Design and synthesis of dihydrofolate reductase inhibitors encompassing a bridging ester group. Evaluation in a mouse colitis model

Article abstract of DOI:10.1021/jm021062y

Crohn's disease is a chronic inflammatory bowel disease characterized by inflammation of both the small and large intestines. Methotrexate (MTX), a classical dihydrofolate reductase (DHFR) inhibitor, has been used as a therapeutic agent in the treatment of patients with Crohn's disease in recent years. We sought to develop antifolates similar in structure to MTX that would be effective in reducing inflammation in a mouse disease model of colitis. Four classical DHFR inhibitors encompassing ester bridges in the central parts of the molecules were synthesized. These antifolates were efficient inhibitors of the DHFR enzyme derived from rat. They were also tested in vitro for their ability to inhibit induced proliferation of lymphocytes from mouse spleen. Inhibition of cell proliferation was achieved only in the micromolar range, whereas MTX was effective at low nanomolar concentrations. One of the DHFR inhibitors (1), with an IC₅₀ value for rlDHFR approximately 8 times higher than that of methotrexate, was selected for in vivo experiments in an experimental colitis model in mice. This compound demonstrated a clear antiinflammatory effect after topical administration, comparable to the effect achieved with the glucocorticoid budesonide. Three parameters were evaluated in this model: myeloperoxidase activity, colon weight, and inflammation scoring. A favorable in vivo effect of compound 1 (15 mg/(kg·day)) was observed in all three inflammatory parameters. However, the results cannot be explained fully by DHFR inhibition or by inhibition of lymphocyte cell proliferation, suggesting that other yet unidentified mechanisms enable reduction of inflammation in the colitis model. The mechanism of action of methotrexate analogues encompassing a bridging ester group is not well understood in vivo but seems to lend itself well to further development of similar compounds.

Full text of DOI:10.1021/jm021062y

J . Med. Chem. 2003, 46, 3455-3462
3455
Articles
Design a n d Syn th esis of Dih yd r ofola te Red u cta se In h ibitor s En com p a ssin g a
Br id gin g Ester Gr ou p . Eva lu a tion in a Mou se Colitis Mod el
Malin Graffner-Nordberg,†,# Meredith Fyfe,‡ Ralph Brattsand,§ Bjo¨rn Mellga˚rd,‡ and Anders Hallberg*,†
Department of Medicinal Chemistry, Uppsala Biomedical Center, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden,
AstraZeneca R&D Mo¨lndal, SE-431 83 Mo¨lndal, Sweden, and AstraZeneca R&D Lund, Box 34, SE-221 00 Lund, Sweden
Received October 10, 2002
Crohn’s disease is a chronic inflammatory bowel disease characterized by inflammation of both
the small and large intestines. Methotrexate (MTX), a classical dihydrofolate reductase (DHFR)
inhibitor, has been used as a therapeutic agent in the treatment of patients with Crohn’s disease
in recent years. We sought to develop antifolates similar in structure to MTX that would be
effective in reducing inflammation in a mouse disease model of colitis. Four classical DHFR
inhibitors encompassing ester bridges in the central parts of the molecules were synthesized.
These antifolates were efficient inhibitors of the DHFR enzyme derived from rat. They were
also tested in vitro for their ability to inhibit induced proliferation of lymphocytes from mouse
spleen. Inhibition of cell proliferation was achieved only in the micromolar range, whereas
MTX was effective at low nanomolar concentrations. One of the DHFR inhibitors (1), with an
IC50 value for rlDHFR approximately 8 times higher than that of methotrexate, was selected
for in vivo experiments in an experimental colitis model in mice. This compound demonstrated
a clear antiinflammatory effect after topical administration, comparable to the effect achieved
with the glucocorticoid budesonide. Three parameters were evaluated in this model: myelo-
peroxidase activity, colon weight, and inflammation scoring. A favorable in vivo effect of
compound 1 (15 mg/(kg‚day)) was observed in all three inflammatory parameters. However,
the results cannot be explained fully by DHFR inhibition or by inhibition of lymphocyte cell
proliferation, suggesting that other yet unidentified mechanisms enable reduction of inflam-
mation in the colitis model. The mechanism of action of methotrexate analogues encompassing
a bridging ester group is not well understood in vivo but seems to lend itself well to further
development of similar compounds.
