Syntheses, biological evaluation of some novel substituted benzoic acid derivatives bearing hydrazone as linker

Article abstract of DOI:10.1007/s11164-021-04555-y

On the basis of rational drug design fourteen novel compounds having benzoic acid as acidic head,?hydrazone?as?linker?and?substituted?diaryl?sulfanyl/aryl-cyclohexylsulfanyl?as?a?hydrophobic?tail?were synthesized?and?characterized by physicochemical and spectrophotometric (FTIR, Mass, ¹HNMR and ¹³CNMR) analysis. The spectral data were satisfactory with their structures. The?designed compounds were docked against peroxisome proliferated activated receptors (PPARγ) and further evaluated for in vitro PPARγ agonist activity and in vivo hypoglycemic activity in wistar strain of albino rats.?Compound 3k and 3m exhibited potent?anti-diabetic activity without ulcerogenic toxicity and minimum side effects as weight gain. Therefore these compounds would be considered as promising agents for the development of novel antidiabetic agents.

Full text of DOI:10.1007/s11164-021-04555-y

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-021-04555-y
Syntheses, biological evaluation of some novel substituted
benzoic acid derivatives bearing hydrazone as linker
Ganesh Prasad Mishra1,2 · Rajesh Sharma1 · Mukul Jain3 ·
Debdutta Bandyopadhyay3
Received: 18 May 2021 / Accepted: 22 July 2021
© The Author(s), under exclusive licence to Springer Nature B.V. 2021
Abstract
On the basis of rational drug design fourteen novel compounds having benzoic acid
as acidic head, hydrazone as linker and substituted diaryl sulfanyl/aryl-cyclohex-
ylsulfanyl as a hydrophobic tail were synthesized and characterized by physico-
1
chemical and spectrophotometric (FTIR, Mass, HNMR and 13CNMR) analysis.
The spectral data were satisfactory with their structures. The designed compounds
were docked against peroxisome proliferated activated receptors (PPARγ) and fur-
ther evaluated for in vitro PPARγ agonist activity and in vivo hypoglycemic activ-
ity in wistar strain of albino rats. Compound 3k and 3m exhibited potent anti-dia-
betic activity without ulcerogenic toxicity and minimum side efects as weight gain.
Therefore these compounds would be considered as promising agents for the devel-
opment of novel antidiabetic agents.
Keywords PPARγ agonists · Benzoic acid derivatives · Hydrazone · Thioether ·
Anti-diabetic · Ulcerogenic toxicities
Introduction
PPAR(peroxisome proliferator activated receptor) γ is a member of the peroxi-
some proliferator activated receptor family and has been the subject of exten-
sive research for mechanistic importance in glucose and lipid homeostasis [1].
The receptor is widely distributed in the spleen, colon, adipose tissue and
macrophages, and found to a lesser extent in the liver, pancreas and skeletal
*
Ganesh Prasad Mishra
gm25mishra@gmail.com
1
2
3
School of Pharmacy, Devi Ahilya Vishwavidyalaya, Takshshila Campus, Khandwa Road ,
Indore, M.P. 452001, India
Kharvel Subharti College of Pharmacy, Swami Vivekanand Subharti University, Subhartipuram,
NH-58, Delhi-Haridwar Bypass Road, Meerut, U.P. 250002, India
Zydus Research Centre, Sarkhej-Bavla N.H 8A, Moraiya, Ahmedabad 382210, India
Vol.:(0123456789)
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G. P. Mishra et al.
muscle [2]. PPARγ agonist actions are known to improve hyperglycemia and
hyperlipidemia, and to reduce cardiovascular risk factors such as atherosclero-
sis, arterial hypertension and infammatory mediators. Hyperlipidemia is usually
associated with hyperglycemia and frequently leads to the development of type-2
diabetes mellitus (T2DM) and cardiovascular disease [3], thus posing a substan-
tial worldwide economic burden [4].
PPARγ agonists, such as thiazolidinediones (TZDs) have proven to be ef-
cacious as insulin sensitizing agents in the treatment of persistent hyperglyce-
mia [5–9]. Due to unwanted adverse efects of long term administered TZDs,
such as weight gain and fuid retention, search of new PPARγ agonists without
adverse efects is essential in the development of new antidiabetic agents.
Literature was reported that benzoic acid derivatives stimulated expression
and exhibited PPARγ agonistic activity [10–12]. Benzoic acid possesses a sim-
ple skeleton that belong to open chain acidic group in place of close chain acidic
TZD and that binds comfortably in the active site of PPARγ [13, 14]. These fnd-
ings prompted us to search novel benzoic acid derivatives containing diferent
hydrophobic tail part as PPARγ agonists.
Design
Compounds having trifunctional unit (acidic head part, hydrophobic tail part
and a connecting part as linker) have been shown good PPARγ agonistic activ-
ity [15]. Keeping these structural features in mind novel benzoic acid deriva-
tives were designed. The diferent hydrophobic tail parts are conveniently
selected according to physicochemical properties such as hydrophobicity and
electronic distribution. The hydrophobic tail part which difers in the com-
pounds is of major interest. In designed compound, the benzoic acid as acidic
head part is linked with substituted aryl/cyclohexylsulfanyl via hydrazone moi-
ety as linker (Fig. 1). Earlier hydrazone was efectively evaluated as anti-dia-
betic agents [16, 17].
HOOC
HC=NHNOC
R2
Linker
Acidic head group
Hydrophobic tail group
R1
R1 = R2 = Substituted aryl/cyclohexylsulfanyl
Fig. 1 Design of benzoic acid derivatives
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Syntheses, biological evaluation of some novel substituted…
Experimental section
Molecular docking
The Program Molegro Virtual Docker (MVD 2012. 5.5, Molegro Bioinformatics, Aar-
chus C, Denmark) was employed to generate grid, calculate dock score and evaluate
conformers [18]. The compounds were docked using PPARγ protein coordinates. Pro-
tein coordinates were downloaded from the Protein Data Bank, accession code 2PRG
[19]. Chain A was prepared for docking within the MVD by removing chain B and all
water molecules. Maximum number of cavities was fxed to 5 for detection of possible
binding cavities, grid resolution was 0.60 Å with center at coordinates x=(−17.45),
y=(−16.26) and z=(18.04) Å, and the binding site radius was set to 16 Å, while
other parameters was set as default. All compounds were stored in a MVD fle and
an original conformation was generated for each compound. Compounds were docked
into the protein coordinates and the highest scoring pose was selected for each of the
compounds. The best docking poses are predicted to be the most stable conformation
of each compound for binding to the PPARγ receptor. The validation of the dock-
ing process was performed and determines whether the molecular docking algo-
rithm is able to recover the crystallographic.
position with root mean square distance value less than 2.0 Å [see Supplementary
File 1].
Synthetic materials and methods
All synthetic starting material, reagents, and solvents were procured from Sigma
Aldrich and Merck and used without further purifcation. Thin-layer chromatography
(TLC) and column chromatography was used to reach the completion of the reaction
and purity of the compounds synthesized respectively. Melting points were recorded
using an open capillary tube electrothermal melting point apparatus and are uncor-
rected. IR spectra were obtained in KBr discs on a Shimadzu 8400S Fourier- transform
1
infrared (FTIR) spectrophotometer. H NMR spectra were recorded on a FT NMR
(400 MHz) and 13C NMR were recorded at 100 MHz on a BRUKER AVANCE II 400
NMR spectrometer; DMSO-d6 was used as a solvent. Chemical shifts are reported as δ
(ppm). The Mass spectra were recorded on a Waters Q-TOF Micro mass spectrometer.
Elemental analysis was performed using EURO Vector EA 3000 analyzer. The spot on
sample loaded TLC (E-Merck pre-coated plates) plates were identifed by exposing to
UV light and iodine vapour. The CsF-Celite reagent was prepared by stirring an aque-
ous solution of CsF with celite 521 at room temperature for 20 min [20].
Preparation of intermediates
Procedure for preparation of ester
A mixture of substituted benzoic acid (1.0 mmol), methanol (10 mmol) and few
drops of Conc. sulfuric acid as catalyst was refuxed at 70 °C for 4–5 h. and reaction
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G. P. Mishra et al.
was monitored by TLC. After completion of reaction, the reaction mixture was
cooled and fltered. The solid residue (IM1) was washed with distilled water and
dried.
Procedure for preparation of thioether
A mixture of aryl/acylthiol (100 mmol) and CsF-Celite as a solid base (150 mmol)
in 20 ml of acetonitrile containing substituted ester (200 mmol) was refuxed at
80 °C for 03–18 h and the progress of the reaction was monitored by TLC. After
completion of reaction, the reaction mixture was cooled at room temperature and
fltered. The CsF-Celite was separated as residue and thioether as fltrate. The fltrate
was evaporated to get desired product (IM2).
Procedure for preparation of hydrazide
Hydrazide intermediates were obtained from reaction of the substituted thioether
(10 mmol) with excess of hydrazine 90% (10 mmol) under refux at 110 °C for
45 min. The solid was obtained by cooling the reaction vessels, fltered of and
washed with distilled water and dried to get desired products (IM3).
General method for the preparation of compounds (3a–3n)
The compounds were synthesized by reacting equimolar proportion of substituted
aryl hydrazides (IM3; 10 mmol) and p-formyl benzoic acid (10 mmol) in absolute
ethanol (25 mL) under refux at 78 °C for 40 min. The precipitate was fltered of
and washed with distilled water and fnally recrystallized with dimethylformamide
to obtain 3a–3n.
(1) 4‑{[2‑(4‑Chloro‑phenylsulfanyl)‑benzoyl]‑hydrazonomethyl}‑benzoic acid (3a)
White solid; Yield: 83%; mp: 219–221 °C; IR (KBr, υ cm−1): 3348(NH), 3059(CH),
2988(OH), 1749(C=O), 1680 (C=O), 1530(C=C), 1058(C–Cl), 680(C–S). 1HNMR
(DMSO-d6): δ 12.13(s, 1H, COOH), 11.56(s, 1H, NH), 10.10 (s, 1H, –N=CH),
7.95–7.92(d, J=7.37 Hz, 2H), 7.73–7.70 (d, J=6.70 Hz, 2H), 7.59–7.54 (m, 6H),
7.39–7.35. (d, J=8.62 Hz, 2H). 13C NMR (DMSO-d6): δ 192.35, 171.53, 164.98,
145.32, 138.15, 137.46, 135.33, 129.92, 125.70, 125.19, 125.30, 125.21, 125.19,
125.09, 124.90, 124.84, 124.53, 124.31, 124.17, 124.11, 124.05. MS: m/z. 412.70
(M+ +2). Anal. Calcd for C21H15ClN2O3S: C, 61.40; H, 3.69; N, 6.83; S, 7.80.
Found: C, 61.37; H, 3.53; N, 6.70; S, 7.47.
(2) 4‑{[2‑(4‑Fluoro‑phenylsulfanyl)‑benzoyl]‑hydrazonomethyl}‑benzoic acid (3b)
White solid; Yield: 93%; mp: 229–231 °C; IR(KBr, υ cm−1): 3391(NH),
3077(CH), 3019(OH), 1747(C=O), 1698(C=O), 1527(C=C), 1157(C–F),
683(C–S). 1HNMR (DMSO-d6): δ 12.14(s, 1H, COOH), 11.65(s, 1H, NH), 10.13
1 3

