Investigation of Dipicolinic Acid Isosteres for the Inhibition of Metallo-β-Lactamases

Article abstract of DOI:10.1002/cmdc.201900172

New Delhi metallo-β-lactamase-1 (NDM-1) poses an immediate threat to our most effective and widely prescribed drugs, the β-lactam-containing class of antibiotics. There are no clinically relevant inhibitors to combat NDM-1, despite significant efforts toward their development. Inhibitors that use a carboxylic acid motif for binding the Zn^II ions in the active site of NDM-1 make up a large portion of the >500 inhibitors reported to date. New and structurally diverse scaffolds for inhibitor development are needed urgently. Herein we report the isosteric replacement of one carboxylate group of dipicolinic acid (DPA) to obtain DPA isosteres with good inhibitory activity against NDM-1 (and related metallo-β-lactamases, IMP-1 and VIM-2). It was determined that the choice of carboxylate isostere influences both the potency of NDM-1 inhibition and the mechanism of action. Additionally, we show that an isostere with a metal-stripping mechanism can be re-engineered into an inhibitor that favors ternary complex formation. This work provides a roadmap for future isosteric replacement of routinely used metal binding motifs (i.e., carboxylic acids) for the generation of new entities in NDM-1 inhibitor design and development.

Full text of DOI:10.1002/cmdc.201900172

DOI: 10.1002/cmdc.201900172
Full Papers
Investigation of Dipicolinic Acid Isosteres for the Inhibition
of Metallo-b-Lactamases
Allie Y. Chen,[a] Pei W. Thomas,[b] Zishuo Cheng,[c] Nasa Y. Xu,[b] David L. Tierney,[c]
Michael W. Crowder,*[c] Walter Fast,*[b] and Seth M. Cohen*[a]
New Delhi metallo-b-lactamase-1 (NDM-1) poses an immediate
threat to our most effective and widely prescribed drugs, the
b-lactam-containing class of antibiotics. There are no clinically
relevant inhibitors to combat NDM-1, despite significant efforts
toward their development. Inhibitors that use a carboxylic acid
motif for binding the ZnII ions in the active site of NDM-1
make up a large portion of the >500 inhibitors reported to
date. New and structurally diverse scaffolds for inhibitor devel-
opment are needed urgently. Herein we report the isosteric re-
placement of one carboxylate group of dipicolinic acid (DPA)
to obtain DPA isosteres with good inhibitory activity against
NDM-1 (and related metallo-b-lactamases, IMP-1 and VIM-2). It
was determined that the choice of carboxylate isostere influen-
ces both the potency of NDM-1 inhibition and the mechanism
of action. Additionally, we show that an isostere with a metal-
stripping mechanism can be re-engineered into an inhibitor
that favors ternary complex formation. This work provides a
roadmap for future isosteric replacement of routinely used
metal binding motifs (i.e., carboxylic acids) for the generation
of new entities in NDM-1 inhibitor design and development.
Introduction
b-Lactam-containing antibiotics have been one of the most
successful and popular class of antibiotics for combating a
wide range of Gram-positive and -negative bacterial infections.
Unsurprisingly, since the introduction of b-lactam containing
antibiotics (beginning with penicillin in the 1940s), the wide-
spread use of this class of antibiotics has led to the emergence
of various resistance mechanisms. Resistance mechanisms that
bacteria employ include mutation of penicillin binding proteins
(PBPs), modification of outer membrane proteins, production
of efflux pumps, and expression of b-lactamases.[1,2] b-Lacta-
mases use either an active site serine residue (Ambler classes
A, C, and D) or ZnII metal ion(s) (Ambler class B, also known as
metallo-b-lactamases, MBLs) to hydrolyze the b-lactam ring of
the target antibiotic and render the drug ineffective.[1,3] A dedi-
cated b-lactamase database provides an up-to-date compila-
tion of the biochemical and structural data of all MBLs (http://
www.bldb.eu/).[4] First observed in 1966 by Sabath and Abra-
ham (merely two decades after the introduction of penicillin),
there are now >80 reported unique MBL families.[3,5] MBLs
have become one of the most problematic bacterial resistance
mechanisms due to their wide substrate profile, with the abili-
ty to hydrolyze virtually all clinically used bicyclic b-lactam anti-
biotics.[6,7]
MBLs are divided into B1, B2, and B3 subclasses depending
on sequence identity and the number of ZnII ion(s) (either one
or two) in the active site. Description of subclasses and their
mechanism of action are reviewed elsewhere.[6–9] Commonly
observed and clinically relevant members of the MBLs belong
to subclass B1,[10] of which New Delhi metallo-b-lactamase
(NDM) is a prominent representative. NDM bears a dinuclear
ZnII active site, with Zn1 ligated by H116, H118, H196, and a
bridging hydroxide, and Zn2 ligated by D120, C221, H263, the
bridging hydroxide, and an apical H2O (standard BBL number-
ing).[11] The active site is flanked between two flexible loops, al-
lowing the protein to accommodate a wide range of antibiotic
substrates.[12,13] Plasmids that carry the blaNDM gene can under-
go horizontal gene transfer between different species of micro-
organisms, leading to an increase in the prevalence of blaNDM
-
[a] A. Y. Chen, Prof. S. M. Cohen
bearing pathogens.[14,15] Additionally, the threat of NDM is exa-
cerbated (relative to the two other most prevalent members of
the B1 MBLs, IMiPenemase, IMP, and Verona Integron-encoded
metallo-b-lactamase, VIM) by the ability of NDM to anchor to
the cellular membrane, leading to higher protein stability and
secretion.[16,17] Resistance to a broad spectrum of b-lactams
and the high horizontal gene transfer ability have allowed for
rapid propagation from nosocomial infections to infections
within the general population.[18–20] Furthermore, blaNDM is
often carried on plasmids containing other genes that encode
various resistance factors (including macrolides, aminoglyco-
sides, rifampicin, and sulfamethoxazole),[14] resulting in bacteri-
Department of Chemistry and Biochemistry, University of California San
Diego, La Jolla, CA 92093 (USA)
E-mail: scohen@ucsd.edu
[b] Dr. P. W. Thomas, N. Y. Xu, Prof. W. Fast
Division of Chemical Biology & Medicinal Chemistry, College of Pharmacy,
University of Texas Austin, Austin, TX 78712 (USA)
E-mail: walt.fast@austin.utexas.edu
[c] Z. Cheng, Prof. D. L. Tierney, Prof. M. W. Crowder
Department of Chemistry and Biochemistry, Miami University, Oxford, OH
45056 (USA)
E-mail: crowdemw@miamioh.edu
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under:
https://doi.org/10.1002/cmdc.201900172.
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al infections that are resistant to many different classes of anti-
microbials.
boxylic acid motif.[37–39] The ionizable nature of the carboxylic
acid under physiological conditions (pH 7.4) makes it a useful
handle for generating strong inhibitor-target interactions (i.e.,
electrostatic and hydrogen bonds). In metalloenzyme inhibi-
tion, the carboxylic acid motif routinely participates in metal
coordination, as seen in inhibitors for neutral endopeptidase
(NEP), class II fructose-1,6-bisphosphate aldolase (FBP-aldolase),
angiotensin-converting enzyme (ACE), and many others.[40] De-
spite the efficacy of this functional group, its usefulness can be
limited in drug development due to poor cell permeability,
metabolic instability, and off-target effects.[37,39] In an attempt
to overcome such liabilities, chemists turn to the use of pro-
drugs or isosteric replacement. Recent studies have begun to
explore the effect of MBP isosteric replacement in metalloen-
zyme inhibition and pharmacokinetic profile.[41,42]
It has been ten years since the first report of NDM-1, with
>24 NDM variants currently identified.[4,14,21] Some NDM var-
iants exhibit an increase in thermal stability, ZnII affinity, and
mononuclear ZnII activity that may provide an evolutionary ad-
vantage when ZnII availability is low.[22–24] Unfortunately, even
with the rapid spread of the blaNDM gene and the evolution of
NDM variants, little advancement has been made in inhibitor
development. There are currently ꢀ525 inhibitors reported in
literature (representative structures shown in Figure 1),[25] but
In this study, we report the development and evaluation of
10 DPA isosteres (Figure 2) as inhibitors of NDM-1 and related
MBLs. A selection of carboxylic acid isostere motifs was chosen
to span a range of acidity (pKa) values and metal coordination
preferences. Varying the identity of the isostere impacts both
inhibition value and inhibition mechanism. Additionally, we
show the re-engineering of an isostere from a metal-stripping
mechanism to one that favors the formation of a ternary com-
plex. This study provides a roadmap for the isosteric replace-
ment of current and future metal-binding motifs for the gener-
ation of new entities in NDM-1 inhibitor design and may be
adopted for the inhibitor development of MBLs and other met-
alloenzymes.
Figure 1. Representative inhibitors of NDM-1.[28–36]
many inhibitors share similar structural features and none have
progressed to clinical trials. The flexible active site of NDM-1,
the diversity of related MBLs, a lack of understanding for inhib-
ition mechanisms, and misinterpretation of structural data
have all contributed to delayed inhibitor development.[26,27]
A
survey of current NDM-1 inhibitors reveals a large portion of
inhibitors bear a carboxylic acid motif.[25,26] In line with this, our
lab previously used a fragment-based drug discovery (FBDD)
approach to identify dipicolinic acid (DPA) as a lead metal-
binding pharmacophore (MBP) for NDM-1 inhibitor develop-
ment.[28] Derivatization of DPA was performed to obtain 4-(3-
Figure 2. DPA isosteres reported herein.
aminophenyl)pyridine-2,6-dicarboxylic acid (Figure 1) as
a
highly selective inhibitor for NDM-1 (IMP-1 and VIM-2) that in- Results and Discussion
hibits by forming a stable NDM-1:ZnII:inhibitor ternary com-
Isostere syntheses
plex. In this study, we further investigate the DPA MBP as an
inhibitor for NDM-1 using the concept of isosteric replace-
ment.
The syntheses of isosteres 1–10 are presented in Schemes 1–3.
