阅读说明：本技术 制造高镜像选择性二级醇的方法 (Method for producing secondary alcohol with high mirror image selectivity ) 是由 游丞德 谢义簧 林书顗 赵晋升 谢殷程 赖明添 于 2020-02-21 设计创作，主要内容包括：本发明提供一种合成化合物OBI-3424R-form及S-form产物的新方法。通过在后续阶段再引进不稳定的磷酸盐结构,由化合物OBI-3424-5首次合成“R-form”化合物OBI-3423时,总产率为48％。利用五步骤化学酶合成反应的组合建立立体化学结构,以使光学纯度达99％。接着,由化合物OBI-3424-5制备“S-form”化合物OBI-3424时,总产率提升至54％。利用四步骤化学酶合成反应的组合建立立体化学结构,以使光学纯度达极佳的99％。(The invention provides a novel method for synthesizing a compound OBI-3424R-form and an S-form product. The first synthesis of the "R-form" compound OBI-3423 from the compound OBI-3424-5 by reintroducing the labile phosphate structure at a later stage gave an overall yield of 48%. The stereochemistry was established using a combination of five-step chemoenzymatic synthesis reactions to achieve 99% optical purity. Then, when the compound OBI-3424-5 is used to prepare the compound OBI-3424 of "S-form", the total yield is increased to 54%. The stereochemical structure is established by a combination of four-step chemoenzymatic synthesis reactions to achieve an excellent optical purity of 99%.)

1. A process for preparing a compound of formula 1, comprising:

step (1): in the presence of Borane (BH)₃) B-diisopinocampheylchloroborane (B-Chlorodiisopinocamphylborane) (DIP-chloride), (S) - (-) -1,1 '-Bi-2-naphthol ((S) - (-) -1,1' -Bi-2-naphthol), or sodium borohydride (NaBH)₄) Reacting a compound of formula 2 with a compound of formula 3 a; and

step (2): reacting the product of step (1) with:

(A) lipase acrylic resin (lipase acrylic resin), sodium carbonate (Na)₂CO₃) And one of isopropyl acetate (isopropenyl acetate) and 2,2,2-trifluoroethyl butyrate (2,2,2-trifluoroethyl butyrate); or

(B) Protease, sodium carbonate, and one of isopropyl acetate and 2,2,2-trifluoroethyl butyrate; and reacting the product of step (B) with sodium methoxide (sodium methoxide) in a methanol-containing environment,

wherein R isAn aliphatic chain or Rx;

wherein Rx is hydrogen, an unsubstituted or substituted cyclic group, an electron withdrawing group, or an electron withdrawing group.

2. The method of claim 1, wherein the cyclic group is an aromatic group, a cyclic saturated or partially unsaturated group, or a heterocyclic group; the electron withdrawing group is a halogeno group, a nitroso group, an aminocarbonyl group, a carboxyl group, an alkoxycarbonyl group, a formyl group, an acyl group, a haloformyl group, a trihalomethyl group, a cyano group, a nitrogen group, an ammonium group, an azido group, or a sulfonyl group; and the electron-pushing group is an alkyl group, a vinyl group, a phenyl group, an acyloxy group, an amido group, an alkylthio group, a mercapto group, a hydroxyl group, an alkoxy group or an amido group.

3. The method of claim 1, wherein the compound of formula 1 is a compound of formula 1a and the compound of formula 2 is a compound of formula 2 a:

4. the method of claim 3, wherein the product of step (1) is a compound of formula 5:

5. a process for preparing a compound of formula 1, comprising:

step (1 a): reacting a compound of formula 2 with a compound of formula 3a in an environment comprising borane, DIP-chloride, (S) - (-) -1,1' -bi-2-naphthol, or sodium borohydride;

step (1 b): reacting the product of step (1a) with acetic anhydride (Ac)₂O) reaction: and

step (2): reacting the product of step (1b) with:

(A) lipase acrylic resin and sodium carbonate; and reacting the product of step (a) with sodium methoxide in a methanol-containing environment; or

(B) A protease and sodium carbonate, wherein the protease is a sodium carbonate,

wherein R isAn aliphatic chain or Rx;

wherein Rx is hydrogen, an unsubstituted or substituted cyclic group, an electron withdrawing group, or an electron withdrawing group.

6. The method of claim 5, wherein the cyclic group is an aromatic group, a cyclic saturated or partially unsaturated group, or a heterocyclic group; the electron withdrawing group is a halogeno group, a nitroso group, an aminocarbonyl group, a carboxyl group, an alkoxycarbonyl group, a formyl group, an acyl group, a haloformyl group, a trihalomethyl group, a cyano group, a nitro group, an ammonium group, an azido group or a sulfonyl group; and the electron-pushing group is an alkyl group, a vinyl group, a phenyl group, an acyloxy group, an amido group, an alkylthio group, a mercapto group, a hydroxyl group, an alkoxy group or an amido group.

7. The method of claim 5, wherein the compound of formula 1 is a compound of formula 1a and the compound of formula 2 is a compound of formula 2 a:

8. the method of claim 7, wherein the product of step (1b) is a compound of formula 6:

9. a process for preparing a compound of formula 4, comprising:

step (1): reacting a compound of formula 2 with a compound of formula 3b in an environment comprising borane, DIP-chloride, (S) - (-) -1,1' -bi-2-naphthol, or sodium borohydride; and

step (2): reacting the product of step (1) with:

(A) lipase acrylic resin, sodium carbonate, and one of isopropyl acetate and 2,2, 2-trifluoroethylbutyrate; and reacting the product of step (a) with sodium methoxide in a methanol-containing environment; or

(B) Protease, sodium carbonate, and one of isopropyl acetate and 2,2, 2-trifluoroethylbutyrate,

wherein R isAn aliphatic chain or Rx;

wherein Rx is hydrogen, an unsubstituted or substituted cyclic group, an electron withdrawing group, or an electron withdrawing group.

10. The method of claim 9, wherein the cyclic group is an aromatic group, a cyclic saturated or partially unsaturated group, or a heterocyclic group; the electron withdrawing group is a halogeno group, a nitroso group, an aminocarbonyl group, a carboxyl group, an alkoxycarbonyl group, a formyl group, an acyl group, a haloformyl group, a trihalomethyl group, a cyano group, a nitro group, an ammonium group, an azido group or a sulfonyl group; and the electron-pushing group is an alkyl group, a vinyl group, a phenyl group, an acyloxy group, an amido group, an alkylthio group, a mercapto group, a hydroxyl group, an alkoxy group or an amido group.

11. The method of claim 9, wherein the compound of formula 4 is a compound of formula 4a and the compound of formula 2 is a compound of formula 2 a:

12. the method of claim 11, wherein the product of step (1) is a compound of formula 5:

13. a process for preparing a compound of formula 4, comprising:

step (1 a): reacting a compound of formula 2 with a compound of formula 3b in an environment comprising borane, DIP-chloride, (S) - (-) -1,1' -bi-2-naphthol, or sodium borohydride;

step (1 b): reacting the product of step (1a) with acetic anhydride (Ac)₂O) reaction: and

step (2): reacting the product of step (1b) with:

(A) lipase acrylic resin and sodium carbonate; or

(B) Protease and sodium carbonate; and reacting the product of step (A) with sodium methoxide in an environment containing methanol,

wherein R isAn aliphatic chain or Rx;

wherein Rx is hydrogen, an unsubstituted or substituted cyclic group, an electron withdrawing group, or an electron withdrawing group.

14. The method of claim 13, wherein the cyclic group is an aromatic group, a cyclic saturated or partially unsaturated group, or a heterocyclic group; the electron withdrawing group is a halogeno group, a nitroso group, an aminocarbonyl group, a carboxyl group, an alkoxycarbonyl group, a formyl group, an acyl group, a haloformyl group, a trihalomethyl group, a cyano group, a nitro group, an ammonium group, an azido group or a sulfonyl group; and the electron-pushing group is an alkyl group, a vinyl group, a phenyl group, an acyloxy group, an amido group, an alkylthio group, a mercapto group, a hydroxyl group, an alkoxy group or an amido group.

15. The method of claim 5, wherein the compound of formula 4 is a compound of formula 4a and the compound of formula 2 is a compound of formula 2 a:

16. the method of claim 15, wherein the product of step (1b) is a compound of formula 6:

17. an improved compound (R) -1- (3- (3-N, N-dimethylaminocarbonyl) phenoxy-4-nitrophenyl) -1-ethyl-N, N '-bis (ethylene) phosphoramide (1- (3- (3-N, N-dimethylamino) phenyl-4-nitrophenyl) -1-ethyl-N, N' -bis (ethylene) phosphoramidate)

And the compound (S) -1- (3- (3-N, N-dimethylaminocarbonyl) phenoxy-4-nitrophenyl) -1-ethyl-N, N' -bis (ethylene) phosphoramide

A method of yield or optical purity of (a), comprising:

step (a): asymmetric reduction;

step (b): lipase/protease selective protection;

step (c): adding phosphate; and

step (d): aziridine formation.

18. The method of claim 17, wherein step (b) further comprises:

step (i): transesterification of lipase;

step (ii): hydrolyzing with lipase;

step (iii): performing transesterification on protease; or

Step (iv): and (4) hydrolyzing by using protease.

Technical Field

The invention relates to synthesis and corresponding identification data of a compound OBI-3424 bulk drug designed as an anticancer small molecule prodrug.

Background

Cancer is one of the leading causes of illness and death in humans. Cancer therapy is challenging because it is difficult to kill cancer cells without also destroying or killing normal cells. Destroying or killing normal cells during cancer treatment is a cause of side effects in the patient and may limit the dose of anti-cancer drugs administered to cancer patients.

