Novel Mercapto Propionamide Derivatives with Potent New Delhi Metallo-β-Lactamase-1 Inhibitory Activity and Low Toxicity

Article abstract of DOI:10.1021/acsinfecdis.8b00366

The emergence and worldwide prevalence of New Delhi metallo-β-lactamase 1 (NDM-1) expressing Gram-negative bacteria with resistance against most β-lactam antibiotics pose a serious threat to human health. However, no NDM-1 inhibitors are clinically approved at present. Herein, based on the lead compound captopril, a series of compounds were designed, synthesized, and evaluated for NDM-1 inhibitory activities. All designed compounds showed single digit micromolar or submicromolar NDM-1 inhibitory activities, which were much more potent than that of captopril. Among them, compounds 14a and 14m exhibited excellent NDM-1 inhibitory activities, with IC₅₀ values of 0.10 and 0.12 μM, respectively. Further studies demonstrated that compound 14m displayed low cytotoxicity, good water solubility, high metabolic stability, and low acute toxicity in mice. Importantly, compound 14m exhibited potent synergistic antimicrobial activities with Meropenem (MEM) for the treatment of clinically isolated NDM-1-expressing strains.

Full text of DOI:10.1021/acsinfecdis.8b00366

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Article
Novel Mercapto Propionamide Derivatives with Potent New Delhi
Metallo-#-Lactamase-1 (NDM-1) Inhibitory Activity and Low Toxicity
Zhi Meng, Mei-Lin Tang, Liting Yu, Yongxi Liang, Jilai Han,
ChenChen Zhang, Fupin Hu, Jian-Ming Yu, and Xun Sun
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00366 • Publication Date (Web): 06 Mar 2019
Downloaded from http://pubs.acs.org on March 7, 2019
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Novel Mercapto Propionamide Derivatives with Potent New Delhi
Metallo-β-Lactamase-1 (NDM-1) Inhibitory Activity and Low
Toxicity
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Zhi Meng,† Mei-Lin Tang,†,# Liting Yu,† Yongxi Liang,† Jilai Han,† ChenChen
†
§,
†
,†,‡
‖
Zhang, Fupin Hu, Jian-Ming Yu and Xun Sun*
†Department of Natural Products Chemistry, School of Pharmacy, Fudan University, 826
Zhangheng Road, Shanghai 201203, China
‡Institute of Chinese Integrative Medicine, Fudan University, 826 Zhangheng Road, Shanghai
201203, China
§Institute of Antibiotics, Huashan Hospital, Fudan University, 12 Wulumuqi Zhong Road,
Shanghai 200040, China
‖Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, 12 Wulumuqi
Zhong Road, Shanghai 200040, China
#State Key Laboratory of Molecular Engineering and Institutes of Biomedical Sciences, Fudan
University, 220 Handan Road, Shanghai 200433, China
*Corresponding author: Xun Sun, E-mail: sunxunf@shmu.edu.cn
The emergence and worldwide prevalence of New Delhi metallo-β-lactamase 1 (NDM-1)
expressing Gram-negative bacteria with resistance against most β-lactam antibiotics pose a serious
threat to human health. However, no NDM-1 inhibitors are clinically approved at present. Herein,
based on the lead compound captopril, a series of compounds were designed, synthesized and
evaluated for NDM-1 inhibitory activities. All designed compounds showed single digit
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micromolar or submicromolar NDM-1 inhibitory activities, which were much more potent than
that of captopril. Among them, compounds 14a and 14m exhibited excellent NDM-1 inhibitory
activities, with IC50 values of 0.10 and 0.12 μM, respectively. Further studies demonstrated that
compound 14m displayed low cytotoxicity, good water solubility, high metabolic stability and low
acute toxicity in mice. Importantly, compound 14m exhibited potent synergistic antimicrobial
activities with meropenem (MEM) for the treatment of clinically isolated NDM-1-expressing
strains.
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Keywords: NDM-1, Gram-negative bacteria, resistance, inhibitors, captopril, inhibitory activities,
meropenem
Antibiotics play a crucial role in the treatment of infectious disease.1 However, the emergence
of antimicrobial resistance (AMR) presents a threat to public health worldwide: even worse, it has
been exacerbated by the abuse of antibiotics.2,3 Carbapenem-resistant Enterobacteriaceae (CRE)
consists of multiple Gram-negative bacteria that exhibit AMR to many antibiotics.4,5 CRE
infections have become a serious threat to human health and have caused the eventual death of
half of the patients who receive CRE-associated bloodstream infections.6,7 A list of AMR “priority
pathogens” recently published by the World Health Organization (WHO) demonstrated that CRE
are among the most critical infection-causing bacteria that urgently necessitate the development of
new antibiotics.8
One of the main resistance mechanisms of CRE is the generation of β-lactamases, which can
be divided into two types on the basis of their catalytic mechanisms.9 One type is the
serine-β-lactamases (SBLs; classes A, C and D); these SBLs hydrolyze β-lactam rings by a serine
nucleophilic mechanism. The other type is the metallo-β-lactamases (MBLs; class B); MBLs
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utilize active-site zinc to coordinate a nucleophilic hydroxide to breakdown the β-lactam ring.10
Combination therapy has been identified as a more economical and effective option compared to
monotherapy, and it has been widely adopted in clinical practice.11,12 Several SBL inhibitors, such
as clavulanic acid, sulbactam, tazobactam and avibactam, have been used in combination with
appropriate β-lactam antibiotics to treat infections caused by SBL-expressing bacteria.13-15 In
contrast, Me1071 (CP-3242) was the only MBL inhibitor in phase I/II clinical trials for the
treatment of bacterial infection. However, no recent development has been reported for this
compound.16
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According to amino acid sequence and metal ion features, MBLs could be further grouped into
three subclasses (i.e., B1, B2 and B3).17 Among the B1 subfamily, Imipenemase-1 (IMP-1),
Verona integron-encoded MBL (VIM) and New Delhi metallo-β-lactamase-1 (NDM-1) are the
most prevalent MBLs observed in the clinic.18 In particular, NDM-1 represents one of the most
troublesome MBLs. NDM-1 can inactivate nearly all β-lactam antibiotics, including carbapenems,
which are known as the “last resort” for antibiotic treatment.19 Additionally, NDM-1 has been
widely expressed by a variety of clinically relevant Gram-negative species and spreads worldwide.
Furthermore, the NDM-1 gene is carried on easily transmissible plasmids that contribute to its
wide spread, and plasmids with the NDM-1 gene are often relevant to other MBL genes.18,20,21 To
overcome the prevalence of AMR caused by NDM-1, a number of inhibitors have been discovered
over the last decade, such as the natural product aspergillomarasmine A (AMA),22 metal chelator
tris-picolylamine (TPA) derivative,3 2,6-dipicolinic acid (DPA),18 cyclic boronate,23 covalent
inhibitor cefaclor,24 triazolylthioacetamide,21 rhodanine derivative ML30225 and L-captopril26
(Figure 1).
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N
N
HO
O
N
O
O
H
N
H
OH
N
N
HO
N
H
O
H2N
HN
HO
HO
H
NH2
O
O
N
B
O
COOH
O
OH
7
N
NH2
HOOC
N
COOH
H
O
8
1 (AMA)
2 (TPA derivative)
3 (DPA)
4 (cyclic boronate)
9
NH2
H
10
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Cl
O
N
H
N
S
NH2
O
H
Cl
H
N
O
S
N
N
HS
N
N
N
S
Cl
COOH
H
O
S
N
O
Cl
N
N
COOH
O
8 (L-captopril)
6 (triazolythioacetamide)
5 (cefaclor)
7 (ML302)
Figure 1. Representative NDM-1 inhibitors
Among the reported NDM-1 inhibitors, sulfur-containing compounds were found to be one of
the most promising categories, exemplified by the anti-hypertension drug captopril.27-29 Both
L-captopril and D-captopril demonstrated only moderate NDM-1 inhibitory activity, with IC50
values of 202.0 μM and 7.9 μM, respectively.30 However, very limited studies designing and
testing captopril analogues as NDM-1 inhibitors were reported in the literature.31-37 Importantly,
L-captopril was successfully cocrystallized with NDM-1 (PDB: 4EXS), which laid the foundation
for the rational design of more potent NDM-1 inhibitors.38 The crystal structure of L-captopril
with NDM-1 reveals that the thiol group could coordinate to zinc ions and that the carboxyl group
could form hydrogen bond with Asn220, suggesting that both thiol and carboxyl groups were
pivotal for NDM-1 inhibitory activities and should be maintained in the new designed analogues.
