Design, Synthesis and Biological Evaluation of Bengamide Analogues as ClpP Activators

Article abstract of DOI:10.1002/cjoc.202000133

To combat multidrug-resistant Gram-positive bacteria, new antimicrobials particularly those with novel mechanism of action are badly needed. Different with conventional antibiotics which are typical inhibitors, small-molecule activators of bacterial ClpP represent a new class of antibiotics. No ClpP activator has been developed for clinical trial. Herein, we conducted a screening on our library of bengamide-like ring-opened analogues and found that L472-2 possesses a low minimum inhibitory concentration (MIC) against S.aureus and shows no activity for ClpP activation in vitro, but it displayed reduced antibacterial activity against S. aureus with clpP deletion. In order to obtain bengamide analogues that activate ClpP in vitro as well as possess antibacterial activity, we perform further structural modifications starting from L472-2. Compound 37 remains the antimicrobial activity and activation of ClpP protein in vitro, which could be viewed as a new chemical scaffold for ClpP activators and worthy of further investigation.

Full text of DOI:10.1002/cjoc.202000133

Chinese Journal
of Chemistry
CJC 中 国 化 学 - An International Journal
Accepted Article
Title: Design, Synthesis and Biological Evaluation of Bengamide Analogues as ClpP
activators
Authors: Xue-Qing Kong, Bing-Yan Wei, Chen-Xi Yu, Xiang-Na Guan, Wei-Ping Ma,
Gang Liu, Cai-Guang Yang,* and Fa-Jun Nan*
This manuscript has been accepted and appears as an Accepted Article online.
This work may now be cited as: Chin. J. Chem. 2020, 38, 10.1002/cjoc.202000133.
The final Version of Record (VoR) of it with formal page numbers will soon be
published online in Early View: http://dx.doi.org/10.1002/cjoc.202000133.
ISSN 1001-604X • CN 31-1547/O6
mc.manuscriptcentral.com/cjoc
www.cjc.wiley-vch.de

Design, Synthesis and Biological Evaluation of Bengamide Analogues
as ClpP activators
¶
,a,b
¶, a,b
,a
a,b
a,b
Xue-Qing Kong, Bing-Yan Wei,
Chen-Xi Yu, Xiang-Na Guan, Wei-Ping
a
a
,a,c
Ma, Gang Liu, Cai-Guang Yang, * and Fa-Jun Nan*
a
Chinese National Center for Drug Screening, CAS Key laboratory of Receptor Research, State Key
Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
5
55 Zuchongzhi Road, Shanghai, 201203, China
bUniversity of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, P. R. China
c
Yantai Key Laboratory of Nanomedicine & Advanced Preparations, Yantai Institute of Materia
Medica, No. 39, Science and Technology Avenue, High-tech Industrial Development Zone, Yantai
City, Shandong, 264000, China
Summary of main observation and conclusion To combat multidrug-resistant Gram-
positive bacteria, new antimicrobials particularly those with novel mechanism of action
are badly needed. Different with conventional antibiotics those are typical inhibitors,
small-molecule activators of bacterial ClpP represent a new class of antibiotics. No ClpP
activators have been developed for clinical trial. Herein, we conducted a screening on
our library of bengamide-like ring-opened analogues and found that L472-2 possesses
a low minimum Inhibitory Concentration (MIC) against S.aureus and shows no activity
for ClpP activation in the in vitro study but displayed reduced antibacterial activity
against S. aureus with clpP deletion. In order to obtain bengamide analogues that
activate ClpP in vitro as well as possess antibacterial activity, we perform further
structural modifications starting from L472-2. Compound 37 remains the antimicrobial
activity and activation of ClpP protein in vitro, which could be viewed as a new chemical
scaffold for ClpP activators and worthy of further investigation.
