Synthesis and evaluation of novel fluorinated methotrexate derivatives for application to rheumatoid arthritis treatment

Article abstract of DOI:10.1021/jm970085n

In ongoing search for new antifolate drugs useful against rheumatoid arthritis (RA) led us to prepare new methotrexate (MTX) derivatives containing enantiomerically pure L-erythro- or L-threo-γ-fluoroglutamic acid. The derivatives in which the phenyl ring was replaced by a 3'- substituted phenyl or methylthiophene ring showed potent immunosuppressive activities, including in vitro inhibition of mitogen responses of both T and B cells and in vivo inhibition of antibody production in mice. These compounds also exhibited inhibitory activity in adjuvant arthritis in rats. Their toxicity was lower than that of MTX, which was probably due to the strong electronegativity of fluorine, which increases the acidity of the γ- carboxyl group and thereby decreases polyglutamylation in normal cells. These results revealed the potential of the fluorinated MTX derivatives as candidate drugs for the treatment of RA.

Full text of DOI:10.1021/jm970085n

3280
J . Med. Chem. 1997, 40, 3280-3291
Syn th esis a n d Eva lu a tion of Novel F lu or in a ted Meth otr exa te Der iva tives for
Ap p lica tion to Rh eu m a toid Ar th r itis Tr ea tm en t
Yoshitsugu Kokuryo, Kyozo Kawata, Takuji Nakatani, Akira Kugimiya, Yoshinori Tamura, Kenji Kawada,*
Mitsunobu Matsumoto, Ryuji Suzuki, Kenji Kuwabara, Yozo Hori, and Mitsuaki Ohtani
Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553, J apan
Received February 10, 1997X
An ongoing search for new antifolate drugs useful against rheumatoid arthritis (RA) led us to
prepare new methotrexate (MTX) derivatives containing enantiomerically pure L-erythro- or
L-threo-γ-fluoroglutamic acid. The derivatives in which the phenyl ring was replaced by a 3′-
substituted phenyl or methylthiophene ring showed potent immunosuppressive activities,
including in vitro inhibition of mitogen responses of both T and B cells and in vivo inhibition
of antibody production in mice. These compounds also exhibited inhibitory activity in adjuvant
arthritis in rats. Their toxicity was lower than that of MTX, which was probably due to the
strong electronegativity of fluorine, which increases the acidity of the γ-carboxyl group and
thereby decreases polyglutamylation in normal cells. These results revealed the potential of
the fluorinated MTX derivatives as candidate drugs for the treatment of RA.
In tr od u ction
Methotrexate (MTX, 1, Figure 1), a potent antifolate
in wide clinical use against various types of cancers, has
also been used in the treatment of rheumatoid arthritis
(RA) for more than 2 decades. During the 1980s, clinical
trials of low-dose and weekly treatment have verified
the efficacy for patients not treatable by conventional
drugs such as nonsteroidal anti-inflammatory drugs.1
Moreover, significant improvement of painful and swol-
len joints has been observed in such trials.2 The Food
and Drug Administration approved MTX for treating
RA in 1988 and in recent years it has become a major
therapeutic agent used in early stages of the disease in
the United States and Europe. Now in J apan, phase
III studies are being conducted in order to determine
the adequate dosage.
The continued use of MTX in the treatment of RA has
been implicated in a variety of adverse effects including
F igu r e 1.
gastrointestinal toxicity, stomatitis, hematologic toxic-
ity, hepatotoxicity, and pulmonary toxicity. In addition,
modifications at the glutamic acid moiety, such as
γ-carboxyl substitution with amide or peptide groups7
other undesirable side effects such as central nervous
and replacement of glutamic acid by other amino acids,8
system3 and cutaneous reactions4 have been reported.
have been reported, aimed at generating a new class of
Therefore, although MTX can be ranked among the
antitumor agents.
most effective agents for the treatment of RA, particular
We previously prepared several MTX derivatives
caution and careful monitoring of the patients are
containing R- or γ-substituted glutamic acid and found
that γ-fluoromethotrexate (FMTX, 2i) showed favorable
features for high-dose treatment of MTX-resistant can-
needed during MTX therapy. These critical toxicities
have been pointed out by only a few reports on the
development of MTX derivatives useful for treating RA5
cers.9 These effects were probably due to the extreme
compared with the huge number of derivatives aimed
electronegativity of fluorine causing acidity enhance-
at application to cancer treatment.
ment of the γ-carboxylic acid group and hence decreased
its in vivo polyglutamate formation.10 Since adverse
effects of MTX in the treatment of RA are also assumed
to be related to the accumulation of poly-γ-glutamate
metabolites in normal tissues, we decided to extend this
approach to the development of less toxic drugs to treat
RA.
Recently, poly-γ-glutamates of MTX derivatives bio-
logically synthesized by folylpoly-γ-glutamate syn-
thetase have been recognized as important determi-
nants of cytotoxicity and therapeutic selectivity against
malignant as compared with normal tissues in the
treatment of cancers, because poly-γ-glutamates, having
greater inhibitory activity against thymidylate synthase
and aminoimidazolecarboxamide ribotide formyltrans-
ferase, exhibit prolonged cellular retention in compari-
son with the parent drug.6 In order to reduce the
formation of poly-γ-glutamates in normal cells, various
RA has been recognized as a chronic systemic au-
toimmune inflammatory disease, and immunological
effects have been suggested to play an important role
in the pathogenesis.11 For example, proliferation of T
cells triggering the release of tissue-damaging mediators
from synovial cells, activation of T cells producing a
variety of cytokines and eicosanoids, and antibody
X Abstract published in Advance ACS Abstracts, August 15, 1997.
S0022-2623(97)00085-X CCC: $14.00 © 1997 American Chemical Society

Novel Fluorinated Methotrexate Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3281
Sch em e 1a
a
(a) SOCl2, i-PrOH; (b) see Table 1; (c) see Table 1; (d) see Table 1; (e) Ba(OH)2, aq EtOH.
production by B cells resulting in formation of immune
complexes and complement activation have been re-
ported to be associated with the disease.
containing D-glutamic acid has been reported to be a
poor inhibitor of cell growth.14 Therefore, we decided
to synthesize FMTX derivatives in enantiomerically
pure L-form. The synthesis of enantiomerically pure
L-threo-FGlu (3t) from L-4(R)-hydroxyproline has been
reported by Hudlicky.15 Recently, using this method,
L-threo-FMTX (2it) has been prepared by Coward et al.
and found to be a potent inhibitor of dihydrofolate
reductase but inactive as a substrate for FPGS;16 its in
vivo activity has not been described. Consequently, in
order to obtain the large amount of FMTX derivatives
necessary for in vivo evaluation, we developed a practi-
cal way of preparing enantiomerically pure L-erythro-
and L-threo-FGlu (3e and 3t) using aminoacylase.17
With substantial supplies of both enantiomerically
pure glutamates 3e and 3t in hand, we sought to
prepare both L-erythro- and L-threo-FMTX derivatives,
for which purpose an efficient method would be to couple
their protected derivatives 4e and 4t with a 4-ami-
nopteroic acid derivative at a late stage of the synthesis.
However, all efforts to use this approach were unre-
warding.18 Therefore, L-erythro- and L-threo-FGlu were
incorporated at an early stage and the resulting com-
pounds were manipulated using a procedure similar to
that described in our previous report9 regarding racemic
FMTX 2i as shown in Scheme 1.
Consequently, although the precise mechanism for the
efficacy of MTX in RA still remains obscure, we adopted
immunological assays, i.e., an in vitro inhibitory test
against responses of both T and B cells to mitogens12
and an in vivo inhibitory test against antibody produc-
tion in mice for exploring new antifolate drugs useful
for treating RA. Along this line, a number of MTX
derivatives containing enantiomerically pure L-erythro-
or L-threo-γ-fluoroglutamic acid (L-erythro-, L-threo-
FGlu) were prepared. Among them, various compounds
in which the phenyl ring was replaced by a 3′-
substituted phenyl or a methylthiophene ring showed
potent immunosuppressive activities and were selected
for further evaluations such as against adjuvant arthri-
tis in rats. The present paper describes the synthesis
of these fluorinated MTX derivatives and their biological
activities.
Ch em istr y
In considering the significant biological effect of
chirality,13 enantiomerically pure fluorinated MTX de-
rivatives for the treatment of rheumatoid arthritis are
of particular interest. Moreover, the MTX analogue

3282 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Kokuryo et al.
Sch em e 2a
Ta ble 1. Reagents of Scheme 1
compd
step b
step c
step d
i
KHCO3, H2O, CH2Cl2 HBr, HOAc DMA
ii
iii
iv
v
Et3N, DME
Zn, HOAc
excluded
excluded
excluded
H2, PtO2,
EtOAc
DMA
DMA
DMA
DMA
Proton-Sponge,
DMA
DEPC, Et3N, DME
DEPC, Et3N, DME
DEPC, Et3N, DME
Et3N, CH2Cl2
vi
vii
viii
ix
DEPC, Et3N, DME
DEPC, Et3N, DME
DEPC, Et3N, DME
excluded
excluded
CF3CO2H,
CH2Cl2
KI, DMA
KI, DMA
DMA
a
(a) I2, NaHCO3, aq MeOH; (b) Ac2O, Py; (c) CuCN, DMF; (d)
concd HCl.
Ta ble 2. In vitro Mitogen Responses and Primary Antibody
Responses in Mice
Sch em e 3a
antibody production
mitogen response
IC50 (ng/mL)
suppressive
lethal
dosed
safety
indexe
compd T cella
B cellb
dosec
MTX
2ie
2it
2iie
2iit
2iiie
2iiit
2ive
2ve
6.1
25.9
64.6
7.8
10.7
3.8
3.2
5.3
8.0
16.8
10.0
9.4
4.3
2.1
12.5
21.0
61.5
9.9
16.5
9.7
0.625
40
64
12
-
40
50
>400
0.31
600
f
-
12.5
100
1.25
80
0.625-1.25
1.25-2.5
5.0-10.0
5.0
2.5-5.0
1.25
100
150
80-160
60-120
20-40
-
-
160
160
100
>40-80
30-60
>18
9.2
22.2
14.3
19.5
29.3
22.7
11.8
8.2
200
a
f
(a) DPPA, Et3N, t-BuOH; (b) NaH, MeI, DMF; (c) LDA, CO2,
-
-
f
THF.
2vt
2vie
2vit
2viie
2viiie
2ixe
2ixt
200
200
80
>200
150
1.25
0.8
The utmost concern in the previous synthetic scheme
was racemization. The diastereomeric composition of
FMTX 2 was determined by HPLC using a reversed-
phase octadecylsilica (ODS) column coated with L-
stearoylcarnitine.19 Since the analysis of enantiomers
of 2 was rather difficult, the enantiomeric purity was
determined on a chiral column after converting 2 to a
dimethyl ester. These HPLC analyses suggested that
several percent of racemization at the γ-position of FGlu
occurred during hydrolysis of the isopropyl ester 9 with
sodium or lithium hydroxide to give a mixture of
diastereomers, while no racemization took place during
the other steps, including the amide bond formation of
step b. Although the use of a methyl ester would be
desirable at the hydrolysis step, protection of the
carboxyl group as isopropyl ester 4e and 4t was needed
in order to prevent cyclization to a 2-pyrrolidone deriva-
tive in step b due to the inductive effect of fluorine. This
racemization problem was overcome by using barium
hydroxide at the hydrolysis step e.20
2.5-5.0
3.8
11.5
5.1
14.9
2.5-5.0
10.0
>180
a
Inhibitory activity against Con A-stimulated T cell prolifera-
b
tion. Inhibitory activity against LPS-stimulated B cell prolifera-
tion. c 75% suppression (mg/kg). One of the mice died (mg/kg).
d
e Ratio of lethal dose/suppressive dose value. f Not determined
owing to insolubility.
Other 3-substituted 4-aminobenzoic acids derivatives
were commercially available or prepared according to
known procedures.24
Biologica l Eva lu a tion a n d Discu ssion
For the primary screening test of immunological
activities of the fluorinated MTX derivatives, the in vitro
effects of MTX and these derivatives on the responses
of both T and B cells to mitogens were examined.12 As
shown in Table 2, these compounds inhibited the
proliferative responses of mouse spleen cells to T cell
and B cell mitogens, concanavalin A (Con A) and
lipopolysaccharide (LPS), respectively. Although the
activity of FMTX 2ie and 2it was several times weaker
than that of MTX, the derivatives modified in the phenyl
ring showed approximately the same potencies as MTX.
These results suggested that introduction of a substitu-
ent at the 3′-position of the phenyl ring or replacement
of the phenyl ring by the methylthiophene ring led to
higher immunosuppressive activity. Moreover, the
steric effects of this position seemed to be more impor-
tant than the electronic factors for the activity, since
electon-donating (2ii, 2iii, 2iv) and -withdrawing (2v,
2vi, 2vii, 2viii) groups made no significant difference.
As for the biological effect of the stereochemistry of
fluorine substitution, both diastereomers showed almost
equal potency, though the erythro diastereomers were
a little more potent than the threo diastereomers in
some cases, such as 2i and 2ix.25 Therefore, the effects
Preparation of 4-amino-3-propylbenzoic acid (5iv) was
accomplished using a procedure similar to that de-
scribed for the synthesis of 3-ethyl and 3-trifluoro-
methyl derivatives 5iii21 and 5v22 as shown in Scheme
2, i.e., iodination of 2-propylaniline (10), protection of
the primary amino group of 11 as an acetamide,
conversion of iodide 12 to nitrile 13, and hydrolysis of
both amide and nitrile groups.
