Structural studies on bioactive compounds. 34.1 Design, synthesis, and biological evaluation of triazenyl-substituted pyrimethamine inhibitors of Pneumocystis carinii dihydrofolate reductase

Article abstract of DOI:10.1021/jm0108698

The triazenyl-pyrimethamine derivative 3a (TAB), a potent and selective inhibitor of Pneumocystis carinii DHFR, was selected as the starting point for a lead optimization study. Molecular modeling studies, corroborated by a recent crystal structure determination of the ternary complex of P. carinii DHFR-NADPH bound to TAB, predicted that modifications to the acetoxy residue of the lead inhibitor could exploit binding opportunities in the vicinity of an active site pocket bounded by residues Ile33, Lys37, and Leu72. Substitutions in the benzyl moiety with electron-donating and electron-withdrawing groups were predicted to probe face-edge interactions with amino acid Phe69 unique to the P. carinii enzyme. New triazenes 10a-v and 12a-f were prepared by coupling the diazonium tetrafluoroborate salt 6b of aminopyrimethamine with substituted benzylamines or phenethylamines. The most potent of the new inhibitors against P. carinii DHFR was the naphthylmethyl-substituted triazene 10t (IC₅₀: 0.053 μM), but a more substantial increase in potency against the rat liver DHFR led to a reduction in selectivity (ratio rat liver DHFR IC₅₀/P. carinii DHFR IC₅₀: 5.36) compared to the original lead structure 3a (ratio rat liver DHFR IC₅₀/P. carinii DHFR IC₅₀: 114).

Full text of DOI:10.1021/jm0108698

J . Med. Chem. 2001, 44, 2555-2564
2555
Str u ctu r a l Stu d ies on Bioa ctive Com p ou n d s. 34.1 Design , Syn th esis, a n d
Biologica l Eva lu a tion of Tr ia zen yl-Su bstitu ted P yr im eth a m in e In h ibitor s of
P n eu m ocystis ca r in ii Dih yd r ofola te Red u cta se
David C. M. Chan,† Charles A. Laughton,† Sherry F. Queener,‡ and Malcolm F. G. Stevens*,†
Cancer Research Laboratories, School of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, U.K., and
Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202
Received March 7, 2001
The triazenyl-pyrimethamine derivative 3a (TAB), a potent and selective inhibitor of Pneu-
mocystis carinii DHFR, was selected as the starting point for a lead optimization study.
Molecular modeling studies, corroborated by a recent crystal structure determination of the
ternary complex of P. carinii DHFR-NADPH bound to TAB, predicted that modifications to
the acetoxy residue of the lead inhibitor could exploit binding opportunities in the vicinity of
an active site pocket bounded by residues Ile33, Lys37, and Leu72. Substitutions in the benzyl
moiety with electron-donating and electron-withdrawing groups were predicted to probe face-
edge interactions with amino acid Phe69 unique to the P. carinii enzyme. New triazenes 10a -v
and 12a -f were prepared by coupling the diazonium tetrafluoroborate salt 6b of amino-
pyrimethamine with substituted benzylamines or phenethylamines. The most potent of the
new inhibitors against P. carinii DHFR was the naphthylmethyl-substituted triazene 10t
(IC50: 0.053 µM), but a more substantial increase in potency against the rat liver DHFR led to
a reduction in selectivity (ratio rat liver DHFR IC50/P. carinii DHFR IC50: 5.36) compared to
the original lead structure 3a (ratio rat liver DHFR IC50/P. carinii DHFR IC50: 114).
In tr od u ction
Pneumocystis carinii (PC) is an ubiquitous microor-
ganism that causes life-threatening pneumonia (PCP)
in individuals with severe immunodeficiency. In the
early stages of the AIDS epidemic, up to 75% of
individuals infected with HIV developed PCP. At the
end of 1998, the number of individuals carrying HIV
had soared to 33.4 million with 6 million new infections
reported every year.2
The dihydrofolate reductase (DHFR) inhibitor trime-
thoprim 1 (Figure 1) is a first-line therapy for PCP but
its moderate potency and poor species selectivity toward
P. carinii DHFR render it effective only in combination
F igu r e 1. Structures of inhibitors of Pneumocystis carinii
dihydrofolate reductase.
with the sulfa drug sulfamethoxazole. Many patients
are unable to tolerate the combined therapy due to life-
threatening adverse drug reactions.3 Recently, trime-
In 1997 we reported the exquisite species selectivity
trexate 2, a potent lipophilic quinazoline inhibitor of
both human and P. carinii DHFR, has been shown to
be an effective but toxic alternative treatment, requiring
expensive concomitant leucovorin rescue therapy to
counter the antifolate effects on the host.4 Despite
enormous efforts to improve existing compounds from
various research groups, the goal of identifying an agent
possessing high potency and species selectivity for the
clinical management of PCP remains unfulfilled.
In addition to P. carinii, a coccidian protozoan Toxo-
plasma gondii causes an opportunistic infection associ-
ated with AIDS5 which can lead to encephalitis,6 and
treatment regimes for T. gondii infections are similar
to those for PCP.
(ratio rat liver DHFR IC50/P. carinii DHFR IC50: 114)
displayed by a triazenyl-pyrimethamine derivative (TAB;
3a ); this compound was also selectively toxic to the T.
gondii DHFR enzyme (ratio rat liver IC50/T. gondii
IC50: 28).7 This agent emerged from an analogue
development program based on the dimethyltriazene 3b
with modest species selectivity (ratio rat liver IC50/P.
carinii IC50: 6.75). Although the IC50 value for 3a of
0.17 µM toward the P. carinii enzyme is an order of
magnitude less than that of the currently used agent
trimetrexate 2 (IC50 0.042 µM), there are no more potent
PC DHFR-inhibitory chemical entities (i.e., IC50 value
< 1 µM) reported in the literature possessing a better
species selectivity profile than that exhibited by TAB.7
In a search for more potent and selective P. carinii
DHFR inhibitors than the existing compounds, we have
exploited new structural insights developed from mo-
lecular modeling and crystallographic studies to guide
* Tel: (115) 951 3414. Fax: (115) 951 3412. E-mail:
malcolm.stevens@nottingham.ac.uk.
† University of Nottingham, Nottingham.
‡ Indiana University School of Medicine.
10.1021/jm0108698 CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/30/2001

2556 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Chan et al.
Sch em e 1a
F igu r e 2. Comparison of the modeled (colored gray) and X-ray
derived structures10 (colored blue) of the P. carinii DHFR-TAB-
NADPH ternary complex. Backbone CR atoms are shown as
the yellow ribbon. Side chains from the modeled structure are
depicted colored by atom type.
a program of further lead optimization based on the
structure of TAB 3a .
Molecu la r Mod elin g
The design process commenced with a manual dock-
ing study of TAB bound to the P. carinii DHFR utilizing
the only X-ray determined coordinates of P. carinii
DHFR available at the time this program was initiated.8
The benzyl function of TAB was predicted to form a
face-edge aromatic-aromatic interaction with the ac-
tive site residue Phe69. Such an interaction, which is a
common phenomenon in proteins and protein-ligand
complexes, requires in this case the rotation of the
N(2)-N(3) triazenyl bond by 47° from the planar
conformation. This simultaneously steers the acetoxy
branch of TAB toward the side chain of Lys37 which is
located on the surface of the active site cleft. Phe69 and
Lys37 are two of the most significant points of difference
between the human and P. carinii DHFR active sites.
In a hydrophobic reversal, P. carinii Phe69 is replaced
by Asn64 in the human variant; also Lys37 (P. carinii)
is substituted with Gln35 (human). Favorable interac-
tions with Phe69 have been implicated as the basis of
species selectivity of other P. carinii DHFR inhibitors
of the 2,4-diamino-5-substituted furo[2,3-d]pyrimidine
class.9
a
Reagents: (i) 3 M HCl/NaNO2 (6a ) or HBF4/NaNO2 (6b); (ii)
R1CHO/MeOH/NaBH4; (iii) RCO2H/HCl or RCOCl/HCl; (iv) aq
Na2CO3/0 °C.
We anticipated that, by modifying the alkyl branches
attached to the terminal triazenyl N(3) atom in a new
cycle of synthesis/evaluation, a structure with enhanced
potency and selectivity toward the P. carinii DHFR
enzyme might be discovered.
In this work we have concentrated on two strategies:
(i) exploring binding opportunities in the vicinity of a
potential binding pocket delineated by Ile33, Lys37, and
Leu72 through new variants at the acetoxy group of
TAB and (ii) probing the proposed face-edge interaction
of the benzyl group of TAB with Phe69 by incorporating
electron-donating and electron-withdrawing substi-
tutents in the benzyl moiety.
The validity of the manual docking model was con-
firmed unequivocally by our subsequent crystal struc-
ture determination of the P. carinii DHFR-TAB-NADPH
ternary complex.10 Least-squares superimposition of the
two sets of CR atoms gave an RMS deviation of 0.94 Å
(Figure 2). The modeled structure (colored gray) pre-
dicted accurately the orientations of several important
active site residues (Glu32, Phe36, Phe69, and Leu72)
and particularly the crucial dispositions of the benzyl
and acetoxyethyl substituents of the flexible TAB ligand.
Ch em istr y
Aminopyrimethamine 5, available by reduction of the
precursor nitro compound,11 was the starting material
for the synthesis of new triazenyl-pyrimethamine de-
rivatives 10 (Scheme 1). Diazotization of 5 in aqueous
HCl gave solutions of the diazonium chloride 6a suitable
for coupling reactions, but in practice it proved to be
more efficient to prepare (and store at 4 °C) the
diazonium tetrafluoroborate 6b as a stable hydrotet-

Synthesis of P. carinii DHFR Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2557
Sch em e 2a
a
Reagents: (i) aq Na2CO3/0 °C; (ii) BzCl/Pyridine/DMAP; (iii) AcOH/HCl.
rafluoroborate salt.12 N-Substituted ethanolamines 8
were usually synthesized from ethanolamine 7 by initial
formation of a Schiff base with appropriate benzalde-
hydes followed by sodium borohydride reduction.13
Alternatively, the nitrobenzyl-ethanolamines 8r ,s were
prepared by direct alkylation of ethanolamine with the
appropriate nitrobenzyl chlorides, compound 11c from
ethanolamine and 2-phenethyl chloride, and the chiral
ethanolamines 11d ,e from the alkylation of R-(+)-R-
methylbenzylamine and S-(-)-R-methylbenzylamine,
respectively, with 2-chloroethanol.
shifts can be compared directly with those of related
first generation compounds,7 simple 3-alkyl-3-benzyl-
triazenes of the triazenyl-pyrimethamine series 13a ,b,
and model reference triazenes 13c-e prepared from
diazotized 2-chloroaniline. Although the terminal ni-
Esterification of secondary amines 8 was achieved
either by treatment with a carboxylic acid-HCl gas, or
by interaction with an acid chloride. The water-soluble
hydrochloride salts 9 were coupled with diazonium salts
6 in an aqueous sodium carbonate medium at 0 °C to
afford the required triazenes 10 in high yields.
