N9-substituted 2,4-diaminoquinazolines: Synthesis and biological evaluation of lipophilic inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase

Article abstract of DOI:10.1021/jm800694g

N9-substituted 2,4-diaminoquinazolines were synthesized and evaluated as inhibitors of Pneumocystis carinii (pc) and Toxoplasma gondii (tg) dihydrofolate reductase (DHFR). Reduction of commercially available 2,4-diamino-6- nitroquinazoline 14 with Raney n

Full text of DOI:10.1021/jm800694g

J. Med. Chem. 2008, 51, 6195–6200
6195
N9-Substituted 2,4-Diaminoquinazolines: Synthesis and Biological Evaluation of Lipophilic
Inhibitors of Pneumocystis carinii and Toxoplasma gondii Dihydrofolate Reductase
Aleem Gangjee,*,† Ona O. Adair,† Michelle Pagley,† and Sherry F. Queener‡
DiVision of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne UniVersity, Pittsburgh, PennsylVania 15282,
Department of Pharmacology and Toxicology, Indiana UniVersity School of Medicine, Indianapolis, Indiana 46202
ReceiVed June 6, 2008
N9-substituted 2,4-diaminoquinazolines were synthesized and evaluated as inhibitors of Pneumocystis carinii
(pc) and Toxoplasma gondii (tg) dihydrofolate reductase (DHFR). Reduction of commercially available
2,4-diamino-6-nitroquinazoline 14 with Raney nickel afforded 2,4,6-triaminoquinazoline 15. Reductive
amination of 15 with the appropriate benzaldehydes or naphthaldehydes, followed by N9-alkylation, afforded
the target compounds 5-13. In the 2,5-dimethoxybenzylamino substituted quinazoline analogues, replacement
of the N9-CH3 group of 4 with the N9-C2H5 group of 8 resulted in a 9- and 8-fold increase in potency
against pcDHFR and tgDHFR, respectively. The N9-C2H5 substituted compound 8 was highly potent, with
IC50 values of 9.9 and 3.7 nM against pcDHFR and tgDHFR, respectively. N9-propyl and N9-cyclopropyl
methyl substitutions did not afford further increases in potency. This study indicates that the N9-ethyl
substitution is optimum for inhibitory activity against pcDHFR and tgDHFR for the 2,4-diaminoquinazolines.
Selectivity was unaffected by N9 substitution.
Introduction
Opportunistic infections, such as Pneumocystis pneumonia
(PCPa) and toxoplasmosis, remain a major cause of mortality
in acquired immunodeficiency syndrome (AIDS) patients.1-3
Trimethoprim (TMP) (Figure 1) is a relatively weak inhibitor
(Ki ) 25 ( 7 nM) of dihydrofolate reductase (DHFR) from P.
jiroVecii, the form of Pneumocystis found in humans; TMP is
even less effective (Ki ) 770 ( 340 nM) against the DHFR
found in P. carinii (pc), the organism that infects rats. In humans
and in the drug testing model in rats, TMP must be coadmin-
istered with sulfonamides to provide synergistic antifolate
effects.4,5 Similarly, when pyrimethamine, a rather nonselective
DHFR inhibitor, is used to treat toxoplasmosis, it is necessary
to combine the weak DHFR inhibitor with a sulfonamide to
achieve synergistic effects. In both of these situations, the side
effects associated with sulfonamides often result in discontinu-
Figure 1
ation of therapy.6
Lipophilic, nonclassical antifolates, such as trimetrexate
(TMQ) (Figure 1), can passively diffuse into the pathogenic
cells, as well as host cells. TMQ is 286-fold and 159-fold more
potent against pcDHFR and tgDHFR, respectively, than TMP,
but TMQ is not selective for the mammalian DHFR and thus
must be coadministered with the classical folate leucovorin (5-
formyltetrahydrofolate) due to lack of selectivity.7-9 Host tissues
can be selectively rescued because leucovorin is only taken up
by the mammalian transport system(s). Drawbacks of the TMQ-
leucovorin regimen are the high cost and variable effectiveness
of leucovorin.10 Thus, the search for single agents that are potent
and selective against pcDHFR and/or tgDHFR continues in
several laboratories.
