Compounds, compositions and methods for cancer treatment
阅读说明：本技术 用于癌症治疗的化合物、组合物和方法 (Compounds, compositions and methods for cancer treatment ) 是由 T.A.刘易斯 吴晓筠 H.格鲁利克 M.迈耶森 M.埃勒曼 P.利瑙 K.艾斯 A. 于 2018-02-01 设计创作，主要内容包括：本发明涉及改进的化合物,尤其是具有结构(I)的化合物。使用与药物敏感性相关的生物标记(例如,PDE3A、PDE3B、SLFN12和/或CREB3L1)鉴定患者的组合物和方法,且因此用本发明的药物治疗分级的患者群体。<Image he="517" wi="597" file="DDA0002155520520000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The present invention relates to improved compounds, particularly compounds having structure (I). Compositions and methods for identifying patients using biomarkers associated with drug sensitivity (e.g., PDE3A, PDE3B, SLFN12, and/or CREB3L1), and thus treating a graded population of patients with a drug of the invention.)
1. A compound of formula (I)
Wherein R is1In each case the same and is Cl or F or a pharmaceutically acceptable salt or prodrug thereof.
2. The compound according to claim 1, having the structure:
or a pharmaceutically acceptable salt or prodrug thereof.
3. A pharmaceutical composition comprising one of the compounds selected from the group consisting of:
or a pharmaceutically acceptable salt or prodrug thereof, and one or more pharmaceutically acceptable carriers or excipients.
4. A method of killing or reducing survival of a cancer cell selected to respond to a phosphodiesterase 3A (PDE3A) and/or (PDE3B) modulator comprising contacting the cell with one or more PDE3A and/or PDE3B modulators having the structure:
wherein the cell is selected to have an increased level of PDE3A and/or PDE3B or Schlafen12(SLFN12) polypeptide or polynucleotide, or a combination thereof, relative to a reference, thereby reducing survival of the cancer cell.
5. A method of reducing cancer cell proliferation in a subject pre-selected for a cancer that is responsive to one or more PDE3A and/or PDE3B modulators having the structure:
comprising administering the PDE3A/PDE3B modulator to a subject, wherein the subject is pre-selected by: detecting an increase in the level of PDE3A and/or PDE3B and Schlafen12(SLFN12) polypeptide or polynucleotide, or a combination thereof, in cells derived from the cancer of the subject, relative to a reference, thereby reducing cancer cell proliferation in the subject.
6. The method of claim 4 or 5, wherein the PDE3A and/or PDE3B modulator reduces the activity of PDE3A and/or PDE 3B.
7. The method of claim 6, wherein the cells derived from the cancer of the subject are from a biological sample of the subject, wherein the biological sample is a tissue sample comprising cancer cells.
8. A method of treating a hyperproliferative disorder with one of the compounds selected from:
9. the method according to claim 8, wherein the hyperproliferative disease is cancer.
10. The method of claim 9, wherein the cancer is bone cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, gastrointestinal stromal tumor (GIST), head and neck cancer, hematopoietic cancer, kidney cancer, leiomyosarcoma, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, soft tissue sarcoma, thyroid cancer, or urinary tract cancer.
11. The composition of claim 3 wherein said compound is
12. The method of any one of claims 4 to 10 further comprising detecting a deletion that reduces the level of expression of a polypeptide or polynucleotide relative to a reference CREB3L 1.
13. The method of claim 12, further comprising detecting a decrease in SLFN12 levels.
14. The method according to any one of claims 4-13, wherein the PDE3A and/or PDE3B modulator is
15. A method of administration wherein the PDE3A and/or PDE3B modulator according to claim 1 is administered orally or intravenously.
16. A kit for reducing cancer cell proliferation in a subject pre-selected for response to a PDE3A/PDE3B modulator comprising one of the compounds selected from the group consisting of
Or a pharmaceutically acceptable salt or prodrug thereof.
17. A kit for identifying a subject having a cancer that is resistant to PDE3A/PDE3B modulation of a compound according to claim 1, the kit comprising a capture reagent that binds to a CREB3L1 polypeptide or polynucleotide.
18. The kit of claim 17, further comprising a capture reagent that binds to SLFN12 polypeptide or polynucleotide.
Use of a PDE3A and/or PDE3B modulator in the manufacture of a medicament for the treatment of cancer, wherein the PDE3A and/or PDE3B modulator is one of the compounds selected from
Or a pharmaceutically acceptable salt or prodrug thereof.
20. The use of claim 19, wherein the cancer is bone cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, gastrointestinal stromal tumor (GIST), head and neck cancer, hematopoietic cancer, kidney cancer, leiomyosarcoma, liver cancer, lung cancer, lymphoma, skin cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, soft tissue sarcoma, thyroid cancer, or cancer of the urinary tract.
21. The use of claim 20, wherein the cancer is melanoma or cervical cancer.
22. A process for preparing compound 1, said process comprising the steps of
Reacting a compound of formula (IV)
With pure morpholine at elevated temperature, or with morpholine and a base, such as an amine or carbonate, especially N, N-diisopropylethylamine, optionally in a polar aprotic solvent such as an alcohol or CH3CN, at reflux temperature to obtain compound (V)
Then reacting it with a strong base in polarReaction in aprotic solvent at low temperature, e.g., -78 ℃ to-60 ℃, followed by addition of (C) neat or in polar aprotic solvent1-C4Alkyl) bromoacetate or (C)1-C4Alkyl) chloroacetate, warming the mixture from initial-78 ℃ to room temperature, optionally isolating the crude product, and then adding hydrazine or hydrazine hydrate in a polar protic organic solvent at reflux temperature to give racemic compound 1c
Subsequently separating the enantiomers of Compound 1c to obtain Compound 1 and Compound (1a)
Wherein optionally compound (1a) is converted to racemic compound (1c), which can then be separated again to obtain a minor fraction of the initial amount of compound 1 and compound 1a separated from the enantiomer.
23. A process for preparing Compound 1, wherein Compound (IV)
Reacting with a strong base in a polar aprotic solvent at a low temperature of-78 ℃ to-60 ℃, and then adding (C) neat or in a polar aprotic solvent1-C4Alkyl) bromoacetate or (C)1-C4-alkyl) chloroacetate, warming the mixture from initial-78 ℃ to room temperature, optionally isolating the crude product, and then adding hydrazine or hydrazine hydrate in a polar protic organic solvent at reflux temperature to prepare compound (VII)
And further reacting compound (VII) with pure morpholine at elevated temperature, or with morpholine and a base in a polar aprotic solvent at reflux temperature to give compound 1c
Subsequently separating the enantiomers of Compound 1c to obtain Compound 1 and Compound (1a)
Wherein optionally compound 1a is converted to a racemic material which can then be separated to give compound 1 and a minor portion of the initial amount of compound 1 a.
24. The use of compounds (IV), (V), (VI), (VII) according to claims 22 and 23 for the preparation of compound 1,
compound 1 has the formula
Background
Cancer causes more than 550,000 deaths in the united states and more than 800 million deaths worldwide each year. New drugs, including small molecules, molecules that affect tissue-specific growth requirements, and immunomodulators, have been shown to be beneficial in subgroups of patients with cancer having unique genomic mutations or other characteristics. Unfortunately, many cancer patients still have no effective treatment options.
One approach to identifying new anticancer agents is to perform phenotypic screens to find novel small molecules that show strong selectivity between cancer cell lines, and then identify cellular features associated with drug responses by predictive chemogenomics. During the 90's of the 20 th century, Weinstein and colleagues demonstrated that the cytotoxic characteristics of compounds could be used to identify cellular features associated with drug sensitivity, such as gene expression profiles and DNA copy number. In recent years, the ability to identify the characteristics of cancer cell lines that mediate their response to small molecules has greatly increased through automated high-throughput chemical sensitivity testing of large-scale cell lines, as well as comprehensive genomic and phenotypic characterization of cell lines. Phenotypic observations of small molecule sensitivity may be associated with expression patterns or somatic changes, as in the case of trastuzumab-sensitive HER 2-amplified breast cancer or erlotinib-sensitive EGFR mutant lung cancer.
Savai et al (Expert Opinion on innovative Drugs, vol. 19,
Methods for characterizing malignant tumors at the molecular level can be used to stratify patients and thereby rapidly guide them to effective treatment. There is an urgent need for improved methods for predicting responsiveness of a subject suffering from cancer.
Disclosure of Invention
The present invention relates to compounds, methods for their preparation and methods for cancer treatment, as described below.
The compounds are suitable for treating a patient having a cancer that is sensitive to treatment with a phosphodiesterase 3A (PDE3A) and/or phosphodiesterase 3B (PDE3B) modulator (e.g.,
In one aspect, the present invention provides compounds having the structure
Wherein R is1In each case the same and is Cl or F or a pharmaceutically acceptable salt or prodrug thereof.
In another aspect, the present invention provides compounds having the structure:
or a pharmaceutically acceptable salt or prodrug thereof.
In another aspect, the present invention provides compounds having the structure:
or a pharmaceutically acceptable salt or prodrug thereof.
In another aspect, the present invention provides a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers or excipients and a compound of formula (I)
Wherein R is1In each case the same and is Cl or F or a pharmaceutically acceptable salt or prodrug thereof.
In another aspect, the present invention provides a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers or excipients and one of the compounds selected from the group consisting of:
or a pharmaceutically acceptable salt or prodrug thereof.
In one aspect, the invention provides a method of killing or reducing survival of a cancer cell selected to respond to a phosphodiesterase 3A (PDE3A) and/or phosphodiesterase 3B (PDE3B) modulator comprising contacting the cell with a PDE3A and/or PDE3B modulator having the structure:
wherein R is1In each case the same and is Cl or F or a pharmaceutically acceptable salt or prodrug thereof.
In some embodiments the cell is selected to have an increased level of PDE3A and/or PDE3B or Schlafen12(SLFN12) polypeptide or polynucleotide, or a combination thereof, relative to a reference, thereby reducing survival of the cancer cell.
In another aspect, the invention provides a method of reducing cancer cell proliferation in a subject pre-selected for a cancer that is responsive to a PDE3A and/or PDE3B modulator, comprising contacting the cell with a PDE3A and/or PDE3B modulator having the structure:
wherein R is1In each case the same and is Cl or F or a pharmaceutically acceptable salt or prodrug thereof, and wherein the subject is pre-selected by: detecting an increase in the level of a polypeptide or polynucleotide relative to a reference PDE3A and/or PDE3B or Schlafen12(SLFN12) or a combination thereof, thereby reducing cancer cell proliferation in the subject.
In some embodiments, the subject is pre-selected by detecting an increase in the level of PDE3A and/or PDE3B polypeptide or polynucleotide relative to a reference and detecting an increase in the level of SLFN12 polypeptide or polynucleotide, thereby reducing cancer cell proliferation following treatment with a compound of formula (I) in the subject. In some embodiments, the subject is pre-selected by detecting an increase in the level of PDE3A and/or PDE3B polypeptide or polynucleotide relative to a reference and detecting an increase in the level of SLFN12 polypeptide or polynucleotide, thereby reducing cancer cell proliferation in the subject following treatment with the compound of formula (I).
In another aspect, the present invention provides a method of treating a hyperproliferative disease, particularly cancer, comprising administering to a subject in need thereof a compound of formula (I) having the structure
Wherein R is1In each case the same and is Cl or F or a pharmaceutically acceptable salt or prodrug thereof.
In another aspect, the present invention provides a method of treating a hyperproliferative disease, particularly cancer, comprising administering to a subject in need thereof a compound of formula (I) having the structure
Wherein R is1Identical in each case and is Cl or F; or a pharmaceutically acceptable salt or prodrug thereof, wherein the cancer is responsive to a PDE3A and/or PDE3B modulator.
In another aspect, the present invention provides a method of treating a hyperproliferative disease, particularly cancer, comprising administering to a subject in need thereof a compound of formula (I) having the structure
Wherein R is1Identical in each case and is Cl or F; or a pharmaceutically acceptable salt or prodrug thereof, wherein the subject has been diagnosed as havingCancers that respond to PDE3A and/or PDE3B modulators.
In another aspect, the present invention provides a method of treating a hyperproliferative disease, particularly cancer, comprising administering to a subject in need thereof a compound of formula (I) having the structure
Wherein R is1In each case the same and is Cl or F, or a pharmaceutically acceptable salt or prodrug thereof, wherein the cancer is bone cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, gastrointestinal stromal tumor (GIST), head and neck cancer, hematopoietic cancer, kidney cancer, leiomyosarcoma, liver cancer, lung cancer, lymphatic cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, soft tissue sarcoma, thyroid cancer, urinary tract cancer.
In another aspect, the invention provides a kit for reducing cancer cell proliferation in a subject pre-selected for response to a PDE3A and/or PDE3B modulator, the PDE3A and/or PDE3B modulator comprising a compound having the structure
Wherein R is1Identical in each case and is Cl or F; or a pharmaceutically acceptable salt or prodrug thereof.
