Antirheumatic agents: Novel methotrexate derivatives bearing a benzoxazine or benzothiazine moiety

Article abstract of DOI:10.1021/jm9605288

Novel methotrexate (MTX) derivatives bearing dihydro-2H-1,4- benzothiazine or dihydro-2H-1,4-benzoxazine were synthesized and tested for in vitro antiproliferative activities against human synovial cells (hSC) and human peripheral blood mononuclear cells (hPBMC) obtained from patients with rheumatoid arthritis and healthy volunteers, respectively. In vivo antiarthritic activities of these derivatives were also evaluated in a rat adjuvant arthritis model. N-[[4-[(2,4-Diaminopteridin-6-yl)methyl]-3,4- dihydro-2H-1,4-benzothiazin-7-yl]carbonyl]-L-glutamic acid (3c) exhibited more potent antiproliferative activities in hSC and hPBMC than MTX in vitro. Antiproliferative activities of N-[[4-[(2,4-diaminopteridin-6-yl)methyl]- 3,4-dihydro-2H-1,4-benzoxazin-7-yl]carbonyl]-L-homoglutamic acid (3b) and N- [[4-[(2,4-diaminopteridin-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7- yl]carbonyl]-L-homoglutamic acid (3d) (MX-68) were comparable to that of MTX in these in vitro assays. Compounds 3b,d (MX-68) significantly suppressed progression of the adjuvant arthritis in a dose-dependent manner ranging from 0.5 to 2.5 mg/kg (po). In addition, 3d (MX-68) completely suppressed this progression at the dose of 2.5 mg/kg (po). Importantly, 3d (MX-68) having benzothiazine and homoglutamate, as expected, did not undergo polyglutamation, a process which may be responsible for the associated side effects of MTX. These results suggest that 3d (MX-68) is a potent and safe candidate antirheumatic agent, absent of the side effects of MTX.

Full text of DOI:10.1021/jm9605288

J . Med. Chem. 1997, 40, 105-111
105
An tir h eu m a tic Agen ts: Novel Meth otr exa te Der iva tives Bea r in g a Ben zoxa zin e
or Ben zoth ia zin e Moiety
Hiroharu Matsuoka,* Nobuhiro Ohi, Masahiko Mihara, Hiroshi Suzuki, Katsuhito Miyamoto,
Noriaki Maruyama, Keiichiro Tsuji, Nobuaki Kato, Toshio Akimoto, Yasuhisa Takeda, Keiichi Yano, and
Toshio Kuroki
Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Company, Ltd., 135, 1-Chome Komakado,
Gotemba City, Shizuoka 412, J apan
Received J uly 22, 1996X
Novel methotrexate (MTX) derivatives bearing dihydro-2H-1,4-benzothiazine or dihydro-2H-
1,4-benzoxazine were synthesized and tested for in vitro antiproliferative activities against
human synovial cells (hSC) and human peripheral blood mononuclear cells (hPBMC) obtained
from patients with rheumatoid arthritis and healthy volunteers, respectively. In vivo
antiarthritic activities of these derivatives were also evaluated in a rat adjuvant arthritis model.
N-[[4-[(2,4-Diaminopteridin-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]carbonyl]-L-
glutamic acid (3c) exhibited more potent antiproliferative activities in hSC and hPBMC than
MTX in vitro. Antiproliferative activities of N-[[4-[(2,4-diaminopteridin-6-yl)methyl]-3,4-
dihydro-2H-1,4-benzoxazin-7-yl]carbonyl]-L-homoglutamic acid (3b) and N-[[4-[(2,4-diaminop-
teridin-6-yl)methyl]-3,4-dihydro-2H-1,4-benzothiazin-7-yl]carbonyl]-L-homoglutamic acid (3d )
(MX-68) were comparable to that of MTX in these in vitro assays. Compounds 3b,d (MX-68)
significantly suppressed progression of the adjuvant arthritis in a dose-dependent manner
ranging from 0.5 to 2.5 mg/kg (po). In addition, 3d (MX-68) completely suppressed this
progression at the dose of 2.5 mg/kg (po). Importantly, 3d (MX-68) having benzothiazine and
homoglutamate, as expected, did not undergo polyglutamation, a process which may be
responsible for the associated side effects of MTX. These results suggest that 3d (MX-68) is a
potent and safe candidate antirheumatic agent, absent of the side effects of MTX.
In tr od u ction
Methotrexate (MTX; Figure 1) was synthesized about
50 years ago and is still used as an antileukemic agent
because of its high anti-folate activity. Based on its
biological profile, it is effective for the treatment of
rheumatoid arthritis (RA),1 psoriasis,2 and other au-
toimmune diseases3,4 and has been improved over recent
years.5,6 However, long term MTX therapy is associated
with some serious side effects, such as hepatic dysfunc-
tion7 and lung fibrosis.8
MTX via its metabolites is thought to exert both its
biological action and side effects because it acts as a
substrate for folylpolyglutamate synthetase (FPGS),
which catalyzes the formation of intracellular poly-
glutamates at the γCOOH group extending from
glutamate within MTX.9 This potentially leads to
inclusion bodies that elicit cellular disruption and
subsequent inflammation.10 Thus we initially at-
tempted to suppress this FPGS substrate activity and
enhance dihydrofolate reductase (DHFR) binding by
designing a compound which is not metabolically al-
tered. This was done by synthesizing the MTX deriva-
tive MX-33 (2), which has a homoglutamate and an
indoline ring in place of the glutamate and aminobenzoic
acid, respectively (Figure 1).11 MX-33 did not act as a
substrate for FPGS because polyglutamation could not
occur in the absence of the γCOOH. The use of the
indoline was based on our interpretation of Oefner’s
model which examined binding between MTX and
DHFR.12 According to this model, we hypothesized the
existence of a hydrophobic open space between the
DHFR and the aminobenzoic acid moiety of MTX. We
F igu r e 1.
speculated that filling this space with a fixed hydro-
phobic substituent would cause tighter and energetically
more favorable binding to DHFR and thus enhance the
anti-DHFR activity.
MX-33 potently inhibited proliferation of human
synovial cells (hSC) and human peripheral blood mono-
nuclear cells (hPBMC) obtained from patients with RA
and healthy volunteers, respectively. It also signifi-
cantly suppressed progression of adjuvant arthritis at
a dose of 5.0 mg/kg (po).
To further enhance DHFR binding, considerable at-
tention was paid to the indoline moiety. After consider-
ing both Oefner’s model and our binding studies,13 we
predicted that we could potentiate binding to DHFR
without changing the direction and orientation of the
pteridine ring if we replaced the indoline ring of MX-
33 with the slightly enlarged dihydro-2H-1,4-benzox-
azine or dihydro-2H-1,4-benzothiazine. In our model,
the carbon atom adjacent to the phenyl ring in this
indoline moiety was partially exposed to the solvent
X Abstract published in Advance ACS Abstracts, December 1, 1996.
S0022-2623(96)00528-6 CCC: $14.00 © 1997 American Chemical Society

106 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 1
Matsuoka et al.
