Substituted naphthalene diimide and its use

Abstract

The present invention relates to naphthalene diimide, NDI and methods for synthesizing them. NDI has DNA-quadruplex binding and stabilizing activity and has the potential to treat pancreatic cancer, prostate cancer and other human cancers. NDI is a compound of formula I:

Description

Field of the Invention

[0001] The present invention relates to naphthalene diimide, NDI and methods for synthesizing the same. NDI has DNA-quadruplex binding and stabilizing activity and has the potential to treat pancreatic cancer, prostate cancer and other human cancers. Background of the Invention

[0003] In WO2009 / 068916, we describe tri- and tetra-substituted naphthalene diimides and methods for producing them. None of the tri-substituted exemplified products have different amino functional ligands at equal and polar positions on the core ligand. The method said to be suitable for producing tri-substituted compounds is based on the following scheme:

[0004]

[0005] Where R 1 is optionally substituted alkyl or aryl and n is 0 or 1. In practice, a mixture of tetrasubstituted (n=1) and trisubstituted (n=0) compounds is produced. All substituents (i.e., R 1 groups) are the same.

[0006] The specification describes a process for producing tetrasubstituted compounds starting from the dichloro-substituted analogs of the dibromo compounds used above. The process is carried out in one step, in this case the same H2NR 1 The reagent reacts at two anhydride groups and two chloro-substituted carbons to provide four identical R 1 substituents, or in two steps, wherein in the first step, the first reagent H2NR 2 The reaction takes place at two anhydride groups and in a second step, a second reagent H2NR 3 The reaction occurs at two chloro-substituted carbon atoms. Compounds with basic substituents on the imide substituents and / or on the aromatic rings have strong DNA quadruplex binding properties.

[0007] In WO2017 / 103587, we described trisubstituted naphthalene diimides and methods for producing them. The method said to be suitable for producing trisubstituted compounds is based on the following scheme:

[0008]

[0009] Where Y is H or Br, the group R 12 are the same and are selected from the group consisting of straight chain and branched chain C 1-6 The group consisting of alkanediyl, R 13 Select from H and C 1-6 The group consisting of alkyl groups, R14 Selected from straight chain and branched chain C 1-6 Alkanediyl and C 7-12 Aranediyl group, X 2 Selected from halogen, R 11 NR 15 2. CONR 16 2. COOR 17 , SH and COR 18 The group composed of R 11 Selected from H, optionally substituted C 1-6 Alkyl, optionally substituted C 5-7 Cycloalkyl, C 5-7 A group consisting of a heterocycloalkyl group and an aryl group, each R 15 Choose from H, C 1-6 Alkyl, aryl and C 7-12 The group consisting of aralkyl groups, or the group R 15 Together with the nitrogen atom to which they are attached, they form a saturated heterocyclic ring of 5-7 atoms. 16 Select from H and C 1-6 The group consisting of alkyl groups, or the group R 16 Together with the nitrogen atom to which they are attached, they form a 5-7 membered heterocyclic ring. 17 Selected from optionally substituted C 1-6 Alkyl, C 7-12 A group consisting of an aralkyl group and an aryl group, R 18 Selected from optionally substituted C 1-6 Alkyl, C 7-12 A group consisting of an aralkyl group and an aryl group, and wherein the Br atom or one or each of the Br atoms is substituted with a nucleophilic amine nitrogen of an amine reagent to form a substituted NDI compound.

[0010] In WO2009 / 068916, US2014-0275065A, and Hampel Tetrasubstituted products (including those with residues corresponding to the group R) have been tested in SM et al., Bioorg. Med. Chem. Lett. (2010) 20, 6459-6463, Micco. M. et al., J. Med. Chem. (2013) 56, 2959-2974, Collie, GW et al., JACS (2012) 134, 2723-2731, Gunaratnam, M. et al., J. Med. Chem. (2009) 52, 3774-3783, Gunaratnam, M. et al., Bioorg. Med. Chem. (2011) 19, 7151-7157 and Mitchell, T. et al., Biochemistry (2013) 52, 1429-1436.3 Different groups R 2 The binding properties of the imide products) to telomere quadruplexes and those found in the promoter regions of some genes. The data show effective downregulation of multiple proteins whose genes' promoters were targeted by the imide and thus resulted in growth inhibition of multiple cell lines from a panel of cancer cell lines. We propose in these publications to further investigate the effects of varying the nature of the substituents and the basicity of the tertiary amine group in the cationic substituent on binding specificity and strength, and to investigate the potential of the compounds in cancer therapy through test models of cancer, including pancreatic cancer.

[0011] In Scientific Reports (2015) 5:11385, Ohnmacht, SA et al. disclosed the in vivo activity of 4,9-bis((3-(4-methylpiperazin-1-yl)-propyl)amino)-2,7-bis(3-morpholinopropyl)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (also known as MM41) in a mouse model of human pancreatic cancer.

[0012] Nadai, M. et al. disclose trisubstituted naphthalene diimide compounds in Int. J. Oncol. (2015) 46, 369-380, which have 2-dimethylaminoethyl substituted at each imide nitrogen atom and 2-(4-hydroxy-3-dimethylaminomethylphenyl)ethylamino substituted at the 4-position on the NDI core as the third substituent. It has the activity of stabilizing telomeric G-quadruplex (GQ), thereby leading to telomere dysfunction and telomerase downregulation. Global gene expression on a panel of cell lines showed regulation of genes related to telomere function and mechanism of cancer. However, the authors concluded that direct evidence of the biological relevance of G-4s in a cellular context is still lacking (Marchetti et al., J Med Chem, 2018, 61 (6), pp. 2500-2517).

[0013] The synthesis of the trisubstituted compounds reported by Nadai et al. is disclosed in Doria et al., Org Biomol. Chem, (2012) 10, 2798-2806. SUMMARY OF THE INVENTION

[0015] The present inventors have surprisingly found that specific groups of the side chains on the tetra-substituted naphthalene diimide compounds lead to improved binding of the diimide compounds to GQ, thereby leading to improved anticancer activity.

