Potentially antibreast cancer enamidines via azide–alkyne–amine coupling and their molecular docking studies

Abstract

An attempt to prepare a new triazole series by Cu catalyzed multicomponent reactions of tosyl azide, propargyl bromide and secondary amines led to the synthesis of enamidines. All the synthesized enamidines were evaluated for antiproliferative activities. The four molecules 4a–c and 4h showed higher anticancer activity with GI₅₀ values less than the standard drug doxorubicin against human breast cancer cell line MCF-7. The virtual analysis ascertains the mode of action of these compounds via inhibition of human cell division protein kinase7 (CDK7).

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Introduction

The applications of multicomponent reactions (MCRs) for the synthesis of privileged scaffolds is of prime importance in diversity oriented synthesis, combinatorial chemistry and medicinal chemistry.¹ This is primarily because of the inherent advantages in the formation of multiple chemical bonds from three or four components thereby leading to products of high molecular complexity.² The importance of molecular complexity has been clearly recognized in identifying specific bioactive molecules for the elucidation of mysterious biological processes.³ A key feature of MCR chemistry is the very large chemical space which is amenable to the discovery of potential drug candidates.⁴ Among the various MCRs developed for this purpose, the copper catalyzed reaction of azides, alkynes and amines represents one of the most stimulating, dynamic and synthetically powerful process in contemporary organic synthesis as it allows rapid creation of molecular complexity under environmentally benign conditions.⁵ The classical copper catalysed coupling of simple azides, terminal alkynes and amines results in the formation of triazoles and other scaffolds which have intriguing structural features and promising biological activities.⁶ The contemporary investigations have revealed that a small structural variation in the substrates can exerts a strong influence on the outcome of the product. The CuI system triggering a variant of the azide–alkyne–amine coupling was reported by Chang and co-workers.⁷ They reported on the unexpected synthesis of N-sulfonyl amidines from CuI catalyzed reaction of p-toluene sulfonyl azide, terminal alkyne and secondary amine. The amidines are commonly seen in bioactive natural products and have received significant attention as synthetic targets due to their intriguing structural features and promising biological activities.⁸ The reaction is proposed to proceed via the formation of ketenimine intermediate, which is generated in situ by Cu catalyzed cycloaddition of sulfonyl azides with terminal alkynes followed by ring cleavage of resultant triazole. A precise reason for such a difference in the output based on computational predictions has been provided separately by Chang et al. and Fokin et al. in their publications.^9,10

In order to extend the scope of copper catalyzed multicomponent reactions for maximizing molecular diversity with high relevance in biological space, we sought to explore the chemistry of copper catalyzed, previously unexplored combination of propargyl bromide, tosyl azide and secondary amines. The key theme of our synthetic strategy is construction of a drug like molecular libraries through the creative reconstruction of core skeletons containing privileged substituents.

Results and discussion

To pursue our objective, the model reaction of tosyl azide (1) propargyl bromide (2), and diethyl amine (3a) was carried out using CuI as catalyst, triethyl amine (TEA) as base and water as solvent. The reaction afforded a white coloured product (4a) which was subjected to ¹H NMR analysis. The observation of two doublets at δ 5.6 and 5.4 ppm (J = 12 Hz) and a doublet of doublet at δ 6.6 (J = 12, 18 Hz) indicated the presence of olefinic double bond. The spectral data could not be reconciled with the expected amidine⁸ or triazole⁹ indicating that a new molecule was formed under applied reaction conditions. This was further confirmed by the presence of nine signals in ¹³C NMR spectrum in the region of δ 120–165 ppm. To gain further insight, we recorded DEPT that displayed a signal at δ 123 ppm indicating the presence of olefinic methylene group. The unexpected signal in the DEPT study led us towards single crystal analysis for precise structure elucidation. The diffusion of petroleum ether into an ethyl acetate solution of 4a afforded colourless single crystal suitable for X-ray diffraction analysis. The details of crystal data, data collection and the refinement are given in the ESI.† The X-ray analyses revealed that the complex 4a crystallizes in the monoclinic space group P21/n. The molecular structure of 4a along with the atom numbering scheme is illustrated in Fig. 1. The single crystal analysis revealed the enamidine nature of 4a that was identified as N,N-diethyl-N′-acrylamidine. Our observations are in concordance with the synthetic strategy reported by Zinic et al.¹¹

Fig. 1 X-ray crystal structure of N,N-diethyl-N′-acrylamidine (displacement ellipsoids are drawn at the 50% probability level).