As shown in Scheme 2, synthesis of intermediate 17
was achieved using a slight modification of Wolfe’s
method.14 Initially, starting material 10 was converted
into benzothiazol 11 by treatment with sodium thiocy-
anate in the presence of bromine. Alkali hydrolysis of
11 gave thiol 12, which was immediately submitted to
S-alkylation with 1-bromo-2-chloroethane in aqueous
solvent to give thioether 13. Esterification of 13 was
achieved by the treatment of HCl-MeOH to give 14,
which was protected with a 4-toluenesulfonyl group to
give sulfonyl amide 15. Subsequently, 15 was cyclized
by the treatment of NaH to give benzothiazine ester 16.
Compound 16 was then hydrolyzed with 1 N NaOH to
produce the key intermediate 17.
As shown in Scheme 3, the synthesis of the target
compounds 3a -d was achieved using one standard
synthetic method.15 The two key intermediates, 9 and
17, were converted to the corresponding acid chlorides.
Subsequently, their coupling with amino diesters was
performed using the Schotten Baumann’s procedure to
give amides 18a -d . The toluenesulfonyl group of
18a -d was then removed by HBr in acetic acid to give
deprotected amines 19a -d , which were effectively alky-
lated with 6-(bromomethyl)-2,4-diaminopteridine16 (20)
to produce 21a -d . Finally, 21a -d were hydrolyzed
with 1 N NaOH to yield the target compounds 3a -d .
F igu r e 2.
Sch em e 1
Resu lts a n d Discu ssion s
The novel MTX derivatives synthesized were tested
for antiproliferative activity in hSC and hPBMC in vitro.
As shown in Table 1, all tested compounds suppressed
proliferation in hSC with differing potency; glutamate
derivatives 3a ,c were more potent than MTX with IC50
values of 32 and 18 nM, respectively, whereas that of
MTX was 61 nM. Antiproliferative effects of homo-
glutamate derivatives 3b,d (MX-68) were comparable
to that of MTX with IC50 values of 87 and 47 nM and
were 3 and 5 times more potent than that of the
homoglutamate derivative MX-33, respectively. These
results may support our hypothesis that DHFR binding
affinity is enhanced after enlargement and insertion of
the heteroatom within the indoline ring. All tested
compounds potently suppressed proliferation of hPBMC;
compounds more potent than MTX were 3c and MX-33
with IC50 values of 12 and 20 nM, respectively. The
calculated relative value in hSC for 3d (MX-68) was
considerably less than that for MX-33, whereas it was
1.5 times greater than that for MX-33 in hPBMC. Thus
3d (MX-68) is more selective for hSC than for hPBMC.
In light of the role played by SC during synovial
inflammation, MX-68’s potent action toward these cells
supports its use as an antirheumatic agent.
In both hSC and hPBMC assays, the inhibitory effects
of the glutamate derivatives 3a ,c were 2-3 times
enhanced over their corresponding homoglutamate coun-
terparts 3b,d (MX-68). These results indicate that this
enhanced action occurs via polyglutamation. We cor-
roborated this concept by using Moran’s method17 to
show the presence and absence of substrate activity for
FPGS by MTX and 3d (MX-68), respectively (Table 2).
Antirheumatic activities of our novel MTX derivatives
were assessed in vivo by their effects on rat adjuvant
arthritis.18 Table 3 compares the effects of these
derivatives to MTX; the suppressive effect of 3a was
comparable at the dose of 0.25 mg/kg (po), whereas the
portion; therefore, insertion of either an oxygen or sulfur
atom between the methylene group and benzene ring
was expected to energetically favor its location toward
the solvent portion and gain a more stable interaction
with solvent water molecules rather than the carbon
atom. Insertion of a sulfur atom was expected to
enhance DHFR binding by van der Waals interactions.
In this paper, we report the design, synthesis, and
biological properties of novel MTX derivatives bearing
a dihydro-2H-1,4-benzoxazine or dihydro-2H-1,4-ben-
zothiazine moiety (Figure 2).
Ch em istr y
Preparations of the key intermediates of N-(4′-tolyl-
sulfonyl)-3,4-dihydro-2H-1,4-benzoxazine-7-carboxylic acid
(9) and N-(4′-tolylsulfonyl)-3,4-dihydro-2H-1,4-benzothi-
azine-7-carboxylic acid (17) are presented in Schemes
1 and 2, respectively. Synthesis of the target com-
pounds 3a -d is presented in Scheme 3.
As shown in Scheme 1, synthesis of intermediate 9
started with O-alkylation of the phenol 4 with 1-bromo-
2-chloroethane to give ether 5, and its following reduc-
tion with zinc powder in acetic acid yielded the corre-
sponding amine 6. The amino group of 6 was then
protected with a 4-toluenesulfonyl group to give sulfonyl
amide 7. Subsequent cyclization of 7 proceeded ef-
fectively by the treatment of NaH to yield benzoxazine
ester 8, and hydrolysis of 8 with 1 N NaOH produced
the key intermediate 9.

MTX Derivatives with a Benzoxazine/ Benzothiazine
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 1 107
Sch em e 2
Sch em e 3
Ta ble 1. Antiproliferative Activities of Novel MTX Derivatives
Ta ble 3. Effects of Novel MTX Derivatives on the
Development of Adjuvant Arthritis in Rats
IC50 (nM)a,b
compd
dose (mg/kg)
% suppression of arthritis
compd
hSC
hPBMC
3a
0.05
0.25
0.25
0.5
1.0
2.5
0.05
0.1
0.25
0.5
1.0
2.5
48
94
18
32
53
84
52
97
20
58
74
94
26
38
75
92
(p < 0.02)
(p < 0.02)
(p < 0.05)
(p < 0.05)
(p < 0.02)
(p < 0.02)
(NS)
(p < 0.02)
(NS)
(p < 0.02)
(p < 0.02)
(p < 0.02)
(NS)
3a
3b
3c
32 (0.52)
87 (1.4)
18 (0.30)
47 (0.77)
230 (3.8)
61 (1.0)
24 (1.0)
45 (1.9)
12 (0.50)
32 (1.3)
20 (0.83)
24 (1.0)
3b
3d (MX-68)
MX-33
MTX
3c
a
b
The results are the mean of triplicate assays. Numbers in
parentheses are normalized relative to the IC50 for each compound
against the IC50 of MTX.
3d (MX-68)
Ta ble 2. Activities of MTX Derivatives as Substrates of FPGSa
compd drug concentration (µM) FPGS activities (nmol/mg/h)
MX-33
MTX
2.5
5.0
0.1
0.25
(NS)
(p < 0.02)
(p < 0.02)
MTX
MX-68
MX-68
100
100
500
2.64
<0.01
0.07
a
FPGS activity was determined as described in ref 17, with
partially purified enzyme from rat liver. The results are the mean
of triplicate assays.
spect, the homoglutamate derivative 3d (MX-68), found
to be dose-dependent and orally active in the rat
adjuvant arthritis and not to undergo polyglutamation,
is a strong candidate antirheumatic agent. MX-68 is
currently under preclinical investigation with the aim
of clinical study in the near future.
effect of 3c was obviously more potent. Furthermore,
homoglutamate derivatives 3b,d (MX-68) suppressed
progression of the arthritis in a dose-dependent manner
from 0.5 to 2.5 mg/kg (po). More importantly, 3d (MX-
68) completely suppressed the arthritis at the dose of
2.5 mg/kg. It was more effective than MX-33 at the
same dose and comparable to MTX at 0.25 mg/kg.
Furthermore, greater in vitro potency of 3d (MX-68)
over MX-33 in hSC is consistent with the effects
observed in vivo.
