Synthesis and antibacterial activity of 3-substituted-6-(3-ethyl-4- methylanilino)uracils

Article abstract of DOI:10.1021/jm050517r

Numerous 3-substituted-6-(3-ethyl-4-methylanilino)uracils (EMAU) have been synthesized and screened for their capacity to inhibit the replication-specific bacterial DNA polymerase IIIC (pol IIIC) and the growth of Gram+ bacteria in culture. Direct alkylat

Full text of DOI:10.1021/jm050517r

J. Med. Chem. 2005, 48, 7063-7074
7063
Synthesis and Antibacterial Activity of
3-Substituted-6-(3-ethyl-4-methylanilino)uracils
Chengxin Zhi,† Zheng-yu Long,† Andrzej Manikowski,† Neal C. Brown,† Paul M. Tarantino, Jr.,† Karsten Holm,†
Edward J. Dix,† George E. Wright,*,† Kim A. Foster,‡ Michelle M. Butler,‡ William A. LaMarr,‡ Donna J. Skow,‡
Irina Motorina,§ Serge Lamothe,§ and Richard Storer§,|
GLSynthesis Inc., One Innovation Drive, Worcester, Massachusetts 01605, Microbiotix Inc.,
One Innovation Drive, Worcester, Massachusetts 01605, and Shire BioChem Inc., Laval, Quebec, Canada
Received June 2, 2005
Numerous 3-substituted-6-(3-ethyl-4-methylanilino)uracils (EMAU) have been synthesized and
screened for their capacity to inhibit the replication-specific bacterial DNA polymerase IIIC
(pol IIIC) and the growth of Gram+ bacteria in culture. Direct alkylation of 2-methoxy-6-amino-
4-pyrimidone produced the N3-substituted derivatives, which were separated from the byproduct
4-alkoxy analogues. The N3-substituted derivatives were heated with a mixture of 3-ethyl-4-
methylaniline and its hydrochloride to effect displacement of the 6-amino group and
simultaneous demethylation of the 2-methoxy group to yield target compounds in good yields.
Certain intermediates, e.g. the 3-(iodoalkyl) compounds, were converted to a variety of (3-
substituted-alkyl)-EMAUs by displacement. Most compounds were potent competitive inhibitors
of pol IIIC (Kis 0.02-0.5 µM), and those with neutral, moderately polar 3-substituents had
potent antibacterial activity against Gram+ organisms in culture (MICs 0.125-10 µg/mL).
Several compounds protected mice from lethal intraperitoneal (ip) infections with S. aureus
(Smith) when given by the ip route. A water soluble derivative, 3-(4-morpholinylbutyl)-EMAU
hydrochloride, given subcutaneously, prolonged the life of infected mice in a dose dependent
manner.
Introduction
the Gram+ bacterium Bacillus subtilis, with strong
selective antibacterial activity against a variety of
Gram+ organisms.3 However, among these derivatives,
some lacked significant antibacterial activity despite
their potent pol IIIC inhibition. Factors that may limit
antibacterial activity could include lack of penetration
of the cell wall or membrane, removal of compound by
active efflux mechanisms, and alteration of the sensitiv-
ity of the target enzyme.
To learn more about the structure-antibacterial
activity relationship and to select compounds for study
in animal infection models, we undertook additional
synthesis and screening programs among this class of
compounds. Specifically, we have prepared additional
derivatives of 6-(3-ethyl-4-methylanilino)uracil, “EMAU”,
the platform structure with the optimal substitution
pattern for potent and selective inhibition of pol IIIC.3,4
We have developed new methods for preparation of key
intermediates to support the synthesis program. In
addition, we report the inhibition of pol IIIC, SAR for
in vitro antibacterial activity, and antibacterial activity
in mice of selected compounds.
The emergence of antibiotic-resistant Gram+ bacte-
ria, notably Staphylococcus aureus, Enterococcus fecalis,
Enterococcus fecium, and Streptococcus pneumoniae, has
prompted development of new 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 organisms.
Interference with pol IIIC function prevents the replica-
tion of the Gram+ host chromosome, thus killing the
host.1,2 Simple 3-(hydroxyalkyl) and 3-(methoxyalkyl)
derivatives of 6-(3-ethyl-4-methylanilino)uracil (1, 2)
protected mice from an intraperitoneal (ip) S. aureus
infection when also administered by the ip route.2
Results
Synthesis of Compounds. 3-Substituted-6-anili-
nouracils and related compounds have been prepared
by conversion of N-alkylbarbituric acids to 6-chlorou-
racils followed by reaction with anilines or other
amines.1,3 This sequence, however, is not suitable for
compounds with N3 substituents that are labile under
the required strongly basic and acidic conditions. An
alternative, two-step method has been developed, il-
lustrated in Scheme 1. The first step is a phase transfer-
catalyzed alkylation of 6-amino-2-methoxy-4-pyrimi-
We recently reported that optimally 3-substituted
6-anilinouracils were potent inhibitors of pol IIIC from
* Author for correspondence: Phone 508 754-6700. Fax 508 754-
7075. E-mail: george.wright@glsynthesis.com.
† GLSynthesis Inc.
‡ Microbiotix Inc.
§ Shire BioChem Inc.
| Present address: Laboratoires Idenix, 1682 Rue de la Valsie`re,
34189 Montpellier, France.
10.1021/jm050517r CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/12/2005

7064 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Zhi et al.
Scheme 1
done, similar to alkylations reported by Mu¨ller et al.5
R-X is a substituted alkyl halide, and PTC is a phase
transfer catalyst such as benzyltriethylammonium chlo-
ride (TBAC) or tetrabutylammonium bromide (TBAB),
and acetone or acetonitrile is used as solvent. Reagents
are heated at reflux for 10-72 h. Chromatography on
silica gel with chloroform:methanol as eluent gives first
the byproduct 6-amino-4-alkoxy-2-methoxypyrimidine 3
and then the desired 6-amino-2-methoxy-3-alkyl-4-py-
rimidone 4, in approximately equal yields. In a variation
on this method, 6-amino-2-methoxy-4-pyrimidone is
treated with sodium hydride in N,N-dimethylformamide
(DMF) at 0 °C. Lithium bromide is added, the mixture
is added dropwise to a solution of the alkylating agent
in DMF at 50-80 °C, and the reaction mixture is stirred
at 50-80 °C for 3-10 h. Workup by chromatography
on silica gel gives first the O-alkylated isomers 3
followed by the N-alkylated isomers 4.
In the second step, a mixture of intermediate 4,
3-ethyl-4-methylaniline hydrochloride, and a few drops
of 3-ethyl-4-methylaniline (ca. 0.1-1 equiv) is heated
at 120-170 °C for between 10 min to 3 h. After addition
of water and extraction with chloroform, the residue is
purified by chromatography on silica gel to give the
target compounds, 3-alkyl-6-(3-ethyl-4-methylanilino)-
uracils 5a-x, in good yields (Scheme 1). This step not
only effects displacement of the 6-amino group but also
demethylation of the 2-methoxy group of 4, yielding the
desired uracil derivatives directly. Numerous examples
of this method are presented in the Experimental
Section and in Supporting Information.
3-(4-acetoxybutyl), or 3-(5-hydroxypentyl)-EMAU with
iodotrimethylsilane in dry chloroform (ref 3 and Scheme
2). Reaction of 3-(4-iodobutyl)- or 3-(5-iodopentyl)-
EMAUs (6a or 6b) with nucleophiles in the presence of
potassium carbonate in a suitable solvent (acetone,
acetonitrile, or DMF) and purification by chromatogra-
phy on silica gel gave several candidate compounds (7a-
e). Several ω-hydroxyalkyl derivatives (8a-d) were
prepared by reduction of the corresponding esters or by
dealkylation of corresponding ethers (Scheme 3). 3-(4-
Aminobutyl)- and 3-(5-aminopentyl)-EMAUs (9a and
9b) were prepared by reduction of the corresponding
nitriles and acylated with carboxylic acid chlorides or
sulfonyl chlorides to give 3-carboxamidoalkyl (10a-c)
and 3-sulfonamidoalkyl derivatives (11a,b), respectively
(Scheme 4). 3-(3-Carboxypropyl)-EMAU (12) was made
by hydrolysis of the corresponding ester, 5o.
DNA Polymerase IIIC Inhibition. The EMAU
derivatives were tested for inhibition of B. subtilis DNA
polymerase IIIC using the “truncated” assay lacking the
competitor dGTP, an assay which yields the Ki value of
competitive inhibitors directly.6 Many of the compounds
were potent inhibitors of the enzyme, with Ki values
ranging from 0.021 to 0.3 µM (Table 1). The common
structural feature of the compounds is the 6-(3-ethyl-
4-methylanilino)uracil core which optimizes binding of
the class to the Gram+ pol IIIC enzymes.4 The standard
deviation of Ki values of inhibitors of pol IIIC is ca. 44%,
indicating that the range of potencies of active 3-sub-
stituted EMAUs in Table 1 is small. [Other pol IIIC
inhibitors with Ki values ranging between 0.024 and 73
µM lacked antibacterial activity (see Supporting Infor-
mation)].
Several useful intermediates for synthesis of 3-(sub-
stituted-alkyl)-EMAUs have been prepared. 3-(ω-Iodo-
alkyl) intermediates 6 were obtained in 80-95% yields
by treatment of 3-(4-methoxybutyl), 3-(4-hydroxybutyl),
The chemical properties of N3 substituents had
moderate to no effect on pol IIIC inhibition potency of