Syntheses, biological evaluation of some novel substituted…
(s, 1H, –N=CH), 7.89–7.86 (d, J = 8.93 Hz, 2H), 7.74–7.71(d, J = 7.05 Hz, 2H),
7.59–7.50 (m, 6H), 7.46–7.42(d, J = 7.23 Hz, 2H). 13C NMR (DMSO-d6): δ
192.96, 171.34, 166.53, 145.76, 140.34, 138.62, 135.77, 129.88, 125.70, 125.10,
124.47, 124.29, 124.23, 124.21, 124.17, 124.11, 124.07, 124.01, 123.83, 123.76,
123.65. MS: m/z 395.43 (M+). Anal. Calcd for C21H15FN2O3S: C, 63.94; H, 3.84;
N, 7.11; S, 8.13. Found: C, 63.85; H, 3.64; N, 7.02; S, 8.08.
(3) 4‑{[2‑(4‑Bromo‑phenylsulfanyl)‑benzoyl]‑hydrazonomethyl}‑benzoic acid (3c)
White solid; Yield: 81%; mp: 230–232 °C; IR(KBr, υ cm−1): 3388(NH),
3113(CH), 3013(OH),1758(C=O), 1683 (C=O), 1514(C=C), 1041(C–Br),
1
669(C–S), H NMR (DMSO-d6): δ 12.13(s, 1H, COOH), 11.64(s, 1H, NH),
10.09(s,1H, –N=CH), 7.84–7.81(d, J = 7.19 Hz, 2H), 7.73–7.70(d, J = 7.81 Hz,
2H), 7.61–7.55(m, 6H), 7.47–7.43 (d, J = 8.01 Hz, 2H). 13C NMR (DMSO-d6): δ
192.21, 171.11, 165.82, 145.47, 139.46, 135.71, 131.19, 126.47, 125.21, 125.13,
125.01, 124.93, 124.61, 124.18, 124.11, 124.03, 124.95, 124.61, 124.33, 124.13,
124.11.MS:m/z 456.63 (M+ + 2). Anal. Calcd for C21H15BrN2O3S: C, 55.39; H,
3.32; N, 6.15; S, 7.08. Found: C, 55.35; H, 3.04; N, 6.12; S, 7.03.
(4) 4‑[(2‑p‑Tolylsulfanyl‑benzoyl)‑hydrazonomethyl]‑benzoic acid (3d)
Of-white solid; Yield: 78%; mp: 224–226 °C; IR(KBr,υcm−1):3331(NH),312
1
1(CH), 3008(OH),1751(C=O), 1689 (C=O), 1521(C=C), 691(C–S). H NMR
(DMSO-d6): δ 2.08(s, 1H, COOH), 11.56(s, 1H, NH), 10.11(s, 1H, –N=CH),
7.88–7.85(d, J = 7.03 Hz, 2H), 7.65–7.63(d, J = 7.12 Hz, 2H), 7.59–7.53(m, 6H),
7.39–7.35 (d, J = 7.33 Hz,2H), 2.22 (s, 3H).13C NMR (DMSO-d6): δ 191.02,
171.51, 165.21, 145.74, 138.52, 135.43, 129.94, 125.71, 125.58, 125.53, 125.33,
125.21,125.10, 125.03, 124.93, 124.77, 124.71, 124.11,123.33, 123.09, 122.70,
21.42. MS: m/z 391.10 (M+ + 1). Anal. Calcd for C22H18N2O3S: C, 67.69; H,
4.65; N, 7.17; S, 8.22. Found: C, 67.35; H, 4.64; N, 7.12; S, 8.18.
(5) 4‑{[2‑(4‑Methoxy‑phenylsulfanyl)‑benzoyl]‑hydrazonomethyl}‑benzoic acid (3e)
Of white solid; Yield: 89%; mp: 261–263 °C; IR (KBr, υ cm−1); 3359(NH),
3100(CH), 2991(OH), 2831(OCH3), 1733(C=O), 1687(C=O), 1530(C=C),
684(C–S).1H NMR (DMSO-d6): δ 12.10(s, 1H, COOH), 11.63(s, 1H, NH), 10.11(s,
1H, –N=CH), 7.84–7.82(d, J=7.83 Hz, 2H), 7.69–7.67(d, J=6.71 Hz, 2H),
7.60–7.50(m, 4H),7.41–7.37(d, J=5.22 Hz, 2H), 3.43(s, 3H). 13C NMR (DMSO-
d6): δ 191.91, 170.40, 167.15, 157.46, 144.97, 138.29, 135.74, 129.52, 125.89,
125.31, 125.28, 125.19, 125.17, 125.14, 125.07, 124.92, 124.71, 124.57, 124.38,
124.17, 124.07, 55.36.MS: m/z 407.08 (M+ +1). Anal. calcd for C22H18N2O4S; C,
65.05; H, 4.48; N, 6.89; S,7.89. Found: C, 64.95; H, 4.34; N, 6.42; S, 7.68.
1 3

