6-anilinouracil-based inhibitors of bacillus subtilis DNA polymerase III: Antipolymerase and antimicrobial structure-activity relationships based on substitution at uracil N3

Article abstract of DOI:10.1021/jm980693i

6-Anilinouracils (6-AUs) are dGTP analogues which selectively inhibit the DNA polymerase III of Bacillus subtilis and other Gram-positive bacteria. To enhance the potential of the 6-AUs as antimicrobial agents, a structure- activity relationship was devel

Full text of DOI:10.1021/jm980693i

J . Med. Chem. 1999, 42, 2035-2040
2035
Brief Articles
6-An ilin ou r a cil-Ba sed In h ibitor s of Ba cillu s su btilis DNA P olym er a se III:
An tip olym er a se a n d An tim icr obia l Str u ctu r e-Activity Rela tion sh ip s Ba sed on
Su bstitu tion a t Ur a cil N3
Paul M. Tarantino, J r.,§ Chengxin Zhi,‡,§ J oseph J . Gambino,§ George E. Wright,§ and Neal C. Brown*,‡
Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical School, 55 Lake Avenue North,
Worcester, Massachusetts 01655, and GLSynthesis, Inc., 222 Maple Avenue, Shrewsbury, Massachusetts 01545
Received December 10, 1998
6-Anilinouracils (6-AUs) are dGTP analogues which selectively inhibit the DNA polymerase
III of Bacillus subtilis and other Gram-positive bacteria. To enhance the potential of the 6-AUs
as antimicrobial agents, a structure-activity relationship was developed involving substitutions
of the uracil N3 position in two 6-AU platforms: 6-(3,4-trimethyleneanilino)uracil (TMAU)
and 6-(3-ethyl-4-methylanilino)uracil (EMAU). Series of N3-alkyl derivatives of both 6-AUs
were synthesized and tested for their ability to inhibit purified B. subtilis DNA polymerase III
and the growth of B. subtilis in culture. Alkyl groups ranging in size from ethyl to hexyl
enhanced the capacity of both platforms to bind to the polymerase, and with the exception of
hexyl, they also significantly enhanced their antimicrobial potency. N3 substitution of the
EMAU platform with more hydrophilic hydroxyalkyl and methoxyalkyl groups marginally
enhanced anti-polymerase III activity but enhanced antibacterial potency severalfold. In sum,
the results of these studies indicate that the ring N3 of 6-anilinouracils can tolerate substituents
of considerable size and structural variety and, thus, can be manipulated to significantly
enhance the antibacterial potency of this novel class of polymerase III-specific inhibitors.
In tr od u ction
pol IIIs that renders them and their respective hosts
exquisitely sensitive to inhibition by the 6-AUs.5,11
We have sought to improve both the anti-pol III and
antimicrobial potency of the 6-AUs. As part of this effort,
we have exploited structure-activity relationships (SARs)
involving the anilino moiety to develop two “platform”
6-AUs: 6-(3,4-trimethyleneanilino)uracil (TMAU, 4) and
6-(3-ethyl-4-methylanilino)uracil (EMAU, 11).6,7,10,11 Al-
though 4 and 11 display favorable potency versus the
pol III target, they have relatively poor antibacterial
potency. This deficiency has prompted us to further
explore the effects of substitution at uracil N3sthe only
ring atom of the 6-AUs which can be substituted without
negatively affecting inhibitor binding to pol III.7 We
have exploited compounds 4 and 11 and a model
Bacillus subtilis system10 to develop a broad-ranging
SAR based on alkyl, hydroxyalkyl, and methoxyalkyl
substitution of N3. The results, which are described
below, indicate that N3 has significant potential as a
site for enhancing the antimicrobial efficacy of these
novel pol III-specific inhibitors.