Synthesis of isosteres 1, 4, 9, and 10 are summarized in
Scheme 1. Isostere 1 was obtained from a palladium-catalyzed
Hirao cross-coupling reaction of commercially available methyl
6-bromopicolinate and diethyl phosphate, followed by acid hy-
drolysis. Isostere 4 was achieved by conversion of the methyl
6-bromopicolinate to a nitrile via the Rosenmund–von Braun
reaction, followed by an azide–nitrile cycloaddition. Isosteres 9
and 10 were synthesized by palladium-catalyzed Stille coupling
of methyl 6-bromopicolinate with the corresponding organotin
reagents, followed by saponification. Syntheses of isosteres 2,
Isosteres bear similar structural and physical properties to
the parent functional group and are used as surrogates in an
effort to improve the overall drug-likeness (i.e., increased per-
meability, better pharmacokinetics, and/or decreased toxicity)
of the original molecule. A prominent example includes substi-
tuting the carboxylic acid functional group with carboxylic acid
isosteres.[37,38] There are an estimated >450 marketed drugs
(including nonsteroidal anti-inflammatory drugs, antibiotics,
anticoagulants, and cholesterol-lowering statins) that use a car-
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Scheme 3. Synthesis of isostere 7: a) Methanesulfonyl chloride, triethylamine,
CH2Cl2, 08C, 16 h, then 1m NaOH/THF, RT, 16 h, two steps 12.4%.
Inhibition assays
A preliminary screen of isosteres 1–10 was performed against
NDM-1 using meropenem as substrate (at a single concentra-
tion of 180 mm, Table 1). Meropenem was observed to have a
Table 1. IC50 values for DPA isostere inhibition of NDM-1 catalyzed mero-
Scheme 1. Synthesis of isosteres 1, 4, 9, and 10: a) Pd2(dba)3, Pd(dppf)Cl2, di-
ethyl phosphate, triethylamine, toluene, 908C, 20 h, then 6m HCl, 1008C,
20 h, two steps 19%; b) CuCN, pyridine, 1168C, 4 h, 21%; c) NaN3, NH4Cl,
DMF, 1308C, 20 h, then 2m HCl, 258C, 1 h, two steps 98%; d) 2-(tributylstan-
nyl)thiazole/2-(tributylstannyl)oxazole, Pd(PPh3)2Cl2, THF, 758C, 18 h, then 3:1
1m NaOH/THF, 708C, 3 h, two steps 8–74%.
penem hydrolysis.
Compound
IC50 [mm][a]
Compound
IC50 [mm][a]
DPA
1
2
3
4
0.84Æ0.04
0.31Æ0.01
0.13Æ0.01
7.7Æ0.6
6
7
8
9
10
>10
>10
>10
>10
>10
7.0Æ0.5
5
>10
3, 5, 6, and 8 are summarized in Scheme 2. Briefly, compound
11 was obtained from commercially available methyl 6-(hy-
droxymethyl)picolinate. Substitution of the alkyl bromide of
compound 11 with various nucleophiles, followed by hydroly-
sis yielded isosteres 2, 3, 5, 6, and 8. Lastly, methyl 6-aminopi-
colinate was treated with methanesulfonyl chloride, followed
by saponification to yield isostere 7 (Scheme 3).
[a] Activity measurements were taken in triplicate using varying inhibitor
concentrations that bracket each IC50 value, with fitting errors listed
above.
K
M,NDM-1 =80Æ7 mm and kcat =15.3Æ0.4 sÀ1. Compounds 1–4
exhibited inhibition against NDM-1 with IC50 values ranging
from 0.13–7.7 mm. Compounds 1 and 2 (both bearing a phos-
phonic acid) exhibited lower IC50 values (130Æ10 and 310Æ
10 nm, respectively) than that of DPA (840Æ40 nm), while 3
and 4 revealed higher IC50 values (7.7Æ0.6 and 7.0Æ0.5 mm, re-
spectively). Interestingly, compound 5, which possesses a simi-
lar acidic motif as 1 and 2 (a sulfonic acid versus a phosphonic
acid) showed no inhibition at concentrations up to 10 mm. No-
tably, all compounds where the substituted moiety was not
acidic (6–10) showed no appreciable inhibition at concentra-
tions up to 10 mm.
To determine the ability of isosteres to inhibit other B1
MBLs, the inhibition of 1–4 against NDM-1, IMP-1, and VIM-2
was investigated. An alternative substrate, fluorocillin
(KM,NDM-1 =460 nm), was used to increase assay sensitivity.[28,43]
Lower IC50 values for fluorocillin relative to meropenem were
expected due to using a smaller [S]/KM ratio in these experi-
ments. IC50 values determined for NDM-1 inhibition via fluoro-
cillin showed the same trends as with meropenem, with 1 and
2 showing the lowest IC50 values, followed by the parent DPA
fragment, with 3 and 4 yielding higher IC50 values among
these five compounds (Table 2). Similar trends, but with IC50
values about an order of magnitude higher, were observed for
IMP-1 and VIM-2 as well.
Scheme 2. Synthesis of isosteres 2, 3, 5, 6, and 8: a) PBr3, CHCl3, 08C, 3 h,
70%; b) P(OEt)3, toluene, 1408C, 2 h, 80%; c) 6m HCl, 1008C, 27 h, 98%;
d) KCN, THF, 508C, 19 h, 49%; e) 12m HCl, 1008C, 12 h, then 1m NaOH,
608C, 6 h, 30%; f) Na2SO3, H2O, then 4m HCl, 1008C, 16 h, two steps 41%;
g) NaSO2CH3, DMF, 1208C, 2 h, then 3:1 1m NaOH/THF, 708C, 3 h, two steps
43%; h) CH3NHSO2CH3, K2CO3, ACN, 758C, 25 h, then 1m NaOH, 708C, 3 h,
two steps 55%.
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enzyme.[28] The spectrum of CoCo-NDM-1 shows an intense
peak between 320–350 nm, which has been assigned to a cys-
teine-to-CoII ligand-to-metal charge transfer (LMCT) band, and
multiple peaks between 500–650 nm, which have been as-
signed to CoII ligand field bands (Figure 4).[22,47] The inclusion
Table 2. IC50 values for isostere inhibition of B1 MBL catalyzed fluorocillin
hydrolysis.
Compound
IC50 [mm][a]
NDM-1
IMP-1
VIM-2
DPA
1
2
3
4
0.33Æ0.04
0.064Æ0.001
0.068Æ0.002
2.05Æ0.09
2.2Æ0.1
2.54Æ0.04
0.85Æ0.06
0.63Æ0.01
59Æ5
2.34Æ0.04
0.85Æ0.02
0.62Æ0.01
35Æ1
22Æ1
27Æ2
[a] Activity measurements were taken in triplicate using varying inhibitor
concentrations that bracket each IC50 value, with fitting errors listed
above.
Equilibrium dialysis
Equilibrium dialysis experiments were performed to give in-
sight into the inhibition mechanism (metal stripping versus ter-
nary complex formation) of DPA and the active isosteres. After
protein purification, NDM-1 was exchanged into ammonium
acetate buffer to facilitate the use of ICP-AES in determining
metal content. Metal analyses of the resulting protein showed
that the enzyme binds ꢀ1.7 equivalents of ZnII (Figure 3).
Figure 3. ZnII content of NDM-1 (8 mm) upon incubation with increasing con-
centrations of captopril, DPA, and 1–4 (16–128 mm).
Figure 4. UV/Vis spectrum of CoCo-NDM-1 with captopril and EDTA (top),
and with DPA and 1–4 (bottom).
While holoNDM-1 is expected to bind 2 equivalents of ZnII, pre-
vious studies have shown that one of the ZnII ions binds more
weakly than the other, resulting in protein that contain less
than the full complement of two ZnII ions.[44,45] As previously
reported, incubation of NDM-1 with l-captopril, a known com-
petitive inhibitor (Figure 1),[46] did not show any evidence of
ZnII removal. In contrast, the ZnII content of NDM-1 was signifi-
cantly decreased when incubated with DPA.[28] Isosteres 1–4
exhibited different levels of ZnII removal from NDM-1. Com-
pounds 3 and 4 behaved more like captopril, removing only
small amounts of ZnII from NDM-1 even at concentrations up
to 128 mm. Conversely, compounds 1 and 2 removed more ZnII
from NDM-1 than the parent DPA.
of captopril to the sample results in large changes in the
ligand field transitions, and an increase in the LMCT band.
These changes are consistent with the formation of a NDM-
1:captopril ternary complex, with the captopril sulfur bridging
the two CoII ions.[48] Incubation of CoCo-NDM-1 with EDTA re-
sulted in a significant decrease of the LMCT and ligand field
peaks, indicating that EDTA strips the metal from NDM-1.[28] In-
cubation of CoCo-NDM-1 with compounds 1–4 and DPA result-
ed in a reduction of absorbance at 500–650 nm (Figure 4),
which indicates that CoII is being removed from the active site
to varying degrees. Compound 2 appears to remove the most
CoII, followed by compound 1, DPA, 3, and then 4. The findings
with CoCo-NDM-1 are wholly consistent with the equilibrium
dialysis results.
UV/Vis spectroscopy
As previously reported, UV/Vis spectrophotometry of CoII-sub-
stituted NDM-1 can be used to probe inhibitor binding to the
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Derivatization of isostere 2
Although 2 was shown to remove ZnII from NDM-1, we sought
to reduce the metal-removal behavior via elaboration using a
fragment-growth strategy. We previously had success using
this approach to optimize DPA.[28] Also during the time of this
study, a structure of 2 in complex with IMP-1 (PDB ID: 5HH4)
was reported.[49] In this structure, the Zn1 of IMP-1 is coordinat-
ed by residues H116, H118, H196, and a bridging hydroxide in
a tetrahedral coordination geometry (Figure 5, standard BBL
Scheme 4. Synthesis of derivatives of isostere 2 (compounds 18a–m):
a) MeOH, H2SO4 (cat.), 708C, overnight, 60%; b) P2O5, TBAB, toluene, 1008C,
3 h, 75%; c) NaBH4, MeOH/CH2Cl2, 08C, 1 h, 80%; d) PBr3, CHCl3, 08C, 1 h,
68%; e) P(OEt)3, toluene, 1408C, 22 h, 92%; f) boronic acid, K3O4P, Pd(PPh3)4,
1,4-dioxanes, 808C, 18 h, 20–90%; g) 6m HCl, 1008C, 24 h, 29–97%.