G. P. Mishra et al.
(6) 4‑{[2‑(2,4‑Dimethyl‑phenylsulfanyl)‑benzoyl]‑hydrazonomethyl}‑benzoic acid
(3f)
White solid;Yield: 81%; mp: 205–207 °C; IR (KBr, υ cm−1); 3369(NH), 3122(CH),
3011(OH), 1724(C=O), 1681(C=O), 1516(C=C), 659(C–S).1H NMR (DMSO-
d6): δ 12.09(s, 1H, COOH), 11.61(s, 1H, NH), 10.12(s, 1H, -N=CH), 7.85–7.83(d,
J=6.22 Hz, 2H), 7.77–7.73(d, J=5.19 Hz, 2H), 7.61–7.56(m, 5H), 7.42–7.39(d,
J=7.57 Hz, 2H), 2.46(s, 6H).13C NMR (DMSO-d6): δ 191.29, 171.23, 165.41,
145.33, 137.43, 135.46, 130.45, 126.91, 125.62, 125.33, 125.30, 125.21, 125.11,
125.01, 124.73, 124.33, 124.24, 124.17, 124.09, 123.82, 123.71, 22.37, 21.13.
MS: m/z 405.13 (M+ +1). Anal. calcd for C23H20N2O3S: C,68.32; H,4.95; N, 6.93;
S,7.94. Found: C, 68.03; H, 4.64; N, 6.62; S, 7.78.
(7) 4‑[(2‑Cyclohexylsulfanyl‑benzoyl)‑hydrazonomethyl]‑benzoic acid (3 g)
White solid; Yield: 67%; mp: 209–211 °C; IR (KBr, υ cm−1): 3311(NH), 3113(CH),
1
2941(OH), 1733(C=O), 1684(C=O), 1525(C=C), 621(C–S). H NMR (DMSO-
d6): δ 11.98 (s, 1H,COOH), 11.41(s, 1H, NH), 10.04(s, 1H, -N=CH), 7.84–7.82(d,
J=7.20 Hz, 2H), 7.66–7.64(d, J=5.28 Hz), 7.36–7.33(d, J=5.43 Hz, 2H),
7.11–7.06(m, 2H), 3.33(m, 1H), 1.63–1.39(m, 10 H) 13C NMR (DMSO-d6): δ
190.08, 170.03, 164.93, 144.42, 137.12, 135.37, 129.41, 125.61, 125.05, 124.39,
124.33, 124.23, 124.13, 124.09, 123.08, 43.15, 39.43, 39.42, 26.45, 25.19, 25.18.
MS: m/z 383.33(M+ +1). Anal. Calcd for C21H22N2O3S: C, 65.94; H, 5.81; N, 7.33;
S, 8.38. Found: C, 65.83; H, 5.64; N, 7.12; S, 8.08.
(8) 4‑{[4‑(4‑Chloro‑phenylsulfanyl)‑benzoyl]‑hydrazonomethyl}‑benzoic acid (3 h)
White solid; Yield: 84%; mp: 278–280 °C; IR (KBr, υ cm−1): 3359(NH), 3101(CH),
1
2989(OH) 1733(C=O), 1697(C=O), 1518(C=C), 1069(C–Cl), 683(C–S). H NMR
(DMSO-d6): δ 12.13(s, 1H, COOH), 11.57(s, 1H, NH), 10.08(s, 1H, –N=CH),
7.93–7.91(d, J=7.02 Hz, 2H), 7.72–7.70(d, J=8.73 Hz, 2H), 7.58–7.54(m, 6H),
7.37–7.33(d, J=6.88 Hz,2H).13CNMR(DMSO-d6): δ 192.32, 171.46, 165.93,
145.72, 139.46, 138.40, 135.33, 129.92, 126.71, 125.60, 125.39, 125.33, 125.21,
125.11, 125.09, 124.48, 124.27, 124.11, 124.03, 123.81, 123.76. MS: m/z
412.94(M+ +2). Anal. Calcd for C21H15ClN2O3S: C, 61.40; H, 3.69; N, 6.83; S,
7.80. Found: C, 61.31; H, 3.64; N, 6.60; S, 7.71.
(9) 4‑{[4‑(4‑Fluoro‑phenylsulfanyl)‑benzoyl]‑hydrazonomethyl}‑benzoic acid (3i)
White solid; Yield: 93%; mp: 270–272 °C; IR(KBr, υ cm−1): 3402(NH),
3049(CH),3023(OH),1741(C=O),
1701(C=O),
1529(C=C),
1148(C–F),
689(C–S).1H NMR (DMSO-d6): δ 12.14 (s, 1H COOH), 11.63. (s, 1H, NH),
10.13(s, 1H, –N=CH), 7.88–7.85(d, J=7.17 Hz, 2H), 7.73–7.71(d, J=7.06 Hz,
2H),7.59–7.54(m, 6H), 7.43–7.39(d, J=8.07 Hz, 2H). 13CNMR (DMSO- d6): δ
192.90, 171.74, 166.51, 145.73, 140.12, 138.40, 135.77, 130.31, 126.70, 125.58,
125.41, 125.30, 125.23, 125.11, 124.59, 124.44, 124.31, 124.25, 124.23, 124.19,
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Syntheses, biological evaluation of some novel substituted…
124.10. MS: m/z 395.14 (M+ +1). Anal. Calcd for C21H15FN2O3S: C, 63.94; H,
3.84; N, 6.83; S, 8.13. Found: C, 63.83; H, 3.74; N, 6.70; S, 8.01.
(10) 4‑{[4‑(4‑Bromo‑phenylsulfanyl)‑benzoyl]‑hydrazonomethyl}‑benzoic acid (3j)
White solid; Yield: 82%; mp: 279–281 °C; IR (KBr, υ cm−1): 3349 (NH), 3101(CH),
2999 (OH), 1719(C=O), 1682(C=O), 1522(C=C), 1051(C–Br), 661(C–S),.1H
NMR (DMSO-d6): δ 12.12(s,1H COOH), 11.62(s, 1H, NH), 10.09(s, 1H, –N=CH),
7.85–7.83(d, J=7.20 Hz, 2H),7.73–7.70(d, J=8.03 Hz, 2H), 7.60–7.54(m,
6H),7.47–7.42(d, J=6.91 Hz,2H).13CNMR(DMSO-d6): δ 192.21, 170.11, 165.41,
145.07, 138.40, 136.72, 135.12, 129.91, 125.91, 125.59, 125.41, 125.30, 125.26,
125.18, 125.15, 124.80, 124.61, 124.55, 124.30, 124.09, 124.01.MS: m/z 456.61
(M+ +2). Anal. calcd for C21H15BrN2O3S: C, 55.39, H, 3.32, N, 6.15, S, 7.08.
Found: C, 55.27, H, 3.14, N, 6.12, S, 7.06.
(11) 4‑[(4‑p‑Tolylsulfanyl‑benzoyl)‑hydrazonomethyl]‑benzoic acid (3 k)
Of-white solid; Yield: 76%; mp: 256–258 °C; IR(KBr, υ cm−1): 3341(NH),
3103(CH), 2943(OH), 1721. (C=O), 1681(C=O), 1527(C=C), 663(C–S). 1H NMR
(DMSO-d6): δ 12.10(s,1H,COOH), 11.58(s, 1H, NH), 10.12(s, 1H, –N=CH),
7.88–7.85(d, J=6.20 Hz, 2H),7.67–7.65(d, J=5.01 Hz, 2H), 7.61–7.56(m, 6H),
7.37–7.34(d, J=4.97 Hz, 2H), 2.23(s, 3H). 13C NMR (DMSO-d6): δ 192.19,
171.10, 166.41, 145.77, 138.40, 135.30, 129.94, 126.71, 125.69, 125.41, 125.33,
125.23, 125.11, 125.05, 124.93, 124.84, 124.71, 124.51, 124.33, 123.19, 123.10,
20.42. MS: m/z 391.02 (M+ +1). Anal. calcd for C22H18N2O3S: C, 67.68; H, 4.65;
N,7.18; S, 8.21. Found: C, 67.51; H, 4.64; N, 7.02; S, 8.05.
(12) 4‑[4‑(4‑Methoxy‑phenylsulfanyl)‑benzylidene‑hydrazinocarbonyl]‑benzoic acid
(3l)
Of white solid; Yield: 88%; mp: 285–287 °C; IR (KBr,
υ
cm−1);
3359(NH), 3107(CH), 2979(OH), 2833(OCH3),1733(C=O), 1687(C=O),
1533(C=C),684(C–S).1HNMR(DMSO-d6): δ 12.10(s, 1H, COOH), 11.61 (s,
1H, NH), 10.15(s, 1H, –N=CH), 7.86–7.82(d, J=7.10 Hz, 2H), 7.70–7.67(d,
J=7.03 Hz, 2H), 7.60–7.57(m, 6H), 7.41–7.38(d, J=6.73 Hz, 2H), 3.44(s, 3H).
13C NMR (DMSO-d6): δ 191.66, 170.40, 166.44, 157.34, 144.77, 138.44, 130.44,
125.61, 125.30, 125.22, 125.20, 125.18, 125.11, 125.05, 124.93, 124.80, 124.71,
124.51, 124.23, 124.09, 123.95, 55.48. MS: m/z 407.21 (M+ +1). Anal.calcd for
C22H18N2O4S: C, 65.05: H, 4.48; N, 6.89; S, 7.90. Found: C, 65.00; H, 4.34; N,
6.42; S, 7.61.
(13) 4‑{[4‑(2,4‑Dimethyl‑phenylsulfanyl)‑benzoyl]‑hydrazonomethyl}‑benzoic acid
(3m)
White solid; Yield: 79%; mp: 259–261 °C; IR (KBr, υ cm−1); 3311(NH), 3094(CH),
2981(OH), 1715(C=O). 1680(C=O), 1531(C=C), 653(C–S).1H NMR (DMSO-d6):
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G. P. Mishra et al.
δ 12.09(s, 1H, COOH), 11.61(s, 1H, NH), 10.11(s, 1H, -N=CH), 7.84–7.82(d,
J=6.77 Hz, 2H),7.79–7.77(d, J=7.03 Hz, 2H), 7.61–7.56(m, 5H),7.51–7.47(d,
J=5.54 Hz, 2H), 2.43(s, 6H). 13C NMR (DMSO-d6): δ 191.11, 171.16, 165.33,
145.75, 138.40, 130.35, 125.94, 125.71, 125.49, 125.33, 125.21, 125.20, 125.11,
125.05, 124.93, 124.74, 124.61, 124.41, 124.23, 124.09, 123.90, 21.62, 20.50.
MS: m/z 405.31(M+ +1). Anal. calcd for C23H20N2O3S: C,68.30; H, 4.95; N,6.92;
S,7.94. Found: C, 68.17; H, 4.64; N, 6.62; S, 7.78.
(14) 4‑[(4‑Cyclohexylsulfanyl‑benzoyl)‑hydrazonomethyl]‑benzoic acid (3n)
White solid; Yield: 63%; mp:252–254 °C; IR (KBr,υ cm−1):3313(NH),3104(CH),
2953(OH), 1742(C=O),1681 (C=O), 1517(C=C), 648(C–S).1H NMR (DMSO-
d6): δ 12.01(s, 1H COOH), 11.44(s, 1H, NH), 10.02(s, 1H, –N=CH), 7.86–7.83(d,
J=4.26 Hz, 2H), 7.68–7.65(d, J=5.73 Hz, 2H), 7.39–7.35(d, J=5.88 Hz, 2H),
7.15–7.09(m, 2H), 3.41(m, 1H), 1.76–1.43(m, 10H).13C NMR (DMSO-d6): δ
190.10, 170.06, 163.53, 145.72, 138.45, 135.33, 129.52, 125.71, 124.28, 124.21,
124.15, 124.01, 123.12, 123.01, 122.77, 44.14, 39.39, 39.40, 26.43, 25.20, 25.21.
MS: m/z 383.43 (M+ +1). Anal. Calcd for C21H22N2O3S: C, 65.90; H, 5.80; N,
7.31; S, 8.40. Found: C, 65.67; H, 5.64; N, 7.23; S, 8.11.
Biological studies
Wistar strain of albino rats(normal and hyperglycemic; 150–190 g) were maintained
under standard conditions, i.e., room temperature (25 2 °C) and photoperiod of
12 h day/night cycles each day for 1 week before and during the experiment. The
rats were allowed free access to tap water and pellet diet. Rats were divided into
following fve groups for evaluation of intraday, interday hypoglycemic activity, bio-
chemical parameters and body weight measurement:
Group
Description
Group I
Normal control rats treated with vehicles (10% Tween 80 suspension)
Group II Hyperglycemic control rats treated with vehicles (10% Tween 80 suspension)
Group III Normal control rats treated with compound 3m (3m in 10% Tween 80 suspension at a dose of
28 mg/kg)
Group IV Hyperglycemic rats treated with rosiglitazone (Rosiglitazone in 10% Tween 80 suspension at
a dose of 10 mg/kg)
Group V Hyperglycemic rats treated with synthesized compounds (Synthesized compounds in 10%
Tween 80 suspension at a dose of 28 mg/kg)
Intraday blood glucose level was measured at 0th, 1st, 3rd and 6th hrs dura-
tion after the administration of vehicle, standard drug (rosiglitazone) and test com-
pounds (3a–3n). For evaluation of the efect of compound 3k and 3m on interday
study of blood glucose, biochemical parameters (TC, TG, HDL, LDL, AP), the same
rats were continued with the same dose of test compounds, standard drug and vehicle
once daily for 3 sweek (21 days). Standard kit (Beacon Diagnostics, India) were used
1 3

