Synthesis, biological evaluation, and molecular docking studies of benzyl, alkyl and glycosyl [2-(arylamino)-4,4-dimethyl-6-oxo-cyclohex-1-ene] carbodithioates, as potential immunomodulatory and immunosuppressive agents

Article abstract of DOI:10.1016/j.bmc.2012.03.003

The immunomodulating properties of functionalized [2-(arylamino)-4,4- dimethyl-6-oxo-cyclohex-1-ene] carbodithioates and 6,6-dimethyl-4-(2-(propan-2- ylidene)hydrazinyl)-6,7-dihydro-2H-indazole-3(5H)-thione compounds have been investigated. Four of them,

Full text of DOI:10.1016/j.bmc.2012.03.003

Bioorganic & Medicinal Chemistry 20 (2012) 3000–3008
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation, and molecular docking studies of benzyl, alkyl
and glycosyl [2-(arylamino)-4,4-dimethyl-6-oxo-cyclohex-1-ene]carbodithio-
ates, as potential immunomodulatory and immunosuppressive agents
El Sayed H. El Ashry a,b, , Mohammad R. Amer a, Omer M. Abdalla c, Aly A. Aly d, Samreen Soomro c,
⇑
Almas Jabeen c, Sobia Ahsan Halim c, M. Ahmed Mesaik c, Zaheer Ul-Haq c
a H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
b Chemistry Department, Faculty of Science, Alexandria University, Alexandria, Egypt
c Dr.Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
d Chemistry Department, Faculty of Science, Benha University, Benha, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The immunomodulating properties of functionalized [2-(arylamino)-4,4-dimethyl-6-oxo-cyclohex-
1-ene] carbodithioates and 6,6-dimethyl-4-(2-(propan-2-ylidene)hydrazinyl)-6,7-dihydro-2H-indazole-
3(5H)-thione compounds have been investigated. Four of them, 13, 18, 19 and 20 inhibited PBMC
proliferation induced by phytohemagglutinin (PHA) in a dose dependent manner with an IC50 of
Received 6 January 2012
Revised 28 February 2012
Accepted 1 March 2012
Available online 15 March 2012
620
cells (PBMCs) is significantly inhibited by 13, 19 and 20 with an IC50 of 8.4 0.4, 5.34 0.15 and
4.9 0.7 M, respectively. They also inhibited the PMA/lipopolysaccharide-induced proinflammatory
cytokines, IL-1b and TNF- production in human monocytic leukemia cells (THP-1), by 86%, 46% and
59.2% for IL-1b and by 83.8%, 48.2% and 58.7% for TNF- , respectively. Only 20 showed significant
suppressive activity against the phagocyte oxidative burst in a dose dependent manner, with an IC50 of
23.8 M. LPS-induced nitrites in mouse macrophages were found to be inhibited by compounds 6, 8,
13–15 and 19 with an IC50, which range between 7.7 and 63 M. The cytotoxicity for the active com-
lM. The Th-1 cytokine, interleukin-2 (IL-2) in PHA/PMA-stimulated peripheral blood mononuclear
Keywords:
Carbodithioate
Dimedone
l
a
a
Enamine
Indazolethione
Immunomodulatory
Immunosuppressive
Docking
l
l
pounds was also studied on Rat Wistar Hepatocyte cell line, CC1 and the Mouse Fibroblast cell line
3T3 NIH in the presence of compounds using a standard MTT assay. Furthermore, structural–activity rela-
tionship using automated docking software revealed that active compounds 7, 13 and 19, adapted the
same binding mode, however the most active compound 20 is found deeply inserted within the ligand
binding site of IL-2, as multiple hydrophobic and hydrophilic key interactions stabilize the compound
inside the binding site, thus contributing higher activity.
Glycosyl
S
O
O
R
S
N
NH
NH
NH
S
N
Y
Y
NH
X
X
20
2-7, 9-13, 15-19
1, 8, 14
Ó 2012 Elsevier Ltd. All rights reserved.
⇑
Corresponding author.
E-mail address: eelashry60@hotmail.com (El Sayed H. El Ashry).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.003

El Sayed H. El Ashry et al. / Bioorg. Med. Chem. 20 (2012) 3000–3008
3001
O
S
1. Introduction
O
R
S
The incorporation of dithioate functionality in a variety of com-
pounds led to structural characteristics which, in turn influence
their physical properties and their metabolic products. Thus wide
spectrums of biological and industrial applications have been re-
ported.1–6 This class of compounds has been demonstrated to pos-
sess insecticidal, antifungal, antibacterial, antiviral and antitumor
activity7–12 as well as inhibitory action against brassinin detoxifi-
cation by phytopathogenic fungus Leptosphaeria maculans (Phoma
lingam)13,14 potential apoptotic inducing character, and inhibition
of replication of rhinovirus, influenza virus and polio virus.10–12
Complexes of the dithioates exhibit higher antibacterial and anti-
fungal activity than free ligand.4 Moreover, the dithioates have
modulated the function of a number of key proteins involved in
apoptosis, oxidative stress, transcription, proteosome function15
and as antioxidants as well as immunosuppressive agents.16–18
However, few members of this class have been thoroughly investi-
gated for immunotoxicity.16,18,19 Thus, this class of dithioates
exhibits complex biological activities including the ability to po-
tently modulate or impair immune function.
CS2
/
DMSO / RX
Cl
NaOH
NH
NH
Cl
Cl
1
Cl
2-7
2: X= Br, R = -CH2(CH2)2CH3
3: X =Br, R = -CH2(CH2)9CH3
4: X =Br, R = -CH2C6H5
5: X =Br, R = -CH2CH2OH
OAc
O
AcO
AcO
6:
X= Br , R=
X= Br , R=
OAc
AcO
AcO
OAc
7:
O
OAc
O
S
O
R
S
CS2
/ NaOH
DMSO / RX
Based on the above considerations and as reported,20–24 we
have examined the effect of a group of enamine carbodithioate
NH
NH
and
hydrazinyl-6,7-dihydro-2H-indazole-3(5H)-thione
com-
pounds 1–20 on the pro-inflammatory cytokines; IL-1b and TNF
a
produced by activated macrophages through toll-like receptors
mediated pathogen recognition. These two cytokines are known
to induce the expression of many inflammatory genes in a syner-
gistic mode.25 It is also reported that IL-1b in synergy with IL-2 or
Cl
8
Cl
9-13
9: X= Br, R = -CH2(CH2)2CH3
10: X =Br, R = -CH2(CH2)9CH3
11: X =Br, R = -CH2C6H5
IL-12 in NK and T-cells induces the production of IFNc 26,27
We
.
12: X =Br, R = -CH2CH2OH
have also investigated their effect on the production of a short-
OAc
lived radical, nitric oxide, produced by the enzymatic oxidation
O
AcO
AcO
13:X= Br , R=
of L
-arginine and the action of nitric oxide synthase.28,29 Activated
OAc
macrophages produce large amounts of nitric oxide, which is con-
sidered an essential mediator for host defence28,29 as well as it is
involved in the pathogenesis of various types of diseases.30,31 On
the other hand, we have examined the effect of enamine carbod-
ithioates compounds on adaptive immune response through their
effect on the proliferation of T cells and production of IL-2 cyto-
kine. Th-cells secrete or stimulate the production of powerful im-
mune factors called cytokines, which are involved in various
cellular responses, such as activation, proliferation and growth.
