HK1212331B - 4-carboxamido-isoindolinone derivatives as selective parp-1 inhibitors - Google Patents

Description

4-Formylamino-isoindolinone derivatives as selective PARP-1 inhibitors

Technical Field

The present invention provides novel substituted 4-formylamino-isoindolinone derivatives that are shown to be potent and selective inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) relative to its poly(ADP-ribose) polymerase-2 (PARP-2) counterpart and are therefore useful in treating cancer, cardiovascular disease, nervous system damage, and inflammation. The present invention also provides methods for preparing these compounds, pharmaceutical compositions containing these compounds, and methods of treating diseases using pharmaceutical compositions containing these compounds.

Background Art

Poly(ADP-ribose) polymerases belong to an 18-member family of enzymes that catalyze the addition of ADP-ribose units to DNA or to various receptor proteins, influencing diverse cellular processes including replication, transcription, differentiation, gene regulation, protein degradation, and spindle maintenance. PARP-1 and PARP-2 are the only PARPs activated by DNA damage and involved in DNA repair.

PARP-1 is a nuclear protein composed of three domains: an N-terminal DNA-binding domain containing two zinc fingers, an automodification domain, and a C-terminal catalytic domain. PARP-1 binds to single-strand breaks (SSBs) in DNA via the zinc-finger domain, cleaves NAD+, and attaches multiple ADP-ribose units to target proteins, such as histones and various DNA repair enzymes. This results in highly negatively charged targets, which in turn lead to the unwinding and repair of damaged DNA via the base excision repair pathway. In mouse models with artificial disruption of specific genes, PARP-1 deficiency impairs DNA repair, but it is not embryonic lethal. However, mice with double knockout of PARP-1 and PARP-2 die early in embryonic development, suggesting that the two enzymes exhibit incompletely overlapping functions. Enhanced PARP-1 expression and/or activity has been shown in various tumor cell lines, including malignant lymphomas, hepatocellular carcinomas, cervical cancers, colorectal cancers, and leukemias. This may allow tumor cells to resist genotoxic stress and increase their resistance to DNA-damaging agents. Thus, it has been shown that inhibition of PARP-1 by small molecules sensitizes tumor cells to cytotoxic therapies such as temozolomide, platinum, topoisomerase inhibitors, and radiation. A significant window appears to exist between the ability of PARP inhibitors to achieve therapeutic benefit and the undesirable side effects. While the therapeutic use of combining PARP inhibitors with DNA damaging agents is not new, the use of these agents as monotherapy, particularly in settings where the genetic background of the tumor is deficient in homologous recombinant DNA repair, represents a novel approach. Individuals with heterozygous germline mutations in the BRCA-1 or BRCA-2 homologous recombination repair genes exhibit a high risk of developing breast and other cancers over their lifetime. Tumors that arise in mutation carriers often lose the wild-type allele and do not express functional BRCA-1 and BRCA-2 proteins.

Thus, loss of these two proteins leads to tumor-specific dysfunction in the repair of double-strand breaks caused by homologous recombination. It is known that when PARP-1 is inhibited, base excision repair is reduced and single-strand breaks generated during the normal cell cycle persist. It is also well established that encountering an unrepaired break by a replication fork can form a double-strand break, which is normally repaired by homologous recombination. Tumor cells deficient in homologous recombination repair (e.g., BRCA-1 and BRCA-2 mutants) are therefore highly sensitive to PARP inhibition compared to wild-type cells. This is consistent with the concept of synthetic lethality, in which defects in both pathways are individually harmless, but their combined defects become lethal: PARP inhibitors may be more effective in patients with tumors that have specific DNA repair defects, while the inhibitors have no effect on normal, heterozygous tissue. In addition to BRCA mutants, which represent the majority of inherited breast and ovarian cancers, the putative patient population also includes a significant proportion of sporadic cancers that have defects in homologous recombination repair, a phenomenon known as "BRCAness." For example, methylation of the promoter of the BRCA-1 or FANCF gene and amplification of the EMSY gene, which encodes a BRCA-2 interacting protein. By rationally inferring the synthetic lethality of PARP and BRCA-1 and BRCA-2, it is possible that any gene deficiency that is not redundant in double-strand break repair should be sensitive to PARP inhibition. For example, ATM deficiency found in patients with T-cell prolymphocytic leukemia and B-cell chronic lymphocytic leukemia and breast cancer, and CHK2 germline mutations identified in sarcomas, breast cancer, ovarian cancer and brain tumors, when combined with PARP deficiency and other known HR pathway proteins (including RAD51, DSS1, RAD54, RPA1, NBS1, ATR, CHK1, CHK2, FANCD2, FANCA, FANCC and pTEN), also show synthetic lethality. FANCC and FANCG mutations have been found in pancreatic cancer. Methylated FANCF promoters have been found in ovarian cancer, breast cancer, cervical cancer and lung cancer. The first clinical evidence that BRCA-mutated cancers could be sensitive to PARP inhibitor monotherapy came from a Phase I trial of the oral small molecule PARP inhibitor Lynparza. In a Phase I population enriched for BRCA mutation carriers, a 47% objective response rate was observed in 19 patients with BRCA mutations and breast, ovarian, and prostate cancers. Other PARP inhibitors, such as Rucaparib and Veliparib, are currently known to be in Phase II clinical trials in combination with single agents. Early indications are that these treatments show low toxicity as single agents. However, compounds with high selectivity for PARP-1 are expected to show even lower toxicity in long-term treatment schedules or combinations.

PARP-1 has also been implicated in angiogenesis. In particular, PARP-1 inhibition appears to result in reduced accumulation of the transcriptional hypoxia-inducible factor 1α, an important regulator of tumor cell adaptation to hypoxia.

Proinflammatory stimuli trigger the release of proinflammatory mediators that result in the production of excess nitrate and hydroxyl residues, which in turn produce single-strand DNA breaks and subsequent activation of PARP-1. Excessive activation of PARP-1 depletes NAD+ and energy stores, ultimately leading to cellular dysfunction and necrosis. This cell suicide mechanism has been implicated in the pathomechanism of conditions such as stroke, myocardial ischemia, diabetes, diabetes-related cardiovascular dysfunction, shock, traumatic central nervous system injury, arthritis, colitis, allergic encephalomyelitis, and various other forms of inflammation. Of particular interest is that PARP-1 enhances nuclear factor kB-mediated transcription, which plays a key role in the expression of inflammatory cytokines, chemokines, and inflammatory mediators.

Substituted dihydro-isoindolones for the treatment of kinase disorders are described in WO 2007/047646, inventors Janssen Pharmaceutica; Wender et al. claim isoindolinone analogs as kinase inhibitors in US Pat. No. 7,232,842. Patent application US 2008/0108659 by Gandhi et al. describes 3-oxo-2,3-dihydro-1H-isoindoles as poly(ADP-ribose) polymerase inhibitors, also reported in Bioorg. Med. Chem. Lett., 2010, 20, 1023-1026. WO 2011/006794 and WO 2011/006803, both inventors Nerviano Medical Sciences, describe 3-oxo-2,3-dihydro-1H-isoindole-4-carboxamides as selective PARP-1 inhibitors.

Summary of the Invention

The present invention provides novel 4-formylamino-isoindolinone derivatives that are demonstrated to be potent and selective inhibitors of PARP-1 relative to PARP-2 and are therefore useful in the treatment of cancer, cardiovascular disease, nervous system damage and inflammation.

The present invention also provides methods for preparing these compounds, pharmaceutical compositions containing these compounds, and methods for treating diseases using pharmaceutical compositions containing these compounds.

Therefore, the first object of the present invention is to provide a compound of formula (I):

in:

R is hydrogen or fluorine; and

n, m, R1 and R2 have the following meanings:

a) n is 0 and m is 0, 1, 2 or 3;

R1 is a 3- to 6-membered cycloalkyl group or a 4- to 6-membered heterocyclyl group; and

R2 is a 3-, 5- or 6-membered cycloalkyl group, a 4- to 6-membered heterocyclyl group, an aryl group or a heteroaryl group;

or

b) n is 1 and m is 0;

R1 is 3- to 6-membered cycloalkyl or aryl, each of which is optionally further substituted by one or more straight-chain or branched (C ₁ -C ₆ )-alkyl groups; and

R2 is absent, 3- to 6-membered cycloalkyl, 4- to 6-membered heterocyclyl, aryl or heteroaryl, each of which is optionally further substituted with one or more straight-chain or branched (C ₁ -C ₆ )-alkyl groups;

or

c) n is 2 or 3, and m is 0;

R1 is 3- to 6-membered cycloalkyl, 4- to 6-membered heterocyclyl, aryl or heteroaryl, each of which is optionally further substituted with one or more straight-chain or branched (C ₁ -C ₆ )-alkyl groups; and

R2 is absent, 3- to 6-membered cycloalkyl, 4- to 6-membered heterocyclyl, aryl or heteroaryl, each of which is optionally further substituted with one or more straight-chain or branched (C ₁ -C ₆ )-alkyl groups;

or

d) n and m are each independently 1, 2 or 3;

R1 and R2 are each independently 3- to 6-membered cycloalkyl, 4- to 6-membered heterocyclyl, aryl or heteroaryl;

or a pharmaceutically acceptable salt thereof.

The compounds of formula (I) as defined above are potent and selective inhibitors of PARP-1 relative to PARP-2 and are therefore useful in the treatment of cancer, cardiovascular disease, nervous system damage and inflammation.

The present invention also provides a process for the synthesis of substituted 4-formylamino-isoindolinone derivatives of formula (I) as defined above, by a process consisting of standard synthetic transformations.

The present invention also provides a method for treating diseases mediated by PARP-1 protein, comprising administering an effective amount of a compound of formula (I) as defined above to a mammal, preferably a human, in need thereof.

A preferred method of the present invention is to treat a disease mediated by the PARP-1 protein selected from the group consisting of cancer, cardiovascular disease, nervous system damage and inflammation.

Another preferred method of the present invention is to treat specific types of cancer, including, but not limited to: carcinomas such as bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, including small cell lung cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, and skin cancer, including squamous cell carcinoma; hematopoietic cancer of the lymphoid lineage; The present invention relates to a group of hematopoietic cancers, including leukemias, acute lymphoblastic leukemias, acute lymphoblastic leukemias, B-cell lymphomas, T-cell lymphomas, Hodgkin's lymphomas, non-Hodgkin's lymphomas, hairy cell lymphomas, and Burkitt's lymphomas; hematopoietic cancers of the myeloid lineage, including acute and chronic myeloid leukemias, myelodysplastic syndromes, and promyelocytic leukemias; tumors of mesenchymal origin, including fibrosarcomas, Ewing's sarcomas, and rhabdomyosarcomas; tumors of the central and peripheral nervous system, including astrocytomas, neurocytomas, gliomas, and schwannomas; and other tumors, including melanomas, seminoma, teratomas, osteosarcomas, xeroderma pigmentosum, keratoxanthoma, thyroid follicle carcinoma, and Kaposi's sarcoma.

Additionally, the methods of the present invention provide for the inhibition of tumor angiogenesis and metastasis.

Another preferred method of the present invention is for treating certain types of cardiovascular disease, including but not limited to myocardial reperfusion injury, cardiomyopathy, and diabetic cardiovascular dysfunction.

Another preferred method of the invention is for treating certain types of nervous system injury including, but not limited to, stroke, brain injury, and neurodegenerative disorders.

Another preferred method of the present invention is for treating certain types of inflammatory diseases including, but not limited to, colitis, arthritis, and uveitis.

The present invention also provides an in vitro method for selectively inhibiting the activity of PARP-1 protein, comprising contacting said protein with an effective amount of a compound of formula (I) as defined above.

The present invention further provides a method of treating a disease comprising a compound of formula (I) as defined above in conjunction with a radiotherapy or chemotherapy regimen for simultaneous, separate or sequential use in anti-cancer treatment.

The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) as defined above or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, carrier or diluent.

In addition to the compound of formula (I), the pharmaceutical composition of the present invention may also contain one or more chemotherapeutic agents - such as cytostatic or cytotoxic drugs, antibiotics, alkylating agents, antimetabolites, hormones, immunotherapies, interferons, cyclooxygenase inhibitors (such as COX-2 inhibitors), matrix metalloproteinase inhibitors, telomerase inhibitors, tyrosine kinase inhibitors, anti-growth factor receptor agents, anti-HER agents, anti-EGFR agents, anti-angiogenic agents (such as angiogenesis inhibitors), farnesyltransferase inhibitors, ras-raf signaling pathway inhibitors, cell cycle inhibitors, other cdks inhibitors, tubulin binding agents, topoisomerase I inhibitors, topoisomerase II inhibitors, etc. Preferably, the chemotherapeutic agent is an alkylating agent. Even more preferably, the alkylating agent is temozolomide.

In addition, the present invention provides a product comprising a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, and one or more chemotherapeutic agents, wherein the product is in the form of a combined preparation, wherein the compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, and the one or more chemotherapeutic agents are used simultaneously, separately, or sequentially for anticancer treatment. Preferably, the chemotherapeutic agent is an alkylating agent. Even more preferably, the alkylating agent is temozolomide.

Furthermore, the present invention provides a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for use as a medicament, preferably as a medicament having anticancer activity.

In still another aspect, the present invention provides a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for use in a method of treating cancer.

Finally, the present invention provides the use of a compound of formula (I) as defined above or a pharmaceutically acceptable salt thereof in the preparation of a medicament having anticancer activity.

The compound of formula (I) can have one or more asymmetric centers and therefore can exist as each optical isomer or racemic mixture or diastereomer.Therefore, all possible isomers of the compound of formula (I) and mixtures thereof all fall within the scope of the present invention.As mentioned above, the salt of the compound of formula (I) also falls within the scope of the present invention.

Unless otherwise specified, when referring to a compound of formula (I) itself and any pharmaceutical composition thereof or any treatment containing the same, the present invention includes all isomers, tautomers, hydrates, solvates, N-oxides and pharmaceutically acceptable salts of the compounds of the invention.

If a chiral center or another form of an isomeric center is present in the compounds of the present invention, all forms of that isomer, including enantiomers and diastereomers, are intended to be included herein. Compounds containing chiral centers can be used as racemic mixtures, and enantiomerically enriched mixtures or racemic mixtures can be separated by well-known techniques, and the individual enantiomers can be used alone. In the case of compounds containing unsaturated carbon-carbon double bonds, both cis (Z) and trans (E) isomers are within the scope of the present invention.

In cases where compounds can exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within the present invention, regardless of whether it exists in equilibrium or exists predominantly in one form.

By the term halogen atom we mean a fluorine, chlorine, bromine or iodine atom.

With the term "straight-chain or branched (C ₁ -C ₆ )-alkyl" we mean any radical such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl etc.

By the term "3- to 6-membered cycloalkyl", unless otherwise indicated, we mean a 3- to 6-membered all-carbon monocyclic ring which may contain one or more double bonds but does not contain a completely conjugated π-electron system. Non-limiting examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, and cyclohexadienyl.

By the term "4- to 6-membered heterocyclyl" we mean a 4- to 6-membered saturated or partially unsaturated carbocyclic ring in which one or more carbon atoms are replaced by a heteroatom such as nitrogen, oxygen or sulfur; the heterocyclyl ring may be optionally fused or linked to aromatic or non-aromatic carbocyclic and heterocyclic rings. Non-limiting examples of heterocyclyl groups are, for example, pyranyl, pyrrolidinyl, pyrrolinyl, imidazolinyl, imidazolidinyl, pyrazolidinyl, pyrazolinyl, thiazolinyl, thiazolidinyl, dihydrofuranyl, tetrahydrofuranyl, 1,3-dioxolane, piperidinyl, piperazinyl, morpholinyl, and the like.

The term "aryl" means a mono-, bi- or poly-carbocyclic hydrocarbon having 1 to 4 ring systems, which are optionally further fused to each other or connected by single bonds, wherein at least one carbocyclic ring is "aromatic", wherein the term "aromatic" means a completely conjugated π-electron bond system. Non-limiting examples of such aryl groups are phenyl, α- or β-naphthyl or biphenyl groups.

As used herein, the term "heteroaryl" means an aromatic heterocycle, typically a 5- to 8-membered heterocycle having 1 to 3 heteroatoms selected from N, O or S; the heteroaryl ring may optionally be further fused or linked to aromatic and non-aromatic carbocyclic and heterocyclic rings. Non-limiting examples of such heteroaryl groups are, for example, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, imidazolyl, thiazolyl, isothiazolyl, pyrrolyl, phenyl-pyrrolyl, furanyl, phenyl-furanyl, oxazolyl, isoxazolyl, pyrazolyl, thienyl, benzothienyl, isoindolinyl, benzimidazolyl, indazolyl, quinolinyl, isoquinolinyl, 1,2,3-triazolyl, 1-phenyl-1,2,3-triazolyl, 2,3-dihydroindolyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl; benzopyranyl, 2,3-dihydrobenzoxazinyl, 2,3-dihydroquinoxalinyl, and the like.

According to the present invention, and unless otherwise provided, when any of the above groups may be optionally substituted, any vacant position thereof may be substituted by one or more linear or branched (C ₁ -C ₆ )alkyl groups.

The term "pharmaceutically acceptable salts" of compounds of formula (I) refers to those salts that retain the biological effectiveness and properties of the parent compound. Thus, pharmaceutically acceptable salts of compounds of formula (I) include acid addition salts formed with inorganic or organic acids, such as nitric, hydrochloric, hydrobromic, sulfuric, perchloric, phosphoric, acetic, trifluoroacetic, propionic, glycolic, (D) or (L) lactic, oxalic, ascorbic, fumaric, malonic, malic, maleic, tartaric, citric, benzoic, cinnamic, mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, isethionic, succinic, and salicylic acids.

