Bis(hydroxymethyl)pyrrolophthalazine hybrid, preparation method and use thereof

Abstract

The present invention relates to novel dual-functional compounds and their use in treating and / or preventing cancer. The dual-functional compounds of the present invention have the structure of formula (I).

Description

[0001] Related Applications

[0002] The present invention claims priority to provisional application No. 62 / 587,484 filed on November 17, 2017, pursuant to 35 U.S.C. § 119(e), the disclosure of which is incorporated herein by reference. Background of the Invention 1. Technical Field

[0004] The present invention generally relates to the field of cancer treatment; in particular, to novel dual-functional compounds that exhibit both anti-angiogenesis and DNA cross-linking activities, and their use in treating and / or preventing cancer. 2. Background technology

[0006] Cancer is one of the leading causes of death worldwide. Treatment of cancer usually involves surgical removal and / or administration of a chemotherapeutic agent or radiation therapy. Many compounds have been developed as anticancer agents, however, most drug developers design anticancer agents by targeting only a specific cellular mechanism of uncontrolled cell proliferation. Instead of focusing on inhibiting enzymes in a specific metabolic pathway, the inventors of this case created a compound with a dual-functional mode by joining two active moieties into one molecule, specifically, one moieties with anti-angiogenic activity and the other with DNA cross-linking activity. Accordingly, it can be expected that the hybrid molecule will become a more potential anticancer agent because it has two functional groups from two completely different pathways to attack cancer cells. Summary of the invention

[0007] The inventors of the present invention designed and synthesized a new class of compounds by combining VEGF and aurora kinase inhibitory clusters with DNA crosslinking clusters. These newly synthesized hybrid compounds exhibit significant antiproliferative activity in various human cancer cells, and therefore, these compounds are potential candidate drugs that can be developed into treatment and / or prevention of cancer.

[0008] Accordingly, one aspect of the present invention is to provide a novel compound having a structure of formula (I), and a pharmaceutically acceptable solvate or stereoisomer thereof,

[0009]

[0010] in,

[0011] A is an unsaturated carbon ring with 6-10 members which may be substituted;

[0012] R 1is an alkyl group which may be substituted with halogen, –OR, –OAc, –OSO2R or –OCONHR, wherein R is hydrogen, alkyl, cycloalkyl or aryl;

[0013] R 2 is hydrogen, alkyl, cycloalkyl, aryl or heteroaryl;

[0014] R 3 Yes – NR A R B , –NHPhR C , a mono- or disaccharide residue, or an amino acid residue, or -NR A R B The present invention relates to a cycloalkylamine or aniline with or without a substituent selected from the group consisting of morpholine, pyrrolidine, piperidine, 1-substituted piperazine, 1,4'-bipiperidine and 4'-substituted 4,4'-bipiperidine; wherein the substituents of piperazine and 4,4'-bipiperidine are alkyl, -(CH2) n CONH(CH2) m NR A R B ,in

[0015] n and m are each an integer between 1 and 5,

[0016] R A and R B are each H or C1-C6 alkyl; and

[0017] R C It is hydrogen, halogen, alkoxy, –CH2OH, –NHCOR a 、–NHC(O)OR a , or an alkyl, alkenyl, alkynyl, heterocyclic or aryl group which may be substituted, and R a It is C 1–6 an alkyl or aryl group; and

[0018] The aryl or heteroaryl group may or may not have at least one substituent, wherein the at least one substituent is selected from C- 1–6 Alkyl, alkoxy, halogen, cyano, nitro, –NH2, –NHR c , –N(R c) 2, a group consisting of cycloalkylamino, methylenedioxy and ethylenedioxy, wherein R c are independently hydrogen or C 1–10 alkyl.

[0019] According to certain embodiments of the present invention, in formula (I), R 3 It is 1H-pyrazol-3-amino, 1H-imidazol-2-amino or pyrimidine-amino.

[0020] According to certain embodiments of the present invention, in formula (I), the cycloalkylamino group is selected from the group consisting of pyrrolidinyl, piperidinyl, morpholino, piperazinyl and 4-piperidopiperidinyl.

[0021] According to certain embodiments of the present invention, a specific compound has a structure of formula (1-A),

[0022]

[0023] in,

[0024] R 1 is an alkyl group which may be substituted with halogen, –OR, –OAc, –OSO2R or –OCONHR, wherein R is hydrogen, alkyl, cycloalkyl or aryl;

[0025] R 2 is hydrogen, alkyl, cycloalkyl, aryl or heteroaryl;

[0026] R 3 Yes – NR A R B , –NHPhR C , a mono- or disaccharide residue, or an amino acid residue, or -NR A R B The phenylamine or cycloalkylamine is combined to form an aniline or cycloalkylamine with or without a substituent selected from the group consisting of morpholine, pyrrolidine, piperidine, 1-substituted piperazine, 1,4'-bipiperidine and 4'-substituted 4,4'-bipiperidine; wherein the substituents of piperazine and 4,4'-bipiperidine are alkyl, -(CH2) n CONH(CH2) m NR A R B ,in

[0027] n and m are each an integer between 1 and 5,

[0028] R A and R B are each H or C1-C6 alkyl; and

[0029] R C is hydrogen, halogen, alkoxy, –CH2OH, –NHCOR a 、–NHC(O)OR a , or an alkyl, alkenyl, alkynyl, heterocyclic or aryl group which may be substituted, and R a It is C 1–6 Alkyl or aryl;

[0030] R 4 and R 5 Each is hydrogen, –OH, alkoxy or –O(CH2) x N(R b )2, where

[0031] x is an integer between 1 and 5; and

[0032] R b It is C 1–10 Alkyl or monocycloalkylamino; and

[0033] The aryl or heteroaryl group may or may not have at least one substituent, wherein the at least one substituent is selected from C- 1–6 Alkyl, alkoxy, halogen, cyano, nitro, –NH2, –NHR c , –N(R c ) 2. a group consisting of cycloalkylamino, methylenedioxy and ethylenedioxy, wherein R c are independently hydrogen or C 1–10 alkyl.

[0034] According to a preferred embodiment, in formula (I-A), R 1 is a methyl group substituted with –OH, R 2 is ethyl, R 3 is morpholine, and R 4 and R 5 Each is hydrogen.

[0035] According to a preferred embodiment, in formula (I-A), R 1 is a methyl group substituted with –OH, R 2 is methyl, R 3 is pyrrolidine, and R 4 and R 5 Each is hydrogen.

[0036] According to another preferred embodiment, in formula (I-A), R 1is a methyl group substituted with –OH, R 2 is methyl, R 3 is 1,4'-bipiperidine, and R 4 and R 5 Each is hydrogen.

[0037] According to a further embodiment, in formula (IA), R 1 is a methyl group substituted by –OCONH(C2H5), R 2 is methyl, R 3 is pyrrolidine, and R 4 and R 5 Each is hydrogen.

[0038] According to certain embodiments of the present invention, a specific compound has a structure of formula (I-B),

[0039]

[0040] in,

[0041] R 1 is an alkyl group which may be substituted with halogen, –OR, –OAc, –OSO2R or –OCONHR, wherein R is hydrogen, alkyl, cycloalkyl or aryl;

[0042] R 2 is hydrogen, alkyl, cycloalkyl, aryl or heteroaryl;

[0043] R 3 Yes – NR A R B , –NHPhR C , a mono- or disaccharide residue, or an amino acid residue, or -NR A R B The phenylamine or cycloalkylamine is combined to form an aniline or cycloalkylamine with or without a substituent selected from the group consisting of morpholine, pyrrolidine, piperidine, 1-substituted piperazine, 1,4'-bipiperidine and 4'-substituted 4,4'-bipiperidine; wherein the substituents of piperazine and 4,4'-bipiperidine are alkyl, -(CH2) n CONH(CH2) m NR A R B ,in

[0044] n and m are each an integer between 1 and 5,

[0045] R A and R B are each H or C1-C6 alkyl; and

[0046] R C is hydrogen, halogen, alkoxy, –CH2OH, –NHCORa 、–NHC(O)OR a , or an alkyl, alkenyl, alkynyl, heterocyclic or aryl group which may be substituted, and R a It is C 1–6 Alkyl or aryl;

[0047] R 4 and R 5 Each is hydrogen, –OH, alkoxy or –O(CH2) x N(R b )2, where

[0048] x is an integer between 1 and 5; and

[0049] R b It is C 1–10 Alkyl or monocycloalkylamino; and

[0050] The aryl or heteroaryl group may or may not have at least one substituent, wherein the at least one substituent is selected from C- 1–6 Alkyl, alkoxy, halogen, cyano, nitro, –NH2, –NHR c , –N(R c ) 2. a group consisting of cycloalkylamino, methylenedioxy and ethylenedioxy, wherein R c are independently hydrogen or C 1–10 alkyl.

[0051] According to a preferred embodiment, in formula (I-B), R 1 is a methyl group substituted with –OH, R 2 is methyl, R 3 is dimethylamine, and R 4 and R 5 Each is hydrogen.

[0052] According to another embodiment, in formula (I-B), R 1 is a methyl group substituted by –OCONH(C2H5), R 2 is methyl, R 3 is dimethylamine, and R 4 and R 5 Each is hydrogen.

[0053] According to a further embodiment, in formula (IB), R 1 is a methyl group substituted by –OCONH(C2H5), R 2 is methyl, R 3 is pyrrolidine, and R 4 and R 5 Each is hydrogen.

[0054] The second aspect of the present invention is to provide a pharmaceutical composition for treating or preventing an individual suffering from or suspected of suffering from cancer. The pharmaceutical composition comprises a therapeutically or preventively effective amount of a compound of formula (I); and a pharmaceutically acceptable carrier.

[0055] When calculated based on the total weight of the pharmaceutical composition, the compound of formula (I) accounts for about 0.1% to 99% of the weight of the pharmaceutical composition. In certain embodiments, the compound of formula (I) accounts for at least 1% of the weight of the pharmaceutical composition. In specific embodiments, the compound of formula (I) accounts for at least 5% of the weight of the pharmaceutical composition. In other embodiments, the compound of formula (I) accounts for at least 10% of the weight of the pharmaceutical composition. In other embodiments, the compound of formula (I) accounts for at least 25% of the weight of the pharmaceutical composition.

[0056] Preferably, the compound has the structure of formula (I-A)

[0057]

[0058] in,

[0059] R 1 is an alkyl group which may be substituted with halogen, –OR, –OAc, –OSO2R or –OCONHR, wherein R is hydrogen, alkyl, cycloalkyl or aryl;

[0060] R 2 is hydrogen, alkyl, cycloalkyl, aryl or heteroaryl;

[0061] R 3 Yes – NR A R B , –NHPhR C , a mono- or disaccharide residue, or an amino acid residue, or -NR A R B The phenylamine or cycloalkylamine is combined to form an aniline or cycloalkylamine with or without a substituent selected from the group consisting of morpholine, pyrrolidine, piperidine, 1-substituted piperazine, 1,4'-bipiperidine and 4'-substituted 4,4'-bipiperidine; wherein the substituents of piperazine and 4,4'-bipiperidine are alkyl, -(CH2) n CONH(CH2) m NR A R B ,in

[0062] n and m are each an integer between 1 and 5,

[0063] R A and R B are each H or C1-C6 alkyl; and

[0064] RC is hydrogen, halogen, alkoxy, –CH2OH, –NHCOR a 、–NHC(O)OR a , or an alkyl, alkenyl, alkynyl, heterocyclic or aryl group which may be substituted, and R a It is C 1–6 Alkyl or aryl;

[0065] R 4 and R 5 Each is hydrogen, –OH, alkoxy or –O(CH2) x N(R b )2, where

[0066] x is an integer between 1 and 5; and

[0067] R b It is C 1–10 Alkyl or monocycloalkylamino; and

[0068] The aryl or heteroaryl may or may not have at least one substituent, wherein the at least one substituent is selected from C- 1–6 Alkyl, alkoxy, halogen, cyano, nitro, –NH2, –NHR c , –N(R c ) 2. a group consisting of cycloalkylamino, methylenedioxy and ethylenedioxy, wherein R c are independently hydrogen or C 1–10 alkyl.

[0069] Optionally or additionally, the compound of formula (IA) is formulated into liposomes.

[0070] According to a preferred embodiment, in formula (I-A), R 1 is a methyl group substituted with –OH, R 2 is ethyl, R 3 is morpholine, and R 4 and R 5 Each is hydrogen.

[0071] According to another preferred embodiment, in formula (I-A), R 1 is a methyl group substituted with –OH, R 2 is methyl, R 3 is pyrrolidine, and R 4 and R 5 Each is hydrogen.

[0072] According to a further embodiment, in formula (IA), R 1 is a methyl group substituted with –OH, R 2 is methyl, R 3 is 1,4'-bipiperidine, and R4 and R 5 Each is hydrogen.

[0073] According to yet a further embodiment, R 1 is a methyl group substituted by –OCONH(C2H5), R 2 is methyl, R 3 is pyrrolidine, and R 4 and R 5 Each is hydrogen.

[0074] According to another preferred embodiment of the present invention, the compound has a structure of formula (I-B):

[0075]

[0076] in,

[0077] R 1 is an alkyl group which may be substituted with halogen, –OR, –OAc, –OSO2R or –OCONHR, wherein R is hydrogen, alkyl, cycloalkyl or aryl;

[0078] R 2 is hydrogen, alkyl, cycloalkyl, aryl or heteroaryl;

[0079] R 3 Yes – NR A R B , –NHPhR C , a mono- or disaccharide residue, or an amino acid residue, or -NR A R B The phenylamine or cycloalkylamine is combined to form an aniline or cycloalkylamine with or without a substituent selected from the group consisting of morpholine, pyrrolidine, piperidine, 1-substituted piperazine, 1,4'-bipiperidine and 4'-substituted 4,4'-bipiperidine; wherein the substituents of piperazine and 4,4'-bipiperidine are alkyl, -(CH2) n CONH(CH2) m NR A R B ,in

[0080] n and m are each an integer between 1 and 5,

[0081] R A and R B are each H or C1-C6 alkyl; and

[0082] R C is hydrogen, halogen, alkoxy, –CH2OH, –NHCOR a 、–NHC(O)OR a , or an alkyl, alkenyl, alkynyl, heterocyclic or aryl group which may be substituted, and Ra It is C 1–6 Alkyl or aryl;

[0083] R 4 and R 5 Each is hydrogen, –OH, alkoxy or –O(CH2) x N(R b )2, where

[0084] x is an integer between 1 and 5; and

[0085] R b It is C 1–10 Alkyl or monocycloalkylamino; and

[0086] The aryl or heteroaryl may or may not have at least one substituent, wherein the at least one substituent is selected from C- 1–6 Alkyl, alkoxy, halogen, cyano, nitro, –NH2, –NHR c , –N(R c ) 2. a group consisting of cycloalkylamino, methylenedioxy and ethylenedioxy, wherein R c are independently hydrogen or C 1–10 alkyl.

[0087] According to a preferred embodiment, in formula (I-B), R 1 is a methyl group substituted with –OH, R 2 is methyl, R 3 is dimethylamine, and R 4 and R 5 Each is hydrogen.

[0088] According to another preferred embodiment, in formula (I-B), R 1 is a methyl group substituted by –OCONH(C2H5), R 2 is methyl, R 3 is dimethylamine, and R 4 and R 5 Each is hydrogen.

[0089] According to a further preferred embodiment, in formula (I-B), R 1 is a methyl group substituted by –OCONH(C2H5), R 2 is methyl, R 3 is pyrrolidine, and R 4 and R 5 Each is hydrogen.

[0090] The present invention also encompasses a method for treating or preventing a subject suffering from or suspected of suffering from cancer, comprising the step of administering to the subject a pharmaceutical composition of the present invention.

[0091] Optionally or additionally, the method further comprises administering a chemotherapeutic agent to the subject prior to, simultaneously with, or after administering the pharmaceutical composition of the present invention.

[0092] Specific chemotherapeutic agents useful in the methods of the present invention include, but are not limited to, deoxyelephantopin (DET), Vemurafenib (PLX4032), docetaxel, paclitaxel, cisplatin, oxaliplatin, betulinic acid, 4-S-cysteaminyl catechol, 4-S-cysteaminylphenol, everolimus, bortezomib, carboplatin, dacarbazine, celecoxib, temozolomide, sorafenib, thalidomide, lenalidomide, valproic acid, acid, vinblastine, imatinib mesylate, bosentan, apomine, arsenic trioxide, carmustine, lambrolizumab, anti-CTLA-4 drug, anti-programmed death receptor-1 (PD-1) drug, ipilimumab, tremelimumab, doxorubicin, MEK inhibitor, capecitabine, poly (ADP-ribose) polymerase (PARP) inhibitor, phosphoinositide 3-kinase 3-kinase (PI3K) inhibitors, mammalian target of rapamycin (mTOR) inhibitors and tamoxifen.

[0093] Cancers that can be specifically treated using the methods of the present invention include, but are not limited to, Hodgkin's disease, non-Hodgkin's lymphomas, acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), Ewing's sarcoma, multiple myeloma, Wilms' tumor, bone tumors, neuroblastoma, retinoblastoma, testicular cancer, thyroid cancer, prostate cancer, larynx cancer, cervical cancer, nasopharynx cancer, breast cancer, colon cancer, cancer, pancreatic cancer, head and neck cancer, esophageal cancer, rectal cancer, small-cell lung cancer, non-small-cell lung cancer, brain cancer, melanoma, non-melanoma skin cancer, and CNS neoplasms.

[0094] The present invention also includes a kit for treating or preventing a subject suffering from cancer. The kit comprises at least a first container containing the compound of formula (I); and a second container containing a chemotherapeutic agent.

[0095] The following description will describe one or more embodiments of the present invention in detail. Other features and advantages of the present invention will be readily apparent after reading the following detailed description and the scope of claims.

