7-(4-Alkylidenylpiperidinyl)-quinolone bacterial topoisomerase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.10.014

Novel antibacterial fluoroquinolone agents bearing a 4-alkylidenylpiperidine 7-position substituent are active against quinolone-susceptible and quinolone-resistant gram-positive bacteria, including Streptococcus pneumoniae and MRSA. Analogs 22b, 23c, and 24 demonstrated superior in vitro and in vivo efficacy to ciprofloxacin against these cocci.

Full text of DOI:10.1016/j.bmcl.2014.10.014

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
7-(4-Alkylidenylpiperidinyl)-quinolone bacterial topoisomerase
inhibitors q
⇑
Eugene B. Grant , Barbara D. Foleno, Raul Goldschmidt, Jamese J. Hilliard, Shu-Chen Lin, Brian Morrow,
Steven D. Paget, Michele A. Weidner-Wells, Xiaodong Xu, Xiaoqing Xu, William V. Murray, Karen Bush,
Mark J. Macielag
Janssen Research & Development, L.L.C., Welsh & McKean Roads, Spring House, PA 19477, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel antibacterial fluoroquinolone agents bearing a 4-alkylidenylpiperidine 7-position substituent are
active against quinolone-susceptible and quinolone-resistant gram-positive bacteria, including
Streptococcus pneumoniae and MRSA. Analogs 22b, 23c, and 24 demonstrated superior in vitro and
in vivo efficacy to ciprofloxacin against these cocci.
Received 13 August 2014
Revised 26 September 2014
Accepted 1 October 2014
Available online xxxx
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Antibacterial agent
Fluoroquinolone-resistant
Alkylidenylpiperidinyl fragment
Since the discovery of nalidixic acid1 over four decades ago, the
quinolones have developed into a clinically useful class of antibac-
terial agents.2 Several of the quinolones, including ciprofloxacin3
and levofloxacin4 are widely used in the clinic to treat a range of
bacterial infections. The quinolones act against bacteria by
selectively inhibiting the type II topoisomerases DNA gyrase and
topoisomerase IV, enzymes that play a critical role in bacterial cell
growth and division.2c However, extensive clinical use of these
agents has led to increasing bacterial resistance to the available
quinolones.2d The incidence of quinolone resistance in the
important respiratory tract pathogen, Streptococcus pneumoniae,
has generally remained low both in the United States5 and
globally.6 However, recent reports of elevated resistance among
pneumococcal isolates in regions with high quinolone usage signal
the potential for further dissemination of resistant strains.7,8 In
contrast, the high rate of quinolone resistance among methicillin-
resistant Staphylococcus aureus (MRSA) isolates has limited the
empiric use of marketed quinolones in the treatment of skin and
skin structure infections.9,10 Thus a continuing challenge is to
discover and develop newer quinolone antibacterial agents with
activity against resistant organisms, including gram-positive
bacteria associated with respiratory tract and skin infections.
Previous studies have demonstrated the importance of the
quinolone C7 substituent for potent in vitro antibacterial activity,
including against resistant gram-positive organisms.11 In particular,
the (3S)-amino-(4R)-ethylpiperidinyl fluoroquinolone 1 was shown
to possess superior activity against MRSA and penicillin-resistant S.
pneumoniae compared to marketed quinolones (Fig. 1).11
Modeling investigations of the C7 substituent of 1 revealed a
low energy conformation that overlapped with the aminoethyl-
pyrrolidine side chain of premafloxacin (2), another quinolone
antibacterial agent with potent gram-positive activity.12 We
hypothesized that the 2-aminoethylidenylpiperidine substituent
5, derived from fragment 4 by shifting the primary amine to the
terminus of the ethyl group and restricting the side chain with
an exocyclic double bond, would position the amino and alkyl
groups in a similar region of three-dimensional space as the
aminoethylpyrrolidine side chain (6) of premafloxacin (Fig. 1). To
test this concept, we launched an investigation of the synthesis
and antibacterial activity of quinolones and naphthyridones bear-
ing this unprecedented 7-position substituent (3).