[0016] Therefore, in the first aspect of the present invention, there is provided a novel compound of formula I:

[0017]

[0018] L is in the meta or para position of the benzene ring and is selected from (CH2) 1-6 and (CH2) 1-5 The group composed of NH;

[0019] R 1 Selected from optionally substituted C 5-7 Cycloalkyl, optionally substituted nitrogen-containing 5-7 membered heterocycloalkyl and NR9R 10 The group composed of;

[0020] R 2 and R 4 Independently selected from linear and branched C 1-6 - a group consisting of alkanediyl;

[0021] R 3 , R 9 and R 10 Independently selected from H or C 1-6 A group consisting of alkyl groups;

[0022] X is selected from halogen, OR 5 NR 6 2. CONR 7 2. COOR 8 , H and COR 8 The group composed of;

[0023] R 5 Choose from H, C 1-6 Alkyl, C 4-7 the group consisting of cycloalkyl, 4-7 membered heterocycloalkyl and aryl;

[0024] R 6 Choose from H, C 1-6 Alkyl, aryl and C 7-12 -aralkyl group, or group R 6 Together with the N atom to which they are attached, they form a N-containing saturated 4-7 membered heterocyclic group;

[0025] Group R 7 Each selected from H and C 1-6 Alkyl, or group R 7 Together with the N atom to which they are attached, they form a 4-7 membered heterocyclic group;

[0026] R 8 Choose from C 1-6 Alkyl, C 7-12 The group consisting of aralkyl and aryl; and

[0027] Its salts, hydrates and solvates.

[0028] The invention further provides novel compounds for use in methods of treating animals to treat cancer or to inhibit the growth or reduce the size of solid tumors, such as pancreatic and prostate tumors.

[0029] The present invention also provides a composition containing the novel compound and a diluent or carrier. The composition is preferably a pharmaceutical composition and the carrier is pharmaceutically acceptable.

[0030] In a second aspect of the present invention, there is provided a method for synthesizing a substituted naphthalene diimide compound according to the first aspect of the present invention, the method comprising the following steps:

[0031] i) reacting a compound of formula IV with a compound of formula V in a nucleophilic substitution reaction:

[0032]

[0033] HN(R 3 )(R 4 X)

[0034] Formula IV

[0035] wherein at least one Br in the compound of formula III is replaced by a nucleophilic amine nitrogen in the compound of formula IV;

[0036] ii) reacting a compound of formula V, which is obtainable from the product resulting from the nucleophilic substitution reaction of formula III and formula IV, with a compound of formula VI:

[0037]

[0038]

[0039] wherein an aryl-aryl bond is formed between the phenyl group of formula VI and the phenyl group to which Br is attached in the compound of formula V, wherein LG and Br are leaving groups, thereby preparing a compound of formula I; and preferably

[0040] iii) separating the compound of formula I from the product resulting from the reaction of formula V and formula VI;

[0041] Among them, L, X and R 1 To R 4 is as defined for formula I of the first aspect of the invention.

[0042] Attached photos

[0043] Figure 1 : shows tumor regression in pancreatic cancer tumors in mice treated with compounds of the invention and comparative compounds. DETAILED DESCRIPTION OF THE INVENTION

[0045] definition

[0046] As used herein, unless otherwise specified, "alkyl", "cycloalkyl", "heterocycloalkyl", "heterocyclyl", "aryl" and "aralkyl" may be monovalent or divalent.

[0047] As used herein, unless otherwise indicated, "aryl" means a monocyclic, bicyclic or tricyclic monovalent or divalent (where appropriate) aromatic group such as phenyl, biphenyl, naphthyl, anthracenyl, which may be optionally substituted with up to three substituents.

[0048] As used herein, unless otherwise specified, "optionally substituted" means having one of the substituents selected from the group consisting of C1-C6 alkyl, hydroxy, C1-C3 hydroxyalkyl, C1-C3 alkoxy, C1-C3 haloalkoxy, amino, C1-C3 monoalkylamino, C1-C3 dialkylamino, C1-C3 acylamino, C1-C3 aminoalkyl, mono(C1-C3 alkyl)aminoC1-C3 alkyl, di(C1-C3 alkyl)amino, C1-C3 alkyl)amino, C1-C3 alkyl, C1-C3-acylamino, C1-C3 alkylsulfonylamino, halogen, nitro, cyano, trifluoromethyl, carboxyl, C1-C3 alkoxycarbonyl, aminocarbonyl, mono-C1-C3 alkylaminocarbonyl, di-C1-C3 alkylaminocarbonyl, -SO3H, C1-C3 alkylsulfonyl, aminosulfonyl, mono-C1-C3 alkylaminosulfonyl and di-C1-C3-alkylaminosulfonyl.

[0049] As used herein, unless otherwise indicated, "heterocycloalkyl" and "heterocyclyl" are carbocyclic groups containing up to 4 heteroatoms selected from oxygen, nitrogen and sulfur. They can be bicyclic or monocyclic. They are preferably saturated. If the heterocycle is a divalent linking group, the heterocycle can be connected to the adjacent group through a carbon atom or through one of the heteroatoms (e.g., N). Examples of heterocycles are pyrrolidine, piperazine and morpholine.

[0050] Preferred groups of the present invention

[0051] In the first aspect of the present invention, L is preferably (CH2) 1-6 , preferably (CH2) 1-4 , more preferably (CH2) 1-3 , still more preferably (CH2) 1-2 , even more preferably (CH2). Preferably L is in the para position of the phenyl group. When R 1 is an optionally substituted nitrogen-containing 5-7 membered heterocycloalkyl or NR9R 10 When R 1 Via R 1 The nitrogen atom of is bonded to L.

[0052] Conceived L or R1 contains a basic nitrogen atom. Therefore, R 1 It can be any group containing a basic nitrogen atom. 1 Preferably, it is a nitrogen-containing 5-7 membered heterocycloalkyl, preferably a nitrogen-containing 5-6 membered heterocycloalkyl, more preferably a nitrogen-containing 5 membered heterocycloalkyl. Preferably, the nitrogen of the nitrogen-containing 5-7 membered heterocycloalkyl is the only heteroatom in the heterocycloalkyl. In another aspect, the nitrogen-containing 5-7 membered heterocycloalkyl contains a second heteroatom, such as an oxygen atom.

[0053] Suitably, the nitrogen-containing 5-7 membered heterocycloalkyl is selected from the group consisting of pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, morpholinyl, azepanyl, diazepanyl, preferably pyrrolidinyl. Suitably, L is (CH2) and R 1 It is pyrrolidino.

[0054] Suitably, R 1 It is NR9R 10 . R 9 and R 10 Independently selected from H or C 1-6 The group consisting of alkyl groups, preferably C 1-6 Alkyl, more preferably C 2-4 Alkyl, even more preferably C 2-3 Alkyl. Suitably, NR9R 10 It is diethylamino, dipropylamino or ethylpropylamino.

