Molecular modeling and snake venom phospholipase A2 inhibition by phenolic compounds: Structure-activity relationship

Article abstract of DOI:10.1016/j.ejmech.2016.03.008

In our earlier study, we have reported that a phenolic compound 2-hydroxy-4-methoxybenzaldehyde from Janakia arayalpatra root extract was active against Viper and Cobra envenomations. Based on the structure of this natural product, libraries of synthetic structurally variant phenolic compounds were studied through molecular docking on the venom protein. To validate the activity of eight selected compounds, we have tested them in in vivo and in vitro models. The compound 21 (2-hydroxy-3-methoxy benzaldehyde), 22 (2-hydroxy-4-methoxybenzaldehyde) and 35 (2-hydroxy-3-methoxybenzylalcohol) were found to be active against venom-induced pathophysiological changes. The compounds 20, 15 and 35 displayed maximum anti-hemorrhagic, anti-lethal and PLA₂ inhibitory activity respectively. In terms of SAR, the presence of a formyl group in conjunction with a phenolic group was seen as a significant contributor towards increasing the antivenom activity. The above observations confirmed the anti-venom activity of the phenolic compounds which needs to be further investigated for the development of new anti-snake venom leads.

Full text of DOI:10.1016/j.ejmech.2016.03.008

Accepted Manuscript
Molecular modeling and snake venom phospholipase A inhibition by phenolic
2
compounds: structure-activity relationship
Md. Iqbal Alam, Mohammed A. Alam, Ozair Alam, Amit Nargotra, Subhash Chandra
Taneja, Surrinder Koul
PII:
S0223-5234(16)30182-9
10.1016/j.ejmech.2016.03.008
EJMECH 8433
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 January 2016
Revised Date: 1 March 2016
Accepted Date: 2 March 2016
Please cite this article as: M.I. Alam, M.A. Alam, O. Alam, A. Nargotra, S. Chandra Taneja, S.
Koul, Molecular modeling and snake venom phospholipase A inhibition by phenolic compounds:
2
structure-activity relationship, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.03.008.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Molecular modeling and snake venom phospholipase A2 inhibition
by phenolic compounds: structure-activity relationship
Md. Iqbal Alama, Mohammed A. Alamb, Ozair Alamc, Amit Nargotrad, Subhash Chandra
Tanejad, Surrinder Kould
Md. I. Alam*
Department of Physiology, Hamdard Institute of Medical Sciences & Research, Jamia Hamdard,
New Delhi – 110062, India
M. A. Alam
National Brain Research Center, Manesar, Gurgaon, India
O. Alam
Department of pharmaceutical chemistry, Jamia Hamdard, New Delhi-110062
Amit Nargotra, Subhash Chandra Taneja, Surrinder Koul
Indian Institute of Integrative Medicine, Canal Road, Jammu – 180001, India
Corresponding Author
*M.I. Alam
E-mail: iqbalasc@yahoo.com
Tel: +91-999 008 3747
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ABSTRACT
In our earlier study, we have reported that a phenolic compound 2-hydroxy-4-
methoxybenzaldehyde from Janakia arayalpatra root extract was active against Viper and Cobra
envenomations. Based on the structure of this natural product, libraries of synthetic structurally
variant phenolic compounds were studied through molecular docking on the venom protein. To
validate the activity of eight selected compounds, we have tested them in in vivo and in vitro
models.
The compound 21 (2-hydroxy-3-methoxy benzaldehyde), 22 (2-hydroxy-4-
methoxybenzaldehyde) and 35 (2-hydroxy-3-methoxybenzylalcohol) were found to be active
against venom-induced pathophysiological changes. The compounds 20, 15 and 35 displayed
maximum anti-hemorrhagic, anti-lethal and PLA2 inhibitory activity respectively. In terms of
SAR, the presence of a formyl group in conjunction with a phenolic group was seen as a
significant contributor towards increasing the antivenom activity. The above observations
confirmed the anti-venom activity of the phenolic compounds which needs to be further
investigated for the development of new anti-snake venom leads.
Key words: Antisnake venom activity; PLA2 inhibitors; Phenolic compounds; 2-hydroxy-3-
methoxybenzyl alcohol; SAR.
Abbreviations used: ASP, Aspartate; CDCl3, Deuterated chloroform; DMSO, Dimethyl
sulfoxide; FT-IR, Fourier transform-Infrared spectroscopy; GLY, Glycine; LD50, Lethal Dose 50
(dose required to kill 50 percent of a population of test animals); MCDP/S,Minimum
clotting
dose of plasma/serum; MDD, Minimum defibrinogenating dose; MHD, Minimum hemorrhagic
dose; MHz, Megahertz; NMR, Nuclear magnetic resonance; PLA2, Phospholipase A2; ppm, part
per million; SAR, Structure-activity relationship; TLC, Thin layer chromatography; TMS,
Tetramethylsilane.
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1. Introduction
Snake envenomation is one of the neglected problems of the world. Antiserum still remains
the only therapeutic agent available for snakebite treatment. In the remote and rural areas of
many developing countries, antiserum is generally not easily available. The antiserum
sometimes does not produce enough protection against venom induced hemorrhage, necrosis,
nephrotoxicity and hypersensitive reactions [1-3]. Further development of antiserum in animal is
time consuming, costly and requires strict cold chain conditions. To overcome these factors
search for natural/synthetic molecules as an alternate source of antidote is an urgent requirement.
The development of stable and effective venom neutralizing drug/antidote is being ventured
throughout the World. One of the strategies could be to develop a molecule from the natural/
synthetic sources to combat the effect of venom partially or completely which may help in
improving the chances of survival of snake bite victims. Venom neutralizing drugs may prove to
be more effective life saver [4]. Development of such molecules is vital to address the cases
when identification of the snake is difficult, specific treatment is unavailable and the health
centre is far away.
Many plants and plant products have been used in folk medicines as antidote against
snakebite [5-8]. Preliminary investigations have suggested that several classes of constituents
such as steroids, terpenoids, alkaloids and glycosides from plant origin are capable of
neutralizing snake venom induced pathophysiological changes [9-16]. More than 800 plant
species have been screened for the anti-snake venom activity [15], however, very few plant
extracts have shown significant protection against snake-venom and none of the pure isolates has
displayed equivalent activity [4,17,14,18,19]. Several workers have also reported natural as well
as synthetic compounds as active inhibitors of snake venom phospholipase A2 (PLA2) [20-22].
