WO2014074883A1 - Novel synthesis of ld101 - Google Patents

Abstract

Novel synthetic methods for preparation of LD101, a compound according to formula (A) are disclosed.

Description

NOVEL SYNTHESIS OF LD101

FIELD OF THE INVENTION

[0001] This invention relates to novel syntheis and manufacturing methods for preparation of 4- amino-8-[3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-oxopyrido[2,3-d]pyrimidine-6-carboxamide, a novel AKT/PKB modulator (LD101).

BACKGROUND OF THE INVENTION

[0002] Akt/PKB, represents a subfamily of the serine/threonine kinase. Akt was first described as the cellular homologue of the product of the v-Akt oncogene (Bellacosa et al. 1991), and it has three members, Aktl/PKBa, ΑΐΛ2/ΡΚΒβ and Akt3/PKBy ((hereinafter referred to as 'Aktl ', 'Akt2' and 'Akt3'), respectively) (Cheng et al. 1992; Jones et al. 1991a; Jones et al. 1991b). Activation of Akt depends on the integrity of the pleckstrin homology (PH) domain, which mediates its membrane translocation, and on the phosphorylation of Thr³⁰⁸ in the activation loop and Ser⁴⁷³ (Konishi et al. 1995). Phosphoinositides, PtdIns-3,4-P2 and PtdIns-3,4,5-P3, produced by PI3 K bind directly to the PH domain of Akt, driving a conformational change in the molecule, which enables the activation loop of Akt to be phosphorylated by PDKl at Thr³⁰⁸ (Datta et al. 1999). Phosphorylation of Aktl occurs on two regulatory sites, Thr³⁰⁸ in the catalytic domain activation loop and on Ser⁴⁷³ near the carboxy terminus (D. R. Alessi et al. EMBO J. 15:6541-6551 (1996) and R. Meier et al. J. Biol. Chem. 272:30491-30497 (1997)).

Equivalent regulatory phosphorylation sites occur in Akt2 (Thr³⁰⁹ and Ser⁴⁷⁴) and Akt3 (Thr³⁰⁶ and Ser⁴⁷²). The upstream kinase, which phosphorylates Akt at the activation loop site has been cloned and termed 3'- phosphoinositide dependent protein kinase 1 (PDKl). PDKl phosphorylates not only Akt, but also p70 ribosomal S6 kinase, p90RSK, serum and glucocorticoid-regulated kinase (SGK), and protein kinase C. Full activation of Akt is also associated with phosphorylation by PDK2 (Peterson et al. 1999, Liu et al. 2005) at Ser⁴⁷³ within a C-terminal hydrophobic motif (Datta et al. 1999). Although the role of PDKl in Thr³⁰⁸ phosphorylation is well established, the mechanism of Ser⁴⁷³ phosphorylation by PDK2 has not been completely elucidated. A number of candidate enzymes designated PDK2 responsible for this phosphorylation have been put forward, including integrin- linked kinase (Persad et al. 2001), PDKl when in a complex with the kinase PRK2 (Wick et al. 2000), Akt itself, through autophosphorylation (Toker et al. 2000), DNA-dependent kinase (Feng et al. 2004), and the rictor-mTOR complex (Sarbassoy et al. 2005). The activity of Akt is negatively regulated by tumor suppressor called phosphatase and tensin homolog (PTEN), which is frequently mutated in human malignancy (Vazquez et Al. 2000). PTEN encodes a dual-specificity protein and lipid phosphatase that reduces intracellular levels of

phosphatidylinositol 3-kinase or PtdIns(3,4,5)P3 (PI3K) by converting them to phophatidylinositol 2- kinase or PtdIns(4,5)P2 (PI2K), thereby inhibiting the PI3K/Akt signaling transduction intracellular pathway (Stambolic et al. 1998). [0003] Apoptosis (programmed cell death) plays essential roles in embryonic development and pathogenesis of various diseases, such as degenerative neuronal diseases, cardiovascular diseases and cancer. Recent work has led to the identification of various pro- and anti-ap opto tic gene products that are involved in the regulation or execution of programmed cell death. Expression of anti-ap opto tic genes, such as Bcl2 or Bcl-x.sub.L, inhibits apoptotic cell death induced by various stimuli. On the other hand, expression of pro-apoptotic genes, such as Bax or Bad, leads to programmed cell death (Adams et al. Science, 281 : 1322-1326 (1998)). The execution of programmed cell death is mediated by caspase- 1 related proteinases, including caspase-3, caspase-7, caspase-8 and caspase-9 etc (Thornberry et al.

Science, 281 : 1312-1316 (1998)).

[0004] The phosphatidylinositol 3'-OH kinase (PI3K)/Akt/PKB pathway appears important for regulating cell survival/cell death (Kulik et al. Mol. Cell. Biol. 17: 1595- 1606 (1997); Franke et al, Cell, 88:435-437 (1997); Kauffmann-Zeh et al. Nature 385:544-548 (1997) Hemmings Science, 275:628-630

(1997) ; Dudek et al., Science, 275:661 -665 (1997)). Survival factors, such as platelet derived growth factor (PDGF), nerve growth factor (NGF) and insulin- like growth factor-1 (IGF-1), promote cell survival under various conditions by inducing the activity of PI3K (Kulik et al. 1997, Hemmings 1997). Activated PI3K leads to the production of phosphatidylinositol (3,4,5)-triphosphatase (Ptdlns (3,4,5)-P3), which in turn binds to, and promotes the activation of, the serine/threonine kinase Akt, which contains a pleckstrin homology (PH)-domain (Franke et al Cell, 81 :727-736 (1995); Hemmings Science, 277:534 (1997); Downward, Curr. Opin. Cell Biol. 10:262-267 (1998), Alessi et al., EMBO J. 15: 6541 -6551 (1996)). Specific inhibitors of PI3K or dominant negative Akt/PKB mutants abolish survival-promoting activities of these growth factors or cytokines. It has been previously disclosed that inhibitors of PI3K (LY294002 or wortmannin) blocked the activation of Akt/PKB by upstream kinases. In addition, introduction of constitutively active PI3K or Akt/PKB mutants promotes cell survival under conditions in which cells normally undergo apoptotic cell death (Kulik et al. 1997, Dudek et al. 1997).

[0005] Three members of the Akt subfamily of second-messenger regulated serine/threonine protein kinases have been identified and termed Aktl/PKBot, ΑΜ2/ΡΚΒβ, and Akt3/PKBy (hereinafter referred to as "Aktl", "Akt2" and "Akt3"), respectively. The isoforms are homologous, particularly in regions encoding the catalytic domains. Akts are activated by phosphorylation events occurring in response to PI3K signaling. PI3K phosphorylates membrane inositol phospholipids, generating the second messengers phosphatidyl-inositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate, which have been shown to bind to the PH domain of Akt. The current model of Akt activation proposes recruitment of the enzyme to the membrane by 3'-phosphorylated phosphoinositides, where

phosphorylation of the regulatory sites of Akt by the upstream kinases occurs (B. A. Hemmings, Science 275:628-630 (1997); B. A. Hemmings, Science 276:534 (1997); J. Downward, Science 279:673-674

(1998) ).

