Synthesis and biological activities of conformationally restricted, tricyclic nonclassical antifolates as inhibitors of dihydrofolate reductases

Article abstract of DOI:10.1021/jm960693n

Seven novel tricyclic pyrimido[4,5-c][2,7]naphthyridones 5-8 and the corresponding naphthyridines 9-11 were synthesized as conformationally restricted inhibitors of dihydrofolate reductase (DHFR) and as antitumor and/or antiinfectious agents. The analogues were designed to orient the side chain trimethoxyphenyl group in different conformationally defined positions in order to explore the effect of the side chain orientation on binding affinity and selectivity for DHFR from various species. The semirigid orientations were achieved by bridging the C5 and N10 of compound 12 with a N-ethyl bridge and by variation of the position of double bonds in rings B and C as well as substitution at the 2',6'- positions of the phenyl ring. The synthesis of compounds 5-11 were accomplished by cyclocondensation of the appropriate keto ester (as the biselectrophile) with 2,4,6-triaminopyrimidine to afford the lactam 5. The dehydrolactams 6 and 7 were prepared by air oxidation and PtO₂-catalyzed dehydrogenation of 7, respectively. The dichloro dehydro lactam 8 was obtained by refluxing lactam 5 and/or 6 in POCl₃ or a mixture of POCl₃/PCI₅. Compounds 9-11 were obtained by two methods, direct borane reduction of lactam 5 or 6 or thiation of the dipivoylated lactam 15 followed by reductive dethiation. Compounds 9-11 were interconverted by air oxidation or PtO₂-catalyzed reduction/oxidation, respectively. The compounds were evaluated as inhibitors of DHFR from Pneumocystis carinii (pc) and Toxoplasma gondii (tg) with rat liver (rl) serving as the reference mammalian enzyme. In the lactam series 5-8, the most unsaturated analogue 7 showed an IC₅₀ of 86 nM against rlDHFR, almost 100- fold more active than 5 and 3-fold more active than 6. The 2',6'-dichloro dehydro lactam 8 was less active than the corresponding dehydro lactam 6 against rlDHFR. In the naphthyridine series 9-11, the dehydro analogue 10 was more active than 9 against rlDHFR. The fully reduced analogue 11 (as a mixture of cis and trans isomers) was the most active in the naphthyridine series. The analogues were, in general, more inhibitory against rlDHFR than against pcDHFR, or tgDHFR, and thus lacked selectivity. In addition, they were less potent than the bicyclic compounds trimetrexate 3 (TMQ) and piritrixim 4 (PTX).

Full text of DOI:10.1021/jm960693n

1930
J . Med. Chem. 1997, 40, 1930-1936
Notes
Syn th esis a n d Biologica l Activities of Con for m a tion a lly Restr icted , Tr icyclic
Non cla ssica l An tifola tes a s In h ibitor s of Dih yd r ofola te Red u cta ses1
Aleem Gangjee,*,† J ufang Shi,† 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 4, 1996X
Seven novel tricyclic pyrimido[4,5-c][2,7]naphthyridones 5-8 and the corresponding naph-
thyridines 9-11 were synthesized as conformationally restricted inhibitors of dihydrofolate
reductase (DHFR) and as antitumor and/or antiinfectious agents. The analogues were designed
to orient the side chain trimethoxyphenyl group in different conformationally defined positions
in order to explore the effect of the side chain orientation on binding affinity and selectivity
for DHFR from various species. The semirigid orientations were achieved by bridging the C5
and N10 of compound 12 with a N-ethyl bridge and by variation of the position of double bonds
in rings B and C as well as substitution at the 2′,6′- positions of the phenyl ring. The synthesis
of compounds 5-11 were accomplished by cyclocondensation of the appropriate keto ester (as
the biselectrophile) with 2,4,6-triaminopyrimidine to afford the lactam 5. The dehydrolactams
6 and 7 were prepared by air oxidation and PtO2-catalyzed dehydrogenation of 7, respectively.
The dichloro dehydro lactam 8 was obtained by refluxing lactam 5 and/or 6 in POCl3 or a
mixture of POCl3/PCl5. Compounds 9-11 were obtained by two methods, direct borane
reduction of lactam 5 or 6 or thiation of the dipivoylated lactam 15 followed by reductive
dethiation. Compounds 9-11 were interconverted by air oxidation or PtO2-catalyzed reduction/
oxidation, respectively. The compounds were evaluated as inhibitors of DHFR from Pneu-
mocystis carinii (pc) and Toxoplasma gondii (tg) with rat liver (rl) serving as the reference
mammalian enzyme. In the lactam series 5-8, the most unsaturated analogue 7 showed an
IC50 of 86 nM against rlDHFR, almost 100-fold more active than 5 and 3-fold more active than
6. The 2′,6′-dichloro dehydro lactam 8 was less active than the corresponding dehydro lactam
6 against rlDHFR. In the naphthyridine series 9-11, the dehydro analogue 10 was more active
than 9 against rlDHFR. The fully reduced analogue 11 (as a mixture of cis and trans isomers)
was the most active in the naphthyridine series. The analogues were, in general, more
inhibitory against rlDHFR than against pcDHFR, or tgDHFR, and thus lacked selectivity. In
addition, they were less potent than the bicyclic compounds trimetrexate 3 (TMQ) and piritrixim
4 (PTX).
Dihydrofolate reductase (DHFR)3 catalyzes the reduc-
tion of folate or 7,8-dihydrofolate (FH2) to tetrahydro-
folate (FH4) and intimately couples with thymidylate
synthase (TS). TS is a crucial enzyme that catalyzes
the reductive methylation of 2′-deoxyuridine 5′-mono-
phosphate (dUMP) to 2′-deoxythymidine 5′-monophos-
phate (dTMP) utilizing 5,10-methylenetetrahydrofolate
as a cofactor which functions as the source of the methyl
group as well as the reductant. This is the exclusive
de novo source of dTMP; hence inhibition of DHFR or
TS activity, in the absence of salvage, leads to “thym-
ineless death”.4,5 Thus DHFR inhibition has long been
an attractive goal for the development of chemothera-
peutic agents.6
DHFR inhibitors, such as trimethoprim (1, TMP) or
pyrimethamine (2), with dihydropteroate synthase in-
hibitors such as sulfonamides. An alternative agent for
P. carinii infection is pentamidine.7 These standard
therapies have significant frequency and severity of
toxicity. Some of the adverse reactions associated with
combined therapy may be antibody-mediated because
the addition of leucovorin does not prevent side effects
such as neutropenia.8 Pentamidine has systemic ad-
verse effects, which in some cases may lead to perma-
nent diabetes.6,7 Trimetrexate 3 (TMQ),9 an antineo-
plastic agent, is a second line drug for P. carinii
pneumonia (PCP) and could be a more effective option
for initial as well as salvage therapy. It is a potent
DHFR inhibitor with an IC50 ) 42 nM against pcDHFR
and is 1000-1500 times more potent than TMP. Be-
cause of its potency and lack of selectivity, TMQ needs
to be administered with leucovorin, which is selectively
taken up by host cells and circumvents DHFR blockade,
leading to selective rescue of host cells. Piritrexim 4
(PTX), which is under phase I clinical trials as an
antineoplastic drug, also inhibits pcDHFR and tgDHFR
Opportunistic infections with Pneumocystis carinii
(pc) and Toxoplasma gondii (tg) remain a principal
cause of death in patients with Acquired Immunodefi-
ciency Syndrome (AIDS) in the United States. The
current therapeutic regimens involve combinations of
† Duquesne University.
