CN106191172A

CN106191172A - Prepared by the enzyme process of cytosine nucleoside analogs

Info

Publication number: CN106191172A
Application number: CN201510226648.5A
Authority: CN
Inventors: M·帕斯卡尔吉拉波特; V·M·德隆赛勒托马斯; R·蒙蒂利亚阿雷瓦洛
Original assignee: Plasmia Biotech SL
Current assignee: Plasmia Biotech SL
Priority date: 2015-05-06
Filing date: 2015-05-06
Publication date: 2016-12-07

Abstract

The present invention relates to by use nucleoside phosphorylase especially pyrimidine-nucleoside phosphorylase (PyNPs) or the novel synthesis of the cytosine nucleoside analogs of the mixture of purine nucleoside phosphorylase (PNPs) and PyNPs.

Description

Enzymatic preparation of cytosine nucleoside analogues

Technical Field

The present invention relates to a novel enzymatic process for the preparation of cytosine Nucleoside Analogues (NAs), in particular for the industrial preparation of cytosine NAs active substances acting as pharmaceutically relevant antiviral and anticancer drugs, intermediates or prodrugs thereof.

Background

Nucleoside Analogues (NAs) are synthetic compounds structurally related to natural nucleosides. In its structural aspect, a nucleoside is composed of three key elements: (i) hydroxymethyl, (ii) heterocyclic nitrogenous base moieties, and (iii) furanose rings that in many instances appear to function to provide spacing of the hydroxymethyl and base in the correct orientation.

NAs are characterized by high structural diversity, as they exhibit various modified carbohydrate and/or aglycone fragments.

NAs are widely used as antiviral and antitumor agents. These molecules have been routinely synthesized by different chemical methods, which typically require time-consuming multistep processes involving protection-deprotection reactions of heterocyclic bases and/or pentose moieties to allow modification of naturally occurring nucleosides (Boryski j 2008.reactions of transduction in the nucleoside chemistry. Curr Org Chem 12: 309-. This time consuming multi-step process often results in low yields and increased costs. Indeed, chemical processes generally increase the difficulty of obtaining products with the correct stereoselectivity and regioselectivity, generating by-products as impurities (Condezo, L.A. et al 2007.Enzymatic synthesis of modified nucleosides, p.401-423.Biocatalysis in the pharmaceutical and Biotechnology industries CRC Press, Boca Raton, FL, Mikhalinopodio, I.A. 2007; Sinisterra, J.V. et al 2010.Enzyme-catalyzed synthesis of non-natural or modified nucleosides, p.1-25.Encyclopedia of biological: Technology, biological: biological, Cell and Cell Technology 2010. John & Cell technologies, Biotechnology & Biotechnology:. Biochemical industries, biological technologies and biological technologies, biological industries, John & biological technologies, biological industries, Biochemical industries, Process, biological technologies, biological industries, Biochemical industries. In addition, chemical methods involve the use of expensive and environmentally hazardous chemical reagents and organic solvents.

Enzymatic synthesis of nucleosides involves the use of enzymes that catalyze the condensation of heterocyclic bases and sugars, thus forming glycosidic linkages. In general, two different enzymes can be used: nucleoside analogs are prepared by base exchange (transglycosylation) of nucleoside phosphorylases (NPs or NPs) and N-2' -deoxyribotransferases (NDTs or NDT). NPs include, based on their substrate specificity for pentofuranosyl donors and nucleobase acceptors (nucleobasicacceptors): pyrimidine nucleoside phosphorylases (e.c.2.4.2.1), purine nucleoside phosphorylases (e.c.2.4.2.2), uridine nucleoside phosphorylases (e.c.2.4.2.3) and thymidine nucleosides (e.c. 2.4.2.4). However, cytosine and its nucleosides (cytidine, 2' -deoxycytidine) or modified cytosine analogues or derivatives and their corresponding nucleosides are not substrates for these NPs enzymes (mikharipulo, i.a et al 2010.New Trends in nucleotide Biotechnology, Acta Naturae, 2(5), 36-58).

In contrast, N-2' -deoxyribosyltransferase (E.C.2.4.2.6.) specifically catalyzes the transfer of deoxyribofuranosyl moieties between a nucleoside and an acceptor base without intermediate formation of deoxyribofuranosyl 2-phosphate. NDTs substrate specificity relates to strict specificity for the 2-deoxyribofuranose moiety, rather than to broad tolerance with respect to modified purines and good substrate activity for cytosine as an acceptor for 2-deoxy-and 2, 3-dideoxyribofuranose residues (mikhailaplolo, i.a et al 2010.New trends in Nucleoside Biotechnology, Acta Naturae, 2(5), 36-58). Freeco-Taboada et al (New observations on NDTs: a versatile biocatalysis for one-pot one-step synthesis of nucleoside analogs; New insights for NDTs: multifaceted biocatalysis for one-pot one-step synthesis of nucleoside analogs), appl.Microbiol.Biotechnol, 2013, 97, 3773-3785) disclose that cytosine is the optimal receptor for the 2' -deoxyribofuranose moiety in all cases.

Thus, the choice of NPs or NDTs depends on the structure of the acceptor base and the donor nucleoside. The complementary NPs and NDTs allow the biocatalytic preparation of natural nucleosides and their modified analogs, most of which are useful in the preparation of many anticancer and antiviral drugs.

However, neither NPs nor NDTs allow the preparation of cytosine ribonucleosides, as cytosine is not the acceptor base of NPs, while ribofuranosyl nucleoside donors are not substrates for NDTs. Thus, the preparation of cytosine nucleoside analogues remains a problem not only because of the above-mentioned substrate specificity but also because certain enzymes commonly present in the preparation of biocatalysts, such as cytosine and cytidine deaminases or cytosine and cytidine deacetylases, degrade the substrate or the end product, resulting in a non-productive process for industrial purposes.

Araki et al (EP1254959, US 7629457) disclose a method for preparing cytosine nucleoside compounds from pentose-1-phosphoric acid and cytosine or derivatives thereof using a nucleoside phosphorylase reactive to cytosine or a bacterium having the enzymatic activity. In particular, the inventors found that Purine Nucleoside Phosphorylase (PNP) derived from a bacterium belonging to the genus Escherichia (Escherichia) which itself should catalyze a reaction involving a purine base as a substrate in place of a pyridine base is capable of catalyzing the production of cytosine nucleosides from cytosine and pentose-1-phosphoric acid. However, it was found that the reaction of cytosine or a derivative thereof disclosed therein with pentose-1-phosphoric acid in the presence of an enzymatically active bacterium produces almost only a deamination product of cytosine or a derivative thereof, and thus the target cytosine nucleoside compound cannot be efficiently accumulated. To minimize the production of deaminated cytosine by-products, the cited patent additionally discloses a method for specifically reducing deaminase activity that degrades a substrate or end product by contacting an enzyme preparation with an organic solvent and heating the enzyme preparation at a temperature of 60 ℃ to 90 ℃ for an effective period of time.

Araki et al (US 2005/0074857) disclose a process for preparing pyrimidine nucleoside compounds and novel pyrimidine nucleoside compounds using pyrimidine base derivatives using an enzyme derived from a microorganism such as escherichia coli (e.coli) which is called Purine Nucleoside Phosphorylase (PNP), and which also has cytosine nucleoside phosphorylase activity. The authors overcome some deacetylation inactivation by heating the bacterial cells of the microorganism or treated enzymes or placing in contact with a suitable organic solvent.

The same authors disclose several methods for the preparation of the above compounds by producing microorganisms lacking both cytosine and cytidine deaminase genes without the application of complex deaminase-inactivating treatments (JP2004344029) or with the aid of zinc salts (JP 2004024086).

Ding q.2010. enzymic synthesis of nucleosides by nucleosides phosphorescenses co-expressed in Escherichia coli (enzymatic synthesis of nucleosides by nucleoside phosphorylases co-expressed in Escherichia coli), j.zheijiang Univ-Sci B (Biomed & Biotechnol)11 (11): 880-888 discloses the synthesis of many types of nucleosides. However, the disclosure fails to make cytosine or arabinoside nucleosides, thus avoiding the use of NPs for the synthesis of this particular type of nucleoside.

Szeker, k.2012. nucleotide phosphorylases from thermophiles, ph.d. academic paper, Berlin Institute of Biotechnology, Germany, discloses that the phospholytic activity of GtPyNP (NP from bacillus thermophilus) is determined for cytidine as a substrate, only the activity of TtPyNP (MP from Thermus thermophilus) is determined to be low (see page 84 above), and cytidine is poorly recognized as a substrate by PNPs and apap (5' -methylthioadenosine phosphorylase from aeromonas thermophilus (aeromonas thermophilus), see page 116 above).

Thus, biocatalytic synthesis of cytosine nucleoside analogs remains a problem and, as explained above, its application on an industrial scale is limited by product degradation due to competing enzymes present in the biocatalyst formulation.

Since many non-natural nucleosides acting as antiviral or anticancer agents contain cytosine as the nucleobase moiety, it is of interest to develop new and efficient industrial enzymatic processes that can overcome the previously mentioned disadvantages and improve the synthesis of this type of cytosine nucleoside analogs.

Capecitabine, a well-known anticancer drug with the chemical structure of cytosine NA, has been prepared in the past according to various methods described in the prior art for the multistep chemical synthesis of this NA (f. hoffmann-La Roche AG inventor, Arasaki et al, EP 0602454; Kamiya et al, EP 0602478).

The above methods suffer from certain disadvantages such as: the solvents or reagents used are toxic, require chromatographic purification steps (not suitable for large scale production), multi-step complex processes including protection and deprotection stages of the hydroxyl groups on the glycosidic subunits, and low to moderate overall yields of capecitabine synthesis. The addition of two additional steps to the protection and deprotection procedures in all processes reduces the overall yield from a commercial production point of view and increases the time and cost of the process.

In WO 2011/104540, a one-step process for the preparation of capecitabine is disclosed. The method is based on the reaction of 5-fluoro-5 '-deoxycytidine with a pentoxycarbonylation reagent, which may avoid the need for conventional protection of the hydroxyl group of the previously formed nucleoside 5-fluoro-5' -deoxycytidine. The reaction takes place in an organic solvent, giving capecitabine moderate yields (50 to 67%) using chemical catalysts (such as bases or corrosive inorganic acids as HCl gas), high temperatures (up to 110 ℃ under solvent reflux) and long reaction times (typically 9 to 10 hours).

The inventors have demonstrated that capecitabine suffers from thermal instability at high temperatures and loses up to 40% of the product after heating the product at neutral pH over 10 hours at 90 ℃. Thus, the process described in WO 2011/104540 suffers from severe losses of the final product.

In contrast, the enzymatic process of the present invention is carried out under milder conditions, using water as solvent and providing yields higher than 70%.

Kanan et al (WO 2010/061402) disclose the use of (CALB) Novozyme 435 (a lipase acrylic resin from Candida antartica) catalyst in the presence of an organic solvent. This method requires at least four steps to obtain capecitabine.

Thereafter, in light of the above disadvantages associated with the prior art and the importance of capecitabine in cancer therapy, there is a great need to develop an improved method for preparing such Active Principle Ingredients (API), which does not include multiple steps, uses relatively inexpensive reagents, and which would impart high yield and purity to nucleoside analog products.

The same results were obtained for cytarabine (cytarabine) synthesis. Like capecitabine, cytarabine is another well-known anticancer agent that also shares the chemical structure of cytosine NA. There are early patents in the name of Merck (NL 6511420 or Salt lake Institute of Biological Studies in 1964 (1969)) which already describe the chemical synthesis of the active compounds, which may also involve the same disadvantages involved with capecitabine.

