CA1152520A - Fortimicin b derivatives and process for production thereof - Google Patents

Abstract

ABSTRACT: .

The present invention relates to certain novel 4-N-substituted derivatives of the antibiotic Fortimicin B and processes for production thereof. Included in the process aspect of the invention, is a novel method for the semi-synthetic production of the antibiotic Fortimicin C.

Description

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BACKGROUND OF THE INVENTION
-The present invention relates ~enerally to 4-N-substituted derivatives of for-timicin B, their pharmaceutically acceptable acid addition salts, and a process for producing the same, as well as novel synthetic processes for producing the compounds fortimicin A and fortimicin C.
Fortimicins (A B and C) are compounds classified as pseudodi.saccharides containing 1,4-diaminocyclitol.
The physical properties and antibacterial activities of these compounds and processes for production thereof by fermentation are described in United States Patents Nos.
3,931,400, 3,976,768 and 4,048,015.
The planar structural formulae of the known fortimicins are shown in the above-mentioned United States specifications. As a result of further studies, it has been found that the structural formulae of fortimicin B, fortimicin A and fortimicin C are given by the following formulae:
Forti~icin B

O~ 1 3 OH

HO ~

pc/ ~) --I Fortimicin A
:. C~3 7 ~ CH-NH2 2 3 ', ~ O \ ~ OC~3 ~ -6 ~O W-CH3 " CH2NH`2J
8 Fortimicin C

~ NH2 CH-NH2 1 \ :
12 ~ \ ~ ~ OCH3 \

\~\ I ' 1l '' O C~2-NH-CNH2 6 Structures of fortimicins A, B and C are described below:
I? l) Fortimicin B
la : Results of mass spectrum of fortimicin B are given 19 below:
m/e 349(M + 1), 348(M ), 331, 313, 305, 235, 217, 21 207, 143, 126 22 It is presumed from the results of the mass spectrum 23 that fortimicin B has the following partial structural formula

2~ containing purpurosamine B.

26 CH~ NH2 27 : C~ X ~C8H17N203 Purpurosamine B

,,~,' ' , ~
. " ~
.

~ L~ S2S2.
`: :
I : The results from the analysis of PMR spectrum and CMR
2 spectrum of fortimicin B in deuterium oxide are given below: :

3 ,'

4 ~ PMR ~(ppm) Internal standard DSS
:~ H-l' 5.03 H-l 2.96 6 H-2' ~2.9 H-2 3.6B
2 . CH2-3'l4 1.2-1.9 H-3 3.62 H-5' 3.6 H-4 3.06 9 H-6' 2.80 H-5 3.96 C83 6 1.00 H-6 3.42 OCH3 3.42 .. NCH3 2.36.
13 Jl~2' 3.8Jl',2 9.5 .

5',6' J2',3 ~3 J4',5 . 4.5 17 ;; J5',6 18 Jl',6 s .
CMR ~(ppm) Internal standard Dioxane (67.4 ppm) 21 C-l' 102.4 C-l 53.8 22 2' 50.6 2 71.1 23 3' 27.1 3 79 9 2~ 4' 27.4 4 60.8 5' 75.1 5 71.1 26 6' 50.4 6 84.1 n 6'-CH3 18.7 OCH3 28 NCH3 36.0 From these data of PMR and CMR, and spectrophotometric 31 data of copper complexes of fortimicin B, diethylthioacetal ;; - 5 -~525;2~

I derivative of purpurosamine B portion, and methyl glycoside 2 derivative prepared according to the conventional processes, 3 the absolute strUcture of fortimicin B has been determined to be: -

6 4' ~ O CH3

7 ~ ~ ~\

13 Other physical data of fortimicin B are:
1~ ~ m.p. 101 - 103C
1s [~]23 = +30 3 (c=1.0, water) 16 ElementarY analysis as C15H32N4Os H2O
l7 Calculated: C, 49.15; H, 8.80; N, 15.29 IS Found: C, 49.36; H, 8.77; N, 15.38 19 2) Fortimicin A
Mass spectrum: m/e 406(M + 1), 405(M ), 388, 370, 21 362, 292, 274, 263, 246, 143, 126 22 PMR (deuterium oxide): ~(ppm), 1.06(3H,a), 1.2-1.9(4H,m), 23 2.8(1H,br), 2.86(1H,m), 3.05(3H,s), 3.44(3H,s), 2~ 3.5(2H,br), 3.52(2H,s), 3.88(lH,q), 4.08(lH,q), 4.16(1H,t), 4.36(1H,t), 4.84(lH,d), 4.95(lH,q) 26 CMR (deuterium oxide): ~(ppm), 18.7, 27.1, 27.3, 32.3, 27 41.6, 50.2, 50.5, 55.0, 56.4, 71.1, 73.0, 73.6, 75.1, 78.4, 100.1, 169.5 29 Other physical data of fortimicin A are:
m.p.: above 200C(dec.) 31 [~]D5 = +26.0 (c=0.2, water) , 6 , I

~ 5~520 Elementary analysiS as C17H35N5O6 7 Calculated: C, 50.35; H, 8.70; N, 17.27 3 Found: C, 50.23; H, 8.67; N, 17.49 4 From the foregoing data, and the fact that hydrolysis of fortimicin A produces fortimicin B and glycine, the structure of 6 fortimicin A has been confirmed to be 4-N-glycylfortimicin ~ (Ia).
9 3) Fortimicin C
Field desorption (FD) mass spectrum*: m/e 449(M + 1), Il Mass spectrum**: m/e 406, 387, 375, 325, 292, 274, 264, l2 246, 235, 228, 217, 207, 200, 143 13 PMR (deuterium oxide): ~(pp~), 1.08~3H,d), 1.2-1.9(4H~m), 1~ ~2.8(1H,br), 2.85(1H,m)~ 3.10(3H,s), 3.43(3H,s), 3.5(2H,br), 3.87(1H,q), 4.00 and 4.04 (jointly 2H, 16 individually s), 4.07(1H,q), 4;18(1H,t), 4.38(1H,t), 17 4.84(lH,d), 4.92(lH,q)

8 CMR (deuterium oxide): ~(ppm), 17.5, 26.3, 27.5, 32.5, 1~ ~ 44.1, 50.0, 51.1, 52.7, 55.5, 56.4, 71.0, 72.7, 73.1, 73.4, 77.9, 99.1, 161.7, 172.7 '^
2l * Mass spectrum where M+ is liable to appear.
22 ** M fails to appear in ordinary mass spectrum (EI mass spectrum) Other physical data of fortimicin C are given below:
t~ m.p.: 153-157C(dec.) 1~]D = +84.3~ (c=0.1, water) 26 Elementary analysiS as C18H36N6O7~2H2o n Calculated: C, 45.00; H, 8.33; N, 17.50 28 Found: C, 44.84; H, 8.19; N, 17.36 29 From the foregoing data, and a fact that hydrolysis of fortimicin C produces fortimicin B and hydantoic acid, the 3I structure of fortimicin C has been found to be 4-N-hydantoyl .

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I fortimicin B (IIb) ~fortimicins A and C have been synthetically 2 derived from fortimicin B as giVen in Examples, and the~ the 3 structures have been confirmed).
4 Fortimicins (A, B and C) all have antibacterial activity, but the antibacterial activity of B is not as good as the other 6 factors; and A and C are slightly unstable under strongly alkaline 7 conditions. Therefore, compounds having more distinguished 8 properties are in demand.

9 As a result of various studies, the present inventors have found that certain 4-N-substituted derivatives of fortimicin Il B have enhanced antibacterial activity and good stability even 12 under strongly alkaline conditions. Furthermore~ the 4-N-alkyl-13 fortimicin B derivatives can be used as starting materials for 1~ further modified derivatives, i.e. introduction of other group(s) to the amino or hydroxy group(s) thereof.

17 Compounds of the present inVention are 4-N-substituted 18 derivatives of fortimicin B represented by the general formula 22 ~ 2' \ I ~ OCH3 3 ~ ~ 5 ~ N-CH3 27 [wherein R shows a group represented by -C-Rl or -CH2-R2, 28 wherein Rl represents an alkyl group having 2 to 8 carbon atoms, 29 a hydroxyalkyl group having 1 to 5 carbon atoms, an aminoalkyl group having 2 to 8 carbon atoms, a carbamoxylaminoalkyl group 3I having 3 to 9 carbon atoms, R2 represents an alkyl group having .
I

2~

1 l to 8 carbon atoms, a hydroxyalkyl group having l to 5 carbon 2 atoms, an aminoalkyl group having l to 8 carbon atoms, a carba-3 moylaminoalkyl group having 2 to 9 carbon atoms, an N-alkylamino-~ alkyl group having 2 to lO carbon atoms, an aminohydroxylalXyl group having 2 ~o 8 carbon atoms, an N-substituted aminoalkyl 6 group (where the aminoalkyl group has 2 to 5 carbon atoms, and 7 the N-substituents is an aminoalkyl group having l to 5 carbon 8 atoms), or an ~-alkylaminohydroxyalkyl group having 2 to 8 carbon 9 atoms1, and their pharmaceutically acceptable non-toxic acid addition salts.
11 4-N-substituted derivatives of fortimicin B derived 12 from the compound represented by the general formula (IV), i.e.
13 the compounds of the present invention, are exemplified in the 1~ following Table l together with their physical values. For reference, the physical properties of fortimicins B and A are also given.
17 ~ In Table 2, the Rf values of thin layer chromatography 18 (TLC) of these compounds developed on a silica gel plate using 19 various solvents are shown. For a developed layer, a silica gel plate (Merck DC-Fertigplatten Kieselgel~ 0 F 254) was used, and 21 colored with ninhydrin or iodine. The solvent system given in 22 the table is as follows:
23 A: isopropanol-28% aqua ammonia-chloroform (2:1:1 by volume) B: methanol: chloroform (5.95 by volume) 26 C: methanol: chloroform (l.9 by volume) 27 The antibacterial activity (MIC) of the compounds of 28 the invention are shown in Tables 3-l and 3-2. Measurement was 29 carried out according to the agar dilution method, using a medium of pH 8.0, or the Japanese Antibiotic Medicament Standard, using 31 a medium of pH 7.2. Units are in mcg/ml.

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Microorganisms used in Table 3-2 and their abbreviations 2 are given below:
3 S.A.: Staphylococcus aureus RY4279 ATCC6538P
~ B.S.: Bacillus subtilis KY4273 E.C.: Eschericia coli KY4271 ATCC26 P.V.: Proteus vulgaris KY4277 ATCC6897 7 S.S.: Shigella sonnei KY4281 ATCC9290 8 S.T.: Salmonella typhosa KY4278 ATCC9992 9 K.P.: Klebsiella pneumoniae RY427~ ATCC10031 0 The identifying numbers of the compounds in Tables 3-1 ll and 3-2 are the same as in Table 1.
12 The stability of 4-N-acylfortimicin B derivatives in 13 aqueous alkaline conditions is poor. For example, if fortimicin l~ A free base was left standing in an aqueous solution (pH 10~ at room temperature for 2 weeks or at 100C for 4 hours, it would be 16 almost completely de~omposed. If 4-N-(y-Amino-~-hydroxybutyryl) 7 fortimicin B and 4-N-(~-amino-n-valeryl)fortimicin B were left 18 standing in aqueous solutions (pH 10), these were almost com-19 pletely decomposed within one hour at room temperature. There-fore, purification of these compounds under basic conditions (for B 21 example, column chromatography with Amberlite CG-50, eluting with 22 aqueous ammonia) is not practical.
23 In contrast with the instability of 4-N-acylfortimicin 2~ B derivatives, 4-N-alkylfortimicin B derivatives are so stable that no decomposition occurs even in aqueous barium hydroxide 26 solution at reflux temperature for 18 hours. Therefore, in the 27 case of 4-N-alkylfortimicin B, any substituents can be introduced 28 at 4-N-position of fortimicin B, even though such substituents 29 can not be introduced as the acyl type to such position due to the aforementioned instability.

