PL248245B1 - Conjugates of hydroxycinnamic acids and 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline, method of obtaining them, medical use and pharmaceutical composition - Google Patents

Abstract

The subject of the application are new amide conjugates of hydroxycinnamic acid or a derivative thereof and 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline, also known as new derivatives of N-(hydroxy)cinnamoyl-9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline, represented by formula I, wherein R-C(O)- is an acyl group derived from hydroxycinnamic acid or a derivative thereof, and pharmaceutically acceptable salts thereof. The invention also includes a method for obtaining the novel hybrid compounds defined above, as well as their medical use and a pharmaceutical composition containing them. The obtained new derivatives with a hybrid structure can be used as active substances in pharmaceutical compositions intended for the treatment of cancer.

Description

Description of the invention

The subject of the invention are amide conjugates of hydroxycinnamic acids (HCA) and 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline, otherwise N-(hydroxy)cinnamoyl-9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline derivatives, a method for obtaining them, medical use and a pharmaceutical composition.

N-(hydroxy)cinnamoyl-9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline derivatives exhibit anticancer and cytostatic activity. They may find use in the pharmaceutical industry as active substances in pharmaceutical compositions for the treatment of pre-cancer diseases, particularly pancreatic cancer.

Based on analysis of GLOBOCAN data, the number of patients requiring first-line chemotherapy per year is estimated to increase from 9.8 million to 15.0 million between 2018 and 2040, representing a relative increase of 53% (Wilson BE et al. Lancet Oncol. 2019, 20(6), 769). For cancers diagnosed in England between 2013 and 2016, 28% of those receiving at least one of the main types of treatment were treated with chemotherapy (PELE, Official Statistics Chemotherapy, Radiotherapy and Surgical Tumor Resections in England, April 2020).

Cytostatics, which directly or indirectly affect DNA, remain a widely used group of chemotherapeutic drugs. These therapeutic agents can bind to DNA as alkylating agents, DNA intercalators, or compounds capable of stabilizing the DNA-intercalatortopoisomerase I/II complex. Although significant progress has been made in recent years in early diagnosis and the introduction of new targeted therapies, there remains a need for new molecules that can overcome drug resistance and increase the efficacy of currently used therapeutic regimens.

As a result of many years of research, a new generation of cytostatics, natural alkaloids, analogues of neocryptolepine with an indolo[2,3-b]quinoline ring were discovered (PL144539B1, Kaczmarek Ł. et al. Pol. J. Pharmacol. Pharm. 1981, 33, 121. Peczynska-Czoch W. et al. in Bushel and Graefe Progress in Industrial Microbiology 1989, 27, 333; Kaczmarek Ł. et al., Archiv. Pharm. 1988, 321, 463).

Among simple indolo[2,3-b]quinoline derivatives, the 5,11-dimethyl-5H-indolo[2,3-b]quinoline molecule (DiMIQ) shows high cytotoxic activity against cancer lines, including human oral epidermoid carcinoma KB with an IC50 of lμM. (Kaczmarek Ł. et al. Bioorganic&Medicinal Chemistry 1999, 7(11), 2457; Kaczmarek Ł. et al. Arch Pharm (Weinheim) 1988, 321(8), 463; Jaromin A. et al. Drug Deliv. 2008, 15(1), 49). DiMIQ intercalates with the DNA strand, and the increase in its strength depends on the pH value, i.e., the presence of DiMIQ in the protonated form (Peczyńska-Czoch W. et al. J. Med. Chem. 1994, 37, 3503). Due to the low solubility in aqueous media and high cytotoxicity of DiMIQ against normal cells, various derivatives of this compound have been synthesized for structure-activity studies in order to find a less toxic derivative that retains strong cytotoxic properties.

Polish patent PL206855 B1 discloses synthetic DiMIQ derivatives with dialkylaminoalkyl substituents linked by amide, amine, or ether bonds to an aromatic system. They have been shown to have cytotoxic activity against KB cells.

In turn, Polish patent descriptions PL202545 B1 and PL206681 B1 and the publication by Godlewska J. et al. (Radiol. Onkol. 2004, 38, 137) describe sugar derivatives of 5,11-dimethyl-5H-indolo[2,3-b]quinoline and 6,11-dimethyl-6H-indolo[2,3-b]quinoline. Sugar substituents, including deoxy-glucose, deoxy-rhamnose, deoxy-lactose, and aminoglycosyl substituents, were introduced at positions 2 and 9 of the aromatic ring of indolo[2,3-b]quinoline. Sugar derivatives were characterized by better solubility in water compared to unsubstituted 5,11-dimethyl-5H-indolo[2,3-b]quinoline and 6,11-dimethyl-6H-indolo[2,3-b]quinoline, while showing moderate activity against KB cell lines (from several to a dozen or so μM).

Another known group of compounds are DiMIQ conjugates with amino acids or peptides. These derivatives showed significantly better solubility in aqueous solutions with higher or equal cytotoxicity in vitro. Furthermore, they were characterized by significantly improved selectivity for healthy cells compared to the starting 5,11-dimethyl-5H-indolo[2,3-b]quinoline (K. Sidoryk et al., J. Med. Chem. 2012, 55 (11), 5077, patent PL 219085). DiMIQ derivatives with a glycine, L-, and D-proline substituent showed the highest cytotoxicity against KB cell lines, with IC50 values ranging from 0.42 to 0.73 μM. The cytotoxic activity of the dipeptide derivatives was similar to or lower than the effect of unsubstituted DiMIQ on cancer cells (IC50 values between 1.54-4.65 μM). In order to reduce the toxicity of DiMIQ and/or its derivatives towards normal (healthy) cells, another group of DiMIQ derivatives – those with a guanyl substituent – was designed, synthesized, and tested in biological tests. These compounds are described in patent application WO2015147666A1 and the scientific publication by Sidoryk K. et al. (Eur J Med Chem. 2015,105, 208). The guanyl derivatives showed high cytotoxic activity in vitro, and some of them showed very high selectivity between normal cells and the cancer cell lines A549 and MCF-7.

Hydroxycinnamic acids (HCAs) are the most common phenolic acids. Acids in this group, such as caffeic, feluric, sinapic, and coumaric, have the ability to bind carcinogens formed as a result of the transformation of carcinogenic compounds. They inhibit the formation of nitrosamines. In addition to the anticancer effect (Zaremba-Czogalla M. et al. Materials (Basel). 2020, 13(24), 5813; Rocha LD et al. Cancer and Clinical Oncology 2012, 1(2), 109) associated with the inhibition of cell proliferation (Yamaguchi M. et al. Oncol Rep. 2015, 34(6), 3304), they affect the level of intracellular reactive oxygen species (ROS), lipid peroxidation, and mitochondrial membrane permeability, which induces apoptosis (Espindola KMM et al. Front Oncol. 2019, 9, 541). They also exhibit antiangiogenic activity and inhibit DNA methylation. Caffeic acid (3,4-dihydroxycinnamic acid, an example of a compound from the HCA group) intercalates with calf thymus DNA (ct-DNA). Thermodynamic parameter analysis suggests that hydrogen bonds and van der Waals forces play a major role in binding. Additional studies confirm that caffeic acid interacts with the minor groove of ct-DNA (Sarwar T et al. Int J Biol Macromol. 2017, 98, 319). Studies on ester derivatives of caffeic acid demonstrate their cytotoxic activity against AsPC-1 and BxPC-3 pancreatic cancer cells, with low toxicity against normal NHDF cells (human skin fibroblasts) (Zaremba-Czogalla M. et al. Materials (Basel). 2020, 13(24), 5813). Furthermore, some ester derivatives of HCA, in combination with curcumin and/or carnosic acid, exhibit synergistic activity against AML leukemia cells (Trachtenberg A et al. Front Pharmacol., 2019, 10, 507).

