Quinazoline antifolate thymidylate synthase inhibitors: γ-linked L-D, D- D, and D-L dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8- dideazafolic acid (ICI 198583)

DOI: 10.1021/jm950471+

Source and publish data:

Journal of Medicinal Chemistry p. 73 - 85 (1996)

Update date:2022-08-03

Topics:

Authors:

Bavetsias

Jackman Jackman

Kimbell Kimbell

Gibson Gibson

Boyle Boyle

Bisset Bisset

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Article abstract of DOI:10.1021/jm950471+

The syntheses of γ-linked L-D, D-D, and D-L dipeptide analogues of 2- desamino-2-methyl-N¹⁰-propargyl-5,8-dideazafolic acid (ICI 198583) are described. The general methodology for the synthesis of these molecules involved the preparation of the dipeptide derivatives employing solution phase peptide synthesis followed by condensation of the dipeptide free bases with the appropriate pteroic acid analogue via diethyl cyanophosphoridate (DEPC) activation. In the final step, tert-butyl esters were removed by trifluoroacetic acid (TFA) hydrolysis. Z-L-Glu-OBu(t)-γ-D-Ala-OBu(t), for example, was prepared from α-tert-butyl N-(benzyloxycarbonyl)-L-glutamate and tert-butyl D-alaninate via isobutyl-mixed anhydride coupling. The Z- group was removed by catalytic hydrogenolysis and the resulting dipeptide free base condensed with 2-desamino-2-methyl-N¹⁰-propargyl-5,8- dideazapteroic acid via DEPC coupling. Finally, tert-butyl esters were removed by TFA hydrolysis to give ICI 198583-γ-D-Ala. The compounds were tested as inhibitors of thymidylate synthase and L1210 cell growth. Good enzyme and growth inhibitory activity were found with y-linked L-D dipeptides, the best examples being the Glu-γ-D-Glu derivative 35 (K(i) = 0.19 nM, L1210 IC₅₀ = 0.20 ± 0.017 μM) and the Glu-γ-D-α-aminoadipate derivative 39 (K(i) = 0.12 nM, L1210 IC₅₀ = 0.13 ± 0.063 μM). In addition, ICI 198583 L-γ-D-linked dipeptides were resistant to enzymatic degradation in mice.

Full text of DOI:10.1021/jm950471+

J . Med. Chem. 1996, 39, 73-85

Qu in a zolin e An tifola te Th ym id yla te Syn th a se In h ibitor s: γ-Lin k ed L-D, D-D, a n d

D-L Dip ep tid e An a logu es of 2-Desa m in o-2-m eth yl-N¹⁰-p r op a r gyl-5,8-d id ea za folic

Acid (ICI 198583)^†,‡

Vassilios Bavetsias,*^,§Ann L. J ackman,^§Rosemary Kimbell,^§William Gibson,^§F. Thomas Boyle,^|and

Graham M. F. Bisset^§,

CRC Centre for Cancer Therapeutics, The Institute of Cancer Research, Cancer Research Campaign Laboratories,

15 Cotswold Road, Sutton, Surrey SM2 5NG, England, and Zeneca Pharmaceuticals, Mereside, Alderley Park,

Macclesfield, Cheshire SK10 4TG, England

Received J une 26, 1995^X

The syntheses of γ-linked L-D, D-D, and D-L dipeptide analogues of 2-desamino-2-methyl-N¹⁰-

propargyl-5,8-dideazafolic acid (ICI 198583) are described. The general methodology for the

synthesis of these molecules involved the preparation of the dipeptide derivatives employing

solution phase peptide synthesis followed by condensation of the dipeptide free bases with the

appropriate pteroic acid analogue via diethyl cyanophosphoridate (DEPC) activation. In the

final step, tert-butyl esters were removed by trifluoroacetic acid (TFA) hydrolysis. Z-^L-Glu-

OBu^t-γ-D-Ala-OBu^t, for example, was prepared from R-tert-butyl N-(benzyloxycarbonyl)-L-

glutamate and tert-butyl ^D-alaninate via isobutyl-mixed anhydride coupling. The Z-group was

removed by catalytic hydrogenolysis and the resulting dipeptide free base condensed with

2-desamino-2-methyl-N¹⁰-propargyl-5,8-dideazapteroic acid via DEPC coupling. Finally, tert-

butyl esters were removed by TFA hydrolysis to give ICI 198583-γ-^D-Ala. The compounds were

tested as inhibitors of thymidylate synthase and L1210 cell growth. Good enzyme and growth

inhibitory activity were found with γ-linked ^L-D dipeptides, the best examples being the Glu-

γ-^D-Glu derivative 35 (K_i) 0.19 nM, L1210 IC₅₀) 0.20 ( 0.017 µM) and the Glu-γ-D-R-

aminoadipate derivative 39 (K_i) 0.12 nM, L1210 IC₅₀) 0.13 ( 0.063 µM). In addition, ICI

198583 ^L-γ-D-linked dipeptides were resistant to enzymatic degradation in mice.

In tr od u ction

catalyze the cleavage of the γ-glutamyl amide bond of

polyglutamates,⁹and may result in normal tissue tox-

icities due to polyglutamate retention. For these rea-

sons, we were interested in designing and synthesizing

potent inhibitors of TS which would not depend on

polyglutamation for antitumor activity. However, as

part of our requirement, compounds were designed to

utilize the reduced folate/MTX carrier (RFC), a trans-

port system commonly used by many classical anti-

folates such as MTX or ZD1694,⁴since use of the RFC

transport system was found to increase the growth

inhibitory potency of many water-soluble antifolates. In

addition, utilization of the RFC transport system has

been reported to offer some tumor selectivity.¹⁰

Polyglutamation of antifolates targeted toward thy-

midylate synthase (TS) can be considered as a desirable

metabolic process since the polyglutamate metabolites

are often more potent inhibitors of TS than the parent

drug, and these molecules are retained within the cells

for prolonged periods due to the polyionic character. In

addition, it is believed that some degree of tumor

selectivity may be achieved since in tumor cells in mice

polyglutamation has been reported to occur more rapidly

than in normal tissues.¹Tomudex (ZD 1694)²is an

example of a polyglutamatable inhibitor of TS which is

currently in phase III clinical study for the treatment

of colorectal cancer.³However, the FPGS substrate

activity of antifolates such as ZD1694 may be of a

disadvantage in tumors expressing low levels of, or an

altered expression of, folylpolyglutamyl synthetase

(FPGS)^4-6or in tumors expressing high levels of

γ-glutamyl hydrolases,^7,8a group of enzymes that

^†Part of this work has been presented in preliminary form; see:

Bavetsias, V.; J ackman, A. L.; Thornton, T. J .; Pawelczack, K.; Boyle,

F. T.; Bisset, G. M. F. Quinazoline Antifolates Inhibiting Thymidylate

Synthase: Synthesis of γ-Linked Peptide and Amide Analogues of

2-Desamino-2-Methyl-N¹⁰-Propargyl-5,8-Dideazafolic Acid (ICI 198583).

In Advances 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 593-596.

^‡Abbreviations: TS, thymidylate synthase; FPGS, folylpolyglutamyl

synthetase; MTX, methotrexate; DEPC, diethyl cyanophosphoridate;

Z, benzyloxycarbonyl; pg, propargyl; Glu, glutamic acid; Ala, alanine;

Phe, phenylalanine; aad, R-aminoadipic acid; TFA, trifluoroacetic acid.

^§Cancer Research Campaign Laboratories.

^|Zeneca Pharmaceuticals.

Present address: Cross Medical Ltd., 30 Skylines Village, Marsh

Wall, London E14 9TS, England.

^XAbstract published in Advance ACS Abstracts, November 1, 1995.

74 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1

Bavetsias et al.

Sch em e 1

ICI 198583 γ-linked L-L dipeptides (in which the

second amino acid was other than glutamate) were the

first class of dipeptides synthesized as inhibitors of TS

that do not undergo polyglutamation.¹¹Although these

γ-linked L-L dipeptides were potent inhibitors of TS,

their utility was limited because when they were

injected into mice they were degraded to ICI 198583,¹²

a compound known to be a substrate for FPGS. This

degradation is believed to be the result of the enzymatic

cleavage of the γ-glutamyl amide bond by γ-glutamyl

hydrolases that have high activity in tissues such as

liver. We therefore envisaged stabilizing the γ-glutamyl

amide bond in γ-linked L-L dipeptide derivatives of ICI

198583 to enzymatic degradation by inverting the

stereochemistry of the amino acids from L to D. It was

also believed that the second amino acid could be

glutamate since it had previously been reported that

natural folates and folate analogues with a D-Glu

instead of an L-Glu were not substrates for FPGS.¹³

With regard to TS inhibition, we believed that L-γ-D-

dipeptide analogues of ICI 198583 could maintain the

high affinity for TS shown by quinazoline antifolate

L-γ-L dipeptides.¹¹During the course of the study, a

computer graphics model of the humanized Escherichia

coli active site of TS was developed and indicated that

the R-carboxyls of the diglutamate chain of ICI 198583-

γ-L-Glu (3) play a crucial role for TS binding. The

R-carboxyl of the first glutamate was shown to interact

with a lysine residue (conserved Lys48) through a

molecule of water, and the R-carboxyl of the second

glutamate interacts electrostatically with an arginine

residue in the active site of the enzyme. Further

examination of the model indicated that the electrostatic

interaction could take place regardless of the stereo-

chemistry of the second (terminal) amino acid and

therefore supported the view that L-D dipeptides may

also maintain high affinity for TS possibly to a level

shown by their L-L counterparts.¹¹We report here the

synthesis of 15 dipeptide analogues of ICI 198583, some

of which include 2′-F and 7-Me substituents, which had

been shown in other series to enhance further TS

inhibition.^14,15

28% yield or by treatment with isobutylene/concentrated

H₂SO₄in CH₂Cl₂in 45% yield. The preparation of 5b

is shown in Scheme 1. Z-D-Phe (8) was converted into

the tert-butyl ester 9 by treatment with isobutylene/

concentrated H₂SO₄in CH₂Cl₂, and the Z-group was

then removed by catalytic hydrogenolysis to afford 5b

in 69% overall yield. Finally, di-tert-butyl D-R-amino-

adipate hydrochloride (5d ) was prepared from D-R-

aminoadipic acid by a method identical with that

described for the L-enantiomer.¹¹

Ch em istr y

The chemistry of dipeptide analogues of various

antifolates has been described in a recent paper from

our laboratories.¹¹Our route to γ-linked L-D dipeptide

analogues of ICI 198583, summarized in Scheme 1, was

identical with that used for the synthesis of L-γ-L

dipeptide derivatives of ICI 198583 and is based on the

strategy used in the synthesis of pteroyloligo-γ-glu-

tamates¹⁶and the polyglutamates of ZD1694.¹⁷

For the synthesis of Z-blocked L-γ-D dipeptides 6

(Scheme 1), the mixed carbonic anhydride coupling

method employing isobutyl chloroformate as the acti-

vating agent was used.¹⁸The carboxyl-protecting group

tert-butyl was selected to avoid the possibility of γ f R

transpeptidation associated with alkaline carboxyl depro-

tection,¹⁹since the tert-butyl esters can be easily cleaved

by treatment with trifluoroacetic acid under mild condi-

tions in a racemization-free process.

Quinazoline antifolate dipeptide tert-butyl esters 19-

26 (Table 1) were prepared by condensing dipeptides 7

(obtained from 6 by catalytic hydrogenolysis, Scheme

1) with the appropriate pteroic acid analogues, i.e., 10¹¹

and 11-13, via DEPC activation²²(Scheme 1). The

general route to pteroic acid analogues 11-13 is shown

in Scheme 2. Compound 11, for example, was prepared

by coupling 6-(bromomethyl)-3,4-dihydro-2,7-dimethyl-

4-oxoquinazoline (45b)²³with tert-butyl 4-(prop-2-ynyl-

amino)benzoate (46a )^24,25followed by TFA hydrolysis.

