Synthesis and Structure-Activity Relationships of Ring-Opened Bengamide Analogues against Methicillin-Resistant Staphylococcus aureus?

Article abstract of DOI:10.1002/cjoc.202000502

Methicillin-resistant Staphylococcus aureus (MRSA) has become a major threat on public health because of the increase of clinically isolated strains that exhibit resistance to many antibiotics. Therefore, development of new antibiotics for the treatment of MRSA infection is a sustained challenge. We have previously identified a ring-opened bengamide analogue L472-2 that displays moderate activity against the growth of S. aureus. In our previous work, we started from L472-2 and identified a class of analogues containing alkynyl groups which have the potential to activate SaClpP activity but moderate antibacterial activity. Herein, we focused on the antibacterial activity of L472-2, and a novel series of ring-opened bengamide analogues were synthesized and their activities were evaluated against MRSA. By conducting a compact analysis of the structure-activity relationships (SAR) of these analogues, we found that an adamantane ethanol ester bengamide 2j showed excellent antibacterial activity towards six S. aureus strains, including MRSA, while it does not activate ClpP. Therefore, these bengamide analogues represent a new class of candidates that suppress MRSA viability.

Full text of DOI:10.1002/cjoc.202000502

Chinese Journal
of Chemistry
CJC
中 国 化 学 - An International Journal
Accepted Article
Title: Synthesis and Structure-Activity Relationships of Ring-Opened Bengamide
Analogues Against Methicillin-Resistant Staphylococcus aureus (MRSA)
Authors: Chen-Xi Yu, Bing-Yan Wei, Xue-Qing Kong, Cai-Guang Yang*, 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.202000502.
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.202000502.
ISSN 1001-604X • CN 31-1547/O6
mc.manuscriptcentral.com/cjoc
www.cjc.wiley-vch.de

For submission: https://mc.manuscriptcentral.com/cjoc
Report
For published articles: https://onlinelibrary.wiley.com/journal/16147065
CJC
中
国 化 学 - An International Journal
Cite this paper: Chin. J. Chem. 2021, 39, XXX—XXX. DOI: 10.1002/cjoc.202100XXX
Synthesis and Structure-Activity Relationships of Ring-Opened
Bengamide Analogues Against Methicillin-Resistant
Staphylococcus aureus (MRSA)
Chen-Xi Yu†,§,¶, Bing-Yan Wei †,‡,¶, Xue-Qing Kong†, §, Cai-Guang Yang†,‡*, Fa-Jun Nan†,#,*
† State Key Laboratory of Drug Research, the National Center for Drug Screening, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China
§ University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, 100049, China
‡ School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, UCAS,
Hangzhou 310024, China
# Yantai Key Laboratory of Nanomedicine & Advanced Preparations, Yantai Institute of Materia Medica,
Shandong, 264000, China
¶These authors contributed equally to this work
Keywords
Bengamides | MRSA | Antibacterial activity | SAR | Pharmacokinetic
Main observation and conclusion
Methicillin-resistant Staphylococcus aureus (MRSA) has become a major threat on public health
because of the increase of clinically isolated strains that exhibit resistance to many
antibiotics. Therefore, development of new antibiotics for the treatment of MRSA infection is a
sustained challenge. We have previously identified a ring-opened bengamide analogue L472-2 that
displays moderate activity against the growth of S. aureus. In our previous work, we started from L472-
2 and identified a class of analogues containing alkynyl groups have the potential to activate SaClpP
activity but moderate antibacterial activity. Herein, we focused on the antibacterial activity of L472-2,
and a novel series of ring-opened bengamide analogues were synthesized and evaluated their activities
against MRSA. By conducting a compact analysis of the structure-activity relationships (SAR) of these
analogues, we found that an adamantane ethanol ester bengamide 2j showed excellent antibacterial
activity towards six S. aureus strains, including MRSA, while it does not activate ClpP. These
bengamide analogues therefore represent a new class of candidates that suppress MRSA viability.
Comprehensive Graphic Content
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.202000502
This article is protected by copyright. All rights reserved.

