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K. Singh et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2985–2990
drug-resistant phenotype.8b,10 Verapamil,
a
calcium channel
deliver intermediate 10, which upon N-methylation in the pres-
ence of methyl iodide and NaH and subsequent deprotection with
TFA, afforded intermediate 11 (Scheme 2). Reaction of compound
11 with intermediate 6 in the presence of K2CO3 in DMF yielded
analogues 12a,b (Scheme 2).
antagonist has been found to be one of the most promising EPIs
for further evaluation as an adjunctive TB agent as it has been
shown to inhibit multiple bacterial efflux pumps in pure culture
as well as in experimental models of infection.8a,11 Verapamil has
been found to restore activity of first-line anti-TB drugs in mice
A number of analogues were synthesized according to Scheme 3
to address SAR 3. Morpholino, piperidinyl as well as substituted
piperidinyl and piperazinyl were used as a heterocyclic amine
groups in this reaction. Compound 6 was reacted with various het-
erocyclic amines 13 in DMF in the presence of K2CO3 to provide the
desired compounds 14a–h in average to good yields (Scheme 3).
The synthetic plan to prepare analogues according to SAR 4
involved alkylation of 3,4-dimethoxyphenylacetonitrile 4 with var-
ious alkyl bromides using n-BuLi at 0 °C to afford intermediate 15
(Scheme 4). This alkylated product was then reacted with
1-bromo-3-chloropropane to afford 16, which upon reaction with
N-methylhomovetrylamine in DMF in the presence of K2CO3
yielded target compounds 17a–f. All the synthesized compounds
were fully characterized using various analytical and spectroscopic
techniques (see Supporting Information).
infected with
a
multidrug-resistant M. tuberculosis strain.12
Similarly, addition of verapamil to the standard TB drug regimen
accelerates both the bactericidal and the sterilizing activity of stan-
dard TB drugs.13 These results strongly support a further study of
verapamil and other efflux pump inhibitors in TB treatment.
In this work, we present the synthesis and preliminary struc-
ture–activity relationship (SAR) studies of new analogues of verap-
amil. All the synthesized compounds were evaluated in
combination with RIF in vitro against the H37Rv strain of M. tuber-
culosis to evaluate the nature of interactions between them. In
addition, docking studies of the synthesized compounds and
verapamil using binding sites of the efflux protein Rv1258c were
performed in an attempt to rationalize the observed interactions
with RIF.
In order to explore the utility of the verapamil scaffold to poten-
tiate the antimycobacterial activity of RIF, new analogues of verap-
amil were synthesized for SAR studies, Figure 1. The initial
exploratory SAR investigation was focused on the replacement of
the methyl substituent on the tertiary nitrogen with other substit-
uents (SAR 1), varying the length of the methylene spacer between
the tertiary nitrogen and aromatic ring (SAR 2), replacing the
2-(3,4-dimethoxyphenyl)-N-methylethanamine group with vari-
ous heterocycles (SAR 3) and replacing the isopropyl group at the
stereogenic carbon centre with a hydrogen atom and other alkyl
substituents (SAR 4).
The synthesis of analogues required for SAR 1 commenced with
the treatment of commercially available 3,4-dimethoxyphenyleth-
ylamine 1 with di-tert-butyl dicarbonate [(Boc)2O] in the presence
of triethylamine to afford intermediate 2, which was alkylated
with various alkyl halides in the presence of NaH and subsequent
deprotection using TFA yielded intermediate 3 (Scheme 1). Alkyl-
ation of commercially available 3,4-dimethoxyphenylacetonitrile
4 with isopropyl bromide using n-BuLi at 0 °C afforded compound
5, which upon reaction with 1-bromo-3-chloropropane using
lithium diisopropylamide (LDA) at low temperature yielded inter-
mediate 6. Compound 6 was then reacted with 3,4-dimethoxyphe-
nylethylamine 1 and intermediate 3 in the presence of K2CO3 to
obtain new analogues of verapamil 7a and 7b–d, respectively
(Scheme 1). The cyclopropyl substituted verapamil analogue 7e
was obtained through a coupling of 7a with cyclopropyl boronic
acid 8 (Scheme 1).
All the synthesized compounds were evaluated for their in vitro
antimycobacterial activity against the laboratory strain, M. tuber-
culosis H37Rv. The minimum concentration of each compound that
resulted in 99% growth inhibition (MIC99) is shown in Table 1. Not
surprisingly, verapamil and its derivatives demonstrated poor anti-
mycobacterial activity, as indicated by the high MIC99 values. How-
ever, compounds 7e, 14e–h and 17e,f displayed better
antimycobacterial activities relative to verapamil with the most
active being 14h and 17f (MIC99, 62.5 lM). The MIC99 of RIF against
M. tuberculosis was determined alone and in combination with
verapamil and its analogues. The compounds were tested at half
of their known MIC99 in order to ensure that they had no effect
on bacterial viability. The MIC99 of RIF was 0.0083
consistent with previous reports.14 The MIC99 of RIF was reduced
by four-fold (0.0021 M) in the presence of verapamil. Potential
lM, which is
l
synergistic interactions of these compounds with RIF were deter-
mined by calculating fractional inhibitory concentration indices
(FICI). Synergism is achieved when the activity of a combination
of two drugs against a given microorganism is greater than the
sum of the individual activities of each member of that combina-
tion.15 According to the strict definition,16 a combination of two
compounds is said to be synergistic when FICI 6 0.5 with FICI P 4
being an antagonistic effect while any value falling in between is
an additive effect or no interaction.17 The combination of verapa-
mil with RIF resulted in a synergistic interaction with FICI value
of 0.5.
Verapamil derivatives 7a–e with various substituents on the
tertiary nitrogen demonstrated varying effects on the antimyco-
bacterial activity of RIF (Table 1). In the presence of RIF, unsubsti-
tuted derivative 7a resulted in a two-fold reduction in the MIC99 of
The synthesis of analogues for SAR 2 commenced with the
protection of commercially available 3,4-dimethoxyaniline 9a
(n = 0) and 3,4-dimethoxybenzyl amine 9b (n = 1) with (Boc)2O to
RIF (0.0042 lM) but did not exhibit any synergistic interactions
(FICI 0.56), but ethyl and propyl substituted derivatives 7b and
7c did not show any reduction in the RIF MIC99 and neither did
they exhibit synergistic interactions with RIF as indicated by FICI
values of these combinations (FICI 0.78–1.0). However, there was
SAR 2
a four-fold (0.0021 lM) and two-fold (0.0042 lM) reduction in
the MIC99 of RIF in the presence of benzyl and cyclopropyl substi-
tuted analogues 7d and 7e, respectively. The RIF-7d combination
also exhibited synergistic effect with a FICI value of 0.3.
O
O
CN
*
O
N
O
Relative to verapamil, the derivatives where the aminoethyl
aromatic group of verapamil was replaced with substituted aniline
(12a) and aminomethyl aromatic group (12b) did not exhibit any
effect on the antimycobacterial activity of RIF when evaluated in
combination (Table 1). The derivatives, 14a–h, obtained by replac-
ing the aminoethyl aromatic group of verapamil with various
heterocyclic groups demonstrated varying effect on the
SAR 1
SAR 4
SAR 3
Figure 1. Proposed SAR study of verapamil.