Surprisingly, it was found that the previously cited drawbacks of biocatalytic synthesis can be avoided and that cytosine NAs can be obtained with a conversion higher than 70% and an anomeric purity higher than 95%. This is possible by applying suitable enzymatic methods based on the use of Nucleoside Phosphorylases (NPs) (purine nucleoside phosphorylases (PNPs), or pyrimidine nucleoside phosphorylases (PyNP), or mixtures of pyrimidine nucleoside phosphorylases (PyNP) and Purine Nucleoside Phosphorylases (PNP)). Surprisingly, the enzymes mentioned are able to recognize chemically modified cytosines properly and to carry out transglycosylation reactions on donor nucleoside analogues without any evidence of deamination or deacetylation impurities. The inventors have demonstrated that the same biocatalytic reaction, except using unmodified cytosine as the nucleoside acceptor, does not lead to the desired end product. For purposes of this specification, a preferred enzyme to be used in the method of the invention is a pyrimidine nucleoside phosphorylase. Another preferred embodiment of the invention is the use of a mixture of pyrimidine nucleoside phosphorylase (PyNP) and Purine Nucleoside Phosphorylase (PNP), especially when a purine nucleoside or derivative or analogue thereof is used as the starting compound.

Compared to the methods used in the prior art for the chemical synthesis of these NAs, and furthermore with respect to the enzymatic synthesis using CALB Novozyme 435, the method of the invention applied as an example to the preparation of capecitabine or cytarabine as cytosine NAs has several advantages, such as:

(i) the number of the steps is reduced, and the method,

(ii) the conversion rate and the yield are higher,

(iii) protection/deprotection strategies for the hydroxyl groups in the sugar are not required,

(iv) mild reaction conditions: environmental protection technology (water or aqueous medium, neutral pH),

(v) organic solvents are avoided in the enzymatic step,

(vi) excellent selectivity: stereoselectivity, enantioselectivity, chemical regioselectivity,

(vii) no side reaction: the impurity profile (reduced by-product content),

(viii) the generation of the total waste is reduced,

(ix) method of productivity

(x) Overall lower manufacturing costs

Furthermore, there are other additional advantages of the biocatalytic process of the present invention over the chemical processes used in the prior art. Of particular relevance is the absence in the process of the present invention of organic solvents used in the chemical processes of Arasaki et al and Kamiya et al, such as pyridine, Dichloromethane (DCM), Acetonitrile (ACN), methanol, Tetrahydrofuran (THF), or the enzymatic processes of Kannan et al (pyridine, Dichloromethane (DCM), etc.).

In particular, it is relevant that no metal catalysts such as tin chloride, toxic agents such as silylating agents, etc. are present in the process of the invention. All those process organic solvents and reagents must be removed or destroyed before any waste is discharged to the environment. Given that the processes described in the prior art involve multi-step manipulations (including protection and deprotection steps), an additional inconvenience of the processes described in the prior art relative to the manipulations of the present invention relative to the simpler enzymatic processes of the present invention is their complexity.

Description of the invention

The inventive method using PyNP or PyNP/PNP enzyme as described herein is outlined as follows:

scheme 1 enzymatic Synthesis of Cytosidine analogs

Applicants have surprisingly found that by using a nucleoside phosphorylase (a native, recombinant or mutein derived from a bacterium or archaea), and in particular a pyrimidine nucleoside phosphorylase (a native, recombinant or mutein derived from a bacterium or archaea), a suitable N⁴Modified cytosine or N⁴The modified cytosine derivatives allow the preparation/manufacture of cytosine nucleoside analogues with high conversion rates (greater than 70%) and complete avoidance of side reactions such as deamination or deacetylation as reported in the prior art cited above.

No evidence is found in the prior art to point to the fact that chemical modification/substitution/protection at this or any other position in the cytosine chemical backbone can alter the specificity of the substrate for nucleoside phosphorylases. For purposes of this specification, the term N⁴The modified cytosine/cytosine derivative is N⁴Substituted cytosine/cytosine derivatives or N⁴Protected cytosine/cytosine derivatives are synonyms.

For the purposes of this patent specification, the terms cytosine or cytosine derivative, nucleoside, intermediate, base or nucleobase should be understood as meaning all of them as compounds derived from the cytosine skeleton. For the purposes of the present invention, in particular as cytosine or cytosine derivative, are represented by the formula II:

suitable chemical modifications, object of the present invention, provide a more convenient, efficient and easier method for nucleoside analogue synthesis, which completely avoids the instability problems associated with side reactions. Particularly preferred is N⁴Modifications include amino modifications in the form of carbamates (-NHCOO-), more specifically carbamates attached to alkyl chains of 1 to 40 carbon atoms, alkenyl chains of 1 to 40 carbon atoms, or alkynyl chains of 1 to 40 carbon atoms (the chains in each case being straight, branched, or substituted with any other functional group), aryl groups, or heterocyclic rings. N of amino group in the form of amide or related acyl derivative (-NHCO-)⁴The modification also allows transglycosylation reactions, especially when the amide group is linked to an alkyl chain of 4 to 40 carbon atoms, an alkenyl chain of 1 to 40 carbon atoms or an alkynyl chain of 1 to 40 carbon atoms (the chains being in each case straight-chain, branched or substituted by any other functional group), an aryl group or a heterocyclic ring. Surprisingly, longer alkyl chains are preferred over short alkyl chains (1 to 3 carbon atoms) because, according to the experiments performed, deacetylation side reactions are also observed in short alkyl chains, unlike what is taught in US 2005/0074857. According to the present invention, the acyl group of the alkyl group having 1 carbon atom cannot prevent deacetylation.

More precise chemical modifications at N according to the present description⁴(nitrogen atom at position 4 in the cytosine heterocycle) to the cytosine backbone. More specifically, those chemical modifications made in the cytosine backbone are those made by R in formula II₂And/or R₃Chemical modification represented in part:

wherein

R¹Is O, CH₂、S、NH；

R²Is hydrogen, optionally substituted C_4-40An alkyl chain, an optionally substituted alkenyl chain, an optionally substituted alkynyl chain, an optionally substituted aryl linked to N through an optionally substituted alkyl, alkenyl or alkynyl chain, an optionally substituted heterocycle linked to N through an optionally substituted alkyl, alkenyl or alkynyl chain, COR⁶、CONR⁶R⁷、CO₂R⁶、C(S)OR⁷、CN、SR⁶、SO₂R⁶、SO₂R⁶R⁷CN, P (O) aryl, P (O) heterocycle, P (S) aryl, P (S) heterocycle, P (O) O₂R⁸；

R³Is hydrogen, optionally substituted C_4-40An alkyl chain, an optionally substituted alkenyl chain, an optionally substituted alkynyl chain, an optionally substituted aryl linked to N through an optionally substituted alkyl, alkenyl or alkynyl chain, an optionally substituted heterocycle linked to N through an optionally substituted alkyl, alkenyl or alkynyl chain, COR⁶、CONR⁶R⁷、CO₂R⁶、C(S)OR⁷、CN、SR⁶、SO₂R⁶、SO₂R⁶R⁷CN, P (O) aryl, P (O) heterocycle, P (S) aryl, P (S) heterocycle, P (O) O₂R⁸；R³And R²Independently of each other; and with the proviso that R₂Or R₃At least one of which is different from hydrogen.

R⁴Is hydrogen, OH, NH₂SH, halogen (preferably F or I); an optionally substituted alkyl chain; an optionally substituted alkenyl chain; optionally substituted alkynyl chain, trihaloalkyl, OR⁶、NR⁶R⁷、CN、COR⁶、CONR⁶R⁷、CO₂R⁶、C(S)OR⁶、OCONR⁶R⁷、OCO₂R⁶、OC(S)OR⁶、NHCONR⁶R⁷、NHCO₂R⁶、NHC(S)OR⁶、SO₂NR⁶R⁷An optionally substituted aryl linked to Y by an optionally substituted alkyl, alkenyl or alkynyl chain, an optionally substituted heterocycle linked to Y by an optionally substituted alkyl, alkenyl or alkynyl chain, andindependent R¹、R²、R³、R⁴Or R⁵Any of (a) or (b) is selected from an optionally substituted heterocycle or an optionally substituted aryl of:

provided that Y is a carbon or sulfur atom, and alternatively, R is provided that Y is a nitrogen atom⁴Is absent;

wherein

X is O, S, N-R^B2、Se；R^B1Is H, OH, NH₂Straight or branched chain C_1-10Alkyl, F, Cl, Br, I, X-R^B2、-C≡C-R^B2、CO₂R^B2；R^B2Is H, OH, NH₂Straight or branched chain C_1-5Alkyl, phenyl;

R⁵is hydrogen, OH, NH₂SH, halogen (preferably F OR I), optionally substituted alkyl chain, optionally substituted alkenyl chain, optionally substituted alkynyl chain, trihaloalkyl, OR⁶、NR⁶R⁷、CN、COR⁶、CONR⁶R⁷、CO₂R⁶、C(S)OR⁶、OCONR⁶R⁷、OCO₂R⁶、OC(S)OR⁶、NHCONR⁶R⁷、NHCO₂R⁶、NHC(S)OR⁶、SO₂NR⁶R⁷；CH₂-heterocycle, CN;

and independently R²、R³、R⁴Or R⁵Any of (a) or (b) is selected from an optionally substituted heterocycle or an optionally substituted aryl of:

wherein

X is O, S, N-R^B2、Se；R^B1Is H, OH, NH₂SH, straight-chain or branched C_1-10Alkyl, F, Cl, Br, I, X-R^B2、-C≡C-R^B2、CO₂R^B2；R^B2Is H, OH, NH₂Straight or branched chain C_1-5Alkyl, phenyl;

R⁶and R⁷Each independently is hydrogen, an optionally substituted alkyl chain, an optionally substituted alkenyl chain, an optionally substituted alkynyl chain, a heterocycle, or an optionally substituted aryl;

R⁸is hydrogen, optionally substituted alkyl chain, optionally substituted alkenyl chain, optionally substituted alkynyl chain, optionally substituted aryl, optionally substituted heterocycle;

y is C, N, S;

according to a comparative example carried out by the applicant and shown in the present specification, nucleoside phosphorylases (natural, recombinant or mutant enzymes), in particular pyrimidine nucleoside phosphorylases, are used, at N⁴Cytosine derivatives that do not contain suitable modifications do not undergo transglycosylation reactions and, therefore, do not yield the desired nucleoside end products.

Furthermore, unprotected cytosines (bases or corresponding nucleosides) undergo side reactions (such as deamination or deacetylation) which some authors have attempted to inactivate by conventional methods such as heating or the use of organic solvents (EP1254959 and US2005/0074857), the addition of zinc salts (JP2004024086) or by using more complex methods such as the use of microorganisms that delete both the cytosine deaminase gene and the cytidine deaminase gene simultaneously (JP 2004344029). All the technical solutions disclosed in the above prior art significantly reduce the overall yield of the corresponding synthesis process. The present invention provides a method for the synthesis of nucleoside analogues to fully and certainly solve the above mentioned technical problems related to the side reactions that reduce the overall yield of the synthetic methods described herein.

Can be prepared specifically according to eachChemical structure of the final product optionally removes N from the final product for modification/substitution/protection⁴Functional group of the site (deprotection of the final product). However, in some cases, such as for capecitabine molecules, the modified/substituted/protected cytosine N is not required⁴The group is deprotected because the modification is still attached to the amino group in the final product (or API). For capecitabine itself, the enzymatic process of the present invention greatly shortens the time required when using chemically conventional preparation methods.