Additionally, the active site of NDM-1 contains a large hydrophobic domain and no obvious
interactions of the pyrrolidine ring in captopril with NDM-1 were observed, implying that the
pyrrolidine ring may not be essential for NDM-1 inhibitory activities (Figure 2A, Figure 2B).38
This hydrophobic channel of the active domain is a long corridor which might be suitable for
chain-like large substituents. Thus, different bulky amino acids could be introduced to maintain
hydrogen bonds by carboxylic acids with protein residues (such as Asn220, Ile210) and increase
the hydrophobic interactions. Meanwhile, due to the relatively large hydrophobic domain near the
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methyl group (Figure 2B), diversified aromatic rings were simultaneously imported to enhance
the hydrophobic interactions with protein residues in the active site of NDM-1 (Figure 2C).
Taken together, twenty-nine novel NDM-1 inhibitors were designed and synthesized, and their
biological activities were evaluated.
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C
R
H
Design
HS
N
HS
N
R1
Zn
COOH
O
O
COOH
14a-q
Asn220
Figure 2. (A and B) Binding modes of L-captopril in the active site of NDM-1(PDB code: 4EXS).
(C) Design of novel NDM-1 inhibitors 14a-q.
RESULTS AND DISCUSSION
Chemistry. Starting from the commercially available boronic acids and ethyl
2-(bromomethyl)-acrylate, intermediates 10a-i were formed via a Suzuki coupling reaction based
on the reported methods39 prior to hydrolysis with aqueous NaOH to afford the acrylic derivatives
11a-i. Next, compounds 11a-i were reacted with thioacetic acid via Michael addition reaction to
obtain intermediates 12a-i, which were coupled with various methyl or ethyl amino acid esters to
yield their corresponding mercapto propionamide derivatives 13a-q. Finally, these esters 13a-q
were subsequently hydrolyzed to obtain target compounds 14a-q (Scheme 1).
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Scheme 1. Synthesis of compounds 14a-qα
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9
HO
OH
a
c
b
R
e
B
R
R
COOH
11a-11i
COOCH2CH3
10a-10i
10
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9a-9i
O
COOR1
O
COOH
R2
COOH
d
R
S
R
N R2
R
N
H
H
O
S
HS
14a-14q
12a-12i
13a-13q
O
R1 = CH3 or CH2CH3
O
O
COOH
COOH
COOH
O
COOH
N
H
N
H
N
H
N
HS
14m
HS
H
HS
14a
14g
O
COOH
O
O
COOH
O
N
H
S
N
N
H
H
N
HS
14n
HS
HS
14h
NH
COOH
14b
O
COOH
O
O
COOH
N
H
N
H
N
N
HS
HS
H
HS
HS
HN
14c
O
14i
14o
O
COOH
O
COOH
N
H
O
COOH
N
H
N
S
H
HS
HS
14p
O
14j
14d
O
COOH
O
COOH
O
COOH
N
N
H
N
H
H
S
F3C
HS
14q
HS
HS
HS
14e
14k
O
COOH
NH
O
COOH
N
N
H
H
N
H
HS
14f
14l
αReagents and conditions: (a) ethyl 2-(bromomethyl)acrylate, Pd(TFA)2, KOH, H2O, 90 °C, 3 h,
47-72% yields; (b) THF-3 M aqueous NaOH (1:6), 90 °C, 5 h, 95-100% yields; (c) thioacetic acid,
Et3N, CH2Cl2, N2, rt, 30 h, 38-57% yields; (d) amino acid methyl/ethyl ester, EDCI, HOBt,
N-methylmorpholine, CH2Cl2, rt, overnight; (e) THF-3 M aqueous NaOH (1:6), N2, rt, 10 h,
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21-63% yields (two steps).
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NDM-1 Inhibitory Activities. Initially, diastereomers 14a-q were tested against NDM-1 by
the fluorescence-based assay using L-captopril as a positive control.18,26,40 As shown in Table 1,
all compounds showed single digit micromolar or submicromolar activity against NDM-1 and
much more potent activity than L-captopril, indicating that most of the substitution patterns (i.e.,
L-phenylalanine, L-homophenylalanine or L-tryptophan at the R1 position and biphenyl, indole, or
2-benzo[b]thiophene at the R position) were well tolerated. L-Phenylalanine analogue 14a was
19-fold more potent than its corresponding O-benzyl-L-tyrosine analog 14d. In general, all
compounds bearing O-benzyl-L-tyrosine at the R1 position (14d, 14 g and 14o) were shown to
exhibit relatively weak activity in this series. One possible reason for this is that the hydrophobic
pocket at the R1 position was smaller and deeper. When the R1 group was substituted with
tryptophan, all compounds (14c, 14n and 14f) exhibited good NDM-1 inhibitory activity, with
IC50 values of 0.98, 0.22 and 0.45 µM, respectively. If the R group was substituted by a bulkier
group (i.e., phenanthrene or dibenzo[b,d]thiophene), compounds 14i-k demonstrated some degree
of decreased activity. Overall, compounds 14a and 14m exhibited the most potent NDM-1
inhibitory activity in this series, with IC50 values of 0.10 μM and 0.12 μM, respectively.
Table 1. NDM-1 Inhibitory Activities of Target Compounds 14a-q.
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ID
Chemical structure
IC50 (μM)
ID
Chemical structure
IC50 (μM)
O
COOH
208.60 ±
12.01
N
N
HS
H
8
14i
1.78 ± 0.59
HS
COOH
O
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O
COOH
O
COOH
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8
N
N
H
H
14a
14b
14c
0.10 ± 0.05
0.18 ± 0.02
0.98 ± 0.10
14j
14k
14l
2.66 ± 0.79
1.33 ± 0.35
1.30 ± 0.24
S
HS
HS
9
O
COOH
O
COOH
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N
H
N
H
S
HS
HS
O
COOH
O
COOH
N
H
N
H
HS
N
HS
H
HN
O
COOH
O
COOH
N
H
N
H
14d
1.90 ± 0.49
14m
0.12 ± 0.02
HS
N
H
HS
O
O
COOH
O
COOH
N
N
H
H
14e
14f
0.39 ± 0.07
0.22 ± 0.09
14n
14o
0.45 ± 0.06
6.28 ± 0.18
N
HS
HS
NH
O
COOH
O
COOH
N
H
N
H
N
HS
HS
NH
O
O
COOH
O
COOH
N
H
N
H
14g
14h
2.41 ± 0.47
0.55 ± 0.27
14p
14q
0.78 ± 0.13
3.90 ± 0.23
HS
HS
O
O
COOH
O
COOH
S
N
N
H
H
F3C
HS
HS
It was interesting to further separate the diastereomers of these analogues. Compounds 14a,
14b, 14e, 14g, 14j and 14m were chosen to be purified again by preparative TLC to obtain their
counterparts. Their optical rotations were measured by a polarimeter. To determine their absolute
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configurations, a single crystal of compound 14j-2 was cultivated, and X-ray crystallographic
analysis showed that the absolute configuration of 14j-2 was R, S (Figure 3). Meanwhile, the
stereochemistry of compound 14j-1 was identified as S, S. Furthermore, the absolute
configurations of compounds 14m-1 and 14m-2 were confirmed by electrostatic circular
dichroism (ECD) analyses.41 The theoretical calculated ECD curves of (S,S)-14m and (R,S)-14m
were consistent with the experimental ECD curves of 14m-1 and 14m-2, respectively (Figure 4).
Therefore, the stereochemical configurations of 14m-1 and 14m-2 were identified as (S,S)-14m-1
and (R,S)-14m-2, respectively. Finally, by the comparison of specific rotations or experimental
ECD curves of compounds 14a, 14b, 14e and 14g to those of compounds 14j-1, 14j-2, 14m-1 and
14m-2, the absolute configurations of diastereomers were also determined (Table S1 and Figure
S2).
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Figure 3. X-ray crystallographic analysis of 14j-2
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Figure 4. (A) Experimental ECD spectra of 14m-1 and calculated ECD spectra of (S,S)-14m. (B)
Experimental ECD spectra of 14m-2 and calculated ECD spectra of (R,S)-14m.
As shown in Table 2, the two most potent compounds, 14a and 14m, displayed similar
NDM-1 inhibitory activities to their diastereomeric counterparts, (R,S)-14a-2 and (R,S)-14m-2,
respectively, and were more potent than their corresponding (S,S)-14a-1 and (S,S)-14m-1
diastereomer. A similar trend could be found within compounds 14b, 14e, 14g and 14j. Therefore,
diastereomers were chosen for follow-up studies.
