Dicyclic and tricyclic diaminopyrimidine derivatives as potent inhibitors of Cryptosporidium parvum dihydrofolate reductase: Structure-activity and structure-selectivity correlations

Article abstract of DOI:10.1128/AAC.45.12.3293-3303.2001

A structurally diverse library of 93 lipophilic di- and tricyclic diaminopyrimidine derivatives was tested for the ability to inhibit recombinant dihydrofolate reductase (DHFR) cloned from human and bovine isolates of Cryptosporidium parvum (J. R. Vasquez et al., Mol. Biochem. Parasitol. 79:153-165, 1996). In parallel, the library was also tested against human DHFR and, for comparison, the enzyme from Escherichia coli. Fifty percent inhibitory concentrations (IC₅₀s) were determined by means of a standard spectrophotometric assay of DHFR activity with dihydrofolate and NADPH as the cosubstrates. Of the compounds tested, 25 had IC₅₀s in the 1 to l0 μM range against one or both C. parvum enzymes and thus were not substantially different from trimethoprim (IC₅₀s, ca. 4 μM). Another 25 compounds had IC₅₀s of <1.0 μM, and 9 of these had IC₅₀s of <0.1 μM and thus were at least 40 times more potent than trimethoprim. The remaining 42 compounds were weak inhibitors (IC₅₀s, < 10 μM) and thus were not considered to be of interest as drugs useful against this organism. A good correlation was generally obtained between the results of the spectrophotometric enzyme inhibition assays and those obtained recently in a yeast complementation assay (V. H. Brophy et al., Antimicrob. Agents Chemother. 44:1019-1028, 2000; H. Lau et al., Antimicrob. Agents Chemother. 45:187-195, 2001). Although many of the compounds in the library were more potent than trimethoprim, none had the degree of selectivity of trimethoprim for C. parvum versus human DHFR. Collectively, the results of these assays comprise the largest available database of lipophilic antifolates as potential anticryptosporidial agents. The compounds in the library were also tested as inhibitors of the proliferation of intracellular C. parvum oocysts in canine kidney epithelial cells cultured in folate-free medium containing thymidine (10 μM) and hypoxanthine (100 μM). After 72 h of drug exposure, the number of parasites inside the cells was quantitated by indirect immunofluorescence microscopy. Sixteen compounds had IC₅₀s of <3 μM, and five of these had IC₅₀s of <0.3 μM and thus were comparable in potency to trimetrexate. The finding that submicromolar concentrations of several of the compounds in the library could inhibit in vitro growth of C. parvum in host cells in the presence of thymidine (dThd) and hypoxanthine (Hx) suggests that lipophilic DHFR inhibitors, in combination with leucovorin, may find use in the treatment of intractable C. parvum infections.

Full text of DOI:10.1128/AAC.45.12.3293-3303.2001

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2001, p. 3293–3303
0066-4804/01/$04.00ϩ0 DOI: 10.1128/AAC.45.12.3293–3303.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 45, No. 12
Dicyclic and Tricyclic Diaminopyrimidine Derivatives as Potent
Inhibitors of Cryptosporidium parvum Dihydrofolate Reductase:
Structure-Activity and Structure-Selectivity Correlations
RICHARD G. NELSON1 AND ANDRE ROSOWSKY2*
Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, California 94143,1
and Dana-Farber Cancer Institute and Department of Biological Chemistry and Molecular Pharmacology,
Harvard Medical School, Boston, Massachusetts 021152
Received 20 April 2001/Returned for modification 8 August 2001/Accepted 28 August 2001
A structurally diverse library of 93 lipophilic di- and tricyclic diaminopyrimidine derivatives was tested for
the ability to inhibit recombinant dihydrofolate reductase (DHFR) cloned from human and bovine isolates of
Cryptosporidium parvum (J. R. Va´squez et al., Mol. Biochem. Parasitol. 79:153–165, 1996). In parallel, the
library was also tested against human DHFR and, for comparison, the enzyme from Escherichia coli. Fifty
percent inhibitory concentrations (IC50s) were determined by means of a standard spectrophotometric assay
of DHFR activity with dihydrofolate and NADPH as the cosubstrates. Of the compounds tested, 25 had IC50s
in the 1 to 10 ␮M range against one or both C. parvum enzymes and thus were not substantially different from
trimethoprim (IC50s, ca. 4 ␮M). Another 25 compounds had IC50s of <1.0 ␮M, and 9 of these had IC50s of <0.1
␮M and thus were at least 40 times more potent than trimethoprim. The remaining 42 compounds were weak
inhibitors (IC50s, >10 ␮M) and thus were not considered to be of interest as drugs useful against this
organism. A good correlation was generally obtained between the results of the spectrophotometric enzyme
inhibition assays and those obtained recently in a yeast complementation assay (V. H. Brophy et al., Antimi-
crob. Agents Chemother. 44:1019–1028, 2000; H. Lau et al., Antimicrob. Agents Chemother. 45:187–195, 2001).
Although many of the compounds in the library were more potent than trimethoprim, none had the degree of
selectivity of trimethoprim for C. parvum versus human DHFR. Collectively, the results of these assays
comprise the largest available database of lipophilic antifolates as potential anticryptosporidial agents. The
compounds in the library were also tested as inhibitors of the proliferation of intracellular C. parvum oocysts
in canine kidney epithelial cells cultured in folate-free medium containing thymidine (10 ␮M) and hypoxan-
thine (100 ␮M). After 72 h of drug exposure, the number of parasites inside the cells was quantitated by
indirect immunofluorescence microscopy. Sixteen compounds had IC50s of <3 ␮M, and five of these had IC50s
of <0.3 ␮M and thus were comparable in potency to trimetrexate. The finding that submicromolar concen-
trations of several of the compounds in the library could inhibit in vitro growth of C. parvum in host cells in
the presence of thymidine (dThd) and hypoxanthine (Hx) suggests that lipophilic DHFR inhibitors, in
combination with leucovorin, may find use in the treatment of intractable C. parvum infections.
Diaminopyrimidine inhibitors of dihydrofolate reductase
(DHFR) such as trimethoprim, pyrimethamine, trimetrexate,
and piritrexim (compounds 1 to 4, respectively, in Fig. 1) are
used in the prophylaxis and treatment of opportunistic infec-
tions in patients whose immune systems are impaired as a
result of human immunodeficiency virus infection or immuno-
suppressive chemotherapy (14, 38). Typically, these agents are
coadministered with a sulfonamide or sulfone inhibitor of di-
hydropteroate synthetase (44, 49), and in the case of trime-
trexate or piritrexim, with leucovorin to minimize the toxic side
effects of the antifolate (13, 74). Although other microbial
parasites are known to cause life-threatening secondary infec-
tions in AIDS patients in other parts of the world, the organ-
isms most frequently found to cause opportunistic disease in
patients in the United States and other industrialized coun-
tries, sometimes at the same time, are Pneumocystis carinii and
Toxoplasma gondii. The rationale for combining sulfa drugs
with DHFR inhibitors is that P. carinii and T. gondii, but not
mammalian cells, can synthesize essential tetrahydrofolate co-
factors de novo with the help of dihydropteroate synthetase
(39). Because of the lack of this mechanism in humans, sulfa
drugs are selective in that they do not affect the folate status of
the patient’s cells. In the case of DHFR inhibitors that are
more potent than trimethoprim, such as trimetrexate or piri-
trexim, leucovorin can be added to the regimen as a rescue
agent to prevent dose-limiting hematological toxicity. In this
scenario, the selectivity of the host-protective effect results
from the fact that the parasites lack an active transport path-
way for reduced folates and thus are impervious to rescue (39).
