Synthetic indole Mannich bases: Their ability to modulate in vitro cellular immunity

Article abstract of DOI:10.1016/j.bioorg.2015.05.003

The synthetic indole Mannich bases 1-13 have been investigated for their ability to modulate immune responses measured in vitro. These activities were based on monitoring their affects on T-lymphocyte proliferation, reactive oxygen species (ROS), IL (inte

Full text of DOI:10.1016/j.bioorg.2015.05.003

Accepted Manuscript
Synthetic indole Mannich bases: Their ability to modulate in vitro cellular im-
munity
M. Ahmed Mesaik, Khalid Mohammed Khan, Fazal Rahim, Muhammad Taha,
Syed Moazzam Haider, Shahnaz Perveen, Ahmed Shukralla Khalid, Omer M.
Abdalla, Samreen Soomro, Wolfgang Voelter
PII:
S0045-2068(15)00041-3
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.05.003
YBIOO 1813
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
17 March 2015
Please cite this article as: M. Ahmed Mesaik, K.M. Khan, F. Rahim, M. Taha, S.M. Haider, S. Perveen, A.S. Khalid,
O.M. Abdalla, S. Soomro, W. Voelter, Synthetic indole Mannich bases: Their ability to modulate in vitro cellular
immunity, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.2015.05.003
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Synthetic indole Mannich bases: Their ability to modulate in vitro
cellular immunity
M. Ahmed Mesaik,a,b* Khalid Mohammed Khan,c∗, Fazal Rahim,d Muhammad Taha,e,f
Syed Moazzam Haider,c Shahnaz Perveen,g Ahmed Shukralla Khalid,a,h † Omer M.
Abdalla,a,h Samreen Soomro,a and Wolfgang Voelteri
aDr. Panjwani Center for Molecular Medicine and Drug Research, International Center
for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
bDrug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan
Malaysia, Kuala Lumpur, Malaysia
cH. E. J. Research Institute of Chemistry, International Center for Chemical and
Biological Sciences, University of Karachi, Karachi-75270, Pakistan
dDepartment of Chemistry, Hazara University, Mansehra, Khyber Pukhthunkhwa,
Pakistan
eAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA
(UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia
fFaculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia
gPCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui,
Karachi-75280, Pakistan
hnstitute of Radiobiology, Ministry of Science and Technology, P. O. Box 3001Khartoum,
Sudan
iInterfakultäres Institut für Biochemie, Universität Tübingen, Hoppe-Seyler-Str. 4, 72076
Tübingen, Germany
ABSTRACT:
The synthetic indole Mannich bases 1-13 have been investigated for their ability to
modulate immune responses measured in vitro. These activities were based on
monitoring their affects on T-lymphocyte proliferation, reactive oxygen
species (ROS), IL (interleukin)-2, IL-4, and nitric oxide production.
Compound 5 was found to be the most potent immunomodulator in this
context. Four of the synthesized compounds, 5, 11, 12, and 13, have significant potent
inhibitory effects on T-cell proliferation, IL-4, and nitric oxide production. However,
none of the thirteen indole compounds exerted any activity against ROS production.
Keywords: Indole Mannich base, ROS, Nitric Oxide, Cytokine Inhibition.
*Corresponding Authors: mmesaik@hotmail.com, Tel 0060392898055; hassaan2@super.net.pk;
khalid.khan@iccs.edu; Tel. 00922134824910; Fax. 00922134819018; † deceased in 2014
1

