Novel alkynyl substituted 3,4-dihydropyrimidin-2(1H)-one derivatives as potential inhibitors of chorismate mutase

Article abstract of DOI:10.1016/j.bioorg.2013.08.002

A series of novel alkynyl substituted 3,4-dihydropyrimidin-2(1H)-one (DHPM) derivatives were designed, synthesized and evaluated in vitro as potential inhibitors of chorismate mutase (CM). All these compounds were prepared via a multi-component reaction (

Full text of DOI:10.1016/j.bioorg.2013.08.002

Bioorganic Chemistry 51 (2013) 48–53
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Preliminary Communications
Novel alkynyl substituted 3,4-dihydropyrimidin-2(1H)-one derivatives
as potential inhibitors of chorismate mutase
V. Mallikarjuna Rao a, P. Mahesh Kumar b, D. Rambabu c, Ravikumar Kapavarapu d, S. Shobha Rani a,
Parimal Misra c, Manojit Pal c,
⇑
a Center for Pharmaceutical Sciences, Institute of Science and Technology, Jawaharlal Nehru Technological University, Hyderabad 500 085, Andhra Pradesh, India
b Custom Pharmaceutical Services, Dr. Reddy’s Laboratories Limited, Bollaram Road Miyapur, Hyderabad 500 049, India
c Dr Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, India
d Doctoral Program in Experimental Biology and Biomedicine, Center for Neuroscience and Cell Biology, University of Coimbra, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 May 2013
Available online 19 August 2013
A series of novel alkynyl substituted 3,4-dihydropyrimidin-2(1H)-one (DHPM) derivatives were designed,
synthesized and evaluated in vitro as potential inhibitors of chorismate mutase (CM). All these com-
pounds were prepared via a multi-component reaction (MCR) involving sequential I2-mediated Biginelli
reaction followed by Cu-free Sonogashira coupling. Some of them showed promising inhibitory activities
Keywords:
Pyrimidinone
Alkyne
Iodine
Palladium
Chorismate mutase
when tested at 30
14.76 0.54 M indicating o-alkynylphenyl substituted DHPM as a new scaffold for the discovery of
promising inhibitors of CM.
lM. One compound showed dose dependent inhibition of CM with IC50 value of
l
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
resistance, (c) co-morbidity with HIV-AIDS and (d) declined effort
in anti-infective drug discovery research. Indeed, TB kills more
than two million people a year worldwide. Thus the discovery
[14], development and introduction of new treatments for tubercu-
losis have become an essential goal of current pharmaceutical re-
search. Mycobacterium tuberculosis chorismate mutase (ÃMtbCM
or CM) catalyzes the rearrangement of chorismate to prephenate
in the biosynthetic pathway to form phenylalanine and tyrosine
after chorismate being formed by the action of chorismate syn-
thase, an enzyme of the shikimate pathway main trunk (Fig. 1).
In bacteria, MtbCM plays a key role in the synthesis of aromatic
amino acids necessary for the survival of organism and therefore
inhibition of MtbCM may hinder the supply of nutrients to the
organism. Due to the absence of this pathway in animals but not
in bacteria CM is considered as a promising target for the identifi-
cation of new drugs [15]. However, only a few small molecules
have been reported to possess inhibitory activity against CM [16–
18]. In continuation of our efforts on the identification of novel
inhibitors of CM [19–24] we became interested in evaluating the
library of small molecules based on 4-substituted 3,4-dihydropyr-
imidin-2(1H)-ones B derived from known DHPM framework A
(Fig. 2). We anticipated that introduction of an alkynyl moiety pos-
sessing an appropriate polar/functional group at the o-position of
the C-4 aryl ring may favor the interaction of the resulting mole-
cule with the CM protein. This was supported by the docking stud-
ies of a representative molecule C (glide score = À17.23) which
showed H-bonding interactions involving the OH group of the
Dihydropyrimidinone (DHPM) framework has been found to be
integral part of several biologically active marine alkaloids [1–4].
Among them, the most potent are the crambine [1] and batzella-
dine [2]. In addition to their potent HIV gp-120 CD4 inhibitory
activities [1,5], these compounds also showed antiviral, antitumor,
antibacterial and anti-inflammatory activities [6,7]. Among various
DHPM derivatives known the 4-substituted 3,4-dihydropyrimidin-
2(1H)-ones have attracted particular attention due to their wide
range of biological activities [7]. They have a similar pharmacolog-
ical profile to that of classical dihydropyridine based calcium chan-
nel modulators [8–10]. Nevertheless, DHPMs have attracted our
attention because of the fact that several DHPMs have been exam-
ined for their antimicrobial properties in the recent years [11–13].
Structurally, all these derivatives differed with respect to the sub-
stituents on the two key positions i.e. C-4 and C-5 positions of the
dihydropyridine ring.
Despite the availability of a range of effective antibiotics and
drugs, the threat of infectious diseases or death due to microbial
infections, still poses considerable health problems. Tuberculosis
(TB) still remains a leading cause of death worldwide due to a
number of factors such as (a) long duration of treatment (6–
9 months), (b) increased incidence of (multi or extensive) drug
⇑
Corresponding author. Fax: +91 40 6657 1581.
E-mail address: manojitpal@rediffmail.com (M. Pal).
0045-2068/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2013.08.002

