2.11 (m, 4H), 2.09 (s, 6H), 2.07 - 2.01 (m, 2H), 1.63 (p, J = 7.4 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H).
Step D: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,3c]pyridazin-8-yl}-5-(3-{4-[3~(dimethylainino)propyl]-2-fluorophenoxy}propyl)-l,3-thiazole4-carboxylic acid
To a solution of the product from Step C (55 mg, 0.08 mmol, 1 eq) in 1,4-dioxane (5 mL) was added lithium hydroxide monohydrate (33.5 mg, 0.8 mmol, 10 eq) and the mixture was heated 20 at reflux overnight. Purification by automated flash column chromatography (CombiFlash Rf, 4g RediSep™ silica cartridge) eluting with a gradient 0 - 20% 7N methanolic ammonia in dichloromethane gave a solid that was triturated with acetonitrile, filtered and dried under vacuum to afford the desired product as a yellow solid (32.8 mg, 0.05 mmol, 62%).
HRMS-ESI (m/z) [M+H]+ calcd for C33H37FN7O3S2: 662.2383, found 662.2402.
Example 37: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyI-5,6-dihydropyrrolo[2,3c]pyridazin-7-yl]-5-[3-[2-fluoro-4-[3-(methylamino)prop-lynyl]phenoxy]propyl]thiazole-4-carboxylic acid
200
Step A: methyl 2-(tert-butoxycarbonyl-(2-(3,6-dichloro-5-methyl-pyridazin-4yl)ethyl]amino]-5-[3-(2-fluoro-4-iodo-phenoxy)propyl]thiazole~4-carboxylat€
Using Mitsunobu General Procedure starting from 5.18 g of Préparation la (Step D) (9.6 5 mmol, 1.0 eq.) as the appropriate carbamate and 2.0 g of Préparation 2c (9.6 mmol, 1.0 eq.) as the appropriate alcohol, 5.6 g of the desired product (80% Yield) was obtained.
Step B: methyl 2-(2-(3,6-dichloro-5-methyl-pyridaziii-4-yl) ethylamino]-5-(3-(2-fluoro-4iodo-phenoxy)propyl]thiazole-4-carboxylate
Using Deprotection with HFIP General Procedure starting from 5.65 g of the product from 10 Step A as the appropriate carbamate, 2.9 g of the desired product (60%) was obtained.
Ή NMR (500 MHz, DMSO-rf?) δ ppm 7.76 (ί, 1H), 7.59 (dd, 1H), 7.45 (dm. 1H), 6.97 (t, 1H), 4.02 (t, 2H), 3.71 (s, 3H), 3.48 (m, 2H), 3.13 (t, 2H), 3.1 (m, 2H), 2.44 (s, 3H), 1.99 (m, 2H); I3C NMR (125 MHz, DMSO-dr,) δ ppm 163.1, 134.2, 125, 117.7, 82.4, 67.9, 51.8,41.6, 30.4, 30.4, 23.2, 16.8; HRMS-ESI (m/z): [M+HJ+ calcd for C2iH2iC12FIN4O3S: 624.9734, 15 found 624.9740.
Step C: methyl 2-(3-chloro-4-methyl-S,6-dihydropyrrolo(2,3-c]pyridazm-7-yl)-5-(3-(2fîuoro-4-iodo-phenoxy)propyl]thiazole-4-carboxylate
A suspension of 3.0 g of the product from Step B (4.79 mmol, 1 eq.) and 1.85 g césium carbonate (9.59 mmol, 2 eq.) were stirred at 80°C for 3 h in 25 mL dry 1,4-dioxane, while full 20 conversion was observed. Reaction mixture directly was evaporated to Celite, and then purified by flash chromatography using DCM-MeOH as eluents to obtain 2.64 g of the desired product (93% Yield).
Ή NMR (400 MHz, DMSO-d6) δ ppm 7.6 (dd, 1H), 7.46 (dm, 1H), 6.99 (t, 1H), 4.35 (t, 2H), 4.1 (t, 2H), 3.79 (s, 3H), 3.3 (m, 2H), 3.3 (m, 2H), 2.28 (s, 3H), 2.12 (m, 2H); I3C NMR (100 25 MHz, DMSO-dû) δ ppm 134.2, 125.0, 117.7,68.1,52.2,49.8,30.7,24.6,23.5, 16.0; HRMS
201 φ ESI (m/z): [M+H]+ calcd for C21H2GCIFIN4O3S: 588.9967, found 588.9959.
Step D: methyl 5-[3-[4-[3-[tert-btdoxycarbonyl(methyl)amino]prop-l-ynyl]-2-fluorophenoxyJpropyl]-2-(3-ch loro-4-methyl-5,6-dihydropyrrolo[2,3-c]pyridazin-7-yl) thiazole-4carboxylate
A 100 mL oven-dried, one-necked, round-bottom flask was equipped with a PTFE-coaled magnetic stirrîng bar and fitted with a reflux condenser. It was charged with 2.6 g the product from Step C (4.48 mmol, 1 eq.), 1.51 g tert-butyl N-methyl-N-prop-2-ynyl-carbamate (8.96 mmol, 2 eq.) and 4 mL DIPEA, then 16 mL dry THF was added and the system was flushed with argon. After 5 minutes stirrîng under inert atmosphère 42 mg mg Pd(PPh3)2C12 (0.224 10 mmol, 0.05 eq.) and 79 mg (0.224 mmol, 0.05 eq.) were added. The resulting mixture was then warmed up to 60°C and stirred at that température for 2 hours to reach complété conversion. Celite was added to the reaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography using Heptane-EtOAc as eluents to give 1.88 g of the desired product (67% Yield).
Step E: methyl 2-/3-(1,3-benzothiazol-2-ylamino)-4-methyl-5,6-dihydropyrrolo[2,3c]pyridazin~7-yl]-5-/3-]4-]3-[tert-butoxycarbonyl(methyl)amino] prop-1 -ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylate
A 4 mL oven-dried vial equipped with a PTFE-coated magnetic stir bar was charged with 80 mg the product from Step D (0.127 mmol, 1.0 eq.), 28 mg l,3-benzothiazol-2-amine (0.19 20 mmol, 1.5 eq.) and 113 uL DIPEA (0.635 mmol, 5 equiv) suspended in 0.5 mL dry 1,4dîoxane. Resulting mixture was flushed with nitrogen, and then 11 mg Pd2(dba)3 (0.012 mmol, 0.1 eq.) and 14 mg XantPhos (0.024 mmol, Ü.2 eq.) were added. The reaction mixture was then warmed up to 120°C and stirred at that température for 2 h, when the reaction reached complété conversion. The réaction mixture directly was evaporated to Celite, and 25 then purified by flash chromatography on using heptane-EtOAc as eluents to give 65 mg of the desired product (68% Yield).
Step F: 2-/3-(1,3-benzothiazol-2-ylamino)-4-methyl-5,6-dihydropyrrolo]2,3-c]pyridazin-7yl]-5-[3-[2-fluoro-4-[3-(methylamino)prop-l-ynyl]phenoxy/propyl]thîazole-4-carboxylic acid
Using Deprotection and Hydrolysis General Procedure starting from the product from Step E as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C31H29FN7O3S2: 630.1752, found 630.1755.
202
Example 38: 2-|3-(l,3-Benzothiazol-2-ylamino)-6-hydroxy-4-methyl-6,7-dihydro-5Bpyrido[2,3-c]pyridazm-8-yl]-5-[3-[2-fluoro-4-[3-(metliylamino)prop-lynyI]phenoxy]propyl]tliiazole-4-carboxylic acid (enantiopure, from Enantiomer 2 of
Préparation 2b, Step A)
Step A: methyl 2-[tert-butoxycarbonyl-[2-[tert-butyl(diphenyl)silyl]oxy-3-(3,6-dichloro-5methyl-pyridazin-4-yl)propyl]amino]-5-[3-[4-[3-[tert-butoxycarbonyl(methyl)amino]prop-lynylf2-fluoro-phenoxy]propyl]thiazole-4-carboxylate(enantiopure,from Enantiomer 2 of Préparation 2b, Step A)
Using Mitsunobu General Procedure starting from 1.91 g Préparation 1b (3.4 mmol, 1.0 eq.) as the appropriate carbamate and 1.6 g Préparation 2b (3.4 mmol, 1.0 eq.) as the appropriale alcohol, 2.2 g of the desired product (63% Yield) was obtained.
*H NMR (500 MHz, DMSO-t/6) 5 ppm 7.54-7.26 (m, 10H), 7.29 (m, 1H), 7.18 (d, 1H), 7.08 (t, 1H), 4.76 (m, 1H), 4.34/3.84 (m, 2H), 4.06 (t, 2H), 3.75 (s, 3H), 3.23 (m, 2H), 2.96/2.89 (m, 2H), 2.86 (bs, 3H), 2.06 (m, 2H), 2.03 (bs, 3H), 1.4 (s, 18H), 1.22 (m, 2H), 0.81 (s, 9H);
13C NMR (125 MHz, DMSO-d&) δ ppm 129.0, 119.2, 115.4, 68.8,67.9, 52.0, 51.1,36.1, 33.9, 30.4, 28.0, 26.9, 22.9, 22.7, 19.1, 16.8; HRMS-ESI (m/z): [M+H]+ calcd for CszHôaCEFNiOsSSi: 1034.3522, found 1034.3519.
Step B: methyl 5-[3-[4-[3-[tert-butoxycarbonyl(methyl)amino]prop-l-ynyl]-2-fluoro20 phenoxyJpropylJ-2-[[2-[tert-butyl(diphenyl) silylJoxy-3-(3,6-dichloro-5-methyl-pyridazin -4yl)propyl]amino]thiazole-4-carboxylate (enantiopure, from Enantiomer 2 of Préparation 2b, Step A)
Using Deprotectîon with HFIPA General Procedure starting from the product from Step A as the appropriate carbamate, 1.6 g of the desired product (81% Yield) was obtained.
203
Φ Ή NMR (500 MHz, DMSO-ώ,) δ ppm 7.76 (t, III), 7.56-7.26 (m, 10H), 7.31 (d, 1H), 7.21 (d, 1H), 7.12 (t, 1H), 4.46 (m, 1H), 4.23 (br„ 2H), 4.06 (t, 2H), 3.68 (s, 3H), 3.38/3.25 (m+m, 2H), 3.11 (t, 2H), 3.05 (m, 2H), 2.86 (br, 3H), 2.11 (s, 3H), 1.98 (quint, 2H), 1.41 (s, 9H), 0.85 (s, 9H); 13C NMR (125 MHz, DMSO-d6) δ ppm 162.9, 157.4, 157.3, 154.9, 151.5, 140.6, 5 139.8, 129.1, 119.3, 115.3, 69.9, 67.9, 51.7, 50.1, 38.5, 36.2, 33.8, 30.5, 28.5, 27.1, 23.2, 17.0;
HRMS-ESI (m/z): [M+H]+ calcd for CivHsiCkFNsObSSi: 934.2997, found 934.2994.
Step C: methyl 5-[3-[4-[3-[tert-butoxy£arbonyl(methyl)amino]prop-l -ynyl]-2-fluorophenoxyJpropyl]-2-[6-[tert-butyl(diph enyl) silyl]oxy-3-chloro-4-methyl-6,7-dihydro-5Hpyrido[2,3-c]pyridazin-8-yl]thiazole-4-carboxylate (enantiopure, from Enantiomer 2 of 10 Préparation 2b, Step A)
A 40 mL oven-dried vial equipped with a PTFE-coated magnetic stir bar was charged with 1.50 g of the product from Step B (1.6 mmol, 1.0 equiv) dissolved in 8 mL 1,4-dioxane, then 620 mg césium carbonate (3.2 mmol, 2.0 equiv) and 560 uL DI PEA (410 mg, 3.2 mmol, 2.0 equiv) were added, and then placed under an inert atmosphère. After addition of the 110 mg 15 Pd(AtaPhos)2C12 (0.16 mmol, 0.10 eq.) the reaction mixture was then warmed up to 80°C and stirred al that température for 30 min, when the reaction reached complété conversion. Celite was added to the réaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography using heptane and EtOAc as eluents to give 550 mg of the desired product (38% Yield).
*H NMR (500 MHz, DMSO-t/6) δ ppm 7.47 (dm, 4H), 7.47/7.43 (tm+tm, 2H), 7.38/7.33 (tm+tm, 4H), 7.3 (dm, 1H), 7.18 (m, 1H), 7.11 (t, 1H), 4.72/3.86 (m+m, 2H), 4.63 (m, 1H), 4.22 (s, 2H), 4.11 (t, 2H), 3.79 (s, 3H), 3.27 (m, 2H), 2.95/2.87 (m+m, 2H), 2.85 (s, 3H), 2.14 (s, 3H), 2.12 (m, 2H), 1.41 (s, 9H), 0.8 (s, 9H); iJC NMR (125 MHz, DMSO-d6) δ ppm 163.0, 156.0, 151.9, 151.6, 151.5, 151.4, 147.5, 142.2, 137.0, 135.6/135.5, 135.0, 25 133.2/133.1, 130.6/130.5, 129.1, 128.4/128.3, 126.8, 119.4, 115.4, 114.8, 85.3, 82.5, 79.8,
68.3, 63.1, 52.0, 51.8, 38.5, 33.8, 32.6, 30.7, 28.5, 26.8, 23.1, 19.1, 15.5; HRMS-ESI (m/z): [M+H]+ calcd for C+yH^ClFNsOôSSi: 898.3231, found 898.3238.
Step D: methyl 2-(3-(1,3-benzothiazol-2-ylamino)-6-[tert-butyl(diphenyl)silyl]oxy-4-methyl6,7-dihydro-5H-pyrido(2,3-c]pyridazin-8-yl]-5-(3-(4‘(3-(tert30 butoxycarbonyl(methyl)amino]prop-l -ynyl]-2-fluoro-phenoxy]propyl]thiazole-4carboxylate (enantiopure, from Enantiomer 2 of Préparation 2b, Step A)
A 4 mL oven-dried vial equipped with a PTFE-coated magnetic stir bar was charged with 179
204 mg of the product from Step C (0.2 mmol, 1.0 eq.), 60 mg l,3-benzothiazol-2-amine (0.4 mmol, 2.0 eq.) and 104 uL DIPEA (0.6 mmol, 3 equiv) dîssolved m 1 mL dry DMF. Resulting mixture was flushed with nitrogen, and then 18 mg Pd2(dba)3 (0.02 mmol, 0.1 eq.) and 23 mg XantPhos (0.04 mmol, 0.2 eq.) were added. The reaction mixture was then warmed up to 140°C and stirred at that température for 30 min, when the reaction reached complété conversion. The reaction mixture was directly injected onto a preconditioned silica gel column, and then it was purified via flash chromatography using heptane and EtOAc as eluents to give 50 mg of the desired product (24% Yield).
Step E: 2-[3-(l,3-Benzothiazol-2-ylamino)-6-hydroxy-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-[3-[2-fluoro-4-[3-(methylamino)prop-l-ynyl]phenoxy]propyl]thiazole-4carboxylic acid (enantiopure, frotn Enantiomer 2 of Préparation 2b, Step A)
Using Deprotection and Hydrolysis General Procedure starting from the product from Step D as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C32H31FN7O4S2: 660.1857, found 660.1847.
Example 39: 2-[(6R)-3-[(l,3-Benzothiazol-2-yl)amino]-4-methyl-6-{[2(methylamino)ethyl]amino}-577,6H,7H,8i/-pyrido[2,3-c]pyridazin-8-yl]-5-(3-{4-[3(diniethylamino)propyl]-2-fluorophenoxy}propy])-l,3-thiazole-4-carboxylic acid
Step A: tert-butyl (4R)-4-(2-hydroxyethyl)-2,2-dimethyl-l,3-oxazolidine-3-carboxylate
To a solution of tert-butyl JV-[(2R)-l,4-dihydroxybutan-2-yl]carbamate (10.5 g, 51.2 mmol, 1 eq) in dîchioromethane (110 mL) was added 2,2-dimethoxypropane (12.5 mL, 102 mmol, 2 eq) and p-toluenesulfonic acid monohydrate (0.51 mL, 5.12 mmol, 0.1 eq) and the mixture was stirred at ambient température overnight. The réaction was quenched with 0.2N aqueous
205 sodium hydroxide (50 mL) and the organic phase was dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 220 g RediSep™ silica cartridge) eluting wîth a gradient of 0 - 60% ethyl aeetate in isoheptane afforded the desired product as a white solid (5.13 g, 20.9 mmol, 41%).
’H NMR (400 MHz, DMSO-d6) δ 4.49 - 4.40 (m, 1H), 3.92 - 3.82 (m, 2H), 3.79 (d, J = 7.8 Hz, 1H), 3.48 - 3.39 (m, 2H), 1.87 - 1.69 (m, 1H), 1.66 - 1.50 (m, 1H), 1.47 (s, 3H), 1.42 (s, 12H).
Step B', tert-butyl (4R)-2,2-dimethyl-4-(2-oxoethyl)-l,3-oxazolidine-3-carboxylate
A solution of the product from Step A (5.13 g, 20.9 mmol, 1 eq) in dichloromethane (100 10 mL) was cooled to 0 °C. Dess-Martin periodinane (9.31 g, 22 mmol, 1.05 eq) was added and the mixture was stirred at ambient température for 2.5 h. The reaction was partitioned between dichloromethane and saturated aqueous sodium bicarbonate, and the organic phase was dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf. 220 g RediSep™ silica cartridge) eluting with a 15 gradient of 0 - 40% ethyl aeetate in zso-heptane afforded the desired product as a clear oil (3.23 g, 13.3 mmol, 64%).
Ή NMR (400 MHz, DMSO-dô) δ 9.65 (d, J = 6.7 Hz, 2H), 4.22 (t, J = 6.0 Hz, 2H), 4.09 3.98 (m, 1H), 3.71 (t, J = 9.0 Hz, 1H), 2.76-2.61 (m, 2H), 1,48 (s, 3H), 1.40 (d, J = 8.1 Hz, 12H).
Step C'. tert-butyl (4R)-4-(3,3-dibromoprop-2-en-i-y 1/-2,2-dimethyl-1,3-oxazolidine-3carboxylate
To a solution of tetrabromomethane (4.04 mL, 39.8 mmol, 3 eq) in dichloromethane (65 mL) was added a solution of triphenylphosphîne (20.9 g, 79.7 mmol, 6 eq) in dichloromethane (100 mL) and the mixture was stirred for 10 min. To a solution of the product from Step B 25 (3.23 g, 13.3 mmol, 1 eq) in dichloromethane (60 mL) was added triethyiamine (16.6 mL, 120 mmol, 9 eq) and the mixture was cooled to 0 °C. The first solution was added portionwise, and the résultant mixture was stirred ai 0 °C for 2 h. The reaction was partitioned between dichloromethane and saturated aqueous sodium bicarbonate and the organic phase was washed with brine, dried (PTFE phase separator) and concentrated in vacuo. The residue was 30 triturated with chîlled diethyl ether, the solids were collected by filtration and washed with diethyl ether. Purification by automated flash column chromatography (CombiFlash Rf, 120 g
206
RediSep™ silica cartridge) eluting with a gradient of 0 - 15% ethyl acetate in tso-heptane afforded the desired product as a clear oil (1.77 g, 4.43 mmol, 33%).
*H NMR (400 MHz, DMSO-d6) δ 6.61 (t, J = 7.5 Hz, IH), 4.08 - 3.87 (m, 2H), 3.83 - 3.70 (m, 2H), 2.45 - 2.28 (m, 2H), 1.50 (s, 3H), 1.41 (d, 12H).
Step D·. tert-butyl (4R)-4-(but-2-yn-l-yl)-2,2-dimethyl-l,3-oxazolidine-3-carboxylate
To a solution of the product from Step C (1.77 g, 4.43 mmol, 1 eq) in tetrahydrofuran (40 mL), cooled to -78 °C, was added n-butyllithium (2.5M in hexanes; 5.32 mL, 13.3 mmol, 3 eq) and the mixture was stirred at this température for 1 h. lodomethane (1.38 mL, 22.2 mmol, 5 eq) was added and the mixture was ailowed to warm to ambient température and stir overnight. The reaction was partitioned between ethyl acetate and saturated aqueous ammonium chloride, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0-15% ethyl acetate in iso-heptane afforded the desired product as a clear oil (1.04 g, 4.11 mmol, 93%).
Ή NMR (400 MHz, DMSOM6) δ 4.02 - 3.92 (m, IH), 3.90 - 3.75 (m, 2H), 2.46 (d, J = 15.8 Hz, IH), 2.37 - 2.20 (m, IH), 1.75 (t, J = 2.6 Hz, 3H), 1.48 (s, 3H), 1.41 (dd, J = 8.2, 3.8 Hz, 12H).
Step E: tert-butyl N-[(2R)-l-hydroxyhex-4-yn-2-yl]carbamate
To a solution of the product of Step D (1.04 g, 4.11 mmol, 1 eq) in methanol (30 mL) was added p-toluenesulfonic acid monohydrate (0.08 mL, 0.82 mmol, 0.2 eq) and the mixture was stirred at ambient température overnight. The reaction was partitioned between ethyl acetate and saturated aqueous sodium bicarbonate, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 60% ethyl acetate in iso-heptane afforded the desired product as a clear oil (724 mg, 3.39 mmol, 83%).
>H NMR (400 MHz, DMSO-d6) δ 6.54 (d, J = 8.3 Hz, IH), 4.67 (t, J = 5.7 Hz, IH), 3.53 3.40 (m, IH), 3.39 - 3.26 (m, 2H), 2.38 - 2.09 (m, 2H), 1.73 (t, J = 2.6 Hz, 3H), 1.39 (s, 9H).
Step F·, tert-butyl N-[(2R)-l-[(tert-butyldiphenylsilyl)oxy]hex-4-yn-2-yl]carbamate
To a solution of the product from Step E (724 mg, 3.39 mmol, 1 eq) in dîchloromethane (30 mL) was added imidazole (0.45 mL, 6.79 mmol, 2 eq) and the mixture was cooled to 0 C then ierr-butyl(chloro)diphenylsilane (0.93 mL, 3.56 mmol, 1.05 eq) was added and the
207 mixture was stirred at ambient température overnight. The reaction was partitioned between dichloromethane and saturated aqueous ammonium chloride, and the organic phase was separated (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 8% ethyl acetate in iso-heptane afforded the desired product as a clear oil (1.67 g, 3.7 mmol, 109%).
LC/MS (C27H37NO3Si) 352 [M-Boc]+; RT 1.28 (LCMS-V-B2)
NMR (400 MHz, DMSO-d6) δ 7.73 - 7.59 (m, 4H), 7.52 - 7.35 (m, 6H), 3.67 - 3.57 (m, 2H), 2.46 - 2.14 (m, 2H), 1.70 (t, J = 2.5 Hz, 3H), 1.39 (s, 9H), 1.37 - 1.29 (m, 1H), 1.00 (s, 9H).
Step G: {[(2R)-2~aminohex-4-yn-l-yl]oxy}(tert-butyl)diphenylsilane
To a solution of the product from Step F (1.67 g, 3.7 mmol, 1 eq) in dichloromethane (35 mL), cooled to 0 °C, was added trifluoroacetic acid (8.07 mL, 105 mmol, 28.5 eq) and the mixture was stirred at ambient température for 1 h. The reaction was partitioned between dichloromethane and IN aqueous sodium hydroxide (120 mL), and the organic phase was separated (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 — 70% ethyl acetate in zso-heptane afforded the desired product as a clear oil (997 mg, 2.84 mmol, 77%).
>H NMR (400 MHz, DMSO-d6) δ 7.68 - 7.61 (m, 4H), 7.53 - 7.40 (m, 6H), 3.54 (d, J = 5.7 Hz, 2H), 2.92 - 2.81 (m, 1H), 2.38 - 2.14 (m, 2H), 1.73 (t, J = 2.6 Hz, 3H), 1.62 - 1.44 (m, 2H), 1.01 (s, 9H).
Step H: benzyl N-(2-{[(2R) ·![(tert-butyldiphenylsilyl) oxy]hex-4-yn-2-yl]amino} ethyl) methylcarbamate
To a solution of the product of Step G (997 mg, 2.84 mmol, 1 eq) in tetrahydrofuran (24 mL) was added a solution of benzyl jV-methyl-7V-(2-oxoethyl)carbamate (647 mg, 3.12 mmol, 1.1 eq) in tetrahydrofuran (11.9 mL) and the mixture was cooled to 0 °C. Sodium triacetoxyborohydride (1.8 g, 8.51 mmol, 3 eq) was added and the mixture was allowed to wann to ambient température and stir for 4 h. The reaction was partitioned between ethyl acetate and IN aqueous sodium hydroxide, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography
208 ^b (CombiFlash Rf. 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 40% ethyl acetate in iso-heptane afforded the desired product as a clear oil (1.46 g, 2.69 mmol, 95%).
LC/MS (C33H42N2O3Si) 543 [M+H]+; RT 2.34 (LCMS-V-C) ’H NMR (400 MHz, DMSO-d6) δ 7.63 (d, J = 6.7 Hz, 4H), 7.52 - 7.39 (m, 6H), 7.37 - 7.24 (m, 5H), 5.03 (d, J = 2.6 Hz, 2H), 3.64 - 3.52 (m, 2H), 3.28 (t, J = 6.5 Hz, 2H), 2.86 (d, J =
4.2 Hz, 3H), 2.74 - 2.60 (m, 2H), 2.35 - 2.23 (m, 2H), 1.68 (t, 3H), 1.63 - 1.50 (m, 1H), 0.99 (s, 9H).
Step I: benzyl N-(2-{[(benzyloxy) carbonyl](methyl)amino}ethyl) -N-[(2R)-1 -[(tertbutyldiphenylsilyl)oxy]hex-4-yn-2-yl]carbamate
To a solution of the product from Step H (1.46 g, 2.69 mmol, 1 eq) in ethyl acetate (30 mL) was added a solution of sodium bicarbonate (497 mg, 5.92 mmol, 2.2 eq) in water (8.5 mL) and the mixture was cooled to 0 °C then benzyl chloroformate (0.46 mL, 3.23 mmol, 1.2 eq) was added and the mixture was allowed to warm to ambient température and stir ovemight. The reaction was partitioned between ethyl acetate and saturated aqueous sodium 15 bicarbonate, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo.
Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 30% ethyl acetate in tso-heptane afforded the desired product as a clear oîl (1.75 g, 2.59 mmol, 96%).
LC/MS (C4iH4sN2O5Si) 677 [M+H]+; RT 1.32 (LCMS-V-B2)
NMR (400 MHz, DMSO-d6) δ 7.67 - 7.50 (m, 4H), 7.49 - 7.22 (m, 16H), 5.16 - 4.90 (m, 4H), 4.19 - 4.07 (m, 1H), 3.79 - 3.51 (m, 2H), 3.44 - 3.34 (m, 2H), 3.28 - 3.15 (m, 2H), 2.92 - 2.71 (m, 3H), 2.45 - 2.17 (m, 2H), 1.64 (s, 3H), 0.95 (d, J = 4.4 Hz, 9H).
Step J: benzyl N-(2-{[(benzyloxy) carbonyl](methyl)amino}ethyl) -N-[(2R)-1 -[(tertbutyldiphenylsilyl)oxy]-3-(3,6-dichloro-5-inethylpyridazin-4-yl)propan-2'yl]carbaniate
To a solution of the product from Step I (1.75 g, 2.59 mmol, 1 eq) in toluene (35 mL) was added 3,6-dichloro-l,2,4,5-tetrazine (1.56 g, 10.3 mmol, 4 eq) and the mixture was heated at 150 ÙC in a sealed flask for 3 days. The reaction was concentrated in vacuo and purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 40% ethyl acetate in zso-heptane afforded the crude desired 30 product as a red gum (1.2 g, 1.5 mmol, 58%) that was used directly in the subséquent step without further purification.
209 φ LC/MS (CwHœNiOsSÎCh) 799 [M+H]·; RT 1.32 (LCMS-V-B2)
Step K·, benzyl N-(2-{[(benzyloxy)carbonyl](methyl)amino}ethyl)-N-[(2R)-l-(3,6-dichloro-5methylpyridazin-4-yl)-3-hydroxypropan-2-yl]carbamate
To a solution of the product from Step J (1.2 g, 1.5 mmol, 1 eq) in methanol (55 mL) was 5 added acetyl chloride (0.54 mL, 7.5 mmol, 5 eq) and the mixture was stirred at ambient température for 3 days. The reaction was concentrated in vacuo then partitioned between dichloromethane and water, and the organic phase was separated (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% methanol in 10 dichloromethane afforded the crude desired product as an orange gum (846 mg, 1.51 mmol, 100%) that was used directly in the subséquent step without further purification.
LC/MS (C27H30CI2N4O5) 561 [M+H]\ RT 0.80 (LCMS-V-B2)
Step L: methyl 2-{[(tert’butoxy)carbonyl]amino}-5-(3‘{4-[3-(dimethylamino)propyl]-2fluorophenoxy}propyl)-I,3-thiazole~4-carboxylate
To a solution of the product from Préparation 4d (652 mg, 3.31 mmol, 1.2 eq) in dimethylformamide (15 mL), cooled to 0 °C, was added sodium hydride (60% dispersion; 264 mg, 6.61 mmol, 2.4 eq) and the mixture was stirred for 30 min. A solution of the product from Préparation Id (1.17 g, 2.75 mmol, 1 eq) in dimethylformamide (15 mL) was added and the mixture was stirred at ambient température for 1 h. The reaction was partitioned between 20 ethyl acetate and brine, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 16% methanol in dichloromethane afforded the desired product as a clear gum (431 mg, 0.87 mmol, 32%).
LC/MS (C24H34FN3O5S) 496 [M+H]+; RT 1.81 (LCMS-V-C) XH NMR (400 MHz, DMSO-d6) δ 11.64 (br s, 1H), 7.13 - 7.00 (m, 2H), 6.97 - 6.89 (m, 1H), 4.04 (t, J = 6.2 Hz, 2H), 3.75 (s, 3H), 3.28 - 3.15 (m, 2H), 2.58 - 2.50 (m, 2H), 2.18 (t, J = 7.2 Hz, 2H), 2.12 (s, 6H), 2.12 - 2.00 (m, 2H), 1.71 - 1.59 (m, 2H), 1.47 (s, 9H).
Step Mt methyl 2-{[(2R)-2-{[(benzyloxy)carbonyl](2{[(benzyloxy) carbonyl](methyl)amino}ethyl)amino}-3-(3,6-dichloro-5-methylpyridazin-430 yl)propyl][ ftert-butoxy)carbonyl]amino}-5-(3-{4-[3-(dimethylamino)propyl]-2fluorophenoxy}propyl)-l,3-thiazole-4-carboxylate
210 φ Το a solution of the product from Step K (422 mg, 0.75 mmol, 1 eq) in toluene (25 mL) was added the product from Step L (431 mg, 0.87 mmol, 1.16 eq), followed by triphenylphosphine (394 mg, 1.5 mmol, 2 eq) and di-terz-butyl azodîcarboxylate (346 mg, 1.5 mmol, 2 eq) and the mixture was stirred at ambient température for 3 h. The reaction was partitioned between dichloromethane and water, and the organic phase was washed with brine, separated (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting with a gradient of 0 — 10% methanol in dichloromethane afforded the desired product as an orange gum (517 mg, 0.5 mmol, 66%).
LC/MS (C51H62CI2FN7O9S) 1040 [M+H]+; RT 2.48 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 7.31 (s, 27H), 7.24 (s, 2H), 7.11 - 6.95 (m, 8H), 6.90 (d, J = 8.6 Hz, 3H), 5.76 (s, 3H), 5.22 (s, 1H), 5.05 (s, 1H), 4.97 (s, 4H), 4.78 (s, 1H), 4.50 (d, J = 12.9 Hz, 1H), 4.00 (s, 7H), 3.69 (s, 6H), 2.94 (s, 3H), 2.82 (s, 3H), 2.76 (s, 1H), 2.37 (s, 1H), 2.30 (s, 1H), 2.20 (s, 7H), 2.13 (s, 17H), 2.09 (s, 1H), 2.05 (s, 3H), 1.96 (s, 5H), 1.64 (d, J = 15 8.1 Hz, 8H), 1.50 (s, 5H), 1.43 (d, J = 10.5 Hz, 6H).
Step N: methyl 2-{[(2R)-2-{[(benzyloxy)carbonyl](2{[ (benzyloxy)carbonyl](methyl)amino}ethyl)amino}-3-(3,6-dichloro-5-methylpyridazin-4yl)propyl]amino}-5-(3-{4-[3-(dimethylamino)propyl]-2-fluorophenoxy}propyl)-l,3-thiazole4-carboxylate
To a solution of the product from Step M (517 mg, 0.5 mmol, 1 eq) in dichloromethane (10 mL) was added trifluoroacetic acid (4 mL, 52.2 mmol, 105 eq) and the mixture was stirred at ambient température for 1 h. The reaction was diluted with dichloromethane and cooled to 0 °C and quenched with IN aqueous sodium hydroxide (60 mL), and the organic phase was washed with brine, separated (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 8% IN methanolic ammonia in dichloromethane afforded the desired product as an orange gum (461 mg, 0.49 mmol, 99%).
LC/MS (C46H54CI2FN7O7S) 939 [M+H]+; RT 2.16 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 7.88 (s, 1H), 7.48 - 7.18 (m, 10H), 7.11 - 6.88 (m, 3H), 30 5.12 - 4.82 (m, 4H), 4.06 - 3.90 (m, 2H), 3.76 - 3.58 (m, 4H), 3.53 - 3.36 (m, 2H), 3.29 -
3.21 (m, 2H), 3.17 - 3.03 (m, 4H), 3.01 - 2.81 (m, 3H), 2.79 - 2.69 (m, 2H), 2.36 - 2.24 (m, 4H), 2.18 (t, 2H), 2.11 (s, 6H), 2.00 - 1.93 (m, 3H), 1.64 (p, J = 7.3 Hz, 2H).
211
Step O: methyl 2-[(6R)-6-{[(benzyloxy)carbonyl](2{[ (benzyloxy) carbonyl](methyl)amino}ethyl)amino}-3-chloro-4-methyl-5H,6Hf 7H,8Hpyrido! 2,3-e]pyrida~indyl]-5-C3d4-l3-(dimethylamino)propyll-2-llu()iophciioxy}pr<)pyl)1,3-thiazole-4-carboxylate
To a solution of the product from Step N (461 mg, 0.49 mmol, 1 eq) in 1,4-dioxane (10 mL) was added césium carbonate (320 mg, 0.98 mmol, 2 eq) and XA-diisopropylethylamine (171 pL, 0.98 mmol, 2 eq). The mixture was sparged with nitrogen (10 min) then bis(di-terL butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(n) (34.8 mg, 0.05 mmol, 0.1 eq) was added and the mixture was heated at 80 °C for 3 h under microwave irradiation. The reaction was partitioned between ethyl acetate and water, and the organic phase was washed wîth brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 12% methanol in dichloromethane afforded the desired product as an orange gum (285 mg, 0.32 mmol, 64%).
LC/MS (C46H53CIFN7O7S) 902 [M+H]+; RT 2.32 (LCMS-V-C) ΣΗ NMR (400 MHz, DMSO-d6) δ 7.52 - 7.21 (m, 9H), 7.15 - 7.00 (m, 4H), 5.21 - 4.91 (m, 4H), 4.84 - 4.63 (m, 1H), 4.39 - 4.22 (m, 1H), 4.07 (t, J = 6.2 Hz, 2H), 4.05 - 3.91 (m, 1H), 3.77 (s, 3H), 3.56 - 3.43 (m, 2H), 3.41 - 3.36 (m, 2H), 3.31 (s, 2H), 3.30 - 3.23 (m, 2H), 3.01 - 2.75 (m, 4H), 2.28 (s, 2H), 2.18 (d, J = 6.9 Hz, 4H), 2.11 (s, 6H), 1.64 (p, J = 7.4 Hz, 2H).
Step P: methyl 2-[(6R)-3-[(l,3-benzothiazol-2-yl)amino]-6-{[(benzyloxy)carbonyl](2{[(benzyloxy) carbonyl](methyl) amino}ethyl)amino}-4-methyl-5H,6H, 7H,8H-pyrido[2,3c]pyridazin-8-yl]-5-(3-{4-[3-(dimethylamino)propyl]‘2'fluorophenoxy}propyl)-l ,3-thiazole4-carboxylate
To a solution of the product from Step O (117 mg, 0.13 mmol, 1 eq) in 1,4-dioxane (5 mL) was added 2-aminobenzothiazole (39 mg, 0.26 mmol, 2 eq), /V^-diisopropylethy lamine (67.9 pL, 0.39 mmol, 3 eq) and Josîphos Pd G3 (24 mg, 0.03 mmol, 0.2 eq) and the mixture was sparged with nitrogen (10 min) then heated at 140 °C for 3 h under microwave irradiation. The reaction was partitioned between ethyl acetate and water, and the organic phase was washed with brine, dried (magnésium sulfate) and concentrated üi vacuo. Purification by reverse phase automated flash chromatography (CombiFlash Rf, C18 13g RediSep column) eluting with a gradient of 5 - 95% acetonitrile in water afforded the crude
212 desired product as a yellow gum (23 mg, 0.02 mmol, 17%) that was used directly in the subséquent step without further purification.
LC/MS (C53H58FN9O7S2) 1017 [M+H]+; RT 2.43 (LCMS-V-C)
Step ()·. 2-[(6R)-3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6-{[2‘ (methylamino) ethyl]amino}-5H,6H, 7H,8H-pyrido[2,3-c]pyridazin-8-yl]-5-(3-{4-[3(dimethylamino)propyl]-2-fluorophenoxy}propyl)-l,3-thiazole-4-carboxylic acid
To a solution of the product from Step P (23 mg, 0.02 mmol, 1 eq) in 1,4-dioxane (0.5 mL)was added concentrated hydrochloric acid (1.5 mL) and the mixture was stirred at ambient température for 3 days. The reaction was concentrated in vacuo and purification by 10 automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 30% 7N methanolic ammonia in dichloromethane gave a solid that was triturated with dîethyl ether, filtered and dried under vacuum to afford the desired solid as a yellow solid (9.5 mg, 0.01 mmol, 57%).
HRMS-ESI (m/z) [M+H]+ calcd for C36H45FN9O3S2: 734.3071, found 734.3096.
Example 40: 2-{3-[(l,3-Benzothiazol-2-yI)amino]-4-methyl-5/f ,617,717,8/f-pyrido[2,3c]pyridazin-8-yl}-5-(3-{4-[3-(dimethylamino)piOp-l-yn-l-yl]-2(trifluoromethyl)phenoxy}propyl)-l,3-thiazole-4-carboxylic acid
Step A: ethyl 5-{3-[4-bromo-2-(trifluoromethyl)phenoxy]propyl}-2-(4-methyl-3-{[(2/)-3-{[220 (trimethylsilyl) ethoxy] methyl} -2,3-dïhydro-l,3-benzothiazol-2-ylidene]amino}5H,6H, 7H,8H-pyrido[2,3-c]pyridazin-8-yl) -1,3-thiazole~4-carboxylate
213
Diisopropyl azodicarboxylate (0.09 ml, 0.47 mmol, 1.5 eq) was added dropwise to a solution of the product from Préparation 3g (200 mg, 0.31 mmol, 1 eq), 4-bromo-2(trifluoromelhyl)phenol (100 mg, 0.41 mmol, 1.33 eq) and triphenylphosphine (123 mg, 0.47 mmol, 1.5 eq) in toluene (10 mL). The mixture was stirred at ambient température for 18 h 5 then concentrated in vacuo. Purification by flash column chromatography (20 g silica) eluting with a gradient of 0 - 25% ethyl acetate in zso-heptane afforded the desired product as a yellow solid (265 mg, 0.26 mmol, 84%).
LC/MS (C37H42BrF3N6O4SiS2) 863 [M+H]*; RT 1.91 (LCMS-V-B1) ‘H NMR (400 MHz, CDCh) δ 7.71 - 7.64 (m, IH), 7.62 - 7.52 (m, 2H), 7.39 - 7.34 (m, 2H), 10 7.24 - 7.17 (m, IH), 6.88 (d, J = 8.9 Hz, IH), 5.84 (s, 2H), 4.50 - 4.30 (m, 4H), 4.12 (t, J = .3 Hz, 2H), 3.82 - 3.68 (m, 2H), 3.39 - 3.30 (m, 2H), 2.87 (t, J = 6.4 Hz, 2H), 2.38 (s, 3H), 2.33 - 2.22 (m, 2H), 2.18 - 2.07 (m, 2H), 1.40 (t, J = 7.1 Hz, 3H), 1.01 - 0.90 (m, 2H), -0.08 (s, 9H).
Step B: ethyl 5-(3-{4-[3-(dimethylamino)prop-I-yn-l-ylj-215 (trifluoromethyl)phenoxy}propyl)-2-(4-methyl-3-{[(2Z) -3f[2-(trimethylsilyl)ethoxyjmethyl}2,3-dihydro-l ,3-benzothiazol-2-ylidene]amino}-5H,6H,7H,8H-pyrido[2,3-c]pyrÎdazin-8-yl)· 1,3-thiazole-4-carboxylate
To a solution of the product from Step A (265 mg, 0.26 mmol, 1 eq) in 2methyltetrahydrofuran (10 mL) was added copper(I) iodide (9.93 mg, 0.05 mmol, 0.2 eq) and 20 tetrakis(triphenylphosphine)palladium(0) (30.1 mg, 0.03 mmol, 0.1 eq) and dimethyl(prop-2yn-l-yl)amine (0.1 ml, 0.93 mmol, 3.5 eq). Λζ/V-diisopropylethylamine (0.14 ml, 0.78 mmol, 3 eq) was added and the mixture was heated at 75 °C for 96 h. The reaction was ailowed to cool to ambient température and concentrated in vacuo. Purification by flash column chromatography (20 g silica) eluting with a gradient of 0 - 100% ethyl acetate in wo-heptane 25 afforded the desired product as a dark yellow gum (170 mg, 0.2 mmol, 75%).
LC/MS (C42H5oF3N704SiS2) 866 [M+H]+; RT 1.57 (LCMS-V-B1) X H NMR (400 MHz, CDCh) S 7.66 (d, J = 2.3 Hz, IH), 7.59 (dt, J = 7.7, 0.9 Hz, IH), 7.54 7.50 (m, IH), 7.40-7.31 (m, 2H), 7.23-7.17 (m, IH), 6.92 (d,./ = 8.6 Hz, 1 H), 5.84 (s, 2H), 4.49 - 4.32 (m, 4H), 4.18 - 4.12 (m, 2H), 3.80 - 3.66 (m, 2H), 3.42 (s, 2H), 3.35 (dd, J = 8.5, 30 6.6 Hz, 2H), 2.87 (t, J = 6.4 Hz, 2H), 2.38 (s, 3H), 2.34 (s, 6H), 2.31 - 2.25 (m, 2H), 2.17 - .08 (m, 2H), 1.40 (t, J = 7.1 Hz, 3H), 0.99 - 0.90 (m, 2H), -0.07 (s, 9H).
214
Step C: 2·{3-[(l,3-benzothiazol-2-yl)aminoJ-4-methyl-5H,6H, 7H,8H-pyrido[2,3c]pyridazin-8-yl}-5-(3-{4‘[3-(dÎmethylamino)prop-l-yn~l-yl]-2(trifluoromethyl)phenoxy}propyl)-l,3-thiazole-4-carboxylic acid
To a solution of the product from Step B (160 mg, 0.18 mmol, 1 eq) in 1,4-dioxane (4.5 5 mL) at 0 °C was added trifluoroacetic acid (0.5 ml, 6.47 mmol, 35 eq) and the mixture was stirred for 36 h. The reaction was diluted with dichloromethane (50 mL) and successively washed with IM aqueous ammonia (20 mL), water (20 mL) and brine (20 mL), dried magnésium sulfate and concentrated in vacuo. To a suspension of the crude material in a mixture of water (1.5 mL) and tetrahydrofuran (1.5 mL) was added lithium hydroxide 10 monohydrate (23.3 mg, 0.55 mmol, 3 eq) and the mixture was stirred for 72 h at ambient température. Water (5 mL) was added and the suspension was neutralised with acetic acid. The solids were collected by filtration, washed with water (10 mL) and dried under vacuum to afford the desired product as a yellow solid (75 mg, 0.11 mmol, 57%).
HRMS-ESI (m/z) [M+HJ+ calcd for C34H33F3N7O3S2: 708.2038, found 708.2058
Exampie 41: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/f-pyrido[2,3c]pyridazin-8-yi]-5-[3-[2-fluoro-4-[3-(4-methylpiperazin-l-yl)but-lynyl]phenoxy]propyl]thiazole-4-carboxylic acid
Step A: methyl 2-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3-cJpyridazin-8-yl)-5-[3-[220 fluoro-4-[3-(4-methylpiperazin-l -yl)but-l -ynylJphenoxylpropyl]thiazole-4-carboxylate
A 24 mL oven-drîed vial was equipped with a PTEE-coated magnetic stirring bar, and was charged with 250 mg 1-methylpiperazine (2.5 mmol, 5.0 eq.) dissolved in 2.5 mL dry THF Then 133 mg 3-bromobut-l-yne (1.0 mmol, 2.0 equiv) was added dropwise via syringe over a period of 5 minutes, and stirred at that température for 30 min. To the resulting mixture 301
215 mg of Préparation 3a (0.50 mmol, 1.0 eq.), 18.15 mg Pd(PPh3)2Ch (0.025 mmol, 0.05 eq.) and 4.76 Cul (0.025 mmol, 0.05 eq.) were added, then it was heated to 60°C and stirred for 2h at that température. The reaction reached complété conversion. Celite was added to the reaction mixture and the volatiles were removed under reduced pressure. Then it was purified 5 via flash chromatography using DCM and MeOH (1.2% NH^) as eluents to give 300 mg (95% Yield) of the desired product.
