L. Gavara et al.
Bioorganic Chemistry 113 (2021) 105024
the corresponding carboxylic acid. Then, the alkylamines R2-NH2, either
commercially available or prepared as described in Supporting Infor-
mation, were treated with DPT (dipyridylthionocarbonate) to yield the
intermediate isothiocyanates, which were directly reacted with hydra-
zides to form the thiosemicarbazide derivatives. Their basic treatment
yielded the expected 1,2,4-triazole-3-thione compounds. Specific syn-
thetic steps carried out for the preparation of several compounds are
presented in Scheme 2. The hydroxamate 4 was prepared by coupling 3
to O-trityl-hydroxylamine, followed by trityl removal in acidic condi-
tions. Compound 8 was obtained from the Boc-protected precursor and
was reacted with: (i) benzoyl chloride and p-tosyl-chloride to yield
compounds 9 and 12, respectively; (ii) DPT as described in Scheme 1,
followed by condensation with benzhydrazide and hot basic treatment
to give the di-triazole-thione compound 15; (iii) phthalic anhydride in
refluxing pyridine to yield the phthalimide 10, which was opened in
basic conditions to afford 11. Compound 38 was obtained from 39
through Boc removal (the same was performed for 36 from 37) and was
condensed to phenylisocyanate to yield the urea 40.
Overall, five compounds displayed Ki values in the micromolar to
submicromolar range against both VIM-2 and VIM-4. Compounds 1 and 2
had a non-functionalized linear butyl and hexyl chain, respectively, while
compounds 3, 5 and 16 contained a butanoic, a butanol or a pyrrolidinone
moiety, respectively. Few other compounds showed more modest activ-
ities (Ki values in the 10–40 μM range) against VIM-2 and/or VIM-4 (i.e. 4,
6, 10, 11, 13, 14, 15, 17–19) with often a preference for VIM-4. Finally, in
this first series, four compounds, 3, 5, 6 and 19, were tested against VIM-1
andshowedmodesttogoodinhibitorypotencies(Ki’s, 39.8± 6.7μM, 1.69
± 0.12 μM, 6.15 ± 0.33 μM, 4.25 ± 0.32 μM, respectively).
According to these first results and as the presence of a carboxylic
group on the substituent at position 4 has already been highlighted in
previous series [38,43], we kept it in a second group of 20 compounds
bearing various alkanoic moieties and a phenyl ring at the 5-position
(Table 2, compound 3 was added for comparison). We varied the
length of the alkanoic chain (20, 22, 23, 26, 27), introduced an amide
bond (21), heteroatoms (O for 24, S for 25), aromatic rings (28–31),
cyclohexyl moieties (32–34), or an amine group, protected (36, 38, 39)
or not (35, 37). As in the case of the first series, all compounds were
inactive or only moderately active against NDM-1 and IMP-1: the best
compounds against NDM-1 displayed longer alkanoic chains (i.e. hex-
2.2. Evaluation of inhibitory potency toward purified MBLs
Compounds were tested against five representative MBLs, the sub-
class B1 enzymes VIM-2, VIM-4, NDM-1 and IMP-1, and the subclass B3
L1. Few compounds were also tested against VIM-1. Initially, testing was
anoic for 26 andoctanoic for 27) and showed Ki values in the 15–20 μM
range. In this series, VIM-2 and VIM-4 inhibition could be retained or
slightly improved. For these enzymes, the alkyl length was important as
compounds with an alkanoic chain shorter than butanoic (acetic 20,
propionic 22) were poorly or not active, whereas those with longer ones
(pentanoic 23, hexanoic 26 and octanoic 27) showed similar or higher
(i.e. submicromolar range) inhibitory potencies compared to compound
performed at one concentration (100 or 200 μM) and Ki values were
measured for compounds showing significant inhibition (typically >
75%). As all compounds evaluated against L1 were no or low inhibitors
(<50% inhibition at 100 μM) of this enzyme, we chose to not include
these results in the Tables.
