Development of a lead inhibitor for the A16V+S108T mutant of dihydrofolate reductase from the cycloguanil-resistant strain (T9/94) of Plasmodium falciparum

Article abstract of DOI:10.1021/jm0009181

The Ala16Val+Ser108Thr (A16V+S108T) mutant of the Plasmodium falciparum dihydrofolate reductase (DHFR) is a key mutant responsible for cycloguanil-resistant malaria due to steric interaction between Val-16 and one of the C-2 methyl groups of cycloguanil. 4,6-Diamino-1,2-dihydrotriazines have been prepared, in which both methyl groups of cycloguanil are replaced by H or by H and an alkyl or phenyl group, and their inhibition constants against wild-type and mutant DHFR determined. The S108T mutation is considered to decrease cycloguanil binding further through the effect on the orientation of the p-chlorophenyl group. By moving the p-chloro-substituent to the m-position in the chlorophenyl group, the activity against the A16V+S108T mutant enzyme is improved, and this effect is reinforced by the p-chloro substituent in the 3,4-dichlorophenyl group. A lead compound has been found with inhibitory activity similar to that of cycloguanil against the wild-type DHFR and about 120-fold more effective than cycloguanil against the A16V+S108T mutant enzyme. The activity of this compound against P. falciparum clone (T9/94 RC17) which harbors the A16V+S108T DHFR is about 85-fold greater than cycloguanil.

Full text of DOI:10.1021/jm0009181

2738
J . Med. Chem. 2000, 43, 2738-2744
Develop m en t of a Lea d In h ibitor for th e A16V+S108T Mu ta n t of Dih yd r ofola te
Red u cta se fr om th e Cyclogu a n il-Resista n t Str a in (T9/94) of P la sm od iu m
fa lcipa r u m †
Yongyuth Yuthavong,‡ Tirayut Vilaivan,§ Netnapa Chareonsethakul,§ Sumalee Kamchonwongpaisan,‡
Worachart Sirawaraporn,| Rachel Quarrell, and Gordon Lowe*,
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency,
Bangkok 10400, Thailand; Department of Chemistry, Faculty of Science, Chulalongkorn University,
Bangkok 10330, Thailand; Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand,
and The Dyson Perrins Laboratory, Oxford University, South Parks Road, Oxford, OX1 3QY, U.K.
Received February 4, 2000
The Ala16Val+Ser108Thr (A16V+S108T) mutant of the Plasmodium falciparum dihydrofolate
reductase (DHFR) is a key mutant responsible for cycloguanil-resistant malaria due to steric
interaction between Val-16 and one of the C-2 methyl groups of cycloguanil. 4,6-Diamino-1,2-
dihydrotriazines have been prepared, in which both methyl groups of cycloguanil are replaced
by H or by H and an alkyl or phenyl group, and their inhibition constants against wild-type
and mutant DHFR determined. The S108T mutation is considered to decrease cycloguanil
binding further through the effect on the orientation of the p-chlorophenyl group. By moving
the p-chloro-substituent to the m-position in the chlorophenyl group, the activity against the
A16V+S108T mutant enzyme is improved, and this effect is reinforced by the p-chloro
substituent in the 3,4-dichlorophenyl group. A lead compound has been found with inhibitory
activity similar to that of cycloguanil against the wild-type DHFR and about 120-fold more
effective than cycloguanil against the A16V+S108T mutant enzyme. The activity of this
compound against P. falciparum clone (T9/94 RC17) which harbors the A16V+S108T DHFR
is about 85-fold greater than cycloguanil.
In tr od u ction
Despite continued efforts aimed at complete eradica-
tion of malaria, the disease remains a major health
threat in many areas of the world, especially in tropical
and subtropical countries including Africa.1 The wide-
spread occurrence of drug-resistant Plasmodium falci-
parum suggests that the effectiveness of the few anti-
malarials currently in use will have a limited life span
and has highlighted the urgent need for the discovery
and development of novel antimalarial agents aimed at
combating the emerging resistant parasites.
Cycloguanil (Cyc) and its closely related compound
pyrimethamine (Pyr) are potent inhibitors of Plasmo-
resistance to Pyr and Cyc is associated with point
mutations in the DHFR.2-7 The mutations in pfDHFR
thus far reported are amino acid residues 16, 51, 59,
108, and 164. Mutant pfDHFRs involving mutations at
residues 51, 59, 108, and 164 confer cross-resistance to
Pyr and Cyc, while those involving mutation at residue
16 (A16V) are resistant to Cyc but susceptible to Pyr.
The importance of residue 16 for binding Cyc has been
investigated using the mutants obtained via mutagen-
esis of a synthetic gene.8 Recently, a three-dimensional
homology model of pfDHFR was constructed to aid
understanding of the structural basis of antifolate
resistance in malaria.9 The studies led to a hypothesis
which proposed that resistance to Cyc is due to a steric
clash for Cyc binding as a result of A16V mutation of
the pfDHFR, and that mutation of residue 108 (S108T)
further reinforces the steric constraint for Cyc binding
through displacement of the p-chlorophenyl group of the
dium falciparum dihydrofolate reductase (pfDHFR), one
of a few well-defined drug targets for malaria therapy.
Both compounds have been extensively employed, either
alone or in combination with sulfa-drugs, as prophylac-
tic agents for the treatment of malaria. Unfortunately,
resistance of the malaria parasite to the drugs has
rapidly emerged and compromised their clinical utility.
Analysis of DHFR sequences of several Pyr- and Cyc-
resistant P. falciparum isolates from different geographi-
cal origins with different drug sensitivities revealed that
† Abbreviations: Cyc, cycloguanil; Pyr, pyrimethamine; DHFR,
dihydrofolate reductase; pf, Plasmodium falciparum.
* Address correspondence to Professor Gordon Lowe. Phone: +44
(0)1865 275649. Fax: +44 (0)1865 275674. E-mail: gordon.lowe@
chem.ox.ac.uk.