[0096] It is to be understood that both the foregoing general description and the following detailed description are exemplary only, and are intended to explain the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] The accompanying drawings are included in the specification and constitute a part of the specification, and are used to illustrate several embodiments, methods and other exemplary implementations of various aspects of the present invention. The present invention will be more clearly understood after reading the following detailed description with reference to the accompanying drawings, in which:

[0098] Figure 1 is a photograph obtained according to one embodiment of the present invention, illustrating the effect of the compound of formula (I) on the formation of DNA interstrand crosslinks (ICL);

[0099] Figure 2 The results obtained according to one embodiment of the present invention illustrate the effect of BO-2768 or cisplatin on DNA interstrand crosslinking in H526 cells; wherein (A) representative images show individual cells with comet tails obtained using a modified comet assay, and (B) a bar graph shows the tail moment of cells treated with cisplatin or BO-2768. A shorter tail moment indicates a stronger DNA interstrand crosslinking effect. The experimental data are the average of 3 independent experiments;

[0100] Figure 3 is a photograph obtained according to one embodiment of the present invention, illustrating the effect of (A) BO-2590, (B) BO-2577 or (C) vatalanib on inhibiting VEGFR-2 activation;

[0101] Figure 4 1 is a result obtained according to an embodiment of the present invention, illustrating the effect of BO-2590, BO-2698 and BO-2768 on the anti-angiogenesis of endothelial cells, wherein (A) is the inhibition of endothelial cell migration, which is measured using a transwell, (B) and (C) are photographs showing the inhibition results detected using a tube formation assay, and (D) is a line graph showing the quantitative results of (A) and (B) panels;

[0102] Figure 5 is the result obtained according to one embodiment of the present invention, which illustrates the situation that BO-2590 interferes with the cell cycle in H460 human lung cancer cells;

[0103] Figure 6The results obtained according to one embodiment of the present invention illustrate that BO-2590 induces apoptotic sub-G1 cells in H460 human lung cancer cells at (A) 24 hours, (B) 48 hours or (C) 72 hours;

[0104] Figure 7 The results obtained according to one embodiment of the present invention illustrate the situation in which BO-2590 or cisplatin induces apoptosis of Annexin V+ cells;

[0105] Figure 8 is the result obtained according to one embodiment of the present invention, which illustrates the situation that BO-2768 induces apoptosis in H526 cells;

[0106] Fig. 9 is a line graph obtained according to an embodiment of the present invention, illustrating the synergistic effect of BO-2768 and cisplatin, which inhibit the proliferation of H211 cells at a ratio of (A) 1:2, (B) 1:4, and (C) 1:8, respectively; a combined index (CI) is then used to calculate the synergistic effect or antagonistic effect between different combined doses, wherein when CI=1, it indicates that the two drugs have an additive effect, when CI<1, it indicates that the effect is better than the additive effect (i.e., synergistic effect), and when CI>1, it indicates that the effect is worse than the additive effect (i.e., antagonistic effect);

[0107] Fig.10 The results obtained according to one embodiment of the present invention illustrate the effects of liposome BO-2590L on (A) tumor volume and (B) body weight changes of nude mice bearing H526 xenografts;

[0108] Fig.11 The results obtained according to one embodiment of the present invention illustrate the effects of liposomal BO-2590L, vatalanib and cisplatin on (A) tumor volume and (B) body weight changes of nude mice bearing H526 xenografts, respectively;

[0109] Fig.12 The results obtained according to one embodiment of the present invention illustrate the effects of micellar BO-2590, BO-2768, BO-2792, irinotecan and cisplatin on (A) tumor volume and (B) body weight changes of nude mice bearing H526 xenografts, respectively;

[0110] Fig.13 is a line graph obtained according to one embodiment of the present invention, illustrating that BO-2768 exhibits a dose-dependent inhibitory effect on (A) tumor volume and (B) body weight change of nude mice with H526 small cell lung cancer (SCLC) xenografts;

[0111] Fig.14 The results obtained according to one embodiment of the present invention illustrate the effects of the combined use of BO-2768 and cisplatin on (A) tumor volume and (B) body weight changes in nude mice bearing H211 SCLC xenografts. BO-2768 was administered at a dose of 20 mg / kg in 100 μl for 5 consecutive days and one day off for a total of two cycles. Cisplatin was administered at a dose of 2 or 4 mg / kg once every 4 days for a total of 3 times. Body weight changes were observed after drug treatment;

[0112] Fig.15 The results obtained according to one embodiment of the present invention illustrate the effect of BO-2590L, cisplatin or vatalanib on the amount of tumor CD31 marker; wherein (A) is a representative IHC staining image using an antibody against CD31 on day 6 after treatment, and (B) quantification of tumor CD31 signal. The experimental data are represented by the mean ± standard deviation of 15 fields of view in each group; and

[0113] Fig.16 The results obtained according to one embodiment of the present invention illustrate the effects of BO-2590L, cisplatin or vatalanib on tumor γ-H2AX; wherein (A) is a representative IHC staining image using an antibody against γ-H2AX on day 6 after treatment, and (B) quantification of tumor γ-H2AX signals. The experimental data are represented by the mean ± standard deviation of three mice in each group. DETAILED DESCRIPTION

[0114] The detailed description and accompanying drawings provided below are intended to make the description of the present invention more detailed and complete, but are not intended to represent the only form for understanding or using the present invention.

[0115] 1. Definition

[0116] When a numerical range is listed, it is intended to include every value and sub-range within that range. For example, “C 1–10 " is intended to cover C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 1–10 , C 1–9 , C 1–8 , C 1–7 , C 1–6 , C 1–5 , C 1–4 , C 1–3 , C 1–2 , C 2–10 , C 2–9 , C 2–8 , C 2–7 , C 2–6 , C 2–5, C 2–4 , C 2–3 , C 3–10 , C 3–9 , C 3–8 , C 3–7 , C 3–6 , C 3–5 , C 3–4 , C 4–10 , C 4–9 , C 4–8 , C 4–7 , C 4–6 , C 4–5 , C 5–10 , C 5–9 , C 5–8 , C 5–7 , C 5–6 , C 6–10 , C 6–9 , C 6–8 , C 6–7 , C 7–10 , C 7–9 , C 7–8 , C 8–10 , C 8–9 and C 9–10 .

[0117] Unless otherwise indicated, the term "alkyl" refers to a straight chain, branched chain, and / or cyclic ("cycloalkyl") hydrocarbon having 1 to 20 (e.g., 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1) carbon atoms. 1–4 Alkyl groups that contain alkyl radicals (i.e., cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl) are referred to as "lower alkyl". Examples of alkyl radicals include methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, isobutyl, 2-isopropyl-3-methylbutyl, pentyl, pent-2-yl, hexyl, isohexyl, heptyl, hept-2-yl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, and dodecanyl. Cycloalkyl radicals may be monocyclic or polycyclic, and examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Unless otherwise indicated, each example of alkyl radical is independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkyl radical") or substituted with one or more substituents (a "substituted alkyl radical"). In certain embodiments, an alkyl radical is a substituted C 2–10 In certain embodiments, a cycloalkyl group is a monocyclic, saturated, ring-forming ring having 3 to 6 carbon atoms ("C 3–6In certain embodiments, the cycloalkyl group has 5 to 6 ring carbon atoms ("C 5–6 (cycloalkyl) 5–6 Examples of cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C6). Unless otherwise indicated, each example of a cycloalkyl group is independently unsubstituted (an "unsubstituted cycloalkyl") or substituted with one or more substituents (a "substituted cycloalkyl"). In certain embodiments, a cycloalkyl group is an unsubstituted C 3–10 In certain embodiments, the cycloalkyl group is a substituted C 3–10 Cycloalkyl.

[0118] "Heterocycloalkyl" refers to a 3- to 10-membered non-aromatic ring system radical having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, phosphorus, and silicon ("3-10 membered heterocycloalkyl"). For heterocycloalkyls containing one or more nitrogen atoms, the point of attachment may be a carbon atom or a nitrogen atom, as valence permits. Unless otherwise indicated, each instance of a heterocycloalkyl radical is independently optionally substituted, i.e., unsubstituted (an "unsubstituted heterocycloalkyl") or substituted with one or more substituents (a "substituted heterocycloalkyl"). In certain embodiments, a heterocycloalkyl radical is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, a heterocycloalkyl is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur. In certain embodiments, the 5-6 membered heterocyclic ring has 1-3 ring heteroatoms selected from nitrogen, oxygen and sulfur. In certain embodiments, the 5-6 membered heterocyclic ring has 1-2 ring heteroatoms selected from nitrogen, oxygen and sulfur. In certain embodiments, the 5-6 membered heterocycloalkyl has one ring heteroatom selected from nitrogen, oxygen and sulfur. Specific 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione. Specific 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, dithiolanyl and Specific 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Specific 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Specific 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dithianyl. Alkyl. Specific 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, triazinanyl. Specific 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Specific 8-membered heterocyclic groups containing one heteroatom include, but are not limited to, azocanyl, oxecanyl, and thiocanyl.

[0119] Unless otherwise indicated, the term "aryl" refers to an aromatic ring or a portion of an aromatic ring system consisting of carbon atoms and hydrogen atoms. Aromatic group clusters may include multiple rings that are bonded or fused together. Examples of aromatic group clusters include phenyl, naphthyl, pyrenyl, anthracenyl, and phenanthrenyl. Unless otherwise indicated, each instance of an aryl group is independently optionally substituted, that is, unsubstituted (an "unsubstituted aryl") or substituted with one or more substituents (a "substituted aryl"). In certain embodiments, the aryl group is a substituted phenyl group (e.g., benzyl).

[0120] Unless otherwise indicated, the term "heteroaryl" refers to an aromatic group in which at least one of its carbon atoms is replaced by a heteroatom (e.g., N, O, or S). In certain embodiments, a heteroaryl is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In certain embodiments, a heteroaryl is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-8 membered heteroaryl"). In certain embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (a "5-6 membered heteroaryl"). In certain embodiments, the 5-6 membered heteroaryl group has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In certain embodiments, the 5-6 membered heteroaryl group has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In certain embodiments, the 5-6 membered heteroaryl group has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently optionally substituted, i.e., unsubstituted (an "unsubstituted heteroaryl") or substituted with one or more substituents (a "substituted heteroaryl"). In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl group. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl group. Specific 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. Specific 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, Azolyl, iso Specific 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Specific 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Specific 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Specific 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Specific 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Specific 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Specific 10-membered heteroaryl groups containing two heteroatoms include, but are not limited to, quinazolinyl.

[0121] Unless otherwise indicated, the term "alkylaryl" or "alkyl-aryl" refers to an alkyl cluster bonded to an aryl cluster.

[0122] Unless otherwise stated, the terms "halogen" and "halo" encompass fluorine, chlorine, bromine and iodine.

[0123] The term "amino" refers to a group of the formula: -N(R c )2 cluster, where R c Each instance of is independently a substituent as described herein, or R c Two examples of are combined to form a substituted or unsubstituted heterocycle. In a specific embodiment, the amino group is an unsubstituted amino group (i.e., -NH2). In a specific embodiment, the amino group is a substituted amino group, wherein R c At least one instance of is not hydrogen.

[0124] The term "saccharide group" refers to a saccharide monogroup covalently linked to another compound or atom through any atom of the saccharide group, such as through the glycoside carbon atom. By way of illustration, representative sugars include hexoses such as D-glucose, D-mannose, D-xylose, D-galactose, vancosamine, 3-demethyl-vancosamine, 3-epi-vancosamine, 4-epi-vancosamine, 3-amino-2,3,6-deoxy-L-arabinose-hexose (acosamine), actinosamine, daunosamine, 3-epi-daunosamine, ristosamine, D-glucosamine, N-methyl-D-glucosamine, D-glucuronic acid, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, sialic acid, iduronic acid, acid), L-fucose, etc.; pentoses such as D-ribose or D-arabinose; ketoses such as D-ribulose or D-fructose; disaccharides such as sucrose, lactose, maltose, 2-O-(α-L-vancosamino)-β-D-glucopyranose or 2-O-(3-desmethyl-α-L-vancosamino)-β-D-glucopyranose; derivatives such as acetals, amines, acylated, sulfated and phosphorylated sugars. For the purpose of this definition, these sugars are represented by the conventional three-letter nomenclature, and the sugars can be in their open form or, preferably, in their pyranose form.

[0125] The term "amino acid group" refers to an amino acid moiety attached to another compound or atom through any atom of the amino acid group, for example, through an amino or carboxyl functional group, or any functional group on a side chain, such as the side chain amino group of lysine.

[0126] Unless otherwise specified, when used to describe a chemical structure or group, the term "substituted" refers to a derivative of the structure or group in which one or more hydrogen atoms, chemical groups or functional groups are replaced by an atom such as, but not limited to, -OH, -CHO, alkoxy, alkanoyloxy (e.g., -OAc), alkenyl, alkyl (e.g., methyl, ethyl, propyl, tert-butyl), aryl, aryloxy, halogen or haloalkyl (e.g., -CCl3, -CF3, -C(CF3)3). Unless otherwise specified, the term "alkoxy" refers to -O-alkyl. Examples of alkoxy include, but are not limited to, -OCH3, -OCH2CH3, -O(CH2)2CH3, -O(CH2)3CH3, -O(CH2)4CH3 and -O(CH2)5CH3. The term "loweralkoxy" refers to -O-(loweralkyl), such as -OCH3 and -OCH2CH3.

[0127] Unless otherwise indicated, one or more adjectives immediately preceding a series of nouns shall be deemed to apply to each noun. For example, the phrase "optionally substituted alky, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl" has the same meaning as "optionally substituted alky, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl".

[0128] The present invention is not limited to any one of the substituents specifically listed above.

[0129] The term "solvate" refers to a form of a compound that is bound to a solvent, usually by a solvolysis reaction. This physical binding includes hydrogen bonds. Conventional solvents include water, methanol, ethanol, acetic acid, dimethylsulfoxide (DMSO), tetrahydrofuran (THF), diethyl ether, and the like. The compounds described herein can be prepared, for example, in crystalline form, and can be in the form of a solvate. Suitable solvates include pharmaceutically acceptable solvates (e.g., hydrates), and further include both stoichiometric solvates and non-stoichiometric solvates. In certain cases, such as when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid, the solvate will be able to dissociate. "Solvate" encompasses both solutions and dissociable solvates.

[0130] It is understood that compounds having the same molecular formula but differing in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space are called "isomers". Isomers whose atoms are arranged differently in space are called "stereoisomers".

[0131] Stereoisomers that are not mirror images of each other are called diastereomers, while those that are non-superimposable mirror images are called enantiomers. When a compound has an asymmetric center, such as when it is bonded to four different groups, a pair of enantiomers is possible. Enantiomers can be identified by the absolute configuration of their asymmetric center, described by the R- and S-sequencing rules of Cahn and Prelog, or by the way in which the molecule rotates the plane of polarized light and named dextrorotatory or levorotatory (i.e., (+) or (–)-isomers, respectively). Chiral compounds can exist as individual enantiomers or as mixtures thereof. A mixture containing equal proportions of enantiomers is called a racemic mixture.

[0132] It should be noted that if the stereochemistry of a structure or portion of a structure is not indicated, for example, by bold or dashed lines, then the structure or portion of the structure is considered to encompass all stereoisomers thereof. Similarly, the names of compounds having one or more chiral centers without indicating the stereochemistry of those centers encompass pure stereoisomers and mixtures thereof. In addition, any atom shown in the figures with unsaturated valences is presumed to be attached to sufficient hydrogen atoms to satisfy the valences.

[0133] For purposes of the present invention, heteroatoms (eg, nitrogen) may have hydrogen substituents and / or any suitable substituent described herein which satisfy the valence of the heteroatom and form a stable cluster.

[0134] Unless otherwise indicated, a "therapeutically effective amount" of a compound is an amount sufficient to produce a therapeutic benefit in treating or managing a disease or condition, or in delaying or reducing one or more symptoms associated with the disease or condition. A therapeutically effective amount of a compound is an amount of a therapeutic agent that, whether administered alone or in combination with other therapies, produces a therapeutic benefit in treating or managing the disease or condition. The term "therapeutically effective amount" can encompass an amount that improves overall treatment, reduces or avoids symptoms or causes of the disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0135] Unless otherwise indicated, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or condition, one or more symptoms associated with the disease or condition, or to prevent its recurrence. A prophylactically effective amount of a compound is an amount of a therapeutic agent that, whether administered alone or in combination with other treatments, produces a prophylactic benefit in preventing the disease. The term "prophylactically effective amount" may encompass an amount that improves overall prevention or enhances the prophylactic efficacy of another prophylactic agent.

[0136] Unless otherwise indicated, the terms "treat," "treating" and "treatment" refer to an action that occurs in a patient suffering from a particular disease or condition that reduces the severity of the disease or condition, or one or more symptoms of the disease or condition, or delays or postpones the progression of the disease or condition.

[0137] It should be noted that if the stereochemistry of a structure or a portion of a structure is not explicitly indicated by bold or dashed lines, the structure or portion of the structure should be interpreted as including its entire stereochemistry. Similarly, the name of a compound with one or more chiral centers should be interpreted as including pure stereoisomers and mixtures of pure stereoisomers of the compound if the chiral centers are not explicitly indicated. In addition, any atom in the diagram that appears to have unsatisfied chemical bond valences should be treated as having hydrogen atoms attached to the unsatisfied chemical bond valences.

[0138] Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate values, the relevant numerical values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations caused by individual testing methods. Here, the word "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range. Alternatively, the word "about" means that the actual value falls within the acceptable standard error of the mean value, depending on the consideration of a person of ordinary skill in the art to which the present invention belongs. Except for the experimental examples, or unless otherwise explicitly stated, it should be understood that all ranges, quantities, values ​​and percentages used herein (such as those used to describe material dosage, time duration, temperature, operating conditions, quantitative ratios and the like) are modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the attached patent scope are approximate values ​​and can be changed as needed. At least these numerical parameters should be understood as the number of significant digits indicated and the values ​​obtained by applying the general rounding method.

[0139] As used herein, the singular forms "a," "an," and "the" include plural references unless the context dictates otherwise.

[0140] 2. Novel compounds of formula (I)

[0141] The present invention encompasses compounds of formula (I), and pharmaceutically acceptable solvates or stereoisomers thereof,

[0142]

[0143] in,

[0144] A is an unsaturated carbon ring with 6-10 members which may be substituted;

[0145] R 1 is an alkyl group which may be substituted with halogen, –OR, –OAc, –OSO2R or –OCONHR, wherein R is hydrogen, alkyl, cycloalkyl or aryl;

[0146] R 2 is hydrogen, alkyl, cycloalkyl, aryl or heteroaryl;

[0147] R 3 Yes – NR A R B , –NHPhR C , a mono- or disaccharide residue, or an amino acid residue, or -NR A R BThe phenylamine or cycloalkylamine is combined to form an aniline or cycloalkylamine with or without a substituent selected from the group consisting of morpholine, pyrrolidine, piperidine, 1-substituted piperazine, 1,4'-bipiperidine and 4'-substituted 4,4'-bipiperidine; wherein the substituents of piperazine and 4,4'-bipiperidine are alkyl, -(CH2) n CONH(CH2) m NR A R B ,in

[0148] n and m are each an integer between 1 and 5,

[0149] R A and R B are each H or C1-C6 alkyl; and

[0150] R C is hydrogen, halogen, alkoxy, –CH2OH, –NHCOR a 、–NHC(O)OR a , or an alkyl, alkenyl, alkynyl, heterocyclic or aryl group which may be substituted, and R a It is C 1–6 Alkyl or aryl; and the aryl or heteroaryl may or may not have at least one substituent, wherein the at least one substituent is selected from C- 1–6 Alkyl, alkoxy, halogen, cyano, nitro, –NH2, –NHR c , –N(R c ) 2. a group consisting of cycloalkylamino, methylenedioxy and ethylenedioxy, wherein R c are independently hydrogen or C 1–10 alkyl.

