Effect of N9-methylation and bridge atom variation on the activity of 5-substituted 2,4-diaminopyrrolo[2,3-d]pyrimidines against dihydrofolate reductases from Pneumocystis carinii and Toxoplasma gondii

Article abstract of DOI:10.1021/jm960717q

The effect of N⁹-mathylation and bridge atom variation on inhibitory potency and selectivity of 2,4-diaminopyrrolo[2,3-d]pyrimidines against dihydrofolate reductases (DHFR) was studied. Specifically three nonclassical 2,4-diamino-5-((N-methylan

Full text of DOI:10.1021/jm960717q

J . Med. Chem. 1997, 40, 1173-1177
1173
Effect of N9-Meth yla tion a n d Br id ge Atom Va r ia tion on th e Activity of
5-Su bstitu ted 2,4-Dia m in op yr r olo[2,3-d ]p yr im id in es a ga in st Dih yd r ofola te
Red u cta ses fr om P n eu m ocystis ca r in ii a n d Toxopla sm a gon d ii1a ,b
Aleem Gangjee,*,† Farahnaz Mavandadi,† and Sherry F. Queener‡
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University,
Pittsburgh, Pennsylvania 15282, and Department of Pharmacology and Toxicology, Indiana University School of Medicine,
Indianapolis, Indiana 46202
Received October 11, 1996X
The effect of N9-methylation and bridge atom variation on inhibitory potency and selectivity
of 2,4-diaminopyrrolo[2,3-d]pyrimidines against dihydrofolate reductases (DHFR) was studied.
Specifically three nonclassical 2,4-diamino-5-((N-methylanilino)methyl)pyrrolo[2,3-d]pyrim-
idines with 2′,5′-dimethoxyphenyl (2), 3′,4′-dichlorophenyl (3), 1′-naphthyl (4), one classical
analogue with a 4′-L-glutamate substituent (10), and four nonclassical 2,4-diamino-5-
((phenylthio)methyl)pyrrolo[2,3-d]pyrimidines with 3′,4′-dimethoxyphenyl (5), 3′,4′-dichloro-
phenyl (6), 1′-naphthyl (7), and 2′-naphthyl (8) substituents were synthesized. The classical
and nonclassical analogues were obtained by displacement of the intermediate 2,4-diamino-
5-bromomethylpyrrolo[2,3-d]pyrimidine, 14, with appropriately substituted N-methylaniline,
thiophenols, or 4-(N-methylamino)benzoyl-L-glutamate. Compounds 2-8 and 10 were evaluated
against Pneumocystis carinii (pc), Toxoplasma gondii (tg), and rat liver (rl) DHFRs. The
N-methyl and thiomethyl analogues were more inhibitory than their corresponding anilino-
methyl analogues (previously reported) against all three DHFRs. The inhibitory potency of
these analogues was greater against rlDHFR than against tgDHFR which resulted in a loss of
selectivity for tgDHFR compared to the N9-H analogues. The classical N9-methyl analogue 10
was more potent and about 2-fold more selective against tgDHFR than its corresponding
desmethyl analogue. All of the analogues, 2-8 and 10, were more selective than trimetrexate
(TMQ) against pcDHFR (except 4) and significantly more selective than TMQ against tgDHFR.
Ta ble 1. Inhibitory Concentrations (IC50, µM) against DHFRs
In tr od u ction
and Selectivity Ratios18,19
Defective cell-mediated immunity renders patients
with Acquired Immunodeficiency Syndrome (AIDS)
susceptible to opportunistic infections, which remains
the principal cause of morbidity and mortality in
patients in the United States.2 The prevention of
cerebral toxoplasmosis, caused by the intracellular
coccidian protozoan Toxoplasma gondii, and of P. carinii
pneumonia (PCP), caused by Pneumocystis carinii, is
an essential objective in the management of patients
with AIDS. The treatment of these infections by anti-
folates takes advantage of the fact that these organisms,
unlike mammalian cells, lack a carrier-mediated active
transport mechanism for the uptake of classical anti-
folates with polar glutamate side chains.3 These organ-
isms, however, are permeable to lipophilic nonclassical
antifolates, which can also penetrate into the central
nervous system where T. gondii infections occur.
