Table of Contents Author Guidelines Submit a Manuscript
Journal of Chemistry
Volume 2018 (2018), Article ID 9242616, 15 pages
https://doi.org/10.1155/2018/9242616
Research Article

Thiadiazoline- and Pyrazoline-Based Carboxamides and Carbothioamides: Synthesis and Inhibition against Nitric Oxide Synthase

1Departamento de Química Farmacéutica y Orgánica, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja, s/n, 18071 Granada, Spain
2Departamento de Farmacología, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja, s/n, 18071 Granada, Spain
3CIBERCV, Granada, Spain

Correspondence should be addressed to M. Encarnación Camacho; se.rgu@ohcamace and M. Dora Carrión; se.rgu@noirracd

Received 13 November 2017; Revised 18 December 2017; Accepted 11 January 2018; Published 20 March 2018

Academic Editor: Fabio Polticelli

Copyright © 2018 Fabio Arias et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Two new families of pyrazoline and thiadiazoline heterocycles have been developed. Their inhibitory activities against two different isoforms of nitric oxide synthase (inducible and neuronal NOS) are reported. The novel derivatives were synthesized combining the arylthiadiazoline or arylpyrazoline skeleton and a carboxamide or carbothioamide moiety, used as starting material ethyl 2-nitrobenzoates or substituted nitrobenzaldehydes, respectively. The structure-activity relationships of final molecules are discussed in terms of the R1 radical effects in the aromatic ring, the Y atom in the heterocyclic system, the X heteroatom in the main chain, and the R2 substituent in the carboxamide or carbothioamide rest. In general, thiadiazolines (5a–e) inhibit preferentially the neuronal isoform; among them, 5a is the best nNOS inhibitor (74.11% at 1 mM, IC50 = 420 μM). In contrast, pyrazolines (6a–r) behave better as iNOS than nNOS inhibitors, 6m being the best molecule of this series (76.86% at 1 mM of iNOS inhibition, IC50 = 130 μM) and the most potent of all tested compounds.

1. Introduction

Heterocyclic rings having two or three heteroatoms in their skeleton have been widely described as part of pharmacological agents with interesting therapeutic applications. In this way, thiadiazoline system is part of structures with antileishmanial activity [1], as well as antimicrobial [2], anti-inflammatory [2, 3], anticonvulsant [4], or antitumoral [5, 6]. In addition, heterocyclic pyrazoline shows a wide spectrum of pharmacological properties such as anticancer [79], anti-inflammatory [10], anticonvulsant [11, 12], antimicrobial [13], antibacterial, antifungal, and antiparasitic [14]. These two types of heterocycles have also been recognized as nitric oxide synthase (NOS) inhibitors that could be useful in neurodegenerative diseases and inflammatory arthritis [1519].

Nowadays, NOS inhibitors represent an important pharmacological group with diverse medical applications since the overproduction of nitric oxide () is involved in several pathological processes.

The enzyme NOS catalyzes the conversion of L-arginine (L-arg) in L-citrulline and , an important molecular messenger involved in several physiological actions in mammals, such as neurotransmission [20], immune function [21], or blood flow [22]. Three isoforms of NOS have been identified; two of them are constitutively expressed: neuronal (nNOS), which takes part in neural signaling, and endothelial (eNOS), involved in the systemic blood pressure control and plateled aggregation inhibition; the third isoform, inducible NOS (iNOS), is expressed during immune activation and plays an important role in inflammatory response [23].

Due to its implication in many biological processes, the endogenous synthesis of is tightly regulated. An overproduction of by iNOS and nNOS is associated with diverse disorders, such as stroke [24], migraine [25], inflammatory arthritis [26], and various neurodegenerative processes like Parkinson’s, Alzheimer’s, or Huntington’s diseases [2729]. Moreover, produced by stimulation of eNOS plays a physiological role in blood pressure and flow, but its underproduction can cause hypertension [30]. Therefore, the search of selective inhibitors for nNOS or iNOS, but not eNOS, represents an important therapeutic goal since it could help the treatment of diseases in which the first two isoforms are involved.

With this purpose, we have previously described the synthesis and biological evaluation of diverse NOS inhibitors with thiadiazoline 1 [31] and pyrazoline 2 [16, 32] scaffolds, bearing different acyl substituents in the heterocyclic ring. In addition, we have synthesized and evaluated the NOS inhibition of compounds with general structures 3 [33] and 4 [34] (Figure 1). These derivatives had more flexible structure containing N,N′-disubstituted thiourea and urea rests, isosterics to the terminal guanidine moiety of L-Arg, responsible for the inhibitors binding to the enzyme substrate region; thus it plays an essential role in the enzymatic inhibition [35, 36].

Figure 1: Structure of thiadiazolines 1, pyrazolines 2, and N,N′-disubstituted urea and thiourea derivatives 3-4 with NOS inhibitory activity.

According to this background, two new families of derivatives containing arylthiadiazoline-based carboxamides 5 and arylpyrazoline-based carboxamides and carbothioamides 6 have been designed and synthesized in this study (Figure 2). Compounds 5 proceed by mix of the aryl-thiadiazoline fragment present in 1, and a residue of carboxamide included in 3 and 4 molecules. Additionally, the derivatives 6 have been designed by combination of an aryl-pyrazoline moiety contained in 2 and a residue of carboxamide or carbothioamide, the terminal rest of the previously described derivatives 3 and 4 (Figure 3). All of them have different substituents in the aromatic ring and in the carboxamide or carbothioamide residues in order to perform structure-activity relationship studies. These compounds could be an interesting starting point for possible new alternatives in neurodegenerative or inflammatory disorders.

Figure 2: General structure of new derivatives (5a–e) and (6a–r).
Figure 3: Design strategy and structure of compounds (5a–e) and (6a–r).

2. Experimental

2.1. Chemistry
2.1.1. Materials and Methods

Melting points were determined using an Electrothermal-1A-6301 apparatus and are uncorrected. 1H NMR and 13C NMR spectra were recorded on a Varian Inova Unity 300 spectrometer operating at 300.20 for 1H and 75.49 MHz for 13C, on a Varian Direct Drive 400 spectrometer operating at 400.17 MHz for 1H and 100.73 MHz for 13C, and on a Varian Direct Drive 500 spectrometer operating at 499.79 MHz for 1H and 125.69 MHz for 13C, in CDCl3 (at concentration of ca 27 mg mL−1 in all cases). The center of each peak of CDCl3 [7.26 ppm (1H) and 77.0 ppm (13C)] was used as internal reference in a 5 mm 13C/1H dual probe (Wilmad, No. 528-PP). The temperature of the sample was maintained at 297 K. The peaks are reported in ppm (δ). High-resolution mass spectroscopy (HRMS) was carried out on a VG AutoSpec Q high-resolution mass spectrometer (Fision Instruments). Small scale microwave-assisted reactions were performed using an Initiator 2.0 single-mode microwave instrument producing controlled irradiation at 2.450 GHz (Biotage AB, Uppsala). Reaction time refers to holding time at 80°C, not to total irradiation time. An IR sensor outside the reaction vessel was used to control the temperature. Flash chromatography was carried out using silica gel 60, 230–240 mesh (Merk), and the solvent mixture reported within parentheses was used as eluent.

2.1.2. General Procedure for the Synthesis of 2,2-Dimethyl-N-substituted-5-(2-nitro-5-phenylsubstituted)-1,3,4-thiadiazole-3(3H)-carboxamide Derivatives (16a–e)

To a solution of the previously described thiadiazole intermediates 14-15 (2 mmol) [31] in dry CH2Cl2, the corresponding isocyanate (3 mmol) and Et3N (2 mmol) were added under argon [37]. The reaction mixture was irradiated under microwave conditions at 80°C for 20 min. The residue was purified by flash chromatography (EtOAc/hexane, 1 : 10).

2,2-Dimethyl-N-ethyl-5-(2-nitrophenyl)-1,3,4-thiadiazole-3(2H)-carboxamide (16a). Yellow oil, yield 407 mg (1.320 mmol) (66%). 1H NMR (499.79 MHz, CDCl3): δ/ppm 7.72 (dd, 1H, H-3′, = 7.9 Hz, = 1.3 Hz), 7.60 (ddd, 1H, H-5′, = = 7.6 Hz, = 1.3 Hz), 7.55 (ddd, 1H, H-4′, = = 7.8 Hz, = 1.6 Hz), 7.52 (dd, H-6′, = 7.6 Hz, = 1.5 Hz), 5.82 (bs, 1H, -CONH-), 3.26 (m, 2H, -CH2-CH3), 2.07 (s, 6H, 2x-CH3), 1.16 (t, 3H, -CH2-CH3, = 7.3 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 154.6 (-CONH-), 148.6 (C-2′), 140.1 (C-5), 132.1 (C-5′), 130.7, 130.6 (C-6′, C-4′), 124.4 (C-1′), 123.9 (C-3′), 82.5 (C-2), 35.1 (-CH2-CH3), 29.8 (2x-CH3), 15.3 (-CH2-CH3). MS (LSIMS): 331.0830 [M + Na]+, Calcd. Mass for C13H16N4O3NaS 331.0841.

2,2-Dimethyl-5-(2-nitrophenyl)-N-propyl-1,3,4-thiadiazole-3(2H)-carboxamide (16b). Yellow oil, yield 387 mg (1.200 mmol) (60%). 1H NMR (499.79 MHz, CDCl3): δ/ppm 7.69 (dd, 1H, H-3′, = 7.8 Hz, = 1.3 Hz), 7.59 (ddd, 1H, H-5′, = = 7.6 Hz, = 1.3 Hz), 7.53 (ddd, 1H, H-4′, = = 7.8 Hz, = 1.6 Hz), 7.49 (dd, H-6′, = 7.6 Hz, = 1.5 Hz), 5.88 (bs, 1H, -CONH-), 3.18 (m, 2H, -CH2-CH2-CH3), 2.05 (s, 6H, 2x-CH3), 1.53 (m, 2H, -CH2-CH2-CH3), 0.93 (t, 3H, -CH2-CH2-CH3   = 7.4 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 154.7 (-CONH-), 148.6 (C-2′), 139.9 (C-5), 132.0 (C-5′), 130.6 (C-6′, C-4′), 124.2 (C-1′), 123.8 (C-3′), 82.5 (C-2), 42.0 (-CH2-CH2-CH3), 29.8 (2x-CH3), 23.3 (-CH2-CH2-CH3), 11.4 (-CH2-CH2-CH3). MS (LSIMS): 323.1169 [M + H]+, Calcd. Mass for C14H19N4O3S 323.1178.

2,2-Dimethyl-5-(2-nitrophenyl)-N-phenyl-1,3,4-thiadiazole-3(2H)-carboxamide (16c). Yellow oil, yield 449 mg (1.260 mmol) (63%). 1H NMR (499.79 MHz, CDCl3): δ/ppm 7.91 (bs, 1H, -NH-), 7.74 (dd, 1H, H-3′, = 7.8 Hz, = 1.3 Hz), 7.64 (ddd, 1H, H-5′, = = 7.6 Hz, = 1.4 Hz), 7.59 (ddd, 1H, H-4′, = = 7.8 Hz, = 1.6 Hz), 7.54 (dd, H-6′, = 7.6 Hz, = 1.5 Hz), 7.49 (dd, 2H, H-2′′, H-6′′, = = 8.6 Hz, = = 1.1 Hz), 7.31 (m, 2H, H-3′′, H-5′′), 7.06 (td, 1H, H-4′′, = = 7.4 Hz, = = 1.1 Hz), 2.11 (s, 6H, 2x-CH3). 13C NMR (125.69 MHz, CDCl3): δ/ppm 151.7 (-CONH-), 148.6 (C-5), 141.0 (C-2′), 138.3 (C-1′′), 132.1 (C-6′), 131.0 (C-5′), 130.6 (C-4′), 129.1 (C-3′′, C-5′′), 123.8 (C-4′′), 123.7 (C-1′), 123.4 (C-3′), 119.4 (C-2′′, C-6′′), 82.4 (C-2), 29.8 (2x-CH3). MS (LSIMS): 379.0837 [M + Na]+, Calcd. Mass for C17H16N4O3NaS 379.0841.