As polyglutamation causes side effects but is promi-
nently implicated for the efficacy of MTX, derivatives
which do not have a capability to undergo this process
must compensate by some other means to achieve
sufficient in vivo pharmacological effects. In this re-
Exp er im en ta l Section
1H-NMR spectra were recorded on a J EOL Model J MN-
FX200 NMR spectrometer with Me4Si as the reference,
infrared spectra were run on a Hitachi Model 270-3 infrared
spectrometer, and EI mass spectra were recorded on a Shi-
madzu GCMS-QP1000 instrument. FAB and HR-FAB mass
spectra were recorded on a VG Analytical VG11-250 instru-
ment. TLC was routinely performed on Merck Kieselgel 60
F254. HPLC analysis were performed on a Hitachi L-3000
(detector) with Hitachi L-6200 (pump); the column was YMC-
Pack A-312 S-5 120A ODS. Melting points were taken on a
Yanaco Model MP apparatus.

108 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 1
Matsuoka et al.
Meth yl 3-(2′-Br om oeth oxy)-4-n itr oben zoa te (5). To a
solution of methyl 3-hydroxy-4-nitrobenzoate (4) (1.0 g, 5.07
mmol) in dimethylformamide (DMF; 10 mL) were added 1,2-
dibromoethane (0.5 mL, 5.80 mmol) and potassium carbonate
(700 mg, 5.07 mmol) at room temperature, and the mixture
was stirred for 2 h at 100 °C. The mixture was then poured
into water and extracted with toluene. The organic layer was
dried over Na2SO4, filtered, and concentrated under reduced
pressure. The obtained residue was chromatographed on silica
gel with n-Hex-AcOEt (3:1) to give 5 (860 mg, 56%) as a
colorless oil. 1H-NMR (CDCl3): δ 3.69 (t, 2H, J ) 6 Hz, -OCH2-
), 3.95 (s, 3H, -OCH3), 4.49 (t, 2H, J ) 6 Hz, -CH2Br), 7.75 (m,
3H, Ar). IR (KBr): cm-1 1720, 1610, 1590. MS: m/ z 306 (M+
+ 2), 304 (M+), 106.
Meth yl 4-Am in o-3-(2′-br om oeth oxy)ben zoa te (6). To a
solution of 5 (100 mg, 0.33 mmol) in acetic acid (2.0 mL) was
added zinc powder (213 mg, 3.26 mmol) at 0 °C, and the
reaction mixture was stirred for 2 h at room temperature and
filtered. The filtrate was concentrated under reduced pressure
to afford a brown crude oil, which was dissolved with CHCl3.
The organic solution was washed with 5% NaHCO3, dried over
Na2SO4, filtered, and concentrated to give pure 6 (75 mg, 83%)
as a colorless oil. 1H-NMR (CDCl3): δ 3.68 (t, 2H, J ) 5.7 Hz,
-OCH2-), 3.86 (s, 3H, OCH3), 4.37 (t, 2H, J ) 5.7 Hz, -CH2Br),
6.68 (d, 1H, J ) 8.3 Hz, Ar), 7.44 (m, 1H, Ar), 7.56 (d, 1H, J
) 8.3 Hz, Ar). IR (KBr): cm-1 3380, 1710, 1620. MS: m/ z
276 (M+ + 2), 274 (M+), 166.
concentrated. The obtained residue was dissolved with CH2-
Cl2 (40 mL). To this solution were added a solution of dimethyl
L-homoglutamate hydrochloride (2.8 g, 12.4 mmol) in water
(40 mL) and potassium carbonate (5.0 g, 36.2 mmol). The
whole mixture was vigorously stirred overnight, poured into
water, and extracted with CHCl3. The organic layer was dried
over Na2SO4 and concentrated under reduced pressure. The
obtained residue was chromatographed on silica gel with
CHCl3-MeOH (100:1) to give 18b (4.3 g, 98%) as a colorless
oil. 1H-NMR (CDCl3): δ 1.6-2.1 (m, 4H, hGlu-â,γ), 2.35 (m,
2H, hGlu-δ), 2.39 (s, 3H, toluene-CH3), 3.66 (s, 3H, hGluR-
OCH3), 3.77 (s, 3H, hGluâ-OCH3), 3.72 (m, 2H, -NCH2-), 3.87
(m, 2H, -CH2O-), 4.74 (m, 1H, hGlu-R), 6.73 (d, 1H, J ) 7.3
Hz, NH), 7.2-7.4 (m, 4H, toluene-Ar2H + benzoxazine-Ar2H),
7.51 (d, 2H, J ) 8.3 Hz, toluene-Ar), 7.93 (d, 1H, J ) 8.8 Hz,
benzoxazine-Ar). IR (neat): cm-1 3400, 2950, 1740, 1650.
MS: m/ z 504 (M+), 91.
Dim eth yl N-[(3,4-Dih yd r o-2H-1,4-ben zoxa zin -7-yl)ca r -
bon yl]-L-h om oglu ta m a te (19b). A mixture of 18b (4.3 g,
8.53 mmol) and anisole (4.3 g, 39.8 mmol) in 30% HBr in acetic
acid (50 mL) was stirred for 4 h at room temperature and
poured into ether (300 mL). Precipitates were collected and
washed by decantation with ether. The obtained residue was
dissolved with CHCl3, and this solution was washed with 5%
NaHCO3, dried over Na2SO4, filtered, and concentrated under
reduced pressure to give 19b (1.45 g, 49%) as a colorless oil.
1H-NMR (CDCl3): δ 1.6-2.1 (m, 4H, hGlu-â,γ), 2.38 (t, 2H, J
) 6.8 Hz, hGlu-δ), 3.46 (m, 2H, -NCH2-), 3.68 (s, 3H, hGluR-
OCH3), 3.77 (s, 3H, hGluδ-OCH3), 4.24 (m, 2H, -CH2O-), 4.72
(m, 1H, hGlu-R), 6.59 (d, 1H, J ) 8.3 Hz, Ar), 7.33 (m, 3H,
NH + Ar2H). IR (neat): cm-1 3600-3400, 2950, 1730, 1640,
1610. MS: m/ z 350 (M+), 162. HR-MS calcd for C17H22N2O6:
M, 350.1478. Found: 350.1488 (M+).
Dim eth yl N-[[4-[(2,4-Dia m in op ter id in -6-yl)m eth yl]-3,4-
d ih y d r o -2H -1,4-b e n zo x a zin -7-y l]c a r b o n y l]-L -h o m o -
glu ta m a te (21b). A mixture of 19b (1.45 g, 4.14 mmol) and
6-(bromomethyl)-2,4-diaminopteridine‚HBr‚i-PrOH14 (20) (1.47
g, 3.71 mmol) in dimethylacetamide (DMA; 23 mL) was heated
at 60 °C for 4 h with stirring. The mixture was poured into
5% NaHCO3 and extracted with CHCl3. The organic layer was
dried over Na2SO4, filtered, and concentrated under reduced
pressure. The obtained residual oil was chromatographed on
silica gel with CHCl3-MeOH (10:1) to give 21b (1.48 g, 68%)
as a yellow powder. 1H-NMR (CDCl3-CD3OD): δ 1.6-2.1 (m,
4H, hGlu-â,γ), 2.36 (t, 2H, J ) 6.8 Hz, hGlu-R), 3.58 (m, 2H,
-NCH2-), 3.66 (s, 3H, hGluR-OCH3), 3.76 (s, 3H, hGluδ-OCH3),
4.39 (m, 2H, -CH2O-), 4.67 (s, 2H, pteridine-CH2), 4.73 (m, 1H,
hGlu-R), 6.66 (d, 1H, J ) 8.3 Hz, benzoxazine-Ar), 6.99 (d, 1H,
J ) 7.3 Hz, NH), 7.29 (m, 2H, benzoxazine-Ar), 8.70 (s, 1H,
pteridine-Ar). IR (KBr): cm-1 3500-3300, 3200, 2950, 1750,
1630, 1580. FAB-MS: m/ z 525 (MH+). HR-FAB-MS calcd
for C24H28N8O6: MH+, 525.2210. Found: 525.2200 (MH+).
