Synthesis and antibacterial activity of 5-deoxy-5-episubstituted arbekacin derivatives

Article abstract of DOI:10.1016/j.bmcl.2007.04.065

5-Deoxy-5-episubstituted arbekacin derivatives have been designed and efficiently synthesized. The synthetic compounds showed potent antibacterial activity against both Staphylococcus aureus, including methicillin-resistant S. aureus, and Pseudomonas aeru

Full text of DOI:10.1016/j.bmcl.2007.04.065

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3540–3543
Synthesis and antibacterial activity of 5-deoxy-5-episubstituted
arbekacin derivatives
Yukiko Hiraiwa,a Takayuki Usui,a,*, Yoshihisa Akiyama,a Kazunori Maebashi,a
Nobuto Minowaa,* and Daishiro Ikedab,*,à
aPharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan
bMicrobial Chemistry Research Center, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
Received 19 February 2007; revised 17 April 2007; accepted 19 April 2007
Available online 25 April 2007
Abstract—5-Deoxy-5-episubstituted arbekacin derivatives have been designed and efficiently synthesized. The synthetic compounds
showed potent antibacterial activity against both Staphylococcus aureus, including methicillin-resistant S. aureus, and Pseudomonas
aeruginosa. In particular, these derivatives were superior to arbekacin against MRSA strains expressing the bifunctional aminogly-
coside-modifying enzyme AAC(60)-APH(200). The antibacterial activity of the 5-deoxy-5-episubstituted arbekacin derivatives against
Pseudomonas aeruginosa was markedly influenced by the efflux system of MexXY/OprM. The 60-N-methyl derivative of the 5-epi
arbekacin was effective against Pseudomonas aeruginosa expressing the aminoglycoside-modifying enzyme AAC(60).
Ó 2007 Elsevier Ltd. All rights reserved.
In recent years, infection caused by methicillin-resis-
tant Staphylococcus aureus (MRSA), including vanco-
mycin-resistant strains, and the multidrug-resistant
Pseudomonas aeruginosa, which has resistance against
carbapenems and quinolones, has become a significant
clinical problem.
adenylyltransferase (AAD), and aminoglycoside phos-
photransferase (APH)1 has been reported. Further-
more, the MRSA strains expressing the bifunctional
aminoglycoside-modifying enzyme AAC(60)-APH(200)
have been found in clinical isolates.2 On the other
hand, efflux systems in P. aeruginosa, such as the tri-
partite resistance nodulation division (RND) efflux sys-
tems, have a role in resistance to various antibiotics.
Thus, it has been recently recognized that aminoglyco-
side antibiotics are effluxed by the MexXY/OprM of
the RND efflux system.3
The aminoglycoside antibiotics, represented by arbeka-
cin (ABK), tobramycin, and amikacin, have been
widely used as the clinically significant agents because
of their potent antibacterial activities against both
Gram-positive and Gram-negative bacteria.1 However
the resistance to aminoglycoside antibiotics is caused
by aminoglycoside-modifying enzymes such as amino-
glycoside acetyltransferase (AAC), aminoglycoside
There are several approaches to the development of a
new class of aminoglycoside antibiotics that show anti-
bacterial activity against resistant bacteria.4 Among
them, 5-deoxy-5-episubstituted aminoglycosides such
as 5-epi sisomicin,5 5-epi isepamycin,6 and 5-deoxy-5-
epifluoro arbekacin7 have been reported to show potent
antibacterial activity against resistant Gram-positive
and Gram-negative bacteria. Therefore, to overcome
infection caused by MRSA and P.aeruginosa, we under-
took synthesis of the 5-deoxy-5-episubstituted ABK
derivatives 3a–g and tested them for antibacterial activ-
ity, including their stability against aminoglycoside-
modifying enzymes and the influence of the MexXY/
OprM efflux pump.
Keywords: Aminoglycoside; Methicillin-resistant Staphylococcus aur-
eus; Pseudomonas aeruginosa; Efflux pump; Aminoglycoside-modifying
enzyme.
