Improved methods for producing cyclic guanosine monophosphate analogs - Patents.com

Abstract

The present invention relates to a process for preparing cyclic guanosine-3',5'-monophosphate analogs.The present invention also relates to new cyclic guanosine monophosphate analogs and intermediates obtainable by this process.

Description

[Technical field]

[0001] The present invention relates to a process for preparing cyclic guanosine-3',5'-monophosphate analogs. The invention also relates to new cyclic guanosine monophosphate analogs and intermediates obtainable by this process. [Background technology]

[0002] Retinitis pigmentosa (RP) is a group of severely disabling genetic neurodegenerative diseases. Typically, rod photoreceptor cells (which allow vision in dim light conditions) degenerate first during the disease. The loss of rods then triggers secondary degeneration of cone photoreceptor cells, the source of high-resolution daylight color vision, ultimately resulting in total blindness.

[0003] Genes mutated in retinitis pigmentosa are usually related to photoreceptor function, but some are related to general cellular functions (Kennan et al. 2005, Trends Genet. 21, 103-110). The molecule cGMP (cyclic guanosine monophosphate) plays a direct role in the phototransduction cascade that occurs in photoreceptor cells when light strikes them. In many cases, retinitis pigmentosa mutations lead to an overaccumulation of cGMP in the photoreceptor (Arango-Gonzalez et al. 2014 PLoS One. 9, e112142), as is the case in situations where genes for enzymes involved in photoreceptor cGMP metabolism are affected. This is the case for mutations in the photoreceptor enzyme phosphodiesterase 6 (whose subunits are encoded by the genes PDE6B, PDE6A, PDE6G, and for cone photoreceptors PDE6C, PDE6H), which hydrolyzes cGMP to GMP. The Pde6b gene is mutated in the rd1 mouse model of retinitis pigmentosa and has been thoroughly studied in many laboratories. In the predicted chain of events, the accumulation of cGMP in the PDE6B mutant retina occurs as a direct consequence of the actual gene defect, and therefore it can be considered an early and mechanistically fundamental component of degeneration. In its next step, the increase in cGMP stimulates four targets: 1) cGMP-dependent protein kinase (protein kinase G, PKG), which phosphorylates specific proteins when activated by cGMP; 2) Na+, which phosphorylates specific proteins when activated by cGMP; + and Ca 2+It can be assumed that the PKG and CNGC have at least one of the following cGMP-gated ion channels, 3) phosphodiesterases (PDEs) that allow cGMP-regulated influx of PKG, 4) hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. The first two cGMP targets are directly related to photoreceptor degeneration (Paquet-Durand et al. 2009, J. Neurochem. 108, 796-810; Paquet-Durand et al. 2011, Hum. Mol. Genet. 20, 941-947), while the others are known cGMP targets and therefore potentially involved in the degeneration process. Due to their direct association with early events, PKG and CNGC can be considered disease drivers, although downstream mechanisms are still less well understood in detail (Trifunovic et al. 2012, Curr. Mol. Med. 12, 598-612).

[0004] cGMP-derived PKG inhibitors, also known as cGMP analogues (e.g., Rp-8-Br-cGMPS), are known to provide protection of rd1 and rd2 photoreceptors in both in vitro and in vivo mouse retinitis pigmentosa models ( Paquet-Durand et al., 2009 ).

[0005] cGMP analogues are known in the art. WO2012130829 describes boranophosphate analogues of cyclic nucleotides. WO2018 / 010965 describes multimeric complexes of cGMP analogues. Butt et al. (FEBS letters, 1990, 263(1):48, DOI:10.1016 / 0014-5793(90)80702-K) describes the inhibition of cGMP-dependent protein kinase by (Rp)-guanosine 3',5'-monothiophosphate. In them, the activation of cGMP-dependent protein kinase and cAMP-dependent protein kinase by the diastereomers of guanosine 3',5'-monothiophosphate (Sp)-cGMPS and (Rp)-cGMPS and 8-chloroguanosine 3',5'-monothiophosphate (Sp)-8-Cl-cGMPS and (Rp)-8-Cl-cGMPS) was investigated. The (Sp)-diastereomers bound to cGMP-dependent protein kinase and stimulated its phosphotransferase activity. In contrast, the (Rp)-isomers bound to the enzyme without stimulating its activity. (Rp)-cGMPS and (Rp)-8-Clc-GMPS antagonized the activation of cGMP-dependent protein kinase. (Rp)-cGMPS also antagonized the activation of cAMP-dependent protein kinase. In contrast, (Rp)-8-Cl-cGMPS was a weak inhibitor of cAMP-dependent protein kinase. (Rp)-8-Cl-cGMPS appears to be a fairly selective inhibitor of cGMP-dependent protein kinase.

[0006] The synthesis of cGMP analogues is known in the art, but known protocols, such as those described by Sekhar et al. (1992, Mol. Pharmacol., 42:103-108) and Miller et al. (1973, Biochemistry 12:5310-5319), are only feasible on a laboratory scale. The reason for this is that the synthesis often involves laborious purification steps, including chromatography. Extensive chromatography may be required to separate the different stereoisomers of cGMP analogues, which may be either the Sp or Rp stereoisomers. In this case, the Rp configuration is the more potent material.

[0007] The synthesis of cGMP analogues involves the introduction of phosphorus followed by cyclisation, as described for example for cAMP analogues in scheme 4 on page 18 of WO 2005 / 123755. As described therein, the initial phosphorylation is supposed to take place at the 5'-OH group of the sugar, but neither the 2'-OH nor the 3'-OH groups were protected. This intermediate is never isolated. Instead, the product must then be directly cyclised in aqueous acetonitrile at high dilution with alkali hydroxide to give the diastereomeric nucleoside-3',5'-cyclic thiophosphate cAMP in an approximately 1:1 ratio, which must be separated by chromatographic techniques. Scheme 9 on page 23 of WO 2005 / 123755 describes how the diastereomeric ratio can instead be favourably shifted to 2:3 (Sp / Rp) using phosphites. This mixture then had to be separated again or simply used as a mixture. In both cases, the known reactions are laboratory scale. Summary of the Invention [Problem to be solved by the invention]

[0008] There is a need for more robust synthetic routes and production methods with improved yields. There is a need for production methods that avoid laborious separation techniques. There is a need for production methods with improved stereoselectivity. There is a need for intermediates with high purity. There is a need for improved separation of different stereoisomers. There is a need for synthetic protocols that allow for reactions on a larger scale. [Means for solving the problem]

[0009] The present invention relates to a method for producing a cyclic guanosine-3',5'-monophosphate (cGMP) analog or a synthetic intermediate thereof, comprising the steps of: i) A guanosine analogue of general formula (I) or a salt thereof: [ka] (wherein h is H, halogen, or Q; X 1 and X 2 are independently selected at each occurrence from H or p', p' is independently selected at each occurrence from a hydroxyl protecting group; R 1 and R 2 are each independently H, -(CH2) n -H, -(CH2) n -C 3~9 Heterocyclyl, -(CH2) n - selected from ar, and ar, where each occurrence of n is independently selected from 0, 1, 2, 3, or 4; or R 1 and R 2 together form -CH=C(ar)- or -(CH2) 1~4 C(=O)-, ar is independently at each occurrence a 5- or 6-membered aromatic or heteroaromatic ring, preferably phenyl or 2-furanyl, optionally substituted at each occurrence individually with halogen, -OH, -SH, -NH2, -NO2, -OCH3, -CH3, -CH2CH3, -CH(CH3)2, or -CF3, and optionally fused at the second occurrence of ar to preferably form a naphthyl moiety, and Q ... n -S-(CH2)n -H, -S-(CH2) n -OH, -S-(CH2) n -NH2, -(CH2) n -O-(CH2) n -H, -O-(CH2) n -OH, -O-(CH2) n -NH2, -OC(CH3)3, -O-CH(CH3)2, -(CH2) n -N(-[CH2] n H)2, -NH-(CH2) n NH2, -NH-(CH2) n -OH, -(CH2) n -Nc 1 c 2 and c 1 and c 2 form a 3- to 8-membered heterocycle together with the N to which they are attached, or 1 is H and c 2 is a 3-8 membered heterocycle, -(CH2) n -H, -N3, -CF3, -(CH2) n -ar, -O-(CH2) n -(ar), -NH-(CH2) n -(ar), -S-(CH2) n -(ar), -(CH2) n -Amide-ar, -O-(CH2) n -Amide-(ar), -NH-(CH2) n -Amide-(ar), -S-(CH2) n -amide-(ar), or a linker moiety, in which any -H may be optionally replaced by halogen, and each occurrence of n is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, or 8. providing a ii) contacting the provided guanosine analog with a phosphorus oxoacid derivative to obtain a guanosine 5'-monophosphorus oxoacid ester analog; iii) isolating the resulting guanosine 5'-monophosphoric acid ester analogue by crystallization; In a preferred embodiment, h is H or halogen or Q, preferably H or Br or Q, more preferably Br; 1 is H and X 2 is p', p' is selected from the group consisting of methoxymethyl (MOM), tetrahydropyranyl (THP), t-butyl (tBu), allyl (all), benzyl (Bn), (tri)alkylsilyl (e.g., t-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), or t-butyldiphenylsilyl (TBDPS)), acyl (e.g., acetyl (Ac), pivaloyl (Pv), or benzoyl (Bz)), preferably THP, (tri)alkylsilyl, and acyl; R 1 and R 2 taken together form -CH=C(ar)-, ar is phenyl, 4-methylphenyl, 3-thiophenyl, or 2-furanyl, preferably phenyl, and / or Q is furanyl, -CF3, -SCH3, -S(isopropylphenyl), -S(phenylamidomethyl), -S(halophenyl), -S(hydroxyphenyl), -S(aminophenyl), -S(nitrophenyl), -S(methoxyphenyl), -S(toluyl), -S(trifluoromethylphenyl), -Nc 1 c 2 (where c 1 and c 2 form a 3- to 8-membered heterocycle together with the N to which they are attached), -S-(CH2) n -OH, -S-(CH2) n -NH2, -NH-(CH2)nNH2, or -NH-(CH2)nOH, preferably furanyl, -CF3, -S(4-hydroxyphenyl), or -S(4-chlorophenyl).

[0010] In a preferred embodiment, the guanosine analogue of general formula (I) or a salt thereof used in step i) is 1 is H and X 2 is p', p' is triisopropylsilyl (TIPS), R 1 and R2 taken together form -CH=C(ar)-, and ar is phenyl. In a preferred embodiment, the phosphorus oxoacid derivative in step ii) is a phosphorylating or phosphonylating agent, preferably the phosphorus oxoacid derivative in step ii) is represented by the general formula (P): [ka] (Wherein, M is S or O, or is absent; o 1 and 2 are each independently halogen, -OC 1~8 Hydrocarbons, -SC 1~8 Hydrocarbons, -NH-C 1~8 Hydrocarbons, borano, methylborano, dimethylborano, cyanoborano, and -N(C 1~8 Hydrocarbon) 2, and 3 is H or o 1 or o 1 and 3 together, preferably C 2~12 (forming a chiral auxiliary that is a hydrocarbon) It is of the following.

[0011] In a preferred embodiment, the guanosine 5'-monophosphoric acid ester analog obtained in step ii) has the general formula (II) or a salt thereof: [ka] (In the formula, o 1 and 3 are each independently -OH or as defined above, and M is S or O. In a preferred embodiment, the guanosine analogue of general formula (I) or a salt thereof is: Ia) an unprotected guanosine analogue of general formula (pI) or a salt thereof: [ka] providing a Ib) contacting the unprotected guanosine analog with a (tri)alkylsilyl halide to obtain the multiply protected guanosine analog and, optionally, isolating the multiply protected guanosine analog by crystallization; Ic) selectively deprotecting a multiply protected guanosine analog to a compound of general formula (I), 1 is H and X 2 is p'), Id) The obtained compound of general formula (I) (wherein X 1 is H and X 2 is p'), optionally isolating the guanosine analogue of formula (I) by crystallization; Provided by.

[0012] In a preferred embodiment, the method comprises the steps of: iv) cyclization of the guanosine 5'-monophosphate oxoacid ester analog obtained in step ii) to obtain a cyclic guanosine-3',5'-monophosphate (cGMP) analog, said cyclization being preferably carried out in the presence of a sterically hindered base; Further includes:

[0013] Preferably, the cGMP analogue has the general formula (III) or a salt thereof: [ka] (wherein preferably X 2 is p' as defined above, and preferably o 3 is H) and the method further comprises: v) contacting the cGMP analog with a sulfurizing agent to produce a compound of general formula (III), 3 -SH or -SC 1~12 Optionally, the method further comprises obtaining a thiolated cGMP analog of the general formula (III) having a thiolated hydrocarbon, preferably -SH. Preferably, the cGMP analog of the general formula (III-Rp): [ka] (wherein preferably X 2 is p', and preferably o 3 is H, and optionally the thiolated cGMP analog is of general formula (III-Rp), preferably X 2 is p' and o 3 -SH or -SC 1~12 Hydrocarbon, preferably -SH) Arbitrarily, X 2 is p', and the method comprises: vi) deprotecting the protected hydroxyl moiety with X2 to obtain a deprotected cGMP analog; vii) optionally triturating the deprotected cGMP analog; viii) optionally converting the deprotected cGMP analog to a pharma- ceutical acceptable salt, preferably the sodium salt; Further includes:

[0014] In another embodiment, a compound of formula (II) or a salt thereof: [ka] (In the formula, h, X1, X 2 , R 1 , and R 2 is as defined above, and 1 and 3 are each independently -OH or as defined above, preferably o 3 is H, or o 1 and 3 together, preferably C 2~12 In a preferred embodiment, the compound is crystalline. Preferably, the compound is crystalline. 3 is H. Preferably, h is Br and X 1 is H and X 2 is p', p' is preferably triisopropylsilyl (TIPS), R 1 and R 2taken together form -CH=C(ar)-, where ar is phenyl, 1 is OH, o 3 is H and M is S or O, preferably O.

[0015] Description of the embodiments The present invention provides an improved method for the synthesis of cGMP analogs. The inventors have surprisingly found that the key intermediate guanosine 5'-monophosphoric oxoacid ester analog (which may also be referred to as H-phosphonic acid monoester), formed after phosphorus is first introduced, is crystallizable. Importantly, isolation of this intermediate allows the subsequent step to produce a product of such purity that chromatographic separation is not required for cGMP synthesis using the method of the present invention. In addition, the use of the guanosine 5'-monophosphoric oxoacid ester analog intermediate allows for more precise control over the reaction conditions of the subsequent cyclization, allowing for improved yields of Rp analogs, and even leading to the production of Rp analogs without the need for chromatography at any point. The method allows for synthesis from as little as 50 mg to over 100 grams. The method is easily scalable due to its homogeneous reaction steps, reactivity at room temperature, and lack of exothermicity.

[0016] Synthesis method The present invention relates to a method for producing a cyclic guanosine-3',5'-monophosphate (cGMP) analog or a synthetic intermediate thereof, comprising the steps of: i) A guanosine analogue of general formula (I) or a salt thereof: [ka] (In the formula, h is H, halogen, or Q; X 1 and X 2 are each independently selected from H or p′; p' is independently selected at each occurrence from a hydroxyl protecting group; R1 and R 2 are each independently H, -(CH2) n -H, -(CH2) n -C 3~9 Heterocyclyl, -(CH2) n - selected from ar, and ar, where each occurrence of n is independently selected from 0, 1, 2, 3, or 4; or R 1 and R 2 together form -CH=C(ar)- or -(CH2) 1~4 Forming C(=O)- ar is, independently at each occurrence, a 5- or 6-membered aromatic or heteroaromatic ring, preferably phenyl or 2-furanyl, optionally substituted at each occurrence individually with halogen, -OH, -SH, -NH2, -NO2, -OCH3, -CH3, -CH2CH3, -CH(CH3)2, or -CF3, and optionally fused to the second occurrence of ar; Q is -(CH2) n -S-(CH2) n -H, -S-(CH2) n -OH, -S-(CH2) n -NH2, -(CH2) n -O-(CH2) n -H, -O-(CH2) n -OH, -O-(CH2) n -NH2, -OC(CH3)3, -O-CH(CH3)2, -(CH2) n -N(-[CH2] n H)2, -NH-(CH2) n NH2, -NH-(CH2) n -OH, -(CH2) n -Nc 1 c 2 and c 1 and c 2 form a 3- to 8-membered heterocycle together with the N to which they are attached, or 1 is H and c 2 is a 3-8 membered heterocycle, -(CH2) n -H, -N3, -CF3, -(CH2) n -ar, -O-(CH2) n-(ar), -NH-(CH2) n -(ar), -S-(CH2) n -(ar), -(CH2) n -Amide-ar, -O-(CH2) n -Amide-(ar), -NH-(CH2) n -Amide-(ar), -S-(CH2) n -amide-(ar), or a linker moiety, in which any -H may be optionally replaced by halogen, and each occurrence of n is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, or 8. providing a ii) contacting the provided guanosine analog with a phosphorus oxoacid derivative to obtain a guanosine 5'-monophosphorus oxoacid ester analog; iii) isolating the resulting guanosine 5'-monophosphoric acid ester analogue by crystallization; The present invention provides a method comprising:

[0017] Such a method is hereafter referred to as the method according to the invention. The method is for producing cGMP analogues, but is also quite suitable for producing intermediates useful for further production of cGMP analogues. In a preferred embodiment, the method is for producing cGMP analogues. In a preferred embodiment, the method is for producing intermediates suitable for further conversion to cGMP analogues. Preferred synthetic intermediates produced by the method according to the invention are compounds of general formula II, compounds of general formula III, compounds of general formula III-Rp, or salts thereof. Definitions for salts and these compounds are provided herein at a later stage. In a preferred embodiment, the intermediate is of general formula II or III-Rp. In a preferred embodiment, the intermediate is of general formula III. In a preferred embodiment, the intermediate is of general formula III-Rp. Compounds of general formula II are the most preferred intermediates.