We were gratified to note the formation of enamidines in our initial investigations. Notably, the enamidines are useful synthetic scaffolds¹² and important skeletons for atropisomerism studies¹³ and considerable progress has been made in their synthesis. The important synthetic routes to enamidines include gold catalyzed intermolecular nitrene transfer to alkynes,¹⁴ Pd catalyzed coupling of an alkenyl bromide, isonitrile and an amine or alkoxide/phenoxide,¹⁵ Bronsted acid catalyzed yne carbamate with aldimines involving 4π electrocyclic ring-opening reaction,¹⁶ Cu catalyzed reaction of ketenimine and in situ generated immonium ion,¹⁷ and 1,3-amino group migration strategy.¹⁸

Intrigued by the facile formation of enamidine, we set out to explore the influence of various parameters on the model reaction. The effect of various solvents such as CHCl₃, THF, toluene, 1,4-dioxane, DMF and water was studied (Table 1, entries 1–6). Among all the screened solvents, water was found to furnish excellent yield of product (92%) (Table 1, entry 6). A variety of bases such as TEA, K₂CO_3, DIPEA, DABCO and K₃PO₄ were screened for the model reaction. The use of bases other than triethyl amine resulted in significantly lower yields (Table 1, entries 10, 12, 15, 16). The screening of copper source other than CuI led to inferior results (Table 1, entries 11–12).

Table 1 Optimization of reaction conditions^a

Entry	Catalyst	Solvent	Base	Time (h)	Yield^b (%)
a Tosyl azide 1 (0.6 mmol), propargyl bromide 2 (0.5 mmol), diethyl amine 3a (0.5 mmol), copper catalyst (0.05 mmol), base (0.6 mmol) and solvent (5 mL) were stirred at rt in an open air.b Isolated yields after column chromatography.
1	CuI	CHCl3	TEA	4.0	74
2	CuI	THF	TEA	4.5	72
3	CuI	Toluene	TEA	5.0	52
4	CuI	Dioxane	TEA	5.0	50
5	CuI	DMF	TEA	2.0	80
6	CuI	Water	TEA	1.0	92
7	CuI	Water	K2CO3	1.0	60
8	CuI	Water	DIPEA	2.0	75
9	CuI	Water	DABCO	2.5	68
10	CuCl	Water	DIPEA	2.5	52
11	CuBr	Water	DABCO	2.0	60
12	CuCl	Water	TEA	1.5	65
13	CuBr	Water	TEA	1.5	70
14	CuSO4, Na-ascorbate	Water	TEA	2.0	60
15	CuSO4, Na-ascorbate	Water	K3PO4	2.0	50

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With the optimized set of reaction conditions, we investigated the scope of the protocol by reacting tosyl azide (1) and propargyl bromide (2) with series of amines (3a–m). Several diversified secondary amines reacted efficiently furnishing the corresponding enamidines without any side products (Scheme 1). A high reactivity was observed with aliphatic secondary amines (Table 2, entries 4a–e). It is interesting to note that cyclic secondary amines have reacted productively giving good yields of respective enamidines (Table 2, entries 4g–j). It is noteworthy that aromatic amines could also be employed as substrates to afford the anticipated enamidines in moderate yields (Table 2, entries 4l and 4m).

Scheme 1 Synthesis of enamidines.

Table 2 Multicomponent synthesis of enamidines^a

Entry	Amine (3)	Product (4)	Yield^b (%)	Mp (°C)
a Tosyl azide 1 (0.6 mmol), propargyl bromide 2 (0.5 mmol), amine 3 (0.5 mmol), CuI (0.05 mmol), TEA (0.6 mmol) and water (5 mL) were stirred at rt in an open air for 60 min.b Isolated yields after column chromatography.
a			92	108–110
b			85	84–86
c			88	102–104
d			90	Liquid
e			93	Liquid
f			86	77–80
g			75	Liquid
h			85	75–76
i			80	120–121
j			65	122–124
k			93	107–108
l			65	132–134
m			60	116–118

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Mechanism

A mechanistic rational for the formation of enamidines on the basis of literature reports is postulated in Scheme 2.¹⁹ The proposed path is related to ynamide chemistry where nucleophilic amine initially reacts with propargyl bromide to form propargyl amine (I) that subsequently react with CuI to form Cu(I) acetylide (II) which further reacts with tosyl azide to form 5-cuprated triazole (III) that extrudes N₂ and undergoes subsequent 4-exo-dig cyclization to generate azitidine (IV) via non isolable N-sulfonyl ketenimine intermediate. Further, E₁CB elimination type ring opening process results in the formation of corresponding enamidine (V).¹⁰

Scheme 2 Proposed mechanism for synthesis of enamidines.