Quinazoline antifolate L-Glu-γ-D-Glu tert-butyl esters

27-31 (Table 1) were obtained by condensing 7c with

the appropriate pteroic acid analogue, i.e., 14,¹¹15-17,

and 18,¹¹again using DEPC (Scheme 1). 4-[N-[(3,4-

Dihydro-2,7-dimethyl-4-oxo-6-quinazolinyl)methyl]-N-

methylamino]benzoic acid (15) was prepared from 48²⁶

D-Alanine tert-butyl ester (5a ) was obtained com-

mercially. Di-tert-butyl D-glutamate (5c) was prepared

from the corresponding acid either by transesterification

with tert-butyl acetate^20,21and 70% perchloric acid in

Quinazoline Antifolate TS Inhibitors

J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 75

Ta ble 1. Preparation of Quinazoline Antifolate L-D Dipeptide Esters

compd

R¹

R²

yield (%)

mp (°C)

m/z (M + H)⁺

formula

anal.

Glu(OBu^t)-OBu^t

Clu(OBu^t)-OBu^t

Ala-OBu^t

116-117

774

810^a

814^a

828^a

660

736

788

820

750

764

768

782

763

C₄₂H₅₅O₉N₅‚0.5H₂O

C₄₃H₅₇N₅O₉‚0.5H₂O

C₄₂H₅₄FN₅O₉‚0.5H₂O

C₄₃H₅₆FN₅O₉‚0.5H₂O

C₃₆H₄₅N₅O₇

C₄₂H₄₉N₅O₇‚1.5H₂O

C₄₃H₅₇N₅O₉‚0.25H₂O

C₄₄H₅₈FN₅O₉

C₄₀H₅₅N₅O₉

C₄₁H₅₇N₅O₉‚0.6H₂O

C₄₀H₅₄FN₅O₉

C₄₁H₅₆FN₅O₉

C₄₁H₅₇N₅O₉‚0.5H₂O

CHN

CHNF

CHN

CHNF

CHN

159-161.5

105-108

149.5-150.5

166-168

115-117.5

108-109

149-150

104.5-108.5

243.5-245

100-102

172-173

110.5-113.5

Phe-OBu^t

aad(OBu^t)-OBu^t

Glu(OBu^t)-OBu^t

CHNF

CHN

(M + Na)⁺.

Sch em e 2

the L-D analogues 32-44, except that R-tert-butyl N-

(benzyloxycarbonyl)-D-glutamate (Z-D-Glu-OBu^t, 60) was

used in place of Z-L-Glu-OBu^t(4) (Scheme 5). R-tert-

Butyl N-(benzyloxycarbonyl)-D-glutamate (60) was syn-

thesized by a route similar to that employed for the

synthesis of its L-enantiomer²⁸(Scheme 5). D-Glutamic

acid was converted to its corresponding γ-methyl ester

hydrochloride 57 by treatment with SOCl₂/MeOH.²⁹

Benzyl chloroformate was then used for the introduction

of the Z-group³⁰and the R-carboxyl of 58 masked as its

tert-butyl ester by treatment with Bu^tOH/POCl₃/pyri-

dine.²⁸In the last step, the γ-methyl ester was hydro-

lyzed under alkaline conditions to afford 60 in 33%

overall yield.³¹

Ster eoch em ica l In tegr ity a n d NMR An a lysis of

Qu in a zolin e An tifola te Dip ep tid es

Since there is no evidence to suggest that epimeriza-

tion takes place in the mixed carbonic anhydride-

coupling, hydrogenation, DEPC-coupling or acid hydrol-

ysis steps, it was expected that the route employed for

the preparation of 32-44 and 64a ,b would give iso-

merically pure antifolates. To confirm this, an HPLC

chromatographic separation of 3 (ICI 198583-γ-L-Glu)

and its three stereoisomers 32 (L-D), 64a (D-L), and 64b

(D-D) using a â-cyclodextrin column was attempted. The

chromatogram of the L-D stereoisomer, compound 32,

contained only one peak suggesting that this methodol-

ogy provides isomerically pure products. However, the

chromatogram of the D-D stereoisomer 64b showed a

14% contamination with the L-D stereoisomer 32, while

the chromatogram of the D-L stereoisomer 64a showed

an 8% contamination with the L-L stereoisomer. These

unexpected results led us to an investigation of the

optical purity of the glutamate 60 which was used as

one of the chiral building blocks for the preparation of

both 64a ,b. Indeed, the specific optical rotation of 60

was found to be +20.0°, significantly lower than that

reported for the L-enantiomer (-26.6°),²⁸suggesting that

this compound was enantiomerically impure. As a

consequence, quinazoline antifolates 64a ,b were ob-

tained as diastereoisomeric mixtures D-L/L-L and D-D/L-

D, respectively. Compounds 64a ,b were not prepared

in isomerically pure form since they were substantially

less potent inhibitors of TS compared with ICI 198583-

γ-D-Glu (32) (8- and 6-fold, respectively).

by removing the POM-group under alkaline conditions

(Scheme 3).

The synthesis of pteroic acid analogues 16 and 17 is

shown in Scheme 4. Alkylation of tert-butyl 4-amino-

2-fluorobenzoate (51)²⁴with 6-(bromomethyl)-3,4-dihy-

dro-2-methyl-4-oxo-3-[(pivaloyloxy)methyl]quinazoline

(49)²⁷or 6-(bromomethyl)-3,4-dihydro-2,7-dimethyl-4-

oxo-3-[(pivaloyloxy)methyl]quinazoline (50)²⁴in DMA,

using 2,6-lutidine as base, afforded compounds 52 and

53 from which 54 and 55 were obtained by treatment

with NaBH₃(CN), HCHO, and AcOH. Removal of the

tert-butyl and POM protecting groups was achieved

upon treatment with TFA and then aqueous NaOH.

Quinazoline antifolate dipeptide tert-butyl esters 19-

31 were deprotected with TFA to give quinazoline

antifolates 32-44 (Table 2) as their TFA salts.

In an effort to determine whether stereochemical

changes at the R-center of the first or both glutamic

residues of ICI 198583-γ-L-Glu make the γ-glutamyl

bond resistant to enzymatic degradation, antifolates

64a ,b were prepared. They were synthesized by a route

identical with that described above for the synthesis of

The proton NMR spectrum of compounds unsubsti-

tuted in the p-aminobenzoate residue, e.g., 32, shows

the amidic hydrogen of the first glutamic residue as a

doublet (J ) 7.5 Hz) due to a vicinal coupling to GluL

76 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1

Bavetsias et al.

Sch em e 3

Sch em e 4

Ta ble 2. Preparation of Quinazoline Antifolate L-D Dipeptides

compd

R¹

R²

yield (%)

mp (°C)

m/z (M + H)⁺

formula

C₃₀H₃₁N₅O₉‚1.4TFA

C₃₁H₃₃N₅O₉‚TFA‚0.5Et₂O‚1.3H₂O

C₃₀H₃₀FN₅O₉‚1.5TFA‚0.6Et₂O‚0.5H₂O

₃₁H₃₂FN₅O₉‚TFA‚0.5Et₂O‚0.6H₂O

anal.

Glu

Ala

Phe

aad

Glu

100

146-148

160 dec

115 dec

155 dec

180 dec

606

642^a

624^a

638

570^a

624

620

652

582

596

600

614

596^a

CHN

CHNF

C₂₈H₂₉N₅O₇‚TFA‚0.7Et₂O

130 dec

₃₄H₃₃N₅O₇‚TFA‚0.7H₂O

145-146

143-144 dec

105 dec

₃₁H₃₃N₅O₉‚0.9TFA‚H₂O

₃₂H₃₄FN₅O₉‚0.8TFA‚0.75H₂O

₂₈H₃₁N₅O₉‚1.5TFA‚0.55Et₂O‚1.1H₂O

₂₉H₃₃N₅O₉‚1.1TFA‚0.2Et₂O‚H₂O

₂₈H₃₀FN₅O₉‚1.3TFA‚Et₂O‚0.75H₂O

₂₉H₃₂FN₅O₉‚TFA‚H₂O

150 dec

145-147

159-161

140 dec

C₂₉H₃₃N₅O₉‚0.95TFA‚0.15Et₂O‚1.3H₂O

(M+Na)⁺.

R-CH. However, in the 2′-F and 2′-F,7-Me derivatives,

e.g., compound 34, 35, 21 or 22, the amidic hydrogen of

the first glutamic residue appears as a double doublet

or triplet. The same phenomenon was observed by

Thornton et al.¹⁵with the 2′-F derivative of ICI 198583.

This is due to an additional long range coupling to the

2′-F, and in the case of 22, the existence of this coupling

was demonstrated by a heteronuclear decoupling ex-

periment. When the 2′-F signal was irradiated, the

corresponding amidic hydrogen signal was collapsed to

a doublet (Figure 1), revealing a coupling of 5.5 Hz

between NH and 2′-F.

L1210:1565 cell growth was also as previously de-

scribed.³³L1210:1565 is a L1210 mutant cell line with

impaired reduced folate/MTX transport carrier. This

cell line was made resistant to CI-90, a compound that

uses the RFC transport system,³⁴and hence is cross-

resistant to MTX.

Resu lts a n d Discu ssion

Replacement of the second glutamic residue of ICI

198583-γ-L-Glu by D-amino acids (Glu, Ala, Phe, aad)

gave compounds 32 and 36-38 which were shown to

be resistant to enzymatic degradation (ref 12; Table 4).

ICI 198583-γ-D-Glu (32) was a potent inhibitor of TS

(IC₅₀) 4.6 nM) and only 2-fold worse then ICI 198583-

γ-L-Glu. However, replacing the propionate side chain

of the D-glutamate of 32 by a methyl or benzyl group

gave dicarboxylates 36 and 37 which were respectively

3- and 7-fold worse inhibitors of TS. On the other hand,

lengthening the propionate side chain of the D-glutamate

of 32 by one methylene gave ICI 198583-γ-aad (38) with

inhibitory activity against TS similar to that of 32. As

indicated by an analysis of the computer graphics model

Biologica l Eva lu a tion

The antifolates listed in Table 3 were tested as

inhibitors of partially purified TS from L1210 mouse

leukemia cells that overproduce TS due to amplification

of the TS gene.³²The partial purification and assay

methods used in this study were as previously described,

and they used (()-5,10-methylenetetrahydrofolic acid at

a concentration of 200 µM.³²Inhibition of L1210 and

Quinazoline Antifolate TS Inhibitors

J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 77

Sch em e 5

derivative 32 affects TS and L1210 cell growth inhibi-

tion, compounds 33-35 were synthesized. Crystal

structures of the ternary complex of E. coli TS with CB

3717 and 5-fluoro-2′-deoxyuridylate (FdUMP) indicate

that CB 3717 binds in a partially folded conformation,

with the p-aminobenzoyl ring inclined at 65° to the

quinazoline ring.^35,36The impact of 7-Me, 5-Me, or 7,9-

di-Me substitution on this conformation was studied by

Boyle et al. using molecular mechanics (SCANOPT) and

semiempirical quantum mechanical energy calculations

(AMPAC).¹⁴It was found that 7-Me substitution rein-

forces this conformation and, as predicted, TS inhibition

was improved.¹⁴With this consideration in mind, a

7-methyl group was introduced into the quinazoline ring

to give compound 33. The substitution of a 2′-F in

dipeptide 32 to give compound 34 originated from the

observation that introduction of a 2′-F in ICI 198583

had a beneficial effect in binding to TS.¹⁵As expected,

in our compounds substitution of a 7-Me into the

quinazoline ring (compound 33) or a 2′-F into the

benzene ring (compound 34) enhanced TS inhibition

(each by ∼2-fold compared with the parent 32). Com-

bining both of these modifications to give compound 35

resulted in 5-fold improvements in TS inhibition (K_i)

0.19 nM). However, compounds 32-35 all showed

similar growth inhibitory activity against the L1210 cell

line (IC₅₀∼ 0.2 µM), which may be a consequence of

marginally slower cellular uptake.

