Analogues of methotrexate in rheumatoid arthritis. 1. Effects of 10- deazaaminopterin analogues on type II collagen-induced arthritis in mice

Article abstract of DOI:10.1021/jm9505526

Carbonation of the dianions (LDA) of 5-methylthiophene-2-carboxylic, 2- methylpyridine-5-carboxylic, and 3-methylpyridine-6-carboxylic acids provided the respective carboxy heteroarylacetic acids. The crude diacids were directly esterified in MeOH-HCl to afford the diesters. Alkylation of the sodio anions with ethyl iodide yielded the appropriate α-ethyl diesters. The anions of the various diester substrates were then alkylated by 2,4-diamino- 6-(bromomethyl)-pteridine followed by ester saponification at room temperature to afford the respective 2,4-diamino-4-deoxy-10-carboxy-10- deazapteroic acids. The 10-carboxyl group was readily decarboxylated by heating in DMSO at temperatures of 110-135 °C to give the diamino 10-deaza heteropteroic acid intermediates. Coupling with diethyl L-glutamate followed by ester hydrolysis afforded the target aminopterins. The analogues were evaluated for antiinflammatory effect in the mouse type II collagen model. The thiophene analogue of 10-ethyl-10-deazaaminopterin was found to be an effective inhibitor in terms of reduced visual evidence of inflammation and swelling as determined by caliper measurement.

Full text of DOI:10.1021/jm9505526

370
J . Med. Chem. 1997, 40, 370-376
An a logu es of Meth otr exa te in Rh eu m a toid Ar th r itis. 1. Effects of
10-Dea za a m in op ter in An a logu es on Typ e II Colla gen -In d u ced Ar th r itis in Mice
J oseph I. DeGraw,*,† William T. Colwell,† J ac Crase,† R. Lane Smith, J ames R. Piper,‡ William R. Waud,‡ and
Francis M. Sirotnak§
Bio-Organic Chemistry Laboratory, SRI International, Menlo Park, California 94025, Orthopedic Research Laboratory,
Stanford University Medical Center, Palo Alto, California 94305, Organic Chemistry Department, Southern Research Institute,
Birmingham, Alabama 35255, and Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Received J uly 24, 1995
Carbonation of the dianions (LDA) of 5-methylthiophene-2-carboxylic, 2-methylpyridine-5-
carboxylic, and 3-methylpyridine-6-carboxylic acids provided the respective carboxy heteroary-
lacetic acids. The crude diacids were directly esterified in MeOH-HCl to afford the diesters.
Alkylation of the sodio anions with ethyl iodide yielded the appropriate R-ethyl diesters. The
anions of the various diester substrates were then alkylated by 2,4-diamino-6-(bromomethyl)-
pteridine followed by ester saponification at room temperature to afford the respective 2,4-
diamino-4-deoxy-10-carboxy-10-deazapteroic acids. The 10-carboxyl group was readily decar-
boxylated by heating in DMSO at temperatures of 110-135 °C to give the diamino 10-deaza
heteropteroic acid intermediates. Coupling with diethyl L-glutamate followed by ester hydrolysis
afforded the target aminopterins. The analogues were evaluated for antiinflammatory effect
in the mouse type II collagen model. The thiophene analogue of 10-ethyl-10-deazaaminopterin
was found to be an effective inhibitor in terms of reduced visual evidence of inflammation and
swelling as determined by caliper measurement.
Rheumatoid arthritis (RA) is an inflammation of the
joints arising from infectious, metabolic, or autoimmune
causes of unknown origin. All forms of arthritis have,
as a final manifestation, loss of joint function due to
erosion of the articular cartilage surface and subsequent
limitation of range of motion due to painful articulation.
Onset of cartilage destruction is linked to a common
biochemical pathway involving release of cartilage pro-
teoglycans from the extracellular matrix.1-3 In infec-
tious and inflammatory arthritis, loss of proteoglycan
precedes any change in collagen levels4 and any visually
apparent effects. A working hypothesis is that early and
effective intervention by a drug that reduces proteogly-
can degradation prior to cartilage breakdown will
potentially prevent progression of disease and provide
for cartilage repair. Nonsteroidal antiinflammatory
drugs have been used most frequently to suppress the
inflammation of rheumatoid arthritis, but these drugs
which inhibit prostaglandin synthesis in chondrocytes,
do not interfere with the underlying immune-based
processes believed to be paramount in rheumatoid
arthritis. Refractory cases have been treated with
strongly cytotoxic agents such as azathioprine,4 chloram-
bucil,5 and cyclophosphamide,6 but these compounds are
renowned for carcinogenic properties and adverse drug
reactions.
inflamed joint counts of 17-35% and pain decreases of
about 26-51% over nonsteroidal antiinflammatory drugs
(NSAIDs) alone. MTX is administered by injection or
orally at doses of 7-15 mg/patient/week for RA, doses
that are considerably below the cancer therapy doses
of 50-60 mg/patient/week. Principal acute toxicities
occur with liver, kidney, and mucosal tissue particularly
in the gastrointestinal tract.10 Liver toxicity is evi-
denced13 by increased levels of SGOT enzyme activity,
but these are of a transient nature even at the high
doses used in cancer therapy. There has been concern
that chronic therapy with MTX in RA could cause liver
damage, but this has not been a significant limitation
in long-term patients.
The ability of MTX to affect the inflammatory condi-
tions of RA may be linked to its cytotoxic behavior. This
is probably in the nature of immune suppression and
could involve attack on inflammatory phagocytic cells
such as macrophages and neutrophils or subsets of
T-helper cells located in the synovial region. Very few
MTX analogues have been evaluated in animal models
of RA, and there is no clear indication whether the
antiarthritic properties are directly proportional to
cytotoxicity or immune suppression.
The antifolate drug methotrexate (MTX) has been
used as an antitumor agent since its approval in 1955,
but in more recent years it has become a prominent if
not favored drug for treatment of RA.7 Several clinical
trials8 have verified the efficacy of MTX for treatment
of advanced and refractory cases of RA. In a summation
of major results seen with MTX in placebo-controlled
clinical trials,9-12 one observes an improvement in
Galivan and co-workers14 showed that adjuvant ar-
thritis and streptococcal cell wall arthritis in rats
responded to doses of MTX relative to those used in man
for treatment of RA. They also found that timing of
† SRI International.
‡ Southern Research Institute.
§ Memorial Sloan-Kettering Cancer Center.
Stanford University Medical Center.
