Synthesis and biological evaluation of 5-benzylidenerhodanine-3-acetic acid derivatives as AChE and 15-LOX inhibitors

Article abstract of DOI:10.3109/14756366.2014.940935

A series of 5-benzylidenerhodanine-3-acetamides bearing morpholino-, 4-arylpiperazinyl-, or 4-benzylpiperidinyl- moieties were synthesized and their inhibitory activities against acetylcholinesterase (AChE) were evaluated. Alteration of amide part and substitution on the benzylidene moiety resulted in change of anti-AChE activity. The most active compound was the 1-benzylpiperidinyl derivative containing 4-(dimethylamino)benzylidene scaffold. Notably, the intermediate compounds, namely 5-arylidene-rhodanine-3-acetic acids (3), showed mild inhibitory activity against 15-lipoxygenase (15-LOX), while the final compound 4 showed no activity against 15-LOX.

Full text of DOI:10.3109/14756366.2014.940935

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–7
2015 Informa UK Ltd. DOI: 10.3109/14756366.2014.940935
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RESEARCH ARTICLE
Synthesis and biological evaluation of 5-benzylidenerhodanine-3-acetic
acid derivatives as AChE and 15-LOX inhibitors
Negah Shafii1, Mehdi Khoobi2, Mohsen Amini2, Amirhossein Sakhteman3, Hamid Nadri3, Alireza Moradi3,
Saeed Emami4, Ebrahim Saeedian Moghadam2, Alireza Foroumadi2, and Abbas Shafiee1,2
2
1Tehran University of Medical Sciences, International Campus (TUMS-IC), Tehran, Iran, Department of Medicinal Chemistry, Faculty of Pharmacy
and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran, 3Department of Medicinal Chemistry, Faculty of
4
Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran, and Department of Medicinal Chemistry and Pharmaceutical Sciences
Research Center, Faculty of Pharmacy, Mazanderan University of Medical Sciences, Sari, Iran
Abstract
Keywords
A series of 5-benzylidenerhodanine-3-acetamides bearing morpholino-, 4-arylpiperazinyl-,
or 4-benzylpiperidinyl- moieties were synthesized and their inhibitory activities against
acetylcholinesterase (AChE) were evaluated. Alteration of amide part and substitution on the
benzylidene moiety resulted in change of anti-AChE activity. The most active compound was
the 1-benzylpiperidinyl derivative containing 4-(dimethylamino)benzylidene scaffold. Notably,
the intermediate compounds, namely 5-arylidene-rhodanine-3-acetic acids (3), showed mild
inhibitory activity against 15-lipoxygenase (15-LOX), while the final compound 4 showed no
activity against 15-LOX.
2-Thioxothiazolidin-4-one, 15-lipoxygenase,
acetylcholinesterase, docking study,
inhibitors, rhodanines
History
Received 14 May 2014
Revised 30 June 2014
Accepted 30 June 2014
Published online 22 June 2015
Introduction
inhibitors potentially useful in the management of Alzheimer’s
disease and related dementias12. Also, amide derivatives of
5-arylidene-rhodanine-3-acetic acid have been reported as anti-
Rhodanines (2-thioxothiazolidin-4-ones) and their analogues
have been known as privileged structures in drug discovery.
Compounds containing the rhodanine ring have demonstrated
a broad range of biological activities1. Particularly, 5-arylidene
rhodanines have been reported as small molecule inhibitors
of diverse enzyme targets such as b-lactamase2, hepatitis C
virus (HCV) NS3 protease3, aldose reductase4, UDP-N-
acetylmuramate/L-alanine ligase5, protein mannosyl transferase,
JNK-stimulating phosphatase, cyclooxygenase, 5-lipoxygenase6
and tyrosinase7.
inflammatory agents13,14
.
In view of these facts, although extensive studies on the diverse
bioactivity of 5-arylidene-rhodanines have been reported, the anti-
acetylcholinesterase (AChE) activities of these prototypes
have never appeared in the literature. Accordingly as part of our
ongoing studies in developing new anti-AChE agents15–18
,
we have synthesized 5-benzylidenerhodanine-3-acetamides and
evaluated their inhibitory activity against AChE. Also, the
intermediate compounds 5-arylidene-rhodanine-3-acetic acids
were screened for 15-lipoxygenase (15-LOX) inhibitory activity.
Historically, rhodanines have become a therapeutic class of
compounds since the introduction of epalrestat (Z,E-[5-(2-methyl-
3-phenyl-allylidene)-4-oxo-2-thioxo-thiazolidin-3-yl]-acetic acid)
into clinical use for the treatment of diabetic complications8.
Experimental
Epalrestat is a rhodanine-3-acetic acid analogue and acts as an Carbonyldiimidazole (CDI), dicyclohexylcarbodiimide (DCC),
aldose reductase inhibitor9. Although, the rhodanine-based com- 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), hydroxyl
pounds are suspected to undergo conjugate addition in vivo, they benzotriazole (HOBt) and other commercially available reagents
are frequently employed in drug design, as there is no adverse were used without further purification. TLC was conducted on
effect (e.g. mutagenicity) correlated to this scaffold10. According silica gel 250 micron, F254 plates. Melting points were measured
to a long-term clinical study with epalrestat, the structure can be on a Kofler hot stage apparatus and are uncorrected. The IR
bioavailable and well-tolerated11. Bulic et al. have been described spectra were taken using Nicolet FT-IR Magna 550 spectrograph
a series of 5-arylidene-rhodanine-3-acetic acids as tau aggregation (KBr disks). 1H NMR spectra were recorded on a NMR
instrument Bruker 500 MHz. The chemical shifts (d) and coupling
constants (J) are expressed in parts per million and Hertz,
respectively. Mass spectra of the products were obtained with an
HP (Agilent Technologies, Santa Clara, CA) 5937 Mass Selective
Address for correspondence: Abbas Shafiee, Department of Medicinal
Detector. Elemental analyses were carried out by a CHN-Rapid
Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research
Heraeus elemental analyzer. The results of elemental analyses
(C, H, N) were within 0.4% of the calculated values.