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Table 2 Com- Solvent Rf Color Number Compound Name Syste~ Valu~ t~on 1 fortimicin B A 0.56 nin-hydrin 2 fortimicin A A 0.47 "
3 fortimicin C A 0.43 "
4 4-N-glycolylforti~icin B A 0.46 iodine 4-N-acetylfortimicin B A 0.43 6 4-N-propionylCo_timicin B A 0.46 7 4-N-(n-butyr~l)rortimicin B A 0.48 8 4-N-tn-valervl)fo~timicin B A 0.49 9 4-N-(~-alanyl)fortimicin B A 0.41 "
4-N-~y-amino-n-butyryl) A 0.44 "

11 4-N-(~-amino-n-valeryl) A 0.47 "
fortimiein B
12 4-N-(-amino-n-caproyl) A 0.42 fortimiein B
13 4-N-glycylglycyl_ortimicin B A 0.42 forti~iein B
14 4-N-~L-~ y-amino-~- A 0.41 hydroxybutyrvl~fortimicin B
4-N-(2-aminoethyl) A 0.41 "
fortimiein B
16 4-N-ethylfortimicin B A 0.60 "
17 4-N-(n-prooyl)fortimiein B A 0.66 18 4-N-~n-butyl)fortir.liein B A 0.71 "
19 4-N-~n-Dentyl)fo-timiein B A 0.72 4-N-~3-amino~ro~yl)- A 0.43 fortimiein B
21 4-N-(4-aminobutyl)- A 0.43 fortir.liein B
22 4-N-(5-aminonentyl)- A 0.47 forti~iein B
.~

- 17 ~

Table 2 (cont'd) ~ 5 ~ ~

Comnd Compound Name Solvent reac-Number System Value tion ___ ._ . _ .. . .
23 4-N-(6-aminohexyl)- A 0.49 iodine fortimiein B
24 4-N-(2-hydroxyethyl)- A 0.46 fortimiein B
4-N-[2-(2-aminoethyl~- A 0.36 aminoethyl]fortimicin B
26 4-N-(2-methylaminoethyl)- A 0.43 fortimicin B
27 4-N-[(S)-4-amino-2- A 0.27 hydroxybutyl]fortimicin B
28 4-N-[(S)-4-methylamino-2- A 0.34 hydroxybutyl]fortimicin B
34 1,2',6'-tri-N-earbobenzo:cy- C 0.47 foxtimiein B
1,2',6'-tri-N-t-butoxv- C 0.31 earbonylfortimiein 3 36 tetra-N-earbobenzoxv- B 0.66 fortimiein A
37 tetra-M-earbobenzoxy- B 0.54 (4-'~-glyeylglyeyl-fortimiein B) 38 tetra-N-earbobenzoxy- B 0.78 {4~ [L-(-)-y-amino-~-hydroxybutyryl]fortimiein B}
39 1,2',6'-tri-N-earbobenzoxy- C 0.49 4-N-ethylfortimiein B
tetra-N-ea_hobenzo:cy- C 0.,0 [4-N- (2-aminoethyl)fortimieinB]
41 tri-N-earbobenzoxy-14-N-(2- C 0.15 methylaminoethyl)fortimiein B]
2~ 4-N-t(R,S)-4-amir.o-2-hydroxy- A 0.27 butyl]fortimiein B
4-N-[(R,S)-4-methylamino-2- A 0.34 hydroxybutyl]fortimiein B
31 4-N-[~S)-3-amino-2-hydroxy- A 0~35 propyl]fortimiein B
32 4-N-[(S)-3-methyiamino-2- A 0.40 hydroxypropvl]fortimiein B
33 4-N-~(S)-5-amino-2-hydroxv- A 0.34 pentyl]fortimiein B
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~ I ~ ~ t~tn ~ u~ ~ ~ ~ u u Z I co co ~ ; ~ In o O O ~ O ~ ~ O v~
~D ~ ~I h O S l ~1 'a h Ql ~3 1 ~$ X nl * OD~ ~ h 0~ 0 0 ~ ~
C: I U~ Q. hh 111 ~0 1 ~ o ~0 ~
~ ~ ~ ~
~1 ~ U~ Q~ O rl U t~ ~1 U ~ 1 U U
01 ~ I U ~ U rl ~1 U r~ ~1 r~ rl ~S
"I ~ E E~ ~e E e ~ u ~ ~
l u ~ ~ . e v l : : : h ~ ~ O O
I ~ a~
I ~ e ~ ~ ~ h~ `
I Z U : O ~ I ~
~ rlG) .. ,...... -- .. ~ U
h O ~ ~ 'I ,~
I ,1 C~ 0 ~ _~ N t~
I h S ~ ' ~h q #
I ~ U~ 1 ~ *
u~ ~

~1~2~iZ~) Table 3-2 Minimum inhibition concentration ~MIC mcg/mQ, pH 8.0) \~licro-~organis~
~ ~ S~ BS ECPV SS ST KP
Compound\
Number \
_ .
1 12.5 12.5 25 25 50 12.5 50 2 0.04 0.04 0.16 0.32 0.63 0.16 0.16 3 0.16 0.32 0.08 0.63 1.25 0.32 0.63 4 1.25 5 1.25 2.5 5 1.25 5 12.5 50 50100 50 50 >200 6 12.5 50 50200 >200 50 >200 7 6.25 12.5 6.25 100 50 12.5 25 8 100 25 >100 >200 >200 >200 >200 9 0.08 0.08 0.63 0.63 1.25 0.32 0.32 0.32 0.16 1.25 1.25 2.5 0.63 2.5 12 >20 >20 >20>20 >20 >20 >20 13 0.32 0.32 1.25 2.5 5 1.25 2.5 14 0.63 5 0.63 10 20 2.5 5 0.16 0.16 0.32 0.64 1.25 0.32 0.63 16 1.56 3.13 3.13 6.25 6.25 3.13 ~ 50 17 .13 12.5 6.25 12.5 12.5 6.25 100 18 12.5 25 25 100 25 12.5 >200 19 25 200 100 >200 100 100 >200 1 ~' 1.25 2.5 5 10 20 1.25 80 21 0.32 0.32 1.25 2.5 2.5 1.25 5 22 1.25 0.63 5 10 5 2.5 20 23 5 5 20 >80 >80 20 >80 24 1.25 >80 5 10 10 1.25 40 0.63 1.25 1.25 5 5 1.25 5 26 - 0.04 0.32 0.16 1.25 5 1.25 5 27 0.04 0.04 0.16 0.32 1.25 0.16 0.32 28 0.04 0.04 0.08 0.32 0.63 0.08 0.32 29 0.08 0.0~ 0.16 0.32 0.63 0.16 0.63 0.08 0.02 0.32 0.16 0.63 0.0~ 0.32 3~ 0.16 0.32 2.5 1.25 2.5 0.63 2.5 32 0.16 0.63 2.5 2.5 2.5 0.63 5 33 0.32 0.32 1.25 2.5 5 1.25 10 .
(l~ote): The compound of compound number 11 was unstable ~ in an alkali condition ~p~l 8.0), and could not be measured.
.

1 As is evident from the foregoin~ data, the compounds of 2 the instant invention exhibit good antibacterial activity against 3 various microorganisms and are, therefore, useful as antibacterial ~ agents or antiseptics.
Similarly, the non-toxic acid addition salts of the 6 instant compounds have a wide antibacterial spectrum and are use-7 ful as antibacterial agents, etc. As used herein, the term non-8 toxic acid addition salts means the mono-, di-, tri- and tetra-9 salts obtained by reaction of one molecule of the compound repre-sented by said general formula (I) with l to 6 equivalents of 11 pharmaceutically acceptable, non-toxic acids. Suitable acids 12 include the inorganic acids such as sulfuric acid, hydrochloric 13 acid, hydrobromic acid, hydriodic acid, phosphoric acid, carbonic 1~ acid, nitric acid, etc., and organic acids such as acetic acid, fumaric acid, malic acid, citric acid, mandelic acid, succinic 16 acid, ascorbic acid, etc., amino acids such as aspartic acid, 17 etc., and the like. Thus, the compositions of matter aspect of 18 the present invention also in clude such pharmaceutically accept-19 able non-toxic acid addition salts.
The processes for synthesizing the compounds of the 21 invention are generally illustrated in the follo~Jing flow sheet I.
22 As illustrated, the compounds are synthesized through tl) step l , 23 step 2 and step 3 , (2) step l , step 5 and step 7 , or 2~ (3) step l , step 2 , step 6 and step 7 .
Nore particularly, when the desired compound represented 26 by the general formula (I) is a compound represented by R=-C-Rl, 27 it is synthesized through step l ~ step 2 ~ step 3 . ~hen 28 the desired compound represented by the general formula (I) is a 29 compound represented by R=-CH2R2, it is synthesized through step l ~ step 2 ~ step 3 ~ step 4 or through step l ~ step 31 5 ~ step 7 , or through step l } step 2 ~ step 6 ~ step 7.

.
, 252~
Among the compounds represented by the general formula 2 (I) thus obtained, the compounds represented by R=CH2R2 are more 3 stable under a strongly alkaline conditions than the compounds 4 represented by R-CORl.
The individual steps of the foregoing process will be 6 described in detail below. In the description, the compounds 7 represented by the general formulae (I), (Ia), (Ib), (II) .... (VII) 8 are sometimes referred to as compounds (I), (Ia), (Ib), (II) 9 (VII), correspondingly;
ID

., 2~

Flow Sheet 1 CH-NH2 . CH
\ NE12NH
~~h~o/~1~,}~ Ste~, 1 C\l3 CH3 (II) P~ ~ ~ CH-NHR3 R3 CIH3 CH-NHR3 CH30C}l3 ~ \ NH NH
\ 13 N- C-R'2 Steo 2 ~ ~ ~OH

R3 ~ ~V~ (III) 3 (IV) OCH3 /~ R' \ CH3 \ (VI) \ ~ CH-NHR3~ CH3 \~ ~ \ IH N-cH2-R 2 \ ~1 o ~ OH
R3 HO ~
\ tV) OCH3 \~
NEi 2 CH-NH~

C\Hc3~-NH2 NH2 ~ ~2 o ~ N CH3 ) ~N-C113 ~ tn O 3~ ¦~

( I ) ~ j~--OC113 H"N , 3 CtH 2R2 (I-b) ~S2~
1 Step l - Synthesis of compounds represented by formula 2 (III) from fortimicin B:
3 A compound represented by formu:La (III) in which one ~ of the hydrogen atoms of the amino group bonded to the carbon atoms at the l-, 2'- and 6'-positions of fortimicin B is masked 6 by an amino masking group (R3), can be obtained by reacting 7 fortimicin B free base with an amino-masking reagent in an ap-8 propriate solvent. For this step, amino-masking reagents usually 9 employed in peptide synthesis can be utilized. Examples of suitable amino masking reagents are:
Il : \
O o 1~ 5 ~ -CH2-O-C-O-N

16 R5 ~ CH2-O-C-Y, 2~ ~3C-C-O-C-S ~ ~ Y

z6 CH3 3 ~ \
O o O
2? Il 28 CH -O-C-Y, C2H5-O-C-Y, R -C!i -C-Y, 239 R7-CH2-C-OH, ~ S-OH, C ~ !

.~ ', . ~ .
.

~i2S2~

1 [wherein R5 and R6 may be same or different, and represent B, Z OH, ~2~ Cl, Br, I, alkyl groups (having l to 5 carbon atoms), 3 alkoxy groups (having l to 5 carbon atoms); R7 represents H, P, ~ Cl, Br, I or an alkyl group (having l to 5 carbon atoms), and Y
S represents Cl, Br or I].
6 Suitable solvents for the reaction include dimethyl-7 formam~ide, dimethylacetamide, tetrahydrofuran, dioxane, 1,2-9 dimethoxyethane, methanol, ethanol, acetone, water, or mixtures 9 thereof. Among these solvents, methanol is particularly prefer-able.
11 The concentration of fortimicin B in the reaction is 12 appropriately l to 250 millimoles/l, and l0 to l00 millimoles/l 13 is particularly preferred. The concentration of the amino-masking reagent is appropriately 4 millimoles/l to l mole/l, with 30 millimoles/l to 400 millimoles/l being preferable. The amount 1~ of the amino-masking reagent utilized in the reaction is appro-1~ priately l to 5 moles, and 3 to 4 moles per mole of fortimicin B
8 is preferred. In that case it is not favorable when the amount 19 of the amino-masking reagent is over 5 moles, because the hydrogen atom of the amino group bonded to the carbon atom at the 4-position 21 of fortimicin B is also masked by the amino-masking group, and the 22 yield of the desired formula (III) compound is lowered. On the 23 other hand, it is not favorable, either, that the amount of the 24 amino-masking reagent is below l mole, because the yield of the compound of formula (III) is lowered.
26 The reaction temperature is 0 - 60C, and preferably 27 0C to room temperature. Under such conditions the reaction time 28 is usually 2 to 18 hours.
29 The compound represented by formula (III) synthesized according to the foregoing process can be utilized directly in 31 the successive step as a reaction mixture or may be isolated and i ~

purified and then used in the next step.
~urification and isolation of the compound represented 3 by formula (III) Erom the reaction mixture can be accomplished ~ according to the following procedure. The solvent is distilled off from the reaction mixture, and a residue is obtained. The 6 residue is triturated with an organic solvent such as chloroform 7 or ethyl acetate to dissolve the extractable matters. Then, the 0 resulting extract solution is subjected to column chromatography 9 using silica gel (for example, Kieselgei 60 made by E. Merck, etc.).
Elution is then carried out with an organic solvent such as Il chloroform~methanol, or ethyl acetate-ethanol, etc., and fractions l2 showing a specific Rf value are collected and concentrated to 13 dryness, whereby the desired material is obtained in the form of 1~ a white powder.
~he compound represented by formula (III) thus obtained 16 can be utilized as a raw material for preparing compounds 17 ~represented by formulae (Ia) and (Ib) useful as antibacterial 18 agents, etc.
9 The compound represented by formula (Ia) is unstable in alkali conditions and, therefore, it is desirable in the prepara-21 tion of the compound represented by formula (III) to select an 22 amino-masking reagent which does not require an alkaline condi-23 tion to remove the masking group. Examples of amino~masking 2~ reagents satisfying these conditions are given below:

zg 6 1 , 3- ' ~
~7. Ii ~ .1 R53$~--CII !--O--C--Y, ~

? C:~3-C-O-C-S ~ ~

(wherein R5, R6 and Y have the same meanings as defined above).
11 The compound represented by formula (Ib) is stable in 12 both acid and alkali conditions, and when the compound represented 13 by formula (III) is utilized as a raw material for preparing the 1~ compound represented by formula (Ib), any amino-masking reagent can be utilized.
16 Step 2 - Preparation of Compound (IV) from Compound 17 (III): ~
Compound (IV) is obtained by acylating compound (III) 19 with an ordinary acylating agent in an appropriate solvent.
The acylating agent used herein includes carboxylic 21 acids represented by the general formula (VI), R'~COOH{wherein 2Z R'2 represents an alkyl group, hydroxyalkyl group, carbamoyl-23 aminoalkyl group, N-alkylaminoalkyl gxoup, N-alkylaminohydroxy-24 alkyl group, substituted aminoalkyl group (the substituent 2s represents an amino-masking group), substituted aminohydroxyalkyl 26 group (the substituent represents an amino-masking group), N-27 substituted aminoalkyl group [the substituent represents a 28 substituted aminomethylcarbonyl group (the substituent represents 29 an amino-masking group)1, where the amino-masking group may be same or different from said R3}, or derivatives of carboxylic 31 acids functionally equivalent to these, i.e., acid anhydrides of !
~- ~ - 28 -.