Table 2. Comparison of NDM-1 Inhibitory Activities of Compounds 14a, 14b, 14e, 14g, 14j and
14m with Their Corresponding Diastereomers
ID
Chemical structure
IC50 (μM)
ID
Chemical structure
IC50 (μM)
O
COOH
O
COOH
(S)
(S)
N
H
(S)
(S)
N
H
14a-1
0.47 ± 0.12 14g-1
3.11 ± 0.54
HS
HS
O
O
COOH
(S)
O
COOH
(S)
(R)
(R)
N
H
N
H
14a-2
0.16 ± 0.04 14g-2
1.67 ± 0.43
HS
HS
O
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O
COOH
(S)
O
COOH
(S)
N
H
N
H
14a
0.10 ± 0.05
14g
2.41 ± 0.47
HS
HS
8
O
9
10
11
12
13
14
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O
O
O
COOH
(S)
O
O
O
COOH
(S)
(S)
(S)
N
H
N
H
14b-1
14b-2
14b
0.33 ± 0.11 14j-1
0.16 ± 0.07 14j-2
5.78 ± 0.61
2.07 ± 0.19
2.66 ± 0.79
0.48 ± 0.06
0.17 ± 0.05
0.12 ± 0.02
S
S
S
HS
HS
HS
HS
HS
HS
COOH
(S)
COOH
(S)
(R)
(R)
N
H
N
H
COOH
(S)
COOH
(S)
N
H
N
H
0.18 ± 0.02
14j
O
O
O
COOH
(S)
O
COOH
(S)
(S)
N
H
N
H
(S)
14e-1
14e-2
14e
1.31 ± 0.15 14m-1
0. 46 ± 0.09 14m-2
N
H
HS
HS
HS
HS
COOH
(S)
O
COOH
(S)
(R)
(R)
N
H
N
H
N
H
HS
COOH
(S)
O
COOH
N
H
N (S)
H
0.39 ± 0.07
14m
N
H
HS
Cytotoxicity and Water Solubility. Cytotoxicity is an important aspect in defining reliable
inhibitors.27,36 Taking account of the inhibition of NDM-1 and structural diversity, the toxicities of
compounds 14a, 14b, 14h, 14i, 14l, 14m and 14p to HUVEC were measured using the CCK-8
assay.42 As shown in Figure 5, the IC50 values of all tested compounds showed inhibitory activity
above 200 μM, which preliminarily suggested that the toxicities of these compounds to HUVEC
were very weak. To further examine the cytotoxicity of compound 14m, cell viability and the
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release of lactate dehydrogenase (LDH) from HUVEC, L02 and HEK293 cells were measured at
concentrations of 100 and 200 μM, respectively. As shown in Figure 6A, the growth of three
cultured human cell lines was barely influenced by compound 14m at the concentration of 100
μM, and the compound only slightly impacted cell proliferation even at 200 μM. In the LDH
release assay, compound 14m did not increase the amount of LDH release at all but could
decrease the LDH level (Figure 6B). These results implied that the cytotoxicity of compound 14m
was quite low. In addition, the aqueous solubility values of compounds 14a and 14m were
measured by reported methods,42 and the results showed that the water solubility of compound
14m (800.3 μM) was far superior to compound 14a (15.6 μM).
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Figure 5. HUVEC viability when treated by different compounds at the concentration of 200 μM.
Figure 6. Cell viability (A) and LDH release (B) of HUVEC, L02 and HEK293 cells treated by 8
and 14m at concentrations of 100 and 200 μM.
Microdilution Broth Minimum Inhibitory Concentrations (MICs). To determine the
synergistic activities of compound 14m in combination with carbapenem class antibiotics to
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resistant clinical strains expressing blaNDM-1, the microdilution broth MICs of compound 14m and
meropenem (MEM) were measured on NDM-1 positive clinical isolates of K. pneumoniae and E.
coli (Table 3). The fractional inhibitory concentration index (FICI), which evaluated the degree of
interaction between antibiotics and adjuvant, was calculated by adding the following two
fractional values: (MIC compound A in combination/MIC of compound A alone) + (MIC
compound B in combination/MIC of compound B alone), and the synergistic interaction was
determined by FICI ≤0.5.43 The NDM-1-harboring isolates were identified to be resistant to MEM,
with MICs ≥64 μg/mL, which was far larger than the breakpoint value (8 μg/mL) for MEM
against Enterobacteriaceae as defined by the European Committee on Antimicrobial
Susceptibility Testing (EUCAST).44 Inhibitor 14m itself displayed little to no influence on the
growth of the tested K. pneumoniae (MICs >128 μg/mL) and E. coli (MIC >64 μg/mL) isolates.
Except K. pneumoniae-13443, the MIC data of MEM for most of the tested strains were decreased
when compound 14m was used in combination, suggesting that 14m could enhance the
susceptibility of clinically resistant strains expressing blaNDM-1 to MEM. Of particular note, the
MIC values of MEM against NDM-1-producing E. coli and K. pneumoniae-2470 isolate were
reduced from 64 to 8 μg/mL and from >128 to 32 μg/mL, respectively. Meanwhile, corresponding
FICIs were calculated to be <0.375 and <0.5, indicating the potent synergistic interaction between
compound 14m and MEM. Furthermore, the inhibitory concentrations of compound 14m to all
three cultured human cell lines (HUVEC, L02 and HEK293) were higher than 200 μM (76.4
μg/mL). This means that the cell viabilities of these cells were not influenced by compound 14m
at 100 μM (38.2 μg/mL), which was higher than the concentrations (16 μg/mL, 32 μg/mL)
relevant to the MIC assay. Thus, compound 14m exhibited an obvious synergism effect with
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MEM toward some of the clinically isolated NDM-1-producing strains when applied within the
safe concentration range, and the concentrations used in MIC studies were lower than those of
most of the previously reported NDM-1 inhibitors.18,25,31,32
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Table 3. Microdilution Broth MICs (μg/mL) of MEM Tested Alone or in Combination with
Compound 14m Against Clinical K. pneumoniae and E. coli Strains Expressing blaNDM-1
.
MIC a (fold change b)
Isolates
MEM
MEM + 14m
K. pneumoniae-2470
K. pneumoniae-2472
>128
32 (>4)
>128
64 (>2)
K. pneumoniae-13443
>128
64
>128
8 (8)
E. coli
aCompound 14m, in combination with MEM, was tested at 32 and 16 μg/mL for K. pneumoniae
and E. coli, respectively. bThe fold change was defined as the MIC of MEM used alone divided by
the MIC of MEM used in combination with compound 14m.
Metabolic Stability in the Presence of Human Liver Microsomes. The in vitro metabolic
stability of compound 14m was measured using the human liver microsomes (HLM) assay. As
shown in Table 4, compound 14m displayed high metabolic stability with the half-life of 135.5
min, which was far superior to the reference substances. After 60 min, 70.4% of compound 14m
remained in this HLM assay. The intrinsic clearance (CL) of 14m was 9.2 mL/min/kg.
Table 4. In Vitro Metabolic Stability of Compound 14m in the Presence of Human Liver
Microsomes
HLM (final concentration: 0.5 mg protein/mL)
Compound
a
b
T1/2
(min)
CLint(liver)
Remaining
Remainingc
R2
(mL/min/kg) (T = 60 min)
(NCF = 60 min)
14m
0.9346 135.5
13.4
9.2
70.4%
4.4%
88.3%
88.1%
Testosterone 0.9687
93.4
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Diclofenac 0.9796
Propafenone 0.9828
10.5
7.7
118.2
162.1
1.9%
0.6%
91.7%
99.8%
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8
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bHalf-time. bIntrinsic clearance. cNo co-factor.
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In Vivo Acute Toxicity. Acute toxicity of compound 14m in vivo was determined by a
single-dose intravenous administration and intraperitoneal injection in ICR mice. Compound 14m
was injected into mice at doses of 50, 100, 200, 300 and 400 mg/kg, respectively, and survival
data were recorded until two weeks after administration. As shown in Table 5, almost all of the
mice survived in the intraperitoneal injection group, except for one mouse that was injected with
compound 14m at the highest concentration (400 mg/kg). Furthermore, the median lethal dose
(LD50) value of the intravenous injection group was calculated to be 350.1 mg/kg. Thus, these
results suggested that 14m possessed a low acute toxicity and was well tolerated in mice.
Table 5. Acute Toxicity of Compound 14m in Mice
Intraperitoneal injection
Intravenous injection
Dose
No. of
mice
(mg/kg)
No. of dead mice
0
Survival
No. of dead mice
0
Survival
50
8
8
8
8
8
100%
100%
100%
100%
87.5%
100%
100%
100%
87.5%
12.5%
100
200
300
400
0
0
0
1
0
0
1
7
CONCLUSION
In summary, the present study provided the design and synthesis of twenty-nine novel
compounds based on the lead compound captopril. All designed compounds showed single digit
micromolar or submicromolar NDM-1 inhibitory activities, which were much more potent than
that of captopril. Among them, compounds 14a and 14m exhibited excellent NDM-1 inhibitory
activities, with IC50 values of 0.10 and 0.12 μM, respectively. Further studies demonstrated that
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compound 14m displayed low cytotoxicity, good water solubility, high metabolic stability and low
acute toxicity in mice. Importantly, compound 14m exhibited strong synergistic antimicrobial
activities with MEM for the treatment of clinically isolated NDM-1-expressing strains. The SAR
studies of captopril demonstrated that the methyl and proline fragments were not essential and
could be replaced by large hydrophobic substituents that can improve NDM-1 inhibitory activities.