Combination therapy is currently the gold standard for many cancer types. The main obstacle to overcome in combination therapy is the differences in the pharmacokinetics of drugs administered at specific time schedules, which in practice can lead to suboptimal drug concentrations (Szumilak M. et al. Molecules. 2021, 26( 9), 2601; Eras A. et al. Front Chem. 2022, 10, 889083).

Treatment of some types of cancer still requires overcoming numerous limitations, primarily due to unsatisfactory survival rates. In the case of pancreatic cancer (seventh most common), the introduction of multidrug therapy with Folfirinox has not yielded satisfactory results in extending patient survival. The systematic increase in cancer incidence and the persistently high mortality rate (Global Burden of Disease 2019 Cancer Collaboration JAMA Oncol. 2022, 8(3), 420) encourage intensive research into new, effective therapies and innovative diagnostic methods.

These problems are being addressed by developing various conjugates of anticancer compounds that simultaneously target the same or different signaling pathways and different structures in cancer cells. This represents a promising new approach to designing new, effective anticancer drugs.

According to the invention, this goal was achieved by introducing a hydroxycinnamic group into the indolo[2,3-b]quinoline core via an amide bond. The resulting conjugate exhibits quasi-synergistic activity against cancer cell lines, particularly pancreatic cancer. In the obtained new compounds, both haptophore fragments potentially contribute to the anticancer properties of the hybrid molecule, through their separate anticancer activity and their ability to bind to DNA and stabilize DNA-topoisomerase complexes.

The subject of the invention are amide conjugates of hydroxycinnamic acids (HCA) and 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline, or derivatives of N-(hydroxy)cinnamoyl-9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline. These are compounds with a hybrid structure with various hydroxycinnamic substituents directly bonded via the nitrogen atom at the C-9 position to 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline (9-amino-DiMIQ) and their pharmaceutically acceptable acid addition salts.

The conjugates of the invention are represented in the Figure by formula (I), wherein the acyl group is derived from hydroxycinnamic acid, also known as a hydroxycinnamoyl group. In addition to the conjugates defined above, the invention also includes pharmaceutically acceptable acid addition salts thereof.

The invention is therefore a hybrid compound resulting from the combination of 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline, defined by formula (II), at the C-9 position by an amide bond with hydroxycinnamic acid (HCA), in the form of a conjugate, and a pharmaceutically acceptable acid addition salt thereof.

In the derivatives according to the invention, the acyl group is derived from hydroxycinnamic acid, preferably from the following acids: ortho-coumaric, meta-coumaric, para-coumaric, 2,4-dihydroxycinnamic, caffeic, 3,4,5-trihydroxycinnamic.

The invention also includes pharmaceutically acceptable salts of the compounds of formula (I). These are addition salts formed with mineral acids or organic acids.

Examples of suitable mineral acids are hydrochloric, hydrobromic, sulfuric, nitric, or phosphoric acid. Examples of suitable organic acids are maleic, fumaric, succinic, itaconic, citraconic, oxalic, benzoic, p-aminobenzoic, ascorbic, acetic, propionic, tartaric, salicylic, citric, gluconic, lactic, mandelic, cinnamic, aspartamic, methanesulfonic, ethanedisulfonic, benzenesulfonic, toluenesulfonic, glycolic, glutamic, stearic, or palmitic acid.

Derivatives of formula (I) are valuable in terms of therapeutic activity: 9-[((4-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline and its pharmaceutically acceptable salts, 9-[((2-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline and its pharmaceutically acceptable salts, 9-[((3-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline and its pharmaceutically acceptable salts, 9-[((2,4-dihydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline and its pharmaceutically acceptable salts, 9-[((3,4-dihydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline and its pharmaceutically acceptable salts.

Particularly valuable in terms of therapeutic activity are: 9-[((2-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline dihydrochloride, 9-[((3-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline dihydrochloride, 9-[((4-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline.

The invention also provides a method for obtaining the above-defined conjugates represented by formula (I), wherein O-protected hydroxycinnamic acid or its acid halide is subjected to a coupling reaction (N-acylation) with 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline or a salt thereof. The derivative thus obtained is then purified and the functional groups from the hydroxycinnamoyl fragment (a molecular fragment derived from hydroxycinnamic acid) are deprotected, and optionally the obtained compound (I) is converted into a pharmaceutically acceptable acid addition salt in a known manner.

In the coupling reaction, 9-amino-dimethyl-5H-indolo[2,3-b]quinoline (formula II) is converted into an O-protected amide, i.e., the conjugate of hydroxycinnamic acid and 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline, and then the compound thus obtained is purified and the hydroxyl protecting groups are deprotected by known methods of solvolysis and catalytic deprotection. As a result, a conjugate with free hydroxyl groups represented by formula (I) is obtained, which is optionally converted into a pharmaceutically acceptable acid addition salt thereof.

More specifically, the invention relates to a method for obtaining derivatives represented by formula (I), which comprises:

(i) hydroxycinnamic acid, represented in the Figure by formula (III), is protected on the hydroxyl group/hydroxyl groups in the phenyl ring of the hydroxycinnamoyl fragment, (ii) the O-protected hydroxycinnamic acid derivative thus obtained in step (i) is optionally converted into an acid halide, preferably a chloride, (iii) then 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline (formula II) or a salt thereof, preferably a hydrochloride, is subjected to a coupling reaction with the O-protected hydroxycinnamic acid derivative obtained in step (i) or with its acid halide obtained in step (ii), (iv) whereupon the O-protected amide obtained in step (iii) constituting the conjugate of hydroxycinnamic acid and 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline, is purified and the functional groups are deprotected from the hydroxycinnamoyl fragment by known methods, (v) and optionally the conjugate with free hydroxyl groups represented by formula (I) thus obtained in step (iv) is converted in a known manner into a pharmaceutically acceptable acid addition salt

The course and conditions of the method for obtaining compounds of formula (I) are illustrated in Scheme 1.

The starting derivative of formula (II) is prepared by a classical modification of the Graebe-Ullmann method, described in Polish patent description PL144539B1 and Holt S., Petrow V. J. Chem. Soc. 1948, 922. The synthesis of the designed conjugates requires the use of protected hydroxycinnamic acids (HCA) as building blocks/elements, in which the hydroxyl groups in the acylating cinnamoyl residue are protected in a manner known in the art.

