Pharmacological evaluation of some novel synthesized compounds derived from spiro(cyclohexane-1,2′-thiazolidines)

Article abstract of DOI:10.1007/s00044-013-0830-y

A series of novel spiro(cyclohexane-1,2′-thiazolidine) derivatives 1-5 and spiro(cyclohexane1,2′-thiazolopyridine) derivatives 6-14 were synthesized. Arylidene thiazolidine derivative 2b was reacted with hydroxylamine hydrochloride, hydrogen peroxide or ethyl cyanoacetate to give spiro(cyclohexane-1,2′-thiazolidine), and spiro(cyclohexane-1,2′- thiazole) derivatives 15-17 . Moreover, some of the newly prepared products were screened for their antibacterial, antioxidant, and anticancer activities, and some of them showed moderate to good biological activities. The results showed that the compounds 9, 7a, and 8 have the highest antioxidant activity (92.52, 85.00, and 84.61%, respectively at 100 μg/mL) when compared with butylated hydroxyl toluene as a standard (93.6%). Also, the compound 9 showed the highest anticancer activity against two cell lines (MCF-7 and HepG2). Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0830-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0830-y
ORIGINAL RESEARCH
Pharmacological evaluation of some novel synthesized compounds
derived from spiro(cyclohexane-1,20-thiazolidines)
•
Eman M. Flefel Hayam H. Sayed
•
•
Ahmed I. Hashem Emad A. Shalaby
•
•
Walaa El-Sofany Farouk M. E. Abdel-Megeid
Received: 1 July 2013 / Accepted: 3 October 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of novel spiro(cyclohexane-1,20-thiazol-
idine) derivatives 1–5 and spiro(cyclohexane1,20-thiazolo-
pyridine) derivatives 6–14 were synthesized. Arylidene thia-
zolidine derivative 2b was reacted with hydroxylamine
hydrochloride, hydrogen peroxide or ethyl cyanoacetate to
give spiro(cyclohexane-1,20-thiazolidine), and spiro(cyclo-
hexane-1,20-thiazole) derivatives 15–17. Moreover, some of
the newly prepared products were screened for their antibac-
terial, antioxidant, and anticancer activities, and some of them
showed moderate to good biological activities. The results
showed that the compounds 9, 7a, and 8 have the highest
antioxidantactivity(92.52,85.00,and84.61 %, respectivelyat
100 lg/mL) when compared with butylated hydroxyl toluene
as a standard (93.6 %). Also, the compound 9 showed the
highest anticancer activity against two cell lines (MCF-7 and
HepG2).
Keywords Spiro(cyclohexane-1,20-thiazolidine) ꢀ
Spiro(cyclohexane-1,20-thiazolo-pyridine) ꢀ Antimicronial ꢀ
Antioxidant ꢀ Anticancer activity
Introduction
The investigational approaches toward structure–activity
relationship focusing the search of optimized candidates
have become immensely important. The literature survey
reveals that thiazolidine is parent of a series of compounds
that are important in medicinal and pharmaceutical chem-
istry; a large majority of drugs and drug candidates
incorporate thiazolidine nucleus and these include impor-
tant drugs such as Pioglitazone, Epalrestat, Letosteine, and
Tidiacic. The nucleus is also known as wonder nucleus
because it gives out different derivatives with all different
types of biological activities. The presence of N–C–S
linkage in the compounds has been shown to exhibit anti-
microbial (Sayed et al., 2010; Mishra et al., 2012; Patela
et al., 2012) and anti-HIV (Abhinit et al., 2009; Balzarini
et al., 2007) activities. Some of 4-thiazolidinones have
been assessed for their antifungal (Ozkirimli et al., 2009).
Also, they exhibited antitubercular (Basawaraj et al.,
2010), hypnotic (Ergenc et al., 1999), antioxidant (Maia
et al., 2009), analgesic (Gurupadayya et al., 2008), anti-
convulsant activities (Archana et al., 2002), anti-inflama-
tory (Kumar et al., 2007), anticancer (Lesyk et al., 2011;
Hongyu 2008), antihyperglycemic (Imaran et al., 2009),
anesthetic (Surray 1949), diuretic (Vagdevi et al., 2006),
E. M. Flefel (&) ꢀ H. H. Sayed (&) ꢀ W. El-Sofany ꢀ
F. M. E. Abdel-Megeid
Photochemistry Department, National Research Centre,
Dokki, Cairo 12622, Egypt
e-mail: emanmflefel@yahoo.com
H. H. Sayed
e-mail: hayamsayed@yahoo.com
A. I. Hashem
Department of Chemistry, Faculty of Science, Ain Shams
University, Cairo, Egypt
`
antihistaminic (Rao et al., 2002), antidiabetic (Ottana et al.,
2011), antihistaminic (Diurno, et al., 1999), cyclooxygen-
ase inhibitors, and lipoxygenase inhibitors (Eleftheriou
et al., 2012) activities. So, the aim of the present study
is to synthesize the biologically important scaffold spiro
E. A. Shalaby
Department of Biochemistry, Faculty of Agriculture,
Cairo University, Giza 12613, Egypt
123

Med Chem Res
(cyclohexane-thiazolidine) derivatives and evaluate their
antimicronial, antioxidant, and anticancer activities.
115.2–133.5 (sp2 carbons), 172.5 (C=O); MS, m/z (%): 326
(M?, 64) and 284 (100 %).
2-[(4-Chlorobenzylidene]-4-(4-halophenyl)-1-thia-4-
Materials and methods
azaspiro[4.5]decan-3-one (2a–f)
Chemistry
A mixture of compounds 1a, b (0.01 mol) and aromatic
aldehydes, 4-chlorobenzaldehyde, 4-methylbenzaldehyde,
or benzaldehyde (0.01 mol) was refluxed in ethanolic
NaOH for 2 h. The product was poured onto ice and neu-
tralized with dilute HCl; then the formed solid was filtered
off and crystallized to give 2a–f.
All melting points are uncorrected and measured using
Electro-thermal IA 9100 apparatus, (Shimadzu, Tokyo,
Japan). IR spectra were recorded as potassium bromide
pellets on a Perkin-Elmer 1,650 spectrophotometer (Per-
kin-Elmer, Norwalk, CT, USA). H NMR and 13C NMR
1
were determined on a Jeol-Ex-300 NMR spectrometer
(JEOL, Tokyo, Japan), and chemical shifts were expressed
as part per million, ppm (d values) against TMS as internal
reference. Mass spectra were recorded on VG 2AM-3F
mass spectrometer (Thermo electron corporation, USA);
halo compounds (Cl, Br) showed the pattern of isotope
abundance. Microanalyses were operated using Mario El
Mentar apparatus, Organic Microanalysis Unit, and the
results were within the acceptable range ( 0.30) of the
calculated values. Follow up of the reactions and checking
the purity of the compounds were made by TLC on silica
gel-pre-coated aluminum sheets (Type 60 F254, Merck,
Darmstadt, Germany).
2-[(4-Chlorobenzylidene]-4-(4-fluorophenyl)-1-thia-4-aza-
spiro[4.5]decan-3-one (2a) MP 196–198 °C (AcOH);
yield 74 %; IR (KBr, t, cm-1): 1679 (C=O); 1H NMR
(DMSO-d6): d (ppm) 1.78-1.96 (m, 4H, 2CH2), 7.10–7.67 (m,
9H,Ar–H?CHmethylene);13CNMR24.2–36.4(5CH2),70.2
(C-spiro), 115.4–135.8 (sp2 carbons), 141.2 (C–S), 163.2 (C–F),
168.8 (C=O); MS, m/z (%): 387(M?, 18.8), 389 (M??2, 6.3).
