17064-9fQK1439202911-

Inventi Rapid: Med Chem Vol. 2015, Issue 4
[ISSN 0976-3821]
2015 pmc 17064 © Inventi Journals (P) Ltd
Published on Web 08/08/2015, www.inventi.in
RESEARCH ARTICLE
INTRODUCTION
Michael adducts displayed a wide range of pharmacological
activities like Anti-cancer, Anti-microbial, Anti-leukemic,
Analgesic etc. [1-4] Michael adducts are the intermediates for
the synthesis of pyrrolines and other heterocyclic
compounds. Michael adducts are synthesized from
chalcones using basic catalyst follows Michael addition
reaction.
Michael Addition [5]
The addition of carbon nucleophiles to conjugate acceptor
systems, which is commonly known as Michael addition.
The Michael reaction is one of the most efficient methods
for effecting carbon – carbon bond formation and has wide
synthetic applications. This reaction and its close variants
have been extensively used in organic synthesis. Generally,
Michael additions are conducted in a suitable solvent in the
presence of a strong base either at room temperature or at
elevated temperatures. Due to the presence of the strong
base, side reaction, such as Multiple Condensations,
Polymerizations, rearrangement and retro-michael
additions are common.
This is 1, 4-addition of resonance stabilized carbanions.
The Michael addition is thermodynamically controlled; the
reaction donors are active methylenes such as malonates
and nitroalkanes and the acceptors are activated olefins
such as α, β – unsaturated carbonyl compounds.
Docking Studies
Molecular Modeling, Docking is a method which predicts
the preferred orientation and binding affinity between two
1Department of Pharmaceutical Chemistry, Creative Educational Society’s
college of Pharmacy, Chinnatekur, Kurnool-518218, Andhra Pradesh,
India.
E-mail: blavanya019@gmail.com
*Corresponding author
molecules. [6] Therefore docking is useful for predicting
both the strength and type of signal produced.
Docking is frequently used to predict the binding
orientation of small molecule drug candidates to their
protein targets in order to in turn predict the affinity and
activity of the small molecule. Hence docking plays an
important role in the rational design of drugs. [7] Given the
biological and pharmaceutical significance of molecular
docking, considerable efforts have been directed towards
improving the methods used to predict docking.
In Medicinal chemistry Drug Design plays a very
important role in synthesizing new compounds by
molecular or chemical manipulation of lead moiety to
produce highly active compounds with minimum steric
effect. [8] The main objective of the drug design is to
synthesized new compounds to improve efficacy, potency
and to eliminate the side effects.
Nowadays, the use of computers to predict the binding
of small molecules to the known target structures is an
important component in the drug discovery process. [9, 10]
There is a wide range of software packages available for
molecular docking like AutoDock, Schrodinger GLIDE etc.
[11] Some online docking sites are also available like mCule.
MATERIALS AND METHODS
4-Methoxy acetophenone, 2-Hydroxy acetophenone, 2-
Chlorobenzaldehyde, 4- Methoxybenzaldehyde, 3,4-
Dimethoxybenzaldehyde, 3,4,5-Trimethoxybenzaldehyde,
4-Isopropylbenzaldehyde, Cinnamaldehyde, Ethanol,
Sodium Hydroxide, n-Hexane, Ethyl acetate, Nitromethane,
Dimethyl formamide, Acetone, Dichloromethane, Dimethyl
Sulfoxide, Chloroform, Petroleum Ether, Potassium
Hydroxide are procured from SD Fine chemicals limited,
Mumbai. TLC Plates are procured from Merck, Diethyl ether
from molychem. All the melting points were recorded in
open glass-capillaries on an Aarson digital melting point
apparatus and are uncorrected. Bruker Nuclear Magnetic
In-silico Design, Synthesis and Biological Evaluation of Some
Michael Adducts from Chalcones
Bhatraju Lavanya Lahari1*, T Rajkumar1, L Shiva Shanker Reddy1, Y Siva Rami Reddy1, G
Sivudu1, P Navya Krishna1
Abstract: Docking of small molecules in the receptor binding site is a vital part of structure based drug design. The current
study deals with the synthesis and evaluation of Nitromethane substituted Chalcone derivatives with various targets of
Mycobacterium Tuberculosis and Antimicrobial activity using in-silico docking studies. In this perspective Nitromethane
substituted Chalcone derivatives are docked with various targets like 1A69 (Purine nucleoside phosphorylase), 3IFZ
(Mycobacterium tuberculosis gyrase), 1TED (Polyketide synthase), 2A7S (Acetyl CoA Carboxylase), 3ORI (Protein Kinase B),
Aspartate aminotransferase (PDB ID: 1AHG), Amidophosphoribosyl transferase (PDB ID: 1AO0) and Dihydropterote synthase
(PDB ID: 1AD4). In-silico docking studies were carried out using mcule online docking. OSIRIS property explorer used to explore
the molecular properties. Metabolic sites are predicted using metaprint 2D. Substituted acetophenones on treatment with
substituted aldehydes affords the corresponding chalcones (1a-1k). Treatment of chalcones with nitromethane under Michael
addition condition furnished the corresponding Michael adducts (2a-2k). The structure was proposed based on their 1H NMR
and IR spectral data. All the synthesized compounds (2a-2k) were screened for their Anti-tubercular, Anthelmentic and Anti-
microbial activities. Among synthesized compounds 2g, 2j and 2k showed highest Anti-tubercular activity at 50μg/ml
concentration. Compounds 2g, 2j and 2k showed highest activity suggesting that electron donating groups aid in anthelmentic
activity. Compounds 2d, 2g and 2j were found to be more effective anti-microbial activity. All the compounds showed significant
Anti-tubercular, Anthelmentic and Anti-microbial activities.
