Improved synthesis of antibacterial 3-substituted 6-anilinouracils

Article abstract of DOI:10.1016/j.bmcl.2008.04.061

3-Substituted 6-anilinouracils, presently the most promising class of inhibitors of the bacterial DNA polymerase in Gram-positive bacteria, have been prepared by a general and straightforward three-step procedure starting from a readily available 1-benzyloxymethyl-protected derivative of 6-chlorouracil.

Full text of DOI:10.1016/j.bmcl.2008.04.061

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3215–3218
Improved synthesis of antibacterial 3-substituted 6-anilinouracils
Niels Svenstrup,* Alexander Kuhl, Kerstin Ehlert and Dieter Habich
¨
Bayer HealthCare AG, Aprather Weg, D-42096 Wuppertal, Germany
Received 4 April 2008; revised 21 April 2008; accepted 22 April 2008
Available online 26 April 2008
Abstract—3-Substituted 6-anilinouracils, presently the most promising class of inhibitors of the bacterial DNA polymerase in
Gram-positive bacteria, have been prepared by a general and straightforward three-step procedure starting from a readily available
1-benzyloxymethyl-protected derivative of 6-chlorouracil.
ꢀ 2008 Elsevier Ltd. All rights reserved.
Bacteria are able to overcome antibiotic pressure by a
variety of smart mechanisms that facilitate the selection
of resistant organisms. Antibiotic-resistant Gram-posi-
tive bacteria represent a growing challenge in present
day healthcare: the efficacy of powerful drugs such as
the b-lactams and the macrolides is continually decreas-
ing due to the emergence of antibiotic-resistant bacte-
ria.1 These pathogens carry resistance genes that
spread in the bacterial population across geographic
and interspecies boundaries.2 Development of resistance
is the inevitable result of antibiotic use, and limits effi-
cacy and life span of every antibiotic. Correct antibiotic
use will slow down resistance, but cannot prevent it.
Only the persistent discovery and development of new
antibiotics will guarantee future therapy. Established
antibiotics utilize a limited set of validated mechanisms.3
Therefore, new structural classes of antibiotics that ad-
dress novel and valid targets are urgently needed. Here,
we report our progress in the novel class of anilinoura-
cils, inhibitors of the Gram-positive DNA polymerase
IIIC.4
pounds targeting delayed-type hypersensitivity.9 In
addition, 6-chlorouracils are themselves important start-
ing materials for the synthesis of 6-anilinouracils, pres-
ently the most promising lead structure for targeting
the Gram-positive DNA polymerase IIIC.10 DNA poly-
merase IIIC is the polymerase required for the replica-
tion of chromosomal DNA in Gram-positive bacteria
with low G+C content,11 and thus represents a highly
interesting target for therapy of some of the microorgan-
isms most commonly associated with nosocomial infec-
tions today.
In a recent project we were interested in synthesizing a
series of 3-substituted 6-anilinouracils for the purpose
of investigating the role of the 3-substituent in the struc-
ture–activity relationship (SAR) of anilinouracils.4
Existing methods for the synthesis of such derivatives
are rather cumbersome, involving four linear steps, in
which the 3-substituent is introduced at the very begin-
ning of the sequence (Scheme 2). Consequently only a
handful of such derivatives have been described so
far.10 This methodology is less suitable for use in a dis-
covery setting, where a high-throughput synthesis and
late-stage structural variation would be preferred to rap-
idly elaborate SAR. Our goal was to develop a synthetic
strategy which is operationally simple, short, high yield-
ing, and amenable to be run on parallel synthesis equip-
ment. The result is an improved synthesis of 3-
substituted 6-anilinouracils by a three-step strategy
using an easily available precursor (Scheme 1).
Anilinouracils and other substituted uracils have found
widespread applications as pharmaceutical drugs and
drug candidates, as have substituted nucleotide and
nucleoside analogues in general. Appropriately substi-
tuted 6-chlorouracils are important intermediates in
the synthesis of several biologically active compound
classes such as antivirals,5 antimalarial agents,6 adeno-
sine receptor ligands,7 anti-cancer agents,8 and com-
Traditionally, 3-alkyl-6-chlorouracils are prepared by a
three-step synthetic route7,10 (Scheme 2) starting from
an appropriately substituted alkylamine, which will
eventually form the 3-substituent in the final product.
Keywords: Antibacterials; DNA polymerase IIIC; Gram-positive bac-
teria; Resistance; Anilinouracils.
*
Corresponding author. Tel.: +49 202 364438; fax: +49 202 364061;
e-mail: NielsSvenstrup@Hotmail.com
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.061