In another aspect, the invention provides the use of a PDE3A and/or PDE3B modulator in the manufacture of a medicament for the treatment of cancer, wherein the PDE3A and/or PDE3B modulator is a compound of formula (I) having the structure
Wherein R is1Identical in each case and is Cl or F; or a pharmaceutically acceptable salt or prodrug thereof.
In another aspect, the invention provides PDE3A and/or PDE3B modulators for use in the treatment of cancer, wherein the PDE3A and/or PDE3B modulator is a compound of formula (I) having the structure
Wherein R is1Identical in each case and is Cl or F; or a pharmaceutically acceptable salt or prodrug thereof.
In other embodiments, the invention provides PDE3A and/or PDE3B modulators for use in the treatment of cancer, wherein the PDE3A and/or PDE3B modulator is a compound of formula (I) having the structure
Wherein R is1Identical in each case and is Cl or F; or a pharmaceutically acceptable salt or prodrug thereof, wherein the cancer is bone cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, gastrointestinal stromal tumor (GIST), head and neck cancer, hematopoietic cancer, kidney cancer, leiomyosarcoma, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, soft tissue sarcoma, thyroid cancer, cancer of the urinary tract.
In various embodiments of any aspect delineated herein, the PDE3A and/or PDE3B modulator reduces the activity of PDE3A and/or PDE 3B.
In various embodiments, the PDE3A and/or PDE3B modulators have the following structures:
in some other embodiments, the present invention provides compounds having the following structure as PDE3A/PDE3B modulators:
or a pharmaceutically acceptable salt or prodrug thereof.
In various embodiments the invention provides the above compositions and methods wherein the PDE3A/PDE3B modulator is Compound 1.
In another aspect, the present invention provides compounds having the structure:
or a pharmaceutically acceptable salt or prodrug thereof.
In various embodiments the invention provides the above compositions and methods wherein the PDE3A and/or PDE3B modulator is
In various embodiments of any aspect delineated herein, the method comprises detecting a deletion that reduces the level of expression of a polypeptide or polynucleotide relative to a
In various embodiments of any aspect delineated herein, the method comprises detecting an increase in SLFN12 levels.
In various embodiments of any aspect delineated herein, the biological sample is a tissue sample comprising cancer cells.
In various embodiments, the level of PDE3A, PDE3B, SLFN12, or CREB3L1 polypeptide is detected by a method selected from the group consisting of: immunoblotting, mass spectrometry and immunoprecipitation.
In various embodiments, the level of PDE3A, PDE3B, SLFN12, or CREB3L1 polynucleotide is detected by a method selected from the group consisting of: quantitative PCR, RNA sequencing, Northern blotting, microarray, mass spectrometry, and in situ hybridization.
In various embodiments of any aspect delineated herein, cancer cells selected to be responsive to phosphodiesterase 3A (PDE3A) and/or phosphodiesterase 3B (PDE3B) modulators express CREB3L1 or lack of CREB3L1 expression relative to a reference.
In various embodiments, the cancer cells selected to be responsive to phosphodiesterase 3A (PDE3A) and/or phosphodiesterase 3B (PDE3B) modulators are bone, breast, cervical, colon, endometrial, gastrointestinal stromal tumors (GIST), head and neck, hematopoietic, kidney, leiomyosarcoma, liver, lung, lymph, melanoma, ovarian, pancreatic, prostate, skin, soft tissue sarcoma, thyroid, urinary tract cancer cells.
Thus in various embodiments of any aspect delineated herein, the methods disclosed above further comprise no reduction in the level of a CREB3L1 polypeptide or polynucleotide relative to a reference.
In various embodiments of any aspect delineated herein, cancer cells resistant to a phosphodiesterase 3A (PDE3A) and/or phosphodiesterase 3B (PDE3B) modulator have reduced expression of CREB3L1 and/or SLFN12 or loss of CREB3L1 and/or SLFN12 expression relative to a reference.
In various embodiments, the cancer cells selected to be responsive to phosphodiesterase 3A (PDE3A) and/or phosphodiesterase 3B (PDE3B) modulators are skin cancer (e.g., melanoma), endometrial cancer, lung cancer, hematopoietic/lymphatic cancer, ovarian cancer, cervical cancer, soft tissue sarcoma, leiomyosarcoma, urinary tract cancer, pancreatic cancer, thyroid cancer, renal cancer, glioblastoma, or breast cancer cells.
In various embodiments, the cancer cells selected to be responsive to phosphodiesterase 3A (PDE3A) and/or phosphodiesterase 3B (PDE3B) modulators are bone, breast, cervical, colon, endometrial, gastrointestinal stromal tumors (GIST), head and neck, hematopoietic, kidney, leiomyosarcoma, liver, lung, lymph, melanoma, ovarian, pancreatic, prostate, skin, soft tissue sarcoma, thyroid, urinary tract cancer cells.
In various embodiments of any aspect delineated herein, the cancer cells selected to be responsive to phosphodiesterase 3A (PDE3A) and/or phosphodiesterase 3B (PDE3B) modulators have increased PDE3A and/or PDE3B and Schlafen12(SLFN12) expression.
In various embodiments of any aspect delineated herein, the cancer cells resistant to a phosphodiesterase 3A (PDE3A) modulator have reduced expression of CREB3L1 and/or SLFN12 or loss of CREB3L1 and/or SLFN12 expression relative to a reference.
By "reference" herein is meant the average expression in a representative group of tumor cells or tumor cell lines.
In various embodiments of any aspect delineated herein, the cancer is responsive to a PDE3A and/or PDE3B modulator.
In various embodiments, the subject has been diagnosed with a cancer that is responsive to a PDE3A and/or a PDE3B modulator.
In various embodiments, the cancer is melanoma, endometrial cancer, lung cancer, hematopoietic/lymphoid cancer, ovarian cancer, cervical cancer, soft tissue sarcoma, leiomyosarcoma, urinary tract cancer, pancreatic cancer, thyroid cancer, renal cancer, glioblastoma, or breast cancer.
In various embodiments, the cancer is a skin cancer (e.g., melanoma) or cervical cancer.
In various embodiments of any aspect delineated herein, the PDE3A and/or PDE3B modulator is administered orally.
In various embodiments of any aspect delineated herein, the PDE3A and/or PDE3B modulator is administered by intravenous injection.
In various embodiments of any aspect delineated herein, the PDE3A/PDE3B modulator is administered orally or intravenously.
The invention provides methods of treating a subject having a cancer identified as responsive to treatment with a PDE3A and/or PDE3B modulator selected from compounds 1-2 by detecting a deletion in the cancer of co-expression of PDE3A and/or PDE3B and Schlafen12(SLFN12) polynucleotides or polypeptides and/or reduced expression of CREB3L1 polynucleotides or polypeptides.
The invention therefore further provides methods of detecting CREB3L1 polynucleotide or polypeptide expression for the stratification of patients treated with
The invention further provides a method of detecting the expression of PDE3A and/or PDE3B and/or Schlafen12(SLFN12) polynucleotides or polypeptides for stratifying patients treated with
The compositions and articles defined by the present invention are isolated or manufactured according to the examples provided below. Other features and advantages of the invention will be apparent from the detailed description and from the claims.
Definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide those skilled in the art with a general definition of many of the terms used in the present invention: singleton et al, Dictionary of Microbiology and Molecular Biology (2 nd edition, 1994); the Cambridge Dictionary of Science and Technology (Walker, eds., 1988); the Glossary of Genetics, 5 th edition, R.Rieger et al (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings assigned to them below, unless otherwise specified.
By "
by "
the drawn structure includes all allowable rotations around the keys.
In some embodiments, any of
In some embodiments, a combination of small molecule phosphodiesterase inhibitors or modulators may be used.
In some embodiments, any combination of compounds 1-2 can be used.
In some embodiments, small molecule phosphodiesterase inhibitors or modulators, particularly compounds 1-2, in combination with anti-cancer agents may be used.
General description of the Synthesis of
There are several methods for preparing
In one embodiment, the present invention provides a process for preparing
reacting a compound of formula (IV)
With pure morpholine at elevated temperature, or with morpholine and a base, such as an amine or carbonate, especially N, N-diisopropylethylamine, optionally in a polar aprotic solvent such as an alcohol or CH3CN, at reflux temperature to obtain compound (V)
Then reacting it with a strong base in a polar aprotic solvent at low temperature, e.g., -78 ° to-60 ℃, and then adding (C) neat or in a polar aprotic solvent1-C4Alkyl) bromoacetate or (C)1-C4Alkyl) chloroacetate, warming the mixture from the initial low temperature (e.g., -78 ℃) to room temperature, optionally isolating the crude product, and then adding hydrazine or hydrazine hydrate in a polar protic organic solvent at reflux temperature to obtain the racemic compound (1c)
Subsequently separating the enantiomers of the compound (1c) to obtain the
Wherein optionally compound (1a) is converted to racemic compound (1c), which can then be separated again to obtain a minor fraction of the initial amount of
In another embodiment, the present invention provides a process for preparing
Reacting with a strong base in a polar aprotic solvent at a low temperature of-78 ℃ to-60 ℃, and then adding (C) neat or in a polar aprotic solvent1-C4Alkyl) bromoacetate or (C)1-C4Alkyl) chloroacetate, warming the mixture from the initial low temperature (e.g., -78 ℃) to room temperature, optionally isolating the crude product, and then adding hydrazine or hydrazine hydrate in a polar protic organic solvent at reflux temperature to prepare compound (VII)
And further reacting compound (VII) with pure morpholine at elevated temperature, or with morpholine and a base in a polar aprotic solvent at reflux temperature to give compound (1c)
Subsequently separating the enantiomers of the compound (1c) to obtain the
Wherein optionally compound (1a) is converted to a racemic material which can then be separated to give a minor portion of the initial amount of
In another embodiment, the present invention provides intermediate compounds (IV), (V), (VI), (VII),
use for the preparation of
Another aspect of the invention is a process for preparing
With morpholine neat, or with morpholine and a base, such as an amine, for example diisopropylamine, triethylamine, diisopropylethylamine or a carbonate, for example sodium carbonate, calcium carbonate, magnesium carbonate, especially N, N-diisopropylethylamine, optionally in a polar solvent, such as an alcohol, for example methanol, ethanol, propanol, isopropanol, butanol (N-butanol, sec-butanol, tert-butanol), methoxyisobutanol, acetonitrile, but especially CH3CN, at reflux temperature to obtain compound (V)
Then reacting it with a strong base, such as sodium hydride, butyllithium: (nBuLi、sBuLi、t-BuLi)), Lithium Diisopropylamide (LDA) or lithium hexamethyldisilazide (LiHMDS), especially LiHMDS, in a polar aprotic solvent, such as tetrahydrofuran, dioxane, hexane, cyclohexane, toluene, especially tetrahydrofuran, at low temperatures, such as-78 ° to-60 ℃, preferably at-78 ℃, followed by addition of pure or in tetrahydrofuran, dioxane, or lithium hexamethyldisilazide (LiHMDS), either alone or in combination with a solvent, such as water, ethanol, or a mixture thereofIn alkanes, hexanes, cyclohexane, or toluene, especially in tetrahydrofuran or other solvents (C)1-C4Alkyl) bromoacetate or (C)1-C4-alkyl) chloroacetate, especially ethyl bromoacetate, the mixture is warmed from initial-78 ℃ to room temperature, the crude product is optionally isolated and then hydrazine or hydrazine hydrate is added at reflux temperature in a polar protic organic solvent, such as water, methanol, ethanol, propanol, isopropanol, butanol or methoxyisobutanol, preferably in ethanol, to obtain racemic compound 1c
Subsequently separating the enantiomers of Compound 1c to obtain
Wherein compound 1a is optionally converted to compound 1c, which can then be separated to give
Another aspect of the present invention is the compounds (V) and/or (VI) or (VII)
Use for
Another aspect of the present invention is a process for preparing
With strong bases, e.g. sodium hydride, butyl lithium: (nBuLi、sBuLi、t-BuLi)), Lithium Diisopropylamide (LDA) or lithium hexamethyldisilazide (LiHMDS), especially LiHMDS, in a polar aprotic solvent, such as tetrahydrofuran, dioxane, hexane, cyclohexane, toluene, especially tetrahydrofuran, at low temperatures, such as-78 ° to-60 ℃, preferably at-78 ℃, followed by addition of pure or (C) in tetrahydrofuran, dioxane, hexane, cyclohexane, or toluene, especially in tetrahydrofuran or other solvents1-C4Alkyl) bromoacetate or (C)1-C4-alkyl) chloroacetate, especially ethyl bromoacetate, the mixture is warmed from initial-78 ℃ to room temperature, the crude product is optionally isolated and then hydrazine or hydrazine hydrate in a polar protic organic solvent, such as water, methanol, ethanol, propanol, isopropanol, butanol or methoxyisobutanol, preferably in ethanol, is added at reflux temperature to give racemic compound 1c to prepare compound (VII)
And further reacting compound (VII) with morpholine neat at elevated temperature, or with morpholine and a base, such as an amine, for example triethylamine, diisopropylamine, N-diisopropylethylamine, triethylamine or a carbonate, for example sodium carbonate, calcium carbonate, magnesium carbonate, especially N, N-diisopropylethylamine, optionally in a polar aprotic solvent, such as an alcohol, for example methanol, ethanol, propanol, isopropanol, butanol (N-butanol, sec-butanol, tert-butanol), methoxyisobutanol or acetonitrile (CH)3CN) at reflux temperature to give Compound 1c
Subsequently separating the enantiomers of Compound 1c to obtain
Wherein compound 1a is optionally converted to compound 1c, which can then be separated to give
Thus a further aspect of the invention is the use of compounds (IV) and (VII) for the preparation of
By "CREB 3L1 polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in GenBank accession No. AAH14097.1, which is cleaved upon endoplasmic reticulum stress and has transcription factor activity. The amino acid sequence provided by GenBank accession No. AAH14097.1 is shown below.
