Folate-Synthesizing Enzyme System as Target for Development of Inhibitors and Inhibitor Combinations against Candida albicans - Synthesis and Biological Activity of New 2,4-Diaminopyrimidines and 4′-Substituted 4-Aminodiphenyl Sulfones

Article abstract of DOI:10.1021/jm030931w

The paper describes the design, synthesis, and testing of inhibitors of folate-synthesizing enzymes and of whole cell cultures of Candida albicans. The target enzymes used were dihydropteroic acid synthase (SYN) and dihydrofolate reductase (DHFR). Several series of new 2,4-diaminopyrimidines were synthesized and tested as inhibitors of DHFR and compared with their activity against DHFR derived from mycobacteria and Escherichia coli. To test for selectivity, also rat DHFR was used. A series of substituted 4-aminodiphenyl sulfones was tested for inhibitory activity against SYN and the I₅₀ values compared to those obtained previously against Plasmodium berghei- and E. coll-derived SYN. Surprisingly, QSAR equations show very similar structural dependencies. To find an explanation for the large difference in the I₅₀ values observed for enzyme inhibition (SYN, DHFR) and for inhibition of cell cultures of Candida, mutant strains with overexpressed efflux pumps and strains in which such pumps are deleted were included in the study and the MICs compared. Efflux pumps were responsible for the low activity of some of the tested derivatives, others showed no increase in activity after pumps were knocked out. In this case it may be speculated that these derivatives are not able to enter the cells.

Full text of DOI:10.1021/jm030931w

240
J . Med. Chem. 2004, 47, 240-253
F ola te-Syn th esizin g En zym e System a s Ta r get for Develop m en t of In h ibitor s
a n d In h ibitor Com bin a tion s a ga in st Ca n d id a a lbica n ssSyn th esis a n d
Biologica l Activity of New 2,4-Dia m in op yr im id in es a n d 4′-Su bstitu ted
4-Am in od ip h en yl Su lfon es
Thomas Otzen,† Ellen G. Wempe,‡ Brigitte Kunz,‡ Rainer Bartels,‡ Gudrun Lehwark-Yvetot,‡ Wolfram Ha¨nsel,†
Klaus-J u¨rgen Schaper,‡ and J oachim K. Seydel*,‡
Division of Structural Biochemistry, Research Center Borstel, Leibniz Center for Medicine and Biosciences,
Parkallee 1-40, D-23845 Borstel, Germany, and Pharmaceutical Institute, University of Kiel, D-24118 Kiel, Germany
Received J une 18, 2003
The paper describes the design, synthesis, and testing of inhibitors of folate-synthesizing
enzymes and of whole cell cultures of Candida albicans. The target enzymes used were
dihydropteroic acid synthase (SYN) and dihydrofolate reductase (DHFR). Several series of new
2,4-diaminopyrimidines were synthesized and tested as inhibitors of DHFR and compared with
their activity against DHFR derived from mycobacteria and Escherichia coli. To test for
selectivity, also rat DHFR was used. A series of substituted 4-aminodiphenyl sulfones was
tested for inhibitory activity against SYN and the I50 values compared to those obtained
previously against Plasmodium berghei- and E. coli-derived SYN. Surprisingly, QSAR equations
show very similar structural dependencies. To find an explanation for the large difference in
the I50 values observed for enzyme inhibition (SYN, DHFR) and for inhibition of cell cultures
of Candida, mutant strains with overexpressed efflux pumps and strains in which such pumps
are deleted were included in the study and the MICs compared. Efflux pumps were responsible
for the low activity of some of the tested derivatives, others showed no increase in activity
after pumps were knocked out. In this case it may be speculated that these derivatives are not
able to enter the cells.
In tr od u ction
from secondary infections by C. albicans. Most of these
drugs act on targets within the ergosterol biosynthesis
at different steps (e.g. inhibition of squalene epoxidase)
of fungi.7 Many attempts have been undertaken to
reduce the frequency of side effects and the problem of
resistance.1
Infections caused by fungi have increased dramati-
cally during the past decades.1 The reasons for this are
manifold. In principle, one has to differentiate between
mycoses of the skin or nails and systemic mycoses.
Systemic mycoses mainly appear concomitant with
other diseases2 or are caused by treatment with chemo-
therapeutics, for instance with cytostatics.3 At risk are
patients after organ transplantation treated with im-
munosuppressives4 or those suffering with a weakened
immune system, for example patients with AIDS.5
Endocarditis caused by fungi has been observed with
drug abuse.
Despite several interesting targets for the therapy of
candidiasis, only four classes of drugs are presently
used,6 namely polyenes (amphotericin B, nystatin)
which can be used systemically, 5-fluorocytosine, azoles
(miconazole, ketoconazole, etc.), and allylamines (naf-
tifine, terbinafine); the latter, however, show limited
efficacy in case of candida infections.4 Also morpholine
derivatives such as amorolfine show activity against
Candida albicans, and thiocarbamates have been used
successfully in the treatment of AIDS patients suffering
The folate-synthesizing enzyme system of fungi could
be an alternative target.8 It is a well-established drug
target in microorganisms.9 Various bacterial infec-
tions9 (e.g. Pneumocystis carinii,10 Toxoplasma gondii11)
and malaria12-14 can successfully be treated by folate
inhibitors. C. albicans also possesses the complete
folate-synthesizing enzyme system, despite being an
eukaryont.15 Therefore, many attempts have lately been
made to develop suitable new inhibitors of the folate-
synthesizing enzyme system of various microorganisms,
such as P. carinii and T. gondii,16-18 Leishmaniasis,19
Plasmodium falciparum,20 and C. albicans.21 So far,
however, none of the new compounds could be intro-
duced into therapy, mainly for two reasons: (i) poor
selectivity and (ii) problems in getting into the cyto-
plasm of fungal cells and/or in gastrointestinal absorp-
tion.
In case of bacterial infections, the simultaneous
inhibition of the two enzymes dihydropteroate synthase
(SYN), and dihydrofolate reductase (DHFR) has been
proven to be synergistic and highly effective. Several
examples for synergism on combining sulfonamides or
* To whom correspondence should be addressed. Present address:
Mu¨hloh 2, D-23845 Borstel, Germany (retired). Phone: +49-4537-417.
Fax: +49-4537-188-745. E-mail: J oachim.Seydel@t-online.de.
† University of Kiel.
‡ Leibniz Center for Medicine and Biosciences.
10.1021/jm030931w CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/02/2003

Inhibitors against Candida albicans
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 241
Ta ble 1. Observed and Calculated Inhibitory Activity, I50 (µM), of Substituted 4-Aminodiphenyl Sulfones against SYN Derived from
C. albicans and Observed Activity against SYN from P. berghei, M. lufu (DDS sensitive/resistant), and E. coli
C. albicans
I50 calcd
I50 obsd
M. lufu
sens/res
R
I50 obsd
(eq 1)
P. berghei
E. coli
∆ppm2/6
fia
4-NH2 (DDS)
4-NHCH3
4-NHC2H5
4-H
1.78
2.81
3.16
13.26
10.11
9.05
2.40
14.33
2.78
3.83
1.22
1.33
2.88
2.99
3.29
12.30
8.55
7.37
2.06
13.33
4.30
3.71
0.89
1.20/1.18
1.29/2.00
2.75/1.48
12.06/12.0
8.08/5.92
7.55/9.33
1.50/1.61
12.99/18.7
3.60/3.17
1.76/2.04
0.87/0.82
2.24/2.63
7.01/4.05
11.75/13.8
12.72/9.60
12.41
26.70
34.36
46.21
41.71
116.50
89.85
128.2
34.92
-0.108
-0.105
-0.098
0
-0.027
-0.038
-0.054
0.006
0
0
0
0
0
0
104.0
48.00
4-CH3
4-OCH3
4-OHb
32.23
0.788
4-Cl
4-COOH
0
1
0
1
0
0
0
0
74.24
0.022
4-N(CH3)2
4-NHCH2COOH
4-NHCH2COOCH3
4-NHCOCH3
4-COOCH3
4-CONHNH2
21.51
17.77
-0.089
-0.095
-0.095
-0.036
0.026
37.05
101.24
75.65
149.29
155.39
33.26
147.0
76.51
0.022
a
b
fi ) 1/(1+10pK -pH). 4-OH: pKa ) 7.83;40 tests were performed at pH ) 7.75 (fi ) 0.454) for E. coli and M. lufu and at pH ) 8.4 (fi
a
) 0.788) for P. berghei and C. albicans.
Ta ble 2. Code, Structure of 2,4-Diaminopyrimidines and I50 Values (µMol/L) against Cell-Free DHFR from Various Sources
compd
TMP
X
Z
R
C. albicans
rat
M. lufu
E. coli
-CH2-
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
H
H
H
3,4,5-(OCH3)3
H
2,3-Benzo
3,4-Benzo
H
30.30
90.45
53.46
4.77
1.24
0.73
3.72
3.10
26.1
2.81
0.43
0.49
23.8
7.94
6.63
13.9
84.8
52.3
125.8
1.39
16.1
3.37
35.4
1.85
1.34
5.72
59.4
30.9
4.20
9.90
190.0
115.3
29.4
61.2
10.1
3.53
56.75
2.57
99.7
39.4
0.27
14.77
0.58
0.57
12.3
3.93
1.22
5.77
7.32
2.98
1.01
0.15
5.07
105.0
8.74
35.2
3.09
11.73
4.88
2.46
1.98
30.7
4.35
4.95
0.39
45.0
24.7
4.42
9.97
0.27
0.0022
0.16
0.16
0.16
3.92
1.48
1.43
5.61
2.59
0.43
0.17
0.04
0.66
11.29
2.69
2.81
1.84
3.18
1.12
0.33
1.29
6.75
1.00
2.83
0.33
10.52
1.09
3.71
6.07
0.62
D.1.1.
D.1.9.
D.1.11.
A.1.1.
A.1.2.
A.1.4.
A.1.5.
A.1.6.
A.1.8.
A.1.9.
A.1.10.
A1.12.
C.1.1
C.1.2.
C.1.3.
C.1.4.
C.1.6.
C.1.7.
C.1.9.
C.1.11.
F .1.1.
Ro12-6099
A.2.1.
A.2.4.
E.1.1.
H.1.1.
J .1.1.
F .2.1.
G.2.1.
-CH2-
-CH2-
-CH2-
-OCH2-
-OCH2-
2-Cl
-OCH2-
-OCH2-
-OCH2-
-OCH2-
3,4-Cl2
2,6-Cl2
4-OCH3
4-OCH2-C6H5
2,3-Benzo
4-Br-2,3-Benzo
4-C(CH3)3
H
-OCH2-
-CH2-
2.63
2.82
106.0
58.2
-OCH2-
-NHCH2-
-NHCH2-
-NHCH2-
-NHCH2-
-NHCH2-
-NHCH2-
-NHCH2-
-NHCH2-
-CH2CH2-
-CH2CH2-
-CH2-
2-Cl
3-Cl
71.53
49.8
3,4-Cl2
4-OCH3
3,4-(OCH3)2
2,3-Benzo
3,4-Benzo
H
3,4-(OCH3)2
H
3,4-Cl2
H
>350a
146.2
197.3
5.9
71.3
39.9
315.3
3.36
4.58
130.6
133.1
110.6
12.8
-OCH2-
-CHdCH-
-CH2NH-
-CH2S-
-CH2CH2-
-(CH2)3-
H
3,4-Benzo
H
CH3
CH3
H
0.82
a
No inhibitory activity up to the indicated concentration.
sulfones, inhibitors of SYN, with inhibitors of DHFR are
documented in the literature. The most prominent and
first example is the combination of sulfisoxazole with
TMP22 (Bactrim, Eusaprim) used mainly against infec-
tions with Gram-negative bacteria. The combination of
dapsone (DDS) or a sulfonamide with pyrimethamine
(e.g. Fansidar) has been used in the treatment of ma-
laria,23 and the combination of DDS with brodimoprim
is synergistically active against Mycobacterium leprae.24
Such a combination approach could also improve treat-
ment of candidiasis by increasing selectivity and avoid-
ing severe side effects. Side effect reduction may be

