Synthesis of substituted 6-anilinouracils and their inhibition of DNA polymerase IIIC and gram-positive bacterial growth

Article abstract of DOI:10.1021/jm020591z

Certain substituted 6-anilinouracils are potent and selective inhibitors of Gram+ bacterial DNA polymerase IIIC (pol IIIC). In addition, analogues with 3-substituents in the uracil ring have potent antibacterial activity against Gram+ organisms in culture. In an attempt to find optimal anilino substituents for pol IIIC binding and optimal 3-substituents for antibacterial activity, we have prepared several series of 3-substituted-6-aminouracils and assayed their activity against pol IIIC from Bacillus subtilis and a panel of Gram+ and Gram- bacteria in culture. The 6-(3-ethyl-4-methylanilino) group and closely related substituent patterns maximized pol IIIC inhibition potency. Among a series of 3-(substituted-butyl)-6-(3-ethyl-4-methylanilino)uracils, basic amino substituents increased pol IIIC inhibition, but decreased antibacterial activity. The most potent antibacterials were simple hydroxybutyl and methoxybutyl derivatives, and hydrophobically substituted piperidinylbutyl derivatives.

Full text of DOI:10.1021/jm020591z

J . Med. Chem. 2003, 46, 2731-2739
2731
Syn th esis of Su bstitu ted 6-An ilin ou r a cils a n d Th eir In h ibition of DNA
P olym er a se IIIC a n d Gr a m -P ositive Ba cter ia l Gr ow th
Chengxin Zhi,† Zheng-Yu Long,† J oseph Gambino,† Wei-Chu Xu,† Neal C. Brown,‡ Marjorie Barnes,‡
Michelle Butler,§ William LaMarr,§ and George E. Wright†,*
GLSynthesis Inc., One Innovation Drive, Worcester, Massachusetts 01605, University of Massachusetts Medical School,
Worcester, Massachusetts 01655, and Microbiotix Inc., One Innovation Drive, Worcester, Massachusetts 01605
Received December 30, 2002
Certain substituted 6-anilinouracils are potent and selective inhibitors of Gram+ bacterial
DNA polymerase IIIC (pol IIIC). In addition, analogues with 3-substituents in the uracil ring
have potent antibacterial activity against Gram+ organisms in culture. In an attempt to find
optimal anilino substituents for pol IIIC binding and optimal 3-substituents for antibacterial
activity, we have prepared several series of 3-substituted-6-aminouracils and assayed their
activity against pol IIIC from Bacillus subtilis and a panel of Gram+ and Gram- bacteria in
culture. The 6-(3-ethyl-4-methylanilino) group and closely related substituent patterns
maximized pol IIIC inhibition potency. Among a series of 3-(substituted-butyl)-6-(3-ethyl-4-
methylanilino)uracils, basic amino substituents increased pol IIIC inhibition, but decreased
antibacterial activity. The most potent antibacterials were simple hydroxybutyl and methoxy-
butyl derivatives, and hydrophobically substituted piperidinylbutyl derivatives.
In tr od u ction
tantly, the pol IIICs strictly conserve structural features
which are responsible for their unique sensitivity to
6-anilinouracils (AUs) and related compounds, the
inhibitor class that is the subject of this work.
The last 15 years have witnessed a resurgence of
bacterial and viral diseases worldwide. Gram-positive
(Gram+) bacteria have been particularly problematic
given the frequency of their involvement in nosocomial
disease and their development of resistance to tradi-
tional antibiotics. Among the Gram+ organisms, Sta-
phylococcus aureus, Enterococcus fecalis, Enterococcus
fecium, and Streptococcus pneumoniae are of greatest
concern. The future of clinical management of diseases
caused by these Gram+ pathogens depends, in large
part, on the development of effective chemotherapeutic
agents that selectively attack new bacterial targets. One
new target which has been validated recently in Gram+
organisms is DNA polymerase IIIC (pol IIIC), a DNA-
dependent DNA polymerase which is specifically re-
quired for replicative DNA synthesis in these organ-
isms.1,2
Gram+ pol IIICs share a unique capacity to bind
certain AU compounds, via a guanine-like “base-pairing
domain” and an enzyme-specific “aryl domain” (see
pharmacophore structure). Through its base-pairing
domain the molecule forms Watson-Crick-like hydro-
gen bonds with an unapposed cytosine residue in the
template strand just distal to the DNA primer terminus;
consequently its action is competitive with dGTP.
Simultaneously, the aryl domain binds an aryl-specific
“receptor” near the enzyme’s active site, causing the
formation of an inactive ternary complex of inhibitor,
DNA, and pol IIIC.5 Structure-activity relationship
(SAR) studies have identified the 3-ethyl-4-methylani-
lino (“EMA”) substituent pattern in AU compounds to
be nearly optimal for binding with pol IIIC.6,7
Va lid ity of th e Gr a m + p ol IIIC a s a n An tim icr o-
bia l Ta r get. Pol IIIC is a member of one of three
distinct classes (pols I, II, and III) of DNA polymerases
found in Gram+ eubacteria.3 Of the three enzymes, pol
IIIC is clearly the most attractive antibiotic target,
because it is essential for replication of the host chromo-
some. Inhibition of its activity prevents replicative DNA
synthesis and, as a consequence, the host cell dies. Pol
IIIC is essentially invariant among relevant Gram+
eubacteria, such as the model enzyme of Bacillus
subtilis and those of Staphylococcus aureus, Myco-
plasma pulmonis, and Enterococcus fecalis.3 These
Gram+ pol IIICs share a unique primary structure,
distinct from the distantly related Gram- pol IIIEs and
the recently described Gram+ pol IIIEs.4 Most impor-
We reported recently that inhibitory activity of AU
compounds against pol IIIC and growth of Gram+
bacteria could be enhanced by introduction of substit-
uents at the 3 position of the uracil ring. For example
3-(methoxyalkyl) (1) and 3-(hydroxyalkyl) derivatives (2)
of 6-(3-ethyl-4-methylanilino)uracil (EMAU) showed
significant activity against Gram+ bacteria in culture,2
and two related 3-(4-hydroxybutyl) compounds showed
activity against experimental S. aureus infection in
mice.8 Therefore, we have continued SAR and lead
* Author for correspondence: Phone 508 754-6700. FAX 508 754-
7075. E-mail: george.wright@glsynthesis.com.
† GLSynthesis Inc.
‡ University of Massachusetts Medical School.
§ Microbiotix Inc.
10.1021/jm020591z CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/15/2003

2732 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Zhi et al.
compound selection studies, and here report the effects
of modification of the basic AU pharmacophore at the 3
and 6 positions of the uracil ring on pol IIIC inhibition
and antibacterial activity in vitro.
products and results of screening against B. subtilis
cultures are summarized in Table A of Supporting
Information. Notably, the most potent antibacterials,
i.e., compounds with lowest MIC, were 1b and the
corresponding 6-(3-iodo-4-methylanilino) analogue “MP-
IMAU”, with MICs of 3.13 and 1.57 µg/mL, respectively.
Other derivatives with MIC < 10 µg/mL were 3,4-diMe,
3,4-diCl, 3-Et, 3,4-diBr, 3-Cl-4-Me, and 3-Me-4-Br com-
pounds. Not surprisingly, the pattern of activity paral-
leled that for pol IIIC inhibition potency of the previ-
ously reported AU compounds, i.e., those without the 3
substituent.6,7
Selection of 3-(4-Meth oxybu tyl)- (1c) a n d 3-(4-
Hyd r oxyb u t yl)-E MAUs (2c) a s Lea d Com p ou n d s
a n d Syn t h et ic P la t for m s. Simple 3-substituted
EMAUs were found to possess potent pol IIIC inhibition
and had enhanced antibacterial activity in vitro against
Gram-positive bacteria.2 To explore the components of
the antibacterial pharmacophore, we undertook syn-
thesis of derivatives modified in the 3 and 6 positions
of the uracil ring.
The RS3 results prompted us to choose the 3-MP
compounds as platforms for further SAR studies, for
example by modifying the hydroxyl groups of the 3-hy-
droxyalkyl compounds 2. However, attempted demeth-
ylation of the 3-methoxyalkyl compounds 1a and 1b
resulted in their facile conversion to inactive byproducts.
