Synthesis and activity study of phosphonamidate dipeptides as potential inhibitors of VanX

Article abstract of DOI:10.1016/j.bmcl.2011.09.020

In an effort to develop inhibitors of VanX, the phosphonamidate analogs of d-Ala-d-Ala dipeptides, N-[(1-aminoethyl) hydroxyphosphinyl]-glycine (1a), -alanine (1b), -valine (1c), -leucine (1d) and -phenylalanine (1e) were synthesized, characterized and ev

Full text of DOI:10.1016/j.bmcl.2011.09.020

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7224–7227
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and activity study of phosphonamidate dipeptides as potential
inhibitors of VanX
⇑
Ke-Wu Yang , Xu Cheng, Chuan Zhao, Cheng-Cheng Liu, Chao Jia, Lei Feng, Jian-Min Xiao,
Li-Sheng Zhou, Hui-Zhou Gao, Xia Yang, Le Zhai
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University,
Xi’an 710069, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2011
Revised 18 August 2011
Accepted 7 September 2011
Available online 10 September 2011
In an effort to develop inhibitors of VanX, the phosphonamidate analogs of
D-ala-D-ala dipeptides,
N-[(1-aminoethyl) hydroxyphosphinyl]-glycine (1a), -alanine (1b), -valine (1c), -leucine (1d) and -phen-
ylalanine (1e) were synthesized, characterized and evaluated using recombinant VanX. The crystal
structure of the intermediate 6d was obtained (Deposition number: CCDC 839134), and structural anal-
ysis revealed that it is orthorhombic with a space group P2(1)2(1)2(1), the bond length of P–N is 1.62 Å
and angle of C–N–P is 123.6°. Phosphonamidate 1(a–e) showed to be inhibitors of VanX with IC50 values
of 0.39, 0.70, 1.12, 2.82 and 4.13 mM, respectively, which revealed that the inhibition activities of the
phosphonamidates were dependent on the size of R-substituent of them, with the best inhibitor 1a hav-
ing the smallest substituent. Also, 1a showed antibacterial activity against Staphylococcus aureus (ATCC
Keywords:
Vancomycin resistance
VanX
Inhibitor
Phosphonamidate
Crystal structure
25923) with a MIC value of 0.25 lg/ml.
Ó 2011 Elsevier Ltd. All rights reserved.
Vancomycin inhibits bacterial cell wall synthesis by binding to
the -ala- -alanine end of the pentapeptide via five hydrogen
bonds, thereby preventing cross-linking of the pentapeptides to
form the bacterial peptidoglycan layer.1 VanX is
Zn(II)
VanX, which showed a high affinity (Kinact/Kirr = 9320 MÀ1 SÀ1) for
the enzyme but poor inhibitory efficiency.5,6
Previously, we synthesized phosphonamidate, phosphothioate,
phosphinate, sulfonate, and sulfonamidate analogues of D-ala-D-
D
D
a
metalloenzyme that is required for high-level vancomycin resis-
tance in bacteria. The intra-cellular role of VanX is to hydrolyze
ala dipeptides as potential inhibitors for VanX and bacterial amino-
peptidase N (PepN)7,8, steady-state kinetic studies indicated that
the phosphonamidate was a partial competitive inhibitor of VanX
vancomycin-binding D-Ala-D-Ala dipeptides that are used to syn-
thesize the normal bacterial peptidoglycan layer.1 Vancomycin
resistant bacteria have acquired the ability to produce
with a Ki of 36 lM. Several studies using thermolysin indicated
that phosphonamidate analogues of the peptides bound 800 times
stronger than the corresponding phosphonate analogue, due to the
fact that the phosphonamidate could form one additional H-bond
to the enzyme.9,10
D-Ala-D-lactate depsipeptides which can be integrated into the
peptidoglycan layer; however, these depsipeptides bind vancomy-
cin 1000 times weaker than their dipeptide counterparts.2 Clearly,
VanX is a key drug target in circumventing glycopeptide antibiotic
resistance, and to produce tight-binding inhibitor of VanX is an
effective way to combat vancomycin resistance in bacteria.
Towards this goal, Walsh and co-workers have examined the
inhibition properties of a number of phosphonate and phosphinate
In order to probe whether the phosphonamidate analogue of
D-Ala-D-Ala had a similar increase in binding affinity to VanX and
to investigate the relationship between the structure and inhibi-
tion activity, based on our previous studies7,8, N-[(1-aminoethyl)
hydroxyphosphinyl]-glycine (1a), -alanine (1b), -valine (1c), -leu-
cine (1d) and -phenylalanine (1e) were synthesized according to
Scheme 1.
Diphenyl 1-(phenylmethoxycarbonylamino) ethyl phosphonate
2 was synthesized using triphenyl phosphite, benzyl carbamate,
and aldehyde according to the method of Oleksyszyn11 and con-
verted by ester exchange in methanol to the dimethyl ester 3. In
previous synthesis, the monomethyl ester 4 was obtained by par-
tial base-catalyzed hydrolysis of dimethyl ester 3, but it resulted
in a low yield. In this study, we chose sodium iodide for partial
analogues of
D-Ala-D-Ala as well as of several dithiol com-
pounds.1,3,4 These analogues were shown to inhibit VanX with Ki
values ranging from 90 nM to 300 mM and exhibited competitive,
mixed, and slow-binding modes of inhibition. Badet and co-work-
ers have reported the pseudodipeptide
D-Ala-D-Gly(SC6H4–p–
CHF2) and its derivatives as the mechanism-based inhibitor for
⇑
Corresponding author. Tel./fax: +8629 8830 2429.
E-mail address: kwyang@nwu.edu.cn (K.-W. Yang).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.020