selectivity
ratio rl/pc T. gondii ratio rl/tg
selectivity
compd P. carinii
rl
156
14.4
59.3
1a 8
1b8
1c8
2
45.7
35.3
307
3.4
0.4
0.2
ND
1.7
1.4
1.1
3.40
1.00
0.87
2.60
92
10.3
53.9
>4.0
3.0
>12.0
28.30
209.0
11.10
58.50
10.60
929.0
0.038
0.044
0.042
0.038
12.00
0.001
>12.0
3
4
5
6
3.00
8.20
16.7
0.11
0.04
1.50
0.09
0.28
0.09
1.2
1.36
0.07
0.04
11.1
3.0
9.43
6.42
0.46
3.70
9.01
0.21
0.40
0.30
0.10
5.30
3.00
82.9
0.044
0.06
0.003
0.001
11.6
7
0.81
9.20
0.21
0.15
0.01
0.01
2.7
8
98
10
TMQ
PTX
TMP
MTX
133.0
0.003
49.0
0.21
0.014
The standard treatment for P. carinii (pc) and T.
gondii (tg) infections, involving combinations of sulfona-
mides and trimethoprim (TMP) or pyrimethamine,4 is
often limited by the toxicity of the sulfonamides, used
for their synergistic effect.5 Trimetrexate (TMQ) and
piritrexim (PTX), which are 100-10000 times more
potent than TMP or pyrimethamine against pc- and
tgDHFR are, however, nonselective and strongly inhibit
DHFR from mammalian sources.6 Thus TMQ is ad-
ministered in conjunction with the reduced folate,
leucovorin ((6R,6S)-5-formyl-5,6,7,8-tetrahydrofolic acid),
to selectively rescue host cells.3,7 Development of
inhibitors, possessing both the high potency of TMQ or
PTX and the high selectivity of TMP for pcDHFR and/
or tgDHFR, is a desirable goal.
In our efforts directed toward the design and synthe-
sis of lipophilic nonclassical antifolates with selectivity
and high potency against pcDHFR and tgDHFR, we
recently reported8 some nonclassical 6-5 fused, 5-sub-
stituted 2,4-diaminopyrrolo[2,3-d]pyrimidines of general
structure 1, with various substituted lipophilic side
chains. These analogues possessed excellent selectivity
against tgDHFR (1a -c, Table 1). However, they were
only moderately potent. The present work attempts to
† Duquesne University.
‡ Indiana University School of Medicine.
X Abstract published in Advance ACS Abstracts, March 15, 1997.
S0022-2623(96)00717-0 CCC: $14.00 © 1997 American Chemical Society

1174 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7
Notes
study the effect of N9-methylation and bridge length
variation on both potency and selectivity of these
analogues.
Ch em istr y
The synthesis of the pyrrolo[2,3-d]pyrimidine ana-
logues 2-4 was initially attempted via the reductive
methylation of the precursor NH analogues 1a -c using
formaldehyde and sodium cyanoborohydride, employing
known procedures.9,13 No N9-methylated product was
obtained under these reaction conditions, which yielded
a complex mixture, probably the result of multiple
methylations and/or degradation of the starting mate-
rial. This led to the exploration of an alternate method.
A versatile intermediate, from which both the N9-
methyl (2-4 and 10) as well as the thio-substituted
analogues (5-8) could be synthesized, was sought.