1. Introduction
The indole moiety is a repeating scaffold in the kingdom of natural products [1]. Several
medicinally-applied indole derivatives showed antitumor activity [2], and also can lead to
vessication and inflammation of human skin [3]. Several indoles derivatives like
thiopyrano-indoles have analgesic effects [4], various indole alkaloids like
aspidospermine and yohimbine possess wide range of bioactivity profiles [5]. The indole
compounds have 5-HT receptor activity as agonists [6] or antagonists [7].
Indoles derivatives have been referred to as fortunate structures since it have the ability to
bind with many receptors [8,9]. The indole derivatives substituted at 3-position are of
great intrest due to their various biological activities [10-13].
β- and γ-Carbolines, having indole moiety are the significant structural units for various
biologically important alkaloids [14,15]. They have potent anticancer, CNS [16] and
antitumor activity [17]. The ellipticine and olivacine are most important anticancer
agents.
The indole alkaloids of the Ergot family are of great importance [18], the most valuable
representative of this family is lysergic acid [19-21]. Nevertheless, other members of this
family have also played important roles in natural product chemistry. Some of these
alkaloids such as setoclavine [22] and rugulovasines showed potent dopamine agonist
activity and reiterates the importance of the indole moiety in natural products [23].
T-cell proliferation and oxidative burst activities are two indicators for compounds
influencing cellular and innate immune response. IL-4 is an important
immunomodulatory cytokine expressed by a highly restricted pattern of cells of distinct
lineages located in diverse locations in the body such as circulating T-cells. IL-4 plays an
essential role in protective immunity as well as in inflammatory diseases, making IL-4 an
attractive target for inhibition to protect the body from inflammatory disorders. NO is a
free radical molecule which is produced in large quantities during inflammatory reaction
and due to its cytotoxic properties to normal cells; its signaling is involved in many
physiological and pathological processes [24-31].
2

Numerous classes of compounds have been reported as immunomodulators. Fukahori et.al.
reported recently the effect of AS2521780, a potent PKCθ inhibitor, on T cell-mediated
immunity. It also suppressed CD3/CD28-induced Interleukin-2 gene transcription in
Jurkat T cells and proliferation of human primary T cells [32]. Acanthocheilonema viteae
product ES-62 a small synthetic molecule have been reported to prevents development of
collagen-induced arthritis [33]. The diterpenoids polyandric acid A (1), has been checked
for inhibition of pro-inflammatory cytokine production and other inflammatory mediators
and was found to be significantly inhibited interleukin-1β production [34]. New
sesquiterpenoids, and monoterpenoid were isolated from the rhizomes of Curcuma
wenyujin and found to be strongly inhibited the induction of NO production by LPS [35].
In the context to design and discover new immunomodulators, thirteen Mannich bases
were examined for their effects on T-cell proliferation, IL-2 and IL-4 production of
peripheral blood mononuclear cells (PBMCs), reactive oxygen species (ROS) production
on whole blood phagocytes, and nitric oxide production of mouse macrophage cell
line J774.2.
2. Results and Discussion
2.1.Chemistry
In order to extend our research: on bioactive compounds [36]. Herein we are going to
report synthesis of indole Mannich bases as fellow. Indole (3 mmol), formaldehyde (1.2
mmol), secondary amine (3 mmol), and distilled water (15 ml) were added to a reaction
flask and the solution was refluxed for 2 h with vigorous stirring. Completion of the
reaction was periodically monitored by TLC. In most cases the resulting solid mixture
was collected on a filter. If no precipitate formed, the Mannich bases were extracted with
ethyl acetate, purified by column chromatography using a mixture of acetone/hexane
(1:1, v/v) as eluent, yielding pure products 1-13 in yields ranging from 75-89% (Scheme-
1) [37].
Insert Scheme-1 Here
2.2.Bioactivities
3

2.2.1. Effect of compounds on PHA-activated T-cell proliferation
T-cells undergo a clonal proliferation, when stimulated in vitro by mitogens such as
phytohemagglutinin (PHA-P), concanavalin A (Con A) or pokeweed mitogen (PWM).
3
The level of proliferation is determined by adding radioactive H thymidine to the cells
merging into the freshly synthesized DNA of the dividing cells. The amount of
radioactivity as determined in a scintillation counter is related to the number of newly
produced cells. In the current study thirteen synthetic Mannich bases were screened for
their effect on cellular immune responses. T-Cell proliferation was initiated using the
PHA-P mitogen to activate the PBMCs, isolated from healthy human volunteers. Initial
screening was performed using a single dose (10 µg/mL) of each compound, and those
that inhibited cell proliferation by 50% or more were further analyzed for their IC50
activity (Table-2). Out of thirteen compounds only four, 5, 11, 12, and 13 were found to
inhibit T-cell proliferation with significant inhibitory activities with IC50 value of 1.6, 2.0,
1.6, and 3.5 µg/mL, respectively (Table-1, Figure-1).
Insert Table-1 Here
Insert Figure-1 Here
2.2.2. Effect of compounds on Nitrite production:
Nitrous oxide (NO) is a free radical molecule produced in large quantities during
inflammation and due to its cytotoxic properties to normal cells its signaling is involved
in many physiological and pathological processes [38]. Therefore, our indole Mannich
bases were investigated for their potential inhibitory effect on nitrite generation using
stimulated murine macrophages cells (J774.2). In this study, LPS-treated J774.2 cells
were used for the nitrite production via induction of nitric oxide synthase (NOS). Among
the studied compounds 1, 9, and 11 had moderate inhibitory activity with a percentage
inhibition from 40% to 60%, whereas, compounds 5, 12, and 13 showed highly
significant (P ≤ 0.005) inhibitory effects on nitrite production with a percentage of
inhibition > 90 % at a dose of 25 µg/mL (Table-2).
Insert Table-2 Here
4

