Note: Descriptions are shown in the official language in which they were submitted.
~128~S7
1' ;-
I BACKGROUNO OF THE INVENTION:
t The present invention relates to novel derivatives of
3 fortimicin B, the acid addition salts thereof and a process for
~ preparing the same.
Fortimicins (A, 3 and C) are compounds belonging to
6 pseudodisaccharide antibiotics containing 1,4-diaminocyclitol.
7 The physical properties and antibacterial activitles of these
8 compounds, the processes for producing them by using micro-
9 organisms, and processes for separation and ourification thereof
~o from culture li~uors, etc. are described in detail iA United
States Patents Nos. 3,931,400, 3,976,768 and 4,04a,015.
l2 The planar structural formulae of the fortimicins, are
13 illu~trated in said United States Patents and their structural
l4 formulae showing absolute coordination are described in the
specification of Japanese Published Unexamined Patent Aoplication
16 No. 50140/78.
Fortimicins (A, B and C) all have antibacterial activity,
a but the antibacterial activity of fortimicin B i5 not as good as
~9 the other factors; and fortimicin A and fortimicin C are slightly
unstable under stronyly al~aline conditions. Therefore, compounds
21 h~ving more c1istinguishcd propcrties are in demand.
22 ~s a result of various studies, it has been found tllat
23 certain ~-N-substLtuted derivatives of fortimicin B have enhanced
24 - antibacterial act;lvity and good stability under alkaline condl-
t5 tions (Japanese Published Unexamined Patent ~oplication No.
26 501~0/78).
27 ~oreover, it has now been found that certain 4,2'-di-N-
2a substituted derivatives of fortimicin B have good stabilit~ under
29 strongly alkaline conditions and enhanced antibacterial activity
against a broad spectrum of pathogenic microorganisms including
31 strains resistant to known antibiotics.
.. . .
.
1128~S7 .
;~
. ~ n
1 S~ ARY OF THE INVENTION:
2 The present invention relates to 4,2'-di-N-substituted
3 derivatlves of fortimicin ~, represented by the general formula
~ (I):
NH2
H3C ~ ~ OCH3
NH OH NCH3
g
R2 Rl
11 wherein Rl and R2 may be the same or different, and ~l repre-
12 sents a -C-R3 or -CH2-R3 group, R2 represents a -~C-R4 or
13 0 O
1~ -CH2~R4 group and wherein R3 and R4 represents an aminoalkyl
having l to 8 carbon atoms, a hydroxyalkyl group having l to 8
16 carbon atoms, a carbamoylaminoalkyl grou~ having 2 to 9 carbon
atoms or an aminohydroxylalkyl group having 2 to 9 carbon atoms.
The invention also pertains to a process for preparing the same.
Included in the composition of matter aspect of the
zo invention are the non-toxic acid addition salts of the compounds
21 of the above general formula.
22
23 D~'A~.~3 C~C~
2~ - Compounds of the pre~ent inv~ntion are 4,2'-di-N-
substituted derivatives of fortimicin B, represented by the
26 general formula (I):
27 \ 2
28 H3C ~ NH2 /OH
29 Q_ O ~ - OC~3
31 NH OH INCH3
R2
l28~57 `-~
I ,
I wherein Rl and R2 may be the same or different, and Rl represents
2 a -C-R3 or -CH2-R3, group R2 represents a -ICl-R4 or
3 O
~ -CH2-R4, group and wherein R3 and R4 represent an aminoalkyl
group having 1 to 8 carbon atoms, an hydroxyalkyl group having
6 1 to 8 carbon atoms, a carbamoylaminoalkyl group having 2 to 9
7 carbon atoms and aminohydroxyalkyl group having 2 to 9 carbon
8 atoms and the acid addition salts thereof.
9 4,2'-di-N-substituted derivatives of fortimicin B
l represented by the general formula (I), are exemplif~ed in
the following Table 1 together with the values of their physical
IZ properties. For reference, the physical properties of fortimicin
a B, fortimieill A and fortimicin C are also set forth.
The Rf values of the compounds of the invention as
well as the starting compounds on thin layer chromatography (TLC)
6 using various solvents and silica gel plates are set forth in
7 Table 2. In tlle TLC treatment, DC-Fertigplatten Kieselgel
60 F25~ (produced by E. Merclc 6 Co.) was used as a silica gel
plate and in color reaction, ninhydrin or iodine ~as used. The
solvent systems given in Table 2 are as ollows:
21 A: isopropanol - 28~ aqueous ammonia-chloroform
22 (2:1:1 by vol~1me)
7.3 B: isopropanol - 28~ aqueous c~mmonia-chloroform
z~ - (4:1:1 by volume)
C: the lower layer of methanol - 28~ aqueous ammonia-
26 chloroform ~1:1:1 by volume)
27 D: chloroform : mcthanol (90:10 by volume)
2a E: chloroform : methanol (95:5 by volume)
29 Furthermore in the following Table 3, the anti-
bacterial activity (MIC) of the compounds are-set forth.
31
-- 4 --
- ':
~lZ~3~S7
.,
1 The measurement was carried out according to the Japanese Anti-
2 biotic Medicament Standard using a medium having a p~ of 7 . 2.
3 Numbers of the compounds in Table 3 correspond to those in
4 Tables 1 and 2.
12
.
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17
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19
21
22
23
26
27
29
29
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1128~57
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1 As is evident from the foregoing Table 3, the compounds
2 of the present invention exhibit good antibacterial activity
3 against various microorganisms including resistant strains and
~ are, therefore, useful as antibacterial agents or antise?ties.
The non-toxic acid addition salts of the present com-
6 pounds also exhibit a broad antibacterial spectrum and are like-
I wise useful as antibacterial agents. The non-toxic acid addition
B salts include the mono-, di-, tri-, tetra-, penta- and hexa-salts
9 obtained by reacting one molecule of the compound represented by
said general formula (I) with one to six equivalents of a oharma-
~1 ceutically acceptable, non-toxic acid. Suitable acids include
12 ino~ganic acids such as sulfuric acid, hydrochloric aeid, hydro-
13 bromic acid, hydroiodic acid, phosphoric acid, carbonie aeid,
1~ nitrie acid, ete., organie acids sueh as acetic aeid, fumarie
aeid, malie aeid, eitrie aeid, mandelic aeid, succinie aeid,
ascorbie aeid, etc., and amino aeids such as aspartie aeid and
the like.
18 Generally speaking, the present compounds are synthe-
19 sized according to the processes in the following Flow Sheet I.
21
22
23
2~ -
26
27
28
29
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~lZB~57
1 More particularly, when the desired compound is
2 Compound I-a represented by the general formula (I) wherein ~l
3 is -C-R3 and R2 is -C-R4, the Compound is synthesized by
O O
Step l ~ Step 2 ~ Step 5 + Step 6, or by Ste~ ~ ~ Stap 4 ~ Step 5
6 Step 6.
7 When the desired compound is Compound I-b represented
8 by the general formula (I) wherein Rl is -CH2-R3 and R2 is
9 -C-R~ and Compound I-c represented by the general formula (I~
0
1t wherein Rl is -CH2-R3 and R2 is -CH2-R4, the compound is synthe-
sized by Step 7 using Compound I-a as the starting material, or
by Step 8 ) Step 9 or Step 8 ~ Step lO using a precursor (Compound
14 IX) of Compound I-a as the starting material.
Among the compounds represented by the general formula
~6 (I) thus obtained, Compound I-a is slightly unstable under
17 strongly alkaline conditions~as is fortimicin A and fortimicin C.