Center, Tehran University of Medical Sciences, Tehran 14176, Iran. Tel:
+98 21 66406757. Fax: +98 21 66461178. E-mail: ashafiee@ams.ac.ir

2
N. Shafii et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
General procedure for the synthesis of compounds 3a-l
353 (20), 325 (40), 265 (11), 224 (100), 209 (89), 181 (19.7), 166
(12.6), 86 (21.2), 70 (14.1), 56 (13.3). Anal. (C19H22N2O6S2): C,
H, N, Calcd 52.04, 5.06, 6.39. Found. 51.86, 5.25, 6.14.
Compounds 3a–l were prepared according to the literature6.
General procedure for the synthesis of compounds 4a–n
(Z)-3-(2-Oxo-2-(4-phenylpiperazine-1-yl)ethyl)-2-thioxo-5-
(3,4,5-trimethoxybenzylidene) thiazolidin-4-one (4e)
A solution of compound 3 (1.0 mmol), EDC (1.1 mmol) and
HOBt (1.0 mmol) in acetonitrile (10 ml) was stirred for 1 h at
room temperature. Then, the corresponding amine (1.0 mmol)
was added and stirred for 24 h at ambient temperature. After
completion of the reaction (monitored by TLC), the solvent was
evaporated and the residue was crystallized from chloroform-
petroleum ether to give compound 4.
Yield 67%, yellow solid, m.p. 192–194 ꢀC, IR (cmꢁ1, KBr): ꢀmax
2932 and 2833 (C–H aliphatic), 1651 and 1599 (C¼O); 1H NMR
(CDCl3, 500 MHz): ꢁ 7.71 (s, 1H, vinylic H), 7.34 (m, 2H,
phenyl), 6.97 (m, 3H, phenyl), 6.75 (s, 2H, H2,6), 5.02 (s,
2H,CH2CO), 3.93 (s, 9H, OMe), 3.81 and 3.26 (m, 8H,
piperazine). EI-MS (m/z, %) 513 (M+, 42.5), 480 (45.6), 375
(18), 352 (15.7), 324 (14), 265 (11.8), 224 (28.3), 209 (41.7), 181
(16.5), 160 (62.7), 132 (100), 120 (15), 104 (35.4), 91 (11.8), 77
(19.7), 56 (56). Anal. (C25H27N3O5S2): C, H, N, Calcd 58.46,
5.30, 8.18. Found. 58.22, 5.51, 8.01.
(Z)-5-(4-Cholorobenzylidene)-3-(2-morpholino-2-
oxoethyl)-2-thioxothiazolidin-4one (4a)
Yield 69%, yellow solid, m.p. 225–227 ꢀC, IR (cmꢁ1, KBr): ꢀmax
3325 (C–H aromatic), 2926 and 2850 (C–H aliphatic), 1716 and
1
1668 (C¼O); H NMR (CDCl3, 500 MHz): ꢁ 7.73 (s, 1H, vinylic
(Z)-3-(2-(4-(2-Fluorophenyl)piperazin-1-yl)-2-oxoethyl)-5-
(4-methoxybenzylidene)-2-thioxothiazolidin-4-one (4f)
H), 7.48 and 7.45 (2 d, 4H, H2,3,5,6, J ¼ 8.7 Hz), 4.96 (s, 2H,
CH2CO), 3.8 and 3.73 (2t, 4H, CH2O, J ¼ 4.45 Hz), 3.64 and 3.59
(2t, 4H, CH2N, J ¼ 4.45 Hz). EI-MS (m/z, %) 382 (M+, 19.6), 349
(26.7), 297 (34.6), 268 (37.7), 224 (13.3), 168 (100), 143 (14),
133 (23.6), 114 (14), 99 (31.4), 86 (98.4), 72 (53.5), 56 (77.9).
Anal. (C16H15ClN2O3S2): C, H, N, Calcd 50.19, 3.95, 7.32.
Found. 50.32, 3.71, 7.12.
Yield 49%, yellow solid, m.p. 259–260 ꢀC; 1H NMR (CDCl3,
500 MHz): ꢁ 7.76 (s, 1H, vinylic H), 7.49 (d, 2H, H2,6
,
J ¼ 8.85 Hz), 7.14–6.90 (m, 6H, fluorophenyl and H3,5 methox-
yphenyl), 5.01 (s, 2H, benzylic H), 3.89 (s, 3H, OMe), 3.82-3.10
(m, 8H, piperazine). EI-MS (m/z, %) 471 (M+, 34.6), 438 (56.7),
315 (28.3), 292 (18.9), 264 (30.7), 220 (12.6), 178 (100), 164
(69.3), 150 (80.3), 138 (15), 122 (48), 109 (11.8), 95 (11.8), 77
(17.3), 56 (67). Anal. (C23H22FN3O3S2): C, H, N, Calcd 58.58,
4.70, 4.03. Found 58.39, 4.93, 4.36.