Chen-Xi-Yu & Bing-Yan Wei et al.
Report
Background and Originality Content
Staphylococcus aureus (S. aureus) is a type of Gram-positive bacteria that can cause a wide range
of infectious diseases, such as skin and soft tissue infections (SSTIs), bacteremia, lethal pneumonia and
heart valve infections.[1] Following the discovery of penicillin in the 1940s, S. aureus infections seemed
to be well-controlled: however, the misuse and overuse of antibiotics have led to a dramatic worldwide
increase in the proportion of infections caused by methicillin-resistant Staphylococcus aureus (MRSA).[2]
MRSAis resistant to many kinds of antibiotics, and the resistance mechanisms are very complex.[3] When
first-line antibiotics are no longer effective against MRSA, the health care costs are significantly
increased. In the United States, community-associated MRSA imposes an annual commitment of $1.4 to
$13.8 billion on society, which is a higher cost than many other acute infectious diseases.[4]
In 2017, the World Health Organization published a list of global priority pathogens (PPL) of 12
bacteria with critical, high, and medium antibiotic resistance.[5] MRSA was graded and classified as a
high priority. Regrettably, many pharmaceutical industries have withdrawn research and development
(R&D) for new antibiotics since 2004.[6] Given the rigorous situation of MRSA, the search for new
antibiotic molecules (especially for new chemical structures and novel mechanisms of action) is an urgent
need.[7]
Natural product-derived bengamides have been reported to display an extensive spectrum of
biological activities including antitumoral and anthelmintic properties, as well as antitubercular activity
against Mycobacterium tuberculosis.[8] Bengamide A, one of the most thoroughly studied members of
this family, has furnished many novel and promising analogues (Figure 1). As a new effective antitumor
candidate, LAF389 was approved for clinical trials in 2002. Unfortunately, unanticipated cardiotoxicity
and other off-target effects resulted in clinical discontinuation.[9] In our previous work, we utilized a
linear peptidic chain to replace the caprolactam ring to obtain innovative ring-opened bengamide
analogues.[10] These analogues generally maintained potent antiproliferative activity on MDA-MB-435
human breast carcinoma cells and simultaneously improved water solubility. This profound modification
dramatically simplified the routes of synthesis and provided convenience for further structure-activity
www.cjc.wiley-vch.de
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
Chin. J. Chem. 2021, 39, XXX－XXX
This article is protected by copyright. All rights reserved.

Synthesis and Structure-Activity Relationships of Ring-Opened Bengamide Analogues Against
M thi illi i t t St h l (MRSA)
R
Chin J Chem
relationship research. We found that some of these analogues display potential antibacterial activity on
wild-type S. aureus (8325-4), especially for L472-2. In our previous work, we conducted a screening on
our library of ring-opened bengamide 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
displayed reduced antibacterial activity against S. aureus with clpP deletion.[11] Through further research
on L472-2, we acquired a class of ring-opened bengamide analogues containing alkynyl groups have
potential activate SaClpP activity and antibacterial activity. In order to improve the antibacterial activity
of L472-2, a novel series of ring-opened bengamide analogues were synthesized and evaluated their
activities against MRSA, and conducted a compact analysis of the structure-activity relationships (SAR)
of these analogues against MRSA, while the antibacterial activity of these compounds were not directly
related to ClpP.
work:
previous
a)
O
OH O
OH OH O
H
N
R2
OH O
O
O
H
ring
opening
NH
N
n
O
R1
N
R4
5'
H
O
R1
R3
OH OH O
O
Ring-opend bengamides
R2
=
water solubility mostly
1mg/mL
:
=
=
2
-
natural
S
)
A R
R
5'(
CH3
,
H,
R
Bengamide
(CH2)12
1
:
=
=
-
5'(R
)
synthetic
LAF389 R1
CH3
,
,
CH(CH2)5
2
b)
OH O
OH OH O
O
OH O
O
H
N
H
N
O
O
N
H
N
Cl
H
F
OH OH O
Hit
L472-2
MDA-MB-435
compound
F
µ
=
IC50 0.718
M,
µ
=
aureus
MIC 2.2-5
WT
µ
S.
=
=
on
M
SaClpP
EC50 3.13-5.25
g/mL,
(8325-4)
F
aureus
MIC 6.25-12.5mg/mL, WT
S.
(8325-4)
work:
this
c)
OH O
OH OH O
O
OH O
H
O
H
N
N
O
O
O
O
N
N
H
H
OH OH O
Hit
L472-2
compound
µ
=
IC50 0.718
=
MDA-MB-435
µ
M,
=
on
Newman
g/mL
MIC 0.078-0.156
2j
µ
aureus
MIC 2.2-5
WT
S.
g/mL,
(8325-4)
Figure 1. Derivation process of bengamide A to the hit compound L472-2
The polyhydroxy scaffold is the most prominent structural feature of bengamides that makes these
compounds more water-soluble, especially for ring-opened series.[10] In the following structure-activity
relationship study, we modified the alkenyl, alanine residue, and ester group to explore their effects on
anti-MRSA activity. Taking L472-2 as the hit compound, we synthesized a series of ring-opened
Chin. J. Chem. 2021, 39, XXX－XXX
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
www.cjc.wiley-vch.de
This article is protected by copyright. All rights reserved.

