Structural studies on bioactive compounds. 28. Selective activity of triazenyl-substituted pyrimethamine derivatives against Pneumocystis carinii dihydrofolate reductase

Article abstract of DOI:10.1021/jm970050n

Triazenyl-substituted pyrimethamine derivatives 10a-s have been prepared by coupling diazotized 2,4-diamino-5-(3-amino-4-chlorophenyl)-6-ethyl pyrimidine (1c) with a series of secondary amines in aqueous sodium carbonate solution. The triazenes which are

Full text of DOI:10.1021/jm970050n

1886
J . Med. Chem. 1997, 40, 1886-1893
Str u ctu r a l Stu d ies on Bioa ctive Com p ou n d s. 28.1 Selective Activity of
Tr ia zen yl-Su bstitu ted P yr im eth a m in e Der iva tives a ga in st P n eu m ocystis ca r in ii
Dih yd r ofola te Red u cta se
Malcolm F. G. Stevens,*,† Keith S. Phillip,‡ Daniel L. Rathbone,§ Dennis M. O’Shea,§ Sherry F. Queener,|
Carl H. Schwalbe,‡ and Peter A. Lambert‡
Cancer Research Laboratories, Department of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD,
U.K., Pharmaceutical Sciences Institute, Aston University, Aston Triangle, Birmingham B4 7ET, U.K., Aston Molecules Ltd,
10 Holt Court South, Aston Science Park, Birmingham B7 4EJ , U.K., and Department of Pharmacology and Toxicology,
Indiana University School of Medicine, Indianapolis, Indiana 46202
Received J anuary 22, 1997X
Triazenyl-substituted pyrimethamine derivatives 10a -s have been prepared by coupling
diazotized 2,4-diamino-5-(3-amino-4-chlorophenyl)-6-ethyl pyrimidine (1c) with a series of
secondary amines in aqueous sodium carbonate solution. The triazenes which are stable and
poorly soluble as free bases form more soluble, but unstable, salts with alkanesulfonic acids.
The lead dimethyltriazene 2,4-diamino-5-[4-chloro-3-(3,3-dimethyltriazen-1-yl)phenyl]-6-eth-
ylpyrimidine (4a ) forms a crystalline ethanesulfonic acid salt (solvated with 2-propanol), which
is protonated at the pyrimidine N-1 position as determined by X-ray crystallography. The
ability of these new triazenes to inhibit Pneumocystis carinii dihydrofolate reductase in vitro
has been compared to that of triazene 4a . The most potent and selective compound, 2,4-diamino-
5-[3-[3-[2-(acetyloxy)ethyl]-3-benzyltriazen-1-yl]-4-chlorophenyl]-6-ethylpyrimidine (14a ), has
an IC50 value of 0.17 µM against the microbial enzyme and potentially useful selectivity (rat
liver IC50/P. carinii IC50 ) 114).
In tr od u ction
The emergence of the HIV pandemic has created a
population of immune-supppressed patients who are
vulnerable to opportunistic microorganisms. Infections
by Pneumocystis carinii elicit a pneumonia (PCP) which
is the leading cause of mortality in AIDS sufferers in
the USA.2,3 P. carinii, like most microorganisms,
synthesizes folates from PABA4 and generates reduced
folates by employing a dihydrofolate reductase enzyme
(DHFR) which is structurally distinct from the mam-
malian variant.5,6 Combinations of sulfamethoxazole
and trimethoprim are considered standard chemo-
therapy for PCP, but their use is associated with
unpredictable, and occasionally severe, side effects.7
Although the search for effective treatments for PCP
has broadened to include a range of diverse structures
of different biochemical mechanisms,8 there is still scope
for the development of selective inhibitors of P. carinii
DHFR. Most recently, new series of lipophilic 2,4-
diamino-5-deaza-9 and 2,4-diamino-8-deazapteridines10
have been shown to be superior to the benchmark
trimetrexate 2 as inhibitors of P. carinii and Toxo-
plasma gondii DHFR.
withdrawn from clinical trial because it elicited unpre-
dictable neurotoxicity.13 An alternative lipophilic py-
rimethamine derivative, methylbenzoprim 3, is one of
the most potent known inhibitors of mammalian (rat
liver) DHFR (Ki 0.009 nM),14 possibly because it is
competitive with NADPH as well as dihydrofolate.15
Among a large series of related structures submitted
to the NCI Developmental Therapeutics Program for
antitumor evaluation, including the above, the dimeth-
yltriazenyl-substituted pyrimethamine 4a was subse-
quently compared with pyrimethamine as an inhibitor
of P. carinii and T. gondii DHFR. The useful selectivity
(rat liver IC50/P. carinii IC50 ) 6.7516 and rat liver IC50/
T. gondii IC50 ) 6117) and confirmation that the triaz-
ene-inhibited P. carinii growth in culture with an IC50
In the past, we have utilized the diaminopyrimidine
pyrimethamine 1a as a starting material to prepare the
corresponding nitro (1b) and amino analog (1c) in order
to develop synthetic routes to lipophilic inhibitors of
mammalian DHFR with potential antitumor activity.
Thus the m-azidopyrimethamine 1d ,11 used clinically
as the ethanesulfonic acid salt (MZPES),12 had a shorter
biological half-life in humans than etoprim 1e but was
† University of Nottingham, Nottingham.
‡Aston University, Birmingham.
§Aston Molecules Ltd, Birmingham.
|Indiana University School of Medicine.
X Abstract published in Advance ACS Abstracts, May 1, 1997.
S0022-2623(97)00050-2 CCC: $14.00 © 1997 American Chemical Society

Structural Studies on Bioactive Compounds
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1887
Sch em e 1
Sch em e 2
value of 5.5 µM18 marked out compound 4a as a suitable
lead compound on which to base a synthesis effort for
further structure-activity evaluation. Interestingly,
the related pyrimethamine 4b, with a dimethylforma-
midine side chain (which is presumably protonated at
physiological pH), is less potent and selective againstthe
P. carinii enzyme (rat liver IC50/P. carinii IC50 ) 0.33).16
The literature on 1,1-dialkyl-3-aryltriazenes related
in structure to the dimethyltriazene 4a alerted us to
the concern that methyl- and ethyltriazenes might be
mutagenic.19 Thus, in those 1-alkyl-1-methyl-3-aryl-
triazenes of general structure 5, where the alkyl group
has a free H atom on the R-carbon, metabolic oxidation
can generate unstable (hydroxyalkyl)triazenes 6,20 which
readily lose a carbonyl fragment (formaldehyde from a
hydroxymethyltriazene)21 to liberate a mutagenic monom-
ethyltriazene 7 (Scheme 1). In the case of the dimeth-
yltriazene 4a , metabolic demethylation would afford the
known monomethyltriazene 4c.11 Although monometh-
yltriazenes are relatively stable above pH 7.4,22 gener-
ally in acid media they undergo proteolytic fragmenta-
tion to generate an arylamine 8 (amine 1c from the
monomethyltriazene 4c)11 and the fugitive methyldia-
zonium species 9 which methylates O6- and N7-positions
of guanine residues of DNA.23 Monoethyltriazenes can
similarly ethylate DNA and the aforementioned chem-
istry is, largely, independent of the nature of aryl
substituent X.20 Although in the present study our
main target was limited to identify inhibitors of P.
