SPECIFIC INHIBITION OF DIHYDROFOLATE REDUCTASE AND COMPOUNDS THEREFOR

INHIBITION SPECIFIQUE DE LA DIHYDROFOLATE-REDUCTASE ET COMPOSES UTILISES

English Abstract

2095518 9208461 PCTABS00013
Compounds derived from pyrimidines having improved activity
against fungi such as Pneumocystis carinii, and improved
selectivity for P. carinii dihydrofolate reductase over human
dihydrofolate reductase, are disclosed. P. carinii pneumonia is
advantageously treated with the disclosed compounds.

Claims

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What is Claimed:
1. A method for treating fungal infection in a mammal
which method comprises:
administering to a mammal infected with a fungal pathogen an
effective amount of a compound of formula I:
<IMG>
wherein
R1 is 3-R3-4-R4-5-R5-benzyl or (N-R6)-8-azabicyclo[3.2.1]oct-3-yl;
and R2 is H, or R1 and R2 together form <IMG>;
where R3 and R5 are independently selected from the group consist-
ing of H. lower alkoxy, lower alkylthio, lower alkylsulfinyl, vinyl, carboxy-lower
alkyl, carboxy-lower alkoxy, dicarboxy-lower alkyl, dicarboxy-lower alkoxy, aryl-
lower alkoxy, arylsulfonyl-lower alkoxy, arylsulfamido-lower alkoxy, and radicals
of formula -O(CH2)n-COR8, where n is an integer from 0 to 6 and R8 is an amino
acid;

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R4 is selected from the group consisting of lower alkoxy, aryl-lower
alkoxy, lower alkylthio, halo, lower alkenyl, lower alkenyloxy, and pyrrolyl;
with the proviso that R3, R4, and R5 are not simultaneously methoxy;
R6 is selected from the group consisting of unsubstituted aryl and
aryl substituted with one to three radicals selected from the group consisting of
halo, lower alkyl, lower alkoxy, lower alkylthio, carboxy, carbamido, carboxy-
lower alkyl, and carbamido-lower alkyl; and
R7 is selected from the group consisting of aryl and aryl-lower alkyl,
where aryl may be substituted with one to three radicals selected from the groupconsisting of halo, lower alkyl, lower alkoxy, lower alkylthio, carboxy, carbamido,
carboxy-lower alkyl, and carbamido-lower alkyl;
and lower alkyl esters, amides thereof, and pharmaceutically
acceptable addition salts.
2. The method of claim 1, wherein R2 is H and R1 is
3-R3-4-R4-5-R5-benzyl.
3. The method of claim 2, wherein R3 is methoxy.
4. The method of claim 3, wherein R4 is bromo.
5. The method of claim 4, wherein R5 is
-O(CH2)2CH(COOH)(CH2)2COOH, or a pharmaceutically acceptable mono- or
diester thereof.
6. The method of claim 4, wherein R5 is -O(CH2)n-COR5.
7. The method of claim 6, wherein n is 2 and R8 is
-NHCH(COOH)CH2CH2COOH or a mono- or diester thereof.

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8. The method of claim 6, wherein n is 2 and R8 is
-NHCH2COOH or an ester thereof.
9. The method of claim 3 wherein R5 is methoxy.
10. The method of claim 9, wherein R4 is 2-hydroxyprop-2-yl.
11. The method of claim 9, wherein R4 is propen-2-yl.
12. The method of claim 9, wherein R4 is N-pyrrolyl.
13. The method of claim 9, wherein R4 is methylthio.
14. The method of claim 3, wherein R4 is benzyloxy.
15. The method of claim 14, wherein R5 is benzyloxy.
16. The method of claim 3, wherein R4 is methoxy, and R5 is
-OCH2CH2NHSO2(C6H6)NHCOCH3.
17. The method of claim 2 wherein R3 is H.
18. The method of claim 9 wherein R4 is methoxy.
19. The method of claim 10 wherein R5 is -O(CH2)n-COR8.
where n is 2 and R8 is -NHCH2COOH or an ester thereof.
20. The method of claim 9, wherein R4 is benzyloxy and R5
is H.

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21. The method of claim 2, where R3 and R5 are each ethoxy.
22. The method of claim 21, wherein R4 is pyrrolyl.
23. The method of claim 21, wherein R4 is propen-2-yl.
24. The method of claim 2, where R3 and R5 are each vinyl.
25. The method of claim 2, where R3 is methylthio, R4 is
methoxy, and R5 is methylsulfinyl.
26. The method of claim 1, wherein R2 is H and R1 is (N-R6)-
8-azabicyclo[3.2.1]oct-3-yl.
27. The method of claim 26, wherein R6 is selected from the
group consisting of 2-naphthyl, 3,5-dimethoxyphenyl, and 4-carboethoxyphenyl.
28. The method of claim 1, wherein R1 and R2 together form
<IMG>
29. The method of claim 28, wherein R7 is benzyl.
30. The method of claim 28 where R7 is 3,5-dimethoxyphenyl.

