Inhibition of Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium dihydrofolate reductases by 2,4-diamino-5-[2-methoxy-5-(ω-carboxyalkyloxy)benzyl]pyrimidines: Marked improvement in potency relative to trimethoprim and species selectivity relative to piritrexim

Article abstract of DOI:10.1021/jm010407u

A series of previously undescribed 2,4-diamino-5-[2-methoxy-5-alkoxybenzyl]pyrimidines (3a-e) and 2,4-diamino-5-[2-methoxy-5-(ω-carboxyalkyloxy)benzyl]pyrimidines (3f-k) with up to eight CH₂ groups in the alkoxy or ω-carboxyalkyloxy side chain were synthesized and tested for the ability to inhibit partially purified dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), Mycobacterium avium (Ma), and rat liver in comparison with two standard inhibitors, trimethoprim (1) and piritrexim (2). The latter drug is known to be extremely potent but shows a marked preference for binding to mammalian DHFR, whereas the former is very selective for the parasite enzymes but is a much weaker inhibitor. The underlying strategy for the synthesis of compounds 3a-k was that a hybrid structure embodying some features of both 1 and 2 might possess a more favorable combination of potency and selectivity than either parent drug. The choice of analogues 3f-k was based on the idea that the acidic ω-carboxyl group might interact preferentially with a basic center in the active site of DHFR from any of the parasite species relative to the active site of mammalian DHFR. In addition, the ω-carboxyl group was expected to improve water solubility relative to 1 or 2. In standardized spectrophotometric assays with dihydrofolate as the substrate and NADPH as the cofactor, 2,4-diamino-5-[(2-methoxy-4-carboxybutyloxy)benzyl]pyrimidine (3g) inhibited Pc DHFR with an IC₅₀ of 0.049 μM and rat DHFR with IC₅₀ of 3.9 μM. Its potency against Pc DHFR was 140-fold greater than that of 1 and close to that of 2, and its selectivity index, defined as the ratio IC₅₀(rat liver)/IC₅₀(P. carinii), was 8-fold higher than that of 1 and > 10⁴fold higher than that of 2. Although it was less potent and less selective against Tg than Pc DHFR, it was very potent as well as highly selective against Ma DHFR, with an IC₅₀ of 0.0058 μM and an IC₅₀(rat liver)/IC₅₀(M. avium) ratio of > 600. Because of this favorable combination of potency and selectivity relative to 1 and 2, compound 3g may be viewed as a promising lead in the search for new antifolates with potential clinical activity against P. carinii and other opportunistic pathogens in patients with AIDS.

Full text of DOI:10.1021/jm010407u

J . Med. Chem. 2002, 45, 233-241
233
In h ibition of P n eu m ocystis ca r in ii, Toxopla sm a gon d ii, a n d Mycoba cter iu m
a viu m Dih yd r ofola te Red u cta ses by
2,4-Dia m in o-5-[2-m eth oxy-5-(ω-ca r boxya lk yloxy)ben zyl]p yr im id in es: Ma r k ed
Im p r ovem en t in P oten cy Rela tive to Tr im eth op r im a n d Sp ecies Selectivity
Rela tive to P ir itr exim
Andre Rosowsky,*,† Ronald A. Forsch,† and Sherry F. Queener‡
Dana-Farber Cancer Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,
Boston, Massachusetts 02115, and the Department of Pharmacology and Toxicology, Indiana University School of Medicine,
Indianapolis, Indiana 46202
Received August 28, 2001
A series of previously undescribed 2,4-diamino-5-[2-methoxy-5-alkoxybenzyl]pyrimidines (3a -
e) and 2,4-diamino-5-[2-methoxy-5-(ω-carboxyalkyloxy)benzyl]pyrimidines (3f-k ) with up to
eight CH2 groups in the alkoxy or ω-carboxyalkyloxy side chain were synthesized and tested
for the ability to inhibit partially purified dihydrofolate reductase (DHFR) from Pneumocystis
carinii (Pc), Toxoplasma gondii (Tg), Mycobacterium avium (Ma), and rat liver in comparison
with two standard inhibitors, trimethoprim (1) and piritrexim (2). The latter drug is known to
be extremely potent but shows a marked preference for binding to mammalian DHFR, whereas
the former is very selective for the parasite enzymes but is a much weaker inhibitor. The
underlying strategy for the synthesis of compounds 3a -k was that a hybrid structure embodying
some features of both 1 and 2 might possess a more favorable combination of potency and
selectivity than either parent drug. The choice of analogues 3f-k was based on the idea that
the acidic ω-carboxyl group might interact preferentially with a basic center in the active site
of DHFR from any of the parasite species relative to the active site of mammalian DHFR. In
addition, the ω-carboxyl group was expected to improve water solubility relative to 1 or 2. In
standardized spectrophotometric assays with dihydrofolate as the substrate and NADPH as
the cofactor, 2,4-diamino-5-[(2-methoxy-4-carboxybutyloxy)benzyl]pyrimidine (3g) inhibited Pc
DHFR with an IC50 of 0.049 µM and rat DHFR with IC50 of 3.9 µM. Its potency against Pc
DHFR was 140-fold greater than that of 1 and close to that of 2, and its selectivity index,
defined as the ratio IC50(rat liver)/IC50(P. carinii), was 8-fold higher than that of 1 and >104-
fold higher than that of 2. Although it was less potent and less selective against Tg than Pc
DHFR, it was very potent as well as highly selective against Ma DHFR, with an IC50 of 0.0058
µM and an IC50(rat liver)/IC50(M. avium) ratio of >600. Because of this favorable combination
of potency and selectivity relative to 1 and 2, compound 3g may be viewed as a promising lead
in the search for new antifolates with potential clinical activity against P. carinii and other
opportunistic pathogens in patients with AIDS.
In tr od u ction
cotrimoxazole, is popularly known as Bactrim). A num-
ber of clinical studies evaluating a variety of TMP-sulfa
drug combinations for treatment or prevention of life-
threatening P. carinii and T. gondii infections in AIDS
have been reported in the past decade.2,3 Cotrimoxazole
is relatively inexpensive and thus plays an especially
important role in controlling AIDS opportunistic infec-
tions in economically disadvantaged countries. Initially
developed as an anticancer drug,4 PTX has recently
shown some promise against head-and-neck squamous
cell carcinoma and bladder carcinoma5 and has been the
subject of one limited clinical study against P. carinii
pneumonia in patients with AIDS.6 Both TMP and PTX
have as their target the enzyme dihydrofolate reductase
(DHFR), which plays a ubiquitous role in one-carbon
metabolism by mediating the biosynthesis of DNA,
RNA, and the essential amino acid methionine.7
Trimethoprim (1, TMP) and piritrexim (2, PTX) are
lipid-soluble antifolates that have been used clinically
for the prophylaxis and treatment of Pneumocystis
carinii and Toxoplasma gondii infections in patients
with AIDS.1 TMP was developed over 40 years ago as
an antibacterial drug and continues to be widely pre-
scribed, generally in combination with a sulfonamide
such as sulfamethoxazole (this combination, also called
* To whom correspondence should be addressed. Phone: 617-632-
3117. Fax: 617-632-2410. E-mail: andre_rosowsky@dfci.harvard.edu.
† Harvard Medical School.
