7-Methyl trimethoprim analogues as inhibitors of the folate metabolizing enzymes

Article abstract of DOI:10.1002/jhet.5570400315

A series of 5-(1-phenylethyl)pyrimidines 2-10 (Table I) were designed and synthesized as potent and selective inhibitors of Pneumocystis carinii (P. carinii), Toxoplasma gondii (T. gondii) and Mycobacterium avium (M. avium) dihydrofolate reductases (DHFR)

Full text of DOI:10.1002/jhet.5570400315

507
7-Methyl Trimethoprim Analogues as Inhibitors of the
Folate Metabolizing Enzymes [1]
May-Jun 2003
,a
a
b
Aleem Gangjee,* Xin Lin and Sherry F. Queener
a
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences
Duquesne University, Pittsburgh, PA 15282
b
Department of Pharmacology and Toxicology, School of Medicine,
Indiana University, Indianapolis, IN 46202
Received January 17, 2003
A series of 5-(1-phenylethyl)pyrimidines 2-10 (Table I) were designed and synthesized as potent and
selective inhibitors of Pneumocystis carinii (P. carinii), Toxoplasma gondii (T. gondii) and Mycobacterium
avium (M. avium) dihydrofolate reductases (DHFR). The structure of 2-10 incorporates a 7–methyl group
to increase the potency of monocyclic trimethoprim (TMP). The target compounds were synthesized by an
acid catalyzed condensation of ethyl cyanoacetate and appropriately substituted benzaldehydes followed
by a Michael addition using methyl copper-lithium. The resulting adduct was cyclocondensed with
guanidine to afford 2,6-diamino-4-hydroxy-5-(1-phenylethyl)pyrimidines 2-7. Both amino moieties of 2-4
were protected with pivaloyl groups and their 4-hydroxy group chlorinated with phosphorus oxychloride.
The resulting intermediates were subjected to hydrogenation and deprotection to afford 8-10. Compound 7
was a good inhibitor of DHFR, however the other compounds were poor inhibitors of P. carinii, T. gondii
and M. avium DHFR.
J. Heterocyclic Chem., 40, 507 (2003).
Opportunistic infections, especially those caused by
(DHFR) and T. gondii DHFR, and has to be administered
with sulfonamides to provide synergistic effects.
Pneumocystis carinii (P. carinii) and Toxoplasma gondii
(T. gondii) are the leading cause of death in patients with
acquired immunodeficiency syndrome (AIDS) [2,3]. In
addition, Mycobacterium avium complex, a group of organ-
isms that is responsible for disseminated infections in AIDS
patients, decreases the quality of life of patients with AIDS.
P. carinii infection is treated with antifolates like trimetho-
prim along with sulfamethoxazole or with pentamidine.
Successful as they are in prolonging the period of survival
of AIDS patients, current therapies are effective in only
50-75% of cases with considerable loss of efficacy in
patients with two or more episodes of infection [4-6]. For
the treatment of T. gondii infection, a combination of the
antifolate pyrimethamine and sulfadiazine is used. In the
current regimens for both infections, relapse rates are high
and the side effects are severe enough that in 50% of the
cases, the treatment has to be discontinued [7,8].
With potencies 100-10000 times that of trimethoprim
against P. carinni and T. gondii DHFR, trimetrexate
(TMQ) and the pyrido[2,3-d]pyrimidine piritrexim (PTX)
[11,12] would appear to be better choices for treatment.
However, due to a complete lack of selectivity resulting in
unacceptable toxicities, these drugs have to be used along
with leucovorin for host rescue. This provides the host but
not P. carinii or T. gondii (because it can not be taken up
efficiently by these organisms) with sufficient reduced
folate to circumvent the dihydrofolate reductase inhibition.
Up to 60 percent of patients being treated with TMQ suffer
from relapse within 3 months [13].
Thus, the dihydrofolate reductase inhibitors currently
used clinically are either selective but weak (e.g. TMP), or
potent but non-selective (e.g. TMQ) against these patho-
genic dihydrofolate reductases. Thus the discovery of
dihydrofolate reductase inhibitors that are both potent and
selective is a most desirable goal.