By "CREB 3L1 polynucleotide" is meant any nucleic acid molecule, including DNA and RNA, that encodes a CREB3L1 polypeptide or fragment thereof. Exemplary CREB3L1 nucleic acid sequences are described in NCBI reference numbers: NM _ 052854.3. NCBI reference number: the sequence provided by NM _052854.3 is reproduced as follows:
by "PDE 3A polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI reference NP _000912.3, which catalyzes the hydrolysis of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). An exemplary human full-length PDE3A amino acid sequence is provided below:
three PDE3A isoforms are known: PDE3a1, PDE3a2 and PDE3 A3. PDE3A1 comprises amino acids 146-1141 of the full length PDE3A amino acid sequence,
By "PDE 3A polynucleotide" is meant any nucleic acid molecule, including DNA and RNA, that encodes a PDE3A polypeptide or fragment thereof. Exemplary PDE3A nucleic acid sequences are described in NCBI reference numbers: NM _000921.4 provides:
by "Schlafen 12(SLFN12) polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided at NCBI reference NP _060512.3, which when bound to one of the compounds described herein interacts with PDE 3A. An exemplary human SLFN12 amino acid sequence is provided below:
by "Schlafen 12(SLFN12) polynucleotide" is meant any nucleic acid molecule, including DNA and RNA, encoding a SLFN12 polypeptide or fragment thereof. Exemplary SLFN12 nucleic acid sequences are described in NCBI reference numbers: NM _018042.4 provides:
by "PDE 3B polynucleotide" is meant any nucleic acid molecule, including DNA and RNA, that encodes a PDE3B polypeptide or fragment thereof. Exemplary PDE3B nucleic acid sequences are described in NCBI reference numbers: NM _000922.3 provides:
by "PDE 3B polypeptide" is meant a protein or fragment thereof having at least 85% amino acid sequence identity to the sequence provided in NCBI reference NP _ 000913.2. An exemplary human PDE3B amino acid sequence is provided below:
in some aspects, the compound is an isomer. "isomers" are different compounds having the same molecular formula. "stereoisomers" are isomers that differ only in the way the atoms are arranged in space. As used herein, the term "isomer" includes any and all geometric isomers and stereoisomers. For example, "isomers" include the geometric double bond cis-and trans-isomers, also known as E-and Z-isomers; r-and S-enantiomers; diastereomers, (d) -isomers and (l) -isomers, racemic mixtures thereof; and other mixtures thereof also fall within the scope of the invention
Symbol
Substituents around a carbon-carbon double bond may alternatively be referred to as "cis" or "trans," where "cis" represents the substituent on the same side of the double bond and "trans" represents the substituent on the opposite side of the double bond. The arrangement of substituents around a carbocyclic ring may also be designated as "cis" or "trans". The term "cis" represents substituents on the same side of the ring plane and the term "trans" represents substituents on opposite sides of the ring plane. Mixtures of compounds in which the substituents are located on the same and opposite sides of the ring plane are designated "cis/trans".
The term "enantiomer" refers to a pair of stereoisomers that have mirror images of each other that are not superimposable. Atoms with an asymmetric set of substituents can give rise to enantiomers. Mixtures of a pair of enantiomers in any ratio may be referred to as "racemic" mixtures. The term "(±)" is used to refer to a racemic mixture, where appropriate. "diastereoisomers" are stereoisomers having at least two asymmetric atoms, and are not mirror images of each other. Absolute stereochemistry is specified according to the Cahn-Ingold-prelogR-S system. When the compounds are enantiomers, the stereochemistry of each chiral carbon may be represented as R or S. Resolved compounds of unknown absolute configuration can be designated (+) or (-) depending on the direction (dextro-or levorotatory) in which they rotate plane-polarized light of wavelength sodium D-line. Certain compounds described herein contain one or more asymmetric centers and thus can give rise to enantiomers, diastereomers, and other stereoisomeric forms, which can be defined as (R) -or (S) -based on the absolute stereochemistry at each asymmetric atom. The chemical entities, pharmaceutical compositions and methods of the present invention are meant to include all such possible isomers (including racemic mixtures, optically substantially pure forms and mixtures therebetween).
Optically active (R) -isomers and (S) -isomers can be prepared using, for example, chiral synthons or chiral reagents, or resolved using conventional techniques. Enantiomers can be separated from racemic mixtures by any method known to those skilled in the art, including chiral High Pressure Liquid Chromatography (HPLC), formation and crystallization of chiral salts, or preparation by asymmetric synthesis.
Optical isomers can be obtained by resolution of the racemic mixture according to conventional methods, for example by treatment with an optically active acid or base to form diastereomeric salts. Examples of suitable acids are tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid and camphorsulfonic acid. Separation of the mixture of diastereomers by crystallization followed by release of the optically active base from these salts provides separation of the isomers. Another method involves the synthesis of covalent diastereomeric molecules by reacting the disclosed compounds with an optically pure acid or an optically pure isocyanate in activated form. The synthesized diastereomers may be separated by conventional means, such as chromatography, distillation, crystallization, or sublimation, followed by hydrolysis to provide the enantiomerically enriched compound. Optically active compounds can also be obtained by using active starting materials. In some embodiments, these isomers may be in the form of the free acid, free base, ester, or salt.
In certain embodiments, the compounds of the present invention may be tautomers. As used herein, the term "tautomer" is a type of isomer that includes two or more interconvertible compounds resulting from the migration of at least one form of a hydrogen atom and at least one change in valence (e.g., single bond to double bond, triple bond to single bond, or vice versa). "tautomerism" includes proton shift or proton shift tautomerism, which is considered to be a subset of acid-base chemistry. "proton shift tautomerism" or "proton shift tautomerism" relates to proton migration with a change in bond order. The exact ratio of tautomers depends on several factors including temperature, solvent and pH. When tautomerism is possible (e.g., in solution), the chemical equilibrium of the tautomer can be reached. Tautomerism (i.e., the reaction that provides a tautomeric pair) can be catalyzed by an acid or a base, or can occur in the absence or presence of an external agent. Exemplary tautomers include, but are not limited to, keto-enols; amide to imide; lactam to lactam; enamines to imines; and enamine to (different) enamine tautomerism. A specific example of keto-enol tautomerism is the interconversion of pentane-2, 4-dione and 4-hydroxypent-3-en-2-one tautomers. Another example of tautomerism is phenol-ketone tautomerism. One specific example of phenol-ketone tautomerism is the interconversion of pyridin-4-ol and pyridin-4 (1H) -one tautomers.
All chiral, diastereomeric, racemic, and geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. All methods for preparing the compounds of the present invention and intermediates prepared therein are considered to be part of the present invention. All tautomers of the compounds shown or described are also considered to be part of the present invention.
"agent" refers to any small molecule chemical compound, antibody, nucleic acid molecule or polypeptide, or fragment thereof.
By "improving" is meant reducing, inhibiting, attenuating, reducing, arresting or stabilizing the development or progression of a disease.
"alteration" refers to a change (increase or decrease) in the level of expression or activity of a gene or polypeptide, as detected by methods known in the art, such as those described herein. As described herein, in one embodiment, the alteration comprises a change in expression level of about 10%, preferably about 25%, more preferably about 40%, and most preferably about 50% or greater. In certain embodiments, alteration comprises a change of 10% or less (including 10%) of the expression level, preferably a change of 25% or less (including 25%), more preferably a change of 40% or less (including 40%), and most preferably a change of 50% or less (including 50%) or greater of the expression level. In other embodiments, the alteration comprises a change in the expression level of 9% to 11% (including 9% and 11%), preferably a change of 10% to 25% (including 10% and 25%), more preferably a change of 25% to 40% (including 25% and 40%), and most preferably a change of 40% to 50% (including 40% to 50%) or greater than 50% (including 50%) of the expression level. In other certain embodiments, the alteration comprises a change in the expression level of 9% to 11% (including 9% and 11%), preferably a change of 22% to 28% (including 22% and 28%), more preferably a change of 35% to 45% (including 35% and 45%), and most preferably a change of 45% to 55% (including 45% to 55%) or greater than or equal to 55% of the expression level.
By "analog" is meant a molecule that is not identical but has similar functional or structural characteristics. For example, a polypeptide analog retains the biological activity of the corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the function of the analog relative to the naturally-occurring polypeptide. Such biochemical modifications can increase the protease resistance, membrane permeability, or half-life of the analog without altering, for example, ligand binding. Analogs can include unnatural amino acids.
In the present disclosure, "comprise," "include," "contain," and "have" and the like may have the meaning attributed to them by U.S. patent law, and may mean "include" and the like; likewise, "consisting essentially of …" has the meaning assigned by U.S. patent law, and the term is open-ended, allowing the presence of other elements than the recitation of items, provided that the essential or novel features of the recitation are not changed by the presence of elements other than the recitation, but exclude prior art embodiments.
"detecting" refers to identifying the presence, absence, or amount of an analyte to be detected. In particular embodiments, the analyte is a PDE3A or PDE3AB or SLFN12 polypeptide.
By "disease" is meant any condition or disorder that impairs or interferes with the normal function of a cell, tissue or organ. Examples of diseases include melanoma, adenocarcinoma, lung cancer, cervical cancer, liver cancer, and breast cancer.
By "effective amount" is meant the amount of a compound described herein that is needed to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound for use in practicing the present invention to therapeutically treat a disease will vary with the mode of administration, the age, weight, and general health of the subject. Finally, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such amounts are referred to as "effective" amounts. In other embodiments, the PDE3A and/or PDE3B modulator is
The present invention provides a number of targets that can be used to develop highly specific drugs for the treatment of disorders characterized by the methods described herein. In addition, the methods of the invention provide a convenient way to identify therapies that are safe for use by a subject. Furthermore, the methods of the invention provide a pathway for analyzing the effects of almost any number of compounds on the diseases described herein, with high volumetric throughput, high sensitivity and low complexity.
"fragment" refers to a portion of a polypeptide or nucleic acid molecule. The portion comprises, preferably, at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the entire length of the reference nucleic acid molecule or polypeptide. In certain embodiments, this portion comprises, preferably, at least 9% -11% (including 9% and 11%), 18% -22% (including 18% and 22%), 27% -33% (including 27% and 33%), 36% -44% (including 36% and 44%), 45% -55% (including 45% and 55%), 54% -66% (including 54% and 66%), 63% -77% (including 63% and 77%), 72% -88% (including 72% and 88%), or 81% -99% (including 81% and 99%) of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may comprise about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 nucleotides or amino acids. In some embodiments, a fragment may comprise 9-11, about 18-22, 27-33, 36-44, 45-55, 54-66, 63-77, 72-88, 81-99, 90-110, 180-220, 270-330, 360-440, 450-550, 540-660, 630-770, 720-880, 810-990, or 900-1100 nucleotides or amino acids (including each of the limits, e.g., for "9-11" means including 9 and 11).
"hematological tumors" include aggressive and indolent forms of leukemia and lymphoma, i.e., non-hodgkin's disease, chronic and acute myelogenous leukemia (CML/AML), Acute Lymphoblastic Leukemia (ALL), hodgkin's disease, multiple myeloma, and T-cell lymphoma. Also included are myelodysplastic syndromes, plasmacytomas, paraneoplastic syndromes, cancers with unknown primary site and AIDS-related malignancies.
"hybridization" refers to hydrogen bonding, which can be Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonding between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds.
"hyperproliferative diseases" include, for example, psoriasis, keloids and other hyperplasias which affect the skin, benign hyperproliferative diseases, hematopoietic hyperproliferative diseases, cancers (e.g., metastatic or malignant tumors, solid tumors and hematological tumors).
"benign hyperproliferative diseases" include, for example, endometriosis, leiomyoma, and benign prostatic hyperplasia.
