Synthesis and evaluation of small libraries of triazolylmethoxy chalcones, flavanones and 2-aminopyrimidines as inhibitors of mycobacterial FAS-II and PknG

Article abstract of DOI:10.1016/j.bmc.2012.07.009

A synthetic strategy to access small libraries of triazolylmethoxy chalcones 4{1-20}, triazolylmethoxy flavanones 5{1-10} and triazolylmethoxy aminopyrimidines 6{1-17} from a common substrate 4-propargyloxy-2-hydroxy acetophenone using a set of different

Full text of DOI:10.1016/j.bmc.2012.07.009

Bioorganic & Medicinal Chemistry 20 (2012) 5150–5163
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Bioorganic & Medicinal Chemistry
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Synthesis and evaluation of small libraries of triazolylmethoxy chalcones,
flavanones and 2-aminopyrimidines as inhibitors of mycobacterial
FAS-II and PknG
Namrata Anand a, Priyanka Singh a,b, Anindra Sharma a, Sameer Tiwari c, Vandana Singh c,
Diwakar K. Singh c, Kishore K. Srivastava c, B. N. Singh c, Rama Pati Tripathi a,
⇑
a Medicinal & Process Chemistry Division, Central Drug Research Institute, CSIR, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226001, India
b National Institute of Pharmaceutical Education and Research, Raebareli 229010, India
c Microbiology Division, Central Drug Research Institute, CSIR, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A synthetic strategy to access small libraries of triazolylmethoxy chalcones 4{1–20}, triazolylmethoxy
Received 9 May 2012
Revised 2 July 2012
Accepted 3 July 2012
Available online 14 July 2012
flavanones 5{1–10} and triazolylmethoxy aminopyrimidines 6{1–17} from
a common substrate
4-propargyloxy-2-hydroxy acetophenone using a set of different reactions has been developed. The
chalcones and flavanones were screened against mycobacterial FAS-II pathway using a recombinant
mycobacterial strain, against which the most potent compound showed ꢀ88% inhibition in bacterial
growth and substantially induction of reporter gene activity at 100 lM concentration. The triazolylmeth-
Keywords:
oxy aminopyrimdines were screened against PknG of Mycobaceterium tuberculosis displaying moderate to
Triazolylmethoxy chalcones
2-Aminopyrimidines
Triazolylmethoxy flavanones
Mycobacterium tuberculosis
FAS-II
good activity (23–53% inhibition at 100
lM), comparable to the action of a standard inhibitor.
Ó 2012 Elsevier Ltd. All rights reserved.
PknG
1. Introduction
including 1,2,3-triazoles (which have been used in potent anti-TB
compounds16,17 via blocking the biosynthesis of certain bacterial
lipids), flavanones (which exhibit a wide range of relevant biological
activities18–23), and aminopyrimidines (important as inhibitors of
different types of kinases24–26 among many other effects27–29). Very
recently we have shown a 2-aminopyrimidine derivative to possess
potent antitubercular activity.30 Furthermore, a comparison of the
antitubercular activity of triazolylmethoxy chalcones with their
cyclic counterparts, the flavanones, was of interest. Figure 1 shows
the approach to a small library of the above motifs starting from a
common 4-propargyloxy acetophenone precursor. No sophisticated
instrumentation, conditions, or reagents are required for the trans-
formations, all of which take place under mild conditions.
The concept of ‘molecular hybridization’ in drug design aims
primarily to combat drug resistance and to enrich existing arsenals
of anti-infective agents.1,2 It usually involves the combination of
two or more pharmacophores or chemical entities either linked
with one another or fused together to create a new molecule.3
The selection of the pharmacophores is based upon their known
bioprofiles, with the hope that the resulting hybrid molecules
may exhibit synergistic or additive pharmacological activities.4,5
The development of efficient synthetic strategies to access such
molecules is therefore warranted.
Chalcones are versatile molecular scaffolds in nature and the lab-
oratory, exhibiting numerous beneficial biological activities such as
cytotoxicity to pathogenic organisms6–10 and cancer cells,11 anti-
inflammatory properties,12 inhibition of key enzymes,13 and antiox-
idant behavior.14,15 Of particular interest to us is the antitubercular
activity of natural and synthetic chalcones, via inhibition of FAS-II
pathway enzymes such as enoyl-ACP-reductase, b-ketoacyl-ACP
reductase and b-hydroxyacyl-ACP-dehydratase (ACP = acyl carrier
protein).4 We sought to augment the activity of chalcones against
FAS-II enzymes by pairing them with other pharmacophores,
The resulting triazolylmethoxy chalcones and flavanones were
screened for their antitubercular activity via FasII pathway inhibi-
tion while triazolylmethoxy aminopyrimidines were evaluated for
mycobacterial serine-threonine protein kinase (STPK) inhibitory
activity followed by in vitro evaluation against M. tuberculosis H37Rv.
2. Results and discussion
2.1. Chemistry
The intermediate 2-hydroxy 4-propynyloxy acetophenone (1)
was prepared by the reaction of 2,4-dihydroxy acetophenone (A)
⇑
Corresponding author.
E-mail address: rpt.cdri@gmail.com (R.P. Tripathi).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.009

N. Anand et al. / Bioorg. Med. Chem. 20 (2012) 5150–5163
5151
Figure 1. Preparation of three different prototypes from a common scaffold 2-hydroxy-4-propynyloxy acetophenone.
Scheme 1. Preparation of 2-hydroxy-4-(1-araalkyl/alkyl-1H-1,2,3-triazol-4-yl)-methoxy acetophenone.
with propargyl bromide, in the presence of anhydrous K2CO3 and
tetrabutylammonium bromide (20 mol %) in acetone at ambient
temperature as recently reported by us31 in quantitative yield.
2-Hydroxy-4-propynyloxy acotophenone (1) was elaborated with
azides (B){1–3} and aromatic aldehydes (C){1–8}in standard trans-
formations to give triazoles 2{1–3} and triazolylmethoxy chalcones
4{1–20}. The ‘click reaction’ of 2-hydroxy 4-propynyloxy acetophe-
none (1) with appropriate azides viz n-hexyl azide, benzyl azide
and 4-bromobenzyl azide A{1–3} at ambient temperature in pres-
a mixture of 1:1 tert-BuOH:H2O, led to the formation of respective
triazolylmethoxy acetophenones 2{1–3} regioselectively in very
good yields (Scheme 1). The presence of the group 1,2,3-triazole
was indicated by a singlet in the 1H NMR spectra at approximately
7.6 ppm, and signals for triazolyl C-4 and C-5 at approximately 143
and 132 ppm, respectively, in 13C NMR.16,31
2.1.1. Synthesis of triazolylmethoxy chalcones
There are two possible routes to prepare triazolylmethoxy chal-
cones, In our experiments with route (a) reaction of propynyloxy
ence of CuSO4.5H2O (2 mol %) and sodium-L-ascorbate (5 mol %) in
Scheme 2. Preparation of triazolylmethoxy chalcones via route (a).
Scheme 3. Synthesis of 1-[2-hydroxy-4-(1-araalkyl/alkyl-1H-1,2,3-triazol-4-yl)-methoxyl phenyl]-3-phenylprop-2-en-1-one.

5152
N. Anand et al. / Bioorg. Med. Chem. 20 (2012) 5150–5163
Table 1
Table 2
Library of 1-[2-hydroxy-4-(1-araalkyl/alkyl-1H-1,2,3-triazol-4-yl)-methoxyl phenyl]-
Synthesized triazolylmethoxy flavanone
3-phenylprop-2-en-1-ones
Compound
R
R0
Mp (°C)
Yield (%)
Compound
R
Ar
Mp (°C)
Yield (%)
5{1}
5{2}
5{3}
5{4}
5{5}
5{6}
5{7}
5{8}
5{9}
5{10}
n-Hexyl
n-Hexyl
n-Hexyl
n-Hexyl
Benzyl
Benzyl
Benzyl
4-Br-benzyl
4-Br-benzyl
4-Br-benzyl
Phenyl
120–123
133–135
128–130
115–117
118–120
135–138
123–125
125–127
132–135
120–124
79
75
85
82
85
50
87
80
60
88
4{1}
4{2}
4{3}
4{4}
4{5}
4{6}
4{7}
4{8}
n-Hexyl
n-Hexyl
n-Hexyl
n-Hexyl
n-Hexyl
n-Hexyl
n-Hexyl
n-Hexyl
benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Phenyl
4-Br-phenyl
4-Cl-phenyl
2-Cl,6-F-phenyl
4-OCH3-phenyl
4-Isopropylphenyl
4-Propargyloxyphenyl
Naphthyl
144–146
154–156
150–153
156–158
150–152
146–148
149–152
142–145
144–146
152–154
156–158
148–150
142–145
150–152
151–153
146–148
156–158
157–159
144–146
150–152
91
92
93
93
93
92
82
92
90
91
92
88
91
94
94
94
93
92
93
92
4-Br-phenyl
4-OCH3-phenyl
4-Isopropylphenyl
Phenyl
4-Cl-phenyl
4-Isopropylphenyl
Phenyl
4-Br-phenyl
4-Isopropylphenyl
4{9}
Phenyl
4-Cl-phenyl
4{10}
4{11}
4{12}
4{13}
4{14}
4{15}
4{16}
4{17}
4{18}
4{19}
4{20}
2-Cl,6-F-phenyl
4-OCH3-phenyl
4-Isopropylphenyl
4-Propargyloxyphenyl
Naphthyl
Phenyl
4-Br-Phenyl
2-Cl,6-F-phenyl
4-Isopropylphenyl
Naphthyl
presence of electron deficient groups 4{2, 10 and 17} decreased
the yield of corresponding flavanones 5{2, 6 and 9} as compared
to unsubstituted 4{1, 9, and 16} aryl ring (Table 2). Structures of
these flavanones were established on the basis of their spectro-
scopic data and microanalyses. The characteristic benzylic protons
(OCH) of the dihydrochromanone moiety were visible as dd at
around d 5.4 with with J1 = 3.0 Hz and J2 = 13.0 Hz; while the two
methylene protons of the same were observed as dd at two differ-
ent field strengths at round d 3.0 (J1 = 13.0 Hz and J2 = 17.0 Hz), and
at d 2.87 (J1 = 3.0 Hz and J2 = 17.0 Hz) respectively. The other pro-
ton and carbon signals were observed as usual.
Benzyl
Benzyl
4-Br-benzyl
4-Br-benzyl
4-Br-benzyl
4-Br-benzyl
4-Br-benzyl
acetophenone (1) with aromatic aldehydes followed by click
reaction of the preformed 1-(2-hydroxy-4-(propynyloxy) aryl)-
3-phenylpropen-1-one 3{1–2} with n-hexyl azide B{1} gave triazol-
ylmethoxy chalcone 4{1} in 73% and 4{2} in 71% yields along with
several minor products which could not be isolated and character-
ized (Scheme 2). With route (b) Claisen-Schmidt condensation of
1-(4-((1-hexyl-1H-1,2,3-triazole-4-yl) methoxy)-2-hydroxyphenyl)
ethanone 2{1} with the benzaldehyde C{1} in presence of 10% aque-
ous KOH in ethanol at 30 °C followed by acidification with cold 3 N
HCl led to the formation of respective triazolylmethoxy chalcone
4{1} in 91% isolated yield. No side products were observed during
reaction.
Similarly, triazolylmethoxy acetophenones 2{2–3} were con-
densed with electron rich and electron deficient aromatic alde-
hydes C{1–8} under the above conditions for different intervals
of time to give the respective triazolylmethoxy chalcones 4{2–
20} in good yields (Scheme 3,Table 1). These synthons have been
utilized to prepare the corresponding flavanones and 2-
aminopyrimidines.
2.1.3. Synthesis of triazolylmethoxy amino-pyrimidines
The triazolylmethoxy aminopyrimidines 6{1–17} were pre-
pared in a simple and straight forward manner by the reaction of
their corresponding triazolylmethoxy chalcones 4{1}, 4{3–13},
4{15–17} and 4{19–20} with guanidine hydrochloride in presence
of NaH in anhydrous DMF as a solvent in moderate to good yields
(Scheme 5). It was observed that aromatic substitutents in the
chalcones do not have any substantial effect on the yields of the
resulting 2-aminopyrimidines (Table 3).
All these compounds were characterized on the basis of their
spectroscopic (IR, ESI-MS, 1H NMR and 13C NMR) data and microa-
nalyses. In general, the IR spectra of the compounds exhibited
characteristic strong absorbance in the range of 3490–3200 cmꢁ1
for the -NH2 group. In the 1H NMR spectra of these compounds a
singlet at around d 7.80 accounted the triazolyl proton while the
two exchangeable NH2 protons were observed at around d 6.20.
The trans-(E) geometry of the chalcone double bond was evi-
dent by the large olefinic coupling constant between the relevant
signals in the 1H NMR spectrum (J = 15 Hz). Details of these syn-
thetic methods and spectroscopic data are given in Supplementary
data.
2.2. Biology
2.2.1. FAS-II inhibitory activity of triazolyl methoxy chalcones
and flavanones
2.1.2. Synthesis of triazolylmethoxy flavanone
FAS-II inhibitory activity was analyzed using a recombinant
non-pathogenic mycobacterial strain, Mycobacterium aurum, which
contains the M. tuberculosis H37Rv kas operon promoter in fusion
with an E. coli lacZ reporter gene.32,33 The recombinant strain
shows continued expression of the reporter gene under the influ-
ence of the kas operon promoter during basal conditions, but the
promoter responds to the inhibition of FAS-II pathway by inducing
the additional quantifiable expression of the reporter gene. The
inducibility rendered by the promoter is pathway specific and is
not singularly dependent on the selective inhibition of a candidate
The reaction of the above selected triazolylmethoxy chalcones
4{1}, 4{2}, 4{5}, 4{6}, 4{9}, 4{10}, 4{13}, 4{16}, 4{17} and 4{19} sep-
arately with sodium acetate in ethanol and water (1:1) at 70–80 °C
for different time intervals gave the respective triazolylmethoxy
phenyl chromanone 5{1–10} in good yields (Scheme 4). The effect
of substituents on yields of the products 5{1–10} is dependent on
the nature of substitution in aryl ring at C-3 in the chalcone. In gen-
eral, presence of electron releasing group 4{5, 6, 13 and 19} in aryl
ring enhanced the yield of products 5{3, 4, 7 and 10} whereas
Scheme 4. Synthesis of 7-(1-araalkyl-1H-1,2,3-triazol-4-yl)methoxy-2-phenylchroman-4-one.

