Synthesis of imipridone derivatives and evaluation of their anticancer activity

Abstract

The present application relates to a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and stereoisomers thereof, including enantiomers, racemic mixtures, mixtures of enantiomers, or a combination thereof, for use in the treatment of cancer diseases. The present application also relates to a pharmaceutical composition comprising the above-mentioned compound.

Description

TECHNICAL FIELD

[0001] The present invention relates to a compound of Formula (I)

[0002]

[0003] or pharmaceutically acceptable salts thereof, and stereoisomers thereof, including single enantiomers, racemic mixtures, mixtures of enantiomers, or combinations thereof, for use in the treatment of cancer diseases. The present invention also relates to a pharmaceutical composition comprising the above-mentioned compound. BACKGROUND

[0004] Imipridones are first-in-class small molecule anticancer compounds that contain a strongly basic angular tricyclic heterocyclic framework with two backbone nitrogen atoms bearing substituents [cf. general structure represented by Formula (I)]. This well-defined angular condensation backbone structure contains an ideal number of basic centers in appropriate positions and a lactam group with an aromatic ring to be tuned as a homogeneously distributed potential binding site, which endows imipridones with multi-target properties and ideal drug properties that are essential for their unique mechanism of action. Specific G-protein coupled receptors (GPCRs) that control key signaling pathways in cancer cells are important targets for ONC201 (Reference Compound 1), the first imipridone that is directly antagonizing the GPCR known as DRD2 dopamine receptor, which is in clinical development [1].

[0005]

[0006] The molecule has also been an effective activator of the pro-apoptotic protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors with a wide therapeutic index [2-4]. Kline et al. disclosed that ONC201 elicited dual inhibition of AKT and extracellular signal-regulated kinase (ERK) pathways in several malignant cell lines (e.g., HCT-116, HEPG-2, MCF-7, and MDA-MB-468) and demonstrated that ONC201 induced cell cycle arrest in cell lines tested as early as 24 hours after treatment in addition to apoptosis measured by sub-G1 fraction and caspase activation [2].

[0007] The above cited references [1-4] only relate to ONC201 without disclosing other imipridone derivatives.

[0008] The authors also demonstrated that cell proliferation was inhibited by ONC201 through BrdU labeling experiments, and that the response to this epristeride treatment was a significant decrease in the number of viable cells within 48 hours, even including those that did not undergo apoptosis (e.g. A-549 and SNV-449) [2]. Preclinical studies have shown that it is a very promising apoptotic anticancer agent with significant activity against a variety of cancer cell lines, including, for example, PANC-1, HCT116, MDA-MB-23, U87, HFF, MRC5 and WI-38 [5-8]. In addition, in phase II clinical trials, this compound has been shown to be beneficial in the treatment of patients with various advanced malignancies [9].

[0009] With regard to the above references [5-9], it can also be said that they only refer to ONC201 and do not disclose other epristeride derivatives. In addition to ONC201, Wagner et al. disclose an isomer of ONC201, which has a straight chain [4,3-d] structure, and therefore said isomer is not covered by the scope of the present application [5]. Zhe-Zhu Jin et al. disclose the use of ONC201 with AZD-8055, which is a pyrido[2,3-d]pyrimidine derivative, and therefore it is not covered by the scope of the present application [7].

[0010] In-depth studies of analogs

[10] identified a trifluoromethylated derivative named ONC212 (Reference Compound 2) as a more potent epristeride with potentiation and selective involvement in GPCR targeting and tumor cell death.

[0011]

[0012] This compound produced significantly enhanced activity in nanomolar concentrations against a number of different malignant cell lines, solid tumors and hematological malignancies

[10] . The cited reference

[10] does not disclose ONC201 derivatives with a substituted benzyl group at position N-7.

[0013] It is also important to note that ONC212 showed better preclinical efficacy in some in vivo models of pancreatic cancer, melanoma and hepatocellular carcinoma, including ONC201-resistant tumors, such as PANC-1 and Capan-2 human pancreatic cancer xenograft models

[11] . The above reference

[11] only refers to ONC201 and its derivative ONC212, and it does not disclose other epristeride. Graves et al. demonstrated that ONC201 and some related analogs are highly potent activators of ClpP

[12] . The cited reference

[12] does not disclose ONC201 derivatives with a disubstituted or trisubstituted benzyl group at position N-7.

[0014] Since there is compelling preclinical evidence published on the interaction between TRAIL and redox signaling pathways implicated in cancer

[17] , the inventors of the present application in earlier work

[21] sought to determine whether anti-proliferative epratuzumab with ferrocene-containing substituents is capable of generating reactive oxygen species (ROS), such as nitric oxide, superoxide anion and other forms of free radicals [18, 19], which have been shown to be involved in the biological regulation process leading to programmed cell death (apoptosis)

[20] . Based on reference

[21] , a small collection of ferrocene-containing derivatives and pure organic analogs, including ONC201 and ONC212, were synthesized and tested starting from ferrocene-containing primary amines and selected benzyl amines as reference models without ROS-generating potential

[21] . The results of the in vitro anti-proliferative tests indicated that, although organometallic epratuzumab (particularly compounds with two ferrocene units such as 7de) showed significant cytotoxicity against human malignant cell lines HT-29, HEPG2, PANC1, COLO205, A2058 and EBC1 compared to ONC201, its effect was much lower than those produced by ONC212. On the other hand, it was proven that the effect of organic epratuzumab 7ah and 7ai was comparable to those of ONC212

[21] .

[0015] US Patent No. US 10,239,877 discloses a new class of 4,7-benzyl substituted epratuzumab derivatives, including the lead compound known as ONC201 and some additional substituted analogs thereof. The disclosed compounds are strong TRAIL inducers useful for cancer treatment.

[0016] US Patent No. US 9,376,437 discloses new substituted epratuzumab derivatives of the following formula (Reference Formula 1).

[0017]

[0018] wherein R1and R2independently represent hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, aralkoxy, aralkylthio, alkanoyl, thiol, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl and heterocyclic groups, and wherein, when R1represents CH2-Ph, R2does not represent CH2-(2-CH3-Ph). In preferred compounds,

[0019] R1is CH2-Ph and R2is CH2-(2-Cl-Ph),

[0020] R1 is CH2-Ph and R2 is CH2-(2-thienyl)

[0021] R1 is CH2-Ph and R2 is CH2CH2-Ph

[0022] R1 is CH2-Ph and R2 is CH2CH2-(4-N-benzyl-piperazine).

[0023] R1 is CH2-Ph and R2 is CH2-(2,4-bis-F-Ph).

[0024] R1 is H and R2 is CH2-(2-CH3-Ph).

[0025] R1 is CH3 and R2 is CH2-(2-CH3-Ph).

[0026] R1 is CH2CH2-Ph and R2 is CH2-(2-CH3-Ph).

[0027] US Patent No. 9,845,324 discloses other novel substituted imiridone derivatives of the aforementioned Formula 1, wherein R1 is selected from H, alkyl, alkylphenyl, alkylphenyl ketone, benzylpiperazine, alkylthiophene, alkylpyridinyl, alkylisooxazolyl, alkylmorpholinyl, alkylthiazolyl, and alkylpyrazinyl, wherein the alkyl, alkylphenyl, alkylphenyl ketone, benzylpiperazine, alkylthiophene, alkylpyridinyl, alkylisooxazolyl, alkylmorpholinyl, alkylthiazolyl, and alkylpyrazinyl are optionally substituted with alkyl, alkoxy, hydroxyl, perhalogenated alkyl, or halogen, and wherein R2 is a substituted or unsubstituted heterocyclic alkylalkyl, preferably morpholinoalkyl or piperazinylalkyl, or wherein R2 is a substituted heteroarylalkyl, preferably pyridinylalkyl or isoxazolylalkyl. Furthermore, the cited documents disclose compounds of Formulas 2 and 3, as defined in the cited documents:

[0028]

[0029] U.S. Patent Application No. US2016 / 0264574 discloses novel substituted imiridone derivatives of the aforementioned Reference Formula 1, wherein R1 and R2 independently represent hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, carboxyl, haloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, aralkyl, hydroxyalkyl, alkoxy, aryloxy, alkoxyalkyl, alkoxycarbonyl, arylalkoxy, arylalkylthio, alkanoyl, mercapto, alkylthio, arylthio, alkylsulfinyl, arylsulfinyl, alkylsulfonyl, arylsulfonyl, heteroaryl, acyl, and heterocyclic groups, and wherein, when R1 represents CH2-Ph, R2 does not represent CH2-(2-CH3-Ph). In preferred compounds,

[0030] R1is CH2-Ph and R2is CH2-(2-Cl-Ph),

[0031] R1is CH2-Ph and R2is CH2-(2-Cl-Ph),

[0032] R1is CH2-Ph and R2is CH2-(2-Cl-Ph),

[0033] R1is CH2-Ph and R2is CH2-(2-Cl-Ph),

[0034] R1is CH2-Ph and R2is CH2-(2-Cl-Ph),

[0035] R1is CH2-Ph and R2is CH2-(2-Cl-Ph),

[0036] R1is CH2-Ph and R2is CH2-(2-Cl-Ph),

[0037] R1is CH2-Ph and R2is CH2-(2-Cl-Ph),

[0038] R1is CH2-Ph and R2is CH2-(2-Cl-Ph),

[0039] R1is CH2-Ph and R2is CH2-(2-Cl-Ph).

[0040] US Patent No. US10,266,533 discloses substituted imipridone derivatives of the above formula (with reference to Formula 1, Formula 2 and Formula 3), wherein in Formula 1, R1and R2are independently selected from H, alkyl, alkylphenyl, alkylphenylketone, benzylpiperazine, alkylthienyl, alkylpyridyl, alkylisoxazolidinyl, alkylmorpholinyl, alkylthiazolyl and alkylpyrazinyl, wherein alkyl, alkylphenyl, alkylphenylketone, benzylpiperazine, alkylthienyl, alkylpyridyl, alkylisoxazolidinyl, alkylmorpholinyl, alkylthiazolyl and alkylpyrazinyl are optionally substituted with alkyl, alkoxy, hydroxy, perhalogenated alkyl or halogen, and wherein R2is substituted or unsubstituted heteroarylalkyl; or, wherein in Formula 2, R1is hydrogen; and R a1 , R a2 , R a3 , R a4 and R a5 are each independently selected from hydrogen, X, -CH3, -NO2, -OCH3, -CN, -CXH2, -CX2H, C2-C4alkyl, -CX3, -CH2(CX3), -CH(CX3)2, -C(CX3)3, -C p X 2p+1, -OCX3, -OC p H 2p+1 , -OC p X 2p+1 , OR m , SR m , NR m R n , NR m C(O)R n , SOR m , SO2R m , C(O)R m and C(O)OR m ; wherein R m and R n are independently selected from hydrogen or C1-C4 alkyl; and X represents halogen; or a derivative of formula 4

[0041]

[0042] wherein R a1 , R a4 and R a5 are all hydrogen; and R a2 and R a1 are both chlorine, or

[0043] R a1 , R a3 and R a5 are all hydrogen; and wherein R a2 and R a4 are fluorine, or

[0044] R a2 , R a4 and R a5 are all hydrogen; and wherein R a1 and R a3 are chlorine, or

[0045] R a2 , R a4 and R a5 are all hydrogen; and wherein R a1 is methyl and R a3 is fluorine, or

[0046] R a2 , R a4 and R a5 are all hydrogen; and wherein R a1 is fluorine and R a3 is CF3.

[0047] U.S. Patent No. US2019 / 0194201 is a divisional application of U.S. Patent No. US10,266,533 and discloses substituted imipridone derivatives substantially identical to its parent application.

[0048] U.S. Patent No. US2018 / 0141946 discloses imidazopyrimidinones of reference formula 5

[0049]

[0050] wherein n = 0 or 1; R is selected from H, mono- or polyhalogen, Ci-C6alkyl, Ci-C6alkoxy, halogenated Ci-C6alkyl, nitrogen or oxygen hetero substituents and six-membered heterocycles with 0, 1 or 2 heteroatom substitutions; Ar is selected from mono- or disubstituted aryl, wherein at least one substituent is selected from halogen, Ci-C6alkyl and halogenated Ci-C4alkyl; and wherein, when n = 1 and R is H, Ar is not phenyl, 2-chlorophenyl, 2,4-difluorophenyl or o-methylphenyl. Preferred meanings of R are F, Cl, Br, methyl, isobutyl, methoxy, trifluoromethyl, morpholinyl or piperazinyl. The cited document discloses the preparation and efficacy data of 58 specific compounds.

[0051] International Patent Application No. WO 2018 / 031987 discloses further substituted 4,7-dibenzyl- and 4-benzyl-7-(thiophenyl-methyl)-imipridones of formula (reference formula 6)

[0052]

[0053] wherein V means substituted benzyl, (thiophen-2-yl)-methyl or (thiophen-3-yl)-methyl and the other substituents are as defined in the cited document. The application includes the preparation and efficacy data of 27 specific compounds.

