2,4-Diaminothieno[2,3-d]pyrimidine lipophilic antifolates as inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase

Article abstract of DOI:10.1021/jm970399a

Ten previously unreported 2,4-diaminothieno[2,3-d]pyrimidine lipophilic dihydrofolate reductase inhibitors were synthesized as potential inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase. Pivaloylation of 2,4-diamino-5-methy

Full text of DOI:10.1021/jm970399a

3694
J . Med. Chem. 1997, 40, 3694-3699
2,4-Dia m in oth ien o[2,3-d ]p yr im id in e Lip op h ilic An tifola tes a s In h ibitor s of
P n eu m ocystis ca r in ii a n d Toxopla sm a gon d ii Dih yd r ofola te Red u cta se
Andre Rosowsky,*,† Andrew T. Papoulis,† and Sherry F. Queener‡
Dana-Farber Cancer Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,
Boston, Massachusetts 02115, and Department of Pharmacology and Toxicology, Indiana University School of Medicine,
Indianapolis, Indiana 46202
Received J une 16, 1997X
Ten previously unreported 2,4-diaminothieno[2,3-d]pyrimidine lipophilic dihydrofolate reductase
inhibitors were synthesized as potential inhibitors of Pneumocystis carinii and Toxoplasma
gondii dihydrofolate reductase. Pivaloylation of 2,4-diamino-5-methylthieno[2,3-d]pyrimidine
followed by dibromination with N-bromosuccinimide in the presence of benzoyl peroxide gave
2,4-bis(pivaloylamino)-6-bromo-5-(bromomethyl)thieno[2,3-d]pyrimidine, which after condensa-
tion with substituted anilines or N-methylanilines and deprotection with base yielded 2,4-
diamino-6-bromo-5-[(substituted anilino)methyl]thieno[2,3-d]pyrimidines. Removal of the
6-bromo substituent was accomplished with sodium borohydride and palladium chloride. The
reaction yields were generally good to excellent. The products were tested as inhibitors of
dihydrofolate reductase (DHFR) from P. carinii, T. gondii, and rat liver. Although the IC50
could not be reached for the 6-unsubstituted compounds because of their extremely poor
solubility, three of the five 6-bromo derivatives were soluble enough to allow the IC50 to be
determined against all three enzymes. 2,4-Diamino-5-[3,5-dichloro-4-(1-pyrrolo)anilino]methyl]-
6-bromothieno[2,3-d]pyrimidine was the most active of the 6-bromo derivatives, with an IC50
of 7.5 µM against P. carinii DHFR, but showed no selectivity for either P. carinii or T. gondii
DHFR relative to the enzyme from rat liver.
Pneumocystis carinii and Toxoplasma gondii are
prevalent life-threatening opportunistic microbes in
individuals with compromized immune systems. For
this reason, AIDS patients, immunosuppressed organ
transplant recipients, and patients receiving cancer
chemotherapy are at high risk of contracting these
infections.1-3 Recent advances in the treatment of AIDS
with two- and three-drug cocktails combining nucleo-
sides and protease inhibitors are very promising, but
these new regimens are not universally effective and
the durability of their antiviral effect is not yet estab-
lished. In addition, the high cost of these treatments
and the fact that they have to be given frequently and
over a long period with close medical supervision are
impediments to their use in developing and underde-
veloped countries, where new AIDS cases continue to
be reported with alarming frequency and now greatly
exceed the number of cases in the industrialized world.
Thus, until a practicable approach to worldwide control
of HIV-1 by means of antiviral vaccination or chemo-
therapy is achieved,4 the development of new drugs for
the management P. carinii and T. gondii opportunistic
infections in AIDS patients remains an important goal.
Trimethoprim (TMP, 1) and pyrimethamine (PM, 2)
are clinically approved lipophilic dihydrofolate reductase
(DHFR) inhibitors for the treatment of infection by P.
carinii, T. gondii, and other opportunistic parasites.5-8
TMP is most often used against P. carinii pneumonia
(PCP), whereas PM is most often prescribed for toxo-
plasmosis. While these drugs have a high degree of
binding selectivity for P. carinii and T. gondii DHFR
versus mammalian DHFR, they are not very potent or
effective when used as single agents, and thus are
generally used in combination with a sulfa drug such
as sulfadiazine, sulfamethoxazole, or dapsone. Re-
cently, epiroprim (EPM, 3), a second generation ana-
logue of TMP, has shown promise,9-11 and another
analogue, brodimoprim (4), has been advocated as an
alternative to TMP because of its tighter DHFR binding
and more favorable pharmacokinetics.12,13 Analogues
of epiroprim containing phenyl substituents other than
ethoxy at the 3- and 5-position have also been described,
such as the 3,5-dichloro analogue 5, which had an IC50
of 23 µM against P. carinii DHFR and showed 13-fold
selectivity for this enzyme relative to human DHFR.14
Two other lipophilic antifolates, trimetrexate (TMQ, 6)
and piritrexim (PTX, 7), which were originally developed
as anticancer drugs, have been used to treat P. carinii
and T. gondii infections in AIDS patients.15-17 Unlike
TMP and PM, these dicyclic molecules are very potent,
but unfortunately bind better to mammalian DHFR
species than they do to the P. carinii or T. gondii
enzyme. For this reason, the rescue agent leucovorin
(5-formyl-5,6,7,8-tetrahydrofolate) had to be used to
prevent myelosuppression in the clinical trials with
TMQ and PTX. Lipophilic DHFR inhibitors combining
the potency of TMQ and PTX with the selectivity of TMP
and PM would presumably have avoided the need for
† Dana-Farber Cancer Institute.