Chen-Xi-Yu & Bing-Yan Wei et al.
Report
bengamide analogues and tested these derivatives for their anti-MRSA activity on six different kinds of
MRSA.
Results and Discussion
To assess the contribution of the alanine residue to anti-MRSA activity, bengamide analogues of 1a-
1g were prepared. The synthetic routes for the preparation of 1a-1g are depicted in Scheme 1. (Tert-
butoxycarbonyl)glycine was reacted with cyclohexanol to afford cyclohexyl (tert-
butoxycarbonyl)glycinate, and then the Boc-protecting group was removed to afford 2-(cyclohexyloxy)-
2-oxoethan-1-aminium chloride 5. Coupling reactions between 5 and different kinds of Boc-protected
amino acids 6 proceeded smoothly to give the cyclohexyl ester in good yield. Deprotection of the tert-
butoxycarbonyl gave aminohydrochloride 7a-7g. Amide formation between 7a-7g and the known lactone
8 was accomplished under neutral pH conditions.[12] Subsequent treatment of acetonide-protected amide
with an aqueous HCl (1 M) and THF (1:1) mixture brought about the corresponding bengamide
analogues 1a-1g.
Scheme 1. Synthesis of bengamide analogues 1a-1g
O
BocHN
OH
O
6
R
O
O
ii
i,
H3N
O
Cl
+
HO
N
BocHN
Cl
H3N
H
OH
iii, iv
O
R
O
vi
3
4
5
7a-7g
O
O
O
v,
O
O
OH
O
O
8
H
N
O
N
H
R
OH OH
O
O
1a-1g
:
:
:
:
=
=
=
=
=
7a, 1a
7b, 1b
7c, 1c
7d, 1d
7e, 1e
R
R
R
R
R
H
R
(
)-CH3
S
OH
(
)-CH2
S
CCH
)-CH2
(
:
S
(
)-CH(CH3)2
C6H5
)-CH2
:
=
7f, 1f
R
:
S
(
=
1g
R
H5
S
7g,
(
)-C6
Reagents and conditions: (i) EDCI, DMAP, DCM, rt, 12 h; (ii) 2N HCl in EtOAc, 4 h; (iii) EDCI, HOBt, DIPEA, DCM,
-20℃ to rt,12 h; (iv) 2M HCl in EtOAc, 4 h; (v) Sodium 2-ethylhexanoate, THF, 24 h; (vi) 1M HCl (aq)/THF = 1/1, 4
h.
After substitute alanine with a series of amino acids, we further substituted cyclohexanol with
various alcohols and prepared bengamide analogs 2a-2m (Scheme 2). The coupling reaction of (tert-
butoxycarbonyl)-L-alanine 9 with 2-methoxy-2-oxoethan-1-aminium chloride 10 acquired Boc-
protected methyl L-alanylglycinate, followed by ester hydrolysis in the presence of lithium hydroxide,
www.cjc.wiley-vch.de
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
Chin. J. Chem. 2021, 39, XXX－XXX
This article is protected by copyright. All rights reserved.

Synthesis and Structure-Activity Relationships of Ring-Opened Bengamide Analogues Against
M thi illi i t t St h l (MRSA)
R
Chin J Chem
to afford carboxylic acid 11. Subsequently, we used various alcohols or amine to couple with compound
11 and then deprotected the tert-butoxycarbonyl to give aminohydrochloride 13a-13n. The synthetic
routes of bengamide analogues 2a-2n from 13a-13n refers to the synthesis of 1a-1g in Scheme 1.
Scheme 2. Synthesis of bengamide analogues 2a-2n
12
O
O
O
R
X
O
ii
i,
BocHN
OH
H3N
X
Cl
+
BocHN
Cl
H3N
N
N
R
OH
O
iii, iv
H
H
O
O
9
10
11
13a-13n
O
O
O
vi
v,
O
O
8
OH
O
O
H
N
X
N
R
H
OH OH
O
O
2a-2n
:
:
:
:
:
R
:
:
=
CH3
=
=
13a, 2a
13b, 2b
13c, 2c
13d, 2d
13e, 2e
R
X
X
X
X
X
X
X
X
X
X
X
X
X
X
O
O
=
=
=
=
R
R
R
R
CH2CH2CH3
=
=
=
=
=
=
=
=
=
=
=
=
CH
CH
2C(CH3)3
O
O
O
O
O
O
O
O
O
O
O
2CH(CH3)2
CH2C6H5
:
=
13f, 2f
CH(CH2)3
=
CH(CH2)4
R
R
13g, 2g
13h, 2h
=
2-adamantane
:
=
=
13i, 2i
R
R
:
CH
2(3-
CH2CH
adamantane
)
:
adamantane)
13j, 2j
2(3-
=
13k, 2k
R
CH2CH2CH
adamantane)
2(3-
e)
2(3,5-dimethyladamantan
:
=
13l, 2l
R
CH
:
R
=
CH
13m, 2m
R
2(3,5,7-trimethyladamantane)
2(3-
:
=
NH
13n, 2n
CH2CH
adamantane)
Reagents and conditions: (i) EDCI, HOBt, DIPEA, DCM, -20℃ to rt,12 h; (ii) LiOH·H2O, 10 min, MeOH/H2O = 1/1,
then 1N HCl (aq); (iii) EDCI, DMAP, DCM, rt, 12 h; (iv) 2N HCl in EtOAc, 4 h; (v) Sodium 2-ethylhexanoate, THF,
24 h; (vi) 1N HCl (aq)/THF = 1/1, 4 h.
We also evaluate the function of tert-butyl and the double bond of the bengamide analogues on anti-
MRSA activity. We thus synthesized 3a and 3b (Scheme 3). The following synthetic strategy to afford
16 was promoted by the work of Alam and Dhimane on the use of the Eastwood olefination.[13] We
initiated our synthesis from the known vicinal diol 14,[12] which was converted to the corresponding 2-
methoxy-1,3-dioxolane 15; then, decomposition of cyclic orthoformate of 15 in refluxing acetic
anhydride led to the desired olefin 16. The subsequent reaction between olefin 16 and
aminohydrochloride 13j to afford bengamide analogue 3a was performed using the same protocol as
described in Scheme 1. To evaluate the importance of the double bond to anti-MRSA activity, we
hydrogenated 2j by hydrogen in the presence of palladium, which gave 3b in good yield. We also
prepared the carboxylic acid form of the bengamide analogue by hydrolyzed 2j to afford 2o.
Scheme 3. Synthesis of bengamide analogues 3a, 3b and 2o
Chin. J. Chem. 2021, 39, XXX－XXX
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
www.cjc.wiley-vch.de
This article is protected by copyright. All rights reserved.