[0054] U.S. Patent Application No. US2018 / 016277 relates to new deuterated imidazo[l,2- a]pyrido[3,4-e]pyrimidin-5(lH)-one compounds, such as ONC201 and analogs. The document also discloses compositions comprising the compounds of the cited document, and the use of such compositions, alone or in combination with other treatments, in the treatment of diseases and conditions that are beneficially treated by the administration of an inducer of a gene encoding a member of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) superfamily 10. In addition, the cited patent document discloses the use of imipridone derivatives in the treatment of cancer. So, the use of the compound ONC201 in the treatment of urogenital system cancer is disclosed in U.S. Patent No. US10,456,402.

[0055] US Patent No. 9,688,679 discloses the use of ONC201 in the treatment of leukemia.

[0056] International Patent Application No. WO 2017 / 132661 discloses compounds of the aforementioned Formula 1 for the treatment or prevention of diseases, symptoms, or conditions in subjects requiring selective regulation of class A G protein-coupled receptor (GPCR) or class A GPCR signaling pathway activity, particularly for the treatment of cancers selected from: central nervous system tumors, brain tumors, peripheral nervous system tumors, pheochromocytoma, paragangliomas, neuroendocrine tumors, pancreatic cancer, prostate cancer, endometrial cancer, hematologic malignancies, and lymphatic system tumors. Preferred compounds are ONC 201, 206, 212, 2013, and 236.

[0057] US Patent Nos. 10,172,862 and 10,369,154 disclose the use of compound ONC201 in the treatment of midline gliomas.

[0058] The problem to be solved by the present invention

[0059] There is a need for new compounds that possess enhanced anticancer activity compared to existing technologies, a property that makes these compounds suitable for pharmaceutical use.

[0060] According to the discovery of the present invention

[0061] By carefully examining the structural features of the remaining members of the imirone family, we discovered that the novel imirone derivatives related to this invention have enhanced anticancer activity, said imirone derivatives comprising a 3,5-disubstituted benzyl group attached to the 7th position. Attached Figure Description

[0062] Figure 1 The IC was established based on the in vitro test results of the PC3 human prostate cancer cell line. 50 curve.

[0063] Figure 2 The IC was established based on in vitro testing results of the LNCap human prostate cancer cell line. 50 curve.

[0064] Figure 3 IC50 was established based on in vitro testing results of the BxPC3 pancreatic cancer cell line. 50 curve.

[0065] Figure 4 The IC50 was established based on in vitro testing results of the MiaPaCa2 pancreatic cancer cell line. 50 curve.

[0066] Figure 5IC50: IC established from the results of in vitro tests performed on the Panc 1 pancreatic carcinoma cell line 50 curve.

[0067] Figure 6 IC50: IC established from the results of in vitro tests performed on the A549 lung carcinoma cell line 50 curve.

[0068] Figure 7 IC50: IC established from the results of in vitro tests performed on the HCC827 lung carcinoma cell line 50 curve.

[0069] Figure 8 IC50: IC established from the results of in vitro tests performed on the H1993 lung carcinoma cell line 50 curve.

[0070] Figure 9 IC50: IC established from the results of in vitro tests performed on the H520 lung carcinoma cell line 50 curve.

[0071] Figure 10 IC50: IC established from the results of in vitro tests performed on the MDA-MB-453 breast carcinoma cell line 50 curve.

[0072] Figure 11 IC50: IC established from the results of in vitro tests performed on the MDA-MB-231 breast carcinoma cell line 50 curve.

[0073] Figure 12 IC50: Dose-response curve obtained on the DU 145, LNCaP and PC-3 cell lines for compound I / 1 (ONC 212) (reference).

[0074] Figure 13 IC50: Dose-response curve obtained on the DU 145, LNCaP and PC-3 cell lines for compound I / 7 (ABB-011).

[0075] Figure 14 IC50: Dose-response curve obtained on the DU 145, LNCaP and PC-3 cell lines for compound I / 3 (CZT-021) (reference).

[0076] Figure 15 IC50: Dose-response curve obtained on the DU 145, LNCaP and PC-3 cell lines for compound I / 124 (TBP-333).

[0077] Figure 16Dose-response curves obtained for Compound I / 6 (TBP-218) (reference) on DU 145, LNCaP and PC-3 cell lines.

[0078] Figure 17 Dose-response curves obtained for Compound I / 111 (TBP-272) on DU 145, LNCaP and PC-3 cell lines.

[0079] Figure 18 Dose-response curves obtained for Compound I / 149 (TBP-353) (reference) on DU 145, LNCaP and PC-3 cell lines.

[0080] Figure 19 Dose-response curves obtained for Compound I / 133 (TBP-400) on DU 145, LNCaP and PC-3 cell lines.

[0081] Figure 20 Dose-response curves obtained for Compound I / 30 (TBP-301) on DU 145, LNCaP and PC-3 cell lines.

[0082] Figure 21 Dose-response curves obtained for Compound I / 107 (CZT-136) on DU 145, LNCaP and PC-3 cell lines.

[0083] Figure 22 Dose-response curves obtained for Compound I / 1 (ONC 212) (reference) on Panc-1, Capan-1 and MIA PaCa-2 cell lines.

[0084] Figure 23 Dose-response curves obtained for Compound I / 7 (ABB-011) on Panc-1, Capan-1 and MIA PaCa-2 cell lines.

[0085] Figure 24 Dose-response curves obtained for Compound I / 3 (CZT-021) (reference) on Panc-1, Capan-1 and MIA PaCa-2 cell lines.

[0086] Figure 25 Dose-response curves obtained for Compound I / 124 (TBP-333) on Panc-1, Capan-1 and MIA PaCa-2 cell lines.

[0087] Figure 26 Dose-response curves obtained for Compound I / 6 (TBP-218) (reference) on Panc-1, Capan-1 and MIA PaCa-2 cell lines.

[0088] Figure 27 Dose-response curves obtained for Compound I / 111 (TBP-272) on Panc-1, Capan-1 and MIA PaCa-2 cell lines.

[0089] Figure 28 Dose-response curves obtained for Compound I / 149 (TBP-353) (reference) on Panc-1, Capan-1 and MIA PaCa-2 cell lines.

[0090] Figure 29 Dose-response curves obtained for Compound I / 133 (TBP-400) on Panc-1, Capan-1 and MIA PaCa-2 cell lines.

[0091] Figure 30 Dose-response curves obtained for Compound I / 30 (TBP-301) on Panc-1, Capan-1 and MIA PaCa-2 cell lines.

[0092] Figure 31 Dose-response curves obtained for Compound I / 107 (CZT-136) on Panc-1, Capan-1 and MIA PaCa-2 cell lines.

[0093] Figure 32 Dose-response curves obtained for Compound I / 1 (ONC 212) (reference) on Detroit 562, SCC-25 and Fadu cell lines.

[0094] Figure 33 Dose-response curves obtained for Compound I / 7 (ABB-011) on Detroit 562, SCC-25 and Fadu cell lines.

[0095] Figure 34 Dose-response curves obtained for Compound I / 3 (CZT-021) (reference) on Detroit 562, SCC-25 and Fadu cell lines.

[0096] Figure 35 Dose-response curves obtained for Compound I / 124 (TBP-333) on Detroit 562, SCC-25 and Fadu cell lines.

[0097] Figure 36 Dose-response curves obtained for Compound I / 6 (TBP-218) (reference) on Detroit 562, SCC-25 and Fadu cell lines.

[0098] Figure 37 Dose-response curves obtained for Compound I / 111 (TBP-272) on Detroit 562, SCC-25 and Fadu cell lines.

[0099] Figure 38 Dose-response curves obtained for Compound I / 149 (TBP-353) (reference) on Detroit 562, SCC-25 and Fadu cell lines.

[0100] Figure 39 Dose-response curves obtained for Compound I / 133 (TBP-400) on Detroit 562, SCC-25 and Fadu cell lines.

[0101] Figure 40 Dose-response curves obtained for Compound I / 30 (TBP-301) on Detroit 562, SCC-25 and Fadu cell lines.

[0102] Figure 41 Dose-response curves obtained for Compound I / 107 (CZT-136) on Detroit 562, SCC-25 and Fadu cell lines.

[0103] Figure 42 Dose-response curves obtained for Compound I / 1 (ONC 212) (reference) on EBC-1, MDA-MB-231 and MDA-MB-453 cell lines.

[0104] Figure 43 Dose-response curves obtained for Compound I / 7 (ABB-011) on EBC-1, MDA-MB-231 and MDA-MB-453 cell lines.

[0105] Figure 44 Dose-response curves obtained for Compound I / 3 (CZT-021) (reference) on EBC-1, MDA-MB-231 and MDA-MB-453 cell lines.

[0106] Figure 45 Dose-response curves obtained for Compound I / 124 (TBP-333) on EBC-1, MDA-MB-231 and MDA-MB-453 cell lines.

[0107] Figure 46 Dose-response curves obtained for Compound I / 6 (TBP-218) (reference) on EBC-1, MDA-MB-231 and MDA-MB-453 cell lines.

[0108] Figure 47Dose-response curves obtained for Compound I / 111 (TBP-272) on EBC-1, MDA-MB-231 and MDA-MB-453 cell lines.

[0109] Figure 48 Dose-response curves obtained for Compound I / 149 (TBP-353) (reference) on EBC-1, MDA-MB-231 and MDA-MB-453 cell lines.

[0110] Figure 49 Dose-response curves obtained for Compound I / 133 (TBP-400) on EBC-1, MDA-MB-231 and MDA-MB-453 cell lines.

[0111] Figure 50 Dose-response curves obtained for Compound I / 30 (TBP-301) on EBC-1, MDA-MB-231 and MDA-MB-453 cell lines.

[0112] Figure 51 Dose-response curves obtained for Compound I / 107 (CZT-136) on EBC-1, MDA-MB-231 and MDA-MB-453 cell lines.

[0113] Figure 52 Cytotoxic effect of Compound I / 1 (ONC 212) (reference) on Panc-1 cell line.

[0114] Figure 53 Cytotoxic effect of Compound I / 3 (CZT-021) (reference) on Panc-1 cell line.

[0115] Figure 54 Cytotoxic effect of Compound I / 6 (TBP-218) (reference) on Panc-1 cell line.

[0116] Figure 55 Cytotoxic effect of Compound I / 149 (TBP-353) (reference) on Panc-1 cell line.

[0117] Figure 56 Cytotoxic effect of Compound I / 30 (TBP-301) on Panc-1 cell line.

[0118] Figure 57 Cytotoxic effect of Compound I / 7 (ABB-011) on Panc-1 cell line.

[0119] Figure 58 Cytotoxic effect of Compound I / 124 (TBP-333) on Panc-1 cell line.

[0120] Figure 59 Cytotoxic effect of compound I / 111 (TBP-272) on Panc-1 cell line.

[0121] Figure 60 Cytotoxic effect of compound I / 133 (TBP-400) on Panc-1 cell line.

[0122] Figure 61 Cytotoxic effect of compound I / 107 (CZT-136) on Panc-1 cell line.

[0123] Figure 62 Changes in body weight in animals in the experiment disclosed in Example 23.

[0124] Figure 63 Changes in tumor volume in the experiment disclosed in Example 23 [*: P < 0.05 (Student's t-test)].

[0125] Figure 64 Tumor size (weight) at the end of the experiment in the experiment disclosed in Example 23 [*: p < 0.05 (Student's t-test)].

[0126] Figures 1-11 Tumor at the end of the experiment in the experiment disclosed in Example 23. SUMMARY

[0127] 1. A compound of formula (I) or a stereoisomer, an enantiomer, a mixture of enantiomers, a mixture of diastereomers thereof, or a pharmaceutically acceptable salt thereof

[0128]

[0129]

[0130] wherein

[0131] if Y is phenyl and Z is H, then X is 3-fluorophenyl-methyl (compound I / 44), 3- (aminomethyl)phenyl (compound I / 58), 4-(aminomethyl)phenyl (compound I / 62), 3- azetidinyl (compound I / 90), 4-piperidinyl (compound I / 96), 3-azidophenyl (compound I / 102), 4-azidophenyl (compound I / 104), (4-diferrocenyl-1H-1,2,3-triazol-1- yl)phenyl (compound I / 121), or:

[0132] or

[0133] If Y is 3-fluorophenyl and Z is H, then X is 2-iodoferrocenyl (Compound I / 52), 3- (aminomethyl)phenyl (Compound I / 60), 4-(aminomethyl)phenyl (Compound I / 63), 3- azidophenyl (Compound I / 105), or 4-azidophenyl (Compound I / 106); or

[0134] If Y is 3,5-difluorophenyl and Z is H, then X is 3-(aminomethyl)phenyl (Compound I / 61), 4-(aminomethyl)phenyl (Compound I / 64), 4-azidophenyl (Compound I / 107), 3-azidophenyl (Compound I / 127), (4-ferrocenyl-lH-l,2,3-triazol-l-yl)phenyl (Compound I / 132); or

[0135] If Y is 4-(trifluoromethyl)phenyl and Z is H, then X is 3-(aminomethyl)phenyl (Compound I / 68); or

[0136] If Y is 3-fluoro-4-(trifluoromethyl)phenyl and Z is H, then X is 3-(aminomethyl)phenyl (Compound I / 73); or

[0137] If Y is 3-fluorophenylmethyl and Z is H, then X is 2-methylphenyl (Compound I / 45); or