242 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Otzen et al.
Ch a r t 1. Nomenclature of the Newly Synthesized Substituted 2,4-Diaminopyrimidines
observed because the single doses of the combination
partners could be reduced. Therefore, the aim of this
study was:
(1) to evaluate the activity of sulfones against SYN
derived from C. albicans; (2) in case of favorable activity,
to optimize the inhibitory activity; and (3) to find new
lead structures for a selective inhibition of DHFR
derived from C. albicans, which could then be used in
combination with inhibitors of SYN.
2,4-diamino-5-(3,4-dimethoxyphenylethyl)pyrimidine
(Ro12-6099, Table 2), which is not described elsewhere.
Weinstock et al.29 described a 2,4-diaminopyrimidine
with a -NHCH2- bridge between the heterocycle and
the phenyl ring. The phenyl ring is substituted only by
a carboxyl group. Other references of procedures for the
synthesis of the compounds to be synthesized within this
project could not be found in the literature.
The following nomenclature has been introduced to
describe the newly synthesized substituted 2,4-diamino-
pyrimidines:
The first letter indicates the type of bridge; the first
number describes the substituent in 6-position of the
pyrimidine and the second the substitution at the bridge
moiety (see Chart 1).
The synthetic routes for the 2,4-diaminopyrimidines
bearing different bridging groups are exemplified in
Schemes 1-4 (for details see the Experimental Section).
Syn th esis of 5-Ben zyl-2,4-d ia m in op yr im id in es
(Ser ies D). The general procedure (Scheme 1) describes
the synthetic route on the example of unsubstituted
5-benzyl-2,4-diaminopyrimidine (D.1.1.). The acrylo-
Ma ter ia ls a n d Meth od s
Ch em istr y. The pathway for synthesis of 2,4-diamino-
pyrimidines, the so-called morpholino-anilino proce-
dure, has been reported by Kompis and co-workers.25
For the synthesis of the planned derivatives, however,
only very few examples are found in the literature.
Stogryn26 described the synthesis of a TMP analogue
with a -NHCH2- bridge and Ponsdorf27 a TMP ana-
logue with a -OCH2- bridge. In a review, Kompis et
al.28 mentioned a 5-phenylethyl analogue of TMP [2,4-
diamino-5-(3,4,5-trimethoxyphenylethyl)pyrimidine] and

Inhibitors against Candida albicans
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 243
Sch em e 1a
Sch em e 3a
a
(a) Ph-CHO; CH3ONa/DMSO; (b) Ph-NH2; HCl/EtOH; (c)
guanidine hydrochloride; CH3ONa/EtOH.
a
(a) Guanidine hydrochloride; EtONa/EtOH; (b) H2/Pd/C; 2 N
HCl; 3 h, 60 °C, 4.2 bar; (c) Ac2O/DMF; (d) Ph3P-CH2Ph+Cl-/DMF;
+ DBN; (e) KOH/CH3OH; (f) H2/Pd/C.
Sch em e 2a
Sch em e 4a
a
(a) Guanidine hydrochloride; EtONa/EtOH; (b) ammonium
peroxodisulfate; 5 N NaOH; (c) 5 N HCl; (d) 1-chloromethylnaph-
thalene; EtONa/EtOH.
the corresponding 5-benzylaminopyrimidines (Scheme
2). Lithium borohydride resulted in the highest yield.
a
Syn t h esis of 2,4-Dia m in o-5-st yr ylp yr im id in e
(E.1.1.) a n d 2,4-Dia m in o-5-p h en yleth ylp yr im id in e
(F .1.1.). After many unsuccessful attempts, the deriva-
tives could be synthesized following the synthetic route
proposed by Kompis31 (Scheme 3). A solution of guani-
dine in sodium/ethanol was treated with ethoxymeth-
ylenemalononitrile to give 5-cyano-2,4-diaminopyrimi-
dine. The latter was reduced to the aldehyde by a
modified reduction method. The reaction mixture was
heated and the intermediately formed imine trans-
formed to the aldehyde. The subsequent steps were
performed as described by Baker et al.34 for derivatives
with longer chains in the 5-position. The amino groups
in the 2- and 4-positions were acetylated to increase the
carbonyl reactivity of the aldehyde. This was followed
by a Wittig reaction using benzyltriphenylphosphonium
chloride. 1,5-Diazabicyclo[4.3.0]non-5-ene (DBN) was
used as the base. E.1.1. was obtained by removing the
acetyl groups in alkaline solution. Reduction with H2/
Pd/C led to derivative F .1.1. The synthetic route given
in Scheme 3 was used to synthesize also the derivatives
F .2.1. and G.2.1.
(a) POCl3/Ph-NMe2; (b) NH3/EtOH; (c) Zn/HCl/H2O; (d) R-Ph-
CHO/EtOH; (e) LiBH4 or KBH4/EtOH.
nitrile intermediates are mostly present as a mixture
of E/Z-isomers. â-Alkoxy30 or â-aminopropionitriles25,31
were heated with the substituted aldehydes in the
presence of sodium methylate. The resulting R-benzyl-
â-alkoxy or aminoacrylonitriles were cyclized with
guanidine to the corresponding substituted 5-benzyl-
diaminopyrimidines. Higher yields were obtainable
when â-morpholinopropionitrile (see Scheme 1) was
used with subsequent exchange of the amino group of
the morpholine moiety by aniline instead of using
â-ethoxypropionitrile.31,32
Syn t h esis of 5-Ben zylid en ea m in o-2,4-d ia m in o-
p yr im id in es (Ser ies B). The general synthetic path-
way is shown in Scheme 2. 5-Nitrouracil was reacted
with POCl3. The very unstable intermediate 2,4-dichloro-
5-nitropyrimidine reacted under heat and pressure with
ethanolic ammonia solution to give the 2,4-diamino-5-
nitropyrimidine. This compound was reduced by zinc
powder33 and HCl/H2O to 2,4,5-triaminopyrimidine,
which was then treated with substituted benzaldehydes
according to Zhang et al.33 to give the colored azo-
methines.
Syn th esis of 5-Ben zyloxy-2,4-diam in opyr im idin es
(Ser ies A). Many unsuccessful reactions were per-
formed before this class of derivatives could be obtained.
This was done by following the procedure of Cavalieri
and Bendich.35 We started with the cyclization of
diethoxypropionitrile and guanidine to obtain 2,4-di-
aminopyrimidine, which was further reacted with am-
Syn th esis of 5-Ben zyla m in o-2,4-d ia m in op yr im -
id in es (Ser ies C). The azomethines of series B were
reduced by alkali borohydrides according to Zhang33 to

244 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Otzen et al.
monium peroxodisulfate and subsequently with HCl
according to Elbs to yield the corresponding phenol36,37
(Scheme 4). This was then reacted with the appropriate
benzyl chloride to the final products (Williamson ether
synthesis). For this step many variations have been
tested. The best yields were obtained using sodium
ethylate in ethanol or potassium carbonate in acetone
as reaction medium. Compounds had to be purified by
column chromatography according to a method devel-
oped in our laboratories (see Experimental Section).
E. coli:
log 1/I50 ) -4.157((1.454)∆ppm2/6 +
0.239((0.191)fi +3.933((0.101) (2)
n ) 12 r ) 0.920 s ) 0.110 q ) 0.824
M. lufu (DDS sensitive):
log 1/I50 ) -7.230((1.519)∆ppm2/6 +
0.535((0.216)fi + 4.997((0.108) (3)
Biology
n ) 15 r ) 0.957 s ) 0.133 q ) 0.919
P. berghei:
Deter m in a tion of In h ibitor y Activity a ga in st C.
a lbica n s-Der ived DHFR. The isolation and purifica-
tion of Candida albicans-derived DHFR followed the
procedures described previously for E. coli ATCC 11775,
Mycobacterium lufu L209, and P. berghei.38,39 The
DHFR from rats was commercially available (Sigma D
5519). The inhibition of reduction of dihydrofolate to
tetrahydrofolate was followed photometrically. The
concentration of inhibitor (I50) leading to 50% reduction
in the reaction rate was determined. Results are listed
in Table 2.
In h ibition of SYN. The isolation and purification of
SYN derived from C. albicans was performed in ac-
cordance with the general procedure developed for its
isolation from E. coli, M. lufu, M. leprae, and P.
berghei.38,40 An HPLC-technique was developed to de-
termine quantitatively the inhibition of dihydropteroate
synthesis by sulfones.39
log 1/I50 ) -6.287((1.874)∆ppm2/6 +
4.119((0.127) (4)
n ) 10 r ) 0.939 s ) 0.126 q ) 0.892
For DDS-resistant M. lufu, an almost identical equation
(not shown) was obtained as for the DDS-sensitive
strain (eq 3). In eqs 1 and 2, the 4-NHCH2COOCH3
derivative was omitted (as an outlier because of partial
hydrolysis of the ester group in the enzyme test). In the
regression equations, n is the number of compounds
considered, r is the correlation coefficient, s is the
standard error of the estimate, and q is the cross-
validated correlation coefficient derived from the predic-
tive residual sum of squares (PRESS, leave-one-out
method). Regression coefficients are given with their
95% confidence intervals. ∆ppm2/6 is the 1H NMR
chemical shift of the 2/6 protons of the phenyl group
bound to the sulfonyl group, and fi is the fraction of
ionized sulfone (i.e. 4-OH, 4-COOH, or 4-NHCH2COOH)
at the pH of the enzyme test (pH 8.4 for SYN from C.
albicans, otherwise pH 7.75). In eq 4 the descriptor fi
was not significant, but in this case only two of 10
derivatives bear an ionizable substituent (Table 1).
As for the larger data set,40 no influence of lipophi-
licity (capacity factors log k′ from RP-HPLC40) could be
detected, a fact which presents the possibility to influ-
ence transport, absorption functions, and elimination
pharmacokinetics without losing binding affinity for the
receptor protein.
The similar dependence of the inhibition of SYN on
molecular properties of the sulfones for various classes
of microorganisms is supported by a principal compo-
nent analysis (PCA). In a previous publication40 we had
reported PCA results including I50 values of cell-free
SYN from DDS-sensitive E. coli, M. lufu, and Mycobac-
terium smegm. and of cell-free SYN derived from a M.
lufu strain resistant to sulfones. Furthermore, also I25
values for E. coli whole cell cultures had been included.
In the present study, a PCA analysis with similar
results was performed (details not shown) using the I50
data of Table 1 together with the I25 values of whole
cell E. coli reported in our previous paper.40 P. berghei
data of Table 1 had to be excluded, because the number
of identical derivatives was too small, and thus, the data
set would become too small. As before,40 two principal
components were derived containing 97% of the infor-
mation inherent in the five original variables. The first
principal component (PC) is loaded by log 1/I50 data
Resu lts a n d Discu ssion
In h ibition of SYN. I50, the concentration leading to
50% decrease in dihydropteroic acid production as
compared to the uninhibited control, was determined.
The sulfone derivatives used have been described previ-
ously as well as their activities against SYN derived
from E. coli, mycobacteria, and plasmodia.39,40 Physico-
chemical data and I50 values used in the derivation of
regression equations are summarized in Table 1. I50
data for E. coli, M. lufu, and P. berghei have been taken
from our previous papers,39,40 including a larger data
set of sulfone derivatives. For better comparison with
the new data on inhibition of SYN derived from C.
albicans, the QSAR (eqs 2-4) was determined from
selected identical derivatives.
For the investigated sulfone derivatives, QSAR equa-
tions could be derived that may guide their further
optimization as inhibitors of C. albicans SYN. The
QSAR results point to a very similar and conservative
enzyme structure of SYN in prokaryotic and eukaryotic
cells. For all investigated microorganisms the inhibitory
effect depends solely on the electronic nature of the
1
substituents (described by H NMR shifts: ∆ppm2/6)
and on the degree of ionization (fi) in the case of
ionizable substituents. Regression equations with high
predictive power were obtained.
C. albicans:
log 1/I50 ) -5.843((1.851)∆ppm2/6 +
0.585((0.206)fi - 4.910((0.146) (1)
n ) 11 r ) 0.955 s ) 0.122 q ) 0.856

