WO2021220178A1 - Antagonistes d'iap et leurs applications thérapeutiques - Google Patents

Antagonistes d'iap et leurs applications thérapeutiques Download PDF

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Publication number
WO2021220178A1
WO2021220178A1 PCT/IB2021/053506 IB2021053506W WO2021220178A1 WO 2021220178 A1 WO2021220178 A1 WO 2021220178A1 IB 2021053506 W IB2021053506 W IB 2021053506W WO 2021220178 A1 WO2021220178 A1 WO 2021220178A1
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Prior art keywords
thiazol
fluorobenzoyl
piperidin
cyclohexyl
compounds
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PCT/IB2021/053506
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English (en)
Inventor
Béla BERTÓK
György Dormán
Ferenc Darvas
Alex DRIJVER
Csaba MAGYAR
Miklós SZÁVULY
Barbara BERKES
Alexandra WACHTLER
Gellért SIPOS
Viktória UJJ
László KŐHIDAI
Orsolya LÁNG
Angéla TAKÁCS
Diana MEZŐ
Eszter LAJKÓ
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Cominnex Zrt.
Semmelweis Egyetem
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Priority to HU2200468A priority Critical patent/HUP2200468A1/hu
Publication of WO2021220178A1 publication Critical patent/WO2021220178A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to novel IAP (Inhibitor of Apoptosis Proteins) antagonist compounds including their isomers, tautomeric forms and rotamers, salts, pharmaceutical compositions containing them together with their manufacturing processes and their therapeutic/pharmacological use alone or in combination with other active ingredients for the treatment of cell and tissue malignances caused diseases, including uncontrolled cell or tissue growth, proliferation and cancer.
  • IAP Inhibitor of Apoptosis Proteins
  • apoptosis (programmed cell death) plays a critical role in protection against tumorigeneses removing the abnormal cells.
  • the abnormal cells can not be eliminated, it promotes uncontrolled cell proliferation leading to tumor initiation.
  • the control of the cell division and the programmed cell death has opposite regulations, thus, they mutually inhibit each other’s signaling pathways. Consequently, the signals that activate cell growth inhibit apoptosis, and apoptosis signals stop the cell division. On the other hand, uncontrolled apoptosis causes serious damage of the tissues, which ultimately leads to death of the person. Thus, proper regulation has a key role for the healthy and normal functioning of the living systems.
  • IAPs Natural anti-apoptotic proteins (Inhibitors of Apoptosis Proteins - IAPs) play important roles in the regulation of the programmed cell death (apoptosis). Those proteins regulate the activation of caspases. IAPs inhibit initiator (Caspase-9) and effector caspases (Caspase-3 and - 7), while XIAP (IAP) antagonists such as SMAC (second mitochondria-derived activator of caspases)/DIABLO (direct IAP-binding protein with low pi) could temporarily suspend the caspase inhibition.
  • SMAC secondary mitochondria-derived activator of caspases
  • DIBLO direct IAP-binding protein with low pi
  • XIAPs X-chromosome linked inhibitors of apoptosis proteins
  • XIAPs X-chromosome linked inhibitors of apoptosis proteins
  • the resistance is a typical outcome of the chemotherapy treatment and reduces the success rate of the medication.
  • AVPI Ala-Val-Pro-Ile
  • BIR3 and BIR2 BIR domains
  • Kd 580 nM
  • the particular advantage of LCL161 is that it can be administered per os either as a pill or a capsule. Presently, the Phase-II clinical trials are going on.
  • LCL161 requires relatively high dose, which leads to the release of pro- inflammatory cytokines.
  • the initial 1800 mg/patient/week dose could not be raised over 3000 mg/patient/week since such high dose led to definite side-effects.
  • J.Clin.Oncol., 2014, 32(28), 3103-3110 J.Clin.Oncol., 2014, 32(28), 3103-3110.
  • in vivo investigations confirmed that combination with other anti-cancer agents and treatments was fairly effective in many cases for example it led to positive “breaking” the resistance in resistant lymphomas. (Blood Advances, 2018, 2(23), 3516-24).
  • further increase of the effectiveness of such compound family would be highly desired in order to reduce the therapeutic dose (Appl.
  • FIG. 1 The major structural elements of the SMAC binding motif (AVPI - tetrapeptide) according to their positions (P1-4)
  • the abbreviation the AVPI inhibitor tetrapeptide stands for: Alanine, Valine, Proline and Isoleucine.
  • the P3 unit tolerates only slight structural modifications based on the available data.
  • SMAC mimetic compounds contain either the pyrrolidine ring alone or condensed with other rings. Few publications discussed the potential feasibility of the O-substitution of the pyrrolidine ring at the 3 position, otherwise no other modifications were reported.