3216
N. Svenstrup et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3215–3218
nucleophilic replacement of the chlorine under thermal
O
O
conditions with an aniline.10 Wright et al. addressed
some of these shortcomings in a much improved two-
step synthesis via alkylation of 2-methoxy-6-amino-4-
pyrimidone and following one-pot arylamination and
demethylation.14 The yield and regioselectivity in the
first step is generally poor, however, and a demanding
chromatographic separation is usually involved, making
this method less useful for medium-throughput medici-
nal chemistry.
Deprotection
Alkylation
HN
R
N
R
O
N
PG
Cl
O
N
PG
Cl
O
O
R´
H2N
R
N
N
R´
O
N
H
Cl Arylamination
O
N
H
N
H
1
2
For medicinal chemistry purposes, the ideal synthetic
access to 3-substituted 6-anilinouracils optimized would
be by N3-alkylation of a preformed 6-chlorouracil.
Depending on the alkylating agent, the steric and elec-
tronic characteristics of the uracil ring system favor
alkylation of N-1 in most cases, although with varying
degrees of selectivity. We took advantage of this fact
by employing 1-benzyloxymethyl-6-chlorouracil 3, eas-
ily prepared from commercially available 6-chlorouracil,
as starting material (Scheme 3).15
Scheme 1. New synthetic strategy for the preparation of 3-substituted
6-anilinouracils.
a
b
R
R
NH
NH2
R
NH2
O
O
Cl
O
O
c
R
N
N
R
N
1-Benzyloxymethyl-6-chlorouracil 3 can be alkylated
with alkyl halides in DMF using cesium carbonate as
the base in good yields (Scheme 3).
+
O
N
H
O
N
H
Cl
O
N
H
O
Must be separated
by chromatography
Deprotection of derivatives 4a–f is achieved with non-
aqueous TFA under conditions that selectively hydro-
lyze the benzyloxymethyl group without affecting esters
and other hydrolytically sensitive groups. The resulting
3-substituted 6-chlorouracils 1a–f can conveniently be
isolated by crystallization. In the final step of the reac-
tion, the aniline moiety is introduced by nucleophilic
substitution (Scheme 3) as performed in the traditional
synthesis.
R´
O
H2N
R
N
R´
O
N
H
N
H
Scheme 2. Traditional synthesis of anilinouracils by chlorination of
barbituric acid derivatives. Reagents: (a) KOCN, HCl, H2O; (b)
Diethyl malonate, NaOEt, EtOH, reflux; (c) POCl3, heating.
By this synthetic methodology, a large number of struc-
turally diverse anilinouracils 2 were prepared, many of
which were previously synthetically inaccessible (repre-
sentative examples are shown in Table 1). Compound
In the first step the amine is converted to the corre-
sponding urea by treatment with aqueous hydrochloric
acid and potassium cyanate, and the resulting urea is
then cyclized to an N1-substituted barbituric acid using
either (a) a base-promoted cyclization employing diethyl
malonate and sodium ethoxide in refluxing ethanol,10 or
(b) an acid-promoted cyclization using malonic acid and
acetic anhydride in glacial acetic acid at 90 ꢁC.7,12 Both
reactions share certain disadvantages: they are incom-
patible with many functional groups, thus limiting the
scope of the reaction to derivatives insensitive to either
acid/acid anhydrides or bases. Additionally, the acidic
cyclization is often accompanied by the formation of
the corresponding 5-acetylated barbituric acid in signif-
icant amounts,13 thus lowering overall yields and com-
plicating isolation of the desired product. In the third
step of this synthetic sequence the barbituric acid is
transformed into the desired 3-alkyl-6-chlorouracil
using phosphoryl chloride at elevated temperature.7,10
In principle, two regioisomers can be formed in this
reaction (Scheme 2), but inherent selectivity often favors
the formation of the desired isomer. Again, conditions
are rather harsh, and we were concerned about the
safety issues relating to the very exothermic quenching
of the reaction mixtures resulting from these reactions,
especially on a larger scale. The final step consists of a
O
N
O
N
a
R
N
O
HN
O
Cl
O
Cl
O
4a-f
R´
3
O
b
R
N
H2N
O
N
H
Cl
1a-f
O
R
N
R´
O
N
H
N
H
2a-f
Scheme 3. Novel and improved synthesis of antibacterial anilinoura-
cils. Reagents: (a) RCH2X, Cs2CO3, DMF; (b) TFA, heating.