To evaluate the anticancer potential of synthesized enamidines, we tested in vitro anti-proliferative activities of compounds 4a–m against human breast cancer cell line MCF-7 by employing the sulforhodamine B (SRB) assay method (Table 3).²⁰ It is noteworthy that all the compounds except 4i, 4k–m displayed significant cytotoxicity as compared to the standard drug i.e. doxorubicin,²¹ with the GI₅₀ values ranging from 3.24 to 45.2 μM. The compounds 4a–c and 4h were found to be most active with GI₅₀ values of 3.56, 3.96, 3.24, and 3.42 μM respectively which are less than the standard anticancer agent doxorubicin (18.4 μM). The higher activities of enamidines obtained from aliphatic secondary amines as compared to others indicated that linear alkyl frame work is required for the anticancer activity of the compounds.

Table 3 In vitro cytotoxicities of enamidines against human breast cancer cell line MCF-7^a

Compound	LC50^b	TGI^c	GI50^d
a Concentration in μM.b Concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning calculated from [(Ti − Tz)/Tz] × 100 = −50.c Drug concentration resulting in total growth inhibition (TGI) calculated from Ti = Tz.d Growth inhibition of 50% (GI50) calculated from [(Ti − Tz)/(C − Tz)] × 100 = 50.
4a	68.1	27.8	3.56
4b	>80	31.7	3.96
4c	73.1	33.2	3.24
4d	>80	70.3	29.2
4e	>80	68	23.3
4f	>80	71.1	30
4g	>80	61	26.2
4h	76.9	32	3.42
4i	>80	>80	>80
4j	>80	>80	45.2
4k	>80	>80	>80
4l	>80	>80	>80
4m	>80	>80	>80
(ADR) doxorubicin^21	73.2	<18.4	<18.4

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The master regulators cyclin dependent kinases (CDKs) have significant contribution in cancer development and progression which makes them a promising target for novel therapeutic interventions.²² CDKs are the controllers of the two major steps in the cell division; as promoters of DNA synthesis in the S phase and entry into mitosis. They also serve as actual driving force behind the progression of the cell cycle.²³ Activity of these kinases is regulated by the number of factors like periodic synthesis and degradation of positive regulators, cyclins, negative regulators and most important cyclin kinase inhibitors (CKIs). CDK regulates each phase in the cell cycle. Deregulation of these is the key factor in the tumor growth and progression in number of cancers.²⁴ Thus, targeting CDKs has become an important mode to develop new anticancer agents. Cyclin depenedent protein kinase7 (CDK7) is associated with cyclin H to give a complex known as CDK activating kinase (CAK) which gives phosphorylation of the CDK activation loop or T loop resulting into activation of number of CDK/cyclins. CDK7 also plays vital role in transcription via contribution in phosphorylation of RNA polymerase II. Biological importance of the CDK7 makes it a promising target for anticancer therapy as number of CDK7 inhibitors like roscovitine is in clinical trials.²⁵

In order to substantiate the activity profiles of synthesized enamidines, docking simulations were performed on the binding sites of CDK7. The efficiency of the docking procedure was assessed by using docking analysis of reference ATP molecule in the active site of CDK7 (pdb id: 1UA2) using the BIOPREDICTDA module in V life MDS 4.3. All synthesized compounds (4a–m) were subsequently docked into the binding site of CDK7. The results obtained revealed that enamidines obtained from aliphatic secondary amines have excellent binding affinity with the CDK7 than those obtained from alicyclic and aromatic amines.

The computed binding energies of 4a–m for CDK7 range between −56.9 to −65.14 kcal mol⁻¹, clearly suggesting the favourable binding towards CDK7. Compound 4c exhibited the strongest binding affinity with CDK7 followed by 4h and 4a (Table 4) which is in agreement with in vitro anti-proliferative activity data. The docked enzyme inhibitor compounds were further analyzed to get a deeper understanding of their interaction relationship. All docked compounds exhibited both hydrogen bond and hydrophobic interactions with the CDK7. The predicted docking pose of most active compound 4c formed one hydrogen bond with carbonyl oxygen of ASN142 and displayed hydrophobic and van der Waals interactions with the GLY19, GLN22, LYS139 and SER161 (Fig. 4). The bonded conformation of 4h showed three hydrogen bonds with ASN142 and displayed hydrophobic and van der Waals interactions with SER161, LYS160, ALA24, PHE23, GLN22, LYS41, VAL26, and LEU18 (Fig. 5). Compound 4b showed hydrogen bond interaction with nitrogen of LYS41 and hydrophobic and van der Waals interactions with GLU20, GLY21, GLN22, PHE23, ALA24 (Fig. 3). Similarly 4a showed H bond interaction with ASN142 with the distance of 2.5 A° (Fig. 2). The docking virtual analysis (Fig. 2–5) revealed that presence of smaller aliphatic alkyl groups on nitrogen exhibit high influence on anti-proliferative activity which might be due to approaching similar conformation with respect to binding site of CDK7.

