The synthesis of novel methotrexate-like compounds

Article abstract of DOI:10.1007/s00706-007-0716-0

Methotrexate (MTX) is a folate antagonist used in treatment of several chronic inflammatory and neoplastic conditions. In this study, new MTX-like compounds that may-be potential anticancer agents were synthesized and their structures were determined by IR, UV, GC-MS, ¹H NMR, and ¹³C NMR spectra. Also, in this study, a series structurally related to MTX or folate analogous compounds were evaluated whether they have inhibitory properties on the dihydrofolate reductase activity (DHFR).

Full text of DOI:10.1007/s00706-007-0716-0

Monatshefte fu¨r Chemie 138, 1283–1287 (2007)
DOI 10.1007/s00706-007-0716-0
Printed in The Netherlands
The Synthesis of Novel Methotrexate-like Compounds
Naki C°
olak1, Yılmaz Yıldırır1;ꢀ, Mustafa Kavutcu2, and Nilhan Nurlu2
1
2
Department of Chemistry, Faculty of Science and Arts, Gazi University, Ankara, Turkey
Department of Medical Biochemistry, Faculty of Medicine, Gazi University, Ankara, Turkey
Received April 18, 2007; accepted (revised) May 15, 2007; published online September 24, 2007
# Springer-Verlag 2007
High doses (>3 g=m2) have been prescribed as a cy-
Summary. Methotrexate (MTX) is a folate antagonist used
in treatment of several chronic inflammatory and neoplastic
conditions. In this study, new MTX-like compounds that may-
be potential anticancer agents were synthesized and their
tostatic agent in oncology since 1953 [5]. Low doses of
methotrexate (5–25mg=m2 per week) have been used
increasingly for the treatment of patients with rheu-
matoid arthritis (RA) [6, 7], or psoriasis [8–12] and
steroid-dependent asthma [13–15]. At the same time
MTX is widely utilized in the treatment of acute leu-
kemia, carcinoma of the breast, osteosarcoma, ovari-
an cancer, and other malignant diseases [16].
1
structures were determined by IR, UV, GC-MS, H NMR,
and 13C NMR spectra. Also, in this study, a series structurally
related to MTX or folate analogous compounds were evaluated
whether they have inhibitory properties on the dihydrofolate
reductase activity (DHFR).
Keywords. Methotrexate; p-Aminobenzoic acid; DMAP;
DCC; Amino acid esters.
Introduction
Methotrexate (MTX; 2,4-diamino-N10-methylpteroyl-
glutamic acid; I) is a small molecule inhibitor of the
ubiquitous enzyme dihydrofolate reductase (DHFR),
which is a requisite component of the biosynthesis
pathway for purine and pyrimidine. MTX is a classi-
cal type of inhibitor of dihydrofolate reductase and
is known to be metabolized to polyglutamyl forms
after its incorporation into cells [1]. MTX is also a
potent folic acid antagonist which inhibits the reduc-
tion of folic acid to tetrahydrofolic acid, thereby
leading to the suppression of the nucleic acid bio-
synthesis pathway, and cell death. It has been used in
the treatment of neoplastic disorders since the late
1940s [2, 3]. As a consequence, MTX is used as a
drug for the treatment of a wide range of diseases,
e.g., for cancer treatment since its development and
the identification of its biological activities [4].
Therapeutic drug monitoring is of importance in
order to improve MTX–RA therapy because of the
severe side-effects of MTX, e.g., marrow suppres-
sion, gastro-intestinal lesion, renal insufficiency, he-
patic failure, hypoalbuminemia, and pancytopenia
[17, 18].
MTX is known for its severe serious side effects
although it is used in a wide range in medicine. Be-
cause of these shortcomings the synthesis of MTX-like
compounds is pursued. Aim of this work is the synthe-
sis of compounds which can be used instead of MTX
and having less side effects than MTX. Therefore we
use some aromatic aldehydes instead of the pterin ring,
but did not alter the PABA and glutamic acid parts.