Pharmaceutically acceptable salts of the compounds of formula (I) also include salts formed with inorganic or organic bases, such as hydroxides, carbonates or bicarbonates of alkali metals or alkaline earth metals, in particular sodium, potassium, calcium, ammonium or magnesium, and acyclic or cyclic amines, preferably methylamine, ethylamine, diethylamine, triethylamine, piperidine and the like.

In a first preferred embodiment, the present invention provides compounds of formula (I) as defined above, characterized in that:

R is hydrogen or fluorine; and

n, m, R1 and R2 have the following meanings:

a) n is 0 and m is 0 or 1;

R1 is a 6-membered heterocyclyl; and

R2 is a 3- or 6-membered cycloalkyl group, a 6-membered heterocyclyl group, an aryl group, or a heteroaryl group;

or

b) n is 1 and m is 0;

R1 is aryl, which is optionally substituted by one or more linear or branched (C ₁ -C ₆ )-alkyl groups; and

R2 does not exist;

or

c) n is 2 or 3, and m is 0;

R1 is 6-membered heterocyclyl, aryl or heteroaryl, each of which is optionally further substituted by one or more straight or branched (C ₁ -C ₆ )-alkyl chains; and

R2 does not exist, is a 6-membered heterocyclic group or an aryl group;

or

d) n is 2 or 3, and m is 1;

R1 is a 6-membered heterocyclyl; and

R2 is an aryl group;

or a pharmaceutically acceptable salt thereof.

In a more preferred embodiment, the present invention provides a compound of formula (I) as defined above, characterized in that:

R is hydrogen or fluorine; and

n, m, R1 and R2 have the following meanings:

a) n is 0 and m is 0 or 1;

R1 is a 6-membered heterocyclyl; and

R2 is a 3- or 6-membered cycloalkyl group, a 6-membered heterocyclyl group, an aryl group, or a heteroaryl group;

or

c) n is 2 or 3, and m is 0;

R1 is 6-membered heterocyclyl, aryl or heteroaryl, each of which is optionally further substituted by one or more straight-chain or branched (C ₁ -C ₆ )-alkyl groups; and

R2 does not exist, is a 6-membered heterocyclic group or an aryl group;

or a pharmaceutically acceptable salt thereof.

Even more preferably, the present invention provides compounds of formula (I) as defined above, characterized in that:

R is hydrogen or fluorine; and

n, m, R1 and R2 have the following meanings:

a) n is 0 and m is 0 or 1;

R1 is a 6-membered heterocyclyl; and

R2 is a 3- or 6-membered cycloalkyl group, a 6-membered heterocyclyl group, an aryl group, or a heteroaryl group;

or a pharmaceutically acceptable salt thereof.

Most preferably, the present invention provides compounds of formula (I) as defined above, characterized in that:

R is hydrogen or fluorine; and

n, m, R1 and R2 have the following meanings:

a) n is 0 and m is 0 or 1;

When m is 0, R1 is a piperidine ring, and R2 is a cyclohexyl ring;

When m is 1, R1 is a piperidine ring, and R2 is a pyridine ring;

or a pharmaceutically acceptable salt thereof.

Particularly preferred compounds (cpd) of the present invention are listed below:

1. 2-Benzyl-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

2. 3-Oxo-2-phenylethyl-2,3-dihydro-1H-isoindole-4-carboxamide;

3. 2-[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

4. 3-Oxo-2-(2-piperidin-1-yl-ethyl)-2,3-dihydro-1H-isoindole-4-carboxamide;

5. 2-(2-Morpholin-4-yl-ethyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

6. 2-(3-Morpholin-4-yl-propyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

7. 2-[2-(3,4-Dihydro-2H-quinolin-1-yl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

8. 3-Oxo-2-(1-pyridin-4-ylmethyl-piperidin-4-yl)-2,3-dihydro-1H-isoindole-4-carboxamide;

9. 3-Oxo-2-(1-thiophen-2-ylmethyl-piperidin-4-yl)-2,3-dihydro-1H-isoindole-4-carboxamide;

10. 3-Oxo-2-(1-pyridin-3-ylmethyl-piperidin-4-yl)-2,3-dihydro-1H-isoindole-4-carboxamide;

11. 2-(1-cyclohexyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

12. 2-(1-Furan-2-ylmethyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

13. 3-Oxo-2-(1-thiophen-3-ylmethyl-piperidin-4-yl)-2,3-dihydro-1H-isoindole-4-carboxamide;

14. 2-(1-Furan-3-ylmethyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

15. 3-Oxo-2-(1-pyridin-2-ylmethyl-piperidin-4-yl)-2,3-dihydro-1H-isoindole-4-carboxamide;

16. 3-Oxo-2-[1-(1H-pyrrol-2-ylmethyl)-piperidin-4-yl]-2,3-dihydro-1H-isoindole-4-carboxamide;

17. 3-Oxo-2-(3-phenyl-propyl)-2,3-dihydro-1H-isoindole-4-carboxamide;

18. 3-Oxo-2-(2-pyridin-2-yl-ethyl)-2,3-dihydro-1H-isoindole-4-carboxamide;

19. 2-[3-(3,4-Dihydro-1H-isoquinolin-2-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

20. 2-[3-(3,4-Dihydro-2H-quinolin-1-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

21. 2-[3-(4-Methyl-piperazin-1-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

22. 3-Oxo-2-[3-(4-phenyl-piperazin-1-yl)-propyl]-2,3-dihydro-1H-isoindole-4-carboxamide;

23. 6-Fluoro-2-(3-morpholin-4-yl-propyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

24. 2-(1-Cyclopropylmethyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

25. 3-Oxo-2-(3-piperidin-1-yl-propyl)-2,3-dihydro-1H-isoindole-4-carboxamide;

26. 2-(3-[1,4']bipiperidin-1'-yl-propyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

27. 2-[3-(2,6-Dimethyl-piperidin-1-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

28. 3-Oxo-2-[1-(tetrahydro-pyran-4-yl)-piperidin-4-yl]-2,3-dihydro-1H-isoindole-4-carboxamide;

29. 2-(1-Cyclohexyl-piperidin-4-yl)-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

30. 2-(1-Benzyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

31. 2-[2-(1-Benzyl-piperidin-4-yl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

32. 2-[3-(4-Benzyl-piperidin-1-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

33. 2-[1-(4,4-Dimethyl-cyclohexyl)-piperidin-4-yl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

34. 2-[1-(4,4-Dimethyl-cyclohexyl)-piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide;

35. 6-Fluoro-3-oxo-2-(1-spiro[2.5]octan-6-yl-piperidin-4-yl)-2,3-dihydro-1H-isoindole-4-carboxamide;

36. 3-Oxo-2-(1-spiro[2.5]octan-6-yl-piperidin-4-yl)-2,3-dihydro-1H-isoindole-4-carboxamide;

or a pharmaceutically acceptable salt thereof.

The present invention also provides a process for preparing the compounds of formula (I) as defined above.

Therefore, the method of the present invention comprises one of the following steps:

Sequence A (when R is fluorine, Scheme 1):

Step a) Halogenation of 4-fluoro-2-methyl-phenylamine (XI):

Step b) cyano-de-aminating the obtained compound of formula (X):

wherein Hal is a halogen, such as Cl, Br, and I;

Step c) hydrolysis to obtain a compound of formula (IX):

where Hal is as defined above, and

Step d) hydrolysis to obtain a compound of formula (VIII):

wherein Hal is as defined above;

or

Step e) Halogenation of 4-fluoro-2-methyl-benzoic acid (XII):

Then:

Step f) esterifying the compound of formula (VII) obtained in step d) or e),

wherein Hal is as defined above;

Step g) cyano-dehalogenation of the obtained compound of formula (VI):

wherein T is (C ₁ -C ₆ )-alkyl or aryl-(C ₁ -C ₆ )-alkyl, and Hal is as defined above;

Step h) cyclization of the obtained compound of formula (V) by reaction with a suitable amine of formula (XIII):

wherein T is as defined above, the suitable amine of formula (XIII) is:

X-R1-[CH ₂ ] _n -NH ₂ (XIII)

wherein R1 and n are as defined above, and X is R2-[CH ₂ ] _m -, wherein R2 and m are as defined above; or when R1 is a nitrogen-containing heterocyclic group, X is a suitable nitrogen protecting group;

Step c') hydrolysis to obtain a compound of formula (IV):

wherein R1, n and X are as defined above, so as to obtain

A compound of formula (I) as defined above, wherein X is R2-[ _CH2 ] _m- , wherein R2 and m are as defined above; or

A compound of formula (III), wherein R1 is a nitrogen-containing heterocyclic group and X is a suitable nitrogen protecting group,

wherein n is as defined above, R1 is a nitrogen-containing heterocyclic group, and X is a suitable nitrogen protecting group;

Step i) deprotecting the compound of formula (III) as defined above to obtain

A compound of formula (I) as defined above, or

Compounds of formula (II):

wherein R1 and n are as defined above;

Step 1) alkylating the obtained compound of formula (II) as defined above with a suitable alkylating agent of formula (XIV),

R2-[CH ₂ ] _m-1 -Y(XIV)

wherein Y is a formyl group or, when m=1, an oxygen atom (=O) connected to R2 via a double bond, so as to obtain a compound of formula (I).

Sequence B (when R is hydrogen, Scheme 2):

Step m) Reductive amination of furan-2-carbaldehyde (XV) with a suitable amine of formula (XIII):

Suitable amines of formula (XIII) are:

X-R1-[CH ₂ ] _n -NH ₂ (XIII)

wherein R1 and n are as defined above, and X is R2-[CH ₂ ] _m -, wherein R2 and m are as defined above, or when R1 is a nitrogen-containing heterocyclic group, X is a suitable nitrogen protecting group;

Step n) subjecting the obtained compound of formula (XVI) to a Diels-Alder reaction:

wherein R1, n and X are as defined above;

Step o) aromatizing the obtained compound of formula (XVII):

wherein R1, n and X are as defined above;

Step p) Amidation of the obtained compound of formula (XVIII):

wherein R1, n and X are as defined above, so as to obtain

A compound of formula (I) as defined above, wherein X is R2-[ _CH2 ] _m- , wherein R2 and m are as defined above; or

A compound of formula (XX), wherein R1 is a nitrogen-containing heterocyclic group and X is a suitable nitrogen protecting group,

wherein n is as defined above, R1 is a nitrogen-containing heterocyclic group, and X is a suitable nitrogen protecting group;

Step i') deprotecting the compound of formula (XX) as defined above;

Step 1') alkylating the obtained compound of formula (XXI) with a suitable alkylating agent of formula (XIV):

wherein R1 and n are as defined above, suitable alkylating agents of formula (XIV) are:

R2-[CH ₂ ] _m-1 -Y(XIV)

wherein Y is formyl or, when m=1, an oxygen atom (=O) connected to R2 via a double bond, so as to obtain a compound of formula (I) as defined above.

In the case of step o, the compound obtained by aromatization of the compound of formula (XVII) is a compound of formula (XIX), that is, when X is an unstable nitrogen protecting group, the following step q is performed:

Step q) installing a suitable nitrogen protecting group on the obtained compound of formula (XIX):

wherein R1 and n are as defined above, so as to obtain a compound of formula (XVIII), wherein R1 and n are as defined above, and X is a suitable nitrogen protecting group, and then carrying out the sequence of reactions p), i') and l') to obtain a compound of formula (I) as defined above.

If necessary or desired, the above methods comprise converting a compound of formula (I) into a different compound of formula (I) by known chemical reactions; and/or, if desired, converting a compound of formula (I) into a pharmaceutically acceptable salt thereof or converting a salt into a free compound of formula (I).

Known chemical reactions which can transform a compound into a different compound include, for example, reductive amination (Cv1).

All of the above methods are similar methods that can be performed according to well-known methods and under suitable conditions known in the art.

The synthesis of compounds of formula (I) according to the above-described synthetic methods can be carried out in a stepwise manner, whereby each intermediate is isolated and purified by standardized purification techniques, such as column chromatography, and then subjected to subsequent reactions. Alternatively, two or more steps of the synthetic sequence can be carried out according to a so-called "one-pot" method as is well known in the art, whereby only the compounds resulting from the two or more steps are isolated and purified.

The following Schemes 1-2 show the preparation of compounds of formula (I) as defined above.

Solution 1

Sequence A

Option 2

Sequence B

According to step a), the compound of formula (X) can be obtained by halogenating 4-fluoro-2-methyl-phenylamine (XI) according to various methods and experimental conditions known in the art. Preferably, the reaction is carried out in the presence of N-bromosuccinimide, N-iodosuccinimide, N-chlorosuccinimide, bromine, iodine, hydrobromic acid/hydrogen peroxide in a suitable solvent such as acetonitrile, N,N-dimethylformamide, dioxane, dimethyl sulfoxide, acetic acid or water at a temperature of about room temperature to reflux for a period of time of about 1 h to about 96 h.

According to step b), a compound of formula (IX) can be obtained from a compound of formula (X) by a two-step reaction sequence according to various methods and experimental conditions known in the art. The first step is preferably carried out in the presence of sodium nitrite/hydrochloric acid or tert-butyl nitrile in a suitable solvent such as tetrahydrofuran, dimethoxyethane, dimethyl sulfoxide, acetic acid or water at a temperature of about -20°C to room temperature for different time periods of 10 min to about 24 h. The second step is preferably carried out in the presence of sodium cyanide, copper cyanide or potassium cyanide, usually in the presence of an additive such as copper chloride or potassium chloride, in a suitable solvent such as tetrahydrofuran, dimethoxyethane, dimethyl sulfoxide, acetic acid, toluene or water at a temperature of about -20°C to reflux for a time period of 10 min to about 96 h.

According to step c), the compound of formula (IX) is hydrolyzed into the compound of formula (VIII), which can be carried out in various ways according to the conventional method of converting the cyano group into an amide. Preferably, the reaction is carried out in a suitable solvent such as methanol, ethanol, butanol, 1,4-dioxane, toluene, water or a mixture thereof, in the presence of a suitable acid or base such as sulfuric acid, methanesulfonic acid, hydrochloric acid, trifluoroacetic acid, sodium hydroxide, sodium carbonate or a suitable reagent such as hydrogen peroxide, sodium perborate or or indium (III) salt, in the presence of acetaldehyde oxime. Typically, the reaction is carried out at a temperature from room temperature to reflux for a different time of about 1h to about 96h.

According to step d), the compound of formula (VIII) is converted into the compound of formula (VII) according to a conventional method. Preferably, the reaction is carried out as follows: in the presence of water, by treating with a base such as potassium carbonate or sodium carbonate, potassium hydroxide or sodium hydroxide, in a suitable solvent, such as methanol or ethanol, at a temperature from room temperature to reflux, the reaction is carried out for a time of about 30min-about 96h. Alternatively, the reaction is carried out for a time of about 1h-about 96h at a temperature from room temperature to reflux in the presence of sodium nitrite/acetic acid, sulfuric acid or phosphoric acid.

According to step e), 4-fluoro-2-methyl-benzoic acid (XII) is halogenated into the compound of formula (VII) in a variety of ways, according to the conventional method for halogenation. Preferably, the reaction uses tert-butylammonium bromide and/or iodine in the presence of phenyl iodide (III) bis (trifluoroacetate) or phenyl iodide (III) diacetate as a halogen source, in a suitable solvent such as N,N-dimethylformamide or ethylene dichloride, at a temperature of room temperature to reflux, the different times of about 1h-about 48h are carried out. Catalyst is typically a metal, and the most common is a palladium derivative, such as palladium chloride (II) or palladium acetate (II).

According to step f), the compound of formula (VII) is converted into the compound of formula (VI) according to a conventional method. Preferably, the reaction is carried out at a temperature of room temperature to reflux for a period of about 1 h to about 96 h using methanol, ethanol, water or a mixture thereof as a solvent in the presence of salt solution, sulfuric acid or acetic acid. Alternatively, the reaction can be carried out at a temperature of room temperature to reflux for a period of about 1 h to about 96 h using alkyl iodide, bromide or toluenesulfonate in the presence of a suitable base such as sodium carbonate or potassium carbonate and sodium hydroxide, lithium hydroxide or potassium hydroxide.

According to step g), the compound of formula (VI) is converted into the compound of formula (V) in various ways according to conventional methods for cyanation reactions. Preferably, the reaction is carried out in the presence of copper (I) cyanide or potassium hexacyanoferrate (II) as a cyano source, in a suitable solvent such as methanol, ethanol, tetrahydrofuran, 1,4-dioxane, toluene, xylene, N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide or a mixture thereof, at a temperature between room temperature and reflux, for a time varying from about 1 h to about 96 h. If a catalyst is required, it is generally a metal, most commonly a palladium derivative such as tetrakis(triphenylphosphine)palladium(0), palladium(II) chloride or palladium(II) acetate, in the presence of a suitable base such as sodium carbonate, potassium carbonate or cesium carbonate and cesium fluoride.

According to step h), a compound of formula (IV) can be obtained by a two-step reaction sequence according to various methods and experimental conditions known in the art from a compound of formula (V) in the presence of a compound of formula (XIII). The first step is preferably carried out in the presence of N-bromosuccinimide and a free radical initiator such as benzoyl peroxide or azobisisobutyronitrile in a suitable solvent such as carbon tetrachloride, chloroform, dichloromethane or methyl pivalate at a temperature of about room temperature to reflux for a period of time ranging from 10 min to about 24 h. The second step can be carried out under alkaline or acidic conditions, for example in the presence of sodium carbonate or potassium carbonate, 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylamine, diisopropylethylamine, pyridine or acetic acid, hydrochloric acid in a suitable solvent such as tetrahydrofuran, dimethoxyethane, 1,4-dioxane or toluene at a temperature of about room temperature to reflux for a period of time ranging from 1 h to about 96 h.