[0006] Phosphorylation of Aktl occurs on two regulatory sites, Thr³⁰⁸ in the catalytic domain activation loop and on Ser⁴⁷³ near the carboxy terminus (D. R. Alessi et al. EMBO J. 15:6541 -6551 (1996) and R. Meier et al. J. Biol. Chem. 272:30491-30497 (1997)). Equivalent regulatory phosphorylation sites occur in Akt2 and Akt3. The upstream kinase, which phosphorylates Akt at the activation loop site has been cloned and termed 3'-phosphoinositide dependent protein kinase 1 (PDK1). PDK1 phosphorylates not only Akt, but also p70 ribosomal S6 kinase, p90RSK, serum and glucocorticoid-regulated kinase (SGK), and protein kinase C. The upstream kinase phosphorylating the regulatory site of Akt near the carboxy terminus has not been identified yet, but recent reports imply a role for the inte grin- linked kinase (ILK- 1), a serine/threonine protein kinase, or autophosphorylation.

[0007] Akt phosphorylates and/or interacts with a number of molecules to exert its normal cellular functions, which include roles in cell proliferation, survival, migration and differentiation (Cheng et al. 2001). Many lines of evidence demonstrate that Akt is a critical player in tumor development and progression. In addition, overexpression of Akt and /or aberrant hyperactivation of Akt pathway has been detected in up to 50% all human tumors (Sun et al. 2001 ; Cheng et al. 1997) and is closely associated with chemoresistance (West et al. 2002). Therefore, Akt has been an attracting target for anti-cancer drug discovery (West et al. 2002). A recent study identified a recurring somatic mutation within PH domain of AKT1 in human breast, colorectal, lung and ovarian cancers that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the lipid-binding pocket (18) which led to hyperactivated and constitutive ly active Aktl . Lys 17 alters the electrostatic interactions of the pocket and forms new hydrogen bonds with a phosphoinositide ligand. This mutation activates A T1 through aberrant pathological localization to the plasma membrane, transforms cells and induces leukemia in mice (18). Further, the E17K substitution reduces the sensitivity to an allosteric Akt kinase inhibitor (18), but not to API- 1 (or LD- 101) which inhibits all isoforms of Akt in a non-PH domain dependent manner. Thus, the pharmacodynamic inhibitory activities of the compound of instant invention is not dependent of the length of amino acid chain, the order of amino acid sequence or the amount of amino acids on the PH-domain of the Akt.

[0008] Analysis of Akt levels in human tumors showed that Aktl is hyperactivated in gastric cancer {Hill, MM., and Hemmings B.A. 2002. Pharmacol. Therap. 93, 243), while Akt2 is overexpressed in a significant number of ovarian (J. Q. Cheng et al. Proc. Natl. Acad. Sci. U.S.A. 89:9267-9271 (1992)) and pancreatic cancers (J. Q. Cheng et al. Proc, Natl. Acad. Sci. U.S.A. 93:3636-3641 (1996)). Similarly, Akt3 was found to be overexpressed in breast and prostate cancer cell lines (Nakatani et al. J. Biol. Chem. 274:21528-21532 (1999).

[0009] The tumor suppressor PTEN, a protein and lipid phosphatase that specifically removes the 3 ' phosphate of Ptdlns(3,4,5)-P3, is a negative regulator of the PI3K/Akt pathway (Li et al. Science 275: 1943- 1947 (1997), Stambolic et al. Cell 95:29-39 (1998), Sun et al. Proc. Natl. Acad. Sci. U.S.A. 96:6199-6204 (1999)). Germline mutations of PTEN are responsible for human cancer syndromes such as Cowden disease (Liaw et al. Nature Genetics 16:64-67 (1997)). PTEN is deleted in a large percentage of human tumors and tumor cell lines without functional PTEN show elevated levels of activated Akt (Li et al. supra, Guldberg et al. Cancer Research 57:3660-3663 (1997), Risinger et al. Cancer Research 57:4736-4738 (1997)).

[0010] These observations demonstrate that the PI3K Akt pathway plays important roles for regulating cell survival or apoptosis in tumorigenesis. [0011] Inhibition of Akt activation and activity can be achieved by inhibiting PI3 with inhibitors such as LY294002 and or wortmannin. However, PI3K inhibition has the potential to indiscriminately affect not just all three Akt isozymes but also other PH domain-containing signaling molecules that are dependent on Pdtlns(3,4,5)-P3, such as the Tec family of tyrosine kinases, hence accompanied toxicities. Moreover, it has been disclosed that Akt can be activated by growth signals that are independent of PI3K.

[0012] Alternatively, Akt activity can be inhibited by blocking the activity of the upstream kinase PDK1. Some PDK1 inhibitors have been disclosed such as BX-795, BX-912, and BX-320 (Berlex Biosciences), A 12 (Arno Therapeutics) and 7-hydroxystaurosporine (UCN-01 - Abbott Labs). Again, inhibition of PDK1 would result in inhibition of multiple protein kinases whose activities depend on PDK1, such as AGC family protein kinases including PKC isoforms, SGK, and S6 kinases, thus accompanied by toxicities (Williams et al. Curr. Biol. 10:439-448 (2000).

[0013] In the last several years, through combinatorial chemistry, high-throughput and virtual screening, and traditional medicinal chemistry, a dozen inhibitors of the Akt pathway have been identified. Lipid-based inhibitors of Akt were the first to be developed, including perifosine (Kondapaka et Al. 2003), PX-316 (Meuillet et al. 2004) and phosphatidylinositol ether lipid analogues (PLAs) (Castillo et al. 2004), which were designed to interact with the PH domain of Akt. In addition, several Akt antagonists have been identified using high-throughput screening of chemical libraries and rational design. These inhibitors include 9-methoxy-2-methylellipticinium acetate (Jin et al. 2004), the indazole- pyridine A-443654 (Luo et al. 2005), isoform-specific allosteric kinase inhibitors (Lindsley et al. 2005) and Akt/PKB signaling inhibitor-2 (API-2), also called triciribine/TCN (Yang et al. 2004). API-2/TCN is a tricyclic nucleoside that previously showed antitumor activity in phase I and phase II trials conducted, but multiple toxicities, including hepatotoxicity, hyperglycemia, thrombocytopenia, and

hypertriglyceridemia, precluded further development (Feun et al. 1993; Hoffman et al. 1996). By screen of the NCI diversity set, Yang et al have previously shown that API-2 inhibit Akt kinase activity and stimulate apoptosis of xenografts of human cancer cells exhibiting high Akt activity (Yang et al. 2004). This finding has provided new interest in studying this drug and raises the possibility that lower doses may inhibit Akt and induce tumor cell apoptosis without the previously associated side effects (Yang et at. 2004; Cheng et ct. 2005).

[0014] Small molecule inhibitors of Akt are useful in the treatment of tumors, especially those with activated Akt (e.g. PTEN null tumors and tumors with ras mutations). PTEN is a critical negative regulator of Akt and its function is lost in many cancer cases, including breast and prostate carcinomas, glioblastomas, and several cancer syndromes including Bannayan-Zonana syndrome (Maehama, T. et al. Annual Review of Biochemistry, 70: 247 (2001)), Cowden disease (Parsons, R.; Simpson, L. Methods in Molecular Biology (Totowa, N.J., United States), 222 (Tumor Suppressor Genes, Volume 1): 147 (2003)), and Lhermitte-Duclos disease (Backman, S. et al. Current Opinion in Neurobiology, 12(5): 516 (2002)). Akt3 is up-regulated in estrogen receptor-deficient breast cancers and androgen- independent prostate cancer cell lines and Akt2 is over-expressed in pancreatic and ovarian carcinomas. Aktl is amplified in gastric cancers (Staal, Proc. Natl. Acad. Sci. USA 84: 5034-7 (1987) and upregulated in breast cancers (Stal et al. Breast Cancer Res. 5: R37-R44 (2003)). Therefore a small molecule Akt inhibitor is expected to be useful for the treatment of these types of cancer as well as other types of cancer. Akt inhibitors are also useful in combination with further chemotherapeutic and anticancer agents.