Trimethoprim was reported [9,10] as a weak but
selective inhibitor of P. carinii dihydrofolate reductase
Figure 1

508
A. Gangjee, X. Lin and S. F. Queener
Vol. 40
Both TMQ and PTX (Figure I) have a 6-6 ring system
and possess a 5-methyl group. Gangjee et al. [14] designed
and synthesized a 5-desmethylpyrido[2,3-d]pyrimidine
analogue of TMQ, 1, whose potency was decreased com-
pared to the 5-methyl analogue. These results suggest that
the 5-methyl group in TMQ and perhaps PTX is important
for high potency. On the basis of X-ray crystal structure
[15,16] and molecular modeling, the 5-methyl group is
shown to interact with amino acid residues in dihydrofo-
late reductase and perhaps also affect the conformation of
the 6-side chain. Thus we designed a series of 7-methyl
TMP analogues and their derivatives 2-10 (Table I). As in
the case of TMP, TMQ, and PTX, all but one of these ana-
logues contains electron donating di- or tri-methoxy sub-
stitutions in the phenyl side chain. Compound 7 has elec-
tron withdrawing 2,4-dichloro substitutions and was
included for comparison. The addition of a methyl group
at the 7-position of TMP not only introduces a certain
degree of conformational restriction to the side chain (thus
decreasing the number of accessible low energy conforma-
tions), but should also increase the hydrophobic interac-
tion with dihydrofolate reductase like the 5-methyl moiety
of TMQ and PTX. We anticipated that this modification
could afford increased potency with retention of selectivity
in TMP analogues.
The initial synthetic strategy was to condense barbituric
acid and substituted acetophenones to afford the desired
heterocycle shown in Scheme I. The condensation product
11 could be converted to the desired target compounds
using a literature procedure reported by Chan and Roth
[17]. While the barbituric acid readily condensed with
benzaldehydes, the acetophenones were extremely inert
and did not condense.
The failure of the previous methodology prompted a
total synthetic strategy shown in Scheme II. The synthesis

May-Jun 2003 7-Methyl Trimethoprim Analogues as Inhibitors of the Folate Metabolizing Enzymes
509
of 2 started with the condensation reaction of 3,4-dimeth-
oxybenzaldehyde with ethyl cyanoacetate, the reaction
was carried out in toluene and catalyzed by ammonium
acetate and acetic acid. Compound 12 was washed with
water and extracted with ethyl acetate. This condensation
product was then subjected to a Michael addition with one
equivalent of methyl copper lithium which was in turn
generated in situ by adding two equivalents of 1.5 M
methyl lithium solution in ethyl ether to one equivalent of
copper (I) iodide. The reaction proceeded in dry THF at
–78 °C for two hours and was quenched with saturated
aqueous ammonium chloride, and the product extracted
with ethyl ether which was purified with column chro-
matography (hexane/ethyl acetate: 6/1, silica gel). Without
further purification, the resulting compound was subjected
to a condensation reaction with guanidine free base that
was in turn obtained from equal amounts of guanidine
hydrochloride and sodium methoxide. The reaction
occurred in ethoxyethanol and required 3 to 6 hours to
reach completion. The resulting reaction mixture was then
chromatographed with chloroform/methanol (20/1) on a
silica gel column to afford the 4-hydroxy analogue 2.
The 2,6-diamino groups of 2 were protected with pival-
oyl chloride. The reaction was carried out in 1,4-dioxane
with triethylamine as the base to afford, after chromato-
graphic purification, a 60% yield of the pivaloyl protected
analogue 13. The protected compound 13 was reacted with
phosphorus oxychloride, which afforded the 4-chloro
analogue 14. Catalytic hydrogenation with 5% palladium-
charcoal and 50 psi of hydrogen afforded compound 15.
Final depivaloylation was carried out in closed vessel at
150 °C with liquid ammonia to afford compound 8.
The target compounds 3-7 and 9-10, were synthesized
using the same procedure as in Scheme I without isolation
of the intermediates as shown in Scheme III. Compounds
3-7 were obtained in 10-20% overall yield. Compounds 9
and 10 were synthesized from 3 and 5 in 9% and 6%
yield, respectively.
