WO2025186751A1 - A quinoline based aniline nitrogen mustard conjugates as anti-cancer agent - Google Patents

Abstract

The present invention is about a quinoline based aniline nitrogen mustard conjugates as anti-cancer agent. The present invention particularly relates to quinoline based aniline nitrogen mustard conjugates with anti-cancer activity and process for the preparation thereof. Formula (1)

Description

A QUINOLINE BASED ANILINE NITROGEN MUSTARD CONJUGATES AS ANTI-CANCER AGENT FIELD OF THE INVENTION The present invention is about a quinoline based aniline nitrogen mustard conjugates as anti-cancer agent. The present invention particularly relates to quinoline based aniline nitrogen mustard conjugates with anti-cancer activity and process for the preparation thereof. BACKGROUND OF THE INVENTION Cancer refers to any one of a large number of diseases characterized by the development of abnormal cells that divide uncontrollably and have the ability to infiltrate and destroy normal body tissue. Cancer often has the ability to spread throughout the body. There are many anti-cancer drugs are available in market. However, they are non-selective, usually damage healthy cells and tissues with rapid turnover causing severe toxic effects. Among the many possible strategies for improving the therapeutic effectiveness of anti-cancer drugs, one of the most important is the synthesis of new derivatives with modified structure. Such modifications appear to be a promising way for improvement of biological properties in comparison with those of the parent anticancer compounds, because such modifications exhibited lower toxicity and were found to be highly cytotoxic to several neoplastic cell lines with multidrug resistance. Quinoline is a heterocyclic aromatic organic compound having molecular formula C9H7N, characterized by a double-ring structure that contains a benzene ring fused to pyridine at two adjacent carbon atoms. Quinoline is also known as, benzopyridine, benzo[b]pyridine, 1-benzazine and benzazine. It is a hygroscopic, yellowish oily liquid, slightly soluble in water, soluble in alcohol, ether and many other organic solvents. Quinoline and its derivatives possess many types of biological activities and have been reported to show significant anticancer activity. Quinoline compounds play an important role in anticancer drug development as they have shown excellent results through different mechanism of action such as growth inhibitors by cell cycle arrest, apoptosis, inhibition of angiogenesis, disruption of cell migration and modulation. DNA-directed alkylating agents (such as N- mustard) play an important role in the development of anticancer drugs. DNA-alkylating agents were the first chemotherapeutic drugs developed for the treatment of cancer. Although the classic, highly reactive DNA-alkylating agents (e.g., melphalan, chlorambucil, and platinum-based agents) are potent chemotherapeutic agents, they have limited clinical benefits mainly because of their toxicity, which results in severe adverse side effects. The toxicity associated with DNA-alkylating agents highlights the need to develop anticancer agents with greater selectivity. To overcome the drawbacks of alkylating agents, there is need to synthesize a DNA alkylating agents containing quinoline based aniline nitrogen mustard by coupling various DNA- affinic carriers (a DNA-intercalating agent or a DNA minor groove binder) that directly target DNA with the N-mustard pharmacophore. These DNA-directed alkylating agent is increased chemical stability and improved anticancer activity. The conjugation of quinoline N mustard with targeting molecules enables targeted delivery to cancer cell. This targeted approach enhance drug accumulation at the tumor site, thereby maximizing therapeutic efficacy and decreased systemic toxicity. Aniline nitrogen mustard, known for their alkylating activity against DNA, are a well-established class of anti-cancer drugs. The introduction of aniline nitrogen mustard functionality into quinoline derivatives is a potent DNA alkylating agent to enhancing the compound’s ability to induce cell death in cancer cells. Therefore, the development of Quinoline-N mustard conjugates as anticancer agents represents a promising strategy for improving cancer treatment outcome through enhanced selectivity, efficacy and reduced side effect. Furthermore, the ability to modify the chemical structure of quinoline N mustard conjugate allow for fine-tuning