2,2-Dimethyl-N-ethyl-5-(5-methoxy-2-nitrophenyl)-1,3,4-thiadiazole-3(2H)-carboxamide (16d). White solid, yield 419 mg (1.240 mmol) (62%). Mp: 164–167°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 7.94 (d, 1H, H-3′, = 8.9 Hz), 7.01 (d, 1H, H-4′, = 2.7 Hz), 6.99 (s, 1H, H-6′), 5.88 (bs, 1H, -CONH-), 3.92 (s, 3H, -OCH3), 3.32–3.22 (m, 2H, -CH2-CH3), 2.10 (s, 6H, 2x-CH3), 1.16 (t, 3H, -CH2-CH3, = 7.2 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 162.7 (C-5′), 154.8 (-CONH-), 141.7, 141.2 (C-2′, C-5), 127.9 (C-1′), 127.1 (C-3′), 116.2 (C-6′), 115.3 (C-4′), 82.8 (C-2), 56.3 (-OCH3), 35.1 (-CH2-CH3), 29.7 (2x-CH3), 15.4 (-CH2-CH3). MS (LSIMS): 361.0954 [M + Na]+, Calcd. Mass for C14H18N4O4NaS 361.0946.

2,2-Dimethyl-5-(5-methoxy-2-nitrophenyl)-N-propyl-1,3,4-thiadiazole-3(2H)-carboxamide (16e). Yelow oil, yield 458 mg (1.300 mmol) (65%). 1H NMR (499.79 MHz, CDCl3) δ/ppm 7.91 (d, 1H, H-3′, = 9.0 Hz), 7.00 (d, 1H, H-4′, = 2.8 Hz), 6.98 (d, 1H, H-6′, = 2.9 Hz), 5.93 (bs, 1H, -CONH-), 3.91 (s, 3H, -OCH3), 3.19 (dd, 2H, -CH2-CH2-CH3, = 13.2 Hz, = 6.8 Hz), 2.09 (s, 6H, 2x-CH3), 1.54 (dd, 2H, -CH2-CH3, = 14.5 Hz, = 7.3 Hz), 0.92 (t, 3H, -CH2-CH3, = 7.4 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 162.6 (C-5′), 154.9 (-CONH-), 141.7, 141.1 (C-2′, C-5), 127.8 (C-1′), 126.9 (C-3′), 116.1 (C-6′), 115.3 (C-4′), 82.7 (C-2), 56.3 (-OCH3), 42.0 (-CH2-CH2-CH3), 29.7 (2x-CH3), 23.4 (-CH2-CH2-CH3), 11.5 (-CH2-CH2-CH3). MS (LSIMS): 353.1309 [M + H]+, Calcd. Mass for C15H21N4O4S 353.1284.

2.1.3. General Procedure for the Synthesis of 3-(2-nitro- or 6-nitrophenyl-substituted)-N-substituted-4,5-dihydro-1H-pyrazole-1-carboxamide or Carbothioamide Derivatives (34a–r)

Thioisocyanate or isocyanate (3 mmol) was added, under argon, to a solution of previously described pyrazole derivatives 30–33 (2 mmol) [16] in dry CH2Cl2 and Et3N (an equimolar amount) [37]. The reaction mixture was irradiated under microwave conditions at 80°C for 20 min. The crude mixture was purified by flash chromatography (EtOAc/hexane, 1 : 1).

3-(5-Chlorophenyl-2-nitro-)-N-ethyl-4,5-dihydro-1H-pyrazole-1-carboxamide (34a). White solid, yield 368 mg (1.240 mmol) (62%). Mp: 123–125°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 7.78 (d, 1H, H-3′, = 8.6 Hz), 7.51 (m, 2H, H-4′, H-6′), 5.78 (bs, 1H, -CONH-), 4.05 (t, 2H, H-5, = 10.2 Hz), 3.34 (m, 2H, -CH2-CH3), 3.12 (t, 2H, H-4, = 10.2 Hz), 1.19 (t, 3H, -CH2-CH3, = 7.2 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 154.8 (-CONH-), 148.0 (C-3), 146.7 (C-2′), 138.7 (C-5′), 129.9, 129.8 (C-4′, C-6′), 128.2 (C-1′), 125.5 (C-3′), 45.4 (C-5), 35.0 (-CH2-CH3), 33.5 (C-4), 15.5 (-CH2-CH3). MS (LSIMS): 297.0757 [M + H]+, Calcd. Mass for C12H14ClN4O3 297.0754.

3-(5-Chlorophenyl-2-nitro)-N-propyl-4,5-dihydro-1H-pyrazole-1-carboxamide (34b). White solid, yield 462 mg (1.220 mmol) (61%). Mp: 110–112°C. 1H NMR (300.20 MHz, CDCl3): δ/ppm 7.69 (d, 1H, H-3′, = 8.6 Hz), 7.43 (m, 2H, H-4′, H-6′), 5.76 (bs, 1H, -CONH-), 3.98 (t, 2H, H-5, = 10.4 Hz), 3.19 (m, 2H, 2H, -CH2-CH2-CH3), 3.05 (t, 2H, H-4, = 10.4 Hz), 1.50 (m, 2H, -CH2-CH2-CH3), 0.88 (t, 3H, -CH2-CH2-CH3, = 7.4 Hz). 13C NMR (125.69 MHz, CDCl3): δ/pp 154.9 (-CO-), 147.9 (C-3), 146.6 (C-2′), 138.7 (C-5′), 129.9, 129.8 (C-4′, C-6′), 128.1 (C-1′), 125.5 (C-3′), 45.4 (C-5), 41.8 (-CH2-CH2-CH3), 33.4 (C-4), 23.4 (-CH2-CH2-CH3), 11.3 (-CH2-CH2-CH3). MS (LSIMS): 311.0917 [M + H]+, Calcd. Mass for C13H16ClN4O3 311.0911.

3-(5-Chlorophenyl-2-nitro)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide (34c). Yellow solid, yield 414 mg (1.200 mmol) (60%). Mp: 119–121°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 7.82 (d, 1H, H-3′, = 8.7 Hz), 7.53 (m, 3H, H-4′, H-2′′, H-6′′), 7.29 (m, 3H, H-3′′, H-5′′, H-6′), 7.05 (t, 1H, H-4′′, = = 7.3 Hz), 4.14 (t, 2H, H-5, = 10.5 Hz), 3.21 (t, 2H, H-4, = 10.5 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 151.8 (-CONH-), 149.1 (C-3), 146.6 (C-2′), 138.9 (C-5′), 138.1 (C-1′′), 130.2, 130.1 (C-4′, C-6′), 129.0 (C-3′′, C-5′′), 127.8 (C-1′), 125.6 (C-4′′), 123.2 (C-3′), 119.1 (C-2′′, C-6′′), 45.1 (C-5), 33.9 (C-4). MS (LSIMS): 367.0569 [M + Na]+, Calcd. Mass for C16H13ClN4O3Na 367.0574.

3-N-Ethyl-(5-methoxyphenyl-2-nitro)-4,5-dihydro-1H-pyrazole-1-carboxamide (34d). Yellow solid, yield 520 mg (1.780 mmol) (89%). Mp: 128–130°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 8.03 (d, 1H, H-3′, = 8.9 Hz), 6.99 (dd, 1H, H-4′, = 8.9, = 2.7 Hz), 6.97 (d, 1H, H-6′, = 2.7 Hz), 5.81 (bs, 1H, -CO-) 4.05 (t, 2H, H-5, = 10.4 Hz), 3.92 (s, 3H, -OCH3), 3.33 (m, 2H, -CH2-CH3), 3.10 (t, 2H, H-4, = 10.4 Hz), 1.17 (t, 3H, -CH2-CH3, = 7.2 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 163.2 (-CONH-), 155.5 (C-3), 151.4 (C-5′), 141.4 (C-2′), 130.5 (C-1′), 127.4 (C-3′), 115.8 (C-6′), 114.7 (C-4′), 56.3 (-OCH3), 45.5 (C-5), 35.1 (-CH2-CH3), 34.9 (C-4), 15.7 (-CH2-CH3). MS (LSIMS): 293.1232 [M + H]+, Calcd. Mass for C13H17N4O4 293.1250.

3-(5-Methoxyphenyl-2-nitro)-N-propyl-4,5-dihydro-1H-pyrazole-1-carboxamide (34e). Yellow solid, yield 441 mg (1.440 mmol) (72%). Mp: 89–92°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 8.02 (d, 1H, H-3′, = 8.9 Hz), 6.99 (dd, 1H, H-4′, = 9.0, = 2.7 Hz), 6.97 (d, 1H, H-6′, = 2.7 Hz), 5.86 (bs, 1H, -CO-), 4.05 (t, 2H, = 10.4 Hz), 3.92 (s, 3H, -OCH3), 3.25 (m, 2H, -CH2-CH2-CH3), 3.11 (t, 2H, H-4, = 10.4 Hz), 1.55 (m, 2H, -CH2-CH2-CH3), 0.93 (t, 3H, -CH2-CH2-CH3, = 7.4 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 163.2 (-CONH-), 155.5 (C-3), 151.4 (C-5′), 141.4 (C-2′), 130.4 (C-1′), 127.3 (C-3′), 115.8 (C-6′), 114.7 (C-4′), 56.3 (-OCH3), 45.5 (C-5), 42.0 (-CH2-CH2-CH3), 34.8 (C-4), 23.6 (-CH2-CH2-CH3), 11.5 (-CH2-CH2-CH3). MS (LSIMS): 307.1422 [M + H]+, Calcd. Mass for C14H19N4O4 307.1406.

3-(5-Methoxyphenyl-2-nitro)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide (34f). Yellow solid, yield 476 mg (1.400 mmol) (70%). Mp: 150–152°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 8.01 (d, 1H, H-3′, = 8.8 Hz), 7.80 (s, 1H, -CO-), 7.43 (d, 2H, H-2′′, H-6′′, = 7.6 Hz), 7.23 (m, 2H, H-3′′, H-5′′), 6.97 (m, 3H, H-4′, H-6′, H-4′′), 4.08 (t, 2H, H-5, = 10.3 Hz), 3.88 (s, 3H, -OCH3), 3.13 (t, 2H, H-4, = 10.3 Hz). 13C NMR (100.73 MHz, CDCl3): δ/ppm 163.3 (-CONH-), 152.6 (C-3), 152.3 (C-5′), 141.3 (C-2′), 138.5 (C-1′′), 130.1 (C-1′), 129.1 (C-2′′, C-6′′), 127.5 (C-3′), 123.1 (C-4′′), 119.1 (C-3′′, C-5′′), 116.0 (C-6′), 114.9 (C-4′), 56.3 (-OCH3), 45.2 (C-5), 35.2 (C-4). MS (LSIMS): 341.1249 [M + H]+, Calcd. Mass for C17H17N4O4 341.1250.

3-(4,5-Dimethoxy-2-nitrophenyl)-N-ethyl-4,5-dihydro-1H-pyrazole-1-carboxamide (34g). Brown solid, yield 464 mg (1.440 mmol) (72%). Mp: 146-147°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 7.57 (s, 1H, H-3′), 6.91 (s, 1H, H-6′), 5.82 (m, 1H, -NH-), 4.04 (t, 2H, H-5, = 10.3 Hz), 4.00 (s, 3H, 5′-OCH3), 3.97 (s, 3H, 4′-OCH3), 3.33 (m, 2H, -CH2-CH3), 3.07 (t, 2H, H-4, = 10.3 Hz), 1.17 (t, 3H, -CH2-CH3, = 7.2 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 155.44 (-CONH-), 153.1 (C-3); 152.1 (C-5′), 149.7 (C-4′), 141.1 (C-2′), 122.4 (C-1′), 112.0 (C-6′), 108.0 (C-3′), 56.8 (5′-OCH3), 56.7 (4′-OCH3), 45.5 (C-5), 35.1 (C-4), 34.9 (-CH2-CH3), 15.7 (-CH2-CH3). MS (LSIMS): 323.1348 [M + H]+, Calcd. Mass for C14H19N4O5 323.1355.

