Synthesis and antibacterial activity of 5-methoxy- and 5-hydroxy-6-fluoro- 1,8-naphthyridone-3-carboxylic acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2005.04.007

A series of 5-methoxy- and 5-hydroxy-6-fluoro-1,8-naphthyridone-3- carboxylic acid derivatives were prepared and evaluated for cell-free bacterial protein synthesis inhibition and whole cell antibacterial activity. When compared to the analogous 5-hydroge

Full text of DOI:10.1016/j.bmcl.2005.04.007

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2716–2719
Synthesis and antibacterial activity of 5-methoxy- and
5-hydroxy-6-fluoro-1,8-naphthyridone-3-carboxylic acid derivatives
T. Matthew Hansen,* Yu-Gui Gu, Tamara M. Rehm, Peter J. Dandliker,
Linda E. Chovan, Mai H. Bui, Angela M. Nilius and Bruce A. Beutel
Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA
Received 7 March 2005; revised 31 March 2005; accepted 4 April 2005
Available online 4 May 2005
Abstract—A series of 5-methoxy- and 5-hydroxy-6-fluoro-1,8-naphthyridone-3-carboxylic acid derivatives were prepared and evalu-
ated for cell-free bacterial protein synthesis inhibition and whole cell antibacterial activity. When compared to the analogous
5-hydrogen compounds, the presence of the 5-OH group negatively affects biochemical potency. However, a tolerance of the 5-meth-
oxy group is indicated. Only moderate whole cell antibacterial activity is seen, but this could be due to poor cellular penetration.
Because only a few 7-position variants were made for this study, further investigation into this novel series combining a broader
range of 7-amino derivatives with these 5-position modifications is warranted.
Ó 2005 Elsevier Ltd. All rights reserved.
Antibacterial resistance is a major public health issue.
Streptococcus pneumoniae is the most common pathogen
responsible for community-acquired pneumonia and its
resistance to penicillin and macrolide antibiotics is on
the rise.1 Studies have shown that 80% of methicillin-
resistant Staphylococcus aureus (MRSA) in the United
States are now resistant to ciprofloxacin,2a–f and about
40% of hospital-acquired staphylococci are resistant to
all forms of therapy except vancomycin.2e,f Nearly two
million patients in the United States get an infection in
the hospital each year, of those patients about 90,000
die as a result of their infection. This number is up from
13,300 patient deaths in 1992.2g The overall global emer-
gence of antimicrobial resistance is severely limiting the
effectiveness of current drugs. Along with a need for
societal restraint in antibiotic usage, the development
of novel antibiotics is one way to maintain lower levels
of resistance and are urgently needed.1
eukaryotic) and broad-spectrum antibacterial activity.
Despite being structurally similar to the quinolone class
of antibiotics, which inhibit DNA gyrase/topoisomerase
IV, NRIs undoubtedly provide antibiotic action via a
different mechanism. A complete description of the dis-
covery of these novel antibacterials and the correspond-
ing mechanism of action studies have recently been
reported.3
Through the course of an extensive medicinal chemistry
effort at Abbott, it was demonstrated that the antibacte-
rial activity of this class of compounds is profoundly
influenced by the combination of substituents at the
C3–C7 and N1 positions.4a–c Despite intense interest in
1,8-naphthyridone-3-carboxylic acids in antibacterial re-
search, a survey of the literature produced no examples
of 5-hydroxy- or 5-methoxy-6-fluoro-1,8-naphthyri-
dones which had been evaluated as antibacterial agents.
There have been, however, a few examples of 5-hydroxy-,
5-methoxy- and 5-amino-quinolones reported.5 Consis-
tent with our efforts to improve the overall potency
of our NRI series, we wanted to evaluate the potential
of introducing a hydrogen bonding group at C5 and
gaining a valuable synthetic handle to further probe
the SAR at the 5-position. We therefore undertook
We recently discovered a novel class of ribosome inhibi-
tors by screening a compound library to identify inhibi-
tors of cell-free bacterial transcription or translation.3
As a result of this screening, A-72310 (1) emerged as
the lead compound representing a novel ribosome inhib-
itor (NRI) class which showed selective (prokaryotic vs
the synthesis of
a
few NRIs substituted with
R1 = OMe (2) or R1 = OH (3) combined with three dif-
ferent C7 amino substituents (R2 = azetidine, pyrrolidine
and aminopyrrolidine).
Keywords: Novel ribosome inhibitors; 1,8-Naphthyridone antibiotics.