3-Substituted-6-(3-ethyl-4-methylanilino)uracils
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 7065
Scheme 2
Scheme 3
Scheme 4
the derivatives (Table 1). Within the homologous series,
e.g. C2-C8 hydroxyalkyl, C2-C5 acetoxyalkyl, C2-C4
methoxyalkyl compounds, there was up to a 4-fold
increase in potency with higher chain length. However,
no increase was observed with the longer C9-10 chains
of the hydroxynonyl and hydroxydecyl compounds, 5c
and 5d. Compounds with polar (CN, CO2Et), acidic
(CO2H, 12), and basic (NH2, NRH, NR2) substituents,
had Ki values that generally were between 0.02 and 0.1
µM. The most potent inhibitors were the most hydro-
philic, i.e., the hydroxyalkyl tertiary amino compounds
7a and 7b. It appears that little change in binding
affinity to the pol IIIC:DNA complex occurs after
introduction of small substituents at N3, and the

7066 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Zhi et al.
Table 1. Pol IIIC Inhibition and Antibacterial Activity in Vitro of 3-Substituted-EMAUs

3-Substituted-6-(3-ethyl-4-methylanilino)uracils
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 7067
Table 1. (Continued)
*SD (44%. **SD (50%. ***MIC+ and MIC-, MIC values in the presence and absence, respectively, of added fetal calf serum.
significant activity of compounds bearing rather large
3-substituents, e.g. 7d,e, demonstrates the high steric
tolerance of substituents at this position in the drug:
polymerase:DNA complex.
Among Gram+ strains, B. subtilis was more sensitive
to active compounds than S. aureus and enterococcal
strains. However, active compounds were uniformly
active against all staphylococci and enterococci, ir-
respective of their sensitivity or resistance to known
antibiotics. For example, MRSA B42876 and E. fecium
are resistant to the fluoroquinolones norfloxacin and
ciprofloxacin, and VRE, as expected, is resistant to
vancomycin (Table 1).
Efficacy of antibiotics in vivo has generally been
correlated to ratio of plasma concentration of free drug
to MIC or “area under the curve” to MIC.8 Because
plasma protein binding can reduce free drug concentra-
tions in vivo, and considering that the plasma concen-
tration of an effective antibacterial drug should exceed
MIC for some time, we retested several potent EMAU
derivatives against two representative bacteria, S. au-
reus (Smith) and E. faecalis, in the presence of 50% fetal
calf serum as a means of mimicking binding to plasma
proteins. The results in Table 1 show that all compounds
tested showed a “serum effect”, i.e., higher MIC in the
presence of serum, but the extents differed widely.
Compounds with hydrophobic substituents had the
greatest serum effect, whereas compounds with hydro-
philic substituents, e.g. several morpholinylalkyl deriva-
tives, had low serum effects. In particular, compound
Antibacterial Activity in Vitro. The EMAU de-
rivatives were screened for antibacterial activity against
a panel of normal and antibiotic-resistant Gram+
bacteria and, as negative control, the Gram- organism
E. coli. Table 1 summarizes the results for several
related series of compounds that showed both potent
enzyme inhibition and potent to moderate Gram+
antibacterial activity. (Results for many compounds that
showed weak or no antibacterial activity are collected
in Supporting Information.) The Gram- organism E.
coli was resistant, as expected, to all compounds at the
highest concentrations tested (data not shown).
Keeping in mind that reported MIC values may vary
(2-fold, 3-substituted-EMAU derivatives bearing hy-
droxyalkyl, methoxyalkyl, and acetoxyalkyl groups were
most active. Derivatives with other neutral substituents,
e.g. CN, CO2R, retained good activity, but those with
ionizable groups (CO2H, NHR) were weaker. Amides
and sulfonamides had intermediate activity. As found
for the enzyme inhibitory activity, there was no direct
steric effect of bulky 3-substituents in reducing anti-
bacterial activity.

7068 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Zhi et al.
Table 2. Effect of Ip Dosed Pol IIIC Inhibitors on Survival of
S. aureus (Smith) ip-Infected Mice
Table 3. Pharmacokinetic Parameters of 1c and 7c in Mice
1ca (mg/kg)
7cb (mg/kg)
survivors (n ) 5)b
iv
sc
iv
sc
po
compd (vehiclea)
10 mg/kg
20 mg/kg
20
20
60
20
20
200 400 200
controls (a or b)
vancomycin (a or b)
1c (a)
0
5
3
4
2
2
2
4
-
5
Cmax (µg/mL)c
Tmax (min)
t1/2 (min)
53
-
5.1
15
13 27.4 4.6 15.1 24.1 7.3
15
nd
-
5
30
30
60
5
33 29.3
39
149 132 373 111
8d (a)
nd
nd
nd
4
AUC (µg‚min/mL) 288 466.5 1214 234 406 1613 3042 922
5k (a)
(0-t) (min)
(60) (120) (120) (120) (180) (180) (180) (180)
5m (a)
a 2 mg/mL in 30% HP-â-cyclodextrin in water. b 4 mg/mL in
13 (a)
PBS. c Calculated with Win-Nonlin software.
7c (b)
5
a Dissolved in 10% DMSO/peanut oil (a) or phosphate buffered
saline (PBS) (b). b Groups of five mice were infected ip with 108
CFU Smith strain in 0.5 mL of LB broth and dosed ip 15 min later;
survivors were counted at 72 h post infection. nd, not done.
efficacy in the ip/ip mouse infection model, and high
water solubility of 7c prompted a detailed pharmaco-
kinetic study of this compound. The uptake and distri-
bution of 7c in saline solution by the iv and subcuta-
neous (sc) routes in mice were compared with the
corresponding parameters for 1c. The latter compound
is not sufficiently soluble in water or saline, however,
and studies were done with 1c dissolved in 30% aqueous
HP-â-cyclodextrin (CDx). Before initiating pharmaco-
kinetic studies, the acute toxicity of both compounds in
their formulations was evaluated. Tail vein iv doses of
1c higher than 20 mg/kg and sc doses higher than 60
mg/kg were lethal to mice, but sc doses of 7c up to 400
mg/kg were well tolerated by mice.
HPLC was used to measure the time-dependence of
plasma concentrations of 1c and 7c following dosing by
several routes (see Experimental Section for details).
The results of Table 3 show that sc injection of 7c
resulted in dose-dependent increases in Cmax, apparent
plasma half-life, and exposure (area under the curve,
AUC). Comparison between properties of 20 mg/kg doses
of 7c given iv and sc suggests complete uptake of the
compound from the sc route and prolongation of the
apparent elimination half-life, t1/2. Oral dosing of 7c
resulted in about half the peak concentration compared
with sc dosing (Table 3). Although the CDx formulation
of 1c permitted dosing at 20 mg/kg iv and 60 mg/kg sc,
the low plasma concentrations and short t1/2 of this
compound are likely responsible for lack of efficacy of
either regimen in ip infections of mice with S. aureus
(Smith) (data not shown).
In addition to the peak corresponding to the parent
compound 7c, a second major peak in the HPLC of
plasma samples from 7c-dosed animals was observed
at 4.17 min. The peak paralleled the rise and fall of
parent compound and was a greater proportion of parent
peak after oral than parenteral dosing. Fractions en-
riched in the second peak were concentrated and
subjected to MS with an ion trap instrument. The
metabolite had M + 1 ) 361, compared with the M + 1
) 387 for 7c, and both gave a MS2 peak at M + 1 )
300. On the basis of the likelihood that oxidation of the
morpholine ring of 7c would result in the 3-oxo inter-
mediate, followed by elimination of acrolein, it was
predicted that the metabolite was 3-[4-(2-hydroxyethyl)-
aminobutyl]EMAU, 7a. This prediction was proved by
comparison of the HPLC and MS properties of the
metabolite with those of authentic 7a.
7c, the hydrochloride of 3-(N-morpholinylbutyl)-EMAU,3
was a potent antibacterial compound with little effect
of serum on its MIC values (Table 1). Because this
compound was also highly water soluble, it became of
interest to consider 7c for parenteral testing in vivo.
In Vivo Studies in Mice. The choice of EMAU
derivatives to evaluate for antibacterial activity in vivo
could depend on several properties. Obviously the lowest
MIC value is important, but the extent of protein
binding, solubility in suitable vehicles, plasma half-life,
and acute toxicity of test doses in a suitable animal
species are important issues. In addition, breadth of
activity against antibiotic-resistant organisms is a
requirement for further development of an effective
compound. We previously demonstrated activity of pol
IIIC inhibitors in a mouse model of intraperitoneal (ip)
infection with S. aureus.2,7 Thus we selected the “best”
antibacterial compounds from Table 1 to study further
in this model.
Swiss-Webster mice were infected ip with 108 colony
forming units (CFU) of S. aureus (Smith strain), and
the survival was monitored for 72 h. Typically animals
die within 10-18 h of the infection, and the positive
control drug vancomycin, given ip or intravenously (iv),
completely protects all animals for the 72 h observation
period (Table 2). Test compounds were given by routes
dependent upon both a suitable vehicle for that route
and by results of dosing and distribution studies.
Because of the poor water solubility of most compounds
of Table 1, initial studies were done via the ip route with
a vehicle consisting of 10% DMSO in peanut oil, a
vehicle that we used previously to show efficacy of 1a
and 1b and related compounds against ip S. aureus
(Smith).2 Table 2 summarizes the response of infected
mice to ip doses of several pol IIIC inhibitors. At
screening doses of 10 and 20 mg/kg, HB-EMAU (1c)
protected 3/5 and 5/5 mice, respectively, consistent with
its reported activity.2 Potent antibacterials 7c and 8d
protected 4/5 mice at 10 mg/kg, and 7c completely
protected mice at 20 mg/kg. Other potent enzyme inhib-
itors 5k and 5m were less effective, protecting 2/5 ani-
mals each. Compound 13 protected 4/5 animals at a dose
of 20 mg/kg. The significant activity of the morpholinyl
derivative 7c, in contrast to the weaker activity of the
hydrophobic compounds 5k and 5m, may be a result of
the high water solubility of the former compound in
peritoneal fluid, and, thus, its better distribution.
Pharmacokinetics of 7c. The combination of mod-
erate antibacterial potency, low serum effect, good
Antibacterial Efficacy of 7c in Vivo. Efficacy
studies of 7c were carried out in S. aureus (Smith) ip
infected mice, based on the findings of the pharmaco-
kinetic studies. The results of single oral (po) and sc
doses are summarized in Figure 1. A po dose of 400 mg/

