New convenient reagents for chemoselective N-alkoxycarbonylation of (S)-isoserine: Application in the isepamicin synthesis

Article abstract of DOI:10.1080/00397910701569320

A synthesis of a series of N-alkoxycarbonyl mercaptobenzothiazoles (MBTs) and their application as reagents for chemoselective protection of amino group are presented herein. It was shown that all new reagents, Z-MBT, Fmoc-MBT, Phoc-MBT, and Tec-MBT, are highly effective in the selective N-alkoxycarbonylation of (S)-isoserine. The transformation is a simple, fast, and low-cost protocol, which is applicable in scale-up experiments. The starting MBT was fully recovered at the end of the process, which is an additional advantage of the method. The efficiency of the Z-reagent was also demonstrated by the selective protection of both gentamicin B and (S)-isoserine before their peptide-type coupling in the synthesis of the aminoglycoside antibiotic isepamicin. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/00397910701569320

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New Convenient Reagents for Chemoselective
N‐Alkoxycarbonylation of (S)‐Isoserine:
Application in the Isepamicin Synthesis
Konstantin Doktorov a , Velichko Tarpanov a & Pepa Mechkarova a
a Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian
Academy of Science, Sofia, Bulgaria
Published online: 19 Oct 2007.
To cite this article: Konstantin Doktorov , Velichko Tarpanov & Pepa Mechkarova (2007): New Convenient
Reagents for Chemoselective N‐Alkoxycarbonylation of (S)‐Isoserine: Application in the Isepamicin Synthesis,
Synthetic Communications: An International Journal for Rapid Communication of Synthetic Organic Chemistry,
37:21, 3709-3718
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Synthetic Communicationsw, 37: 3709–3718, 2007
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910701569320
New Convenient Reagents
for Chemoselective N-Alkoxycarbonylation
of (S)-Isoserine: Application in the
Isepamicin Synthesis
Konstantin Doktorov, Velichko Tarpanov,
and Pepa Mechkarova
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian
Academy of Science, Sofia, Bulgaria
Abstract: A synthesis of a series of N-alkoxycarbonyl mercaptobenzothiazoles
(MBTs) and their application as reagents for chemoselective protection of amino
group are presented herein. It was shown that all new reagents, Z-MBT, Fmoc-
MBT, Phoc-MBT, and Tec-MBT, are highly effective in the selective N-alkoxycarbo-
nylation of (S)-isoserine. The transformation is a simple, fast, and low-cost protocol,
which is applicable in scale-up experiments. The starting MBT was fully recovered
at the end of the process, which is an additional advantage of the method. The efficiency
of the Z-reagent was also demonstrated by the selective protection of both gentamicin
B and (S)-isoserine before their peptide-type coupling in the synthesis of the aminogly-
coside antibiotic isepamicin.
Keywords: chemoselective, gentamicin B, isepamicin, mercaptobenzothiazole,
N-protection, (S)-isoserine
INTRODUCTION
Isepamicin is an aminoglycoside antibiotic with activity against bacteria
producing aminoglycoside-inactivating enzymes.[1,2] Its molecule is con-
structed by a peptide-type bonded (S)-isoserine and gentamicin B building
Received in Poland February 15, 2007
Address correspondence to Konstantin Doktorov, Institute of Organic Chemistry
with Centre of Phytochemistry, Bulgarian Academy of Science, Acad. G. Bonchev
Str., Bl. 9, 1113 Sofia, Bulgaria. E-mail: kdoktorov@orgchm.bas.bg
3709

3710
K. Doktorov, V. Tarpanov, and P. Mechkarova
blocks. Thus, the chemoselective N-protection of both moieties are important
steps in the isepamicin synthesis.