18 That is, Compound I-a is almost decomposed in an alkaline solution
19 with a pH of 10 at room temperature for 2 weeks to form compounds
having low activity. compound I-b, on the other hand, i: more
2~ stable than Compound I-a, and is bare1y decomposed in an alkaline
22 solution with a pH of lO a~ room temperature for 2 weeks. Com-
23 pound I-c is even more stable, and is not decomposed on heating
24 at a temperature of 100C for several hours in an alka1ine solu-
tion saturated with barium hydroxide.
26 The individual steps of the foregoing process are as
27 fol1Ows,
28
29
~o
31
- 15 -
'~: - - . .
llZ8~S7
I Ste~ 1.
2 Preparation of Compound IV from fortimicin B
3 (Compound II):
t Fortimicin s is acylated in a suitable solvent by
using an acylating agent to form Compound IV in which the amino
6 group at the 2'-position of fortimicin B is acylated.
7 As an acylating agent, derivatives of a carboxylic
8 acid represented by the general formula (III) R4'COOH [wherein
9 R4' is a carbamoylaminoalkyl group, substituted aminoal~vl grouo
(the substituent represents an amino-protecting group), hydro:cy-
alkyl group or substituted aminohydroxyalkyl group (the substitu-
. 12 ent represents an amino-protecting group), where said amino-
protecting group may be the same as or different to the amino-
14 protecting group R5 of Compound V obtained in Step 2 or Step a
mentioned below]. For example, acid anhydrides of carbo.xylic
6 acids repres~nted by the general formula (III), active esters of
17 said carboxylic acids with a compound selected from a group of
18 O~ N2
19 HO-N ~ , HO ~ NO2, HO ~ NO2, HN ~ and
N
2~ O
222 N ~ ~ , preferab1y HO-N ~ or acid hydrides of
said carboxylic acids may be used.
Fortimicin 3 is used in a concentration of l to 250 m~
26 preferably l0 to l00 mM. Acylating agents are used in a concen-
27 tration of 0.5 to 2 moles, preferablv l to- 1.5 moles per mole of
28 fortimicin ~.
29 As the solvent, dimethylformamide, dimethvlacetamide,
tetrahydrofuran, dioxane, l,2-dimethoxyethane, methanol, ethanol,
l1 water and a mixture thereof are used.
- . .
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.
- ~128~57
.
1 Reaction is carried out at a temperature of 0C to
2 room temperature for 15 minutes to 20 hours.
3 The reaction mixture containing Compound IV prepared
~ by the foregoing process can be utilized as it is or, after
isolation, in the succecsive step to prepare Compound V.
6 Purification an2 isolation of Compound IV from the
7 reaction mixture can be carried out in the following manner. The
8 solvent is distilled off from the reaction mixture to obtain a
g residue. The residue is subjected to partition between an organic
solvent such as chloroform and water. The water layer obtained
is subjected to column chromatography using an ion exchange resin
r,l ,~ .
12 such as ~mberlite1CG-50 (product of Rollm & Haas Co.) to adsorb
13 the desired compound thereon. Then, elution is carried out with
14 a suitable solvent. Eractions containing Compound IV are combined
and the solvent is distilled off to obtain Compound IV.
16
St.ep 2.
8 Preparation of Compound V from Compound IV:
Compound IV and an amino-protecting reagent are
z0 reacted in a suitable solvent to obtain Compound V in which the
21 amino groups at the l- and 6'-po;itions of Compound IV are
22 protected with an amino-protecting group R5.
z3 As an ami1lo-protecting roaye1lt, those generally used
24 in peptide-synthesis may be used. Examples of proEerable amino-
~5 protecting re~ayents are:
26
27 7~,~ C112-o-C-o-N~ P`~CH2-0-C-Y,
29
~O
31
':
, 1128~57 - I
-c-o-c-~3, ~,C-C-o_C_9 ~ ~ C~3
~CH3 CH3
5O O O O
11 il 11 ~
6CH3-O-C-Y, C2H5-O-C-Y, R8-CH2-c-Y~ R8-CH2 C OH~
9~ S-OH, C2H5-O-C-
10 N02 1~
o
~z [wherein ~6 and R7 may be the same or different and are ~, OH,
13 NO2, Cl, Br, I, an alkyl group having 1 to 5 carbon atoms or an
al~oxy group having l to 5 carbon atoms, R8 is H, F, Cl, Br, I
or an alkyl group having l to 5 carbon atoms and Y is Cl, Br
6 or I].
17 As the solvent, dimethylformamide, dimethylacet-~mide,
13 tetrahydrofuran, dioxane, l,2-dimethoxyethane, methanol, ethanol,
acetone, water or a mixture thereof are used in the reaction.
Among these solvents, methanol is more preferable.
21 Compound IV is used in a concentration of l to 250 m~.,
2z preferably l0 to l00 m~1. The amount of amino-protectincl reagent
23 utili~ed in the reaction i9 àppropriately l to ~ moles, more
t4 preerahly 2 to 2.5 moles per mole of Compound IV.
Reaction is carried out at a temperature of 0 to 60C,
z6 preferably 0C to room temperature for 2 to 18 hours.
27 1'he reaction mixture containing Compound V prenared by
2~3 the foregoing process can be utili7ed as it is or after isolation
2i in the successive step. To isolate and purify Compound V from the
reactior. mixture, the solvent is distilled off from the reaction
31 mixture to obtain a residue. An organic solvent such as chloro-
- 18 -
. l~Z8~S7
.
1 form or ethyl acetate is added to the residue to extract the
2 soluble portions. The extract is subjected to column chromatog-
3 raphy using silica gel such as Kiesel gel 60 tproduct of E. ~lerck
~ & Co.). In this case, elution is carried out with an organic
solvent such as chloroform-methanol or ethylacetate-ethanol and
6 fractions showing a specific Rf value are collected and concen-
7 trated to dryness, whereby a white powder of the desired compound
8 is obtained.
9 Compound V thus obtained can be u-tilized as a starting
material for preparing Compound I-a, Compound I-b and Compound
11 I-c.
12 In the preparation of Compound I-a or Compound I-b
13 from Compound V, since Compound I-a and Compound I b are less
1~ stable than Compound I-c in strongly alkaline conditions, it is
desirable to use Compound V as a starting material which has
16 amino protecting groups eliminable under conditions other than
17 an alkaline condition.
18 E~amples of the amino-protecting reagents of such
19 Compound V are:
O O O
tt R6 ~ CEE2-O-C-O-N ~ C1{2-O-C-Y,
~ ~C-Y, N3C-C;o-3-N3, N3c-f-o-c-S~ .
26 ~ CH3 CIE3 CEE3
27
28
29
31
. .
,~ ~
-` 1128Q57 -
.
I (wherein R6, R7 and Y have the same significance as defined
2 above).
3 Compound I-c is stable in both acidic and alkaline
7 conditions, and when Compound V is utilized as a raw material
for preparing Compound I-c, any amino-protecting reag-nt can be
6 utilized.
8 SteP 3-
9 Preparation of Compound VII from Compound VI:
A 4-W-acylfortimicin B derivative (Compound VI) such as
fortimicin A or fortimicin C (the process of preparing the
derivative is described in Japanese Published Unexamined Patent
l3 Application No. 50140/78) is maintained in an alkaline condition
1~7 in a suitable solvent to form 2'-N-acylfortimicin derivative
(Compound VII) wherein the acyl group at the 4-position of 4-~-
6 acylfortimicin derivativo is transferred to the 2'-position.
11 As the solvent, water, methanol, ethanol, dimethylform-
18 amide, dimethylacetamide, tetrahydrofuran, dioxane, 1,2-dimethoxy-
19 ethane and a mixture thereof are used. When the solvent includes
water, the reaction is carried out at a p~,7 of 7 to 12, preferably
21 g to 11, and at a temperature of from room temperature to 100C
22 for one hour to 2 weeks. Compound VI is used in a concentration
23 of 1 to 250 mM, preferably 10 to 100 m~.