The aldehyde-ketone reductase family 1 member C3(AKR1C3) is an enzyme encoded by the human AKR1C3 gene. This gene encodes a member of the aldehyde-ketone reductase superfamily consisting of more than 40 known enzymes and proteins. The enzymes use NADH and/or NADPH as cofactors to catalyze the conversion of aldehydes and ketones to their corresponding alcohols.

Many Cancer cells overexpress AKR1C3 reductase (e.g., Cancer Res.2010,70: 1573-1584; Cancer Res.2010,66:2815-2825) relative to normal cells. There remains a need for compounds suitable for use in the treatment of cancer patients, including prodrugs activated by selective AKR1C3 reductase for use in the treatment of cancer patients. PCT patent application publication No. WO 2017/087428a1 discloses compounds of formula I or formula II:

or a salt, isotopic variant, pharmaceutically acceptable solvate or hydrate thereof. The aforementioned compounds have an enantiomeric excess (enantiomeric excess) of not less than 80%, not less than 90%, or not less than 95%.

PCT patent publication No. WO 2019/062919A1 discloses compounds (OBI-3424) and methods for treating leukemia. The properties of compound OBI-3424 are listed in Table 1.

Table 1: properties of Compound OBI-3424

Disclosure of Invention

It is an object of the present invention to provide a method for the synthesis and corresponding identification of the compound OBI-3424 drug substance, which is designed as a small molecule prodrug for anticancer.

In one aspect, the present invention provides a process for preparing a compound of formula 1, comprising:

step (1): in the presence of Borane (BH)₃) B-diisopinocamphinochlorilborane (B-chlorodiisophinochlorineborane) (DIP-chloride), (S) - (-) -1,1 '-Bi-2-naphthol ((S) - (-) -1,1' -Bi)-2-naphthol) or sodium borohydride (NaBH)₄) Reacting a compound of formula 2 with a compound of formula 3 a; and

step (2): reacting the product of step (1) with:

(A) lipase acrylic resin (lipase acrylic resin), sodium carbonate (Na)₂CO₃) And one of isopropyl acetate (isopropenyl acetate) and 2,2,2-trifluoroethyl butyrate (2,2,2-trifluoroethyl butyrate); or

(B) Protease, sodium carbonate, and one of isopropyl acetate and 2,2,2-trifluoroethyl butyrate; and reacting the product of step (B) with sodium methoxide (sodium methoxide) in a methanol-containing environment,

wherein R isAn aliphatic chain or Rx;

wherein Rx is hydrogen, an unsubstituted or substituted cyclic group, an electron withdrawing group or an electron withdrawing group.

In another aspect, the present invention provides a process for preparing a compound of formula 1, comprising:

step (1 a): reacting a compound of formula 2 with a compound of formula 3a in an environment comprising borane, B-diisopinocampheylchloroborane (DIP-chloride), (S) - (-) -1,1' -bi-2-naphthol, or sodium borohydride;

step (1 b): reacting the product of step (1a) with acetic anhydride (Ac)₂O) reaction: and

step (2): reacting the product of step (1b) with:

(A) lipase acrylic resin and sodium carbonate; and reacting the product of step (a) with sodium methoxide in a methanol-containing environment; or

(B) A protease and sodium carbonate, wherein the protease is a sodium carbonate,

wherein R isAn aliphatic chain or Rx;

wherein Rx is hydrogen, an unsubstituted or substituted cyclic group, an electron withdrawing group, or an electron withdrawing group.

In yet another aspect, the present invention provides a process for preparing a compound of formula 4, comprising:

step (1): reacting a compound of formula 2 with a compound of formula 3b in an environment comprising borane, DIP-chloride, (S) - (-) -1,1' -bi-2-naphthol, or sodium borohydride; and

step (2): reacting the product of step (1) with:

(A) lipase acrylic resin, sodium carbonate, and one of isopropyl acetate and 2,2,2-trifluoroethyl butyrate; and reacting the product of step (a) with sodium methoxide in a methanol-containing environment; or

(B) Protease, sodium carbonate, and one of isopropyl acetate and 2,2,2-trifluoroethyl butyrate,

wherein R isAn aliphatic chain or Rx;

wherein Rx is hydrogen, an unsubstituted or substituted cyclic group, an electron withdrawing group, or an electron withdrawing group.

In yet another aspect, the present invention provides a method of preparing a compound of formula 4, comprising:

step (1 a): reacting a compound of formula 2 with a compound of formula 3b in an environment comprising borane, DIP-chloride, (S) - (-) -1,1' -bi-2-naphthol, or sodium borohydride;

step (1 b): reacting the product of step (1a) with acetic anhydride (Ac)₂O) reaction: and

step (2): reacting the product of step (1b) with:

(A) lipase acrylic resin and sodium carbonate; or

(B) Protease and sodium carbonate; and reacting the product of step (A) with sodium methoxide in an environment containing methanol,

wherein R isAn aliphatic chain or Rx;

wherein Rx is hydrogen, an unsubstituted or substituted cyclic group, an electron withdrawing group or an electron withdrawing group.

Drawings

FIG. 1: compounds OBI-3423(R-form) and OBI-3424(S-form) were prepared.

FIG. 2: chiral HPLC data for Compound OBI-3423-6: (a) a mixture of OBI-3424-6R/S after treatment with CBS reduction, R/S84.25/15.22 (69.4% ee); (b) compound OBI-3424-6-LR (99.8% ee) after lipase enrichment treatment.

FIG. 3: chiral HPLC data obtained by asymmetric reduction of the S-form based compound OBI-3423-6 using (R) -CBS and borane in THF.

FIG. 4: chiral HPLC analysis data of the compound OBI-3423-6-LS enzyme reaction after lipase selective esterification with the compound OBI-3423-6-R/S mixture.

Detailed Description

Definition of

The following definitions are provided to assist the reader. Unless defined otherwise, technical terms, symbols, and other scientific or medical terms used herein have the same meaning as commonly understood by one of ordinary skill in the chemical and medical arts. In some instances, the general meanings defined herein are for ease of understanding and/or reference, and the definitions contained herein should not be construed as substantially different from the definitions understood by those of ordinary skill in the art.

All values (e.g., pH, temperature, time, concentration, weight, etc.) and ranges of values are approximations that may generally be increased (+) by minus (-)0.1, 1.0, or 10.0, as desired. All numerical values may be understood to include the antecedent "about". The reagents described herein are exemplary and equivalents thereof are known in the art to which this invention pertains.

Unless otherwise indicated, the terms "a" and "an" and "the" and the like herein include a plurality of the indicated objects. Thus, for example, a compound refers to one or more compounds, or at least one compound. Thus, the terms "a", "an", "one or more" and "at least one" may be used interchangeably herein.

As used herein, "comprising" means that the compositions and methods include the listed elements, but do not exclude other elements. "consisting essentially of, when used to define compositions and methods, shall mean excluding other elements of any importance to the composition or method. "combined from" shall mean that other ingredients above trace elements are excluded in the claimed compositions and substantial method steps. The embodiments defined by the foregoing transition words are all within the scope of the present invention. Thus, the methods and compositions may include other steps and ingredients ("comprising") or otherwise include unimportant steps and compositions ("consisting essentially of.. in combination") or refer only to the method steps or compositions ("consisting of.. in combination").

The terms "optically active" and "enantiomerically active" refer to a series of molecules having an enantiomeric excess (ee) of not less than about 10%, not less than about 20%, not less than about 30%, not less than about 40%, not less than about 50%, not less than about 60%, not less than about 70%, not less than about 80%, not less than about 90%, not less than about 91%, not less than about 92%, not less than about 93%, not less than about 94%, not less than about 95%, not less than about 96%, not less than about 97%, not less than about 98%, not less than about 99%, not less than about 99.5%, not less than about 99.8%, or not less than about 99.9%. In certain embodiments, the optically or enantiomerically active compound has an enantiomeric excess of not less than about 90%, not less than about 95%, not less than about 98%, or not less than about 99%.

The prefixes R and S used to describe optically active compounds are used to refer to the absolute configuration with respect to the chiral center of the molecule. The prefixes (+) or (-) are used to refer to the optical rotation (i.e., the direction of rotation of a plane of polarized light that is rotated by an optically active compound) of a compound. The prefix (-) means that the compound is left-handed, i.e., the compound rotates the plane of polarized light to the left-hand or counterclockwise. The prefix (+) refers to a compound that is dextrorotatory, i.e., the compound rotates the plane of polarized light to the right or clockwise. However, the signs (+) and (-) of optical rotation are independent of the absolute configuration of the molecules R and S.

The terms "optically pure" and "enantiomerically pure" refer to a series of molecules having an enantiomeric excess of not less than about 80%, not less than about 90%, not less than about 91%, not less than about 92%, not less than about 93%, not less than about 94%, not less than about 95%, not less than about 96%, not less than about 97%, not less than about 98%, not less than about 99%, not less than about 99.5%, not less than about 99.8%, or not less than about 99.9%. In certain embodiments, the optically or enantiomerically pure compound has an enantiomeric excess of not less than about 90%, not less than about 95%, not less than about 98%, or not less than about 99%. Enantiomeric excess values of compounds can be determined by standard methods by those of ordinary skill in the art, including, but not limited to, chiral chromatography (gas chromatography (GC), High Performance Liquid Chromatography (HPLC), Thin Layer Chromatography (TLC)) using an optically active stationary phase (isotopic dilution), electrophoresis, calorimetry (calorimetry), polarimetry (polarimetry), nuclear magnetic resonance resolution methods (NMR methods) using a chiral derivatization (chiral derivatization), and nuclear magnetic resonance methods using a chiral solvating agent (chiral solvating agent) or a chiral shifting agent (chiral shifting agent).