Replacing the N¹⁰-propargyl substituent in compound

32 by methyl (compound 40) or ethyl (compound 44)

gave poorer inhibitors of TS and L1210 cell growth

(Table 3). However, substitution of 7-Me or 2′-F into

dipeptide 40 gave compounds 41 and 42 with respec-

tively a 7- and 4-fold increase in TS inhibition. Sub-

stitution of a 2′-F into the benzene ring and a 7-Me into

the quinazoline ring of dipeptide 40 gave compound 43,

which was a 17-fold better inhibitor of TS. Since the

2′-F,7-Me substitution pattern strengthened binding to

TS in both the N¹⁰-methyl and N¹⁰-propargyl series,

these substituents were introduced into ICI 198583-γ-

D-aad (38). The resulting compound 39 showed similar

inhibitory activity against TS and L1210 cell growth to

of the humanized E. coli active site of TS (see Introduc-

tion), the L-D dipeptide derivatives 32 and 36-38 would

be expected to exhibit similar TS inhibition to their L-L

counterparts (Table 5).

As inhibitors of cell growth, 36 and 38 showed similar

activity to that of 32, but 37 was less active. Growth

inhibition was generally similar to the L-L counterparts

(Table 5). It appears that changing the stereochemistry

of the second amino acid does not prevent uptake via

the RFC since compounds 32, 36, and 38 were poorly

active in the L1210/1565 cell line (Table 3).

Replacing the first glutamic residue of ICI 198583-

γ-L-Glu (3) by its D-enantiomer gave compound 64a and

resulted in a substantial decrease (∼20-fold) in TS

inhibition (IC₅₀) 37 nM). The same trend was observed

when both glutamic residues of 3 were replaced by their

D-enantiomers (compound 64b, TS IC₅₀) 27 nM).

These results highlight the importance of the R-carboxyl

for binding to TS and suggest that by changing the

stereochemistry of the first glutamic residue of ICI

198583-γ-L-Glu from L to D, the R-carboxyl is no longer

able to bind to the lysine residue in the active site of

the enzyme. The lack of this interaction may account

for the low inhibitory activity of 64a ,b for TS. The D-L

dipeptide 64a was unstable to hydrolysis when injected

into mice, but the D-D analogue 64b was apparently

stable to enzymatic degradation.¹²

that of 35 (TS IC₅₀) 0.56 ( 0.34 nM, L1210 IC₅₀

0.13 ( 0.063 µM).

)

Some compounds were tested as inhibitors of the

growth of L1210:R^D1694cell line which has a marked

polyglutamation defect and is consequently cross-

resistant (18-fold) to ICI 198583-γ-L-Glu.³⁷Compound

32 had good activity against this cell line (IC₅₀) 0.68

( 0.35, Table 5). This is in agreement with other

findings which show that natural folate and folate

analogues possessing a D-glutamate are not substrates

for FPGS.¹³If the second amino acid is Ala, then the

stereochemistry is unimportant as neither can be poly-

glutamated. This also indicates that although hydroly-

sis of the L-L bond occurs in mice, it does not occur in

tissue culture to any significant extent (the breakdown

product ICI 198583 has poor activity against this cell

line because of its requirement for polyglutamation³⁷).

The cytotoxocity of ICI 198583-γ-L-aad (L1210:R^D1694

IC₅₀) 2.9 ( 0.40, Table 5) was interesting in that the

level of cross-resistance seen was quite high but was

reduced when the terminal L-aad was replaced with the

D-aad (compound 38; Table 5). This may suggest that

In an attempt to study how other structural modifica-

tions of the N¹⁰-propargyl quinazoline L-Glu-γ-D-Glu

78 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1

Bavetsias et al.

F igu r e 1. (a) H NMR spectrum of 22 and (b) heteronuclear-decoupled spectrum of 22 obtained by irradiating the 2′-F signal

and then recording the ¹H NMR spectrum.

Ta ble 3. L1210 TS and Cell Growth Inhibition Data for Quinazoline Antifolate γ-Linked Dipeptides^a

inhibition of cell growth, IC₅₀(µM)

inhibition of

L1210 TS, IC₅₀(nM)

compd

R¹

R²

L1210

W1L2

L1210:1565

L-Glu-L-Glu

L-Clu-D-Glu

L-Glu-D-Glu

L-Glu-D-Ala

L-Glu-D-Phe

L-Glu-D-aad

L-Glu-D-Glu

D-Glu-L-Glu

D-Glu-D-Glu

2.0

4.6

1.9

0.16 ( 0.069

0.33 ( 0.25

0.21

0.09, 0.074

0.15, 0.23

0.23

0.22

0.17, 0.093

0.15

4.6, 5.0

15, 16

7.8

6.8, 8.7

64a

64b

1.9

0.21

0.4, 0.92^b

0.20 ( 0.017

0.43 ( 0.13

2.1 ( 0.36

0.08, 0.11

0.13 ( 0.063

3.2

0.58

0.65

0.26

3.6

>10

10, 4.2

0.22

0.19, 0.3

0.56 ( 0.34^c

8.1, 12

4.6

8.8

2.0

0.28

0.9

3.4

1.3

2.4

1.4

∼100

Methodologies for the inhibition of mouse L1210 TS and L1210, L1210:1565 (impaired RFC), and human W1L2 cell growth are as

described in refs 32 and 33. K_i) 0.19 nM. ^cK_i) 0.12 nM.

the L-Glu-γ-L-aad is a substrate for FPGS. Further

work using isolated FPGS may clarify this.

potent inhibitors of TS and also stable to hydrolysis

when injected into mice. The best examples were the

Glu-γ-D-Glu derivative 35 and the Glu-γ-D-aad deriva-

tive 39. Reduced activity against the L1210:1565 cell

line compared to the parental line indicated that L-D

dipeptides utilize the RFC transport system to enter

cells. Equivalent activity in the L1210:R^D1694line

suggests that they were not substrates for FPGS. These

compounds therefore represent an interesting group of

potent, water-soluble, folate-based TS inhibitors that do

not undergo polyglutamation but have antitumor activ-

ity.

Recent data demonstrate that cellular TS is only

inhibited while the compounds, e.g., 32 and 35, are

present in the medium. This is contrast to ICI 198583-

γ-L-Glu that continues to inhibit the enzyme even after

removal from the extracellular environment^37,38which

is consistent with the formation of slowly effluxable

higher chain polyglutamates.

Compounds 35 and 39 were also tested as inhibitors

of tumor growth in mice. Both compounds had activity

against the L57789 TK^-/-and TK^+/-tumors and the

HX62 human ovarian tumor xenograft.^39,40

Exp er im en ta l Section

In conclusion, replacement of the second amino acid

of γ-linked L-L dipeptides of ICI 198583 by their D-

enantiomers produced dipeptide derivatives that were

Thin layer chromatography (TLC) was performed on pre-

coated sheets of silica gel 60F₂₅₄(Merck art. 5735). Visualiza-

Quinazoline Antifolate TS Inhibitors

J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 79

Ta ble 4. Stability of Quinazoline Antifolate γ-Linked

Gen er a l P r oced u r es. P r oced u r e A: P r ep a r a tion of

Z-Block ed Dip ep tid e ter t-Bu tyl Ester s. To a stirred solu-

tion of R-tert-butyl N-(benzyloxycarbonyl)-^L-glutamate (1.0

mmol) in dry THF (3.0 mL) and N-methylmorpholine (1.0

mmol) cooled to -20 °C was added isobutyl chloroformate (1.0

mmol) (a white precipitate had formed). In method A1 stirring

was continued for 10 min at -20 °C, and then a suspension of

the appropriate amino acid tert-butyl ester hydrochloride salt

(1.0 mmol) in dry THF (3.0 mL) and N-methylmorpholine (1.0

mmol) was added to the reaction mixture. In method A2

stirring was continued for 10 min at -20 °C, and then a

solution of the appropriate amino acid tert-butyl ester free base

(1.0 mmol) in dry THF (3.0 mL) was added into the reaction

mixture. Stirring was continued at -20 °C for 10 min and

then for 1.5 h at room temperature. N-Methylmorpholine

hydrochloride was filtered off, and the filtrate was concen-

trated in vacuo to give the crude product which was purified

by column chromatography.

P r oced u r e B: Hyd r ogen olysis of Z-Block ed Dip ep tid e

ter t-Bu tyl Ester s. To a solution of the Z-protected dipeptide

(1.0 mmol) in EtOAc (60 mL) was added 10% Pd/C (10-15%

of the dipeptide’s weight). The resulting black mixture was

degassed and then stirred at room temperature for 3 h under

a hydrogen atmosphere (balloon). The catalyst was filtered

off and the filtrate evaporated to provide the dipeptide free

base which was immediately taken forward into the next step

used without further purification.

Dipeptides in Mice^a

plasma concentration (µM)

dipeptide

(parent

compound)

monoglutamate

(hydrolysis

product)

compd R¹R²

L-Glu-L-Glu

27 ( 5

14 ( 2 (from ref 12)

L-Glu-D-Glu 34 ( 6

ND^b(from ref 12)

Me pg

L-Glu-D-Glu 32 ( 11 ND

L-Glu-D-aad 31 ( 4

Me H ^L-Glu-D-Glu 17 ( 4

Liver only contained the dipeptide. Methodology is described

in ref 12. ND ) not detected.

Ta ble 5. L-D and L-D Dipeptides: Inhibition of L1210 TS and

of L1210 and L1210:R^D1694Cell Growth

P r oced u r e C: P r ep a r a tion of Qu in a zolin e An tifola te

Dip ep tid e Ester s. To a stirred solution of the dipeptide free

base (1.2 mmol) in dry DMF (14 mL) cooled to 0 °C was added

the appropriate pteroic acid analogue, trifluoroacetate salt (1.0

mmol) followed by DEPC (2.2 mmol) and Et₃N (2.2 mmol).

Stirring was continued at 0 °C for 10 min and then for 2 h at

room temperature under an argon atmosphere and in the dark.

The solution was then diluted with EtOAc (100 mL) and H₂O

(100 mL); the two layers were separated, and the aqueous layer

was extracted with EtOAc (2 × 100 mL). The combined

organic extracts were successively washed with 10% aqueous

citric acid (2 × 100 mL), saturated aqueous NaHCO₃(200 mL),

dilute aqueous NaCl (150 mL), and H₂O (150 mL), dried (Na₂-

SO₄), and concentrated in vacuo to leave the crude product.

P r oced u r e D: Acid Hyd r olysis of th e ter t-Bu tyl Ester s

w ith Tr iflu or oa cetic Acid . A solution of the appropriate

antifolate dipeptide tert-butyl ester (1.0 mmol) in TFA (30 mL)

was stirred at room temperature for 1.25 h with protection

from the light. The solution was then concentrated in vacuo,

and the oily residue was triturated with Et₂O. The solid was

collected by filtration, washed with Et₂O (40 mL), and dried

in vacuo over P₂O₅.

