Synthesis of novel 1,4-disubstituted 1,2,3-triazolo-bosentan derivatives – evaluation of antimicrobial and anticancer activities and molecular docking

Abstract

Novel 1,4-disubstituted 1,2,3-triazolo bosentan derivatives 1a–n from bosentan 2 were synthesized in good yields by sequential chlorination, azidation followed by Cu(I) catalyzed 1,3-dipolar cycloaddition. All obtained compounds 1a–n were evaluated for their antimicrobial and in vitro anticancer activities and by in silico docking studies. Among all tested compounds 1e,f and 1h–j show better antimicrobial activities against the tested bacteria and fungi. When subjected to anticancer testing, compounds 1g–j and 1n show significant activities against both A549 and SKOV-3 cell lines with IC₅₀ values at 7.81 μg mL⁻¹ and among them compound 1i exhibited very potent activity. In addition, no toxicity was calculated up to 2 mg mL⁻¹ in Vero cells. In silico studies were conducted to investigate the possible bonding modes of 1a–n with target receptors namely DNA topoisomerase IV (4 EMV) and anaplastic lymphoma kinase (2XP2). Among them, compounds 1e and 1h show maximum binding energies with 4EMV and 2XP2 receptors, respectively which also exhibited good antimicrobial and potent anticancer activities.

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Introduction

As per the WHO, multidrug-resistant organisms (MDRO) are an increasing threat to human life as well as the animal kingdom.^1–5 Microorganisms gain resistance by mutation of their DNA and tolerate the effect of standard antimicrobial drugs resulting in uncured diseases/infections. Cancer is another major threat to human life. When in the early years of a person’s life, normal body cells divide faster to allow him/her to grow well. The division of cell rate decreases when the person becomes an adult and it happens only to replace exhausted cells or to heal injuries. Abnormal growth of cells results in the formation of cancers which are very difficult to cure and end with death in most cases. Most anticancer drugs have side effects on normal cells and result in some abnormal health conditions. Hence, this situation stresses the need for the recurrent development of new drugs that are more efficient and less expensive⁶ than the existing drugs.

Endothelin A (ET_A) and B (ET_B) receptors, have been implicated in the development of several cancers through activation of pathways involved in cell proliferation, migration, invasion, osteogenesis and angiogenesis.⁷ Targeting the ET receptor and its antagonism constitute an attractive and challenging approach for cancer therapy.^8,9 A large number of ET antagonists have been developed and studied in clinical trials for chronic heart failure, hypertension, cancer and fibrosis.^10–13 Bosentan, a dual ET_A/ET_B receptor antagonist is approved for the treatment of pulmonary arterial hypertension^14–16 and used in combination chemotherapy with cisplatin¹⁷ and paclitaxel for ovarian cancer.¹⁸ The development of a potent bosentan derivative as a specific, selective and dual ET receptor antagonist represents an effective treatment for cancer as a mono drug. Structural modification of bosentan may lead to an increase in selective dual activity with the ET receptor and may cause less or no hepatotoxic (damage to liver – a side effect of bosentan) and anemia.^19,20 The current research activity focuses on the preparation of various 1,4-disubstituted 1,2,3-triazoles linked with the pyrimidine ring of bosentan to give triazolo-bosentan derivatives in order to test their anticancer and antimicrobial activities and to carry out molecular docking studies.

Generally, having two is better than one and the same concept is applicable to hybrid drugs which can have enhanced biological activity and can address issues of drug resistance.^21–23 Many hybrid drugs are known to have advantages, as they can potentially overcome pharmacokinetic drawbacks better than their precursors and conventional drugs.^24–26 These can be prepared by adjoining to two different drugs by a covalent bond or by an adequate fusion.²⁷ Hybrid drugs such as NOSH-aspirin,²⁸ artemisinin-quinine,²⁹ reversed chloroquine³⁰ are well known (Fig. 1).

Fig. 1 Hybrid drugs.

Hybrid compounds of pyrimidines which are associated with other heterocycles exhibit good biological activities.³¹ Presently, the interest in developing new hybrid molecules from pyrimidine derivatives is increasing with vast applications in medicinal chemistry.^32–37 Click chemistry is a brilliant concept in organic chemistry which involves synthesizing complex molecules in high yields with simple isolation techniques.^38,39 Various 1,4-disubstituted 1,2,3-triazole derivatives have been reported in the literature using this well known click chemistry approach by Cu(I) catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC)^40–45 which have broad applications in medicinal chemistry.^46–49

Results and discussion

Chemistry

The key intermediate azide 4 was prepared by heating bosentan 2 with thionyl chloride in chloroform followed by treating with sodium azide in N,N-dimethylacetamide (DMA) (Scheme 1). The targeted new bis-pyrimidinyl linked 1,2,3-triazolo-bosentan derivatives 1a–n were prepared via one pot synthesis by reacting azide 4 with various terminal alkynes 5a′–k′ and 5l–n in the presence of CuI catalyst and N,N-diisopropylethylamine (DIPEA) in aqueous n-butanol medium at ambient temperature using a regular click chemistry approach (Scheme 2). Various conditions have been tried to optimize the method (Table 1). Terminal alkynes 5a′–f′ were obtained in situ by heating their corresponding amines 5a–f with propargyl chloride in the presence of a base (Scheme 2). Terminal alkynes 5g′–k′ were also made in situ by converting their corresponding acids 5g–k to acid chlorides followed by treatment with propargyl alcohol (Scheme 2). Terminal alkynes 5l–n were procured commercially and used as such. About 78–95% of overall yields (Scheme 4 and Table 2) were obtained when the optimized reaction conditions mentioned in this article were followed. Compounds 1a–n were obtained as pale brown to brown amorphous solids and further purification also resulted in amorphous solids hence single crystal XRD studies were not performed.

Scheme 1 Synthesis of azide 4 from bosentan 2.

Scheme 2 Overview of the synthesis of novel 1,4-disubstituted 1,2,3-triazoles 1a–n.

Scheme 3 Synthesis of 1l – reaction optimization.

Table 1 Optimization of reaction conditions^a

Entry	Base	Catalyst (mol%)	Solvent	Temp (°C)	Time (h)	Yield^b (%)
a DIPEA – diisopropylethylamine, TEA – triethylamine.b Isolated yields.
1	DIPEA	CuI (10)	n-BuOH	25–30	10	75
2	DIPEA	CuBr (10)	n-BuOH	25–30	10	15
3	DIPEA	CuCl (10)	n-BuOH	25–30	10	Trace
4	DIPEA	CuI (5)	n-BuOH	25–30	10	46
5	DIPEA	CuI (20)	n-BuOH	25–30	10	93
6	DIPEA	CuI (10)	H2O	25–30	10	48
7	DIPEA	CuI (10)	n-BuOH/H2O (1 : 1)	25–30	10	95
8	DIPEA	CuI (10)	EtOH	25–30	10	Trace
9	DIPEA	CuI (10)	EtOH	45–50	20	15
10	DIPEA	CuI (10)	EtOH/H2O (1 : 1)	45–50	20	27
11	DIPEA	CuI (10)	THF	25–30	20	60
12	TEA	CuI (10)	n-BuOH	25–30	10	31
13	TEA	CuI (10)	THF	25–30	10	46
14	KOH	CuI (20)	n-BuOH	25–30	10	Trace
15	KOH	CuI (20)	n-BuOH	50–55	20	Trace

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Scheme 4 One pot synthesis of 1,4-disubstituted 1,2,3-triazoles 1a–n from azide 4 using click chemistry.