ll~Z5Z~0 I l~the carhoxylic acids represented by the general formula (VI), Z j active esters of said carboxylic acids with a compound selected 3 l~from the group consisting of ~O - N~ , t~
6 j ~ NO2~ ~O ~ N02, HNL==~ ~ and HO - N

i preferably HO - ~ , or acid halogenides, etc. of said lo carboxylic acids.
11 ~ When the acylating agent used herein contains a f~ee amino group, it is necessary to mask the amino group with an appropriate amino-masking group according to known procedures.
Of course, it is preferable to utilize the same masking groups as the amino-masking groups at the 1-, 2'- and 6'-positions of the 6 ;compound (III). The amino groups are masked in the same manner 17 ~as in said step 1.
18 The concentration of the compound ~III) used is in the 19 range of from 1 to 250 m~, preferably 10 to 100 mM. Equal or Z more moles of the acylating agent is used to the compound (III).
~1 When an acid anhydride is used as the acylating agent, it is 22 ` preferable to use 1 to 5 moles of the acid anhydride per mole of 23 the compound (III). When an active ester is used as the acylating 2~ agent, it is preferable to use 1 to 1.5 moles of the active ester per mole of the compound ~III).
26 Suitable solvents include dimethylformamide, dimethyl ~ acetamide, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, methanol, 2~ ethanol, water or mixtures thereof. Tetrahydrofuran is preferably used whatever acylating agent is used.
The reaction is carried out at a temperature of from 0 ~1 ' to 70C, preferably 0C to room temperature, for 15 minutes to 20 . I
~LlS2~2C~

I I hours and preferably 1 to 18 hours. - ~
2 ll In addition to the foregoing procedure, a DCC method, 3 j or the like can be applied to the acylation step.
~ Thus, compound (IV) is formed in the reaction solution, , and the reaction solution as such can be used in the preparation 6 of compound (Ia), or the compound (IV) can be isolated and then 7 used in the preparation of the compound (Ia~.
8 , Isolation and purification of the compound (IV) from 9 the reaction solution is carried out by first distilling off the 0 i solvent from the reaction solution. The resulting residues are 11 admixed with an organic solvent such as chloroform, ethyl acetate, l2 etc. to extract the soluble portions. The extract is then sub-l3 jected to column chromatography, using a column Eilled with 14 silica gel such as Kieselgel 60 (trade mark by E. Merck), or the ~5 like. Elution is carried out using an organic solvent system of 16 chloroform-methanol, ethyl acetate-ethanol, etc., and fractions 17 containing the compounds (IV) are collected. The solvent is then 8 removed whereby the compound (IV) is obtained.
~9 Step 3 - Preparation of Compound ~Ia) from Compound (IV) 21 The masking group R3 of the amino group of the compound 2Z (IV) obtained by step 2 is removed according to known procedure 23 to obtain compound (Ia). For example, when the masking group is 2~ a ben~yloxycarbonyl group, the masking group can be removed by catalytic hydrogenolysis in the presence of a metal catalyst of 26 palladiumcarbon, platinum, rhodium, Raney nickel, etc., and in ;the presence of an acid such as hydrochloric acid, hydrobromic 28 acid, acetic acid, etc. in a solvent of water, tetrahydrofuran, 29 j dimethylacetamide, dimethylformamide, lower alcohols, dioxane, ethyleneglycoldimethylether, or combinations thereof, etc., 31 preferably in methanol at room temperature and atmospheric pressure, ,"

, ,' ' ~L~5Z52~
,, .
, . .
I while passing hydrogen gas through the reaction mixture.
i Usually 1 to 10% by weight of the metal catalyst is 3 used on the basis of the compound (IV), and the concentration of ~ compound (IV) is usually 1 to 200 mM, preferably about 50 mM.
5 l; The acid is added to the reaction mixture so that pEI is 6 maintained 4 or less. The end of the reaction can be confirmed 7 by the completion of generation of carbon dioxide or by thin a layer chromatography, and the like.
9 When the masking group is a tertiary butoxycarbonyl lo group, its removal can be carried out in the presence of hydro-1l chloric acid or trifluoroacetic acid in a non-aqueous solvent, 12 for example, dichloromethane, chloroform, trichloroethylene, and 13 ethyl acetate. In such instance, compound (IV) is used at a 14 concentration of 1 to 200 m~, preferably about 50 ~l, and an equivalent amount or more of the acid is used. The completion 6 of the reaction is confirmed by thin layer chromatography, etc.
17 When the masking group is a triphenylmethyl group the 1~ masking group can be removed by treatment with acetic acid or 19 trifluoroacetic acid according to known procedure; and when the masking group is an orthonitrophenylsulfenyl group, the treatment tl is carried out with acetic acid or hydrochloric acid according to 22 known procedure to remove the masking group.
23 Separation and purification of the desired product are 2~ carried out according to known procedure using an ion exchange resin, silica gel column chromatography or the li~e. For exam-26 ple! according to a procedure using ion exchange resin, the t7 reaction mixture is filtered, if necessary, and the resulting 28 filtrate is evaporated to dryness. The residues are dissolved in 29 water and after the pH is adjusted to about 6 by an al~ali, for example, sodium hydroxide, the resulting solution is passed 31 through a column of, for example, ~mberlite~CG-50 (ammonium salt ., .
,l - 31 -l' l ~ .

s~o r~

~ form) to adsorb the desired product. Then, the column is sub-2 jected to elution with an appropriate concentration of ammonia 3 i solution to divide the eluate into fractions. The fractions ~ ,having an antibacterial activity are combined and evaporated to remove the solvent. The desi-red product is obtained as a powder.
6 Step 9 - Preparation of Compound (Ib) from Compound 1 (Ia) 8 The compound (Ia) obtained in step 3 , or fortimicin A
9 and fortimicin C obtained according to known methods is reduced in an appropriate solvent in the presence of a reducing agent for 11 ; converting the carbonyl group in the amide group to a methylene 12 group at room temperature or a solvent reflux temperature, whereby 13 compound (Ib) is obtained.
1~ As the solvent, tetrahydrofuran, dioxane, diethylether, etc. are appropriate. As the reducing agent, an excess amount, 16 usually l0-fold or more of lithium aluminum hydride, diborane, 17 etc. is used.
18 Purification of the desired product is carried out, for 19 example, with ion exchange resin in the following manner.
After the excess reducing agent in the reaction mixture is decom-21 posed by ethyl acetate, water, or the like, most of the solvent 22 is distilled off under reduced pressure. The resulting residues 23 in a semi-solid state are admixed with water to extract water-2~ soluble components, and the resulting extract is subjected to column chromatography in a column filled with weakly acidic ion 26 exchange resin (for example, Amberlite~CG-50). The column is 27 washed with waterj and then eluted with aqueous ammonia. Frac-28 tions containing the compound (Ib) are collected, and ammonia is 29 removed by evaporation whereby the compound (Ib) is obtained as a white powder.
- 31 The separation and purification can be also carried out ' ;

i - 32 - ~
,i , : . .

' ~ 5252~) .
I ilaccording to other ~nown procedures, such as silica gel chroma-t ,tography, etc.
3 l, Step 5 - Preparation of Compound (V) from Compound ~ , (III) Compound (V~ can be prepared by reacting compound (III) 6 with a compound represented by the general formula (VII), R2'CH~X
7 (wherein R2' has the same meaning as defined above, and X repre-8 sents chlorine, bromine, iodine, a methanesulfonylester group or 9 p-toluenesulfonylester group) in an appropriate solvent to alkylate o the compound (III). The concentration of the compound (III) used ll in the reaction is in a range of 1 to 250 mkl/l, preferably 10 to l2 100 mM/l. The amount of the compound (VII) used is 0.5 to 2 l3 moles, preferably 0.8 to 1.2 moles per mole of compound tIII).
1~ Suitable solvents include methanol, ethanol, propanol, butanol, tetrahydrofuran, acetone or their mixtures, and ethanol 16 is preferably used.
17 . The reaction is carried out at a temperature range of 18 0 to 120C, preferably 10 to 80C, for 2 to 24 hours, preferably 19 10 to 20 hours. The desired product thus obtained can be used, as such, in the next reaction without isolation or maybe first 21 isolated and purified in the following manner.
22 After the completion of the reaction, the solvent is 23 distilled off from the reaction mixture, and the residue is 2~ dissolved in an organic solvent such as ethyl acetate, chloroform, 2~ etc. After the organic solution is washed with water and dried, 26 the solvent is evaporated and then additional water is added 27 thereto. After washing by agitation, an aqueous ]ayer is removed, 28 the residue is applied to silica gel column chromatography and 29 the portions soluble in ~he organic solvent are extracted. The extract is subjected to column chromatography in a column filled 3~ with silica gel using, for example, (Kieselgel 60, trade mark of , . .
,l - 33 -!! !
.,,: i 1~1 5;~:5ZO
1 E. Merck Co.). EIution is then carried out with an organic 2 solvent such as chloroform-methanol, ethyl acetate-ethanol, etc., 3 and fractions containing compound (V~, checked by Rf values, ~ are collected. The solvent is removed by distillation, whereby compound (V) is obtained as a white powder.
6 Step 6 - Preparation of Compound (V) from Compound (IV) 7 Compound (V) can be obtained by reducing compound (IV) B obtained in step 2 in the presence of a reducing agent for 9 converting the carbonyl group in the amide group to a methylene group in an appropriate non-aqueous solvent at room temperature Il or a solvent reflux temperature.
12 For this step, suitable solvents include tetrahydrofuran, 13 dioxane, diethylether, etc. and combinations thereof. As the 1~ reducing agent, diborane, lithium aluminum hydride, etc. are IS used. In this reaction, compound (IV) is used at a concentration 16 of l to 250 m~l, preferably 10 to 100 m~l, and usually 10-fold or 17 more equivalents of the reducing agent is used. The reaction is 18 usually completed in from 10 minutes to 18 hours.
19 When the amino group of compound (IV) used in this step is masked by a benzyloxycarbonyl group, it is preferable to use 2I diborane as the reducing agent because the carbonyl group in the 22 amide group is converted to the methylene group without impairing 23 the benzyloxycarbonyl group of the compound tIV). Consequently 24 compound (V) can be obtained in good yield. [W.V. Curran and R.B. Angier: J. Org. Chem., 31, 3a67 (1966)].
26 When R2' of compound (IV) used has a masked amino group 27 in this step 6 , similar compounds, in addition to compound (V), 28 are formed, depending upon the masking group, reaction conditions, 29 and reducing agent. That is, when diborane is used as the reduc-ing agent, and the masking group at the masked amino group of R2' 31 of compound (IV) is a benzyloxycarbonyl group or a t-butyloxycar-_ 34 _ .

1 bonyl group, compound ~V), and compounds in which the masking 2 group at the amino group of R2' of the compound (V) i5 reduced to 3 a methyl group, are obtained. If the reaction time is shorter, ~ the former is principally formed and if the reaction time is prolonged, the yield of the latter is increased.
6 The resulting reaction product can be used as a raw 7 material for step 7 , as such, without isolating co~pound (V).
B Alternatively compound (V) can be isolated in the following 9 manner. The solvent is distilled off from the reaction mixture, and then the residue is admixed with water to decompose the 11 remaining hydride. Then, an organic solvent such as ethyl acetate, 12 chloroform, etc. is added thereto to extract the soluble compo-13 nents. After separation of the aqueous layer, the organic solvent 1~ layer is washed with water, dried with anhydrous sodium sulfate, etc., and the solvent is distilled off. The residue is dissolved 16 in an organic solvent such as chloroform, etc., and the desired 17 product is obtained by silica gel column chromatography.
18 When two end products are involved, these two products 19 can be separately obtained by fractionating the eluates, and if necessary by changing the eluting solvent.
21 Step 7 - Preparation of Compound (Ib) from Compound (V) 22 According to this step, the amino-masking group, R3, 23 of l, 2', 6'-tri-N-masked-4-N-alkyl (or substituted alkyl)fortimi-2~ cin B [compound (V)] obtained in said step 5 or 6 is removed . according to the known procedure based on step 3 in which the 26 compound (V) is used in place of the compound (IV), whereby 4-N-27 alkyl (or substituted alkyl)fortimicin B !compound (Ib)] is ob-28 tained.
29 An acid addition salt of compound (I) thus prepared can be obtained according to the following procedure. The compound 31 is first dissolved in water, and then admixed with an acid.

~. -- 35 --.. . .

I Then, a solvent capable of lowering the solubility of the compound2 (I), for example, ethanol, etc. is added to form a precipitate.
3 The precipitate is filtered and dried, whereby a white or grey ~ powder of the acid addition salts of compound (I) is obtained.
Certain specific embodiments of ~he invention are 6 illustrated by the follOwing representation examples wherein 7 Examples 1 - 2 illustrate embodiments for carrying out step 1 , B Examples 3-10 illustrate embodiments for carrying out step 2 , 9 Examples 11-18 illustra-te embodiments for carrying out step 5 , Examples 23 and 27 illustrate embodiments for carrying out step 6 , and Examples 24, 25, 26, 28, and 29 illustrate embodiments 1~ for carrying out step 7 .