Step B: methyl 2-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7~dihydro-5H-pyrido[2,3cjpyridazîn -8-yl]-5-[3-[2-fluoro-4-[3-(4-methylpiperazin-l -yl) but-1 ynyl]phenoxy]propyl]thiazole-4-carboxylate
Using Buchwald General Procedure II starting from 300 mg of the product from Step A (0.47 mmol, 1.0 eq.) and 140 mg l,3-benzothiazol-2-amine (0.94 mmol, 2.0 eq.), 150 mg (42%) mg of the desired product was obtained.
Step C: 2-[3-(l,3-benzothtazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-cJpyridazin8-yl]-5-[3-[2-fluoro-4-[3-(4-methylpiperazin-l -yl) but-1 -ynyl]phenoxy]propyl]thiazole-4- earboxylic acid
Using Hydrolysis General Procedure starting from the product from Step B as the appropriate methyl ester, the desired product was obtained.
4l NMR (500 MHz, DMSO-d6) δ ppm 7.87 (d, 1H), 7.49 (d, 1 H), 7.36 (t, 1H), 7.26 (dd, 1H), 7.2 (t, 1H), 7.16 (dd, 1H), 7.13 (t, 1H), 4.27 (t, 2H), 4.12 (t, 2H), 3.65 (q, 1H), 3.27 (t, 2H), 20 2.87 (t, 2H), 2.62-2.21 (brm, 8H), 2.14 (s, 3H), 2.13 (qn, 2H), 2.04 (qn, 2H), 1.33 (s, 3H),
1.25 (d, 3H); 13C NMR (125 MHz, DMSO-d6) δ ppm 164.3, 155.4, 151.5, 151.4, 148.6, 147.2, 145.1, 140.2, 136.3, 130.2, 129.0, 129.0, 127.6, 126.5, 122.5, 122.3, 119.2, 116.4, 115.5, 115.4, 88.4, 84.1, 68.5, 51.7, 46.3, 46.1, 31, 23.9, 23.0, 20.3, 19.6, 12.9;
HRMS-ESI (m/z) [M+Hf calcd for C37H40FN8O3S2: 727.2649, found 727.2630
Example 42: 2-[3-(l,3-Benzothiazol-2-ylaniino)-4-methyl-6,7-dihydro-5//-pyrido[2,3c]pyridazin-8-yl]-5-[3-[2-fluoro-4-(3-pyrrolidin-l-yIprop-lynyl)phenoxy]propyl]tliiazole-4-carboxylic acid
216
Step A: methyl 2-[3-(l,3-benzothiazol-2-ylammo)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-[3-[2-fluoro-4-(3-pyrrolidin-l -ylprop-I -ynyl)phenoxy]propyl]thiazole-4carboxylate
Using Propargylic amine préparation General Procedure starting from 258 mg of Préparation 3d (0.40 mmol, leq.) as the appropriate propargylic alcohol and pyrrolidine (20 eq, 670 mg), 120 mg of the desired product (43%) was obtained.
Step B: 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3-c]pyridazin8-yl]-5-[3-[2-fluoro-4-(3 -pyrrolidin -1 -ylprop-l -ynyl)phenoxy]propyl]thiazole-4-carboxylic 10 acid
Using Hydrolysis General Procedure starting from the product from Step A as the appropriate methyl ester, the desired product was obtained.
Ή NMR (500 MHz, DMSO-d6) δ ppm 7.88 (d, 1 H), 7.49 (d, 1H), 7.37 (t, 1H), 7.29 (dd, 1H), 7.2 (dd, 1H), 7.19 (t, 1H), 7.14 (t, 1H), 4.27 (t, 2H), 4.14 (t, 2H), 3.52 (s, 2H), 3.27 (t, 2H), 15 2.88 (t, 2H), 2.52 (t, 4H), 2.34 (s, 3H), 2.13 (qn, 2H), 2.04 (qn, 2H), 1.69 (t, 4H); 13C NMR (125 MHz, DMSO-d6) δ ppm 151.5, 151.4, 148.6, 147.3, 145.1, 140.1, 136.7, 130.2, 129.0, 129.0, 127.5,126.5,122.5, 122.3, 119.2, 116.5, 115.5,115.4, 85.9, 83.3, 68.6,52.3,46.3, 43.3, 31.1, 23.8, 23.8, 23.0, 20.4, 12.9; HRMS-ESI (m/z): [M+H]+ calcd for C35H35FN7O3S2: 684.2221, found 684.2209.
Exampie 43: 2-(3-(1,3-Benzothiazol-2-ylamino)-4-methyl-6î7-dihydro-5f/-pyrido[2,3c]pyridazin-8-yl]‘5»[3-[2-fluoro-4-(3-pyrrolidin-l-ylbut-l-ynyl)phenoxy]propyl]thiaz<»le4-carboxylic acid
217
Sien A: niethyl 2-(3-chloro-4-methyl-6,7-dihydro-5H~pyrido[2,3-c]pyridazin-8-yl)-5-[3-[2fluoro-4-(3-pyrrolidin-J -ylbut-1 -ynyl)phenoxy]propylJthiazole-4-carboxylate
A 24 mL oven-dried vial was equipped with a PTFE-coated magnetic stirring bar, and was 5 charged with 177 mg pyrrolidine (2.500 mmol, 5.0 eq.) dissolved in 2.5 mL dry THF Then 133 mg 3-bromobut-l-yne (1.0 mmol, 2.0 equiv) was added dropwîse via syringe over a period of 5 minutes, and stirred at thaï température for 30 min. To the resulting mixture 301 mg of Préparation 3a (0.50 mmol, 1.0 eq.), 18.15 mg PdiPPhQsCh (0.025 mmol, 0.05 eq.) and 4.76 Cul (0.025 mmol, 0.05 eq.) were added, then it was heated to 60°C and stirred for 2h 10 at lhat température. The reaction reached complété conversion. Celite was added to the reaction mixture and the volatiles were removed under reduced pressure. Then it was purified via flash chromatography using DCM and MeOH (1.2% NH^) as eluents to give 220 mg (73% Yield) of the desired product.
*H NMR (500 MHz, DMSO-d6) δ ppm 7.41 (d. J H), 7.28 (d, 1H), 7.18 (t, 1H), 4.51 (br., 1H), 15 4.26 (m, 2H), 4.13 (t, 2H), 3.77 (s, 3H), 3.5-2.97 (br., 4H), 3.25 (t, 2H), 2.88 (t, 2H), 2.32 (s,
3H), 2.11 (quint, 2H), 2.04 (m, 2H), 1.89 (br, 4H), 1.51 (brd, 3H); 13C NMR (125 MHz, DMSO-dû) δ ppm 163.1, 129.4, 119.6, 115.4, 68.3, 52.4/50.2, 52.0, 51.7, 46.3, 30.7, 24.2, 23.6, 23.0, 19.7, 19.3, 15.7.
Step B: methyl 2-[3-(l,3-benzothiazol-2-ylamino)-d-methyl-ôJ-dihydro-SH-pyridoftJ· 20 c]pyridazÎn-8-yl]-5-[3-[2-fluorO‘4-(3-pyrrolidin-J-ylbut-l-ynyl)phenoxy]propyl]thiazole-4carboxylate
Using Buchwald General Procedure II starting from 220 mg of the product from Step A (0.47 mmol, 1.0 eq.) and 100 mg l,3-benzothiazol-2-amine (0.668 mmol, 2.0 eq.), 150 mg (63% Yield) mg of the desired product was obtained.
218 φ Step C: 2-[3-(1,3-Benzothmzol-2-ylami.no) -4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-[3-[2-fluoro-4~(3-pyrrolidin-l-ylbut-l-ynyl)phenoxy]propyl]thiazole-4carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step B as the 5 appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C36H37FN7O3S2: 698.2377, found 698.2368.
Exampie 44: 2-{3-[( 1,3-Benzothiazol-2-yl)amino]-4-nieth\Ι-5/Λ6//,7//.8H-pyrido[2,3c]pyridazin-8-yl}-5-(3-{4-[2-(dimethylamino)ethoxy]phenoxy}propyl)-l,3-tliiazole-4carboxylic acid
Step A: ethyl5-(3-{4-[2-(dimethylamino)ethoxy]phenoxy}propyl)-2-(4-methyl-3-{[(2Z)-3{[2-(trimethylsilyl)ethoxy)methyl}-2,3-dihydro-l,3-benzothiazol-2-ylideue]amino}5H,6H,7H,8H-pyrido[2,3-c]pyridazin,-8-yl)-l,3-thiazole-4-carboxylate
To a solution of the product from Préparation 3g (100 mg, 0.16 mmol, 1 eq) and the product from Préparation 4e (56.6 mg, 0.31 mmol, 2 eq) in tetrahydrofuran (5 mL) was added triphenylphosphine (81.9 mg, 0.31 mmol, 2 eq) and di-tert-butyl azodicarboxylate (71.9 mg, 0.31 mmol, 2 eq) and the mixture was heated at 50 °C for 8 h. The reaction was partitioned between ethyl acetate and brine, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 11% methanol in dichloromethane afforded the desired product as a brown gum (35 mg, 0.04 mmol, 28%).
LC/MS (C4oH53N705SiS2) 804 [M+H]+; RT 2.72 (LCMS-V-C) ‘H NMR (400 MHz, DMSO-d6) δ 7.82 (dt, J = 7.7, 0.9 Hz, 1H), 7.48 - 7.38 (m, 2H), 7.28 25 7.20 (m, 1H), 6.92 - 6.83 (m, 4H), 5.85 (s, 2H), 4.33 - 4.22 (m, 4H), 4.03 - 3.89 (m, 4H), 3.76
219 • - 3.66 (m, 2Η), 3.30 - 3.19 (m, 2H), 2.88 (t, J = 6.2 Hz, 2H), 2.54 (dt, J = 11.9, 5.9 Hz, 2H), 2.37 (s, 3H), 2.16 (s, 6H), 2.14 - 2.00 (m, 4H), 1.30 (t, J = 7.1 Hz, 3H), 0.96 - 0.86 (m, 2H), 0.11 (s, 9H).
Step B: ethyl 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,35 c]pyridazin-8-y l}-5-(3-{4-[2-(dimethylamino)ethoxy]phenoxy}propy l) -1,3-thiazole-4carboxylate
To a solution of the product from Step A (35 mg, 0.04 mmol, 1 eq) in dichloromethane (2.7 mL), cooled to 0 °C, was added trifluoroacetic acid (0.3 mL, 3.92 mmol, 90 eq) and the mixture was allowed to warm to ambient température and stir ovemight. The reaction was 10 diluted with dichloromethane, cooled to 0 °C and neutralised with aqueous ammonia, then the organic phase was separated (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% methanol in dichloromethane afforded the desired product as a yellow solid (21 mg, 0.03 mmol, 72%).
LC/MS (C34H39N7O4S2) 674 [M+H]+; RT 2.12 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) Ô 11.48 (br s, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.38 (td, J = 8.1, 7.7, 1.3 Hz, 1H), 7.24 - 7.15 (m, 1H), 6.93 - 6.82 (m, 4H), 5.77 (s, 1H), 4.33 - 4.23 (m, 4H), 4.02 - 3.90 (m, 4H), 3.31 - 3.23 (m, 2H), 2.89 (t, J = 6.3 Hz, 2H), 2.58 - 2.51 (m, 2H), 2.35 (s, 3H), 2.17 (s, 6H), 2.13 - 2.01 (m, 4H), 1.31 (t, J = 7.1 Hz, 3H).
Step C: 2-{3-[(l,3-beiizofhiazpl-2-yl)ainitio/-4-niethyl-5H,6H,7H,8H-pyrido[2,3c]pyridazin-8-yl}-5-(3-{4-[2-(dimethylamino)ethoxy]phenoxy}propyl)-l,3-thiazole-4carboxylic acid
To a solution of the product from Step B (21 mg, 0.03 mmol, 1 eq) in 1,4-dioxane (2 mL) was added lithium hydroxide monohydrate (13.1 mg, 0.31 mmol, 10 eq) and the mixture was 25 heated at reflux for 15 h. The reaction was allowed to cool to ambient température and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 20% 7N methanolîc ammonia în dichloromethane afforded the desired product as a white solid (14.6 mg, 0.02 mmol, 73%).
HRMS-ESI (m/z) [M+H]+ calcd for C32H36N7O4S2: 646.2270, found 646.2292
220 φ Example 45: 5-(3-{4-[3-(Azetidin-l-yl)prop-l-yn-l-yl]-2-fluorophenoxy}propyl)-2-{3[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7//,8H-pyrido[2,3-e]pyridazin-8-yl}-l,3thiazole-4-carboxylic acid
Step A: methyl5-[3-[4-[3-(azetidin~l-yl)prop-l-ynyl]-2-fluoro-phenoxy]propyl]-2-[3-(l ,3benzothiawl-2-ylamino)-4-methyl-6,7-dihydra-5H-pyrido[2,3-c]pyridazin-8-yl]thiazole-4carboxylate
Using Propargylic amine préparation General Procedure starting from 258 mg of Préparation 3d (0.40 mmol, leq.) as the appropriais propargylic alcohol and azetidine (456.8 10 mg, 20 eq.), 36 mg of the desired product (36%) was obtained.
Step B: 5-(3-{4-[3~(azetidin-l~yl)prop-l-yn-1 -yl]-2~fluorophenoxy}propyl)-2-/3-/(1,3benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl}-l,3-thiazole4-carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step A as the 15 appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C34H33FN7O3S2: 670.2064, found 670.2065.
Example 46: 2-[3-(l,3-Benzothiazol-2-ylamΐno)-4-methyl·6,7-dihydro-5/7-pyrido[2,3c]pyridazin-8-yl]-5-[3-[2-fluoro-4-[3-(l-pÎperidyl)prop-l-ynyl]phenoxy]propyl]thiazole4-carboxylic acid
221
Step A: methyl 2-[3-(1,3-benzothiazol-2-ylamino)-4-methy 1-6,7~diliydt(>-5H-pyrido[2,3c]pyridazin -8-yl]-5-[3~[2-fluoro-4-[3-(l -piperidyl)prop-l-ynyl]phenoxy}propyl]thiazole-4carboxylate
Using Propargylic amine préparation General Procedure starting from 100 mg of Préparation 3d (0.155 mmol, leq.) as the appropriate propargylic alcohol and piperidine (264.2 mg, 20 eq.), 55 mg of the desired product (50%) was obtained.
Step B: 2-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin8-yl]-5-[3-[2-fluoro-4-[3-(l -piperidyl)prop-l -ynyl]phenoxy]propyl]thiazole-4-carboxylic acid
Using Hydrolysis General Procedure starting from the product of Step A as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C36H37FN7O3S2: 698.2377, found 698.2373.
Example 47: 2-(3-(1,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido [2,315 c]pyridazin-8-yl]-5-[3-[2-fiuoro-4-(3-morpholinoprop-l-ynyl)phenoxy]propyl]thiazole-4carboxylic acid
222 φ Step A: methyl2-/3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3cjpyridazin -8-yl]-5 f3-[2-fluoro-4-(3-morpholinoprop-l-ynyl)phenoxy]propyl]thiazole-4carboxylate
Using Propargylic amine préparation General Procedure starting from 100 mg of 5 Préparation 3d (0.155 mmol, leq.) as the appropriate propargylic alcohol and morpholine (270.3 mg, 20 eq.), 191 mg of the desired product (86%) was obtained.
Step B: 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin8~yl]-5-(3-[2-fluoro-4-(3-morpholinoprop-l-ynyl)phenoxy]propyl]thiazole-4-carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step A as the 10 appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C35H35FN7O4S2: 700.2170, found 700.2163.
Example 48: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-[3-[2-fluoro-4-[3-(4-methyIpiperazin-l-yl)prop-lynyl]plienoxy]propyl] thiazole-4-carboxylic acid
Step A: methyl 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3c]pyridazin-8-yl]-5-(3-[2-fluoro-4-f3-(4-methylpiperazin-I -yl)prop-l ynyl]phenoxy]propyl]thiazole-4-carboxylate
Using Propargylic amine préparation General Procedure starting from 100 mg of 20 Préparation 3d (0.155 mmol, leq.) as the appropriate propargylic alcohol and 1methylpiperazine (310.7 mg, 20 eq.), 150 mg of the desired product (79%) was obtained.
223
StepJi: 2-(3-(1,3-benzothiazol-2-ylamino) -4-methyl-6,7-dihydro-5H-pyrido(2,3-c]pyridaziu8-yl]-5-(3-(2-fluoro-4-(3-(4-methylpiperazin-l-yl)prop-l-ynyl]phenoxy]propyl] thiazole-4carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step A as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C36H38FN8O3S2: 713.2486, found 713.2474.
Example 49: 2-{3-[(1,3-BenzothiazoI-2-yl)amino]-4-methyl-5H,6H,ΊΗ,SH-pyrido[2,3c]pyridazin-8-yl}-5-(3-{4-[3-(4,4-difluoropiperidÎn-l-yl)prop-l-yn-l-yl]-2fluorophenoxy}propyl)-l,3-thiazole-4-carboxylic acid
Step A: methyl 2-(3-(1,3-benzothiazol-2-ylamind)-4-methyl-6,7-dihydro-5H-pyrido(2,3c]pyridazin-8-yl]-5-(3-(4-(3-(4,4-difluoro-l -piperidyl)prop-l -ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylate
Using Propargylic amine préparation General Procedure starting from 100 mg of Préparation 3d (0.155 mmol, leq.) as the appropriate propargylic alcohol and 4,4difluoropîperidine (20 eq.), 120 mg of the desired product (72%) was obtained.
Step B: 2-{3-((l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido(2,3c]pyridazin-8-yl}-S-(3-{4-(3-(4,4-difluoropiperidin -1 -yl)prop-l -yn-1 -yl]-2fluorophenoxy}propyl)-l,3-thiazole-4-carhoxylic acid
Using Hydrolysis General Procedure starting from the product from Step A as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C36H35F3N7O3S2: 734.2189, found 734.2185.
224 φ Example 50: 2-{3-[(1,3-BenzothiazoI-2-yl)amino]-4-methyΊΗ,8/Lpyrido[2,3c]pyridazïn-8-yI}-5-(3-{4-[3-(3,3-difluoropiperidin-l-yl)prop-l-yn-l-ylJ-2fluorophenoxy}propyl)-l,3-thiazole-4-carboxylic acid
Step A: methyl 2-[3-(l,3~benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3cjpyridazin -8-ylJ-5-[3-[4-[3-(3t3-difluoro-l -piperidyl) prop-1 -ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylate
Using Propargylic amine préparation General Procedure starting from 100 mg of Préparation 3d (0.155 mmol, leq.) as the appropriate propargylic alcohol and 3,ΒΙΟ difluoropiperidine, hydrogen chloride (1:1) (488.9 mg, 20 eq.), 30 mg of the desired product (26%) was obtained.
Step B: 2-{3-[(l,3-Benzothiazol-2‘yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,3c]pyridazin-8-yl}-5-(3~{4-[3-(3,3-difluoropiperidin -1 -ybprop-l ~yn-l -ylJ-2fluorophenoxy}propyl)-l,3-thiazole-4-carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step A as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C36H35F3N7O3S2: 734.2189, found 734.2186.
Example 51: 2“{3-[(l,3-Benzothiazol-2-yl)amino]-4-methyl-5Ef,6/7,7H,8H-pyrido[2,3e|pyridazin-8-yl}-5-(3-{2-chloro-4-[3-(dimethylammo)prop-l-yn-l-yl]phenoxy}propyl)20 l,3-thiazole-4-carboxylic acid
225
StepA: methyl5-[3-(2-chloro-4-iodophenoxy)propyl]-2-(4-methyl-3-{[(2Z)-3-{[2(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3-benzothiazol-2-ylidene]amino}5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl)-l,3-thiazole-4-carboxylate
To a solution of product from Préparation 3b (225 mg, 0.32 mmol, 1 eq) in toluene (10 mL) was added 2-chloro-4-iodophenol (100 mg, 0.39 mmol, 1.22 eq), followed by triphenylphosphine (127 mg, 0.48 mmol, 1.5 eq) and diisopropyl azodicarboxylate (0.1 ml, 0.48 mmol, 1.5 eq) and the mixture was stirred at ambient température for 3 h. Purification by flash column chromatography (20 g siiica) eluting with a gradient of 0 - 30% ethyl acetate in 10 zso-heptane afforded the desired product as a yellow solid (235 mg, 0.23 mmol, 72%).
LC/MS (C35H40CIIN6O4S1S2) 863 [M+H]+; RT 1.48 (LCMS-V-B2)
Ή NMR (400 MHz, CDCI3) 5 7.65 (d, J = 2.1 Hz, 1H), 7.59 (dt, J = 7.7, 0.9 Hz, 1H), 7.47 (dd, J = 8.6, 2.2 Hz, 1H), 7.39 - 7.35 (m, 2H), 7.24 - 7.17 (m, 1H), 6.67 (d, J = 8.6 Hz, 1H), 5.84 (s, 2H), 4.47 - 4.35 (m, 2H), 4.13 - 4.06 (m, 2H), 3.90 (s, 3H), 3.80 - 3.70 (m, 2H), 3.43 15 - 3.34 (m, 2H), 2.87 (t, J = 6.3 Hz, 2H), 2.38 (s, 3H), 2.34 - 2.22 (m, 2H), 2.19 - 2.06 (m,
2H), 1.02-0.91 (m, 2H), -0.07 (s, 9H)^
Step B: methyl 5-(3f2-chloro~4-[3-(dimethylamiiio)prop-l-yn-1 -yl]phenoxy}propyl)-2-(4methyl-3-{[(2Z)-3-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3-benzothÎazol-2ylidenejamino} -5H,6H, 7H,8H-pyrido[2,3-c]pyridazin -8-yl) -1,3-thiazole-4-carboxylate
Dimethyl(prop-2-yn-l-yl)amine (0.1 ml, 0.93 mmol, 4.0 eq) was added to a solution of the product from Step A (235 mg, 0.23 mmol, 1 eq), copper(I) iodide (8.81 mg, 0.05 mmol, 0.2 eq) and tetrakis(triphenylphosphine)palladinm(0) (26.7 mg, 0.02 mmol, 0.1 eq) in 2methyltetrahydrofuran (10 mL), then AQV-diisopropylethylamine (0.15 ml, 0.69 mmol, 3 eq) was added and the mixture was heated at 75 °C for 18 h. The reaction was aliowed to cool
226 to ambient température and purification by flash column chromatography (20 g silica) eluting with a gradient of 0 - 100% ethyl acetate in iso-heptane afforded the desired product as a brown gum (75 mg, 0.09 mmol, 40%).
LC/MS (C4oH48ClN704SiS2) 818 [M+H]+; RT 1.56 (LCMS-V-B1)
H NMR (400 MHz, CDCb) δ 7.70 - 7.63 (m, 1H), 7.59 (dt, J = 7.6, 0.9 Hz, 1H), 7.48 - 7.44 (m, 1H), 7.40 - 7.35 (m, 2H), 7.23 - 7.16 (m, 1H), 6.84 (d, J = 8.6 Hz, 1H), 5.84 (s, 2H), 4.46 - 4.36 (m, 2H), 4.18 - 4.05 (m, 2H), 3.90 (s, 3H), 3.79 - 3.69 (m, 2H), 3.49 - 3.34 (m, 4H), 2.87 (t, J = 6.5 Hz, 2H), 2.39 (s, 3H), 2.35 (s, 6H), 2.32 - 2.25 (m, 2H), 2.18 - 2.08 (m, 2H), 1.01-0.93 (m, 2H), -0.07 (s, 9H).
Step C: 2-(3-/1l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H~pyrido[2,3cjpyridazin -8-yl}-5-(3-{2-chloro-4-[3-(dimethylamino)prop-l -yn-1 -yl]phenoxy}propyl)-l ,3thiazole-4-carboxylic aeid
To a solution of the product from Step B (75 mg, 0.09 mmol, 1 eq) in dichloromethane (1.8 mL), cooled to 0 °C, was added trilluoroacetic acid (0.2 ml, 2.75 mmol, 30 eq) and the mixture was stirred for 36 h. The reaction was diluted with dichloromethane (20 mL), successively washed with IM aqueous ammonia (10 mL), water (10 mL) and brine (10 mL), dried (magnésium sulfate) and concentrated in vacuo. The crude material was suspended in a mixture of water (1.5 mL) and methanol (0.5 mL), lithium hydroxide monohydrate (11.5 mg, 0.27 mmol, 3 eq) was added, and the suspension was heated at 80 °C for 18 h. The reaction was neutralised with acetic acid and the solids were collected by filtration and washed with water (20 mL). Purification by préparative HPLC (HPLC-V-A2) afforded the desired product as a pale yellow solid (7 mg, 0.01 mmol, 11%).
HRMS-ESI (m/z) [M+HJ+ calcd for C33H33CIN7O3S2: 674.1775, found 674.1796.
Example 52: 2-{3-|( 1.3-Benzoth iazol-2-yl (amino)-4-metlivl-5//.6//,7//,8//-pyrido [2.3c]pyridazin-8-yl}-5-(3-{4-[2-(pyrrolidin-l-yl)ethoxyîphenoxy}propyl)-l,3-thiazole-4carboxylic acid
227
Step A: ethyl 2-(4-methyl-3-{[(2Z)-3-{/2-(trimethylsilyl)ethoxy}methyl}-2>3-dihydro-lr3benzothiazol‘2-ylideneJamino}-5Ht6H,7H,8H-pyrido[2>3-eJpyridazin-8-yl)-5-(3-{4-(2(pyrrolidin-l-yl) ethoxy]phenoxy}propyl) -l,3-thiazole-4-carboxylate
To a solution of the product from Préparation 3g (150 mg, 0.23 mmol, 1 eq) and the product from Préparation 4f (97 mg, 0.47 mmol, 2 eq) in toluene (6 mL) was added triphenylphosphine (123 mg, 0.47 mmol, 2 eq) and di-ierr-butyl azodicarboxylate (108 mg, 0.47 mmol, 2 eq) and the mixture was heated at 50 °C overnight. The reaction was partitioned between dichloromethane and brine, and the organic phase was dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFIash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 12% methanol in dichloromethane afforded the desired product as a yellow gum (165 mg, 0.2 mmol, 85%).
LC/MS (C42H55N7O5S1S2) 831 [M+H]+; RT 2.75 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 7.82 (d, 1H), 7.48 - 7.39 (m, 2H), 7.28 - 7.20 (m, 1H), 15 6.92 - 6.82 (m, 4H), 5.84 (s, 2H), 4.34 - 4.21 (m, 4H), 4.02 - 3.91 (m, 4H), 3.76 - 3.66 (m,
2H), 3.26 (t, 2H), 2.87 (t, J = 6.2 Hz, 2H), 2.71 (t, 1H), 2.48 - 2.42 (m, 4H), 2.37 (s, 3H), 2.13 - 2.00 (m, 4H), 1.71 - 1.60 (m, 4H), 1.30 (t, J = 7.1 Hz, 3H), 0.95 - 0.86 (m, 2H), -0.11 (s, 9H).
Step B: ethyl 2‘{3-[(l,3-benzothiazol-2-yl)amino]-4-tnethyl-5H,6H)7H>8H-pyrido[2,320 c]pyridazin-8-yl}-5-(3-{4-[2-(pyrrolidin-l-yl)ethoxy]phenoxy}propyl)-l,3-thiazole-4carboxylate
To a solution of the product from Step A (165 mg, 0.2 mmol, 1 eq) in dichloromethane (8.1 mL), cooled to 0 °C, was added trifluoroacetic acid (0.91 mL, 11.9 mmol, 60 eq) and the mixture was stirred at ambient température overnight. The reaction was diluted with 25 dichloromethane, cooled to 0 °C, neutralised with aqueous ammonia and the organic phase was separated (PTFE phase separator) and concentrated in vacuo. Purification by automated
228 φ flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 14% methanoi in dichloromethane afforded the desired product as a yellow solid (84 mg, O.f2 mmol, 60%).
LC/MS (C36H41N7O4S2) 700 [M+H]+; RT 2.14 (LCMS-V-C) ‘H NMR (400 MHz, DMSO-d6) δ 11.48 (br s, IH), 7.89 (d, J = 7.7 Hz, IH), 7.50 (d, J = 7.9 Hz, IH), 7.42 - 7.34 (m, IH), 7.20 (td, J - 7.5, 1.2 Hz, IH), 6.94 - 6.83 (m, 4H), 4.34 - 4.21 (m, 4H), 3.98 (t, J = 6.2 Hz, 4H), 3.32 - 3.23 (m, 2H), 2.89 (t, J = 6.4 Hz, 2H), 2.85 - 2.74 (m, 2H), 2.65 - 2.53 (m, 4H), 2.35 (s, 3H), 2.16 - 1.99 (m, 4H), 1.70 (s, 4H), 1.31 (t, J = 7.1 Hz, 3H).
Step C: 2-{3~[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2t3c]pyridazin-8-yl}-5-(3-{4-[2-(pyrrolidin-l -yl) ethoxy]phenoxy}propyl) -l,3-thiazole-4carboxylic acid
To a solution of the product from Step B (84 mg, 0.12 mmol, 1 eq) in 1,4-dioxane (4 mL) was added lithium hydroxîde monohydrate (50.4 mg, 1.2 mmol, 10 eq) and the mixture was heated 15 at reflux overnight. The reaction was allowed to cool to ambient température and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 15% 7N methanolic ammonia in dichloromethane gave a solid that was trîturated with diethyl ether, filtered and dried under vacuum to afford the desired product as a yellow solid (52.9 mg, 0.08 mmol, 66%).
LC/MS (C34H37N7O4S2) 672 [M+H]+; RT 1.93 (LCMS-V-C)
Έ NMR (400 MHz, DMSO-d6) δ 7.89 (dd, J = 7.9, 1.2 Hz, IH), 7.50 (d, J = 8.1 Hz, IH), 7.38 (td, J = 7.7, 1.3 Hz, IH), 7.20 (td, J = 7.6, 1.2 Hz, IH), 6.94 - 6.81 (m, 4H), 4.28 (dd, J = 7.3, 4.3 Hz, 2H), 4.05 - 3.91 (m, 4H), 3.35 - 3.17 (m, 6H), 2.89 (t, J = 6.2 Hz, 2H), 2.76 (t, J = 5.9 Hz, 2H), 2.35 (s, 3H), 2.18 - 1.96 (m, 4H), 1.73 - 1.62 (m, 4H).
HRMS-ESI (m/z) [M+H]+ calcd for C34H38N7O4S2: 672.2427, found 672.2449
Example 53: 2-{3-[(^,3-Benzothΐazol·2-yl)aIllino]-4-methyl·5ff,6H,7/f,8H-pyridü[2,3c]pyridazin-8-y]}-5-(3-{4-[2-(dimethyIamino)ethyi]-2-fluorophenoxy}propyl)-l,3thiazole-4-carboxylic acid
229
Step A: methyl 5-(3-{4-[2-(dimethylaminô)ethyl]-2-fiuorophenoxy}propyl)-2-(4-methyl-3{[(2Z)-3-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3-beiizothiazol-2-ylidene]amino}5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl)-l,3-thiazole-4-carboxylate
To a solution of the product from Préparation 3b (95 mg, 0.15 mmol, 1 eq) and the product from Préparation 4g (41 mg, 0.22 mmol, 1.48 eq) in toluene (5 mL) was added di-ter+butyl azodicarboxylate (69.8 mg, 0.3 mmol, 2 eq) and triphenylphosphine (79.5 mg, 0.3 mmol, 2 eq) and the mixture was heated at 50 °C for 20 h. The reaction was partitioned between dichloromethane and water, and the organic phase was separated (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 5% methanol in dichloromethane afforded the desired product as a yellow oil (83 mg, 0.1 mmol, 69%).
LC/MS (C39H50FN7O4S1S2) 792 [M+H]*; RT 2.69 (LCMS-V-C) ‘H NMR (400 MHz, DMSO-d6) δ 7.82 (dd, J = 7.3, 1.1 Hz, 1H), 7.48 - 7.38 (m, 2H), 7.28 15 7.20 (m, 1H), 7.14 - 7.02 (m, 2H), 6.98 - 6.92 (m, 1H), 5.85 (s, 2H), 4.26 (dd, J = 7.2, 4.4 Hz,
2H), 4.08 (t, J = 6.2 Hz, 2FI), 3.79 (s, 3H), 3.75 - 3.67 (m, 2H), 3.31 - 3.23 (m, 2H), 2.88 (l, J = 6.4 Hz, 2H), 2.65 - 2.56 (m, 2H), 2.46 - 2.37 (m, 2H), 2.37 (s, 3H), 2.12 (s, 6H), 2.11 - 2.07 (m, 2H), 2.07 - 2.00 (m, 2H), 0.96 - 0.88 (m, 2H), -0.11 (s, 9H).
Step B: methyl 2-{3-[(1,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyridol2,320 c]pyridazin-8-yl}-5-(3-{4-[2-(dimethylamino)ethyl]-2-fluorophenoxy}propyl)-l,3-thiazole-4carboxylate
To a solution of the product from Step A (83 mg, 0.1 mmol, 1 eq) in dichloromethane (5 mL) was added trifluoroacetic acid (1 mL, 12.99 mmol, 124 eq) and the mixture was stirred at ambient température overnight. The reaction was diluted with dichloromethane, cooled to 0 25 °C, neutralised with aqueous ammonia, and the organic phase was separated (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column
230 φ chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 8% methanol in dichloromethane afforded the desired product as a yellow solid (50 mg, 0.08 mmol, 72%).
LC/MS (C33H36FN7O3S2) 662 [M+H]+; RT 2.04 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) δ 11.48 (br s, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.40 - 7.34 (m, 1H), 7.19 (td, J = 7.6, 1.2 Hz, 1H), 7.13 - 7.02 (m, 2H), 6.98 - 6.92 (m, 1H), 4.30 - 4.21 (m, 2H), 4.08 (t, 2H), 3.78 (s, 3H), 3.32 - 3.24 (m, 2H), 2.87 (t, J = 6.3
Hz, 2H), 2.62 (dd, J = 8.6, 6.6 Hz, 2H), 2.46 - 2.38 (m, 2H), 2.34 (s, 3H), 2.14 (s, 6H), 2.13 2.09 (m, 2H), 2.08 - 2.01 (m, 2H).
Step C: 2-{3-[(1,3-b(nizolhi(i-ol-2-yl)amnwl-4-nuthyl-5H,6)H,711,8ll-pyrid<>l2,3c]pyridazui-8-yl}-5-(3-{4-[2-(dimethylamino)ethyl]-2-fluorophenoxy}propyl)-l,3’thiazole‘4carboxylic acid
To a solution of the product from Step B (50 mg, 0.08 mmol, 1 eq) in 1,4-dioxane (3 mL) was added lithium hydroxide monohydrate (31.7 mg, 0.76 mmol, 10 eq) and the mixture was 15 heated at 70 °C overnight. The reaction was allowed to cool to ambient température and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 15% 7N melhanolic ammonia in dichloromethane afforded the desired product as a yellow solid (24.68 mg, 0.Ü4 mmol, 50%).
LC/MS (C32H34FN7O3S2) 648 [M+H]+; RT 1.87 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.89 (d, J = 7.8 Hz, 1H), 7.50 (d, J = 8.1 Hz, 1H), 7.38 (td, J = 8.1, 7.7, 1.3 Hz, 1H), 7.20 (td, J = 7.5, 1.2 Hz, 1H), 7.14 - 7.02 (m, 2H), 6.95 (dt, J = 8.4, 1.4 Hz, 1H), 4.32 - 4.25 (m, 2H), 4.09 (t, J = 6.3 Hz, 2H), 3.31 - 3.23 (m, 2H), 2.89 (t, J = 6.3 Hz, 2H), 2.63 (t, J = 7.6 Hz, 2H), 2.44 (dd, J = 8.5, 6.6 Hz, 2H), 2.35 (s, 3H), 2.17 (s, 6H), 25 2.15-2.08 (m, 2H), 2.08 - 2.00 (m, 2H)
HRMS-ESI (m/z) [M+H]+ calcd for C32H35FN7O3S2: 648.2227, found 648.2269.
Example 54: 3-[2-r3-[4-[3-[2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5J/pyrido[2,3-c]pyridazin-8-yl]-4-carboxy-thiazol-5-yl]propoxy]-3-fluorO-phenyl]prop-2ynylammo]ethyl-dimethyl-ammonio]propane-l-sulfonate
231
Step A: 3-[2-[tert-butoxycarbonyl-[3-[4-[3-[2-(3-chloro-4-methyl-6,7~dihydro-5Hpyrido[2,3-c]pyridazin-8-yl)-4-methoxycarbonyl-thiazol-5-yl]propoxy]-3-fluorophenyl]prop-2-ynyl]amino]ethyl~dimethyl-animonio]propane-l-sulfonate
A mixture of 384 mg of Example 76 (Step Α)φ.55 mmol, 1 eq.) and 1944 mg of oxathiolane 2,2-dioxide (15.92 mmol, 30 eq.) in acetonîtrîle (4 mL/mmoI) and DMF (1 mL/mmol) was stirred at rt. After reaching appropriate conversion, the volatiles were removed under reduced pressure and purified via flash column chromatography (SiO2, EtOAc : 0.6 M NHj in MeOH) to obtain 94 mg (29%) of the desired product.
LC-MS-ESI (m/z): [M+H]+ calcd for C37H49CIFNÛO8S2: 823.3, found 823.2.
Step B: 3-[2-[3-[4-[3-[2-[3-(l,3-benzothiazol-2-ylamino)-4-methyl~6,7-dihydro-5H· pyrido[2,3-c]pyridazin-8-yl]-4-methoxycarbonyl‘thiazol-5~yl]propoxy]-3-fluorophenyl]prop-2-ynyl-tert-butoxycarbonyl-amino]ethyl-dimethyl-ammonio]propane-lsulfonate
Using Buchwald General Procedure II starting from the product from Step A and 1,3benzothiazol-2-amine, the desired product was obtained.
Ή NMR (500 MHz, DMSO-rfc) δ ppm 7.88 (d, 1H), 7.49 (d, 1H), 7.37 (t, 1H), 7.35 (d, 1H), 7.25 (dd, 1H), 7.20 (t, 1H), 7.18 (t, 1H), 4.27 (s, 2H), 4.26 (t, 2H), 4.15 (t, 2H), 3.77 (s, 3H), 3.69 (t, 2H), 3.46 (t, 2H), 3.45 (t, 2H), 3.28 (t, 2H), 3.07 (s, 6H), 2.88 (t, 2H), 2.46 (t, 2H), 20 2.34 (s, 3H), 2.13 (qn, 2H), 2.04 (qn, 2H), 2.02 (qn, 2H), 1.44 (s, 9H); 13C NMR (125 MHz,
DMSO-ify) δ ppm 129.2, 126.5, 122.6,122.4, 119.3, 116.9, 115.5, 85.4, 82.5, 68.4, 63.3, 60.1, 52.0, 50.8, 48.0, 46.4, 40.8, 37.9, 31.0, 28.4, 23.9, 23.2, 20.4, 19.5, 13.0; HRMS-ESI (m/z): [M+H]+ calcd for C44H54FN8O8S3: 937.3205, found 937.3209.
232
5/ep C: 3-[2-[3-[4-[3-[2-[3-(l,3-Benzothiazol-2-ylamino) -4-methyl-6,7-dihydro-5Hpyrido[2,3-c]pyridazin-8-yl]-4-carboxy-thiazol-5-yl]propoxy]-3-fluoro-phenyl]prop-2ynylaminojethyl-diinethyl-ammon io]propane~l -sulfonate
Using Deprotection and Hydrolysis General Procedure followed by repurification via 5 reverse phase préparative chromatography (C18, 25 mM NH4HCO3 in water : MeCN) starting from the product from Step B, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C38H44FN8O6S3: 823.2524, found 823.2523.
Example 55: 2-|3-(l,3-Benzothiazol-2-ylammo)-4-methyl-6,7-dîhydro-5/f-pyrido[2,3cJpyridazin-8-yl]-5-[3-[2-fluoro-4-[3-(3-hydroxy-l-piperidyl)prop-l10 ynyl]phenoxy]propyl]thiazole-4-carboxyIic acid
Step A: methyl 2-[3-(l,3-benzothiaz.ol-2-ylaniino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-[3-[2-fluoro-4-[3-(3-hydroxy-l-piperidyl)prop-Iynyl]phenoxy]propyl]thiazole-4-carboxylate
Using Propargylic amine préparation General Procedure starting from 100 mg of Préparation 3d (0.155 mmol, leq.) as the appropriate propargylic alcohol and piperidin-3-ol (313.8 mg, 20 eq.), 70 mg of the desired product (62%) was obtained.
Step B: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro~5H-pyrido[2,3c]pyridazin’8-yl]-5-[3-[2-fluoro-4-[3-(3-hydroxy-l-piperidyl)prop~l· ynyl]phenoxy]propyl]thiazole-4-carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step A as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C36H37FN7O4S2: 714.2327, found 714.2323.
233
Example 56: 5-[3-[4-[3-[(lS,5R)-3-Azabicyclo[3.1.0]hexan-3-yl]prop-l-ynyl]-2-fluorophenoxy]propyl]-2-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5Hpyrido[2,3-c]pyridazin-8-yl]thiazole-4-carboxylic acid hn s
Step A: methyl5-[3-[4-[3-[(IRflSj-d-azabicyclolS.l.Ojhexan-S-ylJprop-l-ynylj^-fluorophenoxy]propyl]-2-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-SH-pyrido[2,3c]pyridazin-8-yl]thiazole-4~carboxylate
Using Propargylic amine préparation General Procedure starting from 100 mg of Préparation 3d (0.155 mmol, leq.) as the appropriate propargylic alcohol and (lR,5S)-310 azabicyclo[3.1.0]hexane (20 eq.), 150 mg of the desired product (81%) was obtained.
Step B: 5-[3-[4-[3-[(lS,5R)-3-Azabicyclo[3.1.0]hexan-3-yl]prop-l-ynyl]-2-fluorophenoxy]propyl]-2-[3-(l,3-benzothiazol-2‘ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3~ cjpyridazin -8-yl]thiazole-4-carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step A as the 15 appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+HJ+ calcd for C36H35FN7O3S2: 696.2221, found 696.2227.
Exampie 57: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-57/-pyrido[2,3c]pyridazin-8-yl]-5-[3-[2-fluorO'4-[3-[4-(l-piperidyl)-l-piperidylJprop-lynyljphenoxy]propyl] tliiazole-4-carboxylic acid
234
Step A: methyl 2-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3cJpyridazin-8-yl]~5-[3-[2-jluoro-4-[3-[4-(l -piperidyl) -1 -piperidyl]prop-l ynyl]phenoxy]propyl]thiazole-4-carboxylate
Using Propargylic amine préparation General Procedure starting from 100 mg of Préparation 3d (0.155 mmol, leq.) as the appropriate propargylic alcohol and l-(4piperidyl)piperidine, hydrogen chloride (1:2) (748.3 mg, 20 eq.), 100 mg of the desired product (81%) was obtained.
Step 13: 2-[3-(1,3-benzothiazol-2-ylamino) -4-methyl-6,7-dihydro-5H-pyrido[2f3-c]pyridazin10 8-yl]-5-[3-[2-fluoro-4-]3-[4-(l-piperidyl)-!-piperidyl]prop-1 -ynyl]phenoxy]propyl] thiazole4-carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step A as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C41H46FN8O3S2; 781.3112, found 781.3112.
Example 58: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-57/-pyrido[2,3cJpyridazin-8-yl]-5-[3-[2-fluoro-4-[3-(3-oxo-2,8-diazaspiro[4.5]decan-8-yl)prop-l-ynyl] phenoxy]propyl]thiazole-4-carboxylic acid
235
Step A: methyl2-(3-(1,3-benzothiazol~2-ylamind)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-(3-(2-fluoro-4-(3-(3-oxo-2,8-diazaspiro(4.5]decan-8-yl)prop-lynyl]pheuoxy]propyl]thiazole-4-carboxylate
Using Propargylîc amine préparation General Procedure starting from 100 mg of Préparation 3d (0.155 mmol, leq.) as the appropriate propargylîc alcohol and 2,8dîazaspiro[4.5]decan-3-one (478.4 mg, 20 eq.), 125 mg of the desired product (82%) was obtained.
Step B: 2-(3-(1 r3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3-c]pyridazin10 8-yl]-5-(3-(2-fluoro-4-(3-(3-oxo-2,8-diazjaspiro[4.5]decan-8-yl)prop-l -ynyl] phenoxy]propyljthiazole-4-carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step A as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+HJ+ calcd for C39H40FN8O4S2: 767.2592, found 767.2588.
Example 59: 2-{3-[(l,3-BenzothÎazol-2-yl)amÎno]-4-methyI-5H,6H,7/7,8H-pyrido[2,3c]pyridazin-8-yl}-5-(3-{2-bromo-4-[3-(dimethy]amino)prop-l-yn-l-yl]phenoxy}propyl)l,3-thiazole-4-carboxyIic acid
236
Steo A : methyl 5-[3-(2-bromo-4-iodophenoxy)propyl]-2-(4-methyl-3-{[(2Z)-3-{[2(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3-benzothiazol-2-ylidene]amino}5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8~yl)-l,3-thiazole-4-carboxylate
To a solution of the product from Préparation 3b (35Ü mg, 0.5 mmol, 1 eq) and 2-bromo-4iodophenol (200 mg, 0.67 mmol, 1.33 eq) in toluene (10 mL) was added triphenylphosphine (198 mg, 0.75 mmol, 1.5 eq) and diisopropyl azodicarboxylate (0.15 ml, 0.75 mmol, 1.5 eq) and the mixture was stirred at ambient température for 18 h. The reaction was concentrated in vacuo and purification by flash column chromatography (20 g silica) eluting with a gradient 10 of 0 — 30% ethyl acetate in iso-heptane afforded the desired product as a yellow solid (555 mg, 0.49 mmol, 97%).