Table 4 Calculated binding energy with CDK7 receptor of 4a–c and 4h

Enamidine	Docking score	Key interactions
4a	−52.76	ASN142 2.5 hydrogen bond interaction
4b	−46.34	LYS41 2.3 hydrogen bond interaction
4c	−62.90	MET94 1.7 hydrogen bond interaction
4h	−55.77	ASN142 1.8 hydrogen bond interaction
		ASN142 1.7 hydrogen bond interaction
		ASN142 2.4 hydrogen bond interaction
Roscovitine^26	−87.44	ASP97 2.5 hydrogen bond interaction
		MET94 1.5 hydrogen bond interaction
		LYS41 2.5 hydrogen bond interaction

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Fig. 2 Binding mode of 4a with CDK7.

Fig. 3 Binding mode of 4b with CDK7.

Fig. 4 Binding mode 4c with CDK7.

Fig. 5 Binding mode 4h with CDK7.

Conclusions

In conclusion, the disclosed method offers an efficient and broad-in-scope means of accessing enamidines from multicomponent reaction of tosyl azide, propargyl bromide and amines. The significantly higher antibreast cancer activities of some of the synthesized enamidines open new avenue for designing new chemical entities with impressive anti-proliferative activity. This approach should provide an opportunity for creation of combinatorial libraries and allow rapid access to these scaffolds paving the way for future studies of their reactivity in other synthetic applications.

Experimental section

General

Unless otherwise stated all reagents and solvents were purchased from commercial sources and used without further purification. Analytical thin layer chromatography (TLC) was performed on precoated silica gel 60F 254 plates and spots were visualized under UV light. ¹H, ¹³C NMR spectra were recorded on 300/75 MHz spectrometer in CDCl₃ as solvent and tetramethylsilane (TMS) as an internal standard. Infrared spectra were recorded on FTIR spectrometer. The samples were examined as KBr discs ∼5% w/w. Elemental analyses were performed on EURO EA3000 vectro model. Melting points were determined on melting point apparatus and are uncorrected. Column chromatography was performed on silica gel (60–120 mesh) using ethyl acetate/petroleum ether.

X-ray diffraction studies

Diffusion of pet ether into an ethyl acetate solution of compound 4a afforded colourless crystals suitable for X-ray diffraction analysis. The intensity data were collected from a diffractometer in omega and phi scan mode, Mo [Kα] = 0.71073 Å at room temperature, with scan width of 0.3° at φ (0°, 90° and 180°) by keeping the distance at 40 mm between sample to fixed detector at 24°. The X-ray generator was operated at 50 kV and 30 mA. The X-ray data collection was monitored by SMART program. All the data were corrected using SAINT and SADABS programs. SHELX-97 was used to solution of structure and full matrix least-squares refinement on F2. Molecular and packing diagrams were generated using ORTEP-3 and Mercury-3. The positions of all the atoms were obtained by direct methods. All non-hydrogen atoms were refined anisotropically. The remaining hydrogen atoms were placed in geometrically constrained positions and refined with isotropic temperature factors.

General procedure for synthesis of enamidines 4a–m

To an oven-dried round-bottom flask containing propargyl bromide (0.5 mmol), amine (0.5 mmol) and triethyl amine (0.6 mmol) in water (5 mL) was added tosyl azide (0.6 mmol) followed by CuI (0.05 mmol). The resultant mixture was stirred at room temperature until the reactants were consumed (TLC). After reaction was completed, the mixture was poured in water (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic layer was filtered through anhydrous Na₂SO₄ and the solvent removed under reduced pressure. The crude residue was purified by column chromatography on silica gel with an appropriate eluting solvent system (EtOAc/Pet ether, 20–40 : 80–60 depending on substrates).

N,N-Diethyl-N′-[(4-methylphenyl)sulfonyl]prop-2-enamidamide (Table 2, product 4a). Colorless solid; 138 mg; 92% yield; R_f 0.46 (pet. ether/EtOAc 9 : 1); mp 108–110 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.76 (d, J = 7.8 Hz, 2H), 7.26 (d, J = 12.6.8 Hz, 2H), 6.57 (dd, J = 16, 12 Hz, 1H), 5.63 (d, J = 0.9 Hz, 1H), 5.46 (d, J = 3.6 Hz, 1H), 3.57 (q, 4H), 2.38 (s, 3H), 1.25 (t, 6H) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 164.0, 141.6, 141.3, 129.5, 128.9, 128.8, 126.3, 123.5, 44.3, 42.5, 21.4, 13.7, 12.0 ppm; FT-IR (KBr, thin film): ν 3073, 2955, 1600, 1537, 1426, 1392, 1352, 1233, 1182, 1080, 956, 866, 737 cm⁻¹; anal calc. for C₁₄H₂₀N₂O₂S: % C, 59.97; % H, 7.1; % N, 9.99, observed: % C, 59.79; % H, 7.29; % N, 9.89.