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Phenolic compounds such as phenolic acids (hydroxy benzoic acid or benzaldehydes, cinnamic
acid), flavonoids, isoflavonoids, coumarins, pterocarpans, phenolic glycosides and tocopherols
have been reported to display protection against variety of snake venoms [9,17,23]. In our
earlier study, a phenolic compound 2-hydroxy-4-methoxy-benzoic acid was shown to possess
anti-venom and antioxidant activity [4,17]. Through in silico approach we identified a natural
compound 2-hydroxy-4-methoxy-benzaldehyde (21) as snake venom PLA2 inhibitors [IOXL]
[24]. The natural product (21) was isolated from the root extracts of Janakia arayalpathra which
is used in folk medicine in Western India as an antidote of snake bite. It was also reported in the
literature as an immunomodulator [5,6] and potent inhibitor of tyrosinase, which is essentially
indicative of its binding capabilities to specific receptor proteins [25]. In the present study, we
examined several analogs of phenolic compounds against Viper and Cobra venom induced
pathophysiological changes which were found to be effective both in wet lab and in in silico
models.
2. Materials and Methods
2.1.Materials
Unless otherwise stated, all reagents for chemical synthesis were purchased from Sigma-
Aldrich and used without further purification. The solvents used in reactions were distilled and
dried before use. All the material either natural or synthetic was in pure form. The purity of the
natural as well as synthetic compounds was found to be more than 95%. All reactions were
monitored by TLC on 0.25 mm silica gel 60 F254 plates coated on Aluminum sheet (E. Merck).
13
1H NMR and C NMR spectra were recorded on Brucker Avance DPX-200 instrument at 200
MHz and 500 MHz respectively using CDCl3 as solvent with TMS as internal standard.
Chemical shift is expressed in δ (ppm) and coupling constant in Hertz.
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2.2.Chemistry
The melting point of the compounds are uncorrected. Reagents, chemicals and solvents were
purchased from Sigma Aldrich and E Merck. TLCs were run on 0.25 mm silica gel 60 F254 plates
(E. Merck) using UV light or Ceric ammonium sulfate solution for detection/visualization of the
spots. The purification of the products was carried out by column chromatography over silica
gel (60-120 mesh). NMR spectra were run on Bruker Avance DPX-200 at 200MHz, IR on
Bruker 270-30 FT-IR and Mass spectra on JEOL MSD 300. Elemental analysis was performed
on ElementarVario EL-III. Anisaldehyde, 2-hydroxy-3-methoxybenzaldehyde, 3-methoxy-4-
hydroxy-benzaldehyde, 3-hydroxy-4-methoxy-benzaldehyde, 1,2-dihydroxybenzene, 3,4-
dihydroxybenzaldehyde, 2-hydroxybenzaldehyde, 3-ethoxy-4-hydroxy-benzaldehyde were
purchased from Sigma-Aldrich.
2.2.1. Isolation and characterization of natural and synthetic compounds
The plant material of Janakia arayalpatra (root) was obtained from local market and was
identified in the Department of Taxonomy, IIIM, Jammu.
2.2.1.1. Isolation of 2-hydroxy–4-methoxybenzaldehyde
Crushed dry roots of Janakia arayalpatra (500g) was steam distilled for 6 h to get a pale
yellowish liquid (1.4 ml, 0.28%) which on cooling afforded a solid mp 40-41°C (Lit mp 40-42
°C). IR (KBr pellet): 3260, 3030, 2944, 1644, 1630, 1576, 1504, 144, 1366, 1336, 1296, 1222,
1
1204, 1164, 1136, 1020 and 798 cm-1. H NMR (200 MHz, CDCl3): δ 3.82 (3H, s, Ar-OMe),
6.30 (1H,d, J=2.2 Hz, Ar-H) 6.34 (1H, dd, J=8Hz & 2.2 Hz. Ar-H) 7.26(1H,d, J=8Hz, Ar-H),
9.72 (1H,s,-CHO), 11.33 (1H,s,OH). MS M+ m/z (%) 152(11), 137(33), 123(27), 121(34),
82(10), 53(24).
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2.2.1.2. Preparation of 2-hydroxy–4-ethoxybenzaldehyde
Anhydrous potassium carbonate (2g) and ethyl iodide (0.5 ml) were added to 2,4-dihydroxy
benzaldehyde (0.7g, 5mmol) acetone (30 ml) and the contents stirred for 12h at room
temperature. After the completion of the reaction as indicated by TLC, the mixture concentrated
in vacuo and re-dissolved in benzene (3×10 ml). The organic layer concentrated and the residue
chromatography on silica gel (60-120 mesh) using n-hexane: ethyl acetate mixture (46:4) as
eluent to give JA-2 a crystallized solid (0.75g, 45%) melting point 38-39 °C analyzed C9H10O3.
IR (KBr pellet): 3340, 3284, 1674, 1604, 1566, 1444, 1408, 1328, 1252, 1230, 1216, 1180, 1086,
1
1026, 916 and 846 cm-1. H NMR (CDCl3): δ1.45(3H,t, J=6.8Hz, CH3-CH2O), 4.17 (2H,q, J=
6.8 Hz, CH3-CH2O), 6.71 (1H,s, Ar-H),7.03 (1H,d, J=8.5Hz, Ar-H),7.41(1H,dd, J=8.5 Hz &
2.2Hz, Ar-H),9.8 (1H,s, Ar-CHO). MS M+ m/z (%) at 166(100), 149(5), 137(9), 109(14),
108(13), 80(40), 79(31), 63(44).