*
Corresponding author. Tel.: +1 847 935 6879; fax: +1 847 938
3403; e-mail: matthew.hansen@abbott.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.007

T. M. Hansen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2716–2719
2717
Our strategy for the construction of the 5-methoxy- and
5-hydroxy-1,8-naphthyridone core was to install the 5-
methoxy group early in the synthesis and then use well-
known quinolone chemistry to complete the construction
of a common intermediate which would serve as a point
of departure for C7 modification. Late-stage cleavage of
the methyl ether would be a key transformation to pro-
vide the 5-hydroxy-1,8-naphthyridone derivatives.
able to choose three of the better variants for this preli-
minary study. The 7-position azetidine (a), pyrrolidine
(b) and aminopyrrolidine (c) were selected and synthe-
sized from our common core (9) shown in Scheme 2. Dis-
placement of the 7-fluorine by the requisite amine (a, b or
c) proceeded smoothly in the presence of HunigÕs base in
CH3CN to provide 10a–c in high yield. Concomitant
deprotection of the cyanoethyl group and saponification
followed by acidification provided the hydrochloride salts
of 11a–c. In the case of 11c, the Boc group was removed
using 30% trifluoroacetic acid (TFA) in CH2Cl2 to give
TFA salt 11d. The C5-hydroxyl was liberated by treating
compounds 11a,b and d with an excess of pyridine hydro-
chloride (ca. 140 °C, 10 min). Both the 5-methoxy- and
5-hydroxy-compounds were submitted for biological
evaluation (Table 1) and each structure was confirmed
by 1H NMR and mass spectroscopy.
We chose commercially available 3-chloro-2,4,5,6-tetra-
fluoropyridine (4) as a convenient starting material.
Illustrated in Scheme 1 is the selective displacement of
the 4-position fluoride with NaOMe to provide the cor-
responding methoxy derivative. This material was subse-
quently dechlorinated by hydrogenation to furnish
compound 5.6 The lithium anion of 5 reacted smoothly
with benzyl cyanoformate to provide benzyl ester 6 in
high yield. Exposure of 6 to hydrogenolysis afforded carb-
oxylic acid 7 in near-quantitative yield. With 7 in hand,
we were able to use quinolone chemistry to construct
our novel 5-methoxy-1,8-naphthyridone core. Accord-
ingly, the carboxylic acid was converted to the acyl chlo-
ride using standard oxalyl chloride/DMF conditions.
The crude acyl chloride intermediate was then acylated
using the magnesium malonate complex derived from
potassium ethyl malonate to provide b-oxo ester 8.7
Combining 8 with Ac2O and (EtO)3CH formed the cor-
responding ethoxymethylene derivative which was used
directly in the next step without further purification.
Treatment of the crude ethoxymethylene with 3-amino-
propionitrile and subsequent cyclization of the enamine
by treatment with NaH gave the desired 5-methoxy-6-
fluoro-1,8-naphthyridone core (9).8a,b
The biochemical assays used for this study have previ-
ously been described.3,9 Compounds were subjected to
a panel of cell-free translation assays. Reported here
are the concentrations causing 50% inhibition of bacte-
rial protein synthesis (IC50 values) measured in ribo-
some-containing S. pneumoniae cell extracts translating
a luciferase mRNA. In order to confirm bacterial pro-
tein synthesis inhibition in extracts from a Gram-nega-
tive bacterium and rule out direct luciferase inhibition,
a second assay was implemented using cell-free extracts
from Escherichia coli charged with mRNA encoding b-
galactosidase. In this case, the two assays were found
to be in agreement, confirming that these compounds
are inhibitors of bacterial protein synthesis. It should
also be pointed out that quinolone antibiotics, including
ciprofloxacin and levofloxacin, did not inhibit bacterial
protein synthesis in these assays (data not shown). Com-
pounds were also evaluated for antibacterial activity
Because diversification of C7 has been thoroughly investi-
gated within the context of our NRI research,4a–c we were
Scheme 1. Reagents and conditions: (a) NaOMe, MeOH–THF (1:1), 0 °C to rt, 20 h; (b) H2, 10% Pd–C, Et3N, MeOH, 3 atm, 24 h, 54% over two
steps; (c) n-BuLi (2.5 M hexane), THF, À78 °C, 1 h, then benzyl cyanoformate À78 °C to rt, 1 h, 74%; (d) H2, 10% Pd–C, EtOH, rt, 1.5 h, 98%; (e)
oxalyl chloride, DMF (cat.), CH2Cl2, 0 °C to rt, 1.5 h; (f) potassium ethyl malonate, MgCl2, Et3N, CH3CN, 10 °C to rt, 2.5 h, 78% two steps; (g)
(EtO)3CH, Ac2O, 130 °C, 15 h; (h) 3-aminopropionitrile, CH2Cl2, rt, 1 h; (i) NaH (60% oil dispersion), THF, 0 °C to rt, 1 h, 51% over three steps.