*
Corresponding authors. Tel.: +81 45 924 5188 (T.U.); tel.: +81 45 541
2521; fax: +81 45 543 9771 (N.M.); tel.: +81 55 924 0601; fax: +81 55
922 6888 (D.I.); e-mail addresses: usuit@isl.titech.ac.jp; nobuto_
minowa@meiji.co.jp; bdiked@bikaken.or.jp
Present Address: Imaging Science and Engineering Laboratory,
Tokyo Institute of Technology, 4259 Nagatsuda, Midori-ku, Yoko-
hama 226-8503, Japan.
à
Present Address: Numazu Bio-Medical Research Institute, Microbial
Chemistry Research Center, 18-24 Miyamoto, Numazu city, Shi-
zuoka 410-0301, Japan.
Synthesis of 5-deoxy-5-episubstituted ABK derivatives
from ABK8 is shown in Scheme 1. The amino groups
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.065

Y. Hiraiwa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3540–3543
3541
Scheme 1. Synthesis of 5-deoxy-5-epi arbekacin derivatives. Reagents and conditions: (a) (Boc)2O, Et3N, DMF, H2O, rt; (b) Ac2O, pyridine, rt; (c)
MsCl, DMAP, CH2Cl2, rt; (d) CsOAc, DMF, 100 °C, (2a); or LiCl, DMF, 100 °C, (2b); (e) NaOMe, MeOH, CH2Cl2, rt; (f) 90% TFA, rt; (g)
MeNH2, MeOH, sealed tube, 60 °C, (3d); or NH2(CH2)2NH2, sealed tube, DMF, 80 °C, (3f); (h) NaN3, DMF, 100 °C; (i) H2, 10% Pd–C, H2O, rt; (j)
formaldehyde, Et3N, MeOH, 1,4-dioxane, rt, then NaBH4, MeOH, 1,4-dioxane, rt; (k) benzyl bromide, K2CO3, DMF, rt.
of ABK were protected with t-butoxycarbonyl (Boc)
groups, and then the hydroxy groups, except for 5-
OH, were protected with acetyl (Ac) groups to provide
the penta-N-Boc-tetra-O-Ac ABK derivative, which
was subsequently treated with methansulfonyl chloride
(MsCl) to afford 1. Treatment of 1 with CsOAc or LiCl,
and then removal of the Ac and Boc protecting groups
of the resulting compounds 2a or 2b gave the 5-epi
ABK 3a or 5-deoxy-5-epichloro ABK 3b, respectively.
Treatment of 1 with NaN3 gave the 5-epiazide derivative
5. Stepwise removal of the Ac and Boc groups of 5 and
then reduction of the azide group to an amino group
gave the 5-deoxy-5-epiamino ABK 3c.
groups gave 3d or 3f, respectively. Alternatively, com-
pound 5 was transformed to compound 6 by removal
of the Ac groups and reduction of the C-5 azide group
to an amino group. Methylation of the 5-NH2 of 6 with
formaldehyde in the presence of NaBH4, followed by
removal of the Boc groups gave a mixture of the 5-
deoxy-5-epidimethylamino ABK 3e, 5-deoxy-5-epimeth-
ylamino ABK, and 5-deoxy-5-epiamino ABK. The
desired product 3e could be readily purified by column
þ
chromatography on CM Sephadex (NH4 form). Treat-
ment of 6 with benzyl bromide in the presence of K2CO3
and then removal of the Boc groups afforded 3g.
The antibacterial activities of the 5-deoxy-5-episubstitut-
ed ABK derivatives are shown in Table 1.13
Next, we tried to introduce substituted amino groups at
the C-5 position of ABK. Removal of the Ac groups of
compound 1 by NaOMe in MeOH–CH2Cl2 gave 4. Effi-
cient introduction of substituted amino groups at the
hindered C-5 position of 4 was achieved by treatment
with MeNH2 or H2NCH2CH2NH2 at 60 or 80 °C in a
sealed tube, which followed by removal of the Boc
Compounds 3a–c and 3f were comparable with ABK in
antibacterial activity against S. aureus 209P JC-1.