3-Substituted-6-(3-ethyl-4-methylanilino)uracils
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 7069
pounds have potent activity against Gram+ bacteria in
culture. Together with our previous work1,3 and that of
other groups,9,10 the present results confirm the targeted
nature of 6-anilinouracils and related compounds as
inhibitors of Gram+ DNA polymerase IIIC and the
potential of this class of compounds for development as
antibacterial agents.
Potency of 6-anilinouracils as inhibitors of pol IIIC is
largely dictated by the structure of the 6-anilino group.
The 3-ethyl-4-methylanilino group provides maximum
enzyme affinity,3,4 while the 3-substituents often en-
hance that activity, but to a limited extent (Table 1).
However, potent pol IIIC inhibition is not always
translated into potent antibacterial activity. Derivatives
with highly polar or charged substituents show high
enzyme inhibition potency but reduced or absent anti-
bacterial activity. Derivatives with hydrophobic or less
polar substituents show both potent enzyme and anti-
bacterial activity. However, derivatives with hydropho-
bic substituents showed the highest apparent protein
binding, a property that would reduce the free concen-
tration of drug in plasma. In a simple antibacterial
screen in mice, representative compounds with moder-
ately polar substituents (1c, 8d) were more potent than
those with hydrophobic 3 substituents (5k, 5m) in
protecting animals from lethal S. aureus infection (Table
2). Paradoxically, the morpholinyl compound 7c was
highly active in this model (Table 2). Compound 7c had
a moderate level of antibacterial activity and low “serum
effect” (Table 1), and because it was highly water
soluble, it was studied by parenteral dosing in vivo. The
compound was readily dissolved in saline and was well
absorbed by the sc route, but less so by the oral route
(Table 3). A comparison of single 400 mg/kg doses of 7c
in S. aureus (Smith) infected mice showed greater delay
of death after sc than oral dosing (Figure 1). In addition,
a consistent increase in delay of death was observed
with increasing sc doses of 7c (Figure 2). Demonstrating
activity in vivo of the better antibacterial analogues of
Table 1 which are not water soluble, however, will
require development of formulations better suited for
parenteral dosing.
Figure 1. Comparison of oral (po) and sc dosing on antibacte-
rial efficacy of 7c in S. aureus (Smith) ip-infected mice.
Vancomycin was given ip at 10 mg/kg.
Figure 2. Effect of sc doses of 7c on survival of S. aureus
(Smith) ip-infected mice. Vancomycin was given ip at 10 mg/kg.
Experimental Section
kg prolonged the life of mice about 3-fold relative to un-
treated animals, but a sc dose of 400 mg/kg significantly
prolonged the life of the mice, although it was not cura-
tive. Subcutaneously dosed animals lived an average of
30 h, and untreated and orally dosed animals lived an
average of only 6 and 18 h, respectively. The vancomy-
cin-treated mice survived past the 48 h observation
period. A dose-response to single sc doses of 7c is ob-
served in the results of Figure 2. Doses of 200 and 400
mg/kg were clearly more effective in prolonging life than
100 mg/kg, but again complete cures were not obtained.
The inability of 7c to provide cures may be a result
of its moderate MIC (Table 1) but may also result from
its rapid conversion to the weaker hydroxyethylamino
analogue 7a in vivo. Thus, although the high solubility
and low protein binding of 7c were major advantages of
the compound, its rapid conversion to a relatively inac-
tive compound limits its promise as an antibacterial drug.
Materials. Most reagent chemicals and solvents were
obtained from commercial sources. 3-Ethyl-4-methylaniline
was prepared as described.4 Preparation of several starting
materials and reference compounds was published previ-
ously.1,3 Proton NMR spectra were obtained in Me2SO-d6
solution with a Bruker Avance 300 spectrometer, unless
indicated otherwise. Chemical shifts (δ) are in ppm from
internal TMS. Melting points were determined on a Mel-temp
apparatus and are uncorrected. Elemental analyses were done
by the Microanalysis Laboratory, University of Massachusetts,
Amherst. Values for C, H, and N were within 0.4% of
calculated values except as noted. MS and LC-MS were
obtained with a ThermoFinnigan LCQ Advantage instrument.
HR-MS data were obtained with a FAB source on a Kratos
MS50TCTA instrument equipped with a peak matching unit.
General synthesis methods were also applied to preparation
of compounds listed in Supporting Information.
General Method for the Preparation of 3-Substituted
EMAUs. Step 1. 6-Amino-2-methoxy-3-substituted-4-py-
rimidones. A mixture of 6-amino-2-methoxy-4-pyrimidone (1
equiv), potassium carbonate (1.2-2 equiv), benzyltriethylam-
monium chloride (0.2-1 equiv) and alkylating agent (1-5
equiv) in Me2CO or MeCN was heated at 50-100 °C for 10-
72 h. After cooling to room temperature, the insoluble salts
Discussion
Many 3-substituted EMAU derivatives are potent
inhibitors of Gram+ pol IIIC, and some of these com-