Table 2 Novel 1,4-disubstituted 1,2,3-triazoles from bosentan

Compound	Terminal alkynes prepared from	R	Mp^a (°C)	Yield^b (%)
a Uncorrected melting points.b Obtained yields without any purification.c Obtained from commercial sources and used as such.
1a	Ephedrine	[N-Methyl-(1-hydroxy-1-phenyl)-2-propylamino]	122–125	88
1b	Norephedrine	[(1-Hydroxy-1-phenyl)-2-propylamino]	140–143	78
1c	Selegiline	[1-Phenyl-2-propylamino]	99–102	90
1d	Phenylephrine	[N-Methyl-2-(3-hydroxyphenyl)-2-hydroxyethylamino]	148–150	90
1e	1-Aminoindane	[2,3-Dihydro-1H-inden-1-amino]	102–105	88
1f	Noralfentanil	[4-(Methoxymethyl)-4-(N-phenyl-N-propionamide)piperidino]	180–181	91
1g	Benzoic acid	[Benzoyloxy]	95–98	92
1h	2-Bromo-5-methoxybenzoic acid	[2-Bromo-5-methoxybenzoyloxy]	160–164	84
1i	4-Nitrobenzoic acid	[4-Nitrobenzoyloxy]	168–171	88
1j	Veratroic acid	[3,4-Dimethoxybenzoyloxy]	128–132	90
1k	3-Methylbenzoic acid	[4-Methylbenzoyloxy]	140–144	87
1l	Propargyl alcohol^c	[Hydroxymethyl]	168–170	95
1m	1-Hexyne^c	[n-Butyl]	101–103	89
1n	1-Ethynyl-1-cyclohexanol^c	[(1-Hydroxy)cyclohexyl]	105–107	87

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Optimization of reaction conditions

Various solvents, bases, catalysts, reaction temperature and time have been explored to optimize the reaction conditions during this research work (Table 1) using Scheme 3 as a model experiment. During the study, it was noticed that better results were obtained when CuI (10 mol%), DIPEA and n-BuOH/water mixture were used at room temperature for about 10 h (Table 1, entry 6). Usage of excess CuI did not improve the yield (Table 1, entry 5, 14 & 15) and usage of other copper salts resulted in very poor conversions (Table 1, entry 2 & 3). It can be seen that reaction can proceed in other polar solvents but did not go to completion (Table 1, entries 6–11 & 12). The reaction did not proceed when other bases such as triethylamine and potassium hydroxide were used instead of DIPEA (Table 1, entries 12–15).

The structures of all novel 1,2,3-triazole 1a–n compounds were elucidated with the help of IR, ¹H NMR, ¹³C NMR and mass spectra as exemplified for compound 1e. Characteristic IR bands of 1e: the broad band at 3422 cm⁻¹ represents the –NH stretching. The medium band at 2963 cm⁻¹ indicates the aliphatic stretching. The sharp bands that appear at 1674, 1605 & 1564 cm⁻¹ show the presence of benzenoid rings. The band present at 1393 cm⁻¹ confirms the presence of S=O stretching. The bands at 1252, 1219 & 1173 cm⁻¹ support the presence of –C–N and –C–O stretching. The characteristic bands at 864, 762 & 694 cm⁻¹ represent the existence of para- & ortho-disubstituted and monosubstituted aromatic rings respectively. The signals of ¹H & ¹³C NMR of compound 1e are explained in Fig. 2. A distinguishing peak observed at m/z: 748 in the mass spectrum of 1e corresponds to the protonated molecular ion [M + H]⁺.

Fig. 2 Characterization of 1e using NMR spectroscopy.

Pharmacology

Antimicrobial activity. The synthesized novel bis-pyrimidinyl linked 1,2,3-triazolo-bosentan derivatives 1a–n were screened for antimicrobial activity⁵⁰ using the well method⁵¹ at 1000 μg per well against eleven bacteria and two fungi (Table 3). Minimum inhibitory concentration (MIC) studies of the synthesized compounds 1a–n were performed according to the standard reference method for bacteria, filamentous fungi⁵² and yeasts⁵³ (NCCLS/CLSI, 2002). The MIC values of the synthesized compounds 1a–n were calculated (Table 3). Standard antimicrobial drugs, streptomycin, ciprofloxacin and ketoconazole were used as positive controls against bacteria and fungi respectively in both antimicrobial and MIC study (Table 4).

Table 3 Antimicrobial activity of synthesized compounds 1a–n using the well method (zone of inhibition in mm) (1000 μg per well)^a

Organism	Compound
	1a	1b	1c	1d	1e	1f	1g	1h	1i	1j	1k	1l	1m	1n	C
a C: streptomycin – standard antibacterial agent; C: ketoconazole – standard antifungal agent.
Gram positive bacteria
Bacillus subtilis	16	18	—	14	12	14	18	20	19	19	14	—	14	18	24
Micrococcus luteus	15	12	—	14	20	12	17	15	21	16	12	—	14	18	26
Staphylococcus aureus	13	—	—	12	13	13	12	15	11	14	10	—	17	16	14
Staphylococcus epidermidis	14	16	—	10	14	14	22	21	24	19	14	—	15	20	26
Gram negative bacteria
Klebsiella pneumoniae	13	14	—	15	12	17	21	18	19	18	10	—	13	16	20
Salmonella typhimurium	12	13	—	—	16	16	14	—	—	—	—	—	—	14	24
Proteus vulgaris	13	16	—	14	18	13	17	20	15	20	10	—	16	18	30
Shigella flexneri	15	—	—	15	19	12	18	25	21	20	12	—	18	19	30
Enterobacter aerogenes	16	15	—	14	15	16	20	18	18	17	10	—	16	17	22
Pseudomonas aeruginosa	14	18	—	15	16	13	20	22	21	18	13	—	16	18	30
Staphylococcus aureus MRSA	15	12	—	13	18	10	23	22	19	17	16	—	14	16	30
Fungi
Candida albicans	16	15	—	—	—	—	15	—	14	16	—	—	10	14	28
Malassezia pachydermatis	—	13	—	—	—	—	—	—	—	12	—	—	13	—	26

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Table 4 Minimum inhibitory concentration (MIC) of synthesized compounds 1a–n^a

Organism	Compound
	1a	1b	1d	1e	1f	1g	1h	1i	1j	1k	1m	1n	C-1	C-2
a C-1: streptomycin and C-2: ciprofloxacin – standard antibacterial agents.
Gram positive bacteria
Bacillus subtilis	62.5	62.5	125	62.5	125	62.5	31.25	31.25	62.5	125	250	125	25	<0.78
Micrococcus luteus	250	500	250	62.5	125	500	62.5	62.5	125	500	250	125	6.25	>100
Staphylococcus aureus	250	—	500	250	250	250	250	125	250	500	125	125	6.25	<0.78
Staphylococcus epidermidis	250	125	500	250	62.5	250	62.5	31.2	62.5	250	250	62.5	25	6.25
Gram negative bacteria
Klebsiella pneumonia	250	250	250	125	62.5	125	62.5	125	62.5	500	250	125	6.25	6.25
Salmonella typhimurium	500	250	—	125	250	125	125	—	—	—	—	250	30	>100
Proteus vulgaris	250	125	250	125	125	250	125	250	62.5	500	125	125	6.25	6.25
Shigella flexneri	250	—	250	125	125	500	125	62.5	62.5	500	125	125	6.25	<0.78
Enterobacter aerogenes	125	250	250	250	125	125	62.5	62.5	125	500	125	125	25	<0.78
Pseudomonas aeruginosa	125	62.5	125	62.5	125	31.25	31.25	31.25	62.5	250	125	125	25	6.25
Staphylococcus aureus MRSA	250	500	500	125	31.2	500	31.2	125	125	250	250	125	6.25	<0.78

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The obtained results disclosed good antimicrobial activity of the synthesized compounds 1a–n when compared with the standard antimicrobial drugs used in this study. P. aeruginosa has emerged as one of the most problematic Gram-negative pathogens, with an alarmingly high antibiotic resistance rate. Even with the most effective antibiotics against this pathogen, namely carbapenems (imipenem and meropenem), the resistance rate was found to be 15–20.4% amongst aeruginosa strains.⁵⁴ Our current study showed that the synthesized compounds were active against P. aeruginosa. Compounds, 1e,f & h–j show better activity when compared with others against tested bacteria and fungi. The best MIC values were observed for compounds 1h–j. The grounds for this better activity in 1h and 1j may be due to the presence of methoxy substitutions on the benzoic acid which can increase the in vitro activity against Gram positive bacteria when compared to other compounds.^55,56 The reason for the best activity of 1i may be due to the presence of para-nitro substitution on the benzoic acid.

