NZ751740B2

NZ751740B2 - Macrocyclic deaza-purinones for the treatment of viral infections

Info

Publication number: NZ751740B2
Application number: NZ751740A
Authority: NZ
Inventors: Eric Pierre Alexandre Arnoult; Jeanfrancois Bonfanti; Frederic Marc Maurice Doublet; Jerome Michel Claude Fortin; Philippe Muller; Pierre Jeanmarie Bernard Raboisson; Jean Francois Bonfanti; Pierre Jean Marie Bernard Raboisson
Original assignee: Janssen Sciences Ireland Uc
Priority date: 2013-03-29
Filing date: 2014-03-28
Publication date: 2021-03-02

Abstract

This invention relates to macrocyclic deaza-purinones derivatives of formula (I), processes for their preparation, pharmaceutical compositions, and their use in treating viral infections.

Description

Macrocyclic deaza-purinones for the treatment of viral infections This invention and the invention of NZ 711344, from which this application was divided, relate to macrocyclic deaza-purinones derivatives, processes for their preparation, pharmaceutical compositions, and their use in treating viral infections.

The present invention relates to the use of macrocyclic purinones derivatives in the treatment of viral ions, immune or inflammatory disorders, whereby the modulation, or agonism, of toll-like-receptors (TLRs) is involved. Toll-Like Receptors are primary transmembrane proteins characterized by an extracellular leucine rich domain and a asmic extension that contains a conserved . The innate immune system can recognize pathogen- associated molecular patterns via these TLRs expressed on the cell surface of certain types of immune cells. Recognition of foreign pathogens activates the production of cytokines and upregulation of mulatory molecules on phagocytes. This leads to the modulation of T cell behaviour.

It has been estimated that most mammalian species have between ten and fifteen types of Toll- like receptors. Thirteen TLRs (named TLR1 to TLR13) have been identified in humans and mice together, and equivalent forms of many of these have been found in other mammalian species.

However, equivalents of certain TLR found in humans are not present in all mammals. For example, a gene coding for a protein analogous to TLR10 in humans is t in mice, but appears to have been damaged at some point in the past by a retrovirus. On the other hand, mice express TLRs 11, 12, and 13, none of which are represented in humans. Other mammals may express TLRs which are not found in humans. Other non-mammalian species may have TLRs distinct from mammals, as demonstrated by TLR14, which is found in the Takifugu fish.

This may complicate the process of using experimental s as models of human innate immunity.

For detailed reviews on toll-like ors see the following journal articles. Hoffmann, J.A., , 426, , 2003; Akira, S., Takeda, K., and , T., Annual Rev. Immunology, 21, p335-376, 2003; Ulevitch, R. J., Nature Reviews: logy, 4, p512-520, 2004.

Compounds indicating activity on Toll-Like receptors have been previously described such as purine derivatives in , adenine derivatives in WO 98/01448 and WO 99/28321, and pyrimidines in . r, there exists a strong need for novel Toll-Like receptor modulators having preferred selectivity, higher potency, higher metabolic stability, and an improved safety profile compared to the compounds of the prior art.

In the ent of certain viral infections, regular injections of interferon (IFN-alfa) can be administered, as is the case for hepatitis C virus (HCV). For more ation see Fried et al.

Peginterferon-alfa plus ribavirin for chronic hepatitis C virus infection, N Engl J Med 2002; 347: 975-82. Orally available small molecule IFN inducers offer the ial advantages of reduced immunogenicity and convenience of administration. Thus, novel IFN inducers are potentially effective new class of drugs for treating virus infections. An example of a small molecule IFN inducer having antiviral effect see De Clercq, E.; Descamps, J.; De Somer, P. Science 1978, 200, 563-565.

IFN-alfa is also given in combination with other drugs in the ent of certain types of cancer (Eur. J. Cancer 46, 2849–57, and Cancer Res. 1992, 52, 1056). TLR 7/8 agonists are also of interest as vaccine adjuvants because of their ability to induce pronounced Th1 response (Hum. es 2010, 6, 322-335, and Hum. Vaccines 2009, 5, 381−394).

In a particular aspect, the present invention provides a method for the preparation of a compound according to formula (I) and pharmaceutically accepted salts f, wherein X is O, NH or S, Y ents an aromatic ring or heterocyclic ring comprising at least a nitrogen, optionally substituted by one or more substituents independently selected from C1-6alkyl, C1-4alkoxy, trifluoromethyl or halogen, Z represents C1-10 saturated or unsaturated alkyl optionally tuted by an alkyl or alkylhydroxyl; or Z represents C1-6alkyl-NH-C(O)-C1-6alkyl-, C1-6 alkyl-NH- or C1-6alkyl-NH-C(O)-C1-6alkyl-O-; or Z represents C1-10alkyl-O- n said alkyl is unsaturated or saturated and can optionally be substituted by an alkyl or alkylhydroxyl , or Z represents C1-6alkyl-O-C1-6alkyl- wherein said alkyl is unsaturated or saturated and can optionally be substituted by an alkyl or ydroxyl or Z represents C1-6alkyl-O-C1-6alkyl-O- wherein said alkyl is unsaturated or saturated and can optionally be substituted by an alkyl or alkylhydroxyl; (followed by page 2a) -2a- wherein said method comprises the steps of: (a) providing a compound according to formula (II): N O X N (II) wherein n, X, and Y are as for formula (I); Q represents C1-10 saturated or unsaturated alkyl optionally substituted by an alkyl or alkylhydroxyl; or Q represents kyl-NH-C(O)-C1-6alkyl-, kyl-N(tert-butyloxycarbonyl)- or C1-6alkyl-NH-C(O)-C1-6alkyl-O-; or Q represents C1-10alkyl-O- wherein said alkyl is rated or saturated and can optionally be substituted by an alkyl or ydroxyl, or Q represents C1-6alkyl-O-C1-6alkyl- wherein said alkyl is unsaturated or saturated and can optionally be substituted by an alkyl or alkylhydroxyl, or Q ents C1-6alkyl-O-C1-6alkyl-O- wherein said alkyl is unsaturated or saturated and can optionally be substituted by an alkyl or alkylhydroxyl; (b) contacting the compound of formula (II) with acetic acid; or TiCl3 and tetrahydrofuran; and wherein for compounds of formula (II) wherein Q represents C1-6alkyl-N(tertbutyloxycarbonyl )-, said method further comprises the step of (c) ting the compound obtained in step (b) with trifluoroacetic acid and dichloromethane.

[FOLLOWED BY PAGE 2b] -2b- In accordance with the present ion a compound of formula (I) is provided and pharmaceutically accepted salts thereof, wherein X is oxygen, nitrogen or sulfur Y represents an aromatic ring or heterocyclic ring comprising at least a nitrogen, optionally substituted by one or more substituents independently selected from C1-6alkyl, C1-4alkoxy, trifluoromethyl or halogen, Z represents C1-10 saturated or unsaturated alkyl optionally substituted by an alkyl or alkylhydroxyl; or Z ents C1-6alkyl -NH-C(O)- C1-6alkyl-, C1-6alkyl-NH- or kyl -NH-C(O)- C1-6alkyl -O-; or Z represents C1-10alkyl -O- wherein said alkyl is unsaturated or saturated and can optionally be substituted by an alkyl or ydroxyl , or Z represents C1-6alkyl-O-C1-6alkyl- wherein said alkyl is unsaturated or saturated and can optionally be substituted by an alkyl or alkylhydroxyl or Z represents C1-6alkyl-O-C1-6alkyl-O- wherein said alkyl is unsaturated or saturated and can optionally be substituted by an alkyl or ydroxyl.

Preferred nds having one of the following formula’s according to the invention were ed from the group of: [FOLLOWED BY PAGE 3] Part of the invention is also a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt, solvate or rph thereof together with one or more pharmaceutically acceptable excipients, diluents or carriers.

Furthermore to the invention belongs a compound of formula (I) or a pharmaceutically acceptable salt, solvate or polymorph thereof or a pharmaceutical ition above mentioned for use as a medicament.

The invention also relates to a nd of formula (I) or a pharmaceutically acceptable salt, solvate or polymorph thereof or a pharmaceutical ition above ned for use in the treatment of a disorder in which the modulation of TLR7 is involved.

The term "alkyl" refers to a straight-chain or branched-chain mostly saturated (but in specific compounds ing to the invention being unsaturated) aliphatic hydrocarbon containing the speciﬁed number of carbon atoms.

The term "halogen" refers to ﬂuorine, chlorine, e or iodine.

The term "alkoxy" refers to an alkyl (carbon and en chain) group singular bonded to oxygen like for ce a y group or ethoxy group.

Pharmaceutically acceptable salts of the compounds of formula (I) include the acid addition and base salts thereof Suitable acid addition salts are formed from acids which form non-toxic salts.

Suitable base salts are formed from bases which form non-toxic salts.

The compounds of the invention may also exist in unsolvated and solvated forms. The term "solvate" is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol.

The term "polymorph" refers to the ability of the compound of the invention to exist in more than one form or crystal structure.

The compounds of the invention can be present in a led "tautomer(s)" formation refering to isomers of organic compounds that y interconvert by a chemical reaction called tautomerization. This reaction s in the formal migration of a hydrogen atom or proton, anied by a switch of a single bond and adjacent double bond.

The compounds of the present invention may be administered as crystalline or amorphous products. They may be obtained for example as solid plugs, powders, or ﬁlms by methods such as precipitation, crystallization, freeze drying, spray drying, or evaporative . They may be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drugs. Generally, they will be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term "excipient" is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient s y on factors such as the particular mode of administration, the effect ofthe excipient on solubility and ity, and the nature of the dosage form.

The compounds of the present invention or any subgroup fmay be ated into various ceutical forms for administration purposes. As appropriate compositions there may be cited all compositions usually employed for systemically administering drugs. To prepare the pharmaceutical itions of this invention, an effective amount of the particular compound, optionally in addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for stration. These pharmaceutical compositions are desirably in unitary dosage form suitable, for example, for oral, , or percutaneous administration. For example, in ing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed such as, for example, water, s, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, s, emulsions, and solutions; or solid carriers such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules, and tablets. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage 3O unit forms, in which case solid pharmaceutical carriers are obviously employed. Also included are solid form preparations that can be converted, shortly before use, to liquid forms. In the compositions suitable for percutaneous administration, the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions. These compositions may be administered in various ways, e. g., as a transdermal patch, as a spot-on, as an ointment. The compounds of the present invention may also be administered via inhalation or insufﬂation by means of methods and formulations employed in the art for administration via this way. Thus, in l the compounds of the present ion may be administered to the lungs in the form of a solution, a suspension or a dry powder.

It is especially advantageous to ate the aforementioned ceutical compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient ated to produce the desired therapeutic effect in association with the required ceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, powder packets, wafers, suppositories, injectable solutions or suspensions and the like, and segregated multiples thereof Those of skill in the treatment of infectious diseases will be able to determine the ive amount from the test results ted hereinafter. In general it is contemplated that an effective daily amount would be from 0.01 mg/kg to 50 mg/kg body weight, more preferably from 0.1 mg/kg to 10 mg/kg body weight. It may be appropriate to administer the required dose as two, three, four or more sub-doses at riate intervals throughout the day. Said ses may be formulated as unit dosage forms, for example, containing 1 to 1000 mg, and in particular 5 to 200 mg of active ingredient per unit dosage form.

The exact dosage and frequency of administration depends on the particular compound of formula (I) used, the particular condition being treated, the severity of the condition being treated, the age, weight and general physical condition of the particular patient as well as other medication the individual may be taking, as is well known to those skilled in the art.

Furthermore, it is evident that the effective amount may be lowered or increased depending on the response of the treated t and/or depending on the tion of the physician prescribing the compounds of the instant invention. The effective amount ranges ned above are therefore only guidelines and are not intended to limit the scope or use of the invention to any extent.