7070 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Zhi et al.
were removed by filtration and the solvent was evaporated.
The residue was purified by chromatography on silica gel with
CHCl3:MeOH as eluent to give both 6-amino-2-methoxy-4-
alkoxypyrimidine and 6-amino-2-methoxy-3-substituted-4-py-
rimidone. Typical examples are shown below.
Chromatography gave 632 mg (75% yield) of product. Crystal-
lization from EtOH gave white crystals. Mp: 185-186 °C. 1H
NMR: 1.11-1.19 (m, 6H, 2×CH3), 1.49 (m, 4H, 2×CH2), 2.24
(s, 3H, CH3Ar), 2.30 (t, 2H, CH2CO2Et), 2.57 (q, 2H, CH2Ar),
3.69 (t, 2H, CH2N), 4.04 (q, 2H, CO2CH2), 4.72 (s, 1H, 5-H),
6.92-7.15 (m, 3H, ArH), 8.16 (s, 1H, NH), 10.48 (s, 1H, NH).
Anal. (C20H27N3O4) C, H, N.
3-(4,5-Dihydroxypentyl)-6-(3-ethyl-4-methylanilino)-
uracil, 5a. Yield: 79%. Mp: 189 °C. 1H NMR: 1.12 (t, 3H,
ArCH2CH3), 1.30-1.68 (m, 4H, CH2CH2), 2.13 (s, 3H, ArCH3),
2.56 (q, 2H, ArCH2), 3.12-3.30 (m, 3H, CH2O, CHO), 3.70 (t,
2H, NCH2), 4.42 (t, 2H, 2×OH), 4.76 (s, 1H, 5-H), 6.90-7.14
(m, 3H, ArH), 8.12 (s, 1H, NH), 10.35 (s, 1H, NH). Anal.
(C18H25N3O4) C, H, N.
3-(8-Hydroxyoctyl)-6-(3-ethyl-4-methylanilino)uracil,
5b. Yield: 78%. Mp: 155-157 °C. 1H NMR: 1.14 (t, 3H,
CH3CH2Ar), 1.20-1.30 (m, 8H, 4×CH2), 1.37-1.52 (m, 4H,
2×CH2), 2.21 (s, 3H, CH3Ar), 2.57 (q, 2H, CH2Ar), 3.35 (m,
2H, CH2O), 3.64 (t, 2H, CH2N), 4.30 (t, 1H, OH), 4.69 (s, 1H,
5-H), 6.92-7.15 (m, 3H, ArH), 8.05 (s, 1H, NH), 10.35 (s, 1H,
NH). Anal. (C21H31N3O3) C, H, N.
3-(9-Hydroxynonyl)-6-(3-ethyl-4-methylanilino)uracil,
5c. Yield: 74%. Mp: 139-141 °C. 1H NMR: δ 1.14 (t, 3 H,
CH3CH2Ar), 1.25 (m, 10 H, 5×CH2), 1.35-1.51 (m, 4 H,
2×CH2), 2.24(s, 3 H, CH3Ar), 2.57 (q, 2 H, CH2Ar), 3.29-3.39
(m, 2H, CH2OH), 3.66 (t, 2 H, CH2N), 4.33 (t, 1 H, OH), 4.72
(s, 1 H, 5-H), 6.92-7.15 (m, 3 H, Ar), 8.09 (br, 1 H, NH), 10.39
(s, 1 H, NH). Anal. (C22H33N3O3) C, H, N.
6-Amino-2-methoxy-3-(3-cyanopropyl)-4-pyrimidone,
4a. 4-Bromo-1-butyronitrile gave 6-amino-2-methoxy-4-(3-cyano-
propoxy)pyrimidine (3a), which eluted first in 27% yield, Mp:
121-123 °C, followed by 6-amino-2-methoxy-3-(3-cyanopropyl)-
4-pyrimidone (4a) (9.73 g, 66%), isolated as a white solid, Mp:
139-141 °C. 3a: 1H NMR: 1.95 (quintet, 2H), 2.50 (t, 2H),
3.75 (s, 3H), 4.21 (t, 2H), 5.38 (s, 1H), 6.23 (s, 2H). Anal.
(C9H12N4O2) C, H, N. 4a: 1H NMR: 1.78 (m, 2H, CH2), 2.50
(t, 2H, CH2CN) 3.84 (t, 2H, CH2N), 3.88 (s, 3H, CH3O), 4.83
(s, 1H, 5-H), 6.44 (s, 2H, NH2). Anal. (C9H12N4O2) C, H, N.
6-Amino-2-methoxy-3-(4-acetoxybutyl)-4-pyrimidone,
4b. Using 4-acetoxybutyl bromide the O4-isomer 3b was
obtained in 35% yield, Mp: 71-73 °C, and 4b was obtained
in 54% yield as a white solid, Mp: 135-136 °C. 3b: 1H NMR:
1.68 (m, 4H), 2.00 (s, 3H), 3.74 (s, 3H), 4.03 (t, 2H), 4.16 (t,
2H), 5.35 (s, 1H), 6.58 (s, 2H). Anal. (C11H17N3O4) C, H, N. 4b:
1H NMR: 1.52 (m, 4H, 2×CH2), 2.0 (s, 3H, CH3CO) 3.76 (t,
2H, CH2O), 3.88 (s, 3H, CH3N), 4.0 (t, 2H, CH2O), 4.82 (s, 1H,
5-H), 6.41 (s, 2H, NH2). Anal. (C11H17N3O4) C, H, N.
6-Amino-2-methoxy-3-(8-hydroxyoctyl)-4-pyrimidone,
4c. 8-Bromooctanol gave the O4 isomer 3c in 32% yield, Mp:
86-87 °C, and 4c in 34% yield, Mp: 96-97 °C. 3c: 1H NMR:
1.18-1.50 (m, 10 H, 5×CH2), 1.64 (m, 2H, CH2), 3.35 (m, 2 H,
CH2OH), 3.75 (t, 2 H, CH2N), 4.13 (t, 2 H, CH2O), 4.31 (t, 1H,
OH), 5.38 (s, 1H, 5-H), 6.55 (br, 2 H, NH2). Anal. (C13H23N3O3)
C, H, N. 4c: 1H NMR: 1.14-1.58 (m, 12 H, 6×CH2), 3.35 (m,
2 H, CH2OH), 3.73 (t, 2 H, CH2N), 3.88 (s, 3 H, CH3O), 4.30 (t,
1 H, OH), 4.82 (s, 1 H, 5-H), 6.34 (br, 2 H, NH2). Anal.
(C13H23N3O3) C, H, N.
3-(10-Hydroxydecyl)-6-(3-ethyl-4-methylanilino)uracil,
1
5d. Yield: 94% yield. Mp: 105-106 °C. H NMR: δ 1.14 (t, 3
H, CH3CH2Ar), 1.24 (m, 12 H, 6×CH2), 1.30-1.50 (m, 4 H,
2×CH2), 2.23(s, 3 H, CH3Ar), 2.57 (q, 2 H, CH2Ar), 3.36 (m, 2
H, CH2OH), 3.66 (t, 2 H, CH2N), 4.32 (t, 1 H, OH), 4.71 (s, 1
H, C5-H), 6.92-7.12 (m, 3 H, Ar), 8.07 (br, 1 H, NH), 10.37
(s, 1 H, NH). Anal. (C23H35N3O3) C, H, N.
6-Amino-2-methoxy-3-[(4-ethoxycarbonyl)butyl]-4-py-
rimidone, 4d. Ethyl 5-bromovalerate gave the O4 isomer 3d
in 34% yield, Mp: 69-71 °C, and 4d in 37% yield, Mp: 75-
76 °C. 3d: 1H NMR: 1.17 (t, 3 H, CH3CH2), 1.58-1.64 (m, 4
H, 2×CH2), 2.33 (t, 2 H, CH2CO2), 3.74 (s, 3 H, CH3O), 4.04