Gangjee et al.,11-16 as well as others,17-21 have reported
several structural classes of DHFR inhibitors. Selectivity is
reported as the ratio of the IC50 value of mammalian DHFR/
IC50 value of pcDHFR or tgDHFR. A series of 2,4-diaminopy-
rido[2,3-d]pyrimidines were reported by Gangjee et al.9 as two
atom-bridged variations of the potent nonclassical antifolate
piritrexim (PTX).22 Compound 1 (see Figure 2) showed moder-
ate DHFR inhibitory potency with IC50 values of 3.80 and 0.31
µM against pcDHFR and tgDHFR, respectively. N9-methylation
of 1 to afford 2 resulted in a 45-fold and 49-fold increase in
potency against pcDHFR (IC50 ) 84 nM) and tgDHFR (IC50
) 6.3 nM), respectively. The selectivity ratio for tgDHFR versus
recombinant human (rh) DHFR of compound 2 was 304. Thus,
N9-methylation in the reverse-bridged analogue significantly
increased potency against pcDHFR and tgDHFR, as well as
selectivity for tgDHFR versus rhDHFR. However, 2 was 447-
times less potent against T. gondii cells in culture compared to
isolated tgDHFR. These results suggest inadequate cell
penetration.
* To whom correspondence should be addressed. Phone: 412-396-6070.
Fax: 412-396-5130. E-mail: gangjee@duq.edu.
†
Division of Medicinal Chemistry, Graduate School of Pharmaceutical
Sciences, Duquesne University.
‡
Department of Pharmacology and Toxicology, Indiana University
School of Medicine.
a Abbreviations: pc, Pneumocystis carinii; tg, Toxoplasma gondii; DHFR,
dihydrofolate reductase; HIV, human immunodeficiency virus; PCP, Pneu-
mocystis pneumonia; AIDS, acquired immunodeficiency syndrome; TMP,
Trimethoprim; PTX, piritrexim; TMQ, trimetrexate; ma, Mycobacterium
aVium.
10.1021/jm800694g CCC: $40.75
2008 American Chemical Society
Published on Web 09/05/2008

6196 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Gangjee et al.
not interact with Ile123, it was of interest to increase the size
of the N9-alkyl substitution in the quinazoline series. It was
anticipated that this increase would provide both potent and
selective inhibitors.
Thus, the N9-alkyl 2,4-diaminoquinazolines 5-13 were
designed and synthesized as analogues of compound 4. Com-
pound 5 was synthesized as a phenyl ring unsubstituted
analogue. Compounds 6 and 7 contain R- and ꢀ-naphthyl
moieties. A naphthyl group has been shown by Gangjee et al.28
to interact with Phe69 in pcDHFR. Compound 9 contains 2,3,5-
trichloro substituents to determine the effect of electron
withdrawing groups on biological activity. The N9-propyl
analogues 10 and 11 and the cyclopropyl methyl analogues 12
and 13 were synthesized to determine the importance of the
size of the N9-alkyl moiety on biological activity. The increase
in the number of carbons at the N9-position contributes to the
lipophilicity of the analogues and also serves as a site for
possible hydrophobic interactions with Val115 of hDHFR,
Ile123 of pcDHFR, and/or hydrophobic residues in tgDHFR.
The N9-ethyl substituted compounds 5-9 were designed to
determine whether potency and/or selectivity can be increased
by decreasing the distance between the N9-alkyl moiety and
the hydrophobic residues in the DHFR binding site. The N9-
propyl and -cyclopropyl methyl analogues 10-13 were designed
to determine whether the increase in the number of carbons from
2 to 3 or 4 would increase the potential hydrophobic interactions
or exceed the optimum distance between the DHFR residues
and the N9-moiety.
Figure 2
To explore the effect on potency, selectivity, and cell
penetration, Gangjee et al.23 synthesized a series of analogues
of compound 2 with an isosteric replacement of the N8 with a
carbon to yield 2,4-diaminoquinazolines with increased lipo-
philicity. A comparison of the potency and selectivity of the
quinazoline analogues with the pyrido[2,3-d]pyrimidines with
identical side chain substituents revealed different structure-
activity/selectivity relationships for the two classes. Contrary
to the pyrido[2,3-d]pyrimidines, in most cases, the N9-H
quinazoline analogues showed better selectivity for tgDHFR
versus rlDHFR and tgDHFR versus hDHFR than their corre-
sponding N9-methyl analogues. The N9-methyl quinazolines
were, however, considerably more potent against both pcDHFR
and tgDHFR than the N9-H quinazolines. Three trimethox-
yphenyl substituted N9-H quinazolines were selected for
evaluation in T. gondii cell culture studies. The cell culture/
enzyme ratio (IC50 value in T. gondii cell culture/IC50 value in
tgDHFR) ranged from 3 to 142, which was significantly better
than the ratio of 447 for compound 2. This increase in inhibition
of T. gondii cells in culture was attributed, in part, to the
increased lipophilicity of the quinazolines versus the pyrido-
[2,3-d]pyrimidines and provided the impetus to explore further
modifications of quinazolines to improve selectivity and po-
tency.23
X-ray crystal structures of 5-deaza-5-methyl antifolates show
a hydrophobic interaction between the 5-methyl moiety and
Val115 in hDHFR.24 In the crystal structure of PTX complexed
with pcDHFR, a hydrophobic interaction between the 5-methyl
moiety and Ile123 is observed.25 The fact that the 5-methyl
moiety of PTX has hydrophobic interactions in both pcDHFR
and hDHFR may explain, in part, the lack of selectivity of PTX.