According to the results, compound 14m could represent a promising lead compound for
developing clinically useful NDM-1 inhibitors. Further structural optimization, mechanistic
studies and biological evaluations are in progress.
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EXPERIMENTAL SECTION
Chemistry
Unless otherwise noted, all reagents and solvents were purchased from commercial sources
and used without further purification. Thin-layer chromatography (TLC) analysis was carried out
on GF254 silica gel plates (QindaoHaiyang Chemical, China). Silica gel column chromatography
1
was performed with 60G silica gel (QindaoHaiyang Chemical, China). H NMR and 13C NMR
spectra were recorded on Varian Mercury Plus400 or Bruker AVANCE600 spectrometers, with
TMS as an internal standard and CDCl3, CD3COCD3 or DMSO-d6 as solvents. Chemical shifts
and coupling constants (J) were expressed in parts per million (ppm, δ) and Hz, respectively.
Abbreviations applied for proton coupling patterns are described as follows: singlet (s), doublet
(d), doublet of doublets (dd), triplet (t), quadruplet (q) and multiplet (m). Low-resolution mass
spectra (ESI) were recorded on an Agilent 1100 LC/MSD, and high resolution mass spectra
(HRMS) were collected on an AB Sciex Triple TOF® 5600+. All of the target compounds
exhibited purity greater than 95% by HPLC.
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General procedure for the synthesis of 10a-i exemplified by 10a. To a suspension of ethyl
2-(bromomethyl)acrylate (1.0 mL, 7.25 mmol, 1 eq) in distilled H2O (50 mL), 4-biphenylboronic
acid (1.7 g, 8.70 mmol, 1.2 eq), Pd(TFA)2 (2.4 mg, 7.25 μM, 0.001 eq) and KOH (610.2 mg,
10.88 mmol, 1.5 eq) were added. The reaction mixture was heated to 90 °C for 3 h before being
extracted with CH2Cl2 (3 × 100 mL). The combined organic layer was washed with saturated
NaCl, dried over Na2SO4, and evaporated. The residue was purified by silica gel column
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chromatography (PE:EA = 10:1) to afford compound 10a (1.0 g, 53% yield) as a white solid. H
NMR (400 MHz, CDCl3) δ: 7.58 (dd, J = 8.0, 8.0 Hz, 4H), 7.41 (dd, J = 4.0, 4.0 Hz, 2H), 7.31
(dd, J = 8.0, 8.0 Hz, 3H), 6.31 (s, 1H), 5.51 (s, 1H), 4.20 (q, J = 7.1 Hz, 2H), 3.71 (s, 2H),
1.20-1.33 (m, 3H).
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Ethyl 2-(dibenzo[b,d]thiophen-2-ylmethyl)acrylate (10b). Yield 72%, white solid. H NMR
(400 MHz, CDCl3) δ: 8.03-8.13 (m, 1H), 7.95 (s, 1H), 7.76 (dd, J = 8.0, 8.0 Hz, 2H), 7.22-7.44
(m, 3H), 6.26 (s, 1H), 5.48 (s, 1H), 4.08-4.21 (m, 2H), 3.77 (s, 2H), 1.23 (t, J = 6.6 Hz, 3H).
1
Ethyl 2-(phenanthren-9-ylmethyl)acrylate (10c). Yield 66%, white solid. H NMR (400 MHz,
CDCl3) δ: 8.60 (dd, J = 8.0, 8.0 Hz, 2H), 7.90 (dd, J = 4.0, 4.0 Hz, 1H), 7.73-7.81 (m, 1H),
7.46-7.63 (m, 5H), 6.23 (s, 1H), 5.19 (s, 1H), 4.19-4.32 (m, 2H), 4.06 (s, 2H), 1.21-1.35 (m, 3H).
1
Ethyl 2-(4-(trifluoromethyl)benzyl)acrylate (10d). Yield 47%, colourless oil. H NMR (400
MHz, CDCl3) δ 7.56 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 7.8 Hz, 2H), 6.43 (s, 1H), 5.65 (s, 1H), 3.68
(s, 2H).
1
Ethyl 2-([1,1'-biphenyl]-3-ylmethyl)acrylate (10e). Yield 63%, white solid. H NMR (400
MHz, CDCl3) δ: 7.56 (d, J = 7.6 Hz, 2H), 7.40 (dd, J = 7.0, 7.0 Hz, 4H), 7.28-7.35 (m, 2H), 7.17
(d, J = 7.5 Hz, 1H), 6.25 (s, 1H), 5.48 (s, 1H), 4.14-4.19 (m, 2H), 3.68 (s, 2H), 1.25 (t, J = 6.0,
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3H).
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Ethyl 2-(benzo[b]thiophen-2-ylmethyl)acrylate (10f). Yield 51%, white solid. H NMR (400
MHz, CDCl3) δ: 7.68 (t, J = 7.5 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.17-7.27 (m, 2H), 6.99 (d, J =
7.1 Hz, 1H), 6.27 (d, J = 7.2 Hz, 1H), 5.59 (d, J = 7.0 Hz, 1H), 4.13-4.19 (m, 2H), 3.84 (s, 2H),
1.13-1.26 (m, 3H).
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Ethyl 2-((1H-indol-5-yl)methyl)acrylate (10g). Yield 68%, white solid. H NMR (400 MHz,
CDCl3) δ: 8.26 (s, 1H), 7.43 (s, 1H), 7.13 (d, J = 8.3 Hz, 1H), 6.94-6.99 (m, 2H), 6.40 (s, 1H),
6.20 (s, 1H), 5.42 (s, 1H), 4.11 (q, J = 7.1 Hz, 2H), 3.71 (s, 2H), 1.18(t, J = 6.0 Hz, 3H).
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Ethyl 2-((1-methyl-1H-indol-5-yl)methyl)acrylate (10h). Yield 49%, white solid. H NMR
(400 MHz, CDCl3) δ: 7.42 (s, 1H), 7.18 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 8.0 Hz, 1H), 6.94 (t, J =
4.0 Hz, 1H), 6.38 (s, 1H), 6.19 (s, 1H), 5.40 (s, 1H), 4.12-4.17 (m, 2H), 3.71 (s, 2H), 3.64 (s, 3H),
1.23 (t, J = 6.0 Hz, 3H).
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Ethyl 2-(naphthalen-2-ylmethyl)acrylate (10i). Yield 71%, white solid. H NMR (400 MHz,
CDCl3) δ: 7.78 (dd, J = 10.6, 8.8 Hz, 3H), 7.64 (s, 1H), 7.41-7.45 (m, 2H), 7.32 (d, J = 8.4 Hz,
1H), 6.26 (s, 1H), 5.48 (s, 1H), 4.14-4.20 (m, 2H), 3.78 (s, 2H), 1.23 (t, J = 6.0 Hz, 3H).
General procedure for the synthesis of 11a-i exemplified by 11a. To a solution of 10a (980.0
mg，3.68 mmol) in THF (10 mL), 3 M NaOH (60 mL) was added. The resulting mixture was
stirred for 5 h at 90 °C. Upon completion, the solvent was removed, and the residual aqueous
solution was adjusted to pH 3-4 with 3 N HCl. After extraction with ethyl acetate (3 × 100 mL),
the combined organic layer was washed with brine, dried over Na2SO4, filtered and concentrated.
The obtained residue was purified by silica gel column chromatography (DCM:CH3OH = 50:1) to
1
afford target compound 11a (849.7 mg, 97% yield) as a white solid. H NMR (400 MHz,
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DMSO-d6) δ: 12.57 (s, 1H), 7.62 (dd, J = 8.0, 8.0 Hz, 4H), 7.45 (t, J = 7.4 Hz, 2H), 7.25-7.39 (m,
3H), 6.14 (s, 1H), 5.64 (s, 1H), 3.60 (s, 2H).
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2-(Dibenzo[b,d]thiophen-2-ylmethyl)acrylic acid (11b). Yield 97%, white solid. 1H NMR (400
MHz, DMSO-d6) δ: 12.60 (s, 1H), 7.90-8.42 (m, 4H), 7.35-7.52 (m, 3H), 6.17 (s, 1H), 5.63 (s,
1H), 3.76 (s, 2H).
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2-(Phenanthren-9-ylmethyl)acrylic acid (11c). Yield 100%, white solid. H NMR (400 MHz,
DMSO-d6) δ: 12.68 (s, 1H), 8.84 (dd, J = 8.0, 8.0 Hz, 2H), 7.96 (dd, J = 3.6, 3.6 Hz, 2H),
7.62-7.67 (m, 5H), 6.17 (s, 1H), 5.32 (s, 1H), 4.07 (s, 2H).