2.2.3. Effect of Mannich Bases 5, 11, 12, and 13 on ROS production in human whole
blood
Phagocytes upon activation by zymosan or phorbol 12-myristate 13-acetate (PMA)
release reactive oxygen species (ROS) quantifiable by a luminol-enhanced
chemiluminescence assay [38]. Compounds 1-13 (Table-2) were screened for production
of ROS using whole blood and luminol for ROS detection. While compound 4 had a
moderate inhibitory activity (IC50 27.1 µg/mL), compounds 3, 11, and 12 showed effects
with IC50 values ranging between 41.9-47.8 µg/mL. The rest of the studied compounds
did not show any significant activity up to the highest concentration (100 µg/mL) tested
(Figure-2).
Insert Figure-2 Here
2.2.4. Effect of Mannich Bases 5, 11, 12, and 13 on IL-4 production of peripheral blood
mononuclear cells
Production of IL-4 which is of critical importance for Th2 cell activation and
differentiation, was also studied for the four Mannich bases having significant inhibitory
effect against T-cell proliferation, and, indeed, compounds 5, 12, and 13 suppressed IL-4
production significantly with IC50 values of 5.5, 6.3, and 8.4 µg/mL, respectively (Figure-
3).
Insert Figure-3 Here
2.2.5. Effect of compounds on IL-2 production:
To evaluate the effect of indole Mannich bases 5, 11, 12, and 13 (inhibiting T-cell
proliferation, see above) on the production of interleukin 2 (IL-2) by Jurkat T-cells, we
found that only two compounds, 5 and 12, have moderate IL-2 inhibitory activity with
IC50 values of 11.8 and 13 µg/mL (Figure-4).
Insert Figure-4 Here
2.2.6. Cytotoxicity of compounds 5, 11, 12, and13
5

Cytotoxicity of compounds 5, 11, 12, and13 was tested on the NIH 3T3 mouse
embryonic fibroblast cells at concentrations of 1, 5 and 25 µg/mL which showed viability
between 80-100 % at the highest concentration tested (25 µg/mL) except compound 12
with an IC50 value f 12.5 µg/mL.
2.2.7. Statistical analysis
All data are reported as mean SD of the mean and the student t-test was used to
determine the difference between test- and control preparations significance was
attributed to probability values P 6 0.05. The IC50 values were calculated using Excel
based program.
3. Experimental
NMR experiments were performed on Avance Bruker AM 300 MHz. CHN analysis was
done on a Carlo Erba Strumentazion-Mod-1106, Italy. Electron impact mass spectra (EI
MS) were recorded on a Finnigan MAT-311A, Germany. Thin layer chromatography was
accomplished on pre-coated silica gel aluminum plates (Kieselgel 60, 254, E.Merck,
Germany). Chromatograms were visualized by UV at 254 and 365 nm. Ultraviolet (UV)
spectra were recorded on Perkin-Elmer Lambda-5 UV/VIS spectrometer in MeOH.
Infrared (IR) spectra were recorded on JASCO IR-A-302 spectrometer as KBr (disc).
3.1.T-Cell proliferation assay
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous
blood of healthy adult donor by Ficoll-Hypaque gradient centrifugation [39]. Cells were
proliferated following a method reported by Nielsen et al [40]. Briefly cells were cultured
at a concentration of 5 · 105/mL in a 96 well round bottom tissue culture plate (Nalge
Nunc. Inter.). Cells were stimulated with 1.25 µg/mL of phytohemagglutinin (Sigma
Co.,USA). Various concentrations of oxazolone compounds were added to give final
concentrations of 7.5, 16, 25 and 50 µg/mL each in triplicate. The plate was incubated for
72 h at 37 °C in 5% CO2 incubator. After 72 h, cells were pulsed with (0.5 µCi/well)
6