Background and Originality Content The large-scale use of antibiotics
and even abuse in clinical and non-clinical settings have led to the rapid emergence of
[
1]
antibiotic resistance . Multidrug-resistant strains of Gram-positive bacteria,
particularly methicillin-resistant Staphylococcus aureus (MRSA), remain a threat to
[2]
public health in many parts of the world . In addition, many determinant factors have
[
3]
contributed to the decline of new antibiotics . Thus, we are facing severe antimicrobial
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process
which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1002/cjoc.202000133
This article is protected by copyright. All rights reserved.

resistance and approaching the post-antibiotic era. Therefore, the research of new
antimicrobial agents, particularly those with new scaffolds and novel mechanisms is
urgent to tackle the antibiotic crisis.
Bacterial Clp protease is an energy-dependent serine protease which plays a
[
4]
pivotal role in the bacterial pathogenesis . The ClpP protease itself is able to degrade
short peptides, while it requires the assistance of the chaperone proteins such as ClpX
for the proteolysis of polypeptides and proteins. The S. aureus ClpP (SaClpP) has been
established to regulate bacterial virulence and pathogenesis, which represents a novel
[
5]
target for antibacterial drug discovery .
We are interested in the structural and mechanistic studies on SaClpP , as well as
[
6]
[
7]
chemical intervention on SaClpP activity . Both inhibitors and activators for ClpP
[
8]
were developed in the past decade , and particularly activators were extensively
studies. The first activator was ADEPs (Figure 1A), which contended with the ClpX
ATPase in complex with ClpP, thus bypassing ClpX’s regulation. ACPs displayed a
similar mechanism of action to ADEPs, which were identified in high-throughput
screening and named after activators of self-compartmentalizing proteases (Figure 1B).
In a fluorescence-based assay, a non-peptide natural product called sclerotiamide could
activate E. coli ClpP, which possesses a distinct three-dimensional bicyclo-[2.2.2]-
diazoctane motif (Figure 1C).
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H
H
F
F
O
O
A
N
N
O
O
O
O
O
O
O
O
O
NH
N
O
NH
N
O
O
H
H
HN
N
N
HN
N
N
H
H
O
O
ADEP1
ADEP4
O
N
H
CF3
N
B
O
C
HO
H
O
O
O
H
H
N
N
OH
S
O
S
O
N
N
H
O
O
H
N
O
O
O
O
H
Sclerotiamide
ACP1
ACP2
Figure 1. Chemical structures of ClpP activators.
The natural products bengamides and their derivatives exhibited remarkable
[
9]
biological properties, including antitumor, anthelmintic and antibacterial activities .
Previously, we carried out systematic structural optimization and simplification of
bengamides with regards to the antitumor activities[10], thus establishing a bengamide-
like ring-opened focused library. In this study, we have screened our library and
performed structural optimization with the aim to obtain antibacterial bengamide-like
ring-opened analogues as new activators for SaClpP.
Results and Discussion
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Bengamide skeleton Alanine Tail with linker can be modified
OH
O
O
H
N
O
N
H
OH OH
O
O
Linker
L472-2
OH
O
O
OH
O
O
H
H
N
O
O
N
O
N
N
R1
H
H
OH OH
O
OH OH
O
O
library 1
-8
L472-2
4
OH
O
O
H
N
OH
O
O
H
N
N
H
Cl
F
N
OH OH
O
F
H
OH OH
O
3
7
11
F
F
OH
O
O
OH
O
O
H
H
N
N
N
H
N
H
OH OH
O
R2
OH OH
O
R3
library 2
3-19
library 3
2-35
F
1
2
Figure 2. Design of bengamide derivatives as ClpP activators.
In the initial screening of our bengamide-like derivatives, L472-2 displayed
antibacterial activity towards S. aureus (Figure 2). To evaluate whether the antibacterial
activity of L472-2 is associated with SaClpP activation, we determined the MIC values
of L472-2 against different strains, including the wild type S. aureus (8325-4), the S.
aureus clpP mutant strain (ΔclpP), the clpP complementary strain (ΔclpP::clpP), and
[
11]
the 8325-4 strain overexpressing clpP (OEclpP) . As shown in Table 1, compound
L472-2 displayed a lower MIC of 0.78-1.56 μg/ml and 1.56-3.13 μg/ml for against
8
325-4 and ΔclpP::clpP respectively. Interestingly, this compound showed weak
activity against the strain ΔclpP with a MIC of 25.0-50.0 μg/ml, while it still showed
potent activity against OEclpP strain with a MIC of 0.78-1.56 μg/ml. These results
indicate that L472-2 is active against the growth of S. aureus, which is related to ClpP.