Overall Scheme in the preparation of ﬁnal products: method 1 A[/ O\/\ I \ O\/\ J): O K2003 acetone NHs aq 3'21.on)LO N / NH2 NH2 HON R OZN / Grubbs-Hoveyda gzN / N 2nd generation I Fe, ACOH N 0*N \ HO , I o —> / \ NaH, THF Oi\O/\O N o DCE ) water / 0 KO/O o SOCI2 o CHZCIZ HO CI (31 B1 Synthesis of intermediate B1 SOC12 (80 mL; 1.11 mol) was added drop wise to a mixture of G1 (19.1 g; 111 mmol) in CH2C12 (230 mL) at rt. The mixture was stirred at rt for 16 h. The solvent was ated and the residue was solubilized in CH2C12 and treated with a saturated s solution of NaHC03 until basic pH. The layers were separated and the aqueous layer was extracted with CH2C12 (twice). The combined organic layers were dried over MgSO4, ﬁltered and concentrated in vacuo to give 20.1 g of a brown oil. The crude compound was used in the next step t further puriﬁcation.

O\/\ 2BO/JEHIZO WE/OH/ cho3N:|cetone 5E1?) Synthesis of intermediate C1 2014/056270 The reaction was performed on two batches in parallel (13.6 mmol and 27.1 mmol ofA1).

To a solution of A1 (5.0 g; 13.6 mmol) and K2C03 (3.75 g; 27.1 mmol) in acetone (100 mL) were added B1 (4.46 g; 24.4 mmol) and NaI (2.23 g; 14.9 mmol) at rt. The mixture was stirred at rt for 16 h. The mixture was ﬁltered and the e was ated in vacuo to give a brown oil.

The two batches were combined and puriﬁed by preparative LC (Irregular SiOH 15-40 um, 220 g Grace, mobile phase gradient: CHzClz/EtOAc from 100/0 to 80/20). The fractions containing product were combined and the solvent was removed in vacuo to give 18.6 g of intermediate C1 (89% yield).

JOAN O m, CAN ' 2 Br N Br KI\ Br N NH2 D1 Synthesis of intermediate D1 To a solution of C1 (18.6 g; 36.1 mmol) in THF (300 mL) was added an s solution of NH3 (30%) (290 mL) at rt, and the mixture was stirred at rt for 16 h. The e was taken up with EtOAc and saturated NaCl solution, the layers were separated and the organic layer was dried over MgSO4, ﬁltered and the solvent was removed under reduced pressure to give 16.7 g of a yellow-orange oil. The crude was dried under high vacuum to give 16.5 g of a sticky yellow- orange solid, which was used directly in the next step. 0\/\ i HOV OZN o N —’ o \ N o/V/ 2 No2 NaH THF / A /\ | O O Br N NH2 Synthesis of intermediate E1 NaH (60% in oil) (1.75 g; 43.7 mmol) was added portion wise to allyl alcohol (50 mL) at rt. The mixture was stirred at rt for 30 min before being added drop wise to a solution of D1 (5 g; 11.1 mmol) in THF (124 mL) at 0°C. The resulting mixture was then stirred at rt for 1 h and was poured in saturated NH4C1 aqueous solution. EtOAc and saturated NaCl aqueous solution were added, the layers were separated and the aqueous layer was ted with EtOAc (once). The combined organic layers were dried over MgSO4, d and the solvent was removed under reduced pressure to give a yellow oil. The crude nd was puriﬁed by preparative LC (Irregular SiOH 15-40 um, 120 g Grace, liquid injection, mobile phase gradient: from Heptane/EtOAc 100/0 to 50/50) to give 4.04 g of intermediate E1 as a yellow oil (79% yield). k OzNﬂ Grubbs-HoveydaD | 2nd tion \ ON/ 0 A DCE o O/\ Synthesis of intermediate Fl The reaction was performed in 2 batches of 850 mg and 2 s of 1 g of El.

Herein is the procedure for one batch of 850 mg: In a schlenk ﬂask, a solution of El (0.85 g; 1.98 mmol) and chlorodicyclohexylborane (1M solution in hexane) (400 uL; 400 umol) in dichloroethane (570 mL) was stirred at 80°C under N2 atmosphere for 1 h. Grubbs-Hoveyda catalyst 211d generation (124 mg; 198 umol) was added and the mixture was d at 120°C for 16 h. The mixture was degassed by N2 bubbling for 10 min and further Grubbs-Hoveyda catalyst 2Ild generation (124 mg; 198 umol) and chlorodicyclohexylborane (1M solution in hexane) (400 uL; 400 umol) were added. The mixture was stirred at 120°C for 20 h.

The 2 batches were mixed and a ruthenium scavenger (SiliaBond DMT from SiliCycle) (10.4 g; 6.35 mmol) was added and the mixture was stirred at rt for 20 h. The reaction mixture was ﬁltered through a pad of celite and the solvent was removed under d pressure to give a brown residue.

The residue was combined with the residue obtained from the two batches of 1 g of E1. The resulting brown residue was puriﬁed by preparative LC (Irregular SiOH 15-40 um, 120 g Grace, dry loading, mobile phase nt: from Heptane/EtOAc 100/0 to 0/100) to give 1.19 g of a brown solid. The brown solid was further puriﬁed by preparative LC onary phase: irregular bare silica 40 g, mobile phase gradient: from CHzClz/EtOAc 90/10 to 80/20) to give 705 mg of a yellow solid. The yellow solid was r puriﬁed by achiral SFC (stationary phase: Amino 6um 150x21.2mm), mobile phase: Gradient from 85% C02, 15% MeOH to 65% C02, 35% MeOH) to give 660 mg of intermediate F1 as a yellow solid (19% yield, E isomer).

OzN/ N o N / A \l Fe,AcOH N O "0%m 3 N ?/ ~\ 0? Synthesis of ﬁnal compound 1 A e of F1 (570 mg; 1.42 mmol) and iron (795 mg; 14.2 mmol) in AcOH (21 mL) and water (4.2 mL) was stirred at 50°C for 2 h. The mixture was concentrated until dryness. DMF was added, the e was sonicated, heated and d through a pad of celite and the celite was rinsed with hot DMF. An iron scavenger (SiliaBond ole from SiliCycle) (25.4 g; 29.5 mmol) was added to the ﬁltrate and the mixture was stirred at rt for 16 h. The mixture was ﬁltered through celite, the celite was rinsed with DMF and the ﬁltrate was concentrated in vacuo to give 620 mg of a brown solid. The crude was puriﬁed by preparative LC (irregular SiOH, 15- 40 um, 30 g Merck, mobile phase gradient: from CHzClz/MeOH/NHgaq 98/2/02 to 85/15/ 1.5) to give 360 mg of ﬁnal compound 1 as an off-white solid (75% yield). 2): O PPh3—>/DIAD DAN HF 2 / Br N Br Alternative synthesis of intermediate C1 At 0°C, diisopropylazodicarboxylate (DIAD) (3.0 mL,15.0 mmol) was added drop wise to a mixture ofAl (3.70 g, 10.028 mmol), G1 (1.98 g, 12.0 mmol) and PPh3 (3.94 g, 15.0 mmol) in THF (70 mL). The mixture was stirred at rt for 12 h. EtOAc and water were added. The layers were ed. The organic layer was washed with water, dried over MgSO4, ﬁltered and the solvent was evaporated. The crude was d by preparative LC on (Irregular SiOH 20-45 um 450 g Matrex), mobile phase (85% Heptane, 15% AcOEt) to give 4.5 g of intermediate C1 (87% yield). -1 0- Overall Scheme in the preparation of ﬁnal products: method 2 NH2 NH2 02N / Wlkinson'scatalyst 02N / oi N N THF/MeOH O FeyAcoH \ Ho— Synthesis of ﬁnal compound 2 Compound 2 was obtained using the procedure to e compound 1 (54 mg, 44% yield).

Overall Scheme in the preparation of ﬁnal products: method 3 AL / 0 O NH K2C03Y acetone OJLN NH3 aq )fINO —’ IN/ N | + Nal 02 Br \N / Br CI \ | Br N Br \ A1 '1 Br N NH2 NH2 NH2 0 N2 HOW \ IN Grubbs catalyst 002N/ N OiN N / 2nd Generation I Fe, ACOH N , N O/\/\ | 0 HO— | N02 + Nal 2 / Br N Br CI I Br N Br A1 I1 Synthesis of intermediate J 1 11 (5.9 g; 35.6 mmol) was added to a solution of A1 (7.3 g; 19.8 mmol), K2C03 (5.5 g; 39.6 mmol) and NaI (3.3 g; 21.8 mmol) in acetone (145 mL). The mixture was stirred at rt for 20 h.

The mixture was ﬁltered through a pad of celite and the ﬁltrate was evaporated in vacuo to give an orange solid. The residue was taken up in CHzClz. The precipitate was ﬁltered and the ﬁltrate was concentrated in vacuo to give 13 g of a yellow oil. The crude nd was puriﬁed by preparative LC ular SiOH 15-40 um, 300 g Interchim, mobile phase gradient: from Heptane/EtOAc 100/0 to 80/20). The ons containing product were combined and the solvent was d in vacuo to give 7.1 g of intermediate J 1 (72% yield) as a yellow oil.

JL 0 NH3aq )3 N —’ B/ENIB o N / THF 2ﬂ\ No2 r r Br N NH2 Synthesis of intermediate K1 In a schlenk ﬂask, a solution of J 1 (7.1 g; 14.2 mmol) in THF (130 mL) and an aqueous solution ofNH3 (30%) (130 mL) was stirred at rt for 16 h. The mixture was taken up with EtOAc and a saturated water on of NaCl, layers were separated. The organic layer was dried over MgSO4, ﬁltered and concentrated in vacuo to give 6.4 g of a yellow oil (100% yield). 0 02N / N o N \ om 2 N / NaH,THF | A /\ o 0 Br N NH2 K1 L1 Synthesis of intermediate Ll NaH (2.2 g; 54.2 mmol) was added portion wise at rt and under N2 atmosphere to 3-buten—l-ol (76 mL). The mixture was stirred at rt for 30 min before being added drop wise at 0°C to a solution of K1 (5.9 g; 13.6 mmol) in THF (150 mL). The resulting mixture was stirred at 0°C for 1 h. The mixture was poured into an aqueous saturated NH4Cl solution. EtOAc and satured aqueous NaCl solution were added, the layers were ted. The organic layers was dried over MgSO4, ﬁltered and concentrated in vacuo to give a yellow residue which was azeotropically distilled with e (once) to give 6.6 g of a yellow oil. The crude compound was d by preparative LC (Irregular SiOH 15-40 um, 220 g Grace, mobile phase gradient: from Heptane/EtOAc 100/0 to 50/50). The fractions containing product were ed and the solvent was removed in vacuo to give 4.46 g of intermediate L1 (77% yield) as a yellow oil.

NH2 NH2 0 N O N 2 ﬂ O2 / \ Grubbs catalyst |N \ 2nd enerat'on \ 9 ' N O/\/\ CAN 0 CH CI / sis of intermediate M1 The reaction was performed in 2 batches.

Typical procedure for one batch: A on ole (2.45 g; 5.75 mmol) in dry CHzClz (1.7 L) was degassed by N2 bubbling for 15 min. Grubbs catalyst 211d generation (488 mg; 574 umol) was added and the mixture was stirred at rt for 72 h. SiliaBond DMT (7.66 g; 4.59 mmol) was added and the mixture was stirred at rt for 16 h. The 2 batches were combined and ﬁltered through celite. The ﬁltrate was concentrated in vacuo to give a black solid. The crude compound was puriﬁed by preparative LC (Irregular SiOH 15-40 um, 150 g Merck, mobile phase gradient: from Heptane/EtOAc 100/0 to 50/50).

The fractions containing product were combined and the solvent was removed in vacuo to give -1 3- 230 mg of fraction 1 and 2.3 g of fraction 2. on 2 was re-puriﬁed by preparative LC (Stationary phase: irregular SiOH 40 um 120 g, mobile phase: e/CHzClz/MeOH 55/43/2).