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31. The method of claim 2 where R3 and R5 are propoxy and
R4 is N-pyrrolyl.
32. The method of claim 1 wherein said fungal pathogen is
Pneumocystis carinii.
33. The method of claim 1, further comprising coadminstering
a dihydropteroate synthase inhibitor.
34. The method of claim 33, wherein said dihydropteroate
synthase inhibitor is selected from the group consisting of dapsone and sulfa drugs.
35. The method of claim 1, wherein said compound of
formula I is selected from the group consisting of 2,4-diamino-5-(4-benzyloxy-
benzyl)pyrimidine; 2,4-diamino-5-(3,4-dimethoxy-5-benzyloxybenzyl)pyrimidine;
2,4-diamino-5-(3,4-dibenzyloxy-5-methoxybenzyl)pyrimidine; 2,4-diamino-5-[3,5-
dimethoxy-4-(2-hydroxyprop-2-yl)benzyl]pyrimidine; 2,4-diamino-5-(3,5-
dimethoxy-4-N-pyrrolylbenzyl)pyrimidine; 2,4-diamino-5-(3,5-diethoxy-4-N-
pyrrolylbenzyl)pyrimidine; 2,4-diamino-5-(3,5-divinyl-4-vinyloxybenzyl)-
pyrimidine; 2,4-diamino-5-[3-(4-N-acetaminophenyl)sulfonaminoeethoxy-4,5-di-
methoxybenzyl]pyrimidine; 2,4-diamino-5-[3-(4-aminophenyl)sulfonaminoethoxy-
4-methoxybenzyl]pyrimidine; 2,4 diamino-5-[3-(4-N-
acetaminophenyl)sulfonaminoethoxy-4-bromo-5-methoxybenzyl]pyrimidine; 2,4-di-
amino-5-[3-(4-aminophenyl)sulfonaminoethoxy-4-bromo-5-methoxybenzyl]-
pyrimidine; 2,4-diamino-5-[3,5-diethoxy-4-(propen-2-yl)benzyl]pyrimidine; 2,4-
diamino-5-[3,5-dimethoxy-4-(propen-2-yl)benzyl]pyrimidine; 2,4-diamino-5-(3,5-
dimethoxy-4-methylthiobenzyl)pyrimidine; 2,4-diamino-5-(3-methylsulfinyl-4-
methoxy-5-methylthiobenzyl)pyrimidine; 2,4-diamino-5-[3-(4,6-
dicarboxyhexyloxy)-4-bromo-5-methoxybenzyl]pyrimidine; 2,4-diamino-5-[3-(3-

WO 92/08461 PCT/US91/08515
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carboxymethylamino-3-oxopropoxy)-4-methoxybenzyl]pyrimidine; 2,4-diamino-5-
(3-[3-(1,3-dicarboxypropyl)amino-3-oxopropoxy]-4-bromo-5-methoxybenzyl)-
pyrimidine; 2,4-diamino-5-[3,5-dimethoxy-4-((2-phenylsulfonyl)acetyl)benzyl]-
pyrimidine; 2,4-diamino-5-[3-amino-4-methyl-5-(N-pyrrolyl)benzyl]pyrimidine;
2,4-diamino-5-(3,5-di-N-pyrrolyl-4-methoxybenzyl)pyrimidine; 2,4-diamino-5-[3,5-
di-methoxy-4-(3-hydrocarboxy-1-oxopropylamino)benzyl]pyrimidine; 2,4-diamino-
5-[3,5-dimethoxy-(4-acetaminophenylsulfonamino)benzyl]pyrimidine; 2,4-diamino-
5-(3,5-dimethoxy-4-propylbenzyl)pyrimidine; 2,4-diamino-5-(3,5-dichloro-4-N-
pyrrolylbenzyl)pyrimidine; 2,4-diamino-5-[3,5-dimethoxy-4-(2-(2-(2-methoxy)-
ethoxy)ethoxy)ethoxybenzyl]pyrimidine; 2,4-diamino-5-[3-(3-benzyloxycarbonyl-
methylamino-3-oxopropoxy)-4-bromo-5-methoxybenzyl]pyrimidine; 2,4-diamino-5-
[3-(3-carboxymethylamino-3-oxopropoxy)-4-bromo-5-methoxybenzyl]pyrimidine;
2,4-diamino-5-[3-methoxy-4-bromo-5-(4-methylaminobenzamidoethoxy)benzyl]pyr-
imidine; 3-(2,4-diaminopyrimidin-5-ylmethyl)-8-(3,5-dimethoxyphenyl)-8-aza-
bicyclo[3.2.1]octane; 2H,3H-dihydro-5-(2,4-diaminopyrimidin-5-ylmethyl)-6,7-di-
methoxybenzofuran; 5-(2,4-diaminopyrimidin-5-ylmethyl)-7-methoxy-8-bromo-1,2-
benzopyran; 5-(2,4-diaminopyrimidin-5-ylmethyl)-7,8-dimethoxy-1,2-benzopyran;
2,4-diamino-5-[3-phenyl-5-(3-methoxypropoxy)benzyl]pyrimidine; 2,4-diamino-7-
(3,5-dimethoxybenzyl)pyrrolo[2,3-f]quinazoline; 2,4-diamino-5-[6-(4-methoxy-
butoxy)naphth-1-yl]pyrimidine; 2,4 diamino-5-(4,5,6-trimethoxy-2,3-dihydroinden-
1-yl)pyrimidine; 2,2-dimethyl-5-(2,4-diaminopyrimidin-5-ylmethyl)-7-methoxy-
benz[b]dioxolane; 2,4-diamino-5-(3,5-diethoxy-4-carboethoxybenzyl)pyrimidine;
and 2,4 diamino-5-(2,7-dimethylbenzpyrazol-5-ylmethyl)pyrimidine.