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To confirm whether L472-2 directly activates ClpP, we performed a α-casein
degradation assay. L472-2 failed to activate ClpP in α-casein degradation assay in vitro.
L472-2 showed no activity for ClpP activation in the in vitro study but displayed
reduced antimicrobial activity against S. aureus with clpP deletion, just like the positive
control ADEP4. There could be some underlying mechanisms affecting the ClpP
pathway in an indirect manner. The possibility of alternative target and/or off-target
effect needs to be addressed in future study.
Next, we performed further structural modifications beginning with L472-
2
(Figure 2). By remaining the skeleton of bengamide-like ring-opened and modifying
the R , R and R , a series of compounds were designed. The types of rings and the
1
2
3
linker on L472-2 was firstly screened in the library 1. After the introduction of
substituted benzene into L472-2, the compound 8 was acquired. Then the ester group
replaced with alkynyl group for avoiding potential metabolic problems yielded the
compound 11. Replacing the methyl in the compound 11 with different types of
substituents with various amino acids, provided library 2 for further optimization. The
emergence of SaClpP activation in vitro had attracted our great interest among the
compound of library 2. Then, library 3 was gained by systematic structural
modification of the substituted benzene ring. Given the activation of ClpP protein and
the antibacterial activity, the alignment of compound 19 and 30 produced 37 with
further improved activity.
In general, the synthesis of the bengamide skeleton was started from a
commercially available lactone that was converted to the key intermediate 1g (Scheme
1
) [12], which reacted with various amines (3a-3e, 10, 12a-12g, 21a-21n) respectively
by a coupling reaction and then followed by a acid-catalyzed acetonide deprotection[13]
.
Throughout various reaction routes, the reaction of amines with lactone 1g was a
versatile process for the preparation of target compounds.
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O
O
OH
O
O
O
OH
O
O
H
Ref.[12]
HO
OH
m
n
N
R
O
N
O
H
OH OH
OH OH
O
R2
D-glycero-D-gulo-
heptono-1,4-lactone
,
,
,
4
-8, 11 13-19 22-35 37
1g
O
a
O
c
b
d
H N
O
R1 OH
HCl H
N
R
HCl
2
2
1
N
H
R1
O
O
2a-2e
3
a-3e
F
I
F
O
e
f
+
NHBoc
g
h
HCl HN
N
H
NH2 HCl
R2
9
,
10 12a-12g
F
O
R3
R3
HCl H N
2
i
j
N
H
l
k
NHBoc
I
+
NH2 HCl
R3
20a-20n
21a-21n
Scheme 1. Synthesis of bengamide derivatives 4-8, 11, 13-19, 22-35 and 37.
(
a) L-glycine, EDCI, DMAP, DCM; (b) HCl/EA; (c) L-alanine, EDCI, DMAP, DCM;
d) HCl/EA; (e) Pd(PPh Cl , CuI, Et N, rt; (f) HCl/EA; (g) various amino acids, EDCI,
Cl , CuI, Et N, rt; (j) HCl/EA; (k) L-
phenylalanine, EDCI, DMAP, DIPEA, DCM; (l) HCl/EA; (m) 3a-3e, 10, 12a-12g, 21a-
(
3
)
2
2
3
DMAP, DIPEA, DCM; (h) HCl/EA; (i) Pd(PPh
3
)
2
2
3
2
1n, 36, sodium 2-ethylhexanoate, THF, rt; (n) 1 N HCl, THF.
The alcohols were condensed with L-glycine to give esters 2a-2e in high yields,
which was further condensed with L-alanine providing diverse amines (3a-3e).
