Design and synthesis of potent non-polyglutamatable quinazoline antifolate thymidylate synthase inhibitors

Article abstract of DOI:10.1021/jm9803727

The synthesis is described of a series of analogues of the potent thymidylate synthase (TS) inhibitor, N-[4-[N-[(3,4-dihydr0-2,7-dimethyl-4- oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]2-fluorobenzoyl]-L-glutamic acid (4, ZM214888), in which the glutami

Full text of DOI:10.1021/jm9803727

J . Med. Chem. 1999, 42, 3809-3820
3809
Articles
Design a n d Syn th esis of P oten t Non -P olyglu ta m a ta ble Qu in a zolin e An tifola te
Th ym id yla te Syn th a se In h ibitor s
Peter R. Marsham,*,† J . Michael Wardleworth,† F. Thomas Boyle,† Laurent F. Hennequin,§ Rosemary Kimbell,‡
Melody Brown,‡ and Ann L. J ackman‡
AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, England, AstraZeneca, Centre de recherches,
Z. I. La Pompelle, BP 401, 51064 Reims, France, and Drug Development Section, Institute of Cancer Research,
15 Cotswold Road, Sutton, Surrey SM2 5NG, England
Received J une 18, 1998
The synthesis is described of a series of analogues of the potent thymidylate synthase (TS)
inhibitor, N-[4-[N-[(3,4-dihydro-2,7-dimethyl-4-oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]-
2-fluorobenzoyl]-L-glutamic acid (4, ZM214888), in which the glutamic acid moiety is replaced
by homologous amino acids and R-amino acids where the ω-carboxylate is replaced by
acylsulfonamides and acidic heterocycles. In general these modifications when compared to 4
gave compounds with increased potency as inhibitors of isolated TS and as cytotoxic agents
against murine tumor cell lines. The new compounds require transport by the reduced folate
carrier for entry into cells but are not converted intracellularly into polyglutamated species.
Agents with this profile are expected to show activity against tumors that are resistant to
classical antifolates due to low expression of folylpolyglutamate synthetase. The analogue (S)-
2-[4-[N-[(3,4-dihydro-2,7-dimethyl-4-oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]-2-fluo-
robenzamido]-4-(1H-1,2,3,4-tetrazol-5-yl)butyric acid (35, ZD9331) has been selected as a clinical
development candidate and is currently undergoing phase I studies.
Ch a r t 1
In tr od u ction
Tomudex (ZD1694, 1) (Chart 1),1,2 a new quinazoline-
based inhibitor of thymidylate synthase (TS), has
recently been introduced in a number of European
territories for the treatment of advanced colorectal
cancer. It is a highly potent cytotoxic agent in vitro and
shows in vivo antitumor activity in a range of preclinical
models3 without the unacceptable kidney toxicity4 as-
sociated with its predecessor, CB 3717 (2). In interna-
tional phase III studies in patients with previously
untreated advanced colorectal cancer, the efficacy and
acceptable safety profile of Tomudex was confirmed.5
Response rates, time to progression, and survival are
consistent with the published literature for 5-fluoro-
uracil and leucovorin.5 In addition there were reductions
in the incidence of certain potentially serious adverse
events and benefits in terms of improvements in quality
of life, performance status, and weight gain. All the
evidence suggests that the high cytotoxicity of Tomudex
is due to rapid intracellular localization via the reduced
folate carrier protein (RFC) and then extensive metabo-
lism by folylpolyglutamate synthetase (FPGS) to poly-
glutamates which are retained within cells and are 60-
70 times more potent as inhibitors of TS.6 Tumor cells
however may be resistant to classical folate-based
antimetabolites through reduced expression of FPGS7
or upregulation of the polyglutamate-hydrolyzing en-
zyme γ-glutamyl hydrolase.8 This fact has prompted the
search for a complementary class of agents which would
not be substrates for FPGS and hence insensitive to
γ-glutamyl hydrolase but would also still rely on the
RFC for cellular uptake. This requirement for RFC
† AstraZeneca, Alderley Park.
§ AstraZeneca, Reims.
‡ Institute of Cancer Research.
10.1021/jm9803727 CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/26/1999

3810 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Marsham et al.
Sch em e 1a
Schemes 2-5 illustrate variants of the general method,
and the synthesis of the remaining examples is de-
scribed in detail in the Experimental Section. The
corresponding ω-acylsulfonamide analogues 8-16 were
prepared from N-(benzyloxycarbonyl)-L-glutamic acid
R-benzyl ester (39) and the similarly protected R-ami-
noadipic, R-aminopimelic, and R-aminosuberic acids.
Reaction of 39 with methanesulfonamide in the pres-
ence of dicyclohexylcarbodiimide (DCCI) and 4-(di-
methylamino)pyridine (DMAP) afforded the γ-acylsul-
fonamide R-ester 40 (method B). The Cbz and benzyl
ester protecting groups were removed by catalytic
hydrogenation, and the resulting amino acid 41 was
condensed with 38. The target γ-acylsulfonamide ana-
logue was finally obtained by saponification of 42. An
alternative procedure11 (method F), used to remove the
POM protecting group from a number of these acylsul-
fonamide analogues (e.g. 11), involved treatment with
a saturated solution of ammonia in MeOH.
a
(method A): (a) (S)-2-aminoadipic acid dimethyl ester, Et3N,
DMF; (b) 2 N aq NaOH, MeOH then 2 N HCl.
substrate affinity stemmed in part from the observation
of the Sirotnak group that in murine tumor models9
compounds with favorable kinetic parameters for the
RFC may offer a tumor-selective advantage. Agents
with this biochemical profile should therefore be active
in tumors expressing (1) low levels or modified FPGS
expression and (2) high levels of γ-glutamyl hydrolase
and which are therefore resistant to folate-based anti-
metabolites that require polyglutamation for their cy-
totoxicity. The lack of prolonged drug retention through
polyglutamation may also allow for greater control over
the duration of TS inhibition.
A compound not subject to metabolic activation
through polyglutamation needs to have a high intrinsic
potency as a TS inhibitor. As a starting point modifica-
tions to the quinazoline antifolate ICI198583 (3)10 (the
more soluble, less toxic C2-methyl analogue of CB 3717)
were undertaken. The combined effect of the incorpora-
tion of 7-methyl and 2′-fluoro substituents gave the
analogue ZM214888 (4), a compound showing enhanced
inhibition of TS and an overall retention of growth
inhibition in cell lines.11 Moreover, the cytotoxicity of 4
results entirely from the parent monoglutamate since
7-methyl-substituted N10-propargyl quinazoline anti-
folates are not substrates for FPGS.12 Molecular model-
ing studies13 based on an analysis of the X-ray struc-
tures of E. coli TS enzyme ternary complexes of CB
371714,15 and its tetraglutamate derivative16 suggested
that tighter binding inhibitors may be available in this
class of compounds through extension of the glutamate
moiety into the dipeptide region of the ternary complex.
This paper describes the synthesis and biological activi-
ties of new analogues of ZM214888 that were prepared
to explore these hypotheses. This program has resulted
in compounds with improved TS and growth inhibitory
properties leading to the next generation of antitumor
TS inhibitors, which are not substrates for FPGS but
which still require RFC-mediated uptake into cells.
Reversal of the acylsulfonamide unit produced two
sets of analogues having two (17, 18) and three (20, 21)
methylene links, respectively. The racemic sulfonyl
chloride, 2-(benzyloxycarbonyl)-4-(chlorosulfonyl)bu-
tanoic acid benzyl ester (43), readily prepared18 from
DL-homocystine, was elaborated into 17 and 18. The pair
of homologous reversed acylsulfonamides 20 and 21 was
prepared (Scheme 2) as the pure S-enantiomers starting
from N-BOC-L-glutamic acid R-benzyl ester (47) which
was reduced by a modification of the method of Koko-
tos19 to the alcohol 48. Conversion of 48 to the pivotal
intermediate sulfonamide 51 was achieved via chlorina-
tion of the thioacetate 49 to the sulfonyl chloride 50
followed by treatment with ammonia in CHCl3. The
BOC protecting group was removed (CF3CO2H) and the
amino ester 52 condensed with 38 to give 53. Saponi-
fication of 53 yielded the δ-sulfonamide analogue 19.
Acylation of 53 with CH3COCl and PhCOCl (method I)
gave the δ-acylsulfonamides 20 and 21.
Incorporation of azole sulfides, sulfoxides, and sul-
fones as potential ω-acid mimics was achieved in four
series with one to four methylene spacer units. Our
initial attempt to prepare a system with a single
methylene spacer unit involved the reaction of the
disodium salt of 4-mercapto-1,2,3-triazole with either
the tosylate 5520 of Cbz-L-serine or the acrylate 5620
(Scheme 3). Both reactions gave the same racemic Cbz-
amino acid 57, implying that the reaction of 55 occurred
through the intermediacy of 56. Removal of the Cbz
protecting group with aqueous HBr was followed by
coupling to 38 and hydrolysis to the triazolyl sulfide 22.
This underwent oxidation with peracetic acid (method
J ) to the sulfoxide 23 as the RS,RS-diastereomeric
mixture. To avoid this racemization in the formation of
S-linked azole analogues, we investigated the reaction
of commercially available â-chloroalanine (60) with a
sulfur nucleophile, 5-mercaptotetrazole21 sodium salt
(61). Although the yield of the required product 62 was
only 21%, the product was the pure R-enantiomer,
confirming that the chlorine is displaced via an SN2
mechanism. The tetrazolyl sulfide analogue 26 with a
dimethylene spacer and its sulfoxide 27 were synthe-
sized as the pure S-enantiomers via the key intermedi-
ate 66 which was obtained by the nucleophilic displace-
ment of 5-chloro-1-tert-butyl-1H-tetrazole22 with the
Ch em istr y
The general method for the synthesis of these new
TS inhibitors containing chain-extended homologues of
glutamic acid is outlined in Scheme 1. The pentafluo-
rophenyl ester 3811 was condensed with dialkyl esters17
of the appropriate R-aminodicarboxylic acids followed
by alkaline hydrolysis of the ester function (method A).