LC/MS (C35H4oBrIN6O4SiS2) 907 RT 1.50 (LCMS-V-B2) *H NMR (400 MHz, CDCh) δ 7.82 (d, J = 2.1 Hz, 1H), 7.59 (dt, J = 7.6, 0.9 Hz, 1H), 7.50 (dd, J = 8.6, 2.1 Hz, 1H), 7.39 - 7.34 (m, 2H), 7.24 - 7.16 (m, 1H), 6.65 (d, J = 8.7 Hz, 1H), 15 5.84 (s, 2H), 4.47 - 4.36 (m, 2H), 4.17 - 4.05 (m, 2H), 3.90 (s, 3H), 3.80 - 3.69 (m, 2H), 3.39 (dd, J - 8.4, 6.6 Hz, 2H), 2.87 (t, J = 6.4 Hz, 2H), 2.38 (s, 3H), 2.34 - 2.23 (m, 2H), 2.18 2.07 (m, 2H), 1.00 - 0.92 (m, 2H), -0.07 (s, 9H).
Step B: methyl 5-(3-{2-bromo-4-[3-(dimethylamino)prop-l-yn-l-yl]phenoxy}propyl)-2-(4methyl-3-{[ (2Z) -3-{[2-(trimethylsilyl) ethoxy]methyl}-2,3~dihydro-l,3-benzoihiazol~220 ylidene]amino}-5H,6H, 7H, 8H -pyrido[2,3-c]pyridazin-8-yl) -1,3-thiazole‘4-carboxylate
To a solution of the product from Step A (550 mg, 0.52 mmol, 1 eq), copper (I) iodide (18.5 mg, 0.1 mmol, 0.2 eq) and tetrakis(triphenylphosphine)palladium(0) (56 mg, 0.05 mmol, 0.1 eq) in 2-methyl tetrahydrofuran (10 mL) was added dimelhyl(prop-2-yn-l-yl)amine (0.3 mL, 2.79 mmol, 5.75 eq) followed by ΛζΑ-diisopropylethy lamine (0.3 ml, 1.45 mmol, 3 eq) and
237 the mixture was heated at 75 °C for 3 h. The reaction was ailowed to cool to ambient température and concentrated in vacuo. Purification by flash column chromatography (20 g silica) eluting with a gradient of 0 - 100% ethyl acetate in zso-heptane afforded the desired product as a dark orange gum (220 mg, 0.25 mmol, 53%).
LC/MS (C4oH48BrN704SiS2) 862 [M+H]+; RT 1.54 (LCMS-V-B2) *H NMR (400 MHz, CDCh) 8 7.64 (d, J = 2.0 Hz, IH), 7.60 (dt, J = 7.7, 0.9 Hz, IH), 7.38 7.37 (m, 2H), 7.34 - 7.30 (m, IH), 7.23 - 7.16 (m, IH), 6.81 (d, J = 8.6 Hz, IH), 5.84 (s, 2H), 4.48 - 4.35 (m, 2H), 4.16 - 4.07 (m, 2H), 3.90 (s, 3H), 3.78 - 3.69 (m, 2H), 3.50 - 3.35 (m, 4H), 2.87 (t, J = 6.3 Hz, 2H), 2.38 (s, 3H), 2.35 (s, 6H), 2.33 - 2.26 (m, 2H), 2.18 - 2.07 (m, 2H), LOI -0.88 (m, 2H), -0.07 (s, 9H).
Step C: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,3c]pyridazin-8~yl}-5-(3-{2~bromo-4-[3~(dimethylamino)prop-l -yn-1 -yl]phenoxy}propyl) -1,3thiazole-4-carboxylic acid
To a solution of the product from Step B (210 mg, 0.24 mmol, 1 eq) in dîchloromethane (4.5 mL), cooled to Ü °C, was added trifluoroacetic acid (0.5 ml, 6.08 mmol, 25 eq) and the mixture was stirred for 24 h at ambient température. The reaction was diluted with dîchloromethane (40 mL), successîveiy washed with IM aqueous ammonia (20 mL), water (2 x 20 mL) and brine (20 mL), dried (magnésium sulfate) and concentrated in vacuo. To a solution of the crude product în methanol (1 mL) was added water (2 mL) and lithium hydroxîde monohydrate (30.6 mg, 0.73 mmol, 3 eq) and the suspension was heated at 75 °C for 72 h. The reaction was ailowed to cool to ambient température, then neutralised with acetic acid and the solids were collected by filtration and washed with water (2 x 10 mL). Purification by préparative HPLC (HPLC-V-A2) afforded the desired product as a green solid (15 mg, 0.02 mmol, 9%).
LC/MS (C33H32BrN7O3S2) 718 [M+H]+; RT 1.18 (LCMS-V-B1)
Hd NMR (400 MHz, DMSO-d6) 8 7.87 (d, J = 7.8 Hz, IH), 7.63 (d, J = 2.0 Hz, IH), 7.49 (d, J = 8.0 Hz, IH), 7.42 - 7.33 (m, 2H), 7.20 (td, J = 7.6, 1.1 Hz, IH), 7.09 (d, J = 8.6 Hz, IH), 4.26 (t, J = 5.5 Hz, 2H), 4.13 (t, J = 6.1 Hz, 2H), 3.34 - 3.25 (m, 2H), 2.86 (t, J = 6.1 Hz, 2H), 2.54 (s, 2H), 2.33 (s, 3H), 2.17 (s, 6H), 2.16 - 2.09 (m, 2H), 2.08 - 1.95 (m, 2H).
238
Φ Example 60: 2-{3-[(l,3-Benzothiazol-2-yl)ammo]-4-methyI-5/7,6H,7H,8/7-pyrido[2,3c|pyridazin-8-yl}-5-{3-[2-fluoro-4-(4“methylpiperazJn-l-yl)phenoxy]propyl}-l,3-thiazole4-carboxylic acid
Step A: methyl 5-[3-(2-fluoro-4-iodophenoxy)propyl]-2-(4-methyl-3-{[(2Z)-3-{[2(trimethylsilyl)ethoxy/methyl}-2,3-dihydro~l,3-benzothiazol-2-ylidene]amino}5H,6H, 7H,8H-pyrido[2,3-c]pyridazin -8-yl) -1,3-thiazole~4-carboxylate
To a solution of the product from Préparation 3b (200 mg, 0.32 mmol, 1 eq) and 2-fluorû-4iodophenol (152 mg, 0.64 mmol, 2 eq) in toluene (6 mL) was added triphenylphosphine (167 10 mg, 0.64 mmol, 2 eq) and diisopropyl azodicarboxylate (147 mg, 0.64 mmol, 2 eq) and the mixture was stirred at 50 °C for 17 h. The reaction was partitioned between dichloromethane and water, and the organic phase was separated (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartrîdge) eiuting with a gradient of 0 - 100% ethyl acetate in iso-heptane 15 afforded the desired product as a yellow gum (161 mg, 0.19 mmol, 60%).
LC/MS (C35H40FIN6O4S1S2) 847 [M+H]+; RT 3.33 (LCMS-V-C) *H NMR (400 MHz, DMSO-d6) Ô 7.82 (dt, J = 7.6, 0.9 Hz, IH), 7.61 (dd, J = 10.8, 2.1 Hz, IH), 7.49 - 7.39 (m, 3H), 7.24 (ddd, J = 8.3, 6.6,1.9 Hz, IH), 7.01 (t, J = 8.8 Hz, IH), 5.85 (s, 2H), 4.26 (t, J = 5.7 Hz, 2H), 4.11 (t, J = 6.2 Hz, 2H), 3.78 (s, 3H), 3.76 - 3.67 (m, 2H), 3.27 20 (t, 2H), 2.88 (t, J = 6.3 Hz, 2H), 2.37 (s, 3H), 2.18 - 2.09 (m, 2H), 2.08 - 2.01 (m, 2H), 0.95 0.86 (m, 2H), -0.12 (s, 9H).
Step B: methyl 5-{3-[2-fluoro-4‘(4~methylpiperazin-l-yl)phenoxy]propyl}-2-(4-methyl-3{[(2Z)~3-{[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3-benzothiazol~2-ylidene]amino}5H,6H,7H,8H-pyrido[2i3-c]pyridaziti-8-yl)-l,3-thiazole-4-carboxylate
1-Methylpiperazine (14.5 pL, 0.13 mmol, 1.5 eq) was added to a solution of the product from Step A (74 mg, 0.09 mmol, 1 eq), copper(I) iodide (1.66 mg, 0.01 mmol, 0.1 eq), potassium
239 phosphate tribasic (37.1 mg, 0.17 mmol, 2 eq) and [(2,6-dimethylphenyl)carbamoyl]fonnic acid (3.38 mg, 0.02 mmol, 0.2 eq) in DMSO (2 mL) and the mixture was heated at 120 °C for 2 h under microwave irradiation. The reaction was allowed to cool to ambient température, partitioned between ethyl acetate and water, and the organic phase was washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 8% methanol in dichloromethane afforded the desired product as a brown gum (30 mg, 0.04 mmol, 42%).
LC/MS (C4oH5iFN804SiS2) 819 [M+H]+; RT 2.71 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.82 (d, 1H), 7.48 - 7.38 (m, 2H), 7.27 - 7.20 (m, 1H), 7.03 (l, 1H), 6.84 (dd, 1H), 6.67 - 6.61 (m, 1H), 5.84 (s, 2H), 4.26 (t, J = 5.7 Hz, 2H), 4.02 (t, J = 6.2 Hz, 2H), 3.79 (s, 3H), 3.76 - 3.66 (m, 2H), 3.26 (t, 2H), 3.04 - 2.95 (m, 4H), 2.92 2.82 (m, 2H), 2.42 - 2.32 (m, 7H), 2.18 (s, 3H), 2.12 - 1.99 (m, 4H), 0.95 - 0.86 (m, 2H), 0.11 (s, 9H).
Step C: methyl 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,3c]pyridazin-8-yl}-5-{3-[2-fluoro-4-(4-methylpiperazhi-l-yl)phenoxy]propyl}-l,3-thiazole-4carboxylate
To a solution of the product from Step B (116 mg, 0.14 mmol, 1 eq) in dichloromethane (7.5 mL), cooled to 0 °C, was added trifluoroacetic acid (1.52 mL, 19.8 mmol, 140 eq) and the mixture was stirred for 24 h at ambient température. The reaction was diluted with dichloromethane (40 mL), washed with aqueous ammonîa, dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% methanol in dichloromethane afforded the desired product as a yellow gum (72 mg, 0.1 mmol, 74%).
LC/MS (C34H37FN8O3S2) 689 [M+H]+; RT 2.01 (LCMS-V-C)
NMR (400 MHz, DMSO-d6) δ 11.46 (br s, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.56 - 7.44 (m, 1H), 7.38 (ddd, J = 8.3, 7.3, 1.3 Hz, 1H), 7.24 - 7.16 (m, 1H), 7.04 (dd, J = 9.9, 9.0 Hz, 1H), 6.85 (dd, J = 14.6, 2.8 Hz, 1H), 6.69 - 6.61 (m, 1H), 4.27 (t, J = 5.8 Hz, 2H), 4.08 - 3.99 (m, 2H), 3.80 (s, 3H), 3.31 - 3.23 (m, 2H), 3.05 - 2.96 (m, 4H), 2.89 (t, J = 6.3 Hz, 2H), 2.42 2.36 (m, 4H), 2.35 (s, 3H), 2.19 (s, 3H), 2.13 - 2.00 (m, 4H).
240
Step D: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,3cjpyridazin -8-yl}-5-{3-[2-fluoro-4-(4-methylpiperaziu-l -yl)phenoxy]propyl}-l J-thiazole-dcarboxylic acid
To a solution of the product from Step C (72 mg, 0.1 mmol, 1 eq) in 1,4-dioxane (3 mL) was 5 added lithium hydroxide monohydrate (43.9 mg, 1.05 mmol, 10 eq) and the mixture was heated at reflux for 3 h. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 15% 7N methanolic ammonia in dichloromethane gave a solid that was triturated with diethyl ether, fîltered and dried under vacuum to afford the desired product as a yellow solid (35 mg, 0.05 mmol, 50%).
HRMS-ESI (m/z) [M+H]+ calcd for C33H36FN8O3S2: 675.2336, found 675.2364
Example 61: 2-{3-[(l,3-Benzotliiazol-2-yl)amino]-4-methyl-5ET,6H,77Z,8H-pyrido[2,3c]pyridazm-8-yl}-5-[3-({3-[(dimethylamino)methyl]-5-fluoro-l-methyl-LH-indol-6yl}oxy)propyl]-l,3-thiazole-4-carboxyIic acid
Step A: methyl 5-[3-({3-[(dimethylamino)methyl]-5-fluoro-l-methyl-IH-indol-6yl}oxy)propyl]-2-(4-methyl-3-{[(2Z) -3-{[2-(trimethylsilyl) ethoxy]methyl}-2,3-dihydro-l,3benzothiazol-2-ylidene]amino}-5H,6H, 7H,8H-pyrido[2,3-c]pyridazin -8-y!) -1,3-thiazole-4carboxylate
The product from Préparation 3b (120 mg, 0.19 mmol, 1 eq) was taken up în toluene (15 20 mL) and the product from Préparation 4h (67 mg, 0.26 mmol, 1.35 eq) was added, followed by triphenylphospbine (100 mg, 0.38 mmol, 2 eq) and di-terr-butyl azodicarboxylate (88.2 mg, 0.38 mmol, 2 eq) and the mixture was stirred at 50 °C overnight. The réaction was partitioned between dichloromethane and water, and the organic phase was washed with brine, dried (PTFE phase separator) and concentrated in vacuo. Purification by automated
241 flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 — 10% methanol in dichloromethane afforded the desired product as a beige solid (18 mg, 0.02 mmol, 11%).
LC/MS (C4iH5]FNsO4SiS2) 831 [M+HJ+; RT 2.73 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.81 (d, J = Ί.Ί Hz, 1H), 7.47 - 7.43 (m, 2H), 7.40 - 7.33 (m, 1H), 7.28 - 7.21 (m, 1H), 7.15 (d, J = 11.6 Hz, 1H), 6.84 (d, J = 7.4 Hz, 1H), 5.85 (s, 2H), 4.28 (t, 2H), 4.16 (t, J = 6.3 Hz, 2H), 3.79 (s, 3H), 3.76 - 3.71 (m, 2H), 3.69 (s, 2H), 3.63 (s, 3H), 3.48 - 3.41 (m, 4H), 3.88 (t, 2H), 2.38 (s, 3H), 2.08 - 2.00 (m, 2H), 1.46 (s, 6H), 1.35 0.95 - 0.86 (m, 2H), -0.11 (s, 9H).
Step B: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-tnethyl-5H,6H, 7H,8H-pyrido[2,3c]pyridazin-8-yl}-5-[3-({3-[(dimethylamino)methyl]-5-fluoro-l -methyl-lH-indol-6yl}oxy)propyl]-l,3-thiazole-4-carboxylic acid
A solution of the product from Step A (18 mg, 0.02 mmol, 1 eq) in dichloromethane (1 mL) was cooled to 0 °C and trifluoroacetic acid (1 mL, 13 mmol, 600 eq) was added and the 15 mixture was stirred for 4 h at ambient température. Dichloromethane (10 mL) was added and the solution was cooled to 0 °C, washed with aqueous ammonia, dried (PTFE phase separator) and concentrated in vacuo. The residue was suspended in 1,4-dioxane (2 mL), lithium hydroxide monohydrale (9.1 mg, 0.22 mmol, 10 eq) was added and the mixture was heated at 70 °C overnight. Purification by préparative HPLC (HPLC-V-B1) afforded the desired 20 product as a yellow solid (4.5 mg, 0.01 mmol, 30%), as a formic acid sait.
HRMS-ESI (m/z) [M+H]+ calcd for C34H36FN8O3S2: 687.2336, found 687.2362
Exampie 62: 2-{3-[(1,3-BenzothiazoI-2-yl)amino]-4-methy\-5H,6//,7//,8//-pyrido[2,3c]pyridazin-8-yl}-5-(3-{4-[4-(dimethylainino)butyl]-2-fluorophenoxy}propyl)-l,3thiazole-4-carboxylic acid
S
HN N
H OH
242
Step A: methyl 5-(3-{4-[4-(dimethylamino)butyl]-2-fluorophenoxy}propyl)-2-(4-methyl-3{[(2Z)-3f[2-(trimethylsilyl)ethoxy]methyl}-2,3-dihydro-l,3-benzothiazol-2-ytidene]amino}5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl)-l,3-thiazole-4-carboxylate
To a solution of the product from Préparation 3b (120 mg, 0.19 mmol, 1 eq) in toluene (5 5 mL) was added the product from Préparation 4i (107 mg, 0.51 mmol, 2.65 eq), di-rert-butyl azodicarboxylate (88 mg, 0.38 mmol, 2 eq) and trîphenylphosphîne (100 mg, 0.38 mmol, 2 eq) and the mixture was heated at 50 C for 20 h. The reaction was partitioned between dîchioromethane and water, and the organic phase was dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash 10 Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 — 8% methanol in dîchioromethane afforded the desired product as a yellow oil (133 mg, 0.16 mmol, 85%).
LC/MS (C14H54FN7O4SÎS2) 821 [M+H]+; RT 2.74 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.85 - 7.78 (m, IH), 7.48 - 7.38 (m, 2H), 7.27 - 7.20 (m, IH), 7.11 - 7.01 (m, 2H), 6.95 - 6.89 (m, IH), 5.84 (s, 2H), 4.26 (t, J = 6.0 Hz, 2H), 4.07 (t, 15 2H), 3.78 (s, 3H), 3.71 (dd, 2H), 3.28 (dd, J = 15.9, 8.4 Hz, 2H), 2.87 (t, J = 6.2 Hz, 2H), 2.37 (s, 3H), 2.21 - 2.08 (m, 4H), 2.08 - 2.01 (m, 8H), 1.55 - 1.42 (m, 2H), 1.41 - 1.27 (m, 2H), 0.95 - 0.87 (m, 2H), -0.11 (s, 9H).
Step B: methyl 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-SH,6H,7H,8H-pyrido[2,3c]pyridazin-8~yl}-5-(3-{4-[4-(dimethylamino)butyl]-2-fluorophenoxy}propyl)-l,3‘thiazole‘4· 20 carboxylate
A solution of the product from Step A (133 mg, 0.16 mmol, 1 eq) in dichloromethane (6 mL) was cooled to 0 °C and trifluoroacetic acid (1.24 mL, 16.2 mmol, 100 eq) was added and the mixture was stirred for 24 h at ambient température, dichloromethane (40 mL) was added and the solution was washed with saturated aqueous ammonium chloride, dried (PTFE phase 25 separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 14% methanol in dichloromethane afforded the desired product as a yellow gum (81 mg, 0.12 mmol, 72%).
LC/MS (C35H40FN7O3S2) 690 [M+H]+; RT 2.14 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.87 (dd, J = 7.8, 1.2 Hz, IH), 7.49 (d, J = 8.0 Hz, IH), 7.37 (ddd, J = 8.2, 7.2, 1.3 Hz, IH), 7.19 (td, J = 7.5, 1.2 Hz, IH), 7.09 - 7.01 (m, 2H), 6.94 6.87 (m, IH), 4.25 (dd, J = 7.3, 4.5 Hz, 2H), 4.07 (t, J = 6.2 Hz, 2H), 3.78 (s, 3H), 3.27 (t, J =
243 φ 7.7 Hz, 2Η), 2.87 (t, J = 6.3 Hz, 2H), 2.48 - 2.43 (ni, 2H), 2.33 (s, 3H), 2.18 - 2.Ü7 (m, 4H), 2.08 - 1.98 (m, 8H), 1.55 - 1.41 (m, 2H), 1.40 - 1.26 (m, 2H).
Step C: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H, 7H,8H-pyrido[2,3c]pyridazin-8-yl}-5-(3-{4-[4-(dimethylamino)butyl]-2-fluorophenoxy}propyl)-l,3-thiazole-45 carboxylic acid
To a solution of the product from Step B (81 mg, 0.12 mmol, 1 eq) in 1,4-dioxane (3 mL) was added lithium hydroxide monohydrate (49.3 mg, 1.17 mmol, 10 eq) and the mixture was heated at 70 °C overnight. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 20% 7N 10 methanolic ammonia in dichloromethane afforded the desired product as a yellow solid (48.3 mg, 0.07 mmol, 61%).
HRMS-ESI (m/z) [M+H]+ calcd for C34H39FN7O3S2: 676.2540, found 676.2569
Example 63: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5if-pyrido[2,3c]pyridazin-8-yl]-5-[3-[4-[3-[(lS,5R)-6,6-difluoro-3-azabicyclo[3.1.0]hexan-3-yl]prop-l15 ynyl]-2-fluoro-phenoxy]propyl]thiazole-4-carboxylic acid
Step A: methyl 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3c]pyridazin-8-yl]-5-[3-[4-[3-[(lS,5R)-6,6-difluoro-3-azjabicyclo[3.1.0]hexan-3-yl]prop-lynyl]-2-fluoro-phenoxy]propyl] thiazole-4-carboxylate
Using Propargylic amine préparation General Procedure starting from 100 mg of Préparation 3d (0.155 mmol, leq.) as the appropriate propargylic alcohol and (lS,5/ï)-6,6difluoro-3-azabicyclo[3.1.0]hexane (20 eq.), 61 mg of the desired product (52%) was obtained.
244
Step B: 2-(3-(l,3-Benzothiazol-2-ylamino)-4-methy 1-6,7-dihydro-5H-pyrido[2,3c]pyndazui-8-yl]-S-[3-[4-[3-[ (lS,5R)-6,6-difluoro-3-azabicyclo[3.1.0]hexan-3-yl]prop-l ynyl]-2-fluoro-phenoxy]propyl]thiazole-4-carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step A as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C36H33F3N7O3S2: 732.2033, found 732.2023.
Example 64: 2-{3-[(l,3-Benzothiazol-2-yI)amino]-4-methyl-5//,6/7,7//,8//-pyridoL2,3c]pyridazin-8-yl}-5-(3-{2-fluoro-4-[2-(methylamino)ethyl]phenoxy}prüpyI)-l,3-thiazole4-carboxylic acid
ΙΓ OH
Nx S
HN^
StepA: methyl 5-{3-(4-(2-{((tert-butoxy)carbonyl](methyl)amino}ethyl)-2fluorophenoxy}propyl}-2-(4-methyl-3-{( (2Z) -3-{/2-(trimethylsilyl) ethoxy]methyl}-2,3dihydro-l,3-benzothiazol-2-ylidene]amino}-5H,6H, 7H,SH -pyrido(2,3-cJpyridazin -8-y 1)-1,3thiazole-4-carboxylate
To a solution of the product from Préparation 3b (80 mg, 0.13 mmol, 1 eq) in toluene (5 mL) was added the product from Préparation 4j (48 mg, 0.18 mmol, 1.4 eq), di-ter/-butyl azodicarboxylate (58.8 mg, 0.26 mmol, 2 eq) and triphenylphosphine (67 mg, 0.26 mmol, 2 eq) and the mixture was heated at 50 °C overnight. The reaction was partitîoned between dichloromethane and water, separated (phase separator) and the organic phase was concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 — 100% ethyl acetate in isoheptane afforded the desired product as a colourless gum (77 mg, 0.09 mmol, 69%).
LC/MS (C4.3H56FN7O6SiS2) 878 [M+H]+; RT 3.28 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.82 (dt, J = 7.6, 0.9 Hz, IH), 7.48 - 7.38 (m, 2H), 7.24 (ddd, J = 8.3, 6.7, 1.8 Hz, IH), 7.13 - 7.01 (m, 2H), 6.96 - 6.86 (m, IH), 5.84 (s, 2H), 4.27 (t,
245 φ J = 5.7 Hz, 2H), 4.08 (t, 2H), 3.78 (s, 2H), 3.75 - 3.67 (m, 2H), 3.31 - 3.22 (m, 4H), 2.88 (t, J = 6.2 Hz, 2H), 2.71 (s, 3H), 2.69 - 2.61 (m, 2H), 2.37 (s, 3H), 2.17 - 2.06 (m, 2H), 2.05 - 2.00 (m, 2H), 1.24 (s, 9H), 0.95 - 0.86 (m, 2H), -0.12 (s, 9H).
Step B: methyl 2-{3-[(l,3-benzothiazob2-yl)amino]-4-methyl-5H,6H,7H,8H-pyndo[2,35 c]pyridazin-8-yl}-5-(3-{2-fluoro-4-[2-(methylamino)ethyl]phenoxy}propyI)-l,3-thiazole-4carboxylate
A solution of the product from Step A (77 mg, 0.09 mmol, 1 eq) în dichloromethane (4 mL) was cooled to 0 °C and trifluoroacetic acid (0.81 mL, 10.5 mmol, 120 eq) was added and the mixture was stirred for 24 h al ambient température. The reaction was diluted with 10 dichloromethane, washed with aqueous ammonia, dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFIash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 18% methanol in dichloromethane afforded the desired product as a yellow solid (39 mg, 0.06 mmol, 69%).
LC/MS (C32IIM'NO 2) 648 [M+H]4; RT 2.02 (LCMS-V-C) XH NMR (400 MHz, DMSO-d6) δ 7.91 - 7.84 (m, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.41 - 7.33 (m, 1H), 7.19 (td, J = 7.5, 1.2 Hz, 1H), 7.12 - 7.03 (m, 2H), 6.94 (dd, J = 8.8, 1.9 Hz, 1H), 4.27 (dd, J = 7.1,4.5 Hz, 2H), 4.09 (t, J = 6.2 Hz, 2H), 3.79 (s, 3H), 3.31 - 3.21 (m, 2H), 2.88 (t, J = 6.2 Hz, 2H), 2.68 - 2.57 (m, 4H), 2.35 (s, 3H), 2.25 (s, 3H), 2.17 - 2.01 (m, 4H).
Step C: 2-{3-[(l ,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H, 7H,8H-pyrido[2,320 c]pyridazin-8-yl}-5-(3-{2-fluoro-4'[2-(methylamino) ethyl]phenoxy}propyl)-l,3-thiazole-4carboxylic acid
To a solution of the product from Step B (39 mg, 0.06 mmol, I eq) in 1,4-dîoxane (2 mL) was added lithium hydroxide monohydrate (25.3 mg, 0.6 mmol, 10 eq) and the mixture was heated at reflux for 4 h. Purification by automated flash column chromatography (CombiFIash Rf, 4 25 g RediSep™ silica cartridge) eluting with a gradient of 0 - 25% 7N methanolic ammonia in dichloromethane afforded a solid that was suspended in ethyl acetate (2 mL) and hydrochloric acid (4M in 1,4-dioxane; 68.9 pL, 0.28 mmol, 4.58 eq) was added. The mixture was stirred for 10 mins before collecting the solids by filtration and drying under vacuum afforded the desired product as a yellow solid (17.2 mg, 0.03 mmol, 45%), as a hydrochloric acid sait.
HRMS-ESI (m/z) [M+H]+ calcd for C31H33FN7O3S2: 634.2070, found 634.2093.
246
Example 65: 2-{3-[(l,3-Benzothiazol-2-yl)aniino]-4-methyl-6-[2-(methylamino)ethôxy]5//,6//,7W,8//-|)yrido[2,.w-]pyridaz.in-8-yl}-5-(3-{4-i3-(dini<.‘thylaminojprop-l-yn-l-'vl|-2fluorophenoxy}propyI)-l,3-thiazole-4-carbox.ylic acid
Step A: 4-methylmorpholin-3-one
A solution of 2-(methylamino)ethanoI (5.32 mL, 66.6 mmol, 1 eq) in éthanol (100 mL) and 35% aqueous sodium hydroxîde (6.25 mL) was cooled to 15-20 ÛC and chloroacetyl chloride (13.3 mL, 166 mmol, 2.5 eq) and 35% aqueous sodium hydroxîde (22 mL) were added simultaneously with vigorous stirrîng over 1 h. The mixture was stirred for 20 min, then neutralised with aqueous hydrochloric acid and extracted with dichloromethane (3 x 100 mL). The combined organic extracts were washed with water, dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 80 g RediSep™ silica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in isoheptane afforded the desired product as a colourless oil (4.4 g, 38.2 mmol, 58%).
*H NMR (400 MHz, DMSO-d6) δ 4.00 (s, 2H), 3.84 - 3.78 (m, 2H), 3.36 - 3.29 (m, 2H), 2.86 (s, 3H).
Step B: 2-(but-2-yn-l-yl)-4-methylmorpholin-3-one
To a solution of diisopropylamine (6.45 mL, 45.9 mmol, L2 eq) in tetrahydrofuran (130 mL), cooled to -78 °C, was added H-butyllithîum (2.06M in hexanes; 20.4 mL, 42 mmol, 1.1 eq) dropwîse. After 1 minute a solution of the product from Step A (4.4 g, 38.2 mmol, 1 eq) in tetrahydrofuran (30 mL) was added dropwîse. After 15 minutes a solution of 1-bromo2-butyne (4.02 mL, 45.9 mmol, 1.2 eq) in tetrahydrofuran (15 mL) was added dropwîse and
247 φ the mixture was stirred at -78 °C for 1 h then aliowed to warm to ambient température.
Saturated aqueous ammonium chloride was added and the mixture was extracted with ethyl acetate (x3), and the combined organic extracts were dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash 5 Rf, 80 g RediSep™ siiica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in isoheptane afforded the desired product as a yellow oil (5.15 g, 30.8 mmol, 81%).
XH NMR (400 MHz, DMSO-d6) δ 4.09 (dd, J = 7.6, 3.5 Hz, 1H), 4.01 - 3.94 (m, 1H), 3.76 (ddd, J = 11.9, 10.0, 3.6 Hz, 1H), 3.52-3.41 (m, 1H), 3.26-3.18 (m, 1H), 2.86 (s, 3H), 2.67 - 2.58 (m, 1H), 2.57 - 2.44 (m, 1H), 1.73 (t, J = 2.6 Hz, 3H).
1θ Step (· 2-[2-(methylamino)ethoxy]hex-4-ynoic acid
To a solution of the product from Step B (3.25 g, 19.4 mmol, 1 eq) in methanol (110 mL) was added IM aqueous lithium hydroxide (60.3 mL, 60.3 mmol, 3.1 eq) and the mixture was heated at reflux overnight. The reaction was concentrated in vacuo to afford the desired product as an orange gum (5.15 g, 27.8 mmol, 100%) that was used directly in the subséquent 15 step without further characterisation.
Step D: 2-/2-(// (9H-fluoren-9-yl)methoxy]carbonyl}(methyl)amino)ethoxy]hex~4-ynoic acid
To a solution of the product from Step C (5.15 g, 27.8 mmol, 1 eq) in 1,4-dioxane (45 mL) and water (160 mL) was added potassium carbonate (15.4 g, 111 mmol, 4 eq) at 0 °C, 20 followed by 9/f-fluoren-9-yLmethyl chloroformate (7.19 g, 27.8 mmol, 1 eq) and the mixture was aliowed to warm to ambient température and stir for 2 h. The reaction was partitioned between water and ethyl acetate, and the aqueous phase was acidified with aqueous hydrochloric acid to pH 2-3 and extracted with ethyl acetate (3 x 300 mL). The combined organic extracts were washed with brine, dried (magnésium sulfate) and concentrated in 25 vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 120 g
RediSep™ siiica cartridge) eluting with a gradient of 0 - 20% methanol in dichioromethane afforded the desired product as a dark yellow gum (7.06 g, 17.3 mmol, 62%).
LC/MS (C24H25NO5) 408 [M+H]+; RT 0.74 (LCMS-V-B2) ‘H NMR (400 MHz, DMSO-d6) Ô 7.90 (t, J = 6.8 Hz, 2H), 7.65 (dd, J = 7.5, 1.1 Hz, 2H), 30 7.42 (td, J = 7.4, 3.0 Hz, 2H), 7.34 (td, J = 7.4, 1.3 Hz, 2H), 4.43 - 4.22 (m, 3H), 3.50 - 3.42 (m, 1H), 3.39 - 3.28 (m, 1H), 3.26 - 3.15 (m, 3H), 2.90 - 2.82 (m, 3H), 2.51 - 2.44 (m, 2H), 1.71 (dt, J = 13.8, 2.5 Hz, 3H).
248
Step Et (9H-fluoren-9-yl)methyl N-{2-[(1 -hydroxyhex-4-yn-2-yl)oxy]ethyl}-Nmethylcarbamate
A solution of the product from Step D (7.06 g, 17.33 mmol, 1 eq) in tetrahydrofuran (120 mL) was cooled to -10 °C, then triethylamine (2.65 mL, 19.1 mmol, 1.1 eq) and isobutyl 5 chloroformate (2.7 mL, 20.8 mmol, 1.2 eq) in THF (40 mL) were added dropwise. The precipitate was removed by filtration and the solution was cooled to -10 °C. Sodium borohydride (2.62 g, 69.3 mmol, 4 eq) in water (40 mL) was added dropwise and the mixture was stirred for 1 h at -10 °C. The pH of the solution was adjusted to pH 5 using IN aqueous hydrochloric acid, and then adjusted to pH 10 using saturated aqueous sodium bicarbonate. 10 The layers were separated and the organic phase was successively washed water (100 mL) and brine (50 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 80 g RediSep™ silica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in /so-heptane afforded the desired product as a colourless gum (4.64 g, 11.8 mmol, 68%).
LC/MS (C24H27NO4) 394 [M+H]+; RT 0.77 (LCMS-V-B2)
NMR (400 MHz, DMSO-d6) δ 7.90 (d, J = 7.5 Hz, 2H), 7.65 (dt, J = 7.4, 0.9 Hz, 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.35 (td, J = 7.4, 1.2 Hz, 2H), 4.68 - 4.60 (m, 1H), 4.39 (d, J = 6.0 Hz, 1H), 4.34 (d, J = 6.7 Hz, 1H), 4.28 (t, J = 6.4 Hz, 1H), 3.60 - 3.51 (m, 1H), 3.46 - 3.36 (m, 2H), 3.34 - 3.28 (m, 2H), 3.19 (dd, J = 16.6, 5.5 Hz, 2H), 2.84 (d, J = 10.8 Hz, 3H), 2.38 20 - 2.15 (m, 2H), 1.71 (t, J = 2.5 Hz, 3H).
Step F: (9H-fluoren -9-yl) methyl N-[2-({l -[(tert-butyldiph enylsilyl)oxy]h ex-4-yn-2yl}oxy)ethylj-N-methylcarbamate
To a cooled solution of the product from Step E (4.64 g, 11.8 mmol, 1 eq) and imidazole (1.56 mL, 23.6 mmol, 2 eq) in dichloromethane (200 mL) was added tert25 butyl(chloro)diphenylsilane (6.13 mL, 23.6 mmol, 2 eq) dropwise and the mixture was allowed to warm to ambient température and stir overnight. The reaction was quenched with 2M aqueous ammonium chlorîde and the mixture was extracted with dichloromethane (3 x 200 mL). The combined organic extracts were washed with brine, dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column 30 chromatography (CombiFlash Rf, 120 g RediSep™ silica cartridge) eluting with a gradient of 0- 25% ethyl acetate in i'so-heptane afforded the desired product as a colourless gum (5.86 g, 9.27 mmol, 79%).
249
Φ LC/MS (C40H45NO4S1) 632 [M+HJ+; RT 1.38 (LCMS-V-B2)
NMR (400 MHz, DMSO-d6) 5 7.87 (dd, J = 20.0, 7.5 Hz, 2H), 7.67 - 7.56 (m, 6H), 7.53 - 7.39 (m, 7H), 7.39 - 7.22 (m, 3H), 4.38 (t, J = 4.8 Hz, 1H), 4.31 (s, 1H), 4.24 (t, J = 5.7 Hz, 1H), 3.73 - 3.61 (m, 1H), 3.60 - 3.44 (m, 2H), 3.34-3.29 (m, 2H), 3.29 - 3.18 (ni, 1H), 3.16 5 - 3.06 (m, 1H), 2.81 (d, J = 14.1 Hz, 3H), 2.43 - 2.26 (m, 2H), 1.69 (t, J = 2.4 Hz, 3H), 0.98 (S, 9H).
Step G: (9H-fluoren-9-yl)methyl N-)2-((1 ~[(tert-butyldiphenylsilyl)oxy]-3-(3,6-dichloro-5methylpyridazin-4-yl)propan-2-yl}oxy)ethyl]‘N-methylcarbamate
A solution of the product from Step F (5.86 g, 9.27 mmol, 1 eq) and 3,6-dîchloro-l,2,4,510 tetrazine (5.6 g, 37.1 mmol, 4 eq) in toluene (130 mL) was heated at 150 °C overnîght in a sealed flask. The reaction was concentrated in vacuo and purification by automated flash column chromatography (CombiFlash Rf, 120 g RediSep™ silica cartridge) eluting with a gradient of 0 - 30% ethyl acetate in iso-heptane afforded the desired product as a pink foam (2.99 g, 3.97 mmol, 43%).
LC/MS (C42H45C12N3O4Si) 754 [M+HJ+; RT 1.37 (LCMS-V-B2)
Ή NMR (400 MHz, DMSO-d6) δ 7.90 (d, J = 7.7 Hz, 1H), 7.78 (d, J = 7.4 Hz, 1H), 7.68 7.59 (m, 5H), 7.57 - 7.50 (m, 1H), 7.47 - 7.41 (m, 6H), 7.45 - 7.37 (m, 1H), 7.36 - 7.28 (m, 2H), 7.23 (t, J = 7.5 Hz, 1H), 4.30 (d, J = 5.7 Hz, 1H), 4.27 - 4.11 (m, 2H), 3.81 - 3.60 (m, 3H), 3.55 - 3.45 (m 1H), 3.20 - 2.98 (m, 4H), 2.89 - 2.77 (m, 1H), 2.58 (d, J = 23.0 Hz, 3H), 20 2.39 (d, J = 13.1 Hz, 3H), 1.01 (s, 9H).
Stepjl: 4-{3-[(tert-butyldiphenylsilyl)oxy]‘2-[2-(inethyÎamino)ethoxyJpropyl}-3>6-dichloroS-methylpyridazine
A solution of the product from Step G (2.79 g, 3.7 mmol, 1 eq) and diethylamine (0.77 mL, 7.39 mmol, 2 eq) in acetonitrile (60 mL) was stirred at ambient température overnîght. Water 25 was added and the mixture was extracted with ethyl acetate (3 x 70 mL). The combined organic extracts were washed with brine (100 mL), dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 16% methanol in dichloromethane afforded the desired product as an orange/ pink gum (1.9 g, 3.57 mmol, 30 96%).
LC/MS (C27H35CI2N3O2S1) 532 [M+H]+; RT 0.84 (LCMS-V-B2)
250 φ ΣΗ NMR (400 MHz, DMSO-d6) δ 7.69 - 7.62 (m, 4H), 7.54 - 7.41 (ni, 6H), 3.83 - 3.60 (m, 3H), 3.42 - 3.36 (m, 1H), 3.16 - 2.97 (m, 3H), 2.45 (s, 3H), 2.39 - 2.23 (m, 2H), 2.06 (s, 3H), 1.02 (s, 9H).
Step I: tert-butyl N-[2-({l-[(tert-butyldiphenylsilyl)oxy]-3-(3,6-dichloro-5-methylpyridazin5 4-yl)propau-2-yl}oxy) ethyl]-N-methylcarbaniate
To a solution of the product from Step H (1.9 g, 3.57 mmol, 1 eq) in dichloromethane (100 mL) was added di-ieri-butyl dicarbonate (1.53 mL, 7.14 mmol, 2 eq) followed by triethylamine (1.99 mL, 14.3 mmol, 4 eq) and the mixture was stirred at ambient température for 4 h. The reaction was partitioned between dichloromethane and water, and the aqueous 10 phase was acîdified to pH 4 and extracted with dichloromethane (3 x 80 mL). The combined organic extracts were washed with brine, dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep™ silica cartridge) eluting with a gradient of 0 - 25% ethyl aeetate in iso-heptane afforded the desired product as a colourless gum (1.83 g, 2.9 mmol, 81%).
LC/MS (C32H43CI2N3O4SÎ) 532 [M-Boc+H]+; RT 1.33 (LCMS-V-B2)
Ή NMR (400 MHz, DMSO-d6) δ 7.69 - 7.62 (m, 4H), 7.54-7.41 (m, 6H), 3.76 (qd, J = 10.7, 4.7 Hz, 2H), 3.66 (d, J - 5.5 Hz, 1H), 3.44 (q, J = 7.9, 6.3 Hz, 1H), 3.20 - 3.10 (m, 3H), 3.04 (dd, J = 14.0, 4.1 Hz, 2H), 2.58 (s, 3H), 2.44 (s, 3H), 1.31 (d, J = 22.6 Hz, 9H), 1.02 (s, 9H).
Step J: tert-butyl N-(2-{[l-(3,6-dichloro-5-methylpyridazin-4~yl)-3-hydroxypropan-2yl]oxy}ethyl)-N-methylcarbamate
A solution of the product from Step I (1.83 g, 2.9 mmol, 1 eq) în tetrahydrofuran (75 mL) was cooled to 0 °C before the addition of letrabutylammonium fluoride (IM in tetrahydrofuran; 2.9 mL, 2.9 mmol, 1 eq) and stimng at 0 °C for 30 min, then at ambient température for 1 h. 25 The reaction was partitioned between dichloromethane and water, and the aqueous phase was extracted with dichloromethane (x2). The combined organic extracts were washed with brine, dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep™ silica cartridge) eluting wîth a gradient of 0 - 100% ethyl aeetate in iso-heptane afforded the desired product as a pale 30 orange gum (0.73 g, 1.86 mmol, 64%).
251 φ *Η NMR (400 MHz, DMSO-d6) δ 4.93 (t, J = 5.5 Hz, 1H), 3.62 - 3.44 (m, 4H), 3.23 (dt, J =
9.6 , 6.0 Hz, 1H), 3.11 (d, J = 23.9 Hz, 2H), 3.02 (dd, J = 6.5, 2.0 Hz, 2H), 2.60 (d, J = 8.1 Hz, 3H), 2.45 (s, 3H), 1.35 (d, J = 13.0 Hz, 9H).
Step K: methyl 2-{[(tert-butoxy)carbonyl][2-(2-{[(tert5 butoxy)earbonyl](methyl)amino}ethoxy)-3~(3,6-dichloro-5-methylpyridazin-4yl)propyl]amino}-5-(3-{4-[3-(dimethylamino)prop-l -yn-1 -yl!-2-fluorophenoxy}propyl)-l,3thiazole-4-carboxylate
To a solution of the product from Step J (125 mg, 0.32 mmol, 1 eq) in toluene (20 mL) was added the product from Préparation le (171 mg, 0.35 mmol, 1.1 eq), di-ierZ-butyl 10 azodicarboxylate (146 mg, 0.63 mmol, 2 eq) and triphenylphosphine (166 mg, 0.63 mmol, 2 eq) and the mixture was stirred at 50 °C for 1 h. The reaction was partitioned between dichloromethane and water, and the aqueous phase was extracted with dichloromethane (x2), and the combined organic extracts were washed with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash 15 Rf. 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 100% ethyl acetate in isoheptane afforded the desired product as a pale yellow gum (282 mg, 0.32 mmol, 102%).
LC/MS (CwH53C12FN6O8S) 867 [M+H]+; RT 0.97 (LCMS-V-B2) »H NMR (400 MHz, DMSO-d6) δ 7.30 (dd, 1H), 7.23 - 7.17 (m, 1H), 7.12 (t, 1H), 4.29 (dd, J = 13.9, 5.7 Hz, 1H), 4.10 (t, J = 6.0 Hz, 2H), 3.96 - 3.87 (m, 1H), 3.74 (s, 3H), 3.61 - 3.48 20 (m, 1H), 3.42 (s, 3H), 3.32 (s, 2H), 3.25 (dt, J = 7.1, 3.9 Hz, 3H), 3.16 - 2.99 (m, 2H), 2.97 -
2.89 (m, 1H), 2.58 (d, J = 11.6 Hz, 2H), 2.45 (s, 3H), 2.23 (s, 6H), 2.10 (t, J = 6.9 Hz, 2H), 1.52 (s, 9H), 1.31 (d, J = 39.6 Hz, 9H).
Step L: methyl 2-{[2-(2-{[(tert-butoxy)carbonylj(methyl)amino}ethoxy)-3-(3,6-didii<)ro-5methylpyridazin-4-yl)propyl]amino}-5-(3-/4-[3-(dimethylamino)prop-I-yn-l-yl]-225 fluorophenoxyjpropyl) -1,3-thiazole-4-carboxylate
A solution of the product from Step K (275 mg, 0.32 mmol, 1 eq) in l,l,l,3,3,3-hexafluoro-2propanol (2.5 mL, 23.7 mmol, 74.7 eq) was heated at 100 °C for 60 min under inicrowave irradiation. The reaction was concentrated in vacuo and purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a 30 gradient of 0 - 7% methanol in dichloromethane afforded the desired product as a white solid (154 mg, 0.2 mmol, 63%).
LC/MS (C35H45C12FN6O6S) 767 [M+H]+; RT 0.70 (LCMS-V-B2)
252 JH NMR (400 MHz, DMSO-d6) δ 7.83 (br s, IH), 7.30 (dd, J = 11.9, 2.0 Hz, IH), 7.24 - 7.17 (m, IH), 7.12 (t, J = 8.7 Hz, IH), 4.08 (t, J = 6.1 Hz, 2H), 3.82 (dt, J = 9.0, 4.5 Hz, IH), 3.70 (s, 3H), 3.60 - 3.49 (m, IH), 3.46 - 3.39 (m, 4H), 3.33 (s, 2H), 3.29 - 3.18 (m, IH), 3.14 (t, 2H), 3.10 - 3.02 (m, 2H), 2.98 (dd, J = 13.9, 3.8 Hz, IH), 2.64 - 2.53 (m, 2H), 2,44 (s, 3H), 5 2.23 (s, 6H), 2.07 - 1.95 (m, 2H), 1.32 (d, J = 30.8 Hz, 9H).