[0151] According to certain embodiments of the present invention, in formula (I), R 3 is 1H-pyrazole-3-amino, 1H-imidazole-2-amino or pyrimidine-amino.

[0152] According to a further embodiment of the present invention, in formula (I), the cycloalkylamino group is selected from the group consisting of pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl and 4-piperidinylpiperidinyl.

[0153] According to certain embodiments of the present invention, the specific compound is of formula (1-A),

[0154]

[0155] in,

[0156] R 1 is an alkyl group which may be substituted with halogen, –OR, –OAc, –OSO2R or –OCONHR, wherein R is hydrogen, alkyl, cycloalkyl or aryl;

[0157] R 2 is hydrogen, alkyl, cycloalkyl, aryl or heteroaryl;

[0158] R 3 Yes – NR A R B , –NHPhR C , a mono- or disaccharide residue, or an amino acid residue, or -NR A R B Combined to form an aniline or cycloalkylamine with or without a substituent selected from the group consisting of morpholine, pyrrolidine, piperidine, 1-substituted piperazine, 1,4'-bipiperidine and 4'-substituted 4,4'-bipiperidine; wherein the substituents of piperazine and 4,4'-bipiperidine are alkyl, -(CH2) n CONH(CH2) m NR A R B ,in

[0159] n and m are each an integer between 1 and 5,

[0160] R A and R B are each H or C1-C6 alkyl; and

[0161] R C is hydrogen, halogen, alkoxy, –CH2OH, –NHCOR a 、–NHC(O)OR a , or an alkyl, alkenyl, alkynyl, heterocyclic or aryl group which may be substituted, and R a It is C 1–6 Alkyl or aryl;

[0162] R 4 and R 5 Each is hydrogen, –OH, alkoxy or –O(CH2) x N(R b )2, where

[0163] x is an integer between 1 and 5; and

[0164] R b It is C 1–10 Alkyl or monocycloalkylamino; and

[0165] The aryl or heteroaryl group may or may not have at least one substituent, wherein the at least one substituent is selected from C- 1–6 Alkyl, alkoxy, halogen, cyano, nitro, –NH2, –NHR c , –N(R c) 2. a group consisting of cycloalkylamino, methylenedioxy and ethylenedioxy, wherein R c are independently hydrogen or C 1–10 alkyl.

[0166] Exemplary compounds of formula (IA) include, but are not limited to, the following compounds:

[0167]

[0168]

[0169]

[0170] According to certain embodiments of the present invention, a specific compound is of formula (IB),

[0171]

[0172] in,

[0173] R 1 is an alkyl group which may be substituted with halogen, –OR, –OAc, –OSO2R or –OCONHR, wherein R is hydrogen, alkyl, cycloalkyl or aryl;

[0174] R 2 is hydrogen, alkyl, cycloalkyl, aryl or heteroaryl;

[0175] R 3 Yes – NR A R B , –NHPhR C , a mono- or disaccharide residue, or an amino acid residue, or -NR A R B The phenylamine or cycloalkylamine is combined to form an aniline or cycloalkylamine with or without a substituent selected from the group consisting of morpholine, pyrrolidine, piperidine, 1-substituted piperazine, 1,4'-bipiperidine and 4'-substituted 4,4'-bipiperidine; wherein the substituents of piperazine and 4,4'-bipiperidine are alkyl, -(CH2) n CONH(CH2) m NR A R B ,in

[0176] n and m are each an integer between 1 and 5,

[0177] R A and R B are each H or C1-C6 alkyl; and

[0178] R C is hydrogen, halogen, alkoxy, –CH2OH, –NHCOR a、–NHC(O)OR a , or an alkyl, alkenyl, alkynyl, heterocyclic or aryl group which may be substituted, and R a It is C 1–6 Alkyl or aryl;

[0179] R 4 and R 5 Each is hydrogen, –OH, alkoxy or –O(CH2) x N(R b )2, where

[0180] x is an integer between 1 and 5; and

[0181] R b It is C 1–10 Alkyl or monocycloalkylamino; and

[0182] The aryl or heteroaryl group may or may not have at least one substituent, wherein the at least one substituent is selected from C- 1–6 Alkyl, alkoxy, halogen, cyano, nitro, –NH2, –NHR c , –N(R c ) 2. a group consisting of cycloalkylamino, methylenedioxy and ethylenedioxy, wherein R c are independently hydrogen or C 1–10 alkyl.

[0183] Exemplary compounds of formula (IB) include, but are not limited to, the following compounds:

[0184]

[0185]

[0186]

[0187] The compounds of the present invention contain one or more stereocenters, so there may be a racemic mixture of mirror image isomers or a mixture of non-mirror image isomers. The present invention therefore encompasses the form of pure stereoisomers of such compounds, as well as mixtures of these forms. Stereoisomers can be prepared by asymmetric synthesis, or by using conventional techniques such as crystallization, chromatography, and the use of optical splitting agents. A preferred way to separate mirror image isomers from a racemic mixture is to prepare by using high performance liquid chromatography (HPLC). Alternatively, in a solvent, a racemic compound is separated into its mirror image isomer by reacting with an optical splitting agent in an optically active form. Depending on the optical form of the optical splitting agent, one of the two mirror image isomers can be separated into an insoluble salt with a high yield and high optical purity, while retaining the other mirror image isomer in the solution.

[0188] Therefore, the present invention further encompasses stereoisomeric mixtures of the compounds of the present invention. Also encompassed are configurational isomers (e.g., cis and trans isomers, whether or not involving a double bond) of the compounds of the present invention, either in admixture or in pure or substantially pure form.

[0189] 3. Usage

[0190] The present invention encompasses a method for treating or preventing a subject suffering from cancer. The method comprises the step of administering to the subject a therapeutically or prophylactically effective amount of a compound of formula (I) of the present invention, thereby inhibiting cancer growth.

[0191] In certain embodiments, the method further comprises the step of administering to the subject a chemotherapeutic agent prior to, concurrently with, or after administering the compound of formula (I). The chemotherapeutic agent may be selected from the group consisting of deoxycholate, vemurafenib, docetaxel, paclitaxel, cisplatin, oxaliplatin, betulinic acid, 4-S-cysteamine catechol, 4-S-cysteamine phenol, everolimus, bortezomib, carboplatin, dacarbazine, celecoxib, temozolomide, sorafenib, thalidomide, lenalidomide, valproic acid, vinblastine, imatinib mesylate, bosentan, apometamide, arsenic trioxide, carmustine, laplatin, anti-CTLA-4 drugs, anti-PD-1 drugs, ipilimumab, tripelimumab, doxorubicin, MEK inhibitors, capecitabine, PARP inhibitors, PI3K inhibitors, mTOR inhibitors, and tamoxifen.

[0192] In the present invention, the cancer can be any one of Hodgkin's disease, non-Hodgkin's lymphoma, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, Ewing's sarcoma, multiple myeloma, Wilms' tumor, bone tumor, neuroblastoma, retinoblastoma, testicular cancer, thyroid cancer, prostate cancer, laryngeal cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, esophageal cancer, rectal cancer, small cell lung cancer, non-small cell lung cancer, brain cancer, melanoma, non-melanoma skin cancer or central nervous system tumor. According to a preferred embodiment, the cancer that can be treated by the compound of formula (I) of the present invention is small cell lung cancer. According to another preferred embodiment, the cancer that can be treated by the compound of formula (I) of the present invention is acute lymphocytic leukemia.

[0193] The amount, route of administration and time of administration of the compound of formula (I) will depend on various factors, such as the specific indication to be treated, prevented or addressed, the age, sex and condition of the patient. The role played by such factors is well known in the art and can be adjusted by routine experiments.

[0194] 4. Pharmaceutical preparations

[0195] The present invention encompasses a pharmaceutical composition for treating or preventing cancer, which comprises a therapeutically or prophylactically effective amount of a compound of formula (I) of the present invention.

[0196] The compound of formula (I) is present in an amount of about 0.1% to 99% by weight, based on the total weight of the pharmaceutical composition. In certain embodiments, the compound of formula (I) is present in an amount of at least 1% by weight, based on the total weight of the pharmaceutical composition. In a specific embodiment, the compound of formula (I) is present in an amount of at least 5% by weight, based on the total weight of the pharmaceutical composition. In yet other embodiments, the compound of formula (I) is present in an amount of at least 10% by weight, based on the total weight of the pharmaceutical composition. In yet other embodiments, the compound of formula (I) is present in an amount of at least 25% by weight, based on the total weight of the pharmaceutical composition.

[0197] In certain preferred embodiments, the pharmaceutical composition further comprises a chemotherapeutic agent. The chemotherapeutic agent can be selected from the group consisting of deoxycholate, vemurafenib, docetaxel, paclitaxel, cisplatin, oxaliplatin, betulinic acid, 4-S-cysteamine catechol, 4-S-cysteamine phenol, everolimus, bortezomib, carboplatin, dacarbazine, celecoxib, temozolomide, sorafenib, thalidomide, lenalidomide, valproic acid, vinblastine, imatinib mesylate, bosentan, apometamide, arsenic trioxide, carmustine, laplatin, anti-CTLA-4 drugs, anti-PD-1 drugs, ipilimumab, tripelimumab, doxorubicin, MEK inhibitors, capecitabine, PARP inhibitors, PI3K inhibitors, mTOR inhibitors and tamoxifen.

[0198] A particular pharmaceutical composition may be in single unit dosage form suitable for oral, mucosal (eg, nasal, sublingual, vaginal, buccal or rectal), parenteral (eg, subcutaneous, intravenous, bolus injection, intramuscular or intraarterial), or transdermal administration to a patient. Examples of dosage forms include, but are not limited to, tablets, caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; pastes (catties); poultices; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

[0199] The formulation should be compatible with the mode of administration. For example, oral administration requires an enteric coating to prevent degradation of the compounds of the invention in the gastrointestinal tract. Similarly, the formulation may contain ingredients that promote delivery of the active ingredient to the site of action. For example, the compound may be administered in the form of a liposome formulation to prevent degradation by degradative enzymes, promote transport within the circulatory system, and effectively cross the cell membrane to reach the intracellular site of delivery.

[0200] Likewise, poorly soluble compounds can be incorporated into liquid dosage forms (and dosage forms suitable for reconstitution) with the aid of solubilizers, emulsifiers, and surfactants, such as, but not limited to, cyclodextrins (e.g., α-cyclodextrin or β-cyclodextrin), and non-aqueous solvents such as, but not limited to, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, dimethyl sulfoxide (DMSO), biocompatible oils (e.g., cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, fatty acid esters of sorbitan and mixtures thereof (e.g., DMSO: corn oil), fats such as egg yolk phosphatidylcoline (EPC), soybean lecithin (Soybean phosphatidylcholine), and mixtures thereof. phosphatidylcholine (SPC), 1,2-oleoyl-sn-glyceryl-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glyceryl-3-phosphocholine (DSPC), cholesterol (CHO), dipalmitoylphosphatidylcholine (DPPC) and PEG-2000. According to a preferred embodiment, the compound of formula (I) (i.e., BO-2590) is incorporated into fat to form liposomes suitable for oral or parenteral administration.

[0201] The composition, shape and type of dosage form will vary depending on its use. For example, a dosage form used in the acute treatment of a disease may include a larger amount of one or more active ingredients contained therein than a dosage form used in the chronic treatment of the same disease. Similarly, a non-oral dosage form may include a smaller amount of one or more active ingredients contained therein than an oral dosage form used to treat the same disease. Such and other ways in which the specific dosage forms encompassed by the present invention differ from one another will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).

[0202] 4.1 Oral Dosage Form

[0203] Pharmaceutical compositions of the present invention suitable for oral administration can be provided as discrete dosage forms, such as, but not limited to, tablets (e.g., chewable tablets), lozenges, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain a predetermined amount of active ingredient and can be prepared by pharmaceutical methods well known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).

[0204] Typical oral dosage forms can be prepared by combining the active ingredient with at least one excipient in intimate admixture according to conventional pharmaceutical synthesis techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration.

[0205] Tablets and capsules represent the most advantageous oral unit dosage forms because they can be easily administered. If necessary, tablets can be coated by conventional aqueous or non-aqueous techniques. Such dosage forms can be prepared by conventional pharmaceutical methods. Overall, pharmaceutical compositions and dosage forms are prepared by uniformly and thoroughly mixing the active ingredient with a liquid carrier, a finely dispersed solid carrier, or both, and then, if necessary, forming the product into a desired appearance. A solubilizing agent can be incorporated into the solid dosage form for rapid dissolution. A lubricant can also be incorporated to facilitate the manufacture of dosage forms (e.g., tablets).

[0206] 4.2 Non-oral dosage forms

[0207] Non-oral dosage forms can be administered to a patient by a variety of different routes, including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intra-arterial. Because their mode of administration essentially bypasses the patient's natural defense mechanisms against contaminants, non-oral dosage forms must be sterile or capable of being sterilized prior to administration to a patient. Examples of non-oral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable carrier for injection, suspensions ready for injection, and emulsions.

[0208] Suitable carriers that can be used in the non-oral dosage form of the present invention are well known to those skilled in the art. Examples include, but are not limited to, water; aqueous carriers such as, but not limited to, sodium chloride solution, Ringer's solution, and dextrose; water-miscible carriers such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol; and non-aqueous carriers such as, but not limited to, fats, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0209] 4.3 Transdermal, topical and mucosal dosage forms

[0210] Transdermal, topical and mucosal dosage forms include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions or other forms known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 18th eds., Mack Publishing, Easton PA (1990). Transdermal dosage forms include "reservoir type" or "matrix type" patches, which can be applied to the skin and used for a specific period of time to allow the desired amount of active ingredient to penetrate.

[0211] Suitable excipients (eg, carriers and diluents) and other materials for transdermal, topical and mucosal dosage forms are well known to those skilled in the pharmaceutical arts and depend on the particular tissue to which a given pharmaceutical composition or dosage form is to be administered.

[0212] Depending on the specific tissue to be treated, additional components may be used before, simultaneously with, or after treatment with the active ingredients of the present invention. For example, penetration enhancers may be used to aid in delivering the active ingredients to the tissue.

[0213] The pH of the tissue to which the pharmaceutical composition or dosage form or the pharmaceutical composition or dosage form are applied can also be adjusted to improve the delivery of one or more active ingredients. Similarly, the polarity, ionic strength or tension of the solvent carrier can be adjusted to improve delivery. Compounds (such as stearates) can also be added to pharmaceutical compositions or dosage forms to facilitate the hydrophilicity or lipophilicity of one or more active ingredients to improve delivery. For this part, stearates can be used as the fat carrier of preparations, as emulsifiers or surfactants, and as delivery enhancers or penetration enhancers. Salts, hydrates or solvates of different active ingredients can be used to further adjust the properties of the resulting composition.

[0214] 5. Kit

[0215] The invention also encompasses kits for treating or preventing cancer in a subject.

[0216] According to the present invention, the kit comprises at least a first container containing the compound of formula (I) of the present invention, a second container containing the above-mentioned chemotherapeutic agent; and instructions related to the kit to guide the user how to use the kit. The instructions can be in the form of a brochure, a tape, a CD, a VCD or a DVD. Examples of containers include, but are not limited to, vials, test tubes, etc.

[0217] The present invention will be described in more detail with reference to the following embodiments, which are for exemplary purposes rather than for limiting purposes. Although these embodiments are typically conventional, other steps, methodology or techniques well known to those skilled in the art can also be used.

[0218] Example

[0219] Materials and Methods

[0220] Cell culture

[0221] Each cell type used in this study was cultured at 37°C in a 5% CO2 humidified incubator in the manufacturer's recommended medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units of penicillin per ml, and 100 μg of streptomycin per ml.

[0222] Experimental animals

[0223] Experimental animal care was approved and followed the regulations of the Institutional Animal Care and Use Committee.

[0224] Athymic nude mice with nu / nu gene were obtained from the Experimental Animal Center. Male nude mice (older than 6 weeks or weighing 20-24 g or more) were used for human tumor xenografts. Test compounds were administered intravenously through the tail vein. Tumor volume was detected by measuring length × width × height (or width) with a caliper. The carrier used for the test compound was physiological saline (0.9% NaCl solution, etc.). The % body weight change of nude mice with tumors during the experiment was calculated as follows: (total weight on the day of reading / total weight on the day of treatment start) × 100.

[0225] Example 1 Chemical Synthesis of 1,2-Bis(hydroxymethyl)pyrrolo[2,1-a]-phthalazine Derivatives (Formula (IA)) (Scheme 1)

[0226] The compounds of formula (IA) were synthesized according to the steps described in Scheme 1. Commercially available 1-phthalazinone 19 was treated with phosphorus oxychloride to prepare compound 20, which was further treated with various ω-N,N-dialkylalkylamines, cyclic amines, anilines, 1-methylpiperazine, 1-ethylpiperazine, 1-methyl-4,4'-bipiperidine or 1-ethyl-4,4'-bipiperidine in ethanol to prepare compound 21. Compound 21 can then be reacted with trimethyl silylcyanide (TMSCN) and an alkyl or aryl chloride in dichloromethane to produce compound 22, which was converted to the hydrofluoroborate salt by treatment with tetrafluoroboric acid (HBF4) in diethyl ether and subsequently with dimethyl acetylenedicarboxylate (DMAD) to prepare the diester derivative 23. The diester functionality of derivative 23 was reduced to the corresponding bis(hydroxymethyl) derivative 24 by reaction with LiAlH4 (in a mixture of ether / CH2Cl2) under ice-bath. Similarly, the bis(hydroxymethyl) derivative 24 was converted to its corresponding bis(alkylcarbamate) congener by reaction with various isocyanates to prepare compound 25 (where R 1 is -CH2OCONHR, R is alkyl or aryl), or treated with anhydride (in pyridine) to prepare compound 25 (where R 1 is -CH2OCOR), or by reacting with toluene- or methanesulfonyl chloride / Et3N to prepare compound 25 (wherein R 1 is –CH2OSO2R, R is Me or 4-MePh), and other good leaving groups.

