Quinazoline antifolates thymidylate synthase inhibitors: Lipophilic analogues with modification to the C2-methyl substituent

Article abstract of DOI:10.1021/jm950645n

Modification of the potent thymidylate synthase (TS) inhibitor 1-[[N-[4- [N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl]-N-prop-2- ynylamino]benzoyl]amino]methyl]-3-nitrobenzene (4a) has led to the synthesis of quinazolinone antifolates bearing functionalized alkyl substituents at C2. A general synthetic route was developed which involved coupling the appropriate 1-[[N-[4-(alkylamino)benzoyl)amino]methyl]-3-nitrobenzene 20-22 with a 6-(bromomethyl)-2-(acetoxymethyl)-3,4-dihydro-4-oxoquinazoline 9 or 10. Replacement of the 2-acetoxy group by a chlorine atom followed by the displacement of the halogen of 25a-c by various nucleophiles led to compounds 26-40. Good TS (IC₅₀ <1 μM) and growth inhibition (IC₅₀ 0.1-1 μM) were found with most of these new antifolates. TS inhibitors in this series do not apparently require the reduced folate carrier (RFC) for cell entry (they most likely penetrate the cell membrane by passive diffusion) and are not polyglutamated. N, O, S, Cl, and CN as well as large amino and mercapto substituents were tolerated by the enzyme. The simultaneous incorporation of 7-methyl and 2'-F substituents gave a series of highly potent agents inhibiting cell growth at concentrations <1 μM (24, 27bc; 30-32b, 35b). The incorporation of suitable C2 substituents has overcome the decrease in aqueous solubility observed with lipophilic quinazoline antifolates. This is best illustrated by compound 31a, where up to a 54-fold increase in solubility has been achieved by the incorporation of an N-methylpiperazine nucleus into the C2-methyl group of 4a.

Full text of DOI:10.1021/jm950645n

J . Med. Chem. 1996, 39, 695-704
695
Qu in a zolin e An tifola tes Th ym id yla te Syn th a se In h ibitor s: Lip op h ilic An a logu es
w ith Mod ifica tion to th e C2-Meth yl Su bstitu en t
Laurent F. Hennequin,*,† F. Thomas Boyle,‡ J . Michael Wardleworth,‡ Peter R. Marsham,‡
Rosemary Kimbell,§ and Ann L. J ackman§
Zeneca Pharma, Centre de recherches, Z. I. La Pompelle, BP 401, 51064 Reims, France, Zeneca Pharmaceuticals,
Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, England, and CRC Centre for Cancer Therapeutics at the Institute
of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, England
Received August 30, 1995X
Modification of the potent thymidylate synthase (TS) inhibitor 1-[[N-[4-[N-[(3,4-dihydro-2-
methyl-4-oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]benzoyl]amino]methyl]-3-nitroben-
zene (4a ) has led to the synthesis of quinazolinone antifolates bearing functionalized alkyl
substituents at C2. A general synthetic route was developed which involved coupling the
appropriate 1-[[N-[4-(alkylamino)benzoyl)amino]methyl]-3-nitrobenzene 20-22 with a 6-(bro-
momethyl)-2-(acetoxymethyl)-3,4-dihydro-4-oxoquinazoline 9 or 10. Replacement of the 2-ac-
etoxy group by a chlorine atom followed by the displacement of the halogen of 25a -c by various
nucleophiles led to compounds 26-40. Good TS (IC50 <1 µM) and growth inhibition (IC50 0.1-1
µM) were found with most of these new antifolates. TS inhibitors in this series do not
apparently require the reduced folate carrier (RFC) for cell entry (they most likely penetrate
the cell membrane by passive diffusion) and are not polyglutamated. N, O, S, Cl, and CN as
well as large amino and mercapto substituents were tolerated by the enzyme. The simultaneous
incorporation of 7-methyl and 2′-F substituents gave a series of highly potent agents inhibiting
cell growth at concentrations <1 µM (24, 27bc; 30-32b, 35b). The incorporation of suitable
C2 substituents has overcome the decrease in aqueous solubility observed with lipophilic
quinazoline antifolates. This is best illustrated by compound 31a , where up to a 54-fold increase
in solubility has been achieved by the incorporation of an N-methylpiperazine nucleus into
the C2-methyl group of 4a .
Despite being withdrawn from clinical studies because
of unpredictable nephrotoxicity, the quinazolinone-
based antifolate CB3717 (1) established the principle
of antitumor chemotherapy with a specific inhibitor of
thymidylate synthase (TS) by showing responses in
phase I/II1-5 clinical trials against breast, ovarian, and
liver cancers. A search for more soluble analogues6-11
devoid of renal toxicity led to the discovery of the
2-methylquinazolinone analogue ICI 198583 (2)12 and
more recently Zeneca ZD1694 (Tomudex, 3).13-15 The
latter compound has completed phase I, phase II, and
phase III clinical trials.16-18
To inhibit tumor cell growth ICI 198583 and ZD1694
require rapid transport into cells by the reduced folate
carrier (RFC) and subsequent conversion to poly-
glutamated forms catalyzed by the enzyme folylpoly-
glutamate synthetase (FPGS).12,13,15 The resultant
accumulated polyglutamates are up to 100 times more
potent as inhibitors of TS than the parent drugs19,20 and
are not readily effluxed from cells.12,13,21
The potential for tumor cells to acquire resistance to
folate-based antimetabolites22,23 including ZD1694 by
deletion or modification of either the RFC24 or FPGS24
prompted the search for new TS inhibitors which
require neither of these processes to express cell growth
inhibition in vitro.
We recently reported the design of molecules that
satisfy this profile in which the glutamic acid residue
(cf., e.g. 198583) was replaced by lipophilic alkyl, benzyl,
and heterocyclic benzyl amides.25 This early work
clearly showed that molecules such as 4a ,b (Scheme 1,
Table 1) were excellent inhibitors of the isolated TS
enzyme and were growth inhibitory to cells in culture.
Nevertheless, because of their relatively high lipophi-
licity (4a , log P ) 3.7) and the absence of a potentially
† Zeneca Pharma.
‡ Zeneca Pharmaceuticals.
§ Institute of Cancer Research.
X Abstract published in Advance ACS Abstracts, J anuary 1, 1996.
0022-2623/96/1839-0695$12.00/0 © 1996 American Chemical Society

696 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3
Hennequin et al.
Sch em e 1a
a
(a) 27-34a , method A; 27b, 30b, 32b, 34b, method B; 27c, method C; 35a -38a , method D; 35b, method E; 26a , method F; 39a ,
method G; 40a , method H.
Ta ble 1. In Vitro Activities of C2-Substituted Antifolates
IC50 (µM)
inhibn of
protection L1210:1565
L1210 cell at 2/10 IC50 relative
inhibn growth in % of control resistance
compd
R1
R2 R3
R4
mp, °C
formulaa
of TS
culture
growthb
ratiob
24a
24b
24c
25a
26a
27a
27b
27c
28a
29a
30a
30b
31a
31b
32a
32b
33a
OH
OH
OH
Cl
H
H
F
F
H
H
H
F
CH2CtCH 201-203 C27H23N5O5‚2.75H2Oc
0.30
0.072
0.08
1.21
0.64
0.62
0.14
0.084
4.2
4.8
0.7
0.7
2.4
6.7
73/75
92/71
CH3
CH3
H
H
H
CH3
CH3
H
H
H
CH3
H
CH3
H
CH3
H
CH2CtCH 254-256d C28H24FN5O5‚2NaCl
0.17
CH3
CH2CtCH ND1e
CH2CtCH 145-148 C30H29N5O6‚0.66H2O
CH2CtCH NDe
C29H28N6O4‚1.65TFA
CH2CtCH 136-138f C30H29FN6O4‚H2O
166-168 C26H24FN5O5‚0.5H2O
C27H22ClN5O4
68/6
O(CH2)2OCH3
N(CH3)2
N(CH3)2
N(CH3)2
NHCH3
2.7
0.68
0.58
7.8
>20
5
0.95
5.3
0.58
1.9
0.34
9
93/76
0.76
F
CH3
172-173 C28H29FN6O4‚0.8H2O
H
H
H
F
H
F
H
F
H
CH2CtCH 144d
CH2CtCH NDe
CH2CtCH NDe
CH2CtCH 120d
C28H26N6O4‚1.5TFA
NH2
C27H24N6O4‚1.5TFA‚H2O >1.7
N(CH3)(CH2CH2)N(CH3)2
N(CH3)(CH2CH2)N(CH3)2
N(CH2CH2)2NCH3
N(CH2CH2)2NCH3
N(CH2CH2)2O
N(CH2CH2)2O
C32H35N7O4‚1.1H2O
0.82
0.2
0.90
0.11
0.38
0.1
g
C33H36FN7O4‚0.7CHCl3
CH2CtCH 116-138 C32H33N7O4‚2H2O
76/6
CH2CtCH NDe
C33H34FN7O4‚2H2Oh
1.1
1.1
CH2CtCH 122-128 C31H30N6O5‚1.2H2O
CH2CtCH 146-148i C32H31FN6O5‚2H2O
CH2CtCH 148-150 C31H30N6O5‚1.5H2Oj
87/51
84/73
0.6
NCH2CH(OH)CH2CH2
34b
35a
35b
36a
37a
38a
39a
40a
4a m
4bm
4c
N(CH2CH2OH)2
S-2-pyrimidine
S-2-pyrimidine
S-2-pyrimidine-4-NH2
S-2-pyrimidine-4-OH
S-2-pyrimidine-4-NH2-6-OH
CN
NHSO2CH3
CH3
H
CH3
H
H
H
H
H
H
CH3
CH3
F
H
F
H
H
H
H
H
H
F
CH2CtCH 192-193 C32H33FN6O6‚1.5H2O
CH2CtCH 110-115 C31H25N7O4S‚0.66H2Ok
0.16
0.48
0.11
0.84
3.3
1.04
0.38
0.18
0.11
0.044
0.056
7.8
2
0.64
2.2
12
14
11
>20
1.4
0.26
0.076
85/63
81/80
CH2tCH
230-234 C32H26FN7O4S‚1.75H2O
0.65
CH2CtCH 130-144 C31H26N8O4S‚1.5H2O
CH2CtCH 181-185 C31H25N7O5S‚1.25H2O
CH2CtCH 212-216 C31H26N8O5S‚2.25H2Ol
CH2CtCH NDe
CH2CtCH NDe
C28H22N6O4‚0.9H2O
C28H26N6O6S‚0.25TFA
H
H
H
CH2CtCH 240-243 C27H23N5O4‚1.1H2O
CH2CtCH 256-258 C28H24FN5O4‚0.75H2O
85/60
-/66
92/83
0.30
1.0
F
CH3
C26H24FN5O4‚H2O
a
b
d
Anal. C, H, N except where stated otherwise. See biological evaluation section. c N: calcd, 12.80; found, 12.30. Decomposes at
this temperature. e ND ) not determined. f Softens >120 °C. N: calcd, 14.06; found, 13.6. N: calcd, 15.14; found, 14.4. Softens >136
°C. N: calcd, 14.16; found, 13.5. N: calcd, 16.25; found, 15.7. N: calcd, 16.90; found, 16.2. See ref 25.