Abstract published in Advance ACS Abstracts, December 15, 1996.
S0022-2623(95)00552-8 CCC: $14.00 © 1997 American Chemical Society

Analogues of Methotrexate in RA. 1
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 371
Sch em e 1
dosing was most important for reduction of inflamma-
tion. MTX and aminopterin were found to inhibit
inflammation, but other antifolates lacking a 2,4-
diaminopyrimidine moiety or a benzoylglutamate side
chain were ineffective. Krumdieck et al.15 have inves-
tigated in patients with RA, the activity of 10-dea-
zaaminopterin (10-DAM), an antitumor drug that was
developed in our laboratories16,17 as part of a series of
10-deaza analogues. In a limited clinical study, the
compound was found to have an effect comparable to
MTX. A comparative study18 of 10-DAM versus MTX
was conducted in a model of spontaneous autoimmune
disease in the MRL/1pr mouse. The compounds also
had similar effects in this model for skin lesion-
proteinuria scores and toxicity.
-30 °C with 2,4-diamino-6-(bromomethyl)pteridine to
yield the 10-carboxypteroate dimethyl ester intermedi-
ate (5Aa ) in 88% yield. The diester was saponified with
1 N NaOH in 2-methoxyethanol at room temperature
followed by careful acidification to pH 5 to precipitate
the diacid 6Aa in 77% yield. Compound 6Aa was then
dissolved in DMSO and the solution heated at 135 °C
for 45 min to effect decarboxylation to the 4-amino-4-
deoxypteroic acid thiophene analogue 7Aa in 84% yield.
Precipitation of the 10-COOH diacids (e.g, 6Aa ) at a pH
lower than 5 frequently caused the decarboxylation
process to proceed with difficulty, possibly because the
free 4-NH2 group may be assisting in the decarboxyla-
tion process. The procedure is based upon our previous
work20 in the 8,10-dideazaaminopterin series where
decarboxylation was achieved with ester cleavage by
prolonged heating with NaCN-DMSO at 180 °C. That
method was erratic and usually produced dark-colored,
impure products.
The thienylpteroic acid intermediate was coupled with
diethyl L-glutamate in the usual manner with activation
of the thienyl carboxylate with isobutyl chloroformate
in DMF to afford the glutamate diester 8Aa in 32% yield
following purification by chromatography on silica gel.
Saponification at room temperature readily yielded the
target 10-deazaaminopterin thienyl analogue 9Aa . The
general procedure was also applied to the synthesis of
the 10-ethyl analogue 9Ab. Alkylation of the anion of
thiophene diester 2A with ethyl iodide afforded the
R-ethyl diester 3A, readily separable from any unreacted
2A by column chromatography on silica gel. Decar-
boxylation of the 10-ethyl-10-COOH intermediate 6Ab
was even more facile, requiring a temperature of only
125 °C for 30 min.
The 3′-pyridyl analogue (9Ba ) of 10-deazaaminopterin
and its 10-ethyl analogue 9Bb were prepared in a
similar fashion. 6-Methylnicotinic acid (1B) was con-
verted to its dianion with LDA, carbonated, and esteri-
fied (HCl-MeOH) to give the (5-carboxy-2-pyridyl)acetic
acid dimethyl ester (2B) in 30% yield after chromatog-
raphy. Alkylation of 2B with ethyl iodide afforded the
R-ethyl diester 3B in 51% yield. Decarboxylation of the
10-COOH diacid 6Ba occurred over a 20 min period at
110 °C, reflecting participation of the adjacent pyridine
nitrogen atom. This assistance rendered by the pyridine
nitrogen was even more evident in decarboxylation of
the R-ethyl 10-COOH diacid 6Bb. The process took
place at room temperature over 20 min in a DMF
solution to give 7Bb in essentially quantitative yield.
As part of an extensive program for synthesis and
evaluation of new analogues of MTX in rheumatoid
arthritis, we report the synthesis and evaluation of some
10-deazaaminopterin compounds featuring replacement
of the benzoate moiety with heteroaroyl units.
Ch em istr y
We have previously reported17 the synthesis of 10-
alkyl-10-deazaaminopterin analogues by an abbreviated
procedure based on condensation of 2,4,5,6-tetraami-
nopyrimidine with an R-bromoaldehyde moiety contain-
ing the benzoate side chain. However, we have since
adopted an alternate process19 as outlined in Schemes
1 and 2. The key steps in the procedure are alkylation
of a suitable (carbomethoxyaryl)acetic acid ester with
2,4-diamino-6-(bromomethyl)pteridine followed by hy-
drolysis to a 10-carboxypteroic acid intermediate and
subsequent facile decarboxylation to the appropriate
pteroic acid. We have found the general method to be
convenient and adaptable to the synthesis of virtually
any 10-deazapterin analogue.
As shown in Scheme 1, 5-methylthiophene-2-carboxy-
lic acid (1A) was converted to its dianion by treatment
with 2 equiv of lithium diisopropylamide (LDA) in dry
THF at -30 °C. The resulting dianion was then treated
with gaseous CO2 to generate the 2-(carboxymethyl)-
thiophene-5-carboxylate salt. Following collection of the
precipitated salt, the crude material was treated directly
with methanolic hydrogen chloride to yield the dimethyl
ester 2A in 51% yield. HPLC of the salt prior to
esterification indicated a mixture 78% diacid and 22%
starting acid (1A). The diester 2A was converted to its
anion with 1 equiv of NaH in DMF and alkylated at

372 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
DeGraw et al.
Sch em e 2
The 2′-pyridyl analogue of 10-deazaaminopterin was
synthesized starting from 5-methylpicolinic acid (1C).
After dianion formation with LDA and carbonation, the
dimethyl ester of (2-carboxy-5-pyridyl)acetic acid (2C)
was obtained in 49% yield via treatment of the diacid
salt with HCl-MeOH. Since there was concern that
the carboxyl on the pyridine ring would also be lost in
the decarboxylation step, we prepared the methyl ben-
zhydryl mixed diester 4C. Selective alkaline hydrolysis
of 2C gave the ester acid 3C which was converted to
4C by esterification with diphenyldiazomethane. Alky-
lation of 4C with 2,4-diamino-6-(bromomethyl)pteridine
to 5C and cleavage of the benzhydryl ester with trif-
luoroacetic acid in MeCl2 afforded the 10-carboxy-2′-
pyridylpteroate ester 6C. Decarboxylation (130 °C in
DMSO) gave the pteroate ester 7C which was saponified
at room temperature to the pyridylpteroic acid analogue
7Ca . Coupling with glutamate and saponification gave
the target aminopterin 9Ca .
of the control mice show acute inflammation by day 37.