Then we use ꢀ-alanin instead of glutamic acid.
ꢀ
Corresponding author. E-mail: yildirir@gazi.edu.tr

1284
N. C°olak et al.
Scheme 1
Results and Discussion
(mean), were the most potent inhibitors among the
compounds.
Compounds 3a–3j were synthesized as depicted in
Scheme 1. The ꢁCOOH functionalized imines 1
were reacted with amino acids (ꢀ-alanine and L-
glutamic acid) with DCC and DMAP as coupling
agent.
Experimental
Melting points were measured on a Gallenkamp apparatus
using a capillary tube. Infrared absorption spectra were re-
corded from a Mattson 1000-FTIR spectrometer, using KBr
pellets. 1H NMR and 13C NMR spectra were obtained with a
Bruker DPX FT-NMR (300 MHz) spectrometer. The chemical
schifts are reported in ꢁ=ppm with external TMS. Mass spec-
tra were determined on a Thermo Finnigan Trace DSQ. The
elemental analyses were performed on a LECO CHNS-932 C,
H, N analyzer. All were found to agree favourably with
the calculated values. All reagents were of analytical grade.
Salicylaldehyde, 2-hydroxy-4-methylbenzaldehyde, thio-
phene-2-carbaldehyde, pyrrole-2-carbaldehyde, 2-hydroxy-1-
naphtaldehyde, 4-aminobenzoic acid, glycine, ꢀ-alanine,
L-glutamic acid, dichloromethane, thionyl chloride, 4-
(dimethylamino)pyridin, and N,N0-dicyclohexylcarbodiimid
were purchased from Aldrich Chemical Co. Ethanol, metha-
nol, and CH2Cl2 were purchased from Merck (Germany).
Dichloromethane was distilled from P2O5 before use. Amino
acid esters were prepared according to published procedures
[19, 20]. An improved preparation of these two esters is
reported below.
The IR spectra of these compounds show the C¼N
bond stretching vibration in the 1625–1635cmꢁ1 and
the Ar-H bond stretching vibration in the 3101–
3037 cmꢁ1 region and the strong sharp absorption
bands of the amide (NH) appeared at 3307–
1
3435 cmꢁ1. The H NMR spectra of the compounds
1
were recorded in DMF-d7. The H NMR assign-
ments are detailed in the following. The imine pro-
tons for compounds 3a–3j are observed as singlets
of 9.08, 9.00, 9.08, 8.60, 9.88, 9.26, 9.00, 9.08, 8.34,
and 9.87 ppm.
Also this series of compounds was evaluated wheth-
er they have inhibitory properties on the dihydrofolate
reductase activity (DHFR).
The result of screening regarding the effect of
methotrexate related compounds on DHFR activ-
ity is shown in Table 1, listing the compounds in
their rank order of potency as inhibitors of DHFR
activity. As shown in Table 1, compounds 3g, 3e,
3j, and 3d, with inhibitions of 84, 83, and 82%
ꢀ-Alanine Methyl Ester
Thionyl chloride (16.7 g, 0.14 mol) was added dropwise to a
suspension of 8.91g ꢀ-alanine (0.10mol) in 75 cm3 methanol
at0ꢂC. The mixturewas stirred atroomtemperaturefor 18 h and
then concentrated using a rotary evaporator. The residual oil
occurred was triturated with cold ether and ꢀ-alanine methyl
ester precipitated as a white powder. The powder was air-dried
12.38 g (89%), mp 103–104ꢂC (Ref. [21] 103–104ꢂC).