Methylthiophenecarboxylic acid 5ix was prepared
using a procedure similar to that described for the
synthesis of a thiophene antifolate,23 as shown in
Scheme 3, i.e., conversion of the carboxyl group of 14 to
Boc-protected aminothiophene 15, alkylation with io-
domethane, and lithiation of 16 by LDA followed by the
addition of carbon dioxide, though the yield of the last
step c was rather low, probably because of the instability
of the lithiomethylthiophene.

Novel Fluorinated Methotrexate Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3283
Ta ble 3. Secondary Antibody Responses in Mice
2vit by EL4 cells was evaluated by exposing the cells
to the test compounds for only 1 day. If these com-
pounds were polyglutamylated, they should retain the
ability to block cellular proliferation even after a 1-day
exposure. While MTX inhibited the growth of EL4 cells
with an IC50 of 1.57 µM under these conditions, the IC50
of the FMTX derivatives was >100 µM. These results
suggested that the FMTX derivatives were not metabo-
lized to the polyglutamates and were not retained
within the cells, but still exhibited potent immunosup-
pressive and antirheumatic activities.
antibody titer (log2)a
compd
0.625 mg/kgb
1.25 mg/kgb
2.5 mg/kgb
MTX
2iiie
2iiit
2vie
2vit
5.4 ( 0.6
4.8 ( 0.8
5.4 ( 0.9
6.0 ( 0.7
6.2 ( 0.8
4.2 ( 1.1
3.8 ( 1.1
5.2 ( 0.8
5.4 ( 0.6
6.8 ( 0.5
4.0 ( 0.7
3.2 ( 1.3
4.6 ( 1.8
4.8 ( 0.8
5.2 ( 0.5
a
b
Mean titer ( SD. control, 7.5 ( 1.1. Dose (5 days a week
for 3 weeks).
Ta ble 4. Adjuvant Arthritis in Rats
compd
ED50 (mg/kg)a
LD50 (mg/kg)
safety indexb
Con clu sion
MTX
2iiie
2iiit
2vie
2vit
0.058
0.8
0.89
1.6
0.62
0.43
0.8
0.82
20.0
10.9
40.5
29.0
20.0
46.0
14.1
25.0
12.2
25.3
46.8
46.5
57.5
In this report, we have described the preparation of
new MTX derivatives containing enantiomerically pure
L-erythro- or L-threo-γ-fluoroglutamic acid as part of an
ongoing search for less toxic drugs to treat RA. Several
derivatives, in which the phenyl ring was replaced by a
3′-substituted phenyl or methylthiophene ring, showed
potent immunosuppressive activities and inhibitory
effects on adjuvant arthritis in rats and had a higher
safety index than MTX. Their lower toxicity was prob-
ably due to the presence of the strongly electronegative
fluorine atom, which increases the acidity of the neigh-
boring γ-carboxyl group and is unfavorable for poly-
glutamylation.
From these results, the FMTX derivatives revealed
their potential as new drug candidates for the treatment
of RA. These studies also indicated that immunological
assays such as the in vitro inhibitory test against
responses of both T and B cells to mitogens and the in
vivo inhibitory test against antibody production in mice
are useful for evaluating the potential of MTX as
antiarthritis drugs.
2viiie
2ixe
a
b
Inhibition of uninjected paw volume vs vehicle control. Ratio
of LD50/ED50 value.
of the stereochemistry of the γ-position did not seem to
be as significant as that of the R-position.
Next, to evaluate the primary immune responses to
T cell dependent antigen, mice were immunized with
sheep red blood cells (SRBC), and antibody production
was determined after oral administration of the test
compounds daily for 3 days. As shown in Table 2, the
FMTX derivatives exhibited potent suppressive effects
on the antibody production, though FMTX 2ie and 2it
had much lesser effects. The trends obtained in this in
vivo assay and in the in vitro antimitogenic assay were
similar. The lethal doses and safety index (lethal dose/
suppressive dose) values obtained in the in vivo assay
are also listed in Table 2. Several derivatives such as
2iit, 2iiie, 2iiit, 2vie, 2vit, 2viie, and 2viiie had a
greater safety index than MTX.
Exp er im en ta l Section
Gen er a l. Unless otherwise stated, all reactions were
carried out under a nitrogen atmosphere with anhydrous
solvents that had been dried over type 4A molecular sieves.
Drying of an organic phase over anhydrous Na2SO4 is simply
indicated by the word “dried”. Column chromatography using
Merck Silica gel 60 or a Merck Lobar column is referred to as
“chromatography on silica gel”. Melting points are uncor-
rected. 1H and 13C NMR data are presented in Table 5 and 6,
respectively. 1H NMR spectra were determined at 200 or 300
MHz. 13C NMR spectra were determined at 50.3 MHz.
Coupling values to fluorine of 13C NMR signals are given in
parentheses. Diastereomers of 2 were analyzed by HPLC
using an L-column ODS (Waters, 15 × 0.46 cm) coated with
L-stearoylcarnitine with 98:2 10 mM phosphate buffer (pH
7.0)-acetonitrile at 40 °C.19 Enantiomers of the dimethyl
esters of 2ie and 2it, which were prepared by thionyl chloride
in MeOH, were analyzed by HPLC using Ultron ES-OVM
(Shinwa, 15 × 0.46 cm) with 95:5 50 mM phosphate buffer
(pH 6.0)-MeOH or using Ultron ES-CD (Shinwa, 15 × 0.46
cm) with 70:30 50 mM phosphate buffer (pH 6.0)-MeOH.
Exact mass was determined from high-resolution liquid sec-
ondary ion mass spectra (HR-LSIMS) or fast atom bombard-
ment mass spectra (HR-FABMS). Fractional moles of water
found in analytical samples of antifolates could not be pre-
vented in spite of drying in vacuo and were confirmed by the
Karl Fischer method.
To evaluate secondary immune responses, mice were
immunized with a low dosage of SRBC at 1-week
intervals for 3 weeks, and antibody production was
determined after oral administration of the test com-
pounds 2iiie, 2iiit, 2vie, and 2vit 5 days a week for 3
weeks. As shown in Table 3, these derivatives showed
almost the same suppressive effects on antibody produc-
tion as MTX. These results suggested that the FMTX
derivatives also retained the potent immunosuppressive
activity in secondary processes.
The effects of 2iiie, 2iiit, 2vie, 2vit, 2viiie, and 2ixe
on RA models were examined using adjuvant arthritis
in rats. Although the potency of these derivatives was
lower than that of MTX, they showed a significant
reduction in paw swelling as well as an increase in body
weight. The ED50 and LD50 values of the compounds
and their safety index (LD50/ED50) are listed in Table
4. Compared with MTX, the FMTX derivatives exhib-
ited a greater safety index. In addition, the inhibitory
activity and toxicity of these derivatives gradually
increased in a dose-dependent manner, whereas those
of MTX rather abruptly increased over a narrow range.
Since the potency of MTX is strong enough (dosage for
RA, 7.5 mg/week),26 the safety profile would be the most
important factor for new drugs.
4-Iod o-2-p r op yla n ilin e (11). To a solution of 5.0 g (37.0
mmol) of 10 in 25 mL of MeOH was added a solution of 5.0 g
(59.5 mmol) of NaHCO3 in 25 mL of water. After adding 8.4
g (33.1 mmol) of iodine portionwise over a 70-min period at
10 °C, the mixture was stirred for 30 min. The mixture was
diluted with water and extracted with EtOAc. The organic
solution was washed with 5% Na2S2O3 solution and saturated
In order to confirm that the lower toxicity of these
compounds was due to the decreased polyglutamate
formation, the accumulation of 2iiie, 2iiit, 2vie, and

3284 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Kokuryo et al.
Ta ble 5. 1H NMR Data
compd
δ
11
(CDCl3) 0.99 (t, J ) 7.4 Hz, 3H), 1.53-1.69 (m, 2H), 2.40 (t, J ) 7.8 Hz, 2H), 3.63 (br s, 2H), 6.45 (d, J ) 8.0 Hz, 1H),
7.26-7.33 (m, 2H)
12
13
(CDCl3) 0.98 (t, J ) 7.4 Hz, 3H), 1.55-1.66 (m, 2H), 2.19 (s, 3H), 2.48 (t, J ) 7.8 Hz, 2H), 6.97 (br s, 1H), 7.48-7.56 (m, 3H)
(CDCl3) 1.01 (t, J ) 7.4 Hz, 3H), 1.61-1.72 (m, 2H), 2.24 (s, 3H), 2.57 (t, J ) 7.8 Hz, 2H), 7.26 (br s, 1H), 7.46-7.53
(m, 2H), 8.18 (s, 1H)
5iv
15
16