[0227] Process 1

[0228]

[0229] Reagents and conditions: a) POCl3, reflux; b) R 3 NH2, ethanol, reflux; c) TMSCN, AlCl3, R 2 COCl, DCM; d) HBF4, AcOH; e) DMAD, DMF, reflux; f) LiAlH4, THF; g) RNCO, THF

[0230] Exemplary obtainable compounds of formula (IA) include the following compounds:

[0231]

[0232]

[0233]

[0234] 1.1 (3-methyl-6-morpholinylpyrrolo[2,1-a]phthalazine-1,2-diyl)-dimethanol (BO-2571) and (3-methyl-6-morpholinylpyrrolo[2,1-a]phthalazine-1,2-diyl) bis(methylene) bis(ethylcarbamate) (BO-2573)

[0235] (1) 1-Chlorophthalazine

[0236] A mixture of commercially available phthalazin-1(2H)-one (5.0 g, 34.0 mmol) and phosphorus oxychloride (POCl3) (25 mL) was heated and stirred at 100°C for 2 h. After cooling to room temperature, the excess POCl3 was completely distilled off under reduced pressure. The residue was triturated with toluene (2×25 mL) and then with THF (100 mL), and the solid product was collected by filtration and washed with THF. Then, it was dissolved in DCM, washed with saturated aqueous NaHCO3 solution, dried over sodium sulfate and evaporated under reduced pressure to prepare 1-chlorophthalazine. Yield 4.6 g (82%); melting point 119–121°C (literature reported value: melting point 132–134°C).

[0237] 1 H NMR (DMSO-d6) δ 8.20 (2H, t, J = 7.2 Hz, ArH), 8.33 (2H, t, J = 7.6 Hz, ArH), 9.73 (1H, s, ArH).

[0238] 13 C NMR (DMSO–d6) δ126.1, 128.4, 128.7, 155.3. HRMS[ESI + ]: Calculated for C8H5ClN2, 165.0220 [M+H] + , the measured value is 165.0212.

[0239] (2) 4-(Phthalocyanine-1-yl)morpholine

[0240] Morpholine (1.89 mL, 22.0 mmol) was added dropwise to a solution of 1-chlorophthalazine (3.64 g, 20.0 mmol) in ethanol (120 mL) containing triethylamine (6.96 mL, 50.0 mmol). The reaction mixture was heated under reflux for 18 h, and the solvent was removed to dryness under reduced pressure. The reaction was cooled to room temperature and the solvent was evaporated. The crude product was diluted with water and then extracted twice with DCM. The separated organic layer was dried over sodium sulfate and evaporated in vacuo to prepare 4-(phthalazin-1-yl)morpholine. Brown solid; yield 3.8 g (80%); melting point 125-127°C (literature reported value: melting point 82°C).

[0241] 1 H NMR (DMSO–d6) δ3.40 (4H, t, J=4.8Hz, 2×CH2), 3.88 (4H, t, J=4.4Hz, 2×CH2), 7.94–7.97 (2H, m, ArH), 8.09–8.14 (2H, m, ArH), 9.31 (1H, s, ArH).

[0242] 13 C NMR (DMSO–d6) δ51.4, 51.7, 66.5, 120.7, 124.4, 124.5, 127.5, 128.5, 132.4, 132.5, 148.4, 159.7. HRMS[ESI + ]: Calculated as C 12 H 13 N3O, 216.1137[M+H] + , the measured value is 216.1094.

[0243] (3) 2-Acetyl-4-morpholinyl-1,2-dihydrophthalazine-1-carbonitrile

[0244] Me3SiCN (2.32 mL, 18.6 mmol) was added dropwise to a solution of 4-(phthalazin-1-yl)morpholine (2.0 g, 9.3 mmol) in DCM (30 mL) containing a catalytic amount of AlCl3. Acetyl chloride (1.0 mL, 14.0 mmol) was then added dropwise to the mixture at room temperature and stirred for 4 hours. The reaction mixture was poured into ice-water and the organic layer was washed with water, 5% NaOH solution and water in sequence. The solution was dried over sodium sulfate and concentrated in vacuo to prepare 2-acetyl-4-morpholinyl-1,2-dihydrophthalazine-1-carbonitrile. Yield 2.36 g (89%); m.p. 140-142°C.

[0245] 1H NMR (CDCl3) δ2.32 (3H, s, CH3), 3.13–3.18 (2H, m, CH2), 3.44–3.49 (2H, m, CH2), 3.81–3.86 (2H, m, CH2), 3.94–3.99 (2H, m, CH2), 6.70 (1H, s, CH), 7.43 (1H, d, J = 7.6Hz, ArH), 7.51–7.59 (3H, m, ArH).

[0246] 13 C NMR (CDCl3) δ16.9, 49.8, 55.8, 66.7, 116.4, 121.5, 124.0, 123.7, 129.2, 130.3, 132.8, 155.5, 169.6. HRMS[ESI + ]: Calculated as C 15 H 16 N4O2, 285.1352[M+H] + , the measured value is 285.1341.

[0247] (4) Dimethyl-3-methyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-dicarboxylic acid

[0248] HBF4 (1.55 mL) was added dropwise to a solution of 2-acetyl-4-morpholino-1,2-dihydrophthalazine-1-carbonitrile (2.0 g, 7.0 mmol) in warm acetic acid (50 mL). The mixture was stirred at 50-60°C for 30 min. After cooling to room temperature, the yellow solid salt was collected by filtration and the filter cake was washed with dry ether. The solid salt was dissolved in DMF (20 mL) and DMAD (1.6 mL, 13 mmol) was slowly added to the solution. The reaction mixture was heated at 90-100°C for 16 h. The solvent was removed by evaporation in vacuo. The residue was crystallized from MeOH to prepare dimethyl-3-methyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-dicarboxylic acid. Yield 1.5 g (56%); m.p. 182-183°C.

[0249] 1 H NMR(DMSO–d6)δ2.64(3H,s,CH3), 3.30–3.31(4H,m,2×CH2), 3.80(3H,s,COOCH3), 3.87(3H,s,COOCH3), 3.87–3.88(4H,m , 2×CH2), 7.64 (1H, t, J=8.0Hz, ArH), 7.81 (1H, t, J=7.5Hz, ArH), 8.06 (1H, d, J=8.0Hz, ArH), 8.25 (1H, d, J=8.5Hz, ArH).

[0250] 13 C NMR (DMSO–d6) δ13.9, 52.4, 52.7, 55.6, 66.2, 106.7, 115.8, 117.7, 121.0, 124.6, 126.7, 128.9, 130.3, 133.0, 156.2, 159.7, 165.6, 165.9; HRMS [ESI + ]: Calculated as C 20 H 21 N3O5, 406.1379[M+Na] + , the measured value is 406.1385.

[0251] (5) Dimethyl-3-methyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-dicarboxylic acid

[0252] HBF4 (1.55 mL) was added dropwise to a solution of dimethyl-3-methyl-6-morpholinylpyrrolo[2,1-a]phthalazine-1,2-dicarboxylic acid (2.0 g, 7.0 mmol) in warm acetic acid (50 mL). The mixture was stirred at 50-60°C for 30 min. After cooling to room temperature, the yellow solid salt was collected by filtration and the filter cake was washed with dry ether. The solid salt was dissolved in DMF (20 mL) and DMAD (1.6 mL, 13 mmol) was slowly added to the solution. The reaction mixture was heated at 90-100°C for 16 h. The solvent was removed by evaporation in vacuo. The residue was crystallized from MeOH to prepare dimethyl-3-methyl-6-morpholinylpyrrolo[2,1-a]phthalazine-1,2-dicarboxylic acid. Yield 1.5 g (56%); m.p. 182-183°C.

[0253] 1 H NMR(DMSO–d6)δ2.64(3H,s,CH3), 3.30–3.31(4H,m,2×CH2), 3.80(3H,s,COOCH3), 3.87(3H,s,COOCH3), 3.87–3.88(4H,m , 2×CH2), 7.64 (1H, t, J=8.0Hz, ArH), 7.81 (1H, t, J=7.5Hz, ArH), 8.06 (1H, d, J=8.0Hz, ArH), 8.25 (1H, d, J=8.5Hz, ArH).

[0254] 13C NMR (DMSO–d6) δ13.9, 52.4, 52.7, 55.6, 66.2, 106.7, 115.8, 117.7, 121.0, 124.6, 126.7, 128.9, 130.3, 133.0, 156.2, 159.7, 165.6, 165.9; HRMS [ESI + ]: Calculated as C 20 H 21 N3O5, 406.1379[M+Na] + , the measured value is 406.1385.

[0255] (6) (3-methyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-diyl) dimethanol (BO-2571)

[0256] A solution of dimethyl-3-methyl-6-morpholinylpyrrolo[2,1-a]phthalazine-1,2-dicarboxylic acid (1.5 g, 3.9 mmol) in DCM (50 mL) was added dropwise to a stirred suspension of LAH (0.37 g, 9.7 mmol) in ether (20 mL) at 0–5°C. After 2 h of reaction, water (2 mL) and NH4OH (2 mL) were added to decompose the excess LAH. The reaction mixture was filtered through a calcite pad and washed thoroughly with DCM. The combined filtrate and washings were evaporated to dryness in vacuo. The residue was recrystallized from ethanol to prepare (3-methyl-6-morpholinylpyrrolo[2,1-a]phthalazine-1,2-diyl) dimethanol (BO-2571). Yield 1.0 g, (80%), m.p. 184–186°C.

[0257] 1 H NMR(DMSO–d6)δ2.46(3H,s,CH3), 3.23(4H,s,2×CH2), 3.87(4H,s,2×CH2), 4.58(3H,s,1×OCH2 and 1×OH), 4.78–4.83(3H,m,1×OCH2 and 1×OH), 7.45 (1H, t, J=7.5Hz, ArH), 7.73 (1H, t, J=7.5Hz, ArH), 7.97 (1H, d, J=8.0Hz, ArH), 8.29 (1H, d, J=7.5Hz, ArH).

[0258] 13 C NMR (DMSO–d6) δ9.3, 51.9, 53.8, 54.4, 66.5, 114.4, 115.7, 118.3, 121.6, 123.2, 123.6, 125.3, 126.0, 130.2, 132.3, 154.1. HRMS[ESI+ ]: Calculated as C 18 H 21 N3O3, 310.1556 [M+H–H2O] + , the measured value is 310.1569.

[0259] (7) (3-methyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-diyl)bis(methylene)bis(ethylcarbamate) (BO-2573)

[0260] A mixture of 3-methyl-6-morpholinylpyrrolo[2,1-a]phthalazine-1,2-diyl)-dimethanol (0.15 g, 0.5 mmol), TEA (0.25 mL, 2.0 mmol) and ethyl isocyanate (0.15 mL, 2.0 mmol) in dry DMF was stirred at room temperature under argon for 24-48 hours. After the reaction was complete, the reaction mixture was evaporated to dryness in vacuo. The residue was triturated with diethyl ether and the desired product was collected by filtration to prepare (3-methyl-6-morpholinylpyrrolo[2,1-a]phthalazine-1,2-diyl)bis(methylene)bis(ethylcarbamate) (BO-2573). Yield 0.16 g (75%); m.p. 171-173°C.

[0261] 1 H NMR (DMSO–d6) δ0.97 (6H, t, J=6.5Hz, 2×CH3), 2.46 (3H, s, CH3), 2.97–2.98 (4H, m, 2×CH2), 3.22 (4H, s, 2×CH2), 3.85 (4H, s, 2×CH2), 5.17 (2H, s, OCH2), 5.38 (2H, s, OCH2), 7.01 (2H, brs, 2×NH), 7.49 (1H, t, J=7.5Hz, ArH), 7.76 (1H, t, J=7.0Hz, ArH), 7.99 (1H, d, J=8.0Hz, ArH), 8.10 (1H, d, J=7.0Hz, ArH).

[0262] 13 C NMR (DMSO–d6) δ9.35, 15.5, 35.4, 51.9, 56.5, 57.3, 66.4, 109.5, 116.1, 117.6, 119.2, 122.9, 125.2, 126.2, 129.6, 132.7, 154.6, 156.5, 156.6. HRMS[ESI + ]: Calculated as C 24 H 31 N5O5, 294.1606[M+H–2(OCONHC2H5)]+ , the measured value is 294.1602.

[0263] 1.2 (3-methyl-6-(pyrrolidin-1-yl)pyrrolo[2,1-a]phthalazine-1,2-diyl) dimethanol (BO-2686) and (3-methyl-6-(pyrrolidin-1-yl)pyrrolo[2,1-a]phthalazine-1,2-diyl) bis(methylene) bis(ethylcarbamate) (BO-2716)

[0264] (1) 1-(Pyrrolidin-1-yl)phthalazine

[0265] Pyrrolidine (20.0 mL, 240.0 mmol) was slowly added to a solution of 1-chlorophthalazine (10.0 g, 60.0 mmol) in ethanol (200 mL) containing TEA (30.0 mL, 200.0 mmol). The reaction mixture was stirred at room temperature for 48 hours. After the reaction was complete, the solvent was evaporated and the residue was diluted with water and then extracted with DCM (2 x 200 mL). The organic layer was dried over sodium sulfate and then evaporated in vacuo to prepare the desired 1-(pyrrolidin-1-yl)phthalazine product. Yield 10.0 g (83%); m.p. 90-91 °C.

[0266] 1 H NMR (DMSO–d6)δ 1 H NMR (DMSO–d6) δ1.95–1.98 (4H, m, 2×CH2), 3.82 (4H, t, J=6.5Hz, 2×CH2), 7.80–7.88 (2H, m, ArH), 7.94–7.95 (1H, m, ArH), 8.28 (1H, d, J = 8.5Hz, ArH), 8.97 (1H, s, ArH).

[0267] 13 C NMR (DMSO–d6) δ 25.8, 51.1, 119.2, 125.2, 126.5, 128.7, 130.9, 131.6, 144.1, 155.7. HRMS [ESI+]: Calculated for C 12 H 13 N3, 200.1188[M+H] + , measured value 200.1209.

[0268] (2) 2-Acetyl-4-(pyrrolidin-1-yl)-1,2-dihydrophthalazine-1-carbonitrile

[0269] Me3SiCN (6.3 mL, 50.0 mmol) was added dropwise to a solution of 1-(pyrrolidin-1-yl)phthalazine (5.0 g, 25.0 mmol) in DCM (30 mL) containing a catalytic amount of AlCl3. Acetyl chloride (2.7 mL, 37.5 mmol) was then added dropwise to the mixture at room temperature and stirred for 4 hours. The reaction mixture was poured into ice-water and the organic layer was washed with water, 5% NaOH solution and water in sequence. The solution was dried over sodium sulfate and then concentrated in vacuo to prepare 2-acetyl-4-(pyrrolidin-1-yl)-1,2-dihydrophthalazine-1-carbonitrile. Yield 5.7 g (85%); m.p. 123-125°C.

[0270] 1 H NMR(DMSO–d6)δ1.83(2H,t,J=6.8Hz,CH2)2.00(2H,s,CH2),2.21(3H,s,CH3),3.28–3.34(2H,m,C H2), 3.70–3.75 (2H, m, CH2), 7.06 (1H, s, CH), 7.59–7.66 (2H, m, ArH), 7.81 (2H, d, J = 7.4Hz, ArH).

[0271] 13 C NMR (DMSO–d6) δ20.8, 25.3, 49.8, 116.9, 122.7, 126.7, 127.3, 129.7, 130.4, 132.3, 153.5, 170.8. HRMS[ESI + ]: Calculated as C 15 H 16 N4O, 269.1402[M+H] + , the measured value is 269.1416.

[0272] (3) Dimethyl-3-methyl-6-(pyrrolidin-1-yl)pyrrolo[2,1-a]phthalazine-1,2-dicarboxylic acid

[0273] HBF4 (3.6 mL) was added dropwise to a solution of 2-acetyl-4-(pyrrolidin-1-yl)-1,2-dihydrophthalazine-1-carbonitrile (5.0 g, 18.6 mmol) in warm acetic acid (50 mL). The mixture was stirred at 50-60°C for 30 min. After cooling to room temperature, the yellow solid salt was collected by filtration and the filter cake was washed with dry ether. The solid salt was dissolved with DMF (20 mL) and DMAD (4.5 mL, 37.0 mmol) was slowly added to the solution. The reaction mixture was heated at 90-100°C for 16 h. The solvent was removed by evaporation in vacuo. The residue was crystallized from MeOH to prepare dimethyl-3-methyl-6-(pyrrolidin-1-yl)pyrrolo[2,1-a]-phthalazine-1,2-dicarboxylic acid. Yield 2.5 g (48%); m.p. 176-178°C.

[0274] 1 H NMR(DMSO–d6)1.92(4H,s,2×CH2), 2.57(3H,s,CH3), 3.65(4H,s,2×CH2), 3.78(3H,s,COOCH3), 3.86(3H , s, COOCH3), 7.55 (1H, t, J = 7.7Hz, ArH), 7.73 (1H, t, J = 7.7Hz, ArH), 8.14 (1H, d, J = 8.2Hz, ArH), 8.20 (1H d, J=8.1Hz, ArH).

[0275] 13 C NMR (DMSO–d6) δ10.5, 25.7, 51.3, 51.9, 52.7, 106.7, 111.7, 117.8, 120.1, 123.0, 127.4, 127.7, 128.1, 129.4, 132.4, 153.7, 165.0, 167.3. HRMS[ESI + ]: Calculated as C 20 H 21 N3O4, 368.1610[M+H] + , the measured value is 368.1581.

[0276] (4) (3-methyl-6-(pyrrolidin-1-yl)pyrrolo[2,1-a]phthalazine-1,2-diyl) dimethanol (BO-2686)

[0277] A solution of dimethyl-3-methyl-6-(pyrrolidin-1-yl)pyrrolo[2,1-a]-phthalazine-1,2-dicarboxylic acid (2.2 g, 6.23 mmol) in DCM (50 mL) was added dropwise to a stirred suspension of LAH (0.6 g, 15.5 mmol) in ether (50 mL) at 0–5°C. After 2 h of reaction, water (2 mL) and NH4OH (2 mL) were added to decompose the excess LAH. The reaction mixture was filtered through a calcite pad and washed thoroughly with DCM. The combined filtrate and washings were evaporated to dryness in vacuo. The residue was recrystallized from ethanol to prepare (3-methyl-6-(pyrrolidin-1-yl)pyrrolo[2,1-a]phthalazine-1,2-diyl)dimethanol (BO-2686). Yield 1.6 g (83%), mp 160–162°C.

[0278] 1 H NMR (DMSO–d6) δ1.94 (4H t, J=6.5Hz, 2×CH2), 2.42 (3H, s, CH3), 3.58 (4H, t, J=6.4Hz, 2×CH2), 4.51–4.56 (3H, m, 1×OH and 1×OCH2), 4.72 (1H, t, J=5.2Hz, OH), 4.80 (2H, d, J=5.1Hz, OCH2), 7.38–7.41 (1H, m, A rH), 7.68–7.71 (1H, m, ArH), 8.05 (1H, d, J = 8.1Hz, ArH), 8.26 (1H, d, J = 8.0Hz, ArH).