N. Anand et al. / Bioorg. Med. Chem. 20 (2012) 5150–5163
5153
Scheme 5. Synthesis of 2-(2-amino-6-phenylpyrimidine-4-yl)-5-(1-araalkyl/alkyl-1H-1,2,3-triazol-4-yl)-methoxy phenol.
Table 3
Table 4
Synthesized triazolylmethoxy aminopyrimidines
Percent inhibition of bacterial growth by synthesized triazolyl -methoxy chalcones
and flavanones with CC50 values of the potent compounds
Compound
R
Ar
mp (°C)
Yield (%)
Compound
% Inhibition (50
l
M)
% Inhibition (100
l
M)
CC50 (lM)
6{1}
6{2}
6{3}
6{4}
6{5}
6{6}
6{7}
6{8}
n-Hexyl
n-Hexyl
n-Hexyl
n-Hexyl
n-Hexyl
n-Hexyl
n-Hexyl
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Phenyl
4-Cl-phenyl
155–156
192–194
185–187
180–182
164–166
172–175
178–180
185–187
200–202
>200
180–182
178–180
196–198
190–193
210–213
183–185
188–190
55
54
51
52
59
49
56
54
51
48
52
54
49
60
59
56
51
1
2.0 1.0
93.1 2.7
23.7 7.9
70.4 2.4
16.8 4.6
69.6 3.6
6.1 4.2
71.6 0.8
6.2 0.2
0
39.6 10.2
5.5 0.2
49.9 4.7
11.6 1.9
17.6 3.2
25.5 2.0
0
35.6 3.0
91.8 1.7
75.9 0.3
73.7 1.2
57.7 2.0
76.5 4.0
ND
ND
ND
ND
ND
>100.0
ND
>100.0
ND
ND
ND
ND
80.0 13.27
ND
ND
ND
ND
ND
ND
71.7 5.77
ND
65.8 2.58
ND
ND
ND
20.3 2.57
57.5 25.98
ND
ND
ND
23.8 3.33
ND
87.5 17.68
ND
2-Cl,6-F-phenyl
4-OCH3-phenyl
4-Isopropyl phenyl
4-Propargyloxyphenyl
Naphthyl
Phenyl
4-Cl-phenyl
2-Cl,6-F-phenyl
4-OCH3-phenyl
4-Isopropyl phenyl
Naphthyl
2{1}
2{2}
2{3}
4{1}
4{2}
4{3}
4{4}
4{5}
4{6}
4{7}
4{8}
4{9}
4{10}
4{11}
4{12}
4{13}
4{14}
4{15}
4{16}
4{17}
4{18}
4{19}
4{20}
5{1}
5{2}
5{3}
5{4}
5{5}
5{6}
5{7}
5{8}
5{9}
5{10}
59.0 1.9
79.1 4.2
44.7 5.0
31.4 5.9
56.0 7.2
18.8 6.5
88.0 0.8
28.0 0.6
74.5 1.5
68.7 7.0
12.7 3.0
22.0 4.3
51.6 5.7
85.3 5.5
28.0 0.6
83.0 6.5
17.8 3.2
63.2 3.2
70.2 6.8
75.1 1.7
65.7 1.0
56.4 3.0
74.8 2.5
79.8 0.8
79.3 5.1
66.1 2.5
70.6 4.6
72.5 7.2
6{9}
6{10}
6{11}
6{12}
6{13}
6{14}
6{15}
6{16}
6{17}
Benzyl
4-Br-Benzyl
4-Br-Benzyl
4-Br-Benzyl
4-Br-Benzyl
Phenyl
4-Br-phenyl
4-Isopropyl phenyl
Naphthyl
0
2.6 0.1
53.8 2.5
11.6 1.9
53.1 0.4
0
gene product. Notably, the FAS-II pathway in mycobacteria is
inhibited by isoniazid (INH), ethionamide (ETH) and thiolactomy-
cin (TLM). INH and ETH target inhA while TLM targets KasA, but
all three drugs were shown to induce the reporter gene expression
by virtue of their inhibition of the FAS-II pathway.32
Therefore, this screening system allows for the identification of
compounds that inhibit the mycobacterial FAS-II pathway at any of
several points. The cytotoxicity of the potent compounds was
determined according to method reported by O’Brien et al with a
slight modification.
In the present study, preliminary screening with triazolylmeth-
oxy chalcones and flavanones was performed to score the inhibi-
tion of bacterial growth by measuring the decline in colony
forming units (cfu) at different concentrations of drugs (Table 4,
Fig. 2B).
0
19.0 7.2
68.9 1.9
55.9 0.8
34.1 2.5
27.0 2.3
46.9 3.6
54.7 3.8
15.8 2.3
50.0 6.4
31.0 3.3
CC50 = cytotoxic concentration of compounds, ND = not done.
As shown in Table 4, eleven compounds showed P50% inhibi-
tion of recombinant M. aurum growth at 50
l
M. These compounds
than FAS-II inhibition. Although the inhibitory concentrations used
here are high, these tests provide lead structures with a potential
unique mechanism of action that can be considered for further
development into more potent antitubercular compounds.
were assessed by the b-gal enzyme assay to monitor the inducibil-
ity under treated conditions. Enhanced level of b-gal enzyme activ-
ity was observed after treatment with seven of these compounds,
[4{4}, 4{9}, 4{16}, 4{18}, 5{2}, 5{3} and 5{7}] with respect to the
untreated control, with the response somewhat greater at higher
Analysis of structure–activity correlations of these molecules
shows that triazolylmethoxy acetophenones having an n-hexyl
group as the triazol-1-yl substitutent exhibited greater growth
inhibition of recombinant M. aurum than those with benzyl or
4-bromobenzyl groups. The presence of more than one halogen
(Cl and F) in the flavanone C-2 aryl ring also appeared to impart
better activity (compounds 4{4}, 4{11} and 4{18}) than the other
substituents. No other general trends could be discerned.With
respect to other substituents. In general, the growth inhibitory
activity and b-gal inducing power of the flavanones 5 were better
than their precursor chalcones. The triazolylmethoxy acetophe-
nones 2{2–3} were found to inhibit bacterial growth without
inhibiting the FAS-II pathway, whereas their corresponding chal-
cones and flavanones showed more potent FAS-II pathway inhibi-
tion. This suggests that the pharmacophore hybridization concept
may be quite fruitful in antitubercular development.
concentration (100 lM) (Fig. 2A). This enhanced reporter gene
activity is indicative of FAS-II pathway inhibition, which is sensed
by the kas promoter. Conversely, when the same experiment was
performed using another M. aurum recombinant strain carrying
the hsp60 promoter, no reporter gene inducibility was observed
under similar conditions (Fig. 2A). The hsp60 promoter is nonre-
sponsive to inhibition of the FAS-II pathway33 and the decline in
reporter gene activity in the treated samples is in line with the
diminishing viability of bacterial cells as the treatment dose in-
creased. Isoniazid (INH), a known FAS-II pathway inhibitor was
used as a positive control in both conditions. Four compounds
[2{1}, 2{3}, 4{2}, 5{9}] which inhibited the bacterial growth did
not show enhanced levels of b-gal enzyme in comparison to the
untreated control (Fig. 2A), suggesting activity by a pathway other

5154
N. Anand et al. / Bioorg. Med. Chem. 20 (2012) 5150–5163
Figure 2. Reporter gene expression and viability assay after treatment with the indicated compounds. (A) Induced levels of b-gal activity in treated samples of recombinant
strain M. aurum- kaspr:lacZ with respect to untreated control. INH as a positive control shows maximum inducibility. (B) Induced levels of b-gal activity in treated samples of
recombinant M. aurum strain with hsp60 promoter, M. aurum-hsp60pr:lacZ, showing a decline in b-gal activity in all cases, including INH, with respect to untreated control.
Both recombinant strains exhibit similar declines in overall viability; greater inhibition is observed with increasing concentration of compounds.
2.2.2. Screening of compounds against kinase activity
PknG with myelin basic protein (MBP) as a substrate36 using
AX20017 as positive control (Fig. 3).
Serine/threonine protein kinases (STPKs) have been shown to
be important virulence factors in various pathogenic bacteria.
Reversible protein phosphorylation by these STPKs plays a key role
in regulating many cellular processes including stress response,
regulation of cell cycle, and development. This regulatory phenom-
enon is unambiguously preserved during the course of evolution in
all forms of life. The genome sequence of Mycobacterium tuberculo-
sis H37Rv revealed the presence of 11 STPKs. Some of these kinases
have been implicated in the pathogenesis and survival of the tuber-
cle bacillus within host, particularly PknA, PknB and PknG
(Pkn = mycobacterial serine-threonine protein kinase). The last is
of particular interest, since its structure contains inhibitor-binding
pocket that is not present in any human kinase.34The compound
AX20017 specifically binds to this region of PknG, and is therefore
a useful tool for comparison to new candidates in screens for active
compounds.34,35 The compounds prepared above were therefore
also screened as inhibitors of the enzymatic activity of purified
PknG enzymatic activity was determined by quantification of
the ADP generated by the kinase using the ADP–Glo luciferase re-
porter kit (Promega, USA). The triazolylmethoxy acetophenones
and chalcones proved to be inactive, but aminopyrimidine deriva-
tives 6{2}, 6{8}, 6{9}, 6{11}, 6{13}, 6{14} and 6{17} showed respec-
tively 43%, 29%, 53%, 43%, 35%, 41% and 34% inhibition against
(STPK), while the standard inhibitor AX20017 (41%) at 100
(Table 5) However, only the aminopyrimidine 6{1} was able to in-
hibit the growth of M. tuberculosis H37Rv (MIC = 50 g/mL), sug-
lm.
l
gesting that the level of PknG inhibition exhibited by the other
compounds was insufficient, or that they could not reach the en-
zyme in the organism.
A careful SAR of these aminopyrimidine reveals that the com-
pounds 6{1} and 6{2} having n-hexyl as triazolyl ring subtituent
and phenyl/4-Cl-phenyl as 6-aryl substituent were found to be
moderate activity against mycobacterial PknG. Other compound

N. Anand et al. / Bioorg. Med. Chem. 20 (2012) 5150–5163
5155
Figure 3. ADP-Glo kinase assay on PknG with MBP as substrate, in the presence of aminopyrimidines at different concentration (Dose Response). AX20017 was used as a
positive control.
sets of reactions. Several chalcone and flavanone derivatives
showed moderate to good inhibition of the mycobacterial FAS-II
pathway. These compounds were ineffective against protein kinase
G, another attractive mycobacterial target, but several triazolyl-
methoxy aminopyrimidines showed moderate anti-PknG activity.
Further studies on the application of these methods in the synthe-
sis of biologically relevant compounds are in progress.
Table 5
Bio-evaluation of compounds against mycobacterial PknG
Compound
% Inhibition % Inhibition CC50
at 100 at 12.5
(Mean SE) (Mean SE)
(lM)
lm
lm
6{1}
6{2}
6{3}
23 1.86
43 2.94
11 0.32
0
ND
ND
ND
30.3 2.57
ND
6{4}
6{5}
6{6}
6{7}
6{8}
6{9}
6{10}
6{11}
6{12}
6{13}
6{14}
6{15}
6{16}
6{17}
4
5
0.35
0.78
ND
ND
ND
ND
18 5.22
5
ND
10 6.49
ND
ND
ND
ND
76.5 16.26
66.0 29.46
ND
42.7 8.52
>100.0
71.3 1.77
>100.0
ND
4. Experimental
4.1. Chemistry
17 0.27
11 0.76
29 3.03
53 0.61
12 0.34
43 0.61
28 4.20
35 5.97
41 0.78
19 5.84
6.32
Commercially available reagent grade chemicals were pur-
chased from Sigma–Aldrich or Spectrochem Pvt Ltd and were used
as received. All reactions were followed by TLC on E. Merck Kiesel-
gel 60 F254, with detection by UV light. Column chromatography
was performed on silica gel (60–120 mesh, E. Merck). IR spectra
were recorded as thin films or in chloroform with a Perkin–Elmer
Spectrum RX-1 (4000–450 cmꢁ1) spectrophotometer. 1H and 13C
NMR spectra were recorded on a Brucker DRX-300 in CDCl3. Chem-
ical shift values are reported in ppm relative to SiMe4 as internal
reference and J in hertz. MS were performed using a mass Spec-
trometer Jeol SX-102 and ESIMS were performed using Quattro II
(Micromass). Elemental analyses were performed on a Perkin–
Elmer 2400 II elemental analyzer
0
11 3.38
0
0
ND
0
5
0.41
ND
>100.0
34 6.10
AX20017
41 0.02
12 0.07
CC50 = cytotoxic concentration of compounds. ND = not done.
6{8}, 6{9}, 6{11}, 6{13}, 6{14} and 6{17} with benzyl group as triaz-
olyl substituent coupled with phenyl, naphthyl and 4-Cl-phenyl as
6-aryl substituent also displayed only moderate inhibition of the
enzyme.
4.1.1. 4-Propargyloxy-2-hydroxyacetophenone (1)
To a stirring mixture of 2,4-dihydroxy acetophenone (1 g,
6.5 mmol) and K2CO3 (1.07 g, 1.2 equiv) in acetone (15 mL), prop-
argyl bromide (0.59 mL, 6.3 mmol) was slowly added at ambient
temperature. Catalytic amount of TBAB (0.41 g, 20 mol %) was
added to the reaction mixture and stirred for 6 h. The reaction mix-
ture was extracted with ethylacetate, dried over anhydrous Na2SO4
and solvent was evaporated under reduced pressure. Crude prod-
uct was purified by column of silica gel using 9:1 Hexane/EtOAc
as eluent to give compound 1. White solid, mp 197 °C, yield
1.11 g (89.1%); IR mmax cmꢁ1 3411, 2357, 1640.6; 1H NMR
(300 MHz, CDCl3) d 12.6 (s, 1H, OH), 7.63 (dd, J = 7.05 and 2.3 Hz,
3. Conclusion
The FAS-II pathway in mycobacteria is responsible for the elon-
gation of carbon chain length of mycolic acid, and is absent in hu-
mans. It is therefore an excellent target for the development of
small molecules as new chemotherapeutic agents against tubercu-
losis. We describe the synthesis of new triazolylmethoxy hybrid
molecules in good yields from a common scaffold using different

