Quinazolin-2-ylamino-quinazolin-4-ols as novel non-nucleoside inhibitors of bacterial DNA polymerase III

Article abstract of DOI:10.1016/j.bmcl.2008.12.038

High throughput screening led to the discovery of a novel series of quinazolin-2-ylamino-quinazolin-4-ols as a new class of DNA polymerase III inhibitors. The inhibition of chromosomal DNA replication results in bacterial cell death. The synthesis, struct

Full text of DOI:10.1016/j.bmcl.2008.12.038

Bioorganic & Medicinal Chemistry Letters 19 (2009) 800–802
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Quinazolin-2-ylamino-quinazolin-4-ols as novel non-nucleoside inhibitors
of bacterial DNA polymerase III
a
a
a
a
a
Joseph Guiles a, , Xicheng Sun , Ian A. Critchley , Urs Ochsner , Ming Tregay , Kim Stone ,
Jennifer Bertino a, Louis Green a, Rob Sabin a, Frank Dean a, H. Garry Dallmann b,
Charles S. McHenry b, Nebojsa Janjic a
*
a Replidyne, Inc., 1450 Infinite Dr., Louisville, CO 80027, USA
b University of Colorado, Department of Chemistry and Biochemistry, Boulder, CO 80309, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
High throughput screening led to the discovery of a novel series of quinazolin-2-ylamino-quinazolin-4-
ols as a new class of DNA polymerase III inhibitors. The inhibition of chromosomal DNA replication
results in bacterial cell death. The synthesis, structure–activity relationships and functional activity are
described.
Received 14 August 2008
Revised 3 December 2008
Accepted 4 December 2008
Available online 13 December 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Bacterial
Polymerase
Inhibitors
Non-nucleoside
DNA
Bacterial infections caused by the rapid emergence of antibi-
otic-resistant strains have become a serious threat to public health
worldwide1 and have fostered the search for new antibiotic agents
directed against unexploited drug targets.2 A major pathogen of
concern is Staphylococcus aureus wherein both intermediate and
fully resistant isolates to vancomycin3 and methicillin (MRSA)4
have been identified. More recently, MRSA has been recognized
as a community-acquired pathogen causing disease in hosts with
few of the risk factors associated with nosocomial MRSA.5 Research
efforts have focused on identifying new agents to combat this sce-
nario. Bacterial DNA replication systems have been considered an
attractive target system due to their multitude of targets6 that
comprise an essential process7 and have a dissimilar homology
from eukaryotic counterparts.8 Seminal studies by Cozzarelli,
Wright and Brown first arose in the early 1970s that elucidated
the utility of DNA polIII and identified a class of anilinouracil-based
inhibitors.9 Subsequent studies of the 6-anilinouraclis (AUs) re-
vealed very good enzyme inhibition (Ki < 100 nm) and reasonable
activity against sensitive and drug-resistant strains of S. aureus
The known DNA polIII inhibitors reveal one general class (AU),
including citations in the patent literature12 and a recent citation
of anilinopyrimidinediones (APs)
a close regiosomer of AUs
(Fig. 1). The target having been validated in vivo presented an
opportunity from our perspective to improve upon the properties
of the AUs and address some of their intrinsic weaknesses (e.g.,
moderate whole cell activity, and low solubility). Our approach
was to search for new structurally divergent chemotypes using
high throughput screening replicative DNA polIII holoenzyme
screening systems as a lead finding tool. The holoenzyme screening
systems were configured from several pathogens including Esche-
richia coli, Yersinia pestis, S. aureus, and S. pyogenes. The screens
were configured in either 96 or 384 well format and detection of
duplex DNA formation via fluorescent readout was measured. Test
compounds were evaluated for their ability to prevent the forma-
tion of duplex DNA.6,13 The high throughput screening of our
OH
O
and Enterococci in culture (MIC = 2.5–5.0 l
g/mL).10 Proof of con-
R
O
R
O
N
N
cept in vivo studies were achieved with AUs in which several dif-
ferent compounds displayed the ability to rescue mice from a
lethal peritoneal cavity infection of S. aureus.10,11
N
H
N
N
H
N
H
H
AP
AU
* Corresponding author. Tel.: +1 303 817 7753.