However, when overproduced they can cause serious damage,
including inflammation and injury in the joints during the RA
process.32 Also abnormal Th1 immune responses have been impli-
cated in graft rejection and other autoimmune diseases.33,34 The
effect of compounds on the production of reactive oxygen species
by phagocytic cells has also been investigated. The nicotinamide
adenine dinucleotide phosphate (NADPH) oxidase, located in the
plasma membrane of these cells and in the membrane of specific
granules, produces superoxide anion from which other reactive
oxygen species (ROS) are derived. One quick and sensitive meth-
od of measuring the generation of these metabolites is chemilu-
minescence (CL), as described by Pavelkova & Kubala.35 Many
drugs have been developed over the last decades, including cyclo-
O
S
O
R
S
CS2
DMSO
/ NaOH
/ RX
NH
NH
Br
14
Br
15-19
15: X= Br, R = -CH2(CH2)2CH3
16: X =Br, R = -CH2(CH2)9CH3
17: X =Br, R = -CH2C6H5
18: X =Br, R = -CH2CH2OH
OAc
O
AcO
19:X= Br , R=
AcO
OAc
O
S
N
S
Ph
NH
20
S
N
NH
1- NH2NH2 , EtOH
2- acetone, reflux
NH
Cl
sporin A (CsA), rapamycin FK506, anti-IL-2R
a antibodies (anti-
Tac),36–39 in addition to corticosteroids that up-regulates the
expression of anti-inflammatory proteins, cyclophosphamides a
nitrogen mustard alkylating agent, and methotrexate which acts
by inhibiting the metabolism of folic acid. However, the clinical
use of these immunosuppressants is hampered by side effects,
and the major disadvantage being their toxicity.40 Therefore, we
studied the effects of compounds 1–2020,41 (Fig. 1) on different
component of the immune system in order to find a lead com-
pound as immunosuppressant.
Cl
4
Figure 1. Structures of the investigated compounds.
Moreover, docking is frequently used to predict the binding
orientation of a molecule to their protein targets which in turn

3002
El Sayed H. El Ashry et al. / Bioorg. Med. Chem. 20 (2012) 3000–3008
predict the affinity and activity of the small molecule. Moreover,
the binding interaction of all the compounds in the ligand
binding site of IL-2 was predicted by molecular docking
approach.
1.55 (m, 2H, CH2-20), 2.25 (s, 2H, CH2-3), 2.37 (s, 2H, CH2-5), 3.04
(t, 2H, J = 7.3 Hz, SCH2), 7.37–7.47 (m, 2H, ArH), 7.74–7.78 (m,
1H, ArH), 12.12 (s, 1H, NH). 13C NMR (DMSO-d6, 125 MHz) d:
13.8 (CH3-110), 21.9 (C-100), 27.3 (2CH3), 26.4, 28.4, 28.6, 28.7,
28.8, 28.9, 30.9 (8CH2), 31.2 (C-5), 35.9 (SCH2), 41.2 (C-4), 50.7
(C-6), 118.4 (C-1), 128.4, 129.4, 130.5, 131.5, 132.5, 133.9 (ArH),
162.7 (C-2), 191.2 (C@S), 222.9 (C@O). MS-EI: m/z (%) = 513 (4),
358 (100), 292 (26), 186 (5). HRMS-EI (M+): m/z: Calcd for
2. Experimental section
2.1. Chemistry
C26H37Cl2NOS2: 513.1694, found 513.1714.
Melting points were determined with a melt-temp apparatus
(SMP10) in open capillaries and are uncorrected. TLC was per-
formed on Merck silica gel 60 F254 with detection by UV light
absorption. 1H NMR spectra were recorded on Bruker Avance AV
NMR spectrometer at 300 or 400 MHz, whereas the 13C NMR was
done on the same instrument at 75 or 100 MHz, respectively, with
TMS as internal standard. Mass spectra were recorded on a
Finnigan (MAT312) and Jeol (JMS.600H), HRMS were recorded with
Thermo Finnegan (MAT 95XP). Solvents used were purified by
simple distillation.
2.1.4. Benzyl [2-(2,4-dichlorophenylamino)-4,4-dimethyl-6-
oxo-cyclohex-1-ene]carbodithioate (4)
Red crystals, yield (31%), mp 208 °C, TLC (n-hexane/EtOAc 6:4):
Rf 0.32, 1H NMR (CDCl3, 300 MHz) d: 1.01 (s, 6H, 2CH3), 2.33 (s, 2H,
CH2-3), 2.40 (s, 2H, CH2-5), 4.32 (s, 2H, SCH2), 7.14–7.54 (m, 7H,
ArH), 7.55 (s, 1H, ArH), 15.41 (s, 1H, NH). 13C NMR (DMSO-d6,
100 MHz) d: 27.3 (2CH3), 30.8 (C-5), 41.3 (C-4), 41.5 (C-6), 50.8
(SCH2), 117.6 (C-2), 127.2, 128.4, 128.5, 129.5, 129.6, 130.4,
131.4, 133.0, 133.5, 135.3 (ArH), 165.8 (C-1), 192.1 (C@S), 219.2
(C@O). MS-EI: m/z (%) = 449 (2), 416 (2), 358 (23), 292 (24), 91
(74). HRMS-EI (M+): m/z: Calcd for C22H21Cl2NOS2: 449.0442, found
449.0401.
2.1.1. General procedure for preparation of the target
compounds 1–19
A cooled solution of 3-(arylamino)-5,5-dimethyl-cyclohex-2-
en-1-one (0.01 mol) and sodium hydroxide (0.4 g, 0.01 mol) in
DMSO (20 mL) and water (1 mL), was treated with carbon disulfide
(0.03 mol). After 20 min, alkyl halide derivatives (0.012 mol) were
added and the reaction mixture was left for 24 h, then diluted with
water (100 mL) and acidified with 10% hydrochloric acid. The prod-
uct was purified on silica gel column chromatography (ethyl ace-
tate:n-hexane 3:7) and crystallized from methanol to give a
series of carboditioates.20,41 Some of the investigated compounds
have been reported in earlier publications. Thus, (20,30,40,60-tetra-
2.1.5. 2-Hydroxyethyl [2-(2,4-dichlorophenylamino)-4,4-
dimethyl-6-oxo-cyclohex-1-ene]carbodithioate (5)
Red crystals, yield (34.5%), mp 151 °C, TLC (MeOH/CHCl3 5:95):
Rf 0.61, 1H NMR (DMSO-d6, 300 MHz) d: 0.95 (s, 6H, 2 CH3), 2.27
(s, 2H, CH2-3), 2.36 (s, 2H, CH2-5), 3.21 (t, 2H, J = 6.4 Hz, SCH2),
3.62 (q, 2H, CH2O), 4.96 (t, 1H, J = 5.2 Hz, OH), 7.41–7.51 (m,
2H, ArH), 7.80 (m, 1H, ArH), 12.21 (s, 1H, NH). 13C NMR (CDCl3,
125 MHz) d: 27.9 (2CH3), 30.9 (C-5), 38.8 (C-4), 42.2 (C-6), 51.7
(SCH2), 59.5 (CH2O), 118.5 (C-2), 128.2, 129.1, 130.4, 132.2,
133.2, 134.0 (ArH), 166.7 (C-1), 193.1 (C@S), 219.3 (C@O). MS-
EI: m/z (%) = 403 (3), 359 (31), 326 (69), 282 (4), 219 (3), 185
(28). HRMS-EI (M+): m/z: Calcd for C17H19Cl2NO2S2: 403.0234,
found 403.0235.
