Synthesis of new C-dimethylated chalcones as potent antitubercular agents

Article abstract of DOI:10.1007/s00044-018-2183-z

A new class of C-dimethylated-chalcones (9a–q) were synthesized by using 2-hydroxy-3,5-dimethyl-4,6-dimethoxy acetophenone as a key intermediate. The compounds were screened for anti-tubercular activity against Mycobacterium tuberculosis strain (H₃₇Rv) by Microplate Alamar Blue assay (MABA) method at a concentration of 100–0.8 μg/mL. The chalcones, 9a, 9b, 9c, 9k, 9o, and 9p were found to have higher antitubercular activity than the standard drugs, while the remaining compounds showed moderate activity. The antitubercular activity of the chalcones, 9b (MIC₉₀ = 3.98 μM) and 9o (MIC₉₀ = 3.84 μM) was found to be more than two-fold more active than the standard drugs, streptomycin (MIC₉₀ = 10.75 μM) and ciprofloxacin (MIC₉₀ = 9.43 μM), while their antitubercular activity was found to be more than six-fold more active than pyrazinamide (MIC₉₀ = 25.38 μM). Further, the molecular docking studies employing Mycobacterium tuberculosis protein tyrosine phosphatase (MtbPtp) was carried out to observe docking scores.

Full text of DOI:10.1007/s00044-018-2183-z

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2183-z
ORIGINAL RESEARCH
Synthesis of new C-dimethylated chalcones as potent antitubercular
agents
Rambabu Anandam1 Surender Singh Jadav Vasu Babu Ala Mohamed Jawed Ahsan Hari Babu Bollikolla1
2 ●
3 ●
4 ●
●
Received: 6 February 2018 / Accepted: 16 April 2018
©
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
A new class of C-dimethylated-chalcones (9a–q) were synthesized by using 2-hydroxy-3,5-dimethyl-4,6-dimethoxy
acetophenone as a key intermediate. The compounds were screened for anti-tubercular activity against Mycobacterium
tuberculosis strain (H Rv) by Microplate Alamar Blue assay (MABA) method at a concentration of 100–0.8 µg/mL. The
37
chalcones, 9a, 9b, 9c, 9k, 9o, and 9p were found to have higher antitubercular activity than the standard drugs, while the
remaining compounds showed moderate activity. The antitubercular activity of the chalcones, 9b (MIC = 3.98 µM) and 9o
9
0
(
MIC = 3.84 µM) was found to be more than two-fold more active than the standard drugs, streptomycin (MIC = 10.75
9
0
9
0
µM) and ciprofloxacin (MIC = 9.43 µM), while their antitubercular activity was found to be more than six-fold more active
90
than pyrazinamide (MIC = 25.38 µM). Further, the molecular docking studies employing Mycobacterium tuberculosis
90
protein tyrosine phosphatase (MtbPtp) was carried out to observe docking scores.
●
●
●
●
●
Keywords Chalcones Mycobacterium tuberculosis Alamar Blue assay Anti-tuberculosis H Rv Tyrosine phosphatase
3
7
Introduction
2002), antimicrobial (Sivakumar et al. 2009), antioxidant
Mukherjee et al. 2001), antidiabetic (Mahapatra et al. 2015),
(
The naturally occurring chalcones are bioactive aromatic
ketones and serving as a class of intermediates for several
targets based pharmacophore in medicinal chemistry (Zhuang
et al. 2017). The widespread biological activities of chalcone
analogs associated with anticancer (Leon-Gonzalez et al.
antiviral (Vasu Babu et al. 2014), and anti-inflammatory
effects (Chen et al. 2013; Kasthuri et al. 2017) have been
successfully investigated till date. The multi-bioactive role of
chalcone analogs had drawn the attention of many researchers
towards exploring their pharmacological properties (Zhang
2017). The mechanism of action and ability of chalcone
analogs to interfere with a variety of enzymes including
hydrolases, kinases, topoisomerases, MDM2, oxidor-
eductases, and several antimicrobial enzymes have been listed
by Zhou et al. (Zhou and Xing 2015). Tuberculosis is one of
the most dangerous respiratory diseases caused by Myco-
bacterium tuberculosis (Mtb) that has high mortality rate in
affected individuals. HIV positive people are more prone to
tuberculosis. Another complications arise today is multidrug
resistant tuberculosis due to indiscreet use of antibiotics. The
chalcone derivatives posses’ outstanding biological activity
against Mtb is shown in Fig. 1 (Chiaradia et al. 2012).
One of the anti-tuberculosis mechanisms of chalcone is
associated with altering the fundamental role of protein tyr-
osine kinases (Ptks) in controlling of phosphorylation/
dephosphorylation cascades in cells, is now becoming an
acceptable strategy to design the promising inhibitors (Zhou
et al. 2010; Vintonyak et al. 2009; Greenstein et al. 2005).
The tremendous investigational efforts to identify the
2
2
015; Sharma et al. 2013), antitubercular (Vasu Babu et al.
014; Chiaradia et al. 2012; Sivakumar et al. 2007; Lin et al.
Electronic supplementary material The online version of this article
https://doi.org/10.1007/s00044-018-2183-z) contains supplementary
material, which is available to authorized users.
(
*
Hari Babu Bollikolla
dr.b.haribabu@gmail.com
1
2
3
4
Department of Chemistry, Acharya Nagarjuna University,
Guntur, Andhra Pradesh 522510, India
Department of Chemistry, School of Technology, GITAM
University, Hyderabad, Telangana 502102, India
Department of Science & Humanities, QIS College of Engineering
&
Technology, Prakasam District, Ongole, Andhra Pradesh, India
Department of Pharmaceutical Chemistry, Maharishi Arvind
College of Pharmacy, Ambabari Circle, Jaipur, Rajasthan 302039,
India