Table 1. Maximal inhibition of DHFR by selected com-
pounds 3a–3j related to methotrexate
Test substance
Maximal inhibition in % (mean)
Dimethyl L-Glutamate Hydrochloride
Freshly distilled 6.5 cm3 thionyl chloride (75 mmol) were
added dropwise with stirring to cold (at 0ꢂC) anhydrous
20cm3 methanol. Dry L-glutamic acid (2.4g, 14mmol) was
added in portions to this mixture and stirred for 18h at ambi-
ent temperature. On completion of the reaction confirmed by
TLC analysis (n-butanol:acetic acid:H2O ¼ 6:2:2), the reaction
mixture was concentrated on a rotary evaporator to give a
heavy syrup. The syrup was crystallized by the addition of
dry diethyl ether to give a fine white powder. Recrystallization
from acetone-diethyl ether gave 2.55 g (87%) dimethyl L-
glutamate hydrochloride as a colorless solid, mp 77–79ꢂC
(Ref. [22] 78–80ꢂC).
Methotrexate
84
64
72
78
82
83
70
84
74
78
82
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j

Novel Methotrexate-like Compounds
1285
N-[4-[[(2-Hydroxy-4-methoxyphenyl)methylene]amino]-
Synthesis of 4-[(2-Hydroxy-4-methoxybenzylidene)amino]-
benzoic acid] (3b). Typical Procedure
benzoyl]-ꢀ-alanin (3b)
A mixture of 1.52 g 2-hydroxy-4-methoxybenzaldehyde
(10 mmol) in 20 cm3 EtOH and 1.37 g 4-aminobenzoic acid
(10 mmol) in 20 cm3 EtOH was refluxed for 2 h and then cooled
to room temperature. The solid which precipitated was col-
lected, washed with cold ethanol, and recrystallized from
ethanol to give 2.00 g (74%) of a yellow solid, mp 289–290ꢂC.
All imine compounds were prepared according to this
procedure.
Yield 32%, mp 212–215ꢂC; MS (EI): m=z ¼ 343.06 (molecu-
lar weight 342.35); 1H NMR (300MHz, DMF-d7): ꢁ ¼ 2.67 (t,
2H, H-70), 3.68 (q, 2H, H-60), 3.91 (s, 3H, OCH3), 6.56 (d, 1H,
H-3), 6.63 (d, 1H, H-6), 7.53 (d, 2H, H-20, J ¼ 8.60Hz), 7.64
(d, 1H, H-5), 8.05 (d, 2H, H-30, J ¼ 8.60Hz), 8.63 (t, 1H, NH),
9.00 (s, 1H, CH¼N), 12.62 (b, 1H, Ar–OH), 13.60 (s, 1H,
COOH) ppm; 13C NMR (75 MHz, DMF-d7): ꢁ ¼ 35.78 (C-70),
36.05 (C-60), 55.65 (OCH3), 107.80 (C-6), 112.80 (C-5),
113.36 (C-1), 121.16 (C-20), 128.77 (C-30), 132.60 (C-40),
134.63 (C-3), 150.91 (C-10), 152.17 (C-4), 163.83 (C-2),
163.93 (CH¼N), 166.01 (C-50), 173.18 (C-80) ppm; IR (KBr):
ꢂꢀ¼ 3333s (CONH), 3056s (Ar–H), 2921w (C–H), 1734s
Coupling of Amino Acids to Imines. General Procedure
A mixture of 4-[(2-hydroxybenzylidene)amino]benzoic acid,
dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)pyri-
dine in dichloromethane was stirred at 0ꢂC for 3 h. Then ꢀ-
alanine methyl ester hydrochloride was added to the mixture.
After 2 h, temperature of the solution was allowed to rise to
room temperature and the solution was stirred at this temper-
ature for 48 h. The precipitated N,N0-dicyclohexylurea was
removed by filtration and the filtrate was extracted with water,
then cold dilute HCl solution, and then 10% NaHCO3 solu-
tion. Evaporation gave a residue which was dissolved in 2 cm3
ethanol and then hydrolyzed at room temperature with 12cm3
cold aqueous alcoholic sodium hydroxide (0.10M) to afford
the required acid as a sodium salt. The solution of acid salt
was acidified with a dilute hydrochloric acid solution (0.10 M)
to obtain the product as acid. The precipitate occurred was
filtrated off and crystallized from DMF-water.