Non-Polyglutamatable TS Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3811
Sch em e 2a
a
(a) EtOCOCl, Et3N, THF, -10 °C; (b) NaBH4, MeOH; (c) MsCl, Et3N, CH2Cl2; (d) KSCOCH3, acetone; (e) Cl2, HOAc, NaOAc, H2O; (f)
NH3, CHCl3; (g) CF3CO2H, CH2Cl2; (h) pentafluorophenyl ester 38, HOBT, Et3N, DMA; (i) aq NaOH, EtOH (method H); (j) CH3COCl,
Pri2EtN, DMAP, DMF (method I).
sodium salt of L-homocysteine (prepared in situ from
L-homocystine). Removal of the tert-butyl group using
HCl/anisole was accompanied by some rearrangement
to the 2-tert-butyl isomer 68, but separation of the
required amino acid 67 was readily achieved by pre-
parative reverse-phase chromatography.
analytical HPLC of 35 and 36 using a Chirobiotic T
In the trimethylene series sulfur-linked tetrazole (28,
29) and 1,2,4-triazolin-3-one (30-32) substituents were
introduced as ω-acid mimics. The key intermediate
mesylate 71 was prepared from (S)-2-phthalimidoglu-
taric acid 1-ethyl ester (69)23 via the alcohol 70 (Scheme
4). Reaction of 71 with 5-mercaptotetrazole21 and Et3N
followed by removal of the phthalimide (N2H4‚H2O)
gave the amino ester 73 which afforded 74 on coupling
with pentafluorophenyl ester 38 (method K). Oxida-
tion of 74 with 3-chloroperbenzoic acid (method L)
yielded the sulfoxide 75 as the S,RS-diastereomeric
mixture. Esters 74 and 75 were finally saponified
(method M) to 28 and 29. The sulfur-linked triazolinone
system was incorporated by treatment of mesylate 71
with the disodium salt of 5-mercapto-2H-1,2,4-triazol-
3(4H)-one. The tetramethylene-linked tetrazolyl sulfide
33 and its sulfoxide 34 were prepared via the amino
ester 82 from the ꢀ-bromobutyl acetamidomalonate ester
(79).24
(Teichoplanin) column27 demonstrated that both were
>99.7% enantiomerically pure. The higher homologue
37 with a δ-tetrazole as the acid mimic was elaborated
from the mesylate 86, which on treatment with KCN
gave the nitrile 87. Formation of the tetrazole ring by
heating 87 with NaN3 in the presence of NH4Cl in DMF
caused almost complete racemization of the asymmetric
center, since on completion of the synthesis (Scheme 5),
via the amino ester 89, the product 37 was shown by
chiral HPLC to be a 53:47 mixture of the S- and
R-enantiomers. Although the precise stage at which
racemization occurred was not confirmed, it is likely
that the conditions used for the nitrile to tetrazole
conversion were responsible, since the same racemiza-
tion occurred when the glutamine derivative 83 was
reacted with NaN3/NH4Cl in DMF under the same con-
ditions. Presumably the formation of traces of dimeth-
ylamine from DMF at the elevated temperature of the
reaction causes a deprotonation in the R-position of the
protected amino esters. In the event, the biological
activity of 37 was insufficiently interesting to justify
investigation of potential methods for the synthesis of
the pure S-enantiomer.
For the synthesis of the γ-tetrazolylglutamic acid
isostere ZD9331 (35), the pentafluorophenyl ester 38
was condensed with the γ-tetrazolyl amino ester 8425,26
and the product hydrolyzed with 2 N NaOH. To confirm
the enantiomeric purity of 35, the R-enantiomer 36 was
also prepared, utilizing the R-amino ester 85 (derived
from D-glutamine by the literature methods).25,26 Chiral

3812 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Marsham et al.
Sch em e 3a
tion times for the 5-mL cultures were 48 h (L1210 and
L1210:RD1694) and 72 h (L1210:1565). The initial cell
concentration was 5 × 104 mL-1. For the thymidine
protection experiments on compounds 4, 6, and 7 the
L1210 cells were co-incubated with the compound at a
concentration 10 times the IC50 values and 10 µM
thymidine. Under these conditions the cells had the
same rates of growth to those obtained in a drug-free
medium. In Table 1, the L1210:1565 and L1210:RD1694
relative resistance ratios are calculated as the ratio of
the mutant line IC50 over the L1210 IC50. All cell counts
were performed with a model ZM Coulter counter. The
cell doubling times were 12 h (L1210 and L1210:RD1694
)
and 18-24 h (L1210:1565).
Resu lts a n d Discu ssion
Extension of the chain length in a series of glutamate
homologues of 411 led to the R-aminoadipic acid (5),
R-aminopimelic acid (6), and R-aminosuberic acid (7)
containing homologues. These showed enhanced TS
inhibitory potencies when compared to 4 and were
particularly marked for 6 and 7 (6-fold). These results
support our hypothesis that extension of the glutamic
acid chain would give rise to enhanced binding to TS.
Growth inhibitory potency is retained in these homo-
logues, and the TS locus of this activity was confirmed
for three examples, 4, 6, and 7, by thymidine protection
studies. The relatively low activity of the three homo-
logues 5-7 in the L1210:1565 line supports the view
that uptake by the RFC remains the predominant mode
of cell entry. The low relative resistance ratios in the
L1210:RD1694 line, determined for 5-7, suggest that, as
expected, these homologues are not substrates for FPGS
and therefore do not require polyglutamation in order
to exhibit potent growth inhibition.
Replacement of the ω-carboxylic acids in this series
by acylsulfonamides also gives potent TS and growth
inhibitors. This replacement gives pKa values close to
those of the carboxylic acids but renders this end of the
molecule less liable to metabolism. These analogues (8-
16) retain at least the TS and growth inhibitory poten-
cies of the parent ω-carboxylic acids when the relatively
small methanesulfonamide or trifluoromethanesulfona-
mide is incorporated, but there is in general a reduction
in these potencies with the larger arylsulfonamides.
Most potent of these analogues is the methanesulfona-
mide 15 which shows equivalent activity to the ami-
nopimelate 6. The high IC50 values of representative
analogues 11 and 15 in the L1210:1565 line suggest that
a high affinity for the RFC plays an important role in
their cytotoxicities.
Reversal of the acylsulfonamide function in com-
pounds 17-21 appears to retain the TS inhibitory
potency. Again the small acetamido group gives better
binding than the larger benzamide. However compounds
in this series appear to have poorer cell penetration than
the conventional acylsulfonamides since their growth
inhibitory activities do not reflect their potencies against
the enzyme. This conclusion is borne out by the fact that
the IC50 value of 20 in the L1210:1565 line is only 10-
fold higher than that for the parent.
a
(a) MeOH, 45-50 °C; (b) 48% aq HBr; (c) pentafluorophenyl
ester 38, HOBT, Et3N; (d) method E; (e) CH3CO3H, CHCl3, MeOH
(method J ).
Biologica l Eva lu a tion
The compounds prepared are listed in Table 1 and
were tested as inhibitors of isolated TS partially purified
from L1210 mouse leukemia cells that overproduce the
enzyme.28 The partial purification and assay methods
used were as previously described and used a (()-5,10-
methylenetetrahydrofolate concentration of 200 µM.28
Results are expressed as the IC50, which is the concen-
tration of compound that inhibits the control reaction
rate by 50%. Compounds were also tested for their
ability to inhibit the growth of L1210 cells in culture,
and the results are expressed as the concentration of
compound required to inhibit cell growth by 50% (IC50).
The L1210:1565 cell line29 has acquired resistance tothe
antitumor antibiotic CI-920.30 Evidence suggests that
this agent enters cells via the RFC and that the L1210:
1565 line is resistant due to a very much reduced drug
uptake; hence it is cross-resistant to MTX (∼200-fold).31
The L1210:RD1694 cell line has acquired resistance to
Tomudex and is unable to polyglutamate this drug and
related folate analogues.32-34 These cell lines were
grown by suspension culture in RPMI medium without
sodium bicarbonate but containing 20 mM HEPES35 and
supplemented with 10% horse serum (L1210 and L1210:
RD1694) and 10% fetal calf serum (L1210:1565). Incuba-
A series of analogues of 4-7 has also been prepared
in which the ω-carboxylate has been replaced by sulfur-
linked heterocycles. Precedent for this approach was

Non-Polyglutamatable TS Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3813
Sch em e 4a
a
(a) EtOCOCl, Et3N, CH2Cl2; (b) NaBH4, MeOH; (c) MsCl, Et3N, CH2Cl2; (d) 5-mercapto-1H-1,2,3,4-tetrazole, Et3N, EtOH, DMF; (e)
N2H4‚H2O, EtOH; (f) CF3CO2H; (g) pentafluorophenyl ester 38 (method K); (h) 2 N NaOH, MeOH (method M); (i) 3-Cl-C6H4CO3H, CH2Cl2
(method L).
Sch em e 5a
proach was useful in our series of TS inhibitors. The
incorporation of sulfur-linked heterocycles of this type
as ω-acid isosteres (compounds 22-34) has in general
produced potent inhibitors of TS regardless of the nature
of the acid isostere or its position relative to the
R-carboxylate. No clear structure-activity relationship
has emerged for compounds in this series as there
appears to be a complex interplay of the spatial and
electronic parameters. However the most potent TS
inhibitors (28, 29, 31, and 32) have a trimethylene link,
but the precise pKa value of the heterocycle apparently
causes little variation in binding as measured by
enzyme inhibition. The TS inhibition of these com-
pounds translates well into growth inhibitory activity
although the L1210:1565 values suggest variable reli-
ance on the RFC for uptake into cells. For example the
thiotetrazole analogue 24 has a higher growth inhibitory
potency and a relative resistance ratio against the
L1210:1565 line than the corresponding thiotriazole 22
suggesting that it can be better internalized by the RFC.
a
(a) KCN, DMSO; (b) NaN3, NH4Cl, DMF, 90 °C; (c) N2H4‚H2O;
(d) CF3CO2H.
established by Chenard et al.24 who investigated ben-
zylthiotriazoles, benzylthiotriazolones, and their corre-
sponding sulfoxides and sulfones to give compounds of
equivalent activity to the corresponding carboxylic acids
as N-methyl-D-aspartic acid inhibitors. They clearly
established that not only were these functional groups
effective surrogates of carboxylic acids but also a wide
range of pKa values can be achieved through the choice
of combination of heterocycle and oxidation level of the
sulfur link. For a given heterocycle, the pKa can be
varied by up to 3 units between the sulfide and the
sulfone. We elected to evaluate whether such an ap-
It is also well-known that tetrazole analogues of
amino acids have pKa values that agree closely with
those of the corresponding amino acids.36 We found that
replacement of the γ-carboxylate in 4 with a tetrazole

3814 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Marsham et al.