[0018] Preferred cGMP analogs produced by the present method are described below. In general, they are governed by the target binding protein to be modulated by application of the cGMP analog. In a preferred embodiment, the cGMP analog is directed to the activation of cyclic guanosine-3',5'-monophosphate-dependent protein kinase, and preferred cGMP analogs are represented by the general formula III, 3 is OH) or of the general formula III-Sp, 3 is SH, borano, methylborano, dimethylborano, or cyanoborano). Note that the boron analogs are referred to as Rp analogs due to the lower priority of boron compared to oxygen within the Cahn-Ingold-Prelog nomenclature rules.

[0019] In a preferred embodiment, the cGMP analog is for the inhibition of cyclic guanosine-3',5'-monophosphate dependent protein kinase, and the preferred cGMP analog has the general formula III-Rp, 3 are SH, borano, methylborano, dimethylborano, and cyanoborano. However, due to the lower priority of boron compared to oxygen within the Cahn–Ingold–Prelog nomenclature rules, the boron analogues are referred to as Sp analogues.

[0020] In a preferred embodiment, the cGMP analog is for the activation of cyclic guanosine-3′,5′-monophosphate-gated ion channels, and the preferred cGMP analog is represented by the general formula III, 1 and R 2 do not together form -CH=C(ar)-.

[0021] In a preferred embodiment, the cGMP analog is for the simultaneous activation of cyclic guanosine-3',5'-monophosphate-dependent protein kinase and cyclic guanosine-3',5'-monophosphate-gated ion channels, and the preferred cGMP analog is of the general formula III-Sp, 3is SH, borano, methylborano, dimethylborano, or cyanoborano, or optionally OH, and wherein R 1 and R 2 do not together form -CH=C(ar)-.

[0022] In a preferred embodiment, the cGMP analog is for the simultaneous inhibition of cyclic guanosine-3',5'-monophosphate-dependent protein kinase and cyclic guanosine-3',5'-monophosphate-gated ion channels, and the preferred cGMP analog is of the general formula III-Rp, 3 is S, borano, methylborano, dimethylborano, or cyanoborano), or more preferably, preferred cGMP analogs are of the general formula III-Rp, 3 is S, and in the formula R 1 and R 2 together form -CH=C(ar)- or -(CH2) 1~4 Preferred cGMP analogues are of the general formula III-Rp, where: 3 is borano, methylborano, dimethylborano, or cyanoborano, and wherein R 1 and R 2 Together, they form -CH=C(ar)- and -(CH2) 1~4 (It does not form C(=O)- either).

[0023] Further preferred examples of cyclic guanosine-3',5'-monophosphate analogs are: 1. 8-Bromoguanosine-3',5'-cyclic monophosphate (8-Br-cGMP) or its thiophosphate (8-Br-cGMPS), 2. 8-(2,4-dihydroxyphenylthio)guanosine-3',5'-cyclic monophosphate (8-o,pDHPT-cGMP) or its thiophosphate 8-o,pDHPT-cGMPS; 3. 8-(2-aminophenylthio)guanosine-3',5'-cyclic monophosphate (8-APT-cGMP) or its thiophosphate 8-APT-cGMPS; 4. 8-(4-hydroxyphenylthio)guanosine-3',5'-cyclic monophosphate (8-pHPT-cGMP) or its thiophosphate 8-pHPT-cGMPS; 5. 8-(4-aminophenylthio)guanosine-3',5'-cyclic monophosphate (8-pAPT-cGMP) or its thiophosphate 8-pAPT-cGMPS; 6.8-(4-Chlorophenylthio)-β-phenyl-1,N 2 - ethenoguanosine-3',5'-cyclic monophosphate (8-pCPT-PET-cGMP) or its thiophosphate 8-pCPT-PET-cGMPS, 7. 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP) or its thiophosphate 8-pCPT-cGMPS; 8. 8-(2,4-dichlorophenylthio)guanosine-3',5'-cyclic monophosphate (8-o,pDClPT-cGMP) or its thiophosphate 8-o,pDClPT-cGMPS; 9. 8-(4-methoxyphenylthio)guanosine-3',5'-cyclic monophosphate (8-pMeOPT-cGMP) or its thiophosphate 8-pMeOPT-cGMPS; 10.8-Bromo-β-phenyl-1,N 2 - ethenoguanosine-3',5'-cyclic monophosphate (8-Br-PET-cGMP) or its thiophosphate 8-Br-PET-cGMPS, 11. 8-Bromo-(2-naphthyl-1,N 2 -etheno)guanosine-3',5'-cyclic monophosphate (8-Br-(2-N)ET-cGMP) or its thiophosphate 8-Br-(2-N)ET-cGMPS; 12. 8-(4-hydroxyphenylthio)-β-phenyl-1,N 2 - ethenoguanosine-3',5'-cyclic monophosphate (8-pHPT-PET-cGMP) or its thiophosphate 8-pHPT-PET-cGMPS, 13. 8-(4-chlorophenylthio)-β-phenyl-1,N 2 - ethenoguanosine-3',5'-cyclic monophosphate (8-pCPT-PET-cGMP) or its thiophosphate 8-pCPT-PET-cGMPS, 14.2-Naphthyl-1,N 2 -ethenoguanosine-3',5'-cyclic monophosphate ((2-N)ET-cGMP) or its thiophosphate (2-N)ET-cGMPS, 15.β-Phenyl-1,N 2 - ethenoguanosine-3',5'-cyclic monophosphate (PET-cGMP) or its thiophosphate PET-cGMPS, 16. 4-Methoxy-β-phenyl-1,N 2 - ethenoguanosine-3',5'-monophosphate (pMeO-PET-cGMP) or its thiophosphate pMeO-PET-cGMPS, 17.β-1,N 2 -Acetyl-8-bromoguanosine-3',5'-cyclic monothiophosphate (β-1,N 2 -Ac-8-Br-cGMPS and its phosphate (β-1,N 2 -Ac-8-Br-cGMP), 18. 8-Bromo-δ-1,N 2 -Butyrylguanosine-3',5'-cyclic monothiophosphate (8-Br-δ-1,N 2 -But-cGMPS and its phosphate (8-Br-δ-1,N 2 -But-cGMP), 19. 8-Bromo-(4-methyl-β-phenyl-1,N 2 -etheno)guanosine-3',5'-cyclic monothiophosphate (8-Br-pMe-PET-cGMPS) and its phosphate (8-Br-pMe-PET-cGMP), 20.-Bromo-(3-thiophene-yl-1,N 2 -etheno)guanosine-3',5'-cyclic monothiophosphate (8-Br-(3-Tp)ET-cGMPS) and its phosphate (8-Br-(3-Tp)ET-cGMP), 21. 1-Benzyl-8-bromoguanosine-3',5'-cyclic monothiophosphate (1-Bn-8-Br-cGMPS) and its phosphate (1-Bn-8-Br-cGMP), 22. 8-Thioguanosine-3',5'-cyclic monothiophosphate (8-T-cGMPS) and its phosphate (8-T-cGMP), 23. 8-(4-isopropylphenylthio)guanosine-3',5'-cyclic monothiophosphate (8-pIPrPT-cGMPS) and its phosphate (8-pIPrPT-cGMP), 24. 8-Phenylamidomethylthioguanosine-3',5'-cyclic monothiophosphate (8-PAmdMT-cGMPS) and its phosphate (8-PAmdMT-cGMP), 25.β-Phenyl-1,N 2 -Etheno-8-phenylamidomethylthioguanosine-3',5'-cyclic monothiophosphate (PET-8-PAmdMT-cGMPS) and phosphate (PET-8-PAmdMT-cGMP) 26. 8-(4-isopropylphenylthio)-β-phenyl-1,N 2 -Ethenoguanosine-3',5'-cyclic monothiophosphate (8-pIPrPT-PET-cGMPS) and its phosphate (8-pIPrPT-PET-cGMP), 27. 8-(2-aminophenylthio)-β-phenyl-1,N 2 Ethenoguanosine 3',5' cyclic monothiophosphate (8-oAPT-PET-cGMPS) and its phosphate (8-oAPT-PET-cGMP) 28.β-Phenyl-1,N 2 -Etheno-8-thioguanosine-3',5'-cyclic monothiophosphate (PET-8-T-cGMPS) and its phosphate (PET-8-T-cGMP), 29.8-Methylthio-β-phenyl-1,N 2 -Ethenoguanosine-3',5'-cyclic monothiophosphate (8-MeS-PET-cGMPS) and its phosphate (8-MeS-PET-cGMP), 30. 8-Methylthio-guanosine-3',5'-cyclic monothiophosphate (8-MeS-cGMPS), preferably the sodium salt, and its phosphate (8-MeS-cGMP); 31. 8-Phenylguanosine-3',5'-cyclic monothiophosphate (8-Phe-cGMPS) and its phosphate (8-Phe-cGMP), 32. 8-(2-Furyl)guanosine-3',5'-cyclic monothiophosphate (8-(2-Fur)-cGMPS) and its phosphate (8-(2-Fur)-cGMP), 33. 8-(4-chlorophenyl)guanosine-3',5'-cyclic monothiophosphate (8-pCP-cGMPS) and its phosphate (8-pCP-cGMP), 34. 8-Phenyl-β-phenyl-1,N 2 -Ethenoguanosine-3',5'-cyclic monothiophosphate (8-Phe-PET-cGMPS) and its phosphate (8-Phe-PET-cGMP), and 35. 8-(4-chlorophenyl)-β-phenyl-1,N 2 -Ethenoguanosine-3',5'-cyclic monothiophosphate (8-pCP-PET-cGMPS) and its phosphate (8-pCP-PET-cGMP), and pharma- ceutically acceptable salts thereof. More preferred cGMP analogs are 8-Br-cGMP, 8-Br-PET-cGMP, and 8-Br-(2-N)ET-cGMP, 8-Br-δ-1,N2-But-cGMP, 8-Br-δ-1,N2-But-cGMP, 8-Br-(3-Tp)ET-cGMP, 1-Bn-8-Br-cGMP, and thiophosphates thereof, preferably thiophosphates thereof. Other more preferred cGMP analogs are 8-pCPT-PET-cGMP, 8-Br-PET-cGMP, 8-pHPT-PET-cGMP, 8-pCPT-PET-cGMP, PET-cGMP, 8-Br-(2-N)ET-cGMP, (2-N)ET-cGMP, 8-Br-pMe-PET-cGMP, 8-B r-(3-Tp)ET-cGMP, PET-8-PAmdMT-cGMP, 8-pIPrPT-PET-cGMP, 8-oAPT-PET-cGMP, PET-8-T-cGMP, 8-MeS-PET-cGMP, 8-Phe-PET-cGMP, 8-pCP-PET-cGMP, and pMeO-PET-cGMP and their thiophosphates, preferably their thiophosphates, more preferably 8-pCPT-PET-cGMP, 8-Br-PET-cGMP, 8-pHPT-PET-cGMP, 8-pCPT-PET-cGMP, PET-cGMP, 8-Br-pMe-PET-cGMP, PET-8-PAmdMT-cGMP, 8-pIPrPT-PET-cGMP, 8-oAPT-PET-cGMP, PET-8-T-cGMP, 8-MeS-PET-cGMP, 8-Phe-PET-cGMP, 8-pCP-PET-cGMP, and pMeO-PET-cGMP and their thiophosphates, preferably their thiophosphates. Other preferred cGMP analogs are β-1,N2-Ac-8-Br-cGMP and 8-Br-δ-1,N2-But-cGMP and their thiophosphates, preferably their thiophosphates. The most preferred cGMP analog is 8-Br-PET-cGMP or its thiophosphate, preferably its thiophosphate.Another group of highly preferred cGMP analogs consists of 8-Br-PET-cGMP, 8-Br-pMe-PET-cGMP, 8-Br-(3-Tp)ET-cGMP, 8-PAmdMT-cGMP, PET-8-PAmdMT-cGMP, 8-pIPrPT-PET-cGMP, and 8-oAPT-PET-cGMP, and their thiophosphates, preferably their Rp isomers, preferably their thiophosphates, most preferably the Rp isomers of their thiophosphates.

[0024] The above cGMP analogs are preferably the Rp isomers (for ease of reference, for example, the Rp isomer of PET-cGMP is designated Rp-PET-cGMP and the corresponding thiophosphate is designated PET-cGMPS).

[0025] Step i) Providing a guanosine analog The first step of the method is the provision of a guanosine analog. As used herein, guanosine itself can also be considered as a guanosine analog and is not listed separately for ease of reading. Those skilled in the art will understand that guanosine can also be used to practice the present invention and obtain cGMP. The guanosine analog can be synthetically prepared as part of the method, or can be obtained commercially, or can be sourced elsewhere. As part of the method, phosphorus is introduced at the 5' position of the guanosine analog, which is then cyclized to form a cGMP analog. Variables such as h, R1, R2, etc., as defined later herein, are preferably kept constant starting from the end of step i).

[0026] The guanosine analogue (or guanosine as explained above) may also be a salt. In the context of the present invention, the salt is preferably a pharma- ceutically acceptable salt. Pharmaceutically acceptable salts are known in the art. Preferred salts are acid addition salts. Other preferred salts are base addition salts. In the context of the present invention, pharma- ceutically acceptable salts preferably include salts derived from inorganic bases such as Li, Na, K, Ca, Mg, Fe, Cu, Zn, Mn, and salts of organic bases such as N,N'-diacetylethylenediamine, glucamine, triethylamine, choline, dicyclohexylamine, benzylamine, (tri)alkylamines, thiamine, guanidine, diethanolamine, alphaphenylethylamine, piperidine, morpholine, pyridine, hydroxyethylpyrrolidine, hydroxyethylpiperidine, and the like. Such salts also include salts of amino acids such as glycine, alanine, cystine, cysteine, lysine, arginine, phenylalanine, guanidine, and the like. Such salts may include, for example, sulfates, nitrates, phosphates, perchlorates, borates, hydrohalides such as HCl and HBr, acetates, trifluoroacetates, tartrates, maleates, citrates, succinates, palmoates, methanesulfonates, tosylates, benzoates, salicylates, hydroxynaphthoates, benzenesulfonates, ascorbates, glycerophosphates, ketoglutarate salts, and the like, as appropriate, and acid addition salts. Preferred salts are HCl, formates, acetates, triethylammonium (TEAH+), sodium, and trifluoroacetates. More preferred salts are TEAH+ and sodium salts. For cGMP analogs produced by the methods of the invention, the preferred salts are sodium salts. For intermediates produced by or as part of the methods of the invention, the preferred salts are TEAH+ salts.

[0027] The phrase "pharmaceutical acceptable" refers to a compound or composition that is physiologically tolerable and typically does not cause allergic or similar adverse reactions, including but not limited to stomach upset or dizziness, when administered to a mammal. What is or is not pharmaceutical acceptable is within the ability of one of ordinary skill in the art.

[0028] The guanosine analogue may also be a hydrate or a solvate. In the context of the present invention, a hydrate refers to a solvate when the solvent is water. The term solvate, as used herein, refers to a crystalline form of a substance that contains a solvent. The solvate is preferably a pharma-ceutically acceptable solvate, and may be a hydrate or may include other crystallization solvents such as alcohol, ether, etc. Thus, the crystal may be a hydrate or a solvate, and the crystallization may produce a hydrate or a solvate.

[0029] The guanosine analogue is preferably of the general formula (I) or a salt thereof: [ka] (In the formula, h is H, halogen or Q, preferably h is H, F, Br, Cl or Q, more preferably H, Br or Q, in a highly preferred embodiment h is H, in a highly preferred embodiment h is Br, in a highly preferred embodiment h is H or Br, in a highly preferred embodiment h is Q, and in a most preferred embodiment h is H or Br or Q; X 1 and X 2 are each independently selected from H or p', preferably X 1 is H, more preferably H prior to the step in which the cyclic phosphorus oxo acid diester is formed as described later herein, preferably prior to the formation of the 5′-phosphorus oxo acid ester, and X 2 is p', and most preferably, X 1 is H and X 2is p', p' is independently selected in each occurrence from hydroxyl protecting groups, which are known in the art and capable of protecting the oxygen atom of the hydroxyl moiety. Examples of suitable hydroxyl protecting groups are extensively described in the art, for example by PGM Huts and TW Greene in Greene's Protective Groups in Organic Synthesis, Fourth Edition, 2006 (ISBN: 978-0-471-69754-1). Those skilled in the art will be able to select suitable protecting groups for use in accordance with the present invention. Examples of suitable hydroxyl protecting groups are methoxymethyl (MOM), tetrahydropyranyl (THP), t-butyl (tBu), allyl (all), benzyl (Bn), (tri)alkylsilyl (e.g., t-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), or t-butyldiphenylsilyl (TBDPS), where the preferred (tri)alkylsilyl is trialkylsilyl), and acyl (e.g., acetyl (Ac), pivalyl (Pv), or benzoyl (Bz)), more preferably triisopropylsilyl (TIPS). Preferred groups for p' are THP, (tri)alkylsilyl, and acyl. More preferred groups for p' are THP and (tri)alkylsilyl (preferably TIPS), most preferably p' is (tri)alkylsilyl (preferably TIPS). As known in the art, p' is always bonded to oxygen, and multiple instances of p' can be combined to form a single protecting group that protects two or more hydroxyl groups. Examples of such polyvalent protecting groups are acetonide and benzylidene acetal. In a preferred embodiment, when the two moieties protected by p' are bonded to the same carbon atom or adjacent carbon atoms, the two instances of p' are combined to form a single polyvalent protecting group.