[0138] If Y is 3-(aminomethyl)phenyl and Z is H, then X is 4-iodophenyl (Compound I / 59), ferrocenyl (Compound I / 67), 4-(trifluoromethyl)phenyl (Compound I / 69), or ferrocenylmethyl (Compound I / 70); or

[0139] If Y is ferrocenyl and Z is H, then X is 3-(aminomethyl)phenyl (Compound I / 65); or

[0140] If Y is ferrocenylmethyl and Z is H, then X is 3-(aminomethyl)phenyl (Compound I / 66); or

[0141] If Y is 3-(methoxycarbonylamino-methyl)phenyl and Z is H, then X is ferrocenylmethyl (Compound I / 71) or ferrocenyl (Compound I / 72); or

[0142] If Y is 4-aminophenyl and Z is H, then X is 3-azetidinyl (Compound I / 91) or 4-piperidinyl (Compound I / 93); or

[0143] If Y is 3-azetidinyl and Z is H, then X is 2-methylphenyl (Compound I / 92); or

[0144] If Y is 4-piperidinyl and Z is H, then X is 2-methylphenyl (compound I / 94) or 4-fluorophenyl (compound I / 95); or

[0145] If Y is 3-pyrrolidinyl and Z is H, then X is 2-methylphenyl (compound I / 97) or 4-fluorophenyl (compound I / 98); or

[0146] If Y is 2-pyrrolidinyl and Z is H, then X is 2-methylphenyl (compound I / 99) or 4-fluorophenyl (compound I / 100); or

[0147] If Y is 4-azidophenyl and Z is H, then X is 2-methylphenyl (compound I / 101), 3-fluorophenyl (compound I / 108), 4-fluorophenyl (compound I / 109), 4-(trifluoromethyl)phenyl (compound I / 110), 3-fluoro-4-(trifluoromethyl)phenyl (compound I / 114), 4-iodophenyl (compound I / 115), 3,4,5-trimetoxyphenyl (compound I / 116), 4-azidophenyl (compound I / 117), 3-azetidinyl (compound I / 118), or 4-piperidinyl (compound I / 119); or

[0148] If Y is 3-azidophenyl and Z is H, then X is 2-methylphenyl (compound I / 103), 4-(trifluoromethyl)phenyl (compound I / 111), 3-fluoro-4-(trifluoromethyl)phenyl (compound I / 112), 4-iodophenyl (compound I / 113), or 4-chlorophenyl (compound I / 133); or

[0149] If Y is (4-diferrocenyl-lH-l,2,3-triazol-l-yl)phenyl and Z is H, then X is 2-methylphenyl (compound I / 120); or

[0150] If Y is

[0151]

[0152] then X is 2-methylphenyl (compound I / 122); or

[0153] If Y is 3,5-diazidophenyl and Z is H, then X is 4-chlorophenyl (compound I / 138) or 4-(trifluoromethyl)phenyl (compound I / 139); or

[0154] If Y is 3-cyanothiophenyl and Z is H, then X is 4-chlorophenyl (compound I / 142) or 4-(trifluoromethyl)phenyl (compound I / 143); or

[0155] If Y is 3-cyanothiophenyl and Z is H, then X is 4-chlorophenyl (compound I / 145) or 4-(trifluoromethyl)phenyl (compound I / 146).

[0156] 2. The compound according to point 1, or a stereoisomer, an enantiomer, a mixture of enantiomers, a mixture of diastereomers thereof, or a pharmaceutically acceptable salt thereof, wherein

[0157] If Y is 3-cyanothiophenyl and Z is H, then X is 4-chlorophenyl (compound I / 145) or 4-(trifluoromethyl)phenyl (compound I / 146).

[0158] If Y is 3,5-dicyanothiophenyl and Z is H, then X is 4-chlorophenyl (compound I / 138) or 4-(trifluoromethyl)phenyl (compound I / 139).

[0159] If Y is 3-cyanothiophenyl and Z is H, then X is 4-chlorophenyl (compound I / 145) or 4-(trifluoromethyl)phenyl (compound I / 146).

[0160] If Y is 3,5-dicyanothiophenyl and Z is H, then X is 4-chlorophenyl (compound I / 138) or 4-(trifluoromethyl)phenyl (compound I / 139).

[0161] 3. The compound according to point 1, or a stereoisomer, an enantiomer, a mixture of enantiomers, a mixture of diastereomers thereof, or a pharmaceutically acceptable salt thereof, wherein

[0162] If Y is 3,5-dicyanothiophenyl and Z is H, then X is 4-chlorophenyl (compound I / 138) or 4-(trifluoromethyl)phenyl (compound I / 139).

[0163] If Y is 3-cyanothiophenyl and Z is H, then X is 4-chlorophenyl (compound I / 145) or 4-(trifluoromethyl)phenyl (compound I / 146).

[0164] 4. A compound of formula (I), or a stereoisomer, an enantiomer, a mixture of enantiomers, a mixture of diastereomers thereof, or a pharmaceutically acceptable salt thereof, for use as a medicament,

[0165]

[0166] wherein

[0167] If Y is phenyl and Z is H, then X is 4-fluorophenyl (compound I / 5), 3-fluorophenyl (compound I / 8), 3,4,5-trifluorophenyl (compound I / 9), 2,3,4-trifluorophenyl (compound I / 29), 2-fluoro-4-nitrophenyl (compound I / 48) or 3-aminophenyl (compound I / 75); or

[0168] if Y is 3,5-difluorophenyl and Z is H, then X is 4-fluorophenyl (Compound I / 7), 4-(trifluoromethyl)phenyl (Compound I / 30), 4-iodophenyl (Compound I / 38), 4-bromophenyl (Compound I / 39), 2-fluoro-4-nitrophenyl (Compound I / 50), 4-chlorophenyl (Compound I / 124), or 3-aminophenyl (Compound I / 126) or 2-methylphenyl (Compound I / 37); or

[0169] if Y is 3,5-difluorophenyl and Z is H, then X is 4-fluorophenyl (Compound I / 7), 4-(trifluoromethyl)phenyl (Compound I / 30), 4-iodophenyl (Compound I / 38), 4-bromophenyl (Compound I / 39), 2-fluoro-4-nitrophenyl (Compound I / 50), 4-chlorophenyl (Compound I / 124), or 3-aminophenyl (Compound I / 126) or 2-methylphenyl (Compound I / 37); or

[0170] if Y is 2-fluorophenyl and Z is H, then X is 3-fluoro-4-(trifluoromethyl)phenyl (Compound I / 26) or 4-iodophenyl (Compound I / 54); or

[0171] if Y is 4-(trifluoromethyl)phenyl and Z is H, then X is 3-fluoro-4-(trifluoromethyl)phenyl (Compound I / 27); or

[0172] if Y is 4-fluorophenyl and Z is H, then X is 3-fluoro-4-(trifluoromethyl)phenyl (Compound I / 28) or 4-iodophenyl (Compound I / 55); or

[0173] if Y is 4-aminophenyl and Z is H, then X is 4-(trifluoromethyl)phenyl (Compound I / 81), 4-fluorophenyl (Compound I / 86), 4-iodophenyl (Compound I / 87), or 3,4,5-trimethoxyphenyl (Compound I / 89); or

[0174] if Y is 3,5-difluorophenyl and Z is hydroxymethyl, then X is 4-chlorophenyl (racemic) (Compound I / 128), 4-fluorophenyl (R-enantiomer) (Compound I / 129(R)), or 4-fluorophenyl (S-enantiomer) (Compound I / 129(S));

[0175] if Y is 3,5-dicyanophenyl and Z is H, then X is 4-chlorophenyl (Compound I / 136) or 4-(trifluoromethyl)phenyl (Compound I / 137); or

[0176] if Y is phenyl and Z is H, then X is 2-iodophenyl (Compound I / 11).

[0177] 5. A compound of Formula (I), or a stereoisomer, an enantiomer, a mixture of enantiomers, a mixture of diastereomers thereof, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer,

[0178]

[0179] wherein

[0180] if Y is phenyl and Z is H, then X is 4-fluorophenyl (compound I / 5), 3-fluorophenyl (compound I / 8), 3,4,5-trifluorophenyl (compound I / 9), 2,3,4-trifluorophenyl (compound I / 29), 2-fluoro-4-nitrophenyl (compound I / 48), or 3-aminophenyl (compound I / 75); or

[0181] if Y is 3-fluorophenyl and Z is H, then X is 3-fluoro-4-(trifluoromethyl)phenyl (compound I / 31), 4-iodophenyl (compound I / 46), 2-fluoro-4-nitrophenyl (compound I / 49), 2-methylphenyl (compound I / 53), or 4-aminophenyl (compound I / 84); or

[0182] if Y is 3,5-difluorophenyl and Z is H, then X is 4-fluorophenyl (compound I / 7), 4-(trifluoromethyl)phenyl (compound I / 30), 4-iodophenyl (compound I / 38), 4-bromophenyl (compound I / 39), 2-fluoro-4-nitrophenyl (compound I / 50), 4-chlorophenyl (compound I / 124), or 3-aminophenyl (compound I / 126) or 2-methylphenyl (compound I / 37); or

[0183] if Y is 2-fluorophenyl and Z is H, then X is 3-fluoro-4-(trifluoromethyl)phenyl (compound I / 26) or 4-iodophenyl (compound I / 54); or

[0184] if Y is 4-(trifluoromethyl)phenyl and Z is H, then X is 3-fluoro-4-(trifluoromethyl)phenyl (compound I / 27); or

[0185] if Y is 4-fluorophenyl and Z is H, then X is 3-fluoro-4-(trifluoromethyl)phenyl (compound I / 28) or 4-iodophenyl (compound I / 55); or

[0186] if Y is 4-aminophenyl and Z is H, then X is 4-(trifluoromethyl)phenyl (compound I / 81), 4-fluorophenyl (compound I / 86), 4-iodophenyl (compound I / 87), or 3,4,5-trimethoxyphenyl (compound I / 89); or

[0187] if Y is 3,5-dicyano-phenyl and Z is H, then X is 4-chloro-phenyl (compound I / 136) or 4- (trifluoromethyl)-phenyl (compound I / 137); or

[0188] if Y is 3,5-dicyano-phenyl and Z is H, then X is 4-chloro-phenyl (compound I / 136) or 4- (trifluoromethyl)-phenyl (compound I / 137); or

[0189] if Y is phenyl and Z is H, then X is 2-iodo-phenyl (compound I / 11).

[0190] 6. The compound for use according to point 5, wherein the cancer is selected from the group consisting of prostate cancer, pancreatic cancer, lung cancer, breast cancer, glioma, head and neck cancer, colon cancer, skin cancer.

[0191] 7. The compound for use according to points 4 to 6, or a stereoisomer, an enantiomer, a mixture of enantiomers, a mixture of diastereomers thereof, or a pharmaceutically acceptable salt thereof, wherein

[0192] if Y is 3,5-dicyano-phenyl and Z is H, then X is 4-chloro-phenyl (compound I / 136) or 4- (trifluoromethyl)-phenyl (compound I / 137); or

[0193] if Y is 3,5-dicyano-phenyl and Z is H, then X is 4-chloro-phenyl (compound I / 136) or 4- (trifluoromethyl)-phenyl (compound I / 137); or

[0194] if Y is 3,5-dicyano-phenyl and Z is H, then X is 4-chloro-phenyl (compound I / 136) or 4- (trifluoromethyl)-phenyl (compound I / 137); or

[0195] if Y is phenyl and Z is H, then X is 2-iodo-phenyl (compound I / 11).

[0196] 8. The compound for use according to points 4 to 6, or a stereoisomer, an enantiomer, a mixture of enantiomers, a mixture of diastereomers thereof, or a pharmaceutically acceptable salt thereof, wherein

[0197] if Y is 3,5-dicyano-phenyl and Z is H, then X is 4-chloro-phenyl (compound I / 136) or 4- (trifluoromethyl)-phenyl (compound I / 137); or

[0198] 9. The compound according to points 1-3 for use as a medicament.

[0199] 10. The compound according to bullets 1-3 for use in the treatment of cancer.

[0200] 11. The compound for use according to bullet 10, wherein the cancer is selected from the group consisting of prostate cancer, pancreatic cancer, lung cancer, breast cancer, glioma, head and neck cancer, colon cancer, skin cancer. DETAILED DESCRIPTION

[0201] The present application relates to a compound of formula (I) as described in item 1 above, and pharmaceutically acceptable salts, stereoisomers, including single enantiomers, racemic mixtures, mixtures of enantiomers, or combinations thereof thereof.

[0202] One group of compounds of the present application are compounds of formula (I) as described above, and pharmaceutically acceptable salts, stereoisomers, including single enantiomers, racemic mixtures, mixtures of enantiomers, or combinations thereof thereof, wherein the identifiers in the formula are as defined in item 2.

[0203] Another group of compounds of the present application are compounds of formula (I) as described in item 3 above, and pharmaceutically acceptable salts, stereoisomers, including single enantiomers, racemic mixtures, mixtures of enantiomers, or combinations thereof thereof, wherein the identifiers in the formula are as defined in item 3.