P r ep a r a tion of Z-Block ed Dip ep tid e ter t-Bu tyl Ester s:

Di-ter t-bu tyl N-[N-(Ben zyloxyca r bon yl)-^L-γ-glu ta m yl]-D-

a la n in a te (6a ). The general procedure A1 was followed using

R-tert-butyl N-(benzyloxycarbonyl)-^L-glutamate (2.02 g, 6.0

mmol), N-methylmorpholine (0.606 g, 6.0 mmol), dry THF (10

mL), isobutyl chloroformate (0.816 g, 6.0 mmol), and a suspen-

sion of ^D-alanine R-tert-butyl ester hydrochloride (1.09 g, 6.0

mmol) in dry THF (10 mL) and N-methylmorpholine (0.606 g,

6.0 mmol). Purification of the crude product by column

chromatography, on elution with 2:1 (v/v) CH₂Cl₂/EtOAc, gave

the title compound 6a (2.30 g, 83%) as a white solid: mp 78-

80 °C; NMR (Me₂SO-d₆) δ 1.21 (d, J ) 7.3 Hz, 3H, Ala â-CH₃),

1.38, 1.39 (2 × s, 18H, 2 × C(CH₃)₃), 1.74, 1.90 (2 × m, 2H,

â-CH₂), 2.18 (t, J ) 7.5 Hz, 2H, γ-CH₂), 3.88 (m, 1H, Glu R-CH),

4.06 (m, 1H, Ala R-CH), 5.02 (ABq, J ) 12.5 Hz, 2H, ArCH₂),

7.35 (m, 5H, ArH), 7.64 (d, J ) 7.7 Hz, 1H, Glu NH), 8.17 (d,

J ) 7.0 Hz, 1H, Ala NH); MS (FAB): m/ z 465 (M + H)⁺. Anal.

(C₂₄H₃₆N₂O₇) C, H, N.

inhibition of cell

inhibition of

growth, IC₅₀(µM)

L1210 TS,

IC₅₀(nM)

compd

L1210

L1210:R^D1694

L-Glu-L-Glu

L-Glu-D-Glu

L-Glu-L-Ala

L-Glu-D-Ala

L-Glu-L-Phe

L-Glu-D-Phe

L-Glu-L-aad

L-Glu-D-aad

2.0

4.6

0.16 ( 0.069 2.7, 2.9 (18)

0.33 ( 0.25 0.68 ( 0.35 (2)

0.62, 0.79

2.1, 1.6 (2)

0.43 ( 0.13 0.88 (2)

4.4

8.9 (2)

10, 4.2

2.1 ( 0.36

0.30 ( 0.031 2.9 ( 0.40 (10)

0.08, 0.11

0.44, 0.42 (4)

tion was achieved by UV or chlorine-tolidine reagent. Merck

silica gel 60 (art. 15111) was used in low-pressure column

chromatography. High-performance liquid chromatography

(HPLC) analyses were performed using a Waters model 510

solvent delivery system, model 680 automated gradient con-

troller, model U6K injector, and model 490 programmable

wavelength detector set to monitor at 230 and 280 nm.

Retention times were determined on a Trivector Trilab 3000

multichannel chromatography system. Separation of stereo-

isomers was performed on a 25 cm × 0.46 cm Astec cyclobond

I (B) column (Technical Ltd., U.K.), and they were eluted

isocratically with 82% 50 mM Na₂HPO₄/50 mM NaH₂PO₄(1:

1, v/v)-18% CH₃CN at a flow of 1 mL/min. Electron impact

(EI) and chemical ionization (CI) mass spectra were deter-

mined with a VG 7070H spectrometer and a VG 2235 data

system using the direct-insertion method, an ionizing voltage

of 70eV, a trap current of 100 µA, and an ion-source temper-

ature of 160 °C. Fast atom bombardment (FAB) mass spectra

were determined with a VG ZAB-SE spectrometer. Electro-

spray ionization (ESI) mass spectra were recorded using a TSQ

700 triple-quadrupole mass spectrometer (Finnigan MAT)

fitted with an electrospray ionization source (Analytica).

Samples were dissolved in methanol:water (50:50, v/v) con-

taining 1% acetic acid and infused into the mass spectrometer

using a Harvard infusion pump (Cambridge) at 1 µL/min.

Masses were scanned from 200 to 800 amu at a scanning speed

of 3 s/scan. Proton NMR spectra were recorded using a Bruker

AC250 spectrometer. Field strengths are expressed in units

of δ (ppm) relative to tetramethylsilane, and peak multiplici-

ties are designated as follows: s, singlet; d, doublet; dd, doublet

of doublets; dm, doublet of multiplets; t, triplet; q, quartet, br

s, broad singlet; m, multiplet. Optical rotations were obtained

using a Perkin-Elmer model 141 polarimeter. A sodium lamp

was used as radiation source. Melting points were detemined

on a Kofler block and are uncorrected. Elemental analyses

were determined by C.H.N. Analysis Ltd., Leicester, U.K.

r-ter t-Bu tyl N-(Ben zyloxyca r bon yl)-^D-p h en yla la n in a te

(9). N-(Benzyloxycarbonyl)-^D-phenylalanine (2.99 g, 10.0 mmol)

was dissolved in CH₂Cl₂(83 mL) in a 500 mL pressure bottle.

To this solution was added concentrated H₂SO₄(0.37 mL)

followed by liquid isobutylene (41 mL) at -20 °C. The

stoppered reaction vessel was shaken at room temperature for

28 h, and then the reactants were neutralized with a saturated

solution of NaHCO₃. EtOAc (150 mL) was added; the two

layers were separated, and the aqueous layer was washed with

80 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1

Bavetsias et al.

more EtOAc (100 mL). The combined organic extracts were

then washed with a saturated solution of NaHCO₃(2 × 100

mL) and H₂O (2 × 100 mL), dried (MgSO₄), and concentrated

in vacuo to give a white solid. Purification by column

chromatography, on elution with 5% EtOAc in CH₂Cl₂, gave

the title compound 9 (2.53 g, 71%) as a white solid: mp 80-

81 °C; NMR (Me₂SO-d₆) δ 1.33 (s, 9H, C(CH₃)₃), 2.90 (m, 2H,

â-CH₂), 4.13 (m, 1H, R-CH), 4.99 (m, 2H, ArCH₂OCO), 7.29

(m, 10H, 2 × ArH), 7.72 (d, J ) 7.7 Hz, 1H, NH); MS (CI) m/ z

356 (M + H)⁺. Anal. (C₂₁H₂₅NO₄) C, H, N.

ter t-Bu tyl ^D-P h en yla la n in a te (5b). The general proce-

dure B was followed using 9 (2.45 g, 6.9 mmol), EtOAc (220

mL), and 10% Pd/C (0.260 g). The reaction mixture was stirred

at room temperature for 15 h under a hydrogen atmosphere.

Compound 5b was obtained as a colorless oil (1.50 g, 98%):

NMR (Me₂SO-d₆) δ 1.31 (s, 9H, C(CH₃)₃), 2.78 (m, 2H, â-CH₂),

3.43 (t, J ) 6.8 Hz, 1H, R-CH), 7.21 (m, 5H, ArH); MS (CI)

m/ z 222 (M + H)⁺.

ration with hexanes afforded the title compound 6c as a white

solid. This was collected by filtration, washed with hexanes,

and dried in vacuo (1.36 g, 78%): mp 110 °C; NMR (Me₂SO-

d₆) δ 1.39 (s, 27H, C(CH₃)₃), 1.73, 1.89 (2 × m, 4H, â-CH₂),

2.23 (m, 4H, γ-CH₂), 3.89 (m, 1H, GluL R-CH), 4.10 (m, 1H,

Glu^DR-CH), 5.03 (ABq, J ) 14.0 Hz, 2H, PhCH₂), 7.36 (m,

5H, ArH), 7.63 (d, J ) 7.7 Hz, 1H, Glu^LNH), 8.13 (d, J ) 7.7

Hz, 1H, Glu^DNH); MS (FAB) m/ z 579 (M + H)⁺. Anal.

(C₃₀H₄₆N₂O₉) C, H, N.

Di-ter t-bu tyl ^D-r-Am in oa d ip a te Hyd r och lor id e (5d ).

D-R-Aminoadipic acid (1.0 g, 6.2 mmol), tert-butyl acetate (85

mL), and 70% aqueous HClO₄(0.99 g) were stirred at room

temperature for 24 h. The mixture was then cooled in an ice-

water bath and extracted with 0.5 N HCl (2 × 50 mL). The

combined aqueous extracts were immediately neutralized with

solid NaHCO₃. The aqueous solution was extracted with Et₂O

(2 × 150 mL); the ether extracts were pooled and dried

(MgSO₄), and the solvent was reduced in volume to ca. 50 mL.

This solution was acidified by addition of a few drops of dry

ethereal HCl and then cooled in an ice-water bath. The

resulting white solid was isolated by filtration, washed with

ether (2 × 10 mL), and dried in vacuo: 0.93 g (48%); mp 133-

134 °C; NMR (Me₂SO-d₆) δ 1.40, 1.47 (2 × s, 18H, C(CH₃)₃),

1.54, 1.64 (2 × m, 2H, â-CH₂), 1.73 (m, 2H, γ-CH₂), 2.24 (t, J

) 7.2 Hz, 2H, δ-CH₂), 3.88 (t, J ) 5.9 Hz, 1H, R-CH), 8.19 (s,

3H, NH₃).

Tr i-ter t-bu tyl N-[N-(Ben zyloxyca r bon yl)-^L-γ-glu ta m yl]-

D-2-a m in oa d ip a te (6d ). The general procedure A1 was

followed using R-tert-butyl N-(benzyloxycarbonyl)-^L-glutamate

(4.65 g, 14.0 mmol), N-methylmorpholine (1.39 g, 14.0 mmol),

THF (60 mL), isobutyl chloroformate (1.88 g, 14.0 mmol), and

a suspension of 5d (4.27 g, 14.0 mmol) in dry THF (30 mL)

and N-methylmorpholine (1.39 g, 14.0 mmol). Purification of

the crude product by column chromatography, on elution with

1% MeOH in CH₂Cl₂, gave the required product which was

recrystallized from petroleum ether (100 mL), collected by

filtration, and washed with petroleum ether (3 × 50 mL): 7.46

g, (90%); mp 87-88 °C; NMR (Me₂SO-d₆) δ 1.39 (s, 27H,

C(CH₃)₃), 1.53, 1.64, 1.78, 1.91 (4 × m, 6H, Glu â-CH₂, aad

â-CH₂, aad γ-CH₂), 2.20 (m, 4H, Glu γ-CH₂, aad δ-CH₂), 3.89

(m, 1H, Glu R-CH), 4.06 (m, 1H, aad R-CH), 5.04 (ABq, J )

12.4 Hz, 2H, ArCH₂), 7.35 (m, 5H, ArH), 7.57 (d, J ) 7.7 Hz,

1H, Glu NH), 8.08 (d, J ) 7.6 Hz, 1H, aad NH); MS (ESI) m/ z

615 (M + Na)⁺. Anal. (C₃₁H₄₈N₂O₉‚0.25H₂O) C, H, N.

Hyd r ogen olysis of Z-Block ed Dip ep tid e ter t-Bu tyl

Ester s: Di-ter t-bu tyl ^L-γ-Glu ta m yl-D-a la n in a te (7a ). The

general procedure B was followed using 6a (0.696 g, 1.5 mmol),

EtOAc (40 mL), and 10% Pd/C (0.125 g). Compound 7a was

obtained as a white sticky solid (0.480 g, 97%) and used

immediately in the next experiment without further purifica-

tion: NMR (Me₂SO-d₆) δ 1.21 (d, J ) 7.3 Hz, 3H, Ala CH₃),

1.38, 1.41 (2 × s, 18H, 2 × C(CH₃)₃), 1.57, 1.77 (2 × m, 2H,

Glu â-CH₂), 2.18 (t, J ) 7.9 Hz, 2H, Glu γ-CH₂), 3.17 (dd, J )

5.2, 8.2 Hz, 1H, Glu R-CH), 4.07 (m, 1H, Ala R-CH), 8.16 (d, J

) 6.9 Hz, 1H, Ala NH); MS (FAB) m/ z 331 (M + H)⁺.

