2
D. Zeng et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
H
N
aldehyde as the electrophile exhibited even better activity
(IC50 < 0.5
M).13 In recent years, our lab discovered a series of
H
N
O
O
l
H
N
O
O
cyanohydrin derivatives as potent and selective inhibitors of
EV71 3Cpro (e.g., 3, 4).14 More importantly, we obtained the
co-crystal structure of 3/EV71 3Cpro, which revealed the interac-
O
H
N
H
N
O
OH
a
R1
N
H
b
R1
N
H
Boc
O
O
N
H
tions between the cyanohydrin group and 3Cpro
. a-Keto amide, a
O
11
mild electrophilic functional moiety with good druggability, has
been used widely in cysteine and serine protease inhibitors, for
example boceprevir for HCV NS3/4A inhibitor.15 Additionally,
F
F
12a R'=Cbz
13a R'=Cbz
13b R'=Boc
12b R'=Boc
H
N
H
O
N
O
a
-keto amides offer an opportunity to extend interaction with
S10 pocket and to study structure–activity relationship (SAR) of
P10. Herein a series of -keto amides as EV71 3Cpro inhibitors were
reported and SAR was discussed.
Synthesis of
O
O
O
H
N
H
c
d
H
a
N
R
R1
N
H
R1
N
N
H
H
O
O
a
-keto amide from aldehyde via cyanohydrin was
reported.16 However, the toxic potassium cyanide was used in
the reported approach and the overall yield was only as low as
20% due to its lengthy steps. On the basis of previously reported
F
F
9 R'=Cbz R=isopropyl
10 R'=Boc R=cyclopropyl
14a R'=Cbz
14b R'=Boc
methods,17 an improved synthesis of
a-keto amides was accom-
Scheme 2. Synthesis of
(a) (i) TFA, DCM, rt; (ii) Boc-
a
-keto amide inhibitors 9 and 10. Reagents and conditions:
-Phe(4-F)-OH or Cbz- -Phe(4-F)-OH, EDCI, HOBt, TEA,
DCM; 65–74%; (b) NaBH4, MeOH, rt, 85–89%; (c) Dess–Martin periodinane, DCM, rt,
90–92%; (d) (i) RNC, AcOH, DCM, rt; (ii) LiOH, MeOH/H2O, rt; (iii) Dess–Martin
periodinane, DCM, rt; 72–75%.
L
L
plished via Passerini reaction. As illustrated in Scheme 1, aldehyde
5, which was synthesized according to the literature,14 was treated
with isocyanide and acetic acid to give ester 6. Then alcohol 7,
obtained by removal of acetyl group of 6 under basic condition,
was oxidized with Dess–Martin periodinane to give a-keto amide
8. As a result of shorter steps, high conversion ratio and only one
purification process, the overall yield reached as high as 70%.
activities indicated that P1 (S)-d-lactam ring bearing analogs
8f–8j presented approximately 2- to 6-fold better activities than
8a–8e containing (S)-c-lactam ring at P1 position in both the
enzyme and cellular assays. This result was consistent with the
activity comparison between aldehyde 2 and 5. It was clear that
Synthesis of a-keto amides 9 and 10 started from key interme-
diate 11 which was prepared similarly to literature.16 As illustrated
in Scheme 2, removal of the Boc group of 11 with TFA followed by
an amide bond formation using EDCI as coupling reagent resulted
in 12. Alcohol 13, obtained by the reduction of ester 12 with
NaBH4, was finally oxidized to aldehyde 14 with Dess–Martin peri-
replacement of (S)-
improve the potency of inhibitors against EV71 3Cpro. Additionally,
low toxicity (CC50 > 100 M) was observed for all the -keto amide
inhibitors. Hence, the (S)-d-lactam ring as standard P1 residue for
-keto amide inhibitors were investigated in the following studies.
