Synthesis of 1,3,4-trisubstituted pyrrolidines as meropenem adjuvants targeting New Delhi metallo-β-lactamase

Article abstract of DOI:10.1039/d0nj06090a

The effective strategies to neutralize the New Delhi metallo-β-lactamase (NDM-1) activity offer unique opportunities to combination therapy because NDM-1 inactivates all classes of carbapenem antibiotics, which are widely regarded as the last resort of drugs for treating serious bacterial infections. Here we describe the efficient construction of a series of trans-1,3,4-trisubstituted pyrrolidines via boric acid-catalyzed 1,3-dipolar cycloaddition of N-benzylazomethine ylide with methyl ferulate for the biological evaluation of their cytotoxicity and synergistic activity in combination with meropenem towards NDM-1 positive carbapenem-resistant Enterobacteriaceae (CRE). The cell-based screens generated one promising hit, namely compound 10e, which exhibited low cytotoxicity (IC50 > 128 μM), moderate NDM-1 enzyme inhibition (IC50 = 51 μM), and potent synergistic activity against a panel of clinically isolated NDM-1 positive CRE with fractional inhibitory concentration indexes ranging from 0.01 to 0.25. Structure-activity relationship studies revealed that the zinc-chelating moiety of 2-(bis(pyridin-2-ylmethyl)-amino)acetyl group of compound 10e plays a pivotal role for potent activity. Regarding the inhibition mechanism, a series of biochemical assays revealed that compound 10e may inactivate NDM-1 activity by displacing both zinc ions from the active site of the enzyme. Altogether, our studies indicate that compound 10e represents an important pyrrolidine-type scaffold targeting NDM-1, providing a promising starting point to be further developed as carbapenem antibiotic adjuvants. This journal is

Full text of DOI:10.1039/d0nj06090a

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Synthesis of 1,3,4-trisubstituted pyrrolidines as
meropenem adjuvants targeting New Delhi
metallo-b-lactamase†
Cite this: New J. Chem., 2021,
45, 3515
Wen Bin Jin,‡ab Chen Xu,‡ac Xiao Lin Qi,a Ping Zeng,a Wei Gao,a Ki Hon Lai,a
Jiachi Chiou,a Edward W. C. Chan,a Yun-Chung Leung,a Tak Hang Chan,ad
a
a
Kwok-Yin Wong,
Sheng Chen*e and Kin-Fai Chan
*
The effective strategies to neutralize the New Delhi metallo-b-lactamase (NDM-1) activity offer unique
opportunities to combination therapy because NDM-1 inactivates all classes of carbapenem antibiotics, which
are widely regarded as the last resort of drugs for treating serious bacterial infections. Here we describe the
efficient construction of a series of trans-1,3,4-trisubstituted pyrrolidines via boric acid-catalyzed 1,3-dipolar
cycloaddition of N-benzylazomethine ylide with methyl ferulate for the biological evaluation of their
cytotoxicity and synergistic activity in combination with meropenem towards NDM-1 positive carbapenem-
resistant Enterobacteriaceae (CRE). The cell-based screens generated one promising hit, namely compound
10e, which exhibited low cytotoxicity (IC50 4 128 mM), moderate NDM-1 enzyme inhibition (IC50 = 51 mM),
and potent synergistic activity against a panel of clinically isolated NDM-1 positive CRE with fractional inhibitory
concentration indexes ranging from 0.01 to 0.25. Structure–activity relationship studies revealed that the zinc-
chelating moiety of 2-(bis(pyridin-2-ylmethyl)-amino)acetyl group of compound 10e plays a pivotal role for
potent activity. Regarding the inhibition mechanism, a series of biochemical assays revealed that compound
10e may inactivate NDM-1 activity by displacing both zinc ions from the active site of the enzyme. Altogether,
our studies indicate that compound 10e represents an important pyrrolidine-type scaffold targeting NDM-1,
providing a promising starting point to be further developed as carbapenem antibiotic adjuvants.
Received 15th December 2020,
Accepted 28th January 2021
DOI: 10.1039/d0nj06090a
rsc.li/njc
such as bloodstream infections and pneumonia.1 Unfortunately,
the worldwide dissemination of carbapenem-resistant Entero-
Introduction
Carbapenem antibiotics are highly effective ‘‘last-resort’’ b-lactam bacteriaceae (CRE) over the past decade severely limits their
antibiotics commonly used for treating severe bacterial infections therapeutic options, rendering severe clinical infections diffi-
cult to treat.2 The World Health Organization has recently
a State Key Laboratory of Chemical Biology and Drug Discovery and Department of
Applied Biology and Chemical Technology, The Hong Kong Polytechnic University,
Hung Hom, Kowloon, Hong Kong SAR, China. E-mail: kf.chan@polyu.edu.hk
b Faculty of Chinese Materia Medica, Yunnan University of Chinese Medicine,
Kunming, Yunnan, China
c Shenzhen Key Laboratory for Food Biological Safety Control, Food Safety and
Technology Research Centre, The Hong Kong PolyU Shenzhen Research Institute,
Shenzhen, China
d Department of Chemistry, McGill University, Montreal, Quebec, H3A 2K6, Canada
e Department of Infectious Diseases and Public Health, Jockey Club College of
Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon,
Hong Kong SAR, China. E-mail: shechen@cityu.edu.hk
† Electronic supplementary information (ESI) available: 1H and 13C NMR spectra
of compounds 4–10; MIC screening of compound alone and MRM in the presence
of the compound at 100 mM against E. coli BL21 (NDM-1), calculated c log P,
topological polar surface area (tPSA) and reduction fold (RF); X-ray crystal
structure of 9b; HPLC chromatogram of 6a; crystal data and structure refinement
for compound 9b. CCDC 2046394. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/d0nj06090a
prioritized CRE as one of the Priority 1 (‘‘CRITICAL’’) micro-
organisms for which new drugs are urgently needed.3 CRE are a
large family of Gram-negative bacteria that acquire carbapenem
resistance by overexpressing carbapenemases, which are power-
ful b-lactamases capable of inactivating all carbapenem anti-
biotics by breaking down the b-lactam ring of the molecules.
Worse still, these b-lactamases are not only encoded by genes
that are horizontally transferable by plasmids but also asso-
ciated commonly with genes encoding for other resistant
determinants, rendering CRE a veritable ‘‘superbug’’ which
exhibits drug resistance to all current antibiotics.
Metallo-b-lactamases (MBLs) constitute one of the major
groups of clinically important carbapenemases, particularly
New Delhi metallo-b-lactamase (NDM-1). NDM-1 is one of the
zinc-dependent b-lactamases that can inactivate b-lactam anti-
biotics without proceeding via a covalent intermediate. There-
fore, NDM-1 is generally regarded as the MBLs of greatest
‡ These authors contributed equally to this work.
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Fig. 1 The chemical structures of reported NDM-1 inhibitors and molecular design strategies from captopril.
concerns as it is not only the most easily communicable but also
Among the reported sulfur-containing NDM-1 inhibitors,
capable of compromising the clinical efficacy of almost all b- both D- and L-captopril (Fig. 1) have been documented as the
lactam antibiotics except aztreonam. There are no inhibitors of most promising candidates for inhibition of NDM-1 upon
MBLs in current clinical use. Therefore, effective strategies to imipenem hydrolysis with IC50 values of 7.9 and 202.0 mM,
neutralize the activity of NDM-1 are highly desirable. Over the respectively.21,22 The thiol and carboxylic acid moieties of
past decade of research efforts, two major classes of promising captopril are likely to interact with the dinuclear zinc
NDM-1 inhibitors including covalent inhibitors and zinc chelat- center of NDM-1 by displacing the catalytic hydroxyl ions.23
ing inhibitors have been reported in the literature throwing light Pioneering studies of captopril modifications by other
on the importance of metal chelation and anchor residue bind- research groups have identified a series of chiral mercapto
ing features.4–6 Meanwhile, docking platform also shed light on propionamides 14m-2 and other derivatives with potent NDM-1
the key interaction of molecules with the amino acids of NDM-1 inhibition (Fig. 1).24–26 However, adverse effects associated
active site.7 As depicted in Fig. 1, disulfiram,8 ebselen (Eb)9 as with thiol-containing compounds, such as rashes and loss of
well as its derivatives 11a_3810 and ebsulfur derivative 3a11,12 are taste, may present a substantial barrier for further develop-
examples of potent covalent NDM-1 inhibitors targeting impor- ment.27,28 In the present study, we describe our strategy to
tant amino acid residue Cys221 of NDM-1 enzyme. On the other further modify captopril by constructing a small library of
hand, zinc chelating inhibitors, such as AMA and its amino 1,3,4-trisubstituted pyrrolidine derivatives without bearing
carboxylic acid analog AMB,13,14 tris-picolylamine (TPA)-based thiol groups via boric acid-catalyzed 1,3-dipolar cycloaddition
chelators,15 H2dedpa derivatives,16,17 thiourea derivatives,18,19 of N-benzylazomethine ylide with methyl ferulate. Subsequent
and dipicolinic acid derivatives20 are also reported to potently biological evaluation of their cytotoxicity and synergistic
inhibit NDM-1 with the half-maximal inhibitory concentration activity in combination with meropenem (MRM) towards
(IC50) at single-digit micromolar concentrations via a zinc- NDM-1 positive CRE allows us to identify a promising lead
depletion mechanism.
targeting NDM-1.
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Results and discussion
1. Chemical synthesis
envisioned that the R2 and R3 groups should also be arranged
similarly for better fitting into the NDM-1 substrate-binding site.
Inspired by previous works,31,32 we initiated our study by
constructing a 1,3,4-trisubstituted pyrrolidine library via a key
intermediate pyrrolidine 7a, which was obtained from a concise
synthetic method with four steps in good chemical yield. As
shown in Scheme 1, esterification of ferulic acid (4a) with
methanol in the presence of a catalytic amount of sulphuric
acid provided methyl ferulate (4b). The phenol group of ester 4b
was protected with benzyl bromide under the basic medium to
afford benzyl ether 5a in high yield.33 Subsequent intermolecular
1,3-dipolar cycloaddition of 5a as a dipolarophile with nonstabi-
lized N-benzylazomethine ylide, which was generated in situ from
N-(methoxymethyl)-N-(trimethylsilylmethyl)benzyl-amine in the
presence of a catalytic amount of boric acid, furnished desired
Our molecular design strategy is shown in Fig. 1. The core
pyrrolidine ring is retained for maintaining the hydrophobic
interaction with Trp64 of NDM-1.29 Functional group R1 and
carboxylate R2 are selectively installed at positions 1 and 3 of
the pyrrolidine ring respectively. Oxygen-rich R1 groups that
may provide a stronger interaction with the dinuclear zinc
center in the active site of the NDM-1 enzyme will be selected.
The substituted phenyl ring R3 is installed at position 4 as this
aromatic group may enhance p–p interaction with the amino
acid residues such as histidine and tryptophan in the active site
of the NDM-1 enzyme. Moreover, the substitutes at positions
3 and 4 of the 1-pyrroline ring of hydrolyzed MRM are in trans
configuration (please refer to the graphical abstract).30 We pyrrolidine 6a in good yield.34,35 The chemical structure of the
Scheme 1 Reagents and reaction conditions: (a) MeOH, conc. H2SO4 (cat.), reflux, 6 h, 62%; (b) for 5a, BnBr, K2CO3, acetone, reflux, 3 h, 80%; for 5b,
allyl bromide, K2CO3, acetone, reflux, 3 h, 67%; for 5c, mesyl chloride, Et3N, DCM, 0 1C, 4 h, 66%; for 5d, acetic anhydride, pyridine, rt, 3 h, 54%; (c) B(OH)3
(cat.), DCM, rt, 48 h, for 6a, 53%, for 6b, 50%, for 6c, 57%, for 6d, 40%; (d) (i) a-chloroethyl chloroformate, DCM, reflux, 3 h; (ii) MeOH, reflux, 3 h, for 7a,
73%, for 7b, 68%, for 7c, 49%, for 7d, 65%; (e) dimethyl chlorophosphate, NEt3, DMAP, DCM, 0 1C to rt, 14 h, for 8a, 76%, for 8b, 73%, for 8c, 75%, for 8d,
53%; (f) ethyl chloroformate, NEt3, DCM, 0 1C, 4 h, 73%; (g) 7e, KOH, H2O, rt to 60 1C, 16 h, 35%; (h) (1) ethyl chloroformate, NEt3, THF, 0 1C to rt, 16 h;
(2) NH4OH, THF, 0 1C to rt, 16 h, 43%; (i) 7e, 10% Pd/C, H2, THF, rt, 16 h, 49%; (j) LiCl, NaBH4, EtOH, THF, 0 1C to rt, 2 h, 44%. (k) KOH, H2O, rt to 60 1C, 16 h, 43%.
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various bromoesters, such as t-butyl 2-bromoacetate, i-propyl
2-bromoacetate, and ethyl 3-bromopropanoate, or 3-chloro-
propane-1,2-diol under the basic medium provided pyrrolidine
8e–f and 8h–i respectively. Subsequent acidic treatment for
removing the tert-butyl group of 8e with TFA using DCM as
solvent delivered carboxylic acid 8g in moderate yield. To obtain
more pyrrolidine derivatives bearing a carboxylic acid group at
position 1 for the biological study, treatment of pyrrolidine 7a
with a series of cyclic anhydrides, including 2,2-dimethylglutaric
anhydride, 3,3-dimethylglutaric anhydride, maleic anhydride,
succinic anhydride, and phthalic anhydride in the presence of
trimethylamine afforded acids 8j–n in an operationally simple
and scalable way. Similarly, treatment of pyrrolidine 7a with
mesyl chloride or N,N-dimethylsulfamoyl chloride using DCM as
Fig. 2 X-Ray crystal structure of 9b.