Description

WO 92/0&16t PCI`/US91/08515
209~
SPECD~C IN~r~oN OF D~DROFOLATE REDUCTAS~
AND COMPOUNDS T~EREFOR
Descri~tion
Technical Field
This invendon relaus to ph~nracology a~d the inhibi~ion of enzymes
specific to pathogens. More pa~icularly, the invendon relates to methods for spe-
cifically inhibiting the enzyme dihydrofolate reductase in fungal pathogens, andcompounds therefor.
Back~round of the Invention
Pneumocvstis carinii pneumonia (PCP) is a significant, life-threatening
infecdon in immunocompromised subjects, and is a leading cause of mosbidity
and mor~ality in patients presenting acquired immunodeficiency syndrome (A~S~.
Since the onset of the AIDS epidemic, the incidence of PCP has risen from
approximately 200 cases per year to more than 25,000 cases pe~ year in the
United States.
Due to the lack of a conunuous in vitro c~ture system, and the cumber-
some nature of the rat model of PCP, anti-P. cannii therapy has been developed
largely on the assumption that P. carinii was a species of protozoa, and thus that
an~-psotozoal agents were likely to be e~Eective. 'rhe two principle thesapeuùc
modalities, trimethoprinlsulfame~oxazole and pentamidine, wese developed
empirically. Howevèr, P. carinii has recerltly ~een suggested to belong instead to
the Kingdom Fungi (J.C. Edman et al., Nature (1988) 334:519-22).

wo 9V08461 pcr/us91/o8sls
2 ~ 1 8
Trimethoprim (U.S. Pat. No. 2,909,522) and pyrimethamine, and other
dihydrofolate reductase (DHFR) inhibitors are known tO be effective anti-bacterial.
and anti-protozoal agents due to the central role played by DHFR in the metabolic
synthesis of nucleic acid precursors. Despite their efficacy when used in con-
5 junction with a sulfonarnide, trimethoprim and pyrimetharnine are alone poorinhibitors of P. carinii DHFR. For example, trimethopnm and pyrimethamine
exhibit 50% inhibition concentrations (IC50) of 8 and 2,500 nM for E. coli DH~;R,
while IC50s for P. car~ii DHFR are 39,600 and 2,4~0 nM, respectively. Other
anti-folates have been shown to be more effective inhibitors of P. carinii DHFR,10 but require concornitant administration of leucovorm to prevent toxicity to the
host. Allegra et al. (U.S. Pat; No. 4,694,007) suggested treatrnent of P. cannii and
Toxoplasmosis gondii with 2,~diarnino-5-methyl-6-[(3,4,5-trimethoxyanilino)-
methyl]quinazoline (trimetrexate~, on the theory that the DH~ enzyme in this
pathogens is more similar to mammalian DHF~ than to prokaryotic DHF~.
Prior to the AIDS epidemic, these types of agents were sufficient for
treatment of the rare cases of P. carinii pneumonia However, in the HIV-positivepatient, therapy and prophylaxis with the standard anti-P. carinii agents are com-
plicated by frequent toxic and allergic side effects. New compounds that surpassthe efficacy of the hlown antifolates in treating PCP are desirable. especially
20 inhibitors having greater selectivity for P. carinii DHFR relative to the host (par-
ticularly human) DHFR than known inhibitors such as trimethoprim.
Commonly-owned copending U.S. Patent Applicasion Serial No. 447,181,
filed 7 December 1989 disclosed several DHFR inhibitors exhibiting good
selectivity for P. carinii DHFR.

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2~9 ,~1~
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Disclosure of the Invendon
We have now found compounds which exhibit superior activity against
D~;R from fungal pathogens. such as P. carinii. and which exhibit much higher
selectivitY for the fungal en~me as compared with the mammalian (human)
S enzyme. These compounds are represented generally by For~nula I:
Formula I:
R
~ ,q
NHq
20 wherein
R1 is 3-R3-4 R4-5-R5-benzyl or (N-R6)-8-azabicyclo~3.2.1]oct-3-yl; and
,~\N-R,
R~ is H; or R1 and R, together form f~ ~/ where R~ and R5 are
LldepeIldently selected from the group consisting of H, lower aL~coxy, lower
aLkylthio, lower alkylsulfinyl, vinyl, carboxy-lower aL~cyl, carboxy-lower aL~coxy,
25 dicarboxy-lower alkyl, dicarboxy-lower aLtcoxy, aryl-lower aL~oxy, arylsulfonyl-
lower aL"oxy, arylsulfamido-lower alkoxy, and radicals of formula
-O(CH2)"-COR8, where n is an integer from 0 to 6 and R~, is an amino acid: R4 isselected from the group consisting Gf lower alkoxy, aryl-lower aL~coxy, lower
aLkylthio, halo, lower alkenyl, lower aLIcenyloxy, and pyrrolyl; wi~ the proviso

