Synthesis and biological activity of 7-oxo substituted analogues of 5-deaza-5,6,7,8-tetrahydrofolic acid (5-DATHF) and 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF)

Article abstract of DOI:10.1021/jm990411u

We recently described the syntheses of 12a-c, 4-amino-7-oxo substituted analogues of 5-deaza-5,6,7,8-tetrahydrofolic acid (5-DATHF), and 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF), in six steps from commercially available p-substituted methyl benz

Full text of DOI:10.1021/jm990411u

2366
J . Med. Chem. 2001, 44, 2366-2369
Syn th esis a n d Biologica l Activity of 7-Oxo Su bstitu ted An a logu es of
5-Dea za -5,6,7,8-tetr a h yd r ofolic Acid (5-DATHF ) a n d
5,10-Did ea za -5,6,7,8-tetr a h yd r ofolic Acid (DDATHF )
J ose´ I. Borrell,* J ordi Teixido´, J osep Llu´ıs Matallana, Blanca Mart´ınez-Teipel, Carles Colominas, Marta Costa,
Merche Balcells, Elisabeth Schuler, and Mar´ıa J ose´ Castillo
Departament de Quı´mica Orga`nica, Institut Quı´mic de Sarria`, Universitat Ramon Llull, Via Augusta 390,
E-08017 Barcelona, Spain
Received August 12, 1999
We recently described the syntheses of 12a -c, 4-amino-7-oxo substituted analogues of 5-deaza-
5,6,7,8-tetrahydrofolic acid (5-DATHF), and 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF),
in six steps from commercially available p-substituted methyl benzoates in 20-27% overall
yields. Such analogues were tested in vitro against CCRF-CEM leukemia cells and showed
that they are completely devoid of any activity, the IC50 being higher than 20 µg/mL for all
cases. To clarify if the presence of the carbonyl group in position C7, the distinctive feature of
our synthetic methodology, is the reason for this lack of activity, we have now obtained the
7-oxo substituted analogues of 5-DATHF and DDATHF, 18a -c, in 10-30% overall yield. Testing
of 18a -c in vitro against CCRF-CEM leukemia cells revealed that these compounds are totally
inactive. A molecular modeling study of 18b inside the active site of the complex E. coli
GARTFase-5-DATHF-GAR pointed to an electronic repulsion between the atoms of the 7-oxo
group and the carbonyl group of Arg90 as a possible explanation for the inactivity of 18a -c.
In tr od u ction
For over 40 years Methotrexate (MTX, 1)1 has been
the antifolate most widely used, alone or in combination
with other agents, in the treatment of cancer2,3 (Figure
1). MTX acts as a competitive inhibitor of dihydrofolate
reductase (DHFR) enzyme that takes part in the folic
acid (FA, 2) metabolism catalyzing the reduction of 7,8-
dihydrofolic acid (H2FA) to 5,6,7,8-tetrahydrofolic acid
(H4FA)4,5 (Figure 1). Folic acid and its derivatives are
responsible for the de novo biosynthesis of nucleotides,
participating as coenzymes in the transfer, oxidation,
and reduction of carbon units in numerous biochemical
routes, such as the thymidylate cycle, de novo synthesis
of purines, methionine regeneration from homocysteine,
and the interconversion of serine and glycine.4,5
Despite the good results, MTX presents, among oth-
ers, a limited antitumor spectrum and acquisition of
drug resistance through four main mechanisms:3,6 mem-
brane impaired transport, impaired polyglutamation,
DHFR increase activity, and decrease in the enzyme
affinity. Due to these deficiencies, great efforts have
been made in searching for new compounds that over-
come, entirely or partially, these drawbacks.7-10
In this context, E. C. Taylor11 et al. obtained 5,10-
dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF, 3)12 and
5-deaza-5,6,7,8-tetrahydrofolic acid (5-DATHF, 4)13 (Fig-
ure 2). These compounds inhibit glycinamide ribonucleo-
tide transformylase (GARTFase),14 responsible for the
transformation of glycinamide ribonucleotide (GAR) into
R-N-formylglycinamide ribonucleotide (FGAR), therefore
inhibiting the biosynthesis of purines. The 6R-diaste-
reomer of DDATHF15 (Lometrexol, LY264618), topologi-
cal analogue of natural 6S-H4FA,16 has recently finished
phase II clinical trials.17-19
F igu r e 1.
F igu r e 2.
egy for the formation of the 5,6,7,8-tetrahydropyrido-
[2,3-d]pyrimidin-7-one moiety (Scheme 1).20 Thus, cy-
clization with guanidine of 2-methoxy-6-oxo-1,4,5,6-
tetrahydropyridine-3-carbonitriles (8a -c), obtained by
reaction of an R,â-unsaturated ester 7a -c and malono-
nitrile in NaOMe/MeOH, afforded the corresponding
compounds 9a -c. After the hydrolysis of the ester group
present in 9a -c, the resulting carboxylic acids 10a -c
were treated with diethyl cyanophosphonate in Et3N/
DMF and coupled with L-glutamic acid dimethyl ester
to give 11a -c. Finally, the basic hydrolysis of 11a -c
yielded the desired 4-amino-7-oxo substituted analogues
12a -c in 20-27% overall yield. Formation of key
intermediates 7a -c was achieved from commercially
available benzoates 5a -c and methyl 2-(bromomethyl)-
acrylate (6). Unfortunately, biological evaluation of
analogues 12a -c showed no activity against CCRF-
More recently, our group has obtained compounds
12a-c, 4-amino-7-oxo substituted analogues of DDATHF
and 5-DATHF, employing an alternative shorter strat-
10.1021/jm990411u CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/12/2001