In order to explore the SAR of P10, various
-keto amide inhibi-
tors containing different groups were synthesized and evaluated
(Tables 1 and 2). The results showed that most -keto amides with
short terminal chains (less than 5 carbons) at P10 gave satisfactory
activities, with IC50 values from 1.34 0.33 M to 8.21 1.96
and EC50 values from 1.66 0.45 M to 11.6 3.96 M. -Ketoa-
mides 8p, 8q and 8r with long alkyl chains showed dissatisfactory
activities (IC50 > 20 -keto
M) against EV71 3Cpro. Moreover,
amides with small branched alkyls at P10 showed improved activ-
ities, compared with the corresponding -keto amides with
c-lactam ring by (S)-d-lactam ring could
odinane.
a
-Keto amides 9 and 10 were obtained using the method
l
a
similarly to 8.
The inhibitory activities (IC50) of the
a
-keto amide inhibitors
a
against EV71 3Cpro were studied using a fluorescence resonance
energy transfer (FRET)-based enzyme assay.18 The anti-EV71 activ-
ities of these inhibitors were evaluated by an in vitro cell-based
assay with the EV71 replicon cell system,14a,19 and the results
were expressed as EC50 values for antiviral activity and CC50
values for cytotoxicity.
a
a
l
lM
l
l
a
With aldehyde 2 (IC50 = 3.81 0.19
and 5 (IC50 = 0.54 0.02 M, EC50 = 0.26 0.07
antiviral activities of EV71 3Cpro inhibitors containing P1 modifica-
tions ((S)- -lactam ring vs (S)-d-lactam ring) were compared
(Table 1). Overall, the activities of -keto amide inhibitors were
less potent than that of aldehyde inhibitors, probably due to the
lM, EC50 = 3.07 0.20
lM)
l
l
M) as Ref. 16, the
l
a
c
a
a
straight-chain alkyls. For example, 8f and 8m with isopropyl and
cyclopropyl displayed 2–4 fold better activities than 8l, with IC50
mild electrophilic reactivity of
a
-keto amide. The biological
value of 1.34 0.33
respectively. Similar results could be found in comparison of the
activities of inhibitors 8g (IC50 = 8.21 1.96 M) and 8n
(IC50 = 5.07 0.89
M). However, 8o containing t-Bu at P10, exhib-
ited poor anti-EV71 3Cpro activity (IC50 > 20
M), suggesting that
lM, 3.32 0.43 lM and 6.22 1.07 lM,
l
H
N
O
l
O
NH
n
l
steric effect needed to be considered at P10. Additionally, for inhi-
bitors 8i, 8s and 8t, the presence of phenyl group and substituted
phenyl groups were apparently responsible for the loss of activity
n
O
O
O
H
N
H
N
R
H
a
N
H
N
H
N
H
O
O
O
OAc
(IC50 > 20 l
M). Structurewise, the entrance of S10 pocket in EV71
X
3Cpro was rather narrow,10a which may result in the poor
tolerance of sterically rigid and bulky phenyl groups and t-Bu
group. However, it was interesting to find that 8j and 8u
containing aryl methylene groups displayed satisfactory anti-
X
5
6
H
N
H
N
O
O
n
n
O
O
O
O
H
H
N
N
R
R
c
b
N
H
N
H
N
H
N
H
EV71 3Cpro activities (IC50 = 7.83 1.23
lM and 6.30 1.12 lM,
O
OH
O
O
respectively). The addition of methylene group made the aryl
moieties more flexible, which led to aryl moieties better fit to the
X
X= H, F
n=1, 2
X
8
7
S10 pocket. Moreover, all the
toxic, with CC50 values >100
With the para-fluoro benzyl group instead of benzyl group
at P2, 8v (IC50 = 1.32 0.26 M) showed comparable activity
a-keto amide inhibitors were low
lM.
Scheme 1. Improved synthesis of
tions: (a) RNC, AcOH, DCM, rt; (b) LiOH, MeOH/H2O, rt; (c) Dess–Martin period-
inane, DCM, rt, 70–75% from 5 to 8.
a
-keto amide inhibitors. Reagents and condi-
l