The terms "substantially pure", "substantially homogeneous", and the like, refer to sufficiently homogeneous so as to exhibit no immediately detectable impurities as determined by one of ordinary skill in the art using standard analytical methods, including, but not limited to, Thin Layer Chromatography (TLC), Gel Electrophoresis (GE), High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Nuclear Magnetic Resonance (NMR), and Mass Spectrometry (MS); or sufficiently pure that further purification does not result in a measurable change in the physical, chemical, biological and/or pharmacological properties (e.g., enzymatic and biological activity) of the substance. In certain embodiments, "substantially pure" or "substantially homogeneous" refers to a series of molecules in which at least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 99.5% by weight percent are individual stereoisomers of a compound, as determined by standard analytical methods.

The term "isotopic variation" refers to a compound comprising an unnatural proportion of an isotope at one or more of the atoms making up the compound. In certain embodiments, an "isotopic variant" of a compound comprises one or more isotopes in unnatural proportions, including, but not limited to, hydrogen (h), and (h)¹H) Deuterium (1)²H) Tritium (a)³H) Carbon-11 (C)¹¹C) Carbon-12 (C)¹²C) Carbon-13 (C)¹³C) Carbon-14 (C)¹⁴C) Nitrogen-13 (¹³N), nitrogen-14 (¹⁴N), nitrogen-15 (¹⁵N), oxygen-14 (¹⁴O), oxygen-15 (¹⁵O), oxygen-16 (¹⁶O), oxygen-17 (¹⁷O), oxygen-18 (¹⁸O), fluorine-17 (¹⁷F) Fluorine-18 (¹⁸F) Phosphorus-31 (³¹P), phosphorus-32 (³²P), phosphorus-33 (³³P), sulfur-32 (³²S), sulfur-33 (³³S), sulfur-34 (³⁴S), sulfur-35 (³⁵S), sulfur-36 (³⁶S), chloro-35 (³⁵Cl), chloro-36 (³⁶Cl), chloro-37 (³⁷Cl), bromo-79 (⁷⁹Br), bromo-81 (⁸¹Br), iodine-123 (¹²³I) Iodine-125 (¹²⁵I) Iodine-127 (¹²⁷I) Iodine-129 (¹²⁹I) And iodine-131 (¹³¹I) In that respect In certain embodiments, an "isotopic variant" of a compound is in a stable form, i.e., is not radioactive. In certain embodiments, an "isotopic variant" of a compound comprises one or more isotopes in unnatural proportions, including, but not limited to, hydrogen (h), and (h)¹H) Deuterium (1)²H) Carbon-12 (C)¹²C) Carbon-13 (C)¹³C) Nitrogen-14 (¹⁴N), nitrogen-15 (¹⁵N), oxygen-16 (¹⁶O), oxygen-17 (¹⁷O), oxygen-18 (¹⁸O), fluorine-17 (¹⁷F) Phosphorus-31 (³¹P), sulfur-32 (³²S), sulfur-33 (³³S), sulfur-34 (³⁴S), sulfur-36 (³⁶S), chloro-35 (³⁵Cl), chloro-37 (³⁷Cl), bromo-79 (⁷⁹Br), bromo-81 (⁸¹Br and iodine-127: (¹²⁷I) In that respect In certain embodiments, an "isotopic variant" of a compound is in an unstable form, i.e., radioactive. In certain embodiments, an "isotopic variant" of a compound comprises one or more isotopes in unnatural proportions, including, but not limited to tritium (tritium: (ii) (iii))³H) Carbon-11 (C)¹¹C) Carbon-14 (C)¹⁴C) Nitrogen-13 (¹³N), oxygen-14 (¹⁴O), oxygen-15 (¹⁵O), fluorine-18 (¹⁸F) Phosphorus-32 (³²P), phosphorus-33 (³³P), sulfur-35 (³⁵S), chloro-36 (³⁶Cl), iodine-123 (¹²³I) Iodine-125 (¹²⁵I) Iodine-129 (¹²⁹I) And iodine-131 (¹³¹I) In that respect It is to be understood that in any of the compounds provided herein, any hydrogen can be, for example, as judged to be feasible by one of ordinary skill in the art²H. For example, any carbon can be¹³C, or for example anyNitrogen can be¹⁵N, and any oxygen can be¹⁸And O. In certain embodiments, an "isotopic variation" of a compound comprises deuterium in an unnatural proportion.

"isotopic variations thereof"; or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof, and "an" isotopic variant of the compound described therein "; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug "of said compound.

"administering" or "administering" a drug to a patient (or other equivalent expression) refers to direct administration, administration to the patient by a physician or by the patient himself, and/or indirect administration, which may be the act of prescribing the drug. For example, if the physician orders the patient to self-administer the medication and/or prescribe the patient, the medication is administered to the patient as well.

The terms "patient," "subject," and "subject in need thereof" are used interchangeably to refer to a mammal in need of treatment for cancer, particularly leukemia, more particularly T-cell acute lymphocytic leukemia. Generally, the patient is a human. Generally, the patient is a human diagnosed with cancer. In certain embodiments, the terms "patient," subject, "and" subject in need thereof, can refer to a non-human mammal, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat, that is used to screen, characterize, and evaluate drugs and therapies.

The term "effective amount" as used herein, means the amount of each active agent required to produce the desired therapeutic effect in a subject, either alone or in combination with one or more other active agents. It will be appreciated by those skilled in the art that the effective amount will vary with the condition being treated, such as the severity of the condition or individual parameters of the patient, including age, physical condition, size, sex and weight, time of treatment, type of concurrent course of treatment (if any), particular route of administration or factors known to and within the skill of the art. These factors are well known to those of ordinary skill in the art and need only be determined by routine experimentation. When administered, it is generally preferred to use the maximum dose of the individual components or combination, i.e., the highest safe dose according to strict medical judgment. However, one of ordinary skill in the art will appreciate that the patient may insist on using a lower or tolerable dose based on medical, psychological or any other factor.

"treatment" or "therapy" for a condition or disorder refers to the step of obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired results include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer; slowing the severity of the disease; delay or slow the progression of the disease; ameliorating, soothing, or stabilizing the condition; or other beneficial results. In some cases, cancer treatment may result in partial response or no change in the condition.

By "pharmaceutically acceptable" ingredient (e.g., carrier or excipient), it is meant a compound or composition suitable for administration to a subject for treatment as described herein, without causing undue or deleterious side effects on the severity of the disease and the need for treatment. "carrier" refers to a material that does not cause significant irritation to an organism and does not destroy the biological activity and properties of a compound. "excipient" refers to an inert substance added to a pharmaceutical composition to facilitate administration of the compound.

Pharmaceutically acceptable carriers or Excipients that can be used have been disclosed in a number of documents, including the Handbook of Pharmaceuticals Excipients codified by Raymond Crowe, Paul J Sheskey and Marian E Quinn. In a non-limiting example, the pharmaceutically acceptable carrier or excipient may be selected from the group consisting of inert diluents, dispersing agents and/or granules, surfactants and/or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and/or oils. The aforementioned composition optionally further comprises at least one additional biologically active compound or agent.

Pharmaceutical compositions may include, but are not limited to, a unit dose of an active ingredient (e.g., a compound of the invention). For therapeutic purposes, a unit dose may be provided in bulk form, such as, but not limited to, tablets or capsules, or may be a volumetrically measurable solution, suspension, or the like, containing the unit dose of the active ingredient. The term "unit dose" as used herein refers to a unit amount of active ingredient, and is not limited to treating a patient suffering from a disease or disorder including, but not limited to, alcohol addiction, opiate addiction, pain relief or others, by oral, intravenous, intramuscular, transdermal, subcutaneous, intramedullary, transdermal, implant, sublingual, buccal, rectal, intravaginal, ocular, aural, nasal, inhalation or spray administration. Treatment may entail administering a unit dose of a compound of the invention periodically, e.g., twice or more a unit dose a day, one unit dose per meal, one unit dose every four hours or at other intervals, or only one unit dose per day.

Examples

In one embodiment, a method of preparing a compound of formula 1 comprises the steps of:

step (1): in the presence of Borane (BH)₃) B-diisopinocampheylchloroborane (B-Chlorodiisopinocamphylborane) (DIP-chloride), (S) - (-) -1,1 '-Bi-2-naphthol ((S) - (-) -1,1' -Bi-2-naphthol), or sodium borohydride (NaBH)₄) Reacting a compound of formula 2 with a compound of formula 3 a; and

step (2): reacting the product of step (1) with:

(A) lipase acrylic resin (lipase acrylic resin), sodium carbonate (Na)₂CO₃) And one of isopropyl acetate (isopropenyl acetate) and 2,2,2-trifluoroethyl butyrate (2,2,2-trifluoroethyl butyrate); or

(B) Protease, sodium carbonate, and one of isopropyl acetate and 2,2,2-trifluoroethyl butyrate; and reacting the product of step (B) with sodium methoxide (sodium methoxide) in a methanol-containing environment,

wherein R isAn aliphatic chain or Rx;

wherein Rx is hydrogen, an unsubstituted or substituted cyclic group, an electron withdrawing group or an electron withdrawing group.

In one embodiment, the reaction may be as follows:

in one embodiment, a method of preparing a compound of formula 1 comprises the steps of:

step (1 a): reacting a compound of formula 2 with a compound of formula 3a in an environment comprising borane, DIP-chloride, (S) - (-) -1,1' -bi-2-naphthol, or sodium borohydride;

step (1 b): reacting the product of step (1a) with acetic anhydride (Ac)₂O) reaction: and

step (2): reacting the product of step (1b) with:

(A) lipase acrylic resin and sodium carbonate; and reacting the product of step (a) with sodium methoxide in a methanol-containing environment; or

(B) A protease and sodium carbonate, wherein the protease is a sodium carbonate,

wherein R isAn aliphatic chain or Rx;

wherein Rx is hydrogen, an unsubstituted or substituted cyclic group, an electron withdrawing group or an electron withdrawing group.