Further variations in substituents at the triazene
N(3) position 12 were obtained by coupling diazonium
salts 6 with the free bases of N-benzylethanolamine 11a ,
N-benzylpropanolamine 11b, N-phenethylethanolamine
11c, and the N-hydroxyethyl derivatives of (R)- and (S)-
R-methylbenzylamines 11d ,e (Scheme 2). Esterification
of 12a with benzoyl chloride-pyridine-DMAP gave the
benzoate 10c. Because of difficulties experienced in
synthesizing the benzoate salt 9c required for the direct
coupling method (Scheme 1), this was the preferred
route to 10c. Esterification of the triazenyl-substituted
N-benzylpropanolamine 12b with acetic acid-HCl gas
afforded the acetate 12f.
trogen atom in 1-aryl-3,3-dialkyltriazenes is (formally)
tetrahedral, X-ray crystal studies have shown it has sp2
character and the terminal N-N linkage is intermediate
in length between a single and a double bond.14 This
leads to restricted rotation about the N(2)-N(3) triaz-
enyl bond and significant temperature-dependent line
broadening of the alkyl protons in 3,3-dialkyltriazenes
is often observed.15,16 Thus the 1H NMR spectrum of 13a
in DMSO-d6 at 289 K shows two broadened singlets for
the methyl protons at δ 3.13 and 3.52 whereas the
methylene absorption of the benzyl group is a slightly
The triazenes 10a -v and 12b-f were purified either
by flash chromatography (CHCl3-MeOH, 10:1) or by
crystallization from acetone or ethanol, and they were
isolated as stable solids which effervesced vigorously
(-N2) at their melting points. In common with many
other diamino-pyrimidines,11,12 several compounds gave
unsatisfactory elemental analyses because of tenacious
entrapment of solvent. However, an analytically pure
sample of the monoethanesulfonic acid salt of 3a was
prepared (98%) from the free base and ethanesulfonic
acid (1 mol equiv) in ice-cold propanol-2-ol. This salt was
surprisingly stable, being undegraded in boiling ethanol
after 2 weeks.
1
broadened singlet at δ 5.01. However, in the H NMR
spectrum of the simple model triazene 13c, only one
sharp methyl resonance is observed, indicating that
N-N rotation is too fast to be observed by this technique
at ambient temperature. The methylene signals in the
1H NMR spectrum of TAB free base (3a ; Figure 3)
compare closely with those of the model compound 13e;
the methylene absorptions of the acetoxyethyl group of
3a appear as three multiplets between δ 3.91 and 4.28
(δ 3.91-4.30 for 13e).
Biologica l Resu lts
In all cases, triazenes were characterized by 1H NMR
The assay methods used in this study were as
previously described.17-19 Although a full sequence of
1
and 13C NMR spectroscopy and MS. The H chemical

2558 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Chan et al.
F igu r e 3. 1H NMR spectrum of compound 13a in DMSO-d6 at 289 K. The methylene protons of the benzyl group are at δ 5.01,
and the methylene protons of the acetoxyethyl group are between δ 3.91 and 4.28. Numbers above the chemical shift (δ) scale
refer to integral ratios.
Ta ble 1. Structure-Activity Relationship of
the rat liver DHFR has never been published, it has
Triazenyl-Substituted 2,4-Diaminopyrimidines toward P. carinii
been accepted as a reasonable in vitro model for the
human DHFR.20 The ability of the triazenyl-substituted
pyrimethamine derivatives of general structure 10 and
12 to inhibit P. carinii and rat liver DHFR was
compared with that of the lead triazene TAB 3a and
other reference compounds. These results are presented
as inhibitory concentrations (IC50) in Table 1.
The three compounds 10a -c were designed to explore
the bulk tolerance of the pocket created by the P. carinii
enzyme residues Lys37, Leu72, and Arg75. Leaving the
benzyl substituent constant, propionyl, isobutyryl, and
benzoyl moieties replace the acetyl function of 3a .
Compared to TAB these modifications resulted in a 5-
to 8-fold decrease in activity toward P. carinii DHFR.
Similarly, the hydroxyalkyltriazenes 12a -e were less
potent as inhibitors of the P. carinii enzyme; increasing
the length of the hydroxyalkyl chain from methylene
12a to propylene 12b also led to a reduction in potency.
As all the aforementioned compounds were marginally
more potent against the mammalian enzyme than TAB,
this conjunction of activities reduced their selectivity
(rat liver/P. carinii (rl/pc)) ratio in comparison to the
starting structure 3a .
Methoxy groups were introduced into the phenyl
substituent of 3a in the expectation that these bulky
functions would better occupy the hydrophobic pocket
bounded by P. carinii enzyme residues Phe36, Pro66,
and Phe69, thus enhancing potency. However, none of
these compounds 10d -g displayed any increased po-
tency over the lead compound 3a or increase in selectiv-
ity. From manual docking studies of TAB, face-edge
aromatic interactions with P. carinii DHFR Phe69 have
been identified as one of the factors contributing to its
species selectivity. We hypothesized that, as this type
of interaction is partially electrostatic, an electron-
withdrawing group (EWG) on the phenyl ring of TAB
would give rise to an optimal interaction with the π
electrons of Phe69. Therefore EWGs and electron-
donating groups were introduced onto the phenyl ring
(pc), T. gondii (tg), and M. avium (ma) DHFR
IC50 (µM) vs DHFRa selectivity ratiob
rat T.
P.
cpd carinii liver
M.
gondii avium
rl/pc
rl/tg
rl/ma
2c
0.042
0.17
2.8
3.65
1.3
1.2
0.85
2.1
0.003 0.01
0.071
114
6.8
0.003
3a d
3bd
4d
19.4
18.9
2.3
0.69
0.31
0.39
0.7
28
61
5.9
0.63
2.54
3.08
0.81
1.00
0.28
0.98
0.30
0.16
1.74
2.01
1.79
2.23
1.91
1.24
1.10
2.26
4.90
1.24
2.75
5.36
1.04
1.79
10a
10b
10c
10d
10e
10f
10g
10h 92
10i
10j
10k
10l
3.3
4.71
5.61
3.14
4.77
4.88
2.24
6.43
1.91
3.10
0.87
1.38
1.11
2.25
1.93
1.08
6.14
2.61
1.93
3.94
1.44
0.58
9.72
3.7
0.69
2.1
0.39
0.66
0.22
0.44
0.08
1.4
0.86
1.5
0.845 0.378
4.36
0.19
0.45
14.9
0.07
7.8
2.22
1.91
4.7
0.58
0.94
3.87
3.83
8.4
1.25
4.4
6.1
4.07
7.46
0.95
0.51
1.29
1.8
1.16
0.8
10m
10n 11.08
11.23
5.8
0.83
5.39
0.63
2.54
0.83
0.85
0.20
1.05
0.64
24.54
25.2
4.43
10o
10p
10q
10r
10s
10t
10u
10v
12a d
12b
12c
12d
12e
5.29
1.7
1.35
1.3
1.21
0.053
0.59
3.46
0.26
0.68
0.48
1.09
1.26
0.23
0.87
3.85
6.62
1.61
3.33
0.28
0.62
6.19
7
3.69
5.34
0.44
0.85
2.03
3.05
27
7.35
4.67
6.44
6.51
2.01
1.79
1.47
2.7
3.9
5.42
10.87
9.71
5.91
5.17
3.66
2.61
4.38
2.41
2.01
0.86
a
In vitro enzymatic assays were performed according to previ-
ously described methods.17-19 Selectivity is defined by ratio of
b
IC50 (rat liver DHFR)/IC50 (P. carinii, T. gondii, or M. avium
DHFR); values of >1 indicate a preferential binding to the
corresponding enzyme in the denominator. c Data from Chio et al.18
Data from Stevens et al.7
d
of the benzyl group 10h -s to evaluate both electronic
and steric effects on potency and selectivity. However,
confounding the original hypothesis, all three fluoroben-

Synthesis of P. carinii DHFR Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2559
zyltriazenes 10h -j were less potent against the P.
carinii enzyme than their unfluorinated precursor 3a .
The general trend showing that potency in the isomeric
fluoro series is in the order 2-F < 3-F or 4-F was
repeated in the chlorobenzyltriazenes 10k -m and the
methyl-substituted benzyltriazenes 10n -p . We con-
clude that the steric effect imposed by the 2′-substitution
impedes an optimal aromatic interaction between Phe69
and the substituted benzyl moiety. Substitution of the
benzyl moiety by trifluoromethyl 10q or nitro substit-
uents 10r ,s gave compounds which were 10-fold less
active than 3a .
Overall, all new compounds were less potent inhibi-
tors of P. carinii DHFR than TAB 3a with the exception
of 10t where the benzyl substituent has been replaced
by a naphth-1-ylmethyl moiety (IC50 0.053 µM). The
potency of this analogue is comparable to that of the
quinazoline trimetrexate 2 but because 10t is less
selective for the mammalian inhibitor, it shows a
superior rl/pc selectivity ratio (5.36) to 2 (0.071). The
isomeric naphth-2-ylmethyl triazene 10u was 10-fold
less active (IC50 0.59 µM) than the isomer 10t.
The triazenyl-pyrimethamines were also tested for
activity against T. gondii DHFR (Table 1). Overall they
were more active toward the DHFR enzyme from this
protozoan than P. carinii, exhibiting a potency predomi-
nantly in the submicromolar range. Those compounds
with a 2′-substituted phenyl ring were again generally
less active than their 3′- and 4′-substituted isomers.
Where supplies of compound allowed, new triazenes
were evaluated against the DHFR from Mycobacterium
avium (Table 1). IC50 values observed for these com-
pounds spanned only a 10-fold inhibitory range (2.03
to 25.2 µM).
data indicated little room to further optimize interaction
with Phe69. However, replacement of the benzyl sub-
stituent of 3a with a naphth-1-ylmethyl group 10t
resulted in a 3-fold improvement in potency toward the
P. carinii DHFR, placing this analogue in an activity
range similar to that of trimetrexate 2.
All the triazenyl analogues were tested for activity
against T. gondii DHFR. Generally, the new triazenyl-
pyrimethamines were more potent toward the DHFR
enzyme from this protozoa than P. carinii, exhibiting
IC50 values predominantly in the submicromolar range;
the most potent compound with the bulky trimethoxy-
benzyl substituent 10g gave an IC50 value of 0.053 µM.
Selected triazenyl analogues of TAB were less effective
against the DHFR from M. avium than P. carinii and
T. gondii, suggesting that the size of the active site
pocket in the mycobacterium DHFR is more restricted.
Exp er im en ta l Section
Molecu la r Mod elin g. The docking study was performed
using the DISCOVER module within the molecular graphics
program INSIGHT II21 utilizing the AMBER 3.5 force field22
for energy minimization. Full details have been given else-
where.10
Syn th etic Ch em istr y. Melting points were determined in
open capillaries using a Gallenkamp melting point apparatus
and are reported uncorrected. 1H and 13C NMR spectra of
synthetic intermediates and final products were recorded in
DMSO-d6 solutions on a Bruker spectrometer observing 1H at
13
250.13 MHz and
C at 62.9 MHz. Chemical shifts (δ) are
reported in parts per million (ppm) with tetramethysilane as
an internal standard; s ) singlet, d ) doublet, dd ) doublet
of doublets, t ) triplet, brs ) broad singlet, m ) multiplet.
Mass spectra (MS) in the atmospheric pressure chemical
ionization (APCI), electrospray ionization (ES), or fast atom
bombardment (FAB) mode were recorded using an ANI MS-
902, a VG Micromass 7070E, or a VG platform spectrometer.
Optical rotations were recorded on a Bellingham Stanley
ADP220 polarimeter. Elemental analyses (C, H, N) were
performed using either a Perkin-Elmer PE240B elemental
analyzer or an Exeter Analytical CE-440 elemental analyzer
by the Microanalysis service at the School of Chemistry,
University of Nottingham, and element compositions are
within (0.4% of the calculated values unless otherwise stated.
To monitor reaction mixtures by TLC, precoated silica gel
60F254 plates were used with the developing solvent being
either chloroform-methanol or hexanes-ethyl acetate mix-
tures; TLC spots were visualized with UV irradiation. All new
triazenyl-pyrimethamines and model compounds were purified
initially by flash column chromatography using silica gel C60H
from Merck, and crystallized unchanged from acetone, metha-
nol, or ethanol. Samples were dried in vacuo overnight over
P2O5 at room temperature prior to submission for elemental
analysis. No other special procedures were undertaken to
remove residues of solvents, and fractional moles of water and/
or organic solvents were found in some analytical samples. The
presence of these contaminations was detected in the 1H NMR
spectra but are not recorded in the listed spectral data.