2-[(4-Methylbenzylidene)]-4-(4-fluorophenyl)-1-thia-4-
azaspiro[4.5]decan-3-one (2b) MP 218–220 °C (diox-
ane), yield 78 %, IR (KBr, t, cm-1): 1679.3 (CO); 1H NMR
(DMSO-d6): d (ppm) 1.43–1.54 (m, 6H,3CH2), 1.76–1.95
(m, 4H, 2CH2), 2.38 (CH3), 7.17–7.76 (m, 9H, Ar–H?CH
13
methylene); CNMR: 21.4 (CH3), 24.2–39.3 (5CH2), 71
(C-spiro), 117–135 (sp2 carbons), 138 (C-methyl), 142
4-(4-Halophenyl)-1-thia-4-azaspiro[4.5]decan-
(C–S), 164 (C–F), 167.2 (C=O); MS, m/z (%): 367(M?, 12.0).
3-one (1a, b)
A mixture of cyclohexanone (0.98 mL, 0.01 mol), aro-
matic amine, namely, 4-flouroaniline or 4-bromoaniline
(0.01 mol), and thioglycolic acid (0.92 mL, 0.01 mol) in
dry benzene (50 mL) was refluxed for 10 h. The solution
was concentrated; the formed solid was filtered off, dried,
and crystallized to give compounds 1a and b.
(2-Benzylidene-4-(4-fluorophenyl)-1-thia-4-azaspiro[4.
5]decan-3-one (2c) MP 183–185 °C (MeOH), yield
75 %, IR (KBr, t, cm-1): 1677.9 (CO); 1H NMR (DMSO-
d6): d (ppm) 1.40–1.52 (m, 6H, 3CH2), 1.78–1.98 (m, 4H,
2CH2), 6.96–7.66 (m, 9H, Ar–H?CH methylene);
13
CNMR: 23.3–32 (5CH2), 73 (C-spiro), 116–134.3 (sp2
carbons), 138.6 (C–S), 163.5 (C–F), 167 (C=O); MS, m/z
(%): 353.12 (M?, 54).
4-(4-Flourophenyl)-1-thia-4-azaspiro[4,5]decan-3-one
(1a) MP 143–145 °C (MeOH), yield 70 %; IR (KBr, t,
cm-1): 682 (C–S–C), 1688 (CO); H NMR (DMSO-d6): d
(ppm) 1.45–1.59 (m, 6H, 3CH2), 1.72–1.98 (m, 4H, 2CH2),
13
1
4-(4-Bromophenyl)-2-[(4-chlorobenzylidene]-1-thia-4-aza-
spiro[4.5]decan-3-one (2d) MP 216–218 °C (dioxane),
1
yield 76 %, IR (KBr, t, cm-1): 1685.6 (CO); H NMR
3.52 (s, 2H, CH2), 7.00–7.35 (m, 4H, Ar–H);
CNMR
23.4–39.5 (5CH2), 43.7 (CH2), 74.1 (C-spiro), 115.4–138.2
(sp2 carbons), 164 (C–F), 172.18 (C=O); MS, m/z (%): 265
(M?, 100).
(DMSO-d6): d (ppm) 1.42–1.55 (m, 6H, 3CH2), 1.76–1.98
(m, 4H, 2CH2), 7.20–7.81 (m, 9H, Ar–H?CH methylene);
13C NMR: 23.5–38 (5CH2), 69 (c-spiro), 124–135 (sp2
carbons), 135.8 (C–S), 167 (C=O), MS, m/z (%): 450(M?,
22.4), 452 (M??2, 22.9).
4-(4-Bromophenyl)-1-thia-4-azaspiro[4,5]decan-3-one
(1b) MP 131–133 °C (MeOH); yield 72 %; IR (KBr, t,
1
cm-1): 680 (C–S–C), 1682 (CO); H NMR (DMSO-d6): d
(ppm) 1.42–1.52 (m, 6H, 3 CH2), 1.73–1.96 (m, 4H,
4-(4-Bromophenyl)-2-[(4-methylbenzylidene]-1-thia-4-
azaspiro[4.5]decan-3-one (2e) MP 265–267 °C (diox-
ane), yield 80 %, IR (KBr, t, cm-1): 1676.8 (CO) 1H NMR
(DMSO-d6): d (ppm) 1.42–1.53 (m, 6H, 3CH2), 1.75–1.94
2CH2), 3.48 (s, 2H, CH2), 7.01–7.58 (m, 4H, Ar–H);
13
CNMR 23.2–39 (5CH2), 41.2 (CH2), 74.10 (C-spiro),
123

Med Chem Res
(m, 4H, 2CH2), 2.38 (s, 3H, CH3), 7.20–7.76 (m, 8H, Ar–
H), 8.1 (s,1H, CH methylene); 13C NMR 21.3 (CH3),
24.1–39.7 (5CH2), 69.5 (C-spiro), 118.1–134.5 (sp2 car-
bons), 139.5 (C–S), 167.3 (C=O), MS, m/z (%): 429.06
(M?, 26.5), 431(M??2, 25).
162.6 (C=N), 169.9 (C=O); MS m/z (%): 474.04 (M?,
15.2), 476.03 (M?, 6.0).
4-(4-Bromophenyl)-2-[2-(4-fluorophenyl)hydrazono]-1-
thia-4-azaspiro[4.5]decan-3-one (3d) MP 179–181 °C
(MeOH), yield 60 %, IR (KBr, t, cm-1): 1679 (CO), 3135
1
2-Benzylidene-4-(4-bromophenyl)-1-thia-4-azaspiro[4.5]
(NH); H NMR (DMSO-d6): d (ppm) 1.45–1.53 (m, 6H,
3CH2), 1.65–1.96 (m, 4H, 2CH2), 3.8 (s, 1H, NH, D2O
13
decan-3-one (2f) MP 253–254 °C (dioxane), yield 76 %,
1
IR (KBr, t, cm-1): 1674.8 (CO); H NMR (DMSO-d6):
exchangeable), 7.3–7.65 (m, 8H, Ar–H);
CNMR 23.
d (ppm) 1.46–1.55 (m, 6H, 3CH2), 1.72–1.96 (m, 4H,
2CH2), 7.1–7.63 (m, 9H, Ar–H), 8.0 (s, 1H, CH methylene);
7–33.1 (5CH2), 68.2 (C-spiro), 115.2–139.4 (sp2 carbons),
160.1 (C=N), 161.9 (C–F), 168.6 (C=O); MS, m/z (%):
447.04(M?, 17.4), 449.04 (M?, 7.1).
13
CNMR 23.2–38.5 (5CH2), 69.6 (C-spiro), 118.5–134.0
(sp2 carbons), 140.1 (C–S), 168.2 (C=O); MS, m/z (%):
415.04 (M?, 12.3), 417.04 (M??2, 13.8).
General procedure for the synthesis of hydrazone
derivatives 4a–d
General procedure for the synthesis of compounds 3a–d
A mixture of compound 1a or 1b (0.01 mol) and hydrazine
hydrate or phenylhydrazine (0.02 mol) was refluxed in
absolute ethanol (30 mL) for 6 h. The obtained solid sub-
stance was filtered off, dried, and crystallized from the
proper solvents to give compounds 4a–d.