According to step c'), the hydrolysis of the compound of formula (IV) to obtain the compound of formula (I) or the compound of formula (III) can be carried out in various ways and experimental conditions. Preferably, it is carried out in a manner similar to that reported for step c).

According to step i), in the compound of formula (III), when X is a protecting group such as tert-butoxycarbonyl, 4-methoxybenzyl, 2,4-dimethoxybenzyl, and triphenylmethyl protecting groups, the compound of formula (I) or the compound of formula (II) can be obtained by removing these protecting groups under acidic conditions, preferably in the presence of an inorganic or organic acid, such as hydrochloric acid, trifluoroacetic acid or methanesulfonic acid, boron tribromide or aluminum trichloride, in a suitable solvent such as dichloromethane, dichloroethane, dioxane or a lower alcohol such as methanol or ethanol, at a temperature from room temperature to reflux. In the compound of formula (III), when X is a nitrogen protecting group such as benzyloxycarbonyl, the compound of formula (I) or the compound of formula (II) can be obtained by removing these protecting groups under reducing conditions, such as in the presence of hydrogen and a hydrogenation catalyst, in a suitable solvent such as methanol, ethyl acetate, or a mixture thereof. The catalyst is usually a metal, most commonly a palladium derivative such as palladium on carbon, palladium hydroxide, or palladium black. In the compound of formula (III), when X is a nitrogen protecting group such as methoxycarbonyl, ethoxycarbonyl, 9-fluorenylmethoxycarbonyl, etc., the compound of formula (I) or the compound of formula (II) can be obtained by the following steps: under basic conditions, such as sodium carbonate, potassium carbonate or cesium carbonate, sodium hydroxide, potassium hydroxide or barium hydroxide, hydrazine, piperidine, morpholine, etc., in a suitable solvent such as methanol, ethanol, water, N,N-dimethylformamide, N,N-dimethylacetamide, etc., at room temperature to reflux temperature, removing these protecting groups.

According to step 1), the compound of formula (II) is subjected to reductive alkylation in the presence of a compound of formula (XIV) to obtain a compound of formula (I). This process can be carried out in various ways according to conventional methods for carrying out reductive amination. Preferably, the reaction is carried out in a suitable solvent such as methanol, N,N-dimethylformamide, dichloromethane, tetrahydrofuran, benzene, toluene or a mixture thereof, in the presence of a suitable reducing agent such as sodium borohydride, tetraalkylammonium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, tetramethylammonium triacetoxyborohydride, in the presence of an acid or base catalyst such as acetic acid, trifluoroacetic acid, zinc chloride, zinc bromide, tin (IV) chloride, titanium (IV) chloride, boron trifluoride or triethylamine, diisopropylethylamine or pyridine, at a temperature of about 0°C to reflux for a period of time ranging from about 1 h to about 96 h.

According to step m), furan-2-carbaldehyde (XV) can be subjected to reductive amination in the presence of a compound of formula (XIII) to obtain a compound of formula (XVI). Preferably, the reaction is carried out in a suitable solvent such as methanol, N,N-dimethylformamide, dichloromethane, tetrahydrofuran, benzene, toluene or a mixture thereof, in the presence of a suitable reducing agent such as sodium borohydride, tetraalkylammonium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride or tetramethylammonium triacetoxyborohydride, in the presence of an acid or base catalyst such as acetic acid, trifluoroacetic acid, zinc chloride, zinc bromide, tin (IV) chloride, titanium (IV) chloride, boron trifluoride or triethylamine, diisopropylethylamine or pyridine, at a temperature of about 0° C. to reflux for a period of time varying from about 1 h to about 96 h.

According to step n), the compound of formula (XVI) is subjected to a Diels-Alder reaction to obtain a compound of formula (XVII), which can be carried out in various ways according to conventional methods for carrying out such reactions. Preferably, the reaction is carried out in a suitable solvent such as tetrahydrofuran, benzene, toluene or o-xylene in the presence of maleic anhydride at room temperature to reflux temperature for a period of time varying from about 1 h to about 96 h.

According to step o), the compound of formula (XVII) is converted into a compound of formula (XVIII) or a compound of formula (XIX), which can be carried out in various ways according to conventional methods. Preferably, the reaction is carried out in a suitable solvent such as tetrahydrofuran, toluene or water in the presence of hydrochloric acid, p-toluenesulfonic acid or phosphoric acid at room temperature to reflux temperature for a period of time varying from about 1 h to about 24 h.

According to step p), the compound of formula (XVIII) is reacted in various ways and experimental conditions widely known in the field of condensation reactions to obtain the compound of formula (I) or the compound of formula (XX). Preferably, the compound of formula (XVIII) is reacted with ammonia or an ammonia source such as an ammonium salt as follows: in the presence of an activator such as carbonyldiimidazole, benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, dicyclohexylcarbodiimide, di Isopropylcarbodiimide, 1-ethyl-3-(3'-dimethylamino)carbodiimide hydrochloride, optionally in the presence of hydroxybenzotriazole. Preferably, the reaction is carried out in a suitable solvent such as N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, dichloromethane or 1,4-dioxane and in the presence of a proton scavenger such as pyridine, triethylamine or diisopropylethylamine at room temperature to reflux for a period of about 30 min to about 96 h.

According to step i'), the compound of formula (XX) can be deprotected to give the compound of formula (XXI) in various ways and experimental conditions. Preferably, it is carried out in a manner similar to that reported for step i).

According to step 1'), in the presence of a compound of formula (XIV), the reductive alkylation of the compound of formula (XXI) gives a compound of formula (I), which can be carried out in various ways and under various experimental conditions. Preferably, it is carried out in a manner similar to that reported for step 1).

According to step q), the compound of formula (XIX) can be protected in various ways and experimental conditions to obtain a compound of formula (XVIII), wherein X is a suitable nitrogen protecting group. Preferably, when the protecting group is tert-butoxycarbonyl, the reaction can be carried out in the presence of di-tert-butyl dicarbonate in different solvents such as methanol, acetonitrile, tetrahydrofuran or dichloromethane, in the presence of a base such as pyridine, N,N-dimethylaminopyridine, triethylamine, diisopropylethylamine, sodium carbonate or potassium carbonate at room temperature to reflux temperature for different times of about 1 h to about 96 h.

According to transformation 1 (Cv1), the compound of formula (I) can be subjected to reductive alkylation to give the compound of formula (I) in various ways and experimental conditions. Preferably, it is carried out in a manner similar to that reported for step 1).

Substituted isoindolinone derivatives can be prepared using standard methods in organic synthesis, such as those reported in Smith, Michael-March's Advanced Organic Chemistry: Reaction Mechanisms and Structure - 6th Edition, Michael B. Smith and Jerry March, John Wiley & Sons Inc., New York (NY), 2007. It is known to those skilled in the art that the conversion of a chemical functional group into another chemical functional group may require that one or more reactive centers in the compound containing the functional group be protected to avoid undesirable side reactions. Protection of such reactive centers and subsequent deprotection at the end of the synthetic transformation can be carried out according to standard methods, such as those described in Green, Theodora W. and Wuts, Peter G.M. - Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons Inc., New York (NY), 1999.

In the case where the compound of formula (I) contains one or more asymmetric centers, the compound can be separated into single isomers by methods known to those skilled in the art. Such methods include standard chromatographic techniques, including chromatography or crystallization using a chiral stationary phase. For example, general separation methods for compounds containing one or more asymmetric centers are reported in the following literature: Jacques, Jean; Collet, André; Wilen, Samuel H. - Enantiomers, Racemates and Resolutions, John Wiley & Sons Inc., New York (NY), 1981.

The compounds of formula (I) can be converted into pharmaceutically acceptable salts according to standard methods well known to those skilled in the art. Alternatively, the compounds of formula (I) obtained as salts can be converted into free bases or free acids according to standard methods well known to those skilled in the art.

The starting materials for the process of the present invention, namely 4-fluoro-2-methyl-phenylamine (XI), 4-fluoro-2-methyl-benzoic acid (XII), furan-2-carbaldehyde (XV) and compounds of formula (XIII) and (XIV), are commercially available or can be prepared by using well-known methods.

Pharmacology

PARP-1 is a DNA damage-induced polymerase that catalyzes the cleavage of NAD+ into nicotinamide and ADP-ribose, which is then used to synthesize the branched, nucleic acid-like polymer poly(ADP-ribose). The most abundant poly(ADP-ribosylated) protein in the body is PARP-1 itself, followed by histones. PARP-1 is responsible for 90% of this DNA damage-induced activity, while PARP-2 is responsible for the remaining 10%.

Biochemical assays

Affinity evaluation of the test compounds and their selectivity towards different PARP isoforms of interest were assessed in a displacement assay.

A screening method is provided for identifying compounds capable of binding to several PARP proteins, the method comprising the following steps:

a) providing a reaction mixture comprising:

The PARP protein isoforms under study,

Compounds of formula (IP):

wherein R ₁₁ is hydrogen or methyl, B is a (CH ₂ ) _n -NH group, wherein n is 2-6; m is 0 or 1, and ^X is a counterion; and

serial dilutions of test compounds;

b) comparing the polarization signal generated in the absence of the test compound to the polarization signal generated in the presence of varying concentrations of the test compound; and

c) evaluating the ability of the test compound to displace the compound of formula (IP) as defined above as indicated by a decrease in the level of fluorescence polarization.

Preferably, for the above screening method, the PARP protein and the 5H-phenanthridin-6-one-derivatized probe of formula (IP) are premixed, or the PARP protein and the test compound are premixed. In another preferred screening method, the PARP protein is PARP-1, PARP-2 and PARP-3. The term "PARP protein" includes the full-length native protein and fragments thereof. More preferably, R ₁₁ is hydrogen or methyl, m is 0 or 1; when m is 1, n is 3 or 6, and X ^--- is trifluoroacetate. 5H-phenanthridin-6-one-derivatized probes (IP) having the ability to bind to the PARP protein are selected, including the full-length native protein and fragments thereof.

For example, the polarization signal is measured using a plate reader such as Saphire2 (Tecan). Data analysis is performed, for example, using Dynafit software. For example, the displacement data are also fitted to a 4-parameter logistic model (4PL) or a Hill-slope model using an Excel spreadsheet program (Microsoft Inc. Seattle, USA). This assay is used to test compounds of the present invention. The displacement ability of a test compound of formula (I) is related to the affinity of the compound for the NAD pocket of the enzyme. The specific probe of formula (IP) used in the assay is:

P1.9-Dimethylamino-11,11-dimethyl-1-(3-{methyl-[(6-oxo-5,6-dihydro-phenanthridin-2-ylcarbamoyl)-methyl]-carbamoyl}-propyl)-2,3,4,11-tetrahydro-naphtho[2,3-g]quinolinium trifluoroacetate;

P2.9-Dimethylamino-11,11-dimethyl-1-[3-(3-{[(6-oxo-5,6-dihydro-phenanthridin-2-ylcarbamoyl)-methyl]-amino}-propylcarbamoyl)-propyl]-2,3,4,11-tetrahydro-naphtho[2,3-g]quinolinium trifluoroacetate;

P3.9-Dimethylamino-11,11-dimethyl-1-[3-(6-{[(6-oxo-5,6-dihydro-phenanthridin-2-ylcarbamoyl)-methyl]-amino}-hexylcarbamoyl)-propyl]-2,3,4,11-tetrahydro-naphtho[2,3-g]quinolinium trifluoroacetate.

Compounds of formula (IP) as defined above may be prepared as described in WO 2010/133647.

The assay is based on the use of a probe of formula (IP) that binds to the NAD binding pocket and exploits the significant changes in polarization signals observed when the probe binds to PARP-1, -2, and -3. The ability of the probe of formula (IP) to bind to full-length PARP-1, -2, and -3 has been previously reported (WO 2010/133647). The assay was validated as described in WO 2010/133647.

The affinity binding constant (Kd) and _DC50s (the concentration of compound which reduces the polarization signal by 50% compared to untreated controls) of the test compounds can be determined as explained in WO 2010/133647.

Assays using probe P1 or probe P3 were used to evaluate the biochemical potency of compounds of formula (I), as reported in Table 1.

Table 1

*Assay using Compound P3 as probe. In all other cases, Compound P1 was used as probe.

Assay sensitivity limit based on fitting error <50%.

From the above data, it is obvious to those skilled in the art that the compounds of formula (I) of the present invention are highly potent as PARP-1 inhibitors and have great selectivity relative to PARP-2 and PARP-3 (compare the PARP-1, PARP-2 and PARP-3 DC ₅₀ and Kd values in Table 1 above).

Cell assay

PAR assay

The cellular activity of PARP-1 inhibitors was evaluated by measuring the inhibition of hydrogen peroxide-induced PAR formation in HeLa cells (ECACC). Cellular PAR levels were measured by immunocytochemistry and quantified using an ArrayScan vTi instrument (Cellomics Thermo Scientific).

The study was performed as follows: 6,000 cells/well were plated in 96-well plates (Perkin Elmer) in MEM/10% FCS and incubated at 37°C, 5% CO2 for 24 hours. Test compounds were then added at the desired concentration over a 30-minute period. DNA damage was then induced by the addition of 0.1 mM hydrogen peroxide over a 15-minute period. Concentration curves were prepared using compound stocks in DMSO in MEM/10% FCS, with a final DMSO concentration of 0.002% (v/v). A typical peak compound concentration of 20 μM and a 1:3 serial dilution were used, with duplicate wells prepared at each concentration point. Plates were dried and fixed with cold methanol-acetone (70:30) for 15 minutes at room temperature. The fixative solution was aspirated, the wells air-dried for 5 minutes, and then dehydrated in PBS. Nonspecific binding sites were blocked by incubating the wells in PBS containing 5% (w/v) FBS and 0.05% tween 20 for 30 minutes. The wells were then incubated for 1 hour at room temperature in PBS containing an anti-PAR mouse monoclonal antibody (Anti-PAR, Mouse mAb 10H, Tulip Cat No. 1020) diluted 1:200 in blocking solution. After washing three times in PBS, the wells were incubated in PBS (w/v) 5% FBS 0.05% Tween 20 containing 2 μg/ml Cy2-conjugated goat anti-mouse secondary antibody (Amersham Pharmacia Biotech cat. No. PA 42002) (maximum absorption 489 nm, maximum fluorescence 506 nm) and 1 μg/ml DAPI (maximum absorption 359 nm, maximum fluorescence 461 nm) (4',6-diamidino-2-phenylindole lactate) (Sigma cat. No. D9564), a highly sensitive nucleotide staining dye. After washing three more times in PBS, cells were evaluated for PAR immunoreactivity using an ArrayScan vTi instrument equipped with a Zeiss 10X 0.5NA objective and the Cytotoxicity.V3 algorithm (Cellomics/Thermo Fisher) with an XF100 filter. At least 10 fields of view, corresponding to at least 900 cells, were read in each well. The _IC50 value represents the compound concentration at which the PAR signal in the cells is reduced by 50% compared to untreated controls.

Use the following formula:

_IC50 = bottom + (top - bottom) / (1 + 10^(( _LogEC50 - X)));

X is the logarithm of the concentration, and _IC50 is the response value; _IC50 starts at the bottom and reaches the top in an S-shaped pattern.

In the above assay, the compounds of formula (I) of the present invention inhibited PAR formation with _IC50 values below 5 μM, as shown in Table 2.

Table 2

Colony formation assay

MDA-MB-436 breast cancer cells harboring a BRCA-1 mutation were grown at a density of 600 cells/ ^cm² in RPMI medium supplemented with 10% fetal bovine serum. After 24 hours, various doses of the compound were added in duplicate, starting at a concentration of 10 μM. After 10 days, the cells were fixed and stained with crystal violet. Colonies were counted using an infrared scanner (Odyssey Li-Cor). Antiproliferative _IC₅₀ was calculated using Prism.

Pharmacokinetics

In a dedicated (ad hoc) pharmacokinetic study, mice (Balb, Nu/Nu, Harlan, Italy) were used to investigate the pharmacokinetic properties and oral bioavailability of the compound. Compounds were formulated in 10% Tween 80/glucose for intravenous bolus administration, while compounds formulated in 0.5% methylcellulose were used for oral administration. A single administration of 10 mg/kg dose was administered using 3 male animals for each approach. All blood samples were taken from the retroorbital vein at 5 min, 30 min, 1 h, 3 h, 6 h, 24 h after intravenous administration and at 15 min, 30 min, 1 h, 3 h, 6 h, 24 h after oral administration. Plasma samples were prepared by adding 200 μL acetonitrile to the plasma protein precipitation of 20 μL of plasma in 96-well plates. After capping and vortex mixing, the plate was centrifuged at 4000 rpm for 15 min. The supernatant was considered the final extract and injected into an LC-MS-MS system (UPLC system: Waters Acquity, using a BEH C1850*2.1mm 1.7μm analytical column; MS instrument: Waters TQD equipped with an electrospray source operating in positive ion mode). The lower limit of quantification was 5.0ng/ml and the upper limit of quantification was 5000ng/mL. A non-compartmental method (linear trapezoidal rule and linear regression analysis of natural log-transformed plasma concentrations versus time data) was used. Absolute bioavailability (F) was calculated based on the ratio of mean oral-IV (intravenous) dose-normalized plasma AUC (area under the curve) values.