Brief Articles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 14 2367
Sch em e 1
CEM leukemia cells, the IC50 being higher than 20 µg/
mL for all cases. Both DDATHF (3) and Methotrexate
(1) demonstrated potent growth inhibitory activity
against the CCRF-CEM cells, the IC50 of DDATHF and
Methotrexate being 0.007 µg/mL and 0.004 µg/mL,
respectively.
mental conditions, such as phase transfer catalysis in
toluene using potassium carbonate in the presence of
benzyltriethylammonium chloride, gave lower yields
and allowed the isolation and characterization of the
corresponding Michael bisadducts (19a -c) (Figure 3).
Compounds 13 (G ) COOMe) were obtained as diaster-
eomers and used without further separation. The sub-
sequent treatment of 13a -c with guanidine in MeOH
at reflux afforded, in one step, the 2-amino-4-oxopyrido-
[2,3-d]pyrimidines 15a -c in 52-87% yield (Scheme 1).
This methodology was also applicable to the cyclization
with guanidine of the Michael adducts 14a -c (G ) CN),
obtained by reaction of R,â-unsaturated esters 7a -c
with malononitrile, which led to the 2,4-diaminopyrido-
[2,3-d]pyrimidines 9a -c, common precursors of the
previously described 4-amino-7-oxo substituted ana-
logues 12a -c (7 f 8 f 9 f 10 f 11 f 12) (Scheme 1).
Hydrolysis of the ester group present in 15a -c was
achieved in 0.5 N aqueous NaOH to yield the carboxylic
Compounds 12 present a 2,4-diaminopyrimidine ring,
so they are primarily targeted at DHFR, and it is known
that oxygenation at the 7 position is in general not good
for DHFR binding. Consequently, taking into account
that the 4-amino substituted analogue of DDATHF was
capable of inhibiting bovine liver DHFR with an IC50 of
71 nM,12 it has to be concluded that our 4-amino-7-oxo
substituted analogues of DDATHF are inactive precisely
due to the presence of the carbonyl group in position
C7, the distinctive feature of our synthetic methodology.
The only remaining question is if such negative effects
of the carbonyl group would be also present in 7-oxo
substituted analogues of DDATHF, which would be
targeted against glycinamide ribonucleotide transformy-
lase (GARTFase).
Now, we report the synthesis of 18a -c, 7-oxo substi-
tuted analogues of DDATHF (3) and 5-DATHF (4), and
the determination of their biological activity.
Resu lts a n d Discu ssion
Ch em istr y. Adducts 13a -c (G ) COOMe) were
obtained in 41-80% yield, after column chromatography
purification, by reaction of the previously described R,â-
unsaturated esters 7a -c with methyl cyanoacetate in
NaOMe/MeOH (Scheme 1).21 The use of other experi-
F igu r e 3.

