1-Substituted carbamoyl and thiocarbamoyl-4,5-dihydro-1H-pyrazoles as possible cytotoxic and antimicrobial agents

Abstract

Two series of 1-substituted carbamoyl and thiocarbomoyl derivatives were prepared by either treating the corresponding pyrazole with the appropriate isocyanate and isothiocyanate respectively, or alternatively by condensing the appropriate diketone with the proper substituted semicarbazide or thiosemicarbazide. The structures of the prepared compounds were fully determined by analytical and spectral methods. Preliminary biological screening of the prepared compounds revealed significant antibacterial and cytotoxic activities for some compounds. Compounds 4a₂ and 4a₃ were found to be the most active against the human colon carcinoma HT29 (11.8 and 7.5 μg/mL, respectively) and human breast cancer MCF 7 (3.4 and 2.6 μg/mL, respectively) cell lines. The structure–activity relationship (SAR) and in silico drug relevant properties (HBD, HBA, tPSA, cLog P, molecular weight, % ABS, drug-likeness and drug score) further confirmed that the compounds are potential lead compounds for future drug discovery study.

• Antimicrobial and cytotoxic
• carbamoyl pyrazoles
• drug-likeness
• in silico molecular properties
• thiocarbamoyl pyrazoles

Introduction

The worldwide-ongoing efforts of research on treatment of malignancy are focused on the discovery of novel potent and effective antineoplastic agents, particularly those interacting with novel biological targets. Nevertheless, in spite of the large number of available chemotherapeutic agents, the medical need is still largely unmet due to many factors among which the lack of selectivity of conventional drugs leading to toxicity, the metastatic spreading, and the intrinsic or acquired resistance to chemotherapy developed after few therapeutic cycles (multi-drug resistance; MDR). It is well known that the onset of drug resistance is one of the factors responsible for the failure of chemotherapeutic treatment of cancer patients. This also represents the main driver for the research of new lead molecules with anticancer activity able to overcome the known mechanisms of resistance. At the same time, random screening remains one of the essential means to discover new structure leads with antineoplastic activity.

Pyrazole compounds besides showing an array of biological activities have been characterized by their significant antiproliferative and antitumor activities1–5. The discovery of the naturally occurring C-glycoside antibiotic pyrazofurin; 4-hydroxy-3-β-D-ribofuranosyl-1H-pyrazole-5-carboxamide6, however, has provided a basis for a rational design of new pyrazoles as potential antimicrobial7^,8, antiviral9^,10 and anticancer agents8^,11–13. It has also been reported that isoquinoline-1-carboxaldehyde thiosemicarbazone and its several congeners which possess the N-N-S or O-N-S tridentate ligand system, exhibit substantial antineoplastic activity14^,15. Furthermore, the azomethine linkage in this class that plays significant role in certain biological transformations which includes vitamin B₆-catalysed reactions16^,17 paved the way to study pyrazoline derivatives having a carbamoyl or thiocarbamoyl moiety at position-1. This modification carries two fold objective of preparing compounds with possible antineoplastic and antimicrobial activities. In the present work we tried to optimize this chemical series by introducing carbamoyl and thiocarbamoyl moieties at position-1 in the pyrazole ring. The substituent effect is further extended by introducing substituted phenyl groups at nitrogen of carbamoyl and thiocarbamoyl moieties.

Furthermore, molecular descriptors, such as partition coefficient (log P), molecular weight (MW), and hydrogen bond acceptors/donors are usually associated with molecular properties such as membrane permeability and oral bioavailability. Using these descriptors, Lipinski18 formulated a rule for drug design which states that the compounds are more likely to be orally bioavailable if they obey the following criteria: log p ≤ 5, molecular weight ≤500, hydrogen bond acceptors ≤10, and hydrogen bond donors ≤5. We therefore, decided to carry out studies in silico for Lipinski parameters, topological polar surface area (tPSA) and the percentage of absorption (% ABS). Furthermore, according to Veber et al., good bioavailability19 is more likely for compounds with ≤10 rotatable bonds and TPSA of ≤140 Å2. For this reason we have also included the measurement of the number of rotatable bonds in this series of compounds, drug-likeness and drug scores in order to rationalize how physicochemical properties influence the molecular behavior in vivo.

Experimental

Chemistry

Melting points were determined on a Gallenkamp melting point apparatus (London, UK) and are uncorrected. The infrared (IR) spectra were recorded on Shimadzu FT-IR 8400S infrared spectrophotometer (Tokyo, Japan) using the KBr pellet technique. ¹H and ¹³C NMR spectra were recorded on a Bruker DPX-400 FT NMR spectrometer (Bruker BioSpin AG, Fällanden, Switzerland) using tetramethylsilane as the internal standard and a mixture of CDCl₃ and DMSO-d₆ as a solvent (Chemical shifts in δ, ppm). Splitting patterns were designated as follows: s: singlet; d: doublet; m: multiplet. Elemental analyses were performed on a 2400 Perkin Elmer Series 2 analyzer (Waltham, MA) and the found values were within ±0.4% of the theoretical values. Follow up of the reactions and checking the homogeneity of the compounds were made by TLC on silica gel-protected aluminum sheets (Type 60 F254, Merck, Darmstadt, Germany) and the spots were detected by exposure to UV-lamp at λ 254. The starting chalcones 2 were prepared according to a literature procedure20.

3-(Indol-3-yl)-1-substituted propenones (2a–c)

A solution of the indole-3-carboxaldehyde 1 (1.31 g, 0.01 mol) in ethanol (20 mL) was added to a solution of the appropriate ketone (0.01 mol) in 20% ethanolic KOH (20 mL) with stirring. The stirring was continued at room temperature for 6 h. The reaction mixture was then poured onto water (200 mL) and left overnight. The precipitated solid product was collected by filtration, washed with water, dried and recrystallized from ethanol as pale yellow needles. IR (cm⁻¹): 1654–1660(conjugated C=O), 1604–1611(C=C).