Di-ter t-bu tyl ^L-γ-Glu ta m yl-D-p h en yla la n in a te (7b). The

general procedure B was followed using 6b (0.700 g, 1.30

mmol), EtOAc (90 mL), and 10% Pd/C (0.096 g). The title

compound 7b was obtained as a colorless oil (0.514 g, 98%):

NMR (Me₂SO-d₆) δ 1.32, 1.40 (2 × s, 18H, 2 × C(CH₃)₃), 1.70

(m, 2H, Glu â-CH₂), 2.15 (m, 2H, Glu γ-CH₂), 2.90 (m, 2H, Phe

â-CH₂), 3.11 (dd, J ) 8.0, 5.1 Hz, 1H, Glu R-CH), 4.33 (m, 1H,

Phe R-CH), 7.23 (m, 5H, CH₂Ph), 8.24 (d, J ) 7.7 Hz, 1H, Phe

NH); MS (CI) m/ z 407 (M + H)⁺.

Di-ter t-bu tyl N-[N-(Ben zyloxyca r bon yl)-^L-γ-glu ta m yl]-

D-p h en yla la n in a te (6b). The general procedure A2 was

followed using R-tert-butyl N-(benzyloxycarbonyl)-^L-glutamate

(1.051 g, 3.12 mmol), dry THF (5 mL), N-methylmorpholine

(0.315 g, 3.12 mmol), isobutyl chloroformate (0.424 g, 3.12

mmol), and a solution of tert-butyl ^D-phenylalaninate (5b; 0.69

g, 3.12 mmol) in dry THF (5 mL). Purification by column

chromatography, on gradient elution with 1:9-1:4 (v/v) EtOAc/

CH₂Cl₂, gave the title compound 6b as a colorless oil (1.45 g,

86%) which solidified on standing for a few weeks, giving a

white solid: mp 79-80 °C; NMR (Me₂SO-d₆) δ 1.31, 1.38 (2 ×

s, 18H, 2 × C(CH₃)₃), 1.69, 1.85 (2 × m, 2H, Glu â-CH₂), 2.16

(t, J ) 7.0 Hz, 2H, Glu γ-CH₂), 2.90 (m, 2H, Phe â-CH₂), 3.87

(m, 1H, Glu R-CH), 4.32 (m, 1H, Phe R-CH), 5.02 (m, 2H,

PhCH₂OCO), 7.23 (m, 5H, Phe ArH), 7.35 (m, 5H, PhCH₂OCO),

7.62 (d, J ) 7.7 Hz, 1H, Glu NH), 8.24 (d, J ) 7.7 Hz, 1H, Phe

NH); MS (CI) m/ z 541 (M + H)⁺. Anal. (C₃₀H₄₀N₂O₇) C, H,

Di-ter t-bu tyl ^D-Glu ta m a te (5c). a . Tr a n sester ifica tion

Meth od . ^D-Glutamic acid (1.3 g, 8.84 mmol), tert-butyl acetate

(100 mL), and 70% aqueous HClO₄(1.4 g, 9.7 mmol) were

stirred at room temperature for 48 h. The solution was then

cooled in an ice-water bath and extracted with 0.5 N HCl (2

× 60 mL). The combined aqueous extracts were immediately

neutralized with solid NaHCO₃. The aqueous solution was

extracted with Et₂O (2 × 140 mL); the ether extracts were

pooled, dried (Na₂SO₄), and concentrated in vacuo to a colorless

oily residue. This was diluted with Et₂O (25 mL), and then

gaseous HCl was passed through the solution. The solution

was left in a freezer overnight, and then the precipitated white

solid was collected by filtration, washed with Et₂O (20 mL),

and dried in vacuo over P₂O₅. The title compound 5c was thus

obtained as its hydrochloride salt (0.74 g, 28%): mp 121-122

°C; NMR (Me₂SO-d₆) δ 1.40, 1.45 (2 × s, 18H, 2 × C(CH₃)₃),

1.94, 2.27-2.50 (2 × m, 4H, â-CH₂, γ-CH₂), 3.89 (t, J ) 6.6

Hz, 1H, R-CH), 8.38 (s, 3H, NH₃); MS (CI) m/ z 260 (M + H)⁺.

b. Isobu tylen e Meth od . ^D-Glutamic acid (1.90 g, 13.0

mmol) was suspended in CH₂Cl₂(94 mL) in a 500 mL pressure

bottle. To this suspension was added concentrated H₂SO₄(0.58

mL) followed by liquid isobutylene (41 mL) at -20 °C, and

the bottle was tightly stoppered. The reaction mixture was

shaken at room temperature for 72 h and then neutralized

with a saturated solution of NaHCO₃. EtOAc (100 mL) was

added; the two layers were separated, and the aqueous layer

was washed with more EtOAc (2 × 100 mL). The combined

organic extracts were dried (MgSO₄) and then concentrated

in vacuo to give the title compound 5c as a pale yellow oil (1.50

g, 45%): NMR (Me₂SO-d₆) δ 1.40 (s, 18H, 2 × C(CH₃)₃), 1.75

(m, 2H, â-CH₂), 2.27 (m, 2H, γ-CH₂), 3.15 (dd, J ) 8.1, 5.1 Hz,

1H, Glu R-CH); MS (CI) m/ z 260 (M + H)⁺.

Tr i-ter t-bu tyl ^L-γ-Glu ta m yl-D-glu ta m a te (7c). The gen-

eral procedure B was followed using N-[N-(benzyloxycarbonyl)-

L-γ-glutamyl]-D-glutamate (6c; 2.4 g, 4.15 mmol), EtOAc (240

mL), and 10% Pd/C (0.330 g). The product 7c was obtained

as a colorless oil (1.84 g, 100%) and used immediately without

further purification: NMR (Me₂SO-d₆) δ 1.39, 1.41 (2 × s, 27H,

3 × C(CH₃)₃), 1.56-1.87 (m, 4H, 2 × â-CH₂), 2.22 (m, 4H, 2 ×

γ-CH₂), 3.14 (dd, J ) 5.1, 8.1 Hz, 1H, GluL R-CH), 4.11 (m,

1H, Glu^DR-CH), 8.13 (d, J ) 7.6 Hz, 1H, GluD NH); MS (CI)

m/ z 445 (M + H)⁺.

Tr i-ter t-bu tyl N-[N-(Ben zyloxyca r bon yl)-^L-γ-glu ta m yl]-

D-glu ta m a te (6c). The general procedure A2 was followed

using R-tert-butyl N-(benzyloxycarbonyl)-^L-glutamate (1.011 g,

3.0 mmol), N-methylmorpholine (0.303 g, 3.0 mmol), dry THF

(10 mL), isobutyl chloroformate (0.408 g, 3.0 mmol), and a

solution of di-tert-butyl ^D-glutamate (0.855 g, 3.3 mmol) in dry

THF (10 mL). The crude product was purified by column

chromatography using 2% MeOH in CH₂Cl₂as eluent. Tritu-

Tr i-ter t-bu tyl ^L-γ-Glu ta m yl-D-2-a m in oa d ip a te (7d ). The

general procedure B was followed using 6d (5.38 g, 9.08 mmol),

Quinazoline Antifolate TS Inhibitors

J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 81

EtOAc (300 mL), and 10% Pd/C (0.82 g). Standard workup

afforded 7d as a colorless oil (4.2 g, 100%): NMR (Me₂SO-d₆)

δ 1.39, 1.40 (2 × s, 27H, 3 × C(CH₃)₃), 1.50-1.80 (m, 6H, Glu

â-CH₂, aad â-CH₂and γ-CH₂), 2.20 (m, 4H, Glu γ-CH₂, aad

δ-CH₂), 3.13 (dd, J ) 5.1, 8.0 Hz, 1H, Glu R-CH), 4.05 (m, 1H,

aad R-CH), 8.13 (d, J ) 7.5 Hz, 1H, aad, NH); MS (FAB) m/ e

459 (M + H)⁺.

quinazoline 8-H), 7.68 (m, 2H, quinazoline 5-H, 6′-ArH); MS

(ESI) m/ z 380 (M + H)⁺; HRMS (FAB) found 380.1410, calcd

for C₂₁H₁₈FN₃O₃380.1414.

ter t-Bu t yl 4-[N-[(3,4-Dih yd r o-2,7-d im et h yl-4-oxo-6-

qu in azolin yl)m eth yl]-N-pr op-2-yn ylam in o]ben zoate (47a).

The method followed that used to prepare 47b but used

6-(bromomethyl)-3,4-dihydro-2,7-dimethyl-4-oxoquina zo-

line²³(45b; 5.0 g, 18.7 mmol), tert-butyl 4-(prop-2-ynylamino)-

benzoate^24,25(46a ; 4.87 g, 21.1 mmol), anhydrous DMF (95

mL), and 2,6-lutidine (6.10 mL, 52.45 mmol). The reaction

mixture was stirred under argon at 85 °C for 20 h. The desired

product was obtained as a white solid: 4.32 g (55%); mp 232-

233 °C; NMR (Me₂SO-d₆) δ 1.49 (s, 9H, C(CH₃)₃), 2.43, 2.50 (2

× s, 6H, quinazoline 2-CH₃and 7-CH₃), 3.23 (s, 1H, CtCH),

4.31 (s, 2H, CH₂CtC), 4.69 (s, 2H, quinazoline 6-CH₂), 6.78

(d, J ) 8.9 Hz, 2H, 3′,5′-ArH), 7.43 (s, 1H, quinazoline 8-H),

7.67 (s, 1H, quinazoline 5-H), 7.71 (d, J ) 8.7 Hz, 2H, 2′,6′-

ArH), 12.09 (s, 1H, lactam NH); MS (CI) m/ z 417 (M⁺). Anal.

(C₂₅H₂₇N₃O₃‚0.25H₂O) C, H, N.

4-[N-[(3,4-Dih yd r o-2,7-d im eth yl-4-oxo-6-qu in a zolin yl)-

m eth yl]-N-p r op -2-yn yla m in o]ben zoic Acid , Tr iflu or oa c-

eta te Sa lt (11). The method followed that used to prepare

12 but used tert-butyl 4-[N-[(3,4-dihydro-2,7-dimethyl-4-oxo-

6-quinazolinyl)methyl]-N-prop-2-ynylamino]benzoate (47a; 2.31

g, 5.55 mmol) in TFA (21 mL). The required product was

obtained as a pale yellow solid: 2.9 g (97%); mp >310 °C; NMR

(Me₂SO-d₆) δ 2.46, 2.50 (2 × s, 6H, quinazoline 2-CH₃and

7-CH₃), 3.25 (s, 1H, CtCH), 4.34 (s, 2H, CH₂CtC), 4.74 (s,

2H, quinazoline 6-CH₂), 6.79 (d, J ) 9.0 Hz, 2H, 3′,5′-ArH),

7.50 (s, 1H, quinazoline 8-H), 7.73 (s, 1H, quinazoline 5-H),

7.77 (d, J ) 8.8 Hz, 2H, 2′,6′-ArH); MS (ESI) m/ z 362 (M +

H)⁺. Anal. (C₂₁H₁₉N₃O₃‚1.5TFA‚0.5H₂O) C, H, N, F.