Table 3. IC50 values for compounds 18a–m against NDM-1 catalyzed
meropenem hydrolysis.
R[a]
IC50 [mm][b]
para
meta
ortho
H
18a 0.41Æ0.02
18b 0.20Æ0.01
18e 0.48Æ0.01
18h 0.30Æ0.01
18k 0.40Æ0.01
–
–
OCH3
OH
NH2
Cl
18c 0.42Æ0.02
18 f 0.28Æ0.01
18i 0.40Æ0.02
18l 1.29Æ0.07
18d 0.30Æ0.01
18g 0.28Æ0.01
18j 0.24Æ0.01
18m 0.95Æ0.05
[a] R group positioning is shown in Scheme 4. [b] Activity measurements
were taken in triplicate using varying inhibitor concentrations that brack-
et each IC50 value, with fitting errors listed above.
Figure 5. 6-(Phosphonomethyl)picolinic acid (2) was reported as a potent in-
hibitor of B1 and B3 MBLs and able to restore b-lactam activity in MIC
assays. Shown above is 2 complexed with IMP-1 (PDB ID: 5HH4).[49] ZnII ions
are shown in orange, coordinating ligands and protein ribbon are shown in
grey, 2 is shown in green, and ligand–protein interactions are shown with a
yellow dashed line. The image was rendered with Molecular Operating Envi-
ronment (MOE).
um bromide (TBAB) and phosphorus pentoxide (P4O10). Com-
pound 13 was treated with sodium borohydride (NaBH4) at
08C to obtain 14, which was further converted into intermedi-
ate 15 by phosphorous tribromide (PBr3). Compound 16 was
obtained by heating 15 and triethyl phosphite in toluene at
1408C for 22 h. Intermediates 17a–m were obtained via Suzuki
cross-coupling procedures using the corresponding boronic
acid, (Pd(PPh3)4), and K3PO4. Compounds 18a–m were ob-
tained by hydrolysis.
numbering). The bridging hydroxide also coordinates to Zn2,
which is further ligated by residues D120, C221, and H263, as
well as by the pyridine nitrogen donor and carboxylic acid of 2
(in an overall octahedral coordination geometry). The phos-
phonic acid motif of 2 is shown to hydrogen bond with the
bridging hydroxide ion and a nearby S80 residue. Guided by
this crystallographic data and the homology of IMP-1 and
NDM-1 enzymes, a small library of 2 derivatives (18a–m) were
designed, synthesized, and tested for NDM-1 inhibition.
Compounds 18a–m did not yield greater inhibitory activity
than that of 2 (IC50 =0.13Æ0.01 mm). A simple aryl ring (18a),
or an aryl ring with methoxy (18b–d) or hydroxy substituent
(18e–g) were generally well-tolerated, with IC50 =0.20Æ0.01–
0.48Æ0.01 mm, but did not lead to a marked decrease in IC50
value compared with that of 2. Given the structural similarity
of 18h–j (bearing the aniline substituent) to the previous re-
ported inhibitor, (4-(3-aminophenyl)pyridine-2,6-dicarboxylic
acid) (Figure 1), no improvement in inhibition was observed
relative to that of 2 (IC50 =0.24Æ0.01–0.40Æ0.02 mm). Notably,
a chlorine substituent in the ortho- and meta- position of the
aryl ring yield a 2- to 3-fold loss in potency. Due to the similari-
The synthesis of 18a–m is presented in Scheme 4 and the
IC50 values (measured for NDM-1 catalyzed hydrolysis of mero-
penem) are reported in Table 3. This library consisted of simple
aryl derivatives substituted at the 4-position of the pyridine
ring. The aryl ring bears substituents (methoxy, hydroxy, amine,
and chlorine) in the para, ortho, and meta positions of the ring
in attempt to generate interactions with nearby residue side
chains. To prepare these compounds, 4-hydroxypyridine-2,6-di-
carboxylic acid was esterified to 12 with MeOH and catalytic
H2SO4. Next, 12 was converted into 13 with tetrabutylammoni-
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ties between the observed inhibition values, no structure–ac-
tivity relationship (SAR) could be obtained. Although isosteric
replacement of the carboxylate group maintains the ability to
bind metal ions, the relative positioning of attached substitu-
ents is likely altered, possibly due to changes in metal coordi-
nation geometries. In the case of DPA and NDM-1, this pre-
vents a simple group-swapping approach, and may necessitate
re-optimization starting with the new isostere fragment. IC50
values of 18a–m against IMP-1 were also obtained (Table S1,
Figure S1).
In an attempt to decrease the propensity to strip metal from
the NDM-1 active site, a small library of derivatives of com-
pound 2 were synthesized (compounds 18a–m). It was pre-
dicted that 18i, bearing the same amine backbone as a report-
ed inhibitor that uses DPA as a metal-binding group, would
result in a decrease in the observed IC50 value. Unfortunately,
18i (and other derivatives) did not yield a lower IC50 value
than that of 2, suggesting that the isosteres are not exactly
equivalent substitutions of their parent fragment. Isosteric re-
placement may alter the relative positioning of distant sub-
stituents and would necessitate further optimization. Addition-
ally, compounds 18a–m were screened against IMP-1
(Table S1, Figure S1); however, no structure activity relationship
was observed, with relatively flat IC50 values spanning 1.20Æ
0.05–4.1Æ0.2 mm. Dialysis experiments of derivatives 18b and
18i against NDM-1 reveal a lower tendency to remove ZnII
from the NDM-1 active site, albeit with no decrease in the IC50
value, relative to that of isostere 2 (Figure S2).
Despite the understanding that compounds can inhibit
MBLs through a number different mechanisms (covalent inhibi-
tion, metal-chelation, reversible competitive inhibition, alloste-
ric inhibition, etc.), the mechanism of action of most reported
MBL inhibitors is not well defined.[26] Many current studies
focus on achieving low IC50 values, with little consideration re-
garding the mechanism of action. Here, we clearly demon-
strate how the mechanism of inhibition can significantly differ
between similar isosteres and homologous enzymes and illus-
trate challenges to optimizing inhibitors with metal-stripping
mechanisms. In the design of future MBL inhibitors, a loss of
potency may be preferred when selecting compounds with a
desired mechanism of action for further optimization (i.e., se-
lection of 4 over 2). This kind of early stage evaluation of how
the inhibitor interacts with the active site will crucial for accel-
erating NDM (and other MBLs) inhibitor development.
Although lower IC50 values for compounds 18a–m (relative
to that of isostere 2) were not observed, previous evaluation
of DPA isosteres 1–4 has shown that an increase in IC50 value
may be due to a shift in inhibitor mechanism (from metal strip-
ping to ternary complex formation). To evaluate if this was the
case for 18a–m, two candidates (18b and 18i) were selected
for further inhibition mechanism analysis. Indeed, dialysis ex-
periment revealed 18b and 18i did not perturb the ZnII con-
tent of NDM-1 at concentrations as high as 16 mm, with only
slight ZnII removal at concentration of 32 mm (Figure S2). In ad-
dition, one equivalent of ZnII was retained at the highest
tested concentration (128 mm) for 18b and 18i, while less than
one equivalent was retained when performing the same ex-
periment with compound 2. This further supports our previous
findings and shows the possibility to re-engineer a compound
which removes metal from the active site of NDM-1 to one
which favors ternary complex formation.
Herein, we investigate the isosteric replacement of a carbox-
ylic acid group of DPA with various surrogate structures. Com-
pounds 1–10 yield a range of chemical diversity (various acidic
or metal-binding groups) and show that the choice of the car-
boxylate isostere not only impacts the IC50 value, but also the
propensity for metal removal. Replacement of one carboxylic
acid with a phosphonic substituent (2) results in a MBP with
an extraordinarily low IC50 value, but also with a greater ten-
dency for NDM-1 ZnII removal. Replacement of the carboxylic
acid with a tetrazole motif (4) yield a higher IC50 value, but de-
creases NDM-1 ZnII removal. Equilibrium dialysis data, in con-
junction with UV/Vis spectroscopy of CoCoNDM-1, reveals dif-
ferences in inhibition mechanism: inhibition by 2 occurs pri-
marily by ZnII removal, likely forming the inactive mono-ZnII
NDM-1, while inhibition by 4 is mainly via formation of a terna-
ry complex at the dinuclear ZnII site. Because 4 forms a ternary
complex at the active site, we can use IC50 and the Cheng–
Prusoff relationship for competitive inhibitors to calculate a Ki
of 2.2Æ0.5 mm.[50] It is important to note that calculating Ki
values for metal-stripping inhibitors (such as 1 or 2) is not ap-
propriate because changes in IC50 may not reflect changes in
binding affinity. Available data suggesting potential varying in-
hibition mechanisms between homologous MBLs and 2 (terna-
ry complex formation with IMP-1,[49] but metal-stripping with
NDM-1) was unexpected. This difference may be due to a
lower binding affinity for ZnII in the Zn2 site of NDM-1 (Kd =
2 mm)[45] relative to that of IMP-1 (Kd =0.3 mm),[51,52] resulting in
more facile metal removal. Future studies are required to eluci-
date and conclude the observed mechanistic differences of 2
against IMP-1 and NDM-1.
Conclusions
In summary, we report the synthesis and inhibitory activity of
ten DPA isosteres and investigated the mechanism of action of
the most active compounds. We show that the exchange of
the carboxylic acid with other acidic isosteres not only drasti-
cally affects the IC50 value but also the mechanism of action.
The compounds which showed double-digit nanomolar IC50
are predicted to act via a metal-stripping mechanism, while
compounds which displayed single-digit micromolar IC50 are
predicted to form a ternary complex with the enzyme. Prelimi-
nary data suggests differences in inhibition mechanism be-
tween homologous NDM-1 and IMP-1 and isostere 2. Addition-
ally, we demonstrate the potential to morph metal-stripping
compounds into inhibitors which favor ternary complex forma-
tion. This study demonstrates the importance of considering
the inhibition mechanism, and the utility of bioisosteric re-
placement for routinely used metal binding motifs (i.e., carbox-
ylic acid) in NDM-1 inhibitor development.