2a: ¹H NMR (DMSO-d₆/CDCl₃): δ 6.67 (d, (J = 10 Hz), 1H, H-α), 7.54 (d (J = 10 Hz), 1H, H-β), 7.02–7.65 (m, 8H, Ar-H), 10.1 (s, NH); ¹³C NMR: 129.3 (C-α), 142.8 (C-β), 102.4, 111.2, 120.3, 121.2, 122.3, 127.4, 128.2, 128.5, 135.6, 135.8, 144.4, 155.5 (Ar-C), 180.7 (C=O).

2b: ¹H NMR (DMSO-d₆/CDCl₃): δ 6.45 (d, (J = 10 Hz), 1H, H-α), 7.34 (d (J = 10 Hz), 1H, H-β), 6.64–7.72 (m, 8H, Ar-H), 9.98 (s, NH); ¹³C NMR: 128.6 (C-α), 141.5 (C-β), 100.6, 110.1, 119.8, 121.6, 122.5, 124.5, 127.0, 128.4, 131.5, 136.6, 148.8, 153.2 (Ar-C), 175.2 (C=O).

2c: ¹H NMR (DMSO-d₆/CDCl₃): δ 6.45 (d, (J = 10 Hz), 1H, H-α), 7.34 (d, (J = 10 Hz), 1H, H-β), 6.64–7.72 (m, 8H, Ar-H), 9.98 (s, NH); ¹³C NMR: 128.6 (C-α), 141.5 (C-β), 108.4, 112.3, 118.9, 120.4, 121.9, 124.7, 127.2, 128.3, 132.1 135.6, 151.3, 156.8 (Ar-C), 175.0 (C=O).

3-(3-Substituted-4,5-dihydro-1H-pyrazol-5-yl)-1H-indole (3a--c)

Hydrazine hydrate (0.015 mol, 99%) was added to a cooled solution of the appropriate chalcone (0.01 mol) in ethanol (25 ml) over 15 min. The mixture was then refluxed for 1 h on a boiling water-bath. The resulting reaction mixture was concentrated and allowed to attain room temperature. The precipitated product was filtered off, dried and recrystallized from ethanol as colorless needles.

1-Substituted thiocarbamoyl-4,5-dihydro-1-H-pyrazole derivatives (4a--c)

Method A

A mixture of the corresponding pyrazoline (3a–c, 0.05 mol), anhydrous K₂CO₃ (0.1 mol) in dry acetone (20 ml) was stirred at room temperature. Subsequently, a solution of the appropriate isothiocyanate (0.07 mol) in dry acetone (20 ml) was added dropwise to the reaction mixture. After stirring and refluxing the mixture for 6 h, acetone was removed under reduced pressure. The resulting solid residue which was precipitated upon acidification with 20% hydrochloric acid was purified by recrystallization from ethanol as colorless/pale needles (4a–c).

Method B

A solution of the corresponding chalcone 2 (0.05 mol) in ethanol (30 ml) was treated with the appropriate thiosemicarbazide (0.05 mol). The mixture was heated on a boiling water-bath for 1 h. The crude product thus separated on cooling, was purified by recrystallization from ethanol as colorless/pale needles of compounds 4a–c.

1-Substituted carbarmoyl-4,5-dihydro-1-H-pyrazole derivatives (5)

Method A

A mixture of the corresponding pyrazolines (0.05 mol), anhydrous K₂CO₃ (0.1 mol) in dry acetone (20 ml) was refluxed with stirring. To this refluxing reaction mixture a solution of the appropriate isocyanate (0.075 mol) in dry acetone (20 ml) was added dropwise. After stirring and refluxing the reaction mixture overnight, acetone was removed under reduced pressure. The crude product was separated on cooling which was purified by recrystallization from ethanol-benzene (2:1) as needles.

Method B

A solution of the appropriate chalcone 2 (0.05 mol) in ethanol (30 ml) was treated with the corresponding semicarbazide (0.05 mol). The mixture was allowed to condense at moderate temperature on a steaming water-bath for 1 h. The crude product thus separated on cooling, was purified by recrystallization from ethanol:benzene (2:1) as needles.

Method C

A mixture of the appropriate thiocarbamoyl derivative (1.05 mol), yellow HgO (0.1 mol) in ethanol (50 ml) was first stirred for few minutes and then refluxed for 3 h. The resulting reaction mixture was filtered. The filtrate was concentrated and allowed to crystallize. The crude crystals formed was filtered and recrystallized from ethanol:benzene (2:1) as needles. The products obtained following this technique were identical with those prepared by methods A and B as confirmed by their m.p., mixed m.p. and TLC.

Biological evaluation

Procedure for anti-microbial activity

The preliminary anti-microbial activities of the synthesized chalcones, pyrazoline carbamoyl and thiocarbamoyl derivatives 2–5 were measured in a concentration of 50 µg/mL by disc diffusion method21^,22. The prepared compounds were tested for their antimicrobial activity against two types of bacteria: Staphylococcus aureus (ATCC 25923) as Gram positive and Escherichia coli (ATCC 25922) as Gram negative bacteria. The antifungal activity was tested using the pathogenic yeast strain Candida albicans (ATCC 10231) and Aspergillus niger (ATCC 16404). DMSO was used as a solvent control and the standard drugs used were Ampicillin23 and Griseofulvin24. The disc diffusion method was performed using Muller–Hinton agar (Hi-Media) medium. The inhibition zones were measured in mm at the end of an incubation period of 24 h at 37 °C for bacteria and 72 h at 24° C for fungi.

Procedure for anti-microbial activity using UV (366 nm) light

This test was performed as mentioned before but the Petri-discs containing micro-organisms and the testing compounds were subjected to UV light (366) for 3 h before transferring to the incubation period.

In vitro MTT cytotoxicity assay

All the following procedures were done in a sterile area using a Laminar flow cabinet biosafety class II level (Baker, SG403INT, Stanford, ME). Cells were batch cultured for 10 days, then seeded at concentration of 10 × 10³ cells/well in fresh complete growth medium in 96-well microtiter plastic plates at 37 °C for 24 h under 5% CO₂ using a water jacketed carbon dioxide incubator (Sheldon, TC2323, Cornelius, OR). Media was aspirated, fresh medium (without serum) was added and cells were incubated either alone (negative control) or with different concentrations of the test compounds to give a final concentration of 100, 50, 25, 12.5, 3.125, 1.56, 0.78 μg/mL. DMSO was employed as a vehicle for dissolution of the tested compounds and its final concentration on the cells was less than 0.2%. Cells were suspended in RPMI 1640 medium (for HePG2 and HT29 cell lines) and DMEM (for MCF 7 cell line), 1% antibiotic-antimycotic mixture (10 000 IU/mL penicillin potassium, 10 000 μg/mL streptomycin sulfate and 25 μg/mL amphotericin B), and 1% l-glutamine in 96-well flat bottom microplate at 37 °C under 5% CO₂.