Intermediate 9 was prepared from p-iodofluorobenzene using copper-palladium
catalyzed Sonogashira coupling[14], thenceforth reacting with varieties of amino acids
commercially available to yield varieties of amines (10, 12a-12g). In order to get
intermediates 20a-20n, the coupling of the derivatives of iodobenzene with N-Boc-
propargylamine was applied, which then reacted with L-phenylalanine to afford a range
of amines (21a-21n). Then the bengamide analogues 4-8, 11, 13-19, 22-35 were
steadily synthesized from 1g and diverse amines (3a-3e, 10, 12a-12g, 21a-21n)
correspondingly. Inspired by earlier screening for R
2
and R , the suitable moieties of
3
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1
9 and 30 were kept to form bengamide derivative 37.
These bengamide derivatives 4-8, 11, 13-19, 22-35 and 37 were evaluated for their
antibacterial activity and activation of ClpP through in vitro assays. The ClpP activator
ADEP4 was assayed as the positive control and vancomycin as the negative control.
Compounds 4-8 remained the antibacterial activity through modification of the
cyclohexane in L472-2 (Table 1). Unfortunately, these derivatives are still unable to
promote SaClpP to degrade α-casein substrate in vitro. These data suggest that R are
1
not crucial for the antibacterial effect. Carney reported that N-
acyldifluorophenylalanine fragments of ADEPs function via the identical mechanism
of action, as evidenced by its requisite and sufficiency for the performance of ClpP
activation and antibacterial activity [15]. Inspired by the benzene ring with halogen
substitutions in N-acyldifluorophenylalanine fragments, we try to further exploration
from compound 8.
Table 1. MICs of library 1 against S. aurues strains
OH
O
O
H
N
O
N
H
R1
OH OH
O
O
MIC(μg/ml)
R
1
ID
8
325-4
0.78-1.56 25.0-50.0
1.56-3.13 >50.0
ΔclpP
ΔclpP::clpP
1.56-3.13
3.13-6.25
0.39-0.78
<0.39
OEclpP
0.78-1.56
L472-2
4
5
6
7
8
1.56-3.13
0.78-1.56
0.39-0.78
<0.39
0.78-1.56 25.0-50.0
0.39-0.78 6.25-12.5
0.39-0.78 12.5-25.0
0.78-1.56 25.0-50.0
3.13-6.25
0.39-0.78
0.39-0.78
0.78-1.56
F
<0.39
ADEP4
Vancomycin
0.39-0.78
>50.0
0.39-0.78
0.78-1.56
0.78-1.56 0.78-1.56
By considering the instability of esters and the sensitivity of the linker in
metabolism, we replaced the flexible ester in compound 8 with linear alkynyl motif,
which gave compound 11 for further evaluation of the biological activity (Figure 3). It
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is good to find that compound 11 largely kept the antibacterial activity. So we kept the
alkyne group and substituted benzene in compound 11 for further improvement.
F
OH
O
O
H
OH
O
O
H
N
N
O
N
N
H
H
OH OH O
OH OH
O
O
1
1
8
F
MIC=0.78-1.56µ
g/ml
MIC=1.56-3.13 g/ml
µ
Figure 3. Modification of the linker based on 8.
Next, we investigated the effect of R substituents on the antibacterial activity
Table 2). The comparison of antibacterial activity between compound 13 and 14
2
(
clearly revealed that the configuration of L-amino acid is necessary for remaining
antibacterial activity. To our delight, compound 13 with moderate antibacterial activity
was able to activate ClpP to degrade α-casein with an EC50 of 50 μM (Table 3).
Compounds 15, 16, 17 displayed comparable antibacterial activities with compound 11
with low MICs and no appearance of SaClpP activation in degrading α-casein substrate
in vitro. Compound 18 with a MIC of 12.5-25.0μg/ml showed very weak activity for
SaClpP activation in vitro consistently. Compound 19 with 3, 5-F substituted benzyl
exhibited comparable antibacterial activity with compound 13. Interestingly, compound
1
9 displayed much stronger activity than 13 for SaClpP activation in vitro (Table 3).