[0279] 13 C NMR (DMSO–d6) δ9.3, 25.3, 51.3, 53.8, 54.5, 113.8, 116.6, 117.9, 120.8, 122.4, 123.3, 124.7, 126.7, 130.3, 131.8, 152.5. HRMS[ESI + ]: Calculated as C 18 H 21 N3O2 294.1606[M+H–H2O] + , the measured value is 294.1627.

[0280] (5) (3-methyl-6-(pyrrolidin-1-yl)pyrrolo[2,1-a]phthalazine-1,2-diyl)-bis(methylene)bis(ethylcarbamate) (BO-2716)

[0281] A mixture of (3-methyl-6-(pyrrolidin-1-yl)pyrrolo[2,1-a]phthalazine-1,2-diyl)-dimethanol (0.15 g, 0.5 mmol) (0.16 g, 0.5 mmol), ethyl isocyanate (0.2 mL, 2.0 mmol) and TEA (0.3 mL, 2.0 mmol) in anhydrous DMF was stirred at room temperature under argon for 24-48 hours. After the reaction was complete, the reaction mixture was evaporated to dryness in vacuo. The residue was triturated with ether and the desired product was collected by filtration to prepare (3-methyl-6-(pyrrolidin-1-yl)pyrrolo[2,1-a]phthalazine-1,2-diyl)bis(methylene)bis(ethylcarbamate) (BO-2716). Yield 0.13 g, (70%); m.p. 140-142 °C.

[0282] 1 H NMR (DMSO–d6) δ0.96–0.99 (6H, m, 2×CH3), 1.93 (4H, t, J=6.5Hz, 2×CH2), 2.43 (3H, s, CH3), 2.96–3.00 (4H, m, 2×CH2), 3.60 (4H, s, 2×CH2), 5.15 (2H, s, OCH2) , 5.36 (2H, s, OCH2), 6.98–7.03 (2H, m, 2×NH), 7.46 (1H, t, J=7.3Hz, ArH), 7.72 (1H, t, J=7.3Hz, ArH), 8.06 (1H, d, J=8.4Hz, ArH), 8.10 (1H, d, J=8.2Hz, ArH).

[0283] 13 C NMR (DMSO–d6) δ9.3, 15.5, 25.4, 35.5, 51.3, 56.6, 57.5, 108.8, 116.8, 117.0, 118.8, 122.6, 125.6, 127.1, 129.7, 132.2, 152.9, 156.6, 156.7. HRMS[ESI + ]: Calculated as C 24 H 31 N5O4, 278.1657[M+H–2(OCONHC2H5)] + , the measured value is 278.1660.

[0284] 1.3(6-([1,4'-bipiperidinyl]-1'-yl)-3-methylpyrrolo[2,1-a]phthalazine-1,2-diyl)dimethanol (BO-2590)

[0285] (1) 1-([1,4'-bipiperidinyl]-1'-yl)phthalazine

[0286] 1,4'-Bipiperidine (20.0 g, 120.0 mmol) was slowly added to a solution of 1-chlorophthalazine (10.0 g, 60.0 mmol) in ethanol (200 mL) containing TEA (34 mL, 240.0 mmol). The reaction mixture was stirred at room temperature for 48 hours. After the reaction was completed, the solvent was evaporated and the residue was diluted with water and extracted with DCM (2 x 200 mL). The organic layer was dried over sodium sulfate and evaporated in vacuo to prepare the desired 1-([1,4'-bipiperidinyl]-1'-yl)phthalazine product. Yield 10.2 g (57%); melting point 140-142 °C.

[0287] 1 H NMR (DMSO–d6) δ 1.37–1.42 (2H, m, CH2), 1.48–1.53 (4H, m, CH2), 1.75–1.81 (2H, m, CH2), 1.88–1.91 (2H, m, CH2), 2.43 (1H, s, CH), 2.57 (4H, s, 2×CH2), 2.94–3.00 (2H, m, CH2), 3.87–3.90 (2H, m, CH2), 7.92–7.94 (2H, m, ArH), 8.04–8.06 (2H, m, ArH), 9.25 (1H, s, ArH). HRMS [ESI+]: calcd for C 18 H 24 N4, 297.2079 [M+H] + , the measured value is 297.2092.

[0288] (2) 4-([1,4'-bipiperidinyl]-1'-yl)-2-acetyl-1,2-dihydrophthalazine-1-carbonitrile

[0289] Me3SiCN (1.69 mL, 13.5 mmol) was added dropwise to a solution of 1-([1,4'-bipiperidinyl]-1'-yl)phthalazine (2.0 g, 6.7 mmol) in DCM (30 mL) containing a catalytic amount of AlCl3. Acetyl chloride (0.88 mL, 10.0 mmol) was then added dropwise to the mixture at room temperature and stirred for 4 hours. The reaction mixture was poured into ice-water and the organic layer was washed with water, 5% NaOH solution and water in sequence. The solution was dried over sodium sulfate and concentrated in vacuo to prepare 4-([1,4'-bipiperidinyl]-1'-yl)-2-acetyl-1,2-dihydrophthalazine-1-carbonitrile. Yield 1.45 g, (60%); m.p. 160-162°C.

[0290] 1H NMR(DMSO–d6)δ1.42–1.45(1H,m,CH2), 1.70–1.82(6H,m,3×CH2), 2.10–2.26(6H,m,3×CH2), 2.65(3H,s,CH3), 2.66–2.73(1H,m,CH), 2.95–3 .00 (3H, m, CH2), 3.76 (1H, d, J = 7.0Hz, CH2), 3.88 (1H, d, J = 4.5Hz, CH2), 7.00 (1H, s, CH), 7.61–7.69 (3H, m, ArH), 7.82 (1H, d, J = 7.2Hz, ArH). HRMS[ESI + ]: Calculated as C 21 H 27 N5O, 366.2294 [M+H] + , the measured value is 366.2308.

[0291] (3) Dimethyl-6-([1,4'-bipiperidinyl]-1'-yl)-3-methylpyrrolo[2,1-a]phthalazine-1,2-dicarboxylic acid

[0292] HBF4 (0.7 mL) was added dropwise to a solution of 4-([1,4'-bipiperidinyl]-1'-yl)-2-acetyl-1,2-dihydrophthalazine-1-carbonitrile (1.3 g, 3.5 mmol) in warm acetic acid (50 mL). The mixture was stirred at 50-60°C for 30 min. After cooling to room temperature, the yellow solid salt was collected by filtration and the filter cake was washed with dry ether. The solid salt was dissolved with DMF (20 mL) and DMAD (0.8 mL, 6.5 mmol) was slowly added to the solution. The reaction mixture was heated at 90-100°C for 16 h. The solvent was removed by evaporation in vacuo. The residue was crystallized from MeOH to prepare dimethyl-6-([1,4'-bipiperidinyl]-1'-yl)-3-methylpyrrolo[2,1-a]-phthalazine-1,2-dicarboxylic acid. Yield 1.0 g (70%); mp 189-191°C.

[0293] 1H NMR(DMSO–d6)δ1.43–1.46(3H,m,CH2), 1.68–1.72(2H,m,CH2), 1.88(2H,s,CH2), 2.02–2.04(2H , m, CH2), 2.14–2.16 (2H, m, CH2), 2.65 (3H, s, CH3), 3.01 (4H, m, 2 × CH2), 3.17 (1H, s, CH), 3.49–3. 51(3H,m,CH2), 3.81(3H,s,COOCH3), 3.86–3.88(2H,m,CH2), 3.89(3H,s,COOCH3), 7.66(1H,t,J =8.0Hz, ArH), 7.83 (1H, t, J = 7.2Hz, ArH), 8.00 (1H, d, J = 8.4Hz, ArH), 8.27 (1H, d, J = 8.4Hz, ArH).

[0294] 13 C NMR (DMSO–d6) δ12.6, 24.5, 26.2, 28.1, 50.4, 51.5, 58.2, 58.9, 67.5, 70.4, 1 11.6, 118.1, 123.4, 127.6, 128.5, 129.8, 132.8, 133.5, 147.4, 165.9, 175.7. HRMS[ESI + ]: Calculated as C 26 H 32 N4O4, 465.2502[M+H] + , the measured value is 465.2501.

[0295] (4) (6-([1,4'-bipiperidinyl]-1'-yl)-3-methylpyrrolo[2,1-a]phthalazine-1,2-diyl) dimethanol (BO-2590)

[0296] A solution of dimethyl-6-([1,4'-bipiperidinyl]-1'-yl)-3-methylpyrrolo[2,1-a]-phthalazine-1,2-dicarboxylic acid (0.65 g, 1.31 mmol) in DCM (50 mL) was added dropwise to a stirred suspension of LAH (0.10 g, 4.5 mmol) in ether (50 mL) at 0-5°C. After the reaction was completed in 2 hours, water (2 mL) and NH4OH (2 mL) were added to decompose the excess LAH. The reaction mixture was filtered through a calcite pad and then washed thoroughly with DCM. The combined filtrate and washings were evaporated to dryness in vacuo. The residue was recrystallized from ethanol to prepare (6-([1,4'-bipyridinyl]-1'-yl)-3-methylpyrrolo[2,1-a]phthalazine-1,2-diyl) dimethanol. Yield 0.42 g, (78%), melting point 183-185°C.

[0297] 1 H NMR (DMSO–d6) δ1.41 (2H, s, CH2), 1.52 (4H, s, 2×CH2), 1.76–1.88 (4H, m, 2×CH2), 2.43 (3H, s, CH3), 2.50–2.5 3(4H, m, 2×CH2), 2.83 (2H, t, J=12.0Hz, CH2), 3.33 (1H, m, CH), 3.63 (2H, d, J=15.0Hz, CH2), 4.55 (3H, s, 1×OH and 1×OCH2), 4.74 (1H, s, OH), 4.80 (2H, s, OCH2), 7.43 (1H, t, J=7.2Hz, ArH), 7.71 (1H, t, J=7.2Hz, ArH), 7.89 (1H, d, J=8.4Hz, ArH), 8.26 (1H, d, J=8.4Hz, ArH).

[0298] 13 C NMR (DMSO–d6) δ13.5, 16.5, 24.4, 25.9, 27.2, 49.6, 50.8, 53.0, 53.8, 61.7, 66.8 , 113.6, 115.6, 117.5, 120.2, 123.0, 124.7, 125.4, 128.1, 129.6, 131.5, 153.8. HRMS[ESI + ]: Calculated as C 24 H 32 N4O2, 409.2604[M+H] + , the measured value is 409.2638.

[0299] 1.4(3-ethyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-diyl)-dimethanol (BO-2577)

[0300] (1) 4-(Phthalocyanine-1-yl)morpholine

[0301] Morpholine (1.89 ml, 22.0 mmol) was added dropwise to a solution of compound 11 (3.64 g, 20.0 mmol) in ethanol (120 ml) containing triethylamine (6.96 ml, 50.0 mmol). The reaction mixture was heated under reflux for 18 hours, and the solvent was removed to dryness under reduced pressure. The reaction was cooled to room temperature and the solvent was evaporated. The crude product was diluted with water and then extracted twice with DCM. The separated organic layer was dried over sodium sulfate and then evaporated in vacuo to prepare 4-(phthalazin-1-yl)morpholine. Brown solid; yield 3.8 g (80%); melting point 125-127°C (literature reported value: melting point 82°C).

[0302] 1 H NMR (DMSO–d6) δ3.40 (4H, t, J=4.8Hz, 2×CH2), 3.88 (4H, t, J=4.4Hz, 2×CH2), 7.94–7.97 (2H, m, ArH), 8.09–8.14 (2H, m, ArH), 9.31 (1H, s, ArH).

[0303] 13 C NMR (DMSO–d6) δ51.4, 51.7, 66.5, 120.7, 124.4, 124.5, 127.5, 128.5, 132.4, 132.5, 148.4, 159.7. HRMS[ESI + ]: Calculated as C 12 H 13 N3O, 216.1137[M+H] + , the measured value is 216.1094.

[0304] (2) 4-Morpholinyl-2-propionyl-1,2-dihydrophthalazine-1-carbonitrile

[0305] Me3SiCN (2.32 mL, 18.6 mmol) was added dropwise to a solution of 4-(phthalazin-1-yl)morpholine (2.0 g, 9.3 mmol) in DCM (30 mL) containing a catalytic amount of AlCl3. Propionyl chloride (1.2 mL, 14.0 mmol) was then added dropwise to the mixture at room temperature and stirred for 4 hours. The reaction mixture was poured into ice-water and the organic layer was washed with water, 5% NaOH solution and water in sequence. The solution was dried over sodium sulfate and concentrated in vacuo to prepare 4-morpholinyl-2-propionyl-1,2-dihydrophthalazine-1-carbonitrile. Yield 2.46 g (89%); m.p. 146-148°C.

[0306] 1 H NMR (DMSO–d6) δ1.05 (3H, t, J=5.6Hz, CH3), 2.53–2.58 (1H, m, CH2), 2.71–2.75 (1H, m, CH2), 3.03 (2H, s, CH2 ), 3.35(2H,s,CH2), 3.70(2H,s,CH2), 3.87(2H,s,CH2), 7.04(1H,s,CH), 7.64(3H,s,CH), 7.80(1H,s,ArH).

[0307] 13C NMR (DMSO–d6) δ49.8, 51.8, 65.7, 116.4, 121.5, 124.0, 123.7, 129.2, 130.3, 132.8, 155.5, 169.6. HRMS[ESI + ]: Calculated as C 16 H 18 N4O2, 299.1508[M+H] + , the measured value is 299.1519.

[0308] (3) Dimethyl 3-ethyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-dicarboxylic acid

[0309] HBF4 (1.30 mL) was added dropwise to a solution of 4-morpholino-2-propionyl-1,2-dihydrophthalazine-1-carbonitrile (2.0 g, 7.0 mmol) in warm acetic acid (50 mL). The mixture was stirred at 50-60°C for 30 min. After cooling to room temperature, the yellow solid salt was collected by filtration and the filter cake was washed with dry ether. The solid salt was dissolved in DMF (20 mL) and DMAD (1.5 mL, 12.0 mmol) was slowly added to the solution. The reaction mixture was heated at 90-100°C for 16 h. The solvent was removed by evaporation in vacuo. The residue was crystallized from MeOH to prepare dimethyl-3-methyl-6-morpholinopyrrolo[2,1-a]-phthalazine-1,2-dicarboxylic acid. Yield 1.6 g (60%); m.p. 182-183°C.

[0310] 1 H NMR (DMSO–d6) δ1.21 (3H, s, CH3), 3.15 (2H, d, J=6.0Hz, CH2), 3.30 (4H, s, 2×CH2), 3.80 (3H, s, COOCH3), 3.89 (7H, s, 2×CH2 and 1×COOCH3), 7.63 (1H, s, ArH), 7.80 (1H, s, ArH), 8.06 (1H, d, J = 7.0 Hz, ArH), 8.26 (1H, d, J = 7.0 Hz, ArH).

[0311] 13 C NMR (DMSO–d6) δ13.9, 23.0, 52.4, 52.7, 55.6, 66.2, 106.7, 115.8, 117.7, 121.0, 124.6, 126.7, 128.9, 130.3, 133.0, 156.2, 159.7, 165.6, 165.9. HRMS[ESI + ]: Calculated as C 21 H 23N3O5, 420.1535[M+Na] + , the measured value is 420.1552.

[0312] (4) (3-ethyl-6-morpholinopyrrolo[2,1-a]phthalazine-1,2-diyl) dimethanol (BO-2577)

[0313] A solution of dimethyl 3-ethyl-6-morpholinylpyrrolo[2,1-a]-phthalazine-1,2-dicarboxylic acid (1.4 g, 3.5 mmol) in DCM (50 mL) was added dropwise to a stirred suspension of LAH (0.43 g, 10.5 mmol) in ether (20 mL) at 0–5°C. After 2 h of reaction, water (2 mL) and NH4OH (2 mL) were added to decompose the excess LAH. The reaction mixture was filtered through a calcite pad and washed thoroughly with DCM. The combined filtrate and washings were evaporated to dryness in vacuo. The residue was recrystallized from ethanol to prepare (3-ethyl-6-morpholinylpyrrolo[2,1-a]phthalazine-1,2-diyl) dimethanol (BO-2577). Yield 1.2 g (90%); m.p. 180–182°C.

[0314] 1 H NMR (DMSO–d6) δ1.21 (3H, t, J = 7.5Hz, CH3), 2.93–2.98 (2H, m, CH2), 3.21 (4H, t, J = 4.5Hz, 2×CH2), 3.86 (4H, t, J = 4.5Hz, 2×CH2), 4.57 (3H, s, 1×OCH2 and 1×OH), 4.76 (1H, t, J=5.5Hz, OH), 4.82 (2H, d, J=5.0Hz, OCH2), 7.43 (1H, t, J=8.5Hz, Ar H), 7.72 (1H, t, J=8.0Hz, ArH), 7.95 (1H, d, J=7.5Hz, ArH), 8.29 (1H, d, J=8.0Hz, ArH).

[0315] 13 C NMR (DMSO–d6) δ 13.5, 16.5, 51.3, 53.1, 53.8, 65.8, 113.7, 115.2, 117.5, 120.4, 123.0, 124.7, 125.4, 128.3, 129.7, 131.7, 153.4. HRMS [ESI+]: calculated as C 19 H 23 N3O3, 364.1637[M+Na]+, measured value 364.1638.

[0316] Example 2 Chemical Synthesis of Benzo[g]pyrrolo[2,1-α]phthalazine Derivatives (Formula (IB)) (Scheme 2)

[0317] The compounds of formula (I-B) were synthesized by the steps shown in Scheme 2. Commercially available 2,3-naphthalenedicarboxylic anhydride 26 was treated with hydrazine hydrate (in acetic acid) to produce compound 27, which was reacted with phosphorus oxychloride to produce compound 28. Compound 28 was treated with various ω-N,N-dialkylalkylamines, cyclic amines, anilines, 1-methylpiperazine, 1-ethylpiperazine, 1-methyl-4,4'-bipiperidine or 1-ethyl-4,4'-bipiperidine and potassium carbonate (in acetonitrile) to produce compound 29, which was reacted with 10% Pd / C in methanol and H2 to produce compound 30. Compound 30 was further reacted with trimethylsilyl cyanide and alkyl or aryl chloride (in dichloromethane) to produce compound 31. Similarly, compound 31 was converted to the diester derivative 32 by treatment with fluoroboric acid / dimethyl butynedioate (DMAD) as described above. The diester functionality of derivative 32 was reduced to the corresponding bis(hydroxymethyl) derivative 33 (Formula I-B) by reaction with LiAlH4 (in a mixture of ether / CH2Cl2) under ice-bath. The bis(hydroxymethyl) derivative 33 can then be converted to its corresponding bis(alkylcarbamate) analog by treatment with various isocyanates to prepare compound 34 (where R 1 is -CH2OCONHR, R is alkyl or aryl), or treated with anhydride (in pyridine) to prepare compound 34 (where R 1 is -CH2OCOR), or by reacting with toluene- or methanesulfonyl chloride / Et3N to prepare compound 34 (wherein R 1 is –CH2OSO2R, R is Me or 4-MePh), and other good leaving groups.