24 - The reaction mixture containlng Compound VII thus
produced can be utilized as it is or, after isol~ltion, as a
26 starting material for preparing Compound V.
27 To isolate and ~urify Compound VII from the reaction
28 mixture, the reaction solution, as it is or after dilution with
29 wateF, is charged ~ nto a column packed w~th an ion-exchanye resin
such as Amberlite1C~7-50, to adsorb the desired compound thereon.
31 Then, elution is carried out with a suitable solvent. Fractlons
.
- 20 -
- , ~
-- llZ8~57
I containing Compound VII are combined and the solvent is distilled
2 off to obtain Compound VII.
~ Step 4.
Preparation of Compound V from Compound VII:
6 Compound VII and an amino-~rotecting rea~ent are
7 reacted in a suitable solvent to obtain Compound V wherein the
8 amino groups at the 1- and 6'-positions of Compound VII are
g protected with amino-protecting groups and, when E~4 has an amino
lo group, such group is also protected.
sl The amino-protecting reagents and solvents are selected
t2 from those as used in Step 2 and the reaction temperature and
13 reaction time are also similar to those in Step 2.
1~ Compound VII is used in a concentration of 1 to 250 mM,
preferably 10 to 100 mM. The amount of amino-protecting reagents
16 used in the reaction is 2 to 5 moles per mole of Compound VII.
l7 When Compound VII has an R4 group not having any àmino group or
la an R4 group wherein an amino group is already protected, the
V amount of amino-protectLng reagent is preferably 2 to 2.5 moles
per mole of Compound VII. When Compound VII has an R~ group
2I having Eree amino groups, 1 to 1.5 moles of amino-protecting
22 reagent per one amino group are additionally added.
2~
24 - Step 5.
PreparatLon of Compound IX from Compound V:
26 Compound V is acylated using an acylating reagent in a
27 suitable solvent to obtain Compound IX wherein the aMino-group
29 at the 4-position of Compound V is acylated.
29 As an acylating reagent, derivat~ives of carbo~ylic
acids represented by the general formula- R3'COOE~ herein P3'
31 represents a carbamoylaminoalkyl group, hydroxyalkyl group,
- 21 -
.
.
.
, ~ ';
.
~28~57 - "
.; .
I substituted aminoalkyl group (the substituent represents anamino-protecting group), substituted aminohydroxyalkyl group (the
3 substituent represents an amino-protecting group) may be used.
4 The amino-protecting groups may be the same as or different from
the amino-protecting group ~5 in Compound v. ~or example, acid
6 anhydrides of carboxylic acids represented by the general formula
7 tVIII), active esters of said carboxylic acids with a compound
a selected from the group consisting of:
g O
Z HO-N ~ ~ ~ 2 HO ~ -~2
O N02
12
3 HN 3 and HO-N _ ~ , preferablY HO- ~ or
6 acid hydrides o said carboxylic acids are appropriate.
17 Compound V is used in a concentration of l to 250 m.~,
1~ preferably l0 to l00 mM, and the acylating reagents are used in a
v ConCentratiOn of l to l.5 moles per mole of Compound V.
io As the solvent, dimethylformamide, dimethylacetamide,
21 tetrahydrofuran, dioxane, l,2-dimethoxyethQne, methanol, ethanol,
22 water, and mixtures thereo~ are used. Tctrahydrofuran is pre-
2~ ferred.
t4 - The reaction is carried out at a temperature of 0C to
2s 70C, preferably 0C to room temperature, for lS minutes to 20
26 hours, preferably l to 18 hours.
27 In addition to this orocedure, the DCC methcd can also
Z9 be used for the acylation.
29 Compound IX, formed in the reaction solution according
to the above procedure, or the reaction mixture containing Com-
31 pound IX can be used for preparing Compound I-a, X and XI.
- 22 -
- llZ8~57-` .
1 To isolate and purify Compound IX from the reaction
t solution, the solvent is distilled off from the reaction solution
3 to obtain a residue. The residue is mixed with an organic solvent
~ such as chloroform, ethylacetate, or the like to dissolve extract- -
able substances. Then, the resulting extract is subjected to
6 column chromatography packed with silica crel such as Kieselgel 60
7 ~E. Merck & Co.). Elution is carried out with an orsanic solvent
8 such as chloroform-methanol, ethylacetate-ethanol, and fractions
9 containing Compound IX are combined and the solvent is removed
therefrom to obtain Compound IX.
11
Step 6.
13 ~ Preparation of Compound l-a from Compound IX:
1~ The amino-protecting groups R5 o~ Compound IX obtained
in Step 5 as well as the amino-protecting groups in R3' and R4',
16 if such are present, are removed by a method Xnown oer ~e to
obtain Compound I-a.
a For example, when the protecting group is a benzyloxy-
carbonyl group, the protecting grouo can be removed by catalytic
hydrogenolysis in the presence of a metal catalyst such as
21 palladium-carbon, platinum, rhodium, etc., and in the presence
zz of an acid such as hydrochloric acid, hydrobromic acid, acetic
23 acid, etc. in a solvent such as water, tetra1~ydrofuran, dimethyl-
2~ acetamide, di~ethylformamide, lower alcohols, dioxane, ethylene-
glycoldimethyleth~r, or combinations thereof, etc., preferably in
26 rnethanol at room temperature and atmospheric pressure, while
i7 passing hydrogen gas through the reaction ~ixture.
z8 ~sually l to lO~ by weight of the metal catalyst is
Z9 used on the basis of Compound I~, and the-concentration of Com-
oound IX is usually l to 200 r~M, preferably about S0 mt~.
3t The acid is added to the reaction mixture so that pH
'
llZ8QS~ ~
.
I is maintained at 4 or less. The end of the reaction can be
2 confirmed by the cGmpletion of generation of carbon dioxide or by
3 thin layer chromatography, and the like.
~ When the protecting group is a tertiary butoxycarbonyl
s group, its removal can be carried out in the presence of hydro-
6 chloric acid or trifluoroacetic acid in a non-aqueous solvent
7 such as dichloromethane, chloroform, trichloroethylene or
8 ethylacetate.
g Compound IX is used in a concentration of l to 200 mM,
lo preferably about 50 mM.
s~ In such manner, the desired product is formed in the
2 reaction mixture. It can then be separated and purified by known
13 methods using ion-exchange resins, silica gel chromatography,
1~ or the like. For example, according to a method using an ion-
exchange resin, the reaction mixture, after filtration, if neces-
16 sary, is evaporated to dryness. The resulting residue is dis-
solved in water and the pH is adjusted to about 6 by addition of
8 an alkali, such as sodium hydroxide. ~hereafter, the resulting
19 solution ~ charyed into a column packed with, for example,
B 20 Amberlite~CG-50 to adsorb the desired product. Then, elutlon i3
carried out with a suitahle solyent and the eluate is taken in
22 fractions. Fractions having activity are combined and the solvent
23 is distilled off to obtain the desired product.
2~ - Compound I-a after isolation, or the re.sidue containing
Compound I-a obtained by evaporation of the reaction solution, can
26 be used to produce Compound I-b and Compound I-c.
27
28 Ste~ 7.
29 Preparation of Compound I-b and-Compound I-c from
Compound I-a:
31 Compound I-a obtained in Step 6 is reduced in an
- 2~ -
.~ llZ8~57
.
I appropriate solvent in the presence of a reducing agent for con-
2 verting the carbonyl group in the amide to a methylene group at
3 a temperature of room temperature to reflux temperature of the
~ solvents to obtain Compound I-b and Compound I-c.
As the solvent, tetrahydrofuran, dioxane, diethylether,
6 etc. are appropriate. As the reducing agent, an excess amount,
7 usually lO-fold or more of lithium aluminum hydride, diborane,
. a etc. to Compound I-a is used.