"hematopoietic hyperproliferative diseases" also referred to as myoproliferative diseases include, for example, polycythemia vera, essential thrombocythemia, primary myelofibrosis, and the like.
By "marker" or "biomarker" is meant any protein or polynucleotide that has an alteration in expression level or activity (e.g., at the protein or mRNA level) associated with a disease or disorder. In a specific embodiment, the marker of the invention is PDE3A or PDE3B or SLFN12 or
"modulator" refers to any agent that binds to a polypeptide and alters the biological function or activity of the polypeptide. Modulators include, but are not limited to, agents that reduce or eliminate a biological function or activity of a polypeptide (e.g., "inhibitors"). For example, a modulator can inhibit the catalytic activity of a polypeptide. Modulators include, but are not limited to, agents that increase or decrease binding of a polypeptide to another agent. For example, a modulator may facilitate binding of a polypeptide to another polypeptide. In some embodiments, the modulator of a PDE3A/PDE3B polypeptide is DNMDP. In some other embodiments, the modulator of a PDE3A/PDE3B polypeptide is anagrelide or zadaviline. In other embodiments, the modulator of the PDE3A/PDE3B polypeptide is
The term "prodrug(s)" means compounds that may be biologically active or inactive by themselves, but which are converted (e.g., metabolized or hydrolyzed) to the compounds of the invention within their residence time in the body. Derivatives (bioprecursors or prodrugs) of
By "reference" is meant a standard or control condition.
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule encoding a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule encoding a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence is typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule.
"hybridization" refers to the pairing under various stringent conditions to form a complementary polynucleotide sequence (such as the gene described herein) or its part between double-stranded molecules. (see, e.g., Wahl, G.M.and S.L.Berger (1987) Methods enzymol.152: 399; Kimmel, A.R, (1987) Methods enzymol.152: 507).
For example, stringent salt concentrations are typically less than about 750mM NaCl and 75mM trisodium citrate, preferably less than about 500mM NaCl and 50mM trisodium citrate, and more preferably less than about 250mM NaCl and 25mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvents, such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Harsh temperature conditions will generally include temperatures of at least about 30 ℃, more preferably at least about 37 ℃, and most preferably at least about 42 ℃. Varying other parameters, such as hybridization time, concentration of detergents, e.g., Sodium Dodecyl Sulfate (SDS), and inclusion or exclusion of vector DNA, are well known to those skilled in the art. Various levels of harshness are achieved by combining these various conditions as needed. In a preferred embodiment, hybridization occurs at 30 ℃ in 750mM NaCl, 75mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization occurs at 37 ℃ in 500mM NaCl, 50mM trisodium citrate, 1% SDS, 35% formamide, and 100. mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization occurs at 42 ℃ in 250mM NaCl, 25mM trisodium citrate, 1% SDS, 50% formamide, and 200. mu.g/ml ssDNA. Useful variations of these conditions will be readily apparent to those skilled in the art.
For most applications, the washing steps after hybridization also vary in stringency. Wash severity conditions may be defined in terms of salt concentration and temperature. As above, the wash severity can be increased by reducing the salt concentration or by increasing the temperature. For example, the harsh salt concentration used for the washing step will preferably be less than about 30mM NaCl and 3mM trisodium citrate, and most preferably less than about 15mM NaCl and 1.5mM trisodium citrate. The harsh temperature conditions for the washing step will generally include a temperature of at least about 25 ℃, more preferably at least about 42 ℃, and even more preferably at least about 68 ℃. In a preferred embodiment, the washing step takes place in 30mM NaCl, 3mM trisodium citrate and 0.1% SDS at 25 ℃. In a more preferred embodiment, the washing step occurs at 42 ℃ in 15mM NaCl, 1.5mM trisodium citrate and 0.1% SDS. In a more preferred embodiment, the washing step occurs at 68 ℃ in 15mM NaCl, 1.5mM trisodium citrate and 0.1% SDS. Further variations of these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); grunstein and Hogness (proc.natl.acad.sci., USA 72:3961, 1975); ausubel et al (Current Protocols in molecular Biology, Wiley Interscience, New York, 2001); berger and Kimmel (Guide to molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al, Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
"solid tumors" include tumors such as breast, respiratory, brain, bone, central and peripheral nervous systems, colon, rectum, anus, reproductive organs (e.g., cervix, ovary, prostate), gastrointestinal tract, urogenital tract, endocrine glands (e.g., thyroid and adrenal cortex), thyroid, parathyroid, esophagus, endometrium, eye, germ cells, head and neck, kidney, liver, larynx and hypopharynx, lung, mesothelioma, pancreas, prostate, rectum, kidney, small intestine, skin, soft tissues, stomach, testis, ureter, vagina and vulva and connective tissue, and metastases of these tumors. Malignant tumors include hereditary cancers such as retinoblastoma and Wilms' tumor.
Treatable "breast tumors" include, for example, breast cancers with positive hormone receptor status, breast cancers with negative hormone receptor status, Her-2-positive breast cancers, hormone receptor-and Her-2-negative breast cancers, BRCA-associated breast cancers, and inflammatory breast cancers.
Treatable "respiratory tumors" include, for example, non-small cell bronchial and small cell bronchial carcinomas, non-small cell lung cancer, and small cell lung cancer.
Treatable "brain tumors" include, for example, gliomas, glioblastomas, astrocytomas, meningiomas, and medulloblastomas.
Treatable "tumors of the male reproductive organs" include, for example, prostate cancer, malignant epididymal tumors, malignant testicular tumors, and penile cancer.
"tumors of the female reproductive organs" that can be treated include, for example, endometrial, cervical, ovarian, vaginal, and vulvar cancers.
Treatable "gastrointestinal tumors" include, for example, colorectal cancer, anal cancer, gastric cancer, pancreatic cancer, esophageal cancer, gallbladder cancer, small intestine cancer, salivary gland cancer, neuroendocrine tumors, and gastrointestinal stromal tumors.
Treatable "genitourinary tumors" include, for example, bladder cancer, renal cell carcinoma, and cancers of the renal pelvis and urinary tract.
Treatable "ocular tumors" include, for example, retinoblastoma and intraocular melanoma.
Treatable "liver tumors" include, for example, hepatocellular carcinoma and cholangiocarcinoma.
Treatable "skin tumors" include, for example, malignant melanoma, basal cell tumors, spinolioma, kaposi's sarcoma, and merkel cell carcinoma.
Treatable "head and neck tumors" include, for example, laryngeal cancers as well as pharyngeal and oral cancers.
Treatable "sarcomas" include, for example, soft tissue sarcomas, synovial sarcomas, rhabdoid sarcomas, and osteosarcomas.
Treatable lymphomas include, for example, non-hodgkin's lymphoma, cutaneous lymphoma, central nervous system lymphoma, and AIDS-related lymphoma.
Treatable leukemias include, for example, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, and hairy cell leukemia.
By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such sequences are at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using Sequence Analysis Software (e.g., Sequence Analysis Software Package of the genetics Computer Group, University of Wisconsin Biotechnology Center,1710University Avenue, Madison, Wis.53705, BLAST, BESTFIT, GAP or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, casein(ii) an amino acid. In an exemplary method for determining the degree of identity, the BLAST program can be used, at e-3And e-100The probability scores in between indicate closely related sequences.
By "subject" is meant a mammal, including but not limited to a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
Ranges provided herein are to be understood as shorthand for all values falling within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the following group: 1.2, 3,4,5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
As used herein, the term "treatment" or the like refers to a reduction or amelioration of a disorder and/or symptoms associated therewith. It is understood that, although not excluded, treating a condition or disorder does not require complete elimination of the condition, disorder, or symptoms associated therewith.
The term "or" as used herein is to be understood as being inclusive unless explicitly stated or otherwise evident from the context. The terms "a" and "an" and "the" as used herein are to be construed as singular or plural unless expressly stated or apparent from the context.
Unless explicitly stated or otherwise clear from context, the term "about" as used herein is understood to be within the normal tolerance in the art, e.g., within 2 standard deviations of the mean. About may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. Unless otherwise clear from the context, all numbers provided herein are modified by the term "about".
As used herein, if a range is provided, it is always meant to include both upper and lower limits unless specifically stated or otherwise evident from the context.
Recitation of a collection of chemical groups in any definition of a variable herein includes any single group or combination of groups that define the variable as listed. Recitation of embodiments of variables or aspects herein includes such embodiments as any single embodiment or in combination with any other embodiments or portions thereof.
Any of the compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
Brief Description of Drawings
The compositions and articles defined by the present invention are isolated or otherwise prepared in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description and from the claims.
Figure 1 provides dose response curves of
Figure 2 provides dose response curves for
FIG. 3 is an immunoblot showing the lack of endogenous PDE3A protein expression in the compound sensitive cell lines HuT78 and RVH421, in contrast to the high expression of PDE3A in HeLa cells. Focal adhesion proteins were tested as loading controls.
FIG. 4 is an immunoblot showing deletion of PDE3A expression in PDE3A-CRISPR A2058 cells. Focal adhesion proteins were tested as loading controls.
Fig. 5 shows the dose response curve of
Detailed Description
The present invention is based, at least in part, on the discovery that compounds 1 and 2 do have sensitivity to phosphodiesterase 3A modulation (PDE3A modulation) and/or phosphodiesterase 3B PDE3B modulation, and do have increased stability in human hepatocytes and/or reduced clearance in dogs.
Accordingly, the present invention provides a method of selecting a subject having a cancer that is responsive to a PDE3A/PDE3B modulator, in
In a specific embodiment, expression of CREB3L1 and/or SLFN12 polynucleotides or polypeptides is reduced or undetectable in cancer cells that have acquired resistance to PDE3A/PDE3B modulators.
PDE3A/PDE3B modulators
The identification of PDE3A/PDE3B modulators was associated with a phenotypic screen designed to identify small cytotoxic molecules in a background of mutant tp 53. Predictive chemogenomics approaches complement the target-driven drug discovery program, which consists of extensive in vitro and in vivo target validation, and can also be referred to as reverse chemogenomics (Zheng et al,
Phenotypic screening allows the discovery of new targets for cancer therapy, the specific molecular mechanisms of which are often elucidated by future studies (Swinney et al, Nat Rev Drug Discov 10,507-519, 2011). In recent years, two classes of anticancer drugs discovered by unbiased phenotypic screening efforts have been approved by the FDA. Lenalidomide and pomalidomide were found to be modulators of the E3-ligase, which alter the affinity of its target, leading to degradation of lineage-specific transcription factors (
Alterations in tumor suppressor genes are suitable targets for phenotypic screening because they cannot be directly targeted with small molecules, although synthetic lethal methods such as olaparib have been shown to be effective for treating BRCA1/BRCA2 mutant cancers. To date, the tp53 tumor suppressor gene is the most frequently occurring mutation in human cancers, with somatic mutations detected in 36% of 4742 cancers sequenced through the entire exome. Despite many attempts, no compound was found that selectively killed tp53 mutant cells.
Phenotypic screens developed to identify small molecules causing synthetic lethality in tp53 mutant cancer cells have fortuitously discovered a class of cancer selective cytotoxic agents that act as phosphodiesterase 3A (PDE3A) and phosphodiesterase 3B (PDE3B) modulators, as described below. Cyclic nucleotide phosphodiesterase catalyzes the hydrolysis of the second messenger molecules cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) and is important in many physiological processes. Several phosphodiesterase inhibitors have been approved for clinical treatment, including the PDE3 inhibitors milrinone, cilostazol and levosimendan for cardiovascular indications and inhibition of platelet coagulation, and the PDE3 inhibitor anagrelide for thrombocythemia. Other PDE3A inhibitors are known from WO 2014/164704. PDE5 inhibitors such as vardenafil are useful in smooth muscle disorders including erectile dysfunction and pulmonary arterial hypertension, and the PDE4 inhibitor roflumilast reduces exacerbations of Chronic Obstructive Pulmonary Disease (COPD).
Phosphodiesterase inhibitors act by directly inhibiting their target or by allosteric modulation; for example, structural analysis of PDE4 has led to the design of PDE4D and PDE4B allosteric modulators (Burgin et al, Nat Biotechnol 28,63-70,2010; Gurney et al, Neurotheliaceae 12,49-56,2015). The data provided below indicate that the cancer cytotoxic phosphodiesterase modulator DNMDP may act through a similar allosteric mechanism.
Accordingly, the present invention provides methods of identifying subjects having a malignancy that is likely to respond to treatment with a PDE3A/PDE3B modulator, particularly treatment with
In particular embodiments, the invention provides methods of identifying subjects having a malignancy that is resistant to treatment with a PDE3A modulator, particularly
Compound forms and salts
The compounds of the invention include the compounds themselves, as well as salts and prodrugs thereof, if applicable.