‡ Indiana University School of Medicine.
X Abstract published in Advance ACS Abstracts, May 15, 1997.
S0022-2623(96)00693-0 CCC: $14.00 © 1997 American Chemical Society

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1931
at IC50 ) 19.3 and 17.0 nM, respectively, similar to that
observed for TMQ. P. carinii and T. gondii cell culture
inhibition in vitro by PTX and TMQ occurs at concen-
tration of 0.1 -1.0 and 0.1 mg/ml, respectively. In
animal models, PTX was also effective and prophylactic
against PCP in rats.10
Despite their impressive potencies against pcDHFR
and tgDHFR, both TMQ and PTX are nonspecific DHFR
inhibitors and significantly inhibit all other sources of
DHFR including human. Therefore TMQ and PTX,
when used alone to treat opportunistic infections, have
the potential of severe toxicity.
of 12 by bridging the 5-CH3 and 10-CH3 of 12 to afford
tricyclic analogues 5, 6, 7, 9, and 11 was a logical and
worthwhile goal.
Ch em istr y
The synthesis of angular 5,6-disubstituted pyrido[2,3-
d]pyrimidones has been reported by Grivsky et al.15 and
Hurlbert et al.,16 and later by Gangjee et al.17 and
DeGraw et al.18 via the cyclocondensation of substituted
6-aminopyrimidines with appropriate â-keto esters,
followed by the conversion of the resulting pyrido[2,3-
d]pyrimidones to the corresponding pyrido[2,3-d]-
pyrimidines. Adoption of this procedure involved the
synthesis of the keto ester 14, which was accomplished
(Scheme 1) by the Dieckmann cyclization of 13, which
in turn was obtained from the Michael addition19 of
3,4,5-trimethoxyaniline with methyl acrylate. The cy-
clocondensation of 14 with 2,4,6-triaminopyrimidine in
diphenyl ether at reflux (190-200 °C) afforded 6. The
FAB MS spectrum of the product 6 showed that the
molecular weight was two units less than that of the
lactam 5, which suggested that the product could be the
dehydrogenated lactam 6 or its isomer 6a . Assignment
of the structure of the product as the dehydro lactam 6
was based on its 1H NMR spectrum, which showed a
downfield nonexchangeable signal at 9.09 ppm (s, 1H),
which was assigned to the olefinic proton Hc. The
AA′BB′ portion of the spectrum at 4.03 (t, 2 H) and 3.27
(t, 2 H) was assigned to the vicinal methylene protons
Ha and Hb.
The cyclocondensation of keto ester 14 and triami-
nopyrimidine in glacial HOAc at 90-95 °C afforded the
lactam 5 as the major product. The reaction tempera-
ture (90-95 °C) was critical since temperatures over 95
°C afforded lower yields of 5 and temperatures lower
than 90 °C afforded incomplete reaction.
Assignment of 5 as the angular structure rather than
the linear isomer was achieved by comparison of similar
structures in the literature.20,21 The 13C NMR of 5
showed the carbonyl carbon signal at 165.59 ppm, which
was in good agreement with the chemical shifts ob-
served for R-pyridones and similar lactams.19,20 Further
confirmation of the angular nature of 5 was provided
from NOE studies. In difference NOE studies a positive
NOE was observed between the 10-CH2 (t, 2H, 3.15
ppm) and the 1-NH2 (5.92 ppm) protons.
Attempts were made at this stage to convert the
lactam 5 to the corresponding pyrido[2,3-d]pyrimidine
9 via one of the two-step literature reactions i.e. either
chlorination-dechlorination or thiation-dethiation pro-
cedures, reported by Hurlbert et al.29 and Grivsky et al.28
A modified procedure of Grivsky et al. for chlorination
of the lactam 5 using thionyl chloride in DMF afforded
only the dehydrolactam 6, which was confirmed by FAB
MS. Several procedures involving time and tempera-
ture variations and different chlorinating reagents were
attempted for the chlorination of the lactam 5, including
POCl3 and POCl3:PCl5 (1:1).22 The latter reaction
afforded a mixture. This mixture showed three fluo-
rescent spots very close to each other on TLC. The FAB
MS of the mixture showed three clusters ([M + 1] ions)
at m/ z 463, 499, 533; each cluster had its own isotope
peaks (M + 2, M + 4, M + 6), indicating that multiple
chlorination had occurred. This mixture, upon being
stirred in 1 N NaOH-MeOH (1:1) solution, afforded 8
TMP is a bacterial DHFR selective inhibitor, and its
binding to various DHFR has been characterized in
considerable detail both biochemically and crystallo-
graphically.11 The trimethoxyphenyl group of TMP is
bound to bacterial and vertebrate DHFR in two very
different conformations, which result from the different
geometry of DHFR active sites and accounts, in part,
for the preferential binding and hence the selectivity of
TMP for E. coli (ec) DHFR compared to chicken liver
(cl) DHFR.