carinii DHFR with enhanced potency and selectivity
relative to the lead dimethyltriazene 4a , our ultimate
objective is to select modified dialkyltriazenes which
could not be metabolized to mutagenic methylating or
ethylating agents. Furthermore, by incorporating bulky
substituents containing polar oxygen substituents at the
terminal triazene N-atom, we hoped to block protonation
at this site, thereby enhancing acid stability and solu-
bility. In fact, in all the examples studied, there is
provision of an alternative site for protonation at N-1
on the diaminopyrimidinyl residue.24
carbonate solution at 0 °C with a range of dialkylamines
to yield the triazenylpyrimidines 10a -s in yields of 75-
85% (Scheme 2). Interaction of the diazonium salt 1f
with tetrahydroisoquinoline gave the triazene 11, which
can be considered as a modified N-benzyl-N-ethyltria-
zene. The triazene 10p formed (70%) by coupling
diazonium tetrafluoroborate 1f with N-benzylethanola-
mine 12 furnished an O-acetyl derivative 14a (35%)
when treated with acetyl chloride in pyridine at 25 °C
with a catalytic amount of DMAP: this acetate and the
corresponding propionate 14b were formed more ef-
ficiently by coupling the diazonium chloride derived
from the arylamine 1c and the hydrochloride salts of
N-benzylethanolamine esters 13a ,b in aqueous sodium
carbonate in 75 and 80% yields, respectively (Scheme
3).
All of the aforementioned triazenes could be crystal-
lized unchanged from aqueous polar solvents (acetone,
methanol, or ethanol); they were characterized as tria-
zenes by their production of red azo-dye colorations
when heated with 2-naphthol in acetic acid. Examina-
1
tion of the H NMR spectra of “pure” (single-spot TLC)
samples of some of the triazenes revealed the presence
of solvents which were tenaciously held even on pro-
longed vacuum drying. Solvation is a characteristic
feature of other diaminopyrimidines of the pyri-
methamine class.11,14
Resu lts a n d Discu ssion
Syn th etic Ch em istr y. The starting material for the
synthesis of the required triazenes 10 was the diazo-
nium tetrafluoroborate 1f, which was prepared from the
corresponding amine 1c by diazotization in aqueous
tetrafluoroboric acid. We have shown previously that
this diazonium salt, isolated as a hydrotetrafluoroborate
(hemihydrate) salt,11 can be stored safely in bulk
quantity (25 g) for periods >10 years at 25 °C. The
water-soluble salt was coupled in aqueous sodium
P h ysica l P r op er ties of Tr ia zen es. The solubility
of the triazene derived from diethanolamine 10q in
water at 25 °C was disappointingly low (<0.1 mg/mL)
although solubility was enhanced (to 3.85 mg/mL) at pH
4 in citrate buffer in which the triazene is protonated.
However qualitative stability experiments monitored by

1888 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Stevens et al.
rms deviation of 0.01 Å; the pyrimidine ring, the
benzene ring, and the dimethyltriazene chain. The first
two intersect at an angle of 70.7(1)°, and the last two
at 24.8(4)°. A structural feature common to simple
1-aryl-3,3-dimethyltriazenes26 is the large C12-C13-
N17 bond angle [124.1(3)°], which alleviates interference
between the triazene and aryl groups at the expense in
the present structure of a close contact [2.925(5) Å]
between N17 and Cl22 which is not relieved by any
similar expansion of the C13-C14-Cl22 bond angle
[119.7(3)°]. The terminal triazene N atom exhibits sp2
character, and N17-N18 at 1.271(5) Å shows more
double-bond character than N18-N19 at 1.322(5) Å.
As is typical for antifolate drugs,27 the protonated
pyrimidine ring is heavily hydrogen bonded, donating
hydrogen bonds to counterions at the 1 and 2 positions,
forming a centrosymmetric dimer at 3 and 4, and
directing one H atom from each amino group toward
the 2-propanol O29 atom. No triazene N atom is
involved in hydrogen bonding.
Biologica l Resu lts. Results of the in vitro assays
of triazenyl-substituted pyrimethamine derivatives
against P. carinii and rat liver DHFR are recorded in
Table 1. Included in this table are previously published
results from one of us16 which allows for an analysis of
the influence of substitution at the 3′-position of the
pyrimethamine nucleus. Dealing with the reference
compounds first, pyrimethamine 1a and its nitro (1b)
and azido analog (1d ) are relatively weak inhibitors of
P. carinii DHFR and more selectively inhibit the rat
liver enzyme, in particular the nitropyrimethamine 1b.
The reactive diazonium tetrafluoroborate 1f, which,
presumably, can react covalently with nucleophilic
components of the target enzyme, is the most potent of
the four simple pyrimethamine congeners with an IC50
of 0.19 µM against P. carinii enzyme but is still
selectively active against the mammalian enzyme. Tri-
metrexate 2 and methylbenzoprim 3 (the structure of
which was incorrectly assigned in earlier reports)16,17
are 1000-fold more potent than pyrimethamine against
the mammalian enzyme: they differ in that trimetrex-
ate discriminates little between the P. carinii and
mammalian enzymes whereas methylbenzoprim 3 is an
exquisitely selective inhibitor of the rat liver variant.
Indeed, this compound is one of the most active known
nonclassical inhibitors of mammalian DHFR and the
nitro group (cf. 1b) is an essential requirement for high
potency in the series.14,15
F igu r e 1. ORTEP drawing of 2,4-diamino-5-[4-chloro-3-(3,3-
dimethyltriazen-1-yl)phenyl]-6-ethylpyrimidine (4a ) as the
ethanesulfonic acid salt (2-propanol solvate) and the crystal-
lographic numbering scheme.
HPLC showed that ethanol solutions of three triazenes
10p , 10q, and 14a were rapidly degraded by 1 M HCl,
1 M NaOH, and aqueous H2O2 solutions. A detailed
evaluation of the stability of 10q at pH 4 showed that
the compound was essentially undegraded when main-
tained at -20 °C for 1 h, but suffered >25% decomposi-
tion at 4 °C over 1 h. Although the degradation
products have not been identified, it is probable that
heterolytic fission of the triazene linkage generates an
unstable diazonium species which then suffers reduction
by the ethanol.
These results notwithstanding, methanesulfonic and
ethanesulfonic acid salts of some of the triazenes were
prepared successfully from the corresponding bases with
methanesulfonic or ethanesulfonic acids (1 molar equiv)
in 2-propanol and were shown to be monoprotonated
pyrimidines (1H NMR) although they gave unreliable
C, H, N analyses because of association with nonsto-
ichiometric quantities of solvents. Crystals of the
ethanesulfonic acid salt of the lead compound 4a suit-
able for an X-ray analysis were grown from 2-propanol
to confirm the site of protonation and to facilitate future
molecular modeling studies and interpretation of the
biological data.