In tr od u ction
nearly all drugs absorbed from the bowel pass the liver
before entering systemic circulation provides an op-
portunity to improve the therapeutic index of drugs
aimed for topical therapy of the intestinal mucosa. Thus,
budesonide (Figure 1), designed by one of us to undergo
an efficient first-pass metabolism in the liver, is now
used in the clinic. When delivered in an appropriate
pharmaceutical formulation, thereby reaching the in-
flamed gut segment, a significantly improved ratio of
the designed topical actions to the adverse systemic
action is achieved.1 In recent years, immunosuppressive
agents, e.g., azathioprine, 6-mercaptopurine, cyclosporin
A, and methotrexate (Figure 2) as well as monoclonal
antibodies against TNFR,2 have found an increasing and
widespread use for the treatment of patients with IBD.3
The first reports on methotrexate (MTX), a potent
inhibitor of dihydrofolate reductase, and its potential
role in IBD therapy appeared in 1989.4 During the
1990s, further support for a beneficial role of MTX was
reported, and more recently, it was demonstrated that
patients with Crohn’s disease who entered remission
after a low dose of MTX also seemed to maintain
remission after long-term therapy.5-9 MTX has also
Crohn’s disease (CD) and ulcerative colitis (UC) are
chronic inflammatory bowel diseases (IBD). CD affects
both the small and large intestines, whereas UC only
involves the colon. The etiology of IBD is unclear, and
no curative treatments are currently available. Phar-
macological treatments can induce remissions, but most
often these are followed by relapses. The standard
treatments for IBD have been aminosalicylates and
glucocorticoids in the past decades. Sulfasalazine and
5-aminosalicylates are used in the management of mild
to moderate disease, whereas the glucocorticoids remain
the primary therapy for patients suffering from moder-
ate to severe disease. Of these therapies, the gluco-
corticoids are less effective in maintaining remission in
UC patients and are in addition often associated with
severe side effects in long-term treatment. The fact that
* To whom correspondence should be addressed. Telephone:
+46 18 4714284. Fax: +46 18 4714976. E-mail: anders.hallberg@
farmkemi.uu.se.
† Uppsala University.
#
Present address: Department of Medicinal Chemistry, Biovitrum
AB, SE-751 82 Uppsala, Sweden.
‡ AstraZeneca R&D Mo¨lndal.
§ AstraZeneca R&D Lund.
10.1021/jm021062y CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/01/2003

3456 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Graffner-Nordberg et al.
F igu r e 1.
F igu r e 2. rlDHFR inhibition (IC50) indicated for each substance.
been demonstrated to decrease the need for glucocorti-
coids in glucocorticoid-dependent CD.6,10
impact of an ester-containing bridge on the bioactivity
in vivo. We previously identified 1 and 2, consisting of
a quinazoline and a pteridine core, respectively, as
inhibitors of DHFR.12
The most potent DHFR inhibitor (1) was found to
exert an impressive activity after local administration
in an experimental inflammatory bowel disease model
in mice. This effect was similar to that obtained with
systemic administration of a moderate dose of the potent
glucocorticoid budesonide.
Our long-term perspective is to develop soft drug
analogues of antifolates that, like budesonide, are
efficiently transformed locally in the intestinal mucosa,
in serum or in the liver, to nontoxic metabolites after
having exerted their antiinflammatory effects in the
intestinal tissues. While budesonide undergoes an ef-
ficient oxidative deactivation in liver mediated by the
cytochrome P450 system1 (Figure 1), other soft drugs,
e.g., of cyclosporin A, are designed to undergo deactiva-
tion mediated by esterases.11 We postulated that the
methylenamino bridge in MTX could constitute a suit-
able site for a diversity of structural manipulations
including the incorporation of an appropriate ester
feature to provide good substrates for the endogenous
esterases while still retaining the inhibitory activity of
dihydrofolate reductase (DHFR) (Figure 1).
As a first step toward soft drug analogues of MTX, it
was essential to probe the tolerance for structural
alterations in (a) the heterocyclic core12 and (b) the
hydrophilic side chain of the new ester-containing
antifolates. Would (a) the in vitro inhibitory activity and
the ability to inhibit cell proliferation remain and (b)
the in vivo activity in complex animal models remain
after the methyleneamino group in the classical anti-
folates was substituted for an ester group? To address
these issues, we examined a small set of short-bridge,
metabolically stable ester analogues and evaluated the
Resu lts
Syn th eses. Compounds 1-4 were synthesized with
the reaction sequence outlined in Scheme 1. We have
previously prepared compounds 1 and 2 by an alterna-
tive procedure that was partly based on solid-phase
chemistry.12 Di-tert-butyl L-R-aminoadipate13 (5) was
coupled to 4-formylbenzoic acid using (benzotriazol-1-
yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP) and N,N-diisopropylethylamine, affording 8.
Compounds 614 and 715 were synthesized in a similar
manner starting from the commercially available di-tert-
butyl esters of L- and D-glutamic acid hydrochloride,
respectively. The aldehydes were oxidized immediately
after purification to the corresponding carboxylic acids
9-11, using sodium chlorite in tert-butyl alcohol with
2-methyl-2-butene as scavenger.16-18 2,4-Diaminoquinazo-
line-6-methanol19 was subjected to 30% hydrobromic
acid in glacial acetic acid,20 providing compound 12.12

Dihydrofolate Reductase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3457
Sch em e 1a
a
(a) PyBOP, Et3N, or N,N-diisopropylethylamine, CH2Cl2; (b) NaClO2, NaHPO4‚1H2O, 2-methyl-2-butene, tert-butyl alcohol; (c) K2CO3,
DMF, or DMAc; (d) HCO2H or CF3CO2H.