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2Q95~1~
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that R3, R~, and R5 are not simultaneously methoxy; R6 is selected from the group
consisting of unsubstituud aryl and aryl substituted with one to three radicals
selected from the group consisting of halo, lower alkyl, lower aL~oxy, lower alkyl-
thio, carboxy. carbamido. carboxy-lower alkyl, and carbamido-lower alkyl; and R75 is selected from the group consisting of aryl and aryl-lower alkyl, where aryl may
be substituted with one to three radicals selected from the group consisting of
halo, lower alkyl, lower alkoxy, lower allcylthio, carboxy, carbamido, carboxy-
lower alkyl, and carbamido-lower aL~cyl; and lower alkyl esters. amides thereof,and pharmaceutically acceptable addition salts.
One aspec~ of the inven~on is a method for treating a fungal infection
(such as P. cannii) in a marnmal by administe~ing an effective amount of a
compound of foqmula I.
Another aspect of the invendon is a composition for treadng a fungal
infection (such as P. carinii) in a mammal compnsing an effecdve amount of a
15 compound of formula I in combinadon with a pha~naceudcally acceptable
excipient.
Another aspect of the invendon is the use of a compound of formula I to
prepare a composidon for treating a fungal infecdon (such as P. carinii) in a mam-
mal comprising an effective amount of a compound of formula I in combinadon
20 with a pharmaceudcally acceptable excipient.
Modes of Carrying Out The Invendon
A. Definitions
The terms "fungal infecdon" and "fungal pathogen" refer to the infecdon
25 af a mammal with an organism of the Kingdom Fungi, for example Pneumocvsds
i, Aspergillus, Candida, Fusanum, and the lilce. The presently preferred
method of the invention is the treatment of Pneumocvstis ca~ ii using the
compounds of the invendon.

! Wo 9Z/08461 2 ~ 9 S ~ 1 8 Pcr/US91/08515
The term "pharmaceutically acceptable" refers to compounds and compo-
sitions which may be administered to mammals without undue toxicity. Exem-
plaIy pharmaceutically acceptable salts include mineral acid salts such as hydro-
chlorides. hydrobromides, phosphates. sulfates. and the like; and the salts of
5 organic acids such as acetates, propionates, malonates, benzoates, and the like.
The term "effective amount" refers to an amount of compound sufficient
to exhibit a detectable therapeutic effect. The therapeutic effect may include, for
example, without limit~tion, inhibiting the growth of pathogens, inhibitmg or pre-
venting the release of toxins by pathogens, killing pathogens, and preventing the
10 establishmeM of infection (prophyla~cis). The precise effecdve amount for a sub-
ject will depend upon the subject's size and health, the nature of the pathogen, ~e
severity of the infection, and the like. Thus, it is not possible to specify an exact
effective amount in advance. However, the effective amount for a given situa~ioncan be determined by routine experimentation based on the information provided
15 herein.
The term "lower alkyl" refers to saturated straight or branched-chain rad-
icals consisting of carbon and hydrogen having from 1 to 6 carbon atoms, inclu-
sive. such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-hexyl,
and the like. '~ower al~oxy" refers to a radical of the form R-O-, where '~" is
20 lower alkyl as defined above. Suitable examples include methoxy, ethoxy, prop-
oxy, butoxy, and the like. Similarly, "lower aL~cylthio" refes to radicals of the
form R-S-, and 'qower aLkylsulfinyl" refes to groups of the form R-S(-O)-. For
example, one may employ methylthio, ethylthio, methylsulfi~yl, t-butylsulfinyl,
and the li~e. 'I,ower alkenyl" refers to stIaight or branched-chain radicals consist-
25 ing of carbon and hydrogen having 2-6 carbon atoms and at least one double bond
between a pair of carbon atoms, such as ethenyl (vinyl), 2-propenyl, l-methyl-
- ethenyl, 2-butenyl, 3-butenyl, and the like.

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2ass~18
- 6 -
The term "carboxy-lower aL~cyl" refers to radicals havirlg the form
-(CH2)n-COOH, where n is an integer from 1 to 6 inclusive. 'l)icarboxy-lower
alkyl" indicates lower alkyl chains having two COOH groups attached.
"Aryl" denotes cyclic hydrocarbon radicals of 6-10 carbon atoms which
5 exhibit aromatic character, for example phenyl and napthyl.
The term "halo" refers to fluoro, chloro, bromo, and iodo. The term
"amino acid" refers to any of the 20 or so cornmonly occmring amino acids, for
exarnple, glycine. alanine, arginine, phenylalanine, glutamic acid, vali~e, histidine,
prolinè. ornilhine, norleucine. and the lilce. When attached as Ro in a radical of
10 the form -O(CH2)n-COR~, the amino acid will preferably be attached via a pepude
bond l e. by a bond between the amino group of the amino acid and tne acyl car-
bon of the radical.
Tne term "coadministering" means ad~ninistration of a compound of the
invention in combination with a second therapeutic agent. The second therapeutic15 agent is a dihydropteroau synthase inhibitor, preferab}y dapsone or a sulfa drug.
Suitable sulfa drugs include, without lLtnitation, sulfadiazine, sulfamethoxazole,
and the like. Coadm~Iustration may be simultaneous, for example by administer-
ing a mixture of the therapeutic agents, or may be accomplished by adminis~ationof the agents separately within a short time period.
B. General Method
The compounds of the invendon are struct3Ially relaud to the compound
tnmelhoprim (2,4 diamislo-5-(3,4,5-trimetho~cybenzyl)pylinudine), the synthesis for
which is known ifl the att. See for e~ample U.S. Pat. No, 2,909,522, which des-
25 cribes the synthesis of tTimethop~im and related compounds. Compounds of for-mula I may similarly be synthesized by those of ordinaIy sldll ifl the art.
Syntheses of such compounds are described in the following U.S. patents:
Hitchings et aL (2,658,897); Hitchings et al. (2,909,522); Hoffer (3,341,541);
Roth (3,772,289); Roth et al. (3,819,629); Roth (3,822,264); Kompis et aL
.
\