The MTX analogues were applied intraperitoneally 2
days before the initial collagen II challenge, and a dosing
schedule of every 2 days was maintained thereafter.
The data presented in Tables 1 and 2 demonstrate
that derivatives of methotrexate are functional in al-
leviating collagen-induced arthritis. The compounds 10-
DAM, 10-Et-10-DAM, and 9Aa ,Ab,Ba ,Ca decreased the
incidence and severity of the arthritis over a variable
range of doses. As shown in Table 1, the 10-ethyl-10-
deazaaminopterin thienyl analogue 9Ab was the most
effective compound in terms of decreased incidence of
disease when compared with MTX. Only 12% of ani-
mals treated with 9Ab showed visual disease even at
day 44, while MTX was clearly showing indications of
inflammation at day 44. Compound 9Ab was also quite
effective at reduction of swelling in the foot pad at 10-
15 mg/kg, while MTX was equally effective at a dose of
9 mg/kg. 10-DAM which has shown clinical efficacy15
was effective in reduction of swelling, but a significant
percentage of animals had visible inflammation. 10-
Et-10-DAM was a little more effective than its thienyl
counterpart 9Ab for reduction of swelling. The 2′-
pyridyl analogue 9Ca was apparently very effective at
a dose of 12 mg/kg, but this was clearly an unacceptably
toxic dose. Unfortunately, the supply of compound and
scheduling limitations did not permit further evaluation
at lower doses. Compound 9Bb was ineffective in
decreasing onset of disease or severity at the test doses.
Biologica l Eva lu a tion
The analogues were evaluated both for efficacy in an
animal model of rheumatoid arthritis and for their
cytotoxicity in cellular systems in vitro. The animal
model selected is the mouse collagen II-induced arthritis
assay described by Courtenay.21 Methotrexate com-
pounds have been assayed previously by the classical
rat adjuvant method. We chose the collagen-induced
assay because it represents an inflammatory process
that more closely resembles the rheumatoid arthritis
condition in humans. In this procedure, DBA/1 mice, a
genetically conserved strain, are injected with bovine
II collagen to induce disease. In our modification of the
Courtenay procedure, a booster dose of the antigen is
administered at day 21. Visual evidence of inflamma-
tion is observed after about day 23, while measurable
swelling is seen by days 23-30. In most runs, 75-100%
We have also measured the general cytotoxicity of the
compounds in cell culture with L1210 cells and a
representative human liver cell line, namely, the Chang
immortalized liver cell.22 The 3′-pyridyl analogues
9Ba ,Bb were substantially less cytotoxic to L1210 cells
than MTX by a factor of 4.4 and 13.4, respectively. Both
were less cytotoxic than MTX versus the Chang cell line.

Analogues of Methotrexate in RA. 1
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 373
Ta ble 1. Effects of 10-Deaza Analogues of Methotrexate on
Incidence of Mouse Type II Collagen Arthritis
Ta ble 3. Cytotoxicity of 10-Deazaaminopterin Analogues in
L1210 and Chang Liver Cellsa
onset and incidence
growth inhibition IC50 (nM)b
of diseasea,b (% animals)
dose
compd
9Aa
9Ab
9Ba
9Bb
10-DAM
MTX
L1210
Chang liver
compd
(mg/kg, ip) day 23 day 30 day 37 day 44
8.1
3.7
88
3.1
1.6
36
untreated
MTX
0
12
9
15
12
9
18
12
18
12
15
10
8
0
0
0
25
12
0
75
14c
0
90
0
25
38
62
50
12
50
25
50
12
38
50
100
88
100
88
75
100
d
268
3.7
35
98
10-DAM
0
38
50
38
0
25
0
0
0
0
38
57
50
25
63
25
38
0c
50
50
38
12
38
38
50
12
25
25
71
63
88
75
75
75
0c
12
25
12
25
0
0
0
0
0
20
14
a
b
See ref 17 for methods. Average of three runs.
10-Et-10-DAMf
9Aa e
9Ab
9Ba
to L1210 and fairly active against the arthritis at the
highest dose tested. The agents that were highly
cytotoxic to L1210 such as 10-DAM, 10-Et-10-DAM, and
9Aa ,Ab were considerably more effective than their less
cytotoxic counterparts 9Ba ,Bb.
4
2
0
0
Acute toxicity measurements were carried out in four
rodent species for compound 9Ab vs MTX. The LD10
values in DBA mice for 9Ab and MTX were >15 and
12 mg/kg and in BD2F mice 12 and 7.5 mg/kg, respec-
tively. In the Lewis rat the comparison was striking
with a value of 15 mg/kg for 9Ab against 1.5 mg/kg for
MTX. In CD rats the ratio was 4.0 mg/kg for 9Ab vs
1.0 mg/kg for MTX. A 90-day semichronic toxicity study
in CD rats showed a maximum tolerated dose of 1.5 mg/
kg for 9Ab and 0.75 mg/kg for MTX. All of these assays
were conducted with intraperitoneal administration.
Except for the Lewis rat case, the toxicity advantages
of 9Ab over MTX are moderate and do not suggest that
a major therapeutic advantage would be expected.
The mechanism of action by which methotrexate
decreases the onset and severity of mouse type II
collagen arthritis is unknown. The disease process in
susceptible mice is characterized by high titers of
anticollagen antibody, and there are reports that the
antibodies include the isotypes IgG1 and IgG2a, both
capable of fixing complement.23-25 In addition, the
collagen arthritic mouse exhibits a T-cell proliferative
response to type II collagen, and a delayed-type hyper-
sensitivity reaction occurs in these animals with a peak
response prior to onset of arthritis.26-28 These observa-
9Bb
12
5
2.5
1.25
12
12
12
0
12
0
9Ca
a
Incidence of disease was scored as the percentage of animals
within each experimental group that exhibited joint or limb
b
inflammation. Untreated animals typically show an incidence of
arthritis of 80-100%. c One animal died in these groups. Three
d
animals died. e Nair and co-workers have also synthesized 9Aa
by a different route and showed it to have antiarthritic proper-
ties.32 f 10-Et-10-DAM was also shown to be effective in a rat
adjuvant model.33
However, the two thienyl analogues 9Aa ,Ab were highly
cytotoxic in both cell lines.