The isolated compound was combined with ﬁaction 1 and puriﬁed by achiral SFC onary phase: Chiralpak IC 5 um 250x20 mm, mobile phase: 70% C02, 30% iPrOH) to give 1.51 g of intermediate Ml (33% yield, isomer E) as a yellow solid.

OZN / N 0 N / \l Fe AcOH N A ’ I N o , Ho— Overall Scheme in the preparation of ﬁnal products: method 4 NH2 NH2 NH2 OZN / N Wilkinson'scatalyst OZN / / O I o N AGOH AN \ THF/MeOH ‘N szPd/C :2" / Ho— OAN \ ’ I \ o \ 0 N o / H2 EtOH O | | M1 N1 01 -1 4- Synthesis of intermediate Nl Wilkinson’s catalyst (46 mg; 50.2 umol) was added to a solution of M1 (200 mg; 502 umol) in THF/MeOH (50/50) (50 mL) purged by N2 bubbling for 15 min. The mixture was enated (7 bars) at rt for 20 h. The mixture was purged by N2 bubbling for 15 min, further Wilkinson’s catalyst (46 mg; 50.2 umol) was added and the reaction mixture was hydrogenated (7 bars) at rt for 16 h. The on mixture was concentrated in vacuo to give a green oil. The oil was puriﬁed by preparative LC (Irregular SiOH 15-40 um, 25 g Merck, dry loading, mobile phase gradient: from e/EtOAc 100/0 to 70/30) to give 130 mg of intermediate N1 as a yellow solid (66% yield).

Synthesis of intermediate 01 In a pressure vessel reactor, N1 (110 mg; 275 umol) was hydrogenated in EtOH (5 mL) with Pd/C (10%) (30 mg; 28.5 umol) as catalyst at 40°C (3 bars) for 6 h. The catalyst was d by ﬁltration over celite, the celite was washed with EtOH and the ﬁltrate was evaporated under vacuum to give 100 mg of a yellow residue (98% yield). Intermediate 01 was used in the next step without r puriﬁcation.

Synthesis of ﬁnal compound 4 In a sealed tube, 01 (100 mg; 270 umol) in pure acetic acid (5 mL) was stirred at rt for 90 min.

The solvent was removed under reduced pressure to give a yellow residue. The residue was taken up with CHzClz and the solvent was removed under d pressure (twice) to give 87 mg of a yellow-green solid. The solid was azeotropically distilled with e (four times), and was then triturated and sonicated in EtzO. The mixture was ﬁltered off (glass frit n05) to give 75 mg of ﬁnal compound 4 (77% yield, acetate salt). -1 5- Overall Scheme in the preparation of ﬁnal ts: method 5 I / ON\ 0V | N \ 0V 2 K2C03 acetone NH3 aq f1 OJLN O/\/ Nal EHZOZ 2 / N02 \L NH2 NH2 NH2 ON Grubbs—Hove da OZN / N / N N HOW 2 / N O .y o Ho— I / N\ 0V I o N\ 0 O/\/ OJLN NH—3>aq 2 N02 THF )ﬁi Br \N Br -1 5- Synthesis of intermediate R1 In a k ﬂask, to a solution of Q1 (3.28 g; 6.36 mmol) in THF (52 mL), was added an aqueous solution ofNH3 (30%) (52 mL) at rt. The mixture was stirred at rt for 26 h and further aqueous solution of NH3 (10 mL) was added and the mixture was stirred at rt for 4 h. The e was taken up with EtOAc and saturated aqueous solution of NaCl, the layers were separated and the organic layer was dried over MgSO4, ﬁltered and the solvent was removed under reduced pressure to give 2.74 g of intermediate R1 as a yellow oil (87% yield).

N‘ 0V o \h NH2 OJLN Ho/\/\ 02N / 0 IN 2 N02 —> \ OW / / N | NaH’THF I Br \N \ N GAGA Synthesis of ediate Sl NaH (60% in oil) (888 mg; 22.2 mmol) was added portion wise to 3-buten—l-ol (30 mL; 354 mol) at rt. The mixture was stirred at rt for 30 min before being added drop wise to a solution ofR1 (2.74 g; 5.63 mmol) in THF (62 mL) at 0°C. The ing mixture was stirred at rt for l h and was poured in NH4Cl saturated aqueous solution. EtOAc and NaCl ted aqueous solution were added, the layers were separated and the aqueous layer was extracted with EtOAc (once). The combined organic layers were dried over MgSO4, ﬁltered and the solvent was removed under reduced pressure to give a yellow oil. The oil was puriﬁed by preparative LC (Irregular SiOH 15-40 um, 80 g Grace, dry loading, mobile phase gradient: from Heptane/EtOAc 100/0 to 20/80) to give 1.06 g of intermediate S1 as a yellow e (42% yield).

\ NH2 0 2Nﬂ g"Ebb:::tleydaL 82 n genera |0n / N OW L0AM /N|\Nﬁko Synthesis of intermediate T1 The reaction was med in two batches of 480 mg of intermediate S l.

Herein is reported the procedure for one batch: -1 7- In a schlenck ﬂask, a solution of S1 (480 mg; 1.08 mmol) and chlorodicyclohexylborane (1M in hexane) (216 uL; 216 umol) in dry dichloroethane (300 mL) was stirred at 80°C and under N2 here for 1 h. Grubbs-Hoveyda catalyst 2Ild generation (68 mg; 108 umol) was added and the mixture was stirred at 120°C for 2 h.

The two batches were mixed, SiliaBond DMT (2.84 g; 1.73 mmol) was added and the mixture was stirred at rt for 20 h.

The mixture was ﬁltered through a pad of celite, the celite was washed with EtOAc and the ﬁltrate was evaporated in vacuo to give a brown solid. The brown solid was puriﬁed by preparative LC (Irregular SiOH 15-40 um, 40 g Grace, dry loading, mobile phase gradient: CHzClz/EtOAc from 100/0 to 20/80) to give 610 mg of a yellow residue (mixture of E and Z isomers, intermediate U1). 310 mg of intermediate U1 was puriﬁed by Reverse phase (Stationary phase: Nucleodur-Sphinx rp 5um 21x150 mm, mobile phase: nt from 70% formic acid 0.1%, 30% MeCN to 0% formic acid 0.1%, 100% MeCN) to give 195 mg ofintermediate T1 (E isomer) as a yellow solid (22% yield).

NH2 NH2 OZN / N / N N / O m Fe,AcOH HO— The residue was diluted in DMF and ﬁltered through a pad of celite. SiliaBond imidazole (12.7 g; 14.7 mmol) was added to the ﬁltrate and the mixture was stirred at rt for 16 h. The mixture was ﬁltered through a pad of celite and the ﬁltrate was evaporated in vacuo to give a brown solid. The brown solid was d by preparative LC (irregular SiOH 15-40 um, 12 g Grace, dry loading, mobile phase gradient: from /MeOH/NHgaq 97/3/03 to 80/20/2) to give 65 mg of an off-white solid. The solid was puriﬁed by Reverse phase (Stationary phase: X-Bridge- C18 5um 30*150mm, mobile phase gradient: from H20(0.5%NH4C03)/MeOH 70/30 to 0/100) to give 43 mg of ﬁnal compound 5 as a white solid (31% yield, E isomer). -1 8- Overall Scheme in the ation of ﬁnal products: method 6 NH2 NHz OZN / O Ho—eAGOH NQON| 02kN water N THF/MeOH \ IDA)» grvi ofl) Synthesis of intermediate V1 Wilkinson’s catalyst (58 mg; 62.6 umol) was added to a solution of U1 (Z/E mixture, 260 mg; 626 umol) in THF/MeOH (50/50) (66 mL) purged by N2 bubbling for 15 min. The mixture was hydrogenated (7 bars) at rt for 16 h. Further Wilkinson’s catalyst (58 mg; 62.6 umol) was added and the mixture was hydrogenated (7 bars) at rt for 6 h. The reaction mixture was concentrated in vacuo to give a brown solid. The solid was puriﬁed by preparative LC ular SiOH 15-40 pm, 25 g Merck, dry loading, mobile phase gradient: from Heptane/EtOAc 100/0 to 50/50) to give 250 mg of intermediate V1 as a yellow oil (54% yield).

Synthesis of ﬁnal compound 6 A mixture ofVl (238 mg; 359 umol) and iron (120 mg; 2.16 mmol) in acetic acid (20 mL) and water (2.2 mL) was stirred at 80°C for 6 h. Further iron (120 mg; 2.16 mmol) was added and the mixture was stirred at 80°C for 20 h. r iron (120 mg; 2.16 mmol) was added and the mixture was stirred at 80°C for 5 h. The mixture was concentrated in vacuo to give a residue.

The residue was diluted in DMF and ﬁltered through a pad of celite. SiliaBond imidazole (11.1 g; 12.9 mmol) was added to the ﬁltrate and the mixture was stirred at rt for 16 h. The mixture was ﬁltered through a pad of celite and the ﬁltrate was evaporated in vacuo to give a brown solid. The solid was puriﬁed by preparative LC ular SiOH 15-40 pm, 12 g Grace, dry loading, mobile phase gradient: from CHzClz/MeOH/NHgaq 97/3/03 to 80/20/2) to give 32 mg of an off-white solid. The solid was taken up with water, triturated and ted. The resulting sion was ﬁltered off (glass frit n°5) and washed with EtZO (twice) to give 19 mg of ﬁnal compound 6 as an off-white solid (15% yield). -1 9- Overall Scheme in the preparation of ﬁnal products: method 7 O | CAN "0% Enrzbbsnzia'z: ) 0 M351\ O/W 9—» / N02 N l NaH,THF CH CI2 2 \ GAGA X1 10 O\/\ O I OJLN HOW /J —————————> 0201b: NC» o \\ N O/"\\//§§ | NaH, THF \ A O O/\ Br N NH2 D1 W1 Synthesis of ediate W1 NaH (60% in oil) (2.1 g; 52.1 mmol) was added portion wise to 3-buten—l-ol (74 mL) at rt. The mixture was stirred at rt for 30 min before being added drop wise to a solution of D1 (5.97 g; 13.2 mmol) in THF (150 mL) at 0°C. The resulting mixture was then d at rt for 2 h 30 min and was poured in aqueous saturated on of NH4Cl. EtOAc and saturated aqueous solution ofNaCl were added, the layers were separated and the aqueous layer was extracted with EtOAc (once). The combined organic layers were dried over MgSO4, ﬁltered and the solvent was removed under reduced pressure to give 6.77 g of a yellow oil. The crude was puriﬁed by preparative LC (Irregular SiOH 15-40 urn, 120 g Grace, liquid ion, mobile phase gradient: from Heptane/EtOAc 100/0 to 50/50) to give 5.12 g of intermediate W1 as a yellow oil (83% yield).

K OzNﬂ 0 N Grubbs st 02m | 2nd 9eneration CAN \ 0 O \ 0W U2N 9 \ o O/\ Synthesis of intermediate X1 To a solution of W1 (3 g; 6.78 mmol) in CHzClz (1.3 L) degassed by N2 bubbling for 15 min was added Grubbs catalyst 211d tion (578 mg; 678 umol) at rt. The solution was stirred at rt for h. SiliaBond DMT (8.89 g; 5.42 mmol) was added and the mixture was stirred at rt for 20 h.

The on mixture was ﬁltered through a pad of celite and the solvent was removed under reduced pressure to give a brown residue, which was combined with another batch (0.226 mmol scale). The combined residue was taken up with MeOH, sonicated and heated to give a precipitate which was ﬁltered off to give 3.2 g of a brown solid. The crude was puriﬁed by preparative LC (irregular SiOH, 15-40 um, 220 g grace, liquid injection, mobile phase gradient: from CHzClz/EtOAc 100/0 to 50/50) to give 1.7 g of ﬁaction l as a pale brown solid. Fraction 1 was taken up with MeOH, sonicated and heated to give a precipitate which was ﬁltered off to give 820 mg of fraction 2 as a pale brown solid.

