84 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1
Bavetsias et al.
compound 64a was obtained as a yellow solid (0.204 g, 88%):
mp 90 °C dec; NMR (Me2SO-d6) δ 1.74, 1.92 (2 × m, 4H, 2 ×
â-CH2), 2.23 (m, 4H, 2 × γ-CH2), 2.45 (s, 3H, quinazoline
2-CH3), 3.24 (s, 1H, CtCH), 4.18 (m, 1H, GluL R-CH), 4.36
(m, 3H, CH2CtC, GluD R-CH), 4.82 (s, 2H, quinazoline 6-CH2),
6.83 (d, J ) 8.8 Hz, 2H, 3′,5′-ArH), 7.61 (d, J ) 8.4 Hz, 1H,
quinazoline 8-H), 7.77 (m, 3H, quinazoline 7-H, 2′,6′-ArH), 8.02
(s, 1H, quinazoline 5-H), 8.16 (d, J ) 7.7 Hz, 1H, GluL NH),
8.33 (d, J ) 7.4 Hz, 1H, GluD NH); MS (FAB) m/ z 606 (M +
H)+. Anal. (C30H31N5O9‚1.7TFA‚0.25Et2O‚0.5H2O) C, H, N.
Tr i-ter t-bu tyl N-[N-(Ben zyloxycar bon yl)-D-γ-glu tam yl]-
D-glu ta m a te (61b). The general procedure A1 was followed
using R-tert-butyl N-(benzyloxycarbonyl)-D-glutamate (1.43 g,
4.24 mmol), THF (6 mL), N-methylmorpholine (0.428 g, 4.24
mmol), isobutyl chloroformate (0.577 g, 4.24 mmol), and a
solution of di-tert-butyl D-glutamate (1.25 g, 4.24 mmol) in THF
(6 mL). The crude product was purified by column chroma-
tography using a gradient of EtOAc in CH2Cl2 (10-20%) as
eluent. Trituration with petroleum ether afforded the title
compound 61b (1.50 g, 61%) as a white solid: mp 84-86 °C;
NMR (Me2SO-d6) δ 1.38, (s, 27H, 3 × C(CH3)3), 1.72, 1.89 (2 ×
m, 4H, 2 × â-CH2), 2.26 (m, 4H, 2 × γ-CH2), 3.89, 4.10 (2 × m,
2H, 2 × GluD R-CH), 5.03 (m, 2H, ArCH2), 7.35 (m, 5H, ArH),
7.64, 8.12 (2 × d, J ) 7.7 Hz, 2H, 2 × GluD NH); MS (FAB)
m/ z 579 (M + H)+. Anal. (C30H46N2O9) C, H, N.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectral
data of quinazoline γ-linked dipeptide tert-butyl esters 19-
22 and 24-31 and quinazoline γ-linked dipeptides 32-35 and
37-44 (2 pages). Ordering information is given on any current
masthead page.
Refer en ces
(1) Sirotnak, F. M.; DeGraw, J . I. In Folate Antagonists as Thera-
peutic Agents; Sirotnak, F. M., Burchall, J . J ., Ensminger, W.
D., Montgomery, J . A., Eds.; Academic Press: New York, 1984;
Vol. 2, pp 64-67.
(2) J ackman, A. L.; Taylor, G. A.; Gibson, W.; Kimbell, R.; Brown,
M.; Calvert, A. H.; J udson, I. R.; Hughes, L. R. ICI D1694, A
Quinazoline Antifolate Thymidylate Synthase Inhibitor That is
a Potent Inhibitor of L1210 Tumour Cell Growth In Vitro and
In Vivo; A New Agent for Clinical Study. Cancer Res. 1991, 51,
5579-5586.
(3) J ackman, A. L.; Farrugia, D. C.; Gibson, W.; Kimbell, R.; Harrap,
K. R.; Stephens, T. C.; Azab, M.; Boyle, F. T. ZD1694 (Tomu-
dex): A New Thymidylate Synthase Inhibitor With Activity in
Colorectal Cancer. Eur. J . Cancer, in press.
(4) J ackman, A. L.; Kelland, L. R.; Kimbell, R.; Brown, M.; Gibson,
W.; Aherne, G. W.; Hardcastle, A.; Boyle, F. T. Mechanisms of
Acquired Resistance to the Quinazoline Thymidylate Synthase
Inhibitor ZD1694 (Tomudex) in one Mouse and Three Human
Cell Lines. Br. J . Cancer 1995, 71, 914-924.
(5) Pizzorno, G.; Chang, Y.-M.; McGuire, J . J .; Bertino, J . R.