Anticancer activity. Based on the antimicrobial results, compounds 1b and 1e–n were taken selectively and screened for anticancer activity against A549 and SKOV-3 cancer cell lines.⁵⁷ All the tested compounds showed good cytotoxicity activity against A549 and SKOV-3 cell lines, however, some of the synthesized compounds showed prominent cytotoxicity activity in vitro against A549 and SKOV-3 cell lines. The anticancer activity against A549 and SKOV-3 cell lines was observed in 250 to 7.81 μg mL⁻¹ concentrations (Table 5, 6 and Fig. 3, 4). The results show that compounds 1e–j & 1l show significant activity against the A549 cell line when compared to other compounds. In particular, 1g, i, j & l exhibit very potent cytotoxicity activity against the A549 cell line with IC₅₀ values at 7.81 μg mL⁻¹. Interestingly significant activity against SKOV-3 cell lines was also noticed for compounds 1e–j & 1l–n when compared with other compounds. Among the tested compounds 1g, i, j & n showed potent cytotoxicity activity against the SKVO-3 cell line with IC₅₀ values at 7.81 μg mL⁻¹. All concentrations used in the experiment decreased the cell viability significantly (P < 0.05) in a concentration-dependent manner. Interestingly, significant anticancer results were seen in triazolo-bosentan derivatives prepared by using aromatic carboxylic acids 5g–j except for 1k. Compound 1b, prepared from an amine 5b was found to be the least active when compared to the others. Compound 1i shows the best activity for both cell lines and the reason might be the presence of an electron withdrawing group (–NO₂) at the para-position of the benzoic acid 5i. Triazolo-bosentans obtained from aliphatic alkynes 1l–n also showed good activity. Toxicity was tested against Vero cells. Interestingly triazolo-bosentan derivatives of 1e–j, 1l, and 1n showed no toxicity up to 2 mg mL⁻¹.

Table 5 Anticancer activity of synthesized compounds against the A549 cancer cell line

Concentration (μg mL^−1)		Compound
		1b	1e	1f	1g	1h	1i	1j	1k	1l	1m	1n
250	%	31.3	89.2	91.5	96.3	94.7	96.2	94.2	47.2	95.6	19	93.7
	Mean ± S.D.	1.345 ± 0.00325	0.211 ± 0.00839	0.166 ± 0.00712	0.072 ± 0.00337	0.103 ± 0.00441	0.074 ± 0.00118	0.114 ± 0.00890	1.033 ± 0.00881	0.087 ± 0.00879	1.586 ± 0.00557	0.123 ± 0.00997
125	%	27	84.9	62.5	95.8	87	95.5	90.9	35.6	90.8	16.7	80.9
	Mean ± S.D.	1.429 ± 0.00412	0.295 ± 0.00559	0.735 ± 0.00660	0.082 ± 0.00559	0.255 ± 0.00578	0.088 ± 0.00229	0.179 ± 0.00773	1.261 ± 0.00836	0.18 ± 0.00706	1.631 ± 0.00967	0.123 ± 0.00997
62.5	%	24.9	51.2	18.2	82.7	81.5	95.3	86.3	25.6	80.6	14.9	56.1
	Mean ± S.D.	1.47 ± 0.00522	0.955 ± 0.00436	1.602 ± 0.00578	0.338 ± 0.00631	0.363 ± 0.00669	0.091 ± 0.00397	0.268 ± 0.00559	1.456 ± 0.00771	0.38 ± 0.00756	1.666 ± 0.00805	0.859 ± 0.00361
31.25	%	16.4	42.9	13.5	71.9	60.6	79.9	75	14.5	77.9	10.2	18.6
	Mean ± S.D.	1.636 ± 0.00632	1.118 ± 0.00947	1.694 ± 0.00309	0.55 ± 0.00779	0.771 ± 0.00330	0.393 ± 0.00433	0.489 ± 0.00397	1.675 ± 0.00743	0.432 ± 0.00801	1.759 ± 0.00598	1.594 ± 0.00772
15.63	%	13.1	30.9	11.9	68.1	51.2	75	71.6	11.1	66.1	8.2	15.5
	Mean ± S.D.	1.701 ± 0.00203	1.352 ± 0.00871	1.725 ± 0.00399	0.625 ± 0.00597	0.956 ± 0.00449	0.489 ± 0.00507	0.556 ± 0.00227	1.741 ± 0.00491	0.663 ± 0.00699	1.797 ± 0.00671	1.654 ± 0.00589
7.81	%	5.2	26.7	8.6	62	30.5	68.2	57.8	8.1	54.1	6.7	9.6
	Mean ± S.D.	1.856 ± 0.00654	1.436 ± 0.00856	1.789 ± 0.00580	0.745 ± 0.00888	1.361 ± 0.00379	0.623 ± 0.00409	0.826 ± 0.01025	1.799 ± 0.00513	0.899 ± 0.00712	1.826 ± 0.00334	1.77 ± 0.00665

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Table 6 Anticancer activity of synthesized compounds against the SKOV-3 cancer cell line

Concentration (μg mL^−1)		Compound
		1b	1e	1f	1g	1h	1i	1j	1lk	1l	1m	1n
250	%	48.9	71.2	81.2	94.3	94	94.7	92.6	39.9	95.3	84.7	91.6
	Mean ± S.D.	0.653 ± 0.00338	0.368 ± 0.00443	0.24 ± 0.00556	0.073 ± 0.00702	0.077 ± 0.00507	0.068 ± 0.00560	0.095 ± 0.00363	0.768 ± 0.00338	0.06 ± 0.00447	0.195 ± 0.00337	0.107 ± 0.00447
125	%	40.5	54	64.9	87.2	92.2	93.7	83.6	34.9	93.9	77.7	81
	Mean ± S.D.	0.76 ± 0.00561	0.588 ± 0.00881	0.448 ± 0.00773	0.164 ± 0.00564	0.1 ± 0.00812	0.08 ± 0.00609	0.21 ± 0.00569	0.831 ± 0.00501	0.078 ± 0.00510	0.285 ± 0.00429	0.242 ± 0.00366
62.5	%	34.1	42	40.4	80.3	89.8	91.6	81.8	23.4	63.8	65.3	86.3
	Mean ± S.D.	0.841 ± 0.00201	0.741 ± 0.00779	0.761 ± 0.00501	0.252 ± 0.00328	0.13 ± 0.00328	0.107 ± 0.00544	0.232 ± 0.00438	0.978 ± 0.00588	0.462 ± 0.00553	0.443 ± 0.00577	0.175 ± 0.00228
31.25	%	31.6	40.1	30.1	69.5	65.8	85.2	77.6	16.2	54.9	50.1	80.7
	Mean ± S.D.	0.874 ± 0.00198	0.765 ± 0.00278	0.893 ± 0.00337	0.389 ± 0.00209	0.437 ± 0.00449	0.189 ± 0.00224	0.286 ± 0.00371	1.07 ± 0.00579	0.576 ± 0.00438	0.637 ± 0.00804	0.246 ± 0.00509
15.63	%	24.6	28.6	22	63.6	57.1	81.9	72.1	9.2	51.1	31.2	75.3
	Mean ± S.D.	0.963 ± 0.00854	0.912 ± 0.00900	0.996 ± 0.00886	0.465 ± 0.00667	0.548 ± 0.00579	0.231 ± 0.00697	0.356 ± 0.00399	1.159 ± 0.00416	0.625 ± 0.00688	0.878 ± 0.00331	0.315 ± 0.00708
7.81	%	6	22.7	9.5	60.1	50.3	76.4	63.4	3.2	33.7	22.2	63.3
	Mean ± S.D.	1.201 ± 0.00337	0.987 ± 0.00458	1.156 ± 0.00927	0.51 ± 0.00345	0.635 ± 0.00670	0.301 ± 0.00555	0.468 ± 0.00608	1.236 ± 0.00399	0.847 ± 0.00701	0.993 ± 0.00209	0.469 ± 0.00884

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Fig. 3 Comparison of anticancer activity of synthesized compounds against A 549 cancer cell line (note: no toxicity was observed up to 2 mg mL⁻¹ in Vero cells).

Fig. 4 Comparison of anticancer activity of synthesized compounds against SKOV-3 cancer cell line.

