Folate-based inhibitors of thymidylate synthase: Synthesis and antitumor activity of γ-linked sterically hindered dipeptide analogues of 2-desamino- 2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583)

DOI: 10.1021/jm960878u

Source and publish data:

Journal of Medicinal Chemistry p. 1495 - 1510 (1997)

Update date:2022-08-02

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Authors:

Bavetsias, Vassilios

Jackman, Ann L.

Marriott, Jonathan H.

Kimbell, Rosemary

Gibson, William

Boyle, F. Thomas

Bisset, Graham M. F.

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Article abstract of DOI:10.1021/jm960878u

In an effort to synthesize inhibitors of thymidylate synthase (TS) that do not undergo polyglutamation, a series of γ-linked sterically hindered dipeptide analogues of 2-desamino-2-methyl-N₁₀-propargyl-5,8-dideazafolic acid (ICI 198583) was pre

Full text of DOI:10.1021/jm960878u

J . Med. Chem. 1997, 40, 1495-1510

1495

F ola te-Ba sed In h ibitor s of Th ym id yla te Syn th a se: Syn th esis a n d An titu m or

Activity of γ-Lin k ed Ster ica lly Hin d er ed 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,^‡J onathan H. Marriott,^‡Rosemary Kimbell,^‡William Gibson,^‡

F. Thomas Boyle,^§and Graham M. F. Bisset^‡,∇

CRC Centre for Cancer Therapeutics at 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 December 30, 1996^X

In an effort to synthesize inhibitors of thymidylate synthase (TS) that do not undergo

polyglutamation, a series of γ-linked sterically hindered dipeptide analogues of 2-desamino-

2-methyl-N¹⁰-propargyl-5,8-dideazafolic acid (ICI 198583) was prepared. A methyl, ethyl, or

propargyl group was incorporated into the γ-glutamyl amide bond of γ-linked ^L,L dipeptide

derivatives of ICI 198583, such as ICI 198583-γ-^L-Glu. In addition, steric bulk was introduced

on either side of the γ-glutamyl bond of ICI 198583-γ-^L-Glu or ICI 198583-γ-L-Ala. The resulting

dipeptide analogues, e.g., ICI 198583-γ-MeGlu and ICI 198583-γ-Aib, were apparently stable

to in vivo hydrolysis but poorer inhibitors of TS and L1210 cell growth. However, introduction

of 7-Me, 2′-F substitution into the quinazoline nucleus gave significant improvement in the

inhibitory activity against thymidylate synthase. Compounds 28-30, the 7-Me, 2′-F derivatives

of ICI 198583-γ-MeGlu, ICI 198583-γ-EtGlu, and ICI 198583-γ-PgGlu, respectively, were potent

inhibitors of TS (K_iapp) 0.21-1.1 nM) and L1210 cell growth (IC₅₀) 0.05-0.34 µM) and were

similar to that seen with the most potent γ-linked ^L,D dipeptide derivatives of ICI 198583

previously synthesized. Furthermore, the low cross-resistance ratios for the L1210:R^D1694/L1210

cell line indicated that 28-30 do not undergo polyglutamation.

In tr od u ction

In a recent paper from our laboratories the synthesis

of γ-linked L,D dipeptide analogues of 2-desamino-2-

methyl-N¹⁰-propargyl-5,8-dideazafolic acid (ICI 198583),

as inhibitors of thymidylate synthase (TS) that do not

undergo polyglutamation, was described.¹The design

of this class of antifolates was based on γ-linked L,L

dipeptide derivatives of ICI 198583, e.g., ICI 198583-γ-

L-Glu and ICI 198583-γ-L-Ala, because of their excellent

TS inhibitory properties.²However, susceptibility of the

γ-glutamyl bond to enzymatic degradation limited the

utility of these dipeptide derivatives as nonpolyglutamat-

able inhibitors of TS in animal models since ICI 198583,

a product of the enzymatic degradation, had been shown

to undergo polyglutamation.³Replacement of the C-

terminal residue of γ-linked L,L dipeptides by D-amino

acids gave L,D dipeptide derivatives that were potent

inhibitors of TS, nonsubstrates for folylpolyglutamyl

synthetase (FPGS), and resistant to enzymatic degrada-

tion¹(Figure 1). A second approach involved the

replacement of the amidic hydrogen of dipeptide ana-

logues of ICI 198583 by an alkyl group (e.g., methyl) or

the introduction of steric bulk on either side of the

F igu r e 1.

γ-glutamyl amide bond. We therefore prepared a

variety of N-alkylated, and in particular N-methylated,

analogues of active dipeptide derivatives with the view

of determing whether incorporation of N-alkyl residues

in quinazoline antifolate dipeptides would prevent

enzymatic degradation and polyglutamation without

compromising potency. Such agents, with activity

independent of FPGS, were expected to display a

different spectrum of activity to Tomudex,^4-6particu-

larly in tumors expressing low levels of FPGS. We now

report here the synthesis of 14 sterically hindered

γ-linked dipeptide analogues of ICI 198583.

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

synthetase; MTX, methotrexate: DEPC, diethyl cyanophosphoridate;

Pg, propargyl; Glu, glutamic acid; Gly, glycine; Ala, alanine; Aib,

R-aminoisobutyric acid; MeGlu, N-methylglutamic acid; EtGlu, N-

ethylglutamic acid; PgGlu, N-propargylglutamic acid; 4-MeGlu, 4-me-

thylglutamic acid; 4,4-diMeGlu, 4,4-dimethylglutamic acid; LDA,

lithium diisopropylamide; LICA, lithium N-isopropylcyclohexylamide;

NMM, N-methylmorpholine; DIEA, diisopropylethylamine; PyBOP,

benzotriazolyloxytris(pyrrolidino)phosphonium hexafluorophosphate;

Tr, trityl.

^‡Cancer Research Campaign Laboratories.

Ch em istr y

^§Zeneca Pharmaceuticals.

^∇Present address: Cross Medical Ltd., Unit 1, The Chase Centre,

8 Chase Rd, Park Royal, London NW10 6QD, England.

^XAbstract published in Advance ACS Abstracts, April 15, 1997.

The synthetic pathway to γ-linked sterically hindered

dipeptides of ICI 198583 involved preparation of the

1496 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10

Bavetsias et al.

Sch em e 1^a

Conditions: (a) aq NaHCO₃, ClCO₂CH₂Ph; (b) DCC, Bu^tOH, DMAP, CHCl₃; (c) (CH₃)₂CdCH₂, concd H₂SO₄, CH₂Cl₂; (d) paraldehyde,

TsOH‚H₂O, benzene, reflux; (e) Et₃SiH, TFA, CHCl₃, rt; (f) CHtCCH₂Br, K₂CO₃, DMF, rt; (g) H₂, 10% Pd/C, EtOAc, rt; (h) ClCO₂CH(CH₃)₂,

NMM, THF, -20 °C to rt; (i) TFA-CH₂Cl₂, rt; (j) DEPC, Et₃N; (k) TFA.

function of MeGly or MeGlu was protected as a tert-

butyl ester by first blocking the amino function with a

benzyloxycarbonyl (Z) group and then esterifying the

carboxyls using DCC / Bu^tOH⁷or isobutylene/H₂SO₄,

respectively. Removal of the Z group by catalytic

hydrogenolysis afforded 8a ,b.

The synthesis of Z-EtGlu (7) was accomplished by

applying Freidinger’s two-step procedure for Fmoc-

protected N-alkyl amino acids.⁸In the first step Z-L-

Glu was converted to the oxazolidinone 6 by treatment

with paraldehyde under acid-catalyzed conditions

(TsOH). Reduction of this intermediate with the tri-

ethylsilane-trifluoroacetic acid system afforded N-

(benzyloxycarbonyl)-N-ethyl-L-glutamic acid (7, Z-Et-

Glu). Di-tert-butyl N-ethyl-L-glutamate (8c) was then

prepared by a sequence similar to that described for the

N-Me derivative 8b (Scheme 1). Subsequent condensa-

tion of 8a -c with R-tert-butyl N-(benzyloxycarbonyl)-

L-glutamate via isobutyl mixed anhydride activation⁹

dipeptide derivatives by solution peptide synthesis

followed by condensation with the appropriate pteroic

acid analogue (Schemes 1-4). (The chemistry of peptide

analogues of various antifolates has been described in

a recent paper from our laboratories.²)

The preparation of the dipeptides 10a -d , key inter-

mediates for the synthesis of N-alkyl derivatives 25-

34, is shown in Scheme 1. To prepare 8a ,b, the carboxyl

Folate-Based Inhibitors of TS

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1497

Ta ble 1. Quinazoline Antifolate Sterically Hindered Dipeptide

Esters

Sch em e 2

compd

R¹

R²

59a

59b

MeGlu(OBu^t)-OBu^t

EtGlu(OBu^t)-OBu^t

PgGlu(OBu^t)-OBu^t

MeGly-OBu^t

MeGlu(OBu^t)-OBu^t

Aib-OBu^t

Pro-OBu^t

Ta ble 2. Quinazoline Antifolate Sterically Hindered

Dipeptides

compd

R¹

R²

methyl N-[9-(9-phenylfluorenyl)]-L-glutamate using po-

tassium bis(trimethylsilyl)amide ((KN(SiMe₃)₂) as the

base to generate the γ-enolate ester instead of LDA or

LICA.¹²

60a

60b

MeGlu

EtGlu

PgGlu

MeGly

MeGlu

Aib

Our approach to the synthesis of 37a involved dif-

ferentiation of the two carboxyls of glutamic acid to

enable chemoselectivity in later stages of the synthesis.

The R-carboxyl was masked as its tert-butyl ester,^13,14

while a methyl ester was employed as a temporary

protecting group for the γ-carboxy function of the

glutamate (Scheme 3). Trityl protection of the amino

group was used to prevent R-deprotonation and subse-

quent methylation or possible racemization. The γ-eno-

late ester of 40 was generated at -78 °C using KN-

(SiMe₃)₂as the base, and after quenching with MeI, the

required product 41 was obtained as a mixture of two

diastereoisomers, nonseparable by column chromatog-

raphy. Methyl ester removal by treatment with aque-

ous LiOH in THF/H₂O gave 42 which was condensed

with di-tert-butyl L-glutamate via isobutyl mixed anhy-

dride activation to give the dipeptide derivative 43 as a

mixture of two diastereoisomers, separable by column

chromatography (R_f) 0.33, 0.28-20% EtOAc in hex-

anes). The stereochemistry at the γ-center of the MeGlu

residue has not been determined. Only the diastereo-

isomer with the lower R_f(0.28) was taken forward to

Pro

afforded the Z-protected dipeptides 9a -c, from which

the key intermediates 10a -c were obtained by catalytic

hydrogenolysis (Scheme 1).

A different strategy was applied to prepare the

N-propargyl derivative 10d . Treatment of di-tert-butyl

L-glutamate hydrochloride salt with propargyl bromide

and K₂CO₃as the base in DMF afforded di-tert-butyl

N-propargyl glutamate (12) which was then coupled to

Boc-L-Glu-OBu^tvia isobutyl mixed anhydride activation

to give the dipeptide 13 (Scheme 1). Selective removal

of the Boc group was achieved with TFA-CH₂Cl₂(v/v,

1:4) at room temperature over a 10 min period.

Dipeptides 10a -d were then condensed with the

pteroate analogues 14a -g^1,2using diethyl phosphoro-

cyanidate (DEPC) carboxyl activation¹⁰to give the

quinazoline antifolate dipeptide esters 15-24 (Scheme

1, Table 1), which were converted to the final products

25-34 by treatment with TFA (Scheme 1, Table 2).

The synthesis of quinazoline antifolate sterically

hindered dipeptides 36a ,b, in which one or two methyl

groups were introduced at the γ-center of the first

glutamic residue of ICI 198583-γ-L-Glu (1), required the

key intermediates tri-tert-butyl γ-4-methyl-L-glutamyl-

L-glutamate (37a ) and tri-tert-butyl γ-4,4-dimethyl-L-

glutamyl-L-glutamate (37b), respectively (Scheme 2).

The monomethyl dipeptide derivative 37a was pre-

pared by the multistep sequence outlined in Scheme 3.

We envisaged that introduction of the methyl group at

the γ-center of a glutamate could be achieved through

generation of the γ-enolate ester and quenching with

MeI. Baldwin et al. applied this strategy to synthesize

γ-hydroxyalkylated glutamates.¹¹The γ-enolate of R-tert-

butyl γ-methyl N-trityl-L-glutamate was generated us-

ing either LDA or LICA and then quenched with various

carbonyl compounds to give γ-hydroxyalkylated gluta-

mates. Rapoport et al. extended this and successfully

prepared γ-alkylated glutamates from R-tert-butyl γ-

the next step since H-NMR indicated the presence of

an unidentified impurity in the diastereoisomer with the

higher R_f. A pure sample of this isomer was later

obtained by the γ-allyl ester route described in Scheme

4. We found that the allyl ester was superior to the

methyl ester since it could be efficiently hydrolyzed

under mild conditions (Scheme 4). In addition, the

diastereoisomers of 43 were obtained in pure form.

Finally, the trityl group was removed by catalytic

hydrogenolysis to afford the key intermediate 37a .

Initial attempts to prepare 44, required for the

preparation of dipeptide 37b, directly from 40 using

excess of base (3 equiv) and MeI (4 equiv) gave the

γ-monomethylated glutamate derivative 41 and only

about 3% of the desired product 44 (Scheme 3). The

γ-dimethylated glutamate 44 was finally prepared from

41 by quenching its γ-enolate ester with 4 equiv of MeI.

However, a workable route to 37b from compound 44

was not possible since alkaline hydrolysis (NaOH, H₂O/

THF) of R-tert-butyl γ-methyl N-trityl-4,4-dimethyl-L-

glutamate (44) gave unacceptably low yields of 45 even

when the reaction mixture was heated to reflux for 46

1498 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10

Bavetsias et al.

Sch em e 3^a

Conditions: (a) CH₃CO₂C(CH₃)₃, HClO₄, rt; (b) TrCl, Et₃N, Pb(NO₃)₂, CHCl₃, rt; (c) KN(SiMe₃)₂, MeI, THF, -78 °C; (d) LiOH‚H₂O,

H₂O/THF; (e) HCl‚Glu(OBu^t)-OBu^t, ClCO₂CH₂CH(CH₃)₂, NMM, THF, -20 °C to rt; (f) H₂, 10% Pd/C, EtOAc, rt.

Sch em e 4^a

Conditions: (a) KN(SiMe₃)₂, MeI, THF, -78 °C; (b) Pd(PPh₃)₄, pyrrolidine, CH₂Cl₂, rt; (c) HCl‚Glu(OBu^t)-OBu^t, PyBOP, DMAP, DIEA,

CH₂Cl₂, rt; (d) H₂, 10% Pd/C, EtOAc, rt; (e) HCl‚Glu(OBu^t)-OBu^t, ClCO₂CH₂CH(CH₃)₂, NMM, THF, -20 °C to rt.

In view of this, allyl ester protection of the γ-carboxy

function of the glutamate was utilized (Scheme 4).

R-tert-Butyl γ-allyl N-trityl-L-glutamate (46) was syn-

thesized as previously described.¹⁵Subsequently, the

γ-dimethylated glutamate derivative 48 was prepared

from 47 by generation of the γ-enolate ester at -78 °C

with KN(SiMe₃)₂and quenching with MeI (Scheme 4).

The allyl ester was selectively removed by treatment

with catalytic amounts of Pd(PPh₃)₄and excess of

pyrrolidine in CH₂Cl₂. Coupling of 45 to di-tert-butyl

glutamate required activation of the γ-carboxyl with

PyBOP¹⁶and catalytic amounts of DMAP in CH₂Cl₂

followed by addition of di-tert-butyl L-glutamate to give

the desired dipeptide 50 in 45% yield and the cyclized

byproduct 49 in 30% yield. It should be noted that

attempts to prepare 50 using the isobutyl mixed anhy-

dride method or azide method failed to give any product,

whereas the acid chloride method resulted in the

formation of the undesired pyroglutamic acid derivative

49. This is not an unexpected finding since it is known

that the steric bulk of an R,R-disubstituted carboxylic

acid makes activation and amide bond formation more

difficult.¹⁷Removal of the trityl group by catalytic

hydrogenolysis finally afforded the key dipeptide inter-

mediate 37b.

In order to check if the optical purity at the R-center

of the glutamate was preserved during the synthetic

sequence, the dipeptide 54 (Scheme 5) was synthesized

by a route similar to the one employed for the synthesis

of 43 except that allyl esters were used instead of methyl

esters and the synthesis started with the D-glutamate

derivative 51. If racemization occurred at the R-center

of the glutamate, it was expected that 43 and 54 would

give the same H-NMR spectra. However, 43 and 54,

each obtained as a mixture of two diastereoisomers,

gave different H-NMR spectra. For example, the H-

Folate-Based Inhibitors of TS

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1499

Sch em e 5^a

conformers are distinguishable on the time scale of ¹H-

NMR. This was the case with the MeGlu, EtGlu, PgGlu,

and MeGly amide derivatives made in our study. Thus,

for example, the H-NMR of Z-MeGly (3) in DMSO at

ambient temperature (25 °C) showed that the two

conformers exist in similar populations. Raising the

temperature increased the rate of rotation around the

methylated amide bond resulting in a ¹H-NMR of 3

recorded at 345 K showing one peak for the N-Me, which

represents the average environment between the two

conformers. Similarly, the H-NMR of Z-MeGlu (5) in

DMSO at ambient temperature again showed that both

trans and cis conformers are energetically comparable,

since the ratio of the two singlets at 2.76 and 2.79 ppm

corresponding to N-Me protons is about 3:2. After

raising the temperature to 345 K, however, we observed

one signal (singlet) for the N-Me group. The existence

of the two conformers resulted in rather complicated ¹H-

NMR spectra since a number of peaks are doubled even

at high temperatures.

Conditions: (a) KN(SiMe₃)₂, MeI, THF, -78 °C; (b) Pd(PPh₃)₄,

pyrrolidine, CH₂Cl₂; (c) HCl‚Glu(OBu^t)-OBu^t, ClCO₂CH₂CH(CH₃)₂,

NMM, THF, -20 °C to rt.