There is a rapidly growing crisis in the clinical
management of life-threatening infectious disease caused
by multi-antibiotic-resistant (MAR) strains of patho-
genic Gram-positive (Gr+) bacteria.1 Solving this
problem will depend, in part, on the development of
chemotherapeutic agents which selectively attack new
bacterial targets. One such target is DNA polymerase
III (pol III), an enzyme essential for the replication of
the bacterial chromosome.2-5 We have developed and
characterized a novel class of 6-anilinouracils (6-AUs)
specifically targeted to the pol III of Gr+ bacteria.6-10
These agents act as Gr+ pol III-specific dGTP ana-
logues. As shown in panel A of Figure 1, the 6-AU
nucleus has two “domains”: a base-pairing domain and
an enzyme-specific, aryl domain. Figure 1B summarizes
how these two domains combine to effect enzyme
inhibition. The base-pairing domain of the molecule
forms three H bonds with an unopposed template
cytosine just distal to the DNA primer terminus.
Simultaneously, the aryl substituent binds a unique
aryl-specific site, or “receptor”, within the enzyme’s
dNTP binding site and, thus, forms a nonproductive
ternary complex of enzyme, inhibitor, and primer-
template. It is the presence of this conserved aryl
receptor in the active site region of the bacterial Gr+
Resu lts
Syn th esis a n d P r op er ties of N3-Su bstitu ted 6-
An ilin ou r a cils. All N3-substituted 6-anilinouracils
were prepared as described below. The syntheses of
compounds 4-6, 8, 9, and 11 have been reported
previously.7,12,13
* Author for correspondence: Neal C. Brown. Phone: (508) 856-
2152. Fax: (508) 856-5080. E-mail: neal.brown@ummed.edu.
‡ University of Massachusetts Medical School.
§ GLSynthesis, Inc.
N3-Alkyl-6-anilinouracils were synthesized by the
three-step sequence summarized in Scheme 1. First, the
10.1021/jm980693i CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/07/1999

2036 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Brief Articles
platform for alkyl substitution, generating derivatives
5-10. The effects of these compounds on inhibition of
B. subtilis pol III and on the growth of B. subtilis in
culture are summarized in the upper half of Table 1.
As the data in the left column indicate, none of the
groups reduced affinity for pol III compared with the
unsubstituted analogue 4. Rather, with the exception
of methyl, which was approximately neutral in its
impact, larger groups significantly enhanced anti-pol III
potency. The extent of enhancement did not vary strictly
with increasing group size, although there was a trend
in that direction between ethyl and butyl (6-9). Upon
reaching the size of hexyl substitution (10), this trend
began to reverse.
2. In h ibition of Ba cter ia l Gr ow th . As the data in
the right-hand column of Table 1 indicate, alkyl sub-
stitution also significantly affected antimicrobial po-
tency, but in a manner which differed somewhat from
that seen for enzyme inhibition. With the exception of
hexyl, which essentially destroyed the activity of 4, all
groups from methyl to butyl enhanced antimicrobial
activity. Allyl and ethyl groups appeared to be optimal
to increase potency.
Effects of N3-Alk yl Su bstitu tion of EMAU (11)
on An tip olym er a se a n d An tim icr obia l Activity. We
sought to determine if the biological effects of N3-alkyl
substitution of platform 4 also applied more generally
with another 6-AU platform. We therefore targeted 6-(3-
ethyl-4-methylanilino)uracil (EMAU, 11),13 one of the
most potent pol III inhibitors. N3 of 11 was substituted
with ethyl and allyl, the two substituents which in-
creased the antibacterial activity of the compound 4
platform. The properties of the resulting compounds, 12
(ethyl) and 13 (allyl), are summarized in the middle
section of Table 1.
F igu r e 1. (A) 6-Anilinouracils have two essential domains:
a base-pairing domain which binds template cytosine and an
aryl domain which binds a specific aryl receptor in the target
pol III. (B) Schematic summary of inhibitor bound to DNA.
(The third essential component of the ternary complex, pol III,
is not depicted.)