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RESEARCH ARTICLE
Resonance (NMR) spectrometer was used to record 1H
spectra. Chemical shifts (δ) are reported as downfield
displacements from Dimethyl sulphoxide (DMSO) used as
internal standard. Infrared (IR) spectra were recorded with
Bruker FT-IR Transmission mode spectrophotometer on
KBr pellets.
Molecular Docking
Docking studies was performed using Dell Intel Core3
Processor, Ligand were drawn on drawing window of
Chemsketch [12] and further explored for gross biological
activity, which is comprised of drug likeness and drug score
determined by using OSIRIS Property explorer. [13] Major
possible mechanism and site of metabolism of most potent
derivatives were also performed by Metaprint 2D [14]
software. The protein structure like Purine nucleoside
phosphorylase (PDB ID: 1A69), Mycobacterium
Tuberculosis gyrase (PDB ID: 3IFZ), Type III polyketide
synthase (PDB ID: 1TED), Acetyl CoA Carboxylase (PDB ID:
2A7S) and Protein Kinase B (PDB ID: 3ORI), Aspartate
aminotransferase (PDB ID: 1AHG), Amido phosphoribosyl
transferase (PDB ID: 1AO0) and Dihydropterote synthase
(PDB ID: 1AD4) are taken RCSB [15] protein Bank and was
used for docking studies by mcule. [16]
Figure 1: Protein–ligand interaction
R1
Ar
O
N
+
O
-
O
R2
Figure 2: Structure of ligand
O
CH3
R1 +
40%KOH
at room temperature
Substitued Chalcones
R2CH2NO2, DMF-H2Obase,r.t.
Michael's Adduct
Michael's Addition
ArCHO
Ar
O
R1
Ar
O
R1
NO2R2
Substituted Acetophenones
Figure 3: Scheme of synthesis
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RESEARCH ARTICLE
Ligand Preparation
Using Chemsketch software the structures of the drugs
were sketched draw and generated their SMILES file using
open babel GUI. [17]
Procedure for Synthesis of Chalcones (1a-1k)
0.001M Substituted acetophenone derivatives is dissolved
in aqueous Potassium Hydroxide solution by using
magnetic stirrer then add 0.001M substituted aldehydes
and aqueous Potassium Hydroxide solution are added to
the above mixture and then stirred for complete
dissolution. Then the temperature is maintained between
10-15°C in ice bath and stirred for 4-5 hours and the above
mixture is kept overnight in refrigerator. Then filter off the
precipitated chalcone and wash with little water, dried and
recrystalized from ethanol.
Procedure for Synthesis of Michael Adducts (2a-2k)
Solution of NaOH (1M, 5 ml) was added to the stirred
solution of chalcone (5 mmol) and nitromethane (5 mmol)
at reaction time 10 minutes in DMF (5 ml) and the resulting
mixture was stirred until the reaction was complete as
indicated by TLC and then filters off the precipitated
product dried and recrystalized from ethanol.
Biological Evaluation
1. In-vitro Anti-tubercular Activity by Alamar Blue Dye
[18]
a. The anti mycobacterial activity of compounds were
assessed against Mycobacterium tuberculosis using
Microplate Alamar Blue assay (MABA).
b. This methodology is non-toxic, uses as a thermally
stable reagent and shows good correlation with
proportional and BACTEC radiometric method.
c. Briefly, 200 μl of sterile deionzed water was added to all
outer perimeter wells of sterile 96 wells plate to
minimize evaporation of medium in the test wells
during incubation.
d. The 96 wells plate received 100 μl of the Middlebrook
7H9 broth and serial dilution of compounds were made
directly on plate.
e. The final drug concentration tested were 100 to 0.2
μg/ml.
f. Plates were covered and sealed with parafilm and
incubated at 37 for five days.
g. After the time, 25 μl of freshly prepared 1:1 mixture of
Alamar Blue reagent and 10% tween 80 was added to
the plate and incubated for 24 hrs.
h. A blue colour in the well was interpreted as no
bacterial growth and pink colour was scored as
growth.
i. The MIC was defined as lowest drug concentration
which prevented the colour change from blue to pink.
2. In-vitro Anti-helmentic Activity
The anthelmentic activity was carried out as per the
method of Sharmila Sutradhar et al. [19] The assay was
performed in-vitro using adult earthworm (Pheretima
Posthuma) owing to its anatomical and physiological
resemblance with the intestinal round worm parasites of
human beings for preliminary evaluation of anthelmentic
activity.
Test samples of the synthesized compounds were
prepared at concentrations 1, 2, 5, 10 μg/ml in 3 ml of
DMSO and then make up to 25 ml with DMSO. Each earth
worm i.e., Pheretima posthuma were placed in Petri dish
containing 25 ml of the above test solution of the
synthesized compounds. Albendazole was used as the
reference standard and distilled water as the control. All
the test solution and the standard drug solution were
prepared freshly before starting the experiment.
Observations were made for the time taken for paralysis. It
was noted when no movement of any sort could be
observed except when the worms were shaken vigorously.
Time for death of worms were recorded after ascertaining
that worms neither moved when shaken vigorously nor
when dipped in warm water (50 ).
3. In-vitro Anti-microbial Activity by Agar Cup Plate
Method
a. Preparation of Standard Stock Solution
i. Preparation Penicillin Stock Solution: 4 mg of Penicillin
were dissolved in 4 ml of DMSO i.e., 1000 μg/ml
ii. Preparation Streptomycin stock solution: 4 mg of
Streptomycin were dissolved in 4 ml of DMSO i.e., 1000
μg/ml.
b. Preparation of Sample Stock Solution
4mg of different samples were dissolved in 4 ml of DMSO
i.e., 1000 μg/ml from this solution 500, 250 and 125 μg/ml
were prepared.
c. Media Preparation [20-23]
Nutrient agar medium was used as culture medium to
provide the required nutrients and facilitate the growth of
the microorganisms. It was prepared by dissolving the
following ingredients in 1000 ml of purified water:
i. Peptone: 10 g
ii. Beef Extract: 10 g
iii. Agar: 20 g
iv. Sodium Chloride: 5 g
v. Final pH at 25 - 7.4±0.2.
