Synthesis and antibacterial activity of novel neamine derivatives

Article abstract of DOI:10.1016/j.bmcl.2006.09.007

Synthesis and activity of derivatives at the O5 or O6 positions of 1-N-((S)-4-amino-2-hydroxybutyryl)-3′,4′-dideoxyneamine, which is the neamine moiety of arbekacin, were reported. Among these results, the 5-O-aminoethylaminocarbonyl derivative showed eff

Full text of DOI:10.1016/j.bmcl.2006.09.007

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6351–6354
Synthesis and antibacterial activity of novel neamine derivatives
Nobuto Minowa,a,* Yoshihisa Akiyama,a Yukiko Hiraiwa,a
Kazunori Maebashi,a Takayuki Usuia and Daishiro Ikedab,
aPharmaceutical Research Department, 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 April 2006; revised 1 September 2006; accepted 5 September 2006
Available online 25 September 2006
Abstract—Synthesis and activity of derivatives at the O5 or O6 positions of 1-N-((S)-4-amino-2-hydroxybutyryl)-30,40-dideoxyne-
amine, which is the neamine moiety of arbekacin, were reported. Among these results, the 5-O-aminoethylaminocarbonyl derivative
showed effective activity against Staphylococcus aureus expressing a bifunctional aminoglycoside-modifying enzyme AAC(60)-
APH(200).
Ó 2006 Elsevier Ltd. All rights reserved.
Aminoglycoside antibiotics are clinically important
drugs used to treat various infections caused by
Gram-positive and Gram-negative bacteria.1 It is well
recognized that these antibiotics bind specifically in
the bacterial ribosome to the A-site of the decoding re-
gion of 16S rRNA and interfere with protein biosyn-
thesis, leading to bacterial cell death.2 Recently,
growing resistance to aminoglycosides has been ob-
served among bacterial species such as methicillin-resis-
tant Staphylococcus aureus (MRSA). The major
mechanism of resistance to aminoglycosides is their
inactivation by aminoglycoside-modifying enzymes
(AMEs) such as aminoglycoside acetyltransferase
(AAC), aminoglycoside phosphotransferase (APH),
and aminoglycoside adenylyltransferase (AAD)1 in
the bacterial periplasm. Modification of the antibiotic
by AME reduces its affinity for bacterial 16S rRNA
or decreases the membrane permeability of the drug.
To overcome this problem, the development of a novel
class of AME-resistant aminoglycosides is essential. In
the course of our work on such novel aminoglycosides,
we became interested in 1-N-((S)-4-amino-2-hydroxy-
butyryl)-30,40-dideoxyneamine 1, the neamine moiety
of arbekacin (ABK),3 as a lead substrate, since the nea-
mine core in aminoglycosides plays a critical role in
aminoglycoside binding to A-site RNA.1,2 Further,
neamine derivative 1 would be stable against AMEs
such as APH(30), AAD(40), and the bifunctional en-
zyme AAC(60)–APH(200)4 produced by MRSA. The
antibacterial activities of various neamine derivatives
have been studied to date;5 however, to our knowledge,
little has been known about the antibacterial activities
of derivatives of 1. In this paper, we describe the
synthesis of 1-N-((S)-4-amino-2-hydroxybutyryl)-30,40-
dideoxyneamine derivatives at the O5 or O6 positions
and the antibacterial activity of these derivatives.
Keywords: Aminoglycoside; Neamine; Methicillin-resistant Staphylo-
coccus aureus.
*
Corresponding author. Tel.: +81 455412521; fax: +81 455439771;
e-mail: nobuto_minowa@meiji.co.jp
Present address: Numazu Bio-Medical Research Institute, Microbial
Chemistry Research Center, 18-24 Miyamoto, Numazu city,
Shizuoka 410-0301, Japan.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.007