There is a suggestion in this 10-deazaaminopterin
series that cytotoxic potency and antiarthritic activity
are related, but the conclusion is limited because of the
small number of compounds studied. As stated above
the 10-ethyl-3-pyridyl analogue 9Bb was far less cyto-
toxic to L1210 cells and significantly less cytotoxic to
Chang liver calls than MTX. This compound was clearly
ineffective against the collagen-induced arthritis in
terms of incidence of disease and reduction of inflam-
mation. Compound 9Ba was more cytotoxic than 9Bb
Ta ble 2. Antiinflammatory Effects of 10-Deaza Analogues of Methotrexate on Mouse Type II Collagen Arthritis
paw swellingb (mean ( SD, mm)
dosea
compd
(mg/kg, ip)
day 23
day 30
day 37
day 44
untreated
MTX
0
12c
9
15
12
9
18
12
18
12
15
10
8
2.33 ( 0.21
2.27 ( 0.23
2.14 ( 0.04
2.20 ( 0.11
2.21 ( 0.12
2.19 ( 0.13
2.18 ( 0.11
2.26 ( 0.25
2.14 ( 0.03
2.15 ( 0.04
2.15 ( 0.04
2.13 ( 0.04
2.08 ( 0.07
2.08 ( 0.07
2.13 ( 0.07
2.21 ( 0.15
2.15 ( 0.13
2.15 ( 0.08
2.17 ( 0.18
2.14 ( 0.04
2.43 ( 0.16
2.26 ( 0.17
2.16 ( 0.06
2.31 ( 0.11
2.36 ( 0.13
2.32 ( 0.15
2.24 ( 0.08
2.28 ( 0.18
2.16 ( 0.03
2.15 ( 0.05
2.17 ( 0.03
2.15 ( 0.03
2.22 ( 0.11
2.24 ( 0.15
2.35 ( 0.17
2.20 ( 0.11
2.32 ( 0.15
2.18 ( 0.09
2.24 ( 0.12
2.11 ( 0.05
3.09 ( 0.91
2.28 ( 0.13
2.18 ( 0.02
2.29 ( 0.15
2.31 ( 0.10
2.28 ( 0.14
2.19 ( 0.12
2.34 ( 0.16
2.26 ( 0.17
2.28 ( 0.18
2.21 ( 0.14
2.23 ( 0.17
2.20 ( 0.13
2.57 ( 0.65
2.67 ( 0.69
2.59 ( 0.45
2.56 ( 0.44
2.75 ( 0.87
2.47 ( 0.41
2.07 ( 0.06
3.00 ( 0.54
2.22 ( 0.09
2.24 ( 0.13
2.30 ( 0.18
2.33 ( 0.13
2.32 ( 0.13
2.18 ( 0.16
2.32 ( 0.27
2.38 ( 0.55
2.58 ( 0.74
2.26 ( 0.23
2.25 ( 0.14
2.33 ( 0.52
2.68 ( 0.52
2.88 ( 0.63
2.86 ( 0.92
3.09 ( 0.72
2.75 ( 0.69
2.61 ( 0.56
d
10-DAM
10-Et-10-DAM
9Aa
9Ab
9Ba
4
2
9Bb
9Ca
12
5
2.25
1.25
12
a
Each dose tested consisted of eight animals; the untreated controls represent the average of four experiments for a total of 48 animals.
Paw swelling is expressed as the mean ( standard deviation of hind paw dimensions (mm) for both limbs of all animals in each group.
b
Hind paw dimensions average 2.13 ( 0.09 mm in the absence of collagen challenge; arthritic animals typically exhibit hind paw dimensions
greater than 2.60 ( 0.50 mm. c One death was observed in this group. A dose of 9 mg/kg was adopted as a standard for MTX; lower doses
d
were not as uniformly effective. Three animals died.

374 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
DeGraw et al.
24.8 mmol) in MeOH (14 mL). After 2 h, the solution was
adjusted to pH 6.5 by addition of 2 N HCl. The solution was
concentrated in vacuo to give a tan solid that was a mixture
of both monoesters, the dicarboxylic acid and the starting
diester. HPLC indicated the desired monoester 3C to repre-
sent 57% of the mixture.
tions raise the possibility that the mode of action of
methotrexate and the methotrexate analogues may be
through selective inhibition of cell proliferation. Al-
though our studies do not directly address the mecha-
nism of action of methotrexate, the differential response
of the disease to unique structures of the methotrexate
analogues may provide insights into mechanisms un-
derlying drug effects on disease progression.29,30
This study indicates that modification of the MTX
structure can provide compounds that are as effective
if not more so than MTX against rheumatoid arthritis.
It further suggests that an antiproliferative effect is
paramount in the mechanism of action. Since the target
cell(s) has not been identified, we cannot yet conduct a
detailed structure-activity study that would allow
determination of specific cytotoxicity and influx/efflux
parameters vs sensitive host cells as we have conducted
for cancer cell types. However, acute and subchronic
toxicity studies in rodents for 9Ab do suggest a modest
therapeutic advantage for this compound over MTX. At
this stage we can conclude that the thiophene moiety
offers potential as a side chain substituent for develop-
ment of additional 10-deazaaminopterin-based agents.
The mixture in CHCl3 (100 mL) was cooled to 0° and treated
dropwise with a solution of diphenyldiazomethane (5.27 g, 27.2
mmol) in CHCl3 (50 mL). The resulting purple mixture was
stirred at ambient temperature for 24 h. The solution was
washed with 25 mL of saturated NaHCO3 and 25 mL of H2O.
The organic layer was dried over MgSO4 and concentrated to
a purple syrup. Crystallization from pentane gave the product
as a white solid: 1.86 g (21% yield from 2C); mp 90-92 °C
NMR (CDCl3) δ 8.68 (m, 1H, C3-H), 8.10 (d, 1H, C6-H), 7.75
(m, 1H, C4-H), 7.30 (m, 10H, 2 × C6H5), 6.90 (s, 1H, OCH),
4.05 (s, 3H, OCH3), 3.81 (s, 2H, CH2). Anal. (C22H19
NO4‚0.25H2O) C, H, N.
-
r-Eth yl-2-ca r boxyth iop h en e-5-a cetic Acid Dim eth yl
Ester (3A). A suspension of 0.59 g of 50% sodium hydride in
oil (12.2 mmol of sodium hydride) in 20 mL of dry dimethyl-
formamide was cooled to 0 °C. A solution of 2A (2.60 g, 12.2
mmol) in 20 mL of dry dimethylformamide was added, and
the reaction mixture was stirred for an additional 1 h at 0 °C.