Syntheses, biological evaluation of some novel substituted…
to determined TC, TG, HDL, LDL and AP after period of 21 days. All the procedures
were performed in accordance with guidelines of institutional animal ethics commit-
tee, CPCSEA, Government of India. All values were expressed as mean S.E.M. Sta-
tistical signifcance was estimated by analysis of variance (ANOVA).
Induction of hyperglycemia
Hyperglycemia was induced in overnight fasted rats by a single intraperitoneal injec-
tion of 65 mg/kg streptozotocin in citrate bufer (pH 4.5), 15 min after the intraperito-
neal administration of 110 mg/kg.
of nicotinamide. Blood glucose level was checked after 72 h by one touch Accu-
check glucometer. Rats with blood glucose levels greater than 250 mg/dl were consid-
ered hyperglycemic and were used for the study [21].
Intraday evaluation of hypoglycemic activity of benzoic acid derivatives in normal
and STZ induce rats
After overnight fasted rats, intraday blood glucose level was measured at 0th, 1st, 3rd
and 6th hrs duration after the administration of vehicle, standard drug (rosiglitazone)
and test compounds (3a–3n) through gastric intubation using a force feeding needle.
Blood samples were collected from the tail vein and blood glucose level was deter-
mined with one touch Accucheck glucometer.
Interday evaluation of hypoglycemic activity of benzoic acid derivatives in normal
and STZ induce rats
The vehicle, standard drug and test compounds (3k–3m) were administered into the
rats of the respective groups every day morning for 21 days by gastric intubation
using force feeding needle. Blood samples were collected for the measurement of blood
glucose from the tail vein at 1st day, 7th,14th and 21st day duration after the adminis-
tration of vehicle, standard drug and test compounds (3k and 3m) and blood glucose
levels were determined with basic one touch Accucheck glucometer.
Efects of compound 3k and 3m on biochemical parameters of normal and STZ
induce rats at 21 days
Biochemical parameters such as TC, TG, HDL, LDL and AP were also estimated on
overnight fasted rats at the end of 21st day by using Biochemistry Analyzer Chem-
7(Erba Mannhelm) [22–25].
Efects of compound 3k and 3m on body weight of normal and STZ induce rats
at 21 days
At the beginning (1st day) and at the end of study (21st day), overnight fasted rats
were subjected to weight measurement).
1 3

