Design, synthesis and evaluation of new RDP inhibitors

Article abstract of DOI:10.1016/S0040-4039(03)00082-0

Aminophosphinic acid derivatives were synthesized as potential inhibitors of renal dipeptidase, an enzyme overexpressed in benign and malignant colon tumors. Several compounds showed potent enzyme-inhibitory activity.

Full text of DOI:10.1016/S0040-4039(03)00082-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 1871–1873
Design, synthesis and evaluation of new RDP inhibitors
Hallur Gurulingappa, Phillip Buckhaults, Srinivas K. Kumar, Kenneth W. Kinzler, Bert Vogelstein
and Saeed R. Khan*
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, USA
Received 7 January 2003; accepted 8 January 2003
Abstract—Aminophosphinic acid derivatives were synthesized as potential inhibitors of renal dipeptidase, an enzyme over-
expressed in benign and malignant colon tumors. Several compounds showed potent enzyme-inhibitory activity. © 2003 Elsevier
Science Ltd. All rights reserved.
Colon cancer is the second most common cancer in the
US and kills more than 50,000 people each year, but it
is also one of the most preventable cancers. Screening
provides the best prevention. With regular screening,
precancerous polyps can be detected and removed, thus
preventing the development of colon cancer. Current
screening tests such as sigmoidoscopy, colonoscopy and
detection of fecal occult blood have significant prob-
lems, which have stimulated the search for more specific
non-invasive tests for the early detection of colorectal
cancers. In recent Serial Analysis of Gene Expression
(SAGE) studies performed on normal, adenomatous
and cancerous colonic epithelium, the enzyme Renal
Dipeptidase (RDP) was found to be overexpressed in
both benign and malignant tumor compared with nor-
mal colonic epithelium.1
complex5 has demonstrated that the dipeptidyl moiety
of cilastatin is sandwiched between the negatively
charged and positively charged sidewalls. Both ends of
the moiety are clamped tightly by hydrophobic interac-
tions. Certain aminophosphinic acid derivatives bind to
the active site of RDP similar to dipeptides.12 Based on
these findings we designed and synthesized alkyl-
aminophosphinic acid derivatives with C-terminal
residue mimics incorporating cyclohexyl and p-substi-
tuted phenyl groups and tested their ability to act as
inhibitors of RDP, in order to use them as biomarkers
to detect early stage colon tumors.
RDP has been extensively analyzed with respect to its
catalytic mechanism and inhibition kinetics by a variety
of synthetic inhibitors.2–4 The crystal structure of
human renal dipeptidase showed it to be a homodimer
with each subunit consisting of a 369 amino acid
residue peptide (42 kDa).5 RDP is a zinc-containing
hydrolytic enzyme that shows preference for dipeptide
substrates with dehydro amino acids at the carboxyl
position. Moreover, it can accommodate substrates
We report here the synthesis and biological evaluation
of new RDP inhibitors. The method employed for the
preparation of aminophosphinic acid derivatives is out-
lined in Scheme 1. Compound 1 was prepared by the
condensation of cyclohexylacetaldehyde, hypophospho-
ric acid and aminodiphenylmethanehydrochloride,11
followed by protection of the NH2 and P–OH groups
with Boc and CH2N2,12 respectively. Reaction of 1 with
trimethyl-2-phosphonoacrylate in the presence of
NaOMe as base gave an intermediate, which subse-
quently underwent a Wittig–Horner olefination13 with
aldehydes to give mixtures of E and Z isomers, which
with both D- or L-amino acids at that position, provid-
ing an excellent opportunity for the development of
specific probes for its detection in vivo.6 a-Aminophos-
phinic acids, the phosphorous analogues of natural
occurring a-aminocarboxylic acids, have received
increasing interest in medicine7 and synthetic organic
chemistry.8–10 The crystal structure of RDP–cilastatin
* Corresponding author. Tel.: +1-410-614-0200; fax: +1-410-614-8397;
e-mail: khansa@jhmi.edu
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(03)00082-0

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H. Gurulingappa et al. / Tetrahedron Letters 44 (2003) 1871–1873
Scheme 1. Reagents and conditions: (i) NaoMe, MeOH, 0°C, 10 min; (ii) trimethyl-2-phosphonoacrylate, 0°C, 30 min; (iii)
R-Ph-CHO, rt, 1 h; (iv) cyclo-C6H11CHO, rt, 1 h; (v) CF3COOH, CH2Cl2, rt, 30 min; (vi) conc. HCl, 50°C, 18 h; (vii) NBS,
BzOOBz, CCl4, reflux, 2.5 h; (viii) H2, Pd/C, MeOH, 30 min.
50% (IC50). The data for the synthesized compounds
are shown in Table 1. 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 mM ZnCl2,
0.1% Triton X 100. The extract was clarified by cen-
trifugation at 13,000×g for 5 min at 4°C and, for each
were separated by column chromatography over silica
gel. The yields were 30–40% for the Z isomers and
20–30% for the E isomers. The protecting groups were
removed by treatment with CF3COOH followed by
conc. HCl,12 and the crude products were purified by
column chromatography over silica gel to give 8–11.
Known compounds 6 and 7 were prepared12 for com-
parison. The reaction of 6 with NBS gave a mixture of
products from which 14 was isolated in 17% yield.
Removal of the protective groups of 14 gave the corre-
sponding free acid 15.15 The dihydro derivative 16 was
prepared by catalytic hydrogenation of 12 and 13. All
compounds were purified by column chromatography
over silica gel and their structures were unambiguously
confirmed by spectroscopic methods.16,17
Table 1. RDP inhibition activitya of compounds
Compound
Olefin geometry
IC50 (nM)
8
9
Z
E
Z
E
Z
E
Z
5.5
300
8
25
3.5
60
10
11
12
13
15
16
The RDP inhibition activity of these compounds was
determined using crude lysates prepared from human
colon cancers. The results are expressed as the concen-
tration of inhibitor needed to inhibit enzyme activity by
45
300
a IC50 values were determined using colon cancer lysate.