Step M: methyl 2-[6-(2-{[(tert-butoxy)carbonyl](methyl)amino}ethoxy)-3-chloro-4-methyl5H,6H, 7H,8H-pyrido[2,3-c]pyridazin -8‘yl]-5-(3-{4-[3-(dimethylamino)prop-i -yn -1 -ylj-2fiuorophenoxy}propyl)-l,3-thiazole-4-earboxylate
To a solution of the product from Step L (154 mg, 0.2 mmol, 1 eq) in 1,4-dioxane (14 mL) 10 was added césium carbonate (131 mg, 0,4 mmol, 2 eq), ^V-diisopropylethylamine (0,07 mL, 0.4 mmol, 2 eq) and bis(di-ieri-butyl(4-dimethylaminophenyl)phosphinc) dichloropalladium(II) (14,2 mg, 0.02 mmol, 0,1 eq) and the mixture was heated at 80 °C for 45 min. The reaction was partitioned between dîchloromethane and water, and the aqueous phase was extracted with dîchloromethane (x2). The combined organic extracts were washed 15 with brine, dried (magnésium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep™ silica cartridge) eluting with a gradient of 0 - 8% methanol in dîchloromethane afforded the desired product as a cream solid (136 mg, 0.19 mmol, 93%).
LC/MS (C35H44CIFN6CLS) 731 [M+H]+; RT 0.75 (LCMS-V-B2) *H NMR (400 MHz, DMSO-d6) δ 7.31 (dt, J = 12.0, 1.9 Hz, IH), 7.25 - 7.19 (m, IH), 7.14 (t, IH), 4.86 (dd, IH), 4.25 (s, IH), 4.13 (t, J = 6.2 Hz, 2H), 3.93 (d, J = 13.5 Hz, IH), 3.78 (s, 3H), 3.56 (t, J = 5.6 Hz, 2H), 3.42 (s, 3H), 3.32 (s, 2H), 3.30 - 3.23 (m, 2H), 3.21 - 3.09 (m, 2H), 3.08 - 3.00 (m, IH), 2.58 - 2.52 (m, IH), 2.34 (s, 3H), 2.23 (s, 6H), 2.12 (p, J = 6.7 Hz, 2H), 1.27 (d, J =28.5 Hz, 9H).
Step N: methyl 2-{3-[(l ,3-benzothiazol-2-yl)amino}-6-(2-{[(tertbutoxy)carbonylj(methyl) amino}ethoxy)-4-methyl-5H,6H, 7H,8H-pyrido[2,3-cJpyridazin -8yl}-5~(3-{4-[3-(dimethylamino)prop-l -yn-1 -yl]-2-fluoroph enoxyjpropyl)-l ,3-thÎazok-4carboxylate
To a solution of the product from Step M (136 mg, 0.19 mmol, 1 eq) in cyclohexanol (4.5 30 mL) was added 2-aminobenzothiazole (55.7 mg, 0.37 mmol, 2 eq) and N,Ndiisopropylethylamine (0.1 mL, 0.56 mmol, 3 eq) and the mixture was sparged with nitrogen (10 min). Xantphos (21.5 mg, 0.04 mmol, 0.2 eq) and
253 tris(dibenzylideneacetone)dipalladium(0) (17 mg, 0.02 mmol, 0.1 eq) were added and the mixture was heated at 140 °C for 1 h under microwave irradiation. The reaction was partitioned between dichloromethane and water, and the aqueous phase was extracted with dichloromethane (3 x 40 mL). The combined organic extracts were washed with brine, dried (PTFE phase separator) and concentrated in vacuo. Purification by reverse phase automated flash chromatography (CombiFlash Rf, C18 15.5g Gold RediSep column) eluting with a gradient of 5 - 95% acetonitrile in water afforded the desired product as a yellow solid (70.8 mg, 0.08 mmol, 45%).
LC/MS (C42H49FN8OgS2) 845 [M+H]+; RT 0.86 (LCMS-V-B2) *H NMR (400 MHz, DMSO-d6) δ 11.52 (br s, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 8.1 Hz, 1H), 7.37 (ddd, J = 8.2, 7.3, 1.3 Hz, 1H), 7.31 (dd, J = 11.9, 1.9 Hz, 1H), 7.24 - 7.12 (m, 3H), 4.80 (dd, 1H), 4.22 (s, 1H), 4.15 (t, J = 6.2 Hz, 2H), 3.94 (d, J = 13.4 Hz, 1H), 3.78 (s, 3H), 3.56 (t, J = 5.7 Hz, 2H), 3.44 - 3.37 (m, 1H), 3.31 (s, 2H), 3.28 (d, 1H), 3.24 - 3.14 (m, 2H), 3.12 - 2.97 (m, 2H), 2.58 (d, J = 12.3 Hz, 3H), 2.33 (s, 3H), 2.19 (s, 6H), 2.14 (q, J = 7.0 Hz, 2H), 1.27 (d, 9H).
Step O: methyl 2-{3-[(1,3-benzothiazol-2-yl)amino]-4-methyl-6-[2-(methylamino)ethoxyj5H,6H, 7H,8H-pyrido[2,3-c]pyridazin-8-yl}-5-(3-{4-[3-(dimethylamino)prop-l-yn -1 -yl]-2fîuorophenoxy/propyl) ~1,3-thiazole-4-carboxylate
To a solution of the product from Step N (70.8 mg, 0.08 mmol, 1 eq) in dichloromethane (5 mL) was added trifluoroacetic acid (1 mL) slowly and the mixture was stirred at ambient température for 1 h. The reaction was partitioned between dichloromethane and saturated aqueous sodium bicarbonate and the aqueous phase was extracted with dichloromethane (3 x 30 mL). The combined organic extracts were washed with brine, dried (PTFE phase separator) and concentrated in vacuo to afford the desired product as a bright yellow solid (59.8 mg, 0.08 mmol, 96%).
LC/MS (C37H41FN8O4S2) 745 [M+HJ+; RT 1.07 (LCMS-V-B1)
Ή NMR (400 MHz, DMSO-d6) δ 7.88 (dd, J = 7.8, 1.2 Hz, 1H), 7.49 (d, J = 8.1 Hz, 1H), 7.37 (ddd, J = 8.2, 7.2, 1.3 Hz, 1H), 7.32 (dd, J = 11.9, 1.9 Hz, 1H), 7.24 - 7.12 (m, 3H), 4.79 - 4.69 (m, 1H), 4.26 - 4.19 (m, 1H), 4.15 (t, J = 6.2 Hz, 2H), 4.03 (dd, J = 13.5, 2.4 Hz, 1H), 3.78 (s, 3H), 3.60 (t, J = 5.5 Hz, 2H), 3.39 (s, 2H), 3.32 - 3.27 (m, 2H), 3.15 (d, J = 14.6 Hz, 1H), 3.08 - 2.99 (m, 1H), 2.70 (t, J = 5.5 Hz, 2H), 2.38 (s, 3H), 2.29 (s, 3H), 2.22 (s, 6H), 2.17-2.08 (m, 2H).
254
Step P: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6-[2-(methylaminojethoxy]5H,6H,7H,8H-pyrido[2,3-c]pyridazin‘8-yl}-5-(3-{4-[3-(dimethylamino)prop-l -yn-1 -yl]-2fluorophenoxy}propyl)-l,3-thiazole-4-carboxylic acid
To a solution of the product from Step O (59.8 mg, 0.08 mmol, 1 eq) in 1,4-dioxane (2 mL)was added IM aqueous lithium hydroxide (0.24 mL, 0.24 mmol, 3 eq) and the mixture was heated at 50 °C for 2 h. The solid was collected by filtration and dried under vacuum to afford the desired product as a bright yellow solid (43 mg, 0.06 mmol, 73%), as a lithium sait.
HRMS-ESI (m/z) [M+H]+ calcd for C36H40FN8O4S2: 731.2598, found 731.2623.
Exampie 66: 2-{3-[(l,3-Benzothiazol-2-yl)amino]-4-methyl-5j7,6Z/,71ï,8//-pyrido[2,3c]pyridazm-8-yl}-5-{3-[(6-fluoro-2-methyl-l,2,3,4-tetrahydroisoquinolin-7yl)oxy]propyl}-l,3-thiazole-4-carboxylic acid
Step A: methyl5-{3-[(6-jluoro-2-methyl-l,2,3,4-tetrahydroisoquinolin-7-yl)oxy]propyl}-2(4-methyl~3-{[ (2Z) -3-{[2-(trimethylsilyl) ethoxy]methyl}-2,3-dihydro-l,3 -benzothiazol-2ylidetie]amino}-5H,6H, 7H,8H-pyrido[2,3-c]pyridazin -8-yl) -1,3-thiazole-4-carboxylate
To a solution of 6-fluoro-2-methyl-3,4-dihydro-lFf-isoquinolin-7-ol (52 mg, 0.29 mmol, 1.8 eq) and the product from Préparation 3b (100 mg, 0.16 mmol, 1 eq) in toluene (5 mL) was added triphenylphosphine (83.7 mg, 0.32 mmol, 2 eq) and di-teri-butyl azodîcarboxylate (73.5 mg, 0.32 mmol, 2 eq) and the mixture was stirred at 50 °C overnight. The reaction was partitioned between dichloromethane and water, and the organic phase was washed with brine, dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% methanol in dichloromethane afforded the desired product as a yellow solid (61 mg, 0.08 mmol, 48%).
255 • LC/MS (C39H48FN7O4S1S2) 790 [M+H]+; RT 2.68 (LCMS-V-C)
NMR (400 MHz, DMSO-d6) Ô 7.82 (d, 1H), 7.48 - 7.37 (m, 2H), 7.28 - 7.19 (m, IH), 6.94 (d, J = 12.1 Hz, 1H), 6.82 (d, J = 8.7 Hz, 1H), 5.84 (s, 2H), 4.27 (t, J = 5.8 Hz, 2H), 4.05 (t, J - 6.2 Hz, 2H), 3.79 (s, 3H), 3.76 - 3.68 (m, 2H), 3.42 - 3.34 (m, 4H), 3.27 (t, 2H), 2.88 (t, 5 J = 6.2 Hz, 2H), 2.69 (d, J = 6.0 Hz, 2H), 2.38 (s, 3H), 2.26 (s, 3H), 2.17 - 1.99 (m, 4H), 0.95 -0.87 (m, 2H), -.011 (s, 9H).
Step B: methyl 2-{3-[(1,34)enz()thia7X)l-2-yl)amin<)]-4-methyl-5Il,6Il7H,8H-pyrido[2,3cJpyridazin -8~yl}~5-{3-[(6-fluoro-2-methyl-l ,2,3>4~tetrahydroisoquinolin-7-yl)oxy]propyl}1,3-thiazole-4-carboxylate
A solution of the product from Step A (67 mg, 0.08 mmol, 1 eq) in dichloromethane (4 mL) was cooled to 0 °C and trifluoroacetic acid (1.95 mL, 25.4 mmol, 300 eq) was added and the mixture was stirred for 6 h at ambient température. Dichloromethane (10 mL) was added and the solution was cooled to 0 °C, washed with aqueous ammonîa, and the organic phase was separated (phase separator) and concentrated in vacuo. Purification by automated flash 15 column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 10% methanol in dichloromethane afforded the desired product as a yellow solid (39 mg, 0.06 mmol, 70%).
LC/MS (C33H34FN7O3S2) 660 [M+H]+; RT 2.01 (LCMS-V-C)
NMR (400 MHz, DMSO-d6) 5 7.89 (s, 1H), 7.51 (br s, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.20 20 (t, J = 7.5 Hz, 1H), 6.95 (d, J = 12.1 Hz, 1H), 6.83 (d, J - 8.7 Hz, 1H), 4.27 (t, J = 5.7 Hz,
2H), 4.06 (t, J = 6.3 Hz, 2H), 3.80 (s, 3H), 3.43 - 3.36 (m, 4H), 3.29 (t, 2H), 2.89 (t, J = 6.3 Hz, 2H), 2.73 - 2.66 (m, 2H), 2.35 (s, 3H), 2.27 (s, 3H), 2.17 - 2.10 (m, 2H), 2.09 - 2.00 (m, 2H).
Step C: 2-{3f(1,3-benzothiazol-2-yl)amino]-4-methyl‘5H,6H, 7H,8H-pyrido[2,325 c]pyridazin-8-yl}-5-{3-[(6-fluoro-2-methyl-l,2,3,4-tetrahydroisoquinolin - 7-yl) oxyjpropyl}l,3-thiazole-4-carboxylic acid
To a solution of the product from Step B (39 mg, 0.06 mmol, 1 eq) in 1,4-dioxane (2 mL) was added lithium hydroxide monohydrate (24.8 mg, 0.59 mmol, 10 eq) and the mixture was heated at reflux overnight. The reaction was allowed to cool to ambient température and 30 concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 20% 7N methanolic
256 ammonia in dichioromethane afforded the desired product as an off-white solid (19.4 mg, 0.03 mmol, 51%)
HRMS-ESI (m/z) [M+HJ+ calcd for C32H33FN7O3S2: 646.2070, found 646.2094
Example 67: 5-[3-[4-[3-(Azetidin-l-yl)propyl]-2-tluoro-phenoxy]propyl]-2-[3-(l,35 benzothiazol-2-ylanrino)-4-niethyl-6,7-dihydro-5W-pyrido[2,3-c]pyridazin-8-yl]thiazole4-carboxylic acid
Step A: methyl 5-[3-[4-[3-(azetidin-l-yl)propyl]-2-fluoro-phenoxy]propyl]-2-[3-(l,3ben7,othiazol-2-ylainin())-4-metliyl-6,7-dihydro-5ii-pyri(l(>l2y3-clpyrid(izi.ii-8-yl}l.liiaz<)lf’-410 carboxylate
Using Propargylic amine préparation General Procedure starting from 50 mg of Préparation 3e (0.077 mmol, 1.0 eq.) as the appropria te alcohol and azetidine (88.00 mg, 20 eq.), 35 mg of the desired product (75%) was obtained.
Step B; 5-[3-[4-[3-(azetidin-l-yl)propyl]-2‘fluoro-phenoxy]propyl]-2-[3-(l,3-benzothiazol-2~ 15 ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl]thiazole-4-carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step A as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C34H36FN7O3S2: 674.2377, found 674.2386.
Example 68: 2-[3-(1,3-Benzothiazol-2-ylamino)-4-methy 1-6,7-dihydro-5H-pyrido[2,320 c]pyrîdazin-8-yl]-5-[3-[2-fluoro-4-[3-(4-methylpiperazin-lyI)propyl]phenoxy]propyl]thiazole-4-carboxylic acid
257
Steo A: methyl2-[3-(l,3-benzothiazol~2-ylamino)-4~methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-[3-[2-fluoro-4-[3~(4-methylpiperazin-lyl)propyl]phenoxy]propyl]thiazole-4-carboxylate
Using Propargylic amine préparation General Procedure starting from 50 mg of Préparation 3e (0.077 mmol, 1.0 eq.) as the appropriate alcohol and 1-methylpiperazine (154.4 mg, 20 eq.), 46 mg of the desired product (73%) was obtained.
Step B: 2-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin8-yl]-5-[3’[2-fluoro-4-[3-(4-methylpiperazin-l -yl)propyl]phenoxy]propyl]thiazole-4- carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step A as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C36H42FN8O3S2: 717.2799, found 717.2808.
Example 69: 2-[3-(l,3-Benzothiazol-2-yIamino)-4-methyl·6,7-dihydro-5/Z-pyrido[2,315 c]pyridazin-8’yl]-5-[3-[2-fluoro-4-(3-pyrrolidin-l-ylpropyI)phenoxy]propyl]thiazole-4carboxylic acid
258
Step A: methyl2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3clpyridazin-8-yl]-5-(3-(2-fluoro-4-(3-pyrrolidin-l-ylpropyl)phenoxy]propyl]thiazole-4carboxylate
Using Propargylic amine préparation General Procedure starting from 50 mg of 5 Préparation 3e (0.077 mmol, 1.0 eq.) as the appropriate alcohol and pyrrolîdine (109.6 mg, 20 eq.), 53 mg of the desired product (98%) was obtained.
Step B: 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3-c]pyridazin8-yl]-5-(3-(2-fluoro-4-(3-pyrrolidin-l-ylpropyl)phenoxy]propyl]thiazole-4-carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step A as the 10 appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C35H39FN7O3S2: 688.2534, found 688.2533.
Example 70: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-517-pyrido[23c]pyridazin-8-yl]-5-[3-[2-fluoro-4-(3-morpholinopropyl)phenoxy]propyl]thiazole-4carboxylic acid
Step A; methyl 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-(3-[2-fluoro-4-(3-morpholinopropyl)phenoxy]propyl]thiazole-4carboxylate
Using Propargylic amine préparation General Procedure starting from 50 mg of 20 Préparation 3e (0.077 mmol, 1.0 eq.) as the appropriate alcohol and morphoiine (134.3 mg, 20 eq.), 46 mg of the desired product (83%) was obtained.
Step B: 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3-c]pyridaziu8-ylJ-5-(3-(2-fluoro-4-(3 -morpholinopropyl)phenoxy]propyl]thiazole-4-carboxylic acid
259
Using Hydrolysîs General Procedure starting from the product from Step A as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C35HWFN7O4S2: 704.2483, found 704.2471.
Example 71; 2-[3-(l,3-Benzothiazol·2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,35 c]pyridazin-8-yl]-5-[3-[2-tluoro-4-[3-(l-piperidyl)propyl]phenoxy]propyl]thiazole-4carboxylic acid
Step A: methyl 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3c]pyridazin-8-yl]-S -(3-(2-fluoro-4-(3-(l -piperidyl)propyl]pkenoxy]propyl]thiazole-410 carboxylate
Using Propargylîc amine préparation General Procedure starting from 50 mg of Préparation 3e (0.077 mmol, 1.0 eq.) as the appropriate alcohol and piperîdine (131.2 mg, 20 eq.), 43 mg of the desired product (83%) was obtained.
Step B: 2-(3-(1,3-benzothiazol-2-ylamino)-4-tnethyl-6,7-dihydro-5H-pyrido(2,3-c]pyridazin15 8-yl]-5-(3-(2-fluoro-4-(3-(1 -piperidyl)propyl]phenoxy(propyl]thiazole-4-carboxylic acid
Using Hydrolysîs General Procedure starting from the product from Step A as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]-i- calcd for C36H41FN7O3S2: 702.2690, found 702.2703.
Example 72: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5i7-pyrido[2,320 c]pyridazin-8-yl]-5-[3-[4-[3-[(lff, 55)-6,6-difluoro-3-azabicyclo[3.1.0]hexan-3-yl]propyl]2-fluoro-phenoxy]propyl]thiazole-4-carboxylic acid
26Ü
Step A: methyl 2-(3-(1 >3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3c]pyridazin-8-yl]~5-(3-(4-[3-[(1S ,5R) -6,6-difluoro-3-azabicyclo(3.1.0(hexan-3-yl]propyl]-2fluoro-phenoxy]propyl]thiazole-4-carboxylate
Using Propargylic amine préparation General Procedure starting from 50 mg of Préparation 3e (0.077 mmol, 1.0 eq.) as the appropriate alcohol and (15,5/ï)-6,6-difluoro-3azabicyclo[3.1.0]hexane (1.54 mmol, 20 eq.), 24 mg of the desired product (41%) was obtained.
Step B: 2-(3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3-c]pyridazin10 8-yll-5-l 3-l4-l3-[(lR,5S)-6,6-diJhior()-3-azabîcycl<)l 3.1.0lhexüii-3-yllpr<)pyll-2-fluor()ph enoxy]propyl]thiazole-4-carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step A as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C36H37F3N7O3S2: 736.2345, found 736.2340.
Exampie 73: 2-[3-(l,3-Benzothîazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-[3-[2-fluoro-4-[3-(3-oxo-2,8-diazaspiro[4.5]decan-8yl)propyl]phenoxy] propylJthiazole-4-carboxylic acid
261
Step A: methyl 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3cjpyridazin -8-yl]-5-(3-[2-fluûro-4-[3-(3-oxo-2,8-diazaspiro(4.5]decan-8yl)propyl]phenoxy]propyl]thiazole-4-carboxylate
Using Propargylic amine préparation General Procedure starting from 50 mg of Préparation 3e (0.077 mmol, 1.0 eq.) as the appropriate alcohol and 2,8diazaspiro[4.5]decan-3-one (237.7 mg, 20 eq.), 35 mg of the desired product (58%) was oblained.
Step B: 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6)7-dihydro-5H-pyrido(2,3-c]pyridazin10 8-yl]-5-(3-(2-fluoro-4’(3-(3-oxo-2,8-diazjaspiro(4.5]decan-8-yl)propyl]phenoxy] propyl]thiazole-4-carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step A as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C39H44FN8O4S2: 771.2905, found 771.2922.
Exampie 74: 2-{3-[(l,3-Benzothiazol-2-y])amino]-4-methyl-5/7,6//,7/f,8//-pyrido[2,3c]pyridazin-8-yl}-5-{3-[(7-fluoro-2-methyl-l,2,3,4-tetrahydroisoquinolin-6yl)oxy]propyl}-l,3-thiazole-4-carboxylic acid
262
Step A : methyl 5-{3-[(7-fluoro-2-methyl-l,2,3,4-tetrahydroisoquinolin-6-yl)oxy]propyl}-2· (4-methyl-3-{[(2Z)-3-{[2~(trimethylsilyl)ethoxy]niethyl}-2,3-dihydro-l,3-benzothiazol-2ylidene]amino}-5H,6H,7H,8H-pyrido[2,3-c]pyridazin-8-yl)-ls3~thiazole-4-carboxylate
To a solution of the product from Préparation 3b (120 mg, 0.19 mmol, 1 eq) in toluene (5 mL) was added 7-fluüro-2-methyl-3,4-dihydro-l/Msoquinolin-6-ol (69.4 mg, 0.38 mmol, 2 eq), triphenylphosphine (100 mg, 0.38 mmol, 2 eq) and di-teri-butyl azodicarboxylate (88.2 mg, 0.38 mmol, 2 eq). The mixture was stirred at 50 °C ovemight. The reaction was partitioned between dichloromethane and water, and the organic phase was washed with brine, dried (PTFE phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 14% methanol in dichloromethane afforded the desired product as a yellow gum (111 mg, 0.14 mmol, 73%).
LC/MS (C39H48FN7O4SÎS2) 790 [M+H]+; RT 2.64 (LCMS-V-C) ‘H NMR (400 MHz, DMSO-d6) δ 7.82 (dd, J = 7.6, 1.2 Hz, 1H), 7.48 - 7.38 (m, 2H), 7.28 7.20 (m, 1H), 6.94 - 6.84 (m, 2H), 5.85 (s, 2H), 4.27 (t, J = 5.8 Hz, 2H), 4.06 (q, J = 7.4, 6.8 Hz, 2H), 3.79 (s, 3H), 3.76 - 3.68 (m, 2H), 3.34 - 3.22 (m, 4H), 2.88 (t, J = 6.3 Hz, 2H), 2.73 (t, J = 6.0 Hz, 2H), 2.38 (s, 3H), 2.27 (s, 3H), 2.17 - 1.99 (m, 4H), 0.97 - 0.86 (m, 2H), -0.12 (s, 9H).
Step B: methyl 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H,7H,8H-pyrido[2,3· c]pyridazin-8-yl}-5-{3-[(7-fluoro-2-methyl-l,2,3,44etrahydroisoquinolin-6-yl)oxy]propyl}1,3-thiazole-4-carboxylate
A solution of the product from Step A (111 mg, 0.14 mmol, 1 eq) în dichloromethane (5 mL) was cooled to 0 °C and trifluoroacetic acid (1.02 mL, 13.4 mmol, 95 eq) was added and the mixture was stirred at ambient température ovemight. Dichloromethane (10 mL) was added and the solution was cooled to 0 °C, washed with aqueous ammonia, and the organic
263 phase was separated (phase separator) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 — 7% methanol in dichloromethane afforded the desired product as a yellow gum (79 mg, 0.12 mmol, 85%).
LC/MS (C33H54FN7O3S2) 660 [M+Hf; RT 2.01 (LCMS-V-C)
Ή NMR (400 MHz, DMSO-d6) δ 7.88 (d, J = 7.8 Hz, 1H), 7.50 (br s, 1H), 7.42 - 7.33 (m, 1H), 7.20 (t, J = 7.7 Hz, 1H), 6.95 - 6.85 (m, 2H), 4.27 (t, J = 5.7 Hz, 2H), 4.08 (t, J = 6.2 Hz, 2H), 3.79 (s, 3H), 3.34 - 3.22 (m, 6H), 2.89 (t, J = 6.3 Hz, 2H), 2.74 (t, J = 5.9 Hz, 2H), 2.35 (s, 3H), 2.28 (s, 3H), 2.17 - 2.03 (m, 4H).
Step C: 2‘{3-[(l,3‘benzothiazol-2-yl)amino]‘4‘methyl-5H,6H,7H,8H-pyrido[2,3c]pyridazin-8-yl}-5-{3-[(7-fluoro-2-methyl-l,2,3,4-tetrahydroisoquinolin-6-yl)oxy]propyl}1,3-thiazole-4-carboxylic acid
To a solution of the product from Step B (79 mg, 0.12 mmol, 1 eq) in 1,4-dioxane (3 mL) was added lithium hydroxide monohydrate (50.2 mg, 1.2 mmol, 10 eq) and the mixture was heated at reflux for 4.5 h. The reaction was allowed to cool to ambient température and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 20% 7N methanolic ammonia in dichloromethane afforded a solid that was trîturated with diethyl ether, filtered, washed with diethyl ether and dried under vacuum to afford the desired product as a yellow solid (44.4 mg, 0.07 mmol, 57%).
HRMS-ESI (m/z) [M+H]+ calcd for C32H33FN7O3S2: 646.2070, found 646.2103
Example 75: 2-{3-[(l,3-Benzothiazol-2-yl)amino]-4-methyl-5Ff,6H,7Fr,8.H-pyrido[2,3c]pyridazin-8-yl}-5-(3-{2-fluoro-4-[4-(methylamino)butyl]phenoxy}propyl)-l,3-thiazole4-carboxylic acid
264
Step A: methyl5-/3-/4-(4-//(tert-butoxy) carbonyl] (methyl) amino}butyl)-2fliiorophenoxy]propyl}-2-(4-methyl-3-{[(27.)-3-{[2-(tHmethylsilyl)ethoxy]methyl}-2,3dihydro-l,3-benzothiazol‘2-ylidene]amino}-5H,6H,7H,8H-pyrido[2,3‘c]pyridazin-8-yl)-l ,3thiazole-4-carboxylate
To a solution of the product from Préparation 3b (50 mg, 0.08 mmol, 1 eq) in toluene (5 mL) was added the product from Préparation 4k (32.7 mg, 0.11 mmol, 1.38 eq), di-terr-butyl azodicarboxylate (36.7 mg, 0.16 mmol, 2 eq) and triphenylphosphîne (41.8 mg, 0.16 mmol, 2 eq) and the mixture was heated at 50 °C overnight. The reaction was partitioned between dichloromethane and water, separated (PTFE phase separator) and concentrated in vacuo.
Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 70% ethyl acetate in iso-heptane afforded the desired product as a clear gum (45 mg, 0.05 mmol, 62%).
LC/MS (C45H6oFN706SiS2) 906 [M+H]+; RT 3.51 (LCMS-V-C) ‘H NMR (400 MHz, DMSO-d6) δ 7.82 (d, 1H), 7,48 - 7.38 (m, 2H), 7.24 (ddd, J = 8.3, 6.8, 15 1.8 Hz, 1H), 7.11 - 7.01 (m, 2H), 6.91 (d, J = 8.2 Hz, III), 5.85 (s, 2H), 4.27 (t, J = 5.8 Hz,
2H), 4.07 (t, 2H), 3.78 (s, 3H), 3.76 - 3.68 (m, 2H), 3.32 - 23 (m, 4H), 3.16 - 3.05 (m, 2H), 2.88 (t, J = 6.3 Hz, 2H), 2.70 (s, 3H), 2.38 (s, 3H), 2.17 - 2.01 (m, 4H), 1.51-1.39 (m, 4H), 1.34 (d, J = 16.8 Hz, 9H), 0.95 - 0.86 (m, 2H), -0.11 (s, 9H).
Step B: methyl 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6H, 7H,8H-pyrido[2t320 c]pyridazin-8-yl}-5-(3-{2-fluorO‘4-[4-(methylamino)butyl]phenoxy}propyl)-l,3-thiazole-4~ carboxylate
A solution of the product from Step A (45 mg, 0.05 mmol, 1 eq) in dichloromethane (3 mL) was cooled to 0 °C and trifluoroacetic acid (0.61 mL, 7.95 mmol, 160 eq) was added and the mixture was stirred for 24 h at ambient température. Dichloromethane (40 mL) was added 25 and the solution was washed with aqueous ammonia and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0 - 20% methanol in dichloromethane afforded the desired product as a yellow solid (22 mg, 0.03 mmol, 66%).
LC/MS (C34H3sFN7O3S2) 676 [M+H]+; RT 1.12 (LCMS-V-B1) *H NMR (400 MHz, DMSO-d6) Ô 7.85 (d, J = 7.9 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.16 (t, J = 7.5 Hz, 1H), 7.11 - 7.02 (m, 2H), 6.93 (d, J = 8.8 Hz, 1H), 4.26 (dd, J = 6.9, 4.3 Hz, 2H), 4.09 (t, J = 6.1 Hz, 2H), 3.79 (s, 3H), 3.32 - 3.24 (m, 4H), 2.88 (t, J = 6.3
265 • Hz, 2H), 2.42 (t, 2H), 2.34 (s, 3H), 2.23 (s, 3H), 2.19 - 2.09 (m, 2H), 2.08 - 1.99 (m, 2H), 1.60-1.47 (m, 2H), 1.41 - 1.30 (m, 2H).
Step C: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-5H,6Hi7H,8H-pyrido[2,3c]pyridazin-8-yl}-5-(3-{2-fluoro-4-[4-(methylamino)butyl]phenoxy}propyl)'l,3-thiazole-4~ 5 carboxylic acid
To a solution of the product from Step B (22 mg, 0.03 mmol, 1 eq) în 1,4-dîoxane (3 mL) was added lithium hydroxide monohydrate (13.7 mg, 0.33 mmol, 10 eq) and the mixture was heated at reflux overnight. Purification by automated flash column chromatography (CombiFlash Rf, 4 g RediSep™ silica cartridge) eluting with a gradient of 0—25% 7N 10 methanolîc ammonia in dichloromethane afforded a solid that was suspended în ethyl aeetate (1.5 mL) and hydrochloric acid (4M in 1,4-dioxane; 54.5 pL, 0.22 mmol, 6.7 eq) was added. The mixture was stirred for 10 min, then the solids were collected by filtration and dried under vacuum to afford the desired product as a yellow solid (11.4 mg, 0.02 mmol, 53%), as a hydrochloric acid sait.
HRMS-ESI (m/z) [M+H]+ calcd for C33H37FN7O3S2: 662.2383, found 662.2414
Example 76: 2-[3-(l,3-Benzothiazol-2-ylaniino)-4-inethyl-6,7-dihydro-5if-pyrido[2,3c]pyridazin-8-yl]-5-[3-[4-[3-[2-(dimethy]amino)ethylamino]prop-l-ynylJ-2-fluorophenoxy]propyl]thiazole-4-carboxylic acid
Step A: methyl 5-[3-[4-[3-[tert-butoxycarbonyl-[2-(dimethylamitio)ethyl]amino]prop-lynyl]-2-fluoro-phenoxy]propyl]-2-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl)thiazole-4~carboxylate
266
Using Sonogashira General Procedure starting from 1.00 g of Préparation 3a (1.66 mmol, eq.) and 413 mg of tert-butyl N-(2-(dimethylamino)ethyl]-N-prop-2-yiiyl-carbamate (1.83 mmol, 1.1 eq.) as the appropriate alkyne, the desired product was isolated as yellow solid.
Ή NMR (500 MHz, DMSO-rF) δ ppm 7.30 (d, IH), 7.21 (d, IH), 7.15 (t, IH), 4.27 (brt, 2H), 4.26 (t, 2H), 4.12 (t, 2H), 3.77 (s, 3H), 3.47 (brt, 2H), 3.26 (t, 2H), 2.89 (t, 2H), 2.82 (brs, 2H), 2.45 (brs, 6H), 2.32 (s, 3H), 2.11 (qn, 2H), 2.04 (qn, 2H), 1.43 (s, 9H); 1JC NMR (125 MHz, DMSO-ώ) δ ppm 163.1, 155.4, 151.8, 151.4, 151.4, 147.5, 142.4, 136.2, 135, 129.1, 129.1, 119.2, 115.5,114.8, 82.3, 80.3, 68.3, 56.3, 52.0, 46.4, 46.4, 44.6, 43.1, 30.7, 28.5, 24.2, 23, 19.7, 15.7; HRMS-ESI (m/z): [M+H]+ calcd for C34H43CIFN6O5S: 701.2683, found 701.2678.
Step B; methyl 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro~5H-pyrido(2,3cjpyridazin -8-yl]-5-[3-[4-(3-(tert-butoxycarbonyl-(2-(dimethylamino) ethyl]amino]prop-l ynyl]-2-fluoro-phenoxy]propyl]thiazole-4-carboxylate
Usîng Buchwaid General Procedure II starting from the product from Step A and 1,3benzothiazol-2-amine, the desired product was obtained.
LC-MS-ESI (m/z): [M+H]+ calcd for C41H4SFN8O5S2: 815.3, found 815.4.
Step C: 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridaziu8-yl]-5-[3-(4-[3-[2-(dimethylamino)ethylamino]prop-l-ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylic acid
Using Deprotection and Hydrolysis General Procedure followed by repurification via reverse phase préparative chromatography (Cl8, 25 mM NH4HCO3 in water : MeCN) starting from the product from Step B, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C35H38FN8O3S2: 701.2487, found 701.2483.
Exampie 77: 2-(3-(1,S-Benzothiazol^-ylaniinoM-methyl-ôjV-dihydro-Slf-pyridotZ,3c]pyridazin-8-yI]-5-[3-(2-fluoro-4-iodo-phenoxy)propyl]thiazole-4-carboxyhc acid
267
Step A: methyl 5-[3-(2-fluoro-4-iodo-phenoxy)propyl]-2-[4-methy 1-3-/(Z)-[3-(2trimethylsilylethoxymethyl)-l,3-benzothiazol-2-ylidene]amino]-6,7-dihydro-5H-pyrido]2,3c]pyridazin~8-yl]thiazole-4-carboxylate
Using Mitsunobu General Procedure staring from 2.00 g of Préparation 3b (3.19 mmol, 1 eq.) and 835 mg of 2-fluoro-4-iodo-phenol (3.51 mmol, 1.1 eq.) as the appropriate phénol, 2.31 g (85% Yield) of the desired product was obtained.
Ή NMR (500 MHz, DMSO-î/6) δ ppm 7.81 (dm, IH), 7.6 (dd, IH), 7.45 (dm, IH), 7.43 (dm, IH), 7.41 (m, IH), 7.23 (m, IH), 7 (t, IH), 5.83 (s, 2H), 4.25 (t, 2H), 4.1 (t, 2H), 3.77 (s, 3H), 10 3.71 (m, 2H), 3.26 (t, 2H), 2.84 (t, 2H), 2.34 (s, 3H), 2.11 (m, 2H), 2.03 (m, 2H), 0.9 (m, 2H),
-0.11 (s, 9H); HRMS-ESI (m/z): [M+H]+ calcd for C35H4iHN6O4S2Si: 847.1423, found 847.1396.
Step B: 2-/3-(1,3-benzothiazol-2-ylammo)-4-methyl-6,7-dihydro-5H-pyrido[2,3-cJpyridazin· 8-yl]-5-[3-(2-fluoro-4-iodo-phenoxy)propyl]thiazole-4-carboxylic acîd
Using Deprotection and Hydrolysis General Procedure starting from the product from Step A as the appropriate carbamate, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C28H2;iHN6O3S2: 703.0452, found 703.0427.
Example 78: 2'[3-(l,3-BenzothiazoI-2-ylamino)-4-methyI-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-[3-[5-[3-(dimetliylamino)prop-l-ynyl]-2-fluoro20 phenoxy]propyl]thiazoIe-4-carboxylic acid
268
Step A : methyl 5-[3-(2-fluoro-5-iodo-phenoxy)propylJ-2-[4-methyl-3-[(Z)-[3-(2trimethylsilylethoxymethyl)-l,3-benzothiazol-2-ylidene]amino]-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]thiazole-4-carboxylate
Using Mitsunobu General Procedure staring from 390 mg of Préparation 3b (0.622 mmol, 1 eq.) and 177 mg of 2-flttoro-5-iodo-phenol (0.746 mmol, 1.2 eq.) as the appropriale phénol, 416 mg (79% Yield) of the desired product was obtained.
*H NMR (500 MHz, DMSO-d6) δ ppm 7.81 (dm, 1H), 7.46 (dd, 1H), 7.43 (dm, 1H), 7.41 (m, 1H), 7.27 (m, 1H), 7.23 (m, 1H), 7.05 (dd, 1H), 5.83 (s, 2H), 4.26 (t, 2H), 4.14 (t, 2H), 3.78 (s, 3H), 3.71 (m, 2H), 3.26 (t, 2H), 2.85 (t, 2H), 2.34 (s, 3H), 2.11 (m, 2H), 2.04 (m, 2H), 0.91 (m, 2H), -0.11 (s, 9H); HRMS-ESI (m/z): [M+H]+ calcd for C^H^FINeOiS,: 847.1423, found 847.1416.
Step B: methyl 5-[3-[5-[3~(dimethylamino)prop-l-ynyl]-2-fluoro-phenoxy/propyl]-2‘[4~ methyl-3-[(Z)-[3-(2-trimethylsilylethoxymethyl)~l ,3-benzothiazol-2-ylidene]amino]-6r715 dihydro-5H-pyrido[2,3-c]pyridazin-8-yl]thiazole-4-carboxylate
Using Sonogashira General Procedure starting from 310 mg of the product from Step A (0.366 mmol, 1.0 eq.) and 91 mg N,N-dimethylprop-2-yn-l -amine (1.10 mmol, 3 eq.) as the appropriate acetylene, 251 mg (85% Yield) of the desired product was obtained.
’H NMR (500 MHz, DMSO-d(i) δ ppm 7.81 (dm, 1H), 7.43 (dm, 1H), 7.41 (m, IH), 7.23 (m, 20 1H), 7.23 (m, 1H), 7.22 (dd, 1H), 7.03 (m, 1H), 5.82 (s, 2H), 4.25 (t, 2H), 4.15 (t, 2H), 3.78 (s, 3H), 3.71 (m, 2H), 3.50 (s, 2H), 3.27 (t, 2H), 2.84 (t, 2H), 2.33 (s, 3H), 2.28 (s, 6H), 2.12 (m, 2H), 2.03 (m, 2H), 0.9 (m, 2H), -0.11 (s, 9H); HRMS-ESI (m/z): [M+H]+ calcd for C40H49FN7O4S2Si: 802.3035, found 802.3028.
269
Step C: 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3-c]pyridazin8-y 1/-5-(3-(5-(3-(dimethylamino)prop-l -ynyl]-2-fluoro-phenoxy]propyl]thiazole-4carhoxylic acid
Using Deprotection and Hydrolysis General Procedure startîng from the product from Step B as the appropriate carbamate, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C33H33FN7O3S2: 658.2064, found 658.2045.
Example 79: 2-|3-(l,3-Ben/othiazol-2-ylaniino)-4-methyl-6,7-dihydro-5/7-pyridi)|2.3c]pyridazin-8-yn-5-[3-[2-tluoro-4-(3-piperazin-l-ylprop-l-ynyl)phenoxy]propyl]thiazole4-carboxylic acid
Step A: 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3-c]pyridazin8-yl]-5 -(3-(4-(3-(4-tert-butoxycarbonylpiperazjn-l -yl)prop-l -ynylj-2-fluorophenoxy]propyl]thiazole-4-carboxylic acid
Using Silver catalyzed propargylic amine préparation General Procedure startîng from Préparation 3c, paraformaldéhyde as the aldéhyde and tert-butyl piperazine-l-carboxylate as the appropriate secondary amine, the desired product was obtained.
Step B: 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3-c]pyridazin8-yl]-5-[3-(2-fluoro-4-(3-piperazin-l-ylprop-i-ynyl)phenoxy]propyl]thiazole-4-carboxylic acid
The mixture of the product from Step A (207 mg, 0.25 mmol) and HFxPyr (2.5 mmol, 10 eq.) in acetonitrile (4.3 mL) was stirred at 60 °C for 2.5 h. The product was purified via flash chromatography on 24 g silica gel column using DCM and MeOH (NH3) as eluents to give f 43 mg (79%) of the desired product.
270
HRMS-ESI (m/z): [M+HE calcd for CjsHjcFNsOjSz: 699.2330, found 699.2322.
Example 80: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/i-pyndo[2,3c]pyridazin-8-yl]-5-[3-[4-[3-(methylamino)prop-l-ynyl]phenoxy]propyl]thiazole-4carboxylic acid
Step A: methyl 5-[3-(4-iodophenoxy)propyl]-2-[4-methyl-3-[(Z)-[3-(2trûnethylsilylethoxymethyl)-! ,3~benzothiazol-2-ylidene]amino]-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]thiazole-4-carboxylate
Using Mitsunobu General Procedure staring from 313 mg of Préparation 3b (0.50 mmol, 1.0 eq.) and 110 mg of 4-iodo-phenol (0.50 mmol, 1.0 eq.) as the appropriate phénol, 328 mg (63% Yield) of the desired product was obtained.
3H NMR (500 MHz, DMSO-ώ) δ ppm 7.81 (d, 1H), 7.58 (d, 2H), 7.43 (d, 1H), 7.42 (t, IH), 7.24 (t, 1H), 6.81 (d, 2H), 5.83 (s, 2H), 4.25 (t, 2H), 4.02 (t, 2H), 3.78 (s, 3H), 3.71 (t, 2H), 3.26 (t, 2H), 2.85 (t, 2H), 2.35 (s, 3H), 2.1 (qn, 2H), 2.04 (qn, 2H), 0.9 (t, 2H), -0.11 (s, 9H); 13C NMR (125 MHz, DMSO-d^ δ ppm 138.5, 127.1, 123.3, 123.1, 117.8, 1 11.8, 73, 67.3, 66.7, 52.0, 46.4, 31.1, 23.8, 23.2, 20.4, 17.8, 13.0, -0.9; HRMS-ESI (m/z): [M+H]+ calcd for C35H42lN6O4S2Si: 829.1517, found 829.1517.
Step B: methyl 5-[3-[4-[3-[tert-butoxycarbonyl(methy 1)amino]prop-l-ynyl]phenoxy]propyl]2-[4-methy 1-3-((7)-l3-(2-trimethy4silylethoxy methyl)-1,3-benzothiazol-2-ylidene]amino]6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl]thiazole-4-carboxylate
Using Sonogashira General Procedure starting from 3304 mg of the product from Step A (0.294 mmol, 1.0 eq.) and 100 mg tert-butyl N-methyl-N-prop-2-ynyl-carbamate (0.588 mmol, 2 eq.) as the appropriate acetylene,172 mg (67% Yield) of the desired product was obtained.
271
Hl NMR (500 MHz, DMSO-d6) δ ppm 7.81 (d, 1H), 7.44 (d, 1H), 7.42 (t, 1H), 7.36 (d, 2H),
7.24 (t, 1H), 6.96 (d, 2H), 5.84 (s, 2H), 4.26 (t, 2H), 4.2 (bis, 2H), 4.06 (t, 2H), 3.78 (s, 3H), 3.72 (t, 2H), 3.28 (t, 2H), 2.86 (t, 2H), 2.84 (brs, 3H), 2.36 (s, 3H), 2.11 (qn, 2H), 2.04 (qn, 2H), 1.41 (s, 9H), 0.91 (t, 2H), -0.11 (s, 9H); i3C NMR (125 MHz, DMSO-d6) δ ppm 133.5, 127.1, 123.3, 123.1, 115.3, 111.8, 72.9, 67.3, 66.7, 52.0, 46.3, 38.6, 33.7, 31.0, 28.5, 23.8, 23.2, 20.3, 17.8, 13.0, -0.9; HRMS-ESI (m/z): [M+H]+ calcd for C44H5ûN7O&S2Sî: 870.3497, found 870.349.
Step C: 2-[3-(1,3-benzothiazol-2-ylaniino) -4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin 8-yl]-5-[3-[4-[3-(methylamino)prop~l-ynyl]phenoxy]propyl]thiazole-4-carboxylic acid
Using Deprotection and Hydrolysis General Procedure starting from the product from Step B as the appropriate carbamate, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C32H32N7O3S2: 626.2002, found 626.2004.
Example 81: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/Z-pyrido[2,3c]pyridazm-8-yl]-5-[3-[2-fluoro-4-(3-methyl-3-pyrrolidin-l-yl-but-lynyl)phenoxy]propyl] thiazole-4-carboxylîc acid
Using Silver catalyzed propargylic amine préparation General Procedure starting from Préparation 3c, acetone as the ketone and pyrrolidîne as the appropriate secondary amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C37H39FN7O3S2: 712.2534, found 712.2522.
272
Example 82: 5-[3-[4-[3-(Dimethylamino)prop-l-ynyl]-2-fluoro-phenoxy]propyl]-2-[3L(7-fluoro-l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydro-5f/-pyrfdo[2,3c]pyridazin-8-yl]thiazole-4-carboxylic acid
Step A: methyl 2-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl)-5-[3-[4[3-(dimethylamino)prop-l-ynyl]-2-fluoro-phenoxy]propyl]thiazole-4-carboxylate
Using Sonogashira General Procedure starting from 500 mg of Préparation 3a (0.80 mmol, 1.0 eq.) and 100 mg N,N-dimethylprop-2-yn-l-amine (1.2 mmol, 1.5 eq.) as the appropriate acetylene, 254 mg (50% Yield) of the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for CztHjoCIFNsOsS: 558.1736, found 558.1729.