tritiated thymidine, and further incubated for 18 h. Cells were harvested onto a glass fiber
filter (Cambridge Technology, USA) using cell harvester (SKATRON A.S. Flow Lab.
Norway). The tritiated thymidine incorporation into the cells was measured by a liquid
scintillation counter (1211 LKB WALLAC). CPM results were recorded after 120 s.
3.2.Cytokine production from mononuclear cells
Cytokine from mononuclear cells was assayed using the human cytokine kits (Diaclone,
Besancon Cedex,France). Briefly, freshly prepared mononuclear cells (105/well) were
cultured in 96-well microtiter plate in the presence or absence of 1.25 g/mL PHA. Four
different concentrations (0.5, 1, 4 and 16 µg/mL) of each compound along with PHA
were used in this assay. Plate was incubated at 37 oC in CO2 environment. After 18 h of
incubation, the supernatant was collected and analyzed for IL-2, IL-4 and IFN-γ cytokine
production.
3.3.Cytotoxicity evaluation
The MTT test was performed to evaluate the cytotoxic effect of the compounds according
to the following method described previously [41]. Briefly MTT was dissolved in 1 ·
PBS at 2 mg/mL. Mavin Darby bovine kidney (MDBK) adherent cell (2 · 105) were
incubated with serial concentrations of compounds (0.195–50 lg/mL) for three/five days.
Supernatant was removed and 50 lL of MTT solution was then added. Plates were
o
incubated for an additional 2-3 h at 37 C in a tissue culture incubator. MTT was
aspirated off and 200 lL of DMSO was added to dissolve the formazan crystal
formed by living cells. The plate was agitated at room temperature for 15 min then read
at 540 nm using microplate reader.
3.4.Procedure
Indole (3 mmol), formaldehyde (1.2 mmol), and secondary amine (3 mmol), ml were
mixed in reaction flask and then add distill water 10-15 and refluxed the solution for 2 h
with vigorous stirring. The reaction completion was checked by TLC, and the resulting
solid mixture was then filter. In some cases solid mixture was not formed so it was
extracted with ethyl acetate, purified by column chromatography using acetone and
hexane system in 1:1 ratio, and pure product is obtained in 75-89 % yield [37].
7