g
h
i
j
k
l
m
ionized function, the compounds were relatively in-
soluble at physiological pH. Our attention therefore
focused on improving the aqueous solubility of this
series of molecules while retaining a submicromolar
level of cell growth inhibition in vitro. Our previous
work6-8 on analogues of CB3717 modified at the C2
position26 of the quinazolinone nucleus had given indi-
cations that C2-hydroxymethyl derivatives had im-
proved aqueous solubilities compared to their C2-methyl
counterparts.27 These early compounds were generally

Quinazoline Antifolates Thymidylate Synthase Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3 697
Sch em e 2a
a
(a) ClCH2CN, MeONa, MeOH; (b) CH3COONa, DMF; (c) NBS, CHCl3, (PhCOO)2.
Sch em e 3a
micromolar inhibitors of the TS enzyme but had not
been investigated further because of poor inhibition of
cell growth in vitro. This poor activity against the whole
cell we believe to be a result from either reduced affinity
for the reduced folate carrier (RFC)28 which in turn may
lead to a slow rate of polyglutamate formation. These
processes are not relevant to the activity of lipophilic
TS inhibitors,25 but the potential for improved solubility
of these quinazolinones by C2 modifications persuaded
us to explore this position in our current lipophilic
series. This paper now describes the synthesis, solubil-
ity properties, and biological activities of analogues of
4a ,b modified at the C2 position of the quinazolinone
and compares these new molecules to their glutamate-
containing equivalents and C2-methyl analogues.
Ch em istr y
The antifolates modified at the C2 position, listed in
Table 1 were prepared by the routes described in
Schemes 1 and 4. The strategy adopted involved a late
stage displacement of the benzylic halogen of the
chloromethyl derivatives 25a -c with a range of nucleo-
philes either by direct displacement of the chlorine atom
with amines in methanol at room temperature in the
case of 27-34a -c (methods A, B and C) or by reaction
with thiolates derived from the corresponding mercap-
topyrimidines (as in the case of the 2-[(2-pyrimidinylthi-
o)methyl] antifolate derivatives 35-38a -c (methods D
and E)). Ether 26a was obtained by coupling 25a with
sodium 2-methoxyethoxide, generated in situ by reaction
of 2-methoxyethanol with sodium metal (method F). In
preparing the nitrile 39a the solvent employed was
dimethyl sulfoxide (method G). The sulfonamide 40a
was obtained by direct sulfonylation of antifolate 29a
using methanesulfonyl chloride in dichloromethane at
room temperature (method H).
a
(a) Propargyl bromide, 2,6-lutidine, DMF, 95 °C; (b) (CF3CO)2O,
Na2CO3; (c) Na2CO3, MeI, THF; (d) NaHCO3, MeOH, H2O; (e)
CF3COOH, CH2Cl2; (f) DPPA, DMF, Et3N, m-nitrobenzylamine.
The p-aminobenzamides 20-22 were prepared as
described in Scheme 3 or as previously reported.6,11,25
Acylation of 12 with trifluoroacetic anhydride in dichlo-
romethane in the presence of CaCO3 provided 15.
Treatment of 15 with sodium hydride in DMF followed
by alkylation with methyl iodide and deprotection of the
aniline function under basic conditions led to 16.
Subsequent cleavage of tert-butyl esters 13, 14, and 16
was achieved by the classical treatment25 with CF3-
COOH and gave the free acids 17-19. Activation of the
acid function with diphenyl phosphorazidate (DPPA)
followed by reaction with m-nitrobenzylamine provided
anilino amides 20-22.
dimethylanthranilic acid215 with chloroacetonitrile in
methanol in the presence of catalytic amounts of sodium
methoxide at 0 °C proved to be an extremely clean
reaction when compared to the classical acid conditions6
and provided the 2-(chloromethyl)quinazolinone 5 and
7 in high yields. Temporary replacement of the chlorine
atom of 5 and 7 by an acetoxy group to give 6 and 8
was achieved in almost quantitative yield by using
sodium acetate in DMF. Functionalization of the C6
position of the quinazolinones 6 and 8 was achieved by
radical bromination of the C6-methyl substituent using
N-bromosuccinimide (NBS) in refluxing CHCl3 and
provided 9 and 10, respectively. In the case of 10,
regioselective bromination on the C6-methyl was con-
firmed by the presence of a NOE between H5 and the
C6-methylene hydrogens. Compound 10 proved to be
Chloromethyl derivatives 25a -c were crucial inter-
mediates in our strategy, and it was therefore necessary
to find an efficient route for their preparation (Schemes
2 and 4). Reaction of 5-methylanthranilic acid or 4,5-

698 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3
Hennequin et al.
Sch em e 4a
mM HEPES33 and supplemented with 10% horse serum
(L1210) or 10% foetal calf serum (L1210:1565). Incuba-
tion times for the 5 mL cultures were 48 (L1210) and
72 h (L1210:1565). The initial cell concentration was 5
× 104 mL-1. For the thymidine protection experiments
the L1210 cells were co-incubated with the compound
at concentrations of 2 and 10 times the IC50 values and
10 µM thymidine. In Table 1, the thymidine protection
results are expressed as percentage of the growth of
L1210 cells when grown in a drug-free culture medium
and the L1210:1565 relative resistance ratios are cal-
culated as the ratio of L1210:1565 IC50 over L1210 IC50.
All cell counts were performed with a Model ZM coulter
counter. The cell doubling times were 12 (L1210) and
24 h (L1210:1565).
Resu lts a n d Discu ssion
Early data from the glutamic acid series in which
substitution of the methyl group at the C2 position of
the quinazolinone nucleus led to a minor decrease (2-
6-fold) in the potency against the isolated TS enzyme
is shown by the comparison of 2 with 41 and 426 (Table
2). Other preliminary data (cf. 24a , 35a ; and 41, 42;
Tables 1 and Table 2, respectively) also suggested that
replacement of the glutamic acid side chain by the
m-nitrobenzylamide in these C2-modified analogues
resulted in a general retention of inhibitory activity
against the TS enzyme.