The X-ray crystal structures of 2 complexed with hDHFR23 and
pcDHFR26 showed that the distance between the N9-methyl
moiety of 2 and the Val115 (hDHFR) or Ile123 (pcDHFR)25
was 4.66 Å and 4.20 Å, respectively,27 beyond the range for
van der Waals interaction (e4.00 Å). The Val115 of hDHFR
corresponds to Ile123 in pcDHFR. Because the quinazolines
demonstrated better T. gondii cell to tgDHFR inhibitory ratios
and the N9-methyl moiety of the pyrido[2,3-d]pyrimidines did
Chemistry. The synthesis of target compounds 5-13 began
with commercially available 2,4-diamino-6-nitroquinazoline 14
and is outlined in Scheme 1.23 Starting material 14 was reduced
with hydrogen at 30-35 psi and Raney nickel catalyst for 3 h
to afford the key intermediate 2,4,6-triaminoquinazoline 15. To
the reaction vessel was added the appropriate benzaldehyde or
naphthaldehyde, and the reaction mixture was hydrogenated for
an additional 15 h to afford 16-20, the N9-H precursors to
the target compounds, in yields ranging from 36% to 82%,
depending on the phenyl substitution. Reductive N9-alkylation
with acetaldehyde, proprionaldehyde, or cyclopropanecarbox-
aldehyde using sodium cyanoborohydride afforded the target
compounds 5-13 in yields ranging from 9% to 67%, depending
on the aldehyde and the phenyl substitution.
Biological Activity and Discussion. Compounds 5-13 were
evaluated as inhibitors of pcDHFR, tgDHFR, rlDHFR, and
Mycobacterium aVium (ma) DHFR, and the results are reported
in Table 1. In general, the N9-ethyl substituted analogues were
more potent than the unsubstituted or N9-methyl substituted
compounds against pcDHFR and, in most cases, against tgDHFR
as well. Against pcDHFR, the R-naphthyl compound 6 was the
most potent. Against tgDHFR, the unsubstituted phenyl analogue
5 and the 2,5-diOCH3 phenyl analogue 8 were most potent. The
R-naphthyl analogue 6 and the ꢀ-naphthyl analogue 7 were
8-fold and 3-fold more potent against pcDHFR, respectively,
than the unsubstituted phenyl analogue 5. This suggests a
tolerance for larger side chain rings in the pcDHFR binding
site. Against tgDHFR, compounds 6 and 7 were 2-fold and
3-fold less potent, respectively, than 5, which suggests the
naphthyl rings bind less favorably than the phenyl ring, contrary
to that observed for pcDHFR. Substitution of electron-donating
methoxy groups on the side chain phenyl ring in 8 increased
potency against pcDHFR relative to 5. Against tgDHFR, 5 and
8 were equipotent. Electron-withdrawing chloro groups on the
phenyl ring also resulted in an increase in potency against
pcDHFR for compound 9. The fact that methoxy and chloro

N9-Substituted 2,4-Diaminoquinazolines
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6197
Scheme 1a
a Reagents: (a.) Raney nickel, DMF, H2 at 30-35 psi; (b.) Raney nickel, DMF, CH3COOH, ArCHO, H2 at 30-35 psi; (c.) RCHO, NaCNBH3, HCl,
CH3CN.