For example, 1b gave a compound identified as the
pyrimido-oxazine 5 (see Experimental Section). These
byproducts also formed readily during attempts to
acylate the hydroxyl groups of 2a and 2b with acetic
anhydride or methanesulfonyl chloride. It is likely that
these systems, which bear hydroxyl groups â and γ,
respectively, to the pyrimidine ring, cyclize readily to
the corresponding fused ring compounds. We were also
unable to prepare the corresponding aminoethyl or
aminopropyl derivatives because of their decomposition,
likely also via cyclization to analogous fused ring
compounds. For example, although 3-(2-azidoethyl)-
EMAU was prepared successfully by direct synthesis
from 2a (PPh3,NaN3), attempted catalytic reduction
gave not the expected aminoethyl derivative, but a
Va lid a tion of Ra p id , Sm a ll Sca le Syn th etic “RS3”
Meth od s. 3-Substituted EMAU compounds are pre-
pared by fusion of a 6-chlorouracil with EMA at high
temperature in the absence of solvent.1 To explore the
utility of this procedure to prepare large numbers of
derivatives for screening purposes, we studied the
reaction conditions required to convert a typical sub-
strate 3-(3-methoxypropyl)-6-chlorouracil (3b) to AU
compounds in small scales (Scheme 1). Heating mix-
Sch em e 1. Synthesis of
3-(Methoxyalkyl)-6-anilinouracils
tures of 3b with EMA for as little as 10 min at 120 °C
resulted in quantitative conversion to MP-EMAU, 1b.
However, optimal conditions for relatively unreactive
anilines involved heating the 6-chlorouracil (2 mg) and
2 equiv of aniline in glass test tubes at 130-150 °C for
20-30 min. Typically, TLC showed consumption of 3b
and appearance of products (confirmed with authentic
samples in several cases). Little or no decomposition
products were noted. The cooled mixtures were dissolved
directly in DMSO to give “20 mM” calculated stock
solutions of products. Stock solutions were diluted into
B. subtilis growth medium to give 2-fold serial dilutions
from 100 to 1.57 µg/mL (see Experimental Section). In
several cases the resulting antibacterial results were
indistinguishable from those obtained with isolated,
purified products. Heating of 3b or anilines alone under
the same conditions did not produce bioactive mixtures.
This rapid, small scale synthesis “RS3” procedure was
applied to reactions between 3b and 40 substituted
anilines in order to discover 6-anilino substituents that
were equivalent or better than the EMA group in
imparting antibacterial potency to the AU compounds.
Anilino substituents ranged from alkyl and polyalkyl,
halo and polyhalo, halo plus alkyl, alkoxy, and nitro,
and involved substitution at 3, 4, and/or 5 positions. The
1
byproduct whose H NMR suggested it to be a pyrimi-
dine-imidazoline (data not shown). The failure to modify
these 3-substituents led us to explore the 3-(4-hydroxy-
butyl) group as a possible stable alternative for synthe-
sis of 3-substituted-6-anilinouracils and related com-
pounds.
Resu lts
Syn th esis of “MB” a n d “HB” 6-An ilin ou r a cils. A
previously developed1 method was improved to give the
key intermediate 3c. The ring synthesis method il-
lustrated in Scheme 2 required synthesis of N-(4-
methoxybutyl)urea. Methylation of 1,3-propanediol with
iodomethane gave 3-methoxy-1-propanol, which reacted
with N-bromosuccinimide in the presence of triphen-
ylphosphine to give 1-bromo-3-methoxypropane.9 This
compound reacted with potassium cyanide in aqueous

Synthesis and Bioactivity of Substituted 6-Anilinouracils
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2733
Sch em e 2. Synthesis of 3c and 3-Substituted-6-(3-ethyl-4-methylanilino)Uracilsa
a
Reagents and conditions: i, diethyl malonate, NaOMe, MeOH, reflux. ii, BnEt3NCl, POCl3, 50 °C; iii. EMA, 150 °C, 30 min. iv, Me3SiI,
CHCl3, rt. v, Me3SiI, CHCl3, reflux. vi, K2CO3, Me2CO, rt.
Ta ble 1. Pol IIIC Inhibition and Antibacterial Activity of 3-(4-Methoxybutyl)-6-anilinouracils
methanol to give 4-methoxybutyronitrile, which was
reduced by lithium aluminum hydride in diethyl ether
to afford 4-methoxybutylamine, purified by distillation,
in high yield. Treatment of 4-methoxybutylamine as the
hydrochloride with potassium cyanate in water gave
N-(4-methoxybutyl)urea in high yield.10 Reaction of the
urea with diethyl malonate in the presence of sodium
methoxide gave 1-(4-methoxybutyl)barbituric acid (6),
and its selective 6-chlorination with POCl3 in the
presence of benzyltriethylammonium chloride at 50 °C
gave the key intermediate 3-(4-methoxybutyl)-6-chloro-
uracil, 3c.
Compound 3c reacted readily with EMA and 3-iodo-
4-methylaniline at high temperatures to give 1c and 4c,
respectively. These compounds were demethylated by
treatment with iodotrimethysilane in chloroform at
room temperature to afford the desired 4-hydroxybutyl
derivatives 2c and 7. The products were accompanied
by byproducts whose relative yield increased during
prolonged treatment with iodotrimethysilane and at
higher reaction temperature (see Experimental Section).
The byproduct from 2c was identified as the 4-iodobutyl
compound 8. Although a nuisance when 4-hydroxybutyl
compounds are the desired products, the 4-iodobutyl
compounds (“IB-AUs”) are excellent intermediates to
prepare substituted derivatives (see below). In addition,
the 3-(4-hydroxybutyl) group of 2c and 7 was more
stable to acids and bases than the hydroxyethyl and
hydroxypropyl groups. Compounds 2c and 7 had high
potency against pol IIIC and Gram+ bacterial growth
(Table 1), and both compounds, given intraperitoneally
(ip), protected mice from ip challenge with S. aureus.8
RS3 w ith 3c. Availability of the 6-chlorouracil inter-
mediate 3c provided further opportunities to apply the
RS3 procedure to give candidate antibacterials with
diverse 6 substituents. Three groups of compounds were
prepared. Thirty eight anilines, some identical to those
described above but others synthesized herein for this
purpose, 30 benzylamines and related amines, and 18
arylalkylamines of various structures were heated with
3c as described above. Results of assay of the reaction
mixtures against B. subtilis pol IIIC and a panel of
seven Gram+ and one Gram- (E. coli) bacteria are
summarized in Tables B-D (Supporting Information).
Several MB-AUs 9 with substituent patterns similar to
EMA were potent inhibitors of pol IIIC, but none was a
better antibacterial compound than 2c (Table B). As in
the case of the MP compounds described above, no

2734 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Zhi et al.
Ta ble 2. Pol IIIC Inhibition and Antibacterial Activity of 3-(4-Substituted-butyl)-6-(3-ethyl-4-methylanilino)uracils
surprises were discovered based on previously reported
enzyme inhibition results.6,7 Among the 6-benzylamino
compounds, MB-BAUs, only 3,4-diCl (compound 10),
3-Br-4-F, and 3-Cl-4-F derivatives had Ki values below
1 µM or MICs below 10 µg/mL (Table C, Supporting
Information). Related monohalo and dihalo and methyl
BAUs had Ki values between 1 and 10 µM, but these
and all other substituted BAUs were devoid of antibac-
terial activity. MB products obtained from diverse
amines, including heteroarylamines, heteroarylmeth-
ylamines, and tryptamines, were devoid of enzyme
inhibitory or antibacterial activity (Table D, Supporting
Information).
produce compounds of series 9 (Table 1) similar to the
3-ethyl-4-methyl pattern of 2c. In the second group of
compounds, substitutions in the 3-(4-hydroxybutyl)
group of 2c were made to give compounds of series 11.
The 4-iodo group of 8 was readily displaced by various
nucleophiles in the presence of potassium carbonate in
a polar, aprotic solvent at room temperature to give
compounds 11 (Table 2). The 3-(4-aminobutyl) derivative
11b was prepared by reduction of the azido compound
11a with hydrogen over 10% palladium on carbon in
methanol. Details of the synthesis and characterization
of all compounds are presented in the Experimental
Section.
Syn th esis a n d Activity of 3-Su bstitu ted -6-a n i-
lin ou r a cils. Compounds closely related to 2c were
synthesized and characterized. In the first series,
modifications were made in the anilino substituents to
Results of enzyme and antibacterial assays of the
compounds are summarized in Tables 1 and 2 and
compared with activity of the reference compound HB-
EMAU (2c) and antibiotics ciprofloxacin and vancomy-

Synthesis and Bioactivity of Substituted 6-Anilinouracils
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2735
cin. HB-EMAU is a potent inhibitor of pol IIIC, with Ki
value of 0.063 µM, and an effective Gram+ antibacterial,
with MIC values of 2.5-5 µg/mL. (B. subtilis is generally
the most sensitive of the Gram+ organisms.) Ciprof-
loxacin potently inhibited growth of all organisms with
the exception of MRSA B42876, a methicillin-resistant
strain of S. aureus with cross-resistance to the fluoro-
quinolones. Vancomycin was selective for all Gram+
organisms except the vancomycin-resistant strain of E.
fecalis. As expected, none of the 6-anilinouracils inhib-
ited growth of the Gram- organism E. coli.