X-r a y Cr ysta llogr a p h y. The contents of the asym-
metric unit, comprising one molecule of 4a protonated
at N-1 of the heterocycle, one ethanesulfonate counter
ion, and a disordered 2-propanol solvent molecule, are
shown with their crystallographic numbering scheme
in an ORTEP25 drawing in Figure 1. The cation of 4a
consists of three moieties, each of which is planar within
The lead dimethyltriazene 4a is approximately equi-
Sch em e 3

Structural Studies on Bioactive Compounds
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1889
Ta ble 1. Inhibition Data for Triazenyl-Substituted
2,4-Diaminopyrimidines and Reference Compounds against P.
carinii and Rat Liver DHFR
Ta ble 2. Activity of Triazenyl-Substituted
2,4-Diaminopyrimidines against P. carinii Microorganisms
Extracted from Immunocompromised Rats
IC50 (µM) vs DHFRa
IC50
IC50
selectivity indexb
(rl/pc)
compound
(µg/mL)a
compound
(µg/mL)a
compound
P. carinii rat liver
10k
10p
10q
49.7
11.1
46.6
10r
14a
30.5
43.9
2.5
1a (pyrimethamine)
1b
1d
1f
2 (trimetrexate)
3 (methylbenzoprim)
4a
4a (EtSO3H salt)
4b
4c
10a
10a (MeSO3H salt)
10b
10b (MeSO3H salt)
10c
10d
10e
10f
10g
10h
10i
10j
10k
3.65
0.85
1.33
0.19
0.042
1.6
2.8
6.0
10.6
4.6
14.1
6.6
10.2
7.6
2.3
0.015
0.33
0.032
0.003
0.0032
0.63c
0.02c
0.25c
0.17c
0.071c
0.002c
6.8c
pentamidine isethionate
Concentration required to inhibit uptake of [3H]-4-aminoben-
zoic acid over 24 h.
a
18.9
P. carinii IC50 ) 15), with the highest favorable index
thus far observed in this series.
7.8
3.5
2.3
1.3
0.33c
0.5
The acyclic triazenes 10l-s, with one or more oxygen
functionalities attached to the triazene moiety, dis-
played a significant divergence of potency and selectiv-
ity. Within the homologous alkyltriazenes bearing
(2-hydroxyethyl) substituents 10l-o no significant struc-
ture-activity trends were discernable and the com-
pounds had only low selectivity against the P. carinii
DHFR. However, replacement of an alkyl group for
benzyl in the benzyl(hydroxyethyl)triazene 10p gave an
agent with potency (IC50 ) 0.26 µM) and selectivity
against the P. carinii enzyme (rat liver IC50/P. carinii
IC50 ) 27). This compares with the low potency and
selectivity of the “cyclic” benzyltriazene 11 derived from
202
0.57
14
0.09
>4d
0.89
1.8
0.12
0.64
1
2.8
18.2
29.3
17.3.
17.7
>11
>11
66.8
16.2
7.2
26.3
1.1
27.9
27
0.44
10.3
7
5
0.6
>11e
>11e
40.4
1.7
1.9
2.2
15
0.23
8
2.3
0.2
2.1
27
11
29
0.9
0.34
114
8.5
3.3
1.7
4.7
3.5
11.5
2.5
4.9
10k (MeSO3H salt)
10l
10m
10n
10o
10p
10q
10r
10s
11
tetrahydroisoquinoline (rat liver IC50/P. carinii IC50
0.34). The acetoxy derivative 14a of the benzyltriazene
10p is the most potent compound in the series (IC50
)
)
0.26
0.17 µM) with a selectivity index (114) which is one of
the highest thus far recorded for all DHFR inhibitors.8
Incorporation of two oxygen atoms in different arms of
the triazene side chain 10q,r also gave compounds with
relatively high potency against the P. carinii DHFR
(IC50 < 1 µM) and, in the case of the bis(2-methoxyeth-
yl)triazene 10r , a favorable selectivity index of 29. This
selectivity was lost when both oxygen atoms were in the
same alkyl group 10s.
0.44
0.91
0.57
5.0
26.1
0.5
1.7
19.4
14a
0.17
a
See ref 16 for method. b IC50 (µM) for rat liver DHFR/IC50 (µM)
for P. carinii DHFR; values of >1 denote compounds selective
toward the enzyme from P. carinii. c Results from ref 16. No
d
inhibition of rat liver DHFR at 3.2 µM. e No inhibition of P. carinii
DHFR at 11 µM.
The activities of the alkanesulfonic acid salts of
certain of the triazenes were generally similar to those
of the free bases toward P. carinii DHFR, but in all cases
the IC50 values of the salts against rat liver DHFR were
markedly reduced (Table 1). The chemical instability
of the triazenes at pH 4 has been noted previously.
Possibly, variations in the experimental conditions may
allow for increased breakdown of the salts in the rat
liver DHFR assays. The product of such (heterolytic)
degradation would be the diazonium species 1f which
active with pyrimethamine 1a as an inhibitor of the P.
carinii enzyme but a weaker inhibitor of the mam-
malian enzyme (Table 1). This results in a 10-fold
enhancement in selectivity for 4a relative to py-
rimethamine 1a . This selectivity is lost in the dimeth-
ylformamidine 4b and the monomethyltriazene 4c.
Overall, with a few exceptions (see later), the new
triazenes are less potent than pyrimethamine against
the P. carinii enzyme: the observed increase in selectiv-
ity (rl/pc) is due to reduced potency against the rat liver
enzyme. Thus, the ethylmethyltriazene 10a is a 4-fold
less potent inhibitor of the P. carinii enzyme than the
corresponding dimethyltriazene 4a but its significantly
weaker activity against rat liver DHFR gives 10a an
improved selectivity index (rat liver IC50/P. carinii IC50
) 14 compared to 6.5 for the dimethyltriazene). In a
consistent pattern the other N,N-dialkyltriazenes 10b-f
displayed only moderate potency against the P. carinii
enzyme (IC50 > 10 µM) with a tendency to weaker
activity as the size of the hydrophobic attachment
increased. When both alkyl groups are constrained
within a heteroalicyclic ring as in 10g-k the role of an
extra nitrogen or oxygen atom in increasing potency
toward P. carinii DHFR in the triazenes is clearly seen
in a comparison between the activities of the piperidino-
(10h ) and morpholinotriazene (10k ). The morpholino
compound is >20-fold more potent against the P. carinii
enzyme and significantly more selective (rat liver IC50/
is a known potent inhibitor of rat liver DHFR (IC50
)
0.032 µM).16
Certain of the significant triazenes identified above
were also tested for their ability to inhibit the viability
of P. carinii organisms extracted from lungs of immu-
nosuppressed (dexamethasone) rats. Microorganism
viability over 24 h was measured by monitoring uptake
of [3H]-4-aminobenzoic acid. The most active compound
was the triazene 10p , although this compound was less
potent than the reference compound pentamidine
isethionate (Table 2).
Some of the more active triazenes against P. carinii
DHFR were also tested against the enzyme from T.
gondii. Results of these in vitro assays, together with
those of related reference compounds, are summarized
in Table 3. Pyrimethamine is inhibitory to the protozoal
enzyme with an IC50 of 0.3 µM and reasonable selectiv-
ity (rat liver IC50/T. gondii IC50 ) 5.9): the dimethyl-

1890 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Stevens et al.