The pH of the medium was then adjusted to 7.2-7.4 and
sterilized by autoclaving at 15lb pressure for 15 min
(121 ).
d. Procedure
i. The sterilized agar medium was inoculated with the
suspension of microorganism at a temperature between
40-50°C and was immediately poured into the petri
plates and allowed it to solidify.
ii. Then holes of 6 mm in diameter were bored in the
medium with a sterile borer.
iii. The holes were then filled with the specified
concentration solution of synthesized compounds and
standard.
iv. Then the plates were incubated at 37 for 48 hrs and
observed for zone of inhibition.
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Table 1: Substitutions of the Synthesized Compounds
Compound Code for
Chalcones
Compound Code for
Micheal Adducts
R1 R2 Ar
1a 2a 4-OCH3 H
Cl
1b 2b 4-OCH3 H
O
CH3
1c 2c 4-OCH3 H
O
CH3
O
CH3
1d 2d 4-OCH3 H
O
CH3
O
CH3
O
CH3
1e 2e 4-OCH3 H
CH3 CH3
1f 2f 4-OCH3 H
1g 2g 2-OH H
Cl
1h 2h 2-OH H
O
CH3
1i 2i 2-OH H
O
CH3
O
CH3
1j 2j 2-OH H Cl
1k 2k 2-OH H
O
CH3
Table 2: Drug Relevant Properties of Ligand Molecules
Compound Code clog P Solubility Mol. Wt TPSA Drug Likeness Drug Score
2a 2.86 -4.75 333.0 72.12 -2.63 0.37
2b 2.18 -4.03 329.0 81.35 -3.29 0.41
2c 2.35 -4.32 317.0 72.12 -2.81 0.4
2d 2.11 -4.05 359.0 90.58 -0.86 0.5
2e 2.04 -4.06 389.0 99.81 0.34 0.5
2f 3.44 -4.88 341.0 72.12 -5.58 0.33
2g 2.58 -4.43 319.0 83.12 -4.57 0.38
2h 1.91 -3.72 315.0 92.35 -3.75 0.42
2i 1.84 -3.73 345.0 101.5 -2.34 0.44
2j 1.77 -3.75 375.0 110.8 -1.08 0.59
2k 2.11 -4.05 359.0 90.58 -0.86 0.52
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RESULTS AND DISCUSSION
In the present work, a series of eleven new chalcone
derivatives from 4-Methoxy acetophenone and 2- Hydroxy
acetophenone and various aromatic aldehydes 1a – 1k
were synthesized. Thus, we have obtained Michael adducts
2a – 2k through the treatment of 1a – 1k with
Nitromethane under Michael addition conditions.
Drug Relevant Properties of Ligand Molecules from
OSIRIS Property Explorer
The drug relevant properties of ligand molecules were
determined by inserting the SMILES file in the OSIRIS
Property explorer.
Prediction of Metabolism Using METAPRINT 2D
Metaprint 2D is a new software tool implementing a data –
mining approach for predicting sites of xenobiotic
metabolism. The algorithm is based on a statistical analysis
of the occurrences of atom centered circular fingerprints in
both substrates and metabolites.
Metabolism of the Compound 2j Most Potent Derivative
The colour highlighting an atom indicates its normalized
occurrence ratio (NOR). A high NOR indicates a more
frequently reported site of metabolism in the metabolite
database.
Predicted Metabolites
1. Reduction at C=O:
O
OH
NO2
O
CH3
O
CH3
O
CH3
Reduction
OH
OH
NO2
O
CH3
O
CH3
O
CH3
+
OH
NO2
O
CH3
O
CH3
O
CH3
Figure 4: Screenshot of OSIRIS property explorer of compound 2j
Table 3: Normalized Occurrence Ratio of Compound 2j
Red 0.66 <= NOR <= 1.00
Orange 0.33 <= NOR < 0.66
Green 0.15 <= NOR < 0.33
White 0.00 <= NOR < 0.15
Grey Little/no data
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2. At Hydroxyl group the following are the predicted
metabolites:
O
OH
NO2
O
CH3
O
CH3
O
CH3
Phosphorylation
O
O
NO2
O
CH3
O
CH3
O
CH3
PO OH
OH
O
O
NO2
O
CH3
O
CH3
O
CH3
CH3O
Acetylation
Sulfation
O
O
NO 2
O
CH3
O
CH3
O
CH3
S OHO
O
O
O
NO2
O
CH3
O
CH3
O
CH3
O
OH
OH
OH
O
OH
Glucuronidation
O
OH
NO2
O
CH3
O
CH3
O
CH3
Hydroxylation
Demethyalation
O
OH
NO2
O
CH3
OH
O
CH3
O
OH
NO2
O
CH3
O
O
CH3
OH
3. At trimethoxy group:
O
OH
NO2
O
CH3
O
CH3
O
CH3
Hydroxylation
Demethyalation
O
OH
NO2
O
CH3
OH
O
CH3
O
OH
NO2
O
CH3
O
O
CH3
OH
In-silico Docking Studies by Mcule
In silico docking study showed that two designed
derivatives were having excellent affinity towards the
targets 1A69 (Purine nucleoside phosphorylase) and 1AHG
(Aspartate Aminotransferase). Docking studies were
performed in the online docking software mcule.