Since conformations of the side chain of TMP plays
an important role in the selectivity of TMP for bacterial
DHFR, it was of interest to determine if restricting the
flexible side chain of TMQ in different semirigid orien-
tations would provide for selectivity against pcDHFR
and/or tgDHFR. Tricyclic analogs 5, 6, 7, 9, and 11 were
designed as conformationally restricted analogues of
TMQ in which τ1 and τ2 were partially restricted by
incorporation in a six-membered ring. In addition,
different orientations of the side chain were achieved
by variation of the extent of saturation of the B and C
rings of the resulting tricyclic ring system in 5, 6, 7, 9,
and 11. Molecular modeling using SYBYL12 and its
MAXIMIN and SEARCH options indicated that each of
the tricyclic analogues 5, 6, 7, 9, and 11 provides a
different orientation of the trimethoxyphenyl side chain,
each of which is different from that of the DHFR bound
conformation of TMQ.13 We14 have reported 2,4-di-
amino-5-methyl-6-[(3′,4′,5′-trimethoxy-N-methylanilino)-
methyl]pyrido[2,3-d]pyrimidine (12) as a 8-aza-N10-
methyl analogue of TMQ and found it to be more potent
and significantly more selective than TMQ against
tgDHFR. Thus restricting the torsion angles τ1 and τ2

1932 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Notes
Sch em e 1
as a major component, which gave an [M + H]+ ion of
Desulfuration of 16 to 9 was accomplished by reflux-
ing it in freshly prepared Raney Ni suspended in
anhydrous ethanol. Depivaloylation was carried out in
MeOH-1 N KOH or acetone-1 N KOH under nitrogen
which was necessary to prevent air oxidation of the
1
m/ z 463, 465 in its FAB mass spectrum. Its H NMR
showed the absence of the aliphatic methylene protons
as well as the phenyl aromatic protons; however three
new aromatic protons at 9.35, 8.05, and 7.06 ppm were
observed, suggesting skeletal aromatization and 2,6-
dichlorination on the phenyl ring.
1
product. The H NMR spectrum in DMSO-d6 of com-
pound 9 showed the desired signals, and the spectrum
in TFA-d showed an aromatic proton at 8.83 ppm. The
FAB MS has the required molecular weight (MH+ m/ z
383) for 9. The dehydro compound 10 was obtained via
desulfuration and depivaloylation of 16 (liquid NH3)
followed by air oxidation during workup. The structure
The three lactams 5, 6, and 7 (Scheme 1) were
interconvertible. The dehydro lactam 6, upon hydro-
genation in glacial HOAc and 50 psi of H2 with PtO2,
was reduced back to the lactam 5. The dehydro lactam
6, unlike the lactam 5, was quite stable under harsh
conditions such as refluxing glacial HOAc or 75% HOAc
and in the presence of 5% Pd on charcoal. However,
the dehydro lactam 6 and/or lactam 5 in 75% HOAc or
DMSO in the presence of PtO2 at 80-90 °C afforded
compound 7. Its 1H NMR which showed three new
aromatic signals at 9.32 (s, 1 H), 7.72 (d, 1 H, J ) 6.7
Hz), and 6.52 ppm (d, 1 H, J ) 6.7 Hz). This coupling
pattern, especially the two sets of doublets, along with
elemental analysis and FAB MS confirmed structure 7.
The failure of the chlorination and thiation of lactam
5 was attributed to the poor solubility of 5. Solubility
of some insoluble folic acid analogues were reported23
to be greatly improved by pivaloylation of the amino
groups. The pivaloylated derivative 15 was obtained
(Scheme 2) by refluxing 5 in pivalic anhydride. Thiation
of the pivaloylated lactam 15 by Lawesson’s reagent
[2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphet-
ane 2,4-disulfide] in benzene afforded the desired thi-
olactam 16 in 30% yield. The structure of 16 was
1
of 10 was confirmed by H NMR, elemental analysis,
and FAB MS. The mass spectrum showed an [M + H]
ion of m/ z 381, 2 units less than 9 (m/ z 383, [M + H]),
indicating that a double bond had been introduced. The
1H NMR revealed three aromatic peaks at 9.30 (s, 1H),
7.90 (m, 1H, overlapped with NH2 protons), and 6.94
ppm (d, 1H), corresponding to H-6, H-9, and H-10,
respectively. A methylene singlet at 4.05 ppm was
assigned to the CH2 at C-7, and the peak at 3.25 ppm
was assigned to MeOH, which was confirmed on el-
emental analysis. The coupling pattern and decoupling
experiments confirmed the structure of compound 10.
The doublet of the H-10 was attributed to adjacent
coupling with the H-9, which in turn occurred as a
multiplet due to adjacent coupling with H-10 as well as
with protonated N-8. Irradiation at the H-10 resulted
in the simplification of the signal of H-9 (doublet like),
further supporting the protonated form of N-8.
The hexahydro compound 11 was obtained by cata-
lytic reduction (PtO2/H2) of compound 9. On the basis
of literature reports24 and results from Gangjee et al.,19
1
confirmed by FAB MS, H NMR, and elemental analy-
sis.

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1933
Sch em e 2
Ta ble 1. Inhibitory Concentrations (IC50, nM) against DHFRs
and Selectivity Ratios (IC50 rlDHFR/IC50 pc- or tgDHFR)a
bicyclic pyrido[2,3-d]pyrimidine analogues were known
to undergo benzylic type cleavage during hydrogenation
catalyzed by PtO2; however the tricyclic system 9 was
not subject to benzylic cleavage in the presence of PtO2.
The TLC of the reaction mixture immediately after
hydrogenation showed a single spot. The FAB MS of
the reaction sample immediately after hydrogenation
gave the desired [M + H] ion of m/ z 387. However,
compound 11 was unstable and air-oxidized in the
presence of a trace amount of PtO2 to afford compound
10. After the exposure of the reaction mixture to air
for less than 10 min in the presence of the catalyst, the
FAB MS of the mixture showed a predominant [M +
H] peak at m/ z 381 (10) as well as at 397 (lactam 6),
indicating that three double bonds had been introduced
into 11. Thus the workup following hydrogenation of 9
to 11 needed extreme care. While this work was in
progress, DeGraw et al.17 reported a direct method of
lactam reduction with borane. Using a modification of
DeGraw’s method, the mixture of the lactam 5 and the
dehydrolactam 6 (obtained in Scheme 1) were subjected
to borane reduction. Both lactam and dehydrolactam
disappeared from the TLC, and three new components
were formed (TLC). The EI MS of this mixture, after
workup, showed two clusters of peaks with two molec-
ular ions at m/ z 382 (9) and 386 (11). No molecular
ion for 10 at m/ z 380 was observed. A single product
was obtained by stirring the mixture in HOAc-MeOH.
Under these condition, the tetrahydro compound 11 was
aromatized by the loss of four protons of the B ring and
converted to 9. The product obtained was identical in
all respects to 9 described above.
compd P. carinii (pc) T. gondii (tg) rat liver (rl) rl/pc rl/tg
5
6
7
>5000
4280
2400
>5000
240
1120
520
0.06 0.21
0.04 0.17
86
8
9
>1000
>2270
4600
2200
13.2
2300
500
<0.5
0.2
>2700
10
11
12
TMQ
PTX
760
290
0.85
10
17
470
0.1
0.62
160
7.6
3
1.5
0.07 0.55
0.58 8.94
0.07 0.3
0.05 0.09
42
31
a
These assay were carried out at 37 °C under conditions of
substrate (90 µM dihydrofolic acid) and cofactor (119 µM NADPH)
in the presence of 150 mM KCl.24,25
Biologica l Eva lu a tion a n d Discu ssion
Compounds 5-11 were evaluated as inhibitors of
DHFR from P. carinii and T. gondii. Rat liver (rl)
DHFR served as the mammalian reference. The IC50
values along with the selectivity ratios are listed in
Table 1.25,26 In the lactam series 5-8, the most active
analogue was the naphthyridone 7, with the greatest
extent of unsaturation and consequently planarity.