5156
N. Anand et al. / Bioorg. Med. Chem. 20 (2012) 5150–5163
1H, ArH), 6.46–6.43 (m, 2H, -ArH), 4.71 (d, J = 2.3 Hz, 2H, OCH2),
2.55 (s, 3H, CH3), 2.53 (d, J = 2.28 Hz, 1H, C„CH); MS (ESI+) m/z
(M+H): 191.1; Anal. Calcd for C11H10O3: C, 69.46; H, 5.30. Found
C, 69.41; H, 5.38.
4.1.3.2.
(E)-3-(4-Bromophenyl)-1-{2-hydroxy-4-(prop-2-ynyl-
Colorless solid; mp
oxy)phenyl}prop-2-en-1-one 3{2}.
>200 °C, yield, 1.27 g (65%); IR (KBr) cmꢁ1: 3278, 2368, 1642,
1356, 791; 1H NMR (300 MHz, CDCl3) d 13.21 (s, 1H, OH), 7.82–
7.78 (m, 2H, ArH), 7.65–7.64 (m, 2H, ArH), 7.55–7.52 (m, 5H,
ArH), 6.53 (br s, 2H, ArH), 4.74 (s, 2H, OCH2), 2.55 (s,1H, C„CH);
MS (ESI+) m/z (M+H) 357.3 and 359.3: Anal. Calcd for C18H13BrO3:
C, 60.52; H, 3.67. Found C, 60.57; H, 3.71.
4.1.2. Triazolylmethoxy acetophenones 2{1–3}
4.1.2.1. 1-[4-{(1-Hexyl-1H-1,2,3-triazol-4-yl)methoxy}-2-hydrox
yphenyl]ethanone 2{1}.
A mixture of 2-hydroxy-4-propar-
gyloxy acetophenone (1 g, 5.2 mmol) and n-hexyl azide (0.66 g,
5.2 mmol) in 1:1 tert-BuOH:H2O (10 mL) was stirred at ambient
4.1.4. Triazolylmethoxy chalcones 4{1–20}
temperature. A freshly prepared solution (500
bate (0.05 g, 0.2 mmol) in water was added followed by addition of
freshly prepared aqueous solution (200
L) of CuSO4ꢂ5H2O (0.02 g,
l
L) of sodium ascor-
4.1.4.1.
(E)-1-[4-{(1-Hexyl-1H-1,2,3-triazol-4-yl)methoxy}-2-
To a mix-
hydroxyphenyl]-3-phenyl prop-2-en-1-one 4{1}.
l
ture of compound 2{1} (1 g, 3.0 mmol) and benzaldehyde
(0.38 mL, 3.6 mmol) in ethanol (10 mL), pellets of KOH (1.2 equiv)
was added. Reaction was stirred at ambient temperature for 4 hours
then reaction was carefully neutralized with 3 N HCl and precipitate
so obtained was filtered and recrystallized from ethanol as yellow
crystal; mp 144–146 °C, 1.15 g, yield (90%); IR (KBr) cmꢁ1: 3401.5,
2367.6, 1647.3, 1580.0; 1H NMR (300 MHz, CDCl3) d 13.38 (s, 1H,
OH), 7.92 (d, J = 15.13 Hz, 1H, -COCH=CH-), 7.86 (s, 1H, triazolyl-
CH), 7.66–7.63 (m, 3H, ArH), 7.61 (d, J = 15.5 Hz, 1H, –COCH@CH–
), 7.45–7.43 (m, 3H, ArH), 6.60 (d, J = 6.48 Hz, 2H, ArH), 5.27 (s, 2H,
OCH2), 4.40 (t, J1 = 7.20, J2 = 7.26 Hz, 2H, NCH2), 1.95–1.90 (m, 2H,
CH2CH3), 1.32 (br s, 6H, 3xCH2), 0.91 (d, J = 6.4 Hz, 3H, CH3); MS
(ESI+) m/z (M+H): 406.2; Anal. Calcd for C24H27N3O3: C, 71.09; H,
6.71; N, 10.36. Found C, 71.07; H, 6.80; N, 10.39.
0.09 mmol). This heterogeneous mixture was stirred vigorously for
4 h at room temperature after which the reaction mixture was ex-
tracted with ethyl acetate and water. Ethyl acetate layer was dried
(anhyd Na2SO4) and evaporated under reduced pressure to give a
crude product. This was purified by silica gel (60–120) column
chromatography using hexane/EtOAc (1:1) as eluent to give com-
pound 2{1} as white solid, mp 50–52 °C, yield 1.5 g (90%); IR mmax
cmꢁ1 3376 , 1640.6, 1365 ; 1H NMR (300 MHz, CDCl3) d: 12.6 (s, 1H,
OH), 7.62 (dd, J = 6.09 and 3.81 Hz, 1H, ArH), 7.59 (s, 1H, triazolyl-
CH), 6.50 (dd, J = 5.97, J = 2.34 Hz, 2H, ArH), 5.21 (s, 2H, OCH2), 4.35
(t, J = 7.23 Hz, 2H, NCH2), 2.54 (s, 1H, CH3), 1.94–1.89 (t, J = 6.7 Hz,
2H, CH2), 1.32 (br s, 6H, 3 ꢃ CH2), 0.90- 0.89 (m, 3H, CH3); 13C NMR
(300 MHz, CDCl3) d: 202.1, 165.1, 164.5, 142.9, 132.2, 122.4,
114.28, 107.5, 102.0, 62.1, 50.3, 31.1, 30.2, 26.1, 26.0, 22.4, 13.9;
MS (ESI+) m/z (M+H): 318.0; Anal. Calcd for C17H23N3O3: C,
64.33; H, 7.30; N, 13.24. Found C, 64.9; H, 6.39; N, 13.19.
4.1.4.2. (E)-3-(4-Bromophenyl)-1-(4-{(1-hexyl-1H-1,2,3-triazol-
4-yl)methoxy}-2-hydroxy
phenyl]
prop-2-en-1-one
4{2}. The reaction of 2{1} (1 g, 3.0 mmol) and 4-bromobenzal-
4.1.2.2.
1-[4-{(1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy}-2-
The reaction of (1 g,
dehyde (0.70 g, 3.6 mmol) as described above gave yellow solid; mp
154–156 °C, yield, 1.42 g (93%); IR (KBr) cmꢁ1: 3455.0, 2366.1,
1638.3, 1575.0; 1H NMR (300 MHz, CDCl3) d 13.25 (s, 1H, OH),
7.78 (d, J = 13.95 Hz, 1H, –COCH@CH–), 7.75 (s, 1H, triazolyl-CH),
7.53–7.35 (m, 6H, ArH, –COCH@CH–), 6.54 (s, 2H, ArH), 5.24 (s,
2H, OCH2), 4.36 (s, 2H, NCH2), 1.92 (br s, 2H, CH2CH3), 1.32 (m,
6H, 3xCH2), 0.89 (br s, 3H, CH3); 13C NMR (300 MHz, CDCl3) d
191.2, 166.6, 164.7, 142.9, 133.7, 132.2(2C), 131.3, 129.8(2C),
125.0, 120.9, 114.4, 107.8, 102.3, 93.2, 62.4, 50.5, 31.1, 30.3,
26.1(2C), 22.4, 13.9; MS (ESI+) m/z (M+H): 483.9 and 485.9; Anal.
Calcd for C24H26BrN3O3: C, 59.51; H, 5.41; N, 8.67. Found C, 59.43;
H, 5.45; N, 8.72.
hydroxyphenyl]ethanone 2{2}.
1
5.2 mmol) and 4-bromobenzyl azide (0.70 g, 5.2 mmol) as de-
scribed above gave white solid, 89–92 °C, yield 1.5 g (91%); IR mmax
cmꢁ1 3382, 1638. NMR (300 MHz, CDCl3) d: 12.63 (s, 1H, OH), 7.60
(m, 1H, ArH), 7.54 (s, 1H, triazolyl-CH,), 7.36 (m, 2H, ArH), 7.26 (m,
2H, ArH), 6.46 (d, J = 6.87, Hz, 2H, ArH), 5.51 (s, 2H, OCH2), 5.15(s,
2H, NCH2), 2.52 (s, 1H, CH3), MS (ESI+) m/z (M+H): 324.1; Anal.
Calcd for C18H17N3O3: C, 66.86; H, 5.30; N, 13.00. Found C, 66.79;
H, 5.37; N, 12.91.
4.1.2.3. 1-[4-{(1-Bromobenzyl-1H-1,2,3-triazol-4-yl)methoxy}-
2-hydroxyphenyl] ethanone 2{3}.
The reaction of 1 (1 g,
5.2 mmol) and 4-bromobenzyl azide (0.96 g, 5.2 mmol) as de-
scribed above gave white solid, 120–123 °C, yield 1.9 g (91%); IR
mmax cmꢁ1 3459, 1629.4, 1256 ; 1H NMR (300 MHz, CDCl3) d: 12.6
(s, 1H, OH), 7.62 (m, 1H, ArH), 7.54 (m, 3H, triazolyl-CH, 2ArH),
6.50 (d, J = 7.4 Hz, 2H, ArH), 5.49 (s, 2H, OCH2), 5.20 (s, 2H,
NCH2), 2.55 (s, 1H, CH3); MS (ESI+) m/z (M+H): 402.4 and 404.3;
Anal. Calcd for C18H16BrN3O3: C, 53.73; H, 4.01; N, 10.45. Found
C, 53.69; H, 4.10; N, 10.39.
4.1.4.3. (E)-3-(4-Chlorophenyl)-1-[4-{(1-hexyl-1H-1,2,3-triazol-
4-yl)methoxy}-2-hydroxy
4{3}. The reaction of 2{1} (1 g, 3.0 mmol) and 4-chlorobenzal-
dehyde (0.51 g, 3.6 mmol) as described above gave yellow solid;
mp 150–153 °C, yield, 1.26 g (91%); IR (KBr) cmꢁ1
3428.9,
phenyl]prop-2-en-1-one
:
2363.6, 1640.8, 1568.4; 1H NMR (300 MHz, CDCl3) d: 13.25 (s,
1H, OH), 7.84–7.79 (m, 2H, –COCH@CH–, triazolyl-CH), 7.60–7.57
(m, 3H, ArH), 7.55 (d, J @ 15.7 Hz, 1H, –COCH@CH–), 7.40 (d,
J = 8.07 Hz, 2H, ArH), 6.56–6.54 (d, J = 6.45 Hz, 2H, ArH), 5.26 (s,
2H, OCH2), 4.37 (t, J1 = 7.17 Hz, J2 = 7.14 Hz, 2H, NCH2), 1.93–1.91
(m, 2H, CH2CH3), 1.34 (br s, 6H, 3 ꢃ CH2), 0.92–0.90 (d, J = 6.4 Hz,
3H, CH3); 13C NMR (300 MHz, CDCl3) d: 191.2, 166.6, 164.7,
142.9(2C), 136.6, 133.2, 131.1, 129.6(2C), 129.2(2C), 120.7, 114.4,
107.7, 102.3, 62.1, 50.3, 31.1, 30.2, 26.1(2C), 22.4, 13.9; MS (ESI+)
m/z (M+H): 440.0 and 442.0; Anal. Calcd for C24H26ClN3O3: C,
65.52; H, 5.96; N, 9.55. Found C, 65.48; H, 6.11; N, 9.42.
4.1.3. Propargyloxy chalcones 3{1–2}
4.1.3.1.
prop-en-1-one 3{1}.
(E)-1-{2-Hydroxy-4-(pro-2-ynyloxy)phenyl}-3-phenyl
To a mixture of compound 2{1} (1 g,
5.3 mmol) and benzaldehyde (0.67 mL, 6.3 mmol) in ethanol
(10 mL), pellets of KOH (1.2 equiv) was added. Reaction was stirred
at ambient temperature for 4 h then reaction was carefully neu-
tralized with 3 N HCl and precipitate so obtained was filtered
and recrystallized from ethanol as colorless solid; mp 210 °C, yield,
1.01 g (67%); IR (KBr) cmꢁ1: 3264, 2365, 1633, 1365, 976; 1H NMR
(300 MHz, CDCl3) d 13.28 (s, 1H, OH), 7.90–7.82 (m, 2H, ArH), 7.65–
7.64 (m, 2H, ArH), 7.55 (d, J = 15.4 Hz, 1H, -COCH=CH-), 7.43–7.41
(m, 3H, ArH), 6.53–6.50 (m, 2H, ArH), 4.47 (d, 2H, J = 2.2 Hz,
OCH2), 2.50 (d, J = 2.2 Hz,1H, C„CH); MS (ESI+) m/z (M+H) 279.3:
Anal. Calcd for C18H14O3: C, 77.68; H, 5.07. Found C, 77.57; H, 5.18.
4.1.4.4.
1,2,3-triazol-4-yl)methoxy}-2-hydroxy phenyl] prop-2-en-1-
one 4{4}. The reaction of 2{1} (1 g, 3.0 mmol) and 2-Chloro,
6- flourobenzaldehyde (0.42 g, 3.6 mmol) as described above gave
yellow solid; mp 156–158 °C, yield, 1.32 g (92%); IR (KBr) cmꢁ1
3448.5, 2367.0, 1641.3, 1573.5; 1H NMR (300 MHz, CDCl3) d:
(E)-3-(2-Chloro-6-fluorophenyl)-1-[4-{(1-Hexyl-1H-
:

N. Anand et al. / Bioorg. Med. Chem. 20 (2012) 5150–5163
5157
13.20 (s, 1H, OH), 8.07 (d, J = 15.81 Hz, 1H, –COCH@CH–), 7.81 (s,
1H, triazolyl-CH@), 7.79–7.75 (m, 1H, ArH), 7.61 (s, 1H, ArH),
7.28 (d, J = 1.89 Hz, 2H, ArH), 7.11–7.04 (m, 1H, ArH), 6.54 (d,
J = 1.98 Hz, 2H, ArH), 5.24 (s, 2H, OCH2), 4.38–4.33 (t, J1 = 7.23,
J2 = 7.20 Hz, 2H, NCH2), 1.92–1.790 (m, 2H, CH2CH3), 1.32 (br s,
6H, 3xCH2), 0.90 (d, J = 6.2 Hz, 3H, CH3); MS (ESI+) m/z (M+H):
458.0 and 460.0; Anal. Calcd for C24H25ClFN3O3: C, 62.95; H,
5.50; N, 9.18. Found C, 62.91; H, 5.55; N, 9.09.
J = 8.64 Hz, 2H, ArH), 5.26 (s, 2H, OCH2), 4.36 (t, J = 7.20 Hz, 2H,
NCH2), 1.93–1.90 (m, 2H, CH2CH3), 1.33 (s, 6H, 3 ꢃ CH2), 0.92–
0.90 (m, 3H, CH3); 13C NMR (300 MHz, CDCl3) d 191.4, 166.6,
164.7, 141.3, 133.7, 132.2, 131.8, 131.4, 130.9, 128.7, 127.0(2C),
126.3, 125.3(2C), 125.1, 123.5, 122.8, 114.5, 107.7, 102.3, 62.2,
50.4, 31.1, 30.2, 26.1, 22.4, 14.0; MS (ESI+) m/z (M+H): 456.1; Anal.
Calcd for C28H29N3O3: C, 73.82; H, 6.42; N, 9.22. Found C, 73.83; H,
6.48; N, 9.11.
4.1.4.5.
hydroxyphenyl]-3-(4-methoxy
4{5}. The reaction of 2{1} (1 g, 3.0 mmol) and 4-methoxy
(E)-1-[4-{(1-Hexyl-1H-1,2,3-triazol-4-yl)methoxy}-2-
4.1.4.9.
(E)-1-[4-{(1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy}-2-
The reac-
phenyl)prop-2-en-1-one
hydroxyphenyl]-3-phenyl prop-2-en-1-one 4{9}.
tion of 2{2} (1 g, 3.1 mmol) and benzaldehyde (0.38 mL, 3.7 mmol)
as described above gave yellow solid; mp 144–146 °C, yield,1.20 g
(90.5%); IR (KBr) cmꢁ1: 3408.2, 2366.7, 1645.8, 1581.1; 1H NMR
(300 MHz, CDCl3) d 13.21 (s, 1H, OH), 7.79 (m, 2H, -COCH@CH-,
triazolyl-CH), 7.56–7.52 (m, 3H, ArH), 7.51 (d, J = 15.63 Hz, 1H, -
COCH@CH-), 7.33–7.26 (m, 6H, ArH), 7.19 (d, J = 7.23 Hz, 2H,
ArH,), 6.44 (d, J = 2.13 Hz, 2H, ArH), 5.45 (s, 2H, OCH2), 5.12 (s, 2H,
NCH2); MS (ESI+) m/z (M+H): 412.0; Anal. Calcd for C25H21N3O3: C,
72.98; H, 5.14; N, 10.21. Found C, 72.88; H, 5.21; N, 10.12.
benzaldehyde (0.46 mL, 3.6 mmol) as described above gave yellow
solid; mp 150–152 °C, yield 1.20 g (93%); IR (KBr) cmꢁ1: 3621.8,
2364.8, 1639.8, 1564.0; 1H NMR (300 MHz, CDCl3) d 13.47 (s, 1H,
OH), 7.85 (m, 2H, –COCH@CH–, triazolyl-CH@), 7.60 (m, 3H, ArH),
7.44 (d, J = 15.18, 1H, –COCH@CH–), 6.93 (d, J = 8.13 Hz, 2H, ArH),
6.55 (br s, 2H, ArH), 5.24 (s, 2H, OCH2), 4.36 (t, J = 6.69 Hz, 2H,
NCH2), 1.92 (br s, 2H, CH2CH3), 1.32 (m, 6H, 3 ꢃ CH2), 0.89 (br s,
3H, CH3); 13C NMR (300 MHz, CDCl3) d: 191.7, 166.5, 164.4,
161.8, 144.4(2C), 131.3, 130.4(2C), 127.5, 117.7, 114.6, 114.4(2C),
107.6, 102.3, 62.4, 55.3, 50.6, 31.1, 30.3, 26.2(2C), 22.4, 14.0; MS
(ESI+) m/z (M+H): 436.0; Anal. Calcd for C25H29N3O3: C, 68.95; H,
6.71; N, 9.65. Found C, 68.89; H, 6.75; N, 9.61.
4.1.4.10. (E)-1-[4-{(1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy}-2-
hydroxyphenyl]-3-(4-chlorophenyl)
4{10}. The reaction of 2{2}(1 g, 3.1 mmol) and 4-chlorobenz-
prop-2-en-1-one
aldehyde (0.52 g, 3.7 mmol) as described above gave yellow solid;
mp 152–154 °C, yield 1.32 g (91%); IR (KBr) cmꢁ1: 3444.7, 2367.2,
1618.0, 1577.8; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d 8.00 (s, 1H,
triazolyl-CH@), 7.84 (d, J = 16.4 Hz 1H, COCH@CH–), 7.61–7.39 (m,
4H, ArH, and –COCH@CH–), 7.10–6.98 (m, 7H, ArH), 6.26 (br s, 1H,
ArH), 5.26 (s, 2H, OCH2), 4.87 (s, 2H, NCH2); MS (ESI+) m/z (M+H):
445.9 and 448.0; Anal. Calcd for C25H20ClN3O3: C, 67.34; H, 4.52; N,
9.42. Found C, 67.28; H, 4.55; N, 9.38.
4.1.4.6.
hydroxyphenyl]-3-(4-isopropyl
4{6}. The reaction of 2{1} (1 g, 3.0 mmol) and 4-isopropyl
(E)-1-[4-{(1-Hexyl-1H-1,2,3-triazol-4-yl)methoxy}-2-
phenyl) prop-2-en-1-one
benzaldehyde (0.39 mL, 3.6 mmol) as described above gave yellow
solid; mp 146–148 °C, yield 1.28 g, 92%; IR (KBr) cmꢁ1: 3419.6,
2371.9, 1644.0, 1569.2; 1H NMR (300 MHz, CDCl3) d 13.40 (s, 1H,
OH), 7.89–7.82 (m, 2H, –COCH@CH, triazolyl-CH), 7.62–7.56 (m,
3H, ArH), 7.55 (d, J = 15.5 Hz, -COCH@CH-), 7.28 (d, J = 6.57, 2H,
ArH), 6.54 (br s, 2H, ArH), 5.25 (s, 2H, OCH2), 4.36 (t, J = 7.2 Hz,
2H, NCH2), 2.98–2.94 (m, 1H, –CH–), 1.91 (br s, 2H, CH2CH3),
1.31–1.28 (m, 12H, 3xCH2, 2xCH3), 0.90–0.89 (d, J = 3.7 Hz, 3H,
CH3); 13C NMR (300 MHz, CDCl3) d: 191.8, 166.5, 164.6, 152.1,
144.7(2C), 132.4, 131.3, 128.7(2C), 127.1(2C), 122.6, 119.2, 114.6,
107.6, 102.2, 62.2, 50.5, 34.2, 31.1, 30.2, 26.1(2C), 23.8, 22.4,
13.9; MS (ESI+) m/z (M+H): 448.1; Anal. Calcd for C27H33N3O3: C,
72.46; H, 7.43; N, 9.39. Found C, 72.43; H, 7.48; N, 9.32.
4.1.4.11. (E)-1-(4-{(1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy}-2-
hydroxyphenyl]-3-(2-chloro-6-fluoro phenyl) prop-2-en-1-one
4{11}.
fluorobenzaldehyde (0.58 g, 3.7 mmol) as described above gave
yellow solid; mp 152–154 °C, yield, 1.32 g (91%); IR (KBr) cmꢁ1
The reaction of 2{2} (1 g, 3.1 mmol) and 2-chloro-6-
:
3423.3, 2366.4, 1638.9, 1576.6; 1H NMR (300 MHz,
CDCl3 + DMSO-d6) d: 8.27 (s, 1H, triazolyl-CH@), 7.92 (m, 3H,
ArH, –COCH@CH–), 7.49–7.39 (m, 2H, ArH), 7.37–7.29 (m, 6H,
ArH, and –COCH@CH–), 6.67 (d, J = 1.98 Hz, 1H, ArH), 6.61 (dd,
J1 = 2.1, J2 = 8.9 Hz, 1H, ArH), 5.61 (s, 2H, OCH2), 5.24 (s, 2H,
NCH2); 13C NMR (300 MHz, CDCl3+ DMSO-d6) d: 192.2, 170.5,
170.0, 147.4, 140.9, 140.5, 138.2, 137.4, 137.0, 133.9(2C), 133.3,
133.1(2C), 131.4, 129.9, 126.6, 126.4, 120.7, 120.3119.4, 113.3,
107.1, 66.8, 58.1; MS (ESI+) m/z (M+H): 463.9 and 466.0; Anal.
Calcd for C25H19ClFN3O3: C, 64.73; H, 4.13; N, 9.06. Found C,
64.69; H, 4.21; N, 9.01.
4.1.4.7.
hydroxyphenyl]-3-(4-(prop-2-ynyloxy) phenyl)prop-2-en-1-one
4{7}. The reaction of 2{1} (1 g, 3.0 mmol) and 4-propynyloxy-
(E)-1-[4-{(1-Hexyl-1H-1,2,3-triazol-4-yl)methoxy}-2-
benzaldehyde (0.58 g, 3.6 mmol) as described above gave yellow
solid; mp 149–152 °C, yield, 1.36 g (82%); IR (KBr) cmꢁ1: 3462.0,
2367.3, 1635.7, 1578.5; 1H NMR (300 MHz, CDCl3) d 13.40 (s, 1H,
OH), 7.88–7.83 (m, 2H, –COCH@CH–, triazolyl-CH), 7.64–7.60 (m,
3H, ArH), 7.46 (d, J = 15.36 Hz, 1H, –COCH@CH–), 7.03 (d,
J = 8.67 Hz, 2H, ArH), 6.55 (d, J = 7.26 Hz, 2H, ArH), 5.26 (s, 2H,
OCH2), 4.76 (d, J = 2.28 Hz, 2H, OCH2), 4.36 (t, J1 = 7.20,
J2 = 7.21 Hz, 2H, NCH2), 2.57–2.52 (m, 1H, CH), 1.96–1.92 (m, 2H,
CH2CH3), 1.34 (br s, 6H, 3xCH2), 0.93–0.90 (d, J = 6.5 Hz, 3H, CH3);
MS (ESI+) m/z (M+H): 460.0; Anal. Calcd for C27H29N3O4: C,
70.57; H, 6.36; N, 9.14. Found C, 70.53; H, 6.41; N, 9.10
4.1.4.12. (E)-1-[4-{(1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy}-2-
hydroxyphenyl]-3-(4-methoxy
phenyl)prop-2-en-1-one
4{12}. The reaction of 2{2} (1 g, 3.1 mmol) and 4-methoxy-
benzaldehyde (0.45 mL, 3.7 mmol) as described above gave yellow
solid; mp 148–150 °C, yield, 1.26 g (88%); IR (KBr) cmꢁ1: 3408.2,
2366.7, 1645.8, 1581.1; 1H NMR (300 MHz, CDCl3) d 13.41 (s, 1H,
OH), 7.87 (d, J = 15.21 Hz, 1H, -COCH@CH–), 7.79 (s, 1H, triazolyl-
CH), 7.61–7.58 (d, J = 8.5 Hz, 2H, ArH), 7.52 (s, 1H, ArH), 7.48 (d,
J = 15.3 Hz, 1H, –COCH@CH–), 7.33–7.26 (m, 6H, ArH) 6.97 - 6.88
(m, 2H, ArH,), 6.51 (d, J = 6.42 Hz, 2H, ArH), 5.55 (s, 2H, OCH2), 5.23
(s, 2H, NCH2) 3.91- 3.85 (m, 1H, OCH3); 13C NMR (300 MHz, CDCl3)
d: 192.2, 166.5, 164.3, 144.3, 131.0, 130.3(3C), 129.2(3C),
128.8(2C), 128.0(3C), 127.5, 117.7, 114.6, 114.4(2C), 107.4, 102.3,
62.1, 55.2, 54.2; MS (ESI+) m/z (M+H): 442.0; Anal. Calcd for
4.1.4.8.
hydroxyphenyl]-3-(naphthalene-1-yl)
4{8}. The reaction of 2{1} (1 g, 3.0 mmol) and 1-naphthalde-
hyde (0.51 mL, 3.6 mmol) as described above gave yellow solid;
(E)-1-[4-{(1-Hexyl-1H-1,2,3-triazol-4-yl)methoxy}-2-
prop-2-en-1-one
mp 142–145 °C, yield, 1.35 g (92%); IR (KBr) cmꢁ1
: 3135.8,
2359.9, 1724.8, 1582.9; 1H NMR (300 MHz, CDCl3) d 13.40 (s, 1H,
OH), 8.73 (d, J = 15.18 Hz, 1H, –COCH@CH–), 8.26 (d, J = 8.22 Hz,
1H, ArH), 7.94–7.85 (m, 4H, triazolyl-CH, ArH), 7.67 (d,
J = 15.3 Hz, 1H,–COCH@CH–), 7.61–7.49 (m, 4H, ArH), 6.58 (d,
C26H23N3O4: C, 70.73; H, 5.25; N, 9.52. Found C, 70.69; H, 5.29; N,
9.51.