3-(4,5-Dimethoxy-2-nitrophenyl)-N-propyl-4,5-dihydro-1H-pyrazole-1-carboxamide (34h). White solid, yield 464 mg (1.380 mmol) (69%). Mp: 144-145°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 7.39 (s, 1H, H-3′), 6.80 (s, 1H, H-6′), 5.81 (m, 1H, -NH-), 3.87 (t, 2H, H-5, = 10.3 Hz), 3.86 (s, 3H, 5′-OCH3), 3.82 (s, 3H, 4′-OCH3), 3.08 (m, 2H, -CH2-CH2-CH3), 2.93 (t, 2H, H-4, = 10.3 Hz), 1.40 (m, 2H, -CH2-CH2-CH3), 0.78 (t, 3H, -CH2-CH2-CH3, = 7.4 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 155.2 (-CONH-), 152.6 (C-3), 151.6 (C-5′), 149.3 (C-4′), 140.7 (C-2′), 121.8 (C-1′), 111.7 (C-6′), 107.5 (C-3′), 56.4 (5′-OCH3), 56.3 (4′-OCH3), 45.1 (C-5), 41.6 (-CH2-CH2-CH3), 34.4 (C-4), 23.3 (-CH2-CH2-CH3), 11.1 (-CH2-CH2-CH3). MS (LSIMS): 337.1527 [M + H]+, Calcd. Mass for C15H21N4O5 337.1512.

3-(4,5-Dimethoxy-2-nitrophenyl)-N-methyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (34i). Brown solid, yield 441 mg (1.360 mmol) (68%). Mp: 179–181°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 7.59 (s, 1H, H-3′), 7.17 (bs, 1H, -NH-), 6.89 (s, 1H, H-6′), 4.40 (t, 2H, H-5, = 10.0 Hz), 3.99 (s, 3H, 5′-OCH3), 3.98 (s, 3H, 4′-OCH3), 3.16 (d, 3H, -CH3, = 4.8 Hz), 3.13 (t, 2H, H-4, = 10.0 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 177.50 (-CSNH-), 155.8 (C-3), 153.1 (C-5′), 150.0 (C-4′), 141.0 (C-2′), 121.6 (C-1′), 111.8 (C-6′), 108.0 (C-3′), 56.8 (5′-OCH3), 56.6 (4′-OCH3), 49.2 (C-5), 34.7 (C-4), 31.6 (-CH3). MS (LSIMS): 347.0800 [M + Na]+, Calcd. Mass for C13H16N4O4NaS 347.0790.

3-(4,5-Dimethoxy-2-nitrophenyl)-N-ethyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (34j). Yellow solid, yield 474 mg (1.400 mmol) (70%). Mp: 194-195°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 7.60 (s, 1H, H-3′), 7.12 (m, 1H, -NH-), 6.89 (s, 1H, H-6′), 4.40 (t, 2H, H-5, = 10.2 Hz), 4.00 (s, 3H, 5′-OCH3), 3.98 (s, 3H, 4′-OCH3), 3.69 (m, 2H, -CH2-CH3), 3.13 (t, 2H, H-4, = 10.2 Hz), 1.24 (t, 3H, -CH2-CH3, = 7.2 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 176.4 (-CSNH-), 155.8 (C-3), 153.2 (C-5′), 150.0 (C-4′), 141.0 (C-2′), 121.7 (C-1′), 111.9 (C-6′), 108.0 (C-3′), 56.9 (5′-OCH3), 56.7 (4′-OCH3), 49.1 (C-5), 39.7 (-CH2-CH3), 34.7 (C-4), 14.7 (-CH2-CH3). MS (LSIMS): 339.1141 [M + H]+, Calcd. Mass for C14H19N4O4S 339.1127.

3-(4,5-Dimethoxy-2-nitrophenyl)-N-propyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (34k). Yellow solid, yield 458 mg (1.300 mmol) (65%). Mp: 155-156°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 7.54 (s, 1H, H-3′), 7.16 (m, 1H, -NH-), 6.88 (s, 1H, H-6′), 4.35 (t, 2H, H-5, = 10.0 Hz), 3.97 (s, 3H, 5′-OCH3), 3.95 (s, 3H, 4′-OCH3), 3.55 (m, 2H, -CH2-CH2-CH3), 3.10 (t, 2H, H-4, = 10.0 Hz), 1.61 (m, 2H, -CH2-CH3), 0.92 (t, 3H, CH3, = 7.4 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 176.3 (-CSNH-), 155.5 (C-3), 153.0 (C-5′), 149.9 (C-4′), 140.9 (C-2′), 121.4 (C-1′), 111.8 (C-6′), 107.8 (C-3′), 56.7 (5′-OCH3), 56.6 (4′-OCH3), 48.9 (C-5), 46.5 (-CH2-CH2-CH3), 34.5 (C-4), 22.5 (-CH2-CH2CH3), 11.4 (-CH2-CH2-CH3). MS (LSIMS): 353.1274 [M + H]+, Calcd. Mass for C15H21N4O4S 353.1284.

N-Ethyl-3-(6-nitro-2,3,4-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide, (34l). Yellow solid, yield 472 mg (1.340 mmol) (67%). Mp: 126–128°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 7.40 (s, 1H, H-5′), 5.69 (bs, 1H, -CO-), 4.05 (t, 2H, H-5, = 10.3 Hz), 3.98, 3.96, 3.89 (3s, 9H, 2′-OCH3, 3′-OCH3, 4′-OCH3), 3.30 (m, 2H, -CH2-CH3), 3.18 (t, 2H, H-4, = 10.3 Hz), 1.14 (t, 3H, -CH2-CH3, = 7.2 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 155.25 (C-3), 153.7 (-CONH-), 152.4 (C-4′), 149.1 (C-3′), 146.5 (C-6′), 143.6 (C-2′), 116.6 (C-1′), 104.1 (C-5′), 62.0, 61.0 (2′-OCH3, 3′-OCH3), 56.5 (4′-OCH3), 45.1 (C-5), 36.4 (-CH2-CH3), 34.7 (C-4), 15.3 (-CH2-CH3). MS (LSIMS): 353.1456 [M + H]+, Calcd. Mass for C15H21N4O6 353.1461.

3-(6-Nitro-2,3,4-trimethoxyphenyl)-N-propyl-4,5-dihydro-1H-pyrazole-1-carboxamide (34m). Yellow solid, yield 506 mg (1.380 mmol) (69%). Mp: 132–135°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 7.39 (s, 1H, H-5′), 5.74 (bs, 1H, -CO-), 4.03 (t, H-5, = 10.3 Hz), 3.96, 3.95, 3.88 (3s, 9H, 2′-OCH3, 3′-OCH3, 4′-OCH3), 3.20 (t, H-4, = 10.3 Hz), 3.11 (m, 2H, -CH2-CH2-CH3), 1.50 (m, 2H, -CH2-CH2-CH3), 0.91 (t, 3H, -CH2-CH2-CH3, = 7.4 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 155.9 (C-3), 153.6 (-CONH-), 152.3 (C-4′), 149.4 (C-3′), 146.5 (C-6′), 143.6 (C-2′), 116.4 (C-1′), 104.1 (C-5′), 62.1, 61.1 (2′-OCH3, 3′-OCH3), 56.8 (4′-OCH3), 44.9 (C-5), 41.8 (-CH2-CH2-CH3), 36.2 (C-4); 23.2 (-CH2-CH2-CH3), 11.2 (-CH2-CH2-CH3). MS (LSIMS): 389.1436 [M + Na]+, Calcd. Mass for C16H22N4O6Na 389.1437.

3-(6-Nitro-2,3,4-trimethoxyphenyl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide (34n). Yellow solid, yield 536 mg (1.340 mmol) (67%). Mp: 140–142°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 7.73 (bs, 1H, -CONH-), 7.44 (d, 2H, H-2′′, H-6′′, = 7.7 Hz), 7.42 (s, 1H, H-5′), 7.26 (t, 2H, H-3′′, H-5′′, = 7.9 Hz), 6.99 (t, 1H, H-4′′, = 7.4 Hz), 4.12 (t, 2H, H-5, = 10.3 Hz), 3.97, 3.96, 3.91 (3s, 9H, 2′-OCH3, 3′-OCH3, 4′-OCH3), 3.25 (t, 2H, H-4, = 10.3 Hz). 13C NMR (100.73 MHz, CDCl3): δ/ppm 153.9 (C-3), 152.3 (-CONH-); 152.2 (C-4′); 150.3 (C-3′); 146.7 (C-6′); 143.5 (C-2′); 138.5 (C-1′′); 128.8 (C-3′′, C-5′′), 122.8 (C-4′′), 118.9 (C-2′′, C-6′′), 116.2 (C-1′), 104.2 (C-5′), 62.1, 61.2 (2′-OCH3, 3′-OCH3), 56.6 (4′-OCH3), 44.6 (C-5), 36.5 (C-4). MS (LSIMS): 401.1464 [M + H]+, Calcd. Mass for C15H21N4O6 401.1461.

N-Methyl-3-(6-nitro-2,3,4-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (34o). Yellow solid, yield 482 mg (1.360 mmol) (68%). Mp: 122–125°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 7.44 (s, 1H, H-5′), 7.04 (s, 1H, -CSNH-), 4.43 (t, 2H, H-5, = 9.9 Hz), 3.98, 3.98, 3.90 (3s, 9H, 2′-OCH3, 3′-OCH3, 4′-OCH3), 3.23 (t, 2H, H-4, = 9.9 Hz), 3.14 (d, 3H, -CH3, = 4.7 Hz). 13C NMR (100.73 MHz, CDCl3): δ/ppm 177.1 (-CSNH-), 154.0 (C-3), 153.6 (C-4′), 152.2 (C-3′), 146.8 (C-6′), 143.2 (C-2′), 115.9 (C-1′), 104.2 (C-5′), 62.2, 61.2 (2′-OCH3, 3′-OCH3), 56.6 (4′-OCH3), 48.7 (C-5), 36.0 (C-4), 31.4 (-CH3). MS (LSIMS): 355.1075 [M + H]+, Calcd. Mass for C14H19N4O5S 355.1076.

N-Ethyl-3-(6-nitro-2,3,4-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (34p). Yellow solid, yield 472 mg (1.280 mmol) (64%). Mp: 111–113°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 7.40 (bs, 1H, H-5′), 6.94 (s, 1H, -CSNH-), 4.38 (t, 2H, H-5, = 9.9 Hz), 3.95, 3.94, 3.86 (3s, 9H, 2′-OCH3, 3′-OCH3, 4′-OCH3), 3.59 (m, 2H, -CH2-CH3), 3.18 (t, 2H, H-4, = 9.9 Hz), 1.18 (t, 3H, -CH2-CH3, = 7.3 Hz). 13C NMR (100.73 MHz, CDCl3): δ/ppm 175.92 (-CSNH-), 154.0 (C-3), 153.5 (C-4′), 152.2 (C-3′), 146.8 (C-6′), 143.2 (C-2′), 115.9 (C-1′), 104.2 (C-5′), 62.1, 61.2 (2′-OCH3, 3′-OCH3), 56.6 (4′-OCH3), 48.6 (C-5), 39.5 (-CH2-CH3), 35.9 (C-4), 14.5 (-CH2-CH3). MS (LSIMS): 369.1234 [M + H]+, Calcd. Mass for C15H21N4O5S 369.1233.

3-(6-Nitro-2,3,4-trimethoxyphenyl)-N-propyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (34q). Yellow solid, yield 505 mg (1.320 mmol) (66%). Mp: 94–96°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 7.40 (bs, 1H, H-5′), 7.02 (s, 1H, -CSNH-), 4.39 (t, 2H, H-5, = 10.0 Hz), 3.95, 3.94, 3.86 (3s, 9H, 2′-OCH3, 3′-OCH3, 4′-OCH3), 3.55 (m, 2H, -CH2-CH2-CH3), 3.19 (t, 2H, H-4, = 10.0 Hz), 1.59 (m, 2H, -CH2-CH2-CH3), 0.91 (t, 3H, -CH2-CH2-CH3, = 7.4 Hz). 13C NMR (100.73 MHz, CDCl3): δ/ppm 176.07 (-CSNH-), 154.0 (C-3), 153.4 (C-4′), 152.2 (C-3′), 146.7 (C-6′), 143.2 (C-2′), 115.8 (C-1′), 104.2 (C-5′), 62.1, 61.2 (2′-OCH3, 3′-OCH3), 56.6 (4′-OCH3), 48.6 (C-5), 46.4 (-CH2-CH2-CH3), 35.9 (C-4), 22.4 (-CH2-CH2-CH3), 11.3 (-CH2-CH2-CH3). MS (LSIMS): 383.1388 [M + H]+, Calcd. Mass for C16H23N4O5S 383.13893.