Transformation of the aldehyde 12 (Scheme 1), which
Side chain N-methylation of most 6-6 fused nonclas-
sical pyrido[2,3-d]pyrimidines and, more significantly,
6-5 fused classical furo[2,3-d]pyrimidines substantially
increase the DHFR inhibitory potencies9,10 compared to
their N-H analogues. Thus N9-methylation of the 6-5
fused pyrrolo[2,3-d]pyrimidine analogues 1a -c, the
most potent and/or selective analogues in the previous
series,8 were similarly anticipated to increase the DHFR
inhibitory potency and prompted the synthesis of com-
pounds 2-4.
8
we had previously reported, via the alcohol to the
bromide would provide such an intermediate from which
both series of target compounds could be readily ob-
tained by nucleophilic displacement using the appropri-
ate nitrogen or sulfur nucleophiles.
Thus the aldehyde 12, obtained from the nitrile 11,
was reduced with NaBH4, to afford the alcohol 13.
Compound 13 was converted to the bromide 14, with
30% HBr-AcOH, which had Rf 0.62 on TLC which was,
as expected, higher than that of the alcohol 13 (Rf 0.43).
The hygroscopic HBr salt of 14 was isolated by precipi-
tation with Et2O and, owing to its instability, was used
in subsequent steps without further purification. Nu-
cleophilic displacement of the bromide 14, at room
temperature, with excess N-methyl-2,5-dimethoxy-
aniline,14 N-methyl-3,4-dichloroaniline,15 and N-meth-
ylnaphthylamine16 (16a -c, respectively), in anhydrous
DMF or DMAC, containing K2CO3, afforded the desired
analogues 2-4 in 31-36% yield. The classical analogue
10 was obtained by nucleophilic displacement of the
bromide 14 with diethyl (N-methylamino)benzoyl-L-
glutamate17 (16d ) followed by saponification of the
resulting diester with 1 N NaOH, at room temperature,
and acidification to pH 5 which afforded 10 in 50% yield.
Analogues 5-8 were, similarly, obtained via nucleo-
philic displacement of the bromide 14 with 3,4-dimeth-
oxythiophenol, 3,4-dichlorothiophenol, 1-naphthalene-
thiol, and 2-naphthalenethiol, respectively. The thiols,
in each case, were first activated by stirring with sodium
hydride in DMF/DMAC, at room temperature, followed
by addition of the bromide 14. The displacement of the
bromide, at room temperature, provided products 5-8
in 26-38% yield.
In the classical 6-5 fused pyrrolo[2,3-d]pyrimidines,
reported by Miwa et al.,11,12 the analogue possessing a
three-atom bridge, between the heterocycle and the
benzoyl L-glutamate, was significantly more cytotoxic
than the analogue with a two-atom bridge. Thus,
compounds 5-8 were designed wherein the bridge
nitrogen was replaced by a sulfur which, because of its
larger size, was anticipated to increase the bridge
length, between the heterocyclic ring and the aromatic
side chain, of the nonclassical pyrrolo[2,3-d]pyrimidines
to about a three-atom distance and consequently in-
crease the potency.
Compound 10 was also synthesized and evaluated in
order to study the effect of N9-methylation on the
potency of the classical pyrrolo[2,3-d]pyrimidine ana-
logue 9, which we had reported8 as being 1000-, 5-, and
325-fold more potent than the corresponding nonclas-
sical analogues against pcDHFR, tgDHFR, and rlDHFR,
respectively.
Thus a versatile intermediate bromide 14 was ob-
tained from which the target analogues 2-8 and 10
were easily accessible. This intermediate 14 could also
serve as the precursor for nonclassical carbon-bridged
analogues (via Wittig reactions) which will be the topic
of future reports.
Biologica l Eva lu a tion a n d Discu ssion
Compounds 2-8 and 10 were evaluated as inhibitors
of P. carinii, T. gondii, and rat liver DHFR as described
previously.18,19 The inhibitory concentrations (IC50) and
the selectivity ratios vs rlDHFR are listed in Table 1.