~o 92~08461 PCr/US~1/08515
. 2095:)18
(3,931,181); Herrling (3,980,649); Liebenow et al. (3,992,379); Kompis
(4,024,145); Rosen (4,033,962); Kompis et al. (4,039,543); Jernow et al.
(4,075,209); Perun et al. (4,087,528); Fritschi et al. ~4,180,578); Kompis et al.
(4,203.980); Poe et aL (4,258,045); Daluge et al. (4,438,267); Dall'Asta
(4,485,248); Kompis et al. (4,515,948); Swaringen et al. (4,568,744); Roth et al.
(4,587,341); Kompis et aL (4,590,270); Daluge et al. (4,603,136); Kompis et al.
(4,792,557); and Seydel et aL (4,912,112).
Presently prefelTed compounds of the invention are:
2.4-diamin~5-[3,5-dimethoxy-4 (2-hydro~yprop-2-yl)benzyl]pyr~nidine;
2,~diamino-5-(3,5-dimetho~y-4-N-pyIrolylbenzyl)pyIimidine;
2,4-diamino-5-(3,5 -diethoxy-4-N-pyrrolylbenzyl)pyrimidine;
2,4-diamino-5-(3,5-dipropoxy-4-N-pyrrolylbenzyl)pyrimidine;
2,4-diamino-5-(3,5-dibutoxy-~N-pyrrolylbenzyl)pyrimidine;
2,4-diamino-5-(3,5-diethoxy-4-carboetho~ybenzyl)py~imidine;
2,4-diamino-5-(3,5-divinyl-4-vinyloxybenzyl)pyrimidine;
2,4-diamino-5-[3-(~N-acetaminophenyl)sulfonaminoethoxy-4,5 -dimethoxybenzyl]-
pyrimidine;
2.4-diam~no-5-[3-(~aminophenyl)sulfonaminoethoxy-4-methoxybenzyl]pyrim~dine;
2,4-diamino 5-[3-(~N-acetaminophenyl)sulfonaminoetho~y-4 bromo-5-methoxybenzyl]-
pyIimidine;
2,4-diamino-5-[3-(~aminophenyl)sulfonaminoethoxy-4 bromo-5-methoxybenzyl]-
pyrimidine;
2,4 diamin~5-(3-methoxy-4,5-dibenzyloxybenzyl)pyr~midine;
2,4 diamino-5-(4 benzyloxybenzyl)pynmidine;
2,~diam~no-5-(3,4-dibenzylo~y-5-metho~cybenzyl)pyIimidine;
2,~diamino-5-(3,4 dimethoxy-5-benzylo~cybenzyl)pynmidine;
2,~diamino-5-[3,5-diethoxy-4-(propen-2-yl)benzyl]pyrimidine;
2,4-diamino-5-[3,5-dimethoxy-4-(propen-2-yl)benzyl]pynmidine;
2,~diamino-5-(3,5 -dimethoxy-4-methylthiobenzyl)pyrimidine;

Wo 92/08461 Pcr/US91/08~l5
2~9~j~'i8
- 8 -
2,~diamino-5-(3-methylsulfinyl-4-methoxy-S-methylthiobenzyl)pynmidine;
2,4-dian~no-5-[3-(4,6-dicarboxyhexyloxy) 1 bromo-5-methoxybenzyl]py~imidine;
2,~diamino-5-[3-(3-carboxymethylamino-3-oxopropoxy)-4-methoxybenzyl]-
pyrimidine;
5 2,4-diamino-5-(3-[3-~1,3-dicarboxypropyl)amin~3-oxopropoxy]-4-bromo-5-methoxy-
benzyl)pynmidine;
2,4-diam~no-5-[3 ,5-dimethoxy-4-((2-phenylsulfinyl)acetyl)benzyl]pyrill~idine;
2,~diamino-5-~3-amino-4-methyl-5-(N-pyrrolyl)benzyl]pyrimidine;
- 2.4-diamino-5-(3,5-di-N-pyrrolyl-4-methoxybenzyl)pynmidine;
102~diamino-5-[3,$-di-me~hoxy-4-(3-hydrocarboxy-l-oxopropylamino)benzyl]pyrimidine;
2,4-diamino-5-[3,5-dimethoxy-(4-acetaminophenylsulfonamino)benzyl]pyrin~idine;
2,4-diamino-5-(3 ,5 -dimethoxy-4-propylbenzyl)pyrimidine;
2,4-diamino-5-(3,5-dic'nloro-4-N-pyrrolylbenzyl)pyrimidine;
2,4-diamino-5-[3,5-dimetnoxy-~1 (2-(2-(2-metnoxy)ethoxy)ethoxy)ethoxybenzyl]-
lS pynmidine;
2,4-diamino-S- [3 -(3-benzyloxycarbonylrnethylamino-3-oxopropoxy)-4-bromo-S -methoxy-
benzyl]pyrimidine;
2,4-diamino-5-[3-(3-carboxymethylamino-3-oxopropoxy)-4 bromo-S-methoxybenzyll-
pyrimidine;02.4-diamino-5-[3-methoxy-~bromo-5-(4 methylaminobenzamidoethoxy)benzyl]-
pyrimidine;
3-(2,4-diaminopyrimidin-S-ylmetnyl)-8-(3 ,5-dimethoxyphenyl)-8-
azabicyclo[3.2.1]octane;
2H,3H-dihydro-S -(2,4-diamlnopyrimidin-S-ylme~hyl)-6,7-dimethoxybenzofuran;
25 5-(2,~diaminopyrimi~in-5-yknethyl)-7-methoxy-8-bromo-1,2-benzopyran;
5-(2,4~ninopyrimidin-5-ylmethyl)-7,8-dimethoxy-1,2-benzopyran;
2,4-diamino-5-[3-phenyl-5-(3-metho~cy~opoxy)benzyl]pyrimidine;
2.4-diamino-7-(3 ,5-dimethoxybenzyl)pyrrolo[2,3-flql~inazoline;
2.4-diarnino-S - [6-(4-methoxybutoxy)naphth- 1 -yl]pyrimidine;