(Z)-5-(4-(Dimethylamino)benzylidene)-3-(2-morpholino-2-
oxoethyl)-2-thioxothiazolidin-4-one (4b)
Yield 52%, orange solid, m.p. 5300 ꢀC, IR (cmꢁ1, KBr): ꢀmax
3327 (C–H aromatic), 2926 and 2851 (C–H aliphatic), 1707 and
(Z)-3-(2-(4-(3-Fluorophenyl)piperazin-1-yl)-2-oxoethyl)-
2-thioxo-5-(3,4,5-trimethoxybenzylidene)thiazolidin-
4-one (4g)
1
1644 (C¼O); H NMR (CDCl3, 500 MHz): ꢁ 7.72 (s, 1H, vinylic
H), 7.42 (d, 2H, H2,4, J ¼ 8.4 Hz), 6.75 (d, 2H, H3,5, J ¼ 8.4 Hz),
4.95 (s, 2H, CH2CO), 3.80-3.59 (m, 8H, Morpholine), 3.10 (s, 6H,
N(CH3)2). 13C NMR (DMSO-d6, 125 MHz): ꢁ 193.2, 167.1,
163.4, 152.5, 133.7, 132.0, 120.2, 114.0, 112.7, 66.4, 47.9, 45.4,
45.2, 42.4. EI-MS (m/z, %) 391 (M+, 88), 358 (15.7), 278 (13.3),
224 (14), 177 (100), 161 (13.3), 56 (14). Anal. (C18H21N3O3S2):
C, H, N, Calcd 55.22, 5.41, 10.73. Found. 55.48, 5.19, 10.53.
Yield 52%, yellow solid, m.p. 202–204 ꢀC, IR (cmꢁ1, KBr): ꢀmax
3414 (C–H aromatic), 2928 and 2837 (C–H aliphatic), 1718–1648
1
(C¼O); H NMR (CDCl3, 500 MHz): ꢁ 7.70 (s, 1H, vinylic H),
7.32 (d, 2H, H2,6 fluorophenyl, J ¼ 8.95 Hz), 6.99 (d, 1H, H4
fluorophenyl), 6.77 (dd, 1H, H5 fluorophenyl, J ¼ 2.6 Hz), 6.74
(s, 2H, H2,6), 4.99 (s, 2H, CH2CO), 3.93 (s, 9H, OMe), 3.79 and
3.19 (m, 8H, piperazine). EI-MS (m/z, %) 531 (M+, 8.6), 498 (11),
279 (10.2), 241 (16.5), 224 (25.2), 209 (30.7), 167 (26.7), 149
(68.5), 129 (96), 111 (44), 97 (40), 83 (52.7), 71 (58.3), 57 (100).
Anal. (C25H26FN3O5S2): C, H, N, Calcd 56.48, 4.93, 7.90. Found.
56.19, 4.67, 7.70.
(Z)-5-(4-Methoxybenzylidene)-3-(2-morpholino-2-
oxoethyl)-2-thioxothiazolidin-4-one (4c)
Yield 41%, yellow solid, m.p. 201–203 ꢀC, IR (cmꢁ1, KBr): ꢀmax
2927 and 2850 (C–H aliphatic), 1702 and 1667 (C¼O); 1H NMR
(CDCl3, 500 MHz): ꢁ 7.76 (s, 1H, vinylic H), 7.49 (d, 2H, H2,4
,
J ¼ 8.75 Hz), 7.01 (d, 2H, H3,5, J ¼ 8.85 Hz), 4.95 (s, 2H,
CH2CO), 3.89 (s, 3H, OMe), 3.80 and 3.72 (2t, 4H,CH2O,
J ¼ 4.6 Hz), 3.65 and 3.59 (2t, 4H, CH2N, J ¼ 4.6 Hz). 13C NMR
(DMSO-d6, 125 MHz): ꢁ 193.7, 167.1, 163.2, 162.1, 134.2, 133.5,
(Z)-3-(2-(4-(4-Fluorophenyl)piperazin-1-yl)-2-oxoethyl)-
2-thioxo-5-(3,4,5-trimethoxybenzylidene)thiazolidin-4-
one (4h)
125.8, 119.2, 115.6, 66.4, 56.0, 47.9, 45.5, 45.2. EI-MS (m/z, %) Yield 64%, yellow solid, m.p. 200–202 ꢀC, IR (cmꢁ1, KBr): ꢀmax
378 (M+, 78.7), 345 (59), 293 (63), 264 (46.4), 164 (100), 149 3432 (C–H aromatic), 2922 and 2853 (C–H aliphatic), 1706 and
1
(99.2), 121 (45.6), 99 (20.4), 86 (72.4), 77 (27.5), 70 (41.7), 56 1664 (C¼O); H NMR (CDCl3, 500 MHz): ꢁ 7.71 (s, 1H, vinylic
(55). Anal. (C17H18N2O4S2): C, H, N, Calcd 53.95, 4.79, 7.40. H), 7.00 (t, 2H, H2,6 fluorophenyl, J ¼ 8 Hz), 6.92 (m, 2H, H3,5
Found. 53.68, 4.54, 7.73.
fluorophenyl), 6.74 (s, 2H, H2,6), 5.01 (s, 2H, CH2CO), 3.93 (s,
9H, OMe), 3.76 and 3.17 (m, 8H, piperazine). EI-MS (m/z, %) 531
(M+, 48), 498 (56), 375 (16.5), 352 (19.7), 324 (18), 265 (11.8),
224 (35.4), 209 (50.3), 178 (79.5), 150 (100), 138 (15), 122
(36.2), 109 (10.2), 95 (17.3), 56 (50.4). Anal. (C25H26FN3O5S2):
C, H, N, Calcd 56.48, 4.93, 7.90. Found. 56.75, 4.65, 7.61.
