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Title:
METHODS FOR PRODUCTION OF PF1022A DERIVATIVES
Document Type and Number:
WIPO Patent Application WO/2018/166899
Kind Code:
A1
Abstract:
Provided are processes for the production of PF1022A derivative compounds, including incubating a microorganism strain capable of producing such compounds in a culture medium to produce a resulting culture, said culture medium containing carbon and nitrogen sources, and further providing to said culture medium a feedstock comprising a compound of Formula II, Formula III, Formula IV or Formula V: (II), (III), (IV), (V) thereby producing said PF1022A derivative compound in the resulting culture. Compounds of Formula II, Formula III, Formula IV, or Formula V useful in such a feedstock are also provided.

Inventors:
MOSS, Steven (30 The Rookery, Balsham CB21 4EU, CB21 4EU, GB)
SHEEHAN, Lesley (208E Omotesando Court, 5-5-5 JingumaeShibuya-k, Tokyo ., 〒150-0001, JP)
Application Number:
EP2018/055845
Publication Date:
September 20, 2018
Filing Date:
March 08, 2018
Export Citation:
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Assignee:
ACIDOPHIL LTD (20 Station Road, Suite 103, Cambridge CB1 2JD, CB1 2JD, GB)
International Classes:
C07D413/14; C07C271/22; C07K11/02
Foreign References:
EP1215281A12002-06-19
US6043058A2000-03-28
US7285404B12007-10-23
US5116815A1992-05-26
US20110262969A12011-10-27
Other References:
W WECKWWERTH ET AL.: "Biosynthesis of PF1022A and related cyclooctadepsipeptides", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 275, no. 23, 9 June 2000 (2000-06-09), American Society for Biochemistry and Molecular Biology, pages 17909 - 17915, XP002934784, ISSN: 0021-9258
K YANAI ET AL.: "Para-position derivatives of fungal antihelmintic cyclodepsipeptides engineered with Streptomyces venezuelae antibiotic biosynthetic genes", NATURE BIOTECHNOLOGY, vol. 22, no. 7, 1 July 2004 (2004-07-01), Gale Group Inc., pages 848 - 855, XP002332548, ISSN: 1087-0156
JESCHKE, PARASITOL RES, vol. 97, 2005, pages S11 - S16
OHYAMA ET AL., BIOSCI., BIOTECHNO., BIOCHEM., vol. 75, 2011, pages 1354
SCHERKENBECK ET AL., BIOORG. MED. CHEM., vol. 24, 2016, pages 873 - 876
JOURNAL OF ANTIBIOTICS, vol. 45, 1992, pages 692
Attorney, Agent or Firm:
MARKS & CLERK LLP (Aurora, 120 Bothwell Street, Glasgow G2 7JS, G2 7JS, GB)
Download PDF:
Claims:
We claim:

1. A process for the roduction of a PF1022A derivative com ound of Formula I:

wherein X1, X2, X3 and X4, at each occurrence, are independently selected from N and CH; and

R1 and R2 are each independently selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl; comprising: incubating a microorganism strain capable of producing PF1022A derivative compounds in a culture medium to produce a resulting culture, said culture medium containing carbon and nitrogen sources, and further providing to said culture medium a feedstock comprising a compound of Formula II, Formula III, Formula IV or Formula V:

I I I II IV V wherein the dotted line represents an optional double bond;

X2, X3, and X4 are independently selected from N and CH; and

R1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl; and R3 is selected from the group consisting of hydrogen, amino, and sulfhydryl, thereby producing said PF1022A derivative compound in the resulting culture.

2. The process of claim 1 , wherein the feedstock comprises a compound of Formula II.

3. The process of claim 1 , wherein the feedstock comprises a compound of Formula II, and the compound of Formula II is a compound of Formula Ila:

Ila

wherein X2, X3, and X4 are independently chosen from N and CH; and

R1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.

4. The process of claim 1 , wherein the feedstock comprises a compound of Formula III.

5. The process of claim 1 , wherein the feedstock comprises a compound of Formula IV.

6. The process of claim 1 , wherein the feedstock comprises a compound of Formula V.

7. The process of claim 6, wherein R2 is H.

8. The process of any one of claims 1-7, wherein R1 is selected from the group consisting halo, amino, hydroxyl, nitro, and morpholino.

9. The process of any one of claims 1-7, wherein R1 is halo.

10. The process of any one of claims 1-7, wherein R1 is fluoro.

11. The process of any one of claims 1-7, wherein R1 is chloro.

12. The process of any one of claims 1-7, wherein R1 is bromo.

13. The process of claim 3, wherein the compound of Formula Ila is:

The process of claim 3, wherein the compound of Formula Ila

The process of claim 4, wherein the compound of Formula III

16. The process of claim 5, wherein the compound of Formula IV is:

17. The process of any one of claims 1-16, further comprising recovering said PF1022A derivative compound from said culture.

18. The process of any one of claims 1-17, wherein the carbon source comprises a carbohydrate.

19. The process of any one of claims 1-18, wherein the nitrogen source comprises inorganic or organic nitrogen-containing compounds.

20. The process of any one of claims 1-19, wherein said microorganism strain is a fungal strain.

21. The process of claim 20, wherein said fungal strain belongs to the genus Xylaria or the genus Rosellinia.

22. The process of any one of claims 1-21, wherein said producing comprises accumulating said PF1022A derivative compound in the resulting culture.