N,N-Dimethyl-N′-[(4-methylphenyl)sulfonyl]prop-2-enamidamide (Table 2, product 4b). Colorless solid; 127 mg; 85% yield; R_f 0.46 (pet. ether/EtOAc 9 : 1); mp 84–86 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.79 (d, J = 8.1 Hz, 2H), 7.26 (d, J = 6.9 Hz, 2H), 6.62 (dd, J = 12, 12 Hz, 1H), 5.73 (d, J = 12 Hz, 1H), 5.50 (d, J = 18 Hz, 1H), 3.10 (s, 6H), 2.40 (s, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 164.8, 142.9, 141.8, 141.1, 129.3, 128.9, 128.8, 126.6, 126.5, 124.6, 40.0, 38.2, 21.5 ppm; FT-IR (KBr, thin film): ν 2921, 1638, 1546, 1475, 1399, 1265, 1139, 1109, 1087, 685 cm⁻¹; anal calc. for C₁₂H₁₆N₂O₂S: % C, 57.12; % H, 6.39; % N, 11.10, observed: % C, 57.02; % H, 6.31; % N, 11.16.

N′-[(4-Methylphenyl)sulfonyl]-N,N-dipropylprop-2-enamidamide (Table 2, product 4c). Pale yellow solid; 132 mg; 88% yield; R_f 0.53 (pet. ether/EtOAc 9 : 1); mp 102–104 °C; ¹H NMR (300 MHz, CDCl₃): δ 7.75 (d, J = 7.8 Hz, 2H), 7.24 (d, J = 7.8 Hz, 2H), 6.55 (dd, J = 12, 12.3 Hz, 1H), 5.64 (d, J = 12 Hz, 1H), 5.47 (d, J = 18 Hz, 1H), 3.39 (t, J = 7.5 Hz, 2H), 3.28 (t, J = 7.5 Hz, 2H), 2.41 (s, 3H), 1.64–1.51 (m, 4H), 0.88–0.83 (m, 6H) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 164.4, 143.3, 141.6, 141.3, 139.3, 129.6, 128.9, 126.4, 126.3, 123.8, 51.7, 49.8, 21.7, 21.4, 20.0, 11.3, 11.0 ppm; FT-IR (KBr, thin film): ν 2924, 1634, 1535, 1476, 1399, 1255, 1140, 1087, 867, 673 cm⁻¹; anal calc. for C₁₆H₂₄N₂O₂S: % C, % 62.30; % H, 7.84; % N, 9.08, observed: % C, 62.20; % H, 7.64; % N, 9.18.

N,N-Diisopropyl-N′-[(4-methylphenyl)sulfonyl]prop-2 enamidamide (Table 2, product 4d). Pale yellow liquid; 135 mg; 90% yield; R_f 0.50 (pet. ether/EtOAc 9 : 1); ¹H NMR (300 MHz, CDCl₃): δ 7.78 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 6.61 (dd, J = 12, 12.3 Hz, 1H), 5.62 (d, J = 12 Hz, 1H), 5.47 (d, J = 18 Hz, 1H), 4.28 (bs, 1H), 3.65 (bs, 1H), 2.40 (s, 3H), 1.38–1.21 (m, 12H) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 163.0, 141.5, 130.2, 129.5, 128.9, 128.6, 128.4, 126.2, 122.5, 29.6, 21.6, 21.4, 20.1 ppm; FT-IR (KBr, thin film): ν 2924, 1649, 1540, 1475, 1398, 1274, 1240, 1141, 1121, 1086, 879, 679 cm⁻¹; anal calc. for C₁₆H₂₄N₂O₂S: % C, 62.30; % H, 7.60; % N, 9.09, observed: % C, 62.20; % H, 7.60; % N, 9.09.