(CDCl3) 1.01 (t, J ) 7.4 Hz, 3H), 1.63-1.74 (m, 2H), 2.48 (t, J ) 7.6 Hz, 2H), 6.65 (d, J ) 9.0 Hz, 1H), 7.78-7.82 (m, 2H)
(CDCl3) 1.52 (s, 9H), 2.11 (s, 3H), 6.45 (br s, 1H), 6.69 (d, J ) 3.6 Hz, 1H), 6.84 (d, J ) 3.6 Hz, 1H)
(CDCl3) 1.41 (s, 9H), 2.06 (s, 3H), 3.18 (s, 3H), 6.71 (d, J ) 3.6 Hz, 1H), 7.00 (d, J ) 3.6 Hz, 1H)
(CDCl3) 1.43 (s, 9H), 2.09 (s, 3H), 3.22 (s, 3H), 7.56 (s, 1H)
5ix
6ie
7ie
9ie
2ie
6it
(CDCl3) 1.24 (d, J ) 6.2 Hz, 3H), 1.25 (d, J ) 6.2 Hz, 3H), 1.30 (d, J ) 6.2 Hz, 6H), 2.35-2.85 (m, 2H), 3.36 (s, 3H),
4.80-5.18 (m, 4H), 5.19 (s, 2H), 6.98 (d, J ) 6.6 Hz, 1H), 7.34 (s, 5H), 7.37 and 7.82 (AB q, J ) 8.6 Hz, 4H)
(CDCl3) 1.24 (d, J ) 6.2 Hz, 3H), 1.25 (d, J ) 6.2 Hz, 3H), 1.29 (d, J ) 6.2 Hz, 6H), 2.34-2.87 (m, 2H), 2.89 (s, 3H),
4.80-5.22 (m, 4H), 6.58 and 7.72 (AB q, J ) 8.6 Hz, 4H), 6.83 (d, J ) 6.6 Hz, 1H)
(CDCl3-CD3OD) 1.23 (d, J ) 6.2 Hz, 3H), 1.24 (d, J ) 6.2 Hz, 3H), 1.28 (d, J ) 6.2 Hz, 6H), 2.34-2.85 (m, 2H), 3.20
(s, 3H), 4.76 (s, 2H), 4.80-5.20 (m, 4H), 6.77 and 7.74 (AB q, J ) 9.0 Hz, 4H), 6.99 (d, J ) 6.4 Hz, 1H), 8.65 (s, 1H)
(DMSO-d6) 2.10-2.50 (m, 2H), 3.21 (s, 3H), 4.55 (m, 1H), 4.79 (s, 2H), 5.07 (m, 1H), 6.82 and 7.71 (AB q, J ) 9.0 Hz, 4H),
6.95 (br s, 2H), 7.88 (br s, 2H), 8.38 (d, J ) 7.2 Hz, 1H), 8.58 (s, 1H)
(CDCl3) 1.20 (d, J ) 6.2 Hz, 3H), 1.24 (d, J ) 6.2 Hz, 3H), 1.29 (d, J ) 6.4 Hz, 3H), 1.30 (d, J ) 6.4 Hz, 3H), 2.38-2.85
(m, 2H), 3.36 (s, 3H), 4.84-5.18 (m, 4H), 5.19 (s, 2H), 6.84 (d, J ) 6.6 Hz, 1H), 7.34 (s, 5H), 7.36 and 7.80 (AB q,
J ) 8.6 Hz, 4H)
(CDCl3-CD3OD) 1.19 (d, J ) 6.2 Hz, 3H), 1.24 (d, J ) 6.2 Hz, 3H), 1.28 (d, J ) 6.2 Hz, 6H), 2.25-2.70 (m, 2H), 2.88
(s, 3H), 4.83-5.20 (m, 4H), 6.58 and 7.68 (AB q, J ) 8.6 Hz, 4H), 6.69 (d, J ) 7.4 Hz, 1H)
(DMSO-d6) 2.15-2.45 (m, 2H), 3.23 (s, 3H), 4.51 (m, 1H), 4.82 (s, 2H), 4.90 (m, 1H), 6.84 and 7.75 (AB q, J ) 8.6 Hz, 4H),
7.20 (br s, 2H), 8.08 (br s, 2H), 8.43 (d, J ) 8.6 Hz, 1H), 8.63 (s, 1H)
9it
2it
6iie
7iie
(CDCl3) 1.24 (d, J ) 6.3 Hz, 3H), 1.25 (d, J ) 6.3 Hz, 3H), 1.28 (d, J ) 6.6 Hz, 3H), 1.29 (d, J ) 6.6 Hz, 3H), 2.66 (s, 3H),
2.40-2.90 (m, 2H), 4.80-5.00 (m, 4H), 7.05 (br s, 1H), 7.72-7.84 (m, 2H), 8.02 (d, J ) 8.4 Hz, 1H)
(CDCl3) 1.24 (d, J ) 6.3 Hz, 3H), 1.25 (d, J ) 6.3 Hz, 3H), 1.28 (d, J ) 6.6 Hz, 3H), 1.29 (d, J ) 6.3 Hz, 3H), 2.30 (s, 3H),
2.35-2.90 (m, 2H), 3.95 (br s, 2H), 4.80-5.20 (m, 4H), 6.67 (d, J ) 7.8 Hz, 1H), 6.84 (d, J ) 6.0 Hz, 1H), 7.51-7.60
(m, 2H)
9iie
(DMSO-d6) 1.15-1.18 (m, 12H), 2.22 (s, 3H), 2.23-2.48 (m, 2H), 4.46 (m, 1H), 4.56 (d, J ) 5.7 Hz, 2H), 4.83-4.94 (m, 2H),
5.19 (m, 1H), 6.25 (br s, 1H), 6.58 (br s, 2H), 6.59 (d, J ) 8.4 Hz, 1H), 7.52 (dd, J ) 1.8 and 8.4 Hz, 1H), 7.57 (d, J )
1.8 Hz, 1H), 7.63 (br s, 2H), 8.33 (d, J ) 7.5 Hz, 1H), 8.68 (s, 1H)
2iie
6iit
7iit
9iit
(DMSO-d6) 2.22 (s, 3H), 2.22-2.49 (m, 2H), 4.47 (m, 1H), 4.57 (d, J ) 5.1 Hz, 2H), 5.07 (m, 1H), 6.24 (br s, 1H), 6.58 (d,
J ) 8.1 Hz, 1H), 6.76 (br s, 2H), 7.51-7.59 (m, 2H), 7.80 (br s, 2H), 8.28 (d, J ) 7.8 Hz, 1H), 8.69 (s, 1H)
(CDCl3) 1.24 (d, J ) 6.3 Hz, 3H), 1.25 (d, J ) 6.3 Hz, 3H), 1.28 (d, J ) 6.6 Hz, 3H), 1.29 (d, J ) 6.3 Hz, 3H), 2.66 (s, 3H),
2.40-2.70 (m, 2H), 4.85-5.20 (m, 4H), 6.98 (br s, 1H), 7.70-7.86 (m, 2H), 8.03 (d, J ) 8.4 Hz, 1H)
(CDCl3) 1.24 (d, J ) 6.3 Hz, 3H), 1.25 (d, J ) 6.3 Hz, 3H), 1.28 (d, J ) 6.6 Hz, 3H), 1.29 (d, J ) 6.3 Hz, 3H), 2.19 (s, 3H),
2.30-2.60 (m, 2H), 4.85-5.20 (m, 4H), 6.65-6.70 (m, 2H), 7.45-7.60 (m, 2H)
(DMSO-d6) 1.16-1.23 (m, 12H), 2.23 (s, 3H), 2.24-2.49 (m, 2H), 4.47 (m, 1H), 4.57 (d, J ) 5.7 Hz, 2H), 4.85-5.12 (m, 3H),
6.26 (br s, 1H), 6.58 (br s, 2H), 6.60 (d, J ) 8.4 Hz, 1H), 7.54-7.59 (m, 2H), 7.63 (br s, 2H), 8.43 (d, J ) 8.1 Hz, 1H),
8.68 (s, 1H)
2iit
(DMSO-d6) 2.25 (s, 3H), 2.25-2.52 (m, 2H), 4.51 (m, 1H), 4.65 (d, J ) 4.5 Hz, 2H), 4.92 (m, 1H), 6.34 (br s, 1H), 6.60
(d, J ) 8.4 Hz, 1H), 7.57-7.64 (m, 2H), 7.80 (br s, 2H), 8.38 (d, J ) 8.4 Hz, 1H), 8.78 (br s, 2H), 8.79 (s, 1H)
(CDCl3) 1.52-1.61 (m, 15H), 2.74-2.91 (m, 4H), 4.27 (br s, 2H), 5.12-5.49 (m, 4H), 6.97 (m, 1H), 7.14 (d, J ) 7.4 Hz,
1H), 7.78-7.94 (m, 2H)
6iiie
9iiie
2iiie
6iiit
9iiit
2iiit
6ive
9ive
(DMSO-d6) 1.17-1.22 (m, 15H), 2.28-2.66 (m, 4H), 4.50-4.64 (m, 3H), 4.84-4.97 (m, 2H), 5.22 (m, 1H), 6.40 (m, 1H),
6.59-6.66 (m, 3H), 7.53-7.67 (m, 4H), 8.39 (d, J ) 7.2 Hz, 1H), 8.69 (s, 1H)
(DMSO-d6) 1.23 (t, J ) 7.4 Hz, 3H), 2.00-2.51 (m, 2H), 2.62 (q, J ) 7.4 Hz, 2H), 4.43-4.58 (m, 3H), 4.87 (m, 1H), 6.34
(m, 1H), 6.36-6.63 (m, 3H), 7.53-7.65 (m, 4H), 8.62 (d, J ) 9.0 Hz, 1H), 8.67 (s, 1H)
(CDCl3) 1.15-1.30 (m, 15H), 2.25-2.64 (m, 4H), 3.97 (br s, 2H), 4.79-5.18 (m, 4H), 6.68 (m, 1H), 7.21 (br s, 1H),
7.48-7.65 (m, 2H)
(DMSO-d6) 1.15-1.23 (m, 15H), 2.21-2.43 (m, 2H), 2.62 (q, J ) 7.5 Hz, 2H), 4.41-4.58 (m, 3H), 4.83-5.18 (m, 3H), 6.39
(m, 1H), 6.42-6.63 (m, 3H), 7.53-7.66 (m, 4H), 8.45 (d, J ) 7.6 Hz, 1H), 8.66 (s, 1H)
(DMSO-d6) 1.21 (t, J ) 7.4 Hz, 3H), 2.16-2.27 (m, 2H), 2.61 (q, J ) 7.4 Hz, 2H), 4.44-4.57 (m, 3H), 4.81 (m, 1H), 6.33
(m, 1H), 6.56-6.74 (m, 3H), 7.52-7.65 (m, 4H), 8.23 (d, J ) 7.4 Hz, 1H), 8.66 (s, 1H)
(CDCl3) 1.12 (t, J ) 6.2 Hz, 3H), 1.33 (d, J ) 6.2 Hz, 3H), 1.36 (d, J ) 6.2 Hz, 3H), 1.45 (d, J ) 6.2 Hz, 6H), 1.69-1.90
(m, 2H), 2.47-2.98 (m, 4H), 4.93-5.22 (m, 4H), 6.79 (d, J ) 7.5 Hz, 1H), 6.97 (d, J ) 6.2 Hz, 1H), 7.61-7.70 (m, 2H)
(DMSO-d6) 0.97 (t, J ) 6.5 Hz, 3H), 1.14 (t, J ) 6.2 Hz, 3H), 1.16 (d, J ) 6.2 Hz, 3H), 1.21 (d, J ) 6.2 Hz, 6H), 1.52-1.73
(m, 2H), 2.18-2.64 (m, 4H), 4.44-4.59 (m, 3H), 4.78-4.95 (m, 2H), 5.18 (m, 1H), 6.35 (br s, 1H), 6.54-6.63 (m, 3H),
7.46-7.55 (m, 2H), 7.63 (br s, 2H), 8.32 (d, J ) 8.9 Hz, 1H), 8.63 (s, 1H)
2ive
6ve
(DMSO-d6) 0.99 (t, J ) 7.4 Hz, 3H), 1.58-1.68 (m, 2H), 2.10-2.62 (m, 4H), 4.50-4.57 (m, 3H), 4.99 (m, 1H), 6.36 (br s,
1H), 6.57 (d, J ) 8.4 Hz, 1H), 6.78 (br s, 2H), 7.51-7.56 (m, 2H), 7.73 (br s, 2H), 8.39 (d, J ) 7.2 Hz, 1H), 8.66 (s, 1H)
(CDCl3) 1.24 (t, J ) 6.2 Hz, 3H), 1.25 (d, J ) 6.2 Hz, 3H), 1.30 (d, J ) 6.2 Hz, 6H), 2.43-2.74 (m, 2H), 4.57 (br s, 2H),
4.82-5.16 (m, 4H), 6.76 (d, J ) 8.6 Hz, 1H), 6.90 (d, J ) 6.2 Hz, 1H), 7.77 (dd, J ) 2.0 and 8.6 Hz, 1H), 7.97 (d,
J ) 2.0 Hz, 1H)
(DMSO-d6) 1.14 (t, J ) 6.2 Hz, 3H), 1.15 (d, J ) 6.2 Hz, 3H), 1.18 (d, J ) 6.2 Hz, 6H), 2.24-2.51 (m, 2H), 4.53 (m, 1H),
4.66 (d, J ) 5.2 Hz, 2H), 4.78-4.96 (m, 2H), 5.20 (m, 1H), 6.64 (br s, 2H), 6.90-6.98 (m, 2H), 7.56 (br s, 2H), 7.87
(d, J ) 8.2 Hz, 1H), 8.01 (s, 1H), 8.65 (s, 1H), 8.68 (d, J ) 8.2 Hz, 1H)
9ve
2ve
6vt
9vt
(DMSO-d6) 2.18-2.62 (m, 2H), 4.52 (m, 1H), 4.66 (d, J ) 5.2 Hz, 2H), 4.84 (m, 1H), 6.67 (br s, 2H), 6.87-6.96 (m, 2H),
7.20 (br s, 2H), 7.89 (d, J ) 8.0 Hz, 1H), 8.03 (s, 1H), 8.66 (s, 1H), 9.04 (d, J ) 8.2 Hz, 1H)
(CDCl3) 1.21 (d, J ) 6.2 Hz, 3H), 1.23 (d, J ) 6.2 Hz, 3H), 1.30 (d, J ) 6.2 Hz, 6H), 2.40-2.61 (m, 2H), 4.55 (br s, 2H),
4.84-5.18 (m, 4H), 6.73-6.78 (m, 2H), 7.25 (d, J ) 8.6 Hz, 1H) 7.75 (dd, J ) 2.0 and 8.6 Hz, 1H), 7.96 (d, J ) 2.0 Hz, 1H)
(DMSO-d6) 1.15 (d, J ) 6.2 Hz, 3H), 1.17 (d, J ) 6.2 Hz, 3H), 1.21 (d, J ) 6.2 Hz, 6H), 2.17-2.68 (m, 2H), 4.50 (m, 1H),
4.67 (d, J ) 5.4 Hz, 2H), 4.84-5.21 (m, 3H), 6.65 (br s, 2H), 6.93-6.99 (m, 2H), 7.58 (br s, 2H), 7.91 (d, J ) 8.2 Hz,
1H), 8.04 (s, 1H), 8.66 (s, 1H), 8.76 (d, J ) 8.0 Hz, 1H)
2vt
(DMSO-d6) 2.15-2.38 (m, 2H), 4.53 (m, 1H), 4.66 (d, J ) 5.2 Hz, 2H), 4.85 (m, 1H), 6.66 (br s, 2H), 6.86-6.98 (m, 2H),
7.62 (br s, 2H), 7.90 (d, J ) 8.8 Hz, 1H), 8.04 (s, 1H), 8.58 (d, J ) 7.4 Hz, 1H)

Novel Fluorinated Methotrexate Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3285
Ta ble 5. (Continued)
compd
δ
6vie
7vie
9vie
2vie
6vit
7vit
9vit
2vit
6viie
9viie
(CDCl3) 1.23 (d, J ) 6.2 Hz, 3H), 1.24 (d, J ) 6.2 Hz, 3H), 1.30 (d, J ) 6.2 Hz, 6H), 2.45-2.81 (m, 2H), 4.83-5.18 (m, 4H),
