Synthesis and evaluation of aminophosphinic acid derivatives as inhibitors of renal dipeptidase

Article abstract of DOI:10.1016/j.bmcl.2004.04.057

Renal dipeptidase (RDP) is an enzyme overexpressed in benign and malignant colorectal tumors. In an effort to identify potent inhibitors of this enzyme, a series of aminophosphinic acid derivatives were synthesized. Compounds 3a and 3c in which the phenyl

Full text of DOI:10.1016/j.bmcl.2004.04.057

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3531–3533
Synthesis and evaluation of aminophosphinic acid derivatives
as inhibitors of renal dipeptidase
Hallur Gurulingappa, Phillip Buckhalts, Kenneth W. Kinzler, Bert Vogelstein
and Saeed R. Khan*
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, USA
Received 14 January 2004; revised 14 April 2004; accepted 19 April 2004
Abstract—Renal dipeptidase (RDP) is an enzyme overexpressed in benign and malignant colorectal tumors. In an effort to identify
potent inhibitors of this enzyme, a series of aminophosphinic acid derivatives were synthesized. Compounds 3a and 3c in which the
phenyl ring was para substituted with F and Br and olefin with Z geometry, showed better inhibitory activity against RDP enzyme
(IC50 ¼ 5–6 nM).
Ó 2004 Elsevier Ltd. All rights reserved.
Renal dipeptidase (dehydropeptidase I, EC 3.4.13.11) is
a zinc-containing hydrolytic enzyme that exhibit pref-
erence for dipeptide substrates with dehydro amino
acids at the carboxy position. However, it can accom-
mimic unstable tetrahedral intermediates5;6 or naturally
occurring peptide inhibitors.7 For example, potent
inhibitors of the zinc peptidases thermolysin and carb-
oxypeptidase A are obtained when the scissile carbonyl
group of a substrate is replaced with a phosphonic acid
moiety. In addition to their potency, these compounds
appear to be transition-state analogues.6
modate substrate with both
D or L amino acids at that
position as well.1 This enzyme is responsible for the
hydrolytic scission of the lactam bond in carbapenems,
potent broad-spectrum antibiotics that are resistant to
the action of microbial b-lactamases.2 It has been shown
that enzymatic turnover of carbapenems in vivo poses a
serious obstacle to clinical efficacy of these bactericidal
agents. Therefore, specific inhibitors for this enzyme are
widely sought.3 Serial analysis of gene expression
(SAGE) studies have indicated RDP to be over
expressed in both benign and malignant tumor in com-
parison to normal colonic epithelium,4 providing an
excellent opportunity for the development of specific
probes for its detection in vivo to detect colon cancer at
early stage.
We sought to extend this strategy to inhibitors of RDP
by synthesizing dehydropeptide analogues in which
scissile carboxamide has been replaced with
a
PO(OH)CH2 group (Fig. 1). In continuation of our
studies toward RDP inhibitors,8 this communication
describes design, synthesis and the in vitro evaluation of
novel aminophosphinic acid derivatives.
The aminophosphinic acid analogues 3a–t was synthe-
sized following the synthetic strategy (Scheme 1). (1-
tert-Butoxycarbonylamino-2-cyclohexyl-ethy)phosphinic
acid methyl ester (1) was prepared according to reported
procedure.9 Treatment of 1 with NaOMe followed by
trimethylphosphonoacrylate gave an intermediate,
The synthesis of phosphinic acids and phosphonic acids
are currently an area of great interest due to the ability
of these acids to function as effective bioesters of the
carboxylic acid moiety in certain biological system. A
successful approach for the inhibition of a number of
peptidases has been to utilize phosphorus analogues to
R
R
H
N
OH
P
COO
R2
COO
R2
H3N
H3N
O
O
R1
R1
Dehydropeptide
Aminophosphinic acid analog
Keywords: Aminophosphinic acid derivatives; RDP inhibitors.