After 24 h of incubation, the medium was aspirated; 40 mL of MTT salt (2.5 mg/mL) was added to each well and incubated for further 4 h at 37 °C under 5% CO₂. To stop the reaction and dissolve the formed crystals, 200 mL of 10% sodium dodecyl sulfate in deionized water was added to each well and incubated overnight at 37 °C. The absorbance was then measured using a microplate multiwell reader (Bio-Rad Laboratories Inc., model 3350, Hercules, CA) at 595 nm and a reference wavelength of 620 nm. A statistical significance was tested between samples and negative control (cells with vehicle) using independent t-test by SPSS 11 program. Doxorubicin25 is used as positive control cytotoxic agent. The results of LC₅₀ (mM), which is the lethal concentration of the compound causing death of 50% of the cells in 24 h, are presented in Table 6.

Results and discussion

Chemistry

The starting compounds in this study are 3-(Indol-3-yl)-1- substituted propenones 2a–c could be synthesized via Claisen-Schmidt condensation of indole-3-carboxaldehyde with different heterocyclic ketones 1. These chalcones were reacted with hydrazine hydrate to afford the desired 4,5-dihydro-1-H-pyrazole derivatives 3a–c (Scheme 1). Their IR spectra revealed two absorption bands at 1594–1602 cm⁻¹ and 3365–3388 cm⁻¹ corresponding to C=N and NH functions, respectively. The ¹H-NMR exhibited besides the aromatic protons at δ 6.01–7.41 ppm, three multiplets (each of one proton intensity) at δ 5.25–5.42, 3.49–3.55 and 3.78–3.89. The low-field multiplet is assigned to H-5 of the pyrazoline while the other two multiplets are due to H-4 (Table 2). The structures were further confirmed from their ¹³C NMR spectral data (Table 3). The new 1-thiocarbamoyl and carbamoyl derivatives 4 and 5 were prepared by either treating the corresponding pyrazole with the appropriate isothiocyanate or isocyanate respectively or by condensing the corresponding chalcone with the proper substituted semicarbazide15 or thiosemicabazide26 (Table 1). Furthermore, mild oxidation of the thiocarbamoyl derivative 4 with yellow mercuric oxide afforded the corresponding carbamoyl derivative 5. The IR spectra of the carbamoyl derivative 5 exhibited distinguishable carbonyl absorption at 1668–1672 cm⁻¹, whereas compound 4 revealed distinct thiocarbonyl band at 1060–1155 cm⁻¹. Their structures were further confirmed from their ¹H-NMR and ¹³C NMR spectral data (Tables 2 and 3).

Scheme 1. Synthesis of compounds 2–5.

Table 1. Physical and analytical data of compounds 2–5.

							Calc. %			Found %
Compound	X	R	R′ or R″	Yield (%)	m.p. (°C)	Mol. Formula	C	H	N	C	H	N
2a	S	H		92	196–198	C15H11NOS	71.12	4.38	5.53	71.03	4.40	5.63
2b	O	H		88	188–189	C15H11NO2	75.94	4.67	5.90	76.00	4.78	6.12
2c	O	CH3		90	194–196	C16H13NO2	76.48	5.21	5.57	76.52	5.31	5.60
3a	S	H		89	235–237	C15H13N3S	67.39	4.90	15.72	67.43	5.11	15.81
3b	O	H		85	320–322	C15H13N3O	71.70	5.21	16.72	71.82	5.31	16.78
3c	O	CH3		87	324–326	C16H15N3O	72.43	5.70	15.84	72.54	5.82	15.92
4a1	S	H	C6H5	72	300–302	C22H18N4S2	65.64	4.51	13.92	65.72	4.62	13.84
4a2	S	H	4-ClC6H4	74	200–203	C22H17ClN4S2	60.47	3.92	12.82	60.51	4.05	13.01
4a3	S	H	4-FC6H4	70	332–334	C22H17FN4S2	62.83	4.07	13.32	62.94	4.18	13.50
4a4	S	H	4-CH3C6H4	75	312–314	C23H20N4S2	66.32	4.84	13.45	66.45	4.99	13.56
4a5	S	H	CH3	73	204–206	C17H16N4S2	59.97	4.74	16.46	60.12	4.82	16.52
4a6	S	H	Cyclohexyl	80	248–250	C22H24N4S2	64.67	5.92	13.71	64.77	6.12	13.82
4b1	O	H	C6H5	79	265–268	C22H18N4OS	68.37	4.69	14.50	68.42	4.74	14.66
4b2	O	H	4-ClC6H4	82	288–290	C22H17ClN4OS	62.78	4.07	13.31	62.84	4.18	13.45
4b3	O	H	Cyclohexyl	74	334–336	C22H24N4OS	67.32	6.16	14.27	67.35	6.26	14.42
4c1	O	CH3	C6H5	72	332–334	C23H20N4OS	68.98	5.03	13.99	69.11	5.12	14.08
4c2	O	CH3	4-ClC6H4	76	310–312	C23H19ClN4OS	63.51	4.40	12.88	63.68	4.45	13.01
4c3	O	CH3	Cyclohexyl	78	272–274	C23H26N4OS	67.95	6.45	13.78	67.10	6.56	13.89
5a1	S	H	C6H5	76	337–339	C22H18N4OS	68.37	4.69	14.50	68.42	4.71	14.62
5a2	S	H	4-ClC6H4	78	305–307	C22H17ClN4OS	62.78	4.07	13.31	62.87	4.16	13.44
5a3	S	H	Cyclohexyl	74	228–229	C22H24N4OS	67.32	6.16	14.27	67.33	6.23	14.22
5b1	O	H	C6H5	70	220–222	C22H18N4O2	71.34	4.90	15.13	71.42	5.04	15.18
5b2	O	H	4-ClC6H4	72	262–264	C22H17ClN4O2	65.27	4.23	13.84	65.41	4.28	13.92
5b3	O	H	Cyclohexyl	69	330–332	C22H24N4O2	70.19	6.43	14.88	70.28	6.51	15.04
5c1	O	CH3	C6H5	70	260–262	C23H20N4O2	71.86	5.24	14.57	71.92	5.41	14.65
5c2	O	CH3	4-ClC6H4	72	308–310	C23H19ClN4O2	65.95	4.57	13.38	65.81	4.63	13.52
5c3	O	CH3	Cyclohexyl	68	220–222	C23H26N4O2	70.75	6.71	14.35	70.83	6.85	14.48

Table 2. ¹H NMR spectral data (δ/ppm)^a of compounds 3–5.