[0318] Process 2

[0319]

[0320] Reagents and conditions: a) hydrazine hydrate / acetic acid, 100°C; b) POCl3 / pyridine, reflux; c) R 3 NH, K2CO3, ACN, reflux; d) Pd / C, methanol, reflux; e) TMSCN, AlCl3, R 2 COCl, MDC, room temperature; f) HBF4, AcOH; g) DMAD, DMF, 100°C; h) LiAlH4, ether / MDC, 0-30°C; i) RNCO, TEA, THF, reflux

[0321] Exemplary obtainable compounds of formula (IB) include the following compounds:

[0322]

[0323]

[0324] Compound No. <![CDATA[R 3 ]]> <![CDATA[R 2 ]]> <![CDATA[R 1 ]]> Melting point℃ BO–2768 Dimethylamine Me <![CDATA[–CH2OH]]> 156–158 BO–2762 Pyrrolidine Me <![CDATA[–CH2OH]]> 160–162 BO–2755 Piperidine Me <![CDATA[–CH2OH]]> 173–175 BO–2698 Morpholine Me <![CDATA[–CH2OH]]> 190–192 BO–2792 1,4'-Bispiperidine Me <![CDATA[–CH2OH]]> 181–183 BO–2772 Dimethylamine Me <![CDATA[–CH2OCONHEt]]> 132–134 BO–2763 Pyrrolidine Me <![CDATA[–CH2OCONHEt]]> 142–144 BO–2757 Piperidine Me <![CDATA[–CH2OCONHEt]]> 160–162 BO–2756 Morpholine Me <![CDATA[–CH2OCONHEt]]> 178–180 BO–2793 1,4'-Bispiperidine Me <![CDATA[–CH2OCONHEt]]> 169–171

[0325] 2.1 (6-(dimethylamino)-3-methylbenzo[g]pyrrolo[2,1-a]phthalazine-1,2-diyl) dimethanol (BO-2768) and (6-(dimethylamino)-3-methylbenzo[g]-prolog[2,1-a]phthalazine-1,2-diyl)-bis(methylene)bis(ethylcarbamate) (BO-2772)

[0326] (1) 2,3-Dihydrobenzo[g]phthalazine-1,4-dione

[0327] Hydrazine hydrate (80% solution, 63 mL) was added to a stirred suspension of naphthalene-2,3-dicarboxylic anhydride (39.7 g, 200.0 mmol) in glacial acetic acid (600 mL). The mixture was heated under reflux and stirred for 6 hours. After cooling, the solid product was collected by filtration, washed with water and dried to prepare 2,3-dihydrobenzo[g]phthalazine-1,4-dione. Yield: 40.2 g (94%); melting point 344-346 (literature reported value: melting point 344°C).

[0328] 1 H NMR (DMSO–d6): δ7.75–7.78(2H,m,ArH), 8.29–8.31(2H,m,ArH), 8.76(2H,s,ArH), 11.52(2H,s,2×NH).

[0329] 13 C NMR (DMSO–d6): δ123.8, 126.2, 128.5, 129.1, 134.1. HRMS[ESI + ]: Calculated as C 12 H8N2O2, 213.0664[M+H] + , the measured value is 213.0681.

[0330] (2) 1,4-Dichlorobenzo[g]phthalazine

[0331] A suspension of 2,3-dihydrobenzo[g]phthalazine-1,4-dione (40.0 g, 188.0 mmol) in phosphorus oxychloride (400 mL) containing pyridine (24.0 mL) was heated at 100°C for 5 hours. The reaction mixture was cooled to 40°C and then concentrated to dryness under reduced pressure. The solid residue was triturated with ether, filtered, and washed with ether. The solid product was triturated and stirred with ice-water for 30 minutes, and the solid product was collected by filtration, washed with water, and dried to prepare 1,4-dichlorobenzo[g]phthalazine. Yield 40.8 g (85%); mp 217-219°C (literature reported value: mp 217-220°C).

[0332] 1 H NMR (DMSO–d6): δ7.91–7.93 (2H, m, ArH), 8.50–8.52 (2H, m, ArH), 9.09 (2H, s, ArH).

[0333] 13 C NMR (DMSO–d6) δ124.3, 124.6, 126.2, 126.9, 128.0, 130.1, 137.4, 155.5. HRMS[ESI + ]: Calculated as C 12 H6Cl2N2, 248.9986[M+H] + , measured value 249.0000.

[0334] (3) 4-Chloro-N,N-dimethylbenzo[g]phthalazine-1-amine

[0335] Dimethylamine (30 mL, 60.0 mmol) was slowly added to a stirred suspension of 1,4-dichlorobenzo[g]phthalazine (10 g, 40.0 mmol) and anhydrous potassium carbonate (55 g, 400.0 mmol) in anhydrous acetonitrile (400 mL) at room temperature. The reaction mixture was stirred at room temperature for 72 hours and then filtered to remove the potassium carbonate. The filtrate was evaporated under reduced pressure and the solid residue was recrystallized from diethyl ether to prepare 4-chloro-N,N-dimethylbenzo[g]phthalazin-1-amine. Yield 9.3 g (90%); m.p. 90-92°C.

[0336] 1 H NMR (DMSO–d6) δ3.25 (6H, s, 2×NCH3), 7.79–7.83 (2H, m, ArH), 8.37–8.40 (2H, m, ArH), 8.84 (1H, s, ArH), 8.93 (1H, s, ArH).

[0337] 13C NMR (DMSO–d6) δ42.5, 118.9, 123.2, 125.0, 126.6, 128.6, 128.9, 129.0, 129.3, 133.9, 134.0, 147.8, 159.8. HRMS[ESI + ]: Calculated as C 14 H 12 ClN3, 258.0798[M+H] + , the measured value is 258.0791.

[0338] (4) N,N-dimethylbenzo[g]phthalazine-1-amine

[0339] 10% Pd / C (1.03 g) was added to a solution of 4-chloro-N,N-dimethylbenzo[g]phthalazin-1-amine (5.15 g, 20.0 mmol) in MeOH (200 mL). The mixture was hydrogenated at 35 psi for 4 h at room temperature and then filtered through a pad of calcite. The filter cake was washed thoroughly with MeOH. The combined filtrate and washings were concentrated under reduced pressure and dissolved in DCM (200 mL), washed with saturated aqueous NaHCO3 solution, dried over anhydrous sodium sulfate, and evaporated to dryness in vacuo. The product was purified by chromatography (SiO2, gradient 0-40% ethyl acetate in hexanes) to prepare N,N-dimethylbenzo[g]phthalazin-1-amine. Yield 2.8 g (64%); m.p. 115-117°C.

[0340] 1 H NMR (DMSO–d6) δ3.23 (6H, s, 2×NCH3), 7.72–7.76 (2H, m, ArH), 8.24 (1H, dd, J=6.8and 2.2Hz, ArH), 8.35 (1H, dd, J=6.8and 2.2Hz, ArH), 8.69 (1H, s, ArH), 8.85 (1H, s, ArH), 9.26 (1H, s, ArH).

[0341] 13 C NMR (DMSO–d6) δ42.4, 117.4, 125.1, 125.2, 126.3, 127.7, 128.2, 128.4, 129.4, 133.7, 133.8, 146.8, 159.0. HRMS[ESI + ]: Calculated as C 14 H 13 N3, 224.1188[M+H] + , the measured value is 224.1198.

[0342] (5) 2-Acetyl-4-(dimethylamino)-1,2-dihydrobenzo[g]phthalazine-1-carbonitrile

[0343] Me3SiCN (2.5 mL, 20.0 mmol) was added dropwise to a solution of N,N-dimethylbenzo[g]phthalazine-1-amine (2.22 g, 10.0 mmol) in DCM (30 mL) containing a catalytic amount of AlCl3. Acetyl chloride (1.1 mL, 15.0 mmol) was then added dropwise to the mixture and stirred for 4 h at room temperature. The reaction mixture was poured into ice-water and the organic layer was washed with water, 5% NaOH solution and water in sequence. The solution was dried over sodium sulfate and concentrated in vacuo to prepare 2-acetyl-4-(dimethylamino)-1,2-dihydrobenzo[g]phthalazine-1-carbonitrile. Yield 2.7 g (92%); m.p. 125–127°C.

[0344] 1 H NMR(DMSO–d6)δ2.29(3H,s,COCH3),2.99(6H,s,2×NCH3),7.23(1H,s,CH),7.67–7.73(2H,m,Ar H), 8.01 (1H, d, J = 7.7Hz, ArH), 8.20 (1H, d, J = 7.6Hz, ArH), 8.34 (1H, s, ArH), 8.35 (1H, s, ArH).

[0345] 13 C NMR (DMSO–d6) δ20.6, 40.9, 116.4, 118.2, 125.8, 126.3, 127.3, 127.7, 127.8, 128.8, 129.4, 132.8, 133.7, 155.7, 170.9. HRMS[ESI + ]: Calculated as C 17 H 16 N4O, 293.1402[M+H] + , the measured value is 293.1407.

[0346] (6) Dimethyl 6-(dimethylamino)-3-methylbenzo[g]pyrrolo[2,1-a]phthalazine-1,2-dicarboxylic acid

[0347] HBF4 (2.2 mL, 12.0 mmol) was added dropwise to a solution of 2-acetyl-4-(dimethylamino)-1,2-dihydrobenzo[g]phthalazine-1-carbonitrile (2.95 g, 10.0 mmol) in warm acetic acid (80 mL). The mixture was stirred at 50-60°C for 30 min. After cooling to room temperature, the yellow solid salt was collected by filtration and the filter cake was washed with dry ether. The solid salt was dissolved in DMF (20 mL) and DMAD (3.1 mL, 25.0 mmol) was slowly added to the solution. The reaction mixture was heated at 90-100°C for 16 h. The solvent was removed by evaporation in vacuo. The residue was recrystallized from MeOH to prepare dimethyl 6-(dimethylamino)-3-methylbenzo[g]pyrrolo[2,1-a]phthalazine-1,2-dicarboxylic acid. Yield 1.7 g (43%); melting point 180-182°C.

[0348] 1 H NMR (DMSO–d6) δ2.66 (3H, s, CH3), 3.08 (6H, s, 2×NCH3), 3.81 (3H, s, COOCH3), 3.97 (3H, s, COOCH3), 7.64 (1H, t, J = 7.8Hz, Ar H), 7.71 (1H, t, J = 7.2Hz, ArH), 8.06 (1H, d, J = 8.2Hz, ArH), 8.24 (1H, d, J = 8.3Hz, ArH), 8.65 (1H, s, ArH), 8.73 (1H, s, ArH).

[0349] 13 C NMR (DMSO–d6) δ10.2, 42.6, 51.6, 52.5, 107.9, 111.2, 115.6, 119.9, 121.0, 123. 9, 127.0, 127.9, 128.0, 129.0, 129.4, 130.9, 131.3, 134.0, 156.8, 164.4, 166.9. HRMS[ESI + ]: Calculated as C 22 H 21 N3O4, 392.1610[M+H] + , the measured value is 392.1590.

[0350] (7) (6-(Dimethylamino)-3-methylbenzo[g]pyrrolo[2,1-a]phthalazine-1,2-diyl)dimethanol (BO-2768)

[0351] A solution of dimethyl 6-(dimethylamino)-3-methylbenzo[g]pyrrolo[2,1-a]phthalazine-1,2-dicarboxylic acid (1.6 g, 4.0 mmol) in DCM (50 mL) was added dropwise to a stirred suspension of LAH (0.39 g, 10.0 mmol) in ether (100 mL) at 0-5°C. After 2 hours of reaction, water (2 mL) and NH4OH (2 mL) were added to decompose the excess LAH. The reaction mixture was filtered through a calcite pad and washed thoroughly with DCM. The combined filtrate and washings were evaporated to dryness in vacuo. The residue was recrystallized from ethanol to prepare (6-(dimethylamino)-3-methylbenzo[g]-pyrrolo[2,1-a]phthalazine-1,2-diyl) dimethanol (BO-2768). Yield 1.1 g (80%); mp 156-158°C.

[0352] 1 H NMR (DMSO–d6) δ2.46 (3H, s, CH3), 2.99 (6H, s, 2×NCH3), 4.56 (2H, d, J = 5.1Hz, OCH2), 4.62 (1H, t, J = 5.4Hz, OH), 4.90–4.91 (3H, m, OCH2 and OH), 7.52 (1H, t, J = 7.5Hz, ArH), 7.63 (1H, t, J = 7.3Hz, ArH), 8.00 (1H, d, J = 8.3Hz, ArH), 8.14 (1H, d, J = 8.2Hz, ArH), 8.58 (1H, s, ArH), 8.66 (1H, s, ArH).

[0353] 13 C NMR (DMSO–d6) δ8.9, 42.8, 53.4, 54.2, 115.6, 115.7, 117.5, 120.4, 120.4 , 123.6, 125.5, 126.3, 126.6, 127.5, 128.1, 129.3, 130.1, 134.6, 154.7. HRMS[ESI + ]: Calculated as C 20 H 21 N3O2, 318.1606 [M+H–H2O] + , the measured value is 318.1620.

[0354] (8) (6-(Dimethylamino)-3-methylbenzo[g]pyrrolo[2,1-a]phthalazine-1,2-diyl)-bis(methylene)bis(ethylcarbamate) (BO-2772)

[0355] A mixture of (6-(dimethylamino)-3-methylbenzo[g]pyrrolo[2,1-a]phthalazine-1,2-diyl)dimethanol (0.2 g, 0.6 mmol) (0.16 g, 0.5 mmol), ethyl isocyanate (0.2 mL, 2.4 mmol) and TEA (0.55 mL, 4.0 mmol) in anhydrous DMF was stirred at room temperature under argon for 24-48 hours. After the reaction was complete, the reaction mixture was evaporated to dryness in vacuo. The residue was triturated with diethyl ether and the desired product was collected by filtration to prepare (6-(dimethylamino)-3-methylbenzo[g]prolog-[2,1-a]phthalazine-1,2-diyl)-bis(methylene)bis(ethylcarbamate) (BO-2772). Yield 0.15 g (53%); m.p. 132-134°C.

[0356] 1 H NMR (DMSO–d6) δ 0.98–1.01 (6H, m, 2×CH3), 2.48 (3H, s, CH3), 2.98–3.04 (10H, m, 2×CH2 and 2×NCH3), 5.18 (2H, s, OCH2), 5.49 (2H, s, OCH2), 7.06 (1H, t, J = 5.2 Hz, NH), 7.10 ( 1H, t, J=5.5Hz, NH), 7.56 (1H, t, J=7.9Hz, ArH), 7.66 (1H, t, J=7.4Hz, ArH), 7.97 (1 H, d, J = 8.3 Hz, ArH), 8.17 (1H, d, J = 8.3 Hz, ArH), 8.51 (1H, s, ArH), 8.64 (1H, s, ArH).

[0357] 13 C NMR(DMSO–d6).δ8.9, 15.1, 15.2, 35.0, 35.1, 42.8, 56.2, 57.0, 110.6, 115.5, 116.2, 118.5, 120.3, 125.5, 125.6, 126.0, 127.0, 127.6, 128.4, 129.3, 130.3, 134.5, 155.3, 156.1, 156.3. HRMS[ESI + ]: Calculated as C 26 H 31 N5O4, 302.1657[M+H–2(OCONHC2H5)] + , the measured value is 302.1667.

[0358] 2.2 (3-methyl-6-(pyrrolidin-1-yl)benzo[g]pyrrolo[2,1-a]phthalazine-1,2-diyl) dimethanol (BO-2762) and (3-methyl-6-(pyrrolidin-1-yl)benzo[g]pyrrolo[2,1-a]phthalazine-1,2-diyl)-bis(methylene)bis(ethylcarbamate) (BO-2763)

[0359] (1) 1-Chloro-4-(pyrrolidin-1-yl)benzo[g]phthalazine

[0360] Pyrrolidine (3.5 mL, 42.0 mmol) was slowly added to a stirred suspension of 1,4-dichlorobenzo[g]phthalazine (7.0 g, 28.0 mmol) and anhydrous potassium carbonate (55 g, 400.0 mmol) in anhydrous acetonitrile (400 mL). The reaction mixture was stirred at room temperature for 72 hours and then filtered to remove the potassium carbonate. The filtrate was evaporated under reduced pressure and the solid residue was recrystallized from diethyl ether to prepare 1-chloro-4-(pyrrolidin-1-yl)benzo[g]phthalazine. Yield 6.8 g (85%); m.p. 112-114°C.

[0361] 1 H NMR (DMSO–d6) δ2.02 (4H, brs, 2×CH2), 3.96 (4H, brs, 2×CH2), 7.76–7.79 (2H, m, ArH), 8.33–8.38 (2H, m, ArH), 8.75 (1H, s, ArH), 9.06 (1H, s, ArH).

[0362] 13 C NMR (DMSO–d6) δ25.4, 51.1, 118.7, 123.1, 124.0, 126.8, 128.1, 128.6, 128.9, 129.5, 133.7, 144.6, 155.5. HRMS[ESI + ]: Calculated as C 16 H 14 ClN3, 284.0955[M+H] + , the measured value is 284.0948.

[0363] (2) 1-(Pyrrolidin-1-yl)benzo[g]phthalazine

[0364] 1-Chloro-4-(pyrrolidin-1-yl)benzo[g]phthalazine (5.1 g, 18.0 mmol) in MeOH (200 mL) was added 10% Pd / C (1.03 g). Hydrogenation was carried out at 35 psi for 4 h at room temperature, and the reaction mixture was filtered through a calcite pad. The filter cake was washed thoroughly with MeOH. The combined filtrate and washings were concentrated under reduced pressure and dissolved in DCM (200 mL), washed with saturated aqueous NaHCO3 solution, dried over anhydrous sodium sulfate, and the residue was evaporated to dryness in vacuo. The product was purified by chromatography (SiO2, eluent gradient 0-40% ethyl acetate in hexanes) to prepare 1-(pyrrolidin-1-yl)benzo[g]phthalazine. Yield 3.3 g (73%); m.p. 150-152°C.