Quantitative SAR studies suggested that optimal
6-anilinouracil:pol IIIC interaction required in the
anilino ring a hydrophobic group in the 3-position of the
size of Et or I and a hydrophobic group in the 4 position
of the size of Me.7 Some of the derivatives shown in
Table 1 were assayed to determine if subtle changes in
these positions would enhance enzyme binding and/or
antibacterial activity. 3-(4-Methoxybutyl) derivatives 9d
(3-Cl-4-Me), 9f (3-CH2Cl-4-Me), and 9g (3-CH2SCN-4-
Me) were as potent pol IIIC inhibitors as 1c (3-Et-4-
Me), but only 9d was equivalent in antibacterial activ-
ity. Although the 3-Br (9e) and 3-I (4c) analogues were
somewhat weaker as pol IIIC inhibitors, they were
nearly as potent antibacterials as 1c. The closely related
3-vinyl (9b) and 3-isopropenyl (9c) compounds were
weaker in both assays. Compounds with substituents
of similar size in the 3 position, but with electron-
attracting or hydrophilic properties were considerably
weaker pol IIIC inhibitors and largely devoid of anti-
bacterial activity.
sive study showed that the compounds inhibited the
growth of a wide range of clinical isolates of S. aureus,
E. fecalis, and E. fecium in the range of 4-16 µg/mL.11
These compounds were equally active against drug-
sensitive and drug-resistant strains and were bacteri-
cidal against MRSA and both vancomycin-sensitive and
vancomycin-resistant strains of E. fecalis. Although two
analogues (2c and 7) protected mice from intraperito-
neal challenge with S. aureus when administered at 10
mg/kg by the same route,8 attempted treatment by the
intravenous or subcutaneous route was hampered by
the low water solubility of the compounds. Compound
2c was solubilized in 30% â-hydroxypropylcyclodextrin
in physiological saline, but the compound still did not
protect mice when given in single doses intravenously
or subcutaneously in this vehicle (data not shown).
Results of assay of several series of substituted
6-aminouracils have led us to conclude that the 6-(3-
ethyl-4-methylanilino)uracil (EMAU) scaffold provides
optimal binding affinity to the target enzyme pol IIIC
(Table 1, and see Supporting Information). Using this
scaffold we have found that 3 substituents, preferably
substituted alkyl groups, increase antibacterial activity
against Gram+ organisms. Simple hydroxyalkyl and
methoxyalkyl1,2 and more complex aminoalkyl substit-
uents can increase pol IIIC inhibition potency (Table
2), but do not consistently increase antibacterial po-
tency. Indeed, the most potent 3-substituted-butyl
EMAU derivative 11j has a bulky, hydrophobically
substituted piperidinyl group. In the case of 6-benzyl-
aminouracils, the 3,4-dichlorobenzyl substitution pat-
tern of 10 provided optimal binding to pol IIIC and
moderate antibacterial activity (Table 1) among a
limited series of 6-benzylamino and other 6-amino-
uracils (see Supporting Information).
Modification of the uracil 3 position provided a series
of highly potent pol IIIC inhibitors, but with wide
variation in antibacterial activity (Table 2). Simple
changes in the OMe group of MB-EMAU (1c) to OH (2c),
+
The challenges in developing the family of pol IIIC
inhibitors as antibacterials include improved potency
at the target enzyme and whole bacteria levelssMIC
values of 1 µg/mL or less are desirable, water solubility
to facilitate parenteral formulations, metabolic stability
to prolong blood levels of active compound, and en-
hanced oral bioavailability. In this paper we have
addressed the first of these properties, extending two
series of inhibitors in the search for more potent pol IIIC
inhibitors and antibacterials in vitro.
I (8), N3 (11a ), and NH3 (11b) did not alter pol IIIC
affinity; however, the I and NH3+ derivatives were weak
or inactive as antibacterials. A series of tertiary ami-
nobutyl derivatives, consisting of piperidinyl, morpho-
linyl, and piperazinyl compounds, showed enhanced pol
IIIC inhibition, ranging from 2-fold to 4-fold. The narrow
range of Ki values for these derivatives and the lack of
steric limit for bulky substituents in several of them
suggest that these groups interact minimally with
enzyme or DNA primer:template. In contrast to the
increase in pol IIIC inhibition, many of these compounds
had only modest antibacterial activity or were inactive.
For example, the hydrophilic morpholinyl compounds
11f and 11g and the piperazinyl compounds 11k -n
were weak antibacterials, and the piperazinylbutyl
compound 11o was inactive. Among a series of 4-hy-
droxy-4-phenylpiperidinyl compounds, the 4-Cl-phenyl
derivative (11i) was almost as active as 1c, and the
3-CF3-4-Cl-phenyl derivative (11j) was the most potent
antibacterial among the 6-anilinouracils. That the most
hydrophilic analogues have the weakest antibacterial
activity despite their potent inhibition of pol IIIC
suggests that uptake by the bacteria may limit access
to the target enzyme.
Exp er im en ta l Section
Ma ter ia ls. Most reagent chemicals and solvents were
obtained from commercial sources. 3-Acetyl-4-methylaniline
and 3-(1-hydroxyethyl)-4-methylaniline12 and 3-ethyl-4-meth-
ylaniline7 were prepared as described. Other substituted
anilines, benzylamines, and related amines were available
commercially, except as noted. NMR spectra were obtained in
Me2SO-d6 solutions, unless indicated otherwise, with a Bruker
Avance 300 instrument; chemical shifts are in ppm from
internal TMS, and J values were as expected. Melting points
were determined on a Mel-temp apparatus and are uncor-
rected. Elemental analyses were performed by the Microanaly-
sis Laboratory, University of Massachusetts, Amherst. Values
for C, H, and N were within 0.4% of calculated values except
as noted.
Cycliza tion of 2b. Iodotrimethylsilane (Me2SiI) (132 mg,
0.66 mmol) was added to a stirred solution of 2b (100 mg, 0.33
mmol) in dry CHCl3. The reaction mixture was stirred at room
temperature overnight, until disappearance of starting mate-
rial. MeOH and Na2SO3 were then added to the brown-purple
solution. After being stirred at room temperature for 10 min,
Discu ssion
The prototype pol IIIC inhibitors MB-EMAU (1c) and
HB-EMAU (2c) demonstrate potent and selective anti-
bacterial activity against Gram+ organisms. An exten-

2736 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Zhi et al.
the mixture was filtered and the solvent was removed. The
residue was purified by chromatography on silica gel with
2-5% MeOH in CHCl3 as eluent to give 80 mg (85%) of 5, mp
CH2), 3.32(s, 3H, OCH3), 3.38 (t, 2H, NCH2), 3.92 (t, 2H,
OCH2), 5.84 (s, 1H, 5-H) and 9.70 (s, 1H, 1-H). Anal. (C9H13
ClNO3) C, H, N.
-
1
223-224 °C. H NMR: 1.12 (t, 3H, CH3), 2.11 (m, 2H, CH2),
3-(4-Me t h oxyb u t yl)-6-(3-e t h yl-4-m e t h yla n ilin o)u r a -
cil, 1c. A stirred mixture of 3c (3.5 g, 15 mmol) and 3-ethyl-
4-methylaniline (4.5 g, 33.3 mmol) was heated at 150 °C for
30 min. The mixture was cooled to room temperature, and the
mass was crystallized from EtOH to give 3.6 g (72%) of product
as white crystals, mp 186-188 °C. The filtrate was evaporated,
and the residue was purified by chromatography on silica gel
with 2-4% MeOH in CHCl3 as eluent to give an additional
1.15 g (23%) of product. 1H NMR: 1.14 (t, 3H, PhCH2CH3),
1.50 (m, 4H, 2 × CH2), 2.24 (s, 3H, PhCH3), 2.57 (q, 2H, CH2-
Ar), 3.21 (s, 3H, OCH3), 3.32 (t, 2H, NCH2), 3.68 (t, 2H, OCH2),
4.72 (s, 1H, 5-H), 6.92-7.17 (m, 3H, PhH), 8.10 (s, 1H, 6-NH),
10.40 (s, 1H, 1-H). Anal. (C18H25N3O3) C, H, N.
2.20 (s, 3H, PhCH3), 2.56 (q, 2H, PhCH2), 3.69 (t, 2H, NCH2),
4.29 (t, 2H, OCH2), 5.26 (s, 1H, 5-H), 7.05 (d, 1H, PhH), 7.27
(s, 1H, PhH), 7.46 (d, 1H, PhH), 9.11 (s, 1H, NH). Anal.
(C16H19N3O2‚0.25H2O) C, H, N.
N-(4-Meth oxybu tyl)u r ea . a . 4-Meth oxy-1-bu tyr on itr ile.