‡ NSTDA Bangkok.
§ Chulalongkorn University.
| Mahidol University.
Oxford University.
10.1021/jm0009181 CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/13/2000

Development of a Lead Inhibitor of pfDHFR
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2739
Sch em e 1
strongly influenced by the nucleophilicity of the aniline,
and for this reason we have chosen to use a parallel
synthesis protocol. Previous attempts to prepare the
didemethyl analogue of Cyc (2) were unsuccessful,10,11
and we confirm that the standard protocol using for-
malin or paraformaldehyde does not give the required
product. However, when dimethoxymethane is used as
the formaldehyde equivalent, the required product is
formed in excellent yield. Other Cyc derivatives were
prepared according to the literature methods10,11 with-
out modification, except that in some cases addition of
a miscible water scavenger such as triethyl orthoacetate
was found to give improved results, although this is
rarely necessary when the carbonyl component is an
aldehyde.
In h ibition of Wild -Typ e a n d A16V+108T p fDH-
F Rs. The data in Table 1 summarize the inhibition
constant (Ki) values of Cyc and analogues in which the
substituents at C-2 were varied, while the group at N-1
(p-chlorophenyl) was unmodified. As reported previ-
ously,8 Cyc (1) binds to the wild-type pfDHFR with the
Ki value of ∼1.5 nM, but it binds approximately 876-
fold less tightly to the A16V+S108T mutant enzyme.
On the basis of our working hypothesis, we surmise that
the poor binding of Cyc to A16V+S108T enzyme might
be attributed to one of the methyl substituents at the
C-2 position of Cyc. To test this hypothesis, we designed
and synthesized a number of Cyc derivatives in which
one substituent at C-2 was H and the other varied from
H (2), methyl (3), ethyl (4), n-propyl (5), n-butyl (6),
isopropyl (7), tert-butyl (8), and phenyl (9). The effects
of the substituents on binding to both wild-type and
A16V+S108T DHFRs were assessed by determination
of the ratios of the Ki values for the A16V+S108T
mutant enzyme and the wild-type enzyme (Ki-mut./Ki-
wt) as well as their Ki values relative to Cyc. Table 1
shows that the didemethylcycloguanil (2), in which both
methyl groups at position C-2 were replaced by H, is
∼2-fold more effective against the A16V+S108T pfDH-
FR than Cyc but inhibited the wild-type enzyme ∼16-
fold less effectively than Cyc. The monomethyl analogue
(3) has improved binding affinity (∼10-fold) for the
A16V+S108T DHFR, but was about 2.7-fold less effec-
tive than Cyc for the wild-type enzyme. Since the
compound tested was a racemic mixture, if only one
enantiomer is active, the relative Ki value of the effective
enantiomer could be up to twice that of the value shown.
If this is the case, the Ki value of Cyc analogue (3) for
the wild-type enzyme is within the range reported for
Cyc.8
inhibitor from the nicotinamide ring of the cofactor.
Validation of the hypothesis was achieved by testing
both the wild-type and A16V+S108T mutant pfDHFRs
against Cyc analogues devoid of one or both methyl
groups.9
In this paper, we describe further modifications to the
structure of Cyc in a search for more effective inhibitors
of A16V+S108T pfDHFR which are also effective against
the wild-type enzyme. Given the important roles of
A16V and S108T mutations on Cyc binding, we designed
and synthesized a number of Cyc analogues in which
the groups at C-2 and N-1 were varied, and we tested
them against both wild-type and A16V+S108T pfDH-
FRs. The studies have led to the discovery of a lead
which is as effective as Cyc against the wild-type
pfDHFR, but is over 120-fold more effective than Cyc
against the A16V+S108T mutant enzyme. The relative
effectiveness of the lead was also investigated against
the resistant P. falciparum strain T9/94, a mutant
parasite harboring the A16V+S108T mutant enzyme.
Its IC50 value is 180-fold lower than that of Cyc, and it
retains similar activity to Cyc against the wild-type P.
falciparum.
Resu lts a n d Discu ssion
Ch em ica l Syn th eses. 4,6-Diamino-1,2-dihydrotri-
azines, such as Cyc, are generally made in two steps
from dicyandiamide and an aniline under acidic condi-
tions to first generate arylbiguanides which are then
reacted with an appropriate carbonyl compound in the
presence of an acid catalyst to give the 4,6-diamino-1,2-
dihydrotriazine substituted at N-1 and C-2.10,11 The
arylbiguanide may be isolated before further reaction,
or the two-step procedure may be performed in the same
reaction vessel (Scheme 1). This is an attractive feature
of the chemistry and lends itself to the techniques of
combinatorial chemistry.12 However, one should be
aware that the rate of formation of the biguanides is
Ta ble 1. Inhibition Constants (Ki) of Cyc (1) and Its Analogues (A; X ) Cl, Y ) H) against the Wild-Type and A16V+S108T Mutant
DHFRs of P. falciparum and Their Growth Inhibition (IC50) against P. falciparum Clones with Wild-Type (TM4/8.2) and Mutant
(T9/94 RC17) Enzymes
Ki(wt)a
(nM)
rel. to
Cyc
Ki(mut.)b
(nM)
rel. to
Cyc
Ki(mut.)/
Ki(wt)
IC50 TM4
(nM)
IC50 T9/94
(nM)
IC50 ratio
T9/94:TM4
cmpd
R1
R2
1
2
3
4
5
6
7
8
9
Me
H
H
H
H
H
H
H
H
Me
H
Me
Et
1.5 ( 0.3c
24.4 ( 4.3d
4.1 ( 0.0d
3.6 ( 0.0d
4.6 ( 0.2
1.0
16
2.7
2.4
3.1
1314 ( 16c
646 ( 77d
127 ( 14d
189 ( 37d
107 ( 32
167 ( 6.0
1538 ( 345
82721 ( 9888
49 ( 3
1
0.5
0.09
0.14
0.08
0.1
1.2
63
0.04
876
26
31
52
23
45
25
22
11
40 ( 12
952
348 ( 144
40 ( 2
65
2430 ( 571
313 ( 18
60.75
0.33
1.00
12.15
5.62
2.25
0.97
<2.6
1.63
347 ( 116
486 ( 220
365 ( 116
250 ( 109
2818 ( 438
65386 ( 7516
44 ( 15
Prn
Bun
Pri
But
Ph
3.7 ( 0.1
2.5
111
2908
>25000
60.5 ( 10.1
3838 ( 408
4.5 ( 0.2
40.3
2558
3.0
27 ( 11
a
b
d
Wild-type pfDHFR. A16V+S108T mutant pfDHFR. c Data from ref 8. Data from ref 9.