A solution of hydrochloric acid (6 mL), aromatic amine,
namely, 4-nitroaniline or 4-fluoroaniline (0.01 mol), and an
aqueous solution (3 mL) of sodium nitrite (0.72 g,
0.015 mol) was stirred at 0 °C for 1 h. Followed by, the
addition of (0.01 mol) of compound 1a or b in 10 mL
pyridine and stirring was continued at 0 °C for 2 h. The
resulting product was filtered off, washed with water, dried,
and crystallized to give compounds 3a–d, respectively.
4-(4-Fluorophenyl)-3-hydrazono-1-thia-4-azaspiro[4,5]
decane (4a) MP 142–144 °C (MeOH), yield 55 %, IR
(KBr, t, cm-1): 3210 (NH2); 1H NMR (CDCl3-d6): d (ppm)
1.45–1.51 (m, 6H, 3CH2), 1.62–1.97 (m, 4H, 2CH2), 4.2 (s,
2H, NH2, D2O exchangeable), 7.1–7.6 (m, 4H, Ar–H);
13 CNMR 23.3–32.6 (5CH2), 35.6 (S-CH2), 70.3 (C-spiro),
115.6–139 (sp2 carbons), 158.4 (C=N), 160.1 (C–F); MS,
m/z (%): 279.12 (M?, 100).
4-(4-Fluorophenyl)-2-[2-(4-nitrophenyl)hydrazono]-1-
thia-4-azaspiro[4.5]decan-3-one (3a) MP 148–150 °C
(MeOH), yield 60 %, IR (KBr, t, cm-1): 1682 (CO), 3110
1
(NH); H NMR (DMSO-d6): d (ppm) 1.41–1.55 (m, 6H,
3CH2), 1.70–1.94 (m, 4H, 2CH2), 3.8 (s,1H, NH, D2O
exchangeable), 7.14–7.35 (m, 8H, Ar–H); 13 CNMR: 23–32
(5CH2), 72 (C-spiro), 116–132 (sp2 carbons), 163 (C=N),
172 (C=O); MS, m/z (%): 414 (M?, 50), 416 (M?2, 50).
4-(4-Fluorophenyl)-3-(2-phenylhydrazono)-1-thia-4-aza-
spiro[4.5]decane (4b) MP 192–194 °C (dioxane/MeOH),
yield 58 %, IR (KBr, t, cm-1): 3110 (NH); 1H NMR
(DMSO-d6): d (ppm) 1.35–1.49 (m, 6H, 3CH2), 1.68–1.93
(m, 4H, 2CH2), 4.1 (s, 1H, NH, D2O exchangeable), 7.2–7.4
(m, 9H, Ar–H); 13 CNMR 23.2–32.8 (5CH2), 35.5 (S-CH2),
70.9 (C-spiro), 114.6–143.1 (sp2 carbons), 159.7 (C=N),
160.3 (C–F); MS, m/z (%): 355.15 (M?, 100).
4-(4-Fluorophenyl)-2-[2-(4-fluorophenyl)hydrazono]-1-
thia-4-zaspiro[4.5]decan-3-one (3b) MP 151–153 °C
(MeOH), yield 63 %, IR (KBr, t, cm-1): 1689 (CO), 3125
1
(NH); H NMR (DMSO-d6): d (ppm) 1.43–1.58 (m, 6H,
3CH2), 1.72–1.95 (m, 4H, 2CH2), 3.75 (s, 1H, NH, D2O
13
exchangeable), 7.22–7.56 (m, 8H, Ar–H);
CNMR
4-(4-Bromophenyl)-3-hydrazono-1-thia-4-azaspiro[4,5]
decane (4c) MP 138–140 °C (MeOH), yield 58 %, IR
(KBr, t, cm-1): 3233 (NH2); 1H NMR (CDCl3-d6): d (ppm)
1.41–1.55 (m, 6H, 3CH2), 1.61–1.95 (m, 4H, 2CH2), 4.3 (s,
2H, NH2, D2O exchangeable), 7.2–7.67 (m, 4H, Ar–H);
13 CNMR 23.1–32.7 (5CH2), 35.8 (S-CH2), 71.1 (C-spiro),
113.5–137 (sp2 carbons), 159.4 (C=N); MS m/z (%):
340.04. (M?, 100).
23.8–33.1 (5CH2), 70.8 (C-spiro), 114.5–136.2 (sp2 carbons),
158.5, 160.3 (2 C–F), 162.6 (C=N), 170.1 (C=O); MS, m/z (%
): 386(M-1, 8.3) 387.12 (M?, 7.2).
4-(4-Bromophenyl)-2-[2-(4-nitrophenyl)hydrazono]-1-
thia-4-azaspiro[4.5]decan-3-one (3c) MP 170–172 °C
(MeOH), yield 63 %, IR (KBr, t, cm-1): 1685 (CO), 3140
1
(NH); H NMR (DMSO-d6): d (ppm) 1.42–1.53 (m, 6H,
3CH2), 1.71–1.96 (m, 4H, 2CH2), 3.9 (s,1H, NH, D2O
13
4-(4-Bromophenyl)-3-(2-phenylhydrazono-1-thia-4-azas-
excheangeable), 7.20–7.65 (m, 8H, Ar–H);
CNMR
piro[4.5]decane (4d) MP 188–190 °C (dioxane), yield
23.5–32.9 (5CH2), 70.2 (C-spiro), 121.1–139 (sp2 carbons),
57 %, IR (KBr, t, cm-1): 3120 (NH); H NMR (DMSO-
1
123

Med Chem Res
d6): d (ppm) 1.39–1.5 (m, 6H, 3CH2), 1.65–1.89 (m, 4H,
2CH2), 3.8 (s, 1H, NH, D2O exchangeable), 7.1–7.56 (m,
9H, Ar–H); 13 CNMR 24.1–33.8 (5CH2), 35.1 (S-CH2), 70.
2 (C-spiro), 115–141 (sp2 carbons), 159.1 (C=N); MS, m/z
(%): 416.07 (M?, 100).
(C=O), 172.12 (C-p-chlorophenyl); MS, m/z (%): 452 (M?,
100), 453.89 (M?, 36.9).
General procedure for the synthesis of compounds (7a, b)
A mixture of 1a, b (0.01 mol), malononitrile (0.06 g,
0.01 mol), 4-methylbenzaldehyde (1.2 g, 0.01 mol), and
ammonium acetate (0.02 mol) in glacial acetic acid (40 mL)
was refluxed for 24 h. The reaction mixture was cooled and
poured into water. The formed solid was filtered off, dried,
and crystallized to give compounds 7a and b, respectively.
General procedure for the synthesis of compounds (5a, b)
To a solution of compound 1a (0.01 mol) in absolute eth-
anol (30 mL), thiosemicarbazide or 4-aminophenylsulph-
onamide (0.01 mol), and 2 mL of concentrated HCl was
added. The reaction mixture was refluxed for 3 h, then left
to cool; the formed solid was filtered off, washed with
water, and crystallized to give compounds 5a and b,
respectively.