Although combinations of DHFR inhibitors with sulfa drugs
have a sound theoretical rationale and, indeed, are effective in
a significant number of patients, a subset of patients experience
severe cutaneous allergy to the sulfa drug and thus have to
discontinue this type of treatment (73).
A vigorous program of chemical synthesis was launched by
several groups in the early 1990s with the goal of discovering
new inhibitors of P. carinii and/or T. gondii DHFR that might
be potent enough not to require coadministration of a sulfa
drug while being selective enough not to require leucovorin
rescue. Hundreds of lipophilic condensed diaminopyrimidine
* Corresponding author. Mailing address: Dana-Farber Cancer In-
stitute, 44 Binney St., Boston, MA 02115. Phone: (617) 632-3117. Fax:
(617) 632-2410. E-mail: andre_rosowsky@ dfci.harvard.edu.
3293

3294
NELSON AND ROSOWSKY
ANTIMICROB. AGENTS CHEMOTHER.
most extreme cases, massive exfoliation of the intestinal mu-
cosa (33, 75). Cryptosporidiosis is typically acquired from un-
sterilized drinking water and is usually self-limiting, except in
immunosuppressed individuals. Some success has been re-
ported with pyrimethamine and trimethoprim-sulfamethox-
azole in patients infected with Isospora belli, another opportu-
nistic intestinal parasite whose clinical effects include severe
and unremitting diarrhea and thus resemble those produced by
C. parvum (12, 77). Several other experimental treatments
using non-antifolate drugs have also been reported, some of
which are likely to act primarily on the host cell rather than the
parasite (1, 5, 6, 32, 34, 37, 42, 43, 47, 78). The efficacy of these
regimens is, at best, marginal. Thus, until a consistently safe
and effective drug against acute cryptosporidiosis in AIDS pa-
tients is found, the main treatment of this disease remains
palliation with antidiarrheal drugs such as octeotride (35).
With the availability of a large and structurally diverse li-
brary of lipophilic DHFR inhibitors generated by our synthetic
effort over the years, the opportunity presented itself to test
these compounds as inhibitors of the DHFR activity of the
difunctional C. parvum DHFR-thymidylate synthase (TS) en-
zyme, which was recently cloned and sequenced (76). A num-
ber of the compounds in our library, along with others from the
archives of the National Cancer Institute and the Walter Reed
Army Institute of Research, were recently found to be active in
a yeast complementation assay using Saccharomyces cerevisiae
in which the native yeast gene was replaced with either the
entire difunctional DHFR-TS gene or the discrete DHFR do-
main of C. parvum (7, 40). In the present paper, we report data
on 93 compounds from the archival collection of the Dana-
Farber Cancer Institute (DFCI) as in vitro inhibitors of recom-
binant C. parvum and human DHFR activity in a spectropho-
tometric assay. To our knowledge, this constitutes the largest
published database on lipophilic polycyclic diaminopyrimi-
dines as inhibitors of these enzymes. Also reported for com-
parison are the 50% inhibitory concentrations (IC50s) of the
same compounds against Escherichia coli DHFR. Although E.
coli infection is not among the more dangerous complications
of AIDS, drug-resistant E. coli strains are gradually becoming
more prevalent in the food supply and would almost certainly
pose a greater risk to an AIDS patient than to an individual
whose immune defense system is not impaired.
FIG. 1. Structures of trimethoprim (compound 1), pyrimethamine
(compound 2), trimetrexate (compound 3), and piritrexim (compound
4).
antifolates, embodying a rich spectrum of chemical diversity
(see references in Table 1), were tested as inhibitors of DHFR
in cell-free assays (8, 10) and, in some cases, as inhibitors of the
growth of the intact organisms in vitro or in laboratory animals
(4, 51). However, with the exception of the older agents tri-
methoprim and pyrimethamine, the only newer antifolates
tested in large, controlled clinical trials in AIDS patients suf-
fering from, or at risk of developing, P. carinii pneumonia
and/or toxoplasmosis have been trimetrexate (74) and piri-
trexim (13).
Among the opportunistic pathogens other than P. carinii and
T. gondii that are known to pose a significant risk in the man-
agement of AIDS is the intestinal parasite Cryptosporidium
parvum, which can be life threatening because of its ability to
bring on repetitive episodes of violent diarrhea and, in the
TABLE 1. Condensed diaminopyrimidine ring systems tested as
inhibitors of P. carinii and T. gondii DHFRs
Heterocyclic system
Reference(s)
5/6 systems
Thieno[2,3-d]pyrimidines...................................64, 66, 69
Furo[2,3-d]pyrimidines.......................................16, 17, 19, 20, 21
Pyrrolo[2,3-d]pyrimidines...................................22, 23
Pyrazolo[2,3-c]pyrimidines (purines)................26
MATERIALS AND METHODS
Cyclopenta[d]pyrimidines ..................................70
Test compounds. Data were obtained for a total of 93 compounds, comprising
the nine structural families shown in Fig. 2 to 4. Symmetrically 5,6-disubstituted
pteridines I.1 to I.8 were synthesized from tetraaminopyrimidine and 1,2-dike-
tones (52). Pteridines II.1 to II.5 and pyrido[2,3-d]pyrimidine II.6 were obtained
by N-alkylation of secondary amines with 2,4-diamino-6-bromomethylpteridine
(56) or 2,4-diamino-6-bromomethylpyrido[2,3-d]pyrimidine (60). Pyrido[3,2-
d]pyrimidines III.1 to III.6 were prepared from 2,4-diamino-6-bromometh-
ylpyrido[3,2-d]pyrimidine and anilines or N-methylanilines (58). Quinazolines
IV.1 to IV.3 were obtained from the corresponding anthranilonitriles and chlo-
roformamidine hydrochloride (62). Quinazolines IV.4 to IV.7 were obtained
from 2,4-diamino-5-iodoquinazoline and an alkene or alkyne via a palladium-
catalyzed Heck reaction, followed by catalytic hydrogenation in the case of IV.6
and IV.7 (65). N10-unsubstituted quinazolines IV.8 to IV.12 were obtained from
2,4-diaminoquinazoline-5-carbonitrile and the appropriate substituted anilines
by reductive coupling in the presence of Raney nickel (65). 5-Chloro-6-(arylami-
noalkyl)quinazolines IV.13 to IV.16, IV.18 and IV.19 were derived from 2,4,6-
triamino-5-chloroquinazoline and the appropriate substituted aldehydes by sim-
ilar reductive coupling (67). Compounds IV.11, IV.12, and IV.17 were obtained
from IV.8, IV.9, and IV.16, respectively, by reaction with formaldehyde and
6/6 systems
Quinazolines........................................................28, 29, 41, 61, 64, 65,
67, 68
Pyrido[2,3-d]pyrimidines ....................................15, 18, 24, 27, 60
Pyrido[3,2-d]pyrimidines ....................................30, 31, 58
Pyrido[4,3-d]pyrimidines ....................................63
Pteridines.............................................................36, 48, 56, 59, 61
Tricyclic and other ring systems
Indeno[2,3-d]pyrimidines...................................72
Benzo[f]quinazolines ..........................................72
Pyrimido[4,5-c]isoquinolines..............................61
Benzo[3,4]cycloheptal[1,2-d]pyrimidines..........72
Pyrimido[4,5-c][2,7]naphthyridines...................25
Pyrido[4Ј,3Ј:4,5]furo[2,3-d]pyrimidines.............19
Miscellaneous 5,6-(bicycloalkano)
tetrahydroquinazolines...................................71

VOL. 45, 2001
CRYPTOSPORIDIUM PARVUM DHFR INHIBITORS
3295
FIG. 2. Structures of dicyclic diaminopyrimidines tested as DHFR inhibitors (groups I to IV).