2740 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Yuthavong et al.
Ta ble 2. Inhibition Constants (Ki) of Cyc (1) and Its Analogues (A; X ) Br, Y ) H) against the Wild-Type and A16V+S108T Mutant
DHFRs of P. falciparum and Their Growth Inhibition (IC50) against P. falciparum Clones with Wild-Type (TM4/8.2) and Mutant
(T9/94 RC17) Enzymes
Ki(wt)a
(nM)
rel. to
Cyc
Ki(mut.)b
(nM)
rel. to
Cyc
Ki(mut.)/
Ki(wt)
IC50 TM4
(nM)
IC50 T9/94
(nM)
IC50 ratio
T9/94:TM4
cmpd
R1
R2
10
11
12
13
14
15
Me
H
H
H
H
Me
Me
Et
1.1 ( 0.2
5.7 ( 0.5
2.7 ( 0.3
2.6 ( 0.8
30 ( 4.0
2.9 ( 1.2
0.7
3.8
1.8
1.7
20
1947 ( 366
202 ( 17
99 ( 7.0
127 ( 11
1195 ( 53
90 ( 11
1.5
1770
35
37
49
40
31
132
68
63
638
47 ( 8
2759 ( 153
277 ( 32
220 ( 96
250 ( 110
725 ( 277
180 ( 62
89
2.1
3.24
3.97
1.14
3.86
0.15
0.08
0.1
0.9
0.07
Prn
Pri
Ph
H
1.9
31
a
b
Wild-type pfDHFR. A16V+S108T mutant pfDHFR.
Ta ble 3. Inhibition Constants (Ki) of Cyc (1) and Its Analogues (A; X ) Me, Y ) H) against the Wild-Type and A16V+S108T Mutant
DHFRs of P. falciparum and Their Growth Inhibition (IC50) against P. falciparum Clones with Wild-Type (TM4/8.2) and Mutant
(T9/94 RC17) Enzymes
Ki(wt)a
(nM)
rel. to
Cyc
Ki(mut.)b
(nM)
rel. to
Cyc
Ki(mut.)/
Ki(wt)
IC50 TM4
(nM)
IC50 T9/94
(nM)
IC50 ratio
T9/94:TM4
cmpd
R1
R2
16
17
18
19
20
21
Me
H
H
H
H
Me
Me
Et
1.8 ( 0.2
23.0 ( 1.9
5.9 ( 0.2
13.7 ( 0.8
167 ( 11
7.7 ( 2.0
1.2
15
3.9
9.1
111
5.1
1584 ( 210
185 ( 22
1.21
0.14
0.10
0.14
1.11
0.13
880
7.9
22
14
8.7
22
65
462 ( 35
273
167
8223
136 ( 8.3
3617 ( 337
464 ( 208
517 ( 419
152
3446 ( 759
39 ( 9
55.64
1.00
1.89
0.91
0.42
0.29
128 ( 4.0
188 ( 12
Prn
Pri
Ph
1460 ( 161
170 ( 14
H
a
b
Wild-type pfDHFR. A16V+S108T mutant pfDHFR.
Ta ble 4. Inhibition Constants (Ki) of Cyc (1) and Its Analogues (A; R1 ) R2 ) R, Y ) H) against the Wild-Type and A16V+S108T
Mutant DHFRs of P. falciparum and Their Growth Inhibition (IC50) against P. falciparum Clones with Wild-Type (TM4/8.2) and
Mutant (T9/94 RC17) Enzymes
Ki(wt)a
(nM)
rel. to
Cyc
Ki(mut.)b
(nM)
rel. to
Cyc
Ki(mut.)/
Ki(wt)
IC50 TM4
(nM)
IC50 T9/94
(nM)
IC50 ratio
T9/94:TM4
cmpd
R
X
1
Me
Me
Me
H
H
H
Cl
H
F
Cl
H
F
1.5 ( 0.3c
20.0 ( 5.2
4.6 ( 0.7
24.4 ( 4.3
329 ( 27
270 ( 28
1.0
13
3.0
16
220
180
1314 ( 164c
1375 ( 236
1633 ( 269
646 ( 77
1.0
876
69
355
26
1.8
1.7
40 ( 12
546
294
952
11632
9661
2430 ( 571
445 ( 1
1001 ( 466
313 ( 18
356 ( 57
312 ( 36
60.75
0.82
3.40
0.33
0.03
0.03
22
23
2
24
25
1.05
1.20
0.5
0.4
0.4
585 ( 70
469 ( 71
a
b
Wild-type pfDHFR. A16V+S108T mutant pfDHFR. c Data from ref 8.
The Ki values of monoethyl (4), mono-n-propyl (5), and
mono-n-butyl (6) analogues for the wild-type and
A16V+S108T pfDHFRs were comparable to that for the
monomethyl (3) analogue, implying that the C-2 sub-
stituents of these analogues did not appreciably influ-
ence or improve the binding affinities of the inhibitors
compared with analogue 3. However, as predicted, the
Ki values for both wild-type and A16V+S108T pfDHFRs
were greatly increased when the C-2 substituents were
branched (and therefore bulkier) alkyl groups, as ob-
served with analogues 7 and 8. Interestingly, analogue
9, in which one substituent at the C-2 is H and the other
a phenyl group, inhibited the A16V+S108T pfDHFR
with the Ki value of ∼49 nM, a value which is 27-fold
lower than that observed for Cyc (1). The above results
indicate the crucial role of residue 16 for Cyc binding
and suggest the importance of the phenyl group at
position C-2 for achieving effective inhibition of the
A16V+S108T pfDHFR.