2368 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 14
Brief Articles
Ta ble 1. In Vitro Cytotoxicity Assay (IC50) Conducted in
CCRF-CEM Human Leukemia Cells
similar conformation to 5-DATHF inside the active site
of the enzyme. Then we have replaced the structure of
5-DATHF by the structure of 18b in the active site of
the enzyme and determined the resulting interactions.
The replacement shows that Phe88 would be face-to-
face to the 7-oxo group of 18b at 2.68 Å. This would
produce electronic repulsion between the oxygen atoms
of both carbonyl groups, and as a consequence, the
conformation of amino acids surrounding the bicyclic
unit would be distorted, disrupting any possible H-
bridge interaction. This phenomenon would give a
possible explanation for the total absence of activity of
18a -c.
compd
MTX
DDATHF
18a
X
IC50 (µg/mL)
0.004
0.007
>50
NMe
NH
18b
20
18c
CH2
>50
acids 16a -c in almost quantitative yield. If such hy-
drolysis was carried out in 1 N aqueous NaOH, a
mixture of two products was obtained: the expected acid
16 and a monocyclic compound 20 (Figure 3) resulting
from the opening of the pyridone ring as the 13C NMR
data reveals. Further treatment of this mixture in acetic
acid at reflux reverted to the corresponding acid 16.
The coupling of 16a -c with L-glutamic acid dimethyl
ester was achieved by using diethyl cyanophosphonate
as the activating agent and Et3N in DMF at room
temperature, the corresponding esters 17a -c being
obtained in 53-82% yield (Scheme 1).
The last step for the synthesis of the folic acid
analogues is the basic hydrolysis of the ester groups
present in the glutamate unit of 17a -c. Hydrolyses
carried out using 0.5 N NaOH at room temperature
afforded the final compounds 18a -c in 61-95% yield
(Scheme 1).
To summarize, compounds 18a -c, which are 7-oxo
substituted analogues of 5-DATHF (4) and DDATHF (3),
have been obtained in six steps from commercially
available compounds in 22%, 9%, and 27% global yields,
respectively. The methodology used also allows the
synthesis of the 2,4-diamino-7-oxo analogues 12.
Biologica l Eva lu a tion . Compounds 18a -c were
tested in vitro against CCRF-CEM22 leukemia cells
(Table 1). This is a very common T-cell derived lympho-
blastic leukemia that has been widely used as a dis-
criminatory test. The results obtained indicated that our
7-oxo analogues are completely devoid of any activity,
with IC50 values being higher than 20 µg/mL for all
cases. This lack of activity can be attributed, at first
sight, to electronic considerations due to the presence
of the carbonyl group at the C-7 position. In fact, this
is the only different feature with respect to DDATHF
and 5-DATHF.
Exp er im en ta l Section
All melting points, determined with a Bu¨chi 530 capillary
apparatus, and boiling points, determined during distillation,
are uncorrected. Infrared spectra were recorded in BOMEM
Michelson 100 and Nicolet Magna 560 FTIR spectrophotom-
eters. UV spectra were registered in a Hewlett-Packard 8450
instrument. 1H and 13C NMR spectra were determined in a
Varian Gemini-300 operating at a field strength of 300 and
75.5 MHz, respectively. Mass spectra (m/z (%), EI, 70 eV) were
obtained on a Hewlett-Packard 5995 A spectrometer. Elemen-
tal microanalyses were obtained on a Carlo-Erba CHNS-O/
EA 1108 analyzer and gave results for the elements stated
within (0.4% of the theoretical values.
Gen er a l Meth od for th e Syn th esis of Mich a el Ad d u cts
13. Dim eth yl 2-Cya n o-4-{[(4-m eth oxyca r bon ylp h en yl)-
m eth yla m in o]m eth yl}p en ta n ed ioa te (13a ). To a solution
of 2.20 g (9.5 mmol) of sodium in 100 mL of methanol was
added 9.40 g (9.5 mmol) of methyl cyanoacetate. Next, 5.00 g
(1.9 mmol) of methyl p-[N-(2-methoxycarbonylallyl)-N-methyl-
amino]benzoate (7a ) were added dropwise. The resulting
mixture was heated to 35 °C for 90 min and, after being cooled,
was neutralized with concentrated acetic acid. The solvent was
removed in vacuo, and the residue was treated with 100 mL
of CH2Cl2 and 100 mL of water. The two layers were separated,
and the aqueous layer was extracted with (2 ×25 mL) of CH2-
Cl2. The combined organic extracts were washed with water,
dried (MgSO4), and concentrated in vacuo. The crude material
was purified by column chromatography using AcOEt/hexane
(2:1) as eluent to give 3.5 g (9.7 mmol, 51%) of 13a as a
colorless oil: Anal. (C18H22N2O6) C, H, N.
Gen er a l Meth od for th e Syn th esis of 2-Am in o-4,7-
d ioxo-3,4,5,6,7,8-h exa h yd r op yr id o[2,3-d ]p yr im id in es 15.
Meth yl 4-[N-(2-Am in o-4,7-d ioxo-3,4,5,6,7,8-h exa h yd r op y-
r id o[2,3-d ]p yr im id in -6-ylm et h yl)-N-m et h yla m in o]b en -
zoa te (15a ). A 2.2 g (12.2 mmol) portion of guanidine
carbonate was added to a solution of 0.56 g (24.3 mmol) of Na
in 15 mL of methanol, and the mixture was refluxed for 15
min. After the solution was cooled, the sodium carbonate
formed was filtered, and 0.88 g (2.4 mmol) of dimethyl 2-cyano-
4-{[(4-methoxycarbonylphenyl)methylamino]methyl}pen-
tanedioate (13a ) in 15 mL of methanol were added. The
mixture was heated at reflux for 24 h and cooled to room
temperature. The solid was filtered, washed with MeOH, and
dried in vacuo over P2O5 to give 0.75 g (2.1 mmol, 87%) of 15a
as a white solid: mp >250 °C; Anal. (C17H19N5O4‚H2O) C, H,
N.
In this context, the X-ray structure of the complex E.
coli GARTFase-5-DATHF-GAR23 (Protein
Data
Bank, Brookhaven National Laboratory; http://
pdb.pdb.bnl.gov) allowed us to determine (Weblab Viewer
2.01; Molecular Simulations Inc.; http://www.msi.com)
the closest residues surrounding C7 in 5-DATHF.
Among them, Arg90 forms an H-bond with N8, and
Phe88 is placed near C7 at a distance (3.84 Å) that
avoids steric interactions with the inhibitor.
Consequently, a first explanation for the lack of
activity of 18a -c would imply the existence of amide-
iminol tautomerism in the pyridone ring. The presence
of an iminol tautomer would disable the H-bond inter-
action between Arg90 and N8. Nevertheless, spectro-
scopic data for 18a -c are consistent only with the
existence of amide tautomers. This fact is in accordance
with the rest of the 7-oxopyrido[2,3-d]pyrimidines previ-
ously obtained in our group.
Gen er a l Meth od for th e Hyd r olysis of th e Ester Gr ou p
p r esen t in P yr id o[2,3-d ]p yr im id in es 15. 4-[N-(2-Am in o-
4,7-d ioxo-3,4,5,6,7,8-h exa h yd r op yr id o[2,3-d ]p yr im id in -6-
ylm eth yl)-N-m eth yla m in o]ben zoic Acid (16a ). A suspen-
sion of 0.50 g (1.4 mmol) of methyl 4-[N-(2-amino-4,7-dioxo-
3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-6-ylmethyl)-N-
methylamino]benzoate (15a ) in 7 mL of a 0.5 M aqueous NaOH
was stirred at room temperature until solution occurred. The
resulting solution was filtered through a Lida filter (47 mm
filter membrane, 0.45 µm Nylon, Art NY504700, Lida Manu-
facturing Corp., 9115 26th Avenue, Kenosha, WI), and the
filtrate was acidified with concentrated acetic acid (pH 5-6).
On the other hand, we have proved, by modeling our
7-oxo substituted analogues, that 18a -c could adopt a