3. Compound 27 displayed the best Ki values (0.33 and 0.49 μM,
The results obtained for a first series of 19 compounds are presented
in Table 1. In this series, all compounds possessed a phenyl ring at po-
sition 5 and differed by the alkyl substituent at position 4, itself possibly
carrying additional substituents. Two compounds (1 and 2) contained a
non-functionalized saturated alkyl chain, while others displayed diverse
functional groups such as carboxyl (3), hydroxamate (4), alcohol (5, 6),
sulfonic (7), amine (8, 14), amide (9–11, 16), sulphonamide (12), urea
(13), azide (17), sulfonyl (18) and unsaturated alkyl chain (19). In
particular, compounds 9–14 were derivatives of the amine 8. Finally,
compound 15 can be considered as a dimeric 5-phenyl-1,2,4-triazole-3-
thione where the two monomers were linked by an ethylene group at
their 4-position. Overall, most compounds showed poor or no inhibition
towards any MBL tested. In particular, NDM-1 and IMP-1 were not
significantly inhibited by any compound with the exception of com-
respectively) against VIM-2 and VIM-4 in this series. In addition,
although compound 23 was moderately active against VIM-1 as com-
pound 3, compounds 26 and 27 with a longer alkanoic chain were
micromolar inhibitors of this enzyme. Introducing a sulfur but not an
oxygen in the alkyl chain of compound 23 was favourable for both VIM-
type enzymes. Indeed, whereas the ether analogue 24 was moderately or
not active against VIM-2 and VIM-4, respectively, the thioether 25 dis-
played micromolar to submicromolar Ki values against the same en-
zymes. In addition, this compound was also a micromolar inhibitor of
VIM-1 and moderately inhibited NDM-1, indicating that the sulfur
atom significantly improved MBL inhibition.
Branching a phenyl ring at the β position of the butanoic chain of 3
yielded compounds 28 and 29, which were slightly better inhibitors of
VIM-1, VIM-2 and VIM-4 compared to 3. Constraining the alkanoic
chain with a cyclohexyl moiety as in 32–34 was well tolerated for VIM-2
and VIM-4 inhibition. However, compared to the closest analogues 3
and 26, no improvement was observed, with the exception of 34 which
was a better VIM-1 inhibitor.
pound 1, which showed a Ki of around 30 μM against IMP-1. To note, a
close analogue of the NDM-1-inactive compound 19 differing by the
presence of a 2-hydroxyphenyl ring at position 5 has been previously
reported in the literature as a potent NDM-1 inhibitor [41]. In our hands,
however, this compound was also found inactive against NDM-1 (<20%
As a carboxylate group could be unfavourable for external mem-
brane penetration, the adjunction of a protonated or protonatable ni-
trogen atom in a molecule is a frequent mean to counteract the effect of a
negative charge, leading to a zwitterionic form expected to more
favourably penetrate through porins [45]. Unfortunately, replacing the
phenyl ring of compound 28 by a pyridine (i.e. 30 and 31) was detri-
inhibition at 100 μM), supporting the fact that the allyl group was not
more favourable to NDM-1 inhibition than other substituents placed at
position 4 in this series.
mental. Also, introducing an amine group at the
α position of the
butanoic chain (i.e. 35) abolished VIM-2 inhibition, and the addition of a
Boc group was not favourable either (i.e. 36). The same was true for the
α-amino analogue of compound 23 (i.e. 37). However, in contrast, its
Boc-protected derivative 38 showed similar activities to 23 against VIM-
2 and VIM-4 with Ki values in the low micromolar range, suggesting that
the presence of a hydrophobic and bulky group was suitable at this
position. This was confirmed when attaching a phenyl ring to the amine
via a urea link. Although less potent, the corresponding compound 39
significantly inhibited VIM-type enzymes.
Scheme 1. Synthesis of 4-alkyl-1,2,4-triazole-3-thione derivatives. Reagents
and conditions: (a) EtOH, H2SO4, reflux, 5 h; (b) EtOH, hydrazine hydrate, 100 ◦C,
sealed tube; (c) DPT, DMF, sealed tube, 55 ◦C, 3 h; (d) R1-CONHNH2, DMF, 55 ◦C,
3 h; (e) aqueous KOH or NaHCO3, 100 ◦C, 3 h.
Overall, the presence of a hydrophobic linear or constrained alkyl
chain at position 4 was favourable to inhibition of VIM-type enzymes as
compounds 3, 23, 25, 26, 27 and 32–34 were potent inhibitors of these
3