Analogues 2-10 were evaluated as inhibitors of P. carinii,
T. gondii, Mycobacterium avium and rat liver dihydrofolate
reductase and the results are listed in Table II. Compound 7,
the only analogue that had electron withdrawing chloro
groups on the phenyl ring was the most active and selective
dihydrofolate reductase inhibitor of the series. It inhibited
T. gondii dihydrofolate reductase with an IC of 0.58 µM
50
and had a selectivity ratio, versus rat liver dihydrofolate
reductase, of 15.3. This analogue was 5-fold more potent
and 3-fold less selective as compared to TMP. Compound 7
also inhibited M. avium dihydrofolate reductase with an
IC of 0.29 µM and a selectivity ratio of 30.5. The 4-H
50
compounds 8–10 were in general more potent than their
4-hydroxy counterparts (2, 3 and 5) indicating that the
4-hydroxy moiety was detrimental to dihydrofolate reduc-
tase inhibition. These results suggest that perhaps the
Table II
Inhibitory Concentrations (IC ) in µM and Selectivity Ratios of Analogues 2-10 against P. carinii DHFR, T. gondii DHFR,
50
M. avium DHFR versus Rat Liver DHFR
Compd
P. carinii
Rat liver
Rat liver/P.
T. gondii
Rat liver/T.
M. avium
Rat liver/M.
carinii
gondii
avium
2
3
4
5
6
7
8
9
>95 (10)*
>14 (4)*
>58 (19)*
135
>23 (1)*
23.2
>147 (17)*
>33 (18)*
>16 (8)*
0.042
> 95(11)*
>14(12)*
>58(2)*
>131(0)*
>23(8)*
8.85
>147 (12)*
65.6
>16 (4)*
0.003
ND
ND
ND
ND
ND
0.4
ND
ND
ND
0.07
11.1
89.8
ND
ND
ND
ND
ND
15.3
ND
3.7
>95(9)*
>54(8)*
>58 (14)*
>131(8)*
>23(5)*
0.29
19.3
61.1
5.4
ND
ND
ND
ND
ND
ND
30.5
ND
1.07
ND
ND
ND
>54 (7)*
>58 (14)*
268
>23 (18)*
0.58
50.5
17.5
>16 (13)*
0.01
2.7
10
TMQ
TMP
ND
0.3
49
12
133
ND
* Numbers in parentheses are percent inhibition at the highest concentration.

510
A. Gangjee, X. Lin and S. F. Queener
Vol. 40
and washed with water repeatedly to afford 12.7 g (83%) of a yel-
methyl group on the bridge prevents these molecules from
assuming the biologically active conformation of TMP and
thus renders these compounds (except 7) poorly active
against P. carinii dihydrofolate reductase. However, 3, with
electron donating 2,4-dimethoxy substitutions is much less
potent and selective than the corresponding electron with-
drawing substituents containing analog 7. This indicates
that the nature of the substituent on the phenyl ring does
play a major role in the inhibition of these pathogenic
DHFR for this series of compounds.
In conclusion, based on the fact that the 5-methyl group
is conducive to dihydrofolate reductase inhibitory activi-
ties in the case of some dihydrofolate reductase inhibitors
with 6,6-fused ring systems, we synthesized 7-methyl
trimethoprim analogues 2-10 as novel non-classical
dihydrofolate reductase inhibitors using a total synthetic
strategy. With the exception of analogue 7, all the ana-
logues in the series were poor inhibitors of P. carinii, T.
gondii and M. avium dihydrofolate reductase. Compound 7
with 2,4-dichloro substituents in the phenyl ring was more
potent (compared to TMP) against T. gondii and M. avium
dihydrofolate reductase with some selectivity against T.
gondii and M. avium dihydrofolate reductase.
low powder. mp 153.1-155.0 °C, tlc R =0.29 (hexane/ethyl:
f
1
acetate 4/1, silica gel); H nmr (DMSO-d ): δ 1.29 (t, 3H,
6
COCH CH ), 3.83 (s, 3H, OCH ), 3.90 (s, 3H, OCH ), 4.30 (q,
2
3
3
3
2H, COCH CH ), 7.17 (d, 2H, Ar-H), 7.71-7.76 (m, 2H, Ar-H),
2
3
8.29 (s, 1H, vinyl proton); HRMS (EI): m/e calcd for (M+)
NO 261.1001; found 261.0991.
C
H
14 15
4
2,6-Diamino-5-[(3',4'-dimethoxyphenyl)ethyl]pyrimidin-4-ol
(2).