3.4.1. 3-((4-phenylpiperazin-1-yl)methyl)-1H-indole (1)
3.4.2. 3-((4-phenylpiperazin-1-yl)methyl)-1H-indole-5-carbonitrile (2)
3.4.3. 3-((4-methylpiperazin-1-yl)methyl)-1H-indole-5-carbonitrile (3)
3.4.4. 5-bromo-3-((4-ethylpiperazin-1-yl)methyl)-1H-indole (4)
Representative compound UV, IR, CHN, EI-MS and 1H-NMR data
3.4.5. 2-(1-{4-[(5-Bromo-1H-indol-3-yl)methyl]-1-piperazinyl}vinyl)-2,4-
cyclopentadien-1-ol (5)
Rf: 0.51 (ethyl acetate/hexane, 3:7); UV (MeOH) λmax 283 (log ε = 4.2) nm; IR (KBr):
νmax 3062, 2922, 1603 cm-1; 1H-NMR (300 MHz, CDCl3): δ 10.31 (s, 1H, NH), 7.90
(d, 1H, J4,6 = 1.8 H-4), 7.64 (dd, 1H, J4',2' = 0.6, J4',3' = 2.4 Hz, H-4'), 7.37 (d, 1H, J7,6
=
8.7 Hz, H-7), 7.32 (d, 1H, J2,CH2 = 2.1, H-2), 7.21 (dd, J6,4 = 2.1, J6,7 = 8.7 Hz, H-6),
6.91 (dd, 1H, J2',4' = 0.6, J2',3' = 3.3 Hz, H-2'), 6.53 (q, 1H, J3',2',5'=1.8 Hz, H-4'), 3.70 (s,
6H, Aryl-CH2/ H-3'',5''), 2.49 (br.s, H-2'',6''); MS: m/z (rel. abund. %), 387 (M+, 30), 193
(93), 138 (62), 95 (100). Anal. calcd. For C18H18BrN3O2 (387.06); C, 55.68; H, 4.67;
Br, 20.58; N, 10.82; O, 8.24 Found C, C, 55.67; H, 4.65; Br, 20.56; N, 10.80; O, 8.23
3.4.6. 3-((4-(furan-2-carbonyl)piperazin-1-yl)methyl)-1H-indole-5-carbonitrile (6)
3.4.7. 3-((4-(4-methoxyphenyl)piperazin-1-yl)methyl)-1H-indole-5-carbonitrile (7)
3.4.8. 3-((4-acetylpiperazin-1-yl)methyl)-1H-indole-5-carbonitrile (8)
3.4.9. (4-((1H-indol-3-yl)methyl)piperazin-1-yl)(furan-2-yl)methanone (9
3.4.10. 5-bromo-3-((4-phenylpiperazin-1-yl)methyl)-1H-indole) (10)
3.4.11. 3-((4-(4-methoxyphenyl)piperazin-1-yl)methyl)-1H-indole (11)
3.4.12. 5-methoxy-3-((4-(2-methoxyphenyl)piperazin-1-yl)methyl)-1H-indole (12)
3.4.13. ethyl 4-((5-methoxy-1H-indol-3-yl)methyl)piperazine-1-carboxylate (13)
4.0. Conclusion
The presented studies show that four of the new indole Mannich bases (5, 11, 12, and 13)
have significant potent inhibitory effects on T-cell proliferation, IL-4 and nitric oxide
production. Compounds 5 and 12 exert moderate inhibitory effects against IL-2
production, while none of the thirteen indole compounds showed activity against ROS
8

production. Therefore, these active compounds need to be further investigated for the
development of anti-inflammatory lead compounds. The new immunoactive Mannich
base may serve as lead structures for the development of anti-inflammatory remedies
with increased specificity and less side effects.
Acknowledgements: The authors are thankful to the Higher Education Commission
(HEC) Pakistan, for their financial support for research project No. 20-2073.
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Kokkinopoulos, Planta Med. 64 (1998) 208–211.
Captions
Scheme-1: Synthesis of indole Mannich bases.
Table 1: Inhibition (%) and IC50 values of T-cell proliferation at 10 µg/mL, IC50 of IL-4 and
cytotoxicity results for indole Mannich bases. (-) sign means IC50 not done because inhibition
(%) of the compound is < 50.
Figure 1: Effect of indole Mannich basees 5, 11, 12, and 13 on the proliferation of
phytohemagglutinin (PHA)-activated T-cells. T-Cell proliferation was initiated using the PHA-P
12

mitogen to activate the PBMCs, compounds were added (50, 5, and 0.5 µg/mL) to check their
3
effect on proliferation. Level of proliferation was determined by adding radioactive H
thymidine. Each bar represents the mean value of triplicate reading SD.
Table-2: Effect of compounds 1-13 on nitrite production by mouse macrophages J774.2.
Macrophages were incubated in presence or absence of 25 µg/mL of compounds for 48 h.
The supernatant was collected to detect the presence of nitrite by Griess reagent. (NG-
monomethyl-L-arginine (LNMA) is a known inhibitor of NO.
Figure 2: Effect of indole Mannich bases 1-13 on oxidative burst, ROS production using
whole blood phagocytes. Zymosan activated whole blood cells were incubated in
presence or absence of compounds (100, 10, and 1 µg/mL) for 30 min. Luminol was
added at the end of reaction and relative light units were measured using
chemilluminiscence. Each bar represents the mean value of triplicate reading SD.
Figure 3: Effect of indole Mannich bases 5, 11, 12, and 13 on the production of IL-4 from
human peripheral blood mononuclear cells (PBMCs). PMA activated T cells were incubated with
compounds (25, 5, and 1 µg/mL) for 48 h. The supernatant was collected and analyzed for IL-4. +
C indicates PMA activated cells without compounds. - C indicates base line IL-4 level. Each bar
represents the mean value of triplicate reading SD.
Figure-4: Effect of indole Mannich bases 5, 11, 12, and 13 on the production of IL-2 by
Jurkat cells. PMA activated T cells were incubated with compounds (2, 5, and 0.5 µg/mL) for 48
h. The supernatant was collected to detect the IL-2 using R&D kit. +C indicates activated cells.
Each bar represents the mean value of triplicate reading SD.
13