The reaction mixture was cooled to -30 °C, treated dropwise
with a solution of ethyl iodide (1.9 g, 12.2 mmol) in dry
dimethylformamide (10 mL), and then stirred for 2.5 h at 20
°C. The solution was neutralized (pH 8) by adding solid carbon
dioxide, and then concentrated under high vacuum. The
residue was digested in ether (250 mL) and filtered. The
filtrate was washed with 100 mL of H2O and then 50 mL of
10% sodium bisulfite and 100 mL of H2O. The organic layer
was dried over MgSO4 and evaporated in vacuo. The residue
was chromatographed on flash silica gel (EtOAc-hexane, 1:19)
to yield the product as a clear, colorless oil: 1.7 g (58%); TLC
(10% ethyl acetate in hexane on silica gel plate) Rf 0.35; NMR
(CDCl3) δ 7.59 (d, 1H, Ar 3-H), 7.20 (d, 1H, Ar 4-H), 3.81 (m,
7H, 2 × OCH3 + ArCH), 2.06 (m, 2H, CH2CH3), 0.95 (t, 3H,
CH3). Anal. (C11H14O4S) C, H.
Exp er im en ta l Section
Elemental analyses were obtained from Galbraith Labora-
tories, Knoxville, TN, and Atlantic Microlab, Norcross, GA.
Values were within 0.4% of theory unless noted in parentheses.
Mass spectra were run on an LKB 9000 GC-MS spectrometer
or a Ribermag R10-10C MS system. Ultraviolet spectra were
taken on a Perkin-Elmer 552 or Perkin-Elmer-Coleman 575
instrument. 1H-NMR spectra were recorded on a Varian
Gemini 300 spectrometer. Melting points were determined on
a Thomas-Hoover Unimelt apparatus. HPLC analyses were
conducted on a Spectra Physics 8100 XR unit using a Novapak
C18 column with elution by 25% MeOH in 0.1 M NaH2PO4
buffer, pH 6.5, with detection at 254 nm.
2-Car boxyth ioph en e-5-acetic Acid Dim eth yl Ester (2A).
Freshly distilled diisopropylamine (24.6 g, 0.24 mol) in 250
mL of dry THF was cooled to 0 °C under argon and then
treated dropwise with 98 mL (0.24 mol) of 2.5 M BuLi in
hexane. After 1 h, the LDA solution was added dropwise with
stirring to a -30 °C mixture of 15.0 g (0.11 mol) of 5-meth-
ylthiophene-2-carboxylic acid (1A) in 300 mL of dry THF. The
temperature of the resulting red solution was allowed to rise
to 0 °C and was so maintained for another 2 h. Carbon dioxide
was bubbled through the solution to produce a yellow precipi-
tate. The mixture was stirred at ambient temperature for 2
h and filtered. The yellow filter cake was suspended in 300
mL of MeOH, and the mixture was cooled to 0 °C and treated
with 100 mL of MeOH saturated with dry HCl. The mixture
was stirred at room temperature for 72 h and concentrated in
vacuo, and the residue was partitioned between Et2O (500 mL)
and 250 mL of saturated NaHCO3. The Et2O extract was
washed with H2O (3 × 250 mL), dried over MgSO4, and
evaporated to leave a dark oil (15 g). Chromatography on flash
silica gel (EtOAc-hexane, 1:19) gave 11.4 g of the product
(51%) as a white, waxy solid: NMR (CDCl3) δ 7.61 (d, 1H, 3-H),
6.90 (d, 1H, 4-H), 3.87 (m, 5H, ArCOOCH3 + CH2), 3.82 (s,
3H, CH2COOCH3). Anal. (C9H10O4S) C, H, N.
r-Eth yl(3-ca r boxy-6-p yr id yl)a cetic a cid d im eth yl es-
ter (3B): similarly prepared from 3-carboxypyridine-6-acetic
acid dimethyl ester (2B) in 51% yield as a yellow oil; NMR
(CDCl3) δ 9.13 (m, 1H, 6-H), 8.26 (m, 1H, 4-H), 7.39 (m, 1H,
3-H), 3.83 (m, 7H, 2 × OCH3 + R-CH), 2.10 (m, 2H, CH2CH3),
0.87 (t, 3H, CH3CH2). Anal. (C12H15NO4S) C, H, N.
Met h yl 5-[r-Ca r b om et h oxy-â-(2,4-d ia m in o-6-p t er id i-
n yl)eth yl]th iop h en e-2-ca r boxyla te (5Aa ). A suspension of
50% sodium hydride in oil (0.84 g, 17.5 mmol of sodium
hydride) in 15 mL of dry dimethylformamide was cooled to 0
°C. A solution of the diester 2A (3.73 g, 17.4 mmol) in 15 mL
of dry dimethylformamide was added dropwise. The resulting
mixture was stirred at 0 °C for 1 h and then cooled to -30 °C
and treated with a solution of 2,4-diamino-6-(bromomethyl)-
pteridine hydrobromide (16.1 mmol) in 40 mL of dry dimeth-
ylformamide. The resulting mixture was stirred for 2.5 h while
rising to room temperature and then neutralized (pH 7.5) by
adding solid carbon dioxide. The mixture was concentrated
under high vacuum, and the residue was washed with ether
and then water and dried under high vacuum to give the
product as a yellow solid (1.98 g, 88%): MS 389 (M + H); NMR
(DMSO-d6) δ 8.58 (s, 1H, C7-H), 7.60 (m, 3H, C3′-H + NH2),
7.12 (d, 1H, C4′-H), 6.61 (br s, 2H, NH2), 4.9 (t, 1H, C10-H),
3.75 (s, 3H, C2′-COOCH3), 3.63 (m, 5H, C10-COOCH3 + C9-
H2). Anal. (C16H16N6O4S‚0.5H2O) C, H, N.
3-Ca r boxyp yr id in e-6-a cetic a cid d im eth yl ester (2B):
This diester was similarly prepared from 6-methylnicotinic
acid (1B) as a yellow solid; mp 56-57 °C; NMR (CDCl3) δ 9.10
(m, 1H, 6-H), 8.21 (m, 1H, 4-H), 7.33 (m, 1H, 3-H), 3.84 (m,
8H, CH2COOCH3, ArCOOCH3). Anal. (C10H11NO4) C, H, N.