O-acetyl-b-
dimethyl-6-oxocyclohex-1-ene]carbodithioate (6),20 (20,30,40,60-tet-
ra-O-acetyl-b- -galactopyranosyl) [2-(2,4-dichlorophenylamino)-
D-glucopyranosyl) [2-(2,4-dichlorophenylamino)-4,4-
D
4,4-dimethyl-6-oxocyclohex-1-ene]carbodithioate (7), n-butyl
[2-(4-chlorophenylamino)-4,4-dimethyl-6-oxocyclohex-1-ene]-
carbodithioate (9),20 (20,30,40,60-tetra-O-acetyl-b-
D-glucopyranosyl)
2.1.6. n-Undecanyl [2-(4-chlorophenylamino)-4,4-dimethyl-6-
oxo-cyclohex-1-ene]carbodithioate (10)
[2-(4-chlorophenylamino)-4,4-dimethyl-6-oxocyclohex-1-ene]car-
bodithioate (13),20 n-butyl [2-(4-bromophenylamino)-4,4-di-
Yellow crystals, yield (48%), mp 95 °C, TLC (n-hexane/EtOAc
1
7:3): Rf 0.79, H NMR (CDCl3, 300 MHz) d: 0.84 (t, 3H, CH3-110),
methyl-6-oxocyclohex-1-ene]carbodithioate
(15),20
(20,30,40,
0.95 (s, 6H, 2CH3), 1.23 (s, 16H, 8CH2), 1.56–1.59 (m, 2H, CH2-20),
2.27 (s, 2H, CH2-3), 2.56 (s, 2H, CH2-5), 3.06 (t, 2H, J = 7.4 Hz,
SCH2), 7.37–7.26 (d, 2H, ArH), 7.48 (d, 2H, ArH), 12.94 (s, 1H,
NH). MS-EI: m/z (%) = 479 (5), 384 (6), 324 (100), 292 (73), 260
(7), 236 (9), 188 (11), 55 (75). HRMS-EI (M+): m/z: Calcd for
60-tetra-O-acetyl-b-
D-glucopyranosyl) [2-(4-bromophenylamino)-
4,4-dimethyl-6-oxocyclohex-1-ene]carbodithioate (19)20 were
prepared.
2.1.2. n-Butyl [2-(2,4-dichlorophenylamino)-4,4-dimethyl-6-
oxo-cyclohex-1-ene]carbodithioate (2)
C26H38ClNOS2: 479.2083, found 479.2090.
Yellow crystals, yield (34.5%), mp 128 °C, TLC (n-hexane/EtOAc
8:2): Rf 0.41, 1H NMR (CDCl3, 400 MHz) d: 0.93 (t, 3H, J = 7.2 Hz,
CH3-40), 1.01 (s, 6H, 2CH3), 1.45–1.56 (m, 2H, CH2-30), 1.67–1.75
(m, 2H, CH2-20), 2.31 (s, 2H, CH2-3), 2.40 (s, 2H, CH2-5), 3.14 (t,
2H, J = 7.6 Hz, SCH2), 7.51 (d, 1H, J = 8.4 Hz, ArH), 7.31 (m, 1H,
ArH), 7.51 (d, 1H, ArH), 14.73 (s, 1H, NH). 13C NMR (DMSO-d6,
125 MHz) d: 13.5 (CH3-40), 21.9 (CH2-30), 27.3 (2CH3), 28.5 (CH2-
20), 30.9 (C-5), 35.7 (SCH2), 41.2 (C-4), 50.8 (C-6), 118.4 (C-2),
128.1, 129.4, 130.5, 131.5, 132.5, 133.9 (ArH), 162.7 (C-1), 191.3
(C@S), 223.0 (C@O). MS-EI: m/z (%) = 415 (12), 358 (100), 326
(12), 291 (20), 186 (6), 145 (6). HRMS-EI (M+): m/z: Calcd for
C19H23Cl2NOS2: 415.0598, found 415.0556.
2.1.7. Benzyl [2-(4-chlorophenylamino)-4,4-dimethyl-6-oxo-
cyclohex-1-ene]carbodithioate (11)
Reddish crystals, yield (49%), mp 211 °C, TLC (n-hexane/EtOAc
7:3): Rf 0.79, 1H NMR (CDCl3, 300 MHz) d: 1.06 (s, 6H, 2 CH3),
2.23 (s, 2H, CH2-3), 2.63 (s, 2H, CH2-5), 4.41 (s, 2H, SCH2), 5.99 (s,
1H, NH), 7.19-7.26 (m, 5H, ArH), 7.38 (d, 4H, ArH). MS-FAB: m/
z = 416 [M++1]. HRMS-ESI: m/z Calcd for C22H23ClNOS2 (M++H)
416.0909, found 416.0920.
2.1.8. 2-Hydroxyethyl [2-(4-chlorophenylamino)-4,4-dimethyl-
6-oxo-cyclohex-1-ene]carbodithioate (12)
Red crystals, yield (43%), mp 165 °C, TLC (MeOH/CHCl3 5:95): Rf
0.54, 1H NMR (DMSO-d6, 300 MHz) d: 0.94 (s, 6H, 2CH3), 2.27 (s,
2H, CH2-3), 2.56 (s, 2H, CH2-5), 3.22 (t, 2H, J = 6.5 Hz, SCH2), 3.63
(q, 2H, CH2O), 4.94 (t, 1H, J = 5.3 Hz, OH), 7.20 (d, 2H, ArH), 7.61
(d, 2H, ArH), 12.83 (s, 1H, NH). MS-EI: m/z (%) = 369 (79), 336 (2),
278 (11), 130 (100).