- ~O 92/08461 2 0 3 ~ Pcr/US91/085tS
2,~diamino-5-(2,7-dimethylbenzpyrazol-5-ylmethyl)pynmidine;
2.4-diamino-5-(4,5,6-trimethoxy-2,3-dihydroinden-l-yl)pyrimidine; and
2,2 -dimethyl-5 -(2,4-diaminopyrimidin-5 -ylmethyl)-7-methoxybenz[b]dioxolane .
The most preferred compounds at present are 2~4 diamino-5-(3,5-diethoxy-4 N-
pyrrolylbenzyl)pyrimidine and 2,4 diam~no-j-[3,5-dimethoxy-4-(2-hydroxyprop-2-
yl)benzyl]pyrimidine.
Compositions of the invention for administration will generally include
an effecuve amount of a compound of formula I in addition to a pharmaceutically
acceptable excipient. Suitable excipients include most carriers approved for oral
or parenteal administration. including water. saline. Ringer's solution. Hank's
solution, and solutions of glucose, lactose, dextrose, ethanol, glyceroL albun~in,
and the like. These compositions may optionally include stabili7ers~ antioxidants,
antimicrobials. preservatives, buffering agents, surfactants, and other accessory
additives. A presently preferred vehicle comprises about l mglmL serum alburrun
in phosphate-buffered saline (PBS). A thorough discussion of suitable vehicles for
parenteral administration may be found in E.W. Martin, '~emmgton's Pharrna-
ceutical Sciences" (Mack Pub. Co., current edition).
The precise dosage necessary will vary with the age, size. and condition
of the subject, the nature and severity of the disorder to be treated. and the like:
thus, a precise effective amount carmot be specified in advance. However, appro-priau amounts may be determined by routine experimentation with animal models.
general terms, an effective dose of compound of formula I wi~l range from
about lO ,ug/Kg to about 50 mg/Kg. Suitable animal models include the
mouse model illustrated in the Examples below. Rats and other rodents have
DHFR very similar to the human enzyme, and thus make suitable anirnal models.
A group of exper~nental animals is inoculated with lO-lO0 LD~os of Pneumocvstis
~i, followed by treatment with a soludon of test compound. A negative con-
trol group is left untreated, while a positive control group is treated with a stan-
dard therapy, such as trimethoprim. Administration of t~le compounds is prefer-

wo 92/08461 Pcr/ussl/o8sls
209aal8
- 10-
ably per os ~, using a gavage), but may be parenteral, for example by subcu-
taneous or intramuscular injection, or by inhalation of an aerosol. The animals are
monitored during treatment. and are sacrificed and e~amined after 60 days for
presence of infection.
C. Exam~les
The examples presented below are provided as a further guide to the
pracdtioner of ordinary skill in the a~, and are not to be construed as limiting the
invention in any way.
E~amPle 1
(Demonstration of Activity)
A. Materials
Buffers were prepared as follows:
4xDHFR buffer: 200 mM Tes, 300 mM BME, 4 mM EDTA, pH 7Ø
+DHF buffer. 2.5 mg/mL BSA, 0.25 mM NADPH, 625 tlM dihydrofolate. 2.5x DHFR
buffer.
-DHF buffer: 2.5 mglmL BSA, 0.25 mM NADPH, 2.5x DH~;R buffer.
Enzvme buffer 50 mM Tes. S mM DlT, 1 mM EDTA, 20% glycerol, 1 mg/mL BSA,
pH 7Ø
Dilution buffer. 50 mM Tes, S mM DTT, 1 mM E~DTA, 1 mg/mL BSA, pH 7Ø
PcDHFR: S llg/mL P. carinii DHFR in enzyme buffer.
crude hDHFR: crude recombinant human DHFR (obtained ~om Hoffmann-LaRoche)
in enzyme buffer (9.9 mglmL total protein).
25purifled hDHFR: purified recombinant human DH~;R (obtained from Hoffmann-
LaRoche) in enzyme buffer (3.5 mg/mL total protein).
Test compounds were prepared and provided by Ho~mann-LaRoche.
Stock solutio~s were prepared by dissol~ing 2-8 mg in dimethylsulfoxide (DMSO)