In silico studies. Molecular docking is a useful instrument to acquire knowledge on protein–ligand interactions which are very significant in drug discovery.⁵⁸ All the synthesized pyrimidinyl linked 1,2,3-triazolo-bosentan derivatives 1a–n were investigated by molecular docking studies with DNA topoisomerase IV (PDB ID: 4EMV) and anaplastic lymphoma kinase (ALK) (PDB ID: 2XP2) using the AutoDock Tools (ADT) version 1.5.6 and AutoDock version 4.2.5.1 docking program^59,60 to investigate the potential binding mode of inhibitors. The DNA topoisomerase IV receptor is required for maintenance of proper DNA topology during transcription and replication in bacteria.⁶¹ Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor. Due to its critical role of aberrant signalling in cancer, it is an attractive oncology target for therapeutic intervention.⁶² The obtained free energy of binding (FEB) was computed for all the synthesized compounds 1a–n (Table 7).

Table 7 Free energy of binding (FEB) of 1a–n

Compound	Free energy of binding^a (kcal mol^−1)
	DNA topoisomerase IV (4EMV)	Anaplastic lymphoma kinase (2XP2)
a Calculated by AutoDock, NC – not calculated, inhibitor: co-crystallized inhibitor with protein.
1a	−6.95	NC
1b	−5.23	−8.54
1c	−6.58	NC
1d	−5.53	NC
1e	−9.29	−8.66
1f	−7.97	−8.01
1g	−8.40	−8.32
1h	−6.71	−9.55
1i	−7.09	−7.94
1j	−8.13	−6.70
1k	−7.04	−8.70
1l	−6.08	−8.42
1m	−7.44	−8.78
1n	−7.28	−8.28
Inhibitor	−9.80	−8.42

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The molecular docking outcomes of synthesized compounds 1a–n with the 4EMV receptor established that all the docked compounds 1a–n bind efficiently with the receptor and exhibit free energy of binding values from −5.23 to −9.29 kcal mol⁻¹. Interestingly, among all the compounds docked, compound 1e exhibits very high binding with the 4EMV receptor and forms five polar interactions with three amino acids, namely ASP-78, GLY-82 and ARG-140, resulting in a binding energy of −9.29 kcal mol⁻¹. In compound 1e, the N–H attached to the triazole interacts with ASP-78 and forms two polar interactions with bond lengths of 1.7 and 3.3 Å respectively. Likewise, the two nitrogens of the triazole interact with GLY-82 and form two polar interactions with bond lengths of 3.2 and 3.5 Å respectively. Aside from this, the pyrimidine nitrogen forms a polar interaction with the ARG-140 with a bond length of 2.9 Å (Fig. 5).

Fig. 5 Docking with the 4EMV receptor (A) method validation using crystallised and docked ligand; (B) docking mode of all the compounds; (C) docking mode of the highest binding energy compound 1e.

Docking of the synthesized compounds with 2XP2 revealed that compounds efficiently bind with the active site of the 2XP2 receptor and exhibit free energies of binding from −6.70 to −9.55 kcal mol⁻¹. Compounds interact with the active site amino acids of 2XP2 namely ARG-1120, LEU-1122, GLY-1123, VAL-1130, GLU-1132, ALA-1148, LYS-1150, LEU-1196, GLU-1197, LEU-1198, MET-1199, ALA-1200, GLY-1201, GLY-1202, ASP-1203, SER-1206, PHE-1207, GLU-1210, ARG-1253, ASN-1254, CYS-1255, LEU-1256, GLY-1269 and ASP-1270. Among all the compounds docked, compound 1h exhibited very high binding with the 2XP2 receptor and formed seven polar interactions with five amino acids and resulted in a binding energy of −9.55 kcal mol⁻¹. In compound 1h, the nitrogens of the two pyrimidines interact with C=O of LEU-1122 and ASP-1203 to form three polar interactions with bond lengths of 2.7, 3.2 and 3.4 Å. In addition, the nitrogen and oxygen of the triazole and phenoxy groups interact with the C=O and N–H of MET-1199 and LYS-1150 to form two polar interactions of 2.0 and 3.4 Å respectively. Also, the N–H and S=O interact with the C=O of GLY-1123 to form two polar interactions of 2.0 and 3.4 Å respectively. The binding interaction with the 2XP2 receptor is shown in Fig. 6.

Fig. 6 Docking with 2XP2 receptor (A) method validation using crystallised and docked ligand; (B) docking mode of all the compounds; (C) docking mode of the highest binding energy compound 1h.

Conclusions

In summary, we have described the facile one pot synthesis of novel bis-pyrimidinyl linked 1,4-disubstituted 1,2,3-triazoles 1a–n (triazolo-bosentans) with good yields from bosentan 2 as potent hybrid drugs with dual biological activity (antimicrobial and anticancer) and rationalized the activity with the respective proteins using a docking study. During this work good antimicrobial activities were observed for compounds 1e,f & 1h–j against tested bacteria and fungi, amongst which, 1h and 1j exhibited very good activity. In vitro cytotoxicity (lung and ovarian) studies against A549 and SKOV-3 cell lines revealed prominent cytotoxic activity of compounds 1e–n. Among all triazolo-bosentans screened, (1g, i, j) and (1l, e–j, l–n) showed very significant in vitro cytotoxicity against A549 and SKOV-3 cell lines respectively with an IC₅₀ value at 7.81 μg mL⁻¹. Active compounds 1e–j, 1l and 1n were tested against Vero cells and no toxicity was observed up to 2 mg mL⁻¹. When the structure and activity were correlated, it was concluded that antimicrobial and anticancer activities were enhanced by methoxy substitutions on the benzoic acid ring. Among the tested compounds, the best anticancer activity was observed for p-nitro substituted triazolo-bosentan 1i which also exhibited good antimicrobial activity. Free energies of binding were calculated for the synthesized compounds against 4EMV and 2XP2 receptors. Among them, 1e shows a better binding energy (−9.29 kcal mol⁻¹) against the 4EMV receptor with five polar interactions by three amino acids and 1h shows a better binding energy (−9.55 kcal mol⁻¹) against the 2PX2 receptor with seven polar interactions by five amino acids. Based on the significant anticancer results, this class of compounds can be considered as a good starting point for the development of bosentan 2 based dual endothelin receptor (ET) antagonists for efficient mono therapy for cancer.

Experimental

Chemistry

Melting points were measured using a Veego melting point apparatus model VMP-PM. Thin layer chromatography (TLC) was carried out using pre-coated Merck TLC Silica gel 60 F254 and spots were detected using Ultra-Violet light. IR spectra were recorded (KBr pellet) on a Shimadzu Prestige 21 FTIR instrument in the range of 4000 to 400 cm⁻¹. ¹H NMR and ¹³C NMR spectra were recorded using a Bruker-Avance 300 MHz FT-NMR spectrometer (300 and 75 MHz, respectively) using DMSO-d₆ as solvent and TMS as internal standard. Chemical shifts (δ) are given in parts per million (ppm). The following abbreviations are used: s-singlet, d-doublet, t-triplet, q-quintet and m-multiplet. Low resolution mass spectra were recorded on an Agilent 6110LC/MS mass spectrophotometer using the ESI mode. The elemental analysis was done (sample thoroughly dried under vacuum) using a Thermo Fischer Flash 1112 Series elemental analyzer. For antimicrobial activity, the reference cultures were obtained from the Institute of Microbial Technology (IMTECH), Chandigarh, India-160 036 and the remaining cultures were obtained from the Department of Microbiology, Christian Medical College, Vellore, Tamil Nadu, India.

Experimental procedure for the synthesis of compound (3). Thionyl chloride (8 mL, 0.11 mol) was added to bosentan 2 (55 g, 0.10 mol) in chloroform (500 mL) and heated to 55 to 60 °C for 3 h under nitrogen. Excess thionyl chloride and chloroform were completely removed under vacuum to get a yellow solid which was filtered and washed with acetone (120 mL) to give a solid (48 g) after drying.
4-tert-Butyl-N-(6-(2-chloroethoxy)-5-(2-methoxyphenoxy)-2,2′-bipyrimidin-4-yl)benzene-sulfonamide (3). Yellow solid. Yield 85%. Mp: 198–200 °C; IR: 3252, 2954, 2904, 2835, 1684, 1618, 1570, 1564, 1554, 1499, 1455, 1438, 1397, 1384, 1284, 1253, 1213, 1173, 1162, 1155, 1111, 1083, 1026, 993, 874, 833, 827, 741, 698, 679, 625, 572, and 545 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ_H: 1.36 (s, 9H), 3.88 (s, 3H), 4.51 (t, 2H, J = 8.0 Hz), 5.18 (t, 2H, J = 8.0 Hz), 6.76–10.02 (m, 11H, aromatic) and 11.62 (bs, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ_C: 31.33, 35.41, 37.73, 52.59, 56.27, 113.48, 115.93, 120.76, 123.86, 124.58, 126.04, 127.33, 129.15, 138.24, 141.61, 145.71, 147.01, 149.26, 149.52, 153.73, 155.09, 156.59 and 165.98. ESI-MS: [M + H]⁺ at m/z 570 (Cl³⁵) & 572 (Cl³⁷) in 3 : 1 ratio. Anal. calcd for C₂₇H₂₈ClN₅O₅S: C, 56.89; H, 4.95; N, 12.29. Found C, 57.12; H, 4.99; N, 12.35.