Further, these compounds showed more potent antibac-
terial activity than ABK, against MRSA expressing
the aminoglycoside-modifying enzymes AAD(40) and
Table 1. Antibacterial activities of compounds 3a–3g
Test organism
MIC (lg/ml)
3a
3b
0.1
3c
0.13
3d
3e
3f
3g
ABK
Staphylococcus aureus 209P JC-1
S. aureus MF490 (MRSA)a
S. aureus MSC03571 (MRSA)a
Escherichia coli NIH JC-2
0.06
2
0.25
4
1
0.13
0.13
8
0.06
64
16
3.13
6.25
1.56
2
2
4
32
>64
16
2
4
4
2
2
16
32
4
1
2
2
4
1
Pseudomonas aeruginosa PAO1
P. aeruginosa GN315b
2
2
16
32
32
128
1
2
16
8
0.25
50
0.5
128
NTc
>32
NT
64
NT
P. aeruginosa N101(DmexXY of PAO1)
0.5
0.25
a Possessing AAD(40) and AAC(60)-APH(200).
b Possessing AAC(60)-Ib.
c NT, not tested.

3542
Y. Hiraiwa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3540–3543
AAC(60)-APH(200). By contrast, the 5-N-substituted
derivatives 3e and 3g showed less potent activity against
S. aureus and P. aeruginosa as compared with the 5-
deoxy-5-epiamino ABK 3c. The compounds 3a–c and
3f showed potent activity against P. aeruginosa PAO1
equal to that of ABK.
and measured their antibacterial activity. Synthesis of
the 300-N-methyl derivative 11 from 711 is shown in
Scheme 2.
Stepwise reductive alkylations of the 300-NH2 of 7 with
benzaldehyde and then with formaldehyde afforded 8.
Regioselective acetylation of all of the hydroxyl groups
of 8, except for 5-OH, provided 9. Treatment of 9 with
MsCl and then reaction with CsOAc gave the 5-epiacet-
oxy derivative 10. Stepwise removal of the Boc, Ac,
p-methoxybenzyloxycarbonyl (PMZ), and Benzyl pro-
tecting groups of 10 in three steps afforded 11.
Next, we investigated the effect of the efflux system of
P. aeruginosa by comparing the MICs of the compounds
for P. aeruginosa PAO1 and for P. aeruginosa N101 (D
mexXY/oprM PAO1).9 Interestingly, 3a–c, 3g, and
ABK showed increased activity (4- to 32-fold higher
activity) against P. aeruginosa N101 than P. aeruginosa
PAO1. It is noteworthy that, out of the derivatives,
the lipophilic 5-deoxy-5-epi-N-benzyl derivative 3g was
the most susceptible to elimination via the MexXY/
OprM tripartite efflux system of P. aeruginosa PAO1.
These results seem to indicate that the MexXY/OprM
efflux system is a major mechanism in the resistance of
P. aeruginosa against 5-deoxy-5-episubstituted ABK
derivatives and ABK.
Synthesis of the 60-N-methyl derivative 17 from ABK is
shown in Scheme 3.
Regioselective protection12 of the 60-NH2 of ABK with
N-(benzyloxycarbonyloxy)succinimide in the presence
of Zn(OAc)2 gave 60-N-benzyloxycarbonyl (60-N-Cbz)
ABK in 33% yield afterþ column chromatography on
Amberlite CG 50 (NH4 form). Treatment of 60-N-
Cbz ABK with (Boc)2O afforded 12. The Cbz group of
12 was removed by hydrogenolysis with 10% Pd–C to
provide the 60-NH2 derivative. Stepwise reductive alky-
lations of the 60-NH2 with benzaldehyde and formalde-
hyde gave 13. Removal of the benzyl group of 13,
followed by Boc protection of the 60-NHMe group,
afforded 14, which was regioselectively benzoylated to
Furthermore, to investigate 6010- or 300-N-substituted
derivatives of the 5-deoxy-5-episubstituted ABK deriva-
tives for antibacterial activity and stability against ami-
noglycoside-modifying enzymes, we focused on the 5-epi
ABK 3a that has the most potent antibacterial activity.
We synthesized the 60- and 300-N-methyl derivatives of 3a
Scheme 2. Synthesis of 300-N-methyl-5-epi arbekacin. Reagents and conditions: (a) benzaldehyde, Et3N, MeOH, 1,4-dioxane, rt, then NaBH4,
MeOH, 1,4-dioxane, rt; (b) formaldehyde, Et3N, MeOH, 1,4-dioxane, rt, then NaBH(OAc)3, MeOH, 1,4-dioxane, rt; (c) Ac2O, pyridine, rt; (d)
MsCl, DMAP, CH2Cl2, rt; (e) CsOAc, DMF, 100 °C; (f) NaOMe, MeOH, CH2Cl2, rt; (g) 90% TFA, rt; (h) H2, 10% Pd–C, H2O, rt.