The reaction mixture was triturated with cold diethyl
ether to allow for precipitation, which was easily col-
lected under an atmosphere of nitrogen before it was
dried at 40 °C in vacuo and used in the next step
without further purification. The 6-(bromomethyl)-2,4-
diaminopteridine hydrobromide (13)12,21,22 was prepared
according to the same method as for 12 starting from
commercially available 2,4-diaminopteridine-6-metha-
nol. The esters 14-17 were formed by nucleophilic dis-
placement of the appropriate bromide by the benzoates
9-11. The overall yield for the last two steps varied
between 23% and 51%. Prolonging the reaction time or
heating the reaction mixture did not seem to improve
the yields. A final selective hydrolysis of the tert-butyl
esters with formic acid or trifluoroacetic acid provided
compounds 1-4 in good yields.
Dih yd r ofola te Red u cta se In h ibition . Compounds
1-4 were examined for their inhibitory activity of rat
liver DHFR according to published procedures.23 The
F igu r e 3. rlDHFR inhibition (IC50) indicated for each sub-
stance.
results are presented as IC50 values (Figure 2). Com-
pound 1 was found to be the best inhibitor of rlDHFR
with an IC50 value only 8-fold higher than that of MTX.
However, compounds 2-4 exhibited a greater than 10-
fold loss of potency relative to 1. To assess the impact
of the glutamic acid residue and the methyleneamino
bridge, the IC50 values of nine representative derivatives
were included for comparison (Figure 3). As apparent
from the figure, displacement of the glutamic acid
portion rendered less active inhibitors.
Cell P r olifer a tion Assa y. Because MTX and com-
pounds 1-4 are inhibitors of DHFR of varying potency,
the ability of these compounds to inhibit mitogen-
induced proliferation of mouse spleen cells (lympho-

3458 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Graffner-Nordberg et al.
F igu r e 4. Effect of MTX and compounds 1-4 on ConA-mediated mouse spleenocyte proliferation. CPM is counts per minute.
Proliferation was induced by ConA (5 µg/mL), and compounds were added at concentrations as indicated. Two days after initiation
of the experiment, 3H-thymidine was added and labeling was continued for 18 h. Bars indicate the mean of triplicates. Error bars
indicate SD. Only MTX was tested at 3 nM. Significant difference from control group is indicated: (#) p < 0.001; (O) p < 0.01.
cytes) was compared. Inhibition of lymphocyte prolif-
eration could explain at least part of an antiinflammatory
effect in vivo. Cells were thus purified from mouse
spleens and thereafter stimulated with the T-cell mi-
togen concanavalin A (ConA). Inhibition of proliferation
with MTX and compounds 1-4 was evaluated at various
concentrations (Figure 4). MTX inhibited proliferation
at 30 nM and higher. Similar data were obtained in two
independent experiments. Compound 1 suppressed cell
proliferation at only the highest dose compared (30 µM),
a 1000-fold higher concentration than that of MTX.
Thus, the inhibition of mouse lymphocyte proliferation
was not well correlated with inhibition of the enzyme
DHFR.
Colitis Mod el in Mice. The antiinflammatory effects
of compound 1 and MTX were compared in a mouse
model of inflammatory bowel disease. In this model,
locally instilled DNBS (2,4-dinitrobenzenesulfonic acid)
is used to induce an acute inflammation of the colon.
The effect of the compounds was assessed after rectal
administration once daily for 7 days. As a reference
compound, the potent steroidal soft drug budesonide
was administered once daily by intraperitoneal injec-
tion. It should be noted that systemic administration
of budesonide does not take advantage of the high first-
pass metabolism obtained with this compound follow-
ing oral administration. However, in this experimen-
tal setting, we wanted to ensure significant systemic
glucocorticosteroid effect as a positive reference for the
effect of the treatment. Previous data showed that the
dose of budesonide used, 1 µg/(kg‚day) given intra-
peritoneally, produced a pronounced systemic gluco-
corticosteroid effect.24 The doses of MTX and compound
1 were chosen on the basis of earlier experiments in
rodents with MTX12,25 and the relative potency of the
substances regarding DHFR inhibition.12 Previous work
also demonstrated that the maximal well-tolerated dose
of MTX to mice is 0.5 mg/(kg‚day), and preliminary data
also indicated positive effects of this dose in the mouse
colitis model (data not shown).
In the current study, it was found that the reference
compound, budesonide, suppressed the inflammatory
response significantly (Figure 5). However, MTX at the
given dose (0.5 mg/(kg‚day)) had no effect on inflam-
mation assessed by three different methods. Compound
1 was given to mice at two different doses, 15 and 60
mg/(kg‚day), to meet and even exceed the relative
difference in potency between MTX and compound 1
with respect to DHFR inhibition. Interestingly, only the
lower dose of compound 1 (15 mg/(kg‚day)) reduced the
inflammatory response. The effect of the lower dose was
comparable to that of budesonide, and it also differed
significantly from the inflammation in the control group
of animals. In addition to the parameters measured, it
was noted that the animals receiving the 15 mg/(kg‚
day) dose of compound 1 were in much better general
condition than those receiving the higher dose. There
were no obvious effects on health status of the animals
when compound 1 was given to control animals.