7.14 (d, J ) 6.2 Hz, 1H), 7.71-7.82 (m, 2H), 8.15 (m, 1H)
(CDCl3) 1.23 (d, J ) 6.2 Hz, 3H), 1.24 (d, J ) 6.2 Hz, 3H), 1.30 (d, J ) 6.2 Hz, 6H), 2.28-2.84 (m, 2H), 4.08 (br s, 2H),
4.81-5.17 (m, 4H), 6.74-6.84 (m, 2H), 7.42-7.56 (m, 2H)
(CDCl3-CD3OD) 1.24 (d, J ) 6.2 Hz, 3H), 1.25 (d, J ) 6.2 Hz, 3H), 1.30 (d, J ) 6.2 Hz, 6H), 2.41-2.70 (m, 2H), 4.64
(s, 2H), 4.83-5.16 (m, 4H), 6.73 (m, 1H), 7.10 (d, J ) 6.6 Hz, 1H), 7.50-7.61 (m, 2H) 8.81 (s, 1H)
(DMSO-d6) 2.14-2.51 (m, 2H), 4.52 (m, 1H), 4.57 (d, J ) 5.0 Hz, 2H), 4.98 (m, 1H), 6.81-6.93 (m, 4H), 7.54-7.64 (m, 2H),
7.86 (br s, 2H), 8.63 (d, J ) 7.2 Hz, 1H), 8.72 (s, 1H)
(CDCl3) 1.24 (d, J ) 6.2 Hz, 3H), 1.25 (d, J ) 6.2 Hz, 3H), 1.30 (d, J ) 6.2 Hz, 6H), 2.47-2.66 (m, 2H), 4.86-5.20 (m, 4H),
7.00 (d, J ) 7.4 Hz, 1H), 7.69-7.80 (m, 2H), 8.15 (m, 1H)
(CDCl3) 1.23 (d, J ) 6.2 Hz, 3H), 1.24 (d, J ) 6.2 Hz, 3H), 1.30 (d, J ) 6.2 Hz, 6H), 2.40-2.61 (m, 2H), 4.08 (br s, 2H),
4.85-5.14 (m, 4H), 6.69 (d, J ) 7.0 Hz, 1H), 6.77 (m, 1H), 7.43-7.55 (m, 2H)
(CDCl3-CD3OD) 1.15 (d, J ) 6.2 Hz, 3H), 1.16 (d, J ) 6.2 Hz, 3H), 1.18 (d, J ) 6.2 Hz, 6H), 2.25-2.84 (m, 2H), 4.65 (s, 2H),
4.61-5.15 (m, 4H), 6.74 (m, 1H), 6.96 (d, J ) 6.8 Hz, 1H), 7.48-7.60 (m, 2H) 8.82 (s, 1H)
(DMSO-d6) 2.18-2.33 (m, 2H), 4.53 (m, 1H), 4.57 (d, J ) 5.0 Hz, 2H), 4.83 (m, 1H), 6.81-6.91 (m, 4H), 7.56-7.66 (m, 2H),
7.78 (br s, 2H), 8.49 (d, J ) 8.0 Hz, 1H), 8.71 (s, 1H)
(CDCl3) 1.24 (d, J ) 6.2 Hz, 3H), 1.25 (d, J ) 6.2 Hz, 3H), 1.30 (d, J ) 6.2 Hz, 6H), 2.39-2.79 (m, 2H), 4.41 (br s, 2H),
4.83-5.15 (m, 4H), 6.77 (d, J ) 8.1 Hz, 1H), 6.81 (d, J ) 6.0 Hz, 1H), 7.56 (m, 1H), 7.79 (d, J ) 2.1 Hz, 1H)
(DMSO-d6) 1.16 (d, J ) 6.2 Hz, 3H), 1.17 (d, J ) 6.2 Hz, 3H), 1.19 (d, J ) 6.2 Hz, 6H), 2.30-2.49 (m, 2H), 4.51 (m, 1H),
4.61 (d, J ) 6.0 Hz, 2H), 4.85-4.95 (m, 2H), 5.20 (m, 1H), 6.60 (br s, 1H), 6.74-6.89 (m, 3H), 7.54-7.87 (m, 4H), 8.54
(d, J ) 8.4 Hz, 1H), 8.68 (s, 1H)
(DMSO-d6) 2.18-2.54 (m, 2H), 4.55 (m, 1H), 4.61 (d, J ) 5.2 Hz, 2H), 5.00 (m, 1H), 6.83-6.98 (m, 4H), 7.56-7.68 (m, 2H),
7.85 (br s, 2H), 8.61 (d, J ) 7.0 Hz, 1H), 8.71 (s, 1H)
(CDCl3) 1.15 (d, J ) 6.2 Hz, 3H), 1.17 (d, J ) 6.2 Hz, 3H), 1.21 (d, J ) 6.2 Hz, 6H), 2.21-2.65 (m, 2H), 4.53 (m, 1H),
4.82-4.91 (m, 2H), 5.23 (m, 1H), 5.90 (br s, 2H), 6.78 (d, J ) 9.8 Hz, 1H), 7.61 (m, 1H), 7.96 (d, J ) 2.0 Hz, 1H), 8.51
(d, J ) 7.8 Hz, 1H)
(CDCl3) 1.13 (d, J ) 6.2 Hz, 3H), 1.15 (d, J ) 6.2 Hz, 3H), 1.19 (d, J ) 6.2 Hz, 6H), 2.18-2.54 (m, 2H), 4.55 (m, 1H), 4.64
(d, J ) 6.2 Hz, 2H), 4.79-4.90 (m, 2H), 5.22 (s, 1H), 6.62 (br s, 1H), 6.78-6.87 (m, 3H), 7.64-8.03 (m, 4H), 8.56 (d, J )
8.6 Hz, 1H), 8.71 (s, 1H)
2viie
6viiie
9viiie
2viiie
6ixe
7ixe
9ixe
(DMSO-d6) 2.08-2.52 (m, 2H), 4.55 (m, 1H), 4.61 (d, J ) 5.6 Hz, 2H), 4.93 (m, 1H), 6.57 (br s, 1H), 6.69 (br s, 2H), 6.83
(d, J ) 10.2 Hz, 1H), 7.44-7.78 (m, 3H), 8.04 (d, J ) 2.0 Hz, 1H), 8.67 (br s, 1H), 8.69 (s, 1H)
(CDCl3) 1.26 (d, J ) 6.0 Hz, 3H), 1.27 (d, J ) 6.3 Hz, 3H), 1.29 (d, J ) 6.6 Hz, 3H), 1.30 (d, J ) 6.6 Hz, 3H), 1.42 (s, 9H),
2.07 (s, 3H), 2.38-2.75 (m, 2H), 3.19 (s, 3H), 4.78-5.15 (m, 4H), 6.72 (d, J ) 6.3 Hz, 1H), 7.26 (s, 1H)
(CDCl3) 1.25 (d, J ) 6.6 Hz, 3H), 1.26 (d, J ) 6.6 Hz, 3H), 1.28 (d, J ) 6.6 Hz, 3H), 1.29 (d, J ) 6.3 Hz, 3H), 2.00 (s, 3H),
2.36-2.74 (m, 2H), 2.97 (s, 3H), 4.80-5.15 (m, 4H), 6.48 (d, J ) 6.0 Hz, 1H), 7.20 (s, 1H)
(CDCl3) 1.23-1.30 (m, 12H), 2.18 (s, 3H), 2.35-2.71 (m, 2H), 2.84 (s, 3H), 4.35 (s, 2H), 4.78-5.13 (m, 4H), 6.88 (d, J ) 6.6 Hz,
1H), 7.30 (s, 1H), 8.80 (s, 1H)
2ixe
6ixt
(DMSO-d6) 1.90-2.20 (m, 2H), 2.14 (s, 3H), 2.78 (s, 3H), 4.31 (s, 2H), 4.40 (m, 1H), 4.83 (m, 1H), 7.48 (s, 1H), 8.60 (s, 1H)
(CDCl3) 1.22-1.30 (m, 12H), 1.42 (s, 9H), 2.07 (s, 3H), 2.35-2.57 (m, 2H), 3.19 (s, 3H), 4.87-5.15 (m, 4H), 6.57 (d, J ) 6.3 Hz,
1H), 7.23 (s, 1H)
7ixt
9ixt
2ixt
(CDCl3) 1.22-1.29 (m, 12H), 1.99 (s, 3H), 2.31-2.58 (m, 2H), 2.97 (d, J ) 4.2 Hz, 3H), 3.96 (br s, 3H), 4.87-5.13 (m, 4H),
6.31 (d, J ) 7.5 Hz, 1H), 7.17 (s, 1H)
(CDCl3) 1.20-1.30 (m, 12H), 2.17 (s, 3H), 2.30-2.54 (m, 2H), 2.83 (s, 3H), 4.34 (s, 2H), 4.84-5.13 (m, 4H), 6.70 (d, J ) 8.1 Hz,
1H), 7.26 (s, 1H), 8.80 (s, 1H)
(DMSO-d6) 2.10-2.30 (m, 2H), 2.15 (s, 3H), 2.70 (s, 3H), 4.32 (s, 2H), 4.45 (m, 1H), 4.86 (m, 1H), 7.53 (s, 1H), 8.61 (s, 1H)
NaHCO3 solution and then dried and evaporated to afford 9.4
g (98%) of 11 as a dark colored oil: IR (CHCl3) 3431, 3384
cm-1. Anal. (C9H12IN) C, H, I, N.
°C; IR (KBr) 1668 cm-1; HR-FABMS m/ z 180.1006 (M + H)+
(calcd for C10H14NO2 m/ z 180.0988).
2-[N -(t er t -B u t o x y c a r b o n y l)a m in o ]-3-m e t h y lt h io -
p h en e (15). To a solution of 8.0 g (56.3 mmol) of 14 in 60 mL
of tert-butyl alcohol were added 7.8 mL (59.2 mmol) of
triethylamine and 12.5 mL (58.0 mmol) of diphenyl phos-
phorazidate. The mixture was refluxed for 18 h and then
poured into ice water. The resulting precipitate was filtered
and washed with water. The crude product was chromato-
graphed on silica gel using 1:8 EtOAc-hexane to afford 8.0 g
(67%) of 15 as colorless crystals: mp 76-77 °C; IR (KBr) 1694
cm-1. Anal. (C10H15NO2S) C, H, N, S.
4-Iod o-2-p r op yla ceta n ilid e (12). To a solution of 9.4 g
(36.0 mmol) of 11 in 15 mL of toluene were added 7.0 mL (90
mmol) of pyridine and 7.0 mL (78 mmol) of acetic anhydride
at 0 °C. The mixture was stirred for 14 h at 25 °C. The
resulting precipitate was filtered and washed with toluene and
water and then dried in vacuo to afford 9.4 g (86%) of 12 as
colorless crystals: mp 180-181 °C; IR (KBr) 1688 cm-1; HR-
FABMS m/ z 304.0191 (M + H)+ (calcd for C11H15INO m/ z
304.0198).
4-(Acetyla m in o)-2-p r op ylben zon itr ile (13). To a solu-
tion of 8.9 g (29.4 mmol) of 12 in 60 mL of DMF was added
3.9 g (44.1 mmol) of copper(I) cyanide. The mixture was stirred
for 5 h at 160 °C. After cooling to 25 °C, the mixture was
diluted with 150 mL of EtOAc and then filtered. The filtrate
was washed with 4% aqueous ammonia, 1 N HCl solution, and
water. The organic solution was concentrated and the residue
was recrystallized from 1:4 EtOAc-hexane to afford 5.64 g
(95%) of 13 as colorless crystals: mp 131-133 °C; IR (KBr)
2229, 1670 cm-1. Anal. (C12H14N2O) C, H, N.
4-Am in o-2-p r op ylben zoic Acid (5iv). A mixture of 5.64
g (27.9 mmol) of 13 and 125 mL of concentrated HCl solution
was stirred for 4 h at 100 °C. After cooling to 0 °C, the mixture
was brought to pH 2.0 by adding 50% sodium hydroxide
solution. After being stirred for 30 min, the resulting precipi-
tate was filtered and washed with water and then dried to
afford 4.68 g (94%) of 5iv as colorless crystals: mp 109-111
2-[N-(ter t-Bu t oxyca r b on yl)-N-m et h yla m in o]-3-m et h -
ylth iop h en e (16). To a suspension of 0.50 g (20.6 mmol) of
sodium hydride in 11 mL of DMF was added 4.0 g (18.8 mmol)
of 15 at 0 °C. The mixture was stirred for 30 min at 0 °C, and
1.18 mL (19.0 mmol) of iodomethane was added. The mixture
was stirred for 18 h at 25 °C, diluted with water, and extracted
with ether. The organic solution was washed with brine, dried,
and concentrated. The residue was chromatographed on silica
gel using 1:30 EtOAc-hexane to afford 3.47 g (81%) of 16 as
a colorless oil: IR (Nujol) 1704 cm-1. Anal. (C11H17NO2S) C,
H, N, S.
5-[N-(ter t-Bu toxyca r bon yl)-N-m eth yla m in o]-3-m eth yl-
2-th iop h en eca r boxylic Acid (5ix). To a solution of 2.5 mL
(17.5 mmol) of diisopropylamine in 17 mL of THF was added
9.92 mL (16.0 mmol) of 1.6 M n-butyllithium in hexane at -50
°C. To this solution was added a solution of 13.4 g (15.0 mmol)
of 16 in 17 mL of THF dropwise over a 30-min period at -60

3286 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Kokuryo et al.