In a 250 ml flask was placed 6.4 g (33.6 mmol) of copper(I)
iodide in 30 ml of anhydrous diethyl ether, followed by the addi-
tion of 45 ml of 1.5 M solution of methyl lithium in diethyl ether
(67.2 mmol). The mixture was stirred under nitrogen at –78 °C for
30 min, 8.0 g (30.6 mmol) of 12 dissolved in 30 ml of THF was
then added to the mixture. The reaction was allowed to proceed at
-78 °C for 2 hours. The reaction mixture was diluted with 40 ml of
brine, extracted with ethyl ether (3 x 40 ml), and the extract was
dried over anhydrous sodium sulfate and chromatographed
(Hexane/ethyl acetate: 6/1, 800 ml) to afford a colorless oil (4.0 g,
48%). This product (1.5 g) was treated with 17.5 mmol of guani-
dine free base (from 1.7 g (17.5 mmol) of guanidine hydrochlo-
ride and 0.95 g (17.5 mmol) of sodium methoxide) in 100 ml of
glycol ethyl monoether. The reaction mixture was kept at reflux
for 6 hours. The reaction mixture was filtered and the clear filtrate
was stripped of solvent in vacuo. The residue was then chro-
matographed (chloroform:methanol, 10/1). The fractions contain-
ing the desired spot were pooled and the solvent evaporated in
vacuo. The residue was recrystallized from ethyl acetate to afford
0.4 g (18%) of the product as a white powder; mp 269.8-274.4 °C,
EXPERIMENTAL
Melting points were determined on a Mel-Temp apparatus and
1
1
tlc R =0.57 (chloroform/methanol: 5/1), H nmr (DMSO-d ): δ
are uncorrected. Nuclear Magnetic Resonance Spectra ( H nmr)
f
6
1.47 (d, 3H, CHCH ), 3.69 (s, 6H, 3,4-OMe), 4.07 (q, 1H,
were recorded on a Brucker WH-300 (300 MHz). Internal stan-
dard trimethysilane (TMS); s = singlet, br s = broad singlet, d =
doublet, t = triplet, q = quartet, m = multiplet. High resolution
mass spectra (HRMS) were obtained on a VG7070E-HF instru-
ment. Thin layer chromatography was performed on Aldrich sil-
ica gel plates with a fluorescent indicator and were visualized
with UV-lamp at 254 nm and 366 nm. Column chromatography
was performed with (60 Å, 230-400 mesh) silica gel from
Aldrich Chemical Company WI, employing gravity or flash
columns (2.4 x 15 cm) unless otherwise stated. Elutions were
performed using a gradient (specifically stated in the experimen-
tal) and 10 ml fractions were collected. Solvents for column chro-
matography were purchased from Fisher Scientific, PA. Samples
for microanalysis were dried under vacuum over phosphorous
pentoxide at 75 °C for 24-48 hours utilizing the Chem-Dry appa-
ratus. Analyses were performed by Atlantic Microlabs, Georgia
and are within ± 0.4% of the calculated value. Fractional amounts
of solvents could not be removed from some of the compounds
despite drying under vacuum and were confirmed where possible
3
CHCH ), 5.32 (s, 2H, NH ), 5.95 (s, 2H, NH ), 6.79-6.88 (m, 2H,
3
2
2
Ar-H), 6.95 (s, 1H, Ar-H), 9.77 (s, 1H, 4-OH).
Anal. Calcd. for C N O •0.4H O: C, 56.52; H, 6.37; N,
H
14 18
4
3
2
18.83. Found: C, 56.56; H, 6.15; N, 19.12.
2,4-Dipivaloylamino-5-[(3',4'-dimethoxyphenyl)ethyl]-6-
hydroxypyrimidine (13).
In a 250 ml round-bottom flask was placed 300 mg (1.0 mmol)
of 2, which was dissolved in 30 ml of 1,4-dioxane. Triethylamine
(0.52 g, 0.7 ml, 5.0 mmol) was then added, followed by the addi-
tion of 0.62 g of pivaloyl chloride (0.64 ml, 5.0 mmol). The reac-
tion was allowed to proceed under reflux for 2 hours. The solvent
was then evaporated under reduced pressure. The residue was
then chromatographed (hexane/ethyl acetate: 1/1 and then ethyl
acetate) to afford 0.25 g (52%) of product; tlc R =0.85 (chloro-
f
1
form/methanol: 10/1), H nmr (DMSO-d ): δ 1.20 (m, 18H, 2
6
C(CH ) ), 1.63 (d, 3H, CHCH ), 3.74 (s, 6H, 3,4-OCH ), 3.95 (q,
3 3
3
3
1H, CHCH ), 6.90 (m, 3H, Ar-H), 9.43 (s, 1H, NH), 11.10 (s, 1H,
3
1
NH), 11.98 (s, 1H, OH). The product was used without further
purification.
by their presence in the H NMR.