N-[[4-[(2,4-Dia m in op ter id in -6-yl)m eth yl]-3,4-d ih yd r o-
2H-1,4-ben zoxa zin -7-yl]ca r bon yl]-L-h om oglu ta m ic Acid
(3b). A mixture of 21b (1.48 g, 2.82 mmol) and 1 N NaOH
(8.5 mL) in ethanol (80 mL) was stirred overnight at room
temperature and concentrated to 5 mL under reduced pres-
sure. The residue was diluted to 50 mL with water, and this
aqueous solution was adjusted to pH 4 by 1 N HCl and 3%
(NH4)2CO3. Precipitates were collected by filtration, and
obtained solids were dried in vacuo to give 3b (1.26 g, 90%) as
a yellow powder. 1H-NMR (DMSO-d6): δ 1.5-2.0 (m, 4H,
hGlu-â,γ), 2.14 (t, 2H, J ) 6.8 Hz, hGlu-δ), 3.68 (m, 2H, -NCH2-
), 4.28 (m, 3H, -CH2O- + hGlu-R), 4.71 (s, 2H, pteridine-CH2),
6.80 (d, 1H, J ) 8.3 Hz, benzoxazine-Ar), 7.31 (m, 2H,
benzoxazine-Ar), 8.13 (d, 1H, J ) 7.3 Hz, NH), 8.71 (s, 1H,
pteridine-Ar). IR (KBr): cm-1 3500-3300, 1640, 1620.
FAB-MS: m/ z 497 (MH+). HR-FAB-MS calcd for
C22H25N8O6: MH+, 497.1897. Found: 497.1980 (MH+). Mp:
200-202 °C dec. Anal. (C22H24N8O6‚2H2O) C, H, N. Analysis
of HPLC (solvent, CH3CO2H/CH3CO2Na (pH 5.4):MeCN ) 9:1;
flow rate, 1.0 cm3/min; detection, 254 nm) showed the purity
to be at least 98% (retention time, 18 min).
Meth yl N-(4′-Tolylsu lfon yl)-4-am in o-3-(2′-br om oeth oxy)-
ben zoa te (7). A mixture of 6 (1.2 g, 4.38 mmol) and
4-toluenesulfonyl chloride (1.7 g, 8.90 mmol) in pyridine (10
mL) was stirred at room temperature overnight, and the
mixture was poured into aqueous NH4Cl solution. Precipitated
white solids were collected by filtration, and the obtained solids
were extracted with CHCl3. The CHCl3 layer was dried over
Na2SO4, filtered, and concentrated under reduced pressure.
The residual oil was recrystallized from n-Hex-AcOEt to give
7 (750 mg, 40%) as a white solid. 1H-NMR (CDCl3): δ 2.36 (s,
3H, toluene-CH3), 3.5-3.9 (m, 2H, -OCH2-), 3.87 (s, 3H,
-OCH3), 4.24 (m, 2H, -CH2Br), 7.22 (d, 2H, J ) 8.3 Hz, toluene-
Ar), 7.33 (m, 2H, benzoate-Ar + NH), 7.42 (s, 1H, benzoate-
Ar), 7.62 (s, 1H, benzoate-Ar), 7.72 (d, 2H, J ) 8.3 Hz, toluene-
Ar). IR (KBr): cm-1 3240, 1730, 1610. MS: m/ z 429 (M+
+
2), 427 (M+), 91.
Meth yl N-(4′-Tolylsu lfon yl)-3,4-d ih yd r o-2H-1,4-ben zox-
a zin e-7-ca r boxyla te (8). To a solution of 7 (750 mg, 1.75
mmol) in DMF (23 mL) was added NaH (335 mg, 8.38 mmol,
60% in mineral oil) at 0 °C under nitrogen atmosphere, and
the mixture was stirred for 1 h at the same temperature. The
mixture was poured into aqueous NH4Cl solution and ex-
tracted with toluene. The organic layer was dried over Na2-
SO4 and concentrated under reduced pressure. The obtained
residue was chromatographed on silica gel with n-Hex-AcOEt
(2:1) to give 8 (430 mg, 71%) as a white powder. 1H-NMR
(CDCl3): δ 2.38 (s, 3H, toluene-CH3), 3.80 (m, 2H, -NCH2-),
3.88 (s, 3H, -OCH3), 3.89 (m, 2H, -CH2O-), 7.24 (d, 2H, J ) 8.3
Hz, toluene-Ar), 7.4-7.6 (m, 4H, toluene-Ar2H + benzoxazine-
Ar2H), 7.93 (d, 1H, J ) 8.8 Hz, benzoxazine-Ar). IR (KBr):
cm-1 3600, 2950, 1720, 1620, 1600. MS: m/ z 347 (M+), 192.
N-(4′-Tolylsu lfon yl)-3,4-d ih yd r o-2H-1,4-ben zoxa zin e-7-
ca r boxylic Acid (9). A mixture of 8 (350 mg, 1.01 mmol)
and 1 N NaOH (2.9 mL) in ethanol (15 mL) was stirred for 5
h at room temperature, and the mixture was concentrated
under reduced pressure. The obtained residue was diluted to
5 mL with water, and this solution was acidified to pH 2.0
with 1 N HCl. The precipitated solids were collected by
filtration and dried in vacuo to give 9 (298 mg, 89%) as a white
powder. 1H-NMR (CDCl3): δ 2.38 (s, 3H, toluene-CH3), 3.83
(m, 2H, -NCH2-), 3.92 (m, 2H, -CH2O-), 7.24 (d, 2H, J ) 8.3
Hz, toluene-Ar), 7.5-7.7 (m, 4H, toluene-Ar2H + benzoxazine-
Ar2H), 7.93 (d, 1H, J ) 8.8 Hz, benzoxazine-Ar). IR (KBr):
cm-1 3500-3400, 3100-2900, 1680. MS: m/ z 333 (M+), 178.
Anal. (C16H15NO5S) C, H, N, S.