5158
N. Anand et al. / Bioorg. Med. Chem. 20 (2012) 5150–5163
4.1.4.13. (E)-1-[4-{(1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy}-2-
hydroxyphenyl]-3-(4-isopropyl phenyl)prop-2-en-1-one
4{13}. The reaction of 2{2} (1 g, 3.1 mmol) and 4-isopropyl
J = 8.07 Hz, 2H, ArH), 6.62–6.56 (m, 2H, ArH), 5.59 (s, 2H, OCH2),
5.23 (s, 2H, NCH2); MS (ESI+) m/z (M+H): 568.05, 570.05 and
572.05; Anal. Calcd for C25H19Br2N3O3: C, 52.75; H, 3.36; N, 7.38.
Found C, 52.69; H, 3.42; N, 9.31.
benzaldehyde (0.56 mL, 3.7 mmol) as described above gave yellow
solid; mp 142–145 °C, yield 1.30 g, 91%; IR (KBr) cmꢁ1: 3393.3,
2362.5, 1628.9, 1569.6; 1H NMR (300 MHz, CDCl3) d: 13.5 (s, 1 H,
OH), 7.91 (d, J = 15.7 Hz, 1H, –COCH@CH–), 7.83 (s, 1H, triazolyl-
CH), 7.61–7.58 (m, 2H, ArH), 7.57 (d, 1H, J = 15.4 Hz, –COCH@CH–
), 7.44–7.29 (m, 7H, ArH), 6.57–6,54 (m, 2H, ArH), 5.56 (s, 2H,
OCH2), 5.22 (s, 2H, NCH2) 2.98–2.94 (m, 1H, –CH–), 1.28 (d,
J = 6.9 Hz, 6H, 2 ꢃ CH3); 13C NMR (300 MHz, CDCl3) d: 191.9,
166.5, 164.5, 152.1, 144.7, 134.4, 132.4, 131.3, 129.2(2C), 128.9,
128.8(2C), 128.7(2C), 128.1(2C), 127.1(2C), 122.8, 119.2, 114.5,
107.7, 102.2, 62.0, 54.2, 34.1, 23.7 (2C); MS (ESI+) m/z (M +H):
454.0; Anal. Calcd for C28H27N3O4: C, 74.15; H, 6.00; N, 9.27. Found
C, 74.11; H, 6.07; N, 9.21.
4.1.4.18.
(E)-1-[4-{(1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-
yl)methoxy}-2-hydroxy
prop-2-en-1-one 4{18}.
phenyl]-3-(2-chloro-6-fluorophenyl)
The reaction of 2{3} (1 g, 2.5 mmol)
and 2-chloro-6-fluorobenzaldehyde (0.47 g, 3.0 mmol) as de-
scribed above gave yellow solid; mp 157–159 °C, yield, 1.25 g
(92%); IR (KBr) cmꢁ1: 3441.2, 2374.8, 1638.7, 1573.7; 1H NMR
(300 MHz, CDCl3 + DMSO-d6) d 8.26 (s, 1H, triazolyl-CH), 7.92 (m,
, 3H, COCH@CH–, ArH), 7.53 (d, J = 8.37 Hz, 4H, –COCH@CH–,
ArH), 7.32–7.27 (m, 3H, ArH,), 6.66 (d, J = 2.37 Hz, 1H, ArH), 6.63
(dd, J1 = 8.88, J2 = 2.4 Hz, 1H, ArH), 5.59 (s, 2H, OCH2), 5.23 (s, 2H,
NCH2); 13C NMR (300 MHz, CDCl3+ DMSO-d6) d: 196.2, 170.6,
170.0, 147.4, 140.5, 140.2, 138.3, 137.3(2C), 136.9, 136.8(2C),
135.3(2C), 132.8, 131.3, 130.0, 126.8, 126.6, 120.7, 119.4, 113.3,
107.1, 66.7, 57.4; MS(ESI+) m/z (M+H): 541.8 and 543.8; Anal.
Calcd for C25H18BrClFN3O3: C, 55.32; H, 3.34; N, 7.74. Found C,
55.28; H, 3.39; N, 7.69.
4.1.4.14.
2-hydroxyphenyl]-3-{4-(prop-2-yny loxyl) phenyl} prop-2-
en-1-one 4{14}. The reaction of 2{2} (1 g, 3.1 mmol) and
(E)-1-[4-{(1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy}-
4-propynyloxy benzaldehyde (0.59 g, 3.7 mmol) as described
above gave solid; mp 150–152 °C, yield, 1.29 g (84%); IR (KBr) cm
ꢁ1: 3408.2, 2366.7, 1645.8, 1581.1; 1H NMR (300 MHz, CDCl3) d:
13.42 (s, 1H, OH), 7.89 (d, J = 15.63 Hz, 1H, –COCH@CH–), 7.81 (s,
1H, triazolyl-CH), 7.64–7.48 (m, 3H, ArH), 7.40–7.37 (m, 3H,
ArH,–COCH@CH–), 7.30–7.27 (m, 3H, ArH) 7.03 (d, J = 13.05 Hz,
2H, ArH), 6.53–6.47 (m, 2H, ArH), 5.55 (s, 2H, OCH2), 5.22 (s, 1H,
OCH2), 4.75 (d, J = 3.1 Hz, 2H, NCH2), 2.55 (m, 1H, CH); MS (ESI+)
m/z (M+H): 466.0; Anal. Calcd for C28H23N3O4: C, 72.24; H, 4.98;
N, 9.03. Found C, 72.20; H, 5.01; N, 9.01.
4.1.4.19.
4-yl)methoxy}-2-hydroxy phenyl]-3-(4-isopropyl phenyl) prop-
2-en-1-one 4{19}. The reaction of 2{3} (1 g, 2.5 mmol) and
4-isopropylbenzaldehyde (0.45 mL, 3.0 mmol) as described above
yellow solid; mp 144–146 °C, yield, 1.22 g (93%); IR (KBr) cmꢁ1
(E)-1-[4-{(1-(4-Bromobenzyl)-1H-1,2,3-triazol-
:
3457.9, 2367.8, 1633.7, 1570.2; 1H NMR (300 MHz, CDCl3) d:
13.38 (s, 1H, OH), 7.88 (d, J = 15.3 Hz, 2H, –COCH@CH–, triazolyl-
CH), 7.59–7.51 (m, 5H, ArH, –COCH@CH), 7.29–7.02 (m, 5H,
ArH,), 6.54 (br s, 2H, ArH), 5.51 (s, 2H, OCH2), 5.24 (s, 2H, NCH2)
2.95 (m, 1H, -CH-), 1.30 (d, J = 6.63 Hz, 6H, 2 x CH3); MS (ESI+)
m/z (M+H): 532.2 and 534.2; Anal. Calcd for C28H26BrN3O3: C,
63.16; H, 4.92; N, 7.89. Found C, 63.11; H, 4.98; N, 7.86.
4.1.4.15. (E)-1-[4-{(1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy}-2-
hydroxyphenyl]-3-(naph
thalen-1-yl)prop-2-en-1-one
4{15}. The reaction of 2{2} (1 g, 3.1 mmol) and 1-naphthalde-
hyde (0.50 mL, 3.7 mmol) as described above gave yellow solid;
mp 151–153 °C, yield, 1.32 g (94.4%); IR (KBr) cmꢁ1: 3455.2,
2367.1, 1636.6, 1575.9; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d:
8.64 (d, J = 15.2 Hz, 1H, –COCH@CH–), 8.25 (d, J = 8.28 Hz, 1H,
ArH), 8.17 (m, 3H, triazolyl-CH, ArH), 7.99–7.91 (m, 3H, ArH),
7.61–7.52 (m, 3H, ArH, COCH@CH–), 7.31 (br s, 5H, ArH), 6.61 -
6.57 (m, 2H, ArH), 5.58 (s, 2H, OCH2), 5.21 (s, 2H, NCH2); MS
(ESI+) m/z (M+H): 462.2; Anal. Calcd for C29H23N3O3: C, 75.47; H,
5.07; N, 9.10. Found C, 75.49; H, 5.19; N, 9.07.
4.1.4.20.
methoxy}-2-hydroxy phenyl]-3-(naphthalene-1-yl)prop-2-en-
1-one 4{20}. The reaction of 2{3} (1 g, 2.5 mmol) and 4-bro-
(E)-1-[4-{(1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-yl)
mobenzaldehyde (0.40 g, 3.0 mmol) as described above gave yellow
solid; mp 150–152 °C, yield, 1.25 g (92%); IR (KBr) cmꢁ1: 3449.5,
2372.4, 1632.1, 1576.5; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d:
8.67 (d, J = 15.24 Hz, 1H, –COCH@CH–), 8.25 (d, J = 7.02 Hz, 1H,
ArH), 8.11 (br s, 3H, triazolyl-CH, ArH), 7.98–7.87 (m, 3H, ArH),
7.55–7.48 (m, 5H, ArH, -COCH@CH–), 7.27 (m, 2H, ArH), 6.60 (m,
2H, ArH), 5.56 (s, 2H, OCH2), 5.21 (s, 2H, NCH2); MS (ESI+) m/z
(M+H): 539.9 and 541.9; Anal. Calcd for C29H22BrN3O3: C, 64.45;
H, 4.10; N, 7.78. Found C, 64.41; H, 4.13; N, 7.69.
4.1.4.16.
yl)methoxy}-2-hydroxyphenyl]-3-phenyl)prop-2-en-1-one
4{16}. The reaction of 2{3} (1 g, 2.5 mmol) and benzaldehyde
(E)-1-[4-{(1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-
(0.30 mL, 3.0 mmol) as described above gave yellow solid; mp
146–148 °C, yield, 1.10 g (91%); IR (KBr) cmꢁ1: 3408.2, 2366.7,
1645.8, 1581.1; 1H NMR (300 MHz, CDCl3+ DMSO-d6) d: 7.90 (d,
4.1.5. Triazolylmethoxy flavanones 5{1–10}
4.1.5.1.
7-{(1-Hexyl-1H-1,2,3-triazol-4-yl)methoxy}-2-phenyl
To a stirred solution of triazolymeth-
J = 9.63 Hz,
1 H, ArH), 7.80 (s, 1H, triazolyl-CH), 7.83 (d,
chroman-4-one 5{1}.
J = 15.6 Hz, 1H, –COCH@CH–), 7.65–7.60 (m, 3H, ArH), 7.47–7.38
(m, 5H, ArH, –COCH@CH–), 7.18 (d, J = 8.19 Hz, 2H, ArH), 6.52 (d,
J = 2.07 Hz, 2H, ArH), 5.50 (s, 2H, OCH2), 5.18 (s, 2H, NCH2); MS
(ESI+) m/z (M+H): 490.3, 492.3; Anal. Calcd for C25H20BrN3O3: C,
61.24; H, 4.11; N, 8.57. Found C, 61.18; H, 4.15; N, 8.49.
oxychalcone 4{1} (0.5 g, 1.23 mmol) in minimum amount of a mix-
ture of EtOH/H2O (1:1) and NaOAc (0.40 g, 4.92 mmol) was added.
The reaction mixture was refluxed till the disappearance of the
starting materials (TLC). After completion of reaction, the reaction
mixture was allowed to cool to room temperature. The reaction
mixture was extracted with ethyl acetate and water. The combined
organic phases were washed with brine, dried over anhydrous so-
dium sulphate, and concentrated under reduced pressure. Crude
product was purified by recrystallization in ethanol. light yellow
solid, mp 120–123 °C, yield, 0.38 g (79%); IR (KBr) cmꢁ1 3754,
2834, 1708, 1451, 1252, 697; 1H NMR (300 MHz, CDCl3 + DMSO-
d6) d: 7.88 (d, J = 8.7 Hz, 1H, ArH), 7.62 (s, 1H, triazolyl CH@),
7.47–7.42 (m, 2H, ArH), 6.70 (dd, J1 = 2.1 Hz, J2 = 8.8 Hz, 1H, ArH),
6.64–6.59 (m, 1H, ArH), 5.48 (dd, J1 = 2.7 Hz, J2 = 13.1 Hz, 1H,
OCH), 5.24 (s, 2H, OCH2), 4.37 (t, J = 7.1 Hz, 2H, NCH2), 3.05 (dd,
4.1.4.17.
4-yl)methoxy}-2-hydroxy phenyl]-3-(4-bromo phenyl)prop-
2-en-1-one 4{17}. The reaction of 2{3} (1 g, 2.5 mmol) and
4-bromobenzaldehyde (0.55 g, 3.0 mmol) as described above gave
(E)-1-[4-{(1-(4-Bromobenzyl)-1H-1,2,3-triazol-
yellow solid; mp 156–158 °C, yield,1.30 g (93%); IR (KBr) cmꢁ1
:
3408.2, 2366.7, 1645.8, 1581.1; 1H NMR (300 MHz, CDCl3+ DMSO-
d6) d 8.23–8.19 (m, 2H, ArH, triazolyl-CH), 7.96 (d, J = 15.42 Hz,
1H, COCH@CH–), 7.81–7.73 (m, 2H, ArH, –COCH@CH–), 7.60 (d,
J = 8.28 Hz, 2H, ArH), 7.52 (d, J = 8.19 Hz, 2H, ArH), 7.28 (d,