‡ Indiana University.
X Abstract published in Advance ACS Abstracts, October 1, 1997.
S0022-2623(97)00399-3 CCC: $14.00 © 1997 American Chemical Society

2,4-Diaminothieno[2,3-d]pyrimidine Lipophilic Antifolates
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3695
Sch em e 1a
a
(i) Chloroacetone, KHCO3, DMF (77%); (ii) (Me3CCO)2O, pyridine (80%; (iii) NBS, Bz2O2, CHCl3 (96%); (iv) arylamine (e.g., 3,4,5-
trimethoxyaniline), NaHCO3, DMF (56-88%); (v) NaOH, MeOH-H2O (34-89%); (vi) NaBH4, PdCl2, THF-H2O (52-98%).
leucovorin, but unfortunately such compounds are yet
to be developed.
would give a better approximation of the quinazoline
ring system than is provided by furo[2,3-d]pyrimidines.
Several potent DHFR inhibitors with 6/5-fused het-
erocyclic rings and a classical glutamate side chain have
Ch em istr y
recently been reported to have good antitumor activity,
including the cyclopenta[c]pyrimidines 8,18,19 pyrrolo-
With commercially available 2,4-diamino-6-mercap-
topyrimidine as the starting material (Scheme 1), the
brominated intermediates 16a -e can be synthesized in
five steps, and the final target compounds 17a -e can
[2,3-d]pyrimidines 9 and 10,20-24 pyrrolo[3,2-d]pyrim-
idines 11,25 and furo[2,3-d]pyrimidines 12.26,27 Despite
their very tight binding to DHFR, these hydrophilic
derivatives would not be appropriate to use against P.
carinii or T. gondii because the plasma membrane of
these cells lacks the reduced folate carrier protein whose
natural function in mammalian cells is to take up
exogenous reduced folates, as well as 2,4-diamino an-
tifolates with a glutamate side chain.
be obtained by an additional reductive debromination.
Initial efforts to obtain compounds of type 17 via 2,4-
diamino-5-(chloromethyl)thieno[2,3-d]pyrimidine proved
unpromising, inasmuch as all attempts to generate this
intermediate from 1,3-dichloroacetone and 2,4-diamino-
6-mercaptopyrimidine in DMF in the presence of sodium
bicarbonate unexpectedly yielded a complex mixture of
products, of which none had the desired structure. The
failure of this reaction was in contrast to the analogous
reaction of 2,4-diamino-6-hydroxypyrimidine, which
produces 2,4-diamino-5-chloromethylfuro[2,3-d]pyrimi-
dine in good yield.27,29 Fortunately, a more successful
route to 17a -e was found to be via the known inter-
mediate 2,4-diamino-6-methylthieno[2,3-d]pyrimidine
(18).30 Thus the synthesis began with the reaction of
2,4-diamino-6-mercaptopyrimidine with chloroacetone
in refluxing DMF in the presence of potassium bicar-
bonate, which gave a 77% yield of 18 after recrystalli-
zation from methanol. Protection of the amino groups
Lipophilic 6/5-fused analogues whose uptake is not
expected to require active transport by the reduced
folate carrier protein have also been described, the first
of which were the 2,4-diaminothieno[2,3-d]pyrimidines
13 and 14, which lacked a nitrogen atom in the bridge.28
A related group of nonclassical analogues with a lipo-
philic side chain (15) were also reported.26 In the
present paper we report the synthesis of 10 new 2,4-
diaminothieno[2,3-d]pyrimidines (16, 17) which, to our
knowledge, are the first examples of this 6/5 ring system
with a carbon-nitrogen bridge. Because of the closer
bioisosteric relationship between a sulfur atom and two
carbons, we speculated that thieno[2,3-d]pyrimidines

3696 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Rosowsky et al.
Ta ble 1. Inhibition of P. carinii, T. gondii, and Rat Liver
Dihydrofolate Reductase
with pivalic anhydride in refluxing pyridine gave 19 in
80% yield after recrystallization from ethyl acetate.
Attempted monobromination of 19 with 1 equiv of NBS
in the presence of benzoyl peroxide occurred with
predominant attack on the thiophene ring, as evidenced
by the complete disappearance of the SCHd singlet at
IC50 (µM)b
selectivity ratioc
compda rat liver P. carinii T. gondii P. carinii T. gondii
16a
16b
16c
16d
16e
17
33
28
>10
10
13
>100
31
34
>100
127
>10
26
1.2
0.49
ND
0.22
ND
0.39
48
5.9
ND
0.88
ND
1.4
11
0.62
0.07
1
δ 6.5 in the H-NMR spectrum of the crude product. On
>10
the other hand, dibromination with 2.2 equiv of NBS
in chloroform containing a catalytic amount of benzoyl
peroxide afforded 20 in 96% yield after column chro-
matography on silica gel.