2-(4-(Trifluoromethyl)benzyl)acrylic acid (11d). Yield 95%, white solid. 1H NMR (400 MHz,
CDCl3) δ: 7.56 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 7.8 Hz, 2H), 6.43 (s, 1H), 5.65 (s, 1H), 3.68 (s,
2H).
2-([1,1'-Biphenyl]-3-ylmethyl)acrylic acid (11e). Yield 98%, white solid. 1H NMR (400 MHz,
CDCl3) δ: 7.58 (d, J = 7.3 Hz, 2H), 7.43 (t, J = 7.2 Hz, 4H), 7.32-7.37 (m, 2H), 7.19-7.27 (m, 1H),
6.41 (s, 1H), 5.64 (s, 1H), 3.69 (s, 2H).
1
2-(Benzo[b]thiophen-2-ylmethyl)acrylic acid (11f). Yield 95%, white solid. H NMR (400
MHz, DMSO-d6) δ: 12.73 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.25-7.34 (m,
2H), 7.16 (s, 1H), 6.19 (s, 1H), 5.79 (s, 1H), 3.86 (s, 2H).
1
2-[(1H-indol-5-yl)methyl]acrylic acid (11g). Yield 96%, white solid. H NMR (400 MHz,
DMSO-d6) δ: 12.75 (s, 1H), 11.31 (s, 1H), 7.70 (s, 1H), 7.67 (s, 1H), 7.61 (s, 1H), 7.29 (d, J = 8.1
Hz, 1H), 6.71 (s, 1H), 6.46 (s, 1H), 5.83 (s, 1H), 3.98 (s, 2H).
1
2-[(1-Methyl-1H-indol-5-yl)methyl]acrylic acid (11h). Yield 98%, white solid. H NMR (400
MHz, CDCl3) δ: 7.45 (s, 1H), 7.26-7.27 (m, 1H), 7.03-7.08 (m, 2H), 6.41 (s, 1H), 6.35 (s, 1H),
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5.55 (s, 1H), 3.78-3.80 (m, 3H), 3.72 (s, 2H).
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2-(Naphthalen-2-ylmethyl)acrylic acid (11i). Yield 98%, white solid. H NMR (400 MHz,
CDCl3) δ: 7.77 (d, J = 7.8, 3H), 7.64 (s, 1H), 7.42-7.46 (m, 2H), 7.25-7.35 (m, 1H), 6.41 (s, 1H),
5.61 (d, J = 4.0 Hz, 1H), 3.78 (s, 2H).
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General procedure for the synthesis of 12a-i exemplified by 12a. To a stirred solution of
compound 11a (830 mg, 3.49 mmol, 1.0 eq) and Et3N (0.3 mL, 0.22 mmol, 0.06 eq) in CH2Cl2 (30
mL) a solution of thioacetic acid (320 μL, 4.5 mmol, 1.3 eq) in CH2Cl2 (10 mL) was added in a
dropwise manner, and the reaction mixture was continuously stirred at room temperature for 30 h
under a nitrogen atmosphere. The mixture was evaporated to obtain compound 12a (857.6 mg,
49% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ: 11.15 (s, 1H), 7.30-7.48 (m, 6H), 7.23
(d, J = 8.0 Hz, 3H), 2.80-3.16 (m, 5H), 2.26 (s, 3H).
3-(Acetylthio)-2-(dibenzo[b,d]thiophen-2-ylmethyl)propanoic acid (12b). Yield 42%, white
1
solid. H NMR (400 MHz, CDCl3) δ: 11.83 (s, 1H), 8.05 (s, 1H), 7.90 (s, 1H), 7.75 (s, 1H), 7.66
(s, 1H), 7.35 (s, 2H), 7.18 (s, 1H), 3.02-3.12 (m, 5H), 2.21 (s, 3H).
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3-(Acetylthio)-2-(phenanthren-9-ylmethyl)propanoic acid (12c). Yield 57%, white solid. H
NMR (400 MHz, CDCl3) δ: 8.74 (d, J = 8.0 Hz, 1H), 8.66 (d, J = 8.0 Hz, 1H), 8.10 (d, J = 5.3 Hz,
1H), 7.83 (d, J = 7.4 Hz, 1H), 7.56-7.70 (m, 5H), 3.58 (d, J = 8.0 Hz, 1H), 3.15-3.36 (m, 4H), 2.32
(s, 3H).
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3-(Acetylthio)-2-(4-(trifluoromethyl)benzyl)propanoic acid (12d). Yield 38%, white solid. H
NMR (400 MHz, CDCl3) δ: 10.46 (s, 1H), 7.46 (d, J = 7.7 Hz, 2H), 7.22 (d, J = 7.7 Hz, 2H),
2.84-3.06 (m, 5H), 2.23 (s, 3H).
3-([1,1'-Biphenyl]-3-yl)-2-((acetylthio)methyl)propanoic acid (12e). Yield 54%, white solid.
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1H NMR (400 MHz, CDCl3) δ: 7.58 (d, J = 7.5 Hz, 2H), 7.47 (d, J = 7.5 Hz, 2H), 7.42 (d, J = 8.0
Hz, 2H), 7.35 (d, J = 7.7 Hz, 2H), 7.18 (d, J = 7.5 Hz, 1H), 2.96-3.17 (m, 5H), 2.33 (s, 3H).
3-(Acetylthio)-2-(benzo[b]thiophen-2-ylmethyl)propanoic acid (12f). Yield 40%, white solid.
1H NMR (400 MHz, DMSO-d6) δ: 12.73 (s, 1H), 7.89 (s, 2H), 7.80 (d, J = 8.0 Hz, 1H), 7.43 (s,
1H), 7.32 (s, 1H), 7.20 (s, 1H), 3.10-3.20 (m, 3H), 2.89 (s, 1H), 2.34 (s, 3H).
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2-[(1H-Indol-5-yl)methyl]-3-(acetylthio)propanoic acid (12g). Yield 39%, white solid. H
NMR (400 MHz, DMSO-d6) δ: 12.44 (s, 1H), 11.01 (s, 1H), 7.31 (d, J = 6.6 Hz, 3H), 6.90 (d, J =
8.0 Hz, 1H), 6.34 (s, 1H), 2.73-3.04 (m, 5H), 2.31 (s, 3H).
3-(Acetylthio)-2-((1-methyl-1H-indol-5-yl)methyl)propanoic acid (12h). Yield 44%, white
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solid. H NMR (400 MHz, CDCl3) δ: 11.05 (s, 1H), 7.40 (s, 1H), 7.22 (s, 1H), 7.01 (s, 2H), 6.39
(s, 1H), 3.73 (s, 3H), 2.98-3.12 (m, 5H), 2.29 (s, 3H).
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3-(Acetylthio)-2-(naphthalen-2-ylmethyl)propanoic acid (12i). Yield 51%, white solid. H
NMR (400 MHz, CDCl3) δ: 7.79 (t, J = 9.0 Hz, 3H), 7.65 (s, 1H), 7.45 (t, J = 4.0 Hz, 2H), 7.32 (d,
J = 8.1 Hz, 1H), 3.03-3.24 (m, 5H), 2.33 (s, 3H).
General procedure for the synthesis of 14a-q exemplified by 14a. To a solution of 12a (200.0
mg, 0.64 mmol, 1.0 eq) in anhydrous CH2Cl2 (50 mL), EDCI (159.5 mg, 0.83 mmol, 1.3 eq),
HOBt (112.4 mg, 0.83 mmol, 1.3 eq) and N-methylmorpholine (100 μL, 0.91 mmol, 1.4 eq) were
added. The resulting mixture was stirred for 30 min under nitrogen in an ice-salt bath. A solution
of ethyl L-phenylalaninate hydrochloride (190.7 mg, 0.83 mmol, 1.3 eq) and N-methylmorpholine
(90 μL, 0.85 mmol, 1.3 equiv) in CH2CH2 (20 mL) was then added to the mixture. The reaction
was warmed to room temperature and stirred overnight under nitrogen. After completion, the
reaction mixture was diluted with CH2CH2 (100 mL) and washed with 5% aqueous citric acid and
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brine. The organic layer was dried over anhydrous Na2SO4, filtered and evaporated to yield crude
compound 13a, which was used directly for the next step without further purification. Then,
compound 13a was dissolved in degassed THF (5 mL), and 3 M NaOH (30 mL) was added. The
mixture was stirred for 10 h at room temperature under nitrogen. After removal of most of the
THF, the residual aqueous solution was adjusted to pH 3-4 with 3 N HCl and extracted with ethyl
acetate (3 × 30 mL). The combined organic layer was washed with brine, dried over Na2SO4,
filtered and concentrated. The obtained residue was purified by silica gel column chromatography
(DCM: CH3OH = 50: 1) to afford target compound 14a (85.8 mg, 32% yield) as a white solid. The
racemic 14a was further separated by preparative TLC, developing with CH2Cl2/MeOH/HCOOH
(20:1:0.1) to obtain (S,S)-14a-1 and (R,S)-14a-2.