3-(6-Nitro-2,3,4-trimethoxyphenyl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (34r). Yellow oil, yield 533 mg (1.280 mmol) (64%). 1H NMR (400.17 MHz, CDCl3): δ/ppm 8.80 (bs, 1H, -CSNH-), 7.56 (d, 2H, H-2′′, H-6′′, = 7.4 Hz), 7.44 (s, 1H, H-5′), 7.32 (t, 2H, H-3′′, H-5′′, = 7.4 Hz), 7.15 (t, 1H, H-4′′, = 7.4 Hz), 4.49 (t, 2H, H-5, = 9.9 Hz), 3.97, 3.96, 3.92 (3s, 9H, 2′-OCH3, 3′-OCH3, 4′-OCH3), 3.28 (t, 2H, H-4, = 9.9 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm (125.68 MHz, CDCl3), δ 174.1 (-CSNH-), 154.4 (C-3), 154.2 (C-4′), 152.2 (C-3′), 146.8 (C-6′), 143.4 (C-2′), 138.6 (C-1′′), 128.6 (C-3′′, C-5′′), 125.4 (C-4′′), 124.2 (C-2′′, C-6′′), 115.6 (C-1′), 104.3 (C-5′), 62.2, 61.3 (2′-OCH3, 3′-OCH3), 56.6 (4′-OCH3), 48.6 (C-5), 36.2 (C-4). MS (LSIMS): 417.1229 [M + H]+, Calcd. Mass for C19H21N4O5S 417.1246.

2.1.4. General Procedure for the Synthesis of 5-(2-Amino-5-phenylsubstituted)-2,2-dimethyl-N-substituted-1,3,4-thiadiazole-3(3H)-carboxamide Derivatives (5a–e)

To a solution of each nitroarene 16a–e (0.400 mmol) in ethanol, 453 mg of SnCl2 was added. The resulting mixture was refluxed for 2 h. After this period, the reaction was neutralized with NaHCO3 aqueous solution, extracted with AcOEt (2 × 15 ml), and dried with sodium sulfate [38]. Finally, the solvent was evaporated ad the residue was purified by flash chromatography (EtOAc/hexane, 1 : 10) to give the title derivatives 5a–e.

5-(2-Aminophenyl)-2,2-dimethyl-N-ethyl-1,3,4-thiadiazole-3(2H)-carboxamide (5a). Brown oil, yield 94 mg (0.328 mmol) (84%). 1H NMR (400.17 MHz, CDCl3): δ/ppm 7.16 (t, 2H, H-6′, H-4′, = 7.6 Hz), 6.71 (t, 2H, H-5′, H-3′, = 8.2 Hz), 5.69 (bs, 1H, -CONH-), 4.90 (bs, 2H, -NH2), 3.31 (m, 2H, -CH2-CH3), 2.02 (s, 6H, 2x-CH3), 1.16 (t, 3H, -CH2-CH3, = 7.3 Hz). 13C NMR (100.73 MHz, CDCl3): δ/ppm 154.8 (-CONH-), 147.7 (C-2′), 145.2 (C-5), 131.0, 130.7 (C-6′, C-4′), 117.6, 116.4 (C-5′, C-3′), 113.5 (C-1′), 78.2 (C-2), 35.0 (-CH2-CH3), 29.5 (2x-CH3), 15.6 (-CH2-CH3). MS (LSIMS): 301.1087 [M + Na]+, Calcd. Mass for C13H18N4ONaS 301.1099.

5-(2-Aminophenyl)-2,2-dimethyl-N-propyl-1,3,4-thiadiazole-3(2H)-carboxamide (5b). Brown oil, yield 108 mg (0.372 mmol) (93%). 1H NMR (499.79 MHz, CDCl3): δ/ppm 7.17 (m, 2H, H-6′, H-4′), 6.72 (m, 2H, H-5′, H-3′), 5.77 (bs, 1H, -CONH-), 5.16 (bs, 2H, -NH2), 3.24 (m, 2H, -CH2-CH2-CH3), 2.03 (s, 6H, 2x-CH3), 1.56 (m, 2H, -CH2-CH2-CH3), 0.94 (t, 3H, -CH2-CH2-CH3, = 7.4 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 154.9 (-CONH-), 147.7 (C-2′), 145.3 (C-5), 131.0, 130.7 (C-4′, C-6′), 117.6, 116.3 (C-5′, C-3′), 113.5 (C-1′), 78.2 (C-2), 42.0 (-CH2-CH2-CH3), 29.5 (2x-CH3), 23.5 (-CH2-CH2-CH3), 11.5 (-CH2-CH2-CH3). MS (LSIMS): 293.1440 [M + H]+, Calcd. Mass for C14H21N4OS 293.1436.

5-(2-Aminophenyl)-2,2-dimethyl-N-phenyl-1,3,4-thiadiazole-3(2H)-carboxamide (5c). White solid, yield 114 mg (0.348 mmol) (87%). Mp: 113–116°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 7.79 (s, 1H, -CONH-), 7.49 (d, 2H, H-2′′, H-6′′, = = 8.6 Hz), 7.36 (dd, 2H, H-3′′, H-5′′, = = 8.6 Hz, = = 7.4 Hz), 7.27 (m, 2H, H-6′, H-4′), 7.12 (t, H-4′′, = = 7.4 Hz), 6.80 (m, 2H, H-5′, H-3′), 5.30 (bs, 2H, NH2), 2.15 (s, 6H, 2x-CH3). 13C NMR (100.73 MHz, CDCl3): δ/ppm 151.9 (-CONH-), 148.9 (C-2′), 145.3 (C-5), 138.2 (C-1′′), 131.4, 130.8 (C-4′, C-6′), 129.1 (C-3′′, C-5′′), 123.6 (C-4′′), 119.8 (C-2′′, C-6′′), 117.8, 116.5 (C-3′,C-5′), 113.3 (C-1′), 78.2 (C-2), 29.6 (2x-CH3). MS (LSIMS): 327.1269 [M + H]+, Calcd. Mass for C17H19N4OS 327.1280.

5-(2-Amino-5-methoxyphenyl)-2,2-dimethyl-N-ethyl-1,3,4-thiadiazole-3(2H)-carboxamide (5d). Yelow oil, yield 86 mg (0.280 mmol) (70%). 1H NMR (499.79 MHz, CDCl3): δ/ppm 6.82 (dd, 1H, H-4′, = 8.8 Hz, = 2.7 Hz), 6.72 (d, 1H, H-6′, = 2.7 Hz), 6.70 (d, 1H, H-3′, = 8.8 Hz), 5.74 (bs, 1H, -CONH-), 3.75 (s, 3H, -OCH3), 3.32 (dd, 2H, -CH2-CH3, = 13.1 Hz, = 7.1 Hz), 2.03 (s, 6H, 2xCH3), 1.17 (t, 3H, -CH2-CH3, = 7.2 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 154.8 (-CONH-), 151.7 (C-5), 147.2 (C-5′), 139.4 (C-2′), 118.5 (C-4′), 117.9 (C-3′), 114.4 (C-1′), 114.1 (C-6′), 78.5 (C-2), 56.1 (-OCH3), 35.2 (-CH2-CH3), 29.6 (2x-CH3), 15.6 (-CH2-CH3). MS (LSIMS): 309.1382 [M + H]+, Calcd. Mass for C14H21N4O2S 309.1385.

5-(2-Amino-5-methoxyphenyl)-2,2-dimethyl-N-propyl-1,3,4-thiadiazole-3(2H)-carboxamide (5e). Yelow oil, yield 88 mg (0.272 mmol) (68%). 1H NMR (499.79 MHz, CDCl3): δ/ppm 6.83 (dd, 1H, H-4′, = 8.8 Hz, = 2.8 Hz), 6.73 (d, 1H, H-6′, = 2.8 Hz), 6.70 (d, 1H, H-3′, = 8.8 Hz), 5.80 (bs, 1H, -CONH-), 3.75 (s, 3H, -OCH3), 3.24 (dd, 2H, -CH2-CH2-CH3, = 13.6 Hz, = 6.5 Hz), 2.03 (s, 6H, 2xCH3), 1.56 (dd, 2H, -CH2-CH3, = 14.5 Hz, = 7.3 Hz), 0.94 (t, 3H, -CH2-CH3, = 7.4 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 155.1 (-CONH-), 152.0 (C-5), 147.4 (C-5′), 139.7 (C-2′), 118.8 (C-4′), 118.2 (C-3′), 114.7 (C-1′), 114.4 (C-6′), 78.8 (C-2), 56.3 (-OCH3), 42.3 (-CH2-CH2-CH3), 29.8 (2x-CH3), 23.8 (-CH2-CH2-CH3), 11.7 (-CH2-CH2-CH3). MS (LSIMS): 323.1563 [M + H]+, Calcd. Mass for C15H23N4O2S 323.1542.

2.1.5. General Procedure for the Synthesis of 3-(2-Amino or 6-aminophenyl-substituted)-N-substituted-4,5-dihydro-1H-pyrazole-1-carboxamide or Carbothioamide Derivatives (6a–r)

Aminophenyl pyrazolines 6a–r were prepared according to the same synthetic procedure described before for the thiazodiazoline derivatives 5a–e. Purification of the final compounds was made using (EtOAc/hexane, 1 : 2) as eluent in the flash chromatography.

3-(2-Amino-5-chlorophenyl)-N-ethyl-4,5-dihydro-1H-pyrazole-1-carboxamide (6a). White solid, yield 86 mg (0.324 mmol) (81%). Mp: 215–217°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 7.14 (d, 1H, H-6′, = 2.4 Hz), 7.12 (dd, 1H, H-4′, = 8.6, = 2.4 Hz), 6.68 (d, 1H, H-3′, = 8.6 Hz), 5.54 (bs, 3H, -NH2, -CONH-), 3.96 (t, 2H, H-5, = 10.2 Hz), 3.37 (m, 2H, -CH2-CH3), 3.27 (t, 2H, H-4, = 10.2 Hz), 1.20 (t, 3H, -CH2-CH3, = 10.2 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 154.6 (-CONH-), 153.3 (C-3), 144.4 (C-2′), 130.0 (C-4′), 128.3 (C-6′), 121.3 (C-5′), 116.9 (C-3′), 114.9 (C-1′), 43.1 (C-5), 34.8 (-CH2-CH3), 33.3 (C-4), 15.5 (-CH2-CH3). MS (LSIMS): 267.1021 [M + H]+, Calcd. Mass for C12H16ClN4O 267.1013.

3-(2-Amino-5-chlorophenyl)-N-propyl-4,5-dihydro-1H-pyrazole-1-carboxamide (6b). White solid, yield 94 mg (0.336 mmol) (84%). Mp: 212–214°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 7.15 (d, 1H, H-6′, = 2.3 Hz), 7.11 (dd, 1H, H-4′, = 8.6 Hz, = 2.3 Hz), 6.68 (d, 1H, H-3′, = 8.6 Hz), 5.59 (bs, 3H, -NH2, -CONH-), 3.95 (t, 2H, H-5, = 10.4 Hz), 3.27 (m, 4H, H-4, -CH2-CH2-CH3), 1.59 (m, 2H, -CH2-CH2-CH3), 0.95 (t, 3H, -CH2-CH2-CH3, = 7.4 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 154.9 (-CONH-), 153.5 (C-3), 144.6 (C-2′), 130.8 (C-4′), 128.5 (C-6′), 121.6 (C-5′), 117.2 (C-3′), 115.2 (C-1′), 43.4 (C-5), 41.9 (-CH2-CH2-CH3), 33.5 (C-4), 23.6 (-CH2-CH2-CH3), 11.3 (-CH2-CH2-CH3). MS (LSIMS): 281.1163 [M + H]+, Calcd. Mass for C13H18ClN4O 281.1169.

3-(2-Amino-5-chlorophenyl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide (6c). Yellow solid, yield 98 mg (0.312 mmol) (78%). Mp: 112–115°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 7.47 (d, 2H, H-2′′, H-6′′, = 7.7 Hz), 7.32 (t, 2H, H-3′′, H-5′′, = 7.7 Hz), 7.17 (m, 2H, H-6′, H-4′), 7.07 (t, 1H, H-4′′, = = 7.7 Hz), 6.72 (d, 1H, H-3′, = 8.6 Hz), 5.56 (s, 2H, -NH2), 4.04 (t, 2H, H-5, = 10.3 Hz), 3.35 (t, 2H, H-4, = 10.3 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 154.5 (-CONH-), 151.7 (C-3), 144.8 (C-2′), 138.2 (C-1′′), 130.6 (C-4′), 129.0 (C-3′′, C-5′′), 128.7 (C-6′), 123.3 (C-4′′), 121.7 (C-5′), 119.4 (C-2′′, C-6′′), 117.4 (C-3′), 114.8 (C-1′), 43.1 (C-5), 33.9 (C-4). MS (LSIMS): 315.1013 [M + H]+, Calcd. Mass for C16H16ClN4O 315.1013.