A solution of 1-bromo-3-methoxypropane9 (54 g, 353 mmol) in
MeOH (40 mL) was added dropwise to a stirred solution of
KCN (27 g, 415 mmol) in 50 mL of water and 50 mL of MeOH
at room temperature. The mixture was heated at reflux for
20 h, cooled to room temperature, and treated with 100 mL of
water. The mixture was extracted with EtOAc (4 × 100 mL),
and the extracts were dried over MgSO4. After removal of the
solvent, the residue was distilled to give 30 g (86%) of
4-methoxy-1-butyronitrile as a colorless oil, bp 168-169 °C.
1H NMR (CDCl3): 1.92 (m, 2H, CH2), 2.48 (t, 2H, CH2CN),
3.37 (s, 3H, OCH3), 3.50 (t, 2H, OCH2). b. 4-Meth oxybu tyl-
a m in e Hyd r och lor id e. A solution of 4-methoxy-1-butyroni-
trile (29 g, 292 mmol) in 50 mL of anhydrous Et2O was added
dropwise to a stirred solution of LiAlH4 (13 g) in 150 mL
anhydrous Et2O at 0 °C. After the mixture was stirred for 1 h
at room temperature, 15% aqueous NaOH (50 mL) was added
dropwise. The reaction mixture was filtered, and the solid was
washed with Et2O. The ether phase was separated, and the
aqueous phase was extracted with Et2O and CHCl3. After the
combined organic extracts were dried over MgSO4, the solvents
were removed and the residue was dissolved in 30 mL of
MeOH, which was neutralized with concentrated aqueous HCl
3-(4-Meth oxybu tyl)-6-(3-iodo-4-m eth ylan ilin o)u r acil, 4c.
This compound was prepared by the same procedure as above
using 3-iodo-4-methylaniline. Yield, 88%, mp 214-216 °C. 1H
NMR: 1.47 (m, 4H, 2 × CH2), 2.33 (s, 3H, PhCH3), 3.20 (s,
3H, OCH3), 3.31 (t, 2H, NCH2), 3.67 (t, 2H, OCH2), 4.72 (s,
1H, 5-H), 7.16 (d, 1H, PhH), 7.32 (d, 1H, PhH), 7.63 (s, 1H,
PhH), 8.24 (s, 1H, 6-NH), 10.62 (s, 1H, 1-H). Anal. (C16H20
IN3O3) C, H, N.
-
3-(4-H yd r oxyb u t yl)-6-(3-e t h yl-4-m e t h yla n ilin o)u r a -
cil, 2c. Me3SiI (2.3 mL, 16 mmol) was added to a stirred
solution of 1c (1.3 g, 4 mmol) in dry CHCl3 (60 mL). The
reaction mixture was stirred at room temperature until
disappearance of starting material (TLC). MeOH (10 mL) and
Na2SO3 (0.5 g) were added to the brown-purple solution. After
stirring at room temperature for 10 min, the mixture was
filtered, and the solvent was removed in vacuo. The residue
was purified by chromatography on silica gel with 1-7%
MeOH in CHCl3 as eluent to give, first, 3-(4-iodobutyl)-6-(3-
ethyl-4-methylanilino)uracil, 8 (21%) (see below). 1H NMR:
1.14 (t, 3H, PhCH2CH3), 1.54-1.78 (m, 4H, 2 × CH2), 2.24 (s,
3H, PhCH3), 2.57 (q, 2H, PhCH2), 3.29 (t, 2H, ICH2), 3.72 (t,
2H, NCH2), 4.73 (s, 1H, 5-H), 6.92-7.15 (m, 3H, PhH), 8.15
(s, 1H, 6-NH), 10.45 (s, 1H, 1-H). Anal. (C17H22IN3O2) C, H,
N. The second eluate was 2c (710 mg, 57%), mp 211-212 °C.
1H NMR: 1.14 (t, 3H, PhCH2CH3), 1.38-1.50 (m, 4H, 2 × CH2),
2.24 (s, 3H, PhCH3), 2.57 (q, 2H, PhCH2), 3.34 (t, 2H, NCH2),
3.67 (t, 2H, OCH2), 4.38 (t, 1H, OH), 4.72 (s, 1H, 5-H), 6.92-
7.17 (m, 3H, PhH), 8.08 (s, 1H, 6-NH), 10.38 (s, 1H, 1-H). Anal.
(C17H23N3O3) C, H, N.
1
to give 33 g (81%) of 4-methoxybutylamine hydrochloride. H
NMR: 1.58 (m, 4H, 2 × CH2), 2.80 (m, 2H, NCH2), 3.25 (s,
3H, OCH3), 3.33 (t, 2H, OCH2) and 7.91 (br s, 3H, NH3).
c. 4-Meth oxybu tylu r ea . A mixture of 4-methoxybutylamine
hydrochloride (5.0 g, 35.8 mmol) and KNCO (2.9 g, 35.8 mmol)
in 50 mL of water was heated at reflux for 4 h. The water was
evaporated at reduced pressure, EtOH (100 mL) was added,
and the suspension was heated. The warm mixture was
filtered, and the filtered solid was washed with hot EtOH.
Concentration and cooling of the filtrate gave 5 g (96%) of
4-methoxybutylurea.10 Crystallization from ethyl acetate gave
1
white crystals, mp 86-88 °C. H NMR (CDCl3): 1.60 (m, 4H,
2 × CH2), 3.17 (m, 2H, NCH2), 3.33 (s, 3H, OCH3), 3.40 (t, 2H,
OCH2), 4.60 (br s, 2H, NH2) and 5.01 (br s, 1H, NH).
1-(4-Meth oxybu tyl)ba r bitu r ic Acid , 6. Clean Na (1.6 g,
70 mmol) was added slowly to 60 mL of absolute EtOH. When
all the Na had dissolved, N-(4-methoxybutyl)urea (4.0 g, 27.4
mmol) and diethyl malonate (4.4 g, 27.5 mmol) were added,
and the mixture was heated at reflux for 6 h. The mixture
was allowed to cool, and concd aq HCl was added until the
solution was acidic. The solvent was evaporated at reduced
pressure, and EtOH (100 mL) was added to the residue with
heating. The hot mixture was filtered, and the filtered solid
was washed with EtOH. Concentration of the filtrate gave 5.1
g (87%) of 1-(4-methoxybutyl)barbituric acid as a light yellow
oil. 1H NMR (CDCl3): 1.64 (m, 4H, 2 × CH2), 3.33 (s, 3H,
OCH3), 3.41 (t, 2H, NCH2), 3.64 (s, 2H, 5-H), 3.88 (t, 2H, OCH2)
and 8.94 (s, 1H, 3-H). Anal. (C9H14N2O4) H,N. C, calcd. 50.46;
found 51.17.
6-Ch lor o-3-(4-m eth oxybu tyl)u r a cil, 3c. A stirred mixture
of 1-(4-methoxybutyl)barbituric acid (5.0 g, 23.3 mmol) and
benzyltriethylammonium chloride (11 g, 48.3 mmol) in POCl3
(50 mL) was heated at 50 °C for 3 h. The reaction mixture
was cooled to room temperature and evaporated to dryness in
vacuo. The residue was carefully quenched with 60 g of ice
chips and maintained at 0 °C. The solution was extracted with
CHCl3 (4 × 60 mL), and the combined organic extract was
dried over Na2SO4 and the solvent evaporated. Crystallization
of the residue from EtOH gave 3.8 g (70%) of product as white
crystals, mp 145-147 °C. The filtrate was evaporated, and the
residue was purified by chromatography on silica gel with
1-2% MeOH in CHCl3 as eluent to give an additional 0.9 g
(17%) of product. 1H NMR (CDCl3): 1.58-1.80 (m, 4H, 2 ×
3-(4-Hyd r oxybu tyl)-6-(3-iod o-4-m eth yla n ilin o)u r a cil, 7.
This compound was prepared by demethylation of 4c, by the
same procedure as described above, in 48% yield, mp 184-
185 °C. 1H NMR: 1.30-1.57 (m, 4H, 2 × CH2), 2.35 (s, 3H,
PhCH3), 3.37 (t, 2H, NCH2), 3.68 (t, 2H, OCH2), 4.38 (br s,
1H, OH), 4.73 (s, 1H, 5-H), 7.17 (d, 1H, PhH), 7.32 (d, 1H,
PhH), 7.63 (s, 1H, PhH), 8.25 (s, 1H, 6-NH), 10.60 (s, 1H, 1-H).
Anal. (C15H18IN3O3‚0.5H2O) C, H, N.
3-(4-Iodobu tyl)-6-(3-eth yl-4-m eth ylan ilin o)u r acil, 8. Me3-
SiI (7.1 mL, 50 mmol) was added to a stirred solution of 1c
(3.31 g, 10 mmol) in dry CHCl3. The reaction mixture was
stirred at reflux overnight until disappearance of starting
material (TLC). MeOH and Na2SO3 were then added to the
brown-purple solution. After stirring at room temperature for
10 min, the mixture was filtered and the solvent was removed.