of their pharmacokinetic properties such as stability, solubility, and bioavailability to optimize their therapeutic potential. Therefore, inventors of the present invention have surprisingly developed a quinoline based aniline nitrogen mustard conjugates to provide synergistic potentials of quinoline and aniline nitrogen mustard chemistry to deliver innovative therapeutic agents with improved efficacy, selectivity and safety profiles for the treatment of cancer. OBJECT OF THE INVENTION The main object of the present invention is to provide a quinoline based aniline nitrogen mustard conjugates as anti-cancer agent. Another object of the present invention is to provide a quinoline based aniline nitrogen mustard conjugates as anti-cancer agent to improve efficacy, selectivity and safety of compound. Another object of the present invention is to provide a quinoline based aniline nitrogen mustard conjugates as anti-cancer agent to decrease toxicity, side effect and adverse effect. Another object of the present invention is to provide a quinoline based aniline nitrogen mustard conjugates as anti-cancer agent to enhance drug accumulation at the tumor site that leads to increase therapeutic efficacy. Another object of the present invention is to provide a quinoline based aniline nitrogen mustard conjugates as anti-cancer agent which increases chemical stability and bioavailability of compound. SUMMARY OF THE INVENTION The main aspect of the present invention is to provide a quinoline based aniline nitrogen mustard conjugates as anti-cancer agent. Another aspect of the present invention is to provide a quinoline based aniline nitrogen mustard conjugates as anti-cancer agent with the formula (1) Wherein, R is selected from -H, 4-OMe, 5-F, 5-Cl, 5-Br, 5-OMe, 5-NO2, 6-Cl, 6- Br, 7-Br. R1 is selected from H, 4-CH3, 3-F, 4-F, 3-Br, 4-Br, 3,4-di OMe, 2,4-di Cl, 4-OMe, 4-Cl, 3-Cl. Another aspect of the present invention is related to formula (1) is N-(4-(bis (2- chloroethyl) amino) phenyl)-2-aryllquinoline-4- carboxamide. Another aspect of the invention is to provide a quinoline based aniline nitrogen mustard conjugates as anti-cancer agent and process of preparation thereof BRIEF DESCRIPTION OF FIGURES Figure 1: ¹H NMR spectrum of compound c Figure 2: Mass spectrum of HKQM-2c Figure 3: 1H NMR spectrum of HKQM-2c Figure 4: 13C NMR spectrum of HKQM-2c Figure 5: Mass spectrum of HKQM-2e Figure 6: 1H NMR spectrum of HKQM-2e Figure 7: 13C NMR spectrum of HKQM-2e Figure 8: Mass spectrum of HKQM-2k Figure 9: 1H NMR spectrum of HKQM-2k Figure 10: 13 C NMR spectrum of HKQM-2k Figure 11: Mass spectrum of HKQM-2n Figure 12: 1H NMR spectrum of HKQM-2n Figure 13: 13C NMR spectrum of HKQM-2n Figure 14: Peptide coupling mechanism by T3P Figure 15: Growth control at different concentration DETAILED DESCRIPTION OF INVENTION The present invention overcomes the aforesaid drawbacks of the above, and other objects, features and advantages of the present invention will now be described in greater detail. Also, the following description includes various specific details and is to be regarded as merely exemplary. Accordingly, those of ordinary skill in the art will recognize that without departing from the scope and spirit of the present disclosure and its various embodiments there may be any number of changes and modifications described herein. As defined herein, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any process and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. As used herein, whether in a transitional phase or in the body of a claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least”. When used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a composition, the term “comprising” means that the process includes at least the recited features or components, but may also include additional features or components. As used herein, the singular forms “a,” “an” and “the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. The term "alkoxy" as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The term “methyl group” refer is an alkyl derived from methane, containing one carbon atom bonded to three hydrogen atoms, having chemical formula CH3. The term "halogen" refers to an atom of fluorine, chlorine, bromine, or iodine. The term “Lethal concentration 50 (LC50)” defined as the concentration of a given agent which is lethal to 50% of the cells. The term “tumor growth inhibition (TGI)” refer as medical term pertaining to cancer therapy