2.2.1.3. Preparation of 2-hydroxy–4-propyloxybenzaldehyde
To 2, 4-dihydroxy benzaldehyde (0.7g, 5mmol) in acetone (30 ml) was added anhydrous
potassium carbonate (2g) and n-propyl bromide (0.5 ml) and the contents stirred for 12 h at room
temperature. The contents concentrated in vacuo and re-dissolved in benzene (3×10 ml). The
organic layer concentrated and the residue chromatography on silica gel (60-120 mesh) using n-
hexane: ethyl acetate mixture (46:4) as eluent to give an oily compound (0.46g, 52%) analyzed
for C10H12O3. IR (KBr pellet): 3384, 2932, 1668, 1634, 1578, 1498, 1454, 1428, 1374, 1336,
1
1292, 1260, 1216, 1170, 1114, 998, 826 cm-1. H NMR (200 MHz,CDCl3): δ 1.0 (3H,t,J=7.4 Hz,
CH3-CH2 ),1.77-1.88 (2H,m,OCH2-CH2-CH3), 3.97 (2H,t, J=6.53Hz ,OCH2-CH2), 6.41 (1H,d,
J=2.02Hz, Ar-H),6.5 (1H,dd, J=8.6 & 2.0 Hz, Ar-H ) 7.41 (1H,d, J=8.6Hz, Ar-H ), 9.75
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(1H,s,CHO).MS M+ m/z (%) 180(100), 138(98), 120(16), 110(38), 109(15), 92(18), 82(12),
81(50), 69(35), 65(50).
2.2.1.4. Preparation of 2-hydroxy–4-butyloxybenzaldehyde
To 2, 4-dihydroxy benzaldehyde (0.7g, 5mmol) in acetone (30 ml) was added anhydrous
potassium carbonate (2g) and n-butyl bromide (0.5 ml) and the contents stirred for 12 h at room
temperature. The contents concentrated in vacuo and re-dissolved in benzene (3×10 ml). The
organic layer concentrated and the residue chromatographed on silica gel (60-120 mesh) using n-
hexane: ethyl acetate mixture (46:4) as eluent to give an oily compound (0.53g, 55%) analyzed
for C11H14O3. IR (KBr pellet): 3344, 2957, 1681, 1644, 1573, 1478, 1216, 1178, 1080, 914, 832
1
cm-1. H NMR (200 MHz,CDCl3) : δ 0.98 (3H,t, J=7.28 Hz, CH3-CH2) 1.43-150 (2H, m, CH2-
CH2), 1.71-1,85 (2H, m, OCH2-CH2-CH2), 4.0 (2H,t, J=6.46Hz, OCH2-CH2), 6.41
(1H,d,J=2.1Hz,Ar-H ), 6.65 (1H,dd, J=2.1 & 8.6 Hz, Ar-H), 7.42 (1H, d, J=8.6Hz, Ar-H), 9.7
(1H,s, CHO). MS M+ m/z (%), 194(52), 151(2.6), 139(16), 138(100), 137(98), 110(13), 92(5),
81(16), 69(11), 65(19).
2.2.1.5. Preparation of 2-hydroxy–4-heptyloxybenzaldehyde
To 2, 4-dihydroxy benzaldehyde (0.7g, 5mmol) in acetone (30 ml) was added anhydrous
potassium carbonate (2g) and n-heptyl bromide (0.5 ml) and the contents stirred for 12 h at room
temperature. The contents concentrated in vacuo and redissolved in benzene (3×10 ml). The
organic layer concentrated and the residue chromatography on silica gel (60-120 mesh) using n-
hexane: ethyl acetate mixture (46:4) as eluent to give an oily compound (0.58g, 50%) analyzed
for C14H20O3. IR (KBr pellet): 3330, 3240, 2955, 1677, 1634, 1573, 1322, 1244, 1180, 1084,
1
1026, 914, 846 cm-1. H NMR (200 MHz,CDCl3): δ0.81 (3H,t, J=7.3Hz, CH3-CH2), 1.21-1.34
(8H, m, 4×CH2), 1.70-1.84 (2H,m, OCH2-CH2),3.92 (2H,t, J=6.5Hz, OCH2-CH2 ),6.32 (1H,d,
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J=2.1Hz, Ar-H),6.55 (1H,dd, J=8.65 &2.1 Hz , Ar-H),7.33 (1H,d, J=8.65 Hz, Ar-H), 9.62 (1H,s,
CHO). MS M+ m/z (%) 236(37), 151(2), 139(100), 110(10), 95(2), 81(9), 69(12), 65(16).
2.2.1.6. Preparation of 4-allyloxy-2-hydroxybenzaldehyde
The title compound was prepared from 2,4-dihydroxy benzaldehyde (2.0g, 15mmol) and allyl
bromide (1.4ml, 17mmol) by the method as described for 2-hydroxy–4-ethoxybenzaldehyde to
furnish a semisolid (1.12g, 42%) analyzed for C10H10O3. IR (KBr Pellet) 2932, 1668, 1634,
1578, 1498, 1454, 1428, 1374, 1336, 1292, 1260, 1216, 1170, 1114, 998, 926cm-1. 1H NMR
(200 MHz,CDCl3): δ 4.13 (2H, d, J=6.0 Hz,-OCH2-CH=), 4.75-5.10 (2H, m,-CH=CH2), 5.22-
5.43 (1H, m, -CH=CH2), 6.03(1H, dd, J=8.5 & 2.0Hz, Ar-H), 6.83 (1H,dd,J=8.5& 2.0 Hz, Ar-
H),9.0 (1H, s, Ar-CHO).MS M+ m/z (%) 178(18), 163(8), 149(5), 137(9), 69(5), 54(8), 43(100).
2.2.1.7. Preparation of 2-hydroxy-3-methoxybenzaldehyde
The title compound was prepared from 2,3-dihydroxy benzaldehyde (1.0g, >7mmol) and
methyl iodide (0.5ml, 7mmol) by the method as described for 2-hydroxy–4-ethoxy-benzaldehyde
to furnish a semisolid (0.48g, 46%) analyzed for C8H8O3. IR (KBr Pellet): 3184, 2932, 1668,
1634, 1578, 1498, 1454, 1428, 1374, 1336, 1292, 1260, 1216, 1170, 1114, 998, 926cm-1. 1H
NMR (200MHz, CDCl3): δ3.87 (3H,s,OMe), 6.92(1H,dd,J=7.92 & 7.88Hz,Ar-H),7.08(1H,d,
J=7.92 Hz, Ar-H),7.14 (1H,d,J=7.83Hz,Ar-H),9.87(1H, s, Ar-CHO), 11.07(1H,s,OH). MS M+
m/z (%) 152(18), 137(8), 121(15), 81(8), 53(100).