Experimental procedure for the synthesis of azide (4). N,N-Dimethylacetamide (250 mL) was added to 3 (46 g, 0.08 mol), followed by sodium azide (6.5 g, 0.1 mol) and stirred at ambient temperature for 16 to 20 h. Completion of the reaction was confirmed by TLC (10% methanol in ethyl acetate) after which, 500 mL of water was added to the reaction mass and the obtained solid was filtered and dried under vacuum. The obtained azide 4 (43 g) was preserved in a refrigerator.
N-(6-(2-Azidoethoxy)-5-(2-methoxyphenoxy)-2,2′-bipyrimidin-4-yl)-4-tert-butylbenzenesulfonamide (azide 4). Pale brown solid. Yield 93%. Mp: 172–175 °C. IR: 3245, 3066, 2959, 2903, 2835, 2795, 2090, 1681, 1617, 1563, 1550, 1499, 1455, 1397, 1386, 1333, 1254, 1215, 1179, 1162, 1111, 1084, 926, 873, 833, 824, 768, 749, 625, 574 and 545 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ_H: 1.26 (s, 9H), 3.47 (t, 2H, J = 6.2 Hz), 3.83 (s, 3H), 4.12 (t, 2H, J = 18.3 Hz), 6.67–9.11 (m, 11H, aromatic), 11.30 (bs, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ_C: 31.23, 35.24, 44.34, 49.06, 56.18, 113.35, 115.33, 120.85, 122.84, 123.29, 124.33, 125.60, 128.53, 138.48, 146.14, 146.92, 149.33, 151.31, 156.26, 157.27, 158.38 and 159.58. ESI-MS: [M + H]⁺ at m/z 577. Anal. calcd for C₂₇H₂₈N₈O₅S: C, 56.24; H, 4.89; N, 19.43. Found C, 56.55; H, 4.93; N, 19.52.

General experimental procedure for one pot synthesis of (1a–f). Amine 5a–f (0.010 mol), n-butanol (10 mL), potassium carbonate (1.7 g, 0.012 mol, pre-dried at 100 °C) and propargyl chloride (0.75 g, 0.010 mol) were mixed and heated at 55 to 60 °C for 6 h. The mass was cooled to 20 °C and charged with water (10 mL), DIPEA (1.3 g, 0.02 mol), copper iodide (200 mg, 10 mol%) followed by azide 4 (5.8 g, 0.01 mol) and agitated at ambient temperature for 6 to 10 h. Completion of the reaction was checked by TLC (10% methanol/ethyl acetate). To the mass, 50 mL of water and 50 mL of ethyl acetate were charged, stirred and filtered to remove inorganic. The organic layer was separated, dried over anhydrous sodium sulphate followed by vacuum distillation using a rota evaporator at below 60 °C yielding a semi solid. The title compounds 1a–f were obtained as pale brown to brown amorphous solids in between 78 and 91% yields, when it was triturated with n-heptane, filtered and dried under vacuum at 60 °C.
4-tert-Butyl-N-(6-(2-(4-((((1S,2R)-1-hydroxy-1-phenyl-2-propyl)(methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)-ethoxy)-5-(2-methoxyphenoxy)-2,2′-bipyrimidin-4-yl)-benzenesulfonamide (1a). Pale brown amorphous solid. Yield 6.7 g (88%). Mp: 122–125 °C. IR: 3373, 2961, 2924, 2853, 1658, 1651, 1587, 1562, 1520, 1499, 1454, 1395, 1250, 1217, 1177, 1138, 1103, 1078, 851, 750, 702, 629 and 577 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ_H 0.90 (d, 3H, J = 14.4 Hz), 1.01 (s, 9H), 2.71 (t, 2H, J = 12.2 Hz), 3.29 (s, 2H), 3.81 (s, 3H), 4.19 (s, 3H), 4.64 (t, 2H), 4.98 (m, 1H), 5.79 (bs, 1H), 6.78–7.72 (m, 17H, aromatic) and 8.97 (bs, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ_C 21.59, 26.92, 27.17, 29.75, 30.03, 31.59, 34.73, 48.15, 56.15, 63.48, 113.18, 113.29, 113.55, 113.77, 120.84, 120.86, 124.41, 124.55, 125.18, 125.25, 126.29, 126.32, 126.69, 126.76, 126.79, 127.03, 127.10, 127.89, 128.15, 128.67 and 157.98. ESI-MS m/z 780 [M + H]⁺. Anal. calcd for C₄₀H₄₅N₉O₆S: C, 61.60; H, 5.82; N, 16.16. Found C, 62.20; H, 5.87; N, 16.10.
4-tert-Butyl-N-(6-(2-(4-(((1S,2R)-1-hydroxy-1-phenyl-2-propyl-amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)-5-(2-methoxy-phenoxy)-2,2′-bipyrimidin-4-yl)benzenesulfonamide (1b). Brown amorphous solid. Yield 6.0 g (78%). Mp: 140–143 °C. IR: 3356, 2959, 2924, 2853, 1668, 1603, 1562, 1499, 1456, 1393, 1341, 1252, 1219, 1175, 1140, 1103, 1080, 1022, 908, 852, 750, 702, 627, 575 and 548 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ_H 0.94 (d, 3H, J = 10.5 Hz), 1.24 (s, 9H), 3.18 (s, 2H), 3.83 (s, 3H), 4.11 (t, 2H, J = 12.2 Hz), 4.59 (t, 2H, J = 12.2 Hz), 5.10 (d of d, 1H, J = 56 Hz & J = 20.2 Hz), 5.92 (m, 1H), 6.56–7.59 (m, 17H), 8.94 (bs, 1H) and 9.00 (bs, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ_C 14.33, 22.62, 29.46, 29.90, 31.40, 31.76, 32.02, 34.86, 56.14, 113.23, 114.51, 115.11, 116.05, 120.35, 120.84, 123.19, 123.64, 125.15, 126.31, 127.84, 128.19, 128.21, 128.24, 128.32, 128.66, 128.99, 129.92, 131.86, 133.48, 139.58, 153.83 and 158.22. ESI-MS m/z 766 [M + H]⁺. Anal. calcd for C₃₉H₄₃N₉O₆S: C, 61.16; H, 5.66; N, 16.46. Found C, 61.30; H, 5.69; N, 16.50.
4-tert-Butyl-N-(5-(2-methoxyphenoxy)-6-(2-(4-((methyl(1-phenylpropan-2-yl)-amino)methyl)-1H-1,2,3-triazol-1-yl)-ethoxy)-2,2′-bipyrimidin-4-yl)benzenesulfonamide (1c). Brown amorphous solid. Yield 6.9 g (90%). Mp: 99–102 °C. IR: 3447, 3063, 2961, 2924, 2853, 1653, 1595, 1562, 1499, 1458, 1393, 1250, 1221, 1175, 1140, 1107, 1076, 849, 746, 702, 631, 577 and 546 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ_H1.04 (s, 3H), 1.24 (s, 9H), 2.62 (t, 2H, J = 11.3 Hz), 3.14 (t, 2H, J = 15.3 Hz), 3.48 (s, 3H), 3.83 (s, 3H), 4.18 (1H, m), 4.24 (d of d, 2H), 4.65 (t, 2H), 6.60–7.73 (m, 17H, aromatic) and 9.01 (bs, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ_C 29.47, 29.90, 30.34, 31.39, 31.76, 32.09, 34.99, 36.07, 45.11, 56.11, 113.13, 114.37, 120.84, 122.46, 122.54, 122.80, 124.79, 124.95, 126.92, 127.50, 127.78, 128.08, 128.17, 128.91, 129.08, 129.72, 130.12, 133.33, 153.45 and 158.15. ESI-MS m/z 764 [M + H]⁺. Anal. calcd for C₄₀H₄₅N₉O₅S: C, 62.89; H, 5.94; N, 16.50. Found C, 62.85; H, 5.98; N, 16.55.
4-tert-Butyl-N-(6-(2-(4-(((2-hydroxy-2-(3-hydroxyphenyl)-ethyl)(methyl)amino)-methyl)-1H-1,2,3-triazol-1-yl)ethoxy)-5-(2-methoxyphenoxy)-2,2′-bipyrimidin-4-yl)-benzenesulfonamide (1d). Brown amorphous solid. Yield 7.1 g (90%). Mp 148–150 °C. IR: 3443, 3067, 2963, 2868, 1653, 1693, 1593, 1564, 1499, 1456, 1396, 1252, 1217, 1180, 1138, 1103, 1078, 1047, 906, 837, 752, 702, 630 and 577 cm⁻¹. ESI-MS m/z 782 [M + H]⁺. Anal. calcd for C₃₉H₄₃N₉O₇S: C, 59.91; H, 5.54; N, 16.12. Found C, 60.05; H, 5.57; N, 16.24.
4-tert-Butyl-N-(6-(2-(4-((2,3-dihydro-1H-inden-1-ylamino)-methyl)-1H-1,2,3-triazol-1-yl)ethoxy)-5-(2-methoxyphenoxy)-2,2′-bipyrimidin-4-yl)benzenesulfonamide (1e). Yellowish brown solid. Yield 6.6 g (88%). Mp: 102–105 °C. IR: 3422, 2963, 2868, 1674, 1605, 1564, 1499, 1458, 1393, 1340, 1252, 1219, 1173, 1111, 1084, 1020, 864, 837, 762, 625 and 575 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ_H 1.25 (s, 9H), 2.24 &2.41 (m, 2H, J = 7.5 Hz), 2.87 (m, 2H), 3.14 (m, 2H), 3.82 (s, 3H), 4.22 (s, 2H), 4.25 (t, 2H, J = 8.0 Hz), 4.73 (t, 2H, J = 12 Hz), 6.67–8.24 (m, 16H, aromatic) and 9.75 (bs, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ_C 28.80, 28.90, 30.28, 31.28, 35.21, 36.61, 45.35, 56.14, 61.13, 97.74, 104.52, 108.11, 113.21, 115.20, 120.80, 125.45, 125.50, 125.53, 125.57, 126.47, 126.71, 127.05, 127.16, 128.45, 129.91, 137.73, 138.20, 138.79, 145.41, 149.17, 157.42, 158.36 and 159.24. ESI-MS m/z 748 [M + H]⁺. Anal. calcd for C₃₉H₄₁N₉O₅S: C, 62.63; H, 5.53; N, 16.86. Found C, 62.53; H, 5.59; N, 16.94.
N-(1-(2-(1-((6-(4-tert-Butylphenylsulfonamido)-5-(2-methoxy-phenoxy)-2,2′-bipyrimidin-4-yl)methyl)-1H-1,2,3-triazol-4-yl-oxy)ethyl)-4-(methoxymethyl)piperidin-4-yl)-N-phenylpropionamide (1f). Pale brown solid. Yield 8.1 g (91%). Mp 180–181 °C. IR: 3474, 2965, 2833, 1651, 1589, 1562, 1518, 1499, 1456, 1396, 1377, 1323, 1250, 1229, 1178, 1140, 1107, 1078, 1002, 849, 750, 706, 633, 579 and 552 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ_H 0.79 (t, 3H, J = 7.3 Hz), 1.23 (s, 9H), 1.69, (t, 4H), 1.98 (t, 4H), 3.32 (s, 3H), 3.52 (q, 2H), 3.85 (t, 2H), 3.93 (s, 2H), 4.12 (s, 2H), 4.56 (t, 2H), 6.51–7.74 (m, 17H) and 8.95 (bs, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ_C 9.97, 23.30, 28.29, 28.70, 30.47, 31.53, 31.59, 34.80, 59.28, 76.57, 79.46, 89.08, 113.80, 120.86, 122.40, 124.47, 129.13, 131.52, 133.47, 134.01, 135.10, 136.29, 139.18, 140.51, 141.35, 146.09, 149.15, 150.32, 152.92 and 168.34. ESI-MS m/z 891 [M + H]⁺. Anal. calcd for C₄₆H₅₄N₁₀O₇S: C, 62.00; H, 6.11; N, 15.72. Found C, 62.39; H, 6.15; N, 15.89.