Ta ble 6. 13C NMR Data
compd
δ
11
12
13
5iv
15
16
(CDCl3) 14.58, 22.14, 33.52, 80.36, 117.99, 129.83, 135.94, 138.30, 144.36
(CDCl3) 14.41, 23.18, 24.83, 33.44, 90.06, 90.09, 126.12, 136.19 (2C), 138.67, 168.15
(CDCl3) 13.86, 22.18, 24.69, 32.85, 107.95, 118.98, 122.57, 130.96 (2C), 133.08, 139.60, 168.43
(CDCl3) 14.56, 21.93, 33.48, 114.74, 119.22, 125.80, 130.46, 132.53, 149.94, 172.87
(CDCl3) 12.52, 28.21 (3C), 81.16, 117.38, 127.44, 133.49, 152.92, 158.79
(CDCl3) 12.82, 28.25 (3C), 38.39, 80.44, 121.22, 127.99, 132.04, 140.50, 155.02
(CDCl3) 13.07, 28.19 (3C), 38.37, 81.45, 127.21, 133.94, 136.18, 148.65, 154.19, 167.34
5ix
6ie
(CDCl3) 21.56, 21.66 (2C), 21.77, 34.28 (d, J ) 20.6 Hz), 37.38, 49.89 (d, J ) 1.6 Hz), 67.73, 69.89, 70.25, 85.99 (d, J )
184.2 Hz), 125.12 (2C), 127.80 (2C), 128.00 (2C), 128.19, 128.57 (2C), 130.55, 136.23, 146.53, 155.06, 166.36, 168.72
(d, J ) 23.0 Hz), 170.90
(CDCl3-CD3OD) 21.55, 21.62 (2C), 21.74, 34.39 (d, J ) 20.0 Hz), 39.32, 49.64 (d, J ) 2.4 Hz), 55.91, 69.23, 70.09, 86.10
(d, J ) 183.5 Hz), 111.65 (2C), 121.51, 121.87, 128.94 (2C), 147.31, 149.70, 151.67, 155.05, 162.23, 162.87, 167.02, 168.96
(d, J ) 23.1 Hz), 171.29
9ie
2ie
6it
(DMSO-d6) 33.86 (d, J ) 20.6 Hz), 39.19, 48.71 (d, J ) 1.6 Hz), 54.79, 86.55 (d, J ) 181.7 Hz), 111.01, 111.13, 121.00, 121.42,
128.72, 128.90, 146.50, 149.08, 150.89, 154.05, 162.00, 162.59, 166.04, 170.74 (d, J ) 23.0 Hz), 172.99
(CDCl3) 21.59, 21.63 (2C), 21.73, 34.62 (d, J ) 20.7 Hz), 37.35, 49.80 (d, J ) 1.7 Hz), 67.71, 69.97, 70.02, 86.48 (d, J )
185.9 Hz), 125.06 (2C), 127.85 (2C), 127.93 (2C), 128.19, 128.53 (2C), 130.54, 136.19, 146.44, 155.08, 166.50, 168.71
(d, J ) 23.1 Hz), 170.75
(CDCl3) 21.57, 21.63 (2C), 21.73, 34.75 (d, J ) 20.7 Hz), 39.25, 49.55 (d, J ) 3.9 Hz), 55.88, 69.86, 69.97, 86.47 (d, J )
189.1 Hz), 111.58 (2C), 121.46, 121.95, 128.99 (2C), 147.23, 149.58, 151.64, 155.00, 162.34, 162.95, 167.20, 169.00
(d, J ) 23.1 Hz), 171.32
(DMSO-d6) 33.40 (d, J ) 20.7 Hz), 39.84, 48.40 (d, J ) 1.7 Hz), 54.72, 85.77 (d, J ) 181.9 Hz), 110.99 (2C), 121.00, 121.53,
128.85 (2C), 147.66, 148.92, 150.83, 151.67, 160.34, 162.58, 166.31, 170.80 (d, J ) 22.3 Hz), 173.06
(CDCl3) 20.23, 21.58, 21.68 (2C), 21.78, 34.22 (d, J ) 20.5 Hz), 50.16, 70.07, 70.54, 86.14 (d, J ) 185.2 Hz), 124.99, 125.62,
131.83, 134.08, 137.41, 151.21, 165.30, 168.77 (d, J ) 22.9 Hz), 170.74
(CDCl3) 17.23, 21.57, 21.65 (2C), 21.77, 34.52 (d, J ) 20.4 Hz), 49.72, 69.77, 70.00, 86.11 (d, J ) 184.0 Hz), 114.01, 121.52,
123.03, 126.58, 129.89, 148.48, 167.11, 168.89 (d, J ) 22.9 Hz), 171.34
9it
2it
6iie
7iie
9iie
(DMSO-d6) 17.58, 21.24, 21.34 (2C), 21.41, 33.50 (d, J ) 21.7 Hz), 46.20, 48.67, 68.13, 68.98, 86.32 (d, J ) 182.2 Hz), 108.48,
120.93, 121.00, 121.24, 126.90, 129.32, 146.59, 148.65, 149.47, 155.19, 162.74, 162.82, 166.37, 168.19 (d, J ) 23.5 Hz),
170.83
2iie
6iit
7iit
9iit
(DMSO-d6) 17.58, 33.91 (d, J ) 21.7 Hz), 46.19, 48.80, 86.56 (d, J ) 181.0 Hz), 108.47, 121.04, 121.24, 121.30, 126.90, 129.33,
147.53, 148.49, 149.38, 153.44, 161.61, 162.74, 166.24, 170.73 (d, J ) 22.9 Hz), 173.01
(CDCl3) 20.19, 21.64, 21.68 (2C), 21.76, 34.38 (d, J ) 20.5 Hz), 50.08, 70.11, 70.33, 86.58 (d, J ) 185.8 Hz), 124.96, 125.63,
131.87, 134.03, 137.48, 151.23, 165.34, 168.61 (d, J ) 22.9 Hz), 170.52
(CDCl3) 17.22, 21.61, 21.67 (2C), 21.77, 35.02 (d, J ) 20.4 Hz), 49.56, 69.77, 69.85, 86.57 (d, J ) 184.6 Hz), 113.98, 121.48,
123.03, 126.57, 129.93, 148.43, 167.16, 168.89 (d, J ) 23.5 Hz), 171.23
(DMSO-d6) 17.59, 21.22, 21.34 (2C), 21.41, 33.03 (d, J ) 20.5 Hz), 46.20, 48.60, 68.13, 69.02, 85.80 (d, J ) 182.2 Hz), 108.48,
120.92, 120.98, 121.27, 126.92, 129.33, 146.57, 148.69, 149.45, 155.18, 162.74, 162.81, 166.72, 168.34 (d, J ) 22.8 Hz),
170.96
2iit
(DMSO-d6) 17.63, 33.36 (d, J ) 21.6 Hz), 46.15, 48.38, 85.71 (d, J ) 181.6 Hz), 108.50, 125.33 (3C), 126.96, 129.35, 148.13,
148.37, 149.22, 150.30, 158.00, 162.73, 166.56, 170.56 (d, J ) 22.9 Hz), 173.03
2iiie
6iiit
9iiit
(DMSO-d6) 13.55, 23.85, 34.41 (d, J ) 21.1 Hz), 46.59, 49.35 (d, J ) 1.5 Hz), 87.10 (d, J ) 181.1 Hz), 109.16, 121.51, 121.81,
127.21, 127.30, 127.97, 148.16, 148.52, 149.69, 153.09, 161.54, 163.14, 166.73, 171.47 (d, J ) 22.2 Hz), 173.56
(CDCl3) 13.18, 22.03, 22.10 (2C), 22.19, 24.37, 35.45 (d, J ) 19.6 Hz), 49.95 (d, J ) 1.5 Hz), 70.20, 70.28, 85.43 (d, J )
185.1 Hz), 114.91, 123.73, 126.66, 127.77, 128.50, 147.99, 167.55, 169.29 (d, J ) 23.1 Hz), 171.56
(DMSO-d6) 13.53, 21.79 (3C), 21.88, 23.86, 33.48 (d, J ) 20.1 Hz), 46.62, 49.05 (d, J ) 1.5 Hz), 68.62, 69.49, 86.25 (d, J )
182.6 Hz), 109.17, 121.35, 121.46, 127.21, 127.32, 128.00, 147.17, 148.39, 149.81, 155.55, 163.16, 163.19, 167.24, 168.80
(d, J ) 23.1 Hz), 171.41
2iiit
6ive
9ive
(DMSO-d6) 13.46, 23.77, 33.99 (d, J ) 21.1 Hz), 46.48, 48.95 (d, J ) 1.5 Hz), 86.43 (d, J ) 181.2 Hz), 109.05, 121.31, 121.63,
127.07, 127.20, 127.86, 147.86, 148.11, 149.61, 153.99, 162.11, 163.03, 166.88, 171.59 (d, J ) 21.2 Hz), 173.84
(CDCl3) 14.59, 22.03, 22.12 (3C), 22.23, 33.72, 34.91 (d, J ) 20.6 Hz), 50.14 (d, J ) 1.5 Hz), 70.23, 70.48, 86.48 (d, J )
183.6 Hz), 115.02, 123.53, 126.39, 126.77, 129.51, 148.36, 167.53, 169.33 (d, J ) 22.6 Hz), 171.75
(DMSO-d6) 14.24, 21.58, 21.70 (2C), 21.77, 21.81, 32.88, 33.77 (d, J ) 20.6 Hz), 46.45, 48.90 (d, J ) 3.0 Hz), 68.47, 69.31,
84.60 (d, J ) 181.6 Hz), 109.16, 121.22 (2C), 125.61, 127.15, 128.92, 147.15, 148.33, 149.64, 155.36,163.03 (2C), 166.72,
168.52 (d, J ) 24.1 Hz), 171.19
2ive
6ve
(DMSO-d6) 14.29, 21.85, 32.89, 34.52 (d, J ) 22.1 Hz), 46.47, 49.50 (d, J ) 3.0 Hz), 87.44 (d, J ) 180.6 Hz), 109.14, 121.27,
121.52, 125.67, 127.11, 128.91, 147.59, 148.18, 149.58, 154.60, 162.52, 163.01, 166.50, 171.60 (d, J ) 22.1 Hz), 173.59
(CDCl3) 21.96, 22.05 (2C), 22.15, 34.78 (d, J ) 20.1 Hz), 50.21 (d, J ) 1.5 Hz), 70.30, 70.53, 86.49 (d, J ) 185.1 Hz), 112.98
(q, J ) 30.7 Hz), 117.00, 122.63, 125.10 (q, J ) 254.4 Hz), 127.07 (q, J ) 5.6 Hz), 132.17, 148.10, 166.25, 169.19 (d, J )
23.1 Hz), 171.17
9ve
6vt
(DMSO-d6) 21.19, 21.30 (2C), 21.36, 33.21 (d, J ) 21.1 Hz), 45.77, 48.64 (d, J ) 3.0 Hz), 68.25, 68.94, 86.17 (d, J ) 182.1 Hz),
111.00 (q, J ) 30.4 Hz), 111.86, 120.36, 120.76, 124.62 (q, J ) 272.1 Hz), 126.10 (q, J ) 3.2 Hz), 132.86, 145.59, 147.16,
149.23, 155.13,162.60, 162.80, 165.01, 168.09 (d, J ) 23.1 Hz), 170.53
(CDCl3) 21.98, 22.07 (2C), 22.17, 35.06 (d, J ) 19.6 Hz), 50.18 (d, J ) 1.5 Hz), 70.32, 70.37, 86.87 (d, J ) 165.0 Hz), 113.21
(q, J ) 30.2 Hz), 117.01, 122.60, 124.97 (q, J ) 256.8 Hz), 127.21 (q, J ) 1.5 Hz), 132.24, 148.17, 166.41, 169.28 (d, J )
23.1 Hz), 171.23
9vt
(DMSO-d6) 21.30 (3C), 21.36, 32.87 (d, J ) 20.6 Hz), 45.77, 48.69, 68.28, 69.02, 85.68 (d, J ) 181.6 Hz), 111.04 (q, J )
29.5 Hz), 111.86, 120.36, 120.77, 125.95 (q, J ) 254.3 Hz), 126.30 (q, J ) 3.2 Hz), 132.86, 145.59, 147.16, 149.21, 155.14,
162.60, 162.80, 165.40, 168.25 (d, J ) 23.1 Hz), 170.65
7vie
9vie
2vie
(CDCl3) 21.98, 22.07 (2C), 22.17, 35.06 (d, J ) 19.6 Hz), 50.18 (d, J ) 1.5 Hz), 70.32, 70.37, 86.87 (d, J ) 165.0 Hz), 113.21
(q, J ) 30.2 Hz), 117.01, 122.60, 124.97 (q, J ) 256.8 Hz), 127.21 (q, J ) 1.5 Hz), 132.24, 148.17, 166.41, 169.28 (d, J )
23.1 Hz), 171.23
(DMSO-d6) 21.30 (3C), 21.36, 32.87 (d, J ) 20.6 Hz), 45.77, 48.69, 68.28, 69.02, 85.68 (d, J ) 181.6 Hz), 111.04 (q, J )
29.5 Hz), 111.86, 120.36, 120.77, 125.95 (q, J ) 254.3 Hz), 126.30 (q, J ) 3.2 Hz), 132.86, 145.59, 147.16, 149.21, 155.14,
162.60, 162.80, 165.40, 168.25 (d, J ) 23.1 Hz), 170.65
(DMSO-d6) 34.56 (d, J ) 20.6 Hz), 46.03, 49.94 (d, J ) 2.0 Hz), 87.56 (d, J ) 152.4 Hz), 111.49, 113.53 (d, J ) 31.8 Hz),
121.40, 121.85 (d, J ) 5.6 Hz), 125.19, 139.36 (d, J ) 12.0 Hz), 146.76, 149.92, 150.33 (d, J ) 239.4 Hz), 154.91, 162.81,
163.06, 163.18, 163.41, 165.25

Novel Fluorinated Methotrexate Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3287
Ta ble 6 (Continued)
compd
δ
6vit
7vit
9vit
2vit
(CDCl3) 22.07, 22.11 (2C), 22.18, 34.11 (d, J ) 19.6 Hz), 50.63, 70.66, 70.95, 86.94 (d, J ) 186.2 Hz), 118.31 (q, J ) 23.1 Hz),
123.36 (d, J ) 4.8 Hz), 127.04 (d, J ) 3.2 Hz), 139.58, 140.77 (d, J ) 7.2 Hz), 155.81 (d, J ) 266.5 Hz), 164.25, 168.95
(d, J ) 23.6 Hz), 170.59
(CDCl3) 22.03, 22.19 (2C), 22.30, 35.15 (d, J ) 21.1 Hz), 50.71, 69.76, 71.03, 86.46 (d, J ) 184.6 Hz), 115.51 (d, J ) 33.5 Hz),
115.91 (d, J ) 19.2 Hz), 123.89 (d, J ) 12.0 Hz), 124.32 (d, J ) 7.2 Hz), 138.90 (d, J ) 13.6 Hz), 151.09 (d, J ) 240.2 Hz),
166.41, 169.24 (d, J ) 23.1 Hz), 171.39
(DMSO-d6) 21.69 (3C), 21.77, 33.24 (d, J ) 19.6 Hz), 46.01, 49.05 (d, J ) 2.5 Hz), 68.61, 69.40, 86.05 (d, J ) 183.6 Hz), 111.62,
114.04 (d, J ) 19.2 Hz), 121.26, 121.37, 125.37, 139.65 (d, J ) 12.0 Hz), 146.35, 149.96, 150.31 (d, J ) 238.6 Hz), 155.63,
163.29, 163.37, 165.98, 168.64 (d, J ) 23.1 Hz), 171.13
(DMSO-d6) 34.16 (d, J ) 22.1 Hz), 46.03, 49.34 (d, J ) 6.0 Hz), 86.73 (d, J ) 183.6 Hz), 111.48, 113.91 (d, J ) 19.9 Hz),
121.28, 121.65 (d, J ) 5.5 Hz), 125.01 (d, J ) 7.9 Hz), 139.31 (d, J ) 12.7 Hz), 146.60, 149.80, 150.21 (d, J ) 238.2 Hz),
154.81, 162.70, 162.93 (d, J ) 12.7 Hz), 165.54, 172.01, 173.59
(CDCl3) 22.01, 22.12 (2C), 22.23, 34.82 (d, J ) 20.1 Hz), 50.22, 70.34, 70.64, 86.46 (d, J ) 184.1 Hz), 108.90, 115.05, 124.54,