  • n is 1 or 2;
  • R 1 is hydrogen; or selected from alkyl, alkoxyalkyl and -(CH 2 ) l -dioxolane, where the value of “l” is an integer of 2 to 10, where any of the mentioned groups may be substituted independently from each other; or LNK-LGD;
  • R 2 is hydrogen; or selected from alkyl, alkoxyalkyl, alkyl-NHZ, alkyl-NH-C(NH)NHZ, aryl and -(CH 2 ) e -S-(CH 2 ) f -, where e and f, independently from each other, are integer of 1 to 6, where any of the mentioned groups may be substituted independently from each other; or LNK- LGD;
  • Z represents hydrogen; R 4 ; or -COR 4 ;
  • R 3 is hydrogen; or selected from
  • R 1 is hydrogen; C1-C4 alkyl, preferably methyl; or LNK-LGD;
  • R 2 is C1-C6 alkyl, C3-8 cycloalkyl, preferably C3-6 cycloalkyl; -(CH 2 ) e -S-(CH 2 ) f - where e and f, independently from each other, are integer of 1 to 4, preferably -CH 2 -CH 2 -S-CH 3 , -Bu- NHZ, BuCO 2 Z, wherein Z is H or C1-4 alkyl; or phenyl;
  • R 3 is optionally substituted phenyl, e.g.4-F-phenyl, O-LNK-LGD, NHR 4 or -OR 4, wherein R 4 is H or C1-4 alkyl;
  • R 5 is hydrogen; -(CH 2 ) m -aryl or -(
  • composition comprising a compound of general Formula I and salts, solvates or isomers thereof according to any of points 1 to 5 and a pharmaceutically acceptable carrier and/or diluent.
  • ⁇ refers to saturated, monovalent aliphatic radical including straight or branched chain alkyl, having 1 to 6 carbon atoms, suitably 1 to 4 carbon atoms, where 1 to 4 carbon atoms are most preferred such as methyl, ethyl, propyl, isopropyl, and particularly methyl or ethyl.
  • the termticianalkyl also covers theticiancycloalkyl” groups that means 3-8 carbon atom membered monocylic, saturated aliphatic ring dilemmaC3-8 cycloalkyl”).
  • phrase includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl groups.
  • the term could be derived from the term tauralkyl” in such a way that it is connected via an -O- group (ether linkage) to the rest of molecule (such as CH 3 -O-).
  • the preferred groups correspond to the preferred “alkyl” groups as specified above.
  • the term seekingalkenyl could also be derived from the term corpalkyl” in such a way that 2 hydrogen atoms are removed from theticianalkyl” group forming a double bond.
  • the minimum number of the carbon atoms is 2.
  • the carbon atom number is 2, 3 or 4 (C1-4) or 2 or 3 (C2-3), and the most preferred embodiment is the C2 alkenyl group.
  • crizAryl refers to a group derived from optionally substituted aromatic monocyclic or bicyclic ring systems having carbon atoms 6-10, and preferred group is phenyl.
  • Heteroaryl may refer to a group derived from 5-10 (suitably 5-8) membered aromatic ring containing 1-4 heteroatoms selected from N, O and S.
  • heteroaryl groups may refer to a group derived from an 8-12 membered fused ring system including at least one 5-7 membered heterocyclic ring containing 1, 2 or 3 heteroatoms selected from N, O, and S.
  • heteroaryl groups contain 1 or 2 heteroatom(s) (preferably N and/or O), for example groups derived from pyrazole, imidazole, triazole, tetrazole, pyridine, pyrazine and triazine.
  • the substituent is a group typically applied in organic chemistry for the substitution of the above groups.
  • the preferred examples can be selected from the following ones: halogen (fluoro, chloro, bromo, iodo group), carboxyl, carboxamide, hydroxy, alkyl, cycloalkyl, alkoxy, trifluoromethyl, metanesulfonyl, metansulfonamide, sulfonamide, cyano, nitro, nitrobenzyl, alkoxycarbonyl groups. More preferred examples are as follows: C1-4 alkyl, C1-4 alkoxy, halogen (fluoro, chloro, bromo, iodo), hydroxyl, (C1-4 alkoxy)carbonyl. In case of multiple substitution, the substituents can be selected independently from each other from the above lists.
  • R 1 H; C1-6 alkyl, optionally substituted with hydroxyl or (C1-4 alkoxy)carbonyl; (CH 2 ) a -dioxolane, where a is an integer of 2 to 10, preferably 3 to 8; or LNK- LGD, where more specifically LNK stands for -(CH 2 )p-, where p is an integer of 2 to 30, preferably 2 to 15 or 2 to 10, more preferably 3 to 8, or -(CH 2 ) c -CO 2 -(CH 2 ) d - where c and d, independently from each other, are integer of 2 to 10, preferably 3 to 8; and LGD is a sigma receptor ligand.
  • analogues are the PEG (polyethylene glycol) linkers.
  • R 2 C1-6 alkyl, optionally substituted with (C1-4 alkoxy)carbonyl or amino; C3-8 cycloalkyl, preferably C3-6 cycloalkyl; -(CH 2 ) e -S-(CH 2 ) f - where e and f, independently from each other, are integer of 1 to 6, preferably 1 to 4, e.g. -CH 2 -CH 2 -S-CH 3 .
  • the branched C3-6 alkyl groups like isopropyl, t-Bu) are also advantageous substituents.
  • R 3 phenyl, optionally substituted with 1 to 3 halogen atom(s), preferably monosubstituted with halogen, e.g. with F; amino, optionally substituted with C1-4 alkyl, e.g. ethyl; -OR 4 , wherein R 4 is H or C1-4 alkyl; or LNK-LGD, where more specifically LNK stands for -O-(CH 2 ) e - where e is an integer of 2 to 30, preferably 2 to 15 or 2 to 10, more preferably 3 to 8.
  • R 5 H, phenyl or -(CH 2 ) m -phenyl, where g is an integer of 1 to 4, preferably m is 1 (benzyl).
  • substituents e.g. with 1 halogen, which can be F.
  • the very specific examples for the above substituents can be seen in Table 11.