[0055] In another embodiment, L comprises a basic nitrogen atom. Suitably, L is (CH2) 1-5 NH, preferably (CH2) 1-3 NH, more preferably (CH2) 1-2 NH, even more preferably (CH2)NH and R 1 It is C 5-7 Preferably, L is in the para position of the phenyl group.

[0056] The two R in Formula I 2 The groups are identical to each other. 2 Preferably, it is a straight chain C 2-4 -alkanediyl, most preferably straight-chain C3-alkanediyl. 4 Can be used with R 2 Same as or can be compared with R 2 Different, and preferably linear or branched C 2-4 -alkanediyl, most preferably C2-alkanediyl.

[0057] X preferably comprises an amine group, ie X is preferably NR 6 2 or CONR 7 2. Further optimization of NR 62. Group R 6 and R 7 Together with the nitrogen atom to which they are attached, they preferably form a saturated 4-7-membered heterocyclic group containing nitrogen, and more preferably a saturated 5-membered heterocyclic group containing nitrogen. 6 Those linked to form a heterocyclic ring are preferred as they appear to have useful cytotoxic activity in cancer cell line assays. X is most preferably a saturated pyrrolidinyl group.

[0058] Preferably, Formula I has the structure of Formula II below:

[0059]

[0060] Where L and R 1 is as defined for formula I, wherein any of the preferred groups are as outlined above.

[0061] Suitably, the compound is selected from the group consisting of:

[0062] 2,7-Bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)-9-(4-(pyrrolidin-1-ylmethyl)phenyl)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone;

[0063] 4-(4-(Morpholinylmethyl)phenyl)-2,7-bis(3-morpholinylpropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone;

[0064] 2,7-Bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)-9-(3-(pyrrolidin-1-ylmethyl)phenyl)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone;

[0065] 2,7-Bis(3-morpholinopropyl)-4-(4-(piperidin-1-ylmethyl)phenyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone;

[0066] 4-(4-((diethylamino)methyl)phenyl)-2,7-bis(3-morpholinopropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone;

[0067] 4-(4-((cyclopentylamino)methyl)phenyl)-2,7-bis(3-morpholinopropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone;

[0068] 4-(4-(azepan-1-ylmethyl)phenyl)-2,7-bis(3-morpholinopropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone;

[0069] 4-bromo-2,7-bis(3-morpholinopropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone; and

[0070] Its salts, hydrates and solvates.

[0071] In the second aspect of the present invention, L, X and R of Formula IV to Formula VIII 1 To R 4 Preferably, L, X and R are as described above for Formula I as the first aspect of the present invention. 1 To R 4 defined by the preferred features of.

[0072] The process of the present invention comprises a first step of reacting a brominated naphthalene diimide of formula III with an amine reagent of formula IV in an aromatic nucleophilic substitution reaction, whereby the bromine atom is replaced by an amino group N(R 3 )(R 4 X). The starting imide is a dibromo compound, and the aromatic nucleophilic substitution reaction may result in the replacement of two bromine atoms by amine groups or only one of them being replaced (i.e., a compound of formula V), but it is preferred that only one of the bromines is replaced, and at least one compound of formula V must be produced. It is preferred to separate the mixture of mono- and di-substituted naphthalene diimides, for example by using column chromatography.

[0073] In the second step, the compound of formula V produced in the first step is reacted with a reagent of formula VI in a substitution reaction, whereby the bromine atom is replaced by the phenyl group in formula VI via the carbon atom to which LG (leaving group) was originally attached. In one aspect, LG can be a boronic acid group, however, the skilled person will appreciate that there are many ways to carry out the substitution reaction and form an aryl-aryl bond between formula V and VI. Thus, at least one compound of formula I is produced.

[0074] Preferably, in a further step, the compounds of formula I are isolated by using column chromatography.

[0075] Preferably, the specific form of column chromatography used is selected from gel and flash column chromatography.

[0076] The compounds of the present invention can be provided in the form of pharmaceutical compositions. The compounds of the present invention, especially when present in the form of acid addition salts (e.g., some or all of the basic amine groups are converted to salt form), are water soluble and have a roughly neutral pH. Therefore, these salts are suitable for use in the form of aqueous solutions, which are suitable for intravenous administration. The pharmaceutical aqueous solution preferably contains 1 to 500 mg / l of the compound.

[0077] The compounds of the present invention can be provided in a form suitable for making a pharmaceutical composition, for example, in a dry, rehydratable form, for example, with a carrier or diluent. Such a dry form can be produced by crystallization and / or evaporation. Alternatively, the compound can exist as a concentrate, for example, in water or an organic pharmaceutical solvent for dilution before administration.

[0078] As used herein, a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids (such as hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid or nitric acid) and organic acids (such as citric acid, fumaric acid, maleic acid, malic acid, ascorbic acid, succinic acid, tartaric acid, benzoic acid, acetic acid, methanesulfonic acid, ethanesulfonic acid, salicylic acid, stearic acid, benzenesulfonic acid or p-toluenesulfonic acid). Pharmaceutically acceptable bases include alkali metal (such as sodium or potassium) and alkaline earth metal (such as calcium or magnesium) hydroxides and organic bases (such as alkylamines, arylamines or heterocyclic amines).

[0079] For the avoidance of doubt, the present invention also includes prodrugs which react in vivo to produce the compounds of the present invention.

[0080] The combination according to the invention may also be used in conjunction with other agents to inhibit unwanted and uncontrolled cell proliferation, such as antibodies. The compound may be coupled to the antibody or administered as two separate components.

[0081] The compounds of the present invention and compositions comprising them can be administered by any route. In one embodiment, the pharmaceutical composition comprising the compounds of the present invention can be formulated into a form suitable for oral, rectal, parenteral, intranasal or transdermal administration or by inhalation or by suppository administration. Typical administration routes are parenteral, intranasal or transdermal administration or by inhalation administration. For chemotherapy of tumors, the composition is most conveniently administered intravenously.

[0082] When used as a treatment for existing tumors, the compounds of the invention can be administered using regimens developed for chemotherapeutic agents.

[0083] The compounds and compositions of the present invention can be used to treat subjects suffering from cancer. One particular class of cancers is considered to be solid tumors, in which solid masses of cancerous material can be identified. Another class includes hematological cancers, which are considered to be cancers that affect the blood system.