Medicinal Chemistry Research
R
2
Fig. 1 The chalcone derivatives
posses outstanding biological
activity against Mtb
R
1
R
3
4
O
OH
R
R
5
O
O
9
a-q
O
HN
O
C-dimethylated chalcones
N
N
O
O
CH
3
OH
F
O
Furfural chalcone
O
O
Ciprofloxacin
O
Potent Anti-
Tuberculosis
Inhibitors
Cl
O
N
NH
2
O
N
Halogenated chalcone
Pyrazinamide
O
O
Cl
N
2
H N
NH
2
OH
OH
OCH
3
HO
N
OH
O
O
HO
HO
H C
3
O
O
N
heterocyclic chalcone
O
NH O
NH
2
O
OCH
3
OHC
H N
OH
2
OCH
3
Streptomycin
O
OCH
3
3,4,5-trimethoxy chalcone
functional behavior of Mycobacterium tuberculosis protein
by using Remi melting point apparatus and were uncor-
1
tyrosine-phosphatase (MtbPtpA and MtbPtpB) present in host
cells has revealed their part in blocking the extracellular sig-
naling pathways leading to endorse the infection (Bach et al.
rected. H nuclear magnetic resonance (NMR) spectra were
1
3
recorded on Brucker 400 MHz and C NMR was on
Brucker100 MHz spectrophotometers. Mass spectra recor-
ded on LC-MS Agilent LC-1100 series.
2
008; Koul et al. 2004). The potentials of chalcones to
obstruct the mycobacterium protein tyrosine-phosphatase
PTPs) mediated function in host cells has thoroughly been
(
Synthesis of 2,4,6-trimethoxy phloroglucinol (2)
investigated by a few research groups (Chiaradia et al. 2012;
Vintonyak et al. 2011; Oliveira et al. 2011; Rawls et al. 2009;
Soellner et al. 2007). The protein tyrosine-phosphatases
A solution of anhydrous phloroglucinol (100 g, 0.793 mol)
in dry acetone (1200 mL) was refluxed for 8 h at 60–70 °C
under anhydrous conditions with dropwise addition of
dimethyl sulfate (227.2 mL, 299.9 g, 2.380 mol) in presence
of ignited potassium carbonate (558.5 g, 4.047 mol). The
reaction was monitored by thin layer chromatography
(TLC) using 30% Ethyl acetate in hexane as mobile phase.
After completion of the reaction the acetone was removed
and the concentrated reaction mixture was added into the
crushed-ice with vigorous stirring for 3 h, a white color
solid (trimethoxyphloroglucinol) obtained was filtered and
dried at RT (Vasu Babu et al. 2014). White color crystals
with 80% of yield obtained with melting point 52–54 °C
(Scheme 1).
(PTPs) are a class of Ptks secreted by Mtb in a host cell to
alter the immune system (Zhang 2017; He et al. 2013). The
series of 2-hydroxy substituted chalcones were synthesized to
test against Mycobacterium tuberculosis infections employing
H Rv TB strains. Initially, all the synthesized compounds
3
7
were subjected to docking studies employing both MtbPtpA
and MtbPtpB templates to get enzyme specificity. The 2-
hydroxy position was found to be crucial for making stable
interactions with the active site residues of both enzymes.
Earlier we have reported the antitubercular activity of chal-
cones derivatives (hispolon analogs) with MICs ranging
between 25 and >100 µg/mL (Balaji et al. 2017).
Synthesisof 2,4,6-trimethoxybenzaldehyde (3)
Materials and methods
Dimethylformamide (9.0 mL, 8.4 g, 0.116 mol) was added
to trimethoxy-phloroglucinol (15.0 g, 0.089 mol) and
cooled at 0 °C. Phosphorousoxychloride (9.1 mL, 15.0 g)
was added dropwise to this cooling mixture slowly under
anhydrous conditions. The mixture was then allowed to
Experimental
Phloroglucinol and other chemicals used were of AR grade
(
Merck). Melting points were taken in open glass capillary

Medicinal Chemistry Research
CHO
CH
3
CH
3
Scheme 1 Synthesis of new C-
dimethylated-chalcones (9a–h,
HO
OH H CO
OCH3
H CO
OCH3
3
H
3
CO
OCH
3
3
H
3
CO
OCH
3
(i)
(
ii)
(iii)
(iv)
9
j–m, and 9o–q): Reagents and
OHC
conditions: (i) DMS, Acetone,
OH
1)
OCH
(2)
OCH3
(4)
,
3
OCH
(3)
3
OCH
(5)
3
K2CO3 70 °C, 8 h. (ii, iv) DMF,
(
POCl3, 0 °C, 1 h. (iii, v)
NH2–NH2·H2O, KOH, Ethylene
Glycol, 80–90 °C for 2 h
followed by heating at
(
v)
CH3
R4
CHO
CH
3
CH
3
H CO
OH R5
R3
R2
R5
R4
R1
R2
H
3
CO
OH
CH3
H
3
CO
OCH
3
3
(vi)
1
30–140 °C for 2 h. (vi)
CH3COCl, AlCl3, Diethylether,
°C, 6 h (vii) Ar CHO, Aq
KOH/EtOH, 24 h
(
vii)
H C
H
3
C
3
H C
3
0
OCH
3
H CO
O
R
1
R3
(8)
H
3
CO
(7)
O
3
(
9)
(6)
stand for 1 h at room temperature. The reaction was mon-
itored by TLC using 40% ethyl acetate in Hexane as mobile
phase. After completion the reaction mixture was poured
into crushed-ice and basified with 8 M KOH. The solid
precipitate obtained was filtered and dried at 80 °C (Vasu
Babu et al. 2013). Pale pink color powder with 91.6% of
yield obtained with melting point 119–121 °C (Scheme 1).
95.6% of yield obtained with melting point 85.4–88.3 °C
(Scheme 1).
Synthesis of 2,4-dimethyl-1,3,5-trimethoxy benzene (6)
The above reduction procedure was repeated as reported by
Vasu Babu et al. (2013). 3-Methyl-2,4,6-tri-methoxy ben-
zaldehyde (10.0 g, 0.047 mol) was dissolved in ethylene
glycol (32.0 mL, 35.0 g, 0.476 mol) and hydrazine hydrate
Synthesis of 2,4,6-trimethoxytoluene (4)
(10.5 mL, 10.7 g, 0.214 mol). Then powdered KOH (10.6 g,
2
,4,6-trimethoxybenzaldehyde (10.0 g, 0.05 mol) was dis-
0.190 mol) was added in portion wise to the reaction mix-
ture and heated to 80–90 °C for 2 h, foaming was formed.
The reaction mixture was heated to 135–145 °C for another
2 h. The reaction progress was monitored by TLC using
10% ethyl acetate in hexane as mobile phase. After com-
pletion the reaction mixture was poured into the ice-cold
water, acidified with dil. HCl and stirred for 1 h. The solid
obtained was filtered and dried at RT (Vasu Babu et al.
2013). Lemon yellow color powder with 96.7 % of yield
was obtained with melting point 61.4–65.5 °C (Scheme 1).
solved in ethylene glycol (28.7 mL, 31.6 g, 0.510 mol) and
added hydrazine hydrate (11.1 mL, 11.4 g, 0.229 mol). Then
powdered KOH (11.4 g, 0.204 mol) was added in portion-
wise to the reaction mixture and heated to 80–90 °C for 2 h
then foam is formed. Further, the reaction mixture heated to
1
35–145 °C for another 2 h. The reaction progress was
monitored by TLC using mobile phase (30% ethyl acetate in
Hexane). Then the reaction mixture is poured into ice-cold
water, acidified with dil. HCl and extracted with hexane.
The hexane layer was washed with sodium carbonate,
water, and brine solution (Vasu Babu et al. 2013). This
hexane layer was dried over sodium sulfate and con-
centrated. Pale yellow color liquid with 92% of yield
obtained (Scheme 1).
Synthesis of 2-hydroxy-3,5-dimethyl-4,6-dimethoxy
acetophenone (7)
A solution of 2,4-dimethyl-1,3,5-trimethoxy benzene (10 g,
0
.357 mol) in absolute ether (30 mL) was added to a cold
solution of anhydrous aluminum chloride (10.4 g,
.078 mol) in absolute ether (30 mL). To the cooled (0–5 °
Synthesis of 3-methyl-2,4,6-trimethoxy benzaldehyde (5)
0
The above formylation procedure was repeated as reported
by Vasu Babu et al. (2013). Dimethylformamide (5.5 mL,
C) and stirred solution, acetyl chloride (3.0 mL, 3.36 g,
0.042 mol), was added dropwise for about 1 h. The mixture
is stirred for 4 h more. The whole reaction was done under
anhydrous conditions. The reaction progress was monitored
by TLC using 10% Ethyl acetate in Hexane as mobile
phase. Later, the mixture was left 48 h at room temperature
and treated with crushed ice. Further, it was acidified with
5 N HCl and extracted with ethyl acetate. This ethyl acetate
layer was dried over sodium sulfate and concentrated (Vasu
Babu et al. 2013). Light yellow color crystals with 67.5% of
yield obtained with melting point 57.8–58.6 °C (Scheme 1).
5
.2 g, 0.071 mol) was added to 2,4,6-trimethoxy toluene
(
10 g, 0.054 mol) and cooled at 0 °C. Phosphorousox-
ychloride (5.6 mL, 9.2 g) was added dropwise to this cool-
ing mixture slowly under anhydrous conditions. The
mixture was allowed to stand for 1 h at room temperature.
The reaction progress was monitored by TLC using mobile
phase (20% ethyl acetate in Hexane). After completion of
the reaction the mixture was poured into crushed ice and
basified with 8 N KOH. The solid precipitate obtained was
filtered and dried at RT. Pale brown colored compound with