(CON), 1635s (C¼N) cmꢁ1
.
N-[4-(2-Thiophenealdimino)benzoyl]-ꢀ-alanin (3c)
Yield 33%, mp 168–170ꢂC; MS (EI): m=z ¼ 302.38 (molecu-
lar weight 302.35); 1H NMR (300MHz, DMF-d7): ꢁ ¼ 2.86 (t,
2H, H-70), 3.85 (q, 2H, H-60), 7.43 (dd, 1H, H-3), 7.54 (d, 1H,
H-20), 7.96 (d, 1H, H-2), 8.09 (d, 1H, H-4), 8.23 (d, 2H, H-30),
8.79 (t, 1H, NH), 9.08 (s, 1H, CH¼N), 12.73 (b, 1H, COOH)
13
ppm; C NMR (75 MHz, DMF-d7): ꢁ ¼ 34.08 (C-70), 36.24
(C-60), 113.02 (C-3), 121.15 (C-20), 128.73 (C-30), 131.78 (C-
40), 132.34 (C-2), 134.37 (C-4), 143.03 (C-10), 154.01 (C-1),
155.19 (CH¼N), 166.38 (C-50), 173.28 (C-80) ppm; IR (KBr):
ꢂꢀ¼ 3435s (CONH), 3101s (Ar–H), 2935w (C–H), 1721s
(CON), 1633s (C¼N) cmꢁ1
.
The Investigation of Inhibitory Properties of 3a–3j
on the Dihydrofolate Reductase (DHFR) Activity
N-[4-(2-Pyrrolaldimino)benzoyl]-ꢀ-alanin (3d)
Yield 34%, mp 173ꢂC (decomposed); MS (EI): m=z ¼ 284.80
(molecular weight 285.31); 1H NMR (300 MHz, DMF-d7):
ꢁ ¼ 2.86 (t, 2H, H-70), 3.83 (q, 2H, H-60), 6.48 (dd, 1H, H-
3), 6.99 (d, 1H, H-2), 7.37 (d, 1H, H-4), 7.43 (d, 2H, H-20,
J ¼ 8.45 Hz), 8.17 (d, 2H, H-30, J ¼ 8.45Hz), 8.60 (s, 1H,
CH¼N), 8.73 (t, 3H, CONHCH2), 12.02 (b, 1H, NH), 12.73
(b, 1H, COOH) ppm; 13C NMR (75 MHz, DMF-d7): ꢁ ¼ 35.82
(C-70), 36.04 (C-60), 112.87 (C-3), 121.85 (C-20), 128.84 (C-
30), 132.19 (C-40), 134.21 (C-4), 137.85 (C-2), 144.19 (C-10),
153.85 (C-1), 155.03 (CH¼N), 166.23 (C-50), 173.25 (C-80)
ppm; IR (KBr): ꢂꢀ¼ 3371s (CONH), 3070s (Ar–H), 2940w
DHFR and L-7,8-dihydrofolic acid were purchased from
Fluka (from bovin liver, 9 U=cm3) and Sigma, respectively.
Methotrexate and related compounds were dissolved in DMSO
making a stock solution, which was diluted approx. 100 fold
in the assay. At this concentration DMSO had no effect on
DHFR activity as tested in control experiments. All experi-
ments were done in duplicate with 2 parallel samples. Activity
and inhibition values were estimated by the linear regression
analyses Determination of DHFR activity and the inhibitor
studies were carried out as described by Pastore et al. [23],
and DHFR activities were expressed in IU=cm3. The experi-
mental results are given below.
(C–H), 1676s (CON), 1628s (C¼N) cmꢁ1
.