Chemistry
In the present investigation eleven new chalcone
derivatives (1a-1k) were prepared by the Claisen-Schmidt
condensation of different ketones and appropriately
substituted aldehydes using the above mentioned method
and eleven new intermediates i.e., Michael adducts (2a-2k)
from the above synthesized chalcones by Michael addition.
The compounds were re-crystallized using rectified spirit.
All the compounds were characterized by detailed
spectroscopic (IR, 1H NMR) analyses.
1. (2E)-3-(2-chlorophenyl)-1-(4-methoxyphenyl)prop-
2-en-1-one (1a)
C16H13ClO2, M.P.: 122-124 , M.W.: 272.72, White, 52.9%, IR
(KBr cm-1) C-H Str (Aliph) 2932, C-H Str (Arom) 3002.4, C-
O-C Str 1140, C=O Str 1654.2, C=C Str (Aliph) 1598.91, =C-H
Str 3011.1, C=C Str (Arom) 1572.26, C-Cl Str 757.2, C-H
bend 1421.3.
2. (2E)-1,3-bis(4-methoxyphenyl)prop-2-en-1-one (1b)
C17H16O3, M.P.: 108-110 , M.W.: 268.30, Yellow, 81.6%, IR
(KBr cm-1) C-H Str (Aliph) 2942.4, C-H Str (Arom) 3004.6,
C-O-C Str 1111.4, C=O Str 1655.4, C=C Str (Aliph) 1592.42,
=C-H Str 3011.7, C=C Str (Arom) 1509.26, C-H bend
1419.69.
3. (2E)-3-(3,4-dimethoxyphenyl)-1-(4-methoxyphenyl)
prop-2-en-1-one (1c)
C-O-C Str 1145, C=O Str 1651.5, C=C Str (Aliph) 1595.79,
=C-H Str 3014, C=C Str (Arom) 1511.99, C-H bend 1420.37.
4. (2E)-1-(4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)
prop-2-en-1-one (1d)
C-O-C Str 1120.37, C=O Str 1654.3, C=C Str (Aliph) 1606.86,
=C-H Str 3012, C=C Str (Arom) 1504.05, C-H bend 1420.20.
5. (2E)-1-(4-methoxyphenyl)-3-[4-(propan-2-yl)
phenyl]prop-2-en-1-one (1e)
C19H20O2, M.P.: 92-94 , M.W.: 280.36, White, 95.5%, IR
C-O-C Str 1145, C=O Str 1656.54, C=C Str (Aliph) 1605.81,
=C-H Str 3011.4, C=C Str (Arom) 1605.81, C-H bend 1421.14.
6. (2E,4E)-1-(4-methoxyphenyl)-5-phenylpenta-2,4-
dien-1-one (1f)
C18H16O2, M.P.: 114-116 , M.W.: 264.31, Red, 84.5%, IR
C-O-C Str 1142,C=O Str 1651.49, C=C Str (Aliph) 1601.66,
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=C-H Str 3011.8, C=C Str (Arom) 1583.96, C-H bend
1417.43.
7. (2E)-3-(2-chlorophenyl)-1-(2-hydroxyphenyl)prop-
2-en-1-one (1g)
C15H11ClO2, M.P.: 98-100 , M.W.: 258.69, Red, 98.5%, IR
C=O Str 1640.5, C=C Str (Aliph) 1583.23, =C-H Str 3011.93,
C=C Str (Arom) 1485.70, C-Cl Str 780.2, C-H bend 1441.3,
OH Str 3425.2.
8. (2E)-1-(2-hydroxyphenyl)-3-(4-methoxyphenyl)prop
-2-en-1-one (1h)
(KBr cm-1) C-H Str (Aliph) 2924.23, C-H Str (Arom)
3005.02, C=O Str 1638.7, C=C Str (Aliph) 1568.8, =C-H Str
3012.51, C=C Str (Arom) 1502.23, C-H bend 1468.25, OH
Str 3424.82, C-O-C Str 1163.61.
9. (2E)-3-(3,4-dimethoxyphenyl)-1-(2-hydroxyphenyl)
prop-2-en-1-one (1i)
(KBr cm-1) C-H Str (Aliph) 2923.84, C-H Str (Arom)
3003.51, C=O Str 1634.08, C=C Str (Aliph) 1567.22, =C-H
Str 3011.63, C=C Str (Arom) 1504.18, C-H bend 1476.15,
OH Str 3426.2, C-O-C Str 1151.9.
Table 4: Docking Scores of Ligand Molecules with Various Targets
Compound Code
Docking Scores
Anti-tubercular Activity
Docking Scores
Antimicrobial Activity
1A69 1TED 2A7S 3ORI 3IFZ
1AHG
for E.coli
1AO0
for B.subtilis
1AD4
for S.aureus
2a -7.9 -5.3 -4.5 -5.0 -4.8 -9.2 -7.2 -6.4
2b -8.1 -5.4 -5.4 -5.0 -4.9 -9.1 -7.3 -5.9
2c -8.4 -5.6 -5.6 -5.4 -5.4 -9.3 -7.3 -6.0
2d -8.6 -5.7 -5.8 -5.5 -5.5 -9.2 -7.4 -6.6
2e -7.8 -5.0 -4.9 -5.3 -5.0 -9.5 -7.9 -6.5
2f -7.7 -5.1 -5.2 -5.0 -5.4 -8.7 -7.1 -6.3
2g -8.2 -4.6 -4.5 -4.3 -5.1 -9.8 -7.0 -6.7
2h -8.4 -5.9 -5.0 -4.7 -5.0 -10.1 -7.3 -5.6
2i -8.2 -5.3 -5.1 -4.6 -5.5 -10.3 -7.2 -6.1
2j -8.7 -5.2 -5.5 -5.3 -5.6 -8.9 -7.0 -6.5
2k -7.7 -5.1 -5.2 -5.4 -5.3 -8.9 -7.0 -6.3
Isoniazid -6.5 -6.2 -6.0 -6.3 -6.4 - - -
Penicillin - - - - - -14.4 -13.9 -13.6
Streptomycin - - - - - -14.0 -13.4 -12.5
Figure 5: Screen shot of Compound 2j shows good ligand-protein interactions for Anti tubercular activity
Figure 6: Screen shot of Compound 2i shows good ligand-protein interactions for Anti microbial activity
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10. (2E)-1-(2-hydroxyphenyl)-3-(3,4,5-trimethoxy
phenyl)prop-2-en-1-one (1j)
C18H18O5, M.P.: 138-140 , M.W.: 334.30, Yellow, 90%, IR
C=C Str (Arom) 1498.97, C-H bend 1372.7, OH Str 3453.7,
C-O-C Str 1127.70.
