An efficient and selective 1-N-monoethylation of sisomicin: Process development of netilmicin

Article abstract of DOI:10.1021/op000076f

With a new reagent developed for the selective monoethylation at the 1-amino group of sisomicin (1), a new process suitable for the mass production of netilmicin under conditions less sensitive to air and moisture has also been developed. Three of the amino groups, at the C-3, C-2′, and C-6′ positions of the four amino groups of sisomicin, were selectively protected by using Zn(OAc)₂ and acetic anhydride in methanol. Development efforts focused on optimising the conditions for ethylation to give an improved product (96% yield) according to the new and concise synthetic route.

Full text of DOI:10.1021/op000076f

Organic Process Research & Development 2002, 6, 78−81
An Efficient and Selective 1-N-Monoethylation of Sisomicin: Process
Development of Netilmicin
Ghilsoo Nam,* Sung Hoon Kim, Joong-Hyup Kim, Jung-Hyu Shin,† and Eun-Sook Jang‡
Biochemicals Research Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650,
Korea, School of Chemistry and Molecular Engineering, Seoul National UniVersity, Seoul 151-742, Korea, and Re Yon
Pharmaceutical Co. Ltd. 38-1, Dongbing go-Dong, Yong San-Gu, Seoul, Korea
Abstract:
originally developed by Schering Co., U.S.A. in 19764-6 and
launched onto the U.S. market early in the 1980s, it has been
widely used in Great Britain, Scandinavia, continental
Europe, Southeast Asia, Australia, and New Zealand as a
remedy for respiratory localized infections, urinary tract
infections, and bacteremia originating from Gram-negative
bacteria.
With a new reagent developed for the selective monoethylation
at the 1-amino group of sisomicin (1), a new process suitable
for the mass production of netilmicin under conditions less
sensitive to air and moisture has also been developed. Three of
the amino groups, at the C-3, C-2′, and C-6′ positions of the
four amino groups of sisomicin, were selectively protected by
using Zn(OAc)2 and acetic anhydride in methanol. Development
efforts focused on optimising the conditions for ethylation to
give an improved product (96% yield) according to the new
and concise synthetic route.
Even though there have been several efforts to develop
more economical and effective synthetic routes, problems
still exist in the selective monoethylation at the C-1 amino
group of sisomicin and in the selective protection of the three
amino groups of the C-3, C-2′, and C-6′ positions. Sisomicin
has in total, five amino groups: four primary (C-6′, C-1,
C-3, and C-2′ positions) and one secondary (C-3′′ position).
The reactivities of the four primary amino groups are similar
to each other, but that of the secondary position is slightly
different from these. It is difficult to undergo selective
ethylation at the C-1 amino group. Two methods for selective
ethylation have been reported. First, by controlling the pH
of the reaction media due to the pH-dependent reactivity of
amino groups,4 it was possible to accomplish selective
ethylation of the C-1 amino group through reductive alky-
lation with acetaldehyde and sodium cyanoborohydride. But
the formation of byproducts could not be avoided because
very precise pH control of the reaction media was necessary.
They were identified as the diethylated compound at the C-1
position and an ethylated compound at another amino group.
Second, by selective chelation of particular amino and
hydroxy groups, it was possible to protect the remaining free
amino groups and then to achieve the selective ethylation at
the C-1 amino group.5 However, a large excess of chelation
reagent, such as (Cu(OAc)2, Ni(OAc)2 or Co(OAc)2) was
required, and consequently, environmental and cost problems
arise. In addition, the reductive alkylation with acetaldehyde
and sodium cyanoborohydride produced the diethylated
compound as a byproduct.
Introduction
Sisomicin (1) is a naturally occurring aminoglycoside
antibiotic produced by Micromonospora inyoensis, while
netilmicin (4) is a semisynthetic aminoglycoside derived from
ethylation at the 1-amino group of sisomicin. The introduc-
tion of an ethyl group to the 1-amino group of sisomicin
was known to inhibit the acetylation by acetylase,2 resulting
in an improvement not only in the stability against ami-
noglycoside-resistant strains but also in antibacterial activity.