50-Amino-3-(4-fluorophenyl)-70-p-tolyl-30H-spiro-[cyclo-
hexane-1,20-thiazolo[4,5-b]pyridine]-60-carbonitrile (7a) MP
137–139 °C (MeOH), yield 65 %, IR (KBr, t, cm-1): 2216
1
(CN), 3157 (NH2); HNMR (CDCL3): d (ppm) 1.45–1.54
2-(4-(4-Fluorophenyl)-1-thia-4-azaspiro[4.5]decan-3-yli-
dene)hydrazinecarbothio amide (5a) MP 179–181 °C
(dioxane), yield 40 %, IR (KBr, t, cm-1): 3111 (NH), 3214
(m, 6H, 3CH2), 1.72–1.97 (m, 4H, 2CH2), 2.5 (s, 3H, CH3),
3.66 (br, 2H, NH2, D2O exchangeable), 7.0–7.8 (m, 8H,
13
Ar–H);
CNMR: 22 (CH3), 23.6–38 (5CH2), 74.0 (C-
1
(NH2); H NMR (CDCl3-d6): d (ppm) 1.42–1.55 (m, 6H,
Spiro), 82.2 (C-cyano), 114.0 (C:N), 116.0–132.5 (sp2
carbons), 161.6 (C–N), 163.6 (C–NH2), 171 (C-p-tolyl);
MS, m/z (%): 430.17 (M?, 10).
3CH2), 1.6–1.94 (m, 4H, 2CH2), 3.8 (s, 1H, NH, D2O
exchangeable), 4.3 (s, 2H, NH2, D2O exchangeable), 7.1–7.4
(m, 4H, Ar–H); 13 CNMR : 23.2–33.1 (5CH2), 36.7 (S–CH2),
71.1 (C-spiro), 115.7–138.5 (sp2 carbons), 158.6 (C=N),
160.2 (C–F), 188.0 (C=S); MS, m/z (%): 338.4 (M?, 10.5).
50-Amino-3-(4-bromophenyl)-70-p-tolyl-30H-spiro[cyclohex-
ane-1,20-thiazolo[4,5-b]pyrilidine]-60-carbonitrile (7b) MP
182–183 °C (MeOH), yield 60 %, IR (KBr, t, cm-1): 2218
1
2-(4-(4-Fluorophenyl)-1-thia-4-azaspiro[4.5]decan-3-ylide-
neamino)benzenesulfonamide (5b) MP 158–160 °C (diox-
ane), yield 60 %, IR (KBr, t, cm-1): 3217 (NH2); 1H NMR
(DMSO-d6): d (ppm) 1.40–1.52 (m, 6H, 3CH2), 1.78–2.05
(m, 4H, 2CH2), 4.16 (s, 2H, NH2, D2O exchangeable), 7.0–
7.6 (m, 8H, Ar–H); 13 CNMR 23.8–32.2 (5CH2), 38.3 (S-CH2),
71.8 (C-spiro), 113–142 (sp2 carbons), 160.0 (C–F), 166.1
(C=N); MS, m/z (%): 419.5 (M?, 21.54).
(CN), 3188 (NH2); HNMR (CDCL3): d (ppm) 1.43–1.56
(m, 6H, 3CH2), 1.71–1.96 (m, 4H, 2CH2), 2.6 (s, 3H, CH3),
3.8 (br, 2H, NH2, D2O exchangeable), 7.2–7.68 (m, 8H, Ar–
13
H);
CNMR 21.6 (CH3), 24.1–38.2 (5CH2), 72.8 (C-
Spiro), 82.6 (C-cyano), 113.8 (C:N), 115.7–133.5 (sp2
carbons), 161.4 (C–N), 164.1 (C-NH2), 170.9 (C-p-tolyl);
MS, m/z (%): 491.17 (M?, 11), 493.08 (M?, 7.3).
30-(4-Fluorophenyl)-90-p-tolyl-30H,50H,70H-
spiro(cyclohexane-1,20-thiazolo[5,4-e]pyrido[2,3-
d]pyrimidine]-60,80-dithione (8)
70-(4-Chlorophenyl)-30-(4-flurophenyl)-50-oxo-40,50-
dihydro-30H-spiro-[cyclohexane-1,20-thiazolo
[4,5-b]pyridine]-60-carbonitrile (6)
A mixture of 7a (0.01 mol) and CS2 (excess) in ethanolic
NaOH (10 mL, 4 %) was refluxed for about 9 h. The
reaction mixture was poured onto ice, neutralized with
dilute HCl and the obtained solid was filtered off, dried and
crystallized to give compound 8. MP 170–171 °C (diox-
ane), yield 50 %, IR (KBr, t, cm-1): 3172, 3156 (2 NH),
1,228, 1,230 (2C=S); 1H NMR (DMSO-d6): d (ppm)
1.38–1.5 (m, 6H, 3CH2), 1.73–1.96 (m, 4H, 2CH2), 2.5 (s,
3H, CH3), 7.2–7.4 (m, 8H, A–H), 7.6 (s, 1H, NH, D2O
exchangeable), 11.2 (s, NH, D2O exchangeable); 13C
NMR: 24.1–37.2 (5CH2), 74.05 (C-spiro), 116.8–145.0
(sp2 carbons), 161.2 (C–F), 167.5, 174.13 (2 C=S); MS, m/z
(%): 507 (M?, 6.1).
A
mixture of 1a (0.01 mol), 4-chlorobenzaldhyde
(0.01 mol), ethylcyanoacetate (0.01 mol) and anhydrous
ammonium acetate (0.04 mol) in glacial acetic acid (40 mL)
was refluxed for 24 h. The reaction mixture was cooled and
poured into water. The formed solid was filtered off, dried
and crystallized to give compound 6. MP 150–151 °C
(MeOH), yield 40 %, IR (KBr, t, cm-1): 1689 (CO), 3120
1
(NH); H NMR (DMSO-d6): d (ppm) 1.38–1.50 (m, 6H,
3CH2), 1.79–2.09 (m, 4H, 2CH2), 7.10–7.55 (m, 8H, Ar–H),
13
9.2 (s, NH, D2O exchangeable);
CNMR: 22.8–39.3
(5CH2), 74.06 (C-spiro), 116.36 (C-cyano), 116.54 (C:N),
116.8–134.6 (sp2 carbons), 138 (C–S), 161.59 (C–F), 163.57
123

Med Chem Res
169.1 (C–F), 172.3 (C=O);MS, m/z (%): 595 (M?, 10.5),
597 (M??2, 3.4).
30-(4-Fluorophenyl)-60-methyl-90-p-tolyl-30H-
spiro(cyclohexane-1,20-thiazolo[5,4-e]pyrido[2,3-
d]pyrimidin])- 80(70H)-one (9)
3-(4-Fluorophenyl)-80-hydrazinyl-90-p-tolyl-30H-
spiro[cyclohexane-1,20-thiazolo[4,5-b]pyrido[2,3-
d]pyrimidine]-60(50H)-thione] (12)
A solution of 7a (0.01 mol) in AcOH/AC2O (as a mixture)
was refluxed for 9 h. The reaction mixture was poured into
ice/drops of ammonia, and the formed solid was filtered
off, dried, and crystallized to give compound 9. MP
210–212 °C (dioxane), yield 48 %, IR (KBr, t, cm-1):
A mixture of compound 8 (0.01 mol) and hydrazine hydrate
(0.02 mol) was stirred in absolute ethanol (30 mL) for 12 h.