The ﬁltrate was concentrated in vacuo to give 590 mg of fraction 3 as a brown residue (impure X1). Fraction 2 was puriﬁed by preparative LC onary phase: cal bare silica 5um 150x30.0mm, mobile phase gradient: from Heptane/EtOAc 85/15 to 0/100) to give 435 mg of intermediate X1 as a yellow solid (E , 15% yield).

Fraction 3 was d with another batch.

OZN / O N CAN \ o Fe,AcOH H04 l —> N )K©/0\ O water \ \ Synthesis of ﬁnal compound 10 A mixture ole (430 mg; 1.04 mmol) and iron (579 mg; 10.4 mmol) in acetic acid (43 mL) and water (3 mL) was stirred at 50°C for 4 h. The mixture was concentrated until dryness. DMF was added. The mixture was sonicated, heated and ﬁltered through a pad of celite and the celite was -21 _ rinsed with hot DMF. SiliaBond imidazole (17.9 g; 20.8 mmol) was added to the ﬁltrate and the mixture was stirred at rt for 16 h. The e was ﬁltered through , the celite was rinsed with DMF and the ﬁltrate was concentrated in vacuo to give 670 mg of crude compound. The crude was puriﬁed by preparative LC (irregular SiOH, 15-40 um, 25 g Merck, mobile phase gradient: from CHzClz/MeOH/NHgaq 98/2/02 to 85/15/ 1.5) to give an off-white solid. The solid was dried at 40°C under reduced pressure during 20 h to give 295 mg of ﬁnal compound 10 as an off-white solid (84% yield).

Overall Scheme in the ation of ﬁnal products: method 8 "£1503 Wilkinson'5 catalyst O | HO— Synthesis of ﬁnal compound 14 A mixture on1 (55 mg; 0.132 mmol) and iron (74 mg; 1.32 mmol) in acetic acid (5.5 mL) and water (0.4 mL) was stirred at 50°C for 20 h. More iron (37 mg; 0.66 mrnol) was added and the mixture was stirred at 50°C for 3 h. More iron (37 mg; 0.66 mrnol) was added and the mixture was stirred at 50°C for 20 h. The e was ﬁltered through a pad of celite and the celite was rinsed with acetic acid. More iron (74 mg; 1.32 mmol) was added to the ﬁltrate and the mixture was stirred at 50°C for 88 h. More iron (74 mg; 1.32 mmol) was added to the ﬁltrate and the mixture was stirred at 80°C for 24 h. The cyclisation was not complete. The mixture was concentrated in vacuo to give a brown solid.

TiC13 (8.60 mL; 10.0 mrnol) was added drop wise to a solution of the brown solid in C (19 mL).

The e was stirred at rt ght. The mixture was basiﬁed by addition of K2C03 powder at 0°C. The resulting mixture was ﬁltered through a pad of celite and the celite was washed with a solution of AcOEt/MeOH (8:2). The ﬁltrate was concentrated in vacuo. The crude solid was puriﬁed by preparative LC (irregular SiOH, 15-40 um, 10 g Merck, dry loading, mobile phase gradient: from CHzClz/MeOH/NHgaq 98/2/02 to 85/15/15). The fractions containing t were combined and the solvent was removed in vacuo to give 20 mg of ﬁnal compound 14 (12% yield ) as an off-White solid.

Overall Scheme in the preparation/Jof ﬁnal products: method 9 2CI NEt3 A J CH2CI2 LiCI HO MsO / O\/\\ JOL ’0 OW 0 NH 0 2 N02 K2003 acetone OJLN NH3 aq / ’—> —> l ) 0 N / No2 Nal Br N Br I A / N02 \ | A1 Br N Br Br \N NH2 HO/\/\ OzN / N Grubbs catalyst O | | 2nd generation —* CAN \ O 0\Q:%O/\~\ 0% a NaH,THF CH2CI 0 2 2 0 Kg 3 \ D2 E2 /0 Fe AcOH HO—<’ﬂ —, N \ 0 water \ o\ /O H /o H CH3SOZCI, NEt3 o CHZCIZ’LICI HO M80 21 A2 Synthesis of ediate A2 Methanesulfonyl de (8.4 mL; 108 mmol) was added to a solution of Z1 (14 g; 72.1 mmol), NEt3 (20 mL; 144 mmol) and LiCl (4.6 g; 108 mmol) in dry CHzClz (980 mL). The mixture was stirred at rt for 1 h 30. Water was added and the layers were ted. The organic layer was washed with water (once), dried over MgSO4, ﬁltered and concentrated in vacuo to give 18.8 g ofA2 (96%) as a green oil.

/O O\/\ OANH A2 2 No2 K2003, acetone ii / N Nal )3 N02 Br \N / Br I A1 Br N Br Synthesis of intermediate B2 Intermediate B2 was obtained using the procedure described for intermediate C1 (78% yield as a yellow oil).

A O NH3 aq )3 N —> O N N02 THF I ) N02 \ | Br N Br \ Br N NH2 Synthesis of intermediate C2 Intermediate C2 was obtained using the procedure described for intermediate D1 (quantitative yield as a yellow oil). /0 OW j’ N a o p om / 2 NaH,THF I GAGA Br N NH2 \ D2 Synthesis of intermediate D2 ediate D2 was obtained using the procedure described for intermediate W1 (64% yield as a yellow solid).

I OZN / OzN / N Grubbs catalyst I 2nd 9enerat'onI \ O \ 03K N cm A CH2C|2 O o O/\ "1:131:03 Synthesis of intermediate E2 A solution of D2 (1 g; 2.12 mmol) in CH2C12 (400 mL) was degassed by N2 bubbling for 15 min.

Grubbs catalyst 211d generation (181 mg; 212 umol) was added and the mixture was stirred at rt for 16 h. SiliaBond DMT (2.78 g; 1.69 mmol) was added and the mixture was stirred at rt for 16 h. The mixture was ﬁltered through a pad of celite and the ﬁltrate was concentrated in vacuo to give 1.11 g of a brown oil. The crude was d by ative LC (Irregular SiOH 15-40 um, 50 g Merck, mobile phase gradient: from /EtOAc 100/0 to 90/10). The fractions ning product were combined and the solvent was removed in vacuo to give 386 mg of intermediate E2 (41%, isomer E (96.2%) + isomer Z ) as a yellow foam. 021%: NH2 Ho /N G" 0AM \ Fe,AcOH O \ —> N o 0 water \ 0\ Synthesis of ﬁnal compound 15 Iron (291 mg; 5.21 mmol) was added to a solution of E2 (386 mg; 0.869 mmol) in acetic acid (36 mL) and water (3 mL). The mixture was stirred at 80°C for 6 h. Iron (146 mg; 2.61 mmol) was added and the mixture was stirred at 80°C for 16 h. Iron (146 mg; 2.61 mmol) was added again and the mixture was stirred at 80°C for 5 h. The mixture was concentrated until dryness. DMF was added, the mixture was ﬁltered through celite and the celite was rinsed with DMF. Siliabond Imidazole (18 g; 20.9 mmol) was added to the ﬁltrate and the mixture was stirred at rt for 72 h.

The mixture was ﬁltered through , the celite was rinsed with DMF and the ﬁltrate was concentrated in vacuo to give 428 mg of a brown solid. The solid was taken up in CH3CN leading to precipitation. The precipitate was ﬁltered to give 267 mg of a brown solid. The solid was puriﬁed by preparative LC (Irregular SiOH 15-40 um, 10 g Merck, dry loading, mobile phase gradient: from /MeOH/NHgaq 95/5/05 to 85/15/1.5). The fractions containing product were ed and the solvent was d in vacuo to give 124 mg of an off-white solid. The solid was puriﬁed by Reverse phase (Stationary phase: Sunﬁre-C18 5 um 19x150 mm, mobile phase gradient: from CH3CN/H20 (formic acid 0.1%) 5/95 to 50/50) to give 72 mg of ﬁnal compound 15 (23% yield) as a white solid.

Overall Scheme in the preparation of ﬁnal products: method 10 NH2 NH2 O N / | TCII N - - N / CAN \ 0?O HO—<’ HCI 4N In dioxane | HO—<’ IN N O \ 2 THF / MeOH N O F F 16 17 (HCI salt) Synthesis of intermediate F2 Intermediate F2 was obtained with the procedures described for intermediate F1 (E isomer). sis of ﬁnal compound 16 At rt, TiC13 (12.3 mL; 14.341 mmol) was added drop wise to a mixture of F2 (300 mg; 0.717 mmol) in THF (30 mL). The mixture was stirred at rt for 2 hours. The mixture was cooled down to 0°C and basiﬁed with K2C03 powder. The resulting muddy mixture was ﬁltered h a pad of celite and the celite was washed with a solution of AcOEt/CHgOH 8/2. The ﬁltrate was partially evaporated to give 175 mg of ﬁnal compound 16 after ﬁltration of a white solid and drying under vacuum pressure at 85°C (71% yield).

Synthesis of ﬁnal compound 17 The hloride salt was prepared with 10 eq of HCl 4N in e, which was added to the suspension of compound 16 (100 mg; 0.292 mmol) in CHgOH (10 mL). The precipitate was stirred for 3 h, ﬁltered and dried under vacuum at 90°C overnight. The solid was solubilized in MeOH/CHzClz 50/50, CH3CN was added and the solvent was evaporated up to precipitation of a white solid, which was ﬁltered and dried under vacuum at 90°C to give 47 mg of ﬁnal compound 17 as an HCl salt (0.93 HCl, 0.51 H20 ; 42% yield).

WO 54859 Overall Scheme in the preparation of ﬁnal products: method 11 kit £3on _, KarlaO N K THF K H2 2)]; O NH3aq OJLN PPh3 DIAD BO/KEZOZ 2 / N02 THF I Br N NH2 NH2 NH2 HO/\/ R OzNﬂo/V/ Srgbbs-Hmt/Ieyda 82" / n IN genera Ion CAN \ ONY 0 NaH THF . + map DCE 4 U; 0 34 NH2 NH2 )(OJLN/OYOV _4,O O LiAIH kDAN OH i Hl H2 Synthesis of intermediate H2 At -20°C under a N2 ﬂow, G2 (22.0 g; 72.04 mmol) in THF (100 mL) was added drop wise to a suspension of LiAlH4 (3.28 g; 86.45 mmol) in THF (120 mL). The mixture was d at 0°C for l h. 3.5 mL ofwater was added dropwise, followed by 3.5 mL ofNaOH 3N and 10 mL ofwater.

The resulting mixture was ﬁltered through a pad of celite and the celite was washed with EtOAc.

The ﬁltrate was concentrated under reduced pressure to give 19 g of intermediate H2 as a yellow oil (quantitative . 0Y0)? jﬁzgf 231 Synthesis of intermediate 12 At 0°C, diisopropylazodicarboxylate (4.0 mL; 20.32 mrnol) was added drop wise to a mixture of Al (5.0 g; 13.55 mmol), H2 (4.28 g; 16.26 mmol) and PPh3 (5.33 g; 20.327 mmol) in THF (100 mL). The e was stirred at rt for 12 h. EtOAc and water were added. The layers were decanted. The organic layer was washed with water, dried over MgSO4, ﬁltered and the solvent was evaporated. The crude compound was dissolved in Heptane/EtOAc 80/20, the itate was ﬁltered off (mainly POPh3) and the ﬁltrate was puriﬁed by chromatography. Puriﬁcation was carried out by ﬂash chromatography over silica gel (15-40 um, 220 g, Heptane/EtOAc 80/20) The pure fractions were ted and ated to dryness to give 8.2 g of intermediate 12 (99% yield).