1,3,4-trisubstituted pyrrolidine 6a was fully supported by the a solvent in the presence of trimethylamine afforded pyrrolidines
proton and carbon NMR. Moreover, the stereochemistry of 9a–b in good yield respectively. The X-ray crystallographic struc-
pyrrolidine 6a was initially assigned as a trans configuration ture of compound 9b, crystallized in ethyl acetate and hexane,
based on two reasons: (1) a weaker vicinal coupling constant confirmed the existence of the trans configuration of these
between protons at positions 3 and 4 of the pyrrolidine ring; (2) pyrrolidine derivatives (Fig. 2). Besides, direct amidation of a
the inherent trans configuration of alkene 5a. This result was series of acid chlorides, including 2-(2,6-difluorophenyl)acetyl
subsequently confirmed by a crystal structure of compound 9b, chloride, picolinoyl chloride, 2-chloroacetyl chloride, and methyl
indicating that the protons at positions 3 and 4 of pyrrolidine 6-(chlorocarbonyl)picolinate with pyrrolidine 7a under mild con-
ring are trans to each other (Fig. 2). It should be noted that dition delivered desired pyrrolidines 9c–f respectively in moderate
pyrrolidine 6a was obtained as a racemic mixture (Fig. S44, yield. Deprotection of the benzyl group of 9a by using Pd/C-
ESI†). Alternative reaction conditions to produce nonstabilized catalyzed hydrogenation under hydrogen atmosphere followed
N-benzylazomethine ylide in situ by using trifluoroacetic acid by methylation with iodomethane under basic medium furnished
(TFA), acetic acid, p-toluenesulfonic acid sulphuric acid or smoothly pyrrolidine 10a and 10b. Pyrrolidines 9b and 9f were
potassium fluoride resulted in a low yield of the desired product. further converted to carboxylic acid 10c and diacid 10f respectively
Under a mild reaction condition, selective N-debenzylation of by using the same reaction condition (potassium hydroxide) in
tertiary amine of pyrrolidine 6a by treating with a-chloroethyl moderate yield. Finally, the substitution of the chloride moiety of
chloroformate followed by refluxing in methanol delivered multi- 9e with thioacetic acid or bis(pyridin-2-ylmethyl)amine provided
gram quantities of the key intermediate pyrrolidine 7a for the pyrrolidines 10d and 10e in moderate yield respectively. Collec-
ensuing modification.36 Similarly, by replacing the phenol pro- tively, all compounds were easily accessible from commercially
tecting group in the second step with other groups, such as allyl, available building blocks within three to four synthetic steps,
mesyl, or acetyl groups, pyrrolidines 7b–d were furnished allowing the rapid construction of a compound library for bio-
smoothly without further purification. Having constructed the logical screening. The important parameters of ‘‘drug-likeness’’,
skeletal of pyrrolidines 7, we next targeted modifying the sec- such as clog P and topological polar surface area (tPSA) of all
ondary amine moiety of pyrrolidines 7a–d. Treatment of pyrro- synthesized compounds were calculated as a guide to prioritize
lidines 7a–d with dimethyl chlorophosphate in the presence of the chemical syntheses (Table 1).38
4-(dimethylamino)pyridine (DMAP) as a catalyst furnished pyrro-
lidines 8a–d in good yield. Moreover, pyrrolidines 7a was
2. Biological evaluation
selected to react with ethyl chloroformate using DCM as a solvent
2.1 Cell-based screen using E. coli BL21 (NDM-1) strain
at an ice bath temperature, providing carbamate 7e in good yield. identified compound 10e as a promising MRM adjuvant. With a
The methyl ester group at position 3 of carbamate 7e was sufficient amount of compounds in hand, the next stage was set
sequentially converted into carboxylic acid 7g and amide 7h for a cell-based screen for inhibitors of the NDM-1. To increase
under standard reaction conditions. Additionally, selective the screen sensitivity, a test strain with a clean background of
reduction of the methyl ester group of carbamate 7e by using MRM-resistant E. coli BL21 (NDM-1) carrying only an isopropyl
alcoholic solutions of lithium borohydride afforded alcohol 7f b-D-1-thiogalacto-pyranoside (IPTG)-inducible plasmid pET28b-
without destroying the carbamate group.37 Under the hydrogen blaNDM-1 was produced from a parental E. coli BL21 strain
atmosphere at balloon pressure, the benzyl group of carbamate without producing NDM-1.9 All antimicrobial tests were per-
7e was removed by Pd/C-catalyzed hydrogenation providing formed according to the Clinical and Laboratory Standards
pyrrolidine 7i, which was further converted to the carboxylic Institute (CLSI) approved standard guidelines.39 The minimum
acid 7j by treating with potassium hydroxide in moderate yield. inhibition concentration (MIC) of MRM towards this test strain
As depicted in Scheme 2, the same route could be repurposed of E. coli BL21 (NDM-1) was found to be 128 mg mLÀ1 (Table 1,
to allow diversified functionalization of the secondary amine entry 1), which was 1024-fold higher than the parental E. coli
moiety of pyrrolidine 7a. Amine alkylation of pyrrolidine 7a with BL21 (MIC of Mem = 0.125 mg mLÀ1). This result indicated that
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Scheme 2 Reagents and reaction conditions: (a) for 8e, t-butyl 2-bromoacetate, K2CO3, KI (cat.), DCM, 0 1C to rt, 14 h, 80%; for 8f, i-propyl
2-bromoacetate, K2CO3, KI (cat.), DCM, 0 1C to rt, 14 h, 67%; for 8h, ethyl 3-bromopropanoate, K2CO3, KI (cat.), DCM, 0 1C to rt, 14 h, 53%; for 8i,
(Æ)-3-chloropropane-1,2-diol, NEt3, DCM, 0 1C to reflux, 16 h, 55%; for 8j, 2,2-dimethylglutaric anhydride, NEt3, DCM, 0 1C to rt, 16 h, 71%; for 8k,
3,3-dimethylglutaric anhydride, NEt3, DCM, 0 1C to rt, 16 h, 63%; for 8l, maleic anhydride, NEt3, DCM, 0 1C to rt, 16 h, 59%; for 8m, succinic anhydride,
NEt3, DCM, 0 1C to rt, 16 h, 59%; for 8n, phthalic anhydride, NEt3, DCM, 0 1C to rt, 16 h, 51%; (b) TFA, DCM, 0 1C to rt, 16 h, 46%. (c) For 9a, mesyl chloride,
NEt3, DCM, 0 1C, 4 h, 63%; for 9b, N,N-dimethylsulfamoyl chloride, NEt3, DCM, 0 1C, 4 h, 54%; for 9c, 2-(2,6-difluorophenyl)acetyl chloride, NEt3, DCM,
rt, 4 h, 34%; for 9d, picolinoyl chloride, NEt3, DCM, 0 1C to rt, 12 h, 18%; for 9e, 2-chloroacetyl chloride, NEt3, DCM, 0 1C to rt, 12 h, 50%; for 9f, methyl
6-(chlorocarbonyl)picolinate, NEt3, DCM, 0 1C to rt, 12 h, 41%; (d) KOH, H2O, MeOH, reflux, 6 h, for 10c, 39%; For 10f, 63%; (e) 9a, 10% Pd/C, H2, THF, rt,
16 h, 51%; (f) MeI, K2CO3, acetone, reflux, 16 h, 71%; (g) for 10d, thioacetic acid, NEt3, DCM, 0 1C to rt, 16 h, 71%; for 10e, bis(pyridin-2-ylmethyl)amine,
K2CO3, acetone, 0 1C to reflux, 16 h, 33%.
the test strain was capable of overexpressing the NDM-1 enzyme against the growth of the test strain even at a concentration of
and suitable for the cell-based screen of NDM-1 inhibitors. To 128 mg mLÀ1, implying that these compounds are relatively non-
assess the off-target effect of these compounds, the antimicrobial toxic and well-tolerated to bacterial cells. Moreover, at a concen-
activities of all compounds were tested individually by evaluating tration below 128 mg mLÀ1, this class of compounds is very
their MICs towards this test strain. The summarized results are unlikely to undergo non-specific interaction with drug targets that
presented in Table 1 (see Table S1 for all compounds, ESI†). may induce cellular toxicity, which is the prerequisite of being a
Generally, all compounds exhibited very weak inhibitory activities safe antibiotic adjuvant.40
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Table 1 MIC screening of compound alone and MRM in the presence of the compound at 100 mM towards E. coli BL21 (NDM-1), calculated c log P,
topological polar surface area (tPSA), and reduction fold (RF)
MIC (mg mLÀ1
Cpd
)
Entry
Cpd no.
MRM
10e
R1
R2
R3
c log Pa
N.A.
tPSAa
N.A.
MRMb
RFc
N.A.
256
32
1
2
3
4
N.A.d
N.A.
N.A.
OBn
N.A.
OBn
128
N.A.
0.5
4
CO2Me
N.A.
3.68
93.03
65.26
102.37
4128
11
N.A.
0.53
4128
8k
CO2Me
3.80
4128
8
16
5
6
9d
9c
CO2Me
CO2Me
OBn
OBn
2.97
4.82
77.43
65.07
4128
4128
8
8
16
16
7
8
8l
CO2Me
CO2Me
OBn
OBn
3.15
3.04
102.37
65.07
4128
4128
16
16
8
8
9e
9
8e
8f
CO2Me
CO2Me
CO2H
OBn
OBn
OBn
4.79
4.39
2.37
74.30
74.30
96.38
4128
4128
4128
32
32
32
4
4
4
10
11
9b
12
13
7j
CO2H
OH
1.52
0.68
96.30
4128
4128
32
32
4
4
8d
CO2Me
OAc
100.60
14
10f
CO2H
OBn
2.76
125.73
4128
32
4
15
16
17
8i
CO2Me
CO2Me
CO2Me
OBn
OBn
OBn
2.61
3.31
2.89
88.46
56.79
82.14
4128
4128
4128
32
64
64
4
2
2
7a
9a
18
19
20
21
22
23
24
10a
10b
7e
CO2Me
CO2Me
CO2Me
CO2Me
CO2H
OH
0.64
1.12
4.23
1.24
3.76
4.36
3.66
93.14
82.14
74.30
85.30
85.30
74.30
68.23
4128
4128
4128
4128
4128
4128
4128
64
64
64
64
64
64
64
2
2
2
2
2
2
2
OMe
OBn
OBn
OBn
OBn
OBn
8g
7g
8h
7f
CO2Me
CH2OH
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Table 1 (continued)
MIC (mg mLÀ1
)
Entry
25
Cpd no.
R1
R2
R3
c log Pa
tPSAa
91.09
Cpd
MRMb
RFc
2
7h
CONH2
OBn
3.05
4128
4128
64
26
8j
CO2Me
OBn
3.71
102.37
64
2
27
28
8a
6c
CO2Me
CO2Me
OBn
OMs
3.01
2.83
83.53
82.14
4128
4128
64
64
2
2
29
30
6b
8b
CO2Me
CO2Me
OAllyl
OAllyl
4.25
2.02
48.00
83.53
4128
4128
64
64
2
2
31
8n
CO2Me
OBn
4.07
102.37
4128
64
2
a
b
Compound’s c log P and tPSA values were calculated using the ChemDraw Ultra (version 12.0). MIC value of MRM in the presence of a
c
compound at 100 mM. Reduction fold (RF) was calculated by MIC of MRM alone divided by MIC of MRM in the presence of 100 mM of the test
compound. N.A.: not applicable; N = 1–3 independent experiments.
d
To examine the synergistic activities of these compounds in 4 of the pyrrolidine respectively. Removal of the R1 group of
combination with MRM, the MICs of MRM were systematically compound 10e (compound 7a) dramatically reduced the RF from
evaluated in the presence of compounds at a fixed concen- 256 to 2 (Table 1, entry 16), suggesting that the 2-(bis(pyridin-2-
tration of 100 mM. The reduction folds (RF), which were defined ylmethyl)-amino)acetyl group plays a pivotal role for the potent
as the ratio of MIC of MRM along to MIC of MRM in the activity. Interestingly, 2-(bis(pyridin-2-ylmethyl)-amino)acetic acid
presence of compounds at 100 mM, were employed to compare (11) (Table 1) has been reported to be a zinc chelator, forming a
the compound’s synergistic activity directly. The compound stable zinc–ligand complex via the carboxylic acid group and three
showing a larger RF value has a more prominent effect on nitrogen atoms.41 Therefore, it was synthesized and tested, demon-
reversing the antimicrobial activity of MRM. The screen results are strating a potent synergistic activity with an RF value of 32.
summarized in Table 1, in which only MICs of MRM r64 mg mLÀ1 However, replacements of the R1 group of compound 10e with
are shown. MICs of MRM Z64 mg mLÀ1 are shown in the ESI† other zinc-chelating moieties, such as picolinoyl group (Table 1,
(Table S1). In general, all compounds exhibited relatively weak entry 5) and 6-(hydroxycarbonyl)-picolinoyl group (Table 1, entry
synergistic activity with RF ranged from 2 to 16 (Table 1, entries 14) weakened the synergistic activity. Modification of the R2 and R3
4–31), except compound 10e. We reasoned that may due to several groups also resulted in no improvement in the synergistic activity.
reasons: (1) the weak interactions between the dinuclear zinc Taken together, compound 10e demonstrated the most promising
center of NDM-1 and the R1 groups; (2) the intrinsic low perme- RF value. We reasoned that compound 10e may inhibit NDM-1
ability of compound itself to pass through the cell membrane of activity by acting as a zinc chelator just like compound 11.
E. coli; (3) the active membrane efflux pumps presented in the Compound 10e was, therefore, selected for detailed mechanistic
E. coli. More experiments are required to test these hypotheses. characterization.
Encouragingly, compound 10e was found to display the most
2.2 Compound 10e exhibited low cytotoxicity and moderate
promising synergistic activity with an RF value of 256 (Table 1, inhibition of NDM-1 enzyme via zinc depletion mechanism. The
entry 2). In the presence of 100 mM 10e, the MIC of MRM next question we need to answer is whether the observed
dramatically reduced from 128 mg mLÀ1 to 0.5 mg mLÀ1. Structu- synergism of 10e and MRM combination is due to the inhibition
rally, this compound possesses not only optimum drug-likeness of NDM-1. To address this question, we sought to conduct a
properties (clog P = 3.68, tPSA = 93.03) but also important warheads series of standard biochemical assays using the purified NDM-1
of a 2-(bis(pyridin-2-ylmethyl)-amino)acetyl group (R1), a methyl enzyme and colorimetric b-lactamase substrate nitrocefin as
ester group, and a phenyl benzyl ether group at positions 1, 3 and previously described.9 Enzyme inhibition assay revealed that
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compound 10e inhibited moderately the NDM-1 activity with a a ratio of 1 : 10 led to an obvious mass shift to a peak showing the
half-maximal inhibitory concentration (IC50) of 51 Æ 7 mM with a molecular weight of 25 920 Da (Fig. 3d). The delta mass (132 Da)
calculated Ki of 4.6 Æ 0.8 mM (Fig. 3a, blue line). As mentioned was approximate to the loss of two zinc ions, suggesting that
before, compound 10e may inhibit NDM-1 activity by acting as a compound 10e inhibited NDM-1 activity by complete removal of
zinc chelator. Next, we sought to test whether the compound both zinc ion in the active site. On the other hand, a well-known
could inhibit NDM-1 activity at a higher concentration of zinc zinc chelator, ethylenediaminetetracetic acid (EDTA), has been
content. The enzyme inhibition assay was then conducted in the reported to inhibit NDM-1 activity with an IC50 of 0.4 mM via zinc
presence of 50 mM zinc sulfate. Surprisingly, compared with the depletion mechanism.42,43 Incubating the NDM-1 enzyme with
results without adding zinc sulfate, the calculated IC50 and Ki EDTA also led to a similar mass shift to a peak with the
values of compound 10e were increased by two-fold to 103 Æ 12 mM molecular weight of 25919 Da (Fig. 3e). Collectively, these data
and 9.4 Æ 1.1 mM, respectively (Fig. 3a, red line), resulting in confirmed that compound 10e could extract two zinc ions from
stronger enzyme activity and weaker NDM-1 inhibition of com- the NDM-1 enzyme and therefore inhibit its activity. One of the
pound 10e. These results suggested that compound 10e may potential problems usually associated with zinc chelators is their
form a stable zinc-compound 10e complex probably via the non-specific interactions with other metalloproteins, causing
2-(bis(pyridin-2-ylmethyl)-amino)acetyl group at high zinc ion relatively high toxicity towards eukaryotic cells. To confirm the
concentration, resulting in a weaker NDM-1 inhibition. To in vitro cytotoxicity of compound 10e to normal cells, MTT
further investigate the mechanism of the interaction between (thiazolyl blue tetrazolium bromide) assay was employed
compound 10e and NDM-1 enzyme, nano ESI-MS analysis was next.44 HEK293 (human embryonic kidney) cells viability was
conducted (Fig. 3c–e). The molecular weight of the native NDM-1 assessed in the presence of various concentrations of compound
enzyme was found to be approximately 26052 Da (Fig. 3c). 10e. As shown in Fig. 3b, compound 10e exhibited relatively low
Incubating the NDM-1 enzyme with excessive compound 10e at cytotoxicity with 80% of HEK293 cells survived in the presence of
Fig. 3 (a) Inhibition of NDM-1 enzyme by 10e in the normal buffer (blue line) or with the addition of 50 mM ZnSO4 solution (red line); (b) cytotoxicity of
10e towards HEK cell lines; nano-ESI-MS analysis of native NDM-1 (c), native NDM-1 treated with 10e (d), native NDM-1 treated with ETDA (e) and their
cartoon representations (f–h). Grey balls represent the zinc ions.