An N-formylating agent (2a) has been obtained from mixed acetic–
formic anhydride and 2-mercaptobenzothiazole. We have applied this
reagent in gentamicin B protection, whereas N-formyl isoserine have
formed via a two-step protocol: total formylation of isoserine by a mixed
anhydride followed by O-deprotection.[3,4]
Benzyloxycarbonyl N-protective group was introduced in 1932 by
Bergman and Zervas and is widely applied in the field of peptide
synthesis.[5] Since then, a variety of urethane-type protective groups have
been developed.[6] Among them, fluorenylmethyloxycarbonyl (Fmoc), ben-
zyloxycarbonyl (Z), and trichloroethoxycarbonyl (Tec) are vastly used,
especially for the protection of amino acids. These protective groups are
easily formed and can be readily removed under appropriate conditions.
The latter present the main advantage of these compounds.[7] The classical
Schotten–Baumann reaction,[5,8–10] which involves the higher reactive
alkoxycarbonyl chlorides, leads to many side products. Recently, a variety
of alternative reagents has been developed.[11,12] Particularly where N-
selectivity has been desired, the mixed carbonates containing substituted
succinimidyl groups were used.[13–15] Because the alkoxycarbonyl group
is widely used for amine protection, the development of new reagents for
its insertion is of current interest.
Synthesis of a series of new chemoselective and cost-effective reagents,
based on mercaptobenzothiazol, for introduction of urethane-type N-protec-
tive groups in (S)-isoserine are presented herein. Benzyloxycarbonyl mercap-
tobenzothiazol thiocarbonate was further used for selective N-protection in
gentamicin B as a step in isepamicin synthesis.
RESULTS AND DISCUSSION
The known formylating agent 2-formylmercaptobenzothiazole (2a) was
prepared in a new way by condensation of formic acid and 2-mercaptoben-
zothiazole (1) with dicyclohexylcarbodiimide (DCC), and the obtained
product was successfully used in the next step, a direct N-formylation of
(S)-isoserine (3) (Scheme 1).[17]
Scheme 1. Preparation of N-formyl-(S)-isoserine: (i) formic acid (1.05 equiv), DCC
(1.05 equiv), ethyl acetate; (ii) formic acid (2 equiv), 2a (2 equiv), acetonitrile.

N-Alkoxycarbonylation of (S)-Isoserine
3711
Scheme 2. Preparation of R-oxycarbonylmercaptobenzothiazoles: (i) alkylchlorofor-
mate (1.1 equiv), Na2CO3 (2 equiv), THF; (ii) reagent 2b–e (1.1 equiv), Na2CO3 (1
equiv), H2O/THF.
The use of ethyl acetate in the first step allowed the product dicyclohex-
ylcarbamide to be quantitativily removed. The yields and reaction conditions
are summarized in Table 1. At the end of the reaction, the staring material 1
and the unreacted reagent 2a were almost fully recovered.
Further urethane-type protective reagents, Z-, Fmoc-, Phoc-, and Tec-
mercaptobenzothiazolthiocarbonates (2b–e), were prepared in high yields
throughout the acylation of 1 using the corresponding chloroformates in
THF in the presence of dry sodium carbonate (Scheme 2).
Mercaptobenzothiazolthiocarbonates (2b–e) were used to obtain the
desired N-protected (S)-isoserines (4b–e) in good yields (Scheme 2,
Table 1). The starting material (1) was fully recovered because of its lipophi-
licity. The latter is very important in scale-up experiments.
In addition, the amino groups in position 3 and 60 of gentamicin B
(5) were selectively acylated according to the known procedure.[3] The
reagent 2b was used instead of the formylating agent, and a yield of
Scheme 3. Preparation of isepamicin: (i) zinc pivaloate e (3.5 equiv.), DMSO; (ii) 2b
.
(2.5 equiv.); (iii) 4b (1.5 equiv.), HOBT H2O (0.5 equiv.), DCC (1.5 equiv.); (iv) Ra/
Ni, HCl.

3712
K. Doktorov, V. Tarpanov, and P. Mechkarova
83% was achieved. The product 6 was used to prepare the isepamicin (8)
(Scheme 3).
CONCLUSIONS
A synthesis of a series of new reagents for chemoselective N-alkoxycarbonyla-
tion based on the mercaptobenzothiazole scaffold is presented herein. The
N-alkoxycarbonyl mercaptobenzothiazoles, Z-MBT, Fmoc-MBT, Phoc-MBT,
and Tec-MBT, were obtained in excellent yields from the cheap and easily
available mercaptobenzothiazole (MBT). It was shown that all new reagents
are highly effective in the selective N-alkoxycarbonylation of (S)-isoserine.