[0365] 1 H NMR(DMSO–d6)δ2.00–2.03(4H, m, 2×CH2), 3.94–3.97(4H, m, 2×CH2), 7.66–7.73(2H, m, ArH), 8.18(1H , d, J = 8.2 Hz, ArH), 8.31 (1H, d, J = 8.3 Hz, ArH), 8.55 (1H, s, ArH), 8.97 (1H, s, ArH), 9.04 (1H, s, ArH).

[0366] 13 C NMR (DMSO–d6) δ25.5, 50.8, 117.2, 125.3, 125.4, 127.2, 128.0, 128.1, 129.6, 133.5, 144.1, 154.8. HRMS[ESI + ]: Calculated as C 16 H 15 N3, 250.1344 [M+H] + , the measured value is 250.1362.

[0367] (3) 2-Acetyl-4-(pyrrolidin-1-yl)-1,2-dihydrobenzo[g]phthalazine-1-carbonitrile

[0368] Me3SiCN (3.26 mL, 26.0 mmol) was added dropwise to a solution of 1-(pyrrolidin-1-yl)benzo[g]phthalazine (3.25 g, 13.0 mmol) in DCM (50 mL) containing a catalytic amount of AlCl3. Acetyl chloride (1.4 mL, 19.6 mmol) was then added dropwise to the mixture at room temperature and stirred for 4 hours. The reaction mixture was poured into ice-water and the organic layer was washed with water, 5% NaOH solution and water in sequence. The solution was dried over sodium sulfate and concentrated in vacuo to prepare 2-acetyl-4-(pyrrolidin-1-yl)-1,2-dihydrobenzo[g]phthalazine-1-carbonitrile (33b) (BO-2760). Yield 3.2 g (77%); m.p. 162–164°C.

[0369] 1 H NMR (CDCl3) δ1.92–2.00 (2H, m, CH2), 2.08–2.10 (2H, m, CH2), 2.31 (3H, s, COCH3), 3.44–3.47 (2H, m, CH2), 3.83–3.89 (2H, m, CH2), 6 .90 (1H, s, CH), 7.58–7.64 (2H, m, ArH), 7.85 (1H, s, ArH), 7.90 (1H, d, J = 7.9Hz, ArH), 7.93 (1H, d, J = 7.9Hz, ArH), 8.17 (1H, s, ArH).

[0370] 13 C NMR (CDCl3) δ20.7, 15.4, 41.5, 50.1, 115.8, 120.1, 125.7, 126.3, 127.0, 127.7, 127.9, 128.5, 129.0, 133.1, 133.8, 154.1.171.1. HRMS[ESI + ]: Calculated as C 19 H 18 N4O, 319.1559[M+H] + , the measured value is 319.1557.

[0371] (4) Dimethyl 3-methyl-6-(pyrrolidin-1-yl)benzo[g]pyrrolo[2,1-a]phthalazine-1,2-dicarboxylic acid

[0372] HBF4 (2.2 mL, 12.0 mmol) was added dropwise to a solution of 2-acetyl-4-(pyrrolidin-1-yl)-1,2-dihydrobenzo[g]phthalazine-1-carbonitrile (3.2 g, 10.0 mmol) in warm acetic acid (90 mL). The mixture was stirred at 50-60°C for 30 min. After cooling to room temperature, the yellow solid salt was collected by filtration and the filter cake was washed with dry ether. The solid salt was dissolved in DMF (20 mL) and DMAD (3.1 mL, 25.0 mmol) was slowly added to the solution. The reaction mixture was heated at 90-100°C for 16 h. The solvent was removed by evaporation in vacuo. The residue was crystallized from MeOH to prepare dimethyl 3-methyl-6-(pyrrolidin-1-yl)benzo[g]pyrrolo[2,1-a]phthalazine-1,2-dicarboxylic acid. Yield 2.0 g (48%); melting point 190-192°C.

[0373] 1 H NMR (CDCl3) δ2.02–2.05 (4H, m, 2×CH2), 2.68 (3H, s, CH3), 3.76–3.78 (4H, m, 2×CH2), 3.89 (3H, s, COOCH3), 4.04 (3H, s, COOCH3), 7.49 (1H, t, J=7.1Hz, ArH), 7.57 (1H, t, J=6.9Hz, ArH), 7.90 (1H, d, J=8.5Hz, ArH), 7.92 (1H, d, J=8.4Hz, ArH), 8.52 (1H, s, ArH), 8.77 (1H, s, ArH).

[0374] 13 C NMR (CDCl3) δ10.4, 25.6, 51.4, 51.5, 52.4, 107.6, 111.2, 117.1, 120.7, 121.9, 124 .8, 126.3, 127.1, 128.1, 128.1, 128.8, 131.2, 131.4, 134.3, 153.8, 165.6, 168.1. HRMS[ESI + ]: Calculated as C 24 H 23 N3O4, 440.1586[M+Na] + , the measured value is 440.1569.

[0375] (5) (3-methyl-6-(pyrrolidin-1-yl)benzo[g]pyrrolo[2,1-a]phthalazine-1,2-diyl)dimethanol (BO-2762)

[0376] A solution of dimethyl 3-methyl-6-(pyrrolidin-1-yl)benzo[g]pyrrolo[2,1-a]phthalazine-1,2-dicarboxylic acid (1.7 g, 4.0 mmol) in DCM (50 mL) was added dropwise to a stirred suspension of LAH (0.39 g, 10.0 mmol) in ether (100 mL) at 0-5°C. After 2 hours of reaction, water (2 mL) and NH4OH (2 mL) were added to decompose the excess LAH. The reaction mixture was filtered through a calcite pad and washed thoroughly with DCM. The combined filtrate and washings were evaporated to dryness in vacuo. The residue was recrystallized from ethanol to prepare (3-methyl-6-(pyrrolidin-1-yl)benzo[g]prolog-[2,1-a]phthalazine-1,2-diyl)dimethanol (BO-2762). Yield 1.1 g (82%); mp 160-162°C.

[0377] 1 H NMR(DMSO–d6)δ1.98–1.99(4H,m,2×CH2), 2.43(3H,s,CH3), 3.68–3.70(4H,m,2×CH2), 4.52–4.55(3H,m,OCH2 and OH), 4.82 (1H, t, J=5.0Hz, OH), 4.90 (2H, d, J=5.1Hz, OCH2), 7.50 (1H, t, J=7.4Hz, ArH), 7.61 (1H, t, J=7 .3Hz, ArH), 7.98 (1H, d, J = 8.3Hz, ArH), 8.14 (1H, d, J = 8.3Hz, ArH), 8.64 (1H, s, ArH), 8.67 (1H, s, ArH).

[0378] 13 C NMR (DMSO–d6) δ8.9, 25.0, 50.9, 53.4, 54.2, 115.2, 116.4, 117.2, 119.9, 12 0.0, 123.0, 125.3, 126.4, 126.7, 127.3, 128.1, 129.3, 129.9, 134.3, 152.2. HRMS[ESI + ]: Calculated as C 22 H 23 N3O2, 344.1763 [M+H–H2O] + , the measured value is 344.1754.

[0379] (6) (3-methyl-6-(pyrrolidin-1-yl)benzo[g]pyrrolo[2,1-a]phthalazine-1,2-diyl)-bis(methylene)bis(ethylcarbamate) (BO-2763)

[0380] A mixture of (3-methyl-6-(pyrrolidin-1-yl)benzo[g]pyrrolo[2,1-a]phthalazine-1,2-diyl)dimethanol (0.36 g, 1.0 mmol), ethyl isocyanate (0.32 mL, 4.0 mmol) and TEA (0.55 mL, 4.0 mmol) in anhydrous DMF was stirred at room temperature under argon for 24-48 hours. After the reaction was complete, the reaction mixture was evaporated to dryness in vacuo. The residue was triturated with diethyl ether and the desired product was collected by filtration to prepare (3-methyl-6-(pyrrolidin-1-yl)benzo-[g]pyrrolo[2,1-a]phthalazine-1,2-diyl)-bis(methylene)bis(ethylcarbamate) (BO-2763). Yield 0.28 g (56%); m.p. 142-144°C.

[0381] 1 H NMR (DMSO–d6) δ0.95–1.02 (6H, m, 2×CH3), 1.98 (4H, brs, 2×CH2), 2.45 (3H, s, CH3), 2.97–3.0 5(4H, m, 2 × CH2), 3.72 (4H, brs, 2 × CH2), 5.16 (2H, s, OCH2), 5.48 (2H, s, OCH2), 7.02 (1H, t, J= 5.7Hz, NH), 7.07 (1H, t, J = 5.2Hz, NH), 7.54 (1H, t, J = 7.7Hz, ArH), 7.65 (1H, t, J = 7.4Hz, ArH) , 7.94 (1H, d, J = 8.1 Hz, ArH), 8.17 (1H, d, J = 8.3 Hz, ArH), 8.47 (1H, s, ArH), 8.72 (1H, s, ArH).

[0382] 13 C NMR (DMSO–d6) δ8.9, 15.1, 15.2, 25.1, 35.0, 35.1, 51.0, 56.2, 57.1, 110.0, 115.7, 116.3, 1 18.2, 119.8, 124.8, 125.7, 127.1, 127.3, 128.3, 129.3, 130.1, 134.2, 152.6, 156.1, 156.3. HRMS[ESI + ]: Calculated as C 28 H 33 N5O4, 328.1814[M+H–2(OCONHC2H5)] + , the measured value is 328.1816.

[0383] Example 3 Preparation of the compound of formula (I) encapsulated in liposomes

[0384] The compound of formula (I) is substantially hydrophobic. In order to deliver the active compound to specific tumor cells or organ targets at high drug concentration, the compound of formula (I) (particularly BO-2590) is encapsulated in liposomes using a modified dehydration-rehydration method and repeated extrusion as described below to prepare liposome drugs.

[0385] (1) Liposome encapsulation

[0386] Liposome-encapsulated BO-2590 was prepared by mixing soy lecithin (SPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (CHO), and PEG-2000 (at a molar ratio of 50:45:4:1) in CHCl3. The mixture was placed in a round-bottom flask, and BO-2590 was added to the reaction mixture (4 mg / mL). The organic solvent was removed by rotary evaporation under reduced pressure. The resulting dry lipid film was hydrated with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4) and dispersed by hand shaking. The suspension was frozen and thawed several times and then repeatedly extruded at 60° C. using high-pressure extrusion equipment (Lipex Biomembranes, Vancouver, Canada) with polycarbonate membrane filters (Costar, Cambridge, MA) of 0.8, 0.6, 0.4, and 0.2 μm pore sizes. The product solution was stored at 4° C.

[0387] (2) HPLC analysis

[0388] Liposome BO-2590 (BO-2590L) was quantified by high pressure liquid chromatography (Agilent Technologies) using a RP-18 column and mobile phase conditions of [acetonitrile / MeOH / H2O (0.5% TFA) (45 / 50 / 5), (retention time 4.6 min, flow rate 0.5 ml / min)]. Liposome BO-2590 (100 μl) was added to the mobile phase (100 μl), mixed thoroughly by vortexing, and allowed to stand for 10 min. The resulting milky sample was centrifuged and the clear supernatant was used for HPLC quantification. In our preparations (n>5), BO-2590 liposomes contained 3.12 to 2.94 mg / ml (76 to 70% of the encapsulation efficiency of the starting material at a concentration of 4 mg / ml).

[0389] (3) Stability analysis

[0390] The BO-2590L encapsulated in liposomes was stored at 4°C for three weeks and analyzed by HPLC, which showed a decay of about 10% from 76% to 66.3% of the encapsulated BO-2590L.

[0391] Example 4 In vitro characterization of compounds of formula (I)

[0392] 4.1 Compounds of formula (I) have cytotoxicity against cancer cells

[0393] First, all representative compounds of formula (I-A) and (I-B) were evaluated for their cytotoxicity against human lymphoid leukemia cell line (CCRF-CEM) and its vinblastine-resistant derivative cell line (CCRF-CEM / VBL) in vitro (see Table 1). Selected compounds with significant cytotoxicity were further evaluated against a series of solid tumor cell lines, namely, colorectal cancer HCT-116, non-small cell lung cancer H460, small cell lung cancer H526 and pancreatic cancer PacaS1 (Table 2).

[0394] Briefly, the test cells were cultured at 3 × 10 cells / mL in a humidified atmosphere of 5% CO2 at 37°C. 3 The cells were cultured at an initial density in RPMI medium 1640 (GIBCO / BRL) containing penicillin (100 units per ml), streptomycin (100 μg per ml, GIBCO / BRL) and 5% heat-inactivated FBS. After being treated with various concentrations of the newly synthesized compounds for 72 hours, the cells were cultured with The cytotoxic effects on all cell lines were determined using a microplate spectrometer and a 50 μl (Invitrogen) assay. Briefly, at the end of drug treatment, an aliquot of The solution was then incubated for 1–2 h at 37°C. The absorbance at 570 and 600 nm was measured using a microplate reader. The dose–effect relationships of 6 or 7 concentrations of each compound were used to calculate the IC using the half-effect principle developed by Chou and Martin (Pharmacol Rev 2006, 58: 621-681) (CompuSyn software, version 1.0.1; CompuSyn, Inc., Paramus, NJ). 50 Value. IC 50 It was defined as the concentration required to inhibit tumor cell growth by 50%. The experimental data are the mean ± standard deviation of three to six independent experiments for each compound.

[0395] Table 1 summarizes the in vitro cytotoxicity of 1,2-bis(hydroxymethyl)pyrrolo[2,1-a]phthalazine derivatives (Formula I-A) and benzo[g]pyrrolo[2,1-a]phthalazine derivatives (Formula I-B) against human lymphoid leukemia CCRF-CEM and its vinblastine-resistant derivative cell line CCRF-CEM / VBL. The experimental data showed that these compounds exhibited significant cytotoxicity against CCRF-CEM and did not cross-resistance to vinblastine. The selected compounds were evaluated for their cytotoxicity against various types of solid tumors (i.e., colorectal cancer HCT-116, non-small cell lung cancer H460, small cell lung cancer H526, and pancreatic cancer PacaS1). It is noteworthy that the test compounds actively inhibited the cell growth of all solid tumor cell lines (Table 2). Among them, small cell lung cancer H526 cells were the most sensitive to the test compounds.

[0396] Furthermore, the cytotoxicity of the compound of formula (IB) and known antiproliferative agents (e.g., irinotecan, etoposide, cisplatin and carboplatin) on human small cell lung cancer (SCLC) cells was compared, and the results are summarized in Table 3. As shown in the experimental data in Table 3, overall, the compound of formula (IB) of the present invention is more potent than irinotecan, etoposide, cisplatin and carboplatin.

[0397] Table 1. Cytotoxicity of compounds of formula (I-A) and (I-B) against human lymphocytic leukemia (CCRF) and its vinblastine-resistant derivative cell lines (CCRF-CEM / VBL).

[0398]

[0399]

[0400]

[0401]

[0402] a The experimental data are the mean ± standard deviation of three to six independent experiments for each compound.

[0403] Difference. b CCRF–CEM / VBL is a derivative cell line of CCRF–CEM. c Numbers in brackets

[0404] Values ​​are obtained by comparing the corresponding parental cell line IC 50 The resulting resistance factor. d IC 50 Value

[0405] Molar concentration is expressed as volume.

[0406] Table 2. In vitro cytotoxicity of 1,2-bis(hydroxymethyl)pyrrolo[2,1-a]phthalazine derivatives (Formula (I-A)) and benzo[g]pyrrolo[2,1-a]phthalazine derivatives (Formula (I-B)) against human solid tumor cell lines.

[0407]

[0408] Table 3. Comparison of the cytotoxicity of compounds of Formula I-B and therapeutic agents against a panel of human small cell lung cancer (SCLC) cells.

[0409]

[0410]

[0411] 4.2 DNA interstrand crosslinks induced by compounds of formula (I)

[0412] In this example, the DNA cross-linking activity of BO-2590, BO-2577 (Formula I-A) and BO-2698, BO-2755, BO-2762 and BO-2768 (Formula I-B) was tested and the cross-linking activity was analyzed by alkaline agarose gel electrophoresis. Briefly, purified pEGFP-N1 plasmid DNA (1,500 ng) was mixed with various concentrations (1-20 μM) of the test compound in 40 μL of binding buffer (3 mM NaCl / 1 mM sodium phosphate, pH 7.4, and mM EDTA). The reaction mixture was incubated at 37°C for 2 hours. At the end of the reaction, the plasmid DNA was linearized by digestion with BamHI and subsequently precipitated with ethanol. The DNA pellet was dissolved and denatured with an alkaline buffer (0.5 N NaOH-10 mM EDTA). An aliquot of 20 μl of DNA solution (1,000 ng) was mixed with 4 μl of 6× alkaline loading dye and then electrophoresed on a 0.8% alkaline agarose gel containing NaOH–EDTA buffer at 4°C. Electrophoresis was performed at 18 V for 22 hours. The gel was then stained with ethidium bromide solution and the DNA was visualized with UV light. The results are shown in Figure 1 .

[0413] like Figure 1As shown in the photos, BO-2590, BO-2577, BO-2698, BO-2755, BO-2762, and BO-2768 induced DNA interstrand crosslinks, and the effects appeared to be dose-dependent. Furthermore, among these tested compounds, BO-2590 was found to have relatively strong crosslinks compared to melphalan.

[0414] Furthermore, single cell electrophoresis (SCGE, or "comet assay") was performed to assess BO-2768-induced DNA damage, which is a simple method to measure DNA strand breaks in cells. For this purpose, cells were embedded in a low-melting-point agarose suspension on a glass slide, then lysed under neutral or alkaline (pH>13) conditions, and the suspended and lysed cells were subjected to electrophoresis. The term "comet" refers to the pattern of DNA migration through an electrophoresis gel, which usually resembles a comet, and the intensity of the comet tail relative to the comet head reflects the amount of DNA breakage. The experimental results are shown in Figure 2 , confirming that BO-2768, like cisplatin, can induce DNA fragmentation in a dose-dependent manner.

[0415] 4.3 BO-2590 and BO-2577 inhibit angiogenesis

[0416] In this example, Western blot analysis was performed to test the inhibitory effects of BO-2590 and BO-2577 on VEGFR-2 activity. Briefly, EA.hy926 endothelial cells were treated with various concentrations of BO-2590 or BO-2577 for 12 hours. Primary antibodies against VEGFR-2 and p-VEGFR-2 were used to detect total VEGFR-2 and p-VEGFR-2 proteins, respectively. The results are shown in Figure 3 .