U
U

-152S~ I

I Example 1 Preparation of 1,2',6'-tri-N-ben~yloxycarbonyl fortimi-2 cin B:
3 ' In this example, 1.8 g (5.2 millimoles) of fortimicin B
4 ' and 3 ml of triethylamine were dissolved in 100 ml of methanol, and a solution of 4.0 g (16.0 millimoles) of N-(benzyloxycarbonyl-6 oxy)succinimide in 50 ml of tetrahydrofuran was added dropwise 7 thereto with stirring under ice cooling (3-5C) over a period of 8 1.5 hours. After the completion of this addition, the solution 9 was stirred for 2 hours under ice cooling (3-5C), and then the solvent was evaporated under a reduced pressure. The residue Il (solid matter) thus obtained was dissolved in 200 ml of chloro-12 form, and the resulting solution was washed successively with 100 13 ml each of an aqueous 5% sodium bicarbonate solution and water.
4 After the washing, the chloroform solution was dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure.
16 The resulting solid residue was then dissolved in a small amount l7 of chloroform, and the chloroform solution was charged into a 18 column packed with 200 g of silica gel tKieselgel 60, trademark, 19 E. Merck Co.~. In this step, elution was carried out with a mixed solvent of methanol and chloroform (3:97 by volume) and an 21 elute was taken in 60 ml fractions. Fractions Nos. 23-50 con-22 taining compounds having an Rf value 0.47 in solvent system C in 23 Table 2 were combined. These fractions were concentrated to 24 dryness under reduced pressure. As a result 2.3 g of white powder was obtained.
26 Melting point: 79-82C
27 PMR spectrum (methanol-d4): ~(ppm) 1.02 (3H,d), 2~ 1.2-1.6 (4H,m~, 2.30 (3H,s), 2.90 (l~l,t), 29 3.42 (3}1,s), 3.5-4.0 (8H,m), 5.02 (6H,s), 5.36 (lH,d), 7.24 (15H,s) 31 1~]23 = +20.3 (c=l.0, methanol) l - 37 -I
~ '' ' i I Elemental analysis as C39H50N4Oll:
2 Found: C 62.68, H 6.93, N 7.17 (~);
3 Calculated: C 62.38, H 6.71, N 7.46 ~%) 4 From the data above described, it was confirmed that the obtained product was 1,2',6'-tri-N-benzyloxycarbonyl 6 fortimicin B, (3.1 millimoles, yield 59.6~).

8 Example 2 Preparation of 1,2',6'-tri-N-t-butoxycarbonylfortimi-9 cin B:
In this example 348 g (1.0 millimoles) of fortimicin B
was dissolved in 15 ml of methanol, and admixed with 960 mg (4.0 12 millimoles) of t-butyl-S-4,6-dimethylpyrimidin-2-ylthiocarbonate 13 synthesized according to a manner similar to that described in 14 Bull. Chem. Soc., ~apan, 46, 1269, (1973) by T. Nagasawa et al.
The reaction mixture was allowed to stand at room temperature for ~6 5 days with moderate stirring. The reaction mixture thus obtained ~7 was then concentrated to dryness under reduced pressure. There-18 after, 30 ml of ethylacetate and 20 ml of water were added to the ~9 residue and the mixture was stirred. The ethylacetated layer was washed twice with 20 ml.of water and then dried over anhydrous 2~ sodium sulfate. After drying, ethylacetate was evaporated under 22 reduced pressure to obtain a solid residue. The resulting solid 23 residue was dissolved in a small amount of chloroform and the 24 chloroform solution was charged into a column packed with 20 g of silica gel.
26 In this step, elution was carried out with a mixed 27 solvent of methanol and chloroform ~3:97 by volume) and the eluate 2B was taken in 6 ml fractions. Fractions Nos. 23-50 containing 29 compounds having an Rf=0.31 in solvent system C in Table 2 were combined. These fractions were concentrated to dryness under 31 reduced pressure. As a result 175 mg of a whlte powder was -~

, ll`~Z5~

I obtained.
2 PMR spectrum (methanol-d4): ~(ppm) 1.10 (3H,d), 3 1.2-1.6 (4H,m), 1.59 (27H,s), 2.35 (3H,s), ~ 3.5-4.0 (81~,m), 5.33 (lH,d).
Elemental analysis as C30H56~4OLl 6 Found: C 55.24, H 8.70, N 8.59;
? Calculated: C 55.54, H 8.70, ll 8.64 8 From the data above described, it was confirmed that 9 the product was 1,2,6'-tri-N-t-butoxycarbonylfortimicin B. (0.27 lo millimoles, yield 27%) 12 EXample 3 l3 In this example 1 ml of N,N-dimethylformamide, 31 mg 1~ (0.28 millimole) of hydantoic acid and 38 mg (0.28 millimole) of l-hydroxybenzotria~ole were dissolved. 58 mg (0.28 millimole) of 16 N,N'-dicyclohexycarbodiimide was added thereto, and the reaction 1? mixture was stirred under ice cooling (3-5C) for two hours.
18 The resulting solution was adr.lixed with 164 mg (0.25 9 millimole) of 1,2,6'-tri-N-t-butoxycarbonylfortimicin B obtained in Example 2 and allowed to stand at room temperature for 2 days 21 with stirring. Then, deposited insolubles were removed by filtra-22 tion, and the filtrate was concentrated under reduced pressure to 23 obtain a yellowish white solid residue [1,2',6'-tri-N-t-butoxy-2~ carbonyl-4-N-hydantoylfortimicin B].

26 Example 4 27 In this example, 751 mg (1.0 millimole) of 1,2', 6'-28 tri-N-benzyloxycarbonylfortimicin B obtained in a similar method 29 as in Example 1 was dissolved in 20 ml of methanol and 0.5 ml (5.0 millimoles) of acetic anhydride was added thereto. The 31 resulting reaction mixture was allowed to stand at room tempera-, .

115~

1 ture for 16 hoursO The resulting solution was then concentrated - 2 under reduced pressure and the residue wa~ dissolved in 20 ml .
3 of ethyl acetate and the solution was then washed successively e.~4;~ ~ with 10 ml each of an aqueous 5~ sodium bicarbonate solution and water and then dehydrated with anhydrous sodium sulfate. After 6 drying, the ethyl acetate solution was evaporated under reduced 7 pressure and 10 ml o~ n-hexane was added to the residue to wash 8 the residue with stirring. The n-hexane was removed by decan-9 tation to obtain a Iight yellowish white solid residue (1,2',6'-o tri-N-benzyloxycarbonly-4-N-acetylfortimicin B).

12 Example 5 13 . In this example the reactions were carried out using 1~ the method of Example 4 using an acid anhydride shown in Table A
1s to obtain a residue containing the compound (IV) shown in Table A. ~owever, equal molar amounts of the acid anhydride shown in 17 Table A were used in place of the acetic anhydride used in Example lB 4.
19 ", Table A
21 Compound used in place 22 of acetic anhydride Compound (IV) Propionic anhydride 1,2',6'-tri-N-benzyloxycarbonyl-4-23 N-propionylfortimiCin B
24 n-butyric anhydride 1,2',6'-tri-N-benzyloxycarbonyl-4-N-tn-butyryl)fortimicin B
n-valeric anhydride l,2~6~-tri-N-benzyloxycarbonly-4 26 N-(n-valeryl)fortimicin B

,.

~15Z52~
I Example 6 2 In this example 10 ml of tetrahydrofuran, 230 mg (1.1 3 millimoles) of N-benzyloxycarbonylglycine, 148 mg (1.1 millimoles) 4 of l~hydroxybenzotriazole and 227 mg (1.1 millimoles) of N,N'-dicyclohexylcarbodiimide were dissolved and the resulting reaction 6 mixture was stirred under ice cooling (3-5C) for one hour. Then, 7 750 mg (1.0 millimole) of 1,2',6'-tri-N-benzyloxycarbonylforti-8 micin B obtained in a similar manner as in Example 1 was added 9 thereto. The resulting mixture was stirred at room temperature for 18 hours. Then, insolubles were removed by filtration and the solvent in the filtrate was removed under reduced pressure.
12 The resulting concentrate, dissolved in a small portion (2 ml) 13 of chloroform, was charged into a column packed with 50 g of a I4 silica gel. Elution was carried out with a mixed solvent of S chloroform: methanol (2:98 by volume), and the eluate was taken 16 in 6 ml fractions.
17 Fractions Nos. 24-40 were combined in which fractions 18 are contained compounds having an Rf value 0.66 in solvent system 19 B in Table 2. These fractions were concentrated to dryness under reduced pressure. As a result 612 mg of a white powder was ob-21 tained having the following P~IR spectrum data (methanol-d4):
22 ~(ppm), 1.12 (3H,d), 1.2-1.9 (4H,br), 2.98 and 3.06 (3H jointly, 23 s,each), 3.33 (3H,s), 3.2-4.5 (llH,br), 4.95 (lH,d), 5.02 (8H,s), 24 7.20 (20H,s), It was thus confirmed that the product was tetra-N-benzyloxycar-26 bonylfortimicin A. (0.67 millimole, yield 67~).

' :.
.

- 115~
.
1 Example 7 2 - In this example, the same reactions were carried out in 3 a similar manner as in Example 6 using the N-benzyloxycarbonyl ~ derivatives shown in Table B to obtain the compounds (IV) shown in Table B, in which reaction equal molar amounts of N-benzyloxy-6 carbonyl derivatives shown in ~able B were used in place of N-7 benzyloxycarbonylglycine used in Example 6.
3 Table B
9 Compound used in place of N-benzyloxycarbonyl- Compound (IV) glycine 11 N-benzyloxycarbonyl-~- tetra-N-benzyloxycarbonyl-[4-N-(~-12 alanine alanylfortimicin B)]
N-benzyloxycarbonyl-y- tetra-N-benzyloxycarbonyl-t4-N-(y-3 amino-n-butyric acid amino-n-butyrylfortimicin B)]
1~ N-benzyloxylcarbonyl-~- tetra-N-benzyloxycarbonyl-[4-N-(~-amino-n-valeric acid amino-valerylfortimicin B)]
N-benzyloxycarbonyl-~- tetra-N-benzyloxycarbonyl-~4-N-(~-16 amino-n-caproic acid amino-n-caproylfortimicin B)]

IB Example 8 19 In this example 5 ml of tetrahydrofuran, 183 mg (l.l millimoles) of 0-benzylglycolic acid and 148 mg (l.l millimoles) 21 of l-hydroxybenzotriazole were dissolved and the resulting reac-22 tion mixture was stirred under ice cooling (3-5C). Then, 227 mg 23 (l.l millimoles) of N,N'-dicyclohexylcarbodiimide was added thereto, and after stirring under ice cooling (3-5C) for three hours, 75l mg (l.0 millimole) of l,2',6'-tri-N-benzyloxycarbonyl 26 fortimicin B obtained in a similar manner as in Example l was added thereto. The resulting mixture was then stirred at room 28 temperature for 17 hours. Deposited insolubles were removed by 29 filtration and the solvent in the filtrate was removed under reduced pressure. As a result, a light yellowish white solid 31 residue [l,2',6'-tri-N-benzyloxycarbonyl-4-M-(O-benzylglycolyl) . ' ~1525ZO

I fortimicin B] was obtained.

3 Example 9 ~ In this example 30 ml of tetrahydrofuran, 546 mg (2.1 millimoles) of N-benzyloxycarbonylglycylg~ycine and 279 mg (2.1 6 millimoles) of l-hydroxybenzotriazole were dissolved and the 7 resulting reaction mixture was stirred under ice cooling (3-5C).
8 Then, 426 mg (2.1 millimoles) of N,N`-dicyclohexylcarbodiimide g was added thereto, and after stirring under ice cooling (3-5C) lo for 2 hours, 1.50 g. (2.0 millimoles) of 1,2',6'-tri-1~-benzyloxy-11 carbonylfortimicin B obtained in a similar manner as in Example 1 12 was added thereto. The resulting reaction mixture was stirred at 3 room temperature for 18 hours. Deposited insolubles were removed by filtration and the solvent in the filtrate was removed under reduced pressure. The resulting concentrate, dissolved in a 16 small portion (2 ml) of chloroform, was charged into a column 7 packed with 100 g of a silica gel. In this step, 450 ml of a l~ mixed solvent of methanol and chloroform (2:98 by volume) was 9 used for washing the column and elution was also carried out with the same solvent. The eluate was ta~en in 17 ml fractions. Frac-2l tions ilos. 18-60 were combined in which fractions are contained 22 the compounds having an Rf value of 0.54 in solvent system B in ~ Table 2. These fractiGns were concentrated to obtain 1.25 g of a 2~ white powder.
From elemental analysis as C51H62N6O15~ that is~
26 Found: C 61.02, H 6.15, N 8.41 (%), 27 Calculated: C 61.31, H 6.26, N 8.41 (%) 23 it was confirmed that the obtained product was tetra-M-benzyl-29 oxycarbonyl-(4-~i-glycylglycylfortimicin B) (1.3 millimoles, yield 60%)-3l '' ` ` ,,: ~'~"' :

llS~SZ~

I Example 10 2 . In this example 20 ml of tetrahydrofuran, 557 mg (2.2 3 millimoles) of L-(-)-y-benzyloxycarbonylamino-~-hydroxybutyric ~ acid and 297 mg (2.2 millimoles) of l-hydroxybenzotriazole were dissolved and the resulting reaction mixture was stirred under 6 ice cooling (3-5C). Then, 454 mg (2.2 millimoles) of N,N'-7 dicyclohexylcarbodiimide was added thereto, and after stirring a under ice cooling (3-5C) for 1 hour, 1.52 g (2.0 millimoles) of 9 l~2~6~-tri-N-ben7yloxycarbonylfortimicin B obtained in a similar manner ~s in Example 1 was added thereto. The resulting reaction Il mixture was stirred at room temperature for 16 hours. Deposited l2 insolubles were removed by filtration and the solvent in the l3 filtrate was removed under reduced pressure. The resulting concentrate, dissolved in a small portion (2 ml) of chloroform, was charged into a column packed with 100 g of a siIica gel. In 16 this step, 350 ml of a mixed solvent of methanol and chloroform 17 (2:98 by volume) was used for washing the column and elution was lô also carried out with the same solvent. The eluate was taken l9 in 15 ml fractions. Fraction Nos. 15-45 were combined in which to fractions are contained the compounds having an Rf value of 0.78 21 in solvent system B in Tàble 2. These fractions were concentrated 22 to obtain 79 mg of a white powder having the following PMR data 23 (methanol-d4), ~(?Pm)~
2~ 1.12 (3EI,d), 1.2-2.0 (4H,br), 3.03 (3El,s), 3.33 (3H,s), 5.05 (8El,s), 7.26 (20H,s), 26 It was thus confirmed that the product was tetra-N-benzyloxy-27 carbonyl-{4-N-[L-(-)-y-amino-~-hydroxybutyryl]fortimicin B} (0.8 28 millimole, yield 40%).