Dim eth yl N-[[4-(4′-Tolylsu lfon yl)-3,4-d ih yd r o-2H-1,4-
ben zoxazin -7-yl]car bon yl]-L-h om oglu tam ate (18b). A mix-
ture of 9 (2.9 g, 8.71 mmol) and DMF (0.1 mL) in thionyl
chloride (10 mL) was stirred for 2 h at room temperature and
Dieth yl N-[[4-(4′-Tolylsu lfon yl)-3,4-dih ydr o-2H-1,4-ben -
zoxa zin -7-yl]ca r bon yl]-L-glu ta m a te (18a ). Using the same
procedure as described for the preparation of 18b, compound

MTX Derivatives with a Benzoxazine/ Benzothiazine
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 1 109
18a was prepared from compound 9 and diethyl L-glutamate
hydrochloride. The yield of 18a was 95% (colorless oil). 1H-
NMR (CDCl3): δ 1.24 (t, 3H, J ) 6.8 Hz, GluR-OCH2CH3), 1.30
(t, 3H, J ) 6.8 Hz, Gluγ-OCH2CH3), 2.40 (s, 3H, toluene-CH3),
2.0-2.6 (m, 4H, Glu-â,γ), 3.75 (m, 2H, -NCH2-), 3.90 (m, 2H,
-CH2O-), 4.13 (q, 2H, J ) 6.8 Hz, GluR-OCH2CH3), 4.24 (q,
2H, J ) 6.8 Hz, Gluγ-OCH2CH3), 4.74 (m, 1H, Glu-R), 7.00 (d,
1H, J ) 7.3 Hz, NH), 7.2-7.4 (m, 4H, toluene-Ar2H +
benzoxazine-Ar2H), 7.52 (d, 2H, J ) 8.3 Hz, toluene-Ar), 7.95
(d, 1H, J ) 8.8 Hz, benzoxazine-Ar). IR (neat): cm-1 2980,
1740, 1660, 1572. MS: m/ z 518(M+), 133. HR-MS m/ z calcd
for C25H30N2O8S: M, 518.1723. Found: 518.1722 (M+).
Dieth yl N-[(3,4-Dih yd r o-2H-1,4-ben zoxa zin -7-yl)ca r bo-
n yl]-L-glu ta m a te (19a ). Using the same procedure as de-
scribed for the preparation of 19b, compound 19a was pre-
pared from compound 18a . The yield of 19a was 56% (white
dropwise. The precipitated solids were collected by filtration.
The obtained solids were washed with water and suspended
in water (6.0 L). To this suspension was added SnCl2 (20 g,
0.11 mol), and the mixture was heated at 70 °C with stirring.
To the refluxing mixture was added concentrated HCl, until
insoluble solids were dissolved. The mixture was then filtered
with Celite, and to the filtrate was added 1 N HCl (4.0 L)
again. The resulting aqueous solution was stored in a cold
place for 1 day. Precipitated white solids were collected by
filtration and dried in vacuo to give 12 (246 g, 69%) as a white
powder. 1H-NMR (DMSO-d6): δ 6.59 (d, 1H, J ) 8 Hz, H5),
7.3-7.8 (m, 2H, H2 + H6). IR (KBr): cm-1 3360, 1710, 1590.
MS: m/ z 169 (M+), 168. HR-MS calcd for C7H7NO2S: M,
169.0198. Found: 169.0212 (M+).
4-Am in o-3-[(ch lor oeth yl)th io]ben zoic Acid (13). To a
mixture of 12 (334 g, 1.63 mol) and NaOH (195 g, 4.88 mol) in
EtOH (2.4 L) and water (500 mL) was added 1-bromo-2-
chloroethane (542 mL, 6.5 mol) at room temperature, and the
mixture was stirred vigorously for 3.5 h. To the reaction
mixture were added AcOEt (3.0 L) and water (2.0 L), and the
resulting mixture was adjusted to pH 5.0 with 3 N HCl at 0
°C. The organic layer was separated, dried over Na2SO4,
filtered, and concentrated to give pure 13 (270 g, 72%) as a
colorless syrup. 1H-NMR (CDCl3): δ 3.03 (t, 2H, J ) 7.6 Hz,
-SCH2-), 3.58 (t, 2H, J ) 7.6 Hz, -CH2Cl), 6.78 (d, 1H, J ) 8.6
Hz, H5), 7.76 (m, 1H, H6), 8.03 (d, 1H, J ) 2.0 Hz, H2). IR
powder). 1H-NMR (CDCl3):
γ-OCH2CH3), 2.0-2.4 (m, 2H, Glu-â), 2.4-2.6 (m, 2H, Glu-γ),
3.41 (m, 2H, -NCH2-), 4.0-4.3 (m, 6H, GluR, γ-OCH2CH3
δ 1.2-1.4 (m, 6H, GluR,
+
-CH2O-), 4.74 (m, 1H, Glu-R), 6.53 (d, 1H, J ) 8.8 Hz, Ar),
6.94 (d, 1H, J ) 7.8 Hz, NH), 7.22 (m, 2H, Ar). IR (neat): cm-1
3500-3300, 1720, 1640, 1580. MS: m/ z 364 (M+), 162. HR-
MS calcd for C18H24N2O6: M, 364.1634. Found: 364.1634
(M+).
Dieth yl N-[[4-[(2,4-Dia m in op ter id in -6-yl)m eth yl]-3,4-
d ih yd r o-2H -1,4-b en zoxa zin -7-yl]ca r b on yl]-L-glu t a m a t e
(21a ). Using the same procedure as described for the prepa-
ration of 21b, compound 21a was prepared from compound
19a . The yield of 21a was 54% (orange powder). 1H-NMR
(CDCl3-CD3OD): δ 1.23 (t, 3H, J ) 6.8 Hz, GluR-OCH2CH3),
1.29 (t, 3H, J ) 6.8 Hz, Gluγ-OCH2CH3), 2.0-2.3 (m, 2H, Glu-
â), 2.3-2.5 (m, 2H, Glu-γ), 3.60 (m, 2H, -NCH2-), 4.11 (m, 2H,
GluR-OCH2CH3), 4.22 (m, 2H, Gluγ-OCH2CH3), 4.32 (m, 2H,
-CH2O-), 4.6-4.8 (m, 3H, pteridine-CH2 + Glu-R), 6.69 (d, 1H,
J ) 9.0 Hz, NH), 7.29 (d, 1H, J ) 9.0 Hz, benzoxazine-Ar),
7.36 (m, 2H, benzoxazine-Ar), 8.70 (s, 1H, pteridine-Ar). IR
(KBr): cm-1 3330, 3190, 1730, 1630, 1560. FAB-MS: m/ z 539
(MH+). HR-FAB-MS calcd for C25H31N8O6: MH+, 539.2366.
Found: 539.2379 (MH+).
N-[[4-[(2,4-Dia m in op ter id in -6-yl)m eth yl]-3,4-d ih yd r o-
2H-1,4-ben zoxa zin -7-yl]ca r bon yl]-L-glu ta m ic Acid (3a ).
Using the same procedure as described for the preparation of
3b, compound 3a was prepared from compound 21a . The yield
of 3a was 78% (orange powder). 1H-NMR (DMSO-d6): δ 1.9-
2.0 (m, 2H, Glu-â), 2.2-2.4 (m, 2H, Glu-γ), 3.67 (t, 2H, J )
3.8 Hz, -NCH2-), 4.25 (t, 2H, J ) 3.8 Hz, -CH2O-), 4.30 (m,
1H, Glu-R), 4.70 (s, 2H, pteridine-CH2), 6.83 (d, 1H, J ) 8.6
Hz, NH), 7.26 (s, 1H, benzoxazine-Ar), 7.30 (d, 1H, J ) 8.6
Hz, benzoxazine-Ar), 8.09 (m, 1H, benzoxazine-Ar), 8.70 (s, 1H,
pteridine-Ar). IR (KBr): cm-1 3460, 1640, 1510. FAB-MS:
m/ z 483 (MH+). HR-FAB-MS calcd for C21H23N8O6: MH+,
483.1740. Found: 483.1737 (MH+). Mp: 203-205 °C dec.
Anal. (C21H22N8O6) C, H, N. Analysis of HPLC (solvent, CH3-
CO2H/CH3CO2Na (pH 5.4):MeOH ) 82:18; flow rate, 1.0 cm3/
min; detection, 254 nm) showed the purity to be at least 98%
(retention time, 15 min).