Non-Polyglutamatable TS Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3815
pentafluorophenyl ester 38 (350 mg, 0.536 mmol), (S)-2-
aminoadipic acid dimethyl ester hydrochloride (131 mg, 0.58
mmol), and Et3N (800 µL, 5.8 mmol) in DMF (10 mL) was
stirred for 16 h. The resulting slightly cloudy solution was
evaporated to a gum which was purified by chromatography,
eluting with 50-80% v/v EtOAc in isohexane. The pure
product fractions were evaporated to a gum which on tritu-
ration with Et2O yielded the white solid pivaloyloxymethyl
dimethyl ester derivative of 4: 310 mg (84%).
to give 35 surprisingly resulted in an analogue that had
a 6-fold enhancement of TS and a 3-fold enhancement
of growth inhibition. The activity of 35 is highly ste-
reospecific since its enantiomer 36 is at least an order
of magnitude less potent against both parameters.
Interestingly in this case the synthesis of the higher
homologue 37 of 35 resulted in the retention of TS
inhibitory potency and a small drop in potency against
L1210 cell growth, allowing for the fact that 37 is a
racemic mixture.
This dimethyl ester (300 mg, 0.451 mmol) was stirred for
16 h in a mixture of 2 N aq NaOH (2.0 mL, 4.0 mmol) and
MeOH (5 mL). The solvent was evaporated, the residue was
dissolved in H2O (ca. 5 mL), and the solution was filtered into
a centrifuge tube. The filtrate was acidified (2 N HCl) to pH
5.0. The resulting white precipitate was isolated by centrifuga-
tion, washed (4×) with H2O, and vacuum-dried: 157 mg (61%);
NMR δ 1.57 (m, 2 H, CH2CH2CH2), 1.77 (m, 2 H, CHCH2CH2),
2.21 (t, 2 H, CH2CO2H), 2.30 (s, 3 H, CH3), 2.43 (s, 3 H, CH3),
3.20 (t, 1 H, CtCH), 4.28 (m, 3 H, CH2CtC & CH), 4.68 (br s,
2 H, ArCH2N<), 6.65 (m, 2 H, 3′-H & 5′-H), 7.43 (s, 1 H,
quinazoline 8-H), 7.57 (dd, 1 H, 6′-H), 7.70 (s, 1 H, quinazoline
More extensive in vitro studies37,38 on 35 (Zeneca
ZD9331) have shown that it has a 17-fold improvement
in Ki (0.44 nM vs 7.5 nM) against TS compared to its
parent glutamate 4 (ZM214888). The improved cellular
growth inhibition carries over into other cell lines (e.g.
the human lymphoblastoid W1L2).38 Relative resistance
ratios for ZD9331 in the L1210 variants 1565 and RD1694
confirm that it relies predominantly on RFC transport
into cells but not on polyglutamation to exhibit its
growth inhibition. Further support for affinity for the
RFC with ZD9331 is derived from transport competition
studies. In these ZD9331 inhibits the transport of [3H]-
methotrexate into L1210 and W1L2 cells with a Ki of
∼1 µM.37,38 That ZD9331 is not retained in cells is
confirmed in short exposure whole cell TS assay,37,38 and
it therefore only exerts its effects while it is present in
the extracellular medium. This feature, combined with
a rapid plasma clearance in mice,39 gives a greater
degree of control over inhibition of DNA synthesis in
preclinical models. ZD9331 was therefore chosen as the
most promising compound to emerge from the current
series with activity against a number of experimental
tumors.40-42 It is currently undergoing phase II clinical
studies to define more closely the toxicology and opti-
mum administration protocols.
5-H), 7.85 (m, 1 H, CONH); MS m/z 567 [MH]+. Anal. (C27H27
FN4O6‚2.5H2O) C, H, N.
-
N2-(Ben zyloxyca r b on yl)-N5-(m et h a n esu lfon yl)-L-glu -
ta m in e Ben zyl Ester (40). Meth od B. A solution of DCCI
(1.44 g, 7.0 mmol) in CH2Cl2 (5 mL) was added over 1 min to
a stirred mixture of Cbz-L-glutamic acid R-benzyl ester (2.0 g,
5.4 mmol), DMAP (860 mg, 7.0 mmol), and methanesulfona-
mide (660 mg, 7.0 mmol) in CH2Cl2 (20 mL) at 0 °C. The
mixture was then stirred for 24 h and filtered to remove a
white precipitate and the filtrate was evaporated. The crude
product was purified by chromatography, eluting sequentially
with 1:1 EtOAc/isohexane, EtOAc, and 5-10% MeOH in CH2-
Cl2 to give a colorless gum: 1.40 g (58%); NMR δ 1.90 (m, 2
H, CHCH2CH2CO), 2.20 (t, 2 H, CHCH2CH2CO), 2.95 (s, 3 H,
SO2CH3), 4.10 (m, 1 H, CH), 4.15 (br s, 1 H, CONHSO2), 5.05
(s, 2 H, OCH2Ph), 5.15 (s, 2 H, OCH2Ph), 7.32 (m, 10 H, 2 ×
C6H5), 7.82 (d, 1 H, OCONH).
N5-(Meth a n esu lfon yl)-L-glu ta m in e (41). Meth od C. A
solution of 40 (1.40 g, 3.13 mmol) in HOAc (60 mL) was stirred
for 2 h with 10% Pd/C (50 mg) in an atmosphere of H2. The
catalyst was filtered off through Celite. The filtrate was
evaporated to a gum which was triturated with 1:1 CH2Cl2/
Et2O to yield a pale brown solid: 200 mg (29%); NMR (D2O) δ
2.32 (m, 2 H, CHCH2CH2CO2H), 2.60 (t, 2 H, CHCH2CH2-
CO2H), 3.25 (s, 3 H, SO2CH3), 4.0 (m, 1 H, CH).
N2-[4-[N-[[3,4-Dih yd r o-2,7-d im eth yl-4-oxo-3-[(p iva loyl-
oxy)m eth yl]-6-qu in azolin yl]m eth yl]-N-pr op-2-yn ylam in o]-
2-flu or oben zoyl]-N5-(m eth a n esu lfon yl)-L-glu ta m in e (42).
Meth od D. A mixture of the amino acid 41 (150 mg, 0.67
mmol), the pentafluorophenyl ester 38 (300 mg, 0.45 mmol),
HOBT (10 mg), and Et3N (700 µL, 5.0 mmol) in DMF (10 mL)
and DMSO (20 mL) was stirred for 45 min to give a clear
yellow solution. This was concentrated by rotary evaporation
at 0.1 mmHg to a gum which was chromatographed using
5-50% MeOH in CH2Cl2 as eluent to yield a yellow solid: 150
mg (48%); NMR δ 1.10 (s, 9 H, But), 2.00 (m, 4 H, CH2CH2-
CO2H), 2.45 (s, 3 H, CH3), 2.55 (s, 3 H, CH3), 2.70 (s, 3 H,
SO2CH3), 3.20 (t, 1 H, CtCH), 4.15 (m, 1 H, CH), 4.30 (br s, 2
H, CH2CtC), 4.70 (br s, 2 H, ArCH2N<), 6.00 (s, 2 H, OCH2N),
6.65 (dd, 1 H, 3′-H), 6.68 (dd, 1 H, 5′-H),7.50 (s, 1 H,
quinazoline 8-H), 7.65 (dd, 1 H, 6′-H), 7.85 (s, 1 H, quinazoline
5-H).
N2-[4-[N-[(3,4-Dih yd r o-2,7-d im eth yl-4-oxo-6-qu in a zoli-
n yl)m et h yl]-N-p r op -2-yn yla m in o]-2-flu or ob en zoyl]-N5-
(m eth a n esu lfon yl)-L-glu ta m in e (8). Meth od E. A solution
of 42 (150 mg, 0.214 mmol) in EtOH (15 mL) was stirred with
2 N aqueous NaOH (8 mL, 16 mmol) for 30 min. The resulting
clear solution was evaporated to dryness and the residue was
dissolved in H2O (ca. 5 mL). The solution was filtered into a
centrifuge tube and acidified (2 N HCl) to pH 3.0. The pale
yellow precipitate was isolated by centrifugation, washed (4×)
with H2O, and vacuum-dried: 100 mg (60%); NMR δ 2.00 (m,
2 H, CH2CH2CONH), 2.30 (m, 2 H, CH2CH2CONH), 2.32 (s, 3
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) and N,N-
dimethylacetamide (DMA) were purified by azeotropic distil-
lation at 10 mmHg. Flash chromatography was carried out
on Merck Kieselgel 60 (Art. 9385). The purities of compounds
for test were assessed by analytical HPLC on a Hichrom
S5ODS1 Spherisorb column system set to run isocratically
with 60-70% MeOH + 0.2% CF3CO2H in H2O as eluent. TLC
was 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 with a Bu¨chi
melting point apparatus and are uncorrected. Most of the final
products did not give discrete melting points and softened with
decomposition over a wide temperature range. However spec-
tral and microanalytical data of all final products were
consistent with the proposed structures and formulas. The 1H
NMR spectra were determined in Me2SO-d6 solution (unless
stated otherwise) on a Bruker AM 200 (200 MHz) spectrom-
eter. Chemical shifts are expressed in units of δ (ppm), and
peak multiplicities are expressed as follows: s, singlet; d,
doublet; dd, doublet of doublets; t, triplet; br s, broad singlet;
m, multiplet. Fast atom bombardment (FAB) mass spectra
were determined with a VG MS9 spectrometer and Finnigan
Incos data system using Me2SO or MeOH as the solvent and
glycerol as the matrix. With the appropriate mode either
positive or negative ion data could be collected. NMR and mass
spectra were run on all isolated intermediates and final
products and are consistent with the proposed structures.