[0030] R 1 and R 2 are each independently H, -(CH2) n -H, -(CH2) n -C 3~9Heterocyclyl, -(CH2) n - selected from ar, and ar, where each occurrence of n is independently selected from 0, 1, 2, 3, or 4; or R 1 and R 2 together form -CH=C(ar)- or -(CH2) 1~4 When n is 0, there are no CH2 units present, and therefore in a preferred embodiment, -(CH2) n -C3-9 heterocyclyl is C3-9 heterocyclyl. Heterocyclyl in the context of the present invention is an optionally unsaturated heterocycle having 3, 4, 5, 6, 7, 8, or 9 carbon atoms. The heterocycle may be optionally substituted as described for ar, with the carbon atoms in any substitution not being counted towards C3-9. The heteroatoms are preferably selected from O, S, and N. Preferably, the heterocyclyl has at most 3, more preferably at most 2, and most preferably at most 1 heteroatom. The heterocyclyl preferably has at least 1, more preferably at least 2 heteroatoms. Preferred examples of heterocyclyl are piperidinyl, piperazinyl, morpholinyl, tetrahydrofuranyl, pyrazolyl, and pyrrolidinyl. R 1 and R 2 taken together form -CH=C(ar)- or -(CH2) 1~4 When forming C(=O)-, in a preferred embodiment, ar or C(=O) is closest to where R1 is drawn. In another preferred embodiment, ar or (C=O) is closest to where R2 is drawn. Preferably, R 1 and R 2 taken together form -CH=C(ar)- or -(CH2) 1~4 When C(=O)- is formed, a styrylene moiety is formed and therefore ar is phenyl. Most preferably, R 1 and R 2 taken together to form -CH=C(ar)-, -CH=C(phenyl)- is formed with phenyl being closest to R2. 1 and R 2But together, -(CH2) 1~4 When C(=O)- is formed, -CH2-C(=O)- or -(CH2)3-C(=O)- is formed.

[0031] ar is independently in each case a 5- or 6-membered aromatic or heteroaromatic ring, preferably phenyl or 2-furanyl, and each instance of ar is independently optionally substituted with halogen, -OH, -SH, -NH2, -NO2, -OCH3, -CH3, -CH2CH3, -CH(CH3)2, or -CF3, and is optionally fused to a second instance of ar to preferably form a naphthyl moiety, preferably no more than two instances of ar being included in such a fused moiety. In a preferred embodiment, ar is fused to a further instance of ar. In a preferred embodiment, ar is 6-membered. In a preferred embodiment, ar is unsubstituted. Preferred examples of ar are phenyl, furanyl, pyridinyl, imidazolyl, tetrazolyl, triazolyl, thiophenyl, benzothiazolyl, indolyl, 4-methylphenyl, 3-thiophenyl, and naphthyl. A highly preferred embodiment of ar is 4-methylphenyl.

[0032] Q is -(CH2) n -S-(CH2) n -H, -S-(CH2) n -OH, -S-(CH2) n -NH2, -(CH2) n -O-(CH2) n -H, -O-(CH2) n -OH, -O-(CH2) n -NH2, -OC(CH3)3, -O-CH(CH3)2, -(CH2) n -N(-[CH2] n H)2, -NH-(CH2) n NH2, -NH-(CH2)nOH, -(CH2) n -Nc 1 c 2 and c 1 and c 2 form a 3- to 8-membered heterocycle together with the N to which they are attached, or 1is H and c 2 is a 3-8 membered heterocycle, -(CH2) n -H, -N3, -CF3, -(CH2) n -ar, -O-(CH2) n -(ar), -NH-(CH2) n -(ar), -S-(CH2) n -(ar), -(CH2) n -Amide-ar, -O-(CH2) n -Amide-(ar), -NH-(CH2) n -Amide-(ar), -S-(CH2) n -amide-(ar), or a linker moiety, where any -H may be optionally replaced by halogen, and n is independently selected at each occurrence from 0, 1, 2, 3, 4, 5, 6, 7, or 8, and heterocycles are defined above. Linkers are defined below. In a preferred embodiment, Q is -(CH2) n -S-(CH2) n -H, -S-(CH2) n -OH, -S-(CH2) n -NH2, -(CH2) n -O-(CH2) n -H, -O-(CH2) n -OH, -O-(CH2) n -NH2, -OC(CH3)3, -O-CH(CH3)2, -(CH2) n -N(-[CH2] n H)2, -NH-(CH2) n NH2, -NH-(CH2)nOH, -(CH2) n -Nc 1 c 2 and c 1 and c 2 form a 3- to 8-membered heterocycle together with the N to which they are attached, or 1 is H and c 2 is a 3-8 membered heterocycle, -(CH2) n -H, -N3, -CF3, -(CH2) n -ar, -O-(CH2) n -(ar), -NH-(CH2) n-(ar) or -S-(CH2) n -(ar), where any -H is optionally replaced by halogen, and n at each occurrence is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, or 8. -(CH2) n -S-(CH2) n -H is preferably -S-(CH2) n -H or -(CH2) n -SH. -S-(CH2) n In -OH, n is preferably 2, 3, 4, 5, or 6. -S-(CH2) n In -NH2, n is preferably 2, 3, 4, 5, or 6. -(CH2) n -O-(CH2) n -H is preferably -(CH2) n -OH or -O-(CH2) n -H. -O-(CH2) n In -OH, n is preferably 2, 3, 4, 5, or 6. -O-(CH2) n In -NH2, n is preferably 2, 3, 4, 5, or 6. -(CH2) n -N(-[CH2]nH)2 is preferably -(CH2) where n is at least 2. n -NH2, or -(CH2), where n is at least 2 in each case. n In NH-(CH)NH, n is preferably 2, 3, 4, 5, or 6. In -NH-(CH)OH, n is preferably 2, 3, 4, 5, or 6. -(CH) n -Nc 1 c 2 In the preferred embodiment, n is preferably 2, 3, 4, 5, or 6. 1 and c 2 together with the N to which they are attached, form a 3- to 8-membered heterocycle, more preferably a 4- to 6-membered heterocycle, and most preferably a 5- or 6-membered heterocycle. 1 is H and c 2is a 3- to 8-membered heterocycle, more preferably a 4-, 5-, or 6-membered heterocycle, and most preferably a 5- or 6-membered heterocycle. n In -H, n is preferably 0, 1, 2, 3, 4, 5, or 6, more preferably 1, 2, 3, or 4, even more preferably 1 or 2, and most preferably 1. -(CH2) n In -ar, n is preferably 0, 1, 2, 3, 4, 5, or 6, more preferably 0, 1, 2, or 3, even more preferably 0, 1, or 2, and most preferably 0 or 2. n In -(ar), n is preferably at least 2. -NH-(CH2) n In -(ar), n is preferably at least 2. -S-(CH2) n In -(ar), n is preferably at least 2. Preferably, at least one -H may be replaced by halogen, more preferably fluorine, and preferably at most three instances of -H are replaced by halogen. Optionally, all instances of H are replaced by F. Preferably, n is not 0, more preferably not 0 or 1, when this would result in a directly linked heteroatom. Preferably, n is at most 6.

[0033] Linkers are known in the art. The structure of the linker allows the linker to be easily chemically attached to the compounds used in the present invention to form a linker compound, and allows the resulting linker compound to be easily conjugated to further substances, such as polypeptides or surfaces. The choice of linker may affect the stability of such final conjugates in the circulatory system or on a surface. The linker may be cleavable or non-cleavable. Cleavable linkers include moieties that are cleavable, for example, when exposed to lysosomal proteases or an environment with an acidic pH. Suitable cleavable linkers are known in the art and include, for example, di-, tri-, or tetrapeptides, i.e., peptides composed of 2, 3, or 4 amino acid residues. In addition, the cleavable linker may include a self-immolative moiety, for example, an ω-amino aminocarbonyl cyclization spacer (see Saari et al. J. Med. Chem., 1990, 33(1), 97-101), or a -NH-CH2-O- moiety. Cleavage of the linker can increase the availability of the compound to the surrounding medium. A non-cleavable linker can still effectively release the compound of the invention after, for example, the conjugated polypeptide is degraded in the lysosome. Non-cleavable linkers include, for example, succinimidyl-4-(N-maleimidomethyl(cyclohexane)-1-carboxylate and maleimidocaproic acid and analogs thereof.

[0034] In a preferred embodiment, h is H or halogen or Q, preferably H or Br or Q, more preferably Br or Q, most preferably Br; X 1 is H and X 2 is p', p' is selected from the group consisting of methoxymethyl (MOM), tetrahydropyranyl (THP), t-butyl (tBu), allyl (all), benzyl (Bn), (tri)alkylsilyl (e.g., t-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), or t-butyldiphenylsilyl (TBDPS)), acyl (e.g., acetyl (Ac), pivaloyl (Pv), or benzoyl (Bz)), preferably from the group consisting of THP, (tri)alkylsilyl, and acyl; R 1 and R 2 taken together to form -CH=C(ar)-, ar is phenyl, 4-methylphenyl, 3-thiophenyl, or 2-furanyl, preferably phenyl or 2-furanyl, more preferably phenyl; and / or Q is furanyl, -CF3, -SCH3, -S(isopropylphenyl), -S(phenylamidomethyl), -S(halophenyl), -S(hydroxyphenyl), -S(aminophenyl), -S(nitrophenyl), -S(methoxyphenyl), -S(toluyl), -S(trifluoromethylphenyl), -Nc 1 c 2 (where c 1 and c 2 form a 3- to 8-membered heterocycle together with the N to which they are attached), -S-(CH2) n -OH, -S-(CH2) n -NH2, -NH-(CH2)nNH2, or -NH-(CH2)nOH, preferably furanyl, -CF3, -S(4-hydroxyphenyl), or -S(4-chlorophenyl).

[0035] Even more preferably, h is Br and X 1 is H and X 2 is p', p' is triisopropylsilyl (TIPS), R 1 and R 2 are preferably taken together to form -CH=C(ar)-, where ar is closest to R2, and ar is phenyl or 4-methylphenyl.

[0036] The GMP analogue provided is X 2 is p' and X 1 It is highly preferred that X is H, since this facilitates the chemoselective introduction of the 5'-monophosphorus oxo acid ester analogue. The inventors have surprisingly found that protection of X1 has less impact than protection of X2. Therefore, in a preferred embodiment, X 1 is H and X 2 is provided a method according to the present invention, wherein p' is a guanosine analogue of general formula (I) or a salt thereof, provided by the following steps: Ia) An unprotected guanosine analogue of general formula (pI) or a salt thereof: [ka] (In the formula, h, R 1 , and R 2 is as defined for general formula I In this step, a precursor of the guanosine analogue to be converted in step ii) is provided. This precursor is X 1 and X 2 and thus is referred to as an unprotected guanosine analog. Ib) contacting the unprotected guanosine analog with a (tri)alkylsilyl halide to obtain a multiply protected guanosine analog and optionally isolating the multiply protected guanosine analog by crystallization. In this step, the unprotected guanosine analog is protected with a (tri)alkylsilyl halide to give X2 which is a (tri)alkylsilyl protecting group, which is the preferred protecting group for p'. In addition, the 5'-OH group can become protected in this reaction step. Surprisingly, X2 can become protected with a (tri)alkylsilyl halide to obtain a multiply protected guanosine analog by crystallization. 1was found to maintain H. The 5'- and 2'-protected guanosine analogs can be conveniently isolated by crystallization, which is carried out in a preferred embodiment. The reaction is preferably carried out in an aprotic polar solvent such as DMF, pyridine, tetramethylurea, dimethylacetamide, NMP, more preferably in DMF or NMP. The reaction is preferably carried out in the presence of a mild base, more preferably a mild organic base, for example, imidazole, pyridine, trialkylamines such as trimethylamine, N-methylmorpholine, or N-methylimidazole, more preferably imidazole. The progress of the reaction is preferably monitored using analytical techniques such as chromatographic techniques, TLC or HPLC. Substantially all of the X 2 After is protected, the reaction is preferably quenched by the addition of excess water, for example 10 equiv. The quenched reaction is preferably washed with an aqueous phase and then extracted into an aprotic organic solvent, preferably one of low polarity such as toluene, esters such as isopropyl acetate or butyl acetate, ethers such as tert-butyl methyl ether, or benzene, more preferably toluene. The reaction product is preferably isolated by crystallization, preferably from an organic solvent that is not methanol, more preferably from an aprotic organic solvent, even more preferably from a polar one such as ethyl acetate or propyl acetate, most preferably from isopropyl acetate. The crystallization is preferably carried out with the addition of seed crystals, more preferably with the desired product to be crystallized. After the crystallization, which is preferably left for at least 4, more preferably at least 8, even more preferably at least 12 hours, the crystals are preferably washed and / or dried. The washing is preferably carried out with the same solvent as that used for the crystallization. Drying is preferably carried out in vacuum, more preferably at elevated temperature, for example at about 40-80°C, preferably at about 50-70°C. Ic) selectively deprotecting a multiply protected guanosine analog to a compound of general formula (I), 1 is H and X 2is p') to obtain a guanosine analogue of formula (I). In this step, the difference in reactivity of the 2'- and 5'-protected positions is used to selectively deprotect the protected 5'-OH using mild reaction conditions such as aqueous TFA. This step can be carried out using any combination of THF and / or water and acid, preferably TFA, but is preferably carried out in an aprotic organic solvent, preferably a polar one, for example THF or 1,4-dioxane. The deprotection is preferably carried out using a strong acid, preferably TFA, preferably using about 7-13, for example 10 volumes of solvent, about 1-3, for example 2 volumes of water, and about 0.2-1, for example 0.45 volumes of TFA. The progress of the reaction is preferably monitored using a suitable analytical technique such as chromatography, for example HPLC. Monitoring is beneficial as it allows detection of over-deprotection, which may result in the 2'-position also being deprotected. When substantially all of the 5'-protecting groups have been removed, the reaction is preferably quenched by the addition of a base, preferably a mild nitrogen-based base such as ammonia, or a suitable amine base such as a trialkylamine, or an inorganic base such as a carbonate or bicarbonate, more preferably ammonia or an amine base (which avoids gas evolution), most preferably ammonia for volume efficient addition. Preferably, the quenched crude reaction mixture is evaporated and resuspended in a mixture in which the fully deprotected by-product is not soluble, for example in a mixture of dichloromethane or chloroform with a small amount of water (about 10-20, for example 15 volumes of dichloromethane to about 1-3, for example 2 volumes of water), or in a mixture such as acetone. The resulting dilute slurry is selectively filtered to remove possible insoluble contaminants. The organic phase of the filtrate is preferably separated and can then be evaporated to obtain a residue. The residue is preferably triturated with a polar organic solvent, preferably an aprotic one, for example acetonitrile or ethyl acetate, more preferably acetonitrile. The remaining solids are preferably washed with the same solvent used for trituration, and then the product is preferably dried, such as by vacuum drying. Id) The obtained compound of general formula (I) (wherein X1 is H and X 2 is p') by crystallization or, preferably, trituration, where p' is (tri)alkylsilyl and the 2'-protected guanosine analog is conveniently isolated by crystallization. Isolation is preferably carried out as described above in step Ic.

[0037] In step i), the guanosine analog is R 1 and R 2 When both have H, the analog is R 1 and R 2 can be advantageously converted to the analogues, which together form -CH=C(ar)-. For this purpose, R 1 and R 2The analogues where both are H are preferably dissolved in a highly polar aprotic solvent, preferably DMSO, followed by the addition of about 1-3, e.g. 1.5 equiv. of (ar)-C(=O)-CH2Br. As used herein, ar is as defined above, e.g. phenyl or furanyl, preferably phenyl. When ar in (ar)-C(=O)-CH2Br is phenyl, the reactant is phenanthyl bromide. After the addition of (ar)-C(=O)-CH2Br, a strong base, preferably a strong organic base, e.g. DBU or tetramethylguanidine, more preferably DBU, is preferably added in about 1-5, e.g. about 2.5 equiv, preferably over a period of time, e.g. about 10-60 min, e.g. about 30 min. After the reaction is complete, e.g. after about 1 h, the reaction is preferably neutralized, more preferably by the addition of an acid, preferably a weak acid, more preferably an organic acid, e.g. acetic acid. The product is preferably precipitated from the crude reaction mixture by the addition of, for example, water, for example about 10 volumes. The precipitated product is preferably isolated by filtration, preferably washed with, for example, aqueous DMSO or water, and the solids are then preferably dried, such as by vacuum drying. The solids are then preferably triturated with a suitable aprotic polar organic solvent, such as acetonitrile or THF, more preferably acetonitrile, and the product is then preferably dried, such as by vacuum drying. Vacuum drying is preferably carried out at an elevated temperature, for example about 70° C.