Chen-Xi-Yu & Bing-Yan Wei et al.
Report
O
O
O
O
O
OH
O
O
O
O
i
ii
HO
O
O
O
O
O
O
O
O
O
14
15
16
O
H3N
O
Cl
iii, iv
N
H
O
OH
O
O
H
N
13j
O
N
H
OH OH
O
O
3a
OH
O
O
OH
O
O
H
N
H
v
N
O
OH
N
N
H
H
O
OH OH
O
O
OH OH O
2j
2o
OH
O
O
H
N
O
O
vi
N
H
OH OH
O
3b
Reagents and conditions: (i) Trimethyl orthoformate, PPTS, dry THF, rt, 3 h; (ii) Ac2O, reflux, 3 h; (iii) Sodium 2-
ethylhexanoate, THF, 24 h; (iv) 1N HCl (aq)/THF = 1/1, 4 h; (v) LiOH·H2O, MeOH/H2O = 1/1, 30 min, then 1N HCl
(aq); (vi) Pd/C, H2, MeOH, 12 h.
To probe the role of L-alanine on the ring-opened bengamides system, we initially assessed
compounds 1a-1g for their anti-MRSA activity on six different species of MRSA (USA300, NRS-1,
NRS-70, NRS-271, NRS-108 and NRS-100, Newman is methicillin-sensitive Staphylococcus aureus) in
vitro with vancomycin and tetracycline as reference drugs (Table 1). As observed with the D-alanine
derivative 1b, introduction of D-configuration amino acids led to a complete loss of anti-MRSA activity
relative to L472-2. The introduction of polar heteroatoms on the R group decreased the anti-MRSA
activity of 1c. Compounds substituted with aliphatic groups (L472-2, 1a, 1d, 1e) performed as well as
vancomycin, while those with phenyl or benzyl groups (1f, 1g) both had decreased potency. Next, we
kept the L-alanine residue unchanged and studied the effect of the ester group on the antibacterial activity.
Table 1. Anti-MRSA activity of ring-opened bengamide analogues 1a-1g
www.cjc.wiley-vch.de
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
Chin. J. Chem. 2021, 39, XXX－XXX
This article is protected by copyright. All rights reserved.