Brief Articles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 14 2369
The resulting precipitate was filtered, washed with water, and
dried at vacuum over P2O5 to give 0.47 g (1.37 mmol, 98%) of
16a as a white solid: mp >250 °C; Anal. (C16H17N5O4‚0.75H2O)
C, H, N.
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dimethyl N-{4-[N-(2-amino-4,7-dioxo-3,4,5,6,7,8-hexahydropy-
rido[2,3-d]pyrimidin-6-ylmethyl)-N-methylamino]benzoyl}-L-
glutamate (17a ) in 10 mL of 0.5 M NaOH was stirred at room
temperature until solution occurred. The resulting solution
was filtered through a Lida filter (47 mm filter membrane,
0.45µm Nylon, Art NY504700, Lida Manufacturing Corp., 9115
26th Avenue, Kenosha, WI), and the filtrate was acidified with
concentrated acetic acid (pH 5-6). The resulting precipitate
was filtered, washed with water, and dried at vacuum over
P2O5 to give 0.135 g (0.286 mmol, 95%) of 18a as a white
solid: mp > 250 °C; Anal. (C21H24N6O7‚3.9H2O) C, H, N.
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Ack n ow led gm en t. The authors warmly thank Dr.
Chuan Shih (Lilly Research Laboratories, Indianapolis,
IN) for his very useful comments throughout this work.
We also thank Lilly Research Laboratories for the in
vitro cell culture studies. Support of this work by a grant
from the Comisio´n Interdepartamental de Ciencia y
Tecnolog´ıa and the Comissio´ Interdepartamental de
Recerca i Innovacio´ Tecnolo´gica (CIRIT) within the
Programa de Qu´ımica Fina (QFN93-4420) is gratefully
acknowledged. J .L.M. thanks the CIRIT for a grant, and
B.M.-T. thanks the Fundacio´n J uan Salan˜er.
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