and the specific reduction in growth of tumors and oncogene cells by a chemical compound, mechanical therapy (e.g. electroporation) or any other methods. The term “Growth inhibiting concentration 50 (GI50)” defined as inhibits the growth of cells by 50%. The main embodiment of the present invention is to provide a quinoline based aniline nitrogen mustard conjugates as anti-cancer agent. Another main embodiment of the present invention is to provide a quinoline based aniline nitrogen mustard conjugates and derivatives thereof with the formula (1); Wherein, R is selected from -H, 4-OMe, 5-F, 5-Cl, 5-Br, 5-OMe, 5-NO2, 6- Cl, 6- Br, 7-Br. R1 is selected from H, 4-CH3, 3-F, 4-F, 3-Br, 4-Br, 3, 4-di OMe, 2,4-di Cl, 4-OMe, 4-Cl, 3-Cl. The general synthetic route for the preparation of compound having formula (I) involves following general steps wherein the scheme is exemplified with the different R substitutions. Reaction scheme: Step: 1

Synthesis of N, N-bis (2-chloroethyl) benzene-1,4-diamine hydrochloride Synthesis of N- (4- (bis (2-chloroethyl) amino) phenyl) -2-aryllquinoline-4- carboxamide General Procedure for synthesis of 2, 2'-((4-nitrophenyl) azanediyl) diethanol (compound a). a) Weighing 1-fluoro-4-nitrobenzene and N,N-bisethanolamine and mixing; b) Heating the solution of step (a) at 800C for 5-6 hour; c) Cooling the solution of step (b) and adding water in it; d) Filtering the step (c) solution and washing it with water; e) Drying the step (d) to get yellow crystalline solid of 2,2'-((4- nitrophenyl)azanediyl)diethanol; f) Storing step (e) for further use. General Procedure for the synthesis of N,N-bis(2-chloroethyl)-4-nitroaniline (Compound b). a) Adding 2,2'-((4-nitrophenyl)azanediyl)diethanol (compound a) into DMF; b) Adding thionyl chloride into step (a) to chlorination of both hydroxyl groups in compound (a); c) Heating the solution of step (b) at 70⁰C for 4 hour; d) Pouring solution of step (c) into ice; e) Filtering the solid from step (d) and washing it with water; f) Drying the solid of step (e) to get N,N-bis(2-chloroethyl)-4-nitroaniline General Procedure for the synthesis of N,N-bis(2-chloroethyl)benzene-1,4- diamine hydrochloride (compound c) a) Adding N,N-bis(2-chloroethyl)-4-nitroaniline, tin metal into conc. HCl; b) Refluxing the mixture of step (a) for 6 hour till the suspension get clear which indicate the completion of reaction; c) Filtering the suspension of step (b) and basified with NaOH solution by keeping temperature below 10⁰C; d) Extracting the suspension of step (c) with ethylacetate; e) Drying step (d) on sodium Sulphate and acidifying with ethylacetateHCl to precipitate hydrochloride salt; f) Filtering HCl salt of step (e) and washing with ethyacetate to get N,N- bis(2-chloroethyl)benzene-1,4-diamine hydrochloride General procedure for the synthesis of 2-arylquinoline-4-carboxylic acid (compound 1) a) Adding isatin into methanolic NaOH solution in ice bath; b) Adding acetophenone into step (a) mixture; c) Refluxing the mixture of step (b) for 5-6 hour till completion of reaction and pouring into ice-water; d) Filtering the mixture of step (c) and acidifying with con. HCl to get precipitated solid; e) Filtering the precipitated solid of step (d) and washing with water; f) Drying the solid of step (e) and storing for further use. General procedure for the synthesis of N-(4-(bis(2-chloroethyl)amino)phenyl)- 2-aryllquinoline-4- carboxamide (HKQM-2a-o). a) Adding compound 1 and compound c in ethylacetate and mixing; b) Adding T₃P dropwise into step (a) at 10⁰C temperature; c) Adding immediately diisopropylethylamine dropwise into step (b) and stirring for 30 minute; d) Monitoring reaction mixture of step (c) by using TLC(Methanol:MDC 0.5:9.5 ml) till the reaction complete; e) Pouring reaction mixture of step (d) into water and extracting with ethylacetate to get extracted organic layer; f) Drying extracted organic layer of step (e) over sodium Sulphate to get N- (4-(bis(2-chloroethyl)amino)phenyl)-2-aryllquinoline-4- carboxamide; g) Crystalizing the compound of step (f) to get pure compound; As per one embodiment of the present invention, formula (1) is prepared from N, N-bis(2-chloroethyl)benzene-1,4-diamine hydrochloride. As per one embodiment of the present invention, formula (1) is prepared by using peptide coupling mechanism by T3P (propylphosphonic anhydride). The synthesized compounds have shown the potential anti-cancer activities. The process of preparation is simple and provides product with improved yield and purity. Thus, the compounds of Formula (I) may be readily administered, to treat cancer and related conditions in need of such treatment. For this purpose, the compound of Formula (I) may be administered by conventional routes including, but not limited to, oral, sublingual, nasal, buccal, parenteral, transdermal or topical. The invention is further illustrated by the following examples which are provided to be exemplary of the invention and do not limit the scope of the invention. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention. EXAMPLES EXAMPLE 1: Quantity of Ingredient to synthesize N-(4-(bis(2- chloroethyl)amino)phenyl)-2-aryllquinoline-4- carboxamide Ingredient Quantity 1-fluoro-4-nitrobenzene 300 mmol N,N-bisethanolamine 350 mmol 2,2'-((4- 200 mmol nitrophenyl)azanediyl)diethanol DMF (Dimethylformamide) 50 ml Thionyl chloride 250 mmol N,N-bis(2-chloroethyl)-4-nitroaniline 50 mmol Conc. HCl 50 ml Tin metal 200 mmol Ethylacetate 250 mmol N,N-bis(2-chloroethyl)benzene-1,4- 2 mmol diamine hydrochloride Isatin 50 mmol Methanolic NaOH solution 250 mmol NaOH in 50 ml methanol Acetophenone 50 mmol 2-arylquinoline-4-carboxylic acid 2 mmol T3P 2.2 mmol Diisopropylethylamine 4.4 mmol Table 1: Quantity of Ingredient to synthesize N-(4-(bis(2- chloroethyl)amino)phenyl)-2-aryllquinoline-4- carboxamide Example 2: Synthesis of Substituted Quinoline based Aniline Nitrogen mustard Derivatives The Fifteen analogue of Quinoline based Aniline Nitrogen mustard Derivatives were synthesized by substituting of R and R1 in formula (1). The R and R1 was Formula (1) Time Melting Derivatives R R1 min Range (⁰C) HKQM-2a -H -4-CH₃ 15 210-212 HKQM-2b -H -4-F 20 216-218 HKQM-2c -H -3,4-di OMe 20 198-202 HKQM-2d -H 2,4-Di Cl 30 186-188 HKQM-2e -H -4-OMe 15 198-200 HKQM-2f -H -4-Cl 20 162-164 HKQM-2g -H -3-Cl 20 120-122 HKQM-2h -H -4-Br 25 168-170 HKQM-2i -H -3-F 20 172-174 HKQM-2j -6-Cl -CH₃ 15 174-176 HKQM-2k -6-Cl -H 15 210-212 HKQM-2l -6-Cl -4-OMe 15 188-190 HKQM-2m -6-Cl -4-Cl 20 164-166 HKQM-2n -6-Cl -4-Br 25 200-202 HKQM-2o -6-Cl -3,4-Di OMe 30 166-168 Table 2: Synthesis of Substituted Quinoline based Aniline Nitrogen mustard Derivatives The fifteen analogue was synthesized by substituting R and R1 in formula (1). The fifteen analogue structure and IUPAC name were mentioned in below table. HKQM-2a HKQM-2b N-(4-(bis(2- N-(4-(bis(2- chloroethyl)amino)phenyl)-2-(p- chloroethyl)amino)phenyl)-2-(4- tolyl)quinoline-4-carboxamide fluorophenyl)quinoline-4- carboxamide HKQM-2c HKQM-2d N-(4-(bis(2- N-(4-(bis(2- chloroethyl)amino)phenyl)-2-(3,4- chloroethyl)amino)phenyl)-2-(2,4- dimethoxyphenyl)quinoline-4- dichlorophenyl)quinoline-4- carboxamide carboxamide HKQM-2e HKQM-2f N-(4-(bis(2- N-(4-(bis(2- chloroethyl)amino)phenyl)-2-(4- chloroethyl)amino)phenyl)-2-(4- methoxyphenyl)quinoline-4- chlorophenyl)quinoline-4- carboxamide carboxamide HKQM-2g HKQM-2h N-(4-(bis(2- N-(4-(bis(2- chloroethyl)amino)phenyl)-2-(3- chloroethyl)amino)phenyl)-2-(4- chlorophenyl)quinoline-4- bromophenyl)quinoline-4- carboxamide carboxamide HKQM-2i HKQM-2j N-(4-(bis(2- N-(4-(bis(2- chloroethyl)amino)phenyl)-2-(3- chloroethyl)amino)phenyl)-6- fluorophenyl)quinoline-4- chloro-2-(p-tolyl)quinoline-4- carboxamide carboxamide HKQM-2k HKQM-2l N-(4-(bis(2- N-(4-(bis(2- chloroethyl)amino)phenyl)-6-chloro- chloroethyl)amino)phenyl)-6- 2-phenylquinoline-4-carboxamide chloro-2-(4- methoxyphenyl)quinoline-4- carboxamide HKQM-2m HKQM-2n N-(4-(bis(2- N-(4-(bis(2- chloroethyl)amino)phenyl)-6-chloro- chloroethyl)amino)phenyl)-2-(4- 2-(4-chlorophenyl)quinoline-4- bromophenyl)-6-chloroquinoline- carboxamide 4-carboxamide HKQM-2o N-(4-(bis(2- chloroethyl)amino)phenyl)-6-chloro- 2-(3,4-dimethoxyphenyl)quinoline-4- carboxamide Table 3: Fifteen analogue structure and IUPAC name EXAMPLE 3: Spectroscopy Evaluation All synthesized compounds are characterized by Mass, ¹H NMR and ¹³C NMR spectroscopy. 