Con clu sion
A structure-based design process to optimize the
selectivity and potency of a promising lead compound
TAB 3a 7 against DHFR from P. carinii has been
initiated. The approach encompasses molecular model-
ing studies, organic syntheses, and biological evaluation.
Manual docking studies of TAB 3a utilizing the pub-
lished X-ray crystallographically determined coordinates
of the P. carinii enzyme8 have enabled us to investigate
the structural basis for the species selectivity observed.
It was predicted that the benzyl side chain of TAB might
interact favorably with the hydrophobic region occupied
by Phe69 which is unique to P. carinii DHFR. The
veracity of the model was subsequently confirmed by
the X-ray determination of the P. carinii DHFR-TAB-
NADPH ternary complex.10
Both benzyl and acetoxyethyl appendages of the
triazenyl moiety were predicted to be important for
determining affinity and species selectivity and were
therefore investigated through rational structural modi-
fication. Disappointingly, all adjustments to the ac-
etoxyethyl branch, including increasing its length and
steric bulk, or increasing its polarity by deacetylation
had a dyschemotherapeutic effect, despite predictions
from modeling and crystal structure analysis which
suggested that there were opportunities for enhancing
enzyme interactions made by the acyl moiety. Modifica-
tions to the benzyl group were also generally detrimen-
tal, but this was not so unexpected as the structural
2,4-Dia m in o-5-{3-[3-[2-(a cetyloxy)eth yl]-3-ben zyltr ia -
zen -1-yl]-4-ch lor op h en yl}-6-eth ylp yr im id in e eth a n esu l-
fon ic a cid sa lt (3a ): This salt (98%), mp 170-171 °C (efferv.),
was formed from 3a and ethanesulfonic acid (1.1 mol equiv)
in 2-propanol at 0 °C; IR (KBr) 3401, 3144, 1738 (CdO), 1657,
1
1451, 1186, 1038, 740 cm-1; H NMR 1.02 (t, J ) 7.5 Hz, 3H,
CH2CH3), 1.09 (t, J ) 7.5 Hz, 3H, SCH2CH3), 1.90 (s, 3H, Cd
OCH3), 2.23 (q, J ) 7.5 Hz, 2H, CH2CH3), 2.46 (q, J ) 7.5 Hz,
2H, SCH2CH3), 3.93-4.32 (m, 4H, CH2CH2), 5.04 (brs, 2H,
CH2Ph), 7.00-7.61 (m, 11H), 8.20 (s, 1H), 12.21 (brs, NH and
NH+ absorptions); 13C NMR δ 9.9 (CH3), 20.7 (CH3), 23.8 (CH2),
45.4 (CH2), 46.3 (CH2), 50.6 (CH2), 53.2 (CH2), 59.3 (CH2), 61.9
(CH2), 107.5 (C), 120.6 (CH), 127.3 (CH), 128.0 (CH), 128.4

2560 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Chan et al.
(CH), 128.5 (CH), 128.7 (CH), 128.9 (CH), 130.9 (C), 131.2
(CH), 136.2 (C), 136.9 (C), 146.9 (C), 154.6 (C), 154.9 (C), 164.2
(C), 170.3 (C); MS (CI) m/z 468, 470 (M + H, -EtSO3). Anal.
(C25H32ClN7O5S) C, H, N.
The following triazenyl-substituted 2,4-diamino-5-(4-chlo-
rophenyl)-6-ethylpyrimidines were prepared by this general
method.
2,4-Dia m in o-5-{3-[3-ben zyl-3-[2-(p r op ion yloxy)eth yl]-
t r ia zen -1-yl]-4-ch lor op h en yl}-6-et h ylp yr im id in e (10a ):
From 6b and 9a ; 87% (from acetone), mp 127-128 °C (de-
comp.); IR (KBr) 3451, 3322, 3169 (N-H), 1720 (CdO), 1638,
Gen er al Meth od for th e Syn th esis of 2-[N-(Su bstitu ted-
ben zyl)a m in o]-eth a n ol Der iva tives 8a -q, 8t-v, 11a ,b.
The substituted benzaldehyde was dissolved in a solution of
ethanolamine (1.2 mol equiv) in methanol. Sodium hydrogen
carbonate (approximately 2 mol equiv) was added to the
solution which was then heated at reflux for 4 h. The reaction
mixture was cooled to 5 °C, and sodium borohydride (1.2 mol
equiv) was added portionwise during a period of 2 h while
keeping the temperature at 10 °C during which hydrogen gas
was evolved, and an off-white precipitate was formed. The
reaction mixture was maintained at room temperature over-
night. Any insoluble material was filtered off, and the filtrate
was collected and evaporated to dryness. The colored oily
residue was dissolved in chloroform and washed successively
with water and brine. The organic layer was collected and
concentrated in vacuo. The crude 2-[N-(substituted-benzyl)-
amino]ethanol derivatives (8) were purified by distillation
under reduced pressure, or by crystallization from benzene if
solid, and used directly for the next step.
1557, 1449, 1154 cm-1 1H NMR δ 0.88 (t, J ) 7.4 Hz, 3H,
;
CH2CH2CH3), 0.96 (t, J ) 7.4 Hz, 3H, CH2CH3), 2.06-2.21 (m,
4H), 3.92-4.30 (m, 4H, CH2CH2), 5.03 (brs, 2H, CH2Ph), 5.71
(brs, 2H, NH2), 5.92 (brs, 2H, NH2), 6.95-7.49 (m, 8H); 13C
NMR δ 8.9 (CH3), 13.3 (CH3), 26.8 (CH2), 27.7 (CH2), 46.2
(CH2), 50.4 (CH2), 53.1 (CH2), 58.8 (CH2), 59.4 (CH2), 61.7
(CH2), 105.7 (C), 120.7 (CH), 127.2 (CH), 128.3 (CH), 128.5
(CH), 128.8 (CH), 129.1 (CH), 130.5 (CH), 136.3 (C), 136.9 (C),
146.6 (C), 162.2, (C, C), 166.6 (C), 173.5 (C); MS (ES) m/z 482,
484 (M + 1). Anal. (C24H28ClN7O2) C, H, N.
2,4-Dia m in o-5-{3-[3-ben zyl-3-[2-(isobu tyr yloxy)eth yl]-
t r ia zen -1-yl]-4-ch lor op h en yl}-6-et h ylp yr im id in e (10b ):
From 6b and 9b; 78% (from acetone), mp 134-136 °C (de-
comp.); IR (KBr) 3451, 3322, 3169 (N-H), 1720 (CdO), 1630,
1
1557, 1449, 1154 cm-1; H NMR δ 0.99 (m, 9H), 2.08 (q, J )
7.5 Hz, 2H, CH2CH3), 2.33 (m, 1H, CH), 3.94-4.30 (m, 4H,
CH2CH2), 5.04 (brs, 2H, CH2Ph), 5.68 (brs, 2H, NH2), 5.92 (brs,
2H, NH2), 6.94-7.48 (m, 8H); 13C NMR δ 13.3 (CH3), 18.8
(CH3), 27.7 (CH2), 33.3 (CH), 46.1 (CH2), 50.2 (CH2), 53.2 (CH2),
58.7 (CH2), 59.6 (CH2), 61.6 (CH2), 105.7 (C), 120.7 (CH), 127.4
(CH), 128.3 (CH), 128.5 (CH), 129.1 (CH), 130.5 (CH), 135.8
(C), 136.3 (C), 136.9 (C), 146.6 (C), 162.1 (C), 162.3 (C), 166.6
The ethanolamines 8r ,s were prepared from ethanolamine
23
and 3-nitro- and 4-nitrobenzyl chloride, respectively; etha-
nolamine 11c from ethanolamine and 2-phenethyl chloride;24
and ethanolamine 11d ,e from 2-chloroethanol and R-(+)-R-
methylbenzylamine and S-(-)-R-methyl-benzylamine, respec-
tively.25
(C), 176.0 (C); MS (ES) m/z 496, 498 (M + 1). Anal. (C25H30
ClN7O2) C, H, N.
-
Gen er a l Meth od for th e Syn th esis of Hyd r och lor id e
Sa lts of Ester s of 2-[N-(Su bstitu ted -ben zyl)a m in o]eth a -
n ols 9a -v. Esterification of 2-[N-(substituted-benzyl)amino]-
ethanols 8a -v have been achieved by two methods. The
ethanol 8 was dissolved in a minimum of the appropriate
carboxylic acid, and hydrogen chloride gas was bubbled
through the solution for 1 h. The reaction mixture was stirred
at room temperature for a further 24 h to give a pale colored
solution. The off-white solid isolated when the solvent was
removed (vacuum distillation) was washed well with diethyl
ether and collected. Crystallization from either acetonitrile or
ethanol gave pure hydrochloride salts of the required esters
9.
2,4-Dia m in o-5-{3-[3-[2-(ben zoyloxy)eth yl]-3-ben zyltr ia -
zen -1-yl]-4-ch lor o-p h en yl}-6-eth ylp yr im id in e (10c): From
6b and 9c; 89% (from acetone), mp 98 °C (decomp.); IR (KBr)
3451, 3322, 3179 (N-H), 1709 (CdO), 1620, 1553, 1449, 1285
1
cm-1; H NMR δ 0.98 (t, J ) 7.5 Hz, 3H, CH2CH3), 2.06 (q, J
) 7.5 Hz, 2H, CH2CH3), 4.10-4.57 (m, 4H, CH2CH2), 5.57 (brs,
2H, CH2Ph), 5.76 (brs, 2H, NH2), 5.90 (brs, 2H, NH2), 7.08-
7.84 (m, 13H); 13C NMR δ 13.3 (CH3), 27.3 (CH2), 46.3 (CH2),
50.3 (CH2), 53.2 (CH2), 58.7 (CH2), 60.5 (CH2), 62.5 (CH2), 105.9
(CH), 120.8 (CH), 127.3 (CH), 128.5 (CH), 128.7 (CH), 129.0
(CH), 129.2 (CH), 129.5 (C), 130.6 (CH), 133.5 (CH); MS (ES)
m/z 496, 498 (M + 1). Anal. (C28H28ClN7O2) C, H, N.
This compound was also prepared from 2,4-diamino-5-{4-
chloro-3-[3-benzyl-3-(3-hydroxyethyl)triazen-1-yl]phenyl}-6-
ethylpyrimidine 12a and benzoyl chloride (1.5 mol equiv) in
pyridine containing DMAP (0.2 mol equiv) at 50 °C for 2 h.
Alternatively the 2-[N-(substituted-benzyl)amino]ethanol 8
was dissolved in 1,2-dichloro-ethane, and anhydrous hydro-
chloride gas was passed through the solution with stirring for
10 min. The acyl or benzoyl chloride (1 mol equiv) was added,
and the reaction mixture was heated at 60 °C for 4 h. The
precipitated hydrochloride salts (9) were collected by filtration.