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compound 10e at 128 mM after 24 hours treatment, indicating both enantiomers may bind to the NDM-1 enzyme with similar
that compound 10e is relatively non-toxic and well-tolerated to binding affinity.
eukaryotic cells. Moreover, this concentration is much higher
than the effective concentration in the combination study.
2.4 Combination of compound 10e and MRM against
clinically isolated NDM-1 positive CRE. Based on the promising
2.3 Docking study of both enantiomers of compound 10e. results of 10e and MRM combination towards E. coli BL21
To gain more insights into the molecular interaction of com- (NDM-1) strain, we sought to examine whether the NDM-1-
pound 10e with NDM-1 enzyme, computational docking studies mediated resistance to MRM in the screening strain could also
of both enantiomers of compound 10e using previously be reproduced in our in-house collection of clinically isolated
reported crystal structure NDM-1 (PDB ID: 4EXS) was conducted NDM-1-producing CRE strains, including three E. coli, one K.
(Fig. 4).45 The results of docking studies revealed that the highest oxytoca, one C. freundii, one E. cloacae, and two M. morganii
docking score positioned both enantiomers of compound 10e strains. These CRE strains were highly MRM-resistant owing to
into the substrate-binding site of NDM-1. The warheads of the overexpression of the NDM-1 enzyme with half of the CRE
2-(bis(pyridin-2-ylmethyl)-amino)acetyl group of both enantio- strains exhibiting the MICs of MRM Z128 mg mLÀ1 (Table 2,
mers were well-situated in the active site of NDM-1, interacting entries 3–6). In addition to producing NDM-1, four of these strains
with the dinuclear zinc center. For 3R,4S-10e enantiomer, a also co-expressed other b-lactamases such as CTX-M-3, CTX-M-14,
conventional hydrogen-bonding interaction was predicted to TEM-1, and SHV-12. As illustrated in Table 2, compound 10e
occur between the amide group of His122 and the ester group exhibited no antibacterial activity itself (MICs Z 128 mg mLÀ1) but
of 10e (Fig. 4a). For 3S,4R-10e enantiomer, two hydrogen- a strong synergy with the combination of MRM across this panel
bonding interactions were predicted to occur between (1) the of clinical isolates with fractional inhibitory concentration index
amine group of Lys211 and the ester group of 10e; (2) the amide (FICI) ranging from 0.01 to 0.25. Compared with the positive
group of His122 and pyridinyl group of 10e (Fig. 4b). Several control Eb, compound 10e exhibited much stronger synergy,
important amino acid residues of NDM-1 enzyme were predicted meriting further development as carbapenem antibiotic adjuvant.
to be closely adjacent to both enantiomers of 10e, such as
The worldwide dissemination of NDM-1 positive CRE has
Glu152, Met194, Asp223, Gly222, Ala224, Leu221, His189, become a major threat to human health. However, there are
Asn220, Gly219, Leu218, Lys211, His250, Met67, Pro68, Leu65, currently no FDA-approved NDM-1 inhibitors available in clinical
Trp93, Asp124, Gln123, His122, His120, implying that these use. Therefore, it is very important to keep on the development
amino acids may also be involved in the van der Waals inter- of new compounds to meet clinical needs. In this study, we have
action with compound 10e. Importantly, the top-ranked scores of modified the structure of well-known NDM-1 inhibitor captopril
both enantiomers of compound 10e are À79, suggesting that by synthesizing a compound library of trisubstituted pyrrolidines
Fig. 4 Models of compound 10e ((a) 3R,4S-10e, (b) 3S,4R-10e) docked into the substrate-binding site of NDM-1 using the X-ray crystal structure of
NDM-1 (PDB ID: 4EXS) with labeled important amino acid residues. Grey balls and blue dotted lines represent zinc ion and hydrogen-bonding interaction
respectively.
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Table 2 MIC (mg mLÀ1) screening of compound 10e and Eb in combination with MRM against clinically isolated NDM-1 positive CRE strainsa
MIC (mg mLÀ1
)
MIC (mg mLÀ1
)
Entry
CRE strains
Additional b-lactamase determinants
MRM
10e
10e + MRM
FICI
Eb
Eb + MRM
FICI
1
2
3
4
5
6
7
8
EC4-1
EC36-2
EC06
KO03
CF05
EL22
MM23
MM26
None
None
b1, b2, b4
b4
64
32
Z128
Z128
Z128
Z128
64
Z128
Z128
Z128
Z128
Z128
Z128
Z128
Z128
1
0.25
16
4
8
2
0.02
0.01
0.25
0.06
0.13
0.03
0.09
0.19
—
—
—
—
Z128
32
—
—
Z128
Z128
Z128
Z128
Z128
Z128
2.00
0.50
0.25
2.00
1.50
1.50
b4
16
b2, b3, b4
None
None
Z128
64
4
8
64
64
a
EC, Escherichia coli; KO, Klebsiella oxytoca; CF, Citrobacter freundii; EL, Enterobacter cloacae; MM, Morganella morganii. Additional b-lactamase
determinants: b1: CTX-M-3, b2: CTX-M-14, b3: TEM-1, and b4: SHV-12. The synergistic effect is depicted by the FICI, which is calculated as FIC
(cpd) + FIC (MRM), where FIC (cpd) is the (MIC of cpd in combination with MRM)/(MIC of cpd alone) while FIC (MRM) is (MIC of cpd in
combination with MRM)/(MIC of MRM alone). If the MIC value is Z128, then 128 was used for calculating the FICI. The drug combination is
considered synergistic if the FICI r 0.5; —: not tested; N = 1–3 independent experiments.
without bearing thiol groups. These newly developed com- scaffold targeting NDM-1, providing a promising starting point to
pounds are potential inhibitors neutralizing the activity of be further developed as carbapenem antibiotic adjuvants.
NDM-1 metallo-b-lactamase, particularly compound 10e which
demonstrates the ability to restore the activity of meropenem
against clinically isolated NDM-1 positive CRE. The advantage of
Experimental section
these compounds avoids the adverse effects associated with
thiol-containing compounds. Moreover, the scaffold of com-
pound 10e provides a valuable template for deeper exploration
in structural diversity and could be a potential source of new,
more drug-like NDM-1 inhibitors. It is likely that the structural
exploration of other similar classes of pyrrolidine derivatives
without the hydrophobic phenyl rings, especially those capable
of metal coordination, may lead to more active NDM-1 inhibitors
with the improved physiochemical property.
General
All commercially available starting materials, reagents, and
solvents, unless otherwise stated, were used as received without
further purification. All reactions were monitored by using the
thin-layer chromatography (TLC) (silica gel 60-F254, E. Merck) and
visualized under short and long UV light. Flash column chroma-
tography was carried out on the silica-gel 60 (200–300 mesh).
1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were
measured at room temperature on a Bruker Advance-III spectro-
meter (400 MHz FT-NMR System) with tetramethylsilane as an
internal standard. Chemical shifts are expressed in d (ppm) and
coupling constants (J) in Hz. Low-resolution (LRMS) and high-
Conclusion
In this study, based on the rational design on the captopril resolution mass spectra (HRMS) were obtained on a Micromass
scaffold, a total of 36 trans-1,3,4-trisubstituted pyrrolidine deriva- Q-TOF-2 by electron spray ionization (ESI) mode.
tives without bearing thiol group have been synthesized via boric
Synthesis of methyl ferulate (4b). Ferulic acid (4a) (5.0 g,
acid-catalyzed 1,3-dipolar cycloaddition of N-benzylazomethine 26 mmol) was dissolved in MeOH (100 mL) in the presence of
ylide with methyl ferulate for biological evaluation as MRM two drops of concentrated sulfuric acid as a catalyst. The
adjuvant targeting NDM-1. Through a cell-based screen, com- solution was heated under reflux for 6 hours. The reaction
pound 10e bearing a zinc-chelating moiety of 2-(bis(pyridin-2- mixture was cooled to room temperature, concentrated under
ylmethyl)-amino)acetyl group at R1 was found to exhibit low reduced pressure, and redissolved in ethyl acetate (EA) (80 mL).
cytotoxicity (IC50 4 128 mM), moderate NDM-1 enzyme inhibition The reaction mixture was washed with water (200 mL Â 3). The
(IC50 = 51 mM), and potent synergistic activity against a panel of organic portion was dried over anhydrous MgSO4, filtered and
clinically isolated NDM-1 positive CRE with FICI ranged from 0.01 concentrated under reduced pressure to afford the desired
1
to 0.25. A series of biochemical assays further confirmed that product as a pale yellow oil (3.3 g, 62%). H NMR (400 MHz,
compound 10e inhibits NDM-1 activity via displacing two zinc CDCl3) d 7.63 (d, J = 16.6 Hz, 1H), 7.07 (d, J = 7.8 Hz, 1H), 7.02
ions from NDM-1, which was similar to EDTA. Altogether, our (s, 1H), 6.92 (d, J = 7.8 Hz, 1H), 6.30 (d, J = 15.7 Hz, 1H), 3.91
studies indicate that 10e represents an important pyrrolidine-type (s, 3H), 3.80 (s, 3H); 13C NMR (101 MHz, CDCl3) d 167.9, 148.2,
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147.0, 145.1, 126.8, 123.0, 115.0, 114.9, 109.7, 55.9, 51.6; LRMS desired product (0.65 g, 54%). 1H NMR (400 MHz, CDCl3) d 7.61
(ESI) m/z C11H13O4 (M + H+) 209.2.
(d, J = 15.65 Hz, 1H), 7.04–7.11 (m, 2H), 6.97–7.04 (m, 1H), 6.36
Synthesis of (E)-methyl 3-(4-(benzyloxy)-3-methoxyphenyl)acrylate (d, J = 15.65 Hz, 1H), 3.79 (s, 3H), 3.77 (s, 3H), 2.28 (s, 3H); 13C
(5a). Compound 4b (3.3 g, 16 mmol) was dissolved in acetone NMR (101 MHz, CDCl3) d 168.7, 167.2, 151.4, 144.1, 141.4, 133.3,
and subsequently mixed with benzyl bromide (4.1 g, 24 mmol) 123.2, 121.1, 118.0, 111.2, 55.8, 51.7, 20.6; LRMS (ESI) m/z
and potassium carbonate (2.2 g, 16 mmol). The resulting mixture C13H15O5 (M + H+) 251.1.
was heated under reflux for 3 hours. Excess triethylamine (2.4 g,
Synthesis of (Æ)-trans-methyl 1-benzyl-4-(4-(benzyloxy)-3-
24 mmol) was added to the reaction mixture and heated under methoxyphenyl)-pyrrolidine-3-carboxylate (6a). Compound 5a (3.8 g,
reflux for 12 hours. A white solid was precipitated. The crude 13 mmol) was mixed with N-(methoxymethyl)-N-(trimethylsilyl-
reaction mixture was cooled to room temperature and subse- methyl)benzylamine (4.5 g, 19 mmol) in the presence of boric
quently filtered, concentrated under reduced pressure and redis- acid (50 mg) as a catalyst in DCM (60 mL). The solution mixture
solved in EA (80 mL). The crude reaction mixture was washed was stirred at room temperature for 48 hours. The crude reaction
with water (5 mL HCl blended, 200 mL Â 3). The organic portion mixture was purified by using the flash column chromatography
was dried with anhydrous MgSO4, filtered, and concentrated on silica gel with isocratic elution (EA/Hex, 7%) to afford desired
under reduced pressure to afford the desired product as a white product as a pale yellow oil (2.9 g, 53%). 1H NMR (400 MHz,
1
powder (3.8 g, 80%). H NMR (400 MHz, CDCl3) d 7.64 (d, J = CDCl3) d 7.43–7.54 (m, 2H), 7.32–7.43 (m, 7H), 7.27–7.32 (m, 1H),
15.65 Hz, 1H), 7.43–7.49 (m, 2H), 7.40 (t, J = 7.34 Hz, 2H), 7.34 6.94–7.03 (m, 1H), 6.78–6.89 (m, 2H), 5.16 (s, 2H), 3.93 (s, 3H),
(d, J = 6.85 Hz, 1H), 7.02–7.13 (m, 2H), 6.89 (d, J = 8.80 Hz, 1H), 3.61–3.80 (m, 6H), 3.08–3.18 (m, 2H), 2.98 (t, J = 8.80 Hz, 1H), 2.84–
6.33 (d, J = 15.65 Hz, 1H), 5.21 (s, 2H), 3.94 (s, 3H), 3.82 (s, 3H); 2.90 (m, 1H), 2.80 (dd, J = 5.87, 9.29 Hz, 1H); 13C NMR (101 MHz,
13C NMR (101 MHz, CDCl3) d 167.7, 150.3, 149.8, 144.8, 136.6, CDCl3) d 174.7, 149.7, 146.9, 138.9, 137.8, 137.4, 128.7, 128.6,
128.7, 128.0, 127.7, 127.2, 122.4, 115.6, 113.4, 110.2, 70.9, 56.0, 128.3, 127.8, 127.3, 127.1, 119.3, 114.0, 111.2, 71.2, 61.7, 59.9, 57.5,
51.6; LRMS (ESI) m/z C18H19O4 (M + H+) 299.2.
56.0, 52.0, 51.8, 46.6; LRMS (ESI) m/z C27H30NO4 (M + H+) 433.1.
Synthesis of (Æ)-trans-methyl 4-(4-(allyloxy)-3-methoxyphenyl)-
Synthesis of (E)-methyl 3-(4-(allyloxy)-3-methoxyphenyl)acrylate
(5b). This compound (0.8 g, 67%) was prepared from compound 1-benzylpyrrolidine-3-carboxylate (6b). This compound (2.9 g,
4b (1.0 g, 4.8 mmol), allyl bromide (0.7 g, 5.8 mmol), acetone 50%) was prepared from compound 5b (3.8 g, 15 mmol),
(20 mL) and potassium carbonate (1.0 g, 7.2 mmol) according to N-(methoxymethyl)-N-(trimethylsilylmethyl)benzylamine (4.5 g,
the same preparation procedure of compound 5a described above. 19 mmol), and boric acid (50 mg) in DCM (25 mL) according
1H NMR (400 MHz, CDCl3) d 7.61 (d, J = 16.63 Hz, 1H), 6.99–7.10 to the same procedure of compound 6a described above.