The general synthetic route to the protected 4-(2-aminoethy-
lidenyl)piperidines is depicted in Scheme 1.13 Ketone 7 was
subjected to a Horner–Wadsworth–Emmons reaction with triethyl
2-substituted-2-phosphonoacetate
8
to afford substituted
alkylidene ester 9. Reduction to alcohol 10, Mitsunobu reaction
with phthalimide to give 11, and acid deprotection provided
secondary amine 12. Allylic alcohol 13 was converted to the
corresponding amine derivatives 15a, 15b and 15c through a
two-step sequence (Scheme 2).
q
All authors were employed by Janssen Research & Development, L.L.C. at the
Heterocyclic core structures related to marketed and clinical
quinolones and naphthyridones were employed in the synthesis
time the work reported herein was conducted.
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2014.10.014
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Grant, E. B.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.10.014

2
E.B. Grant et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
O
O
N
O
CO2H
CO2H
CO2H
F
F
F
7
7
H2N
N
N
N
N
A
N
R1
R4
OMe
N
OMe
HN
R2
3
1
2
N
A = CH, N, COMe
R4=
R3
R1
1) Shift
amino group
N
A / R4=
2) Restrict
Rotation
H2N
NH2
O
4
5
NH
NH
NH
6
5
R1
NH2
Figure 1. Chemical structures of quinolone antibacterial agents described in the present study.
R1
R1
R1 CO2Et
O
OH
EtO2C
P(O)(OEt)2
8 (R1 = H, F, Cl, CH3)
b
N
N
Boc
10
a
N
Boc
Boc
9
7
O
O
R1
R1
N
N
c
d
O
O
N
Boc
N
H
11
12
Scheme 1. Reagents and conditions: (a) 8 (R1 = H, F, Cl, CH3) NaH, THF or 8 (R1 = F, Cl) K2CO3, EtOH, rt; (b) DIBAL, THF, À78 °C ? 10 °C; (c) PPh3, DIAD,THF, phthalimide; (d) (i)
CF3CO2H, CH2Cl2 and (ii) 10% aq Na2CO3.
of the desired final products (Fig. 2, 16a–f). Naphthyridone deriva-
tives (17a–d) were synthesized by direct condensation of amine 12
with chloronaphthyridine 16a followed by deprotection with
hydrazine (Scheme 3). In contrast, coupling of the amines to the
quinolone core structures (16b–f) demanded activation of the
heterocyclic carboxylic acids with boron trifluoride to yield
the difluoroborate ester14 or with boric acid, acetic anhydride,
and zinc chloride to form the diacetylborate ester (18b–f).13 After
coupling, alcoholysis of the difluoroborate ester was achieved by
refluxing in ethanolic triethylamine, whereas hydrolysis of the
diacetylborate ester was accomplished with 10% hydrochloric
acid in THF. Removal of the phthalimide protecting group under
standard conditions provided the final products 19a–c, 20a–b,
21, 22a–b, 23a–c after purification. Further elaboration of 23c to
the corresponding secondary amine 24 was achieved by protecting
the primary amine of 23c with di-tert-butyl dicarbonate, alkylation
of the intermediate carbamate, and removal of the Boc group with
trifluoroacetic acid.
Analogs 25a, 25b and 25c were synthesized by direct condensa-
tion of amine 15a–c with activated heterocycle 18f followed by
hydrolysis. Quinolone 26 was prepared by debenzylation of 25c
with 1-chloroethyl chloroformate (Scheme 4).
In vitro antibacterial activity was assessed initially against an
abbreviated panel of gram-positive bacteria, which included two
ciprofloxacin-susceptible strains (Staphylococcus aureus OC4172
and Streptococcus pneumoniae ATCC 49619) as well as
three ciprofloxacin-resistant pneumococcal clinical isolates (S.
pneumoniae OC6608, OC6578, and OC5465). Data for representative
Cl
Cl
Cl
R2
R3
OH
Cl
N
a
b
R2 = R3 = Me
15a
N
Boc
N
Boc
N
H
2 = H R3 = iPr 15b
13
14
R
R2 = Et R3 = Bn
15c
Scheme 2. Reagents and conditions: (a) MsCl, Et3N, CH2Cl2; (b) Et3N, CH3CN, R2R3NH (iii) CF3CO2H, CH2Cl2.
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E.B. Grant et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
O
O
O
O
F
CO2H F
Cl
CO2H
F
F
CO2H
F
F
CO2H
Cl
N
N
N
N
N
O
O
16a
16b
16c
16d
O
O
F
CO2H
F
CO2H
F
F
N
F
N
O
MeO
F
16e
16f
Figure 2. Chemical structures of the heterocyclic nuclei employed in the synthesis of 7-(4-alkylidenyl)piperidinyl quinolones and naphthyridones.