2.2.1.8. Preparation of 2,4-dimethoxybenzaldehyde
The compound prepared from 2,4-dihydroxy-benzaldehyde (1.1g, 8mmol) and iodomethane
(1.9ml, 30mmol) in acetone (50ml) containing Potassium carbonate (3.5g). The contents
refluxed (2.5h), concentrated and diluted with water (200 ml), pH adjusted to 6.0 and then
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extracted with solvent ether (3×30ml). The organic layer washed with water (2×10ml) dried
over anhydrous sodium sulfate and concentrated to give 2, 4-dimethoxy benzaldehyde (1.25g,
94%) mp 70 °C (mp 71 °C) 1 analyzed for C9H10O3 [C 65.14 H 6.10 C9H10O3 requires C 65.04
H 6.06 %] IR (KBr pellet): 2984, 1672, 1602, 1582, 1502, 1466, 1408, 1264, 1214, 1110, 1012
1
and 814 cm-1. H NMR (200MHz, Acetone-d6): δ 3.96 (6H, s, 2×Ar-OMe) 6.4 (1H,d, J=8.5 Hz,
Ar-H) 6.5 (1H, s, Ar-H) 7.56 (1H,d, J=8.5 Hz, Ar-H) and 10.1(1H,s, Ar-H-CHO). MS M+ m/z
(%) at 166(100), 149(60), 135(31), 121(28), 106(27), 77(30) and 63(41).
2.2.1.9. Preparation of 1-nitro-3, 4-methylenedioxybenzene
To a solution of 1,3-benzodioxol (10mmol) in acetic acid (8ml) at -5 °C under stirring
conditions added nitrating mixture (1ml) prepared from nitric acid: sulfuric acid (4:1 eq.). The
reaction worked up by dilution with ice water and extraction with DCM, the organic layer
washed with brine solution and dried over CaCl2 and concentrated to give crude product which
on column chromatography over silica gel (60-120 mesh) and elution with hexane: ethyl acetate
(19:1) afforded a yellow compound (1.51g, 90%) mp 150 °C IR (KBr pellet): 2984, 1605, 1582,
1
1543, 1502, 1496, 1440, 1365, 1311, 1264, 1214, 1110, 1044 and 814cm-1. H NMR (200MHz,
CDCl3): δ 6.14 (2H,s, -OCH2O-), 6.77(1H,d, J=8.6Hz, Ar-H), 7.67 (1H,d, J=2.2Hz, Ar-H) and
7.92 (1H, dd, J=8.6 & 2.2 Hz, Ar-H). MS M+ m/z (%) at 167(100), 139(56), 123(36), 111(29),
93(44).
2.2.1.10. Preparation of 2,4-diacetoxybenzaldehyde
The title compound prepared from 2,4-dihydroxy benzaldehyde (2g, 15 mmol) acetic
anhydride (4 ml) and pyridine (0.8 ml) by stirring the contents at room temperature for 15 h. The
reaction worked up is dilution with ice cold water (50 ml), extraction with chloroform (3×20ml),
and washing of organic layer with 0.1M HCl solution followed by water. Drying of the organic
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layer over anhydrous sodium sulfate and crystallization of the product with n-hexane: ethyl
acetate (9:1) to afford 2,4-diacetoxybenzaldehyde (3g, 90%) mp 68 °C (Lit mp 69 °C). IR (KBr
1
pellet): 2960, 1770, 1692, 1660, 1610, 1448, 1428, 1372, 1318, 1188, 1144, 1000 cm-1. H NMR
(200MHz, CDCl3): δ 2.16 (6H,s,2×OCOCH3), 6.5 (2H,bs,Ar-H) 7.26 (1H,d, J=8.5Hz, Ar-H) and
9.4(1H, s, Ar-CHO). MS M+ m/z (%) 222(3), 180(18), 138(20), 81(4), 54(5), 45(100).
2.2.1.11. Preparation of 2,4-dimethoxybenzaldoxime
To a stirring ethanolic solution (20ml) of 2,4-dimethoxybenzaldehyde (2g, 12mmol) added
aqueous solution (3 ml) of hydroxylamine hydrochloride (1.6g, 20mmol) followed by aqueous
solution of sodium hydroxide (5M, 12ml) and stirred the contents for 90 minutes (TLC
monitored). The contents worked up by dilution with water , pH adjusted to 7, extracted with
ethyl acetate (4×25 ml) washed with water and concentrated after drying over sodium sulfate to
give a solid (2.05g, 94% yield ) mp 104 °C. (lit mp 106 °C) analyzed for C9H11NO3 (C 60.01 H
6.19 C9H11NO3 requires C 59.66 H 6.11 N 7.70%). IR (KBr pellet) 2944, 1610, 1504, 1466,
1414, 1270, 1206, 1112, 1026, 922 and 832 cm-1. 1H NMR (200MHz, CDCl3): δ 3.6
(6H,s,2xAr-OMe) 6.06(1H,d, J=8.5Hz, Ar-H) 6.03 (1H,s,Ar-H) 7.0(1H,d, J= 8.5 Hz Ar-H), 7.73
(1H,s,Ar-CH=N.OH). MS M+ m/z (%) 181(58), 163(53), 149(100), 134(36), 121(73), 77(45),
and 46(78).
2.2.1.12. Preparation of 2,4-dimethoxybenzonitrile
To 2,4-dimethoxybenzaldoxime (0.9g, 5mmol) was added acetic anhydride (2ml) and the
contents heated on an oil bath at 150 °C for 1h, the contents cooled and poured into ice-cold
water (100 ml) and the resulting solid filtered washed with water (3×10ml) and dried in vacuo to
give a colorless solid mp 90 °C (lit mp 91 °C), analyzed for C9H9NO2 (C 66.36 H 5.59 N 8.6%)
C9H9NO2 requires C 66.24 H 5.55 N 8.56). IR (KBr) 2948, 2220, 1604, 1502, 1476, 1438, 1422,
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1
1328, 1286, 1214, 1172 and 1006 cm-1. H NMR (200MHz, CDCl3): δ 3.6(6H,s, 2×Ar-OMe)
6.96 (1H,s,Ar-H) 6.03 (1H,d, J=8.5 Hz, Ar-H) 6.86 (1H,d, J=8.5Hz Ar-H). MS M+ (m/z)(%)
177(21), 162(100), 135(38), 115(24).