To investigate the significance of the p-chloro group
on the N-1 substituent of Cyc, two series of Cyc
analogues were synthesized in which the N-1 p-chlo-
rophenyl group is replaced by p-bromophenyl and p-tolyl
groups. Their Ki values against the wild-type and the
mutant pfDHFRs are shown in Table 2 and Table 3.
Inhibition constants for the wild-type and mutant
enzymes by p-bromophenyl (10 to 15) and p-tolyl (16 to
21) analogues showed a trend similar to those where
the N-1 substituent is p-chlorophenyl (Table 1). How-
ever, as the p-substituent changes in polarity and size,
the monophenyl compounds became progressively less
effective against the A16V+S108T mutant enzyme; the
Ki values for the mutant enzyme of analogues 9, 15, and
21 being 49, 90, and 170 nM, respectively.
The contribution of the p-chloro substituent toward
the binding affinities of the inhibitors to pfDHFRs was
investigated. The C-2 substituents of Cyc were either
kept unmodified as dimethyl groups or removed (sub-
stituted with H), and the p-chloro substituent of p-
chlorophenyl group at N-1 in Cyc was replaced with
either H or F. The analogues were then tested against
the wild-type and the A16V+S108T mutant pfDHFRs
(Table 4). The results in Table 4 revealed that replace-
ment of the p-chloro group with H (analogues 22 and
24) did not affect the Ki values for the A16V+S108T
mutant DHFR but substantially increased the Ki values
for the wild-type enzyme. Replacement of the chlorine
by fluorine, however, resulted in a 3-14-fold decrease
in the binding affinities of the inhibitors to the wild-
type enzyme (analogues 23 and 25, Table 4). The data
imply that the p-chloro substituent of the p-chlorophenyl
group is important for binding to the wild-type enzyme
and that replacement of the chlorine by a smaller group
has relatively little effect on the binding of the inhibitor
to the A16V+S108T pfDHFR.
We next investigated the Cyc analogues in which an
additional chlorine atom was placed at the m-position
of the p-chlorophenyl substituent. The importance of

Development of a Lead Inhibitor of pfDHFR
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2741
Ta ble 5. Inhibition Constants (Ki) of Cyc (1) and Its Analogues (A; Y ) Cl) against the Wild-Type and A16V+S108T Mutant DHFRs
of P. falciparum and Their Growth Inhibition (IC50) against P. falciparum Clones with Wild-Type (TM4/8.2) and Mutant (T9/94 RC17)
Enzymes
Ki(wt)a
(nM)
rel. to
Cyc
Ki(mut.)b
(nM)
rel. to
Cyc
Ki(mut.)/
Ki(wt)
IC50 TM4
(nM)
IC50 T9/94
(nM)
IC50 ratio
T9/94:TM4
cmpd
R1
R2
X
26
27
28
29
30
31
Me
Me
H
Me
Me
Me
Me
Ph
H
Cl
H
Cl
H
Cl
3.7 ( 0.6
1.1 ( 0.4
10.2 ( 0.6
1.4 ( 0.2
11.7 ( 2.5
1.6 ( 0.2
2.5
0.73
6.8
0.9
7.8
1.1
340 ( 28
130.7 ( 13.4
38.7 ( 2.9
17.8 ( 0.8
10 ( 7
0.3
92
119
3.8
12.7
0.9
6.9
60 ( 13
4 ( 1
298 ( 54
307 ( 99
28 ( 4
4.97
76.75
NDc
0.1
0.03
0.014
0.008
0.008
NDc
H
35
19 ( 8
0.54
H
H
455 ( 228
24 ( 8
29 ( 2
0.05
0.72
Ph
11 ( 1.8
40 ( 25
a
b
Wild-type pfDHFR. A16V+S108T mutant pfDHFR. c Not determined.
m-chloro substituent was recently shown in Pyr to
improve the effectiveness against the C59R+S108N
mutant pfDHFR.13 As shown in Table 5, the Cyc
analogue with m-chlorophenyl group (26) inhibited the
wild-type pfDHFR with the Ki value ∼2.5 times higher
than that of Cyc (1), but it was about 3-fold more
effective than Cyc against the A16V+S108T mutant
enzyme. Replacement of the p-chlorophenyl group of Cyc
with the 3,4-dichlorophenyl substituent yielded ana-
logue 27, which was about as effective as Cyc against
the wild-type DHFR but inhibited the A16V+S108T
mutant enzyme about 10 times better than Cyc. Indeed,
analogue 27, also known as chlorocycloguanil, is a
potent inhibitor of wild-type pfDHFR and has been used
as an effective agent for the treatment of malaria.14
Replacing one of the C-2 methyl groups of the m-
chlorophenyl analogue by H yielded analogue 28 which
showed a significant decrease in the Ki value for the
A16V+S108T pfDHFR, but the analogue inhibited the
wild-type enzyme ∼7-fold less effectively than Cyc (1).
Addition of a chloro group to analogue 28 gave the
monomethyl analogue with the 3,4-dichlorophenyl group
at position N-1 (29). While the Ki value for analogue 29
was similar to that of Cyc (1) against the wild-type
pfDHFR, the binding affinity for the A16V+S108T
enzyme was dramatically improved, being about 73-fold
more effective than Cyc.
the resistant parasite. The data in Tables 2 and 3 show
the same trend as in Table 1, in that the resistance
factors for the 2-monosubstituted derivatives are all
lower than those for the 2,2-dimethyl parent com-
pounds. All the 2-monosubstituted compounds in Tables
2 and 3 are more effective against the resistant parasite
than the parent compounds. However, none of the
compounds in Tables 2 and 3 are as effective as the
parent compounds against the wild-type parasite.