Compound 2 was insoluble at concentrations greater
than 12 µM, and hence IC50 values could not be
determined against pcDHFR and rlDHFR. Against
tgDHFR N9-methylation decreased potency compared
to the N9-H analogue 1a . N9-Methylation of 1b and 1c
(compounds 3 and 4) increased potency against all three

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7 1175
Sch em e 1a
a
(A) Raney Ni/HCOOH; (B) NaBH4; (C) 30% HBr-AcOH; (D) DMF or DMAC/K2CO3; (E) NaH/DMF or DMAc.
DHFRs but drastically decreased selectivity against
pcDHFR and tgDHFR. The decrease in selectivity of 3
and 4 compared with 1b and 1c is clearly due to the
greater increase in potency against rlDHFR than against
pcDHFR and tgDHFR. Thus N9-methylation did in
general increase inhibitory effects against pcDHFR and
tgDHFR but was detrimental to selectivity compared
to the N9-H analogues.
pc- or tgDHFR and was detrimental to selectivity
compared to the N9-H analogues.
The classical analogue 10 was a more potent inhibitor
of all three DHFRs tested compared to the nonclassical
analogues 2-8. It was similar to the N-desmethyl
analogue, 9, against all three DHFRs and was about
2-fold more selective for tgDHFR than 9.
Both the CH2NCH3 and the CH2S bridge analogues,
2-8 and 10, were more selective than TMQ and PTX
against tgDHFR. The 1′-(N-(methylnaphthyl)amino)-
methyl analogue 4 and the 1′-(naphthylthio)methyl
analogue 7 were about 3-fold more potent than TMP
against tgDHFR and about 67- and 26-fold more selec-
tive than TMQ and PTX, respectively, against tgDHFR.
The ((3′,4′-dimethoxyphenyl)thio)methyl analogue 5
possessed the highest selectivity for pcDHFR (rl/pc: 1.5)
which was about 21- and 38-fold greater than that of
TMQ and PTX, respectively.
Replacement of the nitrogen in the side chain with a
sulfur, as in analogues 5-8, affected inhibitor potency
against all three DHFRs. Compared with 1a and 1c,
analogues 5 and 7, with the 3′,4′-dimethoxyphenyl and
a 1-naphthyl substituent, respectively, were 4- and 29-
fold more inhibitory against pcDHFR and 9- and 20-
fold more inhibitory against rlDHFR, respectively. The
3′,4′-dichlorophenyl analogue, 6, was about 3-fold more
inhibitory against rlDHFR and less active against
pcDHFR and tgDHFR than its corresponding nitrogen-
containing analogue, 1b. Replacing the 1-naphthyl of
7 with a 2-naphthyl moiety, as in analogue 8, resulted
in a decrease in inhibitory potency against all three
DHFRs. Compound 7 was 28-, 88-, and 11-fold more
potent, than 8, against rlDHFR, pcDHFR, and tgDHFR,
respectively. The differential decrease in inhibitory
potency against the three DHFRs for 8 further indicates
the differences in the three DHFRs. Compound 8 was,
however, 3 times more selective for tgDHFR than the
corresponding 1-naphthyl analogue 7. In general, in-
creasing the bridge length by replacement of the N9 with
S increased potency against rlDHFR more so than for
Exp er im en ta l Section
All evaporations were carried out in vacuo with a rotary
evaporator. Analytical samples were dried in vacuo (0.2
mmHg) in an Abderhalden drying apparatus over P2O5 and
refluxing ethanol or toluene. Melting points were determined
on a Fisher-J ohns melting point apparatus and are uncor-
rected. Nuclear magnetic resonance spectra for proton (1H
NMR) were recorded on a Bruker WH-300 (300 MHz) spec-
trometer. Data was accumulated by 16K size with a 0.5 s
delay time and 70° tip angle. The chemical shift values are
expressed in ppm (parts per million) relative to tetramethyl-
silane as internal standard; s ) singlet, d ) doublet, dd )

1176 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7
Notes
doublet of doublet, t ) triplet, q ) quartet, m ) multiplet, bs
) broad singlet. The relative integrals of peak areas agreed
with those expected for the assigned structures. Thin layer
chromatography (TLC) was performed on POLYGRAM Sil
G/UV254 silica gel plates with fluorescent indicator, and the
spots were visualized under 254 and 366 nm illumination.