sodium cyanoborohydride (65, 67). Tetrahydroquinazolines V.1 to V.16 were
synthesized from 4-substituted cyclohexanones by heating with cyanoguanidine
(68). Thieno[2,3-d]pyrimidines VI.1 to VI.12 were derived from 5,6-substituted
2-aminothiophene-3-carbonitriles and chloroform-amidine hydrochloride (64,
66), whereas analogs VI.13 to VI.18, with an arylamine side chain, were synthe-
sized in several steps from preformed 2,4-diamino-5-methylthieno[2,3-d]pyrimi-
dine (69). Pyrido[4,3-d]-pyrimidines VII.1 to VII.4 were made by alkylation of
the parent amine (63). 9H-Indeno[2,1-d]pyrimidines VIII.1 to VIII.4 were syn-
thesized from 3-cyano-2-alkoxy-1H-indenes and guanidine (57). 5,6-Dihydroben-
zo[f]quinazolines VIII.5 and VIII.6 were prepared by heating 2-tetralones with
FIG. 3. Structures of dicyclic diaminopyrimidines tested as DHFR inhibitors (groups V to VII).

3296
NELSON AND ROSOWSKY
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 4. Structures of di- and tricyclic diaminopyrimidines tested as DHFR inhibitors (groups VIII and IX).
cyanoguanidine (55). Seven-membered ring analog VIII.10 was prepared simi-
larly from 6,7-dichloro-2-benzosuberone (53). Fully aromatic benzo[f]quinazo-
lines VIII.7 to VIII.9 were prepared by thermal cyclization of symmetrical N1,N5-
diarylbiguanides, by condensation of 2-aminonaphthalene-1-carbonitriles with
guanidine, or by oxidation of 1,3-diamino-5,6-dihydrobenzo[f]quinazolines with
selenium dioxide (54). Trimethoprim and trimetrexate were obtained from the
National Cancer Institute, Bethesda, Md. All compounds were of recent or
archival origin and were judged to be Ͼ95% pure at the time of assay.
compounds for the ability to inhibit DHFRs from several dif-
ferent species, including C. parvum and humans (7, 40). Fifteen
other compounds provided by the National Cancer Institute
were also tested by direct spectrophotometric assay (7). One of
these, trimethoprim, proved to be Ͼ100 times more potent
against the C. parvum enzyme than against the human enzyme.
Trimetrexate was considerably more potent than trimethoprim
but was completely nonselective. Three other compounds were
also more potent than trimethoprim against both enzymes, but
three of them contained a polar glutamic or aspartic acid side
chain and thus were not considered to be of the lipophilic type.
Trimethoprim emerged as the most selective member of this
initial panel and thus was considered the first promising lead in
our search for anticryptosporidial drugs among lipophilic
DHFR inhibitors. Although interesting patterns of activity
were observed in the DHFR complementation assays (7, 40),
this method lacks the quantitative power of a direct measure-
ment of the kinetics of an enzyme reaction. More importantly,
compounds can only be assumed to be equipotent as enzyme
inhibitors by this method if they achieve the same intracellular
concentration over the time course of the assay, which may not
necessarily be the case, even for lipophilic antifolates. Thus,
the yeast complementation assay should be viewed as only a
preliminary screen. In the present study, the spectrophotomet-
ric assay was used to assess the potency and selectivity of a
larger group of lipophilic antifolates from the DFCI collection
with a view to uncovering additional leads. Some of these
compounds had already been tested in the yeast assay, but
many had not.
As shown in Table 2, the IC50s of trimethoprim against the
Cp-I and Cp-II enzymes were 4.0 and 3.8 ␮M, respectively,
whereas that against human DHFR was 890 ␮M. Thus, as
previously noted (7), trimethoprim was equally active against
the human and bovine strains of C. parvum DHFR and was
remarkably selective for these enzymes relative to human
DHFR. Of the 93 additional diaminopyrimidine antifolates
that have now been tested against the Cp-I and Cp-II DHFRs,
25 had IC50s in the 1 to 10 ␮M range against at least one of the
C. parvum enzymes and thus were not substantially different
from trimethoprim. A second group of 25 compounds had
IC50s of Ͻ1.0 ␮M, and of these, 9 had IC50s of Ͻ0.1 ␮M and
thus were at least 40 times more potent than trimethoprim. A
third group, comprising 42 compounds, afforded Ͻ50% inhi-
Enzyme assays. Recombinant C. parvum and human enzymes were obtained
and used as described earlier (7, 76). The C. parvum type 1 DHFR-TS allele was
cloned from an isolate obtained from an infected AIDS patient, and the C.
parvum type 2 allele was cloned from an isolate obtained from an infected calf.
The designations Cp-I and Cp-II will be used here in order to conform to the
nomenclature used earlier for the two cloned proteins (76; for a discussion of C.
parvum genotypes 1 and 2, see reference 46). The type 1 enzyme is found
exclusively in humans, whereas the type 2 enzyme can infect either humans or
animals. Molecular characterization of the constructs SFGH-1 (from Cp-I) and
NINC-1 (from Cp-I), the latter of which was re-engineered to encode a 22-
residue C-terminal TS domain of Cp-I, was documented earlier (76). Briefly, all
three proteins were expressed from dhfr mutant E. coli strain PA414 after
transfection with the appropriate coding sequence and subcloning into expres-
sion plasmid pTrc99A, which contains a promoter induced with IPTG (isopropyl-
␤-D-thiogalactopyranoside). In the case of the human enzyme, the E. coli mutant
was transfected with previously described plasmid pDFR (50). All three enzymes
were purified to homogeneity from their respective bacterial lysates by chroma-
tography on a methotrexate-Sepharose affinity column. Assays of enzyme activity
were performed at 37°C in a microtiter plate spectrophotometer by monitoring
the change in UV absorbance at 340 nm in a solution containing 50 mM
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (pH 7.0),
1 mM
EDTA, 75 ␮M 2-mercaptoethanol, 0.1% bovine serum albumin, 20 ␮M dihy-
drofolate, and 100 ␮M NADPH. Enzyme concentrations were adjusted to give
linear rates over the 5-min duration of the assay. Reactions were initiated by
adding 100 ␮l of 40 ␮M dihydrofolate in assay buffer to an equal volume of assay
buffer lacking dihydrofolate and containing various concentrations of the inhib-
itor. Each titration was performed twice, and the mean DHFR activity was
plotted against the inhibitor concentration to obtain the IC50. Assays using E. coli
DHFR were performed under the same conditions as those done with the human
enzyme. Stock solutions of all of the test compounds were made up in dimethyl
sulfoxide, and appropriate control experiments were done to ensure that the final
amount of dimethyl sulfoxide in the assay solution was low enough to have no
appreciable effect on the rate of the DHFR-catalyzed reaction or the ability of
cells to grow in culture.