Thus, the present invention provides an improved alternative synthetic method for nucleoside analogues useful as anticancer and/or antiviral products by shortening the conventional multi-step synthesis, increasing overall yield, reducing side reactions and by-product content, and thus improving product purity and quality.

For purposes of this specification, the following terms are further defined as follows.

The term "nucleoside" refers to all compounds in which a heterocyclic base is covalently coupled to a sugar, and particularly preferred couplings of a nucleoside to a sugar include Cl' - (glycoside) linkages of a carbon atom in the sugar to a carbon or heteroatom (typically nitrogen) in the heterocyclic base. Thus, in the context of the present text, the term "nucleoside" means a glycoside of a heterocyclic base. Similarly, the term "nucleotide" refers to a nucleoside in which a phosphate group is conjugated to a sugar.

The term "nucleoside" may be used broadly to include non-naturally occurring nucleosides, and other nucleoside analogs. Illustrative examples of nucleosides are ribonucleosides comprising a ribose moiety and deoxyribonucleosides comprising a deoxyribose moiety. With respect to the base of such a nucleoside, it is understood that it may be any naturally occurring base, such as adenine, guanine, cytosine, thymine, and uracil, as well as any modified variants thereof or any possible non-natural base.

The terms "nucleoside analogue (analogue)", "NA" or "NAs" as used herein refer to all nucleosides in which the sugar is preferably not ribofuranose or arabinofuranose and/or in which the heterocyclic base is not a naturally occurring base (e.g., A, G, C, T, I etc.).

As further used herein, the term "saccharide" refers to all carbohydrates and derivatives thereof, wherein particularly contemplated derivatives include deletions, substitutions, or additions of chemical groups or atoms in the saccharide. For example, specifically contemplated deletions include 2 ' -deoxy, 3 ' -deoxy, 5 ' -deoxy, and/or 2 ', 3 ' -dideoxy-sugars. Particularly contemplated substitutions include replacement of an oxygen on a ring with sulfur or methylene, or replacement of a hydroxyl group with a halogen, azido, amino, cyano, sulfhydryl, or methyl, and particularly contemplated additions include methylene phosphonate groups. Further contemplated sugars also include sugar analogs (i.e., non-naturally occurring sugars), and in particular carbocyclic ring systems. The term "carbocyclic ring system" as used herein refers to any molecule in which a plurality of carbon atoms form a ring, and in particularly contemplated carbocyclic ring systems, a ring is formed from 3, 4, 5, or 6 carbon atoms.

The term "chemoenzymatic synthesis" refers to a method of synthesis of a compound by a combination of chemical and biocatalytic steps. For the purposes of the present invention in particular, in a preferred embodiment of the invention, the sequence of reaction stages must be: 1) chemical modification of cytosine bases; 2) biocatalytic transglycosylation using the NP enzyme; 3) optionally cytosine-N in the final product⁴Further deprotection of (c).

The term "enzymatic synthesis" refers to a method of synthesis of a compound by means of a process comprising only biocatalytic steps by suitable enzymes (NPs). Thus, a further preferred embodiment of the synthetic process described herein is a completely biocatalytic process starting from cytosine derivatives, such as the previously mentioned cytosine derivatives represented by the general formula II, which have been prepared or are commercially available as cytosine derivatives per se, in particular in the N of the cytosine heterocycles according to the invention⁴Position shows R₂And/or R₃And thus step 1 of the above chemical modification of the cytosine skeleton is omitted.

The terms "heterocycle" or "heterocyclic base" or "nucleobase" are used interchangeably herein and refer to any compound in which multiple atoms form a ring through multiple covalent bonds, wherein the ring includes at least one atom other than a carbon atom. Particularly contemplated heterocyclic bases include 5-and 6-membered rings containing at least 1 to 4 heteroatoms each independently selected from nitrogen, oxygen, and sulfur as non-carbon atoms (e.g., imidazole, pyrrole, triazole, dihydropyrimidine). Further contemplated heterocycles may be fused (i.e., covalently bound) to another ring or heterocycle, and thus are referred to as "fused heterocycles" or "fused heterocyclic bases" as used herein. Particularly contemplated fused heterocycles include 5-membered rings fused to a 6-membered ring (e.g., purine, pyrrolo [2, 3-d)]Pyrimidine), and a 6-membered ring fused with another 6-or more-membered ring (e.g., pyrido [4, 5-d ]]Pyrimidine, benzodiazepine □). Examples of these and more preferred heterocyclic bases are given below. It is further contemplated that the heterocyclic base may be aromatic or may contain one or more double or triple bonds. In addition, contemplated heterocyclic bases and fused heterocycles may also be substituted at one or more positions. And any one ring optionally substituted with one, two or three substituents is each independently selected from the group consisting of: halogen, hydroxy, nitro, cyano, carboxy, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy radical C_1-6Alkyl radical, C_1-6Alkylcarbonyl, amino, mono-or di-C_1-6Alkylamino, azido, mercapto, polyhaloC_1-6Alkyl, polyhalo C_1-6Alkoxy, and C_3-7A cycloalkyl group.

The term "nucleobase" encompasses naturally occurring nucleobases as well as non-naturally occurring nucleobases. It will be clear to the skilled person that a plurality of nucleobases which had previously been considered "non-naturally occurring" have subsequently been found in nature. Thus, "nucleobases" includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogs (e.g., N-substituted heterocycles) and tautomers thereof. Illustrative examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, purine, xanthine, 2-chloroadenine, 2-fluoroadenine, (5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamic acid pentyl ester, cytosine N-alkylcarbamate, cytosine N-alkyl ester, 5-azacytosine, 5-bromovinyluracil, 5-fluorouracil, 5-trifluoromethyluracil, 6-methoxy-9H-purin-2-amine and (R) -3, 6, 7, 8-tetrahydroimidazo [4, 5-d ] [1, 3] diazepin □ -8-ol.

The term "nucleobase" is intended to encompass each and all of these examples as well as their analogs and tautomers, and regioisomers. To distinguish these "nucleobases" from other heterocyclic bases also present in the specification, for purposes of this specification, the term "nucleobase" refers primarily to the cytosine base represented by formula II. However, also for purposes of this specification, the term "base" refers primarily to the base present in the nucleoside represented by formula III.

The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert through a low energy barrier. For example, proton tautomers (also referred to as prototropic tautomers) include interconversion by proton migration, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversion via rearrangement of some of the bonding electrons.

The term "regioisomer" in the sense of referring to molecules of the same formula whose atoms are joined in different orders of attachment refers to structural isomers, or constituent isomers.

The term "conversion" refers to the percentage of starting material that is converted to product (the intended end product, by-product, or even degradation product).

The term "yield" is the number of molecules of the synthesized product/number of starting molecules. In a multi-step synthesis, the yield can be calculated by multiplying the yields of all the individual steps.

The term "anomeric purity" refers to the amount of a particular anomer of a compound divided by the total amount of all anomers of that compound present in the mixture, multiplied by 100%.

The term "cytosine modification/substitution/protection" refers to a modification in which at least one position (other than the nitrogen at position 1) on the original cytosine backbone is functionalized, as described for the group R¹、R²、R³、R⁴、R⁵Any nucleoside analogue of those substituted for X and/or Y (each of these groups being independent of the other).

The term "in position N⁴By cytosine modification/substitution/protection "is meant a group in which at least one proton of the amino group at position 4 is replaced by a functional group, as described for the group R₂And/or R₃Each of these substituents being independent of the other, provided that at least one of the above substituents is different from hydrogen.

The term "intermediate" or "intermediates" refers to any nucleoside analogue type compound that can be converted to the Active Pharmaceutical Ingredient (API) of nucleoside structure by means of a suitable additional chemical reaction. Thus, an intermediate is a molecule that can be considered a precursor of an API. For purposes of this specification, when applied to N⁴The term "precursor" when referring to modified cytosine or cytosine derivatives or intermediates means a compound of formula II wherein N is substituted⁴The combined moiety being other than R²Or R³Chemical groups other than those indicated.

The term "prodrug" as used throughout this document means pharmacologically acceptable derivatives such as esters, amides, carbamates and phosphates, whereby the in vivo biotransformation product of the resulting derivative is the active drug as defined in the compound of formula (I). Goodman and Gilman generally describe The prodrug reference (The Pharmacological Basis of therapy, 8 th edition, McGraw-Hill, int. Ed.1992, "Biotransformation of drugs", p 13-15) incorporated herein. The prodrugs preferably have excellent aqueous solubility, increased bioavailability, and are readily metabolized in the body to active inhibitors. Prodrugs of the compounds of the present invention may be prepared by modifying functional groups present in the compounds in a manner that cleaves the modification into the parent compound by conventional manipulation or in vivo.

Preferred prodrugs are pharmaceutically acceptable esters, amides and carbamates of those compounds having hydroxy or amino groups which are hydrolyzable in vivo and are derived from formula I. Esters, amides and carbamates that are hydrolyzable in vivo are ester, amide or carbamate groups that hydrolyze in the human or animal body to produce the parent acid or alcohol. Suitable pharmaceutically acceptable esters, amides and carbamates of amino groups include C, which may be formed at any carboxyl group in the compounds of the present invention_1-6Alkoxymethyl esters, e.g. methoxymethyl, C_1-6Alkanoyloxymethyl esters, e.g. pivaloyloxymethyl, phthalidyl, C_3-8Cycloalkoxy-carbonyloxy group C_1-6Alkyl esters such as 1-cyclohexylcarbonyloxyethyl; 1, 3-dioxolan-2-oxomethyl ester such as 5-methyl-1, 3-dioxolan-2-oxomethyl; and C_1-6Alkoxycarbonyloxyethyl esters such as 1-methoxycarbonyl-oxyethyl.

The in vivo hydrolysable esters of the hydroxy group containing compounds of formula (I) include inorganic esters such as phosphate esters and α -acyloxyalkyl ethers and related compounds, which as a result of in vivo hydrolysis, decompose to give the parent hydroxy group. Examples of α -acyloxyalkyl ethers include acetoxymethoxy and 2, 2-dimethylpropionyloxy-methoxy. The choice of in vivo hydrolysable esters forming the group for the hydroxyl group include alkanoyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl groups, alkoxycarbonyl (to give an alkyl carbonate), dialkylcarbamoyl and N- (dialkylaminoethyl) -N-alkylcarbamoyl (to give a carbamate), dialkylaminoacetyl and carboxyacetyl groups. Examples of substituents on benzoyl include morpholinyl and piperazinyl linked from the ring nitrogen atom via a methylene group to the 3-or 4-position of the benzoyl ring.

For therapeutic use, salts of the compounds of formula (I) are those in which the counter-ion is pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable may also find use, for example, in the preparation or purification of pharmaceutically acceptable compounds. All salts, whether pharmaceutically acceptable or not, are included within the scope of the invention.

The pharmaceutically acceptable acid and base addition salts as described above are intended to include the therapeutically active non-toxic acid and base addition salt forms which the compounds of formula (I) are capable of forming. The pharmaceutically acceptable acid addition salts can be conveniently obtained by treating the base form with such a suitable acid. Suitable acids include, for example, inorganic acids such as hydrohalic acids, e.g., hydrochloric or hydrobromic acids, sulfuric, nitric, phosphoric and the like; or organic acids such as, for example, acetic, propionic, glycolic, lactic, pyruvic, oxalic (i.e., oxalic), malonic, succinic (i.e., succinic), maleic, fumaric, malic (i.e., malic), tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.

Instead, the salt form can be converted to the free base form by treatment with a suitable base.

The compounds of formula (I) containing acidic protons may also be converted into their non-toxic metal or amine addition salt forms by treatment with suitable organic and inorganic bases. Suitable base salt forms include, for example, ammonium salts, alkali and alkaline earth metal salts such as lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases such as benzathine (benzathine), N-methyl-D-glucamine, hydrabamine (hydrabamine) salts, and salts with amino acids such as, for example, arginine, lysine and the like.