In one embodiment, the reaction may be as follows:

in one embodiment, a method of preparing a compound of formula 4 comprises the steps of:

step (1): reacting a compound of formula 2 with a compound of formula 3b in an environment comprising borane, DIP-chloride, (S) - (-) -1,1' -bi-2-naphthol, or sodium borohydride; and

step (2): reacting the product of step (1) with:

(A) lipase acrylic resin, sodium carbonate, and one of isopropyl acetate and 2,2,2-trifluoroethyl butyrate; and reacting the product of step (a) with sodium methoxide in a methanol-containing environment; or

(B) Protease, sodium carbonate, and one of isopropyl acetate and 2,2,2-trifluoroethyl butyrate,

wherein R isAn aliphatic chain or Rx;

wherein Rx is hydrogen, an unsubstituted or substituted cyclic group, an electron withdrawing group or an electron withdrawing group.

In one embodiment, the reaction may be as follows:

in one embodiment, a method of preparing a compound of formula 4 comprises the steps of:

step (1 a): reacting a compound of formula 2 with a compound of formula 3b in an environment comprising borane, DIP-chloride, (S) - (-) -1,1' -bi-2-naphthol, or sodium borohydride;

step (1 b): reacting the product of step (1a) with acetic anhydride (Ac)₂O) reaction: and

step (2): reacting the product of step (1b) with:

(A) lipase acrylic resin and sodium carbonate; or

(B) Protease and sodium carbonate; and reacting the product of step (B) with sodium methoxide in an environment containing methanol,

wherein R isAn aliphatic chain or Rx;

wherein Rx is hydrogen, an unsubstituted or substituted cyclic group, an electron withdrawing group or an electron withdrawing group.

In one embodiment, the reaction may be as follows:

in one embodiment, the aliphatic chain has a carbon number of C₆To C₂₀。

In one embodiment, the cyclic group is an aromatic group, a cyclic saturated or partially unsaturated group, or a heterocyclic group. Preferably, the heterocyclic group includes a heteroatom such as N, O or S.

In one embodiment, the electron withdrawing group is a halide (F, Cl, Br, I), nitroso (-N ═ O), aminocarbonyl (-CONH)₂；-CONHR；-CONR₂Wherein R is alkyl), carboxyl (-CO)₂H) Alkoxycarbonyl (-CO)₂R, wherein R ═ alkyl), formyl (-CHO), acyl (-COR, wherein R ═ alkyl), haloformyl (-COX, wherein X ═ Cl, Br, I), trihalomethyl (-CX), haloformyl (-COX, wherein R ═ Cl, Br, I), and halomethyl (-CX)₃Wherein X ═ F, Cl, Br, I), cyano (-C ≡ N), nitro (-NO ≡ N)₂) Ammonium group (-NR)₃ ⁺Wherein R is alkyl or H), azido (-N)^- ₃) Or sulfonyl (-SO)₂R, wherein R is H, CF₃Alkyl group). Preferably, the alkyl group is C₁-C₅An alkyl group.

In one embodiment, the electron donating group is a lower alkyl (e.g., -CH)₃、-C₂H₅) Vinyl (-CH ═ CH)₂) Phenyl (-C)₆H₅) Acyloxy (-OCOR where R is alkyl), amido (-NHCOR where R is alkyl), alkylthio (-SR where R is alkyl), mercapto (-SH), hydroxy (-OH), alkoxy (-OR where R is alkyl), amino group (-NH)₂；-NHR；-NR₂Wherein R ═ alkyl). Preferably, the alkyl group is C₁-C₅An alkyl group.

In one example, compound OBI-3424(S-form) was successfully synthesized in 54% yield (from compound OBI-3424-5) and 99% optical purity via two combined Corey-Bakshi-Shibata (CBS) asymmetric reduction and lipase esterification steps. Stereochemistry was established by CBS reduction and two chemical enzyme binding steps using lipase to achieve optical purity of at least 99% (fig. 1).

Examples of the invention

The various features and embodiments of the present invention will be described in detail with reference to the following representative examples. The following examples are merely illustrative and are not intended to limit the scope of the present invention. Those skilled in the art will immediately appreciate that the specific examples are provided for illustration only and a more complete description is provided as set forth in the appended claims. The various embodiments and features described herein should be understood to be interchangeable and combinable with other embodiments of the present application.

Clinical test compound OBI-3424 was synthesized first in three steps from "Asymchem" in 19% yield (see route 1 below). To improve yield and reduce possible impurities, the inventors tried to design other synthetic routes (see route 2), and finally chose two approaches to achieve the goal. Firstly, an unstable phosphate structure (labile phosphate structure) is introduced in a later stage, and aziridine is formed in a final stage to avoid nucleophilic damage to the m-benzene derivative. The synthetic route is completed by racemic compound OBI-3424-6, which is prepared using sodium borohydride as a reducing agent and is phosphorylated and aziridine is formed in the subsequent step. Secondly, to improve stereoselectivity, stereogenic centers were designed to be established by the combination of CBS asymmetric reduction and lipase selective protection enrichment (enrichment) (see pathway 3). Combining these methods is expected to yield products with good yields and high optical purity. Thus, the present inventors have developed an alternative synthetic route to obtain compound OBI-3424. CBS reagents were substituted with their enantiomers to prepare S-form based compound OBI-3424-6 mixtures. A small amount of the R-form compound OBI-3424-6 can be selectively acetylated after treatment with lipase. Next, pure S-form compound OBI-3424-6 can be obtained by column purification.

Route 1: GMP Synthesis method (clinical practice) of Compound OBI-3424

Route 2: novel pathway design for the synthesis of compound OBI-3424

Route 3: construction of stereochemistry of Compound OBI-3424-6

Materials and methods

The equipment is listed in table 2.

Table 2: equipment list

Equipment	Manufacturer(s)	S/N	Model number
				Heating plate	IKA	N/A	RCT B S1
Rotary evaporator	Buchi	1000060922	R-210
				Vacuum controller	Buchi	1000064866	V-850
Heating tank	Buchi	1000131345	B-491
				Vacuum pump	Buchi	1000052997	V-700
Vacuum pump	Buchi	1000135289	V-710
				Cooling device	Panchum Scientific Corp.	8503098	UR-8500
Cooling device	HCS	881826	820

The chemical synthesis reagents used in reaction pathways 2 and 3 are listed in table 3.

Table 3: list of reagents

Note that: both CBS reagents were used to prepare a mixture of compound OBI-3424-6R/S: (S) -5, 5-Biphenyl-2-methyl-3, 4-propanol-1, 3,2-oxazaborolidine ((S) -5,5-diphenyl-2-methyl-3,4-propano-1,3,2-oxazaborolidine) (CAS No: 112022-81-8) gives a product based on R-form, whereas (R) -5, 5-Biphenyl-2-methyl-3, 4-propanol-1, 3,2-oxazaborolidine ((R) -5,5-diphenyl-2-methyl-3,4-propano-1,3,2-oxazaborolidine) (CAS No: 112022-83-0) gives a product based on S-form.

Substrate for experiment (substlates)

Substrate 1: 1- (3- (3-N, N-dimethylaminocarbonyl) phenoxy-4-nitrophenyl) ethanol

(1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)ethanol)

¹H NMR(400MHz,CDCl₃)δ7.97(d,J＝0.4Hz,1H),7.41(t,J＝8.0Hz,1H),7.26-7.19(m,2H),7.11-7.09(m,2H),7.03(d,J＝1.2Hz,1H),4.88(q,J＝6.4Hz,1H),3.08(s,3H),2.97(s,3H),1.44(d,J＝6.4Hz,3H)ppm；¹³C NMR(100MHz,CDCl₃)δ170.5(C),156.2(C),154.0(C),150.0(C),140.2(C),138.0(C),130.2(CH),126.0(CH),122.6(CH),120.8(CH),119.9(CH),118.2(CH),117.1(CH),68.9(CH),39.5(CH₃),35.3(CH₃),25.2(CH₃)ppm；HRMS(ESI,M+Na⁺)calcd for C₁₇H1₈N₂O₅Na 353.1108,found353.1108.

Matrix 2: 1- (3-fluoro-4-nitrophenyl) ethan-1-ol

(1-(3-fluoro-4-nitrophenyl)ethan-1-ol)

¹H NMR(400MHz,CDCl₃)δ8.00(t,J＝8.0Hz,1H),7.34(dd,J＝12.0,1.6Hz,1H),7.29-7.27(m,1H),4.99(q,J＝6.8Hz,1H),2.49(s,1H),1.51(d,J＝6.8Hz,3H)ppm；¹³CNMR(100MHz,CDCl₃)δ157.0(C),155.3(C),155.2(C),154.4(C),126.2(CH),121.2(CH),115.2(CH),114.9(CH),68.9(CH),25.3(CH₃)ppm；HRMS(ESI,M+Na⁺)calcd for C₈H₈FNO₃Na 208.0380,found 208.0354.