O
F
CO2H
O
N
O
Cl
N
N
CO2H
F
R1
N
16a
NH2
R1
N
N
O
a, b
N
H
12
R1= H 17a
R1= F 17b
R1= CH3
R1= Cl
17c
17d
O
N
F
CO2BX2
O
O
halogen
A
CO2H
F
R4 18b-f
R1
N
NH2
R1
N
A
N
O
c, b
R4
12
N
H
R1= F
A / R4=
R1= F
A / R4= R1= Cl
21
20a
20b
A = CH
R4
19a
19b
19c
R1= CH3
R1= Cl
R1= CH3
=
O
O
R1= F
R1= F
A = COMe
R4
23a
A = COCHF2
R4 =
22a
22b
R1= CH3
R1= Cl
23b
23c
R1= Cl
=
O
N
CO2H
F
Me
N
NH
Cl
23c
MeO
24
d
O
F
CO2BX2
O
N
R2
R3
CO2H
F
F
N
c
Cl
N
MeO
18f
R3 R2
N
N
MeO
N
H
Cl
R2=H R3 = iPr
R2=H R3 = iPr
15a
15b
25a
25b
R2= R3 = Me
R2= R3= Me
R2= Bn R3 = Et 15c
R2= Bn R3= Et 25c
Scheme 3. Reagents and conditions: (a) 16a, Et3N, CH3CN, reflux; (b) NH2NH2, MeOH, reflux; (c) (i) 18b–18f, Et3N, CH3CN, reflux, (ii) X = F, Et3N,EtOH, reflux or X = OAc, THF,
10%HCl; (d) (i) Boc2O, Et3N, CH2Cl2; (ii) MeI, NaH, DMF; (iii) NaOH, MeOH; (iv) CF3CO2H, CH2Cl2.
compounds and ciprofloxacin are presented in Table 1 as the min-
imal inhibitory concentration (MIC; lowest concentration of com-
pound inhibiting visible growth).15
In the naphthyridone subseries, the analog with an unsubstitut-
ed alkylidenylpiperidine 7-position substituent (17a, R1 = H) had
comparable microbiological activity to ciprofloxacin against
Please cite this article in press as: Grant, E. B.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.10.014

4
E.B. Grant et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
series provided analogs with similar to each other in vitro antibac-
O
N
F
terial profiles, including against ciprofloxacin-resistant S.
pnuemoniae. Consistent with observations from earlier studies,5,12
the 8-methoxyquinolone core structure conferred potent
gram-positive activity (23a–c). Specifically, the combination of an
8-methoxy group and a chloroalkylidenylpiperidine 7-position
substituent provided an analog (23c) with some of the lowest
MIC values against the ciprofloxacin-susceptible and -resistant
bacteria in the primary testing panel (Table 1).
CO2H
Et
25c
N
MeO
NH
Cl
a
26
Scheme 4. Reagents and conditions: (a) (i) 1-chloroethyl chloroformate, 1,2-
dichloroethane, reflux; (ii) satd aq NaHCO3.
Modification of the primary amino group of the 7-position
substituent was investigated using compound 23c as a template
(Table 1). Addition of a methyl group was well-tolerated and affor-
ded an analog (24) with comparable antibacterial activity to 23c.
Increasing steric bulk as in analogs 25a and 26, or introduction of
another methyl group to give the tertiary amine (25b) increased
MIC values, particularly against the resistant isolates.
The attractive in vitro profile of quinolones 22b, 23c, and 24
(Fig. 3) prompted further in vitro susceptibility testing against an
expanded panel of organisms including methicillin-resistant cipro-
floxacin-resistant staphylococci and the gram-negative pathogen,
Escherichia coli (Table 2). All three compounds had MIC values 16
to 64-fold lower than ciprofloxacin against the resistant staphylo-
cocci. In contrast, the alkylidenylpiperidinyl quinolone analogs
were less potent than ciprofloxacin against E. coli.
susceptible strains and had lower MICs against the resistant pneu-
mococcal isolates. The incorporation of a fluoro (17b), methyl
(17c), or chloro (17d) R1 substituent improved microbiological
activity against S. pneumoniae, particularly the resistant isolates.