(E and Z)-3-(2-Morpholino-2-oxoethyl)-2-thioxo-5-(3,4,5-
trimethoxybenzylidene)thiazolidin-4-one (4d)
Yield 70%, yellow solid, m.p. 231–233 ꢀC, IR (cmꢁ1, KBr): ꢀmax
3000 (C–H aromatic), 2935–2853 (C–H aliphatic), 1720 and 1671
(C¼O); 1H NMR (CDCl3, 500 MHz, E/Z ¼ 27/73): ꢁ 7.86 (E) and
7.72 (Z) (2 s, 1H, vinylic H), 6.76 (E) and 6.74 (Z) (2 s, 2H, H2,6
phenyl), 4.96 (E) and 4.55 (Z) (2 s, 2H, CH2CO), 3.94 (E) and
3.93 (Z) (2 s, 3H, OMe), 3.93 (E) and 3.92 (Z) (2 s, 6H, OMe),
(Z)-3-(2-(4-(3,4-Dichlorophenyl)piperazin-1-yl)-2-
oxoethyl)-5-(2,3-dimethoxybenzylidene)-2-
thioxothiazolidin-4-one (4i)
3.80 (E), 3.73 (Z) (2 m, 4H, Morpholine), 3.66 (E) and 3,59 (Z) Yield 25%, yellow solid, m.p. 144–146 ꢀC, IR (cmꢁ1, KBr): ꢀmax
(2 m, 4H, Morpholine). EI-MS (m/z, %) 438 (M+, 82.7), 405 (22), 3431(C–H aromatic), 2929–2841 (C–H aliphatic), 1710 and 1670

DOI: 10.3109/14756366.2014.940935
5-Benzylidenerhodanine-3-acetic acid derivatives
3
(C¼O); 1H NMR (CDCl3, 500 MHz): ꢁ 8.13 (s, 1H, vinylic 2-((Z)-5-(4-(Dimethylamino)benzylidene)-4-oxo-2-
H),7.34 (d, 1H, H2 phenyl, J ¼ 8.8 Hz), 7.17 (t, 1H, H3 phenyl, thioxothiazolidin-3-yl)-N-(1-benzylpiperidin-4-yl)
J ¼ 8 Hz), 7.08 and 7.04 (m, 3H, dicholorophenyl), 6.83 (m, 1H, acetamide (4m)
H4, J ¼ 2.5 Hz), 5.01 (s, 2H, CH2CO), 3.90 (s, 6H, OMe), 3.82
and 3.12 (d, 8H, piperazine). EI-MS (m/z, %) 551 (M+, 30.7), 518
(52.7), 345 (30), 322 (66), 294 (81.8), 270 (25.2), 228 (100), 213
(17.3), 200 (93.7), 179 (42.5), 161 (21.2), 149 (26), 136 (21.2),
121 (18), 108 (12.6), 91 (11), 72 (26), 56 (86.6). Anal.
(C24H23Cl2N3O4S2): C, H, N, Calcd 52.18, 4.20, 7.61. Found.
52.35, 4.01, 7.34.
Yield 40%, orange solid, m.p. 4250 ꢀC. IR (cmꢁ1, KBr): ꢀmax
3292 (NH), 3082 and 3021 (C–H aromatic), 2930 (C–H aliphatic),
1
1698 and 1655 (C¼O); H NMR (DMSO-d6, 500 MHz): ꢁ 8.20
(br, s, 1H, NH), 7.70 (s, 1H, vinylic H), 6.85–7.50 (m, 9H, Ar-H),
4.60 (s, 2H, COCH2N), 3.35–3.50 (br, s, 3H, CHNH and benzylic
CH2), 3.05 (s, 6H, 2 CH3), 2.50–2.95 (m, 4H, CH2N piperidine),
1.40–2.30 (m, 4H, 2 CH2CH2N piperidine). 13C NMR (DMSO-d6,
125 MHz): ꢁ 193.3, 167.1, 164.1, 152.4, 135.2, 133.6, 129.2,
128.6, 127.4, 120.2, 114.3, 112.7, 62.4, 52.1, 46.7, 46.5, 31.8.
EI-MS (m/z, %) 494 (M+,4.5), 465 (8.3), 439 (25), 421 (5.3), 368
(9), 334 (7.5), 292 (11.3), 248 (12), 215 (13.6), 187 (6.8), 172
(96), 148 (43), 105 (41), 91 (100), 82 (53), 77 (18.9), 65 (13.6), 43
(25.5). Anal. (C26H30N4O2S2): C, H, N, Calcd 63.13, 6.11, 11.33.
Found. 63.51, 6.28, 11.03.