(S)-2-[N-[4-[N-[(3,4-Dih yd r o-2,7-d im eth yl-4-oxo-6-qu i-
n a zolin yl)m et h yl]-N-p r op -2-yn yla m in o]-2-flu or ob en z-
oyl]a m in o]a d ip ic Acid (5). Meth od A. A mixture of the

3816 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Marsham et al.
H, CH3), 2.43 (s, 3 H, CH3), 3.05 (s, 3 H, SO2CH3), 3.20 (t, 1 H,
CtCH), 4.30 (m, 3 H, CH & CH2CtC), 4.68 (br s, 2 H,
ArCH2N<), 6.60 (m, 2 H, 3′-H & 5′-H), 7.60 (s, 1 H, quinazoline
8-H), 7.70 (dd, 1 H, 6′-H), 7.92 (s, 1 H, quinazoline 5-H);
MS m/z 584 [M - H]-. Anal. (C27H28FN5O7S‚3NaCl‚2.5H2O)
C, H, N.
6.65 (m, 2 H, 3′-H & 5′-H), 7.47 (s, 1 H, quinazoline 8-H), 7.62
(dd, 1 H, 6′-H), 7.75 (s, 1 H, quinazoline 5-H), 7.90 (dd, 1 H,
CONH); MS m/z 584 [M - H]-. Anal. (C27H28FN5O7S‚1.2NaCl‚
0.5NH4Cl) C, H, N.
(S)-2-[N-(ter t-Bu t oxyca r b on yl)a m in o]-5-h yd r oxyp en -
ta n oic Acid Ben zyl Ester (48). A solution of N-(tert-butoxy-
carbonyl)-L-glutamic acid R-benzyl ester (13.5 g, 40 mmol) in
anhydrous THF (200 mL) was stirred at -10 °C. Et3N (5.61
mL, 40 mmol) was added followed by ethyl chloroformate (3.81
mL, 40 mmol). After 10 min, powdered NaBH4 (4.52 g, 0.12
mol) was added in one portion. MeOH (400 mL) was then
added dropwise over 10 min, keeping the temperature between
-10 and 0 °C. The reaction mixture was stirred below 0 °C
for a further 10 min, allowed to warm to room temperature,
and acidified with 1 N HCl (80 mL). The organic solvents were
removed from the mixture by rotary evaporation and the
residue was partitioned between EtOAc (500 mL) and H2O
(200 mL) with acidification of the aqueous phase to pH 4.0.
The EtOAc solution was washed (2×) with brine, dried, and
evaporated to give crude 48 as a colorless viscous oil (14.28
g): NMR δ 1.35 (s, 9 H, But), 1.30-1.80 (m, 4 H, CH2CH2),
3.35 (t, 2 H, CH2OH), 3.98 (m, 1 H, CH), 5.09 (d, 2 H, OCH2-
Ph), 7.25 (d, 1 H, CONH), 7.36 (br, 5 H, ArH); MS m/z 324
[MH]+. This oil was used without purification for the prepara-
tion of 49.
(S)-5-(Acet ylt h io)-2-[N-(ter t-b u t oxyca r b on yl)a m in o]-
p en ta n oic Acid Ben zyl Ester (49). Et3N (1.15 mL, 8.20
mmol) was added to a stirred solution of 48 (1.36 g, 4.10 mmol)
in CH2Cl2 (15 mL) at 0 °C. Methanesulfonyl chloride (478 µL,
6.15 mmol) was added dropwise over 10 min. Stirring was
continued for 2 h in the ice bath. The solution was washed
with ice-cold H2O, dried, and evaporated to a red oil (1.69 g).
A solution of this oil in acetone (10 mL) was stirred 48 h with
KSCOCH3 (935 mg, 8.2 mmol). The dark brown reaction
mixture was filtered through Celite and the filter cake was
washed well with acetone. The filtrate was evaporated to
dryness and the residue was purified by chromatography,
eluting with 0-10% EtOAc in CH2Cl2 to give a pale brown
gum: 1.348 g (86%); NMR δ 1.38 (s, 9 H, But), 1.45-1.80 (m,
4 H, CH2CH2), 2.30 (s, 3 H, COCH3), 2.82 (t, 2 H, CH2S), 4.00
(m, 1 H, CH), 5.11 (d, 2 H, OCH2Ph), 7.30 (d, 1 H, CONH),
7.35 (br, 5 H, ArH); MS m/z 382 [MH]+.
(S)-5-(Ch lor osu lfon yl)-2-[N-(ter t-bu toxyca r bon yl)a m i-
n o]p en ta n oic Acid Ben zyl Ester (50). A solution of 49 (1.33
g, 3.54 mmol) and NaOAc (2.90 g, 35.4 mmol) in HOAc (20
mL) and H2O (4 mL) was stirred below 10 °C. Liquid Cl2 (2
mL) was condensed into a separate tube at -78 °C. The cooling
bath was removed and the chlorine was slowly blown as a gas
by a stream of argon into the reaction mixture. After 10 min
argon was blown through the mixture for 10 min to remove
excess Cl2 and the solvent was evaporated. The residue was
partitioned between EtOAc (2 × 25 mL) and H2O (20 mL). The
EtOAc solution was washed with brine, dried, and evaporated
to the yellow oily sulfonyl chloride 50, 1.57 g, contaminated
with ca. 5% HOAc: NMR δ 1.37 (s, 9 H, But), 1.50-1.90 (m, 4
H, CH2CH2), 2.50 (t, 2 H, CH2SO2), 3.96 (m, 1 H, CH), 5.11 (d,
2 H, OCH2Ph), 7.30 (d, 1 H, CONH), 7.36 (br, 5 H, ArH). This
product was used without purification in the next stage.
(S)-5-(Am in osu lfon yl)-2-[N-(ter t-bu toxyca r bon yl)a m i-
n o]p en ta n oic Acid Ben zyl Ester (51). A solution of 50 (742
mg, 1.83 mmol) in CHCl3 was added dropwise over 10 min to
a stirred saturated solution of NH3 in CHCl3 (10 mL) below
10 °C. The mixture was stirred for 30 min, allowed to warm
to room temperature, and evaporated to dryness. The residue
was partitioned between EtOAc (2 × 25 mL) and H2O. The
EtOAc solution was washed with brine, dried, and evaporated
to a pale yellow gum: 577 mg (82%); NMR δ 1.37 (s, 9 H, But),
1.60-1.90 (m, 4 H, CH2CH2), 3.03 (t, 2 H, CH2SO2), 4.00 (m,
1 H, CH), 5.12 (d, 2 H, OCH2Ph), 6.76 (br, 2 H, NH2), 7.32 (d,
1 H, CONH), 7.35 (br, 5 H, ArH).
(S )-2-[[4-[N -[(3,4-Dih yd r o-2,7-d im e t h yl-4-oxo-6-q u i-
n azolin yl)m eth yl]-N-pr op-2-yn ylam in o]-2-flu or oben zoyl]-
a m in o]-5-[N -(m e t h a n e su lfon yl)a m in o]ca r b oxyp e n t a -
n oic a cid (11). Meth od F . (S)-2-[[4-[N-[[3,4-Dihydro-2,7-di-
methyl-4-oxo-3-[(pivaloyloxy)methyl]-6-quinazolinyl]methyl]-
N-prop-2-ynylamino]-2-fluorobenzoyl]amino]-5-[N-(methane-
sulfonyl)amino]carboxypentanoic acid (720 mg, 1.0 mmol) was
dissolved in a saturated solution of ammonia in MeOH (200
mL). After 16 h the solution was evaporated to dryness and
the residue was triturated with EtOAc. The white solid product
was dissolved in 1 N aqueous NaOH (5 mL). This solution was
filtered into a centrifuge tube and acidified (2 N HCl) to pH
3.0. The white precipitate was isolated by centrifugation,
washed (3×) with H2O, and vacuum-dried: 560 mg (82%);
NMR δ 1.50 (m, 2 H, CH2CH2CH2CONH), 1.70 (m, 2 H, CH2-
CH2CH2CONH), 2.05 (t, 2 H, CH2CH2CH2CONH), 2.32 (s, 3
H, CH3), 2.42 (s, 3 H, CH3), 2.85 (s, 3 H, SO2CH3), 3.21 (t, 1 H,
CtCH), 4.15 (m, 1 H, CH), 4.27 (br s, 2 H, CH2CtC), 4.69 (br
s, 2 H, ArCH2N<), 6.64 (m, 2 H, 3′-H & 5′-H), 7.41 (s, 1 H,
quinazoline 8-H), 7.61 (dd, 1 H, 6′-H), 7.69 (s, 1 H, quinazoline
5-H), 7.84 (dd, 1 H, CONH); MS m/z 600 [MH]+. Anal. (C28H30
FN5O7S‚0.75NH4Cl‚1.75H2O) C, H, N.
-
(RS)-2-(Ben zyloxyca r bon yla m in o)-4-(a m in osu lfon yl)-
bu ta n oic Acid Ben zyl Ester (44). A solution of the sulfonyl
chloride 4318 (11.0 g, 25.8 mmol) in CHCl3 (110 mL) was added
dropwise to a saturated solution of ammonia in CHCl3 (110
mL) below 10 °C. A white precipitate rapidly formed. After a
further 30 min at room temperature the reaction mixture was
evaporated to dryness and the residue was partitioned between
EtOAc (2 × 250 mL) and H2O (250 mL). The EtOAc solution
was dried and evaporated to a yellow solid. The solid was
stirred for 1 h in isohexane (250 mL), filtered off, washed with
isohexane, and vacuum-dried to an orange solid: 9.95 g (93%);
NMR δ 2.14 (m, 2 H, CH2CH2SO2NH2), 3.04 (m, 2 H, CH2CH2-
SO2NH2), 4.30 (m, 1 H, CH), 5.05 (s, 2 H, CH2Ph), 5.15 (s, 2
H, CH2Ph), 6.85 (s, 2 H, SO2NH2), 7.35 (m, 10 H, C6H5); MS
m/z 407 [MH]+. Anal. (C19H22N2O6S‚0.5H2O) C, H; N: calcd,
6.7; found, 6.2.