2-Am in oben zoth ia zole-6-ca r boxylic Acid Hyd r och lo-
r id e (11). To a solution of sodium thiocyanate (880 g, 10.9
mol) and ethyl 4-aminobenzoate (10) (1650 g, 10 mol) in
methanol (5.1 L) was added bromine (823 g, 5.0 mol) with
stirring below -5 °C during the addition, and the mixture was
stirred for 2 h at the same temperature. Precipitated white
solids were collected by filtration and washed with water. The
obtained solids were suspended in 1 N HCl (4.0 L), refluxed,
and filtered with heating. To the filtrate was added concen-
trated HCl (2.0 L), and this was stored in a cold place to afford
white solids, which were collected by filtration and dried in
vacuo to give 11 (807 g, 32%) as a white powder. 1H-NMR
(CDCl3-CD3OD): δ 6.86 (d, 1H, J ) 9 Hz, H4), 7.7-8.1 (m,
2H, H5 + H7). IR (KBr): cm-1 3300-3000, 1700, 1630, 1600.
MS: m/ z 194 (M+), 169. HR-MS calcd for C8H6N2O2S: M,
194.0150. Found: 194.0131 (M+).
(neat): cm-1 3470, 1670, 1610. HR-MS calcd for C9H10
ClNO2S: M, 231.0121. Found: 231.0155 (M+).
-
Meth yl 4-Am in o-3-[(ch lor oeth yl)th io]ben zoa te Hyd r o-
ch lor id e (14). To a solution of 13 (270 g, 1.17 mol) in MeOH
(5.0 L) was bubbled gaseous HCl until saturation, and the
mixture was stirred overnight at room temperature. To the
reaction mixture were added AcOEt (100 mL) and a large
amount of n-Hex. Precipitated solids were collected by filtra-
tion and dried in vacuo to give 14 (256 g, 78%) as a white
powder. 1H-NMR (CDCl3): δ 3.01 (t, 2H, J ) 7.2 Hz, -SCH2-),
3.55 (t, 2H, J ) 7.2 Hz, -CH2Cl), 3.83 (s, 3H, -OCH3), 6.65 (d,
1H, J ) 9.0 Hz, H5), 7.77 (m, 1H, H6), 8.05 (d, 1H, J ) 2.4 Hz,
H2). IR (KBr): cm-1 3460, 3350, 1690, 1620. MS: m/ z 247
(M+ + 2), 245 (M+), 194, 182. HR-MS calcd for C10H12
-
ClNO2S: M, 245.0277. Found: 245.0284 (M+).
Meth yl 4-(4′-Tolylsu lfon yl)-4-a m in o-3-[(ch lor oeth yl)-
th io]ben zoa te (15). A mixture of 14 (125 g, 0.51 mol) and
4-toluenesulfonyl chloride (108 g, 0.57 mol) in pyridine (500
mL) was stirred at 60 °C for 5 h. The mixture was poured
into aqueous NH4Cl solution and extracted with AcOEt. The
organic layer was dried over Na2SO4, filtered, and concen-
trated. The residue was triturated with n-Hex, and precipi-
tated solids were collected by filtration. The obtained solids
were dried in vacuo to give 15 (189 g, 87%) as a white powder.
1H-NMR (CDCl3): δ 2.33 (s, 3H, toluene-CH3), 2.32 (m, 2H,
-SCH2-), 2.82 (m, 2H, -CH2Cl), 3.81 (s, 3H, -OCH3), 7.0-7.4
(m, 3H, toluene-Ar2H + H5), 7.5-7.9 (m, 4H, toluene-Ar2H +
H2, H6), 7.9-8.1 (m, 1H, NH). IR (KBr): cm-1 3240, 1720,
1600, 1490. MS: m/ z 401 (M+ + 2), 399 (M+), 363, 208, 91.
HR-MS calcd for C17H18ClNO4S2: M, 399.0366. Found:
399.0357 (M+).
Meth yl 4-(4′-Tolylsu lfon yl)-3,4-dih ydr o-2H-1,4-ben zoth i-
a zin e-7-ca r boxyla te (16). To a solution of 15 (188 g, 0.47
mol) in toluene (4.5 L) was added NaH (25 g, 0.63 mol, 60% in
mineral oil). The mixture was stirred at 100 °C for 2 h and
cooled to room temperature. The cooled mixture was neutral-
ized by concentrated HCl and concentrated under reduced
pressure. The obtained residue was poured into aqueous NH4-
Cl solution and extracted with AcOEt. The organic layer was
dried over Na2SO4, filtered, and concentrated to give a brown
crude oil. This oil was recrystallized from n-Hex-AcOEt to
give 16 (116 g, 68%) as a white powder. 1H-NMR (CDCl3): δ
2.40 (s, 3H, toluene-CH3), 2.89 (t, 2H, J ) 5.6 Hz, -CHS2-),
3.89 (s, 3H, -OCH3), 4.03 (t, 2H, J ) 5.6 Hz, -NCH2-), 7.22 (d,
2H, J ) 8.8 Hz, toluene-Ar), 7.49 (d, 2H, J ) 8.8 Hz, toluene-
Ar), 7.75 (m, 3H, benzothiazine-Ar). IR (KBr): cm-1 1720,
1600, 1430. MS: m/ z 363 (M+), 208, 149, 91. HR-MS calcd
for C17H17NO4S2: M, 363.0599. Found: 363.0588 (M+).
4-(4′-Tolylsu lfon yl)-3,4-d ih yd r o-2H-1,4-ben zoth ia zin e-
7-ca r boxylic Acid (17). A mixture of 16 (114 g, 0.31 mol)
4-Am in o-3-m er ca p toben zoic Acid Hyd r och lor id e (12).
A solution of 11 (400 g, 1.74 mol) and KOH (1.74 kg, 31.1 mol)
was refluxed for 4 h and then cooled to room temperature and
filtered. To the filtrate was added concentrated HCl (2.3 L)

110 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 1
Matsuoka et al.
and 1 N NaOH (630 mL) in MeOH (3.4 L) was refluxed for 3
h. The mixture was then cooled to room temperature and
concentrated to 100 mL under reduced pressure. The residue
was diluted with water (2.0 L). This solution was adjusted to
pH 2.0 with 3 N HCl. Precipitates were collected by filtration
and dried in vacuo to give 17 (107 g, 98%) as a white powder.
1H-NMR (CDCl3): δ 2.41 (s, 3H, toluene-CH3), 2.90 (t, 2H, J
) 6.4 Hz, -CH2S-), 4.02 (t, 2H, J ) 6.4 Hz, -NCH2-), 7.25 (d,
2H, J ) 8.7 Hz, toluene-Ar), 7.52 (d, 2H, J ) 8.7 Hz, toluene-
Ar), 7.79 (m, 3H, benzothiazine-Ar). IR (KBr): cm-1 3100-
2900, 1690, 1600. MS: m/ z 349 (M+), 194, 149, 107, 91. Anal.
(C16H15NO4S2) C, H, N, S.