Salts can be formed, for example, between an anion and a positively charged substituent (e.g., amino group) on a compound described herein. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, and acetate. Likewise, salts can also be formed between a cation and a negatively charged substituent (e.g., carboxylate) on a compound described herein. Suitable cations include sodium, potassium, magnesium, calcium and ammonium cations such as tetramethylammonium. Examples of prodrugs include C of a carboxylic acid group1-6An alkyl ester capable of providing an active compound upon administration to a subject.
Pharmaceutically acceptable salts of the compounds of the present invention include those derived from pharmaceutically acceptable inorganic and organic acids and bases. As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by adding a pharmaceutically acceptable acid or base to a compound disclosed herein. As used herein, the phrase "pharmaceutically acceptable" refers to a substance that is acceptable for use in pharmaceutical applications from a toxicological standpoint and does not adversely interact with an active ingredient.
Suitable pharmaceutically acceptable salts of the compounds of the invention may be, for example, acid addition salts of the compounds of the invention which carry a nitrogen atom in the chain or ring, which are, for example, sufficiently basic, for example acid addition salts with inorganic or "mineral acids", such as hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfamic, heavy sulfuric (bisfuric acid), phosphoric or nitric acid, or with organic acids, such as formic, acetic, acetoacetic, pyruvic, trifluoroacetic, propionic, butyric, hexanoic, heptanoic, undecanoic, lauric, benzoic, salicylic, 2- (4-hydroxybenzoyl) -benzoic, camphoric, cinnamic, cyclopentanepropionic, diglucosic, 3-hydroxy-2-naphthoic, nicotine, pamoic, pectinic, 3-phenylpropionic, salicylic, 2- (4-hydroxybenzoyl) -benzoic, camphoric, cinnamic, or succinic acids, Pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, or thiocyanic acid.
Further examples of suitable acidic salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate, and undecanoate. Other acids (e.g. oxalic acid), which are not per se pharmaceutically acceptable, may be used as intermediates in the preparation of salts to obtain the compounds of the invention and their pharmaceutically acceptable acid addition salts.
Furthermore, other suitable pharmaceutically acceptable salts of compounds 1-2, especially of compound 1, which are sufficiently basic are alkali metal salts, such as sodium or potassium salts, alkaline earth metal salts, such as calcium, magnesium or strontium salts, or aluminum or zinc salts, or ammonium salts derived from ammonia or primary, secondary or tertiary organic amines having from 1 to 20 carbon atoms, such as ethylamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, dimethylaminoethanol, diethylaminoethanol, tris (hydroxymethyl) aminomethane, procaine, dibenzylamine, N-methylmorpholine, arginine, lysine, 1, 2-ethylenediamine, N-methylpiperidine, N-methyl-glucamine, N-dimethyl-glucamine, N-ethyl-glucamine, N-methyl-glucamine, N-dimethyl-glucamine, N-ethyl-glucamine, 1, 6-hexanediamine, glucosamine, sarcosine, serinol, 2-amino-1, 3-propanediol, 3-amino-1, 2-propanediol, 4-amino-1, 2, 3-butanetriol or salts with quaternary ammonium ions having 1 to 20 carbon atoms, such as tetramethylammonium, tetraethylammonium, tetra (N-propyl) ammonium, tetra (N-butyl) ammonium, N-benzyl-N, N, N-trimethylammonium, choline or benzalkonium.
In certain embodiments, salts derived from suitable bases include alkali metal (e.g., sodium) salts, alkaline earth metal (e.g., magnesium) salts, ammonium salts, and N- (alkyl)4 +And (3) salt. The present invention also relates to the quaternization of any basic nitrogen-containing group disclosed herein. Water or oil-soluble or dispersible products can be obtained by such quaternization. Salt forms of the compounds of any formula herein may be amino acid salts of the carboxyl group (e.g., L-arginine, L-lysine, L-histidine salts).
A list of suitable salts is described in Remington's Pharmaceutical Sciences, 17 th edition, MackPublishing Company, Easton, Pa., 1985, page 1418; journal of pharmaceutical science, 66, 2 (1977); and "Pharmaceutical Salts: properties, Selection, and Use AHandbook; wermuth, C.G.and Stahl, P.H, (eds.) Verlag Helvetica Chimica Acta, Zurich, 2002[ ISBN 3-906390-26-8], the entire contents of each of which are incorporated herein by reference. Those skilled in the art will further recognize that acid addition salts of the claimed compounds may be prepared by reacting the compounds with the appropriate inorganic or organic acid by any of a number of known methods. Alternatively, the alkali metal and alkaline earth metal salts of the acidic compounds of the invention are prepared by reacting the compounds of the invention with an appropriate base by various known methods.
The present invention includes all possible salts of the compounds of the present invention, either as a single salt, or any mixture of said salts in any proportion.
The neutral form of the compound may be regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties (e.g., solubility in polar solvents), but otherwise the salt is equivalent to the parent form of the compound for purposes of this invention.
In addition to salt forms, the present invention provides compounds in prodrug form. Prodrugs of the compounds described herein are those compounds that undergo a chemical change under physiological conditions to provide the compounds of the present invention. In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical means in an ex vivo environment. For example, prodrugs can be slowly converted to compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical agent. Prodrugs are often useful because, in some cases, they are easier to administer than the parent drug. For example, they may be more bioavailable than the parent drug by oral administration. The prodrugs may also have improved solubility in pharmacological compositions compared to the parent drug. A variety of prodrug derivatives are known in the art, such as those that rely on hydrolytic cleavage or oxidative activation of the prodrug. Examples of prodrugs, without limitation, are compounds of the present invention that are administered as esters ("prodrugs") but which can then be metabolically hydrolyzed to the active entity carboxylic acid. Additional examples include peptidyl derivatives of the compounds of the invention.
The invention also includes various hydrate and solvate forms of the compounds.
The compounds of the present invention may also contain unnatural proportions of one or more of the atoms that make up such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as tritium (A), (B), (C), (D), (C), (D3H) Iodine-125 (125I) Or carbon-14 (14C) In that respect All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention. In particular deuterium containing compounds.
The term "isotopic variant" of a compound or agent is defined as a compound exhibiting an unnatural proportion of one or more isotopes that make up the compound.
The expression "unnatural ratio" refers to a ratio of isotopes above their natural abundance. The natural abundance of isotopes used in this context is described in "Isotopic Compositions of the Elements 1997", Pure appl. chem., 70(1), 217-235, 1998.
Examples of such isotopes include stable and radioactive isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine, bromine and iodine, such as2H (deuterium),3H (tritium),11C、13C、14C、15N、17O、18O、32P、33P、33S、34S、35S、36S、18F、36Cl、82Br、123I、124I、125I、129I and131I。
with respect to the treatment and/or prevention of the diseases described herein, one or more isotopic variations of compounds 1-2 (particularly compound 1) preferably contain deuterium ("deuterium-containing"). In which one or more radioactive isotopes are incorporated, e.g.3H or14Isotopic variations of compounds 1-2 (particularly compound 1) of C are useful, for example, in drug and/or substrate tissue distribution studies. These isotopes are particularly preferred for their ease of incorporation and detection. Positron can be emitted isotopes, e.g.18F or11C incorporates Compound 1-2 (especially Compound 1). These isotopic variations of compounds 1-2 are useful for in vivo imaging applications. In the case of preclinical or clinical studies, containing deuterium and containing13Compound 1-2 of C can be used for mass spectrometry.
Isotopic variations of compounds 1-2 can generally be obtained by methods known to those skilled in the art, for exampleSuch as those described in the schemes and/or examples herein, are prepared by substituting the reagents with isotopic variations of the reagents, preferably with deuterium containing reagents. Depending on the desired site of deuteration, in some cases, one may be from D2Deuterium from O is incorporated directly into the compound or into a reagent useful in the synthesis of such a compound. Deuterium gas is also a reagent that can be used to incorporate deuterium into a molecule. Catalytic deuteration of olefinic and acetylenic bonds is a fast route to incorporation of deuterium. In the presence of deuterium gas, metal catalysts (i.e., Pd, Pt, and Rh) can be used to exchange hydrogen in functional group-containing hydrocarbons directly to deuterium (j.g. atkinson et al, U.S. patent 3966781). Various deuteration reagents and synthetic building blocks are available from, for example, C/D/N Isotopes, Quebec, Canada; companies such as Cambridge Isotope Laboratories Inc., Andover, MA, USA and Combiophos Catalysts, Inc., Princeton, NJ, USA are commercially available.
The term "deuterium containing compound 1-2" is defined as a compound wherein one or more hydrogen atoms are replaced by one or more deuterium atoms and wherein the abundance of deuterium at each deuterated position of any one of compounds 1-2 is greater than the natural abundance of deuterium, which is about 0.015%. In particular, in any of deuterium containing compounds 1-2, the abundance of deuterium at each deuterated position of the compound is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, preferably greater than 90%, 95%, 96% or 97%, even more preferably greater than 98% or 99% at the one or more positions. It is understood that the abundance of deuterium at each deuterated position is independent of the abundance of deuterium at the other deuterated position or positions.
Selective incorporation of one or more deuterium atoms into any of compounds 1-2 can alter the physicochemical properties of the molecule (e.g., acidity [ c.l.perrin, et al, j.am.chem.soc., 2007, 129, 4490], basicity [ c.l.perrin, et al, j.am.chem.soc., 2005, 127, 9641], lipophilicity [ b.testa, et al, int.j.pharm.,1984,19(3),271]) and/or metabolic profile and can result in a change in the ratio of parent compound to metabolite or a change in the amount of metabolite formed. Such changes may result in certain therapeutic advantages and may therefore be preferred in some circumstances. Reduced metabolic rates and metabolic switches have been reported in which the ratio of metabolites is altered (a.e. mutlib et al, toxicol. appl.pharmacol., 2000, 169, 102). These changes in exposure to parent drugs and metabolites may have important consequences for the pharmacodynamics, resistance and efficacy of deuterium containing compounds of general formula (I). In some cases, deuterium substitution reduces or eliminates the formation of undesirable or toxic metabolites and increases the formation of desired metabolites (e.g., nevirapine: a.m. sharma et al, chem. res. toxicol, 2013, 26, 410; efavirenz: a.e. mutlib et al, toxicol. appl. pharmacol., 2000, 169, 102). In other cases, the primary effect of deuteration is to reduce the systemic clearance rate. Thus, the biological half-life of the compound is increased. Potential clinical benefits may include the ability to maintain similar systemic exposure with reduced peak levels and increased trough levels. This may lead to reduced side effects and increased efficacy, depending on the pharmacokinetic/pharmacodynamic relationship of the specific compound. ML-337(c.j.wenthur et al, j.med.chem.,2013,56,5208) and aontacati (k.kassahu et al, WO2012/112363) are examples of such deuteration effects. Other situations have been reported in which decreased metabolic rates lead to increased drug exposure without altering systemic clearance rates (e.g., rofecoxib: f.schneider et al, arzneim. forsch. drug. res.,2006,56, 295; telaprevir: f.maltais et al, j.med.chem.,2009,52, 7993). Deuterated drugs that exhibit this effect may have reduced administration requirements (e.g., reduced number of administrations or reduced dosage to achieve the desired effect) and/or may result in reduced metabolite loading.
Compounds 1-2 may have multiple potential sites of metabolic attack. To optimize the effects of the above physicochemical properties and metabolic profiles, deuterium containing compounds 1-2 can be selected with one or more specific modes of deuterium-hydrogen exchange. In particular, one or more deuterium atoms of one or more deuterium containing compounds 1-2 are attached to a carbon atom and/or are located at those positions of the compounds 1-2 that are attack sites for metabolic enzymes, such as cytochrome P450.
Pharmaceutical composition
Compounds 1-2, especially
For these routes of administration, compounds 1-2 can be administered in a suitable administration form.
For oral administration, compounds 1-2 can be formulated into dosage forms known in the art that rapidly and/or in an improved manner deliver the compounds of the present invention, e.g., tablets (uncoated or coated tablets, e.g., with enteric or controlled release coatings that delay dissolution or are insoluble, orally disintegrating tablets, films/flakes, films/lyophilizates, capsules (e.g., hard or soft gelatin capsules), sugar coated tablets, granules, pills, powders, emulsions, suspensions, aerosols, or solutions.
Parenteral administration may be carried out avoiding or involving an absorption step (e.g. intravenous, intra-arterial, intracardiac, intraspinal or intralumbar) such as intramuscular, subcutaneous, intradermal, transdermal or intraperitoneal. Administration forms suitable for parenteral administration are, in particular, preparations for injection and infusion in the form of solutions, suspensions, emulsions, lyophilisates or sterile powders.
Examples suitable for other routes of administration are pharmaceutical forms for inhalation [ especially powder inhalers, nebulizers ], nasal drops, nasal solutions, nasal sprays; tablets/films/wafers/capsules for lingual, sublingual or buccal administration; suppositories; eye drops, eye ointments, eye baths, eye inserts, ear drops, ear sprays, ear powder, ear lotions, earplugs; vaginal capsules, aqueous suspensions (lotions, shaking cocktails), lipophilic suspensions, emulsions, ointments, creams, transdermal therapeutic systems (e.g. patches), lotions, pastes, foams, powders, implants or stents.