Inherent Resistance of Squamous Carcinoma Cell Lines to
Methotrexate as a Result of Decreased Polyglutamation of This
Drug. Cancer Res. 1989, 49, 5275-5280.
(6) Cowan, K. H.; J olivet, J . A. A Methotrexate Resistant Human
Breast Cancer Cell Line with Multiple Defects Including Di-
minished Formation of MTX Polyglutamates. J . Biol. Chem.
1984, 259, 10793-10800.
(7) Li, W. W.; Tong, W. P.; Waltham, M.; Bertino, J . R. Increased
Folylpolyglutamate Hydrolase Activity as a Novel Mechanism
of Natural Resistance to Methotrexate in a Human Soft Tissue
Sarcoma Cell Line. Proc. Am. Assoc. Cancer Res. 1993, 34, Abstr.
1651.
Tr i-ter t-bu tyl D-γ-Glu ta m yl-D-glu ta m a te (62b). The gen-
eral procedure B was followed using tri-tert-butyl N-[N-
(benzyloxycarbonyl)-D-γ-glutamyl]-D-glutamate (0.718 g, 1.24
mmol), EtOAc (90 mL), and 10% Pd/C (0.100 g). The title
compound 62b was obtained as a colorless oil (0.460 g, 84%)
and immediately used without further purification.
Tr i-ter t-bu tyl N-[N-[4-[N-[(3,4-Dih yd r o-2-m eth yl-4-oxo-
6-qu in a zolin yl)m eth yl]-N-p r op -2-yn yla m in o]ben zoyl]-D-
γ-glu ta m yl]-D-glu ta m a te (63b). The general procedure C
was followed using tri-tert-butyl D-γ-glutamyl-D-glutamate
(0.444 g, 1.0 mmol), 4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-
quinazolinyl)methyl]-N-prop-2-ynylamino]benzoic acid, tri-
fluoroacetate salt (10; 0.461 g, 1.0 mmol), DMF (15 mL), DEPC
(0.359 g, 2.2 mmol), and Et3N (0.222 g, 2.2 mmol). The crude
product was purified by column chromatography using a
gradient of MeOH in EtOAc (0-2%) as eluent. Reprecipitation
from CH2Cl2 (minimum amount)/petroleum ether afforded the
title compound 63b (0.400 g, 52%) as a white solid: mp 110-
116 °C; NMR (Me2SO-d6) δ 1.37, 1.39 (2 × s, 27H, 3 × C(CH3)3),
1.71, 1.89 (2 × m, 4H, 2 × â-CH2), 2.24 (m, 4H, 2 × γ-CH2),
2.33 (s, 3H, quinazoline 2-CH3), 3.23 (s, 1H, CtCH), 4.10, 4.24
(2 × m, 2H, 2 × GluD R-CH), 4.34 (s, 2H, CH2CtC), 4.78 (s,
2H, quinazoline 6-CH2), 6.83 (d, J ) 8.7 Hz, 2H, 3′,5′-ArH),
7.54 (d, J ) 8.4 Hz, 1H, quinazoline 8-H), 7.72 (m, 3H,
quinazoline 7-H, 2′,6′-ArH), 7.96 (s, 1H, quinazoline 5-H), 8.13,
8.32 (2 × d, J ) 7.5 Hz, 2H, 2 × GluD NH), 12.19 (s, 1H, lactam
NH); MS (FAB) m/ z 797 (M + Na)+. Anal. (C42H55N5O9) C,
H, N.
N-[N-[4-[N-[(3,4-Dih ydr o-2-m eth yl-4-oxo-6-qu in azolin yl)-
m et h yl]-N-p r op -2-yn yla m in o]b en zoyl]-D-γ-glu t a m yl]-D-
glu ta m ic Acid (64b). The general procedure D was followed
using 63b (0.208 g, 0.27 mmol) and TFA (15 mL). Compound
64b was obtained as a white solid (0.180 g, 89%): mp 95 °C
dec; NMR (Me2SO-d6) δ 1.74, 1.72 (2 × m, 4H, 2 × â-CH2),
2.23 (m, 4H, 2 × γ-CH2), 2.42 (s, 3H, quinazoline 2-CH3), 3.24
(s, 1H, CtCH), 4.18, 4.36 (2 × m, 2H, 2 × GluD R-CH), 4.30
(s, 2H, CH2CtC), 4.81 (s, 2H, quinazoline 6-CH2), 6.83 (d, J
) 8.8 Hz, 2H, 3′,5′-ArH), 7.60 (d, J ) 8.4 Hz, 1H, quinazoline
8-H), 7.77 (m, 3H, quinazoline 7-H, 2′,6′-ArH), 8.00 (s, 1H,
quinazoline 5-H), 8.13, 8.33 (2 × d, J ) 7.5 Hz, 2H, 2 × GluD
NH); MS (FAB) m/ z 628 (M + Na)+, 606 (M + H)+. Anal.