Table 1. Inhibition Concentrations (IC50, in nM) against DHFR from P. carinii (pc), T. gondii (tg), M. aVium (ma), and Rat Liver (rl) and Selectivity
Ratiosa
R
R′
pcDHFR
rlDHFR
rl/pc
tgDHFR
rl/tg
maDHFR
rl/ma
1
2
3
4
5
6
7
8
9
10
11
12
H
CH3
H
CH3
C2H5
C2H5
C2H5
C2H5
C2H5
C3H7
C3H7
C4H7
C4H7
3800
84
4600
87
350
57
1100
26
4.1
6.5
14.5
4.8
19
5.3
17
9.6
16
0.09
0.7
0.24
0.29
0.2
2.6
2.0
0.5
2.8
0.1
0.6
0.4
0.4
11.1
0.07
310
6.3
160
30
3.6
7.8
12
3.7
19
9.5
19
1.1
9.0
6.88
0.86
1.1
0.8
1.2
1.3
1.0
0.6
0.9
1.3
0.6
H
21
1.1
2.9
2.3
0.98
14
3.3
13
16
3.73
2.24
6.30
4.90
1.36
1.63
1.31
0.60
0.47
2,3-C4H4
3,4-C4H4
2.5
7.2
9.9
6.9
38
27
25
42
2,5-diOCH3
2,3,5-triCl
2,5-diOCH3
3,4-C4H4
2,5-diOCH3
3,4-C4H4
7.3
25
2,700
10
13
34
TMPb
TMQb
12000
42
133000
3.0
49
0.3
a These assays were carried out at 37 °C under saturating conditions of substrate (0.092 mM dihydrofolic acid) and cofactor (0.117 mM NADPH);
rlDHFR and tgDHFR were assayed in the presence of 150 mM KCl. b These values are from ref 28. Most recent quality control values for rlDHFR are
121400 ( 7500 nM for TMP (R2 ) 0.9768), 8.0 ( 0.5 nM (R2 ) 0.9561) for TMQ and 4.4 ( 0.4 nM (R2 ) 0.9386) for PTX. Most recent quality control
values for pcDHFR are 26,820 ( 1700 nM (R2 ) 0.995) for TMP, 47 ( 13 nM (R2 ) 0.923) for TMQ, and 34.3 ( 10 nM (R2 ) 0.9876) for PTX. These
quality control assays used recombinant pcDHFR, as did all the assays for experimental compounds shown above. Published data on TMP, TMQ and PTX
used native pcDHFR; side by side comparisons show no difference in drug susceptibility between the two forms of pcDHFR.
groups both contributed to increased potency against pcDHFR
compared to the unsubstituted phenyl ring suggests that the
increase was not due to electronic factors but was more likely
due to favorable binding conformations induced by the substitu-
tions on the phenyl ring in compounds 8 and 9 compared to 5.
Against tgDHFR, electron-withdrawing chloro substituents on
the side chain phenyl ring results in a 5-fold decrease in
inhibitory potency compared to the unsubstituted phenyl ring,
with IC50 values for compounds 5 and 9 of 3.6 and 19 nM,
respectively.
The effect on inhibitory potency against pcDHFR and
tgDHFR of increasing the number of carbons at the N9-position
in the quinazoline series can be deduced from a comparison of
IC50 values for the 2,5-dimethoxy phenyl substituted analogues
3, 4, 8, 10, and 12. As previously reported,23 the potency against
pcDHFR increased 53-fold upon N9-methylation, with IC50
values of 4600 and 87 nM for 3 and 4, respectively. N9-
substitution with the homologous ethyl group to yield compound
8 resulted in a 9-fold increase in potency against pcDHFR
compared to the N9-methyl analogue 4, with an IC50 value of
9.9 nM for 8. However, propyl substitution at the N9-position
caused the inhibitory potency against pcDHFR to decrease. The
N9-propyl analogue 10 showed an IC50 value of 38 nM, 4-fold
less than 8. The N9-cyclopropyl methyl analogue 12 had a slight
increase in potency against pcDHFR compared to 10, with an
IC50 value of 25 nM. Despite the N9-cyclopropyl methyl group
having one additional carbon compared to the N9-propyl moiety,
the similarity in IC50 values for 10 and 12 was consistent with
the similarity in carbon chain lengths in the two groups. The
increase in inhibitory potency against pcDHFR upon N9-
ethylation is also shown in the R-naphthyl quinazoline ana-
logues. The IC50 values for the previously reported N9-H and
N9-methyl R-naphthyl quinazolines23 were 720 and 17 nM,
respectively, indicating a 42-fold increase in potency against
pcDHFR upon N9-methylation. The N9-ethyl analogue 6
showed an IC50 value of 2.5 nM and was 7-fold more potent
against pcDHFR than the N9-methyl analogue. Also, as seen
in the 2,5-dimethoxy series, in the ꢀ-naphthyl quinazoline
analogues, replacement of the N9-ethyl group with a propyl or
cyclopropyl methyl group resulted in a decrease in potency
against pcDHFR. The N9-ethyl analogue 7 was 4-fold and 6-fold
more potent than 11 and 13, respectively.