11. (2E,4E)-1-(2-hydroxyphenyl)-5-phenylpenta-2,4-
dien-1-one (1k)
C17H14O2, M.P.: 150-152 , M.W.: 250.29, Red, 88.8%, IR
C=C Str (Arom) 1487.11, C-H bend 1444.42, OH Str
3434.08.
12. 3-(2-chlorophenyl)-1-(4-methoxyphenyl)-4-nitro
butan-1-one (2a)
C17H16ClNO4, M.P.: 200-202 , M.W.: 333.76, Light Brown,
60%, IR (KBr cm-1) C-H Str (Aliph) 2974.2, C-H Str (Arom)
3060.5, C-O-C Str 1186.4, C=O Str 1653.9, C-C Str (Aliph)
1251.6,C=C Str (Arom) 1572.2, C-N Str 1316.9, C-H bend
1421.6, NO2 sym 1598.6, C-Cl Str 756.83, 1H NMR ( DMSO,
400 MHz, δ ppm) Ar-H(8H,m)6.67-7.94, OCH3(3H,s)3.86,
Table 5: Results of Anti-tubercular Activity of Synthesized Nitromethane Substituted Chalcones (2a-2k)
S. No. Compound Code
100
μg/ml
50
μg/ml
25
μg/ml
12.5
μg/ml
6.25
μg/ml
3.12
μg/ml
1.6
μg/ml
0.8
μg/ml
1 2a S R R R R R R R
2 2b S R R R R R R R
3 2c S R R R R R R R
4 2d S R R R R R R R
5 2e S R R R R R R R
6 2f S R R R R R R R
7 2g S S R R R R R R
8 2h S R R R R R R R
9 2i S R R R R R R R
10 2j S S R R R R R R
11 2k S S R R R R R R
S = Sensitive, R= Resistant, Here are the standard values for the Anti-TB test which was performed. Pyrazinamide – 3.125 μg/ml, Ciprofloxacin- 3.125
μg/ml, Streptomycin – 6.25 μg/ml
Figure 7: Anti-TB microplate alamar blue assay for standard
Figure 8: Anti-TB microplate alamar blue assay for nitromethane substituted chalcones (2a-2k)
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CH2C=O(2H,d)3.25,4.20, CH-Ar(1H,s)4.11, CH2-
NO2(2H,d)4.65,4.74.
13. 1,3-bis(4-methoxyphenyl)-4-nitrobutan-1-one (2b)
C18H19NO5, M.P.: 237-239 , M.W.: 329.34, Yellowish
Brown, 77%, IR (KBr cm-1) C-H Str (Aliph) 2842.5, C-H Str
(Arom) 3072.2, C-O-C Str 1111.05, C=O Str 1655.22, C-C Str
(Aliph) 1252.6,C=C Str (Arom) 1509.06, C-N Str 1335.09, C-
H bend 1419.5, NO2 sym 1592.46, 1H NMR ( DMSO, 400
MHz, δ ppm) Ar-H(8H,m)6.09-7.69, OCH3(6H,s)3.84,3.76,
CH2C=O(2H,d)3.15,4.22, CH-Ar(1H,s)4.15, CH2-NO2(2H,d)
4.60,4.75.