The term addition salt as used hereinbefore also includes solvates which the compounds of formula (I) and salts thereof are able to form. Such solvates are for example hydrates, alcoholates and the like.

The term "quaternary amine" as used hereinbefore defines a quaternary ammonium salt of a compound of formula (I) formed by reaction between the basic nitrogen of the compound of formula (I) and a suitable quaternising agent such as, for example, an optionally substituted alkyl halide, aryl halide, arylalkyl halide, for example methyl iodide or benzyl iodide. Other reactants having good leaving groups, such as alkyl triflates, alkyl mesylates, and alkyl p-toluenesulfonates, may also be used. Quaternary amines have positively charged nitrogen. Pharmaceutically acceptable counterions include chloro, bromo, iodo, trifluoroacetate and acetate. The selected counter ion can be introduced using an ion exchange resin.

The N-oxide forms of the compounds of the invention are intended to include compounds of formula (I) wherein one or more nitrogen atoms are oxidized to the so-called N-oxide.

It will be appreciated that the compounds of formula (I) may have metal binding, chelating, complex forming properties and may therefore be present as metal complexes or metal chelates. Such metallated derivatives of the compounds of formula (I) are intended to be included within the scope of the present invention.

Some compounds of formula (I) may also exist in their tautomeric forms. Such forms, although not explicitly indicated in the above formula, are intended to be included within the scope of the present invention.

The term "alkyl" as used herein, refers to virtually any straight, branched, or cyclic hydrocarbon in which all carbon-carbon bonds are single bonds.

The terms "alkenyl" and "unsubstituted alkenyl" are used interchangeably herein and refer to any straight, branched, or cyclic alkyl group having at least one carbon-carbon double bond.

Furthermore, the term "alkynyl", as used herein, refers to virtually any straight, branched or cyclic alkyl or alkenyl group having at least one carbon-carbon triple bond.

The term "aryl" as used herein, refers to virtually any aromatic cyclic alkenyl or alkynyl groupAs a group or part of a group is phenyl or naphthyl, each of which is optionally substituted with one, two or three substituents selected from: halogen, hydroxy, nitro, cyano, carboxy, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy radical C_1-6Alkyl radical, C_1-6Alkylcarbonyl, amino, mono-or di-C_1-6Alkylamino, azido, mercapto, polyhaloC_1-6Alkyl, and polyhaloC_1-6An alkoxy group. In the case of an aryl group covalently bonded to an alkyl, alkenyl, or alkynyl group, the term "alkaryl" is employed.

The term "substituted" as used herein refers to an atom or chemical group (e.g., H, NH)₂Or OH) by a functional group, and in particular, by a desired functional group comprising a nucleophilic group (e.g., -NH)₂-OH, -SH, -NC and the like), electrophilic groups (e.g., C (O) OR, C (X) OH and the like), polar groups (e.g., -OH), nonpolar groups (e.g., aryl, alkyl, alkenyl, alkynyl and the like), ionic groups (e.g., -NH₃ ⁺) And halogen (e.g., -F, -Cl), and all chemically reasonable combinations thereof. Thus, the term "functional group" and the term "substituent" are used interchangeably herein and refer to a nucleophilic group (e.g., -NH₂-OH, -SH, -NC, -CN, etc.), electrophilic groups (e.g., C (O) OR, C (X) OH, C (halogen) OR, etc.), polar groups (e.g., -OH), nonpolar groups (e.g., aryl, alkyl, alkenyl, alkynyl, etc.), ionic groups (e.g., -NH)₃ ⁺) And a halogen.

For purposes of this specification, the term "nucleoside phosphorylase" or "NP" or "NPs" includes pyrimidine nucleoside phosphorylase or pynp (PyNPs) enzymes or mixtures of PyNPs with PNPs (purine nucleoside phosphorylase). Those enzymes can be obtained from naturally occurring microorganisms (mesophilic, thermophilic, hyperthermophilic or extreme thermophilic). For purposes of this specification, biological or NPs enzymes, mesophilic organisms or mesophilic are those capable of exerting or effecting NP activity at temperatures in the range of 18 to 60 ℃ (with an optimal temperature range of 40-55 ℃). Biological or NPs enzymes, thermophilic organisms or thermophilic are those capable of exerting or achieving NP activity at temperatures in excess of 60 ℃ and up to 80 ℃. Biological or NPs enzymes, hyperthermophilic organisms or hyperthermophilic are those capable of exerting or achieving NP activity at temperatures in excess of 80 ℃ and up to 100 ℃ (optimal temperature range 80-95 ℃). Furthermore, the enzyme used in the present invention may be a cloned enzyme obtained by genetic recombination techniques and expressed in a host cell transformed with a corresponding vector carrying the respective encoding nucleic acid sequence.

The nucleic acid molecule encoding the NP enzyme according to the invention is preferably selected from: SEQ ID No.1, 2, 5, 7, 9 or 11; or

a) As SEQ id no: 1.2, 5, 7, 9 or 11; or

b) And SEQ id no: 1.2, 5, 7, 9 or 11; or

c) A nucleotide sequence that hybridizes under high stringency conditions with: no: 1.2, 5, 7, 9 or 11; no: 1.2, 5, 7, 9 or 11; or from SEQ id no: 1.2, 5, 7, 9 or 11; or their complements; or

d) And SEQ id no: 1.2, 5, 7, 9 or 11 has at least 80% sequence identity;

e) and SEQ id no: 1.2, 5, 7, 9 or 11 has at least 59% sequence identity;

f) encoding a polypeptide selected from SEQ id no: 3. 4, 6, 8, 10 or 12.

In the sense of the present invention, the conditions for stringent hybridization are as defined by those described in Sambrook et al, Molecular Cloning, A Laboratory Manual (Molecular Cloning, A Laboratory Manual), Cold spring harbor Laboratory Press (1989), 1.1011.104. Accordingly, hybridization under stringent conditions means that a positive hybridization signal is still observed after one hour of washing with 1x SSC buffer and 0.1% SDS at 55 ℃, preferably at 62 ℃ and most preferably at 68 ℃, especially after one hour of washing in 0.2x SSC buffer and 0.1% SDS at 55 ℃, preferably at 62 ℃ and most preferably at 68 ℃.

Furthermore, the invention also encompasses, in the sense of the present specification, nucleotide or amino acid sequences which have, at the nucleotide or amino acid level, a sequence which is identical to SEQ ID No.1 or SEQ ID NO: 2 (nucleotide) or SEQ ID No.3 or SEQ ID NO: 4 (amino acid), particularly preferably at least 80% and most preferably at least 90% identity. Percent identity is determined according to the following equation:

I＝(n/L)x 100

wherein I is the percent identity, L is the length of the base sequence and n is the number of sequence differences in nucleotides or amino acids from the base sequence.

TABLE-1. purine nucleoside phosphorylase (deoD) [ sulfolobus solfataricus ] protein sequence identity to a similar PNP protein in GenBank (http:// www.ncbi.nlm.nih.gov/GenBank /) 15.11.2013.

TABLE-2. identity of the uridine phosphorylase [ Aeropyrum pernix (Aeropyrum pernix) K1] protein sequence to a similar UDP protein in GenBank (http:// www.ncbi.nlm.nih.gov/GenBank /) 15.11.2013.

Login number	Biological organisms	Identity%
			NP_148386.2	Aeropyrum pernix (Aeropyrum pernix) K1(SEQ ID NO: 4)	100％
YP_008604720.1	Aeropyrum camini SY1	93％

A further subject of the present invention is a recombinant vector comprising at least one copy of a nucleic acid molecule as defined above, operatively linked to an expression control sequence. The vector may be any prokaryotic or eukaryotic vector. Examples of prokaryotic vectors are chromosomal vectors such as phages (e.g.lambda phage) and extrachromosomal vectors such as plasmids (see, e.g.Sambrook et al, supra, chapters 1-4). The vector may also be a eukaryotic vector, such as a yeast vector or a vector suitable for use in higher order cells, such as a plasmid vector, a viral vector or a plant vector. Suitable eukaryotic vectors are described, for example, by Sambrook et al, supra, chapter 16. The invention furthermore relates to recombinant cells transformed with a nucleic acid or a recombinant vector as described above. The cell may be any cell, e.g., prokaryotic or eukaryotic. Prokaryotic cells, in particular E.coli cells, are particularly preferred.

For purposes of this specification, the invention also encompasses SEQ ID NOs: 1 to SEQ ID NO: 12, or orthologs (orthologs).

The term "variant" as used throughout the specification should be understood to mean a nucleotide sequence of a nucleic acid or an amino acid sequence of a protein or polypeptide that alters one or more nucleotides or amino acids, respectively. Variants may have "conservative" changes, where a substituted nucleotide or amino acid has similar structural or chemical properties as the substituted nucleotide or amino acid. Variants may also have "non-conservative" changes or deletions and/or insertions of one or more nucleotides or amino acids. The term also includes within its scope any nucleotide or amino acid insertion/deletion for a particular nucleic acid or protein or polypeptide. "functional variant" is understood to mean a variant which retains the function of the nucleotide sequence or protein or polypeptide of reference.

The term "complement" or "complementary" as used herein may mean that each strand of 20 double stranded nucleic acids, such as DNA and RNA, is complementary to the other strand in that the base pairs between them are non-covalently linked via two or three hydrogen bonds. For DNA, the adenine (a) base is complementary to the thymine (T) base, and vice versa; the guanine (G) base is complementary to the cytosine (C) base, and vice versa. The same is true for RNA, except that the adenine (A) base is complementary to the uracil (U) base, rather than the thymine (T) base. Since there is only one 25 complementary base for each base found in DNA and in RNA, one can reconstruct the complementary strand for any single strand.

The term "ortholog" as used throughout the specification should be understood as a homologous gene or miRNA sequence found in different species.

A "recombinant DNA molecule" is understood in the present specification as a DNA molecule which has been subjected to molecular biological treatment. Thus, the term "recombinant DNA" is a form of non-naturally occurring DNA that is created by combining DNA sequences that do not normally occur together.

Once introduced into a host cell, the "recombinant DNA molecule" is replicated by the host cell. By "recombinant protein or enzyme" is meant herein a protein or enzyme prepared by a process employing a "recombinant DNA molecule", and thus, also for the purposes of this specification, the term recombinase or recombinant enzyme should be understood as a protein or enzyme derived from recombinant DNA.

For the purposes of the present specification, mutant enzyme means any enzyme which exhibits at least one mutation, either naturally occurring or induced by artificial treatment including genetic engineering.

In contrast, for purposes of this specification, a native, natural or naturally occurring enzyme, taken as a synonym in its entirety, is intended to mean an enzyme that retains its previous amino acid sequence, as it is found to be generally universal in nature without mutations.

By functional part of the enzyme of the invention is meant any sequence fragment of the original enzyme, or a DNA fragment encoding it, which retains the ability of the complete enzyme sequence to exert its full biocatalytic properties.

For the purposes of this specification, the term cytosine nucleoside analogue means a nucleoside analogue of cytosine or a nucleoside analogue of a cytosine derivative. Likewise, limited to the present patent specification, the term cytosine base means the base cytosine or a derivative thereof. Similarly, as used in the specification, the term modified cytosine base encompasses compounds represented by formula II wherein the backbone of the cytosine base is substituted at position 4, 5 or 6.