  • the term pertains to an ionic compound comprising an ion formed from a compound of the present invention and a counterion. Quaternary ammonium salts are also within the phrase.
  • salts of the compounds of Formula I may be prepared by reacting the compound of Formula I with a suitable amount of acid or base, preferably in an equimolecular ratio followed by precipitation or lyophilization.
  • Suitable salt may be acetate, adipinate, alginate, ascorbate, aspartate, benzoate, benzylsulfonate, hydrogensulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyklopentanpropionate, digluconate, dodecylsulfate, ethylsulfonate, fumarate, glucoheptanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethansulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, tartarate, sulfate, sulfonate, thiocyanate, toluenesulfonate (mesilate), undecanoate, maleate, trifluoroacetate and
  • salts are as follows: hydrochloride (HCl), hydrobromide (HBr), toluenesulfonate (mesilate), maleate and trifluoroacetate (TFA; especially as an intermediate salt).
  • HCl hydrochloride
  • HBr hydrobromide
  • meilate toluenesulfonate
  • TFA trifluoroacetate
  • the general formula I also intends to embrace the solvates (e.g. hydrates), if they are formed.
  • the phrase isomer embraces all possible isomers, e.g. stereoisomers, tautomers and rotamers. Their nomenclature are unambiguously defined and correspond to the teaching of the basic scientific literature (such as March’s Advanced Organic Chemistry, John Wiley & Sohns, inc. USA. ISBN 0-471-58589-0).
  • LNK represents a connecting (linker or spacer) group, which makes connection between the IAP and sigma ligand. Some different known linker group can be applied which can ensure the necessary distance between the IAP and the ligand (which can be e.g. a sigma ligand). “LNK” is preferably a 2-15, e.g. 2 to 10 carbon atom containing alkylene chain or other known linker group as PEG, which linker groups optionally may be substituted on the chain forming carbon atoms.
  • LGD represents a ligand which can facilitate the transfer through a cell membrane (targeting the binding to selected protein which has a role in the internalization).
  • a ligand which is ready to be bound to a sigma receptor (shortly: sigma receptor ligand or simply ligand).
  • the sigma receptor can be sigma-1 or sigma-2 (S1R, S2R) receptor.
  • S1R, S2R sigma-1 or sigma-2
  • the pharmaceutical composition comprising comprising a compound of general Formula I and salts, solvates or isomers thereof may contain a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutically acceptable carrier or diluent refers to usual excipients [e.g., semi-solid and liquid polyols, natural or hydrogenated oils, etc.]; water (e.g.
  • distilled water particularly distilled water for injection, etc.
  • physiological saline e.g., ethanol
  • alcohol e.g., ethanol
  • glycerol e.g., glycerol
  • polyols e.g., aqueous glucose solution, mannitol, plant oils, etc.
  • additives e.g., extending agent, disintegrating agent, binder, lubricant, wetting agent, stabilizer, emulsifier, dispersant, preservative, sweetener, colorant, seasoning agent or aromatizer, concentrating agent, diluent, buffer substance, solvent or solubilizing agent, chemical for achieving storage effect, salt for modifying osmotic pressure, coating agent or antioxidant], and the like.
  • the structural units of the invention compounds are marked with P1-P4 positions as shown below in Figure 7:
  • the main finding of the present invention is to increase the size of the saturated, heterocyclic ring, thus, replacement of the pyrrolidine ring to piperidine or azepane resulted in elevated activity which allowed us to design and develop highly active novel drug candidates.
  • the compounds according to the present inventions are new, there is a long- lasting dogma in the publications that increasing the ring size at the proline position of the IAP ligands leads to a dramatic loss in binding affinity.
  • our activity measurements revealed that the activity of these novel compounds has significantly been increased.
  • the structural modification such as replacement of the proline moiety with a structural element that have higher conformational flexibility, causes significant changes in many ways.
  • the IC 50 values of our piperidine containing analogues show 5-fold decrease at their best performance compared with LCL161, thus, their cytotoxic activity is 5 times higher.
  • the terminal amino acid in P1 has a basic character and its ability for protonation is a critical factor regarding the efficacy of the compound.
  • the efficacy is retained if the compounds are used either as a base or as a salt due to the buffering effect of the biological media.
  • Table 1 the chirality of the chirality centers is given in brackets after the sign of the compounds (see column 1), in the order of the chirality centers (P1, P2 and P3, respectively). .
  • the comparative measurements were performed on various high mortality cancer cell lines, including two pancreatic (PANC-1 and MiaPaca), lung (EBC1), colon (Colo205) and breast (MdaMb231) cancer and melanoma (A2058) cell lines.
  • the cell viability was evaluated after 24, 48, 72 hrs.
  • the cell viability was determined by impedimetric, flow cytometry, and Alamar Blue colorimetric assay methods. Beside the LCL161 compound having “S,S,S” configuration we tested two optically pure forms of our compounds mournS,S,S” - CI10-3), and consumersS,S,R” - CI10-4), as well as their mixtures (CI6-7, CI9-4 and CI10-2).
  • Another remarkable feature of the compounds of the invention is that its cytotoxic activity is amplified on multiresistant (chemotherapy resistant) cell lines. Such resistance evolves by the emergence of specific resistance enhancing factors such as the increasing level of apoptosis inhibitors compared with the sensitive cells.
  • the favorable properties of our compounds and its discovery underline the therapeutic relevance of the compounds in particular that the reference compound (LCL161) to our surprise showed significantly reduced cytotoxic activity on such multiresistant cell lines.