K.-W. Yang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7224–7227
7225
O
CH3
O
OC6H5
OC6H5
a
O
N
P
O
NH2
CH3CHO
P(OC6H5)3
H
O
2
O
CH3
O
CH3
ONa
OCH3
OCH3
b
c
O
N
P
O
N
P
OCH3
H
H
O
O
4
3
O
CH3
O
CH3
H
COOCH3
N
Cl
e
d
f
O
P
O
P
N
H
N
R
OCH3
H
O
O
OCH3
5
6(a-e)
CH3
CH3
H
H
N
O-
N
COOCH3
-
g
CO2
H2N
P
H2N
P
O
R
OCH3
O
R
1(a-e)
7(a-e)
R= -H (1a), -CH3 (1b), -CH (CH3)2 (1c), -CH2CH (CH3)2 (1d), -CH2C6H5 (1e)
Scheme 1. Synthesis of phosphonamidate dipeptides. Reagents and conditions: (a) CH3COOH, rt À85 °C, 3 h; (b) CH3OH, CH3ONa, NaOH, rt, 2 h; (c) NaI, Acetone, reflux, 1 h;
(d) CH2Cl2, SOCl2, rt, 4 h; (e) CHCl3, Et3N, amino acid methyl ester hydrochloride, rt, 24 h; (f) 10% Pd/C, H2, CH3OH, rt, 2 h; (g) LiOH, CH3CN, rt, 3 d.
deprotection of 3 to prepare 4.12 The intermediate 4 was subse-
quently converted by treatment with thionyl chloride in dichloro-
Table 1
Crystallographic data of phosphonamidate 6d
methane to chlorophosphonate 5. The intermediate
5 were
Empirical formula
Formula weight
Crystal system,
Space group
a (Å)
C18H29N2O6P
400.40
coupled with the corresponding amino acid methyl ester using
previously published procedures13–15 to afford compounds
6(a–e). These intermediates were deprotected through two step
reactions to offer the phosphonamidate 1(a–e) as white solid with
yield of 34%, 35%, 30%, 39% and 31%, respectively. 1(a–e) were sta-
ble for over a month at 4 °C and pH >7.0. All phosphonamidates
were characterized by NMR and MS (for details, see Supplementary
data).
Crystals of 6d were obtained through recrystallization of it from
ethyl chloroform/hexane (1:2). A colorless platelet of 0.32 Â
0.27 Â 0.17 mm was chosen for the measurement of crystal struc-
ture, the diffraction intensity data were collected on a Bruker Smart
Orthorhombic
P2(1)2(1)2(1)
9.847(3)
13.196(3)
16.922(4)
90
b (Å)
c (Å)
a
(o)
b (o)
90
90
c
(o)
V (Å3)
2198.7(10)
4
1.210
0.158
856
Z
qcalc (g cmÀ3)
l
(mmÀ1
)
F(000)
Crystal size (mm)
Reflections collected
Reflections unique
0.32 Â 0.27 Â 0.17
Apex II CCD diffractometer with graphite-monochromated Mo-K
a
11050
radiation (k = 0.71073 Å) at 298 K. Empirical absorption corrections