Step B: methyl 5-[3-[4-[3-(dimethylamino)prop-l-ynyl]-2-flu,oro-phenoxy]propyl]-2-l3-[(7fluoro-l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8ylJthiazole-4-carboxylate
Using Buchwald General Procedure II starting from 254 mg of the product from Step A (0.45 mmol, 1.0 eq.) and 153 mg 7A\\xfjm-l,3-benzothiazol-2-amine (0.91 mmol, 2.0 eq.), 161 mg (51% Yield) of the desired product was obtained.
Hl NMR (500 MHz, DMSO-d6) δ ppm 11.59 (brs, IH), 7.41 (dd, IH), 7.4 (t, IH), 7.31 (dd, IH), 7.21 (dd, IH), 7.15 (t, IH), 7.08 (t, IH), 4.26 (t, 2H), 4.13 (t, 2H), 3.78 (s, 3H), 3.49 (s, 2H), 3.28 (t, 2H), 2.87 (t, 2H), 2.34 (s, 3H), 2.27 (s, 6H), 2.13 (qn, 2H), 2.04 (qn, 2H); I3C NMR (125 MHz, DMSO-d6) δ ppm 163.2, 157.0, 155.7, 151.6, 150.4, 149.1, 148.8, 147.5, 141.6, 134.9, 129.0, 128.3, 128.0, 127.9, 119.3, 117.2, 115.5, 115.0, 113.6, 108.4, 84.8, 84.3, 68.3, 51.8, 48.0, 46.4, 43.9, 30.8, 23.9, 22.9, 20.2, 12.8; HRMS-ESI (m/z): [M+H]+ calcd for C34H34F2N7O3S2: 690.2127, found 690.2110.
273
Step C: 5-[3-[4-[3-(dimethylamino)prop-l -ynyl]-2‘fluoro-phenoxy]propyl]-2-[3-[(7-fluorol,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydro-5H-pyrido[2,3-cjpyridazin-8yl]thiazole-4-carboxylic acid
Using Deprotection and Hydrolysîs General Procedure starting from the product from Step 5 B as the appropriate methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C32H31FN7O3S2: 676.1970, found 676.1958.
Example 83: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido[2,3c] pyridazin-8-yl]-5-[3-[4-[3-(dimethylamino)-3-methyl-but-l-ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylic acid
Using Silver catalyzed propargylîc amine préparation General Procedure starting from Préparation 3c, acetone as the ketone and dimethyl amine as the appropriate secondary amine, the desired product was obtained.
HRMS-ESI (m/z): [M+Hf calcd for C35H37FN7O3S2: 686.2377, found 686.2361.
Example 84: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-[3-[4-[2-[l-(dimethylamino)cyclohexyl]ethynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylic acid
274
Using Silver catalyzed propargylic amine préparation General Procedure starting from Préparation 3c, cyclohexanone as the ketone and dimethyl amine as the appropriate secondary amine, the desired product was obtained.
HRMS-ESI (m/z); [M+H]+ calcd for C38H41FN7O3S2: 726.2690, found 726.2676.
Exampie 85: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/f-pyrido[2,3c]pyridazÎn-8-yl]-5-[3-[4-[3-(diethylamino)prop-l-ynyl]-2-fluorophenoxy}propyl]thiazole-4-carboxylic acid
Using Silver catalyzed propargylic amine préparation General Procedure starting from Préparation 3c, paraformaldéhyde as the aldéhyde and diethyl amine as the appropriate secondary amine, the desired product was obtained.
HRMS-ESI (m/z); [M+H]+ calcd for C35H37FN7O3S2: 686.2377, found 686.2386.
Example 86: 2-[3-(I,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5J/-pyrido[2,315 c]pyridazin-8-ylj-5-[3-[4-[3-(diisopropylamino)prop-l-ynyl]-2-tluorophenoxy]propyl]thiazole-4-carboxylic acid
275
Using Silver catalyzed propargylic amine préparation General Procedure starting front Préparation 3c, paraformaldéhyde as the aldéhyde and diisopropyl amine as the appropriate secondary amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C37H41FN7O3S2: 714.2690, found 714.2681.
Example 87: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazm-8-yl]-5-[3-[4-[3-(diisobutylamino)prop-l-ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylic acid
Using Silver catalyzed propargylic amine préparation General Procedure starting from Préparation 3c, paraformaldéhyde as the aldéhyde and jV-isobutyl-2-methyl-propan-l-amine as the appropriate secondary amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C39H45FN7O3S2: 742.3003, found 742.3001.
Example 88: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido[2,315 c]pyridazin-8-yl]-5-[3-[4-[3-[ethyl(methyl)amino]prop-l-ynyl]-2-fluorophenoxy]propyl]thiazole~4-carboxylic acid
276
Using Silver catalyzed propargylic amine préparation General Procedure starting from Préparation 3c, paraformaldéhyde as the aldéhyde and A-methylethanamine as the appropriate secondary amine, the desired product was obtained.
HRMS-ESI (m/z): [M+HJ+ calcd for C34H35FN7O3S2: 672.2221, found 672.2206.
Example 89: 2-(3-(1,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrîdo[2,3e]pyridazin-8-yl]-5-[3-[4-[3-[(3R,55)-3,5-dimethylpiperazin-l-yl]prop-l-ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxyIic acid
Step A: 2-(3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin8-yl]-5-(3-(4-(3-((3R,5S) -4-tert-butoxycarbonyl-3,5-dimethyl-piperaz.in-l -yl]prop-l -ynyl]-2fluoro-phenoxy]propyl]thiazole-4-carboxylic acid
Using Silver catalyzed propargylic amine préparation General Procedure starting from
Préparation 3c, paraformaldéhyde as the aldéhyde and tert-butyl (2Λ,65)-2,615 dimethylpiperazine-l-carboxylate as the appropriate secondary amine, 215 mg (62% Yield) of the desired product was obtained.
Ή NMR (500 MHz, DMSO-d&) δ ppm 7.88 (dm, 1H), 7.49 (brs, 1H), 7.37 (m, 1H), 7.32 (dd.
277
1H), 7.2 (dm, 1H), 7.19 (m, 1H), 7.15 (t, 1H), 4.27 (t, 2H), 4.14 (t, 2H), 3.98 (m, 1H), 3.49 (s,
2H), 3.27 (t, 2H), 2.88 (t, 2H), 2.62/2.25 (dd+dd, 4H), 2.34 (s, 3H), 2.13 (m, 2H), 2.04 (m, 2H), 1.4 (s, 9H), 1.19 (d, 6H); HRMS-ESI (m/z): [M+H]+ calcd for C42H48FN8O5S2: 827.3167, found 827.3186.
Step B: 2-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin· 8-yl]~5~[3-[4-[3~[(3R,5S)-3,5-dimethylpiperazin-l-yl]prop-l-ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylic acid
Using Deprotection and Hydrolysis General Procedure (wîthout LiOH x H2O hydrolysis) starting from the product from Step A as the appropriate carbamate, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C37H40FN8O3S2: 727.2643, found 727.2641.
Example 90: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dîhydro-5H-pyrido[2,3c]pyridazin-8-yll-5-[3-[4-[l-[(dimethylamino)methyl]-3-bicyclo|l.Ll]pentanyl]-2-fluorophenoxy]propyI]thiazole-4-carboxylic acid
Step A: methyl 5-[3-[4-[1 -[(dimethylamino)methyl]-3-bicyclo[l.1.1]pentanyl]-2-fluorophenoxy]propyl]-2-[4-methyl-3-[(Z)-[3-(2-trimethylsilylethoxymethyl)-l,3‘benzothiazol-2ylideiielainiii()l-6.7-dihydr()-51l-pyrid()(2,3-c]pyrida-.'ni-8-yllthia~<)le-4-curb<>xylate
Using Mitsunobu General Procedure starting from Préparation 4a and Préparation 3b as the appropriate alcohol, the crude desired product was isolated and transferred into the next step without further purification.
HRMS-ESI (m/z): [M+H1+ calcd for €43^5^7048281: 844.3505, found 844.3485.
278
Step B: 2-[3-(l,3-benzothiazol-2~ylamino)-4-methyl‘6,7‘dihydro-5H-pyrido[2,3-c]pyridazin8-yl]-5-[3-[4-[1 -[(dimethylamino)methyl]-3-bicyclo[ 1.1 J JpentanylJ-2-fluorophenoxy]propyl]thiazole-4-carboxylic acid
Using Deprotection and Hydrolysis General Procedure folio wed by repurification via 5 reverse phase préparative chromatography (Cl8, 25 mM NH4HCO3 in water : MeCN) starting from the product from Step A, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C36H39FN7O3S2: 700.2534, found 700.2515.
Exampie 91: 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-57f-pyrido[2,3c]pyridazÎn-8-yl]-5-[3-[2-f1uoro-4-[3-methyl-3-(methylamino)but-I10 ynyl]phenoxy]propyl]thiazole-4-carboxylic acid
Step A: methyl 2-(3-chloro-4-methyl-6,7-dihydro~5H-pyrido[2,3-c]pyridazin-8-yl)-5-[3-[2fluoro-4-[3-methyl-3-(methylamino)but-l-ynyl]phenoxy]propyl]thiazole~4-carboxylate
Using Sonogashira General Procedure starting from Préparation 3a and jV,2-dimethylbut15 3-yn-2-amîne, 417 mg of the desired product was obtained.
XH NMR (500 MHz, DMSO-d6) δ ppm 7.23 (dd, 1 H), 7.16 (dd, 1 H), 7.12 (t, 1 H), 4.26 (t, 2 H), 4.11 (t, 2 H), 3.77 (s, 3 H), 3.25 (t, 2 H), 2.89 (t, 2 H), 2.37 (s, 3 H), 2.32 (s, 3 H), 2.1 (m, 2 H), 2.04 (m, 2 H), 1.34 (s, 6 H); I3C NMR (125 MHz, DMSO-d6) δ ppm 163.1, 151.3, 136.2, 129.1, 128.9, 119.1, 115.4, 93.7, 81.5, 68.2, 52, 51, 46.4, 30.7, 30.4, 29, 24.2, 23, 19.7, 20 15.7; HRMS-ESI (m/z): [M+H]+ calcd for C28H32CIFN5O3S: 572.1898; found: 572.1888.
Step B: methyl 2-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-[3-[2-fluoro-4-[3-methyl-3-(methylamino)but-lynyl]phenoxy]propyl]thiazole-4-carboxylate
279
Using Buchwald General Procedure H staring from the product from Step A and 1,3benzothiazol-2-amine, 77 mg of the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C35H37FN7O3S2: 686.2383; found 686.2380
Step C: 2-[3'(lt3-benzothiazol-2-ylamino)-4-inethyl-6,7-dihydro-5H-pyrido[2,3-c]pyridaziu5 8-yl]-5-[3-[2-fluoro-4-[3-methyl-3-(methylamino)but-l -ynyl]phenoxy]propyl]thiazole-4carboxylic acid
Using Hydrolysis General Procedure starting from the product from Step B, 22 mg of the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C34H35FN7O3S2: 672.2227; found: 672.2224.
Example 92: 6-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido[2,3c|pyridazin-8(5H)-yl}-3-(l-{[3-(2-{[(3S)-3,4-dihydroxybutyl]amino}ethoxy)-5,7dimethyladaπlantan-l-yl]methyl}-5-methyl-Lί/-pyrazol·4-yl)pyrΐdΐne-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and 2-((45)-2,2-dimethyM,3-dioxolan-4-yl]ethanamine as the appropriate 15 amine, a compound with a dihydroxy protected amine was obtained. Hydrolysis with a 10%
HCl solution (rt, 1 h) and purification by préparative HPLC (using acetonitrile and 5mM aqueous NH4HCO3 solution as eluents) afforded the desired product.
HRMS-ESI (m/z): [M+H]+ calcd for C44H55N9O5: 822.4125, found: 822.4120.
280
Example 93: 6-{3-[(l,3-benzothiazol-2-yl)amino]-4-inethyl-6,7-dihydropyrido[2,3
c]pyridazin-8(5H)-yl}-3-(l-{[3-(2-{[(3R)-3,4-dihydroxybutyl]amino}ethoxy)-5,7dimethyladamantan-l-ylJmethyl}-5-methyl-lW-pyrazol-4-yl)pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from 5 Préparation 12 and 2-[(4R)-2,2-dimethyl-l,3-dioxolan-4-yl]ethanamine as the appropriate amine, a compound with a dihydroxy protected amine was obtained. Hydrolysis with a 10% HCl solution (rt, 1 h) and purification by préparative HPLC (using acetonitrile and 5mM aqueous NH4HCO3 solution as eluents) afforded the desired product.
HRMS-ESI (m/z): [M+H]+ calcd for C44H56N9O5S: 822.4125, found: 822.4124.
Example 94: 6-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido[2,3c]pyridaziπ-8(5W)-yl}-3-[l-({3,5-dimethyl·7-[2-(methylamino)ethoxy]adamantan-lyl}methyl)-5-methyl-lH-pyrazol-4-yl]pyridine-2-carboxy]Îc acid
281
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and methylamîne as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C41H50N9O3S: 748.3757, found: 748.3746.
Example 95: 6-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido[2,35 c|pyUdazin-8(5W)-yi}-3-[ l-({3-[2-(dimethylamino)ethoxy]-5/7-dimethyladainantan-lyl}methyl)-5-methyl-17f-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and dimethylamine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C42H53N9O3S: 762.3914, found: 762.3912.
282
Example 96: 6-{3-[(l,3-benzothiazol-2-yl)aniino]-4-methyl-6,7-dihydropyrido[2,3c]pyridazin-8(5//)-yl}-3-{l-[(3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}adamantanl-yl)methyl]-5-πlethyl-lJï-pyrazol·4-yl}pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from 5 Préparation 12 and taurine as the appropriate amine, and K2CO3 (10 eq) as base during the substitution step, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C42H52N9O6S2: 842.3482, found: 842.3487.
Example 97: 6-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido[2,3c]pyridazin-8(5//)-yl}-3-{l-[(3,5-dimethyl-7-{2-[methyl(210 sulfoethyl)ammo]ethoxy}adamantan-l-yl)methyl]-5-inethyl-17/-pyrazol-4-yl}pyridine-2carboxylic acid
283
Example 98: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido[2,3c]pyridazin-8(577)-yl}-5-{3-t4-(3-{[(but-3-yn-l-yl)amino]methyl}bicyclo[l.l.l]pentan-lyl)-2-fluorophenoxy]propyl}-l,3-thiazole-4-carboxylic acid
Example 99: 6-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido[2,35 c]pyrîdazin-8(577)-yl}-3-[l-({3-[2-(dimethylamino)ethoxy]adamantan-l-yl}methyl)-5methyl-lH-pyrazol-4-yl]pyridine-2-carboxylic acid
284
Example 100:6-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido[2,3c]pyridazm-8(5/7)-yl}-3-(l-{[3,5-dimethyl-7-(4-methylpiperazin-l-yl)adamantan-lyl]methyl}-5-methyl-17/-pyrazol-4-yl)pyrïdine-2-carboxylic acid
Exampie 101: 5-{3-[4-(3-{[(3-azidopropyl)amino]methyl}bicyclo[l.l.l]pentan-l-yl)-2fluorophenoxy]propyl}-2-{3-[(l,3-benzothiazoi-2-yl)amino]-4-methyl-6,7dihydropyrido[2,3-c]pyridazÎn-8(5f/)-yl}-l,3-thiazole-4-carboxylic acid
285
Example 102: 2-{3-[(l,3-benzothiazol-2-yl)ammo]-4-metliyl-6,7-dihydropyrido[2,3c]pyridazin-8(5T0-yl}-5-(3-{4-[3-(ethylamino)-3-methylbut-l-yn-l-yl]-2tluoroplienoxy)propyl)-l,3-thiazole-4-carboxylic acid
Step A: methyl 5-[3-[4-[3-(tert-butoxycarbonylamino)-3-methyl-but-l -ynyl]-2-fluorophen()xy/propyll-2-C3-chloro-4-melhyl-6J-dihydr<)-5II-pyrido[2.3-clpyridiizin-8-yl)lhia-(>ie4-carboxylate
Using Sonogashira General Procedure starting from 1.00 g of the product from Préparation 3a (1.66 mmol) and 330 mg (1.1 eq) of teri-butyl N-(l,l-dimethylprop-2ynyl)carbamate as the appropriate alkyne, 742 mg (68%) of the desired product was obtained.
XH NMR (500 MHz, DMSO-rfc) δ ppm 7.17 (dd, 1H), 7.12 (t, 1I-I), 7.11 (dd, 1H), 7.07 (brs, 1H), 4.26 (t, 2H), 4.11 (t, 2H), 3.77 (s, 3H), 3.25 (t, 2H), 2.89 (t, 2H), 2.32 (s, 3H), 2.1 (qn, 2H), 2.04 (qn, 2H), 1.50 (s, 6H), 1.40 (s, 9H); 13C NMR (125 MHz, DMSO-Jû) δ ppm 163.1, 155.4, 151.7, 151.5, 151.3,147.1, 142.5,136.2, 134.9,129.1, 128.7, 118.9,115.7,115.4, 94.2, 79.3, 78.7, 68.2, 52.0, 47.1, 46.4, 30.7, 29.8, 28.7, 24.2, 23.1, 19.7, 15.7; HRMS-ESI (m/z): [M+H]+ calcd for C32H38CIFN5O5S : 658.2266, found: 658.2245
Step methyl 5-[3-[4-(3-amino-3-methyl-but-l -ynyl) -2-fluoro-phenoxy]propyl]-2-(3chlorO‘4-methyl-6,7-dihydro-5H‘pyrido[2,3-c]pyridazin-8-yl)thiazole-4-carhoxylate
The mixture of the product from Step A (740 mg, 1.12 mmol) and HFxPyr (3 eq) in acetonitrile (5 mL/mmol) was stirred at 50 °C for 1 h. After the volatiles were removed, purification by column chromatography (silica gel, using EtOAc and MeOH (NH3) as eluents) afforded 560 mg (89%) of the desired product.
*H NMR (500 MHz, DMSO-^) δ ppm 7.18 (dd, 1H), 7.11 (m, 1H), 7.11 (m, 1H), 4.25 (m, 2H), 4.10 (t, 2H), 3.77 (s, 3H), 3.25 (t, 2H), 2.88 (t, 2H), 2.31 (s, 3H), 2.10 (m, 2H), 2.04 (m,
286 • 2H), 1.35 (s, 6H); 13C NMR (125 MHz, DMSO-i/e) δ ppm 163.1, 128.6, 118.9, 115.3, 98.1, 78.4, 68.2, 52.0, 46.3, 46.3, 32.3, 30.7, 24.2, 23.1, 19.7, 15.7; HRMS-ESI (m/z): [M+H]+ calcd for C27H30CIFN5O3S: 558.1742, found: 558.1730.
Step C: methyl 2-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl)-5-[3-[4· 5 (3-(ethylamino)-3-methyl-but-l-ynyl]-2-fluoro-phenoxy]propyl]thiazole-4-carboxylate
The mixture of the product from Step B (550 mg, 0.98 mmol), /V-ethyl-jV-isopropyl-propan-Zamine (0.52 mL, 3 eq) and iodoethane (0.12 mL, 1.5 eq) in A^AMimethylformamide (5 mL/mmol) was stirred at rt for 3 h. After the volatiles were removed, the crude intennediate was purified by column chromatography (silice gel, using EtOAc and MeOH (NH3) as 10 eluents) to gîve 570 mg (99%) of the desired product.
’H NMR (500 MHz, DMSO-tfo) δ ppm 9.07 (brm, 2H), 7.41 (dd, 1H), 7.29 (dd, 1H), 7.20 (t, 1H), 4.27 (t, 2H), 4.15 (t, 2H), 3.78 (s, 3H), 3.27 (t, 2H), 3.17 (m, 2H), 2.90 (t, 2H), 2.33 (s, 3H), 2.12 (qn, 2H), 2.05 (qn, 2H), 1.64 (s, 6H), 1.26 (t, 3H); 13C NMR (125 MHz, DMSOrfô) δ ppm 163.1, 155.4, 151.7, 151.4, 151.3, 148.2, 142.4, 136.3, 135.0, 129.4, 129.1, 119.5, 15 115.5, 113.2, 86.8, 85.3, 68.3, 53.8, 52.0, 46.4, 38.2, 30.7, 26.5, 24.2, 23.1, 19.8, 15.7, 12.2;
HRMS-ESI (m/z): [M+H]+ calcd for C29H34CIFN5O3S: 586.2055, found: 586.2048.
Step Dz methyl 2-(3-(l,3-benzothùizol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3c]pyrldazin-8-yl]-5-[3-[4-[3-(ethylamino)-3-methyl-but-l -ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylat€
Using Buchwald General Procedure I starting from 570 mg of the product from Step C (0.98 mmol) and 292 mg (2 eq) of l,3-benzothiazoI-2-amine, 420 mg (61%) of the desired product was obtained.
*H NMR (500 MHz, DMSO-^) δ ppm 8.98 (m, 2H), 7.88 (brs, 1H), 7.53 (brs, 1H), 7.42 (dd, 1H), 7.38 (m, 1H), 7.29 (dm, 1H), 7.22 (t, 1H), 7.20 (m, 1H), 4.26 (t, 2H), 4.16 (t, 2H), 3.77 25 (s, 3H), 3.29 (t, 2H), 3.14 (m, 2H), 2.88 (t, 2H), 2.34 (s, 3H), 2.15 (m, 2H), 2.04 (m, 2H), 1.60 (s, 6H), 1.23 (l, 3H); HRMS-ESI (m/z); [M+H]+ calcd for C36H39FN7O3S2: 700.2540, found; 700.2532.
Step E: 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin8-yl]-5-(3-(4-(3-(ethylamino)-3-methyl-biit-l-ynyl]-2-fluoro-phenoxy]propyl]thiazole-430 carboxylic acid
287
V To the product from Step D (420 mg, 0.60 mmol) in a 2:1 mixture of 1,4-dioxane and water (7.5 mL/mmol) was added 50 mg (2 eq) of LiOH x H?O, and the mixture was stirred at rt for 3 h. After removal of the volatiles, purification by reverse phase préparative chromatography (C18, 0.1% TFA in water: MeCN) afforded 33 mg (8%) of the desired compound.
HRMS-ESI (m/z): [M+H]+ calcd for C35H37FN7O3S2: 686.2383, found: 686.2378.
Example 103: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido[2,3c]pyridazin-8(5H)-yl}-5-(3-{2-flu(>ro-4-[3-methyl-3-(piperazin-l-yl)but-l-yn-lyl]phenoxy}propyl)-l,3-thiazole-4-carboxylic acid
Example 104:5-[3-(4-{3-[(3-azidopropyl)amino]prop-l-yn-l-yl}-210 fluorophenoxy)propyl]-2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7dihydropyrido[2,3-c]pyridazin-8(5iyr)-yl}-l,3-thiazole-4-carboxylic acid
Example 105: 5-[3-(4-{3-[(3-aminopropyl)amino]prop-l-yn-l-yl}-2fluorophenoxy)propyl]-2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7dihydropyrido[2,3-c]pyridazin-8(5/Z)-yl}-l,3-thiazole-4-carboxylic acid
288
Exampie 106: 2-{3-[(l,3-benzothiazol-2-yl)amino] -4-methy 1-6,7 -dihydropyrido [2,3c]pyridazin-8(5J/)-yl}-5-[3-(2-fluoro-4-{3-[(pent-4-yn-l-yl)amino]prop-l-yn-lyl}phenoxy)propyl]-l,3-thiazole-4-carboxylic acid
Example 107: 2-{3-[(l,3-benzothiazol-2-yl)am ino] -4-methy 1-6,7-dihydropyrido [2,35 c]pyridazin-8(5/7)-yl}-5-{3-[2-fluoro-4-(3-{[2-(2-hydroxyethoxy)ethyl](methyl)amino} prop-l-yn-l-yl)phenoxy]propyl}-l,3-thîazole~4-carboxylic acid
289
Exampie 108: 2-{3-[(l,3benzothΐazol·2-yl)amino]-4-methyl-6,7-dΐhydropyrido[2,3c]pyridazin-8(5H)-yl}-5-[3-(2-11 uoro-4-{3-methy 1-3-[(pent-4-yn-l-yl)ammo]but-l-yii-lyl}phenoxy)propyl]-l,3-thiazole-4-carboxylic acid
Example 109:2-{3-r(1.3-benzûthiazol-2-vl)aminol-4-methvl-6.7-dihvdropvridoi2.3c]pyridazin-8(5J7)-yl}-5-{3-[2-fluoro-4-(3-{[(prop-2-yn-lyl)amino]methyl}bicyclo[l.l.l]pentan-l-yl)phenoxy]propyI}-l,3-thiazole-4-carboxylic acid
Example 110: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-meihyl-6,7-dîhydropyrido[2,310 c]pyridazîn-8(5H)-yI}-5-[3-(2-fluoro-4-{3-[(hex-5-yn-l-yl)amino]prop-l-yn-lyl}phenoxy)propyl]-l,3-thiazole-4-carboxylic acid
290
Example 111: 5-[3-(4-{3-[(4-azidobutyl)aminoJprop-l-yn-l-yl}-2-fluorophenoxy)propyl]2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido[2,3-c]pyridazin-8(517)yl}-l,3-thiazole-4-carboxylic acid
Example 112: 5-[3-(4-{3-[(4-azidobutyl)(methyl)amino]prop-l-yn-l-yl}-25 fluorophenoxy)propyl]-2-{3-[(l,3-benzothiazol·2-yl)aminoJ-4-methyl-6,7dihydropyrido[2,3-c]pyridazin-8(5H)-yl}-l,3-thiazoIe-4-carboxylic acid
Example 113:2-{3-[(l,3-benzothiazol·2-yl)amino]-4-methyl-6,7-dihydropylΊdoE2,3c]pyridazm-8(57/)-y!}-5-[3-(2-fluoro-4-{3-[(hex-5-yn-l-yl)(methyl)amino]prop-l-yn-lyl}phenoxy)propyl]-l,3-thiazole-4-carboxylic acid
291
Example 114: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydiOpyrido[2,3c]pyridazin-8(577)-yl}-5-[3-(2-fluorO-4-{3-[methyl(pent-4-yn-l-yl)amino]prop-l-yn-lyl}phenoxy)propyl]-l,3-thîazole-4-carboxylic acîd
Example 115: 5-[3-(4-{3-[(3-azidopropyl)(methyl)amino]prop-l-yn-l-yl}-25 fluorophenoxy)propyl]-2-{3-[(l,3-benzothiazol-2-yl)aiiiino]-4-methyl-6,7dihydropyrido[2,3-c]pyridazin-8(5H)-yl}-l,3-thiazole-4-carboxylic acid
Example 116: 2-{3-[(l,3-benzothiazoI-2-yI)amino]-4-methyl-6,7-dihydropyrido[2,3c]pyridazin-8(5f7)-yl}-5~{3-[2-fluoro-4-(3-{[methyl(pent-4-yn-l-
292 yl)amino]methyl}bicyclo[ 1.1.1 ]pentan-l-yl)phenoxy]propyl}-l,3-thiazole-4-carboxylic acid
Example 117:5-{3-[4-(3-{[(4-azidobutyl)(methyl)amino]methyl}bicyclo[l.l.l]pentan-l5 yl)-2-fluorophenoxy]propyl}-2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7dihydropyrido[2,3-c]pyridazm-8(5Z/)-yl}-l,3-thiazole-4-carboxylic acid
Exampie 118: 2-{3-[(l,3-benzothiazol-2-yl)aminoJ-4-methyI-6,7-dihydropyrido[2,3c]pyridazin-8(5W-yl}-5-{3-[4-(3-{[(but-3-yn-lyl)(methyl)amino]methyl}bicyclo[l.Ll]pentan-l-yl)-2-fluorophenoxy]propyl}-l,310 thiazole-4-carboxylic acid
Example 119: 5-{3-[4-(3-{[(3-azidopropyl)(methyl)ammo]methyl}bîcyclo[l.l.l]pentaii-lyl)-2-fluorophenoxy]propyl}-2-{3-[(l,3-benzothiazol·2-yl)aminol-4-methyl-6,7dihydropyrido[2,3-c]pyridazin-8(5J7)-yl}-l,3-thiazole-4-carboxylic acid
293
Example 120: 5-{3-[4-(3-{[(4-azidobutyl)amino]methyl}bicyclo[l.l.l]pentan-l-yl)-2fluorophenoxy]propyl}-2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7diliyd!'opyrido|2.3-i’|pyridazin-8(5//)-yl}-l,3-tliiazok‘-4-carl)Oxylic acid
Example 121: 2-{3-[(l,3-benzothiazoI-2-yl)amino]-4-methyl-6,7-dihydropyrido[2,35 c]pyridazin-8(5/0-yl}-5-{3-[2-fluoro-4-(3-{[(pent-4-yn-lyl)amino]methyl}bicyclo[l.l.l]pentan-l-yl)phenoxy]propyl}-l,3-thiazole-4-carboxylic acid
Example 122: 2-[(61ï)-3-[(l,3-benzothiazol-2-yl)amino]-6-(2-hydroxyethyl)-4-methyl-6,7dihydropyrido[2>3-c]pyridazin-8(5//)-yl]-5-(3-{4-[3-(dimethylamino)prop-l-yn-l-yl]-210 fluorophenoxy}propyl)-l,3-thiazole-4-carboxylic acid
294
Example 123: 2-[(6S)-3-[(l,3-benzothiazol-2-yl)amÎno]-6-(2-hydroxyethyl)-4-methyl-6,7dihydropyrido[2,3-c]pyridazm-8(57/)-yl]-5-(3-{4-[3-(diniethylammo)prop-l-yn-l-ylJ-2fluorophenoxy}propyl)-l,3-thiazole-4-carboxyiic acid
Example 124: 2-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/7-pyrido[2,35 c]pyridazin-8-yl]-5-[3-[2-fluoro-4-[3-(prop-2-ynylamino)prop-lynyl]phenoxy]propyl]thiazole-4-carboxylic acid
Step A·, methyl 5-[3-[2-fîuoro-4-[3~(prop-2~ynylamino)prop-l-ynyl]phenoxy]propyl]-2-[4methyl-3-[(Zd-/3-(2-lrimethylsilyleth<)xymethyh-l,3-ben~othiiizol-2-ylidencjaiiiim)l-6,710 dihydroSH-pyrido[2,3~c}pyridazin-8-yl]thiazole-4-carboxylate
Using Sonogashira General Procedure starting from the product from Example 77, Step A
295 (2.30 g, 2.71 mmol, 1.0 eq.) and 1.26 g of ZV-prop-2-ynylprop-2-yn-l-amine (13.58 mmol, 5 eq.) as the appropriate acetylene, 793 mg (36%) of the desired product was obtained.
LC/MS (C4jH47FN7O4S2Si) 812 [M+H]+.
Step B'. 2-[3-(I,3-benzothiazol-2-ylanùno)-4-fnethyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin5 8-yl]-5-[3-[2-fluorO‘4-[3-(prop'2-ynylamino)prop-l-ynyl]phenoxy]propyl]thiazole-4carboxylic acid
Using Deprotection and Hydrolysis General Procedure starting from the product from Step A (900 mg, 1.10 mmol), 222 mg (30%) of the desired product was obtained.
lH NMR (500 MHz, DMSO-rfc) δ ppm 7.88 (d, 1H), 7.48 (br., 1H), 7.37 (t, 1H), 7.28 (dd, 10 1H), 7.19 (d, 1H), 7.18 (t, 1H), 7.14 (l, 1H), 4.27 (br., 2H), 4.14 (t, 2H), 3.55 (s, 2H), 3.39 (d,
2H), 3.27 (t, 2H), 3.09 (t, 1H), 2.87 (ί, 2H), 2.33 (s, 3H), 2.13 (m, 2H), 2.03 (m, 2H); 13C NMR (125 MHz, DMSO-î/ô) δ ppm 128.9, 126.5, 122.5, 122.3, 119.2, 115.5, 87.8,82.8, 82.2, 74.5, 68.5, 46.3, 37.2, 36.6, 31.0, 23.9, 23.1, 20.3, 12.9; HRMS-ESI (m/z): [M+H]+ calcd for C34H3iFN7O3S2: 668.1914, found; 668.1907.
Example 125: 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6/7-dihydro-5H,-pyrido[2,3c]pyridazin-8-yl}-5-{3-[4-(3-{bis[(3S)-3,4-dihydroxybutyl]aniino}prop-l-yn-l-yl)-2fluorophenoxy]propyl}-l,3-thîazole-4-carboxylic acid
Step A·, methyl5-[3~[4-[3-(tert-butoxycarhonylaminô)prop-l-ynyl]-2-fluoro20 phenoxy]propyl]-2-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl)thiazole4-carboxylate
Using Sonogashira General Procedure starting from 3.00 g of Préparation 3a (5.0 mmol) and 1.55 g of iert-butyl N-prop-2-ynylcarbamate (2 eq.) as the appropriate acetylene, 2.79 g of the desired product (89%) was obtained.
296
W *H NMR (500 MHz, dmso-d6) δ ppm 7.34 (brt, 1H), 7.26 (dd, 1H), 7.17 (dm, 1H), 7.13 (t, 1H), 4.25 (t, 2H), 4.11 (t, 2H), 3.95 (brd, 2H), 3.77 (s, 3H), 3.25 (t, 2H), 2.88 (m, 2H), 2.31 (s, 3H), 2.10 (m, 2H), 2.03 (m, 2H), 1.39 (s, 9H); 13C NMR (500 MHz, dmso-d6) δ ppm 128.9, 119.1, 115.4, 68.2, 51.9, 46.3, 30.7, 30.5, 28.7, 24.1, 23.0, 19.7, 15.7; HRMS-ESI (m/z): 5 ΓΜ+Η1+ calcd for CJL.CIFN.CLS: 630.1953, found 630.1945. J DU DD
Step B: methyl 5-[3-[4-(3-aminoprop-l -ynyl)-2-fluoro-phenoxy]propyl]-2-(3-chloro-4inethyl-6y7-dihydr(>-5H-pyrido[2,3-cjpyridaz,iii-8-yl)thiaz(>Ie-4-carb()xylate
The mixture of the product from Step A (2.19 g, 3.47 mmol), pyridine and hydrogen fluoride (1:1) (3.44 g, 10.0 eq) in MeCN (17.3 mL) was stirred at 60 °C for 1.5 h. Purification by flash 10 chromatography (silica gel, DCM and MeOH (1.2% NH3) as eluents) afforded the desired product (1.81 g, 98.5%).
Ή NMR (500 MHz, dmso-d6) δ ppm 7.26 (dd, 1H), 7.18 (dd, 1H), 7.14 (t, 1H), 5.36 (NH3+, br., 3H), 4.25 (m, 2H), 4.12 (t, 2H), 3.77 (s, 3H), 3.61 (s, 2H), 3.25 (t, 2H), 2.88 (t, 2H), 2.31 (s, 3H), 2.10 (m, 2H), 2.04 (m, 2H); 13C NMR (500 MHz, dmso-d6) δ ppm 163.1, 155.3, 15 151.7, 151.5, 151.3, 147.4, 142.5, 136.1, 136.1, 134.9, 128.9, 119.1, 115.5, 115.2, 89.2, 81.9,
68.2 , 51.9, 46.3, 31.1, 30.7, 24.2, 23.0, 19.7, 15.7; HRMS-ESI (m/z): [M+H]+ calcd for C^H^CIFN.CLS: 530.1429, found 530.1410.
Step C: (4S)-4-(2-iodoethyl)-2,2-dimethyl-l ,3-dioxolane
To the mixture of PPh3 (11.84 g, 2.2 eq), imidazole (3.07 g, 2.2 eq) and 2-[(4S)-2,2-dimethyl20 l,3-dioxolan-4-yl]ethanol (2.92 mL, 20.52 mmol) in dichloromethane (103 mL) was added iodine (11.46 g, 2.2 eq) portionwise at 0 °C, then stirred at rt for 18 h. Then, the reaction was quenched with 100 mL of Na2S2O3 solution and the phases were separated, the organic phase was washed with brine, dried, and purified via flash chromatography (silica gel, heptane and heptane-MTBE as eluents) to give the desired compound (2.90 g, 55%).
NMR (500 MHz, dmso-d6) δ ppm 4.06 (m, 1H), 4.01/3.45 (dd+dd, 2H), 3.28/3.21 (dd+dd, 2H), 2.00/1.97 (m+m, 2H), 1.31 (s, 3H), 1.26 (s, 3H); 13C NMR (500 MHz, dmso-d6) Ô ppm 108.7, 75.8, 68.3, 37.9, 27.3, 26.0, 3.5; GC-MS (El, M+): 255.79.
Step D: methyl 5-[3-l4-[3-[bis[2-[(4S)‘2,2-dimethyl-I,3-dioxolan-4-yl]ethyl]amino]prop-lynyl]-2-fluoro-phenoxy]propylJ-2-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,330 c]pyridazin-8-yl)thiazole-4-carboxylate
297
The mixture of the product from Step B (500 mg, 0.94 mmol), the product from Step C (483.2 mg, 2.0 eq) and JV-ethyl-/V-isopropyl-propan-2-amine (1.0 mL, 6 eq) in N,Ndimethylformamide (4.7 mL) was stirred at rt for 6 h. 10 mL of a 2 M solution of dîmethylamine was added and the reaction mixture was further stirred for 1 h. The mixture 5 was diluted with water and saturated solution of NaHCCh and extracted with EtOAc. The combined organic phases were washed with brine, dried, concentrated and purified by préparative HPLC (MeCN, NH4HCO3) to give the desired compound (100 mg, 13%).
Ή NMR (500 MHz, dmso-d6) δ ppm 7.28 (dd, IH), 7.18 (dm, IH), 7.14 (t, IH), 4.27 (t, 2H), 4.11 (t, 2H), 4.07 (m, 2H), 3.99/3.46 (dd+dd, 4H), 3.76 (s, 3H), 3.56 (s, 2H), 2.89 (t, 2H), 2.51 10 (m, 4H), 2.33 (s, 3H), 2.32 (l, 2H), 2.Γ1 (m, 2H), 2.04 (m, 2H), 1.64 (m, 4H), 1.30 (s, 6H),
1.24 (s, 6H); HRMS-ESI (m/z): [M+H]+ calcd for C39H50CIFN5O7S: 786.3104, found 786.3111.
Step E: methyl 2-(3-(1,3-benzothiazol-2-ylamino) -4-methyl-6,7-dihydro-5H-pyrido[2,3cJpyridazin -8-yl]-5-(3-(4-[3-(bis(2-((4S) -2,2-dimethyl-l,3-dioxolan-4-yl]ethyl]amino]prop-l 15 ynyl]-2-fluoro-phenoxy]propyl]thiazole-4-carboxylate
Using Buchwaid General Procedure I starting from 100 mg of the product from Step D (0.127 mmol) and préparative HPLC purification (MeCN, NH4HCO3), 90 mg of the desired product (78%) was obtained.
Ή NMR (500 MHz, dmso-d6) δ ppm 7.90 (brs, 1H), 7.60 (brs, IH), 7.37 (brm, IH), 7.29 (dd, 20 IH), 7.20 (brm, IH), 7.19 (dm, IH), 7.16 (t, IH), 4.45/3.98 (dd+dd, 4H), 4.26 (t, 2H), 4.14 (t, 2H), 4.05 (dd, 2H), 3.78 (s, 3H), 3.53 (s, 2H), 3.28 (t, 2H), 2.88 (t, 2H), 2.5 (m, 4H), 2.35 (s, 3H), 2.14 (m, 2H), 2.05 (m, 2H), 1.63 (m, 4H), 1.3 (s, 6H), 1.24 (s, 6H); HRMS-ESI (m/z): [M+H]+ calcd for C46H55FN7O7S2: 900.3588, found 900.3591.
Step F: 2-(3-(1,3-benzothiazol~2-ylamino) -4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin25 8-yl]-5-(3-(4-[3-(bis( (3S) -3,4-dihydroxybutyl]amino]prop-l -ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylic acid
The mixture of the product from Step E (90 mg, 0.1 mmol) and LiOH x H2O (95 mg, 22.6 eq) in 1,4-dîoxane (1 mL) and water (1 mL) was stirred at rt for 1 h, and at 50 °C for 2 h. After treatment with hydrogen chloride (8 mmol) and stirring at rt for 4 h, a saturated solution of 30 NaHCO3 and a 1:1 mixture of water and brine were added, and the desired product was filtered out (33 mg, 40%).
298 ® HRMS-ESI (m/z) [M+H]+ calcd for C39H45FN7O7S2: 806.2806, found 806.2803.
Example 126: methyl 5-[3-{4-(3-aminoprop-l-ynyl)-2-fluoro-phenoxy]propyl]-2-[3-(l,3beiizothiazol-2-ylaniiiio)-4-methyl-6,7-dihydro-5jyr-pyrido[2,3-i!]pyridazin’8-yl]thiazole4-carboxylate
Step A: methyl 2-(3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c}pyridazin-8-yl]-5-[3-[4-[3-(tert-butoxycarbonylamino)prop-I -ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylate
Using Buchwald General Procedure I starting from 4.60 g of Example 125, Step A and 10 2.20 g (2 eq) of 2-aminobenzothiazole followed by a column chromatography purification (silica gel using heptane, EtOAc, and MeOH (1.2% NHS) as eiuents), 4.02 g (74%) ofthe desired product was obtained.
Ή NMR (500 MHz, dmso-d6) δ ppm 7.90 (br., 1H), 7.61 (br., 1H), 7.37 (brt., 1H), 7.27 (dd, 1H), 7.19 (br., 1H), 7.19 (dd, 1H), 7.15 (t, 1H), 4.25 (t, 2H), 4.14 (t, 2H), 3.94 (d, 2H), 3.77 (s, 15 3H), 3.27 (t, 2H), 2.86 (t, 2H), 2.33 (s, 3H), 2.13 (m, 2H), 2.04 (m, 2H), 1.39 (s, 9H); 13C
NMR (500 MHz, dmso-d6) Ô ppm 163.2, 155.7, 129.0, 126.4, 122.5, 122.2, 119.2, 115.5, 68.4, 51.9, 46.3, 31.0, 30.5, 28.7, 23.9, 23.1, 20.3, 12.9; HRMS-ESI (m/z): [M+H]+ calcd for C37H39FN7O5S2: 744.2438, found: 744.2425.
Step 13; methyl 5-[3-(4-(3-aminoprop-l -ynyl) -2-fluoro-phenoxy]propyl]-2 -(3-(1,320 benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8’yl]thiazole-4carboxylate
The mixture of the product from Step A (4.00 g, 5.38 mmol), pyridine and hydrogen fluoride (1:1) (5.33 g, 10 eq) in MeCN (27 mL) was stirred at 60 C for 16 h. Purification by column chromatography (silica gel, DCM and MeOH (NH?) as eiuents) afforded the desired 25 compound.
HRMS-ESI (m/z): [M+H]+ calcd for C32H31FN7O3S2; 644.1914, found: 644.1913.
299
Example 127: 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyI-6,7-dihydro-5//-pyrido[2,3c] pyrïdazin-8-yl]-3-[l-H3,5-dimethyI-7-(2-pyrrolidin-l-ylethoxy)-l-adamantyl] methyl] 5-inethyl-pyrazül-4-ylJpyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and pyrrolidine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C44H54N9O3S; 788,4070, found: 788.4068.
Example 128: 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5Jt/-pyrido[2,3c]pyridazin-8-yl]-3-[l-[[3,5-dimethyl-7-[2-(4-methylpiperazin-l-yl)ethoxy]-l10 adamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridïne-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and 1-methylpiperazine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z); [M+2H]2+ calcd for C45H58N10O3S: 409.2207, found: 409.2208.
Example 129: 2-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/i-pyrido[2,3c]pyridazin-8-yl]-5-[3-[4-[3-[[(3S)-3,4-dihydroxybutyl]amino]prop-l-ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylic acid
300
The mixture of the product from Préparation 3c (400 mg, 0.67 mmol), paraformaldéhyde (400 mg, 20 eq), (25)-4-aminobutane-l,2-diol, hydrogen chloride (1:1) (754.3 mg, 8 eq), triethylamine (2.3 mL, 25 eq), Cul (127 mg, 1 eq) and molecular sieves (0.5 g) in éthanol (3.3 5 mL) was kept in an Anton-Paar microwave reactor at 120 °C for 1 h. Purification by column chromatography (silica gel, using heptane, EtOAc and MeOH/NH3 (0.6N) as eluents) and RF HPLC (Gemini, using water with 0.1% TFA and acetonitrîle as eluents) afforded 15.3 mg (3%) of the desired product.
HRMS-ESI (m/z): [M+H]+ calcd for C35H37FN7O5S2: 718.2282, found: 718.2266.
Example 130: 6-[3-(l,3-benzothiazol-2-ylanimo)-4-inethyl-6,7-dÎhydro-57/-pyrido[2,3c]pyridazin-8-yl]-3-[l-[[3-[2-[[(3R)-3,4-dihydrnxybutyl]-methyl-amino]ethoxy]-5,7dÎmethyl-l-adamantyllmethyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from 15 Préparation 12 and 2-[(4R)-2,2-dimethyl-l,3-dioxolan-4-yl]-ALmethyl-ethananrine as the appropriate amine, a compound with a dihydroxy protected amine was obtained. Hydrolysis with a 10% HCl solution (rt, 1 h) and purification by préparative HPLC (using acetonitrîle and 5mM aqueous NH4HCO3 solution as eluents) afforded the desired product.