Sch em e 6^a

Biologica l Eva lu a tion

The antifolates listed in Table 2 were tested as

inhibitors of partially purified TS from L1210 mouse

leukemia cells that overproduce TS due to amplification

of the TS gene.²⁰The partial purification and assay

method used in this study was as previously described,

and it used (()-5,10-methylenetetrahydrofolic acid at a

concentration of 200 µM.²⁰Inhibition of L1210 and

L1210:1565 cell growth was also determined as previ-

ously described.²¹L1210:1565 is a L1210 mutant cell

line with impaired reduced folate/MTX transport car-

rier. This cell line was made resistant to CI-920, a

compound that uses the RFC transport system,²²and

hence is cross-resistant to MTX. The methodology for

the in vivo stability was as previously described;²³mice

were administered with 100 mg/kg compound (ip) and

killed 1 h later. Blood, liver, and kidneys were removed

for quantitation by HPLC of the parent dipeptide

derivative of ICI 198583 and any monoglutamate de-

rivative of ICI 198583 present.

Conditions: (a) ClCO₂CH₂CH(CH₃)₂, NMM, THF, -20 °C to

rt; (b) H₂, 10% Pd/C, EtOAc, rt; (c) DEPC, Et₃N, DMF, 0 °C to rt;

(d) TFA.

Resu lts a n d Discu ssion

All compounds were tested as inhibitors of TS and

L1210 cell growth. The results are shown in Table 3.

Replacement of the γ-glutamyl amidic hydrogen of ICI

198583-γ-L-Glu (1) by methyl gave compound 25 which

was approximately 5-fold less potent as an inhibitor of

TS but stable to hydrolysis in mice. The same trend

was observed with ICI 198583-γ-MeGly (31), again

stable in mice but a considerably less potent inhibitor

of TS (∼5-fold) than its parent dipeptide ICI 198583-γ-

Gly (TS IC₅₀) 11 nM, L1210 IC₅₀) 0.11 µM).²

Substitution of 7-Me into the quinazoline ring (com-

pound 26) or 2′-F into the benzene ring of 25 (compound

27) enhanced TS inhibition by 2-4-fold. Combining

both of these structural modifications to give compound

28 resulted in a 10-fold enhancement in TS inhibition

but without any parallel improvement in the L1210 cell

growth inhibition (IC₅₀) ∼0.24 µM).

NMR spectrum of 43 showed one set of two doublets at

7.92 and 8.14 ppm for the amidic protons, whereas the

¹H-NMR spectrum of 54 showed one doublet at 7.99

ppm for the amidic protons. This suggests that the

optical purity at the R-center of the glutamate had been

preserved.

The quinazoline antifolate analogues 36a ,b were then

prepared from the dipeptides 37a ,b, respectively, by the

route employed for the synthesis of the N-alkyl deriva-

tives, i.e., DEPC coupling followed by TFA hydrolysis.

Finally the synthesis of 60a ,b is shown in Scheme 6.

Con for m a tion a l Equ ilibr ia of MeGlu , EtGlu ,

P gGlu , a n d MeGly Am id e Der iva tives

Normally an amide bond adopts the trans planar

conformation. However, it has been reported that

N-alkyl amide bonds tend to adopt both cis and trans

conformations,^18,19due to bulk of the alkyl substituent

compared to that of the replaced amidic hydrogen. Both

conformers normally interconvert, but if rotation around

the N-alkylated amide bond is slowed, then the two

Replacement of the N¹⁰-propargyl substituent by a

methyl in compounds 26-28 generally resulted in

poorer inhibitors of TS and L1210 cell growth (com-

pounds 32-34, Table 3). Only the 7-Me, 2′-F derivative,

compound 34, had an inhibitory activity against TS

1500 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10

Bavetsias et al.

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

inhibition of cell growth, IC₅₀(µM)^a

W1L2

L1210:1565 L1210:R^D1694

inhibition of

L1210 TS,

K_iapp(nM)

stable to

hydrolysis

compd

R¹

R²

L1210

in mice?^b

36a

36b

60a

60b

L-Glu-L-Glu

2.0

0.16 ( 0.069

4.6, 5.0

1.6, 2.3

2.7, 2.9 (18)

2.2, 2.6 (10)

2.0, 2.1 (8)

2.6 (10)

1.4 ( 0.32 (4)

0.22 (6)

0.57 (5)

7.4 (4)

4.0 (6)

9.2 (5)

2.6 (4)

3.4 (10)

1.2, 3.5 (2)

7.1 (2.5)

10 (3)

no (ref 2)

yes

L-Glu-L-MeGlu

L-Glu-L-EtGlu

L-Glu-L-PgGlu

L-Glu-MeGly

L-Glu-L-MeGlu

L-4-MeGlu-L-Glu

L-4,4-diMeGlu-L-Glu

L-Glu-Aib

0.24, 0.22

0.47

Me Pg

2.6

4.0

1.1

0.35

0.21

8.2

3.0

8.6

0.22, 0.24, 0.25 0.64

0.23, 0.27

0.34 ( 0.070

0.07, 0.03

0.11, 0.072

1.1, 2.4

0.44

0.35, 0.70

0.073

0.22, 0.21

0.33

1.8

Me Pg

4, 5.4

9.3, 5.4

4.2

∼70

4.2, 9.8

5.1

5.6

yes

Me Me

yes

0.64, 0.56, 0.78 0.80

2.4, 2.1, 1.5

0.73, 0.60

0.41, 0.25

1.4, 1.0

2.5, 3.1

3.1, 4.2

2.3

0.63

0.50

0.37

0.31

yes

L-Glu-Pro

Methodologies for the inhibition of mouse L1210 TS and L1210, L1210:1565 (impaired RFC), L1210:R^D1694, and human W1L2 cell

growth are as described in refs 20, 21, and 24. Methodology for the stability of these dipeptides in mice is described in ref 23.

comparable to that of the corresponding N¹⁰-propargyl

derivative 28. These data support the previously re-

ported¹view that propargyl is a better N¹⁰-substituent

than methyl and 7-Me, 2′-F substitution pattern en-

hances binding to TS.

line, which exhibits an impaired RFC,²²with cross-

resistance ratios between 8 and 105, except compound

33 (Table 3).

These compounds were designed to overcome the

antifolate-resistant phenotype due to a decreased level

of polyglutamation. The ZD1694-resistant cell line

The impact of different N-substituents on the

γ-glutamyl amide bond from the methyl was next

explored. We chose, as the starting point for this study,

compound 28, a compound bearing the N¹⁰-Pg, 7-Me,

2′-F substitution pattern in which the γ-glutamyl amide

carried a methyl substituent. Replacement by ethyl or

propargyl gave compound 29 or 30, respectively. Rela-

tive to 28 these compounds were up to 5-fold more

potent inhibitors of TS and L1210 cell growth (Table 3)

supporting a degree of tolerance by the enzyme in the

region of the N-substituent of the γ-glutamyl bond.

Both analogues showed improvement in enzyme bind-

ing, but compound 29 was the most potent inhibitor of

the L1210 and W1L2 cell growth (Table 3).

The consequences of introducing one or two methyl

groups onto the γ-center of the first glutamic residue of

ICI 198583-γ-L-Glu (1) were next studied. The mono-

methyl derivative 36a was a 4-fold less potent inhibitor

of TS and ∼2-fold less growth inhibitory than ICI

198583-γ-L-Glu (Table 3). Introduction of two methyl

groups, compound 36b , resulted in a further drop in

potency. The effect of introducing a methyl group on

the other side of the γ-glutamyl amide bond was

revealed by the synthesis of ICI 198583-γ-Aib (60a ). The

compound was a less potent inhibitor of TS and L1210

cell growth compared with its parent compound ICI

198583-γ-L-Ala (IC₅₀) 18 nM, L1210 IC₅₀) 0.56 µM).²

When compounds 36a ,b and 60a were administered to

mice, no hydrolysis products were observed after 1 h,

indicating that the introduction of steric bulk in close

proximity to the γ-glutamyl amide bond significantly

improved the enzymatic stability.

(L1210:R^{D1694 24}

was used to address the question since

)

in this cell line the predominant mechanism of resis-

tance is a decreased ability to polyglutamate synthetic

antifolates. A minor RFC transport defect has also been

shown in this cell line to be a small but contributing

mechanism of resistance. This cell line is >10000-fold

resistant to ZD1694 but only 18-fold cross-resistant to

ICI 198583²⁴reflecting the decreased dependency of the

latter compound on polyglutamation for activity. As

expected, cross-resistance was observed with the γ-linked

L,L dipeptide derivatives of ICI 198583 (compound 1).

Previous studies from our laboratory,²⁴with a number

of compounds that are structurally precluded from

polyglutamation, suggested that compounds which use

the RFC for cell entry but are not substrates for FPGS

give cross-resistance values ranging from ∼2 to 5.

Compounds with a very low affinity of RFC gave values

of ∼1. Using these ratios as precedent we conclude that

four compounds (25-27, 36a ) with high relative resis-

tance ratios (8-10) are poor substrates for FPGS. We

are not able to say clearly whether a low level of

polyglutamation is occurring, and further work with a

cell line expressing a low level of FPGS activity or

radiolabeled studies would be required to establish this.

The two most potent compounds in the L1210:R^D1694cell

line were 29 and 30 (L-Glu-γ-EtGlu and L-Glu-γ-PgGlu,

respectively), which is almost certainly due to their

increased binding to TS.

Replacement of the γ-glutamyl amidic hydrogen by

an alkyl group or introduction of steric hindrance to

either side of this amide bond resulted in compounds

which were stable in vivo but less potent than the

parent γ-linked L,L dipeptide analogues of ICI 198583

in vitro. Reduced activity against the L1210:1565 cell

line compared to the parental line indicated that these

Further characterization of this interesting series of

compounds revealed that the majority utilize the RFC/

MTX transport carrier to enter cells. This is best

illustrated by the poor activity in the L1210:1565 cell

Folate-Based Inhibitors of TS

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1501

base which was immediately taken forward into the next step

used without further purification.

dipeptides utilize the RFC transport system to enter

cells. With regard to polyglutamation the picture is less

clear. L1210:R^D1694cells were partially cross-resistant

to analogues of ICI 198583-γ-L-Glu, such as 25, which

lack 7-Me substitution, and probably reflect a low level

of FPGS substrate activity. However, incorporation of

the 7-Me into these dipeptides was expected to prevent

FPGS substrate activity as previously described when

ICI 198583 was 7-methylated²⁵and gave a low cross-

resistance ratio.²⁴Compounds 28-30 exhibited similar

inhibitory activity against TS and L1210 cell growth as

the most potent γ-linked L,D dipeptide analogues of ICI

198583, a class of compounds that do not undergo

polyglutamation.

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 N-Alk yl Am in o Acid s: N-(Ben zyloxy-

ca r bon yl)-N-m eth ylglycin e (3). N-Methylglycine (7.0 g,

0.078 mol) was added to water (150 mL) and the pH of the

solution adjusted to ∼9 with solid NaHCO₃. The solution was

cooled to 0 °C and stirred vigorously (overhead mechanical

stirring) while benzyl chloroformate (19 mL, 0.13 mol) was

added dropwise over a 20 min period. The mixture was stirred

for a further 2 h at 0 °C and an additional 2 h at room

temperature, during which time the pH was periodically

checked and adjusted to ∼9 with solid NaOH. The reaction

mixture was then extracted with Et₂O (2 × 130 mL), and the

aqueous solution was then acidified to pH 3 with 5 N HCl and

the product isolated by extraction with EtOAc (2 × 130 mL).

The combined organic extracts were dried (Na₂SO₄), and the

solution was concentrated in vacuo to give N-(benzyloxycar-

bonyl)-N-methylglycine as a pale yellow oil (16.2 g, 92%): ¹H-

NMR (DMSO-d₆, 72 °C) 2.91 (s, 3H, N-CH₃), 3.94 (s, 2H,

CH₂CO₂H), 5.07 (s, 2H, PhCH₂), 7.33 (s, 5H, ArH); MS (EI,

m/z) 223 (M⁺).

r-ter t-Bu t yl N-(Ben zyloxyca r b on yl)-N-m et h ylglyci-

n a te (4a ). To a stirred solution of N-(benzyloxycarbonyl)-N-

methylglycine (7.86 g, 0.035 mol) and tert-butyl alcohol (4.68

mL, 0.052 mmol) in CHCl₃(30 mL) cooled in an ice-water

bath was added a solution of DCC (8.0 g, 0.039 mol) in CHCl₃

(30 mL), and then DMAP (0.04 g) was added as catalyst.

Stirring was continued at 0 °C for 2 h; then the mixture was

kept at 4 °C overnight. Dicyclohexylurea was removed by

filtration and the solvent evaporated in vacuo. The residue

was dissolved in EtOAc (110 mL), AcOH (1.10 mL) was then

added, and the solution was stored at 4 °C for 2 h. After

filtration, the ethyl acetate solution was washed with 5%

aqueous NaHCO₃solution (2 × 70 mL) and H₂O (3 × 80 mL),

dried (MgSO₄), and concentrated in vacuo to leave an oily

residue. Purification by column chromatography on elution

with 30% EtOAc in petrol gave the title compound 4a (3.6 g,

37%) as an oil: ¹H-NMR (DMSO-d₆) 1.36, 1.41 (2 × s, 9H,

C(CH₃)₃), 2.88, 2.91 (2 × s, 3H, N-CH₃), 3.91, 3.94 (2 × s, 2H,

CH₂COOH), 5.05, 5.10 (2 × s, 2H, PhCH₂), 7.31, 7.36 (2 × m,

5H, ArH); MS (EI, m/z) 279 (M⁺). Anal. (C₁₅H₂₁NO₄) C, H,

Exp er im en ta l Section

Thin layer chromatography (TLC) was performed on pre-

coated sheets of silica 60F₂₅₄(Merck Art 5735). Visualization

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

60 (Art 15111) was used in low-pressure column chromatog-

raphy. Petrol refers to light petroleum (bp 60-80 °C).

Electron impact (EI) and chemical ionization (CI) mass spectra

were determined with a VG 7070H spectrometer and a VG

2235 data system using the direct-insertion method, an

ionizing voltage of 70 eV, a trap current of 100 µA, and an

ion-source temperature of 160 °C. Fast atom bombardment

(FAB) mass spectra were determined with a VG ZAB-SE

spectrometer. Electrospray 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) containing 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 multiplicities 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 determined on a Kofler block and are

uncorrected. Elemental analyses were determined by C.H.N.

Analysis Ltd., Leicester, U.K.

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 formed).

1. 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.

2. 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 hydrochlo-

ride was filtered off, and the filtrate was concentrated 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

ter t-Bu tyl N-Meth ylglycin a te (8a ). A solution of tert-

butyl N-(benzyloxycarbonyl)-N-methylglycinate (1.60 g, 5.7

mmol) in EtOAc (120 mL) containing 10% Pd/C (0.23 g) was

stirred under hydrogen for 2.5 h. The catalyst was removed

by filtration and the filtrate concentrated in vacuo, yielding

tert-butyl N-methylglycinate (0.71 g, 85%) as an oil: ¹H-NMR

(DMSO-d₆) 1.41 (s, 9H, C(CH₃)₃), 2.25 (s, 3H, N-CH₃), 3.13 (s,

2H, CH₂); MS (EI, m/z) 145 (M⁺).

N-(Ben zyloxyca r bon yl)-N-m eth yl-^L-glu ta m ic Acid (5).

N-Methyl-^L-glutamic acid (5.0 g, 31.0 mmol) was dissolved in

a saturated solution of NaHCO₃(190 mL). The solution was

cooled to 0 °C and stirred vigorously (overhead mechanical

1502 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10

Bavetsias et al.

stirring) while benzyl chloroformate (10.5 g, 62.0 mmol) was

added dropwise over a 20 min period. The mixture was stirred

for a further 2 h at 0 °C and then for a further 20 h at room

temperature and then extracted with Et₂O (2 × 150 mL). The

aqueous solution was acidified to pH 3 with 5 N HCl and the

product isolated by extraction with EtOAc (2 × 150 mL). The

pooled organic extracts were dried over Na₂SO₄, and the

solvent was removed in vacuo to give a yellow oil which

solidified on standing (6.9 g, 75%). Recrystallization from H₂O

gave an analytically pure sample as a white solid: mp 98-

101 °C (lit.²⁶mp 79-80 °C); [R]²⁵) -35.0 (c ) 1, EtOH) (lit.²⁶

[R]²⁷) -25.7 (c ) 1, EtOH)); ¹H-NMR (DMSO-d₆, 67 °C) δ

1.94, 2.14 (2 × m, 2H, â-CH₂), 2.22 (t, J ) 6.3 Hz, 2H, γ-CH₂),

2.79 (s, 3H, N-CH₃), 4.53 (dd, J ) 5.0, 10.4 Hz, 1H, R-CH),

5.08 (s, 2H, ArCH₂), 7.33 (m, 5H, ArH); MS (CI, m/z) 296 (M

+ H)⁺. Anal. (C₁₄H₁₇NO₆) C, H, N.

extracted with EtOAc (150 mL, then 2 × 50 mL), and the

combined organic solution was washed with brine (3 × 50 mL),

dried (Na₂SO₄), and evaporated. The residue was chromato-

graphed on silica gel using hexanes-CH₂Cl₂(2:1, v/v), neat

CH₂Cl₂, and CH₂Cl₂-EtOAc (19:1 and 9:1, v/v) in succession

as eluants. Di-tert-butyl N-(benzyloxycarbonyl)-N-ethyl-^L-

glutamate, 4c, was isolated as an oil (2.97 g, 76%): ¹H-NMR

(DMSO-d₆) δ 1.08 (t, J ) 7.0 Hz, 3H, CH₂CH₃), 1.32, 1.37, 1.39

(3 × s, 18H, C(CH₃)₃), 2.0 (m, 2H, â-CH₂), 2.23 (m, 2H, γ-CH₂),

3.05, 3.42 (2 × m, 2H, CH₂CH₃), 4.10 (m, 1H, R-CH), 5.13 (m,

2H, ArCH₂), 7.35 (m, 5H, ArH); MS (EI, m/z) 422 (M + H)⁺.

Anal. (C₂₃H₃₅NO₆) C, H, N.