[0038] Step ii) Formation of guanosine 5'-monophosphoryl oxoate analogues In a second step, the guanosine analogue provided in step i) is contacted with a phosphorus oxoacid derivative to obtain a guanosine 5'-monophosphorus oxoacid ester analogue. The phosphorus oxoacid derivative is preferably a phosphorylating or phosphonylating agent, and it is envisaged that the term phosphorus oxoacid derivative should not be interpreted narrowly to include only actual oxoacids, but also thio-analogs and derivatives, e.g. PCl3. Such agents are known in the art. In the context of the present invention, the agent is for introducing a phosphorus oxoacid ester at the 5' position of the guanosine analogue to form a GMP analogue. The phosphorylating agent forms an ester in which phosphorus is in the formal oxidation state P(V), e.g. (HO)2P(=O)-guanosine. The phosphonylating agent forms an ester in which phosphorus is in the formal oxidation state P(III), e.g. (HO)HP(=O)-guanosine. In a preferred embodiment, the phosphorus oxoacid derivative is a phosphorylating agent. In an even more preferred embodiment, the phosphorus oxoacid derivative is a phosphonylating agent.

[0039] In a preferred embodiment, the phosphorus oxoacid derivative in step ii) has the general formula (P): [ka] (In the formula, M is S or O or absent, in preferred embodiments M is S or O, more preferably O, in other preferred embodiments M is absent; o 1 and 2 are each independently halogen, -OC 1~8 Hydrocarbons, -SC 1~8 Hydrocarbons, -NH-C 1~8 Hydrocarbons, borano, methylborano, dimethylborano, cyanoborano, and -N(C 1~8 Hydrocarbons) 2; and o 3 is H or o 1 or o 1 and 3together, preferably C 2~12 (forming a chiral auxiliary that is a hydrocarbon) When the compound of formula (P) is a phosphorylating agent, 3 is not H and M is not absent. When the compound represented by formula (P) is a phosphonylating agent, o 3 is H, or M is absent and 1 and 3 together form the chiral auxiliary.

[0040] C 1~8 The hydrocarbon in this context preferably forms a suitable leaving group. The hydrocarbon may contain heteroatoms and may be optionally substituted with halogens, preferably fluorine. In a preferred embodiment, o 1 is a suitable leaving group, for example, halogen, -OC such as -OCF 1~8 Hydrocarbons, -SC such as -SC6F5 1~8 Hydrocarbons, -NH-C 1~8 Hydrocarbons, and -N(C 1~8 In a preferred embodiment, o 2 is a suitable leaving group, e.g., halogen, -OC 1~8 Hydrocarbons, -SC 1~8 Hydrocarbons, -NH-C 1~8 Hydrocarbons, and -N(C 1~8 In a more preferred embodiment, o 1 and 2 Both represent suitable leaving groups. 1 and 2 represent the same moiety. Examples of suitable leaving groups are phenol, pentafluorophenol, imidazole, triazole, diisopropylamine, and halogens, such as chlorine or bromine, more preferably chlorine. N(C 1~8 In hydrocarbon)2, it is understood that two hydrocarbons can together form a cyclic structure including the nitrogen to which they are attached, such as when an imidazole is formed.

[0041] Chiral auxiliaries are moieties that facilitate the final cyclization with a given stereochemical configuration, as they form chiral cyclic structures around the central phosphorus atom of general formula (P), as known in the art (see, for example, Knouse et al., 2018, Science, DOI: 10.1126 / science.aau3369). 1 and 3 An example of a chiral auxiliary depicted herein as a divalent group bridging the positions to which is attached is [ka] (In the formula, C * and C ** together with the carbon atoms to which they are attached, [ka] In compounds of general formula (P) in which a chiral auxiliary is present, M is preferably S or O, more preferably S.

[0042] Preferred compounds of the general formula (P) are dihalophosphites such as diphenylphosphite, dimethylphosphite, diethylphosphite, diisopropylphosphite, dichlorophosphite, di(pentafluorophenyl)phosphite, diphenylthiophosphite, dimethylthiophosphite, diethylthiophosphite, diisopropylthiophosphite, dichlorothiophosphite, di(pentafluorophenyl)thiophosphite, di(diisopropylamino)methoxyphosphite ([(iPr)N]P[OMe]), [(iPr)N]P[OEt], ClP(S)(OMe), ClP(S)(OEt), ClP(O)(SMe), ClP(O)(SEt), (pentafluorophenyl)OP(S)=(chiral auxiliary), P(S)Cl, P(O) Cl3, ClP(S)(OMe)2, ClP(S)(OEt)2, ClP(O)(OMe)2, ClP(O)(OEt)2, (pentafluorophenyl)OP(S)=(OMe)2, (pentafluorophenyl)OP(S)=(OEt)2, (pentafluorophenyl)OP(O)=(OMe)2, (pentafluorophenyl)OP(O)=(OEt)2, ClP(S)(SMe)2, ClP(S)(SEt )2, ClP(O)(SMe)2, ClP(O)(SEt)2, (pentafluorophenyl)OP(S)=(SMe)2, (pentafluorophenyl)OP(S)=(SEt)2, (pentafluorophenyl)OP(O)=(SMe)2, and (pentafluorophenyl)OP(O)=(SEt)2, more preferred are those represented below as more preferred phosphonylation or phosphorylation agents.

[0043] Preferred phosphonylating agents are diphenylphosphite, dimethylphosphite, diethylphosphite, diisopropylphosphite, dihalophosphites such as dichlorophosphite, di(pentafluorophenyl)phosphite, diphenylthiophosphite, dimethylthiophosphite, diethylthiophosphite, diisopropylthiophosphite, dihalothiophosphites such as dichlorothiophosphite, di(pentafluorophenyl)thiophosphite, [(iPr)N]P[OMe], and [(iPr)N]P[OEt], more preferred are diphenylphosphite, diphenylthiophosphite, and [(iPr)N]P[OMe], and most preferred is diphenylphosphite.

[0044] Preferred phosphorylating agents are Cl2P(s)(OMe), Cl2P(s)(OEt), Cl2P(O)(SMe), Cl2P(O)(SEt), (pentafluorophenyl)OP(S)=(chiral auxiliary), P(S)Cl3, P(O)Cl3, ClP(S)(OMe)2, ClP(S)(OEt)2, ClP(O)(OMe)2, ClP(O)(OEt)2, (pentafluorophenyl)OP(S)=(OMe)2, (pentafluorophenyl)OP(S)=(OEt)2, (pentafluorophenyl)OP(O)=(OMe)2, (pentafluorophenyl)OP(O)=(OEt)2, ClP(S)(SMe)2, ClP( and (pentafluorophenyl)OP(O)=(SEt)2, more preferred are ClP(S)(OMe), ClP(O)(SMe), (pentafluorophenyl)OP(S)=(chiral auxiliary), P(S)Cl, P(O)Cl, ClP(S)(OMe), ClP(O)(OMe), ClP(S)(SMe), and ClP(O)(SMe).

[0045] The contacting of the phosphorus oxoacid derivative with the guanosine analog is preferably carried out under conditions conducive to the formation of the guanosine 5'-monophosphorus oxoacid ester analog. The contacting is preferably carried out in an aprotic solvent, more preferably in a relatively low polarity aprotic solvent, such as dichloromethane or chloroform. The contacting is preferably carried out in the presence of a mild base, preferably a non-nucleophilic one, such as pyridine, lutidine, imidazole, or other nitrogenous base having a pKa of about 4-7, more preferably about 5-7. The contacting is preferably carried out with an excess of the phosphorus oxoacid derivative, preferably with about 2 to about 5 equiv., such as about 3 equiv. of reactant. After the reaction has proceeded, the reaction is preferably quenched, for example, with a basic aqueous solution, such as an aqueous (tri)alkylamine solution, such as one volume of water and one volume of trimethylamine, and then preferably left for about 10 to 60 minutes, for example, about 30 minutes. The organic phase is preferably subsequently washed, for example with water. The organic phase is then preferably dried, for example by evaporation of the solvent, preferably followed by co-evaporation, for example with toluene, ethyl acetate, or isopropanol, preferably with ethyl acetate and / or isopropanol.

[0046] The product of such contact is a guanosine 5'-monophosphorus oxoacid ester analogue, which is preferably of the general formula (II) or a salt thereof: [ka] (In the formula, h, X1, X 2 , R 1 , and R 2 is as defined above, h is preferably Br, and X 1 is preferably H, and X 2 is preferably p', more preferably (tri)alkylsilyl, e.g., TIPS; R 1 is preferably H or together with R2 forms -CH=C(ar)-, R2 is preferably taken together with R2 to form -CH=C(ar)- or is H, and most preferably R 1 and R 2 forms -CH=C(ar)-, o 1 and 3 are each independently -OH or as defined above; and M is S or O, preferably O. In the compound of general formula (II), the definition and preferred embodiments of h are preferably as defined for the compound of general formula (I). In the compound of general formula (II), the definition and preferred embodiments of R1 are preferably as defined for the compound of general formula (I). In the compound of general formula (II), the definition and preferred embodiments of R2 are preferably as defined for the compound of general formula (I). In the compound of general formula (II), the definition and preferred embodiments of X1 are preferably as defined for the compound of general formula (I). In the compound of general formula (II), the definition and preferred embodiments of X2 are preferably as defined for the compound of general formula (I).

[0047] In a preferred embodiment, 3 is H and o 1 -OH, -OC 1~8 Hydrocarbons, -SC 1~8 Hydrocarbons, -NH-C 1~8 Hydrocarbon, borano, methylborano, dimethylborano, cyanoborano, or -N(C 1~8 Hydrocarbon)2, more preferably -OC 1~8 Hydrocarbons, -SC 1~8 Hydrocarbons, -NH-C 1~8 Hydrocarbon, or -N(C 1~8 In another preferred embodiment, o 1 and 3 Both are -OH, -OC 1~8 Hydrocarbons, -SC 1~8 Hydrocarbons, -NH-C 1~8 Hydrocarbon, borano, methylborano, dimethylborano, cyanoborano, or -N(C1~8 Hydrocarbon)2, more preferably -OC 1~8 Hydrocarbons, -SC 1~8 Hydrocarbons, -NH-C 1~8 Hydrocarbon, or -N(C 1~8 In a preferred embodiment, o 1 and 3 together form a chiral auxiliary as defined above. Most preferably, 1 is -OH, and O 3 is H.

[0048] The following are preferred compounds of general formula (II): 1. 8-Bromoguanosine 5'-monophosphate, 2. 8-(2,4-dihydroxyphenylthio)guanosine-5'-monophosphate, 3. 8-(2-aminophenylthio)guanosine-5'-monophosphate, 4. 8-(4-hydroxyphenylthio)guanosine-5'-monophosphate, 5. 8-(4-aminophenylthio)guanosine-5'-monophosphate, 6. 8-(4-Chlorophenylthio)-β-phenyl-1,N2-ethenoguanosine-5'-monophosphate 7. 8-(4-chlorophenylthio)guanosine-5'-monophosphate, 8. 8-(2,4-dichlorophenylthio)guanosine-5'-monophosphate, 9. 8-(4-methoxyphenylthio)guanosine-5'-monophosphate, 10. 8-Bromo-β-phenyl-1,N 2 Ethenoguanosine-5'-monophosphate 11. 8-Bromo-(2-naphthyl-1,N 2 etheno)guanosine-5'-monophosphate 12. 8-(4-hydroxyphenylthio)-β-phenyl-1,N 2 Ethenoguanosine-5'-monophosphate 13. 8-(4-chlorophenylthio)-β-phenyl-1,N 2 Ethenoguanosine-5'-monophosphate 14.2-Naphthyl-1,N 2 -Ethenoguanosine-5'-monophosphate 15.β-Phenyl-1,N 2 -ethenoguanosine-5'-monophosphate, 16. 4-Methoxy-β-phenyl-1,N 2 -Ethenoguanosine 5'-monophosphate 17. 8-Bromoguanosine-5'-mono-H-phosphonic acid, 18. 8-(2,4-dihydroxyphenylthio)guanosine-5'-mono-H-phosphonic acid, 19. 8-(2-aminophenylthio)guanosine-5'-mono-H-phosphonic acid, 20. 8-(4-hydroxyphenylthio)guanosine-5'-mono-H-phosphonic acid, 21. 8-(4-aminophenylthio)guanosine-5'-mono-H-phosphonic acid, 22. 8-(4-Chlorophenylthio)-β-phenyl-1,N 2 -ethenoguanosine-5'-mono-H-phosphonate, 23. 8-(4-chlorophenylthio)guanosine-5'-mono-H-phosphonic acid, 24. 8-(2,4-dichlorophenylthio)guanosine-5'-mono-H-phosphonic acid, 25. 8-(4-Methoxyphenylthio)guanosine-5'-mono-H-phosphonic acid, 26. 8-Bromo-β-phenyl-1,N 2 -ethenoguanosine-5'-mono-H-phosphonic acid, 27. 8-Bromo-(2-naphthyl-1,N 2 -etheno)guanosine-5'-mono-H-phosphonic acid, 28. 8-(4-Hydroxyphenylthio)-β-phenyl-1,N 2 -ethenoguanosine-5'-mono-H-phosphonic acid, 29. 8-(4-Chlorophenylthio)-β-phenyl-1,N 2 -ethenoguanosine-5'-mono-H-phosphonic acid, 30.2-Naphthyl-1,N 2-ethenoguanosine-5'-mono-H-phosphonic acid, 31.β-Phenyl-1,N 2 -ethenoguanosine-5'-mono-H-phosphonic acid, 32. 4-Methoxy-β-phenyl-1,N 2 -ethenoguanosine-5'-mono-H-phosphonic acid, 33.β-1,N 2 -acetyl-8-bromoguanosine 5'-monophosphate and its mono-H-phosphonate, 34.8-Bromo-δ-1,N 2 -butyrylguanosine-5'-monophosphate and its mono-H-phosphonate, 35. 8-Bromo-(4-methyl-β-phenyl-1,N 2 -etheno)guanosine-5'-monophosphate and its mono-H-phosphonate, 36. 8-Bromo-(3-thiophene-yl)-1,N 2 -etheno)guanosine-5'-monophosphate and its mono-H-phosphonate, 37. 1-Benzyl-8-bromoguanosine-5'-monophosphate and its mono-H-phosphonate, 38. 8-Thioguanosine-5'-monophosphate and its mono-H-phosphonate, 39. 8-(4-isopropylphenylthio)guanosine-5'-monophosphate and its mono-H-phosphonate, 40. 8-Phenylamidomethylthioguanosine-5'-monophosphate and its mono-H-phosphonate, 41.β-Phenyl-1,N 2 -etheno-8-phenylamidomethylthioguanosine-5'-monophosphate and its mono-H-phosphonate, 42. 8-(4-isopropylphenylthio)-β-phenyl-1,N 2 -ethenoguanosine-5'-monophosphate and its mono-H-phosphonate, 43. 8-(2-aminophenylthio)-β-phenyl-1,N 2 -ethenoguanosine-5'-monophosphate and its mono-H-phosphonate, 44.β-Phenyl-1,N 2-etheno-8-thioguanosine-5'-monophosphate and its mono-H-phosphonate, 45.8-Methylthio-β-phenyl-1,N 2 -ethenoguanosine-5'-monophosphate and its mono-H-phosphonate, 46. ​​8-Methylthio-guanosine-5'-phosphonic acid, preferably the sodium salt and its mono-H-phosphonic acid. 47. 8-Phenylguanosine-5'-monophosphate and its mono-H-phosphonate, 48. 8-(2-Furyl)guanosine-5'-monophosphate and its mono-H-phosphonate, 49. 8-(4-chlorophenyl)guanosine-5'-monophosphate and its mono-H-phosphonate, 50.8-Phenyl-β-phenyl-1,N 2 -ethenoguanosine-5'-monophosphate and its mono-H-phosphonate, 51. 8-(4-chlorophenyl)-β-phenyl-1,N 2 -Ethenoguanosine-5'-monophosphate and its mono-H-phosphonic acid or its salts.

[0049] Reference to the compounds in the above list is intended to also refer to salts of such compounds, where salts are as defined elsewhere herein. In preferred embodiments, reference is made to free base compounds. In preferred embodiments, reference is made to salts. In more preferred embodiments, the compounds are selected from compounds 1-16. In other more preferred embodiments, the compounds are selected from compounds 17-32. In other preferred embodiments, the compounds are selected from 1, 10, 11, 17, 26, and 27, more preferably from 1, 10, and 11, alternatively more preferably from 17, 26, and 27. In other preferred embodiments, the compounds are selected from 6, 10-16, 22, and 26-32, more preferably from 6 and 10-16, alternatively more preferably from 22 and 26-32. In another preferred embodiment, the compounds are selected from 6, 10-13, 15, 16, 22, 26-29, 31, and 32, more preferably from 6, 10-13, 15, and 16, alternatively more preferably from 22 and 26-29, 31, and 32. In another preferred embodiment, the compounds are selected from 1-5, 7-9, 17-21, and 23-25, more preferably from 1-5 and 7-9, alternatively more preferably from 17-21 and 23-25. Very highly preferred compounds are compounds 10 and 26, most preferably compound 26. Other very highly preferred compounds are compounds 10, 35, 36, and 40-43, as well as their mono-H-phosphonic acids, more preferably their mono-H-phosphonic acids.

[0050] Step iii) Crystallization of guanosine 5'-monophosphoryl oxoate analogues The inventors have surprisingly found that the 5'-monophosphorus oxoacid ester analogue produced in step ii) can be crystallized. This intermediate is never isolated in the cGMP production methods known in the art, but purification of this intermediate has been found to have advantageous effects such as increased cyclization yield and improved control over the chirality of the cyclization product. Preferably, the guanosine 5'-monophosphorus oxoacid ester analogue is a salt when crystallized, more preferably a salt whose counterion is a hydrogenated organic base, most preferably a salt whose counterion is a hydrogenated (tri)alkylamine such as TEAH+.