(Z)-3-(2-(4-Benzylpiperidin-1-yl)-2-oxoethyl)-5-
(4-(dimethylamino)benzylidene)-2-thioxothiazolidin-
4-one (4j)
Yield 63%, yellow solid, m.p. 229–230 ꢀC, IR (cmꢁ1, KBr): ꢀmax
2910 and 2853 (C–H aliphatic), 1710 and 1647 (C¼O); 1H NMR
(CDCl3, 500 MHz): ꢁ 7.71 (s, 1H, vinylic H), 7.45 (d, 2H, H2,6
,
J ¼ 8.5 Hz), 7.32 (t, 2H, H3,5 benzyl, J ¼ 8.2 Hz), 7.23 (t, 1H, H4
benzyl, J ¼ 8.2 Hz), 7.17 (d, 2H, H2,6 benzyl, J ¼ 8.2 Hz), 6.89
(d, 2H, H3,5, J ¼ 8.5 Hz), 4.97 and 4.93 (2 d, 2H, CH2CO), 4.54
and 3.85 (2 d, 2H, CH2 benzylic, J ¼ 13.8 Hz), 2.60 (m, 4H,
CH2N), 1.80–1.20 (m, 5H, CH2 and CH). 13C NMR (DMSO-d6,
125 MHz): ꢁ 193.2, 167.1, 162.6, 152.5, 140.4, 135.3, 133.7,
129.4, 128.6, 126.3, 120.2, 114.1, 112.7, 45.6, 44.8, 42.4, 42.3,
37.6, 31.7. EI-MS (m/z, %) 479 (M+, 47.2), 446 (22), 378 (31.4),
345 (22.8), 293 (21.2), 278 (11), 264 (22), 231 (11), 177 (100),
164 (81), 149 (33.8), 121 (14), 91 (19.6), 72 (10.2), 56 (11). Anal.
(C26H29N3O2S2): C, H, N, Calcd 65.11, 6.09, 8.76. Found. 65.34,
6.32, 8.53.
2-((Z)-5-(4-Methylbenzylidene)-4-oxo-2-thioxothiazolidin-
3-yl)-N-(1-benzylpiperidin-4-yl)acetamide (4n)
Yield 38%, yellow solid, m.p. 4250 ꢀC. IR (cmꢁ1, KBr): ꢀmax
3299 (NH), 3082 and 3020 (C–H aromatic), 2924 (C–H aliphatic),
1
1716 and 1663 (C¼O); H NMR (DMSO-d6, 500 MHz): ꢁ 8.05–
8.27 (br, s, 1H, NH), 7.80 (s, 1H, vinylic H), 7.20–7.70 (m, 9H,
Ar-H), 4.62 (s, 2H, COCH2N), 3.30–3.60 (br, s, 3H, CHNH and
benzylic CH2), 2.45–2.95 (m, 4H, 2CH2N piperidine), 2.38 (s, 3H,
CH3), 1.35–2.30 (m, 4H, 2 CH2CH2N piperidine). 13C NMR
(DMSO-d6, 125 MHz): ꢁ 193.9, 167.1, 164.4, 142.1, 134.0, 131.7,
131.2, 130.6, 130.1, 130.0, 129.3, 121.4, 59.5, 51.0, 46.5, 44.7,
29.0, 21.5. EI-MS (m/z, %) 465 (M+, 14.3), 276 (14), 248 (18.9),
172 (100), 148 (35), 132 (11.3), 118 (7.5), 91 (90), 82 (53), 72
(4.5), 56 (2.2), 42 (1.5). Anal. (C25H27N3O2S2): C, H, N, Calcd
64.49, 5.84, 9.02. Found. 64.21, 5.76, 9.28.
(E and Z)-3-(2-(4-Benzylpiperidin-1-yl)-2-oxoethyl)-5-
(3-hydroxy-4-methoxybenzylidene)-2-thioxothiazolidin-
4-one (4k)
Yield 34%, yellow solid, m.p. 278–280 ꢀC, IR (cmꢁ1, KBr): ꢀmax
2923 and 2853 (C–H aliphatic), 1706 and 1646 (C¼O); 1H NMR
(CDCl3, 500 MHz, E/Z ¼ 22/78): ꢁ 7.83 (E) and 7.68 (Z) (2 s, 1H,
vinylic H), 7.37 (E) and 7.32 (Z) (t, 2H, H3,5 benzyl, J ¼ 7.3 Hz),
7.26 (E) and 7.22 (Z) (t, 1H, H4 benzyl, J ¼ 7.3 Hz), 7.17 (E) and
7.08 (Z) (m, 2H, phenyl), 6.95 (E) and 6.58 (Z) (dd, 1H, H6
phenyl, J ¼ 8.2 and 1.9 Hz), 4.95 (E) and 4.53 (Z) (m, 2H,
CH2CO), 4.53 (E) and 4.16 (Z) (m, 2H, CH2N), 3.98 (Z) and 3.97
(E) (2 s, 3H, OMe), 2.60 (E) and 2.43 (Z) (m, 2H, CH2 benzylic),
1.85-1.30 (E and Z) (m, 5H, CH2 and CH Piperidine). EI-MS (m/z,
%) 480 (M+, 7), 466 (26.7), 449 (14.1), 438 (39.3), 413 (15), 384
(45.6), 368 (16.5), 356 (26), 325 (26), 313 (10.2), 298 (15), 280
(15), 265 (19), 251 (14.1), 237 (13.4), 224 (90), 209 (100), 180
(79.5), 165 (48.8), 150 (60.6), 137 (39.3), 122 (33.8), 109 (21.2),
91 (60), 69 (26.7), 56 (43.3). Anal. (C25H26N2O4S2): C, H, N,
Calcd 62.22, 5.43, 5.80. Found. 62.54, 5.27, 5.67.