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[D6]DMSO): d=8.02 (t, J=7.7 Hz, 1H), 7.95 (d, J=7.5 Hz, 1H), 7.67
(d, J=7.6 Hz, 1H), 3.38 (s, 1H), 3.32 ppm (s, 1H); 13C NMR
(126 MHz, [D6]DMSO): d=166.6, 155.4, 148.0, 137.9, 127.8, 122.8,
38.3 ppm. ESI-MS(À) calculated for [C7H8NO5P]À m/z 217.01, found
m/z 216.00 [MÀH]À.
Experimental Section
Chemistry
All reagents and solvents were obtained from commercial sources
and used without further purification. Corning UV-transparent 96-
well microplates (3635), Corning black polystyrene round-bottom
96-well microplates (3792), 3-((3-cholamidopropyl)dimethylammo-
nio)-1-propanesulfonate (CHAPS), 4-(2-hydroxyethyl)-1-piperazinee-
thanesulfonic acid (HEPES), dimethyl sulfoxide (DMSO), l-captopril
and fluorocillin green 495/525 b-lactamase substrate, soluble prod-
uct, were purchased from Thermo Fisher Scientific Inc. (Fair Lawn,
NJ, USA). All other reagents were purchased from Sigma–Aldrich
Inc. (St. Louis, MO, USA). Screening assays were performed on a
PerkinElmer Victor3 V 1420 multilabel counter plate reader. Fluores-
cent assays were performed on a PerkinElmer Victor3 V fluorescent
plate reader. Column chromatography was performed using a Tele-
dyne ISCO CombiFlash Rf system with prepacked silica cartridges.
6-(Carboxymethyl)picolinic acid (3). To a solution of 11 (2.5 g,
10.87 mmol) dissolved in THF (50 mL) was added a pre-dissolved
solution of KCN (1.1 g, 16.30 mmol) in water (9 mL). All supplies in
contact with KCN were quenched with 1m sodium thiosulfate
prior to disposal. The mixture was stirred at 508C for 19 h and
quenched with 1m sodium thiosulfate solution. The mixture was
extracted with CH2Cl2 (25 mLꢁ5). The combined organic layers
were dried over MgSO4, filtered, and concentrated in vacuo. Inter-
mediate methyl 6-(cyanomethyl)picolinate was purified by flash
column chromatography, eluting at 58% ethyl acetate in hexanes
1
to yield yellow crystals in 49% yield (941 mg, 5.34 mmol). H NMR
(400 MHz, [D6]DMSO): d=8.09–7.95 (m, 2H), 7.68 (t, J=5.5 Hz, 1H),
4.30 (s, 2H), 3.88 ppm (s, 3H); ESI-MS(+) calculated for [C9H9N2O2]+
m/z 177.06, found m/z 177.18 [M+H]+.
1
All H NMR and 13C NMR spectra were recorded at either ambient
temperature or at 358C using Varian 400 Mercury Plus or Varian
VX500 instrument located in the Department of Chemistry and Bio-
chemistry at the University of California San Diego. Mass spectrom-
etry data were obtained from the University of California San
Diego Chemistry and Biochemistry Mass Spectrometry Facility
(MMSF). The purity of all compounds used for screening were de-
termined to be ꢁ95% by high-performance liquid chromatogra-
phy (HPLC).
The intermediate, methyl 6-(cyanomethyl)picolinate was dissolved
in 12m HCl and heated at 1008C for 12 h. The solvent was re-
moved in vacuo and the crude was hydrolyzed in 3 mL of 1m
NaOH at 608C for 6 h. The solution was the acidified with 4m HCl
to pH 4, and product 3 was collected by vacuum filtration as a
white solid in 30% yield (37 mg, 0.20 mmol); 1H NMR (400 MHz,
[D6]DMSO): d=7.98–7.86 (m, 2H), 7.56 (d, J=6.3 Hz, 1H), 3.83 ppm
(s, 2H); 13C NMR (126 MHz, [D6]DMSO): d=172.1, 166.6, 155.8,
148.3, 138.3, 128.0, 123.4, 43.6 ppm; ESI-MS(+) calculated for
[C8H8NO4]+ m/z 182.04, found m/z 182.25 [M+H]+.
6-Phosphonopicolinic acid (1). A solution of methyl 6-bromopico-
linate (300 mg, 1.39 mmol), diethyl phosphate (172.0 mL,
1.39 mmol), Pd2(dba)3 (67 mg, 0.69 mmol), Pd(dppf)Cl2 (77 mg,
0.14 mmol), and trimethylamine (387.0 mL, 2.78 mmol) were dis-
solved in toluene (10 mL), and heated at 908C for 20 h. Ethyl ace-
tate (20 mL) was added to the reaction mixture, and the solution
was filtered through a pad of Celite. The collected organic layers
were concentrated in vacuo. The crude mixture was purified by
flash column chromatography, with the intermediate eluting at
50% ethyl acetate in hexanes. The intermediate was heated under
reflux conditions with 6m HCl (3 mL) for 20 h. The excess HCl was
removed in vacuo, and co-evaporated with copious amounts of
methanol and water until a precipitate was observed. The product
was collected by vacuum filtration as a white solid in 19% yield
(54 mg, 0.27 mmol). 1H NMR (400 MHz, CD3OD): d=8.26 (d, J=
7.7 Hz, 1H), 8.19–8.00 ppm (m, 2H); 13C NMR (126 MHz, [D6]DMSO):
d=166.4, 156.2, 149.0, 138.0, 129.4, 126.1 ppm; ESI-MS(À) calculat-
ed for [C6H5NO5P]À m/z 202.10, found m/z 201.98 [MÀH]À.
6-(1H-Tetrazol-5-yl)picolinic acid (4). Methyl 6-bromopicolinate
(3 g, 13.89 mmol) and copper(I) cyanide (2.49 g, 27.77 mmol) were
dissolved in pyridine (120 mL) and heated at 1168C for 4 h. The re-
action was monitored by TLC. Upon completion of the reaction,
the mixture was filtered, and the filtrate was concentrated in va-
cuo. Aqueous NaHCO3 (50 mL) and CH2Cl2 (50 mL) were added to
the crude mixture. The aqueous layer was extracted with CH2Cl2
(50 mLꢁ3), and the combined organic layers were dried over
MgSO4, concentrated in vacuo, and purified with flash chromatog-
raphy. Intermediate methyl 6-cyanopicolinate eluted 40% ethyl
acetate in hexanes as a yellow solid in 20% yield (470 mg,
2.90 mmol). 1H NMR (400 MHz, CDCl3): d=8.34 (d, J=7.9 Hz, 1H),
8.04 (t, J=7.9 Hz, 1H), 7.88 (d, J=7.8 Hz, 1H), 4.04 ppm (s, 3H);
ESI-MS(+) calculated for [C8H7N2O2]+ m/z 163.05, found m/z 163.10
[M+H]+.
In a round-bottom flask, intermediate methyl 6-cyanopicolinate
(200 mg, 1.23 mmol), sodium azide (408 mg, 6.29 mmol), and am-
monia hydrochloride (336 mg, 6.29 mmol) were dissolved in anhy-
drous DMF (10 mL). The reaction was heated under N2 at 1308C for
20 h. After cooling, the inorganic salts were removed by vacuum
filtration, and washed with hot DMF. The organic filtrate was con-
centrated in vacuo to yield a yellow solid. The solid was suspended
in a solution of 2m HCl (3 mL), and stirred at 258C for 1 h. The pre-
cipitate was collected by vacuum filtration and washed with copi-
ous amounts of cold water to afford product 4 as a white solid in
6-(Phosphonomethyl)picolinic acid (2). A solution of 11 (200 mg,
0.87 mmol) and P(OEt)3 (1.6 g, 9.56 mmol) were heated in toluene
(20 mL) at 1408C for 2 h to give a clear liquid solution. Excess
P(OEt)3 and toluene were removed in vacuo, and the intermediate
was purified by flash column chromatography eluting at 100%
ethyl acetate in hexanes as a clear oil in 80% yield (200 mg,
0.70 mmol). 1H NMR (400 MHz, CDCl3): d=8.02 (d, J=8.0 Hz, 1H),
7.80 (td, J=7.8, 2.1 Hz, 1H), 7.62 (d, J=8.0 Hz, 1H), 4.13–4.06 (m,
4H), 3.99 (d, J=2.0 Hz, 3H), 3.53 (d, J=22.0 Hz, 2H), 1.31–
1.25 ppm (m, 6H); ESI-MS(+) calculated for [C12H19NO5P]+ m/z
288.10, found m/z 288.29 [M+H]+.
1
98% yield (232 mg, 1.21 mmol). H NMR (400 MHz, [D6]DMSO): d=
8.37 (d, J=7.5 Hz, 1H), 8.26–8.13 ppm (m, 2H); 13C NMR (126 MHz,
[D6]DMSO): d=165.2, 155.1, 149.1, 144.3, 140.3, 126.8, 126.2 ppm.
ESI-MS(À) calculated for [C7H4N5O2]À m/z 190.04, found m/z 189.25
[MÀH]À.
The intermediate, methyl 6-((diethoxyphosphoryl)methyl)picolinate
(200 mg, 0.70 mmol) was held at reflux in 6m HCl (5 mL) for 27 h.
The excess HCl was removed in vacuo and co-evaporated with co-
pious amounts of methanol and water until a white precipitate
was observed. Compound 2 was collected by vacuum filtration as
6-(Sulfomethyl)picolinic acid (5). In a round-bottom flask, 11
(200 mg, 0.87 mmol) and Na2SO3 (110 mg, 0.87 mmol) were dis-
1
a white solid in 86% yield (130 mg, 0.60 mmol). H NMR (400 MHz,
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solved in H2O (10 mL), and heated at 1008C for 16 h. The reaction
was cooled to room temperature, and H2O was removed in vacuo
until it reached one-third of the original volume. The white precipi-
tate was collected by vacuum filtration as the ester intermediate.