The Cyc analogues in Table 4, in which the p-chloro
substituents had been replaced by sterically less de-
manding groups, have higher IC50 values against the
wild-type parasite than those for the parent compounds,
generally reflecting the higher Ki values. The low values
of resistance factors reflect the poor activities against
the wild-type parasite rather than relatively high activi-
ties against the resistant parasite.
Table 5 shows the effect on the antiplasmodial activi-
ties of Cyc analogues in which dimethyl is changed to a
2-monosubstituted group and the p-chloro is replaced
by an m-chloro or 3,4-dichloro group. All the 2-mono-
substituted compounds show excellent activities against
the resistant parasite, with IC50 values approximately
100 times lower than that of Cyc. Both the 2-monosub-
stituted and the 3,4-dichlorophenyl derivatives (29 and
31) have comparable activities to Cyc against the wild-
type parasite.
The effects of m-chlorophenyl and 3,4-dichlorophenyl
substituents at N-1 were further tested in the most
promising lead compound in which the groups at C-2
were H and phenyl (9) (Table 1). Analogues 30 and 31
were over 120-fold more effective than Cyc against the
A16V+S108T mutant pfDHFR. While analogue 30 was
about 8-fold less effective than Cyc against the wild-
type enzyme, analogue 31 was about as effective as Cyc
(Table 5).
Preliminary results on human DHFR with some
2-monosubstituted analogues of Cyc show relatively
high Ki values (data not shown), suggesting that these
compounds are probably of low toxicity and therefore
might be suitable for further investigation as lead
compounds in the search for new effective antimalarials
against antifolate-resistant P. falciparum.
Con clu sion s
In Vitr o An tip la sm od ia l Activity. The activities of
the Cyc analogues against P. falciparum were tested
in vitro, both in the wild-type clone (TM4/8.2) and the
Cyc-resistant clone, which harbors the A16V+S108T
pfDHFR (T9/94 RC17). The data in Table 1 shows that
some of the 2-monosubstituted analogues of Cyc, namely
ethyl, n-propyl, and phenyl, have IC50 values against
the wild-type parasite which are comparable to that of
the parent compound. Furthermore, all the compounds
have relatively low resistance factors (ratios of IC50 for
T9/94 to TM4) in comparison to Cyc, some with factors
of 1 or less. All compounds except for isopropyl and tert-
butyl derivatives are much more effective than Cyc
against the resistant parasite. The most notable com-
pound in Table 1 is the phenyl derivative, which is
almost twice as effective against the wild-type parasite
and is over 50 times more effective than Cyc against
1-(3′,4′-Dichlorophenyl)-2-monosubstituted-4,6-diamino-
1,2-dihydro-1,3,5-triazines (29 and 31) are useful lead
compounds, being at least as effective against the
mutant resistant P. falciparum strain (T9/94 RC17) as
against the wild-type strain (TM4/8.2). They are as
effective as Cyc against the wild-type strain of P.
falciparum and approximately 100-fold more effective
than Cyc against the resistant P. falciparum strain (T9/
94 RC17).
Exp er im en ta l Section
Meth od s a n d Ma ter ia ls. m-Chloroaniline was distilled
under reduced pressure before use. All other chemicals were
obtained from Sigma-Aldrich Ltd., Lancaster, Avocado Ltd.,
BDH, or other standard suppliers, and they were used without
further purification. Reagent grade acetone and absolute
alcohol were used.

2742 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14
Yuthavong et al.
Ta ble 6. Tabulated Elemental, Mass, and NMR Analysis Data for Cyc Analogues 1-31
cpd
X
Y
H
R1 R2 yd% anal.