Protected derivatives of hydroxycinnamic acids (HCA) are O-acetyl and O-allyl derivatives obtained under standard reaction conditions. O-acyl derivatives of HCA are preferably prepared in the presence of acetic anhydride and pyridine. The reaction product is isolated by adding water to the reaction mixture, acidifying, filtering, and optionally crystallizing the product. This method yields O-acetyl derivatives of HCA (up to 95% yield).

The preparation of O-allyl HCA derivatives involves a three-step procedure, in which the first step is esterification with an alkyl alcohol, preferably methanol and ethanol (Bukowski N. et al. Bioconjug Chem. 2014, 25(12), 2189). After isolation, the product is subjected to an allylation reaction of the free hydroxyl group(s) in the HCA ester derivative. Allylation is preferably carried out with allyl bromide in acetone in the presence of an inorganic or organic base (e.g., K2CO3). After isolation, the ester protected by allyl ether(s) is hydrolyzed under basic conditions, preferably in the following systems: aqueous NaOH/methanol solution, aqueous KOH/ethanol solution, to obtain HCA derivatives protected with allyl group(s) (yield from 75 to 80%).

Methods for coupling amines to form amide bonds are well known in organic chemistry, especially since amide bonds play a central role in the composition and function of biological systems as the key chemical bond linking amino acids into peptides and proteins. Review articles describe classical (Valeur E. & Bradley M. Chem. Soc. Rev. 2009, 38(2), 606) and non-classical (De Figueiredo et al. Chemical Reviews 2016, 116(19), 12029) approaches to amide bond formation. Classical coupling methods utilize, among others, carbodiimides (e.g. DCC, DIC, EDC), carbodiimides with additives (HOBt, HOAt), 1H-benzotriazoles - including uronium/aminium (HATU, HBTU, HCTU, TBTU, TATU etc.), phosphonium (e.g. BOP, PyBOP) and iminium salts (BOMI, BDMP), reagents generating acyl halides (e.g. DAST, DCMT). Halo-uronium, halo-phosphonium, halo-sulfonium, halo-dioxole, halo-thiazolium, halo-pyridinium coupling reagents etc. Other effective coupling reagents include, among others, CDI and COMU – a compound popular in recent years, belonging to the third generation of uronium coupling reagents (based on ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) and a morpholine group). The number of different reagents for activating carboxyl groups to form amide bonds currently exceeds one hundred compounds.

Protected HCA acids or acid halides include in particular: (2-allyloxy)cinnamic acid, (3-allyloxy)cinnamic acid, (4-allyloxy)cinnamic acid, (2,4-diallyloxy)cinnamic acid, (3,4-diallyloxy)cinnamic acid, (2-acetoxy)cinnamic acid, (3-acetoxy)cinnamic acid, (4-acetoxy)cinnamic acid, (2,4-diacetoxy)cinnamic acid, (3,4-diacetoxy)cinnamic acid, (3,4-diacetoxy)cinnamic chloride, (2-acetoxy)cinnamoyl chloride, (3-acetoxy)cinnamoyl chloride, (4-acetoxy)cinnamoyl chloride, (2,4-diacetoxy)cinnamoyl chloride, (3,4-diacetoxy)cinnamic acid, (4-diacetoxy)cinnamoyl chloride, (2,4-diacetoxy)cinnamoyl chloride, (3,4-diacetoxy)cinnamic acid, (4-diacetoxy)cinnamic chloride ... (3,4-diacetoxy)cynamoyl. HCA chlorides are obtained from the corresponding acids using a reagent well known to those skilled in the art, selected from the group: thionyl chloride, oxalyl chloride, etc. HCA chlorides are also formed in situ in the presence of POCl3, as in the protocol described by Quelever et al. (J. Comb. Chem. 2004, 6(5), 695).

The coupling reaction in step (iii) is preferably carried out in the presence of a coupling agent selected from the group: DCC, EDC, TBTU.

Reaction with HCA derivatives containing an allyl protecting group leads to protected conjugates in 68-75% yield. Coupling of 5,11-dimethyl-5H-indolo[2.3-b]quinolin-9-ylamine with O-acetyl-protected hydroxycinnamic acids proceeds with lower yield. Coupling of protected HCA acids is carried out according to one of the well-known procedures in N,N-dimethylformamide, N,N-dimethylacetamide, or mixtures thereof with chloroform, dichloromethane, dichloroethanes as solvents. Reaction with acid chlorides proceeds in the presence of organic amines such as triethylamine, DMAP, etc., preferably pyridine. (Shimma et al. Bioorg. Med. Chem. 2000, 8(7), 1697) in solvents: chloroform, dichloromethane, dichloroethane, N,N-dimethylformamide, N,N-dimethylacetamide, hydrocarbons, aromatic hydrocarbons (toluene, xylene) and their mixtures.

The coupling reaction in step (iii) is also preferably carried out using an acid halide, preferably hydroxycinnamic acid chloride, preferably in the presence of pyridine.

The protected conjugates are isolated and/or purified by known methods. Isolation may involve precipitation of the crude product and drying under reduced pressure or evaporation of the reaction mixture to dryness to yield the crude product. Isolation of the pure products may require purification by column chromatography or preparative plate chromatography and/or purification of the products by other methods commonly known to those skilled in the art, e.g., recrystallization. Acetyl protecting groups of HCA are removed according to one known methods, e.g., under basic conditions, by treatment with potassium carbonate or ammonia. Allyl protecting groups are removed by one of the generally recognized methods (Greene's Protective Groups in Organic Synthesis, 5th edition. Peter G. M. Wuts. ISBN: 978-1-118-05748-3; Chapter 3.), including using tetrakis(triphenylphosphine)palladium, DMSO-I2, DMSO-Nal systems. Deprotection is carried out in DMF, DMA, alcohols and their mixtures with chloroform, dichloromethane, dichloroethanes. Allyl ether-type protections are preferably removed with tetrakis(triphenylphosphine)palladium. This reagent is preferably used in the presence of a free radical scavenger (e.g., triethylsilane) in a standardly rarely used amount of catalyst, i.e. even in an equimolar amount.

The crude final conjugates represented by formula (I) are separated by concentrating the reaction mixture and then purified by a conventional procedure. Purification may involve precipitation of the crude product or evaporation of the reaction mixture to dryness and/or purification of the crude product by column chromatography. The obtained new derivative of formula (I) may, if necessary, be purified by crystallization.

The conjugates of formula (I) and their salts may exist in unsolvated form or in the form of solvates with pharmaceutically acceptable solvents such as water, alcohols, etc. The invention encompasses all forms of the compounds, both solvated and solvent-free.

The invention also provides the medical use of amide conjugates of hydroxycinnamic acids (HCA) and 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline of formula (I) or a pharmaceutically acceptable acid addition salt thereof as active substances in pharmaceutical compositions intended for use in the treatment of cancer. It has been found that the compounds of the invention exhibit anticancer properties and may constitute a promising group of potential therapeutics for use in the treatment of human cancers, particularly pancreatic cancer, breast cancer, cervical cancer, lymphomas, sarcomas, and adenomas.