E-mail address: joe.guiles@comcast.net (J. Guiles).
Figure 1. Examples of known DNA polIII inhibitors.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.038

J. Guiles et al. / Bioorg. Med. Chem. Lett. 19 (2009) 800–802
801
Table 1
compound collection identified a number of low molecular weight
molecules some of which were clustered into chemical series. One
of the chemical series, the quinazolin-2-ylamino-quinazolin-4-ols
(BisQuinols) represented by compounds 1–6, exhibited moderate
biochemical activity and whole cell activity against several of the
major Gram-positive pathogens such as Staphylococci, Strepto-
cocci, and Enterococci (Table 1). A nascent SAR was observed in
which electron donating methyl ethers in the 60 or 80 positions
were clearly superior in activity to a simple methyl group in the
same position. Substitution in the 60 or 80 position also appeared
to be superior to the 70 position, but 60–70 di-substitution appeared
uncompetitive (cooperative). The compounds exhibited a reason-
able correlation of biochemical activity and whole cell potency
and stimulated our further investigation in this series. In this letter,
we detail preliminary investigations of the in vitro and in vivo
characteristics of BisQuinols as a new non-uracil class of DNA polIII
inhibitors.
Representative profile of BisQuinols from HTS
OH
N
5'
6'
N
N
R
N
N
7'
H
8'
Compound
R
IC50 (S. pyo.)
MICb (S. aur.)
MICb (S. pneu.)
a
1
2
3
4
5
6
60-Me
60-OMe
70-OMe
209
4.8
32
0.5
32
4
>64
0.5
16
0.25
32
1
1
0.25
70.0
2.0
27.1
3.1
60,70-Di-MeO
60,70-Di-Me
80-OMe
a
lM.
b
Minimum inhibitory concentration
(l
g/mL) in S. aureus ATCC29213 and
Streptococcus pneumoniae ATCC 49619.14
The BisQuinols structure allows for a hypothetical H-bonding
network with a corresponding cytosine (dGTP analog) in a molecular
recognition similar to AUs. BisQuinols have 2H-bond donors and 1H-
bond acceptor to bind with cytosine on the DNA template. They fur-
ther contain an aromatic group to enable the hydrophobic interac-
tions attributed to the aniline group of AUs (Fig. 2). Two
selectively methylated BisQuinols were designed and synthesized
to test for applicability of the hypothetical H-bonding interaction.
The methylated compounds, 7 and 8, were anticipated to exhibit
lower affinity as a result of deleted H-bond donors. Both compounds
7 and 8 were found to be inactive in the polIII holoenzyme assay. This
is consistent with the proposed molecular recognition system but is
inconsistent with our biochemical investigation of these agents.
Several BisQuinols were investigated for their ability to compete
with nucleotide substrates in DNA synthesis. The BisQuinols were
found to be uncompetitive with nucleotide, whereas a representa-
tive AU compound was competitive to nucleotide in the same assay.
Additionally the BisQuinols were found to be competitive with DNA
and the AU compound was uncompetitive. The biochemical behav-
ior of the BisQuinols toward DNA template is similar in natureto that
of non-nucleoside reverse transcriptase inhibitors.15
BisQuinol
(keto)
AU
R
O
R
N
OMe
N
N
O
N
H
N
O
N
N
N
OMe
H
H
H
H
N
H
O
O
N
N
N
H
H
C
N
N
C
OMe
OMe
OMe
OMe
N
N
N
N
MeO
N
N
N
MeO
N
N
N
A convergent synthetic process was developed that facilitated
small library formats of BisQuinols for rapid SAR exploration.16
The synthetic process features condensation of appropriately
substituted isatoic anhydrides and quinazylguanidines (Scheme 1).
H
7
8
Figure 2. Molecular recognition of AUs and new BisQuinols.
O
HO
H2N
R1
R2
NH2
9
10
b
NH2
NH
a
HN
CN
O
O
R2
O
N
H
OH
12
NH2
N
R1
N
R2
N
R1
NH
N
N
H
N
c
N
N
H
13
11
Scheme 1. Reactions and conditions: (a) i—acetone, tert-butylcatechol, I2 MgSO4, reflux; ii—cyanoguanidine, H2O, HCl; (b) THF, triphosgene, rt; (c) DMF, DIEA, 100 °C.