Synthesis and Structure-Activity Relationships of Ring-Opened Bengamide Analogues Against
M thi illi i t t St h l (MRSA)
R
Chin J Chem
OH
O
O
H
N
O
N
H
R
OH OH
O
O
1a-1g
MIC (μg/mL)
NRS-70
1.00
compound
R
Newman
4.00
USA300
2.00
NRS-1
4.00
NRS-271
4.00
NRS-108
8.00
NRS-100
2.00
(S)-CH3
H
L472-2
1a
12.5-25.0
>50.0
12.5-25.0 6.25-12.5 0.78-1.56 25.0-50.0
6.25-12.5 6.25-12.5
(R)-CH3
(S)-CH2OH
(S)-CH2CCH
(S)-CH(CH3)2
(S)-CH2C6H5
(S)-C6H5
/
>50.0
50.0
>50.0
50.0
>50.0
50.0
>50.0
50.0
>50.0
50.0
>50.0
50.0
1b
25.0-50.0
6.25-12.5
3.13-6.25
12.5-25.0
25.0-50.0
0.78-1.56
0.10-0.20
1c
12.5-25.0 6.25-12.5 6.25-12.5
12.5-25
6.25-12.5 6.25-12.5
0.78-1.56 0.78-1.56
25.0-50.0 12.5-25.0
1d
1.56-3.13 3.13-6.25 1.56-3.13 6.25-12.5
1e
25.0-50.0 25.0-50.0
25.0 25.0-50.0
50.0
12.5-25.0
25.0-50.0
1f
>50.0
50.0
12.5-25.0
1g
Van
Tetra
1.56-3.13 0.78-1.56 0.78-1.56 0.78-1.56
12.5-25.0 25.0 0.39-0.78 0.2-0.39
0.78-1.56 0.78-1.56
0.10-0.20 0.10-0.20
/
Van: Vancomycin; Tetra: Tetracycline
To entirely comprehend the role played by the ester group in the anti-MRSA activity of L472-2 and
to acquire enhanced activity, we designed various analogues 2a-2o. First, we hydrolyzed the ester group
to obtain the acid form of compound 2o. As seen from the data presented in Table 2, acid 2o showed less
activity against all seven bacteria, illustrating that an ester group was essential for the anti-MRSAactivity.
Iso-butyl ester 2c and tert-butyl ester 2d displayed excellent anti-MRSAactivities, as well as benzyl ester
2e, being much more potent than propyl ester 2b, which demonstrated that bulky aliphatic groups were
beneficial for activity. Given this regular pattern, we thought that the bulkier alcohol adamantanol might
be a superior choice. Thus, adamantyl ester 2h was synthesized. Interestingly, compound 2h was indeed
more active than 2c and 2d on both six MRSA. This result proved our hypothesis. Next, we continued to
assess the effect of the carbon chain on anti-MRSA activity. Adamantane methanol ester 2i extended a
carbon atom compared to 2h and showed more potency than 2h. Thus, we synthesized two homologues
of compound 2h (2j and 2k), and at the same time, we prepared two more sterically hindered compounds
2m and 2l. We found that although prolonging the length of the carbon chain improved the anti-MRSA
activity (compared with 2h, 2i and 2j),continuing to extend the carbon chain led to decreased anti-
MRSAactivity (compared with 2j and 2k). Similarly, we continued to increase the steric hindrance based
on compound 2i. Whether two methyl groups (2l) or three methyl groups (2m) were added on adamantyl,
the anti-MRSA activity of 2l and 2m were not improved. We also synthesized an amide analogue 2n
which showed decreased activity, and thus demonstrated that the ester group was crucial for the anti-
Chin. J. Chem. 2021, 39, XXX－XXX
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
www.cjc.wiley-vch.de
This article is protected by copyright. All rights reserved.

Chen-Xi-Yu & Bing-Yan Wei et al.
Report
MRSA activity. Finally, we found a very appropriate bengamide analogue 2j in the aspect of chain length
and steric hindrance, which showed the most potency against MRSA in this series.
Table 2. Anti-MRSA activity of ring-opened bengamide analogues 2a-2o
OH
O
O
H
N
X
N
R
H
OH OH
O
O
2a-2o
MIC（μg/mL）
compound
R
X
Newman
4.00
USA300
2.00
NRS-1
4.00
NRS-70
1.00
NRS-271
4.00
NRS-108
8.00
NRS-100
2.00
CH(CH2)5
CH3
O
O
O
O
O
O
O
O
O
O
L472-2
2a
16.0
32.0
32.0
8.0
32.0
32.0
16.0
CH2CH2CH3
CH2CH(CH3)2
CH2C(CH3)3
CH2C6H5
50.0
50.0
50.0
50
50.0
50.0
50.0
2b
6.25-12.5
6.25-12.5
1.56-3.13
6.35-12.5
12.5-25
1.56-3.13
0.39-0.78
0.078-
3.13-6.25
6.25-12.5
1.56-3.13
12.5-25
12.5-25
0.78-1.56
0.78-1.56
0.156-
3.13-6.25
6.25-12.5
1.56-3.13
12.5-25
6.25-12.5
1.56-3.13
0.39-0.78
0.039-
0.78-1.56
12.5-25
1.56-3.13
12.5-25
0.78-1.56
1.56-3.13
0.78-1.56
0.156-
6.25-12.5
12.5-25
6.25-12.5
12.5-25
25-50
1.56-3.13
12.5-25
1.56-3.13
12.5-25
6.25-12.5
3.13-6.25
0.78-1.56
0.078-
1.56-3.13
6.25-12.5
0.78-1.56
6.25-12.5
6.25-12.5
0.78-1.56
<0.39
2c
2d
2e
CH(CH2)3
2f
CH(CH2)4
2g
2-adamantane
CH2(3-adamantane)
CH2CH2(3-
adamantane)
CH2CH2CH2(3-
adamantane)
CH2(3,5-
3.13-6.25
0.78-1.56
0.156-
2h
2i
0.039-
O
O
2j
2k
2l
0.156
0.313
0.078
0.313
0.313
0.156
0.078
0.78-1.56
0.78-1.56
0.78-1.56
4.00
0.78-1.56
0.78-1.56
0.78-1.56
4.00
0.78-1.56
0.78-1.56
0.78-1.56
4.00
0.78-1.56
1.56-3.13
0.78-1.56
2.00
0.78-1.56
1.56-3.13
1.56-3.13
4.00
0.78-1.56
0.78-1.56
0.78-1.56
4.00
0.39-0.78
0.39-0.78
0.39-0.78
4.00
O
dimethyladamantane)
CH2(3,5,7-
trimethyladamantane)
CH2CH2(3-
adamantane)
H
O
2m
2n
NH
O
/
>50.0
>50.0
>50.0
0.78-1.56
25.0
>50.0
>50.0
>50.0
>50.0
2o
Van
Tetra
/
0.78-1.56
0.10-0.20
1.56-3.13
12.5-25.0
0.78-1.56
0.39-0.78
0.78-1.56
0.20-0.39
0.78-1.56
0.10-0.20
0.78-1.56
0.10-0.20
/
/
Van: Vancomycin; Tetra: Tetracycline
www.cjc.wiley-vch.de
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
Chin. J. Chem. 2021, 39, XXX－XXX
This article is protected by copyright. All rights reserved.