Analysis of the X-ray structures of CB3717/E. coli-
TS enzyme ternary complexes34-37 revealed an impor-
tant network of hydrogen bond interactions between the
quinazolinone nucleus and the enzyme in which the C2-
NH2 moiety of CB3717 binds with the carbonyl oxygen
of Ala263. A molecular surface analysis of this region
of the active site shows that this interaction reduces
access to a potentially large pocket accessible from the
C2 position of the quinazolinone. In the C2-methyl
series, where the hydrogen bond interaction with Ala263
does not exist, molecular modeling studies have shown
that minor rearrangements of the last three C-terminal
residues (Val262, Ala263, and Ile264)38 allow access to
this pocket without compromising the key binding
interactions37 between the C-terminal carboxylate of
Ile264, Arg21 and a conserved molecule of water with
N1 of the quinazoline (Figure 1).
a
(a) DMF, 2,6-lutidine, 80 °C; (b) 2 N NaOH, MeOH; (c) SOCl2,
CH2Cl2.
the only monobrominated product of this reaction
although minor amounts of the 6,6-dibromo derivative
were isolated during the purification. Introduction of
the acetoxymethyl function at the C2 position of the
quinazolinone allowed us to easily differentiate the
methylenes at C2 and C6 during the nucleophilic
displacements of the bromine atoms of 9 and 10 with
the p-aminobenzamides 20-22. This coupling was
conducted at 80 °C in DMF in the presence of 2,6-
lutidine and gave 23a -c in excellent yields (Scheme 4).
After deprotection of the hydroxyl groups, the chloro-
methyl derivatives 25a -c were generated from 24a -c
by treatment at room temperature with SOCl2 in
dichloromethane and used directly in the subsequent
nucleophilic displacement reactions (methods A, B, C,
D, E, and F, Scheme 1).
Biologica l Eva lu a tion
The initial biological results on the 2-hydroxymethyl
derivative were combined with the opportunities sug-
gested by this analysis of the X-ray data and prompted
us to investigate substitution at the C2 position of the
quinazolinone nucleus more broadly. Compounds 4a -c
were used as comparator structures. The C2-methyl
group (Scheme 1) was therefore substituted with O, N,
S, Cl, and CN functionalities selected for their potential
to access and bind in the C2 pocket and to improve the
overall solubility while retaining the m-nitrobenzyla-
mide substituent (Table 1).
In the 7,2′-unsubstituted26 series (Table 1) most of the
structural modifications led to molecules which inhib-
ited the TS enzyme at submicromolar levels (IC50 0.18-
4.2 µM) (Table 1), but when compared to CB3717 (1)
(IC50 0.02 µM)6 were 10-100-fold less potent. This
reduced activity is partially explained by the loss of two
key interactions in the enzyme/inhibitor complex: (1)
the hydrogen bond interaction between the 2-amino
group of 1 and the C-terminal residues6,34 and (2) the
The compounds prepared are listed in Table 1 and
were tested as inhibitors of isolated TS partially purified
from L1210 mouse leukaemia cells that overproduce
TS.29 The partial purification and assay method used
were as previously described and used a (()5,10-
methylenetetrahydrofolate concentration of 200 µM.29
The results are expressed as IC50 values, that is, the
concentration of compound that will inhibit the control
reaction rate by 50%. The compounds were also tested
for their inhibition of the growth of L1210 cells in
culture, and the results are expressed as the concentra-
tion of compound required to inhibit cell growth by 50%
(IC50). The L1210:1565 cell line30 has acquired resis-
tance to the antitumor antibiotic CI-920.31 Evidence
suggests that this agent enters cells via the reduced
folate mechanism and that the L1210:1565 line is
resistant due to a very much reduced drug uptake and
hence it is cross-resistant to MTX (∼200-fold).32 Both
cell lines were grown by suspension culture in RPMI
medium without sodium bicarbonate but containing 20

Quinazoline Antifolates Thymidylate Synthase Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3 699
Ta ble 2. In Vitro Activities of Acid-Containing Antifolates
IC50 (µM)
compd
R1
R2
R3
R5 a
ref
inhibn of TS
inhibn of L1210 cell growth in culture
1
2
41
42
43b
44
NH2
H
OH
H
H
H
H
CH3
H
H
H
H
H
F
(CH2)COOH
(CH2)2COOH
(CH2)2COOH
(CH2)2COOH
(CH2)2COOHc
m-NO2-benzyl
6
6
6
6
39
21
0.02
0.05
0.1
0.24
0.028
0.01
5.0
0.15
5
S-2-pyrimidine
N(CH2CH2)2NCH3
H
100
>20
9
H
a
b
S-isomer except otherwise stated. See ref 43. c R,S.
F igu r e 1. Stereodrawing of compound 35a modeled in the active site of E. coli-TS ternary complex. 35a is depicted with stippled
circles; key hydrogen bonds are depicted with dashes lines. The C-terminal last residues are highlighted in full circles.
hydrogen bond interaction between the R-carboxylic acid
of the glutamate residue and the enzyme.36 The con-
sequences of this latter interaction are illustrated by
comparison of compounds 4a and 44 (Tables 1 and 2),
where 44 is a 10-fold better enzyme inhibitor than 4a .
stituents, as in the ((dimethylamino)ethyl)methylamino
(30b) or bis(2-hydroxyethyl)amino (34b) derivatives
were also well tolerated by the enzyme and gave
compounds with IC50 values of 0.2 and 0.16 µM, respec-
tively. Figure 1 shows the docking of the C2-CH2-S-
pyrimidine derivative 35a into the ternary complex and
illustrates that the pyrimidine nucleus can easily reach
the C2 pocket without disturbing the fundamental
network of hydrogen bond interactions.
As reported by our group, modeling studies39,40 indi-
cated that a 7-methyl substituent could reinforce a
partially folded conformation of the p-aminobenzoate
(PABA) ring inclined at an angle of 65° to the quinazoli-
none, believed to be optimum for binding to the TS
enzyme. Moreover addition of a 2′-fluoro substituent
to such systems gives a further 2-3-fold enhance-
ment7,9,25 in TS inhibition and growth inhibition, pre-
sumably through stabilization of the almost planar
conformation in this region of the molecule due to a
hydrogen bond between the fluorine atom and the amide
N-H of the glutamate. When applied to the C2-modified
series, this double substitution also proved to be ben-
eficial. Comparison of 24b, 27b, 30b, 31b, 32b, and 35b
with 24a , 27a , 30a , 31a , 32a , and 35a , respectively,
clearly indicates that the fully substituted analogues are
consistently 4-8-fold better enzyme inhibitors (IC50
In nitrogen-containing C2 substituents, the tertiary
amine 27a is a 7- to >3-fold more potent TS inhibitor
than the primary or secondary amino derivatives 28a
and 29a . This result can be explained by the increased
solvation of the amine function of the latter two com-
pounds. Interestingly, analogues with large substitu-
ents such as N-methylpiperazine, morpholine, and
hydroxyproline (31-33a ) retained good enzyme inhibi-
tion and were equivalent to the smaller dimethylamino
derivative 27a , indicating that the enzyme can indeed
accommodate fairly bulky substituents in the C2 region
of the quinazolinone. This conclusion was further
substantiated by the mercaptopyrimidine derivatives
35a -b. The level of enzyme inhibition of these com-
pounds (IC50 0.48 and 0.11 µM) is only 2-4-fold lower
than the equivalent C2-methyl analoges (4a ,b, Table 1).
Attempts to gain further interactions with the enzyme
by substituting the pyrimidine nucleus with hydrogen
bond donor substituents (NH2, OH) failed and led to
∼2-7-fold reduction in the level of enzyme inhibition
(35a , 36a , 37a , 38a , Table 1). More flexible C2 sub-

700 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3
Hennequin et al.
Ta ble 3. Physicochemical Properties of C2-Modified
Antifolates
of up to 54-fold (measured at 3 days in phosphate buffer
at 25 °C (pH 7.4)) when compared to the C2-methyl
derivative 4a . Even with bulky substituents, e.g. the
mercaptopyrimidine nucleus 35a , a 10-fold increase in
solubility was achieved despite a slight increase in
lipophilicity. However further functionalization of the
pyrimidine ring by the introduction of hydrogen bond
donor and acceptor moieties reduced the solubility by
10-fold (comparison of 35a with 37a -38a , Table 3). This
is probably due to the combined effect of an increase in
the overall log P resulting from proximity effects
between the heteroatoms of the C2 substituent and the
quinazolinone nucleus, and an increase in the melting
point. C2 substitution with smaller hydrophilic sub-
stituents that easily form hydrogen bonds (e.g. OH in
24a and NH2 in 29a , Table 3) only marginally (2-3-
fold) increased the solubility compared to 4a despite
reducing the log P by ∼1.6 units. Substituents capable
of being protonated at physiological pH (e.g. mono-
amines 27a and 32a ) gave a 5-10-fold increase in
solubility. This improvement is smaller than expected
and is probably explained by the small proportion of the
protonated form present at pH 7 due to the reduced
basicity41 of the C2 amine nitrogen atom resulting from
the strong electron-withdrawing effect of the quinazoli-
none nucleus. The introduction of more basic amines
(e.g. N-methylpiperazine 31a 42), more fully protonated
at physiological pH, resulted in an increase in solubility
of 54-fold compared to their C2-methyl counterpart
(Table 3).
relative
relative
compd ClogPa solubilityb,c compd ClogPa solubilityb,c
4a
24a
27a
29a
31a
3.7d
2.17
NCf
2.22
3.68
1e
2
10
3
54
32a
35a
37a
38a
3.06
4.03
4.60
NCf
5
10
0.5
0.5
a
ClogP have been calculated by substracting from the measured
value of 4a the calculated contribution of 2-methyl-3,4-dihydro-
6-methyloxoquinazoline (calculated using ClogP version 3.64,
Daylight CIS, Irvine CA) and adding the calculated contributions
(using ClogP version 3.64) of the C2-substituted-3,4-dihydro-6-
b
methyl-4-oxoquinazoline. For each compound, relative solubility
is calculated as the ratio of its solubility over the solubility of 4a .
c Equilibrium solubility44 measured at 3 days in 0.01 M aqueous
d
NaH2PO4/0.15 M NaCl buffer at pH 7.4 and 25 °C. Measured
experimentally. e 4a : equilibrium solubility ) 0.12 µg/mL. f NC
) not calculated.