The present specification discloses enzymatic processes for the preparation of cytosine nucleoside analogues, intermediates or prodrugs thereof, which are particularly useful as Active Pharmaceutical Ingredients (APIs), those APIs or intermediates thereof being Nucleoside Analogues (NAs) of formula I:

wherein,

Z₁the method comprises the following steps: o, CH₂、S、NH；

Z₂Independently of Z₁Comprises the following steps: o, C (R)^S2R^S5)、S(R^S2R^S5)、S(R^S2)、S(R^S5) Preference is given to the radicals SO or SO₂；N(R^S2R^S5)、N(R^S2)、N(R^S5)；

R^S1The method comprises the following steps: hydrogen, OH, an ether or ester thereof selected from:

n is 0 or 1, a is oxygen or nitrogen, and each M is independently hydrogen, an optionally substituted alkyl chain, an optionally substituted alkenyl chain, an optionally substituted alkynyl chain, an optionally substituted aryl optionally linked to P by an optionally substituted alkyl, alkenyl or alkynyl chain, an optionally substituted heterocycle optionally linked to P by an optionally substituted alkyl, alkenyl or alkynyl chain, or a pharmaceutically acceptable counterion such as, but not limited to, sodium, potassium, ammonium or alkylammonium;

R^S2is hydrogen, OH or an ether or ester residue thereof, halogen (preferably F), CN, NH₂、SH、C≡CH、N₃；

In the case of NA from 2' -deoxyribonucleoside or arabinonucleoside, R^S3Is hydrogen, or R when NA is from a ribonucleoside^S3Selected from OH and NH₂Halogen (preferably F), OCH₃；

R^S4Is hydrogen, OH or an ether or ester residue thereof, NH₂Halogen (preferably F), CN;

with the proviso that R is when both are ethers or esters of OH residues^S1And R^S4Is different;

when Z is₂When different from oxygen, R^S5Is hydrogen, OH or an ether or ester residue thereof, NH₂Or halogen (preferably F);

R¹is O, CH₂、S、NH；

R²Is hydrogen, an optionally substituted alkyl chain (preferably C)_4-40Alkyl chain), an optionally substituted alkenyl chain, an optionally substituted alkynyl chain, an optionally substituted aryl connected to N by an optionally substituted alkyl, alkenyl or alkynyl chain, byOptionally substituted heterocycle with optionally substituted alkyl, alkenyl or alkynyl chain linked to N, COR⁶、CONR⁶R⁷、CO₂R⁶、C(S)OR⁷、CN、SR⁶、SO₂R⁶、SO₂R⁶R⁷CN, P (O) aryl, P (O) heterocycle, P (S) aryl, P (S) heterocycle, P (O) O₂R⁸；

R³Is hydrogen, an optionally substituted alkyl chain (preferably C)_4-40Alkyl chain), an optionally substituted alkenyl chain, an optionally substituted alkynyl chain, an optionally substituted aryl linked to N through an optionally substituted alkyl, alkenyl or alkynyl chain, an optionally substituted heterocycle linked to N through an optionally substituted alkyl, alkenyl or alkynyl chain, COR⁶、CONR⁶R⁷、CO₂R⁶、C(S)OR⁷、CN、SR⁶、SO₂R⁶、SO₂R⁶R⁷CN, P (O) aryl, P (O) heterocycle, P (S) aryl, P (S) heterocycle, P (O) O₂R⁸；R³And R²Independently of each other; and with the proviso that R₂Or R₃At least one of which is different from hydrogen.

R⁴Is hydrogen, OH, NH₂SH, halogen (preferably F or I); an optionally substituted alkyl chain; an optionally substituted alkenyl chain; optionally substituted alkynyl chain, trihaloalkyl, OR⁶、NR⁶R⁷、CN、COR⁶、CONR⁶R⁷、CO₂R⁶、C(S)OR⁶、OCONR⁶R⁷、OCO₂R⁶、OC(S)OR⁶、NHCONR⁶R⁷、NHCO₂R⁶、NHC(S)OR⁶、SO₂NR⁶R⁷Optionally substituted aryl linked to Y by an optionally substituted alkyl, alkenyl or alkynyl chain, optionally substituted heterocycle linked to Y by an optionally substituted alkyl, alkenyl or alkynyl chain, and independently R¹、R²、R³、R⁴Or R⁵Any of (A) is selected from the group consisting of optionally substituted heterocyclic ring orOptionally substituted aryl:

provided that Y is a carbon or sulfur atom, and alternatively, R⁴Is absent, provided that Y is a nitrogen atom;

wherein

and independently R¹、R²、R³、R⁴Or R⁵Any of (a) or (b) is selected from an optionally substituted heterocycle or an optionally substituted aryl of:

wherein

y is C, N, S;

wherein when the chemoenzymatic preferred embodiment of the process of the present invention is employed, said process for the preparation of a cytosine nucleoside analogue comprises the steps of:

(i) reacting cytosine bases with N for modification in cytosine derivatives⁴Suitable reagents for the amino group at the position are chemically reacted to add the substituent R in the intermediate compounds described as formula II²And R³(ii) said intermediate compound is optionally purified at the end of step (i) by conventional purification methods;

alternatively, the process of the invention can be started directly from a starting product represented by formula II (modified cytosine base), which is either commercially available or previously synthesized.

(ii) Reacting the above-described modified cytosine base with a suitable nucleoside analogue starting material, wherein the reaction comprises adding a Nucleoside Phosphorylase (NPs), preferably a pyrimidine nucleoside phosphorylase (PyNP), to a mixture of starting materials comprising a cytosine derivative of formula II and a nucleoside analogue of formula III in a suitable aqueous reaction medium and under suitable reaction conditions

Wherein,

Z₁is O, CH₂、S、NH；

R^SiIs hydrogen, OH, an ether or ester thereof selected from:

at Z₂In the case of being different from oxygen, R^S5Is hydrogen, OH or an ether or ester residue thereof, NH₂Or halogen (preferably F);

R¹is O, CH₂、S、NH；

R²Is hydrogen, an optionally substituted alkyl chain (preferably C)_4-40Alkyl chain), an optionally substituted alkenyl chain, an optionally substituted alkynyl chain, an optionally substituted aryl linked to N through an optionally substituted alkyl, alkenyl or alkynyl chain, an optionally substituted heterocycle linked to N through an optionally substituted alkyl, alkenyl or alkynyl chain, COR⁶、CONR⁶R⁷、CO₂R⁶、C(S)OR⁷、CN、SR⁶、SO₂R⁶、SO₂R⁶R⁷CN, P (O) aryl, P (O) heterocycle, P (S) aryl, P (S) heterocycle, P (O) O₂R⁸；

R³Is hydrogen, an optionally substituted alkyl chain (preferably C)_4-40Alkyl chain), an optionally substituted alkenyl chain, an optionally substituted alkynyl chain, an optionally substituted aryl linked to N through an optionally substituted alkyl, alkenyl or alkynyl chain, an optionally substituted heterocycle linked to N through an optionally substituted alkyl, alkenyl or alkynyl chain, COR⁶、CONR⁶R⁷、CO₂R⁶、C(S)OR⁷、CN、SR⁶、SO₂R⁶、SO₂R⁶R⁷CN, P (O) aryl, P (O) heterocycle, P (S) aryl, P (S) heterocycle, P (O) O₂R⁸；R³And R²Independently of each other; and with the proviso that R₂Or R₃Is different from hydrogen;

R⁴is hydrogen, OH, NH₂SH, halogen(preferably F or I); an optionally substituted alkyl chain; an optionally substituted alkenyl chain; optionally substituted alkynyl chain, trihaloalkyl, OR⁶、NR⁶R⁷、CN、COR⁶、CONR⁶R⁷、CO²R⁶、C(S)OR⁶、OCONR⁶R⁷、OCO₂R⁶、OC(S)OR⁶、NHCONR⁶R⁷、NHCO₂R⁶、NHC(S)OR⁶、SO₂NR⁶R⁷Optionally substituted aryl linked to Y by an optionally substituted alkyl, alkenyl or alkynyl chain, optionally substituted heterocycle linked to Y by an optionally substituted alkyl, alkenyl or alkynyl chain, and independently R²、R³、R⁴Or R⁵Any of (a) or (b) is selected from an optionally substituted heterocycle or an optionally substituted aryl of:

provided that Y is a carbon or sulfur atom, and alternatively, provided that Y is a nitrogen atom, R⁴Is absent;

wherein

y is C, N, S;

(iii) optionally, N will be in a cytosine nucleoside analogue⁴(ii) deprotection of the amino group at position(s) to recover the free primary amino N on the cytosine nucleoside analogue of formula I further purified by conventional purification methods⁴。

Preferably, the heterocycle of the base in formula III constituting the starting material is selected from: uracil, adenine, cytosine, guanine, thymine, hypoxanthine, xanthine, thiouracil, thioguanine, 9-H-purin-2-amine, 7-methylguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5, 6-dihydrouracil, 5-methylcytosine and 5-hydroxymethylcytosine and any substituted derivatives thereof.

Furthermore, the free cytosine or cytosine derivative to be modified in order to obtain the corresponding nucleobase of formula II is preferably selected from:

furthermore, the cytosine nucleobase in the nucleoside analogue of formula I is preferably selected from:

for the methods described herein, the APIs, intermediates, or prodrugs thereof prepared are selected from: capecitabine (Capecitabine), Decitabine (Decitabine, aza-dCyd or DAC), 5-Azacytidine (5-Azacytidine, aza-Cyd), Cytarabine (Cytarabine, ara-C), Enocitabine (Enocitabine, BH-AC), Gemcitabine (Gemcitabine, dFdC), Zalcitabine (Zalcitabine, ddC), Ibacitabine (Ibacitabine), sapabine (Sapacitabine), 2 '-C-cyano-2' -deoxy-1- β -D-arabino-pentofuranosyl cytosine (CNDAC), Galocitabine (Galocitabine), valopicitabine (Valopritabine, NM283), 2 '-deoxy-4' -thiocytidine, Thiabine (T-arabic), 2 '-aza-4' -thiocytidine (5-azacitidine, or ATCracitabine).

More preferably, the APIs, intermediates or prodrugs thereof prepared are selected from: capecitabine (Capecitabine), Decitabine (Decitabine, aza-dCyd or DAC), 5-Azacytidine (5-Azacytidine, aza-Cyd), Cytarabine (Cytarabine, ara-C), and even more preferably, the described method contemplates, inter alia, the industrial preparation of Capecitabine or Cytarabine.

In yet another preferred embodiment for carrying out the methods described thus far, the API, intermediate or prodrug thereof prepared is capecitabine (scheme 2).

Scheme 2 Synthesis of Capecitabine with the aid of NPs or PyNPs

The biocatalytic step (enzymatic step), enzymatic or chemoenzymatic synthesis of capecitabine is carried out at a temperature preferably ranging from 20 to 120 ℃.

In yet another preferred embodiment for carrying out the process described thus far, the API, intermediate or prodrug thereof prepared is cytarabine (scheme 3).

Scheme 3 Synthesis of Cytarabine with the aid of NPs or PyNPs

For carrying out the biocatalytic process of the present specification, the applicant has selected that the NPs enzyme-containing organism or the isolated NPs enzyme itself, preferably, in both cases, a PyNP enzyme or a PyNP/PNP enzyme mixture, wherein the enzymes or the microorganism containing them are mesophilic or mesophilic in the sense that they are capable of exerting and effecting nucleotidyl transferase activity at temperatures ranging from 18 up to 60 ℃ (optimal temperature range is 40-55 ℃). Biological or NPs enzymes, especially PyNP enzymes, thermophilic organisms or thermophiles are those capable of exerting or achieving nucleotidyl transferase activity at temperatures in the range of above 60 ℃ and up to 80 ℃. Biological or NPs enzymes, especially PyNP enzymes, hyperthermophilic organisms or hyperthermophilic are those capable of exerting or achieving nucleotidyl transferase activity at temperatures in excess of 80 ℃ and up to 100 ℃ (optimal temperature range 80-95 ℃).