2,4-Dia m in o-5-{3-[3-[2-(a cetyloxy)eth yl]-3-(4-m eth oxy-
b e n zyl)t r ia ze n -1-yl]-4-ch lor op h e n yl}-6-e t h ylp yr im i-
d in e (10d ): From 6b and 9d ; 90% (from acetone), mp 146-
148 °C (efferv.); IR (KBr) 3432, 3325, 3187 (N-H), 1730 (Cd
Gen er a l P r oced u r e of th e Syn th esis of Tr ia zen yl-
Su bstitu ted 2,4-Dia m in o-5-(4-ch lor op h en yl)-6-eth ylp y-
r im id in es 10a -v, 12a -e, a n d 13a ,b. (i) A stirred mixture
of aminopyrimethamine 511 (1.32 g, 5 mmol) in 3 M HCl (17.5
mL) was diazotized with a solution of sodium nitrite (375 mg)
in water (2.5 mL) at 0 °C for 1 h. A pale green solution of the
diazonium chloride 6a was obtained. An aqueous solution (5
mL) of the appropriate amine salt 9 (1 mol equiv) was added
dropwise to the above mixture. Solid sodium carbonate was
added to achieve a pH between 9 and 10. The reaction mixture
was stirred for a further 1 h between 0 and 5 °C, and the
creamy precipitate was collected and washed well with copious
amounts of cold water. Analytically pure samples of triazenes
could be obtained either from flash column chromatography
(chloroform-methanol 10:1) and/or crystallization from ac-
etone, methanol, or ethanol.
1
O), 1618, 1559, 1441, 1235 (C-O) cm-1; H NMR δ 0.97 (t, J
) 7.3 Hz, 3H, CH2CH3), 1.91 (d, 3H, CdOCH3), 2.10 (q, J )
7.5 Hz, 2H, CH2CH3), 3.72 (s, 3H, OCH3), 3.88-4.25 (m, 4H,
CH2CH2), 4.94 (brs, 2H, CH2Ph), 5.62 (brs, 2H, NH2), 5.89 (brs,
2H, NH2), 6.90-6.98 (m, 3H), 7.24-7.37 (m, 3H), 7.49 (d, J )
8.3 Hz, 1H); 13C NMR δ 13.4 (CH3), 20.7 (CH3), 27.7 (CH2),
45.9 (CH2), 49.7 (CH2), 52.7 (CH2), 55.2 (CH3), 58.3 (CH2), 59.4
(CH2), 61.9 (CH2), 105.7 (C), 113.9 (CH), 114.3 (CH), 120.7
(CH), 127.4 (C), 128.2 (C), 128.6 (C), 129.1 (CH), 129.8 (CH),
130.1 (CH), 130.6 (CH), 135.8 (C), 146.6 (C), 158.7 (C), 159.1
(C), 162.2 (C), 162.3 (C), 166.7 (C), 170.3 (C); MS (CI) m/z 498,
500 (M + 1). Anal. (C24H28ClN7O3) C, H, N.
2,4-Diam in o-5-{3-[3-[2-(acetyloxy)eth yl]-3-(3,4-dim eth ox-
yb en zyl)-t r ia zen -1-yl]-4-ch lor op h en yl}-6-et h ylp yr im i-
d in e (10e): From 6b and 9e; 73% (from acetone), mp 123-
124 °C (efferv.); IR (KBr) 3445, 3167 (N-H), 1736 (CdO), 1630,
(ii) The diazonium tetrafluoroborate hydrotetrafluoroborate
6b (0.5 g, 1.11 mmol) was dissolved in water (30 mL) at 0 °C
and coupled with an aqueous solution of the appropriate amine
(or amine salt) (1 mol equiv) which was added dropwise.
Sodium carbonate solid was added (to pH 9-10), and the
reaction mixture was then stirred for a further hour between
0 and 5 °C. The creamy precipitate was collected and purified
as above.
1
1572, 1443, 1230 (C-O), 1138 cm-1; H NMR δ 0.97 (t, J )
7.5 Hz, 3H, CH2CH3), 1.91 (d, 3H, CdOCH3), 2.10 (q, J ) 7.6
Hz, 2H, CH2CH3), 3.72 (s, 6H, 2 × OCH3), 3.89-4.26 (m, 4H,
CH2CH2), 4.93 (brs, 2H, CH2Ph), 5.70 (brs, 2H, NH2), 5.94 (brs,
2H, NH2), 6.75-7.34 (m, 5H), 7.50 (d, J ) 8.0 Hz 1H); 13C NMR
δ 13.4 (CH3), 20.7 (CH2), 27.7 (CH3), 46.0 (CH2), 49.9 (CH2),
52.5 (CH2), 55.6 (CH3), 55.7 (CH3), 58.7 (CH2), 59.4 (CH2), 61.9

Synthesis of P. carinii DHFR Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2561
(CH2), 105.7 (C), 111.9 (CH), 112.8 (CH), 120.7 (CH), 121.1
(CH), 127.3 (C), 128.6 (C), 129.0 (CH), 130.6 (CH), 135.8 (C),
146.6 (C), 148.2 (C), 148.8 (C), 162.1 (C), 162.3 (C), 166.6 (C),
170.3 (C); MS (CI) m/z 529, 531 (M + 1). Anal. (C25H30ClN7O4)
C, H, N.
7.5 Hz, 2H, CH2CH3), 3.91-3.44 (brs, 4H, CH2CH2), 5.00 (brs,
2H, CH2Ph), 5.69 (brs, 2H, NH2), 5.91 (brs, 2H, NH2), 6.97 (d,
J ) 7.5 Hz, 1H), 7.16-7.50 (m, 6H); 13C NMR δ 13.4 (CH3),
20.7 (CH3), 27.7 (CH2), 39.9 (CH2), 46.2 (CH2), 49.8 (CH2), 53.1
(CH2), 57.9 (CH2), 59.3 (CH2), 61.9 (CH2), 105.6 (C), 115.1 (CH),
115.4 (CH), 115.8 (CH), 120.7 (CH), 127.4 (C), 129.2 (CH),
130.4 (CH), 130.5 (CH), 132.5 (C), 133.2 (C), 135.8 (C), 146.5
(C), 162.1 (C), 162.3 (C), 166.6 (C), 170.3 (C); MS (CI) m/z 486,
488 (M + 1). Anal. (C23H25ClFN7O2) C, H, N.
2,4-Dia m in o-5-{3-[3-[2-(a cetyloxy)eth yl]-3-(3,5-d im eth -
oxyben zyl)tr ia zen -1-yl]-4-ch lor op h en yl}-6-eth ylp yr im i-
d in e (10f): From 6b and 9f; 46% (from acetone), mp 153-
154 °C (efferv.); IR (KBr) 3408, 3152 (N-H), 1742 (CdO), 1601
(C-O), 1441, 1353, 1231 (C-O), 1055 (C-O) cm-1; 1H NMR δ
0.96 (t, J ) 7.5 Hz, 3H, CH2CH3), 1.97 (d, 3H, CdOCH3), 2.09
(q, J ) 7.5 Hz, 2H, CH2CH3), 3.70 (s, 6H, 2 × OCH3), 3.92-
4.28 (m, 4H, CH2CH2), 4.94 (brs, 2H, CH2Ph), 5.64 (brs, 2H,
NH2), 5.90 (brs, 2H, NH2), 6.40-6.55 (m, 3H), 6.97 (d, J ) 7.8
Hz 1H), 7.22 (brs, 1H), 7.48 (brs, 1H); 13C NMR δ 13.4 (CH3),
20.7 (CH3), 27.7 (CH2), 46.3 (CH2), 50.3 (CH2), 52.9 (CH2), 55.3
(CH3), 58.7 (CH2), 59.4 (CH2), 99.1 (CH), 99.5 (CH), 105.7 (C),
106.2 (CH), 106.5 (CH), 120.8 (CH), 127.4 (C), 129.2 (CH),
130.6 (CH), 135.8 (C), 138.5 (C), 139.4 (C), 146.6 (C), 160.7
(C), 162.2 (C), 162.3 (C), 166.6 (C), 170.3 (C); MS (CI) m/z 529,
531 (M + 1). Anal. (C25H30ClN7O4) C, H, N.
2,4-Dia m in o-5-{3-[3-[2-(a cet yloxy)et h yl]-3-(2-ch lor o-
ben zyl)tr ia zen -1-yl]-4-ch lor op h en yl}-6-eth ylp yr im id in e
(10k ): From 6b and 9k ; 76% (from acetone), mp 181-182 °C
(efferv.); IR (KBr) 3464, 3171 (N-H), 1724 (CdO), 1630, 1553,
1
1441, 1231 (C-O), 1049 (C-O) cm-1; H NMR δ 0.96 (t, J )
7.5 Hz, 3H, CH2CH3), 1.89 (s, 3H, CdOCH3), 2.09 (q, J ) 7.5
Hz, 2H, CH2CH3), 3.67-4.31 (m, 4H, CH2CH2), 5.09 (d, J )
16.8 Hz, 2H, CH2Ph,), 5.64 (brs, 2H, NH2), 5.90 (brs, 2H, NH2),
6.97 (d, J ) 8.0 Hz, 1H), 7.19 (d, J ) 8.0 Hz, 1H), 7.37-7.47
(m, 5H); 13C NMR δ 13.3 (CH3), 20.6 (CH3), 27.6 (CH2), 46.5
(CH2), 49.2 (CH2), 53.4 (CH2), 56.4 (CH2), 59.1 (CH2), 59.4
(CH2), 62.1 (CH2), 105.6 (C), 120.6 (CH), 127.4 (CH), 128.9
(CH), 129.3 (CH), 130.5 (CH), 132.3 (C), 133.2 (C), 134.2 (C),
135.8 (C), 146.4 (C), 162.1 (C), 162.2 (C), 166.6 (C), 170.2 (C);
MS (CI) m/z 502, 504 (M + 1). Anal. (C23H25Cl2N7O2) C, H, N.
2,4-Dia m in o-5-{3-[3-[2-(a cet yloxy)et h yl]-3-(3,4,5-t r i-
m et h oxyb en zyl)-t r ia zen -1-yl]-4-ch lor op h en yl}-6-et h yl-
p yr im id in e (10g): From 6b and 9g; 73% (from acetone), mp
151-152 °C (efferv.); IR (KBr) 3453, 3167 (N-H), 2938, 1740
2,4-Dia m in o-5-{3-[3-[2-(a cet yloxy)et h yl]-3-(3-ch lor o-
ben zyl)tr ia zen -1-yl]-4-ch lor op h en yl}-6-eth ylp yr im id in e
(10l): From 6b and 9l; 75% (from acetone), mp 153-154 °C
(efferv.); IR (KBr) 3464, 3162 (N-H), 1730 (CdO), 1647, 1574,
1
(CdO), 1555, 1443, 1238 (C-O), 1120 cm-1; H NMR δ 0.96
(t, J ) 7.5 Hz, 3H, CH2CH3), 1.91 (d, 3H, CdOCH3), 2.10 (q, J
) 7.5 Hz, 2H, CH2CH3), 3.62 (s, 3H, OCH3), 3.74 (brs, 6H, 2 ×
OCH3), 3.94-4.23 (m, 4H, CH2CH2), 4.93 (s, 2H, CH2Ph), 5.63
(brs, 2H, NH2), 5.88 (brs, 2H, NH2), 6.64-7.22 (m, 4H), 7.49
(d, J ) 8.0 Hz, 1H); 13C NMR δ 13.3 (CH3), 20.7 (CH3), 27.7
(CH2), 46.3 (CH2), 50.5 (CH2), 52.8 (CH2), 56.0 (CH3), 59.0
(CH2), 59.4 (CH2), 60.1 (CH3), 61.9 (CH2), 105.6 (CH), 106.1
(CH), 120.8 (CH), 127.3 (C), 129.1 (CH), 130.6 (CH), 131.9 (C),
132.5 (C), 135.9 (C), 136.9 (C), 146.6 (C), 153.0 (C), 162.2 (C),
162.3 (C), 166.6 (C), 170.3 (C); MS (CI) m/z 559, 561 (M + 1).
Anal. (C26H32ClN7O5) C, H, N.