Synthesis and Structure-Activity Relationships of Ring-Opened Bengamide Analogues Against
M thi illi i t t St h l (MRSA)
R
Chin J Chem
Alkenyl fragment is one of the most typical features of bengamides. To test whether the explored
structural modifications of the alkenyl scaffold could also provide novel systemically active compounds,
we prepared 3a and 3b for further anti-MRSA studies (Table 3). As shown in Table 3, reduction of the
double bond in 2j led to the anti-MRSA activity decreases significantly (3b). This result showed that the
double bond plays an irreplaceable role in the anti-MRSA activity of ring-opened bengamide analogues.
We also attempted to remove the tert-butyl in 2j, which gave the compound 3a. Terminal ene 3a was
dramatically less potent than 2j against MRSA. Through a simple SAR study, we recognized that there
is little room for the alkenyl group to change. We next used compound 2j as a lead compound for further
research.
Table 3. Anti-MRSA activity of ring-opened bengamide analogues 3a and 3b
OH
O
O
OH
O
O
H
H
N
N
O
O
N
N
H
H
OH OH
O
O
OH OH
O
O
3b
3a
MIC (μg/mL)
NRS-70
0.156-0.313 0.039-0.078 0.156-0.313 0.156-0.313 0.078-0.156 0.039-0.078
compound
Newman
0.078-0.156
>50.0
USA300
NRS-1
NRS-271
NRS-108
NRS-100
2j
3a
>50.0
8.00
>50.0
4.00
>50.0
1.00
>50.0
4.00
>50.0
8.00
>50.0
2.00
4.00
3b
Van
Tetra
0.78-1.56
0.10-0.20
1.56-3.13
12.5-25.0
0.78-1.56
25.0
0.78-1.56
0.39-0.78
0.78-1.56
0.2-0.39
0.78-1.56
0.10-0.20
0.78-1.56
0.10-0.20
Van: Vancomycin; Tetra: Tetracycline
The active compounds mentioned above contain an ester group, which is considered unstable and
easily hydrolyzed by carboxylesterases (CEs) in vivo. Our subsequent work evaluated the
pharmacokinetic (PK) parameters of compound 2j in the mouse after oral administration (p.o.) (100
mg/kg) and intraperitoneal administration (i.p.) (100 mg/kg) (Figure 2A). We detected the content of 2j
in the peripheral blood under different time gradients. According to the data, compound 2j was
undetectable and hydrolyzed to the acid 2o from the very beginning. As reported in the literature, the
mouse has high levels of CEs, which caused 2j in the plasma to be hydrolyzed to the corresponding acid
immediately: however, CE expression in the blood varies notably depending upon the species. For
instance, humans are virtually vacant of this activity, whereas small rodents have very high levels of the
circulating enzyme.[14] Given the above results, we used three kinds of plasma (mouse, rabbit and human)
to test the stability of compound 2j (Figure 2B). Compound 2j was extremely unstable in mouse plasma
and was undetectable, while in rabbit plasma and human plasma 2j exhibited high stability. This result
provides the basis for future animal experiments. We next assessed the anti-MRSA activity of 2j on
USA300 in the presence of human plasma (Table 4). Consistent with the results of the stability
Chin. J. Chem. 2021, 39, XXX－XXX
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
www.cjc.wiley-vch.de
This article is protected by copyright. All rights reserved.

Chen-Xi-Yu & Bing-Yan Wei et al.
Report
experiment, the anti-MRSA activity of 2j in the presence of human plasma was well maintained (MIC =
0.5 μg/mL on USA300). While in mouse plasma, 2j completely lost potency (MIC > 256 μg/mL on
USA300). These results show that 2j is stable in human plasma and maintains anti-MRSA activity. We
also conducted a preliminary safety evaluation of compound 2j in vitro. It shows that the compound 2j
has no inhibitory activity on hERG with IC50 > 40μM.
Figure 2. Stability assays of compound 2j. (A) The content of compound 2o in mouse peripheral blood after po
(100 mg/kg) and ip dosing of compound 2j (100 mg/kg). (B) The stability of compound 2j in mouse plasma
(MP), rabbit plasma (RP) and human plasma (HP).
Table 4. Anti-MRSA activity of 2j on USA300 in the presence of human plasma (HP) and mouse
plasma (MP)
addictive
+
-
-
+
-
-
-
+
+
-
+
-
2j
MP
-
+
+
HP
MIC (μg/mL)
on USA300
0.156-0.313
/
/
>256
0.5
Conclusions
A class of novel ring-opened bengamide analogues were designed and synthesized. The
structure−activity relationships were summarized in Figure 3. Most of these analogues exhibited
promising anti-MRSA activity, especially analogue 2j. Furthermore, we assessed the stability and anti-
MRSA activity of 2j in the presence of human plasma. We found that 2j could maintain sufficient anti-
MRSA activity in human plasma, even if 2j was an ester. Further studies to identify the antibacterial
mechanism of action and cytotoxicity are now underway in our lab. We look forward to the development
of a new class of antibiotics through the study of this class of compounds.
www.cjc.wiley-vch.de
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
Chin. J. Chem. 2021, 39, XXX－XXX
This article is protected by copyright. All rights reserved.