9 In this step, when the reaction is carried out at a
higher temperature and for a longer period, Compound I-c is
11 mainly obtained and when at a lower temperature and for a shorter
- 1t period, a mixture of Compound I-b and Compound I-c is obtained.
13 Purification of the desired product is carried out, for
1~ example, with an ion-exchange resin. After the excess reducing
agent in the reaction mixture is decomposed with ethylacetate,
16 water, or the like, most of the solvent is distilled off under
17 reduced pressure. The resulting residue, in a semi-solid state,
8 is admixed with water to extract water-soluble components, and
19 the resulting extract is subjected to column chromatography
B 20 packed with a weakly acidic ion exchange resin, such as Amberlite~
21 CG-S0. The column is washed with water and then elution is
22 carried out with aqueous ammonia. Fractions containing Compound
I-b or Compound I-c are collected, and aqueous ammonia is removed
2~ by distillation to obtain Compound I-b or Compound I-c as a white
powder. Alternatively, separation and purification of the desired
t6 product can be carried out accordiny to known methods such as
27 silica gel chromatography, or the like.
28
ts Step 8.
~o Preparation of Compound X and Compound XI
l from Compound IX: -
.
- 25 -.
,~
,
z~s*
1 Compound IX obtained in step 5 is reduced in a suitable
2 non-aqueous solvent in the presence of a reducing agent for con-
3 verting the carbonyl group in the amide group to a methylene
~ group at a temperature of from room temperature to reflux
temperature of the solvent to obtain Compound X and Compound XI.
6 As the solvent, tetrahydrofuran, dioxane, diethylether,
7 or a mixture thereof is used. As the reducing agent, diborane,
8 lithium aluminum hydride, etc. are used. For the reaction,
g Compound IX is used in a concentration of from l to 250 m~l,
preferably l0 to l00 mM and usually l0-fold or more equivalents
11 of the reducing agent to Compound IX is used. The reaction is
generally completed in 5 minutes to 18 hours.
3 When the amino-protecting group of Compound I~ used in
1~ this step is a ben~yloxycarbonyl group and t-butyloxycarbonyl
~5 group, it is preferable to use diborane as a reducing agent
16 because the carbonyl group in the amide group is converted to
17 the methylene yroup without impairing the ben~yloxycarbonyl group
and t-butoxycarbonyl group of Compound IX. Thus Compound X and
19 Compound XI can be obtained in a good yield. [W.V. Curran and
R.8. Angier: J. Org. Chem., 31, 3867 (1966)].
21 When the reaction is carried out at a higher temperature
22 and for a lon~er period, Compound XI is principally obtained and
23 when at a lower temperature and for a shorter period, Compound X
2~ is princi~ally obtained.
Thus Compound ~ and Compound XI are formed in the
26 reaction solution which can then be used as a starting n~aterial
27 for the following Steps 9 and l0 without isolating.
28 If isolation is desired, the solvent is distilled off
29 from the reaction solution, and then the resulting residue is
~0 admixed with water to decompose the remaining hydride. Then, the
31 solvent in the reaction mixture is distilled off and the resulting
- 26 -
~:
~2~1~S7
.
I residue is admixed with an organic solvent such as ethylacetate,
chloroform, etc. to extract soluble components~ The water layer
3 is divided out and the remaining organic solvent layer is washed
~ with water and dried with a drying agent such as anhydrous sodium
sulfate. The solvent is distilled off and the resulting residue
6 is dissolved in an organic solvent such as chloroform. ~he
7 resulting solution is subjected to silica gel column chromatog-
a raphy to obtain the desired product.
_e~ 9.
~1 Preparation of Compound I-b from Compound X:
2 The amino-protecting groups of Compound X obtained in
s3 Step 8, that is R5 and amino-protecting groups of R3' or R4' in
1~ Compound X, if such are present, are removed according to the
method described in Step 6 wherein Compound X is used in place of
6 Compound IX to obtain Compound I-b.
17
8 Step lO.
Preparation of Compound I-c from Compound XI:
Compound I-c can be obtained by performing the pro-
21 cedure of Step 9 except that Compound XI is used in place of
22 Compound X.
23 When Compound X and Compound XI obtained in Step 8 are
2~ - not separated from each other, the mixture of Compound X and
Compound XI is used in place of Compound X of Step 9 to obtain a
26 mixture of Compound I-b and Compound I-c. Compound I-b and
2i Compound I-c can be separated by known methods such as column
28 chro ~tography using an ion exchange resin, ~or example, A~ber-
29 lite1CG-50, etc., silica gel.
The acid addition salts of Compound I obtained as
31 mentioned above, may be produced in known manner. For example,
- 27 -
:, , .
.~
.
.
;' ' - ' - ' ' .
,
.
llZ8Q57 -
. ; . , ,
I compound I is dissolved in water and an acid is added thereto.
2 Then, a solvent decreasing solubility of acid addition salts of
3 Compound I such as ethanol, etc. is added to the resulting solu-
4 tion to form a precipitate. The precipitate is filtered and
S dried to obtain the acia addition salt of Compound I as a white
6 to gray powder.
7 Certain specific embodiments of the invention are
3 illustrated by the following representative examples. In these
9 examples, Examples 1 to 2 illustrate embodiments for carrying out
Step 1, Examples 3 to 4 for Step 2, Examples 5 to 6 for Step 3,
Il Ex~.mples 7 to 8 for Step 4, Examples 9 to 13 for Step 5, Examples
l~ 14 to 18 for Step 6, Examples 19 to 22 for Step 7, Example 23 for
13 Step 8, Example 24 for Step 9 and Example 25 for Step iO. The
l~ Rf values set forth in the examples are the results when the com-
pounds are developed on a silica yel plate (DC-Fertigplatten
16 Kleselgel 60 F254 made by E. Merc~ & Co.) using the following
17 solvent systems.
8 A: isopropanol-28% aqueous ammonia-chloroform
(2:1:1 by volume)
B: isopropanol-28~ aqueous ammonia-chloroform
21 (4:1:1 by volume)
22 C: the lower layer of methanol-28~ aqueous ~unmonia-
23 chloroform (1:1:1 by volume)
2~ - D: chloroform-methanol ~90:10 by volume)
E: chloroform-methallol (95:5 by volume)
26
27 E~ample 1. Preparation of 2'-N~tS)-4-benzylo~vcarbonyl-amino-2-
2a hydro~ybutyryl]fortimicin B (Compound No. 11):
29 In this e~mple, 8.71 g (25.0 millinoles) of fortimicin
~ is dissolved in 500 ml of methanol and the solution is
stirred under ice cooling (3 to 5C). A solution wherein 13.1 g
- 28 -
. ' ' : '
llZ~3~S7 .
~. , - . .
' ' ,
t (37.5 millimoles) of N-hydroxy~succinimide ester of (S)-4-
2 benzyloxycarbonylamino-2-hydroxy-butyric acid was dissolved in
3 100 ml of tetrahydrofuran is added dropwise to the above solu-
~ tion over a period of 1.5 hours. After the completion of this
s addition, the reaction solution is stirred under ice cooling for
6 one hour and at room temperature for 18 hours, and then the
7 solvent is distilled off. The resulting residue is dissolved in
8 200 ml of water and 100 ml of chloroform and stirred well. The
9 water layer and chloroform layer are divided and the chlorororm
layer is washed with 50 ml of water. The water layer and the
1I washed water are co ~ ined and passed through a column packed with
B 12 400 ml of AmberlitelCG-50 (NH4 form). After the column is
13 washed ~ith 2 L. of water, elution is carried out with 0.1N
l~ aqueous ammonia and the eluate is taken in 20 ml fractions.