V. Mallikarjuna Rao et al. / Bioorganic Chemistry 51 (2013) 48–53
49
Erythrose
Phosphoenol
4-phosphate
pyruvate
OH
OOC
O
O
O
O P
O
O
O
P O
O
H2C
HO
COO
OH
Shikimate
HO
OH
COO
OH
Chorismate
CH2
O
COO
Chorismate mutase
COO
OOC
COO
NH2
O
Prephenate
Anthranilate
OH
COOH
NH2
COO
COO
O
O
HO
N
H
Phenyl pyruvate
4-Hydroxyphenyl pyruvate
Trp
COOH
COOH
NH2
NH2
Phe
HO
Tyr
Fig. 1. Conversion of chorismate to prephanate by CM in the shikimate pathway.
Ar
O
O
OH
R1
EtO2C
RO
NH
R2O
NH
NH
N
H
O
N
H
O
N
H
O
A
B
C
Fig. 2. Design of DHPM based novel inhibitors of chorismate mutase.
alkynyl side chain in addition to the two C@O moieties (of the cen-
tral ring and ester) with Arg49, Lys60, Glu109 and Gln76 residues
of the chorismate mutase protein (Fig. 3). Notably, shifting of the
alkynyl moiety from o- to m- or p- position or its complete removal
decreased the interactions with the protein in silico. These observa-
tions strengthened our initial thought on focusing on B containing
alkynyl moiety at the o-position of the C-4 aryl ring. Herein we re-
port our preliminary findings on the CM inhibiting properties of a
series of alkynyl substituted DHPM derivatives in vitro. To the best
of our knowledge this is the first report that discloses DHPM deriv-
atives as potential inhibitors of CM.
2. Results and discussion
2.1. Chemistry
Generally, DHPM derivatives are conveniently prepared via a
widely used multi component reaction (MCR), called Biginelli reac-