The transformation is a simple, fast, and low-cost protocol, which is applicable
in scale-up experiments. The starting mercaptobenzothiazole was fully
recovered at the end of the process, which presents an additional advantage
of the method. The efficiency of the Z-reagent was also demonstrated by the
selective protection of both gentamicin B and (S)-isoserine before their
peptide coupling in the synthesis of the aminoglycoside antibiotic isepamicin.
EXPERIMENTAL
All reagents were purchased from Aldrich, Merck, and Fluka and were used
without any further purification. Gentamicin B is a gift from Actavis AD,
Sofia, and was purified by column chromatography. Thin-layer chromatography
(TLC) was done on precoated 0.2-mm Merck silica-gel 60 F254 (0.040–
0.060 mm) plates. Melting points (uncorrected) were determined on Mel-
Temp capillary melting-point apparatus, model 1102D-230 VAC (Barnstead
International). The IR spectra were taken on a Bruker IFS 113 v as KBr and
are quoted in centimeters21. The NMR spectra were recorded on a Bruker
Avance DRX 250 spectrometer; the chemical shifts were quoted in ppm in d
values against tetramethylsilane (TMS) as an internal standard, and the
coupling constants were calculated in hertz. The microanalyses were carried
out by the microanalyses service of the Institute of Organic Chemistry,
Bulgarian Academy of Sciences. The optical rotation was recorded with a
Perkin-Elmer 241 polarimeter.
(S)-Formylmercaptobenzothiazole (MBT-CHO, 2a)[3]
To a solution of 1 (44 g, 0.263 mol) in 1000 mL of ethyl acetate, 11.10 mL
(0.289 mol) of formic acid was added and stirred for 15 min. A solution of
57.00 g (0.276 mol) DCC in 300 mL of ethyl acetate was added dropwise at
room temperature, which was vigorously stirring for about 1 hour and

N-Alkoxycarbonylation of (S)-Isoserine
3713
stirred for 5 h. The reaction mixture was filtered. The solvent was evaporated
to dryness to obtain yellow crystals.
Mp 121–1258C; IR (KBr) 1720, 1450, 1300, 1270, 1130, 1000, 750; 1H
NMR (CDCl3) 10.07 (1H, s), 8.56–8.52 (1H, m), 7.48–7.25 (3H, m); MS
(m/z) 195 (Mþ), 167.
N-Formyl-(S)-isoserine (IS-CHO, 3a)[17]
To a suspension of 3 (15 g, 0.143 mol) in 300 mL of acetonitrile, 10.80 mL
(0.290 mol) formic acid was added and stirred until the solution was
formed. Compound 2a 56 g, (0.286 mol) was added at room temperature
with vigorous stirring. It was stirred about 6 h. The reaction mixture was
cooled in an ice bath for 1 h and filtered; mother liqueurs were concentrated
about one-fourth of the volume and filtered again. The solvent was evaporated.
The obtained white oil was boiled with ethyl acetate (2 ꢀ 50 mL) and acetone
(2 ꢀ 50 mL) to remove the formic acid. The obtained hygroscopic white
crystals were filtered and washed with cooled acetone.
20
jajD þ 3.838 (C ¼ 1, H2O); mp 94–968C; IR 3476, 3275, 1700, 1659,
1
1632, 1556; H NMR (MeOD) 8.09 (1H, s), 4.24 (1H, t), 3.54 (2H, m); MS
(m/z) 134 (Mþ), 115, 88.
General Procedure for Preparation of R-Oxycarbonylmercapto
benzothiazolyl-thiocarbonates (2b–e)
To a solution of 1 (5 g, 0.030 mol) in 45 mL of THF, 2 equiv. of sodium
carbonate (6.50 g, 0.060 mol) were added, and the suspension was stirred at
room temperature for 10 min. Solution of 1.1 equiv. of corresponding chloro-
formate (Table 1) in dry THF (15 mL) was added dropwise at room tempera-
ture with vigorous stirring. Sodium carbonate was filtered, and the solvent was
removed by vacuum distillation. The obtained yellow oil was crystallized with
50 mL of petroleum ether. The crude product was recrystallized from
methanol.