.

.

`

: ~Lszsz~

~ Example 11 2 In this example, the solid residues ~1,2',6'-tri~ t-3 butoxycarbonyl-4-N-hydantoylfortimicin B) obtained in Example 3 4 were dissolved in 10 ml of a mi~ed solvent of trifluoroacetic and S dichloromethane (1:1 by volume), and the resulting reaction 6 mixture was allowed to stand with stirring at room temperature 7 for 16 hours. The solvent was removed under reduced pressure.
8 Then 5 ml of water was added to the resulting conce~trate and 9 insolubles were removed by filtration. The filtrate, adjusted to pH 6 by using lN sodium hydroxide, was charged into a column packed with 10 ml of Amberlite CG-50 (NH4 form, trademark, Rohm ~2 and Haas Company). After charging, 50 ml of water was used for l3 washing the column, and then elution was carried out with 0.15N
4 aqueous ammonia. The eluate was taken in 2 ml fractions. Frac-tion Nos. 31-45 were combined in which fractions are contained 16 the compounds having an Rf value of 0.43 in solvent system A in I7 Table 2. These fractions were concentrated to obtain 67 mg of 18 a white powder.
~9 The melting point, P~IR spectrum, mass spectrum, [~]D
and Rf value of thin layer chromatography of the thus obtained 21 compound were completely identical with those of fortimicin C.
22 Thus, it was identified that the compound was 4-N-hydantoyl-23 fortimicin B tfortimicin C). Yield 60~.

!' - 45 -iZ~
.; .
, ' .
I Example 12 2 , In this example concentrated hydrochloric acid (12N) is 3 diluted (60 times) with methanol to make a 0.2N hydrochloric ~ acid-methanol solution. Hydrochloric acid-methanol solutions having various normality hereinaf-ter used are prepared by a 6 similar manner.
7 In 20 ml of 0.2N hydrochloric acid-methanol solution, a 8 solid residue [1,2',6'-tri-~-benzyloxycarbonyl-4-N-acetylfortimicin 9 B] obtained in Example 5 was dissolved and 40 ms of 10~ palladium-charcoal was added thereto. Then, hydrogen gas was bubbled through the reaction mixture at room temperature under atmospheric pressure for 16 hours. After the completion of the hydrogenation 13 reaction, the catalyst was removed by filtra-tion and the solvent l~ in the filtrate was removed under reduced pressure. The resulting l5 concentrate, dissolved in 5 ml of water, was charged into a ~6 oolumn packed with 20 ml of Amberlite~CG-50 (NH4 form) after 17 adjusting the resulting solution to pll 6 using 1l~ sodium hydroxide.
I8 After charging, the column was washed with 60 ml of water, and 19 then elution was carried out with 0.15~ aqueous ammonia. ~he lo eluate was taken in 5 ml fractions. Fractions Nos. 16-31 were 21 combined in which fractions are contained -the compound having an 12 Rf value of 0.43 in solvent system A in Table 2. ~hese fractions 23 were concentrated to obtain 300 mg of a ~7hite powder having the 2~ following physical properties.
2s Mass spectrum m/e 391 (~+ + 1), 390 (M+), 373, 355, 347, 310, ~6 299, 277, 259, 2~9,-231, lal, 143, 97, 43, 27 PMR spectrum (deuterium oxide) ~(ppm): 1.04 (3H,d), 1.2-1.9 28 (4H,m), 2.18 (3H,s), 2.8 (3H,br), 3.12 (3H,s), 3.44 29 (3H,s), 3.5 (2H,br), 3.86 (lH,q), 4.08 (lH,q), 4.16 (l}l,t), 4.36 (lH,t), 4.80 (lH,d), 4.90 (lH,q).

. , ' .

'~

.

~15~

I From the above data, the compound was identified as t 4-N-acetylfortimicin B. Yield 77~. Then 250 mg (0.64 millimole) 3 . of 4-N-acetylfortimicin B obtained above described was dissolved ~ in 2 ml of water and the solution was adjusted to pH 2 with 5 N
S sulfuric acid. The thus obtained solution was poured into 20 ml 6 of ethanol to precipita~e at room temperature. After filtration 7 and drying, 352 mg (0.57 millimole) of 4-N-acetylfortimicin B
8 sulfate was obtained. Yield 85%. [~]25 = ~139.2 ~c=l.0, 9 water) Example 13 In this example, similar reactions, isolations and 13 purifications were repeated as in Example 12, except that resi-1~ dues containing 1,2',6'-tri-N-benzyloxycarbonyl 4-N-propionyl-fortimicin B, 1,2',6'-tri-N-benzyloxycarbonyl-4-N-(n-valeryl) 16 fortimicin B, obtained in Example 5, were used, respectively, in 17 place of the starting material, that is, the residues [1,2',6'-18 tri-N-benzyloxycarbonyl-4-N-acetylfortimicin B] used in Example 12.
9 As a result, 266 mg, 314 mg and 260 mg of white powder were obtained (each free base). The physical properties of the thus ~r 21 obtained compounds are shown below.
22 Product obtained when using 1,2',6'-tri-N-benzyloxy-23 carbonyl-4-N-propionylfortimicin B as the starting material:
2~ Mass spectrum m/e 405 (M + 1), 404 (M ), 387, 369, 361, 331, 324, 299, 291, 273, 263, 245, 227, 212, 195, 26 143, 126, g7 27 PMR (deuterium oxide): ~(ppm) 1.02 (3H,d), 1.08 28 1.2-1.9 (4H,m), 2.46 (2H,q), 2.8 (2H,br), 29 3.13 (3H,s), 3.45 (3H,s), 3.5 (2H,br), 3.87 (lH,q), 4.10 ~lH,q), 4.17 lH,t), 4.33 (lH,t), 4.79 (lH,d), 31 4.91 (lH,q) ~1~ 5Z5~

I Sulfate [~]D5 = +136.8 (c=l.0, water) 2 From these physical properties, the compound was identi-3 fied as 4-N-propionylfortimicin B. (0.66 millimole, yield 66~) ~ Product obtained when using 1,2',6'-tri-N-benzyloxy-carbonyl-4-N-(n-butyryl)fortimicin B as a starting material:
6 Mass spectrum m/e 419 (M + 1), 418 (M ), 338, 305, 299, 7 287, 277, 259, 241, 226, 209, 1~3, 126, 97, 43, 8 PMR (deuterium oxide): ~(ppm) 0.98 (3H,t), 1.00 (3H,d), 9 1.2-1.9 (6H,br), 2.42 (2H,q), 218 (2H,br), 3.02 and 3.14 (3H,s), 3.42 (3H,s), 3.5 (2H,br), 3.85 (lH,q), ll 4.10 (lH,q), 4.16 (lH,m), 4.35 (lH,t), 4.80 (lH,d), 12 4.89 (lH,q) 3 Sulfate [~]25 = +131.0 (c=l.0, water) From these physical properties, the compound was identi-fied as 4-N-(n-butyryl)fortimicin B. (0.75 millimole, yield 6 75%).
17 Product obtained when using 1,2',6'-tri-N-benzyloxy-18 carbonyl-4-N-(n-valeryl)fortimicin B as a starting material.
19 Mass spectrum m/e 433 (M + 1), 432 (M ), 415, 319, 301, 299, 291, 273, 233, 171, 143, 97, 43 21 PMR (deuterium oxide): ~(ppm) 0.96 (3B,t), 1.02 (3H,d), 22 ~ .9 (8H,br), 2.40 (2H,br), 2.90 (2H,br), 3.00 23 and 3.18 (3H,s), 3.43 (3H,s), 3.45 (2H,br), 3.90 2~ (lH,q), 4.18 (lH,m), 4.33 (lH,m), 4.82 (lH;d), 4.90 (lH,q) 2i Sulfate [~]25 = 116.3 (c=l.0, water) 26 From these physical properties, the compound was identi-27 fied as 4-N-(n-valeryl)fortimicin B (0.60 millimole, yield 60%).
2l3 ! ' . .

~ sz~
Example 14 Z In thïs example 20 ml of 0.2 N-hydrochloric acid-3 methanol solution, 500 mg (0.53 millimole) of tetra-N-benzyloxy-~ carbonylfortimicin A obtained in Example 6 was dissolved and about 30 mg of 10~ palladium-charcoal was added thereto. Then, 6 hydrogen gas was bubbled through the reaction mixture at room7 temperature under atmospheric pressure for 18 hours. After the 8 completion of the hydrogenation reaction, the catalyst was removed 9 by filtration, and the solvent in the filtrate was removed under lo reduced pressure. The resulting concentrate, dissolved in 5 ml B 1 of water, was charged into a column packed with 5 ml of Dowex x 4 12 (OH form, product of Dow Chemical Co., Ltd., U.S.A.). After 3 charging, 15 ml of water was used for washing the column.
Combined eluates were concentrated to obtain 212 mg (0.50 millimole) of white powder. This powder was completely 16 i~entical with fortimicin A standard in melting point, PMR spec-~7 trum, mass spectrum, [a]D and Rf value of thin layer chloromatog-18 raphy.
19 In 2 ml of water, 170 mg (1.40 millimoles) of fortimicin A thus obtained was dissolved and adjusted to pH 2 with 5N sul-Zl furic acid. The resulting solution was added dropwise to 20 ml 22 of ethanol and after filtration of the resulting precipitate 23 225 mg (0.33 millimole) of fortimicin A sulfate was obtained.
2~ (Yield 83%) Sulfate []D = +85.9 (c=1.0, water) n ~. 28 i~ , .
.' ' 1.
. .' 1~5Z5Z~
' E~ample 15 2 In this example, similar reactions, isolations and 3 purifications as in Example 14 were used, except that 0.50 milli-4 mole each of tetra-N-benzylo~ycarbonyl-[4-N-(~-alanyl)-fortimicin B], tetra-N-benzyloxycarbonyl-[4-~-(y-amino-n-butyryl~-fortimicin 6 B], tetra-N-benzyloxycarbonyl-[4-N-(~-amino-n-valeryl) fortimicin 7 B] and tetra-N-benzyloxycarbonyl-[4-N-(~-amino-n-caproyl)fortimicin 8 B], obtained in Example 7, were used, respectively, in place of 9 the starting material. As a result, 235 mg (free base), 298 mg o (free base), 310 mg (hydrochloride) (only this compound was isolated as a hydrochloride, since decomposition took place ~2 through the step of preparing the sulfate according to a similar manner disclosed in Example 14) and 208 mg (free base) were 4 obtained respectively. Physical properties of the respective products are shown below.
16 Product obtained when using tetra-N-benzyloxycarbonyl-1? [4-N-(~-alanyl)fortimicin B] as a starting material:
~8 ;lass spectrum m/e 419 (~1+), 402, 306, 288, 278, 271, 260, 19 235, 231, 214, 207, 143, 126, 97186.
Sulfate [~]23 = +80.6 (c=l.0, water) 21 ~ Elemental analysis as C18}137N5O6 2H2SO4 C2 5 2 22 Found: C 32.96, H 7.41, ~ 9.71 23 Calculated: C 33.14, H 7.51, N 9.66.
2~ From these physical properties, the compound was identi-fied as 4-N-(~-alanyl)fortimicin B. (Yield 96~) 26 Product obtained when using tetra-N-benzyloxycarbonyl-n l4-~1-(Y-amino-n-butYryl)fortimicin B] as a starting material:
2B Mass spectrum m/e 434 (M f 1), 433 (M ), 415, 390, 29 349, 331, 320, 302, 274, 235, 217, 207, 202, 189, 143, 126, 97, 86.
o 31 Sulfate [~]23 = +81.8 (c=l.0, water) :
~ - 50 - ~

.

~15252~1 1 ` Elemental anàlysis as Cl9H39N56 2H2S4 C2H5H 4H2O
2 Found. C 33.66, H 7.44, N 9.30.
3 Calculated: C 33.73, H 7.68, N 9.39 4 From these physical properties, the compound was identi-fied as 4-N-(y-amino-n-butyryl)fortimicin B. Yield 93%.
6 Product obtained when using tetra-N-benzylo:cycarbonyl 7 [4-N-(~-amino-n-valeryl)fortimicin B] as a starting material:
8 Hydrochloride [~]23 = +89.5 (c=l.0, water) 9 It was presumed that the compound was 4-N-(~-amino-o n-valeryl)fortimicin B.
Product obtained when using tetra-N-benzyloxycarbonyl 12 [4-N-(s-amino-n-caproyl)fortimicin B] as a starting material:
13 Mass spectrum m/e 461 (M+), 444, 348, 330, 320, 302, 1~ 271, 235, 207, 189, 143, 126, 114, 97, 86.
Sulfate [~]2 = +74.9o (c=l.0, water) 16 Elemental analysis as C21~43~15O6 2H2SO4 2 5 2 17 Found: C 37.20, H 7.62, M 9.45, 18 Calculated: C 37.34, H 7.76, N 9.47.
19 From these physical properties, the compound was identi-fied as 4-N-(s-amino-n-caprQyl)fortimicin B.