0.072-0.2 µM). The improvement in potency seen with
these changes is independent of the nature of the N10
substituent (propargyl or methyl) since 24c and 27c are
almost equipotent to 24b and 27b (Table 1).
In terms of their ability to inhibit in vitro cell growth
of the murine L1210 cell line as illustrated by 24a , 27a ,
32a , and 35a (Table 1), the 7,2′-unsubstituted deriva-
tives were equivalent to CB3717 (1) (IC50 3.5 µM). This
is, despite their being 10-100-fold less potent inhibitors
of the isolated TS enzyme. The 4-8-fold improvements
in enzyme inhibition observed in the C7-Me 2′-F deriva-
tives 24b-c, 27b-c, 30b, 31b, 32b, and 35b also
translates into improved in vitro potency against L1210
and leads to compounds which are ∼5-10-fold better
inhibitors of cell growth than 1. This improved expres-
sion of the enzyme inhibition into cell growth inhibition
is explained by the fact that these non-acid-containing
C2-modified TS inhibitors have lipophilicities in the
range where diffusion through the cell membrane
should be the preferred entry process (Table 3).
The high potency of these new C2-modified TS inhibi-
tors clearly distinguishes them from their glutamic acid
analogues in which, with the exception of the mono- and
the difluoro derivatives,6-8 the C2 modifications gener-
ally led to a dramatic loss in potency against the L1210
cell line. This conclusion is best reinforced by compari-
son of 31b and 43 (Tables 1 and 2), where the non-acid-
containing molecule 31b shows a >35-fold better growth
inhibition of L1210 in vitro despite being a 4-fold less
potent TS inhibitor than 43.
Con clu sion
A series of novel antifolate analogues of 4a in which
the C2-methyl group has been substituted with N, O,
S, Cl, and CN has been synthesized. These modifica-
tions result in compounds which are potent inhibitors
of the isolated TS enzyme, which do not require the RFC
for cell entry and which cannot be substrates for the
FPGS enzyme. The most interesting examples in this
series are active against the murine L1210 cell line with
IC50 values ranging from 0.3 to 1 µM. The advantage
in terms of this series when compared to the glutamic
acid counterparts is clearly demonstrated by the >50-
fold improved potency in cell growth inhibition of 31b
and 35a compared to 43 and 42, respectively.
The low aqueous solubility observed with lipophilic
quinazoline antifolates, which results from the removal
of carboxylic acid functions, has been overcome by the
incorporation of suitable C2 substituents. This is best
illustrated by the incorporation of an N-methylpipera-
zine nucleus into the C2-methyl group of 4a leading to
31a where a 54-fold increase in solubility has been
achieved.
The lack of dependence of these series of compounds
on the RFC to enter cells is strongly suggested by the
consistently low L1210/L1210:1565 IC50 relative resis-
tance ratios, measured on representative examples in
the 7,2′-unsubstituted and 7-methyl-2′-fluoro series
(Table 1).
Thymidine protection studies on representative ex-
amples confirmed that inhibition of TS is the predomi-
nant locus of cell growth inhibition. There are, however,
two exceptions to this: 25a and 31a where thymidine
protects against inhibition of cell growth only at twice
but not at 10 times the IC50 values. This inability to
completely protect the cells from high concentrations of
compound suggests that inhibition of an alternative
locus of action contributes to inhibition of cell growth
(Table 1).
Exp er im en ta l Section
Gen er a l P r oced u r es. All experiments were carried out
under an inert atmosphere and at room temperature unless
otherwise stated. N,N-Dimethylformamide (DMF) was puri-
fied by azeotropic distillation at 10 mmHg. Flash chromatog-
raphy was carried out on Merck Kieselgel 60 (Art. 9385). The
purities of compounds for test were assessed by analytical
HPLC on a Hichrom S5ODS1 Sperisorb column system set to
run isocratically with 60-70% MeOH + 0.2% CF3COOH in
H2O as eluent. TLCs were performed on precoated silica gel
plates (Merck Art. 5715), and the resulting chromatograms
were visualized under UV light at 254 nm. Melting points
were determined on a Kofler Block or with a Bu¨chi melting
As shown in Table 3, introduction of substituents into
the C2-methyl group resulted in an increase in solubility

Quinazoline Antifolates Thymidylate Synthase Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3 701
point apparatus and are uncorrected. The 1H NMR spectra
were determined in Me2SO-d6 solution (unless otherwise
stated) on a Bruker AM 200 (200 MHz) spectrometer or on a
J EOL J NM EX 400 (400 MHz). Chemical shifts are expressed
in unit of δ (ppm), and peak multiplicities are expressed as
follows: s, singlet; d, doublet; dd, doublet of doublet; t, triplet;
br s, broad singlet; m, multiplet. Fast atom bombardment
(FAB) mass spectra were determined with a VG MS9 spec-
trometer and Finnigan Incos data system, using Me2SO as the
solvent and glycerol as the matrix or with a Finnigan SSQ
7000 for the electrospray technique. With the appropriate
mode either positive or negative ion data could be collected.
NMR and mass spectra were run on isolated intermediates
and final products and are consistent with the proposed
structures. 11, 13, and 14 were prepared as described in ref
7.
2-(C h lo r o m e t h y l)-3,4-d i h y d r o -4-o x o -6,7-d i m e t h -
ylqu in a zolin e (7). 2-Amino-4,5-dimethylbenzoic acid hydro-
chloride (4.5 g, 22.3 mmol) was added to a solution of sodium
methoxide, prepared from Na (570 mg) in MeOH (50 mL). This
solution was then poured into a solution of methyl chloroacet-
imidate prepared from chloroacetonitrile (1.88 g, 24.9 mmol)
and sodium methoxide (prepared from Na (100 mg) in MeOH
(50 mL). After stirring for 30 min the reaction mixture was
heated at 80 °C for 2 h. After cooling to 0 °C, the resulting
solid was filtered, washed with MeOH and then water, and
dried under vacuum. The product was isolated as a white
solid: 3.9 g (80%); NMR (Me2SO-d6/CD3COOD) δ 2.35 (s, 3 H,
CH3), 2.37 (s, 3 H, CH3), 4.53 (s, 2 H, CH2), 7.47 (s, 1 H,
quinazoline 8-H), 7.87 (s, 1 H, quinazoline 5-H); MS (CI) m/z
223, 225 [MH]+. Anal. (C11H11ClN2O) C, H, N.
diluted with EtOAc and filtered. After evaporation of the
filtrate to dryness, the resulting oil was dissolved in MeOH
(100 mL) and 7% K2CO3 solution (50 mL) was added. Stirring
was maintained for 3 h, and the mixture was evaporated to
dryness. The resulting solid was partitioned between EtOAc
and H2O. The EtOAc solution was washed with brine, dried,
and evaporated. After purification by flash chromatography
using a gradient 30-50% Et2O in hexane as eluent, 16 was
isolated: 7.4 g (78%); mp 76-78 °C; NMR (Me2SO-d6/CD3-
COOD) δ 1.49 (s, 9 H, But), 2.71 (s, 3 H, CH3), 6.25 (dd, 1 H,
3-H), 6.38 (dd, 1 H, 5-H), 7.55 (dd, 1 H, 6-H); MS (CI) m/z 226
[MH]+. Anal. (C12H16FNO2) C, H, N.
2-F lu or o-4-(m eth yla m in o)ben zoic Acid (19). A solution
of 16 (4 g, 17.8 mmol) in 1/1 CH2Cl2/TFA (60 mL) was stirred
for 3 h. After evaporation of the solvent, the oil was triturated
in ether and the resulting solid was filtered off: 2.5 g (84%);
mp 184-186 °C; NMR (Me2SO-d6/CD3COOD) δ 2.72 (s, 3 H,
CH3), 6.28 (dd, 1 H, 3-H), 6.40 (dd, 1 H, 5-H), 7.65 (dd, 1 H,
6-H); MS (CI) m/z 187 [M + NH4]+. Anal. (C8H8FNO2‚0.16CF3-
COOH) C, H, N.
The same procedure was repeated with 13 and 14 to give
17 and 18, respectively.