23. The process of any one of claims 1-22, wherein said compound of Formula I is enantiomerically pure.

24. A compound of Formula IV:

IV

or a salt thereof,

wherein R is halo. , ι

The compound of claim 24, wherein R is bromo or chloro.

26. The compound of claim 24, wherein R1 is chloro.

27. Use of a compound of Formula IV:

or a salt thereof,

wherein R1 is halo;

in a method of producing a PF1022A derivative compound.

28. The use of claim 27, wherein the PF1022A derivative compound is a compound of Formula I:

wherein X1, X2, X3 and X4, at each occurrence, are independently selected from N and CH; and

R1 and R2 are each independently selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.

Description:
Methods for Production of PF1022A Derivatives

BACKGROUND

Every year the loss of valuable livestock/fishes to invertebrate parasites, both endoparasites (nematodes or helminths) and ectoparasites (flies, ticks, mites and sea lice) totals >£34 billion globally. This is despite the fact that farmers spend ~£4.5 billion globally on compounds to protect their animals from parasites (Parasitol Res (2005) 97:S11-S16, - Jeschke et a!. ). New products are continually needed as new parasites emerge and existing parasites evolve resistance to current treatments.

PF1022A is a fungally-derived non-ribosomal peptide natural product octadepsipeptide anthelmintic agent. Emodepside, a complex semi-synthetic derivative of PF1022A, is a resistance breaking anthelmintic used exclusively for the more profitable companion animal market owing to high cost of production (Ohyama et al, Biosci., Biotechno., Biochem., 2011, 75,

PF1022A Emodepside

The unique and highly complex core structure of the PF1022A natural product has provided challenging opportunities for synthesis. Conversion of PF1022A to the bis-4- morpholino derivative (emodepside) entails low-yielding chemistry such as nitration of the phenyl rings followed by reduction and subsequent functionalization. In addition to the poor chemical yields arising from nitration (or acetylation, another route), the generation of regioisomers further reduces the yield of useful intermediate and necessitates expensive purification of the desired para-regioisomers. Lower cost of goods for emodepside would enable the use of the compound in livestock herds, an application prohibited by its present high cost of manufacture. Additionally, with the increase of insect resistance, new PF1022A derived compounds and methods for their synthesis are needed. The recent demonstration that emodepside may have utility in the treatment of African river disease in humans only sharpens the need for new methods for the preparation of emodepside and related structures.

Semisynthetic routes to the bis-hydroxy PF1022A derivative, PF1022H, have recently been described by Scherkenbeck et al. (Bioorg. Med. Chem. 2016, 24, 873-876). One route proceeds from PF1022A by nitration of the phenyl rings followed by reduction to the amine and diazotization followed by hydrolysis to the phenol. A second route utilizes Friedel-Crafts acylation of the phenyl rings followed by Baeyer-Villiger oxidation and subsequent ester cleavage. The nature of the electrophilic substitution chemistry results in mixtures of para and meta isomers, with para predominating. The para-bis- ydroxy compound PF1022H has been shown to be a useful intermediate for the preparation of lipophilic PF1022A derivatives. (Ohyama 2011).

There remains a need for new methods and compound useful for the preparation of PF1022A derivatives. SUMMARY

It is a purpose of the present invention to provide methods for the regioselective preparation of PF1022A derivatives. In addition, methods disclosed herein may be used in the preparation of new PF1022A derivatives, which may be useful as antihelmintic agents.

Disclosed herein is a process for the production of a PF1022A derivative compound of Formula I:

wherein X 1 , X 2 , X 3 and X 4 , at each occurrence, are independently selected from N and CH; and R 1 and R 2 are each independently selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl; comprising: incubating a microorganism strain capable of producing PF1022A derivative compounds in a culture medium to produce a resulting culture, said culture medium containing carbon and nitrogen sources, and further providing to said culture medium a feedstock comprising a compound of Formula II, Formula III, Formula IV or Formula V:

II III IV V wherein the dotted line represents an optional double bond;

X 2 , X 3 , and X 4 are independently selected from N and CH; and

R 1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl; and R 3 is selected from the group consisting of hydrogen, amino, and sulfhydryl, thereby producing said PF1022A derivative compound in the resulting culture.

In some embodiments, the feedstock comprises a compound of Formula II. In some embodiments, the feedstock comprises a compound of Formula II, and the compound of Formula II is a compound of Formula Ila:

I la

wherein X 2 , X 3 , and X 4 are independently selected from N and CH; and

R 1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl.

In some embodiments, the feedstock comprises a compound of Formula III.

In some embodiments, the feedstock comprises a compound of Formula IV.

In some embodiments, the feedstock comprises a compound of Formula V.

In some embodiments of any of the above, R 2 is H.

In some embodiments of any of the above, R 1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino.

In some embodiments of any of the above, R 1 is halo.

In some embodiments of any of the above, R 1 is fluoro.

In some embodiments of any of the above, R 1 is chloro.

In some embodiments of any of the above, R 1 is bromo.

In some embodiments, the compound of Formula Ila is:

In some embodiments, the compound of Formula Ila is:

In some embodiments, the compound of Formula III is:

In some embodiments, the compound of Formula IV is:

In some embodiments of any of the above, the process further includes recovering the PF1022A derivative compound from said culture.

In some embodiments of any of the above, the carbon source comprises a carbohydrate.