RESULTS AND DISCUSSION
Binding of inhibitors to C. parvum DHFR versus their bind-
ing to human DHFR. The availability of the compounds shown
in Fig. 2 to 4 offered the opportunity to screen a structurally
diverse library of 93 lipophilic antifolates from the DFCI ar-
chive as inhibitors and to determine whether any of them are
selective for the C. parvum enzyme. Yeast complementation
assays had been concurrently used to test a number of these

VOL. 45, 2001
Compound
CRYPTOSPORIDIUM PARVUM DHFR INHIBITORS
TABLE 2. Inhibition of C. parvum and human DHFRs by di- and tricyclic diaminopyrimidines
3297
IC50(␮M)a
Selectivityb
Cp-I
Cp-II
Human
Cp-I
Cp-II
Trimethoprim
I.1
4.0 Ϯ 1.7 (1.0)
1.2 Ϯ 0.32 (3.3)
0.25 Ϯ 0.057 (16)
1.7 Ϯ 0.85 (2.4)
0.82 Ϯ 0.48 (4.9)
1.8 Ϯ 0.84 (2.2)
2.1 Ϯ 1.9 (1.9)
3.8 Ϯ 1.8 (1.0)
1.6 Ϯ 0.38 (2.4)
0.35 Ϯ 0.052 (11)
1.5 Ϯ 0.29 (2.5)
0.75 Ϯ 0.29 (5.1)
1.6 Ϯ 0.88 (2.4)
1.9 Ϯ 0.34 (2.0)
7.2 Ϯ 2.9 (0.53)
1.5 Ϯ 0.76 (2.5)
0.14 Ϯ 0.060 (27)
8.3 Ϯ 2.9 (0.46)
0.049 Ϯ 0.029 (78)
0.029 Ϯ 0.014 (130)
1.9 Ϯ 0.23 (2.0)
0.80 Ϯ 0.095 (4.8)
0.040 Ϯ 0.010 (95)
0.045 Ϯ 0.0064 (84)
0.022 Ϯ 0.0069 (170)
0.011 Ϯ 0.0025 (350)
9.0 Ϯ 0.92 (0.42)
0.13 Ϯ 0.017 (29)
0.65 Ϯ 0.21 (4.2)
0.14 Ϯ 0.026 (27)
0.73 Ϯ 0.14 (5.2)
1.9 Ϯ 0.26 (2.0)
0.28 Ϯ 0.010 (14)
0.69 Ϯ 0.16 (5.5)
0.22 Ϯ 0.064
0.16 Ϯ 0.040 (24)
0.53 Ϯ 0.068 (6.9)
0.34 Ϯ 0.092 (11)
0.26 Ϯ 0.021 (15)
0.76 Ϯ 0.14 (5.0)
0.089 Ϯ 0.00487 (43)
1.7 Ϯ 0.40 (2.2)
8.3 Ϯ 2.9 (0.45)
3.6 Ϯ 0.64 (1.1)
0.65 Ϯ 0.11 (5.8)
8.5 Ϯ 1.6 (0.45)
2.3 Ϯ 0.60 (1.7)
1.4 Ϯ 0.20 (2.7)
0.019 Ϯ 0.0040 (200)
1.4 Ϯ 0.48 (2.7)
0.66 Ϯ 0.22 (5.8)
2.2 Ϯ 0.44 (1.7)
1.2 Ϯ 0.056 (3.2)
0.60 Ϯ 0.16 (6.3)
1.3 Ϯ 0.38 (2.9)
1.3 Ϯ 0.15 (2.9)
890 Ϯ 60 (1)
5.7 Ϯ 0.63 (160)
3.5 Ϯ 5.6 (250)
0.56 Ϯ 0.15 (1,600)
0.23 Ϯ 0.088 (3,900)
0.81 Ϯ 0.40 (1,100)
1.4 Ϯ 0.52 (640)
220
4.8
14
0.33
0.28
0.45
0.67
0.26
0.38
0.12
0.034
1.1
0.019
5.0
2.1
230
3.6
10
I.2
II.2
0.37
0.31
0.51
0.74
0.12
0.33
0.037
0.037
0.55
0.013
1.5
0.94
0.098
0.029
0.45
0.91
1.0
0.17
0.91
0.67
0.32
0.15
0.26
0.28
0.73
0.59
0.72
0.56
0.58
0.41
1.1
0.58
0.077
0.83
2.5
0.86
1.2
0.086
0.63
0.12
0.91
0.86
0.64
3.2
II.3
II.4
II.6
III.1
III.2
III.3
III.4
III.5
III.6
IV.1
IV.2
IV.13
IV.14
IV.17
IV.18
V.1
3.2 Ϯ 3.4 (1.3)
0.83 Ϯ 0.17 (1,100)
0.49 Ϯ 0.12 (1,800)
0.0089 Ϯ 0.0044 (100,000)
0.31 Ϯ 0.20 (2,900)
0.027 Ϯ 0.019 (33,000)
0.00037 Ϯ 0.00027 (2,400,000)
2.8 Ϯ 2.0 (320)
0.75 Ϯ 0.50 (1,200)
0.0039 Ϯ 0.0016 (120,000)
0.0013 Ϯ 00044 (680,000)
0.010 Ϯ 0.0020 (89,000)
0.010 Ϯ 0.00028 (89,000)
9.4 Ϯ 1.0 (95)
0.022 Ϯ 0.0080 (40,000)
0.59 Ϯ 0.20 (1,500)
0.094 Ϯ 0.031 (9,500)
0.23 Ϯ 0.032 (3,900)
0.29 Ϯ 0.068 (3,100)
0.074 Ϯ 0.018 (12,000)
0.19 Ϯ 0.036 (4,700)
0.16 Ϯ 0.052 (5,600)
0.094 Ϯ 0.048 (9,500)
0.38 Ϯ 0.048 (3,400)
0.19 Ϯ 0.092 (4,700)
0.15 Ϯ 0.084 (5,900)
0.31 Ϯ 0.12 (2,900)
0.094 Ϯ 0.022 (9,500)
0.98 Ϯ 0.30 (910)
0.64 Ϯ 0.24 (1,400)
3.0 Ϯ Ͻ0.0001 (300)
1.6 Ϯ 0.31 (560)
7.3 Ϯ 2.3 (120)
2.8 Ϯ 0.356 (320)
0.12 Ϯ 0.056 (7,400)
0.012 Ϯ 0.017 (74,000)
0.17 Ϯ 0.072 (5,200)
0.60 Ϯ 0.13 (1,500)
1.9 Ϯ 0.96 (470)
0.77 Ϯ 0.37 (1,200)
1.9 Ϯ 1.0 (470)
1.3 Ϯ 0.11 (3.1)
0.075 Ϯ 0.039 (53)
9.1 Ϯ 1.6 (0.44)
0.024 Ϯ 0.012 (170)
0.020 Ϯ 0.015 (200)
0.56 Ϯ 0.30 (7.1)
0.35 Ϯ 0.23 (11)
0.023 Ϯ 0.0060 (170)
0.027 Ϯ 0.0025 (150)
0.038 Ϯ 0.011 (105)
0.0065 Ϯ 0.0021 (620)
7.6 Ϯ 2.4 (0.53)
0.21 Ϯ 0.025 (19)
1.0 Ϯ 0.36 (2.9)
0.18 Ϯ 0.040 (22)
1.2 Ϯ 0.36 (3.3)
1.1 Ϯ 0.48 (3.6)
0.33 Ϯ 0.10 (12)
0.40 Ϯ 0.17 (10)
0.56 Ϯ 0.56
0.17
0.048
0.26
1.5
1.2
V.2
0.10
0.59
0.52
0.19
0.26
0.22
0.48
0.29
0.63
0.47
0.42
0.38
0.58
1.4
V.4
V.5
V.6
V.7
V.8
V.9
V.10
V.11
V.12
V.13
V.14
V.15
V.16
VI.1
VI.2
VI.4
VI.5
VI.12
VII.2
VIII.6
VIII.8
VIII.9
IX.1
IX.2
IX.3
IX.4
IX.5
IX.6
0.15 Ϯ 0.010 (27)
0.81 Ϯ 0.34 (3.6)
0.45 Ϯ 0.11 (8.9)
0.40 Ϯ 0.10 (10)
0.53 Ϯ 0.044 (7.5)
0.065 Ϯ 0.018 (62)
2.6 Ϯ 1.1 (1.5)
0.38
0.076
1.3
8.4 Ϯ 2.8 (0.48)
2.3 Ϯ 1.0 (1.7)
0.78 Ϯ 0.28 (5.1)
5.1 Ϯ 2.2 (0.78)
2.1 Ϯ 0.38 (1.9)
0.72 Ϯ 0.52 (5.6)
0.023 Ϯ 0.0078 (170)
1.2 Ϯ 0.88 (3.3)
1.3 Ϯ 0.68 (3.1)
2.4 Ϯ 0.26 (1.7)
1.8 Ϯ 0.44 (2.2)
0.65 Ϯ 0.21 (6.2)
4.7 Ϯ 4.4 (0.85)
3.2 Ϯ 0.92 (1.3)
2.1
1.4
1.3
0.17
0.52
0.14
0.46
0.79
0.43
2.9
0.58 Ϯ 0.26 (1,500)
0.81 Ϯ 0.17 (1,100)
0.12
0.25
0.45
0.62
a Values reported are means Ϯ standard deviations from a minimum of three, and in some cases as many as 7, separate determinations. The values in parentheses
are normalized relative to those for trimethoprim and rounded off to two significant figures. The following compounds had IC50 of Ͼ10 ␮M against one or both C.
parvum enzymes: I.3 to I.8, II.1, II.5, IV.3 to IV.12, IV.15, IV.16, and IV.19 (Fig. 2); V.3, VI.3, VI.6 to VI.11, and VI.13 to VI.18 (Fig. 3); VII.1, VII.3, VIII.1 to VIII.5,