[0015] The US Patent application publication, US 2009/0028855, describes specific pyridopyrimidine derivatives as Akt inhibitors, with anti-tumor activity. The patent application discloses LD101 as a lead compound with excellent anti-tumor activity.

[0016] Accordingly, a need therefore exists for the preparation LD 101, that the present invention is directed.

SUMMARY OF THE INVENTION

[0017] Novel methods for synthesizing LD101 (Formula A) are described herein.

LD-101 (A)

[0018] Accordingly, in one aspect, novel methods to prepare LD101 are provided for LD101 that have formula A.

[0019] In a particular aspect, the invention provides a process for preparing LD101 or a pharmaceutically acceptable salt, solvate, stereoisomer, polymorph or isotopic variant thereof;

comprising the synthetic steps depicted in Scheme 1 :

Scheme 1

^{( ) (5)} LD-101 (A) and wherein each R¹ and R² is independently selected from alkyl; and Steps Al, A2, A3, and A4 are as described herein.

[0020] In one embodiment, with respect to the compound of formula (1), (2), (4), and (5), each

R¹ is Me, Et, i-Pr, n-Pr, i-Bu, n-Bu, or t-Bu.

[0021] In one embodiment, with respect to the compound of formula (1), (2), (4), and (5), each

R¹ is Me or Et.

[0022] In one embodiment, with respect to the compound of formula (2), each R² is Me, Et, i-Pr, n-Pr, i-Bu, n-Bu, or t-Bu.

[0023] In one embodiment, with respect to the compound of formula (2), each R² is independently Me or Et.

[0024] In a further aspect, the invention provides a process for preparing a compound according to formula (1), useful as an intermediate for preparation of LD101 or a pharmaceutically acceptable salt, solvate, stereoisomer, polymorph or isotopic variant thereof;

comprising the synthetic steps depicted in Scheme 2:

Scheme 2

(6) (7) (8)

(1 )

wherein R¹ is alkyl; and Steps B 1 , B2, and B3 are as described herein.

[0025] Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing detailed description.

DETAILED DESCRIPTION OF THE INVENTION

Figures:

[0026] Fig. 1. Typical LD 101 HPLC chromatogram

[0027] Fig. 2. Typical HPLC reagent blank

[0028] Fig. 3. HPLC trace of LD 101 reference material

[0029] Fig. 4. UV Spectrum of LD 101

Definitions

Chemical Definitions

[0001] Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March 's Advanced Organic Chemistry, 5^& Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3^rd Edition, Cambridge University Press, Cambridge, 1987.

[0002] Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et ah, Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et ah, Tetrahedron 33 :2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The invention additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0003] When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example "Ci_₆ alkyl" is intended to encompass, C_h C₂, C₃, C , Q, C₆, Ci_₆, Ci_₅, Q_ 4, Ci_3, Ci_2, C2-6, C2-5, C2-4, C2-3, C3_6, C3 5,

and C5_6 alkyl.

[0004] The following terms are intended to have the meanings presented therewith below and are useful in understanding the description and intended scope of the present invention.

When describing the invention, which may include compounds, pharmaceutical compositions containing such compounds and methods of using such compounds and compositions, the following terms, if present, have the following meanings unless otherwise indicated. It should also be understood that when described herein any of the moieties defined forth below may be substituted with a variety of substituents, and that the respective definitions are intended to include such substituted moieties within their scope as set out below. Unless otherwise stated, the term "substituted" is to be defined as set out below. It should be further understood that the terms "groups" and "radicals" can be considered interchangeable when used herein.

[0005] The articles "a" and "an" may be used herein to refer to one or to more than one (i.e. at least one) of the grammatical objects of the article. By way of example "an analogue" means one analogue or more than one analogue.

[0006] "Alkyl" refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms ("Ci_₂o alkyl"). In some embodiments, an alkyl group has 1 to 12 carbon atoms ("Ci_i2 alkyl"). In some embodiments, an alkyl group has 1 to 10 carbon atoms ("Cno alkyl"). In some embodiments, an alkyl group has 1 to 9 carbon atoms ("Ci_9 alkyl"). In some embodiments, an alkyl group has 1 to 8 carbon atoms ("Ci_8 alkyl"). In some embodiments, an alkyl group has 1 to 7 carbon atoms ("Ci_₇ alkyl"). In some embodiments, an alkyl group has 1 to 6 carbon atoms ("Ci-6 alkyl", also referred to herein as "lower alkyl"). In some embodiments, an alkyl group has 1 to 5 carbon atoms ("Ci_₅ alkyl"). In some embodiments, an alkyl group has 1 to 4 carbon atoms ("Ci^ alkyl"). In some embodiments, an alkyl group has 1 to 3 carbon atoms ("Ci_₃ alkyl"). In some embodiments, an alkyl group has 1 to 2 carbon atoms ("Ci_2 alkyl"). In some embodiments, an alkyl group has 1 carbon atom ("Ci alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C2-6 alkyl"). Examples of Q_6 alkyl groups include methyl (d), ethyl (C₂), n-propyl (C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄), iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl (C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (Q), and n-hexyl (C₆). Additional examples of alkyl groups include n-heptyl (C₇), n-octyl (C₈) and the like. Unless otherwise specified, each instance of an alkyl group is independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") with one or more substituents; e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. In certain embodiments, the alkyl group is unsubstituted Cno alkyl (e.g., -CH₃). In certain embodiments, the alkyl group is substituted Ci_io alkyl.

Other definitions

[0030] "Pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly, in humans.

[0031] "Pharmaceutically acceptable salt" refers to a salt of a compound of the invention that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic may be inorganic or organic acid addition salts and base addition salts. Specifically, such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfbnic acid, 2-naphthalenesulfbnic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-l-carboxylic acid, glucoheptonic acid, 3- phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine and the like. Salts further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the compound contains a basic functionality, salts of non toxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like. The term "pharmaceutically acceptable cation" refers to an acceptable cationic counter-ion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, and the like (see, e.g., Berge, et al, J. Pharm. Sci. 66(1): 1-79 (Jan."77) .

[0032] "Pharmaceutically acceptable vehicle" refers to a diluent, adjuvant, excipient or carrier with which a compound of the invention is administered.

[0033] "Pharmaceutically acceptable metabolically cleavable group" refers to a group which is cleaved in vivo to yield the parent molecule of the structural Formula indicated herein. Examples of metabolically cleavable groups include -COR, -COOR,-CONRR and -CH₂OR radicals, where R is selected independently at each occurrence from alkyl, trialkylsilyl, carbocyclic aryl or carbocyclic aryl substituted with one or more of alkyl, halogen, hydroxy or alkoxy. Specific examples of representative metabolically cleavable groups include acetyl, methoxycarbonyl, benzoyl, methoxymethyl and trimethylsilyl groups.