N. Anand et al. / Bioorg. Med. Chem. 20 (2012) 5150–5163
5159
J = 13.2 Hz, J = 16.8 Hz, 1H, Ha, CH2), 2.85 (dd, J1 = 2.8 Hz,
J2 = 16.8 Hz, 1H, Hb, CH2), 1.92 (br s, 2H, CH2), 1.32 (m, 6H, 3x
CH2), 0.89 (br s, 3H. CH3); 13C NMR (50 MHz, CDCl3 +DMSO-d6) d:
190.2, 164.6, 163.4, 143.06, 138.1, 128.8(3C), 126.1(2C),
122.4(2C), 110.5, 107.6, 101.9, 79.9, 62.3, 50.4, 44.4, 31.1, 30.2,
26.1(2C), 22.4, 13.9; MS (ESI+) m/z (M+H): 406.3; Anal. Calcd for
7.45–7.36(m, 5H, ArH), 7.30–7.26 (m, 2H, ArH), 6.68 (dd,
J1 = 2.3 Hz, J2 = 8.8 Hz, 1H, ArH), 6.60–6.53 (m, 1H, ArH), 5.54 (s,
2H, OCH2), 5.41 (dd, J1 = 3.3 Hz, J2 = 13.0 Hz, 1H, OCH), 5.22 (s,
2H, NCH2), 3.11 (dd, J = 13 Hz, J = 16.9 Hz, 1H, Ha, CH2), 2.87 (dd,
J1 = 3.2 Hz, J2 = 16.9 Hz, 1H, Hb, CH2); MS (ESI+) m/z (M+H): 412;
Anal. Calcd for C25H21N3O3: C, 72.98; H, 5.14; N, 10.21. Found C,
72.92; H, 5.17; N, 10.24.
C24H27N3O3: C, 71.09; H, 6.71; N, 10.36. Found C, 71.11; H, 6.79;
N, 10.28.
4.1.5.6. 7-{(1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy}-2-(4-chlo-
4.1.5.2.
7-{(1-Hexyl-1H-1,2,3-triazol-4-yl)methoxy}-2-(4-
Light yellow solid, mp
rophenyl)chroman-4-one 5{6}.
Light yellow solid, mp 135–
bromophenyl)chroman-4-one 5{2}.
138 °C, yield, 0.30 g (50%); IR (KBr) cmꢁ1 3454, 2934, 1731, 1398,
1282, 691; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d: 7.87 (m, 1H,
ArH), 7.52 (s, 1H, triazolyl CH@, ArH), 7.45–7.28 (m, 9H, ArH),
6.66–6.54 (m, 2H, ArH), 5.50 (s, 2H, OCH2), 5.46 (dd, J1 = 2.5 Hz,
J2 = 13.2 Hz, 1H, OCH), 5.20 (s, 2H, NCH2), 3.02 (dd, J = 13.3 Hz,
J = 16.7 Hz, 1H, Ha, CH2), 2.83 (dd, J1 = 2.6 Hz, J2 = 16.7 Hz, 1H, Hb,
CH2); MS (ESI+) m/z (M+H): 446.0 and 448; Anal. Calcd for
133–135 °C, 0.37 g, yield 75%; IR (KBr) cmꢁ1 3754, 2834, 1698,
1451, 1252, 697; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d: 7.76–
7.71 (m, 1H, ArH), 7.51 (s, 1H, triazolyl CH@), 7.29–7.26 (m, 2H,
ArH), 7.18–7.16 (m, 2H, ArH), 6.55 (dd, J1 = 2.3 Hz, J2 = 8.8 Hz, 1H,
ArH), 5.34–5.29 (m, 1H, ArH), 5.30 (m, 1H, OCH), 5.11 (s, 2H,
OCH2), 4.25 (t, J = 7.2 Hz, 2H, NCH2), 2.94 (dd, J = 13.3 Hz,
J = 16.8 Hz, 1H, Ha, CH2), 2.72 (dd, J1 = 2.7 Hz, J2 = 16.8 Hz, 1H, Hb,
CH2), 1.82–1.79 (m, 2H, CH2), 1.22 (m, 6H, 3 ꢃ CH2), 0.81–0.79
(m, 3H. CH3); 13C NMR (50 MHz, CDCl3+DMSO-d6) d: 190.1,
164.6, 163.3, 143.0, 138.7, 128.8(2C), 126.1(2C), 122.4(2C), 115.3,
110.5, 107.6, 101.9, 79.9, 62.3, 50.4, 44.4, 31.1, 29.6, 26.1(2C),
22.4, 13.9; MS (ESI+) m/z (M+H): 484.3 and 486.3; Anal. Calcd for
C25H20ClN3O3: C, 67.34; H, 4.52; N, 9.42. Found C, 67.27; H, 4.59;
N, 9.36.
4.1.5.7.
7-{(1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy}-2-(4-iso-
Light yellow solid, mp
propylphenyl)chroman-4-one 5{7}.
123–125 °C, yield, 0.44 g (87%); IR (KBr) cmꢁ1: 3454, 2834, 1758,
1398, 1282, 691; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d: 7.88
(m, 1H, ArH), 7.54–7.51 (m, 4H, triazolyl CH@, ArH), 7.40–7.38
(m, 2H, ArH), 7.30 (m, 1H, ArH), 7.18–7.15 (m, 2H, ArH), 6.67 (m,
1H, ArH), 5.34–5.29 (m, 1H, ArH), 5.50 (s, 2H, OCH2), 5.45 (dd,
J1 = 2.3 Hz, J2 = 13.0 Hz, 1H, OCH), 5.21 (s, 2H, NCH2), 3.03 (dd,
J = 13.1 Hz, J = 16.8 Hz, 1H, Ha, CH2), 2.98–2.93 (m, 1H. CH), 2.70
(dd, J1 = 2.6 Hz, J2 = 16.8 Hz, 1H, Hb, CH2), 1.31–1.28(m, 6H,
2xCH3); MS (ESI+) m/z (M+H): 454.0; Anal. Calcd for C28H27N3O3:
C, 74.15; H, 6.00; N, 9.27. Found C, 74.47; H, 6.17; N, 9.16.
C25H27BrN2O3: C, 59.51; H, 5.41; N, 8.67. Found C, 59.47; H, 5.52;
N, 8.58.
4.1.5.3.
7-{(1-Hexyl-1H-1,2,3-triazol-4-yl)methoxy}-2-(4-
Light yellow solid,
methoxyphenyl)chroman-4-one 5{3}.
mp 128–130 °C, yield, 0.42 g 85%; IR (KBr) cmꢁ1 3682, 2821,
1724, 1410, 1232, 692; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d:
7.87 (d, J = 8.8 Hz, 1H, ArH), 7.58 (m, 2H, triazolyl CH@, ArH),
7.39 (d, J = 8.6 Hz, 1H, ArH), 6.95–6.92 (m, 2H, ArH), 6.66 (dd,
J1 = 2.1 Hz, J2 = 8.7 Hz, 1H, ArH), 6.58–6.55 (m, 1H, ArH), 5.40 (dd,
J1 = 2.7 Hz, J2 = 13.0 Hz, 1H, OCH), 5.22 (s, 2H, OCH2), 4.36 (t,
J = 7.1 Hz, 2H, NCH2), 3.83 (s, 3H, OCH3), 3.03 (dd, J = 13.2 Hz,
J = 16.8 Hz, 1H, Ha, CH2), 2.78 (dd, J1 = 2.8 Hz, J2 = 16.8 Hz, 1H, Hb,
CH2), 1.92–1.90 (m, 2H, CH2), 1.33 (m, 12H, 3x CH2), 0.89 (br s,
3H, CH3); 13C NMR (50 MHz, CDCl3 + DMSO-d6) d: 190.3, 164.4,
163.4, 159.9, 144.4, 143.0, 130.7, 130.3, 128.8, 127.5, 122.4,
114.5, 110.4, 107.5, 101.9, 79.7, 62.3, 55.2, 50.4, 44.1, 31.1, 30.2,
26.2, 22.4, 13.9; MS (ESI+) m/z (M+H): 436.; Anal. Calcd for
4.1.5.8. 7-{(1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-yl) methoxy-
2-phenylchroman-4-one 5{8}.
Light yellow solid, mp 125–
127 °C, yield, 0.42 g (83%); IR (KBr) cmꢁ1: 3750, 3453, 2960,
1732, 1562, 1458, 1119, 799; 1H NMR (300 MHz, CDCl3 + DMSO-
d6) d: 7.88 (m, 1H, ArH), 7.66–7.38 (m, 8H, triazolylCH@, ArH),
7.18 (d, J = 8.2 Hz, 2H, ArH), 6.68 (dd, J1 = 2.0 Hz, J2 = 10.8 Hz, 1H,
ArH), 6.59–6.52 (m, 1H, ArH), 5.50 (s, 2H, OCH2), 5.45–5.44 (m,
1H, OCH), 5.21 (s, 2H, NCH2), 3.07 (dd, J = 13.2 Hz, J = 16.8 Hz, 1H,
Ha, CH2), 2.86 (dd, J1 = 2.9 Hz, J2 = 16.9 Hz, 1H, Hb, CH2); MS (ESI+)
m/z (M+H): 490.0 and 492; Anal. Calcd for C25H20BrN3O3: C,
61.24; H, 4.11; N, 8.57. Found C, 61.22; H, 4.07; N, 8.54.
C25H29N3O4: C, 68.95; H, 6.71; N, 9.65. Found C, 68.89; H, 6.69;
N, 5.51.
4.1.5.4. 7-{(1-Hexyl-1H-1,2,3-triazol-4-yl)methoxy}-2-(4-isopro-
pylphenyl)chroman-4-one 5{4}.
Light yellow solid, mp 115–
4.1.5.9. 7-{(1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-yl) methoxy}-
117 °C, 0.41 g, yield 82%; IR (KBr) cmꢁ1 3694, 2832, 1740, 1411,
1232, 697; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d: 7.86 (d,
J = 8.8 Hz, 1H, ArH), 7.59 (s, 1H, triazolyl CH@), 7.55 (m, 1H, ArH),
7.39–7.36 (m, 2H, ArH),7.25 (s, 1H, ArH), 6.65 (dd, J1 = 2.5 Hz,
J2 = 8.8 Hz, 1H, ArH), 6.58–6.54 (m, 1H, ArH), 5.42 (m, 1H, OCH),
5.21 (s, 2H, OCH2), 4.35 (t, J = 7.1 Hz, 2H, NCH2), 3.02 (dd,
J = 13.2 Hz, J = 16.8 Hz, 1H, Ha, CH2), 2.96–2.92 (m, 1H, CH), 2.80
(dd, J1 = 2.6 Hz, J2 = 16.8 Hz, 1H, Hb, CH2), 1.92–1.89 (m, 2H, CH2),
1.32 -1,27(m, 12H, 3x CH2+ 2x CH3), 0.89 (br s, 3H. CH3); 13C
NMR (50 MHz, CDCl3 + DMSO-d6) d: 190.1, 164.6, 163.4, 149.4,
144.6, 143.01, 136.1, 131.2, 128.8, 127.0, 126, 122.4, 119, 115.2,
110.4, 107.5, 101.9, 79.9, 62.3, 50.4, 44.2, 33.9, 31.1, 30.2, 26.1,
23.9, 22.4, 13.9; MS (ESI+) m/z (M+H): 448; Anal. Calcd for C27
H33N3O3: C, 72.46; H, 7.43; N, 9.39. Found C, 72.39; H, 7.33; N, 7.38.
2-(4-bromophenyl) chroman-4-one 5{9}.
Light yellow solid,
mp 132–135 °C, yield, 0.30 g, (60%); IR (KBr) cmꢁ1 3453, 2963,
1725, 1562, 1458, 1215, 799; 1H NMR (300 MHz, CDCl3 + DMSO-
d6) d: 7.89–7.82 (m, 1H, ArH), 7.52 (s, 1H, triazolylCH@), 7.45–
7.36 (m, 8H, ArH), 6.66–6.58 (m, 2H, ArH), 5.54 (s, 2H, OCH2),
5.47–5.42 (m, 1H, OCH), 5.20 (s, 2H, NCH2), 3.06–2.96 (m, 1H, Ha,
CH2), 2.84–2.78 (m, 1H, Hb, CH2); MS (ESI+) m/z (M+H): 567.0,
569.0 and 571; Anal. Calcd for C25H19Br2N3O3: C, 52.75; H, 3.36;
N, 7.38. Found C, 52.72; H, 3.41; N, 7.34.
4.1.5.10. 7-{(1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-yl) meth-
oxy}-2-(4-isopropylphenyl) chroman-4-one 5{10}.
Light
yellow solid, mp 120–124 °C, yield, 0.44 g (88%); IR (KBr) cm
ꢁ1:3750, 3453, 2960, 1625, 1562, 1458, 1119, 799; 1H NMR
(300 MHz, CDCl3 + DMSO-d6) d: 7.87–7.85 (m, 1H, ArH), 7.61–
7.50 (m, 4H, triazolylCH@, ArH), 7.42–7.38 (m, 1H, ArH), 7.30 (d,
J = 7.5 Hz, 2H, ArH), 7.15 (d, J = 8.1 Hz, 2H, ArH), 6.67 (dd,
J1 = 1.8 Hz, J2 = 8.7 Hz, 1H, ArH), 6.58 (m, 1H, ArH), 5.50 (s, 2H,
OCH2), 5.46 (dd, J1 = 2.7 Hz, J2 = 13.3 Hz, 1H, OCH), 5.21 (s, 2H,
NCH2), 3.04 (d, J = 13.1 Hz, 1H, Ha, CH2), 2.97(m, 1H, CH), 2.85
4.1.5.5. 7-{(1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy}-2-phenyl
chroman-4-one 5{5}.
Light yellow solid, mp 118–120 °C,
0.68 g, yield 85%; IR (KBr) cmꢁ1: 3754, 3449, 2923, 1708, 1443,
1252, 696; 1HNMR (300 MHz, CDCl3 + DMSO-d6) d: 7.88 (m, 1H,
ArH), 7.70 (s, 1H, triazolylCH@), 7.63–7.53 (m, 3H, ArH),