General experimental procedure for one pot synthesis of 1g–k. In a nitrogen atmosphere, thionyl chloride (0.8 mL, 0.011 mol) was added to acid 5g–k (0.010 mol) in chloroform (10 mL) at 50 to 55 °C for 3 to 4 h. Then it was cooled to about 10 °C and propargyl alcohol (0.56 g, 0.010 mol) was added and stirred for 4–6 h at ambient temperature. Further it was heated to 50 °C and the solvent was distilled off completely under vacuum and the obtained residue was charged with n-butanol (10 mL), water (10 mL), DIPEA (2.6 g, 0.02 mol) and copper iodide (200 mg, 10 mol%) followed by azide 4 (5.8 g, 0.01 mol) and stirred at ambient temperature for 6 to 10 h. Completion of the reaction was checked by TLC (10% methanol/ethyl acetate). To the mass, 50 mL of water and 50 mL of ethyl acetate were charged, stirred and filtered to remove inorganics. The organic layer was separated, dried over anhydrous sodium sulphate followed by vacuum distillation using a rota evaporator at below 60 °C yielding a semi solid. The title compounds 1g–k were obtained as a pale brown to brownish amorphous solids in between 84 and 92% yield when it was triturated with n-heptane, filtered and dried under vacuum at 60 °C.
(1-(2-(6-(4-tert-Butylphenylsulfonamido)-5-(2-methoxyphenoxy)-2,2′-bipyrimidin-4-yl-oxy)ethyl)-1H-1,2,3-triazol-4-yl)-methyl benzoate (1g). Yellowish brown solid. Yield 6.8 g (92%). Mp: 95–98 °C. IR: 3144, 3067, 2963, 2926, 2868, 2853, 1719, 1676, 1605, 1564, 1499, 1452, 1393, 1340, 1271, 1254, 1219, 1173, 1111, 1084, 1024, 864, 835, 752, 714, 692, 625, 573 and 552 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ_H 1.25 (s, 9H), 3.84 (s, 3H), 4.34 (t, 2H, J = 9.5 Hz), 4.66 (t, 2H, J = 8.0 Hz), 5.32 (s, 2H), 6.70–9.03 (m, 17H, aromatic) and 11.30 (bs, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ_C 31.23, 35.25, 51.34, 56.16, 58.33, 59.58, 113.24 to 165.90. ESI-MS m/z 737 [M + H]⁺. Anal. calcd for C₃₇H₃₆N₈O₇S: C, 60.31; H, 4.92; N, 15.21. Found C, 60.25; H, 4.96; N, 15.20.
(1-(2-(6-(4-tert-Butylphenylsulfonamido)-5-(2-methoxyphenoxy)-2,2′-bipyrimidin-4-yl-oxy)ethyl)-1H-1,2,3-triazol-4-yl)-methyl 2-bromo-5-methoxybenzoate (1h). Pale brown solid. Yield 7.2 g (84%). Mp: 160–164 °C. IR: 3068, 2965, 2905, 1709, 1674, 1602, 1565, 1515, 1500, 1464, 1456, 1419, 1394, 1343, 1290, 1271, 1253, 1220, 1176, 1140, 1111, 1084, 1022, 869, 835, 763, 753, 696, 627, 575 and 547 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ_H 1.26 (s, 9H), 3.77 (s, 3H), 3.83 (s, 3H), 4.32 (t, 2H), 4.67 (t, 2H), 5.32 (s, 2H), 6.69–9.03 (m, 15H, aromatic) and 11.30 (bs, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ_C 31.24, 35.24, 45.69, 45.76, 47.91, 56.20, 58.98, 110.74, 113.29, 115.52, 116.56, 119.56, 120.85, 122.75, 123.23, 124.44, 125.65, 125.91, 128.52, 133.26, 135.26, 138.38, 146.09, 146.69, 149.27, 151.17, 156.31, 157.38, 158.26, 158.85, 159.10 and 165.60. ESI-MS m/z 847 [M + H]⁺. Anal. calcd for C₃₈H₃₇BrN₈O₈S; C, 59.60; H, 4.87; N, 14.63. Found C, 59.69; H, 4.93; N, 14.70.
(1-(2-(6-(4-tert-Butylphenylsulfonamido)-5-(2-methoxyphenoxy)-2,2′-bipyrimidin-4-yl-oxy)ethyl)-1H-1,2,3-triazol-4-yl)-methyl 4-nitrobenzoate (1i). Brown solid. Yield 7.9 g (88%). Mp: 168–171 °C. IR: 3111, 3078, 2965, 2869, 2838, 1726, 1675, 1607, 1565, 1528, 1499, 1456, 1393, 1342, 1267, 1253, 1219, 1172, 1112, 1102, 1084, 1014, 872, 858, 762, 752, 720, 625, 575 and 546 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ_H 1.25 (s, 9H), 3.83 (s, 3H), 4.34 (t, 2H, J = 9.5 Hz), 4.67 (t, 2H, J = 8.0 Hz), 5.38 (s, 2H), 6.69–9.03 (m, 16H, aromatic) and 11.31 (bs, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ_C 31.58, 35.24, 47.82, 56.13, 59.05, 59.26, 124.39, 124.75, 125.46, 125.78, 127.62, 128.51, 131.22, 131.86, 132.12, 135.23, 142.50, 142.81, 146.92, 148.26, 150.77, 158.25, 160.23, 161.22, 161.58, 162.12, 164.45 and 164.56; ESI-MS m/z 891 [M + H]⁺. Anal. calcd for C₃₇H₃₅N₉O₉S: C, 56.84; H, 4.51; N, 16.12. Found C, 56.98; H, 4.55; N, 16.19.
(1-(2-(6-(4-tert-Butylphenylsulfonamido)-5-(2-methoxyphenoxy)-2,2′-bipyrimidin-4-yl-oxy)ethyl)-1H-1,2,3-triazol-4-yl)-methyl 3,4-dimethoxybenzoate (1j). Brown solid. Mp 128–132 °C. Yield 7.2 g (90%). IR: 3605, 3150, 3076, 2964, 2905, 2837, 1707, 1675, 1601, 1565, 1514, 1500, 1457, 1418, 1392, 1343, 1289, 1271, 1253, 1219, 1174, 1111, 1022, 868, 834, 763, 694, 626, 575 and 546 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ_H1.26 (s, 9H), 3.80 (s, 6H), 4.32 (t, 2H), 4.66 (t, 2H), 5.30 (s, 2H), 6.71–9.02 (m, 17H, aromatic) and 11.30 (bs, 1H). ESI-MS: [M + H]⁺ at m/z 797; anal. calcd for C₃₉H₄₀N₈O₉S: C, 58.78; H, 5.06; N, 14.06. Found C, 58.69; H, 5.12; N, 14.04.
(1-(2-(6-(4-tert-Butylphenylsulfonamido)-5-(2-methoxyphen-oxy)-2,2′-bipyrimidin-4-yl-oxy)ethyl)-1H-1,2,3-triazol-4-yl)-methyl 3-methylbenzoate (1k). Brown solid. Mp 140–144 °C. Yield 6.6 g (87%). IR: 3455, 3139, 3067, 2964, 2904, 2869, 1718, 1653, 1562, 1500, 1457, 1397, 1278, 1251, 1228, 1214, 1202, 1179, 1141, 1105, 1081, 1021, 851, 835, 748, 668, 633, 580 and 551 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ_H 1.23 (s, 9H), 2.36 (s, 3H), 3.83 (s, 3H), 4.16 (t, 2H), 4.62 (t, 2H), 5.32 (s, 2H), 6.57–8.07 (m, 15H, aromatic) and 8.95 (bs, 1H). ESI-MS: [M + H]⁺ at m/z 751; anal. calcd for C₃₈H₃₈N₈O₇S: C, 60.79; H, 5.10; N, 14.92. Found C, 60.95; H, 5.14; N, 14.96.