[0034] "Prodrugs" refers to compounds, including derivatives of the compounds of the invention,which have cleavable groups and become by solvolysis or under physiological conditions the compounds of the invention which are pharmaceutically active in vivo. Such examples include, but are not limited to, choline ester derivatives and the like, N- alky lmorpho line esters and the like. Other derivatives of the compounds of this invention have activity in both their acid and acid derivative forms, but in the acid sensitive form often offers advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see, Bundgard, H., Design of Prodrugs, pp. 7-9, 21 -24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well know to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters, amides and anhydrides derived from acidic groups pendant on the compounds of this invention are particular prodrugs. In some cases it is desirable to prepare double ester type prodrugs such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters. Particularly the Ci to Cg alkyl, C2-Cg alkenyl, i- % alkynyl, aryl, C₇-C12 substituted aryl, and C₇-C12 arylalkyl esters of the compounds of the invention.

[0035] "Solvate" refers to forms of the compound that are associated with a solvent or water

(also referred to as "hydrate"), usually by a solvolysis reaction. This physical association includes hydrogen bonding. Conventional solvents include water, ethanol, acetic acid and the like. The compounds of the invention may be prepared e.g. in crystalline form and may be solvated or hydrated. Suitable solvates include pharmaceutically acceptable solvates, such as hydrates, and further include both stoichiometric solvates and non-stoichiometric solvates. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. "Solvate" encompasses both solution-phase and isolable solvates. Representative solvates include hydrates, ethanolates and methanolates.

[0036] A "subject" to which administration is contemplated includes, but is not limited to, humans {i.e., a male or female of any age group, e.g., a pediatric subject (e.g, infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and/or a non-human animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and/or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal. The terms "human", "patient" and "subject" are used interchangeably herein.

[0037] "Therapeutically effective amount" means the amount of a compound that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" can vary depending on the compound, the disease and its severity, and the age, weight, etc. , of the subject to be treated.

[0038] "Preventing" or "prevention" refers to a reduction in risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject not yet exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.

[0039] The term "prophylaxis" is related to "prevention", and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease. Non-limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization; and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.

[0040] "Treating" or "treatment" of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e., arresting the disease or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment "treating" or

"treatment" refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In a further embodiment, "treating" or "treatment" relates to slowing the progression of the disease.

[0041] As used herein, the term "isotopic variant" refers to a compound that contains unnatural proportions of isotopes at one or more of the atoms that constitute such compound. For example, an "isotopic variant" of a compound can contain one or more non-radioactive isotopes, such as for example, deuterium (²H or D), carbon- 13 (¹³C), nitrogen- 15 (¹⁵N), or the like. It will be understood that, in a compound where such isotopic substitution is made, the following atoms, where present, may vary, so that for example, any hydrogen may be ²H/D, any carbon may be ¹³C, or any nitrogen may be ¹⁵N, and that the presence and placement of such atoms may be determined within the skill of the art. Likewise, the invention may include the preparation of isotopic variants with radioisotopes, in the instance for example, where the resulting compounds may be used for drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e., ³H, and carbon- 14, i.e., ¹⁴C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Further, compounds may be prepared that are substituted with positron emitting isotopes, such as ¹¹ C, ¹⁸F, ¹⁵0 and ¹ N, and would be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. All isotopic variants of the compounds provided herein, radioactive or not, are intended to be encompassed within the scope of the invention.

[0042] It is also to be understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed "isomers". Isomers that differ in the arrangement of their atoms in space are termed

"stereoisomers".

[0043] Stereoisomers that are not mirror images of one another are termed "diastereomers" and those that are non-superimposable mirror images of each other are termed "enantiomers". When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a "racemic mixture".

[0044] "Tautomers" refer to compounds that are interchangeable forms of a particular compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of π electrons and an atom (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base.

Another example of tautomerism is the aci- and nitro- forms of phenylnitromethane, that are likewise formed by treatment with acid or base. Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.

[0045] As used herein a pure enantiomeric compound is substantially free from other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). In other words, an "S" form of the compound is substantially free from the "R" form of the compound and is, thus, in enantiomeric excess of the "R" form. The term "enantiomerically pure" or "pure enantiomer" denotes that the compound comprises more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 98.5% by weight, more than 99% by weight, more than 99.2% by weight, more than 99.5% by weight, more than 99.6% by weight, more than 99.7% by weight, more than 99.8% by weight or more than 99.9% by weight, of the enantiomer. In certain embodiments, the weights are based upon total weight of all enantiomers or stereoisomers of the compound.

[0046] As used herein and unless otherwise indicated, the term "enantiomerically pure R- compound" refers to at least about 80% by weight R-compound and at most about 20% by weight S- compound, at least about 90% by weight R-compound and at most about 10% by weight S-compound, at least about 95% by weight R-compound and at most about 5% by weight S-compound, at least about 99% by weight R-compound and at most about 1% by weight S-compound, at least about 99.9% by weight R- compound or at most about 0.1% by weight S-compound. In certain embodiments, the weights are based upon total weight of compound.

[0047] As used herein and unless otherwise indicated, the term "enantiomerically pure S- compound" or "S-compound" refers to at least about 80% by weight S-compound and at most about 20% by weight R-compound, at least about 90% by weight S-compound and at most about 10% by weight R- compound, at least about 95% by weight S-compound and at most about 5% by weight R-compound, at least about 99% by weight S-compound and at most about 1% by weight R-compound or at least about 99.9% by weight S-compound and at most about 0.1% by weight R-compound. In certain embodiments, the weights are based upon total weight of compound.

[0048] In the compositions provided herein, an enantiomerically pure compound or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof can be present with other active or inactive ingredients. For example, a pharmaceutical composition comprising enantiomeric ally pure R- compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure R- compound. In certain embodiments, the enantiomerically pure R-compound in such compositions can, for example, comprise, at least about 95% by weight R-compound and at most about 5% by weight S- compound, by total weight of the compound. For example, a pharmaceutical composition comprising enantiomerically pure S-compound can comprise, for example, about 90% excipient and about 10% enantiomerically pure S-compound. In certain embodiments, the enantiomerically pure S-compound in such compositions can, for example, comprise, at least about 95% by weight S-compound and at most about 5% by weight R-compound, by total weight of the compound. In certain embodiments, the active ingredient can be formulated with little or no excipient or carrier.

[0049] The compounds of this invention may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)- or (S)- stereoisomers or as mixtures thereof.

[0050] Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art.

[0051] One having ordinary skill in the art of organic synthesis will recognize that the maximum number of heteroatoms in a stable, chemically feasible heterocyclic ring, whether it is aromatic or non aromatic, is determined by the size of the ring, the degree of unsaturation and the valence of the heteroatoms. In general, a heterocyclic ring may have one to four heteroatoms so long as the heteroaromatic ring is chemically feasible and stable.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

[0052] In certain aspects, provided herein are synthetic methods to prepare 4-amino-8-[3,4- dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-oxopyrido[2,3-d]pyrimidine-6-carboxamide (LD 101, formula A), a novel AKT/PKB modulator .