1-[[N-[2-F lu or o-4-(p r op -2-yn yla m in o)ben zoyl]a m in o]-
m eth yl]-3-n itr oben zen e (21). To a solution of 18 (2.4 g, 12
mmol) and m-nitrobenzylamine (4.6 g, 25 mmol) in DMF (40
mL) at 0 °C was added dropwise DPPA (2.58 mL) followed by
Et3N (11.7 mL). After stirring for 16 h the solvent was
evaporated. The crude oil was dissolved in EtOAc. The
organic layers were washed with water and brine, dried, and
evaporated. After purification by flash chromatography using
a gradient 30-50% v/v EtOAc in CH2Cl2 as eluent, 21 was
isolated as a solid: 3.1 g (79%); mp 168-170 °C; NMR (Me2-
SO-d6/CD3COOD) δ 3.10 (s, 1 H, CtCH), 3.95 (s, 2 H,
CH2CtC), 4.56 (d, 2 H, NHCH2), 6.45 (dd, 1 H, 3′-H), 6.52 (dd,
1 H, 5′-H), 7.55 (dd, 1 H, 6′-H), 7.62 (dd, 1 H, NO2-benzyl 5-H),
7.80 (d, 1 H, NO2-benzyl 6-H), 8.15 (dd, 1 H, NO2-benzyl 4-H),
8.2 (s, 1 H, NO2-benzyl 2-H), 8.5 (dd, 1 H, HNCO); MS (FAB)
m/z 328 [MH]+. Anal. (C17H14FN3O3) C, H, N.
The same procedure was repeated with 2-amino-5-methyl-
benzoic acid to give 5 in 85% yield.
2-(Ac e t o x y m e t h y l)-3,4-d ih y d r o -4-o x o -6,7-d im e t h -
ylqu in a zolin on e (8). A solution of 7 (22.5 g, 0.10 mol) in
DMF (150 mL) containing sodium acetate (30 g, 0.36 mol) was
heated at 80 °C for 3 h. After evaporation of the solvent the
resulting solid was filtered, washed with petroleum ether and
then water, and dried under vacuum: 23 g (93%); mp 210-
212 °C dec; NMR (Me2SO-d6/CD3COOD) δ 2.13 (s, 3 H, CH3-
CO), 2.34 (s, 3 H, CH3), 2.36 (s, 3 H, CH3), 4.93 (s, 2 H, CH2O),
7.43 (s, 1 H, quinazoline 8-H), 7.86 (s, 1 H, quinazoline 5-H);
MS (CI) m/z 247 [MH]+. Anal. (C13H14N2O3‚0.8H2O) C, H, N.
The same procedure was repeated with 5 to give 6 in 89%
yield.
2-(Acetoxym eth yl)-6-(br om om eth yl)-3,4-dih ydr o-4-oxo-
7-m eth ylqu in a zolin on e (10). A mixture of 8 (2.46 g, 10
mmol), NBS (2.13 g, 12 mmol), and benzoyl peroxide (100 mg)
in CCl4 (75 mL) containing cyclohexene oxide (100 mg, 1 mmol)
was stirred under reflux and light for 2 h. On cooling an off-
white precipitate was obtained which was filtered off. The
organic layer was washed with water and brine, dried, and
evaporated to dryness. The residue was purified by flash
chromatography using a gradient of 30-50% v/v EtOAc in CH2-
Cl2 as eluent. The product was isolated as a white solid: 3.8
g (58%); NMR (CDCl3) δ 2.27 (s, 3 H, CH3CO), 2.57 (s, 3 H,
CH3), 4.61 (s, 2 H, CH2Br), 5.14 (s, 2 H, CH2O), 7.53 (s, 1 H,
quinazoline 8-H), 8.19 (s, 1 H, quinazoline 5-H); MS (CI) m/z
325, 327 [MH]+.
The same procedure was applied to 17 and 19 to give 20
and 22, respectively.
1-[[N-[4-[N-[[2-(Acetoxym eth yl)-3,4-d ih yd r o-7-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 oben zoyl]a m in o]m eth yl]-3-n itr oben zen e (23b).
A
mixture of 21 (1.68 g, 5.3 mmol), the bromomethyl compound
10 (1.73 g, 5.3 mmol), and CaCO3 (445 mg, 7.95 mmol) in DMF
(50 mL) was stirred at 80 °C for 18 h. After evaporation of
the solvent, the solid was triturated with water and filtered.
The crude product was purified by flash chromatography using
a gradient 1:1 to 1:9 v/v CH2Cl2/EtOAc as eluent to give 23b:
1.78 g (60%); NMR δ 2.11 (s, 3 H, CH3CO), 2.44 (s, 3 H, CH3),
3.2 (s, 1 H, CtCH), 4.30 (s, 2 H, CH2CtCH), 4.55 (d, 2 H,
NHCH2), 4.7 (s, 2 H, CH2N), 4.91 (s, 2 H, OCH2), 6.6 (dd, 1 H,
3′-H), 6.7 (d, 1 H, 5′-H), 7.5 (s, 1 H, quinazoline 8-H), 7.58 (t,
1 H, 6′-H), 7.62 (t, 1 H, NO2-benzyl 5-H), 7.72 (s, 1 H,
quinazoline 5-H), 7.75 (d, 1 H, NO2-benzyl 6-H), 8.1 (dd, 1 H,
NO2-benzyl 4-H), 8.15 (s, 1 H, NO2-benzyl 2-H), 8.55 (dd, 1 H,
NH); MS (FAB) m/z 572 [MH]+.
The same procedure was applied to 20 and 22 to give 23a
(78%) and 23c (44%), respectively.
The same procedure was repeated with 6 to give 9 in 64%
yield.
1-[[N-[4-[[3,4-Dih yd r o-2-(h yd r oxym et h yl)-7-m et h yl-4-
oxo-6-qu inazolin yl]-N-pr op-2-yn ylam in o]-2-flu or oben zoyl]-
a m in o]m eth yl]-3-n itr oben zen e (24b). A solution of 23b (50
mg, 0.08 mmol) in 1 N NaOH (5 mL) and MeOH (1 mL) was
stirred for 4 h. After acidification to pH 5 with 2 N HCl, the
suspension was centrifuged. The solid pellet was washed
thoroughly with H2O and freeze dried to give 24b as a white
solid: 40 mg (89%); NMR (Me2SO-d6/CF3COOD) δ 2.55 (s, 3
H, CH3), 3.28 (t, 1 H, CtCH), 4.4 (s, 2 H, CH2 CtC), 4.56 (s,
2 H, NHCH2), 4.70 (s, 2 H, CH2O), 4.80 (s, 2 H, CH2N), 6.65
(d, 1 H, 3′-H), 6.7 (d, 1 H, 5′-H), 7.62 (dd, 1 H, 6′-H), 7.65 (dd,
1 H, NO2-benzyl 5-H), 7.80 (d, 1 H, NO2-benzyl 6-H), 7.82 (s,
1 H, quinazoline 8-H), 7.88 (s, 1 H, quinazoline 5-H), 8.12 (dd,
1 H, NO2-benzyl 4-H), 8.2 (s, 1 H, NO2-benzyl 2-H), 8.62 (dd,
1 H, CONH); MS (FAB) m/z 530 [MH]+. Anal. (C28H24FN5O5‚
2NaCl) C, H, N.
ter t-Bu tyl 2-Flu or o-4-(tr iflu or oacetam ido)ben zoate (15).
To a solution of 127 (10 g, 47 mmol) in CH2Cl2 (250 mL)
containing Na2CO3 (15 g, 0.142 mol) was added trifluoroacetic
anhydride (19.9 g, 95 mmol) over 10 min. After stirring for 2
h, the solid was filtered off. The filtrate was washed with
water and brine and dried, and the solvent was evaporated.
The crude oil was purified by flash chromatography using a
gradient of 15-30% v/v Et2O in hexane as eluent to give 15:
13 g (89%); NMR δ 1.53 (s, 9 H, But), 7.6 (dd, 1 H, 3-H), 7.69
(dd, 1 H, 5-H), 7.86 (t, 1 H, 6-H), 11.63 (s, 1 H, NH); MS (CI)
m/z 325 [MH]+. Anal. (C13H13F4NO3) C, H, N.
ter t-Bu tyl 2-F lu or o-4-(m eth yla m in o)ben zoa te (16). To
a solution of 15 (13 g, 42 mmol) in DMF (200 mL) was added
Na2CO3 (70 g) followed by MeI (86 mL). After stirring
overnight the solvent was evaporated. The crude residue was

702 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3
Hennequin et al.
The same procedure was repeated with 23a ,c to give 24a
(74%) and 24c (70%), respectively.
5′-H), 7.6 (s, 2 H, quinazoline 5-H and 8-H), 7.62 (dd, 1 H,
6′-H), 7.65 (dd, 1 H, NO2-benzyl 5-H), 7.8 (d, 1 H, NO2-benzyl
6-H), 8.1 (d, 1 H, NO2-benzyl 4-H), 8.2 (s, 1 H, NO2-benzyl 2-H);
MS (FAB) m/z 533 [MH]+. Anal. (C28H29FN6O4‚0.8H2O) C,
H, N.