In clinical studies, netilmicin showed significantly less
cytotoxicity than tobramicin, and less nephrotoxicity than
either tobramicin or gentamicin.3
The formation of a diethylated side-product resulted in
intensive purification, lowered the yield, and increased the
cost of production. For this reason, we have focused the
process development of netilmicin in two areas: One is to
optimize the protection conditions of the three amino groups
at the C-3, C-2′, and C-6′ positions with Zn(OAc)2, and the
Antibacterial activity of netilmicin showed a generally
broader antibacterial spectrum than that of sisomicin, to-
bramicin, dibekacin, or gentamicin. Since netilmicin (4) was
* Corresponding author. Fax: +82-2-958-5189. E-mail: Gsnam@kist.re.kr.
† Seoul National University.
(4) Wright, J. J. J. Chem. Soc., Chem. Commun. 1976, 206-208.
(5) Nagabhushan, T. L.; Turner, W. N.; Tsai, H.; Jaret, R. S.; Shumacher, D.;
Chau, J. S. Carbohydr. Res. 1984, 130, 243-249.
‡ Re Yon Pharmaceutical Co. Ltd.
(1) Drugs Future 1982, 3 (7), 527-532.
(2) Antimicrob. Agent Chemother. 1979, 12, 474-478.
(3) Noone, P. Drugs 1984, 27, 548-578.
(6) Tann, C. H.; Heights, B.; Thirvengadam, U. K.; Chiu, J. S. U.S. Patent
4,831,123, 1989.
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10.1021/op000076f CCC: $22.00 © 2002 American Chemical Society and The Royal Society of Chemistry
Published on Web 01/18/2002

Scheme 1
Table 1. Protection of the 3,2′,6′-amino groups using
Zn(OAc)2 and (Ac)2O
Table 2. Result of selective C-1 mono-ethylation of
3,2′,6′-tri-N-acetylsisomicin (2)
equiv of
Zn(OAc)2
equiv of
(Ac)2O
equiv of
TEA
time
(h)
yielda
(%)
aratio of bratio of
temp time yield
entry
entry
AcOH
NaBH4
solvent
(°C)
(h)
(%)
1
2
3
4
5
6
4.0
4.0
4.0
2.5
2.5
2.5
3.2
4.0
4.0
3.2
3.5
3.5
7.0
7.0
7.0
5.0
5.0
-
24.0
48.0
20.0
15.0
18.0
18.0
79
82
92
85
96
51
1
2
3
4
5
6
7
8
9
3.3
3.3
3.3
3.0
3.0
4.0
4.0
4.0
4.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
8.0
8.0
THF
THF
THF
CHCl3
CHCl3
toluene
benzene
THF
rt
96
18
6
48
48
24
48
20
18
NRc
59
28
80
62
18
37
68
96
40
50
40
50
40
40
40
40
a Isolated yield.
CHCl3
other is to develop a selective monoethylation reagent to
avoid diethylation at the C-1 position. In this report, we
describe an optimized, efficient and selective 1-N-ethylation
method which uses a mixture of carboxylic acid and boron
metal hydride.7-9
a Ratio of acetic acid to sodium borohydride. b Ratio of sodium borohydride
to tri-acetylsisomicin. c NR means no reaction. Entry 9 is optimimum condition;
ratio of AcOH/NaBH4 is 4 and ratio of NaBH4/triacetylsisomicin is 8; thus, the
net molar ratio of net AcOH is 32-fold over triacetylsisomicin.
on silica gel to afford the 3,6′,2′-tri-N-acetylated sisomicin
in good yield and purity. (96% chemical yield; 99% chemical
purity.)
Results and Discussion
The synthetic route for netilmicin can be divided into three
steps, as shown in Scheme 1: protection of the amino groups
at the C-3, C-2′, and C-6′ positions, ethylation at the C-1
amino group of the protected sisomicin (2), and deprotection
of 3 to afford 4.