The compounds according to the invention can be incorporated into the administration forms described. This can be achieved in a manner known per se by mixing with pharmaceutically suitable excipients. Pharmaceutically suitable excipients include, inter alia:
fillers and carriers (e.g. cellulose, microcrystalline cellulose (e.g.,
ointment bases (e.g. petrolatum, paraffin, triglycerides, waxes, wool wax, lanolin alcohols, lanolin, hydrophilic ointments, polyethylene glycols),
suppository bases (e.g. polyethylene glycol, cocoa butter, stearin),
solvents (e.g. water, ethanol, isopropanol, glycerol, propylene glycol, medium chain-length triglyceride fatty oils, liquid polyethylene glycols, paraffin wax),
surfactants, emulsifiers, dispersing or wetting agents (e.g. sodium lauryl sulfate), lecithin, phospholipids, fatty alcohols (e.g.,
buffers, acids and bases (e.g. phosphates, carbonates, citric acid, acetic acid, hydrochloric acid, sodium hydroxide solution, ammonium carbonate, tromethamine, triethanolamine),
isotonic agents (e.g. glucose, sodium chloride),
adsorbents (e.g. highly dispersed silica (silicas)),
viscosity-enhancing agents, gel-forming agents, thickeners and/or binders (e.g. polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, carboxymethylcellulose-sodium, starch, carbomers, polyacrylic acids (e.g.,
disintegrants (e.g., modified starch, carboxymethyl cellulose-sodium, sodium starch glycolate (e.g.,
flow modifiers, lubricants, glidants, and mold release agents (e.g. magnesium stearate, stearic acid, talc, highly dispersed silicon dioxide (e.g.,
coating materials (e.g. sugar, shellac) and film formers for films or diffusion films that dissolve rapidly or in a modified manner (e.g. polyvinylpyrrolidone (e.g.,
capsule materials (e.g. gelatin, hydroxypropylmethylcellulose),
synthetic polymers (e.g., polylactides, polyglycolides, polyacrylates, polymethacrylates (e.g.,
plasticizers (e.g.polyethylene glycol, propylene glycol, glycerol triacetate, triacetyl citrate, dibutyl phthalate),
a penetration enhancer,
Stabilizers (e.g., antioxidants such as ascorbic acid, ascorbyl palmitate, sodium ascorbate, butyl hydroxyanisole, butyl hydroxytoluene, propyl gallate),
preservatives (e.g. parabens, sorbic acid, thimerosal, benzalkonium chloride, chlorhexidine acetate, sodium benzoate),
colorants (e.g., inorganic pigments such as iron oxide, titanium dioxide),
flavors, sweeteners, odorants-and/or odor-masking agents.
The invention further relates to pharmaceutical compositions comprising at least one compound 1-2, especially
Thus in one embodiment, the invention relates to compound 1 or
Or a pharmaceutically acceptable salt or prodrug thereof, and one or more pharmaceutically acceptable carriers or excipients.
In another embodiment, the invention relates to compound 1
Or a pharmaceutically acceptable salt or prodrug thereof, and one or more pharmaceutically acceptable carriers or excipients.
In another embodiment, the invention relates to compound 2
Or a pharmaceutically acceptable salt or prodrug thereof, and one or more pharmaceutically acceptable carriers or excipients.
Combination of
According to another aspect, the present invention encompasses a pharmaceutical combination, in particular a medicament, comprising at least one of
In particular, the present invention relates to a pharmaceutical combination comprising:
one or more first active ingredients, in particular one of the
one or more other active ingredients, in particular hyperproliferative diseases, especially cancer
The term "combination" in the present invention is used as known to the person skilled in the art, which combination may be a fixed combination, an unfixed combination or a kit of parts.
In the present invention, "fixed combination" is used as known to the skilled person and is defined as a combination wherein, for example, the first active ingredient, e.g. one or more of compounds 1-2, is present together with the other active ingredients in one unit dose or in a single entity. An example of a "fixed combination" is a pharmaceutical composition, wherein the first active ingredient and the other active ingredient are present in a mixture, e.g. a formulation, which is administered simultaneously. Another example of a "fixed combination" is a pharmaceutical combination, wherein the first active ingredient and the other active ingredients are present in one unit, rather than in a mixture.
In the present invention, non-fixed combinations or "kit of parts" are used as known to the person skilled in the art and are defined as combinations in which the first active ingredient and the further active ingredients are present in more than one unit. An example of a non-fixed combination or kit of parts is a combination wherein the first active ingredient and the other active ingredients are present separately. The components of the non-fixed combination or the kit of parts may be administered separately, sequentially, simultaneously, concurrently or chronologically staggered.
The compounds of the present invention may be administered as the sole agent or in combination with one or more other pharmaceutically active ingredients, wherein the combination does not cause unacceptable side effects.
The invention also encompasses these pharmaceutical combinations. For example, the compounds of the present invention may be combined with known anti-cancer agents and agents that ameliorate the potential side effects these anti-cancer agents may have. Examples of such agents include:
131I-chTNT, abarelix, abiraterone, doxorubicin, adalimumab, trastuzumab antibody-drug conjugate (ado-trastuzumab-Ezetam-Ezetimidol, Evelvetorelin, Evepitazine, Evespidol, Evepitazine, Evepitavastatin, Evepitazine, Evepitavastatin, Evevelvetine, Evepitavastatin, Evepitazine, Evepitavastatin, Evepitamine, Evepitavastatin, Eveclathrin, Evepitavastatin, Evepitamine, Evepitavastatin, Eveclathrin, Evespertine, Evepitamine, Eveclathrine, Evespertisone, Evone, Evespertisone, Evoxil, Evone, Evoxil No, Evone, Evoxil No, Evoxil, Evone, Evoxil, Evone, Evoxil, Evone, Evoxil.
Practicality of use
Another aspect of the invention is
Another aspect of the invention is
Another aspect is a method of preventing and/or treating a hyperproliferative disorder, particularly a method of treating a hyperproliferative disorder, comprising administering an effective amount of
Another aspect is a method of treating a hyperproliferative disease comprising administering to a subject in need thereof one of the compounds selected from
Or a pharmaceutically acceptable salt or prodrug thereof.
In another aspect, the invention relates to the use of one of the compounds selected from
Or a pharmaceutically acceptable salt or prodrug thereof, more particularly wherein the hyperproliferative disease is cancer.
In one aspect of the invention, the cancer is bone cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, gastrointestinal stromal tumor (GIST), head and neck cancer, hematopoietic cancer, kidney cancer, leiomyosarcoma, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, soft tissue sarcoma, thyroid cancer, or urinary tract cancer.
Thus on the other hand hyperproliferative diseases are benign hyperproliferative diseases.
Another aspect of the invention is
Thus another aspect of the invention is a method of treating cancer comprising administering an effective amount of at least one
Another aspect of the invention is a method of treating metastatic or malignant tumors comprising administering an effective amount of
Another aspect of the invention is the use of
Another aspect of the invention is
Another aspect of the invention is a method of treating a solid tumor comprising administering an effective amount of
Another aspect of the invention is the use of
Another aspect of the invention is a method for the treatment of the above-mentioned tumours, comprising administering an effective amount of
In another aspect of the invention,
Another aspect of the invention is
Another aspect of the invention is a method of treating a hematologic tumor comprising administering an effective amount of
Another aspect of the invention is the use of
Another aspect of the invention is the use of
In another aspect of the
Another aspect of the invention is the use of
Another aspect of the invention is the use of
Another aspect of the invention is the use of
Another aspect of the invention is the use of a PDE3A and/or PDE3B modulator in the manufacture of a medicament for the treatment of cancer, wherein the PDE3A and/or PDE3B modulator is one of the compounds selected from the group consisting of
Or a pharmaceutically acceptable salt or prodrug thereof
Another aspect of the invention is the use of a PDE3A and/or PDE3B modulator in the manufacture of a medicament for the treatment of cancer, wherein the PDE3A and/or PDE3B modulator is one of the compounds selected from
The compounds disclosed herein may also be used in methods of reducing cancer cell proliferation in a subject.
In some embodiments, the method of reducing cancer cell proliferation in a subject comprises administering to the subject a PDE3A and/or PDE3B modulator, thereby reducing cancer proliferation in the subject. The subject may be pre-selected (e.g., selected prior to administration) by detecting an increase in PDE3A and/or PDE3B polypeptide or polynucleotide in cells derived from the cancer of the subject relative to a reference level.
In some embodiments, the pre-selection of the subject may be performed by detecting a decrease in SLFN12 in cells derived from the cancer of the subject relative to a reference level. In some embodiments, the pre-selection of the subject may be performed by detecting an increase in SLFN12 in cells derived from the cancer of the subject relative to a reference level.
In some embodiments, the survival of a cancer cell selected to be responsive to a phosphodiesterase 3A (PDE3A) and/or PDE3B modulator involves contacting the cell with one or more PDE3A and/or PDE3B modulators, wherein the cell is selected to have an increased level of a PDE3A and/or PDE3B polypeptide or polynucleotide, or a combination thereof, relative to a reference, thereby decreasing the survival of the cancer cell.
In some embodiments, methods of killing or reducing survival of a cancer cell selected to be responsive to a phosphodiesterase 3A (PDE3A) and/or PDE3B modulator are provided, wherein the methods can comprise contacting the cell with one or more PDE3A and/or PDE3B modulators, wherein the cell is selected to have an increased level of a PDE3A and/or PDE3B polypeptide or polynucleotide, or a combination thereof, relative to a reference, thereby reducing survival of the cancer cell. Typically, the PDE3A and/or PDE3B modulator reduces the enzymatic activity of PDE3A and/or PDE 3B.
In some embodiments, the cancer is melanoma, prostate cancer, or lymphoma.
In some embodiments, the method of reducing cancer cell proliferation in a subject comprises administering to the subject a PDE3A and/or PDE3B modulator, thereby reducing cancer proliferation in the subject. The subject may be pre-selected (e.g., selected prior to administration) by detecting an increase in PDE3A and/or PDE3B polypeptides or polynucleotides and/or Schlafen12(SLFN12) relative to a reference level in cells derived from the cancer of the subject.
In some embodiments, the survival of a cancer cell selected to be responsive to a phosphodiesterase 3A (PDE3A) and/or PDE3B modulator involves contacting the cell with one or more PDE3A and/or PDE3B modulators, wherein the cell is selected to have an increased level of a PDE3A and/or PDE3B polypeptide or polynucleotide or Schlafen12(SLFN12), or a combination thereof, relative to a reference, thereby reducing the survival of the cancer cell.
In some embodiments, there is provided a method of killing or reducing survival of a cancer cell selected to respond to a phosphodiesterase 3A (PDE3A) and/or PDE3B modulator, wherein the method can comprise contacting the cell with one or more PDE3A and/or PDE3B modulators, wherein the cell is selected to have an increased level of a PDE3A and/or PDE3B polypeptide or polynucleotide or Schlafen12(SLFN12), or a combination thereof, relative to a reference, thereby reducing survival of the cancer cell after treatment. Typically, the PDE3A and/or PDE3B modulators reduce the activity of PDE3A and/or PDE 3B.
In other embodiments, the (PDE3A) and/or PDE3B modulator used in the methods of killing or reducing the survival of cancer cells described herein is
Thus in another aspect, the invention relates to a method of reducing cancer cell proliferation in a subject pre-selected for a cancer that is responsive to one or more PDE3A and/or PDE3B modulators having the structure:
comprising administering to a subject a PDE3A/PDE3B modulator, wherein the subject has been pre-selected for reducing cancer cell proliferation in the subject by detecting an increase in the level of a PDE3A or PDE3B or Schlafen12(SLFN12) polypeptide or polynucleotide, or a combination thereof, in cells derived from the cancer of the subject relative to a reference.
In other embodiments, the (PDE3A) and/or PDE3B modulator used in the method reduces the activity of PDE3A and/or PDE 3B.
The pre-selection of a subject in the methods described herein can be performed by obtaining a tumor biological sample (e.g., a tissue sample), including cancer cells.
In another aspect the methods described herein further comprise the step of detecting a deletion that reduces the level of expression of the polypeptide or polynucleotide relative to a
In another aspect the methods described herein further comprise the step of detecting a deletion that reduces the level of expression of a polypeptide or polynucleotide relative to a reference CREB3L1, further comprising the step of detecting a reduction in SLFN12 levels.
In one aspect of the methods disclosed herein, wherein the level of PDE3A, PDE3B SLFN12 or CREB3L1 polypeptide is detected, the detecting is performed by a method selected from the group consisting of: immunoblotting, mass spectrometry and immunoprecipitation.