Matrix 3: 1- (3- (4- (trifluoromethyl) phenoxy) -4-nitrophenyl) ethan-1-ol

(1-(3-(4-(trifluoromethyl)phenoxyl)-4-nitrophenyl)ethan-1-ol)

¹H NMR(400MHz,CDCl₃)δ8.01(d,J＝8.4Hz,1H),7.62(d,J＝8.8Hz,2H),7.31(dd,J＝8.0,1.2Hz,1H),7.18(d,J＝2.0Hz,1H),7.07(d,J＝8.4Hz,2H),4.95(q,J＝6.4Hz,1H),2.02(s,1H),1.48(d,J＝6.4Hz,3H)ppm；¹³C NMR(100MHz,CDCl₃)δ159.2(C),153.7(C),149.0(C),140.7(C),127.43(CH),127.40(CH),126.3(CH),121.6(CH),119.1(CH),117.8(CH),69.1(CH),25.5(CH₃)ppm；HRMS(ESI,M+Na⁺)calcd for C₁₅H₁₂F₃NO₄Na 350.0611,found 350.0607.

Matrix 4: 1- (3- (4- (2-methylphenyl) phenoxy) -4-nitrophenyl) ethan-1-ol

(1-(3-(4-(2-methylphenyl)phenoxyl)-4-nitrophenyl)ethan-1-ol)

¹H NMR(400MHz,CDCl₃)δ7.97(d,J＝8.4Hz,1H),7.32(dd,J＝6.4,2.0Hz,2H),7.27-7.20(m,5H),7.16(d,J＝1.6Hz,1H),7.07(dd,J＝6.8,2.0Hz,2H),4.91(q,J＝6.8Hz,1H),2.29(s,3H),1.47(d,J＝6.4Hz,3H)ppm；¹³C NMR(100MHz,CDCl₃)δ154.8(C),153.2(C),150.8(C),140.8(C),140.2(C),138.1(C),135.4(C),130.8(CH),130.4(CH),129.8(CH),127.4(CH),126.1(CH),125.8(CH),120.1(CH),118.4(CH),117.6(CH),69.3(CH),25.4(CH₃),20.5(CH₃)ppm；HRMS(ESI,M+Na⁺)calcd for C₂₁H₁₉NO₄Na 372.1206,found 372.1204.

Matrix 5: 1- (2-chloro-4-nitrophenyl) ethan-1-ol

(1-(2-chloro-4-nitrophenyl)ethan-1-ol)

¹H NMR(400MHz,CDCl₃)δ8.21(d,J＝2.4Hz,1H),8.16(dd,J＝8.4,2.4Hz,1H),7.85(d,J＝8.4Hz,1H),5.34(q,J＝6.4Hz,1H),2.24(s,1H),1.51(d,J＝6.8Hz,3H)ppm；¹³C NMR(100MHz,CDCl₃)δ150.5(C),147.2(C),132.1(C),127.3(CH),124.6(CH),122.2(CH),66.8(CH),23.6(CH₃)ppm；HRMS(ESI,M+Na⁺)calcd for C₈H₈ClNO₃Na 224.0085,found 224.0085.

Matrix 6: 1- (3-chloro-4-nitrophenyl) ethan-1-ol

(1-(3-chloro-4-nitrophenyl)ethan-1-ol)

¹H NMR(400MHz,CDCl₃)δ7.87(d,J＝8.4Hz,1H),7.58(d,J＝2.0Hz,1H),7.40(dd,J＝8.4,1.6Hz,1H),4.97(q,J＝6.4Hz,1H),2.40(s,1H),1.51(d,J＝6.4Hz,3H)ppm；¹³C NMR(100MHz,CDCl₃)δ152.1(C),146.5(C),128.7(CH),127.3(C),125.9(CH),124.4(CH),68.9(CH),25.4(CH₃)ppm；HRMS(ESI,M+Na⁺)calcd for C₈H₈ClNO₃Na 224.0085,found 224.0065.

Matrix 7: 1- (3-methoxy-4-nitrophenyl) ethan-1-ol

(1-(3-methoxy-4-nitrophenyl)ethan-1-ol)

¹H NMR(400MHz,CDCl₃)δ7.83(d,J＝8.4Hz,1H),7.16(d,J＝1.6Hz,1H),6.97(dd,J＝8.4,1.6Hz,1H),4.96(q,J＝6.4Hz,1H),3.97(s,3H),2.42(s,1H),1.50(d,J＝6.4Hz,3H)ppm；¹³C NMR(100MHz,CDCl₃)δ153.3(C),138.1(C),126.0(CH),117.0(CH),110.1(CH),69.5(CH),56.4(CH₃),25.4(CH₃)ppm；HRMS(ESI,M+Na⁺)calcd for C₉H₁₁NO₄Na 220.0580,found 220.0555.

Matrix 8: 1- (2-methoxy-4-nitrophenyl) ethan-1-ol

(1-(2-methoxy-4-nitrophenyl)ethan-1-ol)

¹H NMR(400MHz,CDCl₃)δ7.86(dd,J＝8.4,2.0Hz,1H),7.71(d,J＝2.0Hz,1H),7.59(d,J＝8.4Hz,1H),5.18(q,J＝6.4Hz,1H),3.96(s,3H),2.43(s,1H),1.49(d,J＝6.4Hz,3H)ppm；¹³C NMR(100MHz,CDCl₃)δ156.4(C),147.9(C),141.3(C),126.3(CH),116.2(CH),105.3(CH),65.5(CH),55.9(CH₃),23.1(CH₃)ppm；HRMS(ESI,M+Na⁺)calcd for C₉H₁₁NO₄Na 220.0580,found 220.0557.

Matrix 9: 1-Phenylethanol

(1-phenylethanol)

¹H NMR(400MHz,CDCl₃)δ7.35-7.30(m,4H),7.27-7.24(m,1H),4.84(q,J＝6.4Hz,1H),2.31(s,1H),1.46(d,J＝6.4Hz,3H)ppm；¹³C NMR(100MHz,CDCl₃)δ145.7(C),128.4(CH),127.4(CH),125.3(CH),70.3(CH),25.0(CH₃)ppm；HRMS(ESI,M+Na⁺)calcd for C₈H₁₀ONa 145.0624,found 145.0621.

Matrix 10: 2-octanol

(2-Octanol)

¹H NMR(400MHz,CDCl₃)δ3.83-3.75(m,1H),1.76(s,1H),1.46-1.26(m,10H),1.18(d,J＝6.0Hz,3H),0.89(t,J＝6.8Hz,3H)ppm；¹³C NMR(100MHz,CDCl₃)δ68.1(CH),39.3(CH₂),31.8(CH₂),29.3(CH₂),25.7(CH₂),23.4(CH₃),22.6(CH--₂),14.0(CH₃)ppm；HRMS(ESI,M+Na⁺)calcd for C₈H₁₈ONa 153.1250,found 153.1237.

Matrix 11: n- [4- (1-hydroxyethyl) phenyl ] acetamide

(N-[4-(1-Hydroxyethyl)phenyl]acetamide)

¹H NMR(400MHz,CDCl₃)δ7.50(d,J＝8.4Hz,2H),7.31(d,J＝8.4Hz,2H),4.79(q,J＝6.4Hz,1H),2.11(s,3H),1.42(d,J＝6.4Hz,3H)ppm；¹³C NMR(100MHz,CDCl₃)δ171.8(C),143.5(C),138.9(C),127.1(CH),121.2(CH),70.6(CH),25.6(CH₃),23.9(CH₃)ppm；HRMS(ESI,M+Na⁺)calcd for C₁₀H₁₃NO₂Na 202.0838,found 202.0809.

Matrix 12: 1-O-acetyl-1- (3- (3-N, N-dimethylaminocarbonyl) phenoxy-4-nitrophenyl) ethane

(1-O-acetyl-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)ethane)

¹H NMR(400MHz,CDCl₃)δ7.96(d,J＝8.4Hz,1H),7.42(td,J＝7.6,2.4Hz,1H),7.24-7.20(m,2H),7.09-7.04(m,3H),5.78(q,J＝6.8Hz,1H),3.10(s,3H),2.98(s,3H),2.06(s,3H),1.48(d,J＝6.8Hz,3H)ppm；¹³C NMR(100MHz,CDCl₃)δ170.3(C),169.9(C),155.9(C),150.0(C),149.4(C),140.6(C),138.4(C),130.2(CH),126.2(CH),122.8(CH),121.2(CH),119.6(CH),118.7(CH),117.1(CH),70.9(CH),39.5(CH₃),35.3(CH₃),22.1(CH₃),21.2(CH₃)ppm；HRMS(ESI,M+Na⁺)calcd for C₁₉H₂₀N₂O₆Na395.1214,found 395.1146.

Matrix 13: 1-O-acetyl-1- (3-fluoro-4-nitrophenyl) ethane

(1-O-acetyl-1-(3-fluoro-4-nitrophenyl)ethane)

¹H NMR(400MHz,CDCl₃)δ8.06(dd,J＝8.4,7.2Hz,1H),7.30-7.25(m,2H),5.87(q,J＝6.8Hz,1H),2.13(s,3H),1.55(d,J＝6.8Hz,3H)ppm；¹³C NMR(100MHz,CDCl₃)δ169.9(C),157.0(C),154.3(C),150.9(C),150.8(C),126.5(CH),121.82(CH),121.79(CH),115.8(CH),115.6(CH),70.6(CH),22.1(CH₃),21.0(CH₃)ppm；HRMS(ESI,M+Na⁺)calcd for C₁₀H₁₀FNO₄Na 250.0486,found 250.0445.