In some embodiments of any of the above, the nitrogen source comprises inorganic or organic nitrogen-containing compounds.

In some embodiments of any of the above, the microorganism strain is a fungal strain.

In some embodiments of any of the above, the fungal strain belongs to the genus Xylaria or the genus Rosellinia.

In some embodiments of any of the above, the producing comprises accumulating said PF1022A derivative compound in the resulting culture.

In some embodiments of any of the above, the compound of Formula I is enantiomerically pure.

Compounds of Formula II, Formula III, Formula IV, or Formula V taught herein that are useful for such feedstocks are also provided.

In some embodiments, the com ound is a compound of Formula IV:

IV

or a salt thereof,

wherein R 1 is halo. In some embodiments, R 1 is bromo or chloro. In some embodiments, R 1 is fluoro. In some embodiments, R 1 is chloro.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts LC-MS chromatograms of the dichloro derivative of PF1022A prepared by the disclosed process. FIG. 2 depicts LC-MS chromatograms of the mono-chloro derivative of PF1022A prepared by the disclosed process.

FIG. 3 depicts mass spectrometry patterns for the mono-chloro (left) and dichloro (right) derivatives of PF1022A prepared by the disclosed process.

DETAILED DESCRIPTION OF EMBODIMENTS

Provide herein are processes and methods useful for the preparation of cyclooctadepsipeptide compounds such as the approved animal anthelmintic compound emodepside.

A. DEFINITIONS

Processes and methods in accordance with the present disclosure include those generally described above and below, and are further illustrated by the embodiments, sub-embodiments, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated.

As described herein, compounds of the invention may be substituted with one or more substituents, such as those generally described herein, such as those illustrated generally herein, or as exemplified by particular classes, subclasses, and species of the invention. In general the term "substituted" refers to the replacement of hydrogen in a given structure with a specified substituent. When a structure is described as "substituted" without further specification, it is understood that said group may be substituted with one or more substituents chosen from the group consisting of hydrogen, halo, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, aryl, heteroaryl, cycloalkyl, and heterocyclyl.

Unless indicated otherwise, nomenclature used to describe chemical groups or moieties as used herein follow the convention where, reading the name from left to right, the point of attachment to the rest of the molecule is at the right hand side of the name. For example, the group "alkylamino" is attached to the rest of the molecule at the amino end, whereas the group "aminoalkyl" is attached to the rest of the molecule at the alkyl end.

Unless indicated otherwise, where a chemical group is described by its chemical formula, including a terminal bond moiety indicated by it will be understood that the attachment is read from left to right. For example, -C(0)Ci_ 6 alkyl is attached to the rest of the molecule at the carbonyl end. "Alkyl" or "alkyl group", as used herein, means a straight-chain (i.e. , unbranched), or branched hydrocarbon chain that is completely saturated. In some embodiments the alkyl has 1 , 2, 3, 4, 5 or 6 carbon atoms. In certain embodiments, alkyl groups contain 1-6 carbon atoms (Ci_ 6 alkyl). In certain embodiments, alkyl groups contain 1-4 carbon atoms (C 1-4 alkyl). In certain embodiments, alkyl groups contain 1-3 carbon atoms (C 1 -3 alkyl). In still other embodiments, alkyl groups contain 2-3 carbon atoms (C 2-3 alkyl), and in yet other embodiments alkyl groups contain 1-2 carbon atoms (C 1-2 alkyl).

"Ar" or "aryl" refer to an aromatic carbocyclic moiety having one or more closed rings. Examples include, without limitation, phenyl, naphthyl, anthracenyl, phenylanthracenyl, biphenyl, and pyrenyl.

"Heteroaryl" refers to a cyclic moiety having one or more closed rings, with one or more heteroatoms (oxygen, nitrogen, or sulfur) in at least one of the rings, wherein at least one of the rings is aromatic, and wherein the ring or rings may independently be fused, and/or bridged. Examples include, without limitation, pyridyl, quinolinyl, isoquinolinyl, indolyl, furyl, thienyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl, quinoxalinyl, pyrrolyl, indazolyl, thiazolyl, oxazolyl, and isoxazolyl.

"Alkoxy" refers to an alkyl or cycloalkyl group, as herein defined, attached to the principal carbon chain through an oxygen atom. Representative examples of "alkoxy" include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, and hexyloxy.

"Hydroxy" or "hydroxyl" refers to an -OH group.

"Halogen" or "halo" refers to one or more of fluoro, chloro, bromo, and iodo.

An "amine" as used herein refers to a primary, secondary or tertiary amine. An "amino" group refers to the primary amine group -NH 2 . Examples of secondary or tertiary amines include, but are not limited to, alkylamino, dialkylamino, alkylaminoalkyl, dialkylaminoalkyl, etc.

"Carboxylate" refers to a salt or ester of a carboxylic acid moiety.

"Carboxyalkyl" refers to a carboxylic acid group attached to the principal carbon chain or molecule through an alkyl group. The carboxylic acid group may be present as the free acid, salt, or an ester.

"Cycloalkyl" as used herein, refers to a saturated cyclic hydrocarbon group containing from 3 to 8 carbons or more. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

"Heteroatom" refers to O, S or N. "Heterocycle" or "heterocyclyl" as used herein, means a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle containing at least one heteroatom in a ring.