(q, 2 H, CH2O), 4.14 (t, 2 H, CH2O), 5.35 (s, 1 H, C5-H), 6.60
(br, 2 H, NH2). Anal. (C12H19N3O4) C, H, N. 4d: 1H NMR 1.17
(t, 3 H, CH3CH2), 1.47 (m, 4 H, 2×CH2), 2.30 (t, 2 H, CH2CO2),
3.73 (t, 2 H, CH2N), 3.87 (s, 3 H, CH3O), 4.04 (q, 2 H, CH2O),
4.81 (s, 1 H, 5-H), 6.39 (br, 2 H, NH2). Anal. (C12H19N3O4) C,
H, N.
3-(4-Hydroxy-2-butynyl)-6-(3-ethyl-4-methylanilino)uracil,
5e. Yield: 41%. Mp: 152 °C (dec). 1H NMR: 1.14 (t, 3H,
ArCH2CH3), 2.23 (s, 3H, ArCH3), 2.57 (q, 2H, ArCH2), 4.02 (d,
2H, OCH2), 4.47 (m, 2H, NCH2), 4.72 (s, 1H, 5-H), 5.12 (t, 1H,
OH), 6.93-7.13 (m, 3H, ArH), 8.19 (S, 1H, NH), 10.60 (s, 1H,
NH). HRMS: calcd for C17H20N3O3 (M + 1), 314.1505; ob-
served, 314.1519.
3-{2-[2-(2-Hydroxyethoxy)ethoxy]ethyl}-6-(3-ethyl-4-
methylanilino)uracil, 5f. Yield: 72%. Mp: 118-119 °C. 1H
NMR: 1.14 (t, 3H, CH3C), 2.24 (s, 3H, CH3Ar), 2.57 (q, 2H,
CH2Ar), 3.32-3.50 (m, 10H, 5×CH2O), 3.87 (t, 2H, CH2N), 4.56
(t, 1H, OH), 4.72 (s, 1H, 5-H), 6.92-7.16 (m, 3H, ArH), 8.16
(s, 1H, NH), 10.50 (s, 1H, NH). Anal. (C19H27N3O5) C, H, N.
3-(2-Acetoxyethyl)-6-(3-ethyl-4-methylanilino)uracil, 5g.
Step 2. 3-Substituted-6-(3-ethyl-4-methylanilino)uracils.
A stirred mixture of the 6-amino-2-methoxy-3-alkyl-4-pyrimi-
done 4 (1 equiv), 3-ethyl-4-methylaniline hydrochloride (1.1-
1.5 equiv), and a few drops of 3-ethyl-4-methylaniline was
heated at 120-170 °C for 10-180 min. After cooling to room
temperature, the residue was either dissolved in MeOH:CHCl3,
or water was added and the mixture was extracted with
CHCl3. The combined organic layers were dried over anhy-
drous MgSO4. The solvent was removed under reduced pres-
sure, and the residue was purified by chromatography on silica
gel with MeOH:CHCl3 as eluent to give target compounds.
3-(4-Acetoxybutyl)-6-(3-ethyl-4-methylanilino)uracil, 5i.
A stirred mixture of 4b (15 g, 59 mmol), 3-ethyl-4-methyla-
niline hydrochloride (12.1 g, 75 mmol), and 3-ethyl-4-methy-
laniline (4.0 g, 29 mmol) was heated in an oil bath at 160 °C
for 15 min. After cooling to room temperature, the residue was
dissolved in 1:1 MeOH:CHCl3, and the solution was evaporated
with silica gel. The material was placed atop a silica gel column
and eluted with 4% MeOH in CHCl3 to give crude product.
Trituration with 1:1 Me2CO:Et2O gave colorless crystals of
product (17.8 g, 84%). Mp: 190-191 °C. 1H NMR: 1.14 (t, 3H,
CH3CH2Ar), 1.53 (m, 4H, 2×CH2), 2.0 (s, 3H, CH3CO), 2.24
(s, 3H, CH3Ar), 2.57 (q, 2H, CH2Ar), 3.71 (t, 2H, CH2O),3.99
(t, 2H, CH2N), 4.73 (s, 1H, 5-H), 6.92-7.15 (m, 3H, ArH), 8.12
(s, 1H, NH), 10.43 (s, 1H, NH). Anal. (C19H25N3O4) C, H, N.
3-(4-Ethoxycarbonylbutyl)-6-(3-ethyl-4-methylanilino)-
uracil, 5p. A mixture of 4d (608 mg, 2.26 mmol), 3-ethyl-4-
methylaniline hydrochloride (430 mg, 2.5 mmol), and a few
drops of 3-ethyl-4-methylaniline was heated at 160 °C for 3 h.
1
Yield: 73%. Mp: 187-188 °C (from MeOH). H NMR: 1.14
(t, 3H, CH3C), 1.94 (s, 3H, CH3CO), 2.24 (s, 3H, CH3Ar), 2.57
(q, 2H, CH2Ar), 3.95 (t, 2H, NCH2), 4.14 (t, 2H, OCH2), 4.73
(s, 1H, 5-H), 6.95-7.15 (m, 3H, ArH), 8.12 (s, 1H, NH), 10.47
(s, 1H, NH). Anal. (C17H21N3O4) C, H, N.
3-(3-Acetoxypropyl)-6-(3-ethyl-4-methylanilino)uracil,
5h. Yield: 54%. Mp: 184-186 °C (from MeOH). 1H NMR: 1.14
(t, 3H, CH3C), 1.81 (quin, 2H, CH2), 1.98 (s, 3H, CH3CO), 2.24
(s, 3H, CH3Ar), 2.55 (q, 2H, CH2C), 3.78 (t, 2H, CH2N), 3.98
(t, 2H, CH2O), 4.73 (s, 1H, 5-H), 6.95-7.14 (m, 3H, ArH), 8.09
(s, 1H, NH), 10.41 (s, 1H, NH). Anal. (C18H23N3O4) C, H, N.
3-(5-Acetoxypentyl)-6-(3-ethyl-4-methylanilino)uracil,
5j. Yield: 81% yield. Mp: 173-175 °C (from MeOH). 1H
NMR: 1.14 (t, 3H, CH3C), 1.29 (quin, 2H, CH2), 1.54 (m, 4H,
2×CH2), 2.00 (s, 3H, CH3CO), 2.24 (s, 3H, CH3Ar), 2.55 (q, 2H,
CH2C), 3.68 (t, 2H, CH2N), 3.97 (t, 2H, CH2O), 4.73 (s, 1H,
5-H), 6.95-7.14 (m, 3H, ArH), 8.06 (s, 1H, NH), 10.37 (s, 1H,
NH). Anal. (C20H27N3O4) C, H, N.
3-[2-(2-Methoxyethoxy)ethyl]-6-(3-ethyl-4-methylan-
1
ilino)uracil, 5k. Yield: 74%. Mp: 160-161 °C. H NMR: δ
1.13 (t, 3H, CH3CH2), 2.24 (s, 3H, CH3Ar), 2.57 (q, 2H, CH2Ar),
3.22 (s, 3H, CH3O), 3.37 (m, 2H, CH2O), 3.49 (m, 4H, 2×CH2O),
3.85 (t, 3H, CH2N), 4.70 (s, 1H, 5-H), 6.92-7.15 (m, 3H, ArH),
8.12 (s, 1H, NH), 10.40 (s, 1H, NH). Anal. (C18H25N3O4) C, H,
N.