[0051] The guanosine 5'-monophosphoric acid ester analogues are isolated by crystallization, preferably from an aprotic organic solvent, more preferably from a polar one, such as ethyl acetate or propyl acetate or THF or tert-butyl methyl ether, most preferably from ethyl acetate. The crystallization is preferably carried out with the addition of seed crystals, more preferably with the desired product to be crystallized. After the crystallization, which is preferably left for at least 4, more preferably at least 8, even more preferably at least 12 hours, the crystals are preferably washed and / or dried. The crystallization is preferably at room temperature. The washing is preferably carried out with the same solvent as that used for the crystallization. The drying is preferably carried out in vacuum, more preferably at elevated temperature, for example at about 40-80°C, preferably at about 50-70°C. In a preferred embodiment, the crystals obtained are solvates. Preferred crystals include substantially pure product, more preferably having a purity of at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, even more preferably at least 95, 96, 97, 98, 99, or 100%, and most preferably at least 97%, optionally greater.

[0052] Step iv) Formation of the cyclic diester The 5'-phosphate monoester isolated in step iii) can advantageously be used to continue the synthesis of a cGMP analogue. Therefore, a preferred method according to the invention further comprises the following steps: iv) cyclization of the guanosine 5'-monophosphate oxoacid ester analog obtained in step ii) (and crystallized in step iii) to obtain a cyclic guanosine-3',5'-monophosphate (cGMP) analog, said cyclization being preferably carried out in the presence of a sterically hindered base. 1 If is not H, it is sensible to preferably precede this step by conversion of X1 to H by deprotecting the corresponding protected hydroxyl moiety.

[0053] The cGMP analogue is preferably of the general formula (III) or a salt thereof: [ka] (In the formula, h, X 2 , R 1 , and R 2 is as defined above, preferably as defined for compounds of general formula (II) Preferably, X 2 is p', more preferably (tri)alkylsilyl such as TIPS. 3 is as defined for compounds of general formula (II), preferably 3 is H. In another preferred embodiment, o 3 -OH, -OC 1~8 Hydrocarbons, -SC 1~8 Hydrocarbons, -NH-C 1~8 Hydrocarbon, borano, methylborano, dimethylborano, cyanoborano, or -N(C 1~8 Hydrocarbon)2, most preferably -OH.

[0054] In the compound of general formula (III), the definition and preferred embodiments of h are preferably as defined for the compound of general formula (II). In the compound of general formula (III), the definition and preferred embodiments of R1 are preferably as defined for the compound of general formula (II). In the compound of general formula (III), the definition and preferred embodiments of R2 are preferably as defined for the compound of general formula (II). In the compound of general formula (III), the definition and preferred embodiments of X2 are preferably as defined for the compound of general formula (II).

[0055] The cyclization can be carried out using methods known in the art. Preferably, the cyclization is carried out in the presence of a coupling reagent. Preferably, the cyclization is carried out in the presence of a sterically hindered base or pyridine, more preferably a mild sterically hindered base or pyridine, even more preferably a mild sterically hindered base. Most preferably, both a coupling reagent and a sterically hindered base are used. The cyclization is preferably carried out under dilute conditions, for example, at a concentration of at most about 200 g / L, preferably at most about 100 g / L, more preferably at most about 80 g / L, even more preferably at most about 60 g / L, for example about 50 g / L, expressed as the weight of 5'-monophosphorus oxo acid ester analogue per liter of solvent. The solvent is preferably an aprotic solvent, more preferably a slightly polar one, for example dichloromethane or chloroform or acetonitrile, even more preferably chloroform or dichloromethane. As one skilled in the art will appreciate, under dilute conditions, the base is preferably present in excess, such as about 2-9 equiv., more preferably about 4-6 equiv., such as about 5 equiv. Similarly, the coupling reagent is preferably present in excess, such as about 1.1-5 equiv., more preferably about 1.2-3 equiv., such as about 1.5 equiv. Preferably, the base is added to the dilute 5'-monophosphorus oxo acid ester analog solution first, followed by the coupling reagent. The reaction is preferably allowed to proceed for at least 30 minutes, more preferably at least 1 hour, and most preferably about 2 hours or more.

[0056] Preferred examples of suitable sterically hindered bases are lutidine, picoline, collidine, N,N-dimethylaniline, N-methylmorpholine, and quinolone, more preferably lutidine. Further preferred sterically hindered bases have a pKa of about 4-7, more preferably about 5-7.

[0057] Suitable coupling reagents are well known in the art.Preferred examples of suitable coupling reagents are arylsulfonic acid derivatives, diester chlorophosphates, carbodiimides, and acyl halides, such as O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), (chloromethylene)dimethyliminium chloride (Vilsmeier's reagent), N-(dimethylaminopropyl)-N'-ethyl-carbodiimide (EDC), carbonyldiimidazole (CDI), propylphosphonic anhydride (T3P), diethyl chlorophosphate, dicyclohexylcarbodiimide (DCC), isobutyl chloroformate, and pivaloyl chloride. More preferably, acyl halides are used, of which acyl chlorides such as propyl chloride, benzoyl chloride, ethyl chloroformate, isobutyl chloroformate, and pivaloyl chloride are most preferred, with pivaloyl chloride being most highly advantageous.

[0058] Preferably, the cGMP analogue can be isolated by washing the organic phase, preferably with water, and then preferably diluting with an aprotic polar solvent, such as acetonitrile. Preferably, the organic phase is evaporated. The resulting crude product is preferably further purified by resuspending the crude solids in an aprotic polar solvent, such as acetonitrile, and then filtering off the solids. The filtrate is then preferably concentrated under vacuum, and then preferably redissolved in an aprotic polar solvent, such as acetonitrile. The crude solution is then preferably treated by adding an excess of an ether solvent, preferably a tert-butyl ether solvent, such as tert-butyl methyl ether (TBME). The excess is preferably at least 5 volumes, more preferably at least 10 volumes, such as about 12 volumes. The resulting composition is preferably left for at least about 4 hours, more preferably at least about 8 hours, and then the solvent is removed, preferably by decantation and / or evaporation. The resulting residue can be an oil. The resulting residue is preferably dried under vacuum and then triturated, preferably with an ether solvent, preferably a tert-butyl ether solvent such as TBME. In another highly preferred embodiment, cyclization should preferably be followed by phosphorothioate formation, and the cGMP analogue is not isolated prior to any further reaction steps.

[0059] The inventors have surprisingly found that the use of a sterically hindered base results in a very desirable stereochemical outcome of the reaction, i.e., a large diastereomeric excess of the Rp stereoisomer, which is the preferred stereoisomer. The Rp and Sp stereoisomers of general formula (III) are shown below and are of general formulae (III-Rp) and (III-Sp), respectively. Without wishing to be bound by theory, the use of a sterically hindered base can result in an overall slower reaction or a reaction under kinetic control, resulting in the preferred formation of the kinetic product, which is the Rp product. [ka]

[0060] In a preferred embodiment, the compound of general formula (III) is of general formula (III-Rp). Since different diastereomers may be appropriate for different applications, in another preferred embodiment, the compound of general formula (III) is of general formula (III-Sp). In the compound of general formula (III-Rp) or (III-Sp), h, X 2 , R 1 , and R 2 is preferably as defined above, more preferably as defined for compounds of general formula (III). 2 is p', more preferably (tri)alkylsilyl, most preferably TIPS. 3 is preferably as defined for compounds of general formula (II), more preferably as defined therein, except that it is not -OH, and even more preferably o 3 is H or SH. The intermediate product produced in step iv is most preferably o 3 is H.

[0061] Preferably, when the compound is of general formula (III-Rp) or general formula (III-Sp), the corresponding diastereomers are present in a diastereomeric excess of at least 2:1, more preferably at least 3:1, even more preferably at least 4:1, other more preferably at least 5:1, other even more preferably at least 6:1, other more preferably at least 7:1, and most preferably at least about 8:1. Preferably, when the compound of general formula (III) and / or general formula (III-Rp) or (III-Sp) is obtained after step iv), the compound has a purity of at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more percent. More preferably, the purity is at least 70%, even more preferably at least 75%, other more preferably at least 80%, other even more preferably at least 85%, and other more preferably at least 90%. Purity can be assayed using known techniques, preferably assayed using HPLC.

[0062] Preferred compounds of general formula (III) are salts thereof, more preferably salts in which the counter ion is a hydrogenated organic base, most preferably counter ion is a hydrogenated (tri)alkylamine such as TEAH+. Preferred compounds of general formula (III-Rp) are salts thereof, more preferably salts in which the counter ion is a hydrogenated organic base, most preferably counter ion is a hydrogenated (tri)alkylamine such as TEAH+. Preferred compounds of general formula (III-Sp) are salts thereof, more preferably salts in which the counter ion is a hydrogenated organic base, most preferably counter ion is a hydrogenated (tri)alkylamine such as TEAH+.

[0063] Step v) Phosphorothioate formation Phosphorothioates are useful cGMP analogues. Therefore, in a preferred embodiment, the method according to the invention comprises the following step v): v) contacting the cGMP analog with a sulfurizing agent to form a compound represented by the general formula (III),3 -SH or -SC 1~12 and obtaining a thiolated cGMP analog of formula (III-Rp, preferably -SH). In a preferred embodiment, the cGMP analog is of formula (III-Rp). In another preferred embodiment, the cGMP analog is of formula (III-Sp). Step v) is advantageous when M is O in the guanosine 5'-monophosphate oxoacid analog of formula (II). When M is S in such precursor of formula (II), step v) is preferably not included in the method.

[0064] Sulfurizing agents are known in the art. Preferred examples of sulfurizing agents are sulfur, phenylacetyl disulfide, and N-(alkyl-thio)-succinimides, such as N-methylthio-succinimide, N-ethylthio-succinimide, and N-propylthio-succinimide. Sulfur is most preferred. Preferred C 1~12 Hydrocarbons are C 1~8 Hydrocarbons, preferably C 1~4 It is a hydrocarbon. Preferred -SC 1~12 Examples of hydrocarbons are -SCH3, -S-CH2CH3, -S-CH(CH3)2, -SC(CH3)3, and -S-phenyl, with -S-CH3 being the most preferred. Suitable sulfur introduction involves the use of electrophilic sulfur, preferably using a sulfurizing agent as described above, followed by oxidation (i.e., increasing the oxidation state), preferably using an oxidizing agent as described below. The use of nucleophilic sulfur is not advantageous.

[0065] Preferably, the contacting is carried out in the presence of a base, more preferably a (tri)alkylamine, such as triethylamine. The reaction is preferably allowed to proceed for at least 30 minutes, more preferably at least 60 minutes, and most preferably at least 90 minutes. The progress of the reaction is preferably assessed using known techniques, such as HPLC.

[0066] Alternatively, it may be useful to convert P(III) to P(V) when this step is carried out starting from a cGMP analogue in which o3 is H. Therefore, in a preferred embodiment, the method according to the invention comprises the following step vO): vO) contacting a cGMP analog with an oxidizing agent to obtain a cGMP analog of general formula (III) in which o3 is -OH. Such oxo derivatives are activators of PKG and not its inhibitors, such as thiophosphates in which o3 contains S. The characteristics and conditions are preferably as described for step v). The oxidizing agents are known in the art. Step vO) is preferably carried out under anhydrous conditions. Examples of oxidizing agents suitable for step vO) are peroxides, such as hydrogen peroxide, bis(trimethylsilyl)peroxide, t-butylhydroperoxide, cumenehydroperoxide, iodine, and alkylamino-oxides, such as N-methylmorpholine N-oxide. Alternatively, the agent can be a catalyst, such as Pd / C powder. Examples of phosphite oxidation to phosphates are known, for example, from Hayakawa et al. (Tet. Lett. 1986, DOI:10.1016 / S0040-4039(00)84946-1) using peroxides and from Nagaosa and Aoyama (Carbon, 2001, DOI:10.1016 / S0008-6223(01)00206-8) using Pd / C.

[0067] Preferably, when the compound of general formula (III) and / or general formula (III-Rp) or (III-Sp) is obtained after step v) or vO), the compound has a purity of at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or more percent. More preferably, the purity is at least 70%, even more preferably at least 75%, other more preferably at least 80%, other even more preferably at least 85%, other more preferably at least 90%. Purity can be assayed using known techniques, preferably assayed using HPLC.

[0068] Step vi) Deprotection of cGMP analogues In order to obtain products with interesting pharmacological properties, the cGMP analogues produced by the method of the invention are preferably deprotected. This means, in the context of the present invention, that the produced cGMP analogues of general formula (III) preferably have X2, which is H. Thus, in a preferred embodiment, X 2 There is provided a method according to the present invention in which p' is p', the method further comprising the steps of: vi) deprotecting the hydroxyl moiety protected by X2 to obtain a deprotected cGMP analogue, which can be represented by the general formula (III), 2is H). Means of deprotection are well known in the art and depend on the nature of p'. In general, deprotection can be carried out under acidic conditions, e.g., at pH<1, optionally at elevated temperature. Deprotection is also preferably carried out in the presence of fluoride, e.g., with HF, (tri)alkylamine·HF, or TBAF. For deprotection of (tri)alkylsilyl protecting groups, deprotection in the presence of fluoride, more preferably with (tri)alkylamine·HF, e.g., triethylamine HF, preferably in a 10-50 vol.-%, more preferably 20-40 vol.-%, e.g., about 33 vol.-% solution, is preferred. Deprotection is preferably carried out in an aprotic solvent, more preferably a polar aprotic solvent that is a cyclic ether, e.g., tetrahydrofuran or 1,4-dioxane. The reaction is preferably allowed to proceed for about 1 day, more preferably about 2 days, even more preferably about 3 days. Standard analytical techniques, such as HPLC, can be used to monitor the reaction progress. The precipitated deprotected product is preferably isolated by filtration, followed by washing, preferably with the solvent used for deprotection. The product is then preferably dried, such as under vacuum. The product obtained in this manner is a fluoride complex of the deprotected cGMP analog. In a preferred embodiment, the cGMP analog produced is in a complex with fluoride. The cGMP analog fluoride complex preferably has a purity of at least 60, 65, 70, 75, or 80 percent, as assayed for cGMP analog. More preferably, the purity is at least 70%, even more preferably at least 75%, and even more preferably at least 80%. Purity can be assayed using known techniques, preferably assayed using HPLC. The compound is preferably a salt, more preferably a (tri)alkylamine salt, and most preferably a TEAH+ salt.

[0069] Step vii) Trituration of the deprotected cGMP analog In a preferred embodiment, the method according to the invention comprises the following step viii): vii) Triturating the deprotected cGMP analogue.

[0070] The deprotected cGMP analog is as described above. This trituration preferably removes fluoride from the cGMP analog fluoride complex. However, the inventors have also surprisingly found that trituration results in improved diastereomeric excess of the isolated compound of general formula (III-Rp). Therefore, the present invention provides a method for producing a cGMP analog, comprising step vi), more preferably comprising steps vi) and vii, wherein the characteristics and definitions are as defined elsewhere herein, and preferably the method also comprises step viii) as described below.

[0071] Preferably, in trituration, the deprotected cGMP analog can be resuspended in a polar solvent, preferably an aprotic polar solvent such as acetonitrile. It is then preferably filtered and washed, more preferably with the resuspended solvent. The resulting cGMP analog preferably has a purity of at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or more percent. More preferably, the purity is at least 75%, even more preferably at least 80%, other more preferably at least 85%, other even more preferably at least 90%, other more preferably at least about 95%. The deprotected cGMP analog is preferably substantially fluoride-free, more preferably completely fluoride-free. The absence of fluoride can be confirmed using 19F NMR. Purity can be assayed using known techniques, preferably using HPLC. The compound is preferably a salt, more preferably a (tri)alkylamine salt, most preferably a TEAH+ salt.

[0072] Step viii) Formation of a Pharmacologically Acceptable Salt For pharmaceutical use, the cGMP analogue is preferably either the free base or a pharma- ceutically acceptable salt, more preferably a pharma- ceutically acceptable salt. Thus, a preferred method according to the invention comprises the following step viii): viii) optionally converting the deprotected cGMP analogue to a pharma- ceutically acceptable salt, preferably a sodium salt, with characteristics and definitions as provided above. The cGMP analogue converted in step viii) is preferably the product of step vi) or step vii), most preferably step vi) followed by step vii). This compound is most preferably a salt, more preferably a (tri)alkylamine salt, most preferably a TEAH+ salt.

[0073] Preferably, the cGMP analogue is mixed with a protic solvent, more preferably a lower alcohol such as methanol. This preferably results in a suspension. This is followed by the addition of an alkoxide salt, preferably an alkoxide salt of a solvent and an alkali metal, such as potassium or sodium, more preferably sodium, whose counterion is the intended counterion for the pharma- ceutically acceptable cGMP salt. A preferred alkoxide salt is sodium methoxide. The resulting solution is preferably concentrated under vacuum to obtain a pharma- ceutically acceptable salt, preferably the sodium salt, of the cGMP analogue.

[0074] After this concentration, the obtained solids are preferably stirred with a lower alcohol, more preferably with ethanol, and then the solids are separated by filtration. The inventors have surprisingly found that after filtration, the mother liquor contains a lower fraction of Sp diastereomers than the filtered solids. Therefore, the present invention provides a method for producing a cGMP analogue, comprising step viii), wherein the characteristics and definitions are as defined elsewhere herein, and preferably the method also comprises step vii) as described above.