Medicinal Chemistry Research
General method for the synthesis of chalcones (9a–q)
Ar–CH3); 13C NMR (CDCl , 100 MHz, δ in ppm): 193.8
3
(C=O), 162.9 (C-4), 161.5 (C-6), 158.5 (C-2), 130.7 (C-β),
To a solution of acetophenone (7) (0.5 g, 0.002 mol) and
substituted benzaldehydes and heterocyclic aldehydes (8a–
q) (0.48 g, 0.0059 mol) in ethanol (10 mL) is added aqueous
potassium hydroxide (0.80 g in 0.8 mL water). The mixture
is stirred at room temperature for 24 h. The reaction was
monitored by TLC using 20% Ethyl acetate in Hexane as
mobile phase. To this reaction mixture added ice cold water
and acidified with dil. HCl. The solid precipitate obtained
was filtered and dried at RT. The solid was purified by
column chromatography using the mobile phase, n-hexane
and further crystallized with aqueous methanol.
123.3 (C-α), 121.3 (C-2′, 6′), 115.5 (C-1′), 115.4 (C-3′, 5′),
112.2 (C-3), 111.9 (C-5), 62.2 (OCH at C-4), 60.1 (OCH3
3
at C-6), 40.2 (N–CH ), 29.8 (N–CH ), 8.8 (Ar–CH at C-5),
3
3
3
8.7 (Ar–CH at C-3); Anal. calc. for C H NO : C, 70.96;
3
21 25
4
+
H, 7.09; found: C, 70.92; H, 7.04; %; LC-MS: (M + H) at
356.
(2E)-3-(1,3-benzodioxol-4-yl)-1-(2-hydroxy-4,6-dimethoxy-
1
3,5-dimethylphenyl)prop-2-en-1-one
(9d)
H
NMR
(CDCl , 400 MHz, δ in ppm): 13.12 (1H, s, Ar–OH), 7.81
3
(2H, s, H-α, β), 7.17 (1H, d, J = 1.6 Hz, H-2′), 7.13 (1H, dd,
J = 1.2, 8.0 Hz, H-6′), 6.85 (1H, d, J = 8.0 Hz, H-5′), 6.03
(2H, s, O–CH –O), 3.76 (3H, s, Ar–OCH ), 3.66 (3H, s,
(
2E)-1-(2-Hydroxy-4,6-dimethoxy-3,5-dimethylphenyl)-3-(4-
2
3
1
methoxyphenyl)prop-2-en-1-one (9a) H NMR (CDCl3,
00 MHz, δ in ppm): 13.18 (1H, s, Ar–OH), 7.86 (2H, s, H-
α, β), 7.61 (2H, d, J = 8.8 Hz, H-2′, 6′), 6.93 (2H, d, J =
Ar–OCH ), 2.17 (3H, s, Ar–CH ), 2.16 (3H, s, Ar–CH );
3 3 3
1
3
4
C NMR (CDCl , 100 MHz, δ in ppm): 193.6 (C=O),
3
161.9 (C-2), 158.3 (C-6), 157.3 (C-4), 149.7 (C-4′), 148.2
(C-3′), 143.8 (C-β), 139.7 (C-1′), 128.9 (C-α), 124.8 (C-6′),
115.3 (C-2′), 114.6 (C-5′), 113.4 (C-1), 106.8 (C-5), 104.2
(C-3), 101.7 (Ar–O–CH –O), 61.8 (Ar–OCH ), 60.3
8
′
.8 Hz, H-3′, 5′), 3.85 (3H, s, Ar′–OCH ), 3.75 (3H, s, Ar
3
–OCH ), 3.66 (3H, s, Ar′–OCH ), 2.17 (3H, s, Ar–CH ),
3
3
3
13
2
.16 (3H, s, Ar–CH3); C NMR (CDCl , 100 MHz, δ in
3
2
3
ppm): 193.9 (C=O), 163.4 (C-2), 161.7 (C-6), 161.5 (C-4),
(Ar–OCH ), 8.8 (Ar–CH ), 8.7 (Ar–CH ); Anal. calc. for
3 3 3
1
1
1
58.7 (C-4′), 143.5 (C-β), 130.3 (C-2′, 6′), 128.1 (C-α),
24.3 (C-1′), 115.5 (C-5), 115.5 (C-3), 114.5 (C-3′, 5′),
11.9 (C-1), 62.3 (OCH ), 60.1 (OCH ), 55.4 (OCH ), 8.9
C H O : C, 67.41; H, 5.66; found: C, 67.43; H, 5.65; %;
20 20 6
LC-MS: (M + H) at 357.2 and (M + Na) at 379.2.
+
+
3
3
3
(
7
Ar–CH ), 8.7 (Ar-CH ); Anal. calc. for C H O : C,
0.+16; H, 6.48; found: C, 70.18; H, 6.41;%; LC-MS: (M +
(2E)-1-(2-Hydroxy-4,6-dimethoxy-3,5-dimethylphenyl)-3-
3
3
20 22
5
1
(3,4-methoxyphenyl)prop-2-en-1-one
(9e)
H
NMR
H) at 343.
(CDCl , 400 MHz, δ in ppm): 13.16 (1H, s, OH at C-2),
3
7
.84 (2H, s, CH=C, H-α, β), 7.26 (1H, m, H-6′), 7.15 (1H,
(
(
2E)-1-(2-Hydroxy-4,6-dimethoxy-3,5-dimethylphenyl)-3-
2,3,4-trimethoxyphenyl)prop-2-en-1-one (9b) H NMR
d, H-2′), 6.90 (1H, d, H-5′), 3.94 (6H, s, OCH at C-3′, 4′),
3.75 (3H, s, OCH at C-6), 3.66 (3H, s, OCH at C-4), 2.17
(6H, s, CH at C-3, 5); C NMR (CDCl , 100 MHz, δ in
3 3
3
1
3
3
1
3
(
CDCl , 400 MHz, δ in ppm): 13.20 (1H, s, Ar–OH), 8.10
3
(
1H, d, J = 15.6 Hz, CH=C, H-β), 7.90 (1H, d, J = 15.6 Hz,
ppm): 193.8 (C=O), 163.3 (C-5), 161.5 (C-3), 158.5 (C-1),
151.3 (C-3′), 149.2 (C-2′), 143.7 (C-β), 128.2 (C-α), 124.3
(C-1′), 123.0 (C-6′), 115.6 (C-4), 115.5 (C-5′), 111.8 (C-
2′), 111.1 (C-6), 110.2 (C-2), 62.2 (OCH ), 60.0 (OCH ),
CH=C, H-α), 7.45 (1H, d, J = 8.0 Hz, H-6′), 6.77 (1H, d, J
=
s, Ar –OCH ), 3.90 (3H, s, Ar –OCH ), 3.78 (3H, s,
1
8.0 Hz, Ar–H, H-5′), 3.97 (3H, s, Ar –OCH ), 3.94 (3H,
3
1
1
3
3
3
3
Ar–OCH ), 3.66 (3H, s, Ar–OCH ), 2.25 (6H, d, Ar–CH );
55.9 (OCH ), 55.9 (OCH ), 8.8 (Ar–CH ), 8.7 (Ar–CH );
3 3 3 3
3
3
3
1
3
C NMR (CDCl , 100 MHz, δ in ppm): 194.2 (C=O),
Anal. calc. for C H O : C, 67.73; H, 6.50; found: C,
21 24 6
3
+
+
1
63.3 (C-2), 161.6 (C-6), 158.6 (C-4), 155.8 (C-2′), 153.8
67.39; H, 6.41;%; LC-MS: (M + H) at 372 and (M + Na)
(
1
C-4′), 142.5 (C-β), 138.7 (C-3′), 125.4 (C-α), 123.6 (C-1′),
at 379.2.
22.3 (C-6′), 115.5 (C-5′), 112.0 (C-1), 107.9 (C-5), 107.5
(
(
C-3), 62.6 (OCH ), 62.3 (OCH ), 61.8 (OCH ), 56.4
OCH ), 56.0 (OCH ), 8.8 (Ar–CH ), 8.7 (Ar–CH ); Anal.
(2E)-3-(4-chlorophenyl)-1-(2-hydroxy-4,6-dimethoxy-3,5-
3
3
3
1
dimethylphenyl)prop-2-en-1-one (9f) H NMR (CDCl3,
400 MHz, δ in ppm): 13.04 (1H, s, Ar–OH), 7.97 (1H, d, J
= 16.0 Hz, H-β), 7.84 (1H, d, J = 16.0 Hz, H-α), 7.58 (2H,
d, J = 8.4 Hz, H-3′, 5′), 7.41 (2H, d, J = 8.4 Hz, H-2′, 6′),
3.76 (3H, s, Ar–OCH ), 3.66 (3H, s, Ar–OCH ), 2.17 (6H,
3
3
3
3
calc. for C H O : C, 65.66; H, 6.51; found: C, 65.46; H,
6
2
2
26
7
+
.41; %; LC-MS: (M + H) at 403.
(
(
2E)-1-(2-Hydroxy-4,6-dimethoxy-3,5-dimethylphenyl)-3-[4-
dimethylamino)phenyl]prop-2-en-1-one (9c)
3
3
1
13
H
NMR
s, Ar–CH3); C NMR (CDCl , 100 MHz, δ in ppm): 193.8
3
(
(
7
CDCl , 400 MHz, δ in ppm): 13.37 (1H, s, Ar–OH), 7.90
1H, d, J = 7.90 Hz, H-β), 7.80 (1H, d, J = 7.80 Hz, H-α),
.56 (2H, d, J = 8.8 Hz, H-2′, 6′), 6.69 (2H, d, J = 8.8 Hz,
(C=O), 163.9 (C-4′), 163.7 (C-2), 161.6 (C-6), 158.6 (C-4),
142.0 (C-β), 131.5 (C-1′), 130.4 (C-2′, 6′), 130.3 (C-3′, 5′),
126.3 (C-α), 116.1 (C-1), 115.7 (C-5), 111.8 (C-3), 62.3
(Ar–OCH ), 60.2 (Ar–OCH ), 8.8 (Ar–CH ), 8.7 (Ar–CH );
3
H-3′, 5′), 3.74 (3H, s, Ar–OCH ), 3.66 (3H, s, Ar–OCH ),
3
3
3
3
3
3
3
.04 (6H, s, N–(CH ) ), 2.17 (3H, s, Ar–CH ), 2.16 (3H, s,
3 2 3