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(3-([1,1'-Biphenyl]-4-yl)-2-(mercaptomethyl)propanoyl)-L-phenylalanine (14a). Yield 62%,
white solid. 1H NMR (400 MHz, CDCl3) δ: 7.53 (d, J = 7.6 Hz, 2H), 7.46 (d, J = 7.9 Hz, 2H), 7.41
(t, J = 7.6 Hz, 2H), 7.32-7.37 (m, 1H), 7.28 (d, J = 5.4 Hz, 3H), 7.16 (s, 2H), 7.14 (s, 2H), 5.73 (d,
J = 7.4 Hz, 1H), 4.76- 4.88 (m, 1H), 3.17-3.21 (m, 1H), 2.88-3.02 (m, 2H), 2.78-2.87 (m, 2H),
2.47-2.57 (m, 2H), 1.33 (t, J = 8.7 Hz, 1H).
((S)-3-([1,1'-Biphenyl]-4-yl)-2-(mercaptomethyl)propanoyl)-L-phenylalanine ((S,S)-14a-1).
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Yield 19%, white solid. [α]25 D +26.4 (c = 0.1, CH3OH). H NMR (400 MHz, DMSO-d6) δ:
12.77 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 4.0 Hz, 2H), 7.54 (d, J = 8.0 Hz, 2H), 7.44 (t, J
= 6.0 Hz, 2H), 7.33 (t, J = 6.0 Hz, 1H), 7.24 (s, 8H), 4.50 (q, J = 8.0 Hz, 1H), 2.88 (t, J = 8.0 Hz,
2H), 2.68-2.70 (m, 2H), 2.53-2.57 (m, 1H), 2.33 (t, J = 8.0 Hz, 1H), 1.81 (t, J = 8.0 Hz, 1H). 13
C
NMR (150 MHz, DMSO-d6) δ: 172.73, 172.30, 139.91, 138.32, 137.83, 137.50, 129.31, 129.00,
128.72, 127.99, 127.02, 126.33, 126.19, 53.09, 50.34, 36.61, 25.39. HRMS m/z calcd for
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C25H25NO3S [M + H]+ 420.1628, found 420.1641.
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((R)-3-([1,1'-Biphenyl]-4-yl)-2-(mercaptomethyl)propanoyl)-L-phenylalanine ((R,S)-14a-2).
Yield 32%, white solid. [α]25 D -15.1 (c = 0.1, CH3OH). 1H NMR (400 MHz, DMSO-d6) δ: 12.78
(s, 1H), 8.37 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.44 (t, J = 8.0
Hz, 2H), 7.33 (t, J = 8.0 Hz, 1H), 7.20 (s, 1H), 7.17 (d, J = 8.0 Hz, 2H), 7.12 (t, J = 8.0 Hz, 4H),
4.46 (q, J = 8.0 Hz, 1H), 2.99 (dd, J = 4.0, 4.0 Hz, 1H), 2.68-2.84 (m, 3H), 2.59-2.63 (m, 2H),
2.35-2.41 (m, 1H), 2.20 (t, J = 8.0 Hz, 1H). 13C NMR (150 MHz, DMSO-d6) δ: 172.84, 172.38,
139.86, 138.26, 137.78, 137.33, 129.27, 128.88, 128.72, 127.93, 127.03, 126.30, 126.17, 53.05,
50.15, 36.59, 25.41. MS m/z 420.0 [M + H]+.
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(2S)-2-(3-([1,1'-Biphenyl]-4-yl)-2-(mercaptomethyl)propanamido)-4-phenylbutanoic
acid
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(14b). Yield 63%, white solid. H NMR (400 MHz, CDCl3) δ 7.52 (d, J = 8.9 Hz, 4H), 7.41 (s, 2H),
7.34 (s, 1H), 7.26 (s, 2H), 7.21 (s, 3H), 7.15 (s, 2H), 5.85 (s, 1H), 4.55 (s, 1H), 2.82-2.99 (m, 3H),
2.56-2.69 (m, 4H), 2.18 (s, 1H), 1.92 (s, 1H), 1.26 (s, 1H).
(S)-2-((S)-3-([1,1'-Biphenyl]-4-yl)-2-(mercaptomethyl)propanamido)-4-phenylbutanoic acid
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((S,S)-14b-1). Yield 22%, white solid. [α]25 D +21.6 (c = 0.1, CH3OH). H NMR (400 MHz,
CDCl3) δ: 7.54 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.41 (t, J = 8.0 Hz, 2H), 7.34 (d, J =
8.0 Hz, 1H), 7.28 (s, 1H), 7.25 (s, 2H), 7.21 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 5.83 (d,
J = 8.0 Hz, 1H), 4.55 (q, J = 5.0 Hz, 1H), 2.93-2.98 (m, 1H), 2.86 (d, J = 8.0 Hz, 2H), 2.64 (t, J =
6.0 Hz, 2H), 2.55-2.59 (m, 2H), 2.15-2.23 (m, 1H), 1.89-1.94 (m, 1H), 1.58 (t, J = 8.0 Hz, 1H).
13C NMR (150 MHz, CDCl3) δ: 177.96, 177.38, 145.48, 144.81, 142.89, 133.82, 133.05, 132.74,
132.57, 131.40, 130.95, 130.16, 55.85, 41.89, 37.71, 36.03, 33.66, 30.33. HRMS m/z calcd for
C26H27NO3S [M + H]+ 434.1784, found 434.1788.
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(S)-2-((R)-3-([1,1'-Biphenyl]-4-yl)-2-(mercaptomethyl)propanamido)-4-phenylbutanoic acid
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((R,S)-14b-2). Yield 36%, white solid. [α]25 D -10.7 (c = 0.1, CH3OH). H NMR (400 MHz,
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DMSO-d6) δ: 12.61 (s, 1H), 8.34 (d, J = 8.0 Hz, 1H), 7.54 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz,
2H), 7.38 (t, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0 Hz, 3H), 7.12 (t, J = 6.0 Hz, 3H), 6.94 (d, J = 8.0
Hz, 2H), 4.05 (q, J = 8.0 Hz, 1H), 2.81-2.87 (m, 3H), 2.71-2.78 (m, 1H), 2.53-2.55 (m, 1H),
2.22-2.6 (m, 3H), 1.80-1.90 (m, 1H), 1.58 (t, J = 8.0 Hz, 1H). 13C NMR (150 MHz, DMSO-d6) δ:
173.47, 172.47, 140.73, 139.81, 138.35, 137.87, 129.31, 128.61, 128.09, 128.00, 126.95, 126.28,
125.59, 51.01, 50.49, 36.93, 32.43, 31.07, 26.20. HRMS m/z calcd for C26H27NO3S [M + H]+
434.1784, found 434.1787.
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(2S)-2-(3-([1,1'-Biphenyl]-4-yl)-2-(mercaptomethyl)propanamido)-3-(1H-indol-5-yl)-propan-
oic acid (14c). Yield 41%, white solid. 1H NMR (400 MHz, DMSO-d6) δ: 12.69 (s, 1H), 10.89 (s,
1H), 8.38 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0
Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.0 Hz, 3H), 7.08 (t, J =
6.0 Hz, 1H), 6.99 (t, J = 6.0 Hz, 1H), 4.58 (q, J = 6.0 Hz, 1H), 3.20 (dd, J = 4.0, 4.0 Hz, 1H),
3.03-3.08 (m, 1H), 2.88 (m, 1H), 2.56-2.77 (m, 3H), 2.34-2.40 (m, 1H), 1.95 (t, J = 8.0 Hz, 1H).
13C NMR (150 MHz, DMSO-d6) δ: 173.19, 172.35, 139.91, 138.31, 137.80, 135.96, 129.32,
128.70, 127.02, 126.30, 123.52, 120.75, 118.17, 118.08, 111.18, 109.76, 52.56, 50.39, 36.72,
27.07, 25.33. HRMS m/z calcd for C27H26N2O3S [M + H]+ 459.1737, found 459.1740.
(2S)-2-(3-([1,1'-Biphenyl]-4-yl)-2-(mercaptomethyl)propanamido)-3-(4-(benzyloxy)phenyl)pr-
opanoic acid (14d). Yield 38%, white solid. 1H NMR (400 MHz, DMSO-d6) δ: 12.76 (s, 1H), 8.34
(d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.43 (t, J = 8.0 Hz, 1H),
7.52 (d, J = 8.0 Hz, 2H), 7.34 (s, 1H), 7.33 (s, 2H), 7.30 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 8.0 Hz,
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2H), 7.02 (d, J = 8.0 Hz, 2H), 6.83 (d, J = 6.0 Hz, 2H), 5.02 (s, 1H), 4.95 (s, 1H), 4.41 (q, J = 8.0
Hz, 1H), 2.95 (dd, J = 4.0, 4.0 Hz, 1H), 2.74-2.85 (m, 2H), 2.54-2.69 (m, 3H), 2.32-2.45 (m, 1H),
2.24 (t, J = 8.0 Hz, 1H). 13C NMR (150 MHz, DMSO-d6) δ: 172.88, 172.38, 156.81, 139.84,
138.81, 137.77, 136.93, 130.04, 129.90, 129.47, 129.30, 128.71, 128.22, 128.17, 127.54, 127.35,
127.02, 126.30, 126.27, 114.34, 68.90, 53.25, 49.99, 36.53, 35.73, 25.54. HRMS m/z calcd for
C32H31NO4S [M + H]+ 526.2047, found 526.2051.