As the data clearly indicate, both ethyl and allyl
enhanced the antipolymerase and antibacterial potency
of 11 as they did with 4. The enhancement of both
parameters was slightly more pronounced than it was
for 4. Antipolymerase potency of 12 and 13 increased
∼5-8-fold relative to 11, compared with increases of
∼3.2-3.8-fold for the analogous derivatives of 4. The
enhancement of antibacterial activity of 12 and 13 was
approximately double that seen with the analogous
derivatives of 4 (i.e., ∼20-fold vs ∼10-fold).
appropriate urea was reacted with diethyl malonate in
refluxing sodium ethoxide/ethanol to form the substi-
tuted barbituric acid as described in the literature.14 The
N-alkylbarbituric acid was specifically chlorinated at C6
by refluxing in POCl3 in the presence of water.15 The
final step employed the resulting substituted 6-chloro-
uracil and the appropriate aniline in refluxing 2-meth-
oxyethanol as described previously.13 Detailed descrip-
tions of the yields and properties of all new inhibitors
are provided in the Experimental Section.
N3-Hydroxyalkyl and N3-methoxyalkyl EMAUs were
synthesized by the four-step sequence summarized in
Scheme 2. 2-(Methoxyethyl)urea (14) and 2-(methoxy-
propyl)urea (15) were condensed with diethyl malonate
in refluxing sodium ethoxide/ethanol to form the N-
substituted barbituric acids.14 The barbituric acids were
selectively chlorinated at the 6 position using benzyl-
triethylammonium chloride and POCl3 at 50 °C to give
18 and 19.16 Synthesis of the two methoxyalkyl EMAUs
(20, 21) employed the 6-chlorouracils and 3-ethyl-4-
methylaniline, which were heated at 150 °C in the
absence of solvent. Each of these compounds was
converted to its respective hydroxyalkyl form (22, 23)
using trimethylsilyl iodide (TMSI) in chloroform at room
temperature. Yields and properties of these compounds
are provided in the Experimental Section.
Effects of N3-Meth oxya lk yl a n d N3-Hyd r oxy-
a lk yl Su bstitu tion of EMAU on An tip olym er a se
a n d An tim icr obia l Activity. To probe the tolerance
of N3 for substituents more hydrophilic than alkyl, we
exploited the EMAU (11) platform and the method
summarized in Scheme 2 to synthesize four relevant
derivatives: N3-(2-hydroxyethyl) (22), N3-(3-hydroxy-
propyl) (23), and their respective methoxy derivatives
(20 and 21). The antipolymerase and antimicrobial
activities of each of these compounds are summarized
in the bottom four rows of Table 1.
At the level of the isolated pol III, none of the four
substituents enhanced inhibitory potency as effectively
as N3-ethyl (12) or N3-allyl (13). Hydroxyethyl (22) and
methoxyethyl (20) groups were the most effective,
enhancing potency approximately 2-fold, while the effect
of the hydroxypropyl (23) and methoxypropyl (21)
groups was essentially neutral. The effect of the four
substituents on antimicrobial activity was both positive
Effects of N3-Alk yl Su bstitu tion of TMAU (4) on
An tip olym er a se a n d An tim icr obia l Activity. 1. P ol
III In h ibition . We exploited 4 (TMAU) as the primary

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 2037
Sch em e 1. Synthesis of N3-Alkyl-Substituted 6-Anilinouracils
Sch em e 2. Synthesis of N3-(Methoxyalkyl)- and N3-(Hydroxyalkyl)-6-anilinouracilsa
a
BTAC, benzyltriethylammonium chloride; TMSI, trimethylsilyl iodide.
and considerably more profound than their respective
effects on anti-pol activity. Potency increases relative
to 11 ranged between 20-fold for the methoxypropyl
derivative 21 and approximately 10-fold for the other
three derivatives.
in size. Groups larger than methyl enhance pol III
binding, but with the exception of butyl (9), the en-
hancement varies no more than 2-fold through hexyl.
These results suggest that the N3 position in the
inhibitor-enzyme complex must approximate an en-
zyme surface or space which can accommodate hydro-
phobic groups that vary considerably in size.