"Monocyclic heterocycle" means a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom, and which is not aromatic. Representative examples of monocyclic heterocycle include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3- dioxanyl, 1,3-dioxolanyl, dihydropyranyl (including 3,4-dihydro-2H-pyran-6-yl), 1,3- dithiolanyl, 1,3-dithianyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, oxadiazolidinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl (including tetrahydro-2H-pyran-4-yl), tetrahydrothienyl, thiadiazolidinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomor- pholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl.

"Bicyclic heterocycle" means a monocyclic heterocycle fused to an aryl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl or cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle. Representative examples of bicyclic heterocycles include, but are not limited to, 3,4-dihydro-2H-pyranyl, 1,3- benzodioxolyl, 1,3-benzodithiolyl, 2,3-dihydro-l,4-benzodioxinyl, 2,3-dihydro-l- benzofuranyl, 2,3-dihydro-l-benzothienyl,2,3-dihydro-lH-indolyl, 3,4-dihydroquinolin- 2(lH)-one and 1,2,3,4- tetrahydroquinolinyl.

"Tricyclic heterocycle" means a bicyclic heterocycle fused to an aryl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl or cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle. Representative examples of tricyclic heterocycles include, but are not limited to, 2,3,4,4a,9,9a-hexahydro- lH-carbazolyl, 5a,6,7,8,9,9a- hexahydro-dibenzo[b,d]furanyl, and 5a,6,7,8,9,9a-hexahydrodibenzo[b,d]thienyl.

"Azido" refers to the group -N 3 .

"Cyano" refers to the group -CN.

"Isonitrile" refers to the group -NC.

"Nitro" refers to the group -N0 2 .

"Vinyl" refers to the group -C=C.

Unless otherwise stated, structures depicted herein are also meant to include all enantiomeric, diastereomeric, and geometric (or conformational) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Tautomeric forms include keto-enol tautomers of a compound. In addition, unless otherwise stated, all rotamer forms of the compounds of the invention are within the scope of the invention. Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or probes in biological assays.

"Isomers" refers to compounds having the same number and kind of atoms and hence the same molecular weight, but differing with respect to the arrangement or configuration of the atoms. It will be understood, however, that some isomers or racemates or others mixtures of isomers may exhibit more activity than others. "Stereoisomers" refers to isomers that differ only in the arrangement of the atoms in space. "Diastereoisomers" refers to stereoisomers that are not mirror images of each other. "Enantiomers" refers to stereoisomers that are non-superimpo sable mirror images of one another.

In some embodiments, enantiomeric compounds taught herein may be "enantiomerically pure" isomers that comprise substantially a single enantiomer, for example, greater than or equal to 90%, 92%, 95%, 98%, or 99%, or equal to 100% of a single enantiomer.

In some embodiments, enantiomeric compounds as taught herein may be stereochemically pure. "Stereochemically pure" as used herein means a compound or composition thereof that comprises one stereoisomer of a compound and is substantially free of other stereoisomers of that compound.

In some embodiments, "R" and "S" as terms describing isomers are descriptors of the stereochemical configuration at an asymmetrically substituted carbon atom. The designation of an asymmetrically substituted carbon atom as "R" or "S" is done by application of the Cahn-Ingold-Prelog priority rules, as are well known to those skilled in the art, and described in the International Union of Pure and Applied Chemistry (IUPAC) Rules for the Nomenclature of Organic Chemistry. B. METHODS FOR PRODUCTION

An objective of the present invention is to provide a method for producing PF1022A derivatives, by a direct fermentation method. Accordingly, provided herein are methods for preparing cyclooctadepsipeptide compounds of Formula I:

I wherein X 1 , X 2 , X 3 and X 4 , at each occurrence, are independently selected from N and

CH;

R 1 and R 2 are independently selected from the group consisting of hydrogen, halo, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl. In some embodiments, one of R 1 and R 2 is hydrogen and the other is not hydrogen. In some embodiments, neither R 1 nor R 2 is hydrogen.

In some embodiments, the present invention provides for the biosynthetic production of cyclooctadepsipeptides of Formula I by providing a feedstock with a compound as taught herein to a microrganism strain capable of producing PF1022A derivative compounds. The microorganism strain may be cultured according to an ordinary method by appropriately selecting a medium, culture conditions, and the like. The medium may be supplemented with a carbon source and nitrogen source that can be utilized by the microorganism strain, inorganic salts, various vitamins, various amino acids such as glutamic acid and asparagine, trace nutrients such as nucleotides, selectable agents such as antibiotics, etc. See, e.g. , U.S.

Patent Nos. 6,043,058 to Ohyama et al. and 7,285,404 to Midoh et al.

Any kind of carbon source and nitrogen source may be used in the medium as long as they can be utilized by the microorganisms of the present invention. As the carbon source, for example, various carbohydrates, such as sucrose, glucose, starch, glycerin, fructose, maltose, mannitol, xylose, galactose, ribose, dextrin, animal and plant oils and the like, or hydrolysates thereof, can be used. The preferable concentration generally is from 0.1% to 5% of the medium. As the utilizable nitrogen source, for example, animal or plant components, or exudates or extracts thereof, such as peptone, meat extract, com steep liquor, and defatted soybean powder, organic acid ammonium salts such as succinic acid ammonium salts and tartaric acid ammonium salts, urea, and other various inorganic or organic nitrogen-containing compounds can be used.