Inhibitors against Candida albicans
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 245
obtained from four cell-free test systems including the
data for SYN derived from sensitive and resistant M.
lufu (Table 1). This remarkable information showed that
the resistance is not due to changes in the target enzyme
but rather to changes in the membrane or to an increase
in the production of transport proteins of the p-gp type.
As before,40 the second PC is loaded by the log 1/I25
values obtained from the whole cell cultures of E. coli
and pointed again to the importance of the bacterial cell
wall permeation for the antibacterial activity of these
chemotherapeutics. The I25 values obtained in whole cell
cultures of E. coli could be correlated with lipophilicity.40
The important influence of cell wall permeation on the
inhibitory activity of sulfones becomes again obvious
when the significant decrease in inhibitory activity
against whole cell C. albicans cultures is considered.
SAR Resu lts. The following ranking in activity
toward DHFR of C. albicans as a function of the “bridge
elements” is observed:
-OCH2- > -CH2CH2- > -CHdCH- >
-NHCH2- > -CH2NH- > -CH2-
The unsubstituted analogue A.1.1. with the -OCH2-
bridge is 20 times more potent than TMP. Compared
to the unsubstituted 5-benzyl derivative D.1.1., it is
even 90 times more potent.
Derivative F .1.1. bearing a -CH2CH2- bridge shows
also increased activity compared to TMP, but the
increase is significantly lower (9 times).
Derivative C.1.1. possessing a -NHCH2- bridge is
about 4 times more active than TMP and the analogue
with the inverse bridge -CH2NH- (derivative H.1.1.)
is less active (than TMP).
The increase in bridge length, G.2.1., led to high
toxicity (compared to F .2.1.): the I50 against rat DHFR
decreased to 0.82 µmol/L.
Effect of Su lfon es on Wh ole Cell C. a lbica n s. The
inhibitory effect on C. albicans ATCC 11651 cultures
was determined by a yeast cell generation technique.
The generation kinetics was determined by a Coulter
counter technique, analogously to previous papers on
E. coli41 and mycobacteria.42 As observed for the effect
of SYN inhibitors on E. coli and M. lufu, a constant lag
phase in onset of inhibition appears. The onset is,
however, not depending on the inhibitor concentration
so that a diffusion controlled effect can be excluded. A
disappointingly low inhibitory effect (data not shown)
was observed even for the most potent sulfone deriva-
tives, which showed low I50 values against extracted
SYN. Also the ratio between the two I50 values was
disappointing (for DDS, 1.70/150 µmol/L).
Develop m en t of New Ben zylp yr im id in es w ith
In h ib it or y Act ivit y a ga in st C. a lbica n s-Der ived
DHFR. Trimethoprim (TMP, Table 2) is one of the best
and most selective inhibitors of bacterial DHFR. It
shows, however, low activity against DHFR isolated
from C. albicans (I50 ) 30 µmol/L) and no activity
against whole cell C. albicans. The main aim of this
study was to develop new lead structures for inhibitors
of C. albicans-derived DHFR by studying
Introduction of a methyl group in the 6-position of the
pyrimidine ring also led to an increase in toxicity (A.1.1./
A.2.1.).
Changes in the substitution pattern of the benzyl ring
led to a further increase in activity. Introduction of the
1-naphthyl group reduced the I50 toward C. albicans
DHFR to 0.5 µM. Substitution by methoxy groups led
to a decrease in activity. This was in contrast to the
positive influence of methoxy groups observed so far on
5-benzyl-2,4-diaminopyrimidines when tested against
other species.
The tendency, however, could not be overlooked that
an increase in inhibitory activity against C. albicans-
derived DHFR was always paralleled by an increase in
activity against rat DHFR. A similar tendency was also
observed by Kuyper et al.21 for new 7,8-dialkyl-1,3-
diaminopyrrolo[3,2-f]quinazolines.
Derivatives possessing larger substituents (i.e. spacer
sequences with polar end groups) are supposed to reach
additional binding sites at the DHFR. This was first
shown by Kuyper44 for 2,4-diamino-5-benzylpyrimidines
bearing long chain fatty acids, in which the carboxylate
group could reach and interact with Arg 57 of the active
center of E. coli-derived DHFR as was also found for
methotrexate. Similarly, additional binding sites had
also been postulated for derivatives substituted by a
diaminodiphenylsulfone connected by a spacer of three
methylene groups to the ether oxygen in 4-position of
the benzyl ring of TMP.45-48 One of these derivatives,
K-130,45 together with the derivatives HH-133, HH-135,
HH-136, HH-15449 and the newly synthesized A.1.30.-
A.1.60., is presented in Table 3 together with the
biological activities against the different DHFRs.
The most active derivative is A.1.40., which is com-
parable to the activity of the benzylpyrimidine HH-133.
It is 15 times more active compared with the basic
structure A.1.1. However, note that the activity of
A.1.40., if compared to the activity of HH-133 (possess-
ing only a -CH2- bridge), is not increased and the
selectivity remains the same. The derivatives with a
sulfonamide group, A.1.50., A.1.60., and HH-154, are
more potent than the phenylpropyl-substituted com-
(a) the influence of the distance between the phar-
macophoric diaminopyrimidine group and the aryl ring
[It has been shown that some substituted 2,4-diamino-
quinazolines are strong inhibitors of the target
DHFR.21,43 It could therefore be hypothesized that an
increase in the distance between the ring systems could
be an advantage. Therefore, compounds were synthe-
sized in which the distance between the rings is bridged
by a -CH2CH2- fragment.],
(b) the influence of changes in bridge atoms [Instead
of -CH2CH2-, other bridges containing different het-
eroatoms such as -OCH2-, -NHCH2-, or -CH2NH-
were synthesized.];
(c) the influence of the variation in the 6-position of
the pyrimidine ring like in pyrimethamine;
and (d) the influence of substitution in 4-position of
the benzyl moiety to test if an additional binding site
may be recognized as found for other TMP derivatives
in the case of M. lufu- and E. coli-derived DHFR.44-48
The newly synthesized compounds were tested against
cell-free DHFR derived from C. albicans and from other
species including DHFR from rats to test for selectivity
(Tables 2 and 3).

246 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Otzen et al.
Ta ble 3. Biological Activity (Cell-Free DHFR, I50, µM/L) of 2,4-Diamino-5-benzyl(oxy)pyrimidines with Extended Substituents at the
Benzyl Ring

Inhibitors against Candida albicans
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 247
F igu r e 2. Checkerboard titration of the antifungal activity
of fluconazole in combination with cyclosporin A.
F igu r e 1. C. albicans generation rate in the absence and
presence of DDS and KC 1308, alone and in combination: (0)
control growth rate: 1.56 × 10-4 sec-1; (O) 100 µM DDS, 1.38
×10-4; (4) 50 µM KC 1308, 0.60 × 10-4; (1) 100 µM DDS + 50
against the isolated enzyme is still not fully understood,
but possibly it is due to problems in cell wall perme-
ation. The quinazolines possess a lower molecular
weight and have a more compact structure. This to-
gether with other properties such as lipophilicity and
charge might be reasons responsible for the higher
activity of 2,4-diaminoquinazolines compared to the 2,4-
diamino-5-benzylpyrimidines. The argument of a too
high molecular weight for the latter as the only reason
for its low activity does, however, not explain the low
whole cell activity observed for the low molecular weight
4-aminodiphenyl sulfones.
µM KC 1308, 0.55 × 10-4
.
pound A.1.20., but they are less potent than the deriva-
tives substituted by the phthalimido group and also less
selective.
It can be assumed that A.1.40. could be a promising
new lead structure. It is about 400 times more active
against candida DHFR than the standard compound
TMP. Its inhibitory activity against rat DHFR is lower
in comparison to the other new derivatives possessing
a -OCH2- bridge, i.e. it shows improved selectivity. The
question remains, however, is the ability of the new
derivatives sufficient to permeate the candida cell wall
and to reach the cytoplasm?
Exp er im en ts To Affect th e Gen er a tion Ra tes of
C. a lbica n s Cells. Growth kinetic experiments were
performed analogously to those reported for E. coli and
M. lufu cultures in the presence and absence of inhibi-
tors.41,42 The results were disappointing. Despite the low
I50 values against isolated DHFR of most of the com-
pounds, only A.1.9. (-OCH2- bridge and 1-naphthyl
group, I50 ) 0.43 µM) showed a significant inhibitory
effect (54%) at 50 µM, whereas the more potent deriva-
tive A.1.40. (I50 ) 0.078 µM) showed no effect at this
concentration.
The effect of a combination of a sulfone (DDS) and a
benzylpyrimidine (KC 1308,47 see Table 3) on the growth
rate of C. albicans cultures is shown in Figure 1. The
combination led to an almost complete stop of growth
and shows at least an additive effect. The high concen-
trations needed for inhibition of growth indicates that
the permeability of the C. albicans cell wall seems to
be the decisive step. This barrier prevents the drugs
from reaching the target enzyme in sufficient time and
amounts. This argument is supported by data published
previously by Chan et al.50 These authors developed 2,4-
diaminoquinazolines as inhibitors of C. albicans DHFR.
Their most potent derivatives possess I50 values of
0.008-0.02 µM, similar to our most potent derivatives
HH-133 and A.1.40. with an I50 of 0.047 and 0.078 µM,
respectively, and with similar selectivity indices. But
in contrast to our compounds, most of the quinazolines
are also highly active against C. albicans cell cultures.
This observation showed that the inhibition of DHFR
of C. albicans cultures is possible. The reason for the
low activity of our compounds despite their high activity
Effect of Efflu x P u m p s on In h ibitor y Activity.
Another reason for inactivity or low activity of many
potential drugs in whole cell cultures could be the
presence of efflux pumps. The recent discovery of drug-
efflux-mediated resistance mechanisms in yeasts opens
up a new therapeutic concept. It had been recognized
that C. albicans expresses multidrug efflux transporter
(MET) proteins belonging to two different classes, the
ATP-binding-cassette transporters and the major facili-
tators.51 METs mediate the efflux of a broad range of
compounds, including azoles such as fluconazole. Dif-
ferent MET genes have been identified in C. albicans,
and the upregulation of some of these genes results in
a decrease of the intracellular level of, for example,
fluconazole in azole-resistant cells. Thus, possibly also
the low activity of our DHFR inhibitors is induced by
an efflux system. Recently Sanglard et al.51 investigated
drugs that, by interfering with the MET-mediated active
efflux of antifungals, could potentiate the activity of
azoles. These authors described that cyclosporin, which
had no intrinsic antifungal activity, showed a potent
antifungal effect in combination with fluconazole. In a
specific test system the combination even seemed to be
synergistic. To investigate whether cyclosporin could
also potentiate the activity of our diaminopyrimidines,
we determined the minimum inhibitory concentrations
(MIC values) of potential antifungals toward C. albi-
cans. Furthermore, this test was extended to enable the
investigation of synergistic, additive, or antagonistic
effects of drug combinations by checkerboard titration
of combination MICs. In a first test of the combination
of cyclosporin A with fluconazole, we could confirm the
findings of Sanglard et al. In Figure 2 the downward
convex transition from growth (3+, growth like unin-
hibited control) to nongrowth clearly demonstrates