  • Table 6. The cytotoxic activity of our lead compound on the multiresistant cell lines are similar to the sigma ligand linked conjugates. Furthermore, there are also significant differences between the compounds on the sensitive cell lines. An additional indicator of this examination is the duration of the effectiveness, and in this context our novel lead compound has additional favorable properties.
  • the piperidine ring containing compound retains it cytotoxic activity (14.9 -> 13.9), namely the recurring proliferation of the surviving cancer cells is suppressed within 5 days.
  • the cytotoxicity results measured on human uterine sarcoma cell lines are shown in Table 6.
  • the compounds showed positive synergism with several chemotherapy agents in broad dose ranges.
  • the application of the compounds in optimized dose and ratio is further possibility to treat cancer cells successfully.
  • the neutralization of the Apoptosis Inhibitor Proteins allows to stimulate the activity of chemotherapy on cancer cells.
  • the derived combination index equation for two mutually non exclusive drugs is: Where D is the dose and (Dx) 1 is for (D) 1 “alone” that inhibits a system x%, and (Dx) 2 is for (D) 2 “alone” that inhibits a system x% whereas in the numerator, (D) 1 + (D) 2 , “in combination” also inhibit x%.
  • D is the dose and (Dx) 1 is for (D) 1 “alone” that inhibits a system x%
  • (Dx) 2 is for (D) 2 “alone” that inhibits a system x% whereas in the numerator, (D) 1 + (D) 2 , “in combination” also inhibit x%.
  • CI Index>1 that means antagonism
  • CI 1 additive effect and CI ⁇ 1 indicates synergistic activity.
  • Tables 7-10 The results are summarized in Tables 7-10.
  • Table 8 Viability and Combination Index values of CI10-3 combined with Paclitaxel after 96h Further biological activity data of some randomly selected compounds are summarized in Table 11 to illustrate the potential substitution possibilities of the clamed structures without limitation of the claimed compounds, the scope and the field of the invention. It is concluded that the claimed structures are valuable molecules for pharmaceutical applications.
  • the general synthetic routes of the claimed compounds are summarized in the Scheme 13.
  • the starting amino acids of general formula 1 are commercially available in optical pure and racemic forms. Synthesis can be carried out similarly using the “S” and the “R” stereoisomers applying stereoselective reactions published in the chemical literature. The coupling of the racemic mixtuture of compound 4 with optically pure forms of amino acids gives mixture of epimers from which the diastereomers can be separated using common chromatographic methods, thus optically pure dipeptides might be isolated. That possibility allows to purify the compounds even if the stereoselectivity of the reactions (amidation, hydrolysis or protecting group cleavage) were not complete. The synthesis of the compounds containing piperidine or azepane ring in P3 position of the final compounds can be performed analogous manner using same reactions or synthetic sequence.
  • Carboxylic acid 1 can be transformed to the corresponding amide 2 applying common amidation reactions.
  • the transformation of the amides with Lawesson’s reagent or phosphorus- pentasulfide gives the thioamide 3.
  • Running the thiolation below 50 °C the reaction is stereoselective, however, at higher temperatures the chiral center may isomerize.
  • Thioamide 3 can be cyclized using ethyl-bromo-pyruvate to thiazole 4. It is important to maintain basic conditions during the reaction otherwise the chiral center may racemize and the protecting group may also cleave off.
  • the stereoselectivity might be monitored using chiral HPLC methods e.g.
  • the position 5 of the thiazol ring of compound 4 can be activated using catalytical amount palladium reagent then can be substituted with reagents of general structure R 5 –X (X: leaving-group) containing classical leaving group -X such as mesylate group or halogen atom in a facilitated or reactive position, such as benzylic, allylic etc. halogenides. Further possibility is based on the lithiation of compound 4 with LDA at -78 oC, which gives the similar metallated derivative in the position 5 of the ring. That intermediate then can be brominated or substituted with R 5 –X reagent similar to the one which was used in the palladium catalyzed CH activation reaction.
  • the lithium “salt” of compound 4 is a highly sensitive compound, thus both the lithiation and the next substitution has to be carried out at law (-78 oC) temperature and quickly, in few minutes, otherwise the substance decomposes.
  • the best solution to have high yield and standard reproduction rate is to run that reaction in flow reactor where the temperature, the heat transfer and the reaction time (resident time) time can be set and controlled easely.
  • the resulting 4b bromo-derivative is a potential starting substance to get further derivatives of the general structure 4a.
  • Using palladium catalyzed cross-coupling reactions such as Suzuki, Sonogashira, Buchwald, Negishi etc. reactions ary. Alkynyl, amino-aryl etc. derivatives can be prepared.
  • compound 1a is commercially available or might be prepared using conventional bromination reaction in high quantities. Starting from that 5-Br thiazol the palladium catalyzed reaction results in the formation of intermediate 2a, which can be reacted then in postion 2 of the ring with the activated amino acid derivative 1b using photo-redox catalyst in Minisci reaction. Reagent 1b can be synthesized from compound 1 by ester formation with N-hydroxy- phtalimide. Compound 4a can be hydrolyzed using classical methods. The formed acid 5 is transformed to Weinreb amide 5a with methoxy-methylamine applying conventional amidation reaction conditions.
  • the formed 8a amines can be also reacted with protected amino acid derivatives of N-(Boc)R 1 such as illustrated with general form 7c.