128.01, 132.65, 147.85, 166.08, 169.23 (d, J ) 23.1 Hz), 171.56
(DMSO-d6) 21.23, 21.32 (2C), 21.40, 33.28 (d, J ) 21.1 Hz), 45.87, 48.71, 68.22, 68.97, 86.21 (d, J ) 183.1 Hz), 110.64, 117.27,
120.87, 121.80, 127.88, 128.37, 145.79, 146.16, 149.45, 149.82, 155.18, 162.68, 162.82, 165.00, 168.14 (d, J ) 22.6 Hz),
170.60
6viie
9viie
2viie
(DMSO-d6) 34.51 (d, J ) 20.6 Hz), 45.84, 49.62 (d, J ) 4.0 Hz), 87.90 (d, J ) 180.6 Hz), 110.56, 110.72, 114.59, 117.29, 120.87,
121.50, 122.26, 127.66, 128.37, 145.90, 149.38, 154.81, 162.63, 164.50, 174.44
6viiie (DMSO-d6) 22.03, 22.12 (2C), 22.23, 34.82 (d, J ) 20.6 Hz), 50.24 (d, J ) 1.5 Hz), 70.34, 70.65, 86.46 (d, J ) 184.6 Hz), 108.91,
115.07, 124.56, 128.03, 132.65, 147.82, 166.08, 169.23 (d, J ) 23.1 Hz), 171.56
9viiie (DMSO-d6) 21.72, 21.82, 21.88, 23.86, 33.74 (d, J ) 21.6 Hz), 46.48, 49.15 (d, J ) 3.5 Hz), 68.73, 69.48, 86.70 (d, J ) 182.6 Hz),
108.08, 111.19, 122.88, 128.60, 128.95, 132.14 (2C), 147.30, 147.52, 149.33, 149.82, 163.13, 165.33, 168.61 (d, J ) 23.1 Hz),
171.06
2viiie (DMSO-d6) 34.46 (d, J ) 20.6 Hz), 46.49, 49.72 (d, J ) 3.5 Hz), 87.47 (d, J ) 181.1 Hz), 108.13, 111.14, 121.39, 123.24, 128.90,
132.17, 146.81, 147.40, 149.84, 154.55, 162.54, 163.11, 165.16, 171.72 (d, J ) 22.3 Hz), 173.50
6ixe
9ixe
2ixe
6ixt
9ixt
2ixt
(CDCl3) 13.09, 21.54, 21.64, 21.67, 21.75, 28.22 (3C), 34.41 (d, J ) 20.5 Hz), 38.39, 49.76, 69.89, 70.29, 81.22, 85.99 (d, J )
184.6 Hz), 130.05, 132.38, 133.54, 145.52, 154.33, 161.63, 168.72 (d, J ) 23.5 Hz), 170.82
(CDCl3) 13.59, 21.22, 21.33 (2C), 21.39, 33.39 (d, J ) 20.4 Hz), 43.22, 48.58, 59.69, 68.33, 69.05, 86.19 (d, J ) 182.8 Hz),
121.24, 124.32, 126.83, 131.65, 144.62, 149.91, 155.26, 156.97, 161.26, 162.78, 162.93, 168.11 (d, J ) 23.4 Hz), 170.47
(DMSO-d6) 13.51, 34.17 (d, J ) 21.5 Hz), 43.16, 49.28, 59.74, 87.19 (d, J ) 181.1 Hz), 121.19, 124.52, 127.43, 131.29, 144.83,
149.86, 154.71, 156.63, 160.96, 162.53, 162.68, 171.21 (d, J ) 22.3 Hz), 172.89
(CDCl3) 13.07, 21.64, 21.70 (2C), 21.76, 28.20 (3C), 34.87 (d, J ) 19.9 Hz), 38.38, 49.66, 70.00, 70.09, 81.18, 86.47 (d, J )
185.7 Hz), 130.02, 132.43, 133.50, 145.54, 154.34, 161.65, 168.74 (d, J ) 23.4 Hz), 170.69
(DMSO-d6) 13.57, 21.33 (2C), 21.39 (2C), 32.94 (d, J ) 19.9 Hz), 43.19, 48.54, 59.72, 68.37, 69.09, 85.68 (d, J ) 182.8 Hz),
121.24, 124.34, 126.71, 131.74, 144.64, 149.94, 155.27, 157.10, 161.61, 162.78, 162.93, 168.27 (d, J ) 22.9 Hz), 170.63
(DMSO-d6) 13.54, 33.67 (d, J ) 21.7 Hz), 43.19, 48.61, 59.78, 85.98 (d, J ) 182.2 Hz), 121.25, 124.52, 127.29, 131.44, 144.90,
149.93, 154.79, 156.82, 161.31, 162.59, 162.76, 170.86 (d, J ) 21.7 Hz), 172.80
°C. After being stirred for 30 min, crushed dry ice was added
at -50 °C. The mixture was allowed to warm to 25 °C and
stirred for 18 h. The mixure was poured into water, brought
to pH 5.0 by adding citric acid, and then extracted with CHCl3.
The organic solution was washed with brine, dried, and
concentrated. The residue was chromatographed on silica gel
using 1:20 MeOH-CHCl3 to afford 1.51 g (37%) of 5ix as
mixture was stirred for 18 h at 50 °C. After adding 300 mL
of water at 0 °C, the mixture was stirred for 5 h and left for
24 h. The resulting precipitate was filtered and dissolved in
1:9 MeOH-CHCl3. The organic solution was washed with
saturated NaHCO3 solution and water and then dried and
concentrated. The residue was chromatographed on silica gel
using 1:10 MeOH-CHCl3 to afford 2.87 g (67%) of 9ie as a
colorless crystals: mp 133-134 °C; IR (KBr) 1715, 1666 cm-1
.
yellow powder: mp 152-154 °C; [R]24 +7.2° (c 1.0, MeOH-
D
CHCl3); IR (KBr) 1735, 1655 cm-1. Anal. (C26H33FN8O5‚0.4H2O)
C, H, F, N.
Anal. (C12H17NO4S) C, H, N, S.
N-[4-[[(Ben zyloxy)ca r b on yl]m et h yla m in o]b en zoyl]-
(rS,γR)-γ-flu or oglu tam ic Acid r,γ-Diisopr opyl Ester (6ie).
To a solution of 2.68 g (9.39 mmol) of 4e‚HCl17 in 20 mL of
CH2Cl2 was added a solution of 7.25 g (75.1 mmol) of potassium
bicarbonate in 40 mL of water at 0 °C. After addition of 3.42
g (11.3 mmol) of 5i in 20 mL of CH2Cl2, the mixture was stirred
for 24 h at 25 °C and then diluted with EtOAc. The organic
solution was washed with brine, dried, and concentrated. The
residue was chromatographed on silica gel using 1:2 EtOAc-
hexane to afford 4.14 g (85%) of 6ie as colorless crystals: mp
78-79 °C; [R]24D +21.4° (c 1.0, CHCl3); IR (CHCl3) 1731, 1702,
1663 cm-1. Anal. (C27H33FN2O7) C, H, F, N.
N-[4-[[(2,4-Diam in o-6-pter idin yl)m eth yl]m eth ylam in o]-
ben zoyl]-(rS,γR)-γ-flu or oglu ta m ic Acid r,γ-Diisop r op yl
Ester (9ie). A solution of 4.04 g (7.82 mmol) of 6ie in 25 mL
of 30% hydrogen bromide in acetic acid was stirred for 4 h at
25 °C. After adding 250 mL of ether at 0 °C, the mixture was
stirred for 1 h and then left for 24 h. The organic layer was
decanted and then the residue was washed with ether three
times. The resulting residue was dissolved in EtOAc. The
organic solution was washed with saturated NaHCO3 solution
and brine and then dried and evaporated to afford N-[4-
(methylamino)benzoyl]-(RS,γR)-γ-fluoroglutamic acid R,γ-di-
isopropyl ester (7ie), which was used for the next reaction
without further purification.
N-[4-[[(2,4-Diam in o-6-pter idin yl)m eth yl]m eth ylam in o]-
ben zoyl]-(rS,γR)-γ-flu or oglu ta m ic Acid (2ie). To a sus-
pension of 1.13 g (2.04 mmol) of 9ie in 25 mL of ethanol was
added a solution of 1.6 g (5.1 mmol) of barium hydroxide
octahydrate in 25 mL of water at 0 °C. After being stirred for
5 h at 25 °C, ethanol was evaporated. The mixture was
brought to pH 3.0 by adding 1 N HCl solution. The precipitate
was filtered and washed with water and then dried in vacuo
at 40 °C to afford 0.84 g (87%) of 2ie as a yellow powder: mp
>253 °C; [R]25 +20.9° (c 1.0, 0.1 N NaOH); IR (KBr) 1639
D
cm-1
. HPLC tR ) 6.3 min; HPLC of the dimethyl ester on
Ultron ES-OVM, tR ) 28.8 min, on Ultron ES-CD, tR ) 17.8
min. Anal. (C20H21FN8O5‚2.3H2O) C, H, F, N.
N-[4-[[(Ben zyloxy)ca r b on yl]m et h yla m in o]b en zoyl]-
(rS,γS)-γ-flu or oglu ta m ic Acid r,γ-Diisop r op yl Ester (6it).
The procedure described for the preparation of 6ie was used:
colorless oil; [R]23.5 +15.5° (c 1.0, CHCl3); IR (CHCl3) 1731,
D
1701, 1665 cm-1. Anal. (C27H33FN2O7) C, H, F, N.
N-[4-[[(2,4-Diam in o-6-pter idin yl)m eth yl]m eth ylam in o]-
ben zoyl]-(rS,γS)-γ-flu or oglu ta m ic Acid r,γ-Diisop r op yl
Ester (9it). The procedure described for the preparation of
9ie was used.
N-[4-(Met h yla m in o)b en zoyl]-(rS,γS)-γ-flu or oglu t a m -
ic a cid r,γ-d iisop r op yl ester (7it) was used for the next
reaction without further purification.
To a solution of the above product in 25 mL of N,N-
dimethylacetamide was added 2.64 g (6.67 mmol) of 8.27 The

3288 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Kokuryo et al.
9it: mp 158-159 °C; [R]25 +9.8° (c 1.0, MeOH-CHCl3);
procedure described for the preparation of 2ie was used: mp
239-242 °C; [R]23D +7.6° (c 0.5, DMSO). HPLC tR ) 13.2 min;
HR-FABMS m/ z 487.1880 (M + H)+ (calcd for C21H24FN8O5
m/ z 487.1854).
D
IR (KBr) 1736, 1633 cm-1
H, F, N.
. Anal. (C26H33FN8O5‚0.5H2O) C,
N-[4-[[(2,4-Diam in o-6-pter idin yl)m eth yl]m eth ylam in o]-
ben zoyl]-(rS,γS)-γ-flu or oglu ta m ic Acid (2it). The proce-
dure described for the preparation of 2ie was used: mp >255
N-(4-Am in o-3-eth ylben zoyl)-(rS,γS)-γ-flu or oglu ta m ic
Acid r,γ-Diisop r op yl Ester (6iiit). To a solution of 2.60 g
(9.08 mmol) of 4t‚HCl17 in 45 mL of DMF was added a solution
of 1.5 g (9.08 mmol) of 5iii. After cooling to -40 °C, 1.7 mL
(10.9 mmol) of diethyl cyanophosphonate (DEPC) and 5.06 mL
(36.3 mmol) of triethylamine were added. The mixture was
allowed to warm to 25 °C and stirred for 14 h. The mixture
was poured into water and extracted with EtOAc. The organic
solution was washed with brine, dried, and concentrated. The
residue was chromatographed on silica gel using 1:2 EtOAc-
hexane to afford 2.63 g (73%) of 6iiit as colorless crystals: mp
°C; [R]24 +11.4° (c 1.0, 0.1 N NaOH); IR (KBr) 1638 cm-1
.
D
HPLC tR ) 11.6 min; HPLC of the dimethyl ester on Ultron
ES-OVM, tR ) 25.6 min, on Ultron ES-CD, tR ) 23.5 min. Anal.
(C20H21FN8O5‚1.8H2O) C, H, F, N.
N-(3-Met h yl-4-n it r ob en zoyl)-(rS,γR)-γ-flu or oglu t a m -
ic Acid r,γ-Diisop r op yl Ester (6iie). The procedure de-
scribed for the preparation of 6ie was done using triethylamine
and DME: mp 70-71 °C; [R]22D +35.0° (c 0.5, CHCl3); IR (KBr)
1749, 1726, 1645, 1528, 1345 cm-1. Anal. (C19H25FN2O7) C,
H, F, N.
N-(4-Am in o-3-m eth ylben zoyl)-(rS,γR)-γ-flu or oglu ta m -
ic Acid r,γ-Diisop r op yl Ester (7iie). To a solution of 904
mg (2.19 mmol) of 6iie in 22 mL of acetic acid was added 1.7
g of zinc dust. The mixture was stirred for 2.5 h at 25 °C and
filtered. The filtrate was concentrated in vacuo. The residue
was poured into saturated NaHCO3 solution and then ex-
tracted with CHCl3. The organic solution was washed with
saturated NaHCO3 solution and brine and then dried and
concentrated. The residue was chromatographed on silica gel
using 1:3 EtOAc-hexane to afford 671 mg (80%) of 7iie as
colorless crystals: mp 121-122 °C; [R]22D +28.2° (c 0.5, CHCl3);
IR (KBr) 1762, 1724, 1638 cm-1. Anal. (C19H27FN2O5) C, H,
F, N.