The formed solid was filtered off, dried, and crystallized to
give compound 12. MP 161–163 °C (MeOH), yield 66 %,
IR (KBr, t, cm-1): 3114 (NH), 3120 (NH), 3210 (NH2);
1H NMR (DMSO-d6): d (ppm) 1.38–1.49 (m, 6H, 3CH2),
1.79–2.00 (m, 4H, 2CH2), 2.44 (s, 3H, CH3), 3.9 (br, 2H,
NH2, D2O exchangeable) 7.11–7.75 (m, 9H, A–H?NH), 9.2
(s, NH, D2O exchangeable); 13C NMR 22.1 (CH3),
25.1–36.4 (5CH2), 71.0 (C-spiro), 118.3–143.2 (sp2 car-
bons), 160.2 (C–F), 164.7 (C-NHNH2), 180.9 (C=S); MS,
m/z (%): 506 (M?, 27).
1
3152 (NH), 1669 (C=O); HNMR (DMSO-d6): d (ppm)
1.41–1.50 (m, 6H, 3CH2), 1.72–1.96 (m, 4H, 2CH2), 2.4
(2CH3) 7.2–7. 58 (m, 9H, Ar–H?NH, D2O exchangeable);
13
CNMR: 21, 23 (2CH3), 25–38 (5CH2), 74.3 (C-spiro),
116.3–145 (sp2 carbons), 161.0 (C-CH3, in pyridine ring),
163.2 (C–F), 171 (C=O); MS, m/z (%): 472 (M?, 35.6).
N-(60-Cyano-30-(4-fluorophenyl)-70-p-tolyl-30H-spiro-
[cyclohexane-1,20-thiazolo[4,5-b] pyridin]-50-yl)]-
acetamide (10)
30-(4-Fluorophenyl)-100-thioxo-110-(p-tolyl)-30,100-
dihydro-spiro[cyclohexane-1,20-thiazolo-[50,40:5,6]
pyrido[2,3-d]thiazolidino[3,2,-a]pyrimidin]-80-one (13)
Compound 7a (0.01 mol) was dissolved in dioxane, and
0.01 mol of K2CO3 was added to the previous solution with
stirring; then 5 mL of ClCOCH3 was added to the mixture
dropwise with stirring for 10 h. The product was poured
into ice/drops of HCl, and the residue was filtered, dried,
and crystallized to give compound 10. MP 231–232 °C
(MeOH), yield 72 %, IR (KBr, t, cm-1): 3162 (NH), 2223
(C:N), 1695 (C=O); 1HNMR (DMSO-d6): d (ppm)
1.45–1.56 (m, 6H, 3CH2), 1.62–1.97 (m, 4H, 2CH2), 2.5
A mixture of 8 (0.01 mol), chloroacetic acid (0.01 mol), and
sodium acetate (0.02 mol) in acetic acid (30 mL)/acetic
anhydride (15 mL) was refluxed for 8 h. The reaction mix-
ture was cooled and poured into water. The formed solid was
filtered off, dried, and crystallized to give compound 13. MP
157–158 °C (MeOH), yield 55 %, IR (KBr, t, cm-1): 1225
(C=S), 1694 (C=O); 1H NMR (DMSO-d6): d (ppm)
1.42–1.53 (m, 6H, 3CH2), 1.76–2.1 (m, 4H, 2CH2), 2.4 (s,
3H, CH3) 3.4 (CH2-thiazole) 7.2–7.5 (m, 8H, Ar–H); 13C
(CH3), 2.7 (CH3), 4.1 (s, 1H, NH, D2O exchangeable),
13
7.1–7. 6 (m, 8H, Ar–H);
CNMR 21.2, 23.6 (2CH3),
24.2–36.6 (5CH2), 72.3 (C-spiro), 95.7 (C–CN), 114.2
(C:N), 118.2–145.8 (sp2 carbons), 150.2 (N–C–N), 153.1
(C-NH), 161.3 (C–F), 170.7 (C=O); MS, m/z (%): 472.17
(M?, 82.6).
0
NMR: 22.2 (CH3), 25.1–35.5 (5CH2), 38.2 (C7 -H2), 70 (C-
spiro), 118.8–148.0 (sp2 carbons), 159.8 (C–F), 164.3 (C–S),
172.1 (CO), 180.2 (C=S); MS, m/z (%): 546.9 (M?, 9.8).
3-(4-Chlorophenyl)-N-(60-cyano-30-(4-fluorophenyl)-70-p-
tolyl-30H-spiro-[cyclohexane-1,20-thiazolo[4,5-b]pyridin]-
50-yl)acrylamide (11)
70-(4-Chlorobenzylidene)-30-(4-fluorophenyl)-100-thioxo-
110-(p-tolyl)-spiro[cyclohexane-1,20-thiazolo[50,40:5,6]
pyrido[2,3-d]thiazolidino[3,2,-a]pyrimidin]-80-one (14)
A mixture of compounds 10 (0.01 mol) and 4-chloro-
benzaldehyde in sodium hydroxide/ethanol solution was
refluxed for 2 h. The product was poured onto ice and
neutralized with HCl; then the solid was filtered off and
crystallized to give compound 11. MP 241–243 °C (diox-
ane), yield 77 %, IR (KBr, cm-1): 2222 (C:N), 3166
Method A
A mixture of 13 (0.01 mol) and 4-chloro-
benzaldehyde (1.4 g, 0.01 mol) in glacial acetic acid/acetic
anhydride (40 mL, 3:1) was refluxed for 3 h, allowed to
cool, then poured onto water. The solid formed was col-
lected by filtration and crystallized to give 14.
1
(NH); HNMR (DMSO-d6): d (ppm) 1.24–1.53 (m, 6H,
3CH2), 1.65–1.89 (m, 4H, 2CH2), 3.5 (CH3), 3.7 (s, 1H,
Method B To a mixture of 8 (0.01 mol), chloroacetic
acid (1.0 g, 0.01 mol) and anhydrous sodium acetate (1.
60 g, 0.01 mol) in glacial acetic acid/acetic anhydride
(40 mL, 3:1 ratio), 4-chloro benzaldehyde (0.01 mol) were
added. The reaction mixture was heated under reflux for
2 h, then cooled, and poured into water. The solid formed
NH, D2O exchangeable), 7.1–7.5 (m, 13H, Ar?CH–CO),
13
8.0 (CH methylene);
CNMR 21.3 (CH3), 25.1–36.4
(5CH2), 72.2 (C-spiro), 93.2 (C–C:N), 114.2 (O=C–
CH=CH–), 115.7 (C:N), 118.6–145.1 (sp2 carbons),
146.3 (O=C–CH=CH–), 149.3 (N–C–N), 152.6 (C–NH),
123

Med Chem Res
was collected by filtration, dried, and crystallized to yield a
product that was identified as compound 14 by m. p., mixed
m. p. and TLC in comparison with authentic sample from
Method A. mp. 278–280 °C (AcOH), yield 64 %, IR (KBr,
cm-1): 1730 (C=O); 1H NMR (DMSO-d6): d (ppm) 1.
42–1.55 (m, 6H, 3CH2), 1.84–2.24 (m, 4H, 2CH2), 2.52 (s,
3H, CH3), 6.85–7.76 (m, 12H, Ar–H), 8.02 (s, CH, meth-
water; the formed solid was filtered off, dried, and crystal-
lized to give compound 17. MP 290–292 °C (AcOH), yield
74 %, IR (KBr, t, cm-1): 1673 (C=O), 2222 (CN); 1H NMR
(DMSO-d6): d (ppm) 1.46–1.58 (m, 6H, 3CH2), 1.77–2.01
(m, 4H, 2CH2), 2.6 (s, 3H, CH3), 7.1–7.5 (m, 8H, Ar–H); 13
C
NMR: 20.79 (CH3), 22-39.3 (5CH2), 72 (C-spiro), 92.2 (C–
C:N), 114.8 (C:N), 115.6–142.8 (sp2 carbons), 161 (C–
F), 168 (C=O); MS, m/z (%): 432.35 (M?, 15).
ylene); 13C NMR 22 (CH3), 25.1.-36.2 (5CH2), 70.6 (C-
2
spiro), 116.8 (C7 =CH), 118.2–145.0 (sp carbons), 148.1
0
0
(C7 =CH), 158.8 (C–F), 171.0 (CO), 184.7 (C=S); MS, m/z
(%): 669.0 (M?, 15.8), 671.3 (M??2,6).