  • protected amino acid derivatives of N-(Boc)R 1 such as illustrated with general form 7c.
  • several 9a derivatives are prepared in a raw using parallel synthesis principle. Cleaving the Boc group of 9a intermediate in acidic conditions serve the salt of the target compounds 10a’ from which the amine 10a can be freed off with basic treatment then reacting it with other pharmaceutically accepted acids novel salt forms of 10a” may be isolated in a stoichiometric manner.
  • the proper form for biological absorption or formulation can be prepared.
  • the 2 linear steps of the amidation reactions can be run in one step as well.
  • Dipeptide 7a prepared separately can be reacted with the cyclic-amine 7 in convergent manner and the intermediate 9a can be isolated from the amine 7 directly.
  • the needed 7a dipeptide is synthesized with common amidation reactions from the protected acid and amino groups such as illustrated with 7c and 7d structures.
  • the protected amino acids and their optical isomers are commercially available in pure form.
  • Special derivatives of intermediate 7c can be prepared from alanine-ester according the following scheme. The synthesis can be applied for the preparation of linker conjugated amino acids of the formula 7i which can be used for the preparation of linker conjugated peptide derivatives according to the methodology described for the transformation of 7c.
  • Target compound number 10 can be synthesized, which may serve as intermediate to prepare derivatives in modified sequence as well.
  • the amino group of compound 10 can be substituted with R 1 L reagents (where L means leaving or amine reactive functional group) in substitution or reductive amination reactions, thus target compounds 10a can be get.
  • Reagent of the general formula R 1 L also comprises the compounds of the formula II of Sigma Receptor Ligands where the “A” group is a “Linker” group and “L” is amino group reactive functional group, thus can be reacted with compound 10 to prepare Linker conjugated Sigma receptor and IAP ligand chimeras.
  • the reaction is preferably reductive amination where L is carbonyl (aldehyde) function, thus the P1 amino acid part of the target compound remains basic, which is essential to bind to the IAP protein.
  • the preparation of the Sigma Receptor ligands II and their analogs is illustrated in the general synthesis summary as follows: Boc protected piperidin-one (compound 12) was cyclized with thiophenyl ethanol 13 in acidic conditions.
  • the resulted spiro compound 14 was brominated with NBS in the position of 2 of the thiophene ring.
  • the isolated bromo compound 15 could be substituted in palladium catalyzed cross coupling reactions (Suzuki, Buchwald, Sonogashira etc.) to get wide range of molecules of general structure 11 having characteristic Sigma receptor binding structure.
  • the synthesis was slightly modied implicitly with re protecting amino group according to apllied cross coupling reaction requirements.
  • the representative sigma ligand 11a could be connected to IAP binding moiety.
  • Compound was 10 is alkylated with bifunctional reagent of the general formula of PG-A-L (where PG is protective group and L is a functional group which is able to react with the amine in compound 10 selectively like e.g. formyl, halogen, mesyl etc.).
  • the received intermediate was deprotected and the prepared reactive intermediate 10c was coupled after activation with Sigma Ligand 11a.
  • Sigma Ligand 11a There are several potential variation possibility of the conjugation where the step sequence is changed. For example, the synthesis is carried out in reverse order. First the Sigma Receptor ligand 10a is reacted with the protected linker moiety PG-A-L and after activation of the conjugate 11c is coupled with IAP ligand 10.
  • the IAP ligand 11 might be connected with the IAP intermediates earlier in the sequence for example to the amino acids or P3 or P4 fragments as well in earlier phase of the synthesis.
  • the IAP ligand of general structure I has several connection possibilities (in position P1 characterized as N-terminal connection point, at P2 in the central part of the chain via R 2 substituent and at P4 characterized as C-terminal), thus applying the same principle many conjugated compounds can be synthetized.
  • the conjugation possibility of the IAP ligand in its central part is illustrated as follows: The synthetic sequence certainly can be modified in that possibility as well like connecting intermediate 9a with linker reagent and connect the prepared intermediate with amine 11a.
  • the conjugation at C-terminal is shown as follows:
  • the C-teminal connection is possible at position 4 of the thiazole ring as well when the R 3 substituent has potential connection group like in compound 9c. That possibility is illustrated as follows:
  • Those possibilities might be applied and selected according to the reactivity, stability or synthetic feasibility of the reaction partners as well as the biological targets.
  • the reactions are carried out according the rules, experiences and prudent practices of the chemical syntheses (for example – if not indicated differently – the reaction were carried out at normal pressure in standard laboratory environment and conditions). According to the listed synthetic routes and reaction examples any skilled chemists is able to prepare all compounds and results claimed in the application.
  • Optical purity was determined by chiral HPLC tests applying YMC CELLULOSE-SB 250*4,6, 5 ⁇ M column and Heptane-IPA 95-5 eluent at ambient temperature. The structures were confirmed by 1H, 13C and 2D NMR tests. The nuclear magnetic resonance spectra were recorded on a Bruker Avance II 300, 400 or 500 MHz instrument. For the calibration of spectra, solvent-peak or tetramethylsilane signals were used.
  • the PANC-1 human pancreatic carcinoma of ductal origin
  • COLO 205 human colorectal adenocarcinoma
  • A2058 human metastatic melanoma
  • the reference cell line was NHDF (normal human fibroblast, Promo Cell, Heidelberg, Germany). The passage number of the applied cell line cultures were between p27 and p61 while in the case of NHDF it was below p10.