Scheme 3. Synthesis of 60-N-methyl-5-epi arbekacin. Reagents and conditions: (a) Zn(OAc)2, N-(benzyloxycarbonyloxy)succinimide, H2O, rt; (b)
(Boc)2O, Et3N, MeOH, 1,4-dioxane, rt; (c) H2, 10% Pd–C, 1,4-dioxane, H2O, rt; (d) benzaldehyde, Et3N, MeOH, 1,4-dioxane, rt, then NaBH4,
MeOH, 1,4-dioxane, rt; (e) formaldehyde, Et3N, MeOH, 1,4-dioxane, rt, then NaBH4, MeOH, 1,4-dioxane, rt; (f) H2, Pd–C, 1,4-dioxane, H2O, rt,
then (Boc)2O, Et3N, MeOH, 1,4-dioxane, H2O, rt; (g) benzoyl chloride, pyridine, 0 °C; (h) MsCl, DMAP, CH2Cl2, rt; (i) CsOAc, DMF, 100 °C; (j)
NaOMe, MeOH, CH2Cl2, rt; (k) 90% TFA, rt.

Y. Hiraiwa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3540–3543
3543
Table 2. Antibacterial activities of compounds 11 and 17
was markedly reduced by the MexXY/OprM efflux sys-
tem in P. aeruginosa. Based on these findings, further
structure–activity relationship studies of this class of
compounds are currently in progress.
Test organism
MIC (lg/ml)
17 3a
0.13 0.06
11
0.13
ABK
Staphylococcus aureus 209P JC-1
S. aureus MF490 (MRSA)a
S. aureus MSC03571 (MRSA)a
Escherichia coli NIH JC-2
Pseudomonas aeruginosa PAO1
P. aeruginosa GN315b
0.06
64
16
1
4
16
2
32
2
2
1
2
8
8
1
4
4
Acknowledgments
2
32
2
16
We thank Prof. Naomasa Gotoh from Kyoto Pharma-
ceutical University for kindly providing us with the
P. aeruginosa PAO1DmexXY/oprM strain. We also
thank Dr. Makoto Oyama and Miss. Shigeko Miki
(Meiji Seika Kaisha Ltd.) for NMR spectroscopic and
mass spectrometric analyses, respectively.
a Possessing AAD(40) and AAC(60)-APH(200).
b Possessing AAC(60)-Ib.
give 15. Treatment of 15 with MsCl provided the 5-OMs
derivative and then reaction with CsOAc gave the 5-epi-
acetoxy derivative 16. Removal of the Boc, Ac, and ben-
zoyl groups of 16 in two steps provided the 60-N-methyl
derivative 17.
References and notes
1. Magnet, S.; Blanchard, J. S. Chem. Rev. 2005, 105, 477.
2. (a) Daigle, D. M.; Hughes, D. W.; Wright, G. D. Chem.
Biol. 1999, 6, 99; (b) Fujimura, S.; Tokue, Y.; Takahashi,
H.; Kobayashi, T.; Gomi, K.; Abe, T.; Nukiwa, T.;
Watanabe, A. FEMS Microbiol. Lett. 2000, 190, 299; (c)
Ishino, K.; Ishikawa, J.; Ikeda, Y.; Hotta, K. J. Antibiot.
2004, 57, 679.
The antibacterial activities of 11 and 17 are shown in
Table 2.13
As expected, the 60-N-methyl derivative 17 was slightly
more active than the 60-NH2 derivative 3a against
P. aeruginosa GN315 expressing the aminoglycoside-
modifying enzyme AAC(60)-Ib. However, 17 showed less
potent antibacterial activity against S. aureus strains
MF490 and MSC03571 expressing AAC(60)-APH(200)
and AAD(40), as compared with 3a. On the other
hand, introduction of a methyl group at the 300-NH2 re-
sulted in decreased activity against S. aureus and P.
aeruginosa (compound 11). These results indicated that
introduction of a methyl group at the 60-NH2 of 5-
deoxy-5-episubstituted derivatives led to enhanced sta-
bility against the aminoglycoside-modifying enzyme
AAC(60)-Ib.