802
J. Guiles et al. / Bioorg. Med. Chem. Lett. 19 (2009) 800–802
Table 2
SAR of the quinazole ring
As a means to assess the efficacy of this new series, com-
pound 17, one of the early and more potent compounds in the
series was studied in a simple murine model. Test animals are
inoculated in the intra-peritoneal cavity with the minimum
lethal dose (MLD) of the test organism. The compound is then
monitored for its ability to protect the mouse from a lethal
infection with the test organism in a concentration-dependent
manner. Compound 17 was effective in protecting 50% of test
subjects from a S. aureus IP infection when treated with a single
dose (25 mg/kg) 1 h post-infection.
In summary, we have discovered a series of quinazolin-2-ylami-
no-quinazolin-4-ols (BisQuinols) as a novel class of non-uracil DNA
polymerase III inhibitors. The series exhibited improved antibacte-
rial potency across the major Gram-positive pathogens including
drug-resistant strains of S. aureus (MRSA) versus the AU or AP
chemotypes reported in the literature. Preliminary SAR revealed
sensitivity to potency and protein binding in the quinazole bearing
ring. A representative example of the series demonstrated in vivo
efficacy in treatment of a lethal S. aureus infection in mice. The pre-
liminary set of compounds demonstrated poor selectivity over the
mammalian polIII enzyme and may warrant further SAR studies to
overcome this issue.
OH
N
8
7
R
6
MeO
N
N
N
N
5
H
Compound
R
IC50 (S. pyo.)
IC50 (Pold)
MICb (S. aur.)
a
a
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
5-OMe
6-F
>100
77.8
>100
3.7
na
1.9
na
2 (32)
64
na
1 (32)
2 (64)
64
1 (4)
1 (16)
1 (8)
2 (8)
6-Cl
6-Me
2.0
1.8
0.4
4.9
0.2
0.4
0.4
5.0
1.7
14.9
1.2
1.0
0.9
0.7
0.6
0.6
2.2
6-OMe
6-NO2
6-NH2
6-NHMe
6-NHEt
6-NMe2
6-NHAc
7-Me
3.8
>100
4.7
0.5
0.4
0.4
5.5
2.8
8.7
0.7
0.6
2 (64)
7-NO2
8-Me
2 (16)
2 (16)
1 (8)
0.25 (16)
1 (4)
8-OMe
8-Cl
6,8-Di-Me
6-NH2, 8-Me
6-NH2, 8-OMe
6,7,8-Tri-MeO
0.56
0.3
0.5
0.5
1.3
0.5 (16)
2 (64)
References and notes
a
l
M.
Minimum inhibitory concentration (
presence of 50% human serum).
1. Richet, H.; Mohammed, J.; McDonald, L. C.; Jarvis, W. R. Emerg. Infect. Dis. 2001,
b
lg/mL) in S. aureus ATCC29213 (MIC in the
7, 319.
2. Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Rosenberg, M. Methods Mol. Biol. 2004,
266, 231.
3. (a) Hiramatsu, K.; Hanaki, H.; Ino, T.; Yabuta, K.; Oguri, Y.; Tenover, F. C. J.
Antimicrob. Chemother. 1997, 40, 135;
Prevention. JAMA 2002, 288, 2116.
4. Michel, M.; Gutmann, L. Lancet 1997, 349, 1901.
5. (a) Enright, M. C. Curr. Opin. Pharmacol. 2003, 3, 474; (b) Herold, B. C. et al JAMA
1998, 279, 593; (c) Zetola, N. et al Lancet 2005, 5, 275.
6. (a) Braithwaite, D.; Ito, J. Nucleic Acids Res. 1993, 21, 787; (b) Seville, M.; West,
A. B.; Cull, M. G.; McHenry, C. S. Biotechniques 1996, 4, 664.
7. (a) Kornberg, A.; Baker, T. A. DNA Replication; W.J. Freeman: New York, 1992;
(b) Barnes, M. H.; Miller, S. D.; Brown, N. C. J. Bacteriol. 2002, 184, 1824.
8. Brown, N. C.; Dudycz, L. W.; Wright, G. E. Drugs Exp. Clin. Res. 1986, 12, 555.
9. (a) MacKenzie, J. M.; Neville, M. M.; Wright, G. E.; Brown, N. C. Proc. Natl. Acad.