In view of the superior activity of the substituted alkylidene ana-
logs in the naphthyridone subseries, the corresponding 7-position
substituents were incorporated into other heterocyclic nuclei. In
the 8-H quinolone subseries, the MIC values of the fluoro (19a)
and chloro (19c) analogs were uniformly lower than the methyl
analog (19b), a trend that was maintained in the tricyclic quino-
lone subseries based on the core structure of levofloxacin (20a–b,
21). It should be noted that the fluoro (20a) and methyl (20b) ana-
logs were racemic, whereas and the chloro analog (21) had the (S)-
stereochemistry of levofloxacin. Nevertheless, the significantly
lower MIC values of 21 were clear evidence of the superiority of
the chloroalkylidene side chain in this subseries. Incorporation of
fluoro (22a) and chloro (22b) R1 substituents in the 8-OCHF2 sub-
Quinolones 22b, 23c, and 24 were tested for efficacy in a murine
lethal systemic infection model, in which Swiss Webster mice were
infected with methicillin-susceptible S. aureus (Smith) and oral
ED50 values (50% effective dose) determined.16 Compound 24
Table 1
Minimum inhibitory concentrations
O
O
N
O
O
N
CO2H
CO2H
CO2H
CO2H
F
F
F
F
R5
N
N
R5
R5
N
R5
O
N
O
O
CO2H
F
17a-d
19a-c
F
20a-b
21
O
N
O
R5=
R2
N
CO2H
CO2H
R1
F
HN
N
R5
F
R5
N
N
Ciprofloxcin
R3
O
MeO
F
22a-b
23a-c, 24, 25a-b, 26
Compd
R1
R2
R3
MIC(l
g/ml)15
A
B
1
C
D
E
Cipro
17a
17b
17c
17d
19a
19b
19c
20a
20b
21
22a
22b
23a
23b
23c
24
0.12
0.12
0.03
0.06
0.06
0.03
0.12
0.03
0.25
0.5
60.015
0.06
0.06
0.03
0.03
60.015
60.015
0.12
0.25
0.12
>16
8
2
0.25
1
ND
8
ND
2
4
0.25
0.25
0.5
0.5
ND
0.25
0.25
ND
8
>16
8
0.5
1
0.25
1
2
0.5
8
>16
0.25
2
1
1
0.25
0.25
0.5
4
>16
ND
1
1
1
ND
8
1
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.5
0.12
0.12
0.12
0.06
1
0.12
0.5
1
0.03
0.25
0.12
0.12
0.12
0.06
0.06
0.5
CH3
Cl
F
CH3
Cl
F
CH3
Cl
F
Cl
F
CH3
Cl
Cl
Cl
Cl
Cl
8
>16
0.5
0.25
0.12
0.5
0.25
0.25
0.25
2
CH3
iPr
CH3
Et
25a
25b
26
0.5
0.25
8
1
2
1
1
(A) Staphylococcus aureus OC4172; (B) S. pneumoniae ATCC49619; (C) Streptococcus pneumoniae OC6608; (D) S. pneumoniae OC6578; (E) S. pneumoniae OC5465; ND: not
determined.
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E.B. Grant et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
5
O
N
O
N
Acknowledgment
F
CO2H
F
CO2H
The authors wish to thank Ellyn Wira for contributions to
in vitro testing.
N
F
N
O
MeO
H2N
H2N
References and notes
F
Cl
Cl
23c
22b
O
N
1. Lesher, G. Y.; Froelich, E. J.; Gruett, M. D.; Bailey, J. H.; Brundage, R. P. J. Med.
Chem. 1962, 1063, 5.
F
CO2H
2. For some reviews, see (a) Ball, P. J. Antimicrob. Chemother. 2000, 46, 17;
(b) Appelbaum, P. C.; Hunter, P. A. Int. J. Antimicrob. Agents 2000, 16, 5; (c)
Andriole, V. T. Clin. Infect. Dis. 2005, 41, S113; (d) Bambeke, F. V.; Michot,
J. M.; Eldere, J. V.; Tulkens, P. M. Clin. Microbiol. Infect. 2005, 11, 256; (e)
Mitscher, L. A. Chem. Rev. 2005, 105, 559.
3. (a) Wise, R.; Andrews, J. M.; Edwards, L. J. Antimicrob. Agents Chemother. 1983,
23, 559; (b) Grobe, K.; Heitzer, H. Liebigs Ann. Chem. 1987, 29.
4. (a) Atarashi, S.; Yokohama, S.; Yamamzaki, K.; Sakano, K.; Imamura, M.;
Hayakawa, I. Chem. Pharm. Bull. 1896, 1987, 35; (b) Une, T.; Fujimoto, T.; Sato,
K.; Osada, Y. Antimicrob. Agents Chemother. 1988, 32, 559.