248 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Otzen et al.
Ta ble 4. MICsa (µg/mL) of 2,4-Diaminopyrimidines against Various Efflux Strains of C. albicans
YEM12
WT
YEM13
BENup
YEM14
WT
YEM15
CDR1CDR2up
YEM30
WT
YEM27
quad deletion
compd
A.1.9.
K-130
K-220
HH-135
HH-136
Ro17-3279
fluconazole
32
>64
>64
>64
>64
16
>64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
32
32
>64
>64
>64
>64
1
1
64
2
e0.25
a
MIC determination was performed in RPMI medium; readings were taken after 48 h.
Ta ble 5. MICsa (µg/mL) of 2,4-Diaminopyrimidines against
Various Efflux Strains of C. glabrata
YEM75
WT
YEM88
YEM216
compd
A.1.9.
K-130
K-220
HH-135
HH-136
Ro17-3279
fluconazole
CgCDR1CgCDR2up pCDR1pCDR2pBEN
16
>64
>64
>64
>64
4
64
>64
>64
>64
>64
32
16
>64
>64
>64
>64
4
4
128
2
a
MIC determination was performed in RPMI medium; readings
were taken after 48 h.
Six selected derivatives, namely A.1.9., HH-135, HH-
136, K-130, K-220, and Ro17-3279 (see Tables 2 and 3
for structures) were tested against these candida strains
and mutants and the results are compared with flu-
conazole results in Table 4.
F igu r e 3. Checkerboard titration of the antifungal activity
of A.1.9. in combination with cyclosporin A.
synergism (MIC of single drugs: cyclosporin > 20 µg/
mL, fluconazole > 5 µg/mL).
For Ro17-3279, showing the lowest I50 in vitro (0.025
µmol/L, 0.009 µg/mL), the MIC against the wild types
YEM12, YEM14, and YEM30 is 16, 64, and 32 µg/mL,
respectively. But when all pumps are knocked out, as
in YEM27, the MIC drops to 1 µg/mL. This means about
a 32-fold increase in effect, whereas all other compounds
(with the exception of A.1.9.) show MICs exceeding 64
µg/mL and no effect is seen when the pumps are
knocked out, despite their similar activity against the
isolated DHFR, e.g. in case of HH-135 and HH-136
(Table 3). This could mean that their inhibitory effect
against the isolated enzyme (their I50 is about 2-20
times higher than that of Ro17-3279) is not sufficient
to influence the growth of C. albicans cultures. However,
considering the small differences in in vitro activity, this
argument does not seem to be very likely. Another factor
that may influence activity differently is the different
rate of drug influx into candida cells in response to
changes in properties and molecular structure. Obvi-
ously, the effect is always a balance between uptake and
efflux. Efflux may be more constant between similar
compounds, since it can be assumed that in a given cell
the pumps tend to operate at a “fixed” rate.
The interpretation of results obtained with A.1.9. is
more problematic. The MIC against the wild-type YEM
12 is 32 µg/mL (120 µmol/L), whereas the MIC of Ro17-
3279 is 16 µg/mL (45 µmol/L), i.e. the molar MICs differ
by a factor of 3 only. In contrast, the I50 values for the
cell free system differ by a factor of about 18. It remains
unclear whether the uptake of A.1.9. is better or
whether it is not as good a substrate as Ro17-3279 for
one of the four pumps that are deleted in YEM27.
Compound A.1.9. is a smaller molecule (MW 266) and
is more lipophilic (log k′ ) 2.14) than Ro17-3279 (MW
355, log k′ ) 1.76). This could point to a higher influx
rate of A.1.9., possibly because of lower molecular
Unfortunately the checkerboard test of the combina-
tion of cyclosporin A with the DHFR inhibitor A.1.9.
showed no potentiating effect (Figure 3). In the tested
concentration range growth levels were almost identical
with those observed in the right-hand column with no
cyclosporin A. This unfavorable finding may be the
result of (a) a different efflux system, which is not
inhibited by cyclosporin A, or (b) an effective barrier
function of the candida cell wall.
To answer this question, a selection of benzylpyrim-
idines was tested against C. albicans mutants possess-
ing either overexpressed efflux pumps or mutants where
certain pumps were deleted. At this stage we got access
to mutants of C. albicans and Candida glabrata pos-
sessing these properties (from Dr. George H. Miller,
Exec. VP, Research and Development, Essential Thera-
peutics Inc., Mountain View, CA).
The strains used (see Tables 4 and 5) are outlined
below.
YEM12 is the wild type of the corresponding YEM13
strain in which the BEN pump is overexpressed. BEN
is a member of the major facilitator family of pumps
whose only known clinical substrate is fluconazole.
YEM14 is the wild type host for YEM15 in which
mutant two coordinately regulated ABC cassette pumps
(CDR1, CDR2) are overexpressed. These pumps are
capable of effluxing not only fluconazole but also all
other clinically used azoles and terbinafine, as well as
a large number of experimental compounds.
YEM30 is the wild type C. albicans for YEM27 in
which four pumps have been deleted, BEN, CDR1,
CDR2, and yet another major facilitator pump, FLU,
which is known to efflux fluconazole in laboratory
strains but is not thought to be a determinant of
clinically observed resistance.

Inhibitors against Candida albicans
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 249
weight. The compound shows about 20 times lower
activity in the isolated system but exhibits similar
inhibitory activity against whole cell candida as Ro17-
3279. This argument would also explain the low whole
cell activity of HH-135 and HH-136, which possess
similar inhibitory activity against DHFR as Ro17-3279.
Both HH derivatives possess higher molecular weight
and more polar substituents. Other derivatives in the
test set, K-130, K-220, A.1.40., and A.1.60., have also
higher MW (MW 450-560) compared to A.1.9. and
Ro17-3279.
ity. These derivatives could be candidates for further
development of antifungal drugs.
Two problems remain unsolved: (i) the still insuf-
ficient selectivity of the new DHFR inhibitors and (ii)
their low activity against whole cell cultures of C.
albicans. It could be shown that the discrepancy be-
tween the degree of inhibition of isolated enzymes and
of whole cell cultures is at least in part due to the
presence of efflux pumps. Other factors as problems in
permeability caused by too high molecular weight, by
charge, and/or by low lipophilicity cannot be excluded.
In light of the negative results with A.1.9. in the
presence of the pump blocker cyclosporin A (Figure 3),
it could be possible that fluconazole and A.1.9. are
transported by different pumps.
Exp er im en ta l Section
1H NMR Sp ectr a . All synthesized derivatives were char-
acterized by 1H NMR at 360 MHz (Bruker A360 spectrometer)
with TMS as internal standard and DMSO-d6 as solvent. NMR
data of newly synthesized compounds are listed in the Sup-
porting Information.
In addition to the studies on C. albicans, also C.
glabrata mutants were used to study the effect of pumps
on the activity of the folate inhibitors (Table 5).
Strain YEM75 is a wild type and YEM88 represents
the glabrata version of overexpressed CDR1CDR2;
YEM216 is the glabrata version in which three of the
pumps are deleted. The overexpression of the CDR1CDR2
pumps led to an 8-fold increase in MICs of Ro17-3279,
but deletion of pump activity did not change the MIC.
This seems paradoxical at first glance. It could mean
that with overexpression Ro17-3279 can be a substrate
of the CDR1CDR2 pumps, but that the basal level of
expression present in YEM75 does not affect MICs,
because there is no further effect when the pumps are
knocked out. Thus, one may conclude that CDR1CDR2
is a potential source of resistance but does not affect
basal activity against C. glabrata. This may support the
tentative explanation why A.1.9. acts slightly better
against C. glabrata if it indeed penetrates better than
the other five compounds. It can of course not be
excluded that A.1.9. is a better inhibitor of the C.
glabrata DHFR enzyme.
C, H, N An a lyses were done by Mikroanalytisches Labor
Ilse Beetz, Kronach, Germany.
Gen er a l P r oced u r e for t h e Syn t h esis of Ben zyloxy-
Su bstitu ted 2,4-Dia m in op yr im id in es (Ser ies A). Some
indicated compounds had to be purified by column chroma-
tography according to a method developed in our laborato-
ries: Crude final products were suspended with silica gel in
dichloromethane and dried, and the mixture was placed on
top of the column [length 10-15 cm, 2 cm diameter; mobile
phase, toluene/2-propanol/freshly distilled diethylamine 60/30/
10 (by vol)].
5-Ben zyloxy-2,4-d ia m in op yr im id in e (A.1.1.). To a solu-
tion of sodium ethylate (140 mg Na in 5-20 mL ethanol) was
added 400 mg (2 mmol) of 2,4-diamino-5-hydroxypyrimidine‚
2HCl and the solution warmed to 60 °C for 30 min. Thereafter
253 mg (2 mmol) of benzyl chloride was added and the mixture
heated to reflux for 48 h. The solvent was then evaporated.
The dry mass was suspended in dichloromethane, 100 mg of
silica gel added, and the solvent again evaporated. The mixture
was purified by column chromatography as described above.
The reaction product was recrystallized two times in ethanol
followed by recrystallization in water: yield 150 mg (35%), mp
128 °C. Anal. (C11H12N4O): C, H, N.
A comparison of the activity of Ro17-3279 against the
deletion strains YEM27 (C. albicans, MIC ) 1 µg/mL)
and YEM216 (C. glabrata, MIC ) 4 µg/mL) indicates
that either the uptake of this compound by C. albicans
is better than by C. glabrata or it is less active against
the DHFR enzyme of the latter species.
5-(2-Ch lor oben zyloxy)-2,4-d ia m in op yr im id in e (A.1.2.).
Instead of benzyl chloride, 413 mg (2 mmol) of 2-chlorobenzyl
bromide was used: yield 400 mg (79%), mp 164 °C (MeOH).
Anal. (C11H11ClN4O): C, H, N.
2,4-Diam in o-5-(4-m eth oxyben zyloxy)pyr im idin e (A.1.6.).
4-Methoxybenzyl chloride (313 mg, 2 mmol) was used: yield
120 mg (24%), mp 156 °C (EtOH). Anal. (C12H14N4O2): C, H,
N.
Con clu sion
2,4-D ia m in o -5-(1-n a p h t h y lm e t h y lo x y )p y r im id in e
(A.1.9.). 1-Chloromethylnaphthalene (440 mg, 2.5 mmol) was
New highly active inhibitors of C. albicans-derived
SYN and DHFR were synthesized. For the sulfones,
inhibitors of SYN, a straightforward QSAR could be
derived. Remarkably, from the results of this QSAR it
can be concluded that a great similarity seems to exist
between the active site of SYN of various microorgan-
isms, including eukariotic C. albicans. Therefore, resist-
ance does not seem to be due to changes in the active
site. In contrast, a great dissimilarity was observed for
the sensitivity of DHFR of prokaryotic microorganisms
on one hand and DHFR derived from eukaryotic cells
such as C. albicans and from rats on the other.
Replacement of the trimethoprim CH2 group by other
bridge atoms as well as introduction of longer spacer
groups bearing polar end groups led in most cases to
derivatives with increased inhibitory activity. The most
active compounds against DHFR (A.1.40. and HH-133,
-135, -136) possess about 400 times lower I50 values as
compared to trimethoprim and also interesting selectiv-
used: yield 400 mg (60%), mp 190 °C (EtOH). Anal. (C15H14
N4O): C, H, N.
-
5-(4-Br om o-1-n a p h th ylm eth yloxy)-2,4-d ia m in op yr im i-
d in e (A.1.10.). 4-Bromo-1-bromomethylnaphthalene (602 mg,
2 mmol) was used: yield 200 mg (29%), mp 185 °C (EtOH).
Anal. (C15H13BrN4O): C, H, N.
For the following derivatives, 140 mg of Na (6 mmol) and 9
mL of ethylene glycol monomethyl ether were used instead of
sodium ethylate, and a small amount of KI was added.
2,4-Dia m in o-5-(3,4-d ic h lor ob e n zyloxy)p yr im id in e
(A.1.4.). 3,4-Dichlorobenzyl chloride (390 mg, 2 mmol) was
used. After 3 days stirring at 20-22 °C, the reaction product
was isolated (addition of 10 mL of H2O) and recrystallized in
methanol: yield 250 mg (44%), mp 163 °C. Anal. (C11H10
Cl2N4O): C, H, N.
-
5-(4-B e n zy lo x yb e n zy loxy )-2,4-d ia m in op y r im id in e
(A.1.8.). 2,4-Diamino-5-hydroxypyrimidine‚2HCl (300 mg, 1.5
mmol) was reacted with 320 mg (1.4 mmol) of 4-benzyloxy-
benzyl chloride under conditions as for A.1.4.: yield 320 mg
(54%), mp 142 °C (EtOH). Anal. (C10H18N4O2): C, H, N.