The intermediate was hydrolyzed by stirring in 4m HCl at 1008C
for 16 h. The excess HCl was removed in vacuo, and the product
was co-evaporated with copious amounts of MeOH and H2O until
white crystals were observed. The precipitate was collected by
vacuum filtration to yield compound 5 as a white crystal in 41%
(s, 3H); 13C NMR (126 MHz, [D6]DMSO): d=166.5, 157.4, 148.5,
138.9, 125.7, 124.1, 54.9, 36.7, 35.2 ppm. ESI-MS(À) calculated for
[C9H11N2O4S]À m/z 243.04, found m/z 243.31 [MÀH]À.
6-(Thiazol-2-yl)picolinic acid (9). In a round-bottom flask, methyl
6-bromopicolinate (300 mg, 1.39 mmol) and 2-(tributylstannyl)thia-
zole (624 mg, 1.67 mmol) were dissolved in anhydrous THF
(15 mL). The solution was purged with N2 for 15 min, followed by
the addition of bis(triphenylphosphine)palladium(II) dichloride
(97 mg, 0.14 mmol). The reaction was heated at 758C for 18 h.
Upon completion, water (20 mL) and ethyl acetate (20 mL) were
added to the brown slurry. The aqueous layer was extracted with
ethyl acetate (20 mLꢁ3), and the combined organic layers were
dried over MgSO4, filtered, and concentrated in vacuo. The ester in-
termediate was purified by column chromatography, eluting at
45% ethyl acetate in hexanes as a yellow solid. The intermediate
was hydrolyzed in 3:1 1m NaOH/THF at 708C for 3 h. THF was re-
moved in vacuo, and the aqueous layer was acidified with 4m HCl
to pH 4. The precipitate was collected by vacuum filtration to
1
yield (77.0 mg, 0.35 mmol). H NMR (400 MHz, [D6]DMSO): d=8.08–
7.88 (m, 2H), 7.79 (d, J=6.0 Hz, 1H), 4.06 ppm (s, 2H); 13C NMR
(126 MHz, [D6]DMSO): d=164.7, 155.0, 145.2, 140.7, 129.9, 124.1,
57.6 ppm; ESI-MS(À) calculated for [C7H6NO5S]À m/z 216.10, found
m/z 216.08 [MÀH]À.
6-((Methylsulfonyl)methyl)picolinic acid (6). In a round-bottom
flask, 11 (300 mg, 1.30 mmol) and sodium methanesulfinate
(266 mg, 2.61 mmol) were dissolved in DMF (15 mL). The solution
was heated at 1208C for 2 h. DMF was removed in vacuo, and the
crude product was purified by flash column chromatography elut-
ing at 90% ethyl acetate in hexanes. The intermediate was hydro-
lyzed by stirring in 3:1 1m NaOH/THF at 708C for 3 h. The THF was
removed in vacuo, and the solution was acidified with 4m HCl
until pH 4. The aqueous layer was extracted with ethyl acetate
(20 mLꢁ3) to afford 6 a white solid in 43% yield over two steps
(120 mg, 0.56 mmol). 1H NMR (400 MHz, [D6]DMSO): d=8.03 (s,
2H), 7.70 (t, J=4.5 Hz, 1H), 4.75 (s, 2H), 3.08 ppm (s, 3H); 13C NMR
(126 MHz, [D6]DMSO): d=166.3, 150.7, 149.2, 138.9, 129.3, 124.5,
61.5, 41.1 ppm; ESI-MS(À) calculated for [C8H8NO4S]À m/z 214.02,
found m/z 214.16 [MÀH]À.
6-(Methylsulfonamido)picolinic acid (7). Commercially available
methyl 6-aminopicolinate (300 mg, 1.97 mmol) was dissolved in a
solution of triethylamine/CH2Cl2 (1 mL:5 mL) and cooled to 08C.
Next, methanesulfonyl chloride (168.0 mL, 2.17 mmol) was added
dropwise to the mixture. The reaction was stirred at room temper-
ature for 16 h. The solvent was removed in vacuo, and the crude
mixture was purified by flash column chromatography. The ester
intermediate eluted at 62% ethyl acetate in hexanes as a white
solid. The intermediate was hydrolyzed by stirring at room temper-
ature for 16 h in 3:1 1m NaOH/THF (8 mL). The excess THF was re-
moved in vacuo, and the solution was acidified with 4m HCl to
pH 4. The aqueous layer was extracted with ethyl acetate (10 mLꢁ
3), and the combined organic layers were dried over MgSO4, fil-
tered, and concentrated in vacuo to afford 7 as a white solid.
1H NMR (400 MHz, CD3OD): d=7.89 (t, J=7.9 Hz, 1H), 7.79 (d, J=
7.5 Hz, 1H), 7.28 (d, J=8.3 Hz, 1H), 3.33 ppm (s, 3H); 13C NMR
(126 MHz, [D6]DMSO): d=166.2, 152.4, 147.1, 140.1, 119.5, 115.9,
42.5 ppm; ESI-MS(À) calculated for [C7H7N2O4S]À m/z 215.10, found
m/z 215.05 [MÀH]À.
afford compound
9 as a tan solid in 74% yield (213 mg,
1
1.03 mmol). H NMR (400 MHz, [D6]DMSO): d=9.18 (s, 1H), 8.68 (s,
1H), 8.22 (d, J=8.0 Hz, 1H), 8.05 (t, J=7.8 Hz, 1H), 7.94 ppm (d, J=
7.7 Hz, 1H); 13C NMR (126 MHz, [D6]DMSO): d=166.1, 157.0, 150.5,
149.0, 142.3, 139.7, 139.1, 124.2, 123.4 ppm; ESI-MS(À) calculated
for [C9H5N2O2S]À m/z 205.01, found m/z 205.18 [MÀH]À.
6-(Oxazol-2-yl)picolinic acid (10). In a round-bottom flask, methyl
6-bromopicolinate (300 mg, 1.39 mmol) and 2-(tributylstannyl)oxa-
zole (596 mg, 1.67 mmol) were dissolved in anhydrous THF
(15 mL). The solution was purged with N2 for 15 min, followed by
the addition of bis(triphenylphosphine)palladium(II) dichloride
(79 mg, 0.14 mmol). The solution was heated at 758C for 18 h.
Upon completion, water (20 mL) and ethyl acetate (20 mL) were
added to the slurry. The aqueous layer was extracted with ethyl
acetate (20 mLꢁ3), and the combined organic layers were dried
over MgSO4, filtered, and concentrated in vacuo. The ester inter-
mediate was purified by column chromatography, eluting at 60%
ethyl acetate in hexanes as a yellow solid. The intermediate was
hydrolyzed in 3:1 1m NaOH/THF at 708C for 3 h. THF was removed
in vacuo, and the aqueous layer was acidified with 4m HCl until
pH 4. The aqueous layer was extracted with ethyl acetate (20 mLꢁ
3), and the combined organic layers were dried over MgSO4, fil-
tered, and concentrated in vacuo to yield 10 as a white solid in 8%
1
yield (20 mg, 0.12 mmol). H NMR (400 MHz, [D6]DMSO): d=8.35 (s,
1H), 8.28 (dd, J=6.4, 2.6 Hz, 1H), 8.16–8.10 (m, 2H), 7.49 ppm (s,
1H); 13C NMR (126 MHz, [D6]DMSO): d=166.2, 160.0, 149.4, 145.9,
142.0, 139.4, 129.4, 126.2, 125.3 ppm; ESI-MS(À) calculated for
[C9H5N2O3]À m/z 189.03, found m/z 189.20 [MÀH]À.
Methyl 6-(bromomethyl)picolinate (11). To a solution of methyl 6-
(hydroxymethyl)picolinate (1.0 g, 5.98 mmol) in chloroform (25 mL)
at 08C was added PBr3 (1.9 g, 7.18 mmol) dropwise over the course
of 15 min. The reaction was stirred at room temperature for 3 h.
The reaction mixture was quenched with saturated sodium carbon-
ate in water and extracted with chloroform (20 mLꢁ4). The com-
bined organic layers were dried over MgSO4, filtered, and concen-
trated in vacuo. The product was purified by flash column chroma-
tography, eluting at 45% ethyl acetate in hexanes to afford 11 as a
6-((N-Methylmethylsulfonamido)methyl)picolinic acid (8). In a
round-bottom flask, 11 (158 mg, 0.69 mmol), N-methylmethanesul-
fonamide (74 mg, 0.69 mmol), and potassium carbonate (48 mg,
0.34 mmol) were dissolved in acetonitrile (10 mL) and heated at
758C for 20 h. The solvent was removed in vacuo and the crude
mixture was purified by flash column chromatography. The ester
intermediate eluted at 3% MeOH in CH2Cl2 as a yellow oil. The in-
termediate was hydrolyzed by stirring in 1m NaOH at 708C for 3 h.
The solution was the acidified with 4m HCl to pH 4, and the aque-
ous layer was extracted with ethyl acetate (20 mLꢁ4). The com-
bined organic layers were dried over MgSO4, filtered, and concen-
trated in vacuo to afford 8 a pale solid in 55% yield over two steps
(92 mg, 0.38 mmol). 1H NMR (400 MHz, [D6]DMSO): d=8.04–7.90
(m, 2H), 7.61 (d, J=7.3 Hz, 1H), 4.46 (s, 2H), 3.08 (s, 3H), 2.75 ppm
1
white crystal in 70% yield (95 mg, 4.14 mmol). H NMR (400 MHz,
CDCl3): d=8.06 (d, J=7.8 Hz, 1H), 7.86 (td, J=7.8, 1.4 Hz), 7.68 (d,
J=7.8 Hz, 1H), 4.64 (s, 2H), 4.01 ppm (s, 3H); ESI-MS(+) calculated
for [C8H9BrNO2]+ m/z 229.98, found m/z 230.24 [M+H]+.
Dimethyl 4-hydroxypyridine-2,6-dicarboxylate (12). Commercial-
ly-available
4-hydroxypyridine-2,6-dicarboxylic
acid
(10.0 g,
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Full Papers
55 mmol) was dissolved in MeOH (500 mL) and H2SO4 (cat.) was
added. The reaction was stirred at 708C for 16 h. The solvent was
removed in vacuo, and the crude mixture was purified by column
chromatography, eluting at 1% MeOH in CH2Cl2 as a yellow solid
General procedures for the synthesis of compounds 17a–m
In a round-bottom flask, compound 16 (1 equiv), the correspond-
ing boronic acid (1.2 equiv) and K3O4P (2 equiv) were dissolved in
1,4-dioxanes (5 mL). The solution was purged under N2 for 20 min,
followed by the addition of tetrakis(triphenylphosphine)palladi-
um(0) (0.1 equiv). The reaction was heated at 808C for 18 h under
N2. The crude mixture was filtered through a pad of Celite, washed
with ethyl acetate, and the combined organic layers were concen-
trated in vacuo. The crude products were purified by flash column
chromatography to afford derivatives 17a–m.