Ethyl-3-(3,4-dimethoxyphenyl)-2-cyanoprop-2-enoate (E/Z
mixture) (12).
4-Amino-6-chloro-2-pivaloylamino-5-[(3',4'-dimethoxyphenyl)-
ethyl]pyrimidine (14).
In a 250 ml round bottom flask was charged 10 g (60 mmol) of
3,4-dimethoxybenzaldehyde, which was followed by the addition
of 30 ml of toluene. To the resulting clear solution was added 4.7
A 100 ml round-bottom flask was charged with 250 mg
(0.53 mmol) of 13, which was then dissolved in 30 ml of phos-
phorus oxychloride. The mixture was kept at reflux for 1.5 hours;
phosphorus oxychloride was then evaporated under reduced pres-
sure. The residue was neutralized with concentrated ammonium
hydroxide and extracted with chloroform (3x40 ml). The extract
g (60 mmol) of NH OAc, 11 g (54 mmol) of AcOH and 6.6 g (58
4
mmol) of ethyl cyanoacetate. The mixture was kept refluxing
under a Dean-Stark apparatus for 21 hours. The reaction was then
cooled to room temperature. The yellow precipitate was collected

May-Jun 2003 7-Methyl Trimethoprim Analogues as Inhibitors of the Folate Metabolizing Enzymes
511
was dried over sodium sulfate and chromatographed (chloro-
form:methanol, 20/1). 164 mg (75%) of the product was obtained
2,6-Diamino-5-[(2',4'-dimethoxyphenyl)ethyl]pyrimidin-4-ol
(3).
as a light brown powder; mp 152.6-159.3 °C, tlc R =0.79 (chloro-
f
This compound was synthesized using general procedure from
1
form:methanol, 10/1), H nmr (DMSO-d ): δ 1.09-1.24 (m, 9H,
6
2,4-dimethoxybenzaldehyde in 16% overall yield; mp 202.0-
C(CH ) ), 1.57 (d, 3H, CHCH ), 3.71 (s, 3H, OMe), 3.72 (s, 3H,
3 3
3
1
204.0 °C; tlc R =0.60 (chloroform/methanol: 5/1), H nmr
f
OMe), 4.55 (q, 1H, CHCH ), 6.53 (br s, 2H, NH ), 6.70-6.81 (m,
3
2
(DMSO-d ): δ 1.45 (d, 3H, CHCH ), 3.71 (s, 3H, OCH ), 3.77 (s,
6
3
3
2H, Ar-H), 6.90 (d, 1H, Ar-H), 9.59 (s, 1H, NH); HRMS (EI): m/e
calcd for (M+) C H ClN O 392.1615; found 392.1618.
3H, OCH ), 4.10 (q, 1H, CHCH ), 5.31 (s, 2H, NH ), 5.93 (s, 2H,
3
3
2
19 25
4 3
NH ), 6.43 (t, 2H, Ar-H), 7.39 (d, 1H, Ar-H), 9.71 (s, 1H, 4-OH).
2
Anal. Calcd. for C
H N O •0.5CH OH: C, 56.85; H, 6.58;
4-Amino-2-pivaloylamino-5-[(3',4'-dimethoxyphenyl)ethyl]-
14 18 4 3 3
N, 18.29. Found: C, 56.66; H, 6.32; N, 18.65.
pyrimidine (15).
2,6-Diamino-5-[(2',3',4'-trimethoxyphenyl)ethyl]pyrimidin-4-ol
(4).
Compound 14, 150 mg (0.51 mmol), was hydrogenated in 40
ml of ethanol over 100 mg of 5% palladium charcoal under 40 psi
pressure for 12 hours. The catalyst was then removed by filtration
and the solvent removed in vacuo, the residue was chro-
This compound was synthesized using general procedure from
2,3,4-trimethoxybenzaldehyde in 14% overall yield; mp 197.6-
matographed to afford 119 mg (87%) of the product as light
1
199.4 °C, tlc R =0.60 (chloroform/methanol: 5/1); H nmr
f
1
brown powder; H nmr (DMSO-d ): δ 1.16 (s, 9H, C(CH ) ),
6
3 3
(DMSO-d ): δ 1.47 (d, 3H, CHCH ), 3.68 (s, 3H, OCH ), 3.71 (s,
6
3
3
1.48 (d, J=7.1 Hz, 3H, CHCH ), 3.70 (s, 3H, OCH ), 3.72 (s, 3H,
3
3
3H, OCH ), 3.74 (s, 3H, OCH ), 4.06 (q, 1H, CHCH ), 5.35 (s,
3
3
3
OCH ) 4.01 (q, 1H, CHCH ), 6.54 (br s, 2H, NH ), 6.84-6.90
3
3
2
2H, NH ), 5.92 (s, 2H, NH ), 6.72 (d, 1H, Ar-H), 7.24 (d, 1H, Ar-
2
2
(m, 1H, Ar-H), 7.91 (d, 1H, Ar-H), 9.29 (s, 1H, NH). The product
was used without further purification.