Inorganic salts, for example, those which can produce sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphate, sulfate, and/or other ions may also be used.

Any medium which contains other components, such as cells, exudates or extracts of microorganisms such as yeasts, and fine plant powders, may be used as long as these other components do not unduly interfere with the growth of the microorganism and the production and accumulation of the PF1022A derivative of the present invention. When a mutant strain having a nutritional requirement is cultured, a substance to satisfy its nutritional requirement is added to the medium. However, this kind of nutrient may not necessarily be added when a medium containing natural substances is used.

The pH of the medium may be, for example, about 6 to 8. Incubation may be carried out by a shaking culture method under an aerobic condition, an agitation culture method with aeration or an aerobic submerged culture method. An appropriate incubation temperature in some embodiments may be 15°C to 40°C, more preferably about 26°C to 37°C.

Efficient production of the PF1022A derivative of the present invention may depend on the medium, culture conditions, or microorganism strain used. However, the maximum accumulation may generally be attained in 2 to 25 days by any culture method. The incubation may be terminated when the amount of the PF1022A derivative compound of the present invention in the medium reaches its peak, at which time the compound may be isolated from the culture and optionally further purified.

The PF1022A derivative compound of the present invention accumulated in the culture thus obtained may be contained in the cells of the microorganism and/or in the culture filtrate. Accordingly, in some embodiments the PF1022A derivative compound may be recovered from both culture filtrate and microorganism cells by separating the culture into fractions, e.g., by centrifugation.

The PF1022A derivative can be recovered from the culture filtrate according to ordinary procedures known to those skilled in the art. The procedures can be carried out singly, in combination in a certain order, or repeatedly. For example, extraction, filtration, centrifugation, salting out, concentration, drying, freezing, adsorption, detaching, means for separation based on the difference in solubility in various solvents, such as precipitation, crystallization, recrystallization, reverse solution, counter- current distribution, and chromatography, can be used.

The PF1022A derivative may be recovered from inside the cells of the microorganism, for example, by cell lysis (e.g., smashing or pressure disruption), cell recovery (e.g., filtration and centrifugation) and lysis, and purification (e.g., salting out and solvent precipitation), according to methods known in the art.

The crude PF1022A derivative obtained may be further purified according to methods known in the art, for example, by column chromatography using a carrier such as silica gel and alumina or reverse-phase chromatography using an ODS carrier.

In some embodiments, the feedstock com rises a compound of Formula II:

I I

wherein the dotted line represents an optional double bond, X 1 , X 2 , X 3 , and X 4 are independently chosen from N and CH; and R 1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl. In some embodiments, R 1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino.

In some embodiments, the feedstock comprises a compound of Formula Ila:

Ila wherein X 2 , X 3 , and X 4 are independently chosen from N and CH; and R 1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl. In some embodiments, R 1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino. In some embodiments, the feedstock com rises a compound of Formula lib:

lib

wherein X 2 , X 3 , and X 4 are independently chosen from N and CH; and R 1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl. In some embodiments, R 1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino. In some embodiments, the carbon in Formula lib marked with an asterisk has the R configuration. In some embodiments, the carbon in Formula lib marked with an asterisk has the S configuration. In some embodiments, the compound of Formula lib is a mixture of R and S isomers. In some embodiments of the formulas, X 1 , X 2 , X 3 and X 4 are CH. In some embodiments, R 1 is methoxy or cyano. In some embodiments, X 1 , X 2 , X 3 and X 4 are CH and R 1 is methoxy or cyano.

In some embodiments, the feedstock comprises a compound of Formula III:

I II

wherein R 1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl. In some embodiments, R 1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino. In some embodiments, R 1 is amino or nitro. In some embodiments, the carbon in Formula III marked with an asterisk has the R configuration. In some embodiments, the carbon in Formula III marked with an asterisk has the S configuration. In some embodiments, the compound of Formula III is a mixture of R and S isomers.

In some embodiments, the feedstock com rises a compound of Formula IV:

wherein R 1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl. In some embodiments, R 1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino. In some embodiments, R 1 is amino or nitro. In some embodiments, R 1 is halo. In some embodiments, R 1 is bromo or chloro. In some embodiments, R 1 is chloro. In some embodiments, the carbon in Formula IV marked with an asterisk has the R configuration. In some embodiments, the carbon in Formula IV marked with an asterisk has the S configuration. In some embodiments, the compound of Formula IV is a mixture of R and S isomers.

In some embodiments, the feedstock com rises a compound of Formula V:

V

wherein R 1 is selected from the group consisting of hydrogen, halo, alkoxy, cyano, isonitrile, carboxylate, carboxyalkyl, nitro, hydroxyl, amine such as amino, protected amino (e.g., protected by standard amino protecting groups such as Boc, CBz, etc.), azido, vinyl, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, and substituted or unsubstituted heterocyclyl; and R 3 is selected from the group consisting of hydrogen, amino, and sulfhydryl. In some embodiments, R 1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino. In some embodiments, R 1 is selected from the group consisting of halo, amino, hydroxyl, nitro, and morpholino and R 3 is H. In some embodiments, R 1 is halo and R 3 is H.