1
in 60% yield (7.0 g, 30 mmol). H NMR (400 MHz, CD3OD): d=7.59
(s, 2H), 3.96 ppm (s, 6H); ESI-MS(+) calculated for [C9H10NO5]+ m/z
212.06, found m/z 212.07 [M+H]+.
Dimethyl 4-bromopyridine-2,6-dicarboxylate (13). Tetrabutylam-
monium bromide (28.623 g, 88.789 mmol) and phosphorus(V)
oxide (12.6 g, 89 mmol) were dissolved in dry toluene (50 mL), and
heated at 1008C for 30 min under N2. Compound 12 (7.5 g,
36 mmol) was added to the solution and the mixture was heated
at 1008C for 3 h. The toluene layer was decanted, and an addition-
al 50 mL of toluene was added to the residual brown oil. The mix-
ture was heated at 1008C for an additional 30 min, and the toluene
layer was decanted again. Addition and removal of toluene was re-
peated three times. The combined toluene layers were dried in va-
cuo, and the crude mixture was purified by column chromatogra-
phy. Compound 13 eluted at 45% ethyl acetate in hexanes as
yellow needles in 75% yield (7.3 g, 27 mmol). 1H NMR (400 MHz,
[D6]DMSO): d=8.42 (s, 2H), 3.91 ppm (s, 6H); ESI-MS(+) calculated
for [C9H9BrNO4]+ m/z 273.97, found m/z 274.26 and 275.25 [M+
H]+.
Methyl
6-((diethoxyphosphoryl)methyl)-4-phenylpicolinate
1
(17a). Yield: 74% (59 mg, 0.16 mmol). H NMR (400 MHz, CD3OD):
d=8.26 (s, 1H), 7.90 (d, J=2.1 Hz, 1H), 7.77 (d, J=6.5 Hz, 2H),
7.65–7.31 (m, 3H), 4.28–4.07 (m, 4H), 3.65 (d, J=22.2 Hz, 2H),
1.28 ppm (t, J=7.0 Hz, 6H); ESI-MS(+) calculated for [C18H23NO5P]+
m/z 364.13, found m/z 364.11 [M+H]+.
Methyl 6-((diethoxyphosphoryl)methyl)-4-(4-methoxyphenyl)pi-
1
colinate (17b). Yield: 79% (120 mg, 0.31 mmol). H NMR (400 MHz,
[D6]DMSO): d=8.14 (s, 1H), 7.87 (s, 1H), 7.79 (d, J=7.3 Hz, 2H),
7.10 (d, J=7.7 Hz, 2H), 4.11–3.94 (m, 4H), 3.89 (s, 3H), 3.82 (s, 3H),
3.56 (d, J=21.7 Hz, 2H), 1.18 ppm (t, J=6.2 Hz, 6H); ESI-MS(+) cal-
culated for [C19H25NO6P]+ m/z 394.14, found m/z 394.28 [M+H]+.
Methyl 4-bromo-6-(hydroxymethyl)picolinate (14). In a round-
bottom flask, compound 13 (1.0 g, 3.65 mmol) was dissolved in a
solution of MeOH/CH2Cl2 (16 mL:4 mL) and cooled to 08C. NaBH4
(138 mg, 3.65 mmol) was added to the mixture and stirred at 08C
for 1 h. The reaction was quenched with aqueous NaHCO3 (10 mL).
The mixture was extracted with CH2Cl2 (10 mLꢁ3), and the com-
bined organic layers were dried in vacuo. The crude mixture was
purified by column chromatography, with 14 eluting at 70% ethyl
acetate in hexanes as a white crystal in 80% yield (689 mg,
2.80 mmol). 1H NMR (400 MHz, CD3OD): d=8.17 (s, 1H), 7.97 (s,
1H), 4.73 (s, 2H), 3.97 ppm (s, 3H); ESI-MS(+) calculated for
[C8H9BrNO3]+ m/z 245.98, found m/z 246.16 and 248.09 [M+H]+.
Methyl 6-((diethoxyphosphoryl)methyl)-4-(3-methoxyphenyl)pi-
colinate (17c). Yield: 58% (86 mg, 0.22 mmol). H NMR (400 MHz,
1
CD3OD): d=8.26 (s, 1H), 7.90 (s, 1H), 7.45 (t, J=8.0 Hz, 1H), 7.34
(d, J=7.8 Hz, 1H), 7.30 (s, 1H), 7.07 (d, J=8.2 Hz, 1H), 4.21–4.07
(m, 4H), 4.00 (s, 3H), 3.88 (s, 3H), 3.66 (d, J=22.2 Hz, 2H),
1.29 ppm (t, J=7.0 Hz, 6H); ESI-MS(+) calculated for [C19H25NO6P]+
m/z 394.14, found m/z 416.13 [M+Na]+.
Methyl 6-((diethoxyphosphoryl)methyl)-4-(2-methoxyphenyl)pi-
1
colinate (17d). Yield: 87% (125 mg, 0.32 mmol). H NMR (400 MHz,
CD3OD): d=8.20 (s, 1H), 7.81 (s, 1H), 7.50–7.36 (m, 2H), 7.15 (d, J=
8.4 Hz, 1H), 7.09 (t, J=7.5 Hz, 1H), 4.24–4.06 (m, 4H), 3.98 (s, 3H),
3.86 (s, 3H), 3.63 (d, J=22.2 Hz, 2H), 1.28 ppm (t, J=7.0 Hz, 6H);
ESI-MS(+) calculated for [C19H25NO6P]+ m/z 394.14, found m/z
416.13 [M+Na]+.
Methyl 4-bromo-6-(bromomethyl)picolinate (15). Compound 14
(400 mg, 1.63 mmol) was dissolved in CHCl3 (4 mL) and cooled to
08C. Next, PBr3 (528 mg, 1.95 mmol) was added dropwise, and the
reaction was stirred at 08C for 1 h. The reaction was tracked by
TLC. Upon completion, the reaction mixture was quenched with
aqueous Na2CO3 (40 mL), and the aqueous layer was extracted
with chloroform (40 mLꢁ4). The combined organic layers were
dried over MgSO4, filtered, and concentrated in vacuo to yield a
yellow oil with white precipitate. The crude product was purified
by flash column chromatography, with compound 15 eluting at
42% ethyl acetate in hexanes as a white solid in 68% yield
(342 mg, 1.11 mmol). 1H NMR (400 MHz, CDCl3): d=8.21 (s, 1H),
7.86 (s, 1H), 4.59 (s, 2H), 4.02 ppm (s, 3H); ESI-MS(+) calculated for
[C8H8Br2NO2]+ m/z 307.89, found m/z 308.07 and 310.00 [M+H]+.
Methyl 6-((diethoxyphosphoryl)methyl)-4-(4-hydroxyphenyl)pi-
1
colinate (17e). Yield: 20% (27 mg, 0.07 mmol). H NMR (400 MHz,
CD3OD): d=8.21 (s, 1H), 7.83 (s, 1H), 7.65 (d, J=6.4 Hz, 2H), 6.92
(d, J=6.7 Hz, 2H), 4.31–4.04 (m, 4H), 3.99 (s, 3H), 3.61 (d, J=
23.0 Hz, 2H), 1.28 ppm (t, J=7.0 Hz, 7H); ESI-MS(+) calculated for
[C18H23NO6P]+ m/z 380.12, found m/z 402.13 [M+Na]+.
Methyl 6-((diethoxyphosphoryl)methyl)-4-(3-hydroxyphenyl)pi-
1
colinate (17 f). Yield: 90% (135 mg, 0.40 mmol). H NMR (400 MHz,
CD3OD): d=8.23 (s, 1H), 7.86 (s, 1H), 7.35 (t, J=7.9 Hz, 1H), 7.22
(d, J=7.9 Hz, 1H), 7.16 (s, 1H), 6.92 (d, J=8.0 Hz, 1H), 4.21–4.06
(m, 4H), 4.00 (s, 3H), 3.65 (d, J=20.8 Hz, 2H), 1.29 ppm (t, J=
6.3 Hz, 6H); ESI-MS(+) calculated for [C18H23NO6P]+ m/z 380.12,
found m/z 402.13 [M+Na]+.
Methyl 4-bromo-6-((diethoxyphosphoryl)methyl)picolinate (16).
In a round-bottom flask, 15 (3.5 g, 11.39 mmol) and triethyl phos-
phite (5.7 g, 34.16 mmol) were dissolved in toluene (30 mL). The re-
action was heated at 1408C for 22 h. Excess triethyl phosphite and
toluene were removed in vacuo, and the crude product was puri-
fied by flash column chromatography. Compound 16 eluted at
70% ethyl acetate in hexanes as a clear oil in 92% yield (3.9 g,
10.52 mmol). 1H NMR (400 MHz, CDCl3): d=8.17 (s, 1H), 7.78 (s,
1H), 4.12 (dq, J=10.2, 3.6 Hz, 4H), 3.99 (s, 3H), 3.50 (d, J=22.0 Hz,
2H), 1.29 ppm (t, J=7.0 Hz, 6H); ESI-MS(+) calculated for
[C12H18BrNO5P]+ m/z 366.01, found m/z 366.03 [M+H]+.
Methyl 6-((diethoxyphosphoryl)methyl)-4-(2-hydroxyphenyl)pi-
1
colinate (17g). Yield: 63% (91 mg, 0.24 mmol). H NMR (400 MHz,
CD3OD): d=8.31 (s, 1H), 7.91 (s, 1H), 7.41 (d, J=7.4 Hz, 1H), 7.28
(t, J=7.8 Hz, 1H), 7.02–6.80 (m, 2H), 4.21–4.05 (m, 4H), 3.98 (s,
3H), 3.63 (d, J=22.1 Hz, 2H), 1.28 ppm (t, J=7.0 Hz, 6H); ESI-
MS(+) calculated for [C18H23NO6P]+ m/z 380.12, found m/z 402.13
[M+Na]+.