Example 1: synthesis of intermediate for Compound OBI-3424 (preparation of Compound OBI-3424-5 from Compound OBI-3424-3)

Synthesis of Compound OBI-3424-3

In a round-bottomed flask, compound OBI-3424-1(9g, 48.6mmol), dimethylformamide (0.1g, mmol) and thionyl chloride (SOCl)₂30mL, mmol) was refluxed at 75 ℃ for 3 hours. During reflux, anhydrous MgCl was added at room temperature (about 25 ℃ C.)₂(2.79g, mmol), dimethyl malonate (5.7mL, mmol) and triethylamine (triethylamine) (14.1mL, mmol) were combined in another round flask. Obtained after mixingThe white suspension was stirred for 1.5 hours to become a white mud (white mud). After refluxing for 3 hours, compound OBI-3424-1 and SOCl were added₂The reaction mixture was cooled to about 40 ℃. The reaction mixture was then concentrated using a rotary evaporator to remove excess solvent. Subsequently, the resulting slurry (syrup) was diluted with 15mL of toluene. The resulting toluene solution was added dropwise to a white sludge mixture prepared in advance. After 1.5 hours of mixing, the reaction was checked using TLC (EtOAc/n-hexane volume ratio 1: 4). The resulting mixture was treated with 30mL of 6N HCl and extracted with 60mL EtOAc. The aqueous layer was extracted again with 60mL EtOAc. The combined organic layers were over MgSO₄Dried, filtered and concentrated using a rotary evaporator (water bath temperature about 35 ℃ C., pressure about 130mbar) to give a reddish slurry. The resulting slurry was mixed with 30mL of 6N HCl and refluxed at 100 ℃ for about 17 hours. The resulting crude reaction product was then cooled to room temperature, diluted with 60mL of EtOAc and extracted. The aqueous layer was extracted again with 60mL EtOAc. Collecting the organic layer, using MgSO₄Dried, filtered and concentrated using a rotary evaporator (water bath temperature about 35 ℃ C., pressure about 130mbar) to give a reddish slurry. The resulting slurry was purified by column chromatography through a silica gel pad (10g of Compound OBI-3424-1 using 200g of gel powder). Mix hexanes/EtOAc/CH with silica gel₂Cl₂12/1/0.5 volume ratio. It is proposed to use an undershoot liquid (fluent) system: Hexane/EtOAc/CH₂Cl₂12/1/0.5(1 column volume), 10/1/1(2-3 column volumes). The collected product was concentrated using a rotary evaporator (water bath temperature about 35 ℃, pressure about 130mbar) and high vacuum (about 25 ℃, about 17 hours) to afford the expected product compound OBI-3424-3 as a yellow solid (5.4g, isolated yield 55%).

Synthesis of Compound OBI-3424-4

M-hydroxybenzoic acid (15g) was dissolved in THF (anhydrous, 300mL) and the reaction was cooled to-30 ℃ under nitrogen. After stirring for 20 minutes, at 15 minutesTriethylamine (67mL) was added to the cooled reaction. After stirring for 15 minutes, the reaction turned into a white suspension. Isobutyl chloroformate (31mL) was added to the resulting suspension over 15 minutes. After stirring for 2 hours, the reaction was checked by TLC (CH)₂Cl₂Volume ratio of/EtOAc 2/1). After the reaction was stopped with M-hydroxybenzoic acid, dimethylamine (2M in THF, 270mL) was added to the reaction over 30 minutes. After the addition, the reaction was stirred for another 30 minutes, then warmed to room temperature (about 25 ℃) and stirring continued for 18 hours. After completion of the reaction, water (90mL) was added to the reaction and THF was removed using a rotary evaporator (bath temperature 35 ℃ C., ca. 100 mbar). The crude reaction product was then extracted with cooled 1N HCl (30mL) and EtOAc (200 mL). The aqueous layer was extracted twice with EtOAc (200 mL). The organic extracts were combined and extracted with MgSO₄Dried, filtered and concentrated to about 30% by volume using a rotary evaporator, some of which crystals precipitated (bath temperature 35 ℃ C., about 120 mbar). The resulting crude sample was treated with hexane (200mL) with stirring (360rpm) to form a white suspension. The resulting solid was filtered and washed with a cooled hexane/EtOAc mixture (vol. 20/1) and dried using a high vacuum system for about 16 hours (25 ℃) to afford the expected compound OBI-3424-4 as a white solid (12.7g, 71% isolated yield).

Synthesis of Compound OBI-3424-5

Compound OBI-3424-3(0.35g) was added to a solution of compound OBI-3424-4(0.25g) in dry THF (4mL) in an appropriate two-necked round-bottomed flask at room temperature (about 23 ℃ C.) under nitrogen. The reaction mixture was cooled for 15 minutes to 0 ℃. Mixing Cs₂CO₃(0.84g) was added to the cooled reaction. After 1 hour of addition, the reaction was checked by TLC (CH)₂Cl₂The volume ratio of EtOAc is 2/1, and the Rf value of compound OBI-3424-5 is 0.3). After compound OBI-3424-4 reaction stopped, the reaction was diluted with 8mL EtOAc and with 4mL saturated NH₄And (4) extracting with Cl. The aqueous layer was extracted twice with 8mL EtOAc. Combination of Chinese herbsAnd the organic layer was washed with 4mL of saturated brine (brine), MgSO₄Drying followed by concentration using a rotary evaporator (water bath about 35 ℃ C., about 130mbar) gave a yellow slurry. The resulting slurry was purified by column chromatography through a silica gel pad. The following flush systems are suggested: (hexane/EtOAc ratio by volume 5/1) hexane/EtOAc 4/1(1 column volume), hexane/EtOAc 1/1(1 column volume), hexane/EtOAc 1/2(3 column volume) until the desired product was collected. The separated liquid was concentrated using a rotary evaporator (water bath temperature about 35 ℃ C., about 140mbar) and high vacuum (about 25 ℃ C., 16 hours) to obtain compound OBI-3424-5 as a pale yellow solid (0.46g, isolated yield 93%).

The inventors first succeeded in synthesizing compound OBI-3424-3 and compound OBI-3424-4 in acceptable yields according to the method described in the literature (PCT patent publication No. WO 2016/145092A 1). Next, compound OBI-3424-3 and compound OBI-3424-4 were coupled to prepare compound OBI-3424-5 in about 80% yield under basic process conditions. Based on the ease of handling and in order to improve the yield, the inventors tested several conditions. After several tests, anhydrous THF and Cs were found₂CO₃Resulting in the highest yield (table 4). THF was used as reaction solvent and Cs₂CO₃The isolation yield is best as a base. Further, it is generally considered that Cs₂CO₃Is a mild alkali which is convenient to operate and is beneficial to mass production in operation.

Table 4: improve the preparation condition of the OBI-3424-5

Test of	Base (1.7)Equivalent weight)	Solvent(s)	Isolated yield
				1	Sodium hydride	DMF	80％
2	Sodium hydride	CH3CN	83％
				3	Sodium hydride	THF	78％
4	Cs2CO3	DMF	80％
				5	Cs2CO3	CH3CN	82％
6	Cs2CO3	THF	93％

Note: all solvents were anhydrous (commercial) and used without further treatment.

Example 2: synthesis of Compound OBI-3424-6 (rac) from Compound OBI-3424-5

Route 4: synthesis of Compound OBI-3424-6 (rac) from Compound OBI-3424-5

To demonstrate whether the designed route is feasible (see route 2), compound OBI-3424-5 was rapidly reduced with sodium borohydride to give compound OBI-3424-6 (racemic) in 85% isolated yield (see route 4), and the reaction was stable in an ice bath. Note that addition of sodium borohydride releases hydrogen gas. The obtained compound OBI-3424-6 (racemic) was further used for the synthesis of compound OBI-3424, and the reaction was observed.

Compound OBI-3424-5(500mg) was dissolved in methanol and cooled to 0 ℃. Sodium borohydride (58mg) was added to this solution (note: gas evolution during the reaction). After stirring for 30 minutes, the reaction was checked by TLC (CH)₂Cl₂The volume ratio of EtOAc is 2/1, the Rf value of compound OBI-3424-5 is 0.3, and the Rf value of compound OBI-3424-6 is 0.2). After completion of the reaction, the reaction was diluted with EtOAc (50mL) and 1N HCl (5 mL). The resulting crude product was isolated. The aqueous layer was extracted again with EtOAc. The organic layer was collected and washed with MgSO₄Drying, filtration and subsequent concentration using a rotary evaporator gave a slurry (water bath temperature 35 ℃ C., ca. 70 mbar). The resulting slurry was purified by column chromatography through a silica gel pad (silica gel ═ 20 g). The following flush systems are suggested: (volume ratio of sealed hexane/EtOAc 3/1), eluent: hexane/EtOAc 1/1(1 column volume), CH₂Cl₂EtOAc 2/1(1 column volume), CH₂Cl₂EtOAc 1/1(3 column volumes) until the desired product was obtained. The resulting product was concentrated using a rotary evaporator (water bath temperature about 35 ℃, about 120mbar) and high vacuum (about 25 ℃, 16 hours) to afford compound OBI-3424-6 (rac) as a yellow slurry (428mg, 85% isolated yield).

Example 3: synthesis of the Compound OBI-3424-6-LR

This approach was first proposed in 1981, s. After a few years, e.j.corey shows that the adapted catalyst can reduce achiral ketones in a highly enantioselective manner in rapid large quantities in the presence of borane/THF. The catalytic oxazaborolidine is known as Corey-Bakshi-Shibata (CBS) reduction. The catalyst is applied to a number of substrates and has a good high enantioselectivity (J.mol.Catal.B Enz.1997,3: 65-72). According to the disclosed experimental results, similar substrates are more prone to form R-form products by S-CBS catalysts. Thus, the inventors tried to use S-CBS in the matrix (compound OBI-3424-5) and found that the selectivity was about 3:1 to 6:1 in terms of R/S volume ratio. To increase the enantiomeric excess ee% of the R-form product, the inventors followed a combination of lipase selective acetylation to increase the R/S ratio (see pathway 2). The lipase selective acetylation of achiral alcohols is a mature technique. According to the published experimental results, lipases tend to react with R-form substrates. As a result of testing two different lipases CAL-B and PAL, it was found that better results were obtained with CAL-B. After the S-CBS reduction method and the lipase acetylation method are combined, the ee% of the final product of the compound OBI-3424-6 in HPLC can be improved to be not less than 97%.