In some embodiments, the microorganism strain is a fungal strain capable of producing compound PF1022A and derivatives thereof. In some embodiments, the fungal strain is a fungal strain belonging to the genus Xylaria or the genus Rosellinia of the family Xylariaceae. One preferred example of a fungal strain capable of producing PF1022A substance or derivatives thereof is the PF1022 strain deposited at the National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Tsukuba-shi, Japan, under the accession number FERM BP-2671. The myco logical properties of BP-2671 are described for example in U.S. Patent No. 5,1 16,815 and the Journal of Antibiotics, 1992, Vol. 45, pg. 692. See also US Patent Application Publication No. 2011/0262969 to Harder et al.

In some embodiments, the PF1022A-producing strains may be labile in their properties. Thus, for example, BP-2671 itself, or any mutant derived from this strain, phenotypic conjugation (either spontaneously generated or artificially induced), or genetic recombinant of said strain may be used in practicing the process of the invention, if it can produce PF1022A and the described derivatives thereof.

In an embodiment, the PF1022A-producing fungal strain is cultivated in a culture medium containing such ordinary carbon source and nitrogen source which can generally be utilized as nutrients for microorganisms, as noted above. Such nutrients may be used which are known to have been used for the cultivation of fungi. Non-limiting examples of carbon sources which may be used with the PF1022A-producing fungal strain include glucose, sucrose, starch syrup, dextrin, starch, glycerol, molasses, animal oils, and vegetable oils. Non-limiting examples of nitrogen sources which may be used with the PF1022A-producing fungal strain include soybean flour, wheat germ, corn steep liquor, cotton seed oil, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, and urea. Inorganic salts may be added to the medium, as previously described herein. In addition, inorganic and organic substances capable of promoting the growth of the fungal strain may also be added in an appropriate amount.

In some embodiments for the methods of cultivation of the PF1022A-producing fungal strain, a cultivation method carried out under aerobic conditions is suitable. In some embodiments, a cultivation method under submerged conditions is preferable. For cultivation of the PF1022A-producing fungal strain, a temperature range of 15-30°C may be suitable. In some embodiments, a temperature of about 26°C is preferable.

In shake- or tank-cultivation, the production of PF1022A derivatives typically arrives at a maximum accumulation in 2-10 days. The skilled worker will appreciate that the incubation period required for maximum accumulation may vary depending on the composition of the culture medium and the cultivation conditions employed. Cultivation may be discontinued when production of the PF1022A derivative substance has reached its peak. The resulting culture may then be separated by filtration or by centrifugation to provide a solid portion (pellet) including the cultured cells and other solid materials, and a broth filtrate. The filtering operation may be performed using filtering aids known in the art, such as diatomaceous earth.

The recovery of PF1022A derivative substances produced by the cultivation of microbial or fungal strains may be effected by the use of one or more techniques for the isolation of biological materials well known to those skilled in biochemistry or microbiology. Non-limiting examples of such techniques suitable for use in the present invention include solvent extraction or adsorption, ion-exchange resin treatment, partition column chromatography, gel filtration, dialysis, and techniques of precipitation. These methods may be used alone or in appropriate combination.

In some embodiments, the PF1022A derivative substances are relatively insoluble in water and thus exist largely in the cultured cells rather than the broth filtrate. PF1022A derivative substances may be extracted from the cultured cells with an organic solvent or an aqueous organic solvent. Organic solvents which may be used for the purpose include by way of non- limiting examples methanol, ethanol, ethyl acetate, acetone, acetonitrile, and halogenated solvents such as dichloromethane and chloroform. Any of these organic solvents or similar organic solvents may be utilized as appropriate as solvent-water mixtures.

Purification methods which may be used to isolate the PF1022A derivative substances of the invention include chromatographic methods with silica gel or alumina as an adsorbent, as well as chromatographic methods with a gel filtration agent such as Sephadex LH-20 (Pharmacia Co.). The PF1022A derivative substances of the present invention may be further purified by recrystallization. Recrystallization may be from single solvent or mixed solvents. Non-limiting examples of single solvents include methanol, ethanol, ethyl acetate, acetone, diethyl ether, methyl tert-butyl ether, and methylene chloride. Examples of mixed solvents include, without limitation, methanol-water, ethanol- water, ethyl acetate-hexanes, and diethyl ether-hexanes.

In some embodiments the process disclosed herein comprises the steps of separating the cultured cells from the produced culture, and extracting the separated cultured cells with an organic solvent or an aqueous solvent, to obtain an extract containing the PF1022A derivative compound. In a further embodiment, the obtained extract is concentrated to provide the PF1022A derivative compound, which may be further purified as described herein.

In an embodiment of the present invention, there is provided a process for the preparation of PF1022A derivatives, the process comprising incubating a PF1022A-producing strain, in a culture medium containing carbon and nitrogen sources and in the presence of a 3-(4-substituted- phenyl)-2-oxopropanoic acid of Formula Ila, or a salt thereof. In some embodiments, the 3-(4- substituted-phenyl)-2-oxopropanoic acid of Formula Ila is present as a sodium salt. In some embodiments, the 3-(4-substituted-phenyl)-2-oxopropanoic acid of Formula Ila is present as a potassium salt. In some embodiments, the 3-(4-substituted-phenyl)-2-oxopropanoic acid of Formula Ila is present as a free acid.

In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium between 6 hours and 96 hours after inoculation of the culture medium with the microorganism. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium 6 hours after inoculation of the culture medium. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium 24 hours after inoculation of the culture medium. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium 48 hours after inoculation of the culture medium. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium 72 hours after inoculation of the culture medium. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium 96 hours after inoculation of the culture medium.