The abbreviations used in this document have the following meanings:

AUC (area under the plasma concentration versus time curve up to the last measured concentration)

Cl (plasma clearance)

Cmax (maximum plasma concentration)

T1/2 (terminal half-life)

Vdss (steady state distribution volume)

Certain representative compounds of formula (I) were evaluated for their pharmacokinetic parameters, which are reported as mean values in Table 3 below.

Table 3

From the above, it is clear to those skilled in the art that the compounds of formula (I) have good to excellent pharmacokinetic properties and oral bioavailability.

In vivo efficacy studies

Balb athymic Nu/Nu male mice from Harlan (Italy) were housed in cages with sterile filter paper lids, food, bedding, and acidified water in accordance with European Communities Council Directive 86/609/EEC on the protection of animals used for experimental or other scientific purposes. Fragments of Capan-1 human pancreatic cancer tumors were implanted subcutaneously. Mice bearing palpable tumors (100-200 mm ³ ) were selected and randomly divided into control and treatment groups. Each group consisted of 7 animals. Treatment began one day after randomization. The compound of formula (I) was administered orally in the form of a methylcellulose suspension at the dose and frequency indicated. Tumor size was measured regularly by caliper during the experiment, and tumor mass was calculated as described in Simeoni M. et al., Cancer Res 64, 1094-1101 (2004). Tumor growth inhibition (TGI, %) was calculated according to the following equation: %TGI=100-(average tumor weight of the treatment group/average tumor weight of the control group)*100.

The antitumor activity of certain representative compounds of Formula (I) as single agents in the Capan-1 BRCA-2 mutant mouse model is evaluated and reported in Table 4. Toxicity was assessed based on body weight loss (no body weight loss was observed in the 7 mice treated).

Table 4

Compound dose Schedule Max TGI (%) toxicity (11) 75 mg/kg 1-8 per day 42% 0/7 (15) 75 mg/kg 1-10 per day 54% 0/7

Representative compounds of formula (I) in combination with temozolomide were evaluated for their antitumor activity in a mouse model of Capan-1 BRCA-2 mutation. Compounds of formula (I) and temozolomide were administered orally. Tumor growth was assessed by caliper. Both diameters were recorded and tumor weight was calculated according to the following formula: length (mm) x width ² /2. The antitumor therapeutic effect was assessed by delaying the onset of exponential growth of the tumor (see Anticancer Drugs 7: 437-60, 1996). This delay (TC value) was defined as the difference in time (in days) required for the tumor to reach a predetermined size (1 g) between the treatment group (T) and the control group (C). Toxicity was assessed based on weight loss and animal survival. When the compound of formula (I) was combined with temozolomide, the observed TC was superior to that expected by simple addition of the TC obtained by monotherapy, indicating a significant synergistic effect.

Accordingly, the present invention provides a compound of formula (I) for use in therapy.

The compounds of formula (I) of the present invention suitable for administration to mammals, such as humans, can be administered through conventional routes, and the dosage level depends on the patient's age, weight, condition and administration route.

For example, a suitable dosage for a compound of formula (I) for oral administration can be in the range of about 1 to about 1000 mg/dose, 1 to 5 times per day. The compounds of the present invention can be administered in various dosage forms, for example, orally in the form of tablets, capsules, sugar-coated or film-coated tablets, liquid solutions or suspensions; rectally in the form of suppositories; parenterally, for example, intramuscularly; or by intravenous and/or intrathecal and/or intraspinal injection or infusion.

As mentioned above, the present invention also includes a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient which may be a carrier or diluent.

Pharmaceutical compositions comprising the compounds of the present invention are generally prepared according to conventional methods and are administered in suitable pharmaceutical dosage forms. For example, solid oral dosage forms, in addition to comprising active compounds, may also include: for example, diluents such as lactose, glucose, sucrose, sucrose, cellulose, corn starch or potato starch; lubricants such as silicon dioxide, talc, stearic acid, magnesium stearate or calcium stearate and/or polyethylene glycol; binders such as starch, gum arabic, gelatin, methylcellulose, carboxymethyl cellulose or polyvinyl pyrrolidone; disintegrants such as starch, alginic acid, alginate or sodium starch glycolate; effervescent mixtures; dyes; sweeteners; wetting agents such as lecithin, polysorbate, lauryl sulfate; and generally non-toxic and pharmacologically inactive substances for pharmaceutical preparations. These pharmaceutical preparations can be prepared in a known manner, for example, by mixing, granulating, tabletting, sugar coating or film coating processes.

Liquid dispersions for oral administration may be, for example, syrups, emulsions and suspensions. Syrups may contain sucrose or sucrose with glycerol and/or mannitol and sorbitol as carriers.

Suspensions and emulsions may contain natural gums, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol as examples of carriers. Suspensions or solutions for intramuscular injection may contain, in addition to the active compound, a pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, glycols such as propylene glycol and, if desired, an appropriate amount of lidocaine hydrochloride.

Solutions for intravenous injection or infusion may contain sterile water as a carrier, or preferably they may be in the form of sterile isotonic saline solutions, or they may contain propylene glycol as a carrier.

Suppositories may contain, in addition to the active compound, a pharmaceutically acceptable carrier such as cocoa butter, polyethylene glycol, polyoxyethylene sorbitan fatty acid ester surfactant or lecithin.

Experimental part

When referring to any specific compound of formula (I) of the present invention, the compound is optionally present in the form of a pharmaceutically acceptable salt, see the experimental section and claims. With reference to the following examples, the compounds of the present invention were synthesized using the methods described herein or other methods well known in the art.

The abbreviations and abbreviations used in this document have the following meanings:

amu (atomic mass unit)

calcd.(theoretical value)

μM (micromolar)

μL (microliter)

μm (micrometer)

mol

mM (millimolar)

mmol (millimolar)

nm (nanometer)

g (gram)

mg (milligrams)

ng (nanogram)

h(hour)

min(minutes)

DC ₅₀ (half-maximal displacement concentration)

IC ₅₀ (half-maximal inhibitory concentration)

PAR (poly(ADP-ribose))

MEM (Minimum Essential Medium)

FCS (fetal calf serum)

FBS (fetal bovine serum)

PBS (phosphate-buffered saline)

LC-MS (liquid chromatography-mass spectrometry)

HPLC (High Performance Liquid Chromatography)

TLC (thin layer chromatography)

MHz (megahertz)

Hz (Hertz)

DMSO-d ₆ (deuterated dimethyl sulfoxide)

CDCl ₃ (deuterated chloroform)

ESI (electrospray ionization)

In order to better illustrate the purpose of the present invention without limiting it in any way, the following examples are given.

The symbols used herein and the conventions used in the methods, schemes, and examples are consistent with those used in contemporary scientific literature, such as the Journal of the American Chemical Society or the Journal of Biological Chemistry.

Unless otherwise noted, all materials were obtained from commercial suppliers of the highest quality and used without further purification. Anhydrous solvents such as N,N-dimethylformamide, tetrahydrofuran, dichloromethane, and toluene were obtained from Aldrich Chemical Company. All reactions involving air- or moisture-sensitive compounds were performed under nitrogen or argon atmosphere.

General purification and analytical methods

Flash chromatography was performed on silica gel (Merck grade 9395). HPLC was performed on a Waters X Terra RP 18 (4.6 x 50 mm, 3.5 μm) column using a Waters 2790 HPLC system equipped with a 996 Waters PDA detector and micromass mode. A ZQ single quadrupole mass spectrometer was used with an electrospray (ESI) ion source. Mobile phase A was 5 mM ammonium acetate buffer (pH 5.5 with acetic acid-acetonitrile 95:5), and mobile phase B was water-acetonitrile (5:95). The gradient was from 10 to 90% B over 8 minutes, with a hold at 90% B for 2 minutes. UV detection was at 220 nm and 254 nm. The flow rate was 1 mL/min. The injection volume was 10 μL. A full scan was performed with a mass range of 100 to 800 amu. The capillary voltage was 2.5 kV; the source temperature was 120°C; and the cone voltage was 10 V. Retention times (HPLC r.t.) are given in min at 220 nm or 254 nm. Masses are given as m/z ratios.

When necessary, compounds were purified by preparative HPLC on a Waters Symmetry C18 (19 x 50 mm, 5 μm) column or on a Waters X Terra RP 18 (30 x 150 mm, 5 μm) column using a Waters Preparative HPLC 600 equipped with a 996 Waters PDA detector and micro mode. ZMD single quadrupole mass spectrometer, electrospray ionization, positive ion mode. Mobile phase A was water-0.01% trifluoroacetic acid, mobile phase B was acetonitrile. The gradient was from 10 to 90% B over 8 minutes, maintained at 90% B for 2 minutes. The flow rate was 20 mL/min. Alternatively, mobile phase A was water-0.1% ammonium hydroxide, mobile phase B was acetonitrile. The gradient was from 10 to 100% B over 8 minutes, maintained at 100% B for 2 minutes. The flow rate was 20 mL/min.

¹ H-NMR spectroscopy was performed in DMSO-d ₆ or CDCl ₃ using a Varian Inova 400 operating at 400.5 MHz and a Varian Mercury 300 operating at 300.0 MHz. ¹³ C NMR spectroscopy was performed in DMSO-d ₆ at 75.0 MHz.

The residual solvent signal was used as a reference (δ = 2.50 or 7.27 ppm). Chemical shifts (δ) are reported in mega-Bess (ppm) and coupling constants (J) are reported in Hz. The following abbreviations are used for diversity: s = singlet; br.s. = broad signal; d = doublet; t = triplet; m = multiplet; dd = two sets of doublets.

ESI(+) high-resolution mass spectra (HRMS) were obtained using a Q-Tof Ultima (Waters, Manchester, UK) directly connected to an 1100 micro-HPLC system (Agilent, Palo Alto, US) as described above (Colombo, M., Sirtori, F. R. and Rizzo, V. (2004) A fully automated method for accurate mass determination using high-performance liquid chromatography with a quadrupole/orthogonal acceleration time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom. 18, 511-517).

DETAILED DESCRIPTION

Example 1

Step a

2-Bromo-4-fluoro-6-methyl-phenylamine (X) [Hal = Br]

A solution of N-bromosuccinimide (18.7 g, 0.105 mol) in 70 mL of N,N-dimethylformamide was added dropwise to a solution of 4-fluoro-2-methyl-aniline (XI) (12.5 g, 0.1 mol) in 70 mL of the same solvent at 20°C. The reaction mixture was stirred overnight. The dark solution was poured into a mixture of water (1000 mL), brine (50 mL), and ethyl acetate (300 mL). The mixture was transferred to a separatory funnel, shaken, and separated. The aqueous phase was extracted with ethyl acetate (4 x 150 mL). The combined organic layers were washed with water (5 x 100 mL), brine ( ₂ x 100 mL), dried over _Na₂SO₄ , filtered, and concentrated. The product was purified by flash chromatography (eluent: ethyl acetate:n-hexane = 1:8). Pure fractions were combined and evaporated to yield 14.9 g of product. The impure fractions were combined, concentrated, redissolved in ether (30 mL), and extracted with 5% hydrochloric acid (5 x 10 mL). The acid phase was basified with aqueous potassium hydroxide solution and extracted with ether to give another 0.8 g of the title compound. The total yield was 15.7 g (77%).

¹ H NMR (400.5MHz, DMSO-d ₆ ) δppm 2.16 (s, 3H), 4.83 (br.s, 2H), 6.91 (dd, J _HF = 9.3Hz, J _HH = 2.9Hz, 1H), 7.16 (dd, J _HF = 8.3Hz, J _HH = 2.9Hz, 1H).

Step b

2-Bromo-4-fluoro-6-methyl-benzonitrile (IX) [Hal＝Br]

A solution of potassium cyanide (16.25 g, 0.25 mol) in 20 mL of water was added to a freshly prepared solution of copper (I) chloride (9.5 g, 0.096 mol) in 40 mL of water. Toluene (30 mL) was then added, and the mixture was cooled to 0°C. 2-Bromo-4-fluoro-6-methyl-phenylamine (X) (15.7 g, 0.077 mol) was added to a mixture of 16.5 mL of 36% aqueous hydrochloric acid and 40 mL of water. The resulting suspension was heated until a solution formed. The solution was cooled to 2°C, and the amine hydrochloride precipitated. A solution of sodium nitrite (5.34 g, 0.078 mol) in 15 mL of water was slowly added, maintaining the reaction mixture temperature below 5°C. Powdered sodium carbonate decahydrate was added in small portions to adjust the reaction mixture's pH to approximately 7. The resulting diazonium salt solution was then slowly added to the previously prepared cyanocuprate reagent, again maintaining the reaction mixture temperature below 5°C. A bright red-orange precipitate formed. The reaction mixture was warmed to 20°C and maintained at this temperature overnight. It was then heated at 70°C for 1 hour. The precipitate dissolved almost completely. The reaction mixture was cooled to 20°C and filtered. The organic phase was separated, and the aqueous phase was extracted with toluene (3 x 70 mL). The combined organic layers were washed with water (2 x 100 mL), brine (2 x 100 mL), dried over Na ₂ SO ₄ , filtered, and concentrated. Crude nitrile (IX) (13.9 g, 84%) was used without further purification.

¹ H NMR (400.5MHz, DMSO-d ₆ ) δppm 2.52 (s, 3H), 7.44 (dd,J _HF = 9.4Hz, J _HH = 2.1Hz, 1H), 7.73 (dd, J _HF = 8.2Hz, J _HH = 2.1Hz, 1H).

¹³ C NMR (75.0MHz, DMSO-d ₆ ) δ 115.8, 112.7 (d, J _CF = 3Hz), 117.0 (d, J _CF = 23Hz), 118.4 (d, J _CF = 27Hz), 126.1 (d, J _CF = 11Hz), 147.8 (d, J _CF =11Hz),163.5(d,J _CF =257Hz).

Step c

2-Bromo-4-fluoro-6-methyl-benzamide (VIII) [Hal＝Br]

2-Bromo-4-fluoro-6-methyl-benzonitrile (IX) (0.428 g, 2 mmol) was heated in 70% aqueous sulfuric acid (2 mL) at 150°C overnight. The reaction mixture was poured into ice and extracted with ethyl acetate (4 x 2 mL). The organic phase was washed with water (4 x 2 mL), brine (2 x 2 _mL ), dried over _Na₂SO₄ , filtered, and concentrated to yield 300 mg of crude 2-bromo-4-fluoro-6-methyl-benzamide (VIII). A pure sample was obtained by recrystallization from benzene.

¹ H NMR (400.5MHz, DMSO-d ₆ ) δppm 3.31 (s, 3H), 7.17 (dd, J _HF = 9.8Hz, J _HH = 2.2Hz, 1H), 7.41 (dd, J _HF = 8.6Hz, J _HH ＝2.2Hz,1H),7.89(br.s,1H),7.65(br.s,1H).

Step d

2-Bromo-4-fluoro-6-methyl-benzoic acid (VII) [Hal＝Br]

2-Bromo-4-fluoro-6-methyl-benzamide (VIII) (0.9 g, 3.9 mmol) was dissolved in 75% aqueous sulfuric acid (4 mL) at 80°C. Sodium nitrite (0.5 g, 7.2 mmol) was carefully added in small portions over 1 hour. The reaction mixture was cooled to 20°C and cold water (15 mL) was added. The product was extracted with ethyl acetate (6 x 2 mL). The organic phase was washed with water (4 x 2 mL), then water (2 x 2 mL), _dried over _Na₂SO₄ , filtered, and concentrated to yield 0.879 g (97%) of pure (VII) as a solid.

¹ H NMR (400.5MHz, DMSO-d ₆ ) δppm 2.31 (s, 1H), 7.22 (dd, J _HF = 9.6Hz, J _HH = 2.2Hz, 1H), 7.47 (dd, J _HF = 8.5Hz, J _HH = 2.4Hz, 1H), 13.7 (br.s, 1H).

¹³ C NMR (75.0MHz, DMSO-d ₆ +CCl ₄ ) δppm 19.5, 116.0 (d, J _CF = 22Hz), 116.8 (d, J _CF = 24Hz), 118.3 (d, J _CF = 10Hz), 134.0 (d, J _CF = 3Hz), 138.4 (d, J _CF =8Hz),163.0(d,J _CF =250Hz),168.0.

Step f

2-Bromo-4-fluoro-6-methyl-benzoic acid methyl ester (VI) [Hal = Br; T = methyl]

A mixture of 2-bromo-4-fluoro-6-methyl-benzoic acid (VII) (1.94 g, 8.33 mmol), anhydrous potassium carbonate (1.72 g, 12.5 mmol), and iodomethane (2.36 g, 17 mmol) in N,N-dimethylformamide (15 mL) was stirred vigorously at 20°C for 23 h. The suspension was poured into 70 mL of water. A thick oil separated. The product was extracted with ethyl acetate (4 x 25 mL). The organic phase was washed with water (5 x 20 mL) and brine (2 x 20 mL), dried over _Na₂SO₄ , filtered, and concentrated to afford 2.07 g (quantitative yield) of _methyl 2-bromo-4-fluoro-6-methyl-benzoate (VI).

¹ H NMR (400.5MHz, CDCl ₃ ) δppm 2.35 (s, 3H), 3.96 (s, 3H), 6.91 (dd, J _HF = 9.0Hz, J _HH = 2.2Hz, 1H), 7.18 (dd, J _HF = 8.1Hz, J _HH = 2.4Hz, 1H).