[0053] In certain aspect, provided herein is a process for preparing LD101 or a compound according to formula A:

LD-101 (A) ·

Larmaceutically acceptable salt, solvate, stereoisomer, polymorph or isotopic variant thereof comprising Steps of:

Al) converting the 4,6-dichloropyrimidine compound of formula (1):

to the 4,6-dichloropyrimidine compound of formula (2):

wherein each R¹ and R² is independently alkyl;

A2) reacting the 4,6-dichloropyrimidine compound of formula (2) with the D-ribofuranosyl derivative (3)

(3) to form the coupled compound of formula (4):

or an isomer thereof;

A3) cyclizing the compound of formula of (4) to form the pyrido[2,3-d]pyrimidine of formula (5):

(5)

A4) converting the pyrido[2,3-d]pyrimidine of formula (5) to form 4-amino-8-[3,4-dihydroxy-5- (hydrox}Tnethyl)oxolan-2-yl]-5-oxop3Tido[2,3-d]pyrimidine-6-carboxamide (LD-101) or a compound of formula A:

LD-101 (A) or a pharmaceutically acceptable salt, solvate, stereoisomer, polymorph or isotopic variant thereof.

[0054] In another aspect, provided herein is a process for preparing LD- 101 or a compound according to formula A:

LD-101 (A) ·

or a pharmaceutically acceptable salt, solvate, stereoisomer, polymorph or isotopic variant thereof comprising the synthetic steps depicted in Scheme 1 :

Scheme 1

and wherein each R¹ and R² is independently selected from alkyl.

[0055] In one embodiment, with respect to Step Al of the above process, the formation of compound of formula (2) occurs in the presence of trialkyl-o-formate.

[0056] In one embodiment, with respect to Step Al of the above process, the formation of compound of formula (2) occurs in the presence of trimethyl-o- formate or triethyl-o-formate.

[0057] In one embodiment, with respect to Step Al of the above process, the formation of compound of formula (2) occurs in the presence of an acid anhydride.

[0058] In one embodiment, with respect to Step Al of the above process, the formation of compound of formula (2) occurs in the presence of acetic anhydride.

[0059] In one embodiment, with respect to Step Al of the above process, the formation of compound of formula (2) occurs at 0-150 °C.

[0060] In one embodiment, with respect to the above process, R² is Me or Et.

[0061] In one embodiment, with respect to Step Al of the above process, the formation of compound of formula (2) occurs in the presence of triethyl orthoformate and acetic anhydride.

[0062] In one embodiment, with respect to Step A2 of the above process, the formation of compound of formula (4) occurs without any presence of a solvent.

[0063] In one embodiment, with respect to the Step A2 of the above process and with respect to the compound of formula (3), the Bz group may be replaced with any suitable hydroxyl protecting group.

[0064] In one embodiment, with respect to the Step A2 of the above process and with respect to the compound of formula (3), the compound may be

wherein Prot group is any suitable hydroxyl protecting group.

[0065] In one embodiment, with respect to the Prot group, Prot may be a suitable hydroxyl protecting group described by T. W. Greene and P. G. M. Wuts in Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991.

[0066] In one embodiment, with respect to the Prot group, Prot may be substituted or unsubstituted benzyl, alkoxyalkyl, trihaloalkyl, trialkylsilylalkyl, t-butyl, trialkylsilyl, or acetyl.

[0067] In one embodiment, with respect to the Step A2 of the above process, the formation of compound of formula (4) occurs at 0- 100 °C.

[0068] In one embodiment, with respect to Step A3 of the above process, the formation of compound of formula (5) occurs in the presence of a base.

[0069] In one embodiment, with respect to Step A3 of the above process, the formation of compound of formula (5) occurs in the presence of a tertiary amine.

[0070] In one embodiment, with respect to Step A3 of the above process, the formation of compound of formula (5) occurs in the presence of trialkyl amine.

[0071] In one embodiment, with respect to Step A3 of the above process, the formation of compound of formula (5) occurs in the presence of triethylamine.

[0072] In one embodiment, with respect to Step A3 of the above process, the formation of compound of formula (5) occurs in the presence of a solvent.

[0073] In one embodiment, with respect to Step A3 of the above process, the formation of compound of formula (5) occurs in the presence of acetonitrile.

[0074] In one embodiment, with respect to Step A3 of the above process, the formation of compound of formula (5) occurs in the presence of acetonitrile and triethylamine.

[0075] In one embodiment, with respect to Step A3 of the above process, the formation of compound of formula (5) occurs at - 10 to 100 °C.

[0076] In one embodiment, with respect to Step A3 of the above process, the formation of compound of formula (5) occurs around 0 °C.

[0077] In one embodiment, with respect to Step A4 of the above process, the formation of LD-

101 or the compound of formula (A) occurs in the presence of ammonia.

[0078] In one embodiment, with respect to Step A4 of the above process, the formation of LD-

101 or the compound of formula (A) occurs in the presence of a solvent.

[0079] In one embodiment, with respect to Step A4 of the above process, the formation of LD-

101 or the compound of formula (A) occurs in the presence of a protic solvent.

[0080] In one embodiment, with respect to Step A4 of the above process, the formation of LD-

101 or the compound of formula (A) occurs in the presence of an alcoholic solvent.

[0081] In one embodiment, with respect to Step A4 of the above process, the formation of LD-

101 or the compound of formula (A) occurs in the presence of methanol or ethanol.

[0082] In one particular embodiment, with respect to the above process, R² is Et.

[0083] In one particular embodiment, with respect to the above process, R¹ is Me or Et. [0084] In one embodiment, with respect to Step A4 of the above process, the formation of LD-

101 or the compound of formula (A) occurs in the presence of ammonia and methanol.

[0085] In one embodiment, with respect to Step A4 of the above process, the formation of LD-

101 or the compound of formula (A) occurs at -10 to 100 °C.

[0086] In one embodiment, with respect to the above process, Bz can be any suitable hydroxyl protecting group. In one particular embodiment, Bz is benzyl.

[0087] In one embodiment, with respect to the above process, the compound 4,6- dichloropyrimidine compound of formula (1) is prepared using a process comprising Steps of :

B l) converting pyrimidine compound of formula (6):

(6) to the 4,6-dichloropyrimidine compound of formula (7):

B2) converting the 4,6-dichloropyrimidine compound of formula (7) to the corresponding acid of formula (8):

B3) converting the 4,6-dichloropyrimidine carboxylic acid compound of formula (8) to the desired compound of formula (1):

(1 )

wherein R¹ is alkyl. [0088] In another embodiment, with respect to the above process, the compound 4,6- dichloropyrimidine compound of formula (1) is prepared following a procedure comprising the synthetic steps depicted in Scheme 2:

(1 )

wherein R¹ is alkyl.

[0089] In one embodiment, with respect to Step B 1 of the above process, the formation of compound of formula (7) occurs in the presence of POCI₃.

[0090] In one embodiment, with respect to Step B 1 of the above process, the formation of compound of formula (7) occurs in the presence of a solvent.

[0091] In one embodiment, with respect to Step B 1 of the above process, the formation of compound of formula (7) occurs in the presence of DMF.

[0092] In one embodiment, with respect to Step B 1 of the above process, the formation of compound of formula (7) occurs at 0-150 °C.

[0093] In one embodiment, with respect to Step B 1 of the above process, the formation of compound of formula (7) occurs at 90-110 °C.

[0094] In one embodiment, with respect to Step B 1 of the above process, the formation of compound of formula (7) occurs around 100 °C.

[0095] In one embodiment, with respect to Step B 1 of the above process, the formation of compound of formula (7) occurs for about 1 - 10 hr.

[0096] In one embodiment, with respect to Step B 1 of the above process, the formation of compound of formula (7) occurs for about 3-7 hr.

[0097] In one embodiment, with respect to Step B 1 of the above process, the formation of compound of formula (7) occurs for about 5 hr.