Therefore, the present invention also relates to a pharmaceutical composition comprising an active substance and a pharmaceutically acceptable and compatible carrier and/or excipients, wherein the active substance is a conjugate of formula (I) as defined above or a pharmaceutically acceptable acid addition salt thereof.

A pharmaceutical composition comprising a therapeutically effective amount of a conjugate of formula (I) or a pharmaceutically acceptable salt thereof is administered to a patient in need of treatment in a convenient pharmaceutical unit dosage form appropriate to the type of agent by route, e.g., intravenously, intramuscularly, subcutaneously or orally. For the purpose of anticancer therapy, the pharmaceutical agent used may be formulated in a variety of pharmaceutical forms well known to those skilled in the art, e.g., according to Remington's Pharmaceutical Sciences, 18th ed., Mack Publ. Co.

The choice of drug dose and dosing regimen depends on the type of disease, age, weight and health condition of the patient and can be determined by a specialist based on known cancer treatment and prevention regimens.

The novel amide conjugates represented by formula (I) have been found to have valuable potential anticancer properties, demonstrated in in vitro studies on selected cancer cell lines, including human pancreatic cancer BxPC-3 (normal line) and AsPC-1 (metastatic line). A quasi-synergy effect induced by the conjugates against BxPC-3 pancreatic cancer cells has been demonstrated. The conjugates were found to have a greater growth inhibitory potential than DiMIQ and the corresponding HCA derivatives administered both separately and in combination. Furthermore, the compounds of the invention also exhibit high antiproliferative activity against hormone-dependent breast cancer (MCF-7) and cervical cancer cells (HeLa). The highest cytotoxic activity among the tested conjugates of formula (I) was demonstrated by the compounds:

9-[((4-hydroxy)cinnamoyl)amino]-5.1l-dimethyl-5H-indolo[2.3-b]quinoline dihydrochloride (1) 9-[((2-hydroxy)cinnamoyl)amino]-5.1l-dimethyl-5H-indolo[2.3-b]quinoline dihydrochloride (2) dihydrochloride 9-[((3-hydroxy)cinnamoyl)amino]-5,1l-dimethyl-5H-indolo[2.3-b]quinoline (3)

Compounds 1 and 2 exhibited particularly high cytotoxic activity against BxPC-3 cells (IC50 805.0 nM, 347.53 nM) and AsPC-1 cells (IC50 2641.5 nM, 377.85 nM). It was shown that combinations of DiMIQ (6) with HCA derivatives: methyl (4-hydroxy)cinnamate (Kwi), methyl (2-hydroxy)cinnamate (Kw2) do not exhibit synergistic activity. Conjugates of these compounds surprisingly exhibit a quasi-synergy effect in vitro, meaning they are more effective than either compound used alone (higher cytotoxicity against cancer cells). For DiMIQ (6) with Kwi and with Kw2, conjugate 1 and conjugate 2 at concentrations of 500 nM (0.5 μM), the viability of BxPC-3 cells decreased from 71.78% to 52.35% and 39.03%, respectively.

The invention is illustrated by non-limiting embodiments.

EXAMPLES

Analytical methods

Thin-layer chromatography (TLC) was performed on aluminum foil plates coated with MERCK silica gel 60 F254. Column chromatography was performed using MERCK silica gel 60 (230-400) mesh, with hexane:ethyl acetate, dichloromethane:methanol, and chloroform:methanol as the eluent.

Abbreviations used:

AcOEt - ethyl acetate

HCA - hydroxycinnamic acid(s).

K2CO3 - potassium carbonate

NaOH - sodium hydroxide

KOH - potassium hydroxide

Nal - sodium iodide

I2 - iodine

DMF - N,N-dimethylformamide,

DMA - N,N-dimethylacetamide

DMSO - dimethyl sulfoxide

BDMP - 5-(1H-benzotriazol-1-yloxy)-3,4-dihydro-1-methyl-2H-pyrroliic hexachloroantimonate

BOMI - benzotriazol-1-yloxy-N,N-dimethylmethaniminium hexachloroantimonate

BOP - benzotriazolyl-N-oxytridimethylaminophosphonium hexafluorophosphate

CDI - carbonyl diimidazole

COMU - (1-cyano-2-ethoxy-2-oxoethylideneaminoxy)dimethylaminomorpholine carbenium hexafluorophosphate

DAST - (diethylamino)sulfur trifluoride

DCC - dicyclohexylcarbodiimide

DCMT - 2,4-dichloro-6-methoxy-1,3,5-triazine

DIC - diisopropylcarbodiimide

DIPEA - N,N-diisopropylethylamine

EDC - 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

HATU - O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

HBTU - O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

HCl - hydrogen chloride

HCTU - (2-(6-chlorobenzol-1-yl))-1,1,3,3-tetramethyluronium hexafluorophosphate

HOAt - 1-hydroxy-7-azabenzotriazole

HOBt - 1-hydroxy-1H-benzotriazole

PyBOP - benzotriazolyl-N-oxytripyrrolidinophosphonium hexafluorophosphate

SiO2 - silicon dioxide, silica gel, silica gel

TATU - O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate

TBTU - O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate

Example 1

Preparation of 9-[((2-allyoxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline

To a solution of (2-allyloxy)cinnamic acid (188 mg, 0.915 mmol, 1.2 eq.) in DMF (4 mL) was added TBTU (1.2 eq.) and DIPEA (3 eq.). Stirring was carried out at room temperature for 15 min. A solution of 5,11-dimethyl-5H-indolo[2,3-b]quinolin-9-ylamine (200 mg, 0.766 mmol, 1 eq.) in 2 mL of DMF was then added and stirring was continued for 24 h. The crude product was precipitated with water, filtered off, washed with water, and dried under reduced pressure. It was then purified by flash chromatography on silica gel eluting with methylene chloride/methanol (15:1 to 1:1). 9-[((2-allyloxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline was obtained in the form of an orange solid. Yield 234 mg (68%); m.p. 196-198°C; 1H NMR (500 MHz, DMSO) δ 10.30 (s, 1H, NH), 8.77 (d, 1H, J = 1.7 Hz), 8.30 (d, 1H, J = 7.4 Hz), 7.95 (d, 1H, J = 8.6 Hz), 7.93 (d, 1H, J = 15.8 Hz), 7.85 (t, 1H, J = 7.1 Hz), 7.70 (dd, 1H, J = 8.5 Hz, J = 1.9 Hz), 7.61 (dd, 1H, J = 7.6 Hz, J = 1.3 Hz), 7.54-7.50 (m, 2H), 7.35 (m, 1H), 7.10 (d, 1H, J = 8.3 Hz), 7.02 (t, 1H, J = 7.5 Hz), 6.90 (d, 1H, J = 15.8 Hz), 6.15-6.12 (m, 1H), 5.47 (m, 1H), 5.33 (m, 1H), 4.70-4.69 (m, 2H), 4.24 (s, 3H), 3.09 (s, 3H); HRMS (ES+) m/z: [M+H]+: calcd for C29H26N3O2: 448.2025, measured: 448.2025.