14. 3-(3,4-dimethoxyphenyl)-1-(4-methoxyphenyl)-4-
nitrobutan-1-one (2c)
C19H21NO6, M.P.: 256-258°C, M.W.: 359.37, Yellowish
Brown, 75%, IR (KBr cm-1) C-H Str (Aliph) 2938.6, C-H Str
(Arom) 3005.9, C-O-C Str 1139.77, C=O Str 1650.48, C-C Str
Table 6: Anthelmentic Effect of Synthesized Nitromethane Substituted Chalcones on Pheretima posthuma
Concentration
(μgm/ml)
Paralysis Time (min)
(Mean±S.E.M)
Death Time (min)
(Mean±S.E.M)
1 Control (Water) 0 0 0
2 Albendazole
1 3.52±0.015 10.34±0.003
2 3.23±0.01 8.25±0.004
5 2.11±0.008 5.45±0.008
10 1.32±0.007 12.35±0.004
3 2a
1 5.27±0.23 12.35±0.79
2 4.71±0.18 10.26±0.67
5 3.71±0.11 8.54±0.53
10 2.83±0.20 5.55±0.22
4 2b
1 6.98±0.62 12.21±0.03
2 6.26±0.51 10.16±0.03
5 4.97±0.40 8.16±0.04
10 3.38±0.17 5.42±0.03
5 2c
1 5.26±0.017 12.35±0.02
2 5.16±0.015 10.15±0.005
5 4.19±0.008 8.17±0.01
10 3.49±0.015 6.12±0.03
6 2d
1 5.19±0.016 11.12±0.02
2 5.06±0.016 9.08±0.016
5 4.12±0.02 6.30±0.013
10 3.41±0.007 5.15±0.03
7 2e
1 5.40±0.002 12.33±005
2 5.31±0.008 10.38±0.02
5 4.23±0.01 8.19±0.02
10 3.50±0.02 6.15±0.05
8 2f
1 6.03±0.016 13.02±0.009
2 5.44±0.02 10.20±0.03
5 4.23±001 8.47±0.01
10 3.21±0.008 6.24±0.01
9 2g***
1 4.10±0.0016 10.42±0.034
2 3.31±0.01 8.21±0.06
5 2.14±0.001 5.46±0.04
10 1.40±0.005 4.33±0.03
10 2h
1 5.10±0.003 11.07±0.015
2 4.43±0.008 9.10±0.004
5 3.26±0.018 6.25±0.02
10 2.39±0.018 4.34±0.04
11 2i
1 5.20±0.003 11.25±0.05
2 4.34±0.02 9.25±0.03
5 3.34±0.03 6.13±0.006
10 2.30±0.02 5.31±0.003
12 2j***
1 4.02±0.007 10.42±0.004
2 3.28±0.017 8.31±0.003
5 2.14±0.005 5.42±0.007
10 1.40±0.008 4.50±0.003
13 2k**
1 4.02±0.7 10.22±0.99
2 3.34±0.13 8.10±0.02
5 2.23±0.2 5.40±0.008
10 1.50±0.03 4.39±0.02
Mean ± SEM = Mean ± Standard Error Mean, *** p value < 0.001, ** p value < 0.01, * p value < 0.1
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H bend 1418.35, NO2 sym 1588.50, 1H NMR ( DMSO, 400
MHz, δ ppm) Ar-H(7H,m)7.52-7.74, OCH3(9H,s)3.87, 3.82,
CH2C=O(2H,d)3.20,4.23, CH-Ar(1H,s)4.13, CH2NO2(2H,d)
4.62,4.73.
15. 3-(3,4,5-trimethoxyphenyl)-1-(4-methoxyphenyl)-
4-nitrobutan-1-one (2d)
Brown, 90.7%, IR (KBr cm-1) C-H Str (Aliph) 2943.12, C-H
Str (Arom) 3004.3, C-O-C Str 1120.47, C=O Str 1656.40, C-C
Str (Aliph) 1268.4,C=C Str (Arom) 1504.02, C-N Str
1309.01, C-H bend 1420.40, NO2 sym 1606.9, 1H NMR (
DMSO, 400 MHz, δ ppm) Ar-H(6H,m)6.70-8.64, OCH3(3H,s)
3.75,3.78,3.85, CH2C=O(2H,d)3.21,4.24, CH-Ar(1H,s)4.10,
CH2-NO2(2H,d)4.63,4.72.
16. 1-(4-methoxyphenyl)-4-nitro-3-[4-(propan-2-yl)
phenyl]butan-1-one (2e)
C20H23NO4, M.P.: 186-188 , M.W.: 341.40, Brown, 92.7%,
IR (KBr cm-1) C-H Str (Aliph) 2963.5, C-H Str (Arom)
3051.3, C-O-C Str 1172.18, C=O Str 1655.6, C-C Str (Aliph)
Figure 9: Anthelmentic activity of albendazole in case of paralysis
A
lb
e
n
d
a
z
o
le
2
a
2
b
2
c
2
d
2
e
2
f
2
g
2
h
2
i
2
j
2
k
0
2
4
6
8
C o m p o u n d s
A lb e n d a z o le
2 a
2 b
2 c
2 d
2 e
2 f
2 g
2 h
2 i
2 j
2 k
2.54
4.12
5.39
4.52
4.45
4.61
4.72
2.73
3.78
3.79
2.71
2.76
A n th e m e n tic a c tiv ity o f c o m p o u n d s - P a r a ly s is T im e
TimeinMin
Figure 10: Anthelmentic activity of synthesized compounds in case of paralysis
Figure 11: Anthelmentic activity of albendazole in case of death
0
5
10
15
1μg/ml 2μg/ml 5μg/ml 10μg/ml
TimeinMin
Concentration
Anthelmentic Activity of Albendazole - Paralysis Time
Control Albendazole
0
2
4
6
8
10
12
1μg/ml 2μg/ml 5μg/ml 10μg/ml
TimeinMin
Concentration
Anthelmentic Activity of Albendazole - Death Time
Control
Albendazole
10

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RESEARCH ARTICLE
1421.24, NO2 sym 1605.7, 1H NMR ( DMSO, 400 MHz, δ
ppm)Ar-H(8H,m)6.74-8.67,OCH3(3H,s)3.03,CH2C=O (2H,d)
3.26,CH-Ar(1H,s)4.14, CH2-NO2(2H,d)4.53,4.69,Isopropyl
(7H,s)1.2,2.82.
17. (4E)-1-(4-methoxyphenyl)-3-(nitromethyl)-5-
phenylpent-4-en-1-one (2f)
Brown, 72.16%, IR (KBr cm-1) C-H Str (Aliph) 2923.9, C-H
Str (Arom) 2965.9, C-O-C Str 1177.2, C=O Str 1651.4, C-C
Str (Aliph) 1251.02,C=C Str (Arom) 1507.31, C-N Str
1351.5,C-H bend 1417.19, NO2 sym 1583.8, C=C Str
(Aliph) 1601.5, 1H NMR ( DMSO, 400 MHz, δ ppm) Ar-
H(9H,m) 6.73-8.67, OCH3(3H,s)3.5, CH2C=O(2H,d)---, CH-
Ar (1H,s) 4.20, CH2-NO2 (2H,d) 4.66, Olefin protons(2H,s)
5.94,6.7.