WO 92/08461 PCI-/US91/08515
. .
2,~95'~
to prepare 50 mM solutions. Compounds which did not dissolve at 50 mM were
diluted serially to 25, 16.6, or 12.5 mM.
B. Assav
Eight compounds (6.7 ,uL each) were added to wells in one column of a
S Falcon~ 96-well micro~iter plate, and diluted with 160 llL of water. The
remairling wells received 126 ~IL of water. The solutions in the f~st column were
then serially diluted (4x) into columns 2-9, with colurnn 10 containing a control
sample, and columns 11-12 containing water blanks. The final compound
concentrations were 1 mM to 15 nM (after the rema~ng reagents were added).
Using an 8~hannel pipet, 100 ,uL of -DHF buffer were added to each
well in columns 11 and 12 (blanks containing only water). Next. 100 ~IL of
+DHF buffer were added to each well in colurnns 1-10. PcDHFR or crude or
pu~ified rhDHFR enzyme was then added (25 pL) to each well, and the plate
contents mLxed using a Titertek shaker. The plates were read on a Molecular
Devices Plate Reader at an absorbance of 340 nm, -0.05 O.D. scale. for 10 min-
utes reading every 10 seconds. The kinedc data was analyzed using Delta Soft
software (Biometallics), and the IC50 and Ki calculated for both human and P.
carinii enzymes. Selectivity was calculated as Ki~n"",~"/Ki,E ~""",; thus, higher
values for selectivity indicate that the compound inhibits the P. carinii enzyme to
20 a greater degree than the human enzyme. The results are shown in Table 1 below.
For purposes of comparison, trimethoprim in this assay e~hibits an IC50 of 20
with a selectivity of 0.1.

wo 92/08461 pcr/us91/o8~l$
2~ -8
TABLE l: Compounds of Fonnula I
R3 R4 R5 IC50 Selectivity
S
-OMe -OMe -O(CH2)2NHSO2~NHAc 2. ~M 350
-OMe -CMe2OH -OMe 25 ~M 350
10-OEt pyrrolyl -OEt 2.4 ~M 310
-OMe pyrrolyl -OMe 29 ~M 30
vinyl vi~yloxy vinyl 3.4 ~M 250
H BzO- H 16 ~M 50
-OMe BzO- BzO- 8.3 ~M 100
20-OMe -C(Me)=CH2 -OMe 24 ~M 160
-OEt -C(Me)=CH2 -OEt 2.5 ~M 160
-OMe -SMe -OMe 19 ~M 145
-SMe -ONe -S(O)Me 18 ~M 490
-OMeBr -O(CH2)2C~(CH2~2COOHO.04 ~M 525
COOH
-OMeBr -O(CH2)2CO(N-Asp) 2 ~M 4300
H-OMe -O(CH2)2CO(N-Gly) 9.3 ~M 80
-OMeBr -O(CH2)2CO(N-Gly) 10 ~M 8
Me -CH3, E~ - -CH2CH3, Bz - -CH2C6k6, N-~ly - -NHCH2COOH, N-Asp -
-NHCH(COOH)CH2COOH, ~- phenyl, Ac - -COCH3
Example 2
Proceeding as described in E~ample l above, the compounds listed below
were assayed and found to exhibit high activity and selectivity, as set for~ in
Table II:
A) 2,~diamino-5-(~benzylo~cybenzyl)pyTimidine;

Wo 92/084~l PCr/US91/08515
" 209~.jl8
- 13 -
B) 2,~dia~r.uno-5-(3,~dimethoxy-5-benzyloxybenzyl)pyrimidine:
C) 2,4 diarr.uno-5-(3,4 dibenzyloxy-5-methoxybenzyl)pyrirr.udine;
D) 2,~diamino-5-[3,5-dimethoxy-~(2-hydroxyprop-2-yl)benzyl~pyrilI~dine;
E) 2,~diamino-5-(3,5-dimethoxy- 1 N-pyrrolylbenzyl)pynmidine:
F) 2,1 diammo-5-(3,5-diethoxy-4-N-pyrrolylbenzyl)pyIimidine;
G) 2,1 diamino-5-(3~5-divinyl-4 vinyloxybenzyl)pyrimidine;
H) 2,4-diamino-5-[3-(l N-acetaminophenyl)sulfonaminoethoxy-4,5 dimeth-
oxybenzyl]pyrim~dine;
I) 2,~dia~runo-5-[3-(4 aminophenyl)sulfonatninoethoxy-~methoxybenzyl]-
pyri~udine;
J) 2,~diamino-5-[3-(4N-acetaminophenyl)sulfona n~noethoxy-~l bromo-5-
methoxybenzyl]pyrimidine;
K) 2,1 diarnino-5-[3-(~aminophenyl~sulfonaminoethoxy-~bromo-5-methoxy-
benzyl]pyrimidine;
L) 2,~diamino-5-[3,5-diethoxy-1 (propen-2-yl)benzyl]pyr~idine;
M) 2,~1 diamino-5-[3,5-dimethoxy-1 (propen-2-yl)benzyl]pyrimidine;
N~ 2,1 diarnino~5-(3,5-dimethoxy-~methylthiobenzyl)pyrimidine;
O) 2,~diamino-5-(3-methylsulfinyl-~methoxy-5-methylthiobenzyl)-
pyrirnidine;
P) 2,4-diamino-5-[3-(4,6-dicarboxyhexyloxy)-4-bromo-5-methoxybenzyl]-
pyrimidine;
Q) 2,4-diamino-5-[3-(3-carboxymethylamino-3-oxopropoxy)-l methoxy-
benzyl]pynmidine;
R) 2,~diamino-5-(3-[3-(1,3-dicarboxypropyl)amino-3-oxopropoxy]-1 bromo-
5-methoxybenzyl)pylimidi~e;
S) 2,~dian~no-5-[3,5-dimetho~cy-~((2-phenylsulfonyl)acetyl)benzyl]-
pyrimidine;
T) 2,~diam~no-5-[3-am~no-4 methyl-5-(N-pyIrolyl)benzyl]pynmidine;
U) 2,4 diamino-5-(3,5-di-N-pyrrQlyl-4 methoxybenzyl)pyrimidine;