Step g

2-Cyano-4-fluoro-6-methyl-benzoic acid methyl ester (V) [T = methyl]

A mixture of methyl 2-bromo-4-fluoro-6-methyl-benzoate (VI) (275 mg, 1.12 mmol), potassium hexacyanoferrate (II) (206 mg, 0.56 mmol), anhydrous sodium carbonate (237 mg, 2.24 mmol), and palladium (II) acetate (5 mg, 0.0224 mmol) in 3 mL of N-methylpyrrolidone was heated at 120° C. in a sealed tube under argon overnight. The reaction mixture was diluted with dichloromethane and filtered through a pad of celite. The organic phase was washed with water (13 x 6 mL), brine (2 x 6 mL), dried over Na ₂ SO ₄ , filtered, and concentrated. Column chromatography (n-hexane/ethyl acetate: 7/3) afforded methyl 2-cyano-4-fluoro-6-methyl-benzoate (76 mg, 35%).

¹ H NMR (400.5MHz, DMSO-d ₆ ) δppm 2.42 (s, 3H), 3.93 (s, 3H), 7.65 (dd, J _HF = 9.6, J _HH = 2.6 Hz, 1H), 7.85 (dd, J _HF = 8.3, 2.6 Hz, 1H).

Step h

6-Fluoro-2-(3-morpholin-4-yl-propyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carbonitrile (IV) [n=3; R1=morpholin-4-yl; X=absent, when m=0 and R2=absent]

To a solution of 2-cyano-4-fluoro-6-methyl-benzoic acid methyl ester (V) (208 mg, 1.07 mmol) in methyl pivalate (2 mL) was added N-bromosuccinimide (310 mg, 1.74 mmol) and benzoyl peroxide (20 mg, 0.097 mmol). The reaction mixture was stirred at 85°C under a nitrogen atmosphere for 3 h. The crude product was filtered through Gooch and washed with toluene. The volatiles were evaporated and the residue was dissolved in acetonitrile (3 mL). Triethylamine (0.41 mL, 2.9 mmol) and 3-morpholin-4-yl-propylamine (XIII) (140 mg, 0.97 mmol) were added and the reaction mixture was stirred at 90°C for 3 h. The crude product was diluted with dichloromethane and washed with 15% ammonium hydroxide. The organic phase was dried _{over Na2SO4} , filtered and evaporated. Column chromatography (chloroform/methanol: 96/4 to chloroform/methanol: 94/6 gradient) gave 6-fluoro-2-(3-morpholin-4-yl-propyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carbonitrile (IV) (130 mg, 40% yield).

¹ H NMR (400.5MHz, CDCl ₃ ) δppm 1.87 (quintet, J = 7.1Hz, 2H), 2.34-2.49 (m, 6H), 3.62-3.74 (m, 6H), 4.45 (s, 2H), 7.42 (dd, J _HF = 7.3Hz, J _HH =2.0Hz,1H),7.47(dd,J _HF =8.3Hz,J _HH =2.0Hz,1H).

Step c’

6-Fluoro-2-(3-morpholin-4-yl-propyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd23

[R＝F; n＝3; R1＝morpholin-4-yl; m＝0; R2＝absent]

A solution of 6-fluoro-2-(3-morpholin-4-yl-propyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carbonitrile (IV) (100 mg, 0.33 mmol) in 1.5 mL of 36% hydrochloric acid was heated at 50°C for 10 h. All volatiles were evaporated, and the residue was dissolved in 2 mL of cold water. The solution was neutralized with solid sodium carbonate. The precipitated solid was dissolved in dichloromethane, and the organic phase was washed with saturated aqueous sodium carbonate (2 x 1 mL), brine (2 x 1 mL), dried over _Na₂SO₄ , filtered, and concentrated to yield 73 mg (73%) of ₆ -fluoro-2-(3-morpholin-4-yl-propyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I).

¹ H NMR (400.5MHz, DMSO-d ₆ ) δppm 1.79 (quintet, J=7.1Hz, 2H), 2.28-2.35 (m, 6H), 3.47-3.52 (m, 4H), 3.59 (t, J=7.1Hz, 2H), 4.58 (s, 2H), 7.68 (dd, J _HF ＝7.8,J _HH ＝2.6Hz,1H),7.83(br.s.,1H),7.89(dd,J _HF ＝10.9,J _HH ＝2.6Hz,1H),10.81(br.s.,1H).

HRMS (ESI+): Calcd. for C ₁₆ H ₂₁ FN ₃ O ₃ [M+H] ⁺ 322.1562; Found 322.1565

Example 2

Step e

4-Fluoro-2-iodo-6-methyl-benzoic acid (VII) [Hal＝I]

A mixture of 4-fluoro-2-methyl-benzoic acid (XII) (20.00 g, 0.130 mol), iodophenyl diacetate (50.15 g, 0.156 mol), iodine (39.52 g, 0.156 mol) and palladium (II) acetate (1.46 g, 0.006 mol) in N,N-dimethylformamide (360 mL) was degassed by cycling vacuum and nitrogen three times and then heated under argon at 100° C. internal temperature for 18 h. The resulting dark mixture was cooled to room temperature, diluted with methyl tert-butyl ether (200 mL), and treated with a solution of sodium metabisulfite (250 g) in water (500 mL) under efficient stirring. The yellow mixture was then acidified by slowly adding concentrated hydrochloric acid (130 mL). The aqueous layer was separated and washed twice with methyl tert-butyl ether (mL 100 x 2). The combined organic extracts were treated with a solution of sodium hydroxide pellets (80 g) in water (300 mL) under stirring. The organic layer containing only iodobenzene was discharged and sodium chloride was added to the aqueous layer, cooled to freezing temperature, and a very low pH was reached with concentrated hydrochloric acid (130 mL). The product was extracted from this aqueous medium with methyl tert-butyl ether (100 mL x 3), and the combined extracts were dried over _{Na SO} and finally concentrated under reduced pressure to give 30.5 g (84%) of 4-fluoro-2-iodo-6-methyl-benzoic acid as a brown solid. The crude material was used in the next step without purification.

¹ H NMR (300.0MHz, CDCl ₃ ) δppm 2.46 (s, 3H), 6.96 (dd,J _HF =9.1, J _HH =2.6Hz, 1H), 7.45 (dd,J _HF =7.9, 2.3Hz, 1H).

Step f

4-Fluoro-2-iodo-6-methyl-benzoic acid methyl ester (VI) [Hal = I; T = methyl]

To a solution of 4-fluoro-2-iodo-6-methyl-benzoic acid (VII) (30.05 g, 0.109 mol) in N,N-dimethylformamide (300 mL) was added anhydrous potassium carbonate (22.0 g, 0.16 mol) under high-efficiency magnetic stirring. After 15 minutes, methyl p-toluenesulfonate (30.7 g, 0.16 mol) was added. The brown suspension was stirred at room temperature for 2 hours. Potassium acetate (12.4 g, 0.13 mol) was then added to destroy unreacted methyl p-toluenesulfonate, and the mixture was stirred overnight. The thick reaction mixture was diluted with methyl tert-butyl ether (100 mL) and washed with water (600 mL); the aqueous layer was separated and extracted twice with methyl tert-butyl ether (70 mL x 2). The combined organic extracts were washed with brine (50 mL), dried over _Na₂SO₄ , _and concentrated under reduced pressure to yield a solid residue. The material was purified by chromatography (eluent n-hexane/ethyl acetate 9:1) to give 26.2 g (81%) of the product as a colorless oil.

¹ H NMR (400.5MHz, DMSO-d ₆ ) δppm 2.27 (s, 3H), 3.86 (s, 3H), 7.25 (dd, J _HF = 9.6, J _HH = 2.4Hz, 1H), 7.63 (dd, J _HF = 8.2, J _HH = 2.4Hz, 1H).

Step g

2-Cyano-4-fluoro-6-methyl-benzoic acid methyl ester (V) [T = methyl]

A solution of methyl 4-fluoro-2-iodo-6-methyl-benzoate (VI) (26.02 g, 88.48 mmol) in 260 mL of N,N-dimethylformamide was treated with copper (I) cyanide (12.18 g; 0.136 mol) and stirred at 110°C for 5 h. The dark mixture was cooled to approximately 60°C, treated with 105 g of crude 560 (Fluka) under efficient stirring, and diluted with ethyl acetate (250 mL). After cooling to room temperature, the mixture was slowly poured into 0.25 N aqueous sodium hydroxide solution (500 mL) and then filtered. The reaction flask and plate were washed with ethyl acetate (100 mL). The aqueous layer was separated and extracted twice with ethyl acetate (250 mL + 100 mL). The combined organic extracts were washed with brine (200 mL), dried _{over Na₂SO₄} , and concentrated under reduced pressure to give 22.00 g of crude product as a yellow solid. This material was crystallized from n-hexane (40 mL). After cooling to room temperature, the solid was collected by filtration and the mother liquor was concentrated under reduced pressure. The solid residue thus obtained was crystallized from n-hexane (20 mL). After filtration of the solid, a second crop of product was obtained. The combined batches (14.15 g) were finally purified by chromatography using a gradient elution from n-hexane/methyl tert-butyl ether 9:1 to n-hexane/ethyl acetate 9:1. After evaporation of the fractions, 12.0 g (70%) of 2-cyano-4-fluoro-6-methyl-benzoic acid methyl ester (V) were obtained.

¹ H NMR (400.5MHz, DMSO-d ₆ ) δppm 2.42 (s, 3H), 3.93 (s, 3H), 7.65 (dd, J _HF = 9.6, J _HH = 2.6 Hz, 1H), 7.85 (dd, J _HF = 8.3, 2.6 Hz, 1H).

Step h

2-(1-Cyclohexyl-piperidin-4-yl)-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carbonitrile (IV) [R = F; n = m = 0; R1 = piperidin-4-yl; R2 = 1-cyclohexyl]

To a solution of 2-cyano-4-fluoro-6-methyl-benzoic acid methyl ester (V) (208 mg, 1.07 mmol) in methyl pivalate (2 mL) were added N-bromosuccinimide (310 mg, 1.74 mmol) and benzoyl peroxide (20 mg, 0.097 mmol). The reaction mixture was stirred at 85°C under a nitrogen atmosphere for 3 h. The crude product was filtered and washed with toluene. The volatiles were evaporated and the residue was dissolved in acetonitrile (3 mL). Potassium carbonate (670 mg, 4.85 mmol) and 1-cyclohexyl-piperidin-4-ylamine dihydrochloride monohydrate (XIII) (265 mg, 0.97 mmol) were added and the reaction mixture was stirred at 90°C for 3 h. The crude product was diluted with dichloromethane and washed with 15% ammonium hydroxide. The organic phase was dried _{over Na2SO4} , filtered and evaporated. Column chromatography (dichloromethane/methanol/ammonia solution, 7N in methanol: 97/2/1) gave 2-(1-cyclohexyl-piperidin-4-yl)-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carbonitrile (IV) (100 mg, 30%).

¹ H NMR (400.5MHz, DMSO-d ₆ ) δppm 1.02-1.13(m,1H),1.16-1.27(m,4H),1.55-1.62(m,1H),1.68-1.80(br.s.,7H) ,2.23-2.39(m,3H),2.87-2.97(m,2H),3.95(br.s.,1H),4.52(s,2H),7.86(dd,J _HF = 8.3, J _HH = 2.2Hz, 1H), 7.98 (dd, J _HF = 9.3, J _HH = 2.2Hz, 1H).

HRMS (ESI+): Calcd. for C ₂₀ H ₂₅ FN ₃ O [M+H] ⁺ 342.1976; Found 342.1988

Step c'

2-(1-Cyclohexyl-piperidin-4-yl)-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd29

[R＝F; n＝m＝0; R1＝piperidin-4-yl; R2＝1-cyclohexyl]

To a stirred solution of 2-(1-cyclohexyl-piperidin-4-yl)-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carbonitrile (IV) (100 mg, 0.3 mmol) in acetic acid (5 mL) was added concentrated sulfuric acid (2.7 mL) dropwise over 30 minutes. The reaction was then warmed at 80°C for 9 hours, cooled to room temperature, and poured into cold water (10 mL). The aqueous phase was then made basic by the addition of concentrated ammonia and extracted with dichloromethane (3 x 10 mL). The combined organic phases were washed with 2N aqueous sodium hydroxide (2 _x 12 mL) and brine, dried over _Na₂SO₄ , and evaporated to dryness in vacuo. Purification by column chromatography (dichloromethane/methanol/ammonia solution, 7N in methanol: 97/2/1) afforded the title compound as a white solid (43 mg, 40%).

¹ H NMR (400.5MHz, DMSO-d ₆ ) δppm 1.00-1.14(m,1H),1.14-1.28(m,4H),1.53-1.61(m,1H),1.67-1.80(m,6H),2. 25-2.36(m,3H),2.88-2.95(m,2H),3.94-4.03(m,1H),4.55(s,2H),7.66(dd,J _HF = 7.7, J _HH = 2.6Hz, 1H), 7.85 (br.s., 1H), 7.89 (dd, J _HF = 10.9, J _HH = 2.6Hz, 1H), 10.78 (br.s., 1H).

HRMS (ESI+): Calcd. for C ₂₀ H ₂₇ FN ₃ O ₂ [M+H] ⁺ 360.2082; Found 360.2098

Example 3

Step m

4-[(Furan-2-ylmethyl)-amino]-piperidine-1-carboxylic acid tert-butyl ester (XVI) [n=0; R1=piperidin-4-yl; X=tert-butoxycarbonyl]

To an equimolar solution of furan-2-carbaldehyde (XV) (250 mg, 2.6 mmol) and tert-butyl 4-amino-piperidine-1-carboxylate (XIII) (473 mg, 2.6 mmol) in dichloromethane (14 mL) was added a 1 M solution of titanium (IV) chloride in dichloromethane (1.3 mL, 1.3 mmol) and triethylamine (0.32 mL, 2.6 mmol). The reaction mixture was stirred under a nitrogen atmosphere for 2 days. A solution of sodium cyanoborohydride (493 mg, 7.8 mmol) in methanol (7 mL) was then added dropwise with stirring, and the solution was stirred at room temperature overnight. 35% sodium hydroxide was added, and the product was extracted with ethyl acetate. The organic phase was separated, washed with brine, dried _{over Na₂SO₄} , and evaporated to dryness in vacuo. The crude product was purified by flash chromatography (dichloromethane/methanol 95:5) to afford the title compound as a red oil (406 mg, 56%).

HRMS (ESI+): Calcd. for C ₁₅ H ₂₅ N ₂ O ₃ [M+H] ⁺ 281.1860; found 281.1867.

Following a similar procedure, but using appropriately substituted starting materials (XIII), the following compounds were obtained:

Benzyl-furan-2-ylmethyl-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₂ H ₁₄ NO [M+H] ⁺ 188.1070; Found 188.1075

Furan-2-ylmethyl-phenethyl-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₃ H ₁₆ NO [M+H] ⁺ 202.1226; Found 202.1230

[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-ethyl]-furan-2-ylmethyl-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₆ H ₂₁ N ₂ O [M+H] ⁺ 257.1648; Found 257.1642

Furan-2-ylmethyl-(2-piperidin-1-yl-ethyl)-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₂ H ₂₁ N ₂ O [M+H] ⁺ 209.1648; Found 209.1650

Furan-2-ylmethyl-(2-morpholin-4-yl-ethyl)-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₁ H ₁₉ N ₂ O ₂ [M+H] ⁺ 211.1441; Found 211.1446

Furan-2-ylmethyl-(3-morpholin-4-yl-propyl)-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₂ H ₂₁ N ₂ O ₂ [M+H] ⁺ 225.1598; Found 225.1590

[2-(3,4-Dihydro-2H-quinolin-1-yl)-ethyl]-furan-2-ylmethyl-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₆ H ₂₁ N ₂ O [M+H] ⁺ 257.1648; Found 257.1652

Furan-2-ylmethyl-(3-phenyl-propyl)-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₄ H ₁₈ NO [M+H] ⁺ 216.1383; Found 216.1387

Furan-2-ylmethyl-(2-pyridin-2-yl-ethyl)-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₂ H ₁₅ N ₂ O [M+H] ⁺ 203.1179; Found 203.1181

[3-(3,4-Dihydro-1H-isoquinolin-2-yl)-propyl]-furan-2-ylmethyl-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₇ H ₂₃ N ₂ O [M+H] ⁺ 271.1805; Found 271.1799

[3-(3,4-Dihydro-2H-quinolin-1-yl)-propyl]-furan-2-ylmethyl-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₇ H ₂₃ N ₂ O [M+H] ⁺ 271.1805; Found 271.1811

Furan-2-ylmethyl-[3-(4-methyl-piperazin-1-yl)-propyl]-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₃ H ₂₄ N ₃ O [M+H] ⁺ 238.1914; Found 238.1912

Furan-2-ylmethyl-[3-(4-phenyl-piperazin-1-yl)-propyl]-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₈ H ₂₆ N ₃ O [M+H] ⁺ 300.2070; Found 300.2077

Furan-2-ylmethyl-(3-piperidin-1-yl-propyl)-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₃ H ₂₃ N ₂ O [M+H] ⁺ 223.1805; Found 223.1802

(3-[1,4']Bipiperidin-1'-yl-propyl)-furan-2-ylmethyl-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₈ H ₃₂ N ₃ O [M+H] ⁺ 306.2540; Found 306.2544

[3-(2,6-Dimethyl-piperidin-1-yl)-propyl]-furan-2-ylmethyl-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₅ H ₂₇ N ₂ O [M+H] ⁺ 251.2118; Found 251.2120

Furan-2-ylmethyl-[1-(tetrahydro-pyran-4-yl)-piperidin-4-yl]-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₅ H ₂₅ N ₂ O ₂ [M+H] ⁺ 265.1911; Found 265.1919

(1-Benzyl-piperidin-4-yl)-furan-2-ylmethyl-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₇ H ₂₃ N ₂ O [M+H] ⁺ 271.1805; Found 271.1807

[2-(1-Benzyl-piperidin-4-yl)-ethyl]-furan-2-ylmethyl-amine (XVI)

HRMS (ESI+): Calcd. for C ₁₉ H ₂₇ N ₂ O [M+H] ⁺ 299.2118; Found 299.21222

[3-(4-Benzyl-piperidin-1-yl)-propyl]-furan-2-ylmethyl-amine (XVI)

HRMS (ESI+): Calcd. for C ₂₀ H ₂₉ N ₂ O [M+H] ⁺ 313.2274; Found 313.2280

(1-Cyclohexyl-piperidin-4-yl)-furan-2-ylmethyl-amine (XVI)

A solution of equimolar amounts of furan-2-carboxaldehyde (XV) (1.3 g, 13.5 mmol) and 1-cyclohexyl-piperidin-4-ylamine (XIII) (2.46 g, 13.5 mmol) in toluene (140 mL) was heated at reflux for 8 h using a Dean-Stark apparatus. The reaction mixture was concentrated in vacuo and rinsed with ethanol (50 mL). Sodium triacetoxyborohydride (3.8 g, 17.93 mmol) was added and the mixture was allowed to stand at room temperature overnight. It was then basified with aqueous ammonia (8%), the aqueous layer separated, and extracted with diethyl ether. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the title compound as a yellow oil which was used in the next step without any additional purification.