250 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Otzen et al.
2,4-Dia m in o-5-(2,6-d ic h lor ob e n zyloxy)p yr im id in e
(A.1.5.). The reaction conditions for this derivative were
slightly changed. 2,4-Diamino-5-hydroxypyrimidine‚2HCl (400
mg, 2 mmol) and 482 mg (2 mmol) of 2,6-dichlorobenzyl
bromide were mixed with 830 mg (60 mmol) of potassium
carbonate in 15 mL of acetone, and a small amount of KI was
added. The mixture was refluxed for 72 h. The purification of
the mixture was done by column chromatography as de-
instead of water: yield 36%, mp 105 °C. Following the general
procedure, the product is reacted with 4-hydroxybenzaldehyde
to the ether. The aldehyde is then reduced to the corresponding
alcohol and finally transformed to the benzyl bromide by
treatment with PBr3 as described above to obtain the raw
product 4-[3-(4-tolylsulfonamido)propoxy]benzyl bromide: yield
67%.
2,4-Dia m in o-5-{3-m et h oxy-4-[3-(4-t olylsu lfon a m id o)-
p r op oxy]ben zyloxy}p yr im id in e (A.1.60.). 2,4-Diamino-5-
hydroxypyrimidine‚2HCl (200 mg, 1 mmol) was mixed with
450 mg (1 mmol) of 3-methoxy-4-[3-(4-tolylsulfonamido)pro-
poxy]benzyl bromide in 10 mL of ethanolate: yield 90 mg
(18%), mp 124 °C (EtOH). Anal. (C22H27N5O5S): C, H, N.
The 3-methoxy-4-[3-(4-tolylsulfonamido)propoxy]benzyl bro-
mide was synthesized analogously to the procedure given
above for the 4-[3-(4-tolylsulfonamido)propoxy]benzyl bro-
mide: yield 64% raw product.
Gen er a l P r oced u r e for th e Syn th esis of Ben zylid en e-
a m in o-Su bstitu ted 2,4-Dia m in op yr im id in es (Ser ies B,
P r ep r od u cts for Ser ies C). 5-Ben zylid en ea m in o-2,4-d i-
a m in op yr im id in e (B.1.1.). Similar to the procedure de-
scribed by Zhang et al.,33 equimolar amounts of 2,4,5-tri-
aminopyrimidine (b.1.3.) were mixed with the corresponding
benzaldehyde and heated in ethanol under reflux temperature
for about 12 h. After cooling in a refrigerator, the precipitate
is isolated and recrystallized in 70-99% ethanol. The com-
pounds are characterized by 1H NMR and C, H, N analysis.
Synthesis of B.1.1.: 250 mg of b.1.3. and 233 mg of benz-
aldehyde in 4 mL of ethanol: yield 250 mg (59%), mp 168 °C.
Anal. (C11H11N5): C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of th e 5-Ben zyl-
a m in o-2,4-d ia m in op yr im id in es (Ser ies C). Similar to the
method used by Zhang,33 alkali borohydrides were used for
reduction of the B-derivatives. However, different alkali boro-
hydrides were used, a larger excess was used, and the time
for reaction was prolonged. Also the purification procedure had
to be modified. The substituted 5-benzylideneamino-2,4-di-
aminopyrimidines (series B) (0.76-1.2 mmol) were used with
the given amounts of alkali borohydrides and heated under
reflux for 3 h. After cooling in a refrigerator, the precipitate
was recrystallized in 70-99% ethanol. 1H NMR (Table 8,
Supporting Information) and C, H, N analysis were used for
characterization.
scribed: yield 150 mg (26%), mp 125 °C (EtOH). Anal. (C11H10
Cl2N4O): C, H, N.
-
5-(4-t er t -B u t y lb e n zy lo x y )-2,4-d ia m in o p y r im id in e
(A.1.12.). 4-tert-Butylbenzyl bromide (456 mg, 2 mmol) was
used: yield 200 mg (37%), mp 157 °C (EtOH). Anal. (C15
20N4O): C, H, N.
All following derivatives of series A were also reacted with
-
H
sodium/ethanol with the addition of a small amount of KI.
Some were obtained only in low yield and had to be purified
by column chromatography. All were identified by NMR and
mostly also by C, H, N analysis.
2,4-Dia m in o-5-[3-m eth oxy-4-(3-p h en ylp r op oxy)ben zyl-
oxy]p yr im id in e (A.1.20). 2,4-Diamino-5-hydroxypyrimidine‚
2HCl (356 mg, 1.7 mmol) was reacted with 600 mg (1.7 mmol)
of 3-methoxy-4-(3-phenylpropoxy)benzyl bromide (Scheme 4).
The latter was obtained by reacting 0.055 mol of NaH in DMF
with 0.05 mol of 4-hydroxy-3-methoxybenzaldehyde and 0.05
mol of 1-bromo-3-phenylpropane in the presence of a small
amount of KI. The benzaldehyde was reduced to the corre-
sponding alcohol with sodium borohydride. The corresponding
benzyl bromide was obtained by dissolving 1.5 mmol of the
alcohol in dichloromethane. To the solution was added 0.5
mmol of PBr3 and the mixture stirred for 1 h. Ice water was
added and the acid removed by washing. The benzyl bromide
was immediately used: yield 200 mg (29%), mp 148 °C (EtOH).
Anal. (C21H24N4O3): C, H, N.
N-{3-[4-(2,4-Diam in opyr im idin -5-yloxym eth yl)ph en oxy]-
p r op yl}p h th a lim id e (A.1.30.). 2,4-Diamino-5-hydroxypyrim-
idine‚2HCl (532 mg, 2.7 mmol) was mixed with 1.0 g (2.7
mmol) of N-[3-(4-bromomethylphenoxy)propyl]phthalimide in
35 mL of ethanolate: yield 350 mg (31%), mp 176 °C (EtOH).
Anal. (C22H21N5O4): C, H, N.
The N-[3-(4-bromomethylphenoxy)propyl]phthalimide was
synthesized by the following reaction steps: 0.021 mol of NaH
was suspended in 60 mL of DMF, and 0.019 mol of 4-methyl-
phenol was added under stirring. After the reaction had
stopped, the corresponding substituted phthalimide (N-(3-
bromopropyl)phthalimide) was added together with a small
amount of KI and heated to reflux for16 h. The reaction
mixture was evaporated to dryness and taken up in dichloro-
methane/1.5 N NaOH, followed by extraction with dichloro-
methane twice. N-[3-(4-methylphenoxy)propyl]phthalimide (3.4
mmol) was then reacted with 3.7 mmol of NBS (N-bromo-
succinimide) in 10 mL of CCl4 under reflux and UV-light and
with repeated addition of Me2C(CN)-NdN-C(CN)Me2: yield
79%, mp 90 °C.
5-Ben zyla m in o-2,4-d ia m in op yr im id in e (C.1.1.). 5-Ben-
zylidenamino-2,4-diaminopyrimidine (250 mg, 1.2 mmol) dis-
solved in 8 mL of ethanol was reduced with 250 mg (4.8 mmol)
of potassium borohydride: yield 128 mg (51%), mp 146 °C
(EtOH). Anal. (C11H13N5): C, H, N.
5-(2-C h lo r o b e n zy la m in o )-2,4-d ia m in o p y r im id in e
(C.1.2.). 5-(2-Chlorobenzylideneamino)-2,4-diaminopyrimidine
(249 mg, 1.4 mmol) dissolved in 9 mL of ethanol was reduced
with 122 mg (5.6 mmol) of lithium borohydride: yield 300 mg
(85%), mp 231 °C (EtOH). Anal. (C11H12ClN5): C, H, N.
5-(3-C h lo r o b e n zy la m in o )-2,4-d ia m in o p y r im id in e
(C.1.3.). 5-(3-Chlorobenzylideneamino)-2,4-diaminopyrimidine
(200 mg, 0.9 mmol) dissolved in 5 mL of ethanol was reduced
with 80 mg (3.6 mmol) of lithium borohydride: yield 80 mg;
(34%), mp 108 °C (EtOH). Anal. (C11H12ClN5). C, H, N.
2,4-Dia m in o-5-(3,4-d ich lor ob en zyla m in o)p yr im id in e
(C.1.4.). 2,4-Diamino-5-(3,4-dichlorobenzylideneamino)pyrim-
idine (271 mg, 0.9 mmol) dissolved in 6 mL of ethanol was
reduced with 206 mg (38 mmol) of potassium borohydride:
N-{4-[4-(2,4-Diam in opyr im idin -5-yloxym eth yl)ph en oxy]-
bu tyl}p h th a lim id e (A.1.40.). 2,4-diamino-5-hydroxypyrimi-
dine‚2HCl (418 mg, 2.1 mmol) was mixed with 800 mg (2.1
mmol) of N-[4-(4-bromomethylphenoxy)butyl]phthalimide in
20 mL of ethanolate: yield 200 mg (22%), mp 157 °C (EtOH).
Anal. (C23H23N5O4): C, H, N.
The N-[4-(4-bromomethylphenoxy)butyl]phthalimide was
obtained following the procedure described for N-[3-(4-bromo-
methylphenoxy)propyl]phthalimide: yield 1.5 g (78%), mp 107
°C.
yield 150 mg (55%), mp 186 °C (70% EtOH). Anal. (C11H11
Cl2N5): C, H, N.
-
2,4-Dia m in o-5-{4-[3-(4-t olylsu lfon a m id o)p r op oxy]-
ben zyloxy}pyr im idin e (A.1.50). 2,4-Diamino-5-hydroxypyrim-
idine‚2HCl (200 mg, 1 mmol) was mixed with 400 mg (1 mmol)
of 4-[3-(4-tolylsulfonamido)propoxy]benzyl bromide in 10 mL
of ethanolate: yield 100 mg (22%), mp 125 °C (EtOH). Anal.
(C21H25N5O4S): C, H, N.
2,4-Dia m in o-5-(4-m e t h oxyb e n zyla m in o)p yr im id in e
(C.1.6.). 2,4-Diamino-5-(4-methoxybenzylideneamino)pyrimi-
dine (300 mg, 1.2 mmol) dissolved in 7 mL of ethanol was
reduced with 258 mg (4.8 mmol) of potassium borohydride:
yield 200 mg (66%), mp 164 °C (EtOH). Anal. (C12H15N5O):
C, H, N.
The 4-[3-(4-tolylsulfonamido)propoxy]benzyl bromide was
synthesized by the following steps. First, N-(3-bromopropyl)-
4-tolylsulfonamide was synthesized using the procedure of
Henning49 with the exception that ethanol is used as solvent
2,4-Dia m in o-5-(3,4-d im e t h oxyb e n zyla m in o)p yr im i-
d in e (C.1.7.). 2,4-Diamino-5-(3,4-dimethoxybenzylidene-
amino)pyrimidine (330 mg, 1.2 mmol) dissolved in 8 mL of
ethanol was reduced with 550 mg (9.7 mmol) of potassium