[0204] Another group of compounds of the present application are compounds of formula (I) as described in item 4 above, and pharmaceutically acceptable salts, stereoisomers, including single enantiomers, racemic mixtures, mixtures of enantiomers, or combinations thereof thereof, wherein the identifiers in the formula are as defined in item 4.

[0205] Another group of compounds of the present application are compounds of formula (I) as described in item 5 above, and pharmaceutically acceptable salts, stereoisomers, including enantiomers, racemic mixtures, mixtures of enantiomers, or combinations thereof. As described above, the compounds of the present application can exist as racemic mixtures as well as as optical isomers, in the form of one enantiomer, either pure or predominantly present. It is to be understood that both the racemic mixtures and the pure form or the enantiomer predominantly present in the mixture are subject matter of the present application.

[0206] The present application also relates to the use of a compound of the present application for the treatment of a cancer disease selected from the group consisting of prostate cancer, pancreatic cancer, lung cancer, breast cancer, glioma, head and neck cancer, colon cancer, skin cancer.

[0207] By carefully examining the structural features of the remaining members of imipridone as documented in the prior art, it was determined that, in addition to the limited diversity of the substituents bound to N-4 and N-7, excluding ONC234 (Reference Compound 1), the known imipridones do not contain disubstituted or trisubstituted benzyl groups attached to position 7, which suggests that the available chemical space has not been fully exploited. This realization led to the identification of new lead compounds with more pronounced potential in chemotherapeutic applications, which, in addition to having stronger anticancer activity, also have additional beneficial properties, such as a superior therapeutic window and bioavailability over the current state-of-the-art collection.

[0208] Accordingly, following the synthetic route outlined in our presentation of Reaction Scheme 1, we performed a diversity-oriented synthesis of new imipridones bearing various mono-, di- and tri-substituted benzyl groups at both ends of the heterocyclic backbone and amine-based molecular fragments (see Table 1).

[0209] To expand the range of functional groups on the substituents of the backbone to be processed, compounds prepared from commercially available building blocks containing Boc-protected aromatic amine residues were converted into azido derivatives (see Table 1) by a one-pot procedure involving simultaneous acid-catalyzed N-deprotection and diazotization followed by diazo→azido exchange (see Reaction Scheme 2). By copper(I)-catalyzed coupling of the two azides with ethynylferrocene and erlotinib, triazole-tethered hybrids were obtained (see Table 1).

[0210] In the course of the creative work, while designing and synthesizing new imipridones displaying enhanced cytotoxic effects, the results of the biological tests revealing characteristic structure-activity relationships (SAR) were constantly taken into account for structure refinement. Since only a limited set of data obtained from biological assays of the known imipridones of the prior art was available (WO 2018031987A1), in addition to ONC212, we prepared three more known halogenated analogs [I / 2 (ONC217), I / 3 (2185824-99-9P) and I / 4 (2185824-98-8P) in Table 1] as references for our biological tests.

[0211] As a result of the stepwise refinement, we identified three new halogenated imipridones [I / 7 (ABB-011), I / 30 (TBP-301) and I / 39 (TBP-302)] as the most effective drug candidates, which displayed extremely strong cytotoxicity against representative cell lines characterized by IC 50Values ​​in the range of approximately 3–8 nM unequivocally demonstrate their superiority over each of the reference compounds known in the art (Tables 2 and 3). We also disclose that the R-enantiomer of the hydroxymethyl-substituted imiridone I / 129(R) [TBP-339(R)] exhibits significantly stronger antiproliferative activity against the PANC1 cell line than the S-enantiomer I129(S) [(TBP-339(S)] (IC50). 50 =15nM vs 265nM: Table 2).

[0212] A gradually increasing trend in the cytotoxicity observed in ONC212, 2185824-99-9P, and I / 30 (TBP-301) established a clear binding-effect relationship, which is related to the stepwise introduction of fluorine substituents at the meta position of the N-7-benzyl group. The same trend was observed in the antiproliferative effects of the I / 5 (TBP-134), I / 6 (TBP-218), I / 7 (ABB-011), I / 41 (TBP-285), and 2185824-98-8PI / 39 (TBP-302) series; the 3,5-difluorobenzyl-substituted derivatives are the most active to date.

[0213] Preparation of the compounds of the present invention

[0214] The aggregation synthesis of new imiridones of general formula (I) (except for compounds containing N3, CN, SCN and SeCN substituents on the side chains at positions 4 and 7) (reaction scheme 1) is based on direct coupling reaction and anullation using readily available precursors such as 2-(methylthio)-4,5-dihydro-1H-imidazolium (1), methacrylates and primary amine types 3 and 5.

[0215]

[0216] Materials and methods

[0217] All fine chemicals were commercially available (Merck, Fluorochem, Molar Chemicals, VWR) and required no further purification before use. Dioxane was distilled off from sodium benzoate. Merck Kieselgel (230-400 mesh) Used for flash column chromatography. Melting point (uncorrected) was determined using a Büchi M-560. All compounds... 1 H NMR and 13 The CNMR spectra were obtained in 5 mm tubes in CDCl3 solution at room temperature on a Bruker DRX-500 spectrometer at 500 nm. 1 H) and 125 ( 13C) MHz recorded with the deuterium signal of the solvent as lockfield, TMS as internal standard. Support 1 H NMR and 13 The accurate assignment of the C NMR signals was obtained by HSQC, HMBC, COSY and NOESY spectra using standard Bruker pulse programs.

[0218] The general procedure for the synthesis steps finally leads to compounds of formula (I) as shown in reaction scheme 1

[0219] 1. Methyl 2-(methylthio)-4,5-dihydro-1 H-imidazole-1 -carboxylate (2)

[0220]

[0221] Commercially available 2-methylthio-4,5-dihydroimidazolium iodide (12.21 g, 50 mmol) and triethylamine (TEA, 16 mL, 11.62 g, 115 mmol) were dissolved in DCM (50 mL). Methyl chloroformate (5 mL, 6.12 g, 65 mmol) was added dropwise to the previously cooled to 0 °C solution. The reaction mixture was allowed to warm to 25 °C and stirred overnight. EtOAc (200 mL) was added and after stirring for 15 minutes the precipitated ammonium salt was filtered off and washed with EtOAc (50 mL). The combined solutions were evaporated to dryness. The solid residue was triturated with water, filtered off and dried under vacuum to give 5 as a white solid. Yield: 5.55 g (64%).

[0222] 2. N-substituted-4,5-dihydro-1 H-imidazol-2-amine (4)

[0223]

[0224] To a solution of primary amine type 3 (2 mmol) dissolved in a mixture of MeOH:AcOH (4 mL: 1 mL) was added methyl 2-(methylthio)-4,5-dihydro-1 H-imidazole-1 - carboxylate 2 (0.47 g, 2.4 mmol) and the resulting solution was stirred at reflux for 20 hours. After cooling, the reaction was concentrated in vacuum and the oily residue was dissolved in DCM (30 mL). The solution was washed with 3 M NaOH (10 mL), brine (10 mL), dried over Na2S04and evaporated to dryness. The colourless oil was crystallized from ether and used for the cyclization to the imipenem framework without further purification.

[0225] 3. Cyclization to imipenem

[0226] A primary amine 5 (1 mmol), dissolved in MeOH (4 mL), was added methyl acrylate (0.23 mL, 2.5 mmol) and the mixture was stirred at room temperature for 24 hours. The resulting crude dipropionic acid ester type 6 was dissolved in dry THF (4 mL). NaH (0.12 g, 5 mmol) was added in small portions to the vigorously stirred solution previously cooled to 0°C under an argon atmosphere. The resulting suspension was stirred for an additional 2 hours at reflux temperature and concentrated to dryness under vacuum. The resulting solid residue containing the crude sodium salt of N-substituted oxopiperidine-3-carboxylic acid methyl ester (7) was dissolved in dry MeOH (5 mL). To this solution was added N-substituted-4,5-dihydro-lH-imidazol-2-amine (4) (1 mmol) prepared in different steps as described above. The basic solution was stirred at reflux temperature under an argon atmosphere for 12 hours and then cooled with ice water. The cooled reaction mixture was stirred for 1 hour and the precipitated solid was collected by filtration, washed with cold methanol and dried to give the pure imipenem product of formula (I).

[0227] 4. Synthesis of amine-containing imipenem.

[0228] To synthesize target compounds having a primary or cyclic secondary amine moiety to be processed, in the general procedure described above, the corresponding diamine (3 or 5) mono-Boc-protected in the X or Y group was used as coupling component. The isolated Boc-protected imipenem (2 mmol) was dissolved in cc HCl (5 mL) and the resulting solution was heated at reflux for 5 minutes and cooled to room temperature. The pH of the solution was then adjusted to about 13-14 with a concentrated aqueous potassium hydroxide solution. The precipitated amine product was collected by filtration, washed thoroughly with cold water and dried in a desiccator with potassium hydroxide pellets.

[0229] 5. Synthesis of azido-benzyl imipenem

[0230] The corresponding amino-benzyl imipenem (1 mmol) was dissolved in cc HCl (5 mL). To this solution, cooled to 0°C, was added dropwise an aqueous solution of NaNO2(137.8 mg, 2 mmol, dissolved in 2.5 mL of water). The diazotization reaction was monitored by TLC. Upon completion, NaN3(324 mg, 5 mmol) was added to the reaction mixture at 0°C and then stirred at room temperature for 1 hour. The pH of the solution was adjusted to 10-11 by careful addition of solid Na2CO3. The resulting mixture was extracted with CH2Cl2(2 x 40 mL) and the organic phase was dried over Na2SO4and then evaporated to dryness. The oily residue was crystallized from n-hexane to give the product as a colorless solid.

[0231] 6. Synthesis of aryl-triazolyl-benzyl imipenem from azido-benzyl imipenem

[0232] The corresponding azidobenzyl imelinide (1 mmol), the terminal alkyne component (1 mmol) and CuI (29.3 mg, 0.15 mmol) were dissolved in DMSO (5 mL). The reaction mixture was stirred at room temperature in a closed vessel for 24 h, then poured onto water (50 mL). The precipitated solid was collected by filtration, washed with water (100 mL) and suspended in an ammonia solution (20 mL). The suspension was stirred for 20 min and filtered. The residue was washed with water (50 mL), dried and dissolved in a 9:1 mixture of CH2Cl2and MeOH (10 mL). The solution was passed through silica and evaporated. The solid residue was crystallized from ether.

[0233] In the following, the application will be illustrated by way of exemplary embodiments, which are, however, not to be understood as limiting the application.

[0234] Example

[0235] Example 1:

[0236] 7-(3,5-difluorobenzyl)-4-(4-(trifluoromethyl)benzyl)-2,4,6,7,8,9-hexahydroimidazo[l,2- a]pyrido[3,4-e]pyrimidin-5(lH)-one (TBP-301) (Compound I / 30)

[0237]

[0238] Yield: 387 mg (81 %). Melting point: 168.0 °C. 1 H-NMR (CDCI3): 7.53 and 7.50 (A and B are part of an AA'BB' spin system, J AB = 8.9 Hz, 2x2H, H-3",5" and H-2",6", resp.); 6.84 (br dt, J ~ 7 Hz and ~ 2 Hz, 2H, H-2',6'); 6.67 (tt, J = 9.0 Hz and 2.3 Hz, 1H, H-4'); 5.06 (s, 2H, H-11); 3.88 (s, 4H, H-1 and H-2); 3.60 (s, 2H, H-10); 3.24 (br s, 2H, H-6); 2.64 (t, 2H, J = 5.7 Hz, H-8); 2.46 (t, 2H, J = 5.7 Hz, H-9). 13C-NMR (CDCl3): 163.1 (dd, J = 250.2Hz and 15.6Hz, C-3', 5'); 161.3 (C-5); 152.9 (C-3a); 145.8 (C-9a); 142.2 (t, J = 8.4Hz, C -1'); 140.8 (C-1"); 129.6 (qa, J=32.5Hz, C-4"); 128.8 (C-2", 6"); 125.3 (qa, J=3.8Hz, C-3", 5"); 124.4 (qa, J=272.5Hz, C F3); 111.3 (dd, J = 19.3Hz and 4.9Hz, C-2', 6'); 102.8 (t, J = 25.8Hz, C-4'); 101.6 (C-5a); 61.4(C-10); 50.6(C-2); 49.3(C-6); 48.4(C-8); 46.9(C-1); 45.0(C-11); 26.8(C-9).

[0239] Example 2:

[0240] 4-(4-bromobenzyl)-7-(3,5-difluorobenzyl)-2,4,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(1H)-one (TBP-302) (Compound I / 39)

[0241]

[0242] Yield: 271 mg (56%). Melting point: 173.5 °C. 1 H-NMR (CDCl3): 7.36 (d, J=8.3Hz, 2H, H-3", 5"); 7.32 (d, J=8.3Hz, 2H, H-2", 6"); 6.84 (br dt,J~7Hz and~2Hz,2H,H-2',6');6.66(tt,J=9.0Hz and 2.3Hz,1H,H-4');4.95(s,2H,H-11);3.87(s,4H,H-1 and H-2);3.60(s,2H,H-10);3.23(br s, 2H, H-6); 2.62 (t, 2H, J = 5.7Hz, H-8); 2.44 (t, 2H, J = 5.7Hz, H-9). 13C-NMR (CDCl3): 163.1 (dd, J = 250.2Hz and 15.6Hz, C-3', 5'); 161.3 (C-5); 152.9 (C-3a); 145 .8(C-9a); 142.2(t,J=8.4Hz,C-1'); 135.9(C-1″); 131.4(C-3″,5″); 130.6(C-2″,6″); 12 1.4 (C-4"); 111.3 (dd, J = 19.3Hz and 4.9Hz, C-2', 6'); 102.8 (t, J = 25.7Hz, C-4'); 101.6 (C-5 a); 61.4 (C-10); 50.6 (C-2); 49.4 (C-6); 48.4 (C-8); 46.9 (C-1); 44.8 (C-11); 26.8 (C-9).

