Biological evaluation of isothiazoloquinolones containing aromatic heterocycles at the 7-position: In vitro activity of a series of potent antibacterial agents that are effective against methicillin-resistant Staphylococcus aureus

Article abstract of DOI:10.1016/j.bmcl.2005.11.064

We synthesized a diverse series of 9H-isothiazolo[5,4-b]quinoline-3,4- diones containing heteroaromatic groups at the 7-position via palladium-catalyzed cross-coupling. Many of these compounds demonstrated potent antistaphylococcal activity (MICs ≤2 μg/mL) against a multi-drug-resistant strain (ATCC 700699) and low cytotoxic activity (CC₅₀ > 100 μM) against the human cell line Hep2 (laryngeal carcinoma).

Full text of DOI:10.1016/j.bmcl.2005.11.064

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1277–1281
Biological evaluation of isothiazoloquinolones containing aromatic
heterocycles at the 7-position: In vitro activity of a series of
potent antibacterial agents that are effective against
methicillin-resistant Staphylococcus aureus
Jason A. Wiles,* Yongsheng Song, Qiuping Wang, Edlaine Lucien, Akihiro Hashimoto,
Jijun Cheng, Christopher W. Marlor, Yangsi Ou, Steven D. Podos, Jane A. Thanassi,
Christy L. Thoma, Milind Deshpande, Michael J. Pucci and Barton J. Bradbury*
Achillion Pharmaceuticals, Inc., 300 George Street, New Haven, CT 06511-6653, USA
Received 27 October 2005; accepted 18 November 2005
Available online 7 December 2005
Abstract—We synthesized a diverse series of 9H-isothiazolo[5,4-b]quinoline-3,4-diones containing heteroaromatic groups at the
7-position via palladium-catalyzed cross-coupling. Many of these compounds demonstrated potent antistaphylococcal activity
(MICs 62 lg/mL) against a multi-drug-resistant strain (ATCC 700699) and low cytotoxic activity (CC50 > 100 lM) against the
human cell line Hep2 (laryngeal carcinoma).
Ó 2005 Elsevier Ltd. All rights reserved.
Isothiazoloquinolones (ITQs) are an under-explored
subclass of fluoroquinolones that were investigated ini-
tially by Chu and coworkers at Abbott.1–3 ITQs repre-
sent one of the few examples of successful replacement
of the archetypal 3-carboxylic acid of fluoroquinolones
(i.e., with a ring-fused isothiazolone, Fig. 1) without
compromising antibacterial activity.4 Compound A-
62824, for example, was reported as ꢀ10-fold more po-
tent in vitro than the corresponding fluoroquinolone
ciprofloxacin.5,6 The ITQs reported by Abbott were lim-
ited to analogues that contained amino substituents at
C-7 attached directly via a C–N bond (nitrogen cou-
pled).7 In addition to inhibiting bacterial topoisomerase
II, these potent antibacterial agents unfortunately inhib-
ited mammalian topoisomerase II8 causing topoisomer-
ase II-mediated DNA breakage, which correlated with
mammalian cellular toxicity.9 We reported recently,10
however, that several carbon-coupled ITQs (1,
Fig. 1)—having functionalized phenyl groups at C-7—
showed reduced cytotoxicity in a human cell line and
O
O
N
O
3
O
5
8
4
F
6
F
NH
NH
7
R
2
S
S
N
N9
1
HN
A-62824
1
O
4
O
O
O
5
8
F
F
6
OH
OH
3
7
2
N
N1
N
N
HN
HO
ciprofloxacin
nadifloxacin
Figure 1. Examples and numbering schemes of known isothiazoloqu-
inolones (top) and fluoroquinolones (bottom).
increased activity against methicillin-resistant Staphylo-
coccus aureus (MRSA) when compared with A-62824
and contemporary fluoroquinolones (e.g., ciprofloxa-
cin). Although analogues of 1 demonstrated potent in vi-
tro activity against MRSA,10 they may not necessarily
perform well in vivo because most—in contrast with
all marketed fluoroquinolones, except the topically
administered nadifloxacin (Fig. 1)—lack the distal
Keywords: Antibacterial; Methicillin-resistant Staphylococcus aureus;
Quinolone.
*
Corresponding authors. Tel.: +1 203 624 7000; fax: +1 203 752 5454
(J.A.W.); e-mail addresses: jwiles@achillion.com; bbradbury@
achillion.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.064

1278
J. A. Wiles et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1277–1281
amine of the amino C-7 substituent that generally af-
fords good in vivo efficacy.11 We believe that substitu-
tion of non-amino functionalized phenyl groups with
nitrogen-containing heteroaromatic groups at C-7 of 1
may address this potential liability. We herein report
the in vitro biological evaluation of such heteroaryl ana-
logues in an effort to fully explore carbon-coupled ITQs
as potential antistaphylococcal agents.