Proportions of solvents used for TLC are by volume. Eluents
used in column chromatography contained 0.1% NH4OH by
volume. Elemental analyses were performed by Atlantic
Microlabs Inc., Norcoss, GA. Analytical results indicated by
element symbols are within (0.4% of the calculated values.
Fractional moles of water or organic solvents frequently found
in some analytical samples of antifolates were not removed in
spite of 24-48 h of drying in vacuo and were confirmed where
possible by their presence in the 1H NMR spectrum. All
solvents and chemicals were purchased from Aldrich Chemical
Co. and Fisher Scientific and were used as received.
6.42 (s, 1 H, 6-CH), 6.87 (m, 1 H, 6′-CH), 7.03 (d, 1 H, 2′-CH),
7.37 (d, 1 H, 5′-CH), 10.56 (s, 1 H, 7-NH). Anal. Calcd for
(C14H14N6Cl2‚0.75H2O‚0.41HCl) C, H, N, Cl.
2,4-Dia m in o-5-[(N-m eth yln a p h th yla m in o)m eth yl]p yr -
r olo[2,3-d ]p yr im id in e (4): yield 0.04 g (31%); mp 172-174
°C dec; TLC Rf 0.65 (CHCl3/MeOH/NH4OH, 10:3:0.1, silica gel);
1H NMR (Me2SO-d6) δ 2.75 (s, 3 H, 9-CH3), 4.25 (s, 2 H, 8-CH2),
5.49 (bs, 2 H, NH2), 6.66 (bs, 2 H, NH2), 6.78 (s, 1 H, 6-CH),
7.33 (m, 1 H, C10H7), 7.43 (m, 1 H, C10H7), 7.47 (m, 2 H, C10H7),
7.62 (m, 1 H, C10H7), 7.90 (d, 1 H, C10H7), 8.16 (d, 1 H, C10H7),
10.58 (s, 1 H, 7-NH). Anal. Calcd for (C18H18N6‚0.4H2O) C,
H, N.
N-[4-[N-[(2,4-Dia m in op yr r olo[2,3-d ]p yr im id in -5-yl)-
m et h yl]m et h yla m in o]b en zoyl]-L-glu t a m ic a cid d iet h yl
1
ester (17): yield 0.08 g (38%); mp 143 °C; H NMR (Me2SO-
d6) δ 1.17 (t, 6 H, CH2CH3), 2.07 (m, 2 H, Glu â-CH2), 2.42 (t,
2 H, Glu γ-CH2), 2.94 (s, 3 H, 9-CH3), 4.06 (q, 4 H, CH2CH3),
4.41 (m, 1 H, Glu R-CH), 4.61 (s, 2 H, 8-CH2), 6.20 (bs, 4 H,
NH2), 6.47 (s, 1 H, 6-CH), 6.89 (d, 2 H, 3′-, 5′-CH), 7.76 (d, 2
H, 2′-,6′-CH), 8.37 (d, 1 H, CONH), 10.95 (bs, 1 H, 7-NH).
Gen er a l P r oced u r e for P r ep a r a tion of 2,4-Dia m in o-
5-[(su bstitu ted p h en ylth io)m eth yl]p yr r olo[2,3-d ]-p yr i-
m id in e (5-8). To a solution of substituted thiophenol (1.70
mmol) in DMF or DMAC (15 mL) was added NaH (0.04 g, 1.70
mmol). The mixture was stirred at room temperature for 1 h.