Step II: Monoethylation of the C-1 Amino Group. The
monoethylation of the C-1 amine is the key step because of
the potential formation of the diethylated byproduct. In
general, a series of N-alkylamine is more reactive than the
free amines for the reductive alkylation reaction. Even though
monoethylation had occurred, it was difficult to maintain
constant reaction conditions, such as pH, the amount of
reagent (especially acetaldehyde), and so on. Therefore, it
was inevitable that the diethylated compound was produced.
Accordingly, a new method for the specific ethylation in the
synthesis of 1-N-ethylsisomicin was employed. Gordon W.
Gribble’s7 reductive alkylation method of amine was applied
to this case. The reaction of sisomicin with a mixture of
sodium borohydride and acetic acid in chloroform gave the
monoethylated product in 96% yield. The results of these
reactions are shown in Table 2. The sodium borohydride was
added in several portions at room temperature, and the
reaction was carried out under the presented conditions at
several temperatures.
Step I: Protection of the Amino Groups at the C-3,
C-2′, C-6′ Positions. For the protection of the amino groups
at the C-3, C-2′, and C-6′ positions, Zn(OAc)2 was used as
the chelating reagent. In early investigations, the desired
product was obtained from the reaction of 1 with Zn(OAc)2
and acetic anhydride in methanol in 51% yield; however, it
contained tetraacetylated byproducts, which made the puri-
fication procedure tedious and lowered the yield. We
therefore made an effort to improve the reaction conditions
and found that the presence of triethylamine in the reaction
inhibits the formation of byproducts. The results are sum-
marized in Table 1. Use of 2.5 equiv of zinc acetate, 3.5
equiv of acetic anhydride, and 5 equiv of triethylamine
significantly improved the yield of the desired product.
Ammonia water removed chelating metal from the
protected sisomicin derivative, followed by chromatography
The optimized method in order to enhance the yield was
the use of a 4 M ratio of acetic acid and an 8 M of sodium
borohydride (see entry 8 in Table 2). Another way to improve
the yield was achieved by using chloroform at 40 or 50 °C.
Using other solvents, THF, toluene, or benzene lowed yields
in the range of 20-60%. A temperature above 50 °C or room
temperature also caused a reduction in the yield.
(7) Gribble, G. D.; Lord, P. D.; Scotnicki, J.; Dietz, S. E.; Eaton, J. T.; Johnson,
J. L. J. Am. Chem. Soc. 1974, 96, 7812-7814.
(8) Marchini, P.; Liso, G.; Reho, A. J. Org. Chem. 1975, 40, 3453-3456.
(9) Kim, J. H.; Kim, S. H.; Nam, G. S.; Kim, H. Y.; Son, H. J. U.S. Patent
5,696,244, 1997.
(10) Reetz, T. J. Am. Chem. Soc. 1969, 82, 5039.
Vol. 6, No. 1, 2002 / Organic Process Research & Development
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79

Table 3. Mono-1-N-ethylation using various reducing agents
ammonia water (1 L) and ethanol (2 L). The organic layer
was extracted with chloroform (2 L × 3) and then washed
with saturated brine (1 L). The organic layer was concen-
trated under vacuum. The resultant residue was subjected to
silica gel column chromatography (70-230 mesh, 1.5 kg)
using an eluting solution comprising chloroform, methanol,
and 14% ammonia water in the ratio of 2:1:1. Evaporation
of the appropriate combined fractions gave the title com-
pound (551 g, 0.96 mol) as an ivory solid: 96% yield. MS
(FAB) 574 (M + H+); 1H NMR (D2O, 300 MHz) δ 1.34 (s,
3H, C-CH3), 1.95 (s, 3H), 2.00 (s, 3H), 2.02 (s, 3H), 2.19
(m, 3H), 2.91 (s, 3H, N-CH3, 3.46-3.51 (m, 3H), 4.19 (dd,
1H, J ) 10.86 Hz, 3.54 Hz), 5.05 (d, 1H, J ) 4.61 Hz),
5.47 (d, 1H, J ) 1.83 Hz); 13C NMR (D2O, 75 MHz) δ
176.88, 176.53, 176.11, 148.04, 103.72, 99.79, 98.72, 86.80,
80.51, 77.66, 72.62, 70.34, 69.03, 66.17, 57.06, 53.18, 49.74,
48.19, 43.82, 37.21, 32.48, 26.41, 24.58, 23.61, 21.78.