O
O
O
a
Cl
Cl
HO
EtO
O
N
N
45
HO
46
TBDMSO
b
c
Cl
Cl
N
N
48
47
We introduced carbon-coupled groups to C-7 of the
ITQ core via palladium-catalyzed (Suzuki–Miyaura
and Stille) cross-coupling of bromide 210 with organobo-
ron and organotin compounds (Scheme 1). The heteroa-
romatic stannanes, boronic acids, and dioxaborolanes
used in this study were either (a) purchased from com-
mercial suppliers, (b) prepared from the corresponding
known12 or commercially available bromides using stan-
dard methods,13,14 or (c) prepared as shown in Schemes
215 and 3.
O
d
B
O
TBDMSO
N
49
Scheme 3. Reagents and conditions: (a) EtOCOCl (1.05 equiv), Et3N
(1.05 equiv), C6H6, rt, 1 h; (b) LAH (1.05 equiv), THF, ꢁ78 °C, 0.5 h,
66% (2 steps); (c) TBDMSCl (1.1 equiv), imidazole (5 equiv), DMF, rt,
15 h, 96%; (d) bis(pinacolato)diboron (1.1 equiv), 15 mol % Pd(OAc)2,
30 mol % 1,3-bis(2,6-diisopropylphenyl)-4,5-dihydroimidazolium chlo-
ride, KOAc (2.5 equiv), THF, reflux, 15 h, used directly in subsequent
Suzuki–Miyaura coupling.
Analogues 3–40 (Fig. 2) were tested against Gram-neg-
ative (Escherichia coli) and Gram-positive (S. aureus)
organisms (Table 1) using standard techniques.16 These
minimum inhibitory concentration (MIC) values were
compared with those of ciprofloxacin (CIP) and moxi-
floxacin (MFX). Although the activities of several ITQs
against Gram-negative bacteria were inferior to those of
contemporary quinolones (CIP and MFX), several ana-
logues displayed activities that were superior against
methicillin-sensitive S. aureus (MSSA, MICs <0.02 lg/
mL) and multi-drug-resistant MRSA (MICs 62 lg/
mL). Strong activities against MSSA tracked well with
the corresponding activities against MRSA. The most
active compound tested against MRSA was 21, with
an MIC of 0.25 lg/mL. Analogues containing represen-
tative 3-pyridinyl, 4-pyridinyl, 5-pyrimidinyl, and
5-quinolinyl groups at C-7 were tested against a panel
of organisms (Table 2), the results of which confirmed
the broad antibacterial coverage of ITQs against many
Gram-positive (e.g., S. aureus and S. pneumoniae) and
specific Gram-negative (i.e., H. influenzae and M.
catarrhalis) pathogens. With these results in mind, we
optimized (a) the potency of ITQs against a clinically
important, multi-drug-resistant strain of S. aureus and
(b) the restricted cytotoxic activity of ITQs against a
mammalian cell line (Hep2).
O
O
O
O
F
F
a or b
NH
NH
S
S
Br
N
R
N
3−40
2
R = heteroaryl
Scheme 1. Reagents and conditions: (a) RB(ORÕ)2 where R = hetero-
aryl and R0 = H or –C(CH3)2– (3–4 equiv), NaHCO3 (10 equiv),
Pd(PPh3)4 (5–10 mol %), DMF/H2O, MWI (130 °C), 10–20 min, 30–
50% yield after HPLC purification; (b) RSnBu3 where R = heteroaryl
(2 equiv), Pd(PPh3)4 (5 mol %), DMF, MWI (130 °C), 20 min, 35%
yield after HPLC purification.
For monocyclic heterocycles, the antibacterial activity
(Table 1) decreased generally in the order 4-pyridi-
nyl < 3-pyridinyl < 5-pyrimidinyl < 2-(1H-pyrrolyl) < 2-
pyridinyl < 2-pyrazinyl. Typically, analogues containing
amino, hydroxy, and methoxy substituents were less po-
tent against MRSA than their parent (unsubstituted)
analogues, which were, in turn, less potent than those
containing methyl and fluorine substituents. Addition
of a single methoxy group to 3-pyridines and 5-pyrimi-
dines did not improve their antibacterial activity against
MRSA (24, 25, and 27); however, addition of another
methoxy group ortho to the biaryl linkage effected im-
proved activity (23 and 28). Installation of methyl sub-
stituents at the C-2/C-6 positions of 3- and 4-pyridinyl
groups generally afforded potent ITQs. There was no
apparent antibacterial advantage in adding fluorine sub-
stituents to the 3- and 4-pyridinyl groups (e.g., 13 and
14); however, the resulting ITQs maintained low cyto-
toxic activity comparable with their parent compounds.