59-61 °C; [R]23 +20.9° (c 0.5, CHCl3); IR (KBr) 1731, 1632
D
cm-1. Anal. (C20H29FN2O5) C, H, F, N.
N-[4-[[(2,4-Dia m in o-6-p ter id in yl)m eth yl]a m in o]-3-eth -
ylben zoyl]-(rS,γS)-γ-flu or oglu ta m ic Acid r,γ-Diisop r op yl
Ester (9iiit). The procedure described for the preparation of
9ie was used: mp 234-236 °C; [R]23 +13.4° (c 0.5, DMSO);
D
IR (KBr) 1737, 1627 cm-1
H, F, N.
. Anal. (C27H35FN8O5‚0.6H2O) C,
N-[4-[[(2,4-Dia m in o-6-p ter id in yl)m eth yl]a m in o]-3-eth -
ylben zoyl]-(rS,γS)-γ-flu or oglu ta m ic Acid (2iiit). The pro-
cedure described for the preparation of 2ie was used: mp 254-
256 °C; [R]23 +15.6° (c 0.5, DMSO); IR (KBr) 1638 cm-1
.
D
HPLC tR ) 15.0 min; HR-FABMS m/ z 487.1876 (M + H)+
(calcd for C21H24FN8O5 m/ z 487.1854).
N -[4-[[(2,4-Dia m in o-6-p t e r id in yl)m e t h yl]a m in o]-3-
m eth ylben zoyl]-(rS,γR)-γ-flu or oglu ta m ic Acid r,γ-Diiso-
p r op yl Ester (9iie). The procedure described for the prepa-
N-(4-Am in o-3-p r op ylben zoyl)-(rS,γR)-γ-flu or oglu ta m -
ic Acid r,γ-Diisop r op yl Ester (6ive). The procedure de-
scribed for the preparation of 6iiit was used: mp 94-95 °C;
[R]22D +24.1° (c 0.5, CHCl3); IR (CHCl3) 1733, 1653 cm-1. Anal.
(C21H31FN2O5) C, H, F, N.
ration of 9ie was used: mp 234-236 °C; [R]23 +10.0° (c 0.5,
D
DMSO); IR (KBr) 1735, 1633 cm-1; HR-LSIMS m/ z 557.2637
(M + H)+ (calcd for C26H34FN8O5 m/ z 557.2634).
N-[4-[[(2,4-Dia m in o-6-p ter id in yl)m eth yl]a m in o]-3-p r o-
p ylben zoyl]-(rS,γR)-γ-flu or oglu ta m ic Acid r,γ-Diisop r o-
p yl Ester (9ive). The procedure described for the preparation
of 9ie was used: mp 227-229 °C; [R]23D +10.2° (c 0.5, DMSO);
N -[4-[[(2,4-Dia m in o-6-p t e r id in yl)m e t h yl]a m in o]-3-
m et h ylb en zoyl]-(rS,γR)-γ-flu or oglu t a m ic Acid (2iie).28
The procedure described for the preparation of 2ie was used:
mp >270 °C; IR (KBr) 1639 cm-1. HPLC tR ) 6.5 min; HR-
LSIMS m/ z 473.1695 (M + H)+ (calcd for C20H22FN8O5 m/ z
473.1695).
IR (KBr) 1735, 1626 cm-1
H, F, N.
. Anal. (C28H37FN8O5‚0.3H2O) C,
N-[4-[[(2,4-Dia m in o-6-p ter id in yl)m eth yl]a m in o]-3-p r o-
p ylben zoyl]-(rS,γR)-γ-flu or oglu ta m ic Acid (2ive). The
procedure described for the preparation of 2ie was used: mp
N-(3-Met h yl-4-n it r ob en zoyl)-(rS,γS)-γ-flu or oglu t a m -
ic Acid r,γ-Diisop r op yl Ester (6iit). The procedure de-
scribed for the preparation of 6ie was done using triethylamine
>270 °C; [R]23 +15.9° (c 0.5, DMSO); IR (KBr) 1638 cm-1
.
D
and DME: colorless oil; [R]23 +19.2° (c 0.5, CHCl3); IR (film)
D
HPLC tR ) 32.0 min; HR-FABMS m/ z 501.2028 (M + H)+
1738, 1652, 1527, 1347 cm-1. Anal. (C19H25FN2O7) C, H, F,
N.
(calcd for C22H26FN8O5 m/ z 501.2010).
N-[4-Am in o-3-(tr iflu or om eth yl)ben zoyl]-(rS,γR)-γ-flu -
or oglu ta m ic Acid r,γ-Diisop r op yl Ester (6ve). The pro-
cedure described for the preparation of 6iiit was used: color-
less oil; [R]23D +15.4° (c 0.5, CHCl3); HR-FABMS m/ z 437.1697
(M + H)+ (calcd for C19H25F4N2O5 m/ z 437.1699).
N-(4-Am in o-3-m eth ylben zoyl)-(rS,γS)-γ-flu or oglu ta m -
ic Acid r,γ-Diisop r op yl Ester (7iit). The procedure de-
scribed for the preparation of 7iie was used: mp 91-92 °C;
[R]22 +24.0° (c 0.5, CHCl3); IR (KBr) 1743, 1717, 1636 cm-1
.
D
Anal. (C19H27FN2O5) C, H, F, N.
N-[4-[[(2,4-Dia m in o-6-p ter id in yl)m eth yl]a m in o]-3-(tr i-
flu or om eth yl)ben zoyl]-(rS,γR)-γ-flu or oglu tam ic Acid r,γ-
Diisop r op yl Ester (9ve). The procedure described for the
N -[4-[[(2,4-Dia m in o-6-p t e r id in yl)m e t h yl]a m in o]-3-
m eth ylben zoyl]-(rS,γS)-γ-flu or oglu ta m ic Acid r,γ-Diiso-
p r op yl Ester (9iit). The procedure described for the prepa-
preparation of 9ie was used: mp 241-243 °C; [R]22 +9.1° (c
ration of 9ie was used: mp 233-235 °C; [R]23 +14.3° (c 0.5,
D
D
0.5, DMSO); HR-FABMS m/ z 611.2379 (M + H)+ (calcd for
DMSO); IR (KBr) 1735, 1633 cm-1; HR-LSIMS m/ z 557.2631
(M + H)+ (calcd for C26H34FN8O5 m/ z 557.2634).
C26H31F4N8O5 m/ z 611.2353).
N-[4-[[(2,4-Dia m in o-6-p ter id in yl)m eth yl]a m in o]-3-(tr i-
flu or om et h yl)b en zoyl]-(rS,γR)-γ-flu or oglu t a m ic Acid
(2ve).28 The procedure described for the preparation of 2ie
was used: mp >270 °C. 1H NMR (DMSO-d6) δ 2.18-2.62 (m,
2H), 4.52 (m, 1H), 4.66 (d, J ) 5.2 Hz, 2H), 4.84 (m, 1H), 6.67
(br s, 2H), 6.87-6.96 (m, 2H), 7.20 (br s, 2H), 7.89 (d, J ) 8.0
Hz, 1H), 8.03 (s, 1H), 8.66 (s, 1H), 9.04 (d, J ) 8.2 Hz, 1H);
HPLC tR ) 17.8 min; HR-FABMS m/ z 527.1424 (M + H)+
(calcd for C20H19F4N8O5 m/ z 527.1415).
N -[4-[[(2,4-Dia m in o-6-p t e r id in yl)m e t h yl]a m in o]-3-
m et h ylben zoyl]-(rS,γS)-γ-flu or oglu t a m ic Acid (2iit).28
The procedure described for the preparation of 2ie was used:
mp >270 °C; IR (KBr) 1638 cm-1. HPLC tR ) 7.1 min; HR-
LSIMS m/ z 473.1694 (M + H)+ (calcd for C20H22FN8O5 m/ z
473.1695).
N-(4-Am in o-3-et h ylb en zoyl)-(rS,γR)-γ-flu or oglu t a m -
ic Acid r,γ-Diisop r op yl Ester (6iiie). The procedure de-
scribed for the preparation of 6iiit was used: mp 58-59 °C;
HR-FABMS m/ z 397.2130 (M + H)+ (calcd for C20H30FN2O5
m/ z 397.2139).
N-[4-[[(2,4-Dia m in o-6-p ter id in yl)m eth yl]a m in o]-3-eth -
ylben zoyl]-(rS,γR)-γ-flu or oglu ta m ic Acid r,γ-Diisop r o-
p yl Ester (9iiie). The procedure described for the preparation
of 9ie was used: mp 231-233 °C; HR-FABMS m/ z 571.2827
(M + H)+ (calcd for C27H36FN8O5 m/ z 571.2793).
N-[4-[[(2,4-Dia m in o-6-p ter id in yl)m eth yl]a m in o]-3-eth -
ylben zoyl]-(rS,γR)-γ-flu or oglu t a m ic Acid (2iiie). The
N-[4-Am in o-3-(tr iflu or om eth yl)ben zoyl]-(rS,γS)-γ-flu o-
r oglu ta m ic Acid r,γ-Diisop r op yl Ester (6vt). The proce-
dure described for the preparation of 6iiit was used: colorless
oil; [R]22 +8.0° (c 0.5, CHCl3); HR-FABMS m/ z 437.1690 (M
D
+ H)+ (calcd for C19H25F4N2O5 m/ z 437.1699).
N-[4-[[(2,4-Dia m in o-6-p ter id in yl)m eth yl]a m in o]-3-(tr i-
flu or om eth yl)ben zoyl]-(rS,γS)-γ-flu or oglu ta m ic Acid r,γ-
Diisop r op yl Ester (9vt). The procedure described for the
preparation of 9ie was used: mp 237-239 °C; [R]22 +9.5° (c
D

Novel Fluorinated Methotrexate Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3289
0.5, DMSO); IR (KBr) 1735, 1622 cm-1; HR-FABMS m/ z
611.2362 (M + H)+ (calcd for C26H31F4N8O5 m/ z 611.2353).
N-[4-[[(2,4-Dia m in o-6-p ter id in yl)m eth yl]a m in o]-3-(tr i-
flu or om et h yl)b en zoyl]-(rS,γS)-γ-flu or oglu t a m ic Acid
(2vt).28 The procedure described for the preparation of 2ie
was used: mp >270 °C; IR (KBr) 1632 cm-1. HPLC tR ) 18.5
min; HR-FABMS m/ z 527.1427 (M + H)+ (calcd for C20H19F4-
N8O5 m/ z 527.1415).
N-(3-F lu or o-4-n it r ob en zoyl)-(rS,γR)-γ-flu or oglu t a m -
ic Acid r,γ-Diisop r op yl Ester (6vie). The procedure de-
scribed for the preparation of 6ie was done using triethylamine
and CH2Cl2. mp 86-87 °C. HR-FABMS m/ z 417.1483 (M +
H)+ (calcd for C18H23F2N2O7 m/ z 417.1473).
N-(4-Am in o-3-flu or oben zoyl)-(rS,γR)-γ-flu or oglu ta m -
ic Acid r,γ-Diisop r op yl Ester (7vie). A solution of 980 mg
(2.35 mmol) of 6vie in 25 mL of EtOAc was hydrogenated using
60 mg of platinum(IV) oxide for 4 h. The mixture was filtered
through Celite, and the filtrate was concentrated. The residue
was chromatographed on silica gel using 1:1 EtOAc-hexane
to afford 818 mg (100%) of 7vie as colorless crystals: mp 102-
104 °C; [R]22D +24.6° (c 0.5, CHCl3); HR-FABMS m/ z 387.1718
(M + H)+ (calcd for C18H25F2N2O5 m/ z 387.1732).
N-(4-Am in o-3-br om oben zoyl)-(rS,γR)-γ-flu or oglu ta m -
ic Acid r,γ-Diisop r op yl Ester (6viiie). The procedure
described for the preparation of 6iiit was used: mp 121-123
°C; [R]22 +25.7° (c 0.5, CHCl3); IR (CHCl3) 1733, 1657 cm-1
.
D
Anal. (C18
H24BrFN2O5) C, H, Br, F, N.
N-[3-Br om o-4-[[(2,4-d ia m in o-6-p ter id in yl)m eth yl]a m i-
n o]ben zoyl]-(rS,γR)-γ-flu or oglu ta m ic Acid r,γ-Diisop r o-
p yl Ester (9viiie). The procedure described for the prepa-
ration of 9ie was done using potassium iodide: mp 213-215
°C; [R]22 +11.3° (c 0.5, DMSO); IR (KBr) 1735, 1635 cm-1
;
D
HR-FABMS m/ z 621.1583 (M + H)
O5 m/ z 621.1585).
+ (calcd for C25H3179BrFN8-
N-[3-Br om o-4-[[(2,4-d ia m in o-6-p ter id in yl)m eth yl]a m i-
n o]ben zoyl]-(rS,γR)-γ-flu or oglu ta m ic Acid (2viiie). The
procedure described for the preparation of 2ie was used: mp
>270 °C; [R]23
+10.5° (c 0.5, DMSO); IR (KBr) 1637 cm-1
.
D
HPLC tR ) 16.9 min; HR-FABMS m/ z 537.0676 (M + H)+
(calcd for C19H1979BrFN8O5 m/ z 537.0646).