2718
T. M. Hansen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2716–2719
Scheme 2. Reagents and conditions: (a) amine (a, b or c), DIPEA, CH3CN, 60 °C, 90–100%, (b) 1.0 N NaOH, EtOH, 80 °C, 1.5 h, then acidification
with HCl(aq), 70–80%, (c) pyridine hydrochloride, 140 °C, 5–10 min, 80–90%.
against a variety of quinolone-resistant and susceptible
strains of bacteria. Minimum inhibitory concentrations
(MICs) for a representative quinolone-resistant strain
and a Gram-negative strain are reported.
As shown in Table 1, the 5-methoxy derivatives (entries
1–3) are more potent protein synthesis inhibitors than
the corresponding 5-hydroxy analogs (entries 4–6).
More importantly, when entries 1–3 are compared to
Table 1. Cell-free bacterial protein synthesis inhibition and antibacterial activity
Entry
Compound
R5
R7
S. pneumoniae translation
IC50 (lM)
S. pneumoniae 7257a
MIC (lg/ml)
H. influenzas GYR 1435b
MIC (lg/ml)
1
2
3
11a
11b
11d
OMe
OMe
OMe
14
32
4
8
32
64
32
64
64
4
5
6
7
8
12a
12b
12d
13
OH
OH
OH
H
200
51
17
5
64
4
64
4
64
4
32
1
14
1
H
H
10
4
1
9
7
32
16
4
Levofloxacin
>100
0.015
a Quinolone-resistant strain.
b Gram-negative.

T. M. Hansen et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2716–2719
2719
2. (a) Yee, Y. C.; Thornsberry, C. Antimicrob. Infect. Dis.
Newslett. 1995, 14, 1–18; (b) Voss, A.; Doebbeling, B. N.
J. Antimicrob. Agents 1995, 101–106; (c) Brumfitt, W.;
Hamilton-Miller, J. M. T. Drugs Exp. Clin. Res. 1994,
20, 215–224; (d) Neu, H. C. Science 1992, 257, 1064–
1073; (e) Begley, S. The End of Antibiotics. Newsweek
1994, 14, 1–18; (f) Stahl, L. Super-Resistant Superbugs
60 Minutes, May 2, 2004; (g) National Institute of
Allergy and Infectious Diseases (NIAID) fact sheet, April
2004.
the previously described 5-hydrogen compounds (entries
7–9),3,4a–c similar in vitro potencies are seen. In particu-
lar, compound 11d (IC50 = 4 lM) is roughly equipotent
to the parent compound 1 (IC50 = 7 lM). Compound
11a (IC50 = 14 lM) where R7 = azetidine, is only 2–3
times less potent than the analogous 5-hydrogen com-
pound (13, IC50 = 5 lM). These data indicate a toler-
ance for this type of substitution at the 5-position.
Also shown in Table 1 is the antibacterial activity of
each compound. Data for levofloxacin, a second-gener-
ation quinolone antibiotic, are included as a reference.
Moderate to good antibacterial activity is seen for com-
pounds 11a,b, 12b and d against S. pneumoniae 7257, a
strain resistant to the fluoroquinolones due to mutations
in the DNA gyrase and topoisomerases IV,10 and Hae-
mophilus influenzae GYR 1435, a representative Gram-
negative pathogen. These MICs, taken together with
the biochemical assay results, provide solid evidence
that these compounds exert their antimicrobial effects
through a non-quinolone mechanism of action. A possi-
ble explanation for the lack of antibacterial activity for
11d and 12d in either of these strains could be poor cel-
lular penetration. No permeabilizer studies were done
with this group of compounds.4c
3. Dandliker, P. J.; Black-Schaefer, C.; Bui, M.; Cai, Y.;
Cao, Z.; Chovan, L. M.; Clark, R. F.; Daly, M. M.;
David, C. A.; Englund, E. E.; Hickman, R. K.; Kakavas,
S. J.; Lerner, C. G.; Merta, P.; Nilius, A. M.; Pratt, S.
D.; Ruan, X.; Saiki, A. Y.; Shen, L. L.; Soni, N. B.;
Wagner, R.; Weitzberg, M.; Xuei, X.; Zhong, P.; Beutel,
B. A. Antimicrob. Agents Chemother. 2003, 47, 3831–
3839.