76%) as a yellowish orange powder. 1H-NMR (DMSO-d6): δ
1.7-2.2 (m, 4H, hGlu-â,γ), 2.50 (t, 2H, J ) 7.2 Hz, hGlu-δ),
3.46 (m, 2H, -CH2S-), 4.23 (m, 2H, -NCH2-), 4.58 (m, 1H, hGlu-
R), 5.05 (s, 2H, pteridine-CH2), 7.09 (d, 1H, J ) 8.9 Hz,
benzothiazine-Ar), 7.73 (m, 2H, benzothiazine-Ar), 8.49 (d, 1H,
J ) 7.6 Hz, NH), 8.95 (s, 1H, pteridine-CH2). IR (KBr): cm-1
3500-3300, 1640, 1590, 1500. FAB-MS: m/ z 513 (MH+). HR-
FAB-MS calcd for C22H25N8O5S: MH+, 513.1669. Found:
513.1675 (MH+). Mp: 200-203 °C dec. Anal. (C22H24N8O5S‚1/
2H2O) C, H, N, S. Analysis of HPLC (solvent, CH3CO2H/CH3-
CO2Na (pH 5.4):MeOH ) 3:1; flow rate, 1.0 cm3/min; detection,
254 nm) showed the purity to be at least 99% (retention time,
22 min).
Dim eth yl N-[[4-(4′-Tolylsu lfon yl)-3,4-d ih yd r o-2H-1,4-
ben zoth ia zin -7-yl]ca r bon yl]-L-h om oglu ta m a te (18d ). A
solution of 17 (1.47 g, 4.21 mmol) in thionyl chloride (10 mL)
was stirred for 2 h at room temperature and concentrated
under reduced pressure. The residue was dissolved with CH2-
Cl2 (15 mL). To this solution was added a solution of dimethyl
homoglutamate hydrochloride (1.4 g, 6.22 mmol) in water (15
mL). To this mixture was added K2CO3 (3.4 g, 24.6 mmol),
and the resulting mixture was vigorously stirred overnight.
The organic layer was separated, washed with 1 N HCl, dried
over Na2SO4, filtered, and concentrated under reduced pres-
sure. The residue was chromatographed on silica gel with
CHCl3-MeOH (100:1) to give 18d (2.2 g, 92%) as a colorless
oil. 1H-NMR (CDCl3): δ 1.6-2.1 (m, 4H, hGlu-â,γ), 2.41 (m,
5H, toluene-CH3 + hGlu-δ), 2.87 (m, 2H, -CH2S-), 3.68 (s, 3H,
hGluR-OCH3), 3.79 (s, 3H, hGluδ-OCH3), 4.00 (m, 2H, -NCH2-
), 4.77 (m, 1H, hGlu-R), 6.78 (d, 1H, J ) 7.6 Hz, NH), 7.1-7.3
(m, 3H, toluene-Ar2H + benzothiazine-Ar1H), 7.4-7.6 (m, 3H,
toluene-Ar2H + benzothiazine-Ar1H), 7.77 (d, 1H, J ) 8.6 Hz,
benzothiazine-Ar). IR (neat): cm-1 3600-3300, 1730, 1640,
1600. MS: m/ z 520 (M+), 91. HR-MS calcd for
C24H28N2O7S2: M, 520.1338. Found: 520.1338 (M+).
Dim et h yl N-[(3,4-Dih yd r o-2H-1,4-b en zot h ia zin -7-yl)-
ca r bon yl]-L-h om oglu ta m a te (19d ). A mixture of 18d (2.2
g, 4.23 mmol) and anisole (2.2 g, 20.3 mmol) in 30% HBr in
acetic acid (25 mL) was stirred for 4 h at room temperature
and poured into ether (200 mL). Precipitates were decanted
with ether. The residue was suspended with 5% NaHCO3 and
extracted with CHCl3. The organic layer was dried over Na2-
SO4, filtered, and concentrated to give pure 19d (700 mg, 45%)
as white powder. 1H-NMR (CDCl3): δ 1.6-2.0 (m, 4H, hGlu-
â,γ), 2.40 (t, 2H, J ) 6.9 Hz, hGlu-δ), 3.03 (m, 2H, -CH2S-),
3.66 (s, 3H, hGluR-OCH3), 3.70 (m, 2H, -NCH2-), 3.77 (s, 3H,
hGluδ-OCH3), 4.77 (m, 1H, hGlu-R), 6.44 (d, 1H, J ) 8.1 Hz,
NH), 6.54 (d, 1H, J ) 8.1 Hz, Ar), 7.38 (m, 1H, Ar), 7.49 (d,
1H, J ) 2.4 Hz, Ar). IR (KBr): cm-1 3320, 1720, 1630, 1590.
MS: m/ z 366 (M+), 178, 122, 59. HR-MS calcd for
C17H22N2O5S: M, 366.1249. Found: 366.1249 (M+).
Dim eth yl N-[[4-[(2,4-Dia m in op ter id in -6-yl)m eth yl]-3,4-
d ih yd r o-2H -1,4-b e n zot h ia zin -7-yl]ca r b on yl]-L -h om o-
glu ta m a te (21d ). A mixture of 19d (640 mg, 1.74 mmol) and
20 (712 mg, 1.80 mmol) in DMA (10 mL) was stirred for 3 days
at 70 °C. The mixture was poured into 5% NaHCO3 and
extracted with CHCl3. The organic layer was dried over Na2-
SO4, filtered, and concentrated. The residue was chromato-
graphed on silica gel with CHCl3-MeOH (10:1) to give 21d
(500 mg, 53%) as a yellow powder. 1H-NMR (CDCl3-CD3-
OD): δ 1.5-2.0 (m, 4H, hGlu-â,γ), 2.36 (t, 2H, J ) 6.9 Hz,
hGlu-δ), 3.14 (m, 2H, -CH2S-), 3.67 (s, 3H, hGluR-OCH3), 3.78
(s, 3H, hGluδ-OCH3), 3.90 (m, 2H, -NCH2-), 4.77 (m, 1H, hGlu-
R), 5.18 (s, 2H, pteridine-CH2), 6.58 (d, 1H, J ) 7.8 Hz, NH),
6.68 (d, 1H, J ) 8.6 Hz, benzothiazine-Ar), 7.41 (m, 1H,
benzothiazine-Ar), 7.60 (d, 1H, J ) 2.2 Hz, benzothiazine-Ar),
8.74 (s, 1H, pteridine-Ar). IR (KBr): cm-1 3500-3200, 1740,
1630. FAB-MS: m/ z 541 (MH+). HR-FAB-MS calcd for
C24H29N8O5S: MH+, 541.1981. Found: 541.1989 (MH+).