(RS)-2-(Ben zyloxyca r bon yla m in o)-4-(N-a cet yla m in o-
su lfon yl)bu ta n oic Acid Ben zyl Ester (45). Meth od G. A
solution of 44 (2.03 g, 5.0 mmol) in DMF (30 mL) was stirred
for 16 h with acetyl chloride (470 µL, 6.6 mmol), DMAP (810
mg, 6.6 mmol), and Pri EtN (1.15 mL, 6.6 mmol). The DMF
2
was removed by rotary evaporation at 0.1 mmHg followed by
azeotropic removal of traces of DMF by rotary evaporation of
added xylene. The residue was stirred for 10 min with 2 N
HCl (50 mL) and extracted with EtOAc (2 × 75 mL). The
EtOAc solutions were washed with brine, dried, and evapo-
rated. The crude oily product was purified by chromatography,
eluting with 3:1 EtOAc/isohexane to yield a viscous yellow
oil: 1.30 g (58%); NMR δ 1.98 (s, 3 H, NHCOCH3), 2.13 (m, 2
H, CH2CH2SO2NH2), 3.46 (m, 2 H, CH2CH2SO2NH2), 4.31 (m,
1 H, CH), 5.05 (s, 2 H, CH2Ph), 5.15 (s, 2 H, CH2Ph), 6.85 (s,
2 H, SO2NH2), 7.35 (m, 10 H, C6H5); MS m/z 449 [MH]+.
(R S )-2-[[4-[N -[(3,4-D ih y d r o -2,7-d im 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]am in o]-4-(N-acetylam in osu lfon yl)bu tan oic Acid (17).
The benzyl ester 45 (1.30 g, 2.90 mmol) was hydrogenated
according to method C to give (RS)-2-amino-4-(N-acetylami-
nosulfonyl)butanoic acid which was condensed (method D) with
38 to yield (RS)-2-[[4-[N-[[3,4-dihydro-2,7-dimethyl-4-oxo-3-
[(pivaloyloxy)methyl]-6-quinazolinyl]methyl]-N-prop-2-yny-
lamino]-2-fluorobenzoyl]amino]-4-(N-acetylaminosulfonyl)bu-
tanoic acid: 680 mg (33%). This was hydrolyzed (method F)
to 17: 504 mg (76%); NMR δ 1.98 (s, 3 H, NHCOCH3), 2.27
(m, 2 H, CH2CH2SO2NH), 2.33 (s, 3 H, CH3), 2.45 (s, 3 H, CH3),
3.08 (t, 1 H, CtCH), 3.44 (m, 2 H, CH2CH2SO2NH), 4.31 (br
s, 2 H, CH2CtC), 4.50 (m, 1 H, CH), 4.72 (br s, 2 H, ArCH2N<),
(S)-5-(Am in osu lfon yl)-2-[N-[4-[N-[[3,4-d ih yd r o-2,7-d i-
m et h yl-4-oxo-3-[(p iva loyloxy)m et h yl]-6-q u in a zolin yl]-
m et h yl]-N-p r op -2-yn yla m in o]-2-flu or ob en zoyl]a m in o]-
p en ta n oic Acid Ben zyl Ester (53). CF3CO2H (1 mL) was

Non-Polyglutamatable TS Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3817
added to a solution of 51 (250 mg, 0.64 mmol) in CH2Cl2 (3
mL). The mixture was swirled to give a homogeneous solution
which was set aside for 1 h. The solvent was evaporated and
the gummy residue was dried for 2 h at 0.1 mmHg. The crude
amino ester CF3CO2H salt 52 was dissolved in DMA (5 mL)
and stirred for 16 h with the pentafluorophenyl ester 38 (330
mg, 0.5 mmol), Et3N (700 µL, 5.0 mmol), and HOBT (10 mg).
The mixture was evaporated to dryness and the residue was
partitioned between EtOAc (2 × 20 mL) and H2O (10 mL), with
acidification (2 N HCl) of the aqueous layer to pH 1.0. The
EtOAc solution was washed with brine, dried, and evaporated.
The residue was chromatographed, eluting with 0-6% EtOH
in CH2Cl2 to yield a white foam: 240 mg (59%); NMR δ 1.11
(s, 9 H, But), 1.65-1.90 (m, 4 H, CH2CH2), 2.46 (s, 3 H, CH3),
2.58 (s, 3 H, CH3), 2.98 (t, 2 H, CH2SO2), 3.22 (t, 1 H, CtCH),
4.30 (br s, 2 H, CH2CtC), 4.45 (m, 1 H, CH), 4.72 (br s, 2 H,
ArCH2N<), 5.14 (s, 2 H, OCH2Ph), 6.00 (s, 2 H, OCH2N), 6.64
(m, 2 H, 3′-H & 5′-H), 6.75 (br, 2 H, NH2), 7.34 (br, 5 H, ArH),
7.48 (s, 1 H, quinazoline 8-H), 7.56 (dd, 1 H, 6′-H), 7.75 (s, 1
H, quinazoline 5-H), 8.20 (dd, 1 H, CONH); MS m/z 762 [MH]+.
Anal. (C39H44FN5O8S‚3H2O) C, H, N.
(RS)-2-Am in o-3-(3H -1,2,3-t r ia zol-4-ylt h io)p r op a n oic
Acid Tr ieth yla m m on iu m Sa lt (58). A mixture of 57 (1.70
g, 5.28 mmol) and 48% aqueous HBr (50 mL) was stirred for
2 h at 25 °C. The reaction mixture was extracted with CH2Cl2
and the aqueous solution was evaporated to dryness at 0.1
mmHg: NMR δ (D2O) 3.67 (m, 2 H, CH2), 4.46 (m, 1 H, CH),
8.22 (br s, 1 H, CH). The residue was dissolved in H2O (25
mL). The solution was filtered, treated with Et3N (2.0 mL, 14.2
mmol), and evaporated at 0.1 mmHg to yield the crude Et3N
salt of 58 (1.5 g), an off-white solid, which was used im-
mediately in the condensation with the pentafluorophenyl
ester 38.
(RS,RS)-2-[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 or oben -
zoyl]am in o]-3-(3H-1,2,3-tr iazol-4-ylsu lfin yl)pr opan oic Acid
(23). Meth od J . Peracetic acid (32% w/w in HOAc, 250 µL)
was added to a stirred solution of 22 (650 mg, 1.18 mmol) in
CHCl3 (100 mL) and MeOH (50 mL). Stirring was continued
for 16 h. The solution was evaporated down to a thick white
slurry which was sucked dry in a sinter. The white solid was
vacuum-dried: 650 mg (70%); NMR (400 MHz) δ (Me2SO-d6/
HOAc-d6) 2.50 (s, 3 H, CH3), 2.60 (s, 3 H, CH3), 3.22 (t, 1 H,
CtCH), 3.50-3.85 (m, 2 H, CH2SO), 4.34 (br s, 2 H, CH2Ct
C), 4.63, 4.84 (2 × m, 1 H, CH), 4.78 (br s, 2 H, ArCH2N<),
6.64 (m, 2 H, 3′-H & 5′-H), 7.60 (dd, 1 H, 6′-H), 7.65 (s, 1 H,
quinazoline 8-H), 7.75 (s, 1 H, quinazoline 5-H), 8.55, 8.62 (2
(S)-5-(Acet yla m in osu lfon yl)-2-[N-[4-[N-[[3,4-d ih yd r o-
2,7-d im eth yl-4-oxo-3-[(p iva loyloxy)m eth yl]-6-qu in a zoli-
n yl]m eth yl]-N-pr op-2-yn ylam in o]-2-flu or oben zoyl]am in o]-
p en ta n oic Acid Ben zyl Ester (54). Meth od I. Redistilled
acetyl chloride (45 µL, 0.63 mmol) was added over 5 min to a
× br, 1 H, triazole-H); MS m/z 566 [MH]+. Anal. (C26H24
-
stirred mixture of 53 (238 mg, 0.312 mmol), Pri EtN (109 µL,
2
FN7O5S‚CHCl3‚CH3OH) H, N; C: calcd, 46.8; found, 47.3.
0.63 mmol), and DMAP (77 mg, 0.63 mmol) in DMA (3 mL).
Stirring was continued for 16 h and the resulting solution was
evaporated to dryness. The residue was partitioned between
EtOAc (2 × 10 mL) and H2O (10 mL). The EtOAc solution was
dried and evaporated. The crude product was purified by
chromatography, eluting with 0-6% EtOH in CH2Cl2 to afford
a sticky off-white solid: 221 mg (88%); NMR δ 1.12 (s, 9 H,
But), 1.65-2.00 (m, 4 H, CH2CH2), 1.95 (s, 3 H, COCH3), 2.46
(s, 3 H, CH3), 2.58 (s, 3 H, CH3), 3.24 (t, 1 H, CtCH), 3.35 (t,
2 H, CH2SO2), 4.32 (br s, 2 H, CH2CtC), 4.45 (m, 1 H, CH),
4.72 (br s, 2 H, ArCH2N<), 5.15 (s, 2 H, OCH2Ph), 6.00 (s, 2
H, OCH2N), 6.63 (m, 2 H, 3′-H & 5′-H), 7.34 (br, 5 H, ArH),
7.50 (s, 1 H, quinazoline 8-H), 7.54 (dd, 1 H, 6′-H), 7.75 (s, 1
H, quinazoline 5-H), 8.22 (dd, 1 H, CONH); MS m/z 804 [MH]+.