2.1.3. n-Undecanyl [2-(2,4-dichlorophenylamino)-4,4-dimethyl-
6-oxo-cyclohex-1-ene]carbodithioate (3)
Yellow crystals, yield (31%), mp 77 °C, TLC (n-hexane/EtOAc
6:4): Rf 0.51, 1H NMR (DMSO-d6, 300 MHz) d: 0.84 (t, 3H,
J = 6.6 Hz, CH3-110), 0.95 (s, 6H, 2CH3), 1.23 (br s, 16H, 8CH2),

El Sayed H. El Ashry et al. / Bioorg. Med. Chem. 20 (2012) 3000–3008
3003
2.1.9. n-Undecanyl [2-(4-bromophenylamino)-4,4-dimethyl-6-
oxo-cyclohex-1-ene]carbodithioate (16)
Scitech. DIV., Ashai Techno glass, Japan), 50
l
L of cell suspension
(2.5 ꢀ 106 cell/mL), 50
l
L of phytohemagglutinin (PHA) for a final
Yellow crystals, yield (38%), mp 137 °C, TLC (n-hexane/EtOAc
7:3): Rf 0.70, 1H NMR (DMSO-d6, 300 MHz) d: 0.81 (t, 3H, CH3-
110), 0.95 (s, 6H, 2CH3), 1.23 (s, 16H, 8CH2), 1.56–1.58 (m, 2H,
CH2-20), 2.27 (s, 2H, CH2-3), 2.56 (s, 2H, CH2-5), 3.04 (t, 2H,
J = 7.3 Hz, SCH2), 7.19 (d, 2H, ArH), 7.60 (d, 2H, ArH), 12.83 (s,
concentration of 5
l
g/mL, 50 L supplemented RPMI-1640 and
l
50
50
l
l
L of test compounds in a final concentration of 0.5, 5 and
g/mL (in triplicates) were added to the culture mixture. Plates
were then incubated at 37 °C in a humidified atmosphere of 5% CO2
in air for 72 h. To each well 0.5
l
Ci [methyl-3H]-thymidine (Amer-
13
1H, NH). C NMR (DMSO-d6, 100 MHz) d: 13.8 (CH3-110), 22.0
sham Place Little Chalfont, Buckinghamshire, UK) was added for
additional 18 h. Cells were harvested using a cell harvester (Ino-
tech, Dottikon, Switzerland), and incorporation was measured by
liquid scintillation counter (Beckman coulter, LS 6500, Fullerton,
CA, USA).
(C-100), 26.4, 27.3, 28.5, 28.6, 28.7, 28.8, 28.9 (7CH2), 30.9 (C-5),
31.2 (C-20), 35.9 (SCH2), 41.4 (C-4), 50.8 (C-6), 118.3 (C-2), 119.5,
127.7, 132.1, 136.4 (ArH), 163.1 (C-1), 191.4 (C@S), 221.4 (C@O).
MS-EI: m/z (%) = 523 (3), 370 (67), 304 (4), 97 (20), 55 (100).
HRMS-ESI: m/z Calcd for C26H39BrNOS2 (M++H) 524.1656, found
524.1645.
2.2.2. Interleukin-2 (IL-2) assay
The PBMNCs were cultured in a 96 well flat-bottomed plate
2.1.10. Benzyl [2-(4-bromophenylamino)-4,4-dimethyl-6-oxo-
cyclohex-1-ene]carbodithioate (17)
(1.0 ꢀ 105 cell/well) in the presence or absence of three concen-
trations of test compounds (0.5, 5, and 50 lg/mL), and phyto-
Yellow crystals, yield (40%), mp 175 °C, TLC (n-hexane/EtOAc
7:3): Rf 0.72, 1H NMR (DMSO-d6, 300 MHz) d: 0.92 (s, 6H, 2CH3),
2.30 (s, 2H, CH2-3), 2.62 (s, 2H, CH2-5), 4.26 (s, 2H, SCH2),
7.26-7.38 (m, 7H, ArH), 7.65 (d, 2H, ArH), 14.18 (s, 1H, NH).
MS-EI: m/z (%) = 459 (2), 370 (96), 336 (30), 245 (6), 91 (100).
HRMS-EI (M+): m/z: Calcd for C22H22BrNOS2: 459.0326, found
459.0316.
hemagglutinin (PHA) Phorbol myristate acetate (PMA) in a
final concentration of 5 ng/mL and 20 ng/mL respectively. After
an incubation period of 18 h at 37 °C in a humidified atmo-
sphere of 5% CO2 in air, supernatant was collected for IL-2
determination. Interleukin-2 levels were measured by using en-
zyme-linked immunosorbent assay (ELISA) development kit
(R&D systems, Minneapolis, MN, USA). In brief the procedure
followed was: 96 well ELISA plates were coated with 4.0
lg/
2.1.11. 2-Hydroxyethyl [2-(4-bromophenylamino)-4,4-
dimethyl-6-oxo-cyclohex-1-ene]carbodithioate (18)
Yellow crystals, Yield (43.5%), mp 136 °C, TLC (MeOH/CHCl3
5:95) Rf 0.46, 1H NMR (DMSO-d6, 300 MHz) d: 0.94 (s, 6H, 2CH3),
2.27 (s, 2H, CH2-3), 2.56 (s, 2H, CH2-5), 3.22 (t, 2H, J = 6.4 Hz,
SCH2), 3.64 (q, 2H, CH2O), 4.94 (t, 1H, J = 5.0 Hz, OH), 7.27 (d, 2H,
ArH), 7.49 (d, 2H, ArH), 12.92 (s, 1H, NH). MS-FAB: m/z = 414
[M++1]. HRMS-EI (M+): m/z: Calcd for C17H20BrNO2S2: 413.0119,
found 413.0097.
mL mouse anti-human IL-2, in PBS, pH 7.4. Then, 100 L re-
l
combinant human IL-2 standards and culture supernatants sam-
ples were added. The plates were incubated for 2 h at room
temperature and washed three times with phosphate buffered
saline (PBS), followed by addition of 100
goat anti-human IL-2 and further incubated for 2 h at room
temperature. The plates were again washed and 100 L/well of
lL/well of biotinylated
l
streptavidin conjugated horse radish peroxidase was added and
incubated for additional 20 min at room temperature. After
three final washes, 100 lL/well of the enzyme substrate solution
2.1.12. 6,6-Dimethyl-4-(2-(propan-2-ylidene)hydrazinyl)-6,7-
dihydro-2H-indazole-3(5H)-thione (20)
of H2O2 and tetra methyl benzidine (1:1 v/v) was added and the
color allowed to develop at room temperature in the dark. Plates
were then read at 450 nm in a plate reader (DIAReader GMBH,
Wr. Neudorf, Austria). Results were analyzed using Microsoft
Excel software.