Ta ble 3. Comparative Inhibition Data for
Triazenyl-Substituted 2,4-Diaminopyrimidines and Reference
Compounds against T. gondii and Rat Liver DHFR
to have a selectivity (human DHFR IC50/P. carinii IC50)
of 200.30 However, it has been shown by one of us8 that,
when this agent was retested in the standard assay
employed in the current work, a selectivity of 44 was
measured for human recombinant DHFR and 28 for rat
liver DHFR. The selectivity of epiroprim can be ex-
plained by its very low potency toward mammalian
DHFR.
IC50 (µM) v DHFRa
selectivity indexb
compound
T. gondii rat liver
(rl/tg)
1a (pyrimethamine)
1b
1d
1f
2 (trimetrexate)
3 (methylbenzoprim)
4a
4b
0.39
0.013
0.69
0.023
0.01
0.091
0.31
1.6
2.3
0.015
0.33
0.032
0.003
0.0032
5.9c
1.1c
0.5c
1.4c
0.3c
0.04c
61c
Exp er im en ta l Section
18.9
Mass spectra were recorded on an AEI MS-902 or a VG
Micromass 7070E spectrometer. IR spectra were determined
in KBr on a Mattson 2020 GALAXY series FT-IR spectropho-
tometer. 1H NMR and 13C NMR spectra were recorded on
Bruker AC250 or ARX250 spectrometers in DMSO-d6 solutions
TLC systems for routine monitoring of reaction mixtures and
confirming the homogeneity of analytical samples employed
Kieselgel 60F254 (0.25 mm) with either CHCl3 or CHCl3-2%
ethanol as developing solvents.
3.5
26.3
26.1
19.4
2.2c
138
3
10k
10r
14a
0.19
8.8
0.69
28
a
b
See ref 17 for method. IC50(µM) for rat liver DHFR/IC50 (µM)
for T. gondii DHFR; values of >1 denote compounds selective
toward the enzyme from T. gondii. c Results from ref 17.
Gen er a l P r oced u r e for 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 yr i-
m id in es. To a stirred solution of the diazonium tetrafluo-
roborate 1f11 in water at 0 °C was added the appropriate
secondary amine (1 molar equiv) and sufficient solid sodium
carbonate to adjust the mixture to pH 10. The suspension was
stirred at 0 °C for 2 h, and the solid triazene was collected
and washed with copious amounts of water. The products
were isolated in yields of 80-95%. The triazenes could be
purified by crystallization from methanol or aqueous acetone,
or by flash chromatographic fractionation on Sorbsil silica gel
C 60-H (40-60 mm), using an ethyl acetate-hexane solvent
mixture. Alternatively, an aqueous solution of the diazonium
chloride hydrochloride salt (prepared in situ by diazotization
of amine 1c in 3 M hydrochloric acid at 0 °C)11 was coupled
with the appropriate amines at pH 10. The following com-
pounds were prepared.
triazene is equipotent against the parasitic enzyme but
10-fold more selective (rat liver IC50/T. gondii IC50
)
61). The morpholinotriazene 10k is the most potent
(IC50 ) 0.19 µM) of the limited number of new triazenes
evaluated in this work and displays potentially useful
selectivity (rat liver IC50/T. gondii IC50 ) 138). The
benzyl(acetoxyethyl)triazene 14a , the most effective
compound (combination of potency and selectivity)
against P. carinii DHFR, also exhibited reasonable
potency and selectivity toward the T. gondii enzyme.
Con clu sion
We have shown previously by 19F and 1H NMR
studies that 2,4-diamino-5-(4-fluoro-3-nitrophenyl)-6-
ethylpyrimidine (1b; Cl ) F)28 and methylbenzoprim 3
2,4-Dia m in o-5-[4-ch lor o-3-(3,3-d im et h ylt r ia zen -1-yl)-
p h en yl]-6-eth ylp yr im id in e (4a ): mp 233-235 °C (efferv)
29
bind to Lactobacillus casei DHFR in two different
1
[lit.11 mp 233-236 °C (efferv)]; H NMR δ 1.00 (t, 3 H), 2.18
conformations which are unequally populated and not
interconvertible in the bound state. These conforma-
tions are generated by restricted rotation about the
pivotal pyrimidine-phenyl bond and position the critical
nitro group in two different environments within the
active site of the enzyme. The X-ray structure of the
dimethyltriazenylpyrimidine 4a (Figure 1), which also
has an asymmetrically-substituted 5-aryl group, sug-
gests that similar steric factors will determine the
binding potency and specificity of the sterically demand-
ing triazenyl moieties to P. carinii DHFR and the rat
liver enzyme.
Basing a synthetic effort on the lead dimethyltriazene
4a , we have achieved the following:
(i) An efficient high-yielding synthesis (three steps
from pyrimethamine 1a ) of a series of triazenyl pyrim-
idines with varying substituents at the triazene termi-
nus.
(ii) Replacement of the potentially mutagenic dim-
ethyltriazene fragment with other groups which do not
have the potential for metabolic conversion to potential
DNA-alkylating monoalkyltriazenes.
(iii) Identification of derivative 14a which is 16-fold
more potent than 4a as an inhibitor of P. carinii DHFR
(IC50 0.17 µM) and 16-fold more selective (rat liver IC50/
P. carinii IC50 ) 114). Intriguingly, compound 14a
shares with methylbenzoprim 3 the presence of an
N-benzyl grouping but the differing selectivity for DHFR
from different sourcessP. carinii DHFR for 14a and rat
liver enzyme for 3sis especially notable. A trimetho-
prim analog epiroprim (Ro11-8958) has been reported
(q, 2 H), 3.25 (s, 3 H), 3.56 (s, 3 H), 5.85 (br, 2 H), 6.02 (br, 2
H), 7.00 (dd, 1 H), 7.29 (d, 1 H), 7.55 (d, 1 H).
2,4-Dia m in o-5-[4-ch lor o-3-(3-eth yl-3-m eth yltr ia zen -1-
yl)p h en yl]-6-eth ylp yr im id in e (10a ): mp 235-236 °C (ef-
ferv); 1H NMR δ 0.95 (t, 3 H), 1.21 (t, 3 H), 2.09 (q, 2 H), 3.17
and 3.45 (2 x s, 3 H), 3.81 (q, 2 H), 5.62 (br, 2 H), 5.88 (br, 2
H), 6.90 (dd, 1 H), 7.15 (d, 1 H), 7.44 (d, 1 H). Anal. (C15H20
ClN7) C, H, N.
-
2,4-Dia m in o-5-[4-ch lor o-3-(3-m eth yl-3-p r op yltr ia zen -1-
yl)p h en yl]-6-eth ylp yr im id in e (10b): mp 189-191 °C (ef-
ferv); 1H NMR δ 0.83 (t, 3 H), 0,95 (t, 3 H), 1.63 (q, 2 H), 2.08
(q, 2 H), 3.18 (s, 3 H), 3.73 (t, 2 H), 5.62 (br, 2 H), 5.87 (br, 2
H), 6.90 (dd, 1 H), 7.15 (d, 1 H), 7.44 (d, 1 H). Anal. (C16H22
ClN7) C, H, N.