Sci. U.S.A. 1973, 70, 512; (b) Gass, K. B.; Low, R. I.; Cozzarelli, N. R. Proc. Natl.
Acad. Sci. U.S.A. 1973, 70, 103; (c) Wright, G. E.; Brown, N. C. J. Med. Chem. 1974,
17, 1277; (d) Low, R. L.; Rashbaum, S. A.; Cozzarelli, N. R. Proc. Natl. Acad. Sci.
U.S.A. 1974, 71, 2973; (e) Clements, J. E.; D’Ambrosio, J.; Brown, N. C. J. Biol.
Chem. 1975, 250, 522; (f) Wright, G. E.; Baril, E. F.; Brown, N. C. Nucleic Acids Res.
1980, 8, 99; (g) Wright, G. E.; Brown, N. C. J. Med. Chem. 1977, 20, 1181; (h)
Coulte, C. L.; Cozzarelli, N. R. J. Mol. Biol. 1975, 91, 329.
b Centers for Disease Control and
In cases where the isatoic anhydride starting material was not
commercially available it was prepared in two standard steps from
the appropriately substituted nitrobenzoic acid. The quinazylgua-
nidine species were assembled from the condensation of an aniline
with acetone to form an intermediate 1,2-dihydroquinoline which
is isolated as its hydrochloride salt and then reacted with aqueous
cyanoguanidine to form species 11.17
The DNA polIII screening systems derived from two of major clin-
ical Gram-positive pathogens (S. aureus and S. pyogenes) along with
whole cell antibacterial activity (MIC) of the major clinical Gram-po-
sitive pathogens was used for SAR exploration. In addition, assays for
eukaryoticpolymerases (d and c) were utilized toassess the specific-
ity of inhibitors for the bacterial target system. Our preliminary lead
optimization effort focused on quinazolinol ring substitution effects.
Electron donating substitution in the 6 or 7 positions was favored
over electron withdrawing. Substitution in the 8 position was unaf-
fected by the nature of the electronic effect and resulted in improved
activity. Substitution in both the 6 and the 8 positions resulted in the
most potent compounds (Table 2).
10. Tarantino, P. M.; Zhi, C.; Wright, G. E.; Brown, N. C. Antimicrob. Agents
Chemother. 1999, 43, 1982.
11. Zhi, C.; Long, Z.-y.; Manikowski, A.; Brown, N. C.; Tarantino, P. M.; Holm, 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.
12. Ali, A.; Taylor, G. E. Expert Opin. Ther. Patents 2005, 15, 947.
13. Dean, F. B.; Gibson, K.; Sabin, R.; Bullard, J. M.; Dallmann, G. H.; Janjic, N.;
McHenry, C. S. 44th Annual Interscience Conference on Antimicrobial Agents and
Chemotherapy, Washington, DC, September 17–20, 2004; American Society for
Microbiology, 2004; F-1517, 223.
14. All organisms were tested by broth microdilution according to the Clinical
Laboratory Standards Institute (CLSI) guidelines.
15. (a) Balzarini, J. Curr. Top. Med. Chem. 2004, 4, 921; (b) McKercher, G.; Beaulieu,
P. L.; Lamarre, D.; LaPlante, S.; Lefebvre, S.; Pellerin, C.; Thauvette, L.; Kukolj, G.
Nucleic Acids Res. 2004, 32, 422.
The compounds demonstrated no selectivity over the mamma-
lian polymerases pold. The affinity for the other eukaryotic poly-
merases, pol
c
, was generally lower in the range of 2–10ꢀ (data
not shown). The MIC in the presence of human serum, an indirect
measure of the protein binding, was reasonably high. Substitution
with basic groups had a beneficial effect on protein binding. In par-
ticular, the least protein bound were the primary, secondary and
tertiary amine compounds 20, 22, 23, and 31, respectively.
16. All new compounds showed purity >95% by HPLC and the structure was
characterized by 1H NMR and HPLC–MS.
17. Webb, T. R.; Lvovskiy, D.; Kim, S.-A.; Ji, X.-d.; Melman, M.; Linden, J.; Jacobson,
K. A. Biorg. Med. Chem. 2003, 11, 77.