were applied using the SADABS program. The structures were
solved by direct methods and refined by the full-matrix least-
squares based on F2 using SHELXTL-97 program.16 All non-hydro-
gen atoms were refined anisotropically and hydrogen atoms were
generated geometrically. Crystal data and structural refinement
parameters of 6d are summarized in Table 1, the selected bond
length and bond angles are listed in Tables 2 and 3, an ORTER plot
and unit cell packing diagram are presented in Figures 1 and 2,
respectively. The crystal structure of 6d is orthorhombic with a
space group P2(1)2(1)2(1). The geometry at the phosphorus centre
is distorted tetrahedral with bond length of P–N = 1.62 Å,
P–O = 1.52 Å, the bond angle of O–P–N = 105.73° and N–P–C =
110.49°.
3889
R1, wR2 [I > 2
R1, wR2 (all data)
max and min (e ÅÀ3
)
r(I)]
0.0585, 0.1235
0.1142, 0.1460
0.205 and À0.197
D
q
Table 2
Relevant bond lengths (Å) of phosphonamidate 6d
P(1)–O(1)
P(1)–O(2)
P(1)–N(1)
P(1)–C(9)
O(5)–C(7)
C(2)–C(7)
1.470(3)
1.560(3)
1.623(3)
1.800(4)
1.176(10)
1.523(10)
N(1)–C(2)
N(2)–C(11)
N(2)–C(9)
O(6)–C(7)
O(6)–C(8)
C(3)–C(4)
1.452(5)
1.324(6)
1.455(5)
1.216(10)
1.583(9)
1.480(7)
O(2)–C(1)
1.450(5)
1.213(5)
1.341(5)
1.451(6)
1.521(6)
1.509(9)
O(3)–C(11)
O(4)–C(11)
O(4)–C(12)
C(2)–C(3)
C(4)–C(6)
VanX was overexpressed and purified as previously described.17
Briefly, an overnight culture of BL21(DE3) Escherichia coli cells con-
taining the MBP-VanX expression plasmid was used to innoculate
collected by centrifugation and ruptured by ultrasonic. The cell ly-
sis was loaded onto an amylose resin column and eluted using
160 ml of 20 mM Tris, pH 7.6, containing 10 mM maltose and
200 mM NaCl. The fractions containing MBP-VanX were pooled,
concentrated and cleaved using thrombin, and the resulting
sample was loaded onto a Q-Sepharose column and eluted with a
4 Â 1 liter of LB containing 25
lg/ml kanamycin. The cells were
shaken at 30 °C until reaching an OD600 of 1.6–1.8, and protein
production was induced by making the cultures 1 mM IPTG. The
cultures were shaken for 75 min at 30 °C, and the E. coli cells were