-
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 (10c): mp 196-197 °C (ef-
ferv); 1H NMR δ 1.06 (t, 3 H), 2.26 (q, 2 H), 3.15 and 3.55 (2 s,
3 H), 5.03 (s, 2 H), 7.32 (m, 10 H), 8.1 (br, 1 H), 12.0 (br, 1 H).
Anal. (C20H22ClN7) C, H, N.
2,4-Dia m in o-5-[4-ch lor o-3-[3-[2-(d iet h yla m in o)et h yl]-
3-m eth yltr iazen -1-yl]ph en yl]-6-eth ylpyr im idin e (10d): mp
1
157-159 °C (efferv); H NMR δ 0.93 (3 t, 9 H), 2.11 (q, 2 H),
2.44 (q, 4 H), 2.65 (m, 2 H), 3.51 (s, 3 H), 3.85 (m, 2H), 5.62
(br, 2 H), 5.90 (br, 2 H), 6.92 (dd, 1 H), 7.19 (d, 1 H), 7.46 (d,
1 H); 13C NMR δ 12.04, 13.39, 27.65, 35.38, 46.54, 51.19, 53.90,
105.79, 120.61, 126.91, 128.22, 130.46, 135.63, 147.16, 162.11,
162.28, 166.54. Anal. (C19H29ClN8) C, H, N.
2,4-Dia m in o-5-[4-ch lor o-3-(3,3-d ieth yltr ia zen -1-yl)p h e-
1
n yl]-6-eth ylp yr im id in e (10e): mp 225-226 °C (efferv); H
NMR δ 0.96 (t, 3 H), 1.21 (t, 6 H), 2.08 (q, 2 H), 3.75 (q, 4 H),
5.63 (br, 2 H), 5.89 (br, 2 H), 6.90 (dd, 1 H), 7.14 (d, 1 H), 7.44
(d, 1 H). Anal. (C16H22ClN7) C, H, N.
2,4-Dia m in o-5-[4-ch lor o-3-(3,3-d iisobu tyltr ia zen -1-yl)-
p h en yl]-6-eth ylp yr im id in e (10f): mp 185-189 °C (efferv);
1H NMR δ 0.83 (m, 12 H), 0.92 (t, 3 H), 2.05 (q, 2 H), 2.10 (m,

Structural Studies on Bioactive Compounds
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1891
2H), 3.51 (d, 4 H), 5.72 (br, 2 H), 5.95 (br, 2 H), 6.86 (dd, 1 H),
7.10 (d, 1 H), 7.41 (d, 1 H). Anal. (C20H30ClN7) C, H, N.
3.88 (m, 4H), 4.89 (br, 2 H), 5.74 (br, 2 H), 5.94 (br, 2 H), 6.94
(dd, 1 H), 7.19 (d, 1 H), 7.49 (d, 1 H). Anal. (C16H22ClN7O2)
C, H, N.
2,4-Dia m in o-5-[4-ch lor o-3-[3,3-bis(2-m eth oxyeth yl)tr i-
a zen -1-yl]p h en yl]-6-eth ylp yr im id in e (10r ): mp 130-132
°C (efferv); 1H NMR δ 0.96 (t, 3 H), 2.09 (q, 2 H), 3.26 (d, 6 H),
3.62 (d, 4H), 3.95 (m, 4 H), 5.63 (br, 2 H), 5.89 (br, 2 H), 6.94
(dd, 1 H), 7.16 (d, 1 H), 7.47 (d, 1 H). Anal. (C18H26ClN7O2)
C, H, N.
2,4-Dia m in o-5-[4-ch lor o-3-[3-(1,3-d ioxol-2-ylm eth yl)-3-
m eth yltr ia zen -1-yl]p h en yl]-6-eth ylp yr im id in e (10s): mp
213-214 °C (efferv); 1H NMR δ 0.97 (t, 3 H), 2.11 (q, 2 H),
3.24 (s, 3 H), 3.9 (m, 6 H), 5.08 (m, 1 H), 5.68 (br, 2 H), 5.91
(br, 2 H), 6.96 (dd, 1 H), 7.18 (d, 1 H), 7.48 (d, 1 H); 13C NMR
δ 13.38, 27.69, 36.59, 57.55, 64.66, 101.93, 105.71, 120.85,
127.16, 128.78, 130.50, 135.73, 146.87, 162.12, 162.30, 166.60.
Anal. (C17H29ClN7O2) C, H, N.
2,4-Dia m in o-5-[4-ch lor o-3-(1,2,3,4-tetr a h yd r oisoqu in o-
lin -2-yla zo)p h en yl]-6-eth ylp yr im id in e (11): mp 170-172
°C (efferv); 1H NMR δ 0.98 (t, 3 H), 2.17 (q, 2 H), 3.01 (s, 2 H),
4.08 (s, 2 H), 4.90 (s, 2 H), 6.98 (dd, 1 H), 7.20 (m, 9 H), 7.52
(d, 1 H). Anal. (C21H22ClN7) C, H, N.
2,4-Dia m in o-5-[4-ch lor o-3-(p yr r olid in -1-yla zo)p h en yl]-
6-eth ylp yr im id in e (10g): mp 247-251 °C (efferv); 1H NMR
δ 0.94 (t, 3 H), 1.96 (m, 4 H), 2.09 (q, 2 H), 3.58 (s, 2 H), 3.88
(s, 2 H), 5.62 (br, 2 H), 5.89 (br, 2 H), 6.89 (dd, 1 H), 7.13 (d,
1 H), 7.44 (d, 1 H). Anal. (C16H20ClN7) C, H, N.
2,4-Dia m in o-5-[4-ch lor o-3-(p ip er id in -1-yla zo)p h en yl]-
6-eth ylp yr im id in e (10h ): mp 250-252 °C (efferv); 1H NMR
δ 1.00 (t, 3 H), 1.63 (m, 6 H), 2.11 (q, 2 H), 3.79 (m, 4 H), 5.63
(br, 2 H), 5.88 (br, 2 H), 6.93 (dd, 1 H), 7.18 (d, 1 H), 7.45 (d,
1 H). Anal. (C17H22ClN7) C, H, N.
2,4-Dia m in o-5-[4-ch lor o-3-(4-m eth ylp ip er a zin -1-yla zo)-
p h en yl]-6-eth ylp yr im id in e (10i): mp 215-216 °C (efferv);
1H NMR δ 0.96 (t, 3 H), 2.10 (q, 2 H), 2.25 (s, 3 H), 3.80 (m, 8
H), 5.63 (br, 2 H), 5.89 (br, 2 H), 6.97 (dd, 1 H), 7.20 (d, 1 H),
7.49 (d, 1 H); 13C NMR δ 13.36, 27.68, 45.72, 105.64, 120.48,
127.40, 129.20, 130.60, 135.79, 146.47, 162.10, 162.32, 166.57.
Anal. (C17H23ClN8) C, H, N.