Compounds 5 and 6 in which the C ring is not planar
and more flexible than that of 7 results in a decrease
in inhibitory potency against all three enzymes. Sub-
stitution of electron-withdrawing chlorine groups in the
phenyl ring as in 8 also leads to a decrease in potency
compared to 7.
For the naphthyridines 9-11, the most potent ana-
logue against all three DHFRs was 11, the most
saturated, least planar, and consequently the most
flexible analogue. This result is in contrast to the
naphthyridones where planarity in the C ring resulted
in the greatest potency.

1934 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Notes
Compound 13 was collected as white crystals (10.00 g, 47%)
which separated (mp 77-78 °C). The crystals were washed
thoroughly with ethyl ether: TLC Rf 0.57 (CHCl3:Et2O 9:2);
1H NMR (60 MHz, CDCl3) δ 2.55 (t, 4 H, 2 CH2COO), 3.47-
3.80 (m, 19 H, 3 CH3O, 2 COOCH3 and 2 CH2N), 5.95 (s, 2 H,
Ar-H); IR (Nujol) 1725, 1250 (COO); MS(CI) m/ z 356 (MH+).
Anal. (C17H25NO7) C, H, N.
Meth yl 4-Oxo-1-(3′,4′,5′-tr im eth oxyp h en yl)p ip er id in e-
3-ca r boxyla te (14). A solution of 13 (10.00 g, 28 mmol) in
dry toluene (30 mL) was added to a suspension of NaOCH3
(3.00 g, 56 mmol) in toluene (70 mL) under gentle reflux which
was continued for 1 h. The reaction mixture was cooled in an
ice bath, and the solid was collected by filtration, washed
thoroughly with ethyl ether, and air-dried for 30 min. The
solid was suspended in ice-H2O (100 mL) and neutralized with
6 N HCl. Compound 14 was obtained on filtration as a white
precipitate (8.20 g, 90% yield): TLC Rf 0.5 (CHCl3:Et2O, 9:2);
mp 105-106 °C; 1H NMR (60 MHz, CDCl3) δ 2.65 (m, 2 H,
CH2CO), 3.48 (t, 2 H, NCH2), 3.78-3.92 (m, 15 H, 3 CH3O and
COOCH3 and NCH2CH-), 4.28 (t, 1 H, CHCOO), 6.32 (s, 2 H,
Ar-H); IR (Nujol) 1670, 1630 cm-1(â-keto ester); CI MS m/ z
324, MH+. Anal. (C16H21NO6) C, H, N.
None of the analogues 5-11 were selective for pcDH-
FR or tgDHFR, and the potency of the analogues was
severalfold less than TMQ. Thus restriction of τ1 and
τ2 of 12 by incorporation into a six-membered ring is
detrimental to potency and does not afford selectivity
for pcDHFR or tgDHFR. The effect of τ1 and τ2
restriction by incorporation within a six-membered ring
also severely restricts the side chain trimethoxyphenyl
moiety; thus the orientation of the side chain is almost
entirely predicted on the conformation of the rings and
the orientation of the nitrogen of the C ring. This is
reflected in the observed variation in potency with the
extent of unsaturation in each of the naphthyridones
and naphthyridines 5-11. Obviously the orientations
of the trimethoxyphenyl ring in the analogues 5-11 are
not conducive to potency or selectivity.
We have reported the 8-aza-N10-methyl analogue
1219 of TMQ. It was more potent than TMQ and
significantly more selective than TMQ against tgDHFR
(IC50 0.8 nM; rl/tg ) 8.14). The activity and selectivity
of 12 suggests that the additional substituents on the
5- and 10-positions of 12 to afford the tricyclic analogues
5-11 are probably not responsible for the decrease in
potency and lack of selectivity and lends further cre-
dence to the idea that the inappropriate orientation of
the side chain trimethoxyphenyl ring in 5-11 is re-
sponsible for the lack of potency and selectivity of these
analogues against pcDHFR and tgDHFR.
1,3-Dia m in o-8-(3′,4′,5′-tr im eth oxyp h en yl)-7,8,9,10-tet-
r a h yd r op yr im id o[4,5-c][2,7]n a p h th yr id in -6-(5H)-on e (5).
A mixture of 2,4,6-triaminopyrimidine (7.50 g, 60.0 mmol) and
keto ester 14 (20.00 g, 61.70 mmol) in glacial HOAc (400 mL)
was stirred under gentle reflux at 90-95 °C for 36 h. After
the mixture was cooled to room temperature, the precipitated
acetate salt (7.30 g containing unreacted triaminopyrimidine)
was collected, dissolved in hot H2O, and left at room temper-
ature for 12 h. The solid which separated was collected by
filtration and recrystallized from MeOH. Compound 5 was
obtained as pale yellow crystals (3.00 g, 12.5% yield), which
were homogeneous on TLC [Rf 0.34 (EtOAc:MeOH, 4:1)]: mp
>300 °C; 1H NMR (DMSO-d6) δ 3.05 (s, 2 H), 3.15 (t, 2 H),
3.62 and 3.73 (2 s, 9 H, 3CH3O), 3.90 (t, 2 H), 5.51 (br s, 2 H,
NH2), 5.92 (br s, 2 H, NH2), 6.58 (s, 2 H, ArH), 8.68 (br s, 1 H,
NH); 13C NMR (decoupled) showed 17 singlets (2 pairs of
symmetric carbons of phenyl 2′ and 6′, and 3′ and 5′ were
overlapped), 165.59 ppm (CdO); CI MS m/ z 399 (MH+). Anal.
(C19H22N6O4‚0.8H2O) C, H, N.
Exp er im en ta l Section
Starting materials used in synthetic procedures were ob-
tained from Aldrich Chemical Co., Milwaukee, WI. Melting
points were determined on a Fisher-J ohns melting point
apparatus and are uncorrected. Catalytic hydrogenation
reactions were performed in a Parr pressure reaction ap-
paratus. Infrared (IR) spectra were determined neat or as
Nujol mulls on a Perkin-Elmer 1430 ratio recording infrared
spectrophotometer and are reported in reciprocal centimeters.