Crystallization of the residue from EtOH gave 3.6 g (84%) of
product as a white solid, mp 216-217 °C. The filtrate was
evaporated, and the residue was purified by chromatography
on silica gel with 1-2% MeOH in CHCl3 as eluent to give an
additional 0.4 g (9.4%) of product. See above for properties.
3-(4-Meth oxybu tyl)-6-a n ilin ou r a cils. Gen er a l P r oce-
d u r e. Compound 3c (465 mg, 2 mmol) and the aniline (2 equiv)
were mixed well in a test tube and stirred at 145 °C for 8-15
min under N2. The reaction mixture typically solidifies after
a few min. The reaction was cooled to room temperature, and
the solid was crystallized from EtOH or purified by column
chromatography on silica gel to obtain the product.

Synthesis and Bioactivity of Substituted 6-Anilinouracils
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2737
3-(4-Meth oxybu tyl)-6-(3,4-d im eth yla n ilin o)u r a cil, 9a .
Reaction with 3,4-dimethylaniline for 15 min yielded 540 mg
(85%) of product as an off-white powder after crystallization,
mp 210-212 °C. 1H NMR: 1.47 (m, 4H, 2 × CH2), 2.18 (s, 3H,
PhCH3), 2.21 (s, 3H, PhCH3), 3.02 (s, 3H, OCH3), 3.30 (t, 2H,
NCH2), 3.65 (t, 2H, OCH2), 4.69 (s, 1H, 5-H), 6.90 (dd, 1H,
PhH), 6.96 (d, 1H, PhH), 7.13 (d, 1H, PhH), 8.01 (s, 1H, 6-NH),
10.35 (s, 1H, 1-H). Anal. (C17H23N3O3) C, H, N.
1H, PhH), 7.22 (d, 1H, PhH), 8.30 (s, 1H, 6-NH), 10.50(s, 1H,
1-H). Anal. (C18H22N4O3) C,H; N, calcd. 16.36, found 15.52.
3-(4-Meth oxybu tyl)-6-(3-m er ca p tom eth yl-4-m eth yla n i-
lin o)u r a cil, 9i. Reaction of NaSH‚H2O (25.5 mg, 0.5 mmol)
and 9f (35 mg, 0.1 mmol), as described for the preparation of
9g, yielded the product (24 mg, 70%) as a white solid, mp 198-
200 °C. 1H NMR: 1.45 (m, 4H, 2 × CH2), 2.25 (s, 3H, CH3),
3.17 (s, 3H, OCH3), 3.28 (t, 2H, NCH2), 3.67 (t, 2H, SCH2),
3.76 (t, 2H, OCH2), 4.76 (t, 1H, SH), 4.84 (s, 1H, 5-H), 7.00
(m, 2H, 2 × PhH), 7.16 (d, 1H, PhH), 8.17 (s, 1H, 6-NH), 10.38
(s, 1H, 1-H). Anal. (C17H23SN3O3‚0.5H2O) C,H. N, calcd 11.72;
found 10.45.
3-(4-Me t h oxyb u t yl)-6-(3-vin yl-4-m e t h yla n ilin o)u r a -
cil, 9b. A mixture of 3c (46.5 mg, 0.2 mmol) and 3-(1-
hydroxyethyl)-4-methylaniline (60.5 mg, 4 mmol) was heated
at 150 °C for 15 min. The mixture was purified by chroma-
tography on silica gel with 1-1.5% MeOH in CHCl3 as eluent
to give 30 mg (45%) of 9b as an off-white powder, mp 155 °C
3-(4-Meth oxybu tyl)-6-[3-(1-ch lor oeth yl)-4-m eth yla n ili-
n o]u r a cil, 9j. Ph3P (157 mg, 0.6 mmol) was added to a solution
of 9m (174 mg, 0.5 mmol) in CCl4 (2 mL) and MeCN (2 mL) at
room temperature. The mixture was stirred at room temper-
ature for 2 h and at 40 °C for 0.5 h. The solvents were removed
in vacuo, and the residue was purified by silica gel chroma-
tography. Elution with 2-3% MeOH in CHCl3 yielded 128 mg
1
(dec). H NMR: 1.47 (m, 4H, 2 × CH2), 2.29 (s, 3H, PhCH3),
3.20 (s, 3H, OCH3), 3.28 (t, 2H, NCH2), 3.69 (t, 2H, OCH3),
4.66 (s, 1H, 5-H), 5.30 (d, 1H, vinyl), 5.62 (d, 1H, vinyl), 6.88
(dd, 1H, vinyl), 6.95 (dd, 1H, PhH), 7.12 (d, 1H, PhH), 7.28 (d,
1H, PhH), 8.10 (s, 1H, 6-NH), 10.50 (s, 1H, 1-H). Anal.
(C18H23N3O3) H,N. C, calcd. 65.63; found 65.12.
1
(70%) of 9j as a white solid, mp 190-192 °C. H NMR: 1.44
3-(4-Meth oxybu tyl)-6-(3-isop r op en yl-4-m eth yla n ilin o)-
u r a cil, 9c. Reaction with 3-isopropenyl-4-methylaniline for 15
min yielded 357 mg (52%) of product as a light yellow powder
(m, 4H, 2 × CH2), 1.76 (d, 3H, CCH3), 2.32 (s, 3H, PhCH3),
3.20 (s, 3H, OCH3), 3.27 (t, 2H, NCH2), 3.66 (t, 2H, OCH2),
4.70 (s, 1H, 5-H), 5.47 (q, 1H, ClCH), 7.06 (dd, 1H, PhH), 7.18
(d, 1H, PhH), 7.32 (d, 1H, PhH), 8.20 (s, 1H, 6-NH), 10.48 (s,
1H, 1-H). Anal. (C18H24ClN3O3) C, H, N.
1
after crystallization, mp 186-188 °C. H NMR: 1.42 (m, 4H,
2 × CH2), 1.98 (s, 3H, CdCCH3), 2.21 (s, 3H, PhCH3), 3.18 (s,
3H, OCH3), 3.28 (t, 2H, NCH2), 3.62 (t, 2H, OCH3), 4.71 (s,
1H, 5-H), 4.81 (d, 1H, dCH), 5.20 (d, 1H, dCH), 6.90 (d, 1H,
PhH), 7.00 (dd, 1H, PhH), 7.17 (d, 1H, PhH), 8.11 (s, 1H,
6-NH), 10.44 (s, 1H, 1-H). Anal. (C19H25N3O3‚0.55 H2O) C,
H, N.
3-(4-Met h oxyb u t yl)-6-(3-a cet yl-4-m et h yla n ilin o)u r a -
cil, 9k . Reaction with 3-acetyl-4-methylaniline for 8 min
yielded 518 mg (75%) of product as an off-white powder after
1
crystallization, mp 182-184 °C (dec). H NMR: 1.47 (m, 4H,
2 × CH2), 2.08 (s, 3H, COCH3), 2.39 (s, 3H, PhCH3), 3.20 (s,
3H, OCH3), 3.28 (t, 2H, NCH2), 3.68 (t, 2H, OCH2), 4.75 (s,
1H, 5-H), 7.29 (m, 2H, 2 × PhH), 7.59 (s, 1H, PhH), 8.30 (s,
1H, 6-NH), 10.62 (s, 1H, 1-H). Anal. (C18H23N3O4‚0.35 H2O)
C, H, N.
3-(4-Met h oxyb u t yl)-6-(3-ch lor o-4-m et h yla n ilin o)u r a -
cil, 9d . Reaction with 3-chloro-4-methylaniline for 15 min
yielded 527 mg (78%) of product as a light yellow powder after
crystallization, mp 220-222 °C. 1H NMR: 1.45 (m, 4H, 2 ×
CH2), 2.27 (s, 3H, PhCH3), 3.18 (s, 3H, OCH3), 3.27 (t, 2H,
NCH2), 3.67 (t, 2H, OCH2), 4.76 (s, 1H, 5-H), 7.08 (dd, 1H,
PhH), 7.23 (d, 1H, PhH), 7.31 (d, 1H, PhH), 8.31 (s, 1H, 6-NH),
10.60 (s, 1H, 1-H). Anal. (C16H20ClN3O3) C, H, N.
3-(4-Met h oxyb u t yl)-6-(3-b r om o-4-m et h yla n ilin o)u r a -
cil, 9e. Reaction with 3-bromo-4-methylaniline for 15 min
yielded 535 mg (70%) of product as a light yellow powder after
crystallization, mp 218-220 °C. 1H NMR: 1.44 (m, 4H, 2 ×
CH2), 2.29 (s, 3H, PhCH3), 3.18 (s, 3H, OCH3), 3.26 (t, 2H,
NCH2), 3.67 (t, 2H, OCH2), 4.75 (s, 1H, 5-H), 7.12 (dd, 1H,
PhH), 7.32 (d, 1H, PhH), 7.39 (d, 1H, PhH), 8.31 (s, 1H, 6-NH),
10.62(s, 1H, 1-H). Anal. (C16H20BrN3O3) C, H, N.