				Pyrazoline H
Compound	X	R	R′ or R″	H-4 (2 m, 2H)	H-5 (m, 1H)	CH3 (s)	N-H (s)	Ar-H (m)	Others
3a	S	H		3.49,3.88	5.28		7.85, 10.21	7.01–7.26 (8H)
3b	O	H		3.54,3.89	5.42		8.01, 10.15	6.43–7.41 (8H)
3c	O	CH3		3.55,3.78	5.34	2.17	7.78, 9.92	6.01–7.41 (7H)
4a1	S	H	C6H5	3.50,3.82	5.23		6.33, 10.30	7.00–7.35 (13H)
4a2	S	H	4-ClC6H4	3.48,3.87	5.30		10.00	6.50–7.28 (13H)^b
4a3	S	H	4-FC6H4	3.51,3.85	5.25		9.89	6.44–7.25 (13H)^b
4a4	S	H	4-CH3C6H4	3.49,3.89	5.23	2.36	6.42, 10.02	6.58–7.24 (12H)
4a5	S	H	CH3	3.48,3.88	5.22	2.48	5.40, 10.11	7.00–7.23 (8H)
4a6	S	H	Cyclohexyl	3.50,3.77	5.26		5.03, 10.13	6.40–7.45 (8H)	1.44-1.60, 2.58 (2 m, 11H, cyclohexyl H)
4b1	O	H	C6H5	3.44,3.87	5.45		4.98, 10.00	6.42–7.40 (13H)
4b2	O	H	4-ClC6H4	3.46,3.85	5.50		4.85, 9.98	6.39–7.32 (12H)
4b3	O	H	Cyclohexyl	3.49,3.90	5.42		4.72, 10.12	6.37–7.41 (8H)	1.42-1.59, 2.60 (2 m, 11H, cyclohexyl H)
4c1	O	CH3	C6H5	3.45,3.88	5.39	2.17	4.90, 10.12	6.22–7.20 (12H)
4c2	O	CH3	4-ClC6H4	3.48,3.87	5.46	2.16	4.78, 10.01	6.03–7.19 (11H)
4c3	O	CH3	Cyclohexyl	3.55,3.79	5.43	2.14	4.62, 9.85	5.98–7.29 (7H)	1.44-1.63, 2.57 (2 m, 11H, cyclohexyl H)
5a1	S	H	C6H5	3.52,3.84	5.38		6.24, 10.21	7.00–7.64 (13H)
5a2	S	H	4-ClC6H4	3.54,3.86	5.39		6.10, 10.16	6.99–7.58 (12H)
5a3	S	H	Cyclohexyl	3.53,3.89	5.35		6.00, 10.04	7.02–7.28 (8H)	1.40-1.58, 2.57 (2 m, 11H, cyclohexyl H)
5b1	O	H	C6H5	3.56,3.77	5.67		6.18, 10.12	6.38–7.32 (13H)
5b2	O	H	4-ClC6H4	3.41,3.86	5.78		6.06, 10.04	6.56–7.28 (12H)
5b3	O	H	Cyclohexyl	3.51,3.79	5.63		6.02, 9.98	6.38–7.19 (8H)	1.42-1.66, 2.58 (2 m, 11H, cyclohexyl H)
5c1	O	CH3	C6H5	3.49,3.84	5.64	2.19	6.31, 10.23	6.45–7.20 (12H)
5c2	O	CH3	4-ClC6H4	3.48,3.86	5.71	2.17	6.12, 10.14	6.39–7.28 (11H)
5c3	O	CH3	Cyclohexyl	3.47,3.87	5.69	2.15	6.08, 9.86	6.35–7.17 (7H)	1.40-1.59, 2.63 (2 m, 11H, cyclohexyl H)

^aSolutions in mixtures of CDCl₃ and DMSO-d₆.

^bAr-H + N-H.

Table 3. ¹³C NMR spectral data (δ/ppm)^a of compounds 2–5.