[0084] Specific types of cancer that can be treated using the compounds and compositions of the present invention include, but are not limited to, prostate cancer, pancreatic cancer, small cell lung cancer, or gastrointestinal cancer. In a preferred embodiment, the cancer is prostate cancer or pancreatic cancer.

[0085] The compounds and compositions of the invention are useful in treatment to inhibit the growth of, or to reduce the size of, a solid tumor, for example where the tumor is a pancreatic or prostate tumor.

[0086] Suitably, the subject to be treated is an animal, preferably a human.

[0087] Thus, there is provided a method of treatment comprising administering to a subject a compound or pharmaceutical composition of the invention to treat cancer, particularly those cancers which have been described above.

[0088] Also provided is the use of a compound or pharmaceutical composition of the invention in the preparation of a medicament for treating cancer, in particular those cancers already described above.

[0089] The present invention is further illustrated in the accompanying examples. Example

[0090] A series of tetrasubstituted naphthalene diimides have been synthesized and evaluated as G-quadruplex ligands and potential anticancer agents.

[0091] Chemical

[0092] Unless otherwise stated, all chemicals, reagents and solvents were purchased from commercial sources and used as received. Solvents were commercial HPLC grade unless anhydrous solvents were specified, in which case Aldrich 'SureSeal' anhydrous solvents were used. Column chromatography was performed on pre-packed silica (230-400 mesh, 40-63 μm) columns using the specified eluents.

[0093] 1 H NMR spectra were acquired on a Bruker Avance III spectrometer at 400 MHz using residual undeuterated solvent as reference.

[0094] Perform analytical LCMS using either acidic or basic methods as follows:

[0095] Acidic, HPLC: Waters X-Select CSH C18, 2.5 μm, 4.6×30 mm column, eluted with a gradient of 0.1% formic acid in MeCN in 0.1% formic acid in water. A gradient of 5-95% 0.1% formic acid in MeCN occurred at 2.5 ml / min at 0.00-3.00 minutes, with a rinse at 4.5 ml / min at 3.01-3.5 minutes. Column re-equilibration to 5% MeCN was performed at 2.5 ml / min at 3.60-4.00 minutes. UV spectra of the elution peaks were measured at 254 nm using an Agilent 1260 Infinity or Agilent 1200 VWD. Mass spectra were measured using an Agilent 6120 or Agilent 1956 MSD running with positive / negative switching or an Agilent 6100 MSD running in positive or negative mode.

[0096] Basic, HPLC: Waters X-Select BEH C18, 2.5 μm, 4.6×30 mm column, eluted with a gradient of MeCN in 10 mM aqueous ammonium bicarbonate. A gradient of 5-95% MeCN occurred at 2.5 ml / min at 0.00-3.00 minutes, with a rinse at 4.5 ml / min at 3.01-3.5 minutes. Column re-equilibration to 5% MeCN was performed at 2.5 ml / min at 3.60-4.00 minutes. UV spectra of the elution peaks were measured at 254 nm using an Agilent 1260 Infinity or Agilent 1200 VWD. Mass spectra were measured using an Agilent 6120 or Agilent 1956 MSD running with positive / negative switching or an Agilent 6100 MSD running in positive or negative mode.

[0097] Alternatively, analytical UPLC / MS was performed using an acidic or basic method as follows:

[0098] Acidic, UPLC: Waters Acquity CSH C18, 1.7 μm, 2.1×30 mm column, eluted with a gradient of 0.1% formic acid in MeCN to 0.1% formic acid in water. The gradient was constructed with a starting point of 5% MeCN held at 0.0-0.11 minutes. The gradient of 5-95% occurred at 0.11-2.15 minutes, with a rinse at 2.15-2.56 minutes. Column re-equilibration to 5% MeCN was performed at 2.56-2.83 minutes. The UV spectrum of the elution peak was measured using an Acquity PDA, and the mass spectrum was recorded using an Acquity QDa detector with ESI positive / negative switching.

[0099] Basic UPLC: Waters Acquity BEH C18, 1.7 μm, 2.1×30 mm column, eluted with a gradient of MeCN in 10 mM aqueous ammonium bicarbonate. The gradient was constructed with a starting point of 5% MeCN held at 0.0-0.11 minutes. The gradient of 5-95% occurred at 0.11-2.15 minutes, with a rinse at 2.15-2.56 minutes. Column re-equilibration to 5% MeCN was performed at 2.56-2.83 minutes. The UV spectra of the elution peaks were measured using an Acquity PDA, and the mass spectra were recorded using an Acquity QDa detector with ESI positive / negative switching.

[0100] Preparative HPLC was performed using a Waters Xselect CSH C18, 5 μm, 19×50 mm column with a gradient of 0.1% formic acid in MeCN in 0.1% formic acid in water or a gradient of MeCN in 10 mM ammonium bicarbonate in water; or a Waters Xbridge BEH C18, 5 μm, 19×50 mm column with a gradient of MeCN in 10 mM ammonium bicarbonate in water. Fractions were collected after UV detection at a single wavelength measured by a variable wavelength detector on a Gilson 215 preparative HPLC or a Varian PrepStar preparative HPLC; fractions were collected after mass and UV detection at a single wavelength measured by a dual wavelength detector on a ZQ single quadrupole mass spectrometer (with positive and negative ion electrospray) and a Waters FractionLynx LCMS.

[0101] Example 1: 2,7-bis(3-morpholinylpropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)-9-(4-(pyrrolidin-1-yl)ethyl)amino (alkyl-1-ylmethyl)phenyl)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone

[0102]

[0103] 4-Bromo-2,7-bis(3-morpholinylpropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (100 mg, 0.141 mmol), (3,5-dimethoxyphenyl)boronic acid (77 mg, 0.422 mmol) or 1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)pyrrolidine (121 mg, 0.422 mmol) and Pd(Ph3P)4 (8.12 mg, 7.03 μmol) were dissolved in THF / 2M K2CO3 (3:1, 2 mL) and degassed, backfilled with nitrogen three times. The mixture was heated (70°C block temperature) for 3 hours with stirring. The reaction was cooled, diluted with DCM (15 mL), washed with water (15 mL), passed through a hydrophobic frit and concentrated in vacuo. The crude product was purified by preparative HPLC (basic, 20-50 MeCN in water) to give the title compound (7.1 mg, 8.34 μmol, 6% yield) as a dark red solid.