3-(4-Meth oxybu tyl)-6-(3-ch lor om eth yl-4-m eth ylan ilin o)-
u r a cil, 9f. Compound 9l (166.5 mg, 0.5 mmol) was treated as
in the preparation of 9j to give the product (109 mg, 62%) as
a white solid, mp 196-198 °C. 1H NMR: 1.45 (m, 4H, 2 ×
CH2), 2.32 (s, 3H, PhCH3), 3.18 (s, 3H, OCH3), 3.27 (t, 2H,
NCH2), 3.67 (t, 2H, OCH2), 4.73 (s, 1H, 5-H), 4.76 (s, 2H,
ClCH2), 7.08 (dd, 1H, PhH), 7.21 (d, 1H, PhH), 7.23 (d, 1H,
3-(4-Meth oxybu tyl)-6-(3-h yd r oxym eth yl-4-m eth yla n ili-
n o)u r a cil, 9l. Reaction with 3-hydroxymethyl-4-methylaniline
for 10 min yielded 333 mg (50%) of product as a light yellow
1
powder after crystallization, mp 190-192 °C (dec). H NMR:
1.45 (m, 4H, 2 × CH2), 2.16 (s, 3H, PhCH3), 3.18 (s, 3H, OCH3),
3.26 (t, 2H, NCH2), 3.66 (d, 2H, OCH2), 4.45 (d, 2H, PhCH2),
4.76 (s, 1H, 5-H), 5.19 (t, 1H, OH), 6.96 (d, 1H, PhH), 7.10 (d,
1H, PhH), 7.20 (s, 1H, PhH), 8.11 (s, 1H, 6-NH), 10.33 (s, 1H,
1-H). Anal. (C17H23N3O3‚0.25H2O) C, H, N.
3-(4-Meth oxybu tyl)-6-[3-(1-h yd r oxyeth yl)-4-m eth yla n i-
lin o]u r a cil, 9m . A solution of 9k (345 mg, 1 mmol) in MeOH
(10 mL) was treated 0 °C with NaBH4 (228 mg, 6 mmol). The
temperature was raised to room temperature, and the mixture
was stirred for 2 h. The reaction mixture was cooled in an ice-
water bath, and aqueous NH4Cl (10 mL) was added slowly.
After stirring the mixture for 0.5 h at room temperature, the
solid was filtered and washed with MeOH (3 × 5 mL). The
combined filtrate was concentrated, and the residue was
applied to a silica gel column and eluted with 6% MeOH in
CHCl3 to yield 330 mg (95%) of 9m as a white solid, mp 204-
PhH), 8.20 (s, 1H, 6-NH), 10.47 (s, 1H, 1-H). Anal. (C17H22
ClN3O3) C, H, N.
-
1
206 °C. H NMR: 1.27 (d, 3H, CCH3), 1.47 (m, 4H, 2 × CH2),
3-(4-Meth oxybu tyl)-6-(3-th iocya n om eth yl-4-m eth yla n i-
lin o)u r a cil, 9g. KSCN (48.5 mg, 0.5 mmol) was added to a
solution of 9f (35.2 mg, 0.1 mmol) in EtOH (5 mL). The mixture
was heated at reflux for 6 h, the solvent was removed in vacuo,
and the residue was applied to a silica gel column. Elution
with 2-3% MeOH in CHCl3 yielded the product (27 mg, 71%)
2.24 (s, 3H, PhCH3), 3.20 (s, 3H, OCH3), 3.30 (t, 2H, NCH2),
3.69 (t, 2H, OCH2), 4.79 (s, 1H, 5-H), 4.87 (m, 1H, PhCH), 5.17
(d, 1H, OH), 6.95 (dd, 1H, PhH), 7.10 (d, 1H, PhH), 7.29 (d,
1H, PhH), 8.16 (s, 1H, 6-NH), 10.37 (s, 1H, 1-H). Anal.
(C18H25N3O4 ‚0.25H2O) C, H, N.
1
as a white solid, mp 170-172 °C. H NMR: 1.45 (m, 4H, 2 ×
3-(4-Met h oxyb u t yl)-6-(3,4-d ich lor ob en zyla m in o)u r a -
cil, 10. A mixture of 3c (111 mg, 0.5 mmol) and 3,4-
dichlorobenzylamine (444 mg, 2.5 mmol) was heated at 125
°C for 4 h. The reaction mixture was cooled to room temper-
ature and dissolved in 20 mL of CHCl3. The solution was
extracted with 2 × 20 mL of 0.1 M aqueous HCl, and the CHCl3
layer was evaporated to leave a solid. This solid was stirred
in EtOAc and filtered to give 125 mg (69%) of product as a
white powder, mp 226-229 °C. 1H NMR: 1.46 (m, 4H, 2 ×
CH2), 3.19 (s, 3H, OCH3), 3.28 (t, 3H, NCH2), 3.64 (t, 2H,
OCH2), 4.29 (d, ΒnCH2), 4.51 (s, 1H, 5-H), 6.68 (t, 1H, 6-NH),
7.31 (dd, 1H, PhH), 7.60 (d, 1H, PhH), 7.62 (d, 1H, PhH), 10.40
(s, 1H, 1-H). Anal. (C16H19Cl2N3O3) C, H, N.
CH2), 2.31 (s, 3H, CH3), 3.18 (s, 3H, OCH3), 3.28 (t, 2H, NCH2),
3.67 (t, 2H, OCH3), 4.38 (s, 2H, NCSCH2), 4.83(s, 1H, 5-H),
7.08 (d, 1H, PhH), 7.19 (s, 1H, PhH), 7.22 (d, 1H, PhH), 8.22
(s, 1H, 6-NH), 10.35 (s, 1H, 1-H). Anal. (C18H22N4O3S) C,H.
N, calcd. 14.96; found 13.52.
3-(4-Meth oxybu tyl)-6-(3-cyan om eth yl-4-m eth ylan ilin o)-
u r a cil, 9h . Reaction of KCN (32 mg, 0.5 mmol) and 9f (35
mg, 0.1 mmol), as described for the preparation of 9g, yielded
the product (20 mg, 60%) as a white solid, mp 200-202 °C.
1H NMR: 1.45 (m, 4H, 2 × CH2), 2.24 (s, 3H, CH3), 3.17 (s,
3H, OCH3), 3.28 (t, 2H, NCH2), 3.67 (t, 2H, OCH2), 3.98 (s,
2H, NCCH2), 4.76 (s, 1H, 5-H), 7.06 (dd, 1H, PhH), 7.18 (d,

2738 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13
Zhi et al.
3-(Su bstitu ted -bu tyl)-EMAUs. Gen er a l P r oced u r e. A
mixture of 8, K2CO3, and the nucleophile in an appropriate
solvent (Me2CO, MeCN, DMF) was stirred at room tempera-
ture. Once the reaction was complete (TLC), the solution was
concentrated in vacuo and water was added. The mixture was
extracted with CHCl3, and the extracts were dried over Na2-
SO4. After removal of solvent, the residue was purified by
chromatography on silica gel using a mixture of MeOH:CHCl3
as eluent to give the product. In all cases, proton NMR
resonances characteristic of the 3-ethyl-4-methylphenyl group
were observed (e.g., see compounds 1c and 2c).
3-(4-Azid obu tyl)-6-(3-eth yl-4-m eth yla n ilin o)u r a cil, 11a .
A mixture of 8 (150 mg, 0.35 mmol) and NaN3 (200 mg, 3
mmol) in DMF (30 mL) was heated at 70-80 °C for 7 h. The
solution was concentrated in vacuo, and water (25 mL) was
added. The mixture was extracted with CHCl3 (3 × 40 mL),
and the combined organic layers were dried over anhydrous
MgSO4. After removal of the solvent, the residue was purified
by chromatography on silica gel using 3-5% MeOH in CHCl3
as eluent, to give 110 mg (92%) of product, mp 209-210 °C.
1H NMR: 1.15 (t, 3H, PhCH2CH3), 1.53 (m, 4H, 2 × CH2), 2.23
(s, 3H, PhCH3), 2.57 (q, 2H, PhCH2), 3.33 (t, 2H, N3CH2), 3.70
(t, 2H, NCH2), 4.73 (s, 1H, 5-H), 6.92-7.18 (m, 3H, PhH), 8.11
(s, 1H, 6-NH), 10.40 (s, 1H, 1-H). Anal. (C17H22N6O2) C, H, N.