N′-[(4-Methylphenyl)sulfonyl]-N,N-di(n-butyl)prop-2-enamidamide (Table 2, product 4e). Sticky liquid; 139 mg; 93% yield; R_f 0.53 (pet. ether/EtOAc 9 : 1); ¹H NMR (300 MHz, CDCl₃): δ 7.77 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 6.57 (dd, J = 12, 12 Hz, 1H), 5.66 (d, J = 12.3 Hz, 1H), 5.52 (d, J = 18 Hz, 1H), 3.44–3.39 (m, 2H), 3.34–3.29 (m, 2H), 2.39 (s, 3H), 1.60–1.40 (m, 4H), 1.34–1.22 (m, 4H), 0.93–0.88 (m, 6H) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 164.1, 141.5, 129.0, 128.8, 126.3, 123.8, 49.8, 47.9, 30.5, 28.8, 21.4, 20.1, 19.8, 13.6 ppm; FT-IR (KBr, thin film): ν 2921, 1638, 1546, 1475, 1399, 1265, 1139, 1109, 1087, 685 cm⁻¹; anal calc. for C₁₈H₂₈N₂O₂S: % C, 57.12; % H, 6.39; % N, 11.10, observed: % C, 57.02; % H, 6.31; % N, 11.16.

N′-[(4-Methylphenyl)sulfonyl]-N,N-di(cyclohexyl)prop-2-enamidamide (Table 2, product 4f). Pale yellow solid; 129 mg; 86% yield; mp 77–80 °C, R_f 0.46 (pet. ether/EtOAc 9 : 1); ¹H NMR (300 MHz, CDCl₃): δ 7.79 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 8.1 Hz, 2H), 6.59 (dd, J = 12.12 Hz, 1H), 5.63 (d, J = 12 Hz, 1H), 5.51 (d, J = 18 Hz, 1H), 3.78 (bs, 1H), 3.16 (bs, 1H) 2.40 (s, 3H), 1.76–1.21 (m, 20H) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 163.1, 141.6, 141.4, 130.3, 128.8, 126.1, 122.6, 26.2, 25.5, 25.1, 21.4 ppm; FT-IR (KBr, thin film): ν 2922, 2853, 1638, 1541, 1476, 1397, 1275, 1246, 1142, 1113, 1086, 878, 683 cm⁻¹; anal calc. for C₂₂H₃₂N₂O₂S: % C, 68.00; % H, 8.30; % N, 7.21, observed: % C, 67.90; % H, 8.32; % N, 7.11.

4-Methyl-N-(1-pyrrolidin-1-ylprop-2-en-1-ylidene)benzenesulfonamide (Table 2, product 4g). Sticky liquid; 112 mg; 75% yield; R_f 0.46 (pet. ether/EtOAc 9 : 1); ¹H NMR (300 MHz, CDCl₃): δ 7.79 (d, J = 8.1 Hz, 2H), 7.27 (d, J = 8.1 Hz, 2H), 6.61 (dd, J = 12, 12.3 Hz, 1H), 5.65 (d, J = 8.1 Hz, 1H), 5.48 (d, J = 18 Hz, 1H), 3.51–3.41 (m, 4H), 2.40 (s, 3H), 1.49–1.36 (m, 4H); ¹³C NMR (75 MHz, CDCl₃): δ 164.8, 130.1, 129.7, 129.5, 129.3, 128.9, 128.8, 128.5, 128.4, 128.0, 126.4, 126.0, 123.5, 43.6, 43.2, 21.6, 13.1, 12.0 ppm; FT-IR (KBr, thin film): ν 2921, 1667, 1554, 1483, 1397, 1273, 1241, 1217, 1143, 1085, 1007, 918, 818, 684 cm⁻¹; anal calc. for C₁₄H₁₈N₂O₂S: % C, 60.41; % H, 6.52; % N, 10.06, observed: % C, 60.38; % H, 6.45; % N, 10.16.

4-Methyl-N-(1-piperidin-1-ylprop-2-en-1-ylidene) benzenesulfonamide (Table 2, product 4h). Colorless solid; 127 mg; 85% yield; mp 75–76 °C; R_f 0.50 (pet. ether/EtOAc 9 : 1); ¹H NMR (300 MHz, CDCl₃): δ 7.75 (d, J = 8.1 Hz, 2H), 7.21 (d, J = 8.1 Hz, 2H), 6.56 (dd, J = 12, 12 Hz, 1H), 5.63 (d, J = 12 Hz, 1H), 5.39 (d, J = 18 Hz, 1H), 3.39–3.28 (m, 4H), 2.38 (s, 3H), 1.65–1.58 (m, 6H) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 164.0, 141.6, 141.3, 129.5, 128.9, 128.7, 127.6, 126.4, 126.3, 123.8, 46.9, 29.6, 25.1, 24.1, 22.6, 21.4 ppm; FT-IR (KBr, thin film): ν 2923, 2855, 1649, 1533, 1483, 1394, 1266, 1136, 1083, 859, 680 cm⁻¹; anal calc. for C₁₅H₂₀N₂O₂S: % C, 61.62; % H, 6.89; % N, 9.58, observed: % C, 61.56; % H, 6.82; % N, 9.49.