Compound	X	R	R′ or R″	CH3	C-4 & C-5	Ar-C & Others
3a	S	H			42.2, 50.1	111.0, 112.3, 119.6, 120.2, 121.4, 122.3, 126.0, 127.1, 127.5, 131.4, 135.8, 155.7
3b	O	H			41.9, 44.9	110.2, 113.6, 118.3, 120.5, 121.7, 122.8, 126.2, 128.1, 131.0, 136.5, 143.4, 152.9
3c	O	CH3		14.7	41.6, 44.4	107.2, 110.8, 112.7, 118.9, 120.6, 121.9, 123.4, 127.1, 131.3, 136.0, 141.1, 154.4
4a1	S	H	C6H5		41.8, 50.3	113.1, 115.2, 119.7, 120.5, 121.2, 122.6, 124.5, 125.2, 126.1, 127.0, 127.6, 128.8, 131.0, 136.2, 139.4, 155.2; 184.6 (CS)
4a2	S	H	4-ClC6H4		41.2, 45.3	112.1, 114.3, 118.8, 120.6, 120.9, 121.9, 122.4, 126.1, 126.4, 126.6, 127.4, 128.9, 129.7, 131.3, 136.0, 137.4, 154.8; 182.4 (CS)
4a3	S	H	4-FC6H4	20.8	41.4, 45.6	111.8, 113.6, 118.9, 120.4, 121.2, 122.8, 125.3, 126.6, 127.0, 127.8, 129.5, 132.1, 133.7, 134.8, 136.4, 156.0; 185.6 (CS)
4a5	S	H	CH3	34.1	41.3, 45.2	110.6, 114.2, 120.1, 120.9, 121.6, 122.2, 125.8, 126.7, 127.8, 128.2, 133.1, 134.8, 155.9; 184.3 (CS)
4b1	O	H	C6H5		40.2, 47.5	109.8, 114.2, 119.6, 120.4, 121.6, 122.9, 124.8, 125.3, 126.1, 127.0, 127.5, 128.8, 131.9, 135.5, 139.6, 155.6; 186.6 (CS)
4b3	O	H	Cyclohexyl		39.8, 46.7	111.7, 112.4, 119.5, 120.4, 121.2, 122.6, 126.1, 127.4, 128.2, 132.0, 136.4, 156.2 (ArC); 22.6, 27.1, 33.6, 51.9 (cyclohexyl-C); 185.6 (CS)
4c2	O	CH3	4-ClC6H4	15.2	39.4, 47.9	112.2, 114.3, 119.8, 121.0, 121.6, 122.7, 123.1, 126.0, 126.7, 129.2, 129.6, 130.1, 132.0, 134.9, 137.5, 153.4; 184.9 (CS)
4c3	O	CH3	Cyclohexyl	14.9	38.7, 47.2	110.9, 112.4, 118.9, 120.2, 121.4, 122.5, 126.2, 127.1, 127.9, 131.5, 134.6, 136.2, 156.4 (ArC); 22.4, 27.3, 33.6, 51.6 (cyclohexyl-C); 185.6 (CS)
5a1	S	H	C6H5		41.2, 45.2	111.2, 112.6, 119.7, 120.4, 120.6, 121.7, 122.8, 124.1, 126.2, 126.8, 127.8, 128.7, 131.6, 136.6, 138.4, 156.2; 159.9 (CO)
5a2	S	H	4-ClC6H4		41.4, 45.7	108.9, 113.4, 117.9, 120.2, 120.6, 121.8, 122.6, 126.2, 127.4, 128.1, 129.3, 129.5, 130.8, 132.4, 136.3, 137.1, 155.4; 160.1 (CO)
5a3	S	H	Cyclohexyl		40.9, 45.2	110.4, 112.2, 119.6, 120.3, 121.6, 122.4, 125.9, 126.2, 127.8, 131.2, 132.1, 135.5, 154.2 (ArC); 22.0, 27.1, 33.6, 47.8 (cyclohexyl-C); 163.3 (CO)
5b1	O	H	C6H5		41.3, 45.2	110.2, 113.4, 115.6, 118.9, 120.3, 120.8, 121.6, 122.9, 124.4, 126.3, 126.6, 127.4, 128.6, 132.0, 135.4, 143.1, 156.0; 162.8 (CO)
5b2	O	H	4-ClC6H4		41.4, 45.6	109.3, 112.8, 114.2, 119.3, 120.2, 121.0, 121.6, 122.8, 125.4, 126.8, 127.3, 129.1, 129.6, 131.6, 136.2, 142.2, 155.4; 161.8 (CO)
5c1	O	CH3	C6H5	14.5	40.8, 44.6	112.4, 114.6, 119.2, 120.3, 120.5, 121.8, 122.2, 122.8, 124.1, 125.8, 126.2, 127.0, 127.6, 128.6, 131.2, 138.2, 156.6; 159.9 (CO)
5c2	O	CH3	4-ClC6H4	14.3	40.5, 44.2	111.8, 112.2, 119.6, 120.3, 121.0, 121.8, 122.6, 125.0, 126.1, 126.7, 127.6, 127.2, 129.1, 129.3, 132.2, 136.3, 154.6; 160.0 (CO)

^aSolution in a mixture of CDCl₃ and DMSO-d₆.

In silico calculations of molecular properties

Molecular descriptors represent the combined physicochemical, pharmacokinetic and pharmacodynamic effects of the synthesized compounds 2–5 in order to verify that these compounds exhibit good (theoretical) oral bioavailability potential. Lipinski rule of five is considered predictive for oral bioavailability; however 16% of oral drugs violate at least one of the criteria and 6% fail in two or more27. The lipophilicity (log P) and topological polar surface area (tPSA) were calculated using the online software Molinspiration28, while the aqueous solubility, drug-likeness, drug score and toxicity risk assessment (TRA) were calculated using the OSIRIS property explorer software29. Molinspiration uses sophisticated Bayesian statistics to compare structures of representative ligands active on the particular target with structures of inactive molecules and to identify substructure features (which in turn determine physicochemical properties) typical for active molecules. For the study of drug-likeness, the OSIRIS program uses a list of 5300 molecular fragments, where the frequency of occurrence of each fragment is determined based on a collection of 3300 drugs and 15 000 commercially available chemicals (Fluka AG, Buchs, Switzerland) that are not drugs. tPSA was used to calculate the percentage of absorption (%ABS) according to the equation: %ABS = 109 − 0.345 × TPSA, as reported by Zhao et al30. Furthermore, according to Veber et al., good bioavailability19 is more likely for compounds with ≤10 rotatable bonds and TPSA of ≤140 Å2. Reduced molecular flexibility, as measured by the number of rotatable bonds, and low polar surface area or total hydrogen bond count (sum of donors and acceptors) are found to be important predictors of good oral bioavailability, independent of molecular weight. The calculation data are shown in Table 4.

Table 4. Molecular Properties Calculation^a: calculated absorption (% ABS), polar surface area (PSA), LogS, Lipinski’s parameters, drug-likeness and drug score of the compounds.