Effective use of TMP against P. carinii and T. gondii
requires coadministration of a sulfa drug because these
parasites are able to synthesize reduced folate cofactors
‡ Indiana University School of Medicine.
10.1021/jm010407u CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/06/2001

234 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Rosowsky et al.
Sch em e 1a
a
Reagents: (a) MeOC(dO)Cl/C5H5N; (b) MeOCHCl2/TiCl4; (c) HCl, 0°C; (d) HCl/MeOH, reflux; (e) Br(CH2)5Me/K2CO3/DMSO; (f)
3-morpholinopropionitrile/NaOMe/DMSO; (g) PhNH2‚HCl/EtOH; (h) H2NC(dNH)NH2/EtOH.
de novo. Indeed, these organisms lack a membrane-
transport protein for reduced folates and thus cannot
rely on their host for their supply of these essential
cofactors.8 However, complete inhibition of one-carbon
metabolism in P. carinii and T. gondii cannot be
achieved by blocking the action of DHFR with a weak
inhibitor like TMP but requires de novo synthesis of
reduced folate cofactors in the parasite to be turned off
as well. An unfortunate disadvantage of sulfa drugs is
that some patients tend to become severely allergic to
them and therefore cannot tolerate chronic treatment
with TMP-sulfa drug combinations.9a Although allergic
side effects can be lessened with corticosteroids, the use
of steroids in AIDS can itself pose significant risk.9b
Thus, a TMP analogue potent enough against P. carinii
or T. gondii to circumvent the need to administer a sulfa
drug would be highly desirable.
Tighter-binding DHFR inhibitors than TMP have the
potential to be effective without coadministration of a
sulfa drug, provided that they bind sufficiently better
to the P. carinii and/or T. gondii enzyme than to the
enzyme of the host. A modest level of species selectivity
for both P. carinii and T. gondii DHFR compared with
mammalian DHFR is shown by TMP, but its potency
as an inhibitor of the enzyme is only in the micromolar
range.10a-d By contrast, PTX inhibits DHFR at nano-
molar concentrations but unfortunately binds much
more tightly to the mammalian enzyme than it does to
the P. carinii or T. gondii enzyme.10a-d For this reason,
in the clinical trial to assess the efficacy of PTX against
P. carinii pneumonitis in AIDS patients, 5-formyl-
5,6,7,8-tetrahydrofolate (leucovorin) was coadministered
in order to prevent life-threatening hematologic toxic-
ity.6
increase potency and species selectivity in tandem. The
present paper describes the synthesis of the previously
unknown compounds 3a -k and also reports data on
their activity as inhibitors of partially purified prepara-
tion of DHFR from rat liver, P. carinii, and T. gondii.
Because of the well-documented prevalence of tubercu-
losis as a life-threatening opportunistic disease in AIDS
patients,14 we also report the activity of these com-
pounds against the enzyme from Mycobacterium avium.
A number of DHFR inhibitors have been found to inhibit
proliferation of M. avium in culture or in an animal
model,15 but the potential benefit of lipophilic antifolates
in combination with leucovorin and sulfa drugs for the
prophylaxis or therapy of opportunistic tuberculosis
infection in AIDS patients appears to be untested at this
point.
Ch em istr y
As shown in Scheme 1, treatment of 4-methoxyphenol
(4) with methyl chloroformate in the presence of pyri-
dine afforded the carbonate ester 5 (92% yield), which
on reaction with MeOCHCl2 and TiCl4 as described
earlier16 was converted to the masked aldehyde 6. On
treatment with HCl at 0 °C for 30 min, compound 6
underwent cleavage to aldehyde 7. More vigorous acid-
olysis of 7 cleaved the carbonate ester to form the phenol
8, and further reaction with 1-bromopentane in the
presence of K2CO3 in DMSO solution afforded the
previously unknown ether 9. Condensation of 9 with
3-morpholinopropionitrile in anhydrous DMSO contain-
ing a catalytic amount of NaOMe17a led to the 3-mor-
pholinoacrylonitrile 10, which on sequential heating
with aniline hydrochloride and guanidine17b was con-
verted into 2,4-diamino-5-[(2-methoxy-5-pentyloxy)ben-
zyl]pyrimidine (3a ) via the 3-anilinoacrylonitrile 11.
Exchange of the morpholino group by an anilino group
was an essential step in the sequence because 10 itself
failed to undergo cyclization with guanidine. Com-
pounds 10 and 11 were each used without purification.
As part of a larger search for lipophilic DHFR inhibi-
tors that would combine the species selectivity of TMP
with the potency of PTX,11 we were interested in
synthesizing analogues of general structure 3, in which
the 2,4-diaminopyrimidine moiety of TMP was retained
but the pattern of substitution in the right-hand benzyl
moiety was modified from that of TMP (i.e., 3′,4′,5′-
trimethoxy) to that of PTX (i.e., 2′,5′-dimethoxy). More-
over, we postulated, on the basis of reported 3D struc-
tures of the ternary complexes of P. carinii and
mammalian DHFR with TMP12 and PTX,13 that re-
placement of the O-methyl group at the 5′-position by
long-chain O-alkyl or O-(ω-carboxyalkyl) groups might

Inhibition of Dihydrofolate Reductases
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 235
Sch em e 2a
a
Reagents: (a) NaOMe/MeOH; (b) RBr/DMSO; (c) 3-morpholinopropionitrile/NaOMe/DMSO; (d) PhNH2‚HCl/EtOH; (e) H2NC(dNH)NH2;
(f) TsOH/MeOH; (g) NaOEt/EtOH; (h) Br(CH2)nCO2Et/DMSO; (i) NaOH/aqueous DMSO.
The overall yield of 3a for the seven steps starting from
4 was 30%. The final product was purified by flash
chromatography on silica gel (10:1 EtOAc/MeOH) fol-
lowed by recrystallization from aqueous EtOH. In
H2O, it was converted to 2,4-diamino-5-[(2-methoxy-5-
hydroxy)benzyl]pyrimidine (15) in 89% yield. The Na
salt 16 was then generated with a stoichiometric
amount of NaOEt in EtOH and was isolated by evapo-
ration of the solvent. After being rigorously dried in a
vacuum, 16 was taken up in dry DMSO and allowed to
react with 1 equiv of ethyl 5-bromopentanoate at room
temperature while monitoring the progress of the reac-
tion by TLC (silica gel, 5:1 EtOAc/MeOH). The resulting
ester 17 (n ) 5) was saponified by addition of a 2-fold
excess of NaOH directly to the alkylation mixture, and
the resulting acid 3g was purified by column chroma-
tography on Dowex 50W-X2 sulfonic acid resin using
1.5% NH4OH as the eluent. Freeze-drying of appropri-
ately pooled fractions followed by redissolution in a
minimal volume of dilute NH4OH and acidification with
10% AcOH afforded the product as an off-white powder,
usually solvated with AcOH and/or H2O. Purification
by ion-exchange chromatography on a DEAE-cellulose
(HCO3- form) was also tried but was found to be much
less satisfactory because of poor solubility. The same
sequence of steps was also used to obtain 3f and 3h -k
from other bromo esters via 17 (n ) 3, 4, 6-8) with an
overall two-step yield of 65-85% based on 15. The
identity of each product was confirmed by 1H NMR and
by microanalysis. As with 3a -e, characteristic singlets
were observed at δ 3.46, 3.72, and 7.37, and where
solvation with AcOH was indicated by microanalysis,
an extra singlet not assignable to protons in the di-
aminopyrimidine derivative was observed at δ1.88. It
may be noted that Scheme 2 should, in principle, be
applicable to the synthesis of 3a -e but was not used
for this purpose because adequate amounts of the simple
O-alkyl analogues for in vitro testing had already been
made via Scheme 1.