4-Methyl-N-(1-morpholin-4-ylprop-2-en-1-ylidene)benzenesulfonamide (Table 2, product 4i). Colorless solid; 120 mg; 80% yield; mp 120–121 °C; R_f 0.53 (pet. ether/EtOAc 9 : 1); ¹H NMR (300 MHz, CDCl₃): δ 7.66 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.1 Hz, 2H), 6.62 (dd, J = 12, 12.3 Hz, 1H), 5.74 (d, J = 18 Hz, 1H), 5.49 (d, J = 18 Hz, 1H), 3.75 (q, J = 6 Hz, 4H), 2.99 (q, J = 6 Hz, 4H), 2.39 (s, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 163.9, 142.1, 140.7, 129.0, 128.1, 126.5, 125.0, 66.3, 29.6, 21.4 ppm; FT-IR (KBr, thin film): ν 2935, 1640, 1517, 1440, 1360, 1260, 1151, 1131, 1113, 1085, 1029, 825, 675 cm⁻¹; anal calc. for C₁₄H₁₈N₂O₂S: % C, 57.12; % H, 6.16; % N, 9.52, observed: % C, 57.07; % H, 6.10; % N, 9.55.

4-Methyl-N-[1-(octahydro-(6H)-pyrrolo[3,4-b]pyridin-6-yl)prop-2-en-1-ylidene]benzene sulfonamide (Table 2, product 4j). White solid; 97 mg; 65% yield; mp 122–124 °C; R_f 0.46 (pet. ether/EtOAc 9 : 1); ¹H NMR (300 MHz, CDCl₃): δ 7.77 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 8.1 Hz, 2H), 6.58 (dd, J = 6.9, 8.1 Hz, 1H), 5.64 (d, J = 12 Hz, 1H), 5.46 (d, J = 18 Hz, 1H), 3.68–3.55 (m, 5H), 3.18–3.13 (m, 2H), 2.71–2.66 (m, 2H), 2.53–2.50 (m, 3H), 2.37 (s, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 163.8, 141.1, 141.0, 129.1, 126.1, 55.3, 55.2, 46.8, 44.8, 28.1, 24.7, 21.4 ppm; FT-IR (KBr, thin film): ν 2922, 1640, 1517, 1462, 1271, 1138, 1087, 846, 718, 694 cm⁻¹; anal calc. for C₁₇H₂₃N₂O₂S: % C, 71.26; % H, 5.98; % N, 6.93, observed: % C, 71.23; % H, 5.91, % N, 6.90.

N′-[(4-Methylphenyl)sulfonyl]-N,N-dibenzylprop-2-enamidamide (Table 2, product 4k). Colorless solid; 139 mg; 93% yield; mp 107–108 °C; R_f 0.56 (pet. ether/EtOAc 9 : 1); ¹H NMR (300 MHz, CDCl₃): δ 7.40 (d, J = 5.4 Hz, 2H), 7.37–7.33 (m, 2H), 7.31–7.27 (m, 2H), 7.26–7.21 (m, 3H), 7.20–7.19 (m, 3H), 7.17–6.98 (m, 2H), 6.48 (dd, J = 12, 18 Hz, 1H), 6.02 (d, J = 12 Hz, 1H), 5.61 (d, J = 18 Hz, 1H), 4.55 (s, 4H), 2.40 (s, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 141.8, 141.1, 139.2, 135.8, 134.9, 129.0, 129.0, 128.8, 128.6, 128.2, 127.9, 127.8, 127.5, 126.7, 126.3, 126.1, 50.4, 50.0, 21.4 ppm; IR (KBr, thin film): ν 2921, 1638, 1546, 1475, 1399, 1265, 1139, 1109, 1087, 880, 685 cm⁻¹; anal calc. for C₂₄H₂₄N₂O₂S: % C, 71.25; % H, 5.98; % N, 6.92, observed: % C, 71.20; % H, 5.92; % N, 6.90.