2,4-Dia m in o-5-[4-ch lor o-3-[(4-h yd r oxyp ip er id in -1-yl)-
a zo]p h en yl]-6-eth ylp yr im id in e (10j): mp 224-225 °C (ef-
1
ferv); H NMR δ 0.98 (t, 3 H), 1.50 (m, 2 H), 1.82 (m, 2 H),
2,4-Dia m in o-5-[3-[3-[2-(a cet yloxy)et h yl]-3-b en zylt r ia -
zen -1-yl]-4-ch lor op h en yl]-6-eth ylp yr im id in e (14a ). A so-
lution of N-benzylethanolamine (2.0 g) in acetic acid (30 mL)
was saturated with dry hydrogen chloride gas at 20 °C for 1
h. The mixture was maintained at 20 °C (24 h) and solvent
removed by vacuum evaporation to yield 2-(N-benzylamino)-
ethyl acetate hydrochloride 13a (98%). The salt was coupled
with the diazonium chloride solution prepared from the
arylamine 1c in the presence of excess aqueous sodium
carbonate to furnish triazene 14a (75%): mp 166-168 °C
(efferv); IR (KBr) 3453, 3326 (NH2), 1723 (CdO) cm-1; 1H NMR
δ 0.98 (t, 3 H), 1.90 (s, 3 H), 2.10 (q, 2 H), 3.9-4.3 (m, 4 H),
5.04 (br, 2 H), 5.80 (br, 2 H), 5.98 (br, 2 H), 6.96 (dd, 1 H),
7.31-7.47 (m, 7 H); 13C NMR δ 14.1, 21.4, 28.3, 51.1, 53.7,
59.4, 60.1, 62.6, 106.4, 121.4, 128.0, 128.1, 129.0, 129.2, 129.6,
129.8, 131.3, 136.4, 137.0, 137.6, 147.2, 162.9, 167.1, 171.0.
Anal. (C23H26ClN7O2) C, H, N.
2.10 (q, 2 H), 3.40-4.10 (m, 5 H), 4.90 (m, 1H), 5.65 (br, 2 H),
5.89 (br, 2 H), 6.91 (dd, 1 H), 7.19 (d, 1 H), 7.46 (d, 1 H); 13C
NMR δ 13.39, 27.69, 48.81, 65.20, 105.73, 120.45, 127.22,
128.87, 130.56, 135.73, 146.76, 162.15, 162.30, 166.58. Anal.
(C17H22ClN7O) C, H, N.
2,4-Dia m in o-5-[4-ch lor o-3-(m or p h olin -4-yla zo)p h en yl]-
6-eth ylp yr im id in e (10k ): mp 255-257 °C (efferv); 1H NMR
δ 0.95 (t, 3 H), 2.09 (q, 2 H), 3.78 (m, 8 H), 5.61 (br, 2 H), 5.88
(br, 2 H), 6.98 (dd, 1 H), 7.20 (d, 1 H), 7.49 (d, 1 H). Anal.
(C16H20ClN7O) C, H, N.
2,4-Dia m in o-5-[4-ch lor o-3-[3-(2-h yd r oxyeth yl)-3-m eth -
yltr ia zen -1-yl]p h en yl]-6-eth ylp yr im id in e (10l): mp 217-
1
218 °C (efferv); H NMR δ 0.97 (t, 3 H), 2.11 (q, 2 H), 3.37 (s,
3 H), 3.66 (d, 2 H), 3.85 (m, 2 H), 4.85 (br, 1 H), 5.66 (br, 2 H),
5.91 (br, 2 H), 6.92 (dd, 1 H), 7.19 (d, 1 H), 7.47 (d, 1 H); 13C
NMR δ 13.41, 27.70, 36.62, 58.26, 59.60, 105.81, 120.72,
126.95, 128.32, 130.47, 135.65, 147.16, 162.13, 162.29, 166.59.
Anal. (C15H20ClN7O) C, H, N.
The same (acetoxyethyl)benzyltriazene (35%) was prepared
by treating the (hydroxyethyl)benzyltriazene 10p with acetyl
chloride (1 molar equiv) and catalytic DMAP in pyridine at
25 °C.
2,4-Dia m in o-5-[4-ch lor o-3-[3-eth yl-3-(2-h yd r oxyeth yl)-
tr ia zen -1-yl]p h en yl]-6-eth ylp yr im id in e (10m ): mp 214-
1
215 °C (efferv); H NMR δ 0.97 (t, 3 H), 1.23 (t, 3 H), 2.11 (q,
2,4-Dia m in o-5-[4-ch lor o-3-[3-[2-(p r op ion yloxy)et h yl]-
3-b en zylt r ia zen -1-yl]p h en yl]-6-et h ylp yr im id in e (14b ).
Similarly prepared, from 2-(N-benzylamino)ethyl propionate
hydrochloride 13b and the diazonium salt prepared from
arylamine 1c, the [(propionyloxy)ethyl]benzyltriazene (80%)
had mp 184-186 °C (efferv): IR (KBr) 3338, 3137 (NH2), 1730
2 H), 3.8 (m, 6 H), 4.86 (br, 1 H), 5.64 (br, 2 H), 5.91 (br, 2 H),
6.92 (dd, 1 H), 7.16 (d, 1 H), 7.47 (d, 1 H); 13C NMR δ 13.4,
27.7, 42.1, 50.7, 57.1, 60.0, 105.8, 120.5, 127.0, 128.3, 130.4,
135.7, 147.0, 162.1, 162.3, 166.5. Anal. (C16H22ClN7O) C, H,
N.
1
(CdO) cm-1; H NMR δ 0.92 (t, 3 H), 1.04 (t, 3 H), 2.18 (q, 2
2,4-Dia m in o-5-[4-ch lor o-3-[3-(2-h yd r oxyeth yl)-3-p r op y-
ltr ia zen -1-yl]p h en yl]-6-eth ylp yr im id in e (10n ): mp 186-
187 °C (efferv); 1H NMR δ 0.88 (m, 3 H), 0.98 (t, 3 H), 1.70 (m,
2 H), 2.12 (q, 2 H), 3.8 (m, 6 H), 4.85 (br, 1 H), 5.70 (br, 2 H),
5.93 (br, 2 H), 6.93 (dd, 1 H), 7.17 (d, 1 H), 7.47 (d, 1 H); 13C
NMR δ 11.20, 11.80, 13.42, 18.38, 21.83, 27.70, 49.57, 50.39,
56.59, 57.23, 59.80, 105.81, 120.45, 127.04, 128.34, 130.44,
135.66, 147.10, 162.15, 162.29, 166.60. Anal. (C17H24ClN7O)
C, H, N.
2,4-Dia m in o-5-[3-[3-ter t-bu tyl-3-(2-h yd r oxyeth yl)tr ia -
zen -1-yl]-4-ch lor op h en yl]-6-eth ylp yr im id in e (10o): mp
224-226 °C (efferv); 1H NMR δ 0.98 (t, 3 H), 1.41 (s, 9 H),
2.10 (q, 2 H), 3.66 (m, 2 H), 3.81 (m, 2 H), 4.78 (br, 1 H), 5.60
(br, 2 H), 5.89 (br, 2 H), 6.91 (dd, 1 H), 7.12 (d, 1 H), 7.46 (d,
1 H); 13C NMR δ 13.52, 27.67, 28.02, 47.22, 57.11, 60.82,
105.85, 120.68, 127.00, 128.26, 130.44, 135.67, 147.50, 162.13,
162.29, 166.59. Anal. (C18H26ClN7O) C, H, N.