3-(2-Amino-5-methoxyphenyl)-N-ethyl-4,5-dihydro-1H-pyrazole-1-carboxamide (6d). White solid, yield 102 mg (0.388 mmol) (97%). Mp: 194-195°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 6.83 (dd, 1H, H-4′, = 8.8, = 2.8 Hz), 6.74 (d, 1H, H-6′, = 2.8 Hz), 6.71 (d, 1H, H-3′, = 8.8 Hz), 5.62 (bs, 1H, -CONH-), 5.20 (bs, 2H, -NH2), 3.93 (t, 2H, H-5, = 10.1 Hz), 3.76 (s, 3H, -OCH3), 3.37 (m, 2H, -CH2-CH3), 3.27 (t, 2H, H-4, = 10.1 Hz), 1.19 (t, 3H, -CH2-CH3, = 7.2 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 155.1 (-CONH-), 154.5 (C-3), 151.5 (C-5′), 140.6 (C-2′), 117.4 (C-4′), 117.4 (C-3′), 114.9 (C-1′), 114.2 (C-6′), 56.1 (-OCH3), 43.4 (C-5), 35.2 (-CH2-CH3), 33.9 (C-4), 15.9 (-CH2-CH3). MS (LSIMS): 263.1490 [M + H]+, Calcd. Mass for C13H19N4O2 263.1508.

3-(2-Amino-5-methoxyphenyl)-N-propyl-4,5-dihydro-1H-pyrazole-1-carboxamide (6e). White solid, yield 100 mg (0.364 mmol) (91%). Mp: 187–190°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 6.82 (dd, 1H, H-4′, = 8.8 Hz, = 2.8 Hz), 6.74 (d, 1H, H-6′, = 2.8 Hz), 6.71 (d, 1H, H-3′, = 8.8 Hz), 5.66 (bs, 1H, -CONH-), 5.22 (bs, 2H, -NH2), 3.93 (t, 2H, H-5, = 10.1 Hz), 3.76 (s, 3H, -OCH3), 3.28 (m, 4H, -CH2-CH2-CH3, H-4), 1.57 (m, 2H, -CH2-CH2-CH3), 0.94 (t, 3H, -CH2-CH2-CH3, = 7.4 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 155.2 (-CONH-), 154.4 (C-3), 151.5 (C-5′), 140.5 (C-2′), 117.4 (C-4′), 117.3 (C-3′), 115.0 (C-1′), 114.2 (C-6′), 56.1 (-OCH3), 43.5 (C-5), 42.0 (-CH2-CH2-CH3), 33.9 (C-4), 23.8 (-CH2-CH2-CH3), 11.5 (-CH2-CH2-CH3). MS (LSIMS): 277.1675 [M + H]+, Calcd. Mass for C14H21N4O2 277.1665.

3-(2-Amino-5-methoxyphenyl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide (6f). Yellow solid, yield 120 mg (0.384 mmol) (96%). Mp: 168–170°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 7.64 (s, 1H, -CONH-), 7.49 (dt, 2H, H-2′′, H-6′′, = = 8.1 Hz, = = 1.1 Hz), 7.31 (dd, 2H, H-3′′, H-5′′, = = 8.1 Hz, = = 7.5 Hz), 7.05 (tt, 1H, H-4′′, = = 7.5 Hz, = = 1.1 Hz,), 6.87 (dd, 1H, H-4′, = 8.8 Hz, = 2.8 Hz), 6.78 (d, 1H, H-6′, = 2.8 Hz), 6.75 (d, 1H, H-3′, = 8.8 Hz), 5.27 (bs, 2H, -NH2), 4.02 (t, 2H, H-5, = 10.3 Hz), 3.78 (s, 3H, -OCH3), 3.36 (t, 2H, H-4, = 10.3 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 155.4 (-CONH-), 152.0 (C-3), 151.6 (C-5′), 140.6 (C-2′), 138.5 (C-1′′), 129.1 (C-2′′, C-6′′), 123.3 (C-4′′), 119.4 (C-3′′, C-5′′), 117.9 (C-4′), 117.6 (C-3′), 114.6 (C-1′), 114.28 (C-6′), 56.2 (-OCH3), 43.2 (C-5), 34.2 (C-4). MS (LSIMS): 311.1516 [M + H]+, Calcd. Mass for C17H19N4O2 311.1508.

3-(2-Amino-4,5-dimethoxyphenyl)-N-ethyl-4,5-dihydro-1H-pyrazole-1-carboxamide (6g). Brown solid, yield 98 mg (0.336 mmol) (84%). Mp: 161–163°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 6.67 (s, 1H, H-6′), 6.28 (s, 1H, H-3′), 5.58 (m, 1H, -NH-), 3.91 (m, 2H, H-5), 3.86 (s, 3H, 4′-OCH3), 3.80 (s, 3H, 5′-OCH3), 3.36 (m, 2H, -CH2-CH3), 3.25 (t, 2H, H-4, = 10.0 Hz), 1.18 (t, 3H, CH3, = 7.2 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 155.3 (-CONH-), 154.6 (C-3), 151.9 (C-4′), 142.3 (C-5′), 141.2 (C-2′), 113.1 (C-6′), 106.0 (C-1′), 99.9 (C-3′), 57.2 (5′-OCH3), 55.9 (4′-OCH3), 43.2 (C-5), 34.1 (C-4), 35.1 (-CH2-CH3), 15.9 (-CH2-CH3). MS (LSIMS): 293.1620 [M + H]+, Calcd. Mass for C14H21N4O3 293.1614.

3-(2-Amino-4,5-dimethoxyphenyl)-N-propyl-4,5-dihydro-1H-pyrazole-1-carboxamide (6h). Brown solid, yield 118 mg (0.388 mmol) (97%). Mp: 197–199°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 6.67 (s, 1H, H-6′), 6.28 (s, 1H, H-3′), 5.63 (bs, 1H, -NH-), 5.41 (bs, 2H, -NH2), 3.91 (t, 2H, H-5, = 10.1 Hz), 3.86 (s, 3H, 4′-OCH3), 3.81 (s, 3H, 5′-OCH3), 3.27 (m, 4H, -CH2-CH2-CH3, H-4), 1.57 (m, 2H, -CH2-CH2-CH3), 0.94 (t, 3H, -CH2-CH2-CH3, = 7.4 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 155.4 (-CONH-), 154.6 (C-3), 151.9 (C-4′), 142.2 (C-5′), 141.2 (C-2′), 113.1 (C-6′), 106.0 (C-1′), 99.9 (C-3′), 57.2 (5′-OCH3), 55.9 (4′-OCH3), 43.2 (C-5), 42.0 (-CH2-CH2-CH3), 34.1 (C-4), 23.8 (-CH2-CH2-CH3), 11.5 (-CH2-CH2-CH3). MS (LSIMS): 307.1775 [M + H]+, Calcd. Mass for C15H23N4O3 307.1770.

3-(2-Amino-4,5-dimethoxyphenyl)-N-methyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (6i). Brown solid, yield 106 mg (0.360 mmol) (90%). Mp: 180–182°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 6.79 (bs, 1H, -NH-), 6.67 (s, 1H, H-6′), 6.28 (s, 1H, H-3′), 4.28 (t, 2H, H-5, = 10.0 Hz), 3.87 (s, 3H, 4′-OCH3), 3.81 (s, 3H, 5′-OCH3), 3.29 (t, 2H, H-4, = 10.0 Hz), 3.21 (d, 3H, CH3, = 4.7 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 175.7 (-CSNH-), 158.1 (C-3), 152.8 (C-4′), 142.8 (C-5′), 141.5 (C-2′), 113.0 (C-6′), 105.1 (C-1′), 99.8 (C-3′), 57.1 (5′-OCH3), 56.0 (4′-OCH3), 47.3 (C-5), 33.7 (C-4), 31.7 (-CH3). MS (LSIMS): 295.1219 [M + H]+, Calcd. Mass for C13H19N4O2S 295.1229.

3-(2-Amino-4,5-dimethoxyphenyl)-N-ethyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (6j). Yelow solid, yield 98 mg (0.316 mmol) (79%). Mp: 180–182°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 6.73 (m, 1H, -NH-), 6.67 (s, 1H, H-6′), 6.27 (s, 1H, H-3′), 5.38 (bs, 2H, -NH2), 4.28 (t, 2H, H-5, = 10.0 Hz), 3.87 (s, 3H, 4′-OCH3), 3.81 (s, 3H, 5′-OCH3), 3.72 (m, 2H, -CH2-CH3), 3.29 (t, 2H, H-4, = 10.0 Hz), 1.26 (t, 3H, -CH2-CH3, = 7.2 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 174.65 (-CSNH-), 158.0 (C-3), 152.8 (C-4′), 142.8 (C-5′), 141.4 (C-2′), 113.0 (C-6′), 105.1 (C-1′), 99.8 (C-3′), 57.1 (5′-OCH3), 56.0 (4′-OCH3), 47.2 (C-5), 39.7 (-CH2-CH3), 39.6 (C-4), 15.0 (-CH2-CH3). MS (LSIMS): 309.1364 [M + H]+, Calcd. Mass for C14H21N4O2S 309.1385.

3-(2-Amino-4,5-dimethoxyphenyl)-N-propyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (6k). Yellow solid, yield 106 mg (0.328 mmol) (82%). Mp: 193–195°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 6.83 (m, 1H, -NH-), 6.67 (s, 1H, H-6′), 6.29 (s, 1H, H-3′), 4.28 (t, 2H, H-5, = 10.0 Hz), 3.87 (s, 3H, 4′-OCH3), 3.81 (s, 3H, 5′-OCH3), 3.64 (m, 2H, -CH2-CH2-CH3), 3.29 (t, 2H, H-4, = 10.0 Hz), 1.67 (m, 2H, -CH2-CH2-CH3), 0.97 (t, 3H, -CH2-CH2-CH3, = 7.4 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 174.8 (-CSNH-), 158.0 (C-3), 152.7 (C-4′), 142.3 (C-5′), 141.5 (C-2′), 112.9 (C-6′), 105.3 (C-1′), 99.9 (C-3′), 57.1 (5′-OCH3), 56.0 (4′-OCH3), 47.2 (C-5), 46.5 (-CH2-CH2-CH3), 33.6 (C-4), 22.9 (-CH2-CH2-CH3), 11.5 (-CH2-CH2-CH3). MS (LSIMS): 323.1540 [M + H]+, Calcd. Mass for C15H23N4O2S 323.1542.

3-(6-Amino-2,3,4-trimethoxyphenyl)-N-ethyl-4,5-dihydro-1H-pyrazole-1-carboxamide (6l). Brown solid, yield 104 mg (0.322 mmol) (80%). Mp: 150–153°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 6.06 (s, H-5′), 5.62 (bs, 3H, -NH2, -CONH-), 3.87 (m, 8H, H-5, 2′-OCH3, 3′-OCH3), 3.76 (s, 3H, 4′-OCH3), 3.35 (m, 4H, H-4, -CH2-CH3), 1.17 (t, 3H, -CH2-CH3, = 7.2 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 155.3, 155.1 (-CONH-, C-3), 154.0 (C-4′), 153.6 (C-2′), 142.9 (C-6′), 134.2 (C-3′), 102.8 (C-1′), 95.1 (C-5′), 61.2, 61.0 (2′-OCH3, 3′-OCH3), 55.7 (4′-OCH3), 43.6 (C-5), 36.4 (-CH2-CH3), 34.9 (C-4), 15.8 (-CH2-CH3). MS (LSIMS): 323.1720 [M + H]+, Calcd. Mass for C15H23N4O4 323.1710.

3-(6-Amino-2,3,4-trimethoxyphenyl)-N-propyl-4,5-dihydro-1H-pyrazole-1-carboxamide (6m). Brown solid, yield 112 mg (0.332 mmol) (83%). Mp: 156–158°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 6.06 (s, 1H, H-6′), 5.69 (bs, 3H, -NH2, -CONH-), 3.88 (m, 8H, H-5, 2′-OCH3, 3′-OCH3), 3.76 (s, 3H, 4′-OCH3), 3.35 (t, 2H, H-4, = 10.2 Hz), 3.26 (m, 2H, -CH2-CH2-CH3), 1.55 (m, 2H, -CH2-CH2-CH3), 0.93 (t, 3H, -CH2-CH2-CH3, = 7.40 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 155.4, 155.1 (-CONH-, C-3), 154.0 (C-4′), 153.6 (C-2′), 142.8 (C-6′), 134.3 (C-3′), 102.9 (C-1′), 95.2 (C-5′), 61.2, 61.0 (2′-OCH3, 3′-OCH3), 55.7 (4′-OCH3), 43.7 (C-5), 41.8 (-CH2-CH2-CH3), 36.4 (C-4), 23.6 (-CH2-CH2-CH3), 11.3 (-CH2-CH2-CH3). MS (LSIMS): 337.1870 [M + H]+, Calcd. Mass for C16H25N4O4 337.1876.