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(3-([1,1'-Biphenyl]-3-yl)-2-(mercaptomethyl)propanoyl)-L-phenylalanine (14e). Yield 47%,
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white solid. H NMR (400 MHz, DMSO-d6) δ: 12.77 (s, 1H), 8.36 (d, J = 7.8 Hz, 1H), 7.64 (d, J =
7.7 Hz, 2H), 7.43-7.53 (m, 4H), 7.30-7.38 (m, 2H), 7.03-7.15 (m, 6H), 4.42-4.47 (m, 1H), 2.90-2.99 (m,
1H), 2.71-2.85 (m, 3H), 2.65-2.68 (m, 2H), 2.37-2.45 (m, 1H), 2.19 (t, J = 7.8 Hz, 1H).
((S)-3-([1,1'-Biphenyl]-3-yl)-2-(mercaptomethyl)propanoyl)-L-phenylalanine
((S,S)-14e-1).
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Yield 15%, white solid. [α]25 D +17.1 (c = 0.2, CH3OH). H NMR (400 MHz, DMSO-d6) δ:
12.78 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 2H), 7.47 (d, J = 4.0 Hz, 2H), 7.44 (d,
J = 8.0 Hz, 2H), 7.37 (d, J = 4.0 Hz, 1H), 7.32 (t, J = 6.0 Hz, 1H), 7.15 (s, 1H), 7.14 (s, 2H), 7.07
(d, J = 8.0 Hz, 3H), 4.44 (q, J = 6.7 Hz, 1H), 2.93 (dd, J = 4.0, 4.0 Hz, 1H), 2.72-2.81 (m, 3H),
2.61-2.68 (m, 2H), 2.37-2.43 (m, 1H), 2.20 (t, J = 8.0 Hz, 1H). 13C NMR (150 MHz, DMSO-d6) δ:
172.78, 172.38, 140.14, 140.09, 139.92, 139.64, 137.21, 128.94, 128.85, 128.69, 128.60, 127.89,
127.18, 126.55, 126.15, 124.38, 53.04, 50.32, 37.26, 36.70, 25.31. MS m/z 420.0 [M + H]+.
((R)-3-([1,1'-Biphenyl]-3-yl)-2-(mercaptomethyl)propanoyl)-L-phenylalanine ((R,S)-14e-2).
Yield 26%, white solid. [α]25 D -23.7 (c = 0.2, CH3OH). 1H NMR (400 MHz, DMSO-d6) δ: 12.76
(s, 1H), 8.38 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 2H), 7.46 (s, 3H), 7.44 (s, 1H), 7.35 (d, J =
8.0 Hz, 2H), 7.23 (s, 4H), 7.15 (t, J = 6.0 Hz, 2H), 4.50 (q, J = 6.7 Hz, 1H), 3.10 (dd, J = 4.0, 4.0
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Hz, 1H), 2.85-2.92 (m, 2H), 2.68-2.77 (m, 2H), 2.51-2.55 (m, 1H), 2.27-2.34 (m, 1H), 1.84 (t, J =
8.0 Hz, 1H). 13C NMR (150 MHz, DMSO-d6) δ: 172.71, 172.32, 140.13, 140.02, 139.68, 137.44,
129.00, 128.69, 128.63, 127.98, 127.91, 127.18, 127.14, 126.59, 126.19, 124.40, 53.06, 50.23,
37.06, 36.70, 25.21. HRMS m/z calcd for C25H25NO3S [M + H]+ 420.1628, found 420.1631.
(3-([1,1'-Biphenyl]-3-yl)-2-(mercaptomethyl)propanoyl)-L-tryptophan (14f). Yield 36%,
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white solid. H NMR (400 MHz, DMSO-d6) δ: 12.64 (s, 1H), 10.87 (s, 1H), 8.38 (d, J = 4.0 Hz,
1H), 7.64 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 1H), 7.50 (s, 1H), 7.46 (t, J = 6.0 Hz, 3H), 7.33
(d, J = 8.0 Hz, 3H), 7.16 (t, J = 8.0 Hz, 2H), 7.07 (t, J = 8.0 Hz, 1H), 6.99 (t, J = 8.0 Hz, 1H), 4.59
(q, J = 6.7 Hz, 1H), 3.21 (dd, J = 4.0, 4.0 Hz, 1H), 3.03-3.10 (m, 1H), 2.90-2.97 (m, 1H),
2.68-2.83 (m, 3H), 2.56-2.63 (m, 1H), 1.94 (t, J = 8.0 Hz, 1H). 13C NMR (150 MHz, DMSO-d6) δ:
173.17, 172.43, 140.13, 140.02, 139.69, 135.93, 128.69, 128.62, 127.17, 127.05, 126.59, 124.40,
123.48, 123.31, 120.75, 118.17, 111.17, 109.69, 52.56, 50.20, 37.21, 27.18, 25.14. HRMS m/z
calcd for C27H26N2O3S [M + H]+ 459.1737, found 459.1737.
(2S)-2-(3-([1,1'-Biphenyl]-3-yl)-2-(mercaptomethyl)propanamido)-3-(4-(benzyloxy)phenyl)-
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propanoic acid (14g). Yield 54%, white solid. H NMR (400 MHz, DMSO-d6) δ: 12.72 (s, 1H),
8.30 (d, J = 7.9 Hz, 1H), 7.65 (d, J = 7.4 Hz, 2H), 7.29-7.54 (m, 11H), 7.08 (d, J = 7.4 Hz, 1H), 6.94 (d,
J = 8.3 Hz, 2H), 6.76 (d, J = 8.4 Hz, 2H), 4.95 (s, 2H), 4.40 (d, J = 5.0 Hz, 1H), 2.62-2.90 (m, 6H),
2.43 (s, 1H), 2.21 (t, J = 8.1 Hz, 1H).
(S)-2-((S)-3-([1,1'-Biphenyl]-3-yl)-2-(mercaptomethyl)propanamido)-3-(4-(benzyloxy)phenyl
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propanoic acid ((S,S)-14g-1). Yield 17%, white solid. [α]25 D +25.5 (c = 0.1, CH3OH). H NMR
(400 MHz, DMSO-d6) δ: 12.71 (s, 1H), 8.29 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.45 (t,
J = 8.0 Hz, 4H), 7.36 (s, 4H), 7.31 (t, J = 8.0 Hz, 3H), 7.07 (d, J = 8.0 Hz, 1H), 6.93 (d, J = 8.0 Hz,
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2H), 6.73 (d, J = 8.0 Hz, 2H), 4.93 (s, 2H), 4.39 (q, J = 6.7 Hz, 1H), 2.78-2.86 (m, 2H), 2.60-2.75
(m, 4H), 2.38-2.45 (m, 1H), 2.20 (t, J = 8.0 Hz, 1H). 13C NMR (150 MHz, DMSO-d6) δ: 172.81,
172.35, 156.73, 140.11, 139.88, 139.64, 136.97, 129.96, 129.87, 129.27, 128.69, 128.57, 128.21,
127.89, 127.56, 127.35, 127.20, 126.56, 124.36, 114.20, 68.86, 53.24, 50.27, 37.28, 35.84, 25.44.
MS m/z 526.2 [M + H]+.
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(S)-2-((R)-3-([1,1'-Biphenyl]-3-yl)-2-(mercaptomethyl)propanamido)-3-(4-(benzyloxy)phenyl
) propanoic acid ((R,S)-14g-2). Yield 29%, white solid. [α]25 D -3.4 (c = 0.1, CH3OH). 1H NMR
(400 MHz, DMSO-d6) δ: 12.70 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.47 (s,
2H), 7.45 (s, 2H), 7.40 (t, J = 6.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.32 (t, J = 4.0 Hz, 2H), 7.31
(s, 1H), 7.13 (t, J = 6.0 Hz, 3H), 6.87 (t, J = 8.0 Hz, 2H), 5.02 (s, 2H), 4.43 (q, J = 6.7 Hz, 1H),
3.00 (dd, J = 4.0, 4.0 Hz, 1H), 2.88-2.93 (m, 1H), 2.63-2.81 (m, 3H), 2.55-2.60 (m, 1H), 2.29-2.35
(m, 1H), 1.89 (t, J = 8.0 Hz, 1H). 13C NMR (150 MHz, DMSO-d6) δ: 172.79, 172.36, 156.89,
140.17, 140.06, 139.75, 137.08, 130.09, 129.56, 128.74, 128.67, 128.28, 127.98, 127.62, 127.44,
127.23, 127.18, 126.64, 124.44, 114.37, 69.01, 53.35, 50.24, 37.10, 35.93, 25.31. HRMS m/z
calcd for C32H31NO4S [M + H]+ 526.2047, found 526.2049.