Dissolve (0.3 mmol) of benzyl [2-(2,4-dichlorophenylamino)-
4,4-dimethyl-6-oxocyclohex-1-ene]carbodithioate 4 in (5 mL) of
methanol, and hydrazine hydrate (0.5 mL). The mixture was
heated under reflux for 6 h., the product was dissolved in acetone
(10 mL), and refluxed for 0.5 h., and the product was recrystallized
from methanol. Yield (60%), yellow crystals, mp 262 °C; TLC (5:95
MeOH/CHCl3): Rf 0.61; 1H NMR (DMSO-d6, 300 MHz): d 1.03 (s, 6H,
2CH3), 2.12, 2.13 (2s, 6H, 2CH3), 2.46 (s, 2H, CH2-5), 2.91 (s, 2H,
CH2-7), 12.51(s, 1H, NH), 13.99 (s, 1H, NH); 13C NMR (DMSO-d6,
125 MHz): d 18.9, 25.1 (2CH3), 27.9 (2CH3), 35.7 (C-6), 37.7 (C-7),
39.0 (C-5), 107.1 (C-9), 153.2 (C@N), 162.4 (C-8), 163.2 (C-4),
164.6 (C@S); EIMS: m/z (%) = 250 (93), 209 (6), 194 (42), 178
(24), 152 (13), 138 (8), 108 (7), 82 (19), 56 (100); ESIMS: Calcd
for C12H19N4S: m/z 251.1330, found 251.1324.
2.2.3. TNF-a and IL-1b cytokines
Human monocytic leukemia cells (THP-1) were obtained from
European Collection of Cell Cultures, UK (ECACC) and grown in
RPMI-1640 supplemented with 10% FBS, 2 mmol/L
L-glutamine,
5.5 mmol/L glucose, 50 mol/L mercaptoethanol, 1 mmol/L sodium
l
pyruvate, and 10 mmol/L HEPES (MP Biomedicals lllkirch, France),
until they attained 70% confluency. Cells were then plated in 24-
well tissue culture plates at a concentration of 2.5 ꢀ 105 cells/mL
and were differentiated into macrophage like cells by using phor-
bol myristate acetate (PMA) (SERVA (Heidelberg, Germany)) at a fi-
nal concentration of 20 ng/mL and incubated for 24 h at 37 °C in 5%
CO2. Cells were then stimulated with LPS (bacterial lipopolysacch-
ride) purchased from DIFCO Laboratories (USA) at a final concen-
2.2. Biological activities
2.2.1. T-cell proliferation assay
Fresh venous blood from a healthy donor was mixed with equal
volume of incomplete RPMI-1640 media (Mediatech Inc., Herndon,
tration of 50 ng/mL and treated with compounds (25
incubated for 4 h at 37 °C in 5% CO2. The supernatant collected
after 4 h incubation was analyzed for TNF- and after 18 h incuba-
tion were analyzed for IL-1b level. Cytokines quantification in
supernatants were performed by human TNF- and IL-1b Duo set
lg/mL) and
a
VA, USA) containing 2 mM L-glutamine and 1% penicillin/strepto-
mycin. The diluted blood was then layered onto a lymphocyte sep-
aration medium (MP Biomedicals, Inc., OH, USA) and centrifuged at
400 g for 20 min at 25 °C. The mononuclear cell layer was col-
lected, washed with incomplete RPMI-1640 and centrifuged at
300 g for 10 min at 4 °C. The peripheral blood mononuclear cells
(PBMNCs) were resuspended in RPMI-1640 complete media con-
taining 10% fetal bovine serum from PAA laboratories GmbH Pas-
a
Kits (R&D systems, Minneapolis, USA) and according to manufac-
turers instructions.
2.2.4. Nitrite concentration in mouse macrophage culture
medium
The mouse macrophage cell line J774.2 (UK ECACC) was cul-
tured in T75 flasks in DMEM that contained 10% fetal bovine
ching, Austria. In
a 96 wells round-bottomed plate (IWAKI,

3004
El Sayed H. El Ashry et al. / Bioorg. Med. Chem. 20 (2012) 3000–3008
serum supplemented with streptomycin/penicillin 1%. Flasks
were kept at 37 °C in atmosphere of humidified air containing
5% CO2. Cells (106 cells/mL) were then, the seeded in a 24-well
plate and the nitric oxide synthase (NOS-2) in macrophages was
2.3. Molecular docking
All computational studies were performed on IntelÒ Xeon Quad
Core processor runing open SUSE 10.3 OS. The crystal structure of
the Interleukin-2 in complex with phenylalanine methyl ester
derivative, (PDB entry code 1M4844) was retrieved from the RCSB
Brookhaven Protein Data Bank45 and used for docking calculations.
All water molecules were removed from the original PDB crystal
structure. Hydrogen atoms were added to the protein using the
Biopolymer module in SYBYL 7.3 software package.46 The struc-
tures of the ligands were constructed on ChemDraw and converted
into 3D using babel 2.2. The 3D structures were imported into SYB-
YL 7.3 and optimized. Geometry optimizations were performed
using the Tripos force field47 with a distance-dependent dielectric
and the Powell conjugate gradient algorithm. Gasteiger–Huckel
charges were used for the ligands.
induced by 20
Laboratories, USA). The test compounds were added at a con-
centration of 25 g/mL soon after LPS stimulation and incubated
lg/mL Escherichia coli lipopolysaccharide (DIFCO
l
at 37 °C in 5% CO2. Cell culture supernatant was collected after
24 h. Nitrite accumulation in J774.2 cell culture supernatant
was measured using the Griess method,42 where 50
l
L of 1%
sulfanilamide in 2.5% phosphoric acid, followed by 50 L of
0.1% naphtyl-ethylene diamine dihydrochloride in 2.5% phos-
phoric acid were added to 50 of culture medium. After
l
l
L
10 min of incubation at 23 °C the absorbance at 550 nm was
read. Micromolar concentrations of nitrite were calculated from
standard curve constructed with sodium nitrite as a reference
compound. Results were expressed as means SD of duplicate
readings.
GOLD 3.048 docking suite was employed for docking studies.
GOLD Score and ChemScore were chosen as a fitness function
and the standard default settings were used in all calculations. De-
fault cut off values of 2.5 Å for H bonds and 4.0 Å for Van der Waal
interactions were employed. A 10.0 Å radius active site was drawn
on the original position of the co-crystallized ligand and automated
cavity detection was used. After docking thirty poses were saved
for each ligand.
2.2.5. Chemiluminescence assay
Release of the reactive oxygen species (ROS) from whole blood
phagocytes during the oxidative burst was measured by the
luminol-enhanced chemiluminescence assay procedure.35 In brief;
three concentrations of each compound (1.0, 10 and 100
lg/mL)
were prepared in 25
l
L of Hank’s balanced salt solution (HBSS++
)
with calcium chloride and magnesium sulfate in half area 96 well
white flat-bottomed plate (Sigma Aldrich, Germany) with a final
3. Results and discussion
3.1. Synthesis
well volume of 100 lL. Then 25 lL of whole blood diluted 1:50
in suspension of Hanks balance salt solution (HBSS++) (Sigma, St.