[0104] 1H NMR (400 MHz, chloroform-d) δ 10.24 (t, J = 5.3 Hz, 1H), 8.48 (s, 1H), 8.32 (s, 1H), 7.52 (d, J = 7.9 Hz, 2H), 7.38-7.29 (m, 2H), 4.34-4.25 (m, 2H), 4.21-4.07 (m, 2H), 3.87 (s, 2H), 3.7 5 (q, J = 6.2 Hz, 2H), 3.62 (dt, J = 16.8, 4.7 Hz, 8H), 2.95 (t, J = 6.5 Hz, 2H), 2.78 (s, 4H), 2.71-2.64 (m, 4H), 2.53 (t, J = 7.0 Hz, 2H), 2.50-2.35 (m, 10H), 2.02-1.78 (m, 12H). 1H NMR in CDCl31863-70-prep2 is consistent with the product structure at 93% purity. LCMS, basic, 1863-70B-prep, m / z 792.4 [M+H] + , at 4 min, 96% purity @254 nm. Contains 4% CMO3 by LC @254 nm.

[0105] Example 2: 4-(4-(morpholinylmethyl)phenyl)-2,7-bis(3-morpholinylpropyl)-9-((2-(pyrrolidine-1- (2H,7H)-tetraone)-1,3,6,8-(2H,7H)-dione

[0106]

[0107] A stirred mixture of 4-bromo-2,7-bis(3-morpholinylpropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (202 mg, 0.284 mmol) and (4-(morpholinylmethyl)phenyl)boronic acid (188 mg, 0.852 mmol) in dioxane (4 mL) was treated with potassium carbonate (568 μL of a 2M aqueous solution, 1.135 mmol) and degassed. S-Phos Pd G3 (6.64 mg, 8.52 μmol) was added and the mixture was degassed again, then the whole was heated to 80° C. (block temperature, preheated). After 18 hours, the mixture was cooled, then diluted with water (10 mL) and a saturated aqueous solution of NaHCO3 (10 mL) and extracted with DCM (2×20 mL). The combined organic matter was dried and evaporated with Na2SO4. Column chromatography (12 g Buchi FlashPure, pre-adsorbed, 10-70% [9:1 (1:1 THF:DCM): 7M NH3 in MeOH]) gave two fractions of medium pure product. The center portion of the product band was evaporated and dissolved in MeCN (2 mL). After about 48 hours, it was filtered and the solid was discarded. Meanwhile, the material from the edge of the product band was evaporated and slurry was formed again by isohexane. The material was merged with the MeCN liquid from the above batch, and the resulting product was purified by column chromatography (12 g RediSep Gold, 30-70% (9:1 DCM: 0.7M NH3 in MeOH) in DCM, loaded into DCM). The center fraction of the band was evaporated to give the product, which was a bright red glassy solid (50 mg, 22%).

[0108] LCMS: found m / z 808.3 (C 45 H 58 N7O7(MH + )Required 808.4)@6.68min. 1H NMR (500MHz, chloroform-d) δ10.24(t,J=5.3Hz,1H),8.50(s,1H),8.33(s,1H),7.45(d,J=8.0Hz,2H),7.32(d,J=8.0Hz,2H),4.30(t,J=7.4Hz,2H),4.15(t ,J=7.4Hz,2H),3.83-3.71(m,6H),3.65-3.59(m,10H),2.95(t,J=6.4Hz, 2H),2.70-2.67(m,4H),2.58-2.50(m,6H),2.47-2.40(m,10H),1.96(app p,J=7.1Hz,2H),1.90-1.84(m,6H).

[0109] Example 3: 2,7-bis(3-morpholinylpropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)-9-(3-(pyrrolidin-1-yl)ethyl)amino (alkyl-1-ylmethyl)phenyl)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone

[0110]

[0111] A stirred mixture of 4-bromo-2,7-bis(3-morpholinylpropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (149 mg, 0.209 mmol) and 1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)pyrrolidine (180 mg, 0.628 mmol) in dioxane (4 mL) was treated with potassium carbonate (419 μL of a 2M aqueous solution, 0.837 mmol) and degassed. S-Phos Pd G3 (4.90 mg, 6.28 μmol) was charged, the mixture was degassed again and the whole was heated to 80°C. After 16 hours, the mixture was cooled, then diluted with water (10 mL) and NaHCO saturated aqueous solution (10 mL) and extracted with DCM (2 × 20 mL). The combined organic Na SO Drying and evaporation. Column chromatography (12 g RediSep Gold, 30-60% (9:1 DCM: 0.7M NH in MeOH) in DCM) was loaded in DCM) to obtain a product of medium purity. Residue was purified by reverse phase column chromatography (12 g Reveleris C-18, 75-100% (70 mM NH in MeOH) in water) in DMSO) to obtain a better but still unsatisfactory product of purity. The residue was purified again by reverse phase column chromatography (12 g Reveleris C-18, 75-100% (70 mM NH3 in MeOH) in water, loaded into DMSO) to give the product as a bright red glassy solid (16 mg, 10%).

[0112] LCMS: found m / z 792.4: (C 45 H 58 N7O6(MH + )Required 792.4)@6.42min. 1 H NMR (500 MHz, dichloromethane-d2) δ 10.26 (t, J = 5.5 Hz, 1H), 8.47 (s, 1H), 8.35 (s, 1H), 7.47-7.37 (m, 2H), 7.35 (brs, 1H), 7.26 (dt, J = 7.2, 1.7 Hz, 1H), 4.30 (t, J = 7.4, 2H), 4.1 3(t,J=7.4Hz,2H),3.82-3.65(m,4H),3.52-3.58(m,8H),2.95(t,J=6.2Hz,2H),2 .69-2.66(m,4H),2.61-2.30(m,16H),1.93(p,J=6.9Hz,2H),1.89-1.75(m,10H).

[0113] Example 4: 2,7-bis(3-morpholinylpropyl)-4-(4-(piperidin-1-ylmethyl)phenyl)-9-((2-(pyrrolidine- 1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone

[0114]

[0115] A stirred mixture of 4-bromo-2,7-bis(3-morpholinylpropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (155 mg, 0.218 mmol) and 1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)piperidine (197 mg, 0.653 mmol) in dioxane (4 mL) was treated with potassium carbonate (436 μL of a 2M aqueous solution, 0.871 mmol) and degassed. S-Phos Pd G3 (5.10 mg, 6.53 μmol) was charged, the mixture was degassed again and the whole was heated to 80°C. After 16 hours, the mixture was cooled, then water (10mL) and NaHCO saturated aqueous solution (10mL) was diluted and extracted with DCM (2×20mL). The organic matter was combined with Na SO Drying and evaporation. Column chromatography (12g RediSep Gold, 30-60% (9:1DCM: 0.7M NH in MeOH) in DCM) was loaded into DCM) to obtain the product of medium purity. Residue was purified by reverse phase column chromatography (12g Reveleris C-18, 75-100% (70mM NH in MeOH) in water, loaded into DMSO) to obtain the product, which was a bright red glassy solid (61mg, 35%).