50
V. Mallikarjuna Rao et al. / Bioorganic Chemistry 51 (2013) 48–53
Fig. 3. Docking of molecule C into CM (PDB code-2FP2).
tion [25] that involves the cyclocondensation of an aldehyde, a b-
ketoester, and urea (or thiourea) to give dihydropyrimidin-2(1H)-
ones (or thio analogues) [6,7]. A large number of reports are now
available on Biginelli reaction [9] to improve the reaction condi-
tions and product yields along with variations in all three reac-
tants. Recently, we have explored further application of Biginelli
reaction beyond three component limit. Thus, an overall four com-
ponent reaction was developed involving a sequential phospho-
rous acid-mediated Biginelli followed by Cu-free Sonogashira or
Heck or Suzuki reaction [26]. The use of elemental iodine on the
other hand has been explored for successful Biginelli reaction ear-
lier [27]. In view of the easy availability and inexpensive as well as
safer nature of I2 we modified our earlier strategy by replacing the
phosphorous acid with I2 and found that the new strategy worked
well (vide infra). We therefore used this modified method to syn-
thesize our target alkynyl derivatives B (or 5, Scheme 1) that were
designed for the potential inhibition of CM. Thus, a mixture of alde-
hyde 1 (1.0 mmol), ethyl or t-butyl or methyl acetoacetate 2
(5.0 mL), urea 3 (1.25 mmol) and I2 (10 mol%) was stirred at room
temperature for 5 min. Subsequently, the reaction mixture was
heated to 100–120 °C for 1–2 h and then cooled to 50 °C. To this
was added pyrrolidine (3 mL), PdCl2(PPh3)2 (2 mol%) and the al-
kyne 4 (1.5 mmol) with stirring and the mixture was stirred at
80–85 °C for 1–2 h. After usual work up and purification the de-
sired product 5 was isolated in good yield. All the compounds syn-
thesized were well characterized by IR absorbsions near 2227
(AC„CA), 1700 (C@O) and 1650 (C@O) cmÀ1 and appearance of
a peak near d 5.6 corresponding to C-4 hydrogen [24].
2.2. Pharmacology
All the synthesized DHPM derivatives were tested for their
inhibitory potential against Mycobacterium tuberculosis H37Rv
chorismate mutase (CM). The assay [28,29] involved determination
of activity of enzyme CM which catalyzes the conversion of choris-
mate to prephenate. Thus determination of activity of CM is based
on the direct observation of conversion of chorismic acid to pre-
phenate spectrophotometrically at OD274. This reaction was per-
formed in the presence of test compounds to determine their CM
inhibiting activities. A known inhibitor of CM i.e. 4-(3,5-dimeth-

V. Mallikarjuna Rao et al. / Bioorganic Chemistry 51 (2013) 48–53
51
Table 1 (continued)
CHO
R2O
Entry Compound 5
% inhibition @
30 lM
I
I2
100-120 o
C
R2O2C
R1
O
O
+
NH
R1 ( )
4
O
2
1
N
H
O
(PPh3)2PdCl2
Pyrrolidine
80-85 oC
H2N
NH2
EtO2C
EtO2C
EtO2C
NH
OH
5
3
N
H
O
R1
n-Hexyl
n-Pentyl
R2
5
% yield
5d
Et 5a
Et 5b
Et 5c
Et 5d
Et 5e
Et 5f
Et 5g
85
87
84
80
83
85
90
86
81
85
87
82
84
82
81
84
83
5
63
-CH2OH
-CH(OH)CH3
-(CH2)2OH
-(CH2)3OH
1-cyclohexenyl
OH
NH
O
N
H
1-hydroxycyclohexanyl Et 5h
phenyl
4-(n-pentyl)phenyl
n-Pentyl
1-cyclohexenyl
1-hydroxycyclohexanyl t-Bu 5m
1-hydroxycyclohexanyl Me 5n
Et 5i
Et 5j
t-Bu 5k
t-Bu 5l
5e
6
57
OH
NH
1-cyclohexenyl
Me 5o
Me 5p
Me 5q
O
n-Hexayl
N
H
4-Methyl phenyl
5f
Scheme 1. Synthesis of o-alkynylphenyl substituted dihydropyrimidin-2(1H)-one
derivatives (5) via a MCR involving sequential iodine-mediated Biginelli reaction
followed by Cu-free Sonogashira coupling.
7
27
EtO2C
EtO2C
EtO2C
EtO2C
NH
Table 1
Inhibition of chorismate mutase by compound 5 in vitro.
O
N
H5g
Entry Compound 5
% inhibition @
30
lM
8
20
1
31
HO
NH
EtO2C
NH
O
N
O
N
H5h
H
5a
9
17
2
29
EtO2C
NH
NH
O
N
O
N
H 5i
H5b
10
19
3
60
O
NH
NH
EtO
OH
N
H
O
O
N
H
5j
5c
(continued on next page)
4
41
oxyphenethylamino)-3-nitro-5-sulfamoylbenzoic acid [16] was
prepared and used as a reference compound the IC50 value of
and 5f showed 60%, 41%, 63% and 57% inhibition of CM compared
to other molecules when tested (see ESI for the procedure) at
which was found to be less than 10
l
M. Compounds 5c, 5d, 5e
30 lM (Table 1) whereas rest of the compounds were either less