Data
(S)-1,3-Benzothiazol-2-yl-O-benzyl-thiocarbonate (Z-MBT, 2b)
Mp. 91–938C; Rf 0.45 (hexane–EtOAc, 9:1 v/v); IR 3417, 1742, 1454, 1423,
1368, 1313, 1255, 1213, 1142, 1076, 1022, 1011, 929, 912, 808, 753, 746, 726,
1
699, 667, 603, 492, 430; H NMR (CDCl3) 8.02 (ddd, J ¼ 7.9, 1.5, 0.6 Hz,
1H), 7.88 (ddd, J ¼ 7.9, 1.4, 0.6 Hz, 1H), 7.49 (ddd, J ¼ 7.9, 7.2, 1.4 Hz,
1H), 7.41 (ddd, J ¼ 7.9, 7.2, 1.5 Hz, 1H), 7.39 (m, 5H Ar-H), 5.36 (s, 2H,

3714
K. Doktorov, V. Tarpanov, and P. Mechkarova
-CH2-Ph); 13C NMR (62.89 Hz) 166.18, 157.76, 151.99, 136.42, 134.01,
129.02, 128.99, 128.83, 128.80, 128.77, 128,70, 126.39, 126.58, 123.02,
121.11, 70.64. Anal. calcd. for C15H11NO2S2:C, 59.78; H, 3.68; N, 4.65; S,
21.28. Found: C, 59.26; H, 3.58; N, 4.59, S, 21.01.
(S)-1,3-Benzothiazol-2-yl-O-(9H-fluoren-9-ylmethyl)-thiocarbonate
(Fmoc-MBT, 2c)
Mp. 98–1008C; Rf 0.48 (heptane–EtOAc, 5:1 v/v); IR 3050, 2944, 2870,
2280, 1700, 1545, 1418, 1311, 1224, 1144, 1070, 1016, 1003, 936, 829,
748, 732, 723, 621, 547, 541; 1H NMR (CDCl3) 8.04 (d, J ¼ 8.13 Hz, 1H, ben-
zothiazole), 7.88 (d, J ¼ 8.0 Hz, 1H, benzothiazole), 7.73 (d, J ¼ 8.0 Hz, 2H,
fluoren), 7.54 (d, J ¼ 7.5 Hz, 2H, fluoren), 7.43–7.29 (m, 2H, Ar-H, ben-
zothiazole), 4.62 (d, 2H, -CH22), 4.29 (tr, 1H,55CH- from fluoren); 13C
NMR (62.89 Hz) 128.05, 127.25, 126.45, 125.71, 125.00, 123.16, 121.17,
120.12, 70.68, 46.47. Anal. calcd. for C22H15NO2S2:C, 67.84; H, 3.88; N,
3.60; S, 16.47. Found: C, 68.10; H, 3.72; N, 3.65; S, 16.38.
(S)-1,3-Benzothiazol-2-yl-O-phenylthiocarbonate (Phoc-MBT, 2d)
Mp. 92–948C (MeOH); Rf 0.40 (hexane–EtOAc, 9:1 v/v); IR 3060, 1735,
1589, 1489, 1455, 1426, 1314, 1242, 1178, 1161, 1124, 1020, 1003, 906,
856, 757, 737, 725, 681, 651, 598, 492, 429; 1H NMR (CDCl3) 8.21
(d, J ¼ 8.00 Hz, 1H, benzothiazole), 7.90 (d, J ¼ 8.00 Hz, 1H, benzothia-
zole), 7.30–7.21 (m, 7H, 5 Ar-H, 2H benzothiazole); 13C NMR (62.89 Hz)
166.1, 157.8, 152.7, 151.5, 137.2, 130.4, 127.5, 127.2, 126.4, 123.8, 121.9,
121.8, 121.7, 121.6. Anal. calcd. for C14H9NO2S2:C, 58.528; H, 3.16; N,
4.87; S, 22.32. Found: C, 58.71; H, 3.03; N, 4.82; S, 22.30.