Inhibitors against Candida albicans
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 251
borohydride: yield 220 mg (66%), mp 190 °C (EtOH). Anal.
(C13H15N5O2): C, H, N.
G.2.1. 2-Am in o-4-ch lor o-6-m eth yl-5-(2-p h en yleth yl)p yr -
im id in e a n d 2-Am in o-4-ch lor o-6-m eth yl-5-(3-p h en ylp r o-
p yl)p yr im id in e. R-Substituted acetylacetic acid esters54 (Ac-
CHR-COOEt, R ) (CH2)2Ph, (CH2)3Ph) were heated under
reflux for 4 h with guanidine carbonate in ethanol to obtain
2-imido-6-methyl-5-(2-phenylethyl)uracil and 2-imido-4-meth-
yl-5-(3-phenylpropyl)uracil: yield ∼27%, mp 226 and 230 °C,
respectively.
Following the procedure of Roth et al.,55 0.025 mol of the
imido product was heated to reflux with 100 mL of POCl3 for
3 h. The main fraction of POCl3 was evaporated under vacuum,
and the residue was treated with ice and extracted with
dichloromethane. The solution was washed twice with NaHCO3/
H2O and then dried over Na2SO4 and filtered. The solvent was
evaporated, and 2-amino-4-chloro-6-methyl-5-(2-phenylethyl)-
pyrimidine or the corresponding 2-amino-4-chloro-6-methyl-
5-(3-phenylpropyl)pyrimidine was obtained as oily products:
yield 34% and 78%, respectively. The products were im-
mediately used for synthesis of F .2.1. and G.2.1.: The corre-
sponding preproducts were treated in an autoclave in ethanolic
ammonia for 10 h at 170 °C. The reaction mixture was
evaporated to dryness, taken up with 3 N NaOH, and extracted
with dichloromethane; the organic phase was dried with
Na2SO4, filtered, and evaporated. The residue was recrystal-
lized in ethanol.
2,4-Dia m in o-5-(1-n a p h t h ylm et h yla m in o)p yr im id in e
(C.1.9.). 2,4-Diamino-5-(1-naphthylideneamino)pyrimidine (200
mg, 0.7 mmol) dissolved in 6 mL of ethanol was reduced with
328 mg (6 mmol) of potassium borohydride: yield 100 mg
(50%), mp 175 °C (EtOH). Anal. (C15H15N5): C, H, N.
2,4-Dia m in o-5-(2-n a p h t h ylm et h yla m in o)p yr im id in e
(C.1.11.). 2,4-Diamino-5-(2-naphthylideneamino)pyrimidine
(200 mg, 0.7 mmol) dissolved in 5 mL of ethanol was reduced
with 132 mg (6 mmol) of lithium borohydride: yield 150 mg
(74%), mp 214 °C (70% EtOH). Anal. (C15H15N5): C, H, N.
Syn th esis of 5-(Su bstitu ted -ben zyl)-2,4-d ia m in op yr im -
id in es (Ser ies D). The synthesis was performed using the
procedure of Kompis et al.31 and according to the modification
of Hachtel.32 The 2-substituted morpholinoacrylonitriles were
prepared in DMSO with solid sodium methylate. Used amounts
were between 0.03 and 0.05 mol. In all cases the product was
a red oil and was directly used to prepare the 2-substituted
anilinoacrylonitriles as described by Hachtel32 in a 2-propanol/
HCl solution with aniline and with heating to reflux. The
2-substituted 3-anilinoacrylonitriles were mixed with solid
sodium methanolate and guanidine hydrochloride and heated
in ethanol for 36 h under reflux condition.
5-Ben zyl-2,4-d ia m in op yr im id in e (D.1.1.): yield 2.1 g
(79%), mp 192 °C (EtOH).
2,4-Dia m in o-5-(1-n a p h th ylm eth yl)p yr im id in e (D.1.9.):
yield 3.2 g (90%), mp 227 °C (EtOH). Anal. (C15H14N4): C, H,
N.
2,4-Dia m in o-5-(2-n a p h th ylm eth yl)p yr im id in e (D.1.11.):
yield 1.3 g (74%), mp 226 °C (EtOH). Anal. (C15H14N4): C, H,
N.
Syn th esis of 2,4-Dia m in o-5-styr ylp yr im id in e (E.1.1.).
The synthesis (Scheme 3) was performed in accordance with
a synthesis scheme given by Kompis.52 Procedures for the
different steps were taken from the literature and modified if
necessary.
5-Cyano-2,4-diaminopyrimidine was prepared according to
the method of Huber53 and reduced to the corresponding 2,4-
diamino-5-formylpyrimidine by H2/Pd/C at 4.2 bar in HCl/
water. Following the procedure of Baker,34 225 mg of 2,4-
diamino-5-formylpyrimidine was heated to 100 °C in 1.8 mL
of acetanhydride and 2 mL of DMF under stirring. After
cooling to -5 °C the precipitate was recrystallized in DMF/
toluene: yield 250 mg (69%) 2,4-diacetylamino-5-formyl-
pyrimidine.
2,4-Dia cetyla m in o-5-styr ylp yr im id in e. A Wittig reaction
was applied as described by Baker31 for the synthesis of similar
compounds. Some modifications were introduced. 2,4-Diacetyl-
amino-5-formylpyrimidine (100 mg) was mixed with 175 mg
of benzyltriphenylphosphonium chloride in 1 mL of DMF.
Within 10 min, 56 mg of DBN was added and the solution color
was changing to dark red. After 10 min, 10 mL of toluene was
added and the solution was stirred for 12 h. The solution was
evaporated under reduced pressure. The oily residue was
dissolved in acetonitrile. The crystals formed were recrystal-
lized in DMF: yield 80 mg (60%).
2,4-Dia m in o-5-st yr ylp yr im id in e (E .1.1.). 2,4-Diacetyl-
amino-5-styrylpyrimidine (1.4 g) was heated in 50 mL of 3 N
methanolic potassium hydroxyde solution (8.4 g of KOH/50 mL
of methanol) for 2 h under reflux. The solvent was evaporated
under vacuum and the residue taken up in water. The
remaining yellow powder was recrystallized in ethanol: yield
850 mg (85%), mp 179 °C (EtOH). Anal. (C12H12N4): C, H, N.
2,4-Diam in o-5-(2-ph en yleth yl)pyr im idin e (F.1.1.). E.1.1.
(481 mg, 2.3 mmol) and 68 mg of 10% Pd/C were suspended
in 40 mL of ethanol and hydrogenated under 1.5 bar for 2.5 h.
The catalyst was filtered off and the solvent evaporated. The
residue was suspended in water under vigorous stirring and
the pH changed with ammonia. The precipitate was recrystal-
lized in water: yield 390 mg (80%), mp 139 °C. Anal.
(C12H14N4): C, H, N.
2,4-Dia m in o-6-m e t h yl-5-(2-p h e n yle t h yl)p yr im id in e
(F .2.1.). 2-Amino-4-chloro-6-methyl-5-(2-phenylethyl)pyrimi-
dine (1.0 g) was autoclaved in 100 mL of NH3/EtOH: yield
200 mg (23%), mp 141 °C (EtOH). Anal. (C13H16N4): C, H, N.
2,4-Dia m in o-6-m et h yl-5-(3-p h en ylp r op yl)p yr im id in e
(G.2.1.). 2-Amino-4-chloro-6-methyl-5-(3-phenylpropyl)pyrimi-
dine (2.4 g) was heated in an autoclave in 160 mL of NH3/
EtOH: yield 500 mg (23%), mp 130 °C (EtOH). Anal.
(C14H18N4): C, H, N.
5-An ilin om eth yl-2,4-d ia m in op yr im id in e (H.1.1.). Fol-
lowing the procedure of Tieckelmann et al.,56 500 mg of 2,4-
diamino-5-formylpyrimidine were suspended in 20 mL of water
and treated with a solution of 100 mg of sodium borohydride
as reported. For the following steps the synthetic route of
Weinstock29 was followed but with several modifications. The
obtained 2,4-diamino-5-hydroxymethylpyrimidine was heated
for 3 h in 30% HBr/acetic acid to reflux. The mixture was
evaporated to dryness and, because of instability, the 5-bromo-
methyl-2,4-diamino-pyrimidine‚HBr intermediate (314 mg)
was further reacted with 205 mg of freshly distilled aniline in
10 mL of dry DMF; after 6 h DMF was removed by distillation
under vacuum. The residue was extracted with 1.5 N NaOH/
dichloromethane. The organic phases were combined and dried
over Na2SO4, and again the solvent was removed. The oily
residue was treated with a small amount of diethyl ether and
kept at -8 °C. Crystals were recrystallized in ethanol: yield
45 mg (19%), mp 165 °C (EtOH). Anal. (C11H13N5): C, H, N.
2,4-Dia m in o -5-(2-n a p h t h ylt h iom e t h yl)p y r im id in e
(J .1.11.). NaH (206 mg) was suspended in 20 mL of DMF
and 1.058 g of thionaphthol added. After gas development had
stopped, 620 mg of 5-bromomethyl-2,4-diaminopyrimidine‚HBr
(see above) was added. The mixture was stirred at 20-22 °C
for 1 week. The solvent was removed by distillation under
vacuum. The residue was treated with 3 N NaOH and
extracted with dichloromethane. The organic phases were
combined, reduced to a volume of about 5 mL, a small amount
of silica gel was added, and the mixture evaporated and put
on a column for chromatography as described above: yield ca.
15 mg (2.4%), mp 130 °C (EtOH). Anal. (C15H14N4S): C, H, N.
Syn t h esis of 5-Ben zyloxy-6-m et h yl-2,4-d ia m in op yr -
im id in es (A.2.1. a n d A.2.4.). 2,4-Dia m in o-6-m et h ylp yr -
im id in e 5-Hyd r ogen Su lfa te. The synthesis of 2,4-diamino-
6-methylpyrimidine 5-hydrogen sulfate was performed as
described by Hull36 and Hurst.37 The intermediate was then
hydrolyzed in 8 mL of 5 N HCl to the 5-OH derivative. After
heating, the reaction mixture was then titrated to pH 6 with
11 N NaOH and NH4Cl to obtain the 2,4-diamino-5-hydroxy-
6-methylpyrimidine hydrochloride.
Gen er a l P r oced u r e for th e Syn th esis of 5-P h en yla lk yl-
su bstitu ted 6-m eth yl-2,4-d ia m in op yr im id in es F .2.1. a n d