Biological assay
60-(4-Fluorophenyl)-30-p-tolyl-30-60-dihydro-20H-
spiro[cyclohexane-1,50-isoxazolo[5,4-d]thiazole] (15)
Assessment of antioxidant activity
2,2-Diphenyl-1-picrylhydrazyl (DPPH) method
A mixture of 2b (3.6 g, 0.01 mol) and hydroxyl amine
hydrochloride (0.5 g, 0.01 mol) was refluxed in pyridine
(20 mL) for 10 h. The reaction mixture was cooled, poured
into 100 mL water and neutralized with dilute HCl. The
product was filtered off, dried, and crystallized to give
compound 15. MP 241–243 °C (AcOH), yield 65 %, IR
(KBr, t, cm-1): 3163 (NH); 1H NMR (DMSO-d6): d (ppm)
1.44–1.58 (m, 6H, 3CH2), 1.79–2.09 (m, 4H, 2CH2), 2.6 (s,
3H, CH3), 7.2–7.6 (m, 9H, Ar–H?CH oxazole), 8.6 (s, 1H,
NH, D2O exchangeable); 13C NMR: 21.5 (CH3), 23–38
(5CH2), 73.2 (C-spiro), 118.0-142.0 (sp2 carbons), 161.3
(C–F); MS, m/z (%): 382.15 (M?, 18.9).
The DPPH tests were carried out for evaluation of activity
of the newly synthesized compounds as described by Burits
and Bucar (2000). One mL of the tested compounds at
different concentrations (50 and 100 lg/mL) was mixed
with 1 mL DPPH reagent [0.002 % (w/v)/methanol water
solution]. After an incubation period (30 min), the absor-
bance was measured at 517 nm, and the antioxidant
activity of tested compounds was compared with butylated
hydroxyl anisole (BHA) as synthetic antioxidant standard.
% Antioxidant activity ðEÞ ¼ ððAc ꢁ AtÞ=AcÞ ꢂ 100
where Ac is the absorbance of methanolic DPPH solution
(control), and At is the absorbance of the extract sample.
2-(3-(p-Tolyl)oxiran-2-yl)-4-(4-fluorophenyl)-1-thia-4-
azaspiro[4.5]decan-3-one (16)
Anticancer assay
To a cold mixture of arylidene spiro-thiazolidinone 2b
(0.01 mol) in acetone:methanol (30:10) containing 1 g
NaOH, hydrogen peroxide (5 mL, 36 %) was added por-
tionwise with stirring for 1 h. The solvent was concentrated
under reduced pressure. The solid formed was filtered off,
washed with water, and crystallized to give compound 16.
MP 229–231 °C (EtOH), yield 70 %, IR (KBr, t, cm-1):
The cytotoxicity of the evaluated compounds was tested
against HepG-2 (Hepatoma cell line) and MCF-7 cells
Table 1 Antioxidant activity (%) of different synthetic compounds at
50 and 100 lg/m against DPPH radical
1
1698 (CO); H NMR (DMSO-d6): d (ppm) 1.40-1.58 (m,
Compound no.
Concentration (lg/mL)
6H, 3CH2), 1.73–1.99 (m, 4H, 2CH2), 2.5 (s, 3H, CH3), 3.4
(s,1H, CH-oxirane), 7.2–7.6 (m, 8H, Ar–H); 13C NMR: 21.4
(CH3), 24.3–38 (5CH2), 67.1 (C-spiro, thiazolone), 71.8 (C–
O, oxirane), 73.7 (C-spiro, oxirane), 115.8-134.5 (sp2 car-
bons), 162 (CF), 173.2 (C=O); MS, m/z (%): 383.2 (M?, 22).
50
100
2b
41.06 1.02
54.62 2.65
60.05 1.00
65.0 2.52
49.87 0.58
50.21 1.85
41.02 0.78
82.4 5.62
0.97
66.78 2.65
85.0 3.04
84.61 3.40
92.52 4.65
79.85 2.74
78.20 4.85
60.45 1.62
93.6 4.10
1.25
7a
8
9
30-(4-Flourophenyl)-50-oxo-70-p-tolyl-30,50-
dihydrospiro[cyclohexane-1,20-pyrano[2,3-d]thiazole]-
’6-carbonitrile (17)
10
12
15
BHA (as antioxidant stander)
LSD
A mixture of compound 2b (3.6 g, 0.01 mol) and ethyl cy-
anoacetate (0.01 mol) in glecial acetic acid (15 mL) con-
taining anhydrous sodium acetate (0.7 g, 0.01 mol) was
refluxed for 20 h. The reaction mixture was poured into
Each value is presented as mean of triplet treatments, LSD least
significant difference at P B 0.01 according to Duncan’s multiple
range test
123

Med Chem Res
{sulforhodamine B,2-(3-diethylamino-6-diethylazaniumy-
lidene-xanthen-9-yl)-5-sulfo-benzenesulfonate} solution for
10 min in dark place and subsequently washed with 1 %
glacial acetic acid. After drying overnight, Tris–HCl was used
to dissolve the SRB-stained cells and color intensity was
measured at 540 nm.
Table 2 Anticancer activity (%) of different synthetic compounds at
100 lg/mL against two cell lines (HepG2 and MCF-7)
Compound no.
Cell lines
HepG2
MCF-7
2b
7a
8
50.32 1.45
45.0 2.30
50.21 3.6
78.85 6.0
50.63 2.52
15.65 0.65
45.60 1.45
3.45
61.20 1.7
55.84 2.51
64.20 1.9
84.0 4.5
65.0 1.45
45.62 0.8
56.62 2.0
1.92
Antimicrobial assay
9
The antibacterial activities of the newly synthesized com-
pounds were tested against Escherichia coli NRRL B-210
(Gram -ve bacteria), Bacillus subtilis NRRL B-543, and
Staphylococcus aureus NRRL B-313 (Gram ?ve bacteria)
using nutrient agar medium. The antifungal activity of these
compounds was also tested against Candida albicans NRRL
Y-477 using Sabouraud dextrose agar medium (Tables 1, 2).
10
12
15
LSD
Each value is presented as mean of triplet treatments, LSD least
significant difference at P B 0.01 according to Duncan’s multiple
range test
Agar diffusion medium
(Breast cancer) by SRB assay (Skehan et al., 1990). Expo-
nentially, growing cells were collected using 0.25 % Tryp-
sin–EDTA and plated in 96-well plates at 1,000–2,000 cells/
well. Cells were exposed to each test compound for 72 h and
subsequently fixed with TCA (10 %) for 1 h at 4 °C. After
several washings, cells were exposed to 0.4 % SRB
The synthesized compounds were screened in vitro for
their antimicrobial activity against S. aureus, B. subtilis,
E. coli, C. albicans, and Aspergillus niger, by agar diffu-
sion method Cruickshank et al. (1975). A suspension of
each of the aforementioned microorganisms (0.5 mL) was
Table 3 Antimicrobial activity
Compound no.