Methyl 4-(4-acetamidophenyl)-6-((diethoxyphosphoryl)methyl)-
picolinate (17h) Yield: 43% (72 mg, 0.170 mmol); 1H NMR
(400 MHz, CD3OD): d=8.36 (s, 1H), 8.02 (s, 1H), 7.87–7.72 (m, 4H),
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4.23–4.09 (m, 4H), 4.03 (s, 3H), 3.72 (d, J=22.3 Hz, 2H), 2.16 (s,
3H), 1.29 ppm (t, J=7.0 Hz, 6H); ESI-MS(+) calculated for
[C20H26N2O6P]+ m/z 421.15, found m/z 421.20 [M+H]+.
[D6]DMSO): d=166.7, 160.4, 156.2, 149.4, 148.6, 138.8, 131.0, 125.3,
120.4, 119.6, 115.7, 112.7, 55.8, 38.4 ppm; ESI-MS(À) calculated for
[C14H13NO6P]À m/z 322.05, found m/z 322.02 [MÀH]À.
Methyl 4-(3-acetamidophenyl)-6-((diethoxyphosphoryl)methyl)-
picolinate (17i). Yield: 39% (60 mg, 0.14 mmol); H NMR (400 MHz,
4-(2-Methoxyphenyl)-6-(phosphonomethyl)picolinic acid (18d).
Yield: 67% (67 mg, 0.21 mmol). H NMR (400 MHz, CD3OD): d=8.32
1
1
CDCl3): d=8.22 (s, 1H), 8.16 (s, 1H), 7.85 (d, J=6.8 Hz, 2H), 7.68 (d,
J=7.0 Hz, 1H), 7.43–7.32 (m, 2H), 4.28–4.05 (m, 4H), 4.00 (s, 3H),
3.67 (d, J=21.9 Hz, 2H), 2.22 (s, 3H), 1.30 ppm (t, J=7.1 Hz, 6H);
ESI-MS(+) calculated for [C20H26N2O6P]+ m/z 421.15, found m/z
421.19 [M+H]+.
(s, 1H), 7.96 (s, 1H), 7.59–7.38 (m, 2H), 7.17 (d, J=8.2 Hz, 1H), 7.11
(t, J=7.4 Hz, 1H), 3.88 (s, 3H), 3.55 ppm (d, J=21.7 Hz, 2H);
13C NMR (126 MHz, [D6]DMSO): d=166.7, 156.7, 155.5, 147.9, 147.2,
131.2, 130.6, 127.7, 126.6, 123.3, 121.5, 112.5, 56.1, 38.3 ppm; ESI-
MS(À) calculated for [C14H13NO6P]À m/z 322.05, found m/z 322.04
[MÀH]À.
Methyl 4-(2-acetamidophenyl)-6-((diethoxyphosphoryl)methyl)-
picolinate (17j). Yield: 84% (136 mg, 0.32 mmol). 1H NMR
(400 MHz, CD3OD): d=8.17 (s, 1H), 7.80 (s, 1H), 7.64–7.29 (m, 4H),
4.32–4.08 (m, 4H), 4.02 (s, 3H), 3.74 (d, J=22.3 Hz, 2H), 1.99 (s,
3H), 1.31 (t, J=7.0 Hz, 6H); ESI-MS(+) calculated for [C20H26N2O6P]+
m/z 421.15, found m/z 421.21 [M+H]+.
4-(4-Hydroxyphenyl)-6-(phosphonomethyl)picolinic acid (18e).
1
Yield: 82% (16 mg, 0.06 mmol). H NMR (400 MHz, CD3OD): d=8.38
(s, 1H), 8.08 (s, 1H), 7.81 (d, J=8.2 Hz, 2H), 6.96 (d, J=8.0 Hz, 2H),
3.56 ppm (d, J=21.4 Hz, 2H); 13C NMR (126 MHz, [D6]DMSO): d=
166.7, 159.5, 155.9, 148.8, 148.7, 128.7, 127.5, 124.0, 119.4, 116.6,
38.2 ppm; ESI-MS(À) calculated for [C13H11NO6P]À m/z 308.03,
found m/z 308.02 [MÀH]À.
Methyl 4-(4-chlorophenyl)-6-((diethoxyphosphoryl)methyl)picoli-
nate (17k). Yield: 60% (91 mg, 0.23 mmol). 1H NMR (400 MHz,
CD3OD): d=8.28 (s, 1H), 7.91 (s, 1H), 7.80 (d, J=8.7 Hz, 2H), 7.55
(d, J=8.6 Hz, 2H), 4.23–4.08 (m, 5H), 4.00 (s, 3H), 3.66 (d, J=
22.2 Hz, 2H), 1.28 ppm (t, J=7.1 Hz, 6H); ESI-MS(+) calculated for
[C18H22ClNO5P]+ m/z 398.09, found m/z 398.24 [M+H]+.
4-(3-Hydroxyphenyl)-6-(phosphonomethyl)picolinic acid (18 f).
1
Yield: 82% (77 mg, 0.30 mmol). H NMR (400 MHz, CD3OD): d=8.47
(s, 1H), 8.21 (s, 1H), 7.48–7.33 (m, 2H), 7.29 (s, 1H), 7.01 (d, J=
7.4 Hz, 1H), 3.73 ppm (d, J=22.1 Hz, 2H); 13C NMR (126 MHz,
[D6]DMSO): d=166.6, 158.6, 156.2, 148.9, 148.9, 138.5, 131.0, 125.0,
120.2, 118.0, 117.0, 113.9, 38.3 ppm; ESI-MS(À) calculated for
[C13H11NO6P]À m/z 308.03, found m/z 308.05 [MÀH]À.
Methyl 4-(3-chlorophenyl)-6-((diethoxyphosphoryl)methyl)picoli-
nate (17l). Yield: 42% (63 mg, 0.16 mmol). 1H NMR (400 MHz,
CD3OD): d=8.28 (s, 1H), 7.93 (s, 1H), 7.84 (s, 1H), 7.78–7.68 (m,
1H), 7.61–7.47 (m, 2H), 4.24–4.08 (m, 4H), 4.01 (s, 3H), 3.67 (d, J=
22.0 Hz, 2H), 1.29 ppm (t, J=6.8 Hz, 6H); ESI-MS(+) calculated for
[C18H22ClNO5P]+ m/z 398.09, found m/z 398.15 [M+H]+.
4-(2-Hydroxyphenyl)-6-(phosphonomethyl)picolinic acid (18g).
1
Yield: 76% (56 mg, 0.18 mmol). H NMR (400 MHz, CD3OD): d=8.87
(s, 1H), 8.48 (s, 1H), 7.68 (d, J=6.7 Hz, 1H), 7.45 (t, J=7.1 Hz, 1H),
7.13–7.01 (m, 2H), 3.86 ppm (d, J=22.4 Hz, 2H); 13C NMR
(126 MHz, [D6]DMSO): d=166.8, 155.5, 155.3, 147.8, 147.6, 130.9,
130.5, 127.2, 124.5, 123.1, 120.2, 116.8, 38.4 ppm; ESI-MS(À) calcu-
lated for [C13H11NO6P]À m/z 308.03, found m/z 308.09 [MÀH]À.
Methyl 4-(2-chlorophenyl)-6-((diethoxyphosphoryl)methyl)picoli-
nate (17m). Yield: 71% (114 mg, 0.29 mmol). 1H NMR (400 MHz,
CD3OD): d=8.11 (s, 1H), 7.74 (s, 1H), 7.61–7.40 (m, 4H), 4.23–4.07
(m, 4H), 3.99 (s, 3H), 3.67 (d, J=22.2 Hz, 2H), 1.28 ppm (t, J=
7.1 Hz, 6H); ESI-MS(+) calculated for [C18H22ClNO5P]+ m/z 398.09,
found m/z 398.13 [M+H]+.
4-(4-Aminophenyl)-6-(phosphonomethyl)picolinic acid (18h).
1
Yield: 56% (14 mg, 0.05 mmol). H NMR (400 MHz, CD3OD): d=8.68
(s, 1H), 8.43 (s, 1H), 8.14 (d, J=8.2 Hz, 2H), 7.56 (d, J=8.2 Hz, 2H),
3.86 ppm (d, J=22.4 Hz, 2H); 13C NMR (126 MHz, [D6]DMSO): d=
166.6, 155.5, 151.1, 149.2, 148.6, 128.2, 123.1, 123.0, 118.5, 114.6,
38.0 ppm; ESI-MS(À) calculated for [C13H12N2O5P]À m/z 307.05,
found m/z 307.09 [MÀH]À.
General procedures for the synthesis of 18a–m
Compounds 17a–m were dissolved the in a solution of 6m HCl
and heated at 1008C for 24 h. Excess HCl was removed in vacuo
followed by co-evaporation with copious amounts of water and
MeOH until precipitate was observed. The precipitate was collected
by vacuum filtration and washed with cold water to afford prod-
ucts 18a–m.
4-(3-Aminophenyl)-6-(phosphonomethyl)picolinic acid (18i).
1
Yield: 96% (48 mg, 0.16 mmol). H NMR (400 MHz, CD3OD): d=8.35
(s, 1H), 8.01 (s, 1H), 7.96–7.47 (m, 4H), 3.59 ppm (d, J=21.9 Hz,
2H); 13C NMR (126 MHz, [D6]DMSO): d=166.4, 156.3, 148.9, 148.2,
138.3, 137.9, 131.1, 125.1, 123.6, 122.4, 120.3, 119.4, 38.3 ppm; ESI-
MS(À) calculated for [C13H12N2O5P]À m/z 307.05, found m/z 307.09
[MÀH]À.
4-Phenyl-6-(phosphonomethyl)picolinic acid (18a). Yield: 29%
(14 mg, 0.05 mmol). 1H NMR (400 MHz, CD3OD): d=8.65 (s, 1H),
8.39 (s, 1H), 8.14–7.57 (m, 5H), 3.82 ppm (d, J=22.3 Hz, 2H); ESI-
MS(À) calculated for [C13H11NO5P]À m/z 292.03, found m/z 292.04
[MÀH]À.