1
addition to other alkyl and aryl proton signals, the H
NMR spectrum of 3a in DMSO-d6 solution featured the
expected singlets at δ 3.47 for the CH2 group between
the phenyl and pyrimidine rings, δ 3.73 for the 2-OMe
group, and δ 7.39 for the C6 proton on the pyrimidine
ring.
Although the synthesis of 3a as described above was
workable, we felt that an improvement would be to use
NaOMe for the deprotection of 7. We reasoned that
cleavage of the carbonate protecting group would afford
a DMSO-soluble Na salt of the phenol that could be
alkylated directly. Thus, 7 was treated with 1 equiv of
NaOMe in MeOH at room temperature, the solution was
evaporated to dryness, and the residue was triturated
with Et2O to obtain the Na salt (12) as a solid. After
being dried thoroughly under vacuum, the solid was
dissolved in dry DMSO and allowed to react with
1-bromohexane at room temperature to obtain the
O-hexyl derivative of 8, which was then subjected to the
same sequence of steps as 9 (cf. Scheme 1) to obtain 2,4-
diamino-5-[(2-methoxy-5-hexyloxy)benzyl]pyrimidine (3b)
with a five-step yield of 58% starting from 8. The same
improved sequence using other bromoalkanes afforded
the longer-chain analogues 3c-e in overall yields of 63%
(3c), 58% (3d ), and 47% (3e).
Because of a concern that the strongly basic conden-
sation reactions in Scheme 1 might cause side reactions
involving the R-CH2 or carbonyl group, a somewhat
different sequence of steps was followed to obtain the
carboxy analogues 3f-k . Thus, as shown in Scheme 2,
compound 8 was converted to 12 with NaOMe in MeOH,
the dried salt was condensed with benzyl bromide in
DMSO, and the resulting O-benzyl derivative 13a was
converted to the previously unknown diaminopyrimi-
dine 14a (59% yield), from which we had intended to
cleave the O-benzyl group by catalytic hydrogenolysis
over Pd/C. In the event, this debenzylation proved to
be quite difficult, and we therefore abandoned 14a in
favor of the O-(4-methoxybenzyl) derivative 14b. When
14b was left to stand for 2 days at room temperature
in MeOH solution containing a 2-fold excess of TsOH‚
En zym e In h ibition
Compounds 3a -k were tested for their ability to
inhibit DHFR from P. carinii, T. gondii, M. avium, and
rat liver as described in earlier papers,10b,c and selectiv-
ity index (SI) values were determined from the ratio
IC50(rat liver)/IC50(P. carinii, T. gondii, or M. avium).
The results are shown in Table 1, along with previously

236 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Rosowsky et al.
Ta ble 1. Inhibition of P. carinii, T. gondii, M. avium, and Rat
Liver DHFR by 2,4-Diamino-5-[2-methoxy-5-(long-chain alkoxy
and ω-carboxyalkoxy)benzyl]pyrimidines
than that of 3g, confirming that the optimal value of n
against P. carinii DHFR was 4. The carboxyalkyl
derivatives 3f-k were also more potent than 3a -e
against both T. gondii and M. avium DHFR. With the
exception of 3c, this was also true for inhibition of the
rat enzyme. As in the case of the P. carinii enzyme, 3g
was the best inhibitor of T. gondii and M. avium DHFR
but was also a potent inhibitor of the rat enzyme.
A gratifying aspect of the data in Table 1 was the high
selectivity of 3g for P. carinii versus rat DHFR. Al-
though a somewhat lower SI was obtained with 3h , both
compounds were superior to 1 and 2. The selectivity
index of 3g for this enzyme was 7-fold greater than that
of 1 and >104 times greater than that of 2. Compound
3g also displayed the best selectivity index of any of the
compounds in both the 5′-O-alkyl and 5′-O-(ω-carboxy-
alkyl) series against the M. avium enzyme. Interest-
ingly, the potency of 3g was 33-fold higher than that of
1 against both the M. avium and rat DHFR, and thus,
there was no difference in SI for the M. avium enzyme
between these two compounds.
It was of interest to note that the pattern of selectivity
of 3f-k for T. gondii versus rat DHFR differed mark-
edly from the pattern against the P. carinii and M.
avium enzymes in that the most selective compound was
3i rather than 3g and that the SI was relatively
insensitive to the length of the side chain, with 3i (SI
) 65) being only twice as selective as the other con-
geners (SI ) 28-35). Also worth noting is the greater
similarity in potency between 3g and 2 against P. carinii
and T. gondii DHFR than against rat DHFR, which
suggests that the active site of these two enzyme may
have features not present in the rat enzyme. While all
three of the parasite enzymes have been cloned and
sequenced,18 only the 3D structure of the P. carinii
enzyme is published.12b,c
There was a striking similarity between our results
and those published about 20 years ago by Kuyper and
co-workers21 in one of the earliest examples of rational
structure-based design of DHFR inhibitors. In a ho-
mologous series of TMP analogues in which the 3′-OMe
group was replaced by O-(ω-carboxyalkyl) substituents
containing up to six CH2 groups, inhibition assays
against E. coli DHFR revealed a progressive decrease
in the Ki values from 2.6 to 0.035 nM as the number of
CH2 groups was increased from 1 to 3, followed by
stabilization of the K1 in the 0.025-0.066 nM range as
this number was increased from 3 to 6. The highest
affinity for E. coli DHFR was observed with the O-(5-
carboxypentyl) analogue 18. On the basis of the results
a
IC50 (µM)
SIb
P.
T.
M.
rat
P.
T.
M.
cmpd carinii gondii avium
liver carinii gondii avium
1
2
12
2.7
0.19
130
11
44
680
ND
140
74
0.031 0.017 ND
19
14
5.6
44
51
0.25
0.049 0.11
0.80
2.6
0.0015 0.048
0.088
6.8
3.1
2.1
2.0
2.7
14
35
35
65
33
3a
3b
3c
3d
3e
3f
3g c
3h
3i
6.5
9.2
5.0
15
32
0.18
0.31
0.39
0.89
3.3
44
29
11
30
86
2.3
2.1
1.9
0.69
1.7
11
80
21
4.5
1.7
2.5
12
9.1
2.7
560
660
110
63
32
0.0048 2.6
0.0058 3.9
0.15
0.19
0.13
0.15
0.48
0.18
0.36
0.43
17
12
12
12
3j
3k
7.1
4.8
92
80
28
a
Results for trimethoprim (1) and piritrexim (2) were obtained
under the same standardized assay conditions as 3a -k , and are
b
taken from ref 11. ND ) not determined. SI ) IC50(rat liver)/
IC50(P. carinii, T. gondii, or M. avium). c In a separate experiment
to confirm the results for 3g against P. carinii and rat liver DHFR,
the IC50 values were found to be 0.055 and 5.4 µM, respectively,
giving a calculated SI of 98.
reported data for trimethoprim (1) and piritrexim (2)
also presented for comparison.
The most potent of the O-alkyl derivatives against P.
carinii, T. gondii, and rat liver DHFR was 3c (n ) 6),
with IC50 values of 5.6, 5.0, and 11 µM, respectively.