52
V. Mallikarjuna Rao et al. / Bioorganic Chemistry 51 (2013) 48–53
Table 1 (continued)
Entry Compound 5
% inhibition @
30
lM
11
30
O
O
NH
O
N
H
5k
12
22
O
O
Fig. 4. Dose dependent inhibition of CM by the compound 5e.
NH
others. A hydrophobic chain or a bulky group at the alkynyl moiety
was found to be unfavorable for activities. In general, a bulky ester
group attached to the central DHPM ring (e.g. 5k-m) was also
found to be unfavorable. Based on the preliminary data generated
compound 5e was chosen for further evaluation in vitro. In a dose
response study, compound 5e showed inhibition of CM in a dose
O
N
H
5l
13
18
OH
O
O
dependent manner with an IC50 value of 14.76 0.54 lM (Fig. 4).
NH
Thus, alkynyl substituted DHPM framework appeared as a new
scaffold for the development of novel inhibitors of chorismate mu-
tase. Since tuberculosis is a leading cause of death worldwide, the
present classes of compounds are of further interest as potential
antitubercular agents.
O
N
H
5m
14
25
OH
3. Conclusions
MeO2C
NH
In conclusion, we have described the design, synthesis and
in vitro evaluation of novel o-alkynylphenyl substituted 3,4-dihy-
dropyrimidin-2(1H)-one derivatives as potential inhibitors of
chorismate mutase. A series of small molecules synthesized via a
MCR involving sequential iodine-mediated solvent-free Biginelli
reaction followed by copper-free Sonogashira coupling in a single
pot were screened against CM. Some of them showed promising
N
O
H5n
15
21
inhibitory activities when tested at 30
showed dose dependent inhibition of CM with IC50 value of
14.76 0.54 M. Overall, this research has identified alkynyl
substituted DHPM as a new scaffold for the development of new
inhibitors of chorismate mutase.
lM and one compound
MeO2C
NH
l
N
O
H5o
16
28
Acknowledgment
CH3
The authors thank the management of Dr. Reddy’s Institute of
Life Sciences for continuous support and encouragement.
MeO2C
NH
N
H
O
Appendix A. Supplementary material
5p
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2013.
08.002.
17
16
MeO2C
NH
References
CH3
N
H
O
[1] B.B. Snider, Z. Shi, J. Org. Chem. 58 (1993) 3828–3839.
[2] A.D. Patil, N.V. Kumar, W.C. Kokke, M.F. Bean, A.J. Freyer, C. DeBrosse, S. Mai, A.
Trunesh, D.T. Faulkner, B. Carte, A.L. Breen, R.P. Hertzberg, R.K. Johnson, J.W.
Westley, B.C.M. Potts, J. Org. Chem. 60 (1995) 1182–1188.
[3] Y. Kashman, S. Hirsh, O.J. McConnel, I. Ohtani, K. Takenori, H. Kakisawa, J. Am.
Chem. Soc. 111 (1989) 8925–8926.
5q
aData presented are average of three experiments.
[4] I. Ohtani, T. Kusumi, H. Kakisawa, Y. Kashman, J. Am. Chem. Soc. 114 (1992)
8472–8479.
[5] B.B. Snider, J. Chen, A.D. Patil, A. Freyer, Tetrahedron Lett. 37 (1996) 6977–
6980.
[6] C.O. Kappe, Tetrahedron 49 (1993) 6937–6963.
active or inactive. Notably, compounds containing alkynyl side
chain possessing a hydroxy group showed superior activities than