N-[[4-[(2,4-Dia m in op ter id in -6-yl)m eth yl]-3,4-d ih yd r o-
2H-1,4-ben zoth ia zin -7-yl]ca r bon yl]-L-h om oglu ta m ic Acid
(3d ). A mixture of 21d (500 mg, 0.93 mmol) and 1 N NaOH
(2.8 mL) in EtOH (23 mL) was stirred overnight at room
temperature and concentrated to 2 mL under reduced pres-
sure. The residue was diluted to 5 mL with water and
adjusted to pH 3.7 with 1 N HCl. Precipitated solids were
collected by filtration and dried in vacuo to give 3d (360 mg,
Dieth yl N-[[4-(4′-Tolylsu lfon yl)-3,4-dih ydr o-2H-1,4-ben -
zoth ia zin -7-yl]ca r bon yl]-L-glu ta m a te (18c). Using the
same procedure as described for the preparation of 18d ,
compound 18c was prepared from compound 17 and diethyl
glutamate hydrochloride. The yield of 18c was 95% (colorless
oil). 1H-NMR (CDCl3): δ 1.25 (t, 3H, J ) 7.3 Hz, GluR-
CH2CH3), 1.31 (t, 3H, J ) 6.8 Hz, Gluγ-CH2CH3), 1.9-2.6 (m,
7H, Glu-â,γ + toluene-CH3), 2.87 (m, 2H, -CH2S-), 4.00 (m,
2H, -NCH2-), 4.1-4.3 (m, 4H, GluR, δ-CH2CH3), 4.76 (m, 1H,
Glu-R), 7.03 (d, 1H, J ) 7.3 Hz, NH), 7.23 (2H, d, J ) 7.8 Hz,
toluene-Ar), 7.52 (m, 4H, toluene-Ar2H + benzothiazine-
Ar2H), 7.77 (d, 1H, J ) 8.4 Hz, benzothiazine-Ar). IR (neat):
cm-1 3370, 2980, 1730, 1650. MS: m/ z 535 (M+ + 1), 176.
Dieth yl N-[(3,4-Dih yd r o-2H-1,4-ben zoth ia zin -7-yl)ca r -
bon yl]-L-glu ta m a te (19c). Using the same procedure as
described for the preparation of 19d , compound 19c was
1
prepared from compound 18c. The yield of 19c was 62%. H-
NMR (CDCl3): δ 1.23 (t, 3H, J ) 6.8 Hz, GluR-CH2CH3), 1.30
(t, 3H, J ) 6.8 Hz, Gluδ-CH2CH3), 2.0-2.5 (m, 4H, Glu-â,γ),
3.01 (t, 2H, J ) 4.9 Hz, -CH2S-), 3.68 (t, 2H, J ) 4.9 Hz, -NCH2-
), 4.11 (m, 2H, GluR-CH2CH3), 4.22 (m, 2H, Gluδ-CH2CH3),
4.76 (m, 1H, Glu-R), 6.43 (d, 1H, J ) 8.6 Hz, Ar), 6.74 (d, 1H,
J ) 7.3 Hz, NH), 7.34 (d, 1H, J ) 8.1 Hz, Ar), 7.48 (s, 1H, Ar).
IR (neat): cm-1 3400, 2980, 1720, 1630, 1600. MS: m/ z 380
(M+), 178.
Dieth yl N-[[4-[(2,4-Dia m in op ter id in -6-yl)m eth yl]-3,4-
d i h y d r o -2 H -1 ,4 -b e n z o t h i a z i n -7 -y l ] c a r b o n y l ] -L -
glu ta m a te (21c). Using the same procedure as described for
the preparation of 21d , compound 21c was prepared from
compound 19c. The yield of 21c was 60%. 1H-NMR
(CDCl3-CD3OD): δ 1.22 (t, 3H, J ) 7.3 Hz, GluR-CH2CH3),
1.29 (t, 3H, J ) 7.0 Hz, Gluδ-CH2CH3), 2.0-2.3 (m, 2H, Glu-
â), 2.4-2.5 (m, 2H, Glu-γ), 3.12 (m, 2H, -CH2S-), 3.89 (m, 2H,
-NCH2-), 4.10 (m, 2H, GluR-CH2CH3), 4.22 (m, 2H, Gluδ-CH2-
CH3), 4.6-4.8 (m, 3H, pteridine-CH2 + Glu-R), 6.66 (d, 1H, J
) 7.1 Hz, benzothiazine-Ar), 7.11 (d, 1H, J ) 7.3 Hz, NH),
7.40 (d, 1H, J ) 8.6 Hz, benzothiazine-Ar), 7.58 (d, 1H, J )
1.9 Hz, benzothiazine-Ar), 8.67 (s, 1H, pteridine-Ar). IR
(KBr): cm-1 3320, 1730, 1630, 1590. FAB-MS: m/ z 555
(MH+). HR-FAB-MS calcd for C25H31N8O5S: MH+, 555.2138.
Found: 555.2115 (MH+).
N-[[4-[(2,4-Dia m in op ter id in -6-yl)m eth yl]-3,4-d ih yd r o-
2H-1,4-ben zoth ia zin -7-yl]ca r bon yl]-L-glu ta m ic Acid (3c).
Using the same procedure as described for the preparation of
3d , compound 3c was prepared from compound 21c. The yield
of 3c was 96%. 1H-NMR (DMSO-d6): δ 1.8-2.2 (m, 2H, Glu-
â), 2.30 (m, 2H, Glu-γ), 3.18 (m, 2H, -CH2S-), 3.95 (m, 2H,
-NCH2-), 4.37 (m, 1H, Glu-R), 4.76 (s, 2H, pteridine-CH2), 6.79
(d, 1H, J ) 8.8 Hz, benzothiazine-Ar), 7.42 (m, 1H, benzothi-
azine-Ar), 7.59 (d, 1H, J ) 2.0 Hz, benzothiazine-Ar), 8.22 (d,
1H, J ) 7.3 Hz, NH), 8.67 (s, 1H, pteridine-Ar). IR (KBr):
cm-1 3400, 1640, 1590. FAB-MS: m/ z 499 (MH+). HR-FAB-
MS calcd for C21H23N8O5S: MH+, 499.1512. Found: 499.1521
(MH+). Mp: 204-207 °C dec. Anal. Calcd for C21H22N8O5S‚
3H2O: C, 45.65; H, 5.11; N, 20.28; S, 5.84. Found: C, 45.97;
H, 4.82; N, 20.26; S, 6.70. Analysis of HPLC (solvent, CH3-
CO2H/CH3CO2Na (pH 5.4):MeOH ) 3:1; flow rate, 1.0 cm3/
min; detection, 254 nm) showed the purity to be at least 97%
(retention time, 15 min).
P er ip h er a l Blood Mon on u clea r Cell Cu ltu r e. PBMC
from healthy donors were separated by centrifugation on
Ficoll-Paque (Pharmacia, Uppsala, Sweden). Cells were re-
suspended in RPMI 1640 medium containing 5% fetal bovine

MTX Derivatives with a Benzoxazine/ Benzothiazine
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 1 111
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serum (FBS; Hyclone Laboratories Inc., Logan, UT), glutamine,
penicillin G, and streptomycin. Cells (1 × 105 cells/well) were
cultured in 0.2 mL in 96-well microtiter plates (Corning
#25870) with phytohemagglutinin (0.3 µg/mL) (PHA; Welcome
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Syn ovia l Cell Cu ltu r e. Synovial tissues were obtained
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In d u ction of Ad ju va n t Ar th r itis. Induction of adjutant
arthritis was done as previously reported.18 Briefly, male rats
(Lewis, 6-week-old) were inoculated in the base of the tail with
50 µL of liquid paraffin containing 35 µg of heat-killed
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% suppression )
{(control group - treated group)/control group} × 100
Statistical significances of differences from the control were
analyzed by means of Wilcoxon’s rank sum test.
Ack n ow led gm en t. The authors are indebted to Dr.
P. Sanders and Dr. N. Kubodera for their help in the
preparation of this paper and for the many useful
suggestions for its improvement.
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