  • the multi drug-resistant cell line MES-SA/Dx5 was derived from the human uterine sarcoma cell line MES-SA (Sigma Catalogue number. 95051030) which was originally obtained from a tumour from a 56-year-old Caucasian female at the time of hysterectomy.
  • the Dx5 variant exhibits a 100-fold resistance to doxorubicin and has a reported doubling time of 30 hours.
  • the two additional marker chromosomes indicate clonal selection during drug selection.
  • MES- SA/Dx-5 cells exhibit marked cross-resistance to a number of chemotherapeutic agents (including daunorubicin, dactinomycin, vincristine, taxol, colchicine) and moderate cross- resistance to mitomycin C and melphalan. Cross resistance to bleomycin, cisplatin, carmustine, 5-fluorouracil or methotrexate was not observed.
  • the application special reagents and instruments are described in the examples.
  • HATU (1.47 eq.) was added to the mixture at 0°C then the mixture was stirred for 18 h at room temperature. The reaction was monitored by LCMS and TLC. After completion, the reaction mixture was diluted with water (10 mL/mmol) and extracted with EtOAc (3x10 mL/mmol). The combined organic phases were washed with 10% Na 2 CO 3 solution (2x3 mL/mmol), brine (2x3 mL/mmol) and dried over anhydrous Na 2 SO 4 . After filtration and concentration, the title compound was purified by flash chromatography (eluent: Heptane or Cyclohexane/EtOAc, 0-40 % EtOAc).
  • tert-Butyl-(2S)-2-carbamoylpiperidine-1-carboxylate (2-1) The title compound was prepared according to Example 1 starting from tert-Butyl-(2S)- 2-piperidine carboxylic acid (1-1). White powder, Y: 85%. APCI MS, m/z 229 [M+H]+ , HPLC- MS (ELSD signal) 100% (AUC). 1H NMR (400 MHz, CDCl 3 ) identical with WO2005115986 p105.
  • tert-Butyl-(2R)-2-carbamoylpiperidine-1-carboxylate (2-2) The title compound was prepared according to Example 1 starting from tert-Butyl-(2S)- 2-piperidine carboxylic acid (1-2). White powder, Y: 88%. APCI MS, m/z 229 [M+H]+ , HPLC- MS (ELSD signal) 100% (AUC). 1H NMR (400 MHz, CDCl 3 ) identical with WO2005115986 p103.
  • tert-Butyl-2-carbamoylazepane-1-carboxylate (2-3) The title compound was prepared according to Example 1 starting from tert-Butyl-2- azepane carboxylic acid (1-3). White powder, Y: 86%. APCI MS, m/z 242 [M+H]+ , HPLC-MS (ELSD signal) 100% (AUC).
  • thioamide 3-2 was prepared according to general Example 2 starting from tert-butyl- (2R)-2-carbamoylpiperidine-1-carboxylate (2-2).
  • thioamide 3-3 was prepared according to general Example 2 starting from tert-butyl- 2-carbamoylazepane-1-carboxylate (2-3).
  • Example 4 General procedure for the preparation of 2,5-disubstituted 1,3-thiazole carboxylates 4a
  • the starting 2,4-disubstituted thiazole 4 (1.0 eq.) was dissolved in dry THF (5 ml/mmol), cooled to -78°C then LDA solution (1.2 eq.) was added and stirred for 3 min then reagent (1.2 eq.) were added.
  • the reaction mixture was stirred for 3 min at -78°C, then warmed to room temperature, quenched with water and extracted with DCM.
  • the combined organic phases were evaporated and purified by column chromatography (eluent: Heptane or Cyclohexane/EtOAc, 0- 20% EtOAc).
  • Enantiomer purity ee 73% AMYLOSE-SA 250 mm*4,6 mm; 5 ⁇ M; A1B150:50; AMBIENT TEMP.; 5 ⁇ l; A1B150:50; A1: 1000ml H 2 O + 1ml TFA, B1: 1000ml ACN +1ml TFA.
  • Example 6 General procedure for the preparation of Weinreb Amides 5a
  • the thiazole carboxylic acid 5 was dissolved in THF (3 mL/mmol) and EDCI (1.1 eq.), HOBt (1.1 eq.), DIPEA (3.0 eq.) and N,O-dimethyl hydroxylamine hydrochloride (1.2 eq.) were added then stirred at room temperature overnight. After completion, the solvent was evaporated and the crude product was purified by flash chromatography (eluent: n-Heptane or Cyclohexane/EtOAc, 0-50% EtOAc).
  • Example 8 General procedure for N-Boc deprotection, preparation of compounds 7
  • the starting N-Boc protected derivative (4a, 6) was dissolved in DCM (4 ml/mmol) and treated with TFA (4 ml/mmol) or saturated solution of dry HCl in dioxane (2.5 mL/mmol), the mixture was stirred at room temperature for 2-4 h. After completion the reaction mixture was evaporated at reduced pressure. After concentration the salt was triturated with n-heptane, then diisopropylether, and the precipitated off-white solid was filtered off and dried.
  • Example 11 General procedure for the deprotection of N-Boc amino acid derivatives 8, preparation of compounds 8a
  • the title compound 8a-6 was prepared according to general Example 11 starting from Methyl-(5S) ⁇ 5 ⁇ [(tert ⁇ butoxy)carbonyl]amino ⁇ 6 ⁇ [(2S) ⁇ 2 ⁇ [4 ⁇ (4 ⁇ fluorobenzoyl) ⁇ 1,3 ⁇ thiazol ⁇ 2 ⁇ yl]piperidin ⁇ 1 ⁇ yl] ⁇ 6 ⁇ oxohexanoate (8-6). Y: 99%.