[0098] In one embodiment, with respect to Step B 1 of the above process, Step Bl) further comprises treatment of water.

[0099] In one embodiment, with respect to Step B2 of the above process, the formation of compound of formula (8) occurs in the presence of an oxidizing agent. [00100] In one embodiment, with respect to Step B2 of the above process, the formation of compound of formula (8) occurs in the presence of NaHP0₄.

[00101] In one embodiment, with respect to Step B2 of the above process, the formation of compound of formula (8) occurs in the presence of NaOClO.

[00102] In one embodiment, with respect to Step B2 of the above process, the formation of compound of formula (8) occurs in the presence of NaHP0₄ and NaOClO.

[00103] In one embodiment, with respect to Step B2 of the above process, the formation of compound of formula (8) occurs in a solvent.

[00104] In one embodiment, with respect to Step B2 of the above process, the formation of compound of formula (8) occurs in a protic solvent.

[00105] In one embodiment, with respect to Step B2 of the above process, the formation of compound of formula (8) occurs in t-BuOH.

[00106] In one embodiment, with respect to Step B2 of the above process, the formation of compound of formula (8) occurs in the presence of water.

[00107] In one embodiment, with respect to Step B2 of the above process, the formation of compound of formula (8) occurs at 0- 100 °C.

[00108] In one embodiment, with respect to Step B2 of the above process, the formation of compound of formula (8) occurs at 20-30 °C.

[00109] In one embodiment, with respect to the Step B3 of the above process, the formation of compound of formula (1) comprises an initial step of forming an intermediate wherein the OH is replaced with any suitable leaving group known to one skilled in the art.

[00110] In one particular embodiment, with respect to Step B3 of the above process, the formation of compound of formula (1) comprises an initial step of forming the corresponding acid chloride according to formula (9):

[00111] In one embodiment, with respect to Step B3 of the above process, the formation of the acid chloride (9) occurs in the presence of thionyl chloride.

[00112] In one embodiment, with respect to Step B3 of the above process, the formation of the acid chloride (9) occurs in the presence of oxazyl chloride.

[00113] In one embodiment, with respect to the Step B3 of the above process, the formation of compound of formula (1) comprises reaction of the acid chloride (9) or a compound of formula (9) wherein the CI group is replaced with any other suitable leaving group, with HO-C(=0)-CH₂-(C=0)-OR¹ (10).

[00114] In one embodiment, with respect to Step B3 of the above process, the formation of compound of formula (1) comprises reaction of the acid chloride (9) with HO-C(=0)-CH₂-(C=0)-OR¹ (10): ΗΟ_

O O

(10)

wherein R¹ is alkyl..

[00115] In one embodiment, with respect to Step B3 of the above process, the formation of the compound of formula (1) occurs in the presence of a base.

[00116] In one embodiment, with respect to Step B3 of the above process, the formation of the compound of formula (1) occurs in the presence of NaOH, or KOH.

[00117] In one embodiment, with respect to Step B3 of the above process, the formation of the compound of formula (1) occurs in the presence of Na₂C0₃ or K₂C0₃.

[00118] In one embodiment, with respect to Step B3 of the above process, the formation of the compound of formula (1) occurs in the presence of MgCl₂.

[00119] In one embodiment, with respect to Step B3 of the above process, the formation of the compound of formula (1) occurs in the presence of a solvent.

[00120] In one embodiment, with respect to Step B3 of the above process, the formation of the compound of formula (1) occurs at 0-100 °C.

[00121] In one embodiment, with respect to Step B3 of the above process, the formation of the compound of formula (1) occurs for 1-6 hr.

[00122] All patents, publications and pending patent applications identified are hereby incorporated by reference.

[00123] Abbreviations used in the description of the chemistry and in the Examples that follow are: AEBSF (p-aminoethylbenzenesulfonyl fluoride); BSA (bovine serum albumin); BuLi (n-Butyl lithium); CDC1₃ (chloroform-d); Cul (copper iodide); CuS0₄ (copper sulfate); DCE (dichloroethane); DCM (dichloromethane); DEAD (diethyl azodicarboxylate); DMF (N,N-dimethylformamide); DMSO (dimethyl sulfoxide); DTT (dithiothreitol); EDTA (ethylene-diamine-tetra-acetic acid); EGTA (ethylene-glycol- tetra-acetic acid); EtOAc (ethyl acetate); EtOH (ethanol); HOAc (acetic acid); HPLC (high-performance liquid chromatography); HRMS (high resolution mass spectrum); LCMS (liquid chromatograph-mass spectrometer); LHMDS (lithium bis(trimethylsilyl)amide); LRMS (low resolution mass spectrum); MeOH (methanol); MP-B(CN)H₃ (Macroporous cyanoborohydride); NaHC0₃ (sodium bicarbonate); Na₂S0₄ (sodium sulfate); Na(OAc)₃BH (sodium triacetoxyborohydride); NH₄OAc (ammonium acetate); NBS (N- bromosuccinamide); NMR (nuclear magnetic resonance); PBS (phosphate buffered saline); PCR

(polymerase chain reaction); Pd(dppf) ([l,l'-bis(diphenylphosphino)ferrocene]palladium); Pd(Ph3)4 (palladium(O) tetrakis-triphenylphosphine); POCl₃ (phosphorous oxychloride); PS-DIEA (polystyrene diisopropylethylamine); TBAF (tetrabutylammonium fluoride); THF (tetrahydrofuran); TFA

(trifluoroacteic acid); and TMSCH₂N₂ (trimethylsilyldiazomethane).

[00124] The compounds of this invention may be prepared by employing reactions as shown in the following section (General Synthetic Procedure, Specific (Novel) Synthesis), in addition to other standard manipulations that are known in the literature or exemplified in the experimental procedures. The illustrative Reaction Schemes below, therefore, are not limited by the compounds listed or by any particular substituents employed for illustrative purposes. Substituent numbering as shown in the General Synthetic Procedure does not necessarily correlate to that used in the claims and often, for clarity, a single substituent is shown attached to the compound where multiple substituents are optionally allowed under the definitions of the compound of invention hereinabove.

[00125] Reactions used to generate the compounds of this invention are prepared by employing reactions as shown in the General Synthetic Procedure (next section below), in addition to other standard manipulations such as ester hydrolysis, cleavage of protecting groups, etc., as may be known in the literature or exemplified in the experimental procedures.

GENERAL SYNTHETIC PROCEDURES

[00126] The compound LD101 can be prepared from readily available starting materials using the following general methods and procedures. See, e.g., Synthetic Scheme, below. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

[00127] Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and P. G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein.

[00128] The compound of this invention, for example, may be prepared by the reaction of a chloro derivative with ammonia and the product isolated and purified by known standard procedures. Such procedures include (but are not limited to) recrystallization, column chromatography or HPLC. The following schemes are presented with details as to the preparation of representative fused heterocyclics that have been listed hereinabove. The compounds of the invention may be prepared from known or commercially available starting materials and reagents by one skilled in the art of organic synthesis.

[00129] The compounds of the present invention may be prepared by a variety of processes well known for the preparation of compounds of this type, for example reaction schemes, and general procedures as described below.

[00130] The syntheses of the compounds of this invention are carried out in accordance with the methods set forth above and using the appropriate reagents, starting materials, and purification methods known to those skilled in the art. All starting materials in the following general syntheses may be commercially available or obtained by conventional methods known to those skilled in the art.