1
1444, 1375, 1230 (C-O) cm-1; H NMR δ 0.96 (t, J ) 7.5 Hz,
3H, CH2CH3), 1.89 (s, 3H, CdOCH3), 2.10 (q, J ) 7.5 Hz, 2H,
CH2CH3), 3.95-4.30 (m, 4H, CH2CH2), 4.99 (brs, 2H, CH2Ph),
5.65 (brs, 2H, NH2), 5.90 (brs, 2H, NH2), 6.97 (d, J ) 8.3 Hz,
1H), 7.21-7.48 (m, 6H); 13C NMR δ 13.4 (CH3), 20.7 (CH3),
46.6 (CH2), 50.4 (CH2), 53.5 (CH2), 58.0 (CH2), 59.4 (CH2), 61.9
(CH2); 105.6 (C), 120.8 (CH), 126.9 (CH), 127.2 (CH), 127.5
(C), 128.4 (CH), 129.3 (CH), 130.3 (CH), 130.6 (CH), 133.1 (C),
135.9 (C), 138.9 (C), 146.4 (C), 162.2 (C), 162.3 (C), 166.6 (C),
170.3 (C); MS (CI) m/z 502, 504 (M + 1). Anal. (C23H25Cl2N7O2)
C, H, N.
2,4-Dia m in o-5-{3-[3-[2-(a cet yloxy)et h yl]-3-(2-flu or o-
ben zyl)tr ia zen -1-yl]-4-ch lor op h en yl}-6-eth ylp yr im id in e
(10h ): From 6b and 9h ; 68% (from acetone), mp 183-184 °C
(efferv.); IR (KBr) 3459, 3185 (N-H), 1724 (CdO), 1628, 1555,
2,4-Dia m in o-5-{3-[3-[2-(a cet yloxy)et h yl]-3-(4-ch lor o-
ben zyl)tr ia zen -1-yl]-4-ch lor op h en yl}-6-eth ylp yr im id in e
(10m ): From 6b and 9m ; 82% (from acetone), mp 154-156 °C
(efferv.); IR (KBr) 2917, 2769, 1730 (CdO), 1562, 1441, 1246
(C-O), 1065 (C-O), 770 cm-1; 1H NMR δ 0.96 (t, J ) 7.4 Hz,
3H, CH2CH3), 1.90 (brs, 3H, CdOCH3), 2.10 (q, J ) 7.4 Hz,
2H, CH2CH3), 3.92-4.29 (m, 4H, CH2CH2), 5.00 (brs, 2H, CH2-
Ph), 5.70 (brs, 2H, NH2), 5.92 (brs, 2H, NH2), 6.97 (d, J ) 7.3
Hz, 1H), 7.22 (s, 1H), 7.38-7.49 (m, 5H); 13C NMR δ 13.4 (CH3),
20.7 (CH3), 27.7 (CH2), 46.8 (CH2), 50.9 (CH2), 53.8 (CH2), 57.9
(CH2), 59.5 (CH2), 62.0 (CH2), 105.6 (C), 120.8 (CH), 123.6 (CH),
127.6 (C), 129.0 (CH), 129.5 (CH), 130.6 (CH), 135.8 (C), 144.6
(C), 146.6 (C), 162.2 (C), 162.3 (C), 166.6 (C), 170.3 (C); MS
(CI) m/z 502, 504 (M + 1). Anal. (C23H25Cl2N7O2) C, H, N.
2,4-Dia m in o-5-{3-[3-[2-(a cet yloxy)et h yl]-3-(2-m et h yl-
b e n zyl)t r ia ze n -1-yl]-4-ch lor op h e n yl}-6-e t h ylp yr im i-
d in e (10n ): From 6b and 9n ; 61% (from acetone), mp 175-
176 °C (efferv.); IR (KBr) 3459, 3177 (N-H), 1723 (CdO), 1626,
1553, 1441, 1233 (C-O), 1045 (C-O) cm-1; 1H NMR δ 0.96 (t,
J ) 7.5 Hz, 3H, CH2CH3), 1.90 (brs, 3H, CdOCH3), 2.10 (q, J
) 7.5 Hz, 2H, CH2CH3), 2.28 (s, 3H, CH3), 3.90-4.00 (m, 2H,
NCH2), 4.26 (t, J ) 4.9 Hz, 2H, CH2O), 5.09 (brs, 2H, CH2Ph),
5.64 (brs, 2H, NH2), 5.89 (brs, 2H, NH2), 6.96 (d, J ) 7.5 Hz,
1H), 7.14-7.47 (m, 6H); 13C NMR δ 13.4 (CH3), 19.0 (CH3),
20.7 (CH3), 27.7 (CH2), 45.9 (CH2), 48.5 (CH2), 52.5 (CH2), 57.1
(CH2), 59.3 (CH2), 61.9 (CH2), 105.6 (C), 120.9 (CH), 126.2 (CH),
127.3 (CH), 127.9 (CH), 128.1(CH), 129.1 (CH), 130.6 (CH),
135.8 (C), 146.6 (C), 162.2 (C), 162.3 (C), 166.6 (C), 170.3 (C);
MS (CI) m/z 482, 484 (M + 1). Anal. (C24H28ClN7O2) C, H, N.
1
1447, 1236 (C-O), 1047 (C-O) cm-1; H NMR δ 0.96 (t, J )
7.5 Hz, 3H, CH2CH3), 2.0 (s, 3H, CdOCH3), 2.09 (q, J ) 7.5
Hz, 2H, CH2CH3), 3.95-4.31 (m, 4H, CH2CH2), 5.04 (d, 2H,
CH2Ph), 5.64 (brs, 2H, NH2), 5.91 (brs, 2H, NH2), 6.96 (d, J )
7.8 Hz), 7.19-7.47 (m, 6H); 13C NMR δ 13.4 (CH3), 20.6 (CH3),
27.7 (CH2), 45.2 (CH2), 46.4 (CH2), 52.9 (CH2), 53.4 (CH2), 59.4
(CH2), 62.0 (CH2), 105.6 (C), 115.2 (CH), 115.5 (CH), 120.7
(CH), 123.0 (C), 124.3 (C), 127.5 (CH), 129.3 (CH), 130.6 (CH),
130.8 (CH), 135.8 (C), 146.4 (C), 158.7 (C), 162.2 (C), 162.3
(C), 162.5 (C), 166.6 (C), 170.3 (C); MS (CI) m/z 486, 488 (M +
1). Anal. (C23H25ClFN7O2) C, H, N.
2,4-Dia m in o-5-{3-[3-[2-(a cet yloxy)et h yl]-3-(3-flu or o-
ben zyl)tr ia zen -1-yl]-4-ch lor op h en yl}-6-eth ylp yr im id in e
(10i): From 6b and 9i; 74% (from acetone), mp 146-147 °C
(efferv.); IR (KBr) 3464, 3187 (N-H), 1720 (CdO), 1624, 1555,
1
1443, 1235 (C-O), 1045 (C-O) cm-1; H NMR δ 0.96 (t, J )
7.5 Hz, 3H, CH2CH3), 1.89 (brs, 3H, CdOCH3), 2.10 (q, J )
7.5 Hz, 2H, CH2CH3), 3.95-4.3 (m, 4H, CH2CH2), 5.03 (brs,
2H, CH2Ph), 5.66 (brs, 2H, NH2), 5.91 (brs, 2H, NH2), 6.96-
7.49 (m, 7H); 13C NMR δ 13.4 (CH3), 20.6 (CH3), 27.7 (CH2),
46.5 (CH2), 50.3 (CH2), 53.4 (CH2), 58.1 (CH2), 59.4 (CH2), 61.9
(CH2), 105.6 (C), 113.9 (CH), 114.2 (CH),114.6 (CH), 114.9
(CH), 115.3 (CH), 120.8 (CH), 124.3 (CH), 124.3 (CH), 127.5
(C), 129.3 (CH), 130.6 (CH), 135.9 (C), 139.3 (C), 140.0 (C),
146.4 (C), 160.4 (C), 162.2 (C), 162.3 (C), 164.3 (C), 166.6 (C),
170.3 (C); MS (CI) m/z 486, 488 (M + 1). Anal. (C23H25ClFN7O2)
C, H, N.
2,4-Dia m in o-5-{3-[3-[2-(a cet yloxy)et h yl]-3-(4-flu or o-
ben zyl)tr ia zen -1-yl]-4-ch lor op h en yl}-6-eth ylp yr im id in e
(10j): From 6b and 9j; 84% (from acetone), mp 161-162 °C
(efferv.); IR (KBr) 3476, 3173 (N-H), 1730 (CdO), 1605, 1562,
2,4-Dia m in o-5-{3-[3-[2-(a cet yloxy)et h yl]-3-(3-m et h yl-
b e n zyl)t r ia ze n -1-yl]-4-ch lor op h e n yl}-6-e t h ylp yr im i-
d in e (10o): From 6b and 9o; 76% (from acetone), mp 155-
156 °C (efferv.); IR (KBr) 3451, 3181 (N-H), 1724 (CdO), 1620,
1572, 1443, 1235 (C-O), 1045 (C-O) cm-1; 1H NMR δ 0.97 (t,
J ) 7.5 Hz, 3H, CH2CH3), 1.90 (d, 3H, CdOCH3), 2.10 (q, J )
1
1437, 1225 (C-O), 1059 (C-O) cm-1; H NMR δ 0.96 (t, J )
7.5 Hz, 3H, CH2CH3), 1.89 (brs, 3H, CdOCH3), 2.10 (q, J )

2562 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Chan et al.
7.5 Hz, 2H, CH2CH3), 2.27 (s, 3H, CH3), 3.90-4.27 (m, 4H,
CH2CH2), 4.98 (brs, 2H, CH2Ph), 5.65 (brs, 2H, NH2), 5.90 (brs,
2H, NH2), 6.98-7.49 (m, 7H); 13C NMR δ 13.4 (CH3), 20.7
(CH3), 21.2 (CH3), 27.7 (CH2), 46.2 (CH2), 50.4 (CH2), 53.0
(CH2), 58.8 (CH2), 59.4 (CH2), 61.9 (CH2); 105.7 (C), 120.8 (CH),
125.4 (CH), 127.4 (C), 128.0 (CH), 128.0 (CH), 128.6 (CH),
129.1 (CH), 130.6 (CH), 135.8 (C), 136.2 (C), 136.8 (C), 137.5
(C), 138.0 (C), 146.6 (C), 162.2 (C), 162.3 (C), 166.6 (C), 170.3
(C); MS (CI) m/z 482, 484 (M + 1). Anal. (C24H28ClN7O2) C, H,
N.
2,4-Dia m in o-5-{3-[3-[2-(a cet yloxy)et h yl]-3-(4-m et h yl-
b e n zyl)t r ia ze n -1-yl]-4-ch lor op h e n yl}-6-e t h ylp yr im i-
d in e (10p ): From 6b and 9p ; 72% (from acetone), mp 167-
169 °C (efferv.); IR (KBr) 3437, 3410, 3154 (N-H), 1728
(CdO), 1579, 1433, 1229 (C-O), 1063 (C-O) cm-1; 1H NMR δ
0.97 (t, J ) 7.5 Hz, 3H, CH2CH3), 1.86 (d, 3H, CdOCH3), 2.08
(q, J ) 7.5 Hz, 2H, CH2CH3), 2.27 (s, 3H, CH3), 3.88-4.26 (m,
4H, CH2CH2), 4.97 (brs, 2H, CH2Ph), 5.67 (brs, 2H, NH2), 5.92
(brs, 2H, NH2), 6.97-7.50 (m, 7H); 13C NMR δ 13.4 (CH3), 20.7
(CH3), 20.9 (CH3), 27.7 (CH2), 46.0 (CH2), 50.0 (CH2), 52.8
(CH2), 58.6 (CH2), 59.4 (CH2), 61.9 (CH2), 105.7 (C), 127.4 (C),
127.7 (CH), 128.3 (CH), 128.5 (CH), 129.1 (CH), 129.4 (CH),
130.6 (CH), 133.2 (C), 133.8 (C), 135.8 (C), 136.5 (C), 137.3
(C), 146.6 (C), 162.2 (C), 162.3 (C), 166.6 (C), 170.3 (C); MS
(CI) m/z 482, 484 (M + 1). Anal. (C24H28ClN7O2) C, H, N.