Fraction Nos. 68 to 84 containing a compound having a Rf value of
~6 0.57 in solvent system B are combined. The solvent is distilled
17 off to obtain 5.90 g of a white solid.
18 P~R (methanol-d4) spectra of the product is as
19 follows:
c~ 1.09 (3~, d), 1.2-1.9 t61~, m), 2.40 (3H, s),
21 5.04 (2~, s), 7.33 (2H, s)
22 On the basis of thc data, it i9 conEirmed that the
23 product is 2'-N-~(9)-~-benzyloxycarbonylamlno-2-hyclroxy-butyryl~-
24 fortimicin B.
Yield
26
22 Example 2. P e~~rat~_n of 2'-N-(O-benzvl~lycolyl)fortimicin_B
28 (Com~ound No. 12):
; 29 In this example, 2.67 g (16.1 millimoles) of O-ben~yl-
glycolic acid and 1.85 g (16.1 millimoles) of N-hydroxysuccinimide
31 are dissolved in 100 ml of tetrahydrofuran and the solution is
- 29 -
.
. . . . . .
.: , . ' '' ' ' ' .
. . .
~128~S7
,
stirred under ice cooling (3 to 5C). Then, 3.33 g (16.l milli-
moles) of N,N'-dicyclohexylcarbodiimide is added and the resulting
3 solution is stirred under ice cooling for 2 hours. The thus pre-
pared solution of N-hydroxysuccinimide ester of O-benzylglycolic
acid is added to a solution of 4.00 g (ll.5 milliFloles) of forti-
6 micin B in l00 ml of methanol and stirred at room temperature for
7 l3 hours. The solvent in the reaction solution is distilled off
8 to obtain a solid residue. To the residue 100 ml of ethylacetate
9 is added and then l00 ml of water and the mixture ~as well stirred.
lo Insoluble matters in the reaction mixture are filterRd off and
11 the çthylacetate layer of the filtrate is divided and washed with
12 50 ml of water. The water layer of the filtrate and the washed
13 water are combined and passed through a column packed with 1;0 ml
E~ of Amberlite1CG-50 (NH4 form). After the column is washed with
700 ml of water, elution is carried out with 0.075N aqueous
16 ammonia. The eluate is taken in 20 ml fractions. Fraction Nos.
17 26 to 47 containing a compound having a Rf value of 0.33 in
la solvent system C are combined and the solvent Ls distille~ off to
19 obtain l.30 g of a white solid. PMR (methanol-d4) spectra of the
product is as follows:
21 ~ 1.05 (3EI, d), 1.2-l.9 (4H, m), 2.40 (3EI, s),
22 3.49 (3H, s), 3.92 (2H, s), 5.10 (lH, d),
23 7.33 (SEI, s).
2~ On the basis of the data, it is confirmed that the product is
2'-N- (O-benzyl~lycolyl)Eortimicin B.
26 Yield 23~.
27
23 Example 3. Preparation of lf6'-di-N-benzyloxYcarbonvl-2~ -[(s)
29 -bcnzyloxycarbonyla ino-2-hydroxybutyry1]~ fortir~licin B (compound
i No. 13):
31 In thLs example, 3 90 y (6.7 millimoles) of 2'-N- [(S)-
~ . .
-- 30 --
.
:
': ~
1~28~)S7
.
i 4-benzyloxycarbonylamino-2-hydroxybutyryl]fortimicin B and 2 ml
2 (14.4 millimoles) of triethylamine are dissolved in 200 ml of
3 methanol and the solution is stirred under ice cooling (3 to 5C).
4 A solution of 4.00 g (16.0 millimoles) of N-(benzyloxycarbonyloxy)-
succinimide in 50 ml of tetrahydrofuran is then added dropwise to
6 the above solution over a period of one hour. After the completion
7 of this addition, the solution is stirred under ice cooling for
8 one hour and at room temperature for 16 hours. The solvent is
9 distilled off from the reaction solution and the resulting solid
residue is dissolved in 250 ml of chloroform. The thus obtained
Il solution is then washed twice with 100 ml of water and dried with
12 anhydrous sodium sulfate. Then the chloroform solution is concen-
13 trated to dryness under reduced pressure to obtain a solid residue.
The solid residue is dissolved in a small amount of chloroform
and the chloroform solu-tion is charged into a column packed with
t6 200 g of silica c;el (Xieselgel 60 made by E. Merck s Co.). E~lution
7 is carried out with a solvent system of chloroform-methanol (95:5
18 by volume). The eluate is taken in 16 ml fractions. Fraction
Nos. 121 to 370 containing a compound having a Rf value of 0.31 in
solvent system D are combined and the solvent is distilled off to
21 obtain 3.12 g of a white solid. PMR (mcthanol-d4) spectra of the
22 product is as follows:
23 ~ 1.03 ~3H, d), 1.2-1.9 (6Ei, m), 2.37 ~3EI, s),
24 3.48 (3Ei, s), 5.06 (611, s), 7.30 (15H, ~3~
On the basis of the data, it is confirmed that. the
26 product is 1,6'-di-N-benzyloxycarbonyl-4-N--~(S)-4-benzyloxycar-
27 bonylamino-2-hydroxybutyryl]fortimicin B. -
28
29 Example 4. Preparation of 1,6'-di-M-benzyloxycarbonyl-2~-h-
(O-benzylqlylcoly])fortimicin B (Compound No. 14):
31 In this example, 1.30 g (2.62 millimoles) of 2'-N-~O-
.
,
. . .
`.. ': ',
' , :
llZ8Q57
1 benzylglycolyl)fortimiein B obtained in Example 2 is dissolved in
t 50 ml of rnethanol and the solution is stirred under iee eooling
3 (3 to 5C). To the solution, 1.43 g (5.75 millimoles) of N-
~ (benzylo~yearbonyloxy) succinimide is added and the solution isstirred at room temperature for la hours. The solvent in the
6 reaetion solution is then distilled off to obtain a solid residue.
7 The solid residue is dissolved in 50 ~1 of chloroform, and the
8 solution is washed twice with 30 ml of water and dried with
9 anhydrous sodium sulfate. The chloroform solution is coneen-
trated to dryness under reduced pressure to obtain a solid resi-
11 due. The solid residue is dissolved in a small amount of ehloro-
12 Eorm and charged into a eolumn paeked with 60 g of siliea gel as
l3 in Example 3. Elution is earried out with a solvent system of
1~ chloroform-methanol (95:5 by voiume). The eluate is taken in 16
S ml fractions. Fraction Nos. 14 to 66 containing a compound
16 having a Rf value of 0.41 in solvent system D are eombined and17 the solvent is distilled off to obtain 1.30 g of 1,6'-di-N-
l~ benzyloxyearbonyl-2'-N-(0-benzylglycolyl) fortimiein B as a white
solid.
Yield 65~.
2~
22 Example S. Preparation of 2'-N-ctlveylEortimiein B ~Comoound
2j No. 15):
.. . .
24 In this e~ample, lO0 cJ of the sulEate of fortimiein A,
whlch corresponds to 61 g (]50 millimoles) of Eortimiein A, is
6 dissolved in 2 L. oE water. The solution is adjusted to a pH
27 oE about 9 with potassium carbonate and then to a pH of 10 with
5N sodium hydroxide solution and heated under reflux for 4 hours.
29 After eooling to room temperature, the reaction solution is
~o adjusted to a pH of 7 with eoneentrated hydroehloric a~ d and
31 passed through a column packed with 2 L. of Amberlite IRC-50
,
- 32 -
.r
' ~
.
:
.: '
1128~S7
(NH"+ form). lrhe column is washed with 10 L. of water and then
elution is carried out with 0.lN aqueous ammonia. Then, 5 r,. of
early eluate is collected and the successive eluate is taken in
0.5 L. fractions. Fraction Nos. 3 to 27 containinq a com~ound
having a Rf value of 0.45 in solvent system A are combined. The
solvent is distilled off to obtain 37.6 g of a white solid.