H), 9.70 (s, 1H, 4-OH).
Anal. Calcd. for C
H N O •0.2CH COOC H : C, 56.15; H,
15 20 4 4 3 2 5
6.44; N, 16.58. Found: C, 56.00; H, 6.35; N, 16.61.
2,4-Diamino-5-[(3',4'-dimethoxyphenyl)ethyl]pyrimidine (8).
2,6-Diamino-5-[(2',4',5'-trimethoxyphenyl)ethyl]pyrimidin-4-ol
(5).
Compound 15, 116 mg (0.48 mmol), was placed in a closed ves-
sel, followed by the addition of 30 ml of liquid ammonia. The
container was kept in an oil bath with external temperature at 160 °C
overnight. The liquid ammonia was allowed to evaporate at room
temperature, the residue was first neutralized with acetic acid and
then purified with chromatography (chloroform:methanol, 10/1) to
This compound was synthesized using general procedure from
2,4,5-trimethoxybenzaldehyde in 20% overall yield; mp 227.2-
1
232.2 °C, tlc R =0.60 (chloroform/methanol: 5/1), H nmr
f
(DMSO-d ): δ 1.50 (d, 3H, CHCH ), 3.64 (s, 3H, OCH ), 3.70 (s,
6
3
3
afford 27 mg (31%) of 8 as a white powder; mp 201.6-205.5 °C; tlc
3H, OCH ), 3.74 (s, 3H, OCH ), 4.05 (q, 1H, CHCH ), 5.39 (s,
3
3
3
1
Rf=0.51 (chloroform:methanol, 5/1); H nmr (DMSO-d ): δ 1.40 (d,
6
2H, NH ), 5.96 (s, 2H, NH ), 6.59 (s, 1H, Ar-H), 7.24 (s, 1H, Ar-
2
2
3H, CHCH ), 3.70 (s, 3H, OCH ), 3.72(s, 3H, OCH ), 3.92 (q, 1H,
3
3
3
H), 9.73 (s, 1H, 4-OH).
CHCH ), 6.41 (s, 2H, NH ), 6.71 (s, 2H, NH ), 6.77 (s, 1H, Ar-H),
3
2
2
Anal. Calcd. for C
H N O : C, 56.23; H, 6.29; N, 17.49.
15 20 4 4
6.87 (d, 2H, Ar-H), 7.60 (s, 1H, 6-H); HRMS (EI): m/e calcd for
(M+) C H N O 274.1430; found 274.1434.
Found: C, 56.08; H, 6.36; N, 17.44.
14 18
4 2
2,6-Diamino-5-[(2',4',6'-trimethoxyphenyl)ethyl]pyrimidin-4-ol
(6).
General Procedure for the Synthesis of Analogues 3-7.
A round bottom flask was charged with 1 equivalent of the
appropriately substituted benzaldehyde, followed by the addition
of 30 ml of toluene. To the resulting clear solution was added 1
This compound was synthesized using general procedure from
2,4,6-trimethoxybenzaldehyde in 11% overall yield; mp 208.9-
1
212.6 °C; tlc R =0.56 (chloroform/methanol: 5/1); H nmr
f
equivalent of NH OAc, 3 equivalents of AcOH and 1 equivalent of
4
(DMSO-d ): δ 1.37 (d, 3H, CHCH ), 3.70 (s, 6H, 2, 6-OCH ),
6 3 3
ethyl cyanoacetate. The mixture was kept at reflux under a Dean-
Stark apparatus for 21 hours. The reaction was then cooled to room
temperature. The precipitate that formed was collected and washed
with water repeatedly and added to a ethyl ether solution of 1
equivalent of methyl copper lithium (from 1 equivalent of cop-
per(I) iodide and 2 equivalents of methyl lithium) without purifi-
cation. The reaction was allowed to proceed at –78 °C for 2 hours.