151 °C (efferv.); IR (KBr) 3445, 3162 (N-H), 1738 (CdO), 1624,
1
1553, 1445, 1341, 1233 cm-1; H NMR δ 0.98 (t, J ) 7.5 Hz,
3H, CH2CH3), 1.79 (brs, 3H, CdOCH3) 2.09 (q, J ) 7.5 Hz,
2H, CH2CH3), 3.94-4.23 (m, 4H, CH2CH2), 5.50 (brs, 2H, CH2-
naphthyl), 5.67 (brs, 2H, NH2), 5.91 (brs, 2H, NH2), 6.99 (dd,
J ) 2.0, 8.3 Hz, 1H), 7.26-8.16 (m, 9H); 13C NMR δ 13.4 (CH3),
20.5 (CH3), 27.7 (CH2), 45.8 (CH2), 47.9 (CH2), 51.9 (CH2), 57.0
(CH2), 59.2 (CH2), 61.8 (CH2), 105.6 (C), 120.7 (CH), 121.0 (C),
123.7 (CH), 123.8 (CH), 125.7 (CH), 126.2 (CH), 126.6 (CH),
127.3 (C), 127.4 (C), 127.9 (CH), 128.2 (CH), 128.8 (CH), 129.2
(CH), 130.6 (CH), 131.1 (C), 131.5 (C), 132.1 (C), 133.6 (C),
133.7 (C), 135.9 (C), 146.6 (C), 162.2 (C), 162.3 (C), 166.6 (C),
170.2 (C); MS (CI) m/z 518, 520 (M + 1); HR-MS (FAB) calcd
for C27H29ClN7O2 (M + H) m/z 518.207126, found 518.207048.
2,4-Dia m in o-5-{3-[3-[2-(a cet yloxy)et h yl]-3-(n a p h t h -2-
ylm et h yl)t r ia zen -1-yl]-4-ch lor op h en yl}-6-et h ylp yr im i-
d in e (10u ): From 6b and 9u ; 84% (from acetone), mp 77-78
°C (efferv.); IR (KBr) 3449, 3163 (N-H), 1736 (CdO), 1632,
1
1555, 1445, 1339, 1233 (C-O) cm-1; H NMR δ 0.96 (t, J )
7.5 Hz, 3H, CH2CH3), 1.86 (brs, 3H, CH3), 2.09 (q, J ) 7.5 Hz,
2H, CH2CH3), 3.96-4.32 (m, 4H, CH2CH2), 5.19 (brs, 2H, CH2-
naphthyl), 5.64 (brs, 2H, NH2), 5.90 (brs, 2H, NH2), 6.95 (t,
1H), 7.27 (d, 1H), 7.50 (brs, 4H), 7.89 (brs, 4H); 13C NMR δ
13.4 (CH3), 20.6 (CH3), 27.7 (CH2), 46.3 (CH2), 50.7 (CH2), 53.2
(CH2), 59.0 (CH2), 62.0 (CH2), 105.7 (C), 120.8 (CH), 126.1 (CH),
126.2 (CH), 126.4 (CH), 127.1 (CH), 127.5 (C), 127.8 (CH),
128.1 (CH), 128.6 (CH), 129.2 (CH), 130.6 (CH), 132.4 (C),
132.6 (C), 133.1 (C), 133.9 (C), 134.4 (C), 135.9 (C), 146.6 (C),
162.2 (C), 162.3 (C), 166.6 (C), 170.3 (C); MS (CI) m/z 518, 520
(M + 1); HR-MS (FAB) calcd for C27H29ClN7O2 (M + H) m/z
518.207126, found 518.207393.
2,4-Dia m in o-5-{3-[2-(a cet yloxy)et h yl]-3-(4-t r iflu or o-
m eth ylben zyl)tr iazen -1-yl]-4-ch lor oph en yl}-6-eth ylpyr im i-
d in e (10q): From 6b and 9q; 75% (from ethanol), mp 142-
143 °C (efferv.); IR (KBr) 3455, 3183 (N-H), 1726 (CdO), 1628,
1555, 1443, 1325, 1067 (C-O) cm-1 1H NMR δ 0.95 (t, J )
,
7.5 Hz, 2H, CH2CH3), 1.87 (s, 3H, CdOCH3), 2.09 (q, J ) 7.5
Hz, 2H, CH2CH3), 3.96-4.32 (m, 4H, CH2CH2), 5.11 (m, 2H,
CH2Ph), 5.64 (brs, 2H, NH2), 5.90 (brs, 2H, NH2), 6.95-7.76
(m, 7H); 13C NMR δ 13.8 (CH3), 21.1 (CH3), 28.5 (CH2), 46.7
(CH2), 51.3 (CH2), 53.5 (CH2), 59.4 (CH2), 60.6 (CH2), 62.8
(CH2), 76.5 (CF), 77.0 (CF), 77.5 (CF), 107.2 (C), 120.6 (CH),
125.4 (CH), 126.1 (CH), 128.3 (CH), 128.8 (CH), 129.3 (C),
130.9 (CH), 134.4 (C), 139.9 (C), 140.5 (C), 146.8 (C), 161.8
(C), 162.0 (C), 168.3 (C), 170.6 (C); MS (CI) m/z 536, 538 (M +
1). Anal. (C24H28F3ClN7O2) C, H, N.
2,4-Dia m in o-5-{3-[3-[2-(a cetyloxy)eth yl]-3-(4-p yr id in -4-
ylm et h yl)t r ia zen -1-yl]-4-ch lor op h en yl}-6-et h ylp yr im i-
d in e (10v): From 6b and 9v; 74% (from acetone), mp 168-
170 °C (efferv.); IR (KBr) 3462, 3322, 3163 (N-H), 1740 (Cd
1
O), 1632, 1555, 1443, 1235 (C-O) cm-1; H NMR δ 0.96 (t, J
) 7.5 Hz, 3H, CH2CH3), 1.89 (s, 3H, CdOCH3), 2.10 (q, J )
7.5 Hz, 2H, CH2CH3), 4.01-4.32 (m, 4H, CH2CH2), 5.02 (brs,
2H CH2-pyridyl), 5.70 (brs, 2H, NH2), 5.91 (brs, 2H, NH2),
6.96-7.47 (m, 5H), 8.51 (brs, 2H); 13C NMR δ 13.4 (CH3), 20.6
(CH3), 27.6 (CH2), 47.0 (CH2), 50.4 (CH2), 53.8 (CH2), 57.5
(CH2), 59.4 (CH2), 62.0 (CH2), 105.6 (C), 120.8 (CH), 122.8 (CH),
127.5 (C), 129.4 (CH), 130.6 (CH), 135.8 (C), 145.4 (C), 146.3
(C), 146.3 (C), 149.6 (CH), 162.1 (C), 162.2 (C), 166.5 (C), 170.3
(C); MS (CI) m/z 469, 471 (M + 1); HR-MS (FAB) calcd for
2,4-Dia m in o-5-{3-[3-[2-(a ce t yloxy)e t h yl]-3-(3-n it r o-
ben zyl)tr ia zen -1-yl]-4-ch lor op h en yl}-6-eth ylp yr im id in e
(10r ): From 6b and 9r ; 79% (from ethanol), mp 167-168 °C
(efferv.); IR (KBr) 3484, 3150 (N-H), 1736 (CdO), 1601, 1549
1
(NO2), 1437, 1349 (NO2), 1240 cm-1; H NMR δ 0.95 (t, J )
C
22H26ClN8O2 (M + H) m/z 469.186725, found 469.187214.
2,4-Dia m in o-5-{3-[3-ben zyl-3-(2-h yd r oxyeth yl)tr ia zen -
7.5 Hz, 3H, CH2CH3), 1.89 (s, 3H, CdOCH3), 2.09 (q, J ) 7.5
Hz, 2H, CH2CH3), 3.99-4.33 (m, 4H, CH2CH2), 5.08 and 5.17
(2 brs, 2H, CH2Ph), 5.63 (brs, 2H, NH2), 5.89 (brs, 2H, NH2),
6.97 (d, J ) 7.5 Hz, 1H), 7.20 (brs, 1H), 7.45 (d, J ) 8.0 Hz,
1H), 7.64 (t, J ) 7.3 Hz, 1H), 7.83 (d, J ) 8.0 Hz, 1H), 8.12 (d,
J ) 8.0 Hz, 1H), 8.24 (s, 1H); 13C NMR δ 13.4 (CH3), 20.7 (CH3),
27.7 (CH2), 46.8 (CH2), 50.7 (CH2), 53.8 (CH2), 57.7 (CH2), 59.4
(CH2), 62.0 (CH2), 105.6 (C), 120.8 (CH), 122.2 (CH), 123.1
(CH), 127.5 (C), 129.5 (CH), 129.9 (CH), 130.6 (CH), 135.0
(CH), 138.9 (C), 146.3 (C), 147.9 (C), 162.2 (C), 162.3 (C), 166.6
1-yl]-4-ch lor op h en yl}-6-eth ylp yr im id in e (12a ): Prepared
(68%) from 6b and ethanolamine (11a ), this triazene had
identical physical characteristics to an authentic sample.7
2,4-Diam in o-5-{3-[3-ben zyl-3-(3-h ydr oxypr opyl)tr iazen -
1-yl]-4-ch lor op h en yl}-6-eth ylp yr im id in e (12b): From 6b
and 11b; 53% (from ethanol), mp 147-148 °C (efferv.); IR
(KBr) 3329, 3181 (N-H), 1620, 1555, 1443, 1260, 1053, 698
1
cm-1; H NMR δ 0.98 (t, J ) 7.5 Hz, 3H, CH2CH3), 1.81 (brs,
(C), 170.3 (C); MS (CI) m/z 513, 515 (M + 1). Anal. (C23H25
ClN8O4) C, H, N.
-
2H, NCH2CH2), 2.11 (q, J ) 7.5 Hz, 2H, CH2CH3), 3.39-3.47
(m, 2H, NCH2), 3.69-3.88 (m, 2H, CH2OH), 4.53 (t, J ) 5.1
Hz, 1H, OH,), 4.99 (brs, 2H, CH2Ph), 5.62 (brs, 2H, NH2), 5.89
(brs, 2H, NH2), 6.94 (d, J ) 8.3 Hz, 1H), 7.25-7.49 (m, 7H);
13C NMR δ 13.5 (CH3), 27.7 (CH2), 28.4 (CH2), 32.0 (CH2), 44.7
(CH2), 50.3 (CH2), 51.7 (CH2), 58.2 (CH2), 58.8 (CH2), 105.8
(C), 120.6 (CH), 127.3 (CH), 128.0 (CH), 128.2 (CH), 128.5
(CH), 128.7 (CH), 130.5 (CH), 135.7 (C), 146.9 (C), 137.2 (C),
136.4 (C), 162.2 (C), 162.3 (C), 166.6 (C); MS (CI) m/z 440, 442
(M + 1). Anal. (C22H26ClN7O) C, H, N.