Louis, USA) was added. Positive and negative controls and blank
wells were included. Cells and compounds were incubated for
The enaminones 1, 8 and 14 were prepared by reaction of dime-
done with the respective aromatic amines.21 Their reaction with
electrophilic reagent as carbon disulfide in presence of sodium
hydroxide was investigated, in earlier publications, to give the car-
bodithioate whose alkylation or glycosylation gave the respective
30 min at 37 °C, then 25
(Research Organics Cleveland, OH, USA), was added into each well
and 25 L serum opsonized zymosan (Saccharomyces cerevisiae ori-
lL luminol [3-aminophthalhydrazide]
l
esters.20,41 Thus, (20,30,40,60-tetra-O-acetyl-b-
D
-glucopyranosyl) [2-
(arylamino)-4,4-dimethyl-6-oxocyclohex-1-ene] carbodithioates
and (20,30,40,60-tetra-O-acetyl-b-
-galactopyranosyl) [2-(arylami-
gin) purchased from (Fluka, Buchs, Switzerland) was added except
for negative and blank wells. The ROS chemiluminescence kinetic
was monitored with luminometer from Labsystems Luminoskan,
(Helsinki, Finland) for 50 min in the repeated scan mode. Peak
and total integral chemiluminescence reading were expressed in
the relative light unit.
D
no)-4,4-dimethyl-6-oxocyclohex-1-ene]carbodithioates were pre-
pared. The reaction was extended in the present work to include
a variety of alkyl or glycosyl derivatives, 2–7, 9–13 and 15–19.
The structures can be readily confirmed by studying their 1H
NMR spectra. Thus, a signal at the low field region at d 12.12–
15.41 ppm confirmed the presence of the NH group in the respec-
tive compounds. The 13C NMR spectra of the carbodithioates
showed signals at dc 191.2–193.1 ppm which confirm the presence
of C@S in their structures. The X-ray analysis has confirmed the
structures of some representatives.20,41
2.2.6. Cytotoxicity assessment using MTT assay and two cell-
lines
In vitro cytotoxicity assays were performed as described by
Scholz et al.,43 using the 3T3-L1 mouse embryo fibroblast cell line
(American Type Culture Collection ‘ATCC’, Manassas, VA 20108,
USA), and CC-1 cells, a Rat Wistar Hepatocyte cell line (European
Collection of Cell Cultures, Salisbury, UK). The CC-1 cells were sus-
pended in Minimum Essential Medium Eagle (MEM) supplemented
with 10% FBS, 2 mM glutamine, 1% non-essential amino acids and,
20 mM HEPES. While the 3T3-L1 cells were suspended in
Dulbecco’s Modified Eagle’s Medium (DMEM) formulated with
10% FBS. Using flat bottomed plates, both cell-lines were plated
at a concentration of 6 ꢀ 104 cells/mL and incubated for 24 h at
37 °C and 5% CO2 environment. After removal of media, cells were
challenged with three different concentrations (1.0, 5.0, and
The hydrazone 20 was prepared by reaction of benzyl [2-(2,4-
dichlorophenylamino)-4,4-dimethyl-6-oxocyclohex-1-ene]carbod-
ithioate 4 with hydrazine hydrate followed by reaction of the
product with acetone.
3.2. Biological activity
The inflammation process is a complex of cellular and biochem-
ical events for the elimination of infectious agents. However,
uncontrolled or repeated inflammation contributes to tissues dam-
age eventually results in chronic inflammatory disorders. In the
current work, we investigated in vitro the effects of series of enam-
ine carbodithioates on the generation of ROS, reactive nitrogen
species (RNS) and several cytokines in activated immune cells.
Among 20 different compounds three compounds 13, 19 and 20
were found to suppress the proliferation of T-cell, and the produc-
20 lg/mL) of compounds in triplicates and were then further
incubated for 48 h at 37 °C in CO2 incubator. Following exposure
to each compound, cells viability was assessed by using 0.5 mg/mL
of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) for 4 h followed by removal of supernatant and addition
of DMSO to solubilize the formazan complex. Plates were read at
540 nm after one minute shaking and readings were processed
using MS Excel software. Results were expressed as means SD
of triplicate readings.
tion of IL-2, TNF-a, IL-1b and NO. This implies that these com-
pounds may non-specifically suppress the immune system either

El Sayed H. El Ashry et al. / Bioorg. Med. Chem. 20 (2012) 3000–3008
3005
by forming aggregates with signaling proteins or with the activa-
tors. The potential of compound 20 could be due to the presence
of an additional –NH group on 7th carbon. In addition to this we
have observed that ROS was selectively inhibited by compound
20. This inhibition could be either at the cell receptor level or
due to interference with enzymes involved in the ROS generation.
Moreover, these compounds also down regulated the production of
PHA mitogen-induced cell proliferation. Compounds 13, 18, 19
and 20 were found to have a dose dependent inhibition on the
proliferation of human PBMC stimulated by PHA with an IC50 of
120
13
100
80
60
TNF-a and IL-1b which are the key inflammatory cytokines pro-
18
19
20
duced after stimulation of monocytes/macrophage. Beside the
aforementioned compounds we also found that compounds 6, 8
and 14 effectively decreased NO production in LPS stimulated
J774.2 mouse macrophage cell line. Nitric oxide (NO) mediates a
variety of physiological and pathological processes including
inflammatory reaction. The increased production of NO is harmful
to the host, leading to chronic inflammation condition or autoim-
mune diseases. Therefore NO inhibition by these compounds could
have therapeutic implication in inflammatory diseases. These com-
pounds possibly work through inhibition or competition with the
enzyme inducible nitric oxide synthase (iNOS), as it is known that
LPS is used to trigger these pathways of NO production in macro-
phages.28 We have also used the same model in our investigation.
The activity of these compounds may be based on the presence of
oxygen on the additional benzene ring. To validate this result, we
examined the cytotoxic potential of the potent compound on two
cell lines, Rat Wistar Hepatocyte cell line CC1 and Mouse Fibroblast
cell line 3T3, and the tested compounds were found to be non-
toxic. Moreover structure–activity relationship by docking re-
vealed that, the most active compound 20 is located at the hydro-
philic groove of IL-2 ligand binding site and is involved in multiple
hydrogen bonding, hydrophobic interactions and hydrophilic
interactions that stabilize the compound inside binding site. In
contrast, although compound 7, 13 and 19 are located inside the
binding site, due to the lack of interaction with neighboring amino
acids their activity is lower compared to compound 20.
40
20
0
0.5 µg/mL
5 µg/mL
50 µg/mL
Figure 2. Dose–response curves for 13, 18, 19 and 20 on lymphocyte proliferation.
PBMC were stimulated with PHA in the presence of increasing concentrations of the
compounds. Cell proliferation was evaluated as indicated under Materials and
Methods. Results shown are the mean SD of triplicate measurement expressed as
percentage of stimulated control cells in the absence of compounds.
120
13
100
19
80
20
60
40
20
0
3.2.1. Dose–response effect on cell proliferation
To determine the immunosuppressive activity of enamine
dithioate compounds, we first determined their capacity to inhibit
1.0 µg/mL
5.0 µg/mL
20 µg/mL
Figure 3. Dose–response curves for IL-2 production. PHA/PMA-stimulated PBMCs
were incubated with three different concentrations of 13, 19 and 20. After 18 h the
supernatant was used for the estimation of IL-2. Data are mean SD expressed as
percentage of stimulated control cells in the absence of compounds.
Table 1
Effect of enamine dithioate compounds on T-cell proliferation, IL-2 production and on
the proliferation of 3T3-L1 and CC-1 cell-lines
Table 2
Compound
No.