Moreover, there was an increase in potency against all
three enzymes as the length of the 5′-O-alkyl group
increased from n ) 4 to n ) 6, followed by a decrease in
potency as this length increased from n ) 6 to n ) 8.
However, this pattern was slightly different in the case
of the M. avium enzyme, which tended to be more
sensitive than the others. The most potent of the 5′-(O-
alkyl) derivatives against this enzyme were 3a (n ) 4)
and 3b (n ) 5), with IC50 values of 0.31 and 0.39 µM.
Interestingly, while 3a -e were only marginally selective
for P. carinii and T. gondii DHFR relative to the rat
enzyme, 3a was somewhat selective for M. avium
DHFR, with SI values of 140. Although the SI of 3a
against M. avium DHFR was not as high as that of
trimethoprim (1), which had an SI of 680, these results
suggested that efforts directed toward the discovery
of more selective analogues than 3a might be of
interest.
As can be seen from Table 1, the potency of 3c, the
best of the 5′-(O-alkyl) derivatives, was slightly greater
against P. carinii DHFR than that of 1. However,
because 3c inhibited the rat enzyme 12-fold better than
1, its SI was lower. Moreover, 3c showed even lower
selectivity for the T. gondii enzyme. On the other hand,
because of the very high potency of piritrexim (2)
against rat DHFR, the selectivity of 3c for both the P.
carinii and the T. gondii enzymes relative to 2 was
improved by at least 10-fold.
The most potent of the 5′-O-(ω-carboxyalkyl) ana-
logues 3f-k against P. carinii DHFR was 3g (n ) 4),
with an IC50 of 0.049 µM compared with 12 µM for 1
and 0.031 µM for 2. Thus, 3g approached the potency
of 2 against this enzyme and was 245 times more potent
than 1. As in the 5-(O-alkyl) series, the potency of the
5′-O-(ω-carboxyalkyl) derivatives decreased when n was
greater than 4. Moreover, the potency of 3f was lower
presented here, it would appear (a) that P. carinii and
M. avium DHFR resemble E. coli DHFR in showing a
binding preference for derivatives with a medium-length
O-(ω-carboxyalkyl) group at the 3′ position and (b) that
this feature is retained even when the OMe groups at
the 3′ and 4′ positions are replaced by a single OMe
group at the 2′ position.

Inhibition of Dihydrofolate Reductases
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 237
Lancaster Synthesis (Windham, NH). Elemental analyses were
performed by Quantitative Technologies, Inc. (Whitehouse, NJ )
and were within (0.4% of theoretical values.
2-Meth oxy-5-p en tyloxyben za ld eh yd e (9). Step 1. A
stirred solution of 4 (24.8 g, 0.2 mol) and pyridine (16.6 g, 17
mL, 0.21 mol) in CH2Cl2 (150 mL) was cooled in an ice bath
and treated dropwise with MeOCOCl (19.9 g, 16.2 mL, 0.21
mol) over a period of 15 min. The mixture was then stirred at
room temperature for 45 min. After extraction with 1 N HCl,
H2O, 0.5 N NaOH, and H2O, the organic solvent was removed
by rotary evaporation and the residue distilled under vacuum
to obtain 4-(methoxycarbonyloxy)anisole (5) as a colorless
liquid that crystallized on prolonged standing and was used
directly in the next step: yield 33.4 g (92%); bp 112 °C/0.2
Torr; mp 33-34 °C (lit.27 mp 34.5-36 °C); 1H NMR (200 MHz,
CDCl3) δ 3.73 (s, 3H, MeO), 3.83 (s, 3H, MeOCO), 6.85 (d, J )
9 Hz, 2H, aryl 3- and 5-H), 7.11 (d, J ) 9 Hz, 2H, aryl 2- and
6-H).
Step 2. To a stirred solution of 5 (20 g, 0.11 mol) in CH2Cl2
(250 mL) at 0 °C was added TiCl4 (49.2 g, 28.5 mL, 0.26 mol)
in CH2Cl2 (50 mL). Stirring was continued at 0 °C while a
solution of MeOCHCl2 (14.6 g, 11.5 mL, 0.127 mol) was added
dropwise over 30 min. When the addition was complete, the
solution was warmed to room temperature, left to stand for
30 min, and concentrated to half-volume by rotary evaporation.
The resulting solution, containing the R-chloro ether 6, was
poured into a mixture of ice (250 g) and 12 N HCl (10 mL),
and then EtOAc (200 mL) was added. The two-phase mixture
was stirred vigorously for 30 min, the layers were separated,
the aqueous layer was back-extracted with EtOAc, and the
combined EtOAc extracts were evaporated to dryness. Recrys-
tallization of the crude solid (24.2 g) from a mixture of
isooctane and absolute EtOH afforded 2-methoxy-5-(methoxy-
carbonyloxy)benzaldehyde (7) as white needles (17.7 g, 77%):
mp 88-89 °C (lit.16 mp 88-90 °C); 1H NMR (CDCl3) δ 3.90 (s,
3H, MeO), 3.92 (s, 3H, MeO), 6.99 (d, J ) 9 Hz, 1H, aryl 3-H),
7.38 (dd, J ) 9 Hz, J ) 3 Hz, 1H aryl 4-H), 7.63 (d, J ) 3 Hz,
1H, aryl 6-H), 10.45 (s, 1H, CHdO). Evaporation of the
supernatant from the first crop afforded an additional 5.3 g
(23%), bringing the total to 23 g (100%). The two fractions were
combined and used without further purification.
St ep 3. A suspension of the carbonate ester 7 (5.28 g, 5
mmol) in a mixture of MeOH (25 mL) and 2 N HCl (50 mL)
was refluxed for 6 h, during which most of the solid dissolved.
A small amount of insoluble tar was filtered off, the MeOH
was evaporated under reduced pressure, and the residue was
extracted with EtOAc. Evaporation of the EtOAc extract and
recrystallization from H2O containing a small amount of EtOH
afforded 8 as a yellow powder (2.49 g, 65%) that was used
directly in the next step: mp 109-110 °C (lit.16 mp 111-113
°C); TLC Rf ) 0.2 (silica gel, 2:1 isooctane/EtOAc).
Step 4. A solution of 8 (760 mg, 5 mmol) and K2CO3 (828
mg, 6 mmol) in dry DMSO (5 mL) was treated with 1-bromo-
pentane (744 µL, 906 mg, 6 mmol). After 20 h at room
temperature and then 10 min at 60 °C, the mixture was diluted
with H2O and extracted with EtOAc. Evaporation under
reduced pressure left a solid (1.1 g), which was chromato-
graphed on silica gel (15 g, 2 cm × 12 cm) with 2:1 isooctane/
EtOAc as the eluent. Appropriately pooled fractions were
evaporated to obtain 2-methoxy-5-pentyloxybenzaldehyde (9)
as a yellow oil (1.12 g, 100%): TLC bluish spot, Rf ) 0.6 (silica
gel, 2:1 isooctane/EtOAc); IR (thin film) ν 2960, 2930, 2870,
2750, 1680, 1605, 1580, 1495, 1425 cm-1; 1H NMR (200 MHz,
CDCl3) δ 0.85 (m, 3H, pentyl Me), 1.35 (m, 4H, pentyl 3- and
4-CH2), 1.73 (p, J ) 7 Hz, 2H, pentyl 2-Me), 3.85 (s, 3H, MeO),
3.90 (t, J ) 7 Hz, 2H, OCH2), 6.90 (d, J ) 9 Hz, 1H, aryl H-3),
7.09 (dd, J ) 9 Hz, J ) 3 Hz, 1H, aryl H-4), 7.28 (d, J ) 3 Hz,
1H, aryl H-6), 10.40 (s, 1H, CHdO). Anal. (C13H18O3) C, H.