	Lipinski’s Parameters												Toxicity Risk Assessment
Compound	miLogP^b	MW^c	nON^d	nOHNH^e	nviolations^f	tPSA^g	% ABS^h	nrotb^i	volume^j	Solubility^k	Druglikeness^l	Drug- Score^m	MUTA^n	TE^o	IRR^p	RE^q
2a	3.68	253.33	2	1	0	32.86	97.66	3	221.54	−4.38	4.1	0.74	−^r	−	−	−
2b	3.04	237.26	3	1	0	46.00	93.13	3	212.39	−4.05	2.29	0.77	−	−	−	−
2c	3.26	251.28	3	1	0	46.00	93.13	3	228.96	−4.42	1.38	0.68	−	−	−	−
3a	3.37	267.36	3	2	0	40.18	95.14	2	233.59	−4.37	4.98	0.75	−	−	−	−
3b	2.72	251.29	4	2	0	53.32	90.60	2	224.45	−4.05	3.18	0.80	−	−	−	−
3c	2.95	265.32	4	2	0	53.32	90.60	2	241.01	−4.41	2.22	0.72	−	−	−	−
4a1	4.86	402.55	4	2	0	43.42	94.02	5	345.65	−5.92	4.21	0.15	+^s	−	−	+
4a2	5.54	436.99	4	2	1	43.42	94.02	5	359.18	−6.66	6.28	0.12	+	−	−	+
4a3	5.03	420.54	4	2	1	43.42	94.02	5	350.58	−6.24	4.44	0.14	+	−	−	+
4a4	5.31	416.57	4	2	1	43.42	94.02	5	362.21	−6.27	4.27	0.13	+	−	−	+
4a5	3.80	340.48	4	2	0	43.42	94.02	4	290.79	−4.41	6.04	0.26	+	−	−	+
4a6	5.71	408.60	4	2	1	43.42	94.02	5	364.23	−5.99	0.02	0.12	+	−	−	+
4b1	4.22	386.48	5	2	0	56.56	89.49	5	336.50	−5.59	2.61	0.18	+	−	−	+
4b2	4.90	420.92	5	2	0	56.56	89.49	5	350.04	−6.33	4.75	0.14	+	−	−	+
4b3	5.07	392.53	5	2	1	56.56	89.49	5	355.09	−5.66	−1.54	0.11	+	−	−	+
4c1	4.44	400.51	5	2	0	56.56	89.49	5	353.06	−5.96	1.59	0.15	+	−	−	+
4c2	5.12	434.95	5	2	1	56.56	89.49	5	366.60	−6.7	3.69	0.12	+	−	−	+
4c3	5.29	406.55	5	2	1	56.56	89.49	5	371.65	−6.03	−2.56	0.09	+	−	−	+
5a1	4.96	386.48	5	2	0	60.49	88.13	3	336.77	−5.84	4.96	0.47	−	−	−	−
5a2	5.64	420.92	5	2	1	60.49	88.13	3	350.30	−6.57	7.29	0.37	−	−	−	−
5a3	5.83	392.53	5	2	1	60.49	88.13	3	355.35	−5.90	1.82	0.44	−	−	−	−
5b1	4.32	370.41	6	2	0	73.63	83.60	3	327.62	−5.51	3.46	0.32	+	−	−	−
5b2	4.99	404.86	6	2	0	73.63	83.60	3	341.16	−6.25	5.87	0.26	+	−	−	−
5b3	5.19	376.46	6	2	1	73.63	83.60	3	346.21	−5.58	0.38	0.26	+	−	−	−
5c1	4.54	384.44	6	2	0	73.63	83.60	3	344.18	−5.89	2.40	0.28	+	−	−	−
5c2	5.22	418.88	6	2	1	73.63	83.60	3	357.72	−6.61	4.76	0.23	+	−	−	−
5c3	5.41	390.49	6	2	1	73.63	83.60	3	362.77	−5.94	−0.72	0.19	+	−	−	−
Ampicillin^t	−0.95	363.44	7	4	0	112.73	70.11	4	315.10	−1.67	8.86	0.90	−	−	−	−
Griseofulvin^u	1.57	352.77	6	0	0	71.08	84.48	3	293.59	−3.96	0.71	0.39	−	+	−	−
Doxorubicin^v	0.57	543.52	12	7	3	206.08	37.90	5	459.18	−4.97	4.57	0.31	−	−	+	−

^amipc – Molinspiration Property Calculator; ^bMolinspiration (miLogP2.2 – November 2005); ^cMolecular Weight; ^dNumber of Hydrogen-Bond Acceptors (O and N atoms); ^eNumber of Hydrogen-Bond Donors (OH and NH groups); ^fNumber of Rule of 5 Violations; ^gTopological Polar Surface Area; ^hPercentage of Absorption: % ABS = 109 − (0.345 × TPSA); ⁱNumber of Rotatable Bonds; ^jMolecular Volume; ^kSolubility Prediction (LogS); ^lFragment Based Druglikeness; ^mDrug score combines druglikeness, cLogP, logS, molecular weight and toxicity risks in one handy value; ⁿMutagenic risk; ^oTumorigenic risk; ^pIrritant effect; ^qReproductive effect; ^rNo risk; ^sPossibility of Risk; ^tPositive control antimicrobial agent; ^uPositive control antifungal agent; ^vPositive control cytotoxic agent.

The calculation results show that more than half of compounds meet the Lipinski rules of the five, suggesting that these compounds theoretically would not have problems with oral bioavailability. Compounds which had scores of less than 5 for lipophilicity, ranging from 2.72 to 4.99 for 15 compounds (2a–c, 3a–c, 4a₁, 4a₅, 4b₁, 4b₂, 4c₁, 5a₁, 5b₁, 5b₂ and 5c₁) and 12 compounds (4a_2–4, 4a₆, 4b₃, 4c₂, 4c₃, 5a₂, 5a₃, 5b₃, 5c₂ and 5c₃) had scores more than 5 ranging from 5.00 to 5.83. Most of the compounds also showed a tPSA much less than 140 Ų, indicating a good permeability of the drug in the cellular plasma membrane. All compounds have shown very high percentage of absorption (% ABS) calculated ranged from 83.60 to 97.66%, which is an indication of good bioavailability by oral route (Table 4). Table 4 shows that all compounds have Log S value less than −4.00, ranging between −4.05 and −6.66.

The toxicity risk assessment (TRA) indicators, namely, mutagenic effect (MUTA), tumorigenic effect (TE), irritant effect (IRR) and reproductive effect (RE) are the predictors for potential toxicity risks. These calculations are based on a precomputed set of structural fragment(s) that give rise to toxicity alerts in case they are encountered in the structures currently undertaken. The TRA suggested that compounds 2a–c, 3a–c and 5a did not show any toxicity risk profile. However, compounds 4a–c showed the possibility of mutagenic and reproductive effects and compounds 5b–c have shown the possibility of mutagenic effect. None of the compounds have shown the risk of tumorigenic or irritant effects (Table 4).