22 Example 16 23 In this example, the solid residue {1,2',6'-tri-N-24 benzyloxycarbonyl-[4-N-(O-benzylglycolyllfortimicin B]} obtained in Example 8 was dissolved in 10 ml of O.lN hydrochloric acid 26 method of solution and 40 mg of 10% palladium-charcoal was added 27 thereto. Then, hydrogen gas was bubbled through the reaction 28 mixture at xoom temperature under atmospheric pressure for 8 29 hours. After completion of the hydrogenation reaction, the catalyst was removed by filtration, and the solvent in the 31 fiLtrate was removed under reduced pressure. The resulting .

, s%~

I concentrate, dissolved in 5 ml of water, was charyed into a 2 column packed with 15 ml of Amberlite~CG-50 (NH4 form) after 3 adjusting the resulting solution to pH 6 using lN sodium hydroxide.
~ ; After charging, 40 ml of water ~as used for washing the column, and then, elution was-carried out with 0.2N aqueous ammonia. The 6 eluate was taken in 5 ml fractions. Fractions Nos. 8-14 were ? combined in which fractions are contained the compounds having an 8 Rf 0.46 in solvent system A in Table 2. These fractions were 9 concentrated to obtain 154 mg of a white powder.
From Mass spectrum data, Il ; m/e 406 (~1+), 389, 331, 247, 235, 207, 143, 126, It 97, 86.
I3 the product was identified as 4-N-glycolyl-fortimicin B.
l4 In 2 ml of water, 103 mg (0.25 millimole) of the 4-N-glycolylfortimicin B was dissolved and adjusted to p~ 2 with 5N
16 sulfuric acid. The resulting solution was added dropwise to 20 17 ml of ethanol and after filtration of the resulting precipitate, 18 91 mg (0.16 millimole) of 4-N-glycolylfortimicin B sulfate was l9 obtained.
Sulfate [~]23 = +89.3 (c=l.0, water) tl Elemental analysis as C17H34N4O7 1.5H2SO4 2 5 2 22 Found: C 36.70, H 7.44, N 8.93 23 Calculated: C 36.95, H 7.34, N 9.07 2~
Example 17 26 In this example, 1.22 gram of tetra-N-benzyloxycarbonyl-27 (4-N-glycylglycylfortimicin B) obtained in Example 9 was dis-28 solved in 35 ml of 0.2N hydrochloric acid-method solution, and 29 about 50 mg of 10% palladium-charcoal was added thereto. Then, hydrogen gas was bubbled through the reaction mixture at room 3I temperature under atmospheric pressure for 16 hours. ~fter , . . .

S25~
, , . .
I completian of the hydrogenation reaction, the catalyst was removed 2 by fil~ration, and the solvent in the filtrate was removed under 3 `reduced pressure. The resulting concentrate, dissolved in 10 ml ~ ;of water, and adjusted to pH 6 using lN sodium hydroxide, was S charged into a column packed with 30 ml of Amberlite~CG-S0 (NH4 6 form). After charging, 150 ml of water was used for washing the 7 column, and then, elution was carried out with 0.3N aqueous 8 ammonia. The eluate was taken in 5 ml fractions. Fractions Nos.
9 13-24 were combined in which fractions are contained the com-pounds having an Rf 0.42 in solvent system A in Table 2. T`nese fractions were concentrated to obtain 514 mg of white powder.
In 2 ml of water, 100 mg (0.25 millimole) of the com-l3 pound thus obtained was dissolved and adjusted to pH 2 with 5N
14 sulfuric acid. The resulting solution was added dropwise to 20 1S ml of ethanol and after filtration of the resulting precipitate, 16 123 mg of white powder was obtained. Physical properties of the 7 compound are shown below.
8 [~]25 = +70.1 (c=l.0, water) Found: C 33.27, H 7.11, N 10.81.
21 Calculated: C 33.24, H 7.17, N 11.07.
22 From these physical properties, the compound was identi-23 fied as 4-1l-glycylglycylfortimicin B sulfate. Yield 78~.

Example 18 26 In this example, 100 mg (0.19 millimole) of tetra-N-27 benzyloxycarbonyl {4-N-lL-(-)-y-amino-~-hydroxybutyryl]fortimicin 2B B) was dissolved in 10 ml of O.lN hydrochloric acid-methanol 29 solution and about 10 mg of 10~ palladium-charcoal was added thereto. Then, hydrogen gas was bubbled through the reaction 31 mixture at room temperature under atmospheric pressure for 7 , .
!, - 53 - ~

~.

~L~IS2~2~

., .
I hours. -After completion of the hydrogenation reaction, the 2 catalysL was removed by filtration and the solvent in the filtrate 3 was removed under reduced pressure to obtain 62 mg (0.14 milli-~ mole) of 4-N-~L-(-)-y-amino-~-hyclroxybutyryl]fortimicin B hydro-S chloride.

7 Example 19 8 In this example, 2.0 grams (4.9 millimoles) of forti-9 micin A obtained by a similar manner as in Example 14 was sus-lo pended in 200 ml of tetrahydrofuran and 2~0 grams (53.0 milli-ll moles) of lithium aluminum hydride was added thereto. The 12 resulting reaction mixture was heated at a reflux temperature 13 with stirring for 46 hours. After the completion of the reaction, 1~ the reaction mixture was cooled to room temperature and 30 ml 1S of ethyl acetate was added in drops to decompose the excess 6 lithium aluminum hydride. The ethyl acetate was removed under 17 reduced pressure. To the resulting residue, S0 ml of water was 18 added and the insolubles in the resulting mixture were removed by l9 filtration. The filtrate and water used for washing the fiitrate were combined. The solvent of the combined solution was removed 2l under reduced pressure and then 200 ml of an aqueous saturated 22 barium hydroxide solution was added to the concentrate, and the 23 solution was heated under reflux for one hour.
2~ The resulting solution was allowed to stand to cool, 2s and then was neutralized by adding dry ice. Then, the solution 26 was filtered, and the filtrate was charged into a column packed 27 with 100 ml of Amberlite~CG-50 (N~4 form). After charging, 28 400 ml of water and 600 ml of 0.3~1 aqueous ammonia were used for 29 washing the column, and then elution was carried out with 0.5N
aqueous ammonia. An eluate was taken in 20 ml fractions. Frac-3~ tion ~los. 6-20 were combined, in which fractions are contained - i - 54 -, .
, .
.

.~ . f ~lS~S2~

~ the compounds having an Rf value of 0.41 in solvent system A in 2 Table 2. These fractions were concentr,ated to obtain 608 mg of 3 white powder. Physical properties of the compound are shown ~ below.
~ Mass spectrum m/e 392 ~M + 1), 341, 282, 278, 250, 143, 126 6 P~iR (deuterium oxide): ~(ppm) 1.02 (3H,d), 1.2-1.9 (4H,m), 7 2.38 (3H,s), 2.4 (lH,br), 2.70 (~H,s?, 2.76 (lI~,mj, 8 3.09 (lH,q?, 3.16 (lH,t), 3.40 (3H,s), 3.5 (lH,br), 9 3.74 (lH~t); 3.85 (lH,q), 4.06 (lH,q), 4.16 (lH,q), lo 4.92 (lH,d).
Il CMR (deuterium oxide), ~(ppm): 18.6, 27.0, 27.3, 39.2, 40.6, l2 50.3, 50.5, 54.9, 57.5, 58.0, 60.7, 71.3, 71.~, 75.1, 13 76.9, 80.6, 100.6 14 From the foregoing data, the compound was identified as 4-N-(2-aminoethyl)fortimicin B. Yield 32.7%.
6 In 2 ml of water, 390 mg (1.0 millimole) of 4-N-(2-17 aminoethyl)fortimicin B was dissolved and adjusted to pH 2 with 18 5N sulfuric acid. The resulting solution was added dropwise to 19 20 ml of ethanol and after filtration of the resulting precipitate, 637 mg (0.91 millimole) of 4-N-(2-aminoethyl)fortimicin B sulfate 21 was obtained.
22 [ ]25 = +77 8 (c=l.0, water) 23 ElementarY analysis as C17H37NsOs 22H2 4 2 ;. 2 2~ Found: C 32.55, H 7.19, N 9.93 Calculated: C 32.56, H 7.21, N 9.99.

27 Example 20 In this example, 200 mg (0.49 millimole) of fortimicin 29 A obtained in a similar manner as in Example 14 was suspended in 10 ml of tetrahydrofuran and 10 ml of a tetrahydrofuran 31 solution containing 1 mole/l of diborane (10.0 millimoles).

1~525~

I Then, the resulting reaction mix~ure was s-tirred at room 2 temperature for two hours. After the completion of the reaction, 3 1 ml of water was added thereto in order to decompose excess 4 diborane. The resulting reaction mixture was concentrated to dryness under reduced pressure. Then 20 ml of 80~ hydrazine 6 solution was added to the residue, and the reaction mixture 7 was heated under reflux for 16 hours and then was concentrated 8 to dryness under reduced pressure. The resulting concentrate, 9 dissolved in 10 ml of water and adjusted to pH 6 with lN hydro-l chloric acid, was charged into a column packed with 10 ml of Amberlite~CG-50 (NH4 form).
After charging, 50 ml of water and 90 ml of 0.3N
13 aqueous ammonia were used for washing the column, and then, l4 elution was carried out with 0.5N aqueous ammonia. The elute was taken in 2 ml fractions. Fractions Nos. 9-36 were combined l6 and concentrated to obtain 146 mg of white powder. Physico-l7 chemical properties of the powder were identical with the 18 compound obtained in Example 18. The compound thus obtained was 19 4-N-(2-aminoethyl)fortimicin B. Yield 75.5%.
zo Zl Example 21 22 In this example, the procedures of Example 20 were 23 repeated, except that 0.5 millimole each of 4-N-acyl (or sub-24 stituted acyl)fortimicin B shown in the following Table D was used in place of fortimicin A.

2~

~3 !

1 ~able D
2 ` Example No. Compound used 3 ' 21-1 4-N-acetylfortimicin B
4 ~, 21-2 4-N-propionylfortimicin B
21-3 4-N-(n-butyryl)fortimicin B
6 21-4 4-N-(n-valeryl)fortimicin B
7 21-5 4-N-(~-alanyl)fortimicin B
8 21-6 4-N-(y-amino-n-butyryl)fortimicin B
9 21-7 4-N-(~-amino-n-valeryl-fortimicin B
lo 21-8 4-N-(s-amino-n-caproyl)fortimicin B
1l 21-9 4-N-glycolylfortimicin B
12 ; 21-10 4-N-glycylglycylfortimicin B
13 It was identified from the respective physical properties thus 14 that the following products were obtained.
~5 (1) name of the compound, (2) amount, (3) yield and (4) physical 16 properties of the powder thus obtained are given below.

18 Example 21-1 (1) 4-N-ethyl fortimicin B
(2) 125 mg (0.33 millimole) 21 ., (3) 66%
22 (4) Mass spectrum:
23 m/e 377 (M + 1), 376 (M ), 359, 344, 327, 314, 24 299, 286, 273, 263, 235, 217, 215, 202, 143, 114 PMR (deuterium oxide):
26 ~(ppm): 1.02 (3H,d), 1.08 (3H,t), 1.2-1.9 (4H,m), 27 2.40 (3H,s), 2.6-3.0 (4H,m), 3.12 (lH,q), 3.18 (lH,t), 3.42 (3H,s), 3.40 (lH,br), 3.74 (lH,t), 29 3.85 (lH,q), 4.08 (lH,q), 4.17 (lH,t), 4.92 (lH,d).
', ,, 1~ - 57 -.

I Example 21-2 2 ~1) 4-N-(n-propyl)fortimicin B
3 (2) 136 mg (0.35 millimole) ~ (3) 70%
(4) Mass spectrum:
6 . m/e 390 (M+), 373, 361, 358, 344, 341, 328, 7 287, 277, ~49, 231, 229, 219, 202, 143, 128 9 Example 21-3 (1) 4-N-(n-butyl)fortimicin B
Il . (2) 137 mg ~0.34 millimole) 12 (3) 68%
13 (4) Mass spectrum:
m/e 404 (M+), 372, 361, 342, 301, 291, 286, IS . 263, 219, 202, 143, 142 16 :~
17 Example 21-4 s (1) 4-N-tn-pentyl)fortimicin B
l9 (2) 167 mg (0.40 millimole) (3) 80%
(4~ Mass spectrum:
22 m/e 419 (N + 1), 418 (M ), 386, 369, 361, 356, ~ 344, 331, 326, 315, 305, 277, 259, 219, 207, 2~ 202, 156, 143 26 Example 21-5 2? (1) 4-N-(3-aminopropyl)fortimicin B
2~ (2) 117 mg (0.29 millimole) 29 (3) 58%
(4) Mass spectrum:
31 m/e 406 (M + 1), 373, 338, 328, 292, 264, .