OYOJ/ OYOJ/ N\/\\ i 0 NH3aq j N NO / 2 Br N Br jﬁiﬂ: Synthesis of intermediate J2 12 (8.2 g; 13.349 mmol) was stirred in NH4OH (100 mL) and THF (100 mL) at rt for 24 h. The mixture was half evaporated under reduced pressure. The residue was taken up with EtOAc. The organic layer was washed with water, dried over MgSO4, ﬁltered and the solvent was evaporated to give 8.15 g of intermediate J2 (quantitative yield). The crude compound was used directly in the next reaction step. )1 HOV K o N —> OZNﬂ \ j/EINOZ OYN N o/V/ NaH,THF Br N +0 \GﬂoAOA NH2 K2 Synthesis of intermediate K2 Under a N2 ﬂow, NaH (60% in oil) (1.15 g; 28.64 mrnol) was added n wise to allyl alcohol (35 mL) at rt. The mixture was stirred at rt for 30 min before being added drop wise to a solution of J2 (4.0 g; 7.26 mmol) in THF (80 mL) at 0°C. The resulting mixture was then stirred at rt for 2 h 30 min and was poured in a saturated solution of NH4Cl. EtOAc and a saturated aqueous solution ofNaCl were added, the layers were separated and the aqueous layer was extracted with EtOAc (once). The combined c layers were dried over MgSO4, ﬁltered and the solvent was removed under reduced re to give 4.7 g of a yellow oil. Puriﬁcation was carried out by ﬂash chromatography over silica gel (15-40 um, 80 g, CH2Cl2/Heptane . The pure fractions were collected and evaporated to dryness to give 2.65 g of intermediate K2 (69% yield).

NH2 NH2 k OZNﬂ Grubbs-Hoveyda gZN / | IN 2nd generation \ O/V/ OYN N DAN \ DCE 0:? +o A /\ J N O O )\ o 3< sis of intermediate L2 Prior to the reaction, the dichloroethane was degassed by bubbling N2 through.

In a Slenck tube, a solution of K2 (1.3 g; 2.464 mmol) and chlorodicyclohexylborane (l M in hexane) (493 uL; 0.493 mmol) in dichloroethane (600 mL) was stirred at 80°C under N2 for l h.

Grubbs-Hoveyda catalyst 2Ild tion (609 mg; 0.493 mrnol) was added and the mixture was d at 120°C for 16 h. Siliabond DMT (2.98 g; 1.82 mmol) was added and the mixture was stirred at rt for 16 h. The reaction mixture was ﬁltered through celite and the ﬁltrate was evaporated in vacuo to give 16 g which was combined with another reaction (2.46 mmol scale) before puriﬁcation (total weight to purify 3.2 g). Puriﬁcation was carried out by ﬂash chromatography over silica gel (15-40 um, 80 g, CHzClz/CHgOH: 99.5/0.5). The pure fractions were collected and evaporated to dryness to give 0.99 g of Fl (E/Z mixture of expected compound, 40% yield) and 0.65 g of F2 ing material K2).

Fl was r puriﬁed by achiral SFC (Stationary phase: NH2 5 um 150*30 mm), Mobile phase: 92% C02, 8% MeOH) to give 664 mg of intermediate L2 (E isomer, 27% yield).

NH NH2 §2N / N N / FeAcOH N Ho— Synthesis of ﬁnal compound 18 At 0°C, CF3C02H (0.455 mL) was added drop wise to a mixture of M2 (100 mg, 0.236 mmol) in CH2C12 (1 mL). The mixture was stirred at rt overnight, and then basiﬁed with a 10% solution of K2C03 in water. The precipitate was ﬁltered off, washed with water and CH3CN, and ﬁnally dried under vacuum to afford 35 mg of ﬁnal compound 18 (E isomer, 46% yield).

Overall Scheme in the preparation of ﬁnal products: method 12 NH2 NH2 HO— Synthesis of ﬁnal nd 23 At 0°C, H (0.281 mL, 3.643 mrnol) was added drop wise to a mixture of N2 (62 mg, 0.146 mrnol) in CH2C12 (1 mL). The mixture was d at rt overnight. The mixture was basiﬁed with a 10% on of K2C03 in water. The mixture was extracted twice with CHzClz and CH30H (80/20). The organic layer was dried over MgSO4, ﬁltered and the solvent was evaporated. The crude compound was taken up with DMF, 2 g of SiOz 60-200 urn was added and the resulting suspension was evaporated until dryness. This residue was put on the top of a chromatography column (solid deposit). Puriﬁcation was carried out by ﬂash chromatography over silica gel (15-40 um, 25 g, CHzClz/CHgOH/NH4OH: 95/5/05). The pure fractions were ted and evaporated to dryness to give 20 mg. The fraction was taken up with CH3CN, the precipitate was ﬁltered off and dried under vacuum to afford 18 mg of ﬁnal compound 23 (38% yield). -31 _ Overall Scheme in the preparation of ﬁnal products: method 13 8N / TiCI N / N HO ("Hf/KNHZ \T:’ :INEg HCI6MIn IPrOH HO 3Q N o CHgCN/MeOH 03 22 (HCI salt) Synthesis of intermediate 02 Intermediate 02 was obtained with the procedures described for intermediate X1 (E isomer).

Synthesis of ﬁnal compound 21 At rt, TiC13 (51.5 mL; 60.128 mrnol) was added drop wise to a e of 02 (1.3 g; 3.006 mrnol) in THF (130 mL). The mixture was stirred at rt for 2 h. The mixture was cooled down to 0°C and then basiﬁed with K2C03 powder. The resulting muddy mixture was ﬁltered through a pad of celite and the celite was washed with a solution of AcOEt/CHgOH 8/2. The ﬁltrate was partially evaporated to give 380 mg of ﬁnal compound 21 (35% yield) after ﬁltration of a white solid and drying under vacuum at 85°C.

Synthesis of ﬁnal compound 22 Compound 21 (118 mg, 0.331 mrnol) in CH30H (2 mL) + CH3CN (2 mL) was cooled down to °C. HCl (6M in isopropanol) (0.16 mL, 0.993 mrnol) was added drop wise and the e was stirred at rt for 1 h. The itate was ﬁltered off, washed with EtzO and dried under vacuum to give 109 mg of ﬁnal compound 22 as an HCl salt (0.76 HCl 0.81 H20, 83% yield).

Overall Scheme in the preparation of ﬁnal products: method 14 NH2 NH2 02N / O Wlkinson's OzN / IN TiCI3 ,N / N CAN \ caaysH t | O A \ HO—< JKCEF O N 03 N O \ THF o THF/CI2-|30H 2 KC?) L F : .o HC|6MInIPrOH Ho— Synthesis of ﬁnal compound 19 At rt, TiC13 (9.5 mL; 11.049 mmol) was added drop wise to a mixture of P2 (0.24 g; 0.552 mrnol) in THF (25 mL). The mixture was stirred at rt for 2 h. The mixture was cooled down to 0°C and then basiﬁed with K2C03 powder. The resulting muddy mixture was ﬁltered through a pad of celite and the celite was washed with a solution of AcOEt/CHgOH 8/2. The ﬁltrate was partially evaporated to give 100 mg of ﬁnal nd 19 (50% yield) after ion of a white solid and drying under vacuum at 85°C.

Synthesis of ﬁnal compound 20 Compound 19 (58 mg; 0.162 mrnol) in CH3OH (2 mL) + CH3CN (4 mL) was cooled down to °C. HCl (6M in isopropanol) (81 ML; 0.486 mmol) was added drop wise and the mixture was stirred at rt for 1 h. The itate was d off, washed with diisopropylether and dried under vacuum at 90°C to give 57 mg of ﬁnal compound 20 as an HCl salt (0.88 HCl 0.04 H20, 89% yield).

Overall Scheme in the preparation of ﬁnal products: method 15 O / 0 2ft PPh3/DIAD OJLN 231m\ | Br N Br O \ 2 NHsaq HOV O2N / N o N THF —’ 2 \l /V/ NO2 O N O /| NaH,THF \ DADA Br N NH2 82 T2 Grubbs—Hoveyda NH2 catalyst / . IN N 2nd generatlon AK603\ 0 Fe, AcOH HO— O N / —> N \ O DCE 2 water l 03 2): PPh3/DIAD OJLN FF 2f1 A1 Br N Br Synthesis of intermediate R2 At 0°C, diisopropylazodicarboxylate (3.8 mL; 19.107 mrnol) was added drop wise to a mixture of A1 (4.7 g; 12.738 mmol), Q2 (2.27 g; 12.738 mmol) and PPh3 (5 g; 19.107 mmol) in THF (100 mL). The e was stirred at rt for 12 h. EtOAc and water were added. The layers were decanted. The organic layer was washed with water, dried over MgSO4, d and the solvent was evaporated. The crude compound was puriﬁed by column chromatography over silicagel (15-40 pm; 220 g) in Heptane/AcOEt 85/15 to 5.3 g of intermediate R2 (79% yield).

ON ON 0 O OJLN NH3 aq O N 2 THF / N02 2 / No2 \ | Br N Br Br N NH2 sis of intermediate S2 R2 (5.3 g; 10.015 mmol) was stirred in THF (80 mL) and NH4OH (80 mL) at rt for 24 h. The mixture was concentrated under reduced pressure. The residue was taken up with CHzClz, the precipitate (mineral) was ﬁltered off and the ﬁltrate was concentrated under reduced pressure.

The crude compound was puriﬁed by column chromatography over silica gel (15-40 um; 220 g) in e/AcOEt 85/15 to give 3.65 g of intermediate S2 (78% .

O \ NH2 JL "0M 0 N —, WW | 2 N02 O MK)"\ /V/ NaH,THF N O 82 T2 Synthesis of intermediate T2 NaH (1.35 g; 33.88 mmol) was added portion wise to allyl alcohol (41 mL) at rt. The mixture was stirred at rt for 30 min before being added drop wise to a solution of S2 (4 g; 8.597 mmol) in THF (100 mL) at 0°C. The resulting mixture was then stirred at rt for 2 h 30 min and was poured in an saturated s solution of NH4Cl. EtOAc and a saturated aqueous solution of NaCl were added, the layers were separated and the aqueous layer was extracted with EtOAc (once).

The combined organic layers were dried over MgSO4, ﬁltered and the solvent was removed under reduced pressure to give a yellow oil. The crude compound was d by preparative LC (Irregular SiOH 15-40 um, 120 g Grace, liquid ion, mobile phase gradient: Heptane/EtOAc 85/15) to give 3.2 g of intermediate T2 as a yellow oil (84% yield).

\ NH2 Grubbs--Hoveyda catalyst 2 / | 2nd generation O OZNﬂ \ N O/\// OkN \ DADA DCE J U:©/NHNOE/O Synthesis of intermediate U2 A solution of T2 (1 g; 2.26 mmol) and chlorodicyclohexylborane (1M in hexane) (904 uL; 904.013 umol) in dry roethane (540 mL) was stirred at 80°C and under N2 atmosphere for 1 h. The mixture was degassed by N2 bubbling for 15 min, Grubbs-Hoveyda catalyst 211d generation (141.6 mg; 226 umol) was added, the mixture was degassed again by N2 bubbling for min and then stirred at 120°C for 16 h. 0.25 eq of catalyst were added again and mixture was stirred at 120°C for 16 h. Siliabond DMT (5.9 g; 3.616 mmol) was added and the mixture was stirred rt for 16 h. The mixture was ﬁltered through a pad of celite and the e was concentrated under vacuum to give a black oil. The crude compound was puriﬁed by preparative LC ular SiOH 15-40 um, 80 g Merck, mobile phase: CH2Cl2/AcOEt 97/3). The fractions containing product were combined and the solvent was removed under vacuum to give 335 mg of intermediate U2 (E isomer, 36% yield).

U2 25 sis of ﬁnal nd 25 Iron (0.45 g; 8.084 mmol) was added to a e of U2 (0.335 g; 0.808 mmol) in acetic acid (24 mL) + water (5 mL). The mixture was stirred vigorously at 50°C for 5 h.

CH2Cl2 was added and the reaction mixture was ﬁltered through a pad of celite, and then washed with acetic acid. The solvent was removed under d pressure. The crude was puriﬁed by chromatography over silicagel column (SiO2 15-40 um, 25 g) in CH2Cl2/CH30H/NH4OH 96/4/05 to give 154 mg of ﬁnal compound 25 (56% yield). The compound was crystallized in CHgOH, ﬁltered and dried under vacuum at 90°C to give 70 mg (25% yield).