(S)-1,3-Benzothiazol-2-yl-O-(2,2,2-trichloropropyl)-thiocarbonate
(Tec-MBT, 2e)
Mp. 99–1028C (MeOH); Rf 0.52 (hexane–EtOAc, 9:1 v/v); IR 3051, 3017,
2957, 2213, 1720, 1450, 1420, 1357, 1310, 1260, 1064, 1020, 1003, 840,
800, 757, 710, 650, 564, 480; 1H NMR (CDCl3) 8.05 (d, J ¼ 7.50 Hz, 1H, ben-
zothiazole), 7.90 (d, J ¼ 7.50 Hz, 1H, benzothiazole), 7.54–7.26 (m, 2H, Ar-
H, benzothiazole), 4.94 (s, 2H, -CH2-); MS (m/z) 343 (Mþ); 13C NMR
(62.89 Hz) 166.6, 156.6, 152.8, 137.4, 127.3, 126.6, 124.0, 121.6, 94.3,
76.9. Anal. calcd. for C10H6Cl3NO2S2:C, 35.05; H, 1.76; Cl, 31.04; N, 4.09;
S, 18.72. Found: C, 35.28; H, 1.92; Cl, 31.18; N, 4.23; S, 19.00.

N-Alkoxycarbonylation of (S)-Isoserine
3715
General Procedure for Preparation of N-Protected-(S)-isoserines
(4b–e)
To a mixture of 3 (0.40 g, 0.0048 mol) in 8 mL of water and 1 equiv. of
sodium bicarbonate, a solution of corresponding R-oxycarbonylmercapto-
benzothiazolthiocarbonates (2b–e) (1.10 equiv.) in 24 mL THF was added
dropwise at 508C. After 1.5–2 h (Table 1) of vigorous stirring, the presence
of 4 was not established (TLC). The reaction mixture was diluted with
30 mL of 20% aqueous sodium bicarbonate. The organic solvent was evapor-
ated, and the precipitate of 1 was filtered off. The additional amount of starting
material (1) was extracted with ethyl acetate (3 ꢀ 30 mL). Later, the aqueous
layer was acidified (pH ¼ 3) with 20% sulphuric acid and extracted with ethyl
acetate (5 ꢀ 20 mL). The organic layer was dried (sodium sulphate) and
filtered, and solvent was evaporated. The obtained N-protected-(S)-isoserine
was recrystallized.
Data
(2S)-3-f[(Benzyloxy)carbonyl]aminog-2-hydroxypropanoic
Acid (Z-IS, 4b)
Mp. 125–1278C (acetone); Rf 0.46 (CHCl3–MeOH–100% CH3COOH,
18:3:1 v/v/v); [a]2D0þ3.188; IR 3432, 3340, 2950, 2129, 1745, 1690, 1535,
1495, 1456, 1377, 1271, 1186, 1149, 1116, 1088, 1079, 1001, 985, 950,
1
930, 911, 974, 780, 747, 731, 696, 636, 576, 466, 409; H NMR (DMSO)
7.24–7.36 (m, 6H, Ar-H þ -NH), 5.01 (s, 2H, Ar-CH2-O-), 4.02 (dd, 1H, J
7.0, 4.8 Hz), 3.31 (m, 1H, 1/2 of -CH2-N), 3.15 (m, 1H, 1/2 of -CH2-N);
13C NMR (62.89 Hz) 174.0, 156.2, 137.2, 128.4, 127.8, 127.7, 69.3, 65.3.
Anal. calcd. for C11H13NO5:C, 55.23; H, 5.48; N, 5.86. Found: C, 55.29; H,
5.46; N, 5.62.
Table 1. The yields and reaction conditions for the preparation of 2 and 4
Product 2
Product 4
Reagent
R
Time, h No Yield, % Time, h
No
Yield, %
HCOOH/DCC CHO
6
2a
2b
2c
2d
2e
94
97
98
98
93
7
4a
4b
4c
4d
4e
80
93
91
94
92
Z-Cl
Z
1.5
2
1.5
2
Fmoc-Cl
Phoc-Cl
Tec-Cl
Fmoc
Phoc
Tec
1.5
2
1.5
2
aBefore recrystalization.