H), 2.20 (q, 2 H), 3.94-4.32 (m, 4 H), 5.00 (br, 2 H), 6.46 (br,
2 H), 6.70 (br, 2 H), 7.01 (dd, 1 H), 7.34-7.52 (m, 7 H). Anal.
(C24H28ClN7O2) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Alk a n esu lfon -
ic Acid Sa lts 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 yr im id in es. The triazene (0.5 g) in
a minimum of 2-propanol at 50 °C was treated with the
alkanesulfonic acid (1.1 molar equiv) and immediately cooled
to 5 °C. The crystalline salts were collected and washed with
a minimum of ice-cold 2-propanol. The following salts were
prepared.
Eth a n esu lfon ic a cid sa lt of 2,4-d ia m in o-5-[4-ch lor o-3-
(3,3-dim eth yltr iazen -1-yl)ph en yl]-6-eth ylpyr im idin e (4a),
which crystallized as a 2-propanol solvate, mp 160 °C (dec)
(see Crystallography Section).
Meth a n esu lfon ic a cid sa lt of 2,4-d ia m in o-5-[4-ch lor o-
3-(3-et h yl-3-m et h ylt r ia zen -1-yl)p h en yl]-6-et h ylp yr im i-
d in e (10a ): mp 155 °C (dec); 1H NMR δ 0.98 (t, 3 H), 1.10 (t,
3 H), 2.15 (q, 2 H), 2.37 (s, 3 H), 2.85 (q, 2 H), 3.14 (s, 3 H),
3.77 (q, 2 H), 6.95 (dd, 1 H), 7.21 (m, 1 H), 7.50 (m, 1 H), 7.6
(br s, 2 H), 8.1 (br s, 1 H), 8.3 (br s, 1 H), 12.25 (br s, 1 H).
Meth a n esu lfon ic a cid sa lt of 2,4-d ia m in o-5-[4-ch lor o-
3-(3-m eth yl-3-p r op yltr ia zen -1-yl)p h en yl]-6-eth ylp yr im i-
d in e (10b): mp 150 °C (dec); 1H NMR δ 0.94 (t, 3 H), 1.08 (t,
3 H), 1.67 (q, 2 H), 2.24 (q, 2 H), 2.49 (s, 3 H), 2.88 (q, 2 H),
2,4-Dia m in o-5-[3-[3-ben zyl-3-(2-h yd r oxyeth yl)tr ia zen -
1-yl]-4-ch lor op h en yl]-6-eth ylp yr im id in e (10p ): mp 112-
1
114 °C (efferv); H NMR δ 0.98 (t, 3 H), 2.12 (q, 2 H), 3.71-
4.10 (m, 4 H), 4.91 (br, 1 H), 5.05 (br, 2 H), 5.68 (br, 2 H), 5.91
(br, 2 H), 6.96 (dd, 1 H), 7.4 (m, 7 H). Anal. (C21H24ClN7O)
C, H, N.
2,4-Dia m in o-5-[4-ch lor o-3-[3,3-bis(2-h yd r oxyeth yl)tr ia -
zen -1-yl]p h en yl]-6-eth ylp yr im id in e (10q): mp 219-220 °C
1
(efferv); H NMR δ 0.98 (t, 3 H), 2.12 (q, 2 H), 3.71 (m, 4H),

1892 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Stevens et al.
3.25 (s, 3 H), 3.81 (q, 2 H), 7.05 (dd, 1 H), 7.29 (m, 1 H), 7.60
(m, 1 H), 7.7 (br s, 2 H), 8.2 (br s, 1 H), 8.4 (br s, 1 H), 12.4 (br
s, 1 H).
Meth a n esu lfon ic a cid sa lt of 2,4-d ia m in o-5-[4-ch lor o-
3-(m or p h olin -4-yla zo)p h en yl]-6-eth ylp yr im id in e (10k ):
mp 205 °C (efferv); 1H NMR δ 0.97 (t, 3 H), 2.19 (q, 2 H), 2.34
(s, 3 H), 3.06 (m, 2 H), 3.73 (m, 6 H), 7.03 (dd, 1 H), 7.25 (d, 1
H), 7.53 (m, 1 H), 7.55 (br s, 2 H), 8.1 (br s, 1 H), 8.7 (br s, 1
H), 12.0 (br s, 1 H).
Cr ysta llogr a p h y. The ethanesulfonate salt of 4a crystal-
lized from 2-propanol at 5 °C as rosettes, from which an
irregular brown specimen crystal 0.7 × 0.5 × 0.3 mm was
obtained. Crystal data: unit cell dimensions a ) 8.438(1) Å,
b ) 9.933(1) Å, c ) 16.969(2) Å, a ) 99.86(1)°, b ) 92.92(1)°,
g ) 113.92(2)°, V ) 1269.4(2) Å3, C14H19ClN7+ C2H5O3S-‚C3H8O,
FW ) 490.03, Z ) 2, Dc ) 1.282 g cm-3, triclinic, P1h, Mo KR
radiation (λ ) 0.7107 Å), 4420 reflections measured by ω-2θ
scans, 2906 observed [I > 2σ(I)].
Refer en ces
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Structural studies on bio-active compounds. Part 8. Synthesis,
crystal structure, and biological properties of a new series of 2,4-
diamino-5-aryl-6-ethylpyrimidine dihydrofolate reductase inhibi-
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Meek, M. A.; Griffin, R. J .; Stevens. Structural studies on bio-
active compounds. Part 8. Molecular modeling and crystal-
lographic studies on methylbenzoprim, a potent inhibitor of
dihydrofolate reductase. J . Med. Chem. 1992, 35, 2315-2320.
(16) Broughton, M. C.; Queener, S. F. Pneumocystis carinii dihydro-
folate reductase used to screen potential antipneumocystis drugs.
Antimicrob. Agents Chemother. 1991, 35, 1348-1355.
(17) 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.
(18) Bartlett, M. S.; Shaw, M. M.; Navaran, P.; Smith, J . W.; Queener,
S. F. Evaluation of potent inhibitors of of dihydrofolate reductase
in a culture model for growth of Pneumocystis carinii. Antimi-
crob. Agents Chemother. 1995, 39, 2436-2441.
Str u ctu r e Deter m in a tion a n d Refin em en t. The struc-
31
ture was solved by direct methods with MULTAN
and
refined by the full-matrix least-squares technique with
SHELXL.32 Two alternative positions were found for one of
the methyl groups of the 2-propanol solvent molecule. Posi-
tions and anisotropic displacement parameters were refined
for all non-hydrogen atoms. Hydrogen atoms were located in
difference electron density maps and freely refined with
isotropic temperature factors, except for the C20 and C21
methyl groups, which were constructed and refined as rotating
rigid groups, the C27-C28 ethyl group, for which a riding
model was used, and the H atoms attached to 2-propanol C
atoms, which were omitted entirely. The weighting scheme
w ) 1/[σ2(Fo2) + (0.1193P)2 + 0.7850P] was applied, where P
2
) (Fo + 2Fc2)/3. At termination no shift exceeded 0.008 esd,
discrepancy indices were R ) 0.061 for observed reflections,
wR(F2) ) 0.217 for all reflections. No feature on a difference
electron density map exceeded +0.48 or -0.38 e Å-3
. Final
non-hydrogen fractional coordinates, bond distances, and bond
angles have been deposited as supplementary material.