HRMS-ESI (m/z): [M+2H]2+ calcd for C45H59N9O5S: 418.7180, found: 418.7167.
301
Example 131: 2-[3-(1,3-ϋοηζοίΗίρζοΕ2-γ1ρ mino)-4-methyl-6,7-(1 iliydro-51ï-py rîdo [2,3qpyridazin-8-ylJ-5-[3-[4-[3-[[(3R)-3,4-dihydroxybutyl]amino]prop-l-ynyl]-2-fluoiOphenoxy]propylJthiazole-4-carboxylic acid
The mixture of the product from Préparation 3c (200 mg, 0.33 mmol), paraformaldéhyde (200 mg, 20 eq), (2/ï)-4-aminobutane-l,2-diol, hydrogen chloride (1:1) (471 mg, 10 eq), triethylamine (1.2 mL, 25 eq), Cul (64 mg, 1 eq) and molecular sieves (0.25 g) in éthanol (1.6 mL) was kept in an Anton-Paar microwave reactor at 120 °C for 1 h. Purification by column chromatography (silica gel, using heptane, EtOAc and MeOH/NH3 (0.6N) as eluents) and RF HPLC (Gemini, using water with 0.1% TFA and acetonitrile as eluents) afforded 43 mg (18%) of the desired product.
HRMS-ESI (m/z): [M+H]+ calcd for C35H37FN7O5S2 : 718.2282, found: 718.2281.
Example 132: 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3cJpyridazm-8-yl]-3-[l-[[3-[2-(4-liydroxybutylamino)ethoxy]-5,7-dimethyl-ladamantylJmethyl]-5-methyl-pyrazol-4-yI]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and 4-aminobutan-l-ol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C44H56N9O4S: 806.4176, found: 806.4174.
302
Example 133: 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-S-yl]-3-[l-[[3-[2-[[3-hydroxy-2-(hydroxymethyl)propyl]amino]ethoxy]-5,7dimethyI-l-adamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and (2,2-dimethyl-l,3-dioxan-5-yI)methanamine as the appropriate amine a compound with a dihydroxy protected amine was obtained. Hydrolysis with a 10% HCl solution (rt, 1 h) and purification by préparative HPLC (using acetonitrile and 5mM aqueous NH4HCO3 solution as eluents) afforded the desired product.
HRMS-ESI (m/z): [M+H]+ calcd for C44H56N9O5S: 822,4125, found: 822.4099.
Example 134: 6-[3-( l,3-benzotbiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-3-[l-[[3-[2-[bis(2-hydroxyethyl)amino]ethoxyl-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
OH
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and 2-(2-hydroxyethylamino)ethanol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C44H56N9O5S: 822,4125, found: 822.4123.
303
Exampie 135: 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyI-6,7-dihydro-5Z7-pyrido[2,3c]pyridazin-8-yl]-3-[l-[[3-[2-[[2-hydroxy-l-(hydroxymethyl)ethylJaniino]ethoxy]-5,7dimetliyl-l-adamantyl]metliyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and 2-aminopropane-l,3-diol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): (M+H]+ calcd for C43H54NgOsS: 808.3969, found: 808.3965.
Example 136: 6-(3-(1,3-benzothiazol-2-ylamino)-4-methy 1-6,7-dihydro-5H-pyrido[2,310 e]pyridazin-8-yl]-3-[l-[[3-[2-[2-(2-hydroxyethoxy)ethylamino]etlioxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from
Préparation 12 and 2-(2-aminoethoxy)ethanol as the appropriate amine, the desired product 15 was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C44H56N9O5S: 822.4125, found: 822.4116.
304
Exampie 137: 6-[3-(l,3-benzothiazoi-2-ylainino)-4-methyI-6,7-dihydro-5H-pyrido[2,3i,]pyridazin-8-yl]-3-[l-[[3-[2-[bis(3-hydroxypropyl)amino]ethoxyl-5,7-dÎmethyl-ladamantyl]methyl]-5-methyl-pyrazoI-4-yl]pyridine-2-carboxylîc acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and 3-(3-hydroxypropylamino)propan-l-ol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H1+ calcd for Cd,H,„NqO.S: 850.4438, found: 850.4436. x x * J -ID (jL? 7 D
Example 138: 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/f-pyrido[2,310 c]pyridazin-8-yl]-3-[l-([3-[2-(3-hydroxypropylamino)ethoxy]-5,7-dimethyl-ladamantyI]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and 3-aminopropan-l-ol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C43H54N9O4S: 792.4019, found: 792.4012.
Example 139: 5-[3-I4-[3-[acetyl(methyl)amino]prop-l-ynyl]-2-fluoro-phenoxy]propyl]-2[3-(l,3-benzothiazol-2-ylammo)-4-methyI-6,7-dihydro-5Z7-pyrido[2,3-c]pyrÎdazin-8yl]thiazole-4-carboxylic acid
305
Step A: methyl S-[3-[4-[3-[tert-butoxycarbonyl(methyl)amino]prop-l -y nyl/-2-fluorophenoxy]propylJ-2-(3-ch loro-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin -8 -yl)thiazole4-carboxylate
Using Sonogashira General Procedure starting from 4.00 g of Préparation 3a (6.63 mmol) and 2.24 g tert-butyl N-methyl-N-prop-2-ynyl-carbamate (13.3 mmol, 2 eq) as the appropriate acetylene, 2.40 g (55%) of the desired product was obtained.
‘H NMR (500 MHz, dmsoM6) δ ppm 7.30 (dd, 1H), 7.20 (dm, IH), 7.13 (t, 1H), 4.24 (m, 2H), 4.23 (brs, 2H), 4.11 (t, 2H), 3.77 (s, 3H), 3.24 (t, 2H), 2.87 (m, 2H), 2.86 (s, 3H), 2.30 (s, 10 3H), 2.10 (m, 2H), 2.03 (m, 2H), 1.41 (s, 9H); I3C NMR (500 MHz, dmsü-d6) δ ppm 129.1,
119.3, 115.4, 85.2, 82.4, 68.2, 51.9, 46.3, 38.6, 33.8, 30.7, 28.5, 24.1, 23.0, 19.7, 15.7; HRMS-ESI (m/z): [M+H]+ calcd for CsiH^CIFNsOîS: 644.2110, found: 644.2094.
Step B: methyl 2~(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2>3-c]pyridazin-8-yl)-5-(3-(2fluoro-4-[3-(methylamino)prop-l -ynyl]phenoxy]propyl]thiazole-4-carboxylate
To 322 mg product from Step A (0.5 mmol) in 2.5 mL acetonitrile was added 0.9 mL of hydrogen fluoride in pyridine (20 eq). The reaction mixture was stirred at 60 °C until no further conversion was observed. Purification via flash chromatography (silica gel, using DCM and MeOH (1.2% NH3)) afforded 258 mg (95%) of desired product.
*H NMR (500 MHz, dmso-d6) δ ppm 7.25 (dd, 1 H), 7.17 (dd. 1H), 7.12 (t, 1H), 4.25 (t, 2H), 20 4.11 (t, 2H), 3.77 (s, 3H), 3.46 (s, 2H), 3.25 (t, 2H), 2.88 (t, 2H), 2.32 (s, 3H), 2.31 (s, 3H),
2.10 (qn, 2H), 2.03 (qn, 2H), 1.99 (brs, 1H); 13C NMR (500 MHz, dmso-d6) δ ppm 163.1, 155.4, 151.7, 151.6, 151.3,147.2, 142.5,136.2, 134.9, 129.0,128.8, 119.1, 115.7, 115.4, 88.7, 82.1, 68.3, 52.0, 46.4, 40.5, 35.4, 30.8, 24.2, 23.1, 19.7, 15.7; HRMS-ESI (m/z): [M+H]+ calcd for C26H2SCIFN5O3S: 544.1585, found: 544.1570.
Step C: methyl 5-(3-(4-[3-(acetyl(methyl)amino]prop-J-ynyl]-2-fluoro-phenoxy]propyl]-220573
306 (3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl)thiazole-4-carboxylate
Το 220 mg of the product from Step B (0.41 mmol) and 0.085 mL of TEA (1.5 eq) in 2 mL of dîchloromethane was added 0.031 mL of acetyl chloride (1.1 eq). The reaction mixture was stirred untii no further conversion was observed. Purificationvia flash chromatography (silica 5 gel, using DCM and MeOH (1.2% NH3) as eluents) afforded 174 mg (73%) of the desired product.
Ή NMR (500 MHz, dmso-d6) δ ppm 7.34/7.31 (dd/dd, IH), 7.23/7.20 (brd/brd., IH), 7.14/7.13 (t/t, IH), 4.38/4.34 (s/s, 2H), 4.25 (m, 2H), 4.12 (t, 2H), 3.77 (s, 3H), 3.25 (t, 2H), 3.05/2.88 (s/s, 3H), 2.88 (t, 2H), 2.31 (s, 3H), 2.10 (m, 2H), 2.09/2.02 (s/s, 3H), 2.03 (m, 2H);
13C NMR (500 MHz, dmso-d6) δ ppm 170.2/170.1, 163.0, 155.4, 151.3, 142.4, 134.9,
129.2/129.1, 119.4/119.3, 115.4, 85.3/84.7, 82.9/81.9, 68.2, 51.9, 46.3, 40.6/36.3, 35.4/33.1, 30.7, 24.1, 23.0, 21.9/21.8, 19.7, 15.7. HRMS-ESI (m/z): [M+H]+ calcd for C28H30CIFN5O3S: 586.1691, found: 586.1690.
Step D: methyl 5-[3-[4-[3-[acetyl(methyl)atnino]prop-l -ynyl]-2-fluoro-phenoxy]propyl]-215 [3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8yljthiazole-4-carboxylate
Using Buchwald General Procedure I starting from 170 mg (0.29 mmol) of the product from Step C and 87 mg (2 eq) of l,3-benzothiuzol-2-amine, 220 mg (98%) of the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C35H35FN7O4S2: 700.2176, found: 700.2180.
Step E: 5-[3-[4-[3-[acetyl(methyl)amino]prop-l -ynyl]-2-fluoro-phenoxy]propyl}-2-[3-(l ,3benzothiazol-2‘ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-ylJthiazole-4carboxylic acid
Using Hydrolysis General Procedure starting from the product of Step D as the appropriate 25 methyl ester, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C34H33FN7O4S2; 686.2023, found: 686.2019.
Exampie 140: 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3clpyridazin-8-yl]-3-[l-[|3-[2-[bis(4-hydroxybutyl)ammo]ethoxy]-5,7-dimethyl-ladamantyl]niethyl]-5-methyl-pyrazol-4-yl]pyridme-2-carboxylic acid
307
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and 4-(4-hydroxybutylamino)bulan-l-ol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C4gH64N9O5S: 878.4751, found: 878.4752.
Example 141: 2-[3-(l,3-benzothiazol·2-ylamino)-4-methyl-6,7-dihydro-5/î-pyrido[2,3c]pyridazm-8-yl]-5-[3-[4-[3-(dimethylammo)prop-l-ynyl]phenoxy]propyl]thiazole-4carboxylïc acid
Step A: 4-[3-(dimethylamino)prop-l-ynyljphenol
Using Sonogashira General Procedure starting from 10.0 g of 4-iodophenol (45.45 mmol) and 4.91 g (1.3 eq) of N,N-dimethyIpiOp-2-yn-I-amine, 3.29 g (41%) of the desired product was obtained.
Ή NMR (400 MHz, DMSO-d6) δ ppm 9.83 (brs, 1H), 7.25 (d, 2H), 6.74 (d,2H), 3.44 (s, 15 2H), 2.26 (s, 6H); LC/MS (C11H14NO) 176[M+H]+.
Step B: methyl 2-(tert-butoxycarbonylamino)-5-[3-[tertbutyl(diphenyl)silyl]oxypropyl]th iazole-4-carboxylate
To the product of Préparation la, Step C (77.0 g, 243.7 mmol), imidazole (33.14 g, 2 eq) and DMAP (1.49 g, 0.05 eq) in DMF (973 mL) was added dropwise tert
308 butyl(chloro)diphenylsilane (93.5 mL, 1.5 eq) and the reaction mixture was stirred at rt for 16 h. After removal of the volatiles, purification by column chromatography (silica gel, using heptane and EtOAc as eluents) afforded 13.56 g (99%) of the desired product.
Ή NMR (500 MHz, DMSO-tfc) δ ppm 11.63 (s, IH), 7.60 (d, 4H), 7.45 (l, 2H), 7.42 (t, 4H), 5 3.74 (s, 3H), 3.67 (t, 2H), 3.20 (t, 2H), 1.87 (qn, 2H), 1.47 (s, 9H), 0.99 (s, 9H); 13C NMR (125 MHz, DMSO-dô) δ ppm 162.8, 156.0, 142.6, 135.6, 135.5, 133.5, 130.3, 128.3, 81.8, 62.9, 51.9, 34.0, 28.3, 27,1, 23.2, 19.2; HRMS-ESI (m/z): [M+H]+ calcd for C29H39N2O5SS1: 555.2349, found: 555.2336.
Step Ç: methyl 2-[tert~butoxycarbonyl-[3-(3,6-dichtoro-5-methyl-pyridazin-410 y l) propyl]amino] ~5-[3 ·[tert~butyl(diphenyl)silyl]oxypropyl]thiazole-4-carboxylate
Using Alkylation General Procedure starting from 34.95 g (63 mmol) of the product from Step B and 25.0 g (1.2 eq) of 3,6-dichloro-4-(3-iodopropyl)-5-methyl-pyridazine as the appropriate iodine compound, 51.0 g (quantitative yield) of the desired product was obtained.
NMR (500 MHz, DMSO4) δ ppm 7.63-7.37 (m, 10H), 4.09 (t, 2H), 3.75 (s, 3H), 3.67 (t, 15 2H), 3.20 (t, 2H), 2.82 (m, 2H), 2.40 (s, 3H), 1.87 (m, 2H), 1.87 (m, 2H), 1.50 (s, 9H), 0.97 (s,
9H); 13C NMR (125 MHz, DMSO-dô) δ ppm 62.9, 52.0, 46.1, 33.9, 28.1, 27.5, 27.1, 25.9, 23.8, 16.4; HRMS-ESI (m/z): [M+H]+ calcd for C37H47CJ2N4O5SS1: 757.2413, found: 757.2395.
Step D: methyl 5-[3-[tert-butyl(diphenyl)silyl]oxypropyl]-2 -[3-(3,6-dichU>ro-5-methyl20 pyridazin-4-yl)propylamino]thiazole-4-carboxylate
Using Deprotection with HFIP General Procedure starting from 51.70 g of the product from Step C (68 mmol), 36.32 g (81 %) of the desired product was obtained.
*H NMR (500 MHz, DMSO-de) δ ppm 7.71 (t, IH), 7.63-7.37 (m, 10H), 3.69 (s, 3H), 3.67 (t, 2H), 3.30 (m, 2H), 3.10 (t, 2H), 2.85 (m, 2H), 2.83 (s, 3H), 1.79 (m, 2H), 1.78 (m, 2H), 0.98 25 (s, 9H); 13C NMR (125 MHz, DMSO-rfc) δ ppm 62.9, 51.7, 44.1, 34.2, 28.0, 27.1, 27.0, 23.4,
16.4; HRMS-ESI (m/z): [M+H]+ calcd for CTzH^CLNaCLSSi: 657.1889, found: 657.1875.
Step E: methyl 5-[3-[tert-butyl(diphenyl)silyl]oxypropyl]-2-(3-chloro-4-methyl-6,7-dihydro5H-pyrido[2,3-c]pyridazin-8-yl)thiazole-4-carboxylate
The mixture of 36.0 g (54.7 mmol) of the product from Step D and 35.7 g (2 eq) of CS2CO3 in 30 1,4-dioxane (383 mL) was stirred at 90 °C for 18 h. After dilution with water, the precipitated
309 solid was filtered off, washed with dielhylether, and dried to give 34.0 g (99%) of the desired product.
’H NMR (500 MHz, DMS0-î/6) δ ppm 7.61 (d, 4H), 7.43 (t, 2H), 7.42 (t, 4H), 4.26 (t, 2H), 3.77 (s, 3H), 3.70 (t, 2H), 3.23 (t, 2H), 2.90 (t, 2H), 2.33 (s, 3H), 2.04 (qn, 2H), 1.90 (qn, 2H), 1.00 (s, 9H); 13C NMR (125 MHz, DMSO-^) δ ppm 163.1, 155.3, 151.8, 151.4, 143.2, 136.2, 135.5, 134.7, 133.6, 130.3, 129.0, 128.3, 63.1, 51.9, 46.3, 34.1, 27.1, 24.2, 23.1, 19.8, 19.2, 15.7; HRMS-ESI (m/z): [M+H]+ calcd for C32H38ClN4O3SSi: 621.2122, found: 621.2097.
Step F', methyl 2-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl)-5-(3hydroxypropyl)thiazole-4-carboxylate
The mixture of 23.36 g (37.6 mmol) of the product from Step E and 45 mL (1.2 eq.) of 1 M TBAF solution in THF (5 mL/mmol) was stirred at rt for 2 h. After the removal of the volatiles, purificationby column chromatography (silica gel, using EtOAc and MeOH/NH3 as eluents) afforded 12.88 g (89%) of the desired product.
rH NMR (500 MHz, DMSO-de) δ ppm 4.54 (br., 1H), 4.25 (m, 2H), 3.80 (s, 3H), 3.45 (t, 2H), 3.11 (m, 2H), 2.88 (t, 2H), 2.31 (s, 3H), 2.04 (m, 2H), 1.77 (m, 2H); i3C NMR (125 MHz, DMSO-rfc) δ ppm 163.1, 155.2, 151.2, 143.8, 136.1, 134.5, 129.0, 60.5, 52.0, 46.3, 34.6, 24.2, 23.2, 19.7, 15.7; HRMS-ESI (m/z): [M+H]+ calcd for C16H20CIN4O3S: 383.0945, found: 383.0937.
Step G: methyl 2-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl)'5-[3-[4[3-(dimethylamino)prop-l-ynyl/phenoxy]propylJthiazole-4-carboxylate
Using Mitsunobu General Procedure starting from 0.65 g (1.2 eq) of the product from Step F and 250 mg (1.43 mmol) of 4-[3-(dimethylamino)prop-l-ynyl]phenol in THF (9 mL/mmol), 0.28 g (37%) of the desired product was obtained.
*H NMR (500 MHz, DMSO-de) δ ppm 7.34 (d, 2H), 6.91 (d, 2H), 4.26 (t, 2H), 4.03 (t, 2I-I), 3.78 (s, 3H), 3.40 (s, 2H), 3.25 (t, 2H), 2.88 (t, 2H), 2.31 (s, 3H), 2.22 (s, 6H), 2.08 (qn, 2H), 2.03 (qn, 2H); 13C NMR (125 MHz, DMSO-rfe) δ ppm 163.1, 158.9, 155.3, 151.7, 151.3, 142.7, 136.2, 134.9, 133.3, 129.0, 115.2, 115.0, 85.2, 84.1, 67.1, 52.0, 48.3, 46.3, 44.3, 30.8, 24.1, 23.1, 19.7, 15.7; HRMS-ESI (m/z): [M+H]+ calcd for C27H31CIN5O3S: 540.1836, found: 540.1834.
310
Step H: methyl 2-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-[3-[4-[3-(dimethylamino)prop-I -ynyl]phenoxy]propyl]thiazole-4carboxylate
Using Buchwald General Procedure I starting from 0.27 g of the product from Step G (0.5 mmol), 0.29 g (89%) of the desired product was obtained.
Ή NMR (500 MHz, DMSO-ώ) δ ppm 7.83 (dm, 1H), 7.50 (dm, 1H), 7.36 (m, 1H), 7.35 (m, 2H), 7.18 (m, 1H), 6.94 (m, 2H), 4.28 (m, 2H), 4.09 (t, 2H), 3.80 (s, 3H), 3.39 (s, 2H), 3.29 (t, 2H), 2.88 (t, 2H), 2.35 (s, 3H), 2.23 (s, 6H), 2.13 (m, 2H), 2.07 (m, 2H); HRMS-ESI (m/z): [M+H]4 calcd for C34H36N7O3S2: 654.2321, found: 654.2322.
Step 1' 2-[3-(l ,3-benzothiazol-2-ylamino) -4-methyl-6f 7-dihydro-5H-pyrido[2,3-c]pyridazin8-yl]-5-[3-[4-[3-(diinethylamino)prop-l-ynyl]phenoxy]propyl]thiazole-4-carboxylic acid
To the product from Step H (280 mg, 0.43 mmol) in a 1:1 mixture of THF and water (10 mL/mmol) was added 90 mg (5 eq) of LiOHxHzO, and the reaction mixture was stirred at 50 °C for 18 h. After the removal of the volatiles, purificationby reverse phase préparative chromatography (Cl8, 0.1% TFA in water and MeCN as eluents) afforded 132 mg (48%) of the desired compound.
HRMS-ESI (m/z): [M+H]4 calcd for C33H34N7O3S2 : 640.2165, found: 640.2160.
Example 142: 6-[3-( l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-3-[l-[[3,5-dimethyl-7-(2-morpholinoethoxy)-l-adamantyl]methyl]-5methyl·pyrazol-4-yl]pyridiπe-2-carboxylic acid
O. OH.N LJ *».
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and morpholine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+HJ+ calcd for C44H54NgO4S: 804.4019, found: 804.4012.
311 φ Exampie 143: fi-fS-iGS-benzothiazol^-ylaminoH-niethyl-ôJ-dihydro-SH-pyndoU^c]pyridazin-8-yI]-3-[l-[[3,5-dimethyl-7-[2-(l-piperidyl)ethoxy]-l-adamantyl]methyl]-5methyl-pyrazol-4-yl]pyrîdine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and piperidine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C45H56N9O3S: 802.4227, found: 802.4223.
Example 144: 6-[3-(l,3-benzothÎazol-2-ylamino)-4-methyl-6,7’dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-3-[l-[[3,5-dimethyl-7-(2-pÎperazin-l-ylethoxy)-l-adamantyl]methyl]-510 methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
O.OH^N
L N H
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and piperazine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C44H55N[0O3S; 803.4179, found: 803.4177.
Example 145: 3-[l-l[3-[2-(azepan-l-yl)ethoxy]-5,7-dimethyl·l-adamantyl]methyl]-5niethyl-pyrazol-4-yl]-6-[3-(l,3-benzothiazol-2-ylamino)-4-metliyl-6,7-dihydro-5Zfpyrido[2,3-c]pyrÎdazin-8-yl]pyridine-2-carboxylic acid
312
Using the Amine substitution and Hydrolysis General procedure startîng from Préparation 12 and azepane as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C46H58N9O3S: 816.4383, found: 816.4379.
Example 146: 6-[3-( l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-3-[l-[[3-[2-(4-isopropylpiperazin-l-yl)ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyI-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure startîng from 10 Préparation 12 and 1-isopropylpiperazine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C47H6]N)0O3S: 845.4649, found: 845.4646.
Example 147: 2-[3-(l,3-benzothiazoI-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido[2,3c]pyridazin-8-yl]-5-[3-[2-fluoro-4-[3-(3-hydroxypropylamino)prop-l15 ynyl]phenoxy]propyl]thiazole-4-carboxylic acid
313
Step A: 3-[tert-butyl(dimethyl)silyl]oxy-N-prop-2-ynyl-propan-l-amine
The mixture of 0.70 mL (3.0 mmol) of 3-bromopropoxy-Zert-butyl-dimethyl-silane, 1.9 mL (10 eq) of propargylic amine and 1.6 mL (3 eq) of DIPEA in acetonitrile (15 mL ) was stirred 5 at 50 °C untîl no further conversion was observed. The réaction mixture was concentrated, diluted with DCM, and extracted with saturated NaHCO3 and brine. The combined organic layers were dried and concentrated to give the desired product in quantitative yield.
NMR (500 MHz, dmso-d6) δ ppm 3.62 (t, 2H), 3.27 (d, 2H), 3.02 (t, IH), 2.59 (t, 2H), 2.19 (brs, IH), 1.57 (m, 2H), 0.86 (s, 9H), 0.02 (s, 6H); 13C NMR (500 MHz, dmso-d6) δ ppm 73.9, 61.5, 45.2, 37.9, 32.7, 26.3, -4.8; HRMS (El) (m/z): [M-CH3]+ calcd for CuH22NOSi: 212.1471, found: 212.1467.
Step B: ethyl 5-(3-(4-[3-[3-[tert-butyl(dimethyl)silyl]oxypropylamino]prop-l-ynyl]-2-fluorophenoxy]propyl]-2-(3-chloro-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl)thîazole4-carboxylate
Using Sonogashira General Procedure starting from 1.0 g (1.64 mmol) of the product of Préparation 15 and 737 mg (2 eq.) of the product from Step A as the appropriate acetylene, 1.16 g (96%) of the desired product was obtained.
NMR (500 MHz, dmso-d6) δ ppm 45.2 (t, 2H), 7.24 (dd, IH), 7.17 (dd, IH), 7.14 (t, IH), 4.27 (br., 2H), 4.25 (q, 2H), 4.12 (t, 2H), 3.65 (t, 2H), 3.6 (s, 2H), 3.25 (t, 2H), 2.89 (t, 2H),
2.32 (s, 3H), 2.11 (m, 2H), 2.04 (m, 2H), 1.63 (m, 2H), 1.28 (l, 3H), 0.84 (s, 9H), 0.02 (s, 6H);
13C NMR (500 MHz, dmso-d6) δ ppm 128.8, 119.1, i 15.4, 68.3, 61.3, 60.7, 46.3, 45.2, 38.4, 32.4, 30.8, 26.3, 24.2, 23.1, 19.7, 15.7, 14.6, -4.8; HRMS-ESI (m/z): [M+H]+ calcd for C35H48ClFN5O4SSi: 716.2869, found: 716.2868.
Step C: ethyl 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,325 cjpyridazin -8-yl]-5-(3-(4-(3-(3-(tert-butyl(dimethyl) silyl]oxypropylamino]prop-l -ynyl]-2 20573
314 fluoro-phenoxy]propyl]thiazole-4-carboxylate
Using Buchwald General Procedure I starting from 1.16 g (1.57 mmol) of the product from Step B and 730 mg (2 eq) of l,3-benzothiazol-2-amine, 598 mg (45%) of the desired product was obtained.
3H NMR (500 MHz, dmso-d6) δ ppm 7.87 (d, IH), 7.49 (d, IH), 7.37 (td, III), 7.25 (dd, IH), 7.19 (t, IH), 7.17 (t, IH), 7.17 (m, IH), 4.26 (br., 2H), 4.25 (q, 2H), 4.14 (t, 2H), 3.63 (t, 2H), 3.57 (s, 2H), 3.27 (t, 2H), 2.87 (t, 2H), 2.69 (t, 2H), 2.34 (s, 3H), 2.13 (m, 2H), 2.04 (m, 2H), 1.61 (m, 2H), 1.28 (t, 3H), 0.84 (s, 9H), 0.02 (s, 6H); I3C NMR (500 MHz, dmso-d6) δ ppm 128.9, 126.5, 122.5, 122.3, 119.1, 116.3, 115.5, 68.4, 61.3, 60.6, 46.3, 45.2, 38.4, 32.4, 31.1, 26.3, 23.9, 23.2, 20.3, 14.6, 12.9, -4.9; HRMS-ESI (m/z): [M+H]+ calcd for C42H53FN7O4S2Si: 830.3354, found: 830.3347.
Step D: 2-(3-(1 t3-benzothiazol-2-ylafnino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin8-yl]-5-(3-(2-fluoro-4-[3-(3-hydroxypropylamino)prop-l-ynyl]phenoxy]propyl]thiazole-4carboxylic acid
The mixture of 590 mg (0.71 mmol) of the product from Step C and 298 mg of LiOHxH2O (10 eq) în 7 mL of THF / water (1:1) was stirred at 60 °C until no further conversion was observed. The reaction mixture was treated with 0.71 mL (12 eq) of concentrated hydrogen chloride at 0°C (pH = 2-3) and stirred until no further conversion was observed. After the réaction mixture was concentrated to remove THF and lyophilization, the solid was dîssolved in a 6N NH3 solution in MeOH and purified by reverse phase chromatography (using 25 mM NH4HCO3 and MeCN as eluents) to give 100 mg (21%) of the desired product.
HRMS-ESI (m/z): (M+H]+ calcd for QhHssFNvOïSz: 688.2176, found: 688.2179.
Example 148: 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-[3-[4-[3-[[(3S)-3,4-dihydroxybutylJ-methyl-amino]prop-l-ynylJ-2tluoro-phenoxy]propyl]thïazole-4-carboxylic acid
315
Step A : 2-[(4S)~2,2-dimethyl-lf3-dioxolan~4-yl]ethyl 4-methylbenzenesulfonate
To 1.0 g (6.8 mmol) of 2-[(45)-2,2-dimethyl-l,3-dioxoIan-4-y[]ethanol and 3.8 mL (4 eq) of triethylamine in 34 mL of DCM was added 4.5 g (2 eq) of p-tolylsulfonyl 45 methylbenzenesulfonate at 0 °C. The reaction mixture was stirred until no further conversion was observed, concentrated and treated with diisopropyl ether. Then, the precipitated hydrochloric sali was filtered off and the mother liquour was concentrated and purified via flash chromatography (silica gel, using heptane and EtOAc as eluents) to give 1.6 g (81%) of desired product.
*H NMR (500 MHz, dmso-d6) δ ppm 7.79 (dm, 2H), 7.49 (dm, 2H), 4.08 (m, 2H), 4.00 (m, 1H), 3.91/3.44 (dd+dd, 2H), 2.42 (s, 3H), 1.83/1.77 (m+m, 2H), 1.24/1.20 (s+s, 6H); 13C NMR (500 MHz, dmso-d6) δ ppm 132.7, 132.7, 130.7, 128.1, 108.6, 72.3, 68.7, 68.4, 32.9, 27.2/25.9, 21.6; HRMS-ESI (m/z): [M+H]+ calcd for C14H21O5S: 301.1110, found: 301.1107.
Step B: N-[2-[(4S)-2,2-dimethyl-l,3-dioxolan-4-yl]ethylJprop-2-yn-l-amine
The mixture of the product from Step A (7.6 g, 25.3 mmol), prop-2-yn-l-amine (16 mL, 10 eq) and DIPEA (13.22 mL, 3 eq) in 127 mL of MeCN was stirred at 50 °C for 16 h. After concentration, taken up in DCM and extraction with cc. NaHCO3 solution and brine, the combined organic layers were dried and concentrated to give 5.0 g (107%) of the desired 20 product, which was used without any further purification.
Ή NMR (500 MHz, dmso-d6) δ ppm 4.07 (m, 1H), 3.98/3.43 (dd+t, 2H), 3.28 (m, 2H), 3.05 (t, 1H), 2.62/2.55 (m+m, 2H), 2.23 (brs, 1H), 1.63/1.59 (m+m, 2H), 1.30 (s, 3H), 1.25 (s, 3H); 13C NMR (500 MHz, dmso-d6) δ ppm 108.2, 83.4, 74.6, 74.1, 69.2, 45.1, 37.8, 33.6, 27.3, 26.2; HRMS (El) (m/z): [M]+ calcd for Ci0Hi7NO2: 183.1259, found: 183.1260.
Step C: N-[2-[(4S)-2,2-dimethyl-l ,3-dioxolan -4-yl]ethyl]-N-methyl-prop-2-yn-l -amin e
316
To the product from Step B (500 mg, 2.73 mmol) in Λ,Α-dimethylformarnide (14 mL) was added portionwise sodium hydride (120 mg, 1.1 eq) at 0°C. After stirring at 0 °C for 0.5 h, the mixture was treated with iodomethane (0.17 mL, 1 eq) and stirred at rt for 18 h. After quenching with a saturated solution of NH4CI and water, the mixture was extracted with Et2O. 5 The combined organic phases were dried and concentrated to give the desired product (362 mg, 67%). GC/MS (C11H19NO2) 197 [M+].
Step D: ethyl 2-(3-ehloro~4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl)-5-[3-[4-[3[2-[(4S) -2,2-dimethyl-l ,3-dioxolan -4-yl]ethyl-methyl-amino]prop-l -ynyl]-2-fluorophetioxy]propyl]thiazole-4-earboxylate
Using Sonogashira General Procedure starting from 0.548 g (0.89 mmol) of the product of Préparation 15 and 350 mg (2 eq) of the product from Step C as the appropriate acetylene, 510 mg (82%) of the desired product was obtained. LC/MS (C34H42CIFN5O5S) 686 [M+H]+.
Step E‘. ethyl 2-(3-(1,3-benzothiazol‘2-ylamiiio)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-5-[3-[4-[3-[2-[(4S) -2,2-dimethyl-l,3-dioxolan-4-yl]ethyl-methyl15 amino]prop-l -ynyl]-2-fluoro-phenoxy]propytfthiazole~4-carboxylate
Using Buchwald General Procedure I starting from 510 mg (0.52 mmol) of the product from Step D and 234 mg (3 eq) of l,3-benzothiazol-2-amine, 200 mg (48%) of the desired product was obtained.
Ή NMR (500 MHz, dmso-d6) δ ppm 7.88 (dm, 1H), 7.49 (brd, 1 H), 7.37 (m, 1H), 7.3 (dd, 20 1H), 7.20 (dm, 1H), 7.19 (m, 1H), 7.16 (t, 1H), 4.26 (m, 2H), 4.25 (q, 2H), 4.14 (t, 2H), 4.04 (m, 1H), 3.98/3.45 (dd+dd, 2H), 3.46 (s, 2H), 3.28 (m, 2H), 2.87 (t, 2H), 2.45/2.39 (m+m, 2H), 2.34 (s, 3H), 2.21 (s, 3H), 2.13 (m, 2H), 2.04 (m, 2H), 1.63 (m, 2H), 1.29 (t, 3H), 1.29 (s, 3FI), 1.24 (s, 3H); HRMS (ESI) (m/z): [M+H]+ calcd for C41H47FN7O5S2: 800.3064, found: 800.3064.
Step F: 2-(3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin8-yl]-5-[3-[4-[3-[[ (3S) -3,4-dihydioxybulyll-methyl-amino]prop-l -ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylic acid
The mixture of 200 mg (0.25 mmol) of product from Step E and 53 mg of LiOHxH2O (5 eq) in 5 mL of THF / water (1:1) was stirred at 60 °C for 18 h. The reaction mixture was treated 30 with 0.125 mL (6 eq) of concentrated hydrogen chloride at 0 °C (pH = 2-3) and stirred at rt, then at 60 °C for 0.5 h. After the reaction mixture was concentrated to remove THF and lyophilization, the solid was dissolved in 6 N NH3 solution in MeOH and purified by reverse
317 φ phase chromatography (using 5 mM NH4HCO3 and MeCN as eluents) to give 47 mg (25%) of the desired product.
HRMS (ESI) (m/z): [M+H]+ calcd for C36H39FN7O5S2: 732.2438, found: 732.2441.
Example 149: 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,35 c]pyridazin-8-yl]-3-[l-[[3-[2-[(4-hydroxyphenyl)methylamino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysîs General procedure starting from Préparation 12 and 4-(aminomethyl)phenol as the appropriate amine, the desired product 10 was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C47H54N9O4S: 840.4019, found: 840.4016.
Example 150: 6-[3-(l, 3-benzothîazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3cJpyridazin-8-yl]-3-[l-[[3-[2-[2-hydroxyethyl(methyl)amino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
HO^ .Ο
OH
Using the Amine substitution and Hydrolysîs General procedure starting from Préparation 12 and 2-(methylamino)ethanol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C43H54N9O4S: 792.4019, found: 792.4019.
318
Example 151: 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-3-[I-[[3-[2-[3-methoxypropyl(inethyl)ammo]ethoxy]-5,7-dimethyl-Iadamantyl]methyi]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and 3-inethûxy-N-methyl-propan-l-amine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C45H58N9O4S: 820.4332, found: 820.4328.
Example 152: 3-(1-((3,5-dimethyl-7-(2-pyrrolidin-l-ylethoxy)-l-adamantyl]methyl]-510 methyl-pyrazol-4-yl]-6-[3-[(5-hydroxy-l,3-benzothiazol-2-yl)amino]-4-methyl-6,7dihydro-5H-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
Step A: 5-[tert-butyl(dimethyl)silyl]oxy-l,3-benzothiazol-2-amine
To a mixture of 2-amino-l,3-benzothiazol-5-ol (750 mg, 4.51 mmol), DM AP (110 mg, 0.2 15 eq), and imidazole (399 mg, 1.3 eq) in DMF (23 mL) was added tertbutyl(chloro)diphenylsilane (816 mg, 1.2 eq) and the reaction mixture was stirred for 18 h. After quenching with water and extraction with EtOAc, the combined organic phases were dried, concentrated, and purified by column chromatography (siiica gel, heptane and EtOAc as eluents) to give the desired product (1.07 g, 84.5%).
319 φ ‘H NMR (400 MHz, DMSO-d6): δ ppm 7.46 (d, 1H), 7.44 (s, 2H), 6.78 (d, 1H), 6.53 (dd, 1H), 0.95 (s, 9H), 0.17 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ ppm 168.1, 154.5, 154.1, 124.0, 121.5, 114.0, 109.6, 26.1, 18.4, -4.0; 1SN NMR (100 MHz, DMSO-d6) δ ppm 237, 79.
Step B: methyl 6-[3-[[5-[tert-butyl(dimethyl)silyl]oxy-l,3-benzothiazol-2-yl]amino]-45 methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl]-3-[l-[[3-(2-hydroxyethoxy)-5,7dimethyl-l-adamantyl]methyl]’5-methyl-pyrazol-4-yl]pyridine-2-carboxylate
Using Buchwald General Procedure I at 130 C for 1 h, starting from 1.0 g (1.57 mmol) of the product from Préparation 12, Step C and 883 mg (2 eq) of the product from Step A, 1.1 g (80%) of the desired product was obtained.
Ή NMR (400 MHz, DMSO-d6): δ ppm 7.95 (d, 1H), 7.7 (d, 1H), 7.65 (br, 1H), 7.38 (s, 1H), 6.95 (br, 1H), 6.71 (brd, 1H), 4.45 (t, 1H), 4.00 (t, 2H), 3.88 (s, 2H), 3.70 (s, 3H), 3.41 (q, 2H), 3.35 (t, 2H), 2.85 (t, 2H), 2.31 (s, 3H), 2.16 (s, 3H), 1.98 (qn, 2H), 1.39 (s, 2H), 1.32/1.25 (d+d, 4H), 1.18/1.12 (d+d, 4H), 1.08/1.00 (d+d, 4H), 0.97 (s, 9H), 0.87 (s, 6H), 0.21 (s, 6H); 13C NMR (100 MHz, DMSO-d6) δ ppm 139.9, 137.5, 122.3,119.1,115.3, 62.1, 61.5,
58.9, 52.6, 50.1, 47.0, 46.1, 45.4, 43.3, 30.2, 26.1, 24.3, 21.7, 12.6, 10.9, -4.0; HRMS-ESI (m/z): [M+H]+ calcd for C47H63NsO5SSi: 879.4411, found: 879.4412.
Step C: methyl 6-[3-[[5-[tert-butyl(dimethyl)silyljoxy-l,3-benzothiazol-2-yl]amino]-4methyl-6,7-dihydro-5H-pyrido[2,3~c]pyridazin-8-yl]-3-[l -[[3,5-dimethyl- 7-[2-(p~ tolylsulfonyloxy)ethoxy]-l -adamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine~2-carboxylate
To the product from Step B (1.1 g, 1.26 mmol) and triethylamine (0.53 mL, 3 eq) in DCM (13 mL) was added p-tolylsulfonyl 4-melhyIbenzenesulfonate (618 mg, 1.5 eq) and the reaction mixture was stirred for 2 h. Purîficationby column chromatography (silica gel, DCM and EtOAc as eluents) afforded the desired product (590 mg, 45%).
‘H NMR (400 MHz, DMSO-d6): δ ppm 7.96 (d, 1H), 7.90-6.40 (brs, 3H), 7.70 (d, 10H), 7.70 25 (m, 2H), 7.46 (m, 2H), 7.38 (s, 1H), 4.07 (m, 2H), 4.00 (m, 2H), 3.85 (s, 2H), 3.69 (s, 3H),
3.49 (m, 2H), 2.85 (t, 2H), 2.40 (s, 3H), 2.31 (s, 3H), 2.15 (s, 3H), 1.98 (m, 2H), 1.33-0.91 (m, 12H), 0.97 (s, 9H), 0.84 (s, 6H), 0.21 (s, 6H); HRMS-ESI (m/z): [M+H]+ calcd for C54H69N8O7S2Si: 1033.4500, found: 1033.4504.
Step Dt methyl 6-[3-[[5‘[tert-butyl(dimethyl)silyl]oxy-l,3-beuzûthiazol-2-yl]amino]-430 methyl-6,7-dihydro-5H-pyrido[2,3~c]pyridazin-8-yl]-3-[l -[[3,5-dimethyl- 7-(2-pyrroHdin-l ylethoxy)-l-adamantyl]methyl]-5-inethyl-pyrazol-4-yl]pyridine-2-carboxylate
32Ü φ To the product from Step C (180 mg, 0.17 mmol) in MeCN (1.7 mL) and NMP (1.0 mL) was added pyrrolidine (0.10 mL, 7 eq) and the reaction mixture was stirred at 60 °C for 18 h. Purification by column chromatography (silica gel, DCM and 0.6 M NH3 in MeOH as eluents) afforded the desired product (144 mg, 89%).
rH NMR (400 MHz, DMSO-d6): δ ppm 11.24 (brs, 1H), 7.95 (d, 1H), 7.69 (d, 1H), 7.63 (d, 1H), 7.37 (s, 1H), 6.92 (br, 1H), 6.70 (dd, 1H), 4.00 (t, 2H), 3.87 (s, 2H), 3.70 (s, 3H), 3.43 (t, 2H), 2.85 (t, 2H), 2.46 (t, 2H), 2.41 (t, 4H), 2.31 (s, 3H), 2.16 (s, 3H), 1.98 (qn, 2H), 1.63 (t, 4H), 1.38 (s, 2H), 1.30/1.25 (d+d, 4H), 1.19/1.12 (d+d, 4H), 1.08/0.99 (d+d, 2H), 0.97 (s, 9H), 0.86 (s, 6H), 0.21 (s, 6H); ,3C NMR (100 MHz, DMSO-d6) δ ppm 139.8, 137.5, 122.6, 10 119.0, 115.3, 59.5, 58.9, 56.6, 54.5, 52.6, 50.1, 47.0, 46.0, 46.0, 43.3, 30.2, 26.0, 24.2, 23.6,
21.7, 12.6, 10.9, -4.0; HRMS-ESI (m/z): [M+H]+ calcd for C51H7nN9O4SSi: 932.5041, found: 932.5014.
Step E: 3-[l-[[3,5-dimethyl-7-(2-pyrrolidin-l-ylethoxy)~l-adatnantyl]inethyl]-5-methylpyrazol-4-yl]-6-[3-[ (5-hydroxy-l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydro-5H15 pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
To the product of Step D (70 mg, 0.075 mmol) in THF (1.2 mL) and water (0.30 mL), was added LiOHxH2O (25.2 mg, 8 eq) and the reaction mixture was stirred at 60 °C for 1.5 h. Purification by préparative reversed-phase HPLC (Cl8, 5 mM NH4HCO3 (aqueous) and 1PA as eluents) afforded the desired product (45 mg, 74%).
HRMS-ESI (m/z): [M+H]+ calcd for C44H54N9O4S: 804,4019, found: 804.4019.
Example 153: 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-57/-pyrido[2,3c]pyridazin-8-yl]-3-[l-[[3-[2-[4-hydroxybutyl(methyl)amino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazoL4-yl]pyridine-2-carboxylic acid
321
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 12 and 4-(methylamino)butan-l-ol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C45H58N9O4S: 820.4332, found: 820.4339.
Exampie 154: 6-[3-(l,3-benzothiazol-2-ylammo)-4-methyl-6,7-dihydro-57/-pyrido[2,3c]pyridazin-8-ylJ-3-[l-[[3-[2-(dimethyIamino)ethoxyJ-l-adamantyl]methyl]-5-methylpyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from 10 Préparation 14 and dimethylamine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C40H48N9O3S; 734.3601, found: 734.3589.
Example 155: 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-57/-pyrido|2J3cJpyridazin-8-yl]-3-[5-methyl-l-[[3-(2-pyrrolidin-l-ylethoxy)-l15 adamantyl]methyl]pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 14 and pyrrolidine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C42H50N9O3S: 760.3757, found: 760.3730.
322
Example 156: 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5W-pyrido[2,3i,]pyridazm-8-yl]-3-[5’methyl-l-[[3-[2-(4-methylpiperazin-l-yl)ethoxy]-ladamantyl]methylJpyrazol-4-yl]pyridme-2-carboxylic acid
HO.
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 14 and 1-methylpiperazine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C43H53N10O3S: 789.4017, found: 789.4023.
Example 157: 6-[3-(l,3-benzothiazol-2-ylaniino)-4-methyl-6,7-diliydro-5/Z-pyrido[2,310 c]pyridazin-8-yl]-3-[5-methyl-l-[[3-(2-morpholinoethoxy)-l-adamantyl]methyl]pyrazol4-ylJpyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 14 and morpholine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C42H,0N9O4S: 776.3706, found: 776.3697.
Example 158: 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-3-[l-[[3-[2-(3-hydroxypropylammo)ethoxy]-l-adamantyl]methyl]-5methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
323
H
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 14 and 3-aminopropan-l-ol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C4]H50N9O4S: 764.3706, found: 764.3700.
Example 159: 6-(3-( l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5W-pyrido[2,3e|pyridazin-8-yl]-3-[l-[[3-[2-(4-hydroxybutylammo)ethoxy]-l-adamantyl] methyl]-5methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 14 and 4-amînobutan-l-ol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]4 calcd for C42H52N9O4S: 778.3863, found: 778.3859.