Discu ssion
The underlying mechanism behind the effects of
methotrexate in IBD therapy is still not fully elucidated.
It has been proposed that the antiinflammatory action
of methotrexate is due to accumulation of adenosine,26
and adenosine A2 receptor antagonists have been shown
to suppress the antiinflammatory effect of MTX.27
However, the role of adenosine as an important media-
tor of the antiinflammatory effect of MTX has been
questioned28 and is not yet proven in a clinical situa-
tion.29 AICARtf (5-aminoimidazole-4-carboxamide ribo-
nucleotide transformylase), the last enzyme in de novo
purine synthesis, is inhibited by methotrexate poly-
glutamates and is suggested as another candidate.

Dihydrofolate Reductase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3459
F igu r e 5. Effects of compound 1, MTX, and budesonide in the DNBS mouse colitis model. Effect of treatment was assessed by
three different parameters: colon weight, MPO (myeloperoxidase), and IS (inflammation scoring). The animals were treated
with test compounds once daily over 7 days, and the effects were analyzed 1 day after end of treatment. The results are expressed
as the percent of response in untreated control animals (mean values). Significant differences from control group are indicated:
(/) p < 0.05; (//) p < 0.01; (///) p <0.001.
Purines, as well as the pyrimidines, are required for
lymphocyte proliferation. Hence, inhibition of AICARtf
has been proposed as one of the mechanisms for the
immunosuppressive effects of MTX.
experiments were performed in mice, rat and mouse
DHFR are highly homologous and show similar sensi-
tivity to MTX. It is notable that a comparative phar-
macokinetic study showed that the bioavailability of
compound 1 after oral administration was only slighter
lower than that for methotrexate, according to LC-
MS.12,30
All four ester analogues (1-4) exhibit fair inhibitory
activity of DHFR. Despite the modifications of the
hydrophilic side chain, the 2,4-diaminoquinazolines 1,
3, and 4 still exhibited better inhibitory activity of
recombinant hDHFR than the corresponding pteridine
analogue 2.30 The structure of the side chain has
apparently less impact on the binding of the ligand to
the enzyme although derivatives 1 and 2, comprising
the L-glutamic acid residue, should more likely serve
as substrates for polyglutamation than 3 and 4.31
Regarding MTX and compounds 1-4, the in vitro data
demonstrate a discrepancy between DHFR inhibition
and the ability of these compounds to inhibit cell
proliferation. While MTX exerted an inhibitory effect
at a minimum of 30 nM, no signs of such inhibition were
found with any of the compounds 1-4. With respect to
the DHFR inhibition value for compound 1, one may
have expected to see inhibition of lymphocyte prolifera-
tion at approximately 8 times the dose of MTX. Unex-
pectedly, the only inhibitory effect on proliferation with
compound 1 observed was at the highest dose tested,
30 µM. At this concentration of compound 1, inhibition
of cell proliferation was comparable to that of MTX at
30 nM. The observed discordance between DHFR bind-
ing and cell growth inhibition may reflect differences
in cellular influx via the reduced folate carrier.
The colitis model used in this study is quite complex,
involving several components of the inflammatory re-
sponse. We have earlier also noted some experimental
variation concerning the degree of inflammation from
experiment to experiment. An important part of the
defense to mucosal injury is the regeneration of the
epithelial layer. We propose that high concentrations
of metabolically stable antifolates may negatively affect
the regeneration of the epithelium and thus the healing
process. This could possibly explain the better effect of
a lower dose, as observed with compound 1. Negative
effects would be of particular importance in experiments
where the animals have a high degree of inflammation
and thereby are more susceptible to interference with
healing mechanisms. The data suggest that compound
1, devoid of ability to inhibit proliferation, has beneficial
antiinflammatory properties compared to MTX, al-
though the molecular mechanisms remain to be eluci-
dated.
Con clu sion
Analogues of methotrexate in which the C9-N10
bridge has been replaced by an ester group retain much
of the DHFR inhibitory activity. One of the antifolates
synthesized (compound 1) exerted a clear antiinflam-
matory effect in a mouse model of colitis as assessed by
three different parameters. This potent ester-based
antifolate analogue of methotrexate did not inhibit
mouse spleen cell proliferation, implicating that the
effect of this compound in vivo is less likely to be related
to inhibition of lymphocyte proliferation. We conclude
that the mechanism of action of this ester analogue of
MTX may be different from that of MTX. The results
A significant positive effect was noted in vivo with
compound 1 but surprisingly only at the lower dose of
15 mg/(kg‚day). The inhibition of DHFR may play an
important role for the therapeutic effect, but this is not
supported by the in vivo data with compound 1 given
at a 30-fold higher dose than MTX. The inverse dose
response observed in vivo with compound 1 could point
toward potential side effects using MTX, or analogues
thereof, at the indicated doses in vivo. However, one
ought to take into account that although the in vivo

3460 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Graffner-Nordberg et al.