Molecular modelling studies
All docking studies were performed using Autodock Vina
(ver. 1.1.1, Florida, CA)19. The crystal structures of AChE
complexed with E2020 (code ID: 1EVE) and soybean lypoxygen-
ase complexed with 13(S)-hydroproxy-9(Z)-2,11(E)-octadecadie-
noic acid (code ID: 1IK3) were retrieved from protein data bank.
In both cases, the co-crystallized ligand and water molecules were
removed. Then the Fe in the structure of soybean lipoxygenase
was modified to Fe(III)–OH and both proteins were converted to
pdbqt format using Autodock Tools (1.5.4, Florida, CA)20.
The 2D structures of ligands were sketched using
MarvinSketch 5.8.3, 2012, ChemAxon (http://www.chemaxon.
com) and then converted to 3D and pdbqt format by Openbabel
(ver. 2.3.1, Pittsburgh, PA)21.
The docking parameters were as follows:
LOX: size_x ¼ 20; size_y ¼ 20; size_z ¼ 20; center_x ¼ 19.693;
2-((Z)-5-(4-Chlorobenzylidene)-4-oxo-2-thioxothiazolidin-
3-yl)-N-(1-benzylpiperidin-4-yl)acetamide (4l)
center_y ¼ 0.054;
num_modes ¼ 15.
center_z ¼ 17.628;
exhaustiveness ¼ 100;
AChE: center_x ¼ 2.023; center_y ¼ 63.295; center_z ¼ 67.062;
size_x ¼ 25; size_y ¼ 25; size_z ¼ 25; exhaustiveness ¼ 80;
num_modes ¼ 15. The other parameters were left as default for
both of target enzymes. Finally, the conformations with the most
favorable free energy of binding were selected for analyzing
the interactions between the target enzyme and inhibitor. All the
3D models are generated using the Chimera 1.6 software (San
Francisco, CA)22.
Yield 43%, yellow solid, m.p. 4 250 ꢀC. IR (cmꢁ1, KBr): ꢀmax
3300 (NH), 3088 and 3023 (C–H aromatic), 2930 (C–H aliphatic),
1
1714 and 1656 (C¼O); H NMR (DMSO-d6, 500 MHz): ꢁ 8.50-
8.60 (br, s, 1H, NH), 7.70 (s, 1H, vinylic H), 8.00–8.30 (m, 4H,
Ar-H), 7.40–7.80 (m, 5H, Ar-H of benzyl), 4.80 (s, 2H,
COCH2N), 3.35–3.65 (br, s, 3H, CHNH & benzylic CH2),
2.40–2.85 (m, 4H, 2CH2N piperidine), 1.40–2.20 (m, 4H, 2
CH2CH2N piperidine). EI-MS (m/z, %) 485 (M+, 27), 385 (5),
314 (7.7), 297 (6.9), 268 (7.5), 230 (3.8), 203 (12.3), 172 (93.8),
165 (31.5), 146 (6), 133 (16.9), 125 (23), 99 (3), 91 (98), 82 (100),
AChE inhibition assay
65 (7.5), 42 (10). Anal. (C24H24ClN3O2S2): C, H, N, Calcd 59.31, The inhibitory potency of target compounds on AChE was
4.98, 8.65. Found. 59.65, 4.76, 8.91.
determined using Ellman’s method23. The test compounds were

4
N. Shafii et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
dissolved in DMSO (1 ml) and phosphate buffer (9 ml, pH ¼ 8) inhibitory activity against AChE in concentrations less than
and then diluted using phosphate buffer to achieve five concen- 64 mM.
Among the morpholine compounds 4a–d, 4-(N,N-dimethyla-
trations in the range from 10ꢁ3 to 10ꢁ7 M. The reaction mixture
included 100 ml of DTNB 0.1 M, 50 ml of enzyme (2 U/ml) and mino)-benzylidene derivative 4b exhibited higher activity. As
50 ml of inhibitor solution. The changing of the absorbance was evidenced from IC50 values of 4a, 4c and 4d, the introduction of
measured at 412 nm for 2 min after addition of 10 ml of substrate 4-chloro-, 4-methoxy- and 3,4,5-trimethoxy- substituents dramat-
(acetylthiocholine iodide) 0.15 M to the reaction mixture. ically diminished the activity. In the N-phenylpiperazine ser-
The IC50 values were determined graphically from inhibition ies 4e–i, the unsubstituted analogue 4e showed weak activity
curves (log inhibitor concentration versus percent of inhibition). (IC50 value ¼ 285 mM), while remaining congeners 4f–i showed
All experiments were performed on a UV-2100 Rayleigh Double good activities in the range of 22.4–61.9 mM. The comparison
Beam Spectrophotometer in triplicate.
of IC50 values of unsubstituted compound 4e with 3-fluoro- and
4-fluoro- derivatives (4g and 4h, respectively) revealed that
fluoro- group increased the anti-AChE activity. Moreover, by
comparing compound 4e with 4d or 4f with 4c demonstrated that
N-phenylpiperazine moiety is more favorable than morpholine
attached to the acetamide linker. In the case of compounds
containing 4-(N,N-dimethylamino)- substituent, morpholine
derivative 4b and 4-benzylpiperidine analogue 4j showed nearly
same activity, and 1-benzylpiperidin-4-yl derivative 4m was more
potent than the latter compounds.