Microorganisms
Antibacterial activity
of the tested compounds
(in vitro) by agar diffusion
method
Antibacterial activity
Staphylococcus
aureus
Bacillus
subtilis
Escherichia
coli
Candida
albicans
Aspergillus
niger
1a
1b
2a
2d
3a
4a
4b
5b
7a
15
17
??ve
?ve
???ve
??ve
-ve
???ve
??ve
?ve
???ve
??ve
?ve
???ve
?ve
-ve
-ve
-ve
?ve
-ve
-ve
-ve
-ve
?ve
-ve
-ve
?ve
-ve
?ve
??ve
?ve
??ve
?ve
-ve
?ve
-ve
?ve
?ve
-ve
-ve
?ve
??ve
?ve
?ve
-ve
?ve
-ve
-ve
?ve
Highly active, ???ve;
moderately active, ??ve; slightly
active, ?ve; inactive, -ve
-ve
-ve
-ve
?ve
???ve
???ve
-ve
??ve
Table 4 MIC (in lg/mL of the
most active compounds)
Compound no.
Staphylococcus
aureus
Bacillus
subtilis
Escherichia
coli
Candida
albicans
Aspergillus
niger
1a
1b
3a
4a
17
0.150
0.300
0.175
0.400
0.125
0.125
0.200
0.175
0.400
0.120
0.125
0.200
0.125
0.200
0.450
0.435
0.200
0.125
0.300
0.450
0.300
123

Med Chem Res
Fig. 1 The suggested
CH3
CH3
mechanism for the promising
compound 9 as antioxidant with
DPPH radical
O
N
OH
S
N
S
N
N
N
DPPH radical
- DPPH2
N
N
9
N
CH3
CH3
F
F
Scheme 1 Synthetic route
for compounds 1–7
Ar
N
S
N
H
S
N
CH-Ar
O
N
O
S
H
N
R'
N
N
x
3a-d
a: X=F, Ar=C6H4NO2
x
4a-d
a: X=F, R'=H
b: X=F, R'=C6H5
c: X=Br, R'=H
2a-f
X
b: X=F, Ar=C6H4F
c: X=Br, Ar=C6H4NO2
d: X=Br, Ar=C6H4F
a: X=F, Ar=4-C6H4Cl
b: X=F, Ar=4-C6H4CH3
c: X=F, Ar=C6H5
-nitro or
-fluoroaniline
p
p
d:X=Br, Ar=4-C6H4Cl
e: X=Br, Ar=4-C6H4CH3
f: X=Br, Ar=C6H5
NaNO2
d: X=Br, R'=C6H5
ArCHO
Na/ethanol
NH2NH2 or
PhNHNH2
S
N
O
dry benzene
- H2O
O
+
R
NH2
+
SHCH2COOH
malononitrile
OHC CH3
thiosemicarbazide
X
1a: X=F
1b: X=Br
or
1a,b
4-aminophenyl
sulphfonamide
CH3COONH4
CH3
CNCH2COOEt
CH3COONH4
OHC
Cl
S
Cl
CN
S
N
R
N
N
N
NH2
7a,b
CN
O
S
N
F
a: X=f
b: X=Br
X
5a,b
N
H
a: R=NHCSNH2
b: R=4-C6H4SO2NH2
6
F
123

Med Chem Res
added to sterile nutrient agar media at 45 °C, and the
mixture was transferred to sterile Petri dishes and allowed
to solidify. Holes of 0.9 cm in diameter were made using a
cork borer. Amounts of 0.1 mL of the synthesized com-
pounds were poured inside the holes. A hole filled with
DMSO was also used as control. The plates were left for
1 h at room temperature as a period of pre-incubation
diffusion to minimize the effects to variation in time
between the applications of the different solutions. The
diameters of the inhibition zone of the tested compounds
were measured and compared with that of the standard and
the values were tabulated. The same method was carried
out using Sabouraud dextrose agar medium on using C.
albicans NRRL Y-477. The plates were then incubated at
30 °C for 24 h and observed for antibacterial activity. The
diameters of inhibition zone were measured and compared
with that of the standard, the values were tabulated. Cip-
rofloxacin (50 lg/mL) and Fusidic acid (50 lg/mL) were
used as standard for antibacterial and antifungal activity,
respectively. The observed zone of inhibition is presented
in Table 3.
reference drugs solutions were prepared using the proper
nutrient broth. The final concentration of the solutions
varied between 500 and 100 lg/mL with the concentration
of DMSO not exceeding 2.5 %. The tubes were then
inoculated with the test organisms, grown in their suitable
broth at 37 °C for 24 h for bacteria and 48 h for fungi
(about 1 9 106 cells/mL), each 5 mL received 0.1 mL of
the above inoculum and were incubated at 37 °C for 48 h.
The lowest concentration showing no growth was taken as
the minimum inhibitory concentration (MIC) (Table 4).
Statistical analysis
Data were subjected to an analysis of variance, and the
means were compared using the ‘‘least significant differ-
ence (LSD)’’ test at 0.01 levels, as recommended by
Snedecor and Cochran (1982).
Conclusion
In this study, 31 new compounds derived from the key
starting compounds 1a, b{40(4-fluorophenyl- or 40(4-chlo-
rophenyl)spiro(cyclohexane-1,20-thiazolidine)} were syn-
thesized. The starting compounds were prepared (by one
pot reaction) by condensation between cyclohexanone,
4-fluroaniline or 4-bromoaniline and mercaptoacetic acid.
The ring systems of the new novel products 8, 9, 12, 13,
Minimal inhibitory concentration (MIC) measurements
The bacteriostatic activity of the active compounds (having
highly active inhibition zones) was then evaluated using
the two-fold serial dilution technique Scott (Collee et al.,
1989). Two-fold serial dilutions of the test compounds and
CH3
CH3
CH3
ClCOCH3
CN
CS2
S
S
N
CN
S
S
N
NH
NH2
N
N
NHCOCH3
N
N
N
H
S
10
7a
CH3
8
F
F
CHO
F
CH3
Ac2O
CN
Cl
S
N
O
N
S
NH
N
NHCOCH=CH
N
N
CH3
11
9
F
Cl
F
Scheme 2 Synthetic route for compounds 8–11
123

Med Chem Res
and 14 are hitherto unknown ring systems. The antioxidant
activity and anticancer activity of compounds 2b, 7a, 8, 9,
10, 12, and 15 were evaluated. Compound 9 is the most
active compound having antioxidant and anticancer activ-
ity. Also, the antimicrobial activity of compounds 1a, b,
2a, d, 3a, 4a, b, 5b, 7a, 15, and 17 were evaluated (Fig. 1).
mercaptoacetic acid in dry benzene to afford 3-fluoro- or
3-bromo-phenyl)-spiro(cycloheane-1,20-thiazolidine)-5-one
1a, b. The structure of compound 1a was confirmed on the
basis of its IR spectrum which showed peaks at 682 cm-1
indicating the presence of C–S–C stretching and
1,688 cm-1 corresponding to the thiazolidinone carbonyl.
1H NMR spectrum of 1a displayed a singlet signal at
d 3.52 ppm attributed to methylene protons of the thiazo-
lidinone ring, also, signals at 1.45 and 1.98 ppm attributed
to cyclohexane protons. Its 13C NMR spectrum showed
characteristic peak at d 172.18 ppm, thiazolidine carbonyl
carbon, and 43.7 ppm attributed to the methylene carbon,
besides the signals corresponding to aromatic carbons (cf.