Synthesis and Structure-Activity Relationships of Ring-Opened Bengamide Analogues Against
M thi illi i t t St h l (MRSA)
R
Chin J Chem
Figure 3. Structure−activity relationships for ring-opened bengamide analogues
Experimental
Experimental procedures are available in the Supporting information.
Supporting Information
The supporting information for this article is available on the WWW under https://
Acknowledgement
This work was supported by the Science and Technology Commission of Shanghai Municipality
(16ZR1407000), and the National Natural Science Foundation of China (81861138046 and 21725801).
References
[1]
Lowy., F. D. Staphylococcus aureus infections. N. Engl. J. Med. 1998, 339, 520-532.
[2]
(a) Sengupta, S., Chattopadhyay, M. K., Grossart, H. P. The multifaceted roles of antibiotics and antibiotic resistance
in nature. Front. Microbiol. 2013, 10, 47-47; (b) Mediavilla, J. R., Chen, L., Mathema, B., Kreiswirth, B. N. Global
epidemiology of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA). Curr. Opin.
Microbiol. 2012, 15, 588-595; (c) Lakhundi, S., Zhang, K. Methicillin-Resistant Staphylococcus aureus: Molecular
Characterization, Evolution, and Epidemiology. Clin. Microbiol. Rev. 2018, 31, e00020-00018; (d) Tiemersma, E.
W., Bronzwaer, S. L., Lyytikainen, O., Degener, J. E., Schrijnemakers, P., Bruinsma, N., Monen, J., Witte, W.,
Grundman, H. Methicillin-resistant Staphylococcus aureus in Europe, 1999-2002. Emerg. Infect. Dis. 2004, 10,
1627-1634.
[3]
(a) Lee, A. S., de Lencastre, H., Garau, J., Kluytmans, J., Malhotra-Kumar, S., Peschel, A., Harbarth, S. Methicillin-
resistant Staphylococcus aureus. Nat. Rev. Dis. Primers. 2018, 4, 1-23; (b) Guo, Y., Song, G., Sun, M., Wang, J.,
Chin. J. Chem. 2021, 39, XXX－XXX
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
www.cjc.wiley-vch.de
This article is protected by copyright. All rights reserved.