´
3. (a) Aires, J. R.; Ko¨hler, T.; Nikaido, H.; Plesiat, P.
Antimicrob. Agents Chemother. 1999, 43, 2624; (b) Masu-
da, N.; Sakagawa, E.; Ohya, S.; Gotoh, N.; Tsujimoto, H.;
Nishino, T. Antimicrob. Agents Chemother. 2000, 44, 2242;
(c) Masuda, N.; Sakagawa, E.; Ohya, S.; Gotoh, N.;
Tsujimoto, H.; Nishino, T. Antimicrob. Agents Chemother.
2000, 44, 3322; (d) Sobel, M. L.; Mckay, G. A.; Poole, K.
Antimicrob. Agents Chemother. 2003, 47, 3202.
4. (a) Kondo, S.; Iinuma, K.; Yamamoto, H.; Maeda, K.;
Umezawa, H. J. Antibiot. 1973, 26, 412; (b) Kondo, S.;
Iinuma, K.; Yamamoto, H.; Maeda, K.; Umezawa, H.
J. Antibiot. 1973, 26, 705.
5. (a) Kabins, S. A.; Nathan, C. Antimicrob. Agents Chemo-
ther. 1978, 14, 391; (b) Fu, K. P.; Neu, H. C. Antimicrob.
Agents Chemother. 1978, 14, 194.
6. Daniels, P. J. L.; Cooper, A. B.; McCombie, S. W.;
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Finally, we tested the antibacterial activity of the 5-
deoxy-5-episubstituted derivatives 3a and 3c against 54
clinical isolates of MRSA. Against these 54 strains, the
MIC50 and MIC90 of 3a (MIC50, 0.5 lg/mL, MIC90,
1.0 lg/mL) and 3c (MIC50, 0.5 lg/mL, MIC90, 0.5 lg/
mL) indicated that these compounds were more potent
than ABK (MIC50, 1.0 lg/mL, MIC90, 2.0 lg/mL).13
7. Shitara, T.; Umemura, E.; Tsuchiya, T.; Matsuno, T.
Carbohydr. Res. 1995, 276, 75.
8. Arbekacin (ABK) was launched for clinical use in Japan
as the first anti-MRSA drug (see Ref. 4).
9. Masuda, N.; Sakagawa, E.; Ohya, S.; Gotoh, N.; Tsujim-
oto, H.; Nishino, T. Antimicrob. Agents Chemother. 2000,
44, 2242.
In summary, we have designed and synthesized several
5-deoxy-5-episubstituted ABK derivatives in order to
investigate the novel aminoglycoside antibiotic agents
having the antibacterial activities against aminoglyco-
side resistant bacteria. The compounds 3a–c and 3f
showed good antibacterial activity against S. aureus
and P. aeruginosa. In particular, the 5-epi ABK 3a
and the 5-deoxy-5-epiamino ABK 3c showed potent
activity against S. aureus, including MRSA expressing
the bifunctional aminoglycoside-modifying enzyme
AAC(60)-APH(200). Introduction of a methyl group at
the 60-NH2 of 3a led to enhanced activity against
P. aeruginosa expressing the aminoglycoside-modifying
enzyme AAC(60)-Ib. The antibacterial activity of the
5-deoxy-5-episubstituted ABK derivatives and ABK
10. Introduction of
a
methyl group at the 60-NH2 of
kanamycin B resulted in increased stability against ami-
noglycoside-modifying enzyme AAC(60) (a) Umezawa, H.;
Nishimura, Y.; Tsuchiya, T.; Umezawa, S. J. Antibiot.
1972, 25, 743; (b) Umezawa, H.; Iinuma, K.; Kondo, S.;
Hamada, M.; Maeda, K. J. Antibiot. 1975, 28, 340; (c)
Umezawa, H.; Iinuma, K.; Kondo, S.; Maeda, K.
J. Antibiot. 1975, 28, 483.
11. Iinuma, K.; Murai, Y. Yuki Gosei Kagaku Kyokaishi 1999,
57, 368.
12. Tsuchiya, T.; Takagi, Y.; Umezawa, S. Tetrahedron Lett.
1999, 51, 4951.
13. MICs were determined by the twofold agar dilution
method according to NCCLS.