Preparation of 1-N-Ethyl-3,2′,6′-tri-N-acetylsisomicin
(3). To glacial acetic acid (1.8 L, 32 mol) in chloroform (4
L) was slowly added sodium borohydride (300 g, 8 mol) at
20 °C. The addition rate was adjusted by observing the
generation rate of hydrogen gas. To this solution, a solution
of 3,2′,6′-tri-N-acetylsisomicin (573 g, 1 mol) solution in
chloroform (5 L) was added, and the mixture was stirred
for 20 h at 40 ( 5 °C. After being cooled to room
temperature, the resulting solution was neutralized with
saturated sodium hydroxide solution. This neutralized solu-
tion was diluted with ethanol (5 L), extracted with chloroform
(5 L), and distilled in vacuo. The residue was chromato-
graphed on a column of silica gel (2.5 kg) with 30:10:1
chloroform:methanol:14% ammonia water as eluant. Frac-
tions were collected, concentrated, and lyophilized to give
the title compound 3 as an ivory powder (578 g, 96%). MS
(FAB) 602 (M + H+); 1H NMR (D2O, 300 MHz) δ 1.27 (t,
3H, J ) 6.79 Hz, CH2CH3) 1.34 (s, 3H, C-CH3), 1.68-
1.78 (m, 2H), 1.96 (s, 3H), 2.00 (s, 3H), 2.02 (s, 3H), 2.12
(m, 1H), 2.31 (m, 1H), 2.91 (m, 1H), 2.92 (s, 3H, N-CH3),
3.08 (m, 1H), 3.26 (m, 1H), 3.47-3.51 (m, 3H), 3.64-4.00
(m, 8H), 4.23-4.27 (dd, 1H, J ) 3.36 Hz, 10.73 Hz), 5.05
(d, 1H, J ) 3.41 Hz, H-1′′), 5.48 (d, 1H, J ) 1.82 Hz, H-1′);
13C NMR (D2O, 75 MHz): δ 174.53, 174.20, 173.74, 145.74,
101.87, 97.44, 96.36, 84.26, 77.98, 75.30, 70.27, 68.04,
66.89, 64.04, 57.11, 54.77, 47.40, 45.85, 41.47, 41.14, 35.04,
27.86, 22.56, 22.36, 22.22, 21.19, 11.25.; Anal. for
C27H47N5O10‚15H2O. Calcd: C, 51.88; H, 8.02; N, 11.14.
Found: C, 51.50; H, 8.13; N, 11.40.
entry
reducing agent
solvent temp (°C) yielde (%)
1
2
3
4
NaBH(OAc)3a (7 equiv) CHCl3
NaBH(OAc)3b (7 equiv) CHCl3
40
40
40
40
87
78
72
96
KBH4/AcOHc
NaBH4/AcOHd
CHCl3
CHCl3
a It was synthesized in our laboratory by reference 10 and also contained
NaBH2(OAc)2 and NaBH3OAc. b It was purchased from Aldrich Chemicals.c The
same ratio as entry 9 in Table 2. d The same ratio as entry 9 in Table 2. e Isolated
yield.
Potassium borohydride or triacetoxy sodium borohydride
was used in place of sodium borohydride as the reducing
agent (Table 3). These reagents helped to reduce the
environmental problems compared to sodium cyanoborohy-
dride.