5160
N. Anand et al. / Bioorg. Med. Chem. 20 (2012) 5150–5163
(dd, J1 = 2.6 Hz, J2 = 16.6 Hz, 1H, Hb, CH2), 1.30 (s, 3H, CH3), 1.28(s,
3H, CH3); MS (ESI+) m/z (M+H): 532.0 and 534.0 ; Anal. Calcd for
2H, NCH2), 3.85 (s, 3H, OCH3), 1.88–1.86 (m, 2H, CH2CH3), 1.30
(br s, 6H, 3xCH2), 0.86 (br s, 3H, CH3); 13C NMR (300 MHz,
CDCl3 + DMSO-d6) d: 169.8, 167.6, 166.7 166.6, 166.4, 165.9,
147.8, 134.7 , 133.8, 133.5(2C), 128.3, 118.7(2C), 116.1, 111.7,
107.7, 103.7, 66.5, 60.5, 54.7, 35.8, 34.9, 30.8, 27.1, 18.8; MS
(ESI+) m/z (M+H): 475.2 Anal. Calcd for C26H30N6O3: C, 65.80; H,
6.37; N, 17.71. Found C, 65.77; H, 6.39; N, 17.74.
C28H26BrN3O3: C, 63.16; H, 4.92; N, 7.89. Found C, 63.12; H, 5.01;
N, 17.84.
4.1.6. Triazolylmethoxy aminopyrimidines 6{1–17}
4.1.6.1.
2-(2-Amino-6-phenylpyrimidin-4-yl)-5-{(1-hexyl-1H-
A solution of gua-
1,2,3-triazol-4-yl) methoxy} phenol 6{1}.
nidine hydrochloride (0.13 g, 1.35 mmol) dissolved in DMF (1 mL)
was added into a slurry of NaH (0.32 g, 1.35 mmol) in DMF (1 mL)
at 0 °C, followed by the addition of chalcone 4{1} (0.50 g,
1.23 mmol) in DMF (1 mL). The whole reaction mixture was stirred
at 0 °C for about half an hour and after at 100 °C until reaction got
completed (The reaction progress and completion was monitored
by TLC). Reaction mixture was filtered over cellite pad using Ethyl-
acetate as solvent. Organic layer was washed three times with
water, dried over Na2SO4 and concentrated in vacuum. The viscous
crude mass (sometimes solid) thus obtained was purified by column
chromatography using ethylacetate/hexane as eluent in the 3:10 ra-
4.1.6.5.
2-{2-Amino-6-(4-isopropylphenyl)pyrimidin-4-yl}-5-
The
{(1-hexyl-1H-1,2,3-triazol-4-yl) methoxy} phenol 6{5}.
reaction of 4{6} (0.5 g, 1.11 mmol) and Guanidine hydrochloride
(0.116 g, 1.21 mmol) as described above gave yellow solid; mp
164–166 °C, yield, 0.32 g (59%); IR (KBr) cmꢁ1: 3488.8, 3333.7,
2365.3, 1580.6; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d: 8.05 (d,
J = 8.64 Hz, 2H, ArH), 7.83 (d , J = 8.07 Hz, 2H, ArH, triazolyl CH@),
7.51 (s, 1H, ArH), 7.33 (d, J = 8.07 Hz, 2H, ArH), 6.85 (br s, 2H,
NH2), 6.56 (d, J = 6.03 Hz, 2H, ArH), 5.16 (s, 2H, OCH2), 4.36 (t,
J1 = J2 = 7.08 Hz, 2H, NCH2), 2.98 -2.93 (m, 1H, -CH-), 1.86–1.82
(m, 2H, CH2), 1.27–1.25 (m, 12H, 3xCH2, 2xCH3), 0.85 (m, 3H,
CH3); MS (ESI+) m/z (M+H): 487.1; Anal. Calcd for C28H34N6O2: C,
69.11; H, 7.04; N, 17.27. Found C, 69.08; H, 7.10; N, 17.31.
tio, yellow solid, mp 155–156 °C, yield 0.30 g (55%); IR (KBr) cmꢁ1
:
3416.4,. 3315.0, 2336.2, 1571.1; 1H NMR (300 MHz, CDCl3 + DMSO-
d6) d: 8.03–8.01 (m, 2H, ArH), 7.80 (s, 1H, triazolyl CH@), 7.76 (s, 1H,
ArH), 7.44–7.36 (m, 4H, ArH), 6.52–6.50 (m, 2H, ArH), 6.24 (s, 2H,
NH2), 5.16 (s, 2H, OCH2), 4.34 (t, J1 = 7.08, J2 = 7.2 Hz, 2H, NCH2),
1.87 (m, 2H, CH2), 1.28 (br s, 6H, 3x-CH2), 0.84 (br s, 3H, CH3); 13C
NMR (300 MHz, CDCl3 + DMSO-d6) d: 170.3, 167.6, 166.6, 165.9,
147.8, 142.4, 135.2.2, 133.3(2C), 131.9(2C), 128.0, 121.2, 116.1,
111.8, 107.6, 104.6, 66.4, 54.9, 36.6. 35.8, 34.9, 30.8, 27.1, 18.7; MS
(ESI+) m/z (M+H): 445.0 Anal. Calcd for C25H28N6O2: C, 67.55; H,
6.35; N, 18.91. Found C, 67.51; H, 6.39; N, 18.94.
4.1.6.6.
2-{2-Amino-6-(4-propynyloxy)pyrimidin-4-yl}-5-{(1-
The
hexyl-1H-1,2,3-triazol-4-yl) methoxy) phenol 6{6}.
reaction of 4{7} (0.5 g, 1.0 mmol) and Guanidine hydrochloride
(0.11 g, 1.20 mmol) as described above gave yellow solid; mp
172–175 °C, 0.27 g, yield 49%; IR (KBr) cmꢁ1: 3443.5, 3314.8,
2367.2, 1584.2; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d: 8.12–
8.10 (m, 4H, ArH, triazolyl CH@), 7.44–7.38 (m, 1H, ArH), 7.16–
7.01 (m, 2H, ArH,), 6.56–6.54 (m, 4H, NH2, ArH), 5.18 (s, 2H,
OCH2), 4.79 (s, 1H, OCH2), 4.38 (t, J1 = J2 = 6.93 Hz, 2H, NCH2), 3.02
(m, 1H, -CH), 1.88 (m, 2H, CH2), 1.31 (m, 6H, 3xCH2), 0.88 (m, 3H,
CH3); MS (ESI+) m/z (M+H): 499.1; Anal. Calcd for C28H30N6O3: C,
67.42; H, 6.06; N, 16.86. Found C, 67.38; H, 6.16; N, 16.84.
4.1.6.2. 2-(2-Amino-6-(4-chlorophenyl)pyrimidin-4-yl)-5-{(1-
hexyl-1H-1,2,3-triazol-4-yl) methoxy} phenol 6{2}.
The
reaction of 4{3} (0.5 g, 1.13 mmol) and Guanidine hydrochloride
(0.19 g, 1.24 mmol) as described above gave yellow solid, mp
192–194 °C, yield 0.29 g (54%), IR (KBr) cmꢁ1: 3505.2, 3386.1,
2367.1, 1609.3; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d: 8.06 (d,
J = 8.4 Hz, 2H, ArH) 7.83–7.80 (m, 2H, triazolyl CH@, ArH), 7.42–
7.38 (m, 3H, ArH), 6.52–6.49 (m, 4H, ArH, NH2), 5.15 (s, 2H,
OCH2), 4.36 (t, J1 = J2 = 7.1 Hz, 2H, NCH2), 1.89–1.85 (m, 2H, CH2),
1.29 (br s, 6H, 3xCH2), 0.87 (d, J = 6.2 Hz, 3H, CH3); MS (ESI+) m/z
(M+H): 479.1 and 481.1; Anal. Calcd for C25H27ClN6O2: C, 62.69;
H, 5.68; N, 17.55. Found C, 62.64; H, 5.73; N, 17.51.
4.1.6.7. 2-{2-Amino-6-(4-napthalen-1-yl)pyrimidin-4-yl}-5-((1-
hexyl-1H-1,2,3-triazol-4-yl) methoxy) phenol 6{7}.
The
reaction of 4{8} (0.5 g, 1.09 mmol) and Guanidine hydrochloride
(0.115 g, 1.20 mmol) as described above gave yellow solid; mp
178–180 °C, yield, 0.27 g, 56%; IR (KBr) cmꢁ1: 3488.8, 3333.7,
2362.3, 1575.6; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d: 8.12(m,
1H, ArH), 7.97–7.76 (m, 2H, ArH, triazolyl CH@), 7.68–7.60 (d ,
2H, ArH,), 7.56–7.45 (m, 3H, ArH), 7.20 (s, 1H, ArH), 6.85 (br s,
2H, NH2), 6.48 (dd, J1 = 2.34 Hz, J2 = 8.82 Hz, 1H, ArH), 5.15 (s, 2H,
OCH2), 4.36 (t, J1 = J2 = 7.08 Hz, 2H, NCH2), 1.86–1.78 (m, 2H, CH2),
1.28 (m, 12H, 3xCH2, 2xCH3), 0.85–0.77 (m, 3H, CH3); MS (ESI+)
m/z (M+H): 495.1; Anal. Calcd for C29H30N6O2: C, 70.42; H, 6.11;
N, 16.99. Found C, 70.38; H, 6.19; N, 16.94.
4.1.6.3.
yl)-5-{(1-hexyl-1H-1,2,3-triazol-4-yl)methoxy}
6{3}. The reaction of 4{4} (0.5 g, 1.09 mmol) and Guanidine
2-(2-Amino-6-(2-chloro-6-fluorophenyl)pyrimidin-4-
phenol
hydrochloride (0.11 g, 1.19 mmol) as described above gave yellow
solid; mp 185–187 °C, yield, 0.24 g, 51%; IR (KBr) cmꢁ1: 3427.0,
3354.6, 2366.4, 1577.2; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d:
7.82 (m, 1H, ArH), 7.67–7.59 (m, 2H, ArH), 7.41–7.30 (m, 2H,
ArH), 6.98–6.48 (m, 3H, ArH, NH2), 5.18 (s, 2H, OCH2), 4.35 (t,
J = 6.80 Hz , 2H, NCH2), 1.88–1.89 (m, 2H, CH2CH3), 1.31 (br s, 6H,
3xCH2), 0.87 (br s, 3H, CH3); MS (ESI+) m/z (M+H): 497.0 and
499.1; Anal. Calcd for C25H26ClFN6O2: C, 62.42; H, 5.27; N, 16.91.
Found C, 62.38; H, 5.33; N, 16.84.
4.1.6.8. 2-(2-Amino-6-phenylpyrimidin-4-yl)-5-{(1-benzyl-1H-
1,2,3-triazol-4-yl)methoxy} phenol 6{8}.
The reaction of
4{9} (0.5 g, 1.2 mmol) and Guanidine hydrochloride (0.127 g,
1.33 mmol) as described above gave yellow solid, mp 185–
186 °C, yield, 0.30 g (54%); IR (KBr) cmꢁ1: 3491.3. 3356.5, 2366.2,
1572.1; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d: 8.07–8.06 (m,
2H, ArH), 7.82 (s, 1H, triazolyl CH@), 7.66 (s, 1H, ArH), 7.48–7.28
(m, 9H, ArH), 6.55–6.53 (m, 2H, ArH), 6.27 (s, 2H, NH2), 5.56 (s,
2H, OCH2), 5.18 (s, 2H, NCH2); MS (ESI+) m/z (M+H): 451.1; Anal.
Calcd for C26H22N6O2: C, 69.32; H, 4.92; N, 18.66. Found C, 69.29;
H, 4.96; N, 18.64.
4.1.6.4. 2-(2-Amino-6-(4-methoxyphenyl)pyrimidin-4-yl)-5-{(1-
hexyl-1H-1,2,3-triazol-4-yl) methoxy} phenol 6{4}.
The
reaction of 4{5} (0.5 g, 1.14 mmol) and Guanidine hydrochloride
(0.12 g, 1.25 mmol) as described above gave yellow solid; mp
180–182 °C, yield, 0.28 g (52%); IR (KBr) cmꢁ1: 3427.0, 3354.6,
2366.4, 1577.2; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d: 8.05 (d,
J = 8.6 Hz, 2H, ArH), 7.83 (d, J = 8.07 Hz, 2H, ArH), 7.36 (s, 1H,
ArH), 6.97 (d, J = 7.8 Hz, 2H, ArH), 6.54–6.52 (m, 2H, ArH), 6.38
(br s, 2H, NH2), 5.17 (s, 2H, OCH2), 4.34 (t, J1 = 7.10, J2 = 6.90 Hz ,
4.1.6.9. 2-{2-Amino-6-(4-chlorophenyl)pyrimidin-4-yl}-5-{(1-
benzyl-1H-1,2,3-triazol-4-yl)methoxy} phenol 6{9}.
reaction of 4{10} (0.5 g, 1.10 mmol) and Guanidine hydrochloride
(0.115 g, 1.21 mmol) as described above gave yellow solid, mp
200–203 °C, yield, 0.28 g, (52%); IR (KBr) cmꢁ1: 3496.9, 3370.8,
The