Overall Scheme in the preparation of ﬁnal products: method 16 oY?0 N\/\\ NH2 ’f1 OYN\©ﬂN \ OW +° 0*" Br N NH2 V2 NH2 NH2 iesneration 0*N \ O + O \ CAN \ CHZCIZ } KO»; 2 0 34 KG";o % W2 X2 Fe,AcOH HO—<’N / IN CFscozH / HO_ —’ \ \ N N 0 water 0% CHch2 "3 B: N Fe,AcOH Ho— \ \ N O N 0 water CHZCIZ EL KG"H ’ 22 29 OYOJ/ NA NH2 0 Ho/\/\ H OZN / CAN —> \ O/W N H THFa" OYN "KI +0 Geo" Br N NH2 v2 Synthesis of intermediate V2 ediate V2 was synthesized using the procedure described for intermediate K2 with 3- butenol as starting material (3.9 g, 44% .

Synthesis of intermediates W2 and X2 Grubbs catalyst 211d generation (236 mg, 0.277 mrnol) was added to a e of V2 (1.5 g, 2.77 mmol) in dry CHzClz (400 mL). The mixture was stirred at rt under a N2 ﬂow for 24 h. Siliabond DMT (3.6 g, 2.216 mmol) was added and the mixture was stirred at rt for 12 h. The e was ﬁltered through celite, the celite was washed with CH2C12 and the ﬁltrate was evaporated.

Puriﬁcation was carried out by ﬂash chromatography over silica gel (15-40 um, 80 g, CHzClz/CHgOH: 99.5/0.5) pure fractions were collected and ated to dryness to give 0.98 g of a mixture of W2 and X2. The two isomers were separated by achiral SFC (Stationary phase: CHIRALPAK 1C 5 um 250x20 mm), Mobile phase: 70% C02, 30% CHgOH) to give 0.805 g of intermediate W2 (E isomer, 57% yield) and 0.14 g of intermediate X2 (Z isomer, 10% yield).

Synthesis of ﬁnal compound 26 Final compound 26 was synthesized with the procedures described for ﬁnal compound 18 (1St step: Y2, 0.68 g, 99% yield; 211d step: 52 mg, 27% yield).

Synthesis of ﬁnal compound 29 -38— Final nd 29 was synthesized with the procedures described for ﬁnal compound 18 (1St step: ZZ, 0.12 g, 100% yield; 2Ild step: 8 mg, 9% yield).

Overall Scheme in the preparation of ﬁnal products: method 17 /\/Br F H/ DIBAI-H o O K2003 CH3CN O CHchZ A3 03 F O\/\\ r F M 0 NH i 0 j NHsaq / No2 PPh3/DIAD o N | —’ 0 N NO THF \N THF } 2 Br Br /| j N02 \ /| A1 Br N Br Br \N NH2 ﬂow OZN / N HOW O@:O//\N| OzNﬁ Grubbs catalyst i | 2nd tIon' \ NaH,THF CH2CI2 )0 Kg0:;0 G3 F NH2 NH2 N N HCI (6M . . / In IPrOH) N Fe,AcOH —» HO_ Synthesis of intermediate C3 Under N2, DIBAL-H (1.2 M in toluene) (97 mL; 116.5 mmol) was added to a solution of B3 (9.8 g; 46.6 mmol) in dry CHzClz (250 mL) at 0°C. The reaction e was stirred at 0°C for 1 h, and then 1 h at rt. Water was added, The organic layer was ted from the aqueous layer, dried over MgSO4, ﬁltered and concentrated under vacuum to give 8.4 g of intermediate C3 (99% yield). The crude compound was used directly in the next reaction step.

F oA OJLNH C3 0 j No2 PPh3/DIAD / oiN | 2 NO2 Br \N THF / Br | A1 Br N Br Synthesis of intermediate D3 Intermediate D3 was synthesized using the procedure described for intermediate R2 with C3 as starting material (1.9 g, 88% yield).

F OW F OW JL 0 NH3aq j N —> O N N02 THF I 2 N02 \ | Br N Br \ Synthesis of ediate E3 Intermediate E3 was synthesized using the ure described for intermediate S2 with D3 as starting material (1.8 g, 93% yield). 2014/056270 F OA 0 HOW OZN / 0 N O \ OW 2 N02 F / A /\ | 0 O Br N NH2 F Synthesis of intermediate F3 Intermediate F3 was synthesized using the procedure described for intermediate Wl with E3 as starting material (0.65 g, 66% yield).

OzN / N Grubbs catalyst O | 2nd generation O \ CAN \:HINOE N OW A CHZCIZ O o O/\ Synthesis of intermediate G3 ediate G3 was synthesized using the procedure described for intermediate X1 with intermediate F3 as starting material (E isomer, 520 mg, 19% yield).

NH2 NH2 own N / Fe AcOH H04m N \ 0 water \ Synthesis of ﬁnal compound 27 Final compound 27 was synthesized using the procedure described for ﬁnal compound 10 with intermediate G3 as starting material (174 mg, 42% yield).

W0 2014/154859 NH2 NH2 Flo—(ﬂN / N . . N HC|(6M m IPrOH) / N H04 I N \ N \ o —> o \ CH3CN o \ 27 HCIsaIt F 28 F Synthesis of ﬁnal compound 28 Final compound 28 was synthesized using the procedure described for ﬁnal compound 20 with compound 27 as starting material (101 HCl 0.89 H20, 95 mg, 69% yield).

Overall Scheme in the preparation of ﬁnal products: method 18 NH2 NH2 Wilkinson's N N / CAN \gzNﬁ Fe/AcOH st gzNﬁ/ HO— Synthesis of ﬁnal compound 32 A mixture of H3 (150 mg; 0.345 mmol) and iron (190 mg; 3.45 mmol) in acetic acid (13 mL) and water (1.5 mL) was stirred at 50°C for 5 h. CH2C12 was added and the reaction mixture was ﬁltered through a pad of celite and concentrated under vacuum. The crude compound was taken up with DMF, ﬁltered through a pad of celite and concentrated. The solid was pre-puriﬁed by chromatography over silicagel column (SiOz 63-200 um. 80g) in CHzClz/CHgOH/NH4OH( 98/2/01 to 90/10/05). A second puriﬁcation by achiral SFC (Stationary phase: Whelk Ol (S,S) um 250*2l.l mm), Mobile phase: 60% C02, 40% CH3OH (0.3% iPrNH2)) afforded 27 mg of ﬁnal compound 32 (22% yield).

Overall Scheme in the preparation of ﬁnal ts: method 19 F O\/\ 0 R OZNﬂ / Grubbs—Hoveyda O2N 2)]; \ Z HO/\/ | catalyst o \ . I 2 d genera Iont N O n CAN \ NaH THF 0 o/\ CH20|2 } O$\ I3 \q 1/0 J3 F HCI (6M m IPrOH) Tim Ho— o THF \ CH3OH/CH3CN \ \ 0 o F 31 F HCIsaIt F O\/\ 0 R 02N / N / 2): Ho/\/ | N O NaH THF 0 o/\ Synthesis of intermediate 13 Intermediate 13 was synthesized using the procedure described for intermediate T2 (4.2 g, 83%).

NH2 NH2 | OZN / Grubbs—Hoveyda | f catalyst o \ N 0 2nd generation §2Nﬁ I3 Jifigj> Synthesis of intermediate J3 Intermediate J3 was sized using the procedure described for intermediate Fl (isomer E, 125 mg, 17%). 2014/056270 NH2 NH2 O2N / N O N N I / IN . . / 2K \ TiCI3 Ho— Synthesis of ﬁnal compound 31 Final compound 31 was synthesized using the procedure described for ﬁnal compound 22 (0.98 HC10.15 H20, 72 mg, 59% yield).

Overall Scheme in the preparation of ﬁnal ts: method 20 o/ 0/ H/ OH WE" o KZCO3/CH3CN HO K3 L3 0 NH i O o 0 2 L3 / N02 NHsaq 0 N OJLN | PPh /D|AD NO THF Br \N Br THF3 /| 2 2 N02 \ /| A1 Br N Br Br \N NH2 / NH O NHZ 2 Grubbs—Hoveyda HOV OZN / 2 / N catalyst N O | 2nd generation I —. \ N O/V/ —> O>\\N \ O NaH, THF T OAOA dry DCE ) 1\ 03 P3 N / TICI3. N H04<, I O 36 OH /\/E"r KZCO3/CH3CN HO K3 Synthesis of intermediate L3 A11y1 bromide (1.7 mL; 19.6 mmol) was added to a solution ofK3 (3 g; 17.8 mmol) and K2C03 (2.7 g; 19.6 mmol) in CH3CN (90 ml). The mixture was stirred at 90°C for 20 h, and then ﬁltered. The ﬁltrate was concentrated under vacuum. The crude t was taken up with CH2C12 and an aqueous solution ofNaOH 5%. The layers were separated and the organic layer was dried over MgSO4, ﬁltered and the solvent was removed under reduced pressure to give 3 .9 g of intermediate L3 (quantitative . The crude compound was used directly in the next reaction step. o NH O 0 ’II L3 0*"ICE f PPh/DMD NO2 3 Br ‘\N Br THF "J//Efl:ﬂ:\ A1 Br N Br Synthesis of intermediate M3 At 0°C, diisopropylazodicarboxylate (4.8 mL; 24.36 mrnol) was added drop wise to a mixture of Al (6 g; 16.2 mmol), L3 (3.2 g; 15.36 mmol) and PPh3 (6.4 g; 24.36 mmol) in THF (120 mL).

The mixture was stirred at rt for 12 h. EtOAc and water were added. The layers were decanted.

The organic layer was washed with water, dried over MgSO4, ﬁltered and the solvent was evaporated. 20 mL of Heptane/AcOEt 70/30 were added to precipitate a large part of the formed PPth, which was removed by ﬁltration. The crude product was puriﬁed by ative LC (Irregular SiOH 15-40 um, 120 g him, mobile phase Heptane/EtOAc 80/20) to give 8 g of intermediate M3 (88% yield).

' I ft 0 O fgo NH3 aq 0 N _, OJLN /) No2 THF /, /) N02 | // \ | Br N Br Br \N NH2 Synthesis of intermediate N3 M3 (8.8 g; 15.7 mmol) was stirred in THF (120 mL) and NH4OH (120 mL) at rt for 24 h. The mixture was trated under reduced pressure. The residue was taken up with CH2C12, the precipitate (mineral) was ﬁltered off and the e was dried over MgSO4, ﬁltered through a pad of celite and concentrated under vacuum. The crude product was d by preparative LC (Irregular SiOH 15-40 um, 120 g Interchim, mobile phase Heptane/EtOAc 80/20) to afford 3 g of intermediate N3 (38% yield). of / o o o NH2 on W JG; «N )ﬁNOZ O \ CV NaH,THF Br \N NH2 3 / DADA Synthesis of intermediate 03 NaH (60% in oil) (0.93 g; 23 mmol) was added n wise to allyl alcohol (28 mL) at rt. The mixture was stirred at rt for 30 min before being added drop wise to a solution ofN3 (2.9 g; 5.85 mmol) in THF ( 70 mL) at 0°C. The ing mixture was then d at rt for 2 h 30 min and was poured into a saturated aqueous solution ofNH4Cl. EtOAc and a saturated aqueous solution ofNaCl were added, the layers were separated and the s layer was extracted with EtOAc (once). The combined organic layers were dried over MgSO4, ﬁltered and the solvent was removed under reduced pressure to give a yellow oil. The crude t was puriﬁed by preparative LC (Irregular SiOH 15-40 um, 120 g Grace, liquid injection, mobile phase heptane/EtOAc 80/20) to give 2.4 g of intermediate 03 (87% yield).