3716
K. Doktorov, V. Tarpanov, and P. Mechkarova
(2S)-3-f[(9H-Fluoren-9-ylmethoxy)carbonyl]aminog-2-hydroxypropanoic
Acid (Fmoc-IS, 4c)
Mp. 166–1688C (MeOH); Rf 0.51 (CHCl3–MeOH–100% CH3COOH,
15:4:1 v/v/v); [a]2D0 22.358 (C ¼ 10, MeOH); IR 3274, 3061, 3026, 2928,
2868, 1946, 1908, 1742, 1695, 1606, 1477, 1445, 1418, 1371, 1363, 1314,
1208, 1190, 1152, 1097, 1072, 1049, 1025, 1006, 937, 867, 783, 757, 740,
1
726, 647, 621, 592, 562, 537, 429, 413; H NMR (DMSO) 12.54 (brs, 1H,
-COOH), 7.88 (d, 2H, J 7.5, from fluoren), 7.71 (d, 2H, J 7.5, from
fluoren), 7.37 (m, 5H, from fluoren and -NH-), 4.25 (m, 3H, -CH2-and
55CH-from fluoren), 3.33 (m, 2H, 1/2 of -CH2-N and OH), 3.17 (m, 1H, 1/
2 of -CH2-N); 13C NMR (62.89 Hz) 174.0, 156.2, 143.9, 140.7, 127.6,
127.1, 125.2, 120.1, 69.3, 65.5, 46.6, 44.2. Anal. calcd. for C18H17NO5:C,
66.05; H, 5.23; N, 4.28. Found: C, 66.15; H, 5.14; N, 4.42.
(2S)-2-Hydroxy-3-[(phenoxycarbonyl)amino]propanoic
Acid (Phoc-IS, 4d)
Mp. 89–918C (EtOAc); Rf 0.41 (CHCl3–MeOH–100% CH3COOH, 18:4:1
v/v/v); [a]2D0 þ5.298 (C ¼ 10, MeOH); IR 3432, 3349, 2926, 1744, 1707,
1535, 1492, 1455, 1383, 1263, 1225, 1168, 1113, 1078, 942, 881, 749, 686,
1
640; H NMR 12.10 (brs, 1H, -COOH), 7.77 (t, 1H, J 6.0, -NH-), 7.37
(t, 2H, J 7.5, m-Ar-H), 7.20 (t, 2H, J 7.5, p-Ar-H), 7.08 (d, 2H, J 7.5, o-Ar-
H), 4.09 (dd, 1H, J 7.5, 4.8), 3.39 (m, 1H, 1/2 of -CH2-N), 3.21 (m, 1H, 1/2
of -CH2-N); 13C NMR (62.89 Hz) 173.9, 154.5, 151.1, 129.2, 124.9, 121.7,
69.1, 44.4. Anal. calcd. for C10H11NO5:C, 53.33; H, 4.92; N, 6.22. Found: C,
53.40; H, 4.97; N, 6.09.
(2S)-2-Hydroxy-3-f[(2,2,2-trichloroethoxy)carbonyl]aminogpropanoic
Acid (Tec-IS, 4e)
Mp. 77–798C (H2O–MeOH, 5:1, v/v); Rf 0.47 (EtOAc–MeOH, 3:2 v/v);
[a]2D0 08 (C ¼ 1, MeOH); IR 3421, 3405, 3315, 3101, 2954, 1743, 1690,
1564, 1533, 1430, 1374, 1353, 1282, 1245, 1205, 1169, 1145, 1120, 1080,
1
1061, 1049, 1011, 954, 870, 813, 773, 727, 660, 570; H NMR 12.57 (brs,
1H, -COOH), 7.70 (t, 1H, J 6.0, -NH-), 4.78 (s, 2H), 4.04 (dd, 1H, J 7.0,
5.0), 3.33 (m, 1H, 1/2 of -CH2-N), 3.17 (m, 1H, 1/2 of -CH2-N); 13C-NMR
(62.89 Hz) 173.8, 154.5, 96.1, 73.4, 69.0, 44.4. Anal. calcd. for
C6H8Cl3NO5:C, 25.69; H, 2.87; Cl, 37.92; N, 4.99. Found: C, 25.67; H,
3.03; Cl, 38.03; N, 5.15.