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N. Minowa et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6351–6354
Derivatives 8–11 possessing an amino alkyl side chain at
the O5 or O6 positions were prepared from 2 (Scheme
1). Selective deprotection4b,4c of 2 prepared from nea-
mine gave 3,20,60-tri-N-benzyloxycarbonyl-30, 40-dide-
oxyneamine. Then, N-tert-butoxycarbonyl (Boc)
protection of the amino group followed by treatment
of the resulting diol with 1,10-carbonyl diimidazole
(CDI) afforded cyclic carbonate 3. Reaction of 3 with
N-(benzyloxycarbonyl)ethylenediamine (N-Cbz-ethylene-
diamine) gave carbamates 4 and 5 which could be
separated by column chromatography on silica gel
(CHCl3/MeOH, 80:1), while the reaction of 3 with N-
(benzyloxycarbonyl)propanediamine gave carbamates 6
and 7 which could be separated by flash chromatogra-
phy on silica gel (CH2Cl2/MeOH, 50:1). Deprotection
of the N-Boc group of 4 with formic acid followed by
condensation with the N-hydroxysuccinimide ester of
(S)-4-p-methoxybenzyloxycarbonylamino-2-hydroxybu-
tanoic acid (PMZ-AHB) gave the corresponding amide,
which was deprotected by Pd–C catalyzed hydrogena-
tion to afford the desired 86,7. In a similar procedure,
5, 6, and 7 were converted to 10,7 9, and 11,
respectively.6
nas aeruginosa, including resistant strains, were tested.
Lead compound 1, the neamine moiety of ABK, showed
a good antibacterial spectrum compared to 30,40-dide-
oxyneamine (DN). Introduction of the aminoethylami-
nocarbonyl group at the O5 position of 1 resulted in
increased activity against both sensitive and resistant
S. aureus (compound 8). In particular, compound 8
showed effective activity against gentamicin (GM) and
ABK-resistant strains such as S. aureus RN4220/
pMF490 and MF490 expressing AAC(60)-APH(200)
and AAD(40). It is noteworthy that compound 8 is more
active than 1 and GM against P. aeruginosa GN4925
expressing AAC(60)-Ib. On the other hand, compounds
9, 10, and 11 showed significantly reduced activity
against S. aureus, E. coli, and P. aeruginosa. These re-
sults seem to indicate that the introduction of the 5-O-
aminoethylaminocarbonyl group led to enhanced stabil-
ity against modification by AMEs such as AAD(40)-I,
AAC(60)-Ib, and AAC(60)-APH(200).
Further, to obtain information on how the O5 or O6
side chains of the derivatives bind to the A-site RNA,
docking studies were performed for these derivatives.
Structural models of compounds 8–11 bound to the A-
site RNA were generated based on the crystal structure
of the A-site RNA, in complex with paromomycin2c and
Antibacterial activities of 8–11 are shown in Table 1.8
Staphylococcus aureus, Escherichia coli, and Pseudomo-
Scheme 1. Reagents and conditions: (a) NaH, DMF, 0 °C; (b) Ba(OH)2, dioxane, H2O, 80 °C; (c) (Boc)2O, Et3N, THF, H2O, rt, 51% from 2; (d)
CDI, THF, rt, 91%; (e) N-Cbz-ethylenediamine, CH2Cl2, rt, 4: 49%, 5: 30%; (f) N-Cbz-propanediamine, CH2Cl2, rt, 6: 24%, 7: 20%; (g) 1—HCOOH,
rt; 2—PMZ-AHB, N-hydroxysuccinimide, DCC, THF, rt; 3—10% Pd–C, EtOH, H2O, rt, 8: 52%, 9: 42%, 10: 15%, 11: 32%.