~s~o I 231, 228, 219, 202, 196, 172, 143, 126, 1~0, 2 89, 58 3 PMR (deuterium oxide):
~ ~(ppm): 1.04 ~3H,d), 1.2-1.9 (6H,m), 2.42 (3H,s), ~ 2.5-3.0 (6H,m), 3.14 (lH,q), 3.20 (lH,t), 3.44 6 (3H,s), 3.4 (lH,m), 3.79 (lH,t), 3.88 (lH,q), 7 4.06 (lH,q), 4.18 (lH,t), 4.94 (lH,d) Sulfate: [~]D3 = +71.9 (c=l.0, water) 9 Elementary analysis as C18 39 5 2 4 2 5 2 Io Found: C 32.04, H 7.80, N 8.99, II Calculated: C 31.99, H 7.52, N 9.33 13 Example 21-6 I~ (1) 4-N-(4-aminobutyl)fortimicin B
I5 (2) 75 mg (0.18 millimole) 16 (3) 35%
7 (4) Mass spectrum:
18 m/e 240 (M + 1), 419 (M ), 370, 352, 342, 306, 19 278, 219, 210, 207, 186, 157, 143, 103, 72 PMR (deuterium oxide):
21 ~(ppm): 1.02 (3H,d), 1.2-1.9 (8H,m), 2.42 (3H,s), 22 2.5-3.0 (6H,m), 3.06 (lH,t), 3.09 (lH,t), 3.44 23 (3H,s), 3.4 (lH,m), 3.78 (lH,q), 3.86 (lH,q), 24 4.04 (lH,q), 4.16 (lH,t), 4.93 (lH,d) Sulfate [~]23 = +72.8 (c=l.0, water) 26 ElementarY analysis as ClgH41 5 5 2 4 2 5 2 27 Found: C 32.74, H 7.69, N 8.91 28 Calculated: C 32.98, H 7.64, N 9.16 :

- 1~525~:~

1 Example 21-7 (1) 4-N-(5-aminopentyl)fortimicin 3 (2) 22 mg (0.05 millimole) 4 ~3) 10%
(4) Mass spectrum:
6 m/e 434 (M + 1), 433 (M ), 384, 366, 320, 292, 7 271, 224, 219, 171, 143, 126, 117, 89, 86 8 P~IR ~deuterium oxide): -9 ~(ppm): 1.04 (3H,d), 1.2-1.9 (lOH,m), 2.44 Io (3H,s), 2.5-3.0 ~6H,m), 3.14 (lH,t), 3.20 II (lH,t), 3.40 (lH,m), 3.44 (3H,s), 3.80 ~lH,tj, 3.84 (lH,q), 4.07 (lH,q), 4.16 (lH,t), 4.96 I3 (lH,d), Sulfate [~123 = +67.3 (c=l.O, water) Example 21-8 7 (1) 4-N-(6-aminohexyl)fortimicin B
la (2) 147 mg ~0.33 millimole) 9 (3) 66%
(4) Mass spectrum:
2I m/e 448 (M + 1), 447 (M ), 429, 402, 398, 380, 22 370, 361, 334, 320, 306, 288, 285, 238, 222, 23 219, 199, 185, 143, 131, 126, 112, 98 2~ PMR (deuterium oxide);
u ~(ppm): 1.02 (3H,s), 1.2 1.9 (12H,m), 2.40 26 (3H~s), 2.5-3.0 (6H,m), 3.12 (lH,t), 3.16 27 (lH,t), 3.43 (3H,s), ~3.4 (lH,m), 3.73 (lH,t), 18 3.84 (lH,q), 4.04 (lH,q), 4.14 (lH,t), 4.86 (lH,d) 29 Sulfate [~]2~3 = +71.3 (c=l.O, water) 30 ElementarY analysis as C21H45N55 2 5~I2504 2 4 2 3I Found: C 35.74, H 7.77, N 8.78, "
1.

' ' ~ ,.

' 1 Calculated: C 35.65, H 7.80, N 9.04 3 Example 21-9 4 ~1) 4-N-(2-hydroxyethyl)fortimicin B
(2) 106 mg (0.27 millimole) 6 (3) 54~
7 (4) Mass spectrum:
8 m/e 393 (M + 1), 374, 361, 344, 331, 279, 259, g 251, 235, 219, 207, 202, 143, 130, 126, 100, Io 97, 86 11 PNR (deuterium oxide):
12 ~(ppm): 1.01 (3H,d), 1~2-1.9 (4H,m), 2.44 (3B,s), 3 2.78 (2H,t), 2.4-3.0 (2H,m), 3.14 (lH,t), 3.i8 I~ (lH,t), 3.4 (lH,m), 3.44 (lH,s), 3.64 (2H,t~, 3.76 (lH,t), 3.87 (lH,q), 4.06 (lH,q), 4.16 16 (lH,t), 4.96 (lH,d) Sulfate [~]24 = +77 40 (c=1.0, water) ~9 Example 21-10 (1) 4-N-[2-t2-aminoethyl)aminoethyl]fortimicin B
2I (2) 121 mg (0.28 millimole) 22 (3) 57~
23 (4) Mass spectrum:
2~ m/e 435 (M + 1), 417, 404, 361, 344, 219, 143, 126, 100 26 PMR (deuterium oxide):
27 ~(ppm): 1.00 (3H,d), 1.2-1.9 (4H,m), 2.40 (3H,s), 2/3 2.6-3.0 (lOH,m), 3.08 (lH,q), 3.16 (lH,t), 3.40 29 (3H,s), - 3.4 (lH,m), 3.74 (lH,t), 3.86 (lH,q), 4.08 (lH,q), 4.16 (lH,t), 4.94 (lH~d) i - 61 -,~

:

~L~5;~S2~
.

I Example 22 ~, .
2 In this example, 151 mg (0.20 millimole) of 1,2',6'-3 tri-N-benæyloxycarbonylfortimicin B was dissolved in 10 ml ~ l of ethanol and 0.02 ml (0.25 millimole) of ethyl iodine was added thereto. The reaction mixture was heated under reflux 6 for 17 hours and thereafter was concentrated to dryness 7 under reduced pressure. The resulting concentrate was dissolved 8 ' in 20 ml of ethylacetate and 10 ml each of an aqueous 5~ sodium 9 bicarbonate solution and water was added thereto. After shaking, l the ethyl acetate layer separated by using a funnel was Il dried with anhydrous sodium sulfate, and then concentrated ~2 to dryness under reduced pressure.
13 The resulting solid residue was dissolved in a small 14 amount of chloroform, and the chloroform solution was charged into a column packed with 25 g of silica gel 1Kieselgel~60].
6 In this step, elution was carried out with a mixed solvent of 17 methanol and chloroform (2:98 by volume) and the elute was 8 taken in 6 ml fractions. Fractions Nos. 29-54 were combined 19 in which fractions are contained the compounds having an Rf value 0.49 in solvent system C in Table 2. ~hese fractions 21 were concentrated to dryness under reduced pressure whereby 22 30 mg of white powder was obtained.
23 Physical properties of the compound are shown below.
2~ P~IR (methanol-d4~:
~tppm): 1.08 (3H,t), 1.02 (3H,d), 1.2-1.9 (4H,m), 26 2.48 (3H,s), 2.90 (2H,q), 3.43 (3H,s), 5.02 (6H,s), 27 ~ 7.28 (15H,-s) 28 From the physical properties above described, the compound 29 was identified as 1,2',6'-tri-N-benzyloxycarbonyl-4-N-ethylfortimicin B. Yield 19~.

. . .

ii , I
. .~ ~ .

:~.3 S~SZ~
Example 23 !! In this example, 942 mg ~1.0 millimole) of tetra-3 N-benzyloxycarbonylEortimicin A obtained by a similar method ~ j,as described in Example 6 was dissolved in 10 ml of tetrahydro-3 ijfuran and 10 ml of diborane in tetrahydrofuran (concentration:
6 1 mole/l) was added thereto. Then, the resulting reaction 7 mixture was stirred for 2 hours at room temperature.
After the completion of the reaction, 1 ml of water 9 was added to the reaction mixture in order to decompose any.excess lo diborane and the reaction mixture was concentrated to dryness under reduced pressure. The resulting concentrate was dissolved in 20 ml of ethylacetate and then 10 ml of 5~ aqueous sodium ~ bicarbonate was added thereto. After shaking the resulting ~ mixture, the ethyl acetate layer was washed twice with 10 ml of ~5 ; water. The èthyl acetate layer was then separated and dried ~6 with anhydrous sodium sulfate and concentrated to dryness under l? ~,reduced pressure. The resulting concentrate, dissolved in a 18 small portion of chloroform, was charged into a column packed 19 with 40 g of a silica gel ~Kieselgel~60).
lo Elution was carried out with a mixed solvent of 21 methanol-chloroform (2:98 by volume), and the elute was 22 taken in 6 ml fractions. Fractions Nos. 6-19 were combined in 23 which fractions the compound having an Rf value of 0.70 in solvent 2~ system C in Table 2 and these fractions were concentrated under reduced pressure to obtain 318 mg of white powder.
Physical properties of the compound are shown below.
27 ! E~MR spectrum ~methanol-d4):
23 ~ppm): 1.08 (3H,d), 1.2-1.9 (4H,m), 2.35 (3H,s), 29 3.34 (3H,s), 5.02 (8H,s), 7.28 (20H,s).
~ Prom the data above described, the compound was identified as 31 tetra-benzyloxycarbonyl-[4-N-~2-aminoethyl)fortimicin B~.
., .

li !l - 63 -~,, 1i ~5~52~

.
I An additional elution was carried out with 250 ml of Z a mixed solvent of methanol-chloroform ~1:9 by volume) to 3 ` obtained fractions in which the compouncl having an Rf value ~ of 0.15 iD solvent system C in Table 2. The fractions were concentrated under reduced pressure to obtain 266 mg of white 6 powder.
7 The physical properties are shown below:
8 PMR spectrum (methanol-d4):
9 ~(ppm): 1.08 (3H,d), 1.2-1.9 (4H,m), 2.37 (3H,s), l 2.42 (3H,s), 3.40 (3H,s), 5.08 (6H,s), 7.28 and ll 7.34 (15H total; s, respectively) 12 From the data above described, the compound was 13 identified as 1,2',6'-tri-N-benzyloxy-carbonyl-4-N-(2-1~ methylaminoethyl)fortimicin B.

16 Example 24 17 In this example, 30 mg (0.039 millimole) of 1,2',6'-18 tri-~-benzyloxycarbonyl-(4-N-ethyl)fortimicin B obtained in 19 Example 22 was dissolved in 10 ml of O.lN hydrochloric acid-methanol solution and about 2 mg of palladium charcoal 21 was added thereto. Then, hydrogen gas was bubbled through 22 the reaction mixture at room temperature under atmospheric 23 pressure for 8 hours. After the completion of the hydrogenation 2~ reaction, the catalyst was removed by filtration. The solvent of the filtrate was removed under reduced pressure.
26 As a result, 21 mg of white powders were obtained.
27 Physical properties of thus obtalned compound were 28 identical with those of the compound obtained in Example 21-1, 29 and it was identified that the compound was 4-r~-ehtylfortimicin ~ B hydrochloride. Yield 97%.

,: ~

.

. . . . .. .
.

: . - ~ . , . . ~ . , . ;

i~SZ52 `, .
I Example 25 2 . In this example, 310 mg (0.33 millimole) of tetra-3 ' N-benzyloxycarbonyl- [4-N- (2-aminoethyl)fortimicin B] obtained 4 ; in Example 23 was dissolved in 20 ml of O.lN hydrochloric acid-S methanol solution and about 20 mg of 10~ palladium-charcoal was 6 added thereto. Then, hydrogen gas was bubbled through the 7 reaction mixture, at room temperature under atmospheric pressure 8 for 18 hours. After the completion of the hydrogenation reaction, 9 the catalyst was removed by filtration. The solvent of the filtrate was removed under reduced pressure. The resulting Il concentrate, dissolved in 5 ml of water, was changed into a 12 column packed with Amberlite~CG-50 (NH4 form) after being 13 adjusted to pH 6 using lN sodium hydroxide. After charging, l4 50 ml of water and 90 ml of 0.3N aqueous ammonia was used for washing the column, and thsn elution was carried out with 0.5N
16 aqueous ammonia. The eluate was taken in 5 ml fractions.
17 Fractions Nos. 6-43 were combined in which fractions are ~B contained the compound having an Rf 0.4 in solvent system A
19 in Table 2. These fractions were concentrated to obtained 123 mg of white powder.
21 Physical properties of thus obtained compound were 22 identical with those of the compound obtained in Example 19, 23 and was identified as 4-N-(2-aminoethyl)-fortimicin B.
24 Yield 94~.

26 Example 26 27 In this example, 261 mg (0.32 millimole) of 1,2',6'-- 2B tri-N-benzyloxycarbonyl-4-N-(2-methylaminoethyl)fortimicin B
29 obtained in Example 23 was dissolved in 15 ml of O.lN hydrochloric acid-methanol solution and about 20 mg of 10% palladium-3I charcoal was added thereto. Then, hydrogen gas was bubbled j - 65 -.

- ` ` 11S25~

I through the reaction mixture at room temperature under atmospheric 2 pressure for 17 hours. After completion of the hydrogenation 3 reaction, the catalyst was removed by filtration and the solvent ~ of the filtrate was removed under reduced pressure. The resulting , concentrate, dissolved in 5 ml of water and adjusted to pH 6 6 with lN sodium hydroxide, was charged into a column packed 7 with 10 ml of Amberlite~CG-50 (NH4 form). After charging, 8 50 ml of water and 90 ml of 0.3N aqueous ammonia was used for 9 washing the column, and then, elution was carried out with lo 0.5N aqueous ammonia. The eluate was taken in 5 ml fractions.
Fractions Nos. 6-18 were combined in which fractions are It contained the compounds having an Rf 0.43 in solvent system A in l3 Table 2. These fractions were concentrated to obtain 92 mg s of white powder.
Physical properties of thus obtained compound are shown below.
1~ Mass spectrum:
18 m/e 406 (M + 1), 375, 355, 296, 292, 264, 219, 19 143, 126, 100 PMR (deuterium oxide):
2l ~(ppm): 1.02 (3H,d), 1.2-1.9 (4Ei,m), 2.12 (31i,s), n 2.41 (3H,s), 2.5-3.0 (6H,m), 3.08 (lH,q), 3.16 (lH,t), 23 3.4 (lH,m), 3.41 (3H,s), 3.73 (lH,t), 3.84 (lH,q), 24 4.05 (lH,q), 4.15 (lH,t), 4.92 (lH,d) Mass spectrum:
26 m/e 436 (M + 1), 417, 400, 387, 361, 344, 330, 2t ~ 332, 294, 259, 245, 235, 219, 207, 202, 143, 126, 28 119, 100 29 CMR (deuterium oxide~:
~(ppm): 18.6, 27.0, 27.3, 35.6, 40.6, 49.1, 50.4, 31 50.5, 54,7, 54.9, 57.4, 60.5, 71.3, 71.8, 75.2, I, - 66 -il . -! .