[0075] In a preferred embodiment, the solids are stirred with a slightly moist lower alcohol, more preferably with a slightly moist ethanol, most preferably with 96% ethanol (the remaining 4% is preferably essentially water, both vol.-%) for at least 1, more preferably at least 2 hours. After this, the filtrate is preferably concentrated under vacuum. The purity of the obtained solids is preferably further increased by further suspension in anhydrous lower alcohol, preferably absolute ethanol, after which the suspension is filtered and the solids are preferably washed with additional absolute alcohol, preferably the same alcohol used for resuspension. The obtained solids are then preferably dried, such as by vacuum drying.

[0076] Preferably, the pharma- ceutically acceptable salt of the cGMP analog contains at most 10%, more preferably at most 5%, even more preferably at most 4, 3, or 2%, even more preferably at most 1%, for example at most 0.5% of the undesired diastereomer. Preferably, the pharma- ceutically acceptable salt of the cGMP analog has a purity of at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more percent. More preferably, the purity is at least 90%, even more preferably at least 94%, other more preferably at least 95%, other more preferably at least 96%, other more preferably at least about 97%. Purity can be assayed using known techniques, preferably using NMR.

[0077] In a highly preferred embodiment, X 2 is provided a method according to the invention in which p' is a vi) deprotecting the protected hydroxyl moiety with X2 to obtain a deprotected cGMP analog; vii) optionally triturating the deprotected cGMP analog; viii) optionally converting the deprotected cGMP analog to a pharma- ceutical acceptable salt, preferably the sodium salt; Further includes:

[0078] Products and intermediates produced by the process Besides providing advantageous routes to cGMP analogues, the method of the present invention also allows for the efficient formation of several key intermediates. In another aspect, the present invention provides several such compounds, preferably compounds of general formula (II) or salts thereof, wherein h, X1, X 2 , R 1 , and R 2 is as defined above, and 1 and 3 are each independently -OH or as defined for compounds of general formula (P), preferably o 3 is H, or o 1 and 3 together, preferably C 2~12 A chiral auxiliary is formed which is a hydrocarbon, M is S or O, and preferably the compound is a salt. The product can be used for the manufacture of a pharmaceutical agent, preferably a pharmaceutical agent for the treatment of retinitis pigmentosa.

[0079] Further features and definitions are as described above in the first aspect for compounds of general formula (II). Thus, in a preferred embodiment within this aspect, 3 is H and o 1 -OH, -OC 1~8 Hydrocarbons, -SC 1~8 Hydrocarbons, -NH-C 1~8 Hydrocarbon, borano, methylborano, dimethylborano, cyanoborano, or -N(C 1~8 Hydrocarbon)2, more preferably -OC 1~8 Hydrocarbons, -SC 1~8 Hydrocarbons, -NH-C 1~8 Hydrocarbon, or -N(C 1~8 In another preferred embodiment, o 1 and 3 Both are -OH, -OC 1~8 Hydrocarbons, -SC 1~8 Hydrocarbons, -NH-C 1~8Hydrocarbon, borano, methylborano, dimethylborano, cyanoborano, or -N(C 1~8 Hydrocarbon)2, more preferably -OC 1~8 Hydrocarbons, -SC 1~8 Hydrocarbons, -NH-C 1~8 Hydrocarbon, or -N(C 1~8 In a preferred embodiment, o 1 and 3 together form a chiral auxiliary as defined above. Most preferably, 1 is -OH, and o 3 is H.

[0080] Preferably, the compound within this aspect is crystalline. The intermediate may also be a hydrate or solvate. In a preferred embodiment, the crystal is a solvate, more preferably an isopropyl acetate solvate. In another embodiment, the crystal is a hydrate. In a highly preferred embodiment, the crystal is neither a solvate nor a hydrate. In a preferred embodiment, the crystal comprises a fluoride. In a preferred embodiment, the crystal comprises a substantially pure intermediate, more preferably having a purity of at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, even more preferably at least 95, 96, 97, 98, 99, or 100%, most preferably at least 97%, or optionally more.

[0081] In a preferred embodiment, the present invention relates to a compound of general formula (II) or a salt thereof (wherein h, X1, X 2 , R 1 , and R 2 is as defined above, and 1 is -OH or as defined for compounds of general formula (P), o 3 is H, M is S or O, and preferably the compound is a salt, more preferably a TEAH+ salt. Further features and definitions are as described in the first aspect above for compounds of general formula (II).

[0082] In a preferred embodiment, the present invention relates to a compound of general formula (II) or a salt thereof (wherein h, X1, X 2 , R 1 , and R 2 is as defined above, or 1 and 3 together, preferably C 2~12 and M is S or O, and preferably the compound is a salt. Further features and definitions are as described above in the first aspect for compounds of general formula (II).

[0083] In a preferred embodiment, the present invention relates to a compound or a salt thereof of general formula (II), 1 is H and X 2 is p', p' is preferably (tri)alkylsilyl, more preferably triisopropylsilyl (TIPS), R 1 and R 2 are preferably taken together to form -CH=C(ar)-, where ar is closest to R2, and ar is phenyl or naphthyl, preferably phenyl; 1 is OH, o 3 is H and M is S or O, preferably O).

[0084] The desired products of the present process are those of general formula (III), more preferably those of general formula (III-Rp), 3 is -SH (or optionally -S if the compound is a sodium salt), and X 2 is H, h is -Br, and R 1 and R 2 are taken together to form -CH=C(ar)-, preferably with ar being closest to R2 and ar being 4-methylphenyl.

[0085] general definition In this document and in the claims, "to comprise" is used in its open-ended sense, meaning to include the items that follow the word, but not to exclude items not specifically listed. Additionally, a reference to "an element" by the indefinite article "a" or "an" does not exclude the possibility that there is more than one element, unless the context clearly requires that only one element be present. Thus, the indefinite article "a" or "an" typically means "at least one."

[0086] The words "about" or "approximately" when used in connection with numerical values ​​(e.g., about 10) preferably mean that the given value can be 1% greater or less than the given value.

[0087] Oxoacids, also known as oxyacids, are compounds that contain hydrogen, oxygen, and at least one additional element, and where at least one hydrogen atom bonded to the oxygen is capable of dissociating to produce an H+ cation and the anion of the acid. Phosphorus oxoacids are oxoacids where the additional element is phosphorus.

[0088] The molecules provided herein can be optionally substituted. A preferred optional substitution is the replacement of -H with a halogen. Preferred halogens are F, Cl, Br, and I. Further preferred optional substitutions are the replacement of one or more -H with -NH2, -OH, =O, alkyl, alkoxy, haloalkyl, haloalkoxy, alkene, haloalkene, alkyne, haloalkyne, and cycloalkyl. The alkyl group can be represented by the general formula C n H 2n+1 and may alternatively be linear or branched. Unsubstituted alkyl groups may also contain cyclic moieties, and therefore may have the additional general formula C n H 2n-1Optionally, the alkyl group is substituted with one or more substituents as more particularly described in this document. Examples of alkyl groups include methyl, ethyl, propyl, 2-propyl, t-butyl, 1-hexyl, 1-dodecyl, and the like.

[0089] Unless otherwise specified, -H is C1-C 12 Alkyl groups, C2-C 12 Alkenyl groups, C2-C 12 Alkynyl groups, C3-C 12 Cycloalkyl groups, C5-C 12 Cycloalkenyl groups, C8-C 12 Cycloalkynyl groups, C1-C 12 Alkoxy group, C2-C 12 Alkenyloxy group, C2-C 12 -Alkynyloxy group, C3-C 12 and optionally substituted with one or more substituents independently selected from the group consisting of cycloalkyloxy groups, halogens, amino groups, oxo, and silyl groups, where the silyl group is represented by the formula (R s ) 3Si-, where Rs is C1 to C 12 Alkyl groups, C2-C 12 Alkenyl groups, C2-C 12 Alkynyl groups, C3-C 12 Cycloalkyl groups, C1-C 12 Alkoxy group, C2-C 12 Alkenyloxy group, C2-C 12 Alkynyloxy groups and C3-C 12 and cycloalkyloxy groups, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy, and cycloalkyloxy groups are optionally substituted, and the alkyl, alkoxy, cycloalkyl, and cycloalkoxy groups are optionally interrupted by one or more heteroatoms selected from the group consisting of O, N, and S.

[0090] A structural formula or chemical name is understood by those skilled in the art to have chiral centers, although when no chirality is indicated, individual reference is made to each chiral center, to all three: racemic mixture, pure R enantiomer, and pure S enantiomer. When two moieties are said to form a bond together, this suggests the absence of atoms of these moieties, and valence compliance is satisfied by the replacement electron bond. Dashes in moiety representations serve primarily to provide guidance to the reader, and include, as a non-limiting example, -NH-(CH2) n -OH and -NH-(CH2) n OH means the same moiety. All of this is known in the art.

[0091] Whenever a parameter of a substance is discussed in the context of the present invention, it is assumed that the parameter is determined, measured, or expressed under physiological conditions, unless otherwise specified. Physiological conditions are known to those of skill in the art and generally include an aqueous solvent system, atmospheric pressure, a pH value of 6 to 8, a temperature in the range of room temperature to about 37° C. (about 20° C. to about 40° C.), and suitable concentrations of buffer salts or other components. Charge is often understood to be associated with an equilibrium. A moiety that is said to carry or carry a charge is one that will be found in a state in which it carries or carries such a charge more frequently than one that neither carries nor carries such a charge. Thus, as will be understood by those of skill in the art, atoms suggested in the present disclosure to be charged may be uncharged under specific conditions, and neutral moieties may be charged under specific conditions.

[0092] In the context of the present invention, a decrease or increase in the assessed parameter means a change of at least 5% in the value corresponding to that parameter. More preferably, a decrease or increase in value means a change of at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In this latter case, it may be the case that there is no longer a detectable value associated with that parameter.

[0093] The use of a substance as a pharmaceutical agent described in this document can also be interpreted as the use of said substance in the manufacture of a pharmaceutical agent. Similarly, whenever a substance is used for treatment or as a pharmaceutical agent, it can also be used to manufacture a pharmaceutical agent for treatment. A product for use is suitable for use in a treatment method.

[0094] Filtration can be carried out using any method known in the art, for example, preferably using a Buchner funnel or an agitated Nutsche filter.

[0095] The present invention has been described above with reference to several exemplary embodiments. Modifications and alternative realizations of several parts or elements are possible and fall within the scope of protection defined in the appended claims. All citations of literature and patent documents are hereby incorporated by reference. [Brief description of the drawings]

[0096] [Figure 1] Synthetic pathway leading to Rp-8-Br-PET-cGMPS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0097] Working Example Example 1 - Synthesis of Rp-8-Br-PET-cGMPS 1.1 PET formation 152 g 8-bromoguanosine (90% assay, remainder water) was stirred in 5 vol DMSO (760 ml) and 1.5 equiv. phenacyl bromide was added to the suspension. Then 2.5 equiv. DBU was added for 30 min. After 1 h, 3 equiv (65 ml) acetic acid was added. The product was precipitated by slow addition of 10 vol water (1520 ml). The slurry was filtered and washed with 2000 ml DMSO:water 1:2 followed by 200 ml water. The cake was dried in vacuum and resuspended in 2500 ml MeCN and stirred overnight. After filtration and MeCN washing, it was dried in vacuum at 70° C. to give 130 g (74%) of intermediate 1.

[0098] 1.2 Protection of guanosine analogues 130 g Br-PET-guanosine (intermediate 1) was suspended in 5 vol DMF (650 ml). 5 equiv. imidazole (96 g) and 2.5 equiv. TIPS-Cl (151 ml) were added. After 48 h the reaction was analyzed (HPLC showed 1.08% 5'-mono TIPS, 67.76% 5'2'-di TIPS, 29.52% 5'3'-di TIPS, and 1.64% 5'2'3'-tri TIPS). The reaction was quenched with 50 ml water (10 equiv.) and stirred for 1 h. After dilution with 15 vol toluene (1900 ml), the reaction was washed with 3 x 5 vol water (650 ml each). The organic phase was evaporated and the sticky residue was dissolved in 20 volumes i-propyl acetate (2300 ml), seeded and left overnight to crystallize. Filtration and washing with i-propyl acetate gave, after drying in vacuum, 81 g (37%) of intermediate 2 (98.5% purity by HPLC).

[0099] 1.3 Selective deprotection 150g 5'-,2'-diTIPS-Br-PET-guanosine (intermediate 2) was dissolved in 10vol THF (1500ml). Then 2vol water (300ml) and 0.45vol TFA (67.5ml) were added. After stirring overnight (the reaction was checked and HPLC showed 3.88% double deprotection, 94.75% desired 2'-TIPS product, and 1.37% unconverted product), 0.4vol 28% ammonia (aq.) (60ml) was added (pH became 5) and the mixture was concentrated. 15vol CH2Cl2 (2250ml) and 2vol water (300ml) were added and the resulting diluted slurry was filtered (this removes the perhydrolyzed material, i.e. Br-PET-guanosine). The organic phase was separated and evaporated. To the residue was added 3 vol acetonitrile (450 ml) and the slurry was stirred for 1 h, followed by filtration and washing with 2 vol acetonitrile (300 ml). Drying in vacuum gave 96 g (78%) of intermediate 3, which was assayed to be 100% pure by HPLC.

[0100] 1.4 Formation of 5'-monoester 111 g of 2'-TIPS-Br-PET-guanosine (intermediate 3) was dissolved in a mixture of 19 vol CH2Cl2 (1945 ml) and 1 vol pyridine (110 ml). Then, 3 equiv. (102 ml) of diphenyl phosphite was added while stirring. After 2 hours, 1 vol water (110 ml) and 1 vol triethylamine (110 ml) were added, the reaction was left for 30 min and then washed with 2 x 10 volumes (2 x 1100 ml) of water. The organic phase was evaporated and the residue was co-evaporated with 500 ml i-propanol followed by 500 ml ethyl acetate. The residual oil was then dissolved in 1500 ml ethyl acetate, seeded and left overnight to crystallize. Filtration and washing with ethyl acetate gave 106 g (76%) of intermediate 4 after drying in vacuum. The product does not behave well in a reverse phase liquid chromatography system, giving a broad, slow peak (H2O:MeCN, 0.1% formic acid), but assays at 97.56%.

[0101] 1.5 Formation of cGMP analogues by cyclization 50.5 g of 5'-phosphonate-2'-TIPS-BrPET guanosine was dissolved in 1000 ml CHCl (20 vol.) followed by the addition of 37 ml 2,6-lutidine (5 equiv.). This was followed by the addition of 11.8 ml pivaloyl chloride. The reaction was allowed to proceed for 2 hours.

[0102] 1.6 Phosphorothioate formation To the crude reaction solution of Example 1.5, 5.9 g of sulfur (3 equiv.) was added followed by 26 ml of triethylamine. After 1.5 h (HPLC showed 74.05% Rp diastereomer and 9.72% Sp diastereomer, the rest was impurities), the solution was washed with 2×200 ml water and the organic phase was diluted with 200 ml MeCN and evaporated. The residue (100 g) was slurried in 400 ml MeCN for 10 min and filtered. The filtrate was evaporated and redissolved in 100 ml MeCN. 1200 ml TBME was added slowly and stirred overnight, giving some oily sticky precipitate. After decantation and evaporation and dissolution, the residual oil was thoroughly dried by high vacuum to form a sticky foam. The foam was stirred and triturated with 700 ml TBME, yielding 38 g yellow powder after filtration and drying. The assay was 68% calculated as the TEAH salt of the desired diastereomer giving a corrected yield of 50%. HPLC purity was 80% (260 nm) with a diastereomeric ratio of 8:1.

[0103] 1.7 Deprotection of cGMP analogues 35g of intermediate 5 (Rp diastereomer with 68% assay) was dissolved in 4vol THF (140ml) and 2vol TEA·3HF (70ml) was added. The reaction was stirred for 3 days (HPLC showed 70.68% deprotection), filtered, washed with 3vol THF (105ml) and dried in vacuum. 17.5g of product with 83% assay (76% yield) was obtained. The material is some kind of fluoride-containing complex (as seen by 19F NMR and evident from the low assay). The material was slurried in 300 ml MeCN, filtered, and washed with 150 ml MeCN (this MeCN reslurry removed the fluoride seen by NMR and improved overall purity; HPLC showed 97.13% desired Rp product and only 0.78% undesired Sp product, while the mother liquor contained 76.83% Rp product and 14.53% undesired Sp product). This gave 13.5 g of material with a 95% assay, 67% yield. The material contained no fluoride based on 19F NMR.

[0104] 1.8 Sodium salt formation 13g intermediate 6 (8-Br-PET-cGMP TEAH salt, 95% NMR assay) was suspended in 500ml MeOH. 1 equiv (1.095g) sodium methoxide was added to bring the suspension into solution, which was concentrated on a rotary evaporator. The solution converted to a gel when approximately 150mL remained. Evaporation was continued and the gel converted to a powder. The solid was stirred with 950ml 96% EtOH for 1 hour and filtered. This gave 9g of material after drying. The filtrate gave 2.9g of material after evaporation and drying. HPLC showed that the material from the mother liquor contained less of the undesired diastereomer than the 9g portion (0.25% vs. 0.85), so the material was pooled and reslurried in 950ml 96% ethanol for 2 hours and then filtered. The mother liquor (containing most of the product) was evaporated and reslurried in 170ml absolute ethanol. Filtration and washing with 100 ml absolute ethanol gave a gel-like cake which was dried in vacuum to give 8.1 g of 8-Br-PET-cGMP sodium salt with 0.5% undesired diastereomer (97% NMR assay, 72% yield).