(m, 2H), 6.84 (d, J = 7.82 Hz, 1H), 6.30 (d, J = 15.65 Hz, 1H), 5.96– 1H NMR (400 MHz, CDCl3) d 7.39–7.47 (m, 2H), 7.34 (t, J = 7.34
6.14 (m, 1H), 5.33–5.46 (m, 1H), 5.29 (d, J = 8.80 Hz, 1H), 4.62 (d, Hz, 2H), 7.25–7.31 (m, 1H), 6.96 (d, J = 1.96 Hz, 1H), 6.78–6.88
J = 4.89 Hz, 2H), 3.88 (s, 3H), 3.78 (s, 3H); 13C NMR (101 MHz, (m, 2H), 6.03–6.16 (m, 1H), 5.35–5.48 (m, 1H), 5.24–5.33 (m, 1H),
CDCl3) d 167.6, 150.1, 149.5, 144.7, 132.8, 127.5, 122.4, 118.3, 4.60 (d, J = 4.89 Hz, 2H), 3.90 (s, 3H), 3.73–3.81 (m, 1H), 3.67–3.73
115.5, 112.8, 110.0, 69.7, 55.9, 51.6; LRMS (ESI) m/z C14H17O4 (M + (m, 4H), 3.61–3.67 (m, 1H), 3.08–3.20 (m, 2H), 2.99 (t, J = 8.80 Hz,
H+) 249.1.
1H), 2.76–2.92 (m, 2H); 13C NMR (101 MHz, CDCl3) d 174.7,
Synthesis of (E)-methyl 3-(3-methoxy-4-((methylsulfonyl)oxy)- 149.5, 146.7, 138.9, 137.6, 133.6, 128.7, 128.3, 127.1, 119.2, 117.8,
phenyl)acrylate (5c). Compound 4b (1.0 g, 4.8 mmol) was dis- 113.4, 111.0, 70.0, 61.7, 59.9, 57.5, 55.9, 52.0, 51.9, 46.6; HRMS
solved in DCM (20 mL) and subsequently mixed with mesyl m/z calcd for C23H28NO4 (M + H)+ 382.1940, found 382.1945.
chloride (0.7 g, 5.8 mmol) and triethylamine (5 mL) in an ice bath
Synthesis of (Æ)-trans-methyl 1-benzyl-4-(3-methoxy-4-((methyl-
for 4 hours. The crude reaction mixture was warmed to room sulfonyl)oxy)phenyl)pyrrolidine-3-carboxylate (6c). This compound
temperature and subsequently washed with water (50 mL Â 2). (3.1 g, 57%) was prepared from compound 5c (3.7 g, 13 mmol),
The organic portion was dried over anhydrous MgSO4, filtered and N-(methoxymethyl)-N-(trimethylsilylmethyl)benzylamine (4.5 g,
concentrated under reduced pressure to furnish a crude mixture, 19 mmol), and boric acid (50 mg) in DCM (25 mL) according to
1
which was subjected to purification by using the flash column the same procedure of compound 6a described above. H NMR
chromatography on silica gel with isocratic elution (EA/Hex, 10%) (400 MHz, CDCl3) d 7.25–7.45 (m, 5H), 7.18–7.24 (m, 1H), 7.09
1
to afford the desired product (0.9 g, 66%). H NMR (400 MHz, (s, 1H), 6.93 (dd, J = 1.96, 8.80 Hz, 1H), 3.90 (s, 3H), 3.75–3.83 (m,
CDCl3) d 7.59 (d, J = 15.65 Hz, 1H), 7.26 (d, J = 7.83 Hz, 1H), 7.06– 1H), 3.63–3.73 (m, 5H), 3.09–3.29 (m, 5H), 2.92–3.03 (m, 1H), 2.78–
7.14 (m, 2H), 6.38 (d, J = 15.65 Hz, 1H), 3.88 (s, 3H), 3.77 (s, 3H), 2.92 (m, 2H); 13C NMR (101 MHz, CDCl3) d 174.0, 151.4, 136.9,
3.18 (s, 3H); 13C NMR (101 MHz, CDCl3) d 167.0, 151.7, 143.4, 128.8, 128.4, 127.4, 124.3, 119.9, 112.1, 61.1, 59.7, 57.3, 56.0, 52.1,
139.5, 134.6, 124.8, 121.1, 119.0, 111.9, 56.1, 51.8, 38.5, 31.6; LRMS 51.7, 46.5, 38.2; HRMS m/z calcd for C21H26NO6S (M + H)+
(ESI) m/z C12H15O6S (M + H+) 287.1.
420.1481, found 420.1473.
Synthesis of (E)-methyl 3-(4-acetoxy-3-methoxyphenyl)acrylate
Synthesis of (Æ)-trans-methyl 4-(4-acetoxy-3-methoxyphenyl)-
(5d). To a well-stirred solution of compound 4b (1.0 g, 4.8 mmol) 1-benzylpyrrolidine-3-carboxylate (6d). This compound (0.40 g,
in pyridine (10 mL) was added acetic anhydride (0.6 g, 5.8 mmol) 40%) was prepared from compound 5d (0.65 g, 2.6 mmol),
and stirred at room temperature for 3 hours. The crude reaction N-(methoxymethyl)-N-(trimethylsilylmethyl)benzylamine (0.81 g,
mixture was purified by using the flash column chromatography 3.4 mmol), and boric acid (50 mg) in DCM (25 mL) according to
on silica gel with isocratic elution (EA/Hex, 10%) to afford the the same procedure of compound 6a described above. 1H NMR
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(400 MHz, CDCl3) d 7.31–7.44 (m, 4H), 7.25–7.31 (m, 1H), 7.04 desired compound as a reddish oil (0.33 g, 73%). 1H NMR
(s, 1H), 6.89–7.00 (m, 2H), 3.86 (s, 3H), 3.59–3.81 (m, 6H), 3.08– (400 MHz, CDCl3) d 6.59–6.75 (m, 3H), 5.85–6.02 (m, 1H), 5.19–
3.23 (m, 2H), 2.91–3.01 (m, 1H), 2.78–2.89 (m, 2H), 2.33 (s, 3H); 5.32 (m, 1H), 5.09–5.18 (m, 1H), 4.45 (d, J = 4.89 Hz, 2H), 3.69–
13C NMR (101 MHz, CDCl3) d 174.6, 169.2, 151.0, 143.7, 138.8, 3.78 (m, 3H), 3.62 (s, 3H), 3.58–3.61 (m, 3H), 3.54–3.58 (m, 2H),
138.2, 128.7, 128.4, 127.1, 122.6, 119.5, 111.6, 61.6, 59.8, 57.5, 55.8, 3.52 (s, 3H), 3.46–3.51 (m, 1H), 3.32–3.43 (m, 1H), 3.12–3.21
52.0, 51.8, 46.6, 20.7; HRMS m/z calcd for C22H26NO5 (M + H)+ (m, 1H), 2.97–3.08 (m, 1H); HRMS m/z calcd for C18H27NO7P
384.1733, found 384.1742.
(M + H)+ 400.1525, found 400.1524.
Synthesis of (Æ)-trans-methyl 4-(4-(benzyloxy)-3-methoxyphenyl)-
Synthesis of (Æ)-trans-methyl 1-(dimethoxyphosphoryl)-4-(3-
pyrrolidine-3-carboxylate (7a). Compound 6a (1.0 g, 2.3 mmol) methoxy-4-((methylsulfonyl)oxy)phenyl)pyrrolidine-3-carboxylate
was dissolved in DCM and mixed with 1-chloroethyl chlorofor- (8c). Compound 7c (1.2 g, 49%) was prepared from 6c (3.1 g,
mate (0.5 g, 3.5 mmol). The solution was heated under reflux for 7.4 mmol) and a-chloroethyl chloroformate (1.6 g, 11 mmol)
3 hours. After that, the solution was dried under high vacuum. according to the same preparation procedure of compound 7a
The crude product was redissolved in MeOH and refluxed for described above. A mixture of compound 7c (0.31 g, 1.0 mmol),
3 hours. The mixture was purified by using the flash column dimethyl chlorophosphate (0.22 g, 1.5 mmol) and excess triethy-
chromatography on silica gel with gradient elution (MeOH/DCM, lamine (0.30 g, 3 mmol) in dry DCM (25 mL) was stirred at 0 1C
3% to 10%) to afford desired product as a brown oil (0.58 g, for 30 minutes in the presence of catalytic amount of DMAP. The
73%). 1H NMR (400 MHz, CDCl3) d 7.39–7.45 (m, 2H) 7.34 (s, 2H) solution was allowed to warm to room temperature and stir for
7.28 (s, 1H) 6.78–6.85 (m, 2H) 6.72 (d, J = 6.6 Hz, 1H) 5.10 (s, 2H) 14 h. The organic solvent was evaporated to dryness to give a crude
3.87 (s, 3H) 3.64 (s, 3H) 3.48 (br. s, 2H) 3.39 (d, J = 11.5 Hz, 1H) mixture. Subsequent purification of the residue was performed by
3.32 (br. s, 1H); 13C NMR (101 MHz, CDCl3) d 174.9, 149.8, 147.2, using the flash column chromatography on silica gel with gradient
137.2, 134.5, 128.5, 127.8, 127.3, 119.2, 114.3, 111.4, 71.1, 56.1, elution (EA/Hex, 10% to 30%) to afford the desired compound as a
1
55.3, 52.3, 52.0, 51.4, 49.9. HRMS (ESI) calcd for C20H24NO4 (M + H+) reddish oil (0.31 g, 75%). H NMR (400 MHz, CDCl3) d 7.10 (d,
342.1700, found 342.1715.
J = 8.80 Hz, 1H), 6.82 (d, J = 1.96 Hz, 1H), 6.71–6.79 (m, 1H), 3.72–
Synthesis of (Æ)-trans-methyl 4-(4-(benzyloxy)-3-methoxyphenyl)- 3.84 (m, 3H), 3.64 (s, 3H), 3.61 (s, 3H), 3.58 (d, J = 7.83 Hz, 2H),
1-(dimethoxyphosphoryl)pyrrolidine-3-carboxylate (8a). A mixture 3.55 (s, 3H), 3.35–3.44 (m, 1H), 3.15–3.23 (m, 1H), 3.03–3.10
of compound 7a (0.33 g, 1.0 mmol), dimethyl chlorophosphate (m, 4H); 13C NMR (101 MHz, CDCl3) d 174.0, 151.4, 136.9, 128.8,
(0.22 g, 1.5 mmol) and excess triethylamine (0.30 g, 3.0 mmol) in 128.4, 127.4, 124.3, 119.9, 112.1, 77.5, 77.1, 76.8, 61.1, 59.7, 57.3, 56.0,
dry DCM (25 mL) was stirred at 0 1C for 30 min with a little 52.1, 51.7, 46.5, 38.2; HRMS m/z calcd for C16H25NO9PS (M + H)+
amount of DMAP as the catalyst. The solution was allowed to 438.0988, found 438.0982.
warm to room temperature and stir for 14 h. The organic solvent
Synthesis of (Æ)-trans-methyl 4-(4-acetoxy-3-methoxyphenyl)-
was evaporated to dryness to give a crude residue. Subsequent 1-(dimethoxyphosphoryl)pyrrolidine-3-carboxylate (8d). Com-
purification of the residue was performed by using flash column pound 7d (0.40 g, 65%) was prepared from 6d (0.80 g, 2.1 mmol)
chromatography on silica gel with gradient elution (EA/Hex, 10% and a-chloroethyl chloroformate (0.45 g, 3.1 mmol) according to
to 30%) to afford the desired compound as a reddish oil (0.33 g, the same preparation procedure of compound 7a described
76%). 1H NMR (400 MHz, CDCl3) d 7.35–7.44 (m, 2H), 7.31 (t, above. A mixture of compound 7d (0.29 g, 1.0 mmol), dimethyl
J = 7.34 Hz, 2H), 7.21–7.27 (m, 1H), 6.73–6.85 (m, 2H), 6.65–6.73 chlorophosphate (0.22 g, 1.5 mmol) and excess triethylamine
(m, 1H), 5.08 (s, 2H), 3.83 (s, 3H), 3.54–3.75 (m, 12H), 3.42–3.52 (0.30 g, 3.0 mmol) in dry DCM (25 mL) was stirred at 0 1C for
(m, 1H), 3.25 (dt, J = 2.93, 8.80 Hz, 1H), 3.02–3.16 (m, 1H); 13C 30 minutes in the presence of catalytic amount of DMAP. The
NMR (101 MHz, CDCl3) d 171.1, 150.0, 147.8, 137.0, 130.1, 128.5, solution was allowed to warm to room temperature and stir for
127.9, 127.2, 119.6, 114.2, 111.2, 71.0, 56.1, 53.2, 53.2, 52.3, 51.0, 14 h. The organic solvent was evaporated to dryness to give a
50.0, 47.5, 47.3; HRMS m/z calcd for C22H29NO7P (M + H)+ crude mixture. Subsequent purification of the mixture was
450.0124, found 450.0112.
performed by using the flash column chromatography on silica
Synthesis of (Æ)-trans-methyl 4-(4-(allyloxy)-3-methoxyphenyl)- gel with gradient elution (EA/Hex, 10% to 30%) to afford the
1-(dimethoxyphosphoryl)pyrrolidine-3-carboxylate (8b). Com- desired compound as a reddish oil (0.21 g, 53%). 1H NMR
pound 7b (1.5 g, 68%) was prepared from 6b (2.9 g, 7.6 mmol) (400 MHz, CDCl3) d 6.94 (d, J = 7.82 Hz, 1H), 6.77–6.87 (m, 2H),
and a-chloroethyl chloroformate (1.7 g, 12 mmol) according to 3.78 (s, 3H), 3.62–3.74 (m, 12H), 3.43–3.53 (m, 1H), 3.21–3.34
the same preparation procedure of compound 7a described (m, 1H), 3.13 (q, J = 7.83 Hz, 1H), 2.27 (s, 3H); 13C NMR (101 MHz,
above. A mixture of compound 7b (0.33 g, 1.0 mmol), dimethyl CDCl3) d 172.8, 168.9, 151.2, 138.9, 138.8, 122.9, 119.1, 111.6, 55.8,
chlorophosphate (0.22 g, 1.5 mmol) and excess triethylamine 53.3, 53.3, 53.2, 53.2, 53.2, 53.1, 52.1, 51.2, 51.1, 49.7, 49.7, 48.2,
(0.30 g, 3.0 mmol) in dry DCM (25 mL) was stirred at 0 1C for 48.1, 20.6; HRMS m/z calcd for C17H25NO8P (M + H)+ 402.1240,
30 minutes in the presence of catalytic amount of DMAP. The found 402.1236.