Medicinal Chemistry Research
Anal. calc. for C H ClO : C, 65.80; H, 5.52; found: C,
CH=C, H-α), 6.27 (2H, s, Ar–H, H-3′, 5′), 3.95 (6H, s,
1
9
19
4
+
1
6
5.79; H, 5.50; %; LC-MS: (M + H) at 347.
Ar –OCH ), 3.86 (3H, s, Ar–OCH ), 3.78 (3H, s,
3
3
Ar–OCH ), 3.73 (3H, s, Ar–OCH ), 2.18 (3H, s, Ar–CH ),
3
3
3
1
3
(
2E)-3-(4-Fluorophenyl)-1-(2-hydroxy-4,6-dimethoxy-3,5-
2.16 (3H, s, Ar–CH3); C NMR (CDCl , 100 MHz, δ in
3
1
dimethylphenyl)prop-2-en-1-one (9g) H NMR (CDCl3,
00 MHz, δ in ppm): 13.04 (1H, s, Ar–OH). 7.89 (1H, d, J
15.6 Hz, H-β) 7.81 (1H, d, J = 15.6 Hz, H-α), 7.63 (2H,
dd, J = 5.6, 3.2 Hz, H-2′, 6′), 7.10 (2H, t, J = 8.4 Hz, H-3′,
ppm): 195.4 (C=O), 163.2 (C-2), 162.7 (C-6), 161.8 (C-2′,
6′), 161.7 (C-4′), 158.8 (C-4), 135.5 (C-β), 126.0 (C-α),
115.2 (C-1′), 112.2 (C-1), 106.9 (C-3, 5), 90.5 (C-3′, 5′),
62.3 (Ar–OCH ), 60.1 (Ar–OCH ), 56.1 (Ar–OCH ), 55.7
4
=
3
3
3
5
(
′), 3.75 (3H, s, Ar–OCH ), 3.66 (3H, s, Ar–OCH ), 2.17
(Ar–OCH ), 55.5 (Ar–OCH ), 8.9 (Ar–CH ), 8.7 (Ar–CH );
3 3 3 3
Anal. calc. for C H O : C, 65.66; H, 6.51; found: C,
22 26 7
3
3
13
3H, s, Ar–CH ), 2.16 (3H, s, Ar–CH ); C NMR (CDCl3,
3
3
+
100 MHz, δ in ppm): 193.9 (C=O), 165.7 (C-4′), 163.8 (C-
2), 162.4 (C-6), 161.7 (C-4), 158.7 (C-β), 142.2 (C-1′),
131.5 (C-2′, 6′), 130.5 (C-3′, 5′), 126.4 (C-α), 116.1 (C-1),
115.8 (C-5), 111.9 (C-3), 62.4 (Ar–OCH3), 60.2
65.65; H, 6.51; %; LC-MS: (M + H) at 403.
(2E)-1-(2-Hydroxy-4,6-dimethoxy-3,5-dimethylphenyl)-3-(3-
ethoxy-4-methoxyphenyl)prop-2-en-1-one (9k) H NMR
1
(
Ar–OCH ), 8.9 (Ar–CH ), 8.8 (Ar–CH ); Anal. calc. for
(CDCl , 400 MHz, δ in ppm): 13.21 (1H, s, Ar–OH), 7.92
3
3
3
3
C H FO : C, 69.08; H, 5.80; found: C, 69.09; H, 5.79; %;
LC-MS: (M + H) at 331.
(2H, s, H-α, β), 7.28 (2H, m, H-2′, 6′), 6.92 (1H, d, J =
8.1 Hz, H-5′), 4.18 (2H, q, J = 7.2 Hz, Ar–OCH CH ), 3.92
1
9
19
4
+
2
3
(
3H, s, Ar–OCH ), 3.78 (3H, s, Ar–OCH ), 3.64 (3H, s,
3
3
(
2E)-3-(4-Bromophenyl)-1-(2-hydroxy-4,6-dimethoxy-3,5-
Ar–OCH ), 2.18 (6H, s, Ar–CH ), 1.52 (3H, t, J = 6.8 Hz,
CH –CH O–Ar); C NMR (CDCl , 100 MHz, δ in ppm):
3 2 3
3
3
1
13
dimethylphenyl)prop-2-en-1-one (9h) H NMR (CDCl3,
00 MHz, δ in ppm): 13.01 (1H, s, Ar–OH). 7.94 (1H, d, J
15.6 Hz, H-β), 7.72 (1H, d, J = 15.6 Hz, H-α), 7.48 (2H,
d, J = 8.0 Hz, H-3′, 5′), 7.43 (2H, d, J = 8.0 Hz, H-2′, 6′),
.79 (3H, s, Ar–OCH ), 3.68 (3H, s, Ar–OCH ), 2.18 (6H,
4
=
193.9 (C=O), 163.7 (C-2), 161.7 (C-6), 158.7 (C-4), 157.9
(C-4′), 156.7 (C-3′), 142.5 (C-β), 137.1 (C-α), 130.3 (C-1′),
129.9 (C-6′), 127.3 (C-2′), 123.4 (C-5′), 118.4 (C-1), 115.7
(C-5), 111.82 (C-3), 66.4 (Ar–OCH CH ), 62.3
3
3
3
2
3
1
3
s, Ar–CH3); C NMR (CDCl , 100 MHz, δ in ppm): 193.7
(Ar–OCH ), 62.1 (Ar–OCH ), 55.7 (Ar–OCH ), 15.4
3 3 3
3
(
C=O), 163.7 (C-2), 161.6 (C-6), 158.6 (C-4), 141.7 (C-β),
(CH CH ), 8.8 (Ar–CH ), 8.7 (Ar–CH ); Anal. calc. for
2 3 3 3
1
39.4 (C-4′), 134.1 (C-1′), 132.1 (C-3′, 5′), 129.7 (C-2′, 6′),
C H O : C, 68.36; H, 6.78; found: C, 68.35; H, 6.80; %;
22 26 6
LC-MS: (M + H) at 387.
+
1
27.1 (C-α), 124.5 (C-1), 115.7 (C-5), 111.7 (C-3), 62.3
(
Ar–OCH ), 60.1 (Ar–OCH ), 8.8 (Ar–CH ), 8.7 (Ar–CH );
3 3 3 3
Anal. calc. for C H BrO : C, 58.33; H, 4.89; found: C,
(2E)-3-(2-Chlorophenyl)-1-(2-hydroxy-4,6-dimethoxy-3,5-
dimethylphenyl)prop-2-en-1-one (9l) H NMR (CDCl3,
1
9
19
4
+
1
5
8.35; H, 4.88; %; LC-MS: (M + H) at 392.
400 MHz, δ in ppm): 12.99 (1H, s, OH at C-2), 8.24 (1H, d,
(
2E)-1-(2-hydroxy-4,6-dimethoxy-3,5-dimethylphenyl)-3-(1-
J = 15.5 Hz, CH=C, H-β), 7.93 (1H, d, J = 16.0 Hz,
CH=C, H-α), 7.76 (1H, m, H-2′), 7.45 (1H, m, H-5′), 7.34
1
methyl-1H-indol-2-yl)prop-2-en-1-one
(
8
dd, CH=C, H-α), 7.38 (1H, t, C-3′), 7.34 (1H, s, C-6′), 7.31
(
OCH at C-6), 3.71 (3H, s, OCH at C-4), 2.21 (3H, s, CH
3
at C-3), 2.17 (3H, s, CH at C-4); C NMR (CDCl3,
(9i)
H
NMR
CDCl , 400 MHz, δ in ppm): 13.55 (1H, s, OH at C-2),
(2H, m, H-3′, 4′), 3.76 (3H, s, OCH at C-6), 3.66 (3H, s,
3
3
1
3
.19 (1H, s, H- 1′), 8.06 (1H, dd, CH=C, H-β), 7.48 (1H,
OCH at C-4), 2.17 (6H, s, CH at C-3, 5); C NMR
3 3
(CDCl , 100 MHz, δ in ppm): 193.9 (C=O), 163.9 (C-4),
3
2H, t, C-4′, 5′), 3.85 (3H, s, OCH at N–CH ), 3.76 (3H, s,
161.9 (C-6), 138.9 (C-2), 135.8 (C-β), 133.5 (C-2′), 131.2
3
3
(C-1′), 130.5 (C-3′, 4′), 129.2 (C-6′), 127.9 (C-5′), 127.3
3
3
13
(C-α), 115.9 (C-5), 112.0 (C-3), 62.6 (OCH at C-6), 60.3
3
3
1
00 MHz, δ in ppm): 193.9 (C=O), 162.9.9 (C-5), 161.7
(OCH at C-4), 8.9 (CH at C-3), 8.8 (CH at C-5); Anal.
3
3
3
(
1
1
C-3), 158.6 (C-1), 138.4 (C-β), 138.3 (C-8′), 135.0 (C-α),
26.2 (C-2′), 123.3 (C-9′), 121.9 (C-5′), 121.2 (C-4′, 6′),
20.9 (C-4′), 115.5 (C-3′), 113.6 (C-4), 112.1 (C-2), 110.2
calc. for C H ClO : C, 65.80; H, 5.52; found: C, 65.48; H,
19 19 4
5.26; %; LC-MS: (M + H) at 347.
+
(
C-6), 62.4 (Ar–OCH ), 60.2 (Ar–OCH ), 33.4 (N–CH ),
(2E)-3-(2-Fluorophenyl)-1-(2-hydroxy-4,6-dimethoxy-3,5-
3
3
3
1
9
.0 (Ar–CH ), 8.8 (Ar–CH ); Anal. calc. for C H NO : C,
dimethylphenyl)prop-2-en-1-one (9m) H NMR (CDCl3,
3
3
22 23
4
7
%
2.31; H, 6.34; N, 3.83; found: C, 72.45; H, 6.38; N, 3.81;
; LC-MS: (M + H) at 366.
400 MHz, δ in ppm): 13.07 (1H, s, Ar–OH), 7.99 (2H, m,
H-α, β), 7.66 (1H, t, H-4′), 7.35 (1H, d, J = 5.5 Hz, H-6′),
+
7
.16 (2H, m, H-3′), 3.74 (3H, s, OCH at C-4), 3.67 (3H, s,
3
13
(
(
2E)-1-(2-Hydroxy-4,6-dimethoxy-3,5-dimethylphenyl)-3-
2,4,6-trimethoxyphenyl)prop-2-en-1-one (9j)
OCH at C-6), 2.15 (6H, s, CH at C-3, 5); C NMR
3 3
1
H
NMR
(CDCl , 100 MHz, δ in ppm): 194.1 (C=O), 161.9 (C-3),
3
(
(
CDCl , 400 MHz, δ in ppm): 13.60 (1H, s, Ar–OH), 8.45
1H, d, J = 15.2 Hz, CH=C, H-β), 8.42 (1H, d, J = 15.2 Hz,
163.9 (C-5), 158.9 (C-2′), 135.8 (C-1), 131.8 (C-β), 131.7
(C-4′), 129.3 (C-6′), 129.1 (C-5′), 129.0 (C-α), 124.6
3