Table 2. MICs of library 2 against S. aureus strains
OH
O
O
H
N
N
H
OH OH
O
R2
F
MIC (μg/ml)
R
2
ID
8325-4
ΔclpP
ΔclpP::clpP
OEclpP
1
1
1
3
1.56-3.13 1.56-3.13
6.25-12.5 25-50
1.56-3.13
0.78-1.56
3.13-6.25
6.25-12.5
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14
15
16
>50
>50
>50
25-50
0.78-1.56 6.25-12.5
3.13-6.25 6.25-12.5
0.78-1.56
3.13-6.25
1.56-3.13
3.13-6.25
1
1
1
7
8
9
0.78-1.56 3.13-6.25
12.5-25.0 25.0-50.0
6.25-12.5 12.5-25.0
0.78-1.56
12.5-25.0
6.25-12.5
0.78-1.56
6.25-12.5
6.25-12.5
NHCbz
NH
F
F
ADEP4
Vancomycin
0.39-0.78
>50.0
0.39-0.78
0.78-1.56
0.39-0.78
0.78-1.56
0.78-1.56 0.78-1.56
Table 3. EC50 values of derivatives activating SaClpP
Compound
EC50 (μM)
ADEP4
L472-2
3.13-6.25
>100
2
2
1
2
3
2
1
3
3
7
3
4
0
9
9
7
>100
50-100
50-100
50-100
25-50
12.5-25
6.25-12.5
3.13-6.25
The derivatives in Table 4 were prepared by changing the para-fluorine group in
compound 13 to varying substituents at different sites on the phenyl ring. Compound
2
8 with the ortho-fluorine substitution and compound 32 bearing ortho-isopropyl
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substitution completely lost the antibacterial activity. The MICs of the compounds (13,
3-27, 29-30) with halogen substitutions at other sites rather than the ortho-position
were moderate. The properties of electrons in R group minimally affected the
antibacterial activity in compounds 31-35. Only those compounds with halogen
substitution display agonistic activity on ClpP enzyme. The halogen substituents in R
2
3
3
had different effects on activation of SaClpP degrading protein substrate. The activity
for SaClpP activation also depends on both the halogen atoms and the substituted
positions. Compound 29 and 30 bearing both fluorine and chlorine groups displayed
moderate activities for SaClpP activation in vitro (Table 3).
Table 4. MICs of library 3 against S. aureus strains
OH
O
O
H
N
N
H
OH OH
O
R3
MIC (μg/ml)
R
3
ID
8
325-4
ΔclpP
ΔclpP::clpP
12.5-25.0
12.5-25.0
6.25-12.5
6.25-12.5
3.13-6.25
6.25-12.5
12.5-25.0
>50.0
OEclpP
6.25-12.5
12.5-25.0
25.0-50.0
6.25-12.5
6.25-12.5
6.25-12.5
12.5-25.0
25.0-50.0
p-F
-
13
22
23
24
25
26
27
28
29
30
31
32
33
34
35
12.5-25.0 25.0-50.0
12.5-25.0 25.0-50.0
12.5-25.0 12.5-25.0
6.25-12.5 12.5-25.0
6.25-12.5 12.5-25.0
6.25-12.5 12.5-25.0
25.0-50.0 25.0-50.0
m-Br
m-Cl
p-Br
p-Cl
m-F
o-F
>50.0
>50.0
m-F, p-F
12.5-25.0 25.0-50.0
6.25-12.5 25.0-50.0
25.0-50.0 25.0-50.0
6.25-12.5
6.25-12.5
12.5-25.0
>50.0
6.25-12.5
6.25-12.5
12.5-25.0
>50.0
m-Cl, p-F
o-OCF
3
o-CH(CH
p-CN
3
)
2
>50.0
>50.0
12.5-25.0 25.0-50.0
25.0-50.0 25.0-50.0
12.5-25.0 25.0-50.0
12.5-25.0
25.0-50.0
12.5-25.0
0.39-0.78
0.78-1.56
12.5-25.0
25.0-50.0
6.25-12.5
0.39-0.78
0.78-1.56
o-CH
m-NO , p-F
ADEP4
Vancomycin
3
2
0.39-0.78
>50.0
0.78-1.56 0.78-1.56
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Finally, compound 37 was prepared on the basis of the balanced enzyme
activation(Table 3) and antibacterial activity(Table 5) as observed in compounds 19
and 30. To our delight, 37 exhibited significantly improved activation on the
degradation of protein substrate by SaClpP with the in vitro EC50 of 3.13-6.25 μM
(Table 3), while this compound is still active against S. aureus in a ClpP-dependent
way (Table 3). Therefore, the bengamide derivatives could function as a new class of
antibiotics by actively targeting the S. aureus ClpP protease.