[0116] LCMS: found m / z 806.3: (C 46 H 60 N7O6(MH + )Required 805.5)@7.38min. 1 H NMR (500 MHz, dichloromethane-d2) δ 10.25 (t, J = 5.1 Hz, 1H), 8.47 (s, 1H), 8.34 (s, 1H), 7.43 (d, J = 8.1 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H), 4.29 (t, J = 7.4 Hz, 2H), 4.14 (t, J = 7.3 Hz, 2H), 3.75 (q, J =5.9Hz,2H),3.64-3.50(m,10H),2.95(t,J=6.2Hz,2H),2.69-2.66(m,4H),2.56-2.29( m,16H),1.93(p,J=7.0Hz,2H),1.89-1.82(m,6H),1.68-1.62(m,4H),1.53-1.49(m,2H).

[0117] Example 5: 4-(4-((diethylamino)methyl)phenyl)-2,7-bis(3-morpholinylpropyl)-9-((2-(pyrrole (alkyl-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone

[0118]

[0119] A stirred mixture of 4-bromo-2,7-bis(3-morpholinylpropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (155 mg, 0.218 mmol) and N-ethyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)ethanamine (189 mg, 0.653 mmol) in dioxane (4 mL) was treated with potassium carbonate (436 μL of a 2M aqueous solution, 0.871 mmol) and degassed. S-Phos Pd G3 (5.10 mg, 6.53 μmol) was charged, the mixture was degassed again and the whole was heated to 80°C. After 16 hours, the mixture was cooled, then water (10mL) and NaHCO saturated aqueous solution (10mL) was diluted and extracted with DCM (2×20mL). The organic matter Na sO dried and evaporated. Column chromatography (12g Buchi Flash Pure, 30-60% (9:1 DCM: 1.4M NH in MeOH) in DCM) was loaded in DCM) to obtain the product of medium purity. Residue was purified by reverse phase column chromatography (12g Reveris C-18, 75-100% (70mM NH in MeOH) in water, loaded in DMSO) to obtain the product, which was a bright red glassy solid (81mg, 47%).

[0120] LCMS: found m / z 794.2: (C 45 H 60 N7O6(MH + )Required 794.5)@6.93min. 1H NMR (500 MHz, dichloromethane-d2) δ 10.24 (t, J = 5.2 Hz, 1H), 8.46 (s, 1H), 8.32 (s, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 4.28 (t, J = 7.4 Hz, 2H), 4.13 (t, J = 7.4 Hz, 2H), 3.79-3.71 (m, 2H), 3.69 (s, 2 H),3.58-3.53(m,8H),2.94(t,J=6.2Hz,2H),2.69-2.66(m,4H),2.61(q,J=7.1Hz,4H),2.50(t,J =6.8Hz,2H),2.47-2.30(m,10H),1.93(p,J=6.9Hz,2H),1.88-1.82(m,6H),1.12(t,J=7.1Hz,6H).

[0121] Example 6: 4-(4-((cyclopentylamino)methyl)phenyl)-2,7-bis(3-morpholinylpropyl)-9-((2-(pyrrole (alkyl-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone

[0122]

[0123] A stirred mixture of 4-bromo-2,7-bis(3-morpholinylpropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (153 mg, 0.215 mmol) and N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)cyclopentylamine (194 mg, 0.645 mmol) in dioxane (4 mL) was treated with potassium carbonate (430 μL of a 2M aqueous solution, 0.860 mmol) and degassed. S-Phos Pd G3 (5.03 mg, 6.45 μmol) was charged, the mixture was degassed again and the whole was heated to 80°C. After 16 hours, the mixture was cooled, then water (10mL) and NaHCO saturated aqueous solution (10mL) was diluted and extracted with DCM (2×20mL). The organic matter Na sO dried and evaporated. Column chromatography (12g Buchi Flash Pure, 30-60% (9:1 DCM: 1.4M NH in MeOH) in DCM) was loaded in DCM) to obtain the product of medium purity. Residue was purified by reverse phase column chromatography (12g Reveris C-18, 75-100% (70mM NH in MeOH) in water, loaded in DMSO) to obtain the product, which was a bright red glassy solid (29mg, 17%).

[0124] LCMS: found m / z 806.4: (C46 H 60 N7O6(MH + )Required 805.5)@6.57min. 1 H NMR (500 MHz, dichloromethane-d2) δ 10.25 (t, J = 5.1 Hz, 1H), 8.46 (s, 1H), 8.34 (s, 1H), 7.45 (d, J = 7.9 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 4.29 (t, J = 7.3 Hz, 2H), 4.13 (t, J = 7.4, Hz, 2H), 3.88 (s, 2H), 3.75 (q, J = 5.9 Hz, 2H), 3.58-3.53 (m, 8H), 3.23 (p, J = 6.4 Hz, 1H), 2.95 (t, J = 6.2 Hz, 2H), 2.69-2.66 (m, 4H), 2.50 (t, J = 6.8 Hz, 2H), 2.47-2.30 (m, 10H), 1.96 -1.89 (m, 4H), 1.88-1.80 (m, 6H), 1.79-1.72 (m, 2H), 1.65-1.58 (m, 2H), 1.51-1.42 (m, 2H), CH2NHCH was not observed.

[0125] Example 7: 4-(4-(azepan-1-ylmethyl)phenyl)-2,7-bis(3-morpholinylpropyl)-9-((2- ((pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone

[0126]

[0127] A stirred mixture of 4-bromo-2,7-bis(3-morpholinylpropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (223 mg, 0.313 mmol) and 1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)azepane (296 mg, 0.940 mmol) in dioxane (4 mL) was treated with potassium carbonate (627 μL of a 2M aqueous solution, 1.253 mmol) and degassed. S-Phos Pd G3 (7.33 mg, 9.40 μmol) was charged, the mixture was degassed again and the whole was heated to 80°C. After 16 hours, the mixture was cooled, then water (10mL) and NaHCO saturated aqueous solution (10mL) was diluted and extracted with DCM (2×20mL). The organic matter Na sO dried and evaporated. Column chromatography (12g Buchi FlashPure, 30-60% (9:1DCM: 1.4M NH in MeOH) in DCM) was loaded in DCM) to obtain the product of medium purity. Residue was purified by reverse phase column chromatography (12g Reveris C-18, 75-100% (70mM NH in MeOH) in water, loaded in DMSO) to obtain the product, which was a bright red glassy solid (130mg, 51%).