A positive value for drug-likeness indicates that the compound contains predominantly fragments that are often present in most currently used drugs. The drug score combines drug-likeness, lipophilicity, solubility, molecular weight and the risk of toxicity into a single numerical value that can be used to predict a global value for each compound as a potential new drug candidate. The results in the calculations show that most of the compounds gave values for drug-likeness between 1.38 and 7.29. Compounds 4a₆ and 5b₃ have shown drug-likeness 0.02 and 0.38, respectively. However, only three compounds, namely, 4b₃, 4c₃ and 5c₃ have displayed negative values of druglike-ness (−1.54, −2.56 and −0.72 respectively). For comparisons we have done similar calculations of reference drugs as well.

All compounds showed positive values in the drug score calculation, the values ranged from 0.09 to 0.80. Even those compounds which have shown negative druglike-ness have displayed positive drug scores (Table 4). The results show that these compounds, especially compounds 2, 3 and some members from 4 and 5, have potential as new drug candidates. Moreover, non-violations of Lipinski’s “Rule-of-Five” and Veber’s “criteria for good bioavailability” also confirm the suitability of these compounds to be used as a template for the design of dual-target–directed drugs.

Anti-microbial activity

Compounds 2–5 were screened in vitro for their antimicrobial and antifungal activity against Escherichia coli, Staphylococcus aureus, Aspergillus niger and Candida albicans. The zones of inhibition formed for the compounds against bacteria and fungi are summarized in Table 5. All compounds were found to possess mild to moderate activity. Compounds 3a, 3b, 4a₂, 4a₃, 4b₂, 4c₂, 5a₂, 5b₂, 5c₂ were however, significantly active when compared with rest of the series. Moreover, after using UV-Visible light, most of the tested compounds showed an additional activity especially towards Escherichia coli and Candida albicans (Table 5). The anti-microbial activity of compounds 3a, 3b, 4a₂, 4a₃, 4b₂, 4c₂, 5a₂, 5b₂ and 5c₂ was further corroborated by their high values of drug-likeness (4.98, 3.18, 6.28, 4.44, 4.75, 3.69, 7.29, 5.87 and 4.76 respectively) and significant drug scores (0.75, 0.80, 0.12, 0.14, 0.14, 0.12, 0.37, 0.26 and 0.23 respectively). All test data in Tables 5 and 6 were of average values from triplicate runs and the test compounds showed reduced antimicrobial activities when compared with their respective standards. Compounds containing thiocarbamoyl part showed enhanced activity in the present series. The most active compound 4a₃ which contains 4-fluorophenyl substituent at the thiocarbamoyl end showed best antimicrobial activity (Table 5) and this was further supported by the value of its drug-likeness (4.44) and a quite favorable percentage of absorption (94.02%) (Table 4). In compounds 4a₆, 4b₃, 4c₃, 5a₃, 5b₃ and 5c₃ where the aromatic ring was replaced by a cyclohexyl ring, a marked reduction in their antimicrobial activity were seen and this was further substantiated by the values of their drug-likeness which was found to be negative in compounds 4b₃, 4c₃ and 5c₃.

Table 5. Anti-bacterial and anti-fungal data of compounds 2–5.

	Zone of inhibition in mm				Zone of inhibition in mm
	Anti-bacterial activity				Anti-fungal activity
	Escherichia coli (ATCC 25922)		Staphylococcus aureus (ATCC 25923)		Aspergillus niger (ATCC 16404)		Candida albicans (ATCC 10231)
Compound	IZ^a	IZ (UV 366)^b	IZ	IZ (UV 366)	IZ	IZ (UV 366)	IZ	IZ (UV 366)
2a	16	17	15	–	16	–	17	18
2b	18	–	17	18	19	–	18	–
2c	12	–	14	–	12	–	14	–
3a	19	20	18	–	20	21	19	20
3b	20	–	21	22	18	19	20	–
3c	13	14	12	–	10	–	8	–
4a1	19	20	16	17	18	19	16	17
4a2	22	23	21	–	20	–	20	–
4a3	27	28	24	25	22	23	16	17
4a4	13	–	12	–	11	–	11	–
4a5	14	–	13	–	12	–	14	15
4a6	12	–	12	–	13	14	12	14
4b1	14	16	14	15	10	12	12	13
4b2	21	22	20	–	18	–	19	–
4b3	17	–	16	–	17	–	18	–
4c1	14	15	12	14	14	–	15	16
4c2	21	–	23	–	22	–	20	21
4c3	14	–	12	–	10	11	10	–
5a1	12	13	12	–	10	12	14	–
5a2	20	21	21	22	19	10	18	–
5a3	11	–	10	–	9	–	8	–
5b1	13	–	13	14	12	14	12	12
5b2	21	–	20	–	19	20	20	–
5b3	12	–	14	15	14	–	13	15
5c1	11	13	13	–	15	–	15	–
5c2	20	21	22	–	20	21	19	20
5c3	14	–	13	15	13	–	14	16
Ampicillin^c	32	32	28	28	–	–	–	–
Griseofulvin^d	–	–	–	–	30	30	29	29

^aInhibition zone (mm); ^bInhibition zone (mm) using UV (366) light; ^cAmpicillin trihydrate, antibacterial standard; ^dGriseofulvin, antifungal reference.

Table 6. Cytotoxic effects (LC₅₀; μg/mL)^a of the active compounds on some human tumor cell lines using the MTT assay.

	Human colon carcinoma	Human hepatocellular carcinoma	Human breast cancer
Compound	HT29	HepG2	MCF 7
3a	20.5	36.4	45.5
3b	18.4	25.2	36.3
4a2	11.8	22.9	3.4
4a3	7.5	21.3	2.6
4a6	80.2	–^b	–
4b1	40.1	37.3	–
4b2	14.2	28.4	10.6
4c2	37.7	45.2	54.6
5a2	62.3	48.1	–
5b2	56.3	50.7	66.0
5c2	76.2	–^b	–
Doxorubicin^c	12.1	1.69	2.14

^aLC₅₀: Lethal concentration of the compound which causes death of 50% of cells in 24 h (μg/mL); ^btotally inactive against this cell line; ^cPositive control cytotoxic agent.