(2S)-2-(3-(Benzo[b]thiophen-2-yl)-2-(mercaptomethyl)propanamido)-4-phenylbutanoic acid
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(14h). Yield 21%, white solid. H NMR (400 MHz, DMSO-d6) δ: 12.66 (s, 1H), 8.48 (d, J = 8.0
Hz, 1H), 7.85 (t, J = 6.0 Hz, 1H), 7.01 (t, J = 6.0 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.25 (d, J =
8.0 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H), 7.13 (d, J = 8.0 Hz, 3H), 6.86 (d, J = 8.0 Hz, 2H), 4.07 (q, J
= 5.3 Hz, 1H), 3.11-3.20 (m, 2H), 2.92-2.97 (m, 1H), 2.75-2.79 (m, 1H), 2.58-2.63 (m, 1H),
2.29-2.43 (m, 3H), 1.74-1.93 (m, 2H). 13C NMR (150 MHz, DMSO-d6) δ: 173.45, 172.06, 142.59,
140.61, 139.52, 138.65, 128.00, 125.59, 124.03, 123.53, 122.69, 121.97, 51.12, 50.08, 32.34,
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31.76, 31.05, 26.23. HRMS m/z calcd for C22H23NO3S2 [M + H]+ 414.1192, found 414.1205.
(2S)-2-(3-Mercapto-2-(phenanthren-9-ylmethyl)propanamido)-4-phenylbutanoic acid (14i).
Yield 38%, white solid. 1H NMR (400 MHz, DMSO-d6) δ: 12.61 (s, 1H), 8.90 (d, J = 4.0 Hz, 1H),
8.80 (d, J = 8.0 Hz, 1H), 8.51 (d, J = 8.0 Hz, 1H), 8.27 (s, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.73 (s,
3H), 7.63 (t, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 7.18 (s, 3H), 4.20 (s, 1H), 3.39-3.44 (m,
1H), 3.25-3.29 (m, 1H), 3.01-3.10 (m, 1H), 2.85 (q, J = 8.0 Hz, 1H), 2.58-2.72 (m, 3H), 2.40 (q, J
= 8.0 Hz, 1H), 1.86-2.04 (m, 2H). 13C NMR (150 MHz, DMSO-d6) δ: 173.15, 172.76, 140.73,
132.83, 131.01, 130.50, 129.97, 129.00, 127.96, 127.87, 127.81, 126.93, 126.35, 126.23, 125.91,
125.82, 125.39, 124.11, 122.75, 121.86, 51.17, 49.22, 34.47, 32.83, 31.30, 26.36. HRMS m/z
calcd for C28H27NO3S [M + H]+ 458.1784, found 458.1788.
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(3-(Dibenzo[b,d]thiophen-2-yl)-2-(mercaptomethyl)propanoyl)-L-phenylalanine (14j). Yield
46%, white solid. 1H NMR (400 MHz, DMSO-d6) δ: 12.86 (s, 1H), 8.45 (d, J = 8.0 Hz, 1H), 8.34 (s,
1H), 8.20 (s, 1H), 8.05 (s, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.55 (d, J = 2.9 Hz, 2H), 7.35 (d, J = 7.9 Hz,
1H), 7.17-7.27 (m, 5H), 4.54 (d, J = 3.8 Hz, 1H), 3.04-3.20 (m, 2H), 2.85-2.95 (m, 3H), 2.63 (s, 1H),
2.40 (s, 1H), 1.90 (s, 1H).
((S)-3-(Dibenzo[b,d]thiophen-2-yl)-2-(mercaptomethyl)propanoyl)-L-phenylalanine ((S,S)-
14j-1). Yield 14%, white solid. [α]25 D +23.3 (c = 0.2, CH3OH). 1H NMR (400 MHz, DMSO-d6)
δ: 12.82 (s, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.30 (s, 1H), 8.16 (s, 1H), 8.01 (s, 1H), 7.88 (d, J = 8.0
Hz, 1H), 7.51 (t, J = 6.0 Hz, 2H), 7.31 (d, J = 8.0 Hz, 1H), 7.22 (s, 3H), 7.18 (d, J = 8.0 Hz, 2H),
4.50 (q, J = 8.0 Hz, 1H), 3.03-3.13 (m, 2H), 2.81-2.91 (m, 3H), 2.56-2.63 (m, 1H), 2.33-2.40 (m,
1H), 1.86 (t, J = 8.0 Hz, 1H). 13C NMR (150 MHz, DMSO-d6) δ: 172.76, 172.29, 138.68, 137.52,
136.16, 135.77, 134.95, 134.78, 128.99, 128.11, 127.96, 126.77, 126.17, 124.46, 122.85, 122.49,
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121.93, 121.77, 53.17, 50.43, 36.91, 36.63, 25.34. HRMS m/z calcd for C25H23NO3S2 [M + H]+
450.1192, found 450.1199.
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((R)-3-(Dibenzo[b,d]thiophen-2-yl)-2-(mercaptomethyl)propanoyl)-L-phenylalanine ((R,S)-
14j-2). Yield 26%, white solid. [α]25 D -5.7 (c = 0.2, CH3OH). 1H NMR (400 MHz, DMSO-d6) δ:
12.83 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.28 (s, 1H), 8.10 (s, 1H), 8.01 (s, 1H), 7.86 (d, J = 8.0 Hz,
1H), 7.51 (s, 2H), 7.23 (d, J = 4.0 Hz, 1H), 7.01 (s, 5H), 4.42 (q, J = 8.0 Hz, 1H), 2.67-2.94 (m,
5H), 2.63-2.65 (m, 1H), 2.42-2.47 (m, 1H), 2.22 (t, J = 8.0 Hz, 1H). 13C NMR (150 MHz,
DMSO-d6) δ: 172.79, 172.34, 138.71, 137.21, 136.18, 135.72, 134.90, 134.78, 128.74, 128.03,
127.74, 126.77, 126.02, 124.44, 122.87, 122.48, 121.88, 121.70, 53.10, 50.61, 37.12, 36.61, 25.55.
(2S)-2-(3-(Dibenzo[b,d]thiophen-2-yl)-2-(mercaptomethyl)propanamido)-4-phenylbutanoic
acid (14k). Yield 21%, white solid. 1H NMR (400 MHz, DMSO-d6) δ: 12.61 (s, 1H), 8.45 (d, J =
8.0 Hz, 1H), 8.32 (d, J = 8.0 Hz, 1H), 8.21 (s, 1H), 8.00 (d, J = 6.0 Hz, 1H), 7.92 (d, J = 8.0 Hz,
1H), 7.50 (d, J = 6.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 1H), 7.25 (t, J = 6.0 Hz, 2H), 7.16 (t, J = 8.0
Hz, 3H), 4.18 (q, J = 8.0 Hz, 1H), 3.09-3.15 (m, 1H), 2.87-2.92 (m, 2H), 2.70-2.76 (m, 2H),
2.55-2.68 (m, 2H), 2.28 (t, J = 8.0 Hz, 1H), 1.98-2.04 (m, 1H), 1.84-1.90 (m, 1H). 13C NMR (150
MHz, DMSO-d6) δ: 173.35, 172.57, 140.90, 138.70, 136.20, 135.74, 134.98, 134.80, 128.22,
128.14, 126.77, 125.73, 124.46, 122.85, 122.51, 121.99, 121.78, 51.11, 50.39, 37.21, 32.74, 31.25,
25.34. HRMS m/z calcd for C26H25NO3S2 [M + H]+ 464.1349, found 464.1353.
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(2S)-2-(2-((1H-Indol-5-yl)methyl)-3-mercaptopropanamido)-4-phenylbutanoic acid
(14l).
Yield 26%, white solid. 1H NMR (400MHz, DMSO-d6) δ: 10.96 (s, 1H), 8.28 (d, J = 8.0 Hz, 1H),
7.36 (s, 1H), 7.28 (s, 1H), 7.26 (s, 1H), 7.19 (t, J = 8.0 Hz, 2H), 7.11 (d, J = 8.0 Hz, 1H), 6.95 (d, J
= 8.0 Hz, 1H), 6.89 (d, J = 8.0 Hz, 2H), 6.32 (s, 1H), 4.02 (q, J = 8.0 Hz, 1H), 2.78-2.90 (m, 4H),
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