Di-ter t-bu tyl N-Eth yl-^L-glu ta m a te (8c). The general

procedure B was followed using 4c (1.761 g, 4.18 mmol), EtOAc

(125 mL), and 10% Pd/C (0.24 g). The reaction mixture was

stirred under hydrogen at room temperature for 3 h, and the

crude product was chromatographed with CH₂Cl₂-EtOH

(100:0 to 97:3, v/v) as eluant. Di-tert-butyl N-ethyl-^L-glutamate,

8c, was isolated as a colorless oil (1.132 g, 94%): ¹H-NMR

(CDCl₃) δ 1.08 (t, J ) 7.1 Hz, 3H, CH₂CH₃), 1.44, 1.47 (2 × s,

18H, C(CH₃)₃), 1.83 (m, 2H, â-CH₂), 2.33 (m, 2H, γ-CH₂), 2.49,

2.63 (2 × m, 2H, CH₂CH₃), 3.10 (dd, J ) 7.4, 6.4 Hz, 1H, R-CH);

MS (EI, m/z) 287 (M)⁺.

Di-ter t-b u t yl

N-(Ben zyloxyca r b on yl)-N-m et h yl-^L-

glu ta m a te (4b). To a suspension of N-(benzyloxycarbonyl)-

N-methyl-^L-glutamic acid (1.85 g, 6.3 mmol) in CH₂Cl₂(52 mL)

in a 500 mL pressure bottle was added concentrated H₂SO₄

(0.28 mL) followed by liquid isobutylene (25 mL) at -20 °C.

The stoppered reaction vessel was shaken vigorously at room

temperature for 28 h and then cooled, and a saturated solution

of NaHCO₃(100 mL) and EtOAc (200 mL) were added. The

organic layer was separated and washed with a saturated

NaHCO₃solution (2 × 160 mL) and H₂O (100 mL), then dried

(Na₂SO₄), and concentrated in vacuo. Purification by column

chromatography using 10% EtOAc in CH₂Cl₂as eluant gave

the title compound 4b (1.8 g, 70%) as an oil: ¹H-NMR (DMSO-

d₆, 67 °C) δ 1.37, 1.38 (2 × s, 18H, 2 × C(CH₃)₃), 1.90, 2.07 (2

× m, 2H, â-CH₂), 2.20 (m, 2H, γ-CH₂), 2.79 (s, 3H, N-CH₃),

4.44 (dd, J ) 5.3, 10.2 Hz, 1H, R-CH), 5.11 (m, 2H, ArCH₂),

Di-ter t-bu tyl N-P r op -2-yn yl-^L-glu ta m a te (12). Di-tert-

butyl ^L-glutamate hydrochloride (3.0 g, 10.1 mmol), anhydrous

K₂CO₃(2.94 g, 21.3 mmol), DMF (15 mL), and propargyl

bromide (80% solution in toluene, 1.2 mL, 10.6 mmol) were

stirred together under argon at ambient temperature with

protection from light. After 3 days the reaction mixture was

concentrated and the residue partitioned between CH₂Cl₂(100

mL) and H₂O (100 mL). The aqueous layer was extracted with

CH₂Cl₂(2 × 20 mL), and the combined CH₂Cl₂solution was

washed with H₂O (5 × 30 mL), dried (MgSO₄), and evaporated.

Two portions of toluene were added and evaporated, and the

residue was chromatographed on silica gel, using CH₂Cl₂-

hexanes (1:2 to 3:1), neat CH₂Cl₂, and CH₂Cl₂-EtOAc (9:1) in

succession as eluants. Di-tert-butyl N-prop-2-ynyl-^L-glutamate,

12 (major, more polar product), was obtained as an oil (1.77

g, 59%): ¹H-NMR (CDCl₃) δ 1.44, 1.48 (2 × s, 18H, C(CH₃)₃),

1.84 (m, 2H, â-CH₂), 2.19 (t, J ) 2.5 Hz, 1H, CtCH), 2.35 (t,

J ) 7.6 Hz, 2H, γ-CH₂), 3.27 (dd, J ) 7.7, 5.5 Hz, 1H, R-CH),

3.39 (m, 2H, CH₂-CtC); MS (EI, m/z) 298 (M + H)⁺.

7.33 (m, 5H, ArH); MS (CI, m/z) 408 (M + H)⁺. Anal. (C₂₂H₃₃

NO₆) C, H, N.

Di-ter t-bu tyl N-Meth yl-^L-glu ta m a te (8b). The general

procedure B was followed using di-tert-butyl N-(benzyloxycar-

bonyl)-N-methyl-^L-glutamate (1.6 g, 3.9 mmol), EtOAc (120

mL), and 10% Pd/C (0.22 g). Workup as described gave the

title compound 8b (1.04 g, 98%) as an oil: ¹H-NMR (DMSO-

d₆) δ 1.39, 1.42 (2 × s, 18H, C (CH₃)₃), 1.67 (m, 2H, â-CH₂),

2.19 (s, 3H, N-CH₃), 2.23 (m, 2H, γ-CH₂), 2.91 (dd, J ) 6.1,

7.8 Hz, 1H, R-CH); MS (CI, m/z) 274 (M + H)⁺.

N-(Ben zyloxyca r bon yl)-N-eth yl-^L-glu ta m ic Acid (7). A

stirred mixture of N-(benzyloxycarbonyl)-^L-glutamic acid (10

g, 35.5 mmol), p-toluenesulfonic acid monohydrate (0.7 g, 3.7

mmol), and benzene (700 mL) was heated under reflux in a

flask fitted with a Dean-Stark trap. After 80 min, paralde-

hyde (11 mL) was added to the mixture in portions during 20

min. Two further portions of paraldehyde (6.5 mL each) were

added after total times of 2.5 and 5 h. After a further 30 min

the mixture was cooled, decanted from separated tar, washed

with water (200 mL followed by 3 × 100 mL), dried (MgSO₄),

and evaporated. The residue was chromatographed on silica

gel using increasingly polar mixtures of CH₂Cl₂and EtOAc

(100:0 to 60:40) as eluants to give a mixture of two dia-

stereoisomeric products, 6, as an oil (2.17 g). TFA (6 mL) and

triethylsilane (3.3 mL, 20.5 mmol) were added to a solution of

6 (2.1 g, 6.8 mmol) in dry CHCl₃(6 mL), and the solution was

stirred under nitrogen at room temperature. Two further

portions of triethylsilane (1.1 mL each) were added after total

times of 16 and 40 h. After 64 h (total) the solution was

concentrated and the residual gum chromatographed on silica

gel with a gradient of MeOH-H₂O (95:5) in CH₂Cl₂(0-10%).

N-(Benzyloxycarbonyl)-N-ethyl-^L-glutamic acid, 7, was isolated

as a gum (1.825 g, 17%): ¹H-NMR (DMSO-d₆-D₂O) δ 1.09 (t,

J ) 6.8 Hz, 3H, CH₂CH₃), 1.98, 2.14 (2 × m, 2H, â-CH₂), 2.28

(m, 2H, γ-CH₂), 3.13, 3.35 (2 × m, 2H, CH₂CH₃), 4.24 (m, 1H,

R-CH), 5.11 (m, 2H, ArCH₂), 7.34 (m, 5H, ArH); MS (EI, m/z)

309 (M)⁺. Anal. (C₁₅H₁₉NO₆‚0.25H₂O) C, H, N.

P r ep a r a t ion of r-ter t-Bu t yl N-Tr it yl-4-m et h yl-^L-

glu ta m a te (42) a n d r-ter t-Bu tyl N-Tr ityl-4,4-d im eth yl-^L-

glu ta m a te (45): r-ter t-Bu tyl γ-Meth yl ^L-Glu ta m a te (39).

γ-Methyl ^L-glutamate hydrochloride (12.88 g, 0.08 mol), tert-

butyl acetate (500 mL), and 70% aqueous HClO₄(12.6 g, 0.08

mol) were stirred at room temperature for 88 h. The mixture

was then cooled in an ice bath and extracted with cooled 0.5

N HCl (3 × 130 mL). The combined aqueous extracts were

immediately neutralized with solid NaHCO₃and then ex-

tracted with Et₂O (3 × 200 mL). The ether extracts were

combined, dried (Na₂SO₄), and filtered, and the solution was

concentrated in vacuo to give R-tert-butyl γ-methyl ^L-glutamate

(14.5 g) as a colorless oil. This was immediately used in the

next experiment without further purification.

r-ter t-Bu tyl γ-Meth yl N-Tr ityl-^L-glu ta m a te (40). To a

stirred solution of R-tert-butyl γ-methyl ^L-glutamate (13.71 g,

63.0 mmol) in dry CHCl₃(50 mL) was added Et₃N (8.49 g, 84.0

mmol) followed by lead nitrate (8.34 g, 25 mmol) and then a

solution of trityl chloride (11.67 g, 42.0 mmol) in dry CHCl₃

(20 mL). After stirring at room temperature for 28 h a white

solid had formed. This was removed by filtration, and the

filtrate was concentrated in vacuo to an orange residue.

Purification by column chromatography, on gradient elution

with CH₂Cl₂in hexanes (10-100%), gave a semisolid product,

a mixture of the desired product 40 and triphenylmethanol.

This was treated with petrol; the white solid (triphenylmetha-

nol, 3.9 g) was filtered off, and the filtrate was concentrated

in vacuo to give the title compound 40 (9.46 g, 49%) as an oil,

which solidified on standing at -20 °C for 4 weeks: mp 79-

80 °C (hexanes); [R]²⁵) +23.22° (c ) 0.818, CHCl₃) (lit.¹¹[R]²⁰

) +22.5° (c ) 0.75, CHCl₃)); ¹H-NMR (DMSO-d₆) δ 1.13 (s,

9H, C(CH₃)₃), 1.80, 1.92 (2 × m, 2H, â-CH₂), 2.18, 2.44 (2 ×

m, 2H, γ-CH₂), 2.79 (d, J ) 9.0 Hz, 1H, NH), 3.17 (m, 1H,

Di-ter t-bu tyl N-(Ben zyloxyca r bon yl)-N-eth yl-^L-glu ta -

m a te (4c). A solution of 7 (2.878 g, 9.3 mmol) in CH₂Cl₂(74

mL), contained in a pressure bottle, was cooled in acetone-

dry ice, and concentrated H₂SO₄(0.44 mL) and liquid iso-

butylene (39 mL) were added. The mixture was shaken at

room temperature for 52 h, then cooled, and poured into

saturated aqueous NaHCO₃(150 mL). The products were

Folate-Based Inhibitors of TS

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1503

R-CH), 3.60 (s, 3H, OCH₃), 7.16-7.41 (m, 15H, TrH); MS (CI,

m/z) 460 (M + H)⁺. Anal. (C₂₉H₃₃NO₄) C, H, N.

then MeI (2.79 g, 1.22 mL, 19.68 mmol) was added under

argon. Stirring was continued at -78 °C for 3 h, then the

white slurry was poured into a saturated solution of NH₄Cl

(150 mL), and the mixture was extracted with Et₂O (2 × 200

mL). The organics were combined, dried (Na₂SO₄), and

concentrated in vacuo. Purification by column chromatogra-

phy, on gradient elution with EtOAc in hexanes (3-9%), gave

the desired product 44 contaminated with the starting mate-

rial 41 (NMR indicated a ratio of 2:5 monomethyl:dimethyl).

It was possible, however, to isolated the desired product 44

by recrystallization of the crude product from MeOH/hexanes

at -20 °C (the product was recrystallized three times from

MeOH/hexanes (10:4, v/v) and twice from MeOH/hexanes (4:

2, v/v)). The product was obtained as a white solid (0.850 g,

28% from 40: mp 110-113 °C; ¹H-NMR (DMSO-d₆) δ 1.91,

1.93 (2 × s, 6H, 2 × γ-CH₃), 1.20 (s, 9H, C(CH₃)₃), 1.30 (dd, J

) 3.8, 14.3 Hz, 1H, â-CH), 2.05 (dd, J ) 8.7, 14.2 Hz, 1H,

â-CH), 2.77 (d, J ) 8.9 Hz, 1H, NH), 3.07 (m, 1H, R-CH), 3.56

(s, 3H, CO₂CH₃), 7.15-7.39 (m, 15H, TrH); MS (CI, m/z) 386

(M - CO₂Bu^t)⁺, 243 (Tr⁺). Anal. (C₃₁H₃₇NO₄) C, H, N.

r-ter t-Bu tyl γ-Allyl N-Tr ityl-4-m eth yl-^L-glu ta m a te (47).

To a stirred solution of KN(SiMe₃)₂(23.68 mL, 11.84 mmol,

0.5 M in toluene) in dry THF (22 mL) at -78 °C was added a

solution of R-tert-butyl γ-allyl N-trityl-^L-glutamate (46) (3.59

g, 7.4 mmol) in dry THF (26 mL) over a 5 min period under

an argon atmosphere. The resulting pale yellow solution was

stirred at -78 °C for 55 min, and then MeI (2.10 g, 0.92 mL,

14.8 mmol) was added. Stirring was continued at -78 °C for

30 min, then the white slurry was poured into a saturated

solution of NH₄Cl (200 mL), and the resulting mixture was

extracted with Et₂O (3 × 150 mL). The ether extracts were

combined, dried (Na₂SO₄), and concentrated in vacuo. Puri-

fication by column chromatography, on gradient elution with

EtOAc in petrol (6-8%), gave the title compound 47 (3.26 g,

88%), a colorless oil, as a mixture of two diastereoisomers

(2S,4S and 2S,4R): ¹H-NMR (DMSO-d₆) δ 1.00, 1.07 (2 × d, J

) 7.0 Hz, 3H, γ-CH₃), 1.12, 1.15 (2 × s, 9H, C(CH₃)₃), 1.38,

1.57 (2 × m, 1H, â-CH), 1.95, 2.15 (2 × m, 1H, â-CH), 2.45 (m,

1H, γ-CH), 2.80 (t, J ) 9.4 Hz, 1H, NH), 3.12 (m, 1H, R-CH),

4.37, 4.48 [2 × dd, J ) 13.7, 5.4 Hz, 4.60 (d, J ) 5.3 Hz), 2H,

CH₂CHdCH₂], 5.10-5.35 (m, 2H, CHdCH₂), 5.70-6.00 (m,

1H, CHdCH₂), 7.16-7.40 (m, 15H, TrH); MS (CI, m/z) 500 (M

+ H)⁺. (C₃₂H₃₇NO₄‚0.5H₂O) C, H, N.

r-ter t-Bu t yl γ-Met h yl N-Tr it yl-4-m et h yl-^L-glu t a m a t e

(41). To a stirred solution of 0.6 M KN(SiMe₃)₂in THF (13.85

mL, 8.25 mmol) at -78 °C was added a solution of R-tert-butyl

γ-methyl N-trityl-^L-glutamate (2.52 g, 5.5 mmol) in dry THF

(25 mL) over a 5 min period under argon. The resulting

yellowish solution was stirred at -78 °C for 50 min under

argon, and then MeI (1.56 g, 0.68 mL, 11.0 mmol) was added

under argon. Stirring was continued at -78 °C for 1 h and

15 min, and then the whitish slurry was poured into a

saturated solution of NH₄Cl (200 mL). The mixture was

extracted with Et₂O (2 × 200 mL), and the ether extracts were

combined, dried (Na₂SO₄), and concentrated in vacuo. Puri-

fication by column chromatography on gradient elution with

EtOAc in hexanes (3-9%) gave R-tert-butyl γ-methyl N-trityl-

4-methyl-^L-glutamate (1.62 g, 62%), a viscous oil, as a mixture

of two diastereoisomers (2S,4S and 2S,4R): ¹H-NMR (DMSO-

d₆) δ 0.98, 1.04 (2 × d, J ) 7.0 Hz, 3H, γ-CH₃), 1.12, 1.14 (2 ×

s, 9H, C(CH₃)₃), 1.38, 1.54 (2 × m, 1H, â-CH), 1.93, 2.10 (2 ×

m, 1H, â-CH), 2.40 (m, 1H, γ-CH), 2.75, 2.80 (2 × d, J ) 8.9,

9.4 Hz, 1H, NH), 3.10 (m, 1H, R-CH), 3.50, 3.66 (2 × s, 3H,

CO₂CH₃), 7.16-7.39 (m, 15H, TrH), a singlet at 3.55 ppm

indicated that about 3% of 44 was present; MS (CI, m/z) 474

(M + H)⁺. Anal. (C₃₀H₃₅NO₄) C, H, N.

r-ter t-Bu tyl N-Tr ityl-4-m eth yl-^L-glu ta m a te (42). To a

mixture of lithium hydroxide monohydrate (0.117 g, 2.79

mmol) and H₂O (1.86 mL) was added a solution of R-tert-butyl

γ-methyl N-trityl-4-methyl-^L-glutamate (1.1 g, 2.33 mmol) in

THF (27 mL). The resulting mixture was refluxed for 3 h,

then more H₂O (0.6 mL) was added, and refluxing was

continued for 5 h. After that the organic solvent was removed

by evaporation, the residue was treated with a half-saturated

NaHCO₃solution (100 mL), and the resulting mixture was

acidified to pH 6 with 1 N HCl. The mixture was extracted

with EtOAc (2 × 100 mL); the extracts were combined, dried

(Na₂SO₄), and concentrated in vacuo. The residue was dried

in vacuo over P₂O₅to give R-tert-butyl N-trityl-4-methyl-L-

glutamate (1.06 g) as a white foam contaminated with ap-

proximately 10% of the starting material. This was taken

forward in the next step without further purification.