G. P. Mishra et al.
Gastro‑ulcerogenic toxicity studies
Albino rats of wistar strain (150–200gm) of both sexes were divided into diferent
groups, control, test and standard (containing six animals each).The test group, con-
trol group and standard group were received test drug, vehicle and standard drug
respectively. The test compounds and standard compound were suspended in 10%
tween-20 and administered orally to each animal by using gastric gavage needle.
The control group animals, however received the same volume of vehicle. In this
study,the animals were administered a 28 mg/kg (body weight) dose of the test drugs
and 10 mg/kg (body weight) dose of standard drug (indomethacin) [26].
Results and Discussion
Molecular docking
Figure 2 illustrates the best docking pose of the most active compound 3m with
amino acid residues of PPARγ. The compound 3m showed hydrogen bond interac-
tion with Tyr473, Ser289, His323, and Cys285. This hydrogen bonding pattern is
conserved in most PPARγ agonist complex structures and essential for the activ-
ity of the compound [14, 27]. The crystal form of 2PRG [19] consists 2 molecules
in the asymmetric unit, denoted A and B. Chain A was chosen for the PPARγ ago-
nist docking study. The redocking results with chain A gave most reliable docked
conformation for the synthesized compound- protein interactions. This is rea-
son behind selection of chain A over chain B. The crystal structure of the 2PRG
complex was re-docked for validation.
Chemistry of synthesized compounds
A simple and convenient synthetic route has been developed for synthesis of
designed compounds [28–31]. In the frst step, the starting material was esteri-
fed in strongly acidic conditions and obtained intermediates (IMI) were sub-
jected to thioetherifcation (IM2). Thioetherifcation follows an addition–elimi-
nation two-step reaction; in the frst step, aryl/cyclohexylsulfanyl as nucleophile
attacks on aryl ring bearing chloride leaving group. In the second step, elimination
of the chloride leads to generation of thioethers. Here, the utility of cesium fuoride-
celite (CsF-Celite) was to activation of the aryl halide groups by the lewis acid type
efect. In next step IM2 were subjected to ammonolysis to get hydrazides (IM3)
which on further reaction with absolute ethyl alcohol containing 4-formylbenzoic
acid yielded desired compounds 3a–3n. This reaction condition is essential for
nucleophilic addition of the amino group to the carbonyl function of the aldehyde.
The synthesized compounds (3a–3n) were confrmed by FTIR, 1H NMR, 13C NMR,
Mass and Elemental analysis. The FT-IR spectrum of intermediates clearly indi-
cated the desired substitution in the intermediates. The absence of OH group, SH
1 3