Di-ter t-bu tyl N-(4-Ca r boxyben zoyl)-L-R-a m in oa d ip a te
(11). Compound 11 was prepared from 8 (665 mg, 1.64 mmol)
according to the same procedure as described for compound
9, affording after purification by flash chromatography [CHCl3/
MeOH (9:1)] 643 mg (93%) of the pure carboxylic acid: IR
presented herein suggest that compounds such as 1
provide starting points for further development toward
new chemical entities with lower systemic exposure and
better antiinflammatory properties.
1
(CH2Cl2) 1731 (ester), 1655 (amide) cm-1; H NMR (CDCl3) δ
Exp er im en ta l Section
8.09-7.86 (m, 4H, ArH), 7.03 (d, J ) 7.42 Hz, 1H, NH), 4.72-
4.65 (m, 1H), 2.33-2.28 (m, 2H), 2.06-1.61 (m, 4H), 1.51 (s,
9H), 1.44 (s, 9H); 13C NMR (CDCl3) δ 172.74, 171.77, 169.73,
166.49, 138.16, 132.68, 130.20 (2C), 127.13 (2C), 82.73, 80.55,
Ch em istr y. Gen er a l In for m a tion . 1H and 13C NMR
spectra were recorded on a J EOL J NM-EX270 spectrometer
at 270 and 67.8 MHz, respectively, and a J EOL J NM-EX400
spectrometer at 400 and 100 MHz, respectively. Thin-layer
chromatography (TLC) was performed by using aluminum
sheets precoated with silica gel 60 F254 (0.2 mm) type E, Merck.
Chromatographic spots were visualized by UV light or an
ethanolic solution of ninhydrin or an acidic ethanolic solution
of p-anisaldehyde followed by heating. Column chromatogra-
phy was conducted on silica gel S (0.032-0.063 mm, Riedel-
deHae¨n) and silica gel 60 (0.040-0.063 mm, E. Merck) unless
otherwise noted. Centrifugal chromatography was carried out
on a Harrison Research Chromatotron (model 7924T) with
silica gel PF254 containing gypsum (E. Merck) as solid phase.
Melting points (uncorrected) were determined in open glass
capillaries on an Electrothermal apparatus. Infrared (IR)
spectra were recorded on a Perkin-Elmer 1605 FT-IR spec-
trophotometer and are recorded in νmax (cm-1). The elemental
analyses were performed by Mikro Kemi AB, Uppsala, Swe-
den, or Analytische Laboratorien, Gummersbach, Germany,
and were within (0.4% of the calculated values. All com-
mercial chemicals were used without further purification.
Di-ter t-bu tyl L-R-Am in oa d ip a te (5). See refs 13 and 30.
Di-ter t-bu tyl N-(4-F or m ylben zoyl)-L-glu ta m a te (6). See
refs 14 and 30.
53.06, 34.77, 31.57, 28.05 (3C), 27.98 (3C), 20.67. Anal. (C22H31
-
NO7‚0.5H2O) C, H, N.
6-(Br om om eth yl)-2,4-d ia m in oqu in a zolin e Hyd r obr o-
m id e (12). See refs 12 and 30.
6-(Br om om eth yl)-2,4-d ia m in op ter id in e Hyd r obr om id e
(13). See refs 12, 21, 22, and 30.
Di-ter t-bu tyl N-[4-(2,4-Dia m in oqu in a zolin e-6-yl)m eth -
yl]ben zoyl]-L-glu ta m a te (14). Freshly prepared 12 (0.53
mmol) was added to a mixture of 9 (322 mg, 0.79 mmol) and
K2CO3 (146 mg, 1.06 mmol) in dry DMAc (15 mL). The reaction
mixture was stirred at room temperature for 24 h before it
was filtered and evaporated to dryness on silica gel under
reduced pressure. The silica plug was added on top of a silica
column and the crude product was purified by repeated flash
chromatography [CHCl
3/MeOH + NH3(aq) (9:1)], providing 116
mg (38% over two steps) of the pure ester: IR (KBr) 1723
(ester), 1655 (amide) cm-1; H NMR (DMF-d6) δ 8.88 (d, J )
1
7.42 Hz, 1H, NH), 8.26 (d, J ) 1.32 Hz, 1H, H-5), 8.16-8.09
(m, 4H, ArH), 7.72 (dd, J ) 8.58, 1.65 Hz, 1H, H-7), 7.53 (br
s, 2H, NH2), 7.33 (d, J ) 8.58 Hz, 1H, H-8), 6.16 (br s, 2H,
NH2), 5.43 (s, 2H), 4.58-4.49 (m, 1H), 2.47 (app t, 2H), 2.26-
1.99 (m, 2H), 1.45 (s, 9H), 1.42 (s, 9H); 13C NMR (DMF-d6) δ
172.11, 171.42, 166.63, 165.76, 163.43, 161.69, 152.81, 138.90,
133.37, 132.79, 129.69 (2C), 128.22, 128.07 (2C), 124.96,
124.21, 110.55, 81.18, 80.18, 67.25, 53.40, 31.83, 27.69 (3C),
27.62 (3C), 26.63. Anal. (C30H37N5O7‚0.75H2O) C, H, N.