N-[4-[[(2-Hydroxy-1-naphtyl)methylene]amino]benzoyl]-ꢀ-
alanin (3e)
N-[4-[[(2-Hydroxyphenyl)methylene]amino]benzoyl]-ꢀ-
alanin (3a)
Yield 41%, mp 235–237ꢂC; MS (EI): m=z ¼ 363.53 (molec-
ular weight 362.39); 1H NMR (300 MHz, DMF-d7): ꢁ ¼
2.66 (t, 2H, H-70), 3.63 (q, 2H, H-60), 7.08 (d, 1H, H-5),
7.40 (t, 1H, H-6), 7.58 (t, 1H, H-7), 7.78 (d, 2H, H-20,
J ¼ 7.50 Hz), 7.88 (d, 1H, H-8), 8.02 (d, 1H, H-4), 8.11
(d, 2H, H-30, J ¼ 7.50 Hz), 8.59 (d, 1H, H-3), 8.68 (t, 1H,
CONHCH2), 9.88 (s, 1H, CH¼N), 15.72 (b, 1H, Ar–OH)
ppm; 13C NMR (75 MHz, DMF-d7): ꢁ ¼ 34.04 (C-70), 36.24
(C-60), 109.39 (C-10), 120.59 (C-6), 120.65 (C-20), 122.16
(C-7), 123.96 (C-5), 127.57 (C-9), 128.50 (C-8), 128.99
(C-30), 129.40 (C-4), 137.47 (C-3), 157.02 (CH¼N),
132.68 (C-1), 133.76 (C-40), 147.50 (C-10), 166.09 (C-2),
170.73 (C-50), 173.23 (C-80) ppm; IR (KBr): ꢂꢀ¼ 3352s
Yield 36%, mp 158–160ꢂC; MS (EI): m=z ¼ 313.27 (molecu-
lar weight 312.33); 1H NMR (300MHz, DMF-d7): ꢁ ¼ 2.71 (t,
2H, H-70), 3.65 (q, 2H, H-60), 7.11 (dd, 2H, H-4, H-5), 7.49 (t,
1H, H-6), 7.57 (d, 2H, H-20, J ¼ 8.51Hz), 7.74 (dd, 1H, H-3),
8.09 (d, 2H, H-30, J ¼ 8.51Hz), 8.65 (t, 1H, NH), 9.08 (s, 1H,
CH¼N), 12.62 (b, 1H, Ar–OH), 13.01 (s, 1H, COOH) ppm;
13C NMR (75 MHz, DMF-d7): ꢁ ¼ 33.87 (C-70), 36.08 (C-60),
112.80 (C-5), 116.83 (C-4), 117.27 (C-1), 121.41 (C-20),
128.78 (C-30), 133.11 (C-6), 133.75 (C-3), 150.94 (C-40),
152.19 (C-10), 161.11 (C-2), 165.12 (CH¼N), 166.71 (C-50),
173.20 (C-80) ppm; IR (KBr): ꢂꢀ¼ 3352s (CONH), 3056s
(Ar–H), 2929w (C–H), 1740s (CON), 1625s (C¼N) cmꢁ1
.

1286
N. C°olak et al.
(CONH), 3050s (Ar–H), 2935w (C–H), 1740s (CON), 1631s
(C¼N) cmꢁ1
8.39Hz), 8.34 (s, 1H, CH¼N), 8.58 (d, 1H, CONH), 11.85 (b,
1H, NH), 12.42 (b, 2H, COOH) ppm; 13C NMR (75 MHz,
DMF-d7): ꢁ ¼ 26.44 (C-70), 30.88 (C-80), 52.15 (C-60), 110.99
(C-3), 112.88 (C-2), 120.98 (C-20), 129.19 (C-30), 120.91 (C-
40), 129.48 (C-4), 130.95 (C-10), 152.29 (C-1), 151.86 (CH¼N),
166.96 (C-50), 174.01 (C-90), 174.43 (C-100) ppm; IR (KBr):
ꢂꢀ¼ 3377s (CONH) , 3048s (Ar–H), 2947w (C–H), 1727s
.