ter t-Bu t yl 4-[N-[[3,4-Dih yd r o-2-m et h yl-4-oxo-3-[(p iv-

a loyloxy)m et h yl]-6-q u in a zolin yl]m et h yl]a m in o]-2-flu o-

r oben zoa te (52). A mixture of 6-(bromomethyl)-3,4-dihydro-

2-methyl-4-oxo-3-[(pivaloyloxy)methyl]quinazoline²⁷(49; 9.15

g, 25.0 mmol), tert-butyl 4-amino-2-fluorobenzoate²⁴(51; 5.80

g, 27.5 mmol), 2,6-lutidine (2.94 g, 0.0275 mol), and a few

crystals of NaI in dry DMA (30 mL) was stirred under argon

at 95 °C for 10 h. The reaction mixture was then allowed to

cool to room temperature; the DMA was removed in vacuo,

and the residue was partitioned between EtOAc (300 mL) and

H₂O (200 mL). The two layers were separated, and the organic

layer was washed with H₂O (200 mL), dried (MgSO₄), and

concentrated in vacuo to a yellow oil which crystallized

overnight. This was dissolved in CH₂Cl₂, and to the resulting

solution was added Merck silica gel (art. 7734; 100 g). The

solvent was then removed under reduced pressure to give a

free-running pale yellow powder which was placed on a silica

gel column made up in 1:2 (v/v) EtOAc/hexanes. Elution of

the column with a gradient of EtOAc in hexanes (50-100%)

afforded the required product as a pale yellow solid: 10.3 g

(83%); mp 149-150 °C; NMR (Me₂SO-d₆) δ 1.14 (s, 9H, POM

C(CH₃)₃), 1.46 (s, 9H, CO₂C(CH₃)₃), 2.60 (s, 3H, quinazoline

2-CH₃), 4.50 (d, J ) 5.9 Hz, 2H, CH₂NH), 6.05 (s, 2H, POM

CH₂), 6.33 (d, J ) 14.4 Hz, 1H, 3′-ArH), 6.47 (d, J ) 8.6 Hz,

1H, 5′-ArH), 7.40-7.61 (m, 3H, CH₂NH, 6′-ArH, quinazoline

8-H), 7.80 (d, J ) 8.5 Hz, 1H, quinazoline 7-H), 8.06 (s, 1H,

quinazoline 5-H); MS (ESI) m/ z 498 (M + H)⁺.

ter t-Bu t yl 4-[N-[[3,4-Dih yd r o-2-m et h yl-4-oxo-3-[(p iv-

aloyloxy)m eth yl]-6-qu in azolin yl]m eth yl]-N-m eth ylam in o]-

2-flu or oben zoa te (54). To a stirred solution of 52 (10.3 g,

0.021 mol) in glacial AcOH (150 mL) was added 37% aqueous

formaldehyde (17 mL). Stirring was continued at room

temperature for 10 min, and then sodium cyanoborohydride

(2.83 g, 0.046 mol) was added over a period of 5 min (caution:

evolution of HCN takes place). The AcOH was removed in

vacuo, and the resulting residue was partitioned between

EtOAc (300 mL) and H₂O (200 mL). The two layers were

separated, and the organic layer was extracted with EtOAc

(150 mL). The combined EtOAc extracts were washed with

saturated aqueous NaHCO₃(300 mL) and H₂O (300 mL), dried

(MgSO₄), and concentrated in vacuo to a yellow oil. Purifica-

tion by column chromatography, on gradient elution with

EtOAc in CH₂Cl₂(0-30%), gave the required product as a

yellow oil: 8.4 g (80%). A small amount of the product (0.320

P r ep a r a tion of P ter oic Acid An a logu es: ter t-Bu tyl

4-[N-[(3,4-Dih ydr o-2-m eth yl-4-oxo-6-qu in azolin yl)m eth yl]-

N-p r op -2-yn yla m in o]-2-flu or oben zoa te (47b). To a sus-

pension of 6-(bromomethyl)-3,4-dihydro-2-methyl-4-oxoquinazo-

line⁴¹(45a ; 5.0 g, 19.8 mmol) and tert-butyl 2-fluoro-4-(prop-

2-ynylamino)benzoate²⁴(46b; 4.43 g, 17.8 mmol) in anhydrous

DMF (60 mL) was added 2,6-lutidine (5.14 mL, 44.2 mmol).

The reaction mixture was stirred under argon at 80 °C for 19

h, then it was allowed to cool to ∼45 °C and concentrated in

vacuo. The residue was partitioned between EtOAc (700 mL)

and H₂O (250 mL); the two layers were separated, and the

aqueous layer was extracted with more EtOAc (200 mL). The

combined organic extracts were successively washed with

aqueous NH₄OH (H₂O/NH₃, v/v 10:1, 2 × 200 mL), a dilute

aqueous solution of NaCl (200 mL), and H₂O (200 mL), dried

(MgSO₄), and concentrated in vacuo. The residue was dis-

solved in CH₂Cl₂(250 mL), and to this solution was added

Merck silica gel (art. 7734; 50 g). The solvent was removed

in vacuo, and the resulting free running powder was placed

on a silica gel column made up in EtOAc. Elution of the

column with EtOAc afforded the required product as a white

solid: 2.9 g (39%); mp 226-229 °C; NMR (Me₂SO-d₆) δ 1.48

(s, 9H, C(CH₃)₃), 2.33 (s, 3H, quinazoline 2-CH₃), 3.28 (s, 1H,

CtCH), 4.37 (s, 2H, CH₂CtC), 4.81 (s, 2H, quinazoline 6-CH₂),

6.62 (m, 2H, 3′,5′-ArH), 7.56 (m, 2H, 6′-ArH, quinazoline 8-H),

7.67 (dd, J ) 2.1, 7.9 Hz, 1H, quinazoline 7-H), 7.93 (s, 1H,

quinazoline 5-H), 12.21 (s, 1H, lactam NH); MS (CI) m/ z 421

(M⁺). Anal. (C₂₄H₂₄FN₃O₃) C, H, N, F.

4-[N -[(3,4-Dih yd r o-2-m e t h yl-4-oxo-6-q u in a zolin yl)-

m eth yl]-N-p r op -2-yn yla m in o]-2-flu or oben zoic Acid , Tr i-

flu or oa ceta te Sa lt (12). A solution of 47b (2.2 g, 5.2 mmol)

in TFA (21 mL) was stirred at room temperature for 45 min

with protection from the light. The TFA was then removed

in vacuo, and the residue was triturated with EtOAc. The

product, a white solid, was collected by filtration, washed with

petroleum ether, and dried in vacuo over P₂O₅: 2.4 g (96%);

mp >300 °C; NMR (Me₂SO-d₆) δ 2.43 (s, 3H, quinazoline

2-CH₃), 3.30 (s, 1H, CtCH), 4.40 (s, 2H, CH₂CtC), 4.85 (s,

2H, quinazoline 6-CH₂), 6.63 (m, 2H, 3′,5′-ArH), 7.63 (m, 2H,

6′-ArH, quinazoline 8-H), 7.76 (d, J ) 8.5 Hz, 1H, quinazoline

7-H), 7.99 (s, 1H, quinazoline 5-H); MS (ESI) m/ z 366 (M +

H)⁺. Anal. (C₂₀H₁₆FN₃O₃‚TFA‚0.25H₂O), C, H, N. F.

ter t-Bu t yl 4-[N-[(3,4-Dih yd r o-2,7-d im et h yl-4-oxo-6-

qu in a zolin yl)m eth yl]-N-p r op -2-yn yla m in o]-2-flu or oben -

zoa te (47c). To a suspension of 6-(bromomethyl)-3,4-dihydro-

2,7-dimethyl-4-oxoquinazoline²³(45b; 4.0 g, 15.0 mmol) and

tert-butyl 2-fluoro-4-(prop-2-ynylamino)benzoate²⁴(46b; 3.73

g, 15.0 mmol) in anhydrous DMF (110 mL) was added 2,6-

lutidine (3.6 mL, 29.9 mmol). The reaction mixture was stirred

under argon at 70 °C for 16 h; then more 2,6-lutidine (1.3 mL,

11.17 mmol) was added, and stirring was continued at 80 °C

for 8 h. Workup and purification as described for 47b afforded

the required compound as a white solid: 2.3 g (35%); mp >310

°C; NMR (Me₂SO-d₆) δ 1.49 (s, 9H, C(CH₃)₃), 2.32, 2.43 (2 × s,

6H, quinazoline 2-CH₃and 7-CH₃), 3.26 (s, 1H, CtCH), 4.32

(s, 2H, CH₂CtC), 4.71 (s, 2H, quinazoline 6-CH₂), 6.60 (m, 2H,

3′,5′-ArH), 7.44 (s, 1H, quinazoline 8-H), 7.65 (m, 2H, 6′-ArH,

quinazoline 5-H), 12.09 (s, 1H, lactam NH); MS (EI) m/ z 435

(M⁺). Anal. (C₂₅H₂₆FN₃O₃) C, H, N, F.

4-[N-[(3,4-Dih yd r o-2,7-d im eth yl-4-oxo-6-qu in a zolin yl)-

m eth yl]-N-p r op -2-yn yla m in o]-2-flu or oben zoic Acid , Tr i-

flu or oa ceta te Sa lt (13). The method followed that used to

prepare 12 but used tert-butyl 4-[N-[(3,4-dihydro-2,7-dimethyl-

4-oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]-2-fluoroben-

zoate (47c; 2.1 g, 4.8 mmol) in TFA (21 mL). The required

compound was obtained as a white solid: 2.55 g; mp >310 °C;

NMR (Me₂SO-d₆) δ 2.39, 2.45 (2 × s, 6H, quinazoline 2-CH₃

and 7-CH₃), 3.26 (s, 1H, CtCH), 4.33 (s, 2H, CH₂CtC), 4.74

(s, 2H, quinazoline 6-CH₂), 6.59 (m, 2H, 3′,5′-ArH), 7.46 (s, 1H,

82 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1

Bavetsias et al.

g) was rechromatographed on a silica gel column using 35%

EtOAc in hexanes as eluent to yield a yellow gum that turned

into a pale yellow solid on drying in vacuo over P₂O₅: mp 74-

77 °C (softens); NMR (Me₂SO-d₆) δ 1.13 (s, 9H, POM C(CH₃)₃),

1.48 (s, 9H, C(CH₃)₃), 2.60 (s, 3H, quinazoline 2-CH₃), 3.13 (s,

3H, N¹⁰-CH₃), 4.82 (s, 2H, CH₂NCH₃), 6.04 (s, 2H, POM CH₂),

6.53 (dd, J ) 15.2, 2.3 Hz, 1H, 3′-ArH), 6.60 (dd, J ) 9.0, 2.4

Hz, 1H, 5′-ArH), 7.62 (m, 3H, quinazoline 7-H and 8-H, 6′-

ArH), 7.88 (d, J ) 1.3 Hz, 1H, quinazoline 5-H); MS (ESI) m/ z

534 (M + Na)⁺, 512 (M + H)⁺. Anal. (C₂₈H₃₄FN₃O₅) C, H, F;

N: calcd, 8.21; found, 7.66.