Me Me 62 CHN
formula
MH+ a
NMR details (200 MHz unless otherwise specified)
1
Cl
C
11H15N5Cl2
252 (iii)
1H (D2O, 400 MHz) 1.38 (6H, s, 2 × Me), 7.35 and 7.54 (2 × 2H,
AB doublet, J ) 8 Hz, aromatic C-H)
2
Cl
H
H
H
79 CHN C9H11N5Cl2
224 (i)
1H (DMSO-d6) 4.71 (2H, s, CH2), 6.98 (1H, s br ex, NH), 7.48 (4H,
dd, J AB ) 8, Ar-C-H), 7.65 (2H, s, NH2), 7.85 (1H, s br ex, NH),
8.74 (1H, s, NH+)
3
4
Cl
Cl
H
H
H
H
Me
Et
49 CHN
92 CHN
C
C
10H13N5Cl2
11H15N5Cl2
238 (i)
1H (D2O) 1.20 (3H, d, J ) 6 Hz, Me-2), 5.06 (1H, q, J ) 6 Hz,
H-2), 7.22 and 7.40 (2 × 2H, AB doublet, J ) 8 Hz, Ar-C-H)
1H (D2O) 0.94 (3H, J ) 6.5 Hz, CH3), 1.52 (2H, m, CH2), 4.84 (1H,
dd, J ) 6.5, 4 Hz, H-2), 7.22 and 7.38 (2 × 2H, AB doublet,
J ) 8 Hz, Ar-C-H)
252 (ii)
5
6
7
Cl
Cl
Cl
H
H
H
H
H
H
Prn 86 HNb C21H17N5Cl2‚
266 (ii)
280 (iii)
266 (i)
1H (D2O) 0.65 (3H, t, CH3), 1.15 (2H, m, CH2), 1.55 (2H, m, CH2),
4.94 (1H, dd, J ) 6, 4 Hz, H-2), 7.25 and 7.42 (2 × 2H,
AB doublet, J ) 8 Hz, aromatic C-H)
H2O
Bun 89 CHN C13H19N5Cl2‚
1H (D2O) 0.60 (3H, t J )6.5 Hz, Me), 1.08 (4H, m, 2 × CH2), 1.55
(2H, m, CH2), 4.96 (1H, dd J ) 6.5, 4 Hz, H-2) 7.25, 7.40 (2 × 2H,
AB doublet, J ) 8 Hz, Ar-C-H)
3.5H2O
Pri
59 CHN C12H17N5Cl2
1H (D2O) 0.65 and 0.75 (6H, 2 × d J ) 6.8 Hz, 2 × Me), 1.88 (1H,
m, CHMe2), 4.74 (1H, d J ) 2.8 Hz, H-2), 7.24, 7.40 (2 × 2H,
AB doublet, J ) 8 Hz, Ar-C-H)
8
9
C
C
H
H
H
H
H
H
H
But 31 CHN C13H19N5Cl2
280 (i)
300 (iii)
246 (iii)
282,
284 (iii)
296,
298 (iii)
1H (D2O) 0.66 (9H, s, 3 × Me), 4.65 (1H, s, H-2), 7.31 (4H, m,
aromatic C-H)
Ph
55 CHN
78 CHN
53 CHN
93 CHN
C
C
C
C
15H15N5Cl2
1H (D2O) 5.84 (1H, s, H-2), 6.90 (2H, part of AB doublet, J ) 8 Hz,
Ar-C-H), 7.05 (7H, m, Ar-C-H)
10 Br
11 Br
12 Br
Me Me
11H15N5BrCl
10H13N5BrCl
11H15N5BrCl
1H (D2O) 1.28 (6H, s, 2 × Me), 7.16 and 7.58 (2 × 2H, AB doublet,
J ) 8 Hz, aromatic C-H)
H
H
Me
Et
1H (D2O) 1.16 (3H, d, J ) 6 Hz, Me-2), 5.00 (1H, q, J ) 6 Hz,
H-2), 7.14 and 7.55 (2 × 2H, AB doublet, J ) 8 Hz, aromatic C-H)
1H (D2O) 0.74 (3H, t, J ) 6.5 Hz, CH3), 1.50 (2H, m, CH2), 4.92
(1H, dd, J ) 6.5, 4 Hz, H-2), 7.20 and 7.50 (2 × 2H, AB doublet,
J ) 8 Hz, aromatic C-H)
13 Br
14 Br
H
H
H
H
H
Prn 74 CHN C12H17N5BrCl‚
310,
312 (iii)
1H (D2O) 0.68 (3H, t, J ) 6 Hz, CH3), 1.18 (2H, m, CH2), 1.60
(2H, m, CH2), 4.92 (1H, dd, J ) 6, 4 Hz, H-2), 7.20 and 7.50
(2 × 2H, AB doublet, J ) 8 Hz, aromatic C-H)
H2O
Pri
Ph
71 CHN C12H17N5BrCl
310,
312 (iii)
1H (D2O) 0.66 and 0.76 (6H, 2 × d, J ) 7 Hz, 2 × Me), 1.88 (1H,
m, CHMe2), 4.78 (1H, d, J ) 3 Hz, H-2), 7.18 and 7.56 (2 × 2H,
AB doublet, J ) 8 Hz, aromatic C-H)
15 Br
16 Me
17 Me
H
H
H
93 CHN
59 CHN
27 CHN
C
C
C
15H15N5BrCl
12H20N5OCl
344,
346 (iii)
232 (iii)
1H (D2O) 5.88 (1H, s, H-2), 6.88 and 7.34 (2 × 2H, AB doublet,
J ) 8 Hz, aromatic C-H), 7.16 (5H, m, aromatic C-H)
1H (D2O) 1.26 (6H, s, 2 × Me), 2.20 (3H, s, 4′-Me), 7.08 and 7.25
(2 × 2H, AB doublet, J ) 8 Hz, Ar-C-H)
Me Me
H
H
H
H
H
Me
Et
11H16N5Cl‚
HCl‚0.7H2O
218 (iii)
1H (D2O) 1.18 (3H, d, J ) 6.5 Hz, Me-2), 2.15 (3H, s, Me-4′), 5.05
(1H, q, J ) 6.5 Hz, H-2), 7.08 and 7.20 (2 × 2H, AB doublet,
J ) 8 Hz, aromatic C-H)
18 Me
19 Me
20 Me
21 Me
H
H
H
H
77 CHN
C
12H18N5Cl
232 (iii)
246 (iii)
1H (D2O) 0.72 (3H, t, J ) 6 Hz, CH3), 1.65 (2H, m, CH2), 2.18
(3H, s, Me-4′), 4.94 (1H, dd, J ) 6, 5 Hz, H-2), 7.15 and 7.25
(2 × 2H, AB doublet, J ) 8 Hz, aromatic C-H)
Prn 87 CHN C13H20N5Cl‚
1H (D2O) 0.64 (3H, t, J ) 7 Hz, CH3), 1.15 (2H, m, CH2CH3), 1.60
(2H, m, CH2CH2), 2.18 (3H, s, Me-4′), 4.94 (1H, dd, J ) 6, 4 Hz,
H-2), 7.12 and 7.22 (2 × 2H, AB doublet, J ) 8 Hz, aromatic C-H)
1H (D2O) 0.65 and 0.75 (6H, 2 × d, J ) 7 Hz, 2 × Me), 1.88 (1H,