Mean SD of IC50 in lg/mL
Effect of enamine dithioates (25
produced by macrophages
lg/mL) on proinflammatory cytokines (IL-1b, TNF-a)
T-cell
IL-2
Cytotoxic effect
3T3-L1 cell-line
proliferation
production
CC-1 cell-line
Compound No.
% Inhibition
1
2
3
4
17.6 0.4
>50
>50
>50
>50
>20
>20
>20
>20
—
—
—
—
—
—
—
—
IL-1b
38.5 8.8
TNF-a
1
2
3
6.3 5.9
ꢁ14.7 5.9
29.3 3.0
ꢁ22.0 10.7
5
>20
—
—
30.3 1.0
6
7
8
9
15.8 0.3
3.9 0.6
15.0 0.7
>50
>20
2.1 0.1
>20
—
>20
—
—
>20
—
—
4
5
6
7
ꢁ119.0 5.8
16.5 1.0
ꢁ50.3 8.9
ꢁ50.3 3.0
ꢁ48.2 17.8
83.8 3.0
ꢁ273.1 37.0
>20
—
86.0 1.9
10
11
12
13
14
15
16
17
18
19
20
>50
>50
21.0 0.6
9.9 0.3
>50
>50
>50
>50
>20
>20
>20
3.5 0.1
>20
>20
>20
>20
>20
—
—
—
>20
—
—
—
—
—
—
—
—
>20
—
—
—
—
8
9
17.2 5.8
ꢁ4.3 3.1
ꢁ92.9 11.7
ꢁ15.8 13.6
13.1 0.0
10.5 0.0
10
11
12
13
14
15
16
17
18
19
ꢁ56.6 0.0
ꢁ21.0 3.0
0.0 14.8
48.1 8.8
46.8 18.5
ꢁ26.8 3.9
ꢁ244.9 10.7
ꢁ22.7 1.9
ꢁ158.2 4.9
5.5 1.0
48.2 5.9
ꢁ31.4 35.6
ꢁ31.4 17.8
ꢁ58.7 3.0
ꢁ50.3 8.9
ꢁ33.5 14.8
58.7 8.9
8.3 1.0
8.8 1.3
13.2 1.7
—
3.4 0.5
14.7 1.4
>20
—
19.8 0.28
—
59.2 1.0
Results are presented as means SD of triplicate measurements for IC50 of test
compounds in each experiment. — = not determined.
Results are expressed as means SD of three determinations.

3006
El Sayed H. El Ashry et al. / Bioorg. Med. Chem. 20 (2012) 3000–3008
15.6 2.4, 15.1 0.5, 20 2.4 and 12.6 1.4
l
M, respectively
were found to inhibit the IL-2 production in a dose dependent
manner with IC50 values of 8.4 0.4, 5.34 0.15, >48.4 and
4.86 0.72 lM, respectively (Fig. 3 and Table 1). The false cytotoxic
effect of these compounds was excluded by measuring the prolifer-
ation of two cell lines using standard MTT assay. No growth inhibi-
tion of the mouse embryo fibroblast cell line 3T3-L1, and a Rat
(Table 1 and Fig. 2). The rest of the compounds did not show signi-
ficant suppressive activity. As the IL-2 cytokine is essential for
T-cell proliferation, we studied the anti-proliferative effect of 13,
18, 19 and 20 on production of IL-2 by T-cells. These 4 compounds
Wistar Hepatocyte cell line CC-1 could be observed up to 20
mL concentration tested as highest dose (Table 1).
lg/
Table 3
Effect of enamine dithioate compounds on phagocyte oxidative burst activity and
nitrite production by mouse macrophages
3.2.2. Effect on proinflammatory cytokines
Compound No.
IC50
(
l
g/mL) for
% of NO inhibited by
25 g/mL test compound
We also observed anti-proliferative action on monocytes, and
we determined the influence of the enamine thiocarbazate com-
pounds on proinflammatory cytokines induced by LPS (Table 2).
For this purpose THP-1 cells were differentiated into macrophage
like cells by the action of phorbol myristate acetate (PMA) and fur-
ther stimulated with LPS in the presence or absence of test com-
pounds. The supernatants collected after 4 h incubation were
phagocytes ROS
l
1
2
3
4
5
6
7
8
>100
>100
>100
>100
>100
>100
5.94 0.07
>100
>100
>100
>100
>100
90
>100
>100
>100
>100
>100
>100
42.07
35.17
39.68
39.51
44.81
57.18
66.82
62.43
23.61
16.48
10.43
43.33
77.31
62.71
18.92
32.10
38.53
36.97
68.41
analyzed for the level of TNF-
bation were analyzed for IL-1b level by using ELISA technique.
When used in a concentration of 25 g/mL, 13, 19 and 20 reduced
the induction of TNF- cytokine by 83.8%, 48.2% and 58.7%, respec-
a and that collected after 18 h incu-
9
l
10
11
12
13
14
15
16
17
18
19
a
tively and reduced the production of IL-1 b by 86%, 46.8% and 59.2%
(Table 2).
3.2.3. Effect on the macrophage oxidative burst ROS and nitrites
Phagocytes play an essential role in inflammation processes.
The effects of the enamine dithioate compounds on functional
characteristics of human phagocytic cells have been studied and
their reactive oxygen species production through a chemilumines-
cence assay, where certain molecules can enhance the light
ROS produced by human blood phagocytes and NO produced by mouse macro-
phages. Results are expressed as means SD of three determinations.
Figure 4. The docked poses of active IL-2 inhibitors: (a) 20 (pink), (b) 19 (cyan), (c) 13 (olive) and (d) 7 (purple). The interacting residues are presented in yellow stick.

El Sayed H. El Ashry et al. / Bioorg. Med. Chem. 20 (2012) 3000–3008
3007
Figure 5. The binding mode of inactive compounds (a) 14 (purple), 8 (yellow), and 1 (blue) (b) 2 (blue), 15 (yellow), 9 (light-green), (c) 11 (yellow), 17 (pink) and 4 (blue). The
GOLD predicted binding mode shows that all inactive compounds are surface exposed.
production to a detectable level by luminometer were determined.
However, the compound 20 showed significant suppressive activ-
ity against the phagocyte oxidative burst response and ROS pro-
duction in a dose dependent manner as shown in Table 3. The
the N
e
and Ng2 of Arg38, respectively. Moreover para substituted
–cation interaction with the side
benzene moiety is stabilized via
p
chain of Arg38. The carbonyl of the acetyl moiety at the C6 of Glu-
cose molecule is engaged in hydrogen bonding with the amide
nitrogen on Lys43 at a distance of 1.68 Å. The binding pattern of
19 in the ligand binding site of IL-2 is presented in Figure 4b.