Di-ter t-bu tyl N-(4-F or m ylben zoyl)-D-glu ta m a te (7). See
refs 15 and 30.
Di-ter t-b u t yl N-(4-F or m ylb en zoyl)-L-R-a m in oa d ip a t e
(8). A mixture of 4-formylbenzoic acid (295 mg, 1.96 mmol),
PyBOP (929 mg, 1.79 mmol), N,N-diisopropylethylamine (0.68
mL, 3.90 mmol), and CH2Cl2 (12 mL) was stirred together for
15 min. Di-tert-butyl L-R-aminoadipate (5) in CH2Cl2 (13 mL)
was added, and the reaction mixture was allowed to stir
overnight. Workup was performed as described for compound
6, yielding an oil, which was purified with centrifugal chro-
matography [EtOAc/isohexane (1:7)] affording 665 mg (92%):
1H NMR (CDCl3) δ 10.06 (1H, CHO), 7.99-7.92 (m, 4H, ArH),
7.05 (d, J ) 7.26 Hz, 1H, NH), 4.69-4.62 (m, 1H), 2.30-2.24
(m, 2H), 2.01-1.59 (m, 4H), 1.49 (s, 9H), 1.42 (s, 9H); 13C NMR
(CDCl3) δ 191.53, 172.56, 171.32, 165.81, 139.16, 138.24,
129.76 (2C), 127.79 (2C), 82.60, 80.44, 53.04, 34.73, 31.59,
28.05 (3C), 27.98 (3C), 20.50. Anal. (C22H31NO6) C, H, N.
Di-ter t-bu tyl N-(4-Ca r boxyben zoyl)-L-glu ta m a te (9).
Sodium chlorite (2.29 g, 25.3 mmol) and NaH2PO4‚1H2O (2.71
g, 19.6 mmol) in water (30 mL) was added dropwise to a
solution of 6 (1.10 g, 2.81 mmol), tert-butyl alcohol (50 mL),
and 2-methyl-2-butene (15 mL). The reaction was finished
after 3 h according to TLC. The reaction mixture was extracted
between 1 M HCl and CHCl3 followed by washing with 10%
Na2S2O3 and water, respectively. Subsequent drying (MgSO4),
filtration, concentration under reduced pressure, and purifica-
tion by flash chromatography [CHCl3/MeOH (19:1)] provided
a white foam: yield, 1.07 g (93%); IR (CH2Cl2) 1725 (ester),
1649 (amide) cm-1; 1H NMR (CDCl3) δ 9.35 (br s, 1H, COOH),
8.09-7.85 (m, 4H, ArH), 7.32 (d, J ) 7.59 Hz, 1H, NH), 4.71-
Di-ter t-bu tyl N-[4-(2,4-Dia m in op ter id in e-6-yl)m eth yl]-
ben zoyl]-L-glu ta m a te (15). Compound 15 was prepared from
freshly prepared 13 (0.52 mmol) under the same conditions
as described above for the synthesis of 14. Purification by
repeated flash chromatography [CHCl3/MeOH + NH3(aq) (29:1
and 39:1)] yielded 71 mg (23% over two steps) of a light-yellow
solid: IR (KBr) 1720 (ester), 1670 (amide) cm-1 1H NMR
;
(DMF-d6) δ 9.00 (s, 1H, H-7), 8.88 (d, J ) 7.42 Hz, 1H, NH),
8.21-8.01 (m, 4H, ArH), 7.85 (br s, 2H, NH2), 6.80 (br s, 2H,
NH2), 5.54 (s, 2H), 4.59-4.51 (m, 1H), 2.48 (app t, 2H), 2.22-
1.95 (m, 2H), 1.46 (s, 9H), 1.43 (s, 9H); 13C NMR (DMF-d6) δ
172.11, 171.43, 166.49, 165.66, 164.28, 163.87, 156.59, 150.73,
143.31, 139.04, 132.44, 129.85 (2C), 128.15 (2C), 122.24, 81.18,
80.18, 66.13, 53.48, 31.90, 27.73 (3C), 27.68 (3C), 26.72. Anal.
(C28H35N7O7‚0.5H2O) C, H, N.
Di-ter t-bu tyl N-[4-(2,4-Dia m in oqu in a zolin e-6-yl)m eth -
yl]ben zoyl]-D-glu ta m a te (16). Compound 16 was prepared
from 10 (270 mg, 0.66 mmol) and as described above for
compound 14. Workup as for 14 with repeated flash chroma-
tography [CHCl3/MeOH + NH3(aq) (39:1 with a gradient to 29:
1)] and further isolation of the product with centrifugal
chromatography [CH2Cl2/MeOH + NH3(aq) (49:1)] afforded 157
mg (51% over two steps) of the desired compound: IR (KBr)
1725 (ester), 1637 (amide) cm-1; 1H NMR (CDCl3) δ 8.00-7.78
(m, 4H, ArH), 7.73 (d, J ) 1.65 Hz, 1H, H-5), 7.59 (dd, J )
8.58 Hz, 1.65, 1H, H-7), 7.44 (d, J ) 7.59 Hz, 1H, NH), 7.40
(d, J ) 8.91 Hz, 1H, H-8), 6.14 (br s, 2H, NH2), 5.33 (s, 2H),
5.15 (br s, 2H, NH2), 4.68-4.60 (m, 1H), 2.51-2.05 (m, 4H),
1.48 (s, 9H), 1.40 (s, 9H); 13C NMR (CDCl3) δ 172.17, 171.31,
166.30, 165.67, 162.63, 160.28, 152.18, 137.82, 133.70, 132.47,
129.66 (2C), 128.89, 127.15 (2C), 125.60, 122.62, 110.10, 82.61,
81.06, 66.85, 53.19, 31.77, 28.00 (6C), 27.05. Anal. (C30H37N5O7‚
1.5H2O) C, H, N.