N-[4-[[(2-Hydroxyphenyl)methylene]amino]benzoyl]-L-
glutamic acid (3f)
Yield 40%, mp 165–167ꢂC; MS (EI): m=z ¼ 371.53 (molecu-
1
lar weight 370.37); H NMR (300 MHz, DMF-d7): ꢁ ¼ 2.41
(CON), 1689s (COO), 1628s (C¼N) cmꢁ1
.
(dm, 2H, H-70), 2.74 (t, 2H, H-80), 4.87 (m, 1H, H-60), 7.21
(dd, 2H, H-4, H-5), 7.67 (dd, 1H, H-6), 7.76 (d, 2H, H-20,
J ¼ 8.46Hz), 7.91 (dd, 1H, H-3), 8.31 (d, 2H, H-30, J ¼
8.46Hz), 8.90 (d, 1H, CONH), 9.26 (s, 1H, CH¼N), 12.88
(b, 1H, Ar–OH), 13.26 (b, 2H, COOH) ppm; 13C NMR
(75 MHz, DMF-d7): ꢁ ¼ 26.90 (C-70), 30.84 (C-80), 52.88
(C-60), 112.93 (C-4), 117.01 (C-5), 119.51 (C-6), 119.73 (C-
1), 121.59 (C-20), 129.46 (C-30), 133.95 (C-3), 133.29 (C-40),
151.34 (C-10), 159.66 (C-2), 165.38 (CH¼N), 173.78 (C-50),
174.33 (C-90), 174.41 (C-100) ppm; IR (KBr): ꢂꢀ¼ 3384s
(CONH), 3080s (Ar–H), 2933w (C–H), 1734s (CON),
N-[4-[[(2-Hydroxy-1-naphtyl)methylene]amino]benzoyl]-L-
glutamic acid (3j)
Yield 40%, mp 181–183ꢂC; MS (EI): m=z ¼ 420.93 (mo-
lecular weight 420.43); 1H NMR (300 MHz, DMF-d7):
ꢁ ¼ 2.23 (dm, 2H, H-70), 2.57 (t, 2H, H-80), 4.70 (m, 1H,
H-60), 7.11 (d, 1H, H-4), 7.08 (d, 1H, H-5), 7.44 (t, 1H, H-
6), 7.61 (t, 1H, H-7), 7.80 (d, 2H, H-20, J ¼ 8.60 Hz), 7.88
(d, 1H, H-8), 8.19 (d, 2H, H-30, J ¼ 8.60 Hz), 8.61 (d, 1H,
H-3), 8.77 (d, 1H, NH), 9.87 (s, 1H, CH¼N), 12.80 (b, 2H,
COOH), 15.69 (b, 1H, Ar–OH) ppm; 13C NMR (75 MHz,
DMF-d7): ꢁ ¼ 26.75 (C-70), 30.68 (C-80), 52.71 (C-60),
109.41 (C-10), 112.92 (C-6), 121.49 (C-20), 122.16 (C-7),
123.97 (C-8), 124.63 (C-5), 128.50 (C-4), 129.41 (C-30),
127.58 (C-9), 132.23 (C-40), 133.77 (C-1), 137.51 (C-3),
147.74 (C-10), 157.10 (CH¼N), 166.46 (C-2), 170.75 (C-
50), 173.78 (C-90), 174.22 (C-100) ppm; IR (KBr): ꢂꢀ¼ 3423s
(CONH), 3063s (Ar–H), 2947w (C–H), 1721s (CON),
1715s (COO) cmꢁ1
.
N-[4-[[(2-Hydroxy-4-methoxyphenyl)methylene]amino]-
benzoyl]-L-glutamic acid (3g)
Yield 36%, mp 222–223ꢂC; MS (EI): m=z ¼ 401.26 (mo-
lecular weight 400.39); 1H NMR (300 MHz, DMF-d7):
ꢁ ¼ 2.22 (dm, 2H, H-70), 2.54 (t, 2H, H-80), 3.19 (s, 3H,
OCH3), 4.69 (m, 1H, H-60), 6.56 (d, 1H, H-3), 6.63 (dd,
1H, H-5), 7.53 (d, 2H, H-20, J ¼ 8.53 Hz), 7.64 (d, 1H, H-6),
8.14 (d, 2H, H-30, J ¼ 8.53 Hz), 8.72 (d, 1H, CONH), 9.00
(s,1H, CH¼N), 12.84 (b, 2H, COOH), 13.58 (b, 1H, Ar–
1625s (C¼N) cmꢁ1
.