1-[[N -[4-[N -[[2-(Ch lor om e t h yl)-3,4-d ih yd r o-4-oxo-6-
qu in a zolin yl]m eth yl]-N-p r op -2-yn yla m in o]ben zoyl]a m i-
n o]m eth yl]-3-n itr oben zen e (25a ). A solution of 24a (200
mg, 0.40 mmol) in CH2Cl2 (5 mL) containing SOCl2 (86 µL)
was stirred for 1 h. After evaporation to dryness the solid was
triturated with ether and filtered off. The solid was purified
by flash chromatography using a gradient of 1:1 to 1:9 v/v CH2-
Cl2/EtOAc as eluent to give 25a : 180 mg (86%); NMR δ 3.25
(t, 1 H, CtCH), 4.35 (s, 2 H, CH2CtC), 4.52 (s, 2 H, CH2Cl),
4.55 (d, 2 H, NHCH2), 4.8 (s, 2 H, CH2N), 6.85 (d, 2 H, 3′-H
and 5′-H), 7.65 (dd, 1 H, NO2-benzyl 5-H), 7.67 (d, 1 H,
quinazoline 8-H), 7.80 (d, 4 H, 2′-H, 6′-H, quinazoline 7-H and
NO2-benzyl 6-H), 8.0 (s, 1 H, quinazoline 5-H), 8.15 (d, 1 H,
NO2-benzyl 4-H), 8.17 (s, 1 H, NO2-benzyl 2-H), 8.85 (t, 1 H,
NH); MS (FAB) m/z 516 [MH]+. Anal. (C27H22ClN5O4‚1.1H2O)
C, H, N.
The same procedure was applied to 24b,c to give 25b,c,
respectively, which were used in the next step without
purification. In the case of 25b and 25c the NMR and mass
spectra are consistent with those of the assigned structures.
1-[[N-[4-[N-[[3,4-Dih yd r o-2-[(d im eth yla m in o)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]a m in o]m eth yl]-3-n itr oben zen e (27a ). Meth od A. To
a saturated solution of dimethylamine in MeOH (15 mL) was
added 25a (150 mg, 0.29 mmol). After 2 h the solvent was
evaporated. The residue was triturated with ether, and the
resulting solid was filtered off and washed with water.
Purification was achieved by preparative HPLC using a
gradient 20:80 to 40:60 of CH3CN/H2O (0.1% CF3COOH) v/v.
The appropriate fractions were combined and the CH3CN was
removed by rotary evaporation. Lyophilization of the residual
solution gave 27a as a white solid: 71 mg (47%); NMR δ 2.98
(s, 6 H, NMe2), 3.15 (t, 1 H, CtCH), 4.3 (s, 4 H, CH2NMe2 and
CH2CtC), 4.55 (d, 2 H, NHCH2), 4.8 (s, 2 H, CH2N), 6.85 (d,
2 H, 3′-H and 5′-H), 7.62 (t, 1 H, NO2-benzyl 5-H), 7.65 (d, 1
H, quinazoline 8-H), 7.75 (d, 4 H, 2′-H, 6′-H, quinazoline 7-H
and NO2-benzyl 6-H), 8.05 (d, 1 H, quinazoline 5-H), 8.1 (d, 1
H, NO2-benzyl 4-H), 8.15 (s, 1 H, NO2-benzyl 2-H), 8.85 (t, 1
H, NH), 12.5 (s, 1 H, NH); MS (FAB) m/z 525 [MH]+. Anal.
(C29H28N6O4‚1.65CF3COOH) C, H, N.
1-[[N-[4-[N-[[3,4-Dih yd r o-7-m et h yl-4-oxo-2-[(2-p yr im -
id in ylt h io)m e t h yl]-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 oben zoyl]a m in o]m eth yl]-3-n itr oben -
zen e (35b). Meth od E. 2-Mercaptopyrimidine (61 mg, 0.54
mmol) was added to 60% NaH (27 mg) in DMF (15 mL). After
stirring for 30 min 25b (290 mg, 0.54 mmol) was added, and
stirring was continued for 24 h. After evaporation of the
solvent the residue was partitioned between EtOAc/water. The
organic layers were washed with water and brine, dried, and
evaporated. The residue was purified by flash chromatography
on silica using a gradient 25:75 to 60:40 v/v EtOAc/CH2Cl2 as
eluent: 60 mg (84%); NMR δ 2.43 (s, 3 H, CH3), 3.2 (s, 1 H,
CtCH), 4.29 (s, 2 H, CH2CtC), 4.38 (s, 2 H, SCH2), 4.57 (d, 2
H, NHCH2), 4.69 (s, 2 H, CH2N), 6.62 (d, 1 H, 5′-H), 6.7 (s, 1
H, 3′-H), 7.22 (t, 1 H, pyrimidine 5-H), 7.47 (s, 1 H, quinazoline
8-H), 7.5-7.65 (m, 2 H, 6′-H and NO2-benzyl 5-H), 7.72 (s, 1
H, quinazoline 5-H), 7.8 (d, 1 H, NO2-benzyl 6-H), 8.1 (d, 1 H,
NO2-benzyl 4-H), 8.2 (s, 1 H, NO2-benzyl 2-H), 8.5-8.6 (m, 1
H, NH), 8.65 (d, 2 H, pyrimidine 4-H and 6-H); MS (FAB) m/z
624 [MH]+. Anal. (C32H26FN7O4S‚1.75H2O) C, N; H: calcd,
4.54; found, 4.1.
The same procedure was repeated with 25a in place of 25b
to give 35a (66%) (method D). Applied to the appropriate
thiols, method D gave 36a (63%), 37a (56%), and 38a (66%).
1-[[N-[4-[N-[[3,4-Dih ydr o-2-[(2-m eth oxyeth oxy)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]a m in o]m eth yl]-3-n itr oben zen e (26a ). Meth od F . To
a suspension of 60% NaH (64 mg) in DMF (4 mL) was added
2-methoxyethanol (3 mL). After stirring for 30 min 25a (206
mg, 0.4 mmol) in DMF (2 mL) was added, and stirring was
continued for 18 h. After evaporation to dryness, the residue
was triturated with water and the resulting solid was filtered
off. Purification by flash chromatography using a gradient
2:98 to 4:96 v/v MeOH/CHCl3 as eluent gave 26a : 41 mg (18%);
NMR δ 3.15 (t, 1 H, CtCH), 3.25 (s, 3 H, OCH3), 3.5 (dd, 2 H,
OCH2), 3.65 (dd, 2 H, OCH2), 4.33 (d, 2 H, CH2CtC), 4.39 (s,
2 H, CH2O), 4.55 (d, 2 H, NHCH2), 4.8 (s, 2 H, ArCH2N), 6.85
(d, 2 H, 3′-H and 5′-H), 7.6 (d, 2 H, quinazoline 8-H and NO2-
benzyl 5-H), 7.75 (dd, 1 H, quinazoline 7-H), 7.8 (d, 1 H, NO2-
benzyl 6-H), 7.85 (dd, 2 H, 2′-H and 6′-H), 8.0 (d, 1 H,
quinazoline 5-H), 8.1 (d, 1 H, NO2-benzyl 4-H), 8.2 (s, 1 H,
NO2-benzyl 2-H), 12.0 (s, 1 H, NH); MS (FAB) m/z 556 [MH]+.
Anal. (C30H29N5O6‚0.66H2O) C, H, N.
The same procedure was repeated with the appropriate
amines to give 28a (39%), 29a (27%), 30a (32%), 31a (65%),
32a (53%), and 33a (31%).
1-[[N-[4-[N-[[3,4-Dih yd r o-2-[(d im eth yla m in o)m eth yl]-
7-m eth yl-4-oxo-6-qu in azolin yl]m eth yl]-N-pr op-2-yn ylam i-
n o]-2-flu or oben zoyl]am in o]m eth yl]-3-n itr oben zen e (27b).
Meth od B. To a solution of 25b (210 mg, 0.38 mmol) in MeOH
(2 mL) was added a saturated solution of dimethylamine in
MeOH (5 mL). After stirring at 80 °C for 30 min the solvent
was evaporated, and the residue was purified by flash chro-
matography on silica using 3:97 v/v MeOH/CHCl3 as eluent
to yield 27b: 120 mg (56%); NMR (Me2SO-d6/CF3COOD) δ 2.55
(s, 3 H, CH3), 2.97 (s, 6 H, (CH3)2N), 3.25 (s, 1 H, CtCH), 4.38
(s, 4 H, N-CH2CtC and Me2NCH2), 4.6 (s, 2 H, NHCH2), 4.8
(s, 2 H, CH2N), 6.65 (d, 1 H, 3′-H), 6.68 (d, 1 H, 5′-H), 7.60 (s,
1 H, quinazoline 8-H), 7.62 (dd, 1 H, 6′-H), 7.65 (dd, 1 H, NO2-
benzyl 5-H), 7.78 (s, 1 H, quinazoline 5-H), 7.85 (d, 1 H, NO2-
benzyl 6-H), 8.1 (d, 1 H, NO2-benzyl 4-H), 8.2 (s, 1 H, NO2-
benzyl 2-H); MS (ESI) 557 [MH]+. Anal. (C30H29FN6O4‚H2O)
C, H, N.