Syntheses, biological evaluation of some novel substituted…
Fig. 2 The most active compound 3m (ball and stick tube) is interacted with Phe226, Glu291, Glu295,
Met329, Ser 342 and Glu343 of amino acid residues in the active binding sites of the receptor. H-bond
interactions with key amino acids Tyr473, Ser289, His323 (not appeared) and Cys285 are shown in
magenta dashed lines
group absorption and sharp decrease in C=O stretching of hydrazide in the FT-IR
spectra of the IM1, IM2 and IM3 respectively confrmed the reactions of esterifca-
tion, thioetherifcation and ammonolysis (Scheme 1).
The FT-IR spectra of synthesized compounds 3a–3n showed absorp-
tion bands around 3023–2943 cm−1 for OH (COOH), 3402–3311 cm−1 for
NH hydrazone, while the distinguished absorption peaks C=O for acid was
observed in the range 1758–1715 cm−1, C=O for hydrazone was observed in the
range 1701–1680 cm−1and C–S for the thioether was observed in the range of
691–621 cm−1.1H NMR spectrum revealed the lack of –SH signal and the presence
of characteristic singlets around δ 12.14–11.98 for carboxylic acid protons, around
δ 11.65–11.41 for the NH protons of hydrazone while condensation of p-formyl
benzoic acid with various substituted aryl hydrazides was confrmed by downfeld
region of δ 10.15–10.02 observed as singlet for=CH group at phenyl ring. In addi-
1
tion, the H NMR spectra of compounds 3a–3n showed aromatic group protons as
three doublets around δ 7.93–7.82, 7.77–7.63 and 7.51–7.33. On the other hand, the
spectrum of 3d, 3f, 3k and 3m exhibited singlets for CH3 groups at δ 2.23, 2.43,
1
2.22 and 2.46 respectively. The H NMR spectroscopic data of compound 3e and
3 l showed the presence of singlet at δ 3.44 and 3.43 respectively for three protons
in the methoxy group. The compound 3g and 3n showed the presence of multiplets
around δ 7.15–1.39 for aromatic and aliphatic protons. Moreover, compounds 3a–3f
and 3h–3 m showed the presence of a multiplets around δ 7.61–7.50 for aromatic
protons. 13C NMR spectra, which were in conformity with the assigned structures,
displayed most characteristic signals appearing at around δ 192.96–190.10 ppm
for carbonyl carbon peak, δ 171.74–170.03 ppm for carboxylic acid carbon peak, δ
1 3