Against tgDHFR, compound 4 was 5-fold more potent than
3, illustrating that just as for pcDHFR, N9-ethylation resulted
in an increase in potency against tgDHFR compared to the N9-
methyl analogue. Compound 8 was 8-fold more potent than 4
against tgDHFR. However, the N9-propyl homologue 10
showed a 3-fold decrease in potency compared to 8. The N9-
cyclopropyl methyl analogue 12 showed a very similar potency
against tgDHFR compared to 10. Potency against tgDHFR also
increased with N9-ethylation in the R-naphthyl quinazoline
analogues. The N9-ethyl analogue 6 showed a 3-fold increase
in potency compared to the N9-methyl analogue against
tgDHFR. In the ꢀ-naphthyl quinazoline analogues 7, 11, and
13 reported herein, the increase in the number of carbons at the
N9-position caused about a 2-fold reduction in potency against
tgDHFR.
Selectivity for pcDHFR in the quinazoline analogues is
generally low. N9-substituents with increasing numbers of
carbons did not significantly improve selectivity for pcDHFR.
Compounds 3 and 4 both showed increased potency against
rlDHFR compared to pcDHFR with selectivity ratios of 0.24
and 0.29, respectively. N9-ethyl, -propyl, or -cyclopropyl methyl
substitutions (compounds 8, 10, and 12), in general, did not
improve selectivity for pcDHFR. Only compounds 6, 7, and 9
showed greater potency against pcDHFR compared to rlDHFR

6198 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Gangjee et al.
1
and are uncorrected. H NMR spectra were recorded on a Bruker
WH-300 (300 MHz)NMR spectrometer. The chemical shift (δ)
values are reported as parts per million (ppm) relative to tetram-
ethylsilane as internal standard: s ) singlet, d ) doublet, t ) triplet,
q ) quartet, m ) multiplet, br ) broad peak, exch ) exchangeable
protons by addition of D2O. Elemental analyses were performed
by Atlantic Microlabs, Inc., Norcross, GA. Elemental compositions
were within ( 0.4% of the calculated values. Fractional moles of
water or organic solvents frequently found in some analytical
samples of antifolates could not be removed despite 24 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 Co. and were used
as received except where anhydrous solvents were needed, which
were freshly dried in the laboratory.
with marginal increase in selectivity ratios. N9-propylation and
N9-cyclopropylmethylation of the ꢀ-naphthyl quinazoline re-
sulted in a decrease in selectivity for pcDHFR compared to 7.
Selectivity for tgDHFR in the N9-H quinazoline analogues
was generally higher than selectivity for pcDHFR, with selectiv-
ity ratios of 7 and 0.2 for tgDHFR and pcDHFR, respectively,
for the 2,5-dimethoxyphenyl analogue 3. N9-alkylation resulted
in a decrease in selectivity for tgDHFR; compounds 8, 10, and
12 showed selectivity ratios of 1.3, 0.6, and 1.3, respectively,
for tgDHFR. The selectivity for tgDHFR of the N9-ethyl,
-propyl, and -cyclopropyl analogues was low, indicating a
minimal but consistent decrease in selectivity for tgDHFR with
a number of carbons greater than one in the N9-position.
Compounds 5-13 were also evaluated as inhibitors of
maDHFR. The N9-ethyl dimethoxyphenyl analogue 8 was the
most potent with an IC50 value of 0.98 nM. Compound 8 was
equipotent compared to the unsubstituted phenyl analogue 5.
The R- and ꢀ-naphthyl analogues 6 and 7 were slightly less
potent. Compound 9, with the electron-withdrawing 2,3,5-
trichloro substituents on the side chain phenyl ring, was 13-
fold less potent than 5 against maDHFR. N9-propylation of the
2,5-dimethoxy quinazoline resulted in a 3-fold decrease in
potency for 10 against maDHFR compared to 8. Cyclopropyl
methylation at the N9-position decreased potency against
maDHFR by 5-fold for 12 compared to 10. N9-propylation of
the ꢀ-naphthyl quinazoline resulted in a 6-fold decrease in
potency against maDHFR for 11 compared to 7. The N9-
cyclopropyl methyl ꢀ-naphthyl quinazoline 13 was 3-fold less
potent compared to 11.
The unsubstituted phenyl analogue 5 was selective for
maDHFR versus rlDHFR with a selectivity ratio of 3.7. The
R-naphthyl analogue 6 was less selective compared to 5 with a
selectivity ratio of 2.2. However, the ꢀ-naphthyl analogue 7 was
more selective for maDHFR compared to 5 with a selectivity
ratio of 6.3 and was the most selective analogue for maDHFR
reported herein. Compound 8, with electron-donating 2,5-
dimethoxy substituents on the phenyl ring, was more selective
for maDHFR compared to 5. The decrease in selectivity for
maDHFR of 9 (selectivity ratio ) 1.4), with electron-withdraw-
ing 2,3,5-trichloro substituents on the phenyl ring, suggests that
selectivity for maDHFR versus rlDHFR may be affected by the
electronic nature of the phenyl ring substitutions. Selectivity
for maDHFR was generally greater for the N9-ethyl quinazoline
analogues and decreased with an increase in the number of
carbons at the N9-position. N9-propyl analogues 10 (selectivity
ratio ) 1.6) and 11 (selectivity ratio ) 1.3) were 3-fold and
5-fold less selective compared to 8 and 7, respectively. N9-
cyclopropylmethylation resulted in analogues that were more
selective for rlDHFR versus maDHFR with selectivity ratios
for 12 and 13 of less than one.