General experimental procedure for the synthesis of 1l–n. Aliphatic terminal alkyne 5l–n (0.01 mol), n-butanol (6 mL), water (6 mL), DIPEA (2.6 g, 0.02 mol) and copper iodide (200 mg, 10 mol%) were mixed followed by the addition of azide 4 (5.8 g, 0.01 mol) and agitated at ambient temperature for 6 to 10 h. Completion of the reaction was checked by TLC (10% methanol in ethyl acetate). To the mass, 50 mL of water and 50 mL of ethyl acetate were charged, stirred and filtered to remove inorganics. The organic layer was separated, dried over anhydrous sodium sulphate followed by vacuum distillation using a rota evaporator at below 60 °C yielding a semi solid. The title compounds, 1l–n were obtained as pale brown to brown amorphous solids in between 87 and 95% yield when the semi solid was triturated with n-heptane, filtered and dried under vacuum at 60 °C.
4-tert-Butyl-N-(6-(2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)-ethoxy)-5-(2-methoxyphenoxy)-2,2′-bipyrimidin-4-yl)-benzenesulfonamide (1l). Yellowish brown solid. Mp 168–170 °C. Yield 6.0 g (95%). IR: 3447, 3134, 2965, 2870, 2835, 1678, 1661, 1609, 1568, 1499, 1427, 1394, 1348, 1250, 1219, 1196, 1167, 1113, 1084, 1026, 856, 833, 756, 692, 625, 573 and 546 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ_H 1.27 (s, 9H), 3.84 (s, 3H), 4.30 (t, 2H, J = 9.5 Hz), 4.45 (t, 2H, J = 9.5 Hz), 4.61 (s, 2H), 5.13 (bs, 1H), 6.70–9.05 (m, 12H, aromatic) and 11.27 (bs, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ_C 22.41, 28.69, 47.77, 55.46, 69.75, 71.11, 111.40, 113.65, 115.24, 118.88, 123.45, 126.56, 128.54, 128.68, 129.04, 134.68, 136.43, 143.13, 143.91, 147.00, 148.41, 150.75, 157.66 and 161.20. ESI-MS: [M + H]⁺ at m/z 633; anal. calcd for C₃₀H₃₂N₈O₆S: C, 56.95; H, 5.10; N, 17.71. Found C, 57.15; H, 5.14; N, 17.85.
4-tert-Butyl-N-(6-(2-(4-butyl-1H-1,2,3-triazol-1-yl)ethoxy)-5-(2-methoxyphenoxy)-2,2′-bipyrimidin-4-yl)-benzenesulfonamide (1m). Brown solid. Mp 101–103 °C. Yield 5.9 g (89%). IR: 3136, 3069, 2959, 2870, 1676, 1605, 1564, 1499, 1456, 1393, 1340, 1252, 1219, 1173, 1111, 1084, 1045, 1022, 864, 837, 752, 694, 625, 575 and 546 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ_H 0.88 (t, 3H, J = 6.8 Hz), 1.27 (m, 13H, aromatic), 1.51 (t, 2H, J = 7.5 Hz), 3.83 (s, 3H), 4.31 (t, 2H, J = 9.5 Hz), 4.57 (t, 2H, J = 9.5 Hz), 6.71–9.04 (m, 12H, aromatic) and 11.27 (bs, 1H). ESI-MS: [M + H]⁺ at m/z 659. Anal. calcd for C₃₃H₃₈N₈O₅S: C, 60.17; H, 5.81; N, 17.01. Found C, 60.85; H, 5.86; N, 17.15.
4-tert-Butyl-N-(6-(2-(4-(1-hydroxycyclohexyl)-1H-1,2,3-triazol-1-yl)ethoxy)-5-(2-methoxyphenoxy)-2,2′-bipyrimidin-4-yl)-benzenesulfonamide (1n). Brown solid. Mp 105–107 °C. Yield 5.9 g (87%). IR: 3418, 3140, 3069, 2936, 2860, 1674, 1607, 1566, 1499, 1456, 1393, 1340, 1285, 1252, 1219, 1171, 1111, 1084, 1047, 1020, 866, 835, 752, 702, 625, 575 & 546 cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆): δ_H 1.27 (m, 11H), 1.41 (m, 2H), 1.64 (m, 4H, J = 5.2 Hz), 1.78 (m, 2H), 3.85 (s, 3H), 4.29 (t, 2H, J = 5.6 Hz), 4.60 (m, 2H, J = 6.4 Hz), 4.77 (bs, 1H), 6.74–9.06 (m, 12H, aromatic), 11.27 (bs, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ_C 22.06, 25.72, 31.24, 35.24, 38.19, 45.89, 47.42, 56.21, 68.41, 113.30, 115.49, 120.90, 121.89, 122.84, 123.22, 124.42, 125.62, 128.51, 138.43, 146.15, 146.80, 147.52, 149.29, 151.25, 156.25, 157.41, 158.32 & 159.15; ESI-MS: [M + H]⁺ at m/z 683; anal. calcd for C₃₅H₄₀N₈O₆S: C, 59.98; H, 5.75; N, 15.99. Found C, 60.12; H, 5.79; N, 16.11.