4-[N -[(3,4-Dih yd r o-2-m e t h yl-4-oxo-6-q u in a zolin yl)-

m eth yl]-N-m eth yla m in o]-2-flu or oben zoic Acid , Tr iflu o-

r oa ceta te Sa lt (16). A solution of 54 (6.58 g, 0.0192 mol) in

TFA (35 mL) was stirred at room temperature for 1 h with

protection from the light and then concentrated in vacuo to a

brown oil which was treated with EtOH (30 mL) and Et₂O (30

mL). The precipitated white solid was collected by filtration,

washed with Et₂O, and dried in vacuo over P₂O₅: 4.54 g. This

solid (4.34 g) was treated with EtOH (200 mL), H₂O (60 mL),

and aqueous NaOH (1 N, 34 mL, 34 mmol), and the resulting

solution was stirred at room temperature for 4 h and then

acidified to pH 4 with 2 N HCl. The precipitated pale yellow

solid was collected by filtration, washed with H₂O, and dried

in vacuo over P₂O₅: 3.20 g (84% from 54). To obtain the

trifluoroacetate salt, part of this solid (1 g, 2.93 mmol) was

dissolved in TFA (40 mL). The solution was allowed to stand

at room temperature for 10 min and then concentrated in

vacuo, and the resulting residue was triturated with Et₂O (50

mL). The required product was collected by filtration as a pale

yellow solid and dried in vacuo over P₂O₅: 1.35 g; mp 297-

298 °C; NMR (Me₂SO-d₆) δ 2.39 (s, 3H, quinazoline 2-CH₃),

3.13 (s, 3H, N¹⁰-CH₃), 4.80 (s, 2H, CH₂NCH₃), 6.52 (d, J ) 15.4

Hz, 1H, 3′-ArH), 6.60 (d, J ) 9.0 Hz, 5′-ArH), 7.58 (d, J ) 8.5

Hz, 1H, quinazoline 8-H), 7.66 (m, 2H, quinazoline 7-H, 6′-

ArH), 7.87 (s, 1H, quinazoline 5-H); MS (ESI) m/ z 342 (M +

H)⁺. Anal. (C₁₈H₁₆FN₃O₃‚TFA) C, H, N; F; calcd, 16.69; found,

16.28.

ter t-Bu tyl 4-[N-[[3,4-Dih ydr o-2,7-dim eth yl-4-oxo-3-[(piv-

a loyloxy)m et h yl]-6-q u in a zolin yl]m et h yl]a m in o]-2-flu o-

r oben zoa te (53). The method followed that used to prepare

52 but used 6-(bromomethyl)-3,4-dihydro-2,7-dimethyl-4-oxo-

3-[(pivaloyloxy)methyl]quinazoline²⁴(50; 5.7 g, 15.0 mmol),

tert-butyl 4-amino-2-fluorobenzoate²⁴(51; 3.48 g, 16.5 mmol),

2,6-lutidine (1.76 g, 15.0 mmol), and a few crystals of NaI in

dry DMA (30 mL). The crude product was dissolved in CH₂-

Cl₂(200 mL), and to the resulting solution was added Merck

silica gel (art. 7734; 60 g). The mixture was concentrated in

vacuo to a fine powder which was placed on a silica gel column

made up in 1:2 (v/v) EtOAc/hexanes. Elution with a gradient

of EtOAc in hexanes (30-50%) afforded the required product

as a yellow solid: 5.75 g (75%); mp 131-132 °C; NMR (Me₂-

SO-d₆) δ 1.12 (s, 9H, POM C(CH₃)₃), 1.48 (s, 9H, CO₂C(CH₃)₃),

2.47 (s, 3H, quinazoline 7-CH₃), 2.58 (s, 3H, quinazoline

2-CH₃), 4.44 (d, J ) 5.3 Hz, 2H, CH₂NH), 6.02 (s, 2H, POM

CH₂), 6.34 (d, J ) 14.4 Hz, 1H, 3′-ArH), 6.49 (d, J ) 8.8 Hz,

1H, 5′-ArH), 7.35 (t, 1H, CH₂NH), 7.48 (s, 1H, quinzaoline 8-H),

7.55 (t, J ) 8.9 Hz, 1H, 6′-ArH), 7.90 (s, 1H, quinazoline 5-H);

MS (ESI) m/ z 512 (M⁺).

ter t-Bu tyl 4-[N-[[3,4-Dih ydr o-2,7-dim eth yl-4-oxo-3-[(piv-

aloyloxy)m eth yl]-6-qu in azolin yl]m eth yl]-N-m eth ylam in o]-

2-flu or oben zoa te (55). The method followed that used to

prepare 54 but used 53 (9.15 g, 17.9 mmol), glacial AcOH (150

mL), 37% aqueous formaldehyde (15 mL), and sodium cy-

anoborohydride (2.44 g, 39.4 mol). Workup as described for

54 afforded a yellow foam which was triturated with Et₂O.

The required product was then collected by filtration as a white

solid, washed with Et₂O and hexanes, and dried in vacuo over

P₂O₅: 6.21 g (66%); mp 174-175 °C; NMR (Me₂SO-d₆) δ 1.12

(s, 9H, POM C(CH₃)₃), 1.48 (s, 9H, CO₂C(CH₃)₃), 2.44, 2.57 (2

× s, 2H, quinazoline 7-CH₃and 2-CH₃), 3.12 (s, 3H, N¹⁰-CH₃),

4.73 (s, 2H, CH₂NH), 5.99 (s, 2H, POM, CH₂), 6.50 (d, J )

14.6 Hz, 1H, 3′-ArH), 6.55 (dd, J ) 2.3, 8.5 Hz, 1H, 5′-ArH),

7.48, 7.50 (2 × s, 2H, quinazoline 8-H and 5-H), 7.61 (t, J )

8.8 Hz, 1H, 6′-ArH); MS (ESI) m/ z 526 (M + H)⁺. Anal.

(C₂₉H₃₆FN₃O₅) C, H, N, F.

4-[N-[(3,4-Dih yd r o-2,7-d im eth yl-4-oxo-6-qu in a zolin yl)-

m eth yl]-N-m eth yla m in o]-2-flu or oben zoic Acid , Tr iflu o-

r oa ceta te Sa lt (17). A solution of 55 (3.0 g, 5.7 mmol) in

TFA (30 mL) was stirred at room temperature for 1 h with

protection from the light and then concentrated in vacuo to a

dark green oil. Trituration with Et₂O (50 mL) afforded a buff

solid which was dried in vacuo over P₂O₅(2.5 g) and then

treated with EtOH (100 mL), H₂O (30 mL), and aqueous NaOH

(1 N, 17 mL). The resulting solution was stirred at room

temperature for 3 h and then acidified to pH 4 with 2 N HCl.

The precipitated solid was collected by filtration, washed with

H₂O (50 mL), dried in vacuo over P₂O₅, and then dissolved in

TFA (30 mL). The purple solution was allowed to stand at

room temperature for 10 min and then concentrated in vacuo

to an oily residue. Trituration with Et₂O (50 mL) gave a light

purple solid which was collected by filtration, washed with

Et₂O, and dried in vacuo over P₂O₅: 1.73 g (69% from 55); mp

312-314 °C dec; NMR (Me₂SO-d₆) δ 2.37, 2.45 (2 × s, 6H,

quinazoline 2-CH₃and 7-CH₃), 3.13 (s, 3H, N¹⁰-CH₃), 4.73 (s,

2H, CH₂NCH₃), 6.51 (d, J ) 15.1 Hz, 1H, 3′-ArH), 6.55 (d, J )

8.9 Hz, 1H, 5′-ArH), 7.47, 7.48 (2 × s, 2H, quinazoline 5-H

and 8-H), 7.68 (t, J ) 8.7 Hz, 1H, 6′-ArH); MS (ESI) m/ z 356

(M + H)⁺. Anal. (C₁₉H₁₈FN₃O₃‚0.7TFA‚0.4H₂O) C, H, N, F.

4-[N-[(3,4-Dih yd r o-2,7-d im eth yl-4-oxo-6-qu in a zolin yl)-

m eth yl]-N-m eth yla m in o]ben zoic Acid , Tr iflu or oa ceta te

Sa lt (15). A mixture of 6-(bromomethyl)-3,4-dihydro-2,7-

dimethyl-4-oxo-3-[(pivaloyloxy)methyl]quinazoline²⁴(50; 3.0 g,

7.89 mmol), 4-(methylamino)benzoic acid (2.38 g, 15.78 mmol),

and CaCO₃(1.18 g, 11.83 mmol) in dry DMA (25 mL) was

heated at 60 °C for 21 h; then it was allowed to cool to room

temperature, filtered, and washed with DMA (30 mL). The

filtrate was concentrated in vacuo to a brown residue which

was triturated with EtOAc. The precipitated brown solid was

collected by filtration and treated with EtOH (60 mL), H₂O

(50 mL), and aqueous NaOH (1 N, 30 mL, 30 mmol). The

resulting solution was stirred at room temperature for 1.5 h

and acidified to pH 4, and the precipitated solid was collected

by filtration and dried in vacuo over P₂O₅. Then it was

suspended in TFA (30 mL), and the resulting cloudy solution

was concentrated in vacuo to a brown residue which was

triturated with Et₂O. The precipitated brown solid was

collected by filtration and then dried in vacuo over P₂O₅: 2.86

(80%); mp >270 °C; NMR (Me₂SO-d₆) δ 2.43, 2.47 (2 × s, 6H,

quinazoline 2-CH₃and 7-CH₃), 3.15 (s, 3H, N¹⁰-CH₃), 4.74 (s,

2H, quinazoline 6-CH₂), 6.72 (d, J ) 9.0 Hz, 2H, 3′,5′-ArH),

7.51 (s, 2H, quinazoline 5-H and 8-H), 7.74 (d, J ) 8.9 Hz,

2H, 2′,6′-ArH); MS (ESI) m/ z 338 (M + H)⁺; HRMS (FAB)

found 338.1509, calcd for C₁₉H₁₉N₃O₃338.1505.

P r epar ation of Qu in azolin e An tifolate γ-Lin ked Dipep-

tid e ter t-Bu tyl Ester s: Di-ter t-bu tyl N-[N-[4-[N-[(3,4-Di-

h 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]-^L-γ-glu ta m yl]-D-a la n in a te (23). The

general procedure C was followed using 7a (0.480 g, 1.45

mmol), 4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-

N-prop-2-ynylamino]benzoic acid, trifluoroacetate salt (10;

0.461 g, 1.0 mmol), DMF (20 mL), DEPC (0.326 g, 2.0 mmol),

and Et₃N (0.202 g, 2.0 mmol). Purification by column chro-

matography eluting with EtOAc gave a pale yellow solid.

Subsequent reprecipitation from CH₂Cl₂(minimum amount)/

petroleum ether yielded the title compound 23 (0.290 g, 44%)

as a white solid: mp 166-168 °C; NMR (Me₂SO-d₆) δ 1.19 (d,

J ) 7.3 Hz, 3H, Ala â-CH₃), 1.37, 1.40 (2 × s, 18H, 2 ×

C(CH₃)₃), 1.96 (m, 2H, â-CH₂), 2.23 (m, 2H, γ-CH₂), 2.32 (s,

3H, quinazoline 2-CH₃), 3.23 (s, 1H, CtCH), 4.07 (quintet, J

) 7.3 Hz, 1H, Ala R-CH), 4.24 (m, 1H, Glu R-CH), 4.34 (s, 2H,

CH₂CtC), 4.78 (s, 2H, quinazoline 6-CH₂), 6.83 (d, J ) 8.8

Hz, 2H, 3′,5′-ArH), 7.54 (d, J ) 8.4 Hz, 1H, quinazoline 8-H),

7.69 (d, J ) 9.6 Hz, 1H, quinazoline 7-H), 7.73 (d, J ) 8.9 Hz,

2H, 2′,6′-ArH), 7.96 (s, 1H, quinazoline 5-H), 8.19 (d, J ) 7.0

Hz, 1H, Ala NH), 8.31 (d, J ) 7.2 Hz, 1H, Glu NH), 12.19 (s,

1H, lactam NH); MS (FAB) m/ z 660 (M + H)⁺. Anal.

(C₃₆H₄₅N₅O₇) C, H, N.

The procedure C was repeated with the appropriate dipep-

tide tert-butyl ester free bases 7b-d and the appropriate

pteroic acid analogues 10-18 to give the coupled quinazoline

γ-linked dipeptide tert-butyl esters 19-22 and 24-31. Yields

Quinazoline Antifolate TS Inhibitors

J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 1 83

and mass spectral and analytical data of these products are

given in Table 1. The ¹H NMR spectra of these compounds

were consistent with the assigned structures.