N. Svenstrup et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3215–3218
3217
Table 1. Synthesis of 3-alkylated 6-chlorouracils 1 and their corresponding 3-alkyl-6-anilinouracils 2
Entry
Starting material
Chlorouracil 1
Yield (%)a
Anilinouracil 2
Yield (%)
Br
Br
O
O
Br
N
N
1
94
88
68
78
51
37
49
O
O
Br
O
N
H
2a
N
H
O
N
H
Cl
O
1a
O
O
N
N
Br
2
3
4
5
6
73
96
68
78
67
O
O
N
H
Cl
O
N
H
N
H
1b
2b
O
O
O
N
N
O
Br
O
O
O
N
Cl
O
N
N
H
O
H
H
2c
1c
O
O
O
O
N
N
N
O
O
N
O
O
N
H
Cl
O
N
H
N
H
Cl
Cl
N
N
N
N O
O
O
2d
1d
O
O
O
O
O
N
O
O
N
O
O
N
H
N
H
O
N
H
Cl
O
1e
2e
O
O
HO
N
HO
N
HO
Br
O
N
H
N
H
O
N
H
Cl
1f
2f
a Overall yield for the alkylation and deprotection steps.
2f (Table 1, entry 6), a known DNA polymerase IIIC
inhibitor,10 was synthesized by the new method for the
purpose of comparison (the published synthesis involves
five synthetic steps).
The 3-substituent has a significant effect on the antibac-
terial potency of anilinouracils. Small substituents such
as substituted alkyl or heteroarylalkyl seem to be pre-
ferred. The antibacterial effect correlates well with target
activity (Table 2), except for compounds in which very
polar functionality prevents the compound from pas-
sively penetrating the bacterial cell wall and membrane
(sulfonic acids and certain amines and carboxylic acids).
The most potent compound of this series is 2b, which in
terms of antibacterial potency is equipotent or superior
to the known anilinouracil 2f against all tested
organisms.
Optimal yields are achieved with activated alkylating
agents such as alpha-bromo esters and ketones (Table
1, entries 1 and 3), or with halomethyl-heterocycles (Ta-
ble 1, entry 4), but the reaction is not limited to such
substrates. Non-activated alkylating agents also react
well, as demonstrated by the synthesis of 3-hydroxypro-
pyl-6-chlorouracil in 67% combined yield for the alkyl-
ation and deprotection (Table 1, entry 6); such
reactions generally require longer reaction times. The
general synthetic sequence, as illustrated by the synthesis
Still, despite good in vitro antibacterial properties, 2b
exhibited no efficacy in vivo at a dose of 100 mg/kg after
intraperitoneal application in a S. aureus lethal chal-
lenge model 30 min post infection with 106 CFU/mouse.
of
3-(cyclopropylmethyl)-6-(2,3-dihydro-1H-inden-5-
ylamino)uracil 2b, is included in the Reference section.16