Proton nuclear magnetic resonance (1H NMR) spectra were
recorded on a Varian EM-360 (60 MHz) or a Bruker WH-300
(300 MHz) spectrometer. Only 60 MHz spectra are indicated
in the experimental text, and all others unspecified (1H NMR)
are 300 MHz. The 13C NMR spectra were obtained on a
Bruker WH-300 instrument at 75.46 MHz; chemical shift
values are expressed in δ values (parts per million) relative
to tetramethylsilane as an internal standard; s ) singlet, d )
doublet, t ) triplet, m ) multiplet. Mass spectra (MS) were
recorded on a Varian MATCH-311 mass spectrometer in a fast-
atom-bombardment (FAB) mode or as indicated. Thin layer
chromatography (TLC) was performed on Eastman Kodak
chromatogram sheets (silica gel) with florescent indicator.
Proportions of solvents stated and used for TLC are by volume.
Spots on TLC were detected by UV light at either 254 or 350
nm. Elemental analyses were carried out by Atlantic Microlab,
Inc., Necross, GA, and/or Galbraith Laboratories, Knoxville,
TN. Element compositions are within (0.4% of the calculated
values. Fractional moles of water or organic solvents fre-
quently found in some analytical samples of antifolates could
not be removed in spite of drying in vacuo and were confirmed
by their presence in the 1H NMR spectrum.
1,3-Dia m in o-8-(3′,4′,5′-tr im eth oxyph en yl)-9,10-d ih yd r o-
p yr im id o[4,5-c][2,7]n a p h t h yr id in -6(5H ,8H )-on e
(6).
Meth od A. In a flask equipped with a Dean-Stark trap, a
mixture of the keto ester 14 (4.85 g, 15 mmol), 2,4,6-triami-
nopyrimidine (1.88 g, 15 mmol), and diphenyl ether (50 mL)
was heated rapidly with vigorous stirring to 195 °C and
maintained at 195-200 °C until no additional water-ethanol
mixture distilled (1 h). The reaction mixture was cooled to
room temperature, methanol (40 mL) was added, and the solid
that precipitated (2.10 g) was collected by filtration. The crude
product was suspended in boiling H2O (100 mL) and filtered,
and the residue was washed with hot water, followed by
methanol (40 mL) and ether (50 mL), to afford a brown solid.
Recrystallization from methanol and concentrated HCl af-
forded 6 as its hydrochloride salt (0.74 g, 12% yield): mp >300
°C (295 °C dec); TLC Rf 0.17 (EtOAc:MeOH, 4:1); 1H NMR
(DMSO-d6) δ 3.27 (t, 2 H, CH2CH2N), 3.67 (s, 3 H, 4′-OCH3),
3.76 (s, 6 H, 3′,5′-OCH3), 4.03 (t, 2 H, CH2), 6.75 (s, 2 H, Ar-
H), 7.49-7.65 (br peak, 2 H, NH2), 8.63 (br s, 2H, NH2), 9.09
(s, 1 H, C7H); MS m/ z 397 (MH+). Anal. (C19H20N6O4‚1.0HCl‚
1.0H2O) C, H, N.
Method B. This dehydro lactam 6 was also obtained by
gently heating 5 at 80 °C in HMPT (also in solvents or reagents
such as DMSO, POCl3, or glacial HOAc) for 12 h. Workup as
described above afforded a pure product in 50% yield without
additional purification, which was identical in all respects to
6 obtained by method A.
1,3-Dia m in o-8-(3′,4′,5′-tr im eth oxyp h en yl)p yr im id o[4,5-
c][2,7]n a p h th yr id in -6-on e (7). To a solution of the dehydro
lactam 6 (0.10 g, 0.25 mmol) in 75% HOAc (50 mL) was added
PtO2 (0.016 g). The reaction mixture was stirred and refluxed
for 7 h. After the mixture was cooled to room temperature,
the catalyst was filtered and the filtrate evaporated to dryness.
The residue was dissolved in MeOH (50 mL) and clarified by
filtration through Celite. The filtrate was concentrated to 5
Dim eth yl 3′,4′,5′-Tr im eth oxya n ilin e-â,â′-d ip r op ion a te
(13). A mixture of 3,4,5-trimethoxylaniline (10.90 g, 59.60
mmol), methyl acrylate (25.80 g, 300 mmol), and glacial HOAc
(15 mL) was heated under reflux with stirring for 24 h. Excess
methyl acrylate and HOAc were removed under reduced
pressure (H2O aspirator, bath temperature 70 °C). To the
residue was added CH2Cl2:hexane (1:1) (500 mL). The solution
was filtered through a funnel column (3.5 in. in diameter)
containing 80 g of silica gel. The filtrate was concentrated to
a viscous oil, diluted with ethyl ether (200 mL), and kept at
room temperature for 2 h and then in a freezer overnight.

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1935
mL and left in the freezer overnight. The dark brown solid
which formed was collected. The solid was then dissolved in
TFA and carefully filtered through Celite and the filtrate
evaporated. To the residue was added MeOH and the solution
kept in a freezer overnight. The dark-red solid which sepa-
rated was collected by filtration to afford 0.07 g (66% yield) of
6.59 (s, 2 H, Ar-H), 9.72 (s, 1 H, NH), 9.82 (s, 1 H, NH), 10.12
(s, 1 H, NH); MS m/ z 583 (MH+). Anal. (C29H38N6O5S‚0.5H2O)
C, H, N, S.
1,3-Dia m in o-8-(3′,4′,5′-tr im eth oxyp h en yl)-7,8,9,10-tet-
r a h yd r o-2H -p yr im id o[4,5-c][2,7]n a p h t h yr id in e
(9).
Meth od A. A mixture of compound 16 (0.50 g, 0.86 mmol)
and anhydrous Raney Ni (3 g) were suspended in anhydrous
EtOH (20 mL) and slowly heated to reflux for 1 h. The reaction
mixture was cooled, the insoluble material filtered, and the
filtrate evaporated to dryness. The TLC (CHCl3:CH3OH, 9:2,
with 3 drops of NH4OH) of the residue showed three prod-
ucts: a mixture of 9 and monopivoylated and dipivoylated
derivatives which precipitated on addition of ether. The crude
material (0.45 g) was collected by filtration and used for the
next step without further purification.
1
7: mp >300 °C; H NMR (DMSO-d6-D2O) δ 3.71 (s, 3 H, 4′-
OCH3), 3.77 (s, 6 H, 3′,5′-OCH3), 6.83 (s, 2 H, Ar-2′,6′), 6.53
(d, 1H, H9, J ) 6.7 Hz), 7.72 (d, 1H, H10, J ) 6.7 Hz), 9.32 (s,
1H, H7); MS m/ z 395 (MH+). Anal. (C19H18O4N6‚0.8CF3-
COOH‚1.6H2O) C, H, N.
