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best moiety in the R1 position, interacting probably with the S10
subsite of the enzyme, is p-methoxybenzyl, for example, Ih and
Ik. The combination of the two gives the best inhibitor, Ih, with
IC50 = 0.25 lM (Ki = 143 nM, see Fig. 2) as the racemic mixture. It
is interesting to see the clear additive effect of both optimal substi-
tutions at the S1 and S10 subsite of the enzyme. This could suggest
an easier development of future inhibitors. The thiocarbamoyl-
phosphinates Ia–Ik are comparable to the previously reported,3
corresponding oxo analogs, derivatives of carbamoyl phosphinates.
Competitive inhibition of APN by Ih proves the binding to the ac-
tive site of enzyme and the competition with substrate (Fig. 2).
As reported here (as well as earlier),3 the compound Ih, with
Ki = 143 nM (Fig. 2), is the best inhibitor with the new thiocarba-
moyl-phosphinate ZBG. Bestatin 7 has IC50 = 2.1 lM under the con-
ditions of our assay. The compound Ih, even as racemic mixture, is
almost ten times better an inhibitor than bestatin, an accepted
anti-cancer drug, active in vivo. Therefore, it is a good candidate
for the cell culture or in vivo studies to establish the role of APN
activity in cancer development.
General procedure for synthesis of compound I (a–k). 0.5 mM of compound 5
(a–k) was dissolved in 5 ml 2% HBr/AcOH. After 2 h mixture was evaporated,
dissolved in 1 ml MeOH and propylene oxide was added to pH 6. Compounds
were crystallized as a white solid.
Compound Ia: yield 25.6%; C11H17N2O2PS; MW: 272.301/mol; LC–MS 273.2
(M+1); 31P NMR (D2O): 28.10; 1H NMR (D2O): 0.77 (dd, 3 H, J = 14.7; 7.2 Hz,
CH3), 2.65–2.70 (t, 2H, CH2CH2Ph) 2.79–2.84 (m, 1H, NH2CHP), 3.49–3.64 (m,
2H, CH2CSNH), 6.39–7.06 (m, 5H, Ar–H).
Compound 1b: yield 55.6%; C11H17N2O3PS; MW: 288.31/mol; LC–MS 289.3
(M+1); 31P NMR (D2O): 28.16; 1H NMR (D2O): 0.90 (dd, 3H, J = 14.7; 7.5 Hz,
CH3), 3.00–3.05 (m, 1H, NH2CHP), 3.54 (s, 3H, OCH3), 4,45–4,50 (m, 2H,
CH2CSNH), 6.70–7.04 (m, 4H, Ar–H).
Compound Ic: yield 21.3%; C13H21N2O2PS; MW: 300.351/mol; LC–MS 301.3
(M+1); 31P NMR (D2O): 27.81; 1H NMR (D2O): 0.45–0.50 (t, 3H, CH3), 0.78–0.92
(m, 2H, CH2CH3), 1.06–1.18 (m, 2H, CH2CHP), 2.59–2.66 (m, 3H, NHCHP,
CH2Ph), 3.43–3.56 (m, 2H, CH2CSNH), 6.88–7.00 (m, 5H, Ar–H).
Compound Id: yield 49.0%; C13H21N2O3PS; MW: 316.351/mol; LC–MS: 317.3
(M+1); 31P NMR (D2O): 27.89; 1H NMR (D2O): 0.62 (d, 3H, J = 7.2 Hz, CH3),
1.06–1.08 (m, 2H, CH2CH3), 1.26–1.36 (m, 2H, CH2CHP), 2.88–2.94 (m, 1H,
NH2CHP), 3.56 (s, 3H, PhOCH3), 4.50 (d, 2H, J = 2.70 Hz, CH2CSNH), 6.71–7.17
(m, 4H, Ar–H).
Compound Ie: yield 43.5%; C9H21N2O2PS; MW: 252.311/mol; LC–MS 253.2
(M+1); 31P NMR (D2O): 29.22; 1H NMR (D2O): 0.99–1.07 (m, 12 H, 4 ꢀ CH3),
2.10–2.18 (m, 2H, 2 ꢀ CH), 3.23 (dd, J = 9.6; 4.2 Hz, 1H, NH2CHP), 3.51–3.63 (m,
2H, CH2NHCS).
Compound If: yield 46.2%; C11H25N2O2PS; MW: 280.361/mol; LC–MS 281.3
(M+1); 31P NMR (D2O): 26.61; 1H NMR (D2O): 0.78–0.81 (t, 3H, CH2CH3), 0.92
(dd, J = 29.7; 6.6 Hz, 6H, 2 ꢀ CH3), 1.21–1.25 (m, 2H, CH2(CH2)2CH3), 1.28–1.31
(m, 4H, 2 ꢀ CH2), 1.60–1.65 (m, 2H, CH2CH2NH), 2.01–2.04 (m, 1H, CH(CH3)2),
3.10 (dd, J = 9.0; 4.2 Hz 1H, NHCHP), 3.56–3.65 (m, 2H, CH2NHCS).
Compound Ig: yield 63.3%; C14H23N2O2PS; MW: 314.411/mol; LC–MS: 315.3
(M+1); 31P NMR (D2O): 27.31; 1H NMR (D2O): 0.51–0.55 (t, 3H, CH3), 0.85–1.28
(m, 6H, 3 ꢀ CH2), 2.32 (d, 1H, J = 1.5 Hz, NH2CHP), 2.59–2.67 (m, 2H, CH2Ph),
3.44–3.47 (m, 2H, CH2NHCS), 6.89–7.00 (m, 5H, Ar–H).
Acknowledgments
This work was supported by Ministry of Science and Higher
Education, Grants N405 008 31/0559 and N401 188 32/3917.