γ-Allyl ester protection provides a simpler and most efficient

route to 42: To a stirred solution of 47 (0.244 g, 0.49 mmol)

in anhydrous CH₂Cl₂(2.5 mL) under argon was added Pd-

(PPh₃)₄(0.020 g) followed by pyrrolidine (0.09 mL). The

resulting yellow solution was stirred at room temperature

under argon for 4 h; then more catalyst (0.010 g) was added,

and stirring was continued at room temperature overnight

before the mixture was partitioned between Et₂O (50 mL) and

0.5 N HCl (40 mL). The aqueous layer was extracted with

more Et₂O (2 × 50 mL), and the combined extracts were dried

(Na₂SO₄) and concentrated in vacuo. The residue was treated

with Et₂O (10 mL), the insoluble material was filtered off, and

the filtrate was concentrated in vacuo. The product was

further purified by column chromatography on elution with

3% MeOH in EtOAc and then dried in vacuo over P₂O₅. The

title compound 42 was obtained as a white, solid foam (0.152

r-ter t-Bu tyl γ-Allyl N-Tr ityl-4,4-d im eth yl-^L-glu ta m a te

(48). To a stirred solution of KN(SiMe₃)₂(16.2 mL, 0.5 M in

toluene, 8.1 mmol) in dry THF (16 mL) at -78 °C was added

a solution of R-tert-butyl γ-allyl N-trityl-4-methyl-L-glutamate

(47) (2.24 g, 4.5 mmol) in dry THF (16 mL) over a 5 min period

under argon. The resulting yellow solution was stirred at -78

°C for 55 min, and then MeI (1.12 mL, 2.56 g, 18 mmol) was

added. Stirring was continued at -78 °C for 2 h, and then

the yellowish slurry was poured into a saturated solution of

NH₄

Cl (200 mL). This was then extracted with Et₂O (3 × 150

mL); the ether extracts were combined, dried (Na₂

SO₄), and

concentrated in vacuo. Purification by column chromatogra-

phy, on gradient elution with EtOAc in petrol (3-6%), gave

the title compound 48 (2.08 g, 90%) as a colorless oil: ¹H-NMR

(DMSO-d₆

) δ 0.94, 0.96 (2 × s, 6H, 2 × γ-CH₃), 1.20 (s, 9H,

g, 68%): mp 62-65 °C (softens); H-NMR (DMSO-d₆) δ 0.95,

1.04 (2 × d, J ) 7.0 Hz, 3H, γ-CH₃), 1.11, 1.14 (2 × s, 9H,

C(CH₃)₃), 1.30, 1.50 (2 × m, 1H, â-CH), 1.90-2.15, 2.30 (2 ×

m, 2H, â-CH, γ-CH), 2.76 (t, J ) 9.2 Hz, 1H, NH), 3.10 (m,

1H, R-CH), 7.15-7.41 (m, 15H, TrH); MS (FAB, m/z) 482 (M

+ Na)⁺, 460 (M + H)⁺.

C(CH₃)₃), 1.34 (dd, J ) 14.2, 3.7 Hz, 1H, â-CH), 2.06 (dd, J )

14.3, 8.6 Hz, 1H, â-CH), 2.78 (d, J ) 8.9 Hz, 1H, NH), 3.09

(m, 1H, R-CH), 4.41, 4.59 (2 × ddd, J ) 15.0, 5.0, 1.2 Hz, 2H,

CH₂

CHdCH₂), 5.17 (dm, J ) 11.6 Hz, 1H, CHdCH₂cis), 5.23

(dm, J ) 17.3 Hz, 1H, CHdCH₂trans), 5.87 (m, 1H, CHdCH₂),

7.16-7.40 (m, 15H, TrH), a doublet at 1.07 ppm indicated that

about 4% of 47 was present; MS (CI, m/z) 514 (M + H)⁺. Anal.

(C₃₃H₃₉NO₄‚0.5H₂O) C, H, N.

r-ter t-Bu tyl γ-Meth yl N-Tr ityl-4,4-dim eth yl-^L-glu tam ate

(44). To a stirred solution of 0.6 M KN(SiMe₃)₂in THF (18.10

mL, 10.85 mmol) at -78 °C was added a solution of 40 (2.84

g, 6.19 mmol) in dry THF (28 mL) over a 5 min period under

argon. The resulting solution was stirred at -78 °C for 50

min, then MeI (1.76 g, 12.38 mmol) was added, and stirring

was continued at -78 °C for 1 h and 20 min. Workup and

purification as described above afforded the γ-methyl glutamate

derivative 41 as a colorless viscous oil (2.46 g, 84%). To a

stirred solution of 0.6 M KN(SiMe₃)₂in THF (16.0 mL, 9.60

mmol) at -78 °C was added a solution of 41 (2.33 g, 4.92 mmol)

in dry THF (20 mL) over a 5 min period and under argon. The

resultant yellow solution was stirred at -78 °C for 50 min;

r-ter t-Bu tyl N-Tr ityl-4,4-d im eth yl-^L-glu ta m a te (45). To

a stirred solution of R-tert-butyl γ-allyl N-trityl-4,4-dimethyl-

L-glutamate (1.4 g, 2.72 mmol) in dry dichloromethane (11 mL)

under argon was added tetrakis(triphenylphosphine)palladium-

(0) (0.091 g) followed by pyrrolidine (0.35 mL, excess). The

resulting yellow solution was stirred at room temperature for

20 min, and then Et₂O (100 mL) and 1 N HCl (50 mL) were

added. The two layers were separated, and the aqueous layer

was washed with more Et₂O (100 mL). The ether extracts

were combined, dried (Na₂SO₄), and concentrated in vacuo. The

1504 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10

Bavetsias et al.

residue was treated with diethyl ether (30 mL); the yellow

precipitate was filtered off, and the filtrate was concentrated

in vacuo yielding the title compound 45 (1.25 g, 97%) as a white

solid foam: mp 66-68 °C; ¹H-NMR (DMSO-d₆) δ 0.88, 0.90 (2

× s, 6H, 2 × γ-CH₃), 1.20 (m, 10H, â-CH, C(CH₃)₃), 2.01 (dd,

J ) 14.2, 8.5 Hz, 1H, â-CH), 2.76 (d, J ) 8.4 Hz, 1H, NH),

3.06 (m, 1H, R-CH), 7.17-7.48 (m, 15H, TrH); MS (CI, m/z)

474 (M + H)⁺.

P r ep a r a tion of Z-, Tr -, or Boc-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]-N-m eth ylglycin a te (9a ). To a stirred solution

of R-tert-butyl N-(benzyloxycarbonyl)-^L-glutamate (1.63 g, 4.8

mmol) and NMM (0.487 g, 4.83 mmol) in dry THF (8 mL)

cooled to -20 °C was added isobutyl chloroformate (0.657 g,

4.83 mmol). After 10 min a solution of tert-butyl N-methylg-

lycinate (0.70 g, 4.83 mmol) in THF (8 mL) was added.

Stirring was continued for 10 min at -20 °C and then at room

temperature for 4 h. N-Methylmorpholine hydrochloride was

filtered off and the filtrate concentrated in vacuo. The residue

was purified by column chromatography, on elution with a

gradient of EtOAc in CH₂Cl₂(5-25%) affording the title

compound 9a (1.73 g, 77%) as a yellow oil: ¹H-NMR (DMSO-

d₆) δ 1.32, 1.38, 1.39, 1.41 (4 × s, 18H, 2 × C(CH₃)₃), 1.77,

1.87 (2 × m, 2H, â-CH₂), 2.24, 2.38 (2 × m, 2H, γ-CH₂), 2.79,

2.95 (2 × s, 3H, N-CH₃), 3.93(m) and 4.08(s) (3H, CH₂CO₂Bu^t,

Glu R-CH), 5.03 (m, 2H, ArCH₂), 7.36 (m, 5H, ArH), 7.64 (t, J

) 7.9 Hz, 1H, NH); MS (CI, m/z) 465 (M + H)⁺. Anal.

(C₂₄H₃₆N₂O₇‚0.25H₂O) C, H, N.

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

N-m eth yl-^L-glu ta m a te (9b). The general procedure A2 was

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

(1.31 g, 3.67 mmol), NMM (0.37 g, 3.67 mmol), dry THF (5

mL), isobutyl chloroformate (0.50 g, 3.67 mmol), and a solution

of di-tert-butyl N-methyl-^L-glutamate (1.00 g, 3.67 mmol) in

dry THF (5 mL). The reaction mixture was stirred at -20 °C

for 10 min and then at room temperature for 4 h. The crude

product was purified by column chromatography using a

gradient of EtOAc in CH₂Cl₂(5-20%) as eluant. Trituration

with Et₂O gave a white solid, the title compound 9b. This was

isolated by filtration, washed with petrol, and dried in vacuo

(1.62 g, 74%): mp 92-93 °C; ¹H-NMR (DMSO-d₆) δ 1.32, 1.37,

1.38 (3 × s, 27H, 3 × C(CH₃)₃), 1.86, 2.03 (2 × m, 4H, 2 ×

â-CH₂), 2.12, 2.38 (2 × m, 4H, 2 × γ-CH₂), 2.60, 2.79 (2 × s,

3H, N-CH₃), 3.94 (m, 1H, Glu R-CH), 4.49, 4.76 (2 × dd, J )

4.8, 10.8 Hz, 1H, MeGlu R-CH), 5.03 (m, 2H, PhCH₂), 7.35 (s,

5H, ArH), 7.64 (t, J ) 7.7 Hz, 1H, NH); MS (CI, m/z) 593 (M

+ H)⁺. Anal. (C₃₁H₄₈N₂O₉) C, H, N.

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

N-eth yl-^L-glu ta m a te (9c). Isobutyl chloroformate (0.26 mL,

2 mmol) was added to a stirred solution of R-tert-butyl

N-(benzyloxycarbonyl)-^L-glutamate (0.675 g, 2.0 mmol) and

NMM (0.22 mL, 2.0 mmol) in THF (2 mL) at -20 °C under

argon. After 10 min a solution of di-tert-butyl N-ethyl-^L-

glutamate (0.575 g, 2 mmol) in THF (3 mL) was added. After

a further 10 min at -20 °C the mixture was allowed to come

to room temperature, and stirring was continued at this

temperature for 20 h. The reaction mixture was then parti-

tioned between CH₂Cl₂(40 mL) and saturated aqueous NaH-

CO₃(30 mL). The aqueous layer was extracted with CH₂Cl₂

(3 × 10 mL), and the combined organic solution was washed

successively with saturated aqueous NaHCO₃(20 mL) and

H₂O (3 × 20 mL), dried (MgSO₄), and evaporated. The residue

was chromatographed on silica gel using hexanes-CH₂Cl₂

(1:1, 1:2, and 1:3), neat CH₂Cl₂, and CH₂Cl₂-EtOAc (19:1, 9:1,

4:1, and 3:1) in succession as eluants. The title compound 9c

was isolated as a colorless gum (1.023 g, 84%): ¹H-NMR

(DMSO-d₆, 383 K) δ 1.14 (t, J ) 7.1 Hz, 3H, CH₂CH₃), 1.41,

1.42, 1.43 (3 × s, 27H, 3 × C(CH₃)₃), 2.01 (m, 4H, 2 × â-CH₂),

2.23, 2.41 (2 × m, 4H, 2 × γ-CH₂), 3.19, 3.40 (2 × m, 2H, CH₂-

CH₃), 4.02 (m, 1H, R-CH), 4.21 (m, 1H, R-CH), 5.06 (s, 2H,

PhCH₂), 6.98 (d, J ) 7.7 Hz, 1H, NH), 7.34 (m, 5H, ArH); MS

(FAB, m/z) 607 (M + H)⁺. Anal. (C₃₂H₅₀N₂O₉) C, H, N.

Tr i-ter t-bu tyl N-[N-(ter t-Bu tyloxycar bon yl)-^L-γ-glu tam -

yl]-N-p r op -2-yn yl-^L-glu ta m a te (13). Isobutyl chloroformate

(1.35 mL, 10.4 mmol) was added to a stirred solution of R-tert-

butyl N-(tert-butyloxycarbonyl)-^L-glutamate (3.2 g, 10.4 mmol)

and NMM (1.15 mL, 10.4 mmol) in THF (13 mL) at -20 °C

under argon. After 10 min a solution of di-tert-butyl N-prop-

2-ynyl-^L-glutamate (12) (3.089 g, 10.4 mmol) in THF (18 mL)

was added. After a further 10 min at -20 °C the mixture was

allowed to warm to ambient temperature. Stirring was

continued at this temperature for 27 h, and then the reaction

mixture was partitioned between CH₂Cl₂(200 mL) and satu-

rated aqueous NaHCO₃(150 mL). The aqueous layer was

extracted with CH₂Cl₂(3 × 50 mL), and the combined CH₂Cl₂

solution was washed successively with saturated aqueous

NaHCO₃(100 mL) and H₂O (3 × 50 mL), dried (MgSO₄), and

evaporated. The residue was chromatographed on silica gel

using petrol-CH₂Cl₂(1:1 and 1:2 in succession) followed by

CH₂Cl₂-EtOAc (100:0 to 85:15) as eluants. The title com-

pound 13 was isolated as a gum and crystallized from hexanes

(4.104 g, 68%): mp 111-112 °C; H-NMR (DMSO-d₆) δ 1.38,

1.39, 1.40 (3 × s, 36H, 4 × C(CH₃)₃), 1.93, 2.07, 2.23, 2.33 (4

× m, 8H, 2 × â-CH₂, 2 × γ-CH₂), 3.02 (m, 1H, CtCH), 3.84

(m, 1H, R-CH), 4.12 (m, 2H, CH₂-CtC), 4.52 (m, 1H, R-CH),

7.07 (m, 1H, NH); MS (FAB, m/z) 583 (M + H)⁺. Anal.

(C₃₀H₅₀N₂O₉) C, H, N.

ter t-Bu tyl r-Meth yla la n in a te (55). R-Aminoisobutyric

acid (6.18 g, 60.0 mmol), tert-butyl acetate (200 mL), and 70%

aqueous HClO₄(9.45 g, 66.0 mmol) were shaken at room

temperature for 7 days in a 500 mL conical flask. The mixture

was then cooled in an ice-water bath and extracted with cold

0.5 N HCl (4 × 50 mL). The combined extracts were washed

with EtOAc (100 mL) and then immediately neutralized with

solid NaHCO₃while cooling in an ice bath. The neutralized

solution was extracted with Et₂O (3 × 200 mL); the extracts

were combined, dried (Na₂SO₄), and concentrated in vacuo

(take care, the product is volatile). The product was obtained

as a colorless oil 4.98 g (52%): ¹H-NMR (DMSO-d₆) δ 1.16 (s,

6H, C(CH₃)₂), 1.40 (s, 9H, C(CH₃)₃); MS (CI, m/z) 160 (M +

H)⁺.

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

r-m eth yla la n in a te (57a ). The general procedure A2 was

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

(3.033 g, 9.0 mmol), NMM (0.909 g, 9.0 mmol), dry THF (10

mL), isobutyl chloroformate (1.224 g, 9.0 mmol), and a solution

of tert-butyl R-methylalaninate (55) (2.0 g, 12.5 mmol) in dry

THF (5 mL). The reaction mixture was stirred at -20 °C for

10 min and then at room temperature for 1 h. The crude

product was purified by column chromatography using 2%

MeOH in CH₂Cl₂as eluant. Reprecipitation from Et₂O/petrol

gave a white solid, the title compound 57a . This was isolated

by filtration, washed with petrol, and dried in vacuo (3.86 g,

90%): mp 109-110 °C; ¹H-NMR (DMSO-d₆) δ 1.27 (s, 6H,

C(CH₃)₂), 1.34, 1.39 (2 × s, 18H, 2 × C(CH₃)₃), 1.73, 1.90 (2 ×

m, 2H, â-CH₂), 2.13 (t, J ) 7.3 Hz, 2H, 2 × γ-CH₂), 3.90 (m,

1H, Glu R-CH), 5.03 (m, 2H, PhCH₂), 7.36 (m, 5H, ArH), 7.64

(d, J ) 7.5 Hz, 1H, Glu NH), 8.06 (s, 1H, R-methylalanine NH);

MS (FAB, m/z) 501 (M + Na)⁺. Anal. (C₂₅H₃₈N₂O₇) C, H, N.

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

L-p r olin a te (57b). The general procedure A2 was followed

using R-tert-butyl N-(benzyloxycarbonyl)-^L-glutamate (3.15 g,

9.3 mmol), NMM (0.909 g, 9.0 mmol), dry THF (10 mL),

isobutyl chloroformate (1.22 g, 9.0 mmol), and a solution of

tert-butyl ^L-prolinate (1.54 g, 9.0 mmol) in dry THF (10 mL).

The reaction mixture was stirred at -20 °C for 10 min and

then at room temperature for 4 h. The crude product was

purified by column chromatography using a gradient of EtOAc

in CH₂Cl₂(10-30%) as eluant. Trituration with petrol gave

a white solid, the title compound 57b. This was isolated by

filtration, washed with petrol, and dried in vacuo (2.86 g,

65%): mp 106-108 °C; ¹H-NMR (DMSO-d₆) δ 1.37, 1.39 (2 ×

s, 18H, 2 × C(CH₃)₃), 1.62-2.10 (m, 6H, Glu â-CH₂, Pro 3-

and 4-CH₂), 2.33 (m, 2H, Glu γ-CH₂), 3.42 (m, 2H, Pro 5-CH₂),

3.93 (m, 1H, Glu R-CH), 4.13, 4.40 (2 × dd, J ) 8.8, 3.9 Hz,

1H, Pro 2-CH), 5.03 (m, 2H, PhCH₂), 7.35 (m, 5H, ArH), 7.59

(t, 1H, Glu NH); MS (CI, m/z) 491 (M + H)⁺. Anal.

(C₂₆H₃₈N₂O₇) C, H, N.

Tr i-ter t-b u t yl N-(N-Tr it yl-γ-4-m et h yl-^L-glu t a m yl)-L-

glu ta m a te (43). To a stirred solution of R-tert-butyl N-trityl-

4-methyl-^L-glutamate (42) (0.760 g, purity 90%, approximately

1.50 mmol) and NMM (0.151 g, 1.50 mmol) in dry THF (3.5

Folate-Based Inhibitors of TS

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1505

mL) cooled to -20 °C was added isobutyl chloroformate (0.204

g, 1.50 mmol). After 10 min a suspension of di-tert-butyl

L-glutamate hydrochloride (0.442 g, 1.50 mmol) in dry THF

(4.5 mL) and NMM (0.151 g, 1.50 mmol) was added. Stirring

was continued at -20 °C for 10 min and then for 1.5 h while

the mixture was allowed to warm up to room temperature.