3-(6-Amino-2,3,4-trimethoxyphenyl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide (6n). Brown solid, yield 110 mg (0.298 mmol) (74%). Mp: 163–165°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 7.69 (bs, 1H, -CONH-), 7.49 (d, 2H, H-2′′, H-6′′, = 7.7 Hz), 7.31 (t, 2H, H-3′′, H-5′′, = 7.7 Hz), 7.04 (t, 1H, H-4′′, = 7.7 Hz), 6.08 (s, 1H, H-5′), 3.97 (t, 2H, H-5, = 10.3 Hz), 3.90, 3.86, 3.79 (3s, 9H, 2′-OCH3, 3′-OCH3, 4′-OCH3), 3.44 (t, 2H, H-4, = 10.3 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 155.5 (C-3), 154.6 (-CONH-), 154.1 (C-4′), 152.1 (C-2′), 143.3 (C-6′), 138.5 (C-1′′), 134.1 (C-3′), 128.9 (C-3′′, C-5′′), 122.9 (C-4′′), 119.1 (C-2′′, C-6′′), 102.4 (C-1′), 95.0 (C-5′), 61.2, 61.1 (2′-OCH3, 3′-OCH3), 55.8 (4′-OCH3), 43.4 (C-5), 36.7 (C-4). MS (LSIMS): 371.1706 [M + H]+, Calcd. Mass for C19H23N4O4 371.1719.

3-(6-Amino-2,3,4-trimethoxyphenyl)-N-methyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (6o). Brown solid, yield 106 mg (0.326 mmol) (82%). Mp: 110–112°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 6.83 (bs, 1H, -CSNH-), 6.01 (s, 1H, H-5′), 4.21 (t, 2H, H-5, = 9.9 Hz), 3.85, 3.82, 3.74 (3s, 9H, 2′-OCH3, 3′-OCH3, 4′-OCH3), 3.39 (t, 2H, H-4, = 9.9 Hz), 3.17 (d, 3H, -CH3, = 4.8 Hz). 13C NMR (100.73 MHz, CDCl3): δ/ppm 175.9 (-CSNH-), 157.6 (C-3), 155.9 (C-4′), 154.3 (C-3′), 143.8 (C-6′), 134.1 (C-2′), 101.8 (C-1′), 94.8 (C-5′), 61.2, 61.0 (2′-OCH3, 3′-OCH3), 55.7 (4′-OCH3), 47.6 (C-5), 36.2 (C-4), 31.5 (-CH3). MS (LSIMS): 347.1156 [M + Na]+, Calcd. Mass for C14H20N4O3NaS 347.1154.

3-(6-Amino-2,3,4-trimethoxyphenyl)-N-ethyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (6p). Brown solid, yield 110 mg (0.324 mmol) (81%). Mp: 131–133°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 6.79 (bs, 1H, -CSNH-), 6.01 (s, 1H, H-5′), 4.21 (t, 2H, H-5, = 9.9 Hz), 3.85, 3.82, 3.74 (3s, 9H, 2′-OCH3, 3′-OCH3, 4′-OCH3), 3.68 (m, 2H, -CH2-CH3), 3.38 (t, 2H, H-4, = 9.9 Hz), 1.23 (t, 3H, -CH2CH3, = 7.2 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 174.83 (-CSNH-), 157.6 (C-3), 155.9 (C-4′), 154.3 (C-3′), 143.7 (C-6′), 134.1 (C-2′), 101.9 (C-1′), 94.9 (C-5′), 61.2, 61.0 (2′-OCH3, 3′-OCH3), 55.8 (4′-OCH3), 47.6 (C-5), 39.5 (C-4), 36.2 (-CH2-CH3), 14.8 (-CH2-CH3). MS (LSIMS): 339.1482 [M + H]+, Calcd. Mass for C15H23N4O3S 339.1477.

3-(6-Amino-2,3,4-trimethoxyphenyl)-N-propyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (6q). Brown solid, yield 118 mg (0.334 mmol) (83%). Mp: 144–146°C. 1H NMR (400.17 MHz, CDCl3): δ/ppm 6.86 (bs, 1H, -CSNH-), 6.01 (s, 1H, H-5′), 4.21 (t, 2H, H-5, = 9.9 Hz), 3.85, 3.82, 3.74 (3s, 9H, 2′-OCH3, 3′-OCH3, 4′-OCH3), 3.61 (m, 2H, -CH2-CH2-CH3), 3.38 (t, 2H, H-4, = 9.9 Hz), 1.64 (m, 2H, -CH2-CH2-CH3), 0.94 (t, 3H, -CH2-CH2-CH3, = 7.4 Hz). 13C NMR (100.73 MHz, CDCl3): δ/ppm 174.94 (-CSNH-), 157.5 (C-3), 155.9 (C-4′), 154.3 (C-3′), 143.7 (C-6′), 134.1 (C-2′), 101.9 (C-1′), 94.8 (C-5′), 61.2, 61.0 (2′-OCH3, 3′-OCH3), 55.7 (4′-OCH3), 47.6 (C-5), 46.4 (-CH2-CH2-CH3), 36.2 (C-4), 22.7 (-CH2-CH2-CH3), 11.4 (-CH2-CH2-CH3). MS (LSIMS): 353.1642 [M + H]+, Calcd. Mass for C16H25N4O3S 353.1647.

3-(6-Amino-2,3,4-trimethoxyphenyl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (6r). Brown solid, yield 138 mg (0.358 mmol) (89%). Mp: 150–152°C. 1H NMR (499.79 MHz, CDCl3): δ/ppm 8.62 (bs, 1H, -CSNH-), 7.58 (d, 2H, H-2′′, H-6′′, = 8.1 Hz), 7.38 (t, 2H, H-3′′, H-5′′, = 7.9 Hz), 7.21 (dd, 1H, H-4′′, = 10.6 Hz, = 4.2 Hz), 6.05 (s, 1H, H-5′), 4.33 (t, 2H, H-5, = 9.8 Hz), 3.92, 3.87, 3.79 (3s, 9H, 2′-OCH3, 3′-OCH3, 4′-OCH3), 3.50 (t, 2H, H-4, = 9.8 Hz). 13C NMR (125.69 MHz, CDCl3): δ/ppm 172.9 (-CSNH-), 158.5 (C-3), 156.2 (C-4′), 154.5 (C-3′), 144.0 (C-6′), 138.8 (C-1′′), 134.2 (C-2′), 128.6 (C-3′′, C-5′′), 125.5 (C-4′′), 124.6 (C-2′′, C-6′′), 101.6 (C-1′), 94.9 (C-5), 61.2, 61.1 (2′-OCH3, 3′-OCH3), 55.8 (4′-OCH3), 47.5 (C-5), 36.4 (C-4). MS (LSIMS): 387.1490 [M + H]+, Calcd. Mass for C19H23N4O3S 387.1491.

2.2. Biology
2.2.1. nNOS and iNOS Activity Determination

All the experiments were performed in vitro, using reagents obtained mainly from Sigma-Aldrich Química and Merk (Spain). The radioactive L-[H3]-arginine was obtained from Amersham Biosciences (Perkin Elmer) (Spain) and Calmodulin along with nNOS and iNOS recombinant enzymes from Enzo Life Sciences, Group Taper, Seville (Spain).

The NOS activity was measured by controlling the L-[3H]-arginine conversion to L-[3H]-citrulline in three experiments performed in triplicate, following the Bredt and Snyder method [39].

The reaction took place in a final incubation volume of 100 μL which includes 10 μL of an aliquot of recombinant enzyme added to 50 μL of a buffer with a final concentration of 25 mM tris-(hydroxymethyl)-aminometane hydrochloride (Tris-HCl), 1 mM DL-dithiothreitol (DTT), 4 μM 5,6,7,8-tetrahydro-L-biopterin dihydrocloride (H4-biopterin), 10 μM flavin-adenine dinucleotide (FAD), 0,5 mM hypoxantine-9-β-D-ribofuranoside (inosine), 0,5 mg/mL bovine serum albumin (BSA), 0,1 mM CaCl2, 10 μM L-arginine, 10 μg mL−1 calmodulin (only for nNOS), and 0,05 μM L-[3H]-arginine, at pH 7.6, 10 μL of a 10 mM in ethanol solution of each thiadiazoline 5a–e or pyrazoline 6a–r derivative, and enough water to reach 100 μL. In order to start the reaction, 10 μL of a 7.5 mM NADPH was added. NADPH was omitted in control incubations. All the samples were stirred and incubated for 30 minutes at 37°C. At the end of the incubation, 400 μL of a cold solution 0.1 M N-(2-hydroxymethyl)piperazine-N′-(2-ethanesulfonic acid) HEPES, 10 mM ethylene glycol-bis-(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), and 0.175 mg/mL L-citrulline at pH 5.5 stopped the reaction. This mixture goes through a column packed with Dowex-50W ion-exchange resin (Na+ form) and was eluted with 1.2 mL of water losing around 98% of radioactivity. Finally, 50 μL of each sample was diluted with scintillation liquid and the measurements were taken. The nNOS activity was expressed in picomoles of L-[3H]-citrulline produced/mg of protein/min.

2.2.2. eNOS Inhibition

Experiments were conducted in male Wistar rats obtained from Harlan Laboratories (Barcelona, Spain) weighing 200–250 g. The descending thoracic aortic rings were dissected and cut into rings which were mounted in organ chambers by means of two L-shaped stainless-steel wires inserted into the lumen and attached to the chamber and to an isometric force-displacement transducer coupled to a signal amplifier (Dynamometer UF1, Cibertec, Madrid) and connected to a computer via an A/D interface. Contractile tension was recorded by a Power-Lab 800 (AD Instruments, Cibertec, Madrid), as previously described [40]. The chamber was filled with Krebs solution (composition in mmol/L: NaCl, 118; KCl, 4.75; NaHCO3, 25; MgSO4, 1.2; CaCl2, 2; KH2PO4, 1.2; and glucose, 11) at 37°C and gassed with 95% O2 and 5% CO2. Rings were stretched to 2 g of tension and equilibrated for 90–120 min. After equilibration, aortic rings with a functional endothelium were contracted with phenylephrine (1 μmol/L) and a concentration-response curve was constructed by cumulative addition of acetylcholine (basal curve). Then, to evaluate the inhibition of eNOS activity, the rings were washed and incubated with vehicle (DMSO), 6m (0.1, 0.5, 1 mmol/L), or L-NAME (-nitro-L-arginine methyl ester hydrochloride) (1 mmol/L) for 30 min, and again a concentration-response curve to acetylcholine was constructed in phenylephrine-contracted rings. Results are expressed as percentage of phenylephrine-evoked contraction and area under curve (AUC) of the relaxant response was calculated as an index of eNOS activity. Data are expressed as the mean ± SEM and reflects the number of aortic rings from different rats.

2.3. Statistical Analysis

Data are expressed as the mean ± SEM. Statistically significant differences between groups were calculated by Students’ -test for unpaired observations or for multiple comparisons. ANOVA, followed by the Newmane-Keuls multiple range test, was used. A was considered statistically significant.

3. Results and Discussion

3.1. Synthetic Chemistry

The general synthetic pathways followed to obtain the new compounds 5a–e and 6a–r are represented in Schemes 1 and 2, respectively.

Scheme 1: Synthetic route of the final compounds (5a–e).
Scheme 2: Synthetic pathway of the final compounds (6a–r).