3-Substituted-6-(3-ethyl-4-methylanilino)uracils
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 7071
3-[4-(N-Morpholinylcarbonyloxy)butyl]-6-(3-ethyl-4-
3-[2-(2-Benzyloxyethoxy)ethyl]-6-(3-ethyl-4-methylan-
ilino)uracil, 5l. Yield: 72%. Mp: 151-152 °C. 1H NMR: 1.13
(t, 3H, CH3CH2), 2.24 (s, 3H, CH3Ar), 2.57 (q, 2H, CH2Ar), 3.53
(m, 6H, 3×CH2), 3.88 (t, 2H, CH2N), 4.47 (s, 2H, PhCH2), 4.72
(s, 1H, 5-H), 6.92-7.15 (m, 3H, ArH), 7.25-7.36 (m, 5H, Ph-
H), 8.16 (s, 1H, NH), 10.49 (s, 1H, NH). Anal. (C24H29N3O2) C,
H, N.
1
methylanilino)uracil, 5x. Yield 82%. Mp: 211-212 °C. H
NMR: 1.10 (t, 3H, ArCH2CH3), 1.55 (m, 4H, CH2CH2), 2.18
(s, 3H, ArCH3), 2.56 (q, 2H, ArCH2), 3.30 (m, 4H, 2×CH2), 3.54
(m, 4H, 2×CH2), 3.71 (t, 2H, NCH2), 4.03 (t, 2H, CH2COO),
4.74 (s, 1H, 5-H), 6.92-7.14 (m, 3H, ArH), 8.16 (s, 1H, NH),
10.45 (s, 1H, NH). Anal. (C22H30N4O5) C, H, N.
3-(4-Hydroxybutyl)-6-(3-ethyl-4-methylanilino)uracil,
1c. A stirred suspension of the acetoxy compound 5i (10.5 g,
24 mmol) in MeOH (150 mL) was treated with concentrated
NH4OH (150 mL) at room temperature. After 30 min all solid
dissolved, and the solution was stirred for 72 h. The solvent
was removed, and the solid was coevaporated three times with
MeOH and filtered from MeOH to give the product as a
colorless solid (9.0 g, 97%), identical with an authentic sample.3
3-(3-Cyanopropyl)-6-(3-ethyl-4-methylanilino)uracil, 5m.
Yield: 81% yield. Mp: 265-267 °C. 1H NMR: 1.14 (t, 3H,
CH3CH2Ar), 1.79 (m, 2H, CH2), 2.24 (s, 3H, CH3Ar), 2.50 (t,
2H, CH2CN), 2.57 (q, 2H, CH2Ar), 3.79 (t, 2H, CH2N), 4.74 (s,
1H, 5-H), 6.92-7.15 (m, 3H, ArH), 8.12 (s, 1H, NH), 10.47 (s,
1H, NH). Anal. (C17H20N4O2) C, H, N.
3-(4-Cyanobutyl)-6-(3-ethyl-4-methylanilino)uracil, 5n.
1
Yield: 78%. Mp: 217-218 °C. H NMR: 1.14 (t, 3H, CH3C),
3-[2-(2-Hydroxyethoxy)ethyl]-6-(3-ethyl-4-methylanili-
no)uracil, 8d. A mixture of the benzyl ether 5l (150 mg, 0.35
mmol) and 10% Pd/C (60 mg) in MeOH (40 mL) was stirred at
room temperature under an atmosphere of H2 for 12 h. 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 2-4% MeOH in
CHCl3 as eluent, to give 109 mg (92% yield) of product. Crystal-
lization from EtOH gave white crystals. Mp: 153-154 °C. 1H
NMR: 1.14 (t, 3H, CH3CH2), 2.24 (s, 3H, CH3Ar), 2.57 (q, 2H,
CH2Ar), 3.53-3.40 (m, 6H, 3×CH2), 3.87 (t, 2H, CH2N), 4.56
(t, 1H, OH), 4.72 (s, 1H, 5-H), 6.92-7.16 (m, 3H, ArH), 8.16
(s, 1H, NH), 10.50 (s, 1H, NH). Anal. (C17H23N3O4) C, H, N
3-(5-Hydroxypentyl)-6-(3-ethyl-4-methylanilino)uracil,
8a. Method a. A solution of 1.0 M LiAlH4 in tetrahydrofuran
(THF) (1.5 mL) was added dropwise to a stirred solution of
the ester 5p (160 mg, 0.43 mmol) in anhydrous THF (30 mL)
at room temperature. After stirring for 20 min, MeOH (5 mL)
was added dropwise, and the solvents were removed. EtOH
was added, the mixture was filtered, and the solid was washed
carefully with EtOH. The solvent was removed, and the
residue was purified by chromatography on silica gel with
2-4% MeOH in CHCl3 as eluent to give 141 mg (99% yield) of
product. Crystallization from 50% aqueous EtOH gave white
crystals. Mp: 198-199 °C. 1H NMR: 1.14 (t, 3H, CH3CH2Ar),
1.20-1.30 (m, 2H, CH2), 1.37-1.52 (m, 4H, 2×CH2), 2.24 (s,
3H, CH3Ar), 2.57 (q, 2H, CH2Ar), 3.34 (t, 2H, CH2O), 3.67 (t,
2H, CH2N), 4.35 (t, 1H, OH), 4.72 (s, 1H, 5-H), 6.92-7.15 (m,
3H, ArH), 8.18 (s, 1H, NH), 10.48 (s, 1H, NH). Anal.
(C18H25N3O3) C, H, N. Method b. A suspension of the acetoxy
compound 5j (12.0 g, 32.1 mmol) in a mixture of MeOH (300
mL) and concentrated NH4OH (200 mL) was stirred at room
temperature for 65 h. Evaporation of solvents gave 9.2 g (86%)
of product as a white powder, identical with the sample
prepared by method a.
3-(6-Hydroxyhexyl)-6-(3-ethyl-4-methylanilino)uracil,
8b. Reduction of the ester 5w by method a used above for 8a
gave the product in 94% yield. Mp: 145-147 °C. 1H NMR:
1.14 (t, 3H, CH3CH2Ar), 1.17-1.48 (m, 8H, 4×CH2), 2.24 (s,
3H, CH3Ar), 2.57 (q, 2H, CH2Ar), 3.34 (m, 2H, CH2O), 3.67 (t,
2H, CH2N), 4.35 (t, 1H, OH), 4.72 (s, 1H, C5-H), 6.92-7.15
(m, 3H, ArH), 8.08 (s, 1H, NH), 10.39 (s, 1H, NH). Anal.
(C19H27N3O3‚0.25H2O) C, H, N.
3-(3-Carboxypropyl)-6-(3-ethyl-4-methylanilino)uracil,
12. A mixture of the ester 5o (80 mg, 0.22 mmol) and NaOH
(10 mg, 0.25 mmol) in 10 mL of water and 10 mL of MeOH
was stirred at reflux for 3 h. Evaporation of solvents and
mixing the residue with 6 N hydrochloric acid gave a precipi-
tate, which was filtered and washed with water. Drying gave
68 mg (92% yield) of product as white crystals. Mp: 229-230
°C. 1H NMR: 1.14 (t, 3H, CH3CH2Ar), 1.17 (m, 2H, CH2), 2.17
(t, 2H, CH2CO2H), 2.24 (s, 3H, CH3Ar), 2.57 (q, 2H, CH2Ar),
3.72 (t, 2H, CH2N), 4.73 (s, 1H, 5-H), 6.92-7.15 (m, 3H, ArH),
8.42 (s, 1H, NH), 10.80 (s, 1H, NH), 12.05 (s, 1H, CO2H). Anal.
(C17H21N3O4‚0.25H2O) C, H, N.
1.50-1.61 (m, 4H, 2×CH2), 2.24 (s, 3H, CH3Ar), 2.58 (m, 4H,
CH2CN, CH2Ar), 3.72(t, 2H, CH2N), 4.73 (s, 1H, 5-H), 6.92-
7.15 (m, 3H, ArH), 8.12 (s, 1H, NH), 10.45 (s, 1H, NH). Anal.
(C18H22N4O2) C, H, N.
3-(3-Ethoxycarbonylpropyl)-6-(3-ethyl-4-methylanili-
1
no)uracil, 5o. Yield: 92%. Mp: 171-173 °C. H NMR: 1.16
(m, 6H, 2×CH3), 1.75 (m, 2H, CH2), 2.24 (s, 3H, CH3Ar), 2.27
(t, 2H, CH2CO2Et), 2.57 (q, 2H, CH2Ar), 3.72 (t, 2H, CH2N),
4.03 (q, 2H, CO2CH2), 4.72 (s, 1H, C5-H), 6.92-7.16 (m, 3H,
ArH), 8.09 (s, 1H, NH), 10.40 (s, 1H, NH). Anal. (C19H25N3O4)
C, H, N.
3-(2-Oxopropyl)-6-(3-ethyl-4-methylanilino)uracil, 5q.
Yield: 59%. Mp: 212 °C (dec). 1H NMR: 1.14 (t, 3H,
ArCH2CH3), 2.14 (s, 3H, ArCH3), 2.24 (s, 3H, CH3CO), 2.56
(q, 2H, ArCH2), 4.54 (s, 2H, NCH2), 4.72 (s, 1H, C5-H), 6.94-
7.16 (m, 3H, ArH), 8.20 (s, 1H, NH), 10.59 (s, 1H, NH). Anal.
(C16H19N3O3) H,N. C, calcd 63.77; found 64.43.
3-(4-Oxopentyl)-6-(3-ethyl-4-methylanilino)uracil, 5r.
Yield: 32%. Mp: 223-225 °C. 1H NMR: 1.17 (t, 3 H,
CH3CH2Ar), 1.90 (t, 2 H, CH2), 2.09(s, 3 H, CH3CO), 2.28 (s, 3
H, CH3Ar), 2.45 (t, 2 H, CH2CO), 2.58 (q, 2 H, CH2Ar), 3.87 (t,
2 H, CH2N), 5.09 (s, 1 H, 5-H), 6.89-7.11 (m, 3 H, Ar), 7.46
(br, 1 H, NH), 10.12 (s, 1 H, NH). Anal. (C18H23N3O3) C, H, N.
3-[2-(N-Morpholinyl)ethyl]-6-(3-ethyl-4-methylanilino)-
1
uracil, 5s. Yield: 75%. Mp: 221-223 °C. H NMR: 1.14 (t,
3H, ArCH2CH3), 2.23 (s, 3H, ArCH3), 2.40 (m, 4H, CH2NCH2),
2.58 (q, 2H, ArCH2), 3.24 (m, 2H, CH2N), 3.47 (m, 4H,
CH2OCH2), 3.82 (m, 2H, NCH2), 4.72 (s, 1H, 5-H), 6.90-7.18
(m, 3H, ArH), 8.14 (S, 1H, NH), 10.48 (s, 1H, NH). Anal.
(C19H26N4O3) C, H, N.
3-[3-(N-Morpholinyl)propyl]-6-(3-ethyl-4-methylanili-
1
no)uracil, 5t. Yield: 78%. Mp: 220-222 °C. H NMR: 1.16
(t, 3H, CH3C), 1.68 (m, 2H, CH2), 2.23 (s, 3H, ArCH3), 2.22-
2.38 (m, 6H, 3xNCH2), 2.60 (q, 2H, ArCH2), 3.59 (m, 4H,
CH2OCH2), 3.78 (t, 2H, NCH2), 4.75 (s, 1H, 5-H), 6.95-7.18
(m, 3H, ArH), 8.12 (s, 1H, NH), 10.40 (s, 1H, NH). Anal.
(C20H28N4O3‚0.5H2O) C, H, N.
3-[8-(N-Morpholinyl)octyl]-6-(3-ethyl-4-methylanilino)-
1
uracil, 5u. Yield: 47%. Mp: 175-176 °C. H NMR: 1.12 (t,
3H, ArCH2CH3), 1.20-1.52 (m, 12H, 6×CH2), 2.20-2.32 (m,
7H, 2×CH2, ArCH3), 2.56 (q, 2H, ArCH2), 3.30 (m, 2H, CH2),
3.54 (m, 4H, 2×CH2), 3.70 (t, 2H, NCH2), 4.74 (s, 1H, 5-H),
6.90-7.14 (m, 3H, ArH), 8.12 (s, 1H, NH), 10.35 (s, 1H, NH).
Anal. (C25H38N4O3) C, H, N.
3-[2-(4-Benzoylpiperazinyl)ethyl]-6-(3-ethyl-4-methyl-
1
anilino)uracil, 5v. Yield: 81%. Mp: 104-106 °C. H NMR:
1.16 (t, 3H, ArCH2CH3), 2.24 (s, 3H, ArCH3), 2.42-2.60 (m,
6H, NCH2 × 2, ArCH2), 3.30 (m, 4H, NCH2 × 2), 3.59 (m, 2H,
CH2N), 3.83 (t, 2H, NCH2), 4.72 (s, 1H, 5-H), 6.92-7.45 (m,
8H, ArH), 8.12 (S, 1H, NH), 10.45 (s, 1H, NH). Anal.
(C26H31N5O3‚0.75H2O) C, H, N.
3-[(5-Ethoxycarbonyl)pentyl]-6-(3-ethyl-4-methylanili-
no)uracil, 5w. Yield: 75%. Mp: 160-161 °C. 1H NMR: 1.16
(t, 6 H, 2×CH3CH2Ar), 1.22 (m, 2 H, CH2), 1.42-1.52 (m, 4 H,
2×CH2), 2.21 (s, 3 H, CH3Ar), 2.24(t, 2 H, CH2COO), 2.57 (q,
2 H, CH2Ar), 3.64 (t, 2 H, CH2N), 4.02 (q, 2 H, CH2O), 4.69 (s,
1 H, 5-H), 6.92-7.15 (m, 3 H, Ar), 8.08 (br, 1 H, NH), 10.39 (s,
1 H, NH). Anal. (C21H29N3O4) C, H, N.
3-(4-Hydroxy-2-butenyl)-6-(3-ethyl-4-methylanilino)uracil,
8c. A solution of 1.0 M BCl3 in CH2Cl2 (3 mL) was added
dropwise during 10 min to a stirred solution of 3-(4-benzyloxy-
2-butenyl)-6-(3-ethyl-4-methylanilino)uracil (200 mg; see Sup-