F igu r e 1. Structures of P. carinii DHFR inhibitors reported
to be more potent as well as more selective than trimethoprim
(1).
The published literature contains hundreds of mono-,
di-, and tricyclic 2,4-diaminopyrimidines that are more
potent than 1 as inhibitors of P. carinii versus rat
DHFR, but a careful search reveals only a handful
whose selectivity and potency are both greater than
those of 1 (Figure 1). These include the trimethoprim
analogue 19 (epiroprim, Ro11-8958),19,20 the pyri-
methamine analogues 20 and 21,22 the purine 22,23 the
furo[2,3-d]pyrimidines 23 and 24,24 and the pteridines
25 and 26.25,26 Thus, compound 3g, which most nearly
approaches 21 in terms of having a highly favorable
combination of potency and selectivity, joins this small
group and thus may be viewed as a promising lead for
structure-activity optimization.
Exp er im en ta l Section
IR spectra were obtained on a Perkin-Elmer model 781
double-beam recording spectrophotometer. Only peaks with
wavenumbers above 1400 cm-1 are reported. 1H NMR spectra
were recorded at 200 MHz on a Varian VX200 instrument, or
in some instances at 60 MHz on a Varian EM360 instrument
with Me4Si as the internal standard. Each peak is denoted as
a singlet (s), broad singlet (br s), doublet (d), doublet of doublets
(dd), triplet (t), or pentet (p). TLC analyses were on Whatman
MK6F silica gel plates with UV illumination at 254 nm.
Column chromatography was on Baker 7024 flash silica gel
(40 µM particle size). Melting points were measured in Pyrex
capillary tubes in a Mel-Temp “electrothermal” apparatus
(Fisher Scientific, Pittsburgh, PA) and are not corrected.
3-Morpholinopropionitrile was prepared by adding acrylonitrile
dropwise to an equimolar amount of morpholine in an ice bath
and stirring the mixture at room temperature for 1 h. The
resulting light-yellow oil was used directly to prepare 13 and
other 2-arylmethyl-3-morpholinoacrylonitriles. Other chemi-
cals were purchased from Sigma-Aldrich (St. Louis, MO) or
2,4-Dia m in o-5-(2-m eth oxy-5-p en tyloxyben zyl)p yr im i-
d in e (3a ). Step 1. A solution of NaOMe was prepared by
dissolving clean metallic Na (23 mg, 1 mmol) in absolute
MeOH (3 mL). The solvent was evaporated under reduced
pressure, the solid was taken up in DMSO (1.5 mL), and to
the solution was added 3-morpholinopropionitrile (728 mg, 5.2

238 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Rosowsky et al.
mmol). The solution was placed in an oil bath at 100 °C, and
to it was slowly added a solution of 9 (1.05 g, 4.73 mmol) in
DMSO (1.5 mL). After 20 min of heating, the reaction mixture
was cooled and partitioned between EtOAc and H2O that had
been slightly acidified with dilute aqueous citric acid to prevent
the formation of an emulsion. TLC analysis (silica gel, 1:1
isooctane/EtOAc) showed an I2-absorbing spot with Rf ) 0.3
(not UV-absorbing), along with a major UV-absorbing spot (Rf
) 0.4) corresponding to the product (10) and a faint UV-
absorbing impurity (Rf ) 0.7). Flash chromatography on silica
gel (20 g, 2 cm × 17 cm) afforded 2-(2-methoxy-5-pentyloxy-
benzyl)-3-morpholinoacrylonitrile (10) as a yellow gum that
(see above). The following analogues were prepared by this
general method.
2,4-Dia m in o-5-(2-m et h oxy-5-h exyloxyb en zyl)p yr im i-
d in e (3b). Yield 950 mg (58%); glistening white plates, mp
139-140 °C; IR (KBr) ν 3430, 3330, 3250, 3130, 3010, 2950,
2870, 1675, 1645, 1610, 1575, 1545, 1510, 1490, 1475, 1450,
1435, 1400 cm-1; 1H NMR (200 MHz, DMSO-d6) δ 0.86 (t, J )
7 Hz, 3H, hexyl Me), 1.30 (m, 6H, hexyl 3- to 5-CH2), 1.64 (p,
J ) 7 Hz, 2H, hexyl 2-CH2), 3.47 (s, 2H, bridge CH2), 3.73 (s,
3H, OMe), 3.83 (t, J ) 7 Hz, 2H, OCH2), 5.65 (br s, 2H, NH2),
6.02 (br s, 2H, NH2), 6.64 (d, J ) 3 Hz, 1H, aryl H-6), 6.70
(dd, J ) 8 Hz, J ) 3 Hz, 1H, aryl H-4), 6.87 (d, J ) 8 Hz, 1H,
aryl H-3), 7.39 (s, 1H, pyrimidine H-6). Anal. (C18H26N4O2) C,
H, N.
2,4-Dia m in o-5-(2-m eth oxy-5-h ep tyloxyben zyl)p yr im i-
d in e (3c). Yield 1.09 mg (63%); glistening white plates, mp
134-135 °C; IR (KBr) ν 3400, 3310, 3210, 3110, 2990, 2920,
2850, 1665, 1635, 1605, 1575, 1540, 1500, 1485, 1455, 1440,
1415 cm-1; 1H NMR (200 MHz, DMSO-d6) δ 0.86 (t, J ) 7 Hz,
3H, heptyl Me), 1.28 (m, 8H, heptyl 3- to 6-CH2), 1.65 (p, J )
7 Hz, 2H, heptyl 2-CH2), 3.47 (s, 2H, bridge CH2), 3.73 (s, 3H,
OMe), 3.83 (t, J ) 7 Hz, 2H, OCH2), 5.66 (br s, 2H, NH2), 6.03
(br s, 2H, NH2), 6.65 (d, J ) 3 Hz, 1H, aryl H-6), 6.72 (dd, J )
8 Hz, J ) 3 Hz, 1H, aryl H-4), 6.87 (d, J ) 8 Hz, 1H, aryl
H-3), 7.40 (s, 1H, pyrimidine H-6). Anal. (C19H28N4O2) C, H,
N.
2,4-Dia m in o-5-(2-m et h oxy-5-oct yloxyb en zyl)p yr im i-
d in e (3d ). Yield 1.03 mg (58%); mp 135-136 °C; IR (KBr) ν
3400, 3300, 3110, 2980, 2910, 2840, 1660, 1645, 1600, 1565,
1535, 1500, 1480, 1455, 1435, 1415 cm-1; 1H NMR (200 MHz,
DMSO-d6) δ 0.85 (t, J ) 7 Hz, 3H, octyl Me), 1.27 (m, 10H,
octyl 3- to 7-CH2), 1.66 (p, J ) 7 Hz, 2H, octyl 2-CH2), 3.49 (s,
2H, bridge CH2), 3.75 (s, 3H, OMe), 3.85 (t, J ) 7 Hz, 2H,
OCH2), 5.67 (br s, 2H, NH2), 6.05 (br s, 2H, NH2), 6.66 (d, J )
3 Hz, 1H, aryl H-6), 6.74 (dd, J ) 8 Hz, J ) 3 Hz, 1H, aryl
H-4), 6.88 (d, J ) 8 Hz, 1H, aryl H-3), 7.41 (s, 1H, pyrimidine
H-6). Anal. (C20H30N4O2) C, H, N.