Example 160: 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-diliydro-5H-pyrido[2,315 c]pyridazin-8-yl]-3-[l-((3-(2-(((35)-3,4-dihydroxybutyl]amino]ethoxy]-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
324
Using the Amine substitution and Hydrolysis General procedure starting front Préparation 14 and 2-((45)-2,2-dimethyl-l,3-dioxolan-4-yl]ethanamine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C42H52N9O5S: 794.3812, found: 794.3807.
Example 161: 6-[3-(l,3-benzothiazoI-2-ylamino)-4-methyI-6,7-dihydro-5H-pyrido[2,3c] pyridazin-8-yl]-3-[1-((3-(2-((3-hydroxy-2-(hydroxy methyl) propyl] amino] ethoxy J-1adamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 14 and 2-(aminomethyl)propane-l,3-diol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): (M+H]+ calcd for C42H52N9O5S: 794.3812, found: 794.3808.
Example 162: 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,315 c]pyridazm-8-yl]-3-[l-[[3-[2-[4-hydroxybutyl(methyl)amino]ethoxy]-ladamantyl]methyl]-5-methyl-pyrazol-4-yI]pyridine-2-carbüxylic acid
325
\
Using the Amine substitution and Hydrolysis General procedure startîng from Préparation 14 and 4-(methylamino)butan-l-ol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C43H54N9O4S: 792.4019, found: 792.4020.
Example 163: 6-[3-( l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-3-[l-[[3-I2-[3-hydroxypropyl(methyl)amino]ethoxy]-ladamantyl]methyl]-5-niethyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure startîng from Préparation 14 and 3-(methylamino)propan-l-oI as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C42H52N9O4S: 778.3863, found: 778.3858.
Example 164: 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,315 c]pyridazin-8-yl]-3-[l-[[3-[2-[bis(3-hydroxypropyl)amino]ethoxy]-l-adamantyl]metliyl]5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
326
^OH
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 14 and 3-(3-hydroxypropylamino)propan-l-ol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C44H56NgO5S: 822.4125, found: 822.4121.
Example 165: 6-[3-(l,3-benz.othia/Ol-2-ylainmo)-4-metliyl-6,7-dihydiO-5//-pyrido[2,3c]pyridazin-8-yl]-3-[5-methyl-l-[[3-(2-piperazin-l-ylethoxy)-ladamantyl]methylJpyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 14 and piperazine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C44H56N9O5S: 775.3866, found: 775.3859.
Example 166: 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido[2,315 c]pyridazin-8-yl]-3-[l-[[3,5-dimethyl-7-(3-pyrrolidin-l-ylpropyl)-l-adamantyl]methyl]5-inethyl-pyrazol-4-yl]pyridine-2-carboxylic acid
327
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 13 and pyrrolidine as the appropriate amine, the desired product was obtained. HRMS-ESI (m/z): [M+H]+ calcd for C45H%N9O2S: 786.4278, found: 786.4273.
Example 167: 6-[3-(l,3-benzothiazol-2-ylanlino)-4-methyl·6,7-dihydro-5frΓ-pyrido[2,3c|pyridazin-8-yl]-3-ll-[[3-[3-(dimethylamino)pΓopyl]-5,7-dimethyl·ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from 10 Préparation 13 and dimethylamine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C43H54NQO2S: 760.4115, found; 760.4121.
Exampie 168: 6-[3-(l,3-benzothiazol-2-ylamino)’4-metliyl-6,7-dihydro-5jff-pyrido[2,3c]pyridazin-8-ylJ-3-[l-[[3,5-dimethyl-7-[3-(4-methylpiperazin-l-yl)propyl]-l15 adamantylJmethyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
328
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 13 and 1 -methylpiperazine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C46H59N1(|O2S: 815.4543, found: 815.4534.
Exampie 169: 6-[3-(l, 3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-3-[l-[[3,5-dimethyl-7-(3-morpholinopropyl)-l-adamantyl]methyl]-5niethyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 13 and morpholine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C45H56N9O3S: 802.4227, found: 802.4221.
Example 170: 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,315 c]pyridazin-8-yl]-3-[l-[[3-[3-(3-hydroxypropylamino)propyn-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
329
H
Using the Amine substitution and Hydrolysîs General procedure starting from Préparation 13 and 3-aminopropan-l-ol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C44H56N9O3S: 790.4227, found: 790.4220.
Example 171: 6-[3-( l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3c]pyridazin-8-yl]-3-[l-[[3-[3-(4-hydroxybutylamino)propyI]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysîs General procedure starting from Préparation 13 and 4-(amino)butan-l-ol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C45H58N9O3S: 804.4383, found: 804.4377.
Example 172: 6-[3-( l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,315 c]pyridazin-8-yl]-3-[l-[[3-[3-[[(3S)-3,4-dihydroxybutyl]amino]propyl]-5,7-dimethyl-ladamantyl]methyll-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
330
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 13 and 2-[(4S)-2,2-dimethyl-l,3-dioxolan-4-yl]ethanamine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C42H;2N9O5S: 820.4332, found: 820.4328.
Example 173: 6-[3-(l,3-benzothiazol·2-ylamino)-4-methyl·6,7-dihydro-5/7-ρyrido[2,3e]pyridazin-8-yl]-3-[l-[[3-[3-[[3-hydroxy-2-(hydroxymethyl)propyl]amino]propyl]-5,7dimethyl-l-adamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 13 and 2-(aminomethyl)propane-l,3-diol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C42H52NQO5S: 820.4332, found: 820.4329.
Example 174: 6-[3-(l,3-benzothiazol-2-ylaniino)-4-methyl-6,7-dihydro-5JH-pyrido[2,315 c]pyridazin-8-yl]-3-[l-[[3-[3-[4-hydroxybutyl(methyI)amino]propyl]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
331
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 13 and 4-(methylamino)butan-l-ol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C43H54N9O4S: 818.4540, found: 818.4536.
Example 175: ô-tS-ll^-benzothiazol-l-ylaniinoM-niethyl-ôJ-dihydro-SH-pyndo^ScJpyridazin-8*yl]-3-[l-[[3-[3-[3-hydroxypropyl(methyl)aininoJpropyl]-5,7-dimethyl-ladamantyl]methyl]-5-metliyl-pyrazol-4-yl]pyridine-2-carboxyIic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 13 and 3-(methylamino)propan-l-ol as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C42H52NqO4S: 804.4383, found: 804.4380.
Example 176: 6-[3-(l,3-benzothiazol·2-ylamino)-4-methyl-6,7-dihydro-5f^-pyrido[2,3c]pyridazin-8-yl]-3-[l-[[3-[3-[bis(3-hydroxypropyl)amino]propyl]-5,7-dimethyl-ladamantyl]methyl]-5-methyI-pyrazol-4-yl]pyridme-2-carboxylic acid
332
OH
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 13 and 3-(3-hydroxypropylamino)propan-l-oI as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C44H56N9O5S: 848.4645, found: 848.4645.
Example 177: 6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-57f-pyrido[2,3clpyridazin-8-yl]-3-[l-[[3,5-dimethyl-7-(3-piperazin-l-ylpropyl)-l-adamantylJmethyl]-5methyl-pyrazoI-4-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from Préparation 13 and piperazine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C44H%N9O5S: 801.4387, found: 801.4370.
Example 178: 3-[l-[[3,5-dimethyl-7-(2-pyrrolidin-l-ylethoxy)-l-adamantyI]methyl]-5methyl-pyrazol-4-yl]-6-[3-[(7-fluoro-l,3-benzothiazo]-2-yl)aniino|-4-methyl-6,7-dihydro15 5H-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
333
Step A: methyl 6-(3-/ (7-fluoro-l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydro-5Hpyrido(2>3-c]pyridazin-8-yl]-3-[l-[[3-(2-hydroxyethoxy)-5,7-dimethyl-l-adamantyl]methyl]5-methyl-pyrazol-4-yl]pyridine-2-carboxylate
Using Buchwald General Procedure I at 130 °C for 2 h, starting from 130 mg (0.2 mmol) of the product from Préparation 12, Step C and 52 mg (1.5 eq) of the 7-fluoro-l,3benzothiazol-2-amine, 139 mg (88%) of the desired product was obtained.
1H NMR (500 MHz, dmso-d6) δ ppm 7.95 (d, IH), 7.71 (d, IH), 7.45-7.35 (m, IH), 7.457.35 (br., IH), 7.38 (s, IH), 7.05 (m, IH), 4.46 (br., IH), 4 (t, 2H), 3.88 (s, 2H), 3.71 (s, 3H), 10 3.41 (q, 2H), 3.35 (t, 2H), 2.87 (t, 2H), 2.33 (s, 3H), 2.16 (s, 3H), 1.99 (m, 2H), 1.39 (s, 2H),
1.30/1.25 (d+d, 4H), 1.18/1.12 (d+d, 4H), 1.07/1.00 (d+d, 2H), 0.87 (s, 6H); 13C NMR (500 MHz, dmso-d6) δ ppm 157.1, 140.0, 137.5, 127.7, 119.3, 108.3, 62.1, 61.5, 59.0, 52.7, 50.1, 47.0, 46.0, 45.5, 43.3, 30.2, 24.3, 21.6, 12.5, 10.9; HRMS-ESI (m/z): [M+H]+ calcd for C4iH4sFN8O4S: 767.3482, found: 767.3503.
Step B: methyl 3-[l -[[3,5-dimethyl- 7-[2-(p-tolylsulfonyloxy) ethoxy]-! -adamantyl(methyl]-5methyl-pyrazol-4-yl]-6-[3-((7-fluoro-l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydro5H-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylate
To the product from Step A (130 mg, 0.17 mmol) and triethy lamine (0.071 mL, 3 eq) in DCM (2 mL) was added p-tolylsulfonyl 4-methylbenzenesulfonate (83 mg, 1.5 eq) and the reaction 20 mixture was stirred for 1 h. Purifîcationby column chromatography (silica gel, DCM and
EtOAc as eluents) afforded the desired product (54 mg, 34%).
Ή NMR (500 MHz, dmso-d6) δ ppm 7.96 (d, IH), 7.77 (d, 2H), 7.71 (d, IH), 7.63-7.26 (br., IH), 7.46 (d, 2H), 7.40 (br., IH), 7.39 (s, IH), 7.05 (br., IH), 4.06 (m, 2H), 4.00 (t, 2H), 3.85 (s, 2H), 3.69 (s, 3H), 3.49 (m, 2H), 2.87 (t, 2H), 2.41 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H), 1.99 25 (m, 2H), 1.28 (s, 2H), 1.20-1.06 (m, 4H), 1.20-1.06 (m, 4H), 1.02/0.97 (d+d, 2H), 0.84 (s,
6H); 13C NMR (500 MHz, dmso-d6) δ ppm 140.0, 137.6, 130.6, 128.1, 127.6, 119.3, 108.3,
334
71.5, 58.9, 58.4, 52.6, 49.9, 46.6, 45.9, 45.5, 43.0, 30.1, 24.3, 21.6, 21.6, 12.5, 10.9; HRMSESI (m/z): [M+H]+ calcd for C4iH48FN8O4S: 921.3592, found: 921.3567.
Step C; 3-[l -[[3,5-dimethyl-7-(2-pyrrolidin-l ~ylethoxy)-I-adamantyl]methyl]-5-methylpyrazol-4-yl]-6-[3-[(7-fluoro-l,3-betizolhiazol-2-yl)amino]-4-methyl-6,7-dihydro-5H· pyrido[2,3-c]pyridazin -8-yl]pyridine-2-carboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from the product from Step B and pyrrolidine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C44H53FN9O3S: 806.3976, found; 806.3974.
Example 179: 3-[l-[[3,5-dimethyl-7-(2-pyrroIidin-l-ylethoxy)-l-adamantyl]methyl]-5methyl-pyrazol-4-y]]-6-[4-methyl·3-[(5-methyl·l,3-benzothiazol-2-yl)amiπoJ-6,7dihydro-5H-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
Step A: methyl 3-[l-[[3-(2-hydroxyethoxy)-5,7-dimethyl-l-adamantyl]methyl]-5-methylpyrazol-4-yl]-6-[4-methyl-3-[ (5-methyl-l ,3 -benzothiazol-2-yl)amino]-6,7-dihydro-5Hpyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylate
Using Buchwald General Procedure 1 at 130 “C for 1.5 h, starting from 140 mg (0.22 mmol) of the product from Préparation 12, Step C and 54.3 mg (1.5 eq) of the 5-methyl-l,3benzothiazol-2-amîne, 126 mg (75%) of the desired product was obtained.
Ή NMR (500 MHz, dmso-d6) δ ppm 12.08/10.89 (brs/brs, 1H), 7.95 (d, 1H), 7.69 (d, III), 7.67 (br, 1H), 7.38 (s, 1H), 7.30 (br, 1H), 7.00 (d, 1H), 4.46 (brs, 1H), 4.00 (t, 2H), 3.88 (s, 2H), 3.70 (s, 3H), 3.41 (t, 2H), 3.35 (t, 2H), 2.85 (t, 2H), 2.39 (s, 3H), 2.32 (s, 3H), 2.16 (s, 3H), 1.98 (qn, 2H), 1.39 (s, 2H), 1.30/1.25 (d+d, 4H), 1.18/1.12 (d+d, 4H), 1.08/1.02 (d+d, 2H), 0.87 (s, 6H); 13C NMR (500 MHz, dmso-d6) δ ppm 139.8, 137.5, 123.6, 121.6, 119.0, 62.1, 61.5, 59.0, 52.7, 50.1, 47.0, 46.0, 45.4, 43.3, 30.2, 24.3, 21.7, 21.6, 12.6, 10.9; HRMSESI (m/z): [M+H]+ calcd for C42H5jN8O4S: 763.3760, found: 763.3754.
335 φ Step B: methyl 3-[1 -/[3,5-dimethyl-7-[2-(p-tolylsulfonyloxy)ethoxy]-l-adamanty l]methyl]-5methyl-pyrazol-4-yl]-6-[4-methyl-3-[(5-methyl-l,3-benzothiazol-2-yl)amino]-6,7-dihydroSH-pyrido[2,3-c]pyridazin -8-yl]pyridine-2-carboxylate
To the product from Step A (119 mg, 0.16 mmol) and triethy lamine (0.066 mL, 3 eq) in DCM 5 (2 mL) was added p-tolylsulfonyl 4-methylbenzenesulfonate (76 mg, 1.5 eq) and the reaction mixture was stirred for 1 h. Purificationby column chromatography (silica gel, DCM and EtOAc as eiuents) afforded the desired product (93 mg, 65%).
Πΐ NMR (500 MHz, dmso-d6) δ ppm 12.17/10.83 (brs/brs, 1H), 7.95 (d, 1H), 7.77 (d, 2H), 7.7 (d, 1H), 7.69 (br, 1H), 7.46 (d, 2H), 7.42 (br, 1H), 7.39 (s, 1H), 7.00 (d, 1H), 4.07 (t, 2H), 10 4 (t, 2H), 3.96 (s, 3H), 3.85 (s, 2H), 3.49 (t, 2H), 2.85 (t, 2H), 2.40 (s, 3H), 2.39 (s, 3H), 2.32 (s, 3H), 2.15 (s, 3H), 1.99 (qn, 2H), 1.29 (s, 2H), 1.17/1.1 (d+d, 4H), 1.12/1.1 (d+d, 4H), 1.02/0.97 (d+d, 2H), 0.84 (s, 6H); I3C NMR (500 MHz, dmso-d6) δ ppm 139.8, 137.6, 130.6, 128.1, 123.6, 119.0, 71.5, 58.8, 58.4, 52.7, 49.9, 46.6, 45.9, 45.4, 43.0, 30.1, 24.3, 21.6, 21.6, 21.6, 12.6, 10.9; HRMS-ESI (m/z): [M+H]+ calcd for C49H57N8O6S2: 917.3842, found: 15 917.3840.
Step C: 3-[1 -][3,5-dimethyl- 7-(2-pyrrolidin-l -ylethoxy)-! -adamantyl]methyl]-5-methylpyrazol-4-yl]~6-[4-methyl-3-[(5-methyl-l,3-benzothiazol-2-yl)amino]-6,7-dihydro-5H pyrido]2,3-c]pyridazin-8-yl]pyridine‘2-earboxylic acid
Using the Amine substitution and Hydrolysis General procedure starting from the product 20 from Step B and pyrrolidine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C45H56N9O3S: 802.4227, found: 802.4220.
Example 180: 3-[l-[[3,5-dimethyl-7-(2-pyrroïidin-l-ylethoxy)-l-adamantyl]methyl]-5mι+hyl-pyrazol·4-yl|-6-|3-|(5-Inetlwxy-L3-benz.(>thillzol-2-yl)aInino]--4-meίllyl-6,7dihydro-57f-pyrido[2,3-e]pyridazin-8-yl]pyridine-2-carboxylic acid
336
Step A; methyl 3-[l-[[3-(2-hydroxyethoxy)-5,7-dimethyl-l-adamantyl]methyl]-5-methylpyrazol-4-yl]-6-[3-[(5-methoxy-l ,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydro-5l1pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylate
Using Buchwald General Procedure I at 130 °C for 2.5 h, starting from 140 mg (0.22 5 mmol) of the product from Préparation 12, Step C and 60 mg (1.5 eq) of the 5-methyl-1,3benzothiazol-2-amine, 129 mg (75%) of the desired product was obtained.
*H NMR (500 MHz, dmso-d6) δ ppm 7.95 (d, IH), 7.69 (d, IH), 7.67 (br., IH), 7.38 (s, IH), 7.02 (br., IH), 6.80 (dd, IH), 4.46 (br., IH), 4.00 (t, 2H), 3.88 (s, 2H), 3.80 (s, 3H), 3.70 (s, 3H), 3.41 (t, 2H), 3.35 (t, 2H), 2.85 (t, 2H), 2.32 (s, 3H), 2.16 (s, 3H), 1.98 (m, 2H), 1.39 (s, 10 2H), 1.30/1.25 (d+d, 4H), 1.18/1.12 (d+d, 4H), 1.08/1 (d+d, 2H), 0.87 (s, 6H); 13C NMR (500
MHz, dmso-d6) δ ppm 139.8, 137.5, 122.6, 119.0, 110.5, 62.1, 61.5, 58.9, 55.8, 52.6, 50.1, 47.0, 46.0, 45.4, 43.3, 30.2, 24.3, 21.7, 12.6, 10.9; HRMS-ESI (m/z): [M+H]+ calcd for C42H51N8O5S; 779.3703, found: 779.3687.
Step B: methyl 3-[l -[[3,5-dimethyl-7-[2-(p-tolylsulfonyloxy) ethoxy]-! -adamantyl]methyl]-515 methyl-pyrazol-4-yl]-6-[3-[(5-methoxy-l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydro5H-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylate
To the product from Step A (122 mg, 0.16 mmol) and triethylamine (0.066 mL, 3 eq) in DCM (2 mL) was added p-tolylsulfonyl 4-methylbenzenesulfonate (77 mg, 1.5 eq) and the reaction mixture was stirred for 1 h. Purificationby column chromatography (silica gel, DCM and 20 EtOAc as eluents) afforded the desired product (79 mg, 54%).
XH NMR (500 MHz, dmso-d6) δ ppm 12.17/10.83 (brs/brs, IH), 7.95 (d, IH), 7.77 (d, 2H), 7.72 (d, IH), 7.67 (brd, IH), 7.46 (d, 2H), 7.39 (s, IH), 7.02 (br, IH), 6.80 (d, IH), 4.07 (t, 2H), 4.00 (t, 2H), 3.86 (s, 2H), 3.80 (s, 3H), 3.69 (s, 3H), 3.49 (t, 2H), 2.86 (t, 2H), 2.41 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H), 1.99 (qn, 2H), 1.29 (s, 2H), 1.17/1.1 (d+d, 4H), 1.12/1.10 25 (d+d, 4H), 1.02/0.97 (d+d, 2H), 0.84 (s, 6H); 13C NMR (500 MHz, dmso-d6) δ ppm 139.9,
137.6, 130.6, 128,1, 119.0, 110.6, 71.5, 58.8, 58.4, 55.9, 52.6, 49.9, 46.6, 45.9, 45.8, 43.0, 30.1, 24.3, 21.6, 21.6, 12.7, 10.9; HRMS-ESI (m/z): [M+H]+ calcd for C49H57N8O7S2: 933.3792, found: 933.3794.
Step C: 3-[1 -U 3,5-di methyl- 7-(2-pyrrolidin-l -ylethoxy) -1 -adamantyl] methyl]-5-methyl30 pyrazol-4-yl]-6-[3-[(5-methoxy-l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydro-5Hpyrido[2,3-c]pyridazin -8-yl]pyridine-2-carboxylic acid
337
Using the Amine substitution and Hydrolysis General procedure startîng from the product from Step B and pyrrolidine as the appropriate amine, the desired product was obtained.
HRMS-ESI (m/z): [M+H]+ calcd for C45H57N9O4S: 818.4176, found: 818.4172.
The compounds of the foliowing Examples 181-219 are synthesised using the Amine 5 Substitution and Hydrolysis General procedure startîng from one ofthe Préparation 12, 13,14 or analogs benzothîazole dérivatives and the appropriate amine.
Example 181: 3-[l-[[3,5-dimethyl-7-[2-(methylamino)ethoxy]-l-adamantyl]methyl]-5methyl-pyrazol-4-yl]-6-[3-[(7-fluoro-l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydro5//-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
Example 182: 3-[l-[[3-[2-(dimethylamino)ethoxy]-5,7-dimethyl-l-adamantyl]methyll-5methyl·pyrazol-4-yl]-6-[3-[(7-fluoro-l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydro5//-pyrido[2,3’t?]pyridazin-8-yl]pyridine-2-carboxylic acid
338
Example 183: 3-[l-[[3,5-dimethyl-7-[2-(4-methylpiperazin-l-yl)ethoxy]-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]-6-[3-[(7-fluoro-l,3-benzothiazol-2-yI)amino]4-methyl-6,7-dihydro-5/f-pyrido[2;i3-c]pyridazin-8-yI]pyridine-2-carboxylÎc acid
Example 184: 6-[3-[(7-fluoro-l,3-benzothiazoI-2-yl)amino]-4-methyl-6,7-dihydro-5Hpyrido[2,3-c]pyridazin-8-yl]-3-[l-[[3-[2-(3-hydroxypropylamino)ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Example 185: 6-[3-[(7-fluoro-l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydro-51ï10 pyrido[2,3-c]pyridazin-8-ylJ-3-[l-[[3-[2-(4-hydroxybutylamino)ethoxy]-5,7-diniethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxyIic acid
339
Example 186: 3-[l-[[3-[2-[[(3R)-3,4-dihydroxybutyl]amino]ethoxy]-5,7-dimethyl-ladainantylJinethyll-S-methyl-pyrazol'M-yll-ô-tS-KV-fluoro-ljS-benzothiazol-Z-yOaniino]· 4-methyl-6)7-dihydro-5ff-pyrido[2,3-c]pyridazin-8-yI]pyridine-2-carboxylic acid
Example 187: a-El-t^-^-ttiSSj-S^-dihydroxybutyllaniinolethoxyl-S^-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]-6-[3-[(7-fluoro-l>3-benzothiazol-2-yl)amino]4-methyl-6,7-dihydro-5H-pyrido[2i3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
340
Exampie 188: 3-[l-[[3,5-dimethyl-7-(2-piperazin-l-ylethoxy)-l-adamantyl]methyl]-5methyl-pyrazol-4-yl]-6-[3-[(7-fluoro-l,3-benzothiazol-2-yl)amino]-4-methyl-6J7-dihydi'o57f-pyrido[2.,3-c]pyridazin-8-ylJpyrîdine-2-carboxylic acid
Exampie 189: 3-[l-[[3,5-dimethyl-7-(2-morpholinoetlioxy)-l-adamantyl]methyl]-5methyl-pyrazol-4-yl]-6-[3-[(7-fluoro-l:(3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydro51/-pyrido[2,3-c]pyrÎdazin-8-yl]pyridine-2-carboxylic acid
Example 190: 6-[3-[(7-fluoro-l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydro-5/f10 pyrido[2,3-c]pyridazin-8-yl]-3-[l-[[3-[2-[3-hydroxypropyl(methyl)amino]ethoxy]-5,7dimethyl-l-adamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
341
Example 191: 6-^3-[(7-fluoro-l,3-benzothίazol·2-yl)amino]-4-methyl-6,7-dihydro-5J7py rido [2,3-c]pyridazin-8-y 1]-3-[1-[[3-[2-[4-liydroxybutyl (methyl) amino] ethoxy]-5,7dimethyl-l-adamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid
Example 192: 6-[3-[(7-fluoro-l,3-benzothiazoi-2-yl)amino]-4-methyl-6,7-dihydro-5//pyrido[2,3-c]pyridazin-8-yl]-3-[l-[[3-[2-[[3-hydroxy-2(hydroxymethyl)propyl]amino]ethoxy]-5,7-dimethyl-l-adamantyl]methyl]-5-methylpyrazol-4-yl|pyridine-2-carboxylic acid
342
Exampie 193: S-ll-dS-P-nbisfS-hydroxypropyOaminoJethoxyl-SJ-dimethyl-ladamantyI]methyl]-5-methyl-pyrazoI-4-yl]-6-[3-[(7-fluoro-l,3-benzothiazol-2-yI)amino]4-methyl-6,7-dihydro-5//-pyrido[213-c]pyridazin-8-yl]pyridine-2-carboxylic acid
Example 194: 3-[l-[[3,5-dimethyl-7-[2-(methylamino)ethoxy]-l-adamantyl]methyl]-5methyl-pyrazol-4-yl]-6-[4-methyl-3-[(5-methyl-l,3-benzothiazol-2-yl)amino]-6,7dihydro-5ff-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
Example 195: 3-[l-[[3-[2-(dimethylamino)etlioxy]-5,7-dimethyl-l-adamantyl]methyl]-5 methy!-pyrazol-4-yl]-6-[4-methyl-3-[(5-methyl-l,3-benzothiazol-2-yl)amino]-6,7dihydro-5i/-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
343
Example 196: 3-[l-[[3,5-dimethyl-7-[2-(4-methylpiperazin-l-yl)ethoxy]-ladamantyllmethyll-S-methyl-pyrazolU-yll-ô-^-methyl-S-EtS-methyl-l^-benzothiazol-lyl)amiiio]-6,7-dihydro-5/Z-pyrido(2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
Example 197: 3-[l-[[3-[2-(3-hydroxypropylamino)ethoxy]-5,7-diinethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]-6-[4-methyl-3-[(5-methyl-l,3-benzothiazol-2yl)amino]-6/7-dÎhydro-5//-pyrido[2,3-c]pyridazm-8-yl]pyridme-2-carboxyIic acid
344 φ Exampie________198: 3-(l-[[3-[2-(4-hydroxybutylamino)ethoxy]-5,7-dimetliyMadamantyl]methyl]-5-methyl-pyrazol-4-yl]-6-[4-methyl-3-[(5-methyl-l,3-benzothÎazol-2yl)amino]-6,7-dihydro-5/Î-pyrido[2,3-c]pyridazin-8-yl]pyndine-2-carboxyIic acid
Exampie 199: 3-[l-[[3-[2-[[(31ï)-3,4-dihydroxybutyl]amino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]-6-[4-methyl-3-[(5-methyl-l,3-benzothiazol-2' yl)amino]-6,7-dihydro-5//-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
Example 200: 3-[l-[[3-[2-[[(3S)-3,4-dihydroxybutyl]amino]ethoxy]-5,7-dimethyl-l10 adamantyljmethylJ-5-methyl-pyrazol-4-yl]-6-[4-methyl-3-[(5-methyl-l,3-benzothiazol-2yl)amino]-6,7-dihydro-51/-pyrido[2,3-c]pyridaziii-8-yl]pyridine-2-carboxylic acid
345
Exampie 201; 3-[l-[[3,5-dimethyl-7-(2-piperazin-l-ylethoxy)-l-adamantyl]methyl]-5methyl·pyrazol·4’yl]-6-[4-methyl-3-[(5-methyl·l,3-benzothiazol-2-yI)amino]-6,7dihydro-5/7-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
Example 202: 3-[l-[[3,5-dimethyl-7-(2-morpholinoethoxy)-l-adamantyllmethyl]-5methyl-pyrazol-4-yl]-6-[4-methyl-3-[(5-methyl-l,3‘benzothiazol-2-yl)amino]-6,7dihydro-5/f-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
346
Example 203: 3-[l-[[3-[2-[3-hydroxypropyl(methyl)aminoJethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]-6-[4-methyl-3-[(5-methyM,3-benzothiazoI-2yl)amino]-6,7-dihydro-5H-pyrido[2,3-c|pyridazin-8-ylJpyridine-2-carboxylic acid
Example 204: 3-[l-[[3-[2-[4-hydroxybutyl(methyl)amino]ethoxy]-5,7-dimethyl-ladamantyl]methyI]-5-methyl-pyrazol-4-yl]-6-[4-methyl-3-[(5-methyl-l,3-benzothiazol-2ybaminol-ô^-dihydro-S/f-pyridotZjS-clpyridazin-S-ylJpyridine-Z-carboxylic acid
Example 205: 3-[l-[[3-[2-[[3-hydroxy-2-(hydroxymethyl)propyl]amino]ethoxy]-5/710 dimetllyl-l-adamantyl]methyl]-5-methyl-pyrazol-4-yl]-6-[4-methyl·3-[(5-methyl-l,3benzothiazol-2-yl)amino]-6,7-dihydro-5/f-pyrido[2,3-c]pyridazin-8-yl]pyridine-2carboxylic acid
347
Example 206: 3-[l-[[3-[2-[bis(3-hydroxypropyl)amino]ethoxy]-5,7-dimethyl-ladainantyl]methyl]-5-methyl-pyrazol-4-yl]-6-[4-methyl-3-[(5-methyl-l,3-benzothiazol-2· yl)amino]-6,7-dihydro-57/-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
Example 207: 3-[l-[[3,5-dimethyl-7-[2-(methylamino)ethoxy]-l-adamantyl]methyI]-5methyl-pyrazol-4-yl]-6-[3-[(5-methoxy-l,3-benzothiazol-2-yI)amino]-4-methyl-6,7dihydiO-5//-pyrido[2,3-c]pyndazin-8-yl]pyridme-2-carboxylic acid
348 φ Exampie 208: 3-[l-[[3-[2-(dimethylamino)ethoxyJ-5,7-dimethyl-l-adamantyl]methyl]-5methyi-pyrazol-4-yl]-6-[3-[(5-methoxy-l,3-benzothiazol-2-yl)amino]-4-methyl-6,7dihydro-5/7-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
O —
Exampie 209: 3-[l-[[3,5-dimethyl-7-[2-(4-methylpiperazin-l-yl)ethoxy]-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]-6-[3-[(5-methoxy-l,3-benzüthiaz(>l-2yl)ainino]-4-methyl-6,7-dihydrO5H-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
Example 210: 3-[l-[[3-[2-(3-hydroxypropylamino)ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-ylJ-6-[3-|(5-methoxy-l,3-benzothiazol-2yl)amino]-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
349
Example 211: 3-[l-[l3-[2-(4-hydroxybutylamino)ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yll-6-[3-[(5-methoxy-l,3-benzothiazol-2yl)amino]-4-methyl-6,7-dihydro-5f/-pyrido[2,3-c]pyridazin-8-yl]pyridine'2-carboxylic acid
Example 212: 3-[l-[[3-[2-[[(31f)-3,4-dihydroxybutyl]amino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl‘pyrazol-4-yl]-6-[3-[(5-methoxy-l,3-benzothiazol-2yl)amino]-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yI]pyridine-2-carboxylic 10 acid
350
Example 213: 3-[l-[[3-[2-[[(3S)-3,4-dihydroxybutyl]amino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]-6-[3-[(5-methoxy-l>3-benzothiazol-2yl)amino]-4-methyl-6,7-dihydro-5/f-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic 5 acid
Example 214: 3-[l-[[3,5-dimethyl-7-(2-piperazm-l-ylethoxy)-l-adamantyl]inethyl]-5methyl-pyrazol-4-yl]-6-[3-[(5-methoxy-l,3-benzoihiazol-2-yl)amino]-4-methyl-6,7dihydro-5/7-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
351
Example 215: 3-[l-[[3f5-dimethyl-7-(2-morpholinoethoxy)-l-adamantyl]methy!]-5methyl-pyi'azol-4-yI]-6-[3-[(5-methoxy-l,3-benzothiazol-2-yl)amino]-4-methyl-6,7dihydro-5H-pyrido[2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid
Example 216: 3-[l-[[3-12-[3-hydroxypropyI(methyl)amino]ethoxy]-5,7-dimethyl-ladamantyllmethyl]-5-metllyl-pyrazol-4-yl]-6-[3·[(5-meth()xy'-l,3-benzothiazol·2yl)amino]-4-methyl·6,7-dihydro-5H-pyrido[2,3-c]pyridazin-8-yl]ρyridine-2-carboxylic acid
352
Example 217: 3-[l-E(3-[2-[4-hydroxybutyl(methyl)amino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yll-6-[3-[(5-methoxy-l,3-benzothiazol-2yl)ammo]-4-methyl-6,7-dihydro-51T-pyrÎdo[2,3-c]pyridazm-8-yllpyridine-2-carboxylic 5 acid
O—
Example 218: 3-[l-[[3-[2-[[3-hydroxy-2-(hydroxymethyl)propyl]amino]ethoxy]-5,7dimethyl-l-adamantyl]methyl]-5-methyl·pyrazol-4-yll-6-[3-[(5-methoxy-l,3benzothiazol-2-yl)amino]-4-methyl-6/7-dihydro-57î-pyrido[2,3-c]pyridazin-810 yl]pyridine-2-carboxylic acid
353
Example 219: 3-[l-[[3-[2-[bis(3-hydroxypropyl)aminolethoxy]-5,7-dimethyl-ladamantyllmethyll-S-methyl-pyrazol^-yll-ô-P-KS-methoxy-l^-benzothiazol^yl)aininoP4-mcthvl-6.7-dihv(liX)-5/Fpyrido|2,3-e|pyHdazm-8-yl|pvridine-2-carboxylic 5 acid
Example 220: 5-[3-[4-[3-(dimethylamino)prop-l-ynyl]-2-nuoro-phenoxy]propyl]-2-[4inethyl-3-[(5-methyl-l,3-benzothiazol-2-yl)amino]-6,7-dihydro-5H-pyrido[2,3~ c]pyridazin-8-yl]thiazole-4-carboxylic acid
354
Example 221: 5-[3-[4-[3-(dimethylamino)prop-l-ynyl]-2-tluoro-phenoxylpropyl]-2-[3[(5-methoxy-l,3-benzotliiazol-2-yl)aminoJ-4-methyl-6,7-dihydro-5//-pyrido[2,3c]pyridazin-8-yl]thiazole-4-carboxylic acid
355 φ PHARMACOLOGICAL STUDY
EXAMPLE A: Fluorescence Polarisation Assay Data
Fluorescence polarization measures the rotation of a fluorescing species in solution, the larger the molécule the more polarized the fluorescence émission.
The fluorescent PUMA (UniProtKB® primary accession number Q9BXH1 — SEQ ID:01) based probe Fluorescein-betaAla-Ahx-AREIGAQLRRMADDLNAQY-OH from Biopeptides binds to GST(1-218)-ÎFACTOR XAPhsBCLXL(2-209) having an aminoacid sequence (SEQ
ID:02):
[MSPILGYWK1KGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPY 10 Y1DGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDF
ETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTFIPDFMLYDALDVVLYMDPMCL DAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLIEGRGIPE FEFSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEMETPSAINGNPS WHLADSPAVNGATGHSSSLDAREV1PMÂAVKQALREAGDEFELRYRRAFSDLTSQL 15 hitpgtayqsfeovvnelfrdgvnwgrivaffsfggalcvesvdkemqvlvsriaaw
MATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQER] (GST UniProtKB® primary accession number P08515 and BCLXL UniProtKB® primary accession number Q07817-1), resulting în an increase in anisotropy. If a compound is added which competitively binds to 20 the same site as the probe, thereby releasing it, anisotropy decreases due to the increased amount of free probe.
An 11-point serial dilution of each compound was prepared in DMSO, the final buffer conditions were 10 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid [HEPES], 150 mM NaCI, 0.05% Tween 20, pH 7.4 and 5% DMSO. The final protein concentration in the 25 assay was 20 nM with the fluorescent probe present at 10 nM. The experiments were incubated for 2 hours at 23 °C before fluorescence polarization was measured on a Biotek SynergyNeo plate reader (Excitation 485nm, émission 525nm, parallel and perpendicular reads). The dose response curves were plotted with XL-Fit software using a 4-Parameter Logistic Model (Sigmoidal DoseResponse Model) and the inhibitory concentrations that gave 30 a 50% increase in fluorescence intensîty was determined (IC50). The Ki values were
356 determined from the IC'su values according to Cer et al, Nucleic Acids Res, 2009, Jul l;37(WebServer issue): W441-W445.
The results are summarised in Table 1. They show that the compounds of the invention inhibit the interaction between the Bcl-xL protein and the fluorescent peptide described hereinbefore.
Table 1
Example Ki(M) 12 3.0E-08 24 < 1E-09
1 3.7E-05 13 9.2E-09 25 1.2E-07
2 > 1E-05 14 5.5E-09 26 1.5E-05
3 3.6E-05 15 2.3E-09 27 < 1E-09
4 > 1E-05 16 1.2E-07 28 1.4E-08
5 > 1E-05 17 2.0E-09 29 9.6E-09
6 > 1E-05 18 5.0E-06 30 < 1E-09
7 9.9E-06 19 3.3E-06 31 < 1E-09
8 1.1E-06 20 8.5E-08 32 < 1E-09
9 1.3E-08 21 1.6E-06 33 5.5E-09
10 2.1E-07 22 3.0E-09 35 < 1E-09
11 9.2E-09 23 1.1E-09 36 < 1E-09
357
EXAMPLE B: Quench Assay Data
Fluorescence quenching assay measures the change rn fluorescence intensity of C-terminally Cy5-labeIIed BCL-xL protein, His-His-(EK)-/rtBÇLXL(2-197)[N197C] (UniProtKB® primary accession number Q07817-1) having an amino acid sequence (SEQ ID:03):
[MHHHHHHHHG ATG STAGSGTAGSTG ASG ASTGGTGATH H H HHHHHD D DD KS PM G SQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEMETPSAINGNPSWH LADSPAVNGATGHSSSLDAREVIPMAAVKQALREAGDEFELRYRRAFSDLTSQLHITP GTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMAT YLNDHLEPWIQENGGWDTFVELYG] which is linked in C-termînal région to the amino acid X which corresponds to a cysteine labelled on the sulfur with Sulfo-Cyanine5 from Lumiprobe GmbH catalogue number 13380, upon binding of a C-terminally labelled peptide derived from PUMA (UniProtKB® primary accession number Q9BXH1) having an aminoacid sequence (SEQ ID:04): [QWAREIGAQLRRMADDLNAQY] which is linked in C-terminal région to the amino acid X’ where X’ is cysteine labelled on the sulfur wîth TQ5WS from AAT Bioquest catalogue number 2079.
The addition of a compound which binds competitively to the same site as the peptide will resuit in an increase in the fluorescence intensity of the protein due to displacement of the fluorescence quencher.
An 11-point serial dilution of each compound was prepared in DMSO, the final buffer conditions were 10 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid [HEPES], 150 mM NaCl, 0.05% Tween 20, pH 7.4 and 5% DMSO. The final protein concentration in the assay was 1 nM with the peptide présent at 400 nM. The experiments were incubated for 2 hours at 23°C before fluorescence intensity was measured on a Biotek SynergyNeo plate reader (Excitation 620nm, émission 680nm). The dose response curves were plotted with XLFit software using a 4-Parameter Logistic Model (Sigmoidal DoseResponse Model) and the inhibitory concentrations that gave a 50% increase in fluorescence intensity was determined (IC50). The Ki values were determined from the IC50 values according to Cer et al, Nucleic Acids Res, 2009, Jul l;37(WebServer issue): W441-W445.
The results are summarised in Table 2.
358
Table 2
Example Ki (M)
22 2.5E-11
23 5.1E-11
24 8.0E-12
27 8.5E-12
30 3.6E-12
31 8.2E-12
32 4.6E-12
33 1.4E-09
34 1.9E-12
35 7.7E-12
36 1.7E-12
37 7.7E-12
38 2.9E-12
39 9.8E-12
40 5.7E-11
41 2.5E-12
42 2.8E-12
43 3.1E-12
44 1.5E-12
45 4.9E-12
46 1.5E-11
47 8.9E-12
48 1.6E-12
49 6.4E-11
50 7.1E-11
51 1.2E-11
52 1.3E-12
53 6.6E-12
54 1.2E42
55 9.2E-12
56 4.9E-11
57 3.0E-12
58 4.8E-12
59 2.3Ε-Γ1
60 8.9E-12
61 3.3E-11
62 3.4E-12
63 8.1E-11
64 5.2E-12
65 5.0E-12
66 1.2E-11
67 3.2E-12
68 3.8E-12
69 3.7E-12
70 1.7E-11
71 4.5 E-12
72 8.0E-11
73 2.5E-12
74 1.0E-11
75 3.3E-12
76 1.8E-1.2
77 1.7E-1Ü
78 2.3E-11
79 5.2E-12
80 6.5E-12
81 1.5E-11
82 2.3E-11
83 2.6E-I1
84 5.3E-11
85 4.4E-11
86 1.9E-11
87 4.4E-11
359
88 l.OE-11
89 2.8E-12
90 2.2E-12
91 5.7E-12
92 1.7E-12
93 2.4E-12
94 1.5E-12
95 5.6E-12
96 2.2E-12
102 4.5E-12
125 6.7E-12
126 6.7E-11
127 2.1E-12
128 1.8E-12
129 4.7E-12
130 2.3E-12
131 4.5E-12
132 2.0E-12
133 2.1E-12
134 2.8E-12
135 1.8E-12
136 1.4E-12
137 9.9E-13
138 7.1E-13
139 2.0E-11
140 3.2E-12
141 8.5E-12
142 2.6E-12
143 1.4E-12
144 1.4E-12
145 1.8E-12
146 3.2E-12
147 4.6E-12
148 5.9E-12
149 3.7E-12
150 1.5E-12
151 1.5E-12
152 1.7E-12
153 1.2E-12
154 #N/A |
155 #N/A
156 #N/A
157 #N/A
158 #N/A
159 #N/A
160 #N/A
161 #N/A
162 #N/A
163 #N/A
164 #N/A
165 #N/A
166 2.7E-12
167 1.9E-12
168 2.9E-12
169 #N/A
170 2.7E-12
171 #N/A
172 2.0E-12
173 #N/A
174 #N/A
175 #N/A
176 #N/A
177 #N/A
178 2.2E-12
179 2.9E-12
180 1.2E-12 ~ _ -----
360
The results of Tables 1 and 2 show that most of the compounds of the invention are potent inhibitors of the Bcl-xL protein.
EXAMPLE C: Effect of Bcl-xL inhibitors in MOLT-4 or H146 cell viability using MTT assay
MTT colorimétrie assay is based on the mitochondrial réduction of tétrazolium sait by living cells. The viable cell number is proportîonal to the production of formazan salts, which can be read spectrophotomelrically at 540 nm.
MOLT-4 and H146 cells were purchased from ATCC and cultivated in RPMI 1640 supplemented with 10% heat inactivated fêtai bovine sérum, penicillin (100 lU/ml), 10 streptomycin (100 pg/ml) and L-glutamine (2 mM). Cells were cultured at 37°C in a humidified atmosphère containing 5% CO2.. Cells were seeded in 96 microwell plates (150pL per well) and exposed to the compounds for 48h (3,16 fold serially diluted; 9 concentrations each, triplicates). At the end of incubation time, 15 pL of MTT solution (5mg/ml) were added per well and the cells were incubated for another 4h. Then, 100 pL of 10% Sodium Dodecyl 15 Sulfate (SDS)/HC1 lOmM were added per well and the plate was incubated overnight, before measurement of optical density at 540 nm. IC50S were calculated using standard fourparametric curve fitting. IC50 is defined as the compound concentration at which the MTT signal is reduced to 50% of that measured for the control. Results represent the mean of at least 2 independent experiments and are presented in Table 3 below.
361
Table 3
Example H146 IQo (M) MOLT-4 IC50 (M)
9 2.34E-06 #N/A
10 >1.5E-05 >1.5E-05
11 6.36E-06 2.43E-06
13 7.9E-07 1.26E-07
14 3.08E-06 4.53E-07
15 2.6E-06 1.05E-06
17 3.93E-07 1.28E-07
20 3.1ΕΌ6 1.62ΕΌ7
21 >1.5E-05 4.23E-06
22 1.27E-08 3.42E-09
23 2.77E-09 9.48E-10
24 3,27 E-08 4,85E-09
26 >1.5E-05 >=1.34E-05
27 6,53E-O9 l,46E-09
28 3,14E-06 8,80E-07
30 6.55E-09 7.75E-10
31 8.82E-08 1.48E-08
32 #N/A 9.87E-10
35 6.28E-08 3.02E-08
36 5.51E-09 #N/A
37 1.02E-08 4.61E-09
38 2.21E-08 5.94E-08
39 3.36E-08 3.71E-08
40 2.08E-08 8.16E-09
41 3.67E-09 3.6E-10
42 3.02ΕΌ9 3.27E-10
43 1.17E-08 1.71E-09
44 1.07E-08 4.04E-09
45 1.06E-08 5.51E-1O
46 1.11E-08 1.2E-O9
47 7.31E-09 9.57E-10
48 2.25E-09 3.09E-10
49 3.34E-08 2.13E-09
50 8.77E-08 3.71E-09
51 9.6E-09 2.54E-09
52 1.07E-08 2.42E-09
53 __ 1.49E-08 9.74E-09
362
54 5.95 E-07 4.72E-08
55 5.65E-09 9.49E-1O
56 1.2 9 E-08 1.46E-09
57 5.54E-09 5.92E-10
58 6.65E-09 8.17E-10
59 1.73E-08 3.36E-09
60 8.86 E-09 2.75E-09
61 1.2E-07 1.22E-07
62 6.9E-08 2.O2E-O8
63 2.02E-08 #N/A
64 1.18E-08 3.05E-08
65 3.98 E-09 2.66E-08
66 8.22E-09 2.01E-09
67 1.46E-08 7.4E-09
68 1.04E-08 2.91E-09
69 #N/A 4.11E-09
70 1.69 E-08 3.08E-09
71 9.3E-09 2.11E-09
72 #N/A 1.39E-08
73 1.47E-08 6.54E-09
75 #N/A 9.78E-09
76 #N/A 7.4E-10
77 #N/A 2.26E-O7
78 #N/A 2.46E-09
79 #N/A 1.01E-09
80 #N/A 5.75E-10
81 #N/A 1.75E-10
82 #N/A 1.21E-09
83 #N/A 5.73E-10
84 #N/A 1.83E-09
85 #N/A 4.16E-10
86 #N/A 1.5E-O9
88 #N/A 1.81E-10
89 #N/A 5.7E-10
90 #N/A 4.6E-10
91 #N/A 3.59E-10
These data show that the majority of these compounds are active in cells and can induce a dose dépendent decrease in the viability of H146 and MoIt-4 cell lines.