solution was allowed to warm to room temperature and stir for
Synthesis of (Æ)-trans-1-ethyl 3-methyl 4-((4-benzyloxy)-3-
14 h. The organic solvent was evaporated to dryness to give a methoxyphenyl)pyrrolidine-1,3-dicarboxylate (7e). To a mixture
crude mixture. Subsequent purification of the mixture was of compound 7a (1 g, 3 mmol) and trimethylamine (0.87 g,
performed by using the flash column chromatography on silica 8.6 mmol) in dry DCM (25 mL) was added ethyl chloroformate
gel with gradient elution (EA/Hex, 10% to 30%) to afford the (0.49 g, 4.5 mmol) at 0 1C with stirring for 4 h. Removal of the
3526 | New J. Chem., 2021, 45, 3515À3534
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organic layer under reduced pressure gave a brownish residue. 71.1, 61.3, 56.2, 52.9, 14.8; HRMS m/z calcd for C22H27N2O5 (M + H)+
Subsequent purification of the residue was performed by using 399.4654, found 399.4635.
the flash column chromatography on silica gel with gradient
Synthesis of (Æ)-trans-ethyl 3-(4-(benzyloxy)-3-methoxyphenyl)-
elution (EA/Hex, 20% to 50%) to afford the desired compound 4-(hydroxymethyl)pyrrolidine-1-carboxylate (7f). To a 50 mL round-
as a brown oil (0.90 g, 73%). 1H NMR (400 MHz, CDCl3) d 7.39– bottomed flask was added a mixture of LiCl (0.025 g, 0.6 mmol)
7.46 (m, 2H), 7.34 (t, J = 7.34 Hz, 2H), 7.23–7.30 (m, 1H), 6.82 and NaBH4 (0.023 g, 0.6 mmol) in EtOH and THF at a ratio of 5 : 3
(d, J = 8.80 Hz, 1H), 6.77 (s, 1H), 6.67–6.75 (m, 1H), 5.10 (s, 2H), (25 mL) at 0 1C followed by the addition of compound 7e (0.12 g,
4.08–4.23 (m, 2H), 3.88–3.96 (m, 1H), 3.82–3.88 (m, 4H), 3.55– 0.3 mmol). The solution was allowed to warm to room tempera-
3.67 (m, 5H), 3.35–3.52 (m, 1H), 3.05–3.23 (m, 1H), 1.26 (q, J = ture and stir for 2 h. The organic solvent was removed under
6.85 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 172.6, 154.7, 149.8, reduced pressure and the crude mixture was purified by the flash
147.5, 137.1, 128.5, 127.8, 127.2, 119.2, 114.2, 111.2, 71.0, 61.2, column chromatography on silica gel with gradient elution
56.0, 52.3, 52.1, 50.5, 49.0, 48.6, 47.4, 46.6, 14.8; HRMS m/z (MeOH/DCM, 5% to 10%) to afford the desired compound as a
calcd for C23H28NO6 (M + H)+ 414.1917, found 414.1912.
Synthesis of (Æ)-trans-4-(4-(benzyloxy)-3-methoxyphenyl)-1- (m, 5H), 6.62–6.92 (m, 3H), 5.13 (s, 2H), 4.07–4.21 (m, 2H), 3.74–
(ethoxycarbonyl)-pyrrolidine-3-carboxylic acid (7g). well- 3.97 (m, 5H), 3.62–3.72 (m, 1H), 3.47–3.59 (m, 1H), 3.27–3.46
colorless oil (0.058 g, 44%). 1H NMR (400 MHz, CDCl3) d 7.24–7.54
A
stirred mixture of compound 7e (1.0 g, 3.0 mmol) and KOH (m, 2H), 3.09 (d, J = 7.83 Hz, 1H), 2.47 (br. s, 1H), 2.31 (m, 2H),
(0.34 g, 8.6 mmol) in water (25 mL) was heated at 60 1C for 16 h 1.17–1.43 (m, 3H); 13C NMR (101 MHz, CDCl3) d 155.2, 149.9,
until the TLC showed the disappearance of starting material. 147.3, 137.2, 133.1, 128.5, 127.9, 127.3, 119.6, 114.3, 111.3, 71.2,
The resulting solution was poured into 6 M HCl ice water 62.5, 61.1, 56.1, 53.2, 49.1, 14.8; HRMS m/z calcd for C22H28NO5
(80 mL) and extracted with EA (100 mL Â 3). The organic layers (M + H)+ 386.1975, found 385.1991.
were dried over anhydrous MgSO4 and filtered. Removal of the
Synthesis of (Æ)-trans-1-ethyl 3-methyl 4-(4-hydroxy-3-
organic layers under reduced pressure gave a brownish residue methoxyphenyl)-pyrrolidine-1,3-dicarboxylate (7i). To a solution
and subsequent purification was performed by using the flash of compound 7e (0.70 g, 1.7 mmol) in dry THF (35 mL) was
column chromatography on silica gel with gradient elution (EA/ added 10% palladium on carbon (0.22 g, 2.1 mmol) at room
Hex, 30% to 50%) to afford the desired compound as a brown temperature with stirring for 16 h under hydrogen atmosphere at
oil (0.42 g, 35%). 1H NMR (400 MHz, CDCl3) d 10.22 (br. s, 1H), balloon pressure. The catalyst was filtered and washed with THF
7.23–7.53 (m, 5H), 6.78–6.94 (m, 2H), 6.74 (d, J = 7.82 Hz, 1H), (30 mL) and the filtrate was removed under reduced pressure.
5.12 (br. s, 2H), 4.17 (d, J = 5.87 Hz, 2H), 3.87 (br. s, 5H), 3.54– The residue was purified by using the flash column chromato-
3.72 (m, 2H), 3.34–3.54 (m, 1H), 3.07–3.28 (m, 1H), 1.28 (d, J = graphy on silica gel with gradient elution (EA/Hex, 20% to 50%)
6.85 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 176.1, 155.1, 149.8, to afford the desired compound as a colourless oil (0.27 g, 49%).
147.5, 137.1, 132.1, 128.5, 127.9, 127.3, 119.3, 114.2, 111.3, 71.1, 1H NMR (400 MHz, CDCl3) d 6.84 (d, J = 8.80 Hz, 1H), 6.66–6.76
61.6, 56.1, 52.4, 50.3, 49.4, 48.9, 48.5, 47.2, 46.4, 14.7; HRMS m/z (m, 2H), 5.82 (s, 1H), 4.15 (q, J = 6.85 Hz, 2H), 3.81–3.97 (m, 5H),
calcd for C22H26NO6 (M + H)+ 400.1760, found 400.1769.
3.52–3.67 (m, 5H), 3.33–3.52 (m, 1H), 3.13 (t, J = 8.31 Hz, 1H),
Synthesis of (Æ)-trans-ethyl 3-(4-(benzyloxy)-3-methoxyphenyl)- 1.17–1.33 (m, 3H); 13C NMR (101 MHz, CDCl3) d 172.6, 154.9,
4-carbamoylpyrrolidine-1-carboxylate (7h). To a 50 mL round- 146.8, 145.0, 130.7, 130.7, 119.8, 114.7, 110.0, 61.3, 55.9, 52.4,
bottomed flask was added a mixture of compound 7g (0.36 g, 52.4, 52.1, 50.6, 49.7, 49.0, 48.7, 47.5, 46.8, 14.7; HRMS m/z calcd
0.9 mmol) and excess triethylamine (0.25 g, 2.5 mmol) in THF for C16H22NO6 (M + H)+ 324.1447, found 324.1440.
(25 mL) at 0 1C followed by the addition of ethyl chloroformate
Synthesis of (Æ)-trans-1-(ethoxycarbonyl)-4-(4-hydroxy-3-
(0.13 g, 1.2 mmol). The solution was allowed to warm to room methoxyphenyl)pyrrolidine-3-carboxylic acid (7j). A well-stirred
temperature and stir overnight. The organic solvent was removed mixture of compound 7i (0.26 g, 0.8 mmol) and KOH (0.08 g,
under reduced pressure to yield the desired intermediate with 2 mmol) in a mixture of water and MeOH (25 mL) was heated
sufficient purity to be used directly for the next step. To this for 16 h until the TLC showed the disappearance of starting
intermediate in THF (25 mL) was slowly added ammonium material. The resulting solution was poured into 6 M HCl ice
hydroxide solution (20 mL) at 0 1C with stirring. The reaction water (80 mL), extracted with EA (100 mL Â 3), dried over
mixture was allowed to warm to room temperature slowly and anhydrous MgSO4 and filtered. Removal of the organic layers
stirred for 16 h. Finally, removal of the organic solvent under under reduced pressure gave a brownish residue and subse-
reduced pressure furnished a brownish residue and subsequent quent purification of the residue was performed by using the
purification of the residue was performed by using the flash flash column chromatography on silica gel with gradient elu-
column chromatography on silica gel with gradient elution tion (EA/Hex, 30% to 50%) to afford the desired compound as a
(MeOH/DCM, 5% to 10%) to afford the desired compound as a brown oil (0.11 g, 43%). 1H NMR (400 MHz, CDCl3) d 6.83–6.95
1
yellowish oil (0.15 g, 43%). H NMR (400 MHz, CDCl3) d 7.23– (m, 1H), 6.68–6.82 (m, 2H), 4.18 (q, J = 6.85 Hz, 2H), 3.78–4.00
7.52 (m, 5H), 6.70–6.90 (m, 3H), 5.98 (br. s, 1H), 5.73 (br. s, 1H), (m, 5H), 3.55–3.75 (m, 2H), 3.34–3.54 (m, 1H), 3.18 (t, J = 8.31 Hz,
5.11 (s, 2H), 4.14 (d, J = 6.85 Hz, 2H), 3.74–3.99 (m, 5H), 3.66 (br. 1H), 1.28 (d, J = 5.87 Hz, 4H); 13C NMR (101 MHz, CDCl3) d 176.7,
s, 1H), 3.50 (br. s, 1H), 3.42 (br. s, 1H), 2.99 (br. s, 1H), 1.26 (t, J = 155.0, 146.7, 145.0, 130.6, 119.9, 114.7, 109.9, 61.5, 55.9, 52.5,
6.36 Hz, 3H); 13C NMR (101 MHz, CDCl3) d 173.5, 154.9, 150.0, 48.9, 48.5, 47.4, 46.6, 14.7; HRMS m/z calcd for C15H20NO6 (M + H)+
147.6, 137.0, 131.9, 128.6, 127.9, 127.4, 127.3, 119.4, 114.5, 111.4, 310.3320, found 310.3302.
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Synthesis of (Æ)-trans-methyl 4-(4-(benzyloxy)-3-methoxyphenyl)-
NJC
Synthesis of (Æ)-trans-methyl 4-(4-(benzyloxy)-3-methoxyphenyl)-
1-(2-(tert-butoxy)-2-oxoethyl)pyrrolidine-3-carboxylate (8e). To a 1-(3-ethoxy-3-oxopropyl)pyrrolidine-3-carboxylate (8h). A mixture
mixture of compound 7a (1.0 g, 3 mmol) and K2CO3 (1.2 g, of compound 7a (1 g, 3 mmol), ethyl 3-bromopropionate (0.81 g,
8.6 mmol) in dry DCM (25 mL) with a small amount of KI as 4.5 mmol) and K2CO3 (0.62 g, 6.0 mmol) in dry DCM (25 mL) was
catalyst was added t-butyl 2-bromoacetate (0.88 g, 4.5 mmol) at stirred in the presence of catalytic amount of KI at 0 1C for
0 1C. The reaction mixture was stirred at room temperature for 30 minutes. The solution was allowed to warm to room tem-
14 hours. The crude reaction mixture was washed with water perature and stir for 14 h. The resulting solution was then
(200 mL Â 3). The organic portion was dried with anhydrous diluted with DCM (40 mL), washed with water (50 mL), dried
MgSO4 and filtered. Removal of the organic layer under reduced over anhydrous MgSO4 and filtered. The organic layers were
pressure gave a brownish residue and subsequent purification of evaporated to dryness giving a brownish crude residue. Sub-
the residue was performed by using the flash column chromato- sequent purification was performed by the flash column chroma-
graphy on silica gel with gradient elution (EA/Hex, 20% to 50%) tography on silica gel with gradient elution (EA/Hex, 10% to 30%)
to afford the desired compound as a colourless oil (1.1 g, 80%). to afford the desired compound as a brown oil (0.70 g, 53%).
1H NMR (400 MHz, CDCl3) d 7.41 (d, J = 7.82 Hz, 2H), 7.33 (t, J = 1H NMR (400 MHz, CDCl3) d 7.40–7.50 (m, 2H), 7.35 (t, J = 7.34 Hz,
7.34 Hz, 2H), 7.27 (d, J = 7.82 Hz, 1H), 6.92 (s, 1H), 6.80 (s, 2H), 2H), 7.22–7.31 (m, 1H), 6.90 (d, J = 1.96 Hz, 1H), 6.73–6.84 (m, 2H),
5.10 (s, 2H), 3.88 (s, 3H), 3.60–3.69 (m, 4H), 3.31–3.42 (m, 1H), 5.12 (s, 2H), 4.07–4.22 (m, 2H), 3.89 (s, 3H), 3.67 (s, 3H), 3.53–3.63
3.15–3.24 (m, 2H), 3.00–3.14 (m, 3H), 2.96 (dd, J = 7.34, 9.29 Hz, (m, 1H), 3.00–3.17 (m, 2H), 2.92–3.00 (m, 1H), 2.81–2.92 (m, 2H),
1H), 1.46 (s, 9H); 13C NMR (101 MHz, CDCl3) d 174.3, 169.7, 2.70–2.81 (m, 2H), 2.53 (t, J = 7.34 Hz, 2H), 1.26 (t, J = 7.34 Hz, 3H);
149.7, 147.0, 137.4, 136.6, 128.5, 127.7, 119.3, 114.2, 111.4, 81.1, 13C NMR (101 MHz, CDCl3) d 174.5, 172.2, 149.8, 147.0, 137.4,
71.1, 61.1, 56.9, 56.8, 56.1, 51.9, 51.6, 46.9, 28.2; HRMS m/z calcd 137.3, 128.5, 127.7, 127.2, 119.3, 114.2, 111.4, 71.1, 61.6, 60.3, 57.4,
for C26H34NO6 (M + H)+ 456.2386, found 456.2377.
56.0, 51.9, 51.6, 50.9, 46.6, 34.1, 14.2; HRMS m/z calcd for
Synthesis of 2-((Æ)-trans-3-(4-benzyloxy)-3-methoxyphenyl)- C25H32NO6 (M + H)+ 442.2230, found 442.2221.