15-LOX inhibition assay
LOX inhibitory activity of target compounds was studied by a -
spectrophotometric assay24. The tested compounds were dissolved
in DMSO (1 ml) and phosphate buffer (9 ml, 0.1 M, pH ¼ 8).
This stock solution was added to test solution containing enzyme
(final concentration: 167 U/ml), phosphate buffer (pH ¼ 8) to
achieve concentrations in range of 10ꢁ3 to 10ꢁ6. After incubation
of test solution for 4 min, substrate (linoleic acid, final concentra-
tion: 134 mM) was added and change in the absorbance was
measured for 60 s at 234 nm. The enzyme solution was kept in ice
In vitro anti-15-LOX activity
and controls were measured at intervals throughout the experi- Since the 5-LOX inhibitory activity of 5-benzylidenerhodanine-3-
mental periods to ensure that the enzyme activity was constant. acetic acid has been reported,6 thus we investigated the 15-LOX
All experiments were performed and then analyzed as mentioned inhibitory potential of the intermediate compounds 3a–e against
above for AChE assay.
soybean 15-LOX enzyme (Table 2). For structure–activity
relationships study, the 5-benzylidenerhodanine-3-acetic acid
series was completed by synthesizing compounds 3f–l and
evaluating their 15-LOX inhibitory activity. The results in
comparison with quercetin were summarized in Table 2. All
Results and discussion
Chemistry
In this work, we describe synthesis of a novel series of 2-((Z)- compounds with the exception of compounds 3b and 3g showed
5-benzylidene-4-oxo-2-thioxothiazolidin-3-yl)acetamide
4a–n. mild inhibitory activity against 15-LOX (IC50 values ¼ 147–
As outlined in Scheme 1, initially, an aldol condensation of 421 mM). The 2-hydroxy-4-bromo and 2,4-dihydroxy derivatives
rhodanine-3-acetic acid 1 with different aldehydes 2 in the (compounds 3f and 3h, respectively) with IC50 values ꢂ149 mM,
presence of catalytic amount of S-proline yielded intermediate 3. were the most active compounds. The comparison of
Under these conditions, (Z)-configuration was obtained as major unsubstituted compound 3l with substituted benzylidene
product. In the 1H NMR spectroscopy, the vinylic proton compounds 3c, 3g, and 3i revealed that the introduction of
located on the exocyclic double bond was assigned as a singlet 4-(N,N-dimethylamino), 4-methoxy, 3-bromo-4,5-dimethoxy
ranging from 7.71 to 8.13 ppm. According to the literature reports, and 2-hydroxy substituents decreased the anti-LOX activity.
vinilic proton in the (Z)-isomers appeared at 7.7–8.5 ppm In contrast, the insertion of 3,4,5-trimethoxy, 2,3-dimethoxy,
downfield compared to the (E)-isomers25,26. It would be due to 2-hydroxy-4-methoxy, 2,4-dihydroxy, 2-methoxy, and 2-bromo
the deshielding effect of the carbonyl group. Finally, the target substituents on the benzylidene moiety of compound 3l resulted in
compounds 4a–n were obtained via condensation of the more active compounds. Interestingly, the inhibitory activity of
carboxylic acids
coupling agents including CDI, DCC, EDC and HOBt in different of 4-methoxy derivative 3c. Also, compound 3 h having 2,4-
3
with appropriate amines. Several 3,4,5-trimethoxy analogue (compounds 3d) was superior than that
solvents were investigated, but the best result was obtained by dihydroxy group showed higher inhibition than 2-hydroxy con-
using EDC/HOBt in acetonitrile.
gener 3i. These findings demonstrated that the second hydroxyl
substitution on the 4-position of 2-hydroxybenzylidene compound
3i improved the inhibitory activity against 15-LOX. By compar-
ing the IC50 values of compounds 3j and 3i, it is revealed that the
O-methylation of the 2-OH is favorable. Notably, formation of
amides with various amines that yield final compounds 4a–n, led
to inactive compounds toward lipoxygenase enzyme.
In vitro anti-AChE activity
The potential of the target compounds 4a–n to inhibit AChE
activity was assessed by the Ellman method. The inhibitory
activity (IC50 values, mM) of the test compounds in comparison to
tacrine as reference drug was listed in Table 1. The most active
compound was 1-benzylpiperidinyl derivative 4m with IC50 value
of 21.0 mM. The N-(3-fluorophenyl)piperazinyl derivative 4g with
Docking studies
IC50 value of 22.4 mM was as potent as compound 4m. Molecular docking study was performed using the most active
Furthermore, compounds 4b, 4f, 4h–l, and 4n showed remarkable compound 4m to investigate the possible interactions with amino
O
O
O
Scheme 1. Synthesis of 5-benzylidenerhoda-
nine-3-acetamides (4).
O
appropriate
amine
Ar
H
Ar
2
N
Ar
N
N
OH
S
OH
S
NR'R"
S
EDC, HOBt,
CH3CN
(S)-proline,
EtOH, reflux
O
O
S
S
O
S
4
3
1

DOI: 10.3109/14756366.2014.940935
5-Benzylidenerhodanine-3-acetic acid derivatives
5
Table 1. Chemical structure and eelAChE inhibition data of target Table 2. Chemical structure and soybean 15-LOX inhibition activity data
compounds 4a–n.
of 5-arylidene-rhodanine-3-acetic acids 3a–l.