VIII.7, and VIII.10 (Fig. 4). Of these, however, the following had IC50s of Ͻ10 ␮M against human DHFR: I.5, 5.3; I.7, 3.7; II.1, 7.2; IV.4, 1.9; IV.5, 6.5; IV.10, 1.9;
V.3, 5.3; VIII.1, 7.3; VIII.2, 9.9; VIII.5, 7.2; VIII.10, 0.0029. The other compounds with IC50s of Ͼ10 ␮M against Cp-I and/or Cp-II also had IC50 of Ͼ10 ␮M against
human DHFR.
b Selectivity is defined as the ratio IC50 for human DHFR/IC50 for Cp-I or IC50 for human DHFR/IC50 for Cp-II. Selectivity ratios were calculated only for
compounds whose IC50s were Ͻ10 ␮M against both the C. parvum and human DHFRs.
bition at 10 ␮M, the concentration arbitrarily chosen as a
reasonable upper limit for the screen, and thus are merely
footnoted in Table 2. The fact that roughly half of the com-
pounds in our library were at least as potent as trimethoprim
raises doubt about a recent comment in the literature that C.
parvum DHFR may be intrinsically resistant to 2,4-diaminopy-
rimidine inhibitors (11).
The best compound tested was IV.18, whose IC50 of 0.0065
␮M against the Cp-I enzyme approached the value obtained
earlier for trimetrexate (7). Like trimetrexate, however, this
compound was nonselective. With the exception of I-2, which
had 14-fold selectivity for the Cp-I enzyme and 10-fold selec-
tivity for the Cp-II enzyme, almost all of the compounds were
more like trimetrexate than trimethoprim, in that they inhib-

3298
NELSON AND ROSOWSKY
ANTIMICROB. AGENTS CHEMOTHER.
ited the C. parvum and human DHFRs with about the same
potency or were actually better inhibitors of the human en-
zyme. For the purpose of this discussion, we have arbitrarily
chosen a 10-fold difference in IC50s as the minimum threshold
for selectivity. This places I.1, IV.1, IV.2, and IX.4 in the
nonselective category even though they are, in fact, slightly
selective for the Cp-I enzyme. It is of interest that five of the
nine compounds with IC50s of Ͻ0.1 ␮M against at least one of
the C. parvum enzymes contained either the 3,4,5-trimethoxy-
phenyl substitution of trimethoprim and trimetrexate (cf. III.3,
IV.14, and IV.17) or the 2,5-dimethoxyphenyl substitution of
piritrexim (cf. IV.13 and IV.18), while four others contained 3-
and/or 4-chloro substitutions on the phenyl ring (cf. III.5, III.6,
V.16, and VIII.8). However, 10 of the other 13 compounds
with a 3,4,5-trimethoxyphenyl ring in the side chain and 5 of
the other 9 compounds with a 2,5-dimethoxyphenyl ring in the
side chain had IC50s of Ͼ10 ␮M, indicating that these substi-
tutions are not necessarily favorable with every diaminopy-
rimidine ring system.
Structural differences between pairs of analogs with widely
divergent potencies as DHFR inhibitors were, in some cases,
astonishingly small. Among the pyrido[3,2-d]pyrimidine ana-
logs, for example, methylation of the bridge nitrogen increased
potency by approximately 1 order of magnitude (cf. III.2 and
III.3) and replacement of a 4-chlorophenyl or 3,4-dichlorophe-
nyl group with a 3-chlorophenyl group in the side chain had a
comparable effect (cf. IV.4 to IV.6). However, moving a sub-
stituent from the para to the meta position of the phenyl ring
was not necessarily favorable and appeared to depend on the
nature of the heterocyclic moiety and the bridge (cf. V.6 and
V.7 versus III.4 and III.5 and V.14 and V.15 versus III.4 and
III.5). Insertion of an extra CH2 group into the bridge of
quinazoline analogs IV.13 and IV.14 caused potency to de-
crease by 2 orders of magnitude (cf. IV.15 and IV.16). In other
cases, there was remarkably little variation in binding among
compounds with considerably different patterns of aromatic
substitution (cf. V.4 to V.15). Even though some of the indi-
vidual structure-activity correlations in Table 2 were intriguing,
the limited data obtained did not yield any clear guidelines as
to how selective inhibitors of C. parvum DHFR might be de-
signed. Moreover, none of the compounds approached the
dramatic selectivity reported earlier for trimethoprim as an
inhibitor of the C. parvum enzyme (7). A better understanding
of how an optimal combination of high potency and high se-
lectivity might be designed into an inhibitor of C. parvum
DHFR will no doubt be easier to obtain once the first three-
dimensional structure of a ternary complex of the enzyme has
been solved, e.g., by X-ray crystallography or nuclear magnetic
resonance analysis. High-resolution crystallographic structural
analysis of several such complexes is in progress (R. G. Nelson,
personal communication).
asite growth was due to an antifolate effect on the host cell, the
medium was supplemented with 10 ␮M dThd and 100 ␮M Hx.