2,4-Dia m in o-5-(2-m et h oxy-5-n on yloxyb en zyl)p yr im i-
d in e (3e). Yield 877 mg (47%); mp 134-135 °C; IR (KBr) ν
3410, 3320, 3120, 2920, 2850, 1665, 1635, 1605, 1565, 1540,
1500, 1485, 1455, 1440, 1420 cm-1; 1H NMR (200 MHz, DMSO-
d6) δ 0.86 (t, J ) 7 Hz, 3H, nonyl Me), 1.25 (m, 12H, nonyl 3-
to 8-CH2), 1.64 (p, J ) 7 Hz, 2H, nonyl 2-CH2), 3.47 (s, 2H,
bridge CH2), 3.74 (s, 3H, OMe), 3.83 (t, J ) 7 Hz, 2H, OCH2),
5.65 (br s, 2H, NH2), 6.03 (br s, 2H, NH2), 6.65 (d, J ) 3 Hz,
1H, aryl H-6), 6.72 (dd, J ) 8 Hz, J ) 3 Hz, 1H, aryl H-4),
6.88 (d, J ) 8 Hz, 1H, aryl H-3), 7.40 (s, 1H, pyrimidine H-6).
Anal. (C21H32N4O2) C, H, N.
2,4-Dia m in o-5-[2-m eth oxy-5-(4-ben zyloxy)ben zyl]p yr -
im id in e (14a ). This compound was prepared by the same
procedure as for 3b-e except that the scale was increased
2-fold, the alkylation step with benzyl bromide was performed
in MeOH instead of DMSO, and the reaction mixture was
refluxed for 18 h. Yield 1.99 g (59%); beige powder, mp 157-
158 °C; IR (KBr) ν 3510, 3480, 3420, 3360, 3300, 3160, 3110,
3050, 3030, 1675, 1640, 1605, 1575, 1505, 1460 cm-1; 1H NMR
(200 MHz, DMSO-d6) δ 3.49 (s, 2H, bridge CH2), 3.74 (s, 3H,
OMe), 4.99 (s, 2H, OCH2), 5.67 (br s, 2H, NH2), 6.05 (br s, 2H,
NH2), 6.75-6.95 (m, 3H, aryl protons on the anisole), 7.40 (m,
6H, pyrimidine H-6 and aryl protons on the benzyloxy group).
Anal. (C19H20N4O2) C, H, N.
2-Methoxy-4-[(4-methoxybenzyl)oxy]benzaldehyde (13b).
Compound 8 (2.10 g, 0.01 mol) was added to a solution of
NaOMe prepared by dissolving metallic Na (0.23 g, 0.01 mol)
in absolute MeOH (40 mL). After 5 min, the solvent was
evaporated and the residue triturated with Et2O until a
powder formed, at which point the Et2O was decanted. The
powder was dried in vacuo and taken up in dry DMF (2 mL),
and to the solution was added 4-methoxybenzyl chloride
(freshly synthesized from 4-methoxybenzyl alcohol and SOCl2
in toluene). After 20 h at room temperature, the DMF was
evaporated under reduced pressure and the residue was
partitioned between EtOAc and 1 N NaOH. The organic layer
was evaporated, and the residue was recrystallized from EtOH/
1
was used directly the next step: yield 1.16 g (71%); H NMR
(200 MHz, CDCl3) δ 0.88 (m, 3H, pentyl CH3), 1.41 (m, 4H,
pentyl 3- and 4-CH2), 1.75 (p, J ) 7 Hz, 2H, pentyl 2-CH2),
3.0-3.8 (m, 15H, morpholine CH2, MeO, pentyl OCH2, and
benzylic CH2), 6.79 (m, 4H, vinyl and aryl protons).
Step 2. A solution of 10 (0.835 g, 2.43 mmol) and aniline
hydrochloride (0.47 g, 3.64 mmol) in absolute EtOH (10 mL)
was refluxed for 1 h. In a separate flask, guanidine hydro-
chloride (1.09 g, 11.4 mmol) was added to a solution of NaOEt
prepared by dissolving clean metallic Na (345 mg, 15 mmol)
in absolute EtOH (15 mL), and the flask was swirled manually
for 5 min. The entire contents of the second flask (including
the NaCl) were added to the first, and the combined mixture
was refluxed for 18 h and then filtered while hot. TLC analysis
of the filtrate (silica gel, 10:1 EtOAc/MeOH) showed the
product (3a ) as a major UV-absorbing spot (Rf ) 0.3), along
with several fast-moving impurities. Flash chromatography
(silica gel, 18 g, 2 cm × 15 cm) with 10:1 EtOAc/MeOH as the
eluent afforded an amorphous solid, which on recrystallization
from aqueous EtOH afforded 3a as white flakes (540 mg,
70%): mp 140-141 °C; IR (KBr) ν 3410, 3330, 3230, 3120,
2990, 2950, 2910sh, 2870, 2830, 1665, 1635, 1605, 1565, 1540,
1505, 1480, 1460, 1440, 1420 cm-1; 1H NMR (200 MHz, DMSO-
d6) δ 0.87 (m, 3H, pentyl Me), 1.33 (m, 4H, pentyl 3- and
4-CH2), 1.64 (p, J ) 7 Hz, 2H, pentyl 2-CH2), 3.47 (s. 2H, bridge
CH2), 3.73 (s, 3H, OMe), 3.83 (t, J ) 7 Hz, 2H, OCH2), 5.65
(broad s, 2H, NH2), 6.02 (br s, 2H, NH2), 6.64 (d, J ) 3 Hz,
1H, aryl H-6), 6.70 (dd, J ) 8 Hz, J ) 3H, 1H, aryl H-4), 6.87
(d, J ) 8 Hz, 1H, aryl H-3), 7.39 (s, 1H, pyrimidine H-6). Anal.
(C17H24N4O2) C, H, N.
Gen er a l P r oced u r e for a Mor e Dir ect Syn th esis of 2,4-
Diam in o-5-(2-m eth oxy-5-alkoxyben zyl)pyr im idin es. Com-
pound 8 (1.05 g, 5 mmol) was added to a solution of NaOMe
prepared by dissolving clean metallic Na (115 mg, 5 mmol) in
absolute MeOH (5 mL). After 5 min, the intensely yellow
solution of the Na salt 12 was evaporated to dryness and the
residue was triturated with Et2O until a solid formed. The
Et2O was decanted and the powder dried with aid of a vacuum
pump and taken up in DMSO (5 mL). The alkyl halide (5
mmol) was then added and the solution allowed to stand at
room temperature overnight. In a separate flask, 3-morpho-
linopropionitrile (700 mg, 5 mmol) was added to a solution of
freshly prepared NaOMe (1 mmol) in DMSO (2 mL), and the
mixture was placed in an oil bath preheated to 100 °C. To this
solution were then added the contents of the first flask, and
the combined reaction mixture was heated for 20 min. After
being cooled to room temperature, the mixture was partitioned
between EtOAc and dilute aqueous citric acid. TLC analysis
of the organic layer gave predominantly one spot with an Rf
of ca. 0.5 (silica gel, 1:1 EtOAc/isooctane) corresponding to the
desired 2-arylmethyl-3-morpholinoacrylonitrile. The EtOAc
was evaporated and replaced with absolute EtOH (20 mL), and
after addition of aniline hydrochloride (0.97 g, 7.5 mmol), the
mixture was refluxed for 1 h. Separately, guanidine hydro-
chloride (1.19 g, 12.5 mmol) was added to a solution of NaOEt
obtained by dissolving Na metal (460 mg, 20 mmol) in absolute
EtOH (15 mL). After being stirred for 5 min, the contents of
the flask (including the precipitated NaCl) were transferred
to the flask containing the 2-arylmethyl-3-anilinoacrylonitrile.