G. P. Mishra et al.
COOH
COOH
COOCH3
CONHNH2
R1
COOCH3
CH
R1
a
R1
R1
d
b
c
CONHN
R1
R2
R2
R1=R2= Cl
Ortho or para-
chlorobenzoic
acid ester (IM1)
R2
R2
(IM2)
R1=R2= Cl
Ortho or para-
chlorobenzoic
acid
(IM3)
R2
(3a - 3n)
(1) IM2, IM3 and 3a : R1= 4-Cl-C6H4S- , R2 = H
(2) IM2, IM3 and 3b : R1= 4-F-C6H4S- , R2 = H
(3) IM2, IM3 and 3c : R1= 4-Br-C6H4S- , R2 = H
(8) IM2, IM3 and 3h : R1 = H, R2 = 4-Cl-C6H4S-
(9) IM2, IM3 and 3i : R1 = H, R2 = 4-F-C6H4S-
(10) IM2, IM3 and 3j : R1 = H, R2 = 4-Br-C6H4S-
(4) IM2, IM3 and 3d : R = 4-CH -C6H4S- , R = H
(11) IM2, IM3 and 3k : R1 = H, R2 = 4-CH -C6H4S-
3
3
1
2
(5) IM2, IM3 and 3e : R = 4-OCH -C6H4S-, R = H
(12) IM2, IM3 and 3l : R1 = H, R2 = 4-OCH -C6H4S-
3
3
1
2
(6) IM2, IM3 and 3f : R = 2,4-(CH ) -C6H3S-, R = H (13) IM2, IM3 and 3m : R1 = H, R2 = 2,4-(CH ) -C6H3S-
3
3
1
2
2
2
3g : R =
, R = H
3n : R = H, R2
=
;
C6H11S-
(7) IM2, IM3 and
C6H11S-
(14) IM2, IM3 and
1
2
1
Scheme 1 Reaction pathways, Reagents and Conditions: a Esterifcation; absolute Methanol, conc.
H2SO4, Refux, 4–5 h; b Thioetherifcation; substituted aryl/cyclohexylthiols, CsF-Celite, CH3CN,
Refux, 3–18 h; c Ammonolysis; NH2NH2(90%), Refux, 45 min.; d Condensation; 4-Formylbenzoic
acid, absolute ethanol, Refux, 40 min.
167.12–163.53 ppm for C=N peak, aromatic carbons around δ 145.77–122.70 ppm
and aliphatic carbon around δ 78.67–25.18 ppm. Two carbons of the aromatic ring
(3e and 3l) displayed chemical shifts at δ 157.34 and 157.46 due to carbon attached
with methoxy group. Mass spectra of all the newly synthesized compounds exhib-
ited a prominent molecular ion peak and isotopic peaks with diferent intensities. All
the newly synthesized compounds were also characterized by elemental chemi-
cal analysis and gave satisfactory experimental values and acceptable error range
( 0.4%) compared to calculated values.
PPARγ in vitro activity and structure–activity relationships
The synthesized compounds were screened on full length PPAR receptor trans-
fected in HepG2 cells (Table 1) following the procedure described in our earlier
1 3

Syntheses, biological evaluation of some novel substituted…
publication [32]. Two compounds 3 k (80.21%) and 3 m (94.60%) showed signif-
cant PPARγ transactivation activity as fold activation and rest of the compounds
were exhibited moderate to weak activity in the range of 67.26–17.26% (Table 1).
The efect of substituents at R1 and R2 position of the compound on hPPARγ
transactivation activity was determined. Dimethyl substitution (3m) provides
more hPPARγ transactivation activity as compare to monomethyl substitu-
tion (3k) at para position of the phenyl ring(Hydrophobic tail part). Potency
of the compounds also indicates that para phenylsufanyl substituents are more
efective than ortho phenylsufanyl substituents. The potency for PPARγ agonis-
tic activity was signifcantly increased by the introduction of electron donating
groups at the phenyl ring of the tail part of the compounds. The compounds (3g,
3n) having unsubstituted cyclohexyl moiety in place of substituted phenyl ring
were least active. These fndings may be due to, the bulky moieties containing
tail part comprise at least one substituted phenyl group capable of vander waals
interaction to the receptor surface and one substituted phenylsulfanyl moiety for
hydrophobic bonding interactions. Moreover, it has been hypothesized that two
phenyl groups as bulky moieties exhibited more potency than one phenyl plus a
cyclohexyl ring. This would imply that two fat- surfaced benzene rings are bet-
ter than one and their combined vander walls forces and hydrophobic interactions
important for increased biological potency.
Table 1 In vitro PPARγ
Compound
In vitro activation(Mean SEM)*
agonistic activity as hPPARγ
transactivation activities#
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
0.81 0.51 (29.13%)
0.99 0.22 (35.61%)
1.45 0.21 (52.15%)
1.54 0.40 (55.39%)
1.00 0.42 (35.95%)
1.87 0.42 (67.26%)
0.48 0.41 (17.26%)
0.90 0.51 (32.37%)
0.76 0.35 (27.33%)
1.23 0.18 (44.24%)
2.23 0.38 (80.21%)
1.13 0.18 (40.64%)
2.63 0.50 (94.60%)
0.62 0.41 (22.30%)
2.78 0.20 (100.00%)
Rosiglitazone
*A mean from three determinations at concentration 0.02 µM.
#Activities are presented as fold induction of hPPARγ activation.
EC50 of the compounds were not determined. Fold activation relative
to maximum activation obtained with rosiglitazone (100%)
1 3