General Procedure for the Synthesis of Compounds 5-13. To
the 2,4-diamino[(N-substituted benzyl)amino]quinazoline suspended
in CH3CN was added acetaldehyde, proprionaldehyde, or cyclo-
propanecarboxaldehyde, followed by NaCNBH3. The suspension
was pH adjusted to 2-3 by dropwise addition of concentrated HCl.
As the mixture was acidified, the suspended starting material began
to dissolve into solution, followed by precipitation of the crude
product. In some cases, dissolution was not seen but a visible color
change occurred. The suspension containing the crude product was
stirred for an additional 0.5 h. The crude product was filtered, stirred
in 2 N Na2CO3, and washed with H2O, C2H5OH, and C2H5OC2H5.
The purity of each product was checked by TLC using a solvent
system of 5:2:0.1 (A) or 5:1:0.1 (B) by volume of CHCl3:CH3OH:
NH4OH. Crude products were dissolved in CH3OH, pH adjusted
to 8 with NH4OH, added to silica gel, followed by the evaporation
of the solvent to afford a silica gel plug. The dry plug containing
the product was placed on a column of wet silica gel and eluted
with solvent B or a solvent system of 10:1 (C) by volume of CHCl3:
CH3OH. Pure fractions, identified as single spots by TLC, were
collected and the solvent evaporated to yield analytically pure solids.
2,4-Diamino-6-[N-(benzyl)-N-ethylamino]quinazoline 5. Com-
pound 5 was synthesized from 16 (0.20 g, 0.75 mmol), acetaldehyde
(0.20 mL, 3.6 mmol), and NaCNBH3 (0.125 g, 2.0 mmol) in 50
mL of CH3CN. Compound 5 was filtered from the reaction mixture
to yield an analytically pure bright-yellow solid (0.10 g, 45%): mp
1
280-282 °C; TLC (solvent A) Rf 0.46. H NMR (DMSO-d6) δ
1.11 (t, 3 H, N-CH2-CH3), 3.48 (q, 2 H, N-CH2-CH3), 4.60 (s, 2 H,
10-CH2), 7.24-7.38 (m, 10 H, Ar, NH2), 8.6 (br, 2 H, NH2). Anal.
(C17H19N5 ·1.8HCl·0.6CH3OH) C, H, N.
2,4-Diamino-6-[N-(r-naphthyl)-N-ethylamino]quinazoline
6. Compound 6 was synthesized from 17 (0.47 g, 1.50 mmol),
acetaldehyde (0.40 mL, 7.2 mmol), and NaCNBH3 (0.25 g, 4.0
mmol) in 50 mL of CH3CN. Compound 6 was filtered from the
reaction mixture to yield an analytically pure solid (0.20 g, 39%):
1
mp 266-267 °C; TLC (solvent B) Rf 0.40. H NMR (DMSO-d6)
δ 1.16 (t, 3 H, N-CH2-CH3), 3.49-3.56 (q, 2 H, N-CH2-CH3), 5.05
(s, 2 H, 10-CH2), 7.18-7.21 (m, 2 H, Ar), 7.27-7.30 (m, 1 H,
Ar), 7.39-7.62 (m, 4 H, Ar, 2 H, NH2), 7.82-7.85 (d, 1 H, Ar),
7.96-7.99 (m, 1 H, Ar), 8.08-8.11 (m, 1 H, Ar), 8.60 (s, 2 H,
NH2), 8.92 (s, 2 H, NH2). Anal. (C21H21N5 ·1.5HCl) C, H, N.