252 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Otzen et al.
5-Ben zyloxy-2,4-d ia m in o-6-m eth ylp yr im id in e (A.2.1.).
2,4-Diamino-5-hydroxy-6-methylpyrimidine‚HCl (600 mg) and
468 mg of benzyl chloride were mixed with 986 mg of K2CO3
in 30 mL of acetone. After the reaction was finished, the
solvent was removed by distillation under vacuum and the
residue treated with diethyl ether. Crystals were recrystallized
in H2O/EtOH: yield 580 mg (74%), mp 135 °C (10% EtOH).
Anal. (C12H14N4O): C, H, N.
2,4-Dia m in o-5-(3,4-d ich lor oben zyloxy)-6-m eth ylp yr im -
id in e (A.2.4.). 2,4-Diamino-5-hydroxy-6-methylpyrimidine‚
HCl (400 mg) was dissolved in alcoholate made from 150 mg
of sodium and 10 mL of ethylene glycol monomethyl ether,
and 430 mg of 3,4-dichlorobenzyl chloride and a small amount
of KI were added. The mixture was stirred at 20-22 °C for 48
h, then water was added and the precipitate recrystallized
Samples were taken over 15 h, and the number of cells counted
in a Coulter counter using a capillary equipped with a 38 µm
orifice.
Lin ea r Regr ession An a lysis a n d P r in cip a l Com p on en t
An a lysis. Both types of analysis were performed using
standard in-house computer programs (K.J .S.).
Ack n ow led gm en t. The authors are grateful to Dr.
George H. Miller and Mrs. K. Lolans of Exec. VP
Research and Development, Essential Therapeutics Inc.,
Mountain View, CA, for performing the MIC determina-
tions on their C. albicans and C. glabrata strains with
overexpressed and deleted efflux pumps.
Su p p or tin g In for m a tion Ava ila ble: 1NMR data (chemi-
cal shifts, ppm) and shift assignment of substituted 2,4-
diaminopyrimidines (series A-J ). This material is available
free of charge via the Internet at http://pubs.acs.org.
from ethanol: yield 250 mg (38%), mp 181 °C. Anal. (C12H12
Cl2N4O): C, H, N.
-
Ro12-6099 a n d Ro17-3279 were a generous gift of Hoff-
mann-LaRoche, Basel, Switzerland. The derivatives HH-133,
HH-135, HH-136, a n d HH-154 of Table 3 were synthesized
as described by Henning.49
Refer en ces
(1) Sternberg, S. The emerging fungal threat. Science 1994, 266,
1632-1634.
(2) Edwards, J . F. Invasive candida infections. Evolution of a fungal
pathogen. N. Engl. J . Med. 1991, 324, 1060-1062.
(3) Horn, R.; Wong, B.; Kiehn, T. E.; Armstrong, D. Fungemia in a
cancer hospital: Changing frequency, earlier onset, and results
of therapy. Rev. Infect. Dis. 1985, 7, 646-655.
(4) Mutschler, E. Arzneimittelwirkungen; Wissenschaftliche Ver-
lagsgesellschaft: Stuttgart, Germany, 1991.
(5) Leoung, G.; Mills, J ., Eds. Opportunistic Infections in Patients
with the Acquired Immunodeficiency Syndrome; Marcel Dek-
ker: New York, 1989.
Syn th esis of Su bstitu ted 4-Am in od ip h en yl Su lfon es.
The synthesis has been described elsewhere.40
Biology. For the preparation of partially purified enzyme
extracts with dihydropteroate synthase (SYN) activity from
E. coli ATCC 11775, M. lufu L209, and P. berghei, see Coats
et al.,40 Wiese et al.,39 and Bartels and Bock.38 This procedure
was also used to obtain partially purified extracts from
Candida albicans ATCC 11651.
In h ibition Stu d ies of SYN by Su lfon es. The rate of folate
production with respect to time was determined by periodic
sampling of the incubation mixtures and quenching of the
reaction by trichloroacetic acid. Amounts of folate produced
were determined using folate requiring Streptococcus faecium.
The incubation temperature was 37 and 31 °C for E. coli and
M. lufu, respectively. For details, see Coats et al.;40 deviating
from this procedure the inhibitory activity against P. berghei-
and C. albicans-derived SYN was determined by HPLC
analysis of the produced amount of dihydropteroic acid after
oxidation to pteroic acid. For details, see Wiese et al.39
P r ep a r a tion of P a r tia lly P u r ified En zym e Extr a cts
w ith Dih yd r ofola te Red u cta se (DHF R) Activity. The
purification was performed as described in detail38 for E. coli
starting with ion exchange chromatography on a DEAE-
Sepharose Cl-6B column with a salt gradient, followed by
hydrophobic-interaction chromatography on Phenyl-Sepharose
Cl-4B and size-exclusion chromatography on Ultrogel AcA 54.
In h ibition of DHF R. The DHFR activity was assayed in
0.1 M Tris buffer, pH 7.24, by monitoring the decrease in
absorbance photometrically at 340 nm as a function of time.
After incubation of the DHFR with 0.1 mM NADPH and
various amounts of inhibitor for 5 min at 25 °C, the reaction
was started by adding 0.03 mM dihydrofolate. I50 values were
calculated by nonlinear regression analysis as the concentra-
tion of inhibitor required for 50% decrease in velocity of the
enzyme reaction.45,47
(6) Georgopapadakou, N. H. Effects of drugs on lipids and mem-
brane integrity of fungi, In Perspectives in Antiinfective Therapy;
J ackson, G. G., Schlumberger, H. D., Zeilers, H.-J ., Eds.; Bayer
Centenary Symp., Washington, DC, 1988; Springer-Verlag:
Heidelberg, Germany, 1989; pp 100-111.
(7) Ryder, N. S. Mechanism of action and biochemical selectivity of
allylamine antimycotic agents. Ann. N. Y. Acad. Sci. 1988, 544,
208-220.
(8) Albert, A. Selective Toxicity; J ohn Wiley: New York, 1979.
(9) Salter, A. J . Trimethoprim-sulfamethoxazole: An assessment
of more than 12 years use. Rev. Infect. Dis. 1982, 4, 196-236.
(10) Hitchings, G. H. The metabolism of plasmodia and the chemo-
therapy of malaria infections. In Tropical Medicine from Ro-
mance to Reality; Wood, C., Ed., Academic Press: London, 1978;
pp 79-98.
(11) Seydel, J . K.; Wiese, M.; Kansy, M.; Schaper, K.-J .; Walter, R.
Tropical diseases, recent drug developments with special em-
phasis on antimalarial and antileprosy drugs. In Trends in Drug
Research; Timmerman, H., Ed., Elsevier: Amsterdam, 1990;
Vol.13, pp 109-131.
(12) Wiese, M.; Kansy, M.; Kunz, B.; Schaper, K.-J .; Seydel, J . K.;
Walter, R.; Chandra, S. New inhibitors of plasmodial folate
synthesissComparison of cell-free, whole cell and in vivo activi-
ties for single drugs and drug combinations. Proceedings in
Chemistry and Biology of Pteridines 1989; Curtius, H.-C., Ghisla,
S., Blau, N., Eds.; Walter de Gruyter & Co,: Berlin, New York,
1990; pp 535-546.
(13) Falcon, J .; Allegra, C. J .; Kovacs, J . A.; O’Neil, D.; Ogata-
Arakaki, D.; Feuerstein, I.; Polis, M.; Davey, R.; Lane, H. C.;
LaFon, S.; Rogers, M.; Zunich, K.; Zurlo, J .; Tuazon, C.; Parenti,
D.; Simon, G.; Masur, H. Piritexim with leucovorin for the
treatment of Pneumocystis carinii pneumonia in AIDS patients.
Clin. Res. 1990, 38, 361 A.
In Vitr o Wh ole Cell An tifu n ga l Activity Assa y. Whole
cell antifungal activity was obtained by determination of the
minimum inhibitory concentrations (MIC) using the serial
dilution method. The MIC was defined as the lowest concen-
tration of test compounds that resulted in a culture with
turbidity e20% of the value of a control culture. The strains
presented in Tables 4 and 5 were grown in RPMI medium,
whereas results shown in Figures 2 and 3 (C. albicans ATCC
11651) were obtained (at 35 °C, pH 7) in vitamin-free yeast
base medium (Difco) supplemented with 1.5 g of asparagine,
3 mg of thiaminiumdichloride, 0.05 mg of biotin, and 0.2 mL
of Tween 80 per liter. Here readings were taken after ∼30 h.
(14) Kovacs, J . A.; Allegra, C. J .; Masur, H. Characterization of
dihydrofolate reductase of Pneumocystis carinii and Toxoplasma
gondii. Exp. Parasitol. 1990, 71, 60-68.
(15) Baccanari, D. P.; Tansik, R. L.; J oyner, S. S.; Fling, M. E.; Smith,
P. L.; Freisheim, J . H. Characterization of Candida albicans
dihydrofolate reductase. J . Biol. Chem. 1989, 264, 1100-1107.
(16) Rosowsky, A.; Hynes, J . R.; Queener, S. F. Structure-activity
and structure selectivity studies on diaminoquinazolines and
other inhibitors of Pneumocystis carinii and Toxoplasma gondii
dihydrofolate reductase. Antimicrob. Agents Chemother. 1995,
339, 79-86.
(17) Gangjee, A.; Vasudevan, A.; Queener, S. F.; Kisliuk, R. L. 2,4-
Diamino-5-deaza-6-substituted pyrido[2,3-d]pyrimidine anti-
folates as potent and selective nonclassical inhibitors of dihy-
drofolate reductase. J . Med. Chem. 1996, 39, 1438-1446.
(18) Gangjee, A.; Vasudevan, A.; Queener, S. F. Synthesis and
biological evaluation of nonclassical 2,4-diamino-5-methylpyrido-
[2,3-d]pyrimidines with novel side chain substituents as poten-
tial inhibitors of dihydrofolate reductase. J . Med. Chem. 1997,
40, 479-485.
Furthermore, the inhibitory effect on C. albicans ATCC
11651 whole cell cultures was determined quantitatively by a
yeast cell generation technique. The generation kinetics were
determined by a Coulter counter technique analogously as
described in previous papers for E. coli41 and mycobacteria.42

