2
D. R. Banerjee et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
loops, namely loop-I and loop-II, which play crucial roles for the
activity. The structure of holo-FabG4 bound to hexanoyl-CoA
The synthesis of 3-O-propargyl tetrabenzyl epicatechin (16) or cat-
1
1
echin (17) was described previously. The azide components were
synthesized by following the procedure as depicted in Scheme 1A.
Synthesis of 1-(azidomethyl)-4-nitrobenzene (9) was done via the
(
HXC) (PDB ID: 3V1U) shows that the active site of FabG4 could
be accessed from two different sides—namely a major and a minor
portal. The co-factor NADH binds at the relatively wide major por-
tal, while CoA/ACP bound fatty acyl substrate accesses the active
attack of NaN
To obtain 1-(azidomethyl)-3-nitrobenzene (12), 3-nitrobenzalde-
hyde was reduced by NaBH to get the corresponding alcohol. Suc-
displacements led to the azide 12. The synthesis of the
3
on commercially available 4-nitrobenzyl bromide.
1
0
site via minor portal. The NADH binding location comprise three
subsites, namely the nicotinamide binding subsite (N-subsite), the
adenosine binding subsite (A-subsite) and the pyrophosphate
binding subsite (P-subsite). The basic structural features and func-
tion of FabG4 have been represented in Figure 1.
4
2
cessive S
N
remaining azide component, 1-(azidomethyl)-3,5-dinitrobenzene
(15), was carried out starting from 3,5-dinitrobenzoic acid via
reducing the acid group by borane dimethyl sulfide complex in
THF. The resulting benzyl alcohol 13 was converted to the azide
15 in a similar fashion.
We have recently reported a novel class of triazole linked poly-
phenol–gallol hybrids (1–2) as potent inhibitors of Mt-FabG4 at
1
1
low micromolar concentrations for the first time (Fig. 2).
A
The click reaction was performed in presence of Cu(I), made
detailed computational, spectroscopic and thermodynamic analy-
sis depicts that these inhibitors compete with co-factor NADH at
major portal. Docking studies of these compounds with FabG4 sug-
gest that they compete for all three subsites of NADH binding loca-
tion. These findings prompted us to further explore the role of the
gallol subunit by replacing with other electron rich aromatic rings,
namely the aminobenzenes. Since an amino group is capable of
forming hydrogen bonds both as a donor and an acceptor, it would
be worth to replace the hydroxyl group(s) in the gallol moiety by
one or more amino group(s) in an attempt to increase the potency
4
in situ by reduction of CuSO with sodium ascorbate (Scheme 1B).
Debenzylation and nitro group reduction were carried out in a
single step using Pearlman catalyst in presence of high hydrogen
pressure to furnish the final compounds (Scheme 1C). All final com-
pounds were purified by repeated precipitation from methanol—
1
13
ether and characterized by H, C NMR and mass spectra. Purity
of these compounds was checked through reverse-phase analytical
HPLC (traces are included in SI).
FabG4 inhibition assay: Screening of compound 3–8 was carried
out to evaluate their FabG4 inhibition potencies. All six compounds
inhibited FabG4 enzyme at micromolar concentrations (Table 1,
dose–response plots are included in SI). Compounds 3, 5 and 7 of
epicatechin series have shown similarity in their inhibitory poten-
1
2,13
14
of compounds.
This isosteric replacement (bioisosterism)
may also eliminate pharmacokinetic and toxicological limitations
normally associated with phenols as presence of a large number
of phenolic hydroxyl group causes instability due to oxidation by
CYP 450 or O-methylation.15 Based on the above rationale, we
designed and subsequently synthesized a series of triazole linked
polyphenol–amino benzene hybrids (3–8) and screened them for
inhibition against Mt-FabG4. Molecular docking studies of these
new hybrids have given an insight about the binding information
which is different than previously reported compounds 1–2. The
results which are given here, provided a structure–activity relation
cies (IC50 ꢀ70
l
M). Compounds belonging to catechin series (4, 6, 8)
also have similar IC50 values (ꢀ60
lM). Compounds of catechin ser-
ies have shown better inhibition of FabG4.
Structure–activity relations (SAR) study: We have previously
reported that hybrid with catechin unit (2) has been better inhib-
1
1
itor than the hybrid with epicatechin unit (1). This previous find-
ing is also supported by the present study, as the catechin linked
hybrids (4, 6, 8) have shown better inhibitory activities than the
respective epicatechin linked analogues (3, 5, 7). This finding indi-
cates that the stereochemistry in the polyphenolic part of the scaf-
fold plays an important role in case of inhibitory capabilities. On
the other hand, replacement of galloyl fragment by aminobenzyl
unit has resulted in 1.5 to 2-fold decrease in inhibition potencies.
Thus, the aminobenzyl units (in 3–8) are comparatively inferior
bioisostere than galloyl unit (as in 1–2). The decrease in inhibition
by the aminobenzene hybrids may be due to their poor binding as
supported by docking studies. Interestingly, the binding is not
affected by the number or positions of the amino groups as
(
SAR) for substituents linked to N-1 of triazole moiety.
Synthesis: The methodology to obtain compounds 3–8 is similar
to the one described in our previous work.11 In brief, click reaction
was performed between 3-O-propargyl tetrabenzyl epicatechin
(16) or catechin (17) with respective azide counterparts (9, 12,
1
5). The benzyl ether was chosen as the protecting group of poly-
phenol because of its easy deprotection under neutral condition to
provide the final compounds.
The key components for the click reaction, namely alkyne and
azide counterparts, were synthesized prior to click procedure.
Figure 1. (A) Major portal of FabG4 with bound NAD. Catalytic tetrad and loops are shown. (B) Basic function of FabG4.