N-Alkoxycarbonylation of (S)-Isoserine
3717
Preparation of Isepamicin
Preparation of 3,60-N-Di-carbobenzoxygentamicin B[14,16] (7)
To a solution of 5 (4 g, 0.0083 mol) in 50 mL of DMSO, 3.5 equiv. zinc
pivaloate (7.75 g, 0.0290 mole) were added and stirred at room temperature
for 90 min. Compound 2b 6.19 g, (3.5 equiv., 0.0205 mol) was added at
room temperature and stirred for 180 min. The reaction mixture was precipi-
tated into 400 mL of water and filtered through a small pad of Celitew. The
filtrate was diluted with water to a final volume of 700 mL and charged
onto a column containing 400 mL of Amberlite IRC-50 resin, which had
been adjusted to a partial ammonium cycle. The product was eluted with
0.75M ammonium hydroxide. The fractions containing the product were
pooled and lyophilized. Yield 5.17 g (83%).
Rf 0.35 (CH2Cl2–MeOH–NH4OH (25%), 50:35:5 v/v/v); 1H NMR
(D2O) 1.30 (s, 3H, C-CH3), 1.34–1.41 (dd, 1Hax, C2-H), 1.92–2.22 (dd,
1Heq, C2-H), 2.77 (s, 3H, N-CH3), 5.07 (s, 4H, -CH2-), 5.11 (J ¼ 4.2 Hz, d,
0
0
1H, C1 -H), 5.24 (J ¼ 3.6 Hz, d, 1H, C1 -H), 7.33–7.44 (m, 10H, Ar-H).
Preparation of Isepamicin Base[3]
To a solution of 6 (5 g, 0.0093 mol) in 15 mL water, 40 mL of methanol and
0.72 g (0.063 mol) 1-hydroxybenzotriazole hydrate were added. To this
solution, simultaneously solutions of 4.30 G (0.0208 mol) DCC in 35 mL of
methanol and 3.56 G (0.0140 mol) 3b in 35 mL of methanol were added
slowly (for 45 min). The reaction mixture was stirred at room temperature
for 60 min. The presence of 6 was not established (TLC). The reaction
mixture was diluted with 30 mL of methanol and 30 mL of water, stirred
for 20 min, and filtered. The filtrate was diluted with water to a final
volume of 360 mL, and 60 mL of conc. HCl was added. After 5 min of
stirring, 32 g of Ra/Ni in four portions were added over 120 min. The
reaction was stirred for 24 h at room temperature. The catalyst was
removed by filtration, and filtrate was neutralized (pH 7) with 20% NaOH.
The solution containing isepamicin base was filtered and charched onto a
column containing 300 mL of Amberlite CG-50 resin, which has been
adjusted to a partial ammonium cycle. The product was eluted with 0.75M
ammonium hydroxide. The fractions containing the product were pooled
and lyophilized. Yield 3.03 g (82%).
Rf 0.31 (CH2Cl2–EtOH–n-BuOH–NH4OH (25%), 5:5:4:2 v/v/v/v); 1H
NMR (D2O) (1.33, s 3H, C-CH3), (1.74–1.84, dd 1Hax, C2-H), (2.10–2.17, dd
0
1Heq, C2-H), (2.90, s, 3H, N-CH3), (5.16, J ¼ 3.96 Hz, d, 1H, C1 -H), (5.62,
20
J ¼ 3.83 Hz, d, 1H, C1 -H); [a]D þ 1148 (H2O,C ¼ 1).
0

3718
K. Doktorov, V. Tarpanov, and P. Mechkarova
ACKNOWLEDGMENTS
We gratefully thank ACTAVIS AD, Sofia, for supplying us with gentamicin B
and for the financial support.
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