7072 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Zhi et al.
porting Information) in CH2Cl2 (8 mL) at -78 °C under a N2
atmosphere. The reaction mixture was stirred at -78 °C for 5
h, whereupon the reaction was quenched by a cautious addi-
tion of MeOH (5 mL) and 10% ammonia in MeOH (5 mL). The
mixture was allowed to warm to room temperature, and the
insoluble portion was filtered and washed with CH2Cl2:MeOH
(4:1, 4 × 25 mL). The solvents were removed, and the residue
was purified by chromatography on silica gel with 10-25%
MeOH in CH2Cl2 as eluent to give 118 mg (72% yield) of
product. Mp: 185-186 °C. 1H NMR: 1.16 (t, 3H, ArCH2CH3),
2.22 (s, 3H, ArCH3), 2.59 (q, 2H, ArCH2), 4.15 (m, 2H, NCH2),
4.38 (m, 2H, OCH2), 4.66 (t, 1H, OH), 4.72 (s, 1H, C5-H), 5.32
(m, 1H, dCH), 5.60 (m, 1H, dCH), 6.93-7.13 (m, 3H, ArH),
8.12 (s, 1H, NH), 10.46 (s, 1H, NH). Anal. (C17H21N3O5‚0.25H2O)
C, H, N.
3-(4-Aminobutyl)-6-(3-ethyl-4-methylanilino)uracil Hy-
drochloride, 9a. A solution of 0.5 M LiAlH4 in diglyme (3
equiv) was added dropwise to a stirred solution of the nitrile
5m (1 equiv) in anhydrous diglyme at room temperature. The
reaction mixture was stirred at room temperature until
disappearance of the starting material (TLC). MeOH was
added dropwise, and the solvents were removed. EtOH was
added, the mixture filtered, and the solid washed with EtOH.
The solvent was removed, and the residue was purified by
chromatography on silica gel with CHCl3:MeOH as eluent, to
give 3-(4-aminobutyl)-6-(3-ethyl-4-methylanilino)uracil (91%
yield). This compound was dissolved in CHCl3 and MeOH, and
a solution of 4 M HCl in dioxane was added. The mixture was
stirred at room temperature for 1 h, and the solvents were
removed to give the hydrochloride 9a as a white solid, identical
with an authentic sample.3
or 9b (100 mg, 1 equiv) in EtOH (10 mL) containing triethy-
lamine (2.5 equiv) or dry pyridine (10 mL) at room tempera-
ture. The mixture was stirred at room temperature or at 50
°C for 3-15 h. The reaction mixture was concentrated under
vacuum, and the residue was applied to a silica gel column.
Elution with MeOH:CHCl3 yielded products which were
crystallized from MeOH or Me2CO.
3-[4-(4-Toluenesulfonamido)butyl]-6-(3-ethyl-4-methyl-
anilino)uracil, 11a. Yield: 33%. Mp: 197-199 °C. 1H NMR:
1.14 (t, 3H), 1.32 (quintet, 2H), 1.44 (quintet, 2H), 2.03 (s, 3H),
2.37 (s, 3H), 2.58 (q, 2H), 2.68 (q, 2H), 3.63 (t, 2H), 4.71 (s,
1H), 6.92 (d, 1H), 6.97 (s, 1H), 7.14 (d, 1H), 7.38 (d, 2H), 7.50
(t, 1H), 7.65 (d, 2H), 8.10 (s, 1H), 10.41 (s, 1H). Anal.
(C24H30N4O4S‚0.5H2O) C, H, N.
3-[4-(2-Thiophenesulfonamido)butyl]-6-(3-ethyl-4-me-
thylanilino)uracil, 11b. Yield: 52%. Mp: 183-185 °C. 1H
NMR: 1.14 (t, 3H), 1.35 (quintet, 2H), 1.47 (quintet, 2H), 2.23
(s, 3H), 2.56 (q, 2H), 2.79 (q, 2H), 3.65 (t, 2H), 4.72 (s, 1H),
6.93 (dd, 1H), 6.98 (d, 1H), 7.17 (m, 2H), 7.56 (d, 2H), 7.80
(t, 1H), 7.90 (d, 2H), 8.10 (s, 1H), 10.42 (s, 1H). Anal.
(C21H26N4O4S2) C, H, N.
3-(5-Iodopentyl)-6-(3-ethyl-4-methylanilino)uracil, 6b.
A stirred suspension of the hydroxy compound 8a (3.4 g, 10.3
mmol) in CHCl3 (100 mL) was treated with iodotrimethylsilane
(4 equiv) at room temperature. The solid disappeared to give
a yellow solution. The solution was heated under reflux for
14 h, and after cooling to room temperature, the reaction was
quenched with saturated aqueous Na2SO3. The mixture was
extracted with CHCl3 (3 × 50 mL), and the combined organic
extracts were evaporated under reduced pressure to give a
white slurry. The solid was filtered, washed with water and
acetone, and dried to give 4.3 g (95%) of product. Mp: 201-
203 °C (from MeOH). 1H NMR: 1.14 (t, 3H, CH3C), 1.32-1.48
(m, 6H, 3×CH2), 1.77 (quin, 2H, CH2), 2.24 (s, 3H, CH3Ar),
2.54 (q, 2H, CH2C), 3.27 (t, 2H, CH2N), 3.68 (t, 2H, CH2I), 4.73
(s, 1H, C5-H), 6.95-7.14 (m, 3H, ArH), 8.06 (s, 1H, NH), 10.37
(s, 1H, NH). Anal. (C18H24IN3O2) C, H, N.
General Method for the Reaction of 3-(Iodoalkyl)-
EMAUs with Nucleophiles. A mixture of 6a or 6b, K2CO3,
and nucleophile in a suitable solvent (Me2CO, MeCN, DMF)
was stirred at room temperature. Once the reaction was
complete, as monitored by TLC, the solution was concentrated
in vacuo, and water was added. The mixture was extracted
with CHCl3, and the extracts were dried over Na2SO4. After
removal of solvent, the residue was purified by chromatogra-
phy on silica gel using MeOH:CHCl3 as eluent to give the
product.
3-{[4-(2-Hydroxyethyl)amino]butyl}-6-(3-ethyl-4-me-
thylanilino)uracil, 7a. Yield: 77%. Mp: 140 °C (dec). 1H
NMR: 1.10 (t, 3H, CH3CH2Ar), 1.58 (m, 4H, 2×CH2), 2.18 (s,
3H, CH3Ar), 2.56 (q, 2H, CH2Ar), 2.91 (m, 4H, 2×CH2N), 3.30
(s, 1H, NH), 3.60 (m, 2H, CH2O), 3.73 (t, 2H, CH2N), 4.72 (s,
1H, 5-H), 5.22 (t, 1H, OH), 6.88-7.12 (m, 3H, ArH), 8.22 (s,
1H, NH), 10.40 (br s, 1H, NH). HRMS: calcd for C19H29N4O3
(M + 1), 361.2240; found 361.2236.
3-{[4-Bis(2-hydroxyethyl)amino]butyl}-6-(3-ethyl-4-me-
thylanilino)uracil, 7b. Yield: 42%. 1H NMR: 1.12 (t, 3H,
CH3CH2Ar), 1.51 (m, 4H, 2×CH2), 2.21 (s, 3H, CH3Ar), 2.57
(q, 2H, CH2Ar), 3.0 (m, 6H, 3×CH2N), 3.62-3.78 (m, 6H,
2×CH2O, CH2N), 4.72 (s, 1H, 5-H), 4.90 (br, 2H, OH), 6.92-
7.15 (m, 3H, ArH), 8.10 (s, 1H, NH), 10.40 (br s, 1H, NH).
HRMS: calcd for C21H33N4O4 (M + 1), 405.2502; found
405.2488.
3-(5-Aminopentyl)-6-(3-ethyl-4-methylanilino)uracil Hy-
drochloride, 9b. Reduction ofthe nitrile 5n and conversion
to the hydrochloride, as described for 9a, gave the product in
64% yield as a hygroscopic white solid. 1H NMR: 1.12 (t, 3H,
CH3CH2Ar), 1.25-1.70 (m, 6H, 3×CH2), 2.21 (s, 3H, CH3Ar),
2.57 (q, 2H, CH2Ar), 2.76 (m, 2H, CH2NH2), 3.68 (t, 2H, CH2N),
4.75 (s, 1H, 5-H), 6.92-7.15 (m, 3H, ArH), 8.30 (br, 3H, NH3),
9.60 (s, 1H, NH), 10.95 (s, 1H, NH). HRMS: calcd for
C18H27N4O2 (M + 1), 331.2134; observed, 331.2139.
General method for the Preparation of 3-(Carbox-
amidoalkyl)-6-(3-ethyl-4-methylanilino)uracils. The ap-
propriate acyl chloride (1.2-3.3 equiv) was added to a solution
of 9a or 9b (100 mg, 1 equiv) in dry 1,2-dichloroethane or
EtOH. Triethylamine (2.4-10 equiv) was added, and the
solution was stirred at room temperature for 3-3.5 h. The
solvent was removed in vacuo, and the residue was applied to
a silica gel column. Products were eluted with MeOH:CHCl3.
3-[4-(Propionamido)butyl]-6-(3-ethyl-4-methylanilino)-
1
uracil, 10a. Yield: 63%. Mp: 214-215 °C (from MeOH). H
NMR: 0.98 (t, 3H, CH3C), 1.14 (t, 3H, CH3), 1.36, 1.47 (quin,
4H, 2×CH2), 2.04 (q, 2H, CH2C), 2.25 (s, 3H, CH3Ar), 2.58 (q,
2H, CH2NH), 3.02 (q, 2H, CH2NH), 3.68 (t, 2H, CH2N), 4.73
(s, 1H, C5-H), 6.95-7.15 (m, 3H, ArH), 7.74 (t, 1H, NHCH2),
8.10 (s, 1H, NH), 10.41 (s, 1H, NH). Anal. (C20H28N4O3) C, H,
N.
3-[4-(Chloroacetamido)butyl]-6-(3-ethyl-4-methylanili-
no)uracil, 10b. Yield: 50%. Mp: 191-192 °C (from MeOH).
1H NMR: 1.14 (t, 3H, CH3C), 1.38-1.49 (m, 4H, 2×CH2), 2.24
(s, 3H, CH3Ar), 2.55 (q, 2H, CH2N), 3.09 (q, 2H, CH2C), 3.69
(t, 2H, CH2NH), 4.03 (s, 2H, CH2Cl), 4.73 (s, 1H, C5-H), 6.95-
7.15 (m, 3H, ArH), 8.07 (s, 1H, NH), 8.19 (t, 1H, NHCH2), 10.37
(s, 1H, NH). Anal. (C19H25ClN4O3) C, H, N.
3-{4-[(Cyclopropanecarboxamido]butyl}-6-(3-ethyl-4-
methylanilino)uracil, 10c. Yield: 66%. Mp: 224-225 °C.
1H NMR: 0.64 (m, 4H, (CH2)2), 1.13 (t, 3H, CH3Ar), 1.35 (quin,
2H, CH2), 1.48 (m, 3H, CH3C), 2.23 (s, 3H, CH3Ar), 2.55 (q,
2H, CH2N), 3.03 (q, 2H, CH2C), 3.69 (t, 2H, CH2NH), 4.72 (s,
1H, C5-H), 6.93 (d, 1H, CH), 6.97-7.14 (m, 3H, ArH), 8.02 (t,
1H, NHCH2), 8.10 (s, 1H, NH), 10.41 (s, 1H, NH). Anal.
(C21H28N4O3) C, H, N.
3-{5-[1-(4-(4-Fluorophenyl)-1,2,3,6-tetrahydropyridinyl)]-
pentyl}-6-(3-ethyl-4-methylanilino)uracil, 7d. Yield: 76%.
1H NMR (400 MHz, DMSO-d6): 1.1 (t, 3H, ArCH2CH3), 1.25-
1.3 (m, 2H, CH2), 1.49-1.55 (m, 2H, CH2), 1.67 (m, 2H, CH2),
2.2 (s, 3H, ArCH3), 2.53 (q, 2H, ArCH2CH3), 2.63-2.72 (m, 2H,
CH2), 3.12-3.25 (m, 3H, CHN and NCH2), 3.66-3.7 (m, 4H,
2xNCH2), 3.95 (m, 1H, CHN), 4.69 (s, 1H, C5-H), 6.13 (s, 1H,
CH), 6.90-6.94 (m, 2H, ArH), 7.13 (d, 1H, ArH), 7.17-7.21
(m, 2H, ArH), 7.49-7.51 (m, 2H, ArH), 8.07 (s, 1H, NH), 9.44
(s, 1H, NH). HRMS: calcd for C29H36FN4O2, 491.2822; found
491.2835.
General Method for the Preparation of 3-(Sulfonami-
doalkyl)-6-(3-ethyl-4-methylanilino)uracils. The appropri-
ate sulfonyl chloride (1.1 equiv) was added to a solution of 9a