m, CHMe2), 2.18 (3H, s, Me-4′), 4.82 (1H, d, J ) 3 Hz, H-2), 7.12
and 7.22 (2 × 2H, AB doublet, J ) 8 Hz, aromatic C-H)
1H (D2O) 2.16 (3H, s, Me-4′), 5.96 (1H, s, H-2), 6.94 and 7.12
(2 × 2H, AB doublet, J ) 8 Hz, aromatic C-H), 7.30 (5H, m,
aromatic C-H)
HCl
Pri
Ph
85 HNc C13H20N5Cl‚HCl‚ 246 (iii)
CH3OH
58 CHN
67 CNd C11H16N5Cl
93 CHN 11H15N5FCl
C
16H18N5Cl
280 (iii)
22
23
H
F
H
H
Me Me
Me Me
218 (iii)
235 (i)
1H (D2O, 400 MHz) 1.45 (6H, s, 2 × Me), 2.25 (2H, q, J ) 7 Hz,
CH2), 7.40, 7.55 (2 × m, 5H, Ar-C-H)
C
1H (DMSO-d6) 1.31 (6H, s, 2 × Me), 6.42 (1H, s br ex, NH), 7.29-7.47
(6H, m, Ar-C-H and NH2), 7.67 (1H, s br ex, NH), 9.01 (1H, s,
NH+). 13C (DMSO-d6) 27.6 (2C, 2 × Me), 70.1 (1C, CMe2), 117.4 +
117.9 (2C, Ar-C-F ortho), 131.7 (1C, CN3), 133.0 + 133.2 (2C,
Ar-C-F meta), 158.24 (1C, Ar-C-F ipso), 160.1 (1C, CN3), 165.6
(1C, Ar-C-N). 19F (DMSO-d6, 250 MHz) -112.5 (1F, Ar-F)
1H (DMSO-d6) 4.78 (2H, s, CH2), 6.89 (1H, s br ex, NH), 7.34-7.58
(m, 7H, Ar-C-H and NH2), 7.80 (1H, s br ex, NH) 8.58 (1H, s, NH+)
1H (DMSO-d6) 1.31 (6H, s, 2 × Me), 6.42 (1H, s br ex, NH), 7.29-7.47
(6H, m, Ar-C-H and NH2), 7.67 (1H, s br ex, NH), 9.01 (1H, s,
NH+). 13C (DMSO-d6) 27.6 (2C, 2 × Me), 70.1 (1C, CMe2), 117.4 +
117.9 (2C, Ar-C-F ortho), 131.7 (1C, CN3), 133.0 + 133.2 (2C,
Ar-C-F meta), 158.24 (1C, Ar-C-F ipso), 160.1 (1C, CN3), 165.6
(1C, Ar-C-N). 19F (DMSO-d6, 250 MHz) -112.5 (1F, Ar-F)
1H (D2O) 1.30 (6H, s, 2 × Me), 7.10-7.50 (m, 4H, Ar-C-H)
24
25
H
F
H
H
H
H
H
H
85 CHN C9H12N5Cl
95 CHN C9H11N5FCl
190 (i)
208 (i)
26
H
Cl Me Me
60 CHN
71 CHN
C
C
11H15N5Cl2‚
0.4H2O
11H14N5Cl3
252 (iii)
286 (iii)
27 Cl Cl Me Me
1H (D2O, 400 MHz) 1.38 (6H, s, 2 × Me), 7.26 (1H, dd, J ) 8.5,
2.6 Hz, Ar-CH), 7.60 (1H, d, J ) 2.6 Hz, Ar-CH), 7.65 (d, 1H,
J ) 8.5 Hz, Ar-C-H)

Development of a Lead Inhibitor of pfDHFR
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 14 2743
Ta ble 6. Continued
cpd
X
H
Y
R1 R2 yd% anal.
formula
MH+ a
NMR details (200 MHz unless otherwise specified)
28
Cl
H
H
H
H
Me
Me
Ph
Ph
18
76
90
63
CHN
CHN
HNe
C
C
10H13N5Cl2
238 (iii) 1H (D2O) 1.18 (3H, d, J ) 6 Hz, Me-2), 4.98 (1H, q, J ) 6 Hz, H-2),
7.20, 7.35 (m, 4H, Ar-C-H)
29
30
31
Cl Cl
Cl
Cl Cl
10H12N5Cl3‚ 272 (iii) 1H (D2O) 1.18 (3H, d, J ) 6.5 Hz, Me), 5.04 (1H, q, J ) 6.5 Hz,
2HCl‚H2O
H-2), 7.15 (1H, d, J ) 8.5, 2.6 Hz, Ar-C-H), 7.50 (2H, m, Ar-C-H)
H
C15H15N5Cl2‚ 300 (iii) 1H (D2O) 5.95 (1H, s, H-2), 6.90-7.30 (m, 9H, Ar-C-H)
H2O
11H14N5Cl3
CHN
C
334 (iii) 1H (D2O) 5.92 (1H, s, H-2), 6.90 (1H, dd, J ) 8.0, 2.5 Hz, Ar-CH),
7.10-7.30 (7H, m, Ar-CH)
a
b
d
Mass spectrometric methods are (i) APCI+, (ii) ESI, (iii) MALDI-TOF. C calcd 45.0; found 45.6. c C calcd 48.0; found 47.5. H calcd
6.4; found 7.0. e C calcd 46.4; found 46.9.
Parallel synthesis reactions were performed in a Radley
multiple synthesis block of 56 wells, equipped with a “Big Bill”
rotating shaker, a water-cooled condensing stage, and a J -KEM
programmable thermocouple and controller. Reagents were
heated and refluxed in 4 mL capacity ReactiVials equipped
with 10 cm condensing tubes inserted into Teflon-coated seals
and caps. Reactions involving highly volatile solvents were
contained under SubaSeals vented using balloon leaks.
En zym e Assa ys a n d In h ibition by Cyc An a logu es. The
activities of wild-type and A16V+S108T mutant pfDHFRs
were determined spectrophotometrically according to the
method previously described.17 The reaction (1 mL) contained
1× DHFR buffer (50 mM TES, pH 7.0, 75 mM â-mercaptoet-
hanol, 1 mg/mL bovine serum albumin), 100 µM each of the
substrate H2 folate and cofactor NADPH, and an appropriate
amount (0.001-0.005 units) of the affinity-purified enzymes.