4.64 (s, 1H), 2.52-2.02 (m, 4H), 1.49 (s, 9H), 1.42 (s, 9H); 13
C
NMR (CDCl3) δ 172.88, 171.37, 169.97, 166.43, 138.33, 132.06,
130.30 (2C), 127.24 (2C), 82.83, 81.21, 53.18, 31.71, 28.03 (6C),
27.13. Anal. (C21N29NO7‚1.25H2O) C, H, N.
Di-ter t-bu tyl N-(4-Ca r boxyben zoyl)-D-glu ta m a te (10).
Compound 10 was prepared from 7 (0.988 g, 2.52 mmol) as
described above for compound 9. Subsequent workup afforded
a white foam: yield, 0.803 g (78%); IR (CH2Cl2) 1725 (ester),
1655 (amide) cm-1; 1H NMR (CDCl3) δ 8.10-7.86 (m, 4H, ArH),
7.31 (d, J ) 7.42 Hz, 1H, NH), 4.71-4.64 (m, 1H), 2.53-2.02
(m, 4H), 1.50 (s, 9H), 1.42 (s, 9H). Anal. (C21H29NO7‚0.5H2O)
C, H, N.
Di-ter t-bu tyl N-[4-(2,4-Dia m in oqu in a zolin e-6-yl)m eth -
yl]ben zoyl]-L-R-a m in oa d ip a te (17). Compound 17 was pre-
pared from 11 (333 mg, 0.79 mmol) in dry DMF (15 mL) as
described above for compound 14. Purification with flash
chromatography [CHCl3/MeOH + NH3(aq) (19:1)] was followed

Dihydrofolate Reductase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3461
by centrifugal chromatography [CHCl3/MeOH + NH3(aq) (39:
1)] to afford 106 mg (34% over two steps) of the pure ester:
1H NMR (CDCl3) δ 8.11-7.85 (m, 4H, ArH), 7.71-7.67 (m, 2H,
H-5 + H-7), 7.48 (d, J ) 9.57 Hz, 1H, H-8), 6.98 (d, J ) 7.42
Hz, 1H, NH), 5.57 (br s, 2H, NH2), 5.41 (s, 2H), 4.89 (br s, 2H,
NH2), 4.70-4.63 (m, 1H), 2.28 (app t, 2H), 2.00-1.60 (m, 4H),
1.50 (s, 9H), 1.43 (s, 9H); 13C NMR (CDCl3) δ 172.60, 171.71,
166.35, 165.63, 162.67, 160.05, 151.49, 137.96, 133.71, 132.34,
129.63 (2C), 128.94, 127.13 (2C), 125.10, 122.71, 110.01, 82.55,
80.47, 66.80, 53.10, 34.71, 31.51,28.04 (6C), 20.75. Anal.
(C31H39N5O7‚0.5H2O) C, H, N.
N-[4-[(2,4-Dia m in oqu in a zolin e-6-yl)m eth ylen e]oxyca r -
bon yl]ben zoyl L-Glu ta m ic Acid (1). Meth od A. Compound
14 (180 mg, 0.31 mmol) was dissolved in trifluoroacetic acid
(10 mL), and the mixture was stirred at room temperature
for 40 min. Evaporation under reduced pressure was followed
by trituration with diethyl ether, and the precipitate was
filtered. Water (4 mL) was added, and the pH was adjusted to
3.9 with 1 M NaOH. The precipitate was filtered off and
washed consecutively with water, ethanol, acetone, and diethyl
ether, yielding 121 mg (83%) of a white solid: IR (KBr) 1724
(ester), 1652 (amide) cm-1; 1H NMR (DMSO-d6) δ 8.73 (d, J )
7.75 Hz, 1H, NH), 8.20 (app d, 1H, H-5), 8.10-7.98 (m, 4 +
2H, ArH + NH2), 7.83 (br s, 2H, NH2), 7.74 (dd, J ) 8.58, 1.65
Hz, 1H, H-7), 7.32 (d, J ) 8.58 Hz, 1H, H-8), 7.09 (br s, 2H,
NH2), 5.37 (s, 2H), 4.42-4.34 (m, 1H), 2.35 (app t, 2H), 2.16-
1.88 (m, 4H); 13C NMR (DMSO-d6) δ 174.06, 173.84, 165.51,
165.12, 162.65, 158.54, 138.42, 133.98, 131.77, 129.28 (2C),
128.93, 127.80 (2C), 124.29, 121.27, 109.46, 66.52, 52.44, 30.72,
26.31 (one aromatic carbon missing).