N. Anand et al. / Bioorg. Med. Chem. 20 (2012) 5150–5163
5161
2365.8, 1580.0; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d: 8.14 (d,
J = 6.9 Hz, 2H, ArH), 8.04 (s, 1H, triazolyl CH@), 7.94–7.92 (m, 1H,
ArH), 7.50 (d, J = 2.2 Hz, 1H, ArH), 7.45 (d, J = 7.9 Hz, 2H, ArH),
7.33–7.31 (m, 5H, ArH), 6.79 (s, 2H, NH2), 6.52 (d, J = 6.6 Hz, 2H,
ArH), 5.57 (s, 2H, OCH2), 5.14 (s, 2H, NCH2); 13C NMR (300 MHz,
CDCl3 + DMSO-d6) d: 170.3, 170.0, 167.6, 166.6, 166.1, 148.2,
142.5, 139.2, 136.7, 135.2(2C), 134.8(2C), 133.4(2C), 133.3(2C),
132.0 128.7, 127.0, 116.1, 111.7, 107.7, 104.5, 66.4, 57.7; MS
(ESI+) m/z (M+H): 485.0 and 487.1; Anal. Calcd for C26H21ClN6O2:
C, 64.40; H, 4.36; N, 17.33. Found C, 64.37; H, 4.39; N, 17.28.
130.9, 130.5, 130.0, 128.9, 115.9, 111.9, 109.1, 107.8, 66.4, 58.4;
MS (ESI+) m/z (M+H): 501.1; Anal. Calcd for C30H24N6O2: C, 71.98;
H, 4.83; N, 16.79. Found C, 71.92; H, 4.87; N, 16.74.
4.1.6.14. 2-(2-Amino-6-phenylpyrimidin-4-yl)-5-{1-(4-bromob-
enzyl)-1H-1,2,3-triazol-4-yl} methoxy)phenol 6{14}.
The
reaction of 4{16} (0.5 g, 1.02 mmol) and Guanidine hydrochloride
(0.107 g, 1.12 mmol) as described above gave yellow solid, mp
190–193 °C, yield, 0.30 g (60%); IR (KBr) cmꢁ1: 3501, 3349, 1577,
691; 1H NMR (300 MHz, CDCl3+ DMSO-d6) d: 8.04 (br s, 2H, ArH),
7.91 (s, 1H, triazolyl CH@), 7.84–7.77 (m, 1H, ArH), 7.46–7.39 (m,
6H, ArH), 7.21 (d, J = 8.12 Hz, 2H, ArH), 6.47–6.39 (m, 4H, ArH,
NH2), 5.51 (s, 2H, OCH2), 5.13 (s, 2H, NCH2); 13C NMR (300 MHz,
CDCl3+ DMSO-d6) d: 170.3, 170.0, 167.7, 166.6, 166.1, 148.2,
142.5, 139.4, 136.7(2C), 135.2, 134.8(2C), 133.5, 133.3(2C),
132.0(2C), 128.7, 127.0, 116.1, 111.8, 107.7, 104.5, 66.4, 57.7; MS
(ESI+) m/z (M+H): 529, 531; Anal. Calcd for C26H21BrN6O2: C,
58.99; H, 4.00; N, 15.87. Found C, 58.92; H, 4.07; N, 15.84.
4.1.6.10. 2-{2-Amino-6-(2-chloro-6-flourophenyl)pyrimidin-4-
yl}-5-{(1-benzyl-1H-1,2,3-triazol-4-yl)methoxy}
phenol
6{10}. The reaction of 4{11} (0.5 g, 1.07 mmol) and Guanidine
hydrochloride (0.113 g, 1.18 mmol) as described above gave yellow
solid, mp >200 °C, yield, 0.26 g (48%); IR (KBr) cmꢁ1: 3487.2,
3365.3, 2384.1, 1568.0; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d:
13.52 (s, 1H, OH), 7.41 (s, 1H, triazolyl CH@), 7.41–7.12 (m, 1H,
ArH), 6.78 (br s, 7H, ArH), 6.69–6.58 (m, 1H, ArH), 6.45–6.39 (m,
1H, ArH), 6.11–5.98 (s, 2H, NH2), 5.03 (s, 2H, OCH2), 4.62 (s, 2H,
NCH2); MS (ESI+) m/z (M+H): 503 and 505.2; Anal. Calcd for
4.1.6.15. 2-{2-Amino-6-(4-bromophenyl) pyrimidin-4-yl}-5-{1-
(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methoxy}
phenol
C26H21ClFN6O2: C, 62.09; H, 4.01; N, 16.71. Found C, 62.07; H,
6{15}. The reaction of 4{17} (0.7 g, 1.33 mmol) and Guanidine
4.09; N, 16.68.
hydrochloride (0.139 g, 1.46 mmol) as described above gave yellow
solid, mp 210–212 °C, yield, 0.440 g (59%); IR (KBr) cmꢁ1: 3466,
3310, 1621,793; 1H NMR (300 MHz, CDCl3+ DMSO-d6) d: 14.2 (s,
1H, OH), 8.14 (d, J = 7.9 Hz, 2H, ArH), 8.07 (s, 1H, triazolyl CH@),
7.98 (m, 1H, ArH), 7.47 (m, 5H, ArH), 7.24 (d, J = 7.9 Hz, 2H, ArH),
6.77 (br s, 2H, NH2), 6.53 (d, J = 6.6 Hz, 2H, ArH), 5.56 (s, 2H,
OCH2), 5.16 (s, 2H, NCH2); 13C NMR (300 MHz, CDCl3+DMSO-d6)
d: 170.3, 168.8, 167.4, 166.7, 166.1, 147.9, 140.9, 140.7, 140.1,
136.8(2C), 135.3(2C), 134.4, 133.9(2C), 133.8(2C), 129.8, 126.7,
116.0, 112.1, 107.7, 104.3, 66.2, 57.4; MS (ESI+) m/z (M+H):
607.3, 609.3 and 611.1; Anal. Calcd for C26H20Br2N6O2: C, 51.34;
H, 3.31; N, 13.82. Found C, 51.39; H, 3.43; N, 13.78.
4.1.6.11. 2-{2-Amino-6-(4-methoxyphenyl)pyrimidin-4-yl}-5-
{(1-benzyl-1H-1,2,3-triazol-4-yl)
6{11}. The reaction of 4{12} (0.5 g, 1.13 mmol) and Guanidine
methoxy}
phenol
hydrochloride (0.12 g, 1.24 mmol) as described above gave yellow
solid; mp 180–182 °C, 0.28 g, yield (52%); IR (KBr) cmꢁ1: 3496.9,
3370.8, 2365.8, 1580.0; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d:
8.12 (d, J = 6.78 Hz, 1H, ArH), 8.07 (s, 1H, triazolyl CH@), 7.98 (d,
J = 9.51 Hz, 1H, ArH), 7.47(s, 1H, ArH), 7.27–7.24 (m, 5H, ArH), 6.94
(d, J = 8.85 Hz, 2H, ArH), 6.84 (br s, 2H, NH2), 6.47(d, 2H, J = 7.2 Hz,
ArH), 5.51 (s, 2H, OCH2), 5.08 (s, 2H, NCH2) 3.76 (s, 1H, OCH3); 13C
NMR (300 MHz, CDCl3 + DMSO-d6) d: 169.8, 169.6, 167.7, 166.7,
166.5, 166.1, 147.9, 140.9, 134.6, 134.2(2C), 133.9(2C), 133.2(2C),
133.1(2C), 129.7, 118.9(2C), 116.2, 111.9, 107.7, 103.5, 66.4, 60.4,
58.1; MS (ESI+) (M+H): 481.1; Anal. Calcd for C27H24N6O2: C,
67.49; H, 5.03; N, 17.49. Found C, 67.42; H, 5.07; N, 17.44.
4.1.6.16. 2-{2-Amino-6-(4-isopropyl)pyrimidin-4-yl}-5-{(1-(4-
bromobenzyl)-1H-1,2,3-triazol-4-yl)
methoxy}
phenol
6{16}. The reaction of 4{19} (0.5 g, 0.94 mmol) and Guanidine
hydrochloride (0.98 g, 1.03 mmol) as described above gave yellow
solid; mp 183–185 °C, 0.25 g, yield 56%; IR (KBr) cmꢁ1: 3438.6,
3333.7, 2368.2, 1576.5; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d:
8.05–7.91 (m, 3H, ArH), 7.51–7.20 (m, 8H, ArH, triazolyl CH@),
6.73–6.51 (m, 4H, NH2, ArH), 5.57 (s, 2 H, OCH2), 5.15 (s, 2 H,
NCH2); MS (ESI+) m/z (M+H): 571.0 and 573.1; Anal. Calcd for
4.1.6.12. 2-{2-Amino-6-(4-isopropylphenyl)pyrimidin-4-yl}-5-
{(1-benzyl-1H-1,2,3-triazol-4-yl)
6{12}. The reaction of 4{13} (0.5 g, 1.10 mmol) and Guanidine
methoxy}
phenol
hydrochloride (0.115 g, 1.21 mmol) as described above gave yellow
solid; mp 178–180 °C, 0.29 g, yield (54%); IR (KBr) cmꢁ1: 3481.3,
3366.5, 2365.8, 1577.8; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d:
8.11–8.05 (m, 3H, triazolyl CH@), 7.98 (d, J = 7.65 Hz, 1H, ArH),
7.50 (s, 1H, ArH), 7.33–7.31 (m, 7H, ArH), 6.83 (br s, 2H, NH2),
6.54 (d, J = 7.29 Hz, 2H, ArH), 5.58 (s, 2H, OCH2), 5.15 (s, 2H,
NCH2), 2.98–2.91 (m, 1H, -CH-), 1.28–1.22 (m, 6H, 2xCH3); MS
(ESI+) m/z (M+H): 493.5; Anal. Calcd for C29H28N6O2: C, 70.71; H,
5.73; N, 17.06. Found C, 70.69; H, 5.76; N, 17.04.
C29H27BrN6O2: C, 60.95; H, 4.76; N, 14.71. Found C, 60.91; H,
4.81; N, 14.64.
4.1.6.17. 2-{2-Amino-6-(naphthalen-1-yl)pyrimidin-4-yl}-5-{(1-
(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)
methoxy}
phenol
6{17}. The reaction of 4{20} (0.5 g, 0.93 mmol) and Guanidine
hydrochloride (0.98 g, 1.02 mmol) as described above gave yellow
solid; mp 188–190 °C, yield, 0.26 g (51%); IR (KBr) cmꢁ1: 3428.6,
3339.7, 2369.9, 1576.9; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d:
8.22–8.14 (m, 1H, ArH), 8.11 (s, 1H, triazolyl CH@), 7.96–7.90 (m,
2H, ArH), 7.80 (d, J = 9.0 Hz, 1H, ArH), 7.64 (d, J = 6.24 Hz,1H,
ArH), 7.57–7.46 (m, 6H, ArH), 7.26–7.23 (m, 3H, ArH), 7.04 (br s,
2H, NH2), 6.54 (d, J = 0.21 Hz, 1H, ArH), 6.49 (dd, 1H, J1 = 0.21,
J2 = 8.82 Hz, 1H, ArH), 5.55 (s, 2 H, OCH2), 5.14 (s, 2 H, NCH2); MS
(ESI+) m/z (M+H): 579.0 and 581.0; Anal. Calcd for C30H23BrN6O2:
C, 62.18; H, 4.00; N, 14.50. Found C, 62.21; H, 4.07; N, 14.54.
4.1.6.13.
{(1-benzyl-1H-1,2,3-triazol-4-yl)
6{13}. The reaction of 4{15} (0.5 g, 1.08 mmol) and Guanidine
2-{2-Amino-6-(naphthalene-1-yl)pyrimidin-4-yl}-5-
methoxy} phenol
hydrochloride (0.113 g, 1.18 mmol) as described above gave yellow
solid; mp 196–198 °C, yield, 0.30 g (54%); IR (KBr) cmꢁ1: 3473.2,
3305.3, 2371.4, 1572.1; 1H NMR (300 MHz, CDCl3 + DMSO-d6) d:
8.22–8.20 (m, 1H, ArH), 7.97 (s, 1H, triazolyl CH @), 7.93–7.89 (m,
2H, ArH), 7.73 (d, J = 8.85 Hz, 1H, ArH), 7.63 (d, J = 6.15 Hz, 1H,
ArH), 7.56–7.47 (m, 3H, ArH), 7.31–7.30 (m, 5 H, ArH), 7.19 (s, 1H,
ArH), 6.80 (br s, 2H, NH2), 6.54 -6.46 (m, 2H, ArH), 5.55 (s, 2H,
OCH2), 5.14 (s, 2 H, NCH2); 13C NMR (200 MHz, CDCl3 + DMSO-d6)
d: 173.0, 169.7, 167.7, 166.7, 165.9, 148.0, 142.1, 140.3, 138.5,
135.3, 134.3, 133.7(2C), 133.5, 133.2(2C), 132.9, 131.8, 131.4,
4.2. Biological assay
4.2.1. Material and method for Fas-II inhibitory screening
4.2.1.1. FAS-II inhibition assay.
FAS-II inhibitory activity was
assessed using recombinant non-pathogenic mycobacterial
a

5162
N. Anand et al. / Bioorg. Med. Chem. 20 (2012) 5150–5163
strain, Mycobacterium aurum, which contains M. tuberculosis kas
operon promoter in fusion with lacZ reporter gene.33 The strain
shows continued expression of reporter gene under the influence
of kas operon promoter during basal conditions, while an increased
expression of the reporter gene is noticed only after treatment with
FAS-II pathway inhibitors. The preliminary screening of the com-
pounds shows FAS-II inhibitory activity at two different concentra-
5000 ꢃ g for 15 min and resuspended in binding buffer [Sodium
Phosphate 20 mM (pH 7.4), NaCl 50 mM, Imidazole 5 mM, PMSF
1 mM] and sonicated on ice for 2 min. After sonication TritonX-
100 was added in cell lysate at a final concentration of 1% before
centrifugation at 30000 ꢃ g for 30 min at 4 °C. Supernatant was
loaded onto Ni2+-NTA column, washed with 60 mM Imidazole
and 6-His-PknG was eluted with 200 mM Imidazole. Affinity puri-
fied 6-His-PknG was further purified by size exclusion chromatog-
raphy using Sephacryl 200 column and AKTA Prime protein
purification system (GE healthcare).
tions, 50 and 100 lM.
4.2.1.2. Bacterial strains and viability assay.
The generation
of recombinant M. aurum strains was described earlier.33 M. aurum
cultures were grown in Sauton’s medium supplemented with
4.2.2.2. Screening of compounds against Kinase activ-
0.05% Tween-80 and kanamycin (25
l
g/mL) and were plated on
ity.
The compounds were dissolved completely in DMSO.
M concentration
Nutrient-agar plates with 0.05% Tween-80 (NAT) supplemented
with kanamycin. For post treatment viability assay, M. aurum
was grown in Sauton’s medium up to 0.6 OD600 and the culture
was diluted to 0.05 OD with fresh medium. From these diluents,
ꢀ1 ꢃ 105cells were inoculated into different tubes containing
5 ml fresh medium and added varying concentration of com-
pounds. The cultures were allowed to grow for 12 h at 37 °C with
continuous shaking at 180 rpm. The treated and untreated cultures
were plated on NAT-Km plates using 10-fold serial dilution to
count the number of viable cells. % inhibition was scored consider-
ing the number of bacterial colonies in untreated condition as
100%.
For the determination of primary efficacy, 100
l
of each compound was screened using purified PknG as an enzyme
and myelin basic protein as a substrate. The activity and the inhi-
bition were determined by using luciferase activity mediated by
ATP, by ADP–Glo (Promega, USA). Briefly, ADP-Glo™ Kinase Assay
is a luminescent kinase assay that measures ADP formed from a ki-
nase reaction. ADP is converted into ATP, which is converted into
light by Ultra-Glo™ Luciferase.
4.2.3. Cytotoxicity Assay: CC50 Determination
The cytotoxicity assays was performed according to method re-
ported by O’Brien et al.37 with a slight modification. Macrophages
were harvested from subconfluent monolayers. The suspended cells
were seeded in 96-well microplates, at an approximate initial den-
sity of 1 ꢃ 106 cells per well, in RPMI-1640 medium. Compounds
4.2.1.3. Reporter gene expression analysis.
Recombinant M.
aurum strains were grown in Sauton’s medium with Kanamycin at
37 °C to 0.5 OD600 after which culture was diluted to 0.04–0.05 OD
with fresh medium. Ten millilitre of diluted culture were distrib-
uted to separate tubes, equilibrated for 2 h at 37 °C and then vary-
were added at the concentrations from 100 lM to 12.5 lM. Rifam-
picin was taken as positive control for showing viability at concen-
trations from 4 g/ml to 0.5 g/ml. 96-well plates were incubated
l
l
ing concentrations (50 and 100
l
M) of compounds were added to
for 48 h at 5% CO2 at 37 °C. After 48 h, the resazurin was added
and incubated for 4 h. The fluorescence and absorbance were mea-
sured in a spectrophotometer at 535/590 nm. Cytotoxicity was
determined by comparing the resulting fluorescence with the mean
fluorescence of the control wells (untreated cells), and was ex-
pressed as percentage of cell viability. The 50% cytotoxic concentra-
tion (CC50) is defined as the quantity of compound generating 50% of
cell viability, compared to the control. The values of the percentages
of cell viability were plotted against the concentrations, and
CC50 was determined. Experiments were carried out in triplicates.
different tubes. Following 12 h incubation at 37 °C, 5 ml cultures
from each tube were pelleted, washed and resuspended in PBS
(Phosphate Buffer Saline, pH 7.2), sonicated at 4 °C and superna-
tant was collected by centrifugation at 13000 rpm for10 min at
4 °C. Protein contents were quantified using Bradford Assay re-
agent (Sigma B6916) as per manufacturer protocol. b-Gal assay
was performed from total cellular protein as described earlier1.
Briefly, same amount of protein were mixed with 200
(4 mg/ml) and incubated for 30 min at 37 °C. Reaction was stopped
by adding 500 L of 1MNa2CO3 and optical density was measured
lL of ONPG
l
at 410 nm. Experiments were carried out in triplicates for each
treatment and b-galactosidase units were calculated for each set
individually. The culture at each point was also plated to confirm
the decline in viability of cells after drug treatment. The whole
experiment was repeated twice and similar trends in results were
obtained. Mean value and standard deviation were calculated and
plotted for each set of data.
Acknowledgements
Authors thanks UGC, CSIR, and DRDO New Delhi for financial
assistance. Namrata and Anindra are thankful to UGC and CSIR
New Delhi for their SRF. We are also thankful to SAIF Division, CDRI
for providing the spectral data and microanalyses. It is a CDRI com-
munication no. 8276T.
4.2.2. Material and method for PknG inhibitory screening
Supplementary data
4.2.2.1.
PKnG.
Purification
of
Mycobacterium
tuberculosis
The compounds were screened against the mycobacte-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.07.009.
rial serine threonine protein kinase G (PknG). The recombinantly
purified enzyme was used for the study.36 Briefly the Mycobacte-
rium tuberculosis (MTB) genomic DNA was used as a template for
amplification of pknG gene by PCR. The gene was cloned in pTriEx4
vector using the primers containing the desired restriction enzyme
sites. For expression in E. coli, pknG with HindIII flanking sites was
subcloned in pTriEx4 vector. E. coli BL21 (DE3) cells were trans-
formed with pTriEX4-pknG and transformants were grown in LB
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