  • the amino acid derivate 7c (1.1 eq.) was dissolved in dry THF (5 mL/mmol) then HATU (1.2 eq.), DIPEA (3.0 eq.) were added and stirred at room temperature for 30 min. After activation of the amino acid, the amine 8a (1.0 eq.) was added and the mixture was stirred at room temperature and the reaction was monitored by LCMS analysis. After completion, the reaction was diluted with water and extracted with EtOAc. The organic phase was washed with brine and dried over anhydrous Na 2 SO 4 , filtered and evaporated.
  • Example 13 General procedure for the amidation of amino acid derivatives 8a with N-protected alanine, preparation of compounds 9b Under inert atmosphere, the acid derivate 7c (1.1 eq.) was dissolved in dry THF (5 mL/mmol), then HATU (1.2 eq.), DIPEA (3.0 eq.) were added and stirred at room temperature for 30 min. After that, the amine 8a (1.0 eq.) was added and the mixture was stirred at room temperature for overnight. The mixture was monitored by LCMS and TLC analysis. After completion, the reaction mixture was diluted with water and extracted with EtOAc. The organic phase was washed with brine and dried over anhydrous Na 2 SO 4 , then filtered off. The organic phase was concentrated and the crude product was purified by flash or column chromatography (eluent: n-heptane or cyclohexane/EtOAc, 0-50% EtOAc).
  • step (9b-4) was prepared according to general Example 13 starting from Ethyl- 2 ⁇ [(2S) ⁇ 1 ⁇ [(2S) ⁇ 2 ⁇ amino ⁇ 2 ⁇ cyclohexylacetyl]piperidin ⁇ 2 ⁇ yl] ⁇ 5 ⁇ benzyl ⁇ 1,3 ⁇ thiazole ⁇ 4 ⁇ carboxylate (8a-4) and Boc protected amino acid (7c).
  • step (9b-4) was prepared according to general Example 13 starting from Ethyl- 2 ⁇ [(2S) ⁇ 1 ⁇ [(2S) ⁇ 2 ⁇ amino ⁇ 2 ⁇ cyclohexylacetyl]piperidin ⁇ 2 ⁇ yl] ⁇ 5 ⁇ benzyl ⁇ 1,3 ⁇ thiazole ⁇ 4 ⁇ carboxylate (8a-4) and Boc protected amino acid (7c).
  • step (9b-4) was prepared according to general Example 13 starting from Ethyl- 2 ⁇ [(2S) ⁇ 1 ⁇ [(2S) ⁇ 2 ⁇ amino ⁇ 2 ⁇ cyclohexylacetyl]piperidin ⁇ 2 ⁇ yl] ⁇ 5 ⁇ benzyl ⁇ 1,3 ⁇ thiazole ⁇ 4 ⁇ carboxylate (8a-4) and Boc protected amino acid (7c).
  • the title compound 10a-7 was prepared according to general Example 14 starting from Methyl-(5S) ⁇ 5 ⁇ [(2S) ⁇ 2 ⁇ [(tert ⁇ butoxy)carbonyl](methyl)amino ⁇ propanamido] ⁇ 6 ⁇ [(2S) ⁇ 2 ⁇ [4 ⁇ (4 ⁇ fluorobenzoyl) ⁇ 1,3 ⁇ thiazol ⁇ 2 ⁇ yl]piperidin ⁇ 1 ⁇ yl] ⁇ 6 ⁇ oxohexanoate (9a-6).
  • Example 19 General procedure for the photo-redox coupling of thiazole derivatives 2a with activated pipecolinic acid, preparation of compounds 4a
  • 4CzIPN tetrakis(9H-carbazol-9-yl)benzene-1,3- dicarbonitrile
  • the active ester 1b in DMSO was transferred into the photoreactor and stirred under blue light (Blue LED, 450 nm, 32 W) at room temperature. After 3 hours and then in every 2 hours 1 mL of premixed active ester 1b was added to the reaction until total consumption of thiazole 2a. The reaction was monitored by LCMS. After completion the reaction mixture was extracted with DCM.
  • Example 24 General procedure I for the deprotection of linked sigma receptor ligand 11b-1, preparation of compounds 11c-1
  • Starting material 11b-1a was dissolved in MeCN (8 mL/mmol) and aqueous 1M hydrochloric acid (8 mL/mmol) was added. The resulting mixture was stirred at room temperature for 2 hours. The pH was adjusted 7 and the solvent was evaporated. Crude product was purified by flash chromatography using MeOH (0 to 5%) in CHCl 3 .
  • BIOLOGICAL TEST DESCRIPTIONS Example 30 General procedure for the Impedimetry assays The effect of investigated molecules on the viability of the model-cells (tumor and reference) was assessed using the xCELLigence SP System (Roche Applied Science, Indianapolis, IN, USA), which monitors the cellular events by measuring electrical impedance across interdigitating gold microelectrodes integrated on the bottom of tissue culture plates (E- plates). The detected impedance is influenced by the viability and morphology of the attached cells on the surface of the electrodes.
  • the impedance depends on the number of the attached cells and on the dimensional change of the attached cells on the electrodes. More cells attached to the electrode or spreading cause a larger increase in the impedance.