1H (400 MHz), 13C (100 MHz) NMR spectra were recorded on a Bruker AVANCE II spectrometer in CDCl3 and DMSO. Chemical shifts are expressed in δ ppm downfield from TMS as an internal standard. Mass spectra were determined using direct inlet probe on a GCMS-QP 2010 mass spectrometer (Shimadzu). N,N-bis(2-chloroethyl)benzene-1,4-diamine hydrochloride( compound c) Cream colour solid: mp 212-214⁰C; ¹H NMR (DMSO-d6) (δ ppm): 3.73(8H, s, 4(CH₂)), 6.82-6.84 (d,2H, Ar-H, J = 9.04 Hz), 7.20-7.22(d,2H,Ar-H,J = 8.96 Hz), 10.07 (s, 2H, exchangeable, -NH2). N-(4-(bis(2-chloroethyl)amino)phenyl)-2-(p-tolyl)quinoline-4 carboxamide (HKQM-2a):Light yellow solid; Melting range:210-212°C; R_f:0.34 (Methanol:MDC 0.5:9.5 ml); IR (KBr):3229, 2956, 1661, 1613, 1509, 1451, 1417, 1354, 1282, 1251, 1180, 1143, 1064, 1014, 820, 766, 738, 711 cm^-1; ¹H NMR(δ ppm): 2.34(s, 3H, -CH₃), 3.57-3.61(t, 4H,2(-CH₂-)), 3.67-3.70(t, 4H,2(-CH₂-)), 6.64-6.66(d, 2H, Ar-H, J=8.8Hz), 7.20-7.18(d, 2H, Ar-H, J=6.8Hz), 7.35-7.39(t, 1H, Ar-H, J=7.6Hz), 7.57-7.55(d, 2H, Ar-H, J=9.2Hz), 7.64-7.60(t, 1H, Ar-H, J=6.8Hz), 7.71(s, 1H, Ar-H), 7.87-7.85(d, 2H, Ar-H, J=8Hz), 7.99-7.97(d, 1H, Ar- H, J=8.4), 8.05-8.03(d, 1H, Ar-H, J=8.4), 8.14 21.39, 40.59, 53.65, 112.52, 116.20, 122.51, 123.06, 124.99, 127.20, 127.28, 128.29, 129.62, 130.23, 135.64, 140.10, 142.82, 143.71, 146.53, 156.64, 165.47;MS (m/z): 477(M⁺); Anal. Calcd for:C₂₇H₂₅Cl₂N₃O; C, 67.78; H, 5.27; N, 8.78; Found:C, 67.72; H, 5.34; N, 8.83. N-(4-(bis(2-chloroethyl)amino)phenyl)-2-(4-fluorophenyl)quinoline-4- carboxamide (HKQM-2b): Light yellow solid; Melting range:216-218°C; Rf:0.35(Methanol:MDC 0.5:9.5 ml); IR (KBr):3215, 3045, 2986, 1664, 1587, 1525, 1450, 1421, 1353, 1314, 1253, 1176, 1030, 836, 814, 799, 776, 760, 740 cm^-1; ¹H NMR(δ ppm): 3.53-3.54(t, 4H,2(-CH2-)), 3.59-3.61(t, 4H,2(-CH2-)), 6.48-6.50(d, 2H, Ar-H, J=8.8Hz), 7.15-7.18(m, 1H, Ar-H), 7.25-7.31(m, 3H, Ar-H), 7.42- 7.44(m, 3H, Ar-H), 7.47-7.51(m, 1H, Ar-H), 7.71-7.73(d, 1H, Ar-H, J=8), 7.76- 7.78(d, 2H, Ar-H, J=6.8Hz), 7.88-7.90(d, 1H, Ar-H, J=8.4), 8.77(s, 1H, -NH); ¹³C NMR(δppm): 40.55, 53.59, 112.32, 116.18, 122.48, 123.04, 125.01, 127.25, 127.31, 128.34, 128.85, 129.35, 129.87, 130.21, 138.10, 142.68, 143.55, 148.17, 156.29, 165.44; MS (m/z): 481(M⁺); Anal. Calcd for:C₂₆H₂₂Cl₂FN₃O; C, 64.74; H, 4.60; N, 8.71; Found:C, 64.70; H, 4.56; N, 8.68. N-(4-(bis(2-chloroethyl)amino)phenyl)-2-(3,4-dimethoxyphenyl)quinoline-4- carboxamide (HKQM-2c): Light yellow solid; Melting range:198-202°C; Rf:0.36(Methanol:MDC 0.5:9.5 ml); IR (KBr):3214, 3076, 2960, 1663, 1588, 1517, 1456, 1421, 1349, 1316, 1260, 1176, 1145,1025, 872, 809, 768 cm^-1; ¹H NMR(δ ppm): 3.58-3.61(t, 4H,2(-CH₂-)), 3.68-3.72(t, 4H,2(-CH₂-)), 3.82(s, 3H, -OCH₃), 3.87(s, 3H, -OCH3) 6.67-6.69(d, 2H, Ar-H, J=9.2Hz), 6.79-6.82(s, 1H, Ar-H, J=8.4), 7.38-7.44(m, 2H, Ar-H), 7.60-7.72(m, 5H, Ar-H), 8.01-8.03(d, 1H, Ar-H, J=8.4Hz), 8.06-8.08(d, 1H, Ar-H, J=8.4Hz), 8.20 41.20, 52.25, 55.56, 110.32, 111.66, 112.09, 116.33, 120.41, 121.74, 123.05, 125.13, 126.75, 129.11, 129.38, 130.06, 130.80, 143.13, 143.21, 148.83, 149.03, 150.60, 155.52, 164.69 MS (m/z): 523(M⁺); Anal. Calcd for:C₂₈H₂₇Cl₂N₃O₃; C, 64.13; H, 5.19; N, 8.01; Found:C, 64.08; H, 5.25; N, 7.95. N-(4-(bis(2-chloroethyl)amino)phenyl)-2-(2,4-dichlorophenyl)quinoline-4- carboxamide (HKQM-2d): Light yelllow solid; Melting range:186-188°C; Rf:0.38 (Methanol:MDC 0.5:9.5 ml); IR (KBr):3230, 3092, 2960, 1649,1590, 1546, 1513, 1478, 1351, 1244, 1181, 1137, 1040, 921, 816, 756 cm^-1; ¹H NMR(δ ppm): 3.54-3.57(t, 4H,2(-CH₂-)), 3.64-3.67(t, 4H,2(-CH₂-)), 6.57-6.60(d, 2H, Ar-H, J=7.6Hz), 7.26-7.28(dd, 2H, Ar-H, J=2, 2Hz), 7.40-7.43(m, 3H, Ar-H), 7.49- 7.54(m, 2H, Ar-H), 7.67-7.71(m, 1H, Ar-H), 7.75(s, 1H, Ar-H), 7.93(s, 1H, -NH), 8.07-8.09(d, 2H, Ar-H, J=8.4Hz), 8.16-8.18 40.45, 53.58, 112.35, 119.91, 122.58, 123.57, 125.11, 127.70, 127.85, 128.17, 129.96, 130.02, 130.51, 132.56, 132.97, 135.64, 137.17, 141.85, 143.77, 148.59, 155.72, 165.03;MS (m/z):531 (M⁺); Anal. Calcd for:C₂₆H₂₁Cl₄N₃O; C, 58.56; H, 3.97; N, 7.88; Found:C, 58.32; H, 4.06; N, 7.94. N-(4-(bis(2-chloroethyl)amino)phenyl)-2-(4-methoxyphenyl)quinoline-4- carboxamide (HKQM-2e): Light yelllow solid; Melting range:198-200°C; Rf:0.36 (Methanol:MDC 0.5:9.5 ml); IR (KBr):3229, 3044, 2962, 2883, 1662,1586, 1516, 1455, 1421, 1354, 1284, 1254, 1175, 1145, 1030, 837, 760 cm^-1;¹H NMR(δ ppm): 3.57-3.60(t, 4H,2(-CH₂-)), 3.66-3.69(t, 4H,2(-CH₂-)), 3.77(s, 3H, -OCH₃), 6.62-6.64(d, 2H, Ar-H, J=9.2Hz), 6.83-6.85(d, 2H, Ar-H, J=9.6Hz), 7.26-7.30(m, 1H, Ar-H), 7.55-7.58(m, 4H, Ar-H), 7.83-7.88(m, 3H, Ar-H), 7.95-7.97(d, 1H, Ar- H, J=8.4Hz), 8.40(s, 1H, -NH); ¹³C NMR(δppm): 40.52, 53.65, 55.42, 112.49, 114.24, 115.84, 115.97, 122.48, 125.00, 126.94, 128.43, 128.76, 129.26, 130.22, 130.76, 142.76, 143.65, 148.32, 156.07, 161.16, 165.49; MS (m/z):493 (M⁺); Anal. Calcd for:C27H25Cl2N3O2; C, 65.59; H, 5.10; N, 8.50; Found:C, 65.62; H, 5.15; N, 8.46. N-(4-(bis(2-chloroethyl)amino)phenyl)-2-(4-chlorophenyl)quinoline-4- carboxamide (HKQM-2f): Light yelllow solid; Melting range:162-164°C; R_f:0.32 (Methanol:MDC 0.5:9.5 ml); IR (KBr):3308, 3056, 2960, 2892, 1644, 1588, 1519, 1413, 1351, 1239, 1177, 1089, 1012, 927, 836, 813 cm^-1; ¹H NMR(δ ppm): 3.75(m, 8H, 4(-CH2-)), 6.81-6.83(d, 2H, Ar-H, J=9.2Hz), 7.63-7.69(m, 5H, Ar-H), 7.83- 7.87(t, 1H, Ar-H), 8.16-8.22(m, 2H, Ar-H), 8.33(s, 1H, -NH-), 8.40-8.42(d, 2H, Ar- H, J=8.8Hz); ¹³C NMR(δppm): 52.25, 59.73, 112.07, 116.57, 121.81, 123.43, 125.23, 127.44, 128.93, 129.58, 130.33, 134.87, 136.95, 143.19, 143.44, 147.86, 154.53, 164.45; MS (m/z):497(M⁺); Anal. Calcd for:C26H22Cl3N3O; C, 62.60; H, 4.45;; N, 8.42;Found:C, 62.57; H, 4.52; N, 8.34. N-(4-(bis(2-chloroethyl)amino)phenyl)-2-(3-chlorophenyl)quinoline-4- carboxamide (HKQM-2g): Light yelllow solid; Melting range:120-122°C; Rf:0.35(Methanol:MDC 0.5:9.5 ml); IR (KBr):3304, 3060, 2956, 2878, 1642, 1589, 1525, 1244, 1180, 1024, 918, 813, 769, 694 cm^-1; MS (m/z):497 (M⁺); Anal. Calcd for:C₂₆H₂₂Cl₃N₃O; C, 62.60; H, 4.45; N, 8.42; Found:C, 62.54; H, 4.52; N, 8.37. N-(4-(bis(2-chloroethyl)amino)phenyl)-2-(4-bromophenyl)quinoline-4- carboxamide (HKQM-2h): Light yelllow solid; Melting range:168-170°C; R_f:0.34(Methanol:MDC 0.5:9.5 ml); IR (KBr):3308, 3050, 2964, 1641, 1586, 1516, 1411, 1348, 1238, 1181, 1072, 1007, 834, 810, 759 cm^-1; MS (m/z):541 (M⁺); Anal. Calcd for:C26H22BrCl2N3O; C, 57.48; H, 4.08; N, 7.73; Found:C, 57.53; H, 3.92; N, 7.66. N-(4-(bis(2-chloroethyl)amino)phenyl)-2-(3-fluorophenyl)quinoline-4- carboxamide (HKQM-2i): Light yelllow solid; Melting range:172-174°C; R_f:0.32(Methanol:MDC 0.5:9.5 ml); IR (KBr): 3252, 3046, 2913, 1688, 1546, 1508, 1453, 1436, 1366, 1275, 1180, 1008, 856, 805, 754, 718 cm^-1; MS (m/z): 481(M⁺); Anal. Calcd for:C26H22Cl2FN3O; C, 64.74; H, 4.60; N, 8.71; Found:C, 64.70; H, 4.68; N, 8.66. N-(4-(bis(2-chloroethyl)amino)phenyl)-6-chloro-2-(p-tolyl)quinoline-4- carboxamide (HKQM-2j): Light yelllow solid; Melting range:174-176°C; R_f:0.35(Methanol:MDC 0.5:9.5 ml); IR (KBr):3279, 3037, 2963, 1740, 1676, 1589, 1517, 1450, 1349, 1252, 1176, 1017, 885, 814, 750 cm^-1; MS (m/z):511 (M⁺); Anal. Calcd for:C27H24Cl3N3O; C, 63.23; H, 4.72; N, 8.19; Found:C, 63.18; H, 4.68; N, 8.25. N-(4-(bis(2-chloroethyl)amino)phenyl)-6-chloro-2-phenylquinoline-4- carboxamide (HKQM-2k): Light yelllow solid; Melting range:210-212°C; R_f:0.33(Methanol:MDC 0.5:9.5 ml); IR (KBr):3309, 3058, 2963, 2919, 1647, 1587, 1515, 1437, 1347, 1245, 1175, 1083, 1021, 923, 884, 813, 755, 690 cm^-1;¹H NMR(δ ppm): 3.75(m, 8H, 4(-CH2-)), 6.81-6.83(d, 2H, Ar-H, J=9.2Hz), 7.53-7.62(m, 3H, Ar-H), 7.65-7.67(d, 2H, Ar-H, J=9.2), 7.85-7.88(dd, 2H, Ar-H, J=2,2.4), 8.17- 8.20(d, 2H, Ar-H, J=8Hz), 8.24-8.25(d, 1H, Ar-H, J=2), 8.36-8.38(d, Ar-H, J=8.4), 8.39(s, 1H, Ar-H),; ¹³C NMR(δppm): 41.18, 52.21, 112.05, 118.00, 121.93, 123.99, 124.06, 127.40, 128.79, 128.95, 130.18, 130.73, 131.72, 137.78, 141.95, 143.29, 146.45, 156.35, 163.94 MS (m/z): 497(M⁺); Anal. Calcd for:C₂₆H₂₂Cl₃N₃O; C, 62.60; H, 4.45; N, 8.42; Found:C, 62.54; H, 4.48; N, 8.37. N-(4-(bis(2-chloroethyl)amino)phenyl)-6-chloro-2-(4-methoxyphenyl) Quinoline -4-carboxamide (HKQM-2l): Light yelllow solid; Melting range:188- 190°C; Rf:0.36(Methanol:MDC 0.5:9.5 ml); IR (KBr):3274, 3047, 2960, 1676, 1611, 1587, 1516, 1349, 1251, 1175, 1030, 834, 811, 752, cm^-1; MS (m/z): 527(M⁺); Anal. Calcd for:C₂₇H₂₄Cl₃N₃O₂; C, 61.32; H, 4.57; N, 7.95; Found:C, 61.28; H, 4.63; N, 7.96. N-(4-(bis(2-chloroethyl)amino)phenyl)-6-chloro-2-(4-chlorophenyl)quinoline- 4-carboxamide (HKQM-2m): Light yelllow solid; Melting range:164-166°C; Rf:0.33(Methanol:MDC 0.5:9.5 ml); IR (KBr): 3260, 3066, 2961, 1675, 1642, 1589, 1519, 1346, 1250, 1175, 1088, 1013, 887, 832, 747 cm^-1;¹H NMR(δ ppm): 3.76(m, 8H, 4(-CH₂-)), 6.81-6.83(d, 2H, Ar-H, J=9.2Hz), 7.64-7.68(m, 4H, Ar-H), 7.85-7.87(dd, 1H, Ar-H, J=2,2), 8.16-8.18(d, 2H, Ar-H, J=8.8Hz), 8.25(d, 1H, Ar- H, J=2.2), 8.38-8.42(m, 3H, Ar-H), 10.63(s, 1H, -NH-),; ¹³C NMR(δppm):41.17, 52.22, 112.03, 117.83, 121.95, 124.02, 124.12, 128.77, 129.12, 130.83, 131.70, 131.94, 135.14, 136.54, 142.06, 143.30, 146.37, 155.05, 163.84MS (m/z):531 (M⁺); Anal. Calcd for:C26H21Cl4N3O; C, 58.56; H, 3.97; N, 7.88; Found:C, 58.52; H, 4.06; N, 7.96. N-(4-(bis(2-chloroethyl)amino)phenyl)-2-(4-bromophenyl)-6-chloroquinoline- 4-carboxamide (HKQM-2n): Light yelllow solid; Melting range:200-202°C; R_f:0.34(Methanol:MDC 0.5:9.5 ml); IR (KBr):3291, 3080, 3043, 2964, 1673, 1590, 1520, 1347, 1251, 1175, 1076, 1008, 885, 825, 746 cm^-1; MS (m/z):574 (M⁺); Anal. Calcd for:C26H21BrCl3N3O; C, 