Chen-Xi-Yu & Bing-Yan Wei et al.
Report
Wang, Y. Prevalence and Therapies of Antibiotic-Resistance in Staphylococcus aureus. Front. Cell Infect. Microbiol.
2020, 10, 1-11.
[4]
[5]
[6]
Lee, B. Y., Singh, A., David, M. Z., Bartsch, S. M., Slayton, R. B., Huang, S. S., Zimmer, S. M., Potter, M. A., Macal,
C. M., Lauderdale, D. S., Miller, L. G., Daum, R. S. The economic burden of community-associated methicillin-
resistant Staphylococcus aureus (CA-MRSA). Clin. Microbiol. Infect. 2013, 19, 528-536.
Asokan, G. V., Ramadhan, T., Ahmed, E., Sanad, H. WHO Global Priority Pathogens List: A Bibliometric Analysis
of Medline-PubMed for Knowledge Mobilization to Infection Prevention and Control Practices in Bahrain. Oman
Med. J. 2019, 34, 184-193.
(a) Payne, D. J., Gwynn, M. N., Holmes, D. J., Pompliano, D. L. Drugs for bad bugs: confronting the challenges of
antibacterial discovery. Nat. Rev. Drug Discov. 2007, 6, 29-40; (b) Boucher, H. W., Talbot, G. H., Bradley, J. S.,
Edwards, J. E., Gilbert, D., Rice, L. B., Scheld, M., Spellberg, B., Bartlett, J. Bad bugs, no drugs: no ESKAPE! An
update from the Infectious Diseases Society of America. Clin. Infect. Dis. 2009, 48, 1-12.
[7]
[8]
Zhou, L. L., Yang, C. G. Chemical Intervention on Staphylococcus aureus Virulence. Chin. J. Chem. 2019, 37, 183-
193.
(a) Quinoa, E., Adamczeski, M., Crews, P., Bakus, G. J. Bengamides, heterocyclic anthelmintics from a Jaspidae
marine sponge. J. Org. Chem. 1986, 51, 4494-4497; (b) Thale, Z., Kinder, F. R., Bair, K. W., Bontempo, J., Czuchta,
A. M., Versace, R. W., Phillips, P. E., Sanders, M. L., Wattanasin, S., Crews, P. Bengamides Revisited:ꢀ New
Structures and Antitumor Studies. J. Org. Chem. 2001, 66, 1733-1741; (c) Kinder, F. R., Versace, R. W., Bair, K. W.,
Bontempo, J. M., Cesarz, D., Chen, S., Crews, P., Czuchta, A. M., Jagoe, C. T., Mou, Y., Nemzek, R., Phillips, P. E.,
Tran, L. D., Wang, R., Weltchek, S., Zabludoff, S. Synthesis and Antitumor Activity of Ester-Modified Analogues of
Bengamide B. J. Med. Chem. 2001, 44, 3692-3699; (d) Lu, J. P., Yuan, X. H., Ye, Q. Z. Structural analysis of
inhibition of Mycobacterium tuberculosis methionine aminopeptidase by bengamide derivatives. Eur. J. Med. Chem.
2012, 47, 479-484; (e) Quan, D. H., Nagalingam, G., Luck, I., Proschogo, N., Pillalamarri, V., Addlagatta, A.,
Martinez, E., Sintchenko, V., Rutledge, P. J., Triccas, J. A. Bengamides display potent activity against drug-resistant
Mycobacterium tuberculosis. Sci. Rep. 2019, 9, 1-10.
[9]
Dumez, H., Gall, H., Capdeville, R., Dutreix, C., van Oosterom, A. T., Giaccone, G. A phase I and pharmacokinetic
study of LAF389 administered to patients with advanced cancer. Anti-cancer drugs. 2007, 18, 219-225.
Tai, W. Y., Zhang, R. T., Ma, Y. M., Gu, M., Liu, G., Li, J., Nan, F. J. Design, Synthesis, and Biological Evaluation
[10]
of Ring‐Opened Bengamide Analogues. ChemMedChem. 2011, 6, 1555-1558.
[11]
[12]
Kong, X.-Q., Wei, B.-Y., Yu, C.-X., Guan, X.-N., Ma, W.-P., Liu, G., Yang, C.-G., Nan, F.-J. Design, Synthesis and
Biological Evaluation of Bengamide Analogues as ClpP Activators. Chin. J. Chem. 2020, 38, 1111-1115.
Xu, D. D., Waykole, L., Calienni, J. V., Ciszewski, L., Lee, G. T., Liu, W., Szewczyk, J., Vargas, K., Prasad, K.,
Repič, O., Blacklock, T. J. An Expedient Synthesis of LAF389, a Bengamide B Analogue. Org. Process. Res. Dev.
2003, 7, 856-865.
[13]
[14]
Alam, S., Dhimane, H. A Concise Synthesis of Bengamide E and Analogues via E-Selective Cross-Metathesis
Olefination. Synlett. 2010, 2010, 2923-2927.
(a) Hatfield, M. J., Umans, R. A., Hyatt, J. L., Edwards, C. C., Wierdl, M., Tsurkan, L., Taylor, M. R., Potter, P. M.
www.cjc.wiley-vch.de
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
Chin. J. Chem. 2021, 39, XXX－XXX
This article is protected by copyright. All rights reserved.

Carboxylesterases: General detoxifying enzymes. Chem. Biol. Interact. 2016, 259, 327-331; (b) Li, B., Sedlacek, M.,
Manoharan, I., Boopathy, R., Duysen, E. G., Masson, P., Lockridge, O. Butyrylcholinesterase, paraoxonase, and
albumin esterase, but not carboxylesterase, are present in human plasma. Biochem. Pharmacol. 2005, 70, 1673-1684;
(c) Morton, C. L., Iacono, L., Hyatt, J. L., Taylor, K. R., Cheshire, P. J., Houghton, P. J., Danks, M. K., Stewart, C.
F., Potter, P. M. Activation and antitumor activity of CPT-11 in plasma esterase-deficient mice. Cancer Chemother.
Pharmacol. 2005, 56, 629-636; (d) Morton, C. L., Wierdl, M., Oliver, L., Ma, M. K., Danks, M. K., Stewart, C. F.,
Eiseman, J. L., Potter, P. M. Activation of CPT-11 in mice: identification and analysis of a highly effective plasma
esterase. Cancer Res. 2000, 60, 4206-4210; (e) Guemei, A. A., Cottrell, J., Band, R., Hehman, H., Prudhomme, M.,
Pavlov, M. V., Grem, J. L., Ismail, A. S., Bowen, D., Taylor, R. E., Takimoto, C. H. Human plasma carboxylesterase
and butyrylcholinesterase enzyme activity: correlations with SN-38 pharmacokinetics during a prolonged infusion
of irinotecan. Cancer Chemother. Pharmacol. 2001, 47, 283-290.
Chin. J. Chem. 2021, 39, XXX－XXX
© 2021 SIOC, CAS, Shanghai, & WILEY-VCH GmbH
www.cjc.wiley-vch.de
This article is protected by copyright. All rights reserved.