[0417] like Figure 3 As shown in the figure, treatment with BO-2590 or BO-2577 can significantly inhibit the amount of p-VEGFR-2 protein, and BO-2590 is more conducive to reducing p-VEGFR-2 than BO-2577. Furthermore, since BO-2590 or BO-2577 did not significantly change the total amount of VEGFR-2 protein, it is speculated that BO-2590 or BO-2577 may act as an inhibitor of VEGFR activation. This experiment used vatalanib as a control group. Unexpectedly, the dose of BO-2590 that effectively inhibited p-VEGFR-2 was about 10 times lower than that of vatalanib. This shows that BO-2590 is a potential VEGFR-2 inhibitor.

[0418] 4.4BO–2590 reduces endothelial cell migration

[0419] In this example, a migration assay using cell culture inserts was performed to investigate whether compound BO-2590 could inhibit cell migration.

[0420] Briefly, endothelial cells were placed on an upper cell permeable membrane, and a solution containing BO-2590 was placed below the cell permeable membrane. After the incubation period (8 hours), cells that moved through the membrane were stained and counted using fluorescence microscopy. Obviously, the inhibition of the VEGFR pathway by the compound BO-2590 was also accompanied by an impairment of cell migration ( Figure 4 -A).

[0421] 4.5BO-2590 blocks angiogenesis

[0422] Angiogenesis refers to the process of generating new blood vessels by extension from existing blood vessels. Observing blood vessel behavior or monitoring angiogenesis can be used to detect whether a compound can block angiogenesis. In this example, angiogenesis assay was used to study whether the compound of formula (I) blocks angiogenesis.

[0423] Briefly, EA.hy926 cells were treated with various concentrations of BO-2590 for 24 h. 5 Cells were suspended in 100 μl of culture medium containing 1% FCS and then seeded in a 96-well plate pre-coated with matrigel for 1 hour at 37° C. After culturing for 48 hours, the angiogenic ability was detected using a phase contrast microscope.

[0424] like Figure 4 -B shows that before the addition of BO-2590, a sound tubular structure can be formed. After the action of BO-2590 in the nanomolar concentration range, the angiogenesis of the tubular structure is significantly and dose-dependently destroyed. Vatalanib can also inhibit angiogenesis, but at a higher concentration. The experimental results show that the inhibition of angiogenesis by BO-2590 is closely related to the inhibition of VEGFR-2 caused by BO-2590. Therefore, it is reasonable to conclude that BO-2590 triggers anti-angiogenic activity by inhibiting the expression of p-VEGFR2. BO-2768 and BO-2698 also showed similar inhibitory effects on angiogenesis in a dose-dependent manner ( Figure 4 -C, Figure 4 -D).

[0425] 4.6BO–2590 interferes with the cell cycle

[0426] The effect of BO-2590 on cell cycle progression was further investigated in lung cancer H460 cells at concentrations of 0, 0.125, 0.25, 0.5, 1, and 2 μM, and at 24, 48, and 72 hours. Flow cytometry was used to analyze cell cycle distribution. Figure 5 As shown in the figure, BO-2590 interferes with the cell cycle in a dose-dependent manner. When the concentration of BO-2590 is increased, a significant termination of the G2 phase is first observed, followed by termination of the S phase, and finally termination of G1 at the 24-hour mark. However, after 48 or 72 hours of treatment, the cell cycle slows down and a large number of sub-G1 cells begin to appear ( Figure 6 Sub-G1 cells also represent apoptotic cells. Therefore, it was found that BO-2590 induced the generation of sub-G1 cells in a dose-dependent manner, indicating that BO-2590 can trigger apoptotic cell death through DNA damage.

[0427] 4.7BO-2590 induces apoptotic cell death

[0428] In this example, Annexin V staining analysis was used to evaluate BO-2590-induced apoptotic cell death in H460 cell line. Briefly, H460 cells were treated with 0.5, 1, and 2 μM BO-2590 for 48 h and then stained with Annexin V-FITC and propidium iodide (PI). The stained cells were analyzed by flow cytometry. After 48 h of treatment, BO-2590 was found to significantly increase Annexin V expression in a dose-dependent manner. + The proportion of cells ( Figure 7 ).

[0429] 4.8BO-2768 induces apoptotic cell death

[0430] In this example, in addition to using H526 cell lines to detect apoptotic cell death, the effect of BO-2768 was evaluated according to the steps of Example 4.7. Briefly, H526 cells were treated with 0.01, 0.02, 0.04, and 0.08 μM BO-2768 or 2, 4, 8, and 16 μM cisplatin for 24, 48, or 72 hours, and then stained with annexin V-FITC and propidium iodide. The stained cells were analyzed by flow cytometry. After 72 hours of treatment, BO-2768 was found to significantly increase the expression of annexin V in a dose-dependent manner. + The proportion of cells ( Figure 8 ).

[0431] 4.9BO-2768 synergistically inhibits cell proliferation with cisplatin

[0432] In this example, whether BO-2768 and cisplatin have a synergistic effect in inhibiting cell proliferation was investigated. To this end, H211 cells were treated with BO-2768 or cisplatin, either alone or in combination, and PrestoBlue TM The proliferation rate of H211 cells was analyzed. The experimental results are shown in Fig. 9 .

[0433] like Fig. 9 As shown in the experimental data, the combined administration of BO-2768 and cisplatin at a ratio of 1:2, 1:4 and 1:8, respectively, can synergistically reduce the proliferation of H211 cells.

[0434] Example 5 In vivo confirmation of the properties of the compound of formula (I)

[0435] As described above in Example 4, the compounds of formula (I) are substantially cytotoxic to the tumor cell lines tested. In this example, the therapeutic efficacy of selected compounds of formula (I) in nude mice bearing human SCLC was investigated.

[0436] 5.1 The BO-2590 (BO-2590L) liposomes of Example 3 can effectively inhibit the SCLC H526 allogeneic transplantation

[0437] Since BO-2590 has poor solubility, this example uses the liposomes of BO-2590 (ie, BO-2590L) of Example 3 to study its therapeutic efficacy on nude mice bearing human SCLC H526 xenografts.

[0438] In all experiments, tumor cells were implanted subcutaneously in nude mice, and BO-2590L was administered via the tail vein when tumor volume reached approximately 100 mm3. Mice bearing H526 xenografts were first treated with BO-2590L at doses of 2.5, 5, and 10 mg / kg, administered every other day for a total of six times (Q2D×6). Fig.10 As shown in Figure 2A, 10 mg / kg of BO-2590L inhibited H526 tumor volume by 55% on day 30. BO-2590L did not cause body weight loss at all doses tested, indicating that BO-2590L has low toxicity ( Fig.10 -B).

[0439] To confirm the therapeutic activity of BO-2590L, a comparative study was conducted with known chemotherapeutic agents such as vatalanib and cisplatin. Briefly, mice bearing H526 xenografts were divided into 4 groups and treated with vehicle, BO-2590L (10 mg / kg, QD×9), vatalanib (100 mg / kg, QD×9), and cisplatin (4 mg / kg, Q2D×3), respectively. The experimental results are shown in Figure 11.

[0440] The results showed that BO-2590L was far more potent than vatalanib and was almost as effective as cisplatin in inhibiting the growth of H526 xenografts on day 33. BO-2590L was observed to inhibit approximately 60% of the tumors ( Fig.11 -A). Of note, administration of 10 mg / kg of BO-2590L for 9 consecutive days did not cause weight loss ( Fig.11 -B), supporting previous findings that BO-2590L has low toxicity. In contrast, cisplatin at a dose of 4 mg / kg resulted in severe weight loss in the experimental animals tested.

[0441] 5.2 BO-2590, BO-2768 or BO-2792 micelles can effectively inhibit SCLC H526 allogeneic transplantation

[0442] The therapeutic efficacy of BO-2590, BO-2768, BO-2792, irinotecan and cisplatin on nude mice with human SCLC H526 xenografts was investigated according to the procedures similar to those in Example 5.1. BO-2590, BO-2768, BO-2792, irinotecan and cisplatin were prepared in 20% The micelles were formed in a solution of 10% TWEEN80, 10% PEG400, 30% ethanol and 40% D5W. Fig.12 and 13 .

[0443] The experimental results showed that BO-2590 and BO-2792 were as effective as irinotecan and cisplatin in reducing tumor volume ( Fig.12 -A); however, the 20 mg / kg (twice daily) dose of BO-2768 unexpectedly was more potent than any of BO-2590, BO-2792, irinotecan, or cisplatin alone, completely inhibiting tumor growth (i.e., no growth) throughout the study period, while tumors in the other groups (i.e., groups treated with BO-2590, BO-2792, irinotecan, or cisplatin) slowly increased in size over time. Further, none of the tested compounds (i.e., BO-2590, BO-2768, BO-2792, irinotecan, and cisplatin) exhibited any deleterious effects on the body weight of the test animals ( Fig.12 -B).

[0444] 5.3BO-2768 inhibits SCLC H526 allogeneic transplantation in a dose-dependent manner

[0445] Reviewing the findings in Example 5.2, the antitumor efficacy of BO-2768 was further studied in detail. As expected, BO-2768 inhibited tumor growth in a dose-dependent manner, and a dose of 20 mg / kg (twice daily) completely inhibited tumor growth during the entire study period ( Fig.13 -A) and did not show any adverse effects on the body weight of the test animals ( Fig.13 -B).

[0446] 5.4BO-2768 and cisplatin synergistically reduce the size of SCLC H211 allogeneic transplants without adversely affecting the body weight of the test subjects

[0447] In this example, the synergistic effect of BO-2768 and cisplatin on inhibiting tumor volume and body weight changes of the test subjects was investigated. For this purpose, aliquots of H211 cells (5×10 6 , 50 μl) were implanted subcutaneously into nude mice. Then, the test animals were treated with BO-2768 or cisplatin, alone or in combination. BO-2768 (20 mg / kg dissolved in 20% HS15, 10% Tween80, 10% PEG400, 30% ethanol and 40% distilled water) for 5 consecutive days, followed by a one-day break; and the cycle was repeated once (i.e., two cycles in total). Cisplatin at a dose of 2 or 4 mg / kg was given once every 4 days for a total of 3 times. The experimental results are shown in Fig.14 .

[0448] like Fig.14 As shown in the experimental data, the combined administration of BO-2768 and cisplatin can synergistically reduce tumor volume ( Fig.14 -A), and had no adverse effect on the body weight of the test animals ( Fig.14 -B).

[0449] 5.5 Immunohistochemical staining demonstrated the anti-angiogenic and DNA damage activities of BO-2590L (Formula IA) in SCLC H526 tissue sections

[0450] The tumor xenograft tissue sections on the slides were deparaffinized with xylene for 7 minutes each time, twice. The slides were then rehydrated with a gradient of ethanol (from 100% to 70%) and then wetted with dH2O. Antigen retrieval was performed in a cooker with citrate buffer (0.01M, pH 6.0) for 50 minutes and then cooled at room temperature for 30 minutes. The slides were then wetted with dH2O. Immunohistochemical (IHC) staining was performed with primary antibodies (anti-rabbit CD31 antibody and anti-mouse γ-H2AX antibody) according to the manufacturer's instructions (Novolink Polymer Detection system, Leica Biosystems, Wetzlar, Germany). After IHC processing, the slides were digitally scanned using a Panoramic 250Flash II whole slide scanner and analyzed using 3DHISTECH Panoramic viewing software (3DHISTECH Ltd., Budapest, Hungary).

[0451] Tumor vascular density was assessed by immunofluorescence staining using anti-CD31, an endothelial cell marker. Fig.15 -A). Compared with the vehicle control group (or control group), tumors treated with BO-2590L, vatalanib, or cisplatin showed a significant decrease in blood vessels overall ( Fig. 9 -A). After IHC, the slides were scanned with a Panoramic 250 Flash II whole slide scanner. CD31 expression in tissue sections was observed from 15 different fields of view of 3 tumor sections and the count average was calculated using 3DHISTECH Panoramic Observation Software (3DHISTECH Ltd., Budapest, Hungary). Fig.15 As shown in (B), CD31 labeling increased over time in untreated tumors. However, on day 9, CD31 labeling intensity was reduced to 4% (95% CI, 2.3-3.7) in tumors treated with BO-2590L or vatalanib compared to the control group. Fig.15 -B). Since cisplatin significantly inhibited tumor growth at the dose used, a decrease in CD31 labeling was also observed in cisplatin-treated tumors. On day 9, the CD31 labeling intensity in cisplatin-treated tumors was only 19% of that in the control group, which was lower than that of BO-2590L or vatalanib. These results suggest that BO-2590L has a comparable potential to vatalanib in inhibiting angiogenesis in vivo.

[0452] Since BO-2590L is a DNA-damaging anticancer agent, immunohistochemical staining was used to investigate the quantitative effect of BO-2590L on a DNA damage marker (γ-H2AX).

[0453] As expected, tumors treated with cisplatin or BO-2590L were observed to have significantly increased γ-H2AX, whereas tumors treated with vatalanib had very low amounts of γ-H2AXs ( Fig.16 -A). Quantitative analysis showed that almost no γ-H2AX signal was found in the control tumors. However, tumors treated with cisplatin or BO-2590 were found to increase γ-H2AX signal over time. In tumors treated with vatalanib, moderate amounts of γ-H2AX ( Fig.16 -B).

[0454] In summary, BO-2590L can kill cancer cells through DNA damage and inhibition of angiogenesis.

[0455] Although the above embodiments disclose specific embodiments of the present invention, those with ordinary knowledge in the art to which the present invention belongs can make various changes and modifications thereto. The above description, examples and experimental data provide a complete description of the structure and use of the present invention as a specific embodiment. Although the above description of various embodiments of the present invention has a certain degree of characteristics, or refers to one or more individual embodiments, those with ordinary knowledge in the art to which the present invention belongs can still make many modifications to the disclosed embodiments without departing from the spirit and scope of the present invention.

Claims

1. A compound of formula (IA), in, R 1 is -CH2OH, -CH2OCONHEt or –CH2OCONH–i–Pr; R 2 is -Me, -Et or 4'-MeO-C6H4-; R 3 is morpholine, dimethylamine, pyrrolidine, piperidine or 1,4'-bipiperidine; and R 4 and R 5 Each is hydrogen.

2. The compound of claim 1, wherein R 1 is -CH2OH, R 2 is ethyl, R 3 It's morpholine.

3. The compound as claimed in claim 1, wherein R 1 is -CH2OH, R 2 is methyl, R 3 It's pyrrolidine.

4. The compound as claimed in claim 1, wherein R 1 is -CH2OH, R 2 is methyl, R 3 It is 1,4'-bipiperidine.

5. The compound as claimed in claim 1, wherein R 1 Yes -CH2OCONHEt, R 2 is methyl, R 3 It's pyrrolidine.

6. A compound of formula (IB), in, R 1 is -CH2OH, -CH2OCONHEt or –CH2OCONH–i–Pr; R 2 is -Me, -Et or 4'-MeO-C6H4-; R 3 is morpholine, dimethylamine, pyrrolidine, piperidine or 1,4'-bipiperidine; and R 4 and R 5 Each is hydrogen.

7. The compound of claim 6, wherein R 1 is -CH2OH, R 2 is methyl, R 3 It's dimethylamine.

8. The compound of claim 6, wherein R 1 Yes -CH2OCONHEt, R 2 is methyl, R 3 It's dimethylamine.

9. The compound of claim 6, wherein R 1 Yes -CH2OCONHEt, R 2 is methyl, R 3 It's pyrrolidine.

10. Use of the compound according to claim 1 for preparing a drug for treating cancer, wherein the cancer is selected from lymphocytic leukemia, colorectal cancer, non-small cell lung cancer, small cell lung cancer and pancreatic cancer.

11. The use according to claim 10, wherein the compound has the structure of formula (I-A) in, R 1 is -CH2OH, -CH2OCONHEt or –CH2OCONH–i–Pr; R 2 is -Me, -Et or 4'-MeO-C6H4-; R 3 are morpholine, dimethylamine, pyrrolidine, piperidine, 1,4'-bipiperidine; and R 4 and R 5 Each is hydrogen.

12. The use according to claim 11, wherein R 1 is -CH2OH, R 2 is ethyl, R 3 It's morpholine.

13. The use according to claim 11, wherein R 1 is -CH2OH, R 2 is methyl, R 3 It's pyrrolidine.

14. The use according to claim 11, wherein R 1 is -CH2OH, R 2 is methyl, R 3 It is 1,4'-bipiperidine.

15. The use according to claim 11, wherein R 1 Yes -CH2OCONHEt, R 2 is methyl, R 3 It's pyrrolidine.

16. Use of the compound according to claim 6 for preparing a drug for treating cancer, wherein the cancer is selected from lymphocytic leukemia, colorectal cancer, non-small cell lung cancer, small cell lung cancer and pancreatic cancer.

17. The use according to claim 16, wherein the compound has the structure of formula (I-B) in, R 1 is -CH2OH, -CH2OCONHEt or –CH2OCONH–i–Pr; R 2 is -Me, -Et or 4'-MeO-C6H4-; R 3 are morpholine, dimethylamine, pyrrolidine, piperidine, 1,4'-bipiperidine; and R 4 and R 5 Each is hydrogen.

18. The use according to claim 17, wherein R 1 is -CH2OH, R 2 is methyl, R 3 It's dimethylamine.

19. The use according to claim 17, wherein R 1 Yes -CH2OCONHEt, R 2 is methyl, R 3 It's dimethylamine.

20. The use according to claim 17, wherein R 1 Yes -CH2OCONHEt, R 2 is methyl, R 3 It's pyrrolidine.

21. The use according to claim 10 or 16, wherein the drug can be used in combination with a chemotherapeutic agent.

22. The use of claim 21, wherein the chemotherapeutic agent is selected from the group consisting of an anti-CTLA-4 drug, an anti-programmed death receptor-1 (PD-1) drug, a MEK inhibitor, a poly (ADP-ribose) polymerase (PARP) inhibitor, a phosphoinositide 3-kinase (PI3K) inhibitor, and a mammalian target of rapamycin (mTOR) inhibitor.

23. The use of claim 21, wherein the chemotherapeutic agent is selected from the group consisting of deoxyelephantopin (DET), Vemurafenib (PLX4032), docetaxel, paclitaxel, cisplatin, oxaliplatin, betulinic acid, 4-S-cysteaminyl catechol, 4-S-cysteaminylphenol, everolimus, bortezomib, carboplatin, dacarbazine, celecoxib, temozolomide, sorafenib, thalidomide, lenalidomide, valproic acid, acid, vinblastine, imatinib mesylate, bosentan, apomine, arsenic trioxide, carmustine, lambrolizumab, ipilimumab, tremelimumab, doxorubicin, capecitabine, and tamoxifen.

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