HRMS (ESI+): Calcd. for C ₁₆ H ₂₇ N ₂ O [M+H] ⁺ 263.2118; Found 263.2120

Step n

3-(1-tert-Butoxycarbonyl-piperidin-4-yl)-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII) [n＝0; R1＝piperidin-4-yl; X＝tert-butoxycarbonyl]

To a solution of tert-butyl 4-[(furan-2-ylmethyl)-amino]-piperidine-1-carboxylate (XVI) (5.6 g, 21 mmol) in toluene (300 mL) was added maleic anhydride (2.1 g, 21 mmol). The reaction mixture was refluxed for 6 h and stirred at room temperature overnight. The resulting precipitated solid was filtered, washed with diethyl ether, and dried to afford the desired compound (6.5 g, 82%) as a white solid.

¹ H NMR (400.5MHz, DMSO-d ₆ ) δppm 1.40(s,9H),1.40-1.63(m,4H),2.45(d,J＝9.3Hz,1H),2.75(br.s.,2H),2.76(d,J＝9.3Hz,1H),3.59(d,J＝11.6Hz,1H),3.88(d,J＝11.6 0Hz,1H),3.90(m,1H),3.96-4.06(m,2H),4.95(d,J＝1.6Hz,1H),6.42(dd,J＝5.6,1.7Hz,1H),6.55(d,J＝5.6Hz,1H),12.03(br.s.,1H).

HRMS (ESI+): Calcd. for C ₁₉ H ₂₇ N ₂ O ₆ [M+H] ⁺ 379.1864; Found 379.1876

Following a similar procedure, but using the appropriately substituted starting material (XVI), the following compounds were obtained:

3-Benzyl-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₁₆ H ₁₆ NO ₄ [M+H] ⁺ 286.1074; Found 286.1078

4-Oxo-3-phenethyl-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₁₇ H ₁₈ NO ₄ [M+H] ⁺ 300.1230; Found 300.1237

3-[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-ethyl]-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₂₀ H ₂₃ N ₂ O ₄ [M+H] ⁺ 355.1652; Found 355.1657

4-Oxo-3-(2-piperidin-1-yl-ethyl)-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₁₆ H ₂₃ N ₂ O ₄ [M+H] ⁺ 307.1652; Found 307.1660

3-(2-Morpholin-4-yl-ethyl)-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₁₅ H ₂₁ N ₂ O ₅ [M+H] ⁺ 309.1445; Found 309.1446

3-(3-Morpholin-4-yl-propyl)-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₁₆ H ₂₃ N ₂ O ₅ [M+H] ⁺ 323.1601; Found 323.1609

3-[2-(3,4-Dihydro-2H-quinolin-1-yl)-ethyl]-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₂₀ H ₂₃ N ₂ O ₄ [M+H] ⁺ 355.1652; Found 355.1660

4-Oxo-3-(3-phenyl-propyl)-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₁₈ H ₂₀ NO ₄ [M+H] ⁺ 314.1387; Found 314.1392

4-Oxo-3-(2-pyridin-2-yl-ethyl)-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₁₆ H ₁₇ N ₂ O ₄ [M+H] ⁺ 301.1183; Found 301.1179

3-[3-(3,4-Dihydro-1H-isoquinolin-2-yl)-propyl]-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₂₁ H ₂₅ N ₂ O ₄ [M+H] ⁺ 369.1809; Found 369.1811

3-[3-(3,4-Dihydro-2H-quinolin-1-yl)-propyl]-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₂₁ H ₂₅ N ₂ O ₄ [M+H] ⁺ 369.1809; Found 369.1801

3-[3-(4-Methyl-piperazin-1-yl)-propyl]-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₁₇ H ₂₆ N ₃ O ₄ [M+H] ⁺ 336.1918; Found 336.1920

4-Oxo-3-[3-(4-phenyl-piperazin-1-yl)-propyl]-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C _{2 2} H _{2 8} N ₃ O ₄ [M+H] ⁺ 398.2074; Found 398.2079

4-Oxo-3-(3-piperidin-1-yl-propyl)-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₁₇ H ₂₅ N ₂ O ₄ [M+H] ⁺ 321.1809; Found 321.1812

3-(3-[1,4']bipiperidin-1'-yl-propyl)-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C _{2 2} H _{3 4} N ₃ O ₄ [M+H] ⁺ 404.2544; Found 404.2540

3-[3-(2,6-Dimethyl-piperidin-1-yl)-propyl]-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₁₉ H ₂₉ N ₂ O ₄ [M+H] ⁺ 349.2122; Found 349.2119

4-Oxo-3-[1-(tetrahydro-pyran-4-yl)-piperidin-4-yl]-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₁₉ H ₂₇ N ₂ O ₅ [M+H] ⁺ 363.1914; Found 363.1920

3-(1-Benzyl-piperidin-4-yl)-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₂₁ H ₂₅ N ₂ O ₄ [M+H] ⁺ 369.1809; Found 369.1799

3-[2-(1-Benzyl-piperidin-4-yl)-ethyl]-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₂₃ H ₂₉ N ₂ O ₄ [M+H] ⁺ 397.2122; Found 397.2127

3-[3-(4-Benzyl-piperidin-1-yl)-propyl]-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

HRMS (ESI+): Calcd. for C ₂₄ H ₃₁ N ₂ O ₄ [M+H] ⁺ 411.2278; Found 411.2283

3-(1-Cyclohexyl-piperidin-4-yl)-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII)

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 11.98(br.s.,1H),6.58(d,J＝5.6Hz,1H),6.44(dd,J＝5.6,1.6Hz,1H),4.97( d,J＝1.6Hz,1H),4.00-4.11(m,1H),3.96(d,J＝11.0Hz,1H),3.55(d,J＝11.0Hz 1H),3.38-3.48(m,2H),3.04-3.2(m,3H),2.79(d,J＝9.1Hz,1H),2.48(d,J＝9.1Hz,1H),1.55-2.01(m,8H),1.04-1.44(m,6H).

HRMS (ESI+): Calcd. for C ₂₀ H ₂₉ N ₂ O ₄ [M+H]+: 361.2122; Found: 361.2129

Step o

3-Oxo-2-piperidin-4-yl-2,3-dihydro-1H-isoindole-4-carboxylic acid hydrochloride (XIX) [n＝0; R1＝piperidin-4-yl]

3-(1-tert-Butyloxycarbonyl-piperidin-4-yl)-4-oxo-10-oxa-3-aza-tricyclo[5.2.1.0 ^* 1,5 ^* ]dec-8-ene-6-carboxylic acid (XVII) (6.35 g, 16.8 mmol) was dissolved in 37% hydrochloric acid (80 mL), and the resulting solution was refluxed for 3 h. The solvent was removed under reduced pressure, and the residue was diluted with methanol and decanted to give the desired product (XIX) as a white solid (4.06 g, 82%).

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.95-2.12(m,4H),3.01-3.18(m,2H),3.36-3.45(m,2H),4.36-4.46(m,1H),4.72(s,2H),7.85(dd,J＝7.7,7.5Hz ,1H),7.95(dd,J＝7.5,0.8Hz,1H),8.17(dd,J＝7.7,0.8Hz,1H),8.53(br.s.,1H),8.79(br.s.,1H),15.86(s,1H).

HRMS (ESI+): Calcd. for C ₁₄ H ₁₇ N ₂ O ₃ [M+H] ⁺ 261.1234; Found 261.1222

Following a similar procedure, but using the appropriately substituted starting material (XVII), the following compounds were obtained:

2-Benzyl-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₁₆ H ₁₄ NO ₃ [M+H] ⁺ 268.0968; Found 268.0972

3-Oxo-2-phenylethyl-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₁₇ H ₁₆ NO ₃ [M+H] ⁺ 282.1125; Found 282.1131

2-[2-(3,4-Dihydro-1H-isoquinolin-2-yl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₂₀ H ₂₁ N ₂ O ₃ [M+H] ⁺ 337.1547; Found 337.1541

3-Oxo-2-(2-piperidin-1-yl-ethyl)-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₁₆ H ₂₁ N ₂ O ₃ [M+H] ⁺ 288.1547; Found 288.1552

2-(2-Morpholin-4-yl-ethyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₁₅ H ₁₉ N ₂ O ₄ [M+H] ⁺ 291.1339; Found 291.1335

2-(3-Morpholin-4-yl-propyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₁₆ H ₂₁ N ₂ O ₄ [M+H] ⁺ 305.1496; Found 305.1492

2-[2-(3,4-Dihydro-2H-quinolin-1-yl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₂₀ H ₂₁ N ₂ O ₃ [M+H] ⁺ 337.1547; Found 337.1549

3-Oxo-2-(3-phenyl-propyl)-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₁₈ H ₁₈ NO ₃ [M+H] ⁺ 296.1281; Found 296.1290

3-Oxo-2-(2-pyridin-2-yl-ethyl)-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₁₆ H ₁₅ N ₂ O ₃ [M+H] ⁺ 283.1077; Found 283.1080

2-[3-(3,4-Dihydro-1H-isoquinolin-2-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₂₁ H ₂₃ N ₂ O ₃ [M+H] ⁺ 351.1703; Found 351.1706

2-[3-(3,4-Dihydro-2H-quinolin-1-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₂₁ H ₂₃ N ₂ O ₃ [M+H] ⁺ 351.1703; Found 351.1699

2-[3-(4-Methyl-piperazin-1-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₁₇ H ₂₄ N ₃ O ₃ [M+H] ⁺ 318.1812; Found 318.1820

3-Oxo-2-[3-(4-phenyl-piperazin-1-yl)-propyl]-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C _{2 2} H _{2 6} N ₃ O ₃ [M+H] ⁺ 380.1969; Found 380.1971

3-Oxo-2-(3-piperidin-1-yl-propyl)-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₁₇ H ₂₃ N ₂ O ₃ [M+H] ⁺ 303.1703; Found 303.1702

2-(3-[1,4']bipiperidin-1'-yl-propyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C _{2 2} H ₃ 2 N ₃ O ₃ [M+H] ⁺ 386.2438; Found 386.2442

2-[3-(2,6-Dimethyl-piperidin-1-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₁₉ H ₂₇ N ₂ O ₃ [M+H] ⁺ 331.2016; Found 331.2011

3-Oxo-2-[1-(tetrahydro-pyran-4-yl)-piperidin-4-yl]-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₁₉ H ₂₅ N ₂ O ₄ [M+H] ⁺ 345.1809; Found 345.1816

2-(1-Benzyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₂₁ H ₂₃ N ₂ O ₃ [M+H] ⁺ 351.1703; Found 351.1708

2-[2-(1-Benzyl-piperidin-4-yl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₂₃ H ₂₇ N ₂ O ₃ [M+H] ⁺ 379.2016; Found 379.2020

2-[3-(4-Benzyl-piperidin-1-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₂₄ H ₂₉ N ₂ O ₃ [M+H] ⁺ 393.2173; Found 393.2177

2-(1-Cyclohexyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII)

HRMS (ESI+): Calcd. for C ₂₀ H ₂₇ N ₂ O ₃ [M+H] ⁺ 343.2016; Found 343.2019

Step q

2-(1-tert-Butoxycarbonyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII) [n=0; R1=piperidin-4-yl; X=tert-butoxycarbonyl]

To a solution of 3-oxo-2-piperidin-4-yl-2,3-dihydro-1H-isoindole-4-carboxylic acid (3.9 g, 13.2 mmol) in pyridine (15 mL) were added potassium carbonate (3.6 g, 26.5 mmol) and methanol (40 mL) sequentially. Di-tert-butyl dicarbonate (3.16 g, 14.5 mmol) was then added, and the reaction mixture was stirred at room temperature for 4 h until HPLC analysis revealed the disappearance of the starting material. The solvent was removed under reduced pressure, and the residue was dissolved in dichloromethane. The solution was washed twice with 5% potassium bisulfate, and _the organic phase was dried over _Na₂SO₄ and concentrated in vacuo. The resulting crude product was diluted with diethyl ether and decanted to yield the title compound (3.7 g, 78%) as a white solid.

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.42(s,9H),1.04-1.74(m,2H),1.80-1.88(m,2H),2.89(br.s.,2H),4.04-4.12(m,2H),4.23-4.32(m,1H),4.73 (s,2H),7.83(dd,J＝7.5,0.8Hz,1H),7.91(dd,J＝7.5,0.8Hz,1H),8.17(dd,J＝7.7,0.8Hz,1H),16.03(br.s.,1H).

Step p

4-(7-Carbamoyl-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-1-carboxylic acid tert-butyl ester (XX) [n = 0; R1 = piperidin-4-yl; X = tert-butoxycarbonyl]

Method A: To a solution of 2-(1-tert-butoxycarbonyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII) (3.7 g, 10.3 mmol) in N,N-dimethylformamide (60 mL) was added hydroxybenzotriazole ammonium salt (3.15 g, 20.7 mmol), 1-ethyl-3-(3'-dimethylamino)carbodiimide hydrochloride (3.34 g, 20.7 mmol), and diisopropylethylamine (5.3 mL, 30.9 mmol). The reaction mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure, and the residue was dissolved in ethyl acetate. The solution was washed twice with saturated aqueous sodium carbonate solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography (dichloromethane/methanol 97:3) to afford the title compound (2.74 g, 74%) as a white solid.

Method B: A solution of 2-(1-tert-Butoxycarbonyl-piperidin-4-yl)-2,3-dihydro-1H-isoindole-4-carboxylic acid (XVIII) (5.5 g, 15.3 mmol) and carbonyldiimidazole (3.7 g, 22.8 mmol) in anhydrous tetrahydrofuran (80 mL) was stirred at room temperature for 4 h. Concentrated aqueous ammonia (25 mL) was then added, and the reaction mixture was allowed to stand at room temperature until the starting material disappeared (3 h). The solvent was evaporated under reduced pressure, and the crude primary amide (1.1 g, 20%) was used without any further purification.

HRMS (ESI+): Calcd. for C ₁₉ H ₂₆ N ₃ O ₄ [M+H] ⁺ 360.1918; Found 360.1921

Working up according to Method A, but using appropriately substituted starting materials, the following compounds were obtained:

2-Benzyl-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 1

[R＝H; n＝1; R1＝phenyl; m＝0; R2＝absent]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.72-1.88(m,4H),2.04-2.13(m,2H),2.88-2.96(m,2H),3.51(s,2H),4.00-4.11(m,1H),4.56(s,2H),7.20-7.30(m,1H),7.31-7.3 7(m,4H),7.66(br.s.,1H),7.71(dd,J＝7.6,7.4Hz,1H),7.76(dd,J＝7.6,1.5Hz,1H),8.20(dd,J＝7.4,1.5Hz,1H),10.72(br.s.,1H).

HRMS (ESI+): Calcd. for C ₁₆ H ₁₅ N ₂ O ₂ [M+H] ⁺ 267.1128; Found 267.1120

3-Oxo-2-phenylethyl-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 2

[R＝H; n＝2; R1＝phenyl; m＝0; R2＝absent]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 2.97(t,J＝7.6Hz,2H),3.82(t,J＝7.6Hz,2H),4.49(s,2H),7.17-7.24(m,1H),7.24-7.32(m,4H),7.66(br.s., 1H), 7.70 (dd, J＝7.5, 7.3Hz, 1H), 7.74 (dd, J＝7.5, 1.5Hz, 1H), 8.19 (dd, J＝7.3, 1.5Hz, 1H), 10.68 (br.s., 1H).

HRMS (ESI+): Calcd. for C ₁₇ H ₁₅ N ₂ O ₂ [M+H] ⁺ 281.1285; Found 281.1295

2-[2-(3,4-dihydro-1H-isoquinolin-2-yl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 3

[R＝H; n＝2; R1＝3,4-dihydro-1H-isoquinolin-2-yl; m＝0; R2＝absent]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 2.73-2.84(m,6H),3.65(s,2H),3.81(t,J＝6.2Hz,2H),4.65(s,2H),7.00-7.12(m,4H),7.66(br.s.,1H), 7.69(dd,J＝7.6,7.7Hz,1H),7.76(dd,J＝7.6,1.2Hz,1H),8.19(dd,J＝7.7,1.2Hz,1H),10.75(br.s.,1H).