In contrast, addition of methyl groups to the C-2/C-6
positions (e.g., 10 and 11) and methoxy groups to the
O
Br
Br
B
a
b
O
O
N
C
N
C
N
N
N
41
42
43
O
B
c
O
H2N
N
44
Scheme 2. Reagents and conditions: (a) I2 (1.1 equiv), NH4OH (28%
in H2O, excess), THF, rt, 4 h, quant.; (b) bis(pinacolato)diboron
(1.1 equiv), 10 mol % PdCl2(dppf), KOAc (3.4 equiv), DMSO, 80 °C,
25 h, 64%; (c) H2 (1 atm), 10% Pd/C, HOAc, rt, 16 h, quant.

J. A. Wiles et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1277–1281
1279
N
N
N
N
N
N
3
4
5
6
7
N
N
F
N
N
8
9
10
11
F
N
N
F
N
12
13
14
N
H
N
H
HO
N
N
H2N
N
N
N
N
N
15
16
17
18
19
20
N
N
N
MeO
MeO
N
OMe
21
22
23
OMe
MeO
N
N
N
MeO
NH
N
N
MeO
N
OMe
24
25
26
27
28
NH
29
N
H
N
30
31
32
N
N
N
N
33
34
35
N
N
N
N
N
36
37
38
39
40
Figure 2. Heteroaryl groups and numbering of ITQs used in this study.
C-3/C-4 positions (24 and 25) of the heterocyclic substit-
uents usually increased the cytotoxic activity. Further
addition of a methyl group (22) or modification of the
existing methyl group (15 and 20) usually caused re-
duced cytotoxicity at the expense of antibacterial activ-
ity against MRSA.
with the least sterically crowded biaryl linkage (cf. 33,
35, and 36 with 37, 39, and 40).
Several ITQs demonstrated strong inhibition of wild-
type S. aureus topoisomerase IV that corresponded to
strong antibacterial activity against susceptible S. aure-
us. We note, however, the poor correlation of in vitro
enzymatic inhibition and MICs of ITQs. An obvious
example is compound 21, which showed a 64-fold in-
crease in potency against S. aureus (when compared
with ciprofloxacin) without a concurrent improvement
in inhibition of topoisomerase IV. This poor correlation
Of the limited number of bicyclic heterocycles that we
investigated, 6-isoquinolinyl (34) and 6-quinolinyl (35)
groups exhibited the best antibacterial activity, having
MICs of 1 and 0.5 lg/mL, respectively, against MRSA.
We also observed low cytotoxicity, generally, for ITQs

1280
J. A. Wiles et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1277–1281
Table 1. In vitro antibacterial and cytotoxic activities of ITQsa
Compound
R Group
Substitution on
R group
MICs (lg/mL)
Sa
MRSA (MRQRVI)
Topo IV (WT Sa)
Cytotox (72 h)
IC50 (lM)
CC50 (lM)
Ec
CIP
MFX
3
0.02
0.015
0.060
0.028
0.0035
0.12
1.0
0.25
32
1.0
0.8
6.3
2.2
0.5
3.3
64
>100
>100
>100
ND
>100
18
0.06
2.0
16
Phenyl
None
0.0035
0.0035
0.0071
0.025
0.12
4
4-Pyridinyl
3-Pyridinyl
5-Pyrimidinyl
2-pyrazinyl
2-Pyridinyl
2-Pyridinyl
3-Pyridinyl
3-Pyridinyl
3-Pyridinyl
3-Pyridinyl
4-Pyridinyl
3-Pyridinyl
3-Pyridinyl
3-Pyridinyl
3-Pyridinyl
4-Pyridinyl
4-Pyridinyl
4-Pyridinyl
3-Pyridinyl
3-Pyridinyl
3-Pyridinyl
3-Pyridinyl
3-Pyridinyl
5-Pyrimidinyl
5-Pyrimidinyl
2-(1H-Pyrrolyl)
2-(1H-Indolyl)
5-(1H-Indolyl)
5-(1H-Indolyl)
7-Quinazolinyl
6-Isoquinolinyl
6-Quinolinyl
3-Quinolinyl
4-Isoquinolinyl
5-Quinolinyl
5-Isoquinolinyl
8-Quinolinyl
None
1.0
2.0
8.0
>64
32
5
None
6
7b