4-((methoxycarbonyl)pyrrolidin-1-yl)acetic acid (8g). To a well-
Synthesis of (Æ)-trans-methyl 4-(4-(benzyloxy)-3-methoxyphenyl)-
stirred solution of compound 8e (0.9 g, 2.0 mmol) in DCM 1-((R)-2,3-dihydroxypropyl)pyrrolidine-3-carboxylate (8i). Com-
(20 mL), TFA (0.25 g, 2.2 mmol) was added dropwise at 0 1C pound 7a (0.3 g, 0.9 mmol) was dissolved in MeOH (30 mL).
with stirring for 16 h. Removal of the solvent in vacuo followed (Æ)-3-Chloropropane-1,2-diol (0.06 g, 0.6 mmol) and triethyl-
by the flash column chromatographic purification on silica gel amine (0.2 g, 1.7 mmol) were added to the compound 7a solution
using a mixture of 10% MeOH in DCM as eluent afforded the under an ice bath. The reaction mixture was heated under reflux
1
desired compound 8g (0.37 g, 46%) as a colorless oil. H NMR for 16 h. The reaction mixture was concentrated under reduced
(400 MHz, CDCl3) d 9.81 (br. s, 1H), 7.22–7.48 (m, 5H), 6.92 pressure and subsequently purified by using the flash column
(s, 1H), 6.79 (s, 2H), 5.08 (s, 2H), 3.85 (m, 4H), 3.51–3.81 (m, 9H), chromatography on silica gel with isocratic elution (MeOH/DCM,
3.37–3.51 (m, 1H); 13C NMR (101 MHz, CDCl3) d 171.9, 170.0, 4%) to afford the desired product as a yellow oil (0.2 g, 55%).
149.9, 147.7, 137.1, 131.4, 128.5, 127.8, 127.3, 119.5, 114.2, 111.5, 1H NMR (400 MHz, CDCl3) d 7.42–7.49 (m, 2H), 7.38 (t, J = 7.34 Hz,
71.0, 59.9, 57.7, 56.2, 56.2, 52.3, 49.9, 46.7; HRMS m/z calcd for 2H), 7.32 (d, J = 7.82 Hz, 1H), 6.84 (d, J = 8.80 Hz, 2H), 6.74–6.80
C22H26NO6 (M + H)+ 400.1760, found 400.1778.
(m, 1H), 5.15 (s, 2H), 3.91 (s, 3H), 3.74–3.87 (m, 2H), 3.70 (s, 3H),
Synthesis of (Æ)-trans-methyl 4-(4-(benzyloxy)-3-methoxyphenyl)- 3.54–3.66 (m, 2H), 3.14–3.26 (m, 1H), 3.05–3.14 (m, 2H), 2.96–3.05
1-(2-isopropoxy-2-oxoethyl)pyrrolidine-3-carboxylate (8f). A mix- (m, 1H), 2.76–2.90 (m, 2H), 2.71 (dd, J = 7.34, 9.29 Hz, 1H), 2.43–
ture of compound 7a (1.0 g, 3.0 mmol), i-propyl 2-bromoacetate 2.56 (m, 1H); 13C NMR (101 MHz, CDCl3) d 174.5, 149.8, 147.1,
(0.81 g, 4.5 mmol) and K2CO3 (0.62 g, 6.0 mmol) in dry DCM 137.3, 136.2, 128.5, 127.8, 127.3, 119.1, 114.3, 111.4, 71.2, 68.7,
(25 mL) was stirred in the presence of small amount of KI as 65.0, 62.1, 58.0, 57.7, 57.6, 56.1, 52.1, 51.2, 46.8, 46.8; HRMS m/z
catalyst at 0 1C for 30 minutes. The solution was allowed to warm calcd for C23H30NO6 (M + H)+ 416.2068, found 416.2069.
to room temperature and stirred for 14 h. The resulting solution
Synthesis of 5-((Æ)-trans-3-(4-(benzyloxy)-3-methoxyphenyl)-
was diluted with DCM (40 mL), washed with water (50 mL), dried 4-(methoxycarbonyl)pyrrolidin-1-yl)-2,2-dimethyl-5-oxopentanoic
over anhydrous MgSO4 and filtered. The organic layer was acid (8j). A mixture of compound 7a (1.0 g, 3.0 mmol), 2,2-
evaporated to dryness to give a crude residue. Subsequent dimethylglutaric anhydride (0.64 g, 4.5 mmol) and excess triethyl-
purification of the residue was performed by using flash column amine (0.61 g, 6.0 mmol) in dry DCM (25 mL) was stirred at 0 1C
chromatography on silica gel with gradient elution (EA/Hex, 10% for 30 mintues. The solution was allowed to warm to room
to 30%) to afford the desired compound as brown oil (0.89 g, temperature and stir for 16 h. The organic solvent was evapo-
67%). 1H NMR (400 MHz, CDCl3) d 7.24–7.49 (m, 5H), 6.95 rated to dryness to give a crude residue. Subsequent purification
(s, 1H), 6.75–6.88 (m, 2H), 4.97–5.23 (m, 3H), 3.84–3.98 (m, 3H), of was performed by using the flash column chromatography on
3.61–3.75 (m, 4H), 3.40–3.54 (m, 1H), 3.19–3.37 (m, 2H), 3.04–3.18 silica gel with gradient elution (MeOH/DCM, 5% to 10%) to
(m, 3H), 3.00 (dd, J = 6.85, 8.80 Hz, 1H), 1.28 (d, J = 5.87 Hz, 6H). afford the desired compound as a brown oil (1.0 g, 71%). 1H NMR
13C NMR (101 MHz, CDCl3) d 174.3, 169.9, 149.7, 147.0, 137.3, (400 MHz, CDCl3) d 9.13 (br. s, 1H), 7.27–7.56 (m, 5H), 6.62–6.90
136.5, 128.5, 127.8, 127.3, 119.3, 114.2, 111.4, 71.1, 68.2, 61.1, 57.0, (m, 3H), 5.01–5.20 (m, 2H), 3.82–4.11 (m, 5H), 3.39–3.82 (m, 5H),
56.3, 56.1, 52.0, 51.6, 46.9, 21.9. HRMS m/z calcd for C25H32NO6 3.23 (m, 1H), 2.22–2.46 (m, 2H), 1.94 (d, J = 5.87 Hz, 2H), 1.24
(M + H)+ 442.2230, found 442.2244.
(s., 3H), 1.21 (s, 3H); 13C NMR (101 MHz, CDCl3) d 182.4, 171.9,
3528 | New J. Chem., 2021, 45, 3515À3534
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149.9, 137.0, 128.5, 127.9, 127.3, 119.2, 114.2, 111.2, 71.1, 56.2, 7.33–7.43 (m, 2H), 7.15–7.33 (m, 3H), 6.63–6.84 (m, 3H), 4.95–
52.2, 48.8, 47.6, 41.5, 35.0, 30.3, 25.1, 25.0, 24.9; HRMS m/z calcd 5.11 (m, 2H), 3.99 (dd, J = 8.80, 11.74 Hz, 1H), 3.84–3.94 (m, 1H),
for C27H34NO7 (M + H)+ 484.2335, found 484.2332.
3.80 (d, J = 6.85 Hz, 3H), 3.65–3.72 (m, 1H), 3.56 (d, J = 4.89 Hz,
Synthesis of (Æ)-trans-5-(3-(4-(benzyloxy)-3-methoxyphenyl)- 3H), 3.40–3.52 (m, 2H), 3.05–3.26 (m, 1H), 2.59–2.72 (m, 2H),
4-(methoxycarbonyl)pyrrolidin-1-yl)-3,3-dimethyl-5-oxopentanoic 2.47–2.59 (m, 2H); 13C NMR (101 MHz, CDCl3) d 172.5, 172.2,
acid (8k). A mixture of compound 7a (0.33 g, 1 mmol), 3,3- 170.6, 149.8, 147.5, 137.0, 128.5, 128.5, 127.8, 127.8, 127.3, 119.2,
dimethylglutaric anhydride (0.32 g, 2.3 mmol) and excess triethyl- 114.2, 111.2, 71.0, 56.0, 56.0, 52.2, 52.2, 48.8, 31.5, 29.1, 29.0;
amine (0.31 g, 3 mmol) in dry DCM (25 mL) was stirred at 0 1C for HRMS m/z calcd for C24H28NO7 (M + H)+ 442.1866, found
30 minutes. The solution was allowed to warm to room tempera- 442.1863.
ture and stir for 16 h. The organic solvent was evaporated to
Synthesis of 2-(3-(4-(benzyloxy)-3-methoxyphenyl)-4-(meth-
dryness to give a crude residue. Subsequent purification of the oxycarbonyl)pyrrolidine-1-carbonyl)benzoic acid (8n). Phthalic
residue was performed by using the flash column chromato- anhydride (0.20 g, 1.4 mmol) and triethylamine (0.30 g,
graphy on silica gel with gradient elution (MeOH/DCM, 5% to 3.0 mmol) were added to compound 7a (0.30 g, 0.9 mmol) in
10%) to afford the desired compound as a brown oil (0.31 g, 63%). DCM (30 mL) under an ice bath. The mixture was stirred at
1H NMR (400 MHz, acetone-d6) d 7.50 (d, J = 7.82 Hz, 2H), 7.40 (t, room temperature for 16 h. The reaction mixture was then
J = 7.34 Hz, 2H), 7.30–7.37 (m, 1H), 7.08 (dd, J = 1.96, 10.76 Hz, concentrated under reduced pressure and purified by using the
1H), 7.00 (dd, J = 3.42, 8.31 Hz, 1H), 6.90 (dt, J = 1.96, 8.31 Hz, 1H), flash column chromatography on silica gel with isocratic elution
5.11 (d, J = 3.91 Hz, 2H), 4.12–4.21 (m, 1H), 4.05 (dd, J = 8.31, (MeOH/DCM, 5%) to afford the desired product as a yellow oil
1
12.23 Hz, 1H), 3.85 (s, 3H), 3.58–3.76 (m, 2H), 3.31–3.57 (m, 2H), (0.22 g, 51%). H NMR (400 MHz, CDCl3) d 8.08 (dd, J = 7.82,
2.49–2.56 (m, 2H), 2.40–2.48 (m, 2H), 1.10–1.20 (m, 6H); 14.67 Hz, 1H), 7.58 (dd, J = 8.31, 16.14 Hz, 1H), 7.30–7.51 (m, 7H),
13C NMR (101 MHz, acetone-d6) d 171.7, 171.1, 150.1, 147.8, 6.83–6.90 (m, 1H), 6.68–6.82 (m, 2H), 5.11 (d, J = 12.72 Hz, 2H),
147.7, 137.7, 132.2, 128.3, 127.7, 127.6, 127.5, 119.7, 119.5, 4.19–4.30 (m, 1H), 3.82–3.96 (m, 4H), 3.69–3.80 (m, 1H), 3.61–
114.3, 114.2, 111.9, 111.7, 70.6, 55.4, 55.4, 54.1, 52.3, 50.1, 3.66 (m, 2H), 3.56–3.61 (m, 2H), 3.45–3.55 (m, 1H), 3.14–3.37 (m,
50.0, 48.9, 48.3, 47.8, 46.1, 45.9, 45.9, 42.9, 42.7, 33.5; HRMS 2H); 13C NMR (101 MHz, CDCl3) d 172.6, 172.2, 170.4, 168.6,
m/z calcd for C27H34NO7 (M + H)+ 484.1326, found 484.1315.
149.8, 147.6, 138.6, 137.1, 132.9, 131.1, 129.1, 128.5, 127.8, 127.3,
Synthesis of (E)-4-((Æ)-trans-3-(4-(benzyloxy)-3-methoxyphenyl)- 126.6, 119.4, 114.2, 111.3, 71.0, 56.2, 54.9, 52.1, 51.9, 50.6, 49.2,
4-(methoxycarbonyl)pyrrolidin-1-yl)-4-oxobut-2-enoic acid (8l). A 48.7, 47.6, 46.5. HRMS m/z calcd for C28H28NO7 (M + H)+
mixture of compound 7a (0.33 g, 1 mmol), maleic anhydride 490.1860, found 490.1864.
(0.23 g, 2.3 mmol) and excess triethylamine (0.31 g, 3 mmol) in
Synthesis of (Æ)-trans-methyl 4-(4-(benzyloxy)-3-methoxyphenyl)-
dry DCM (25 mL) was stirred at 0 1C for 30 minutes. The solution 1-(methylsulfonyl)pyrrolidine-3-carboxylate (9a). To a solution of
was allowed to warm to room temperature and stir for 16 h. The mesyl chloride (0.52 g, 4.5 mmol) in dry DCM (25 mL) and excess
organic solvent was evaporated to dryness to afford a crude triethylamine (0.87 g, 8.6 mmol) was added dropwise a solution
mixture. Subsequent purification of the residue was performed of compound 7a (1.0 g, 3.0 mmol) at 0 1C with stirring for 4 h.
by using the flash column chromatography on silica gel with Removal of the organic layer under reduced pressure furnished a
gradient elution (MeOH/DCM, 5% to 10%) to afford the desired brownish residue and subsequent purification of the residue was
compound as a brown oil (0.26 g, 59%). 1H NMR (400 MHz, performed by using the flash column chromatography on silica
CDCl3) d 7.30–7.51 (m, 5H), 6.88 (d, J = 8.80 Hz, 1H), 6.79 (d, J = gel with gradient elution (EA/Hex, 30% to 50%) to afford the
1.96 Hz, 1H), 6.69–6.76 (m, 1H), 6.37–6.60 (m, 2H), 5.17 (s, 2H), desired compound as a brown oil (0.79 g, 63%). 1H NMR
4.05–4.24 (m, 2H), 4.00 (t, J = 9.29 Hz, 1H), 3.65–3.94 (m, 8H), 3.21– (400 MHz, CDCl3) d 7.26–7.52 (m, 5H), 6.74–6.89 (m, 3H), 5.14
3.40 (m, 1H). 13C NMR (101 MHz, CDCl3) d 172.5, 172.2, 171.6, (s, 2H), 3.90 (s, 3H), 3.73–3.85 (m, 2H), 3.57–3.72 (m, 5H), 3.50
149.8, 147.7, 137.1, 128.5, 127.9, 127.3, 119.2, 114.2, 111.1, 77.4, (dd, J = 7.83, 9.78 Hz, 1H), 3.17–3.29 (m, 1H), 2.86–2.96 (m, 3H);
77.1, 76.8, 71.1, 56.1, 53.1, 52.2, 50.7, 48.8, 47.6, 46.1, 41.5, 35.0; 13C NMR (101 MHz, CDCl3) d 172.5, 149.9, 147.7, 137.0, 132.0,
HRMS m/z calcd for C24H26NO7 (M + H)+ 440.3564, found 128.6, 127.9, 127.3, 119.1, 114.2, 111.0, 71.0, 56.1, 53.6, 52.4, 50.6,
440.3579.
Synthesis of 4-((Æ)-trans-3-(4-(benzyloxy)-3-methoxyphenyl)-
50.0, 47.5, 35.4; LRMS (ESI) m/z C21H26NO6S (M + H+) 420.2.