54.05; H, 3.66; N, 7.27; Found:C, 54.08; H, 3.59; N, 7.32. N-(4-(bis(2-chloroethyl)amino)phenyl)-2-(3,4-dimethoxyphenyl)-6- chloroquinoline-4-carboxamide (HKQM-2o): Light yelllow solid; Melting range:166-168°C; R_f:0.34(Methanol:MDC 0.5:9.5 ml); IR (KBr):3476, 3383, 3243, 3081, 2964, 1682, 1641, 1606, 1535, 1519, 1467, 1320, 1257, 1157, 1119, 1066, 1018, 939, 844, 791, 755, 711, 664, 428cm^-1; MS (m/z):557 (M⁺); Anal. Calcd for:C₂₈H₂₆Cl₃N₃O₃; C, 60.17; H, 4.69; N, 7.52; Found:C, 60.21; H, 4.76; N, 7.47. EXAMPLE 4: CYTOTOXICITY EVALUATION The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content. The method optimized for the toxicity screening of compounds to adherent cells in a 96-well format. After an incubation period, cell monolayers are fixed with 10% (wt/vol) trichloroacetic acid and stained for 30 min, after which the excess dye is removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye is dissolved in 10 mMTris base solution for OD (optical density) determination at 510 nm using a microplate reader. For IC₅₀ determination, plot a dose–response curve between the compound concentration and percent growth inhibition. IC50 values can be derived using curve-fitting methods with statistical analysis software or IC50 calculation software. % of control cell % growth inhibition = 100 - % of control cell growth It is possible to use the SRB assay to determine the LD50 values of compounds from the dose–response relationship between the compound concentration and the percentage of cells killed, which is calculated using the formula below. % cells killed = 100 ₁₀₀ In vitro cytotoxicity (% growth control) has been carried out with different concentration of compounds. The result of in vitro cytotoxicity (µg/ml) is given in below table. Results were compared with Adreamycin cytotoxicity (ADR) results. LC50, TGI and GI50 of compounds are also given in below table. The figure was shown in figure 15. Cytotoxicity study was performed on Human Prostate Cancer Cell Line PC3. PC-3 LC50 TGI GI50 HKQM-1 >100 >100 >100 HKQM-2 >100 >100 >100 HKQM-3 >100 >100 >100 HKQM-4 >100 >100 98.26102 HKQM-5 >100 >100 97.2 HKQM-6 >100 >100 87.2 HKQM-11 >100 >100 90.4 HKQM-12 >100 >100 >100 HKQM-13 >100 >100 97.1 HKQM-14 >100 >100 >100 ADR 61.2 19.1 <10 Table 4: Result of LC50, TGI, GI50 Drug Concentrations (µg/ml) % Control Growth Compound Experiment 1 Experiment 2 code 10 20 40 80 10 20 40 80 H^KQM-2a 100.0 100.0 100.0 57.8 100.0 100.0 100.0 63.5 HKQM-2b 100.0 100.0 100.0 64.3 100.0 100.0 97.4 66.1 HKQM-2c 100.0 100.0 98.0 60.4 100.0 100.0 90.5 57.7 H^KQM-2d 100.0 100.0 99.6 52.7 100.0 100.0 97.0 53.5 H^KQM-2e 100.0 100.0 100.0 52.5 100.0 100.0 95.2 47.7 HKQM-2f 100.0 100.0 100.0 39.9 100.0 100.0 100.0 43.3 HKQM-2k 100.0 100.0 100.0 40.7 100.0 100.0 100.0 50.1 H_KQM-2l 100.0 100.0 100.0 46.6 100.0 100.0 100.0 63.7 H^KQM-2m 100.0 100.0 97.5 44.5 100.0 100.0 100.0 56.0 HKQM-2n 100.0 100.0 96.8 46.7 100.0 100.0 100.0 54.5 ADR -30.8 -42.4 -44.9 -48.4 -31.3 -39.3 -45.5 -46.3 Table 5: Cytotoxicity data of synthesized compounds (Experiment 1 and 2) Drug Concentrations (µg/ml) % Control Growth Compound Experiment 3 Average value code 10 20 40 80 10 20 40 80 H^KQM-2a 100.0 100.0 96.3 61.1 100.0 100.0 98.8 60.8 H^KQM-2b 100.0 100.0 95.4 72.9 100.0 100.0 97.6 67.8 H_KQM-2c 100.0 100.0 87.3 77.1 100.0 100.0 91.9 65.1 HKQM-2d 100.0 100.0 90.6 54.2 100.0 100.0 95.7 53.5 HKQM-2e 100.0 100.0 100.0 55.5 100.0 100.0 98.4 51.9 HKQM-2f 100.0 100.0 100.0 50.5 100.0 100.0 100.0 44.6 HKQM-2k 100.0 100.0 100.0 50.1 100.0 100.0 100.0 47.0 HKQM-2l 100.0 100.0 100.0 58.1 100.0 100.0 100.0 56.2 H^KQM-2m 100.0 100.0 100.0 54.2 100.0 100.0 99.2 51.6 H^KQM-2n 100.0 100.0 100.0 65.6 100.0 100.0 98.9 55.6 ADR -42.9 -43.2 -46.7 -50.1 -35.0 -41.7 -45.7 -48.3 Table 6: Cytotoxicity data of synthesized compounds (Experiment 3 and average value) As shown in table 4, 5 and 6, it was found that synthesized compound provides satisfied result in lethal concentration, tumor growth inhibition and growth inhibition. Conclusion The developed N-(4-(bis (2-chloroethyl) amino) phenyl)-2-aryllquinoline-4- carboxamide provides good result in mass, 1H NMR and 13C NMR spectroscopy. From the result of cytotoxicity, it reveals that the synthesized compounds are effective at higher concentration. The synthesized compound provides cancer treatment which increased drug accumulation at the tumor site that leads to increase therapeutic efficacy and decreased toxicity, side effect and adverse effect.