363
Example MOLT-4 IC50 (M)
92 4.58E-09
93 1.42E-08
94 6.16E-10
95 2.05E-10
96 8.27E-09
102 2.68E-10
125 9.53 E-09
126 5.33E-08
127 1.37E-10
128 2.84E-10
129 3.11E-09
130 1.94E-09
131 2.75E-10
132 1.32E-09
133 1.68E-O9
134 1.57E-09
Example MOLT-4 ICS0(M)
135 3.05E-09
136 1.25E-09
137 5.78E-09
138 1.01E-09
139 9.95E-10
140 2.85E-09
141 9.8E-11
142 5.13E-11
143 5.46E-11
144 1.32E-08
145 2.65E-1O
146 2.47E-10
147 8.52E-10
148 2.77E-1O
149 9.82E-10
150 2.19E-10
Example MOLT-4 IC50 (M)
151 8.24E-11
152 #N/A
153 2.29E-10
154 4.49E-10
155 5.43E-10
160 >1.0E-07
166 4.29E-10
167 5.32E-11
168 2.91E-10
170 7.87E-10
172 3.25E-09
178 6.41E-10
179 9.16E-11
180 1.3E-10
364
EXAMPLE D: Pharmacodynamies and tumor régression study
The in vivo therapeutic and pharmacodynamie effects of Bcl-xL-targeting small molécules were determined in MOLT-4 T-cell Acute Lymphoblastic Leukemia (T-ALL) model upon intravenous (IV) or oral (PO) administration.
Materials and methods
MOLT-4 cells (ATCC No. CRL-1582) were cultured in RPMI supplemented with 10% FBS.
Cells were re-suspended in 50% matrigel (BD Biosciences) and 0.1 mL containing 5xl06 cells was subcutaneously inoculated into the right flank of female NOD SCID mîce (Charles River). For efficacy studies, when tumors reached the appropriate volume, mice were 10 randomized (7 animais per group) using Easy stat software. Control vehicle (HPBCD/HC1) or
Example 24 (2.5, 5 or 7.5 mg/kg) were injected IV (twice weekly for 3 weeks - O3D6). Mice body weight was monitored three times a week and tumor size was measured using electronic calipers. Tumor volume was eslimated by measuring the minimum and maximum tumor diameters using the formula: (minimum diameter)2(maximum diameter)/2. The last day with 15 at least half of control animais still présent in the study (day 31), tumor growth inhibition was calculated using the formula:
Médian (TV at Dx in treated group) ) χ 10θ Médian (TV at Dx in Control group) )
Response was evaluated as foliows; CR (Complété Response) if tumor size was <25mm3 for at least three consecutive measurements, PR (Partial Response) if tumor size was comprised between 25mm3 and half of the starting size for at least three consecutive measurements. 20 Mice were sacrificed at the first measurement for which tumor volume exceeded 2000 mm3 or at the first signs of animal health détérioration.
For pharmacodynamies studies, when tumors reached the appropriate volume, mice were randomized (3 animais per group) using Easy stat software. Example 24 (7.5 mg/kg) was dosed IV (once per day - QD) in HPBCD/HC1 or PO (once per day - QD) in 25 PEG3()0/EtOH/Phosal (30/10/60). Tumor samples were collected 6 h after dosing and lysed (10 mM HEPES pH 7.4, 142.5 mM KC1, 5 mM MgCh, 1 mM EDTA, 1% NP40, protease and phosphatase inhibitors cocktails - Calbiochem). Cleared lysâtes were prepared for immunodetection of cleaved PARP and Caspase 3 by using the MSD apoptosis panel whole
365 cell lysale kit (MSD) in 96-well plates according to manufacturer’s instructions, and were analyzed on the OuickPlex SQ 120. Whole blood samples were analyzed on the Hematology Analyzer Coulter Ac*T diff (Beckman Coulter).
Ail experiments were conducted in accordance with the French régulations in force after 5 approval by Servier Research Institute (IdRS) Ethical Committee. NOD SCID mice were maintained according to inslitutional guidelines.
Results
Efficacy of Example 24 on MOLT-4 xenografts is illustrated in Figure 1. Treatment was started 12 days post tumor celis inoculation (average size: 235 mm3). Vehicle (E1PBCD/HC1) or Example 24 (2.5, 5 and 7.5 mg/kg) were dosed IV each 3 days for a total of 6 administrations.
On day 31 after treatment start, the Tumor Growth Inhibition (%TGI) induced by Example 24 was of 77.1% at 2.5 mg/kg, 78.7% at 5 mg/kg and 94.7% at 7.5 mg/kg (p<0.05), as depicted in Figure 1 and Table 4. Partial régression (PR) was achieved in 28.6%, 71.4% and 100% of the cases, respectively. Complété régression (CR) was observed in 14.3% of the animais treated at the highest dose.
No clinically relevant body weight loss due to the treatment was observed (Figure 2).
Table 4: MOLT-4 tumor growth inhibition upon treatment with Exampie 24 (2.5, 5 and 7.5 mg/kg, administered IV, Q3D6).
Group Dose (mg/kg) %TGI (d31) %CR %PR
Example 24 2.5 77.1* 0 28.6
Example 24 5 78.7* 0 71.4
Exampie 24 7.5 94.7* 14.3 100
*p value <0.05 compared to control group.
The effect of Example 24 on apoptosis induction in MOLT-4 tumor cells and number of circulating platelets îs illustrated in Table 5. Treatment was started 18 days post tumor cells
366 inoculation (average size: 461 mm3). Example 24 (7.5 mg/kg) was dosed once PO or IV and samples were collected 6h later. The compound showed an induction of apoptosîs markers, namely cleaved PARP (24.1 - 38.6-fold over non-treated control) and cleaved Caspase 3 (7.2 - 18.9-fold over non-treated control) cleavage, independently on the administration route. In 5 addition, given the well-described rôle of Bcl-xL in regulating platelets life-span, it also caused a signifîcant réduction in platelet numbers (to 1% of control values).
Table 5: Cleaved PARP and cleaved Caspase 3 activation in tumor cells and platelet loss in MOLT-4-grafted female NOD SCID mice 6h after treatment (PO or IV) with Example 24 at 7.5 mg/kg.
Compound tested Dose (mg/kg) and route Timepoint (h) Cleaved PARI’ (fold increase over control) Cleaved Caspase 3 (fold increase over control) Platelet count (xl03/pl) %remaining platelets (vs Controls)
Example 24 7.5, PO 6 24.1 7.2 7 1
Example 24 7.5, IV 6 38.6 18.9 8 1
In conclusion, we show here that the Bcl-xL-targeting smail molécule described in Example 24 is active in vivo after intravenous or oral administration. We observed tumor régression, apoptosîs induction in tumor cells and a strong réduction in cîrculating platelets, în agreement with the previously described rôle of Bcl-xL in apoptosîs control and platelets life-span régulation (Youle and Strasser, Nat, Rev. Mol. Cell Biol. 2008 Jan;9(l):47-59; Zhang et al., 15 Cell Death Dijfer. 2007 May; 14(5):943-51; Mason et al., Cell 2007 Mar 23;128(6):1173-86).
In addition, no clinically relevant body weight loss was observed upon treatment with efficacious doses and platelets loss was recovered after treatment discontinuation (data not shown). Altogether, these data indicate that there is a possible therapeutic margin for the use of these Bcl-xL-targeting small molécules in cancer treatment.
367
EXAMPLE E: In vivo pharmacodynamie profile of the compounds of formula (I)
The pharmacokinetic profile of the compounds of formula (I) is evaluated in rodent (mouse, rat) after PO and/or IV route. The formulation is selected based on the physico-chemical properties of the tested drug as well as the route of administration. A single dose of the drug (<5 mg/kg) prepared in the adapted formulation is administered by IV (bolus or 10min infusion) or PO (gavage) route to animais (3 animals/route). Blood samples from each animal (up to 6 samples/animal) are collected over 24 h after dosing and plasma concentrations of the tested compound are determîned after extraction followed by a liquid chromatography coupled with tandem mass spectrometry détection (LC/MS-MS).
In some cases, the following experimental protocol is used to détermine the pharmacokinetic profile of the compounds of the invention in the Wistar rat:
The drug is prepared in a formulation composed by a mixture of polyethylene glycol 300/anhydrous Ethanol/NaCI 0.9% (40/10/50 v/v/v). The formulation is administered by IV route to male Wistar rat (3 animais) at a dose of 0.75mg/kg (10min inf, 5mL/kg). Blood samples are taken at the following time points from each animal: end of infusion (10min), 0.5h, Ih, 3h, 6h and 24h after dosing. The plasma concentrations of the tested compound are determîned after extraction followed by a liquid chromatography coupled with tandem mass spectrometry détection (LC/MS-MS).
The lower lîmit of quantification is 2.5ng/mL.
The results allow to rank the compounds of the invention based on their plasma exposure, élimination rate constant, clearance and volume of distribution in order to evaluate the therapeutic range of the compounds in animal model.

Claims (8)

  1. wherein:
    ♦ n=0,1 or 2, ♦ .....- represents a single or a double bond, ♦ A4and As independently of one another represent a carbon or a nitrogen atom, ♦ Zi represents a bond, -N(R)~, or-O-, wherein R represents a hydrogen or a linear or branched Ci-C6alkyl, ♦ Ri represents a group selected from: hydrogen; linear or branched Ci-Csalkyl optionally substituted by a hydroxyl or a
    Ci-C6alkoxy group; C3-C6cycloalkyl; trifluoromethyl; linear or branched Ci-C6alkyiene-heterocycloalkyl wherein the heterocycloalkyl group is optionally substituted by a a linear or branched Ci-Cealkyl group;
    ♦ R2represents a hydrogen or a methyl;
    ♦ R3 represents a group selected from: hydrogen; linear or branched Ci-C4alkyl; -Xi-NRaRb; -XiN+RaRbRc; -Xi-O-Rc; -XrCOORc; -XrPO(OH)2; -Xi-SO2(OH); -XrN3 and :
    —X-i---H ♦ Ra and Rt independently of one another represent a group selected from: hydrogen; heterocycloalkyl; -SO2-phenyl wherein the phenyl may be substituted by a linear or branched CrC6alkyl; linear or branched CrC6alkyl optionally substituted by one or two hydroxyl groups; Ci-C6alkylene-SO2OH; Ci-C6alkylene-SO2O·; CrC6alkylene-COOH; Ci-Ctalkylene-POiOH):; CiC6alkylene-NRdRe; Ci-C6alkylene-N+RdReRf; Ci-C6alkylene-phenyl wherein the phenyl may be substituted by a Ci-Cgalkoxy group;
    the group:
    369
    or Ra and Rb form with the nitrogen atom carrying them a cycle Bi;
    or Ra, Rt and Rc form with the nitrogen atom carrying them a
    5 bridged Ca-Caheterocycloalkyl, ♦ Rc, Rd, Re, Rf, independently of one another represents a hydrogen or a linear or branched Ci-
    Cealkyl group, or Rd and Re form with the nitrogen atom carrying them a a cycle B2,
    10 or Rd, Re and Rf form with the nitrogen atom carrying them a bridged Cî-Cgheterocycloalkyl, ♦ Heti represents a group selected from:
    ♦ Het2 represents a group selected from:
    370
    ♦ Al is -NH-, -N(Ci-C3alkyl), 0, S or Se, ♦ A2isN,CHorC(R5), ♦ G is selected from the group consisting of:
    -C(0)0Rg3, -C(O)NRGiRG2, -C(0)Rg2, -NRGiC(O)Rg2, -NRGiC(O)NRs1RG2,
    5 -0C(0)NRGiRg2, -NRgiC(0}0RG3, -C(=NORgi)NRgiRG2,
    -NRgiC(-NCN)NRgiRG2, -NRgiS(O)2NRgiRg2, -S(O)2Rg3, -S(O)2NRgiRg2,
    -NRGiS(O)2RG2, -NRgiC(=NRG2)NRgiRg2, -C(=S)NRgiRg2, -C(=NRGi)NRgiRg2, halogen, -NO2, and CN, in which:
    - RGiand RG2at each occurrence are each independently selected from the group consisting 10 of hydrogen, Ci-CeaIkyl optionally substituted by 1 to 3 halogen atoms,
    Cz-Csalkenyi, Cj-Cealkynyl, Gj-CeCycloalkyl, phenyl and -(CH2)i-4-phenyl;
    - Rgj is selected from the group consisting of Ci-C6alkyi optionally substituted by 1 to 3 halogen atoms, C2-CGalkenyl, C2-C6aikynyl, C3-C6cycloalkyl, phenyl and -(CH2)i-4-phenyl; or Rgi and Rg2, together with the atom to which each is attached are combined to form a C3-
    15 Cgheterocycioalkyi ; or in the alternative, G is selected from the group consisting of:
    wherein RG4 is selected from CrCealkyl optionally substituted by 1 to 3 halogen atoms, C220573
    371
    Csalkenyl, Cz-Csalkynyl and Cs-Cecycloalkyl, ♦ R4 represents a hydrogen, fluorine, chlorine or bromine atom, a methyl, a hydroxyl or a methoxy group, ♦ R5 represents a group selected from: Ci-C6alkyl optionally substituted by 1 to 3 halogen atoms;
    5 Cz-Csalkenyl; Cz-Csalkynyl; halogen or-CN, ♦ R6 represents a group selected from:
    hydrogen;
    -CrCsalkenyl;
    -X2-O-R7;
    Ύ Ό—R7 1 /\
    10 ;
    -Xz-NSO2-R7;
    -C=C(R9)-Yi-O-R7;
    CrCecycloalkyl;
    Ca-Csheterocycloalkyl optionally substituted by a hydroxyl group;
    15 C3-Cecycloalkylene-Y2-R7;
    C3-C6heterocycloalky!ene-Yz-R7 group, an heteroarylene-R7 group optionally substituted by a linear or branched Ci-C6a1kyl group, ♦ R7 represents a group selected from: linear or branched Ci-Cealkyl group;
    (Ca-Cejcycloalkylene-Rs; or:
    372
    wherein Cy represents a Ca-Cgcycloalkyl, ♦ Rg represents a group selected from: hydrogen; linear or branched Ci-Csalkyl, -NR'aR't,; -NR'aCO-OR'c; -NR'a-CO-R'c; -N+R'aR'bR'c; -O-R'c; -NH-XThrR'aR'bR'ô -O-X'2-NR'aR'br -X'2-NR'aR'b 5 NR’c-X'rNs and :
    -NR'c—X'2-^CH ;
    ♦ R9 represents a group selected from linear or branched Ci-CsaIkyl, trifluoromethyl, hydroxyl, halogen, Ci-Cealkoxy, ♦ Riq represents a group selected from hydrogen, fluorine, chlorine, bromine, -CF3 and methyl, 10 ♦ Ru represents a group selected from hydrogen, halogen, Ci-Caalkylene-Rg, -OCi-Csalkylene-
    Rs, -CO-NRbRi and -CH=CH-CrC4alky1ene-NRhRi, -CH=CH-CHO, C3-Cscycloalkylene-CH2-Re, C3Csheterocycloalkylene-CHrRs, ♦ R12 and Ris, independently of one another, représenta hydrogen atom or a methyl group, ♦ R14 and RiS, independently of one another, represent a hydrogen or a methyl group, or RM and 15 r15 form with the carbon atom carrying them a a cyclohexyl, ♦ Rn and R,, independently of one another, represent a hydrogen or a linear or branched CiCealkyl group, * Xi represents a linear or branched
    20 Ci-C4alkylene group optionally substituted by one or two groups selected from trifluoromethyl, hydroxyl, halogen, Ci-Cealkoxy, * X2 represents a linear or branched
    CrC6alkylene group optionally substituted by one or two groups selected from trifluoromethyl, hydroxyl, halogen, Ci-Cealkoxy,
    25 ♦ X'2 represents a linear or branched Ci-C6alkylene, ♦ R'a and R'b independently of one another, represent a group selected from: hydrogen; heterocycloalkyl; -SOz-phenyl wherein the phenyl may be substituted by a linear or branched Ci-C6alkyl; linear or branched Ci-C6alky! optionally substituted by one or two hydroxyl or Cr C6alkoxy groups; Ci-C6alkylene-SO2OH; Ci-Csalkylene-SO2O’;
    373
    CrC6alkylene-COOH;
    Ci-C6alkylene-PO(OH)2;
    Ci-Cealkylene-NR'tiR'e;
    CrC6alkylene-N+R eR'c Ci-Cealkylene-O-Ci-Cealkyîene-OH; Ci-C6alkylene-phenyl wherein the phenyl may be substituted by a hydroxyl or a Ci-C6alkoxy group;
    the group:
    or R'a and R't> form with the nitrogen atom carrying them a cycle B3, or R'a, R'b and R'c form with the nitrogen atom carrying them a bridged C3-CBheterocycloalkyl, ♦ R'c, R'd, R'e, R't, îndependently of one another, represents a hydrogen or a linear or branched Ci-Cealkyl group, or R'd and R'e form with the nitrogen atom carrying them a cycle B4, or R'd, R'e and R't form with the nitrogen atom carrying them a bridged Cj-Cgheterocycloalkyi, ♦ Yi represents a linear or branched Ci-C4alkylene, ♦ Y2 represents a bond, -O-, -O-CH2-, -O-CO-, -O-SO2-, -CH2-, -CH2-O, -CH2-CO-, -CH2-SO2-,-C2H5-, -CO-, -CO-O-, -CO-CH2-, -CO-NH-CH2-, -SO2-, -SO2-CH2-,
    - NH-CO-, -NH-SO2-, ♦ m=0, 1 or 2, ♦ p=l, 2, 3 or 4, ♦ Bi, 82, Bs and B4, îndependently of one another, represents a C3-C8heterocycloalkyl group, which group can: (i) be a mono- or bi-cyclic group, wherein bicyclic group includes fused, briged or spiro ring system, (ii) can contaîn, in addition to the nitrogen atom, one or two hetero atoms selected îndependently from oxygen, sulphur and nitrogen, (iii) be substituted by one or two groups selected from: fluorine, bromine, chlorine, linear or branched Ci-C6alkyl, hydroxyl, -NH2,oxoor pîperidinyl, its enantiomers and diastereoisomers, and addition salts thereof with a pharmaceutically acceptable acid or base.
  2. 2, The compound according to claim 1 wherein A4 and As represent each a nitrogen atom.
  3. 3. The compound according to claim 1 or 2 wherein Zi represents -NH- or -O-,
  4. 4, The compound according to any of claims 1 to 3 wherein Ri represents -Xi-NRaRb.
    374
  5. 5. The compound according to claim 1, which îs selected from:
  6. 6. The compound according to claim 5, which is a compound of formula (IB):
    The compound according to any one of claims 1 to 6 wherein Ri represents a hydrogen
    5 atom, a methyl or a cyclopropyl group.
    & The compound according to any one of claims 1 to
  7. 7 wherein Hetj represents:
    375
    9, The compound according to any one of claims 1 to 8 wherein Het2 represents:
    BL The compound according to any one of claims 1 to 8 wherein Het2 represents;
    5 IL The compound according to claim 9 wherein Rf, represents a-X2-O-R7group wherein
    X2 is a propylene group.
    12, The compound according to claim 11 wherein R7 represents the following group;
    The compound according to claim 11 wherein R7 represents the following group:
    14, The compound according to claim 12 or 13 wherein Rs represents a group selected
    10 from: dimethylamino, dielhylamino, diisopropyl amino, diisobutylamino, methylamino, ethylamîno, ethyl(methyl)amino, 4-methyl-piperazin-l-yl, piperazin-l-yl, pyrrolidin-l-yl,
    376 azetidin-l-yl, 1-piperidyl, 4-morpholînyl, 4,4-difluoropiperidin-l-yl, 3,3-difluoropiperidin-lyl, 3-hydroxy-l-piperidyI, (15,5R)-3-azabicyclo[3.1.0]hexan-3-yl, 4-(l-piperidyl)-1-piperidyl, 3-oxo-2,8-diazaspiro[4.5]decan-8-yl, (lS,5Æ)-6,6-difluoro-3-azabicyclo[3.1.0]hexan-3-yl, 2(dimethylamino)ethylamino, 3-piperazin-l-yl, (3Æ,5S)-3,5-dimethylpiperazin-l-yI, (but-3-ynl-yl)amino, (but-3-yn-l-yl)(methyl)amino, (3-azidopropyl)amino, (3azidopropyl)(methyl)amino (3-aminopropyl)amino, (pent-4-yn-l-yl)ammo, methyl(pent-4yn-l-yl)amino, (prop-2-yn-l-yl)amino, (hex-5-yn-l~yl)ammo, 3-[(hex-5-yn-lyl)(methyl)amino, (4-azîdobutyl)amino, (4-azidobutyl)(methyl)amino, [2-(2hydroxyethoxy)ethyl](methyl)amino, and:
    15. The compound according to claim 12 or 13 wherein Rg represents a group selected from: bis[(3S)-3,4-dihydroxybutyl]amino, amino, [(3S)-3,4-dihydroxybutyl]amino, [(3R)-3,4dihydroxybutyljamino, acetyl(methyl)amino, 3-hydroxypropylamino.
    16. The compound according to claim 11 wherein R? represents:
    wherein Ru is selected from 3-(dimethylamino)propyl, 3-(methylamino)propyl, aminomethyl, 2-(dimethylaminü)elhyl, 4-(dimethylamino)butyl, 2-(methylamino)ethyl, 4(methylamino)butyl, 3-(azetidin-l-yl)propyl, 3-(4-methylpiperazin-l-yl)propyl, 3-pyrrolidin1-ylpropyl, 3-morpholinopropyi, 3-(l-piperidyl)propyl, 3-[(lR,5S)-6,6-difluoro-3azabicyclo[3.1.0]hexan-3-yl and 3-(3-oxo-2,8-diazaspiro[4.5]decan-8-yl)propyl.
    17, The compound according to claim 11 wherein R7 represents a group selected from:
    377
    IIL The compound according to claim 10 wherein R& represents:
    19, The compound according to claim 18 wherein R7 represents a group selected from :
    5 wherein Rs represents a group selected from: hydrogen, 2-(methylamino)ethoxy, 2(dimethylamino)ethoxy, 2-[(2-sulfoethyl)amino]ethoxy, 2-[methyl(2sulfoethyl)amino]ethoxy, 4-methylpiperazin-l-yl and:
    The compound according to daim 18 wherein R7 represents a group selected from :
    378
    wherein Rs represents a group selected from: 2-pyrrolidin-l-ylelhoxy, 2-(4-methylpiperazinl-yl)ethoxy, 2-[[(3R)-3,4-dihydroxybutyl]-methyLamino]ethoxy, 2-(4hydroxybutylamîno)ethoxy, 2-[(3-hydroxy-2-(hydroxymethyl)propyl]aminoJethoxy, 2-[bis(2hydroxyethyl)amino]ethoxy, 2-[[2-hydroxy-l-(hydroxymethyl)ethyl]amino]ethoxy, 2-(2-(25 hydroxyethoxy)ethylamino]ethoxy, 2-[bis(3-hydroxypropyi)amino]ethoxy, 2-(3hydroxypropylamino)ethoxy, 2-[bis(4-hydroxybutyl)amino]ethoxy, 2-morpholinoethoxy, 2(l-pîperidyl)ethoxy, 2-piperazin-l-ylethoxy, 2-(azepan-l-yl)ethoxy, 2-(4-isopropyIpiperazinl-yl)ethoxy, 2-[(4-hydroxyphenyI)methylamino]ethoxy, 2-[2hydroxyelhyl(methyl)amino]ethoxy, 2-[3-methoxypropyl(methyi)amino]ethoxy, 2-[410 hydroxybutyl(methyl)amino]ethoxy, 3-pyrrolidin-l-ylpropyl, 3-(dimethylamîno)propyl, 3-(4methylpiperazin-l-yl)propyl, 3-morpholinopropyl, 3-(3-hydroxypropylamino)propyl, 3-(4hydroxybutylamîiio)propyl, 3-[[(35)-3,4-dihydroxybutyl]amino]propyl, 3-hydroxy-2(hydroxymethyl)propyl]amino]propyl, 3-[4-hydroxybutyl(methyl)amino]propyl, 3-[3hydroxypropyl(methyl)amino]propyl, 3-[3-[bis(3-hydroxypropyI)amino]propyl, 3-piperazin15 1-ylpropyl.
    21, The compound according to any one of claims 1, 2 and 6 wherein R3 represents -XiPO(OH)2, -X^SOziOTI), -Xj-NRaRb; -Xi-N+RaRbRc, wherein Ra or Rbj or both of them, represent a group selected from Ci-Côalkylene-SO2OH, Ci-Cbalkylene-SO2O' and CiCbalkylene-PO(OH)2.
    20 22, The compound according to any one of claims 1, 2 and 6 wherein Rs represents NR’aR’bj -N+R’HR’bR’c; -NH-X’2-N+R’aR’bR’c, wherein R’aand R’b, or both of them, represent a group selected from Ci-CGalkylene-SChOH and Ci-Cbalkylene-PO(OH)2.
    23. A compound according to claim 1 selected in the following group:
    - 2-[3-(l,3-Benzothiazol-2-ylamino)-4-methyi-6,7-dihydro-5/7-pyrido[2,3-c]pyridazin20573
    379
  8. 8-yl]-5-[3-[4-[3-(dimethylamino)prop-l-ynyl]-2-fliioro-phenoxylpropyl]thiazole-4carboxyhc acid,
    2-{3-[(l,3-Benzothiazol-2-yl)amino]-4-methyl-5//,6/f,7/7,8if-pyrido[2,3-c]pyridazin8-yl}-5-(3-{2-fluoro-4-[3-(methyIaniino)prop-l-yn-l-yI]phenoxy}propyl)-lf3thiazole-4-carboxyIic acid,
    - 2-{3-[(l,3-Benzothiazol-2-yl)amino]-4-methyl-5/7,6/7,7/f,8/7-pyrido[2,3-c]pyridazin8-yl}-5-(3-{4-[3-(dimethy]amino)propyl]-2-fluorophenoxy}piOpyl)-l,3-thiazole-4carboxylîc acid,
    2-(3-(1,3-Benzothiazol-2-y lamino)-4-methyl-6,7-dihydro-5H-pyrido [2,3-c]pyridazin8-yl]-5-[3-[2-fluoro-4-[3-(4-methylpiperazin-l-yl)but-lynyl]phenoxy]propyl]thiazole-4-carboxyiic acid,
    - 2-(3-(1,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/7-pyrido[2,3-c]pyridazin-
    8-yl]-5-[3-[2-fluoro-4-(3-pyrrolidin-l-ylpiOp-l-ynyl)phenoxy]propyl]thiazole-4carboxylîc acid, . 5-(3-{4-(3-(Azetidin-i-yl)prop-l-yn-l-yl]-2-fluorophenoxy}propyl)-2-{3-[(l,3benzothiazol-2-yl)ammo]-4-methyi-5/fî67f,7//,8H-pyrido[2,3-c]pyridazin-8-yl}-l,3thiazole-4-carboxylic acid,
    - 2-(3-( 1,3-Benzothiazol-2-y lamino)-4-methy 1-6,7-dihydro-5ff-pyrido [2,3-c]pyrîdazin-
    8-yl]-5-(3-[2-fIuoro-4-[3-(4-methylpiperazin-l-yl)prop-l-ynyl]phenoxy]propyl] lhiazole-4-carboxylic acid,
    - 2-{3 -[(1,3-Benzothiazol-2-yl)amino]-4-methy 1-5/7,6H, 7H,8H-pyrido [2,3-c]pyridazin-
    8-yl}-5-(3-{4-[3-(4,4-difluoropiperidin-l-yl)prop-l-yn-l-yl]-2fluorophenoxy}propyl)-l,3-thiazole-4-carboxylic acid,
    - 2-{3-[(l,3-Benzothiazol-2-yl)amino]-4-methyl-5//,6W,7H,8/f-pyrido[2,3-c]pyridazin8-yl}-5-(3-{4-[3-(3,3-difluoropiperidin-l-yl)prop-l-yn-l-yl]-2fluorophenoxy}propyl)-l,3-lhiazole-4-carboxylic acid,
    - 2-(3-(1,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin8-yl]-5-[3-[2-nuoro-4-[3-(3-oxo-2,8-diazaspiro[4.5]decan-8-yl)prop-l-yiiyl] phenoxy]propyl]thiazole-4-carboxylic acid,
    - 2-(3-(1,3-BenzothiazoI-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin8-yl]-5-[3-[4-[3-[(lS,5R)-6,6-difluoro-3-azabicyclo[3.1.0]hexan-3-yl]prop-l-ynyl]-2lluoro-phenoxy]propyl]thiazole-4-carboxylic acid,
    - 2-(3-(1,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[253-c]pyridazin8-yl]-5-[3-[2-fluoro-4-(3-piperazin-l-ylprop-l-ynyl)phenoxy]propyl]thiazoie-420573
    380 carboxylic acid,
    2-(3-(1,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5ff-pyrido[2,3-c]pyridazin8-yl]-5-[3-[4-[3-[(3JÎ,5S)-3,5-dimelhylpiperazin-l-yl]prop-l-ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylic acid,
    - 2-(3-(1,3-Benzothiazol-2-ylaniino)-4-methyl-6,7-dihydiO-51/-pyrido[2,3-c]pyridazin8-yi]-5-[3-[4-[3-(diethyiamino)prop-l-ynyl]-2-fIuoro-phenoxy]propyl]thiazole-4carboxylic acid,
    - 2-(3-(1,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/7-pyrido[2,3-c]pyridazin8-yl]-5-[3-[4-[3’(diisopropylamino)prop-l-ynyl]-2-fluoro-phenoxy]propyl]thiazole-4carboxylic acid,
    - 2-(3-(1,3-Benzothiazol-2-ylanuno)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin8-yl]-5-[3-[4-[3-[2-(dimethylamino)ethylamino]prop-l-ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylic acid,
    2-{3-[(l,3-Benzothiazol-2-yl)ammo]-4-methyl-6-[2-(methylamino)ethoxy]5H,6H,7H,8J¥-pyrido[2,3-c]pyridazin-8-yl}-5-(3-{4-[3-(dimethylamino)prop-l-yn-lyl]-2-fluorophenoxy}propyl)-l,3-thiazole-4-carboxylic acid,
    - 2-(3-(1,3-Benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5W-pyrido[2,3-c]pyridazin8-yl]-5-[3-[4-[l-[(diniethyiamino)methyl]-3-bicyclo[l.l.l]pentanyl]-2-fluorophenoxy]propyi]thiazole-4-carboxylic acid,
    - 2-(3-(1,3-Benzoihiazol-2-ylainino)-4-methyl-6,7-dihydro-5/7-pyrido[2,3-c]pyridazin8-y I]-5-(3-(2-fluoro-4-[3-niethy 1-3-(methylamino)bu t-1ynyl]phenoxy]propyI]thiazole-4-carboxylic acid,
    - 2-(3-(1,3-benzothiazol-2-ylammo)-4-methyl-6,7-dihydro-5ifrpyrido[2,3-c]pyridazin8-yl]-5-[3-[2-fluoro-4-[3-(prop-2-ynylamino)prop-l-ynyl]phenoxy]propyl]thiazole-4carboxylic acid,
    - 6-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido[2,3-c]pyridazin8(5H)-yl}-3-[l-({3-(2-(dimethylamino)ethoxy]-5,7-dimethyladamantan-l-yl}methyI)5-methyl-LH-pyrazol-4-yl]pyridine-2-carboxylic acid, . 6-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido[2,3-c]pyridazin8(5H)-y 1)-3-(1-({3,5-dimethy 1-7-[2-(methy lamino)ethoxy] adaman tan-1-y l}methyl)5-methyl-l/f-pyrazol-4-yl]pyridme-2-carboxylic acid,
    - 2-{3-[(l,3-benzothiazol-2-yl)amino]-4-methyI-6,7-dihydiOpyrido[2,3-c]pyridazin8(5H)-yl}-5-(3-{4-[3-(ethylamino)-3-mcthylbut-l-yn-l-yi]-2-fluorophenoxy)propyl)l,3-thiazole-4-carboxylic acid,
    381
    3-{l-((Adamantan-l-yl)methyl]-5-methyl-l/f-pyrazol-4-yl}-6-{3-[(l,3-benzothiazol2-y l)amino]-4-methy 1-5/7,6/7,7/7,8//-py ri do [2,3-c]pyridazin-8-yl}py ri dine-2carboxylic acid, its enantiomers and diastereoisomers, and addition salts thereof with a pharmaceuticaliy acceptable acid or base.
    2£ A compound according to claim 1 selected in the following group:
    6-{3-[(l,3-benzothiazol-2-yl)ammo]-4-niethyl-6,7-dihydropyrido[2,3-c]pyridazin8(5/7)-yl}-3-[l-({3-[2-(dimethylamino)ethoxy]-5,7-diinethyiadamantan-i-yl}methyI)5-methyl-l/f-pyrazol·4-yl]pyridine-2-carboxylic acid,
    - 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-51/-pyrido[2,3-c]pyridazin8-yl]-3-[l-[[3,5-dimethyl-7-(2-pyrmIidin-l-ylelhoxy)-l-adamantyl]methyl]-5-methylpyrazol-4-yl]pyridine-2-carboxylic acid, 6-[3-(l,3-benzothiazol-2-ylamino)~4-methyl-6,7-dihydro-5H-pyrido[2,3~c]pyridazin8-yl]-3-[ 1-((3,5-dimethyl-7-(2-(4-methylpiperazin-l-yl)ethoxy]-l-adamantyI]methyl]5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid,
    - 6-[3-(l,3-bei]zothiazoI-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazm8-yl]-3-[l-[[3-[2-(3-hydroxypropylamino)ethoxy]-5,7-dimethyl-l-adamantyl]methyl]5-methyl-pyrazol-4-yl]pyridîne-2-carboxylic acid,
    - 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyI-6,7-dihydro-5//-pyrido[2,3-c]pyridazin8-yl]-3-[l-[[3-[2-(4-hydroxybutylamino)ethoxy]-5,7-dimethyl-l-adamantyl]methyl]5-methyl~pyrazol-4-yl]pyridine~2-carboxylic acid,
    - 6-(3-((1,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropyrido(2,3-c|pyridazin8(5//)-yl}-3-(l-{[3-(2-( [(3S)-3,4-dihydroxybutyl]amino}ethoxy)-5,7dimethyladamaiitan-l-yl]methy1}-5-methyl-l7/-pyrazol-4-yl)pyridine-2-carboxylic acid,
    - 6-(3-((1,3-benzothiazol-2-yl)amino]-4-methyl-6,7-dihydropy rido(2,3-c]pyridazin8(5//)-yl}-3-(l-{(3-(2-{[(3R)-3,4-dihydroxybutyl]amino}ethoxy)-5,7dimethyladamantaπ-l-yl]methyl}-5-methyl-lÆ-pyrazol·4-yl)pyridine-2-carboxylic acid, 6-(3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/7-pyrido[2,3-c]pyridazin8-ylJ-3-[l-([3-[2-[2-hydroxyethyl(methyl)amino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid,
    - 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-57/-pyrido[2,3-c]pyridazin20573
    382
    8-yI]-3-[l-[[3-[2-[4-hydroxybutyl(methyl)aminolethoxy]-5,7-dimeLhyI-ladamantyl]methyl]-5-methyl-pyrazol-4-yI]pyridine-2-carboxylic acid,
    6-(3-(1,3-benzothiazol-2-ylamino)-4-methyi-6,7-dihydro-5//-pyrido[2,3-c]pyridazin8-yl]-3-[l-[[3-[2-[[(3À)-3,4-dihydroxybutylJ-methyl-amino]ethoxy]-5,7-dimethyI-l“ adamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid,
    6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5F/-py rido[2,3-c]pyridazin8-yl]-3-(l-[[3,5-dimethyl-7-(2-piperazin-l-ylethoxy)-l-adamantyl]methyl]-5-melhylpyrazol-4-yl]pyridine-2-carboxylic acid,
    6-{3-[(l,3-benzothiazol-2-yl)amino]-4-rnethyl-6,7-dihydropyrido[2,3-c]pyridazm8(57f)-yl}-3-[l-({3,5-dimethyl-7-[2-(methylamino)ethoxy]adamantan-l-yI}methyl)5-methyl-l/7-pyrazol-4-yl]pyridine-2-carboxylic acid,
    - 6-(3-(1,3-benzothiazol2-ylamino)-4-methyl-6,7-dihydro-5W-pyrido[2,3-c]pyridazin-
    8-yl]-3-[l-[[3,5-dimethyl-7-[2-(l-piperidyl)ethoxy]-l-adamantyl]methyl]-5-methylpyrazol-4-yi]pyridine-2-carboxylic acid,
    - 3-[l-[[3-[2-(azepan-l-yl)ethoxy]-5,7-dimethyl-l-adamantyl]methyl]-5-methylpyrazoi-4-yl]-6-[3-(l,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5ffpyrido(2,3-c]pyridazin-8-yl]pyridine-2-carboxylic acid,
    - 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5//-pyrido(2,3-c]pyridazin8-yl]-3-[l-[[3-[2-(4-isopropylpiperazin-l-yl)ethoxyJ-5,7-dimelhyl-ladamanlyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid,
    - 6-(3-(1,3-benzolhiazol-2-ylamino)-4-methyl-6,7-dihydro-5/f-pyrido[2,3-c]pyridazin-
    8-yl]-3-[l-[[3,5-dimethyl-7-(2-morphoIinoethoxy)-l-adamantyl]methyl]-5-methylpyrazol-4-yl]pyrîdine-2-carboxylic acid,
    - 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5if-pyrido[2,3-c]pyridazin-
    8-yl]-3-[l-[[3-[2-[3-methoxypropyl(methyl)amino]ethoxy]-5,7-dimethyl-ladamanlyl]methylj-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid,
    - 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3-c]pyridazin-
    8-yl]-3-[l-[[3-[2-[2-(2-hydroxyethoxy)ethylamino]ethoxy]-5,7-diniethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid,
    - 6-(3-(1,3-benzothiazoi-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido(2,3-c]pyridazin8-yl]-3-[l-[[3-[2-[[2-hydroxy-l-(hydroxymethyl)ethyl]amino]ethoxy]-5,7-dimethyl-ladamantyl]methyI]-5-methyl-pyrazoI-4-yl]pyridine-2-carboxylic acid,
    - 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyI-6,7-dihydro-5//-pyrido[2,3-c]pyridazin8-yl]-3-[l-([3-[2-[[3-hydroxy-2-(hydroxymethyl)propyl]amino]ethoxy]-5,7-dimethyl
    383 l-adamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid,
    6-(3-(1,3-benzothiazol-2-yIamino)-4-methyl-6,7-dihydro-5/f-pyrido[2,3-c]pyridazin8-yl]-3-[l-[[3-[2-[bis(2-hydroxyethyl)amino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid, 6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/f-pyrido[2,3-c]pyridazin8-yl]-3-[l-[(3-[2-(bis(3-hydroxypropyl)amino]ethoxy]-5,7-diinethyl-ladamantyl]methyI]-5-methyI-pyrazol-4-yl]pyridine-2-carboxylic acid,
    6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/Z-pyrido[2,3-c]pyridazin8-yl]-3-(l-[[3-[2-[bis(4-hydroxybutyl)amino]ethoxy]-5,7-dimethyl-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid,
    6-{3-[(l,3-benzothiazol-2-yl)amino]-4-melhyl-6,7-dihydropyrido[2,3-c]pyridazin8(5H)-yl}-3-{ 1-((3,5-dimethyl-7-{2-[(2-sulfoethyl)amino]ethoxy}adamantan-lyl)methyl]-5-methyl-l//-pyrazol-4-yl}pyridme-2-carboxylic acid,
    6-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5/ï-pyrido[2,3-ci|pyridazin8-yl]-3-[l-[[3-[2-[(4-hydroxyphenyl)methyIamino]ethoxy]-5,7-dimethyi-ladamantyl]methyl]-5-methyl-pyrazol-4-yl]pyridine-2-carboxylic acid,
    - 2-(3-(1,3-Benzothiazol-2-yIamino)-4-methyl-6,7-dihydro-5.H-pyrido[253-c]pyridazm8-yl]-5-[3-[4-[3-(dimethylamino)prop-l-ynyl]-2-fluoro-phenoxy]propyl]thiazole-4carboxylic acid,
    - 2-[3-(l,3-benzothiazol-2-yiamino)-4-methyl-6,7-dihydro-5.H-pyrido[2,3-c]pyridazin8-yi]-5-[3-[4-[3-[[(3S)-3,4-dihydroxybutyl]amino]prop-l-ynyl]-2-fluorophenoxy]propyl]thiazole-4-carboxylic acid,
    - 2-(3-(1,3-benzothiazol-2-ylamino)-4-methyl-6,7-dihydro-5H-pyrido[2,3<] pyridazin-8- yl]-5-[3-[2-fluoro-4~[3-(3-hydroxypropylamino)prop-l-ynyl]phenoxy]propyl] thiazole4-carboxylîc acid, its enantiomers and diastereoisomers, and addition salts thereof with a pharmaceutically acceptable acid or base.
    2^ Pharmaceutical composition comprising a compound according to any of daims 1 to 24, or an addition sait thereof with a pharmaceutically acceptable acid or base in combination with one or more pharmaceutically acceptable excipients.
    26. Pharmaceutical composition according to claim 25 for use as pro-apoptotic agents.
    384
    27. Pharmaceutical composition according to claim 25 for use in the treatment of cancers, auto-immune diseases or immune System diseases.
    28. Pharmaceutical composition for use according to claim 27 wherein the cancer is an haematological malignancy or a solid tumor.
    2^ Phannaceutical composition for use according to claim 28 wherein the haematological malignancy is myeloma, lymphoma, or leukemia, .
    30, Pharmaceutical composition for use according to claim 28 wherein the solid tumor is selected from bladder, brain, breast, utérus, œsophagus and liver cancers, colorectal cancer, rénal cancer, melanoma, ovarian cancer, prostate cancer, pancreatic cancer and lung cancer.
    31. Compound according to any of daims 1 to 24, or an addition sait thereof with a pharmaceutically acceptable acid or base, for use in the treatment of a cancer selected from: myeloma, lymphoma, or leukemia, , bladder, brain, breast, utérus, œsophagus and liver cancers, colorectal cancer, rénal cancer, melanoma, ovarian cancer, prostate cancer, pancreatic cancer and lung cancer.
    32. Combination of a compound according to any of daims 1 to 24, with an anti-cancer agent selected from genotoxic agents, mitotic poisons, anti-métabolites, protéasome inhibitors, kinase inhibitors and antibodies.
    33. Pharmaceutical composition comprising a combination according to daim 32 in combination with one or more pharmaceutically acceptable excipients.
    34. Combination according to claim 32 for use in the treatment of cancers.
    35. Compound according to any of daims 1 to 24, for use în the treatment of cancers requiring radiotherapy.
    Pharmaceutical composition according to claim 25 for use in the treatment of diseases
    385 or conditions characterized by an excess or a deregulated activity of platelets.
    37. Synthesis intermediate selected in the following group:
    (VI)
    wherein R? is as defïned in claim 1 and G1 represents a Ci-C&alkyl group or a (4methoxyphenyl)methyl group.
    Synthesis intermediate selected in the following group:
    386
    wherein Rs is as defined in claim 1 and G1 represents a Ci-C6alkyl group or a (4 methoxyphenyl)methyl group.
    1/2
    Figure 1 : Tumor volume (mm3) of MOLT-4-grafted female NOD SCID mice upon treatment with vehicle (HPBCD/HC1) or Example 24 (2.5, 5 and 7.5 mg/kg, administered IV, Q3D6, n=7).
    Vehicle (HPBCD/HCI)
    Example 24 2.5 MK
    Example 24 5 MK
    Example 24 7.5 MK
    Treatement (IV, Q3D6)
    2/2
    Figure 2 : % of body weight loss of MOLT-4-grafted female NOD SCID mice upon treatment with vehicie (HPBCD/HCI) or Example 24 (2.5, 5 and 7.5 mg/kg, administered IV, Q3D6, n=7).
    % Body weight loss Mean +/- SEW
    Time after treatment initiation (days)
    Vehicie (HPBCD/HCI, IV Q3D6) Example 24 2.5 MK
OA1202200028 2019-07-29 2020-07-28 6,7-dihydro-5H-pyrido[2,3-c] pyridazine derivatives and related compounds as Bcl-xL protein inhibitors and pro-apoptotic agents for treating cancer. OA20573A (en)

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