Scheme 1 shows the general synthetic pathway of the final 5-(2-amino-5-phenylsubstituted)-2,2-dimethyl-N-substituted-1,3,4-thiadiazole-3(3H)-carboxamide derivatives 5a–e described in the present report. We have introduced two principal modifications in these derivatives in order to obtain structure-activity relationships: (i) introduction of an electron-donating group at the H-5′ place of the benzene ring and (ii) insertion of an alkyl or aryl carboxamide moiety in the 3-position of the heterocyclic. The synthetic pathway begins with the esterification of the commercial 5-methoxy-2-nitrobenzoic acid 7 which leads to the ethyl benzoate 9 [31]. This one and the ethyl 2-nitrobenzoate 8 were transformed into the 2-nitro-5-substituted-benzohydrazides 10-11 in the presence of hydrazine and ethanol [31]. These two molecules were turned into the corresponding 2-nitro-5-substituted-N′-(1-isopropylidene)benzohydrazides 12-13 in presence of acetone and ethanol at room temperature. The subsequent addition of Lawesson’s Reagent to the hydrazides led to the cyclation, obtaining the 2,3-dihydro-1,3,4-thiadiazole derivatives 14-15 [31]. Nucleophilic addition of alkyl or aryl-isocyanate to these last compounds using microwave gave the nitrophenyl thiadiazole-carboxamides 16a–e [37], which were subjected to reduction in the presence of stannous chloride in refluxing ethanol [38] to give the desired aminoaryl thiadiazole-carboxamides 5a–e.

On the other hand, Scheme 2 represents the synthetic pathway of the final 3-(2-amino or 6-aminophenyl-substituted)-N-substituted-4,5-dihydro-1H-pyrazole-1-carboxamide or carbothioamide derivatives. In these molecules, three major modifications have been made: (i) substitution of one, two, or three hydrogen atoms in the aromatic ring by electron-withdrawing or electron-donating groups; (ii) introduction of a carboxamide or carbothioamide moiety in the 1-position of pyrazoline heterocycle; and (iii) insertion of an alkyl or aryl substituent in the carboxamide or carbothioamide rest. In the synthesis of these compounds we have carried out the transformation of the commercially available 5-chloro-2-nitrobenzaldehyde 18, 5-methoxy-2-nitrobenzaldehyde 19, 4,5-dimethoxy-2-nitrobenzaldehyde 20, and the synthesized 2,3,4-trimethoxy-6-nitrobenzaldehyde 21 (by nitration of 2,3,4-trimethoxybenzaldehyde 17 with a mixture of HNO3 and AcOH), into the corresponding allylic alcohols 22–25, by treatment with vinyl-magnesium bromide [16, 32]. These intermediates were further oxidized (Jones reagent) obtaining the enone derivatives 26–29 [16, 32]. The reaction of the enones with hydrazine in ethanol produced the 4,5-dihydro-1H-pyrazoles 30–33 [16, 32] which, in situ, were converted into the intermediate nitroderivatives 34a–r by reaction with the adequate alkyl or aryl-isocyanates and -isothiocyanates [37]. Finally, reduction of the nitro group with SnCl2 [38] in ethanol gave the final carboxamides and carbothioamides 6a–r.

3.2. Biological Assays
3.2.1. nNOS and iNOS Inhibition

The in vitro biological evaluation of the new heterocycles 5a–e and 6a–r as inhibitors of both inducible and neuronal NOS isoforms has been made in the presence of recombinant isoenzymes. We have used a 1 mM concentration for each final compound. The results of this assessment are shown in Table 1. The pyrazoline 2a (R1 = 2,3,4-(OMe)3; R2 = Ph) previously published [16] has been used as a control.

Table 1: Structure and in vitro iNOS and nNOS inhibition (%) observed in the presence of 1 mM concentration of compounds (5a–e) and (6a–r). Pyrazoline 2a has been included as a control.

The nNOS inhibition shows that the tested molecules display a moderate inhibition percentage versus this isoform, excepting the thiadiazolines 5a–c which exhibit considerable inhibition results. On the whole, thiadiazolines are better inhibitors than pyrazolines, being thiadiazolines with R1 = H more potent that those with R1 = OMe. In this way, the thiadiazole-carboxamides 5a (X = O, Y = S, R2 = Et, 74.11%) and 5c (X = O, Y = S, R2 = Ph, 67.02%) are the best nNOS inhibitors in this series as well as the best ones of all synthesized compounds. In addition, among pyrazoline derivatives, carbothioamides are better nNOS inhibitors than carboxamides. Within the carbothioamides, molecules with three methoxy electron-donating groups in the phenyl moiety (6o–r) are those with the best inhibition values, 6r (R2 = Ph, 47.12%) being the best inhibitor of this group. In this last series, the inhibition percentage increases when the volume of the substituent in the carbothioamide moiety rises, in the Me < Et < Pr < Ph order.

Concernig the iNOS inhibition, the assayed derivatives show better results versus this inducible isoenzyme, because half of the tested molecules have more than 50% of inhibition percentage. As a rule, aryl-pyrazolines exhibit better percentages than aryl-thiadiazolines, showing inhibition percentages in the range of 50.92% and 76.86%. Among molecules with a thiadiazoline moiety, compounds with a methoxy substituent in the aromatic ring (R1) show better percentages of inhibition than the unsubstituted ones, because the only two synthesized derivatives 5d and 5e have more than 50% of inhibition; and, regarding the R2 substituent in the carboxamide residue, propyl is the best one, derivative 5b being the best iNOS inhibitor of this group (58.07%). With respect to the pyrazoline derivatives, about the aromatic ring substitution, the monosubstituted derivatives with an electron-withdrawing group show better iNOS inhibition values than the ones with an electron-donating group. Besides, disubstituted compounds with two donating groups produce lower inhibition than the trisubstituted derivatives. Moreover, there is not much difference between carboxamides and carbothioamides; but for the R1 and R2 substituents, we can see that compounds with the best inhibition values carry three electron-donating groups on the benzene ring and a propyl substituent in the carbothioamide (6q, 71.29%) or carboxamide (6m, 76.86%) residues, this last molecule being the best inhibitor of this series and the most powerful of all tested compounds. In addition, these compounds show a moderate selectivity iNOS/nNOS.

If we compare derivatives 5 and 6 with the previously synthesized 1–4 molecules (Figure 3), we can see that thiadiazoline-carboxamides 5 are better nNOS inhibitors than acyl-thiadiazolines 1 which had better iNOS inhibition values, although a general rule cannot be established. In addition, pyrazoline-carboxamides and carbothioamides 6 inhibit better iNOS than nNOS unlike the acyl-pyrazolines 2. As a consequence, the type of substituent in the heterocyclic ring seems to be important in order to inhibit one isoform or another. Regarding the more flexible derivatives 3 and 4, the new molecules 6 behave like the 3-oxopropyl-alkylureas and thioureas 3 due to the fact that they are also better iNOS inhibitors, unlike the derivatives with a hydroxyl group 4 (which exhibit better nNOS inhibition values).

Table 2 exhibits the IC50 data for the nNOS inhibition of the most interesting thiadiazolines 5a–c, and the IC50 data for the iNOS inhibition of the best pyrazolines 6b, 6m, 6q, and 6r, and the IC50 value of nNOS for the control 2a [16]. Compound 5a behaves as the best nNOS inhibitor (420 μM), and 6m had the best result versus iNOS (130 μM).

Table 2: IC50 values (mM) for the inhibition of nNOS activity of compounds 5a–c and iNOS activity of 6b, 6m, 6q, and 6r. Pyrazoline 2a has been included as a control [16].
3.2.2. eNOS Inhibition

In addition, the best iNOS inhibitor derivative 6m was tested as an eNOS inhibitor in order to check the absence of side effects that could derive from the inhibition of this last isoform. In this way, acetylcholine-induced endothelium-dependent relaxation has been analyzed using endothelium intact rat aortic rings. This classic cholinergic agonist activates eNOS using a calcium-dependent mechanism [41]. Vehicle of 6m (DMSO) was unable to alter the relaxant responses to acetylcholine as compared to basal. Similarly, 6m at 0.1 and 0.5 mmol/L did not modify this response. However, 6m at 1 mmol/L significantly inhibited the relaxation induced by acetylcholine, whereas the nonselective NOS inhibitor L-NAME [42] suppressed this relaxant response (Figure 4(a)). When we measured the area under curve of relaxation to acetylcholine, considered as an indirect index of eNOS activity, we found that 6m inhibited by 33.1% this activity at 1 mmol/L (Figure 4(b)), confirming that this compound did not inhibit eNOS in the range 100–500 μM, although it displayed a poor eNOS inhibitory effect to higher concentration.

Figure 4: Effects of 6m on eNOS activity. (a) Acetylcholine- (Ach-) evoked relaxation in aortic rings with endothelium contracted with 1 μmol/L phenylephrine under basal conditions and after incubation with DMSO, 6m (0.1, 0.5, 1 mmol/L), or L-NAME (1 mmol/L) for 30 min (). (b) Area under relaxant-response curve (AUC) to Ach from experiments to (a). Data are expressed as the mean ± SEM of experiments. versus vehicle group.

4. Conclusions

In summary, the synthesis of five new thiadiazoline- and eighteen pyrazoline-based carboxamides and carbothioamides 5 and 6, each one with different substituents in the aromatic ring and in the carboxamide or carbothioamide moiety, is described. Furthermore, we evaluate the nNOS and iNOS inhibitory activity of all these new derivatives and the eNOS activity for the best iNOS inhibitor 6m. In general, thiadiazolines are better nNOS inhibitors and pyrazolines present better inhibition against iNOS versus nNOS, carbothioamides being with three donating substituents in the aromatic ring the best ones. Thiadiazoline-carboxamide 5a is the most powerful nNOS inhibitor tested, and pyrazoline-carbothioamide 6m is the best iNOS of all them all. In addition, this last compound is approximately 2.5 times more selective to inhibit NOS in inflammatory processes than the constitutive nNOS. Moreover, 6m does not inhibit eNOS at the concentration values necessary to inhibit the other isoforms, which is convenient in order to avoid hypertension as a side effect. Consequently, these novel derivatives could be an interesting starting point to find possible new therapeutic alternatives for neurodegenerative and inflammatory diseases.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This work was partially supported by the Instituto de Salud Carlos III through Grant FI11/00432.