2,4-Diamino-6-[N-(ꢀ-naphthyl)-N-ethylamino]quinazoline
7. Compound 7 was synthesized from 18 (0.32 g, 1.01 mmol),
acetaldehyde (0.27 mL, 4.85 mmol), and NaCNBH3 (0.17 g, 2.7
mmol) in 50 mL of CH3CN. Compound 7 was filtered from the
reaction mixture to yield an analytically pure solid (0.23 g, 67%):
Experimental Section
All evaporations were carried out in vacuo with a rotary
evaporator. Analytical samples were dried in vacuo over P2O5. Thin-
layer chromatography (TLC) was performed on silica gel plates
with fluorescent indicator. Spots were visualized by UV light (254
and 365 nM). All analytical samples were homogeneous on TLC
in at least two different solvent systems. Purification by column
chromatography was carried out using Merck silica gel 60
(200-400 mesh). The amount of silica gel used for column
chromatography was depended upon the chemical nature and
amount of the compounds being separated. Columns were usually
wet-packed unless otherwise noted. Solvent systems are reported
as volume percent mixtures. Melting points were determined on a
Mel-Temp II melting point apparatus with a digital thermometer
1
mp 278-280 °C; TLC (solvent B) Rf 0.45. H NMR (DMSO-d6)
δ 1.12-1.17 (t, 3 H, N-CH2-CH3), 3.56-3.58 (q, 2 H, N-CH2-
CH3) 4.77 (s, 2 H, 10-CH2), 7.27 (s, 2 H, Ar), 7.40-7.50 (m, 4 H,
Ar, 2 H, NH2), 7.72 (s, 1 H, Ar), 7.81-7.89 (m, 3 H, Ar), 8.62 (br,
2 H, NH2). Anal. (C21H21N5 ·1.0HCl) C, H, N.
2,4-Diamino-6-[N-(2,5-dimethoxybenzyl)-N-ethylamino]quinazo-
line 8. Compound 8 was synthesized from 19 (0.24 g, 0.75 mmol),
acetaldehyde (0.20 mL, 3.6 mmol), and NaCNBH3 (0.125 g, 2.0
mmol) in 50 mL of CH3CN. Compound 8 was purified by column
chromatography to yield an analytically pure bright-yellow solid
(0.16 g, 59%): mp 200-204 °C; TLC (solvent B) Rf 0.43. 1H NMR

N9-Substituted 2,4-Diaminoquinazolines
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 6199
(DMSO-d6) δ 1.05 (br, 3 H, N-CH2-CH3), 3.36-3.38 (br, 2 H,
N-CH2-CH3), 3.52 (s, 3 H, OCH3), 3.73 (s 3 H, OCH3), 4.38 (s, 2
H, 10-CH2), 5.66 (br, 2 H, NH2), 6.52 (s, 1 H, Ar), 6.72 (m, 1 H,
Ar), 6.86-6.94 (m, 3 H, Ar), 7.02-7.05 (d, 1 H, Ar), 7.12-7.17
(br, 2 H, NH2). Anal. (C19H23N5O2 ·0.7H2O) C, H, N.
2,4-Diamino-6-[N-(2,3,5-trichlorobenzyl)-N-ethylamino]quinazo-
line 9. Compound 9 was synthesized from 20 (0.20 g, 0.55 mmol),
acetaldehyde (0.15 mL, 2.7 mmol), and NaCNBH3 (0.10 g, 1.65
mmol) in 20 mL of CH3CN. Compound 9 was purified by column
chromagraphy to yield an analytically pure bright-yellow solid (0.05
g, 21%): mp 222-224 °C; TLC (solvent B) Rf 0.46. 1H NMR
(DMSO-d6) δ 1.1-1.14 (t, 3 H, N-CH2-CH3), 3.45-3.49 (q, 2 H,
N-CH2-CH3), 4.56 (s, 2 H, 10-CH2), 5.65 (s, 2 H, NH2), 7.05-7.06
(d, 2 H, Ar), 7.11-7.16 (m, 4 H, Ar, NH2), 7.74-7.75 (d, 1 H,
Ar). Anal. (C21H21N5Cl3 ·0.3H2O) C, H, N, Cl.
References
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Inhibitors. Bioorg. Med. Chem. 2001, 9, 2929–2935.
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of a Potent Dual Inhibitor of Thymidylate Synthase and Dihydrofolate
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Antifolate and Antitumor Activity of Classical 5-Substituted 2,4-
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6-[(monosubstituted anilino)methyl]-pyrido[2,3-d]pyrimidines. J. Med.
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2,4-Diamino-6-[N-(2,5-dimethoxybenzyl)-N-propylamino]quinazo-
line 10. Compound 10 was synthesized from 19 (0.20 g, 0.62 mmol),
propionaldehyde (0.11 g, 1.8 mmol), and NaCNBH3 (0.116 g, 1.8
mmol) in 50 mL of CH3CN. Compound 10 was purified by column
chromatography to yield an analytically pure solid (13%): mp
1
172-173 °C; TLC (solvent B) Rf 0.58. H NMR (DMSO-d6) δ
0.86-0.91 (t, 3 H, N-CH2-CH2-CH3), 1.53-1.58 (m, 2 H, N-CH2-
CH2-CH3), 3.31 (br, N-CH2-CH2-CH3), 3.56 (s, 3 H, OCH3), 3.78
(3.3 H, OCH3), 4.46 (s, 2 H, 10-CH2), 5.71 (s, 2 H, NH2), 6.52-6.53
(d, 1 H, Ar), 6.72-6.76 (m, 1 H, Ar), 6.90-7.00 (m, 2 H, Ar),
7.07-7.14 (d,
2 H, Ar), 7.23 (br, 2 H, NH2). Anal.
(C20H25N5O2 ·0.5H2O) C, H, N.