In some experiments, the medium could be supplemented ef-
fectively with as little as 0.1 ␮M leucovorin alone, instead of
dThd and Hx (R. G. Nelson, results not shown). Parasites were
visualized by an indirect immunofluorescence assay in which
the cells were fixed and treated sequentially with rat polyclonal
anti-C. parvum serum, biotinylated anti-rat serum, and fluores-
ceinated streptavidin. The fixed cells were also stained for
DNA, and the doubly stained cells were enumerated under the
microscope. Photomicrographs of control cells and of cells
incubated for 48 h with trimethoprim, pyrimethamine, trime-
trexate, and tetrahydroquinazolines V.4, V.10, and V.16, all at
the same concentration (4 ␮M), are presented in Fig. 5 and 6.
As can be seen in these figures, trimethoprim (panels E and F)
and pyrimethamine (panels H and I) had little or no effect on
the number of red-stained parasite-containing vacuoles rela-
tive to those in untreated controls (panels B and C), presum-
ably because 4 ␮M is lower than the optimal concentration of
these drugs. In contrast, 4 ␮M trimetrexate led to marked
diminution of the number of parasitemic vacuoles (panels K
and L) and this was accompanied by aberrant nuclear mor-
phology of the parasites in these vacuoles (panels G and J)
compared to that of controls (panel A). Gratifyingly, com-
pounds V.4 (panels M to O), V.10 (panels P to R), and V.16
(panels S to U) appeared to be equipotent with trimetrexate in
blocking parasitemia. A dose-response analysis was then per-
formed by repeating this experiment over a range of drug
concentrations of 0.001 to 30 ␮M. As shown in Fig. 6, tri-
methoprim at 30 ␮M decreased the number of intracellular
parasites by 50% whereas trimetrexate elicited the same result
at 0.1 ␮M, in rough agreement with the 1,000-fold tighter
binding of the latter drug to DHFR. The tetrahydroquinazo-
line analogs had IC50s in an intermediate range (1 to 3 ␮M).
On the basis of the pilot experiment whose results are de-
picted in Fig. 5 and 6, we reasoned that most of the compounds
in the DFCI library would be effective somewhere in the 0.3 to
30 ␮M range, with trimethoprim and pyrimethamine at one
end of the spectrum and trimetrexate at the other. However,
because we felt that a compound would not be worth studying
further unless its potency could be shown to be at least 10-fold
greater than that of trimethoprim, we selected 3 ␮M as a
reasonable upper drug concentration limit for all subsequent
experiments. Of the 93 compounds tested, the majority had
IC50s of Ͼ3.0 ␮M and thus did not meet this rather stringent
criterion. However, nine compounds with IC50s of Յ0.6 ␮M,
i.e., the pteridine I.2, the pyrido[3,2-d]pyrimidines III.5 and
III.6, the 5-chloroquinazolines IV.14, IV.17, and IV.18, the
tetrahydroquinazolines V.2 and V.5, and the benzo[f]quinazo-
line VIII.8 appeared to be somewhat more potent than V.4,
V.10, or V.16 and Ͼ50 times more potent than trimethoprim.
Because our target endpoint was 50% inhibition, which was 0.2
␮M for several of the compounds in the library, concentrations
of Ͻ0.2 ␮M were not tested. As shown in Fig. 6, the IC50 of
trimetrexate was ca. 0.1 ␮M. The four compounds with the
lowest IC50s, the pyrido[3,2-d]pyrimidine III.6, the quinazo-
lines IV.17 and IV.18, and the tetrahydroquinazolines V.2 and
V.5 approached trimetrexate in potency. However, because of
their lack of enzyme selectivity (Table 2), in vivo use of these
Anticryptosporidial activities of DHFR inhibitors in culture.
In order to determine whether the potent inhibition of isolated
DHFR translates into antiparasitic activity in culture, three
arbitrarily chosen examples of the tetrahydroquinazoline type
(V.4, V.10, and V.16) were tested for the ability to block
proliferation of the intracellular forms of the organism in Ma-
din-Darby canine kidney (MDCK) epithelial cell monolayers
grown in microscope chamber slides and infected with C. par-
vum oocysts. To rule out the possibility that inhibition of par-

VOL. 45, 2001
CRYPTOSPORIDIUM PARVUM DHFR INHIBITORS
3299
FIG. 5. Inhibition of the development and multiplication of C. parvum intracellular life-cycle stages by lipophilic antifolates. MDCK epithelial
cells (2.5 ϫ 104/cm2) were plated in eight-well microscope chamber slides and grown to confluence for 48 h in para-aminobenzoic acid and
folate-free RPMI 1640 medium (deficient medium) supplemented with 100 ␮M Hx and 10 ␮M dThd and containing 5% dialyzed fetal calf serum
(dFCS). The cells were transferred to deficient medium–1% dFCS lacking Hx and dThd for 24 h and subsequently infected by incubation with C.
parvum oocysts (3.5 ϫ 103/cm2) for 3 h at 37°C. After three washes to remove unexcysted oocysts and extracellular sporozoites, fresh deficient
medium–1% dFCS containing 4 ␮M drug (or no drug) was added. Drugs and media were renewed at 24 h postinfection, and the experiment was
terminated at 48 h. After formaldehyde fixation, 1% Triton X-100 extraction, and blockade in 0.5% bovine serum albumin, the infected monolayers
were processed for indirect immunofluorescence assay by consecutive 60-min incubations with 1:500 dilutions of rat polyclonal anti-C. parvum
serum, biotin-conjugated anti-rat serum, and fluorophore CY3-conjugated streptavidin containing the DNA stain 4Ј,6Ј-diamidino-2-phenylindole
(DAPI) at 1 ␮M. Panels in the first and second horizontal rows are photomicrographs (magnification, ca. ϫ1,000) of the same microscopic fields
stained for nuclei with DAPI and for parasite-containing vacuoles with the anti-C. parvum polyclonal antibody, respectively. Panels in the third
horizontal row are lower-power photomicrographs (magnification, ca. ϫ400) demonstrating the overall level of C. parvum infection and the
inhibition of parasite multiplication by the antifolates. Results are representative examples of a number of experiments repeated on different days.
Panels: A to C, controls (no drug); D to F, trimethoprim; G to I, pyrimethamine; J to L, trimetrexate; M to O, compound V.10; P to R, compound
V.15; S to T, compound V.16. All drugs were present at a concentration of 4 ␮M.
compounds would presumably require coadministration of leu-
covorin.
ever the exact details of how these compounds actually get into
the parasite, it seems clear that in designing and evaluating
lipophilic antifolates as candidate drugs against cryptosporidi-
osis, account must be taken of their ability to reach the target
enzyme.
An interesting structure-activity correlation that becomes
evident from the results in Tables 2 and 3 is that a low IC50 in
the spectrophotometric assay of DHFR inhibition is not always
accompanied by proportionally high potency in the C. parvum
growth assay. For example, among the 20 compounds whose
IC50s in the spectrophotometric DHFR assay against the Cp-I
enzyme were Ͻ0.6 ␮M, there were 8 (40%) whose IC50s in the
growth assay were at least 50-fold higher (i.e., Ͼ3.0 ␮M).