References

  1. F. Poorrajab, S. K. Ardestani, S. Emami, M. Behrouzi-Fardmoghadam, A. Shafiee, and A. Foroumadi, “Nitroimidazolyl-1,3,4-thiadiazole-based anti-leishmanial agents: Synthesis and in vitro biological evaluation,” European Journal of Medicinal Chemistry, vol. 44, no. 4, pp. 1758–1762, 2009. View at Publisher · View at Google Scholar · View at Scopus
  2. A. A. Kadi, E. S. Al-Abdullah, I. A. Shehata, E. E. Habib, T. M. Ibrahim, and A. A. El-Emam, “Synthesis, antimicrobial and anti-inflammatory activities of novel 5-(1-adamantyl)-1,3,4-thiadiazole derivatives,” European Journal of Medicinal Chemistry, vol. 45, no. 11, pp. 5006–5011, 2010. View at Publisher · View at Google Scholar · View at Scopus
  3. S. Schenone, C. Brullo, O. Bruno et al., “New 1,3,4-thiadiazole derivatives endowed with analgesic and anti-inflammatory activities,” Bioorganic & Medicinal Chemistry, vol. 14, no. 6, pp. 1698–1705, 2006. View at Publisher · View at Google Scholar · View at Scopus
  4. A. Gupta, P. Mishra, S. K. Kashaw, V. Jatav, and J. P. Stables, “Synthesis and anticonvulsant activity of some novel 3-aryl amino/amino-4-aryl-5-imino-Δ2-1,2,4-thiadiazoline,” European Journal of Medicinal Chemistry, vol. 43, no. 4, pp. 749–754, 2008. View at Publisher · View at Google Scholar · View at Scopus
  5. U. F. Mansoor, A. R. Angeles, C. Dai et al., “Discovery of novel spiro 1,3,4-thiadiazolines as potent, orally bioavailable and brain penetrant KSP inhibitors,” Bioorganic & Medicinal Chemistry, vol. 23, no. 10, pp. 2424–2434, 2015. View at Publisher · View at Google Scholar · View at Scopus
  6. C. De Monte, S. Carradori, D. Secci et al., “Synthesis and pharmacological screening of a large library of 1,3,4-thiadiazolines as innovative therapeutic tools for the treatment of prostate cancer and melanoma,” European Journal of Medicinal Chemistry, vol. 105, pp. 245–262, 2015. View at Publisher · View at Google Scholar · View at Scopus
  7. B. Insuasty, A. García, J. Quiroga et al., “Efficient microwave-assisted synthesis and antitumor activity of novel 4,4-methylenebis[2-(3-aryl-4,5-dihydro-1H-pyrazol-5-yl)phenols],” European Journal of Medicinal Chemistry, vol. 46, no. 6, pp. 2436–2440, 2011. View at Publisher · View at Google Scholar · View at Scopus
  8. B. Insuasty, A. Tigreros, F. Orozco et al., “Synthesis of novel pyrazolic analogues of chalcones and their 3-aryl-4-(3-aryl-4,5-dihydro-1H-pyrazol-5-yl)-1-phenyl-1H-pyrazole derivatives as potential antitumor agents,” Bioorganic & Medicinal Chemistry, vol. 18, no. 14, pp. 4965–4974, 2010. View at Publisher · View at Google Scholar · View at Scopus
  9. M.-Y. Zhao, Y. Yin, X.-W. Yu et al., “Synthesis, biological evaluation and 3D-QSAR study of novel 4,5-dihydro-1H-pyrazole thiazole derivatives as BRAFV600E inhibitors,” Bioorganic & Medicinal Chemistry, vol. 23, no. 1, pp. 46–54, 2015. View at Publisher · View at Google Scholar · View at Scopus
  10. F. F. Barsoum and A. S. Girgis, “Facile synthesis of bis(4,5-dihydro-1H-pyrazole-1-carboxamides) and their thio-analogues of potential PGE2 inhibitory properties,” European Journal of Medicinal Chemistry, vol. 44, no. 5, pp. 2172–2177, 2009. View at Publisher · View at Google Scholar · View at Scopus
  11. Z. Özdemir, H. B. Kandilci, B. Gümüşel, Ü. Çaliş, and A. A. Bilgin, “Synthesis and studies on antidepressant and anticonvulsant activities of some 3-(2-furyl)-pyrazoline derivatives,” European Journal of Medicinal Chemistry, vol. 42, no. 3, pp. 373–379, 2007. View at Publisher · View at Google Scholar · View at Scopus
  12. D. Secci, S. Carradori, A. Bolasco, B. Bizzarri, M. D'Ascenzio, and E. Maccioni, “Discovery and optimization of pyrazoline derivatives as promising monoamine oxidase inhibitors,” Current Topics in Medicinal Chemistry, vol. 12, no. 20, pp. 2240–2257, 2012. View at Publisher · View at Google Scholar · View at Scopus
  13. A. Özdemir, G. Turan-Zitouni, Z. Asim Kaplancikli, G. Revial, and K. Güven, “Synthesis and antimicrobial activity of 1-(4-aryl-2-thiazolyl)-3-(2-thienyl)-5-aryl-2-pyrazoline derivatives,” European Journal of Medicinal Chemistry, vol. 42, no. 3, pp. 403–409, 2007. View at Publisher · View at Google Scholar · View at Scopus
  14. J. Ramírez–Prada, S. M. Robledo, I. D. Vélez et al., “Synthesis of novel quinoline–based 4,5–dihydro–1H–pyrazoles as potential anticancer, antifungal, antibacterial and antiprotozoal agents,” European Journal of Medicinal Chemistry, vol. 131, pp. 237–254, 2017. View at Publisher · View at Google Scholar · View at Scopus
  15. L. C. López Cara, M. E. Camacho, M. D. Carrión et al., “Phenylpyrrole derivatives as neural and inducible nitric oxide synthase (nNOS and iNOS) inhibitors,” European Journal of Medicinal Chemistry, vol. 44, no. 6, pp. 2655–2666, 2009. View at Publisher · View at Google Scholar · View at Scopus
  16. M. D. Carrión, M. Chayah, A. Entrena et al., “Synthesis and biological evaluation of 4,5-dihydro-1H-pyrazole derivatives as potential nNOS/iNOS selective inhibitors. Part 2: Influence of diverse substituents in both the phenyl moiety and the acyl group,” Bioorganic & Medicinal Chemistry, vol. 21, no. 14, pp. 4132–4142, 2013. View at Publisher · View at Google Scholar · View at Scopus
  17. M. D. Carrión, L. C. López Cara, M. E. Camacho et al., “Pyrazoles and pyrazolines as neural and inducible nitric oxide synthase (nNOS and iNOS) potential inhibitors (III),” European Journal of Medicinal Chemistry, vol. 43, no. 11, pp. 2579–2591, 2008. View at Publisher · View at Google Scholar · View at Scopus
  18. T. Castaño, A. Encinas, C. Pérez, A. Castro, N. E. Campillo, and C. Gil, “Design, synthesis, and evaluation of potential inhibitors of nitric oxide synthase,” Bioorganic & Medicinal Chemistry, vol. 16, no. 11, pp. 6193–6206, 2008. View at Publisher · View at Google Scholar · View at Scopus
  19. K. Harish, P. G. Satya, A. S. Anees, and K. Vijay, “Advances in design and development of inhibitors of nitric oxide synthases,” Current Enzyme Inhibition, vol. 9, no. 2, pp. 117–141, 2013. View at Publisher · View at Google Scholar · View at Scopus
  20. H. Yamasaki and M. F. Cohen, “Biological consilience of hydrogen sulfide and nitric oxide in plants: Gases of primordial earth linking plant, microbial and animal physiologies,” Nitric Oxide: Biology and Chemistry, vol. 55-56, pp. 91–100, 2016. View at Publisher · View at Google Scholar · View at Scopus
  21. E. Karpuzoglu and S. A. Ahmed, “Estrogen regulation of nitric oxide and inducible nitric oxide synthase (iNOS) in immune cells: Implications for immunity, autoimmune diseases, and apoptosis,” Nitric Oxide: Biology and Chemistry, vol. 15, no. 3, pp. 177–186, 2006. View at Publisher · View at Google Scholar · View at Scopus
  22. J. D. Allen, T. Giordano, and C. G. Kevil, “Nitrite and nitric oxide metabolism in peripheral artery disease,” Nitric Oxide: Biology and Chemistry, vol. 26, no. 4, pp. 217–222, 2012. View at Publisher · View at Google Scholar · View at Scopus
  23. S. Singh and A. K. Gupta, “Nitric oxide: role in tumour biology and iNOS/NO-based anticancer therapies,” Cancer Chemotherapy and Pharmacology, vol. 67, no. 6, pp. 1211–1224, 2011. View at Publisher · View at Google Scholar · View at Scopus
  24. C. Napoli and L. J. Ignarro, “Nitric oxide and pathogenic mechanisms involved in the development of vascular diseases,” Archives of Pharmacal Research, vol. 32, no. 8, pp. 1103–1108, 2009. View at Publisher · View at Google Scholar · View at Scopus
  25. S. Ashina, L. Bendtsen, and M. Ashina, “Pathophysiology of tension-type headache,” Current Pain and Headache Reports, vol. 9, no. 6, pp. 415–422, 2005. View at Publisher · View at Google Scholar · View at Scopus
  26. C. O. Bingham III, “The pathogenesis of rheumatoid arthritis: Pivotal cytokines involved in bone degradation and imflammation,” The Journal of Rheumatology, vol. 29, no. 65, pp. 3–9, 2002. View at Google Scholar · View at Scopus
  27. S. Singh and M. Dikshit, “Apoptotic neuronal death in Parkinson's disease: involvement of nitric oxide,” Brain Research Reviews, vol. 54, no. 2, pp. 233–250, 2007. View at Publisher · View at Google Scholar · View at Scopus
  28. T. Malinski, “Nitric oxide and nitroxidative stress in Alzheimer's disease,” Journal of Alzheimer's Disease, vol. 11, no. 2, pp. 207–218, 2007. View at Publisher · View at Google Scholar · View at Scopus
  29. P. Aguilera, M. E. Chánez-Cárdenas, E. Floriano-Sánchez et al., “Time-related changes in constitutive and inducible nitric oxide synthases in the rat striatum in a model of Huntington's disease,” NeuroToxicology, vol. 28, no. 6, pp. 1200–1207, 2007. View at Publisher · View at Google Scholar · View at Scopus
  30. C. Napoli and L. J. Ignarro, “Nitric oxide and atherosclerosis,” Nitric Oxide: Biology and Chemistry, vol. 5, no. 2, pp. 88–97, 2001. View at Publisher · View at Google Scholar · View at Scopus
  31. L. C. López-Cara, M. D. Carrión, A. Entrena et al., “1,3,4-Thiadiazole derivatives as selective inhibitors of iNOS versus nNOS: Synthesis and structure-activity dependence,” European Journal of Medicinal Chemistry, vol. 50, pp. 129–139, 2012. View at Publisher · View at Google Scholar · View at Scopus
  32. M. E. Camacho, J. León, A. Entrena et al., “4,5-Dihydro-1H-pyrazole derivatives with inhibitory nNOS activity in rat brain: Synthesis and structure - Activity relationships,” Journal of Medicinal Chemistry, vol. 47, no. 23, pp. 5641–5650, 2004. View at Publisher · View at Google Scholar · View at Scopus
  33. M. Chayah, M. D. Carriõn, M. A. Gallo, R. Jiménez, J. Duarte, and M. E. Camacho, “Development of urea and thiourea kynurenamine derivatives: Synthesis, molecular modeling, and biological evaluation as nitric oxide synthase inhibitors,” ChemMedChem, vol. 10, no. 5, pp. 874–882, 2015. View at Publisher · View at Google Scholar · View at Scopus
  34. M. Chayah, M. E. Camacho, M. D. Carrión, M. A. Gallo, M. Romero, and J. Duarte, “N, N -Disubstituted thiourea and urea derivatives: Design, synthesis, docking studies and biological evaluation against nitric oxide synthase,” MedChemComm, vol. 7, no. 4, pp. 667–678, 2016. View at Publisher · View at Google Scholar · View at Scopus
  35. C. Maccallini, M. Montagnani, R. Paciotti et al., “Selective Acetamidine-Based Nitric Oxide Synthase Inhibitors: Synthesis, Docking, and Biological Studies,” ACS Medicinal Chemistry Letters, vol. 6, no. 6, pp. 635–640, 2015. View at Publisher · View at Google Scholar · View at Scopus
  36. P. Mukherjee, M. A. Cinelli, S. Kang, and R. B. Silverman, “Development of nitric oxide synthase inhibitors for neurodegeneration and neuropathic pain,” Chemical Society Reviews, vol. 43, no. 19, pp. 6814–6838, 2014. View at Publisher · View at Google Scholar · View at Scopus
  37. J. S. Fortin, J. Lacroix, M. Desjardins, A. Patenaude, É. Petitclerc, and R. C.-Gaudreault, “Alkylation potency and protein specificity of aromatic urea derivatives and bioisosteres as potential irreversible antagonists of the colchicine-binding site,” Bioorganic & Medicinal Chemistry, vol. 15, no. 13, pp. 4456–4469, 2007. View at Publisher · View at Google Scholar · View at Scopus
  38. C. Janiak, S. Deblon, and H.-P. Wu, “Syntheses of 5,5-disubstituted 2,2'-bipyridines,” Synthetic Communications, vol. 29, no. 19, pp. 3341–3352, 1999. View at Publisher · View at Google Scholar · View at Scopus
  39. D. S. Bredt and S. H. Snyder, “Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme,” Proceedings of the National Acadamy of Sciences of the United States of America, vol. 87, no. 2, pp. 682–685, 1990. View at Publisher · View at Google Scholar · View at Scopus
  40. R. Jiménez, M. Sánchez, M. J. Zarzuelo et al., “Endothelium-dependent vasodilator effects of peroxisome proliferator-activated receptor β agonists via the phosphatidyl-inositol-3 kinase-Akt pathway,” The Journal of Pharmacology and Experimental Therapeutics, vol. 332, no. 2, pp. 554–561, 2010. View at Publisher · View at Google Scholar · View at Scopus
  41. Z. Ungvari, D. Sun, A. Huang, G. Kaley, and A. Koller, “Role of endothelial [Ca2+]i in activation of eNOS in pressurized arterioles by agonists and wall shear stress,” American Journal of Physiology-Heart and Circulatory Physiology, vol. 281, no. 2, pp. H606–H612, 2001. View at Publisher · View at Google Scholar · View at Scopus
  42. S. Pfeiffer, E. Leopold, K. Schmidt, F. Brunner, and B. Mayer, “Inhibition of nitric oxide synthesis by NG-nitro-L-arginine methyl ester (L-NAME): requirement for bioactivation to the free acid, NG-nitro-L-arginine,” British Journal of Pharmacology, vol. 118, no. 6, pp. 1433–1440, 1996. View at Publisher · View at Google Scholar