7226
K.-W. Yang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7224–7227
Table 3
Relevant bond angles (°) of phosphonamidate 6d
O(1)–P(1)–O(2)
O(1)-P(1)–N(1)
N(1)–P(1)–C(9)
C(11)–O(4)–C(12)
N(1)–C(2)–C(3)
C(3)–C(4)–C(6)
O(5)–C(7)–O(6)
N(2)–C(9)–C(10)
116.37(18)
111.97(17)
110.49(19)
114.3(4)
111.1(4)
114.1(6)
O(2)–P(1)–N(1)
O(1)–P(1)–C(9)
O(2)–P(1)–C(9)
C(7)–O(6)–C(8)
C(3)–C(2)–C(7)
C(3)–C(4)–C(5)
O(5)–C(7)–C(2)
N(2)–C(9)–P(1)
105.73(16)
111.07(18)
100.54(19)
102.5(9)
111.7(5)
108.9(6)
C(2)–N(1)–P(1)
C(11)–N(2)–C(9)
C(1)–O(2)–P(1)
N(1)–C(2)–C(7)
C(4)–C(3)–C(2)
C(6)–C(4)–C(5)
O(6)–C(7)–C(2)
C(10)-C(9)–P(1)
123.6(3)
120.6(4)
120.5(3)
110.3(5)
114.9(5)
109.8(6)
115.8(10)
112.5(3)
122.7(11)
110.9(4)
121.2(11)
110.4(3)
Table 4
The inhibition activity (IC50) against VanX and antibacterial activity (MIC) against
clinical Staphylococcus aureus (ATCC 25923) and MRSA of phosphonamide 1a–ea
Compound ATCC 25923
MRSA 1 (MIC, MRSA 2 (MIC, IC50 (mM)
(MIC,
lg/ml)
l
g/ml)
l
g/ml)
Blank
1a
1b
1c
1d
2
0.25
2
2
2
1
1
1
1
1
1
2
1
2
2
2
2
0.39 0.02
0.70 0.03
1.12 0.01
2.82 0.04
4.13 0.03
1e
2
a
The phosphonamide 1(a–e) dilithium salts were converted to corresponding
diacids by ion-exchange resin for the evaluation of inhibitory and antibacterial
activity.
are presented in Table 4. Upon analysis of these data, we found that
the IC50 values of the phosphonamidate analogues were dependent
on the size of R-substituent of them (see Scheme 1), with the best
inhibitor having the smallest substituent (R@H). Also, the inhibitor
1a had best antibacterial activity comparison with the other
phosphonamidate analogues as description below. This struc-
ture–activity relationship implicated that the phosphonamidates
with a large substituent may result in affinity decrease of them
to VanX.
Figure 1. The structure of phosphonamidate 6d.
The in vitro antibacterial activities of the phosphonamidate
1(a–e) were first investigated by the macrodilution (tube) broth
method from Clinical and Laboratory Standards Institute (CLSI).20
Briefly, antibiotic and phosphonamidate 1(a–e) were dissolved in
dd H2O, respectively, to prepared 1280 lg/ml and 2 mM stock
solutions, the three bacteria from clinic, Staphylococcus aureus
(ATCC 25923), methicillin-resistant S. aureus 1 (MRSA 1) and MRSA
2 were used to assess the phosphonamidates. The solutions of van-
comycin of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125 lg/ml were
prepared, 0.4 ml of stock solution of phosphonamidate and 0.9 ml
of the bacterial cultures (ꢀ108 CFU/ml) in medium (Mueller–
Hinton, MH) were added sequentially to 0.7 ml of the solutions
of vancomycin, respectively, and incubated for 24 h at 37 °C, the
MIC values were collected and listed in Table 4. The results
indicated that the antibacterial activity of compound 1a against
S. aureus showed 8 folds stronger than the absence of it, but the
other compounds 1(b–e) had no activity, which is unclear at this
moment.
In this Letter, we reported the synthesis, characterization, inhib-
itory and antibacterial activities of the phosphonamidate ana-
logues of D-ala-D-ala dipeptides 1(a–e) as potential inhibitors of
VanX. The crystal structure of the intermediate 6d was obtained
and structural analysis revealed that the intermediate is ortho-
rhombic with a space group P2(1)2(1)2(1). The target enzyme
VanX was overexpressed, purified and to be used for evaluation
of the inhibition activity of the synthesized phosphonamidates
by determination of IC50, and the results revealed a structure–
activity relationship, which is that inhibition activities of the inhib-
itors are dependent on the size of their R-substituent. Also, the MIC
values of the phosphonamidates indicated that 1a had best anti-
bacterial activity against three bacteria from clinic. The strategy
used in this work to probe the structure/function of VanX inhibi-
Fig. 2. Unit cell packing diagram for phosphonamidate 6d.
plateau elution profile.18 VanX purity was assessed using SDS–
PAGE.
In an effort to develop inhibitors of VanX, we have reported
phosphonamidate 1b, which is a partial competitive inhibitor of
VanX with a Ki of 36 l
M.7 In order to further investigate the struc-
ture–activity relationship, we synthesized phosphonamidate
1(a–e) and determined the IC50 values of these compounds to VanX
using previously reported method.19 The determined IC50 values

K.-W. Yang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7224–7227
7227
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Acknowledgements
We gratefully thank Professor Michael Crowder at Miami Uni-
versity for the gift of pIADL14 plasmid for expression of VanX used
in this work. We also acknowledge the financial support from Na-
tional Natural Science Foundation of China (20972127), Doctoral
Foundation of China (200806970005), National Science Foundation
of Shaanxi Province (2009JM2002) and Key Fund for International
Cooperation of Shaanxi Province (2010KW-16)
Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.020.
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