* Corresponding author. Tel.: +1-410-614-1153; fax: +1-410-614-8397;
e-mail: ghallur1@jhmi.edu
Figure 1. General structures of dehydropeptide and aminophosphinic
acid analogue.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.04.057

3532
H. Gurulingappa et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3531–3533
a
Table 1. RDP inhibition (IC50
)
values of compounds
R2
R3
R1
Compound
R1
R2
R3
Olefin
geometry
IC50 (nM)
H
a, b, c
3a
3b
3c
3d
3e
3f
H
H
H
H
H
H
H
H
I
H
H
H
H
H
H
I
F
Z
E
Z
E
Z
E
Z
E
Z
E
Z
E
Z
E
Z
E
Z
E
Z
E
5
15
O
P
OMe
O
P
OMe
2
Boc
F
NH
H
NH
COOMe
Boc
Br
Br
I
6
30
1
8
25
d, e
I
3g
3h
3i
H
45
I
H
250
100
1000
10
R2
H
H
H
H
Cl
Cl
Cl
Cl
Br
Br
H
H
H
R3
R1
3j
I
H
H
3k
3l
H
H
H
H
H
H
H
H
H
H
CF3
CF3
Cl
Cl
F
O
P
OH
3
200
20
NH2
COOH
3m
3n
3o
3p
3q
3r
3s
3t
200
30
F
350
40
Scheme 1. Reagents and conditions: (a) NaOMe, MeOH, 0 °C, 10 min;
(b) trimethyl-2-phosphonoacrylate, 0 °C, 30 min; (c) (R1R2R3)PhCHO,
rt, 1 h; (d) CF3COOH, CH2Cl2, rt, 30 min; (e) concd HCl, 50 °C, 8 h.
F
F
3000
70
N(Et)2
N(Et)2
150
a IC50 values are determined using colon cancer lysate.
which subsequently underwent a Wittig–Horner type
olefination10 with substituted benzaldehydes to give
mixture of E and Z isomers. These isomers were sepa-
rated by column chromatography over silica gel. The
yields were 40% for Z isomers and 30% for the E iso-
mers. The protecting groups were removed by treatment
with CF3COOH followed by concd HCl9 in 95% yield.
The known compounds 3e and 3f have been prepared8
and used for activity comparison. All the compounds
were purified by column chromatography over silica gel
and their structure were unambiguously confirmed by
spectroscopic methods.11
compare to ortho and meta position. In general com-
pounds with the Z configuration were more active than
their E counterparts.
In conclusion, novel aminophosphinic acid analogues
were synthesized and evaluated for in vitro RDP inhi-
bition activity. Compounds 3a and 3c were found to be
best inhibitors among the tested compounds with IC50
values 5 and 6 nM, respectively. Modification of active
compounds with radiolabel and fluorescence tag to use
them as biomarkers to detect early stage colon tumors
are underway. Structure–activity relationship studies of
this new class of compounds are in progress and will be
reported in future communication.
The RDP inhibition activity of these compounds was
determined using crude lysates prepared from human
colon cancers according to reported procedure.8 Human
colon cancer extracts were prepared by homogenizing
1 cm3 of frozen colon tissue in 10 mL of 20 mM Tris,
pH 8.0, 10 lM ZnCl2, 0.1% Triton X 100. The extract
was clarified by centrifugation at 13,000g for 5 min at
4 °C and, for each measurement, 20 lL was diluted into
158 lL of 20 mM Tris, pH 8.0, 10 lM ZnCl2. The syn-
thesized compounds (20 lL) were added to each reaction
to obtain final concentrations ranging from 0 to 10 lM.