7.5
TMP (1) 130
PM (2)
TMQ (3)
PTX (4)
12
3.7
0.042
0.031
2.7
0.39
0.01
2.3
0.003
0.015
0.30
0.088
0.017 0.048
Reaction of 20 with substituted anilines and N-
methylanilines in DMF containing excess sodium bi-
carbonate gave intermediates 21a -e in yields of 56-
88% depending on the aniline. The coupled products
were isolated by silica column chromatography. Depro-
tection with sodium hydroxide in methanol and water
gave 16a -e in yields of 34-89%. Attempted dehalo-
genation of 16a in 1:1 CHCl3-MeOH solution with H2
(50 lb/in.2) in the presence of 10% Pd/C yielded a
complex mixture of compounds from which the ret-
rosynthetic 3,4,5-trimethoxyaniline was isolated in high
yield. Treatment with 1.1 equiv of tributyltin hydride
in refluxing THF for 3 days gave back 30% of unchanged
21a along with a complex mixture of unidentified
products. However hydrodebromination of 21a -d was
successfully accomplished with NaBH4 and PdCl2 in
aqueous THF, which afforded 17a -d in 52-98% yield.
The commercially unavailable N-methylanilines needed
for the synthesis of 17c and 17d were prepared from
3,4,5-trimethoxy- and 2,5-dimethoxyaniline by acylation
with 98% HCO2H and reduction with LiAlH4 in THF
as previously reported.31 The starting material for the
synthesis of 17e, the hitherto unknown pyrrole 22, was
prepared in excellent yield from commercially available
2,6-dichloro-4-nitroaniline in two steps. Treatment with
2,4-dimethoxytetrahydrofuran in refluxing AcOH af-
forded N-(2,6-dichloro-4-nitrophenyl)pyrrole (22, 82%
yield), and the latter was reduced with SnCl2 to obtain
23 (93% yield).
a
Compounds 17a -e had IC50 values of >10 µM against all three
enzymes with <30% inhibition at 10 µM. Higher concentrations
could not be tested because of insufficient aqueous solubility. Data
shown for TMP, PM, TMQ, and PTX for comparison purposes are
from ref 35. b Enzyme activity was determined spectrophotometri-
cally at 340 nm according to a standardized and highly reliable
method which has been in continuous use in this program for a
number of years.26,27,31-35 As an illustration of the reproducibility
of the assay, the IC50 value (mean ( standard error) obtained by
S.F.Q. over a 5-year period using the pyrimethamine against rat
liver and Pc DHFR has been 1.52 ( 0.32 and 2.39 ( 0.42 µM,
respectively. c IC50 (rat liver)/IC50 (P. carinii or T. gondii).
P. carinii and T. gondii DHFR being a little more
sensitive to this substitution than the mammalian
enzyme. Compound 16b inhibited rat liver DHFR with
an IC50 of 33 µM, whereas the corresponding value for
16a was 17 µM, suggesting that 2,5-dimethoxy substi-
tution was slightly less favorable than 3,4,5-trimethoxy
substitution, as had also been the case with other
thienopyrimidines studied earlier.28 The presence of a
space-filling Br atom at the 6-position was notable in
view of previous reports showing that three other
compounds with a bromine atom adjacent to the bridge,
namely 2,4-diamino-5-(2-bromo-3,4,5-trimethoxybenzyl)-
pyrimidine (24),12 2,4-diamino-6-(2-bromo-3,4,5-trimeth-
oxybenzyl)-5,6,7,8-tetrahydropyrido[4,3-c]pyrimidine
(25),34 and 2,4-diamino-6-(2-bromo-3,4,5-trimethoxyben-
zyl)-5-methylthieno[2,3-d]pyrimidine (26),35 were better
inhibitors of DHFR than their nonbrominated counter-
parts. In the present work, in contrast to compounds
24-26, a space-filling Br atom on the heterocyclic
moiety adjacent to the CH2NH or CH2NMe bridge did
not seem to have a markedly favorable effect on either
potency or selectivity.
En zym e In h ibition Assa ys
Compounds 16a -c and 17a -e were tested as inhibi-
tors of DHFR from P. carinii, T. gondii, and rat liver as
described previously.32,33 Unfortunately, most of the
thienopyrimidines were too insoluble to allow an IC50
to be determined. Thus it was not possible to compare
the DHFR binding affinity of the two series. To our
surprise, however, three of the brominated derivatives
(16a ,c,e) proved to be more soluble than their nonbro-
minated counterparts, allowing an IC50 to be reached.