The nps (pynp) enzyme used in the process of the invention may be isolated from a microorganism selected from mesophilic organisms, by way of example bacteria, in particular escherichia coli, or from mesophilic, thermophilic or hyperthermophilic Archaea (Archaea), in particular from thermomycetes (thermoproteicolas), and more in particular from the genera and species disclosed in WO 201I/076894. Even more preferred nucleoside phosphorylases according to the present invention are derived from Sulfolobus solfataricus and aerothrix cutaneum (Aeropyrum pernix).

Yet another embodiment of the present invention relates to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 or SEQ ID NO: 2 or a fragment thereof in the disclosed method. The two DNA sequences were isolated from archaea, and more particularly, from archaea having a sequence homologous to nucleoside phosphorylase.

As described throughout the present specification, preferred embodiments for carrying out the process of the invention are based on the use of mesophilic, thermophilic or hyperthermophilic NPs enzymes (purine NPs (PNPs), or pyrimidine NPs (PyNPs), mixtures thereof) as biocatalysts for carrying out base transfer one-step one-pot reactions (one step-one pot reactions). Preferred thermophilic or hyperthermophilic NPs enzymes are recombinant, comprising a gene sequence or fragment thereof encoding an NP enzyme activity capable of performing a nucleobase transfer one-step one-pot reaction at temperatures ranging from above 60 ℃ and up to 100 ℃ and more preferably under suitable reaction conditions as described herein.

For the purposes of this specification, preferred enzymes are used in the biocatalytic process of the invention. The methods of cloning them, the vectors carrying the nucleic acid sequences encoding them, and the host cells transformed with the above vectors are all those disclosed in WO2011/076894 in the name of some of the inventors of the present invention, and the entire specification of their patent application is fully incorporated herein by reference.

Suitable conditions for carrying out the various embodiments of the process of the invention include:

a) a temperature in the range of 20-120 deg.C

b) Reaction time in the range of 1-1000h

c) Starting material concentrations in the range of 1-1000mM

d) A stoichiometric nucleoside starting material to nucleobase in the range of 1: 5 to 5: 1.

e) Amount of NP enzyme in the range of 0,001-100mg/ml

f) Nucleobases added to a reaction medium, optionally dissolved in an organic solvent

g) The aqueous reaction medium optionally also containing up to 40% of a suitable organic solvent is preferably up to 20%, and more preferably up to 5%.

Optionally, an organic solvent can be added to the reaction medium or used to dissolve the nucleobases in advance. Preferred are polar aprotic solvents, preferably selected from: tetrahydrofuran, acetonitrile, acetone, Dimethylformamide (DMF) or Dimethylsulfoxide (DMSO).

The process according to the invention further comprises an isolation and/or purification step of the NA prepared by standard operating means selected from precipitation, filtration, concentration or crystallization.

The thermophilic NP enzyme is able to function at higher temperatures which make the nucleobase transfer reaction more efficient in terms of time and overall yield.

The method of the present invention specifically discloses the preparation of capecitabine (scheme 2) as an embodiment of the invention, wherein the ribonucleoside used as starting material is a 5 ' -deoxyribofuranosyl nucleoside selected from 5 ' -deoxyuridine, 5 ' -deoxy-5-methyluridine or 5 ' -chloro-5 ' -deoxyuridine; the nucleobase used as starting material for the transfer by the PyNP enzyme is (5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) pentyl carbamate and PyNP is a naturally occurring mesophilic, thermophilic or hyperthermophilic enzyme isolated from bacteria.

An alternative embodiment of the process of the invention is the one wherein the API prepared is capecitabine (scheme 2), the ribonucleoside used as starting material is 5 '-deoxyuridine, 5' -deoxy-5-methyluridine or 5 '-chloro-5' -deoxyuridine, the nucleobase also used as starting material for the transfer by the PyNP enzyme is pentyl (5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate and PyNP is a recombinant mesophilic, thermophilic or hyperthermophilic enzyme, the cloned DNA of which is isolated from bacteria.

Another alternative embodiment of the process of the invention is the embodiment wherein the API prepared is capecitabine (scheme 2), the ribonucleoside used as starting material is 5 '-deoxyuridine, 5' -deoxy-5-methyluridine or 5 '-chloro-5' -deoxyuridine, the nucleobase also used as starting material for the transfer by the PyNP enzyme is pentyl (5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate and PyNP is a naturally occurring mesophilic, thermophilic or hyperthermophilic enzyme isolated from archaea.

Yet another alternative embodiment of the process of the invention is the embodiment wherein the API prepared is capecitabine (scheme 2), the ribonucleoside used as starting material is 5 '-deoxyuridine, 5' -deoxy-5-methyluridine or 5 '-chloro-5' -deoxyuridine, the nucleobase also used as starting material for the transfer by the PyNP enzyme is pentyl (5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate and PyNP is a recombinant mesophilic, thermophilic or hyperthermophilic enzyme isolated from archaea.

The process of the invention is also represented by the following embodiment, wherein the API prepared is cytarabine (scheme 3), the ribonucleoside used as starting material is arabinofuranosyl uracil or arabinonucleoside, the nucleobase also used as starting material for the transfer by the PyNP enzyme is N- (2-oxo-1, 2-dihydropyrimidin-4-yl) pentanamide and PyNP is a naturally occurring mesophilic, thermophilic or hyperthermophilic enzyme isolated from bacteria.

Another embodiment of the method of the invention is one wherein the prepared API is cytarabine (scheme 3), the ribonucleoside used as starting material is arabinofuranosyl uracil or arabinonucleoside, the nucleobase also used as starting material transferred by the PyNP enzyme is N- (2-oxo-1, 2-dihydropyrimidin-4-yl) pentanamide and PyNP is a recombinant mesophilic, thermophilic or hyperthermophilic enzyme, the cloned DNA of which is isolated from a bacterium.

An additional embodiment of the method of the invention is one wherein the API prepared is cytarabine (scheme 3), the ribonucleoside used as starting material is arabinofuranosyl uracil or arabinonucleoside, the nucleobase also used as starting material transferred by the PyNP enzyme is N- (2-oxo-1, 2-dihydropyrimidin-4-yl) pentanamide and PyNP is a naturally occurring mesophilic, thermophilic or hyperthermophilic enzyme, the cloned DNA of which is isolated from archaea.

Another embodiment of the process of the invention is one wherein the prepared API is cytarabine (scheme 3), the ribonucleoside used as starting material is arabinofuranosyl uracil or arabinonucleoside, the nucleobase also used as starting material for the transfer by the PyNP enzyme is N- (2-oxo-1, 2-dihydropyrimidin-4-yl) pentanamide and PyNP is a recombinant mesophilic, thermophilic or hyperthermophilic enzyme, the cloned DNA of which is isolated from archaea.

To form part of the same inventive concept, the present specification also discloses recombinant nucleoside phosphorylases (PNPs or PyNPs) for use in any of the methods of the present invention as described above. The native or recombinant NP enzyme may be isolated from mesophiles, more preferably from bacteria and archaea; or isolated from thermophilic organisms, more preferably bacteria and archaea; or from hyperthermophilic organisms, more preferably from bacteria and archaea, and more particularly from sulfolobus solfataricus and aeromonas sobria.

Also disclosed in this specification, for the purpose of incorporating a single inventive concept, is the use of a mesophilic, thermophilic or hyperthermophilic nucleoside phosphorylase (NP or PyNP) in the preparation of APIs, intermediates or prodrugs thereof, those APIs, intermediates thereof or prodrugs thereof being cytosine Nucleoside Analogues (NAs) useful as anticancer or antiviral drugs. More preferably, the aforementioned uses are achieved by the methods of preparation of NAs, intermediates or prodrugs thereof as APIs, and variants thereof, also detailed above. In particular, with respect to the aforementioned uses, recombinant thermophilic nucleoside phosphorylases (PNPs, PyNPs or mixtures thereof) are preferred in the preparation of APIs, those APIs or intermediates thereof being Nucleoside Analogues (NAs) particularly useful as anticancer or antiviral drugs.

Among the APIs, intermediates, or prodrugs thereof prepared according to such uses, the following may be found:

capecitabine (Capecitabine), Decitabine (Decitabine, aza-dCyd or DAC), 5-Azacytidine (5-Azacytidine, aza-Cyd), Cytarabine (Cytarabine, ara-C), Enocitabine (Enocitabine, BH-AC), Gemcitabine (Gemcitabine, dFdC), Zalcitabine (Zalcitabine, ddC), Ibacitabine (Ibacitabine), sapabine (Sapacitabine), 2 '-C-cyano-2' -deoxy-1- β -D-arabino-pentofuranosyl cytosine (CNDAC), Galocitabine (Galocitabine), valopicitabine (Valopritabine, NM283), 2 '-deoxy-4' -thiocytidine, Thiabine (T-arabic), 2 '-aza-4' -thiocytidine (5-azacitidine, or ATCracitabine).

More preferably, the APIs, intermediates or prodrugs thereof prepared according to the use of the enzymes disclosed herein are selected from: capecitabine (Capecitabine), Decitabine (Decitabine, aza-dCyd or DAC), 5-Azacytidine (5-Azacytidine, aza-Cyd), Cytarabine (Cytarabine, ara-C), and even more preferably, the described methods contemplate, inter alia, the industrial preparation of Capecitabine or Cytarabine and their respective intermediates or prodrugs.

Also included in the invention are recombinant expression vectors comprising sequences encoding nucleoside phosphorylase activities (NPs) operably linked to one or more control sequences which direct the expression or overexpression of said nucleoside phosphorylase in a suitable host. Preferred recombinant expression vectors according to the invention are any expression vectors which carry and express or overexpress a gene encoding the enzymatic activity of NP, which is present in thermophilic and hyperthermophilic archaea, preferably in Sphaerotheca (Crenarchaeote), and more preferably from Thermoprotei (Thermoprotei) class, such as: a1RW90(A1RW90THEPD), a putative protein from thermomyces ptospira (strain Hrk 5); Q97Y30(Q97Y30SULSO), putative protein from Sulfolobus solfataricus; a3DME1(A3DME1STAMF), a putative protein from Staphylothermus marinus (strain ATCC 43588/DSM 3639/Fl); q9YA34(Q9YA34AERPE), a putative protein from aeromonas agilis (Aeropyrum pernix); a2BJ06(A2BJ06HYPBU), putative protein from Thermus butanosus (Hyperthermus butylicus) (strain DSM 5456/JCM 9403); and putative proteins of D9PZN7(D9PZN7ACIS3), Foliumlessus saccharivorans (Acidinobus saccharovorans) (strain DSM 16705/VKM B-2471/345).

Preferred recombinant expression vectors according to the invention carry and express or overexpress a nucleic acid sequence selected from: SEQ ID No.1, 2, 5, 7, 9 or 11; or

a) As SEQ id no: 1.2, 5, 7, 9 or 11; or

b) And SEQ id no: 1.2, 5, 7, 9 or 11; or

c) A nucleotide sequence that hybridizes under high stringency conditions with: no: 1.2, 5, 7, 9 or 11; and SEQ id no: 1.2, 5, 7, 9 or 11; or with a polypeptide from SEQ id no: 1.2, 5, 7, 9 or 11; or their complements; or

d) And SEQ id no: 1.2, 5, 7, 9 or 11 has at least 80% sequence identity;

e) and SEQ id no: 1.2, 5, 7, 9 or 11 has at least 59% sequence identity;

f) encoding a polypeptide selected from SEQ id no: 3. 4, 6, 8, 10 or 12.