2,4-Dia m in o-5-{3-[3-[2-(a ce t yloxy)e t h yl]-3-(4-n it r o-
ben zyl)tr ia zen -1-yl]-4-ch lor op h en yl}-6-eth ylp yr im id in e
(10s): From 6b and 9s; 72% (from acetone), mp 117-118 °C
(efferv.); IR (KBr) 3455, 3165 (N-H), 1720 (CdO), 1628, 1555,
1
1522 (NO2), 1441, 1344 (NO2), 1240 (C-O) cm-1; H NMR δ
0.95 (t, J ) 7.5 Hz, 3H, CH2CH3), 1.90 (s, 3H, CdOCH3), 2.09
(q, J ) 7.5 Hz, 2H, CH2CH3), 3.98-4.38 (m, 4H, CH2CH2),
5.10-5.18 (m, 2H, CH2Ph), 5.63 (brs, 2H, NH2), 5.90 (brs, 2H,
NH2), 6.95-7.60 (m, 5H), 8.18-8.28 (m, 2H); 13C NMR δ 13.3
(CH3), 20.6 (CH3), 27.6 (CH2), 20.9 (CH2), 53.7 (CH2), 62.0
(CH2), 105.6 (C), 120.7 (CH), 123.6 (CH), 127.5 (CH), 129.0
(CH), 129.4 (CH), 130.6 (CH), 135.8 (C), 144.6 (C), 146.3(C),
146.6 (C), 162.1 (C), 162.3 (C), 166.6 (C), 170.3 (C); MS (CI)
m/z 513, 515 (M + 1). Anal. (C23H25ClN8O4) C, H, N.
The acetoxy-derivative 12f was prepared from 12b in acetic
acid/anhydrous hydrochloric acid according to the general
method described earlier. Flash column chromatography (dichlo-
romethane/methanol 10:1), followed by crystallization from
ethanol, gave 12f as an off-white solid (39%), mp 202-204 °C;
IR (KBr) 3447, 3322, 3189 (N-H), 1721 (CdO), 1624, 1553,
2,4-Dia m in o-5-{3-[3-[2-(a cet yloxy)et h yl]-3-(n a p h t h -1-
ylm et h yl)t r ia zen -1-yl]-4-ch lor op h en yl}-6-et h ylp yr im i-
d in e (10t): From 6b and 9t; 67% (from acetone), mp 149-
1441, 1262 (C-O) cm-1 1H NMR δ 0.97 (t, J ) 7.5 Hz 3H,
;
CH2CH3), 1.95 (d, 5H, CdOCH3 and CH2CH2CH2), 2.10 (q, J
) 7.4 Hz, 2H, CH2CH3), 3.72-3.90 (m, 2H, NCH2), 3.99 (t, J

Synthesis of P. carinii DHFR Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2563
) 5.6 Hz, 2H, CH2O), 4.98 (brs, 2H, CH2Ph), 5.63 (brs, 2H,
NH2), 5.90 (brs, 2H, NH2), 6.95 (d, 2H), 7.21-7.56 (m, 7H);
13C NMR δ 13.4 (CH3), 20.8 (CH3), 24.6 (CH2), 27.5 (CH2), 27.7
(CH2), 44.3 (CH2), 50.1 (CH2), 51.3 (CH2), 58.2 (CH2), 61.5
(CH2), 62.1 (CH2), 105.7 (C), 120.8 (CH), 127.3 (CH), 128.3
(CH), 128.5 (CH), 128.9 (CH), 130.5 (CH), 135.8 (C), 136.3 (C),
146.7 (C), 162.1 (C), 162.3 (C), 166.6 (C), 170.4 (C); MS (CI)
m/z 482, 484 (M + 1). Anal. (C24H28ClN7O2) C, H, N.
Gen er a l P r oced u r e of th e Syn th esis of Tr ia zen yl-
Su bstitu ted 2-Ch lor oben zen es 13c-e. 2-Chloroaniline (1.53
g) in 1 M HCl (30 mL) was diazotized at 0 °C with a solution
of sodium nitrite (1.1 mol equiv). The mixture was maintained
at 0 °C (1 h), and the appropriate secondary amine or amine
salt (1 mol equiv) was added to the vigorously stirred mixture
followed by sodium carbonate to adjust the pH to 9-10. The
triazenes separated as light brown oils which were extracted
into diethyl ether (2 × 25 mL). The organic layer was
separated, washed successively with brine and water (50 mL),
dried (MgSO4), and evaporated to yield a residue which was
purified by flash column chromatography employing hexanes-
ethyl acetate (10:1) as eluent. The viscous triazenes decom-
posed when subjected to vacuum distillation.
2,4-Dia m in o-5-{3-[3-p h en et h yl-3-(2-h yd r oxyet h yl)t r i-
a zen -1-yl]-4-ch lor op h en yl}-6-eth ylp yr im id in e (12c): From
6b and 11c; 69% (from ethanol), mp 87-89 °C (efferv.); IR
(KBr) 3341, 3156 (N-H), 1655, 1610, 1555, 1452, 1053, 700
1
cm-1; H NMR δ 0.98 (t, J ) 7.5 Hz, 3H, CH2CH3), 2.11 (q, J
) 7.5 Hz, 2H, CH2CH3), 3.00-4.08 (m, 8H, 4 × CH2), 4.85 (brs,
1H, OH), 5.69 (brs, 2H, NH2), 5.95 (brs, 2H, NH2), 6.79-7.53
(m, 8H); 13C NMR δ 13.4 (CH3), 27.6 (CH2), 30.5 (CH2), 34.9
(CH2), 50.2 (CH2), 50.5 (CH2), 56.6 (CH2), 56.9 (CH2), 57.4
(CH2), 59.8 (CH2), 105.9 (C), 120.4 (CH), 120.8 (CH), 126.3
(CH), 126.4 (CH), 127.0 (C), 127.3 (C), 128.4 (CH), 128.5 (CH),
128.6 (CH), 128.9 (CH), 129.1 (CH), 130.3 (CH), 130.5 (CH),
135.6 (C), 138.9 (C), 139.4 (C), 147.1 (C), 162.1 (C), 162.2 (C),
166.2 (C); MS (ES) m/z 440, 442 (M + 1). Anal. (C22H26ClN7O)
C, H, N.
The following triazenyl-substituted 2-chlorobenzenes were
prepared by this general method.
2-(3-B e n z y l-3-m e t h y lt r i a z e n -1-y l)c h lo r o b e n z e n e
(13c): From N-methylbenzyl-amine, 77%; IR (NaCl) 3965,
1
1464, 1348, 1179, 1053, 750, 698, 588 cm-1; H NMR δ 3.12
(s, 3H, CH3), 5.02 (s, 2H CH2Ph), 7.13-7.49 (m, 9H); 13C NMR
δ 34.7 (CH3), 59.1 (CH2), 118.9 (CH), 126.4 (CH), 127.7 (CH),
128.0 (CH), 128.3 (C), 128.9 (CH), 130.1 (CH), 136.8 (C), 146.9
(C); HR-MS (FAB) calcd for C14H14ClN3 (M) m/z 259.08762,
found 259.08704.
R-(-)-2,4-Dia m in o-5-{3-[3-(2-h yd r oxyeth yl)-3-(R-m eth -
ylb en zyl)t r ia zen -1-yl]-4-ch lor op h en yl}-6-et h ylp yr im i-
d in e (12d ): From 6a and 11d ; 78% (from acetone), mp 143-
144 °C (efferv.); [R]D -146.7° (c 0.375 mg mL-1, EtOH, 17.6
°C); IR (KBr) 3322, 3169 (N-H), 1632, 1553, 1439, 1381, 1059,
700 cm-1; 1H NMR 0.99 (t, J ) 7.5 Hz, 3H, CH2CH3), 1.7 (d, J
) 6.8 Hz, 3H, CHCH3), 2.12 (q, J ) 7.4 Hz, 2H, CH2CH3),
3.52-3.89 (m, 4H, CH2CH2), 4.81 (t, J ) 5.2 Hz, 1H, OH), 5.2
(q, J ) 6.8 Hz, 1H, CHCH3), 5.67 (brs, 2H, NH2), 5.92 (brs,
2H, NH2), 6.93-6.97 (m, 1H), 7.22-7.39 (m, 6H), 7.48-7.51
(m, 1H); 13C NMR δ 13.5 (CH3), 20.7 (CH3), 27.7 (CH2), 50.4
(CH2), 56.7 (CH2), 63.9 (CH), 105.9 (C), 120.7 (CH), 126.9 (CH),
127.2 (C), 127.8 (CH), 128.6 (C), 128.8 (CH), 130.5 (CH), 135.7
(C), 141.9 (C), 147.3 (C), 162.2 (C), 166.6 (C); MS (CI) m/z 440,
442 (M + 1). Anal. (C22H26ClN7O) C, H, N.
S-(+)-2,4-Dia m in o-5-{3-[3-(2-h yd r oxyeth yl)-3-(R-m eth -
ylb en zyl)t r ia zen -1-yl]-4-ch lor op h en yl}-6-et h ylp yr im i-
d in e (12e): From 6a and 11e; 78% (from acetone), mp 157-
158 °C (efferv.); [R]D +182.1° (c 0.302 mg mL-1, EtOH, 20.3
°C); IR (KBr) 3322, 3169 (N-H), 1632, 1553, 1439, 1381, 1059,
700 cm-1; 1H NMR 0.99 (t, J ) 7.5 Hz, 3H, CH2CH3), 1.7 (d, J
) 6.8 Hz, 3H, CHCH3), 2.12 (q, J ) 7.4 Hz, 2H, CH2CH3),
3.52-3.89 (m, 4H, CH2CH2), 4.81 (t, J ) 5.2 Hz, 1H, OH), 5.2
(q, J ) 6.8 Hz, 1H, CHCH3), 5.67 (brs, 2H, NH2), 5.92 (brs,
2H, NH2), 6.93-6.97 (m, 1H), 7.22-7.39 (m, 6H), 7.48-7.51
(m, 1H); 13C NMR δ 13.5 (CH3), 20.7 (CH3), 27.7 (CH2), 50.4
(CH2), 56.7 (CH2), 63.9 (CH), 105.9 (C), 120.7 (CH), 126.9 (CH),
127.2 (C), 127.8 (CH), 128.6 (C), 128.8 (CH), 130.5 (CH), 135.7
(C), 141.9 (C), 147.3 (C), 162.2 (C), 166.6 (C); MS (ES) m/z 440,
442 (M + 1). Anal. (C22H26ClN7O) C, H, N.
2-(3-Benzyl-3-ethyltriazen-1-yl)chlorobenzene (13d): From
N-ethylbenzyl-amine, 79%; IR (NaCl) 2970, 1468, 1048, 1327,
1173, 1053, 754, 698 cm-1 1H NMR δ 1.10-1.25 (m, 3H,
;
NCH2CH3), 3.70-3.85 (m, 2H, NCH2CH3), 4.98 (s, 2H, CH2-
Ph), 7.02-7.46 (m, 9H); 13C NMR δ 10.3 (CH3), 14.3 (CH3),
41.9 (CH2), 49.1 (CH2), 49.7 (CH2), 57.6 (CH2), 118.7 (CH),
126.3 (CH), 127.2 (CH), 128.1 (CH), 128.4 (CH), 128.7 (CH),
130.0 (CH), 136.4 (C), 137.1 (C), 146.9 (C); HR-MS (EI) calcd
for C15H16ClN3 (M) m/z 273.10327, found 273.10295.
2-[3-(2-Acetyloxyeth yl)-3-ben zyltr iazen -1-yl]ch lor oben -
zen e (13e): From 2-(benzylamino)ethyl acetate hydrochloride,
92%; IR (NaCl) 1742 (CdO), 1463, 1375, 1348, 1236, 1163,
1
1053, 750 cm-1; H NMR δ 1.92 (s, 3H, CH3), 3.91-4.30 (m,
4H, CH2CH2), 5.02 (s, 2H, CH2Ph), 7.16-7.43 (m, 9H); HR-
MS (EI) calcd for C17H18ClN3O2 (M) m/z 331.10876, found
331.10889.