[0128] LCMS: found m / z 820.3: (C 47 H 62 N7O6(MH + )Required 820.5)@7.69min. 1 H NMR (500 MHz, dichloromethane-d2) δ 10.25 (t, J = 5.1 Hz, 1H), 8.47 (s, 1H), 8.34 (s, 1H), 7.47 (d, J = 7.8 Hz, 2H), 7.33 (d, J = 7.8 Hz, 2H), 4.29 (t, J = 7.4 Hz, 2H), 4.14 (t, J = 7.3 Hz, 2H), 3.82-3.7 0(m,4H),3.57-3.53(m,8H),2.95(t,J=6.2Hz,2H),2.78-2.61(m,8H),2.50(t,J=6.8Hz ,2H),2.47-2.30(m,10H),1.93(p,J=7.0Hz,2H),1.88-1.82(m,6H),1.73-1.68(m,8H).

[0129] Example 8: 4-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-2,7-bis(3-morpholinylpropyl)-9-((2- ((pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone

[0130]

[0131] A stirred mixture of 4-bromo-2,7-bis(3-morpholinylpropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (213 mg, 0.299 mmol) and 1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)piperazine (284 mg, 0.898 mmol) in dioxane (4 mL) was treated with potassium carbonate (599 μL of a 2M aqueous solution, 1.197 mmol) and degassed. S-Phos Pd G3 (7.01 mg, 8.98 μmol) was charged, the mixture was degassed again, and the whole was heated to 80°C. After 16 hours, the mixture was cooled, then water (10mL) and NaHCO saturated aqueous solution (10mL) was diluted and extracted with DCM (2×20mL). The organic matter Na sO dried and evaporated. Column chromatography (12g BuchiFlashPure, 30-60% (9:1DCM: 3.5M NH in MeOH) in DCM) was loaded into DCM) to obtain the product of medium purity. Residue was purified by reverse phase column chromatography (12g Reveris C-18, 75-100% (70mM NH in MeOH) in water, loaded into DMSO) to obtain the product, which was a bright red glassy solid (111mg, 45%).

[0132] LCMS: found m / z 821.2: (C 46 H 61 N8O6(MH + )Required 821.5)@6.06min. 1 H NMR (500 MHz, dichloromethane-d2) δ 10.25 (t, J = 5.1 Hz, 1H), 8.46 (s, 1H), 8.34 (s, 1H), 7.43 (d, J = 8.1 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H), 4.29 (t, J = 7.4 Hz, 2H), 4.13 (t, J = 7.3 Hz, 2H), 3.75 (q, J = 5.9 Hz, 2H), 3.62 (s, 2H), 3.57-3.53 (m, 8H), 2.95 (t, J = 6.2 Hz, 2H), 2.77-2.20 (m, 27H), 1.94 (q, J = 7.1 Hz, 2H), 1.89-1.80 (m, 6H).

[0133] Biophysical and cell biology data

[0134] Cell proliferation assay

[0135] CellTiter The AQueous One-Solution Cell Proliferation Assay (Invitrogen) is a colorimetric method used to determine the number of viable cells in proliferation or cytotoxicity assays. AQueous single solution reagent contains a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES). PES has enhanced chemical stability, which allows it to combine with MTS to form a stable solution. The MTS tetrazolium compound (Owen's reagent) is bioreduced by cells to a colored formazan that is soluble in tissue culture medium. The assay was performed by adding a small amount of CellTiter AQueous single solution reagent is added directly to the culture wells, incubated for 1-4 hours, and then the absorbance at 490 nm is recorded using a 96-well plate reader. The amount of product is directly proportional to the number of viable cells in culture. The kit was used according to the manufacturer's instructions. After incubation with each example compound for 96 hours in MIA-PACA2 cells, cell proliferation of each sample was measured using the MTS Cell Titre 96 Aqueous single solution cell proliferation assay (Promega Ltd).

[0136] Percent inhibition was calculated relative to the mean of DMSO-treated control samples.

[0137]

[0138]

[0139] Table 1 - Cell growth inhibition data for the panel of pancreatic cancer cell lines used in Examples 1 to 8; IC from 96 hour MTS assay 50 (nM) values. The data show that Examples 1 to 8 exhibit different abilities to inhibit cancer cell growth. In particular, Example Compound 1 is the most active in the group.

[0140] In vivo xenograft efficacy studies

[0141] For the study, 5-7 week old mice weighing approximately 25-32 g were implanted and purchased from Charles River. The pancreatic tumor cell implantation procedure involved implanting MIA-PACA2 cells (1×10 7 The 100 μg / ml 100 μg / ml 200 μg / ml was implanted subcutaneously in the rear flank of mice. Parameters evaluated included: tumor size and animal weight. Tumor volume was measured three times per week, and body weight was measured at least 3 times per week. For animals in the efficacy study, when tumors reached approximately 50 mm 3 Randomization to treatment groups was performed at 4 hrs. Animals (female athymic nude mice bearing MIA-PACA2 tumors) were dosed intravenously (IV) twice a week for 28 days at doses of 10 mg / kg and 15 mg / kg for C1 and 0.5 mg / kg and 1.0 mg / kg for Example 1 (taking into account its 10-fold greater cellular potency). Each group contained 8 animals. All protocols used in this study were approved by the appropriate animal welfare and ethical review committees, and all procedures were performed in accordance with the guidelines of the UK Animal (Scientific Procedures) Act 1986. The results are shown in the following table and Figure 1 middle.

[0142]

[0143]

[0144] Table 2 - Basic properties and in vitro GQ binding data of Example 1 compared with prior art compound C1 (ref. WO2017 / 103587A1)

[0145]

[0146]

[0147] Table 3 - Cell growth inhibition data of a panel of pancreatic cancer cell lines compared to Example 1 and prior art compound C1 (ref. WO2017 / 103587A1); IC from 96-hour SRB assay 50 (nM) values, as detailed in our previous publications and disclosures. The data demonstrate that Example 1 is a significantly more potent compound in its ability to inhibit cancer cell growth, and that its pharmacological properties are at least comparable.