2.2.1.13. Preparation of 2,4-diacetoxybenzaldoxime
The title compound was prepared from 2,4-diacetoxy benzaldehyde (0.9g, 4mmol) and
hydroxylamine hydrochloride (0.6g, ~7mmol) and sodium hydroxide as for the procedure
described for the preparation of 2,4-dimethoxy benzaldoxime to give the benzaldoxime product
(058g, 60%). IR (KBr) 3376,1702,1626,1602,1522,1490,1448,1364,1258,1210,1168,1116 and
974 cm-1. 1H NMR (200MHz, CDCl3): δ 2.3 & 2.36(3H each, s, 2×OCOCH3) 7.13(1H,d, J=
8.5Hz, Ar-H) 7.2 (2H,s, Ar-H and CH=N.OH),7.7 (1H,d, J= 8.5 Hz, Ar-H). MS M+ (m/z) (%)
237(12), 220(8), 195(34), 145(42), 117(100), 100(82).
2.3.Pharmacology
2.3.1. Snake venom antiserum
The lyophilized snake venoms of Daboia russelii and Naja kaouthia were obtained from
Calcutta snake park, (Kolkata, India) and preserved in a desiccator at 4 °C for further use. It was
dissolved in 0.9% saline and centrifuged at 2000 rpm for 10 min. The supernatant was used as
°
venom and kept at 4 C until further use. Venom concentration was expressed in terms of dry
weight. Lyophilized polyvalent snake venom antiserum (as reference serum) was obtained from
Haffkine Institute Mumbai, Maharastra and antiserum (mg ml-1) was dissolved in 0.9% saline
prior to its use.
2.4. Animals
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Male Swiss albino mice (20±2 g) and Wistar rats (150-200 g) of both sexes and of same age
were used in this study after acclimatization to the laboratory condition. All mice/rats were
housed five/cages and fed standard laboratory diet and water ad libitum with 12-h dark/light
°
cycles at constant temperature of 25±2 C. The animals were allowed 1 week to adapt to their
environment. All animals were cared for and handled in accordance with the guideline of the
Insitutional Ethical Committee. All animal experiments were performed following ‘Principles of
laboratory animal care’ (NIH publication No. 85-23, revised in 1985) as well as Indian laws on
‘Protection of Animals’ under the provision of authorized investigators.
2.5. Sample Preparation
The test compound was dissolved in 5% DMSO-water and mixed with the venom solution.
2.5.1. Venom inhibiting activity
The pure compound (mg ml-1) was dissolved in 0.9% normal saline (warm) and mixed with
the venom solution. The following pharmacological experiments were performed.
2.5.1.1.Neutralization of lethal venom activity
The toxicity of snake venom was assessed by injection of different concentrations of venom
in 0.2 mL physiological saline into tail vein of mice [26] (Table 1). To assess the in vitro
antagonism, various doses of venom (1-15 LD50) were mixed with a fixed amount of compound.
The mixture incubated at 37 °C for 1h, and centrifuged at 2000 rpm for 10 min. The supernatant
was injected (i.v) in mice, ten mice per dose. The median lethal dose (LD50) was calculated 24 h
after injection of the venom-compound mixture. Lethal toxicity was also assessed by sub-
cutaneous (s.c) injection of various doses of venom. The neutralizing potency of each compound
was assessed by injecting (s.c) 1-5 LD50 of venom into group of 10 mice followed immediately
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afterwards by the fixed doses of compound (i.v). In time kinetics study, where D. russelii venom
was injected (MLD) followed by the compound (i.v) at 0 min, 30 min and 60 min into groups of
six mice and then assessed the mortality rate after 24 h venom injection.
2.5.1.2.Neutralization of venom hemorrhagic activity
The minimum hemorrhagic dose (MHD) of venom defined as the least amount of venom
which when injected intradermaly (i.d) into mice resulted in a hemorrhagic lesion of 10 mm
diameter 24 h later [26] (Table 1). Neutralization of the hemorrhagic activity was estimated by
mixing a fixed amount of compound (100 mmol) with different amounts of venom (1-20 MHD).
The compound-venom mixture was incubated at 37 °C for 1h, centrifuged at 2000 rpm for 10
min, and 0.1 mL of supernatant injected intradermally (i.d). The hemorrhagic lesion was
estimated after 24 h (in vitro). To assess the anti-hemorrhagic activity of venom in vivo, various
amount of venom (1-5 MHD) were injected (i.d) followed immediately by the compounds and
the hemorrhagic lesion measured after 24 h.
2.5.1.3.Neutralization of venom defibrinogenating activity
The minimum defibrinogenating dose (MDD) of venom (D. russelii) is defined as the
minimum amount of venom which when injected (i.v) into mice caused in coagulable blood 1h
later [26] (Table 1). Neutralization of this activity was estimated by mixing different amount of
venom (1-20 MDD) with fixed amounts of compound (100 mM), incubating at 37 °C for 1h, and
centrifugation. The supernatant was injected (i.v) into albino mice (20±2 g) as described above
(in vitro). For in vivo studies, the MDD of venom was injected (i.v) followed by the compound
(i.v) and the nature of the blood observed after 1h.
2.5.1.4.Neutralization of coagulant activity
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The minimum clotting dose of plasma (MCDP) was determined as described by Theakston
and Reid [26] (Table 1). Neutralization of this activity was estimated by mixing different
°
amount of venom, with a fixed amount of compound, incubating for 1h at 37 C. Different
concentration of incubate were added to the experimental tube in place of 0.1ml physiological
saline, and the clotting time was recorded.
2.5.1.5.Neutralization of serum inhibitory activity
To examine whether the decreased clotting of D. russelii venom on goat plasma was due to
venom inhibition in serum, goat serum was collected without anticoagulant, incubated in glass
tubes for 2h at 37 °C and then centrifuged at 2000 rpm for 15 min. Pooled sera were kept frozen
at –20 °C. Equal volumes of Tyrode – Hepes buffer (THB) (137 mM NaCl; 2.7 mM KCl; 12
mM NaHCO3; 0.42 mM NaH2PO4, 10 mM Hepes; pH 7.4) or goat serums were incubated with
venom solution of different concentrations for 5 min at 37 °C. Aliquots of 50 µl were then added
to 200 µl of goat plasma (maintained for 2 min at 37 °C) and the clotting time was recorded.
MCDS were then calculated (Table 1). Neutralization of serum inhibitory activity was estimated
by mixing different amount of venom with fixed amount of compound, incubating for 1h at 37
°C and centrifuged. Supernatant was then added to the experimental group and the clotting time
was observed.