HRMS (ESI+): Calcd. for C ₂₀ H ₂₃ N ₃ O ₂ [M+H] ⁺ 336.1707; Found 336.1722

3-Oxo-2-(2-piperidin-1-yl-ethyl)-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 4

[R＝H; n＝2; R1＝piperidin-1-yl; m＝0; R2＝absent]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.33-1.40(m,2H),1.43-1.50(m,4H),2.35-2.43(m,4H),2.54(t,J＝6.3Hz,2H),3.68(t,J＝6.3Hz,2H),4.63(s,2H),7.6 6(br.s.,1H),7.72(dd,J＝7.7,7.4Hz,1H),7.78(dd,J＝7.4,1.2Hz,1H),8.20(dd,J＝7.7,1.2Hz,1H),10.75(br.s.,1H).

HRMS (ESI+): Calcd. for C ₁₆ H ₂₂ N ₃ O ₂ [M+H] ⁺ 288.1707; Found 288.1712

2-(2-Morpholin-4-yl-ethyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 5

[R＝H; n＝2; R1＝morpholin-4-yl; m＝0; R2＝absent]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 2.41-2.46(m,4H),2.59(t,J＝6.3Hz,2H),3.52-3.57(m,4H),3.71(t,J＝6.3Hz,2H),4.64(s,2H),7.66(br.s., 1H), 7.72 (dd, J＝7.7, 7.6Hz, 1H), 7.78 (dd, J＝7.6, 1.3Hz, 1H), 8.20 (dd, J＝7.7, 1.3Hz, 1H), 10.73 (br.s., 1H).

HRMS (ESI+): Calcd. for C ₁₅ H ₂₀ N ₃ O ₃ [M+H] ⁺ 290.1499; Found 290.1507

2-(3-Morpholin-4-yl-propyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 6

[R＝H; n＝3; R1＝morpholin-4-yl; m＝0; R2＝absent]

¹ H NMR (400.5 MHz, DMSO-d ₆ ) δ ppm 1.80 (quintet, J = 7.1 Hz, 2H), 2.28-2.38 (m, 6H), 3.47-3.54 (m, 4H), 3.61 (t, J = 7.1 Hz, 2H), 4.58 (s, 2H), 7.65 (br. s., 1H), 7.71 (dd, J = 7.6, 7.4 Hz, 1H), 7.77 (dd, J = 7.4, 1.2 Hz, 1H), 8.20 (dd, J = 7.6, 1.2 Hz, 1H), 10.76 (br. s., 1H).

HRMS (ESI+): Calcd. for C ₁₆ H ₂₂ N ₃ O ₃ [M+H] ⁺ 304.1656; Found 304.1664

2-[2-(3,4-Dihydro-2H-quinolin-1-yl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 7

[R＝H; n＝2; R1＝3,4-dihydro-2H-quinolin-1-yl; m＝0; R2＝absent]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.79-1.89(m,2H),2.64-2.70(m,2H),3.27-3.31(m,2H),3.56(t,J＝7.1Hz,2H),3.76(t,J＝7.1Hz,2H),4.65(s,2H),6.44-6.49(m, 1H),6.70-6.75(m,1H),6.85-6.89(m,1H),6.92-6.97(m,1H),7.69(br.s.,1H),7.72(dd,J＝7.6,7.6Hz,1H),7.77(dd,J＝7.6,1.3Hz 1H), 8.20 (dd, J＝7.6, 1.3Hz, 1H), 10.68 (br.s., 1H).

HRMS (ESI+): Calcd. for C ₂₀ H ₂₂ N ₃ O ₂ [M+H] ⁺ 336.1707; Found 336.1692

3-Oxo-2-(3-phenyl-propyl)-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 17

[R＝H; n＝3; R1＝phenyl; m＝0; R2＝absent]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.97 (quintet, J=7.9Hz, 2H), 2.64 (t, J=7.9Hz2H), 3.61 (t, J=7.9Hz, 2H), 4.59 (s, 2H), 7.15-7.20 (m, 1H), 7.23-7.31 (m, 4H), 7 .67(br.s.,1H),7.72(dd,J＝7.6,7.4Hz,1H),7.77(dd,J＝7.6,1.5Hz,1H),8.21(dd,J＝7.4,1.5Hz,1H),10.74(br.s.,1H).

HRMS (ESI+): Calcd. for C ₁₈ H ₁₉ N ₂ O ₂ [M+H] ⁺ 295.1441; Found 295.1433

3-Oxo-2-(2-pyridin-2-yl-ethyl)-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 18

[R＝H; n＝2; R1＝pyridin-2-yl; m＝0; R2＝absent]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 3.12(t,J＝7.3Hz,2H),3.96(t,J＝7.3Hz,2H),4.52(s,2H),7.23(ddd,J＝7. 5,4.9,1.2Hz,1H),7.32(ddd,J＝7.8,1.2,0.8Hz,1H),7.65(br.s.,1H),7.7 1(m,1H),7.70(dd,J＝7.6,7.4Hz,1H),7.75(dd,J＝7.6,1.3Hz,1H),8.19(d d,J＝7.4,1.3Hz,1H),8.48(ddd,J＝4.9,1.8,0.8Hz,1H),10.66(br.s.,1H).

HRMS (ESI+): Calcd. for C ₁₆ H ₁₆ N ₃ O ₂ [M+H] ⁺ 282.1237; Found 282.1243

2-[3-(3,4-Dihydro-1H-isoquinolin-2-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 19

[R＝H; n＝3; R1＝3,4-dihydro-1H-isoquinolin-2-yl; m＝0; R2＝absent]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.92 (quintet, J = 7.3Hz, 2H), 2.52 (t, J = 7.3Hz, 2H), 2.64-2.70 (m, 2H), 2.76-2.82 (m, 2H), 3.57 (s, 2H), 3.64 (t, J = 7.3Hz, 2H), 4.60 (s, 2H), 7.0 0-7.70(m,4H),7.66(br.s.,1H),7.70(dd,J＝7.6,7.4Hz,1H),7.75(dd,J＝7.4,1.3Hz,1H),8.19(dd,J＝7.6,1.3Hz,1H),10.76(br.s.,1H).

HRMS (ESI+): Calcd. for C ₂₁ H ₂₄ N ₃ O ₂ [M+H] ⁺ 350.1863; Found 350.1866

2-[3-(3,4-Dihydro-2H-quinolin-1-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 20

[R＝H; n＝3; R1＝3,4-dihydro-2H-quinolin-1-yl; m＝0; R2＝absent]

¹ H NMR (400.5 MHz, DMSO-d ₆ ) δ ppm 1.80-1.87 (m, 2H), 1.90 (quintet, J = 7.2 Hz, 2H), 2.65 (t, J = 7.2 Hz, 2H), 3.22-3.26 (m, 2H), 3.27-3.30 (m overlaps with water signal, 2H), 3.64 (t, J = 7.2 Hz, 2H), 4.59 (s, 2H), 6.42-6.47 (m, 1H), 6.54- 6.58(m,1H),6.83-6.87(m,1H),6.89-6.94(m,1H),7.67(br.s.,1H),7.72(dd,J＝7.6,7 .6Hz,1H),7.77(dd,J＝7.6,1.3Hz,1H),8.20(dd,J＝7.6,1.3Hz,1H),10.72(br.s.,1H).

HRMS (ESI+): Calcd. for C ₂₁ H ₂₄ N ₃ O ₂ [M+H] ⁺ 350.1863; Found 350.1868

2-[3-(4-Methyl-piperazin-1-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 21

[R＝H; n＝3; R1＝4-methyl-piperazin-1-yl; m＝0; R2＝absent]

¹ H NMR (400.5 MHz, DMSO-d ₆ ) δ ppm 1.80 (quintet, J = 7.2 Hz, 2H), 2.11 (s, 3H), 2.15-2.43 (br. s., 8H), 2.33 (t, J = 7.2 Hz, 2H), 3.60 (t, J = 7.2 Hz, 2H), 4.58 (s, 2H), 7.66 (br. s., 1H), 7.72 (dd, J = 7.6, 7.4 Hz, 1H), 7.77 (dd, J = 7.6, 1.3 Hz, 1H), 8.21 (dd, J = 7.4, 1.3 Hz, 1H), 10.79 (br. s., 1H).

HRMS (ESI+): Calcd. for C ₁₇ H ₂₅ N ₄ O ₂ [M+H] ⁺ 317.1972; Found 317.1975

3-Oxo-2-[3-(4-phenyl-piperazin-1-yl)-propyl]-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 22

[R＝H; n＝3; R1＝piperazin-1-yl; m＝0; R2＝4-phenyl]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.85 (quintet, J=7.1Hz, 2H), 2.39 (t, J=7.1Hz, 2H), 3.02-3.10 (m, 4H), 3.63 (t ,J＝7.1Hz,2H),4.60(s,2H),6.75(t,J＝7.3Hz,1H),6.89(d,J＝7.9Hz,2H),7 .19(dd,J＝7.9,7.3Hz,2H),7.66(br.s.,1H),7.70(dd,J＝7.7,7.4Hz,1H),7 .76(dd,J＝7.4,1.2Hz,1H),8.20(dd,J＝7.7,1.2Hz,1H),10.78(br.s.,1H).

HRMS (ESI+): Calcd. for C _{2 2} H _{2 7} N ₄ O ₂ [M+H] ⁺ 379.2129; Found 379.2145

3-Oxo-2-(3-piperidin-1-yl-propyl)-2,3-dihydro-1H-isoindole-4-carboxamide hydrochloride (I), cpd25

[R＝H; n＝3; R1＝piperidin-1-yl; m＝0; R2＝absent]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.29(m,1H),1.50-1.73(m,3H),1.75-1.85(m,2H),2.00-2.09(m,2H),2.79 -2.92(m,2H),3.03-3.14(m,2H),3.40-3.50(m,2H),3.66(t,J＝6.6Hz,2H),4 .59(s,2H),7.71(br.s.,1H),7.74(dd,J＝7.6,7.4Hz,1H),7.80(dd,J＝7.4,1 .1Hz,1H),8.21(dd,J＝7.6,1.1Hz,1H),8.93(br.s.,1H),10.58(br.s.,1H).

HRMS (ESI+): Calcd. for C ₁₇ H ₂₄ N ₃ O ₂ [M+H] ⁺ 302.1863; Found 302.1865

2-(3-[1,4']bipiperidin-1'-yl-propyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide dihydrochloride (I), cpd 26

[R＝H; n＝3; R1＝piperidin-1-yl; m＝0; R2＝4-piperidin-1-yl]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.33-1.49(m,1H),1.57-1.74(m,3H),1.74-1.94(m,4H),1.95-2.13(m, 2H),2.16-2.30(m,2H),2.87-3.72(m,11H),3.66(t,J＝6.5Hz,2H),4.59 (s,2H),7.72(br.s.,1H),7.75(dd,J＝7.6,6.7Hz,1H),7.80(d,J＝6.7Hz,1H),8.21(dd,J＝7.6,1.2Hz,1H),9.38(br.s.,2H),10.58(br.s.,1H).

HRMS (ESI+): Calcd. for C _{2 2} H _{3 3} N ₄ O ₂ [M+H] ⁺ 385.2598; Found 385.2611

2-[3-(2,6-Dimethyl-piperidin-1-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide hydrochloride (I), cpd 27

[R＝H; n＝3; R1＝2,6-dimethyl-piperidin-1-yl; m＝0; R2＝absent]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.25(d,J＝6.3Hz,6H),1.40-1.55(m,3H),1.80-1.91(m,2H),1.95-2.05(m,2H),3.02-3.46(m,4H),3.68(m,2H),4.63(s,2H),7.70(b r.s.,1H),7.74(dd,J＝7.6,7.6Hz,1H),7.80(dd,J＝7.6,1.2Hz,1H),8.21(dd,J＝7.6,1.2Hz,1H),8.72(br.s.,1H),10.58(br.s.,1H).

HRMS (ESI+): Calcd. for C ₁₉ H ₂₈ N ₃ O ₂ [M+H] ⁺ 330.2176; Found 330.2176

3-Oxo-2-[1-(tetrahydro-pyran-4-yl)-piperidin-4-yl]-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 28

[R＝H; n＝m＝0; R1＝piperidin-4-yl; R2＝1-(tetrahydro-pyran-4-yl)]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.38-1.50 (m, 2H), 1.65-1.72 (m, 2H), 1.73-1.81 (m, 4H), 2.18-2.28 (m, 2H), 2.43-2.47 (m, 1H), 2.97-3.04 (m, 2H), 3.30 (m overlaps with water signal, 2H), 3.89 (dd, J = 11.1, 3.9 Hz, 2H), 4.02 (m, 1H), 4.55 (s, 2H), 7.66 (br. s., 1H), 7.71 (dd, J = 7.6, 7.4 Hz, 1H), 7.76 (dd, J = 7.4, 1.5 Hz, 1H), 8.20 (dd, J = 7.6, 1.5 Hz, 1H), 10.74 (br. s., 1H).

HRMS (ESI+): Calcd. for C ₁₉ H ₂₆ N ₃ O ₃ [M+H] ⁺ 344.1969; Found 344.1962

2-(1-Benzyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 30

[R＝H; n＝0; R1＝piperidin-4-yl; m＝1; R2＝phenyl]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.70-1.88(m,4H),1.99-2.13(m,2H),2.89-2.96(m,2H),3.51(s,2H),4.00-4.11(m,1H),4.56(s,2H),7.20-7.30(m,1H),7.30-7.3 6(m,4H),7.66(br.s.,1H),7.71(dd,J＝7.6,7.4Hz,1H),7.76(dd,J＝7.6,1.5Hz,1H),8.20(dd,J＝7.4,1.5Hz,1H),10.72(br.s.,1H).

HRMS (ESI+): Calcd. for C ₂₁ H ₂₄ N ₃ O ₂ [M+H] ⁺ 350.1863; Found 350.1874

2-[2-(1-Benzyl-piperidin-4-yl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide hydrochloride (I), cpd 31

[R＝H; n＝2; R1＝piperidin-4-yl; m＝1; R2＝phenyl]

¹ H NMR (400.5 MHz, DMSO-d ₆ ) δ ppm 1.29-1.41 (m, 2H), 1.43-1.54 (m, 1H), 1.56-1.64 (m, 2H), 1.92-2.00 (m, 2H), 2.83-2.96 (m, 2H), 3.30 (m overlaps with water signal, 2H), 3.61 (t, J = 6.8 Hz, 2H), 4.25 (d, J = 5.1 Hz, 2H), 4. 57(s,2H),7.47(s,5H),7.69(br.s.,1H),7.72(dd,J＝7.6,7.4Hz,1H),7.77(dd,J＝ 7.4,1.3Hz,1H),8.20(dd,J＝7.6,1.3Hz,1H),9.22(br.s.,1H),10.68(br.s.,1H).

HRMS (ESI+): Calcd. for C ₂₃ H ₂₈ N ₃ O ₂ [M+H] ⁺ 378.2176; Found 378.2178

2-[3-(4-Benzyl-piperidin-1-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 32

[R＝H; n＝3; R1＝piperidin-1-yl; m＝1; R2＝phenyl]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 0.93-1.12(m,2H),1.34-1.53(m,3H),1.68-1.82(m,4H),2.22-2.33(m,2H),2.41(d ,J＝6.8Hz,2H),2.75-2.85(m,2H),3.59(t,J＝6.9Hz,2H),4.56(s,2H),7.09-7.14(m ,2H),7.14-7.19(m,1H),7.23-7.29(m,2H),7.65(br.s.,1H),7.72(dd,J＝7.4,7.4H z,1H),7.77(dd,J＝7.4,1.3Hz,1H),8.21(dd,J＝7.4,1.3Hz,1H),10.78(br.s.,1H).

HRMS (ESI+): Calcd. for C ₂₄ H ₃₀ N ₃ O ₂ [M+H] ⁺ 392.2333; Found 392.2346

Step i’

3-Oxo-2-piperidin-4-yl-2,3-dihydro-1H-isoindole-4-carboxamide hydrochloride (XXI)

[n＝0; R1＝piperidin-4-yl]

A solution of tert-butyl 4-(7-carbamoyl-1-oxo-1,3-dihydro-isoindol-2-yl)-piperidine-1-carboxylate (XX) (2.7 g, 7.5 mmol) in 4M hydrochloric acid in dioxane (18 mL, 75 mmol) was stirred at 50°C for 2 h until HPLC analysis revealed the disappearance of the starting material. The solvent was removed under reduced pressure, and the product was dissolved in diethyl ether and filtered to afford the title compound (2.09 g, 95%) as its hydrochloride salt.

¹ H NMR (400.5 MHz, DMSO-d ₆ ) δ ppm 1.93-2.09 (m, 4H), 3.03-3.17 (m, 2H), 3.35-3.48 (m overlaps with water signal, 2H), 4.32-4.45 (m, 1H), 4.56 (s, 2H), 7.71 (br. s., 1H), 7.75 (dd, J=7.5, 7.5 Hz, 1H), 7.82 (dd, J=7.5, 1.1 Hz, 1H), 8.21 (dd, J=7.5, 1.1 Hz, 1H), 8.59 (br. s., 1H), 8.82 (br. s., 1H), 10.58 (br. s., 1H).