3218
N. Svenstrup et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3215–3218
Table 2. DNA polymerase IIIC target activity and in vitro antibacterial activity against selected pathogens for compounds 2a–f
Compound
DNA Pol IIIC
MIC S. aureus
133b (lg/mL)
MIC S. pneumoniae
1707/4b,c (lg/mL)
MIC E. faecalis
ICB 27159b,c (lg/mL)
a
inhibition IC50 (lM)
2a
2b
2c
2d
2e
2f
>13.0
0.3
1.5
>256
4
>256
4
>256
16
64
16
32
32
8
16
16
16
8
1.2
2.0
32
32
0.3
16
a DNA Pol IIIC activity was assayed using Pol. IIIC from S. aureus (see Ref. 4).
b MIC values were determined by the broth microdilution method with an inoculum of 5 · 105 CFU/mL in BHI medium.
c Bovine serum (10%) was added to the medium, incubation was performed under microaerophilic conditions.
14. Zhi, C.; Long, Z.; Manikowski, A.; Brown, N. C.;
Tarantino, P. M., Jr.; Holk, K.; Dix, E. J.; Wright, G.
E.; Foster, K. A.; Butler, M. M.; LaMarr, W. A.; Skow,
D. J.; Motorina, I.; Lamothe, S.; Storer, R. J. Med. Chem.
2005, 48, 7063.
We speculate that poor pharmaceutical properties, espe-
cially poor aqueous solubility, are the primary reasons
for the lack of efficacy.17
References and notes
15. Edstrom, E. D.; Wei, Y. J. Org. Chem. 1994, 59,
6902.
16. 1-(Benzyloxy)methyl-6-chloro-3-cyclopropylmethyl-uracil
1. (a) Coates, A.; Hu, Y.; Bax, R.; Page, C. Nat. Rev. Drug
Disc. 2002, 1, 895; (b) Infectious Disease Society of
America (IDSA): http://www.idsociety.org.
2. (a) Wright, G. D. Curr. Opin. Chem. Biol. 2003, 7, 1; (b)
EARSS Annual Report 2006, http://www.rivm.nl.
3. von Nussbaum, F.; Brands, M.; Hinzen, B.; Weigand, S.;
Ha¨bich, D. Angew. Chem. 2006, 118, 5194; . Angew. Chem.
Int. Ed. 2006, 45, 5072.
4. Kuhl, A.; Svenstrup, N.; Ladel, C.; Otteneder, M.; Binas,
A.; Schiffer, G.; Brands, M.; Lampe, T.; Ziegelbauer, H.;
Haebich, D.; Ehlert, K. Antimicrob. Agents Chemother.
2005, 49, 987.
4b:
1-(benzyloxy)methyl-6-chlorouracil
3
(1.70 g,
6.4 mmol) was dissolved in dry DMF (15 mL) under
argon and cesium carbonate (4.15 g, 12.7 mmol) was
added. The resulting reaction mixture was stirred for
15 min, whereupon bromomethylcyclopropane (680 lL,
7.0 mmol) was added. The reaction mixture was stirred at
rt for 3 h, the solvent was removed in vacuo, and the crude
product was dissolved in DCM (150 mL) and washed with
brine (3· 50 mL). The organic phase was dried (Na2SO4)
and evaporated in vacuo to give the product 4b as a solid,
yield 1.76 g (86%). 6-Chloro-3-cyclopropylmethyluracil
5. Sun, H.; Zhi, C.; Wright, G. E.; Ubiali, D.; Pregnolato,
M.; Verri, A.; Focher, F.; Spadari, S. J. Med. Chem. 1999,
42, 2344.
6. Cowden, W. B.; Halladay, P. K.; Cunningham, R. B.;
Hunt, N. H.; Clark, I. A. J. Med. Chem. 1991, 34, 1818.
7. Muller, C. E.; Shi, D.; Manning, M., Jr.; Daly, J. W.
¨
1b: a suspension of 1-(benzyloxy)methyl-6-chloro-3-
cyclo-propyl-methyl-uracil 4 b (600 mg, 1.9 mmol) was
suspended in TFA (30 mL) and heated at reflux for
45 min. The reaction mixture was cooled to rt and
evaporated in vacuo, whereupon MeOH (10 mL) was
added and subsequently removed in vacuo. Ethylacetate
(5 mL) was added, and the sample was left overnight to
crystallize. The solid was filtered off, washed with ether
and dried in vacuo to give 1b as colorless crystals, yield
320 mg (85%). 3-Cyclopropylmethyl-6-(2,3-dihydro-1H-
J. Med. Chem. 1993, 36, 3341.
8. Focher, F.; Ubiali, D.; Pregnolato, M.; Zhi, C.; Gambino,
J.; Wright, G. E.; Spadari, S. J. Med. Chem. 2000, 43, 2601.
9. Tobe, M.; Isobe, Y.; Goto, Y.; Obara, F.; Tsuchiya, M.;
Matsui, J.; Hirota, K.; Hayashi, H. Biorg. Med. Chem.
2000, 8, 2037.
10. Tarantino, P. M., Jr.; Zhi, C.; Gambino, J. J.; Wright, G.
E.; Brown, N. C. J. Med. Chem. 1999, 42, 2035.
11. Wright, N. E.; Brown, N. C. Curr. Opin. Anti-Infect.
Investig. Drugs 1999, 1, 45.
inden-5-ylamino)uracil 2b:
a mixture of 6-Chloro-3-
(cyclo-propylmethyl)uracil 1b (120 mg, 0.6 mmol) and 5-
aminoindane (320 mg, 2.4 mmol) was heated under argon
at 160 ꢁC for 30 min. The reaction mixture was cooled to
rt, 2-propanol (5 mL) was added, and the resulting
precipitate was filtered off and dried to give a crude
product, which was purified by reverse-phase preparative
HPLC to give the product 2b as a solid, yield 123 mg
(68%).
12. Link, P. A.; van der Plas, H. C.; Muller, F. J. Heterocycl.
¨
Chem. 1985, 22, 873.
13. Jursic, B. S.; Neumann, D. M. Tetrahedon Lett. 2001, 42,
8435.
17. The aqueous solubility of 2b is 3.3 mg/L.