N. Minowa et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6351–6354
Table 1. Minimum inhibitory concentrations (MICs) of compounds 8–11
OH
6353
OH
O
O
HO
H2N
H2N
H2N
O
O
HN
R
NH2
HO
HN
N
H
HO
HO
H2N
HO
O
O
O
O
NH2
HO
NH2
Arbekacin
O
Gentamicin C1: R=CH3
Gentamicin C2: R=H
H2N
Test organism
AME
MIC (lg/ml)
8
10
9
11
16
16
DN
1
GM
0.25
0.25
64
ABK
Staphylococcus aureus RN4220
S.aureus RN4220/pMS520
S.aureus RN4220/pCR1948
S.aureus RN4220/pMF490
S.aureus MF490 (MRSA)
Escherichia coli NIH JC-2
Pseudomonas aeruginosa PAO1
P.aeruginosa GN4925
2
8
16
16
16
32
16
16
4
4
2
4
8
8
8
0.5
0.5
1
AAD(40)-I
AAC(60)-APH(200)
AAC(60)-APH(200)
2
2
2
8
8
8
8
8
8
16
16
16
>128
>128
>128
32
>128
>128
0.5
8
64
AAD(40), AAC(60)-APH(200)
32
32
128
32
64
64
1
4
4
8
>32
>32
>32
32
32
32
>128
64
4
AAC(60)-Ib
AAC(3)-I
128
64
128
128
64
16
128
128
P.aeruginosa GN3054
16
DN, 30,40-dideoxyneamine; GM, gentamicin (gentamicin C1, gentamicin C2); ABK, arbekacin.
the O5 side chain of 8 locates itself in the space near
the hydroxyl group O500 of paromomycin and forms
two hydrogen bonds with O6 and N7 of G14912a,2c
(Fig. 1).
In summary, we chose the neamine derivative 1 as a
lead compound and synthesized several derivatives
with side chains at the O5 and O6 positions. Among
these derivatives, 8 showed effective activity against
S. aureus expressing AAC(60)-APH(200) and P. aerugin-
osa expressing AAC(60)-Ib. This series of derivatives
offers a new perspective for the development of novel
aminoglycosides that will prove effective against resis-
tant bacteria.
Acknowledgments
We thank Mr. T. Watanabe (Meiji Seika Kaisha Ltd)
for molecular modeling studies. We also thank Dr. M.
Oyama and Miss. S. Miki (Meiji Seika Kaisha Ltd)
for NMR spectroscopic and mass spectrometric analy-
ses, respectively.
References and notes
Figure 1. Modeling of compound 8 (blue) and paromomycin (red)
bound to the A-site of 16S rRNA (green).
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1.80 (quin, J = 7.2Hz, 1H, H-300a), 1.88–1.96 (m, 1H, H-
300b), 1.99–2.03 (m, 1H, H-2b), 2.62–2.77 (m, 7H, H-20,
H-60, H-400, H-3000), 2.96–3.02 (m, 1H, H-3), 3.17–3.22 (m,
2H, H-2000), 3.51 (dd, J = 10.1, 8.9 Hz, 1H, H-4), 3.62 (t,
J = 10.1 Hz, 1H, H-6), 3.87–3.94 (m, 2H, H-1, H-50), 4.23
(dd, J = 7.8, 4.2 Hz, 1H, H-200), 4.75–4.79 (1H, H-5, J
value could not be correctly measured by overlapping
with the solvent peaks), 4.92 (d, J = 2.9 Hz, 1H, H-10);
FAB MS: m/z 478 (M+H)+. Compound 10: 1H NMR
(D2O/ND3, 400 MHz) d 1.37–1.46 (m, 1H, H-40a), 1.55
(q, J = 12.5 Hz, 1H, H-2a), 1.63–1.78 (m, 4H, H-30a, H-
30b, H-40b, H-300a), 1.81–1.89 (m, 1H, H-300b), 2.01 (dt,
J = 12.9, 2.9 Hz, 1H, H-2b), 2.62–2.74 (m, 6H, H-60, H-
400, H-3000), 2.88 (dt, J = 12.0, 3.9 Hz, 1H, H-20), 2.94–2.97
(m, 1H, H-3), 3.08–3.14 (m, 1H, H-2000a), 3.17–3.26 (m,
1H, H-2000b), 3.40 (t, J = 9.3 Hz, 1H, H-4), 3.76 (t,
J = 9.3 Hz, 1H, H-5), 3.87–3.89 (m, 1H, H-50), 4.01–
4.06 (m, 1H, H-1), 4.18 (dd, J = 8.6, 3.5 Hz, 1H, H-200),
4.60–4.66 (1H, H-6, J value could not be correctly
measured by overlapping with the solvent peaks), 5.15 (d,
J = 2.7 Hz, 1H, H-10); FAB MS: m/z 478 (M+H)+. NMR
assignments were made by interpretation of COSY
experiments.
6. Compounds 5–8 were purified by column chromatography
on CM Sephadex (NH4 form, gradient elution with 0.05–
+
1.1 N NH4OH).
7. Selected spectral data. Compound 8: 1H NMR (D2O/
ND3, 400 MHz) d 1.37–1.46 (m, 2H, H-2a, H-40a), 1.53–
1.59 (m, 1H, H-30a), 1.66–1.75 (m, 2H, H-30b, H-40b),
8. MICs were detemined by the two-fold agar dilution method
according to NCCLS.