(2-Ca r boxy-5-p yr id yl)a cetic a cid d im eth yl ester (2C):
prepared in a manner similar to 2A from 5-methylpicolinic
acid31 giving the product as an amber oil in 49% yield; NMR
(CDCl3) δ 8.63 (d, 1H, C3-H), 8.15 (d, 1H, C6-H), 7.81 (m, 1H,
C4-H), 4.02 (s, 3H ArCOOCH3), 3.75 (s, 5H, CH2COOCH3).
(2-Ca r bom eth oxy-5-p yr id yl)a cetic Acid Ben zh yd r yl
Ester (4C). A solution of KOH (1.39 g, 24.8 mmol) in 90%
MeOH (100 mL) was treated with a solution of 2C (5.18 g,
Meth yl 5-[r-ca r bom eth oxy-r-[(2,4-d ia m in o-6-p ter id i-
n yl)m eth yl]p r op yl]th iop h en e-2-ca r boxyla te (5Ab): simi-
larly prepared from 3A in 85% yield as a yellow solid; NMR
(DMSO-d6) δ 8.35 (s, 1H, C7-H), 7.78 (br s, 1H, NH), 7.65 (d,
1H, C3′-H), 7.17 (d, 1H, C4′-H), 6.65 (br s, 2H, NH2), 6.52 (br
s, 1H, NH), 3.77 (s, ArCOOCH3), 3.68 (s, CCOOCH3), 2.06
(m, 2H, CH2CH3), 0.76 (t, 3H, CH3CH2); MS m/ e 416 (M +
H). Anal. (C18H20N6O4S) C, H, N.
Meth yl 6-[r-car bom eth oxy-â-(2,4-diam in o-6-pter idin yl)-
eth yl]p yr id in e-3-ca r boxyla te (5Ba ): similarly prepared
from 2B in 99% yield as a yellow solid; NMR (DMSO-d6) δ
9.00 (m, 1H, C7-H), 8.53 (s, 1H, C2′-H), 8.22 (m, 1H, C4′-H),
7.55 (m, 3H, C5′-H + NH2), 6.55 (br s, 2H, NH2), 4.92 (t, 1H,

Analogues of Methotrexate in RA. 1
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3 375
C10-H), 3.85 (s, 3H, COOCH3), 3.75 (m, 4H C9H + COOCH3),
3.38 (m, C9H), Anal. (C17H17N7O4‚1.7H2O) C, H (0.75), N.
Meth yl 6-[r-ca r bom eth oxy-r-[(2,4-d ia m in o-6-p ter id i-
n yl)m eth yl]p r op yl]p yr id in e-3-ca r boxyla te (5Bb): simi-
larly obtained from 3B in 78% yield as a yellow solid; MS m/ e
412 (M + H); NMR (DMSO-d6) δ 9.04 (s, 1H, C7-H), 8.23 (m,
2H, pyr 2′-H + pyr 4′-H), 7.45 (d, 1H, pyr 5′-H), 6.62 (br s, 2H,
A solution of the monocarboxylic acid 6C (0.99 g, crude) in
40 mL of dimethyl sulfoxide was stirred at 130° for 30 min.
HPLC showed disappearance of the starting carboxylic acid
6C (retention time 4.4 min) and the desired monoester to be
present (retention time 15.2 min). The solution was concen-
trated under high vacuum, and the residue was washed with
ether and water. The orange solid was collected and dried in
vacuo at room temperature to afford 505 mg (64%): NMR
(CDCl3) δ 8.60 (m, 2H, C7-H, 6′-H), 8.10 (d, 1H, 3′-H), 7.85 (d,
1H, 5′-H), 7.20 (m, 3H, NH2), 4.00 (s, 3H, OCH3), 3.35 (s, 4H,
CH2CH2); MS m/ e 325 (M + H).
5-[â-(2,4-Dia m in o-6-p t er id in yl)et h yl]p yr id in e-2-ca r -
boxylic Acid (7Ca ). A mixture of the ester 7C (0.49 g, 1.5
mmol) in 2-methoxyethanol (5 mL) was treated with water (5
mL) and then 10% NaOH (2.5 mL). HPLC analysis indicated
saponification was complete after the resulting red solution
had been stirred for 45 min. The solution was neutralized (pH
7.5) with 2 N HCl and concentrated under high vacuum. The
resulting residue was treated with water and stirred. Filtra-
tion gave 0.27 g of product as an orange solid (57%): HPLC
showed 96% purity; MS m/ e 527 (TMS3). Anal. (C14H13N7O2‚0.
8HCl) C, H, N.
Dieth yl N-5-[â-(2,4-Dia m in o-6-p ter id in yl)eth yl]-2-th en -
oyl]-L-glu ta m a te (8Aa ). A solution of the carboxylic acid 7Aa
(0.7 g, 2.2 mmol) in dry DMF (40 mL) was treated with
triethylamine (2.1 g, 21.0 mmol) and stirred at room temper-
ature for 1.25 h. Isobutyl chloroformate (0.63 g, 4.6 mmol)
was added, and the mixture was stirred at room temperature
for 2 h. Isobutyl chloroformate (0.32 g, 2.3 mmol) was again
added, and the mixture was stirred for 1 h followed by addition
of LL-glutamic acid diethyl ester hydrochloride (0.55 g, 2.3
mmol); stirring was continued for another 1 h. The process
was repeated again, and the mixture was stirred at room
temperature overnight. Concentration under high vacuum
gave a dark residue that was washed repeatedly with Et2O.
The residue was then washed with dilute NH4OH, and then
H2O. The resultant orange solid was dried in vacuo. Chro-
matography on flash silica gel (2.5% MeOH in CHCl3) gave
the product as a yellow powder: 0.32 g (32%); mp 206-208
°C; NMR (DMSO-d6 + CDCl3) δ 8.5 (s, 1H, C7-H), 8.31 (d, 1H,
NHC), 7.6 (d, 1H, 3′-H), 6.80 (d, 1H, 4′-H), 6.32 (br s, 2H, NH2),
4.54 (m, 1H, CHNH), 4.18 (m, 4H, 2 × OCH2), 3.28 (m, C9-
H2); 2.42 (t, 2H, Glu C4-H2); 2.13 (m, 2H, Glu C3-H2); 1.28 (m,
6H, 2 × CH3CH2); MS m/ e 502 (M + H). Anal. (C22H27N7O5S‚
H2O) C, H, N.
NH2), 3.87 (s, 3H, ArCOOCH3), 3.62 (m, 5H, C10-COOCH3
+
C9-H2); 2.01 (m, 2H, CH2CH3), 0.80 (t, 3H, CH3CH2). Anal.