Step 1: S-CBS asymmetric reduction (mainly R form)

We then focused on asymmetric reduction and optical purity enhancement. A commercial CBS reagent ((S) -5, 5-biphenyl-2-methyl-3, 4-propanol-1, 3, 2-oxazolylborane, CAS number: 112022-81-8) was known to be developed by Prof.E.J.Corey (J.Am.chem.Soc.1987,109: 7925-7926). The reagent can be used for chiral reduction with ketone to provide good R-form alcohol yield and high stereoselectivity of borane in THF. Treatment of compound OBI-3435-5 with 0.2mol equivalents of CBS reagent and 1.05mol equivalents of borane (1M in THF) gave a 80% yield of the reduced product (compound OBI-3424-6 enantiomeric mixture) in a few hours under nitrogen. The optical resolution was 48% by chiral HPLC analysis (fig. 2). Increasing the amount of CBS to 0.5mol equivalent and lowering the reaction temperature to-20 ℃ increased the optical purity to 70% ee, but the yield decreased to 65% and the reaction time elongated to 22 hours (Table 5).

Table 5: CBS reduction test (R form is the main)

Step 2: selective acyl protection for lipases

Lipases with high R-form selectivity are excellent enzymes for the isolation of compounds OBI-3424-6R/S mixtures, which can actively add acetyl groups to the R-form compound OBI-3424-6 and passively leave the non-acetylated S-form compound OBI-3424-6 (see pathway 5). The enantiomeric mixture is then treated with a lipase to selectively esterify the hydroxyl group of the R-form enantiomer. The optical purity of the obtained acetylation product, compound OBI-3424-6-Ac, can be increased to 99% ee value. And then removing acetyl of the compound OBI-3424-6-Ac under mild alkaline conditions to obtain the home-made compound OBI-3424-6-LR, and successfully converting the compound OBI-3424-LR into the compound OBI-3424-LR, wherein the isolated yield is 77%.

Route 5: preparation of compound OBI-3424-6-LR by CBS reduction method and lipase enrichment combined method

Example 4: synthesis of the Compound OBI-3424-6-LS

Step 1: R-CBS asymmetric reduction (S form is the main part)

Following structural confirmation, the inventors followed modifications of the synthetic pathway to produce compound OBI-3424-6 (see pathway 6). The CBS reagent was replaced with (R) -5, 5-biphenyl-2-methyl-3, 4-propanol-1, 3,2-oxazaborolidine (CAS No: 112022-83-0). After asymmetric reduction of compound OBI-3424-5, a mixture of the enantiomers of compound OBI-3424-6 with an R/S ratio of 1/4 was obtained in an isolated yield of 90% (FIG. 3). The mixture is then treated with an enzyme (lipase) to increase optical purity.

Route 6: from asymmetric reduction of compound OBI-3424-5 to synthesis of compound OBI-3424-6 based on S-form

Step 2: selective acyl protection for lipases

It is appreciated that lipases can selectively acetylate the R-form compound OBI-3424-6 in the R/S mixture. After purification, passively selected and non-acetylated S-form compound OBI-3424-6(OBI-3424-6-LS) was obtained directly. One step is omitted here compared to the step of synthesizing the compound OBI-3424-6-LR (removal of the acetyl group of the compound OBI-3424-6-Ac). After simple column purification using a silica gel pad, compound OBI-3424-6-LS was collected in 77% yield and 99% ee (route 7, FIG. 4).

Route 7: the S-form compound OBI-3424-6 is formed by enzyme catalytic reaction

Example 5: general Synthesis of the Compound OBI-3424-6R/S mixture

Borane (1M in THF at a concentration of 1.05mol eq.) and CBS reagent (0.2mol eq.) were mixed at 0 ℃ under nitrogen (note: R-form based product was obtained from (S) -5, 5-biphenyl-2-methyl-3, 4-propanol-1, 3,2-oxazaborolidine using two different CBS reagents respectively, along with S-form based product using (R) -5, 5-biphenyl-2-methyl-3, 4-propanol-1, 3, 2-oxazaborolidine). After stirring for 5 minutes, compound OBI-3424-5(1.0mol equivalent, i.e., 0.45g of compound dissolved in 2mL of anhydrous THF) was added to the reaction. The reaction was stirred and warmed to 25 ℃ under nitrogen. After stirring the addition for about 1.5 hours, the reaction was checked by TLC (CH)₂Cl₂The volume ratio of EtOAc is 2/1, and the Rf value of compound OBI-3424-6 is 0.3). After the compound OBI-3424-5 stops reacting, the crude reaction product is reactedMixing with EtOAc (^v/_v4 times the crude product reacted) and 1N HCl (25% by volume of EtOAc) was cooled. The crude reaction product was extracted and separated, followed by extraction of the aqueous layer twice with EtOAc. With saturated NaHCO₃The combined organic layers were washed with brine and MgSO₄Drying, filtration and concentration by rotary evaporator (water bath temperature 35 ℃ C., ca. 100mbar) gave a yellow slurry. The resulting slurry was purified by column chromatography through a silica gel pad. The following flush systems are suggested: (volume ratio of sealed hexane/EtOAc 3/1), eluent: hexane/EtOAc 1/1(1 column volume), CH₂Cl₂EtOAc 2/1(1 column volume), CH₂Cl₂EtOAc 1/1(3 column volumes) until the desired product was obtained. The collected product was concentrated using a rotary evaporator (water bath temperature about 35 ℃, about 120mbar) and high vacuum (about 25 ℃, 16 hours) to obtain a yellow slurry of compound OBI-3424-6R/S mixture (isolated yield about 90%, R/S ratio determined using HPLC with chiral column).

In addition, the inventors also tested several chiral reducing agents (such as DIP-chloride, sodium borohydride or (S) - (-) -1,1' -bi-2-naphthol) in place of the CBS catalyst.

Methods of using (R)/(S) -Me-CBS reagents

Solutions containing different ketone substrates and (R)/(S) ME-CBS catalyst (Tokyo Chemical Industry Co., Ltd.) were cooled for 10 minutes to 0 ℃ under nitrogen protection. Borane (1M in THF) (Acros Organics; batch No.: A0400505) was added slowly to the reaction and stirred for 10 minutes. The reaction mixture was then stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was quenched with 1N HCl and water (queued). The aqueous layer was extracted with two portions of EtOAc. Using NaHCO₃And the collected organic layer was washed with saturated brine, over anhydrous MgSO₄Dried, filtered and concentrated using a rotary evaporator to give a yellow crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; batch: HT 80186). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product.

Methods of using (+)/(-) -DIP-chloride reagents

THF solutions (Acros Organics; batch No.: 1850266) containing different ketone substrates were cooled for 10 minutes to 0 ℃ under nitrogen. (+)/(-) -DIP-chloride (Tokyo Chemical Industry Co., Ltd.) was slowly added to the reaction and stirred for 10 minutes. The reaction mixture was then stirred at room temperature for 3 hours. After completion of the reaction, the reaction mixture was quenched with 1N HCl and MeOH. The aqueous layer was extracted with two portions of EtOAc. Using NaHCO₃And the collected organic layer was washed with saturated brine, over anhydrous MgSO₄Dried, filtered and concentrated using a rotary evaporator to give a yellow crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; catalog No. HT 80186). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product.

Using sodium borohydride (NaBH)₄) Method (2)

The MeOH solutions containing the different ketone matrices were allowed to cool to room temperature. Sodium borohydride was slowly added to the reaction. The reaction mixture was then stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was quenched with water. The aqueous layer was extracted with two portions of EtOAc. Using NaHCO₃And the collected organic layer was washed with saturated brine, over anhydrous MgSO₄Drying, filtering and concentrating by a rotary evaporator to obtain a crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; catalog No. HT 80186). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product.

Methods of using (S) - (-) -BINOL reagents

A white precipitate formed after stirring a solution of (S) - (-) -1,1' -bi-2-naphthol (Tokyo chemical industries, Ltd.; batch No. 4HN7B-OD) and trimethylaluminum (trimethylaluminum) (Sigma-Aldrich, Inc.; batch No. STBG1135V) at room temperature under nitrogen for 10 minutes. Ketone substrate 2 and 2-propanol (Tedia Company inc.; batch No. 15040128) were added to the reaction and stirred at room temperature for 16 hours. After completion of the reaction, the reaction mixture was concentrated by a rotary evaporator to obtain a crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; batch: HT 80186). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product.

Table 6 shows the yields obtained using different chiral reducing reagents for compound OBI-3424-6. As shown in the table, CBS catalyst is the most effective reagent for synthesizing compound OBI-3424-6, but sodium borohydride can not be used to produce S-form or R-form specific product.

Table 6: the yields obtained with different compounds OBI-3424-6 chiral reduction reagent

In addition, the inventors also tested several ketone substrates using R-CBS/S-CBS, (-) -DIP-chloride/(+) -chloride, sodium borohydride or (S) - (-) -1,1' -bi-2-naphthol (see Table 7). Table 7 shows the yields obtained using different ketone substrates.

Table 7: mirror selective reduction of different ketones-based on S-form

Note:

1. using a substrateAnd (S) - (-) -1,1' -bi-2-naphthol (Tokyo Chemical Industry Co., Ltd., CAS No. 18531-99-2) reagent in a yield of 11%, 9% ee, S form.