1-[[N -[4-[N -[[2-(Cya n om e t h yl)-3,4-d ih yd r o-4-oxo-6-
qu in a zolin yl]m eth yl]-N-p r op -2-yn yla m in o]ben zoyl]a m i-
n o]m eth yl]-3-n itr oben zen e (39a ). Meth od G. To a solu-
tion of 25a (155 mg, 0.3 mmol) in DMSO (8 mL) was added
KCN (78 mg, 12 mmol). After stirring for 3 h the solvent was
evaporated. The residue was taken into water, and the pH
was adjusted to 5 with acetic acid. The resulting solid was
filtered off and purified by flash chromatography using EtOAc
as eluent to give 39a : 33 mg (22%); NMR δ 3.15 (t, 1 H,
CtCH), 4.11 (s, 2 H, CH2
CN), 4.3 (s, 2 H, CH2CtC), 4.55 (d,
2 H, NHCH2), 4.8 (s, 2 H, CH2N), 6.85 (d, 2 H, 3′-H and 5′-H),
7.55-7.7 (m, 2 H, NO2-benzyl 6-H and NO2-benzyl 5-H), 7.75
(d, 4 H, 2′-H, 6′-H and quinazoline 7-H, 8-H), 8.0 (d, 1 H,
quinazoline 5-H), 8.1 (d, 1 H, NO2
-benzyl 4-H), 8.15 (s, 1 H,
The same procedure was repeated with the appropriate
amines to give 30b (35%), 31b (31%), 32b (63%), and 34b
(54%).
NO2-benzyl 2-H), 8.85 (t, 1 H, NHCO); MS (FAB) m/z 507
[MH]+. Anal. (C28H22N6O4‚0.9H2O) C, H, N.
1-[[N-[4-[N-[[3,4-Dih yd r o-2-[(N-m eth a n esu lfon a m id o)-
m eth yl]-4-oxo-6-qu in a zolin yl]m eth yl]-N-p r op -2-yn yla m i-
n o]ben zoyl]am in o]m eth yl]-3-n itr oben zen e (40a). Meth od
H. Methanesulfonyl chloride (32 µL, 0.4 mmol) was added to
a solution of 29a (200 mg, 0.4 mmol) in CH3CN (15 mL). After
addition of Et3N (56 µL, 0.4 mmol) the reaction mixture was
stirred for 4 h. After filtration to remove the precipitate the
filtrate was evaporated to dryness, the residue was triturated
with H2O, filtered off, and dried under vacuum. Purification
by preparative HPLC using a gradient 30:70 to 70:30 CH3CN/
H2O (0.1% CF3COOH) as eluent gave 40a : 40 mg (17%); NMR
δ 3.0 (s, 3 H, CH3SO2), 3.15 (s, 1 H, CtCH), 4.15 (s, 2 H, SO2-
1-[[N-[4-[N-[[3,4-Dih yd r o-2-[(d im eth yla m in o)m eth yl]-
7-m eth yl-4-oxo-6-qu in a zolin yl]m eth yl]-N-m eth yla m in o]-
2-flu or ob en zoyl]a m in o]m et h yl]-3-n it r ob en zen e (27c).
Meth od C. To a solution of 25c (210 mg, 0.4 mmol) in MeOH
(3 mL) was added 33% Me2NH in MeOH (7 mL). After stirring
2.5 h at 80 °C the solution was evaporated to dryness. The
residue was purified by flash chromatography using a gradient
MeOH/CHCl3 1:99 to 3:97 v/v as eluent: 89 mg (42%); NMR
(Me2SO-d6/CF3COOD) δ 2.5 (s, 3 H, CH3), 2.97 (s, 6 H, Me2N),
3.15 (s, 3 H, CH3N), 4.35 (s, 2 H, Me2NCH2), 4.58 (s, 2 H,
NHCH2), 4.78 (s, 2 H, CH2N), 6.55 (d, 1 H, 3′-H), 6.6 (d, 1 H,

Quinazoline Antifolates Thymidylate Synthase Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3 703
(14) Tomudex is a trademark, the property of ZENECA plc.
(15) J ackman, A. L.; Farrugia, D. C.; Gibson, W.; Kimbell, R.; Harrap,
K. R.; Stephens, T. C.; Boyle, F. T. ZD1694 (Tomudex): A new
Thymidylate Synthase Inhibitor with Activity in Colorectal
Cancer. Eur. J . Cancer, in press.
(16) Clarke, S. J .; J ackman, A. L.; J udson, I. R. The History of the
Development and Clinical Use of CB3717 and ICI D1694. In
Novel Approaches to Selective Treatments of Human Solid
Tumours: Laboratory and Clinical Correlation; Rustum, Y. M.,
Ed.; Plenum Press: New York, 1993: pp 277-287.
NHCH2), 4.3 (s, 2 H, CH2CtC), 4.55 (d, 2 H, NHCH2), 4.8 (s,
2 H, CH2N), 6.85 (d, 2 H, 3′-H and 5′-H), 7.63 (d, 2 H,
quinazoline 8-H and NO2-benzyl 5-H), 7.75 (dd, 1 H, quinazo-
line 7-H), 7.76 (d, 2 H, 2′-H and 6′-H), 7.8 (d, 1 H, NO2 benzyl
6-H), 8.0 (d, 1 H, quinazoline 5-H), 8.1 (d, 1 H, NO2-benzyl
4-H), 8.15 (s, 1 H, NO2-benzyl 2-H), 8.85 (t, 1 H, NH); MS
(FAB) m/z 575 [MH]+. Anal. (C28H26N6O6S‚0.25CF3COOH)
C, H, N.
(17) Zalcberg, J .; Cunningham, D.; Van Cutsem, E.; Francois, E.;
Schornagel, J . H.; Adenis, A.; Green, M.; Starkhammer, H.; Azab,
M. Good Antitumour Activity of the New Thymidylate Synthase
Inhibitor Tomudex (ZD 1694) in Colorectal cancer. Ann. Oncol.
1994, 5 (Suppl. 5), 133.
(18) Burris, H.; Von Hoff, D.; Bowen, K.; Heaven, R.; Rinaldi, D.;
Eckhardt, J .; Fields, S.; Campbell, L.; Robert, F.; Patton, S.;
Kennealey, G. A Phase II Trial of ZD1694, A Novel Thymidylate
Synthase Inhibitor, in Patients with Advanced Non-small Cell
Lung Cancer. Ann. Oncol. 1994, 5 (Suppl. 5), 133.
(19) Ward, W. H. J .; Kimbell, R.; J ackman, A. L. Kinetic Character-
istics of ICI D1694: A Quinazoline Antifolate which Inhibit
Thymidylate Synthase. Biochem. Pharmacol. 1992, 43 (9),
2029-2031.
(20) Bisset, G. M. F.; Paqwelczak, K.; J ackman, A. L.; Calvert, A. H.
Syntheses and Thymidylate Synthase Inhibitory Activity of the
Poly-γ-glutamyl Conjugates of N-[5-[N-(3,4-Dihydro-2-methyl-
4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl]-L-glutam-
ic Acid (ICI D1694) and Other Quinazoline antifolates. J . Med.
Chem. 1992, 35, 859-866.
(21) Gibson, W.; Bisset, G. M. F.; Marsham, P. R.; Kelland, L. R.;
J udson, I. R.; J ackman, A. L. The Measurement of Poly-
glutamate Metabolites of the Thymidylate Synthase Inhibitor,
ICI D1694, in Mouse and Human Cultured Cells. Biochem.
Pharmacol. 1993, 45 (4), 863-869.
(22) Henderson, G. B.; Strauss, B. P. Characteristics of a Novel
Transport System for Folate Compounds in Wild-Type Metho-
threxate Resistant L1210 Cells. Cancer Res. 1990, 50, 1709-
1714.
Ack n ow led gm en t. We would like to thank the
members of the Molecular Modelling group: Tony Slater
and Christine Lambert and the members of the Physical
methods group for the solubility measurements and the
logP calculations: J eff Morris, J et Longridge, Alan Wait,
and Pierre Bruneau. Also particular thanks to the
synthetic chemists involved in this chemistry: Anthony
P. Flynn and Rodney C. Smith.
Refer en ces
(1) Calvert, A. H.; Alison, D. L.; Harland, S. J .; Robinson, B. A.;
J ackman, A. L.; J ones, T. R.; Newell, D. R.; Siddik, Z. H.;
Wiltshaw, E.; McIlwain, T. J .; Smith, I. E.; Harrap, K. R. A
Phase I Evaluation of the Quinazoline Antifolate Thymidylate
Synthase Inhibitor, N10-Propargyl-5,8-dideazafolic Acid, CB3717.