4. (a) Anderson, D.; Beutel, B.; Bosse, T. D.; Cooper, C.;
Dandliker, P. J.; David, C.; Gu, Y.-G.; Hinman, M.;
Kalvin, D.; Lynch, L.; Ma, Z.; Motter, C.; Rosenberg, T.;
Sanders, W.; Tufano, M.; Wagner, R.; Weitzberg, M.;
Yong, H. U.S. Patent 6 818 654, November 16, 2004; (b)
Anderson, D.; Beutel, B.; Bosse, T. D.; Clark, R. F.;
Cooper, C.; Dandliker, P. J.; David, C.; Gu, Y.-G.;
Hansen, T. M.; Hinman, M.; Kalvin, D.; Larson, D. P.;
Lynch, L.; Ma, Z.; Motter, C.; Palazzo, F.; Rosenberg, T.;
Rehm, T.; Sanders, W.; Tufano, M. D.; Wagner, R.;
Weitzberg, M.; Yong, H.; Zhang, T. U.S. Patent Appli-
cation 20030232818, 2003; (c) Clark, R. F.; Wang, S.; Ma,
Z.; Weitzberg, M.; Motter, C.; Tufano, M. D.; Wagner,
R.; Gu, Y.-G.; Dandliker, P. J.; Lerner, C. G.; Chovan, L.
E.; Cai, Y.; Black-Schaefer, C. L.; Lynch, L.; Kalvin, D.;
Nilius, A. M.; Pratt, S. D.; Soni, N.; Zhang, T.; Zhang, X.;
Beutel, B. A. Bioorg. Med. Chem. Lett. 2004, 14, 3299–
3302.
In summary, we have developed a flexible synthetic
entry to novel 5-methoxy- and 5-hydroxy-6-fluoro-1,8-
naphthyridone-3-carboxylic acid antibiotics. Members
of this class of 1,8-naphthyridones had not previously
been evaluated as antibacterial agents. The data indicate
incorporation of a 5-methoxy group does not have a dele-
terious effect on in vitro potency and given the profound
influence the 7-position has in combination with the 5-
position,11 further exploration of this class of NRIs
should be undertaken. The 5-hydroxyl also constitutes a
new synthetic handle for this class of compounds, mak-
ing it possible to thoroughly investigate the advantages
of hydrophilic substitution at the 5-position of 1,8-naph-
thyridone antibacterials. Extension of this work will
include a comprehensive survey of 5-hydroxy, 5-alkoxy,
5-amino derivatives in combination with a wide variety
of C7 amino substitution, allowing further development
and differentiation of this novel class. Detailed accounts
of continuing studies will be reported in due course.
5. (a) Domagala, J. M.; Bridges, A. J.; Culbertson, T. P.;
Gambino, L.; Hagen, S. E.; Karrick, G.; Porter, K.;
Sanchez, J. P.; Sesnie, J. A.; Spense, F. G.; Szotek, D.;
Wemple, J. J. Med. Chem. 1991, 34, 1142–1154; (b)
Hagen, S. E.; Domagala, J. M.; Heifetz, C. L.; Johnson, J.
J. Med. Chem. 1991, 34, 1155–1161.
6. These two steps have been done with tBuONa to provide a
tBu protecting group on the C4 hydroxyl: Li, Q.; Chu, D.;
Claiborne, A.; Cooper, C.; Lee, C.; Raye, K.; Berst, K.;
Donner, P.; Wang, W.; Hasvold, L.; Fung, A.; Ma, Z.;
Tufano, M.; Flamm, R.; Shen, L.; Baranowski, J.; Nilius,
A.; Alder, J.; Meulbroek, J.; Marsh, K.; Crowell, D.; Hui,
Seif L.; Melcher, L.; Henry, R.; Spanton, S.; Faghih, R.;
Klein, L.; Tanaka, K.; Plattner, J. J. Med. Chem. 1996, 39,
3070–3088.
7. Clay, R.; Collom, T.; Karrick, G.; Wemple, J. Synthesis
1993, 290–292.
Acknowledgements
8. (a) Chu, D.; Fernandes, P.; Claiborne, A.; Gracey, E.;
Pernet, A. J. Med. Chem. 1986, 29, 2363–2369; (b) Radl,
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9. Pratt, S. D.; David, C. A.; Black-Schaefer, C.; Dandliker,
P. J.; Xuei, X.; Warrior, U.; Burns, D. J.; Zhong, P.; Cao,
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B.; Nilius, A. M.; Beutel, B. A. J. Biomol. Screen. 2004, 9,
3–11.
The authors thank the Abbott High Throughput Purifi-
cation group for purification of compounds, the Hydro-
genation Laboratory, Xiaolin Zhang and Carmina
Presto for NMR interpretation and Richard Clark,
Qun Li, Moshe Weitzberg and Robert Keyes for valu-
able discussions.
10. Bruggemann, A. B.; Coffman, S. L.; Rhomberg, P.;
Hungh, H.; Almer, L.; Nilius, A. M.; Flamm, R.; Doern,
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References and notes
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11. Rolf Wagner, personal communication.