1,3-Diam in o-8-(3′,4′,5′-tr im eth oxy-2′,6′-dich lor oph en yl)-
p yr im id o[4,5-c][2,7]n a p h th yr id in -6-on e (8). To a solution
of PCl5 (4.00 g) and POCl3 (40 mL) was added the lactam 5
(1.00 g, 2.51 mmol), and the reaction mixture was stirred and
heated at 115-120 °C for 3.5 h. Excess POCl3 was removed
under reduced pressure (H2O aspirator, bath temperature 65
°C). The residue was added in portions to crushed ice and
neutralized carefully with MeOH-1 N NaOH (1:1). After the
mixture was stirred at room temperature for 24 h, a yellow
solid was obtained. The solid was collected by filtration and
suspended in H2O, stirred for an additional 24 h, and adjusted
to pH 3-4 with 6 N HCl. The solid obtained was collected by
filtration and dissolved in a minimum amount of AcOH,
concentrated to dryness, and triturated with MeOH:EtOAc (1:
5) . The solution was kept in a freezer overnight. Compound
8 separated as a brown solid (0.45 g, 36% yield) and was
collected as the HCl salt: mp > 300 °C; TLC Rf 0.5 (MeOH:
EtOAc, 1:9, with a few drops of NH4OH); 1H NMR (DMSO-d6)
δ 3.68 (s, 3 H, 4′-OCH3), 3.80 (s, 6 H, 3′,5′-OCH3), 7.06 (s, 1 H,
9- or 10-H), 8.05 (s, 1 H, 9- or 10-H), 9.35 (s, 1 H, 7H); MS
m/ z 463 (MH+). Anal. (C19H16N6O4Cl2‚1.7HCl‚0.5H2O‚0.3CH3-
COOH) C, H, N.
To a solution of the above mixture (0.125 g) in acetone (15
mL) was added 1 N KOH (10 mL), and the reaction mixture
was bubbled with nitrogen for 5 min, stoppered tightly, and
stirred at room temperature for 10 days. The reaction mixture
was then concentrated to half its volume and cooled in a freezer
for about 8-10 min, and the resulting solid (0.025 g) was
collected. The solid was dissolved in a minimum amount of
glacial HOAc and diluted with ethanol to give 9 (0.020 g, 20%
yield) as an acetate salt: TLC Rf 0.31 (CHCl3:CH3OH 9:2, with
3 drops of NH4OH); mp >300 °C (275 °C dec); 1H NMR(DMSO-
d6) δ 1.89 (s, 6 H, 2CH3COOH), 3.01 (t, 2 H, CH2CH2N), 3.57
(t, 2 H, CH2CH2N, overlapped with singlet at 3.56 ppm), 3.56
(s, 3 H, 4′-OCH3), 3.77 (s, 6 H, 2-OCH3), 4.37(s, 2 H, CH2),
6.29 (s, 2 H, Ar-H), 6.49 (br s, 2 H, NH2), 7.62 (br s, 2 H, NH2,
1
exchangeable), 8.25 (s, 1 H, C6H); H NMR (TFA-d) δ 3.84 (t,
2 H, CH2CH2N), 4.02 and 4.04 (2 s, 9 H, 3′-4′-5′-OCH3), 4.40
(t, 2 H, CH2CH2N), 5.15 (s, 2 H, CH2), 7.02 (s, 2 H, Ar-H) 8.83
(s, 1 H, C6H); MS m/ z 383 (MH+). Anal. (C19H22N6O3‚2CH3-
COOH) C, H, N.
1,3-Bis(p iva loyla m in o)-8-(3′,4′,5-t r im et h oxyp h en yl)-
7,8,9,10-t et r a h yd r op yr im id o[4,5-c][2,7]n a p h t h yr id in -6-
(5H)-on e (15). A mixture of 5 (1.00 g, 2.51 mmol) and pivaloyl
anhydride (9.18 g, 52.7 mmole) was refluxed with stirring
under nitrogen for 2 h (bath temperature 195-210 °C). The
reaction mixture was then cooled to 100 °C and rapidly filtered
through Celite. To the filtrate was added ethyl ether (100 mL),
and the mixture was placed in a freezer overnight. Compound
15 was obtained as a white solid (1.20 g, 84%), which was
collected by filtration. An analytical sample was crystallized
from a mixture of methanol and ether: mp 188-190 °C; TLC
Rf 0.3 (EtOAc); 1H NMR (DMSO-d6) δ 1.18 (2 s, 18 H, 2
(CH3)3C), 2.59 (t, 2 H, CH2CH2N), 3.27 (s, 2 H, CH2), 3.66 (s,
3 H, 4′-OCH3), 3.73 (s, 6 H, 3′,5′-OCH3), 3.75 (t, 2 H, CH2CH2N,
overlapped with peak at 3.73 ppm), 6.56 (s, 2 H, Ar-H), 9.68
(s, 1 H, NH), 9.79 (s, 1 H, NH), 9.89 (s, 1 H, NH); MS m/ z 567
(MH+). Anal. (C29H38N6O6‚0.5H2O) C, H, N.
1,3-Bis(p iva loyla m in o)-8-(3′,4′,5′-t r im et h oxyp h en yl)-
7,8,9,10-tetr a h yd r op yr im id o[4,5-c][2,7]n a p h th yr id in e-6-
(5H)-th ion e (16). A mixture of the pivaloylated lactam 15
(0.50 g, 0.88 mmol), Lawesson’s reagent (0.40 g, 0.1 mmol) and
50 mL benzene in a two-necked flask equipped with a Dean-
Stark trap and a nitrogen inlet was heated slowly with stirring
to 85 °C and maintained at 85-90 °C for 1.5 h. During this
period, about 40 mL of benzene was distilled. The reaction
mixture was diluted with ethyl acetate, transferred to a
separatory funnel, and washed with H2O (50 mL × 4). The
organic layer was dried over MgSO4 and evaporated to dryness.