Compound 13 adopted a similar orientation to 19. The disulfide
IC50 for this compound was found to be 23.8 0.3
rest of the compounds showed no activity up to a concentration
of 400 M. Moreover, effect of these compounds on nitrite accu-
lM, while the
l
mulation by stimulated macrophages was also monitored. In this
assay the LPS treated J774.2 mouse macrophage cell line has been
used to study the mechanisms of nitric oxide synthase (NOS-2)
induction. We studied the effect of enamine thiocarbazate com-
pounds on nitric oxide production, out of 20 different enamines,
only 6 compounds showed the nitric oxide inhibition activity with
more than 50% inhibition at 25 lg/mL concentration (Table 1). Fur-
thermore, the IC50 of these compounds were found to be as
49.1 17.7, 16.1 1.9, 63.8 4.8, 7.7 0.9, 44.3 0.3 and
group of the compound is hydrogen bonded to the side chain Ne
and Ng2 of Arg38 with the distance of 2.83 and 1.98 Å, respec-
tively, as observed in 19 (Fig. 4b). The acetyl moieties substituted
on glucose molecule are surface exposed and not involved in inter-
action. The lack of interaction of these acetyl moieties with the ac-
tive site amino acid residues is the reason for decreased activity of
this compound. The docked orientation of 13 is shown in Figure 4c.
The docked pose of compound 7 revealed that the compound is
located at the same position of 13 and 19. The disulfide group of
36.4 1.6
l
M for compounds 14, 6, 8, 19, 13 and 20, respectively.
the compound is hydrogen bonded to the side chain Ne of Arg38
with the distance of 2.25 Å, while the hydrogen bond interaction
with the Ng2 of Arg38 is lost. Similar to compound 7, 13 and 19
also do not seem to interact with Lys43. Figure 4d reveals the bind-
ing mode of compound 7. The binding mode analysis of compound
7 reveals that although compound is located in the ligand binding
site but its acetyl substituted glucose moiety is completely surface
exposed and the disulfide moiety is oriented towards the inside of
3.3. Molecular docking
Molecular docking is widely used in the prediction of binding
mode and inhibitory mechanism of small ligands with their target
protein.49 To rationalize the structure activity relationship and to
investigate the inhibitory mechanism of Enamine carbodithioate
derivatives, molecular docking was efficiently realized using the
automated GOLD48 docking software. Enamine thiocarbazate
derivatives were docked into the ligand binding site of IL-245 and
their interactions and binding patterns were observed. The ob-
served binding mode of active compounds, 6, 7, 13 and 19 and is
shown in Figure 4.
Table 4
The docking scores of enamine dithioate derivatives
Compound No.
T-cell (IC50
g/mL)
,
IL-2 (IC50
mean SD)
,
ChemScore
GOLD
score
l
The most active compound 20 is located at the hydrophilic
groove of IL-2 ligand binding site and involved in multiple hydro-
gen bonding with the Tyr45 and Glu62, while the side chains of
Phe48, Phe44 and Pro65 offers hydrophobic interaction to the com-
pound. The NH moiety adjacent to the thiol group formed hydro-
1
2
3
4
5
6
7
8
17.6 0.4
>20
>20
>20
>20
>20
>20
2.1 0.1
>20
>20
>20
>20
>20
3.5 0.1
>20
>20
>20
>20
>20
30.29
25.85
22.06
23.02
25.30
33.63
39.51
34.09
20.88
22.98
20.79
28.65
34.38
27.56
24.88
18.90
16.77
36.07
35.36
32.26
78.66
68.66
53.08
58.90
56.30
80.01
86.81
80.56
53.89
53.60
53.23
74.22
81.19
52.01
53.78
53.60
51.99
81.22
81.08
68.81
15.8 0.3
3.9 0.6
15.0 0.7
gen bond with the side chain O
of 2.70 and 1.49 Å, respectively. While di-methyl moiety of com-
pound 20 is stabilized by -CH3 interaction provided by the side
e1 and Oe2 of Glu62 at a distance
9
p
10
11
12
13
14
15
16
17
18
19
20
chain phenyl moieties of Phe42 and Phe44. The compound is fur-
ther stabilized by the hydrophobic interaction provided by Pro65.
The best docked conformation of compound 20 predicted by GOLD
showed that the compound is deeply inserted within the ligand
binding site of IL-2 and multiple hydrophilic and hydrophobic
key interactions stabilize the compound inside the binding site,
contributing to the higher activity of this compound. The observed
binding mode of 20 is represented in Figure 4a.
21.0 0.6
9.9 0.3
—
8.3 1.0
8.8 1.3
3.4 0.5
14.7 1.4
The observed binding pattern of compound 19 reveal that the
disulfide moiety is hydrogen bonded to the side chain N
e and
Docking was performed using GOLD docking program. GOLD Score and ChemScore
are summarized.
N
g
2 of Arg38. The hydrogen bond distance was 2.23 and 3.0 Å with

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El Sayed H. El Ashry et al. / Bioorg. Med. Chem. 20 (2012) 3000–3008
10. Si, X.; McManus, B. M.; Zhang, J.; Yuan, J.; Cheung, C.; Esfandiarei, M.; Suarez,
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the binding cavity. Hence the absence of hydrogen bonding with
Arg38 caused the loss of activity of this compound (Fig. 4d).
To explain the inactivity of compounds 1–5, 8–12, 14–18, all
the inactive compounds were docked in the ligand binding cavity
of IL-2 by GOLD. From the docking pose, it is evident that all the
inactive compounds were shown to poorly occupy the ligand bind-
ing site of IL-2. The carbonyl moiety of 1, 8 and 14 is oriented away
from Arg38 and the di-methyl substituted six-member carbon ring
is completely surface exposed. The docked poses are shown in
Figure 5a. Similarly, the carbonyl and disulfide moieties of com-
pounds 2, 3, 9, 10, 15 and 16 are tilted away from Arg38, while
the alkyl chain at sulfide moiety is solvent exposed. The observed
binding mode of compounds 2, 9 and 15 are shown in Figure 5b.
The carbonyl moiety of 4, 11 and 17 is shown to be located away
from Arg38 while the disulfide moiety is tilted towards the
Arg38, but due to high distance, hydrogen bonding ability of the
sulfone moiety is lost (Fig. 5c). Consequently, lack of interactions
of those inactive compounds with key residues Arg38, Phe42,
Lys43 and Phe44, might be responsible for the inactivity of these
compounds in the IL-2 inhibition assay (Table 4).
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The reaction of enaminones with carbon disulfide in presence of
sodium hydroxide and then glycosylation gave glycosyl carbodi-
thioates. The reaction was extended to include a variety of alkyl
derivatives. A series of alkylated derivatives were tested for their
effect on three immunomodulatory testing systems, T-cell prolifer-
ation, reactive oxygen and nitrogen species and cytokine studies.
Our results demonstrated the potential of these compounds as
anti-inflammatory agents by inhibiting the cellular immune re-
sponse. Eventually, this study will help to bring about a whole
new range of anti-inflammatory agents that effectively and selec-
tively can target the mediators of the over activated immune
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We thank the Higher Education Commission, Pakistan for the
valuable support of this research project (Project No.20-697/R&D/
06/38 and 20/1444/R&D/ 09/2196).
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