V. Mallikarjuna Rao et al. / Bioorganic Chemistry 51 (2013) 48–53
53
[7] C.O. Kappe, Acc. Chem. Res. 33 (2000) 879–888.
[20] K.S. Kumar, R. Adepu, S. Sandra, D. Rambabu, G.R. Krishna, C.M. Reddy, P.
Misra, M. Pal, Bioorg. Med. Chem. Lett. 22 (2012) 1146–1150.
[21] K.S. Kumar, D. Rambabu, S. Sandra, R. Kapavarapu, G.R. Krishna, M.V.B. Rao, K.
Chatti, C.M. Reddy, P. Misra, M. Pal, Bioorg. Med. Chem. 20 (2012) 1711–1722.
[22] R. Adepu, K.S. Kumar, S. Sandra, D. Rambabu, G.R. Krishna, C.M. Reddy, A.
Kandale, P. Misra, M. Pal, Bioorg. Med. Chem. 20 (2012) 5127–5138.
[23] Alinakhi, B. Prasad, R.M. Rao, U. Reddy, S. Sandra, R. Kapavarapu, D. Rambabu,
G.R. Krishna, C.M. Reddy, R. Kishore, P. Misra, J. Iqbal, M. Pal, Med. Chem.
Commun. 2 (2011) 1006–1010.
[8] G.C. Rovnyak, S.D. Kimball, G. Cucinotta, J.D. Dimareo, J. Gougoutas, A. Hedberg,
M. Malley, J.P. McCarthy, R. Zhang, S. Moreland, J. Med. Chem. 38 (1995) 119–
129.
[9] K.S. Atwall, B.N. Swanson, S.E. Unger, D.M. Floyd, S. Moreland, A. Hedberg, B.C.
OReilly, J. Med. Chem. 34 (1991) 806–811.
[10] D.J. Triggle, S. Padmanabhan, Chemtracts Org. Chem. 8 (1995) 191.
[11] S. Chitra, D. Devanathan, K. Pandiarajan, Eur. J. Med. Chem. 45 (2010) 367–371.
[12] R. Kumar, S. Malik, R. Chandra, Ind. J. Chem. 48 (2009) 718–724.
[13] O.M. Singh, S.J. Singh, M.B. Devi, L.N. Devi, N.I. Singh, S.G. Lee, Bioorg. Med.
Chem. Lett. 18 (2008) 6462–6467.
[24] D. Rambabu, S.K. Kumar, B.Y. Sreenivas, S. Sandra, A. Kandale, P. Misra, M.V.B.
Rao, M. Pal, Tetrahedron Lett. 54 (2013) 495–501.
[14] Actions for Life Towards a World Free of Tuberculosis 2006–2015. World
Health Organization 2006 (<www.stoptb.org/assets/documents/global/plan/
globalplanfinal.pdf>).
[25] P. Biginelli, Gazz. Chim. Ital. 23 (1893) 360–413.
[26] P.M. Kumar, K.S. Kumar, S.R. Poreddy, P.K. Mohakhud, K. Mukkanti, M. Pal,
Tetrahedron Lett. 52 (2011) 1187–1191.
[15] E. Haslam, Shikimic Acid Metabolism and Metabolites, Wiley, New York, 1993.
[16] H. Agarwal, A. Kumar, N.C. Bal, M.I. Siddiqi, A. Arora, Bioorg. Med. Chem. Lett.
17 (2007) 3053–3058.
[17] M.E. Hediger, Bioorg. Med. Chem. 12 (2004) 4995.
[18] P.A. Bartlett, Y. Nakagawa, C.R. Johnson, S.H. Reich, A. Luis, J. Org. Chem. 53
(1988) 3195.
[27] R.S. Bhosale, S.V. Bhosale, S.V. Bhosale, T. Wang, P.K. Zubaidha, Tetrahedron
Lett. 45 (2004) 9111–9113.
[28] S.K. Kim, S.K. Reddy, B.C. Nelson, G.B. Vasquez, A. Davis, A.J. Howard, S.
Patterson, G.L. Gilliland, J.E. Ladner, P.T. Reddy, J. Bacteriol. 188 (2006) 8638–
8648.
[29] S. Sasso, C. Ramakrishnan, M. Gamper, D. Hilvert, P. Kast, FEBS J. 272 (2005)
375–389.
[19] B. Prasad, R. Adepu, S. Sandra, D. Rambabu, G.R. Krishna, C.M. Reddy, G.S.
Deora, P. Misra, M. Pal, Chem. Commun. 48 (2012) 10434–10436.