The mixtures were incubated at room temperature for
30 min to allow enzyme–inhibitor complex formation,
and the reactions were initiated by the addition of 2 lL
Acknowledgements
We are grateful for the support provided by the Na-
tional Institute of Health (CA 62924) and an intramural
grant from the Maryland Cigarette Restitution Fund
and FAMRI to S.R.K. We thank the Biophysical
Chemistry Department for NMR studies. K.W.K.
receives research funding from Genzyme Molecular
Oncology (Genzyme). Under a licensing agreement
between the Johns Hopkins University and Genzyme,
RDP-based technology is licensed to Genzyme, and
K.W.K. and B.V. are entitled to a share of royalty
received by the University from sales of the licensed
technology. The terms of these arrangements are being
managed by the University in accordance with its con-
flict of interest policies.
of 1 mM substrate (2DNP-
L-Lys-D
-Amp).12 While
incubating at 37 °C, fluorescence (kex ¼ 320 nm,
kem ¼ 405 nm) measurements were determined at 30 s
intervals and the relative reaction rate was taken as the
rate of increase of fluorescence over time. The results are
expressed as the concentration of inhibitor needed to
inhibit enzyme activity by 50% (IC50). The data for the
synthesized compounds are shown in Table 1.
References and notes
From the data in Table 1, it follows that the substitution
pattern on the phenyl ring has influence on the RDP
inhibition activity. Compounds 3a and 3c showed
potent inhibitory activity against RDP. Iodo substitu-
tion on para position of the phenyl ring had better effect
1. Greenstein, J. P. Adv. Enzymol. Relat. Subj. Biochem.
1948, 8, 117–169.
2. (a) Kropp, H.; Sundelof, J. G.; Hajdu, R.; Kahan, F. M.
Antimicrob. Agents Chemother. 1982, 22, 62–70; (b) Kim,

H. Gurulingappa et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3531–3533
3533
H. S.; Campbell, B. J. Biochem. Biophys. Res. Commun.
1982, 108, 1638–1642; (c) Kahan, J. S.; Kahan, F. M.;
Goegelman, R.; Currie, S. A.; Hendlin, D.; Mochales, S.;
Hernandez, S.; Woodruff, H. B.; Birnbaum, J. J. Antibiot.
1979, 32, 1–12.
11. Representative examples (a) Compound 2a: 1H NMR
(400 MHz, CDCl3): d 7.85 (d, J ¼ 4:4 Hz, 1H), 7.68 (m,
1H), 7.12 (t, J ¼ 8:4 Hz, 1H), 5.06 (d, J ¼ 10 Hz, 1H), 4.18
(m, 1H), 3.86 (s, 3H), 3.70 (d, J ¼ 10 Hz, 3H), 3.14 (m,
2H), 0.71–2.04 (m, 22H); 13C NMR (125 MHz, CDCl3):
167.5, 160.2, 158.6, 134.5, 132.2, 130.3, 128.6, 116.4, 72.3,
58.4, 52.3, 44.3, 32.3, 30.3, 29.4, 28.7, 27.5, 25.3, 24.1; 31P
NMR (162 MHz, CDCl3): d 51.35; LC–MS m=z 497 [M]þ.
(b) Compound 2b: 1H NMR (400 MHz, CDCl3): d 7.64
(m, 1H), 7.24 (t, J ¼ 8:4 Hz, 1H), 6.94 (d, J ¼ 4:4 Hz, 1H),
4.87 (d, J ¼ 10 Hz, 1H), 4.15 (m, 1H), 3.78 (s, 3H), 3.76 (s,
3H), 3.08 (m, 2H), 0.70–2.00 (m, 22H); 13C NMR
(125 MHz, CDCl3): d 166.8, 161.4, 158.3, 134.7, 132.6,
129.7, 128.3, 116.8, 71.5, 59.6, 52.8, 43.4, 32.7, 30.8, 29.6,
28.5, 27.3, 25.6, 23.8; 31P NMR (162 MHz, CDCl3): d
50.79; LC–MS m=z 497 [M]þ. (c) Compound 3a: 1H NMR
(400 MHz, CD3OD): d 7.98 (d, J ¼ 4:4 Hz, 1H), 7.68 (m,
1H), 7.18 (t, J ¼ 8:4 Hz, 1H), 3.67 (m, 1H), 3.27 (m, 2H),
0.87–1.83 (m, 13H); 13C NMR (125 MHz, CD3OD): d
170.6, 163.3, 138.7, 132.3, 129.3, 128.5, 117.2, 46.7, 32.3,
31.8, 29.3, 27.8, 26.4, 24.8; 31P NMR (162 MHz, CD3OD):
3. (a) Kahan, F. M.; Kropp, H.; Sundelof, J. G.; Birnbaum,
J. J. Antimicrob. Chemother. 1983, 12(Suppl. D), 1–35; (b)
D’Amato, C.; Armignacco, O.; Antonucci, G.; Bordi, E.;