The N-methylmorpholine hydrochloride was filtered off, and

the filtrate was concentrated in vacuo. Purification by column

chromatography on gradient elution with EtOAc in petrol (7-

20%) gave in order of elution: (a) diastereoisomer with R_f)

0.33 (20% EtOAc in hexanes) as a gummy solid (0.100 g); ¹H-

NMR (DMSO-d₆) δ 0.96 (d, J ) 6.8 Hz, 3H, 4-MeGlu_Lγ-CH₃),

1.10 (s, 9H, 4-MeGlu_LC(CH₃)₃), 1.34, 1.40 (2 × s, 18H, Glu_L

C(CH₃)₃), 1.60-2.15 (m, 4H, 4-MeGlu_Lâ-CH₂, Glu_Lâ-CH₂),

2.21 (t, J ) 7.8 Hz, 2H, Glu_Lγ-CH₂), 2.53 (m, 1H, 4-MeGlu_Lγ-

CH), 2.77 (d, J ) 9.6 Hz, 1H, 4-MeGlu_LNH), 3.07 (m, 1H,

4-MeGlu_LR-CH), 4.09 (m, 1H, Glu_LR-CH), 7.14-7.40 (m, 15H,

TrH), 7.92 (d, J ) 7.9 Hz, 1H, Glu_LNH), peaks at 0.89 (d),

temperature for 3 h. Standard workup afforded the title

compound 10b (0.61 g, 99%) as an oil: ¹H-NMR (DMSO-d₆) δ

1.38, 1.39, 1.41, 1.42 (4 × s, 27H, C(CH₃)₃), 1.79, 2.05 (2 × m,

4H, 2 × â-CH₂), 2.12, 2.39 (2 × m, 4H, 2 × γ-CH₂), 2.60, 2.84

(2 × s, 3H, N-CH₃), 3.18 (dd, J ) 8.3, 4.9 Hz, 1H, Glu R-CH),

4.53, 4.78 (2 × dd, J ) 10.5, 4.6 Hz, 1H, MeGlu R-CH); MS

(CI, m/z) 459 (M + H)⁺.

Tr i-ter t-bu tyl ^L-γ-Glu ta m yl-N-eth yl-L-glu ta m a te (10c).

The general procedure B was followed using 9c (0.826 g, 1.36

mmol), EtOAc (80 mL), and 10% Pd/C (0.12 g). The reaction

mixture was stirred at room temperature for 4 h. After

standard workup the product was chromatographed on silica

gel using CH₂Cl₂-hexanes, neat CH₂Cl₂, and CH₂Cl₂-EtOAc

(95:5 to 50:50) in succession as eluants. The title compound

10c was isolated as an oil (0.600 g, 93%): ¹H-NMR (DMSO-

d₆, 383 K) δ 1.16 (m, 3H, CH₂CH₃), 1.42, 1.43, 1.45 (3 × s,

27H, 3 × C(CH₃)₃), 1.64, 1.69, 1.91, 2.23, 2.40 (5 × m, 8H, 2 ×

â-CH₂, 2 × γ-CH₂), 2.84 (m, 1H, R-CH), 3.25, 3.39 (2 × m, CH₂-

CH₃), 4.21 (m, 1H, R-CH); MS (FAB, m/z) 473 (M + H)⁺.

3.55 (t), and 3.79 (d) not assigned for, clean H-NMR obtained

when the reaction was repeated using a sample of 42 obtained

through the γ-allyl route (Scheme 4); MS (CI, m/z) 701 (M +

H)⁺, 243 (Tr⁺); and (b) diastereoisomer with R_f) 0.28 (20%

EtOAc in hexanes) as a viscous colorless oil, which solidified

on drying in vacuo over P₂O₅(0.668, 64%), mp 58-66 °C

(softens); ¹H-NMR (DMSO-d₆) δ 0.94 (d, J ) 6.8 Hz, 3H,

4-MeGlu_Lγ-CH₃), 1.14 (s, 9H, 4-MeGlu_LC(CH₃)₃), 1.39 (m,

19H, Glu_LC(CH₃)₃, 4-MeGlu_Lâ-CH), 1.85, 2.11 (2 × m, 3H,

4-MeGlu_Lâ-CH, Glu_Lâ-CH₂), 2.27 (t, J ) 7.6 Hz, 2H, Glu_L

γ-CH₂), 2.38 (m, 1H, 4-MeGlu_Lγ-CH), 2.68 (d, J ) 9.0 Hz, 1H,

4-MeGlu_LNH), 3.04 (m, 1H, 4-MeGlu_LR-CH), 4.13 (m, 1H,

Glu_LR-CH), 7.14-7.41 (m, 15H, TrH), 8.14 (d, J ) 7.4 Hz,

1H, Glu_LNH); MS (CI, m/z) 701 (M + H)⁺. Anal. (C₄₂H₅₆N₂O₇)

C, H, N.

Tr i-ter t-bu tyl N-(N-Tr ityl-γ-4,4-d im eth yl-^L-glu ta m yl)-

L-glu ta m a te (50). To a stirred solution of R-tert-butyl N-tri-

tyl-4,4-dimethyl-^L-glutamate (0.710 g, 1.50 mmol) and PyBOP

(0.935 g, 1.80 mmol) in dry CH₂Cl₂(1.9 mL) was added DIEA

(0.464 g, 3.6 mmol) followed by DMAP (0.109 g, 0.9 mmol) and

di-tert-butyl ^L-glutamate hydrochloride (0.531 g, 1.8 mmol).

Stirring was continued at room temperature for 3 h, and then

the solvent was removed by evaporation. Purification by

column chromatography, on gradient elution with EtOAc in

hexanes (5-20%), gave the following in order of elution.

Title com p ou n d 50: 0.445 g, 42%, viscous oil; ¹H-NMR

(DMSO-d₆) δ 0.80, 0.95 (2 × s, 6H, 2 × γ-CH₃), 1.19 (s, 9H,

4-diMeGlu_LC(CH₃)₃), 1.34 (s, 9H, Glu_LC(CH₃)₃), 1.39 (m, 10H,

Glu_LC(CH₃)₃, 4-diMeGlu_Lâ-CH), 1.78-1.98 (m, 3H, Glu_L

â-CH₂, 4-diMeGlu_Lâ-CH), 2.22 (t, J ) 7.2 Hz, 2H, Glu_Lγ-CH₂),

2.80 (d, J ) 8.2 Hz, 1H, 4-diMeGlu_LNH), 2.98 (m, 1H,

4-diMeGlu_LR-CH), 4.04 (m, 1H, Glu_LR-CH), 7.14-7.40 (m,

15H, TrH); MS (CI, m/z) 715 (M + H)⁺. Anal. (C₄₃H₅₈N₂O₇‚

0.3H₂O) C, H, N.

r-ter t-Bu tyl N-tr itylp yr oglu ta m a te (49): 0.205 g, 30%,

white solid; mp 69-71 °C (softens); ¹H-NMR (DMSO-d₆) δ 0.96,

1.16 (2 × s, 6H, 2 × γ-CH₃), 1.27 (s, 9H, C(CH₃)₃), 1.86 (d, J )

13.5 Hz, 1H, â-CH), 2.50 (t, J ) 12.9 Hz, 1H, â-CH), 4.06 (d,

J ) 10.7 Hz, 1H, R-CH), 7.15-7.31 (m, 15H, TrH); MS (CI,

m/z) 456 (M + H)⁺. Anal. (C₃₀H₃₃NO₃) C, H, N.

P r ep a r a tion of F r ee Ba se Dip ep tid e ter t-Bu tyl Ester s:

Di-ter t-bu tyl ^L-γ-Glu ta m yl-N-m eth ylglycin a te (10a ). The

general procedure B was followed using di-tert-butyl N-[N-

(benzyloxycarbonyl)-^L-γ-glutamyl]-N-methylglycinate (9a) (0.80

g, 1.72 mmol), EtOAc (90 mL), and 10% Pd/C (0.150 g). The

reaction mixture was stirred at room temperature for 6 h. The

catalyst was removed by filtration and the filtrate concentrated

in vacuo, yielding di-tert-butyl ^L-γ-glutamyl-N-methylglycinate

(0.53 g, 95%) as a yellow oil: ¹H-NMR (DMSO-d₆) δ 1.40, 1.41,

1.43 (3 × s, 18H, 2 × C(CH₃)₃), 1.59, 1.77 (2 × m, 2H, â-CH₂),

2.24, 2.40 (2 × m, 2H, γ-CH₂), 2.79, 2.99 (2 × s, 3H, N-CH₃),

3.17 (m, 1H, R-CH), 4.02(s) and 4.10(d) (2H, CH₂CO₂Bu^t); MS

(EI, m/z) 331 (M + H)⁺.

Tr i-ter t-bu tyl ^L-γ-Glu ta m yl-N-p r op -2-yn yl-L-glu ta m a te

(10d ). TFA (17 mL) was added to a stirred solution of 13 (1.70

g, 2.9 mmol) in CH₂Cl₂(68 mL) at 20 °C. After 10 min the

reaction mixture was poured into vigorously stirred, ice-cooled

saturated aqueous NaHCO₃(500 mL). After shaking the CH₂-

Cl₂layer was separated, and the aqueous layer was extracted

with CH₂Cl₂(2 × 25 mL). The combined CH₂Cl₂solution was

washed with H₂O (2 × 100 mL), dried (MgSO₄), and evapo-

rated. The residue was chromatographed using CH₂Cl₂-

EtOAc (100:0 to 33:67) as eluant. The title compound 10d was

isolated as an oil (0.707 g, 50%): ¹H-NMR (CDCl₃-D₂O) δ 1.44,

1.46, 1.47 (3 × s, 27H, 3 × C(CH₃)₃), 1.85, 2.09, 2.33, 2.51,

2.66 (5 × m, 8H, 2 × â-CH₂, 2 × γ-CH₂), 3.33 (m, 1H, CtCH),

3.71-4.44 (8 × d, 3H, R-CH, CH₂-CtC) 4.59, 5.00 (2 × m, 1H,

R-CH); MS (FAB, m/z) 483 (M + H)⁺.

Di-ter t-bu tyl ^L-γ-Glu tam yl-r-m eth ylalan in ate (58a). The

general procedure B was followed using 57a (0.223 g, 0.47

mmol), EtOAc (30 mL), and 10% Pd/C (0.062 g). The reaction

mixture was stirred at room temperature for 2.5 h. Standard

workup afforded 58a (0.158 g, 98%) as a colorless oil: ¹H-NMR

(DMSO-d₆) δ 1.27 (s, 6H, C(CH₃)₂), 1.34, 1.40 (2 × s, 18H, 2 ×

C(CH₃)₃), 1.50, 1.80 (2 × m, 2H, â-CH₂), 2.12 (t, J ) 7.8 Hz,

2H, γ-CH₂), 3.13 (dd, J ) 4.9, 8.5 Hz, 1H, Glu R-CH); MS (LDI,

m/z) 368 (M + Na)⁺.

Di-ter t-bu tyl ^L-γ-Glu ta m yl-L-p r olin a te (58b). The gen-

eral procedure B was followed using 57b (0.98 g, 2.0 mmol),

EtOAc (50 mL), and 10% Pd/C (0.15 g). The reaction mixture

was stirred at room temperature for 8 h. Standard workup

afforded 58b (0.70 g, 98%) as an oil: ¹H-NMR (DMSO-d₆) δ

1.37, 1.40 (2 × s, 18H, 2 × C(CH₃)₃), 1.55-2.10 (m, 6H, Glu

â-CH₂, Pro 3- and 4-CH₂), 2.33 (t, J ) 7.5 Hz, 2H, Glu γ-CH₂),

3.16 (m, 1H, Glu R-CH), 3.48 (t, J ) 6.7 Hz, 2H, Pro 5-CH₂),

4.14, 4.40 (2 × dd, J ) 8.8, 3.9 Hz, 1H, Pro 2-CH); MS (CI,

m/z) 357 (M + H)⁺.

Tr i-ter t-bu tyl γ-4-Meth yl-^L-glu ta m yl-L-glu ta m a te (37a ).

To a solution of tri-tert-butyl N-(N-trityl-γ-4-methyl-^L-glutamyl)-

L-glutamate (0.608 g, 0.87 mmol) in EtOAc (60 mL) was added

10% Pd/C (0.160 g). The resulting mixture was degassed and

then stirred at room temperature for 26 h under a hydrogen

atmosphere. More catalyst (0.035 g) was then added, and

stirring was continued under hydrogen for 20 h. The catalyst

was removed by filtration, and the filtrate was concentrated

in vacuo to give a white semisolid, a mixture of triphenyl-

methane and tri-tert-butyl γ-4-methyl-^L-glutamyl-L-glutamate

(0.450 g). This was taken forward to the next step without

further purification.

Tr i-ter t-b u t yl γ-4,4-Dim et h yl-^L-glu t a m yl-L-glu t a m a t e

(37b). To a solution of 50 (0.430 g, 0.60 mmol) in EtOAc (50

mL) was added 10% Pd/C (0.065 g), and the resulting mixture

was stirred under hydrogen for 26 h. Then more catalyst

(0.075 g) was added, and stirring was continued under

hydrogen for 16 h. The catalyst was removed by filtration,

and the filtrate was concentrated in vacuo to give a white

semisolid (0.400 g), a mixture of triphenylmethane and tri-

tert-butyl γ-4,4-dimethyl-^L-glutamyl-L-glutamate. This was

taken forward to the next step without further purification.

Tr i-ter t-bu tyl ^L-γ-Glu tam yl-N-m eth yl-L-glu tam ate (10b).

The general procedure B was followed using tri-tert-butyl

N -[N -(ben zyloxyca r bon yl)-^L-γ-glu t a m yl]-N -m et h yl-L -

glutamate (9b) (0.80 g, 1.35 mmol), EtOAc (80 mL), and 10%

Pd/C (0.11 g). The reaction mixture was stirred at room

1506 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10

Bavetsias et al.

P r ep a r a tion of Tr i-ter t-bu tyl N-(N-Tr ityl-γ-4-m eth yl-

D-glu t a m yl)-L-glu t a m a t e (54): r-ter t-Bu t yl γ-Allyl N-

Tr ityl-4-m eth yl-^D-glu ta m a te (52). The method followed

that used to prepare 47 but used a solution of potassium

hexamethyldisilazide (4.1 mL, 2.05 mmol, 0.5 M in toluene)

in dry THF (3.0 mL), a solution of R-tert-butyl γ-allyl N-trityl-

D-glutamate¹⁵(51) (0.62 g, 1.28 mmol) in dry THF (5.0 mL),

and MeI (0.363 g, 0.16 mL, 2.56 mmol). Purification by column

chromatography (twice), on gradient elution with ethyl acetate

in hexanes (1-6%), gave the title compound 52 (0.340, 53%),

a colorless oil, as a mixture of two diastereoisomers (2R,4S

and 2R,4R): ¹H-NMR (DMSO-d₆) δ 1.00, 1.06 (2 × d, J ) 7.0

Hz, 3H, γ-CH₃), 1.12, 1.15 (2 × s, 9H, C(CH₃)₃), 1.37, 1.56, 1.99,

2.14 (4 × m, 2H, â-CH₂), 2.44 (m, 1H, γ-CH), 2.80 (t, J ) 9.4

Hz, 1H, NH), 3.12 (m, 1H, R-CH), 4.37, 4.47 [2 × ddd, J ) 1.4,

5.4, 12.3 Hz, 4.60 (dd, J ) 1.3, 5.3 Hz), 2H, CH₂CHdCH₂],

5.10-5.35 (m, 2H, CHdCH₂), 5.69-5.99 (m, 1H, CHdCH₂),

7.15-7.39 (m, 15H, TrH); MS (CI, m/z) 500 (M + H)⁺. Anal.

(C₃₂H₃₇NO₄) C, H, N.

L-γ-glu ta m yl]-N-m eth yl-L-glu ta m a te (16). The general pro-

cedure C was followed using 4-[N-[(3,4-dihydro-2,7-dimethyl-

4-oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]benzoic acid,

trifluoroacetate salt (0.475 g, 1.0 mmol), tri-tert-butyl ^L-γ-

glutamyl-N-methyl-^L-glutamate (0.505 g, 1.1 mmol), dry DMF

(15 mL), DEPC (0.359 g, 2.2 mmol), and Et₃N (0.222 g, 2.2

mmol). Purification by column chromatography, on gradient

elution with MeOH in EtOAc (0-2%), afforded a pale yellow

solid. Reprecipitation from CH₂Cl₂(minimum amount)/petrol

gave the title compound 16 (0.340 g, 42%) as a white solid:

mp 114-116 °C; H-NMR (DMSO-d₆) δ 1.33, 1.34, 1.36, 1.37,

1.40 (5 × s, 27H, 3 × C(CH₃)₃), 1.74-2.20 (m) and 2.42 (m

obscured) (8H, 2 × â-CH₂, 2 × γ-CH₂), 2.31 (s, 3H, quinazoline

2-CH₃), 2.44 (s, 3H, quinazoline 7-CH₃), 2.62, 2.82 (2 × s, 3H,

N-CH₃), 3.15 (s, 1H, CtCH), 4.26 (s, 2H, CH₂CtCH), 4.30 (m

obscured, 1H, Glu R-CH), 4.44, 4.79 (2 × dd, J ) 10.6, 4.9 Hz,

1H, MeGlu R-CH), 4.66 (s, 2H, quinazoline 6-CH₂), 6.81 (d, J

) 8.9 Hz, 2H, 3′,5′-ArH), 7.43 (s, 1H, quinazoline 8-H), 7.74

(d, J ) 7.74 Hz, 3H, quinazoline 5-H, 2′,6′-ArH), 8.22 (t, J )

7.44 Hz, 1H, Glu NH), 12.00 (s, 1H, lactam NH); MS (ESI,

m/z) 802 (M + H)⁺. Anal. (C₄₄H₅₉N₅O₉‚0.25H₂O) C, H, N.