[0243] Example 3:

[0244] 4-(4-Fluorobenzyl)-7-(3,5-Difluorobenzyl)-2,4,6,7,8,9-Hexahydroimidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(1H)-one (ABB-011) (Compound I / 7)

[0245]

[0246] Yield: 290 mg (68%). Melting point: 190.5 °C. 1 H-NMR (CDCl3): 7.44 (dd, J=8.7Hz and 5.7Hz, 2H, H-2", 6"); 6.92 (t, J=8.7Hz, 2H, H-3", 5"); 6.84 (br dt,J~7Hz and~2Hz,2H,H-2',6');6.66(tt,J=8.9Hz and 2.3Hz,1H,H-4');4.97(s,2H,H-11);3.92-3.83(m,4H,H-1 and H-2);3.59(s,2H,H-10);3.23(br s, 2H, H-6); 2.62 (t, 2H, J = 5.7Hz, H-8); 2.44 (t, 2H, J = 5.7Hz, H-9). 13C-NMR (CDCI3): 162.2 (d, J = 162.2 Hz, C-4"); 163.1 (dd, J = 250.0 Hz and 15.2 Hz, C-3',5'); 161.4 (C-5); 152.9 (C-3a); 145.5 (C-9a); 142.2 (t, J = 8.4 Hz, C-l'); 132.7 (C-l"); 130.7 (d, J = 7.9 Hz, C-2",6"); 115.0 (d, J = 21.3 Hz, C-3",5"); 111.4 (dd, J = 19.3 Hz and 4.9 Hz, C-2',6'); 102.7 (t, J = 25.7 Hz, C-4'); 101.6 (C-5a); 61.4 (C-10); 50.6 (C-2); 49.4 (C-6); 48.4 (C-8); 46.9 (C-l); 44.7 (C-l l); 26.7 (C-9).

[0247] Example 4:

[0248] 4-(4-chlorobenzyl)-7-(3,5-difluorobenzyl)-2,4,6,7,8,9-hexahydroimidazo[l,2- a]pyrido[3,4-e]pyrimidin-5(lH)-one (TBP-333) (Compound I / 124)

[0249]

[0250] Yield: 332 mg (75%). Melting point: 162.4 °C. 1 H-NMR (CDCI3): 7.38 (d, J = 8.3 Hz, 2H, H-2",6"); 7.20 (d, J = 8.3 Hz, 2H, H-3",5"); 6.84 (br dt, J ~ 7 Hz and ~ 2 Hz, 2H, H-2',6'); 6.66 (tt, J = 9.0 Hz and 2.3 Hz, 1H, H-4'); 4.97 (s, 2H, H-l l); 3.90-3.83 (m, 4H, H-l and H-2); 3.59 (s, 2H, H-10); 3.22 (br s, 2H, H-6); 2.62 (t, 2H, J = 5.7 Hz, H-8); 2.44 (t, 2H, J = 5.7 Hz, H-9). 13C-NMR (CDCI3): 163.1 (dd, J = 248.2 Hz and 14.6 Hz, C-3',5'); 161.3 (C-5); 152.9 (C-3a); 145.6 (C-9a); 142.2 (t, J = 8.4 Hz, C-l'); 135.4 (C-l"); 133.2 (C-4"); 130.6 (C-2",6"); 128.4 (C-3",5"); 111.7 (dd, J = 19.3 Hz and 4.9 Hz, C-2',6'); 102.7 (t, J = 25.7 Hz, C-4'); 101.6 (C-5a); 61.4 (C-10); 50.6 (C-2); 49.4 (C-6); 48.4 (C-8); 46.9 (C-l); 44.7 (C-l l); 26.7 (C-9).

[0251] Example 5:

[0252] 4-(3-chlorobenzyl)-7-(3,5-difluorobenzyl)-2,4,6,7,8,9-hexahydroimidazo[l,2- a]pyrido[3,4-e]pyrimidin-5(lH)-one (TBP-344) (Compound I / 125)

[0253]

[0254] Yield: 302 mg (68%). Melting point: 204.8 °C. 1 H-NMR (CDCI3): 7.40 (br s, 1 H, H-2"); 7.30 (m, 1 H, H-5"); 7.18-7.15 (m, 2 H, H-4",6"); 6.84 (br dt, J ~ 7 Hz and ~ 2 Hz, 2 H, H-2',6'); 6.66 (tt, J = 8.9 Hz and 2.2 Hz, 1 H, H-4'); 4.98 (s, 2 H, H-l l); 3.87 (br ~ s, 4 H, H-l and H-2); 3.59 (s, 2 H, H-10); 3.24 (br s, 2 H, H-6); 2.63 (t, 2 H, J = 5.6 Hz, H-8); 2.45 (t, 2 H, J = 5.6 Hz, H-9). 13C-NMR (CDCI3): 163.1 (dd, J = 248.6 Hz and 14.0 Hz, C-3',5'); 161.3 (C-5); 152.9 (C-3a); 145.7 (C-9a); 142.3 (t, J = 8.4 Hz, C-1'); 138.8 (C-1"); 134.1 (C-3"); 128.4 (two superimposed lines, C-2" and C-4"); 127.6 (C-6"); 126.8 (C-5"); 111.3 (dd, J = 19.6 Hz and 5.1 Hz, C-2',6'); 102.7 (t, J = 25.3 Hz, C-4'); 101.6 (C-5a); 61.3 (C-10); 50.6 (C-2); 49.4 (C-6); 48.4 (C-8); 46.9 (C-1); 44.9 (C-11); 26.8 (C-9).

[0255] Example 6:

[0256] rac-4-(4-chlorobenzyl)-7-(3,5-difluorobenzyl)-2,4,6,7,8,9-hexahydroimidazo[l,2- a]pyrido[3,4-e]pyrimidin-5(lH)-one (TBP-342) (compound I / 128 (racemate))

[0257]

[0258] Yield: 38 mg (8%). Melting point: 86 °C. 1 H-NMR (CDCI3): 7.28 (d, J = 8.3 Hz, 2H, H-2",6"); 7.23 (d, J = 8.3 Hz, 2H, H-3",5"); 6.84 (br dt, J ~ 7 Hz and ~ 2 Hz, 2H, H-2',6'); 6.67 (tt, J = 9.0 Hz and 2.3 Hz, 1H, H-4'); 6.08 (dd, J = 5.1 Hz and 1.6 Hz, 1H, H-11); 4.31 (dd, J = 12.8 Hz and 5.1 Hz, 1H, H-12 A ); 4.06 (dd, J = 12.8 Hz and 1.6 Hz, 1H, H-12 B ); 3.93-3.85 (m, 3H, H-1 and H-2 A ); 3.78 (m, 1H, H-2 B ); 3.60 (s, 2H, H-10); 3.24 and 3.21 (A and B are part of an AB spin system, J AB = 14.9 Hz, 2x1H, H-6 A and H-6 B); 2.66 (m, 2H, H-8); 2.50 (m, 2H, H-9). 13 C-NMR (CDCI3): 163.1 (dd, J = 248.2 Hz and 13.9 Hz, C-3',5'); 161.6 (C-5); 153.5 (C-3a); 146.0 (C-9a); 142.2 (t, J = 8.4 Hz, C-l'); 135.2 (C-l"); 133.1 (C-4"); 139.0 (C-2",6"); 128.4 (C-3",5"); 111.4 (dd, J = 19.3 Hz and 4.9 Hz, C-2',6'); 102.8 (t, J = 25.7 Hz, C-4'); 102.4 (C-5a); 62.9 (C-12); 61.3 (C-10); 57.6 (C-11); 49.5 (C-2); 49.4 (C-6); 48.4 (C-8); 46.5 (C-l); 26.8 (C-9).

[0259] Example 7:

[0260] R-4-(4-Fluorobenzyl)-7-(3,5-difluorobenzyl)-2,4,6,7,8,9-hexahydroimidazo[l,2- a]pyrido[3,4-e]pyrimidin-5(lH)-one (TBP-339) (Compound I / 129 (R-enantiomer))

[0261]

[0262] Yield: 51 mg (11%). Melting point: 76 °C. 1 H-NMR (CDCI3): 7.36 (dd, J = 8.8 Hz and 5.8 Hz, 2H, H-2",6"); 6.96 (t, J = 8.3 Hz, 2H, H-3",5"); 6.85 (br dt, J ~ 7 Hz and ~ 2 Hz, 2H, H-2',6'); 6.67 (tt, J = 9.0 Hz and 2.3 Hz, 1H, H-4'); 6.09 (dd, J = 5.3 Hz and 1.6 Hz, 1H, H-11) A ); 4.32 (dd, J = 12.8 Hz and 5.3 Hz, 1H, H-12 B ); 3.94-3.85 (m, 3H, H-l and H-2 A ); 3.81 (m, 1H, H-2 B ); 3.61 (s, 2H, H-10); 3.25 and 3.21 (A and B are part of an AB spin system, J AB = 14.9 Hz, 2x1H, H-6A and H-6 B ) ; 2.66 (m, 2H, H-8) ; 2.50 (m, 2H, H-9). 13 C-NMR (CDCI3): 163.1 (dd, J = 248.2 Hz and 13.9 Hz, C-3',5'); 162.0 (d, J = 245.8 Hz, C-4"); 161.6 (C-5); 153.5 (C-3a); 145.8 (C-9a); 142.1 (t, J = 8.4 Hz, C-1'); 132.5 (br s, C-1"); 129.4 (d, J = 7.8 Hz, C-2",6"); 115.1 (d, J = 21.4 Hz, C-3",5"); 111.3 (dd, J = 19.6 Hz and 5.2 Hz, C-2',6'); 102.8 (t, J = 25.6 Hz, C-4'); 102.5 (C-5a); 63.1 (C-12); 61.3 (C-10); 57.6 (C-11); 49.5 (two superimposed lines, C-2 and C-6); 48.3 (C-8); 46.5 (C-1); 26.8 (C-9).

[0263] Example 8:

[0264] 4-(3-(Aminomethyl)benzyl)-7-(3,5-difluorobenzyl)-2,4,6,7,8,9-hexahydroimidazo[l,2- a]pyrido[3,4-e]pyrimidin-5(lH)-one (TBP-324) (Compound I / 61)

[0265]

[0266] Yield: 376 mg (86%). Melting point: 129.5 °C. 1 H-NMR (CDCI3): 7.33 (br s, 1H, H-2"); 7.30 (br d, J = 7.6 Hz, 1H, H-6"); 7.22 (t, J = 7.6 Hz 1H, H-5"); 7.15 (br d, J = 7.6 Hz, 1H, H-4"); 6.84 (br dt, J ~ 7 Hz and ~ 2 Hz, 2H, H-2',6'); 6.66 (tt, J = 8.9 Hz and 2.3 Hz, 1H, H-4'); 5.00 (s, 2H, H-11); 3.92-3.83 (m, 4H, H-1 and H-2); 3.79 (s, 2H, H-12); 3.59 (s, 2H, H-10); 3.24 (br s, 2H, H-6); 2.63 (t, 2H, J = 5.7 Hz, H-8); 2.45 (t, 2H, J = 5.7 Hz, H-9); 1.77 (br s, 2H, NH 2). 13 C-NMR (CDCI3): 163.1 (dd, J = 248.4 Hz and 14.1 Hz, C-3',5'); 161.5 (C-5); 153.2 (C-3a); 145.6 (C-9a); 143.2 (C-3"); 142.3 (t, J = 8.4 Hz, C-1'); 137.2 (C-1"); 128.6 (C-5"); 127.3 (C-4"); 127.1 (C-2"); 126.2 (C-4"); 111.4 (dd, J = 20.0 Hz and 5.0 Hz, C-2',6'); 102.7 (t, J = 25.7 Hz, C-4'); 101.7 (C-5a); 61.4 (C-10); 50.7 (C-2); 49.4 (C-6); 48.4 (C-8); 46.9 (C-1); 46.4 (C-12); 45.4 (C-11); 26.7 (C-9).