Medicinal Chemistry Research
(
C-1′), 123.4 (C-3′), 116.5 (C-6), 115.8 (C-4), 111.9 (C-2),
(2E)-1-(2-Hydroxy-4,6-dimethoxy-3,5-dimethylphenyl)-3-
(3,4,5-trimethoxyphenyl)prop-2-en-1-one (9q) H NMR
1
6
8
5
3
2.4 (OCH at C-3), 60.2 (OCH at C-5), 9.0 (CH at C-6),
3
3
3
.8 (CH at C-4); Anal. calc. for C H FO : C, 69.08; H,
(CDCl , 400 MHz, δ in ppm): 13.10 (1H, s, OH at C-2),
3
19 19
4
3
+
.80; found: C, 69.04; H, 5.71; %; LC-MS: (M + H) at
7.86 (1H, d, J = 15.6 Hz, CH=C, H-β), 7.86 (1H, d, J =
15.6 Hz, CH=C, H-α), 6.87 (2H, s, H-2′, 6′), 3.92 (6H, s,
OCH at C-4, 6), 3.90 (3H, s, OCH at C-4′), 3.76 (3H, s,
31.
3
3
(
2E)-3-(Furan-2-yl)-1-(2-hydroxy-4,6-dimethoxy-3,5-
OCH at C-3′), 3.66 (3H, s, OCH at C-5′), 2.17 (6H, s,
3
3
1
13
dimethylphenyl)prop-2-en-1-one (9n) H NMR (CDCl3,
00 MHz, δ in ppm): 13.18 (1H, s, OH at C-2), 7.87 (1H, d,
J = 15.2 Hz, H-β), 7.67 (1H, d, J = 15.2 Hz, H-α), 7.52
CH at C-3, 5); C NMR (CDCl , 100 MHz, δ in ppm):
3 3
4
193.9 (C=O), 163.7 (C-4),161.7 (C-6), 158.7 (C-2), 143.6
(C-β), 140.7 (C-4′), 130.8 (C-1′), 125.9 (C-α), 115.7 (C-2′),
(
(
1H, d, H-4′), 6.72 (1H, d, H-2′), 6.51 (1H, m, H-3′), 3.75
111.9 (C-3), 105.9 (C-5), 62.3 (OCH at C-4′), 61.1 (OCH3
3
3H, s, OCH at C-4), 3.68 (3H, s, OCH at C-6), 2.17 (6H,
at C-4), 60.2 (OCH at C-6), 56.3 (OCH at C-3′,4′), 8.8
3
3
3
3
1
3
s, CH at C-3, 5); C NMR (CDCl , 100 MHz, δ in ppm):
(Ar–CH ), 8.7 (Ar–CH ); Anal. calc. for C H O : C,
3 3 22 26 7
3
3
1
(
1
93.4 (C=O), 163.5 (C-5), 161.6 (C-3), 158.6 (C-1), 152.0
C-1′), 145.0 (C-4′), 129.9 (C-β), 124.3 (C-α), 115.8 (C-3′),
15.6 (C-2′), 115.5 (C-6), 112.6 (C-4), 111.8 (C-2), 62.3
OCH at C-5), 60.1 (OCH at C-3), 8.8 (CH at C-5), 8.7
65.66; H, 6.51; found: C, 65.35; H, 6.47; %; LC-MS: (M +
H) at 403.
+
(
(
Antitubercular activity using Alamar Blue Dye
3
3
3
CH at C-4); Anal. calc. for C H O : C, 67.54; H, 6.00;
3
17 18
5
+
found: C, 67.55; H, 6.11; %; LC-MS: (M + H) at 303.
The antimycobacterial activity of compounds was assessed
against Mycobacterium tuberculosis using Microplate Ala-
mar Blue assay (MABA). This methodology is non-toxic,
uses a thermally stable reagent and shows good correlation
with proportional and BACTEC radiometric method.
Briefly, 200 µL of sterile deionized water was added to all
outer perimeter wells of sterile 96-wells plate to minimized
evaporation of medium in the test wells during incubation.
The 96-wells plate received 100 µL of the Middlebrook 7H9
broth and serial dilution of compounds was made directly
on the plate. The final drug concentrations tested were
100–0.2 µg/mL. Plates were covered and sealed with par-
affin and incubated at 37 °C for five days. After this time,
25 µL of freshly prepared 1:1 mixture of Almar Blue
reagent and 10% tween 80 was added to the plate and
incubated for 24 h. A blue color in the well was interpreted
as no bacterial growth, and pink color was scored as growth.
The MIC was defined as lowest drug concentration which
prevented the color change from blue to pink (Franzblau
et al. 1998; Collins and Franzblau 1997).
(
2E)-1-(2-Hydroxy-4,6-dimethoxy-3,5-dimethylphenyl)-3-(3-
1
methyl-2,4,6-trimethoxyphenyl)prop-2-en-1-one (9o)
NMR (CDCl , 400 MHz, δ in ppm): 13.43 (1H, s, Ar–OH),
8
H
3
.34 (1H, d, J = 16.0 Hz, H-β), 8.25 (1H, d, J = 16.0 Hz, H-
α), 6.29 (1H, s, H-5′), 3.94 (3H, s, OCH at C-6), 3.89 (3H,
3
s, OCH at C-2′), 3.74 (3H, s, OCH at C-6′), 3.74 (3H, s,
3
3
OCH at C-4′), 3.67 (3H, s, OCH at C-4), 2.16 (3H, s, CH
3
3
3
at C-3), 2.16 (3H, s, CH at C-3′), 2.10 (3H, s, CH at C-5);
3
3
1
3
C NMR (CDCl , 100 MHz, δ in ppm): 195.4 (C=O),
3
1
(
1
(
(
63.8 (C-4), 162.9 (C-6), 161.6 (C-2), 161.0 (C-β), 160.5
C-α), 159.5 (C-1), 158.8 (C-3), 135.7 (C-5), 127.1 (C-1′),
15.2 (C-2′), 112.6 (C-6′), 112.6 (C-6′), 112.3 (C-4′), 110.9
C-3′), 91.3 (C-5′), 62.2 (Ar–OCH ), 61.0 (Ar–OCH ), 60.1
3
3
Ar′–OCH ), 55.8 (Ar′–OCH ), 55.6 (Ar′–OCH ), 8.9 (Ar–
3
3
3
CH ), 8.7 (Ar–CH ), 8.4(Ar′–CH ); Anal. calc. for
C H O : C, 66.33; H, 6.78; found: C, 67.26; H, 6.52; %;
LC-MS: (M + H) at 417.
3
3
3
2
3
28
7
+
(
2E)-1-(2-Hydroxy-4,6-dimethoxy-3,5-dimethylphenyl)-3-(3-
1
phenoxyphenyl)prop-2-en-1-one (9p) H NMR (CDCl3,
00 MHz, δ in ppm): 13.07 (1H, s, OH at C-2), 7.92 (1H, d,
4
Results and discussion
J = 15.5 Hz, H=C, H-β), 7.78 (1H, d, J = 15.5 Hz, CH=C,
H-α), 7.37 (4H, m, H-4′, 5′, 9′, 11′), 7.27 (1H, s, H-2′), 7.14
Chemistry
(
1H, t, H-6′), 7.04 (3H, q, H-8′, 10′, 12′), 3.75 (3H, s,
OCH at C-6), 3.63 (3H, s, OCH at C-4), 2.16 (6H, s, CH
3
Chalcones are versatile molecules and important inter-
mediates for the synthesis of various flavonoids and dif-
ferent heterocyclic compounds (Gomes et al. 2017; Prasad
et al. 2008). Chalcones can be synthesized in different ways
(Gomes et al. 2017; Narender and Reddy 2007; Eddarir
et al. 2003).The present chalcones (9a–q) were synthesized
by Claisen–Schmidt reaction. In this reaction, the con-
densation was achieved between 2-hydroxy-3,5-dimethyl-
4,6-dimethoxy acetophenone (7) and substituted aromatic
3
3
1
3
at C-3, 5); C NMR (CDCl , 100 MHz, δ in ppm): 193.9
3
(
1
C=O), 163.8 (C-5), 161.7 (C-3), 158.7 (C-1), 157.9 (C-7′),
56.8 (C-3′), 142.5 (C-β), 137.1 (C-1′), 130.2 (C-5′), 129.9
(
(
(
C-9′, 11′), 127.4 (C-5′), 123.7 (C-α), 123.3 (C-10′), 120.4
C-5′), 119.1 (C-8′, 12′), 118.3 (C-4′), 115.7 (C-2′), 111.8
C-2), 62.2 (Ar–OCH ), 60.1 (Ar–OCH ), 8.8 (Ar–CH ),
3
3
3
8
.7 (Ar–CH ); Anal. calc. for C H O : C, 74.24; H, 5.98;
3 25 24 5
+
found: C, 74.67; H, 5.87; %; LC-MS: (M + H) at 405.