To this was added the bromide 14 (0.10 g, 0.40 mmol), and
the reaction mixture was stirred for 4 h until no more bromide
was detected on TLC. The solvent was evaporated under
reduced pressure. The residue was dissolved in MeOH (5 mL),
and silica gel (1.00 g) was added to the solution, which was
evaporated to dryness to form a plug which was loaded onto a
dry silica gel column (2.4 cm × 20 cm). The column was eluted
with a gradient of 1% MeOH in CHCl3 to 10% MeOH in CHCl3.
Fractions corresponding to the product (TLC) were pooled and
evaporated to dryness under reduced pressure. The residue
was triturated in cold Et2O, and hexane(s) was added dropwise
until a suspension formed which was refrigerated for 2 h and
filtered to yield products.
2,4-Dia m in o-5-[[(3′,4′-d im eth oxyp h en yl)th io]m eth yl]-
p yr r olo[2,3-d ]p yr im id in e (5). Fractions obtained from the
column were pooled and acidified (pH 4-5) with glacial AcOH
before evaporation and trituration with cold Et2O: yield 0.05
g (38%); mp 230-231 °C; TLC Rf 0.76 (CHCl3/MeOH/NH4OH,
10:3:0.1, silica gel); 1H NMR (Me2SO-d6) δ 3.67 (s, 3 H, OCH3),
3.70 (s, 3 H, OCH3), 4.21 (s, 2 H, 8-CH2), 5.43 (s, 2 H, NH2),
6.12 (s, 2 H, NH2), 6.45 (s, 1 H, 6-CH), 6.85 (m, 3 H, 2′,5′,6′-
CH), 10.48 (s, 1 H, 7-NH). Anal. Calcd for (C15H17N5O2S‚
0.2CH3COOH) C, H, N, S.
2,4-Dia m in op yr r olo[2,3-d ]p yr im id in e-5-ca r b oxa ld e-
h yd e (12). Compound 12 was synthesized from the corre-
sponding nitrile using HCOOH and Raney nickel:8 mp >300
°C; TLC Rf 0.49 (CHCl3/MeOH/NH4OH, 9:3:0.1, silica gel); IR
1
(Nujol) 1600-1650 cm-1; H NMR (Me2SO-d6) δ 5.84 (s, 2 H,
2-NH2), 6.90 (bs, 1 H, NH2), 7.35 (bs, 1 H, NH2), 7.84 (s, 1 H,
6-CH), 9.51 (s, 1 H, CHO), 11.87 (bs, 1 H, 7-NH).
2,4-Dia m in op yr r olo[2,3-d ]p yr im id in e-5-m eth a n ol (13).
To a solution of 12 (0.80 g, 4.5 mmol) in MeOH (25 mL) was
carefully added NaBH4 (1.00 g). The mixture was stirred at
room temperature for 2 h. The MeOH was evaporated under
reduced pressure, and cold water (5 mL) was carefully added
to the residue which was then neutralized with glacial AcOH.
The suspension was refrigerated overnight and filtered to yield
0.46 g (56%) of the alcohol 13: mp >175 °C dec; TLC Rf 0.43
(CHCl3/MeOH/NH4OH, 10:3:0.2, silica gel); 1H NMR (Me2SO-
d6) δ 4.48 (s, 2 H, CH2), 5.43 (m, 3 H, C-OH, NH2), 6.38 (s, 2
H, NH2), 6.56 (s, 1 H, 6-CH), 10.47 (s, 1 H, 7-NH).
2,4-Dia m in o-5-(b r om om e t h yl)p yr r olo[2,3-d ]p yr im i-
d in e (14). The alcohol 13 (1.00 g, 5.60 mmol) was stirred in
30% HBr-AcOH (10 mL) for 12 h at room temperature. The
reaction mixture was evaporated under reduced pressure. Et2O
(15 mL) was added to the residue, which was filtered and
washed with additional Et2O. The bromide 14 was used
immediately in subsequent reactions: TLC Rf 0.62 (CHCl3/
MeOH/NH4OH, 10:3:0.2, silica gel).