[00131] In this specification, especially in 'Synthetic Methods', the following abbreviations can be used:

BEP 2-bromo- 1 -ethylpyridinium tetrafluoroborate BOP benzotriazol- 1 -yloxy-tris(dimethylamino)phosphonium hexafluorophosphate

CDI 2-chloro- 1,3-dimethylimidazolinium chloride

DCC dicyclohexylcarbodiimide

DCM dichloromethane

DME 1 ,2-dimethoxyethane, dimethoxyethane

DMF N,N-dimethylformamide

DMSO dimethyl sulfoxide

EDC l-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrogen chloride

EtOAc ethyl acetate

EtOH ethanol

HOBt 1 -hydroxybenzotriazole

MeOH methanol

NMP N-methyl-2-pyrroliidone

THF tetrahydrofuran

TFA trifluoroacetic acid

uM μΜ

uL

SPECIFIC SYNTHESIS AND MANUFACTURING PROCEDURES FOR LDlOl

[00132] The present invention provides a large scale synthesis of LD-101, a compound of formula A. The representative synthetic methods used or can be used to prepare LD-101 or analogs are described in the following examples.

SYNTHETIC EXAMPLE 1

[00133] The compound LD-101 is prepared using the synthetic pathway shown in the Scheme 1.

Scheme 1

and wherein R¹ and R² are as described herein.

Step Al : Synthesis of the Intermediate according to formula (2)

[00134] The compound according to formula (2) is prepared by reacting the compound according to formula (1) with acetic anhydride, and triethyl orthoformate at around 130 °C. The product obtained is subsequently characterized by LC/MS and 'HNMR and is used as such for the next step.

Step A2: Synthesis of the Intermediate according to formula (4)

[00135] The compound according to formula (4) is prepared by reacting the compound according to formula (2) with tetra-O-acetyl-P-D-ribofuranose (3). The product obtained is subsequently characterized by LC/MS and ¹HNMR.

Step A3: Synthesis of the Intermediate according to formula (5)

[00136] The compound according to is formula (5) prepared by reacting the compound according to formula (4) with triethylamine in acetonitrile at around 0 °C. The product obtained is subsequently characterized by LC/MS and ¹HNMR.

Step A4: Synthesis of LD101 (formula A) [00137] The compound according to formula (A) (LD101) is prepared by reacting the compound according to formula (5) with methanolic ammonia. The product obtained is subsequently characterized by LC/MS (M+l = 698, calcd. 6978.15) and ¹HNMR.

[00138]

SYNTHETIC EXAMPLE 2

Synthesis of an Intermediate according to formula 1

[00139] The compound according to fomula (1) is prepared using the synthetic pathway shown in the Scheme 2.

Scheme 2

(1 )

and wherein R¹ is as described herein.

Step Bl : Synthesis of the Intermediate according to formula (7)

[00140] The compound according to formula (7) is prepared by reacting the compound according to formula (6) with POCI₃ and DMF at 100 °C for 5 hr. The product obtained is subsequently characterized by LC/MS and ¹HNMR.

Step B2: Synthesis of the Intermediate according to formula (8)

[00141] The compound according to formula (8) is prepared by reacting the compound according to formula (7) with NaHP0₄ and NaOClO in tert-butanol at room temparature. The product obtained is subsequently characterized by LC/MS and ^!HNMR.

Step B3: Synthesis of the Intermediate according to formula (1) [00142] The compound according to formula (1) is prepared by reacting the compound according to formula (8) with oxazolyl chloride to form the corresponding acid chloride. The acid chloride is then reacted with the ester according to formula (10, R¹ = Et) in the presence of K₂CO₃ and MgC^ to obtain the desired intermediate of formula (1).

(10)

[00143] The product obtained is subsequently characterized by LC/MS and ¹FTNMR.

[00144] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. From the foregoing description, various modifications and changes in the compositions and methods of this invention will occur to those skilled in the art. All such modifications coming within the scope of the appended claims are intended to be included therein.

[00145] All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

[00146] REFERENCES

US Patent application publication, US 2009/0028855

Rizkalla, B. H., Broom, A. D. (1972) J Org Chem. 37, 3980-3985

[00147] At least some of the chemical names of compounds of the invention as given and set forth in this application, may have been generated on an automated basis by use of a commercially available chemical naming software program, and have not been independently verified. Representative programs performing this function include the Lexichem naming tool sold by Open Eye Software, Inc. and the Autonom Software tool sold by MDL, Inc. In the instance where the indicated chemical name and the depicted structure differ, the depicted structure will control.

[00148] Chemical structures shown herein were prepared using ISIS^® /DRAW. Any open valency appearing on a carbon, oxygen or nitrogen atom in the structures herein indicates the presence of a hydrogen atom. Where a chiral center exists in a structure but no specific stereochemistry is shown for the chiral center, both enantiomers associated with the chiral structure are encompassed by the structure.

Claims

WHAT IS CLAIMED IS:

1. A process for preparing LD- 101 or a compound according to formula A:

LD-101 (A) .

or a pharmaceutically acceptable salt, solvate, stereoisomer, polymorph or isotopic variant thereof comprising Steps of:

Al) converting the 4,6-dichloropyrimidine compound of formula (1):

(1 ) to the 4,6-dichloropyrimidine compound of formula (2):

wherein each R¹ and R² is independently alkyl;

A2) reacting the 4,6-dichloropyrimidine compound of formula (2) with the D-ribofuranosyl derivative (3)

(3) to form the coupled compound of formula (4):

or an isomer thereof;

A3) cyclizing the compound of formula of (4) to form the pyrido[2,3-d]pyrimidine of formula (5):

A4) converting the pyrido[2,3-d]pyrimidine of formula (5) to form 4-amino-8-[3,4-dihydroxy-5- (hydroxymethyl)oxolan-2-yl]-5-oxopyrido[2,3-d]pyrimidine-6-carboxamide (LD-101) or a compound of formula A:

LD-101 (A) or a pharmaceutically acceptable salt, solvate, stereoisomer, polymorph or isotopic variant thereof.

2. A process for preparing LD- 101 or a compound according to formula A: NH₂ O NH₂

LD-101 (A)

or a pharmaceutically acceptable salt, solvate, stereoisomer, polymorph or isotopic variant thereof comprising the synthetic steps depicted in Scheme 1 :

Scheme 1

W ⁽⁵⁾ LD-101 (A) and wherein each R¹ and R² is independently selected from alkyl.

3. The process according either of claims 1 or 2, wherein in step Al) the formation of compound of formula (2) occurs in the presence of trialkyl-o-formate.

4. The process according to either of claims 1 or 2, wherein in step Al) the formation of

compound of formula (2) occurs in the presence of trimethyl-o- formate or triethyl-o-formate.

5. The process according to either of claims 1 or 2, wherein in step Al) the formation of

compound of formula (2) occurs in the presence of an acid anhydride.

6. The process according to either of claims 1 or 2, wherein in step Al) the formation of

compound of formula (2) occurs in the presence of acetic anhydride.

7. The process according to either of claims 1 or 2, wherein in step Al) the formation of

compound of formula (2) occurs at 0-150 °C.

8. The process according to either of claims 1 or 2, wherein R² is Me or Et. 9, The process according to either of claims 1 or 2, wherein in step Al) the formation of compound of formula (2) occurs in the presence of triethyl orthoformate and acetic anhydride.