Tr i-ter t-bu tyl N-[N-[4-[(3,4-Dih yd r o-2-m eth yl-4-oxo-6-

q u in a zolin yl)m e t h yl]-N -p r op -2-yn yla m in o]-2-flu or o-

ben zoyl]-^L-γ-glu ta m yl]-N-m eth yl-L-glu ta m a te (17). The

general procedure C was followed using 4-[N-[(3,4-dihydro-2-

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

fluorobenzoic acid, trifluoroacetate salt (0.479 g, 1.0 mmol),

tri-tert-butyl ^L-γ-glutamyl-N-methyl-L-glutamate (0.506 g, 1.1

mmol), dry 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 eluant. Reprecipitation from EtOAc-Et₂O (3:1, minimum

amount)/petrol gave the title compound (0.430 g, 53%) as a

white solid: mp 110-112 °C; ¹H-NMR (DMSO-d₆) δ 1.33, 1.34,

1.35, 1.36, 1.40 (5 × s, 27H, 3 × C(CH₃)₃), 1.80-2.20, 2.42 (2

× m, 8H, 2 × â-CH₂, 2 × γ-CH₂), 2.33 (s, 3H, quinazoline

2-CH₃), 2.60, 2.80 (2 × s, 3H, N-CH₃), 3.27 (s, 1H, CtCH),

4.29 (m, 1H, Glu R-CH), 4.37 (s, 2H, CH₂CtCH), 4.50 (dd) and

4.81 (dd obscured) (J ) 10.1, 4.8 Hz, 1H, MeGlu R-CH), 4.79

(s, 2H, quinazoline 6-CH₂), 6.61 (d, J ) 14.6 Hz, 1H, 3′-ArH),

6.66 (d, J ) 8.6 Hz, 1H, 5′-ArH), 7.52 (m, 2H, 6′-ArH,

quinazoline 8-H), 7.68 (dd, J ) 8.4, 1.8 Hz, 1H, quinazoline

7-H), 7.95 (s, 1H, quinazoline 5-H), 8.02 (dd obscured) and 8.07

(dd) (J ) 7.0, 4.2 Hz, 1H, Glu NH), 12.21 (s, 1H, lactam NH);

MS (ESI, m/z) 806 (M + H)⁺. Anal. (C₄₃H₅₆FN₅O₉‚0.25H₂O)

C, H, N, F.

r-ter t-Bu tyl N-Tr ityl-4-m eth yl-^D-glu ta m a te (53). The

method followed that used to prepare 45 but used 52 (0.240

g, 0.48 mmol), dry CH₂Cl₂(2.5 mL), tetrakis(triphenylphos-

phine)palladium(0) (0.017 g), and pyrrolidine (0.06 mL, excess).

Workup as described for 45 yielded the title compound 53

(0.190 g, 86%) as a white foam: mp 66-68 °C (softens); H-

NMR (DMSO-d₆) δ 0.94, 1.03 (2 × d, J ) 6.7 Hz, 6H, 2 ×

γ-CH₃), 1.11, 1.14 (2 × s, 9H, C(CH₃)₃), 1.32, 1.50, 1.95, 2.10

(4 × m, 2H, â-CH₂), 2.30 (m, 1H, γ-CH), 2.80 (t, J ) 8.6 Hz,

1H, NH), 3.12 (m, 1H, R-CH), 7.15-7.41 (m, 15H, TrH); MS

(CI, m/z) 460 (M + H)⁺. This was used in the next experiment

without further purification.

Tr i-ter t-b u t yl N-(N-Tr it yl-γ-4-m et h yl-^D-glu t a m yl)-L-

glu ta m a te (54). The method followed that used to prepare

43 but used R-tert-butyl N-trityl-4-methyl-^D-glutamate (53)

(0.180 g, 0.39 mmol), NMM (0.040 g, 0.39 mmol), dry THF (3.0

mL), isobutyl chloroformate (0.053 g, 0.39 mmol), and a

suspension of di-tert-butyl ^L-glutamate hydrochloride salt

(0.115 g, 0.39 mmol) in dry THF (2.5 mL) and N-methylmor-

pholine (0.040 g, 0.39 mmol). Purification by column chro-

matography, on gradient elution with ethyl acetate in petrol

(5-20%), gave the title compound 54 as a viscous colorless oil,

which solidified on drying in vacuo over P₂O₅(0.205, 75%):

mp 55-60 °C (softens); ¹H-NMR (DMSO-d₆) δ 0.91, 0.99 (2 ×

d, J ) 6.2 Hz, 3H, 4-MeGlu_Dγ-CH₃), 1.10, 1.16 (2 × s, 9H,

4-MeGlu_DC(CH₃)₃), 1.37, 1.38 (2 × s, 18H, 2 × Glu_LC(CH₃)₃),

1.50-2.30 (m, 7H, 2 × â-CH₂, 4-MeGlu_Dγ-CH, Glu_Lγ-CH₂),

3.05 (m, 1H, 4-MeGlu_DR-CH), 4.04, 4.18 (2 × m, 1H, Glu_L

R-CH), 7.14-7.41 (m, 15H, TrH), 7.99 (d, J ) 7.8 Hz, 1H, Glu_L

NH); MS (CI, m/z) 701 (M + H)⁺. Anal. (C₄₂H₅₆N₂O₇) C, H,

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

tid e ter t-Bu tyl Ester s: Tr i-ter t-bu tyl N-[4-[N-[(3,4-Dih y-

d r o-2-m e t h yl-4-oxo-6-q u in a zolin yl)m e t h yl]-N -p r op -2-

yn yla m in o]b e n zoyl]-^L-γ-glu t a m yl]-N -m e t h yl-L-glu t a -

m a te (15). The general procedure C was followed using 4-[N-

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

ynylamino]benzoic acid, trifluoroacetate salt (0.461 g, 1.0

mmol), tri-tert-butyl ^L-γ-glutamyl-N-methyl-L-glutamate (0.60

g, 1.3 mmol), dry 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 EtOAc as eluant. Repre-

cipitation from CH₂Cl₂-Et₂O (1:1, minimum amount)/petrol

gave the title compound (0.56 g, 71%) as a white powder: mp

110-113 °C; ¹H-NMR (DMSO-d₆) δ 1.32, 1.33, 1.35, 1.36, 1.39

(5 × s, 27H, 3 × C(CH₃)₃), 1.86 (m, 4H, 2 × â-CH₂), 2.32 (s,

3H, quinazoline 2-CH₃), 2.15, 2.44 (2 × m, 4H, 2 × γ-CH₂),

2.61, 2.80 (2 × s, 3H, N-CH₃), 3.23 (s 1H, CtCH), 4.29 (m,

3H, CH₂CtC, Glu R-CH), 4.48, 4.77 (2 × m, 3H, quinazoline

6-CH₂, MeGlu R-CH), 6.82 (d, J ) 8.7 Hz, 2H, 3′,5′-ArH), 7.54

(d, J ) 8.4 Hz, 1H, quinazoline 8-H), 7.71 (t, J ) 8.6 Hz, 3H,

quinazoline 7-H, 2′,6′-ArH), 7.96 (s, 1H, quinazoline 5-H), 8.28

(t, 1H, Glu NH), 12.19 (s, 1H, lactam NH); MS (FAB, m/z) 810

(M + Na)⁺, 788 (M + H)⁺. Anal. (C₄₃H₅₇N₅O₉‚0.5H₂O) C, H,

Tr i-ter t-b u t yl N-[N-[4-[(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 o-

r oben zoyl]-^L-γ-glu ta m yl]-N-m eth yl-L-glu ta m a te (18). The

general procedure C was followed using 4-[N-[(3,4-dihydro-2,7-

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

2-fluorobenzoic acid, trifluoroacetate salt (1.94 g, 3.93 mmol),

tri-tert-butyl

L-γ-glutamyl-N-methyl-L-glutamate (1.8 g, 3.93

mmol), dry DMF (60 mL), DEPC (1.28 g, 7.86 mmol), and Et₃N

(0.794 g, 7.86 mmol). The crude product was purified by

column chromatography using a gradient of MeOH in EtOAc

(0-3%) as eluant. Subsequent reprecipitation from CH₂

Cl₂

(minimum amount)/petrol gave the title compound 18 (2.60 g,

81%) as a white solid: mp 122-124 °C; H-NMR (DMSO-d₆)

δ 1.33, 1.34, 1.35, 1.40 (4 × s, 27H, 3 × C(CH₃)₃), 1.70-2.20

(m) and 2.40 (m obscured) (8H, 2 × â-CH₂, 2 × γ-CH₂), 2.30

(s, 3H, quinazoline 2-CH₃

), 2.43 (s, 3H, quinazoline 7-CH₃),

2.60, 2.80 (2 × s, 3H, N-CH₃), 3.25 (s, 1H, CtCH), 4.30 (m,

3H, Glu R-CH, CH₂

CtCH), 4.50, 4.79 (2 × dd, J ) 10.7, 4.6

Hz, 1H, MeGlu R-CH), 4.68 (s, 2H, quinazoline 6-CH₂), 6.61

(m, 2H, 3′,5′-ArH), 7.44 (s, 1H, quinazoline 8-H), 7.51 (t, J )

8.7 Hz, 1H, 6′-ArH), 7.68 (s, 1H, quinazoline 5-H), 8.00, 8.08

(2 × dd, J ) 7.0, 4.3 Hz, 1H, Glu NH), 12.10 (s, 1H, lactam

NH); MS (ESI, m/z) 820 (M + H)

⁺. (C₄₄H₅₈FN₅O₉) C, H, N, F.

Di-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]-^L-

γ-glu ta m yl]-N-m eth ylglycin a te (21). The general proce-

dure C was followed using 4-[N-[(3,4-dihydro-2-methyl-4-oxo-

6-quinazolinyl)methyl]-N-prop-2-ynylamino]benzoic acid, tri-

fluoroacetate salt (0.553 g, 1.2 mmol), di-tert-butyl ^L-γ-glutamyl-

N-methylglycinate (0.53 g, 1.6 mmol), dry DMF (15 mL), DEPC

Tr i-ter t-bu tyl N-[N-[4-[N-[(3,4-Dih yd r o-2,7-d im eth yl-4-

oxo-6-qu in azolin yl)m eth yl]-N-pr op-2-yn ylam in o]ben zoyl]-

Folate-Based Inhibitors of TS

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1507

(0.359 g, 2.2 mmol), and then Et₃N (0.222 g, 2.2 mmol). The

crude product was purified by chromatography on a silica gel

column using EtOAc as eluant. The product was reprecipi-

tated from CH₂Cl₂/Et₂O at -20 °C yielding the title compound

(2 × s, 3H, N-CH₃), 3.11 (s, 3H, N¹⁰-CH₃), 4.29 (m, 1H, Glu

R-CH), 4.50, 4.79 (2 × dd, J ) 10.7, 4.5 Hz, 1H, MeGlu R-CH),

4.69 (s, 2H, quinazoline 6-CH₂), 6.51 (d, J ) 14.4 Hz, 1H, 3′-

ArH), 6.56 (d, J ) 8.1 Hz, 1H, 5′-ArH), 7.48 (m, 3H, quinazoline

8-H, 6′-ArH, quinazoline 5-H), 7.88, 7.98 (2 × t, J ) 6.1 Hz,

1H, Glu NH), 12.12 (s, 1H, lactam NH); MS (ESI, m/z) 796 (M

+ H)⁺. Anal. (C₄₂H₅₈FN₅O₉) C, H, N, F.

Tr i-ter t-bu tyl N-[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]-2-flu o-

r oben zoyl]-^L-γ-glu ta m yl]-N-eth yl-L-glu ta m a te (19). The

general procedure C was followed using 4-[N-[(3,4-dihydro-2,7-

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

2-fluorobenzoic acid (0.341 g, 0.9 mmol), tri-tert-butyl ^L-γ-

glutamyl-N-ethyl-^L-glutamate (0.555 g, 1.17 mmol), dry DMF

(5 mL), DEPC (0.30 g, 1.8 mmol), and Et₃N (0.21 g, 2.0 mmol).

21 as a white solid (0.22 g, 28%): mp 104-108 °C; H-NMR

(DMSO-d₆) δ 1.30, 1.39 (2 × s, 18H, 2 × C(CH₃)₃), 1.96 (m,

2H, Glu â-CH₂), 2.33 (s, 3H, quinazoline 2-CH₃), 2.26, 2.45 (2

× m, 2H, γ-CH₂), 2.79, 2.95 (2 × s, 3H, N-CH₃), 3.23 (s, 1H,

CtCH), 3.95 (d, J ) 6.1 Hz) and 4.06 (s) (2H, MeGly R-CH₂),

4.25 (m, 1H, Glu R-CH), 4.33 (s, 2H, CH₂CtC), 4.77 (s, 2H,

quinazoline 6-CH₂), 6.81 (m, 2H, 3′,5′-ArH), 7.54 (d, J ) 8.3

Hz, 1H, quinazoline 8-H), 7.74 (m, 3H, quinazoline 7-H, 2′,6′-

ArH), 7.96 (s, 1H, quinazoline 5-H), 8.28 (t, J ) 8.6 Hz, 1H,

NH), 12.19 (s, 1H, lactam NH); MS (FAB, m/z) 682 (M + Na)⁺.

Anal. (C₃₆H₄₅N₅O₇‚0.25H₂O) C, H, N.

Tr i-ter t-bu tyl N-[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-m eth yla m in o]ben zoyl]-^L-

γ-glu ta m yl]-N-m eth yl-^L-glu ta m a te (22). The general pro-

cedure C was followed using 4-[N-[(3,4-dihydro-2,7-dimethyl-

4-oxo-6-quinazolinyl)methyl]-N-methylamino]benzoic acid,

trifluoroacetate salt (0.450 g, 1.0 mmol), tri-tert-butyl ^L-γ-

glutamyl-N-methyl-^L-glutamate (0.430 g, 0.94 mmol), dry DMF

(15 mL), DEPC (0.359 g, 2.2 mmol), and Et₃N (0.222 g, 2.2

mmol). Purification by column chromatography, on gradient

elution with MeOH in EtOAc (0-4%), afforded a glass.

Reprecipitation from CH₂Cl₂(minimum amount)/petrol gave

the title compound 22 (0.260 g, 36%) as a white solid: mp 139-

140 °C; ¹H-NMR (DMSO-d₆) δ 1.33, 1.34, 1.36, 1.37, 1.40 (5 ×

s, 27H, 3 × C(CH₃)₃), 1.80-2.18 (m) and 2.40 (m obscured)

(8H, 2 × â-CH₂, 2 × γ-CH₂), 2.30 (s, 3H, quinazoline 2-CH₃),

2.43 (s, 3H, quinazoline 7-CH₃), 2.61, 2.81 (2 × s, 3H, N-CH₃),

3.13 (s, 3H, N¹⁰-CH₃), 4.25 (m, 1H, Glu R-CH), 4.48, 4.80 (2 ×

dd, J ) 10.7, 4.7 Hz, 1H, MeGlu R-CH), 4.69 (s, 2H, quinazoline

6-CH₂), 6.70 (d, J ) 8.9 Hz, 2H, 3′,5′-ArH), 7.44, 7.53 (2 × s,

2H, quinazoline 5-H and 8-H), 7.71 (d, J ) 8.8 Hz, 2H, 2′,6′-

ArH), 8.24 (t, J ) 7.51 Hz, 1H, Glu NH), 12.10 (s, 1H, lactam

NH); MS (ESI, m/z) 778 (M + H)⁺. Anal. (C₄₂H₅₉N₅O₉‚0.3H₂O)

C, H, N.

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 azolin yl)m eth yl]-N-m eth ylam in o]-2-flu or oben zoyl]-

L-γ-glu ta m yl]-N-m eth yl-L-glu ta m a te (23). The general pro-

cedure C was followed using 4-[N-[(3,4-dihydro-2-methyl-4-oxo-

6-quinazolinyl)methyl]-N-methylamino]-2-fluorobenzoic acid,

trifluoroacetate salt (0.410 g, 0.9 mmol), tri-tert-butyl ^L-γ-

glutamyl-N-methyl-^L-glutamate (0.410 g, 0.9 mmol), dry DMF

(16 mL), DEPC (0.322 g, 1.98 mmol), and Et₃N (0.200 g, 1.98

mmol). The crude product was purified by column chroma-

tography using a gradient of MeOH in EtOAc (0-2%) as

eluant. Reprecipitation from CH₂Cl₂(minimum amount)/

petrol gave the title compound (0.465 g, 67%) as a white

solid: mp 107-109 °C; ¹H-NMR (DMSO-d₆) δ 1.35 (m, 27H, 3

× C(CH₃)₃), 1.80-2.18, 2.42 (2 × m, 8H, 2 × â-CH₂, 2 × γ-CH₂),

2.32 (s, 3H, quinazoline 2-CH₃), 2.60, 2.80 (2 × s, 3H, N-CH₃),

3.13 (s, 3H, N¹⁰-CH₃), 4.29 (m, 1H, Glu R-CH), 4.49 (m) and

4.79 (m obscured) (1H, MeGlu R-CH), 4.78 (s, 2H, quinazoline

6-CH₂), 6.54 (d, J ) 16.1 Hz, 1H, 3′-ArH), 6.61 (d, J ) 9.3 Hz,

1H, 5′-ArH), 7.56 (m, 3H, 6′-ArH, quinazoline 7-H and 8-H),

7.84 (s, 1H, quinazoline 5-H), 7.87, 7.97 (2 × t, J ) 6.20 Hz,

1H, Glu NH), 12.22 (s, 1H, lactam NH); MS (ESI, m/z) 782 (M

+ H)⁺. Anal. (C₄₁H₅₆FN₅O₉) C, H, N, F.