Bove, G.; Decarli, G.; De Mori, P.; Rosci, M. A.; Visco,
G. J. Chemother. 1990, 2, 100–107.
4. Buckhaults, P.; Rago, C.; Croix, B. S.; Romans, K. E.;
Saha, S.; Zhang, L.; Vogelstein, B.; Kinzler, K. W. Cancer
Res. 2001, 61, 6996–7001.
5. (a) Holmquist, B.; Vallee, B. L. Proc. Natl. Acad. Sci.
U.S.A. 1979, 76, 6216–6220; (b) Jacobsen, N. E.; Bartlett,
P. A. J. Am. Chem. Soc. 1981, 103, 654–657; (c) Thorsett,
E. D.; Harris, E. E.; Peterson, E. R.; Greenlee, W. J.;
Patchett, A.; Ulm, E. H.; Vassil, T. C. Proc. Natl. Acad.
Sci. U.S.A. 1982, 79, 2176–2180; (d) Galardy, R. E.;
Kontoyiannidou-Ostrem, Y.; Kortylewicz, Z. P. Biochem-
istry 1983, 22, 1990–1995; (e) Galardy, R. E.; Grobelny,
D. Biochemistry 1983, 22, 4556–4561.
d
34.46; LC–MS m=z 369 [M]þ; HRMS calcd for
6. Bartlett, P. A.; Marlowe, C. K. Biochemistry 1983, 22,
4618–4624.
C18H25FNO4PNa [MþNa]þ: 392.1397, found: 392.1382.
1
(d) Compound 3b: H NMR (400 MHz, CD3OD): d 7.65
7. Bartlett, P. A.; Kezer, W. B. J. Am. Chem. Soc. 1984, 106,
4282–4283.
8. Gurulingappa, H.; Buckhaults, P.; Kumar, S. K.; Kinzler,
K. W.; Vogelstein, B.; Khan, S. R. Tetrahedron Lett. 2003,
44, 1871–1873.
9. Parsons, W. H.; Hajdu, R.; Schoen, W. R.; Combs, P. L.;
Sundelof, J.; Patchett, A. A. Biochem. Int. 1991, 23, 1107–1115.
10. Schoen, W. R.; Parsons, W. H. Tetrahedron Lett. 1988, 29,
5201–5204.
(m, 1H), 7.25 (t, J ¼ 8:4 Hz, 1H), 6.95 (d, J ¼ 4:4 Hz, 1H),
3.65 (m, 1H), 3.25 (m, 2H), 0.88–1.84 (m, 13H); 13C NMR
(125 MHz, CD3OD): d 170.2, 162.8, 138.2, 131.8, 128.7,
127.4, 116.7, 45.8, 31.6, 31.2, 28.6, 27.5, 25.8, 24.3; 31P
NMR (162 MHz, CD3OD): d 33.58; LC–MS m=z 369
[M]þ; HRMS calcd for C18H25FNO4PNa [MþNa]þ:
392.1397, found: 392.1385.
12. Ian, J. W.; Jill, L.; Carvell, H. W.; Nigel, M. H. Anal.
Biochem. 1999, 268, 245–251.