(R)-2-Am in o-3-(1H-1,2,3,4-tetr a zol-5-ylth io)p r op a n oic
Acid (62). A mixture of â-chloroalanine‚HCl (1.60 g, 10 mmol),
5-mercapto-1H-1,2,3,4-tetrazole21 (1.10 g, 10.8 mmol), and
Bu3P (100 µL) in 2 N aqueous NaOH (20 mL) was stirred 1 h
at 80 °C. The reaction mixture was cooled and acidified (2 N
HCl) to pH 3.0 and the solution was passed through a column
of octadecyl-functionalized silica gel (Aldrich), eluting with
0.2% aqueous CF3CO2H. The fractions containing the product
were pooled and concentrated by rotary evaporation to yield
25
a white solid: 400 mg (21%); mp 150 °C dec; [R]D -64.0°
(MeOH); NMR δ 3.50, 3.57 (2 × dd, 2 × 1 H, CH2), 4.19 (dd, 2
H, CH), 7.65 (br, 3 H, NH2 + NH); MS m/z 188 [M - H]-.
Anal. (C4H7N5O2S‚H2O) C, H, N, S.
(S)-2-Am in o-4-(1-ter t-bu tyl-1H-1,2,3,4-tetr azol-5-ylth io)-
p r op a n oic Acid (66). A mixture of l-homocystine (2.0 g, 7.4
mmol) and Bu3P (4.0 mL, 16.0 mmol) in 2 N aqueous NaOH
(16.0 mL) was stirred for 30 min. 1-tert-Butyl-5-chloro-1H-
1,2,3,4-tetrazole22 (2.60 g, 16.2 mmol) was added and stirring
was continued for 48 h. The reaction mixture was partitioned
between Et2O (100 mL) and H2O (100 mL). The aqueous layer
was acidified (HOAc) to pH 5.0 and passed through a column
of octadecyl-functionalized silica gel, eluting with H2O then
70:30:0.2 MeOH/H2O/CF3CO2H. The fractions containing the
product were pooled and concentrated by rotary evaporation
to afford the white solid CF3CO2H salt: 3.9 g (70%); NMR
(D2O) δ 1.88 (s, 9 H, But), 2.59 (m, 2 H, CHCH2), 3.66 (m, 2 H,
CH2S), 4.22 (m, 1 H, CH); MS m/z 372 [M - H + CF3CO2H]-.
(S)-2-Am in o-4-(1H -1,2,3,4-t et r a zol-5-ylt h io)p r op a n oic
Acid (67) a n d (S)-2-Am in o-4-(2-ter t-bu tyl-2H-1,2,3,4-tet-
r a zol-5-ylth io)p r op a n oic Acid (68). A mixture of the amino
acid CF3CO2H salt 66 (3.9 g, 10.45 mmol), anisole (2.5 mL,
23.0 mmol), and concd HCl (50 mL) was stirred at 80-90 °C
for 2 h and evaporated to dryness. The residue was partitioned
between Et2O (100 mL) and H2O (100 mL). The aqueous
solution was adjusted (2 N NaOH) to pH 3.0 and passed
through a column of octadecyl-functionalized silica gel, eluting
with 0.2% aqueous CF3CO2H. Fractions containing the first
product to be eluted from the column were pooled and
evaporated to dryness and the residue was dissolved in MeOH.
Evaporation of this solution gave 67 as the white amorphous
CF3CO2H salt: 1.40 g (42%); NMR (D2O) δ 2.55 (m, 2 H,
CHCH2), 3.60 (m, 2 H, CH2S), 4.32 (dd, 1 H, CH); MS m/z 202
[M - H]-. The second product to be eluted was 68, obtained
as the CF3CO2H salt: 1.80 g (46%); NMR (D2O) δ 1.89 (s, 9 H,
But), 2.51 (m, 2 H, CHCH2), 3.51 (dd, 2 H, CH2S), 4.25 (d, 1 H,
CH); MS m/z 372 [M - H + CF3CO2H]- and 258 [M - H]-.
(S)-5-(Acet yla m in osu lfon yl)-2-[N-[4-[N-[(3,4-d ih yd r o-
2,7-d im 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]-2-flu or oben zoyl]a m in o]p en ta n oic Acid (20).
Meth od H. The benzyl ester 54 (215 mg, 0.27 mmol) was
stirred for 2 h in a mixture of aqueous NaOH (2.4 mL, 2.4
mmol) and EtOH (6 mL). The reaction mixture was evaporated
to dryness and the residue was dissolved in H2O (4 mL). The
solution was filtered into a centrifuge tube and acidified (2 N
HCl) to pH 3.0. The gelatinous precipitate was isolated by
centrifugation, washed (3×) with H2O, and freeze-dried to a
fluffy white solid: 130 mg (76%); NMR δ 1.60-2.00 (m, 4 H,
CH2CH2), 1.95 (s, 3 H, COCH3), 2.31 (s, 3 H, CH3), 2.43 (s, 3
H, CH3), 3.22 (t, 1 H, CtCH), 3.35 (t, 2 H, CH2SO2), 4.30 (br
s, 2 H, CH2CtC), 4.35 (m, 1 H, CH), 4.69 (br s, 2 H, ArCH2N<),
6.65 (m, 2 H, 3′-H & 5′-H), 7.43 (s, 1 H, quinazoline 8-H), 7.55
(dd, 1 H, 6′-H), 7.70 (s, 1 H, quinazoline 5-H), 7.96 (dd, 1 H,
CONH); MS m/z 598 [M - H]-. Anal. (C28H30FN5O7S‚1.75H2O)
C, H, N.
(RS)-2-[N-(Ben zyloxyca r bon yl)a m in o]-3-(3H-1,2,3-tr i-
a zol-4-ylth io)p r op a n oic Acid (57). A solution of methyl
2-[N-(benzyloxycarbonyl)amino]acrylate (56)20 (22.0 g, 93.6
mmol) in MeOH (50 mL) was stirred with 4-mercapto-1H-1,2,3-
triazole disodium salt hexahydrate (38.0 g, 0.15 mol) at 45-
50 °C for 3 h. The solvent was evaporated and the residue was
partitioned between EtOAc (500 mL) and 2 N HCl (200 mL).
The EtOAc solution was washed with brine, dried, and
evaporated. Chromatography of the residue, eluting with 4:1
EtOAc/MeOH, yielded a complex mixture of high Rf products
followed by 57 as a yellow gum: 4.40 g (15%); NMR δ 3.12,
3.43 (2 × dd, 2 × 1 H, CH2S), 4.02 (m, 1 H, CH), 5.00 (s, 2 H,
OCH2Ph), 6.90 (d, 1 H, CONH), 7.32 (br, 5 H, ArH), 7.79 (s, 1
H, triazole-H); MS m/z 321 [M - H]-.

3818 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Marsham et al.
Eth yl (S)-5-Hyd r oxy-2-p h th a lim id op en ta n oa te (70). A
solution of (S)-2-phthalimidoglutaric acid 1-ethyl ester (69)23-
(60.8 g, 0.20 mmol) in anhydrous THF (250 mL) was stirred
mechanically using a PTFE paddle and cooled to -10 °C. Et3N
(41.1 mL, 0.30 mol) was added followed by ethyl chloroformate
(23.7 mL, 0.25 mol). Stirring was continued for 10 min and
powdered NaBH4 (22.68 g, 0.60 mol) was added in one portion.
MeOH (200 mL) was added dropwise over 10 min, keeping the
temperature between -10 and 0 °C (ca u tion : vigorous
evolution of hydrogen gas occurred). Stirring was continued
while the temperature of the reaction mixture rose to 20 °C.
H2O (80 mL) was carefully added and the pH was adjusted (2
N HCl) to 7.0. The organic solvents were removed by rotary
evaporation and the aqueous residue was partitioned between
EtOAc (2 × 200 mL) and H2O (200 mL). The EtOAc solution
was washed with brine, dried, and evaporated to a yellow oil.
The above procedure was repeated on the same scale and the
crude products (total yield 111 g) from the two experiments
were combined and chromatographed using 4:1 isohexane/
EtOAc as eluent to yield a colorless viscous oil, 50.2 g, which
was shown to be 84% pure 70 by HPLC: NMR (CDCl3) δ 1.23
(t, 3 H, OCH2CH3), 1.58 (m, 2 H, CH2CH2CH2), 2.32 (m, 2 H,
CHCH2), 3.63 (t, 2 H, CH2OH), 4.20 (q, 2 H, OCH2CH3), 4.87
(dd, 1 H, CH), 7.75 (m, 2H, ArH), 7.83 (m, 2 H, ArH); MS m/z
292 [MH]+. The alcohol 70 was used in the next stage without
further purification.
(400 mL) with acidification (2 N HCl) of the aqueous phase to
pH 1.0. The organic solution was washed (2×) with brine and
dried and the solvent was evaporated. Chromatographic
purification, eluting with 5-15% MeOH in CH2Cl2, gave a pale
brown foam: 4.72 g (33%); NMR δ 1.12 (s, 9 H, But), 1.16 (t,
3 H, OCH2CH3), 1.75 (m, 2 H, CH2CH2CH2), 1.86 (m, 2 H,
CHCH2), 2.50 (s, 3 H, CH3), 2.57 (s, 3 H, CH3), 3.11 (t, 2 H,
CH2S), 3.22 (br s, 1 H, CtCH), 4.08 (q, 2 H, OCH2CH3), 4.31
(br s, 2 H, CH2CtC), 4.35 (m, 1 H, CH), 4.71 (br s, 2 H,
ArCH2N<), 6.00 (s, 2 H, OCH2N), 6.60 (dd, 1 H, 3′-H), 6.66
(dd, 1 H, 5′-H), 7.48 (s, 1 H, quinazoline 8-H), 7.55 (dd, 1 H,
6′-H), 7.76 (s, 1 H, quinazoline 5-H), 8.10 (dd, 1 H, CONH).
(S,RS)-2-[N-[4-[N-[[3,4-Dih yd r o-2,7-d im et h yl-4-oxo-3-
[(p iva loyloxy)m eth yl]-6-qu in a zolin yl]m eth yl]-N-p r op -2-
yn ylam in o]-2-flu or oben zoyl]am in o]-5-(1H-1,2,3,4-tetr azol-
5-ylsu lfin yl)p en ta n oic Acid Eth yl Ester (75). Meth od L.