Conversely, there were no compounds whose IC50s in the
growth assay were lower than the IC50s in the enzyme assay. A
reasonable explanation for divergence between DHFR inhibi-
tion and growth inhibition is that the uptake of some of the
lipophilic diaminopyrimidine antifolates into cells, and ulti-
mately into the intracellular parasite, may occur via a mecha-
nism that is more complex than simple diffusion. Evidence has
been presented that shows that trimetrexate and piritrexim are
both substrates for the P-glycoprotein efflux pump in mamma-
lian cells (2). Because of the very unusual nature of the intra-
cellular localization and mode of nutrient uptake of C. parvum
within its host cell (11), very little is known about how drugs
actually get into this organism at different stages of its lifecycle.
However, it is easily conceivable that C. parvum may express
some type of multidrug resistance pump whose affinity for
lipophilic substrates extends to nonclassical antifolates. What-
The relatively low activities of V.9 and V.10 (IC50s, Ͼ3.0
␮M) against C. parvum in culture were of interest because, in
a standard biochemical assay based on selective [3H]uracil
incorporation into the nuclear DNA of T. gondii tachyzoites
cocultured with human embryonic lung cells in folate-contain-
ing medium, these compounds and the 3-trifluoromethoxyben-
zyl analog V.14 had previously been found to have IC50s as low
as 0.1 to 0.3 ␮M (69). Compound V.10 was also active against
P. carinii trophozoites cocultured with rat embryonic lung fi-
broblasts in medium containing 10 ␮M folic acid, but complete
suppression of growth was achieved only at a fairly high con-
centration of 30 ␮M (69). Thus, it appears that lipophilic
antifolates with potent in vitro activity against T. gondii are not
necessarily as potent against either C. parvum or P. carinii. A
compound with the same potency against T. gondii, P. carinii,
and C. parvum would potentially be of therapeutic interest
because AIDS patients are sometimes infected by two or more
of these organisms. However, the likelihood of finding a single
potent inhibitor that binds equally well to the DHFRs of all
three species without also binding tightly to mammalian
DHFR seems remote.

3300
NELSON AND ROSOWSKY
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 3. Growth inhibition of C. parvum intracellular forms
by lipophilic diaminopyrimidine antifolates
Compound
IC50 (␮M)a
I.2................................................................................................... 0.6
III.5................................................................................................ 0.6
III.6................................................................................................ 0.3
IV.4................................................................................................ 2
IV.13.............................................................................................. 2
IV.14.............................................................................................. 0.6
IV.17.............................................................................................. 0.3
IV.18.............................................................................................. 0.2
V.2 ................................................................................................. 0.3
V.5 ................................................................................................. 0.2
V.14 ............................................................................................... 2
V.15 ............................................................................................... 2
V.16 ............................................................................................... 1
VI.6................................................................................................ 2
VIII.8............................................................................................. 0.6
VIII.9............................................................................................. 2
FIG. 6. Inhibition of the development and multiplication of intra-
cellular C. parvum parasites by lipophilic antifolates. MDCK epithelial
cells were plated, grown, and infected as described in the legend to Fig.
5. Following postinfection washes to remove unexcysted oocysts and
extracellular sporozoites, deficient medium–1% dFCS was added to
each well of the chamber slide and 20 mM stock solutions of the
indicated antifolate drugs were serially diluted fivefold across seven
wells. The last well received no drug and served as a control to quantify
uninhibited growth. Drugs and media were renewed at 24 h postinfec-
tion, the experiment was terminated at 48 h, and the monolayers were
processed as described in the legend to Fig. 5. The slides were micro-
scopically examined for epifluorescence, and the numbers of parasite-
containing fluorescent vacuoles were counted in each of five micro-
scopic fields per well (magnification, ca. ϫ400). Results are expressed
as percentages of the control determined as follows: (average number
of parasite-containing vacuoles in drug-treated cells Ϭ average number
of vacuoles in drug-free controls) ϫ 100. Error bars indicate the stan-
dard deviation at each drug concentration. Each drug was tested at
least twice with similar results. Control experiments using uninfected
MDCK cells showed that the drug concentrations used were not toxic
to the cells (data not shown). Symbols: {, trimetrexate; ᮀ, tri-
methoprim; ‚, pyrimethamine; ϫ, compound V.10; ء, compound
V.15; F, compound V.16.
a IC50s for all other compounds in Fig. 2 and 3 were Ͼ3 ␮M, the highest
concentration tested. Concentrations of Ͻ0.2 ␮M were not tested. The assay
protocol used was that described in the legend to Fig. 6.
trimetrexate and leucovorin to treat severe C. parvum infec-
tion, it is conceivable that this approach might be worth trying
when the disease fails to respond to other forms of treatment.
Our finding that several compounds in the test library inhibited
the in vitro growth of C. parvum in mammalian cells at phys-
iologically reasonable low micromolar concentrations compa-
rable to those at which trimetrexate was similarly effective
suggests that lipophilic DHFR inhibitors deserve to be more
extensively tested in combination with leucovorin for the treat-
ment of the most difficult cases of cryptosporidiosis.
It is important to note that the purpose of this study was
simply to explore, at a molecular level (i.e., in a cell-free assay),
whether any particular structural class among the diaminopy-
rimidine DHFR inhibitors we tested might serve as a starting
point for systematic structural modifications aimed at the even-
tual discovery of a drug whose potency and selectivity profile is
superior to that of trimethoprim or pyrimethamine. However,
it should be remembered that DHFR inhibition data, by them-
selves, are only an early step in predicting whether an antifo-
late will be active in an infected cell or a whole animal; indeed,
even screening for antifolate activity in culture can provide
only a preliminary estimate of whether a drug will be an effec-
tive anticryptosporidial agent in patients. Thus, even though
trimethoprim was found in this study to be very selective in its
binding affinity for C. parvum DHFR versus mammalian
DHFR and was also found to suppress parasite growth in a cell
assay, as others have previously noted (79), the ability of this
agent to control cryptosporidial diarrhea in AIDS patients,
even in combination with sulfamethoxazole, has actually been
disappointing. It is axiomatic that the activity of a drug in a
whole animal is determined by a host of factors, only one of
which is binding to the drug’s target. Thus, while we consider
the database reported in this paper to be of interest from the
standpoint of structure-activity correlation at a molecular level,
our results are not intended to be seen as anything more than
a preliminary indication of therapeutic potential.
The enzyme inhibition data for 20 of the compounds in
Table 2 were correlated with the results of the yeast comple-
mentation assay reported earlier (7). Seven compounds (IV.3,
VI.3, VI.4, VI.10, VI.13, VI.14, and VIII.5) were inactive in the
yeast assay, and five of these had IC50s of Ͼ10 ␮M in the
spectrophotometric assay against at least one of the C. parvum
enzymes. Compound VI.4 was anomalous in that it was inef-
fective in the yeast assay even though its IC50 against both C.
parvum enzymes was in the 1 to 5 ␮M range. Low activity in the
yeast assay could occur if drug diffusion through the plate is
slow or if drug penetration through the yeast cell wall is inef-
ficient. Seven compounds (VI.6, VI.7, VII.1, and VII.6) had
some activity in the yeast assay but were less effective than
trimetrexate, and these compounds also had an IC50s of Ͼ10
␮M in the enzyme assay. Five compounds (IV.1, IV.2, V.9,
V.10, and VIII.8) were almost as effective as trimetrexate in
the yeast assay (7), and all of them had IC50s of Ͻ1 ␮M in the
enzyme assay. Thus, with the exception of VI.4, which had an
IC50 in the 1 to 5 ␮M range against both C. parvum enzymes by
spectrophotometric assay but was inactive in the yeast assay,
there was good agreement between the results of the two
methods.