[0267] Example 9:

[0268] 4-(3-Aminobenzyl)-7-(3,5-difluorobenzyl)-2,4,6,7,8,9-hexahydroimidazo[l,2- a]pyrido[3,4-e]pyrimidin-5(lH)-one (TBP-346) (Compound I / 126)

[0269]

[0270] Yield: 347 mg (82%). Melting point: 114.5 °C. 1 H-NMR (CDCI3): 7.03 (t, J = 7.9 Hz, 1 H, H-5"); 6.85 (br dt, J ~ 7 Hz and ~ 2 Hz, 2 H, H-2',6'); 6.82 (br d, J = 7.9 Hz, 1 H, H-6"); 6.75 (br s, 1 H, H-2"); 6.66 (tt, J = 8.9 Hz and 2.3 Hz, 1 H, H-4'); 6.52 (dd, J = 7.6 Hz and 2.2 Hz, 1 H, H-4"); 4.93 (s, 2 H, H-11); 3.92-3.79 (m, 4 H, H-1 and H-2); 3.59 (s, 2 H, H-10); 3.24 (br s, 2 H, H-6); 2.62 (t, 2 H, J = 5.7 Hz, H-8); 2.44 (t, 2 H, J = 5.7 Hz, H-9). 13C-NMR (CDCI3): 163.1 (dd, J = 248.4 Hz and 14.1 Hz, C-3',5'); 161.5 (C-5); 153.0 (C-3a); 146.4 (C-3"); 145.3 (C-9a); 142.3 (t, J = 8.4 Hz, C-l'); 138.0 (C-l"); 129.2 (C-5"); 119.0 (C-6"); 115.2 (C-2"); 114.3 (C-4"); 111.4 (dd, J = 20.1 Hz and 5.4 Hz, C-2',6'); 102.7 (t, J = 25.7 Hz, C-4'); 101.7 (C-5a); 61.4 (C-10); 50.6 (C-2); 49.4 (C-6); 48.4 (C-8); 46.8 (C-l); 45.3 (C-l l); 26.7 (C-9).

[0271] Example 10:

[0272] 4-(4-azidobenzyl)-7-(3,5-difluorobenzyl)-2,4,6,7,8,9-hexahydroimidazo[l,2- a]pyrido[3,4-e]pyrimidin-5(lH)-one (CZT-136)

[0273]

[0274] Yield: 270 mg (60%). Melting point: 155.5 °C. 1 H-NMR (CDCI3): 7.44 (d, J = 8.3 Hz, 2H, H-2",6"); 6.90 (d, J = 8.3 Hz, 2H, H-3",5"); 6.84 (br ~ dt, J ~ 7 Hz and ~ 2 Hz, 2H, H-2',6'); 6.66 (tt, J = 9.0 Hz and 2.3 Hz, 1H, H-4'); 4.97 (s, 2H, H-l l); 3.91-3.84 (m, 4H, H-l and H-2); 3.59 (s, 2H, H-10); 3.23 (br s, 2H, H-6); 2.63 (t, 2H, J = 5.7 Hz, H-8); 2.44 (t, 2H, J = 5.7 Hz, H-9). 13C-NMR (CDCI3): 163.1 (dd, J = 250.2 Hz and 15.6 Hz, C-3',5'); 161.4 (C-5); 152.9 (C-3a); 145.5 (C-9a); 142.2 (t, J = 8.4 Hz, C-l'); 139.1 (C-4"); 133.8 (C-l"); 130.4 (C-2",6"); 118.9 (C-3",5"); 111.3 (dd, J = 19.3 Hz and 4.9 Hz, C-2',6'); 102.7 (t, J = 25.7 Hz, C-4'); 101.7 (C-5a); 61.4 (C-10); 50.6 (C-2); 49.4 (C-6); 48.4 (C-8); 46.9 (C-l); 44.8 (C-l l); 26.8 (C-9).

[0275] Example 11:

[0276] 7-(3-azidobenzyl)-4-(4-(trifluoromethyl)benzyl)-2,4,6,7,8,9-hexahydroimidazo[l,2- a]pyrido[3,4-e]pyrimidin-5(lH)-one (TBP-272)

[0277]

[0278] Yield: 313 mg (65%). Melting point: 128.8 °C. 1 H-NMR (CDCI3): 7.52 and 7.50 (A and B are part of an AA'BB' spin system, J AB = 8.9 Hz, 2 x 2H, H-3",5" and H-2",6", resp.); 7.26 (t, J = 7.7 Hz, 1H, H-5'); 7.06 (d, J = 7.7 Hz, 1H, H-6'); 6.99 (t, J = 1.5 Hz, 1H, H-2'); 6.90 (dd, J = 7.7 Hz and 1.5 Hz, 1H, H-4'); 5.06 (s, 2H, H-l l); 3.87 (s, 4H, H-l and H-2); 3.61 (s, 2H, H-10); 3.24 (br s, 2H, H-6); 2.64 (t, 2H, J = 5.7 Hz, H-8); 2.45 (t, 2H, J = 5.7 Hz, H-9). 13C-NMR(CDCl3): 161.7(C-5); 153.0(C-3a); 145.9(C-9a); 140.8(C-1″); 140.2(C-3′); 140.0(C-1′); 129.8(C-5′); 129.6(qa,J=32.5Hz,C-4"); 128.9(C-2",6"); 125.5(C-6'); 125.3(qa,J=3.5Hz, C-3",5"); 124.4(qa,J=272.5Hz, C F3); 119,4 (C-2'); 118.1 (C4'); 101.7 (C-5a); 61.9 (C-10); 50.6 (C-2); 49.4 (C-6); 48.4 (C-8); 46.9 (C-1); 45.0 (C-11); 26.8 (C-9).

[0279] Example 12:

[0280] 4-(3-Azidebenzyl)-7-(3,5-Difluorobenzyl)-2,4,6,7,8,9-Hexahydroimidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(1H)-one (TBP-347) (Compound I / 127)

[0281]

[0282] Yield: 355 mg (79%). Melting point: 158 °C. 1 H-NMR(CDCl3): 7.23(t,J=7.9Hz,1H,H-5″); 7.18(br~d,J~8Hz 1H,H-6″); 7.10(br s,1H,H-2″); 6.87(br~d,J~8Hz 1H,H-4″); 6.84(br dt,J~7Hz and~2Hz,2H,H-2',6');6.66(tt,J=8.9Hz and 2.3Hz,1H,H-4');5.00(s,2H,H-11);3.90-3.81(m,4H,H-1 and H-2);3.60(s,2H,H-10);3.24(br s, 2H, H-6); 2.63 (t, 2H, J = 5.7Hz, H-8); 2.45 (t, 2H, J = 5.7Hz, H-9). 13C-NMR (CDCI3): 163.1 (dd, J = 248.4 Hz and 13.7 Hz, C-3',5'); 161.3 (C-5); 153.0 (C-3a); 145.6 (C-9a); 142.3 (t, J = 9.0 Hz, C-l'); 140.0 (C-3"); 138.9 (C-l"); 129.7 (C-5"); 125.1 (C-6"); 121.2 (C-2"); 118.0 (C-4"); 111.4 (dd, J = 20.1 Hz and 5.4 Hz, C-2',6'); 102.7 (t, J = 25.7 Hz, C-4'); 101.7 (C-5a); 61.3 (C-10); 50.6 (C-2); 49.4 (C-6); 48.4 (C-8); 46.9 (C-l); 45.0 (C-l l); 26.8 (C-9).

[0283] Example 13:

[0284] 7-benzyl-4-(4-(4-diferrocenyl-lH-l,2,3-triazol-l-yl)benzyl)-2,4,6,7,8,9- hexahydroimidazo[l,2-a]pyrido[3,4-e]pyrimidin-5(lH)-one (ABB-030)

[0285]

[0286] Yield: 443 mg (71%). Melting point: 165.4 °C. 1 H-NMR (CDCI3): 7.81 (s, 1 H, H-12); 7.65 (d, J = 7.9 Hz, 2H, H-3",5"); 7.59 (d, J = 7.9 Hz, 2H, H-2",6"); 7.31-7.26 (m. 4H, H-2',3',5',6'); 7.22 (m, 1 H, H-4'); 5.11 (s, 2H, H-11); 4.74 (t, J = 1.8 Hz, 2H, H-15,18); 4.29 (t, J = 1.8 Hz, 2H, H-16,17); 4.07 (s, 2H, H-10); 3.87 (br s, 4H, H-l and H-2); 3.63 (s, 2H, H-6); 2.63 (t, 2H, J = 5.7 Hz, H-8); 2.44 (t, 2H, J = 5.7 Hz, H-9). 5 -C5 H 5); 3.87 (br s, 4H, H-l and H-2); 3.63 (s, 2H, H-10); 3.27 (br s, 2H, H-6); 2.63 (t, 2H, J = 5.7 Hz, H-8); 2.44 (t, 2H, J = 5.7 Hz, H-9). 13C-NMR (CDCI3): 161.3 (C-5); 153.0 (C-3a); 147.5 (C-4'); 145.9 (C-9a); 130.1 (C-2", 6"); 128.4 (C-2', 6'); 127.4 (C-4'); 126.1 (C-3', 5'); 120.1 (C-3", 5"); 116.6 (C-12); 102.2 (C-5a); 75.0 (C-14); 69.6 (C-16, 17); 66.8 (C-15, 18); 62.3 (C-10); 50.6 (C-2); 49.5 (C-6); 48.3 (C-8); 46.8 (C-1); 44.9 (C-11); 26.8 (C-9). 5 - C 5H5); 68.8 (C-16, 17); 66.8 (C-15, 18); 62.3 (C-10); 50.6 (C-2); 49.5 (C-6); 48.3 (C-8); 46.8 (C-1); 44.9 (C-11); 26.8 (C-9).

[0287] Example 14:

[0288] Synthesis of 4-arylmethyl-substituted 7-(3,5-dicyano-benzyl)-2,4,6,7,8,9- hexahydro-imidazo[l,2-a]pyrido[3,4-e]pyrimidin-5(lH)-ones (compounds I / 136 and I / 137)

[0289] Sodium azide (0.260 g, 4 mmol), sodium ascorbate (0.119 g, 0.60 mmol), N,N- dimethylethylenediamine (0.080 g, 0.9 mmol), NaOH (0.012 g, 0.30 mmol), the corresponding 7-(3,5-dibromobenzyl)emilidones I / 134 or I / 135 (1 mmol) and CuI (0.057 g, 0.30 mmol) were dissolved in 20 mL of a degassed (argon for 30 min) EtOH / H2O solvent mixture (7:3) and heated at reflux temperature for 3 h. The reaction mixture was concentrated under reduced pressure to about one third of its original volume and extracted with CH2Cl2(3 x 4 mL). The combined organic phases were washed with water (3 x 50 mL), dried over Na2SO4, filtered through paper and concentrated. Final purification was achieved by column chromatography using CH2Cl2 / MeOH (99:1) as eluent.

[0290] Example 15:

[0291] Synthesis of 4-arylmethyl-substituted 7-(3,5-dicyano-benzyl)-2,4,6,7,8,9- hexahydro-imidazo[l,2-a]pyrido[3,4-e]pyrimidin-5(lH)-ones (compounds I / 136 and I / 137)

[0292] To a stirred solution of the corresponding 7-(3,5-dibromobenzyl)emiram I / 134 or I / 135 (1 mmol) in DMF (2 mL), zinc cyanide (0.181 g, 1.2 mmol), 1,1’-bis(diphenylphosphino) ferrocene ligand (DPPF, 0.065 g, 0.117 mmol) were added sequentially, followed by Pd2dba3(45.8 mg, 0.05 mmol). The flask was flushed with nitrogen and stirred at 110-120 °C in an oil bath for 20 h. After cooling to room temperature, the reaction mixture was evaporated in vacuo. The crude product was subjected to flash column chromatography on silica gel (eluent, ethyl acetate:hexane (1:4)) to give the pure dicyanobenzyl substituted emiram.

[0293] Example 16:

[0294] Synthesis of 4-arylmethyl-substituted 7-(3-cyanoseleno-benzyl)-2,4,6,7,8,9- hexahydro-imidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(1H)-ones (compounds I / 145 and I / 146)

[0295] A mixture of the corresponding 7-(3-iodobenzyl)emiram (I / 140 or I / 141) (1.0 mmol), CuSCN (0.12 g, 1.0 mmol), KSCN (0.095 g, 1.0 mmol) and DMF (3 mL) was stirred and heated in an oil bath at 140 °C for 12 h under an argon atmosphere. After cooling, the mixture was diluted with toluene (5 mL) and water (5 mL) and then filtered through a Celite bed. The aqueous phase was extracted with toluene (2 x 5 mL), the combined organic phases were washed with water, dried over Na2SO4and concentrated. The residue was chromatographed on silica gel (n-hexane as eluent) to give the thiocyanate product which was further purified by recrystallization from hexane.

[0296] Example 17:

[0297] Synthesis of 4-arylmethyl-substituted 7-(3-cyanoseleno-benzyl)-2,4,6,7,8,9- hexahydro-imidazo[1,2-a]pyrido[3,4-e]pyrimidin-5(1H)-ones (compounds I / 145 and I / 146)

[0298] To a solution of the corresponding 7-(3-iodobenzyl)emilubinide (I / 79 and I / 144) (5 mmol) in dioxane (4 mL) was added 6N HC1 (10 mL). The resulting suspension was cooled to 0 °C, then NaNO2(0.415 g, 6 mmol) in water (2 mL) was added slowly. After stirring for 30 min, saturated NaOAc solution (about 30 mL) was added portionwise to adjust the pH of the reaction mixture to 5-6. The resulting suspension was poured into a solution of KSeCN (0.793 g, 5.5 mmol) in water (25 mL) at 0 °C. After stirring for 30 min, the reaction mixture was warmed to room temperature and extracted with ether. The organic layer was washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified by column chromatography on silica gel using ethyl acetate-hexane (1 :5) as eluent.