G. P. Mishra et al.
In vivo activity
In vivo studies were carried in wistar strain of albino rats. Streptozotocin (STZ)
was used for induction of hyperglycemia in rats. STZ is well known for its selec-
tive pancreatic β-cell cytotoxicity and has been extensively used to induce dia-
betes mellitus in animals and allows a consistent production of hyperglycemia
as well as hyperlipidemia as diabetic like symptoms within a short period of
time [33–35].
The intraday fasting blood glucose level at 6th hour for group I, II, III and IV
were found 92.66, 303.50, 79.83 and 110.00 mg/dl respectively. While the syn-
thesized benzoic acid derivatives (3a–3n) has exhibited intraday fasting blood
glucose level at 6th hour in the range of 94.65 mg/dl to 137.50 mg/dl (Table 2).
Thus, all the synthesized compounds showed signifcant reduction in fasting
blood glucose level in comparison to diabetic control (group II).
The interday fasting blood glucose level at 21st day of group I, II, III and
IV were found 86.66, 337.65, 78.16 and 104.16 mg/dl respectively. The inter-
day fasting blood glucose level at 21st day of 3k and 3m were found 84.33 and
106.33 mg/dl respectively. The fasting blood glucose levels of interday study
showed consistent blood glucose reduction at 1st, 7th, 14th, 21st day (Fig. 3).
Treatment with benzoic acid derivatives in normal rats for 21 days did not pro-
duce hypoglycemic conditions. In the diabetic untreated rats (group II) the glu-
cose levels remained higher without much change in the whole experimental
period.
After 21 days treatment with compound 3k and 3m biochemical parameters
such as serum total cholesterol (TC), triglycerides (TG), high density lipoprotein
(HDL), low density lipoprotein (LDL) and alkaline phosphatase (AP) were also
determined. It was found that the levels of HDL were signifcantly increased and
TC, TG and LDL levels were signifcantly decreased compared to diabetic untreated
rats (Fig. 4). It is interesting to observe in the present study that treatment with ben-
zoic acid derivatives have not only lowered the TG, TC and LDL levels but also
enhanced the cardio protective lipid HDL after 21st day treatment. AP which is a
hepatic toxicity marker was found higher in diabetic rats treated with standard drug
and untreated diabetic control group compared to diabetic rats treated with com-
pound 3k and 3m. Thus, benzoic acid derivatives might have protective efect
against liver toxicity caused by STZ.
Further, at the end of 21st day treatment, the body weights of the overnight fasted
experimental rats were also determined. It was found that, compound treated rats
showed less weight gain compare to rosiglitazone treated rats, whereas the body
weights of untreated rats (control diabetic) decreased signifcantly. Untreated rats
have shown marked reduction in their body weights compared to normal rats, which
could be due to their uncontrolled diabetes (Fig. 5).
At the end of study (21st day), compound 3k and 3m treated rat were sacrifced
and the stomachs and intestines were removed. A longitudinal incision along the
lesser curvature is made. The stomach and intestine of animals was rinsed in run-
ning water and the presence and absence of ulcers were determined in control group,
test group and standard group and data reported in Table 3.
1 3

Syntheses, biological evaluation of some novel substituted…
Table 2 Efects of test compounds on intraday fasting blood glucose levels (mg/dl) of normal and STZ
induce rats
Groups/compounds
0 h
89.50 3.76
1 h
89.16 3.49
3 h
90.66 3.43
6 h
92.66 3.15
Group I
Group II
Group III
309.30 16.65
81.66 2.41
307.00 15.10
76.66 2.96
305.50 16.84
80.40 3.76
303.50 17.75
79.83 3.68
Group IV
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
307.16 16.52
293.17 13.28b
311.52 12.60c
287.66 12.43b
311.66 18.15c
303.34 14.05c
295.56 20.55b
296.00 15.28b
312.00 12.80c
286.66 11.60b
296.83 9.33b
307.06 9.96c
309.83 8.03c
298.83 12.63b
288.83 19.96b
252.16 4.82
189.00 17.76a
164.50 20.31a
205.65 14.56a
199.83 16.40a
163.00 6.44a
193.16 7.82a
175.85 18.23a
248.50 13.05a
205.67 22.82a
194.43 7.71a
206.16 7.03a
220.03 25.15a
182.66 7.00a
180.66 10.96a
130.66 6.19
121.83 0.83a
114.33 4.07a
120.83 0.30a
118.50 0.93a
132.34 2.06a
117.16 0.98a
130.16 3.62a
133.60 3.7a
149.66 5.81a
102.66 3.79a
102.00 5.93a
149.66 5.81a
112.33 3.20 a
150.50 15.96 a
110.00 1.82
119.86 1.02a
112.66 2.37a
120.33 0.33a
116.16 0.47a
119.46 2.65a
115.83 1.06a
118.16 2.27a
117.03 6.30a
137.50 4.93a
94.65 5.46a
96.83 4.62a
127.50 4.23a
109.33 3.01a
132.13 2.16 a
b
aP<0.001 indicates statistically more signifcant when compared with Group II. P<0.05 indicates sta-
c
tistically signifcant when compared with Group II. P>0.05 not signifcant compared with Group II.
Each value in table is represented as (mean SD)
400
Group I
Group II
300
Group III
Group IV
200
3k
3m
100
0
1
7
4
1
2
1
Days
Fig. 3 Efect of the most active compounds on interday fasting blood glucose levels (mg/dl) of normal
and STZ induce rats
1 3

G. P. Mishra et al.
250
200
150
100
50
Group I
Group II
Group III
Group IV
3k
3m
0
P
L
L
C
G
A
D
T
D
T
L
H
Biochemical Parameters
Fig. 4 Efect of the most active compounds on biochemical parameters of normal and STZ induce rats
after 21 days of treatmentChange in body weight of normal and STZ induce rats at day 1 and day 21
Conclusion
Here, we described the design, syntheses and evaluation of novel benzoic acid
derivatives as PPARγ agonists. In this investigation, the phenyl variants were
utilized aiming to enhance the hydrophobicity and the efects of substituents on
the phenyl ring at tail group examined successfully. Among these compounds,
compound 3k and 3m have displayed marked in vitro PPARγ agonist and in vivo
hypoglycemic activity in comparison to rosiglitazone. Further the values of
change in body weight by compound 3k and 3m in comparison to rosiglitazone
250
Group I
Group II
Group III
Group IV
3k
200
150
100
50
3m
0
1
1
2
y
a
y
D
a
D
Observations
Fig. 5 Change in body weight of normal and STZ induce rats at day 1 and day 21
1 3

Syntheses, biological evaluation of some novel substituted…
Table 3 Gastro-ulcerogenic
Compounds
Ulcer score
Ulceration (%)
toxicity of the most active
compounds in rats
Vehicle
3k
3m
0/6
0/6
0/6
6/6
0.0
0.0
0.0
100
Indomethacin
Bold values provides us information whether synthesized com-
pounds are causing gastric ulcer or not
exhibited that the compounds might be devoid of weight gain, the common
adverse efect of TZDs.
Supplementary Information The online version contains supplementary material available at https://doi.
org/10.1007/s11164-021-04555-y.
Acknowledgements The authors are grateful to management of Zydus Research Centre for skilled and
needful support and Mr. Manoj Rathore, School of Pharmacy for animal handling and Dept of Life sci-
ences, Devi Ahilya Vishwavidyalaya for necessary support.
Declarations
Conficts of interest Authors declares no confict of interest.
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