[0105] Example 2 - Diastereomerically pure R P - Chromatography-free synthesis of guanosine-3',5'-cyclic thiophosphate analogues, 2.1 Overview The cyclic guanosine monothiophosphate analog 1a is currently showing potential as a drug for the treatment of inherited retinal neurodegeneration. To support its ongoing preclinical development, we have developed a diastereoselective chromatography-free synthesis for its preparation. Notable features of the synthetic sequence include a silylation step with 80% selectivity of the 2',5'- over the 3',5'-hydroxyl and a S PThe ring closure of the 5'-H-phosphonic acid monoester with 90% selectivity for the diastereomer was involved. Compounds were isolated via crystallization, including the final product 1a, which was obtained as the EtNH salt in 125 g yield and >99.9% HPLC purity.

[0106] 2.2 Introduction Recent studies suggest that photoreceptor death in several animal models of inherited retinal neurodegeneration (IRD) is primarily governed by an alternative non-apoptotic pathway mediated in part by overactivation of guanosine-dependent protein kinase (PKG). This overactivation results from the non-natural accumulation of cyclic guanosine monophosphate (cGMP) in photoreceptors due to disruption of the phototransduction cascade. This sparked interest in exploring cGMP analogs that could potentially act as therapeutic agents for IRD by blocking this PKG-mediated pathway. The result was the discovery of a promising cyclic guanosine monothiophosphate (cGMPS) analog 1a with potent neuroprotective effects in vivo. The compound contains a phenylethenyl (PET) group on the nucleobase and an R group on the thiophosphate. P It is an 8-bromo-cGMPS derivative with the configuration. Cyclic nucleotide monothiophosphates (cNMPS) are known to resist cleavage by phosphodiesterases and therefore survive longer in cells than their phosphate counterparts, adding to the potential of 1a as a therapeutic agent. In contrast to its antagonistic activity against PKG, its S P Both the -diastereomer 1b and its phosphate equivalent 1c were found to be PKG agonists, as was native cGMP (Scheme 1). [ka]

[0107] To further enable preclinical development, larger quantities of this active pharmaceutical ingredient (API) are desired, and our work toward a scalable robust synthetic process for the preparation of high purity 1a is presented here. Published cyclic thiophosphate syntheses include that by Stec et al., which involves the reaction of sulforyl chloride with aniline to give a mixture of phosphoroanilidate diastereomers, which are then separated (Stereospecific Synthesis of Adenosine 3',5'-(Sp)-and-(Rp)-Cyclic Phosphorothioates (cAMPS). Journal of the Chemical Society, Chemical Communications 1979, 940-941). To generate cyclic thiophosphates with complete configurational retention, the desired isomers are treated with strong base and carbon disulfide (Steck reaction). Eckstein et al. used bisnitrophenyl phosphorochloride thioates on unprotected nucleosides to prepare 5'-bisnitrophenyl phosphorothioates. These were directly cyclized by treatment with potassium tert-butoxide. Genieser's group was the first to use a similar approach, in effect synthesizing 1a, by utilizing thiophosphoryl chloride to obtain 5'-thiophosphorodichloridates as precursors for the cyclization step, as described in US Pat. No. 5,625,056. In all the above cases, the cNMPSs (or their precursors) were obtained as diastereomeric mixtures that were subsequently separated by chromatography. In a more recent synthesis, Andrei et al. (Organic & Biomolecular Chemistry 2007,5,2070-2080) were inspired by these methods to develop a stereoselective chlorination-amidation-stick sequence, this time using cyclic adenosine monophosphate as the starting material. However, this tactic requires the use of R POnly the diastereomer was available, and further chromatographic purification was still required. A completely different approach to cGMPS is the H-phosphonate pathway developed by Stawinski's group (Org. Lett. 2013, 15, 4082-4085). It requires internal cyclization of nucleoside-3'-H-phosphonate monoesters to form the corresponding 3',5'-cyclic H-phosphonate diesters, followed by sulfurization to give the cyclic thiophosphates. In that report, it was shown that 3',5'-cyclic nucleoside-H-phosphonates were formed diastereoselectively, and that this selectivity could be directed towards the preferred diastereomer.

[0108] 2.3 Results and Discussion Route development. We found the latter approach to be the most promising and therefore the focus of our development. We noted that in the prior art, the 1,N2-phenylethenyl (PET) group was introduced after the formation of the cyclic phosphorothioate, and it was performed without any protection on the 2'-hydroxyl. The corresponding H-phosphonate approach would be more sensitive to side reactions on the nucleobase and ribose. Therefore, our synthetic design aimed to first introduce the PET group, since it should function similarly to a protecting group, and then introduce a protecting group at 2' to counteract the formation of the 2',3'-cyclic by-product. We also placed emphasis on the avoidance of chromatographic purification in favor of crystallization, as well as the ability of the process to control key impurities such as the undesired PKG agonists 1b and 1c. [ka]

[0109] Phenylethenylation. The reported synthesis of 8-bromoguanosine with 1,N2-phenylethenyl (PET) modification used 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)-DMSO combination, which served as the starting point for our development. Dissolving 8-bromoguanosine in 10 volumes (ml / g) of DMSO and adding 5 equiv. of 2-bromoacetophenone followed by 7 equiv. of DBU caused consumption of starting material within 30 min, with formation of 8-bromoPET guanosine 2 as the major product (approximately 70% LC area). Following neutralization with acetic acid, the product was precipitated by addition of water (15 volumes), but as a slightly colored solid. It was also found that the amounts of 2-bromoacetophenone and DBU could both be reduced to 2.5 equiv. without compromising the conversion. Overall, the reaction appeared to be robust with little variation in product formation when the stoichiometry was changed. Volumetric efficiency and order of loading were then examined. By adding DBU before the bromoketone, 8-bromoguanosine was dissolved in only 4 volumes of DMSO. Adding bromoacetophenone to this mixture as a solution in 1 volume of DMSO gave the product in nearly 50% yield. Additionally, the 2-bromoacetophenone / DMSO solution was not stable as indicated by the constant yellowing of the clear solution as well as the accumulation of by-products on HPLC. This decomposition does not appear to be related to the reduction in yield, since adding bromoacetophenone as a solid to a solution of bromoguanosine and DBU in 5 volumes of DMSO gave no improvement whatsoever. Finally, it was found that vacuum drying of 8-bromoguanosine increased its solubility in DMSO. Commercially available 8-bromoguanosine contains approximately 8% water (Karl Fischer), which was found to be reduceable to >0.5% by vacuum drying at temperatures up to 80° C. without affecting purity. The dried material gave a clear solution in 5 volumes of DMSO within 15 minutes. No improvement in purity or yield was observed with more than 4 volumes of water (when 5 volumes of DMSO were used) with respect to product precipitation.Rapid water addition should be avoided as it will cause sticky mass formation. Interestingly, when the reaction mixture was not neutralized with acetic acid beforehand, the same effect was obtained even if water was added slowly. It was also found that a final cake wash with MeCN removed the color and some minor impurities. Therefore, the reaction was carried out in 5 volumes of DMSO and the product was precipitated with 4 volumes of water, followed by washing with MeCN, affording intermediate 2 as a white solid in 71% yield. The product was found to be crystalline and >98% pure (HPLC).

[0110] Protection strategies. Introducing a protecting group at the 2' position cannot be achieved without introducing one at the 5' position, which then requires the removal of the latter. Besides, achieving selectivity of the 2'-hydroxyl over the 3'-hydroxyl remains a challenge due to the similar reactivity of the two. Usually, this is addressed by separation of the isomers via chromatography, which we aim to avoid. It is possible to bypass this competition with the help of silylating agents, such as 1,3-dihalo-1,1,3,3-tetraalkyldisiloxanes, which simultaneously block the 5' and 3' positions, which can be removed after further protection of the 2' position. Although this is a widespread approach, it generally involves an extra step for 2'-protection, and we have had better success by optimizing traditional silylation for selectivity towards 2' and developing selective crystallization as described below.

[0111] Selective protection. We began by exploring the most common silylation method, a 1:2 silylating agent:base mixture, most commonly 2.5 equiv. t-butyldimethylsilyl chloride (TBDMS) and 5 equiv. imidazole. With this method, disilylation was achieved within 1.5 hours, but there was virtually no selectivity for either of the disilylated isomers. Furthermore, the 2',3',5'-trisilylated by-product made up 44% of the product composition. When TBDMS-Cl was replaced with TIPS-Cl, substitution at the second hydroxyl resulted in an 80:20 ratio in favor of 2', and after 3 days, less than 1% trisubstitution was seen. However, 2'→3' silyl migration occurred over time, and the initial 80:20 isomer ratio equilibrated at 60:40 over several days. Unfortunately, protection of the second hydroxyl position was slow enough that the mixture underwent some isomerization before full conversion. In one trial with 130 g starting material, only 1.5% of 5'-O-TIPS-8-bromoguanosine remained after 2 days, and the disilylated product had a 70:30 isomer ratio. Following extractive workup and evaporation, the resulting oil was subjected to crystallization trials. It was found that crystallization could be induced from several solvents, with complete desaturation requiring several days. MeOH was found to selectively crystallize the 3',5' isomer, while other solvents were found to be selective for the 2',5' isomer. In the crystallization of the 2',5'-isomer, only that isomer over-crystallized, leaving a near 1:1 isomer mixture in the mother liquor. As a result, >98% pure crystalline material was obtained in 33% yield. Encouraged by this, we attempted to improve on the product loss to the filtrate. Attempts to increase the crystallization yield by changing the solvent volume and adding anti-solvent were all futile. The yield increase was always at the expense of purity. A two-step crystallization was then attempted, first removing the undesired isomer by crystallization from MeOH, and after evaporation, recrystallizing the mother liquor residue from isopropyl acetate. This gave the desired isomer in a somewhat improved yield of 40%. Still, this two-step crystallization was not adopted on a scale because it was time consuming.A few attempts to find kinetic conditions under which the 3',5'-isomer would isomerize to the desired 2',5'-isomer upon crystallization (addition of DBU and TEA were tried) were unsuccessful. Neither isomerization nor crystallization occurred. The conclusion from the crystallization trials was that the regioselectivity of silylation must be improved to increase the isolated yield of 3. Increasing the rate of silylation without also increasing the rate of 2'→3' silyl transfer proved to be a challenge. Attempts included the use of other solvents (pyridine, NMP were tried), various bases, and increasing the reaction temperature. Extensive by-product formation was observed with the latter, and although several conditions were found that improved the isomer ratio at full conversion (see entry 3 in Table 1), the most impressive results came from doubling the amount of TIPS-Cl (entry 4 in Table 1). This encouraged the second silylation step to be completed after overnight without significant isomerization. Finally, crystallization of this isomeric mixture afforded 3 in 58% yield.

[0112] [Table 1]

[0113] This step was also explored as the first step in the synthesis since it gave the lowest yield. However, crystallization of the resulting crude was unsuccessful and required chromatography for isolation. Moreover, the strongly basic conditions of the subsequent PET step also caused some silyl migration, so we did not explore this sequence further.

[0114] Selective deprotection. We first investigated AcOH in H2O and TFA / H2O in THF. The starting material was insoluble in the first mixture, and the latter was found to give good results with complete conversion overnight under mild conditions. Still, we explored the effect of solvent, acid strength and concentration, and water content on the ratio of product 4 to perhydrolysis product 2 (see Table 2). Performing alcoholysis with MeOH instead of hydrolysis significantly slowed the conversion, but adding water to the mixture restored some of the reaction rate. Toluene and DCM (CH2Cl2) gave slightly higher reaction rates and lower by-product formation when compared to THF, but a biphasic system was formed and toluene did not dissolve the starting material as well as THF and DCM. DCM is advantageous as it continues to precipitate 2 and facilitates its removal. Finally, despite the poor solubility of the product in most solvents, only MeCN was able to crystallize it from the crude mixture to give the 2′-monosilyloxynucleoside 4 in 80% yield and >98% purity after three nights.

[0115] [Table 2]

[0116] Formation of H-phosphonate monoesters. Diphenyl phosphite (DPP, diphenyl H-phosphonate) is a common reagent for the formation of nucleoside H-phosphonate monoesters. It easily undergoes transesterification with alcohols and nucleosides in pyridine to form mixed phenyl H-phosphonate diesters (e.g., diester 7, Scheme 3). After hydrolysis of the phenyl moiety, the nucleoside monoesters are obtained in good yields. When DPP (1 equiv. to reduce the risk of bis-ester formation, since 4 has two free hydroxyls) was added to a solution of 4 in pyridine, in addition to the desired intermediate 7, several peaks at 0-15 ppm corresponding to the formation of 3',5'-cyclic H-phosphonates, 3'-mixed esters, or dinucleoside esters appeared in the 31P-NMR resonances. Because the system was too reactive, a less basic solvent mixture containing 5% pyridine in DCM was evaluated. With this system, clean formation of mixed ester 7 was seen in the 31P-NMR. Due to the slow conversion rate, we increased the amount of DPP to 3 equiv., giving complete conversion within 1 h without the formation of by-products. After 1 h, the mixed ester 7 was hydrolyzed by addition of water and Et3N to give the triethylammonium 5'H-phosphonate monoester 5. Extractive workup removed most of the residue from the excess DPP, and the resulting crude was screened for crystallization solvents. Solvent exchange into EtOAc crystallized the monoester 5 in 75% yield and >95% purity. [ka]

[0117] An alternative sequence was also briefly explored (Scheme 4). The disilyl intermediate 3 could also be phosphonylated at the 3'-OH to give the monoester 8. Acidic deprotection of the 5'-silyl group afforded the 2'-protected-3'-H-phosphonate 9. Since initial crystallization attempts of intermediate 8 did not work as well as hoped, we did not explore this sequence further. [ka]

[0118] H-phosphonate cyclization. We initially followed the known conditions of a 19:1 mixture of DCM / pyridine using pivaloyl chloride (Pv-Cl) as the coupling agent. In the first NMR spectrum recorded (after about 10 min), we found the S P :R P A 7:3 ratio between the two peaks at 0.1 ppm (1 JPH = 734 Hz) and 5.8 ppm (1 JPH = 704 Hz) was observed, consistent with the formation of a -cyclic H-phosphonate. Treatment of this mixture with elemental sulfur after 20 min gave the corresponding 7:3 ratio. P :S P A mixture of cyclic phosphorothioates was generated (configuration retention, 56.4 and 55.1 ppm, 3 JPH=18 and 24 Hz, respectively). [ka]

[0119] We also observed the isomerization behavior between the cyclic H-phosphonate diastereomers described by Stawinski's group; namely, ring closure left overnight produced the cyclic H-phosphonate in a 1:9 SP:RP ratio (which could produce sulfurized products in an undesired 1:9 RP:SP ratio), and the kinetic product formed early was the desired cyclic SP-H-phosphonate, and the equilibrium showed a preference for the undesired diastereomer. To preserve the desired kinetic product, we explored 2,6-lutidine and DCM, as both have been shown to reduce the rate of epimerization in contrast to pyridine. Ring closure performed in DCM in the presence of 5 equiv. of 2,6-lutidine gave the cyclic H-phosphonate in an initial 19:1 ratio of the desired diastereomer (after about 10 min). The reaction was slower in this system, requiring about 15 min to reach completion, but this was offset by the highly suppressed epimerization evidenced by the 15:1 SP:RP ratio still seen with the cyclic H-phosphonate after standing for 1 h. After 1 h, the reaction was sulfurized to give products in about a 10:1 ratio with the desired diastereomer predominating. Typically, the final sulfurization mixture contained 75-80% of the desired product (based on 31P-NMR), with the remainder being undesired diastereomers and small amounts of impurities in the phosphite region (>110 ppm) and product region (about 50 ppm), as well as a small amount of starting material. Diphenyl chlorophosphate (DPCP), one of the other commonly used coupling agents, was also tried, but gave the same diastereomeric ratio and offered no benefit. Large excesses of coupling agent should be avoided, especially in the case of DPCP, as some by-products become more evident. The use of 1.3 equiv. of Pv-Cl was found to be sufficient for complete conversion and also to give a reaction that was tolerant to adventitious water.

[0120] When sulfurizing SP:RP-H-phosphonates, we found that a 50% excess of sulfur and Et3N was sufficient. No other sulfurizing agents other than elemental sulfur were evaluated. The preferred sulfurization timing was determined by adding reaction aliquots to vials containing sulfur and Et3N at different times after the addition of Pv-Cl. After charging the sulfur, the unreacted intermediate was left for 45 minutes, and no longer time was associated with deterioration of the diastereomeric ratio. However, after the addition of sulfur / Et3N, the reaction mixture was stable for several nights. After extractive workup and solvent exchange to acetonitrile to precipitate the residual sulfur, filtration and evaporation gave the crude product as a viscous oil. Despite screening several solvents, crystallization of the product as the triethylammonium salt from this crude was unsuccessful. We were able to force precipitation of the product by addition of 1M HBr(aq) as an impure and amorphous solid that is easier to handle.