En zym a tic In h ibition Assa ys. Spectrophotometric assays
used to determined the DHFR potency have been described
in detail previously.17-19 Briefly, activity of the target com-
pounds (series 3, 10, and 12) was reported in terms of drug
concentration required to reduce by 50% the rate of enzymatic
reduction of dihydrofolate to tetrahydrofolate in the presence
of cofactor NADPH compared to control (IC50).
Each standard assay mixture was made up of phosphate
buffer (40.7 mM, pH 7.4) to a total volume of 1 mL which
contained the following materials: NADPH (0.117 mM),
dihydrofolate (0.092 mM), 2-mercaptoethanol (8.9 mM), and
DHFR enzyme (0.018 units of activity; under standard condi-
tions each unit of DHFR reduces 1 µM dihydrofolate/min).
2,4-Diam in o-5-[3-(3-ben zyl-3-m eth yltr iazen -1-yl)-4-ch lo-
r op h en yl]-6-eth ylp yr im id in e (13a ): From 6b and N-meth-
ylbenzylamine, 69% (from ethanol), mp 190-191 °C (lit.7 mp
196-197 °C); 1H NMR δ 0.98 (t, J ) 7.5 Hz, 3H, CH2CH3),
2.12 (q, J ) 7.5 Hz, 2H, CH2CH3), 3.13 and 3.52 (2 × brs, 3H,
NCH3), 5.01 (brs, 2H, CH2Ph), 5.62 (brs, 2H, NH2), 5.88 (brs,
2H, NH2), 6.95 (dd, J ) 1.6,8.3 Hz, 1H), 7.27-7.37 (m, 6H),
7.48 (d, J ) 8.5 Hz, 1H).
2,4-Dia m in o-5-[3-(3-ben zyl-3-eth yltr ia zen -1-yl)-4-ch lo-
r op h en yl]-6-eth ylp yr im id in e (13b): From 6b and N-eth-
ylbenzylamine, 75% (from ethanol), mp 153-154 °C (efferv.);
IR (KBr) 3451, 3314, 3165 (NH), 1630, 1561, 1439, 1346, 1173
cm-1; 1H NMR δ 0.97 (t, J ) 7.5 Hz, 3H, CH2CH3), 1.09-1.26
(m, 3H, NCH2CH3), 2.11 (q, J ) 7.5 Hz, 2H, CH2CH3), 3.71-
3.85 (m, 2H, NCH2CH3), 4.98 (brs, 2H, CH2Ph), 5.62 (brs, 2H,
NH2), 5.88 (brs, 2H, NH2), 6.94 (d, J ) 8.3 Hz, 1H), 7.23-7.49
(m, 7H); 13C NMR δ 10.3 (CH3), 13.4 (CH3), 14.3 (CH3), 27.7
(CH2), 42.0 (CH2), 49.3 (CH2), 49.8 (CH2), 57.6 (CH2), 105.7
(C), 120.6 (CH), 127.1 (C), 128.5 (CH), 128.7 (CH), 130.5 (CH),
135.7 (C), 137.1 (C), 146.9 (C), 162.1 (C), 162.3 (C), 166.5 (C);
MS (CI) m/z 410, 412 (M + 1). Anal. (C21H24ClN7) C, H, N.
Refer en ces
(1) Pala´t, K.; Stevens, M. F. G. Structural Studies on Bioactive
Compounds. Part 33. Synthesis of 9-Arylacridines by Palladium-
mediated Couplings. J . Chem. Res. (S), 2000, 136-137.
(2) AIDS Epidemic Update; December 1998. http://www.who.int/
emchiv/december1998/wadr98e.pdf
(3) Kovacs, J . A.; Hiemenz, J . W.; Macher, A. M.; Stover, D.; Murray,
H. W.; Shelhamer, J .; Lane, H. C.; Urmacher, C.; Honig, C.;
Longo, D. L.; Parker, M. W.; Natanson, C.; Parrillo, J . E.; Fauci,
A. S.; Pizzo, P. A.; Masur, H. Pneumocystis carinii Pneumonia:
A Comparison Between Patients with the Acquired Immuno-
deficiency Syndrome and Patients with Other Immunodeficien-
cies. Ann. Intern. Med. 1984, 100, 663-671.
(4) 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 Pneumocyctis carinii in Culture and in a Rat
Model of Infection. Antimicrob. Agents Chemother. 1987, 31,
1323-1327.
(5) Klepser, M.; Klepser, T. B. Drug Treatment of HIV-related
Opportunistic Infections. Drugs 1997, 53, 40-73. Brun-Pascaud,
M.; Chau, F.; Garry, L.; J acobus, D.; Derouin, F.; Girard, P.
Combination of PS-15, Epiroprim, or Pyrimethamine with
Dapsone in Prophylaxis of Toxoplasma gondii and Pneumocystis
carinii Dual Infection in
a Rat Model. Antimicrob. Agents

2564 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Chan et al.
Chemother. 1996, 40, 2067-2070. Torre, D.; Casari, S.; Sper-
anza, F.; Donisi, A.; Gregis, G.; Poggio, A.; Ranieri, S.; Orani,
A.; Angarano, G.; Chiodo, F.; Fiori, G.; Carosi, G.; Group, T. I.
C. S. Randomised Trial of Trimethoprim-Sulphamethoxazole
versus Pyrimethamine-Sulfadiazine for Therapy of Toxoplasmic
Encephalitis in Patients with AIDS. Antimicrob. Agents Chemo-
ther. 1998, 42, 1346-1349.
(14) Edwards, S. L.; Sherfinski, J . S.; Marsh, R. E. The Crystal
Structure of the Antitumor Agent 5-(3,3-Dimethyl-1-triazeno)-
imidazole-4-carboxamide Monohydrate Hydrochloride (NSC-
45388). J . Am. Chem. Soc. 1974, 96, 2593-2597.
(15) Hooper, D. L.; Pottie, I. R.; Vacheresse, M.; Vaughan, K. 1,2-
Bis(1-aryl-3-alkyltriazen-3-yl)ethanes and Related Compounds.
Can. J . Chem. 1998, 76, 125-135.
(16) Akhtar, M. H.; McDaniel, R. S.; Feser, M.; Oehlschlager, A. C.
NMR Study of Hindered Rotation in 1-Aryl-3,3-dimethyltri-
azenes. Tetrahedron 1968, 24, 3899-3906.
(6) Brun-Pascaud, M.; Rajagopalan-Levasseur, P.; Chau, F.; Ber-
trand, G.; Garry, L.; Derouin, F.; Girard, P. M. Drug Evaluation
of Concurrent Pneumocystis carinii, Toxoplasma gondii, and
Mycobacterium avium Complex Infections in
a Rat Model.
Antimicrob. Agents Chemother. 1998, 42, 1068-1072.
(7) Stevens, M. F. G.; Phillip, K. S.; Rathbone, D. L.; O’Shea, D.
M.; Queener, S. F.; Schwalbe, C. H.; Lambert, P. A. Structural
Studies on Bioactive Compounds. 28. Selective Activity of
Triazenyl-Substituted Pyrimethamine Derivatives against Pneu-
mocystis carinii Dihydrofolate Reductase. J . Med. Chem. 1997,
40, 1886-1893.
(8) Champness, J . N.; Achari, A.; Ballantine, S. P.; Bryant, P. K.;
Delves, C. J .; Stammers, D. K. The Structure of Pneumocystis
carinii Dihydrofolate Reductase to 1.9 Å Resolution. Structure
1994, 2, 915-924.
(9) Gangjee, A.; Guo, X.; Queener, S. F.; Cody, V.; Galitsky, N.; Luft,
J .; Pangborn, W. Selective Pneumocystis carinii Dihydrofolate
Reductase Inhibitors: Design, Synthesis, and Biological Evalu-
ation of New 2,4-Diamino-5-substituted-furo[2,3-d]pyrimidines.
J . Med. Chem. 1998, 41, 1263-1271.
(10) Cody, V.; Chan, D.; Galitsky, N.; Rak, D.; Luft, J . R.; Pangborn,
W.; Queener, S. F.; Laughton, C. A.; Stevens, M. F. G. Structural
Studies on Bio-active Compounds. 30. Crystal Structure and
Molecular Modeling Studies on thePneumocystis carinii Dihy-
drofolate Reductase Cofactor Complex with TAB, a Highly
Selective Antifolate. Biochemistry 2000, 39, 3556-3564.
(11) Bliss, E. A.; Griffin, R. J .; Stevens, M. F. G. Structural Studies
on Bio-active Compounds. Part 5. Synthesis and Properties of
2,4-Diaminopyrimidine Dihydrofolate Reductase Inhibitors bear-
ing Lipophilic Azido Groups. J . Chem. Soc., Perkin Trans. I 1987,
2217-2228.
(17) Broughton, M. C.; Queener, S. F. Pneumocystis carinii Dihydro-
folate Reductase Used to Screen Potential Antipneumocystis
Drugs. Antimicrob. Agents Chemother. 1991, 35, 1348-1355.
(18) Chio, L.-C.; Queener, S. F. Identification of Highly Potent and
Selective Inhibitors of Toxoplasma gondii Dihydrofolate Reduc-
tase. Antimicrob. Agents Chemother. 1993, 37, 1914-1923.
(19) Chio, L.-C.; Boylard, L. A.; Nasr, M.; Queener, S. F. Identification
of
a Class of Sulfonamides Highly Active Against Dihy-
dropteroate Synthase from Toxoplasma gondii, Pneumosystis
carinii, and Mycobacterium avium. Antimicrob. Agents Chemo-
ther. 1996, 40, 727-733.
(20) Rosowsky, A.; Cody, V.; Galitsky, N.; Fu, H.; Papoulis, A. T.;
Queener, S. Structure-Based Design of Selective Inhibitors of
Dihydrofolate Reductase: Synthesis and Antiparasitic Activity
of 2,4-Diaminopteridine Analogues with a Bridged Diarylamine
Side Chain. J . Med. Chem. 1999, 42, 4853-4860.
(21) Biosym, Insight II 95.0. 1995. 9685 Scranton Road, San Diego,
CA 9221, USA.
(22) Weiner, S. J .; Kollman, P. A.; Nguyen, D. T.; Case, D. A. An All
Atom Force Field for Simulations of Proteins and Nucleic Acid.
J . Comput. Chem. 1986, 7, 230-252.
(23) Ba´rbie`re, J . Este´rification Nitrique et Nitration d'Amino-alcools.
Bull. Soc. Chim. Fr. 944, 5, 470-480.
(24) Ba´rbie`re, J . Nitration d'Aralcoylamino-alcools. Bull. Soc. Chim.
Fr. 1940, 7, 621-626.
(12) Griffin, R. J .; Schwalbe, C. H.; Stevens, M. F. G.; Wong, K. P.
Structural Studies on Bio-active Compounds. Part 3. Reexami-
nation of the Hydrolysis of the Antimalarial Drug Pyrimethamine
and Related Derivatives and Crystal Structure of a Hydrolysis
Product. J . Chem. Soc., Perkin Trans. I 1985, 2267-2276.
(13) Morie, T.; Kato, S.; Harada, H.; Yoshida, N.; Matsumoto, J . I.
Synthesis and Biological Activities of the Optical Isomers of (()-
4-Amino-5-chloro-2-ethoxy-N-[[4-(4-fluorobenzyl)-2-morpholinyl]-
methyl]benzamide (Mosapride). Chem. Pharm. Bull. 1994, 42,
877-882.
(25) Potapov, V. M.; Dem’yanovich, V. M.; Maleev, V. I. Derivatives
of Optically Active (1-Phenylethyl)amine in Complexes with
Lithium Aluminium Hydride for the Asymmetric Reduction of
a Carbonyl Group. J . Org. Chem. USSR Eng. Trans. 1985, 21,
1606-1609.
J M0108698