N-[5-[(ter t-Bu toxyca r bon yl)m eth yla m in o]-4-m eth yl-2-
th en oyl]-(rS,γR)-γ-flu or oglu ta m ic Acid r,γ-Diisop r op yl
Ester (6ixe). The procedure described for the preparation of
6iiit was used: colorless oil; [R]23 +19.5° (c 0.5, CHCl3); IR
D
N-[4-[[(2,4-Dia m in o-6-p ter id in yl)m eth yl]a m in o]-3-flu o-
r oben zoyl]-(rS,γR)-γ-flu or oglu ta m ic Acid r,γ-Diisop r o-
p yl Ester (9vie). The procedure described for the preparation
(CHCl3) 1731, 1700, 1654 cm-1; HR-LSIMS m/ z 503.2226 (M
+ H)+ (calcd for C23H36FN2O7S m/ z 503.2225).
N-(4-Meth yl-5-(m eth yla m in o)-2-th en oyl)-(rS,γR)-γ-flu -
or oglu ta m ic Acid r,γ-Diisop r op yl Ester (7ixe). To a
solution of 410 mg (0.90 mmol) of 6ixe in 10 mL of CH2Cl2
was added 10 mL of trifluoroacetic acid. The mixture was
stirred for 1 h at 25 °C and concentrated in vacuo. The residue
was poured into saturated NaHCO3 solution and then ex-
tracted with EtOAc. The organic solution was washed with
saturated NaHCO3 solution and brine and then dried and
concentrated. The residue was chromatographed on silica gel
using 7:50 EtOAc-CHCl3 to afford 590 mg (100%) of 7ixe as a
colorless oil: IR (CHCl3) 1733, 1634 cm-1; HR-LSIMS m/ z
403.1705 (M + H)+ (calcd for C18H28FN2O5S m/ z 403.1702).
N-[5-[[(2,4-Diam in o-6-pter idin yl)m eth yl]m eth ylam in o]-
4-m eth yl-2-th en oyl]-(rS,γR)-γ-flu or oglu ta m ic Acid r,γ-
Diisop r op yl Ester (9ixe). The procedure described for the
preparation of 9ie was used: mp 182-183 °C; IR (KBr) 1735,
1628 cm-1; HR-LSIMS m/ z 577.2356 (M + H)+ (calcd for
C25H34FN8O5S m/ z 577.2355).
of 9ie was done using Proton-Sponge: mp 212-214 °C; [R]22
D
+6.1° (c 0.5, DMSO). HR-FABMS m/ z 561.2376 (M + H)+
(calcd for C25H31F2N8O5 m/ z 561.2385).
N-[4-[[(2,4-Dia m in o-6-p ter id in yl)m eth yl]a m in o]-3-flu o-
r oben zoyl]-(rS,γR)-γ-flu or oglu t a m ic Acid (2vie). The
procedure described for the preparation of 2ie was used: mp
>270 °C; [R]22D +26.9° (c 0.5, DMSO); HPLC tR ) 5.7 min; HR-
LSIMS m/ z 477.1450 (M + H)+ (calcd for C19H19F2N8O5 m/ z
477.1446).
N-(3-F lu or o-4-n it r ob en zoyl)-(rS,γS)-γ-flu or oglu t a m -
ic Acid r,γ-Diisop r op yl Ester (6vit). The procedure de-
scribed for the preparation of 6ie was done using triethylamine
and CH2Cl2: mp 88-90 °C; [R]22 +17.0° (c 0.5, CHCl3); IR
D
(KBr) 1730, 1650, 1543, 1353 cm-1. Anal. (C18H22F2N2O7) C,
H, F, N.
N-(4-Am in o-3-flu or ob en zoyl)-(rS,γS)-γ-flu or oglu t a m -
ic Acid r,γ-Diisop r op yl Ester (7vit). The procedure de-
scribed for the preparation of 7vie was used: mp 101-103
N-[5-[[(2,4-Diam in o-6-pter idin yl)m eth yl]m eth ylam in o]-
4-m eth yl-2-th en oyl]-(rS,γR)-γ-flu or oglu tam ic Acid (2ixe).28
The procedure described for the preparation of 2ie was used:
mp >270 °C; IR (KBr) 1626 cm-1. HPLC tR ) 18.4 min; HR-
LSIMS m/ z 493.1418 (M + H)+ (calcd for C19H22FN8O5S m/ z
493.1417).
°C; [R]22 +20.8° (c 0.5, CHCl3); IR (KBr) 1735, 1636 cm-1
.
D
Anal. (C18H24F2N2O5) C, H, F, N.
N-[4-[[(2,4-Dia m in o-6-p ter id in yl)m eth yl]a m in o]-3-flu o-
r oben zoyl]-(rS,γS)-γ-flu or oglu ta m ic Acid r,γ-Diisop r o-
p yl Ester (9vit). The procedure described for the preparation
of 9ie was done using Proton-Sponge: mp 215-217 °C; [R]22
N-[5-[(ter t-Bu toxyca r bon yl)m eth yla m in o]-4-m eth yl-2-
th en oyl]-(rS,γS)-γ-flu or oglu ta m ic Acid r,γ-Diisop r op yl
Ester (6ixt). The procedure described for the preparation of
D
+12.1° (c 0.5, DMSO); IR (KBr) 1735, 1624 cm-1; HR-FABMS
m/ z 561.2394 (M + H)+ (calcd for C25H31F2N8O5 m/ z 561.2386).
N-[4-[[(2,4-Dia m in o-6-p ter id in yl)m eth yl]a m in o]-3-flu o-
r oben zoyl]-(rS,γS)-γ-flu or oglu ta m ic Acid (2vit). The pro-
cedure described for the preparation of 2ie was used: mp >270
6iiit was used: colorless oil; [R]23 +13.4° (c 0.5, CHCl3); IR
D
(CHCl3) 1732, 1700, 1655 cm-1; HR-LSIMS m/ z 503.2223 (M
+ H)+ (calcd for C23H36FN2O7S m/ z 503.2225).
°C; [R]22 +28.5° (c 0.5, DMSO); IR (KBr) 1618 cm-1. HPLC
N-(4-Meth yl-5-(m eth yla m in o)-2-th en oyl)-(rS,γS)-γ-flu -
or oglu ta m ic Acid r,γ-Diisop r op yl Ester (7ixt). The pro-
cedure described for the preparation of 7ixe was used: color-
less solid; IR (KBr) 1739, 1618 cm-1; HR-LSIMS m/ z 403.1703
(M + H)+ (calcd for C18H28FN2O5S m/ z 403.1702).
N-[5-[[(2,4-Diam in o-6-pter idin yl)m eth yl]m eth ylam in o]-
4-m eth yl-2-th en oyl]-(rS,γS)-γ-flu or oglu ta m ic Acid r,γ-
Diisop r op yl Ester (9ixt). The procedure described for the
preparation of 9ie was used: mp 188-189 °C; IR (KBr) 1737,
1628 cm-1; HR-LSIMS m/ z 577.2356 (M + H)+ (calcd for
C25H34FN8O5S m/ z 577.2355).
D
tR ) 6.0 min; HR-FABMS m/ z 477.1461 (M + H)+ (calcd for
C19H19F2N8O5 m/ z 477.1446).
N-(4-Am in o-3-ch lor oben zoyl)-(rS,γR)-γ-flu or oglu ta m -
ic Acid r,γ-Diisop r op yl Ester (6viie). The procedure
described for the preparation of 6iiit was used: mp 124-125
°C; [R]22 +27.0° (c 1.0, CHCl3); IR (KBr) 1765, 1725, 1629
D
cm-1; HR-FABMS m/ z 403.1421 (M + H)+ (calcd for C18H25
ClFN2O5 m/ z 403.1436).
-
N-[3-Ch lor o-4-[[(2,4-d ia m in o-6-p ter id in yl)m eth yl]a m i-
n o]ben zoyl]-(rS,γR)-γ-flu or oglu ta m ic Acid r,γ-Diisop r o-
p yl Ester (9viie). The procedure described for the prepara-
tion of 9ie was done using potassium iodide: mp 224-226 °C;
N-[5-[[(2,4-Diam in o-6-pter idin yl)m eth yl]m eth ylam in o]-
4-m eth yl-2-th en oyl]-(rS,γS)-γ-flu or oglu tam ic Acid (2ixt).28
The procedure described for the preparation of 2ie was used:
mp >270 °C; IR (KBr) 1637 cm-1. HPLC tR ) 18.8 min; HR-
LSIMS m/ z 493.1413 (M + H)+ (calcd for C19H22FN8O5S m/ z
493.1417).
In Vitr o Mitogen Resp on ses. The effects on mitogen
responses were determined as described in our previous
report.12
[R]22 +7.4° (c 0.5, DMSO); IR (KBr) 1736, 1631 cm-1; HR-
D
FABMS m/ z 577.2108 (M + H)+ (calcd for C25H3135ClFN8O5
m/ z 577.2090).
N-[3-Ch lor o-4-[[(2,4-d ia m in o-6-p ter id in yl)m eth yl]a m i-
n o]ben zoyl]-(rS,γR)-γ-flu or oglu ta m ic Acid (2viie). The
procedure described for the preparation of 2ie was used: mp
>270 °C; IR (KBr) 1630 cm-1; HR-FABMS m/ z 493.1148 (M
+ H)+ (calcd for C19H1935ClFN8O5 m/ z 493.1151); HPLC tR
11.8 min.
)
P r im a r y An tibod y Resp on ses in Mice. A procedure
similar to that reported from these laboratories was used.29

3290 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20
Kokuryo et al.
II collagen-induced arthritis in mice. J . Med. Chem. 1997, 40,
370-376. (f) Piper, J . R.; DeGraw, J . I.; Colwell, W. T.; J ohnson,
C. A.; Smith, R. L.; Waud, W. R.; Sirotnak, F. M. Analogues of
methotrexate in rheumatoid arthritis. II: Effects of 5-deazaami-
nopterin, 5,10-deazaaminopterin, and analogues on type II
collagen-induced arthritis in mice. J . Med. Chem. 1997, 40, 377-
384.
Female BDF1 mice (6 weeks old) were immunized intrave-
nously with 1 × 108 SRBC. A sodium salt of test compound
dissolved in water was administered per os from day 1 to day
3. The mice were sacrificed on day 7 and the blood was
collected. Two-fold dilutions of 0.05 mL of test serum were
prepared in phosphate-buffered saline containing 5 × 10-5
M
(6) (a) McGuire, J . J .; Coward, J . K. Pteroylpolyglutamates: Bio-
synthesis, degradation, and function. In Folates and Pterins;
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York, 1984; Vol. 1, pp 135-190. (b) Chabner, B. A.; Allegra, C.
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(7) Bisset, G. M. F.; Bavetsias, V.; Thornton, T. J .; Pawelczak, K.;
Calvert, A. H.; Hughes, L. R.; J ackman, A. L. The synthesis and
thymidylate synthase inhibitory activity of L-γ-L-linked dipeptide
and L-γ-amide analogues of 2-desamino-2-methyl-N10-propagyl-
5,8-dideazafolic acid (ICI 198583). J . Med. Chem. 1994, 37,
3294-3302.
2-mercaptoethanol in 96-well microtiter plates to which 0.05
mL of 1% suspension of SRBC was added, and then the plates
were incubated at 25 °C for 14 h. The hemagglutination titers
were determined visually.
Secon d a r y An tibod y Resp on ses in Mice. Female BDF1
mice (6 weeks old) were immunized intravenously with 1 ×
105 SRBC on day 0, 7, and 14. The sodium salt of the test
compound dissolved in water was administered per os 5 days
a week from day 0 (total, 15 days). The mice were sacrificed
on day 20 and antibody production was determined as de-
scribed above.
(8) (a) Rosowsky, A.; Bader, H.; Wright, J . E.; Keyomarsi, K.;
Matherly, L. H. Synthesis and biological activity of Nω-hemi-
phthaloyl-R,ω-diaminoalkanoic acid analogues of aminopterin
and 3′,5-dichloroaminopterin. J . Med. Chem. 1994, 37, 2167-
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arthritis: How well do the theories fit the evidence? Clin. Exp.
Immunol. 1993, 92, 1-6. (b) Sewell K. L.; Trentham, D. E.
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Symmons, D. P. M. What is rheumatoid arthritis? Br. Med. Bull.
1995, 51, 243-248.
Ad ju va n t Ar th r itis in Ra ts. A procedure similar to that
described by Winder was used.30 Female Lewis rats (7 weeks
old), weighing 140-160 g, were sensitized by a subcutaneous
injection of 0.05 mL of liquid paraffin containing 0.5 mg of
Mycobacterium butyricum in the left hind paw. A 0.6%
suspension of the test compound in arabic gum was adminis-
tered per os five days a week from day 1 (total, 12 days). Paw
volumes were measured until day 17 with a plethysmometer
and the effect of inhibition of uninjected paw volume versus
the vehicle control was evaluated.
Accu m u la tion in EL4 Cells. EL4 thymoma cell line from
C57BL/6 mice was put in each well of a 96-well microtiter plate
in one 0.1-mL scale containing 4 × 104 cells. The test
compound in 0.1 mL of DMSO was added to each well in such
a manner that its final concentration was in the range of 0-50
µg/mL. After 1 day incubation at 37 °C in a humidified
atmosphere of air containing 5% carbon dioxide, the prolifera-
tion response was determined as described in our previous
report.12
Ack n ow led gm en t. We wish to thank Dr. Y. Iken-
ishi and Mr. S. Yoshimatsu, for the determination of
the MS spectra, and Dr. M. Konishi and Mr. H.
Kamimori, for their valuable suggestions on the HPLC
analysis.
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Novel Fluorinated Methotrexate Derivatives
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 20 3291
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J M970085N