HRMS (ESI+): Calcd. for C ₁₄ H ₁₈ N ₃ O ₂ [M+H] ⁺ 260.1394; Found 260.1398

Step 1'

2-(1-Cyclohexyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 11

[R＝H; n＝m＝0; R1＝piperidin-4-yl; R2＝1-cyclohexyl]

Method A: To a suspension of 3-oxo-2-piperidin-4-yl-2,3-dihydro-1H-isoindole-4-carboxamide hydrochloride (56 mg, 0.19 mmol) in dichloromethane (2 mL) was added cyclohexanone (XIV) (27.5 mg, 0.28 mmol), sodium acetate (32 mg, 0.38 mmol), and methanol (0.3 mL). The resulting solution was stirred at room temperature for 5 h. Sodium cyanoborohydride (13 mg, 0.21 mmol) was then added, and the mixture was stirred overnight. The solvent was removed under reduced pressure, and the residue was dissolved in dichloromethane and washed _twice with water. The organic phase was dried over _Na₂SO₄ and concentrated in vacuo. The residue was purified by flash chromatography (dichloromethane/methanol 95:5) to yield 27 mg (40%) of 2-(1-cyclohexyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide.

Method B: To a solution of 2-piperidin-4-yl-2,3-dihydro-1H-isoindole-4-carboxamide hydrochloride (4.4 g, 14.8 mmol) and cyclohexanone (2.2 g, 22.45 mmol) in N,N-dimethylformamide (100 mL) were added glacial acetic acid (4.5 mL) and tetramethylammonium triacetoxyborohydride (11.8 g, 44.85 mmol). The resulting solution was stirred at room temperature overnight.

The solvent was then evaporated under reduced pressure, the resulting residue was diluted with 8% aqueous ammonia solution and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by flash chromatography (dichloromethane/methanol 95:5), then dissolved in a small amount of methanol and precipitated with diethyl ether. The precipitate was filtered and washed with diethyl ether to give 1.77 g of the desired product as a white solid (35%).

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.00-1.14(m,1H),1.14-1.32(m,4H),1.55-1.62(m,1H),1.70-1.80(m,8H),2.25-2.37(m,3H),2.88-2.98(m,2H),3.95-4.06(m,1H),4 .55(s,2H),7.66(br.s.,1H),7.71(dd,J＝7.6,7.6Hz,1H),7.76(dd,J＝7.6,1.5Hz,1H),8.20(dd,J＝7.6,1.5Hz,1H),10.74(br.s.,1H).

HRMS (ESI+): Calcd. for C ₂₀ H ₂₈ N ₃ O ₂ [M+H] ⁺ 342.2176; Found 342.2175

Working according to Method A, but using the appropriately substituted starting material (XIV), the following compounds were obtained:

3-Oxo-2-(1-pyridin-4-ylmethyl-piperidin-4-yl)-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 8

[R＝H; n＝0; R1＝piperidin-4-yl; m＝1; R2＝pyridin-4-yl]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.75-1.92(m,4H),2.11-2.22(m,2H),2.88-2.95(m,2H),3.56(s,2H),4.02-4.14(m,1H),4.58(s,2H),7.33-7.38(m,2H),7.68(br .s.,1H),7.73(dd,J＝7.6,7.6Hz,1H),7.78(dd,J＝7.6,1.51H),8.21(dd,J＝7.6,1.4Hz,1H),8.51-8.55(m,2H),10.72(br.s.,1H).

HRMS (ESI+): Calcd. for C ₂₀ H ₂₃ N ₄ O ₂ [M+H] ⁺ 351.1816; Found 351.1817

3-Oxo-2-(1-thiophen-2-ylmethyl-piperidin-4-yl)-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 9

[R＝H; n＝0; R1＝piperidin-4-yl; m＝1; R2＝thiophen-2-yl]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.73-1.88(m,4H),2.06-2.20(m,2H),2.94-3.03(m,2H),3.73(s,2H),4.01-4.11(m,1H),4.58(s,2H),6.96-7.00(m,2H),7.42-7.4 6(m,1H),7.67(br.s.,1H),7.72(dd,J＝7.6,7.4Hz,1H),7.77(dd,J＝7.6,1.5Hz,1H),8.21(dd,J＝7.4,1.5Hz,1H),10.72(br.s.,1H).

HRMS (ESI+): Calcd. for C ₁₉ H ₂₂ N ₃ O ₂ S [M+H] ⁺ 356.1427; Found 356.1430

3-Oxo-2-(1-pyridin-3-ylmethyl-piperidin-4-yl)-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 10

[R＝H; n＝0; R1＝piperidin-4-yl; m＝1; R2＝pyridin-3-yl]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.70-1.80(m,4H),2.08-2.18(m,2H),2.87-2.96(m,2H),3.55(s,2H),4.01-4.12(m,1H),4.56(s,2H),7.37(dd,J＝7.7,4.8Hz 1H), 7.66 (br.s., 1H), 7.71 (t, J = 7.7, 7.4 Hz, 1H), 7.73 (the signal overlaps with other signals, 1H), 7.76 (dd, J = 7.7, 1.3 Hz, 1H), 8.20 (dd, J = 7.4, 1.3 Hz, 1H), 8.48 (d, J = 4.8 Hz, 1H), 8.52 (s, 1H), 10.71 (br.s., 1H).

HRMS (ESI+): Calcd. for C ₂₀ H ₂₃ N ₄ O ₂ [M+H] ⁺ 351.1816; Found 351.1822

2-(1-Furan-2-ylmethyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 12

[R＝H; n＝0; R1＝piperidin-4-yl; m＝1; R2＝fur-2-yl]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.70-1.87(m,4H),2.07-2.16(m,2H),2.90-2.97(m,1H),3.53(s,2H),3. 98-4.06(m,1H),4.55(s,2H),6.30(d,J＝2.4Hz,1H),6.41(dd,J＝2.4,1.8H z,1H),7.59(br.s.,1H),7.66(br.s.,1H),7.71(dd,J＝7.6,7.4Hz,1H),7.76(dd,J＝7.4,1.3Hz,1H),8.19(dd,J＝7.6,1.3Hz,1H),10.71(br.s.,1H).

HRMS (ESI+): Calcd. for C ₁₉ H ₂₂ N ₃ O ₃ [M+H] ⁺ 340.1656; Found 340.1651

3-Oxo-2-(1-thiophen-3-ylmethyl-piperidin-4-yl)-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 13

[R＝H; n＝0; R1＝piperidin-4-yl; m＝1; R2＝thiophen-3-yl]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.72-1.90(m,4H),2.00-2.12(m,2H),2.90-2.99(m,2H),3.53(s,2H),4. 00-4.09(m,1H),4.57(s,2H)7.08(d,J＝4.6Hz,1H),7.33(br.s.,1H),7.4 9(dd,J＝4.6,2.8Hz,1H),7.67(br.s.,1H),7.72(dd,J＝7.6,7.4Hz,1H),7.75(dd,J＝7.6,1.3,1H),8.21(dd,J＝7.4,1.3Hz,1H),10.73(br.s.,1H).

HRMS (ESI+): Calcd. for C ₁₉ H ₂₂ N ₃ O ₂ S [M+H] ⁺ 356.1427; Found 356.1432

2-(1-Furan-3-ylmethyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 14

[R＝H; n＝0; R1＝piperidin-4-yl; m＝1; R2＝fur-3-yl]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.72-1.89(m,4H),2.02-2.12(m,2H),2.90-3.02(m,2H),3.37(s,2H),4.00-4.10(m,1H),4.56(s,2H),6.45(s,1H),7.58(s,1H),7.6 2(s,1H),7.67(br.s.,1H),7.72(dd,J＝7.6,7.4Hz,1H),7.76(dd,J＝7.4,1.5Hz,1H),8.21(dd,J＝7.6,1.5Hz,1H),10.73(br.s.,1H).

HRMS (ESI+): Calcd. for C ₁₉ H ₂₂ N ₃ O ₃ [M+H] ⁺ 340.1656; Found 340.1649

3-Oxo-2-(1-pyridin-2-ylmethyl-piperidin-4-yl)-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 15

[R＝H; n＝0; R1＝piperidin-4-yl; m＝1; R2＝pyridin-2-yl]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.74-1.93(m,4H),2.12-2.28(m,2H),2.91-3.00(m,2H),3.65(s,2H),4 .02-4.13(m,1H),4.58(s,2H),7.28(dd,J＝6.8,4.8Hz,1H),7.47(d,J＝7. 8Hz,1H),7.67(br.s.,1H),7.73(dd,J＝7.4,7.4Hz,1H),7.76-7.83(m,2H),8.21(dd,J＝7.4,1.3Hz,1H),8.51(d,J＝4.8Hz,1H),10.73(br.s.,1H).

HRMS (ESI+): Calcd. for C ₂₀ H ₂₃ N ₄ O ₂ [M+H] ⁺ 351.1816; Found 351.1815

3-Oxo-2-[1-(1H-pyrrol-2-ylmethyl)-piperidin-4-yl]-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd 16

[R＝H; n＝0; R1＝piperidin-4-yl; m＝1; R2＝1H-pyrrol-2-yl]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 1.70-1.88(m,4H),1.96-2.09(m,2H),2.88-2.99(m,2H),3.44(s,2H),3.9 4-4.09(m,1H),4.55(s,2H),5.89(br.s.,1H),5.94(br.s.,1H),6.65(br.s .,1H),7.67(br.s.,1H),7.72(dd,J＝7.6,7.4Hz,1H),7.77(dd,J＝7.6,1.3 Hz,1H),8.20(dd,J=7.4,1.3Hz,1H),10.65(br.s.,1H),10.73(br.s.,1H).

HRMS (ESI+): Calcd. for C ₁₉ H ₂₃ N ₄ O ₂ [M+H] ⁺ 339.1816; Found 339.1812

2-(1-Cyclopropylmethyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (I), cpd24

[R＝H; n＝0; R1＝piperidin-4-yl; m＝1; R2＝cyclopropyl]

^1H NMR (400.5MHz, DMSO-d ₆ )δppm 0.06-0.12(m,2H),0.44-0.50(m,2H),0.80-0.89(m,1H),1.72-1.88(m,4 H),2.00-2.11(m,2H),2.21(d,J＝6.3Hz,2H),3.04-3.13(m,2H),3.98-4. 09(m,1H),4.56(s,2H),7.66(br.s.,1H),7.72(dd,J＝7.6,7.6Hz,1H),7.77(dd,J＝7.6,1.2Hz,1H),8.20(dd,J＝7.6,1.2Hz,1H),10.73(br.s.,1H).

HRMS (ESI+): Calcd. for C ₁₈ H ₂₄ N ₃ O ₂ [M+H] ⁺ 314.1863; Found 314.1860

Claims (14)

1. Compounds of formula (I): Or its pharmaceutically acceptable salt, in: R is either hydrogen or fluorine; and n, m, R1, and R2 have the following meanings: a) n is 0 and m is 0 or 1; R1 is piperidinyl; and R2 is cyclopropyl or cyclohexyl; Or c) n is 2 or 3 and m is 0; R1 is a 4- to 6-membered heterocyclic, aryl, or heteroaryl group, each optionally further substituted with one or more straight-chain or branched ( _C1 - _C6 )-alkyl groups; and R2 does not exist; or When R is hydrogen, n is 0 and m is 1; R1 is piperidinyl; and R2 is pyrrole or pyridinyl. 2. The compound of formula (I) according to claim 1, characterized in that... c) n is 2 or 3, and m is 0; R1 is a 6-membered heterocyclic group, optionally further substituted with one or more straight-chain or branched ( _C1 - _C6 )-alkyl groups; and R2 does not exist. 3. The compound of formula (I) according to claim 1, characterized in that: a) n is 0 and m is 0 or 1; When m is 0, R1 is piperidinyl and R2 is cyclohexyl ring; When m is 1, R is hydrogen, R1 is piperidinyl, and R2 is a pyridine ring. 4. Compounds of formula (I) selected from: 3-Oxo-2-phenylethyl-2,3-dihydro-1H-isoindole-4-carboxamide; 2-[2-(3,4-dihydro-1H-isoquinoline-2-yl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide; 3-O-2-(2-piperidin-1-yl-ethyl)-2,3-dihydro-1H-isoindole-4-carboxamide; 2-(2-morpholin-4-yl-ethyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide; 2-(3-morpholin-4-yl-propyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide; 2-[2-(3,4-dihydro-2H-quinolin-1-yl)-ethyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide; 2-(1-Cyclohexyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide; 3-O-2-(1-pyridin-2-ylmethyl-piperidin-4-yl)-2,3-dihydro-1H-isoindole-4-carboxamide; 3-Oxo-2-[1-(1H-pyrrolo-2-ylmethyl)-piperidin-4-yl]-2,3-dihydro-1H-isoindole-4-carboxamide; 2-[3-(3,4-dihydro-1H-isoquinoline-2-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide; 2-[3-(3,4-dihydro-2H-quinolin-1-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide; 2-[3-(4-methyl-piperazin-1-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide; 6-Fluoro-2-(3-morpholino-4-yl-propyl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide; 2-(1-Cyclopropylmethyl-piperidin-4-yl)-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide; 3-O-2-(3-piperidin-1-yl-propyl)-2,3-dihydro-1H-isoindole-4-carboxamide; 2-[3-(2,6-dimethyl-piperidin-1-yl)-propyl]-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide; and 2-(1-Cyclohexyl-piperidin-4-yl)-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide; Or its pharmaceutically acceptable salt. 5. A method for preparing the compound of formula (I) of claim 1, the method comprising one of the following steps in sequence: Sequence A: Step h) involves cyclization with a suitable amine of formula (XIII) to obtain a compound of formula (V): Where T is ( _C1 - _C6 )-alkyl or aryl-( _C1 - _C6 )-alkyl, and suitable amines of formula (XIII) are: X-R1-[ _CH2 ] _n - _NH2 (XIII) Wherein R1 and n are as defined in claim 1, and X is R2-[ _CH2 ] _m- , where R2 and m are as defined in claim 1; or when R1 is a nitrogen-containing heterocyclic group, X is a suitable nitrogen protecting group; The compound of formula (IV) obtained by hydrolysis in step c’) is: Where R1, n, and X are defined as above, so that we can obtain: The compound of formula (I) as defined in claim 1, wherein X is R2-[ _CH2 ] _m- , where R2 and m are as defined in claim 1; or The compound of formula (III), where R1 is a nitrogen-containing heterocyclic group and X is a suitable nitrogen protecting group, Where n is defined above, R1 is a nitrogen-containing heterocyclic group, and X is a suitable nitrogen protecting group; Step i) Deprotect the compound of formula (III) as defined above to obtain: Compounds of formula (I) as defined above; or Compounds of formula (II): Where R1 and n are as defined above; Step 1) Alkylate the resulting compound of formula (II) as defined above using a suitable alkylating agent of formula (XIV). R2-[ _CH2 ] _m'-1 -Y (XIV) Where Y is a formyl group; or when m’＝1, in the case of compound (XIV) being R2-Y, Y is attached to the oxygen atom (＝O) of R2 via a double bond to obtain the compound of formula (I) as defined above; or Sequence B: Step m) Reductive amination of furan-2-carbaldehyde (XV) with a suitable amine of formula (XIII): X-R1-[ _CH2 ] _n - _NH2 (XIII) Where R1 and n are as defined above, and X is R2-[ _CH2 ] _m- , where R2 and m are as defined above; or when R1 is a nitrogen-containing heterocyclic group, X is a suitable nitrogen protecting group; Step n) The resulting compound of formula (XVI) is subjected to a Diels-Adel reaction: Where R1, n, and X are as defined above; Step o) Aromatize the resulting compound of formula (XVII): Where R1, n, and X are as defined above; Step p) amidates the resulting compound of formula (XVIII): Where R1, n, and X are defined as above, so as to obtain For compounds of formula (I) as defined above, X is R2-[ _CH2 ] _m- , where R2 and m are as defined above; or Compounds of formula (XX), where R1 is a nitrogen-containing heterocyclic group and X is a suitable nitrogen protecting group. Where n is defined above, R1 is a nitrogen-containing heterocyclic group, and X is a suitable nitrogen protecting group; Step i’) deprotects the compound of formula (XX) as defined above; Step 1') Alkylate the resulting compound of formula (XXI) with a suitable alkylating agent of formula (XIV): Where R1 and n are as defined above, a suitable alkylating agent for formula (XIV) is: R2-[ _CH2 ] _m'-1 -Y (XIV) Where Y is a formyl group; or when m’＝1, in the case of R2-Y in the compound of formula (XIV), Y is attached to the oxygen atom (＝O) of R2 by a double bond in order to obtain the compound of formula (I) as defined above; Furthermore, in the case of step o, the compound obtained by aromatization of the compound of formula (XVII) is the compound of formula (XIX), that is, when X is an unstable nitrogen protecting group, the following step q is performed: Step q) Install a suitable nitrogen protecting group on the resulting compound of formula (XIX): R1 and n are as defined above, in order to obtain a compound of formula (XVIII), wherein R1 and n are as defined above, and X is a suitable nitrogen protecting group, and then the above reactions p), i’) and l’) are carried out in sequence to obtain a compound of formula (I) as defined above. 6. An in vitro method for selectively inhibiting the activity of PARP-1 protein, comprising contacting the protein with an effective amount of a compound of formula (I) as defined in claim 1. 7. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient, carrier or diluent. 8. The pharmaceutical composition of claim 7 further comprises one or more chemotherapeutic agents. 9. The pharmaceutical composition of claim 8, wherein the chemotherapeutic agent is an alkylating agent. 10. The pharmaceutical composition of claim 9, wherein the alkylating agent is temozolomide. 11. A product comprising a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof and one or more chemotherapeutic agents, which are used as a combination formulation for simultaneous, single or sequential use in anticancer therapy. 12. The product of claim 11, wherein the chemotherapeutic agent is an alkylating agent. 13. The product of claim 12, wherein the alkylating agent is temozolomide. 14. Use of a compound of formula (I) as defined in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament having anticancer activity.