Gen er a l P r oced u r e for th e P r ep a r a tion of 2,4-Di-
a m in o-5-[(su bstitu ted N-m eth yla n ilin o)m eth yl]p yr r olo-
[2,3-d ]p yr im id in es (2-4, 17). To a solution of the bromide
14 (0.10 g, 0.40 mmol) in anhydrous DMF or DMAC (5 mL)
containing K2CO3 (0.10 g) was added N-methyl-substituted
aniline 16 (1.20 mmol). The mixture was stirred at room
temperature until 14 disappeared on TLC (5 h). The solvent
was then evaporated under reduced pressure. The residue was
dissolved in MeOH, and silica gel (2.00 g) was added to the
solution which was then evaporated to dryness to form a plug
which was loaded onto a dry silica gel column (2.4 × 20 cm).
The column was eluted with a gradient of 1% MeOH in CHCl3
to 10% MeOH in CHCl3. Fractions corresponding to the
product (TLC) were pooled and evaporated to dryness under
reduced pressure. The residue was triturated in cold Et2O,
and hexane(s) was added dropwise until a suspension formed
which was refrigerated for 2 h and filtered to yield the
products.
2,4-Dia m in o-5-[(2′,5′-d im e t h oxy-N -m e t h yla n ilin o)-
m eth yl]p yr r olo[2,3-d ]p yr im id in e (2): yield 0.04 g (30.7%);
mp 144-145 °C; TLC Rf 0.65 (CHCl3/MeOH/NH4OH, 10:3:0.1,
silica gel); 1H NMR (Me2SO-d6) δ 2.73 (s, 3 H, 9-CH3), 3.69 (s,
3 H, OCH3), 3.77 (s, 3 H, OCH3), 3.97 (s, 2 H, 8-CH2), 5.55 (bs,
2 H, NH2), 6.00 (bs, 2 H, NH2), 6.18 (s, 1 H, 6-CH), 6.61 (m, 1
H, 4′-CH), 6.70 (d, 1 H, 6′-CH), 6.91 (d, 1 H, 3′-CH), 10.59 (s,
1 H, 7-NH). Anal. Calcd for (C16H20N6‚0.5H2O) C, H, N.
2,4-Diam in o-5-[(3′,4′-dich lor o-N-m eth ylan ilin o)m eth yl]-
p yr r olo[2,3-d ]p yr im id in e (3). Fractions obtained from the
column were pooled and acidified (pH 4-5) with 1 N HCl
before evaporation and trituration with cold Et2O: yield 0.05
g (36%); mp >240 °C dec; TLC Rf 0.53 (CHCl3/MeOH/NH4OH,
10:3:0.1, silica gel); 1H NMR (Me2SO-d6) δ 2.87 (s, 3 H, 9-CH3),
4.51 (s, 2 H, 8-CH2), 5.49 (bs, 2 H, NH2), 6.04 (bs, 2 H, NH2),
2,4-Dia m in o-5-[[(3′,4′-d ich lor op h en yl)th io]m eth yl]p yr -
r olo[2,3-d ]p yr im id in e (6): yield 0.045 g (32%); mp 228-229
°C dec; TLC Rf 0.66 (CHCl3/MeOH/NH4OH, 10:3:0.1, silica gel);
1H NMR (Me2SO-d6) δ 4.41 (s, 2 H, 8-CH2), 5.54 (bs, 2 H, NH2),
6.25 (bs, 2 H, NH2), 6.62 (s, 1 H, 6-CH), 7.28 (m, 1 H, 6′-CH),
7.52 (d, 1 H, 2′-CH), 7.58 (d, 1 H, 5′-CH), 10.62 (s, 1 H, 7-NH).
Anal. Calcd for (C13H11N5SCl2) C, H, N, S, Cl.