O
O
N
N
S
R
R
NR'R"
OH
S
S
O
O
S
Compounds
NR0R00
R
IC50 (mM)*
IC50 (mM)*
soybean 15-LOX
Compounds
R
4a
4-Chloro-
285.0 9.1
N
O
O
O
O
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
4-Chloro-
4-(N,N-dimethylamino)-
4-Methoxy-
3,4,5-Trimethoxy-
2,3-Dimethoxy-
2-Hydroxy-4-bromo-
3-Bromo-4,5-dimethoxy
2,4-Dihydroxy
2-Hydroxy
156 3.6
NAy
390 9.1
164 3.8
175 3.9
149 3.4
NA
4b
4c
4d
4e
4-(N,N-dimethylamino)- 52.3 1.7
N
N
N
4-Methoxy-
664.0 21.0
675.0 22.5
285.0 9.2
147 3.6
421 9.7
251 5.9
203 4.5
366 8.5
18 0.5
2-Methoxy
2-Bromo
H
3,4,5-Trimethoxy-
3,4,5-Trimethoxy-
3l
Quercetin
–
N
N
N
*Data are expressed as mean S.E. of at least three different experiments.
yNo activity.
4f
4-Methoxy-
44.1 1.4
22.4 0.7
N
acid residues on the active site of the AChE. The interaction of
4m and the residues in the active site of eelAChE was illustrated
in Figure 1. The compound was spanning along the narrow gorge
of the enzyme from peripheral anionic binding site (PAS) to
catalytic anionic site (CAS). The active site of the enzyme is lined
with aromatic residues, which comprise 40% of the residues in the
binding pocket. The most active compound 4m takes advantage of
these residues by making a variety of interactions. The positively
charged piperidine nitrogen makes a cation–ꢂ interaction with
Phe330 that is essential for ligand recognition. The benzyl ring of
the compound shows ꢂ–ꢂ stacking with the aromatic ring of the
Trp84. In such orientation, the pendant fragment including the
rhodanine is stabilized by making ꢂ–ꢂ stacking with Trp279
through the 4-(dimethylamino)phenyl part of the molecule that is
positioned at the rim of the gorge.
In the case of 15-LOX enzyme, the best LOX inhibitor, 3h,
was docked into the LOX active site pocket while the Fe core
had been modified to Fe(III)-OH. Analysis of docking results
revealed that free carboxylic group of compound 3 h, oriented
toward Fe–OH and interfered with it through a hydrogen bonding.
It is further stabilized through additional hydrogen bonding
formed between 2-hydroxy group of phenyl moiety and Ser510
and His513 (Figure 2). As mentioned above, all final compounds
4a–k were inactive toward lipoxygenase enzyme. This recon-
firmed the importance of free carboxylic group for binding
of these compounds to target enzyme and their LOX inhibitory
activity.
F
4g
3,4,5-Trimethoxy-
N
N
F
4h
4i
F 3,4,5-Trimethoxy-
52.3 1.7
61.9 2.2
N
N
2,3-Dimethoxy-
N
N
CI
CI
4j
4k
4l
4-(N,N-dimethylamino)- 57.9 1.8
N
3-Hydroxy-4-methoxy- 63.0 2.3
N
4-Chloro-
47 2.6
N
N
H
4m
4-(N,N-dimethylamino)- 21 1.1
N
N
H
Conclusion
4n
4-Methyl-
63 3.8
N
We have synthesized a series of 5-benzylidenerhodanine-3-
acetamides bearing morpholino-, 4-arylpiperazinyl-, or 4-benzyl-
piperidinyl- moieties and evaluated their inhibitory activity
against AChE. Alteration of amide part and substitution on the
benzylidene moiety resulted in changing of anti-AChE activity.
The better result was obtained with 4-(3-fluorophenyl)piperazin-
1-yl derivative 4g containing 3,4,5-trimethoxybenzylidene
scaffold and 1-benzylpiperidinyl derivative 4m bearing 4-(N,N-
dimethylamino)benzylidene moiety.
N
H
Tacrine
–
–
0.41
*Data are expressed as mean S.E. of at least three different experiments.

6
N. Shafii et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
Figure 1. A representative model for interaction of compound 4m and the AChE. The residues involved in the interaction are shown.
Figure 2. Representation of the best docked pose of 3h in the active site of 15-LOX enzyme. Hydrogen bonds are shown as red-dotted lines and Fe(III)
also is shown as ball model. (For interpretation of the references to colour in this caption, the reader is referred to the online version of this article.)
Acknowledgements
Besides AChE, most intermediate compounds 3 showed mild
inhibitory activity against 15-LOX. The 2-hydroxy-4-bromo and
2,4-dihydroxy derivatives (compounds 3f and 3h, respectively)
were the most active compounds.
Chimera is developed by the 25 Resource for Biocomputing,
Visualization, and Informatics at the University of California, San
Francisco.

DOI: 10.3109/14756366.2014.940935
5-Benzylidenerhodanine-3-acetic acid derivatives
7
13. Roman OM, Nektegayev IO, Lesyk RB. Bioactive compounds
among 5-arylidenerhodanine 3-carboxylic acids. Pharm J 2002;5:
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inflammatory activity of 3-aryl-5-arylidene-2-thioxothiazolidin-
4-ones. Pharm J 2002;3:56–9 (in Ukrainian).
Declaration of interest
The authors have declared no conflict of interest.
The work was supported by grant from International Campus, Tehran
University of Medical Sciences.
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Supplementary material available online
Supplementary Figures.