Discu ssion
Second, the results, albeit limited only to two related
platforms, suggest that the effect of a given N3-alkyl
substituent on antimicrobial and anti-pol III activity is
likely to be relatively independent of the structure of
the inhibitor’s anilino moiety. Substitution of the TMAU
(4) and EMAU (11) platforms with ethyl or allyl
enhanced, in a similar fashion, the anti-pol III and
antimicrobial activity of both.
The main purpose of this work was to explore sub-
stitution to enhance the antimicrobial potency of the
6-AU inhibitor molecule for Gr+ bacteria. The results
of our previous SARs on the isolated pol III7,13 effectively
excluded manipulation of all sites except onesthe uracil
N3 position. As the results in Table 1 indicate, the N3
position has shown considerable promise as a target for
rational substitution of the inhibitor nucleus. In sum,
our results make four significant points.
Third, the methoxyalkyl and hydroxyalkyl groups,
like the less polar alkyls of comparable size (i.e., ethyl
(12) and allyl (13)), also have a positive impact on
First, as the SAR for the TMAU (4) platform indicates,
the N3 position can accommodate groups ranging widely

2038 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
Brief Articles
Ta ble 1. Antipolymerase and Antimicrobial Activity of
6-Anilinouracils
the results of assays of the effect of relevant N3-alkyl
TMAUs on pol III-dependent DNA and RNA synthesis
in intact, growing B. subtilis17 indicate that they retain
their specificity for inhibition of DNA synthesis.
Exp er im en ta l Section
An a lyses. All new compounds were fully characterized by
1H NMR and elemental analysis (C, H, N). Proton NMR
spectra were obtained at 300 MHz with a Varian Unity 300
instrument, and results are consistent with the proposed
structures. Elemental analyses were performed by the
Microanalysis Laboratory, University of Massachusetts,
Amherst, MA, and agree to within (0.4% of calculated values
unless otherwise noted. Melting points were determined on a
Mel-temp apparatus and are uncorrected.
Ba r bitu r ic Acid s 2a a n d 2b. All N-alkyl-substituted
barbituric acids were synthesized by condensing diethyl
malonate with the substituted urea as described in the
literature.14 Properties of ethyl-,18 allyl-,19 and hexylbarbituric
acids20 have been previously reported.
N3-Alk yl-6-ch lor ou r a cils 3a a n d 3b. The substituted
barbituric acids were reacted with POCl3 in the presence of
H2O to yield the N3-substituted 6-chlorouracils, as described
in the literature.15 Crude products were isolated and identified
by TLC and NMR but not purified. Isolated products were
reacted directly in the next step.
Syn th esis of N3-Su bstitu ted 6-An ilin ou r a cils a n d Rel-
eva n t In ter m ed ia tes. The synthesis of 6-anilinouracils em-
ployed 6-chlorouracil and the appropriate aniline in refluxing
2-methoxyethanol as described previously.13 3-Ethyl-4-methyl-
aniline was synthesized as described.6 Syntheses of 6-(3,4-
trimethyleneanilino)uracil (TMAU) and 6-(3-ethyl-4-methyl-
anilino)uracil (EMAU) were previously reported.6
3-Allyl-6-(3,4-tr im eth ylen ea n ilin o)u r a cil (7). A stirred
mixture of 3-allyl-6-chlorouracil (2.5 g, 13.3 mmol) and 5-
aminoindane (3.75 g, 26.6 mmol) was heated at reflux in 80
mL of 2-methoxyethanol for 17 h under nitrogen. After cooling
to room temperature, the product was crystallized from 80%
acetic acid to give 2.53 g (67% yield) of 3-allyl-6-(3,4-trimeth-
yleneanilino)uracil (7) as yellow crystals: mp 205-208 °C; 1H
NMR (DMSO-d6) δ 2.04 (m, 2H, CH2CH2CH2), 2.86 (m, 4H,
CH2CH2CH2), 4.30 (d, 2H, CH2N), 4.75 (s, 1H, C5-H), 5.12 (dd,
2H, CH2d), 5.80 (m, 1H, CHd), 7.11 (m, 3H, Ar-H), 8.18 (s,
1H, NH), 10.45 (s, 1H, NH). Anal. (C16H17N3O2) C,H,N.