Inhibition of the enzymes by Cyc and its analogues was carried
out by determination of the Ki values of the inhibitors for the
enzymes by fitting to the equation IC50 ) Ki (1 + ([S]/Km)),18
where IC50 is the concentration of inhibitor which inhibits 50%
of the enzyme activity under the standard assay condition and
Km is the Michaelis constant for the substrate H2/folate. The
resistance factors which determine the effectiveness of the
inhibitor against the mutant pfDHFR over the wild-type
enzyme were assessed from the values of the ratios of the Ki
for the A16V+S108T mutant enzyme and the wild-type
enzyme (Ki-mut/Ki-wt).
Dr u g Scr een in g a ga in st P la sm od iu m fa lcipa r u m in
Vitr o. Two clones of P. falciparum, TM4/8.2 (wild-type DHFR)
and T9/94 RC17 (A16V+S108T DHFR),19 from diverse sources
(generous gifts from the Malaria Research Unit, Chula-
longkorn University, Bangkok, Thailand) were maintained
continuously in human erythrocytes at 37 °C under 3% CO2
in RPMI 1640 culture media supplemented with 25 mM
HEPES, pH 7.4, 0.2% NaHCO3, 40 µg/mL gentamicin, and 10%
human serum.20 In vitro antimalarial activity was determined
by using the [3H]-hypoxanthine incorporation method.21
The drugs were initially dissolved in DMSO and diluted
with the culture media. Aliquots (25 µL) of the drug having
different concentrations were dispensed into 96-well plates,
and 1.5% cell suspension of parasitized erythrocytes with
1-2% parasitemia (200 µL) were added. The final concentra-
tion of DMSO (0.1%) did not affect the growth of the parasite.
The mixtures were incubated in a 3% CO2 incubator at 37 °C.
After 24 h of incubation, 25 µL (0.25 µCi) of [3H]-hypoxanthine
was added to each well, and the parasite cultures were further
incubated under the same conditions for 18-24 h prior to
harvesting the parasite DNA onto 96-well microplates with
built-in glass fiber filters (Unifilter TM plates, Packard, USA).
The filters in the plates were air-dried, and then 22 µL of liquid
scintillation fluid (Microscint, Packard) was added. The ra-
dioactivity on the filters was then measured using a microplate
scintillation counter (TopCount, Packard, USA). The concen-
tration of inhibitor which inhibits 50% of the parasite growth
(IC50) was determined from the sigmoidal curve obtained by
plotting the percentages of [3H]-hypoxanthine incorporation
against the concentrations of the drug used.
Positive-ion chemical ionization mass spectrometry was
carried out as APCI in 1:1 methanol/dichloromethane as
carrier solvent (Dyson Perrins Laboratory, University of
Oxford). MALDI-TOF mass spectra were recorded on a Bruker
Biflex mass spectrometer (Institute of Genetic Engineering and
Biotechnology, Chulalongkorn University, Bangkok). ESI spec-
tra were obtained using a Fisons Instrument Trio 2000 mass
spectrometer (Department of Chemistry, Chulalongkorn Uni-
versity, Bangkok) operating in ESI mode. Masses given are
molecular ions and major fragments only, and they are quoted
as m/z unless otherwise stated. Elemental analysis (C, H, N)
was performed by the Oxford University Inorganic Laboratory
service and by Ms. A. Ungpakornkaew on a Perkin-Elmer CHN
analyzer model PE2400 series II (Chulalongkorn Research
Equipment Centre, Bangkok). Routine 1H and 13C NMR
spectra were recorded on a Varian Gemini 200 spectrometer,
a Bruker AMX250 spectrometer (both Dyson Perrins Labora-
tory, Oxford), or a Bruker ACF200 (Chulalongkorn University,
Bangkok) operating at 200 MHz (1H) and 50.28 MHz (13C).
High field NMR experiments were performed on a Bruker
DRX400 (400 MHz) (National Science and Technology Devel-
opment Agency, Bangkok). 1H and 13C chemical shifts are
quoted in ppm relative to tetramethylsilane and were inter-
nally referenced to the residual protonated solvent signal.
Ch em ica l Syn th eses of 4,6-Dia m in o-1,2-d ih yd r otr ia z-
in e An a logu es. The Cyc derivatives bearing gem-dimethyl
groups at the C-2 position were prepared by a three-component
condensation reaction between an aromatic amine, dicyandia-
mide, and acetone in the presence of concentrated aqueous HCl
as described in the literature (Scheme 1).10 When the carbonyl
compound is an aldehyde, a two-component condensation
between the carbonyl compound (1-2 equiv or as solvent) and
an aryl biguanide,11,15,16 obtained from a reaction between an
aromatic amine and dicyandiamide in the presence of HCl as
catalyst, or a one-pot reaction in which the biguanide was
preformed before reacting with the carbonyl compound was
sometimes found to be superior. Derivatives of formaldehyde
were prepared in the same way as other aldehydes, except that
dimethoxymethane was used as a source of formaldehyde. In
most cases the desired products precipitated from the reaction
medium (usually ethanol) as the crystalline hydrochloride salt.
In cases where the product did not crystallize from the
reaction, the solvent was completely removed and the product
was precipitated by addition of lithium picrate. The picrate
salt so obtained was converted back to the hydrochloride salt
by treatment with a strongly basic anion-exchange resin
Amberlite IRA400 (Cl- form). The products were recrystallized
from ethanol, methanol, or aqueous alcohols. All Cyc deriva-
tives were characterized by 1H NMR, mass spectra (APCI, ESI,
or MALDI-TOF), and elemental analysis (CHN). The full
detailed analysis and the data of the Cyc analogues are shown
in Table 6.
Ack n ow led gm en t. This research was supported by
grants from the World Health Organization (TDR) to
W.S., the European Union (INCO-DC IC18CT970223)
to Y.Y. and G.L., the Thailand Research Fund (Contract
No. PDF/57/2540) to T.V., and the Thailand TDR (98-
1-MAL-22-003) to S.K.
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