# US 2024/0199680 A1 · Synthesis of fluorinated cyclic dinucleotides

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- Number: US 2024/0199680 A1
- Document ID: US20240199680A1
- Status: Published application
- Kind: A1
- Application number: 18/552,679
- Filed: 2022-03-28
- Published: 2024-06-20
- Earliest claimed: 2021-04-02
- Assignees: Merck Sharp & Dohme Corp.
- Applicants: Merck Sharp & Dohme LLC
- Inventors: Chihui An; Patrick S. Fier; Kaori Hiraga; Zhijian Liu; Nicholas M. Marshall; John McIntosh; Steven P. Miller; Jeffrey C. Moore; Grant S. Murphy; Jennifer V. Obligacion; Weilan Pan; Feng Peng; Nastaran Salehi Marzijarani; Matthew S. Winston
- Web page: https://patentgrep.com/patent/US20240199680A1

## CPC

- C07H 21/00: Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H 1/00: Processes for the preparation of sugar derivatives
- C07H 1/04: Introducing polyphosphoric acid radicals
- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C12P 19/32: having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
- C12P 19/36: Dinucleotides, e.g. nicotineamide-adenine dinucleotide phosphate
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

The present invention relates to efficient processes useful in the preparation of fluorinated cyclic dinucleosides, such as [P(R)]-2′-deoxy-2′-fluoro-5′-O—[(R)-hydroxymercaptophosphinyl]-P-thio-β-D-arabino-adenylyl-(3′→5′)-3′-deoxy-3′-fluoroguanosine cyclic nucleotide, which is also known as (2R,5R,7R,8S,10R,12aR,14R,15S,15aR,16R)-7-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-15,16-difluoro-2, 10-bis(sulfanyl)octahydro-2H,10H, 12H-2λ5,10λ5-5,8-methanofuro[3,2-1][1, 3,6,9,11,2,10]pentaoxadiphosphacyclotetradecine-2,10-dione. The present invention also encompasses intermediates useful in the disclosed synthetic processes and the methods of their preparation.

## Claims

### Claim 1

A process for preparing a compound of Formula (I), or a hydrate, or solvate thereof:
wherein each R is a cation independently selected from the group consisting of H+, Na+, K+, Mg++, Co++, Zn++, and NH4+, said process comprising
reacting a compound of Formula (I-1) with a compound of Formula (I-2), in the presence of at least one cGAS type enzyme:

### Claim 2 (depends on 1)

The process according to claim 1, wherein the compound of Formula (I) is a compound of Formula (Ia), or a hydrate, or solvate thereof:

### Claim 3 (depends on 1)

The process according to claim 1, wherein the at least one cGAS type enzyme is selected from cGAS type enzymes having an amino acid sequence that is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

### Claim 4 (depends on 1)

The process according to claim 1, wherein the at least one cGAS type enzyme is incubated in at least one Chaotropic Agent selected from the group consisting of sodium dodecyl sulfate, thiourea, guanidine HCl, phenol, phenyl acetyl sulfide, urea, KCl, MgCl2, LiOAc, NaCl, and mixtures thereof.

### Claim 5 (depends on 1)

The process according to claim 1, wherein the reacting further comprises reacting in the presence of at least one Metal Co-Factor A.

### Claim 6 (depends on 5)

The process according to claim 5, wherein the at least one Metal Co-Factor A is selected from the group consisting of KCl, MgCl2, ZnSO4, CoSO4, CoF2, Co(SCN)2, CoBr2, Co(NO3)2, CoCl2, CoCO3, Co(C2O4)2, and Co(OH)2, and mixtures thereof.

### Claim 7 (depends on 1)

The process according to claim 1, wherein the reacting is conducted in the presence of at least one Solvent A, where the at least one Solvent A is selected from the group consisting of organic solvents, organic solvents in combination with water, and mixtures thereof.

### Claim 8 (depends on 7)

The process according to claim 7, wherein the at least one Solvent A is selected from the group consisting of tetraglyme dimethyl ether, MeCN, MeOH, EtOH, DMSO, propyl nitrile, sulfolane, pyrrolidone, 2-ethoxyl acetate, cyclohexanol, methyl pentyl ketone, cyclohexanone, 1,2,3,4-tetrahydronaphthalene, pivolate methyl ester, 2-methyl-3-butene-2-ol, tert-butanol, DMF, tetra-methyl urea, tetramethylene sulfone, N,N-diethyl acetamide, ethylene glycol, NMP, isopropyl alcohol, 1-methoxy-2-propyl acetate (MPA), and mixtures thereof.

### Claim 9 (depends on 8)

The process according to claim 8, wherein the at least one Solvent A is TGDE.

### Claim 10 (depends on 1)

The process according to claim 1, further comprising preparing the compound of Formula (I-1) by reacting a compound of Formula (I-1a) with at least one guanylate kinase type enzyme and at least one acetate kinase enzyme:

### Claim 11 (depends on 10)

The process according to claim 10, wherein the at least one guanylate kinase type enzyme is selected from guanylate kinase type enzymes having an amino acid sequence that is SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

### Claim 12 (depends on 10)

The process according to claim 10, wherein the at least one acetate kinase type enzyme is selected from acetate kinase type enzymes having an amino acid sequence that is SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

### Claim 13 (depends on 10)

The process according to claim 10, wherein said reacting is conducted in the presence of at least one Co-Factor A selected from the group consisting of 2′F-thio-ATP and natural ATP.