N′-[(4-Methylphenyl)sulfonyl]-N-phenylprop-2-enamidamide (Table 2, product 4l). Brown solid; 97 mg, 65% yield; mp 132–134 °C; R_f 0.53 (pet. ether/EtOAc 9 : 1); ¹H NMR (300 MHz, CDCl₃), δ 7.97 (d, J = 8.1 Hz, 2H), 7.83 (d, J = 8.1 Hz, 2H), 7.36–7.27 (m, 5H), 6.40 (d, J = 16.8 Hz, 1H), 6.14 (dd, J = 10.5, 10.2 Hz, 1H), 5.81 (d, J = 10.2 Hz, 1H), 2.44 (s, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 162.5, 135.5, 131.3, 129.7, 129.6, 128.5, 128.4, 126.4, 21.4 ppm; FT-IR (KBr, thin film): ν 2924, 1634, 1535, 1476, 1399, 1255, 1140, 1087, 867, 673 cm⁻¹; anal calc. for C₁₆H₁₆N₂O₂S: % C, 63.98; % H, 5.37; % N, 9.33, observed: % C, 63.92; % H, 5.30; % N, 9.29.

N-(2-Methylphenyl)-N′-[(4-methylphenyl)sulfonyl]prop-2-enamidamide (Table 2, product 4m). Brown solid; 90 mg; 60% yield; mp 116–118 °C; R_f 0.50 (pet. ether/EtOAc 9 : 1); ¹H NMR (300 MHz, CDCl₃): δ 7.90 (d, J = 8.1 Hz, 2H), 7.33–7.16 (m, 4H), 7.00–6.96 (m, 2H), 6.64 (dd, J = 7.2, 7.2 Hz, 1H), 6.32 (d, J = 7.8 Hz, 1H), 5.69 (d, J = 20.1 Hz, 1H), 2.46 (s, 3H), 2.19 (s, 3H) ppm; ¹³C NMR (75 MHz, CDCl₃): δ 166.6, 145.0, 143.2, 139.2, 135.0, 134.9, 131.2, 130.3, 129.5, 128.8, 127.8, 127.1, 126.9, 122.6, 117.3, 21.6, 21.1 ppm; FT-IR (KBr, thin film): ν 2920, 1636, 1544, 1509, 1489, 1248, 1138, 1106, 1085, 875, 670 cm⁻¹; anal calc. for C₁₇H₁₈N₂O₂S: % C, 64.94; % H, 5.77; % N, 8.91, observed: % C, 64.90; % H, 5.72; % N, 8.95.

Procedure of the SRB-assay

Tumor cells (human breast cancer cell line MCF-7) were grown in tissue culture flasks in growth medium (RPMI-1640 with 2 mM glutamine, pH 7.4, 10% fetal calf serum, 100 μg mL⁻¹ streptomycin, and 100 units per mL penicillin) at 37 °C under the atmosphere of 5% CO₂ and 95% relative humidity employing a CO₂ incubator. The cells at subconfluent stage were harvested from the flask by treatment with trypsin (0.05% trypsin in PBS containing 0.02% EDTA) and placed in growth medium. The cells with more than 97% viability (trypan blue exclusion) were used for cytotoxicity studies. An aliquot of 100 μL of cells were transferred to a well of 96-well tissue culture plate. The cells were allowed to grow for one day at 37 °C in a CO₂ incubator as mentioned above. The test materials at different concentrations were then added to the wells and cells were further allowed to grow for another 48 h. Suitable blanks and positive controls were also included. Each test was performed in triplicate. The cell growth was stopped by gently layering of 50 μL of 50% trichloroacetic acid. The plates were incubated at 4 °C for an hour to fix the cells attached to the bottom of the wells. Liquids of all the wells were gently pipette out and discarded. The plates were washed five times with doubly distilled water to remove TCA, growth medium, etc. and were air-dried. 100 μL of SRB solution (0.4% in 1% acetic acid) was added to each well and the plates were incubated at ambient temperature for half an hour. The unbound SRB was quickly removed by washing the wells five times with 1% acetic acid. Plates were air dried, tris-buffer (100 μL of 0.01 M, pH 10.4) was added to all the wells and plates were gently stirred for 5 minutes on a mechanical stirrer. The optical density was measured on ELISA reader at 540 nm. The cell growth at absence of any test material was considered 100% and in turn growth inhibition was calculated. GI₅₀ values were determined by regression analysis.

Virtual analysis

Virtual analysis was carried out using biopredicta module of V life MDS 4.3 suite. Crystal structure of the human cell division protein kinase7 (PDB code: 1UA2) was utilized for docking analysis which was downloaded from the protein database www.rcsb.org. Protein structure was cleaned with retaining native hydrogen to the protein. The grip based flexible docking protocol was utilized in which the ligand was made flexible so that multiple conformation of the ligand can be generated. The best 100 conformations were identified based on total binding energy of the drug receptor complex.

Acknowledgements

Authors gratefully acknowledge ACTREC, TATA Memorial Centre for Anticancer Screening and Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy for docking studies.

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Footnote

† Electronic supplementary information (ESI) available. CCDC 968455. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra20583f

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