[0121] Deprotection of 2'-protected API. Deprotection using Et3N·3HF in MeCN, THF, dioxane, and EtOH as solvents proved to be beneficial. The 2'-silyl was removed without any observable side reactions, and we were pleased to find that the target cGMPS 1a precipitated continuously over a period of 3 days, with the mother liquor enriched with the unwanted diastereomer and other impurities. We chose to explore THF as the solvent, since it gave the least losses to the mother liquor. The crude product was a pale yellow crystalline salt, almost pure by LC. However, its 1H spectrum revealed the presence of two equivalents of triethylammonium, despite vacuum drying. 19F NMR showed a broad peak around -162 ppm indicating the presence of a fluoride complex, likely triethylammonium fluoride. Reslurrying in MeCN removed the fluoride, excess Et3N, as well as any color, and produced the product in crude form (approximately 1% of the undesired diastereomer). Ethanol was found to be a suitable recrystallization solvent, and the product was recovered as a crystalline hemiethanol solvate. This solvate was stable and remained intact despite vacuum drying, and calorimetric analysis showed no endotherm or sample mass loss when heated. The crude product was soluble in 20 volumes of boiling EtOH without apparent decomposition, making cooling recrystallization feasible. This afforded the target compound as a crystalline white powder with LC purity >99.9%. Confirmation of the correct structure and conformation was based on comparison of its spectroscopic data with those of previously prepared material.

[0122] 2.4 Experiments 8-Bromo-β-phenyl-1,N2-ethenoguanosine (2). 8-Bromoguanosine (683 g, 1.86 mol) was dissolved in DMSO (3.41 L, 5 vol.) and DBU (706 g, 2.5 eq., 4.64 mol). 2-Bromoacetophenone (443 g, 1.5 eq., 2.23 mol) was charged dropwise over 30 min to the reaction mixture held at approximately 25° C. The reaction was stirred for 1 h before the addition of concentrated acetic acid (334 g, 3 eq., 5.57 mol). The product was precipitated by the slow addition of water (2.7 L, 4 vol.) to the reaction mixture, filtered and washed with water (5.5 L, 8 vol.) followed by MeCN (5.5 L, 8 vol.). After drying under vacuum at 80 °C for 5 h, nucleoside 2 (613 g, 71.5%) was recovered as a white crystalline solid. MS (mH) m / z: 460.0257, found: 460.0259 (ES-).

[0123] 8-Bromo-β-phenyl-1,N2-etheno-2',5'-ditriisopropylsilyloxyguanosine (3). The starting nucleoside 2 (480 g, 0.99 mol) was suspended in DMF (2.4 L, 5 vol.). Imidazole (336 g, 5 eq., 4.93 mol) was added to the suspension followed by TIPS-Cl (951 g, 5 eq., 0.54 mmol), gradually dissolving the starting material. The mixture was stirred overnight and then quenched with water (178 mL, 10 eq., 9.87 mol), after which the organic phase, diluted with toluene (7.2 L, 15 vol.), was washed with water (3×2.4 L) and evaporated. i-PrOAc (2.4 L, 5 vol.) was added to the resulting crude oil and stirred overnight. The solid was filtered, washed with 1 cake volume (672 mL) of i-PrOAc and dried in vacuum at 35° C. overnight to give the diprotected nucleoside 3 (462.4 g, 58.4%) as a white crystalline solid. MS (mH) m / z: 772.2925, Actual value: 772.2950 (ES-).

[0124] 8-Bromo-β-phenyl-1,N2-etheno-2'-triisopropylsilyloxyguanosine (4). 2',5'-disilylated nucleoside 3 (461 g, 0.57 mol) was dissolved in DCM (4.61 L, 10 vol.). TFA (231 mL, 0.5 vol.) and water (922 mL, 2 vol.) were charged in a vessel and stirred overnight. The mixture was neutralized with 35% aq. ammonia (0.25 vol.) and perhydrolysis by-products were removed by filtering through packed celite and washing with DCM (922 mL, 2 vol.). The organic phase was washed with water (3×100 mL) and evaporated to give a crude solid that was resuspended in MeCN (3.23 L, 7 vol.) overnight. Filtration and washing of the solid with MeCN (461 mL, 1 vol.) followed by drying in vacuum at 35 °C overnight gave the monoprotected nucleoside 4 (257 g, 74.2%) as a white crystalline solid. MS (mH) m / z: 616.1591, found: 616.1615 (ES-).

[0125] Triethylammonium 8-bromo-β-phenyl-1,N2-etheno-2'-triisopropylsilyloxyguanosine-5'-H-phosphonic acid (5). Compound 4 (262 g, 0.42 mol) was dissolved in DCM (4.98 L, 19 vol.) and pyridine (262 mL, 1 vol.). Diphenyl phosphite (292 g, 3 eq. 1.25 mol) was charged and the reaction was mixed. After 2 h the reaction was quenched with water (262 mL, 1 vol.) and Et3N (262 mL, 1 vol.) and stirred for 1 h. The mixture was washed with water (2×2.5 L) and the organic phase was coevaporated with 2-propanol followed by ethyl acetate. The crude material was recrystallized from ethyl acetate (2.62 L, 10 vol.) and the solid was filtered and washed with ethyl acetate (524 mL, 2 vol.) to give the monoester 5 (312 g, 90.0%) as a white crystalline powder. MS (M-EtNH+) m / z: 680.1305, found: 680.1315 (ES-).

[0126] Rp-8-Bromo-β-phenyl-1,N2-etheno-2'-triisopropylsilyloxyguanosine-3',5'-cyclic monophosphorothioic acid (6). 2,6-Lutidine (199 g, 5 eq. 1.86 mol) was added to a solution of 5 (310 g, 0.37 mol) in DCM (6.2 L, 20 vol.), followed by pivaloyl chloride (67 mL, 1.5 eq. 0.56 mol). After stirring for 1 h, sulfur (18 g, 1.5 eq., 0.56 mol) was charged to the vessel, followed by triethylamine (56 g, 1.5 eq., 0.56 mol). After 1 h, the solution was washed with water (2 x 1.24 L) and the organic phase was evaporated. The residue was stirred in MeCN (1.55 L, 5 vol.) to precipitate sulfur as yellow crystals, which were filtered off. Hydrobromic acid (3.1 L, 10 vol., 1 M) was added to the resulting filtrate to precipitate a crude solid, which was filtered off, resuspended in MeCN (1.55 L, 5 vol.), filtered and washed with 1 cake volume of MeCN. Removal of the solvent residues gave the thiophosphoric acid 6 as a crude white solid, which was used in the following step. MS (mH) m / z: 694.0920, found: 694.0978 (ES-).

[0127] Triethylammonium Rp-8-bromo-β-phenyl-1,N2-ethenoguanosine-3',5'-cyclic thiophosphate (1a). Triethylamine trishydrofluoride (620 mL, 2 vol.) was charged to a solution of crude phosphorothioic acid 6 in THF (1.24 L, 4 vol.). The mixture was seeded, stirred, and allowed to precipitate for three nights. The solid was filtered off, then washed with THF (500 mL, 1.6 vol.) and resuspended in MeCN (930 mL, 3 vol.) for 1 h, then filtered and washed with 1 cake volume of MeCN to give crystalline 1a. This material was cooled recrystallized from 20 volumes of 99% EtOH, followed by filtration and washing with 1 cake volume of the same to give the target cGMPS 1a (126.5 g, 49.5%, 2 steps) as a white crystalline powder with a purity of >99.9%. MS (M-Et3NH+) m / z: 537.9586, Found: 537.9581 (ES-).

[0128] 2.5 Conclusion A six-step batch process for the preparation of cGMPS 1a was developed and upscaled without the use of chromatography or chiral auxiliaries. The use of an H-phosphonate approach allowed for the formation of the desired RP cyclic phosphorothioate in a 9:1 ratio relative to the undesired SP diastereomer 1b. A silyl protection strategy on the ribose was devised that could be optimized to give the desired 2',5'-disilylated nucleoside in an impressive 86:14 ratio relative to the 3',5'-isomer. Selective crystallization was possible at all key steps. The process afforded a total of 126 grams of crystalline material 1a with a purity of >99.9% (total yield 13.8%).

Claims

1. 1. A method for producing a cyclic guanosine-3',5'-monophosphate (cGMP) analog or a synthetic intermediate thereof, comprising: i) A guanosine analogue of general formula (I) or a salt thereof: 【Chemical 1】 (In the formula, h is H, halogen, or Q; X １ and X ２ are each independently selected from H or p′; p' is independently selected in each occurrence from a hydroxyl protecting group; R １ and R ２ are each independently H, —(CH ２ ) ｎ -H, -(CH ２ ) ｎ -C ３～９ Heterocyclyl, -(CH ２ ) ｎ -ar, and ar, where each occurrence of n is independently selected from 0, 1, 2, 3, or 4, or R １ and R ２ are taken together to form -CH=C(ar)- or -(CH ２ ) １～４ forming C(═O)—, ar is, independently at each occurrence, a 5- or 6-membered aromatic or heteroaromatic ring, preferably phenyl or 2-furanyl; ar is, independently at each occurrence, halogen, —OH, —SH, —NH ２ , -NO ２ , -OCH ３ , -CH ３ , -CH ２ CH ３ , -CH(CH ３ ) ２ , or -CF ３ and optionally fused in the second instance of ar to preferably form a naphthyl moiety, Q is -(CH ２ ) ｎ -S-(CH ２ ) ｎ -H, -S-(CH ２ ) ｎ -OH, -S-(CH ２ ) ｎ -NH ２ , -(CH ２ ) ｎ -O-(CH ２ ) ｎ -H, -O-(CH ２ ) ｎ -OH, -O-(CH ２ ) ｎ -NH ２ , —O—C(CH ３ ) ３ , —O—CH(CH ３ ) ２ , -(CH ２ ) ｎ -N(-[CH ２ ] ｎ H) ２ , —NH—(CH ２ ) ｎ NH ２ , —NH—(CH ２ ) ｎ -OH, -(CH ２ ) ｎ -Nc １ c ２ and c １ and c ２ together with the N to which they are attached form a 3- to 8-membered heterocycle, or １ is H and c ２ represents a 3- to 8-membered heterocycle, —(CH ２ ) ｎ -H, -N ３ , -CF ３ , -(CH ２ ) ｎ -ar, -O-(CH ２ ) ｎ -(ar), -NH-(CH ２ ) ｎ -(ar), -S-(CH ２ ) ｎ -(ar),-(CH ２ ) ｎ -amide-ar, -O-(CH ２ ) ｎ -amide-(ar), -NH-(CH ２ ) ｎ -amide-(ar), -S-(CH ２ ) ｎ -amide-(ar), or a linker moiety, wherein any -H may be optionally replaced by a halogen, and each occurrence of n is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, or 8. providing a ii) contacting the provided guanosine analog with a phosphorus oxoacid derivative to obtain a guanosine 5'-monophosphorus oxoacid ester analog; iii) isolating the resulting guanosine 5'-monophosphoric acid ester analogue by crystallization; A method comprising:

2. For the guanosine analogue of general formula (I) or a salt thereof used in step i), h is H or halogen or Q, preferably H or Br or Q, more preferably Br; X １ is H and X ２ is p', p' is selected from the group consisting of methoxymethyl (MOM), tetrahydropyranyl (THP), t-butyl (tBu), allyl (all), benzyl (Bn), (tri)alkylsilyl (e.g., t-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), or t-butyldiphenylsilyl (TBDPS)), acyl (e.g., acetyl (Ac), pivaloyl (Pv), or benzoyl (Bz)), preferably from the group consisting of THP, (tri)alkylsilyl, and acyl; R １ and R ２ together form —CH═C(ar)—, ar is phenyl, 4-methylphenyl, 3-thiophenyl, or 2-furanyl, preferably phenyl, and / or Q is furanyl, -CF ３ , -SCH ３ , -S(isopropylphenyl), -S(phenylamidomethyl), -S(halophenyl), -S(hydroxyphenyl), -S(aminophenyl), -S(nitrophenyl), -S(methoxyphenyl), -S(toluyl), -S(trifluoromethylphenyl), -Nc １ c ２ (where c １ and c ２ form a 3- to 8-membered heterocycle together with the N to which they are attached), —S—(CH ２ ) ｎ -OH, -S-(CH ２ ) ｎ -NH ２ , —NH—(CH ２ ) ｎ NH ２ , or -NH-(CH ２ ) ｎ OH, preferably furanyl, —CF ３ , —S(4-hydroxyphenyl), or —S(4-chlorophenyl); The method of claim 1.

3. For the guanosine analogue of general formula (I) or a salt thereof used in step i), h is Br; X １ is H and X ２ is p', p' is triisopropylsilyl (TIPS); R １ and R ２ together form -CH=C(ar)-, and ar is phenyl; The method of claim 1.

4. The method according to any one of claims 1 to 3, wherein the phosphorus oxoacid derivative in step ii) is a phosphorylating agent or a phosphonylating agent.

5. The phosphorus oxoacid derivative of step ii) is represented by the general formula (P): 【Chemistry 2】 (In the formula, M is S or O or absent; o １ and ２ are each independently a halogen, —O—C １～８ Hydrocarbon, —S—C １～８ Hydrocarbon, —NH—C １～８ Hydrocarbons, borano, methylborano, dimethylborano, cyanoborano, and -N(C １～８ hydrocarbons) ２ is selected from o ３ is H or o １ or as defined for o １ and ３ together, preferably C ２～１２ forming a chiral auxiliary that is a hydrocarbon) The method according to any one of claims 1 to 3, wherein

6. The guanosine 5'-monophosphorus oxoate ester analog obtained in step ii) is represented by the general formula (II) or a salt thereof: 【Chemistry 3】 (In the formula, h, X １ , X ２ , R １ , and R ２ is as defined in any one of claims 1 to 3, o １ and ３ are each independently —OH or as defined in claim 5; and M is S or O. The method according to any one of claims 1 to 3, wherein

7. X １ is H and X ２ is p', and the guanosine analogue of general formula (I) or a salt thereof is Ia) An unprotected guanosine analogue of general formula (pI) or a salt thereof: 【Chemistry 4】 (In the formula, h, R １ , and R ２ is as defined in any one of claims 1 to 3) providing a Ib) contacting the unprotected guanosine analog with a (tri)alkylsilyl halide to obtain a multiply protected guanosine analog, and optionally isolating the multiply protected guanosine analog by crystallization; Ic) selectively deprotecting the multiply protected guanosine analog to obtain a compound of general formula (I) １ is H and X ２ is p'); Id) the compound of the general formula (I) obtained above (wherein X １ is H and X ２ is p'), optionally isolating the guanosine analog of formula (I) by crystallization; The method according to any one of claims 1 to 3, provided by

8. iv) cyclizing the guanosine 5'-monophosphate oxoacid ester analog obtained in step ii) to obtain a cyclic guanosine-3',5'-monophosphate (cGMP) analog, wherein the cyclization is preferably carried out in the presence of a sterically hindered base; The method of any one of claims 1 to 3, further comprising:

9. The cGMP analogue is represented by the general formula (III) or a salt thereof: 【Chemistry 5】 (In the formula, h, X ２ , R １ , and R ２ is as defined in any one of claims 1 to 3, preferably X ２ is p' as defined in any one of claims 1 to 3, o ３ is as defined in claim 6, preferably o ３ is H) and The method comprises: v) contacting the cGMP analog with a sulfurizing agent to form a compound represented by the general formula (III) ３ is -SH or -SC １～１２ obtaining a thiolated cGMP analog of 9. The method of claim 8, optionally further comprising:

10. The cGMP analog of general formula (III) has the general formula (III-Rp): 【Chemistry 6】 (In the formula, h, X ２ , R １ , and R ２ is as defined in any one of claims 1 to 3, preferably X ２ is p' as defined in any one of claims 1 to 3, o ３ is as defined in claim 6, preferably o ３ is H) It is of Optionally, the thiolated cGMP analog has the general formula (III-Rp): (In the formula, h, X ２ , R １ , and R ２ is as defined in any one of claims 1 to 3, preferably X ２ is p' as defined in any one of claims 1 to 3, o ３ is -SH or -SC １～１２ a hydrocarbon, preferably —SH) 10. The method of claim 9, wherein

11. X ２ is p', and vi) X ２ deprotecting the protected hydroxyl moiety by vii) optionally triturating the deprotected cGMP analog; viii) optionally converting the deprotected cGMP analogue to a pharmaceutically acceptable salt, preferably the sodium salt; 11. The method of claim 9 or 10, further comprising:

12. A compound of general formula (II) or a salt thereof: 【Chemistry 7】 (In the formula, h, X １ , X ２ , R １ , and R ２ is as defined in any one of claims 1 to 3, o １ and ３ are each independently -OH or as defined in claim 5, preferably o ３ is H, or o １ and ３ together, preferably C ２～１２ forming a hydrocarbon chiral auxiliary, M is S or O; Preferably, the compound is a salt.

13. o ３ The compound of claim 12, wherein is H.

14. h is Br; X １ is H and X ２ is p', p' is preferably triisopropylsilyl (TIPS); R １ and R ２ together form —CH═C(ar)—, ar is phenyl; o １ is OH, o ３ is H, and M is S or O, preferably O; 14. A compound according to claim 12 or 13.

15. h is Br; X １ is H and X ２ is p', p' is preferably triisopropylsilyl (TIPS); R １ and R ２ together form —CH═C(ar)—, ar is 4-methylphenyl; o １ is OH, o ３ is H, and M is S or O, preferably O; 14. A compound according to claim 12 or 13.

16. 13. The compound of claim 12, wherein the compound is crystalline.

17. 14. The compound of claim 13, wherein the compound is crystalline.

18. 15. The compound of claim 14, wherein the compound is crystalline.

19. 16. The compound of claim 15, wherein the compound is crystalline.

20. The compound is represented by the general formula (II) (In the formula, h is H, X １ and X ２ together form an acetonide protecting group, R １ is H and R ２ is H or -CH ３ and o １ is OH, o ３ is H, and M is O) 14. The compound of claim 13, which is not of

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