The mixture was refluxed for 20 h and filtered while hot, and
the product was isolated and purified in the same way as 3a

Inhibition of Dihydrofolate Reductases
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 239
H2O. Drying in a lyophilization apparatus afforded 13b as a
white powder (1.91 g, 70%): mp 75-76 °C; TLC bluish spot,
Rf ) 0.4 (silica gel, 2:1 isooctane/EtOH); IR (KBr) ν 2970, 2940,
2840, 1675, 1610, 1585, 1515, 1490, 1465, 1450, 1435, 1425
cm-1; 1H NMR (200 MHz, CDCl3) δ 3.82 (s, 3H, OMe), 3.90 (s,
3H, OMe), 4.98 (s, 2H benzylic CH2), 6.91 (m, 4H, aryl protons
of 4-methoxybenzyl group), 7.18 (dd, J ) 8 Hz, J ) 3 Hz, 1H,
benzaldehyde H-4), 7.35 (d, J ) 8 Hz, 1H, benzaldehyde H-3),
7.42 (d, J ) 3 Hz, 1H, benzaldehyde H-6), 10.44 (s, 1H, CHdO).
Anal. (C16H16O4) C, H.
drying in vacuo over P2O5. The following compounds were
obtained in this manner.
2,4-Diam in o-5-[(2-m eth oxy-5-(3-car boxypr opyloxy)ben -
zyl]p yr im id in e (3f). White solid (19% yield), mp 244-246
°C; IR (KBr) ν 3340, 3210, 2960 (broad underlying absorbance
at 3600-2450), 1665, 1650, 1630 sh, 1565, 1505, 1465, 1410
cm-1; 1H NMR (200 MHz, DMSO-d6) δ 1.87 (m, 2H, CH2CH2-
CH2), 2.32 (t, J ) 7 Hz, 2H, CH2COOH), 3.47 (s, benzylic CH2
partially overlapping a broad H2O peak), 3.72 (s, OMe partially
overlapping H2O), 3.84 (t, J ) 6 Hz, OCH2 partially over-
lapping H2O), 5.66 (br s, 2H, NH2), 6.03 (br s, 2H, NH2), 6.68
(m, 2H, aryl H-4 and H-6), 6.87 (d, J ) 8 Hz, aryl H-3), 7.37
(s, 1H, pyrimidine H-6). Anal. (C16H20N4O4‚3.2H2O) C, H, N.
2,4-Diam in o-5-[2-m eth oxy-5-(4-m eth oxyben zyloxy)ben -
zyl]p yr im id in e (14b). This compound was prepared from 13b
in 61% yield via the general procedure described above for 3b-
e: white powder, mp 220-221 °C; IR (KBr) ν 3480, 3410, 3340,
3250, 3120, 2950, 3120, 2960, 2920, 2830, 1665, 1620, 1595,
1565, 1510, 1495, 1450, 1415 cm-1; 1H NMR (DMSO-d6) δ 3.48
(br s, >2H, bridge CH2 and partial H2O of solvation), 3.74 (s,
3H, OMe), 3.75 (s, 3H, OMe), 4.89 (s, 2H, OCH2), 5.66 (br s,
2H, NH2), 6.04 (br s, 2H, NH2), 6.72-7.00 (m, 5H, aryl
protons), 7.33 (d, J ) 8 Hz, 2H, aryl protons), 7.40 (s, 1H,
pyrimidine H-6). Anal. (C20H22N4O3‚0.25H2O) C, H, N.
2,4-Dia m in o-5-[(2-m eth oxy-5-(4-ca r boxybu tyloxy)ben -
zyl]p yr im id in e (3g). Beige solid (65% yield), mp 94-100 °C;
IR (KBr) ν 3340, 3170, 2940 (broad underlying absorbance at
3400-2700), 1655, 1500, 1460 cm-1 1H NMR (200 MHz,
;
DMSO-d6) δ 1.64 (m, 4H, CH2(CH2)2CH2), 2.32 (t, J ) 7 Hz,
2H, CH2COOH), 3.46 (s, 2H, benzylic CH2), 3.72 (s, 3H, OMe),
3.83 (t, J ) 6 Hz, 2H, OCH2), 5.75 (br s, 2H, NH2), 6.12 (br s,
2H, NH2), 6.60 (d, J ) 3 Hz, 1H, aryl H-6), 6.73 (dd, J ) 8 Hz,
J ) 3 Hz, 1H, aryl H-4), 6.87 (d, J ) 8 Hz, 1H, aryl H-3), 7.37
(s, 1H, pyrimidine H-6). This compound was purified on DEAE-
cellulose (HCO3- form), but this method was less effective than
the use of Dowex 50W-X2 (H+ form) because of precipitation
on the column. The presence of AcOH was indicated by a
singlet at δ 1.89. Anal. (C17H22N4O4‚0.75AcOH‚0.5H2O) C, H,
N.
2,4-Dia m in o-5-(2-m et h oxy-5-h yd r oxyb en zyl)p yr im i-
d in e (15). A stirred suspension of 14b (183 mg, 0.5 mmol) in
MeOH (5 mL) was treated with TsOH‚H2O (190 mg, 1 mmol),
whereupon the solids dissolved and a new precipitate formed
quickly The solution was left at room temperature for 2 days,
after which the solvent was evaporated and the residue was
suspended in H2O. The mixture was adjusted to pH >10 with
NaOH, a trace of insoluble material was filtered, and the
filtrate was applied onto a column of Dowex 50W-X2 resin (H+
form, 1.5 cm × 15 cm). The column was washed with H2O until
the eluent was neutral and UV-transparent and then with
1.5% NH4OH to remove the product. Appropriately pooled
fractions were concentrated and freeze-dried to obtain 15 as
a light-brown powder (110 mg, 89%): TLC Rf 0.2 (silica gel,
5:1 EtOAc/MeOH). The analytical sample was prepared by
recrystallization from EtOH/H2O: mp 216-218 °C dec; IR
(KBr) ν 3430, 3350, 3210, 3050, 2960, 2930, 2830, 2680 w, 2500
br, 1665 sh, 1640, 1615, 1605 sh, 1565, 1540, 1505, 1470, 1445
2,4-Diam in o-5-[(2-m eth oxy-5-(5-car boxypen tyloxy)ben -
zyl]p yr im id in e (3h ). Beige powder (85% yield), mp 100-104
°C; IR (KBr) ν 3330, 3190, 2930, 2860 (broad underlying
1
absorbance at 3500-2400), 1655, 1560, 1495, 1455 cm-1; H
NMR (200 MHz, DMSO-d6) δ 1.30-1.70 (m, 6H, CH2(CH2)3-
CH2), 2.19 (t, J ) 7 Hz, 2H, CH2COOH), 3.46 (s, 2H, benzylic
CH2), 3.72 (s, 3H, OMe), 3.82 (t, J ) 6 Hz, 2H, OCH2), 5.66
(br s, 2H, NH2), 6.03 (br s, 2H, NH2), 6.64 (d, J ) 3 Hz, 1H,
aryl H-6), 6.72 (dd, J ) 8 Hz, J ) 3 H, 1H, aryl H-4), 6.86 (d,
J ) 8 Hz, 1H, aryl H-3), 7.38 (s, 1H, pyrimidine H-6). The
presence of AcOH of was indicated by a singlet at δ 1.89. Anal.