2.5.1.6.Neutralization of phospholipase A2 (PLA2) activity
For PLA2 activity experiments, egg yolks (obtained from commercially available eggs), 0.5%
EDTA, 50 mM Tris-HCl buffer (pH 7.5), 2% sodium chloride, 1% calcium chloride and 0.9%
sodium chloride were prepared and used.
For carrying out the experiment, 2 ml of egg yolk suspension and 0.2 ml of test material
(venom only or venom-compound mix) were mixed in the test tube and incubated at 37 °C for 1
14

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h. After incubation the time required for coagulation of egg yolk was recorded by placing the
test tube containing test materials (venom or venom-compound mix) on the boiling water bath.
A blank was run with normal saline instead of test material. One unit enzyme activity was
defined as the amount of venom, which increased the coagulation time of the egg yolk control by
one minute [16].
Neutralization of the enzyme activity was estimated on different amount of compound mixed
with the different amount of viper venom. The venom-compound mixture was incubated at 37
°C for 1 h. Centrifuged at 2000 rpm for 10 minutes; supernatant was tested in a total of 0.2 ml
for the enzyme neutralization activity.
2.6. Molecular docking studies
Molecular modeling studies have been carried out using Glide (Grid-based Ligand Docking
with Energetics), software using Schrodinger Maestro 10.1, Glide, running on Linux 64
operating system. In this direction, we docked the ability of the compounds to inhibit the
enzymatic activity of venom PLA2 (PDB code: 1OXL). The molecular docking studies mainly
involve selection and preparation of appropriate protein, grid generation, ligand preparation
followed by docking and its analysis. The docking score, binding free energy and hydrogen
bonds interaction formed with the surrounding amino acids are used to predict their binding
affinities and proper alignment of these compounds at the active site of the PLA2 enzyme.
3. Results and Discussion
Phenolic compounds or polyphenols constitute one of the most numerous and ubiquitously
distributed group of plant secondary metabolites, with more than 8000 phenolic structures
currently known. The natural phenol ranges from simple molecules (phenolic acids, flavonoids)
to highly polymerized compounds (lignin, melanins, tannins). Based on our previous report,
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anti-snake venom activity of phenolic compound 2-hydroxy-4-methoxy benzaldehyde is one of
the major components of the snake venom PLA2 inhibitors [24]. In continuation to our previous
study, we envisaged to investigate the sensitivity response of Daboia russelii and Naja kaouthia
venoms towards the venom-neutralizing activity of simple phenols, mono-, di- and tri-substituted
phenolic compounds by in vivo/in vitro experiments. The neutralizing effect of lethal,
hemorrhagic, coagulant, anticoagulant and PLA2 inhibiting activities of Daboia russelii and
lethal activity neutralization against Naja kaouthia venoms were studied. The experiments were
carried out on four small libraries based on their structures. The first library comprised of simple
phenols and their methyl ethers (compounds 1-9, Fig. 1A) and the second library contained
phenols bearing formyl groups and formyl group substituted alkylated phenolic compounds
(compounds 10-16, Fig. 1B). The third library comprised of formyl group bearing dihydroxy
benzenes or their alkyloxy/acyl derivatives (compounds 17-32, Fig. 1C), and the fourth library
comprised of hydroxymethyl substituted phenols, phenols with aldoximes ornitrile groups
(compounds 33-37, Fig. 1D). The compounds 38 and 39 are known and reported as anti-snake
venom activity and are taken for comparison with our present study. All the four compound
libraries were screened for anti-venom activity.
Compounds 1-8 displayed anti-hemorrhagic activity with compound 2 found to be potent (5-
fold and 3 fold protection in in-vitro and in-vivo experiments) against Daboia russelii venom but
displayed no protection against lethal activity (Tables 2 and 3). Compounds 10-16 exhibited
anti-venom activities in experimental animal and 15 showed 11-fold protection against lethal, 3-
fold against hemorrhagic and 10-fold against defibrinogenating activity of Daboia russelii
venom. Notably, substituted alkylated phenols bearing formyl group displayed much higher
anti-lethal activity than formyl group substituted non-alkylated phenols (displaying higher
16

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hemorrhagic activity) in both the protocols, i.e. in animals with administration of venom and the
drug combination injections (Table 2) or venom injection immediately followed by drug
injection (Table 3). In in vivo experiments, the maximum protection was found in case of
compounds 15 (lethal) and 12 (hemorrhagic) in mice. While 3-formylated phenol 12 (3-
hydroxybenzaldehyde) showed maximum anti-hemorrhagic activity, 3-formylated anisole 15 (3-
methoxybenzaldehyde) displayed maximum anti-lethal activity in in vitro and in vivo study.
With respect to lethal activity, higher degree of protection was found against Daboia russelii
venom and lesser for Naja kaouthia venom (Tables 2 and 3). Among compounds (17-31), 17 (8-
fold), 18 (10-fold), 20 (12-fold), 21 (10-fold), 22 (10-fold), 25 (10-fold) and 28 (8-fold)
exhibited protection against viper venoms induced hemorrhagic activity and the lethal activity
showing maximum fold of protection in compounds 21, 22, 25, 30 and 35 (Table 2). Three fold
of protection was seen against Naja kaouthia venom-induced lethal activity neutralization by all
the compounds. In in vitro study, among phenolic compounds with formyl- and alkyloxy
substituent, maximum lethal protection of 11-fold against Viper, 3-fold against Cobra and 10-
fold against Viper venom-induced hemorrhagic activity was observed. For venom neutralization
activity, when the venom was administered (s.c) for lethal and intradermally (i.d) for
hemorrhagic activity followed by intravenous (i.v) injection of the phenolic molecule, the degree
of protection was dramatically reduced. In general, the degree of protection was always found to
be higher in case of Daboia russelii venom compared to Naja kaouthia venom both in in vitro
and in vivo experiments.
Substantial fall in the inhibitory activity profile of the compounds was observed in in vivo
studies when the venom was injected subcutaneously followed by the drug given by intravenous
route separately (Table 3). The compound 18 showed only 2 fold lethal protection and 1-fold
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hemorrhagic protections (in vivo) against 2-fold and 10-fold protection in in vitro studies. In
compounds of group-IV (Fig. 1D) where aldehydic group was replaced either by a primary
alcohol group or by an aldoximic or a nitrile group, the activity increased dramatically both in
vitro and in vivo (Tables 2 and 3). These changes in the functional group of the compounds
resulted in drastic change in its venom inhibitory upshot. It was indicative of the importance of
formyl and aldehydic groups in these compounds.