received an intracolonic instillation of 100 µL of a solution
containing 4 mg of DNBS (Sigma) dissolved in a vehicle of 30%
ethanol/water. This procedure was similar to what has previ-
ously been described.32,33 For the study, animals were housed
in groups according to treatments. The day after induction of
the colitis, treatment was initiated, and those animals that
were given treatment by rectal administration were anesthe-
tized briefly at each dosing time. Treatments were continued
for 7 days with administration of test compounds once daily.
One day after the end of the treatment, the animals were
euthanized by cervical dislocation. The degree of colitis was
assessed by (a) analysis of tissue myeloperoxidase, (b) by the
weight of the distal 4 cm of the colon, and (c) by blinded
scoring, essentially as previously described.34 The study was
approved by the local ethics committee for animal experimen-
tal studies.
In Vitr o Cell P r olifer a tion Assa y. Spleen cells were
isolated from female Balb/C mice and stimulated with the
T-cell mitogen ConA.35 Briefly, one or two spleens were cut
into small pieces and passed through a nylon mesh. Erythro-
cytes were lysed with ammonium sulfate, and the remaining
cells were counted. The cell proliferation assay was performed
in 96-well microtiter plates, and 4 × 105 cells were added to
each well. One hour prior to addition of ConA (5 µg/mL), MTX
and compounds 1-4 were added. After 48 h incubation, 3H-
thymidine was added, and after another 18 h, cells were
harvested and the incorporation was measured in a scintilla-
tion counter. All samples were tested in triplicate, and data
were obtained in two independent experiments.
Ack n ow led gm en t. We thank Professor Sherry F.
Queener for skillful in vitro binding experiments on rat
liver dihydrofolate reductase and the Swedish Founda-
tion for Strategic Research (SSF) for financial support.
Agneta Karlsson and Maria Fritsch-Fredin at Integra-
tive Pharmacology, AstraZeneca R&D Mo¨lndal, are
acknowledged for their skillful work with the in vivo
and in vitro experiments.
Meth od B. Compound 14 (0.035 mg, 60 µmol) was dissolved
in formic acid (5 mL) and warmed to 40 °C. TLC showed the
absence of starting material after 30 min. Workup was
performed as described in method A, providing 20 mg (71%).
Anal. (C22H21N5O7‚1H2O) C, H, N.
N-[4-[(2,4-Dia m in op t er id in e-6-yl)m et h ylen e]oxyca r -
bon yl]ben zoyl L-Glu ta m ic Acid (2). Compound 2 was
synthesized from compound 15 (47 mg, 81 µmol) as described
above in method B, providing 30 mg (79%) of a light-yellow
solid: 1H NMR (DMSO-d6) δ 8.90 (s, 1H, H-8), 8.79 (d, J )
7.92 Hz, 1H, NH), 8.12-7.98 (m, 4H, ArH), 7.68 (br s, 2H,
NH2), 6.74 (br s, 2H, NH2), 5.48 (s, 2H), 4.45-4.36 (m, 1H),
2.38-2.33 (m, 2H), 2.12-1.91 (m, 2H); 13C NMR (DMSO-d6) δ
173.82, 173.16, 165.59, 165.05, 163.24, 162.80, 155.74, 150.33,
142.35, 138.22, 131.58, 129.35 (2C), 127.85 (2C), 121.60, 65.64,
52.11, 30.53, 25.98. Anal. (C20H19N7O7‚1HCO2H‚0.25H2O) C,
H, N.
N-[4-[(2,4-Dia m in oqu in a zolin e-6-yl)m eth ylen e]oxyca r -
bon yl]ben zoyl D-Glu ta m ic Acid (3). Compound 3 was
prepared from compound 16 (130 mg, 0.22 mmol) as described
above in method B, affording 90 mg (86%) of a white solid:
1H NMR (DMSO-d6) δ 8.66 (d, J ) 7.26 Hz, 1H, NH), 8.17 (s,
1H, H-5), 8.09-7.98 (m, 4H, ArH), 7.78 (br s, 2H, NH2), 7.70
(d, J ) 8.25 Hz, 1H, H-7), 7.29 (d, J ) 8.58 Hz, 1H, H-8), 6.89
(br s, 2H, NH2), 5.37 (s, 2H), 4.41-4.34 (m, 1H), 2.37-2.31
(m, 2H), 2.14-1.88 (m, 4H). Anal. (C22H21N5O7‚0.5H2O) C, H,
N.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails for the preparation of compounds 5-7 and 12-13,
pharmacokinetics of compound 1 and MTX in rat, IC50 values
of compounds 1-4, rec hDHFR. This material is available free
of charge via the Internet at http://pubs.acs.org.
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prepared from compound 17 (107 mg, 0.18 mmol) according
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