2,4-Diamino-6-[N-(ꢀ-naphthyl)-N-propylamino]quinazoline 11.
Compound 11 was synthesized from 18 (0.20 g, 0.63 mmol),
propionaldehyde (0.74 g, 12.7 mmol), and NaCNBH3 (0.12 g, 2.0
mmol) in 50 mL of CH3CN. Compound 11 was purified by column
chromatography to yield an analytically pure solid (46%): mp
1
189.0-190.7 °C; TLC (solvent B) Rf 0.42. H NMR (DMSO-d6)
δ 0.86-0.91 (t, 3 H, N-CH2-CH2-CH3), 1.56-1.66 (t, 2 H, N-CH2-
CH2-CH3), 4.71 (s, 2 H, 10-CH2), 5.67 (br, 2 H, NH2), 7.21-7.29
(m, 4 H, Ar, NH2), 7.40-7.46 (m, 3 H, Ar), 7.72 (s, 1 H, Ar),
7.79-7.86 (m, 3 H, Ar). Anal. (C22H23N5 ·0.3H2O) C, H, N.
2,4-Diamino-6-[N-(2,5-dimethoxybenzyl)-N-cyclopropyl methyl-
amino]quinazoline 12. Compound 12 was synthesized from 19 (0.20
g, 0.62 mmol), cyclopropanecarboxaldehyde (0.86, 12.3 mmol), and
NaCNBH3 (0.12 g, 1.8 mmol) in 50 mL of CH3CN. Compound 12
was purified by column chromatography to yield an analytically
pure solid (21%): mp 122.7-123.5 °C; TLC (solvent B) Rf 0.54.
1H NMR (DMSO-d6) δ 0.18-0.19 (d, 2 H, N-CH2-cyclopropyl),
0.41-0.44 (d, 2 H, N-CH2-cyclopropyl), 1.06-1.08 (m, 1 H,
N-CH2-cyclopropyl), 3.29-3.31 (d, 2 H, N-CH2-cyclopropyl), 3.56
(s, 3 H, OCH3), 3.79 (3.3 H, OCH3), 4.56 (s, 2 H, 10-CH2), 5.80
(br, 1 H, NH2), 6.58 (s, 1 H, Ar), 6.72-6.75 (d, 1 H, Ar), 6.90-6.93
(d, 1 H, Ar), 7.0-7.035 (d, 1 H, Ar), 7.08-7.10 (d, 1 H, Ar), 7.24
(s, 1 H, Ar), 7.29 (br, 1 H, NH2). Anal. (C21H25N5O2 ·0.6H2O) C,
H, N.
2,4-Diamino-6-[N-(ꢀ-naphthyl)-N-cyclopropyl methylamino]-
quinazoline 13. Compound 13 was synthesized from 18 (0.20 g,
0.63 mmol), cyclopropanecarboxaldehyde (0.89 g, 12.7 mmol), and
NaCNBH3 (0.12 g, 2.0 mmol) in 50 mL of CH3CN. Compound 13
was purified by column chromatography to yield an analytically
pure solid (9%): mp 136-138 °C (dec); TLC (solvent B) Rf 0.58.
1H NMR (DMSO-d6) δ 0.20-0.21 (d, 2 H, N-CH2-cyclopropyl),
0.42-0.45 (d, 2 H, N-CH2-cyclopropyl), 1.06-1.11 (m, 1 H,
N-CH2-cyclopropyl), 4.80 (s, 2 H, 10-CH2), 6.00 (br, 1 H, NH2),
7.08 (d, 1 H, Ar), 7.21 (d, 1 H, Ar), 7.32 (s, 1 H, Ar), 7.43 (m, 4
H, Ar), 7.24 (s, 1 H, Ar), 7.74-8.00 (m, 3 H, Ar, NH2). Anal.
(C23H23N5 ·1.1H2O) C, H, N.
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Acknowledgment. This work was supported in part by
grants from the National Institute of Allergy and Infectious
Diseases, NIH, AI069966 (AG) and AI41743 (AG).
Supporting Information Available: Detailed elemental analysis
data for compounds 5-13. This material is available free of charge
via the Internet at http://pubs.acs.org.

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