Medicinal Chemistry Research
aldehydes (8a–q) in the presence of aqueous KOH and
ethanol at room temperature. The intermediate (7) in turn
was prepared from phloroglucinol (1) starting material by
methylation, formylation and wolf-kishner reductions as
reported in the experimental part. The synthetic protocol is
shown in Schemes 1, 2, and 3 and their physical constants
are given in Table 1. The reactions of chalcones were
monitored throughout by thin layer chromatography using
20% ethyl acetate in Hexane as mobile phase. All the
chalcones (9a–q) were purified by column chromatography
using eluant, n-hexane and further recrystallized with
methanol. All the chalcones analogs were synthesized in
satisfactory yields ranging between 50 and 85% and were
characterized by infrared, nuclear magnetic resonance and
mass spectral data, while the purity of compounds was
checked by microanalysis (C, H, and N analysis). All the
O
Scheme 2 Synthesis of new C-
dimethylated heterocyclic
chalcone (9i): Reagents and
conditions: (i) N-methyl indole
aldehyde, Aq KOH/EtOH, 24 h
CH3
H
CH
3
H CO
OH
H
3
CO
OH
3
(
i)
CH3
N
H C
N
3
H C
3
H CO
O
H CO
O
3
3
(
7)
(8i)
(9i)
CH3
CH
3
Scheme 3 Synthesis of new C-
dimethylated heterocyclic
chalcone (9n): Reagents and
conditions: (i) Furfural, Aq
KOH/EtOH, 24 h
H CO
OH
O
H
3
CO
OH
3
(i)
CH3
O
H
O
H C
3
H C
3
H CO
O
H CO
O
3
3
(7)
(8n)
(9n)
Table 1 The physical constant
for C-dimethylated chalcones
(9a–q)
4
1
Me
R
R
Me
Me
5
3
MeO
Me
OH
R
R
MeO
Me
OH
MeO
Me
OH
MeO
M
2
R
N
Me
O
OMe O
OMe O
O
OMe O
9a-c, 9e-h, 9j-m, 9o-q
9i
9d
Compound
R1
R2
H
R3
R4
R5
% Yield
Mp (°C)
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
a
b
c
H
OCH3
OCH3
H
H
H
–
H
H
H
–
80
65
55
56
75
60
68
50
68
75
52
75
55
72
85
63
65
88–90
OCH3
OCH3
98–99
H
H
N-(CH3)2
145–147
122–123
124–126
111–112
107–108
119–121
147–149
208–210
105–107
121–122
68–70
d
e
–
–
–
H
OCH3
OCH3
Cl
H
H
H
H
–
H
H
H
H
–
f
H
H
H
H
–
g
h
i
H
F
H
Br
–
–
j
OCH3
H
OCH3
OCH3
H
H
H
H
H
–
OCH3
k
l
H
OC2H5
H
Cl
H
H
m
n
o
p
q
F
H
H
H
–
–
–
–
188–190
96–98
OCH3
H
CH3
OCH3
H
H
H
OCH3
H
OC H5
74–75
6
H
OCH3
OCH3
OCH3
H
123–124