Tr i-ter t-bu tyl N-[N-[4-[N-[(3,4-Dih yd r o-2,7-d im eth yl-4-

oxo-6-qu in azolin yl)m eth yl]-N-m eth ylam in o]-2-flu or oben -

zoyl]-^L-γ-glu ta m yl]-N-m eth yl-L-glu ta m a te (24). The gen-

eral procedure C was followed using 4-[N-[(3,4-dihydro-2,7-

dimethyl-4-oxo-6-quinazolinyl)methyl]-N-methyl]-2-fluoro-

benzoic acid, trifluoroacetate salt (0.478 g, 1.0 mmol), tri-tert-

butyl ^L-γ-glutamyl-N-methyl-L-glutamate (0.435 g, 0.95 mmol),

dry 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 (0-5%)

as eluant. Subsequent reprecipitation from CH₂Cl₂(minimum

amount)/petrol gave the title compound 24 (0.518 g, 69%) as

a white solid: mp 162-164 °C; ¹H-NMR (DMSO-d₆) δ 1.34,

1.35, 1.36, 1.40 (4 × s, 27H, 3 × C(CH₃)₃), 1.73-2.18 (m) and

2.40 (m obscured) (8H, 2 × â-CH₂, 2 × γ-CH₂), 2.30 (s, 3H,

quinazoline 2-CH₃), 2.42 (s, 3H, quinazoline 7-CH₃), 2.60, 2.80

The crude product was chromatographed with CH₂Cl₂-EtOH

(100:0 to 97:3) as eluant, and the glass obtained was triturated

with hexanes to give the title compound 19 (0.519 g, 69%):

mp 90-100 °C; H-NMR (DMSO-d₆, 383 K) δ 1.13 (t, J ) 7.1

Hz, 3H, CH₂

CH₃), 1.39, 1.41, 1.45 (3 × s, 27H, 3 × C(CH₃)₃),

2.01, 2.22 (2 × m, 6H, â-CH₂, γ-CH₂), 2.34 (s, 3H, quinazoline

CH₃), 2.45 (m, 5H, γ-CH₂, quinazoline CH₃), 2.96 (t, J ) 2.3

Hz, 1H, CtCH), 3.20, 3.40 (2 × m, 2H, CH₂CH₃), 4.22 (m, 3H,

CH₂CtC, R-CH), 4.42 (dd, J ) 13.0, 7.0 Hz, 1H, R-CH), 4.69

(s, 2H, quinazoline 6-CH₂

), 6.61, 6.67, 6.71, 6.75 (4 × d, J )

2.4 Hz, 2H, 3′,5′-ArH), 7.42 (s, 1H, quinazoline 8-H), 7.60 (m,

2H, Glu NH, 6′-ArH), 7.85 (s, 1H, quinazoline 5-H), 11.60 (br

s, 1H, lactam NH); MS (ESI, m/z) 834 (M + H)⁺

. Anal.

(C₄₅H₆₀FN₅O₉) C, H, N.

Tr i-ter t-bu tyl N-[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]-2-flu o-

r oben zoyl]-^L-γ-glu ta m yl]-N-p r op -2-yn yl-L-glu ta m a te (20).

The general procedure C was followed using 4-[N-[(3,4-dihydro-

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

2-fluorobenzoic acid (0.403 g, 1.06 mmol), tri-tert-butyl ^L-γ-

glutamyl-N-prop-2-ynyl-^L-glutamate (0.657 g, 1.36 mmol), dry

DMF (6 mL), DEPC (0.39 g, 2.4 mmol), and Et₃N (0.24 g, 2.4

mmol). The crude product was chromatographed with CH₂-

Cl₂-EtOH (100:0 to 95:5) as eluant. The glass obtained was

triturated with hexanes to give the title compound 20 (0.654

g, 73%): mp 90-97 °C; ¹H-NMR (DMSO-d₆, 383 K) δ 1.39,

1.40, 1.44 (3 × s, 27H, 3 × C(CH₃)₃), 2.01, 2.24, 2.53 (3 × m,

8H, â-CH₂, γ-CH₂), 2.32, 2.43 (2 × s, 6H, quinazoline CH₃and

CH₃), 2.91, 2.96 (2 × m, 2H, CtCH), 4.08 (m) and 4.21 (d, J )

2.2 Hz) (4H, CH₂CtC), 4.41 (dd, J ) 13.6, 7.5 Hz, 1H, R-CH),

4.56 (dd, J ) 9.0, 5.5 Hz, 1H, R-CH), 4.67 (s, 2H, quinazoline

6-CH₂), 6.59, 6.65, 6.69, 6.73 (4 × d, 2H, 3′,5′-ArH), 7.40 (s,

1H, quinazoline 8-H), 7.60 (m, 2H, Glu NH, 6′-ArH), 7.83 (s,

1H, quinazoline 5-H), 8.95 (br s, 1H, lactam NH); MS (ESI,

m/z) 844 (M + H)⁺. Anal. (C₄₆H₅₈FN₅O₉) C, H, N, F.

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]-γ-

4-m eth yl-^L-glu ta m yl]-L-glu ta m a te (35a ). The general pro-

cedure C was followed using tri-tert-butyl γ-4-methyl-^L-glutamyl-

L-glutamate (37a ) (0.430 g, approximately 0.59 mmol), 4-[N-

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

ynylamino]benzoic acid, trifluoroacetic acid salt (0.327 g, 0.71

mmol), dry DMF (6.5 mL), DEPC (0.231 g, 1.42 mmol), and

Et₃

N (0.143 g, 1.42 mmol). The crude product was purified

by column chromatography using a gradient of MeOH in

EtOAc (0-2%) as eluant. Reprecipitation from CH₂Cl₂-Et₂O

(1:1, minimum amount)/petrol at -20 °C afforded the title

compound 35a as a white solid (0.170 g, 28%): mp 102-110

°C; ¹H-NMR (DMSO-d₆) δ 1.03 (d, J ) 6.81 Hz, 3H, 4-MeGlu_L

γ-CH₃), 1.33, 1.36, 1.38 (3 × s, 27H, 3 × C(CH₃)₃), 1.65, 1.83,

2.05 (3 × m, 4H, 2 × â-CH₂), 2.20 (t, J ) 7.7 Hz, 2H, Glu_L

γ-CH₂), 2.32 (s, 3H, quinazoline 2-CH₃), 2.50 (m, DMSO peak,

4-MeGlu_Lγ-CH), 3.22 (s, 1H, CtCH), 4.07, 4.21 (2 × m, 2H,

4-MeGlu_LR-CH, Glu_LR-CH), 4.34 (s, 2H, CH₂CtC), 4.78 (s,

2H, quinazoline 6-CH₂), 6.82 (d, J ) 8.7 Hz, 2H, 3′,5′-ArH),

7.54 (d, J ) 8.4 Hz, 1H, quinazoline 8-H), 7.72 (t, J ) 8.7 Hz,

3H, quinazoline 7-H, 2′,6′-ArH), 7.96 (s, 1H, quinazoline 5-H),

8.06, 8.20 (2 × d, J ) 7.6 Hz, 2H, Glu_LNH, 4-MeGlu_LNH),

12.18 (s, 1H, lactam NH); MS (ESI, m/z) 788 (M + H)⁺. Anal.

(C₄₃H₅₇N₅O₉‚0.5H₂O) C, H, N.

Tr i-ter t-bu tyl N-[N-[4-[N-[(3,4-Dih yd r o-2-m eth yl-4-oxo-

1508 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10

Bavetsias et al.

6-qu in a zolin yl)m eth yl]-N-p r op -2-yn yla m in o]ben zoyl]-γ-

4,4-d im eth yl-^L-glu ta m yl]-L-glu ta m a te (35b). The general

procedure C was followed using tri-tert-butyl γ-4,4-dimethyl-

L-glutamyl-L-glutamate (37b) (0.360 g, approximately 0.43

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

N-prop-2-ynylamino]benzoic acid, trifluoroacetate salt (0.200

g, 0.43 mmol), dry DMF (7.0 mL), DEPC (0.154 g, 0.95 mmol),

and Et₃N (0.095 g, 0.95 mmol). The crude product was purified

by column chromatography using a gradient of MeOH in

EtOAc (0-2%) as eluant. Reprecipitation from Et₂O/petrol at

-20 °C yielded the title compound 35b (0.200 g, 58%) as a

γ-CH₂), 2.65, 2.81 (2 × s, 3H, N-CH₃), 3.24 (s, 1H, CtCH),

4.35 (br s, 3H, CH₂CtC, Glu R-CH), 4.81 (s, 2H, quinazoline

6-CH₂), 4.58, 4.91 (dd, J ) 11.0, 4.5 Hz, 1H, MeGlu R-CH),

6.83 (d, J ) 8.7 Hz, 2H, 3′,5′-ArH), 7.59 (d, J ) 8.4 Hz, 1H,

quinazoline 8-H), 7.75 (m, 3H, 2′,6′-ArH, quinazoline 7-H), 8.00

(s, 1H, quinazoline 5-H), 8.32 (d, J ) 7.3 Hz, 1H, Glu NH);

MS (FAB, m/z) 642 (M

1.1TFA‚0.5Et₂O‚H₂O) C, H, N, F.

Na)⁺. Anal. (C₃₁H₃₃N₅O₉‚

N -[N -[4 -[N -[(3 ,4 -D i h y d r o -2 ,7 -d i m e t h y l -4 -o x o -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]-N-m eth yl-^L-glu ta m ic Acid (26). The general

procedure D was followed using 16 (0.200 g, 0.25 mmol) and

TFA (15 mL). Compound 26 was obtained as a white powder

white powder: mp 101-104 °C; H-NMR (DMSO-d₆) δ 1.11,

1.16 (2 × s, 6H, 2 × 4-diMeGlu_Lγ-CH₃), 1.30, 1.36 (2 × s, 27H,

3 × C(CH₃)₃), 1.95 (m, 4H, 2 × â-CH₂), 2.21 (t, J ) 7.7 Hz, 2H,

Glu_Lγ-CH₂), 2.32 (s, 3H, quinazoline 2-CH₃), 3.22 (s, 1H,

CtCH), 4.08, 4.23 (2 × m, 2H, Glu_LR-CH, 4-diMeGlu_LR-CH),

4.31 (s, 2H, CH₂CtCH), 4.76 (s, 2H, quinazoline 6-CH₂), 6.81

(d, J ) 8.7 Hz, 2H, 3′,5′-ArH), 7.52 (d, J ) 8.4 Hz, 1H,

quinazoline 8-H), 7.66 (m, 4H, quinazoline 7-H, 2′,6′-ArH,

amidic NH), 7.95 (s, 1H, quinazoline 5-H), 8.19 (d, J ) 7.3 Hz,

1H, amidic NH), 12.19 (s, 1H, lactam NH); MS (ESI, m/z) 802

(M + H)⁺. Anal. (C₄₄H₅₉N₅O₉) C, H, N.

(0.180 g, 95%): mp 170 °C dec; H-NMR (DMSO-d₆) δ 1.82-

2.23 (m) and 2.46 (m obscured) (8H, 2 × â-CH₂, 2 × γ-CH₂),

2.31, 2.48 (2 × s, 6H, quinazoline 2-CH₃, quinazoline 7-CH₃),

2.65, 2.81 (2 × s, 3H, N-CH₃), 3.23 (s, 1H, CtCH), 4.31 (m,

3H, CH₂CtCH, Glu R-CH), 4.56, 4.91 (2 × dd, J ) 10.9, 4.4

Hz, 1H, MeGlu R-CH), 4.71 (s, 2H, quinazoline 6-CH₂), 6.79

(d, J ) 8.9 Hz, 2H, 3′,5′-ArH), 7.48 (s, 1H, quinazoline 8-H),

7.76 (m, 3H, quinazoline 5-H, 2′,6′-ArH), 8.33 (m, 1H, Glu NH);

MS (ESI, m/z) 634 (M + H)⁺. Anal. (C₃₂H₃₅N₅O₉‚TFA‚H₂O)

C, H, N.

Di-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]-^L-

γ-glu ta m yl]-r-m eth yla la n in a te (59a ). The general proce-

dure C was followed using 4-[N-[(3,4-dihydro-2-methyl-4-oxo-

6-quinazolinyl)methyl]-N-prop-2-ynylamino]benzoic acid, tri-

fluoroacetate salt (0.461 g, 1.0 mmol), di-tert-butyl ^L-γ-glutamyl-

R-methylalaninate (0.48 g, 1.4 mmol), dry DMF (15 mL), DEPC

(0.359 g, 2.2 mmol), and then Et₃N (0.222 g, 2.2 mmol). The

crude product was purified by chromatography on a silica gel

column using EtOAc as eluant. The product was reprecipi-

tated from CH₂Cl₂/Et₂O yielding the title compound 59a as a

white solid (0.486 g, 72%): mp 115-116 °C; ¹H-NMR (DMSO-

d₆) δ 1.26 (s, 6H, C(CH₃)₂), 1.33, 1.39 (2 × s, 18H, 2 × C(CH₃)₃),

1.91, 1.99 (2 × m, 2H, Glu â-CH₂), 2.14 (m, 2H, γ-CH₂), 2.32

(s, 3H, quinazoline 2-CH₃), 3.27 (s, 1H, CtCH), 4.25 (m, 1H,

Glu R-CH), 4.34 (s, 2H, CH₂CtC), 4.78 (s, 2H, quinazoline

6-CH₂), 6.83 (d, J ) 8.9 Hz, 2H, 3′,5′-ArH), 7.54 (d, J ) 8.4

Hz, 1H, quinazoline 8-H), 7.70 (dd, J ) 2.0 Hz, 1H, quinazoline

7-H), 7.74 (d, J ) 8.8 Hz, 2H, 2′,6′-ArH), 7.96 (d, J ) 1.6 Hz,

1H, quinazoline 5-H), 8.07 (s, 1H, R-methylalanine NH), 8.34

(d, J ) 7.4 Hz, 1H, Glu NH), 12.20 (s, 1H, lactam NH); MS

(FAB, m/z) 674 (M + H)⁺. Anal. (C₃₇H₄₇N₅O₇‚0.5H₂O) C, H,

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

m e t h y l]-N -p r o p -2-y n y la m in o ]-2-flu o r o b e n zo y l]-^L-γ-

glu ta m yl]-N-m eth yl-^L-glu ta m ic Acid (27). The general

procedure D was followed using 17 (0.220 g, 0.27 mmol) and

TFA (15 mL). The title compound 27 was obtained as a white

solid (0.213 g, 97%): mp 120 °C dec; ¹H-NMR (DMSO-d₆) δ

1.82-2.20 (m) and 2.41 (m obscured) (8H, 2 × â-CH₂, 2 ×

γ-CH₂), 2.43 (s, 3H, quinazoline 2-CH₃), 2.63, 2.80 (2 × s, 3H,

N-CH₃), 3.27 (s, 1H, CtCH), 4.37 (m, 3H, CH₂CtCH, Glu

R-CH), 4.54, 4.90 (2 × dd, J ) 10.9, 4.4 Hz, 1H, MeGlu R-CH),

4.82 (s, 2H, quinazoline 6-CH₂), 6.61 (d, J ) 15.0 Hz, 1H, 3′-

ArH), 6.66 (d, J ) 8.1 Hz, 1H, 5′-ArH), 7.52 (t, J ) 8.7 Hz,

1H, 6′-ArH), 7.61 (d, J ) 8.4 Hz, 1H, quinazoline 8-H), 7.77

(d, J ) 8.5 Hz, 1H, quinazoline 7-H), 8.00 (s, 1H, quinazoline

5-H), 8.05 (m, 1H, Glu NH); MS (ESI, m/z) 638 (M + H)⁺. Anal.

(C₃₁H₃₂FN₅O₉‚1.3TFA‚H₂O‚Et₂O) C, H, N, F.

N -[N -[4 -[N -[(3 ,4 -D i h y d r o -2 ,7 -d i m e t h y l -4 -o x o -6 -

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

zoyl]-^L-γ-glu ta m yl]-N-m eth yl-L-glu ta m ic Acid (28). The

general procedure D was followed using 18 (0.215 g, 0.26

mmol) and TFA (16 mL). The title compound 28 was obtained

as a white solid (0.205 g, 98%): mp 150 °C dec; ¹H-NMR

(DMSO-d₆) δ 1.82-2.20 (m) and 2.46 (m obscured) (8H, 2 ×

â-CH₂, 2 × γ-CH₂), 2.45, 2.48 (2 × s, 6H, quinazoline 2-CH₃

and 7-CH₃), 2.63, 2.81 (2 × s, 3H, N-CH₃), 3.25 (s, 1H, CtCH),

4.33 (m, 3H, CH₂CtCH, Glu R-CH), 4.58, 4.90 (2 × dd, J )

10.9, 4.4 Hz, 1H, MeGlu R-CH), 4.74 (s, 2H, quinazoline

6-CH₂), 6.61 (m, 2H, 3′,5′-ArH), 7.49 (s, 1H, quinazoline 8-H),

7.54 (t, J ) 8.7 Hz, 1H, 6′-ArH), 7.73 (s, 1H, quinazoline 5-H),

8.08 (m, 1H, Glu NH); MS (ESI, m/z) 652 (M + H)⁺. Anal.

(C₃₂H₃₄FN₅O₉‚1.3TFA‚H₂O) C, H, N, F.

N -[N -[4 -[N -[(3 ,4 -D i h y d r o -2 ,7 -d i m e t h y l -4 -o x o -6 -

q u in a zolin yl)m e t h yl]-N -p r op -2-yn yla m in o]-2-flu or o-

ben zoyl]-^L-γ-glu tam yl]-N-eth yl-L-glu tam ic Acid (29). Com-

pound 19 (0.461 g, 0.55 mmol) was stirred with TFA (25 mL)

at room temperature under argon for 75 min. The solution

was evaporated and the residue triturated with Et₂O to give

a powder (0.470 g). A solution of this material (0.370 g) in

0.5 M aqueous NaHCO₃(5 mL) was filtered and adjusted to

pH 4 with 1 N HCl. The resulting suspension was centrifuged,

and the precipitate was collected, washed with H₂O, and dried

to give the title compound 29 (0.248 g, 84%): mp 159-162

°C; ¹H-NMR (DMSO-d₆, 383 K) δ 1.13 (t, J ) 7.0 Hz, 3H,

CH₂CH₃), 2.03, 2.26 (2 × m, 6H, â-CH₂, γ-CH₂), 2.34 (s, 3H,

quinazoline CH₃), 2.45 (m, 5H, γ-CH₂, quinazoline CH₃), 2.96

(m, CtCH), 3.24, 3.38 (2 × m, CH₂CH₃), 4.22 (d, J ) 2.2 Hz,

2H, CH₂CtC), 4.39, 4.48 (2 × m, 2H, R-CH), 4.69 (s, 2H,

quinazoline 6-CH₂), 6.62, 6.68, 6.72, 6.75 (4 × d, J ) 2.4 Hz,

2H, 3′,5′-ArH), 7.41 (s, 1H, quinazoline 8-H), 7.63 (m, 2H, Glu

NH, 6′-ArH), 7.85 (s, 1H, quinazoline 5-H); MS (ESI, m/z) 666

(M + H)⁺. Anal. (C₃₃H₃₆N₅O₉F‚0.75H₂O) C, H, N.