(C18H24N4O4‚0.5AcOH) C, H, N.
1
cm-1; H NMR (200 MHz, DMSO-d6) δ 3.45 (s, 2H, benzylic
2,4-Dia m in o-5-[(2-m eth oxy-5-(6-ca r boxyh exyloxy)ben -
zyl]p yr im id in e (3i). Light-brown powder (77% yield), mp
200-202 °C; IR (KBr) ν 3330, 3190, 2930, 2850 (broad
underlying absorbance at 3500-2700), 1655, 1555, 1535, 1495,
1455 cm-1; 1H NMR (200 MHz, DMSO-d6) δ 1.32-1.63 (m, 8H,
CH2(CH2)4CH2), 2.19 (t, J ) 7 Hz, 2H, CH2COOH), 3.46 (s,
benzylic CH2 partially overlapping a broad H2O peak), 3.72
(s, OMe partially overlapping H2O), 3.82 (t, 2H, J ) 6 Hz,
OCH2), 5.69 (br s, 2H, NH2), 6.06 (br s, 2H, NH2), 6.64 (d, J )
3 Hz, 1H, aryl H-6), 6.72 (dd, J ) 8 Hz, J ) 3 H, 1H, aryl
H-4), 6.86 (d, J ) 8 Hz, 1H, aryl H-3), 7.38 (s, 1H, pyrimidine
H-6). Anal. (C19H26N4O4‚0.75H2O) C, H, N.
CH2), 3.71 (s, 3H, OMe), 5.66 (br s, 2H, NH2), 6.00 (br s, 2H,
NH2), 6.44 (d, J ) 3 Hz, 1H, aryl H-6), 6.54 (dd, J ) 8 Hz, J
) 3 Hz, 1H, aryl 4-H), 6.78 (d, J ) 8 Hz, 1H, aryl H-3), 7.39
(s, 1H, pyrimidine H-6), 8.80 (s, 1H, phenolic OH). The
presence of a small amount of EtOH in the sample was
indicated by a small triplet at δ 1.05. Anal. (C12H14N4O2‚
0.2EtOH) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 2,4-Dia m in o-
5-[2-m et h oxy-5-(ω-ca r b oxya lk oxy)b en zyl]p yr im id in es
3f-k . Clean metallic Na (23 mg (1.0 mmol) was dissolved in
absolute EtOH (2 mL), the solvent was evaporated under
reduced pressure, and the residue was redissolved in DMSO
(3 mL). To this solution was then added compound 15 (246
mg, 1.0 mmol), and after the reaction mixture was stirred at
room temperature for 30 min the bromo ester (1.0 mmol) was
added in a single portion. TLC analysis (silica gel, 5:1 EtOAc/
MeOH) revealed gradual loss of the spot with Rf ) 0.2
(unreacted 15) and its replacement by a faster-moving spot
with Rf ) 0.4. The reaction was typically over in 2 h. The
product was saponified instantaneously upon addition of 2 N
NaOH (1.5 mL) followed by addition of H2O to a final volume
of ca. 60 mL. Any insoluble material remaining at this point
was filtered off, and the clear solution was applied onto a
column of Dowex 50W-X2 (H+ form, 2 cm × 20 cm). The column
was washed with H2O until the eluate was neutral and UV-
transparent and then with 1.5% NH4OH to remove the
product. Appropriately pooled eluates were concentrated to
dryness by rotary evaporation followed by lyophilization. The
solid was redissolved in H2O with a small volume of dilute
NH4OH added as needed and was reprecipitated with 10%
AcOH. The collected product was then dried and analyzed.
Microchemical analyses typically indicated the presence of
fractional amounts of AcOH or H2O of solvation despite careful
2,4-Diam in o-5-[(2-m eth oxy-5-(7-car boxyh eptyloxy)ben -
zyl]p yr im id in e (3j). Light-brown powder (74% yield), mp
108-112 °C; IR (KBr) ν 3330, 3180, 2920, 2850 (broad
underlying absorbance at 3500-2700), 1655, 1525 sh, 1495,
1460 cm-1 1H NMR (200 MHz, DMSO-d6) δ 1.29-1.62 (m,
;
10H, CH2(CH2)5CH2), 2.17 (t, J ) 7 Hz, 2H, CH2COOH), 3.46
(s, 2H, benzylic CH2), 3.72 (s, 3H, OMe), 3.82 (t, 2H, J ) 6 Hz,
OCH2), 5.71 (br s, 2H, NH2), 6.08 (br s, 2H, NH2), 6.66 (d, J )
3 Hz, 1H, aryl H-6), 6.73 (dd, J ) 8 Hz, J ) 3 H, 1H, aryl
H-4), 6.86 (d, J ) 8 Hz, 1H, aryl H-3), 7.38 (s, 1H, pyrimidine
H-6). The presence of AcOH was indicated by a singlet at δ
1.89. Anal. (C20H28N4O4‚0.75AcOH) C, H, N.
2,4-Diam in o-5-[(2-m eth oxy-5-(7-car boxyh eptyloxy)ben -
zyl]p yr im id in e (3k ). Light-brown powder (80% yield), mp
142-144 °C; IR (KBr) ν 3330, 3190, 2920, 2850 (broad
underlying absorbance at 3500-2700), 1655, 1555, 1495, 1460
cm-1 1H NMR (200 MHz, DMSO-d6) δ 1.25-1.63 (m, 12H,
;
CH2(CH2)6CH2), 2.17 (t, J ) 7 Hz, 2H, CH2COOH), 3.47 (s,
benzylic CH2 partially overlapping a broad H2O peak), 3.72
(s, OMe, partially overlapping H2O), 3.82 (t, OCH2 partially
overlapping H2O), 5.73 (br s, 2H, NH2), 6.09 (br s, 2H, NH2),
6.68 (d, J ) 3 Hz, 1H, aryl H-6), 6.73 (dd, J ) 8 Hz, J ) 3 H,

240 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Rosowsky et al.
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1H, aryl H-4), 6.88 (d, J ) 8 Hz, 1H, aryl H-3), 7.38 (s, 1H,
pyrimidine H-6). The presence of AcOH was indicated by a
singlet at δ 1.90. Anal. (C21H30N4O4‚0.5AcOH‚0.25 H2O) C, H,
N.
Ack n ow led gm en t. This work was supported in part
by Grant RO1-AI29904 from the National Institute of
Allergy and Infectious Diseases, Department of Health
and Human Services.
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