Scheme-1:
R1
R1
H
N
H2O
O
N R2
HN
N
+
+
R2
N
H
H
H
N
H
R1 = CN, H, Br, OMe,
R2 = alkyl or Aryl
14

Table(s)
Table-1
Structures of Compounds
IL-4 inhibition ( mg/ mL)
% of T-cell Inhibition T-Cell
Compounds
IL-4 inhibition at
IC50
(10 µg/mL)
IC50 µg/mL
10 mg/mL
44.2
-
-
-
-
-
-
1
2
NC
N
N
11.2
18.4
12.5
N
H
NC
Br
N
N Me
-
-
-
-
-
-
3
4
N
H
CH3
N
N
N
H
1.6
-
5
6
98.4
93.9
5.0 ± 1.8
14.3
-
-
NC
NC
N
N
OMe
3.3
-
-
-
-
-
-
7
8
N
H
O
N
N
Me
17.9
N
H

O
N
N
30.9
-
-
-
9
O
N
H
Br
N
N
20.1
-
-
-
10
11
N
H
N
N
OMe
2.0
99.6
82.9
8.4 ± 3.8
N
H
MeO
MeO
MeO
N
N
1.6
3.5
12
13
96.4
83.0
86.6
64.9
6.3 ± 0.5
N
H
O
O
N
N
10.7 ± 3.2
N
H

Table-2:
Compound No.
% inhibition of NO
47.8
15.9
5.8
1
2
3
16.9
97.0
14.3
9.0
4
5
6
7
9.2
8
9
10
11
12
13
LNMA
47.6
11.6
68.3
96.9
92.2
64.5

Figure(s)
Figure-1:
5
0.5
50
35
30
25
20
15
10
*
**
*
**
5
**
**
**
11
**
0
5
12
13
+ C
- C
Figure-2:
400
10
1
100
350
300
250
200
150
100
50
*
*
*
*
*
*
*
*
*
*
*
0
1
2
3
4
5
6
7
8
9
10
11
12
13
+ C

Figure-3:
25
5
1
60
50
40
30
20
10
0
*
*
**
**
**
5
11
12
13
+ C
- C
Figure-4:
450
400
350
300
250
200
150
100
50
2
5.0
0.5
*
**
**
0
5
11
Fr-III-87
12
Fr-III-91
13
Fr-III-93
Fr-III-76
+ C

Synthetic indole Mannich bases: Their ability to modulate in vitro
cellular immunity
M. Ahmed Mesaik,a,b* Khalid Mohammed Khan,c∗, Fazal Rahim,d Muhammad Taha,e,f
Syed Moazzam Haider,c Shahnaz Perveen,g Ahmed Shukralla Khalid,a,h † Omer M.
Abdalla,a,h Samreen Soomro,a and Wolfgang Voelteri
aDr. Panjwani Center for Molecular Medicine and Drug Research, International Center
for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
bDrug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan
Malaysia, Kuala Lumpur, Malaysia
cH. E. J. Research Institute of Chemistry, International Center for Chemical and
Biological Sciences, University of Karachi, Karachi-75270, Pakistan
dDepartment of Chemistry, Hazara University, Mansehra, Khyber Pukhthunkhwa,
Pakistan
eAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA
(UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia
fFaculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia
gPCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui,
Karachi-75280, Pakistan
hnstitute of Radiobiology, Ministry of Science and Technology, P. O. Box 3001Khartoum,
Sudan
iInterfakultäres Institut für Biochemie, Universität Tübingen, Hoppe-Seyler-Str. 4, 72076
Tübingen, Germany
*Corresponding Authors: mmesaik@hotmail.com, Tel 0060392898055; hassaan2@super.net.pk;
khalid.khan@iccs.edu; Tel. 00922134824910; Fax. 00922134819018; † deceased in 2014
15

16

HIGHLIGHTS
• Indole Mannich base derivatives
• ROS
• Nitic Oxide
• Cytokine Inhibition
17