Grubbs-Hoveyda O | n generation % \ N O/V/ —> OWN \ o A dryDCE / \ o O/\ ) Synthesis of intermediate P3 The reaction was d out on three batches.

A solution of 03 (0.8 g; 1.7 mmol) and chlorodicyclohexylborane (1M in hexane) (0.68 mL; 0.68 mrnol) in dry dichloroethane (400 mL) was stirred at 80°C and under N2 atmosphere for l h.

The mixture was degassed by N2 ng for 15 min, Grubbs-Hoveyda catalyst 211d generation (110 mg; 0.17 mol) was added, the mixture was degassed again by N2 bubbling for 15 min and then stirred at 120°C for 16 h. 0.050 eq of catalyst (49 mg, 0.084 mrnol) were added and mixture was stirred at 120°C for 7 h. Siliabond DMT (3.3 g; 2.03 mmol) was added and the mixture was stirred rt for 16 h. The mixture was ﬁltered through a pad of celite and the ﬁltrate was concentrated under vacuum to give a black oil. The crude product was puriﬁed by preparative LC ular SiOH 15-40 um, 80 g Interchim, mobile phase Heptane/EtOAc 65/35) to give 190 mg of intermediate P3 (isomer E, 25% yield).

Synthesis of ﬁnal compound 36 At rt, T1C13 (19.3 mL; 22.5 mmol) was added drop wise to a mixture of P3 (500 mg; 1.125 mmol) in THF (90 mL). The mixture was stirred at rt for 2 h. At 0°C, the mixture was d with K2C03 powder. The resulting muddy mixture was ﬁltered through a pad of celite and celite was washed with a solution of CHzClz/CHgOH (90/10). The ﬁltrate was concentrated under reduced pressure. The residue was taken up in MeOH. The white solid was ﬁltered off and dried under vacuum. The product was puriﬁed by preparative LC (Irregular SiOH 15-40 um, 40 g Interchim, mobile phase CHzClz/CHgoH/NH4OH 98/2/01) to give 140 mg of ﬁnal compound 36 (34% yield).

LCMS s: General grocedure VDR2 (for methods 30xx.0/g) The LC measurement was performed using a UPLC (Ultra Performance Liquid Chromatography) Acquity s) system comprising a binary pump with degasser, an mpler, a diode-array detector (DAD) and a column as speciﬁed in the respective methods below, the column is hold at a temperature of 40°C. Flow from the column was brought to a MS detector. The MS detector was conﬁgured with an electrospray ionization source. The capillary needle voltage was 3 kV and the source temperature was maintained at 130 0C on the Quattro (triple quadrupole mass spectrometer from ). Nitrogen was used as the nebulizer gas. Data acquisition was performed with a Waters-Micromass MassLynx-Openlynx data .

Method V3018V3001 In on to the general procedure VDR2: Reversed phase UPLC was carried out on a Waters Acquity BEH (bridged ethylsiloxane/silica hybrid) C18 column (1.7 um, 21 x 100 mm) with a ﬂow rate of 0.343 ml/min. Two mobile phases e phase A: 95 % 7 mM ammonium acetate / 5 % acetonitrile; mobile phase B: 100 % acetonitrile) were employed to run a gradient condition from 84.2 % A and 15.8 % B (hold for 0.49 minutes) to 10.5 % A and 89.5 % B in 2.18 minutes, hold for 1.94 min and back to the initial conditions in 0.73 min, hold for 0.73 minutes. An injection volume of 2 ul was used. Cone voltage was 20V for ve and negative -48— ionization mode. Mass spectra were acquired by scanning from 100 to 1000 in 0.2 seconds using an interscan delay of 0.1 seconds.

Table 1. Compounds of formula (I).

Mass LCMS Ret Exact Synthesis # STRUCTURE Found Tlme,. NMR Mass method [M+H] Method 1H NMR (500 MHz, 6) 8 10.06 (s, 1H), N / :1 1.96, 7.36 (s, 1H), 7.20 (t, .1: 7.9 HO{/Q V3018V3 Hz, 1H), 6.76 — 6.93 (m, 1 N 0:? 324.1 325 001 Method 1 2H), 6.05 (s, 1H), 5.94 (dt, .1 KQ/O = 6.0, 16.1 Hz, 1H), 5.43 .64 (m, 3H), 4.84 (s, 2H), 4.40 — 4.66 (m, 4H) 1H NMR (400 MHz, DMSO-d6) 8 10.04 (br. s., NH2 1H), 7.49 (s, 1H), 7.17 (t, .1 Nﬂ 2.02, = 7.9 Hz, 1H), 6.69 - 6.83 V3018V3 2 HOK’N \ | 0O? 326.1 (rn, 2H), 6.15 (s, 1H), 5.54 327 Method 2 Kg 001 (s, 2H), 4.89 (s, 2H), 4.28 (t, .1: 6.3 Hz, 2H), 4.14 (t, .1: 6.6 Hz, 2H), 1.34 — 1.62 (m, 1H NMR (500 MHz, DMSO-d6) 8 10.11 (br. s., 1H), 7.22 (t, .1: 7.6 Hz, 1H), 7.12 (br. s., 1H), 7.09 2.3, (d, .1: 7.6 Hz, 1H), 7.05 (d, .1: 7.6 Hz, 1H), 5.74 (s, 3 322‘] 323 V38(1)§V3 Method 3 1H), 5.51 — 5.63 (m, 3H), .03 (td, .1: 7.2, 14.9 Hz, 1H), 4.93 (s, 2H), 4.15 — 4.26 (m, 2H), 3.15 — 3.23 (m, 2H), 2.24 — 2.33 (m, 2H) Mass LCMS Ret Exact Synthesis STRUCTURE Found Time, NMR Mass method [M+H] Method 1H NMR (500 MHz, DMSO-d6) 8 10.20 (br. s., 1H), 7.34 (s, 1H), 7.21 (t, J = 7.3 Hz, 1H), 7.06 (d, .1: 2.36, 7.3 Hz, 1H), 6.97 (d, .1: 7.3 V3018V3 324.2 325 Method 4 Hz, 1H), 5.59 (s, 2H), 4.93 (s, 2H), 3.91 — 4.03 (m, 2H), 2.55 - 2.62 (m, 2H), 1.59 - 1.71 (m, 2H), 1.12 — 1.27 (m, 4H) 1H NMR (400 MHz, DMSO-d6) 8 9.91 (br. s., 1H), 8.05 (d, .1: 4.6 Hz, 1H), 7.38 (d, .1: 8.6 Hz, 1H), 7.27 (dd, .1: 4.6, 8.6 1.85, Hz, 1H), 6.20 (s, 1H), 5.82 V3018V3 339.1 340 Method 5 001 (dt, .1: 7.0, 15.6 Hz, 1H), .68 (dt, .1: 7.1,15.6 Hz, 1H), 5.46 (s, 2H), 4.91 (s, 2H), 4.33 (d, .1: 7.1 Hz, 2H), 4.12 — 4.27 (m, 2H), 2.36 — 2.46 (m, 2H) 1H NMR (400 MHz, DMSO-d6) 8 9.92 (s, 1H), 8.09 (d, .1: 4.0 Hz, 1H), 7.43 (d, .1: 8.1 Hz, 1H), 1.85, 7.30 (dd, .1: 4.0, 8.1 Hz, 3 1H), 6.40 (s, 1H), 5.51 (s, 341.1 342 Method 6 001 2H), 4.96 (s, 2H), 4.15 (t, .1 = 6.1 Hz, 2H), 3.97 — 4.10 (m, 2H), 1.81 — 1.91 (m, 2H), 1.68 — 1.78 (m, 2H), 1.52 — 1.65 (m, 2H) 1H NMR (400 MHz, 1.84, DMSO-d6) 8 10.03 (br. s., V3018V3 1H), 7.36 (d, .1: 2.0 Hz, 354.1 355 Method 1 001 1H), 6.91 (d, .1: 8.1 Hz, 1H), 6.80 (dd, .1: 2.0, 8.1 Hz, 1H), 6.06 (dt, .1: 6.0, 2014/056270 Mass LCMS Ret Exact Synthesis STRUCTURE Found Time, NMR Mass method [M+H] Method 16.2 Hz, 1H), 5.93 (s, 1H), .54 (s, 2H), 5.41 (dt, .1: .6, 16.2 Hz, 1H), 4.78 (s, 2H), 4.50 — 4.67 (m, 2H), 4.28 — 4.48 (m, 2H), 3.71 (s, 1H NMR (400 MHz, DMSO-d6) 8 10.03 (br. s., 1H), 7.54 (br. s., 1H), 6.87 (d, .1: 8.1 Hz, 1H), 6.76 (d, 1.86, J: 8.1 Hz, 1H), 6.24 (br. s., V3018V3 356.1 357 Method 2 001 1H), 5.53 (br. s., 2H), 4.82 (br. s., 2H), 4.19 — 4.32 (m, 2H), 4.01 — 4.16 (m, 2H), 3.68 (br. s., 3H), 1.34 - 1.60 (m, 4H) 1H NMR (400 MHz, DMSO-d6) 5 9.73 (s, 1H), 7.57 (s, 1H), 7.16 (t, .1: 7.6 Hz, 1H), 7.02 (d, .1: 7.6 Hz, 2.36, 1H), 6.89 (d, .1: 7.6 Hz, V3018V3 Method 1 1H), 5.33 (t, .1: 6.6 Hz, 323.2 324 001 Method 2 1H), 5.25 (s, 1H), 5.15 (s, 2H), 4.85 (s, 2H), 2.91 (q, .1 = 6.6 Hz, 2H), 2.58 — 2.72 (m, 2H), 1.59 — 1.85 (m, 2H), 0.96 — 1.21 (m, 4H) 1H NMR (400 MHz, DMSO-d6) 8 10.05 (br. s., 1H), 7.20 (t, .1: 7.8 Hz, 1H), 6.92 (d, .1: 7.8 Hz, 2.22, 1H), 6.89 (s, 1H), 6.77 (d, .1 V3018V3 = 7.8 Hz, 1H), 6.12 (s, 1H), 338.1 339 Method 7 001 5.50 — 5.64 (m, 3H), 5.35 (dt, J=4.5, 16.2 Hz, 1H), 4.87 (s, 2H), 4.58 (d, .1: 4.5 Hz, 2H), 4.24 (t, J: 5.1 Hz, 2H), 2.21 — 2.32 (m, 2H) Mass LCMS Ret Exact , . Synthes1s # STRUCTURE Found Tlme, NMR Mass method [M+H] Method 1H NMR (500 MHz, DMSO-d6) 5 10.07 (s, 1H), 7.32 (s, 1H), 7.18 (t, .1: 7.7 Hz, 1H), 6.82 (d, .1: 7.7 Hz, NH2 1H), 6.79 (dd, .1: 1.7, 7.7 H64N l ‘/N 2.33, Hz, 1H), 5.98 (dt, .1: 7.7, V3018V3 11 <3)? 15.5 Hz, 1H), 5.80 (s, 1H), 3522 353 M61116d 1 U 001 5.56 (s, 2H), 5.51 (td, .1: 58,155 Hz, 1H), 4.84 (s, 2H), 4.71 (d, .1: 5.8 Hz, 2H), 3.96 (t, .1: 7.7 Hz, 2H), 1.92 — 2.07 (m, 2H), 1.49 — 1.64 (m, 2H) NH2 1H NMR (400 MHz, |_|O_ | /N s., 2.21, 22 356'] 357 001 7.07 (dd, .1: 1.9, 8.2 Hz, 1H), 6.85 — 6.99 (m, 1H), 6.64 (s, 1H), 5.80 (dt, .1: Mass LCMS Ret Exact Synthesis STRUCTURE Found Time, NMR Mass method [M+H] Method 7.2, 15.6 Hz, 1H), 5.59 (dt, .1: 5.7, 15.6 Hz, 1H), 4.98 (s, 2H), 4.68 (d, .1: 5.7 Hz, 2H), 4.41 — 4.59 (m, 2H), 2.38 — 2.50 (m, 2H) 1H NMR (500 MHz, DMSO-d6) 8 9.97 (br. s., 1H), 7.05 (s, 1H), 6.90 (t, J = 7.7 Hz, 1H), 6.42 (d, J: 1.83, 7.7 Hz, 1H), 6.38 (d, J: 7.7 V3018V3 Method Hz, 1H), 6.35 (s, 1H), 5.33 - 23 325.2 326 001 1 2 5.58 (m, 3H), 4.77 (s, 2H), 4.17 (t, J: 6.8 Hz, 2H), 3.20 (q, .1: 6.4 Hz, 2H), 1.42 — 1.52 (m, 2H), 1.32 — 1.41 (m, 2H) 1H NMR (500 MHz, 6) 5 10.04 (br. s., 1H), 7.77 (d, J: 7.6 Hz, 1H), 7.10 (dd,.]= 87,112 2.08, Hz, 1H), 6.78 (br. s., 1H), V3018V3 Method 24 344.1 345 6.28 (s, 1H), 5.56 (s, 2H), 001 1 4 4.86 (s, 2H), 4.28 — 4.47 (m, 2H), 4.04 — 4.23 (m, 2H), 1.52 - 1.66 (m, 2H), 1.31 — 1.50 (m, 2H) 1H NMR (500 MHz, DMSO-d6) 8 10.07 (br. s., 1H), 7.18 (t, J: 7.9 Hz, 1H), 7.14 (s, 1H), 6.73 - 2.13, 6.87 (m, 2H), 5.96 (dt, .1: V3018V3 Method 5.0, 15.7 Hz, 1H), 5.90 (s, 338.1 339 001 1 5 1H), 5.57 (s, 2H), 5.37 (dt, .1 = 5.8, 15.7 Hz, 1H), 4.86 (s, 2H), 4.58 (d, J: 5.8 Hz, 2H), 4.22 (t, J: 5.0 Hz, 2H), 2.27 — 2.42 (m, 2H) Mass LCMS Ret Exact Synthesis # STRUCTURE Found Tlme,. NMR Mass method [M+H] Method 1H NMR (500 MHz, DMSO-d6) 5 10.04 (br. s., 1H), 6.95 (t, .1: 7.2 Hz, 1H), 6.51 (d, .1: 7.2 Hz, N "\"2 1H), 6.46 (dd, .1: 1.3, 7.2 H04 l /N 2.02, Hz, 1H), 6.40 (s, 1H), 6.16 N V3018V3 Method 26 kON('13 (5, 1H), 5.97 (t, J: 6.3 Hz, 337.2 338 001 16 1H), 5.59 (s, 2H), 5.43 (dt, .1 = 63,155 Hz, 1H), 5.22 (dt, .1: 5.0, 15.5 Hz, 1H), 4.76 (s, 2H), 4.23 (t, .1: 5.2 Hz, 2H), 3.56 (t, .1: 5.0 Hz, 2H), 2.18 — 2.30 (111,2H) 1H NMR (500 MHz, 6) 5 10.11 (br. s., NH2 1H), 6.77 (d, .1: 9.1 Hz, N \N 1H), 6.71 (s, 1H), 6.63 (dt, .1 Ho— HO— Forty microliters of cells were then dispensed into each well in 384-well plates, where 200 nL of compound in 100% DMSO was already present. Following 6 hours incubation at 37°C, 5% CO2, the rase activity was determined by adding 15 ul of Steady Lite Plus substrate n Elmer) to each well and readout performed on a ViewLux ultraHTS microplate imager (Perkin Elmer). Dose response curves were generated from measurements performed in quadruplicates.