Example 2

Preparation of 9-[((2,4-dialyoxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline

To a solution of (2,4-diallyloxy)cinnamic acid (240 mg, 0.92 mmol, 1.2 eq.) in DMF (4 mL) was added TBTU (1.2 eq.) and DIPEA (3 eq.). Stirring was carried out at room temperature for 15 min. Then a solution of 5,11-dimethyl-5H-indolo[2,3-b]quinolin-9-ylamine (200 mg, 0.766 mmol, 1 eq.) in 2 mL DMF was added and stirring was continued for 24 h. The crude product was precipitated with water, filtered off, washed with water, and dried under reduced pressure. It was then purified by flash chromatography on silica gel eluting with methylene chloride/methanol (20:1 to 2:1). 9-[((2,4-diallyloxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2,3-b]quinoline was obtained as an orange solid. Yield 277 mg (72%); mp 220-222°C; ^1H NMR (500 MHz, DMSO) δ 10.17 (s, 1H, NH), 8.77 (brs, 1H), 8.34 (d, 1H, J = 8.0 Hz), 7.97 (d, 1H, J = 8.6 Hz), 7.87 (t, 1H, J = 7.6 Hz)), 7.81 (d, 1H, J = 15.7 Hz), 7.70 (d, 1H, J = 8.2 Hz), 7.60-7.52 (m, 3H), 6.77 (d, 1H, J = 15.7 Hz), 6.66-6.62 (m, 2H), 6.14-6.05 (m, 2H), 5.47-5.40 (m, 2H), 5.34-5.27 (m, 2H), 4.70-4.69 (m, 2H), 4.62-4.61 (m, 2H), 4.25 (s, 3H), 3.11 (s, 3H); HRMS (ES+) m/z: [M+H]+: calcd for C32H30N3O3: 504.2287, measured: 504.2286.

Example 3

Preparation of 9-[((3,4-diallyoxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline

To a solution of (3,4-diallyloxy)cinnamic acid (240 mg, 0.92 mmol, 1.2 eq.) in DMF (4 mL) was added TBTU (1.2 eq.) and DIPEA (3 eq.). Stirring was carried out at room temperature for 15 min. Then a solution of 5,11-dimethyl-5H-indolo[2,3-b]quinolin-9-ylamine (200 mg, 0.766 mmol, 1 eq.) in 2 mL DMF was added and stirring was continued for 24 h. The crude product was precipitated with water, filtered off, washed with water, and dried under reduced pressure. It was then purified by flash chromatography on silica gel eluting with methylene chloride/methanol (20:1 to 2:1). 9-[((3,4-diallyloxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2,3-b]quinoline was obtained as an orange solid. Yield 217 mg (75%); mp 213-215°C; ^1H NMR (500 MHz, DMSO) δ 10.16 (s, 1H, NH), 8.76 (d, 1H, J = 1.4 Hz), 8.33 (d, 1H, J = 7.7 Hz), 7.96 (d, 1H, J = 8.6 Hz), 7.86 (m, 1H ), 7.71 (dd, 1H, J = 8.5 Hz, J = 1.7 Hz), 7.54-7.51 (m, 3H), 7.24 (d, 1H, J = 1.4 Hz), 7.16 (dd, 1H, J = 8.3 Hz), J = 1.5 Hz), 7.04 (d, 1H, J = 8.5 Hz), 6.74 (d, 1H, J = 15.6 Hz), 6.09-6.06 (m, 2H), 5.47-5.43 (m, 2H), 5.30-5.26 (m, 2H), 4.65-4.64 (m, 2H), 4.62-4.61 (m, 2H), 4.26 (s, 3H), 3.12 (s, 3H); HRMS (ES+) m/z: [M+H]+: calculated for C32H30N3O3: 504.2287, measured: 504.2283.

Example 4

Preparation of 9-[((4-acetyloxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline

5,11-Dimethyl-5H-indolo[2,3-b]quinolin-9-yl-amine (50 mg, 0.19 mmol, 1 eq.) was dissolved in methylene chloride (4 mL) and dry pyridine (1 eq.) and cooled to -20°C. To the stirred solution in an ice-sodium chloride bath was added a solution of 4-acetyloxycinnamoyl chloride (43 mg, 0.19 mmol, 1 eq.) in methylene chloride (2 mL) portionwise over 15 minutes, maintaining the reaction temperature below -5°C. The reaction mixture was stirred at room temperature for 24 hours. SiO2 was added to the mixture and then evaporated to dryness. The crude product was applied to silica gel and purified by flash chromatography on SiO2 using chloroform/methanol (10:1 to 5:1).

9-[((4-acetyloxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2,3-b]quinoline as a dark yellow solid. Yield 30 mg (35%); mp 238-240°C (decomposition); ^1H NMR (500 MHz, DMSO) δ 10.30 (s, 1H, NH), 8.79 (s, 1H), 8.37 (d, 1H, J = 7.9 Hz), 8.02 (d, 1H, J = 8.4 Hz), 7.91 (t, 1H, J = 7.6 Hz), 7.74 (d, 1H, J = 8.3 Hz), 7.68 (d, 2H, J = 8.4 Hz), 7.61 (d, 1H, J = 15.7 Hz), 7.59-7.52 (m, 2H), 7.22 (d, 2H, J = 8.4 Hz), 6.85 (d, 1H, J = 15.7 Hz), 4.29 (s, 3H), 3.15 (s, 3H), 2.29 (s, 3H); HRMS (ES+) m/z: [M+H]+: calcd for C28H24N3O3: 450.1818, measured: 450.1810.

Example 5

Preparation of 9-[((3-acetyloxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline

5,1l-Dimethyl-5H-indolo[2,3-b]quinolin-9-ylamine (150 mg, 0.57 mmol, 1 eq.) was dissolved in methylene chloride (8 mL) and dry pyridine (1 eq.) and cooled to -20°C. To the stirred solution in an ice-sodium chloride bath was added portionwise a solution of (3-acetyloxy)cinnamoyl chloride (129 mg, 0.57 mmol, 1 eq.) in methylene chloride (2 mL) over 15 minutes, maintaining the reaction temperature below -5°C. The reaction mixture was stirred at room temperature for 24 hours. SiO2 was added to the mixture, and then the mixture was evaporated to dryness. The crude product was applied to silica gel and purified by flash chromatography on SiO2 with chloroform/methanol (10:1 to 5:1). 9-[((3-acetyloxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2,3-b]quinoline was obtained as a dark yellow solid. Yield 94 mg (36%); mp 235-237°C (dec.); ^1H NMR (500 MHz, DMSO) δ 10.56 (s, 1H, NH), 8.95 (brs, 1H), 8.60 (brs, 1H), 8.30 (brs, 1H), 8.09 (brs, 1H), 7.89 (m, 1H), 7.79 (brs, 1H), 7.70 (d, 1H, J = 8.3 Hz), 7.63 (d, 1H, J = 15.6 Hz), 7.55-7.49 (m, 2H), 7.41 (s, 1H), 7.20 (d, 1H, J = 7.3 Hz), 6.91 (d, 1H, J = 15.6 Hz), 4.41 (s, 3H), 3.13 (s, 3H), 2.31 (s, 3H); HRMS (ES+) m/z: [M+H]+: calcd for C28H24N3O3: 450.1818, measured: 450.1816.