In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium at more than one time point. For example, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V may be added to the culture medium at times 6 h and 24 h post-inoculation of the culture medium with the microorganism; 6h, 24h and 48h post inoculation; 6h, 24h, 48h and 72 h post-inoculation; 6h, 24h, 48h, 72h and 96 h post-inoculation; 48h and 72h post-inoculation, and 48h, 72h and 96h post-inoculation. These examples are intended as non-limiting examples of embodiments of the present invention, and the skilled worker will understand that choice of time points may depend upon the particulars of the culture conditions.

In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium as a methanol solution feedstock. In some embodiments, the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added to the culture medium as a methanol solution feedstock at a concentration of between 2 and 10 mM. In some embodiments the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V is added at a concentration of 2mM; 4 mM; 6 mM; 8 mM; or 10 mM. Any of these concentrations of the compound of Formula II, Formula Ila, Formula lib, Formula III, Formula IV, or Formula V may be added once, or more than once, to the culture medium following inoculation.

In some embodiments, the microorganism is fungal strain FERM BP-2671 as the microorganism capable of producing PF1022A derivative compounds.

The present invention is further illustrated by the following non-limiting examples. EXAMPLES

Strain PERM BP-2671 (NBRC 33096) was inoculated from agar plugs and cultured in seed medium (2.0 % soluble starch, 1 .0 % glucose, 0.6 % wheat germ, 0.5 % polypeptone. 0.3 % yeast extract, 0.2 % soybean cake (Nutrisoy), 0.2 % calcium carbonate) in a baffled Erienmeyer flask at 120 rpm, 5 cm throw, 26°C for 3 days. A 5 % inoculum was transferred to production medium (6 % starch syrup ( maltose syrup), 2.6 % soluble starch. 2 % wheat germ, 1 % Pharma media (Archer Daniels Midland Co.), 0.2 % magnesium sulfate heptahydratc, 0.2 % calcium carbonate. 0.3 % sodium chloride) and incubated at 200 rpm, 5 cm throw, 26°C for 12-14 days. After incubation, samples were extracted with an equal volume of methanol, mixed for 30 mins, centrifuged and 150 μΐ aiiquots were transferred to HPLC vials for analysis.

Analysis method A :

Analyses were performed on an Agilent 1 1 00 HPLC connected to Waters ZQ single quadrupole MS. the HPLC column was a Waters XSelect CSH C 18 (2.1 mm x 50 mm. 3.5 μηι), fitted with a Waters VanGuard CSH CI 8 precolumn (2.1 mm x 5 mm, 3.5 μηι), and operated at

60 °C, 1.0 mL/min flow rate giving a system back pressure of 230 bar at injection. Mobile Phase Solution A was 10 mM ammonium formate / 0.2 % formic acid (pi 1 2.9), Mobile Phase Solution B was 95 % acetonitrile / 5 % water / 0.17 % formic acid. Sample injection volumes were typically 5 μ L using the following gradient, 0.00 min, 5 % B, 1 .0 mL/min; 0.10 min, 5 % B, 1.0 mL/min; 0.20 min, 50 % B, 1.0 mL/min; 9.30 min, 80 % B, 1.0 mL/min; 9.50 min, 95 % B, 1.0 mL/min; 10.50 min, 95 % B, 1.0 mL/min; 10.60 min, 95 % B, 1.5 mL/min; 1 1.0 min, 95 % B, 1 .5 mL/min; 1 1 .05 min, 5 % B, 1 .5 mL/min; 1 1 .5 min, 95 % B, 1 .5 mL min and a 1 .5 min injection cycle for equilibration. The MS was operated in electrospray positive ion mode gathering data from 400- 1 300 m/z and UV data were gathered betw een 230 - 400 nm and 230 nm used for PF1022A data analysis and calibration.

Analysis method B:

Analyses were performed on an Agilent 1 100 HPLC connected to Waters ZQ single quadrupole MS, the HPLC column was a Waters Atlantis dC 1 , (4.6 mm x 20 mm. 3 urn), fitted with an Acquity in-line column filter, 0.2 urn and operated at 60 °C, 2.0 ml min. Mobile Phase Solution A was 10 mM ammonium formate / 0.2 % formic acid (pH 2.9), Mobile Phase Solution B was 95 % acetonitrile 1 5 % water / 0. 1 7 % formic acid. Sample injection volumes were typically 5 μ L using the following gradient, 0.00 min, 5 % B, 2.0 m I 'm in; 0.05 min, 5 % B, 2.0 ml/min; 0.1 min, 50 % B, 2.0 ml min; 3.9 min, 80 % B, 2.0 ml min; 4.0 min, 95 % B, 2.0 mi min; 5.0 min, 95 % B, 2.0 ml min, 5. 1 min, 5 % B, 2.0 ml min; 6.0 min, 5 % B, 2.0 ml min. The MS was operated in electrospray positive ion mode gathering data from 400- 1 00 m/z and UV data were gathered between 230 400 nm and 230 nm used for PF 1022A data analysis and calibration.

For directed biosynthesis experiments, cultures were grown as described as above. The feedstock compound (as set out in Table 1 below) was dissolved in methanol to a concentration of 280 mM and then added to the production medium at 2 mM final concentration at 72 hours post inoculation. Following the addition of feedstock, cultures were incubated at 200 rpm, 5 cm throw, 26°C for 7- 1 0 days.