J . Clin. Oncol. 1986, 4, 1245-1252.
(2) Calvert, A. H.; Newell, D. R.; J ackman, A. L.; Gumbrell, L. A.;
Sikora, E.; Grzelakowska-Sztabert, B.; Bishop, J . A. M.; J udson,
I. R.; Harland, S. J .; Harrap, K. R. Recent Preclinical and
Clinical Studies with the Thymidylate Synthase Inhibitor,
N10-Propargyl-5,8-dideazafolic Acid (CB3717). NCI Monogr.
1987, 5, 213-218.
(3) Bassendine, M. F.; Curtin, N. J .; Loose, H.; Harris, A. L.; J ames,
D. F. Induction of Remission in Hepatocellular Carcinoma with
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(4) Newell, D. R.; Siddik, Z. H.; Calvert, A. H.; J ackman, A. L.;
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(5) Newell, D. R.; Alison, D. L.; Calvert, A. H.; Harrap, K. R.;
Jarman, M.; Jones, T. R.; Manteuffel-Cymborowska, M.; O’Connor,
P. Pharmacokinetics of the Thymidylate Synthase Inhibitor N10
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(25) Marsham, P. R.; J ackman, A. L.; Barker, A. J .; Boyle, F. T.; Pegg,
S. J .; Wardleworth, J . M.; Kimbell, R.; O’Connor, B. M.; Calvert,
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(26) Numbering shown on generic formula of Table 1.
(27) Solubilities measured in 0.92 M aqueous NaH2PO4 buffer at pH
7.0 and 25 °C: 2, Sol ) 3.2 mg/mL; 41, Sol > 5 mg/mL. Also a
10-fold improvement of the solubility has been observed in favor
of the C2-hydroxymethyl derivative compared to that of the C2-
methyl counterpart in a series of closely related glutamate-
modified TS inhibitors (unpublished data).
(28) J ackman, A. L.; Kimbell, R.; Brown, M.; Brunton, L.; Bisset, G.
M. F.; Batvesias, V.; Marsham, P. R.; Hughes, L. R.; Boyle, F.
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tionship Between Structural Modifications and Polyglutamation.
Br. J . Cancer, in press.
(29) J ackman, A. L.; Alison, D. L.; Calvert, A. H.; Harrap, K. R.
Increased Thymidylate Synthase in L1210 Cells Possessing
Acquired Resistance to N10-Propargyl-5,8-dideazafolic Acid
(CB3717): Development, Characterization and Cross-Resistance
Studies. Cancer Res. 1986, 46, 2810-2815.
(30) The L1210:1565 cell line was the generous gift of Dr. D. W. Fry,
Warner-Lambert, Ann Arbor, MI.
(31) Synonyms: NSC 339638, 5,6-dihydro-6-(3,6,13-trihydroxy-3-
methyl-4-phosphonoxy-1,7,9,11-tridecatetraenyl)-2H-pyran-2-
one monosodium salt.
(6) Hughes, L. R.; J ackman, A. L.; Oldfield, J .; Smith, R. C.;
Burrows, K. D.; Marsham, P. R.; Bishop, J . A. M.; J ones, T. R.;
O’Connor, B. M.; Calvert, A. H. Quinazoline Antifolate Thymidy-
late Synthase Inhibitors: Alkyl, Substituted Alkyl, and Aryl
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(7) J ackman, A. L.; Marsham, P. R.; Thornton, T. J .; Bishop, J . A.
M.; O’Connor, B. M.; Hughes, L. R.; Calvert, A. H.; J ones, T. R.
Quinazoline Antifolate Thymidylate Synthase Inhibitors: 2′-
Fluoro-N10-propargyl-5,8-dideazafolic Acid and Derivatives with
Modifications in the C2 Position. J . Med. Chem. 1990, 33, 3067-
3071.
(8) Marsham, P. R.; Chambers, P.; Hayter, A. J .; Hughes, L. R.;
J ackman, A. L.; O’Connor, B. M.; Bishop, J . A. M.; Calvert, A.
H. Quinazoline Antifolate Thymidylate Synthase Inhibitors:
Nitrogen, Oxygen, Sulfur, and Chlorine Substituents in the C2
Position. J . Med. Chem. 1989, 32, 569-575.
(9) Marsham, P. R.; J ackman, A. L.; Oldfield, J .; Hughes, L. R.;
Thornton, T. J .; Bisset, G. M. F.; O’Connor, B. M.; Bishop, J . A.
M.; Calvert, A. H. Quinazoline Antifolate Thymidylate Synthase
Inhibitors: Benzoyl Ring Modifications in the C2-Methyl Series.
J . Med. Chem. 1990, 33, 3072-3078.
(10) Marsham, P. R.; Hughes, L. R.; J ackman, A. L.; Hayter, A. J .;
Oldfield, J .; Wardleworth, J . M.; Bishop, J . A. M.; O’Connor, B.
M.; Calvert, A. H. Quinazoline Antifolate Thymidylate Synthase
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(11) J ones, T. R.; Thornton, T. J .; Flinn, A.; J ackman, A. L.; Newell,
D. R.; Calvert, A. H. Quinazoline Antifolates Inhibiting Thymidy-
late Synthase: 2-Desamino Derivatives with Enhanced Solubil-
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(12) J ackman, A. L.; Newell, D. R.; Gibson, W.; J odrell, D. I.; Taylor,
G. A.; Bishop, J . A. M.; Hughes, L. R.; Calvert, A. H. The
Biochemical Pharmacology of the Thymidylate Synthase Inhibi-
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(ICI 198583). Biochem. Pharmacol. 1991, 42, 1885-1895.
(13) J ackman, A. L.; Taylor, G. A.; Gibson, W.; Kimbell, R.; Brown,
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(32) Fry, D. W.; Besserer, J . A.; Boriztki, T. J . Transport of the
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(33) Flow laboratories, Irvine, Scotland, U.K.
(34) Montfort, W. R.; Perry, K. M.; Fauman, E. B.; Finer-Moore, J .
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Biochemistry 1990, 29, 6964-6977.
(35) Matthews, D. A.; Villafranca, J . E.; J anson, C. A.; Smith, W.
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(42) 2-[(N-Methylpiperazin-4-yl)methyl]-3,4-dihydro-6-methyl-4-oxo-
quinazoline: pKa (MeN) ) 9.8.
(43) 43 was prepared by coupling 10 and diethyl N-[2-fluoro-4-(prop-
2-ynylamino)benzoyl]-L-glutamate7 as described in ref 7. Sub-
sequent functionalization of the C2 position was achieved as
described for compound 31b. 43: NMR (Me2SO-d6/CD3COOD)
δ 1.9-2.0 (m, 1 H, CH2), 2.0-2.2 (m, 1 H, CH2), 2.25 (t, 2 H, 2
CH2COOH), 2.5 (s, 3 H, CH3), 2.65 (s, 3 H, NCH3), 2.7-2.8 (s, 4
H, CH2N), 3.0-3.1 (s, 4 H, CH2N), 3.15 (s, 1 H, CtCH), 3.55 (s,
2 H, CH2N), 4.3 (s, 2 H, CH2CtC), 4.4 (dd, 1 H, CHCOOH), 4.75
(s, 2 H, NCH2), 6.65 (dd, 1 H, 3′-H), 6.7 (dd, 1 H, 5′-H), 7.52 (s,
(38) The E. coli-TS enzyme C-terminal residues Val-Ala-Ile are
replaced by Met-Ala-Val in the human enzyme.
(39) Marsham, P. R. Quinazoline Inhibitors of Thymidilate Syn-
thase: Potent New Anticancer agents. J . Heterocycl. Chem.
1994, 3, 603-613.
(40) Boyle, F. T.; Matusiak, Z. S.; Hughes, L. R.; Slater, A. M.;
Stephens, T. C.; Smith, M. N.; Brown, M.; Kimbell, R.; J ackman,
A. L. Substituted 2-Desamino-2-methylquinazolinones: A Series
of Novel Antitumor Agents. In Chemistry and Biology of
Pteridines and Folates; Ayling, J . E., Nair, M. G., Baugh, C. M.,
Eds.; Plenum Press: New York, 1993; pp 585-588.
1
H, quinazoline 8-H), 7.65 (dd, 1 H, 6′-H), 7.8 (s, 1 H,
quinazoline 5-H); MS (FAB) m/z 605 [MH]-. Anal. (C31H35
FN6O6‚1.3H2O) C, H, N.
-
(44) The solubilities have been measured by Dr. D. Leahy’s group of
Zeneca Pharmaceuticals.
(41) 2-[(Dimethylamino)methyl]-3,4-dihydro-6-methyl-4-oxoquinazo-
line: pKa (NMe2) ) 6.95.
J M950645N