The solid residue obtained was loaded onto a column (50 g
silica gel) and eluted with ethyl acetate. The fractions with a
Rf 0.61 (silica gel, EtOAc) was a side product (dithiated
compound). The desired fractions with TLC Rf 0.48 (EtOAc)
were combined and concentrated to dryness. The yellow solid
(0.28 g) obtained was washed with ethyl ether. Further
purification was carried out by first dissolving it in MeOH,
followed by evaporation of MeOH. To the residue was slowly
added 20 mL of ethyl ether to afford 16 (0.15 g 29% yield) as
a crystalline solid: TLC Rf 0.48 (EtOAc); mp 249-251 °C; 1H
NMR (DMSO-d6) δ 1.19 (2s, 18 H, 2 C(CH3)3), 2.70 (t, 2 H,
CH2CH2, J ) 7.0 Hz), 3.57 (s, 2 H, CH2), 3.65 (s, 3 H, OCH3)
3.72 (s, 6 H, 3′,5′-OCH3), 3.78 (t, 2 H, CH2CH2, J ) 7.0 Hz),
Meth od B. Borane in THF (1 M, 25 mL) was added to a
suspension of the lactam 5 (2.0 g, 5.01 mmol) in anhydrous
THF (25 mL) under nitrogen. The reaction mixture was
stirred at room temperature for 18 h, cooled in an ice bath,
and adjusted to pH 2 by adding 6 N HCl dropwise. The
mixture was evaporated, water (25 mL) was added, and the
pH was adjusted to 7-8 with 1 N NaOH. The precipitate
which formed (0.2 g) was filtered and the filtrate extracted
with CHCl3 (50 mL × 3). The organic extracts were combined,
dried (MgSO4), and concentrated to 3-5 mL. The resulting
solution was kept in a freezer overnight to afford a solid (0.45
g, 23%), which separated and was collected by filtration. The
TLC of this solid showed two spots, Rf 0.31 and 0.40 (CHCl3:
MeOH, 9:2), with 3 drops of NH4OH). The solid was suspended
in MeOH (10 mL), and glacial HOAc was added dropwise to
the suspension until the solid just dissolved. The solution was
stirred at room temperature (overnight) until the TLC showed
a single spot. Compound 9 (0.35 g, 18% yield) was then
collected by filtration and was identical in all respect to that
obtained from method A.
1,3-Dia m in o-8-(3′,4′,5′-tr im eth oxyp h en yl)-7,8-d ih yd r o-
p yr im id o[4,5-c][2,7]n a p h th yr id in e (10). Compound 16
(0.50 g, 0.86 mmol) and anhydrous Raney Ni (3.0 g) were
suspended in absolute ethanol (20 mL) and gently heated to
reflux for 1 h. The reaction mixture was cooled to room
temperature, the Raney Ni was filtered, and the filtrate was
evaporated to dryness. The TLC (CHCl3:MeOH, 9:2) of this
residue showed three components. The mixture was directly
used for depivoylation without further purification. A solution
of the mixture (0.45 g) in CH2Cl2 was placed in a steel vessel
and NH3 gas allowed to bubbled through at -78 °C for 10 min.
The reaction mixture was sealed and stirred for 72 h at room
temperature, after which the solution was allowed to evaporate
at room temperature overnight in a hood. MeOH was added
to the solid residue, and the solution formed was stirred for 7
days at room temperature. The resulting solid (0.20 g, 61%
yield ) was collected: MS m/ z 381 (contaminated with about
20% m/ z 383 according to the height of the peak in FAB MS),
MH+ for C19H20O3N6. An analytical sample was prepared by
dissolving the solid in TFA followed by filtration through Celite
and evaporation to dryness. To the residue was added MeOH,
and the insoluble material was removed by filtration. The

1936 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Notes
(7) Rosenblatt, J . E. Antiparasitic Agents. Mayo Clin. Proc. 1992,
67, 276-287.
filtrate was kept in a freezer overnight to afford 0.075 g, 22%
yield, of 10 after filtration: TLC of this product showed a
baseline-tailing spot due to its poor solubility in EtOAc:MeOH
(4:1) or CHCl3:MeOH (9:2) with 3 drops of NH4OH: 1H NMR
(DMSO-d6) δ 3.70 (s, 3 H, 4′-OCH3), 3.77 (s, 6 H, 3′,5′-OCH3),
4.05 (s, 2 H, CH2), 6.70 (br s, 2 H NH2), 6.75 (s, 2 H, ArH,
overlapped with 6.70), 6.94 (d, 1 H, H-10), 7.70 (very broad
peak, 2 H, NH2), 7.90 (m, 1 H, H-9), 9.30 (s, 1 H, C7-H). Anal.
(C19H20N6O3‚CF3COOH‚1.5CH3OH). C, H, N.
(8) Queener, S. F. Inhibition of Pneumocystis DHFR by Analogs of
Pyrimethamine, Methotrexate and Trimetrexate. J . Protozool.
1991, 38, 154s-157s
(9) Queener, S. F.; Bartlett, M. S.; J ay, M. A.; Durkin, M. M.; Smith,
J . W. Activity of Lipid-soluble Inhibitors of Dihydrofolate Re-
ductase Against Pneumocystis carinii in Culture and in a Rat
Model of Infection. Antimicrob. Agents Chemother. 1987, 31,
1323-1327.
(10) Matthews, D. A.; Bolin, J . T.; Burdge, J . M.; Filman, K. W.;
Kaufman, B. T.; Bedell, C. R.; Champness, J . N.; Stammers, D.
K.; Kraut, J . Refined Crystal Structures of E. coli and Chicken
Liver DHFR Containing Bound Trimethoprim. J . Biol. Chem.
1985, 260, 381-391.
cis,tr a n s-1,3-Dia m in o-8-(3′,4′,5′-tr im eth oxyph en yl)-5,6,-
6 a , 7 , 8 , 9 , 1 0 , 1 0 a -o c t a h y d r o p y r i m i d o [ 4 , 5 -c ] [ 2 , 7 ] -
n a p h th yr id in e (11). To a solution of 9 (0.10 g, 0.26 mmole)
in CF3COOH/MeOH (1:1, 40 mL) was added platinum oxide
(0.05 g). The suspension was hydrogenated in a Parr ap-
paratus (50 psi) for 8 h at room temperature. To avoid air
oxidation back to the starting material 9, activated charcoal
was added following hydrogenation and the workup performed
under an atmosphere of nitrogen. The reaction mixture was
rapidly filtered through Celite and the filtrate evaporated
under reduced pressure (bath temperature 30 °C). The residue
obtained was suspended in MeOH:acetone (1:1, 10 mL) and
then kept in a freezer overnight. The solid (0.02 g) obtained
was filtered (TLC of which indicated it to be the starting
material 9, and the filtrate was evaporated to dryness. To this
was added acetone (4 mL), and the solution was kept in a
freezer for 4 h. Compound 11 separated as a solid which was
collected (0.035 g, 35% yield): mp 250 °C dec; TLC Rf 0.40
(CHCl3:MeOH, 9:2, with 3 drops of NH4OH); 1H NMR (DMSO-
d6) δ 1.91 (m, 1H), 2.25 (br s, 1 H), 2.40 (m, 1 H), 2.62 (m, 1
H), 2.96 (s, overlapped with a multiplet, 2 H), 3.35 (br s, MeOH
as solvent overlapped with a multiplet, 1 H), 3.45 (s, 1 H), 3.55
(s, 3 H, OCH3), 3.80 (s, overlapped with multiplet (m, 8 H);
MS m/ z 387 (MH+). Anal. (C19H26N6O3‚1.2CF3COOH‚2.85CH3-
OH) C, H, N.