Wo 92/08461 PCr/US91/08~.15
2~9~18
- 14-
V) 2,4-diamino-5-[3,5-di-methoxy-4 (3-hydrocarboxy-1-oxoprowlamino)-
benzyl]pyri~udine;
W) 2,4-dian~ino-5-[3,5-dimethoxy-(~acetaminophenylsulfonarnino)benzyll-
pyr~midine;
5 X) 2,~diamino-5-(3,5-dimethoxy-~propylbenzyl)pyrimidine;
Y) 2,4-diarnino-5-(3,5-dichloro-4-N-pyrrolylbenzyl)pyrimidine;
Z) 2,~diamino-5-[3,5-dimethoxy-4 (2-(2-(2-methoxy)ethoxy)ethoxy)ethoxy-
benzyl]pyrimidine:
AA) 2,4-diarnino-5-[3-(3-benzyloxycarbonylmethylamino-3-oxopropoxy)-~
bromo-S-methoxybenzyl]pyrimidine;
BB) 2,4-diamino-5-[3-(3-carboxymethylamino-3-oxopropoxy)-4-bromo-5-
methoxybenzyl]pyri~udine;
CC) 2,4-diamino-5-[3-methoxy-4-bromo-5-(4-methylaminobenzamidoethoxy)-
benzyl]pyrimidine;
DD) 3-(2,1 diaminopyrimidin-5-ylmethyl)-8-(3,5-dimethoxyphenyl)-8-aza-
bicyclo[3 .2.1 ]octane;
EE~ 2~I,3H-dihydro-5-(2,~diaminopy~imidin-5-ylmethyl)-6,7-dimethoxybenzo-
furan;
FF) 5-(2,4-diaminopyrimidin-5-ylmethyl)-7-methoxy-8-bromo-1.2-benzopyran;
GG) 5-(2,~diaminopyrimidin-5-ylmethyl3-7,8-dimethoxy- 1 ,2-benzopyran;
HH) 2,~diamino-5-[3-phenyl-5-(3-methoxypropoxy)benzyl]pyr~Irudine:
II) 2,'1 diasnino-7-(3,5-dirnethoxybenzyl)pyrrolo[2,3-f~quinazoline;
JJ) 2,1 diamino-5-[~(4 methoxybutoxy)naphth-1-yl]pyrimidine;
KK) 2,4 diamin~5-(4,5,~tnrnethoxy-2,3 dihydroinden-1-yl)pynmidine;
LL) 2,2-dimethyl-5-(2,~diaminopyriIrLidin-S-ylmethyl)-7-methoxybenz[b]di-
oxolane;
MM) 2,~diamino-5-(3,5-diethoxy-1 carboethoxybenzyl)pyrimidine; and
NN) 2,1 diamino-5-(2,7-dimethylbenzpyrazol-5-yLmethyl)pyrirrudine.

WO 92/0&~1 PCT/US91/08515
2~3;~ 1;8
TABLE ~:
¦ Compound IC50 Selectivity ¦
_ _,
A) 16.0 53.2
B) 13.9 3.3
C) 8.3 90.3
¦ D) 24.8 343.5
E) 2.9 29.4
F) 2.4 312.4
G) 3.4 250.5
H) 2.4 248.5
I) 0.57 7.8
J) 3.7 29.5
~) 0.97 12.1
L) 2.5 190.9
M) 24.0 159.8
N) lg.o 143.5
O) 17.5 486.8
P) 0.043 528.0
Q) 9.3 77.7
R) 2.0 4255.0
S) 14.8 36.8
T) 33.5 42.5
U) 4.5 47.7
V) 100.0 85.2
W) 31.0 14.3
X) 20.0 42.6
Y) 10.9 53.2
Z) 36.0 33.1

WO92~08461 PCT/US91/08,C.15
~9 -~8
-16-
TABLE II (continued):
Compound IC50Selectivity
AA) 7.9 14.4
BB) 10.0 13.1
CC) 7.8 14.6
DD) 0.016 31.0
EE) 12.9 26.4
FF) 5.9 6.9
GG) 11.5 266.7
HH) 12.2 11.2
II) 0.039 1.2
JJ) 80.0 106.5
KK) 87.0 97.9
I LL) 13.9 15.0
NM) 8.3 63.6
I NN) 18.0 10.7