In Vitr o E va lu a t ion of Tr ia zen yl-Su b st it u t ed P y-
r im eth a m in e Der iva tives. P. carinii microorganisms were
recovered from the lungs of immunosuppressed rats by ruptur-
ing the host cells, washing away the soluble mammalian
DHFR, and harvesting intact organisms in association with
the mammalian plasma membranes. P. carinii DHFR was
isolated from the 100000g supernatant from sonicated organ-
isms and the inhibitory activities of triazenes and reference
compounds were measured according to an established
method.16
T. gondii RH was obtained in high yield from culture in
RPMI medium on a line of Chinese hamster ovary cells lacking
DHFR activity (ATCC 3952 dhfr-; American Type Culture
Collection). DHFR preparations were obtained from harvested
organisms and the inhibitory activities of triazenes, and
reference compounds were measured according to an estab-
lished method.17
For assessment of the activitity of triazenes against intact
microorganisms in vitro, P. carinii was extracted from the
lungs of rats immunosuppressed for 6-10 weeks with dexam-
ethasone.33 Serial dilutions of a 10 mg/mL stock solution of
each compound in DMSO were made to give a final concentra-
tion range of 0.2-100 µg/mL. The P. carinii and compound
were incubated together in Glasgow Modification of Eagle’s
Medium (GMEM) with [3H]-4-aminobenzoic acid (pABA) at 37
°C for 24 h in alveolar air (5% CO2, 95% air). Uptake of [3H]-
pABA into the folates of P. carinii was utilized as a selective
indicator of the in vitro viability of the organism. The
inhibitory activity of the compounds was expressed as the
concentration required to inhibit the uptake of pABA by 50%
compared with uptake by untreated control cultures (IC50).
Ack n ow led gm en t. We thank Dr. Helen C. J ackson
of (the then) Glaxo Research and Development Ltd for
conducting the P. carinii inhibition assays and British
Technology Group Ltd and Aston Molecules Ltd for
financial support of the project.
(19) Preussman, R.; Druckrey, H.; Ivankovic, S.; von Hodenberg, A.
Chemical structure and carcinogenicity of aliphatic hydrazo-, azo
and azoxy compounds and of triazenes, potential in vivo alky-
lating agents. Ann. N.Y. Acad. Sci. 1969, 163, 697-714.

Structural Studies on Bioactive Compounds
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1893
(20) Connors, T. A.; Goddard, P. M.; Merai, K.; Ross, W. C. J .;
Wilman, D. E. V. Tumour inhibitory triazenes: structural
requirements for an active metabolite. Biochem. Pharmacol.
1976, 25, 241-246. Gescher, A.; Threadgill, M. D. The metabo-
lism of triazene antitumour drugs. Pharmac. Ther. 1987, 32,
191-205.
(21) Gescher, A.; Hickman, J . A.; Simmonds, R. J .; Stevens, M. F.
G.; Vaughan, K. R-Hydroxylated derivatives of antitumour
dimethyltriazenes. Tetrahedron Lett. 1978, 5041-5044; Kolar,
G. F.; Maurer, M.; Wildschu¨tte, M. 5-(3-Hydroxymethyl-3-
methyltriazeno)imidazole-4-carboxamide (DIC, DTIC, NSC 45388).
Cancer Lett. 1980, 10, 241-253.
(22) Vaughan, K.; Stevens, M. F. G. Monoalkyltriazenes. Chem. Soc.
Rev. 1978, 7, 377-397.
(23) Denny, B. J .; Wheelhouse, R. T.; Stevens, M. F. G.; Tsang, L. L.
H.; Slack, J . A. NMR and molecular modeling investigation of
the mechanism of activation of the antitumor drug temozolomide
and its interaction with DNA. Biochemistry 1994, 33, 9045-
9051.
(24) Threadgill, M. D.; Griffin, R. J .; Stevens, M. F. G.; Wong, S. K.
Structural studies on bio-active compounds. Part 6. Determina-
tion of the sites of protonation on three 2,4-diaminopyrimidines
of pharmaceutical importance by proton-coupled 13C and 1H
nuclear magnetic resonance spectroscopy. J . Chem. Soc., Perkin
Trans. 1 1987, 2229-2234.
(25) J ohnson, C. K. ORTEP. Oak Ridge Thermal-Ellipsoid Plot
Program. Report ORNL-5136; Oak Ridge National Laboratory:
Oak Ridge, TN, 1976.
(28) Tendler, S. J . B.; Griffin, R. J .; Birdsall, B.; Stevens, M. F. G.;
Roberts, G. C. K.; Feeney, J . Direct 19F NMR observation of the
conformational selection of optically active rotamers of the
antifolate compound fluoronitropyrimethamine bound to the
enzyme dihydrofolate reductase. FEBS Lett. 1988, 240, 201-
204.
(29) Birdsall, B.; Tendler, S. J . B.; Arnold, J . R. P.; Feeney, J .; Griffin,
R. J .; Carr, M. D.; Thomas, J . A.; Roberts, G. C. K.; Stevens, M.
F. G. NMR studies of multiple conformations in complexes of
Lactobacillus casei dihydrofolate reductase with analogues of
pyrimethamine. Biochemistry 1990, 29, 9660-9667.
(30) Then, R. L.; Hartman, P. G.; Kompis, I.; Santi, D. Selective
inhibition of dihydrofolate reductase from problem human
pathogens. In Chemistry and Biology of Pteridines and Folates;
Ayling, J . E., et al., Eds.; Plenum Press: New York, 1993; pp
533-536.
(31) Main, P.; Germain, G.; Woolfson, M. M. MULTAN 84 - a
computer program for the automatic solution of crystal struc-
tures from X-ray diffraction data. Universities of York and
Louvain, 1984.
(32) Sheldrick, G. M. SHELXL-93. Program for the refinement of
crystal structures from diffraction data. University of Go¨ttingen,
1993.
(33) J ackson, H. C.; Colthurst, D.; Hancock, V.; Marriott, M. S.; Tuite,
M. F. No detection of characteristic fungal protein elongation
factor EF-3 in Pneumocystis carinii. J . Infect. Dis. 1991, 163,
675-677.
(26) Fronczek, F. R.; Hansch, C.; Watkins, S. F. p-(3,3-Dimethyl-1-
triazeno)benzonitrile. Acta Crystallogr. 1988, C44, 1651-1653.
(27) Schwalbe, C. H.; Cody, V. Structural studies of antifolate drugs.
In Chemistry and Biology of Pteridines; Blair, J . A., Ed.; Walter
de Gruyter: Berlin, 1983; pp 511-515.
J M970050N