From the above observations, it became apparent that many of these molecules possess
potent antisnake venom activity and therefore, few of them were considered for further studies.
The short-listed compounds 18, 21, 22, 25, 28, 34, 35, and 36 were taken for viper venom
neutralization effect of lethal, hemorrhagic, coagulant, defibrinogenating and PLA activities. In
2
time kinetic experiments, when the viper and cobra venom was injected via s.c route followed by
the administration of drug by i.v route at 0 min, 30 min and 60 min (after venom injection),
significant lethal protection was observed at 0 min for majority of the test compounds. The
activity got reduced after 30 min and after 60 min only 20% protection of venoms was observed
in mice. In these experiments, mono-alkylated mono-hydroxy benzaldehyde and aldoximic
derivatives (21, 22, 25, 30, 35 and 36) exhibited protections at 0 min, 40-50% after 30 min and
10-30% after 60 min (18, 21, 22, 35 and 36). It was also observed that the degree of protection
against viper venom (D. russelii) was always higher than the cobra venom (N. kaouthia), having
no protection after 30 and 60 min of compounds introduction (Fig. 2). The selected compounds
effectively antagonized the Viper venom induced defibrinogenating activity. In in vitro studies,
the phenolic compounds (100 mmol) showed protection upto 10 fold of minimum
defibrinogenating dose (MDD) in mice. In in vivo studies, venom induced defibrinogenating
activity was antagonized by all the compounds except 18 and 34. The protection was always
higher in in vitro studies than in vivo (Table 4).
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The effects of Daboia russelii venom on coagulant activity and the neutralization effect of
the selected compounds was also studied by plasma recalcification time. The normal value
(0.9% saline + calcium-induced) of minimum clotting dose of plasma (MCDP) / serum (MCDS)
was found to be 25±0.17 sec and venom + calcium-induced was found to be 15±0.20 sec. In the
above experiments the selected molecules (20 mmol) were incubated with different amounts of
venom (1-100 µg) and tested for coagulant activity. The compounds antagonized the venom-
induced coagulant activity was neutralized effectively as evident by plasma recalcification time.
Compound 35 exhibited maximum protection (Fig. 3A). Phospholipase A activity of Daboia
2
russelii venom was assessed by egg yolk coagulation method. One unit of Daboia russelii
venom activity was found to be 2 µg which increased the coagulation time by one minute
(control 0.9% saline, coagulation time was found to be 45 ± 1.16 sec). The compounds 18, 21,
22, 25, 28, 34, 35, and 36 were incubated with different amounts of viper venom (2-100 ꢀg) and
tested for phospholipase A activity. The venom PLA was found to be effectively neutralizing
2
2
the compounds. The compound 35 (20 mmol) significantly neutralized the venom (60 ꢀg)
induced PLA activity. The degree of protection was found to be higher in compound 35 (Fig.
2
3B).
Molecular Docking
PLA enzyme is one of the major and most toxic proteins found in snake venom. In our
2
present study, we tried to search for PLA enzyme inhibitors that may be useful to design
2
antivenom compound(s) to treat snake envenomations. In order to suggest a mode of action of
the compounds, we performed molecular docking study. The docking scores and binding energy
of the said compounds and co-crystal ligand with the active site of PLA is summarized in Table
2
19

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S1 of Supplementary material. The crystal structure of PLA was prepared for docking with the
2
Protein Preparation Wizard workflow of Maestro that allows addition of hydrogen atoms which
were subsequently minimized with OPLS-2005 force field to optimize the protonation state. The
receptor grid was generated by applying a Van der Waals radii of non-polar atoms, which
decreases penalties for close contacts (scaling factor = 1.00 and partial charge cut off = 0.25).
Before docking calculation, all the compounds were subjected to ligand preparation with the
LigPrep tool. Finally, the ligand docking was done using receptor grid file and LigPrep out file
in the Glide tool of Application view. The docking results revealed that two amino acids ASP 49
and GLY 30 located in the binding pocket of enzyme played vital roles in designing the snake
anti-venom compound(s). Among all the titled compounds for PLA , compound 35 was found
2
to be most potent with a high docking score. The compound 35 assumes favorable orientation
within the binding site. The binding mode of compound 35 is exactly same as the co-crystal
ligand (IDA: 501). The docked pose (Fig. 4A) of the compound 35 showed hydrogen bond
interactions with amino acid ASP 49 (H....O=C, 1.90 Å, Acceptor angle 152.75, Donor angle
146.72) and GLY 30 (H.... O=C, 2.21 Å, Acceptor angle 150.38, Donor angle 111.11) and HIP
48 (O....HN, 2.07 Å, Donor angle 96.99). The CYS 45 (H....O=C, 2.73 Å, Acceptor angle
127.32, Donor angle 124.24) makes an additional hydrogen bonding whereas co-crystal ligand
IDA: 501 forms hydrogen bond with ASP 49 (H....O=C, 2.04 Å, Acceptor angle 142.50, Donor
angle 98.75) and GLY 30 (H.... O=C, 1.95 Å, Acceptor angle 155.65, Donor angle 178.22) and
HIP 48 (O....HN, 2.13 Å, Donor angle 113.42) shown in Fig. 4B. The hydrophobic enclosure
visualization pose of Compound 35 was shown in Fig. 4C. The aligned pose of all the
compounds were also shown in Fig. 4D. In addition, the enzyme surface model was showed in
Fig. 5, which revealed that the molecule 35 occupies the binding pocket of PLA2.
Binding Energy Estimation
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Binding energy estimation can be used to calculate ligand binding energies and ligand strain
energies for a set of ligand and a single receptor PLA2 using Prime molecular mechanics-
generalized born surface area (MM-GBSA) method. All the water molecules were deleted from
the designed complexes prior to dG binding free energy calculation using solvation model VSGB
by Prime MM-GBSA, Maestro 10.1. All the compounds are well fitted in the active sites of
PLA2. The binding poses for each compound were analyzed by examining their free energy
scores. The dG binding energy of compounds was found in the range of -26.587 to -78.857
Kcal/PLA2 (Table S1, Supplementary material). All the compounds displayed a good binding
free energy (dG bind) for PLA2 which indicates that compounds may have better selectivity and
affinity towards enzyme.