[0299] Example 18:

[0300] In this example, we list the compounds of formula (I) according to the present application and exemplify the results of in vitro anti-proliferation assays for representative compounds investigated during our systematic experimental work and in the context of the relevant literature known in the art.

[0301] Cell cultures for long-term treatment experiments

[0302] PANC-1 (human ductal-derived pancreatic carcinoma), COLO 205 (human colorectal adenocarcinoma), A2058 (human metastatic melanoma) obtained from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK) and EBC-1 (human lung squamous cell carcinoma) purchased from the Japanese Research Resources Bank (Tokyo, Japan) were used to determine the tumor growth inhibition effect of emilubinide derivatives. PANC-1 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Lonza, Basel, Switzerland); for culturing the COLO-205 cell line, DMEM medium formulated with 4500 mg / L d-glucose was used; EBC-1 cells were maintained in DMEM medium containing 1% non-essential amino acids (NEAA, Lonza, Basel, Switzerland), 2 mM L-glutamine, 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA), 100 U / mL penicillin and 100 pg / mL streptomycin (P / S, Sigma-Aldrich, St. Louis, MO, USA). The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The cells were seeded in 96-well plates at a density of 5000 cells per well in 100 pL of culture medium and allowed to adhere for 24 h. The medium was then replaced with fresh medium containing the test compound at the desired concentration. The cells were incubated for 72 h at 37 °C in a humidified atmosphere containing 5% CO2. The medium was then replaced with fresh medium without the test compound. The cells were incubated for an additional 72 h at 37 °C in a humidified atmosphere containing 5% CO2. The cells were then fixed with cold methanol and stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at room temperature. The plates were washed with water to remove the excess dye and the absorbance of the dye was measured at 590 nm using a microplate reader (Synergy HTX, BioTek, Winooski, VT, USA). The results are shown in Table 1. DMEM medium (Invitrogen Corporation, New York, NY, USA), 1 mM sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA) while A2058 cell line was grown in RPMI 1640 (Lonza, Basel, Switzerland). In all cases of cell lines, the aforementioned basal medium was supplemented with 10% fetal bovine serum (FBS, L-glutamine (2 mmol / L) (Lonza, Basel, Switzerland) and 100 pg / mL penicillin / streptomycin (Invitrogen Corporation, New York, NY, USA). All cell lines were cultured in plastic Petri dishes (Sigma-Aldrich, St. Louis, MO, USA or Eppendorf AG, Hamburg, Germany) under standard conditions (37°C, 5% C02humidified atmosphere).

[0303] Survival assay (long-term treatment experiment)

[0304] Impedance-based assay

[0305] Cytotoxicity experiments on PANC-1 cells were performed using the impedance-based xCELLigence SP system (ACEA Biosciences, San Diego, CA, USA). For the basics of impedance measurements, we gave more detailed explanations in our previous paper

[22] . Monitoring impedance changes, which are directly proportional to the number of adherent cells on the electrode surface, provides a sensitive method for cytotoxicity studies

[23] . Changes in impedance were expressed in the form of cell index (CI), which was calculated by the software integrated into the xCELLigence system (RTCA2.0, ACEA Biosciences, San Diego, CA, USA). To determine IC50(concentration at which cell viability is reduced by 50%), the tested imipridone was dissolved in DMSO and further diluted in supplemented DMEM medium to prepare a concentration range from 2.5 x 10 50 -4 -7 M. The steps of our impedance experiment were the same as indicated in

[24] . Briefly, after obtaining a constant CI value during the background measurement, PANC-1 cells (1.5 x 10 4 ​​​cells / well) into so-called E-plates and monitored for adhesion / spreading for 24 h to stabilize the platform of cell culture. In the last step, cells in this equilibrium state were treated with test compounds (final concentrations: 2.5 x 10 -5 to 5 x 10 -8 M) and CI changes were monitored at 10 kHz for at least 72 h. In case of control wells, DMSO was added in sufficient volume ratio. Three parallel samples were measured per measurement. CI values obtained for each concentration 72 h after treatment were normalized to the value of the DMSO control. IC 50 values of these normalized CI values were calculated by the non-linear regression function of OriginPro 8 (OriginLab Corporation, Northampton, MA, USA) fitting sigmoidal dose-response curves.

[0306] Colorimetric assay

[0307] The anti-proliferative / cytotoxic effects of emiliumide on COLO-205 and A2058 cell lines were measured by alamarBlue-assay. This colorimetric assay proved to be a more suitable method for the analysis of these cell lines than the xCELLigence system, since COLO-205 cells showed weak / negligible adhesion and the A2058 cell line failed to establish a stable platform during impedance analysis. The procedure of cell seeding and alamarBlue-assay was similar to that described in our previous paper

[24] . The main steps were as follows: (i) cell seeding at a concentration of 10 4 cells / well in 96-well plates (Sarstedt AG, Numbrecht, Germany), (ii) treatment with test compounds at final concentrations of 2.5 x 10 -5 to 5 x 10 -8M test substance was treated for 72 hours, (iii) alamarBlue reagent (0.15 mg / mL, Sigma-Aldrich, St. Louis, MO, USA) was added in PBS (Phosphate Buffered Saline, pH = 7.2), (iv) after 6-8 hours incubation with alamarBlue reagent the fluorescence intensity of the samples was read. The LS-50B luminescence spectrometer (Perkin Elmer Ltd., Buckinghamshire United Kingdom) was used for the fluorescence measurements, set as follows: excitation wavelength = 560 nm, emission wavelength = 590 nm. Each measurement was performed in triplicate. Wells containing sufficient volume proportion of DMSO served as controls. The fluorescence intensity of each sample was expressed as a ratio to the fluorescence of the DMSO control. The non-linear regression function of OriginPro 8 (OriginLab Corporation, Northampton, MA, USA) was used to fit sigmoidal dose-response curves to normalize the fluorescence intensity to calculate IC 50 values.

[0308] Statistical evaluation of the data evaluation of the results was performed using the RTCA2.0 (ACEA Biosciences, San Diego, CA, USA), MS Excel, OriginPro 8 (OriginLab Corporation, Northampton, MA, USA) software. The data obtained for each experiment represent the mathematical mean. The standard deviation of the IC 50 parameters was also obtained with the sigmoidal curve fitting.

[0309] Short-term cytotoxicity studies using the MTT assay

[0310] For short-term cytotoxicity studies, A-431 (human squamous carcinoma) and U-87 (human primary glioblastoma) cells were cultured in RPMI-1640 medium supplemented with 10% FCS (Fetal Calf Serum, Sigma Ltd.), 2 mM L-glutamine and 160 mg / mL gentamicin. Cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2. Cells were grown to confluence and distributed in 96-well plates at an initial cell number of 5.0 x 10 3 After 24 hours incubation at 37°C, cells were treated with compounds in a final volume of 200 μΐ, containing 1.0% volume DMSO. The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used at 10 -4 -10 2The cells were incubated with the compound in the μM concentration range for 1 hour. Control cells were treated with serum-free medium (RPMI-1640) or DMSO (c = 1.0 v / v%) at 37°C for 1 hour. After incubation, the cells were washed twice with serum-free medium (RPMI-1640). To determine the in vitro cell inhibition, the cells were further cultured in medium containing 10% serum for 72 hours. MTT solution (45 mL, 2 mg / mL, final concentration: 0.37 mg / mL) was added to each well. The respiratory chain [26,27] and other electron transport systems

[28] reduce MTT, thereby forming a water-insoluble purple methyl ester in the cells. (formazane) crystals

[29] . The number of these crystals can be determined spectrophotometrically and used as an estimate of the number of mitochondria and the number of viable cells in the well

[30] . After 4 hours of incubation, the cells were centrifuged for 5 minutes (900 g) and the supernatant was removed. The obtained formazane crystals were then collected. Crystals were dissolved in DMSO (100 mL), and the optical density (OD) of the samples was measured at λ = 540 and 620 nm using an ELISA reader (iEMS Reader, Labsystems, Finland). The OD620 value was subtracted from the OD540 value. The percentage of cytostasis was calculated using the following equation: Cytostasis effect (%) = [1 - (OD treatment / OD control)] × 100. The OD treatment value and OD control value correspond to the optical density of treated cells and control cells, respectively. In each case, two independent experiments were performed, comprising four parallel measurements. 50% inhibitory concentration (IC50) was used. 50 The half-maximal inhibitory concentration (IC50) was determined using a dose-response curve. The curve was defined using Microcal TMOrigin 1 (version 7.5) software: cell arrest was plotted as a function of concentration, fitted with an S-shaped curve, and the half-maximal inhibitory concentration (IC50) was determined based on this curve. 50 IC value. 50 This represents the concentration required for a compound to achieve 50% inhibition in vitro.

[0311] High-throughput screening of EBC-1 and H2228 lung cancer cell lines was performed using the CellTiter-Glo luminescent cell viability assay.

[0312] EBC-1 (obtained from JRCB, https: / / cellbank.nibiohn.go.jp / english / ) and H2228 (obtained from ATCC, https: / / www.lgcstandards-atcc.org / ) cell lines were maintained at 37°C in a 5% CO2 humidified incubator according to the instructions provided by JRCB and ATCC. CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI, USA) measures the effect of compounds on cell viability. Cells are plated at 1000 cells / well on white flat-bottom 96-well plates (BRAND plates, Cat. No: 781965). After 24 hours, cells are treated with compounds at 100 nM concentration for 72 hours. After treatment, culture medium is removed and CellTiter-Glo® Reagent is added Reagents. Untreated cells are used as control. Luminescent signal is recorded using a microplate reader (BioTek Synergy 2 Multi-Mode Reader, BioTek, Winooski, VT, USA). Cell viability data (%) compared to untreated control cells are evaluated with Microsoft Excel.

[0313] Table 1: Results of in vitro anti-proliferation assay of compounds of formula (I) according to the application and some representative compounds

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330] The results of high-throughput screening (HTS) were presented in lung cancer cell lines EBC-1 and H2228. Subsequent experiments determined the IC50 of compounds that proved effective in HTS. 50 Values ​​(Table 3).

[0331] Example 19:

[0332] Further testing with representative imiridone and reference ONC212 was conducted on the following human malignant tumor cell lines: PC3 and LNCap (prostate cancer); BxPC3, MiaPaCa2 and Panc1 (pancreatic cancer); A549, HCC827, H1993 and H520 (lung cancer); MDA-MB-453 and MDA-MB-231 (breast cancer) (see Table 2 and...). https: / / www.lgcstandards-atcc.org / ).

[0333] Cell lines based on ATCC ( Figures 12-51 The instructions provided state that the incubator should be kept at 37°C in a 5% CO2 humidified incubator. A luminescent cell viability assay (Promega, Madison, WI, USA) was used to measure the effect of compounds on cell viability. Cells were seeded at 1000 cells / well in white flat-bottomed 96-well plates (BRANDplates, catalog number: 781965). After 24 hours, cells were treated with sequentially diluted 3-fold concentrations of the compound (300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.2 nM) for 72 hours. After treatment, the culture medium was removed, and the compound was added... Reagents. The luminescence signal was recorded using a microplate reader (BioTek Synergy 2 Multi-Mode Reader, BioTek, Winooski, VT, USA).

[0334] Cell viability data (%, compared to untreated control cells) were assessed using Microsoft Excel. Dose-response curves were generated (using a nonlinear regression model, logarithmic (inhibitor) vs. response, with a variable slope) and IC50 was determined using GraphPad Prism 5.02 software (GraphPad Software, San Diego, CA, USA). 50 value.

[0335] In these tests, our compound proved to be an extremely effective antiproliferative agent, characterized by an IC50 concentration. 50The values ​​far exceed those of ONC212 in the low nanomolar range. In particular, TBP-301 and TBP-302 can be considered particularly effective anticancer agents. On the other hand, the potent azide derivatives CZT-136 and TBP-272 offer unique possibilities for identifying cellular targets, thus revealing a defined mechanism of action essential for drug approval.

[0336] Table 2: In vitro test results (IC50) of some representative compounds of the present invention and ONC212 (as a reference) on a group of human malignant tumor cell lines. 50 [nM]).

[0337]

[0338]

[0339] Example 20:

[0340] Other data used to demonstrate the effectiveness of the compounds of this invention.