[0148] Figure 1The graph in shows xenograft data in the MIA-PACA2 model after 28 days of intravenous administration followed by 28 days of measurement (performed by AXIS BioServices). The data shown are mean ± SD for n = 8 until day 23 and n = 4 to the end of the study. The data show that even with a once-weekly dosing regimen, the compound of Example 1 significantly inhibited the growth of pancreatic tumors and reduced the size of tumors compared to the comparative compound C1 or the known anticancer drug gemcitabine. In addition, Example 1 and the dosing schedule were well tolerated and showed no signs of adverse effects. The initial tumor volume was 0.4 mm 3 Example 1 was active in both dose regimens examined (once a week and twice a week, both at a dose of 1 mg / kg). At the end of the dosing period, both had a 5 / 8 complete regression in tumor volume. In the complete regression cohort, after 28 days post-dose, the tumors had completely disappeared and no regrowth was seen. The few tumors in the C1 and Example 1 groups did not show complete regression, but did show a reduction in tumor growth, resulting in consistently smaller volumes compared to the vehicle control group.

[0149] XTT assay

[0150] The CyQUANT XTT Cell Viability Assay (Invitrogen) is a complete, optimized assay that produces consistent colorimetric detection of viable mammalian cells. The assay kit consists of two reagents, the XTT reagent (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) and an electron coupling reagent. The XTT reagent is used to assess cell viability as a function of cellular redox potential, while the electron coupling reagent improves the dynamic range of the assay. The kit was used according to the manufacturer's instructions.

[0151]

[0152]

[0153] Table 4 - Cell growth inhibition data for a panel of prostate cancer cell lines of Example 1 compared to prior art compound C1 (reference WO2017 / 103587A1) and clinically approved hormonal prostate cancer therapeutics abiraterone and enzalutamide; IC50 (nM) values ​​from 72-hour XTT assay. The data show that the compound of Example 1 is highly active in a panel of prostate cancer cell lines, especially in the metastatic and androgen-independent PC-3 line, when compared to C1, and even more so when compared to the two drugs used clinically.

[0154] In conclusion, the compounds of the present invention showed antitumor activity in a wide range of cancer cell lines.

Claims

1. A compound of formula I: L is in the meta or para position of the benzene ring; L is (CH2) 1-2 And R 1 Selected from nitrogen-containing 5-7 membered heterocycloalkyl and NR9R 10 or L is (CH2) 1-5 NH and R 1 It is C 5-7 Cycloalkyl; wherein the nitrogen-containing 5-7 membered heterocycloalkyl is unsubstituted or substituted with a C1-C6 alkyl; R 2 It is a straight chain C 2-4 Alkanediyl; R 3 It is H; R 4 It is a straight chain C 2-4 Alkanediyl; R 9 and R 10 Independently C 1-6 alkyl; X is NR 6 2, where the group R 6 Together with the N atom to which they are attached, they form a pyrrolidin-1-yl group; and Its salt.

2. The compound according to claim 1, wherein L is (CH2) 1-2 And R 1 It is a nitrogen-containing 5-7 membered heterocycloalkyl group.

3. The compound according to claim 1, wherein R 1 It is a nitrogen-containing 5-7 membered heterocycloalkyl group.

4. The compound according to claim 3, wherein the nitrogen-containing 5-7 membered heterocycloalkyl group is selected from the group consisting of pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, morpholinyl, azepanyl, and diazepanyl. The compound according to claim 4 , wherein the nitrogen-containing 5-7 membered heterocycloalkyl group is a pyrrolidinyl group.

6. The compound according to claim 1, wherein L is (CH2) 1-5 NH and R 1 It is C 5-7 Cycloalkyl.

7. The compound according to any one of claims 1 to 6, wherein L is in the para position.

8. The compound according to claim 1, wherein the compound is selected from the group consisting of: 2,7-Bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)-9-(4-(pyrrolidin-1-ylmethyl)phenyl)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone; 4-(4-(Morpholinylmethyl)phenyl)-2,7-bis(3-morpholinylpropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone; 2,7-Bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)-9-(3-(pyrrolidin-1-ylmethyl)phenyl)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone; 2,7-Bis(3-morpholinopropyl)-4-(4-(piperidin-1-ylmethyl)phenyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone; 4-(4-((diethylamino)methyl)phenyl)-2,7-bis(3-morpholinopropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone; 4-(4-((cyclopentylamino)methyl)phenyl)-2,7-bis(3-morpholinopropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone; 4-(4-(azepan-1-ylmethyl)phenyl)-2,7-bis(3-morpholinopropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone; 4-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-2,7-bis(3-morpholinopropyl)-9-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone; and Its salt.

9. The compound according to claim 1, wherein the compound is 2,7-bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)-9-(4-(pyrrolidin-1-ylmethyl)phenyl)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone.

10. The compound according to claim 1, wherein Formula I has the structure of the following Formula II: Where L and R 1 As defined in claim 1.

11. A composition comprising a compound according to any one of claims 1 to 10 in combination with a pharmaceutically acceptable diluent.

12. Use of a compound according to any one of claims 1 to 10 or a composition according to claim 11 in the preparation of a medicament for treating or preventing cancer, wherein the cancer is pancreatic cancer or prostate cancer.

13. Use of a compound according to any one of claims 1 to 10 or a composition according to claim 11 in the preparation of a medicament for inhibiting the growth of a solid tumor or reducing the size of a solid tumor, wherein the solid tumor is a pancreatic tumor or a prostate tumor.

14. A method for synthesizing a compound according to any one of claims 1 to 10, comprising the following steps: i) reacting a compound of formula III with a compound of formula IV in a nucleophilic substitution reaction: wherein at least one Br in the compound of formula III is substituted by a nucleophilic amine nitrogen in the compound of formula IV; ii) reacting a compound of formula V, which is obtainable from the product resulting from the nucleophilic substitution reaction of formula III and formula IV, with a compound of formula VI: wherein an aryl-aryl bond is formed between the phenyl group of formula VI and the phenyl group to which Br is attached in the compound of formula V, wherein LG and Br are leaving groups, thereby preparing a compound of formula I; Among them, L, X and R 1 To R 4 is as defined in any one of claims 1 to 10 for formula I.

15. The method according to claim 14, further comprising the steps of: iii) isolating the compound of formula I from the product resulting from the reaction of formula V and formula VI.

Images