3-Hexyl-6-(3,4-tr im eth ylen ean ilin o)u r acil (10). This com-
pound was prepared by the same procedure as above. Crystal-
lization was from ethanol: mp 232-235 °C; yield 55%; 1H
NMR (DMSO-d6) δ 0.85 (t, 3H, CH3), 1.25 (s, 6H, (CH2)3), 1.42
(t, 2H, CH2CH2N), 2.02 (m, 2H, CH2CH2CH2), 2.83 (m, 4H,
CH2CH2CH2), 3.67 (t, 2H, CH2N), 4.71 (s, 1H, C5-H), 7.07 (m,
3H, Ar-H), 8.11 (s, 1H, NH), 10.38 (s, 1H, NH). Anal.
(C19H25N3O2‚0.125H2O) C,H,N.
3-Eth yl-6-(3-eth yl-4-m eth yla n ilin o)u r a cil (12). A stirred
mixture of 3-ethyl-6-chlorouracil (174 mg, 1.0 mmol) and
3-ethyl-4-methylaniline (341 mg, 2.5 mmol) was heated at
reflux in 6.0 mL of 2-methoxyethanol for 8 h. After the mixture
cooled to room temperature, precipitated product was filtered
and crystallized from ethanol to give 230 mg (85% yield) of
3-ethyl-6-(3-ethyl-4-methylanilino)uracil (12) as white crys-
tals: mp 280-282 °C; 1H NMR (DMSO-d6) δ 1.05 (t, 3H, CH3-
CH2N), 1.16 (t, 3H, CH3CH2), 2.25 (s, 3H, CH3Ar), 2.58 (q, 2H,
CH2CH3), 3.78 (q, 2H, NCH2CH3), 4.76 (s, 1H, C5-H), 7.06 (m,
3H, Ar-H), 8.09 (s, 1H, NH), 10.40 (s, 1H, NH). Anal.
(C15H19N3O2) C,H,N.
compd no.
R
3′-R, 4′-R
Ki (µM)a MIC (µM)b
4
5
6
7
8
H
CH3
-(CH2)3-
-(CH2)3-
-(CH2)3-
1.20
1.34
0.31
0.38
0.31
0.09
0.24
1.00
0.12
0.21
0.58
1.00
0.59
0.73
30
5
3
3
11
CH2CH3
CH2CHdCH2 -(CH2)3-
(CH2)2CH3
(CH2)3CH3
(CH2)5CH3
H
CH2CH3
CH2CHdCH2 Et, Me
(CH2)2OCH3
(CH2)3OCH3
(CH2)2OH
-(CH2)3-
-(CH2)3-
-(CH2)3-
Et, Me
9
3
10
11
12
13
20
21
22
23
>100
30
1.5
1.5
3
1.5
3
Et, Me
Et, Me
Et, Me
Et, Me
Et, Me
(CH2)3OH
3
a
All Ki values for inhibition of B. subtilis pol III are the average
of three independent experiments performed as described in the
Experimental Section; average standard deviation for all values
b
was 20.3%. MICs (minimum inhibitory concentrations) against
B. subtilis represent the average of three independent experiments
performed as described in the Experimental Section; average
standard deviation for all values was (15.3%.
inhibitor-pol III binding. However, their impact is
considerably less, ranging from none for methoxypropyl
(21) to approximately 2-fold for hydroxyethyl and meth-
oxyethyl (i.e., 22 and 20). This reduction in potency
relative to that seen with a comparably sized alkyl group
is consistent with the reduced affinity expected between
the polar hydroxy and methoxy substituents and the
hydrophobic enzyme space hypothesized above.