N
MeO
H3C
N
H
Cl
24
Figure 3. Chemical structures of the lead compounds from the 7-(4-alkylide-
nyl)piperidinyl quinolone series.
5. Draghi, D. C.; Jones, M. E.; Sahm, D. F.; Tillotson, G. S. Int. J. Antimicrob. Agents
2006, 28, 525.
6. Jones, R. N.; Fritsche, T. R.; Sader, H. S.; Stilwell, M. G. Diagn. Microbiol. Infect.
Dis. 2007, 58, 9.
Table 2
Minimum inhibitory concentrations against methicillin-resistant staphylococci and
E. coli
7. Adam, H. J.; Hoban, D. J.; Gin, A. S.; Zhanel, G. G. Int. J. Antimicrob. Agents 2009,
34, 82.
8. Song, J.-H.; Jung, S.-I.; Ko, K. S.; Kim, N. Y.; Son, J. S.; Chang, H.-H.; Ki, H. K.; Oh,
W. S.; Suh, J. Y.; Peck, K. R.; Lee, N. Y.; Yang, Y.; Lu, Q.; Chongthaleong, A.; Chiu,
C.-H.; Lalitha, M. K.; Perera, J.; Yee, T. T.; Kumarasinghe, G.; Jamal, F.;
Kamarulzaman, A.; Parasakthi, N.; Van, P. H.; Carlos, C.; So, T.; Ng, T. K.;
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9. Diekema, D. J.; Pfaller, M. A.; Schmitz, F. J.; Smayevsky, J.; Bell, J.; Jones, R. N.;
Beach, M. Clin. Infect. Dis. 2001, 32(Suppl. 2), S114.
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11. Hu, E. X.; Kim, N. K.; Gray, J. L.; Almstead, J.-I. K.; Seibel, W. L.; Ledoussal, B. J.
Med. Chem. 2003, 46, 3655.
Compd
MIC (l
g/ml)15
A
B
C
Cipro
22b
23c
24
16
1
0.25
0.5
8
0.5
0.25
0.5
0.015
0.5
0.25
0.5
(A) MRSA OC2805; (B) methicillin-resistant Staphylococcus epidermidis OC5340; (C)
E. coli ATCC25922.
12. Domagala, J. M.; Hagen, S. E.; Joannides, T.; Kiely, J. S.; Laborde, E.; Schroeder,
M. C.; Sesnie, J. A.; Shapiro, M. A.; Suto, M. J.; Vanderroest, S. J. Med. Chem. 1993,
36, 871.
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Patent 7,732,612.
14. Kimura, Y.; Atarashi, S.; Takahashi, M.; Hayakawa, I. Chem. Pharm. Bull. 1994,
42, 1442.
15. The minimal inhibitory concentrations (MICs) were determined by the broth
microdilution method according to Clinical and Laboratory Standards Institute
(CLSI M07-A9, (CLSI, formerly NCCLS) guidelines. Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically-9th ed.
Modal MIC values are reported in cases where multiple MIC values were
demonstrated the lowest ED50 value at 3.6 mg/kg with compounds
23c (4.1 mg/kg), and 22b (6.4 mg/kg) being superior to ciprofloxa-
cin (ED50 = 11 mg/kg).
In summary, we developed and synthesized
a series of
7-(4-alkylidenyl)piperidinyl fluoroquinolone and naphthyridone
antibacterial agents by capitalizing on a previously reported
conformational analysis approach for design of a novel 7-position
substituent. The spectrum of activity included ciprofloxacin-
susceptible and ciprofloxacin-resistant gram-positive cocci, such
as S. pneumoniae and S. aureus. The best analogs of this series
(compounds 22b, 23c, and 24) also demonstrated superior
in vivo efficacy to the clinically approved quinolone ciprofloxacin.
Further studies of this series of quinolone antibacterial agents will
be reported in the future.
determined for
batches.
a compound, including multiple test dates and multiple
16. The in vivo efficacy of compounds 22b, 23c, and 24 was tested in female Swiss-
Webster mice infected ip with the Smith strain of S. aureus (OC4172). Mice
were dosed orally with test compound one hour following infection. Mortality
was observed over a period of three days. A group of infected mice not treated
with drug served as controls.
Please cite this article in press as: Grant, E. B.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.10.014