Experimental and Scheme 1). Elemental analysis and mass
spectra agree with the proposed structures.
Results and discussion
Chemistry
The starting compounds 1a, b were prepared (by one pot
reaction) by condensation between cyclohexanone, aro-
matic amines namely, 4-fluroaniline or 4-bromoaniline and
Scheme 3 Synthetic route
for compounds 12–14
CH3
NHNH2
NH2NH2
S
N
N
N
N
S
H
CH3
12
CH3
F
S
S
N
O
S
NH
S
ClCH2COOH
S
N
N
N
N
H
N
N
S
8
13
F
F
ClCH2COOH
OHC
Cl
CH3
OHC
Cl
S
N
O
S
N
CH
Cl
N
N
S
14
F
123

Med Chem Res
Condensation of compounds 1a, b with various aromatic
aldehydes namely, 4-chlorobenzaldehyde, 4-methylbenzal-
dehyde, and benzaldehyde in ethanol under reflux in the
presence of a base as catalyst gave the corresponding ary-
lidene derivatives 2a–f in good yields. The structure assigned
for the arylidene derivative 2b was confirmed by spectral
measurements. The IR spectrum of compound 2b as an
example showed absorption band at 1,679 cm-1(CO). The
1H NMR spectra of compounds 2a–f revealed the disap-
pearance of the signals of the methylene protons and
appearance of signals attributed to the olefinic and aromatic
Furthermore, compounds 1a, b were reacted with mal-
ononitrile and 4-methylbenzaldehyde in the presence of
ammonium acetate and acetic acid under reflux to yield the
thiazolo- aminopyridine-carbonitrile derivatives 7a, b. The
IR spectra of compounds 7a, b showed absorption bands
characteristic for NH2 and C:N groups. Also, the 1H NMR
spectra showed signals at d *3.66 ppm (NH2, exchangeable
with D2O). Elemental analysis and Mass spectral data agree
with the proposed structures (cf. Experimental), (Scheme 1).
The aminothiazolo-pyridine-carbonitrile derivative 7a
was utilized for the synthesis of other heterocyclic com-
pounds. It reacted with CS2, AcOH/AC2O, or ClCOCH3 to
afford compounds 8–10, respectively. Also, compound 10
was condensed with 4-chlorobenzaldehyde to give the
corresponding aryl methylene compound 11 (Scheme 2).
Compound 8 was treated with hydrazine hydrate or chlo-
roacetic acid to give compounds 12 and 13, respectively.
The latter compound was further condensed with 4-chlo-
robenzaldehyde to afford the corresponding benzylidene
derivative 14. On the other hand, product 14 was prepared
via one-pot reaction from compound 8 by reaction with
chloroacetic acid and 4-chlorobenzaldehyde in the pre-
sence of anhydrous sodium acetate (Scheme 3). The
structures of these compounds were confirmed by correct
analytical and spectral data (cf. Experimental).
13
protons. CNMR spectrum for 2b showed signals at d71
(spiro carbon), 117–135 (sp2 carbon atoms), and 167.2 (CO).
Diazotization of compounds 1a, b with sodium nitrite
and HCl in the presence of pyridine at 0–5 °C afforded the
corresponding diazonium salts, then coupling with aro-
matic amines (4-nitroaniline or 4-fluoroaniline) gave the
corresponding hydrazono derivatives 3a–d, respectively in
1
good yield. Their H NMR spectra showed the absence of
thiazolomethylene protons and the presence of signals
characteristic of the NH group. Also, compounds 1a,
b were reacted with different nucleophiles such as hydra-
zine hydrate or phenylhydrazine in absolute ethanol to give
the corresponding hydrazones 4a–d.
When compound 1a was treated with thiosemicabazide
or 4-aminophenyl sulfonamide in absolute ethanol and con.
HCl under reflux it afforded hydrazinecarbothioamide and
aminobenzene sulfonamide derivatives 5a, b, respectively.
Compound 1a was reacted with ethyl cyanoacetate and
4-chlorbenzaldehyde in the presence of anhydrous ammo-
nium acetate and refluxing in acetic acid to afford the
thiazolo-pyridinone-carbonitrile derivative 6.
Finally, the arylidene of spirothiazolidine 2b, containing
conjugate exocyclic a,b-unsaturated ketonic function (–CH=
CH–CO–), has been used as a component of Michael addition
with hydroxyl amine hydrochloride to yield the novel spir-
o(cyclohexane-isoxazolo-thiazole) derivative 15.
In addition, a,b-unsaturated ketone 2b was used for the
synthesis of the corresponding oxiranyl derivative 16 by
Scheme 4 Synthetic route for
compounds 15–17
H
S
CNCH2COOEt
CH3COONa
NH2OH.HCl
N
ACONa/ACOH
CH3
O
CH3
2b
F
CH3
S
N
H2O2
NH
O
NaOH
CN
O
S
N
O
O
O
S
15
H3C
F
N
17
F
F
16
123

Med Chem Res
treatment with hydrogen peroxide (30 %) in the presence
of sodium hydroxide. Also, 2b was treated with ethyl
cyanoacetate in the presence of anhydrous sodium acetate
in glacial acetic acid under reflux to afford the corre-
sponding pyrano-thiazole-carbonitrile derivative 17. The
ring systems of the new novel products 8, 9, 12, 13, and 14
are hitherto unknown ring systems.
The role of the antioxidant compounds on growth
inhibition of cancer cells could be explained as sug-
gested mechanisms that the estimated anticancer value
of these compounds was explained in terms of role of
heteroatom’s lone pairs in the chelation process that
promotes the ROS (reactive oxygenated species) pro-
duction and radical formation, causing DNA damage in
cancer cells.
Acknowledgments The authors thank Nayera A. M. Abdelwahad
for antimicrobial evaluation (Chemistry of Natural and Microbial
Product Department, National Research Centre).
Biological activity
Antioxidant activity
The DPPH scavenging assay (Burits, and Bucar 2000) was
performed to determine the antioxidant activity of seven
synthetic compounds (Table 1). Compound 9 showed the
highest antioxidant activity ranged from 65 to 92.52 % at
50 and 100 lg/mL, respectively. Three of seven com-
pounds (7a, 8, 10) recorded more or less lower and com-
parable activities, i.e., 85.0, 84.61, and 79.85 % at100 lg/mL,
respectively. Additionally, compounds 2b, 12, and 15
exhibited very close antioxidant effects ranged between 60
and 78 % at 100 lg/mL. However, the obtained results
revealed that the antioxidant activity of the tested compounds
was lower than BHA as synthetic antioxidant standard
(93.6 % at 100 lg/mL). The antioxidant activity of the
promising compound 9, to the best of our knowledge, may be
due to the formation of the enol form of the pyrmidinone
moiety which is responsible for its antioxidant activity. In this
enol form, the resulting oxygen-centered radical upon the
DPPH radical quenching reaction is more stabilized under the
effect of the extended conjugation. Furthermore, the presence
of electron-donating groups such as methyl, amino, and
thialkyl groups represents an additive rationale to the
enhanced antioxidant activity of compound 9 compared to
other tested compounds. In the same manner, the activity of
compound 8 may be attributed to its thiol form, however, the
less efficient interaction between the resulting sulfur-centered
radical, the p- cloud of the aromatic rings explains the lower
antioxidant activity of compound 8 relative to that of 9.
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