  • the change in impedances represented as “cell index” (CI) which is a relative and dimensionless value.
  • CI cell index
  • the experimental protocol was as follows: to register the background value, 100 ⁇ L of culture medium was added to each well and impedance was recorded for 1 h to gain constant background curves of impedance. In the following step, cells were seeded in 105 cells/mL density.
  • Example 31 General procedure for the Cytotoxicity assay (AlamarBlue) assays Cytotoxicity was tested by AlamarBlue assay (Invitrogen). The tumor model-cells were seeded at a density of 104 cells/well in 200 ⁇ l culture medium in a 96-well plate and allowed to adhere overnight at 37 °C. The cells were incubated with different concentrations of test compounds (10-7-10-4 M) for 24, 48, and 72 h, respectively.
  • test were run using diluted solutions of the test compounds, which were prepared from 5% DMSO stock solutions of the tested material diluted with PBS to 2.5x10-5 M then halving the concentration up to 2.5x10-7 M concentrations.
  • the IC50 values were from the viability values by OriginPro 2016 software.
  • Example 32 General procedure for drug combination tests The potential synergistic effect of the XIAP inhibitor and the classical chemotherapeutic agent (Gemcitabine and Paclitaxel) was tested in constant combinations ratio in the viability test based on Chou-Talalay's Combination Index Theorem.
  • the tested concentration ranges based on the single-agent activities and earlier dose-response assays were the following: the XIAP inhibitors and Paclitaxel were studied at 6,25x10-7 – 2x10 -5 M while gemcitabine was used in double dose.
  • the viability measurements were performed as it is described above, with the difference that cells were treated with both single and combined treatments.
  • the demonstrated CI indices are represented as CI values for each combination data point without simulation.
  • Example 33 General procedure for resistant cell and collateral activity test
  • the human uterine sarcoma cell line MES-SA and its doxorubicin resistant counterpart MES-SA/Dx5 were applied.
  • MES-SA/Dx5 cells express high levels of P-gp, and therefore are multidrug resistant (MDR) cells.
  • the cells were cultured in DMEM supplemented with 10% FBS, 5 mmol/L glutamine and 50 unit/mL penicillin and streptomycin (Life Technologies).
  • Cell survival was measured by the PrestoBlue® Cell Viability Reagent (Life Technologies, USA). The culture was seeded in 100 mL medium at a density of 5000 cells/well in 96-well plates; the serially diluted drug was added the following day. Cells were then incubated for 72 hours at 37oC in 5% CO 2 ; the drug containing medium was removed, and the PrestoBlue® reagent was added according to the manufacturer instructions. Fluorescence of the cells was measured by a Perkin Elmer EnSpire multimode plate reader or Perkin Elmer Victor X3 microplate reader. Data were background corrected and normalized to untreated cells. Curves were fitted with the help of Prism software using the sigmoidal dose-response model.
  • Curve fit statistics were used to determine IC 50 values. Differences between the IC 50 values of the respective cell lines were analysed by two-sided paired unpaired Student’s t-test and the results were considered statistically significant at a P value of ⁇ 0.05 (*) or 0.01 (**).
  • the well was fitted using BD GentestTM Pre-Coated PAMPA (353015).
  • the plates were incubated at 350 vibration/min for 5 hours at 25oC using BioSan Thermo-Shaker PST-60HL shaker tray.
  • the concentration of the solutions was determined by UV-VIS photometer (BioTek Synergy 2 Multimode Reader) at different wavelength at characteristic wave length maximum of the compounds.

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Abstract

La présente invention concerne des composés de formule I, y compris leurs isomères, leurs formes tautomères et leurs rotamères, leurs sels conjointement avec leur procédé de fabrication et leur utilisation pharmacologique seuls ou en combinaison avec d'autres principes actifs pour le traitement de maladies provoquées par des malignités cellulaires et tissulaires, y compris la croissance, la prolifération et le cancer non contrôlés.
PCT/IB2021/053506 2020-04-29 2021-04-28 Antagonistes d'iap et leurs applications thérapeutiques WO2021220178A1 (fr)

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WO2023154309A1 (fr) * 2022-02-08 2023-08-17 Vertex Pharmaceuticals Incorporated Dérivés de 4',5'-dihydrospiro[pipéridine-4,7'-thiéno[2,3-c]pyran] utilisés en tant qu'inhibiteurs de apol1 et leurs procédés d'utilisation
US11866446B2 (en) 2020-08-26 2024-01-09 Vertex Pharmaceuticals Incorporated Inhibitors of APOL1 and methods of using same
US12060346B2 (en) 2018-12-17 2024-08-13 Vertex Pharmaceuticals Incorporated Inhibitors of APOL1 and methods of using same

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12060346B2 (en) 2018-12-17 2024-08-13 Vertex Pharmaceuticals Incorporated Inhibitors of APOL1 and methods of using same
US11866446B2 (en) 2020-08-26 2024-01-09 Vertex Pharmaceuticals Incorporated Inhibitors of APOL1 and methods of using same
WO2023154309A1 (fr) * 2022-02-08 2023-08-17 Vertex Pharmaceuticals Incorporated Dérivés de 4',5'-dihydrospiro[pipéridine-4,7'-thiéno[2,3-c]pyran] utilisés en tant qu'inhibiteurs de apol1 et leurs procédés d'utilisation

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