The reaction mixture was diluted with 40 ml of brine, extracted
with ethyl ether (3 x 40 ml), and the extract was dried over anhy-
drous sodium sulfate and chromatographed (Hexane/ethyl acetate:
6/1) to afford a colorless oil, which was treated with 3 equivalents
of guanidine free base (from 3 equivalents of guanidine hydrochlo-
ride and 3 equivalents of sodium methoxide) in 100 ml of glycol
ethyl monoether. The reaction mixture was kept at reflux for 6
hours. The solid obtained on cooling was filtered and the clear fil-
trate was stripped of solvent in vacuo. The residue was then chro-
matographed (chloroform: methanol, 10/1) on a silica gel column.
The fractions containing the desired spot were pooled and the sol-
vent evaporated in vacuo. The residue was recrystallized from
ethyl acetate to afford the product as a white powder.
3.73 (s, 3H, 4-OCH ), 4.56 (q, 1H, CHCH ), 5.32 (s, 2H, NH ),
3
3
2
5.82 (s, 2H, NH ), 6.19 (s, 2H, Ar-H), 9.59 (s, 1H, 4-OH).
2
Anal. Calcd. for C
H N O •0.1CH COOC H : C, 56.19; H,
15 20 4 4 3 2 5
6.37; N, 17.02. Found: C, 56.23; H, 6.28; N, 16.88.
2,6-Diamino-5-[(2',4'-dichlorophenyl)ethyl]pyrimidin-4-ol (7).
This compound was synthesized using general procedure from
2,4-dichlorobenzaldehyde in 12% overall yield; mp 208.9-212.6
1
°C; tlc R =0.58 (chloroform/methanol: 5/1); H nmr (DMSO-d ):
f
6
δ 1.45 (d, 3H, CHCH ), 4.38 (q, 1H, CHCH ), 5.65 (s, 2H, NH ),
3
3
2
5.95 (s, 2H, NH ), 7.29-7.32 (m, 1H, Ar-H), 7.40 (s, 1H, Ar-H),
2
7.62 (d, 1H, Ar-H), 9.61 (s, 1H, 4-OH).
Anal. Calcd. for C
H Cl N O: C, 48.18; H, 4.04; N, 18.73,
12 12 2 4
Cl, 23.70. Found: C, 48.42; H, 4.09; N, 18.51; Cl, 23.99.
General Procedure for the Synthesis of Analogues 10 and 11.
To a 250 ml flask was added 1 equivalent of 3 or 5 dissolved in
30 ml of 1,4-dioxane. Triethylamine, 5 equivalents, was then
added, followed by the addition of 5 equivalents of pivaloyl chlo-
ride. The reaction was allowed to proceed under reflux for 2 hours.

512
A. Gangjee, X. Lin and S. F. Queener
Vol. 40
The solvent was evaporated under reduced pressure. The residue
was chromatographed (hexane/ethyl acetate: 1/1 and then ethyl
acetate) to afford an oil, which was dissolved directly in 30 ml of
phosphorus oxychloride. The mixture was refluxed for 1.5 hours;
phosphorus oxychloride was then evaporated under reduced pres-
sure. The residue was neutralized with concentrated ammonium
hydroxide and extracted with chloroform (3x40 ml). The extract
was dried over sodium sulfate and chromatographed (chloro-
form:methanol, 20/1), the fractions with the desired spot were
pooled and the solvent evaporated, the oily residue was hydro-
genated in 40 ml of ethanol over 100 mg of palladium charcoal
under 40 psi pressure for 12 hours. The catalyst was then removed
by filtration and the solvent removed in vacuo. The residue was
chromatographed to afford a brown oil, which was transferred to a
closed vessel, followed by the addition of 30 ml of liquid ammo-
nia. The container was heated in an oil bath with external tempera-
ture at 160 °C overnight. The liquid ammonia was allowed to evap-
orated at room temperature, the residue was first neutralized with
acetic acid and then chromatographically purified using silica gel
and chloroform:methanol, 10/1.
Acknowledgement.
This work was supported in part by NIH grants AI41743 (A.
G.) and AI44661 (A. G.) from the National Institute of Allergy
and Infectious Diseases.
REFERENCES AND NOTES
[1] Presented in part at the 220th ACS national meeting,
Washington, DC, Aug 20-24, 2000 (MEDI 080).