References and notes
Compound Ih: yield 66.8%; C14H23N2O3PS; MW: 330.381/mol; LC–MS: 331.4
(M+1); 31P NMR (D2O): 27.90; 1H NMR (D2O): 0.48ꢁ0.52 (t, 3H, CH3), 0.84–1.01
(m, 4H, 2 ꢀ CH2), 1.08–1.16, (m, 2H, CH2CHP), 1.26–1.35 (m, 2H, CH2CHP),
2.76–2.81 (m, 1H, NH2CHP), 3.48 (s, 3H, PhOCH3), 4.40 (d, 2H, J = 2.70 Hz,
CH2CSNH), 6.62–7.00 (m, 4H, Ar–H).
1. Summers, J. B.; Davidsen, S. K. Ann. Reps. Med. Chem. 1998, 33, 131; Sternlicht,
M. D.; Bergers, G. Emer. Ther. Targets 2000, 4, 609; Overall, C. M.; Lopez-Otin, C.
Nat. Rev. Cancer 2002, 2, 657; Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A.
J. H. Chem. Rev. 1999, 99, 2735.
Compound Ii: yield 43.7%; C12H27N2O2PS; MW: 294.391/mol; LC–MS 395.3
(M+1); 31P NMR (D2O): 27.23. 1H NMR (D2O): 0.77–0.78 (m, 9H, 3 ꢀ CH3), 0.84
(d, J = 6.61 Hz, 6H, 2 ꢀ CH3), 1.21–1.30 (m, 8H, 4 ꢀ CH2), 1.59–1.64 (m, 2H,
CH2CH(CH3)2), 1.71–1.74 (m, 1H, CH(CH3)2), 3.14–3.18 (q, 1H, NH2CHP), 3.57–
3.65 (m, 2H, CH2NHCS).
Compound Ij: yield 55%; C7H17N2O2PS; MW: 224.261/mol; LC–MS 225.1
(M+1); 31P NMR (D2O): 27.73; 1H NMR (D2O): 0.81 (dd, J = 35.8; 6.6 Hz, 6H,
2 ꢀ CH3), 1.25–1.32 (m, 2H, CH2), 1.69–1.74 (m, 1H, CH(CH3)2), 3.09 (d, 3H,
J = 1.8 Hz, CH3NHCS), 3.17–3.26 (m, 1H, NH2CHP).
Compound Ik: yield 64.9%; C18H23N2O3PS; MW: 378.431/mol; LC–MS 379.5
(M+1); 31P NMR (D2O): 27.02; 1H NMR (D2O): 1.24–1.33 (m, 2H, CH2CHP),
1.59–1.68 (m, 2H, CH2CHP), 2.23–2.32 (m, 2H, CH2CH2Ph), 2.52–2.61 (m, 2H,
CH2CH2Ph), 2.82–2.89 (m, 1H, NH2CHP), 3.47–3.52 (t, 3H, PhOCH3), 4.43–4.51
(t, 2H, CH2CSNH), 6.58–7.06 (m, 9H, Ar–H).
2. Coussens, L. M.; Fingleton, B.; Matrisian, L. M. Science 2002, 295, 2387.
3. Drag, M.; Grzywa, R.; Oleksyszyn, J. Bioorg. Med. Chem. Lett. 2007, 17, 1516.
4. Grzywa, R.; Oleksyszyn, J. Polish Patent Application P 383944, 2007.
5. Stange, T.; Kettermann, U.; Holzhausen, H. J. Acta Histochem. 1996, 98, 323.
6. Terauchi, M.; Kajiyama, H.; Shibata, K.; Nawa, K.; Mizutani, S.; Kikkawa, F. BMC
Cancer 2007, 7, 140.
7. Bauvois, B.; Dauzonne, D. Med. Res. Rev. 2006, 26, 88.
8. Shim, J. S. et al Chem. Biol. 2003, 10, 695.
9. Aggarwal, B. B.; Kumar, A.; Bharti, A. C. Anticancer Res. 2003, 23(1A), 363.
10. Petrov, K. A.; Nejmysheva, A. K. Z. Obsc. Chim. 1959, 29, 1819; CA 54, 1960,
8630.
11. Renard, P.-Y.; Schwebel, H.; Vayron, P.; Josien, L.; Valleix, A.; Mioskowski, C.
Chem. Eur. J. 2002, 8, 2910.
12. General procedure for synthesis of compounds
5 (a–k). 1.6 mM of N-
13. Aminopeptidase N inhibition studies. The inhibitory effect of compounds Ia–k
and bestatin towards aminopeptidase N (from porcine kidney, Sigma–Aldrich)
was evaluated using Leu-AMC (Sigma–Aldrich) as a fluorogenic substrate. For
the assay, sodium phosphate buffer (pH 7.2) was used. The final concentrations
were 0.2 lg/ml for APN and 12.5 lM for substrate. All inhibitors was measured
for 10 min at 25 °C without preliminary incubation, final DMSO concentration
was 2%. All IC50 values presented in Table 1 are means of two experiments, and
standard deviation is 20%. All compounds are racemic mixture.
benzyloxycarbonyl-a-aminoalkylphosphinate methyl ester was dissolved in
5 ml of dry dichloromethane. In rt and Ar atm 2.4 mM TEA and 1.6 mM of
TMSCl were added. After 30 min of stirring 3.2 mM of appropriate
isothiocyanate was added. Reaction was stired overnight. 5 ml of saturated
NH4Cl was added. After 10 min mixture was dissolved in 20 ml of AcOEt and
washed with saturated NH4Cl, saturated NaHCO3 and brine. The organic layer
was dried and evaporated. The crude oil was purified by silica gel column
chromatography using chloroform/ethyl acetate (2:1) as an eluent.