18. 3-(2-chlorophenyl)-1-(2-hydroxyphenyl)-4-
nitrobutan-1-one (2g)
C16H14ClNO4, M.P.: 188-190 , M.W.: 319.73, Light Green,
83.3%, IR (KBr cm-1) C-H Str (Aliph) 2928.24, C-H Str
(Arom) 2988.2, OH Str 3389.4, C=O Str 1640.3, C-C Str
(Aliph) 1289.1,C=C Str (Arom) 1485.46, C-N Str 1333.9, C-H
bend 1440.9, NO2 sym 1584.1,C-Cl bend 787.61, 1H NMR
(DMSO, 400 MHz, δ ppm) Ar-H(8H,m)6.74-8.06, OH(1H,s)
12.14, CH2C=O(2H,d)3.27,4.25, CH-Ar(1H,s)4.17, CH2-
NO2(2H,d)4.72,4.90.
19. 1-(2-hydroxyphenyl)-3-(4-methoxyphenyl)-4-
nitrobutan-1-one (2h)
C17H17NO5, M.P.: 192-194 , M.W.: 315.32, Green, 81.6 %, IR
OH Str 3443.02, C=O Str 1640.4, C-C Str (Aliph) 1260.9,C=C
Str (Arom) 1492.96, C-N Str 1374.4, C-H bend 1441.2, NO2
sym 1563.9, C-O-C Str 1163.4, 1H NMR ( DMSO, 400 MHz, δ
ppm) Ar-H(8H,m)7.57-8.72, OH(1H,s)12.5, CH2C=O(2H,d)
3.17,4.21, CH-Ar(1H,s)4.10, CH2-NO2(2H,d)4.61,4.86, OCH3
(3H,s)3.77.
20. 3-(3,4-dimethoxyphenyl)-1-(2-hydroxyphenyl)-4-
nitrobutan-1-one (2i)
C18H19NO6, M.P.: 228-230 , M.W.: 345.32, Light Green, 76.5
%, IR (KBr cm-1) C-H Str (Aliph) 2932.9, C-H Str (Arom)
2998.9, OH Str 3396.35, C=O Str 1631.6, C-C Str (Aliph)
1439.8, NO2 sym 1552.2, C-O-C Str 1153.22, 1H NMR
11.25, CH2C=O(2H,d)3.14,4.19, CH-Ar(1H,s)4.12, CH2-NO2
(2H,d)4.63,4.85, OCH3(6H,s)3.75,3.79.
A
lbendazole
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
0
5
10
15
Compounds
Albendazole
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
Anthelmentic activity of compounds- Death Time
TimeinMin
7.12
9.12
8.98
9.19
7.9
9.26
9.48
7.11
7.67
7.98
7.16
7.03
Figure 12: Anthelmentic activity of synthesized compounds in case of death
Figure 13: Anthelmentic activity of synthesized compounds in case of death and paralysis time
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21. 1-(2-hydroxyphenyl)-4-nitro-3-(3,4,5-trimethoxy
phenyl)butan-1-one (2j)
C19H21NO7, M.P.: 262-264 , M.W.: 375.37, Light Green,
83.15 %, IR (KBr cm-1) C-H Str (Aliph) 2954.01, C-H Str
(Arom) 2997.9, OH Str 3442.35, C=O Str 1631.56, C-C Str
H bend 1439.9, NO2 sym 1552.1, C-O-C Str 1154.22, 1H NMR
12.17, CH2C=O(2H,d)3.20,3.79, CH-Ar(1H,s)4.09, CH2-
NO2(2H,d)4.7,4.93, OCH3(9H,s)3.74,3.85.
22. (4E)-1-(2-hydroxyphenyl)-3-(nitromethyl)-5-
phenylpent-4-en-1-one (2k)
C18H17NO4, M.P.: 285-287 , M.W.: 311.33, Brick Red, 70.7
%, IR (KBr cm-1) C-H Str (Aliph) 2998.7, C-H Str (Arom)
3053.1, OH Str 3424.7, C=O Str 1633.8, C-C Str (Aliph)
Table 7: Anti-microbial Study of Synthesized Nitromethane Substituted Chalcones (2a-2k)
Test Organisms Zone of Inhibition (mm)
Gram+ve Gram-ve
B. subtilis S. aureus E. coli K. pneumoniae
A B C D A B C D A B C D A B C D
1 2a 13 11 - - 12 10 - - 14 9 - - 13 10 - -
2 2b 15 13 - - 14 8 - - 17 12 10 - 15 11 - -
3 2c 14 12 - - 13 9 - - 15 11 6 - 14 9 - -
4 2d 17 16 11 - 15 13 10 - 19 15 10 - 18 14 8 -
5 2e 15 13 - - 14 10 - - 17 12 8 - 15 10 - -
6 2f 12 10 - - 14 11 - - 16 11 7 - 15 8 - -
7 2g 19 17 13 - 16 12 11 - 20 15 10 - 22 17 11 -
8 2h 14 12 - - 17 13 - - 18 12 8 - 18 11 - -
9 2i 13 10 - - 16 11 - - 19 13 5 - 18 9 - -
10 2j 20 19 15 - 19 17 14 - 23 18 12 - 21 17 11 -
11 2k 18 16 12 - 14 13 9 - 21 16 11 - 20 15 13 -
12 Penicillin 32 - - - 32 - - - 32 - - - 32 - - -
13 Streptomycin 28 - - - 28 - - - 28 - - - 28 - - -
14 DMSO - - - - - - - - - - - - - - - -
A - 1000 μg/ml, B - 500 μg/ml, C - 250 μg/ml, D - 125 μg/ml
Figure 14: Anti-microbial activity of synthesized compounds against Gram +ve bacteria B. subtilis
Figure 15: Anti-microbial activity of synthesized compounds against Gram +ve bacteria S. aureus
0
5
10
15
20
25
30
35
ZoneofInhibitioninmm
Compounds
1000μg/ml
500μg/ml
250μg/ml
125μg/ml
0
5
10
15
20
25
30
35
Compounds
1000μg/ml
500μg/ml
250μg/ml
125μg/ml
12

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RESEARCH ARTICLE
1444.1, NO2 sym 1563.6, =CH Str 3017.5, C=C Str 1643.7.