A solution of the sulfide 74 (4.71 g, 6.53 mmol) in CH2Cl2 (150
mL) was stirred with 3-chloroperbenzoic acid (1.35 g of 80%,
6.67 mmol) for 16 h. The reaction mixture was applied directly
to a silica gel column which was eluted with 7.5% MeOH in
CH2Cl2. The fractions containing pure product by TLC were
evaporated to give 75 as a white foam: 1.74 g (36%); NMR δ
1.13 (s, 9 H, But), 1.17 (t, 3 H, OCH2CH3), 1.68 (m, 2 H,
CH2CH2CH2), 1.90 (m, 2 H, CHCH2), 2.45 (s, 3 H, CH3), 2.57
(s, 3 H, CH3), 3.08 (dd, 2 H, CH2SO), 3.20 (br, 1 H, CtCH),
4.08 (q, 2 H, OCH2CH3), 4.30 (br s, 2 H, CH2CtC), 4.35 (m, 1
H, CH), 4.70 (br s, 2 H, ArCH2N<), 5.99 (s, 2 H, OCH2N), 6.59
(dd, 1 H, 3′-H), 6.63 (dd, 1 H, 5′-H), 7.47 (s, 1 H, quinazoline
8-H), 7.55 (dd, 1 H, 6′-H), 7.75 (s, 1 H, quinazoline 5-H), 8.13
(dd, 1 H, CONH); MS m/z 737 [MH]+. Evaporation of the less
pure fractions yielded a further 2.66 g of product, 80% pure
by HPLC.
Eth yl (S)-2-P h th alim ido-5-(1H-1,2,3,4-tetr azol-5-ylth io)-
p en t a n oa t e (72). Methanesulfonyl chloride (5.00 mL, 64.4
mmol) was added dropwise to a stirred solution of the alcohol
70 (15.0 g of 84% pure, 43.3 mmol) in CH2Cl2 (250 mL) at 0-5
°C. The reaction mixture was stirred for 2 h at this temper-
ature when TLC indicated complete reaction. The solution was
washed with ice-cold H2O (2 × 125 mL). The aqueous layers
were back-extracted with CH2Cl2. The CH2Cl2 solution was
dried and evaporated below 30 °C to give the crude mesylate
71 (20.2 g). This intermediate was immediately dissolved in a
mixture of EtOH (250 mL) and DMF (20 mL) and stirred for
60 h with 5-mercapto-1H-1,2,3,4-tetrazole21 (5.78 g, 56.7 mmol)
and Et3N (10.70 mL, 77.3 mmol). The EtOH was evaporated
and the residue was partitioned between EtOAc (2 × 100 mL)
and H2O (100 mL), with acidification (2 N HCl) of the aqueous
phase to pH 1.0. The EtOAc solution was washed with 0.5 N
HCl and H2O and dried and the solvent was evaporated. The
crude product was chromatographed using 95:5 v/v CH2Cl2/
MeOH as eluent. Fractions containing 72 were pooled and
evaporated and the semisolid residue triturated with 1:1 Et2O/
isohexane to yield a white solid: 10.57 g (65%); mp 98-99 °C;
NMR δ 1.25 (t, 3 H, OCH2CH3), 1.68 (m, 2 H, CH2CH2CH2),
2.20 (m, 2 H, CHCH2), 3.11 (t, 2 H, CH2S), 4.25 (q, 2 H, OCH2-
CH3), 4.94 (dd, 1 H, CH), 7.91 (m, 4H, ArH); MS m/z 376
[MH]+. Anal. (C16H17N5O4S) C, H, N.
Eth yl (S)-2-Am in o-5-(1H-1,2,3,4-tetr a zol-5-ylth io)p en -
ta n oa te (73). A solution of the phthalimido compound 72 (10.0
g, 26.7 mmol) in EtOH (100 mL) was treated with N2H4‚H2O
(1.42 mL, 29.3 mmol) for 18 h. HPLC indicated that a small
amount of starting material remained. Further N2H4‚H2O (0.28
mL, 5.8 mmol) was added. After 1 h the solution was
evaporated to dryness and the residue was stirred in CF3CO2H
(100 mL) for 4 h. The CF3CO2H was evaporated and the
residue was digested with H2O (ca. 200 mL). The precipitated
phthalazinedione was filtered off and the filtrate was evapo-
rated to dryness to yield a colorless gum (10.0 g), the crude
CF3CO2H salt of 73 which was used without purification.
(S,RS)-2-[N-[4-[N-[(3,4-Dih yd r o-2,7-d im et h yl-4-oxo-6-
qu in a zolin yl)m eth yl]-N-p r op -2-yn yla m in o]-2-flu or oben -
zoyl]a m in o]-5-(1H-1,2,3,4-tetr a zol-5-ylsu lfin yl)p en ta n o-
ic Acid (29). Meth od M. A mixture of 75 (1.74 g, 2.36 mmol),
2 N aqueous NaOH (30 mL, 60 mmol), and MeOH (20 mL)
was stirred for 90 min, warmed to 50 °C for 10 min, and cooled
back to 20 °C. The MeOH was evaporated and the resulting
aqueous solution was filtered into a centrifuge tube. Acidifica-
tion (2 N HCl) to pH 4.0 produced a gelatinous white
precipitate which was isolated by centrifugation and washed
(4×) with H2O, during which process the precipitate became
more granular. The resulting moist white solid was vacuum-
dried overnight and then suspended in toluene which was then
evaporated on a rotary evaporator. The solid was again
vacuum-dried overnight: 1.02 g (67%); NMR δ 1.73 (m, 2 H,
CH2CH2CH2), 1.88 (m, 2 H, CHCH2), 2.38 (s, 3 H, CH3), 2.46
(s, 3 H, CH3), 3.20 (t, 1 H, CtCH), 3.29 (t, 2 H, CH2SO), 4.30
(br s, 2 H, CH2CtC), 4.37 (dd, 1 H, CH), 4.71 (br s, 2 H,
ArCH2N<), 6.60 (dd, 1 H, 3′-H), 6.65 (dd, 1 H, 5′-H), 7.47 (s, 1
H, quinazoline 8-H), 7.54 (dd, 1 H, 6′-H), 7.72 (s, 1 H,
quinazoline 5-H), 7.91 (dd, 1 H, CONH); MS m/z 595 [MH]+.
Anal. (C27H27FN8O5S‚0.5NaCl‚1.2H2O) C, H, N.
Eth yl (S)-2-P h th a lim id o-5-[2H-1,2,4-tr ia zol-3(4H)-on o-
5-th io]p en ta n oa te (77). A mixture of carbethoxythiosemi-
carbazide43 (5.00 g, 30.7 mmol) and NaOH pellets (2.45 g, 61.4
mmol) in EtOH (150 mL) was stirred under reflux for 1 h to
give a clear solution. The cooled solution was evaporated to
dryness to give the crude disodium salt 76 of 5-mercapto-2H-
1,2,4-triazol-3(4H)-one, 6.32 g, as a greenish solid which was
used without purification.
A solution of the mesylate 71 (10.38 g, 28.14 mmol) in DMF
(10 mL) was added over 10 min to a stirred solution of 76 (6.32
g) in EtOH (200 mL) at 50 °C. Stirring was continued under
reflux for 1 h. The EtOH was evaporated and the residue was
partitioned between EtOAc (2 × 100 mL) and H2O (50 mL).
Chromatographic purification of the crude product, eluting
with 9:1 EtOAc/isohexane, yielded a white foam: 4.27 g (39%);
NMR δ 1.17 (t, 3 H, OCH2CH3), 1.60 (m, 2 H, CH2CH2CH2),
2.15 (m, 2 H, CHCH2), 2.96 (t, 2 H, CH2S), 4.10 (q, 2 H, OCH2-
CH3), 4.90 (m, 1 H, CH), 7.89 (m, 4 H, ArH), 11.40 (br, 1 H,
NH), 11.56 (br, 1 H, NH); MS m/z 391 [MH]+.
(S)-2-[N-[4-[N-[[3,4-Dih yd r o-2,7-d im eth yl-4-oxo-3-[(p iv-
a lo y lo x y )m e t h y l]-6-q u in a zo lin y l]m e t h y l]-N -p r o p -2-
yn yla m in o]-2-flu or ob en zoyl]a m in o]-5-(1H -1,2,3,4-t et r a -
zol-5-ylth io)p en ta n oic Acid Eth yl Ester (74). Meth od K.
Et3N (8.42 mL, 60 mmol), 73 CF3CO2H salt (7.20 g, 20 mmol),
and 1-hydroxybenzotriazole (50 mg) were added to a stirred
solution of the pentafluorophenyl ester 38 (8.11 g, 12.3 mmol)
in DMF (80 mL). Stirring was continued for 4 days with the
addition of further portions of 38 (1.0 g at 18 h and 5.5 g at 42
h). The reaction mixture was evaporated to dryness and the
residue was partitioned between EtOAc (2 × 400 mL) and H2O

Non-Polyglutamatable TS Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3819
Eth yl (S)-2-Am in o-5-[2H-1,2,4-tr ia zol-3(4H)-on o-5-th io]-
p en ta n oa te‚CF 3CO2H Sa lt (78). Hydrazine hydrate (604 µL,
12.5 mmol) was added to a stirred solution of 77 (4.41 g, 11.3
mmol) in EtOH (100 mL). After 90 min the solution was
evaporated to dryness to give a white foam (4.51 g) which was
stirred in CF3CO2H (50 mL) for 2 h. The CF3CO2H was
evaporated and the residue was dissolved in H2O (100 mL).
The solution was filtered to remove the insoluble phthala-
zinedione and the filtrate was evaporated to dryness to give
an pale golden gum, 5.15 g (>100%), which was used without
purification in the condensation with pentafluorophenyl ester
38 (method K).