None
None
>91
>100
51
8
4-Me
0.25
0.12
0.51
0.037
0.015
0.030
0.059
0.059
1.0
0.06
16
9
5-Me
0.044
0.13
32
14
10c
11c
12
13
14
15d
16e
17e
18e
19
20f
21
22c
23
24
25
26
27
28
29
30
31
32
33
34c
35
36
37
38
39e
40
2-Me
1.0
1.0
ND
2.0
1.0
32
3.5
8.5
2.4
12
12
6-Me
0.0037
0.015
0.015
0.0074
0.13
31
2-F
>100
>100
>100
>100
>100
6
6-F
2-F
19
6-H2NCH2
6-(1-Pyrrolyl)
5-(2-Piperidinyl)
5-(2-Pyrrolidinyl)
2-Me
0. 7
18
0.029
0.12
4.0
8.0
8.0
0.50
32
0.46
0.12
0.059
0.14
0.031
0.5
5.0
1.1
26
0.06
25
0.0040
0.034
0.0038
0.13
36
2-HOCH2
2,6-Me2
2,6-Me2
2,6-(MeO)2
4-MeO
5-MeO
6-MeO
2-MeO
2,4-(MeO)2
None
4.8
48
>100
50
0.25
>64
8.0
4.0
4.0
16
8.0
16
>100
98
2.0
0.046
0.023
0.03
0.25
0.061
0.015
0.13
0.25
1.0
10
50
21
34
0.023
0.015
0.019
0.06
26
>100
43
8.0
1.0
16
3.3
42
>100
23
9.9
3.3
>40
3.6
29
None
1.0
0.06
4.0
2.0
8.0
>64
0.50
1.0
8.0
8.0
2.0
>64
16
9
None
0.059
1.0
0.004
0.065
0.06
>82
>100
>100
22
1-Me
None
4.0
None
0.13
0.13
0.13
0.48
0.071
0.061
0.75
0.0020
0.0052
0.011
0.028
0.016
0.016
0.13
3.6
3.2
50
None
>100
>100
51
None
None
>38
33
None
ND
45
None
23
None
>40
79
a Abbreviations: CIP, ciprofloxacin; Ec, Escherichia coli ATCC 25922; MFX, moxifloxacin; MIC, minimum inhibitory concentration; MRQRVI,
methicillin- and quinolone-resistant, vancomycin intermediate-resistant; MRSA, methicillin-resistant Staphylococcus aureus ATCC 700699; ND,
not determined; Sa, Staphylococcus aureus ATCC 29213; WT, wild-type.
b Prepared from the commercially available tributylstannane.
c Prepared from the custom-made boronic acid or dioxaborolane derived from the previously described bromide (see Ref. 12).
d Prepared as shown in Scheme 1.
e Prepared from the custom-made boronic acid or dioxaborolane derived from the commercially available bromide.
f Prepared as shown in Scheme 3.
Table 2. In vitro antibacterial activity of selected ITQsa
Compound
MIC (lg/mL)
Gram-positive organisms
Gram-negative organisms
Sa
Efm
Efs
Spy
Spn
Hi
Mc
Sm
St
Kp
Pa
Ec
CIP
MFX
13
0.22
2
0.56
0.28
0.56
0.018
0.013
0.03
0.033
0.28
0.018
0.061
0.125
0.125
0.125
0.5
0.033
0.031
0.125
0.125
0.5
0.25
1
0.015
0.013
0.03
0.061
0.015
0.004
0.021
0.016
0.125
0.11
0.25
0.17
0.25
0.125
0.125
0.125
0.058
0.125
0.061
0.059
0.04
0.125
0.125
0.029
0.079
0.061
0.061
0.031
0.0037
0.0037
0.021
1
1
2
4
0.5
2
19
0.015
0.14
0.059
0.14
28
0.079
0.25
>64
2
38
0.028
0.0081
0.5
0.016
a Abbreviations: CIP, ciprofloxacin; Ec, Escherichia coli ATCC 25922; Efm, Enterococcus faecium ATCC49032; Efs, Enterococcus faecalis ATCC
29212; Hi, Haemophilus influenzae type b ACH-0056; Kp, Klebsiella pneumoniae ATCC 13883; Mc, Moraxella catarrhalis ATCC 8176; MFX,
moxifloxacin; MIC, minimum inhibitory concentration; ND, not determined; Pa, Pseudomonas aeruginosa ATCC 27853; Sa, Staphylococcus aureus
ATCC 29213; Sm, Stenotrophomonas maltophilia ATCC 13637; Spn, Streptococcus pneumoniae ATCC 49619; Spy, Streptococcus pyogenes ATCC
19615; St, Salmonella typhimurium ATCC 14028.