(C30H31N5O9‚1.15TFA‚H2O) C, H, N.
(8) Rhee, M. S.; Wang, Y.; Nair, M. G.; Galivan, J . Acquisition of
Resistance to Antifolates Caused by Enhanced γ-Glutamyl
Hydrolase Activity. Cancer Res. 1993, 53, 2227-2230.
(9) McGuire, J . J .; Coward, J . K. Pteroylpolyglutamates: Biosyn-
thesis, Degradation, and Function. In Folates and Pterins, Vol
1.; Blakley, R. L., Benkovic, S. J ., Eds.; J ohn Wiley & Sons: New
York, 1984; pp 135-190 and references cited therein.
(10) Sirotnak, F. M.; DeGraw, J . I. In Folate Antagonists as Thera-
peutic Agents; Sirotnak, F. M., Burchall, J . J ., Ensminger, W.
D., Montgomery, J . A., Eds.; Academic Press: New York, 1984;
Vol. 2, pp 86-89.
(11) Bisset, G. M. F.; Bavetsias, V.; Thornton, T. J .; Pawelczak, K.;
Calvert, A. H.; Hughes, L. R.; J ackman, A. L. The synthesis and
Thymidylate Synthase Inhibitory Activity of L-γ-L-Linked Dipep-
tide and L-γ-Amide Analogues of 2-Desamino-2-methyl-N10
-
propargl-5,8-dideazafolic acid (ICI 198583). J . Med. Chem. 1994,
37, 3294-3302.
(12) J odrell, D. I.; Gibson, W.; Bisset, G. M. F.; Boyle, F. T.; J udson,
I. R.; J ackman, A. L. The In Vivo Metabolic Stability of Dipeptide
Analogues of the Quinazoline Antifolate, ICI 198583, In Mice.
Biochem. Pharmacol. 1993, 46, 2229-2234.
(13) McGuire, J . J .; Hsieh, P.; Bertino, J . R.; Coward, J . K. Studies
on the Substrate Specificity of Mammalian Folylpolyglutamate
Synthetase. Chemistry and Biology of Pteridines; Walter de
Gruyter & Co.: Berlin, 1983; pp 615-619.
(14) Boyle, F. T.; Matusiak, Z. S.; Hughes, L. R.; Slater, A. M.;
Stephens, T. C.; Smith, M. N.; Brown, M.; Kimbell, R.; J ackman,
A. L. γ-Linked Dipeptide Analogues of 2-Desamino-2-methyl-
N10-propargyl-5,8-dideazafolate as Antitumour Agents. In Ad-
vances in Experimental Medicine and Biology (Chemistry and
Biology of Pteridines and Folates); Ayling, J . E., et al., Eds.;
Plenum Press: New York, 1993; Vol. 338, pp 585-588.
(15) Thornton, T. J .; J ackman, A. L.; Marsham, P. R.; O’Connor, B.
M.; Bishop, J . A. M.; Calvert, A. H. Quinazoline Antifolate
Thymidylate Synthase Inhibitors: Difluoro-Substituted Benzene
Ring Analogues. J . Med. Chem. 1992, 35, 2321-2327.
(16) (a) Godwin, H. A.; Rosenberg, I. H.; Ferenz, C. R.; J acobs, P. M.;
Meienhofer, J . The Synthesis of Biologically Active Pteroyloligo-
γ-L-Glutamates (Folic Acid Conjugates). J . Biol. Chem. 1972,
247, 2266-2271. (b) Meienhofer, J .; J acobs, P. M.; Godwin, H.
A.; Rosenberg, I. H. Synthesis of Hepta-γ-L-glutamic Acid by
Conventional and Solid-Phase Techniques. J . Org. Chem. 1970,
35, 4137-4140.
Ack n ow led gm en t. This work was supported by
grants from the Cancer Research Campaign. We thank
the School of Pharmacy, University of London, for
determining all FAB mass spectra, M. H. Baker for
electron impact and chemical ionization mass spectra,
and Dr. G. Poon for the electrospray ionization mass
spectra.
(17) Bisset, G. M. F.; Pawelczak, K.; J ackman, A. L.; Calvert, A. H.;
Hughes, L. R. Syntheses and Thymidylate Synthase Inhibitory
Activity of the Poly-γ-glutamyl Conjugates of N-[5-[N-(3,4-
Dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-
2-thenoyl]-L-glutamic Acid (ICI D1694) and Other Quinazoline
Antifolates. J . Med. Chem. 1992, 35, 859-866 and references
cited therein.