PMR (deuterium oxide) spectra of the product is as follows:
1.07 (3H, d), 1.2-1.8 (4H, m), 2.41 (3H, s),
3.31 (2H, s), 3.50 (3H, s), 5.20 (lH, d).
On the basis of the data, it is confirmed that the
product is 2'-N-glycylfortimicin B.
Yield 62%.
Example 6. Preparation of 2'-N-hydantoylfortimicin B
(Compound No. 16):
In this example, 2.0 q (4.5 millimoles) of fortimicin C
is dissolved in 20 ml of water and the solution is heated at a
temperature of 60C for 24 hours. After cooling to room tempera-
ture, the reaction solution is adjusted to a p~! of 7 with lM
hydrochloric acid and passed through a column packed with 100 ml
of Amberlite CG-50 (NH4 form). After the column is washed with
300 ml of water, elution is carried out with 0.lN aqueous
ammonia. The eluate is taken in 20 ml fractions. Fraction ~os.
20 to 26 containing a compound having a Rf value of 0.44 in
solvent system A are combined and the solvent is distilled off
to obtain 658 mq of a white solid. PMR (deuterium oxide) spectra
of the product is as follows:
~ 1.12 (3H, d), 1.2-1.8 ~4H, m), 2.38 (3H, s),
3.47 ~3~1, s), 3.78 (2H, s), 5.28 (lH, d).
; On the basis of the data, it is confirmed that the
product is 2'-N-hydantoylfortimicin B.
dm~ 33 -
:
. .
2~3~S7`
.
I Yield 33~.
z
3 Ex~p~e 7. Pre~aration of 1,6'-di-N-benzyloxycarbonvl-2~-~-(N
~ benzvloxycarbonylglycvl)fortimicin B tCom~ound_17):
In this example, 10.1 g (25 millimoles) of 2'-~-
6 glycylfortimicin E3 obtained in Example S and 10 ml (72 millimoles)
7 of triethylamine are dissolved in 800 ml of methanol and the
8 solution is stirred under ice cooling (3 to 5C). A solution of
9 22.4 g (90 millimoles) of N-(benzyloxycarbonyloxy) succinimide in
200 ml of tetrahydrofuran is then added dropwise over a period of
IL 3 hours to the above solution. After the completion of this
addition, -the reaction solution is stirred under ice cooling for
13 one hour and then at roo~ temperature for 16 hours. The solvent
9 1~ in the reaction solution is distilled off and the resulting
residue is dissolved in 300 ml of chloroform. ~he solution is
16 then washed twice with 200 ml of water and dried with anhydrous
7 sodium sulfate. The chloroform solution is concentrated to
a dryness under reduced pressure to obtain a solid residue. The
I9 solid residue is then dissolved in a small a~ount of chloroform
zo and charged into a column packed with 600 g of silica gel.
2I E~lution is carried out with a solvent system of chloroform-
22 methanol (95:5 by volume) and the eluate is taken in 150 ml
23 fractions. Fraction Nos. 23-11 containing a compound having a
2~ ~E value oE 0.43 in solvent syst~n D are combined and the solvent
is distilled off to obtain 9.0 g of a white solid. P~R (methanol-
26 d4) spectra of the product is as follows:
n ~ 1.03 (3E~, d), 1.2-1.9 (4H, m),- 2.36 (3H, s),
L0 3.47 (3H, s), 7.36 ~15H, m).
29 On the basis of the data, it is ~confirmed that the
product is l~6~-di-N-benzyloxycarbonyl-2;-N-(N-ben7~yloxycarbon
3I glycyl)fortimicin E3.
. . ,
- 34 -
,
~ ~ 2 8 C~ 5 7
I Yield 45~.
3 Example 8. Preparation of 1,6'-di-N-benzyloxycarbonyl-2~-N
hvdalltoylfortimicin B ~Compound No. 18):
In this example, 297 mg (0.66 millimole) of 2'-N-
6 hydantoylfortimicin B obtained in Example 6 and 0.2 ml (l.4 milli-
7 moles) of triethylamine are dissolved in 15 ml of methanol and
t the solution is stirred under ice cooling (3 to 5C). A solution
9 of 362 mg (l.46 millimoles) of N-(benzyloxycarbonyloxy) succini-
o mide in 5 ml of tetrahydrofuran is then added dropwise over a
~1 period of one hour to the above solution. After the completion
l2 of the addition, the solution is stirred under ice cooling for
13 one hour and at room temperature for 16 hours. The solvent in
the reaction mixture is distilled off to obtain a solid residue
and the residue is dissolved in 20 ml of chloroform. The chloro-
form solution is then washed with 20 ml of water and dried with
17 anhydrous sodium sulfate. The chloroform solution is concentrated
a to dryness under reduced pressure to obtain a solid residue. The
residue is dissolved in a small amount of chloroform and charsed
into a column pac~ed with 15 ~ of silica gel. Elution is carried
2l out with a solvent system of isopropanol-chloroform-28~ aqueous
22 ammonia (~0:1:1 by volume) and the eluate is ta~en in 6 ml frac-
Z3 tions. Fraction Nos. 13 to 35 containing a co~pound having a
z~ R value of 0.64 in solvent syst~n B are combined and the solvent
is distilled off to obtain 146 mg of a white solid. P~1R (methanol-
26 d4) Spectra of the product i5 a'; follows:
27 ~ 1.13 (3H, d), 1.2-1.9 (4H, m),-2.40 (3H, s),
2a 3.50 (3H, s), 5.06 (4H, br), 5.33 (lH, d),
29 7.35 (lOH, s)
~o On the basis of the data, it is confirmed that the
3~ product is l~6l-di-N-benzyloxycarbonyl-2l-N-hydantoyl-fortimicin
.
- 35 -
- - . . - .
,.
l~Z8QS-~7
B. Yield 31~.
Example 9. Preparation of ],6'~di-N-benzy]oxycarbonyl-4,2'-di-
N-~N-benzyloxycarbonylglycyl)fortimicin B (Compound No. 19):
In this example, 230 mq (1.1 millimoles) of N-benzyl-
oxycarbonylglycine and 149 m~ (1.1 millimoles) of l-hydroxybenzo-
triazole are dissolved in 20 ml of tetrahydrofuran and the
solution is stirred under ice coolin~ (3 to 5C). To the
solution, 227 mq (1.1 millimoles) of N,M'-dicyclohexylcarbo-
:
diimide is added and the solution is then stirred under icecoolin~ for one hour. Then, to the mixed solution there is
added 808 mg (1.0 millimole) of 1,6'-di-N-benzyloxy-carbonyl-2'-
N-(N-benzyloxycarbonylglycyl)-fortimicin B and the solution is
stirred at room temperature for 19 hours. After insoluble
matters in the reaction solution are removed by filtration, the
solvent in the filtrate is clistilled off to obtain a solid
residue and the residue is then dissolved in a small amount of
chloroform. The chloroform solution is charqed into a column
packed with SO g of silica qel. Elution is carried out with a
solvent system of chloroform-methanol (97:3 by volume) and the
~,, .
eluate is taken in 16 ml fractions. Fraction ~os. 22 to 37
; containinq a compound havinn a Rf value of 0.83 in solvent
system n are combined and the solvent is distilled off to obtain
910 mg of a white solid. PMR (methanol-dl,) spectra of the
product is as follows:
` ~ 1.12 (3H, d), 1.3-1.8 (4H, m), 3.05 (3H, s),
. .,
3.35 (3H, s), 5.3 (8H, br), 7.30 (20H, s).