The desired product 2 was purified by extraction, and the
N-deacylation of 2 with aqueous sodium hydroxide gave 4
after purification by silica gel column chromatography in
over 90% yield. Each compound proposed during the
investigation was elucidated by comparing the NMR spectra
and the physical data of authentic samples.
Conclusions
Netilmicin 4 was synthesized from sisomicin (1) in a 73%
overall yield in three steps. Discovery of a monoethylation
reagent and optimization of monoethylation at the C-1 amino
group of 2 was successfully achieved. In addition, reduction
in the quantity of the chelating agent and the use of an
inexpensive reagent (Zn(OAc)2) together with simplification
of the workup led to a successful, economical, and practical
synthesis of netilmicin.
Experimental Section
Commercial reagent grade solvents were used. All reac-
tions were monitored by analytical thin-layer chromatography
(TLC, silica gel Baker-flex IB2-F plates) with fluorescent
indicator (254 nm), and visualized by ultraviolet light and
staining with anisaldehyde or ceric ammonium molybdate.
1H NMR spectra were recorded on a Bru¨ker AMX-300 (300
MHz) or AMX-500 (500 MHz), and δ values are given in
ppm relative to trimethylsilane as the internal standard. 13
C
NMR spectra were obtained with proton decoupling on a
Bru¨ker AMX-300 (75 MHz), or AMX-500 (125 MHz)
spectrometer and are reported in ppm with residual solvent
as the internal standard (77.0 for CDCl3). Mass spectra were
determined with a VG70-VSEQ mass spectrometer at 8 kV
ionizing voltage (FAB-Gunmicrobeam-35 keV, Cs+, ma-
trix: nitrobenzylalchol). Melting points were measured on
a Thomas-Hoover apparatus, and are not corrected.
Preparation of 3,2′,6′-Tri-N-acetylsisomicin (2). Siso-
micin (1) (447 g, 1.00 mol) was dissolved in methanol (5
L), and zinc acetate dihydrate (447 g, 2.5 mol) was added.
After the reaction mixture stirred at room temperature for
15 h, a solution of acetic anhydride (331 mL, 3.5 mol) and
triethylamine (696 mL, 5.0 mol) in tetrahydrofuran (1.5 L)
was added dropwise to the stirred solution at room temper-
ature over 1 h. The reaction mixture was concentrated under
vacuum, and the residue was dissolved in a mixture of 28%
Preparation of 1-N-Ethylsisomicin (4). 1-N-Ethyl-3,2′,6′-
N-acetylsisomicin (3) (330 g, 0.5 mol) was dissolved in 10%
sodium hydroxide solution (1 L), refluxed for 41 h under a
nitrogen atmosphere. After being cooled to room temperature,
the pH of the resulting mixture was adjusted to 9 with 1 N
sulfuric acid in an ice bath. The resulting solution was
concentrated under vacuum. 2-Propanol (4 L) was added to
the resultant residue to precipitate a solid that was filtered
off. The filtrate was subjected to column chromatography
on silica gel using chloroform, methanol, and 7% ammonia