Fourth, the effect of N3 substitution on antibacterial
potency with B. subtilis in culture (i.e., MIC) is, with
the exception of n-hexyl, positive. However, it differs in
several significant respects from that observed with the
isolated enzyme. First, the effect does not vary as
predictably with respect to size or extent. For example,
the methyl group, which has no significant effect on the
anti-pol III potency of 4, lowers the MIC more than
6-fold, and n-hexyl, which increases anti-pol III potency
of 4, essentially destroys its antimicrobial activity.
Similarly, in the case of 11, the hydroxyalkyl and
methoxyalkyl substituents, which enhanced anti-pol III
potency less than 2-fold, increased antimicrobial potency
between 10- and 20-fold.
We have considered two possible explanations for
these apparent discrepancies between the effect of the
N3 substituent on affinity for the isolated target pol III
in vitro and growth of its host bacterial cell in vivo. The
first envisions the possibility that a given N3 substitu-
ent changes the 6-AU molecule such that it acts not only
on its specific pol III target but nonspecifically on
another bacterial target(s). The second, simpler, expla-
nation assumes that the N3 primarily affects access of
the platform to its target rather than changes its target
specificitysi.e., by facilitating inhibitor transport through
the bacterial cell wall and membrane. We favor the
latter transport model for two reasons. First, it readily
explains the lack of antimicrobial efficacy of the very
potent pol III inhibitor, N3-hexyl TMAU (10). Second,
3-Allyl-6-(3-eth yl-4-m eth yla n ilin o)u r a cil (13). This com-
pound was prepared by the same procedure as above: mp
1
253-254 °C; yield 88%; H NMR (CDCl3) δ 1.21 (t, 3H, CH3-
CH2), 2.30 (s, 3H, CH3Ar), 2.62 (q, 2H, CH2CH3), 4.49 (d, 2H,
CH2N), 5.10 (s, 1H, C5-H), 5.15 (dd, 2H, CH2d), 5.87 (m, 1H,
CHd), 6.40 (s, 1H, NH), 7.05 (m, 3H, Ar-H), 9.22 (s, 1H, NH).
Anal. (C16H19N3O2) C,H,N.
(2-Meth oxyeth yl)u r ea (14).21 (2-Methoxyethyl)amine (15.0
g, 0.2 mol) was neutralized with concentrated hydrochloric acid

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11 2039
to give (2-methoxyethyl)amine hydrochloride, which was treated
with potassium cyanate (16.2 g, 0.2 mol) in 100 mL of water.
After heating at reflux for 4 h, the mixture was evaporated in
vacuo. Ethanol (150 mL) was added, and the residue was
heated. The warm mixture was filtered, and the filtered solid
was washed with hot ethanol. Concentration and cooling of
the filtrate gave 22.2 g (94% yield) of 14. Recrystallization from
CH2O), 3.75 (t, 2H, CH2N), 4.74 (s, 1H, C5-H), 7.05 (m, 3H,
Ar-H), 8.12 (s, 1H, NH), 10.42 (s, 1H, NH). Anal. (C17H23N3O3)
C,H,N.
3-(2-H yd r oxyet h yl)-6-(3-et h yl-4-m et h yla n ilin o)u r a cil
(22). Trimethylsilyl iodide (TMSI) (0.3 mL, 2.1 mmol) was
added to a stirred solution of 20 (152 mg, 0.5 mmol) in dry
chloroform (15 mL). The reaction mixture was stirred at room
temperature until disappearance of the starting material
(about 12 h). Methanol (10 mL) and 0.5 g of sodium sulfite
were then added to the brown-purple solution. After stirring
at room temperature for 30 min, the mixture was filtered and
the solvent was removed. The residue was purified by chro-
matography on silica gel with chloroform/methanol (90/10) as
eluent to give 130 mg (90% yield) of 22. Crystallization from
ethanol/water gave white crystals: mp 243-244 °C; 1H NMR
(DMSO-d6) δ 1.14 (t, 3H, CH3CH2), 2.24 (s, 3H, CH3Ar), 2.58
(q, 2H, CH2CH3), 3.44 (m, 2H, CH2O), 3.78 (t, 2H, CH2N), 4.72
(t, 2H, OH, 1H, C5-H), 7.05 (m, 3H, Ar-H), 8.12 (s, 1H, NH),
10.41 (s, 1H, NH). Anal. (C15H19N3O3) C,H,N.