After incubation, samples of broth and cells were extracted with an equal volume of methanol, mi ed for 30 mins, centrifuged and 1 50 μΐ aliquots were transferred to HPLC v ials for analysis. The samples were analysed using the LC-MS method described above (method A or method B), and the results are summarized in Table 1.

Table 1

3-(4-Hydroxyphenyl)-2-oxopropanoic acid

7.3 (Method A) 984.5

(R)-3-(4-Fiuorophenyl)-2-hydroxypropanoic acid

7.3 (Method A) 1002.6

OH

(R)-3-(4-Fluorophenyl)-2-hydroxypropanoic acid

7.3 (Method A) 984.5

3-(4-fluorophenyl)-2-oxopropanoic acid

7.3 (Method A) 1002.6

3-(4-fluorophenyl)-2-oxopropanoic acid 7.9 (Method A) I 1000.5 I Ci I H

These results shown in Table 1 demonstrate that the feedstock compounds can be incorporated into the final products of the fungal synthesis.

Substrate feeds utilized in the present invention may be prepared by methods known to those of skill in the art of organic synthesis. By way of non-limiting examples, embodiments of substrate feeds may be prepared from substituted phenylalanine compounds as shown below in Scheme I.

Scheme I

Synthesis of 3-(4-Chlorophenyl)-2-hydroxypropanoic acid

Sulfuric acid (34 mL) in water (230 mL) was added slowly to 4-chlorophenylalanine (10.0 g, 50 mmol) in water (200 mL) and cooled to 0 °C. A solution of sodium nitrite (11.75 g, 170 mmol) in water (82 mL) was then added, maintaining the internal temperature < 7 °C. The mixture was allowed to warm to room temperature overnight, diluted with ethyl acetate (500 mL) and the layers separated. The aqueous phase was extracted with ethyl acetate (2 x 500 mL), and the combined organic layers were dried over magnesium sulphate and concentrated under reduced pressure. The crude material was purified by reverse phase column chromatography (Biotage Isolera, C-18 3 x 120 g, 20-80% MeCN with formic acid in H 2 0 0.1% formic acid). The relevant fractions were combined and extracted with dichloromethane (3 x 500 mL). The organics were dried over magnesium sulphate, and concentrated under reduced pressure to yield 3-(4-chlorophenyl)-2-hydroxypropanoic acid as a white powder, 1.74 g, 18%>. UPLC (CSH C18, Short acid 2-95%): 0.62 min, 97.0 %, 199.0 (-ve, M-H)

1H NMR (CDCI3, 300 MHz): δ 7.32-7.17 (m, 4H), 4.51 (m, 1H), 3.23-2.90 (m, 2H)

Synthesis of Methyl 3-(4-chlorophenyl)-2-hydroxypropanoate

Concentrated sulfuric acid (5 drops) was added to 3-(4-chlorophenyl)-2- hydroxypropanoic acid (650 mg, 3.2 mmol) in methanol (20 mL) and heated under reflux for 3 hours. The solution was cooled to room temperature then concentrated under reduced pressure. The residue was dissolved in ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate (100 mL) and brine (100 mL), dried over magnesium sulphate, and concentrated under reduced pressure to yield methyl 3-(4-chlorophenyl)-2-hydroxypropanoate as a clear oil, 600 mg, 87 %.

UPLC (CSH_C18, Long acid 2-95 %): 0.72 min, 97.0 %

1H NMR (CDCI3, 300 MHz); δ 7.33-7.09 (m, 4H), 4.44 (t, 1H), 3.77 (s, 3H), 3.16-2.86 (m, 2H), 2.73 (s, 1H)

Synthesis of (2-Acetamidoethyl) 3-(4-chlorophenyl)-2-hydroxypropanethioate

Dicyclohexylcarbodiimide (660 mg, 3.2 mmol) and 1-hydroxybenzotriazole hydrate (432 mg, 3.2 mmol) were added to a solution of 3-(4-chlorophenyl)-2-hydroxypropanoic acid (650 mg, 3.2 mmol) and N-acetylcysteamine (1.16 g, 9.7 mmol) in acetonitrile (26 mL) and stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate (130 mL) and water (65 mL), filtered through celite, and the layers separated. The organics were washed with water (65 mL), then brine (65 mL), dried over magnesium sulphate and concentrated under reduced pressure. The crude material was purified by normal phase chromatography (0-5% methanol in dichloromethane). The fractions containing the desired product were concentrated under reduced pressure, re-dissolved in ethyl acetate, washed with 1M HCl, then brine, dried over magnesium sulphate and concentrated under reduced pressure. The resulting solid was then triturated with diethyl ether to give (2-acetamidoethyl) 3-(4-chlorophenyl)-2- hydroxypropanethioate as a colourless solid, 387 mg, 40%.

UPLC (CSH_C18, Long acid 2-95 %): 1.54 min, 97.0 % 1H NMR (CDCls, 300 MHz); δ 7.32-7.26 (m, 2H), 7.20-7.15 (m, 2H), 5.66 (br s, 1H),

4.50-4.43 (m, 1H), 3.52-3.32 (m, 2H), 3.18-2.90 (m, 4H), 2.73 (d, 1H), 1.95 (s, 3H).