On the basis of the data, is is confirmed that the
product is 1,6'-di-N-benzyloxycarbonyl-4,2'-di-N-(N-benzyloxy-
carbonylglycyl)fortimicin B. Yield 91%.
m~ 36 -
,
1128~57
I Example 10. Preparation of 1,6'-di-N-benzyloxycarbonyl-4-N-[(S)-4-
2 benzyloxycarbonylamino-2-hydroxybutyryl]-2'-N-(M-benzyl_xycarbonyl-
3 glvcyl)fortimicin ~ (Compound No 20):
~ In this example, 205 mg (0.81 millimole) of (S)-4-
benzyloxycarbonylamino-2-hydroxybutyric asid and 110 mg (0.31
6 millimole) of l-hydroxybenzotriazole are dissolved in 10 ml of
7 tetrahydrofuran and the solution is stirred under ice cooling
8 (3 to 5C). To the solution, 167 mg (0.81 millimole) of N,N'-
9 dicyclohexylcarbodii~ide is added and the mixed solution is
stirred under ice cooling for one hour. Then, 435 mg (0.54
millimole) of 1,6'-di-N-benzyloxycarbonyl-2'-N-(N-benæyloxy-
carbonylglycyl)fortimicin ~ is added and the mixed solution is
13 stlrred at room temperature for 18 hours. After the insoluble
l~ matter in the reaction solution is removed by filtration the
solvent is distilled off to obtain a solid residue. The solid
6 residue is dissolved in a small amount of chloroform and the
~7 chloroform solution is charged into a column packed with 25 g of
18 silica gel. Elution is then carried out with a solvent system of
19 chloroform-methanol (97:3 by volume) and the eluate is ta~en in
10 ml fractions. Fraction Nos. 21-35 containlng a compound
21 having a Rf value oE 0.77 in solvent system D are combined and
22 the solvent is distilled off to obtain 344 mg of a white solid.
2J PMR (methanol-d4) spec~ra of the product i5 as follows:
2~ - ~ 1.13 (3H, d), 1.2-2.0 (6H, m), 3.07 ~3TI, s),
3.33 ~3lI, s), 5.07 ~8H, s), 7.30 ~20H, s)
26 On the basis of the data, it is confirmed that the
Z7 product is l~6l-di-N-ben~yloxycarbonyl-4-N [~S)-4-benzyloxy-
28 carbonylamino-2-hydroxybutyryl]-2'-N-(N-benzyloxy-carbonylglycyl)-
29 fortimicin s. Yield 61~.
- 37 -
. :
"
-- llZ8(~57
.
I Example 11. Preparation of l,6'-di-N-benzyloxycarbonvl-4,2'-di-N-
2 [(S)-4-benzvloxYcarbonylamino-2-hvdroxYbutyrYl]fortimicin B
3 (Compound No. 21):
~ In this example, l.0l g (4.0 millimoles) of (S)-4-
benzyloxycarbonylamino-2-hydroxybutyric acid and 0.54 g (4.0
6 millimoles) of l-hydroxybenzotriazole are dissolved in 80 ml
7 of tetrahydrofuran and the solution is stirred under ice cooling
a (3 to 5C). Then 0.83 g (4.0 millimoles) of N,N'-dicyclohexyl-
9 carbodiimide is added and the mixed solution is stirred under ice
cooling for one hour. To the solution, 3.10 g (3.6 millimoles)
11 of l,6'-di-N-ben7yloxycarbonyl-2'-N-t(S)-4-benzyloxycarbonylamino-
2 2-hydroxybutyryl]fortimicin B is added and the mixed solution is
~3 stirred at room temperature for 18 hours. After insoluble matters
t4 in the reaction mixture are removed by filtration, the solvent in
the filtrate is distilled off to obtain a solid residue. The
residue is dissolved in a small amount of chlorofo~n and the
17 chloroform solution is charged into a column packed with 100 g of
1a silica gel. Elution i9 car~ied out with a solvent system of
19 chloroform methanol (95:5 by volume). The eluate is taken in 16
ml fractions. Fraction Nos. 24-66 containlng a compound having a
21 Rf value of 0.53 in solvent system D are combined and the solvent
22 is distilled off to obtain 1.11 g of a white bolid. PM~ (methanol-
21 d4~ spectra of the product is as ~ollows:
2~ ~ l.l3 ~311, d), 1.2-1.9 (811, m), 3.10 (3H, s),
3.35 (3H, s), 7.33 (201~, s)
26 On the basis of the data, it is confirmed that the
27 product is 1,6'-d.i-N-benzyloxycarbonyl-4,2~-di-N-t(S)-4-
2a benzyloxycarbonylamino-2-hydroxybutyryl]fortimicin B.
29 Yield 28~. -
31
,
- 38 -
,
,.
:: :;,
: l~Z8~!57
Example 12. Preparation of 1,6'-di-N-benzyloxycarbonvl-4-N-
2 [ (S) - 4-benzyloxycarbonylamino-2-hydroxybutyryl~-2'-N- (O-
3 benzvlglycolyl)fortimicin B (Com~ound No. 22):
In this example, 500 mg (1.98 millimoles) of
(S)-4-benzyloxycarbonylamino-2-hydroxybutyric acid and 300
6 mg (1.98 millimoles) of l-hydroxybenzotriazole are dissolved
7 in 15 ml of tetrahydrofuran and the solution is stirred
8 under ice cooling (3 to 5CC). To the solution, 400 mg (1. sa
9 millimoles) of N,N'-dicyclohexylcarbodiimice is added and the
~ mixed solution is stirred under ice cooling for one hour. Then,
1.25 g (1.64 millimoles) of 1,6'-di-N-benzyloxycarbonyl-2'-N-(O-
12 benzylglycolyl)fortimicin B obtained in Example 4 is added and the
13 mixed solution is stirred at room temperature for 46 hours.
1~ After the insoluble matters in the reaction solution are removed
by filtration, the solvent is distilled off to obtain a solid
6 residue. The residue is dissolved in a small amount of chloro-
17 form and the chloroform solution is charged into a column packed
~a with 60 g of silica gel. Elution is carried out with a solvent
19 system of chloroform-methanol (97:3 by volume) and the eluate is
taken in 16 ml fractions. Fraction ;\Tos. 43-54 containing a
21 compound hav~ng a Rf value of 0.79 in solvent system E are com-
n bined and concentrated to dryness to obtain 350 mg of 1,6'-di-N-
23 benzylo~cycarbonyl-9-N-[(S)-4-benzylo:cycarbonyla~nino-2-
.
24 hyclroxybutyryl]-2'-N-(O-benzylglycolyl)Eortimicin 8 as a
~hite solid. Yield 21~.
26
27 Æxalllple 13. Preparation Oe 1,6'-di-N-benzyloxycarbonyl-4,2'-
213 di-N-hydantoYlEortin~icin B:
29 In this e~ample, 71 mg (0.60 millimole) of hydantoic
acid and 81 mg (0.60 millimole) of l-hydroxy-benzotriazole
3l are dissolved in 10 ml of dimethylformamide and the solution
.
_
~lZ8~S7 `
1 is stirred under ice cooling (3 to 5C). To the solution, 124
2 mg (0.60 millimole) of N,N'-dicyclohexylcarbodiimide is added
3 and the mixed solution is stirred under ice cooling for one hour.
~ Then, 330 mg (0.46 millimole) of 1,6'-di-N-benzyloxycarbonyl-2'-
N-hydantoylfortimicin B obtained as in Example 8 is added and the
6 mi~ed solution is stirred at room temperature for 41 hours.