13
OH) ppm; C NMR (75 MHz, DMF-d7): ꢁ ¼ 26.76 (C-70),
Acknowledgements
35.66 (C-80), 52.70 (C-60), 55.60 (OCH3), 107.30 (C-5),
100.90 (C-6), 121.34 (C-20), 129.14 (C-30), 134.82 (C-3),
113.52 (C-40), 132.32 (C-10), 151.30 (C-1), 152.39 (C-4),
164.02 (C-2), 164.30 (CH¼N), 164.75 (C-50), 166.92 (C-
90), 174.24 (C-100) ppm; IR (KBr): ꢂꢀ¼ 3371s (CONH),
3044s (Ar–H), 2947w (C–H), 1708s (CON), 1700s (COO),
We are grateful to the Gazi University Research Foundation
for financial support of this work (FEF 05=2001–21). In
addition, we would like to thank the DPT project (No.
2001K120590) for spectral analyses.
1625s (C¼N) cmꢁ1
.
References
N-[4-(2-Thiophenealdimino)benzoyl]-L-glutamic acid (3h)
Yield 33%, mp 190–193ꢂC; MS (EI): m=z ¼ 360.74 (molecu-
[1] Whitehead VM, Perrault MM, Stelcner S (1975) Cancer
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[6] Furst DE (1985) J Rheumatol 12(12): 11
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1
lar weight 360.39); H NMR (300 MHz, DMF-d7): ꢁ ¼ 2.34
(dm, 2H, H-70), 2.74 (t, 2H, H-80), 4.83 (m, 1H, H-60), 7.43 (t,
1H, H-3), 7.54 (d, 2H, H-20, J ¼ 8.34Hz), 7.96 (d, 1H, H-2),
8.10 (d, 1H, H-4), 8.37 (d, 1H, H-30, J ¼ 8.34 Hz), 8.85 (d, 1H,
CONH), 9.08 (s, 1H, CH¼N), 12.56 (b, 2H, COOH) ppm; 13C
NMR (75 MHz, DMF-d7): ꢁ ¼ 26.61 (C-70), 30.94 (C-80),
52.56 (C-60), 112.90 (C-3), 120.58 (C-2), 121.00 (C-20),
129.16 (C-30), 133.30 (C-40), 134.26 (C-4), 144.19 (C-10),
154.08 (C-1), 155.12 (CH¼N), 166.59 (C-50), 174.03 (C-90),
174.22 (C-100) ppm; IR (KBr): ꢂꢀ¼ 3307s (CONH), 3037s
(Ar–H), 2935w (C–H), 1718w (CON) cmꢁ1
.
[8] Boffa MJ, Chalmers RJ (1996) Clin Exp Dermatol 21(6):
399
N-[4-(2-Pyrrolaldimino)benzoyl]-L-glutamic acid (3i)
Yield 35%, mp 179ꢂC (decomposed); MS (EI): m=z ¼ 343.47
ꢁ ¼ 2.03 (dm, 2H, H-70), 2.36 (t, 2H, H-80), 4.40 (m, 1H,
H-60), 6.22 (dd, 1H, H-3), 6.76 (d, 1H, H-2), 7.06 (d, 1H,
H-4), 7.25 (d, 2H, H-20, J ¼ 8.39Hz), 7.90 (d, 2H, H-30, J ¼
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1
(molecular weight 343.34); H NMR (300 MHz, DMF-d7):

Novel Methotrexate-like Compounds
1287
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