3-Substituted-6-(3-ethyl-4-methylanilino)uracils
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 7073
3-[5-(6-Methoxy-2,3,4,9-tetrahydro-1H-â-carboline-2-yl)-
pentyl]-6-(3-ethyl-4-methylanilino)uracil, 7e. Yield: 36%.
1H NMR (400 MHz, CD3OD): 1.20 (t, 3H, ArCH2CH3), 1.42
(m, 2H, CH2), 1.69 (m, 2H, CH2), 1.81 (m, 2H, CH2), 2.29 (s,
3H, ArCH3), 2.63 (q, 2H, ArCH2CH3), 2.94-3.03 (m, 4H, NCH2
and CH2), 3.31 (m, 2H, NCH2), 3.79 (s, 3H, OCH3), 3.87 (m,
2H, NCH2), 4.13 (m, 2H, NCH2), 4.88 (s, 1H, C5-H), 6.74 (dd,
1H, ArH), 6.93-7.0 (m, 3H, ArH), 7.15-7.2 (m, 2H, ArH).
HRMS: calcd for C30H38N5O3, 516.2975; found 516.2965.
determine serum effects were carried out in the presence of
50% fetal calf serum added to the growth medium.
Experiments in Mice. Pathogen-free Swiss-Webster mice
(males, 20-24 g) were purchased from Taconic Farms (Ger-
mantown, NY). The animals were housed at the University of
Massachusetts Medical School (UMMS) Animal Medicine
facility. All animal experiments were approved by the UMMS
institutional animal care and use committee. Mice were
allowed free access to food and water throughout the study.
3-[4-(N-Morpholinyl)butyl]-6-(3-ethyl-4-methylanilino)-
uracil hydrochloride, 7c. A solution of 3-[4-(N-morpholinyl)-
butyl]-6-(3-ethyl-4-methylanilino)uracil3 (13) (772 mg, 2 mmol)
in 5% MeOH:CHCl3 (20 mL) was treated with 1 mL of 4.0 M
HCl in dioxane. The mixture was stirred at room temperature
for 1 h. After removal of the solvent, the residue was dried in
Pharmacokinetics. Compound 1c was dissolved in a 30%
solution of CDx in water. Compound 7c was dissolved in
phosphate buffered saline (PBS), and the pH was adjusted to
neutral with aqueous KOH prior to dosing. Mice were given
1c or 7c as an iv bolus of 20 mg/kg by tail vein in a volume of
5 mL/kg. Blood samples were taken from groups of three
animals before and 2, 5, 15, 30, 60, and 120 min after dosing.
For sc injection, mice were given drug in single doses of 20
and 60 mg/kg (1c) or 20, 200, and 400 mg/kg (7c). Blood
samples were taken from groups of three animals before and
5, 15, 30, 60, 120, and 180 min after dosing. (A 360 min sample
was included for the animals receiving 400 mg/kg). For po
dosing, mice were given drug in a single dose of 200 mg/kg by
gavage. Blood samples were taken before and 15, 30, 60, 120,
and 180 min after dosing. At the specified time-points, mice
were anesthetized with halothane, and blood was collected by
cardiac puncture and placed in heparinized tubes. Plasma was
harvested by centrifugation at 13 000 rpm in a Microfuge 18
Centrifuge (Beckman Instruments) and stored at -20 °C until
analyzed.
A 0.3 mL aliquot of each plasma sample was transferred to
a microcentrifuge tube, and an equal volume of MeCN was
added. The samples were mixed well by vortexing and centri-
fuged to precipitate protein. The supernatant of each sample
was transferred to a new tube and evaporated in a SpeedVac
evaporator. The residue was suspended in 200 µL of MeCN:
H2O (20:80) for analysis by HPLC. The HPLC system was a
Varian Prostar System (Varian Analytical Instruments, Wal-
nut Creek, CA) equipped with two model PS-210 pumps, a
model 410 autosampler, a model 384 UV-Visible variable
wavelength detector, and a computer using the Star Chroma-
tography Workstation (5.52). The samples were analyzed on
a 5 µm C18 reverse phase column, 4.6 × 150 mm (Microsorb,
Varian). The mobile phase consisted of MeCH:H2O:NEt3:AcOH
(25:74.7:0.2:0.1) at a flow rate of 1 mL/min and a detection
wavelength of 282 nm. The concentration of 1c or 7c was
determined by comparison of peak areas with those from a
standard curve generated from plasma samples spiked with
compound. The linear range was 1 to 40 µg/mL.
1
vacuo to provide the product in 98% yield. H NMR: 1.16 (t,
3H, ArCH2CH3), 1.48-1.55 (m, 4H, CH2CH2), 2.26 (s, 3H,
ArCH3), 2.60 (q, 2H, ArCH2), 3.12 (m, 4H, NCH2 × 2), 3.38
(m, 2H, CH2N), 3.80 (m, 4H, CH2OCH2), 3.90 (m, 2H, NCH2),
4.72 (s, 1H, C5-H), 6.93-7.15 (m, 3H, ArH), 8.75 (s, 1H, NH),
10.58 (s, 1H, NH), 10.75 (s, 1H, NH+). HRMS: calcd for
C21H31N4O3(M-HCl+1), 387.2396; found 387.2391.
DNA Polymerase Assays. DNA polymerase IIIC (pol IIIC)
of B. subtilis was the homogeneous recombinant protein
expressed and prepared as described previously.11 The enzyme
was assayed in a 96-well plate format by using activated calf
thymus DNA as a substrate according to the method of Barnes
and Brown.12 Briefly, enzyme was added to a buffered mixture
containing Mg2+, dithiothreitol, glycerol, dATP, dCTP, dTTP,
[3H]dTTP, and activated calf thymus DNA. Serial dilutions of
compounds (in DMSO) were added to the plates. Assays were
initiated by the addition of 0.025-0.06 units of enzyme (1 unit
is the amount required to incorporate 250 pmol of [3H]dTMP
under standard assay conditions), incubated for 10 min at 30
°C, and terminated by the addition of a trichloroacetic acid-
sodium pyrophosphate solution. Precipitated labeled DNA was
collected on glass fiber filter plates, and the plates were
washed sequentially with dilute aqueous HCl and EtOH, dried,
and counted in a liquid scintillation counter. Apparent inhibi-
tion constants (Ki values) were obtained directly in this
“truncated” assay (lacking the competitor dGTP),6 and had
average standard deviation of (44%.
Bacterial Strains. The standard panel for antibacterial
screening included S. aureus 25923, S. aureus 13709 (Smith),
E. faecalis 29212 and E. faecium 19434, all purchased from
the American Type Culture Collection (ATCC, Manassas, VA).
Methicillin-resistant S. aureus (MRSA B42876) and vancomy-
cin-resistant E. faecium (VRE) are clinical isolates provided
by Dr. Richard Ellison, University of Massachusetts Medical
School. B. subtilis (BD54) is a standard laboratory strain. E.
coli (J-53) was provided by Dr. Martin Marinus, University of
Massachusetts Medical School.
Mass spectra were measured on a ThermoFinnigan LCQ
Advantage ion trap instrument by APC ionization. Samples
were introduced by infusion from solutions in water or by
HPLC in the above conditions.
Determination of Minimum Inhibitory Concentra-
tions. Minimum inhibitory concentrations (MIC) of compounds
were measured using a modification of the microdilution
method outlined by the National Committee for Clinical
Laboratory Standards (NCCLS guidelines M7-A4, vol. 17,
1997). Log-phase bacterial cultures were grown in Luria broth
(LB) for B. subtilis and staphylococci, and in brain-heart
infusion broth (BHIB) for enterococci. Diluted cultures were
seeded into 96-well plates (200 µL/well) at a concentration of
(3-7) × 105 colony forming units (CFU)/mL. Each compound
was tested in duplicate assays by diluting stock solutions 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
with shaking, and bacterial growth was determined by mea-
suring optical density (600 nm, 1 cm path length) in a
microplate reader. MIC values for antimicrobial drug-treated
cultures are the lowest concentrations of test compounds at
which growth was not apparent (less than 25% of the DMSO
control), and reported values varied (2-fold. Experiments to
Antibacterial Efficacy. S. aureus (Smith) was grown at
37 °C to log-phase in LB medium, and the CFU were
determined using a nomogram relating CFU to optical density
at 600 nm. Bacteria were washed in fresh cold LB and injected
ip as a suspension in 0.5 mL of LB, ca. 108 CFU/animal.
Groups of five or 10 mice were treated at 15 min postinfection
and/or various times postinfection with vehicle, test compound
in vehicle, or vancomycin hydrochloride (Vancocin, Lilly) in
saline. Mice were returned to their cages and monitored for
mortality for up to 72 h.
Acknowledgment. This work was supported by
small business grant AI41260 from the National Insti-
tutes of Health and by Shire BioChem Inc., Laval,
Canada.
Supporting Information Available: Syntheses, pol IIIC
inhibition, and antibacterial activity of additional compounds;
elemental analyses. This information is available free of charge
via the Internet at http://pubs.acs.org.

7074 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Zhi et al.
(7) Wright, G. E.; Brown, N. C. DNA polymerase III: A new target
for antibiotic development. Curr. Opin. Anti-inf. Invest. Drugs
1999, 1, 45-48.
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JM050517R