Binding of inhibitors to E. coli DHFR. As shown in Table 4,
the IC50 of trimethoprim against E. coli DHFR, determined
While there are, to date, no reported clinical attempts to use

TABLE 4. Inhibition of E. coli DHFR by di- and tricyclic
under the same assay conditions as were used with the C.
parvum and human enzymes, was 0.012 ␮M, in good agree-
ment with previously published results (3, 9). Among the 86
out of 93 compounds tested against the E. coli enzyme, 31
(36%) were found to be better inhibitors than trimethoprim.
Moreover, 12 of these (II.2, III.3, III.5, IV.14, IV.18, V.2, V.8,
V.10, V.13, V.14, V.16, and VI.5) had IC50s in the 0.1 to 1.0
nM range and 4 others (II.3, III.6, IV.13, and IV.17) had IC50s
that could not be accurately determined because they were
Ͻ0.1 nM. However, because the most active compounds
against E. coli DHFR also proved to be very potent against the
human enzyme (cf. Table 2), they did not match the remark-
able selectivity of trimethoprim.
The preferential affinity of trimethoprim for E. coli DHFR
versus human DHFR has been tentatively ascribed to the fact
that, in the case of the bacterial enzyme (at least in the binary
complex without NADPH present), the trimethoxybenzyl
group can interact with two different hydrophobic pockets at
the active site, called the upper and lower clefts, whereas in the
human enzyme the trimethoxybenzyl group binds only to the
upper cleft (45). The decreased ability of our compounds to
discriminate between the E. coli and human DHFRs, relative
to that of trimethoprim, may be due to the increased separa-
tion between the aryl group of the side chain and the diamin-
opyrimidine moiety, as well as to the fact that the diaminopy-
rimidine moiety is part of a di- or tricyclic ring system. This
combination of features apparently causes the diaminopyrimi-
dine derivatives to lose the ability to interact only with the
upper cleft of the human enzyme. It may be noted that tri-
methoprim displays much more selectivity for the E. coli en-
zyme than for the C. parvum enzyme under identical assay
conditions. Thus, we believe that finding di- or tricyclic DHFR
inhibitors with selectivity for the C. parvum enzyme compara-
ble to that of trimethoprim for the E. coli enzyme may prove
very difficult and that greater success might be achieved by
seeking to increase the potency of trimethoprim rather than to
increase the selectivity of trimetrexate or piritrexim.
diaminopyrimidines
Compound
IC50 (␮M)a
Trimethoprim............................................................ 0.012 Ϯ 0.003
I.1................................................................................ 0.026 Ϯ 0.0076
I.2................................................................................ 0.036 Ϯ 0.015
I.3................................................................................
I.4................................................................................
I.5................................................................................
I.6................................................................................
I.7................................................................................
1.2 Ϯ 0.44
3.1 Ϯ 1.5
0.30 Ϯ 0.064
3.1 Ϯ 2.6
0.23 Ϯ 0.27
II.2 ..............................................................................0.00053 Ϯ 0.0022
II.3 ..............................................................................
Ͻ0.0001
II.4 .............................................................................. 0.0029 Ϯ 0.0029
II.5 .............................................................................. 0.024 Ϯ 0.017
II.6 .............................................................................. 0.059 Ϯ 0.044
III.1............................................................................. 0.0084 Ϯ Ͻ0.001
III.2............................................................................. 0.0061 Ϯ 0.0025
III.3.............................................................................0.00045 Ϯ Ͻ0.0002
III.4............................................................................. 0.012 Ϯ 0.0024
III.5.............................................................................0.00011 Ϯ Ͻ0.00002
III.6.............................................................................
Ͻ0.0001
IV.1............................................................................. 0.0034 Ϯ 0.0028
IV.2............................................................................. 0.046 Ϯ 0.015
IV.3............................................................................. 0.087 Ϯ 0.010
IV.4.............................................................................
IV.5.............................................................................
IV.6.............................................................................
8.5 Ϯ 2.5
0.21 Ϯ 0.088
0.28 Ϯ Ͻ0.0001
IV.7............................................................................. 0.068 Ϯ 0.00887
IV.8.............................................................................
IV.9.............................................................................
IV.19...........................................................................
IV.11...........................................................................
IV.12...........................................................................
IV.13...........................................................................
3.1 Ϯ 0.68
7.5 Ϯ 2.2
4.5 Ϯ 2.6
7.9 Ϯ 1.8
2.0 Ϯ 1.3
Ͻ0.0001
IV.14...........................................................................0.00014 Ϯ Ͻ0.0001
IV.15...........................................................................
IV.16...........................................................................
IV.17...........................................................................
1.5 Ϯ 0.20
1.1 Ϯ 0.16
Ͻ0.0001
IV.18...........................................................................0.00012 Ϯ Ͻ0.0001
V.1 .............................................................................. 0.041 Ϯ 0.014
V.2 ..............................................................................0.00084 Ϯ Ͻ0.0004
V.3 .............................................................................. 0.080 Ϯ 0.020
V.4 .............................................................................. 0.011 Ϯ 0.0020
V.5 .............................................................................. 0.0014 Ϯ 0.0011
V.6 .............................................................................. 0.0036 Ϯ 0.0016
V.7 .............................................................................. 0.0066 Ϯ 0.0013
V.8 ..............................................................................0.00054 Ϯ 0.00025
V.9 .............................................................................. 0.0011 Ϯ 0.0010
V.10 ............................................................................0.00016 Ϯ 0.00012
V.11 ............................................................................ 0.0019 Ϯ 0.00026
V.12 ............................................................................ 0.0030 Ϯ 0.00080
V.13 ............................................................................0.00052 Ϯ 0.00032
V.14 ............................................................................0.00025 Ϯ 0.00026
V.15 ............................................................................ 0.0021 Ϯ 0.0016
V.16 ............................................................................0.00024 Ϯ 0.00012
VI.3............................................................................. 0.0036 Ϯ Ͻ0.001
VI.4............................................................................. 0.031 Ϯ 0.016
VI.5............................................................................. 0.018 Ϯ 0.0052
VI.6............................................................................. 0.075 Ϯ 0.015
VI.7............................................................................. 0.031 Ϯ 0.019
VI.12........................................................................... 0.0075 Ϯ 0.,0023
VII.1 ........................................................................... 0.097 Ϯ 0.031
ACKNOWLEDGMENTS
Support of these studies was provided in part by grant RO-AI29904
to A.R., contract UO1-AI40319 to R.G.N., and contract NO1-AI35171
to S.F.Q. from the National Institute of Allergy and Infectious Dis-
eases.
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VII.4 ...........................................................................
VIII.1..........................................................................
VIII.2..........................................................................
VIII.3..........................................................................
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a The values shown are the means Ϯ the standard deviations of several deter-
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