As shown in Table 1, the IC50 varied from 7.5 to 31 µM
against the P. carinii enzyme, from 26 to 127 µM against
the T. gondii enzyme, and from 10 to 33 µM against
the rat liver enzyme. The most potent compound
against each enzyme was the pyrrole derivative 16e,
whereas the least potent was the 3,4,5-trimethoxy
analogue 16c. A methyl group on N10 appeared to
increase potency slightly depending on the enzyme, with
Exp er im en ta l Section
IR spectra were obtained on a Perkin-Elmer Model 781
double-beam recording spectrophotometer. 1H NMR spectra
were recorded at 60 MHz on a Varian Model EM360 instru-
ment using Me4Si as the reference or at 500 MHz on a Varian
VX500 instrument. TLC analyses were done on Whatman

2,4-Diaminothieno[2,3-d]pyrimidine Lipophilic Antifolates
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3697
MK6F silica gel plates, using 254-nm illumination to visualize
the spots. Column chromatography was on Baker 7024 flash
silica gel (40 mm particle size). Solvents for moisture-sensitve
reactions were purchased from Aldrich. Melting points were
determined in Pyrex capillary tubes using a Mel-Temp ap-
paratus (Laboratory Devices, Inc., Cambridge, MA) and are
not corrected. Elemental analyses were performed by QTI
Laboratories, Whitehouse, NJ , or Robertson Laboratories,
Madison, NJ , and were within (0.4% of theoretical values
unless otherwise indicated.
NMR (CDCl3) δ 3.9 (s, 2H, NH2), 6.3 (m, 2H, pyrrole 3-H), 6.7
(m, 2H, pyrrole 2-H), 6.75 (s, 2H, phenyl protons); IR (KBr) ν
3440, 3350, 3125, 3100, 1625, 1500, 1430, 1280, 1200, 1065,
1010, 805 cm-1. Anal. (C10H8Cl2N2) C, H, N.
2,4-Bis(p iva loyla m in o)-5-[(3,4,5-t r im et h oxya n ilin o)-
m eth yl]-6-br om oth ien o[2,3-d ]p yr im id in e (21a ). A mix-
ture of 20 (500 mg, 1.05 mmol), 3,4,5-trimethoxyaniline (125
mg, 0.682 mmol), and NaHCO3 (888 mg, 10.6 mmol) in dry
DMF (4 mL) was stirred at 55 °C with stirring for 1 day. The
DMF was removed by rotary evaporation, and the residue was
triturated with EtOAc (50 mL). The undissolved solid was
filtered off, the filtrate was evaporated, and the residue was
purified by silica gel column chromatography using 1:1
EtOAc-heptanes: yield 232 mg (56% based on the aniline);
mp 211-214 °C dec; 1H NMR (CDCl3) δ 1.1-1.4 (m, 18H,
Me3C), 3.8 (m, 9H, OMe), 4.2-4.4 (m, 3H, NH, CH2), 5.85 (s,
1H, phenyl proton), 6.2 (s, 1H, phenyl proton), 8.2 (s, 1H, NH),
8.8 (s, 1H, NH); IR (KBr) ν 3400, 3215, 2985, 2915, 1715, 1600,
2,4-Dia m in o-5-m et h ylt h ien o[2,3-d ]p yr im id in e
(18).
Chloroacetone (16.4 g, 177 mmol) was added to a stirred
mixture of 2,4-diamino-6-mercaptopyrimidine hemisulfate (33.8
g, 177 mmol) and KHCO3 (18.6 g, 186 mmol) in dry DMF (250
mL). The mixture was heated at reflux overnight under N2
and then allowed to cool to room temperature. The mixture
was concentrated to dryness by rotary evaporation, EtOAc was
added to the residue, and the insoluble portion was filtered
off. Evaporation of the filtrate and recrystallization of the
residue from MeOH afforded a white solid (25.9 g, 77%): mp
213-215 °C (lit.30 mp 210-212 °C); 1H NMR (CDCl3) δ 2.4 (s,
3H, CH3), 5.9 (s, 2H, NH2), 6.4 (s, 2H, NH2), 6.5 (s, 1H, SCHd).
1430, 1235, 1160, 1130, 1005, 780 cm-1
. Anal. (C26H34-
BrN5O5S‚H2O) C, H, N.
2,4-Bis(p iva loyla m in o)-5-[(2,5-d im e t h oxya n ilin o)-
m eth yl]-6-br om oth ien o[2,3-d ]p yr im id in e (21b). A mix-
ture of 20 (0.130 g, 0.273 mol), 2,5-dimethoxyaniline (0.027 g,
0.177 mmol), and NaHCO3 (0.231 g, 2.75 mmol) in dry DMF
(2 mL) was stirred at 60 °C overnight and worked up as in
the synthesis of 21a : yield 74 mg (72% based on the aniline);
2,4-Bis(p iva loyla m in o)-5-(b r om om e t h yl)-6-b r om o-
th ien o[2,3-d ]p yr im id in e (20). A mixture of pivalic anhy-
dride (32.8 g, 16.2 mmol) and 18 (1.34 g, 7.44 mmol) in dry
pyridine (16 mL) was heated at reflux under N2 overnight,
cooled to room temperature, and evaporated to dryness under
reduced pressure. The residue was dissolved in Et2O (500 mL),
the solution was washed with 5% aqueous NaHCO3 (2 × 100
mL), and the organic layer was dried (MgSO4) and evaporated.