In one aspect of the methods disclosed herein, wherein the level of PDE3A, PDE3B, SLFN12 or CREB3L1 polynucleotide is detected, the detection is by a method selected from the group consisting of: quantitative PCR, RNA sequencing, Northern blotting, microarray, mass spectrometry, and in situ hybridization.
In another aspect, the invention relates to a method of reducing cancer cell proliferation in a preselected subject, the method comprising administering to the subject one or more PDE3A and/or PDE3B modulators, wherein the subject is preselected, by detecting an increase in the level of a PDE3A and/or PDE3B polypeptide or polynucleotide in a subject-derived sample relative to a reference, thereby reducing cancer cell proliferation in the subject.
In another aspect, the invention relates to a method of reducing cancer cell proliferation in a preselected subject, the method comprising administering to the subject one or more PDE3A and/or PDE3B modulators, wherein the subject is preselected, by detecting an increase in the level of PDE3A and/or PDE3B polypeptide or polynucleotide in a subject-derived sample relative to a reference, further comprising detecting an increase in SLFN12 levels, thereby reducing cancer cell proliferation in the subject.
In another aspect, the invention relates to a method of killing or reducing survival of a cancer cell comprising contacting the cell with one or more PDE3A and/or PDE3B modulators, wherein the cell has an increased level of a PDE3A and/or PDE3B polypeptide or polynucleotide relative to a reference, thereby reducing survival of the cancer cell.
In another aspect, the invention relates to a method of killing or reducing survival of a cancer cell, comprising contacting the cell with one or more PDE3A and/or PDE3B modulators, wherein the cell has an increased level of a PDE3A and/or PDE3B polypeptide or polynucleotide relative to a reference, further comprising detecting an increase in SLFN12 levels, thereby reducing survival of the cancer cell.
In another aspect, the invention relates to methods of treating PDE3B and SLFN12 sensitive
In another aspect, the invention relates to methods of treating PDE3B and SLFN12 sensitive to melanoma, prostate cancer, cervical cancer, or
Diagnostics
One feature of the invention is a diagnostic assay for characterizing cancer. In one embodiment, the level of PDE3A, PDE3B, Schlafen12(SLFN12), or CREB3L1 polynucleotide or polypeptide is measured in a sample from a subject and used as an indicator of cancer response to treatment with
In another embodiment, the level of CREB3L1 polynucleotide or polypeptide is measured in a biological sample from the subject. Relative to a reference, a loss or decrease in the expression level of a CREB3L1 or SLFN12 polynucleotide or polypeptide in a biological sample (e.g., a biological sample comprising cancer cells) of a subject indicates that the cancer is resistant to treatment with a PDE3A and/or PDE3B modulator. The levels of PDE3A, PDE3B, SLFN12, and/or CREB3L1 polynucleotides can be measured by standard methods, such as quantitative PCR, RNA sequencing, Northern blotting, microarray, mass spectrometry, and in situ hybridization. Standard methods can be used to measure the levels of PDE3A, SLFN12, and/or CREB3L1 polypeptides in a tumor-derived biological sample. These methods include immunoassays, ELISA, Western blots using antibodies that bind PDE3A, PDE3B, SLFN12, and/or CREB3L1, and radioimmunoassays. Elevated levels of PDE3A and SLFN12 polynucleotides or polypeptides relative to a reference are considered positive indicators of cancer response to treatment with PDE3A and/or PDE3B modulators. Decreased levels of CREB3L1 or SLFN12 polynucleotides or polypeptides are considered indicators of cancer resistance to treatment with
Type of biological sample
In characterizing responsiveness of a malignant tumor to treatment with
PDE3A, PDE3B and/or SLFN12 are expressed in a sample obtained from a subject responsive to treatment with a PDE3A and/or PDE3B modulator, at a level higher than in a non-responsive subject. In another embodiment, PDE3A and/or PDE3B and/or SLFN12 is at least about 5-fold, 10-fold, 20-fold, or 30-fold in a subject with a malignancy compared to a healthy control. Fold change values are determined using any method known in the art. In one embodiment, CREB3L1 or SLFN12 expression is reduced or undetectable relative to a reference.
In particular embodiments, CREB3L1 or SLFN12 expression is reduced by about 10%, 25%, 50%, 75%, 85%, 95% or more.
In one embodiment, the change is determined by calculating the difference in expression of PDE3A, PDE3B SLFN12 and/or CREB3L1 in cancer cells from the level present in non-responsive cancer cells or the level present in corresponding healthy control cells.
Selection of treatment methods
As reported below, subjects with hyperproliferative diseases can be tested for PDE3A, PDE3B, SLFN12 and/or CREB3L1 expression in the course of selecting a treatment. Patients characterized as having increased PDE3A and/or SLFN12 relative to a reference level are identified as responsive to treatment with a PDE3A and/or PDE3B modulator, particularly
Reagent kit
The invention provides kits for characterizing responsiveness or resistance of a subject to treatment with a PDE3A and/or PDE3B modulator, particularly
Also provided herein are kits that can include, for example, a therapeutic composition comprising an effective amount of a PDE3A and/or PDE3B modulator in a unit dosage form.
In some embodiments, the kit comprises a sterile container comprising a therapeutic or diagnostic composition; such containers may be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other material suitable for containing a medicament.
In one embodiment, the kits of the invention comprise reagents for measuring the levels of PDE3A, SLFN12, and/or
In one embodiment, the kits of the invention comprise reagents for measuring the levels of PDE3A/PDE3B, SLFN12 and/or
In one embodiment, the kits of the invention comprise reagents for measuring SLFN12 and/or CREB3L1 levels.
In one embodiment, the kits of the invention comprise a capture reagent that binds to a CREB3L1 polypeptide or polynucleotide and/or a capture reagent that binds to a SLFN12 polypeptide or polynucleotide.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the knowledge of one of ordinary skill in the art. These techniques are well explained in the literature, for example, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The polymeraseChemin Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention and, therefore, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments are discussed in the following sections.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the assays, screens, and therapeutic methods of the invention are made and used, and are not intended to limit the scope of the invention.
Examples
Chemical experimental method
LC-MS-method:
the method comprises the following steps:
the instrument comprises the following steps: waters Acquity UPLCMS singleQuad; column: acquity UPLC BEH C181.7 μm, 50 × 2.1mm; eluent A: water +0.1 vol% formic acid (99%), eluent B: acetonitrile; gradient: 1-99% B in 0-1.6 min, 99% B in 1.6-2.0 min; the flow rate is 0.8 ml/min; temperature: 60 ℃; DAD scan: 210-400 nm.
The
the instrument comprises the following steps: waters Acquity UPLCMS singleQuad; column: acquity UPLC BEH C181.7 μm, 50 × 2.1mm; eluent A: water +0.2 vol% ammonia (32%), eluent B: acetonitrile; gradient: 1-99% B in 0-1.6 min, 99% B in 1.6-2.0 min; the flow rate is 0.8 ml/min; temperature: 60 ℃; DAD scan: 210-400 nm.
NMR data
The 1H-NMR data of the selected compounds are listed in the form of a list of 1H-NMR peaks. Where for each signal peak, the δ value in ppm is given, followed by the signal intensity reported in parentheses. The delta values from different peaks-signal intensity pairs are separated by commas. Thus, the peak list is described by the general form: δ 1 (intensity 1), δ 2 (intensity 2),. ·, δ i (intensity i),. ·, δ n (intensity n).
The intensity of the sharp signal correlates with the height (in cm) of the signal in the printed NMR spectrum. This data may be correlated to the actual ratio of signal strengths when compared to other signals. In the case of a wide signal, more than one peak or center of the signal and its relative intensity are displayed compared to the strongest signal displayed in the spectrum. The list of 1H-NMR peaks is similar to the classical 1H-NMR reading and therefore typically contains all the peaks listed in the classical NMR interpretation. Furthermore, similar to classical 1H-NMR printouts, the peak list may show solvent signals, signals from stereoisomers of a particular target compound, impurity peaks, 13C satellite peaks, and/or rotational sidebands. The peaks of stereoisomers and/or impurity peaks are typically shown at lower intensities compared to the peaks of the target compound (e.g., purity > 90%). Such stereoisomers and/or impurities may be typical for a particular manufacturing process, and thus their peaks may help identify a reproduction of the manufacturing process based on a "byproduct fingerprint". An expert calculating the peak of the target compound by known methods (MestReC, ACD simulation or by using empirically estimated expected values) can separate the peak of the
General details
All reactions were carried out under a nitrogen (N2) atmosphere. All reagents and solvents were purchased from commercial suppliers and used as received. At Bruker (300 or 400 MHz)1H, 75 or 101MHz13C) Nuclear Magnetic Resonance (NMR) spectra were recorded on the spectrometer. The proton and carbon chemical shifts are reported in ppm (δ) with reference to the NMR solvent. The data are reported as follows: chemical shift, multiplicities (br broad, s singlet, d doublet, t triplet, q quartet, m multiplet; coupling constant(s) in Hz). 40-60 μm silica gel on Teledyne Isco Combiflash Rf ((R))
Scheme 1: synthesis of 6- [3, 5-difluoro-4- (morpholin-4-yl) phenyl ] -5-methyl-4, 5-dihydropyridazin-3 (2H) -one:
a) morpholine, N, N-diisopropylethylamine, CH3CN, refluxing; b) LiHMDS, THF, -78 ℃, then ethyl bromoacetate, THF, -78 ℃ to room temperature; c) hydrazine, EtOH, reflux.
Step a: 1- [3, 5-difluoro-4- (morpholin-4-yl) phenyl]Propan-1-one.
7.0g of 1- (3,4, 5-trifluorophenyl) propan-1-one (37mmol), 32.5mL of morpholine (372mmol) and 13.2mL of N, N-diisopropylethylamine (77.4mmol) in 70mL of CH3The solution in CN was heated at reflux temperature overnight. The reaction was cooled and concentrated, water was added and CH was used2Cl2And (5) flushing. Will CH2Cl2Drying (MgSO)4) And concentrated. The crude product is dissolved in CH2Cl2And hexane. Rotary evaporation resulted in the formation of a large amount of solid, which was then concentrated completely and evaporation was stopped. The solid was filtered and washed with hexanes to give 6.06g of product as an off-white solid, clean by LC and NMR analysis. The mother liquor was concentrated and recrystallized from hexane to give another 1.67g of product as a yellow solid in a total yield of 7.73g (81%).1H NMR(300MHz，CDCl3)δ7.46(d，J＝10.8Hz，2H)，3.89–3.75(m，4H)，3.41–3.24(m，4H)，2.90(q，J＝7.2Hz，2H)，1.21(t，J＝7.2Hz，3H)。19F NMR(376MHz，CDCl3) Delta-119.79 mass 256(M + 1).
Step b: 4- [3, 5-difluoro-4- (morpholin-4-yl) phenyl]-ethyl 3-methyl-4-oxobutanoate.
LiHMDS (28.8mL, 28.8mmol) in a 1.0M solution in THF was added to 30mL THF and cooled with a dry ice/isopropanol bath. To this was slowly added 7.42g of 1- [3, 5-difluoro-4- (morpholin-4-yl) phenyl via syringe]A solution of propan-1-one (29.2mmol) in 20mL THF. After stirring with cooling for 1h, a solution of 3.85mL (34.6mmol) ethyl bromoacetate in 10mL tetrahydrofuran was slowly added via syringe and the reaction mixture was allowed to warm to room temperature overnight. The next day NH was used for the reaction4Cl (aq) quench, add EtOAc, separate and rinse with brine. After drying and concentration, the product was chromatographed with 0-10% EtOAc in hexane to give 6.20g (63%) of the product as an oil.1H NMR(400MHz，CDCl3) δ 7.51(d, J ═ 10.7Hz, 2H), 4.11(q, J ═ 7.1Hz, 2H), 3.81(dd, J ═ 16.8, 5.0Hz, 5H), 3.33(s, 4H), 2.96(dd, J ═ 16.9, 8.9Hz, 1H), 2.45(dd, J ═ 16.9, 5.3Hz, 1H), 1.27-1.18 (m, 6H). Mass 342(M + 1).
Step c: 6- [3, 5-difluoro-4- (morpholin-4-yl) phenyl]-5-methyl-4, 5-dihydropyridazin-3 (2H) -one.
To 6.20g of 4- [3, 5-difluoro-4- (morpholine-4-)Radical) phenyl]To a solution of ethyl-3-methyl-4-oxobutanoate in 100mL of LEtOH was added 2.84mL of hydrazine (90.5mmol) and the reaction was heated at reflux temperature overnight. The next morning the solution was cooled to room temperature to give white crystals, which were filtered and rinsed with EtOH to give 1.8g of a clean product, which was determined by LC and NMR analysis.1H NMR(400MHz，CDCl3)δ8.84(s，1H)，7.28(d，J＝11.1Hz，2H)，3.91–3.79(m，4H)，3.30–3.26(m，4H)，3.26–3.20(m，1H)，2.72(dd，J＝17.0，6.9Hz，1H)，2.50(d，J＝16.9Hz，1H)，1.25(d，J＝7.4Hz，3H)。19F NMR(376MHz，CDCl3) Delta-119.69. Mass 310(M + 1). The mother liquor was concentrated in half and refluxed for 6 h. The resulting crystals were cooled, filtered and washed with EtOH to give an additional 910mg of product, which contained small amounts of impurities. Total yield 2.71g (48%).