J. A. Wiles et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1277–1281
1281
9. Chu, D. T.-W.; Plattner, J. J.; Shen, L. L.; Klein, L. L.
U.S. Patent 5,071,848, 1991.
is likely a reflection of other contributing variables such
as efflux and the presence of other targets (e.g., topoiso-
merase II).
10. Wiles, J. A.; Wang, Q.; Lucien, E.; Hashimoto, A.; Song,
Y.; Cheng, J.; Marlor, C. W.; Ou, Y.; Podos, S. D.;
Thanassi, J. A.; Thoma, C. L.; Deshpande, M.; Pucci, M.
J.; Bradbury, B. J. Bioorg. Med. Chem. Lett., 2006, 16,
1272.
11. Culbertson, T. P.; Domagala, J. M.; Hagen, S. E.; Hutt,
M. P.; Nichols, J. B.; Mich, T. F.; Sanchez, J. P.;
Schroeder, M. C.; Solomon, M.; Worth, D. F. In
Quinolones. Proceedings of an International Telesympo-
sium; Fernandes, P. B., Ed.; Prous Science Publishers:
Barcelona, Spain, 1989.
12. Procedures to prepare these bromides are cited below with
their corresponding ITQ analogues. 10 and 11: (a)
Pearson, D. E.; Hargrove, W. W.; Chow, J. K. T.;
Suthers, B. R. J. Org. Chem. 1961, 26, 789; 21: (b) Takalo,
H.; Pasanen, P.; Kankare, J. Acta Chem. Scand. 1988,
42B, 373; 22: (c) Talik, T.; Talik, Z.; Ban-Oganowska, H.
Synthesis 1974, 293; 34: (d) Tyson, F. T. J. Am. Chem.
Soc. 1939, 61, 183.
To summarize, we have described novel carbon-coupled
ITQs adorned by heteroaromatic groups at C-7. Several
analogues displayed potent antistaphylococcal activities
(MICs 62 lg/mL against MRSA) that are superior to
contemporary quinolones and showed low cytotoxic
activity against human cells (CC50 > 100 lM). We antic-
ipate that further modification of the heteroaromatic
substituents at C-7 and the ITQ nucleus at C-8, will,
in general, lower the cytotoxicity and increase the anti-
bacterial activity of this class of compounds. Such stud-
ies are now underway in our laboratories.
References and notes
13. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem.
1995, 60, 7508.
1. Chu, D. T. U.S. Patent 4,687,770, 1987.
2. Chu, D. T. U.S. Patent 4,689,325, 1987.
14. Parry, P. R.; Wang, C.; Batsanov, A. S.; Bryce, M. R.;
Tarbit, B. J. Org. Chem. 2002, 67, 7541.
15. Aldehyde 41 was prepared as described previously. See:
Wang, X.; Rabbat, P.; OÕShea, P.; Tillyer, R.; Grabow-
ski, E. J. J.; Reider, P. J. Tetrahedron Lett. 2000, 41,
4335.
16. MICs were determined as described by the NCCLS (see
National Committee for Clinical Laboratory Standards.
2001. Performance standards for antimicrobial suscepti-
bility testing: 11th informational supplement. Vol. 21, no.
1, M100-S11. National Committee for Clinical Laboratory
Standards, Wayne, PA). The MIC was defined as the
lowest concentration of each compound resulting in
inhibition of visible growth of bacteria after incubation
at 37 °C for 18–24 h.
3. Chu, D. T. W.; Fernandes, P. B.; Claiborne, A. K.; Shen,
L.; Pernet, A. G. In Quinolones. Proceedings of an
International Telesymposium; Fernandes, P. B., Ed.;
Prous Science Publishers: Barcelona, Spain, 1989.
4. Domagala, J. M.; Hagen, S. E. In Quinolone Antimicrobial
Agents, 3rd ed.; Hooper, D. C., Rubenstein, R., Eds.;
ASM: Washington, DC, 2003; pp 3–18.
5. Chu, D. T. W.; Fernandes, P. B.; Claiborne, A. K.; Shen,
L.; Pernet, A. G. Drugs Exp. Clin. Res. 1988, 14, 379.
6. Chu, D. T. W.; Lico, I. M.; Claiborne, A. K.; Plattner, J.
J.; Pernet, A. G. Drugs Exp. Clin. Res. 1990, 16, 215.
7. Chu, D. T. U.S. Patent 4,767,762, 1988.
8. Kohlbrenner, W. E.; Wideburg, N.; Weigl, D.; Saldivar,
A.; Chu, D. T. W. Antimicrob. Agents Chemother. 1992,
36, 81.