Example 6

Preparation of 9-[((2,4-diacetyloxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline

5,11-Dimethyl-5H-indolo[2,3-b]quinolin-9-ylamine (150 mg, 0.57 mmol, 1 eq.) was dissolved in methylene chloride (8 mL) and dry pyridine (1 eq.) and cooled to -20°C. To the stirred solution in an ice-sodium chloride bath was added portionwise a solution of (2,4-diacetyloxy)cinnamoyl chloride (162 mg, 0.57 mmol, 1 eq.) in methylene chloride (2 mL) over 15 minutes, maintaining the reaction temperature below -5°C. The reaction mixture was stirred at room temperature for 24 hours. SiO2 was added to the mixture, and then evaporated to dryness. The crude product was applied to silica gel and purified by flash chromatography on SiO2 with chloroform/methanol (10:1 to 5:1). 9-[((2,4-diacetyloxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2,3-b]quinoline was obtained as a dark yellow solid. Yield 172 mg (58%); mp 245-247°C (dec.); ^1H NmR (500 MHz, DMSO) δ 10.53 (s, 1H, NH), 8.87 (s, 1H), 8.44 (d, 1H, J = 7.6 Hz), 8.11 (d, 1H, J = 7.3 Hz), 7.95 (t, 1H, J = 7.3 Hz)), 7.79-7.77 (m, 2H), 7.64-7.57 (m, 2H), 7.55 (d, 1H, J = 15.7 Hz), 7.19 (d, 1H, J = 8.3 Hz), 7.14 (brs, 1H), 6.95 (d, 1H, J = 15.7 Hz), 4.33 (s, 3H), 3.19 (s, 3H), 2.41 (s, 3H), 2.29 (s, 3H); HRMS (ES+) m/z: [M+H]+: calcd for C30H25N3O5: 508.1872, measured: 508.1868.

Example 7

Preparation of 9-[((3,4-diacetyloxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline

5,11-Dimethyl-5H-indolo[2,3-b]quinolin-9-ylamine (100 mg, 0.38 mmol, 1 eq.) was dissolved in methylene chloride (6 mL) and dry pyridine (1 eq.) and cooled to -20°C. To the stirred solution in an ice-sodium chloride bath was added portionwise a solution of (3,4-diacetyloxy)cinnamoyl chloride (106 mg, 0.38 mmol, 1 eq.) in methylene chloride (2 mL) over 15 minutes, maintaining the reaction temperature below -5°C. The reaction mixture was stirred at room temperature for 24 hours. SiO2 was added to the mixture, and then the mixture was evaporated to dryness. The crude product was applied to silica gel and purified by flash chromatography on SiO2 with chloroform/methanol (10:1 to 5:1). 9-[((3,4-diacetyloxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2,3-b]quinoline was obtained as a dark yellow solid. Yield 88 mg (45%); mp 273-275°C (dec.); ^1H NMR (500 MHz, DMSO) δ 10.63 (s, 1H, NH), 8.83 (s, 1H), 8.56 (d, 1H, J = 8.3 Hz), 8.29 (d, 1H, J = 8.7 Hz), 8.04 (t, 1H, J = 7.7 Hz), 7.78-7.75 (m, 2H), 7.58-7.51 (m, 4H), 7.36 (d, 1H, J = 8.2 Hz), 6.80 (d, 1H, J = 15.7 Hz), 4.44 (s, 3H), 3.24 (s, 3H), 2.33 (s, 3H), 2.31 (s, 3H); HRMS (ES+) m/z: [M+H]+: calcd for C30H25N3O5: 508.1872, measured: 508.1872.

Example 8

Preparation of 9-[((2-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline dihydrochloride

9-[((2-allyoxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2,3-b]quinoline (144 mg, 0.32 mmol), triethylsilane (51 μl, 0.32 mmol) and tetrakis(triphenylphosphine)palladium(0) (185 mg, 0.16 mmol) were dissolved in DMF (6 ml) in a dark flask protected from light. The mixture was stirred at room temperature for 5 hours. After completion of the reaction, the solvent was evaporated under reduced pressure on a rotary evaporator. The obtained oil was applied to silica gel and purified by chromatography on a SiO2 column using chloroform-methanol (20:1 to 2:1) as the eluent. 9-[((2-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline was obtained in the form of a red solid. Yield 64 mg (50%); mp 265-267°C (decomposition); ^1H NMR (500 MHz, DMSO) δ 10.22-10.21 (m, 2H, OH, NH),

8.79 (d, 1H, J = 1.4 Hz), 8.32 (d, 1H, J = 8.2 Hz), 7.96 (d, 1H, J = 8.5 Hz), 7.84 (m, 1H), 7.80 (d, 1H,

J = 15.8 Hz), 7.74 (dd, 1H, J = 8.5 Hz, J = 1.8 Hz), 7.53 (d, 1H, J = 8.5 Hz), 7.51 (m, 1H), 7.49 (m, 1H),

7.21 (m, 1H), 6.96 (d, 1H, J = 15.7 Hz), 6.94 (d, 1H, J = 7.7 Hz), 6.86 (t, 1H, J = 7.4 Hz), 4.26 (s, 3H),

3.12 (s, 3H); calcd for C26H22N3O2: 408.1712, measured: 408.1711. The obtained compound was converted into the dihydrochloride salt using a solution of 2M HCl gas in AcOEt.

Example 9

Preparation of 9-[((2,4-dihydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline dihydrochloride

9-[((2,4-diallyoxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2,3-b]quinoline (105 mg, 0.21 mmol), triethylsilane (34 μl, 0.21 mmol) and tetrakis(triphenylphosphine)palladium(0) (241 mg, 0.21 mmol) were dissolved in DMF (5 ml) in a dark flask protected from light. Stirred at room temperature for 1 hour. After completion of the reaction, the solvent was evaporated under reduced pressure on a rotary evaporator. The obtained oil was applied to silica gel and purified by chromatography on a SiO2 column using chloroform-methanol (20:1 to 2:1) as the eluent. 9-[((2,4-dihydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline was obtained in the form of a red solid. Yield 30 mg (34%); mp 258-260°C (decomposition); ^1H NMR (500 MHz, DMSO) δ 10.10 (m, 2H, OH, NH), 9.81 (brs, 1H, OH), 8.80 (d, 1H, J = 1.3 Hz), 8.34 (d, 1H, J = 7.5 Hz), 7.96 (d, 1H, J = 7.5 Hz), 7.85 (m, 1H),

7.74-7.73 (dd, 1H, J = 8.6 Hz, J = 1.7 Hz), 7.71 (d, 1H, J = 15.7 Hz), 7.54-7.50 (m, 2H), 7.30 (d, 1H,

J = 8.5 Hz), 6.76 (d, 1H, J = 15.7 Hz), 6.42 (d, 1H, J = 2.2 Hz), 6.31 (dd, 1H, J = 8.4 Hz, J = 2.2 Hz),

4.27 (s, 3H), 3.13 (s, 3H); calcd for C26H22N3O3: 424.1661, measured: 424.1660. The obtained compound was converted into the dihydrochloride salt using a solution of 2M HCl gas in AcOEt.