(11) Tripos Associates, Inc., 1699 S. Hanley Rd., Suite 303, St. Louis,
MO 63144.
(12) Cody, V.; Wojtczak, A.; Kalman, T. I.; Freisheim, J . H.; Blakley,
R. L. Conformational Analysis of Human Dihydrofolate Reduc-
tase Inhibitor Complexes: Crystal Structure Determination of
Wild Type and F31 Mutant Binary and Ternary Inhibitor
Complexes. Adv. Exp. Med. Biol. 1993, 338, 481-486.
(13) Gangjee, A.; Shi. J .; Queener, S. F.; Barrows, L. R.; Kisliuk, R.
L. Synthesis of 5-Methyl-5-deaza Nonclassical Antifolates as
Inhibitors of Dihydrofolate Reductase and as Potential Anti-
pneumocystis and Antitoxoplasma and Antitumor Agents. J .
Med. Chem. 1993, 36, 3437-3443.
(14) Grivsky, E. M.; Lee, S.; Sigel, C. W.; Duch, D. S.; Nichol, C. A.
Synthesis and Antitumor Activity of 2,4-Diamino-6-(2,5-dimeth-
oxybenzyl)-5-methylpyrido[2,3-d]pyrimidines. J . Med. Chem.
1980, 23, 327-336.
(15) Hurlbert, B. S.; Ledig, K. W.; Stenbuck, P.; Valenti, B. F.;
Hitchings, G. H. Studies on Condensed Pyrimidne System XXIII.
Synthesis of 2,4-Diaminopyrido[2,3-d]pyrimidines from â-Ke-
toester. J . Med. Chem. 1968, 11, 703-707.
(16) Gangjee, A.; Donkor, I. O.; Kisliuk, R. L.; Gaumont, Y.; Thorndike,
J . Synthesis and Biological Activity of 5,11-Methylenetetrahydro-
5-deazahomofolic Acid. J . Med. Chem. 1991, 34, 611-616.
(17) DeGraw, J . I.; Christen, P. H.; Colwell, W. T.; Sirotnak, F. M.
Synthesis and Antifolate Properties of 5,10-Ethano-dideaza
Aminopterin. J . Med. Chem. 1992, 35, 320-324.
(18) Gallagher, M. J .; Mann, F. G. The Structure and Properties of
Certain Polycyclic Indoles and Quinolidones Part XV. Deriva-
tives of 1-Phenyl-4-piperidone and its Phosphrous and Arsenic
Agent. J . Chem. Soc. 1962, 5110-5120.
(19) Ratajezyk, J . D.; Swett, L. R. The Cyclocondensation of 5-Amino-
1,3-dimethylpyrazole with Ethylacetoacetate. Synthesis of Iso-
meric Pyrazolopyridones. J . Heterocycl. Chem. 1975, 12, 517-
522.
(20) Gangjee, A.; O’Donnell, J . K.; Bardos, T. J .; Kalman, T. I.
Synthesis of Some 1,3,8-Trisubstituted Pyrimido[4,5-c][2,7]-
naphthyridine-6-ones as Potential Antitumor Agent. J . Hetero-
cycl. Chem. 1984, 21, 873-875.
(21) Rahman, A.; Busha, A.; Washeed, W.; Ashneld, S. A New Method
for the Reduction of Amides to Amines with Sodium Borohy-
dride. Tetrahedron Lett. 1976, 219-222.
(22) Taylor, E. C.; Soon, C. M. A Convient Synthesis of 6-Formyl-5-
deazapterin. Synth. Commun. 1988, 18, 1187-1191.
(23) Taylor, E. C.; Hamby, J . M.; Shih, C.; Grindey, G. B.; Rinzel, S.
M.; Peter-Beardsley, G.; Moran, R. G. Synthesis and Antitumor
Activity of 5-Deaza-5,6,7,8-tetrahydrofolic Acid and its N10-
Substituted Analogs. J . Med. Chem. 1989, 32, 1517-1522.
(24) Broughton, M. C.; Queener, S. F. Pneumocystis carinii Dihydro-
folate Reductase Used to Screen Potential Antipneumocystis
Drugs. Antimicrob. Agents Chemother. 1991, 35, 1348-1355.
(25) Chio, L.; Queener, S. F. Identification of Highly Potent and
Selective Inhibitors of Toxoplasma gondii Dihydrofolate Reduc-
tase. Antimicrob. Agents Chemother. 1993, 27, 1914-1923.
Ack n ow led gm en t. This work was supported by
NIH Grants GM 40998 (A.G.) and AI 80900 (A.G.) and
NIH Contracts NO1-AI-87240 (S.F.Q.) and NO1-AI-
35171(S.F.Q.) Division of AIDS. The authors also thank
Dr. F.-T. Lin, Department of Chemistry, University of
Pittsburgh, for the high-resolution 1H NMR and 13C
NMR spectra.
Refer en ces
(1) Taken in part from the thesis submitted by J .S. to the Graduate
School of Pharmaceutical Sciences, Duquesne University, in
partial fulfillment of the requirements for the degree of Doctor
of Philosophy, October 1994.
(2) Blakley, R. L. Dihydrofolate Reductase. In Folates and Pterins;
Blakley, R. L., Benkovic, S. J ., Eds.; Wiley: New York, 1984;
Vol. 1, pp 191-253.
(3) Douglas, K. T. The Thymidylate Synthesis Cycle and Anticancer
Drugs. Med. Res. Rev. 1987, 7, 441-445.
(4) Berman, E. M.; Werbel, L. M. The Renewed Potential for Folate
Antagonists in Contemporary Cancer Chemotherapy. J . Med.
Chem. 1991, 34, 479-485.
(5) Kisliuk, R. L. The Biochemistry of Folates. In Folate Antagonist
as Therapeutic Agents; Sirotnak, F. M., Burchall, J . J ., Ens-
minger, W. B., Montgomery, J . A., Eds.; Academic Press:
Orlando, 1984; Vol. 1, pp 2-55.
(6) Borst, P.; Ouellette, M. New Mechanisms of Drug Resistance in
Parasitic Protozoa. Annu. Rev. Microbiol. 1995, 49, 427-460.
J M960693N