Biological assays

Materials and methods for antimicrobial activity study. Streptomycin and ciprofloxacin (Sigma) were used as positive controls against bacteria. Ketoconazole (Himedia, Mumbai) was used as a positive control against fungi.
Microbes. The following bacteria and fungi were used for the experiments. Bacteria: Bacillus subtilis MTCC 44.1, Klebsiella pneumoniae MTCC 109, Staphylococcus epidermidis MTCC 3615, Micrococcus luteus MTCC 106, Salmonella typhimurium MTCC 1251, Proteus vulgaris MTCC 1771, Shigella flexneri MTCC 1457, Enterobacter aerogenes MTCC 111, Staphylococcus aureus MTCC 96, Pseudomonas aeruginosa MTCC 741 and Staphylococcus aureus (MRSA-methicillin resistant). The reference cultures were obtained from the Institute of Microbial Technology (IMTECH), Chandigarh-160036, India; fungi: Candida albicans MTCC 227 and Malassezia pachydermatis. All the remaining cultures were obtained from the Department of Microbiology, Christian Medical College, Vellore, Tamil Nadu, India.
In vitro well method. Petri plates were prepared with 20 mL of sterile Mueller Hinton agar (MHA) (Hi-media, Mumbai). The test cultures were swabbed on top of a solidified media and were allowed to dry for 10 min and a specific amount of synthesised compound at 1 mg per disc was added to each well separately. The negative control was prepared using the respective solvents. Streptomycin was used as positive control against bacteria. Ketoconazole was used as positive control for fungi. The plates were incubated for 24 h at 37 °C for bacteria and for 48 h at 28 °C for fungi. Zones of inhibition were recorded in mm and the experiment was repeated twice. Bacterial inoculums were prepared by growing cells in Mueller Hinton broth (MHB) (Himedia) for 24 h at 37 °C. The filamentous fungi were grown on Sabouraud dextrose agar (SDA) slants at 28 °C for 10 days and the spores were collected using sterile doubled distilled water and were homogenized. Yeast was grown on a Sabouraud dextrose broth (SDB) at 28 °C for 48 h.
Minimum inhibition concentration study. The required concentrations (1000 μg mL⁻¹, 500 μg mL⁻¹, 250 μg mL⁻¹, 125 μg mL⁻¹, 62.5 μg mL⁻¹, 31.25 μg mL⁻¹, 15.62 μg mL⁻¹ and 7.81 μg mL⁻¹) of the compound were dissolved in DMSO (2%), and were diluted to give serial two-fold dilutions that were added to each medium in 96 well plates. An inoculum of 100 μL from each well was inoculated. The antifungal agents, ketoconazole for fungi and streptomycin for bacteria were included in the assays as positive controls. For fungi, the plates were incubated for 48 to 72 h at 28 °C and for bacteria the plates were incubated for 24 h at 37 °C. The MIC for fungi was defined as the lowest extract concentration, showing no visible fungal growth after incubation time. 5 μL of the tested broth was placed on the sterile MHA plates for bacteria and incubated at the respective temperature. The MIC for bacteria was determined as the lowest concentration of the compound inhibiting the visual growth of the test cultures on the agar plate.
Study of cytotoxicity. A549 (lung), SKOV-3 (ovarian) and Vero cells were obtained from ATCC, USA. A549, SKOV-3 and Vero cells were maintained in complete tissue culture medium DMEM with 10% fetal bovine serum and 2 mM L-glutamine, along with antibiotics (about 100 IU mL⁻¹ of penicillin, 100 μg mL⁻¹ of streptomycin) with the pH adjusted to 7.2. The cytotoxicity was determined according to the published method⁵⁷ with some changes. Cells (5 × 10⁵) were seeded in 96 well plates containing medium with different concentrations such as 250, 125, 62.5, 31.25, 15.63 and 7.81 μg mL⁻¹. The cells were cultivated at 37 °C with 5% CO₂ and 95% air in 100% relative humidity. After various durations of cultivation, the solution in the medium was removed. An aliquot of 100 μL of medium containing 1 mg mL⁻¹ of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was loaded on to the plate. The cells were cultured for 4 h and then the solution in the medium was removed. An aliquot of 100 μL of DMSO was added to the plate, which was shaken until the crystals were dissolved. The cytotoxicity against cancer cells was determined by measuring the absorbance of the converted dye at 570 nm in an ELISA reader. The cytotoxicity of each sample was expressed as IC₅₀ value. The IC₅₀ value is the concentration of test sample that causes 50% inhibition of cell growth, averaged from three replicate experiments. The percentage of growth inhibition was calculated using the following formula; inhibition (%) = A − B/A × 100 (A – control group and B – treated group).

Molecular docking study. Molecular docking studies were done using the AutoDock Tools (ADT) version 1.5.6 and AutoDock version 4.2.5.1 docking program. The DNA topoisomerase IV and ALK structures were obtained from the Protein Data Bank (PDB ID: 4EMV and 2XP2). The co-crystallized ligands in the receptors were removed. Then, the polar hydrogen atoms were added, lower occupancy residue structures were deleted, and any incomplete side chains were replaced using the ADT. Further ADT was used to remove crystal water, Gasteiger charges were added to each atom, and the non-polar hydrogen atoms merged to the protein structures. The distance between the donor and an acceptor atoms that form a hydrogen bond was defined as 1.9 Å with a tolerance of 0.5 Å, and the acceptor–hydrogen–donor angle was not less than 120°. The structures were then saved in PDBQT file format, for further studies in ADT. Grid boxes with dimension of 40 × 40 × 40 ų and 60 × 60 × 60 ų with 0.375 Å spacing and centred on 14.860, 29.555, 6.941 and 29.697, 47.794, 8.863 were created around the binding site of co-crystallised ligand on 4EMV and 2XP2 respectively. The centre of the box was set at the co-crystallised ligand centre and grid energy calculations were carried out. In order to verify the reproducibility of the docking calculations, the bound ligand was extracted from the complexes and submitted to a one-ligand run calculation. This reproduced the top scoring conformation falling within root-mean-square deviation (RMSD) value of 0.58 to 1.53 Å and 1.63 to 1.95 Å with bound X-ray conformation for 4EMV and 2XP2 respectively, suggesting this method is valid enough to be used for docking studies of other compounds. Docking of different ligands to protein was performed using AutoDock, following the same protocol used in as that of the validation study. All dockings taken into 2.5 million energy evaluations were performed for each of the test molecules. For each compound, 50 docked conformations were generated. The energy calculations were done using genetic algorithms. Docked ligand conformations were analyzed in terms of energy, hydrogen bonding, and hydrophobic interaction between ligand and receptor. Detailed analyses of the ligand–receptor interactions were carried out, and final coordinates of the ligand and receptor were saved. For display of the receptor with the ligand binding site, PyMOL software⁶³ was used. From the docking scores, the free energy of binding (FEB) of all compounds were calculated.

Acknowledgements

Sincere thanks to the management of Malladi Drugs & Pharmaceuticals Ltd., Chennai, India for their support. This project was supported by King Saud University, Deanship Scientific Research, College of Science, Research Center.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: IR, NMR and mass spectra and docking. See DOI: 10.1039/c5ra18618h

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