Activity Studies
1. In-vitro Anti-tubercular Activity Studies
The results of in-vitro Anti-tubercular activity studies by
Alamar Blue Assay method are shown in Table 5 and Figure
7-8.
2. In-vitro Anthelmentic Activity Studies
The results of in-vitro anthelmentic activity studies were
shown in Table 6 and Figures 9-13.
The results are expressed as Mean±SEM (Standard
Error Mean). The data was analyzed by one – way ANOVA
followed by student t – test for comparing the control and
the test groups, using trial version of Graph Pad Prism 5
software.
The anthelmentic activity of the synthesized
Nitromethane substituted Chalcone derivatives (2a-2k)
were screened against Pheretima Posthuma in DMSO by
using Albendazole as standard drug.
The results of anthelmentic activity revealed that all
compounds were found to show mild to moderate
anthelmentic activity. Compounds 2g, 2j and 2k exhibit
significant activity than all the other derivatives.
3. In-vitro Anti-microbial Studies
The results of in-vitro Anti-microbial studies of
Nitromethane substituted chalcones were shown in Table 7
and Figure 14 to 17. Among all the compounds 2d, 2g, 2j
and 2k were found to be more effective.
SUMMARY AND CONCLUSION
From the docking studies, we can conclude that the target
1A69 and 1AHG show good ligand-protein interactions
than the other targets and give better docking score.
Especially 2j (R1 = 2-OH, Ar = -Ph-3,4,5-(OCH3)3) showed
best score when compared to the standard drug Isoniazid
this may lead for our Anti-tubercular research and 2i (R1 =
2-OH, Ar = -Ph-3,4-(OCH3)2) shows good ligand-protein
interactions than the other compounds and this may lead
for our Anti-microbial research.
The synthesized compounds were monitored by TLC to
ascertain the completion of the reaction. All the compounds
were found to have good yields. Rfvalues and melting
points of the synthesized compounds were different with
each other indicating the difference between the
compounds. The structures were proposed based on 1H
NMR and IR spectral data.
The Anti-tubercular activity of synthesized
Nitromethane substituted Chalcone derivatives were
Figure 16: Anti-microbial activity of synthesized compounds against Gram -ve bacteria E. coli
Figure 17: Anti-microbial activity of synthesized compounds against Gram -ve bacteria K. pneumoniae
0
5
10
15
20
25
30
35
Compounds
1000μg/ml
500μg/ml
250μg/ml
125μg/ml
0
5
10
15
20
25
30
35
Compounds
1000μg/ml
500μg/ml
250μg/ml
125μg/ml
13

[ISSN 0976-3821]
RESEARCH ARTICLE
screened against Mycobacterium tuberculosis H37Rv stain
by using Streptomycin, Pyrazinamide and Ciprofloxacin as
a standard drugs. The results of antitubercular activity
were given in Table 5 and Figure 7, 8. From this it was
found that 2g (R1 = 2-OH, Ar = -Ph-2-Cl), 2j (R1 = 2-OH, Ar
= -Ph-3,4,5-(OCH3)3) and 2k (R1 = 2-OH, Ar = -Ph-CH=CH-)
shown the highest activity at 50 μg/ml against
Mycobacterium tuberculosis pathogen.
Anthelmentic activity was performed by using
earthworms (Pheretima posthuma). Most of the compounds
showed significant activity with p < 0.001 when the data
subjected to one way ANOVA by using Graph pad prism- 5
software. The results of Anthelmentic activity was given in
Table 6 and Figure 9-13. From this it was found that 2g, 2j
and 2k have shown the moderate anthelmentic activity
when compared to the standard drug. The results are
presented in Mean ± SEM.
The synthesized Nitromethane substituted Chalcone
derivatives were also screened for antimicrobial activity
using four bacteria i.e., B. subtilis, S. aureus, E. coli and K.
pneumoniae. Penicillin and Streptomycin as a standard
drugs. The results of Anti-microbial activity studies was
given in Table 7 and Figure 14-17. The compounds 2d (R1 =
4-OCH3, Ar = -Ph-3,4,5-(OCH3)3), 2g (R1 = 2-OH, Ar = -Ph-2-
Cl), 2j (R1 = 2-OH, Ar = -Ph-3,4,5-(OCH3)3) and 2k (R1 = 2-
OH, Ar = -Ph-CH=CH-) were found to be more effective
than the other derivatives.
It is clear from the present study that the presence of
OH group on the aromatic ring may be necessary for Anti-
tubercular and Anthelmentic activity.
The designed Nitromethane substituted Chalcones (2a-
2k) can be further modified into pyrrolines, pyrroles,
reduced Michael adducts etc., can produce further
substituted derivatives. These can be synthesized and
evaluated for different pharmacological activities.
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Cite this article as: Bhatraju Lavanya Lahari, T
Rajkumar, L Shiva Shanker Reddy et al. In-silico Design,
Synthesis and Biological Evaluation of Some Michael
Adducts from Chalcones. Inventi Rapid: Med Chem,
2015(4):1-14, 2015.
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