[0341] Table 3: Other in vitro test results (IC50) of the representative compounds of the present invention and ONC212 (as a reference) against a group of human malignant tumor cell lines. 50 [nM])

[0342]

[0343]

[0344] Example 21: Cell Viability Assay Protocol

[0345] According to the manufacturer's instructions, via A luminescent cell viability assay (Promega, Madison, WI, USA) was used to measure the effect of selected compounds on cancer cell viability. Cells were plated in white flat-bottomed 96-well plates. Catalog No: 781965). The density of cells seeded was optimized according to the size and growth rate of the cells as follows: Panc-1 : 750 cells / well; DU 145, PC-3, Capan-1, MIA PaCa-2, SCC-25, FaDu and EBC-1 : 1000 cells / well; LNCaP, Detroit 562, MDA-MB-231 and MDA-MB-453: 1500 cells / well. After 48 hours of incubation, cells were treated with serially diluted 3-fold compound concentrations (range 300 nM - 1.2 nM) for 72 hours. Untreated cells (incubated in the respective cell culture medium during the 72 hours treatment period) were used as controls. After treatment, luminescent signal was recorded with a microplate reader (BioTek Synergy 2 Multi-Mode Reader, BioTek, Winooski, VT, USA). Dose-response curves were generated by GraphPad Prism 8.4.2. software using a non-linear regression model (variable slope, four parameters), (see Figures 52-61 ).

[0346] Table 4: Cell viability measurements on DU 145 cell line

[0347]

[0348]

[0349] Table 5: Cell viability measurements on LNCaP cell line

[0350]

[0351] Table 6: Cell viability measurements on PC-3 cell line

[0352]

[0353] Table 7: Cell viability measurements on Panc-1 cell line

[0354]

[0355] Table 8: Cell viability measurements on Capan-1 cell line

[0356]

[0357] Table 9: Cell viability measurements on MIA PaCa-2 cell line

[0358]

[0359] Table 10: Cell viability measurements on Detroit 562 cell line

[0360]

[0361] Table 11: Cell viability measurements on SCC-25 cell line

[0362]

[0363] Table 12: Cell viability measurements on FaDu cell line

[0364]

[0365] Table 13: Cell viability measurements on EBC-1 cell line

[0366]

[0367] Table 14: Cell viability measurements on MDA-MB-231 cell line

[0368]

[0369] Table 15: Cell viability measurements on MDA-MB-453 cell line

[0370]

[0371] Example 22:

[0372] This example demonstrates the cytotoxic effect of some compounds according to the present application on Panc-1 human cell line. IC 50 values were determined in an xCELLigence SP instrument (see Figure 62 ). The following table shows the IC 50 values obtained after 24, 48, 72 and 96 hours of treatment.

[0373] Table 16: Cytotoxicity measurements results on Panc-1 cell line

[0374]

[0375] ND: not detected

[0376] Example 23:

[0377] This example demonstrates the antitumor effect of compounds I / 1 (ONC 212), I / 7 (ABB-011), I / 124 (TBP-333) and I / 107 (CZT-136) on MDA-MB-231 tumor xenografts growing subcutaneously in SCID mice (immunsuprimized mice).

[0378] MDA-MB-231 human triple negative breast cancer xenografts were formed by subcutaneous inoculation of tumor cells into the back of immunodeficient (SCID) mice. Animals were injected intraperitoneally every 2-3 days with the material under study for 3 weeks.

[0379] The results of the experiment show that all the compounds under study reduced the tumor volume, however, this effect was significant only in the case of I / 124 (TBP-333).

[0380] Experimental design:

[0381] Compounds tested:

[0382] I / 1 (ONC 212) as reference: 0.022 mg / animal / treatment; dose: 0.88 mg / kg

[0383] 0.036 mg / animal / treatment; dose: 1.466 mg / kg I / 7 (ABB-011): 0.0213 mg / animal / treatment; dose: 0.85 mg / kg

[0384] 0.0355 mg / animal / treatment; dose: 1.416 mg / kg I / 124 (TBP-333) 0.022 mg / animal / treatment; dose: 0.89 mg / kg

[0385] 0.036 mg / animal / treatment; dose: 1.466 mg / kg I / 107 (CZT-136): 0.022 mg / animal / treatment; dose: 0.89 mg / kg

[0386] 0.036 mg / animal / treatment; dose: 1.466 mg / kg

[0387] and, physiological saline solution with 1% DMSO as control.

[0388] Forty SCID mice were included in the experiment. During the experiment, each group of animals was housed in separate cages. Each cage had an identification card with the date of birth, the date of injection of tumor cells, the number and sex of the animals. The injection of each substance was written on the identification card. Eight animals were used for each substance and were identified by ear notching.

[0389] The animals used in these studies were cared for according to the “Guiding Principles for the Care and Use of Animals” based on the Declaration of Helsinki and the studies were approved by the local ethics committee.

[0390] Animals were housed in IVC (Individually Ventilated Cages) system in sterilized cages, under a 12-hour light and 12-hour dark cycle, light phase from 7:00 a.m. to 19:00 p.m. Temperature and humidity were recorded daily throughout the experiment. Sterilized rodent specific quality controlled diet (VRF1, Special Diets Services Ltd, Witham, UK) and acidified (pH=3) sterilized distilled water were available ad libitum throughout the study.

[0391] The diet of each batch was accompanied by a detailed certificate of analysis of the nutritional composition. The health status of the mice was assessed by the animal farm staff.

[0392] MDA-MB-231 human triple negative breast cancer cells were grown in RPMI-1640 medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum (Sigma) and 1 % penicillin-streptomycin (Sigma) at 37°C in a 5% C02humidified atmosphere. Single layer cultured cells were detached with 0.02% EDTA (Sigma), washed twice with serum-free medium and a one-cell suspension was subcutaneously inoculated in the back of SCID mice at a cell number of 1.3 x 10 6 3 The test product was administered intraperitoneally when the tumor volume reached a detectable size (about 100-200 mm

[0393] All the test compounds were provided in powder form. The powder was dissolved in DMSO. For the final treatment concentration, the compounds were diluted in a physiological saline solution 1 :100 (1 % DMSO).

[0394] All the test compounds were planned to be administered by intraperitoneal injection in a volume of 0.3 mL in a physiological saline solution containing 1 % DMSO, three times a week. However, during the experiment, this schedule was modified: from 07.19, intraperitoneal injections were performed daily in a volume of 0.5 mL (Table 17).

[0395] Table 17: Treatment schedule

[0396]

[0397] * : number of cells inoculated: 1.3 x 10 6

[0398] During the experiment, the body weight and the size of the tumor of the animals were recorded. The compounds under study did not induce a body weight loss Figure 63 ).​

[0399] During the experiment, the tumor volume was measured three times a week. All the compounds studied reduced the tumor growth, but only in the case of TBP-333 the effect was significant Figure 64 ).

[0400] At the end of the experiment, the tumor weight was also measured in each group to evaluate the anti-tumor effect of the compounds ​ and 65 ). From the tumor weight, we can conclude that TBP-333 showed the highest and most significant inhibitory effect, but all the compounds studied reduced the tumor weight compared to the control group.

[0401] The results of the experiment show that the drugs tested have an anti-tumor effect, which is shown both in the volume and in the tumor mass at the end of the experiment, however, this effect is only significant in the case of TBP-333. It can be assumed that even more significant effects can be achieved if the solubility of the material can be improved, or the amount delivered in any other way is increased.

[0402] From the results described above, the following should be noted:

[0403] Regarding the influence of the substitution pattern of the benzyl groups attached at position 4 and 7 of the imipridone skeleton, we have unambiguously determined that the [7-(3",5"-difluorobenzyl)]-substituted compounds with the same substituent at position 4' (on the N-4 atom of the benzyl group) exhibit significantly stronger anti-proliferative activity than their [7-(3"-fluorobenzyl)]-substituted counterparts (TBP-301 vs. CZT-021, ABB-011 vs. TBP-218 and TBP-333 vs. TBP-353), which is clearly reflected in the IC 50 values (Table 16) and in the cell viability data obtained in the in vitro assays on human malignant cell lines by different methods in Example 21. It must be emphasized that even all the [7-(3"-fluorobenzyl)]-imipridones, including our original azido benzyl derivatives, were far more active than ONC-212 in each of the in vitro tests compared in the present application. The original mono-azido derivatives also demonstrated a particularly outstanding activity profile on the cell lines studied. Of significant interest and novelty in terms of effectiveness is that, as evidenced by the IC 50 values measured on the PANC-1 cell line (Table 16), the [7-(3"-azidobenzyl)]-imipridones TBP-272 and TBP-400 have a higher degree of cytotoxicity than the corresponding [7-(3"-fluorobenzyl)]-imipridones CZT-021 and TBP-353, respectively. By way of comparison, the IC This trend is also shown in the luminescent cell viability assay in a series of cell lines (see Example 21).

[0404] Finally, taking into account all the measured IC 50 And cell viability data, it can be said that, at in vitro level, the imipridone 4-(4-chlorobenzyl)-7-(3,5-difluorobenzyl)-2,4,6,7,8,9-hexahydroimidazo[l,2- a]pyrido[3,4-e]pyrimidin-5(lH)-one (TBP-333) that we first prepared and studied is the most effective representative of the family of small molecules anticancer imipridones physically identified to date.

[0405] Industrial applicability

[0406] The compounds of the present application have anticancer activity, therefore these compounds are suitable for use in medicine.

[0407] References cited in this specification:

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[0417]

[10] Wagner, J.; Kline, C. L.; Ralff, M. D.; Lev, A.; Lulla, A.; Zhou, L.; Olson, G. L.; Nallaganchu, B. R.; Benes, C. H.; Allen, J. E.; et al. Preclinical evaluation of the imipridone family, analogs of clinical stage anti-cancer small molecule ONC201, reveals potent anti-cancer effects of ONC212. Cell Cycle 2017 16, 1790-1799, doi: 10.1080 / 15384101.2017.1325046.

[0418]

[11] Lev, A.; Lulla, A. R.; Wagner, J.; Ralff, M. D.; Kiehl, J. B.; Zhou, Y.; Benes, C. H.; Prabhu, V. V.; Oster, W.; Astsaturov, I.; et al. Anti-pancreatic cancer activity of ONC212 involves the unfolded protein response (UPR) and is reduced by IGF1-R and GRP78 / BIP. Oncotarget 2017, 8, 81776-81793, doi: 10.18632 / oncotarget.20819.

[0419]

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[13] Xu, Ruo; Liu, Yunyong. Imidazopyrimidone compounds as antitumor agents and their preparation, pharmaceutical compositions and use in the treatment of cancer. PCT Int. Appl. (2016), WO 2016184437 A1 20161124-

[0421]

[14] Allen, Joshua E.; Stogniew, Martin; Prabhu, Varun Vijay, Preparation of imipridone compounds as G protein-coupled receptor modulators for treatment of cancer, infection, and psychiatric disorders. PCT Int. Appl. (2017), WO 2017132661 A2 20170803

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[15] Allen, Joshua E.; Stogniew, Martin; Prabhu, Varun Vijay, Imipridones for treatment of gliomas. U.S. Pat. Appl. Publ. (2018), US 20180221375 A1 20180809.

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[16] Iwanowicz, Edwin J. Preparation of hexahydroimidazopyridopyrimidinones as protein kinase regulators useful in mono- and combination therapy of diseases. PCT Int. Appl. (2018), WO 2018031987 A1 20180215

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Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof, in If Y is phenyl and Z is H, then X is 4-azidophenyl (compound I / 104); If Y is 3-fluorophenyl and Z is H, then X is 3-(aminomethyl)phenyl (compound I / 60); If Y is 3,5-difluorophenyl and Z is H, then X is 3-(aminomethyl)phenyl (compound I / 61), 4-azidophenyl (compound I / 107), or 3-azidophenyl (compound I / 127); If Y is 3-azidophenyl and Z is H, then X is 2-methylphenyl (compound I / 103), 4-(trifluoromethyl)phenyl (compound I / 111), 3-fluoro-4-(trifluoromethyl)phenyl (compound I / 112), 4-iodophenyl (compound I / 113), or 4-chlorophenyl (compound I / 133).

2. A compound of formula (I), or its enantiomers, racemic mixtures, or pharmaceutically acceptable salts thereof, in If Y is 3,5-difluorophenyl and Z is H, then X is 4-fluorophenyl (compound I / 7), 4-(trifluoromethyl)phenyl (compound I / 30), 4-bromophenyl (compound I / 39), or 4-chlorophenyl (compound I / 124); or If Y is 3,5-difluorophenyl and Z is hydroxymethyl, then X is 4-chlorophenyl (racemic) (compound I / 128) or 4-fluorophenyl (R-enantiomer) (compound I / 129(R)).

3. Use of the compound of claim 2 in the preparation of a medicament for treating cancers selected from prostate cancer, pancreatic cancer, lung cancer, breast cancer, and head and neck cancer.

4. The use of the compound according to claim 3, wherein... If Y is 3,5-difluorophenyl and Z is H, then X is 4-fluorophenyl (compound I / 7), 4-(trifluoromethyl)phenyl (compound I / 30), 4-bromophenyl (compound I / 39), or 4-chlorophenyl (compound I / 124) or a pharmaceutically acceptable salt thereof.

5. The use of the compound according to claim 3, wherein... If Y is 3,5-difluorophenyl and Z is H, then X is 4-fluorophenyl (compound I / 7), 4-(trifluoromethyl)phenyl (compound I / 30), or 4-chlorophenyl (compound I / 124) or a pharmaceutically acceptable salt thereof.

6. Use of the compound of claim 1 in the preparation of a medicament for treating the following cancers, wherein the cancer is selected from pancreatic cancer, colon cancer, melanoma, skin cancer, glioblastoma, prostate cancer, lung cancer, head and neck cancer, and breast cancer.

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