3-{4-[(3-Eth oxy-1-m or ph olin yl]bu tyl}-6-(3-eth yl-4-m eth -
yla n ilin o)u r a cil, 11g. The general method gave the product
in 51% yield, mp 162-163 °C. 1H NMR: 1.12 (m, 6H, 2 × CH3),
1.31-1.50 (m, 4H, 2 × CH2), 1.82-2.24 (m, 5H, NCH2, PhCH3),
2.38-2.60 (m, 6H, NCH2, PhCH2), 3.42 (m, 2H, OCH2), 3.70
(m, 4H, NCH2, OCH2), 4.54 (m, 1H, CHO), 4.73 (s, 1H, 5-H),
6.92-7.12 (m, 3H, PhH), 8.06 (s, 1H, 6-NH), 10.42 (s, 1H, 1-H).
Anal. (C23H34N4O4) C, H, N.
3-{4-[(4-H yd r oxy-4-p h en yl)-1-p ip er id in yl]b u t yl}-6-(3-
eth yl-4-m eth yla n ilin o)u r a cil, 11h . The general method
gave the product in 67% yield, mp 203-204 °C. 1H NMR: 1.10
(t, 3H, ArCH2CH3), 1.36-1.42 (m, 6H, 3 × CH2), 1.90 (t, 2H,
CH2), 2.20 (s, 3H, PhCH3), 2.30-2.72 (m, 8H, 3 × CH2, PhCH2),
3.30 (s, 1H, OH), 3.68 (t, 2H, NCH2), 4.73 (s, 1H, 5-H), 6.91-
7.14 (m, 3H, PhH), 7.20 (t, 1H, PhH), 7.31 (t, 2H, PhH), 7.48
(d, 2H, PhH), 8.33 (s, 1H, 6-NH), 10.66 (s, 1H, 1-H). Anal.
(C28H36N4O3) C, H, N.
3{[4-(4-Ch lor op h en yl)-4-h yd r oxy-1-p ip er id in yl]bu tyl}-
6-(3-eth yl-4-m eth yla n ilin o)u r a cil, 11i. The general method
gave the product in 64% yield, mp 235 °C (dec). 1H NMR: 10.48
(s, 1H, 1-H), 8.22 (s, 1H, 6-NH), 7.50 (d, 2H, PhH), 7.31 (d,
2H, PhH), 6.90-7.14 (m, 3H, PhH), 4.73 (s, 1H, 5-H), 3.70 (t,
2H, NCH2), 2.30-2.72 (m, 8H, 3 × NCH2, PhCH2), 2.20 (s, 3H,
PhCH3), 1.90 (t, 2H, CH2), 1.37-1.62 (m, 6H, 3 × CH2), 1.10
(t, 3H, PhCH2CH3). Anal. (C28H35ClN4O3) C,H; N, calcd 10.96,
found 10.37.
3-(4-Am in obu tyl)-6-(3-eth yl-4-m eth yla n ilin o)u r a cil Hy-
d r och lor id e, 11b. A mixture of 11a (100 mg, 0.3 mmol) and
10% Pd/C (45 mg) in MeOH (40 mL) and CHCl3 (10 mL) was
stirred under H2 at 35 psi for 14 h at room temperature. The
mixture was filtered, and the catalyst was washed with MeOH.
The combined filtrates were concentrated, and the residue was
purified by chromatography on silica gel with 10-20% MeOH
in CHCl3 as eluent, to give 78 mg (84%) of 3-(4-aminobutyl)-
6-(3-ethyl-4-methylanilino)uracil. This product was dissolved
in a mixture of MeOH and CHCl3, and a solution of 4.0 M HCl
in dioxane (4 mL) was added. The mixture was stirred at room
temperature for 0.5 h, and the solvents were removed to give
3{[4-(3-Tr iflu or om eth yl-4-ch lor op h en yl)-4-h yd r oxy-1-
p ip er id in yl]bu tyl}-6-(3-eth yl-4-m eth yla n ilin o)u r a cil, 11j.
The general method gave the product in 54% yield, mp 158-
1
160 °C. H NMR: 1.10 (t, 3H, PhCH2CH3), 1.50 (m, 6H, 3 ×
CH2), 1.88(t, 2H, CH2), 2.10 (s, 3H, PhCH3), 2.24-2.38 (m, 4H,
CH2NCH2), 2.52 (q, 2H, PhCH2), 3.40 (m, 2H, NCH2), 3.68 (m,
2H, NCH2), 4.72 (s, 1H, 5-H), 5.11 (s, 1H, OH), 6.90-7.14 (m,
3H, PhH), 7.68-7.90 (m, 3H, PhH), 8.14 (s, 1H, 6-NH), 10.36
(s, 1H, 1-H). Anal. (C29H34ClF3N4O3‚0.5H2O) C, H, N.
3-[4-(4-Ben zyl-1-p ip er a zin yl)bu tyl]-6-(3-eth yl-4-m eth -
yla n ilin o)u r a cil, 11k . K2CO3 (276 mg, 2 mmol) and 1-ben-
zylpiperazine (317 mg, 1.8 mmol) were added to a mixture of
8 (638 mg, 1.5 mmol) in 60 mL of MeCN. The mixture was
stirred at room temperature for 16 h. Once the reaction was
complete (TLC), the mixture was concentrated in vacuo. Water
(20 mL) was added, and the mixture was extracted with CHCl3
(2 × 50 mL). The extracts were dried over Na2SO4, the solvent
was removed, and the residue was purified by chromatography
on silica gel using 5% MeOH in CHCl3 as eluent to give 545
1
the hydrochloride as a white solid, mp 218-219 °C. H NMR:
1.11 (t, 3H, PhCH2CH3), 1.50 (m, 4H, 2 × CH2), 2.21 (s, 3H,
PhCH3), 2.57 (q, 2H, PhCH2), 2.78 (m, 2H, NH3CH2), 3.72 (t,
2H, NCH2), 4.75 (s, 1H, 5-H), 6.92-7.15 (m, 3H, PhH), 7.86
(br s, 3H, NH3), 8.89 (s, 1H, 6-NH), 10.76 (s, 1H, 1-H). Anal.
(C17H25ClN4O2) C, H, N.
3-[4-(1-P ip er id yl)b u t yl]-6-(3-et h yl-4-m et h yla n ilin o)-
u r a cil, 11c. The general method with piperidine gave the
1
product in 76% yield, mp 202-204 °C. H NMR: 1.10 (t, 3H,
1
PhCH2CH3), 1.35-1.62 (m, 10H, 5 × CH2), 2.23-2.33 (m, 9H,
3 × NCH2, PhCH3), 2.56 (q, 2H, PhCH2), 3.72 (m, 2H, NCH2),
4.73 (s, 1H, 5-H), 6.90-7.14 (m, 3H, PhH), 8.32 (s, 1H, 6-NH),
10.48 (s, 1H, 1-H). Anal. (C22H32N4O2) C, H, N.
mg (78%) of product, mp 205-206 °C. H NMR: 1.10 (t, 3H,
PhCH2CH3), 1.26-1.50 (m, 4H, 2 × CH2), 2.16-2.38 (m, 9H,
3 × NCH2, PhCH3), 2.52 (q, 2H, PhCH2), 3.28 (m, 4H, 2 ×
NCH2), 3.40 (m, 2H, NCH2), 3.64 (t, 2H, NCH2), 4.70 (s, 1H,
5-H), 6.88-7.32 (m, 8H, PhH), 8.15 (s, 1H, 6-NH), 10.38 (s,
1H, 1-H). Anal. (C28H37N5O2‚0.75H2O) C, H, N.
3-[4-(1-Mor p h olin yl)bu tyl]-6-(3-eth yl-4-m eth yla n ilin o)-
u r a cil, 11d . The general method with morpholine gave the
1
product in 72% yield, mp 225-226 °C. H NMR: 1.12 (t, 3H,
3-{4-[(4-F lu or op h en yl)-1-p ip er a zin yl]bu tyl}-6-(3-eth yl-
4-m eth yyla n ilin o)u r a cil, 11l. The general method gave the
PhCH2CH3), 1.30-1.52 (m, 4H, 2 × CH2), 2.20-2.30 (m, 7H,
CH2NCH2, PhCH3), 2.55 (q, 2H, PhCH2), 3.30 (m, 2H, CH2N),
3.56 (m, 4H, CH2OCH2), 3.68 (m, 2H, NCH2), 4.72 (s, 1H, 5-H),
6.90-7.14 (m, 3H, PhH), 8.04 (s, 1H, 6-NH), 10.38 (s, 1H, 1-H).
Anal. (C21H30N4O3) C, H, N.
1
product in 57% yield, mp 188-189 °C. H NMR: 1.14 (t, 3H,
PhCH2CH3), 1.42-1.54 (m, 4H, 2 × CH2), 2.21 (s, 3H, PhCH3),
2.33 (m, 2H, NCH2), 2.55 (m, 2H, NCH2), 2.59 (q, 2H, PhCH2),
2.99 (m, 4H, 2 × NCH2), 3.30 (m, 2H, NCH2), 3.71 (t, 2H,
NCH2), 4.73 (s, 1H, 5-H), 6.93-7.15 (m, 7H, PhH), 8.11 (s, 1H,
6-NH), 10.41 (s, 1H, 1-H), Anal. (C27H34FN5O2) C, H; N, calcd
14.60; found 14.15.