water in the ratio of 40:20:7 as eluent. Lyophilizing of the
concentrated fractions gave the title compound 4 (190 g,
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Vol. 6, No. 1, 2002 / Organic Process Research & Development

80%) as an ivory powder; mp 100-103 °C. MS (FAB) 476
500 MHz) δ 1.33 (t, 3H, J ) 7.35 Hz, CH2CH3), 1.40 (s,
3H, 6′-CH3), 2.10 (q, 2H, J ) 12.5 Hz, CH2CH3), 2.42 (m,
1H, 3′-H), 2.65 (m, 1H, 2-H), 2.74 (m, 1H, 3′-H), 2.94 (s,
3H, 3′′-N-CH3), 3.13 (m, 1H, 2-H), 3.34 (m, 1H, 2-H), 3.49-
3.56 (m, 4H), 3.75-3.80 (m, 3H), 3.91-3.95 (m, 2H), 4.00
(d, 1H, J ) 12.8 Hz, 5′′-H), 4.14 (t, 1H, J ) 9.7 Hz), 4.27
(dd, 1H, J ) 10.77 Hz, 2.98 Hz, 2′′-H), 5.15 (dd, 1H, J )
2.75 Hz, 1′′-H), 5.20 (s, 1H, 4′-H), 5.62 (s, 1H, 1′-H); 13C
NMR (D2O, 125 MHz) δ 12.09 (CH2CH3), 21.97 (6′′-C),
24.86 (3′-CH3), 26.48 (2-C), 35.94 (7′′-C), 41.75 (6′-C),
42.20 (CH2CH3), 47.20 (2′-C), 49.34 (3-C), 57.41 (1-C),
64.84 (3′′-C), 67.78 (2′′-C), 68.79 (5′′-C), 71.08 (4′′-C), 74.71
(5-C), 80.01 (4-C), 84.01 (6-C), 99.40 (1′-C), 101.51 (4′-
C), 102.64 (1′′-C), 144.79 (5′-C). Anal. for C21H41N5O7
2.5H2SO4: Calcd C, 34.99; H, 6.43; N, 9.72; S, 11.12. Found
C, 34.70; H, 6.41; N, 9.87; S, 11.34.
1
(M + H+); H NMR (D2O, 500 MHz) δ 1.13 (t, 3H, J )
6.98 Hz, CH2CH3), 1.28 (s, 3H, 6′′-Me), 2.03 (m, 1H, 3′-
H), 2.21-2.25 (m, 3H, 2-H, 3′-H), 2.52 (m, 1H, 2-H), 2.58
(s, 3H, 7′′-N-CH3), 2.64 (dd, 1H, J ) 6.45 Hz, 0.83 Hz,
3′′-H), 2.77-2.84 (m, 4H, 3′-H, -CH2CH3), 3.14 (m, 1H,
2′-H), 3.38 (m, 3H, 6-H, 5′′-H), 3.53 (t, 1H, J ) 9.63 Hz,
4-H)), 3.63 (t, 1H, J ) 9.28 Hz, 5-H), 3.70 (q, 1H, J ) 7.08
Hz), 3.88 (dd, 1H, J ) 4.06 Hz, 3.96 Hz, 2′′-H), 4.06-4.10
(m, 1H, 5′′-H), 4.95 (d, 1H, J ) 3.86 Hz), 5.01 (d, 1H, J )
3.63 Hz, 1′′-H), 5.42 (s, 1H, 1′-H); 13C NMR (D2O, 75 MHz)
δ 11.85 (-CH2CH3), 19.70 (6′′-C), 22.76 (3′-C), 29.91 (2-
C), 34.89 (7′′-C), 40.40 (6′-C), 38.48 (-CH2CH3), 44.63 (2′-
C), 47.44 (3-C), 58.43 (1-C), 61.44 (3′′-C), 67.44 (2′′-C),
65.97 (5′′-C), 70.50 (4′′-C), 72.79 (5-C), 82.24 (4-C), 83.85
(6-HC), 98.01 (1′-C), 94.83 (4′-C), 99.49 (1′′-C), 146.62 (5′-
C).
Preparation of 1-N-Ethylsisomicin Sulfate. 1-N-Ethyl-
sisomicin (4) (130 g, 270 mmol) obtained from the previous
step was dissolved in water (1 L), and the pH of the solution
was adjusted to 4.5 by slow addition of a 1 N aqueous
sulfuric acid solution (682 mL, 680 mmol). To the resulting
solution was added methanol (2.5 L) followed by filtration
of a solid to give stoichiometrically the desired compound
Acknowledgment
We are grateful for the support of this research through
a grant from Re Yon Co. Ltd and Kook Min Bank.
Received for review July 6, 2000.
OP000076F
1
as a white powder; mp 182-183 °C dec. H NMR (D2O,
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