Recrystallization from Et2O yielded the dipivaloyl drivative
19 as a white solid pure enough to use directly in the next
1
mp 225-226 °C dec; H NMR (CDCl3) δ 1.35 (s, 18H, Me3C),
3.78 (s, 3H, OMe), 3.8 (s, 3H, OMe), 4.35 (s, 2H, CH2), 6.4 (d,
1H, phenyl proton), 6.45 (s, 1H, phenyl proton), 6.75 (d, 1H,
phenyl proton), 8.9 (s, 2H, CONH); IR (KBr) ν 3440, 3320,
2950, 1690, 1595, 1550, 1500, 1455, 1285, 1160 cm-1. Anal.
(C25H32BrN5O4S) C, H, N.
2 ,4 -B i s (p i v a l o y l a m i n o )-5 -[(3 ,4 ,5 -t r i m e t h o x y -N -
m e t h yla n ilin o)m e t h yl]-6-b r om ot h ie n o[2,3-d ]p yr im i-
d in e (21c). A mixture of 20 (0.250 g, 0.525 mmol), 3,4,5-
trimethoxy-N-methylaniline (0.067 g, 0.340 mmol), and NaH-
CO3 (0.444 g, 5.28 mmol) was stirred at 55 °C overnight and
worked up as in the synthesis of 21a : yield 163 mg (77% based
on the aniline); mp softening 112-115 °C; 1H NMR (CDCl3) δ
1.2 (s, 9H, Me3C), 1.3 (s, 9H, Me3C), 2.8 (s, 3H, NMe), 3.8 (s,
9H, OMe), 4.35 (s, 2H, CH2), 6.3 (s, 2H, phenyl protons), 8.7
(s, 2H, CONH); IR (KBr) ν 3550, 3230, 2960, 2870, 1700, 1600,
1
reaction: yield 2.06 g (80%); mp 179.5 °C; H NMR (CD3OD)
δ 1.3 (s, 18H, Me3C), 2.5 (s, 3H, Me), 7.2 (s, 1H, SCHd); IR
(KBr) ν 3420, 3210, 2960, 2870, 1685, 1600, 1550, 1470, 1420,
1295, 1160, 925, 750, 730 cm-1
.
To 600 mL of stirred CHCl3, cooled to 0 °C in an ice bath,
were added 19 (2.61 g, 7.48 mmol), NBS (1.62 g, 9.08 mmol),
and Bz2O2 (0.209 g, 0.825 mmol). The ice bath was removed,
and the resulting solution was allowed to warm to room
temperature. After overnight stirring, more NBS (9.10 g, 51
mmol) and Bz2O2 (1.16 g, 4.6 mmol) were added, and stirring
was continued for a total of 6 days. A yellow precipitate was
filtered off, and the filtrate was washed with H2O (2 × 50 mL),
dried (Na2SO4), and evaporated. Silica gel column chroma-
tography using EtOAc-heptanes (1:1) gave a light-yellow solid
(3.64 g, 96%): mp 230-332 °C dec >200 °C; 1H NMR (CDCl3)
δ 1.35 (s, 18H, Me3C), 5.15 (s, 2H, CH2), 8.25 (s, 2H, NH); IR
(KBr) ν 3250, 2850, 2880, 1690, 1660, 1600, 1550, 1440, 1365,
1290, 1165, 940, 780 cm-1. Anal. (C17H22Br2N4O2S) C, N; H:
calcd, 4.38; found, 3.92.
N-(2,6-Dich lor o-4-n itr op h en yl)p yr r ole (22). A solution
of 2,5-dimethoxytetrahydrofuran (3.52 g, 0.0266 mol) and 2,6-
dichloro-4-nitroaniline (5.0 g, 0.0242 mol) in glacial AcOH (150
mL) was heated at reflux overnight under N2 and then cooled
to room temperature. The AcOH was removed by rotary
evaporation, and the crude residue was taken up in Et2O (100
mL). The Et2O solution was washed with concentrated aque-
ous NaHCO3 (30 mL), rinsed with H2O (2 × 20 mL), dried (Na2-
SO4), and evaporated. Silica gel column chromatography using
1:4 EtOAc-heptanes, followed by recrystallization from the
same solvent mixture, gave a yellow-orange solid (5.09 g, 82%);
mp 92.5-93.5 °C; 1H NMR (CDCl3) δ 6.4 (m, 2H, pyrrole 3-H),
6.75 (m, 2H, pyrrole 2-H), 8.35 (s, 2H, phenyl protons); IR
(KBr) ν 3140, 3080, 1530, 1490, 1345, 1155, 1080, 1010, 905,
890, 810, 760, 730 cm-1. Anal. (C10H6Cl2N2O2) C, H, N.
1550 1420, 1395, 1235, 1125, 1000, 785 cm-1. Anal. (C27H36
BrN5O5S) C, H, N.