SO₄), and the solvent was removed in vacuo to give the title

compound 59 (10.5 g, 82%) as a pale yellow oil: NMR (Me₂-

SO-d₆) δ 1.39 (s, 9H, CO₂C(CH₃)₃), 1.80, 1.94 (2 × m, 2H,

â-CH₂), 2.40 (m, 2H, γ-CH₂), 3.58 (s, 3H, CO₂CH₃), 3.93 (m,

1H, R-CH), 5.04 (s, 2H, PhCH₂), 7.36 (s, 5H, ArH), 7.67 (d, J

P r epar ation of Qu in azolin e An tifolate γ-Lin ked Dipep-

tides: N-[N-[4-[N-[(3,4-Dih ydr o-2-m eth yl-4-oxo-6-qu in azoli-

n yl)m eth yl]-N-p r op -2-yn yla m in o]ben zoyl]-^L-γ-glu ta m yl]-

D-a la n in e (36). The general procedure D was followed using

23 (0.100 g, 0.15 mmol) and TFA (8 mL). The title compound

36 was obtained as a white solid (0.084 g, 79%): mp 180 °C

dec; NMR (Me₂SO-d₆) δ 1.21 (d, J ) 7.3 Hz, 3H, Ala â-CH₃),

1.94, 2.03 (2 × m, 2H, â-CH₂), 2.22 (m, 2H, γ-CH₂), 2.38 (s,

3H, quinazoline 2-CH₃), 3.23 (s, 1H, CtCH), 4.16 (m, 1H, Ala

R-CH), 4.35 (m, 3H, Glu R-CH, CH₂CtC), 4.80 (s, 2H, quinazo-

line 6-CH₂), 6.83 (d, J ) 8.3 Hz, 2H, 3′,5′-ArH), 7.57 (d, J )

8.4 Hz, 1H, quinazoline 8-H), 7.74 (d, J ) 8.4 Hz, 3H, 2′,6′-

ArH), 7.99 (s, 1H, quinazoline 5-H), 8.17 (d, J ) 7.3 Hz, 1H,

Ala NH), 8.30 (d, J ) 7.1 Hz, 1H, Glu NH); MS (FAB) m/ z

) 7.8 Hz, 1H, NH); MS (CI) m/ z 352 (M + H)⁺. Anal. (C₁₈H₂₅

NO₆‚0.25H₂O) C, H, N.

r-ter t-Bu tyl N-(Ben zyloxyca r bon yl)-^D-glu ta m a te (60).

To a stirred solution of 59 (9.7 g, 27.6 mmol) in acetone (15

mL), EtOH (15 mL), and H₂O (15 mL) was added aqueous

NaOH (1 N, 35 mL, 35.0 mmol). After stirring at room

temperature for 24 h, the organic solvents were removed in

vacuo, and the remainder was diluted with H₂O (80 mL). The

alkaline solution was acidified to pH 4.5 with 1 N HCl and

then extracted with EtOAc (3 × 100 mL). The organic extracts

were combined and dried (MgSO₄), and the solvent was

evaporated to give a viscous oil (7.6 g, 82%), which solidified

on standing to give a white solid. An analytically pure sample

was obtained after purification by column chromatography

using 5% MeOH in CHCl₃as eluent: mp 80-83 °C (lit.²⁸mp

82-83.5 °C for the ^L-enantiomer); NMR (DMSO-d₆) δ 1.39 (s,

9H, C(CH₃)₃), 1.75, 1.90 (2 × m, 2H, â-CH₂), 2.29 (m, 2H,

γ-CH₂), 3.93 (m, 1H, R-CH), 5.03 (s, 2H, PhCH₂), 7.35 (s, 5H,

ArH), 7.64 (d, J ) 7.2 Hz, 1H, NH); MS (CI) m/ z 338 (M +

H)⁺; [R]²⁰) +20.0° (c ) 1.2, MeOH) (lit.²⁸[R]²⁰) -26.6° (c )

1.2, MeOH) for the ^L-enantiomer). Anal. (C₁₇H₂₃NO₆) C, H,

Tr i-ter t-bu tyl N-[N-(Ben zyloxyca r bon yl)-^D-γ-glu ta m yl]-

L-glu ta m a te (61a ). The general procedure A1 was followed

using R-tert-butyl N-(benzyloxycarbonyl)-^D-glutamate (2.022

g, 6.0 mmol), N-methylmorpholine (0.606 g, 6.0 mmol), THF

(7 mL), isobutyl chloroformate (0.816 g, 6.0 mmol), and a

suspension of di-tert-butyl ^L-glutamate hydrochloride (1.77 g,

6.0 mmol) in THF (8 mL) and N-methylmorpholine (0.606 g,

6.0 mmol). The crude product was purified by column chro-

matography using a gradient of EtOAc in CH₂Cl₂(8-20%) as

eluent. Trituration with petroleum ether gave the title

compound (2.7 g, 78%) as a white solid: mp 108-111 °C; NMR

(Me₂SO-d₆) δ 1.38 (s, 27H, 3 × C(CH₃)₃), 1.72, 1.90 (2 × m,

4H, 2 × â-CH₂), 2.25 (m, 4H, 2 × γ-CH₂), 3.89 (m, 1H, GluD

R-CH), 4.10 (m, 1H, GluL R-CH), 5.04 (m, 2H, ArCH₂), 7.35

(m, 5H, ArH), 7.63 (d, J ) 7.7 Hz, 1H, Glu^DNH), 8.13 (d, J )

7.4 Hz, 1H, Glu^LNH); MS (CI) m/ z 579 (M + H)⁺. Anal.

(C₃₀H₄₆N₂O₉) C, H, N.

570 (M

TFA‚0.7Et₂O) C, H, N.

+ +

Na)⁺, 548 (M H)⁺. Anal. (C₂₈H₂₉N₅O₇‚

The procedure D was repeated with the appropriate quinazo-

line γ-linked dipeptide tert-butyl esters 19-22 and 24-31 to

yield the quinazoline γ-linked dipeptides 32-35 and 37-44,

all of which had H NMR spectra consistent with the assigned

structures. Yields and analytical data are gathered in Table

P r ep a r a tion ^D-Glu -L-Glu a n d D-Glu -D-Glu Der iva tives

of ICI 198583: γ-Meth yl-^D-Glu ta m a te Hyd r och lor id e (57).

To a flask containing MeOH (116 mL) and cooled to -10 °C

was slowly added SOCl₂(17 mL, 23.0 mmol) followed by

D-glutamic acid (25 g, 0.17 mmol). The flask was fitted with

a condenser; the acetone-dry ice bath was removed, and the

solution was stirred for 30 min and then poured into Et₂O (330

mL). The precipitate was filtered off and washed well with

Et₂O (150 mL) to give the title compound 57 (20.50 g, 61%) as

a white solid: mp 171-175 °C (lit.²⁹mp 161 °C for the

L-enantiomer); NMR (Me₂SO-d₆) δ 2.07 (m, 2H, â-CH₂), 2.51

(m, 2H, γ-CH₂), 3.61 (s, 3H, CO₂CH₃), 3.94 (m, 1H, R-CH), 8.44

(br s, 3H, NH₃); MS (CI) m/ z 162 (M - Cl)⁺.

γ-Meth yl N-(Ben zyloxyca r bon yl)-^D-glu ta m a te (58). To

a vigorously stirred solution (overhead stirring) of NaHCO₃

(19.0 g, 0.22 mol) in H₂O (240 mL) cooled to 5 °C was added

γ-methyl ^D-glutamate hydrochloride (57; 22.0 g, 0.11 mol)

followed by addition of benzyl chloroformate (30 mL, 0.21 mol)

over a 15 min period. Stirring was continued at 5 °C for 1.5

h and then at room temperature for 2 h. After extracting with

Et₂O (3 × 30 mL), the reaction mixture was acidified to pH 2

with 1 N HCl. EtOAc (120 mL) was then added into the

mixture; the two layers were separated, and the aqueous layer

was washed with more EtOAc (2 × 120 mL). The organic

extracts were combined and dried (Na₂SO₄), and the solvent

was removed in vacuo to give a pale yellow oil which solidified

on standing. Recrystallization from CCl₄gave the title

compound 58 (12.0 g). Because of the low yield, the pH of the

acidic solution, obtained during the workup, was adjusted to

9 with NaOH. Benzyl chloroformate (30 mL, 0.21 mol) was

added, and the preparation was repeated as described above

to give an additional 14.4 g of the product (81% overall yield)

as a white solid: mp 64-66 °C (lit.³⁰mp 72-73 °C for the

L-enantiomer); NMR (Me₂SO-d₆) δ 1.80, 1.98 (2 × m, 2H,

â-CH₂), 2.39 (m, 2H, γ-CH₂), 3.57 (s, 3H, CO₂CH₃), 3.98 (m,

1H, R-CH), 5.02 (s, 2H, PhCH₂), 7.35 (s, 5H, ArH), 7.62 (d, J

) 8.1 Hz, 1H, CONH); MS (CI) m/ z 296 (M + H)⁺. Anal.

(C₁₄H₁₇NO₆) C, H, N.

Tr i-ter t-bu tyl ^D-γ-Glu ta m yl-L-glu ta m a te (62a ). The gen-

eral procedure B was followed using tri-tert-butyl N-[N-

(benzyloxycarbonyl)-^D-γ-glutamyl]-L-glutamate (0.754 g, 1.3

mmol), EtOAc (90 mL), and 10% Pd/C (0.108 g). Compound

62a was obtained as a colorless oil (0.570 g, 98%) and used

immediately 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-^L-glu ta m a te (63a ). The general procedure C was

followed using tri-tert-butyl ^D-γ-glutamyl-L-glutamate (0.500

g, 1.1 mmol), 4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazoli-

nyl)methyl]-N-prop-2-ynylamino]benzoic acid, trifluoroacetate

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 chromatography using a gradient of MeOH in

EtOAc (0-2%) as eluent. Reprecipitation from CH₂Cl₂(mini-

mum amount)/petroleum ether gave the title compound 63a

(0.470 g, 61%) as a white solid: mp 112-116 °C; NMR (Me₂-

SO-d₆) δ 1.37, 1.39 (2 × s, 27H, 3 × C(CH₃)₃), 1.71, 1.88 (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 (m, 1H, GluL

R-CH), 4.24 (m, 1H, GluD R-CH), 4.34 (s, 2H, CH₂CtC), 4.78

(s, 2H, quinazoline 6-CH₂), 6.83 (d, J ) 8.8 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), 8.16

(d, J ) 7.5 Hz, 1H, Glu^LNH), 8.32 (d, J ) 7.3 Hz, 1H, GluD

NH), 12.19 (s, 1H, lactam NH); MS (FAB) m/ z 774 (M + H)⁺.

Anal. (C₄₂H₅₅N₅O₉) C, H, N.

r-ter t-Bu t yl γ-Met h yl N-(Ben zyloxyca r b on yl)-^D-

glu ta m a te (59). To a stirred solution of γ-methyl N-(benzyl-

oxycarbonyl)-^D-glutamate (11 g, 0.037 mol) in dry Bu^tOH (70

mL, 0.74 mol) and dry pyridine (30 mL, 0.37 mol) cooled to

-5 °C was added POCl₃(3.45 mL, 0.037 mol) over a 30 min

period. Stirring was continued at -5 °C for 15 min and then

at room temperature for 50 min. Evaporation of the solvents

left a residue which was partitioned between H₂O (40 mL) and

Et₂O (40 mL). The two layers were separated, and the aqueous

layer was washed with more Et₂O (40 mL). The organic

extracts were combined, washed with aqueous saturated

NaHCO₃(2 × 40 mL), 10% aqueous citric acid (2 × 80 mL),

brine (2 × 40 mL), and finally H₂O (40 mL), and dried (Na₂-

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]-L-

glu ta m a ic Acid (64a ). The general procedure D was followed

using 63a (0.218 g, 0.28 mmol) and TFA (15 mL). The title

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, Glu^LNH),

8.33 (d, J ) 7.4 Hz, 1H, Glu^DNH); 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 × Glu^DR-CH), 5.03 (m, 2H, ArCH₂), 7.35 (m, 5H, ArH),

7.64, 8.12 (2 × d, J ) 7.7 Hz, 2H, 2 × Glu^DNH); 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 × Glu^DR-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 × Glu^DNH), 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 × Glu^DR-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 × Glu^D

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

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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,

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J M950471+

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Quinazoline antifolate thymidylate synthase inhibitors: γ-linked L-D, D- D, and D-L dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8- dideazafolic acid (ICI 198583)

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