Lowest effective concentrations (LEC) values, deﬁned as the concentration that s an effect which is at least two fold above the standard deviation of the assay, were determined for each compound.

In parallel, a similar dilution series of nd was used (200 nL of compound in 100% DMSO) with 40 ML per well of cells transfected with NFKB-luc reporter construct alone (1.25 x 105 cells/mL). Six hours after tion at 37°C, 5% CO2, the luciferase activity was determined by adding 15 ul of Steady Lite Plus substrate (Perkin Elmer) to each well and readout performed on a ViewLux ultraHTS microplate imager (Perkin Elmer). Counterscreen data is reported as LEC.

Measurement of eron production in human PBMC Activation of human TLR7 results in robust production of interferon by plasmacytoid dendritic cells present in human blood. The potential of compounds to induce interferon was evaluated by determination of interferon in the conditioned media from peripheral blood mononuclear cells (PBMC). The presence of interferon in the samples was determined, using an interferon reporter cell line stably expressing an interferon-stimulated sive elements (ISRE)-luc reporter construct. The ISRE element with sequence TAGTTTCACTTTCCC is highly responsive to the STATl-STAT2-IRF9 transcription factor, which becomes activated upon binding of IFN-I to the IFN receptor. Brieﬂy, PBMCs were prepared from buffy coats of at least two donors using a standard Ficoll centrifugation protocol. Isolated PBMCs were resuspended in RPMI medium supplemented with 10% human AB serum and 2 X 105 cells/well were dispensed into 384-well plates containing compounds (70 uL total volume). After overnight tion of the PBMCs with the compounds, 10 uL of supernatant was transferred to 384-well plates containing 5 x 103 HEK—ISRE-luc cells/well in 30 uL (plated the day before). Following 24 hours of tion, activation of the ISRE elements was measured by assaying luciferase activity using 40 uL/well Steady Lite Plus substrate (Perkin Elmer) and measured with ViewLuX TS microplate imager n Elmer). The stimulating activity of each compound on the HEK—ISRE-luc cells was reported as LEC. The LEC in turn indicates the degree of ISRE activation on transfer of a deﬁned amount of PBMC e medium. Recombinant interferon alfa-2a (Roferon-A) was used as a standard control compound.

The LEC values for the compounds in table 2 on HEK293 TLR8-NFKB-luc and HEK293 NFKB- luc where r than the highest tested concentration (> 10 uM for compound 4 and > 25 uM for all other compounds).

Table 2. nds of formula (I) n represents the number of experiments performed.

HEK293 PBMC URE TLR7-NFKB-luc n HEK-ISRE-luc n (LEC; HM) (LEC; HM) 2.88 8 0.30 8 KC/O 4.47 1 0.93 2 WO 54859 HEK293 PBMC STRUCTURE TLR7—NFKB-luc HEK-ISRE-luc n (LEC; HM) (LEC; HM) 0.27 0.033 4 2.38 2.56 2 MnN / N N \ 0 2.14 0.082 2 ofl) N \ o 1 0.16 2 ofl) N 0 / 3.88 0.29 2 N / N N O 7.1 0.58 2 N N 11.09 10.87 2 2014/056270 HEK293 PBMC STRUCTURE TLR7-NFKB-luc HEK-ISRE-luc n (LEC; uM) (LEC; uM) 0.78 0.25 4 1.25 0.45 2 1.08 0.3 2 1.71 0.15 2 7.01 2.4 2 0.18 0.04 2 -62— # HEK293 PBMC URE TLR7—NFKB-luc HEK-ISRE-luc (LEC; HM) (LEC; HM) HHNNﬂO 16 0:1 2.02 0.47 N / N Ho— N \ N / O 188 037 4 26 H \ ' ' N \ N H04 | N / 27 o 1.35 0.14 2 WO 54859 # HEK293 PBMC STRUCTURE TLR7-NFKB-luc HEK-ISRE-luc n (LEC; HM) (LEC; HM) N \ H04 N N / \ 0.91 0.15 2 ,HCI KQO MnN \ N / O 29 . 0.4 8 2 N / N Ho 33—

Claims

Claims 1.

1. A method for the preparation of a compound according to formula (I) and pharmaceutically accepted salts thereof, wherein X is O, NH or S, Y represents an aromatic ring or heterocyclic ring comprising at least a nitrogen, optionally 10 substituted by one or more tuents independently selected from C1-6alkyl, C1-4alkoxy, trifluoromethyl or halogen, Z represents C1-10 saturated or unsaturated alkyl optionally substituted by an alkyl or alkylhydroxyl; or Z represents C1-6alkyl-NH-C(O)-C1-6alkyl-, C1-6 alkyl-NH- or C1-6alkyl-NH-C(O)-C1O-; or Z represents lkyl-O- wherein said alkyl is unsaturated or saturated and can optionally be substituted by an alkyl or ydroxyl , or Z represents C1-6alkyl-O-C1-6alkyl- wherein said alkyl is unsaturated or saturated and can optionally be substituted by an alkyl or ydroxyl 20 or Z represents C1-6alkyl-O-C1-6alkyl-O- wherein said alkyl is unsaturated or saturated and can optionally be substituted by an alkyl or ydroxyl; wherein said method comprises the steps of: (a) providing a compound according to formula (II): N O X N (II) wherein n, X, and Y are as for formula (I); Q represents C1-10 saturated or unsaturated alkyl optionally substituted by an alkyl or ydroxyl; 5 or Q represents C1-6alkyl-NH-C(O)-C1-6alkyl-, C1-6alkyl-N(tert-butyloxycarbonyl)- or C1-6alkyl-NH-C(O)-C1-6alkyl-O-; or Q represents C1-10alkyl-O- n said alkyl is unsaturated or saturated and can ally be substituted by an alkyl or alkylhydroxyl, or Q represents C1-6alkyl-O-C1-6alkyl- wherein said alkyl is unsaturated or saturated and 10 can optionally be substituted by an alkyl or alkylhydroxyl, or Q represents C1-6alkyl-O-C1-6alkyl-O- wherein said alkyl is unsaturated or saturated and can optionally be substituted by an alkyl or alkylhydroxyl; (b) contacting the compound of formula (II) with acetic acid; or 15 TiCl3 and tetrahydrofuran; and wherein for compounds of a (II) wherein Q represents C1-6alkyl-N(tertbutyloxycarbonyl )-, said method further comprises the step of (c) contacting the compound ed in step (b) with trifluoroacetic acid and dichloromethane. 20

2. The method according to claim 1, wherein step (b) is contacting the compound of formula (II) with acetic acid, iron, and water.

3. The method according to claim 2, wherein n, X, Y, and Q in the compound of a (II) are in ance with n, X, Y, and Z, respectively, as indicated in a corresponding compound of formula (I) selected from the group consisting of: N N N O , , , , 5 , , , , N N N O , , O , , , , , and the method resulting in the preparation of said corresponding compound of formula (I).

4. The method according to claim 1, wherein the compound of formula (I) is 10 , wherein in step (a) the compound of formula (II) is ; wherein step (b) is contacting the nd of formula (II) with acetic acid.

5. The method ing to claim 1, wherein the compound of formula (I) is 5 , wherein in step (a) the compound of formula (II) is ; wherein step (b) is contacting the compound of formula (II) with acetic acid, iron, and water, and wherein the reaction mixture obtained in step (b) is concentrated to yield a solid, and contacting said solid with TiCl3 and tetrahydrofuran.

6. The method according to claim 1, wherein n, X, Y, and Q in the compound of formula (II) are in accordance with n, X, Y, and Z, respectively, as indicated in a corresponding compound of formula (I) selected from the group consisting of , , , , , , , and ; and step (b) is ting the compound of formula (II) with TiCl3 and tetrahydrofuran; the method resulting in the preparation of said corresponding compound of formula (I).

7. The method according to claim 2, wherein the compound of formula (I) is , N N N O , or ; and wherein in step (a) the compound of formula (II) is , , or ; 10 and wherein the reaction t of step (b) is subjected to step (c).

8. The method according to claim 2, wherein the compound of formula (I) is , or ; and n in step (a) the compound of formula (II) is , , or , 5 wherein the method comprises a step (b2) wherein the reaction product of step (b) is contacted with Wilkinson’s catalyst, H2, tetrahydrofuran, and methanol, yielding as a reaction product N N N O , or O ; and subjecting the reaction product of step (b2) to step (c). 10

9. The method according to claim 1, wherein said method is substantially as herein described with reference to any one of the Examples.