2,4-Dia m in o-5-[(1′-n a p h th ylth io)m eth yl]p yr r olo[2,3-d ]-
p yr im id in e (7): yield 0.046 g (35%); mp >250 °C dec; TLC
1
Rf 0.66 (CHCl3/MeOH/NH4OH, 10:3:0.1, silica gel); H NMR
(Me2SO-d6) δ 4.42 (s, 2 H, 8-CH2), 5.47 (bs, 2 H, NH2), 6.17 (s,
2 H, NH2), 6.53 (s, 1 H, 6-CH), 7.46 (m, 1 H, C10H7), 7.56 (m,
1 H, C10H7), 7.59 (m, 2 H, C10H7), 7.77 (m, 1 H, C10H7), 7.91
(d, 1 H, C10H7), 8.18 (d, 1 H, C10H7), 10.53 (s, 1 H, 7-NH). Anal.
Calcd for (C17H15N5S‚0.5H2O) C, H, N, S.
2,4-Dia m in o-5-[(2′-n a p h th ylth io)m eth yl]p yr r olo[2,3-d ]-
p yr im id in e (8): yield 0.035 g (26%); mp >250 °C dec; TLC
1
Rf 0.65 (CHCl3/MeOH/NH4OH, 10:3:0.1, silica gel); H NMR
(Me2SO-d6) δ 4.50 (s, 2 H, 8-CH2), 5.51 (bs, 2 H, NH2), 6.21
(bs, 2 H, NH2), 6.69 (s, 1 H, 6-CH), 7.48 (m, 3 H, C10H7), 7.88
(m, 4 H, C10H7), 10.65 (s, 1 H, 7-NH). Anal. Calcd for
(C17H15N5S‚0.5H2O) C, H, N, S.
N-[4-[N-[(2,4-Dia m in op yr r olo[2,3-d ]p yr im id in -5-yl)-
m eth yl]m eth yla m in o]ben zoyl]-L-glu ta m ic Acid (10). The
pure ester 17 was stirred in 1 N NaOH (5 mL) for 24 h at
room temperature. Acidification with glacial AcOH to pH 5
(the pH briefly dropped below 5 and was readjusted with
NH4OH solution) afforded a light brown suspension which on

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 7 1177
Trial of Trimetrexate-Leucovorin for the treatment of Cerebral
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Novel 2,4-Diamino-5-Substituted pyrrolo-[2,3-d]pyrimidines as
Classical and Nonclassical Antifolate Inhibitors of Dihydrofolate
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L. Synthesis of 5-Methyl-5-deaza Nonclassical Antifolates as
Inhibitors of Dihydrofolate Reductases and as Potential Anti-
pneumocystis, Antitoxoplasma and Antitumor Agents. J . Med.
Chem. 1993, 36, 3437-3443.
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filtration and washing with water followed by Et2O afforded
0.035 g (50%) of an analytically pure light brown solid, 10:
mp 223-224 °C dec; TLC Rf 0.78 (3% NH4HCO3, cellulose);
1H NMR (Me2SO-d6) δ 1.93 (m, 1 H, Glu â-CH2), 2.08 (m, 1 H,
Glu â-CH2), 2.32 (m, 2 H, Glu γ-CH2), 2.91 (s, 3 H, 9-CH3),
4.33 (m, 1 H, Glu R-CH), 4.57 (s, 2 H, 8-CH2), 5.45 (bs, 2 H,
NH2), 5.98 (bs, 2 H, NH2), 6.41 (s, 1 H, 6-CH), 6.90 (d, 2 H, 3′-,
5′-CH), 7.74 (d, 2 H, 2′-,6′-CH), 8.07 (d, 1 H, CONH), 10.53 (s,
1 H, 7-NH). Anal. Calcd for (C20H23N7O5‚0.6H2O‚0.6NH3) C,
H, N.
Ack n ow led gm en t. This work was supported, in
part, by NIH Grants GM40998 (A.G.) and AI30900
(A.G.) and NIH Contract N01-AI-87240 (S.F.Q.) Division
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mational Studies by Dynamic NMR. 32. Enantiomerization of
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