-
2,4-Dia m in o-5-[(3,4,5-t r im e t h oxya n ilin o)m e t h yl]-6-
b r om ot h ien o[2,3-d ]p yr im id in e (16a ). A solution of 21a
(472 mg, 0.758 mmol) in MeOH (100 mL) was treated with 1
M aqueous NaOH (50 mL) and stirred at 35-40 °C under N2
for 1 day. The white solid that precipitated was filtered,
washed with distilled H2O (3 × 10 mL), and dried in air: yield
1
210 mg (63%); mp 214.5-215.5 °C; H NMR (CDCl3) δ 3.8 (s,
3H, 4-OMe), 3.85 (s, 6H, 3- and 5-OMe), 4.35 (d, 2H, CH2), 4.8
(s, 2H, NH2), 5.0-6.0 (broad s, 2H, NH2), 6.1 (s, 2H, aryl
protons), 6.2 (broad s, 1H, NH); IR (KBr) ν 3440, 3320, 3180,
2910, 2830, 1610, 1550, 1500, 1445, 1230, 1125, 1000, 900, 780
cm-1. Anal. (C16H18BrN5O3S.0.25H2O) C, H, N.
2,4-D ia m in o -5-[(2,5-d im e t h o x y a n ilin o )m e t h y l]-6-
br om ot h ien o[2,3-d ]p yr im id in e (16b). Treatment of 21b
(605 mg, 1.05 mmol) with 1 M NaOH (50 mL) in MeOH (100
mL) at 35-40 °C under N2 for 36 h was followed by a workup
similar to that of 16a : yield 235 mg (55%); mp 225 °C; 1H NMR
(CDCl3) δ 3.79 (s, 6H, OMe), 4.33 (s, 2H, CH2), 4.76 (s, 2H,
NH2), 6.18 (broad s, 2H, NH2), 6.37 (d, 1H, phenyl proton),
6.5 (s, 1H, phenyl proton), 6.75 (d, 1H, phenyl proton); IR (KBr)
ν 3480, 3410, 3340, 3120, 2960, 2830, 1605, 1560, 1510, 1220,
1135, 1015, 910, 850 cm-1. Anal. (C15H16BrN5O2S) C, H, N.
2,4-Dia m in o-5-[(3,4,5-t r im et h oxy-N-m et h yla n ilin o)-
m eth yl]-6-br om oth ien o[2,3-d ]p yr im id in e (16c). A solu-
tion of 21c (472 mg, 0.758 mmol) in MeOH (100 mL) was
treated with 1 M aqueous NaOH (50 mL) and stirred at 35-
40 °C under N2 for 1 day. The white precipitate was collected,
washed with distilled H2O (3 × 10 mL), and dried in air, yield
210 mg (61%). The filtrate was extracted with CHCl3 (3 ×
100 mL), and the combined extracts were dried (Na2SO4),
evaporated, and chromatographed on silica gel using MeOH-
CHCl3 (1:9) to recover an additional 91 mg (26%, total yield
N-(2,6-Dich lor o-4-am in oph en yl)pyr r ole (23). SnCl2‚H2O
(18.6 g, 0.098 mol) was added to a stirred solution of 22 (18.6
g, 0.0198 mol) in EtOAc (70 mL), and the mixture was heated
at reflux under N2 for 3.5 h and then cooled to room temper-
ature. The reaction mixture was diluted with EtOAc (100 mL)
and treated with 5% aqueous NaHCO3 (500 mL). A white
precipitate was filtered off, the two layers in the filtrate were
separated, and the organic layer was dried (Na2SO4) and
evaporated to a yellow solid (4.2 g, 93%): mp 172-174 °C; 1H

3698 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Rosowsky et al.
87%): mp 205.5-207.5 °C; 1H NMR (CDCl3) δ 2.75 (s, 3H,
NMe), 3.8 (s, 3H, 4-OMe), 3.85 (s, 6H, 3- and 5-OMe), 4.25 (s,
2H, CH2), 4.78 (s, 2H, NH2), 5.0-6.0 (broad s, 2H, NH2), 6.3
(s, 2H, phenyl protons); IR (KBr) ν 3550, 3470, 3260, 3120,
2940, 2840, 1640, 1600, 1510, 1230, 1130, 990, 800 cm-1. Anal.
MeOH-H2O, the MeOH and THF were removed by rotary
evaporation, and the remaining aqueous phase was extracted
with CHCl3 (2 × 100 mL). The combined extracts were washed
with H2O (2 × 20 mL), dried (Na2SO4), and evaporated. The
residue was purified by preparative TLC on silica gel plates
using 95:5 CHCl3-MeOH to obtain a light-yellow solid: yield
.
(C17H20N5O3 0.4H2O) C, H, N.
1
21 mg (52%); mp 196.5-198.5 °C; H NMR (CDCl3) δ 3.8 (s,
2,4-D ia m in o -5-[(2,5-d im e t h o x y -N -m e t h y la n ilin o )-
m eth yl]-6-br om oth ien o[2,3-d ]p yr im id in e (16d ). A mix-
ture of 20 (0.910 g, 1.91 mmol), 2,5-dimethoxy-N-methylaniline
(0.209 g, 1.25 mmol), and NaHCO3 (1.05 g, 12.5 mmol) in dry
DMF (5 mL) was stirred at 50-55 °C overnight and worked
up as in the synthesis of 21a . The product (21d ) was pure
enough to use directly in the next step: yield 654 mg (86%
6H, OMe), 4.28 (s, 2H, CH2), 4.42 (m, 1H, NH), 4.76 (s, 2H,
NH2), 6.18 (broad s, 2H, NH2), 6.37 (d, 1H, phenyl proton),
6.5 (s, 1H, phenyl proton), 6.74 (d, 1H, phenyl proton), 6.8 (s,
1H, SCHd). Anal. (C15H17N5SO2‚1/2H2O‚1/6CH3OH) C, H, N.