3-(3-H y d r o x y p r o p y l)-6-(3-e t h y l-4-m e t h y la n ilin o )-
u r a cil (23). TMSI (0.2 mL, 1.41 mmol) was added to a stirred
solution of 21 (95 mg, 0.3 mmol) in dry chloroform (10 mL).
After the mixture stirred for 5 h at room temperature,
methanol (10 mL) and 0.5 g of sodium sulfite were then added
to the brown-purple solution. After stirring at room temper-
ature for 30 min, the mixture was filtered and the solvent was
removed. The residue was purified by chromatography on silica
gel with chloroform/methanol (97/3-90/10) as eluent to give
51 mg (56% yield) of 23: 1H NMR (DMSO-d6) δ 1.14 (t, 3H,
CH3CH2), 1.63 (m, 2H, CH2CH2CH2), 2.24 (s, 3H, CH3Ar), 2.57
(q, 2H, CH2CH3), 3.40 (m, 2H, CH2O), 3.74 (t, 2H, CH2N), 4.42
(t, 1H, OH), 4.73 (s, 1H, C5-H), 7.05 (m, 3H, Ar-H), 8.12 (s,
1H, NH), 10.45 (s, 1H, NH). Anal. (C16H21N3O3) C,H,N.
En zym e Assa y. B. subtilis DNA pol III was a homogeneous
recombinant protein expressed and purified as described
previously.23 Pol III was assayed using activated calf thymus
DNA as described,24 and apparent inhibitor constants (Ki’s) of
the 6-AUs were determined as previously described25 using a
truncated assay in the absence of the competitor dGTP.
Deter m in a tion of Min im a l In h ibitor y Con cen tr a tion
(MIC). The test organism was B. subtilis BD54, a standard,
penicillin-sensitive laboratory strain.26 A log-phase culture of
BD54 was diluted to a concentration of 104 cells/mL in Luria
broth,23 and 0.5 mL of this suspension was distributed to each
of 48 wells of a sterile microtiter plate. Inhibitors were
dissolved in DMSO and were added to wells at concentrations
ranging from 0 (control) to 100 µM. The final concentration of
DMSO in all wells was adjusted to 1%. Plates were incubated
for 24 h at 37 °C, and growth was assessed by visual
inspection. MIC is defined as the lowest concentration of
inhibitor at which bacterial growth was not apparent. The
growth of the test organism was not affected by the presence
of 1% DMSO in the medium.
1
ethyl acetate gave colorless needles: mp 74-76 °C; H NMR
(DMSO-d6) δ 3.11 (m, 2H, CH2N), 3.24 (s, 3H, CH3O), 3.31 (t,
2H, CH2O), 5.45 (s, 2H, NH2), 5.95 (s, 1H, NH).
(3-Meth oxypr opyl)u r ea (15).22 (3-Methoxypropyl)urea was
prepared by the same procedure as above: mp 76-78 °C; yield
90%; recrystallization from ethyl acetate gave colorless needles;
1H NMR (DMSO-d6) δ 1.57 (m, 2H, CH2), 2.98 (m, 2H, CH2N),
3.21 (s, 3H, CH3O), 3.31 (t, 2H, CH2O), 5.39 (s, 2H, NH2), 5.92
(s, 1H, NH).
N-(3-Meth oxyeth yl)ba r bitu r ic Acid (16). Sodium (5.75
g, 0.25 mol) was dissolved in 150 mL of superdry ethanol. (2-
Methoxyethyl)urea (11.8 g, 0.1 mol) and diethyl malonate (16.0
g, 0.1 mol) were added, and the mixture was refluxed for 6 h.