Inhibitors against Candida albicans
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 253
(19) Chowdhury, S. F.; Villamor, V. B.; Guerrero, R. H.; Leal, I.; Brun,
R.; Croft, S. L.; Goodman, J . M.; Maes, L.; Ruiz-Perez, L. M.;
Gonzales Pacanowska, D.; Gilbert, I. H. Design, synthesis, and
evaluation of inhibitors of trypanosomal and leishmanial dihy-
drofolate reductase. J . Med. Chem. 1999, 42, 4300-4312.
(20) Yuthavong, Y.; Vilainvan, T.; Chareonsethakul, N.; Kamchon-
wongpalsan, S.; Sirawaraporn, W.; Quarell, R.; Lowe, G. Devel-
opment of a lead inhibitor for the A16V+S108T mutant of
dihydrofolate reductase from the Cycloguanil-resistant strain
(T9/94) of Plasmodium falciparum. J . Med. Chem. 2000, 43,
2738-2744.
(21) Kuyper, L. F.; Baccanari, D. P.; J ones, M. L.; Hunter, R. N.;
Tansik, R. L.; J oyner S. S.; Boytes, Ch. M.; Rudolph, S. K.; Knick,
V.; Wilson, H. R.; Caddell, J . M.; Friedman, H. S.; Comley, J . C.
W.; Stables, J . N. High-affinity inhibitors of dihydrofolate
reductase: Antimicrobial and anticancer activities of 7,8-dialkyl-
1,3-diaminopyrrolo[3,2-f]quinazolines with small molecular size.
J . Med. Chem. 1996, 39, 892-903.
(22) Bushby, S. R. M.; Hitchings, G. H., Trimethoprim, a sulfonamide
potentiator. Br. J . Pharmacol. Chemother. 1968, 33, 72-90.
(23) Seydel, J . K.; Wiese, M.; Kansy, M.; Schaper, K.-J .; Walter, R.,
Tropical diseases with special emphasis on antimalarial and
antileprosy drugs. Pharmacochem. Libr. 1990, 13, 109-131.
(24) Seydel J . K.; Wempe, E. G.; Rosenfeld, M.; J agannathan, R.;
Mahadevan, P. R., Dhople, A. M., In vitro and in vivo experi-
ments with the new inhibitor of Mycobacterium leprae Brodi-
moprim alone and in combination with Dapsone. Arzneim.-
Forsch./ Drug Res. 1990, 40, 69-75.
(25) Kompis, I.; Mu¨ller, W.; Boehni, E.; Then, R.; Montavon, M. 2,4-
Diamino-5-(pyridylmethyl)-pyrimidine als potentielle Chemo-
therapeutica. Eur. J . Med. Chem. 1977, 12, 531-536.
(26) Stogryn, E. L. Synthesis of trimethoprim variations. J . Med.
Chem. 1972, 15, 200-201.
(27) Ponsford, R. J . (Beecham Group Ltd.) U.S. Patent 4,719,562
1979, Chem. Abstr. 1980, 92, 145802; Chem. Abstr. 1980, 92,
76538.
(28) Kompis, I.; Then, R.; Wick, A.; Montavon, M. 2,4-Diamino-5-
benzyl-pyrimidines as inhibitors of dihydrofolate reductase. In
Enzyme Inhibitors; Brodbeck, U., Ed.; Verlag Chemie: Wein-
heim, Germany, 1980; pp 177-189.
(29) Weinstock, L. T.; O’Brien, D. E.; Cheng, C. C. Folic acid
analogues. I. p-{[(2,4-Diamino-5-pyrimidinyl)methyl]amino}-
benzoyl-L-glutamic acid and related compounds. J . Med. Chem.
1968, 11, 1238-1244.
(39) Wiese, M.; Seydel, J . K.; Pieper, H.; Kru¨ger, G.; Noll, K. R.; Keck,
J . Multiple regression analysis of antimalarial activities of
sulfones and sulfonamides in cell-free systems and principal
component analysis to compare with antibacterial activities.
Quant. Struct.-Act. Relat. 1987, 6, 164-172.
(40) Coats, E. A.; Cordes, H.-P.; Kulkarni, V. M.; Richter, M.; Schaper,
K.-J .; Wiese, M.; Seydel, J . K. Multiple regression and principal
component analysis of antibacterial activities of sulfones and
sulfonamides in whole cell and cell-free systems of various DDS
sensitive and resistant bacterial strains. Quant. Struct.-Act.
Relat. 1985, 4, 99-109.
(41) Seydel, J . K.; Wempe, E.; Miller, G. H.; Miller, L. Kinetics and
mechanism of action of trimethoprim and sulfonamides alone
and in combination upon E. coli. Chemotherapy 1972, 17, 217-
258.
(42) Seydel, J . K.; Wempe, E. Bacterial growth kinetics of “Mycobac-
terium lufu” in the presence and absence of various drugs alone
and in combination. A model for the development of combined
chemotherapy against M. leprae? Int. J . Leprosy 1982, 50, 20-
30.
(43) Hynes, J . B.; Hough, L. V.; Smith, A. B.; Gale, G. R. Activity of
selected 2,4-diaminoquinazolines against C. albicans in vitro.
Proc. Soc. Exp. Biol. Med. 1976, 153, 230-232.
(44) Kuyper, L.; Roth, B.; Baccanari, D.; Ferone, R.; Bedell, C. R.;
Campness, J . N.; Stammers, D. K.; Dann, J . G.; Norrington, F.
E. A.; Baker, D. J .; Goodford, P. J . Receptor-based design of
dihydrofolate reductase inhibitors: Comparison of crystallo-
graphically determined enzyme binding with enzyme affinity in
a series of carboxy-substituted trimethoprim analogues. J . Med.
Chem. 1982, 25, 1120-1123.
(45) Kansy, M.; Seydel, J . K.; Wiese, M.; Haller, R. Synthesis of new
2,4-diamino-5-benzylpyrimidines active against various bacterial
species. Eur. J . Med. Chem. 1992, 27, 237-244.
(46) Czaplinski, K.-H.; Kansy, M.; Seydel, J . K.; Haller, R. Design of
a new 2,4-diamino-benzylpyrimidine as inhibitor of bacterial
dihydrofolate reductase assisted by molecular graphics. Quant.
Struct.-Act. Relat. 1987, 6, 70-72.
(47) Czaplinski, K.-H.; Ha¨nsel, W.; Wiese, M.; Seydel, J . K. New
benzylpyrimidines: Inhibitors of DHFR from various species.
QSAR-, CoMFA- and PC analysis. Eur. J . Med. Chem. 1995, 30,
779-787.
(48) Wiese, M.; Schmalz, D.; Seydel, J . K. New antifolate 4,4′-
diaminodiphenyl sulfones substituted 2,4-diamino-5-benzyl-
pyrimidines. Proof of their dual mode of action and autosyner-
gism. Arch. Pharm. Pharm. Med. Chem. 1996, 329, 161-168.
(49) Henning, H. Synthese, Biologische Testung und Quantitative
Struktur-Wirkungs-Analyse von 2,4-Diamino-5-benzyl-pyrimi-
dinen. Dissertation/PhD-Thesis, Universita¨t Kiel,Germany, 1991.
(50) Chan, J . H.; Hong, J . S.; Kuyper, L. F.; Baccanari, D. P.; J oyner,
S. S.; Tansik, R. L.; Boytos, C. M.; Rudolph, S. K. Selective
inhibitors of Candida albicans dihydrofolate reductase: Activity
and selectivity of 5-(arylthio)-2,4-diaminoquinazolines. J . Med.
Chem. 1995, 38, 3608-3616.
(30) Steinbuck, P.; Baltzly, R.; Hood, H. M. A new synthesis of
5-benzylpyrimidines. J . Org. Chem. 1963, 28, 1983-1988.
(31) Kompis, I.; Then, R.; Boehni, E.; Rey-Bellet, G.; Zanetti, G.;
Montavon, M. Synthesis and antibacterial activity of C(4′)-
substituted analogues of trimethoprim. Eur. J . Med. Chem.
1980, 12, 17-22.
(32) Hachtel, G.; Haller, R.; Seydel, J . K. Synthese und antimykobak-
terielle Wirkung einiger lipophil substituierter 2,4-Diamino-5-
benzylpyrimidine. Arzneim.-Forsch./ Drug Res. 1988, 38, 1778-
1783.
(33) Zhang, X.-P.; Zhang, X.-J .; Zheng, X.-H.; Chen, L.; Dai, Z.-R.
Studies on antimalarials. I. Synthesis and antimalarial effects
of 2,4-diamino-5-substituted aminopyrimidines and 2,4-diamino-
6-methyl-5-substituted aminopyrimidines. Yaoxue Xuebao (Acta
Pharm. Sin.) 1980, 15 (12), 711-18. Chem. Abstr. 95, 150575.
(34) Baker, B. R.; Meyer, R. B., J r. Irreversible enzyme inhibitors
CLI. Active site-directed irreversible inhibitors of dihydrofolate
reductase derived from 5-(p-aminophenylbutyl)-2,4-diamino-
pyrimidines with a terminal sulfonyl fluoride. J . Med. Chem.
1969, 12, 224-227.
(51) Marchetti, O.; Moreillon, P.; Glauser, M. P.; Bille, J .; Sanglard,
D. Potent synergism of the combination of fluconazole and
cyclosporine in Candida albicans. Antimicrob. Agents Chemo-
ther. 2000, 44, 2373-2381.
(52) Kompis, I. Personal communication, 1989.
(53) Huber, W. 2,4-Diamino-5-(4-methyl-5-hydroxyethylthiazolium-
chloride)methylpyrimidine. J . Am. Chem. Soc. 1943, 65, 2222-
2226.
(54) Organikum, VEB Deutscher Verlag der Wissenschaften, Berlin,
1981; pp 600-604.
(55) Roth, B.; Baccanari, D. P.; Sigel, C. W.; Hubbell, J . P.; Eaddy,
J .; Kao, J . C.; Grace, M. E.; Rauckman, B. S. 2,4-Diamino-5-
benzylpyrimidines and analogues as antibacterial agents. 9.
Lipophilic trimethoprim analogues as antigonococcal agents. J .
Med. Chem. 1988, 31, 122-129.
(56) Tieckelmann, H.; Guthrie, R.; Nairn, J . G. 2,4-Diamino-5-
formylpyrimidine and 2,4-diamino-5-hydroxymethylpyrimidine.
J . Org. Chem. 1960, 25, 1257-9.
(35) Cavalieri, L. F.; Bendich, A. J . The ultraviolet absorption spectra
of pyrimidines and pteridines. J . Am. Chem. Soc. 1950, 72,
2587-2594.
(36) Hull, R. Pyrimidines. I. The synthesis of some 5-hydroxypyrimi-
dines. J . Chem. Soc. 1956, 2033-2035.
(37) Hurst, D. T. Application of the Elbs persulfate oxidation for the
preparation of 5-hydroxypyrimidines. Aust. J . Chem. 1983, 36,
1285-1289.
(38) Bartels, R.; Bock, L. Improved methods for the purification of
enzymes of the folate pathway in Escherichia coli. 1. Chromato-
graphic methods. J . Chromatogr. 1990, 523, 53-60.
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