(C19H21N7O4‚1.5H2O) C, H, N.
Meth yl 5-[r-[(ben zh yd r yloxy)ca r bon yl]-â-(2,4-dia m in o-
6-p ter id in yl)eth yl]p yr id in e-2-ca r boxyla te (5C): prepared
in a similar manner from 4C in 75% yield as a yellow powder;
NMR (CDCl3) δ 8.80 (m, 1H, C7-H), 8.62 (s, 1H, C3′-H), 8.10
(d, 1H, C6′-H), 7.84 (m, 1H, C4′-H), 7.20 (m, 12H, 2 × C6H5
+
NH2), 6.80 (s, 1H, OCH), 5.20 (br s, 2H, NH2), 4.55 (m, 1H,
C10-H), 4.02 (s, 3H, OCH3), 3.85 (m, 1H, C9-H), 3.30 (m, 1H,
C9-H).
5-[r-Car boxy-â-(2,4-diam in opter idin yl)eth yl]th ioph en e-
2-ca r boxylic Acid (6Aa ). A solution of the diester 5Aa (1.96
g, 5.05 mmol) in 30 mL of 2-methoxyethanol and 30 mL of 2.5
N NaOH was stirred for 1.5 h. The mixture was filtered, and
the filtrate was neutralized (pH 7) with HOAc and concen-
trated under high vacuum. The residue was suspended in
water (30 mL) and adjusted with HOAc to pH 5 to produce a
precipitate. Filtration gave a tan solid that was digested in
95% EtOH and filtered to give a tan solid that was washed
with Et2O and dried in vacuo, yielding 1.31 g (77%) of
product: HPLC showed 92.2% purity; NMR (DMSO-d6) δ 8.51
(s, 1H, C7-H), 7.55 (br s, 2H, NH2), 7.17 (d, 1H, 3′-H), 6.81 (d,
1H, 4′-H), 6.55 (br s, 2H, NH2), 4.40 (t, 1H, C10-H), 3.15 (m,
2H, C9-H2).
5-[â-(2,4-Dia m in o-6-p ter id in yl)eth yl]th iop h en e-2-ca r -
boxylic Acid (7Aa ). A solution of the dicarboxylic acid 6Aa
(1.31 g, 3.64 mmol) in argon-purged DMSO was placed in a
135 °C oil bath for 45 min. The solution was then concentrated
under high vacuum to a residue that was digested in Et2O
(50 mL). Filtration yielded a brown solid that was washed
with ether and dried in vacuo to give 1.31 g of crude product.
The material was suspended in water (75 mL) and treated
dropwise with 1.5 N NH4OH to pH 12. Insoluble material was
removed by filtration and the filtrate adjusted to pH 5 with
HOAc to give a precipitate. Filtration gave a brown solid that
was washed with H2O and dried in vacuo, yielding 0.97 g of
product (84%): UV (0.1 N NaOH) 257 nm (ꢀ 25 305), 372
(6491). Anal. (C13H12N6O2S‚H2O) C, H, N.
5-[r-[(2,4-Diam in o-6-pter idin yl)m eth yl]pr opyl]-2-th en o-
ic Acid (7Ab). The diester 5Ab was saponified via the above
procedure used to prepare 6Aa to yield (58%) 6Ab as a cream-
colored solid: HPLC 97% purity. The material was im-
mediately decarboxylated by heating a solution in DMSO at
125° for 30 min to afford the R-ethylpteroate analogue 7Ab in
70% yield: UV (0.1 N NaOH) 256 nm (ꢀ 28 546), 372 (7300);
MS (DCl-NH3) m/ e 561 (TMS3) corresponds to 345 (M + 1).
Anal. (C15H16N6O2S‚0.6H2O) C, H, N.
6-[â-(2,4-Dia m in o-6-p t er id in yl)et h yl]p yr id in e-3-ca r -
boxylic Acid (7Ba ). Saponification of the diester 5Ba
afforded the diacid 6Ba in 97% yield; HPLC showed 95.3%
purity. The material was directly decarboxylated by heating
a solution in DMSO at 110 °C for 25 min to give 7Ba in 94%
yield as a yellow solid: MS m/ e 527 (TMS3) corresponds to
311 (M + 1). Anal. (C14H13N7O2‚2H2O) C (0.6), H, N.
6-[r-[(2,4-Diam in o-6-pter idin yl)m eth yl]pr opyl]pyr idin e-
3-ca r boxylic Acid (7Bb). Similar hydrolysis of the diester
5Bb gave the diacid 6Bb in 27% yield as a tan solid.
Decarboxylation was effected by allowing a solution in DMF
to stand at room temperature for 20 min to afford 7Bb in 99%
yield; HPLC showed 90% purity.
The other L-glutamate diesters were similarly prepared from
the corresponding “pteroic acid” intermediates.
Dieth yl N-[5-[r-[(2,4-d ia m in o-6-p ter id in yl)m eth yl]p r o-
p yl]-2-th en oyl]-L-glu ta m a te (8Ab): 20% yield from 7Ab,
yellow foam; NMR (CDCl3) δ 0.90 (t, 3H, C10-CH2-CH3), 1.30
(m, 6H, 2 × OCH2CH3), 2.17 (m, 2H, Glu C3-H2); 2.47 (m, 2H,
Glu C4-H2), 3.20 (m, 3H, C9-H2 + C10-H), 4.16 (m, 4H, 2 ×
OCH2), 4.75 (m, 1H, CHNH), 5.45 (br s, NH), 6.55 (m, 1H, C4′-
H), 6.95 (m, 1H, NHCH), 7.30 (d, 1H, C3′-H), 8.41 (d, 1H, C7H).
Anal. (C24H31N7O5S‚0.7H2O) C, H, N.
Dieth yl N-[6-[â-(2,4-d ia m in o-6-p ter id in yl)eth yl]n ico-
tin oyl]-L-glu ta m a te (8Ba ): 50% yield from 7Ba , yellow
crystals from EtOH; MS m/ e 497 (M + H); NMR (DMSO-d6)
δ 8.90 (d, 1H, NHCO), 8.87 (d, 1H, pyr 6′-H), 8.61 (s, 1H, C7-
H), 8.10 (m, 1H, pyr 4′-H), 7.70 (br d, 1H, NH), 7.42 (d, 1H,
pyr 3′-H), 6.65 (br s, 2H, NH2), 4.40 (m, 1H, CHN), 4.05 (m,
4H, 2 × OCH2), 3.30 (CH2CH2 + H2O), 2.45 (t, 2H, CH2CO2),
2.05 (m, 2H, CH2CH), 1.70 (t, 6H, 2 × CH3). Anal. (C23H28N8O5‚
H2O) C, H, N.