The mixture was allowed to cool, and concentrated hydrochlo-
ric acid was added until the solution was acidic. After evapora-
tion at reduced pressure, ethanol (150 mL) was added, and
the residue was heated. The hot mixture was filtered, and the
filtered solid was washed with hot ethanol. Concentration and
cooling of the filtrate gave 16.5 g (88.6% yield) of 16: mp 91-
1
92 °C; recrystallization from ethanol gave white crystals; H
NMR (CDCl3) δ 3.37 (s, 3H, CH3), 3.61 (t, 2H, CH2O), 3.66 (s,
2H, CH2), 4.11 (t, 2H, CH2N), 9.38 (s, 1H, NH). Anal.
(C7H10N2O4) C,H,N.
N-(3-Meth oxyp r op yl)ba r bitu r ic Acid (17). This com-
pound was prepared by the same procedure as above: yield
86%; recrystallization from ethanol gave white crystals; mp
1
90-92 °C; H NMR (CDCl3) δ 1.89 (m, 2H, CH2CH2N), 3.30
(s, 3H, CH3), 3.45 (t, 2H, CH2O), 3.64 (s, 2H, C5-CH2), 3.97 (t,
2H, CH2N), 8.41 (s, 1H, NH). Anal. (C8H12N2O4) C,H,N.
3-(2-Meth oxyeth yl)-6-ch lor ou r a cil (18). A stirred mix-
ture of N-(3-methoxyethyl)barbituric acid (1.5 g, 8.1 mmol) and
benzyltriethylammonium chloride (BTAC) (3.7 g, 16.2 mmol)
in phosphorus oxychloride (25 mL) was heated at 50 °C for 2
h. The reaction mixture was cooled to room temperature and
evaporated to dryness in vacuo. The residue was carefully
quenched with 40 g of ice chips at 0 °C, and the mixture was
extracted with ethyl acetate (3 × 40 mL). The combined
organic layers were washed with saturated aqueous NaHCO3
and dried over MgSO4. After removal of the solvent, 1.35 g
(82% yield) of 18 was obtained as a white precipitate: 1H NMR
(CDCl3) δ 3.36 (s, 3H, CH3), 3.64 (t, 2H, CH2O), 4.15 (t, 2H,
CH2N), 5.87 (s, 1H, C5-H), 9.77 (s, 1 H, NH). Anal. (C7H9N2O3-
Cl) C,H,N.
3-(3-Meth oxyp r op yl)-6-ch lor ou r a cil (19). This compound
was prepared by the same procedure as above: yield 80%; 1H
NMR (CDCl3) δ 1.88 (m, 2H, CH2CH2CH2), 3.29 (s, 3H, CH3O),
3.42 (t, 2H, CH2O), 3.97 (t, 2H, CH2N), 5.82 (s, 1H, C5-H), 10.59
(s, 1H, NH). Anal. (C8H11N2O3Cl) C,H,N
Ack n ow led gm en t. This work was supported by
STTR Phase I Grant AI41260 from the National Insti-
tutes of Health.
3-(2-Met h oxyet h yl)-6-(3-et h yl-4-m et h yla n ilin o)u r a cil
(20). A stirred mixture of 18 (205 mg, 1.0 mmol) and 3-ethyl-
4-methylaniline (271 mg, 2.0 mmol) was heated at 150 °C for
15 min. After cooling to room temperature, the residue was
chromatographed on silica gel with chloroform/methanol (97/
3-95/5) as eluent to give 280 mg (92% yield) of 20. Crystal-
lization from ethanol gave white crystals: mp 226-227 °C;
1H NMR (DMSO-d6) δ 1.14 (t, 3H, CH3CH2), 2.24 (s, 3H, CH3-
Ar), 2.57 (q, 2H, CH2CH3), 3.23 (s, 3H, CH3O), 3.43 (t, 2H,
CH2O), 3.88 (t, 2H, CH2N), 4.72 (s, 1H, C5-H), 7.05 (m, 3H,
Ar-H), 8.17 (s, 1H, NH),10.45 (s, 1H, NH). Anal. (C16H21N3O3‚
0.75H2O) C,H,N.
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2040 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 11
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