2,4-Dia m in o-5-[(3,4,5-t r im et h oxy-N-m et h yla n ilin o)-
m eth yl]th ien o[2,3-d ]p yr im id in e (17c). To a solution of 16c
(100 mg, 0.220 mmol) in THF (25 mL) were added PdCl2 (77
mg, 0.44 mmol) and H2O (25 mL). The mixture was cooled to
0-5 °C, NaBH4 (83 mg, 2.2 mmol) was added, and stirring
was continued for 15 min in the ice bath and for 3.5 h at room
temperature. The rest of the workup was similar to the
preceding experiment except that 8.5:1 CH2Cl2-MeOH (4 ×
100 mL) was used to extract the aqueous phase after removal
of the THF and MeOH. The combined extracts were washed
with H2O (30 mL), dried (Na2SO4), and evaporated: yield 67
mg (81%); mp 185.5-187 °C (crystallized from MeOH); 1H
NMR (CDCl3) δ 2.7 (s, 3H, NMe), 3.85 (s, 3 H, OMe), 3.9 (s, 6
H, OMe), 4.3 (s, 2H, CH2N), 4.9 (s, 2H, NH2), 6.4 (s, 2H, phenyl
proton), 6.6 (s, 2H, NH2), 6.75 (s, 1H, SCHd); IR (KBr) ν 3420,
3310, 3190, 2990, 2915, 2815, 1650, 1600, 1550, 1440, 1230,
1
based on the aniline); mp 149-151.5 °C; H NMR (CDCl3) δ
1.3 (s, 18H, Me3C), 2.8 (s, 3H, NMe), 3.6 (s, 3H, OMe), 3.8 (s,
3H, OMe), 4.3 (s, 2H, CH2), 6.6 (m, 2H, aryl protons), 6.8 (m,
1H, aryl proton), 8.6 (s, 2H, CONH); IR (KBr) ν 3470, 3200,
2950, 2870, 1700, 1690, 1600, 1550, 1415, 1325, 1270, 1220,
1150, 1050, 1025, 940, 795, 785 cm-1
.
A solution of 21d (605 mg, 0.904 mmol) in MeOH (100 mL)
was treated with 1 M NaOH (50 mL) and stirred at 35 °C
under N2 for 1 day. The precipitate was filtered, washed with
distilled H2O (2 × 10 mL), and dried in air, yield 290 mg. The
filtrate was cooled at 5 °C overnight to obtain a second crop:
1
total yield 342 mg (89%); mp 218.5-220 °C; HNMR (CDCl3)
δ 2.63 (s, 3H, NMe), 3.78 (s, 3H, OMe), 3.81 (s, 3H, OMe), 4.22
(s, 2H, CH2), 4.78 (s, 2H, NH2), 6.1-6.3 (broad s, 2H, NH2),
6.62 (d, 1H, phenyl proton), 6.8 (m, 2H, phenyl protons); IR
(KBr) ν 3460, 3280, 3170, 3020, 2910, 2800, 2770, 1620, 1530,
1480, 1425, 1270, 1210, 1160, 1140, 1100, 1035, 1010, 895, 770
cm-1. Anal. (C16H18BrN5O2S) C, H, N.
.
1125, 1000, 960, 790, 770 cm-1. Anal. (C17H21N5SO3 0.5H2O)
C, H, N.
2,4-Dia m in o-5-[(2,5-d im eth oxy-N-m eth yla n ilin o)m eth -
yl]th ien o[2,3-d ]p yr im id in e (17d ). A solution of 16c (100
mg, 0.236 mmol) in 1:1 THF-H2O (50 mL) was cooled in an
ice bath to 0-5 °C and treated with PdCl2 (84 mg, 0.472 mmol)
followed by NaBH4 (89 mg, 2.4 mmol). The rest of the workup
was similar to that of 17b except that the combined CHCl3
extracts were washed first with H2O (20 mL) and then with
brine (20 mL); yield 59 mg (72%). The analytical sample was
purified by preparative tlc on silica gel with 8.5:1 CHCl3-
2,4-Dia m in o-5-[[3,5-d ich lor o-4-(1-p yr r olo)a n ilin o]-
m eth yl]-6-br om oth ien o[2,3-d ]p yr im id in e (16e). A mix-
ture of 20 (0.209 g, 0.440 mmol), 23 (0.100 g, 0.440 mmol),
and NaHCO3 (0.370 g, 4.40 mmol) in dry DMF (3 mL) was
stirred at 55-60 °C overnight and worked up as in the
synthesis of 21a . The product (21e) was pure enough to use
in the next step: yield 253 mg (88% based on the aniline); mp
226-228 °C; 1H NMR (CDCl3) δ 1.4 (s, 18H, Me3C), 4.1 (d 1H,
NH), 4.7 (d, 2H, CH2), 6.35 (m, 2H, pyrrole 3-H), 6.7 (m, 2H,
pyrrole 2-H), 6.75 (m, 2H, aryl protons), 8.3 (s, 2H, CONH);
IR (KBr) ν 3200, 2950, 2850, 1680, 1590, 1535, 1400, 1280,
1
MeOH: mp 191.5-193.5 °C; H NMR (CDCl3) δ 2.59 (s, 3H,
NMe), 3.78 (s, 3H, OMe), 3.83 (s, 3H, OMe), 4.15 (s, 2H, CH2),
4.78 (s, 2H, NH2), 5.4-6.0 (broad s, 2H, NH2), 6.62 (d, 1H,
phenyl proton), 6.71 (s, 1H, SCHd), 6.76 (d, 1H, phenyl
proton), 6.82 (d, 1H, phenyl proton); IR (KBr) ν 3460, 3420,
3290, 3170, 2930, 2820, 1630, 1560, 1510, 1445,1220, 1025,
850 cm-1. Anal. (C16H19N5SO2‚1/8H2O) C, H, N.