Table 5. MICs of the compound 37 against S. aureus strains
MIC (μg/ml)
ID
8
325-4
ΔclpP
>50
ΔclpP::clpP
6.25-12.5
0.39-0.78
OEclpP
6.25-12.5
0.39-0.78
0.78-1.56
3
7
6.25-12.5
0.39-0.78
0.78-1.56
ADEP4
>50.0
Vancomycin
0.78-1.56
0.78-1.56
Conclusion
Confronted with antimicrobial resistance, the strategy for the study of new
antibiotics with novel mechanisms of action is imperative. ClpP has drawn our attention
for its superiority as a new target for the development of novel antibiotics. After
screening on the bengamide-like focused compound library, we found bengamide
analogues L472-2 could be viewed as a hit for further optimizations. Three different
sets of new derivatives of L472-2 were synthesized. The introduction of the alkynyl
group into compounds 8 yielded compound 11, which might be much metabolite-
tolerant and remains antibacterial activity. Further optimization of the L-alanine with
L-phenylalanine provided compound 13, leading to the occurrence of SaClpP activation
in vitro. Furthermore, compound 19 is more active than 13 for SaClpP activation. In
view of the activation of ClpP protein and the antibacterial activity of compound 19
and 30, the arrangement of them generated 37 with further improved activity.
Compound 37 possesses activation of SaClpP with EC50 of 3.13-6.25 μM and
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antibacterial activity with a MIC of 6.25-12.5 μg/ml. These data suggest that the
bengamide-like ring-opened compounds, especially 37, is a potent activator of SaClpP.
Collectively, we have identified that unreported bengamide scaffolds as ClpP activators
for further investigation.
Experimental section
Experimental procedures are available in the Supporting Information.
Acknowledgements
We would like to thank Hanne Ingmer for providing the strains of 8325-4, ΔclpP
and ΔclpP::clpP. This work was supported by Science and Technology Commission
Shanghai Municipality (16ZR1407000 and 17XD1404400), and the National Natural
Science Foundation of China (81861138046).
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Entry for the Table of Contents
OH
O
O
Page No.
OH
O
O
H
N
H
N
O
N
H
Design, Synthesis and Biological
Evaluation of Bengamide Analogues as
ClpP activators
N
Cl
F
H
OH OH
O
F
OH OH
O
O
F
L472-2
325-4 MIC(μg/ml):0.78-1.56
ΔclpP MIC(μg/ml):25.0-50.0
ΔclpP::clpP MIC(μg/ml):1.56-3.13
OEclpP MIC(μg/ml):0.78-1.56
in vitro no activation of SaClpP
37
8
325-4 MIC(μg/ml): 6.25-12.5
ΔclpP MIC(μg/ml)>50.00
ΔclpP::clpP MIC(μg/ml): 6.25-12.5
OEclpP MIC(μg/ml): 6.25-12.5
8
Xue-Qing Kong,Bing-Yan Wei, Chen-Xi
Yu, Xiang-Na Guan, Wei-Ping Ma, Gang
Liu, Cai-Guang Yang*and Fa-Jun Nan*
in vitro activation of SaClpP EC50(μM):3.13-6.25
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