The invention also covers the use of the aforementioned recombinant expression vectors for the preparation of recombinant NPs or for the preparation of Active Pharmaceutical Ingredients (APIs), intermediates or prodrugs thereof, those APIs or intermediates thereof being Nucleoside Analogues (NAs) which are particularly useful as anticancer or antiviral drugs. In particular, the APIs, intermediates or prodrugs thereof prepared according to the above uses are selected from: capecitabine (Capecitabine), Decitabine (Decitabine, aza-dCyd or DAC), 5-Azacytidine (5-Azacytidine, aza-Cyd), Cytarabine (Cytarabine, ara-C), Enocitabine (Enocitabine, BH-AC), Gemcitabine (Gemcitabine, dFdC), Zalcitabine (Zalcitabine, ddC), Ibacitabine (Ibacitabine), sapabine (Sapacitabine), 2 '-C-cyano-2' -deoxy-1- β -D-arabino-pentofuranosyl cytosine (CNDAC), Galocitabine (Galocitabine), valopicitabine (Valopritabine, NM283), 2 '-deoxy-4' -thiocytidine, Thiabine (T-arabic), 2 '-aza-4' -thiocytidine and 5-azacitidine (ATCrabine).

More preferably, the APIs, intermediates or prodrugs thereof prepared are selected from: capecitabine (Capecitabine), Decitabine (Decitabine, aza-dCyd or DAC), 5-Azacytidine (5-Azacytidine, aza-Cyd), Cytarabine (Cytarabine, ara-C); and even more preferably, the expression vectors described herein are particularly suitable for the industrial preparation of capecitabine or cytarabine, intermediates or prodrugs thereof.

More preferably, the aforementioned use for the preparation of APIs, intermediates or prodrugs thereof is achieved by the preparation methods and variants thereof also detailed above.

The present invention also encompasses a host cell comprising the recombinant expression vector described above, especially when the host cell is Escherichia coli. Also contemplated is the use of a host cell comprising a recombinant expression vector as described above, included as part of a single related concept of the same invention, for the preparation of recombinant NPs (or pynps). Similarly, the use of host cells comprising recombinant expression vectors as described above for the preparation of Active Pharmaceutical Ingredients (APIs), intermediates or prodrugs thereof, those APIs or intermediates thereof being Nucleoside Analogues (NAs) useful as anticancer or antiviral drugs, is also part of the present invention. In particular, if those host cells comprise a recombinant expression vector as described previously, it is useful for the preparation of APIs, intermediates or prodrugs thereof selected from: capecitabine (Capecitabine), Decitabine (Decitabine, aza-dCyd or DAC), 5-Azacytidine (5-Azacytidine, aza-Cyd), Cytarabine (Cytarabine, ara-C), Enocitabine (Enocitabine, BH-AC), Gemcitabine (Gemcitabine, dFdC), Zalcitabine (Zalcitabine, ddC), Ibacitabine (Ibacitabine), sapabine (Sapacitabine), 2 '-C-cyano-2' -deoxy-1- β -D-arabino-pentofuranosyl cytosine (CNDAC), Galocitabine (Galocitabine), valopicitabine (Valopritabine, NM283), 2 '-deoxy-4' -thiocytidine, Thiabine (T-arabic), 2 '-aza-4' -thiocytidine and 5-azacitidine (ATCrabine).

More preferably, the APIs, intermediates or prodrugs thereof prepared using the host cells of the invention transformed with the recombinant expression vectors described above are selected from: capecitabine (Capecitabine), Decitabine (Decitabine, aza-dCyd or DAC), 5-Azacytidine (5-Azacytidine, aza-Cyd), Cytarabine (Cytarabine, ara-C); and even more preferably, the host transformed cells described herein are particularly suitable for the industrial preparation of capecitabine or cytarabine, intermediates or prodrugs thereof.

More preferably, the aforementioned uses are achieved by the methods of preparation of NAs, intermediates or prodrugs thereof as APIs, and variants thereof, also detailed above.

Comparative example 1: synthesis of 5-fluoro-5' -deoxycytidine starting from unmodified 5-fluorocytosine and deoxyuridine

A solution of 2.5mM 5-fluorocytosine and 8.0mM 5' -deoxyuridine, pH 7, in 30mM aqueous phosphate buffer and 10% DMSO was heated at 60 ℃ for 30 minutes. Then, PyNP (5.4U/. mu.mol) was added_Base) And the reaction was stirred at 60 ℃ for 4 hours under the same conditions. After this time, the crude reaction was filtered through a 10KDa membrane and a portion was diluted and analyzed by HPLC. The expected product 5-fluoro-5' -deoxycytidine was not detected by UV-DAD (ultraviolet-diode array detection).

Comparative example 2: synthesis of 5-fluoro-5 '-deoxycytidine starting from unmodified 5-fluorocytosine and chloro-5' -deoxyuridine

A solution of 2.5mM 5-fluorocytosine and 8.6mM 5-chloro-5' -deoxyuridine, pH 7, in 30mM aqueous phosphate buffer and 10% DMSO was heated at 60 ℃ for 30 minutes. Then, PyNP (5.4U/. mu.mol) was added_Base) And the reaction was stirred at 60 ℃ for 4 hours under the same conditions. After this time, the crude reaction was filtered through a 10KDa membrane and a portion was diluted and analyzed by HPLC. The expected product 5-fluoro-5' -deoxycytidine was not detected by UV-DAD.

Comparative example 3: synthesis of cytarabine starting from unmodified cytosine and arabinofuranosyl uracil

A solution of 3.0mM cytosine and 10mM 9- (b-D-arabinofuranosyl) uracil in 30mM aqueous phosphate buffer, pH 7, and 10% DMSO was heated at 60 ℃ for 30 minutes. After that, PyNP (4.4U/. mu.mol) was added_Base) And the reaction was stirred at 60 ℃ for 4 hours under the same conditions. After this time, the crude reaction was filtered through a 10KDa membrane and a portion was diluted and analyzed by HPLC. The expected product 1- (b-D-arabinofuranosyl) cytosine (cytarabine) was not detected by UV-DAD, but uracil from cytosine decomposition by deamination was obtained.

Comparative example 4: synthesis of cytarabine starting from unmodified cytosine and arabinofuranosyl cytosine

A solution of 3.0mM cytosine and 7.6mM 9- (b-D-arabinofuranosyl) adenine in 30mM aqueous phosphate buffer, pH 7, and 10% DMSO was heated at 60 ℃ for 30 minutes. After that, PyNP (4.4U/. mu.mol) was added_Base) And the reaction was stirred at 60 ℃ for 4 hours under the same conditions.After this time, the crude reaction was filtered through a 10KDa membrane and a portion was diluted and analyzed by HPLC. The expected product 1- (b-D-arabinofuranosyl) cytosine (cytarabine) was not detected by UV-DAD, but hypoxanthine from adenine deamination and 9- (b-D-arabinofuranosyl) hypoxanthine from substrate deamination were obtained.

Comparative example 5: synthesis of cytarabine starting from unmodified cytosine and arabinofuranosyl hypoxanthine

A solution of 3.0mM cytosine and 7.5mM 9- (b-D-arabinofuranosyl) hypoxanthine in 30mM aqueous phosphate buffer, pH 7, and 10% DMSO was heated at 60 ℃ for 30 minutes. After that, PyNP (4.4U/. mu.mol) was added_Base) And the reaction was stirred at 60 ℃ for 4 hours under the same conditions. After this time, the crude reaction was filtered through a 10KDa membrane and a portion was diluted and analyzed by HPLC. The expected product 1- (b-D-arabinofuranosyl) cytosine (cytarabine) was not detected by UV-DAD.

Comparative example 6: synthesis of cytarabine starting from unmodified cytosine and arabinofuranosyl guanine

A solution of 3.0mM cytosine and 8.6mM9- (b-D-arabinofuranosyl) guanine, pH 7, in 30mM aqueous phosphate buffer and 10% DMSO was heated at 60 ℃ for 30 minutes. After that, PyNP (4.4U/. mu.mol) was added_Base) And the reaction was stirred at 60 ℃ for 4 hours under the same conditions. After this time, the crude reaction was filtered through a 10KDa membrane and a portion was diluted and analyzed by HPLC. The expected product 1- (b-D-arabinofuranosyl) cytosine (cytarabine) was not detected.

Example 7: synthesis of pentyl (5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate

Under an inert atmosphere, pentyl chloroformate (5g, 1.0 equiv.) was added to a stirred solution of 5-fluorocytosine (1.0 equiv.) in anhydrous pyridine, and the reaction was chargedHeat to 60 ℃. After 2 hours, when no higher conversion was observed, the reaction was quenched by cooling at room temperature. Thereafter, the crude reaction was extracted using AcOEt and HCl 1N, and the organic phase was passed over Na₂SO₄Dried and the solvent evaporated. Using SiO₂And AcOEt as the mobile phase, the resulting white solid was chromatographed to give the desired product (88%).

Example 8: synthesis of N- (2-oxo-1, 2-dihydropyrimidin-4-yl) acetamide

Triethylamine (18, 1. mu.L, 0,13mmol) and acetyl chloride (9, 2. mu.L, 0,13mmol) were added to a stirred solution of cytosine (10,0mg, 0,09mmol) in anhydrous DMF at room temperature over the course of 25 hours under an inert atmosphere. Thereafter, the solvent was evaporated, and the resulting pale yellow solid was washed with water, filtered and dried in vacuo to give the desired product in 55% yield.

Example 9: synthesis of pentyl (2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate

Pentyl chloroformate (0.8mmol, 1.0 equiv.) was added dropwise to a stirred solution of cytosine (0.8mmol, 1.0 equiv.) in anhydrous pyridine under an inert atmosphere, and the reaction was heated to 60 ℃. After 2 hours, when no higher conversion was observed, the reaction was quenched by cooling at room temperature. Thereafter, the crude reaction was extracted using AcOEt and HCl 1N, and the organic phase was passed over Na₂SO₄Dried and the solvent evaporated. Using SiO₂And AcOEt as the mobile phase, the resulting white solid was chromatographed to give the desired product.

Example 10: n from uridine⁴Synthesis of-pentoxycarbonyl-5' -deoxy-5-fluorocytidine (capecitabine)

2.7m of pH 7A solution of amyl M (5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate and 8.0mM 5' -deoxyuridine in 30mM aqueous phosphate buffer and 10% DMSO was heated at 60 ℃ for 30 minutes. Then, PyNP (5.0U/. mu.mol) was added_Base) And the reaction was stirred at 60 ℃ for 4 hours under the same conditions. After this time, the crude reaction was filtered through a 10KDa membrane and a portion was diluted and analyzed by HPLC. The expected product N was detected in a yield of 68% compared to reference example 1, in which no final product was formed or detected⁴-pentoxycarbonyl-5' -deoxy-5-fluorocytidine (capecitabine).

Example 11: n from deoxyuridine⁴Synthesis of-pentoxycarbonyl-5' -deoxy-5-fluorocytidine (capecitabine).

A solution of 2.5mM (5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamic acid pentyl ester and 8.6mM 5' -deoxyuridine, pH 7, in 30mM aqueous phosphate buffer and 10% DMSO was heated at 60 ℃ for 30 minutes. Then, PyNP (5.4U/. mu.mol) was added_Base) And the reaction was stirred at 60 ℃ for 4 hours under the same conditions. After this time, the crude reaction was filtered through a 10KDa membrane and a portion was diluted and analyzed by HPLC. The expected product N was detected in a yield of 77% compared to reference example 2, in which no final product was formed or detected⁴-pentoxycarbonyl-5' -deoxy-5-fluorocytidine (capecitabine).