H. Gurulingappa et al. / Tetrahedron Letters 44 (2003) 1871–1873
1873
measurement, 20 ml was diluted into 158 ml of 20 mM
Tris, pH 8.0, 10 mM ZnCl2. 20 ml of the synthesized
compounds were added to each reaction to obtain final
concentrations ranging from 0 to 10 mM. 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 ml of 1 mM
2. Kropp, H.; Sundelof, J. G.; Hajdu, R.; Kahan, F. M.
Antimicrob. Agents Chemother. 1982, 22, 62–70.
3. Kahan, F. M.; Kropp, H.; Sundelof, J. G.; Birnbaum,
J. J. Antimicrob. Chemother. 1983, 12, 1–35.
4. Campbell, B. J.; Shih, Y. D.; Forrester, L. J.; Zahler,
W. L. Biochem. Biophys. Acta 1988, 956, 110–118.
5. Nitanai, Y.; Satow, Y.; Adachi, H.; Tsujimoto, M. J.
Mol. Biol. 2002, 321, 177–184.
substrate (ꢀDNP-L-Lys-D
-Amp).14 While incubating at
37°C, fluorescence (uex=320 nm, uem=405 nm) mea-
surements were determined at 30 s intervals and the
relative reaction rate was taken as the rate of increase
of fluorescence over time.
6. Greenstein, J. P. Adv. Enzymol. Relat. Subj. Biochem.
1948, 8, 117–169.
7. Atherton, F. R.; Hassall, C. H.; Lambert, R. W. J.
Med. Chem. 1986, 29, 29–40.
8. Smith, A. B.; Yager, K. M.; Taylor, C. M. J. Am.
From the data in Table 1, it is apparent that com-
pounds 8, 10 and 12 are potent RDP inhibitors while 15
and 16 are far less active. In general, compounds with
the Z configuration (8, 10, and 12) are significantly
more active than their E counterparts (9, 11 and 13).
Chem. Soc. 1995, 117, 10879–10888.
9. Xiao, J.; Zhang, X.; Yuan, C. Heteroatom Chem. 2000,
11, 536–540.
10. Wu, Y. Q.; Mobashery, S. J. Med. Chem. 1991, 34,
1914–1916.
11. Baylis, E. K.; Campbell, C. D.; Dingwall, J. D. J.
Chem. Soc., Perkin Trans. 1 1984, 2845–2853.
12. Parsons, W. H.; Hajdu, R.; Schoen, W. R.; Combs, P.
L.; Sundelof, J.; Patchett, A. A. Biochem. Int. 1991, 23,
1107–1115.
13. Schoen, W. R.; Parsons, W. H. Tetrahedron Lett. 1988,
29, 5201–5204.
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Biochem. 1999, 268, 245–251.
Our rationale for the preparation of these compounds
was based on the previous studies that the tetrahedral
geometry of the phosphorous in phosphinic acid is
important to mimic the presumed tetrahedral transition
state to inhibit dipeptide hydrolysis.9 We envisioned
that the use of aromatic side chains in such compounds
would substantially stabilize enzyme-inhibitor complex
formation by hydrophobic interactions.
15. Compound 11: 1H NMR (400 MHz, CD3OD): l 7.02
(m, 1H), 3.44 (m, 1H), 3.21 (m, 2H), 0.80–1.85 (m,
23H); 13C NMR (125 MHz, CD3OD): l 168.3, 145.0,
125.6, 43.5, 39.5, 38.5, 35.3, 32.8, 31.6, 30.5, 29.2, 28.2,
26.5, 25.2, 24.5, 22.5, 21.6, 19.5; LC–MS m/z 436 [M]+,
438 [M+2]+.
In conclusion, we have designed small molecules as
inhibitors of RDP. Structure–activity relationship stud-
ies of this new class of compounds are continuing and
will be reported in due course.
16. Compound 4: 1H NMR (400 MHz, CD3OD): l 7.83
(d, 1H, J=4 Hz), 7.78 (d, 2H, J=8 Hz), 7.46 (d, 2H,
J=8 Hz), 3.63 (m, 1H), 3.07 (m, 2H), 0.4–1.82 (m,
13H); 13C NMR (125 MHz, CD3OD): l 168.6, 138.5,
137.4, 136.8, 134.2, 128.3, 127.5, 126.2, 95.8, 46.3, 32.4,
31.8, 30.6, 29.2, 28.4, 25.3, 24.5, 19.6; LC–MS m/z 477
[M]+.
Acknowledgements
We are grateful for the support provided by the
National Institute of Health. We would like to thank
the Biophysical Chemistry Department for NMR
studies.
17. Compound 5: 1H NMR (400 MHz, CD3OD): l 7.67
(d, 2H, J=8 Hz), 7.14 (d, 2H, J=8 Hz), 6.95 (d, 1H,
J=4 Hz), 3.62 (m, 1H), 3.10 (m, 2H), 0.88–1.81 (m,
13H); 13C NMR (125 MHz, CD3OD): l 169.2, 137.5,
137.2, 136.6, 135.2, 127.5, 126.5, 125.8, 95.3, 46.5, 32.4,
30.8, 31.2, 29.3, 28.2, 25.4, 24.8, 23.6; LC–MS m/z 477
[M]+.
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