ˆ ´
P.-A. Paquet-Cote et al.
Bioorganic & Medicinal Chemistry Letters 35 (2021) 127781
and unnatural amino acids, and screened them as potential β-lactamase
inhibitors.16 These previous works demonstrated that sulfahydantoins
bearing a benzyl substituent at position C4 and N5 showed weak inhi-
bition of TEM-1, a well-known β-lactamase. Therefore, we focused the
present investigation on sulfahydantoin compounds starting from un-
natural substituted L-phenylalanines. Herein, we report on the synthesis
of six novel chiral sulfahydantoins (Fig. 1) and our investigation of their
potential as inhibitors of the prominent TEM-1 and TEM-15 Class A
serine β-lactamases.
peroxymonosulfate (Oxone). These two steps yielded the final crude
compounds 1–6, which were purified by High-Performance Liquid
Chromatography (HPLC) to obtain purities ≥ 95%. The isolated yields
for the last two steps and the purification were from 8 to 51%. The low
yields were mainly caused by losses during the HPLC purification. The 6
novel chiral sulfahydantoins were fully characterized. The sulfahy-
dantoin ring was readily confirmed by the 1H and 13C NMR spectra, and
the exact mass was determined by high resolution mass spectrometry
(HRMS). Full spectroscopic data are reported in the SM. The overall
yields vary from 3% to 11%.
Compounds 1–6, shown in Fig. 1, were prepared starting from 4-
fluoro-L-phenylalanine 7 and 4-bromo-L-phenylalanine 8. The synthesis
is summarized in Scheme 1 and details are available in the Supple-
mentary material (SM). The starting amino acid (7 or 8) was protected
by the formation of a methyl ester using thionyl chloride in methanol to
obtain compounds 9 and 10, respectively, with yields > 95%. The key
sulfamide group was introduced using N-Boc-protected chlor-
osulfonamide prepared in situ from chlorosulfonyl isocyanate (CSI) and
t-butyl alcohol and then mixed with 9 or 10 in basic conditions to
cleanly obtain 11 and 12 with isolated yield of 92% and 96%, respec-
tively. An allyl group was added with a Mitsunobu reaction using allyl
alcohol and diisopropyl azodicarboxylate (DIAD). The reaction selec-
tively added the allyl group onto the N-Boc nitrogen, as needed, to
obtain 13 and 14 with yields of 40% and 60%, not optimized.
The inhibition activity of sulfahydantoins 1–6 was investigated using
two clinically relevant β-lactamases. The first was TEM-1, one of the
most widespread β-lactamases.18 The second was TEM-15, the Glu104-
Lys/Gly238Ser double mutant of TEM-1. Those mutations, individually
and combined, have emerged following the clinical application of
cephalosporins and are among the most common substitutions in TEM-
1.19 TEM-15 was thus selected as a representative, prevalent extended-
spectrum β-lactamase that inactivates third generation cephalospo-
rins.20,21 These two enzymes were obtained by overexpression in
E. coli22 followed by a subsequent purification.23 The purity of the en-
zymes used in the following tests was > 83%, as determined by SDS-
PAGE resolution.
The six novel sulfahydantoins were initially tested as inhibitors of
To allow the upcoming cyclization, the Boc moiety was removed
using trifluoroacetic acid giving N-allyl compounds 15 and 16 with 82%
and 72% yields, respectively. The key cyclization step to obtain 17 or 18
was performed with sodium methoxide in anhydrous methanol at reflux
for only 1 h to minimize the reopening of the heterocycle. This key step
gives 17 in 76% yield and 18 in 88% yield. Indeed, longer reaction times
led to the nucleophilic opening of the sulfahydantoin cycle and lower
yields. Noteworthy of mention, the cyclization conditions used do not
lead to epimerization as demonstrated previously.17 Indeed, the syn-
thesis of sulfahydantoin constrained L-Phe-D-Ala dipeptide using the
same conditions was shown to proceed without epimerization at the C4
chiral center. With these key intermediates in hand, three derivatives
each of 17 and 18 were prepared using a standard SN2 reaction with the
N5 as the nucleophile and K2CO3 as the base. Compounds 19, 20, and 21
were obtained by mixing 17 with 4-nitrobenzyl bromide, 4-methoxy-
benzyl chloride, and 4-bromobenzyl bromide, respectively. Com-
pounds 22, 23, and 24 were obtained by mixing 18 with the same
alkylating agents, with yields ranging from 60 to 93%.
TEM-1 using a previously described protocol.24 The assay was per-
formed by measuring the decrease in the hydrolysis of CENTA (300 μM),
a chromogenic substrate of β-lactamases. This concentration is ~ 8 × the
Michaelis-Menten constant (Km) of TEM-1 for CENTA (36 μM) and has
been determined to provide sufficient turnover while allowing for clear
observation of inhibition.24 Sulfahydantoins 1–6 were used at 1 mM for
screening against 116 and 56 nM of TEM-1 and TEM-15 respectively.
Additional details can be found in the SM.
The activity of TEM-1 is mainly unchanged or even slightly
augmented in the presence of compounds 1, 2, 4 or 5 at a concentration
of 1 mM as compared to the control. Activity ranged from 92 to 124%
(Table 1). However, compounds 3 and 6 show an inhibition of TEM-1
with a percentage of activity dropping to 34 ± 7% and 10 ± 8%
respectively at 1 mM concentration. Further tests with compounds 3 and
6, the most potent inhibitors of TEM-1, were performed to assess their
inhibition potential for TEM-15. Compound 3 shows less inhibition of
TEM-15 than TEM-1, with a percentage of activity of 53 ± 7% (Table 1).
On the other hand, in the presence of compound 6 at 1 mM, no signal
was detectable, demonstrating that the activity of TEM-15 is completely
inhibited by sulfahydantoin 6.
To obtain the final desired compounds (1–6), an ozonolysis was
performed followed directly by an oxidation using potassium
Fig. 1. Generic structure of the sulfahydantoin heterocycle and structures of sulfahydantoin derivatives investigated in the present study as potential β-lacta-
mase inhibitors.
2