Example 12: synthesis of 9- (b-D-arabinofuranosyl) uracil (cytarabine) starting from arabinofuranosyl uracil

A solution of 2.5mM (2-oxo-1, 2-dihydropyrimidin-4-yl) carbamic acid pentyl ester and 8.6mM9- (b-D-arabinofuranosyl) uracil in 30mM aqueous phosphate buffer, pH 7, and 10% DMSO was heated at 60 ℃ for 30 minutes. Then, PyNP (5.4U/. mu.mol) was added_Base) And the reaction was stirred at 60 ℃ for 4 hours under the same conditions. In-line with the aboveThereafter, the crude reaction was filtered through a 10KDa membrane, and a portion was diluted and analyzed by HPLC. The expected product N was detected⁴-pentyloxycarbonyl cytidine.

N⁴The position may optionally be deprotected to give 9- (b-D-arabinofuranosyl) uracil (cytarabine).

Example 13: synthesis of 9- (b-D-arabinofuranosyl) uracil (cytarabine) starting from arabinofuranosyl adenine

A solution of 2.5mM (2-oxo-1, 2-dihydropyrimidin-4-yl) carbamic acid pentyl ester and 8.6mM9- (b-D-arabinofuranosyl) adenine in 30mM aqueous phosphate buffer, pH 7, and 10% DMSO was heated at 60 ℃ for 30 minutes. Then, PyNP (5.4U/. mu.mol) was added_Base) And the reaction was stirred at 60 ℃ for 4 hours under the same conditions. After this time, the crude reaction was filtered through a 10KDa membrane and a portion was diluted and analyzed by HPLC. The expected product N was detected⁴-pentyloxycarbonyl cytidine.

Claims

1. A method for preparing cytosine nucleoside analogue of formula I, intermediate or prodrug thereof by chemical-enzymatic method or enzymatic method

Wherein,

Z₁is O, CH₂、S、NH；

R^S1Is hydrogen, OH, an ether or ester thereof selected from:

R^S2is hydrogen, OH or ether or ester residue thereof, halogen, CN, NH₂、SH、C≡CH、N₃；

In the case of NA from 2' -deoxyribonucleoside or arabinonucleoside, R^S3Is hydrogen, or R when NA is from a ribonucleoside^S3Selected from OH and NH₂Halogen, OCH₃；

R^S4Is hydrogen, OH or an ether or ester residue thereof, NH₂Halogen, CN; with the proviso that R is when both are ethers or esters of OH residues^S1And R^S4Is different;

at Z₂In the case of being different from oxygen, R^S5Is hydrogen, OH or an ether or ester residue thereof, NH₂Or halogen;

R¹is O, CH₂、S、NH；

R³Is hydrogen, optionally substituted C_4-40An alkyl chain, an optionally substituted alkenyl chain, an optionally substituted alkynyl chain, an optionally substituted aryl linked to N through an optionally substituted alkyl, alkenyl or alkynyl chain, an optionally substituted heterocycle linked to N through an optionally substituted alkyl, alkenyl or alkynyl chain, COR⁶、CONR⁶R⁷、CO₂R⁶、C(S)OR⁷、CN、SR⁶、SO₂R⁶、SO₂R⁶R⁷CN, P (O) aryl, P (O) heterocycle, P (S) aryl, P (S) heterocycle, P (O) O₂R⁸；R³And R²Independently of each other; and with the proviso that R₂Or R₃Is different from hydrogen;

R₄is hydrogen, OH, NH₂SH, halogen; an optionally substituted alkyl chain; an optionally substituted alkenyl chain; optionally substituted alkynyl chain, trihaloalkyl, OR⁶、NR⁶R⁷、CN、COR⁶、CONR⁶R⁷、CO₂R⁶、C(S)OR⁶、OCONR⁶R⁷、OCO₂R⁶、OC(S)OR⁶、NHCONR⁶R⁷、NHCO₂R⁶、NHC(S)OR⁶、SO₂NR⁶R⁷An optionally substituted aryl linked to Y by an optionally substituted alkyl, alkenyl or alkynyl chain, an optionally substituted heterocycle linked to Y by an optionally substituted alkyl, alkenyl or alkynyl chain, and R independently selected from the group consisting of²、R³、R⁴Or R⁵Any optionally substituted heterocycle or optionally substituted aryl of (a):

provided that Y is a carbon or sulfur atom, and alternatively, R⁴Absent, provided that Y is a nitrogen atom;

wherein

R⁵is hydrogen, OH, NH₂SH, halogen, optionally substituted alkyl chain, optionally substituted alkenyl chain, optionally substituted alkynyl chain, trihaloalkyl, OR⁶、NR⁶R⁷、CN、COR⁶、CONR⁶R⁷、CO₂R⁶、C(S)OR⁶、OCONR⁶R⁷、OCO₂R⁶、OC(S)OR⁶、NHCONR⁶R⁷、NHCO₂R⁶、NHC(S)OR⁶、SO₂NR⁶R⁷；CH₂-heterocycle, CN;

and R is independently selected from the group consisting of²、R³、R⁴Or R⁵Any optionally substituted heterocycle or optionally substituted aryl of (a):

wherein

y is C, N, S;

wherein the process for preparing a cytosine nucleoside analogue comprises the steps of:

(i) make Y, R therein¹、R⁴、R⁵、R⁶And R⁷Precursors of cytosine nucleobases of formula II as defined above and their use for modifying them at N⁴Suitable reagents for the amino group of the position are chemically reacted to add the substituent group R²And R³The modified cytosine nucleobase of formula II formed therefrom is optionally purified by conventional purification methods; or alternatively, the method from the formula II cytosine nucleobases represented by the starting product itself directly,

(ii) subjecting the modified cytosine nucleobase of formula II above to a biocatalytic reaction with a suitable nucleoside analogue substrate of formula III,

wherein Z₁、Z₂、R^S1、R^S2、R^S3、R^S4、R^S5As defined above, and the bases are selected from: uracil, adenine, cytosine, guanine, thymine, hypoxanthine, xanthine, thiouracil, thioguanine, 9-H-purin-2-amine, 7-methylguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5, 6-dihydrouracil, 5-methylcytosine and 5-hydroxymethylcytosine, pteridine, and any substituted derivatives thereof;

wherein the above reaction carried out in step II) comprises adding a nucleoside phosphorylase, which may be a pyrimidine nucleoside phosphorylase, a purine nucleoside phosphorylase or a combination thereof, to a mixture of starting materials comprising a cytosine nucleobase of formula II and a nucleoside analogue of formula III in a suitable aqueous reaction medium and under suitable reaction conditions,

(iii) optionally, N will be in a cytosine nucleoside analogue⁴The amino group at the position is deprotected to recover the free primary amino N on the cytosine nucleoside analogue of formula I which is further purified by conventional purification methods⁴。

2. The method of claim 1, wherein the cytosine nucleobase of formula II transferred by the pyrimidine nucleoside phosphorylase is selected from the group consisting of:

3. the process according to any one of claims 1 or 2, wherein the nucleoside analogue, intermediate or prodrug thereof prepared is selected from the group consisting of: capecitabine, decitabine, 5-azacytidine, cytarabine, enocitabine, gemcitabine, zalcitabine, ibacitabine, sapatibine, 2 '-C-cyano-2' -deoxy-1- β -D-arabino-pentofuranosyl cytosine, galocitabine, valocitabine, 2 '-deoxy-4' -thiocytidine, Thiarabine, 2 '-deoxy-4' -thio-5-azacytidine, and alecitabine.

4. A method according to any one of claims 1 to 3, wherein the source of the native and mutant or variant thereof, or recombinant nucleoside phosphorylase, is a mesophilic, thermophilic or hyperthermophilic organism.

5. A method according to any one of claims 1 to 4, wherein the source of the native and mutant or variant thereof, or recombinant nucleoside phosphorylase is selected from Archaea (Archaea) or bacteria.

6. The method of claim 5, wherein the nucleoside phosphorylase is isolated from an archaea selected from Sulfolobus solfataricus (Sulfolobus solfataricus) or aeromonas agilis (Aeropyrum pernix).

7. The method of claim 1, wherein said nucleoside phosphorylase or functional portion thereof is encoded by a nucleotide sequence selected from the group consisting of SEQ ID No.1, 2, 5, 7, 9 or 11; or by the following code:

a) as SEQ id no: 1.2, 5, 7, 9 or 11; or

b) And SEQ id no: 1.2, 5, 7, 9 or 11; or

d) And SEQ id no: 1.2, 5, 7, 9 or 11 has at least 80% sequence identity;

e) and SEQ id no: 1.2, 5, 7, 9 or 11 has at least 59% sequence identity;

f) encoding a polypeptide selected from SEQ id no: 3. 4, 6, 8, 10 or 12.

8. The process of claim 3, wherein the nucleoside analog prepared is capecitabine and the ribonucleoside used as starting material is selected from the group consisting of: 5 ' -deoxyuridine, 5 ' -deoxy-5-methyluridine or 5 ' -deoxy-5-chlorouridine; and the nucleobase transferred by the pyrimidine nucleoside phosphorylase, which is also used as a starting material, is amyl (5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate.

9. The process according to claim 3, wherein the nucleoside analogue prepared is cytarabine, the ribonucleoside used as starting material is 9- (b-D-arabinofuranosyl) uracil and the nucleobase transferred by the pyrimidine nucleoside phosphorylase, which is also used as starting material, is N- (2-oxo-1, 2-dihydropyrimidin-4-yl) pentanamide.

10. Use of a mesophilic, thermophilic or hyperthermophilic nucleoside phosphorylase, which may be native or a mutant or variant thereof, or a recombinase, or a functional part thereof, or a recombinant expression vector, or a microorganism or host cell containing same, for the preparation of a cytosine nucleoside analogue, intermediate or prodrug thereof for use as an anti-cancer or anti-viral drug, said recombinant expression vector comprising a sequence encoding a native or recombinant, thermophilic or hyperthermophilic nucleoside phosphorylase or a functional part thereof, operably linked to one or more control sequences directing the expression or overexpression of said nucleoside phosphorylase in a suitable host.

11. The use according to claim 10, wherein the mesophilic, thermophilic or hyperthermophilic nucleoside phosphorylase, or a functional part thereof, is encoded by a nucleotide sequence selected from SEQ ID No.1, 2, 5, 7, 9 or 11; or by the following code:

a) as SEQ id no: 1.2, 5, 7, 9 or 11; or

b) And SEQ id no: 1.2, 5, 7, 9 or 11; or

d) And SEQ id no: 1.2, 5, 7, 9 or 11 has at least 80% sequence identity;

e) and SEQ id no: 1.2, 5, 7, 9 or 11 has at least 59% sequence identity;

f) encoding a polypeptide selected from SEQ id no: 3. 4, 6, 8, 10 or 12.

12. Use according to any one of claims 10 or 11, wherein the prepared cytosine nucleoside analogue, intermediate or prodrug thereof is selected from: capecitabine, decitabine, 5-azacytidine, cytarabine, enocitabine, gemcitabine, zalcitabine, ibacitabine, sapatibine, 2 '-C-cyano-2' -deoxy-1- β -D-arabino-pentofuranosyl cytosine, galocitabine, valocitabine, 2 '-deoxy-4' -thiocytidine, Thiarabine, 2 '-deoxy-4' -thio-5-azacytidine, and alecitabine.

13. The use according to claim 12, wherein the prepared cytosine nucleoside analogue, intermediate or prodrug thereof is capecitabine.

14. The use according to claim 12, wherein the prepared cytosine nucleoside analogue, intermediate or prodrug thereof is cytarabine.