10. The process according to either of claims 1 or 2, wherein in step A2) the formation of

compound of formula (4) occurs without any presence of a solvent.

1 1. The process according to either of claims 1 or 2, wherein in step A2) the formation of

compound of formula (4) occurs at 0-100 °C.

12. The process according to either of claims 1 or 2, wherein in step A3) the formation of

compound of formula (5) occurs in the presence of a base.

13. The process according to either of claims 1 or 2, wherein in step A3) the formation of

compound of formula (5) occurs in the presence of a tertiary amine.

14. The process according to either of claims 1 or 2, wherein in step A3) the formation of

compound of formula (5) occurs in the presence of trialkyl amine.

15. The process according to either of claims 1 or 2, wherein in step A3) the formation of

compound of formula (5) occurs in the presence of triethylamine.

16. The process according to either of claims 1 or 2, wherein in step A3) the formation of

compound of formula (5) occurs in the presence of a solvent.

17. The process according to either of claims 1 or 2, wherein in step A3) the formation of

compound of formula (5) occurs in the presence of acetonitrile.

18. The process according to either of claims 1 or 2, wherein in step A3) the formation of

compound of formula (5) occurs in the presence of acetonitrile and triethylamine.

19. The process according to either of claims 1 or 2, wherein in step A3) the formation of

compound of formula (5) occurs at - 10 to 100 °C.

20. The process according to either of claims 1 or 2, wherein in step A3) the formation of

compound of formula (5) occurs around 0 °C.

21. The process according to either of claims 1 or 2, wherein in step A4) the formation of LD- 101 or the compound of formula (A) occurs in the presence of ammonia.

22. The process according to either of claims 1 or 2, wherein in step A4) the formation of LD- 101 or the compound of formula (A) occurs in the presence of a solvent.

23. The process according to either of claims 1 or 2, wherein in step A4) the formation of LD- 101 or the compound of formula (A) occurs in the presence of a protic solvent.

24. The process according to either of claims 1 or 2, wherein in step A4) the formation of LD- 101 or the compound of formula (A) occurs in the presence of an alcoholic solvent.

25. The process according to either of claims 1 or 2, wherein in step A4) the formation of LD- 101 or the compound of formula (A) occurs in the presence of methanol or ethanol.

26. The process according to either of claims 1 or 2, wherein R² is Et.

27. The process according to either of claims 1 or 2, wherein R¹ is Me or Et.

28. The process according to either of claims 1 or 2, wherein in step A4) the formation of LD- 101 or the compound of formula (A) occurs in the presence of ammonia and methanol. The process according to either of claims 1 or 2, wherein in step A4) the formation of LD- 101 or the compound of formula (A) occurs at -10 to 100 °C.

The process according to either of claims 1 or 2, wherein the compound 4,6- dichloropyrimidine compound of formula (1) is prepared using a process comprising Steps of

Bl) converting pyrimidine compound of formula (6):

(6) to the 4,6-dichloropyrimidine compound of formula (7):

(7)

B2) converting the 4,6-dichloropyrimidine compound of formula (7) to the corresponding acid formula (8):

(8) ;

B3) converting the 4,6-dichloropyrimidine carboxylic acid compound of formula (8) to the desired compound of formula (1):

(1 )

wherein R¹ is alkyl.

31. The process according to either of claims 1 or 2, wherein the compound 4,6- dichloropyrimidine compound of formula (1) is prepared following a procedure comprising the synthetic steps depicted in Scheme 2: Scheme 2

(6) (8)

(1 )

wherein R¹ is alkyl.

32. The process according to either of claims 30 or 31, wherein in step Bl) the formation of compound of formula (7) occurs in the presence of POCl₃.

33. The process according to either of claims 30 or 31, wherein in step Bl) the formation of compound of formula (7) occurs in the presence of a solvent.

34. The process according to either of claims 30 or 31, wherein in step Bl) the formation of compound of formula (7) occurs in the presence of DMF.

35. The process according to either of claims 30 or 31, wherein in step Bl) the formation of compound of formula (7) occurs at 0-150 °C.

36. The process according to either of claims 30 or 31, wherein in step Bl) the formation of compound of formula (7) occurs at 90-110 °C.

37. The process according to either of claims 30 or 31, wherein in step Bl) the formation of compound of formula (7) occurs around 100 °C.

38. The process according to either of claims 30 or 31, wherein in step Bl) the formation of compound of formula (7) occurs for about 1 - 10 hr.

39. The process according to either of claims 30 or 31, wherein in step Bl) the formation of compound of formula (7) occurs for about 3-7 hr.

40. The process according to either of claims 30 or 31, wherein in step Bl) the formation of compound of formula (7) occurs for about 5 hr.

41. The process according to either of claims 30 or 31, wherein Step Bl) further comprises treatment of water.

42. The process according to either of claims 30 or 31, wherein in step B2) the formation of compound of formula (8) occurs in the presence of an oxidizing agent.

43. The process according to either of claims 30 or 31, wherein in step B2) the formation of compound of formula (8) occurs in the presence of NaHP0₄.

44. The process according to either of claims 30 or 31, wherein in step B2) the formation of compound of formula (8) occurs in the presence of NaOClO.

45. The process according to either of claims 30 or 31, wherein in step B2) the formation of compound of formula (8) occurs in the presence of NaHP0₄ and NaOClO.

46. The process according to either of claims 30 or 31, wherein in step B2) the formation of compound of formula (8) occurs in a solvent.

47. The process according to either of claims 30 or 31, wherein in step B2) the formation of compound of formula (8) occurs in a protic solvent.

48. The process according to either of claims 30 or 31, wherein in step B2) the formation of compound of formula (8) occurs in t-BuOH.

49. The process according to either of claims 30 or 31, wherein in step B2) the formation of compound of formula (8) occurs in the presence of water.

50. The process according to either of claims 30 or 31, wherein in step B2) the formation of compound of formula (8) occurs at 0-100 °C.

51. The process according to either of claims 30 or 31, wherein in step B2) the formation of compound of formula (8) occurs at 20-30 °C.

52. The process according to either of claims 30 or 31, wherein in step B3) the formation of compound of formula (1) comprises an initial step of forming the corresponding acid chloride according to formula (9):

53. The process according to claim 52, wherein the formation of the acid chloride (9) occurs in the presence of thionyl chloride.

54. The process according to claim 52, wherein the formation of the acid chloride (9) occurs in the presence of oxazyl chloride.

55. The process according to either of claims 30 or 31, wherein in step B3) the formation of compound of formula (1) comprises reaction of the acid chloride (9) with HO-C(=0)-CH₂- (C ^-OR¹ (10):

wherein R¹ is alkyl..

56. The process according to claim 55, wherein the formation of the compound of formula (1) occurs in the presence of a base.

57. The process according to claim 55, wherein the formation of the compound of formula (1) occurs in the presence of NaOH, or KOH.

58. The process according to claim 55, wherein the formation of the compound of formula (1) occurs in the presence of Na₂C0₃ or K₂C0₃.

59. The process according to claim 55, wherein the formation of the compound of formula (1) occurs in the presence of MgCl₂.

60. The process according to claim 55, wherein the formation of the compound of formula (1) occurs in the presence of a solvent.

61. The process according to claim 55, wherein the formation of the compound of formula (1) occurs at 0-100 °C.

62. The process according to claim 55, wherein the formation of the compound of formula (1) occurs for 1-6 hr.