Dieth yl N-[6-[r-[(2,4-d ia m in o-6-p ter id in yl)m eth yl]p r o-
p yl]n icotin oyl]-L-glu ta m a te (8Bb): 48% from 7Bb, yellow
foam; MS m/ e 525 (M + H); NMR (CDCl3) δ 9.01 (br s, 1H,
pyr 6′-H), 8.45 (br s, 1H, 7-H), 7.97 (d, 1H, pyr 4′-H), 7.35 (br
s, 2H, NH2), 7.08 (d, 1H, pyr 3′-H), 5.38 (br s, 2H, NH2), 4.75
Meth yl 5-[â-(2,4-Dia m in o-6-p ter id in yl)eth yl]p yr id in e-
2-ca r boxyla te (7C). A mixture of the diester 5C (1.29 g, 2.4
mmol) in dichloromethane (67 mL) was treated with 99%
trifluoroacetic acid (33 mL). The yellow solution was kept at
room temperature for 50 min and then concentrated at room
temperature under high vacuum. The residue was washed
repeatedly with ether and then dried in vacuo giving a bright
yellow solid. This was suspended in water and neutralized to
pH 8 with 1.5 N ammonium hydroxide. The mixture was
concentrated under high vacuum giving a yellow solid, 0.99 g.
HPLC showed the conversion to 6C.
(m, 1H, CHN), 4.19 (m, 4H, 2 × OCH2), 3.32 (m, 3H, C9-H2
+
C10-H), 2.50 (m, 2H, C10-CH2-CH3), 2.23 (m, 4H, Glu C4-H2
+
Glu C3-H2); 1.26 (m, 6H, 2 × OCH2CH3), 0.83 (t, 3H, C10
CH2CH3).
-
Dieth yl N-[5-[â-(2,4-d ia m in o-6-p ter id in yl)eth yl]p icoli-
n oyl]-L-glu ta m a te (8Ca ): 18% from 7Ca , yellow foam; MS
m/ e 497 (M + H); NMR (CDCl3) δ 8.60 (d, 1H, C7-H), 8.55 (d,
1H, NH), 8.43 (d, 1H, C5′-H), 8.06 (d, 1H, C2-H), 7.70 (m, 1H,
C6′-H), 4.80 (m, 1H, CHNH), 4.20 (m, 4H, 2 × OCH2), 2.30 (m,

376 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 3
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4H, Glu CH2CH2), 1.30 (m, 6H, 2 × OCH2CH3). Anal.
(C23H28N8O5) C, H, N.
N-[5-[â-(2,4-Dia m in o-6-p t er id in yl)et h yl]-2-t h en oyl]-L-
glu ta m ic Acid (9Aa ). A mixture of the diester 8Aa (0.26 g,
0.5 mmol) in 2-methoxyethanol (5 mL) was treated with water
(5 mL) and 10% NaOH (5 mL). The mixture was stirred for 1
h and then adjusted to pH 5.5 with 2 N HCl and concentrated
under high vacuum. The residue was digested in H2O (5 mL),
and the mixture was filtered. The resulting solid was washed
with H2O and dried in vacuo to afford 0.19 g of product (82%):
HPLC showed 96.4% purity; UV (0.1 N NaOH) 258 nm (ꢀ
28 310), 372 (6737); NMR (DMSO-d6) δ 8.67 (s, 1H, C7-H), 8.50
(d, 1H, NHCH), 8.00 (br s, 2H, NH2), 7.65 (d, 1H, 3′-H), 6.90
(br s, 3H, 4′-H + NH2), 4.30 (m, 1H, CHNH), 3.42 (m, C9-H2 +
C10-H2); 2.35 (t, 2H, Glu C4-H2); 1.95 (m, 2H, Glu C3-H2); MS
(DCl-NH3) m/ e 734 (TMS4) (M + H). Anal. (C18H19N7O5S‚-
2H2O) C, H, N.
The other aminopterin analogues were similarly obtained
by saponification of the glutamate esters.
N-[5-[r-[(2,4-Dia m in o-6-p t er id in yl)m et h yl]p r op yl]-2-
th en oyl]-L-glu ta m ic a cid (9Ab): 57% yield from 8Ab, yellow
solid; HPLC 97% purity; UV (0.1 N NaOH) 256 nm (ꢀ 28 139),
371 (6810); MS (DCl-NH3) m/ e 762 for TMS4 (M + H). Anal.
(C20H23N7O5S‚0.75 HCl) C, H, N.
N-[6-[â-(2,4-Dia m in o-6-p ter id in yl)eth yl]n icotin oyl]-L-
glu ta m ic a cid (3′-a za -10-d ea za a m in op ter in ) (9Ba ): 71%
yield from 8Ba , yellow powder; HPLC 95% purity; UV (0.1 N
NaOH) 258 nm (ꢀ 25 000), 275 sh (13 900), 371 (6600); MS
(DCl-NH3) m/ e 729 (TMS4) (M + H). Anal. (C19H20N8O5‚
2.25H2O) C, H (0.5), N.
N-[6-[r-[(2,4-Dia m in o-6-p t er id in yl)m et h yl]p r op yl]n i-
cotin oyl]-L-glu ta m ic a cid (3′-a za -10-eth yl-10-d ea za a m i-
n op ter in ) (9Bb): 39% from 8Bb, yellow powder; HPLC 98.9%
purity; UV (0.1 N NaOH) 256 nm (ꢀ 25 246), 367 (6562). Anal.
(C21H24N8O5‚1.4 H2O) C, H, N.
N-[5-[â-(2,4-Dia m in o-6-p ter id in yl)eth yl]p icolin oyl]-L-
glu ta m ic a cid (2′-a za -10-d ea za a m in op ter in ) (9Ca ): 58%
yield from 8Ca , yellow solid; HPLC 99.3% purity; UV (0.1 N
NaOH) 257 nm (ꢀ 24 000), 371 (6070); MS (DCl-NH3) m/ e
729 (TMS4) (M + H). Anal. (C19H20N8O5‚0.25H2O) C, H, N.
Ack n ow led gm en t. We are indebted to Dr. David
Thomas for mass spectrometric analyses. This work
was performed under the sponsorship of Mitsubishi
Rayon Co.
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