Medicinal Chemistry Research
Table 2 Antitubercular activity (MIC90) of C-dimethylated chalcones (9a–q)
Compound Compound structure
Anti-tuberculosis activity (MIC90)
(
µg/mL)
(µM)
4.67
9
a
1.6
9
9
b
c
1.6
1.6
3.98
4.50
9
9
d
e
6.25
6.25
17.54
16.78
9
9
f
12.5
12.5
36.04
37.84
g
9
h
6.25
15.97
9
9
i
6.25
6.25
17.10
15.53
j

Medicinal Chemistry Research
Table 2 (continued)
Compound
Compound structure
Anti-tuberculosis activity (MIC90)
µg/mL) (µM)
(
9
k
1.6
4.14
9
9
l
6.25
6.25
18.02
18.92
m
9
9
n
o
Me
6.25
1.6
20.67
3.84
MeO
Me
OH
O
OMe O
9
9
p
q
3.12
7.17
–
–
Pyrazinamide
3.125
25.38

Medicinal Chemistry Research
Table 2 (continued)
Compound
Compound structure
Anti-tuberculosis activity (MIC90)
(µg/mL)
(µM)
Streptomycin
6.25
10.75
Ciprofloxacin
3.125
9.43
MICs reported in literature; Pyrazinamide, 50 µg/ml (Sharma et al. 2010), Streptomycin, 0.50 µg/ml (Biava et al. 2009) and 6.25 µg/ml (Balaji et al.
017), Ciprofloxacin, 4.7 µM (Prasanna et al. 2010).
2
chalcones were characterized by nuclear magnetic reso-
nance and mass spectra. The prototype chalcone, 9o showed
a singlet at δ ppm 6.29 corresponding to the aromatic proton
activity of the chalcones (9a–p) was found to be moderate
to high with minimum inhibitory concentrations (MICs)
ranging between 1.6 and 12.5 µg/mL (3.84 and 37.84 µM).
The MIC90 was be defined as the minimum concentration of
drugs required to inhibit 90% mycobacterium growth. The
chalcone analogs 9a, 9b, 9c, 9k, 9o, and 9p were highly
active against Mycobacterium tuberculosis H Rv with
(
H-5′), a singlet at δ ppm 13.43 corresponding phenolic
proton (Ar–OH), two doublet at δ ppm 8.34 and 8.25 cor-
responding to H-β and H-α (Ar–C(=O)CH=CH–Ar)
respectively, a singlet at δ ppm 3.94, 3.89, 3.74, and 3.67
3
7
corresponding to three protons of OCH (at C-6), three
MICs of 4.67, 3.98, 4.50, 4.14, 3.84, and 7.71 µM respec-
tively, while the remaining chalcones (9j, 9h, 9e, 9i, 9d, 9l,
9m, 9n, 9f, and 9g) showed moderate antitubercular activity
with MICs ranging between 15.53 and 37.84 µM. When
compared with the MICs of standard drug streptomycin and
ciprofloxacin the chalcones, 9a, 9b, 9c, 9k, and 9o were
found to more than two-folds more active. Further, the
chalcones, 9b, 9k, and 9o were found to be more than six
folds, while the chalcones, 9a and 9c were found to be more
than five-folds more active than pyrazinamide. The anti-
tubercular activity of chalcone, 9p was found to be more
than three-folds more active than pyrazinamide. The anti-
tubercular activity of most of the chalcones (except chal-
cones, 9f and 9g) was found to be higher than the standard
drug, pyrazinamide. The antitubercular activity of the
chalcones (9a–p) and the standard drugs in the form of
MICs is given in Table 2. The chalcones (9a–p) showed an
MIC90 ranging between 1.6 and 12.5 µg/mL were found to
be highly potent than our previously synthesized chalcones
and their analogs (Vasu Babu et al. 2013; Balaji et al. 2017).
The antitubercular data showed that substitutions with
halogens (chloro and fluoro) at position 2′ (ortho) (9l and
9k) was found to be more favorable than at position 4′
(para) (9f and 9g) of the phenyl ring. The five member
heterocyclic ring (9n) and heterocyclic fused phenyl ring
3
protons of OCH (at C-2′), six protons of OCH (at C-6′ and
3
3
C-4′), and three protons of OCH (at C-4) respectively,
3
while a singlet at δ ppm 2.16, and 2.10 corresponding to six
protons of CH (at C-3 and C-3′) and three protons of CH
3
3
13
(
at C-5), respectively. The C NMR showed peaks at δ
ppm, 195.4 (C=O), 163.8 (C-4), 162.9 (C-6), 161.6 (C-2),
61.0 (C-β), 160.5 (C-α), 159.5 (C-1), 158.8 (C-3), 135.7
C-5), 127.1 (C-1′), 115.2 (C-2′), 112.6 (C-6′), 112.6 (C-
′), 112.3 (C-4′), 110.9 (C-3′), 91.3 (C-5′), 62.2
Ar–OCH ), 61.0 (Ar–OCH ), 60.1 (Ar′–OCH ), 55.8 (Ar
1
(
6
(
3
3
3
′
–OCH ), 55.6 (Ar′–OCH ), 8.9 (Ar–CH ), 8.7 (Ar–CH ),
3
3
3
3
and 8.4(Ar′–CH ). The LC-MS showed a peak at 417 cor-
3
+
responding to (M + H) .
Antitubercular activity
The antimycobacterial activity of compounds was assessed
against Mycobacterium tuberculosis (Mtb) using Microplate
Alamar Blue assay (MABA) (Franzblau et al. (1998);
Collins and Franzblau (1997)). Among the seventeen
chalcones analogs (9a–q), sixteen chalcones analogs (9a–p)
were evaluated for their antitubercular activity against
Mycobacterium tuberculosis H Rv strains using Micro-
37
plate Alamar Blue assay (MABA). The antitubercular