Claims

CLAIMS We Claims, 1. A quinoline based aniline nitrogen mustard conjugates as anti-cancer agent with the formula (1); Wherein, R is selected from -H, 4-OMe, 5-F, 5-Cl, 5-Br, 5-OMe, 5-NO2, 6- Cl, 6- Br, 7-Br. R1 is selected from H, 4-CH3, 3-F, 4-F, 3-Br, 4-Br, 3, 4-di OMe, 2, 4-di Cl, 4-OMe, 4-Cl, 3-Cl.

2. The quinoline based aniline nitrogen mustard conjugates as anti-cancer agent as claimed in claim 1, wherein said formula (1) is N-(4-(bis(2- chloroethyl)amino)phenyl)-2-aryllquinoline-4- carboxamide.

3. The quinoline based aniline nitrogen mustard conjugates as anti-cancer agent as claimed in claim 1, wherein said formula (1) is prepared from N, N-bis(2-chloroethyl)benzene-1,4-diamine hydrochloride.

4. The quinoline based aniline nitrogen mustard conjugates as anti-cancer agent as claimed in claim 1, wherein said formula (1) is prepared by using peptide coupling mechanism by T₃P (propylphosphonic anhydride).

5. The quinoline based aniline nitrogen mustard conjugates as anti-cancer agent as claimed in claim 1, wherein said formula (1) is useful as anti-cancer agent.

6. The process for preparation of quinoline based aniline nitrogen mustard conjugates as anti-cancer agent as claimed in claim 1 comprises the following steps: a) Adding 2-arylquinoline-4-carboxylic acid (compound 1) and N,N-bis (2-chloroethyl)benzene-1,4-diamine hydrochloride (compound c) in ethylacetate and mixing; b) Adding T₃P dropwise into step (a) at 10⁰C temperature; c) Adding immediately diisopropylethylamine dropwise into step (b) and stirring for 30 minute; d) Monitoring reaction mixture of step (c) by using TLC (Methanol:MDC 0.5:9.5 ml) till the reaction complete; e) Pouring reaction mixture of step (d) into water and extracting with ethylacetate to get extracted organic layer; f) Drying extracted organic layer of step (e) over sodium Sulphate to get N-(4-(bis (2-chloroethyl) amino) phenyl)-2-aryllquinoline-4- carboxamide; g) Crystalizing the compound of step (f) to get pure compound; 7. The quinoline based aniline nitrogen mustard conjugates as anti-cancer agent as claimed in claim 1, wherein said derivatives can be selected from;

HKQM-2a HKQM-2b N-(4-(bis(2- N-(4-(bis(2- chloroethyl)amino)phenyl)-2-(p- chloroethyl)amino)phenyl)-2-(4- tolyl)quinoline-4-carboxamide fluorophenyl)quinoline-4- carboxamide HKQM-2c HKQM-2d N-(4-(bis(2- N-(4-(bis(2- chloroethyl)amino)phenyl)-2- chloroethyl)amino)phenyl)-2-(2,4- (3,4-dimethoxyphenyl)quinoline- dichlorophenyl)quinoline-4- 4-carboxamide carboxamide HKQM-2e HKQM-2f N-(4-(bis(2- N-(4-(bis(2- chloroethyl)amino)phenyl)-2-(4- chloroethyl)amino)phenyl)-2-(4- methoxyphenyl)quinoline-4- chlorophenyl)quinoline-4- carboxamide carboxamide HKQM-2h HKQM-2g N-(4-(bis(2- N-(4-(bis(2- chloroethyl)amino)phenyl)-2-(4- chloroethyl)amino)phenyl)-2-(3- bromophenyl)quinoline-4- chlorophenyl)quinoline-4- carboxamide carboxamide HKQM-2i HKQM-2j N-(4-(bis(2- N-(4-(bis(2- chloroethyl)amino)phenyl)-2-(3- chloroethyl)amino)phenyl)-6- fluorophenyl)quinoline-4- chloro-2-(p-tolyl)quinoline-4- carboxamide carboxamide HKQM-2k HKQM-2l N-(4-(bis(2- chloroethyl)amino)phenyl)-6- N-(4-(bis(2- chloro-2-phenylquinoline-4- chloroethyl)amino)phenyl)-6- carboxamide chloro-2-(4- methoxyphenyl)quinoline-4- carboxamide HKQM-2m HKQM-2n N-(4-(bis(2- N-(4-(bis(2- chloroethyl)amino)phenyl)-6- chloroethyl)amino)phenyl)-2-(4- chloro-2-(4- bromophenyl)-6-chloroquinoline- chlorophenyl)quinoline-4- 4-carboxamide carboxamide HKQM-2o N-(4-(bis(2- chloroethyl)amino)phenyl)-6- chloro-2-(3,4- dimethoxyphenyl)quinoline-4- carboxamide