Eth yl 2-Aceta m id o-2-ca r beth oxy-6-(1H-1,2,3,4-tetr a zol-
5-ylth io)h exa n oa te (80). Et3N (3.36 mL, 24.3 mmol) was
added in one portion to a stirred suspension of ethyl 2-acet-
amido-6-bromo-2-carbethoxyhexanoate (79)24 (5.70 g, 16.2
mmol) and 5-mercaptotetrazole21 (1.50 g, 14.7 mmol) in EtOH
(60 mL). The resulting clear solution was kept at 20 °C for 48
h and the EtOH was evaporated to produce a yellow oil. The
oil was partitioned between EtOAc (2 × 100 mL) and H2O (50
mL) and acidified (2 N HCl) to pH 1.0. The EtOAc solution
was washed with H2O, dried, and evaporated to a sticky solid.
Trituration with Et2O produced a free flowing, amorphous
white solid: 2.0 g (36%); NMR (CDCl3) δ 1.26 (t, 2 H, 2 ×
OCH2CH3), 1.31 (m, 2 H, CH2), 1.78 (m, 2 H, CH2CH2S), 2.13
(s, 3 H, COCH3), 2.38 (m, 2 H, CCH2), 3.23 (t, 2 H, CH2S),
4.26 (q, 4 H, 2 × OCH2CH3), 7.04 (s, 1 H, NH); MS m/z 374
[MH]+.
ester 89 (65 g, 0.098 mol) was stirred with 2 N aqueous NaOH
(400 mL, 0.8 mol) to give a clear pale yellow solution. After 3
h the solution was filtered and acidified (concd HCl) to pH 3.5.
The resulting suspension was allowed to stand for 2 h and the
precipitated solid was then filtered off and washed with H2O
(5 × 500 mL) in the sinter. The resulting paste was dried by
rotary evaporation at 0.1 mmHg with the aid of added toluene
to yield a white amorphous solid: 40.3 g (77%); NMR (400
MHz) δ 2.26 (m, 2 H, CHCH2), 2.32 (s, 3 H, CH3), 2.44 (s, 3 H,
CH3), 2.99 (t, 2 H, CH2tetrazole), 3.23 (t, 1 H, CtCH), 4.32
(br s, 2 H, CH2CtC), 4.46 (m, 1 H, CH), 4.71 (br s, 2 H,
ArCH2N<), 6.66 (dd, 1 H, 3′-H), 6.66 (dd, 1 H, 5′-H), 7.44 (s, 1
H, quinazoline 8-H), 7.59 (dd, 1 H, 6′-H), 7.72 (s, 1 H,
quinazoline 5-H), 8.10 (dd, 1 H, CONH); MS m/z 531 [M -
H]-. Anal. (C26H25FN8O4) C, H, N.
Meth yl (S)-2-P h th a lim id o-5-cya n op en ta n oa te (87). Po-
tassium cyanide (7.50 g, 0.115 mol) was added to a stirred
solution of mesylate 86 (13.63 g, 38.4 mmol; prepared by the
same method as for the ethyl ester 71) in DMSO (200 mL).
Stirring was continued overnight and the DMSO was removed
by rotary evaporation at 0.1 mmHg. The orange gummy
residue was partitioned between EtOAc (2 × 200 mL) and H2O
(100 mL). The EtOAc solutions were washed with brine, dried,
and evaporated to an orange oil: 9.52 g (86%); NMR δ 1.57
(m, 2 H, CH2CH2CH2), 2.26 (m, 2 H, CHCH2), 2.50 (t, 2 H,
CH2CN), 3.65 (s, 3 H, OCH3), 4.94 (dd, 1 H, CH), 7.91 (m, 4H,
ArH); MS m/z 287 [MH]+.
Meth yl (RS)-2-P h th a lim id o-5-(1H-1,2,3,4-tetr a zol-5-yl)-
p en ta n oa te (88). A mixture of the nitrile 87 (9.51 g, 33.3
mmol), NaN3 (2.27 g, 34.9 mmol), and NH4Cl (1.87 g, 34.9
mmol) in DMF (100 mL) was stirred at 90 °C for 16 h. HPLC
showed that the mixture contained contained 88 and unreacted
87 in a ratio of 51:35. The DMF was removed by rotary
evaporation at 0.1 mmHg and the orange oily residue was
partitioned between EtOAc (3 × 100 mL) and H2O (100 mL,
acidified to pH 1.0 with concd HCl). The EtOAc solutions were
washed with H2O, dried, and evaporated to a light brown oil
which was chromatographed using 2% MeOH in CHCl3 as
eluent to yield 88 as a colorless oil: 1.18 g (11%); NMR δ 1.70
(m, 2 H, CH2CH2CH2), 2.12 (m, 2 H, CHCH2), 2.90 (t, 2 H,
CH2tetrazole), 3.65 (s, 3 H, OCH3), 4.94 (dd, 1 H, CH), 7.91
(m, 4H, ArH).
(RS)-2-Am in o-6-(1H -1,2,3,4-t et r a zol-5-ylt h io)h exa n o-
ic Acid Hyd r och lor id e (81). The ester 80 (2.00 g, 5.35 mmol)
was stirred under reflux in 6 N HCl (50 mL) for 2 h to give a
clear solution. The solution was cooled and evaporated to
dryness on a rotary evaporator. The residue was dissolved in
H2O (50 mL) and this solution was evaporated to dryness. This
was repeated with a further portion of H2O followed by the
evaporation of added toluene to give crude 81 as a white
foam: 1.53 g; NMR δ 1.56 (m, 2 H, CH2), 1.70 (m, 2 H, CH2),
1.80 (m, 2 H, CH2CH2S), 3.25 (t, 2 H, CH2S), 3.85 (m, 1 H,
CH), 8.41 (br, 3 H, NH3+), 8.60 (br, 1 H, NH). This was used
without purification in the preparation of the methyl ester 82.
Meth yl (RS)-2-Am in o-6-(1H-1,2,3,4-tetr a zol-5-ylth io)-
h exa n oa te Hyd r och lor id e (82). Thionyl chloride (740 µL,
8.58 mmol) was added to a stirred solution of the amino acid
hydrochloride 81 (1.53 g) in MeOH (60 mL). After 18 h, further
thionyl chloride (370 µL, 4.29 mmol) was added and the
solution was boiled under reflux for 5 min. Evaporation of the
solvent yielded a pale pink foam: 1.60 g (100%); NMR δ 1.47
(m, 2 H, CH2), 1.69 (m, 2 H, CH2), 1.77 (m, 2 H, CH2CH2S),
3.23 (t, 2 H, CH2S), 3.74 (s, 3 H, OCH3), 3.99 (m, 1 H, CH),
8.55 (br, 3 H, NH3+); MS m/z 246 [MH]+. This was used without
purification in the condensation with the pentafluorophenyl
ester 38.
(S)-2-[4-[N-[[3,4-Dih ydr o-2,7-dim eth yl-4-oxo-3-[(pivaloyl-
oxy)m eth yl]-6-qu in azolin yl]m eth yl]-N-pr op-2-yn ylam in o]-
2-flu or oben zam ido]-4-(1H-1,2,3,4-tetr azol-5-yl)bu tyr ic Acid
Meth yl Ester (89). A mixture of (S)-2-amino-4-(1H-1,2,3,4-
tetrazol-5-yl)butyric acid methyl ester (84)25,26 (19.4 g, 0.105
mol), pentafluorophenyl ester 38 (67.5 g, 0.102 mol), and
HOBT (20 mg) in DMF (600 mL) was stirred for 60 h. The
DMF was evaporated and the gummy residue was partitioned
between EtOAc (1.5 L) and H2O (1.0 L). The EtOAc solution
was washed with brine, dried, and evaporated to an orange
gum. Chromatographic purification, eluting with EtOAc fol-
lowed by 9:1 CH2Cl2/MeOH, gave a white amorphous solid:
51.4 g (75%); NMR δ 1.22 (s, 9 H, But), 2.25 (m, 2 H, CHCH2),
2.46 (s, 3 H, CH3), 2.58 (s, 3 H, CH3), 2.97 (t, 2 H, CH2-
tetrazole), 3.22 (t, 1 H, CtCH), 3.65 (s, 3 H, OCH3), 4.32 (br
s, 2 H, CH2CtC), 4.48 (m, 1 H, CH), 4.72 (br s, 2 H, ArCH2N<),
6.00 (s, 2 H, OCH2N), 6.62 (dd, 1 H, 3′-H), 6.66 (dd, 1 H, 5′-H),
7.48 (s, 1 H, quinazoline 8-H), 7.55 (dd, 1 H, 6′-H), 7.75 (s, 1
H, quinazoline 5-H), 8.24 (dd, 1 H, CONH).
Met h yl (RS)-2-Am in o-5-(1H -1,2,3,4-t et r a zol-5-yl)p en -
t a n oa t e (89). Hydrazine hydrate (180 µL, 3.68 mmol) was
added to a stirred solution of 88 (1.10 g, 3.34 mmol) in MeOH
(8 mL). The clear pale yellow solution was kept for 18 h and
evaporated to dryness. The white solid residue was stirred with
CF3CO2H (20 mL) for 2 h and the resulting solution was
evaporated to a sticky white solid which was stirred with H2O
(20 mL) until no more material appeared to go into solution.
The solution was filtered to remove the white insoluble
phthalazinedione, the filtrate was evaporated to dryness, and
the residue was azeotroped with toluene (3×) to give crude
89 (1.4 g) which was used without purification in the conden-
sation with the pentafluorophenyl ester 38.
Refer en ces
(1) Tomudex is a trade mark, the property of ZENECA Ltd.
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Oldfield, J .; Wardleworth, J . M.; Bishop, J . A. M.; O’Connor, B.
M.; Calvert, A. H. Quinazoline Antifolate Thymidylate Synthase
Inhibitors: Heterocyclic Benzoyl Ring Modifications. J . Med.
Chem. 1991, 34, 1594-1605.
(3) J ackman, A. L.; J odrell, D. I.; Gibson, W.; Stephens, T. C. In
Purine and Pyrimidine Metabolism in Man; Harkness, R. A., et
al., Eds.; Plenum Press: New York, 1991; pp 19-23.
(4) 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.
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