Synthesis of (Æ)-trans-methyl 4-(4-hydroxy-3-methoxyphenyl)-
4-(methoxycarbonyl)pyrrolidine-1-yl)-4-oxobutanoic acid (8m). 1-(methylsulfonyl)pyrrolidine-3-carboxylate (10a). To a solution
A mixture of compound 7a (0.33 g, 1 mmol), succinic anhydride of compound 9a (0.6 g, 1.4 mmol) in dry tetrahydrofuran (THF)
(0.23 g, 2.3 mmol) and excess triethylamine (0.31 g, 3 mmol) in (25 mL) was added 10% palladium on carbon (0.22 g, 2.1 mmol)
dry DCM (25 mL) was stirred at 0 1C for 30 minutes. The at room temperature with stirring for 16 h under hydrogen
solution was allowed to warm to room temperature with atmosphere at balloon pressure. The catalyst was filtered and
stirring for 16 h. The organic solvent was evaporated under washed with THF (20 mL) and the filtrate was concentrated
reduced pressure to give a crude mixture. Subsequent purifica- under reduced pressure. The crude reaction mixture was purified
tion of the mixture was performed by using the flash column by using flash column chromatography on silica gel with gradi-
chromatography on silica gel with gradient elution (MeOH/ ent elution (EA/Hex, 30% to 50%) to afford the desired com-
DCM, 5% to 10%) to afford the desired compound as a brown pound as a colourless oil (0.23 g, 51%). 1H NMR (400 MHz,
1
oil (0.26 g, 59%). H NMR (400 MHz, CDCl3) d 8.85 (br. s, 1H), CDCl3) d 6.83–6.91 (m, 1H), 6.72–6.82 (m, 2H), 5.76 (br. s, 1H),
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3.88 (s, 3H), 3.74–3.86 (m, 2H), 3.57–3.71 (m, 5H), 3.49 (dd, J = 47.3, 38.0; HRMS m/z calcd for C21H27N2O6S (M + H)+ 435.1516,
7.83, 9.78 Hz, 1H), 3.16–3.26 (m, 1H), 2.93 (s, 3H); 13C NMR found 435.1521.
(101 MHz, CDCl3) d 172.6, 146.9, 145.1, 130.7, 119.8, 114.7, 109.9, Synthesis of (Æ)-trans-methyl 4-(4-(benzyloxy)-3-methoxyphenyl)-
109.7, 56.0, 53.7, 52.4, 50.7, 50.0, 47.7, 35.4; LRMS (ESI) m/z 1-(2-(2,6-difluorophenyl)acetyl)pyrrolidine-3-carboxylate (9c). 2-
C14H20NO6S (M + H+) 330.1.
(2,6-Difluorophenyl)acetic acid (0.8 g, 4.6 mmol) was dissolved
Synthesis of (Æ)-trans-methyl 4-(3,4-dimethoxyphenyl)-1- in DCM (20 mL) and mixed with oxalyl chloride (1.7 g, 13.4 mmol)
(methylsulfonyl)-pyrrolidine-3-carboxylate (10b). A well-stirred and DMF (3 drops) as catalyst under an ice bath. The reaction
mixture of compound 10a (0.20 g, 0.67 mmol), anhydrous mixture was stirred at room temperature for 6 h. The reaction
potassium carbonate (0.28 g, 2.01 mmol) and methyl iodide mixture was concentrated under reduced pressure to remove excess
(0.29 g, 2.01 mmol) in acetone (20 mL) was refluxed for 16 h. oxalyl chloride and a crude product of 2-(2,6-difluorophenyl)acetyl
After cooling, the reaction mixture was filtered. Removal of the chloride was obtained. To a well-stirred solution of compound 7a
solvent under reduced pressure followed by flash column (0.4 g, 1.17 mmol) in DCM (20 mL) under an ice-bath was added
chromatographic purification on silica gel using a mixture of dropwise the crude product of 2-(2,6-difluorophenyl)acetyl chloride
30% EA in Hex as eluent afforded the desired compound 10b and triethylamine (0.4 g, 3.5 mmol). The reaction mixture was
(0.16 g, 71%) as a colorless oil. 1H NMR (400 MHz, CDCl3) d stirred at room temperature for 4 h. The reaction mixture was
6.68–6.91 (m, 3H), 3.85–3.92 (m, 6H), 3.73–3.85 (m, 2H), 3.58– concentrated under reduced pressure and subsequently purified by
3.72 (m, 5H), 3.47–3.57 (m, 1H), 3.21 (q, J = 7.82 Hz, 1H), 2.94 (s, using the flash column chromatography on silica gel with isocratic
3H); 13C NMR (101 MHz, CDCl3) d 172.5, 149.3, 148.5, 131.4, elution (EA/Hex, 50%) to afford the desired product as a yellow oil
119.1, 111.5, 110.4, 56.0, 55.9, 53.6, 52.4, 50.7, 50.0, 47.6, 35.5; (0.19 g, 34%). 1H NMR (400 MHz, CDCl3) d 7.45 (br. s, 2H), 7.34–
LRMS (ESI) m/z C15H22NO6S (M + H+) 344.2.
7.42 (m, 2H), 7.27–7.34 (m, 1H), 7.18–7.26 (m, 1H), 6.86–6.95 (m,
Synthesis of (Æ)-trans-methyl 4-(4-(benzyloxy)-3-methoxyphenyl)- 3H), 6.73–6.86 (m, 2H), 5.15 (d, J = 5.87 Hz, 2H), 4.01–4.13 (m, 2H),
1-(N,N-dimethylsulfamoyl)pyrrolidine-3-carboxylate (9b). To a 3.90 (d, J = 10.76 Hz, 3H), 3.70–3.81 (m, 2H), 3.64–3.70 (m, 5H),
50 mL round-bottomed flask was added a mixture of compound 3.55–3.63 (m, 1H), 3.20 (d, J = 9.78 Hz, 1H); 13C NMR (101 MHz,
7a (1 g, 3 mmol) and excess triethylamine (0.87 g, 8.6 mmol) in CDCl3) d 172.5, 172.2, 167.1, 162.8, 160.4, 149.9, 147.7, 137.1, 131.8,
DCM (25 mL) at 0 1C followed by the addition of N,N- 131.7, 128.9, 128.8, 128.7, 128.6, 127.9, 127.9, 127.3, 119.2, 114.3,
dimethylsulfamoyl chloride (0.65 g, 4.5 mmol). After 4 h, the 111.2, 111.1, 111.0, 110.9, 71.1, 56.1, 53.2, 52.3, 52.2, 50.9, 49.2,
organic solvent was removed under reduced pressure and the 48.9, 47.7, 46.2; HRMS m/z calcd for C28H28F2NO5 (M + H)+
crude mixture was purified by the flash column chromatography 496.1936, found 496.1930.
on silica gel with gradient elution (EA/Hex, 10% to 30%) to afford
Synthesis of (Æ)-methyl 4-(4-(benzyloxy)-3-methoxyphenyl)-1-
the desired compound as pale brown solid (0.73 g, 54%). picolinoylpyrrolidine-3-carboxylate (9d). Picolinic acid (1.0 g,
1H NMR (400 MHz, CDCl3) d 7.25–7.49 (m, 5H), 6.80–6.91 8.1 mmol) was dissolved in DCM (20 mL) and mixed with oxalyl
(m, 2H), 6.76 (d, J = 7.83 Hz, 1H), 5.14 (s, 2H), 3.90 (s, 3H), chloride (1.45 g, 12.2 mmol) and DMF (2 drops) as the catalyst.
3.73–3.85 (m, 2H), 3.63–3.70 (m, 4H), 3.56–3.63 (m, 1H), 3.44 (t, The reaction mixture was heated under reflux for 3 h. The
J = 9.29 Hz, 1H), 3.20 (d, J = 7.83 Hz, 1H), 2.87 (s, 6H); 13C NMR reaction mixture was concentrated under reduced pressure to
(101 MHz, CDCl3) d 172.4, 149.9, 147.6, 137.1, 132.2, 128.5, 127.8, afford a crude product of picolinoyl chloride. To a well-stirred
127.3, 119.2, 114.2, 111.0, 71.1, 56.1, 54.5, 52.2, 50.9, 50.6, 47.4, solution of compound 7a (1.0 g, 3.0 mmol) in DCM (20 mL) in
38.0; HRMS m/z calcd for C22H29N2O6S (M + H)+ 449.1746, found an ice-bath was added dropwise the crude product of picolinoyl
449.1754. CCDC 2046394.†
chloride and triethylamine (0.9 g, 9.0 mmol). The reaction
Synthesis of (Æ)-trans-4-(4-(benzyloxy)-3-methoxyphenyl)-1- mixture was stirred at room temperature for 12 h. The reaction
(N,N-dimethylsulfamonyl)pyrrolidine-3-carboxylic acid (10c). mixture was concentrated under reduced pressure and sub-
A well-stirred mixture of compound 9b (0.45 g, 1.0 mmol) and sequently purified by using the flash column chromatography
KOH (0.08 g, 2 mmol) in a mixture of water and MeOH (25 mL) on silica gel with gradient elution (MeOH/DCM, 1% to 5%) to
was heated for 16 h until the TLC showed the disappearance of afford the desired product as a yellow oil (0.24 g, 18%). 1H NMR
starting material. The resulting solution was poured into 6 M (400 MHz, CDCl3) d 8.54–8.64 (m, 1H), 7.90–7.99 (m, 1H), 7.81–
HCl ice water (80 mL), extracted with EA (100 mL Â 3), dried 7.90 (m, 1H), 7.26–7.47 (m, 6H), 6.74–6.89 (m, 3H), 5.14 (d, J =
over anhydrous MgSO4 and filtered. Removal of the organic 6.85 Hz, 2H), 4.23 (ddd, J = 5.38, 7.58, 12.47 Hz, 2H), 3.92–4.15
layers under reduced pressure afforded a brownish residue and (m, 2H), 3.89 (d, J = 3.91 Hz, 3H), 3.61–3.73 (m, 4H), 3.19–3.33
subsequent purification of the residue was performed by using (m, 1H); 13C NMR (101 MHz, CDCl3) d 172.6, 172.3, 165.6, 153.1,
the flash column chromatography on silica gel with gradient 149.8, 147.5, 137.5, 131.9, 131.5, 128.5, 127.8, 127.2, 125.2,
elution (EA/Hex, 30% to 50%) to afford the desired compound 124.4, 119.4, 114.2, 111.2, 71.1, 56.1, 55.5, 53.2, 52.2, 51.8,
as a brown oil (0.17 g, 39%). 1H NMR (400 MHz, CDCl3) d 7.27– 51.4, 49.9, 48.7, 48.4, 45.7; HRMS m/z calcd for C26H27N2O5
7.54 (m, 5H), 6.70–6.89 (m, 3H), 5.15 (s, 2H), 3.90 (s, 3H), (M + H)+ 447.1914, found 447.1913.
3.81 (td, J = 9.17, 17.85 Hz, 2H), 3.58–3.73 (m, 2H), 3.43 (t, J =
Synthesis of (Æ)-trans-methyl 4-(4-(benzyloxy)-3-methoxyphenyl)-
8.80 Hz, 1H), 3.24 (d, J = 8.80 Hz, 1H), 2.88 (s, 6H); 13C NMR 1-(2-chloroacetyl)pyrrolidine-3-carboxylate (9e). Compound 7a
(101 MHz, CDCl3) d 177.0, 149.9, 147.7, 137.0, 132.0, 128.5, (0.6 g, 1.8 mmol) was dissolved in DCM (20 mL) and mixed with
127.9, 127.3, 119.3, 114.3, 111.1, 71.1, 56.2, 54.7, 50.6, 50.3, 2-chloroacetyl chloride (0.4 g, 3.5 mmol) and trimethylamine
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(0.53 g, 5.2 mmol) under an ice-bath. The reaction mixture was d 172.6, 172.2, 168.9, 158.8, 149.8, 148.9, 147.6, 137.1, 136.6, 131.7,
then stirred at room temperature for 12 h. The reaction mixture 128.6, 127.9, 127.9, 127.3, 123.8, 122.2, 119.2, 114.3, 111.2, 71.1,
was concentrated under reduced pressure and subsequently 60.5, 60.4, 56.2, 56.1, 52.4, 52.2, 51.9, 50.9, 48.8, 48.6, 48.5, 47.7,
purified by using the flash column chromatography on silica 45.8; HRMS m/z calcd for C34H37N4O5 (M + H)+ 581.2758, found
gel with isocratic elution (EA/Hex, 50%) to afford the desired 581.2761.
product as a yellow oil (0.38 g, 51%). 1H NMR (400 MHz, CDCl3)
Synthesis of (Æ)-trans-methyl 6-(-3-(4-(benzyloxy)-3-methoxy-
d 7.42–7.48 (m, 2H), 7.39 (t, J = 7.34 Hz, 2H), 7.33 (d, J = 6.85 Hz, phenyl)-4-(methoxycarbonyl)pyrrolidine-1-carbonyl)picolinate (9f).
1H), 6.86 (br. s, 1H), 6.70–6.81 (m, 2H), 5.16 (s, 2H), 4.05 (d, J = 6-(Methoxycarbonyl)picolinic acid (0.5 g, 2.9 mmol) was dissolved
11.74 Hz, 4H), 3.82–3.95 (m, 4H), 3.75 (d, J = 9.78 Hz, 1H), 3.60– in DCM (20 mL) and mixed with oxalyl chloride (1.1 g, 8.7 mmol)
3.71 (m, 5H), 3.19 (d, J = 7.83 Hz, 1H); 13C NMR (101 MHz, CDCl3) and DMF (2 drops) as catalyst under an ice-bath. The reaction
d 172.3, 171.9, 164.7, 149.9, 147.7, 137.0, 131.6, 131.4, 128.5, mixture was stirred at room temperature for 4 h. The reaction
127.9, 127.3, 119.1, 114.3, 111.2, 77.6, 71.0, 56.1, 52.8, 52.3, 50.8, mixture was concentrated under reduced pressure to remove
49.1, 49.0, 48.7, 47.7, 45.9, 41.7; HRMS m/z calcd for excess oxalyl chloride. A crude product of methyl 6-(chlorocarbonyl)-
C22H25ClNO5 (M + H)+ 418.1416, found 418.1417.
picolinate was obtained. To a well-stirred solution of compound 7a
Synthesis of (Æ)-trans-methyl 1-(2-(acetylthio)acetyl)-4-(4- (0.5 g, 1.5 mmol) in DCM (20 mL) under an ice-bath was added the
(benzyloxy)-3-methoxyphenyl)pyrrolidine-3-carboxylate (10d). crude methyl 6-(chlorocarbonyl)picolinate dropwise. The reaction
To a well-stirred mixture of compound 9e (0.8 g, 1.91 mmol) mixture was stirred at room temperature for 12 h. The reaction
and excess triethylamine (5 mL) in DCM (20 mL) was added mixture was concentrated under reduced pressure and subse-
thioacetic acid (0.22 g, 2.87 mmol) dropwise under an ice bath. quently purified by using the flash column chromatography on
After the addition, the reaction mixture was stirred at room silica gel with gradient elution (EA/Hex, 30% to 50%) to afford the
temperature for 16 h. The reaction mixture was washed with desired product as a yellow oil (0.3 g, 41%). 1H NMR (400 MHz,
water (100 mL Â 3). The organic layers were dried over CDCl3) d 8.20 (dd, J = 7.83, 11.74 Hz, 1H), 8.10–8.15 (m, 1H),
anhydrous MgSO4, filtered and concentrated under reduced 7.96–8.03 (m, 1H), 7.42–7.48 (m, 2H), 7.35–7.41 (m, 2H), 7.32 (dd,
pressure to afford a crude mixture, which was subsequently J = 3.91, 6.85 Hz, 1H), 6.82–6.89 (m, 2H), 6.75–6.81 (m, 1H), 5.15
purified by using the flash column chromatography on silica (d, J = 7.83 Hz, 2H), 4.25–4.33 (m, 1H), 4.07–4.18 (m, 1H), 3.87–
gel with isocratic elution (EA/Hex, 25%) to afford the desired 4.07 (m, 7H), 3.62–3.82 (m, 5H), 3.27 (d, J = 8.80 Hz, 1H); 13C
product as a yellow oil (0.62 g, 71%). 1H NMR (400 MHz, CDCl3) NMR (101 MHz, CDCl3) d 172.6, 172.2, 165.1, 153.5, 153.5, 149.8,
d 7.35–7.47 (m, 2H), 7.31 (t, J = 7.34 Hz, 2H), 7.20–7.27 (m, 1H), 147.6, 146.3, 138.2, 137.1, 128.5, 127.9, 127.4, 127.2, 126.3, 119.4,
6.62–6.88 (m, 3H), 5.07 (d, J = 4.89 Hz, 2H), 3.94–4.03 (m, 2H), 119.3, 114.2, 111.2, 71.1, 71.1, 56.1, 55.4, 53.3, 52.9, 52.2, 51.8, 51.4,
3.83 (d, J = 7.83 Hz, 3H), 3.63–3.71 (m, 3H), 3.57–3.62 (m, 4H), 50.1, 48.7, 48.4, 45.7; HRMS m/z calcd for C28H29N2O7 (M + H)+
3.45–3.56 (m, 1H), 3.06–3.28 (m, 1H); 13C NMR (101 MHz, 505.1969, found 505.1973.