Di-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]-^L-

γ-glu ta m yl]-^L-p r olin a te (59b). The general procedure C was

followed using 4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazo-

linyl)methyl]-N-prop-2-ynylamino]benzoic acid, trifluoro-

acetate salt (0.461 g, 1.0 mmol), di-tert-butyl ^L-γ-glutamyl-L-

prolinate (0.524 g, 0.15 mmol), dry DMF (15 mL), DEPC (0.359

g, 2.2 mmol), and then Et₃N (0.222 g, 2.2 mmol). The crude

product was purified by chromatography on a silica gel column

using EtOAc as eluant. The product was reprecipitated from

cold Et₂O/petrol yielding the title compound 59b as a white

solid (0.192 g, 28%): mp 113-117 °C; H-NMR (DMSO-d₆) δ

1.31, 1.35, 1.39 (3 × s, 18H, 2 × C(CH₃)₃), 1.70-2.10 (m, 6H,

Glu â-CH₂, Pro 3- and 4-CH₂), 2.38 (t, J ) 7.5 Hz, 2H, γ-CH₂),

2.32 (s, 3H, quinazoline 2-CH₃), 3.41 (m, 2H, Pro 5-CH₂), 3.19

(s, 1H, CtCH), 4.15, 4.40 (2 × dd, J ) 9.0, 3.8 Hz, 1H, Pro

2-CH), 4.32 (m, 3H, Glu R-CH, CH₂CtC), 4.77 (s, 2H, quinazo-

line 6-CH₂), 6.83 (d, J ) 8.9 Hz, 2H, 3′,5′-ArH), 7.53 (d, J )

8.4 Hz, 1H, quinazoline 8-H), 7.68 (dd, J ) 8.4, 2.0 Hz, 1H,

quinazoline 7-H), 7.73 (d, J ) 9.0 Hz, 2H, 2′,6′-ArH), 7.97 (d,

J ) 2.0 Hz, 1H, quinazoline 5-H), 8.23 (t, 1H, NH), 12.20 (s,

1H, lactam NH); MS (FAB, m/z) 708 (M + Na)⁺. Anal.

(C₃₈H₄₇N₅O₇‚0.5H₂O) C, H, N.

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

tid es: N-[N-[4-[N-[(3,4-Dih yd r o-2-m eth yl-4-oxo-6-qu in a zo-

lin yl)m eth yl]-N-pr op-2-yn ylam in o]ben zoyl]-^L-γ-glu tam yl]-

N-m eth yl-^L-glu ta m ic Acid (25). The general procedure D

was followed using 15 (0.167 g, 0.21 mmol) and TFA (10 mL).

Compound 25 was obtained as a white powder (0.152 g, 90%):

mp 205 °C dec; ¹H-NMR (DMSO-d₆) δ 1.98 (m, 4H, 2 × â-CH₂),

2.42 (s, 3H, quinazoline 2-CH₃), 2.14, 2.45 (2 × m, 4H, 2 ×

N -[N -[4 -[N -[(3 ,4 -D i h y d r o -2 ,7 -d i m e t h y l -4 -o x o -6 -

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

Folate-Based Inhibitors of TS

J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1509

zoyl]-L-γ-glu ta m yl]-N-p r op -2-yn yl-L-glu ta m ic Acid (30).

Compound 20 (0.544 g, 0.645 mmol) and TFA (29 mL) were

stirred together at room temperature in the dark for 75 min.

The solution was evaporated to a glass which was triturated

with Et₂O and dissolved in 0.5 M aqueous NaHCO₃(8 mL).

The solution was filtered and adjusted to pH 4 with 1 N HCl.

The resulting suspension was centrifuged and the precipitate

washed three times with H₂O by resuspension-centrifuga-

tion-decantation, then collected by filtration, further washed,

and dried to give the title compound 30 (0.387 g, 87%): mp

162-165 °C; ¹H-NMR (DMSO-d₆, 383 K) δ 2.07, 2.18, 2.34,

2.56 (4 × m, 11H, â-CH₂, γ-CH₂, quinazoline CH₃), 2.45 (s,

3H, quinazoline CH₃), 2.89, 2.97 (2 × m, 2H, CtCH), 4.10 (m)

and 4.22 (d, J ) 2.3 Hz) (4H, CH₂CtC), 4.47 (m, 1H, R-CH),

4.69 (s, 3H, R-CH, quinazoline 6-CH₂), 6.62, 6.68, 6.71, 6.75

(4 × d, J ) 2.4 Hz, 2H, 3′,5′-ArH), 7.41 (s, 1H, quinazoline

8-H), 7.64 (m, 2H, Glu NH, 6′-ArH), 7.84 (s, 1H, quinazoline

1H, 3′-ArH), 6.57 (d, J ) 6.9 Hz, 1H, 5′-ArH), 7.48, 7.50 (2 ×

s, 2H, quinazoline 5-H and 8-H), 7.54 ((t obscured), J ) 8.7

Hz, 1H, 6′-ArH), 8.09 (m, 1H, Glu NH); MS (ESI, m/z) 628 (M

+ H)⁺. Anal. (C₃₀H₃₄FN₅O₉‚1.4TFA‚0.5Et₂O‚H₂O) C, H, N,

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

m e t h yl]-N -p r op -2-yn yla m in o]b e n zoyl]-γ-4-m e t h yl-^L-

glu ta m yl]-^L-glu ta m ic Acid (36a ). The general procedure D

was followed using 35a (0.071 g, 0.09 mmol) and TFA (6 mL).

Compound 36a was obtained as a white powder (0.053 g,

76%): mp 160-165 °C dec; H-NMR (DMSO-d₆) δ 1.02 (d, J

) 6.8 Hz, 4-MeGlu_Lγ-CH₃), 1.65, 1.91, 2.09 (3 × m, 4H, 2 ×

â-CH₂), 2.25 (t, J ) 7.6 Hz, 2H, Glu_Lγ-CH₂), 2.39 (s, 3H,

quinazoline 2-CH₃), 2.50 (DMSO peak 4-MeGlu_Lγ-CH), 3.23

(s, 1H, CtCH), 4.19 (m, 2H, Glu_LR-CH, 4-MeGlu_LR-CH), 4.35

(s, 2H, CH₂CtCH), 4.80 (s, 2H, quinazoline 6-CH₂), 6.82 (d, J

) 8.9 Hz, 2H, 3′,5′-ArH), 7.58 (d, J ) 8.4 Hz, 1H, quinazoline

8-H), 7.74 (d, J ) 8.7 Hz, 3H, quinazoline 7-H, 2′,6′-ArH), 7.99

(s, 1H, quinazoline 5-H), 8.10, 8.26 (2 × d, J ) 7.7 Hz, 2H,

Glu_LNH, 4-MeGlu_LNH); MS (ESI, m/z) 620 (M + H)⁺. Anal.

(C₃₁H₃₃N₅O₉‚0.95TFA‚0.4Et₂O‚1.2H₂O) C, H, N, F.

5-H); MS (ESI, m/z) 676 (M

FN₅O₉‚0.75H₂O) C, H, N, F.

+ -

H)⁺. Anal. (C₃₄H₃₄

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]-^L-γ-glu t a m yl]-N-

m eth ylglycin e (31). The general procedure D was followed

using 21 (0.075 g, 0.11 mmol) and TFA (5 mL). Compound

31 was obtained as a white solid (0.60 g, 72%): mp 140 °C

N-[N-[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]ben zoyl]-γ-4,4-d im eth yl-^L-

glu ta m yl]-^L-glu ta m ic Acid (36b). The general procedure D

was followed using 35b (0.068 g, 0.085 mmol) and TFA (6.5

mL). Compound 36b was obtained as a white powder (0.050

g, 82%): mp >140 °C dec; ¹H-NMR (DMSO-d₆) δ 1.12, 1.17 (2

× s, 6H, 2 × 4-diMeGlu_Lγ-CH₃), 1.84-2.13 (m, 4H, â-CH₂),

2.25 (t, J ) 7.4 Hz, 2H, Glu_Lγ-CH₂), 2.38 (s, 3H, quinazoline

2-CH₃), 3.23 (s, 1H, CtCH), 4.20 (m, 1H, R-CH), 4.33 (m, 3H,

CH₂CtCH, R-CH), 4.79 (s, 2H, quinazoline 6-CH₂), 6.82 (d, J

) 8.6 Hz, 2H, 3′,5′-ArH), 7.57 (d, J ) 8.4 Hz, 1H, quinazoline

8-H), 7.71 (m, 4H, quinazoline 7-H, 2′,6′-ArH, amidic NH), 7.99

(s, 1H, quinazoline 5-H), 8.22 (d, J ) 7.30 Hz, 1H, amidic NH);

MS (ESI, m/z) 634 (M + H)⁺. Anal. (C₃₂H₃₅N₅O₉‚0.6TFA‚

0.8H₂O) C, H, N, F.

dec; H-NMR (DMSO-d₆) δ 2.01 (m, 2H, â-CH₂), 2.33, 2.42 (2

× m, 2H, γ-CH₂), 2.42 (s, 3H, quinazoline 2-CH₃), 2.81, 2.97

(2 × s, 3H, N-CH₃), 3.24 (s, 1H, CtCH), 3.98 (d, J ) 8.0 Hz)

and 4.10 (s) (2H, MeGly R-CH₂), 4.36 (s, 3H, CH₂CtC, Glu

R-CH), 4.81 (s, 2H, quinazoline 6-CH₂), 6.84 (d, J ) 8.3 Hz,

2H, 3′,5′-ArH), 7.59 (d, J ) 8.5 Hz, 1H, quinazoline 8-H), 7.76

(m, 3H, quinazoline 7-H, 2′,6′-ArH), 8.01 (s, 1H, quinazoline

5-H), 8.33 (t, J ) 7.0 Hz, 1H, NH); MS (FAB, m/z) 570 (M +

Na)⁺, 548 (M + H)⁺. Anal. (C₂₈H₂₉N₅O₇‚1.1TFA‚0.8H₂O‚

0.8Et₂O) C, H, N, F.

N -[N -[4 -[N -[(3 ,4 -D i h y d r o -2 ,7 -d i m e t h y l -4 -o x o -6 -

q u in a zo lin y l)m e t h y l]-N -m e t h y la m in o ]b e n zo y l]-^L-γ-

glu ta m yl]-N-m eth yl-^L-glu ta m ic Acid (32). The general

procedure D was followed using 22 (0.135 g, 0.174 mmol) and

TFA (12 mL). Compound 32 was obtained as a white powder

(0.120 g, 100%): mp 155 °C dec; ¹H-NMR (DMSO-d₆) δ 1.80-

2.16 (m) and 2.41 (m obscured) (8H, 2 × â-CH₂, 2 × γ-CH₂),

2.40, 2.47 (2 × s, 6H, quinazoline 2-CH₃and 7-CH₃), 2.64, 2.80

(2 × s, 3H, N-CH₃), 3.13 (s, 3H, N¹⁰-CH₃), 4.31 (m, 1H, Glu

R-CH), 4.57, 4.91 (2 × dd, J ) 11.0, 4.3 Hz, 1H, MeGlu R-CH),

4.72 (s, 2H, quinazoline 6-CH₂), 6.71 (d, J ) 8.7 Hz, 2H, 3′,5′-

ArH), 7.47, 7.53 (2 × s, 2H, quinazoline 5-H and 8-H), 7.73 (d,

J ) 8.5 Hz, 2H, 2′,6′-ArH), 8.28 (d, J ) 7.2 Hz, 1H, Glu NH);

MS (ESI, m/z) 610 (M + H)⁺. Anal. (C₃₀H₃₅N₅O₉‚TFA‚1.5H₂O)

C, H, N, F.

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

m eth yl]-N-m eth yla m in o]-2-flu or oben zoyl]-^L-γ-glu ta m yl]-

N-m eth yl-^L-glu ta m ic Acid (33). The general procedure D

was followed using 23 (0.295 g, 0.38 mmol) and TFA (23 mL).

The title compound 33 was obtained as a white solid (0.270 g,

85%): mp 110 °C dec; ¹H-NMR (DMSO-d₆) δ 1.82-2.18 (m)

and 2.40 (m obscured) (8H, 2 × â-CH₂, 2 × γ-CH₂), 2.45 (s,

3H, quinazoline 2-CH₃), 2.63, 2.81 (2 × s, 3H, N-CH₃), 3.13 (s,

3H, N¹⁰-CH₃), 4.34 (m, 1H, Glu R-CH), 4.53, 4.90 (2 × dd, J )

10.9, 4.4 Hz, 1H, MeGlu R-CH), 4.83 (s, 2H, quinazoline

6-CH₂), 6.56 (d, J ) 15.9 Hz, 1H, 3′-ArH), 6.62 (d, J ) 9.1 Hz,

1H, 5′-ArH), 7.53 (t, J ) 9.0 Hz, 1H, 6′-ArH), 7.62 (d, J ) 8.4

Hz, 1H, quinazoline 8-H), 7.70 (dd, J ) 8.4, 1.8 Hz, 1H,

quinazoline 7-H), 7.89 (d, J ) 1.5 Hz, 1H, quinazoline 5-H),

7.97 (t, J ) 5.5 Hz, 1H, Glu NH); MS (ESI, m/z) 614 (M +

H)⁺. Anal. (C₂₉H₃₂FN₅O₉‚1.6TFA‚H₂O‚0.4Et₂O) C, H, N, F.

N -[N -[4 -[N -[(3 ,4 -D i h y d r o -2 ,7 -d i m e t h y l -4 -o x o -6 -

qu in a zolin yl)m eth yl]-N-m eth yla m in o]-2-flu or oben zoyl]-

L-γ-glu ta m yl]-N-m eth yl-L-glu ta m ic Acid (34). The general

procedure D was followed using 24 (0.296 g, 0.37 mmol) and

TFA (23 mL). The title compound 34 was obtained as a white

solid (0.285 g, 92%): mp 155 °C dec; ¹H-NMR (DMSO-d₆) δ

1.82-2.18 (m) and 2.40 (m obscured) (8H, 2 × â-CH₂, 2 ×

γ-CH₂), 2.43, 2.47 (2 × s, 6H, quinazoline 2-CH₃and 7-CH₃),

2.63, 2.81 (2 × s, 3H, N-CH₃), 3.13 (s, 3H, N¹⁰-CH₃), 4.34 (m,

1H, Glu R-CH), 4.54, 4.90 (2 × dd, J ) 10.9, 4.4 Hz, 1H, MeGlu

R-CH), 4.73 (s, 2H, quinazoline 6-CH₂), 6.53 (d, J ) 13.1 Hz,

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]-^L-γ-glu t a m yl]-r-

m eth yla la n in e (60a ). The general procedure D was followed

using 59a (0.100 g, 0.15 mmol) and TFA (10 mL). Compound

60a was obtained as a white solid (0.062 g, 63%): mp 155-

158 °C; ¹H-NMR (DMSO-d₆) δ 1.29 (s, 6H, C(CH₃)₃), 1.89, 2.02

(2 × m, 2H, Glu â-CH₂), 2.18 (m, 2H, γ-CH₂), 2.38 (s, 3H,

quinazoline 2-CH₃), 3.23 (s, 1H, CtCH), 4.29 (m, 1H, Glu

R-CH), 4.34 (s, 2H, CH₂CtC), 4.80 (s, 2H, quinazoline 6-CH₂),

6.83 (d, J ) 8.3 Hz, 2H, 3′,5′-ArH), 7.57 (d, J ) 8.3 Hz, 1H,

quinazoline 8-H), 7.75 (d, J ) 8.1 Hz, 3H, 2′,6′-ArH, quinazo-

line 7-H), 7.98 (s, 1H, quinazoline 5-H), 8.02 (s, 1H, R-methy-

lalanine NH), 8.29 (d, J ) 7.6 Hz, 1H, Glu NH); MS (FAB,

m/z) 584 (M + Na)⁺. Anal. (C₂₉H₃₁N₅O₇‚0.7TFA‚H₂O) C, H,

N, F.

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]-^L-γ-glu t a m yl]-L-

p r olin e (60b). The general procedure D was followed using

59b (0.067 g, 0.098 mmol) and TFA (5 mL). Compound 60b

was obtained as a white solid (0.054 g, 77%): mp 162 °C dec;

¹H-NMR (DMSO-d₆) δ 1.84, 2.08 (2 × m, 6H, Glu â-CH₂, Pro

3- and 4-CH₂), 2.39 (m, 5H, quinazoline 2-CH₃, γ-CH₂), 3.42

(m, 2H, Pro 5-CH₂), 3.23 (s, 1H, CtCH), 4.23, 4.48 (2 × dd, J

) 9.9, 3.5 Hz, 1H, Pro 2-CH), 4.34 (m, 3H, Glu R-CH,

CH₂CtC), 4.80 (s, 2H, quinazoline 6-CH₂), 6.83 (d, J ) 8.8

Hz, 2H, 3′,5′-ArH), 7.57 (d, J ) 8.4 Hz, 1H, quinazoline 8-H),

7.74 (d, J ) 8.7 Hz, 3H, 2′,6′-ArH, quinazoline 7-H), 7.99 (s,

1H, quinazoline 5-H), 8.32 (t, 1H, NH); MS (FAB, m/z) 596 (M

+ Na)⁺. Anal. (C₃₀H₃₁N₅O₇‚1.1TFA‚0.22Et₂O) C, H, N.

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.

1510 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10

Bavetsias et al.

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Coupling Reagent Devoid of Toxic By-Product. Tetrahedron Lett.

1990, 31, 205-208.

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(17) Frerot, E.; Coste, J .; Pantaloni, A.; Dufour, M.; J ouin, P. PyBOP^®

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Thymidylate Synthase Inhibitory Activity of L-γ-L-Linked Dipep-

(18) Calas, B.; Michelot, R.; Lecaer, J . P.; Cave, A.; Parello, J .; Potier,

P. Synthesis of (pGlu-5, MePhe-8, Sar-9) Substance P (5-11)

(DiMe-C7) Using a Polyacrylamide Resin and Biological Activity

on Guinea Pig Ileum and Tracheal Smooth Muscle. Int. J . Pept.

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(19) Liakopoulou-Kyriakides, M.; Galardy, R. E. s-Cis and s-Trans

Isomerism of the His-Pro Peptide Bond in Angiotensin and

Thyroliberin Analogues. Biochemistry 1979, 18, 1952-1957.

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Increased Thymidylate Synthase in L1210 Cells Possessing

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J M960878U

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