Example 10

Preparation of 9-[((4-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline dihydrochloride

9-[((4-acetyloxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline (20 mg, 0.044 mmol) was dissolved in DMF (1 mL). To the stirring solution was added K2CO3 (7.4 mg, 0.053 mmol). Stirring at room temperature was continued for 2 hours. The solvent was then evaporated and the residue was purified by column chromatography on silica gel using dichloromethane-methanol (20:1 to 1:1) as eluents. 9-[((4-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline was obtained as an orange solid. Yield 14 mg (78%); mp 240-242°C (dec.); ^1H NMR (500MHz, DMSO) 7.78-7.76 (m, 2H), 7.55 (d, 1H, J = 8.7 Hz), 7.44 (d, 2H, J = 8.3 Hz), 7.47 (d, 1H, J = 15.6 Hz), 6.86 (d, 2H, J = 8.3 Hz), 6.71 (d, 1H, J = 15.6 Hz), 4.47 (s, 3H), 3.22 (s, 3H); HRMS (ES+) m/z: [M+H]+: calcd for C26H22N3O2: 408.1712, found: 408.1715. The obtained compound was converted into the dihydrochloride salt using a solution of 2M HCl gas in AcOEt.

Example 11

Preparation of 9-[((3-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline dihydrochloride

9-[((3-acetyloxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline (60 mg, 0.133 mmol) was dissolved in DMF (3 mL). To the stirring solution was added K2CO3 (55 mg, 0.40 mmol). Stirring at room temperature was continued for 4 hours. The solvent was then evaporated and the residue was purified by column chromatography on silica gel using dichloromethane-methanol (10:1 to 1:1) as eluents. 9-[((3-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline was obtained as an orange solid. Yield 43 mg (84%); mp 263-265°C (dec.); ^1H NMR (500 MHz, DMSO) δ 10.75 (s, 1H, NH), 9.76 (s, 1H, OH), 8.88 (s, 1H), 8.57 (d, 1H, J = 8.2 Hz), 8.30 (d, 1H, J = 8.7 Hz), 8.06 (m, 1H), 7.82-7.79 (m, 2H), 7.60 (d, 1H, J = 8.6 Hz), 7.48 (d, 1H, J = 15.6 Hz), 7.24 (t, 1H, J = 8.0 Hz), 7.03-7.02 (m, 2H), 6.85-6.82 (m, 2H), 4.47 (s, 3H), 3.25 (s, 3H); HRMS (ES+) m/z: [M+H]+: calculated for C26H22N3O2:

408.1712, found: 408.1713. The obtained compound was converted into the dihydrochloride using a solution of 2M HCl gas in AcOEt.

Example 12

Preparation of 9-[((3,4-dihydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline dihydrochloride

9-[((3,4-Diacetyloxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline (60 mg, 0.118 mmol) was dissolved in DMF (3 mL). To the stirring solution was added K2CO3 (33 mg, 0.24 mmol). Stirring at room temperature was continued for 5 hours. The solvent was then evaporated and the residue was purified by column chromatography on silica gel using dichloromethane-methanol (10:1 to 1:1) as eluents. 9-[((3,4-dihydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline was obtained as an orange solid. Yield 40 mg (80%); mp 203-205°C (decomposition); ^1H NMR (500 MHz, DMSO) δ 10.38 (s, 1H, NH), 9.51 (brs, 1H, OH), 9.30 (brs, 1H, OH), 8.80 (s, 1H), 8.33 (d, 1H, J = 8.1 Hz), 7.97 (d, 1H, J = 8.6 Hz), 7.87 (m, 1H), 7.76 (d, 1H, J = 8.4 Hz), 7.54-7.51 (m, 2H, H-7), 7.42 (d, 1H, J = 15.5 Hz), 7.06 (s, 1H), 6.90 (d, 1H, J = 7.5 Hz), 6.81 (d, 1H, J = 8.0 Hz), 6.70 (d, 1H, J = 15.5 Hz), 4.27 (s, 3H ), 3.12 (s, 3H); HRMS (ES+) m/z: [M+H]+: calcd for C26H22N3O3: 424.1661, found: 424.1659. The obtained compound was converted into the dihydrochloride salt using a solution of 2M HCl gas in AcOEt.

Biological research

In vitro cytotoxic activity

Selected amide conjugates of hydroxycinnamic acid (HCA) derivatives and 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline, also known as N-(hydroxy)cinnamoyl-9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline derivatives, according to the invention, were tested for biological activity against selected cancer cell lines. The studies used human pancreatic cancer cells BxPC-3 (normal line) and AsPC-1 (metastatic line), as well as breast cancer cells (MCF-7) and cervical cancer cells (HeLa), as well as control healthy human fibroblasts (NHDF).

Cell culture

In vitro cell culture was performed under aseptic conditions. Cells were grown in an Innova CO-180 incubator (New Brunswick Scientific, Edison, NJ, USA) supplied with 5% CO2 at 37°C. Cell growth was monitored using a Nikon Eclipse microscope. Pancreatic cancer cell lines: BxPC-3 (from the primary tumor) and AsPC-1 (from ascites) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and an antibiotic-antimycotic drug mixture. HeLa and MCF7 cells were cultured in DMEM, and NHDF cells were cultured in alpha-MEM. Both cell culture media were supplemented with 10% FBS, 2 mM glutamine, and an antibiotic-antimycotic drug mixture.

Measurement of cytotoxicity of conjugates

Cytotoxicity of the compounds of the invention against tumor and control cells was measured using the MTT assay. All cells used belonged to adherent cell lines and were grown as monolayers in 96-well plates (5000 cells per well). Cells were seeded in the appropriate culture medium and incubated for 24 hours. Then, it was replaced with fresh medium supplemented with the test compounds at concentrations ranging from 125 to 4000 nM. As a solvent control, the medium was diluted with an equivalent volume of DMSO. Before dilution in medium, the compounds were dissolved in DMSO by sonication at 55°C. Incubation was continued for a further 72 hours. Then, the medium containing the test substances was removed, and MTT solution was added (50 μl per well of a 10-fold dilution of the stock solution at a concentration of 0.5 mg/ml in medium). After 3 hours of incubation, the MTT solution was replaced with DMSO (50 µL/well) to dissolve the purple formazan crystals. Absorbance was measured at 560 nm with a reference wavelength of 670 nm on an Asys UVM 340 microplate reader (Cambridge, UK). Results were expressed as the percentage of viable cells compared to the control (the viability of untreated cells was taken as 100%) using the formula:

Cell viability (%) = (AT/AC) x 100 where,

AT = Absorbance of treated cells,

AC = Absorbance of cells not treated with test compounds.

Based on the obtained results of the dependence of the concentrations of the tested compounds on the degree of cell survival, IC 50 values (inhibitory concentration 50 - concentration causing a 50% reduction in cell survival) were determined using the GraphPad Prism program.