3-[4-(1-Th iom or p h olin yl)bu tyl]-6-(3-eth yl-4-m eth yla n i-
lin o)u r a cil, 11e. The general method with thiomorpholine
gave the product in 77% yield, mp 228-229 °C. 1H NMR: 1.10
(t, 3H, PhCH2CH3), 1.30-1.51 (m, 4H, 2 × CH2), 2.18 (s, 3H,
PhCH3), 2.45 (m, 8H, 2 × NCH2CH2S), 2.55 (q, 2H, PhCH2),
3.30 (m, 2H, NCH2), 3.62 (m, 2H, NCH2), 4.71 (s, 1H, 5-H),
6.90-7.12 (m, 3H, PhH), 8.10 (s, 1H, 6-NH), 10.03 (s, 1H, 1-H).
Anal. (C20H27SN4O2) C, H, N.
3-{4-[3-(Eth oxyca r bon ylm eth yl)-1-m or p h olin yl]bu tyl}-
6-(3-eth yl-4-m eth yla n ilin o)u r a cil, 11f. The general method
gave the product in 56% yield, mp 160-161 °C. 1H NMR: 1.15
(m, 6H, 2 × CH3), 1.30-1.50 (m, 4H, 2 × CH2), 1.68 (t, 1H,
NCH), 1.82 (t, 1H, NCH), 2.20 (s, 3H, PhCH3), 2.30-2.62 (m,
7H, CH2N, PhCH2, NCH, CO2CH2), 2.70 (d, 1H, NCH), 3.68
(m, 5H, NCH2, OCH2, OCH), 4.06 (q, 2H, COCH2), 4.72 (s, 1H,
5-H), 6.92-7.13 (m, 3H, PhH), 8.04 (s, 1H, 6-NH), 10.42 (s,
1H, 1-H). Anal.(C25H36N4O5) C, H, N.
3-{4-[4-(2-F u r oyl)-1-p ip e r a zin yl]b u t yl}-6-(3-e t h yl-4-
m eth yla n ilin o)u r a cil, 11m . The general method gave the
1
product in 68% yield, mp181-182 °C. H NMR: 1.15 (t, 3H,
PhCH2CH3), 1.40-1.50 (m, 4H, 2 × CH2), 2.24 (s, 3H, PhCH3),
2.30-2.45 (m, 4H, 2 × NCH2), 2.58 (q, 2H, PhCH2), 3.33 (m,
4H, 2 × CH2N), 3.68 (m, 4H, 2 × NCH2), 4.73 (s, 1H, 5-H),
6.62 (s, 1H, furyl-H), 6.96 (m, 3H, 2 × PhH, furyl-H), 7.15 (m,
1H, PhH), 7.83 (s, 1H, furyl-H), 8.12 (s, 1H, 6-NH), 10.42 (s,
1H, 1-H). Anal. (C26H33N5O4‚0.25H2O) C, H, N.
3-{4-[4-(2-P yr im id in yl)-1-p ip er a zin yl]bu tyl}-6-(3-eth yl-
4-m eth yyla n ilin o)u r a cil, 11n . The general method gave the
1
product in 62% yield, mp 199-201 °C. H NMR: 1.14 (t, 3H,
PhCH2CH3), 1.45-1.51 (m, 4H, 2 × CH2), 2.22 (s, 3H, PhCH3),
2.39 (m, 4H, 2 × NCH2), 2.60 (q, 2H, PhCH2), 3.32 (m, 2H,

Synthesis and Bioactivity of Substituted 6-Anilinouracils
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 13 2739
NCH2), 3.70 (m, 6H, 3 × NCH2), 4.73 (s, 1H, 5-H), 6.61 (t, 1H,
pyrimidine-H), 6.93-7.15 (m, 3H, PhH), 8.12 (s, 1H, 6-NH),
compound, in µg/mL, at which growth was not apparent (<25%
of the DMSO control optical density). Reported MIC values
typically vary 2-fold.
8.38 (m, 2H, pyrimidine-H), 10.41 (s, 1H, 1-H). Anal. (C25H33
N7O2) C, H, N.
-
Ack n ow led gm en t. This work was supported by
small business (SBIR) grant AI41260 from the National
Institutes of Health.
3-[4-(1-P ip er a zin yl)bu tyl]-6-(3-eth yl-4-m eth yla n ilin o)-
u r a cil d ih yd r och lor id e, 11o. Treatment of 8 with N-Boc-
piperazine gave 3-{4-[4-(tert-butoxycarbonyl)piperazino]butyl}-
6-(3-ethyl-4-methylanilino)uracil in 62% yield, mp 215-216 °C
(dec). 1H NMR: consistent. A solution of this intermediate (2.4
g) in a mixture of CHCl3 and MeOH (3:1, 20 mL) was treated
with 50 mL of a solution of 4.0 M HCl in dioxane, and the
mixture was stirred at room temperature for 4 h. After removal
of the solvent, the residue was washed with Et2O and dried
in vacuo to give the product as a colorless solid in 94% yield,
mp 210 °C (dec). 1H NMR: 1.16 (t, 3H, PhCH2CH3), 1.50-
1.70 (m, 4H, 2 × CH2), 2.22 (s, 3H, PhCH3), 2.60 (q, 2H,
PhCH2), 3.18 (m, 4H, 2 × NCH2), 3.50 (m, 6H, 3 × NCH2),
3.72 (t, 2H, NCH2), 4.72 (s, 1H, 5-H), 6.93-7.15 (m, 3H, PhH),
8.50 (s, 1H, 6-NH), 10.38 (s, 3H, NH2+, NH+), 10.58 (s, 1H,
1-H). Anal. (C21H32Cl2N5O2‚2.5H2O) C, H, N.
DNA P olym er a se Assa ys. DNA polymerase IIIC (pol IIIC)
of Bacillus subtilis was the homogeneous recombinant protein
expressed and purified as described previously.13 The enzyme
was assayed in 96-well plates with the use of activated calf
thymus DNA as a substrate according to the method of Barnes
and Brown.14 Briefly, enzyme was added to a buffered mixture
containing Mg2+, dithiothreitol, glycerol, dATP, dCTP, dTTP,
[3H]dTTP, and activated calf thymus DNA. Assays were
initiated by the addition of 0.025-0.06 units of enzyme (1 unit
is the amount required to incorporate 250 pmoles of [3H]dTMP
in a standard assay), incubated for 10 min at 30 °C, and
terminated by the addition of a trichloroacetic acid-sodium
pyrophosphate solution. Precipitated labeled DNA was col-
lected on glass fiber filter plates, and the plates were washed
with dilute aqueous HCl followed by EtOH, dried, and counted
in a liquid scintillation counter. Serial dilutions of compounds
(in DMSO) were added to the plates before enzyme addition.
Apparent inhibition constants (Ki values) are obtained directly
in this “truncated” assay, i.e., lacking the competitor dGTP,
as described previously.15,16 Compounds are assayed in dupli-
cate; reported values typically vary (43%.
Ba cter ia l Str a in s. The standard screening panel included
S. aureus 25923, S. aureus 13709 (Smith) and E. fecalis 29212,
all purchased from the American Type Culture Collection
(ATCC, Manassas, VA). Methicillin-resistant S. aureus (MRSA
B42876), E. fecium, and vancomycin-resistant E. fecium (VRE)
are clinical isolates provided by the University of Mas-
sachusetts Medical School. B. subtilis (BD54) is a standard
laboratory strain.17 E. coli (J -53) was provided by Prof. Martin
Marinus, University of Massachusetts Medical School.
Min im u m In h ibitor y Con cen tr a tion s (MIC). Antibacte-
rial activity was measured as the minimum inhibitory con-
centration (MIC) of test compound required to prevent bacte-
rial growth. Log-phase bacterial cultures were grown in Luria
broth. Diluted bacteria cultures were seeded into 96-well plates
(200 µL/well) from stocks containing (3-7) × 105 colony
forming units/mL. Each compound was tested in duplicate
assays by diluting stock solutions of compound in DMSO in
2-fold serial dilutions to give five concentrations from 20 to
1.25 µg/mL plus an untreated control culture. Each well,
including the DMSO controls, contained a final concentration
of 1% DMSO. Plates were incubated at 37 °C for 16 to 24 h,
and bacterial growth was determined by measuring optical
density (600 nm, 1 cm path length) in a microplate reader.
MIC values are the average of the lowest concentration of test
Su p p or tin g In for m a tion Ava ila ble: Tables of biological
activity data. This material is available free of charge via the
Internet at http://pubs.acs.org.
Refer en ces
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J M020591Z