In vitro MTT cytotoxicity assay

Eighteen analogs namely; 3a–c, 4a₁, 4a₂, 4a₃, 4a₄, 4a₆, 4b₁, 4b₂, 4c₁, 4c₂, 5a₁, 5a₂, 5b₁, 5b₂, 5c₁ and 5c₂ were selected to be evaluated for their in vitro cytotoxic effect via the standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method31^,32 against a panel of three human tumor cell lines, namely, Caucasian breast adenocarcinoma MCF7, hepatocellular carcinoma HePG2 and colon carcinoma HT29. The results are presented in Table 6 as LC₅₀ (μg/mL) which is the lethal concentration of the compound that causes death of 50% of the cells in 24 h.

The obtained data revealed that, the three tested human tumor cell lines exhibited variable degree of activity profiles towards 11 of the tested compounds, namely 3a,b, 4a₂, 4a₃, 4a₆, 4b₁, 4b₂, 4c₂, 5a₂, 5b₂, 5c₂. Among these, the human colon carcinoma HT29 cell line showed pronounced sensitivity against compounds 4a₂ and 4a₃ with LC₅₀ values of 11.8 and 7.5 μg/mL, respectively. Moreover, a significant cytotoxic potential was displayed by compound 4b₂ against the same cell line (14.2 μg/mL). However, compounds 3a and 3b revealed an obvious cytotoxicity profile against colon carcinoma HT29 with LC₅₀ values 20.5 and 18.4 μg/mL, respectively. Compounds 4b₁, 4c₂, 5a₁ and 5b₂ were able to exhibit moderate activity against the same cell line with LC₅₀ values range of 37.7-62.3 μg/mL. Mild activity was displayed by compound 4a₆ (LC₅₀, 80.2 μg/mL) against human colon carcinoma (HT29). Furthermore, the growth of the human hepatocellular carcinoma HePG2 cell line was found to be moderately inhibited by nine of the active compounds, namely 3a–b, 4a₂, 4a₃, 4b₁, 4b₂, 4c₂, 5a₂ and 5b₂, with LC₅₀ values in the range of 21.3–50.7 μg/mL. Among these, the highest cytotoxic activity was displayed by compounds 3b, 4a₂ and 4a₃ which were almost equipotent with LC₅₀ values 25.2, 22.9 and 21.3 μg/mL, respectively. On the other hand, human breast cancer MCF 7 was proved to be the least sensitive among the cell lines tested as it was affected by only seven of the test compounds. An outstanding growth inhibition potential was shown by compounds 4a₂ and 4a₃ as evidenced from their LC₅₀ values (3.4 and 2.6 μg/mL, respectively), however compound 4b₂ displayed appreciable activity. The rest four active compounds namely 3a, 3b, 4c₂ and 5b₂ showed moderate to mild activity against the same cell line with LC₅₀ values of 45.5, 36.3, 54.6 and 66.0 μg/mL, respectively. Further interpretation of the results revealed that, compounds 3a, 3b, 4a₂, 4b₂ and 4a₃ showed considerable broad spectrum of cytotoxic activity against the three tested human tumor cell lines. In particular, compounds 4a₂ and 4a₃ proved to be the most active members in this study with a broad spectrum of activity against the tested cell lines, with special effectiveness against the human colon carcinoma HT29 and human breast cancer MCF 7 cell lines (Table 6). The results in the molecular properties calculations showed that most of the compounds gave favorable values for drug-likeness between 1.38 and 7.29. These values were in total agreement with the tested values for compounds 4a₃ and 4a₄ which were found to be most active against the human colon carcinoma HT29 and human breast cancer MCF 7 cell lines with favorable drug-likeness values (6.28 and 4.44 respectively). A close examination of the structure of the active compounds showed that the presence of electron-withdrawing groups (X = Cl or F) in the thiocarbamoyl moiety (4a₂, 4a₃, 4b₂, 4c₂) play a crucial role in enhancing the activity, whereas, the presence of fluorine atom in compound 4a₃ was responsible for the high activity displayed by this analogs. Moreover, it is clear from the results that the 1-carbamoyl derivatives (5) are much less active than the corresponding 1-thiocarbamoyl analogs (4) and the parent pyrazole compounds (3). No compound with a cyclohexyl group at the thiocarbamoyl end could display any cytotoxic activity indicating a negative role of this substituent.

Conclusion

The results obtained from OSIRIS property explorer and MOLINSPIRATION showed that the synthesized series of carbamoyl and thiocarbamoyl derivatives of pyrazole as potential drug molecules with a drug score values ranged from 0.09 to 0.80. All compounds have exhibited good (theoretical) oral bioavailability potential calculated ranged from 83.60 to 97.66%. All compounds have shown number of rotatable bonds much less than 10 (between 2 and 5) which is an indication of good bioavailability by oral route as shown in Table 4. The results in the calculations showed that most of the compounds gave favorable values for drug-likeness between 1.38 and 7.29. Compounds 3a, 3b, 4a₂, 4a₃, 4b₂, 4c₂, 5a₂, 5b₂ and 5c₂ have shown mild to moderate antimicrobial activity. Furthermore compounds 4a₂ and 4a₃ were found to be most active against the human colon carcinoma HT29 and human breast cancer MCF 7 cell lines. The antimicrobial and cytotoxic activities of these compounds were in total agreement with their favorable drug-likeness, drug-scores and oral bioavailability potential as calculated from OSIRIS property explorer and MOLINSPIRATION programs.

Declaration of interest

This work was supported by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant no. (130-045-D1434). The authors, therefore, acknowledge with thanks DSR technical and financial support. Sincere gratitude is conveyed to the staff members of the Department of Health and Human Services, National Cancer Institute (NCI), Bethesda, Maryland, USA, for carrying out the anticancer screening of the newly synthesized compounds. The authors wish also to thank the Department of Biology, Faculty of Science, Alexandria University for carrying out the microbiological screening.