Medicinal Chemistry Research
Table 3 Molecular docking scores of C-dimethyl chalcones (9a–q) and reported potent chalcones against Mycobacterium tuberculosis protein
tyrosine phosphatases (MtbPtp)
Compd. code Structure
Docking scores
Compd. codea Reported chalconesa
Docking scores
MtbPtpA MtbPtpB
MtbPtpA MtbPtpB
9
a
−2.358
−2.628
−5.532
−5.525
8
ND
ND
−4.528
−4.800
9
b
42
14
9
c
−2.759
−5.520
ND
−4.801
9
9
9
9
d
e
f
ND
−5.486
−5.232
−6.445
−5.549
12
30
1
ND
ND
ND
ND
−6.487
−5.245
−5.522
−6.107
−3.187
ND
g
ND
19
9
9
9
h
i
ND
ND
ND
−5.608
−5.477
−5.524
7
ND
ND
ND
−4.365
−5.835
−6.048
23
20
j
9
9
k
l
−3.387
−3.282
−5.758
−6.073
24
39
−4.301
−5.074
−5.420
ND

Medicinal Chemistry Research
Table 3 (continued)
Compd. code Structure
Docking scores
Compd. codea Reported chalconesa
Docking scores
MtbPtpA MtbPtpB
MtbPtpA MtbPtpB
9
m
n
−2.356
−4.480
10
ND
−5.087
9
−2.351
−5.434
13
18
ND
−3.852
9
o
−2.441
−5.392
ND
−5.181
9
9
p
q
−3.417
−2.660
−6.109
−5.726
28
11
ND
ND
−5.866
−5.739
ND Not docked
Fig. 2 The 2D binding mode of
chalcone, 9e against
Mycobacterium tuberculosis
protein tyrosine phosphatases

Medicinal Chemistry Research
Fig. 3 The binding mode of
chalcone, 9e and compound 30
(
Chiaradia et al. 2012) against
Mycobacterium tuberculosis
protein tyrosine phosphatases
Fig. 4 The binding mode of
chalcone, 9n and compound 24
(
Chiaradia et al. 2012) against
Mycobacterium tuberculosis
protein tyrosine phosphatases
(
9d and 9i), showed less antitubercular activity than the
Molecular docking studies
parent substituted phenyl ring. N,N-dimethyl amino sub-
stitution (9c) at para position was also found to be favor-
able. The chalcone with 3′-methyl-2′,4′,6′-trimethoxy
substitution (9o) on the phenyl ring showed the most pro-
mising antitubercular activity than 2′,3′,4′-trimethoxy (9b),
Zhou et al. verified that Mycobacterium tuberculosis protein
tyrosine phosphatases (MtbPtpA and MtbPtpB) are vital for
intracellular bacterial persistence, and therefore suggested
that inactivation of these enzymes reduce the mycobacterial
growth (Zhou et al. 2010). The chalcones have been well
established as inhibitors of MtbPtps (Chiaradia et al. 2008,
3
-ethoxy-4-methoxy (9k), 2′,4′,6′-trimethoxy (9j), and
′,4′-dimethoxy (9e) substitutions.
3

Medicinal Chemistry Research
2
012). Further, the validated docking methods against X-
trimethoxy substitution (9o) on the phenyl ring showed the
most promising antitubercular activity than 2′,3′,4′-tri-
methoxy (9b), 3-ethoxy-4-methoxy (9k), 2′,4′,6′-tri-
methoxy (9j), 3′,4′-dimethoxy (9e) substitutions. Further
molecular docking studies against Mycobacterium tuber-
culosis protein tyrosine phosphatases (MtbPtpA and
MtbPtpB) showed sound interactions with key active site
residues of MtbPtpB. Further, the residues include Arg166,
Ala162, and Lys164 imparted the vital H-bonds, salt-
bridges and π-cation interactions with 2-hydroxy group and
functional ring and the substituted ring exhibited π–π
interactions with Phe98. 2-Hydroxy group of fundamental
ring in the current series is responsible for making stable
interactions with above said key residues.
ray crystal structures of both MtbPtpA and MtbPtpB has
been carried out using Glide module of Schrodinger’s LLC
for chalcones (9a–q). Most of the compounds were found to
have sound interactions with key active site residues of
MtbPtpB. In case of MtbPtpB, the residues include Arg166,
Ala162, and Lys164 imparted the vital H-bonds, salt-
bridges, and π-cation interactions with the 2-hydroxy group
and functional ring. The substituted ring exhibited π–π
interactions with Phe98. To validate our hypothesis, the pre-
validated chalcone MtbPtp inhibitors reported by Chiaradia
et al. were chosen to evaluate our docking studies (Chiar-
adia et al. 2012). The structurally similar dioxolane sub-
stituted compounds from Chiaradia et al. has been
selectively chosen for docking along with our tested com-
pounds (Chiaradia et al. 2012). The comparative binding
studies provided that, 2-hydroxy group of the fundamental
ring in the current series is responsible for making stable
interactions with above said key residues. Whereas, refer-
ence dioxolane derived chalcone has failed to exhibit this
type of interactions with MtbPtpb enzyme. Altering with
different substitutions on aldehyde ring of 2-hydroxy series
or changing with heteroaryl rings has not provided any
fruitful interactions with MtbPtpb enzyme. Similarly,
binding pattern of reference and 2-hydroxy substituted
chalcone with MtbPtpA enzyme were not identical. How-
ever, the acetophenone portions of both analogs were found
to have interactions with Asn47 and Trp48 residues of
MtbPtpA (Chiaradia et al. 2012). The docking interpreta-
tions cleared the probable selectivity (selective to MtbPtpb)
of the tested 2-hydroxy chalcone analogs. The comparison
of docking scores of the reference and chalcones (9a–q) is
given in Table 3. The 2D binding interaction of chalcone
Acknowledgements The authors are highly thankful to Dr. G.V.
Subbaraju, CEO, Natsol Labs, Visakhapatnam for constant support
and Prof. G. Rambabu, Gitam University, Hyderabad for helpful
suggestions in docking studies. The authors also thank UGC for the
financial assistance (F.No.39-752/2011).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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