1150, 1000, 700 cm-1
.
A solution of 21e (95 mg, 0.758 mmol) in MeOH (20 mL)
was treated with 1 M NaOH (20 mL) and worked up as in the
synthesis of 16a : yield 20 mg (34%); mp 260-261 °C; 1H NMR
(CDCl3) δ 4.03 (m, 1H, NH), 4.4 (d, 2H, CH2), 4.8 (s, 2H, NH2),
5.8 (s, 2H, NH2), 6.4 (m, 2H, pyrrole 3-H), 6.7 (m, 2H, pyrrole
2-H), 6.9 (s, 2H, phenyl protons); IR (KBr) ν 3480, 3380, 3230,
3100, 2825, 1600, 1500, 1390, 1290, 1080, 1010, 910, 810, 730
cm-1. Anal. (C17H13BrCl2N6S) C, H, N.
2,4-Dia m in o-5-[[3,5-d ich lor o-4-(1-p yr r olo)a n ilin o]-
m eth yl]th ien o[2,3-d ]p yr im id in e (17e). To a mixture of 1:1
THF-H2O (30 mL) cooled to 0 °C in an ice bath were added
sequentially PdCl2 (160 mg, 0.90 mmol), NaBH4 (173 mg, 4.57
mmol), and 16e (220 mg, 0.454 mmol). The cooling bath was
removed, and the mixture was stirred overnight and then
filtered through Celite. The Celite pad was rinsed with a 1:1
MeOH-H2O and then MeOH alone. The solid which precipi-
tated in the filtrate was collected, washed with H2O (2 × 10
mL), and then dried in air, yield 77 mg. A second crop
weighing 63 mg was also obtained: total yield 140 mg (76%);
mp 231-232 °C; 1H NMR (CDCl3) δ 4.4 (m, 3H, CH2 and NH),
5.2 (s, 2H, NH2), 6.2 (s, 2H, NH2), 6.35 (m, 2H, pyrrole 2-H),
6.65 (m, 2H, pyrrole 3-H), 6.85 (s, 1H, SCHd), 6.9 (s, 2H,
phenyl protons); IR (KBr) ν 3405, 3305, 3195, 2950, 2850, 1625,
1600, 1550, 1395, 1290, 1180 1160, 1020, 1000, 920, 805, 725
cm-1. Anal. (C17H14Cl2N6S) C, H, N.
2,4-Diam in o-5-[(3,4,5-tr im eth oxyan ilin o)m eth yl]th ien o-
[2,3-d ]p yr im id in e (17a ). To a stirred solution of 16a (20 mg,
0.045 mmol) in 1:1 THF-H2O (4 mL) cooled to 0 °C in an ice
bath were added PdCl2 (37 mg, 0.091 mmol) and NaBH4 (17
mg, 0.45 mmol). After 5 min at 0 °C, the bath was removed,
stirring was continued for 7 h, and the THF was removed by
rotary evaporation. The mixture was diluted with H2O (10
mL) and the product extracted with CHCl3 (30 mL). The
organic layer was washed with H2O (2 × 10 mL), dried (Na2-
SO4), and concentrated to dryness. The residue was purified
by preparative TLC on silica gel plates using 92:8 CHCl3-
MeOH: yield 16 mg (98%); mp 223-224.5 °C; 1H NMR (CDCl3)
δ 3.8 (s, 3H, 4-OMe), 3.85 (s, 6H, 3- and 5-OMe), 4.3 (s, 2H,
CH2), 4.8 (s, 2H, NH2), 5.0-6.0 (broad s, 1H, NH), 6.05 (s, 2H,
phenyl protons), 6.2-6.4 (broad s, 2H, NH2), 6.81 (s, 1H,
SCHd); IR (KBr) ν 3420, 3360, 3200, 2960, 2920, 2860, 1600,
Ack n ow led gm en t. This work was supported by
Grant RO1-AI29904 (A.R.) and Contract NO1-AI35171
(S.F.Q.) from the NIAID, Division of AIDS.
1550, 1500, 1290, 1235, 1130, 1000, 955, 920 cm-1
. Anal.
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