Enantiomers were separated by chiral supercritical fluid chromatography: column: ChiralPak AS-H, 250 × 4.6mm, 5um, mobile phase modifier: 100% methanol, gradient: 5 to 50% methanol over 10 minutes, flow rate: 4mL/min, backpressure: 100 bar, column temperature: at 40 ℃. UV detection was 200-400 nm. The more active (R) -enantiomer (retention time 7.08min) was designated
Scheme 2: synthesis of (5R) -6- [3, 5-difluoro-4- (morpholin-4-yl) phenyl ] -5-methyl-4, 5-dihydropyridazin-3 (2H) -one and (5S) -6- [3, 5-difluoro-4- (morpholin-4-yl) phenyl ] -5-methyl-4, 5-dihydropyridazin-3 (2H) -one:
a) morpholine, N, N-diisopropylethylamine, CH3CN, refluxing; b) LiHMDS, THF, -78 ℃, then ethyl bromoacetate, THF, -78 ℃ to room temperature; then hydrazine hydrate and EtOH are refluxed; c) the enantiomers were separated.
Step a: 1- [3, 5-difluoro-4- (morpholin-4-yl) phenyl]Propan-1-one. Two parallel reactions are as followsThe method comprises the following steps: 1- (3,4, 5-trifluorophenyl) propan-1-one (110ml, 740mmol) was dissolved in acetonitrile (1.4l, 27mol) under nitrogen. Morpholine (490ml, 5.6mol) and N, N-diisopropylethylamine (200ml, 1.1mol) were added and the mixture was stirred at 100 ℃ for 4 h. The solvent was removed and the crude products of two such reactions were combined. Dichloromethane (1000mL) was added and washed with H2O (400mL) and saturated aqueous sodium chloride (300mL) were washed 5 times. The organic phase was dried over magnesium sulfate, filtered and dried in vacuo to give the title compound (383.29g, 100% of theory) in 90% purity. 1H-NMR (400MHz, DMSO-d6) delta ppm]1.03(t，J＝7.22Hz，3H)2.72(q，J＝7.18Hz，2H)3.14(m，4H)3.58-3.67(m，4H)7.19-7.34(m，2H)。
Step b: 6- [3, 5-difluoro-4- (morpholin-4-yl) phenyl ] -5-methyl-4, 5-dihydropyridazin-3 (2H) -one.
1,1,1,3,3, 3-hexamethyldisilazane Lithium amide (
Step c: 6- [3, 5-difluoro-4- (morpholin-4-yl) phenyl ] -5-methyl-4, 5-dihydropyridazin-3 (2H) -one (8.0g, 25,86mmol) is isolated as (5R) -6- [3, 5-difluoro-4- (morpholin-4-yl) phenyl ] -5-methyl-4, 5-dihydropyridazin-3 (2H) -one (Compound 1) and (5S) -6- [3, 5-difluoro-4- (morpholin-4-yl) phenyl ] -5-methyl-4, 5-dihydropyridazin-3 (2H) -one (Compound 1 a).
The instrument comprises the following steps: labomatic HD5000, Labocord-5000; gilson GX-241, Labcol Vario 4000, column: YMC Amylose SA 5 μ 250x50 mm; solvent A: dichloromethane; solvent B: ethanol; isocratic: 80% A + 20% B; the flow rate is 100.0 ml/min; UV 325 nm.
(5R) -6- [3, 5-difluoro-4- (morpholin-4-yl) phenyl]-5-methyl-4, 5-dihydropyridazin-3 (2H) -one 3.77g (95% purity, 45% yield). LC-MS (method 1): rt is 0.99 min; ms (esipos): 310[ M + H ] M/z]+。1H-NMR(500MHz，DMSO-d6)δ[ppm]：1.024(15.88)，1.038(16.00)，2.209(3.09)，2.242(3.49)，2.357(0.46)，2.361(0.65)，2.365(0.48)，2.514(2.20)，2.518(1.98)，2.522(1.56)，2.631(0.54)，2.635(0.75)，2.643(2.60)，2.657(2.91)，2.676(2.45)，2.690(2.28)，3.146(6.85)，3.154(9.80)，3.163(7.30)，3.352(1.66)，3.354(1.66)，3.366(2.32)，3.369(2.28)，3.381(1.56)，3.382(1.47)，3.395(0.40)，3.679(11.05)，3.688(11.70)，3.697(10.24)，5.758(1.59)，7.395(0.53)，7.400(1.00)，7.412(7.35)，7.434(7.49)，7.446(0.94)，7.451(0.61)，11.038(8.10)。[α]20＝-377.7°(DMSO)WL＝589nm。
(5S) -6- [3, 5-difluoro-4- (morpholin-4-yl) phenyl]-5-methyl-4, 5-dihydropyridazin-3 (2H) -one. 3.92g (95% purity, 47% yield). LC-MS (method 1): rt is 0.99 min; ms (esipos): 310[ M + H ] M/z]+。1H-NMR(500MHz，DMSO-d6)δ[ppm]：1.024(15.91)，1.038(16.00)，2.209(3.44)，2.242(3.87)，2.361(0.71)，2.518(2.69)，2.522(2.03)，2.635(0.91)，2.643(2.77)，2.657(2.97)，2.676(2.50)，2.690(2.34)，3.154(11.36)，3.352(2.22)，3.366(2.70)，3.381(1.81)，3.394(0.47)，3.679(11.54)，3.688(13.11)，3.697(10.77)，5.758(0.69)，7.395(0.60)，7.400(1.08)，7.412(7.61)，7.434(7.72)，7.445(1.07)，7.451(0.68)，11.038(8.42)。[α]20＝+356.9°(DMSO)WL＝589nm。
Scheme 3: synthesis of 6- [3, 5-difluoro-4- (morpholin-4-yl) phenyl ] -5-methyl-4, 5-dihydropyridazin-3 (2H) -one:
a) LiHMDS, THF, -78 ℃, then ethyl bromoacetate, THF, -70 ℃ to room temperature; b) morpholine, N-diisopropylethylamine, and then hydrazine hydrate, 100 ℃.
Step a: 3-methyl-4-oxo-4- (3,4, 5-trifluorophenyl) butanoic acid ethyl ester.
1,1,1,3,3, 3-hexamethyldisilazane lithium amido (12mL, 1.0M in THF, 12mmol) was added to THF (10mL) and cooled to-70 deg.C, then 1- (3,4, 5-trifluorophenyl) propan-1-one (1.7mL, 12mmol) dissolved in THF (8mL) was added slowly. The reaction was stirred at-70 ℃ for 1.5 h. Ethyl bromoacetate (1.6mL, 14mmol) in THF (3mL) was added slowly. The mixture was stirred at-70 ℃ for 30 minutes. The cooling bath was removed and the mixture was stirred for 16 h. The mixture was added to aqueous hydrochloric acid (200mL, 1M in H)2O) and extracted three times with dichloromethane. All collected organic phases were dried over magnesium sulfate, evaporated and dried in vacuo. Purification by column chromatography (silica gel, hexane/ethyl acetate, gradient) gave the title compound (1.75g, 46% of theory) in 85% purity. LC-MS (method 1): rt is 0.1.31 min; ms (esipos): 275.3[ M + H ] M/z]+。
Step b: 6- [3, 5-difluoro-4- (morpholin-4-yl) phenyl ] -5-methyl-4, 5-dihydropyridazin-3 (2H) -one.
To a solution of ethyl 3-methyl-4-oxo-4- (3,4, 5-trifluorophenyl) butanoate (110mg, 401. mu. mol) in N, N-diisopropylethylamine was added morpholine (70. mu.l, 800. mu. mol).The mixture was stirred at 100 ℃ for 16 h. After cooling to room temperature, hydrazine hydrate (1:1) (240 μ l, 80% purity, 4.0mmol) was added and the mixture was stirred at 100 ℃ for 3 h. Water was slowly added to the warm mixture and stirring was continued for 30 minutes. The precipitate was filtered, washed with water and dried in vacuo to give the title compound (65mg, 50% of theory) in 95% purity. LC-MS (method 1): rt is 0.99 min; ms (esipos): 310[ M + H ] M/z]+。
1H-NMR(400MHz，DMSO-d6)δ[ppm]：1.022(15.91)，1.040(16.00)，2.205(3.14)，2.245(3.67)，2.322(0.60)，2.326(0.84)，2.332(0.60)，2.518(3.03)，2.522(1.99)，2.637(2.53)，2.655(2.96)，2.664(0.77)，2.668(0.96)，2.673(0.86)，2.679(2.51)，2.697(2.25)，3.143(7.04)，3.154(10.24)，3.166(7.62)，3.348(1.74)，3.351(1.77)，3.366(2.35)，3.370(2.34)，3.384(1.61)，3.403(0.41)，3.677(11.35)，3.689(12.18)，3.700(10.28)，7.388(0.58)，7.395(1.07)，7.409(7.78)，7.438(8.13)，7.452(1.03)，7.459(0.68)，11.038(8.33)。
Synthesis of
6- [3, 5-dichloro-4- (morpholin-4-yl) phenyl ] -5-methyl-4, 5-dihydropyridazin-3 (2H) -one
Step 1):
to 200mg (0.984mmol) of (R) -6- (4-aminophenyl) -5-methyl-4, 5-dihydropyridazin-3 (2H) -one dissolved in 1mL of DMF were added 250. mu.L (2.00mmol) of bis (2-bromoethyl) ether and 400mgK2CO3And the mixture was stirred at 60 ℃ overnight. The next day an additional 250. mu.L of bis (2-bromoethyl) ether and 170mg K were added2CO3. After 3h, EtOAc and water were added, the water was washed with EtOAc, and the combined EtOAc washes were dried and concentrated. By CH2Cl2Chromatography in 0-4% MeOH afforded 125mg of product compound 3 (46%). 1H NMR (300MHz, CDCl)3)δ8.61(s，1H)，7.68(d，J＝8.8，2H)，6.92(d，J＝8.8，2H)，3.99–3.76(m，4H)，3.44–3.31(m，1H)，3.29–3.22(m，4H)，2.70(dd，J＝6.7，16.8，1H)，2.46(d，J＝16.7，1H)，1.24(d，J＝7.3，3H)。13C NMR(75MHz，CDCl3)δ166.64，154.05，152.18，127.10，125.33，114.73，66.69，48.33，33.93，27.94，16.36.MS：274(M+1)。C15H19N3O2Analytical calculation of (a): c, 65.91; h, 7.01; n, 15.37; found 65.81, H, 6.66, N, 15.26.
Compound 2a and
A solution of 300mg of compound 3(1.10mmol) dissolved in 5mL HOAc was stirred vigorously and cooled in a cooling water bath at about 10-15 ℃ so that no freezing occurred. A total of 2.2mL of 10-15% naocl (aq) was added to it via syringe over about 30 minutes, then LC indicated
Compound 2 a: 1H NMR (400MHz, CDCl)3)δ8.58(s，1H)，7.80(d，J＝2.2Hz，1H)，7.60(dd，J＝8.2，2.5Hz，1H)，7.04(d，J＝8.4Hz，1H)，4.02-3.76(m，4H)，3.38-3.22(m，1H)，3.23-3.02(m，4H)，2.70(dd，J＝17.0，6.8Hz，1H)，2.48(d，J＝17.6Hz，1H)，1.24(d，J＝7.3Hz，3H)。13C NMR(101MHz，CDCl3) δ 166.65, 152.50, 150.20, 130.02, 128.81, 128.39, 125.25, 119.96, 66.99, 51.40, 33.80, 27.92, 16.27. Mass 308(M + 1). C15H18ClN3O2Analytical calculation of (a): c, 58.54; h, 5.89; and N, 13.65. Measured value: c, 58.30; h, 5.99; n, 13.63.
Compound 2:1H NMR(400MHz，CDCl3)δ8.95(s，1H)，7.67(s，2H)，3.90-3.75(m，4H)，3.35-3.17(m，5H)，2.70(dd，J＝17.0，6.7Hz，1H)，2.49(d，J＝17.0Hz，1H)，1.24(d，J＝7.3Hz，3H)。13C NMR(101MHz，CDCl3) Delta 166.38, 151.17, 145.62, 134.83, 132.35, 126.65, 67.64, 49.97, 33.72, 27.85, 16.19. Mass 342(M + 1). C15H17Cl2N3O2Analytical calculation of (a): c, 52.64; h, 5.01; n, 12.28. Measured value: c, 52.68; h, 4.90; n, 12.28.
The following methods and materials were or may be used to obtain data supporting the activity of
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