2. Using a substrateAnd (S) -Me-CBS catalyst reagent in a yield of>99％、34％ee、R form。

3. Using a substrateAnd yield of (+) -DIP-chloride reagent is 96%, 91% ee, Rform.

Example 6: separation of enantiomers from a mixture of compounds OBI-3426-6R/S by lipase enzyme-catalyzed selection

Compound OBI-3424-6 was dissolved using isopropyl acetate as a premix solution. Lipase (CALB) and sodium carbonate were added to the premix solution and left at room temperature (25 ℃). Note that the reaction time is different from the time chosen for R-form and S-form (section 2.1, R-is chosen to be 4 hours and S-is chosen to be 20 hours). After completion of the reaction, the upper layer solution was filtered (or pipetted) to remove the lipase resin and transferred to a clean tube for further purification (in TLC inspection system, CH)₂Cl₂EtOAc/hexane volume ratio 1/2/1). The enzyme-catalyzed reaction solution was concentrated using a rotary evaporator (water bath temperature about 35 ℃ C., pressure about 50mbar) to give a slurry. The slurry was then placed on a pad of silica gel (sealed n-hexane/EtOAc volume ratio: 5/1), suggesting the following eluent system: n-hexane/EtOAc volume ratio 3/1(1 column volume), followed by 1/1(1.5 column volume), followed by 1/4(3 column volume) until the desired product was collected; check for CH in the eluent System by TLC₂Cl₂EtOAc/hexanes-1/2/1, Rf of compound OBI-3424-6-LS 0.3, Rf of compound OBI-3424-6-Ac 0.5). In this step, the compound OBI-3424-6-LS will be obtained. In addition, the inventors also tested the reverse of different classes of lipases, such as lipase B from Aspergillus oryzae (Aspergillus oryzae) or immobilized lipase from Candida Antarctica (Candida Antarctica)Should be active. A solution of the hydroxyl substance dissolved in isopropyl acetate (Sigma-Aldrich inc.; batch: STBH5768) at room temperature was taken. Sodium carbonate (Showa.; batch: KDL-100W) and lipase acrylic resin (Sigma-Aldrich, Inc.; batch: SLBW1544) or Candida antarctica lipase B immobilized on Immobead 150, or a recombinant taken from Aspergillus oryzae (Sigma-Aldrich, Inc.; batch: BCBZ7604) or immobilized lipase taken from Candida antarctica (Sigma-Aldrich, Inc.; batch: BCBD5551) were added to the solution. The reaction mixture was left at room temperature for 16 hours. The reaction mixture was filtered and concentrated on a rotary evaporator to give the crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; catalog No. HT 80186). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product.

In addition, the inventors also tested another reagent, 2,2,2-trifluoroethyl butyrate (2,2,2-trifluoroethyl butyrate), in place of isopropyl acetate. A solution of the hydroxyl group dissolved in 2,2,2-trifluoroethyl butyrate (Tokyo Chemical Industry Co., Ltd.; lot: CORPC-HQ) at room temperature was obtained. Sodium carbonate (Showa.; batch KDL-100W) and lipase acrylic resin (Sigma-Aldrich, Inc.; batch SLBW1544) were added to the solution. The reaction mixture was left at room temperature for 16 hours. The reaction mixture was filtered and concentrated on a rotary evaporator to give the crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; catalog No. HT 80186). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product. Table 8 shows the yields obtained with the different lipases. As shown in the table, there was no significant difference in the yields obtained using different kinds of lipases.

Table 8: OBI-3424-6-LS yields of compounds obtained using different lipases

Note: the yield using 2,2,2-trifluoroethyl butyrate was 74%, 96% ee, S form.

In addition, the inventors also tested several substrates using different classes of lipase (see table 9). Table 9 shows the yields obtained using several different substrates and lipases.

Table 9: selective transesterification catalyzed by different lipases

Example 7: selective hydrolysis of S-form based lipases

Step A: the acetyl matrix was dissolved in THF (Acros Organics; batch No. 1850266) and 0.1M potassium phosphate buffer pH 7.0 (Sigma-Aldrich, Inc.; batch No. MKBX6388V) at room temperature. Lipase acrylic resin (Sigma-Aldrich, Inc.; batch: SLBW1544) was added to the solution. The reaction mixture was left at room temperature for 16 hours. The reaction mixture was filtered and concentrated using a rotary evaporator to give the crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; catalog No. HT 80186). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product.

And B: the acetyl matrix was dissolved in MeOH (BioSuperStar Co., Ltd.; batch: 14022569) at room temperature. NaOMe was added to the solution. The reaction mixture was then stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was quenched with HCl (dissolved in MeOH, concentration 1N) and concentrated using a rotary evaporator to give the crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; catalog No. HT 80186). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product. Table 10 shows the yield of lipase reagent using acetyl substrate.

Table 10: yield obtained with lipase reagent using acetyl substrate

Example 8: protease-catalyzed selective transesterification from a mixture of compounds OBI-3426-6R/S

Step A: the hydroxyl matrix was dissolved in 2,2,2-trifluoroethyl butyrate (Tokyo Chemical Industry Co., Ltd.; lot: CORPC-HQ) at room temperature. Proteases from different species (Sigma-Aldrich, Inc., from Bacillus licheniformis (Bacillus licheniformis), batch: SLBX 1986; or from Streptomyces griseus, batch: SLCB9815) were added to the solution. The reaction mixture was left at room temperature for 16 hours. The reaction mixture was filtered and concentrated using a rotary evaporator to give the crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; catalog No. SY 350). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product.

And B: the acetyl matrix was dissolved in MeOH at room temperature. NaOMe was added to the solution. The reaction mixture was then stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was quenched with HCl (dissolved in MeOH, concentration 1N) and concentrated using a rotary evaporator to give the crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; catalog No. HT 80186). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product. Table 11 shows the yields obtained with the different proteases. As shown in the table, low yields were obtained using both proteases.

Table 11: yields obtained using different proteases

Example 9: s-form based protease selective hydrolysis

The acetyl matrix was dissolved in ether (Avantr Performance Materials, Inc.; batch # 0000128533) and 0.1M, pH 7.0.0 potassium phosphate buffer (Sigma-Aldrich, Inc.; batch # MKBX6388V) at room temperature. Protease (Sigma-Aldrich, Inc., from Bacillus licheniformis; batch: SLBX1986) was added to the solution. The reaction mixture was left at room temperature for 16 hours. The reaction mixture was filtered and concentrated using a rotary evaporator to give the crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; catalog No. HT 80186). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product. Table 12 shows the yields obtained with the protease using acetyl substrate.

Table 12: yield obtained using protease reagent of acetyl substrate

Example 10: mirror selective reduction of ketones-based on R-form

Step A: the hydroxyl matrix was dissolved in isopropyl acetate (Sigma-Aldrich, Inc.; batch: STBH5768) at room temperature. Sodium carbonate and lipase acrylic resin (Sigma-Aldrich, Inc.; batch: SLBW1544) were added to the solution. The reaction mixture was left at room temperature for 16 hours. The reaction mixture was filtered and concentrated on a rotary evaporator to give the crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; catalog No. HT 80186). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product.

And B: the acetyl matrix was dissolved in MeOH at room temperature. NaOMe was added to the solution. The reaction mixture was then stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was quenched with HCl (dissolved in MeOH, concentration 1N) and concentrated using a rotary evaporator to give the crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; catalog No. HT 80186). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product. Table 13 shows the yields obtained using the lipase reagent with acetyl substrate.

Table 13: yield obtained with lipase reagent using acetyl substrate

Example 11: selective hydrolysis of R-form based lipases

The acetyl matrix was dissolved in diethyl ether (Avantor Performance Materials, Inc.; batch # 0000128533) and 0.1M, pH 7.0.0 potassium phosphate buffer at room temperature. Protease (Sigma-Aldrich, Inc.; batch: SLBW1544) was added to the solution. The reaction mixture was left at room temperature for 16 hours. The reaction mixture was filtered and concentrated using a rotary evaporator to give the crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; catalog No. HT 80186). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product. Table 14 shows the yields obtained using the lipase reagent with acetyl substrate.

Table 14: yield obtained with lipase reagent using acetyl substrate

Example 12: r-form based protease selective hydrolysis

Step A: the acetyl matrix was dissolved in diethyl ether (Avantor Performance Materials, Inc.; batch # 0000128533) and 0.1M, pH 7.0.0 potassium phosphate buffer at room temperature. Protease (Sigma-Aldrich, Inc.; from Bacillus licheniformis, batch: SLBX1986) was added to the solution. The reaction mixture was left at room temperature for 16 hours. The reaction mixture was filtered and concentrated using a rotary evaporator to give the crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; catalog No. HT 80186). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product.

And B: the acetyl matrix was dissolved in MeOH at room temperature. NaOMe was added to the solution. The reaction mixture was then stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was quenched with HCl (dissolved in MeOH, concentration 1N) and concentrated using a rotary evaporator to give the crude product. The residue was purified by column chromatography through a silica gel pad (FUJI Silysia Chemical Ltd.; catalog No. HT 80186). The EtOAc/hexane volume ratio of the eluent system was 1/4 to 3/1. The collected separated liquid was concentrated using a rotary evaporator and high vacuum to obtain the product. Table 15 shows the yields obtained using acetyl matriptase.

Table 15: yields obtained using acetyl matriptase reagent

Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those described herein can be used in the practice of the present invention, the preferred compositions, methods, kits, and means for communicating information are described herein.

All references cited herein are to be understood as falling within the maximum extent permitted by law. The discussion of the foregoing documents is intended merely to summarize the assertions made by the authors. Nothing herein is admitted to be prior art to any document (or any portion thereof). Applicants reserve the right to challenge the accuracy and pertinence of any such documents.