### Claim 14 (depends on 10)

The process according to claim 10, wherein said reacting is conducted in the presence of at least one Metal Co-Factor B selected from the group consisting of MgCl2, MnCl2, and Mg(OH)2, hydrates thereof, and mixtures thereof.

### Claim 15 (depends on 1)

The process according to claim 1, further comprising preparing the compound of Formula (I-2) by reacting a compound of Formula (I-2a) with at least one acetate kinase type enzyme and at least one adenylate kinase enzyme:

### Claim 16 (depends on 15)

The process according to claim 15, wherein the at least one acetate kinase type enzyme is selected from acetate kinase type enzymes having an amino acid sequence that is SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

### Claim 17 (depends on 15)

The process according to claim 15, wherein the at least one adenylate kinase type enzyme is selected from adenylate kinase type enzymes having an amino acid sequence that is SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27.

### Claim 18 (depends on 15)

The process according to claim 15, wherein said reacting is conducted in the presence of at least one Co-Factor A selected from the group consisting of 2′F-thio-ATP and natural ATP.

### Claim 19 (depends on 15)

The process according to claim 15, wherein said reacting is conducted in the presence of at least one Metal Co-Factor B selected from the group consisting of MgCl2, MnCl2, and Mg(OH)2, hydrates thereof, and mixtures thereof.

### Claim 20 (depends on 1)

The process according to claim 1, further comprising simultaneously preparing the compound of Formula (I-1) by (i) reacting a compound of Formula (I-1a) with at least one guanylate kinase type enzyme and at least one acetate kinase enzyme, and preparing the compound of Formula (I-2) by (ii) reacting a compound of Formula (I-2a) with at least one acetate kinase type enzyme and at least one adenylate kinase enzyme:

### Claim 21 (depends on 20)

The process according to claim 20, wherein the at least one guanylate kinase type enzyme is selected from guanylate kinase type enzymes having an amino acid sequence that is SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

### Claim 22 (depends on 20)

The process according to claim 20, wherein the at least one acetate kinase type enzyme is selected from acetate kinase type enzymes having an amino acid sequence that is SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

### Claim 23 (depends on 20)

The process according to claim 20, wherein the at least one adenylate kinase type enzyme is selected from adenylate kinase type enzymes having an amino acid sequence that is SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27.

### Claim 24

A process for preparing a compound of Formula (I), which comprises
(a) simultaneously preparing the compound of Formula (I-1) by (i) reacting a compound of Formula (I-1a) with at least one guanylate kinase type enzyme and at least one acetate kinase enzyme, and preparing the compound of Formula (I-2) by (ii) reacting a compound of Formula (I-2a) with at least one acetate kinase type enzyme and at least one adenylate kinase enzyme; and
(b) reacting a compound of Formula (I-1) with a compound of Formula (I-2), in the presence of at least one cGAS type enzyme.

### Claim 25 (depends on 24)

The process according to claim 24, further comprising
(c) isolating compound I with ammonium salt formation and converting to sodium salt.

### Claim 26 (depends on 24)

The process according to claim 24, wherein the at least one guanylate kinase type enzyme is selected from guanylate kinase type enzymes having an amino acid sequence that is SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

### Claim 27 (depends on 24)

The process according to claim 24, wherein the at least one acetate kinase type enzyme is selected from acetate kinase type enzymes having an amino acid sequence that is SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

### Claim 28 (depends on 24)

The process according to claim 24, wherein the at least one adenylate kinase type enzyme is selected from adenylate kinase type enzymes having an amino acid sequence that is SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27.

### Claim 29 (depends on 24)

The process according to claim 24, wherein the at least one cGAS type enzyme is selected from cGAS type enzymes having the amino acid sequence that is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

### Claim 30 (depends on 25)

The process according to claim 25, wherein said isolating comprises extracting, washing, crystallizing, and drying the compound of Formula (I).

### Claim 31 (depends on 30)

The process according to claim 30, wherein the process comprises reacting the crude compound of Formula (I) with at least one Salt C selected from the group consisting of Na2SO4, NaHSO4, Na2CO3, NaHCO3, K2SO4, KHSO4, K2CO3, KHCO3, and mixtures thereof.

### Claim 32 (depends on 31)

The process according to claim 31, wherein the process further comprises treating the salt mixture with at least one Immiscible Solvent containing at least one Salt D.

### Claim 33 (depends on 32)

The process according to claim 32, wherein the at least one Immiscible Solvent is selected from the group consisting of 2-MeTHF, EtOAc, and mixtures thereof.

### Claim 34 (depends on 32)

The process according to claim 32, wherein the at least one Salt D is selected from salts having a cation selected from the group consisting of
and having an anion selected from Cl−, Br−, I−, HSO4−, SO42−, H2PO4−, HPO42−, PO43−, and mixtures thereof.

### Claim 35

A compound selected from the group consisting of
wherein each R is a cation independently selected from the group consisting of H+, Na+, K+, Mg++, Co++, Zn++, and NH4+.