[2] A. R. Castellano and M. D. Nettleman, J. Am. Med.
Assoc., 266, 820 (1991).
[3] V. S. Georgiev, Med. Res. Rev., 13, 529 (1993).
[4] M. S. Bartlett and J. W. Smith, Clin. Microbiol. Rev., 4,
137 (1991).
[5] F. R. Settler and J. Feinberg, Chest., 101, 451 (1992).
[6] K. M. Jules-Elysee, D. E. Strover, M. B. Zaman, E. M.
Bernard and D. A. White, Ann. Intern. Med., 112, 750 (1990).
[7] H. Masur, N. Engl. J. Med., 327, 1853 (1992).
[8] B. Dannemann, J. A. McCutchan and D. Israeleki, Ann.
Intern. Med., 116, 3343 (1992).
[9] C. J. Allegra, J. A. Kovacs, J. C. Drake, J. C. Swan, B. A.
Chabner and H. Masur, J. Exp. Med., 165, 926 (1987).
[10] C. J. Allegra, J. A. Kovacs, J. C. Drake, J. C. Swan, B. A.
Chabner and H. Masur, J. Clin. Invest., 79, 478 (1987).
[11] J. A. Kovacs, C. J. Allegra, J. C. Swan, J. C. Drake, J. E.
Parrillo, B. A. Chabner and H. Masur, Antimicro. Agents Chemother.,
32, 430 (1988).
[12] S. F. Queener, M. S. Barglett, M. A. Jay, M. M. Durkin and
J. W. Smith, Antimicrob. Agents Chemother., 31, 1323 (1987).
[13] C. J. Allegra, B. A. Chabner, C. U. Tuazon, D. Ogata-
Arakaki, B. Baird, J. C. Drake J. T. Simmons, E. E. Lack, J. H.
Shelhamer, F. Balis, R. Walker, J. A. Kovacs, H. C. Lane and H.
Masur, N. Engl. J. Med., 317, 978 (1989).
2,4-Diamino-5-[(2',4'-dimethoxyphenyl)ethyl]pyrimidine (9).
This compound was synthesized using the general procedure
from 3 in 9% overall yield; mp 182.4-184.5 °C; tlc R =0.56 (chlo-
f
1
roform/methanol: 5/1); H nmr (DMSO-d ): δ 1.35 (d, J=6.8Hz,
6
3H, CHCH ), 3.70 (s, 3H, OCH ), 3.75 (s, 3H, OCH ), 4.07 (q,
3
3
3
1H, CHCH ), 5.71 (s, 2H, NH ), 5.87 (s, 2H, NH ), 6.47-6.53
3
2
2
(m, 2H, Ar-H), 7.02 (d, 1H, Ar-H), 7.47 (s, 1H, 6-H).
Anal. Calcd. for C N O •0.5CH COOH: C, 59.20; H,
H
14 18
4
2
3
6.62; N, 18.41. Found: C, 58.84; H, 6.37; N, 18.52.
2,4-Diamino-5-[(2',4',5'-trimethoxyphenyl)ethyl]pyrimidine
(10).
This compound was synthesized using general procedure from 5
[14] A. Gangjee, R. Devraj and S. F. Queener, J. Med. Chem.,
40, 470 (1997).
in 6% overall yield; mp 267.0-271.9 °C; tlc R =0.35 (chloro-
f
1
form/methanol: 5/1); H nmr (DMSO-d ): δ 1.40 (d, 3H, CHCH ),
[15] V. Cody, A. Wojtczak, T. I. Kalman, J. H. Friesheim and R.
L. Blakley, Adv. Exp. Med. Biol., 338, 481 (1993).
[16] J. N. Champness, A. Achari, S. P. Ballantine, P. K. Bryant,
C. J. Delves and D. K. Stammers, Structure, 2, 915 (1994).
[17] J. H. Chan and B. Roth, J. Med. Chem., 34, 550 (1991).
6
3
3.70 (s, 3H, OCH ), 3.75 (s, 3H, OCH ), 3.78 (s, 3H, OCH ), 4.15
3
3
3
(q, 1H, CHCH ), 6.70 (s, 1H, Ar-H), 6.87 (d, 1H, Ar-H), 7.33 (s,
3
1H, 6-H), 7.37 (s, 2H, NH ), 7.61 (br s, 2H, NH ); HRMS (EI):
2
2
m/e calcd for (M+) C H N O 304.1535; found 304.1548.
15 20
4 3