Structure-activity relationship (SAR)
Phenolic compounds are commonly found in both edible and inedible plants, and they are
known to have multiple biological effects including antioxidant, anti-inflammatory and cardio-
protective. Phenolic compounds are widely accepted and recommended for their antioxidant
properties as the chemical structure of phenolic compound imparts them the ability to serve as
free radical scavengers. A phenolic compound consists of a benzene ring and an alcoholic
hydroxyl group, which are the defining features of its acidic nature. In the present investigation
it became evident that the substituted phenolic analogs of low molecular weight have the
potential to neutralize snake venom and may also possesses potent antioxidants activity like,
aspirin, paracetamol, coramine et cetera [27,28]. The phenolic compounds with a reactive
formyl group may easily interact with the macromolecule toxins (enzymes/proteins) present in
the snake venom through chelation or chemical modification. Furthermore, there are a number
of mechanisms by which phenolic compounds may act as anti-snake venom: via free radical
21

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scavenging, hydrogen donation, singlet oxygen quenching, metal-ion chelation, or as substrates
for attack by superoxide [29]. The other possibility of the potent anti-snake venom effect of
phenolic compounds might be due to blocking of one or more of active sites of the venom toxins
or blocking the receptors which are structurally prone to chemical attack which is also evident
from our docking studies. In this study, we have shown that molecular docking is an important
tool in evaluating the interaction between PLA and polyphenols. Our results demonstrate that
2
the snake venom PLA binds strongly with compounds 21, 22, 25, 28, 35, and 36 by molecular
2
docking (Table S1, Supplementary material). The docking results revealed that two amino acids
ASP 49 and GLY 30 located in the binding pocket of enzyme played vital role in the designing
anti-snake venom activity. The compound 35 assumes favorable orientation within the binding
site, which is exactly same as the co-crystal ligand (IDA: 501). In addition, the enzyme surface
model also revealed that the molecule 35 occupies the binding pocket of PLA . All the active
2
compounds fit well in the active sites of PLA . The binding poses for each compound were
2
analyzed by examining their free energy scores. The dG binding energy of compounds was
found in the range of -26.587 to -78.857 Kcal for enzyme PLA which indicates that compounds
2
may have better selectivity and affinity towards enzyme PLA .
2
It is evident from the above results that the potent anti-snake venom activity is exhibited by
the substituted benzaldehydes. It is also reported that 2-hydroxy-4-methoxybenzaldehyde
possesses potent tyrosinase inhibition activity, strong chelating action, and potent antioxidant
property [25]. In our earlier studies we observed that the phenolic compound 2-hydroxy-4-
methoxy benzoic acid possessed potent anti-snake venom and antioxidant property. It also
showed that the mechanism of anti-venom activity is through venom induced free radical
22

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inhibition. The relationship between chemical structure of phenolic and their anti-snake venom
activity has been of considerable interest. Apart from the general observations made above,
more specific effects have been noted. The antisnake venom activity of phenols increases when
additional hydroxyl group is added and when the hydroxyl or methoxyl group is present at 3-
position of the benzene ring. Rice-Evans et al. [30] showed that ortho-substitution with electron-
donating such as methoxyl group increases the stability of the aryloxyl radical and hence its
antioxidant potential. It is also reported that the antioxidant activity of phenolic compound
depends on the structure and substitution pattern of hydroxyl groups [31]. The essential
requirement for effective free radical scavenging is the 3,4-orthodihydroxy configuration in ring
B and 4-carboxyl group in ring C. The presence of 3-OH group or 3-and 5-OH groups, giving a
catechol like structure in ring C, was also beneficial for the antioxidant activity of the phenolic
compounds [29,32,33]. It also showed a structurally important role of 3-OH group of the
chromane ring responsible for the enhancement of antioxidant activity. In our present study, the
phenolic compounds having hydroxyl and methoxyl group present at 3-position have showed
potent anti-hemorrhagic and anti-lethal effect respectively against snake envenomations. The
presence of a neighbouring hydroxyl or a methoxyl group further helped in stabilizing the
substrate-protein complex which might be forming a Schiff’s base by interaction of CHO group
of the phenolic compound (substrate) with snake venom toxins (PLA enzyme) that might leads
2
to the increase in activity of phenolic compounds [30]. The protection against venom induced
pathophysiological changes by the phenolic compounds may also be due to the formation of a
complex between a venom protein and the compound, reducing the toxic effects of the venom.
In variance to separate the administration of the two (compound injected i.v. followed by venom
s.c) as in case of in vivo experiments where the activities of the compounds were drastically
diminished. Briefly, the results obtained herein re-enforce the idea that the polyphenolic
23

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compounds present a significant inhibiting capacity of the snake-venom PLA2 activity. Possibly,
the phenolic hydroxyls are bound to ASP 49 and GLY 30 that may influence the capacity of
these amino acids to participate in the coordination of calcium atom that is essential to catalytic
enzyme. Apparently, the number of hydroxyls is not a limiting factor for the efficiency in
capacity of PLA inhibition. The presence of methoxyl group in the benzene ring also helps to
2
enhance the activity of the phenolic compound (compound 35, for instance) and it might be
bound with ASP 49 and GLY 30 of PLA much strongly that may increases the inhibitory
2
efficiency of these compounds.
4. Conclusion
In conclusion, several substituted phenols have been shown effective against snake venom
with induction of pathophysiological changes both in silico and in wet lab experiments. In this
study, molecular docking of the phenolic compounds having hydroxyl and methoxyl groups in
their benzene ring showed maximum inhibitory potency as they bound to active site of (ASP 49
and GLY 30) snake venom PLA . The results showed here led us to the opening of a window of
2
opportunity for screening more potent molecules that may be developed through in silico
chemistry which may lead to manifold anti-snake venom activity. Further work is intended to
establish the mechanism of actions of the phenolic compound(s) against venom-induced
pathophysiological changes and to better understand the action of phenolic compounds on snake-
venom PLA activities.
2
Supplementary material
Docking scores and binding free energy of compounds at the active sites of PLA2 (Table S1)
are represented in Supplementary Material available online.
24

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Conflict of Interest
The manuscript was written through contributions of all authors. This manuscript has not
been published and is not under consideration for publication elsewhere and we have no conflicts
of interest to disclose.
Acknowledgments
The work forms a part of the CSIR-Network Project (CMM0017). Partial financial support
by the Department of Biotechnology (Government of India) is also gratefully acknowledged.
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