4-(2-Aminophenyl)-6-(phosphonomethyl)picolinic acid (18j).
1
yield: 60% (60 mg, 0.19 mmol). H NMR (400 MHz, CD3OD): d=8.24
(s, 1H), 7.87 (s, 1H), 7.54–7.32 (m, 2H), 7.25–7.05 (m, 2H), 3.57 ppm
(d, J=21.7 Hz, 2H); 13C NMR (126 MHz, [D6]DMSO): d=166.7, 155.8,
155.7, 148.9, 148.7, 145.9, 130.3, 127.2, 122.6, 122.1, 117.3, 116.3,
38.3 ppm; ESI-MS(À) calculated for [C13H12N2O5P]À m/z 307.05,
found m/z 307.06 [MÀH]À.
4-(4-Methoxyphenyl)-6-(phosphonomethyl)picolinic acid (18b).
1
Yield: 61% (48 mg, 0.15 mmol). H NMR (400 MHz, [D6]DMSO): d=
8.08 (s, 1H), 7.82 (s, 1H), 7.75 (d, J=8.6 Hz, 2H), 7.09 (d, J=8.5 Hz,
2H), 3.81 (s, 3H), 3.30 ppm (d, J=21.4 Hz, 2H); 13C NMR (126 MHz,
[D6]DMSO): d=166.7, 161.0, 156.2, 148.9, 148.3, 129.2, 128.7, 124.3,
119.6, 115.2, 55.8, 38.3 ppm; ESI-MS(À) calculated for [C14H13NO6P]À
m/z 322.05, found m/z 322.10 [MÀH]À.
4-(4-Chlorophenyl)-6-(phosphonomethyl)picolinic acid (18k).
1
Yield: 97% (60 mg, 0.18 mmol). H NMR (400 MHz, [D6]DMSO): d=
8.13 (s, 1H), 7.86 (s, 1H), 7.83 (d, J=8.1 Hz, 2H), 7.62 (d, J=8.1 Hz,
2H), 3.34 ppm (d, J=21.5 Hz, 2H); 13C NMR (126 MHz, [D6]DMSO):
d=166.6, 156.3, 149.1, 147.5, 136.0, 135.0, 129.8, 129.2, 125.0,
4-(3-Methoxyphenyl)-6-(phosphonomethyl)picolinic acid (18c).
1
Yield: 71% (50 mg, 0.15 mmol). H NMR (400 MHz, CD3OD): d=8.33
(s, 1H), 8.01 (s, 1H), 7.53–7.31 (m, 3H), 7.09 (d, J=7.4 Hz, 1H), 3.89
(s, 3H), 3.56 ppm (d, J=21.8 Hz, 2H); 13C NMR (126 MHz,
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120.2, 38.3 ppm; ESI-MS(À) calculated for [C13H10ClNO5P]À m/z
326.00, found m/z 326.01 [MÀH]À.
UV/Visible spectroscopy
To prepare CoII-substituted NDM-1, NDM-1 (150 mm) was dialyzed
twice against 2 mm EDTA, 50 mm HEPES, pH 6.8, containing
150 mm NaCl, and 2 mm EDTA, followed by three separate dialysis
steps against 50 mm HEPES, pH 6.8, containing 150 mm NaCl, and
0.5 gLÀ1 Chelex resin. Buffers were exchanged at approximately
12 h intervals. Metal-free NDM-1 was diluted to 300 mm with
50 mm HEPES, pH 6.8, containing 150 mm NaCI, 10% glycerol, and
2 mm TCEP (tris(2-carboxyethyl)phosphine). CoCl2 (100 mm stock in
water) was added to result in a protein with 2 molar equivalents of
CoII. The resulting CoCo-NDM-1 enzyme was separated into 500 mL
aliquots, and captopril, DPA, and compounds 1–4 (stock solutions
of 50 mm in DMSO) were added to result in samples with 2 molar
equivalents of compound. The EDTA stock was dissolved in water.
The samples were then incubated on ice for 5 min. The samples
were added to a 500 mL quartz cuvette, and UV/Vis spectra were
collected on a PerkinElmer Lambda 750 UV/Vis/NIR spectrometer
measuring absorbance between 300 and 800 nm at 258C. A blank
spectrum of apo-NDM-1 (300 mm) was used to generate difference
spectra. All data were normalized at 800 nm.
4-(3-Chlorophenyl)-6-(phosphonomethyl)picolinic acid (18l).
1
Yield: 90% (47 mg, 0.14 mmol). H NMR (400 MHz, [D6]DMSO): d=
8.14 (s, 1H), 7.95–7.35 (m, 5H), 3.34 ppm (d, J=21.5 Hz, 2H);
13C NMR (126 MHz, [D6]DMSO): d=166.6, 156.4, 149.1, 147.2, 139.4,
134.6, 131.7, 129.8, 127.2, 126.2, 125.2, 120.4, 38.4 ppm; ESI-MS(À)
calculated for [C13H10ClNO5P]À m/z 326.00, found m/z 325.97
[MÀH]À.
4-(2-Chlorophenyl)-6-(phosphonomethyl)picolinic acid (18m).
1
Yield: 72% (67 mg, 0.20 mmol). H NMR (400 MHz, CD3OD) d=8.57
(s, 1H), 8.30 (s, 1H), 7.77–7.48 (m, 4H), 3.89 ppm (d, J=22.5 Hz,
2H); 13C NMR (126 MHz, [D6]DMSO): d=166.5, 155.8, 148.2, 147.5,
137.0, 131.7, 131.4, 131.2, 130.7, 128.4, 127.9, 123.2, 38.3 ppm; ESI-
MS(À) calculated for [C13H10ClNO5P]À m/z 326.00, found m/z 325.98
[MÀH]À.
Determination of IC50 values for MBL inhibition
MBL metalloforms NDM-1, IMP-1, and VIM-2 were over-expressed
and purified as previously described.[28] The IC50 values for which
meropenem was used as a substrate is described. The decrease in
absorption of meropenem at 300 nm (buffer: 50 mm HEPES, 2 mm
CHAPS, pH 7) was monitored in UV-transparent 96 well plates
(Corning product #3635).[45] Briefly, 20 mL of each compound at var-
ious concentrations (final concentration 0–10 mm) was added to
each well, followed by addition of 50 mL NDM-1 (final concentra-
tion of either 5 or 10 nm). The wells were incubated at 258C for
20 min. Next, 30 mL of meropenem (final concentration 180 mm)
was added to each well to initiate the reaction. The plate was then
centrifuged at 600 rpm for 30 s to eliminate air bubbles, and
placed into a Synergy H4 plate reader (BioTek). The absorbance
was monitored at 300 nm over 5 min with 15 s intervals. The
DAbs300nm minÀ1 was calculated from each slope as described
below, fixing the uninhibited value at 100%, and solving for both
the IC50 and Hill coefficient values.[28]
Acknowledgements
We thank Dr. Yongxuan Su for mass spectrometry sample analy-
sis at The Molecular Mass Spectrometry Facility (MMSF) at UC
San Diego. This work was supported by the US National Institutes
of Health (NIH Grant GM111926), the US National Science Foun-
dation (NSF Grant CHE1509285 to M.W.C.), and by the Robert A.
Welch Foundation (Grant F-1572 to W.F.).
Conflict of interest
S.M.C. is a cofounder of, and has an equity interest in, Cleave Bio-
sciences and Forge Therapeutics, companies that can potentially
benefit from the research results. S.M.C. also serves on the Scien-
tific Advisory Board for these companies. The terms of this ar-
rangement have been reviewed and approved by the University
of California San Diego in accordance with its conflict-of-interest
policies.
The IC50 values for which fluorocillin was used as a substrate were
determined as described previously,[28] with minor modifications
made to the volume of reagents used. Briefly, 20 mL of each com-
pound at various concentrations (final concentration 0.001–10 mm)
was added to each well, followed by addition of 50 mL enzyme
(final concentration, NDM-1, 0.2 nm; VIM-2, 2 nm; IMP-1, 0.06 nm).
Each plate was incubated for 20 min at 258C, followed by the addi-
tion of 30 mL fluorocillin (final concentration 87 nm). The hydrolysis
of fluorocillin is monitored at lex/lem of 495/525 nm. Plates were
prepared in parallel to minimize any variability due to compound
storage and handling. The rates of fluorescence increase are deter-
mined and IC50 values determined as described above for the chro-
mogenic assay.
Keywords: dipicolinic acid · imipenemase-1 · isosteres · metal
binding pharmacophores · metallo-b-lactamases · New Delhi
metallo-b-lactamase
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1798.
Equilibrium dialysis
ZnZn-NDM-1 (final concentration 8 mm) in 5 mL of 100 mm ammo-
nium acetate, pH 7.5, was mixed with the compounds at concen-
trations of 0–128 mm. After incubation for 1 h, the solutions were
dialyzed versus 500 mL of metal-free ammonium acetate, pH 7.5,
overnight (dialysis tubing MWCO 6000–8000, Fisherbrand). The ZnII
content in the resulting NDM-1 samples was determined using in-
ductively coupled plasma with atomic emission spectroscopy (ICP-
AES, PerkinElmer Optima 7300DV). The emission wavelength was
set to 213.856 nm, as previously described.[28]
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Manuscript received: March 15, 2019
Revised manuscript received: April 24, 2019
Accepted manuscript online: && &&, 0000
Version of record online: && &&, 0000
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FULL PAPERS
Re-engineering isosteres: Dipicolinic
acid isosteres were evaluated against
metallo-b-lactamases. The concept of
isosteres is used to show that the
A. Y. Chen, P. W. Thomas, Z. Cheng,
N. Y. Xu, D. L. Tierney, M. W. Crowder,*
W. Fast,* S. M. Cohen*
&& – &&
choice of carboxylate isostere impacts
not only inhibition potency, but also the
mechanism of action. This study dem-
onstrates the utility of isosteric replace-
ment for routinely used metal binding
motifs (e.g., carboxylic acids) in metal-
loenzyme inhibitor development.
Investigation of Dipicolinic Acid
Isosteres for the Inhibition of Metallo-
b-Lactamases
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