CDCl3) d 194.5, 172.3, 172.0, 165.8, 165.8, 149.9, 147.6, 137.1,
Synthesis of (Æ)-trans-6-(3-(4-(benzyloxy)-3-methoxyphenyl)-
137.1, 128.5, 128.5, 127.8, 127.3, 119.2, 119.2, 114.2, 111.2, 71.0, 4-carboxypyrrolidine-1-carbonyl)picolinic acid (10f). Compound
56.1, 53.1, 50.8, 47.7, 46.0, 32.5, 32.3, 30.1; HRMS m/z calcd for 9f (0.10 g, 0.2 mmol) was dissolved in MeOH (25 mL). Potassium
C
24H28NO6S (M + H)+ 458.1632, found 458.1639.
hydroxide (0.022 g, 0.39 mmol) was dissolved in a minimum
Synthesis of (Æ)-trans-methyl 4-(4-(benzyloxy)-3-methoxyphenyl)- amount of water and added to the MeOH solution of 9f under an
1-(2-(bis(pyridin-2-ylmethyl)amino)acetyl)pyrrolidine-3-carboxylate ice bath. The reaction mixture was stirred at reflux for 6 h. The
(10e). Compound 9e (0.26 g, 0.62 mmol) was dissolved in acetone reaction mixture was concentrated under reduced pressure and
(30 mL). Bis(pyridin-2-ylmethyl)amine (0.25 g, 1.3 mmol) and redissolved in EA. A yellow solid was deposited on the surface of
potassium carbonate (0.26 g, 1.9 mmol) were added to the the round bottom flask, which was rinsed with 1 M HCl solution.
solution of 9e under an ice bath. The reaction mixture was The solution was then extracted with EA (10 mL Â 2) and the
subsequently heated under reflux for 16 h. The reaction mixture organic portions were dried over anhydrous MgSO4, filtered and
was concentrated under reduced pressure and re-dissolved in EA evaporated under reduced pressure to afford the desired product
(50 mL). The mixture was washed with water (100 mL Â 3). The as a yellow powder (0.06 g, 63%). 1H NMR (400 MHz, MeOH-d4) d
organic layers were dried over anhydrous MgSO4, filtered and 3.65 (d, J = 18.6 Hz, 2H) 3.84 (d, J = 12.7 Hz, 3H) 4.11 (d, J = 6.8 Hz,
concentrated under reduced pressure. The crude mixture was 2H) 4.18 (d, J = 8.8 Hz, 2H) 5.05 (d, J = 11.7 Hz, 2H) 6.84 (br. s, 1H)
subsequently purified by using the flash column chromatography 6.87–7.04 (m, 2H) 7.24–7.38 (m, 3H) 7.39–7.48 (m, 2H) 8.01 (br. s,
on silica gel with gradient elution (MeOH/DCM, 5% to 7%) to 1H) 8.10 (d, J = 10.8 Hz, 1H) 8.23 (d, J = 7.8 Hz, 1H); 13C NMR
1
afford the desired product as a yellow oil (0.12 g, 33%). H NMR (101 MHz, MeOH-d4) d 174.1, 173.8, 165.9, 159.6, 153.1, 149.9,
(400 MHz, CDCl3) d 8.50 (d, J = 4.89 Hz, 1H), 8.55 (d, J = 3.91 Hz, 147.5, 145.3, 138.6, 137.3, 132.6, 128.1, 127.5, 127.3, 126.6, 126.1,
1H), 7.59–7.71 (m, 2H), 7.50–7.59 (m, 2H), 7.41–7.48 (m, 2H), 7.34– 119.5, 119.4, 114.5, 111.6, 70.9, 55.2, 53.2, 51.6, 50.8, 49.8, 45.8.
7.41 (m, 2H), 7.31 (dd, J = 2.93, 6.85 Hz, 1H), 7.10–7.22 (m, 2H), HRMS m/z calcd for C26H25N2O7 (M + H)+ 477.1656, found 477.1658.
6.78–6.88 (m, 1H), 6.72–6.77 (m, 1H), 6.69 (t, J = 7.34 Hz, 1H), 5.13
Biological studies
(d, J = 7.82 Hz, 2H), 3.98 (d, J = 7.83 Hz, 5H), 3.87 (d, J = 7.82 Hz,
3H), 3.78 (t, J = 9.29 Hz, 1H), 3.54–3.71 (m, 5H), 3.44–3.52 (m, 1H),
Bacterial strains. E. coli strain BL21 (NDM-1) carrying only
3.31–3.44 (m, 2H), 3.01–3.20 (m, 1H); 13C NMR (101 MHz, CDCl3) NDM-1 marker was constructed for the screening of test
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NJC
compounds by determining MIC as previously described.9 reading from negative well À corrected reading from the blank
Briefly, the blaNDM-1 gene was synthesized, PCR amplified and well) Â 100%. IC50 was calculated by the software IC50 calculator.
constructed on the IPTG-inducible pET28b vector. Then the con-
ESI-MS analysis
structed plasmid was transformed with E. coli BL21 to form E. coli
BL21 (NDM-1) for MIC determination. E. coli BL21 (NDM-1)
carrying the recombinant plasmid pET28b-blaH6-mNDM-1, which
encoded G36 to R270 and carried an N-terminal His6 tag for the
purification of NDM-1 enzyme. Clinically isolated strains shown in
Table 2 were isolated from the patients’ urine, feces, and sputum
in the Second People’s Hospital of Jiaxing in Zhejiang Province,
China.46
Waters synapt g2-si electrospray ionization/quadrupole-ion
mobility-time-of flight mass spectrometer was employed to
perform Electrospray ionization mass spectrometry (ESI-MS)
experiments. For qualitative detection of the binding between
NDM-1 and compound under non-denaturing conditions, after
incubating 20 mM of mNDM-1 in 20 mM ammonia acetate with
an equal molar of compound 10e in the same buffer system for
20 minutes, the reaction mixture was infused directly into a
nanospray emitter (Econo12, New Objectives, Woburn, USA),
which was mounted onto a nano-ESI source for analysis. The
spray voltage was carefully raised to start the spray process and
the spray was maintained for around 20 minutes at the voltage
of 150 V.49 During data acquisition, positive ion mode was
exhibited in the operation of the mass spectrometer in the m/z
range of 200–5000 for the detection of multiply charged ions.
Transform program (MassLynx 4.1, Waters) was used to analyze
the obtained raw multiply charged mass spectra.
MIC determination
Bacterial cells of E. coli BL21 (NDM-1) were incubated on
Mueller–Hinton agar (MHA) plate at 37 1C overnight under
aerobic conditions. The bacteria were then transferred to
normal saline where the OD600 value of the solution was
measured to 0.08–0.1. The E. coli BL21 (NDM-1) saline solution
was transferred to a 96-well plate and incubated with Mueller–
Hinton broth (MHB), 1 mM of IPTG, and a serial concentration
of MRM alone, the freshly prepared compound alone in DMSO,
or a combination of various concentrations of MRM and test
compound at 100 mM. After being incubated at 37 1C overnight,
the MIC values were determined following the CLSI guidelines.39
Computational docking studies
CLC Drug Discovery Workbench (Version 2.5, QIAGEN) soft-
ware was used for docking study. The 2D structures of both
enantiomers of the compound 10e were generated from
SMILES and imported into the software. The X-ray crystal
structure of the NDM-1 enzyme (PDB ID: 4EXS) obtained from
the Protein Data Bank (https://www.rcsb.org/) was used directly
for docking without any changes. Using the software function
of ‘‘Find Binding Pockets’’, the software was able to identify the
substrate-binding site of NDM-1 as potential binding pockets.
The identification of ligand binding modes was done iteratively
by evaluating 30 000 ligand conformations and estimating the
binding energy of their interactions with these binding pockets.
The binding pose with the top 5% highest scores were returned
for further visual inspection. The highest scores positioned
both enantiomers of the compound 10e into the substrate-
binding site of NDM-1 with the potential binding poses,
important amino acids, and hydrogen bonding interactions
shown in Fig. 4.
NDM-1 enzyme inhibition assay
Expression and purification of NDM-1 protein were prepared
following our previous report.47
Kinetic assay of NDM-1 was exhibited to determine the
inactivation constants of inhibitors as previously published.9
Briefly, followed by the addition of 7 fold of Km of the reporter
substrate called nitrocefin, 1 nM of pure NDM-1 was mixed with
different concentrations of compound 10e in 500 mL of 50 mM
phosphate buffer with or without 50 mM ZnSO4. Bovine serum
albumin (BSA) was then added to stabilize the activity of diluted
mNDM-1. The readout of the velocity can be recorded by the
wavelength change at 482 nm. Three independent assays for
10e were performed in triplicate.
Cytotoxicity assay
This assay was based on the reduction of 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) to dark blue formazan
by succinate dehydrogenase in living cells.48 HEK cells were
seeded into three 96-well plates at a density of 1 Â 104
cells per well in DMEM (10% FBS) and incubated for 12 h at
Abbreviations
37 1C. Then, the cells were exposed to various concentrations of CRE
Carbapenem-resistant Enterobacteriaceae
compound 10e in DMSO for 24 h. Medium containing 0.5% MBLs Metallo-b-lactamases
DMSO was used as a negative control. Medium without cells was NDM-1 New Delhi metallo-b-lactamase-1
used as blank control. After treatment, MTT at a concentration of Eb
0.5 mg mLÀ1 in PBS was added to each well and the cells were IC50
further incubated for 3 h at 37 1C. The medium was removed to TPA
afford the formazan crystals followed by dissolving in DMSO. MRM
The optical density was measured at 490 nm using a Microplate TFA
Ebselen
Half-maximal inhibitory concentration
Tris-picolylamine
Meropenem
Trifluoroacetic acid
Reader (Clariostar, BMG). The percentage of surviving cells was DMAP 4-(Dimethylamino)pyridine
calculated using the following formula: (corrected reading from tPSA
Topological polar surface area
the test well À corrected reading from the blank well)/(corrected IPTG
Isopropyl b-D-1-thiogalacto-pyranoside
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Paper
12 W. B. Jin, C. Xu, Q. Cheung, W. Gao, P. Zeng, J. Liu,
E. W. C. Chan, Y.-C. Leung, T. H. Chan, K.-Y. Wong,
S. Chen and K.-F. Chan, Bioorg. Chem., 2020, 100, 103873.
13 A. M. King, S. A. Reid-Yu, W. Wang, D. T. King, G. De
Pascale, N. C. Strynadka, T. R. Walsh, B. K. Coombes and
G. D. Wright, Nature, 2014, 510, 503–506.
CLSI
MIC
RF
FICI
EDTA
Clinical and Laboratory Standards Institute
Minimum inhibition concentration
Reduction fold
Fractional inhibitory concentration index
Ethylenediaminetetracetic acid
14 K. H. M. E. Tehrani, H. Fu, N. C. Bru¨chle, V. Mashayekhi,
Author contributions
´
A. Prats Lujan, M. J. van Haren, G. J. Poelarends and
N. I. Martin, Chem. Commun., 2020, 56, 3047–3049.
15 C. Schnaars, G. Kildahl-Andersen, A. Prandina, R. Popal,
S. Radix, M. Le Borgne, T. Gjøen, A. M. S. Andresen, A. Heikal,
The manuscript was written through the contributions of all
authors. All authors have approved the final version of the
manuscript.
¨
O. A. Økstad, C. Frohlich, Ø. Samuelsen, S. Lauksund,
L. P. Jordheim, P. Rongved and O. A. H. Åstrand, ACS Infect.
Dis., 2018, 4, 1407–1422.
Conflicts of interest
16 X.-F. Shi, M.-M. Wang, S.-C. Huang, J.-X. Han, W.-C. Chu,
C. Xiao, E. Zhang and S. Qin, Eur. J. Med. Chem., 2019, 167,
367–376.
The authors declare no competing financial interest.
17 D.-Y. Cui, Y. Yang, M.-M. Bai, J.-X. Han, C.-C. Wang,
H.-T. Kong, B.-Y. Shen, D.-C. Yan, C.-L. Xiao, Y.-S. Liu and
E. Zhang, Bioorg. Chem., 2020, 101, 103965.
18 A. U. Khan, A. Ali, M. Danishuddin, G. Srivastava and
A. Sharma, Sci. Rep., 2017, 7, 9207.
19 B. Zhao, X. Zhang, T. Yu, Y. Liu, X. Zhang, Y. Yao, X. Feng,
H. Liu, D. Yu, L. Ma and S. Qin, Acta Pharm. Sin. B, 2021, 11,
203–221.
20 A. Y. Chen, P. W. Thomas, A. C. Stewart, A. Bergstrom, Z. Cheng,
C. Miller, C. R. Bethel, S. H. Marshall, C. V. Credille, C. L. Riley,
R. C. Page, R. A. Bonomo, M. W. Crowder, D. L. Tierney, W. Fast
and S. M. Cohen, J. Med. Chem., 2017, 60, 7267–7283.
21 D. T. King, L. J. Worrall, R. Gruninger and N. C. J. Strynadka,
J. Am. Chem. Soc., 2012, 134, 11362–11365.
Acknowledgements
We acknowledge the support from the Research Grants Council
of Hong Kong (grant no. 15100115, 25100014), the Collabora-
tive Research Fund from the Research Grant Council of the
Government of Hong Kong SAR (C5026-16G), Health and
Medical Research Fund (14130432), the Innovation and Technol-
ogy Commission, and the Hong Kong Polytechnic University. We
also thank University Research Facilities in Life Sciences (ULS) of
the Hong Kong Polytechnic University for providing MS analysis.
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