PL 248245 Β1

It was observed that two of the designed and obtained compounds (1 and 2) exhibited higher or similar IC50 cytotoxic activity against both tested pancreatic cancer cell lines (BxPC-3 and AsPC-1) as 11-dimethyl-5H-indolo[2,3-b]quinoline (DiMIQ 6). It was confirmed that the cells responded in a dose-dependent manner to the used concentrations (250-4000 nM). The most interesting results of cell growth inhibition were observed for concentrations of 2000 nM and 4000 nM compared to the reference DiMIQ (6) (Fig. 1).

ICso values are shown in Table 1.

Table 1

In vitro cytotoxic activity of the new conjugates in the MTT test - ICso

Relationship ICso(nM) ICsu(nM) NHDF SI AsPC-1 BxPC-3 AsPC-1 BxPC-3 1 2641.54286.66 805.0463.63 2301.334157.85 0.87 2.86 2 336.5±85.01 or 377.854107.97 347.53452.39 1714.33±53.01 4.54 or 5.09 4.93 DiMIQ (6) 1622.5 ±131.98 888.77449.52 2332.334354.03 1.44 2.62

Conjugate 2 was found to have lower cytotoxicity towards healthy control cells compared to cancer cells (Fig. 2), which is reflected in the calculated selectivity indices (Table 1). The higher the SI value, the more selective the compound is and, theoretically, the safer it is as a potential anticancer drug.

Dose-dependent inhibition of cancer cell growth was also observed in the case of the hormone-dependent breast cancer line MCF-7 and the cervical cancer line HeLa (Fig. 3). Similarly to the case of pancreatic cancer cells, the best cytotoxic effect is observed for compounds 1 and 2. Furthermore, compound 3, less active against the BxPC- and AsPC-1 cell lines than DiMIQ (6), showed point-wise better activity than DIMIQ against HeLa cells at a concentration of 2000 nM and comparable activity at a higher concentration of 4000 nM. At a concentration of 2000 nM, conjugates 1, 2, 3 caused a better decrease in the viability of HeLa cancer cells than the effect induced by DiMIQ (6). In the case of MCF-7, only conjugates 2 and 3 showed significant activity in inhibiting cancer cell growth.

Measurement of the synergistic effect of DIMIQ (6) with methyl (4-hydroxy)cinnamate (Kw1) and DiMIQ (6) with methyl (2-hydroxy)cinnamate (Kw2) and the quasi-synergism of their conjugates

The pancreatic adenocarcinoma cell line BxPC-3 and the concentration of the compounds 500 nM were chosen as a representative example. In this study, cell growth inhibition induced by conjugates 1 and 2, DiMIQ (6), methyl (4-hydroxy)cinnamate (Kw1), methyl (2-hydroxy)cinnamate (Kw2), and both combinations of DiMIQ with Kw1 and Kw2 was measured. Kw1 and Kw2 derivatives were chosen as simple HCA derivatives included in conjugates 1 and 2 and previously tested against pancreatic cancer lines (Zaremba-Czogalla M. et al. Materials (Basel) 2020, 13(24), 5813). As shown, compounds Kw1 and Kw2 alone at a concentration of 500 nM did not significantly affect cancer cell viability (Fig. 4).

Administration of Kw1 or Kw2 derivatives together with highly cytotoxic DiMIQ (6) induced a reduction in the viability of BxPC-3 cancer cells without any additional effect, i.e. at the same level as for compound 6. A quasi-synergy effect was induced by the administration of conjugates 1 and 2, i.e. compounds with significant activity against BxPC-3, AsPC-1, MCF-7, and HeLa cancer cells. Their administration at a concentration of 500 nM resulted in a reduction in cancer cell viability to 52.35% and 39.03%, respectively. In contrast, for DiMIQ (6) alone or administered together with Kw1 or Kw2 compounds, it was 71.78%.

Claims (15)

Patent claims 1. Amide conjugates of hydroxycinnamic acids and 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline represented by formula (I), wherein the acyl group is derived from hydroxycinnamic acid, and pharmaceutically acceptable acid addition salts thereof. 2. Conjugates according to claim 1, characterized in that the acyl group is derived from the following acids: orthocoumaric, meta-coumaric, para-coumaric, 2,4-dihydroxycinnamic, caffeic, 3,4,5-trihydroxycinnamic. 3. Conjugates according to claim 1. 1, characterized in that it is 9-[((4-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline and pharmaceutically acceptable salts thereof. 4. Conjugates according to claim 1. The composition of claim 1, characterized in that it is 9-[((2-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline and pharmaceutically acceptable salts thereof. 5. Conjugates according to claim 1, characterized in that they are 9-[((3-hydroxy)cinnamoyl)amino]- -5,11-dimethyl-5H-indolo[2.3-b]quinoline and its pharmaceutically acceptable salts. 6. Conjugates according to claim 1. 1, characterized in that it is 9-[(2,4-dihydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline and pharmaceutically acceptable salts thereof. 7. Conjugates according to claim 1. 1, characterized in that it is 9-[(3,4-dihydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline and pharmaceutically acceptable salts thereof. 8. Conjugates according to claim 1. 1, characterized in that it is 9-[((2-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline dihydrochloride. 9. Conjugates according to claim 1. 1, characterized in that it is 9-[((3-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline dihydrochloride. 10. Conjugates according to claim 10. 1, characterized in that it is 9-[((4-hydroxy)cinnamoyl)amino]-5,11-dimethyl-5H-indolo[2.3-b]quinoline dihydrochloride. 11. A method for obtaining amide conjugates of hydroxycinnamic acids and 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline represented by formula (I), characterized in that (i) hydroxycinnamic acid is protected on the hydroxyl group/hydroxyl groups in the phenyl ring of the hydroxycinnamoyl fragment, (ii) the O-protected hydroxycinnamic acid derivative obtained in step (i) is optionally converted into an acid halide, preferably chloride, (iii) then 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline represented by formula (II) or a salt thereof, preferably hydrochloride, is subjected to a coupling reaction with the O-protected hydroxycinnamic acid derivative obtained in step (i) or with its acid halide obtained in step (ii), (iv) and then the O-protected amide conjugate of hydroxycinnamic acid and 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline obtained in step (iii) is subjected to purification and the functional groups from the hydroxycinnamoyl fragment are deprotected, (v) and optionally the conjugate with free hydroxyl groups represented by formula (I) thus obtained in step (iv) is converted into a pharmaceutically acceptable acid addition salt. 12. The method according to claim 11, characterized in that the coupling reaction in step (iii) is carried out in the presence of a coupling agent selected from the group: dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate. 13. The method according to claim 11, wherein the coupling reaction in step (iii) is carried out using hydroxycinnamic acid chloride in the presence of pyridine. 14. Conjugates of hydroxycinnamic acids and 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline represented by formula (I) or pharmaceutically acceptable acid addition salts thereof for use in the treatment of human neoplastic disease, especially pancreatic cancer, breast cancer, cervical cancer, lymphomas, sarcomas and adenomas. 15. A pharmaceutical composition comprising an active substance and a pharmaceutically acceptable and compatible carrier and/or excipients, characterized in that the active substance is the amide conjugate of hydroxycinnamic acid and 9-amino-5,11-dimethyl-5H-indolo[2,3-b]quinoline as defined in claim 1 or a pharmaceutically acceptable acid addition salt thereof.