~ ~ .
- ~ .
..

~ . .
- ~

Z~

1 76.7, 80.5, 100.6 q O
2 Sulfate [~D5 = ~68.8 (c=l.0, water) 3 From the above data, the compound was identified as 4 4-N-~-methylaminoethylfortimicin B. Yield 70%.

6 Example 27 7 In this example the procedures of Example 23 were 8 repeated, except that an equimolar amount of tetra-M-benzyl-9 oxycarbonyl-4-N-[L-(-)-y-amino-~-hydroxybutyrylfortimicin B]
lo obtained in Example 6 was used in place of the starting material, ~1 that is tetra-N-benzyloxycarbonylfortimicin A used in Example 23.
As a result, tetra-N-benzyloxycarbonyl-4-N-[L-(-)-~-3 amino-~-hydroxybutylfortimicin B] and tri-N-benzyloxycarbonyl-14 4-N-[L-(-)-~-methylamino-~-hydroxybutylfortimicin B were obtained.

Example 28 17 In this example the procedures of Example 25 were 18 repeated, except that tetra-N-benzyloxycarbonyl-4-N-[L-(-)-~-9 amino-p-hydroxybutylfortimicin obtained in Example 27 was used in place of the starting material, that is, tetra-N-2l benzyloxycarbonyl-(4-N-~-aminoethylfortimicin B) used in 22 Example 25.
23 Physical properties of thus obtained compound are 24 shown below.
Mass spectrum:
26 m/e 436 (M + 1), 417, 400, 387, 361, 344, 330, 27 322, 294, 259, 245, 235, 219, 20i, 202, 143, 28 126, 119, 100 29 CMR (deuterium oxide):
~(pp~): 18.6, 27.0, 27.3, 37.7, 38.2, 40.4, 50.3, 31 50.5, 54.9, 57.3, 61.5, 61.9, 67.7, 70.1, 71.7, , 5~

1 75.2, 76.5, 80.5, 100.6 2 From the above data, the compound was identified as 3 4-N-lL-(-)-~-amino-~-hydroxybutylfortimicin B]
4 Sulfate [~]23 = +78 6 (c=l.0, water) 6 Example 29 7 In this example the procedures of Example 25 were 8 repeated, except that tri-N-benzyloxycarbonyl-4-N-lL-(-)-9 ~-methylamino-~-hydroxybutylfortimicin B] obtained in Example 27 was used in place of the starting material, that is, tetra-N-1l benzyloxycarbonyl-(4-N-~-amino-ethylfortimicin B) used in Example 1~ 25.
13 Physical properties of thus obtained compound are 14 shown below.
~5 Mass spectrum:
16 m/e 450 (M + 1), 431, 400, 388, 374, 370, 361, l7 344, 336, 330, 308, 259, 245, 219, 207, 202, 18 143, 133, 126, 100 19 CMR (deuterium oxide):
~(ppm): 18.6, 27.1, 27.3, 34.4, 35.4, 40.4, 48.0, 21 50.3, 50.5, 54.9, 57.3, 61.5, 61.8, 67.4, 68.0, 22 70.8, 71.7, 75.2, 76.5, 80.5, 100.6 23 From the above data, the compound was identified as 24 4-N-[L-(-)-~-methylamino-~-hydroxybutylfortimicin B].
Sulfate [~]23 = +77 5o (c=l.0, water) .

.; , .
, . ~ ' .

Claims (37)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for preparing a 4-N-substituted derivative of fortimicin B of general formula:

and pharmaceutically acceptable non-toxic acid addition salts thereof, wherein R represents a group selected from R1CO- and R2CH2-, wherein R1 represents a group selected from alkyl of 2 to 8 carbon atoms, hydroxyalkyl of 1 to 5 carbon atoms and aminoalkyl of 2 to 8 carbon atoms, and wherein R2 represents a group selected from alkyl of 1 to 8 carbon atoms, hydroxyalkyl of 1 to 5 carbon atoms and aminoalkyl of 1 to 8 carbon atoms; said process comprising:
(a) reacting fortimicin B with a mono-N-protecting agent to produce 1,2',6'-tri-N-protected fortimicin B;
(b) when R represents R1CO-, wherein R1 is as defined above, 4-N-acylating the 1,2',6'-tri-N-protected fortimicin B product of step (a) with an appropriate N-acylating agent, followed by de-N-protection of the 4-N-substituted-1,2',6'-tri-N-protected fortimicin to produce the desired 4-N-substituted fortimicin B;

(c) when R represents R2CH2, wherein R2 is as defined above, reducing the 4-N-carbonyl group of An appro-priate 4-N-substituted fortimicin B as produced in step (b) to a methylene group to produce the desired 4-N-pro-tected substituted fortimicin B; or (d) when R represents R2CH2-, wherein R2 is as defined above, reacting the 1,2',6'-tri-N-protected forti-micin B product of step (a) with a reagent of the general formula R2CH2X, wherein R2 is as defined above and X re-presents a group selected from chlorine, bromine, iodine, a methanesulfonylester and a p-toluenesulfonylester, followed by de-N-protection of the 4-N-substituted-1,2',6'-tri-N-protected fortimicin to produce the desired 4-N-substituted fortimicin B; or (e) when R represents R2CH2-, wherein R2 is as defined above, reducing the 4-N-carbonyl group of an appro-priate 4-N-substituted-1,2',6'-tri-N-protected fortimicin as produced in step (b) to a methylene group, followed by de-N-protection of the 4-N-substituted-1,2',6'-tri-N-protected fortimicin to produce the desired 4-N-substituted fortimicin B;
(f) when R represents R2CH2, wherein R2 represents aminomethyl, reducing the 4-N-carbonyl group of fortimicin A to produce 4-N-(2-aminoethyl)fortimicin B; and (g) when desired, producing the acid addition salts of the products of steps (b) to (f).

2. A process for preparing a 4-N-substituted derivative of fortimicin B of general formula:

and pharmaceutically acceptable non-toxic acid addition salts thereof, wherein R1 represents a group selected from alkyl of 2 to 8 carbon atoms, hydroxyalkyl of 1 to 5 car-bon atoms and aminoalkyl of 2 to 8 carbon atoms; said pro-cess comprising:
(h) reacting fortimicin B with a mono-N-protec-ting agent to produce 1,2',6'-tri-N-protected fortimicin B;
(i) 4-N-acylating the 1,2',6'-tri-N-protected fortimicin B product of step (h) with an appropriate N-acylating agent, followed by de-N-protection of the 4-N-substituted-1,2',6'-tri-N-protected fortimicin to produce the desired 4-N-substituted fortimicin B; and, when desired, producing the acid addition salts thereof.

3. The process defined in claim 2, step (i), wherein said acylating agent is propionic anhydride.

4. The process defined in claim 2, step (i), wherein said acylating agent is n-butyric anhydride.

5. The process defined in claim 2, step (i), wherein said acylating agent is n-valeric anhydride.

6. The process defined in claim 2, step (h), wherein said mono-N-protecting agent is N-(benzyloxycar-bonyl)succinimide.

7. The process defined in claim 6, wherein said acylating agent is a reactive derivative of O-benzylgly-colic acid.

8. The process defined in claim 6, wherein said acylating agent is a reactive derivative of N-benzyloxy-carbonyl-.gamma.-amino-n-butyric acid.

9. The process defined in claim 6, wherein said acylating agent is a reactive derivative of N-benzyloxy-carbonyl-.delta.-amino-n-valeric acid.

10. The process defined in claim 6, wherein said acylating agent is a reactive derivative of N-benzyl-oxycarbonyl-.epsilon.-amino-n-caproic acid.

11. The process defined in claim 1, step (a) or claim 2, step (h), wherein said mono-N-protecting agent is t-butyl-S-4,6-dimethylpyrimidin-2-yl-thiocarbamate.

12. The process defined in claim 1, step (b), wherein said acylating agent is propionic anhydride, fol-lowed by step (c) or (e); or wherein step (d) said reagent is of the general formula CH3CH2X.

13. The process defined in claim 1, step (b), wherein said acylating agent is n-butyric anhydride, fol-lowed by step (c) or (e); or wherein step (d) said reagent is of the general formula CH3(CH2)2X.

14. The process defined in claim 1, step (b), wherein said acylating agent is n-valeric anhydride, fol-lowed by step (c) or (e); or wherein step (d) said reagent is of the general formula CH3(CH2)3X.

15. The process defined in claim 1, step (b), wherein said acylating agent introduces the group -CO(CH2)2-NH2, followed by step (c) or (e); or wherein step (d) said reagent is of the general formula NH2(CH2)2CH2X.

16. The process defined in claim 1, step (b), wherein said acylating agent introduces the group -CO(CH2)3NH2, followed by step (c) or (e); or wherein step (d) said reagent is of the general formula NH2(CH2)3CHX.

17. The process defined in claim 1, step (b), wherein said acylating agent introduces the group -CO(CH2)4NH2, followed by step (c) or (e); or wherein step (d) said reagent is of the general formula NH2(CH2)4CH2X.

18. The process defined in claim 1, step (b), wherein said acylating agent introduces the group -CO(CH2)5NH2, followed by step (c) or (e); or wherein step (d) said reagent is of the general formula NH2(CH2)5CH2X.

19. The process defined in claim 1, step (b), wherein said acylating agent introduces the group -COCH2OH, followed by step (C) or (e); or wherein step (d) said reagent is of the general formula HOCHCH2X.

20. A 4-N-substituted derivative of fortimicin B of general formula:

wherein R represents a group selected from R1CO- and R2CH2-, wherein R1 represents a group selected from alkyl of 2 to 8 carbon atoms, hydroxyalkyl of 1 to 5 carbon atoms and aminoalkyl of 2 to 8 carbon atoms, and wherein R2 represents a group selected from alkyl of 1 to 8 carbon atoms, hydroxy-alkyl of 1 to 5 carbon atoms and aminoalkyl of 1 to 8 carbon atoms; and pharmaceutically acceptable non-toxic acid addition salts thereof; when prepared by the process defined n claim 1, or an obvious chemical equivalent thereof.

21. 4-N-substituted derivative of fortimicin B
of general formula:

wherein R1 represents a group selected from alkyl of 2 to 8 carbon atoms, hydroxyalkyl of 1 to 5 carbon atoms and aminoalkyl of 2 to 8 carbon atoms; and pharmaceutically acceptable non-toxic acid addition salts thereof; when prepared by the process defined in claim 2, or an obvious chemical equivalent thereof.

22. 4-N-propionyl fortimicin B, and pharmaceutically acceptable non-toxic acid addition salts thereof; when prepared by the process defined in claim 3, or an obvious chemical equivalent thereof.

23. 4-N-(n-butyryl) fortimicin B, and pharma-ceutically acceptable non-toxic acid addition salts thereof;
when prepared by the process defined in claim 4, or an obvious chemical equivalent thereof.

24. 4-N-(n-valeryl) fortimicin B, and pharma-ceutically acceptable non-toxic acid addition salts thereof;
when prepared by the process defined in claim 5, or an obvious chemical equivalent thereof.

25. 4-N-glycolyl fortimicin B, and pharmaceutically acceptable non-toxic acid addition salts thereof; when prepared by the process defined in claim 7, or an obvious chemical equivalent thereof.

26. 4-N-(.gamma.-amino-n-butyl) fortimicin B, and pharmaceutically acceptable non-toxic acid addition salts thereof; when prepared by the process defined in claim 8, or an obvious chemical equivalent thereof.

27. 4-N-(.delta.-amino-n-valeryl) fortimicin B, and pharmaceutically acceptable non-toxic acid addition salts thereof; when prepared by the process defined in claim 9, or an obvious chemical equivalent thereof.

28. 4-N-(.epsilon.-amino-n-caproyl) fortimicin B, and pharmaceutically acceptable non-toxic acid addition salts thereof; when prepared by the process defined in claim 10, or an obvious chemical equivalent thereof.

29. 4-N-(n-propyl) fortimicin B, and pharma-ceutically acceptable non-toxic acid addition salts thereof;
when prepared by the process defined in claim 12, or an obvious chemical equivalent thereof.

30. 4-N-(n-butyl) fortimicin B, and pharma-ceutically acceptable non-toxic acid addition salts thereof;
when prepared by the process defined in claim 13, or an obvious chemical equivalent thereof.

31. 4-N-(n-pentyl) fortimicin B, and pharma-ceutically acceptable non-toxic acid addition salts thereof;
when prepared by the process defined in claim 14, or an obvious chemical equivalent thereof.

32. 4-N-(3-aminopropyl) fortimicin B, and pharma-ceutically acceptable non-toxic acid addition salts thereof;
when prepared by the process defined in claim 15, or an obvious chemical equivalent thereof.

33. 4-N-(4-aminobutyl) fortimicin B, and pharma-ceutically acceptable non-toxic acid addition salts thereof;
when prepared by the process defined in claim 16, or an obvious chemical equivalent thereof.

34. 4-N-(5-aminopentyl) fortimicin B, and pharmaceutically acceptable non-toxic acid addition salts thereof; when prepared by the process defined in claim 17, or an obvious chemical equivalent thereof.

35. 4-N-(6-aminohexyl) fortimicin B, and pharmaceutically acceptable non-toxic acid addition salts thereof; when prepared by the process defined in claim 18, or an obvious chemical equivalent thereof.

36. 4-N-(2-hydroxyethyl) fortimicin B, and pharmaceutically acceptable non-toxic acid addition salts thereof; when prepared by the process defined in claim 19, or an obvious chemical equivalent thereof.

37. 4-N-(2-aminoethyl) fortimicin B, and pharmaceutically acceptable non-toxic acid addition salts thereof; when prepared by the process defined in claim 1, step (f), or an obvious chemical equivalent thereof.