7 After insoluble matters in the reaction solùtion are removed by
a filtration, the solvent in the filtrate is distilled off to
g obtain a solid residue containing 1,6'-di-N-benzyloxycarbonyl-
4,2'-di-N-hydantoylfortimicin B.
l2 Example 14. Preparation of 2'-N-glycylfortimicin A
13 (Compound No. 23):
In this example, 500 mg (0.50 millimole) of 1,6'-di-
IS N-benzyloxycarbonyl-4,2'-di-M-(N-benzyloxycarbonylglycyl)
6 fortimicin B obtained in Example 9 is dissolved in 24 ml of
0.2N hydrochloric acid-methanol solution wherein 12N hydrochloric
acid was diluted with methanol to make the solution 0.2N
19 (the same as below). To the solution, about 50 mg of 10% palla-
dium-carbon is added and then hydrogen gas is bubbled at room
zl temperature and atmospheric pressure. After the catalyst in the
22 reaction mi:cture is removed by filtration, the filtrate is concen-
z3 trated to dryness under reduced pressure to obtain a residue.
2~ Th~ residue is dissolved in about S ml of water and the solution
is then adjusted to a pH of 7. The solution is pdssed through a
B z6 column packed with 20 ml of Amberllte~ ~.-50 (NH4~ form). .~fter
27 the column is waslled with 100 ml of water,~elution is carried out
2~ with 0.3N aqueous ~nonia and the eluate is taken in S mL frac-
29 tions. Fraction Nos. 12 to 14 containing ~a compound having a Rf
lo value of 0.42 in solvent system A are combined and the solvent is
3I distilled off to obtain 9;' mg of a white solid. Properties of
.
-
.. . .
. , .
:
' ' ~ :
llZ~3Q57
I the product are as follows:
t ~Sass spectra: m/e 463 (M+ ~ 1), 462 (M+), 445, 416, 328,
3 246, 207, 200
~ PM~ (deuterium oxide): ~ 1.13 (3H, d), 1.3-1.9 (4H, m),
3.05 (3H, s), 3.44 (3H, s),
6 4.65 (lH, d-d), 4.95 (lH, d)
7 On the basis of the data, it is confirmed that the
i3 product is 2'-N-glycylfortimicin A. Yield 42~.
Exam~le 15. Prepara_ion of 4-N-[(S?-4-amlno-2-hYdroxybutyryl]-
-
2'-N-qlycylfortimicin B (Compound No. 24):
It In this example, 4.03 g (3.86 millimoles) of
13 1,6'-di-N-benzyloxycarbonyl-4-N-[(S)-4-benzyloxycarbonyl-amino-
1~ 2-hydroxybutyryl]-2'-N-(N-benzyloxycarbonylglycyl)fortimicin B
obtained as in Example 10 is dissolved in 150 ml of methanol
16 solution containing 2.3 ml t31 millimoles) of trifluoroacetic
17 acid. To the solution, about 200 mg of 10~ palladium-carbon is
a added and hydrogen gas is bubbled at room temperature and atmo-
19 spheric pressure. After the catalyst in the reaction mixture is
removed by filtration, the filtrat:e is concentrated to dryness to
Z~ obtain 3.84 g of the triPluoroacetate of 4-N-~(S)-4-amino-2-
2z hydroxybutyryl]-2'-N-glycylfortimicin B as a grayish whitc'solid.
23 The Rf value of the product on TLC in solvent system A is 0.28.
2~
Exam~le 16. Pre~aration of ? ' -N-hydantovlfortimicin c
26 (ComPound No. 4):
2'7 In this example, all of the 1,6'-di-N-ben~yloxycarhonyl-
2~3 4,2'-di-N-hydantoyLfortimicin B obtained in Example 13 is
z9 dissolved in 15 ml of 0.2N hydrochloric acid-methanol solution
and about 30 mg of 10~ palladium-carbon is then added thereto;
n Hydrogen gas at room temperature and atmospheric pressure is then
- 41 -
- l~Z~3~57
.
I bubbled therethrough. The catalyst in the reaction mixture is
2 removed by filtration, and the filtrate is concentrated to
3 dryness under reduced pressure to obtain a residue. The
~ residue is dissolved in about 2 ml of water. The solution is then
s adjusted to a pH of 7 with lN sodium hyd~;oxide and passed through
6 a column packed with 10 ml of Amberlite(~CG-50 (NH4+ form). After
7 the column is washed with 50 ml of water, elution is carried out
8 with 0.3N aqueous ammonia and the eluate is taken in 2 ml frac-
g tions. Fraction Nos. 33 to 51 containing a compound having a Rf
I0 value of 0.31 in solvent system ~ are combined and the solvent is
distilled off to obtain 104 mg of a white solid. P~IR (deuterium
2 oxide) spectra of the product is as follows:
3 ~ 1.04 (3H, d), 1.2-1.9 (4H, m), 3.10 (3H, s),
3.44 (3H, s), 4.87 (lE~, d), 4.95 (lH, d-d).
On the basis of the data, it is confirmed that the
6 product is 2'-N-hydantoylfortimicin C. Yield 41~.
Then 110 mg (0.2 mlllimole) of the thus obtained
18 2'-N-hydantoylfortimicin C is dissolved in 2 ml of water and
I9 the solution is adjusted to a pH of 4 with 5N sulfuric acid.
The solution is then added dropwise to 30 ml of ethanol and the
2~ resulting precipitate is collected and dried to obtain 124 mg
22 (0.18 millimole) o~ thc sulfate of the compound as a white
23 powder. Yield 87'~.
23
24 - t~D = ~58.5 ~c=0.2, water)
Elementary analysis
26 Calculated as C2ll~LloN~3og-H2so4-c2ll5o 2
27 C, 38. a6~ H, 7.09%: N, 15.77'~
28 Found
29 C, 3'3.76~: H, 6.95~: N, lG.03%
31
,
,
.
,
~ 1~2~Q~7
.
1 Example 17. Preparation of 4,2'-di-N-r~S)-4-amino-2-hydroxy-
2 butyryl~fortimicin B (Compound No. 25):
3 In this example, 1.07 g (0.99 millimole) of 1,6'-di-
4 N-benzyloxycarbonyl-4,2'-di-N-[(5)-4-benzyloxycarbonylamino-2-
hydroxybutyryl]fortimicin B obtained in Example ll is dissolved
6 in 60 ml of methanol containing 0.6 ml (8.l millimoles) of tri-
7 fluoroacetic acid and about lO0 mg of 10% palladium-carbon is then
8 added to the solution. Through the reaction mixture hydrogen gas
9 is bubbled at room temperature and atmospheric pressure. After
the catalyst in the reaction mixture is removed by filtration,
the filtrate is concentrated to dryness to obtain l.06 g of the
trifluoroacetate of 4,2'-di-N-[(S)-4-amino-2-hydroxybutyryl]-
13 fortimicin B as a grayish white solid. The product shows a Rf
1~ value of 0.13 on TLC in solvent system A.
6 xample 18. Preparation of 4-N-~(S)-4-amino-2-hydroxybutyryl]-
2'-N-glycolylfortimicin B (Compound No. 26):
9 In this example, 780 mg (0.78 millimole) of 1,6'-
19 di-W-benzyloxycarbonyl-4-N-[(S)-4-benzyloxycarbonylamino-2-
hydroxybutyryl]-2'-N-(O-ben7ylglycolyl)fortimicin B obtained
2~ as in Example 12 is dissolved in 20 ml of methanol containing
z2 0.40 g (3.5 millimoles) of trifluoroacetic acid and about
23 50 mg of lO~ palladium-carbon is added to the solution. Through
2~ - the reaction mixture hydrogen gas is bubbled at room temperature
and atmospheric pre~sure. After the catalyst in the reaction
26 mixture is removed by filtration, the fLltrate is concentrated to
27 dryness to obtain the trifluoroacetate of -4-N-[(S)-4-amino-2-
28 hydroxybutyryl]-2'~N-glycolylfortimicin B as a grayish white
29 solid. The product shows a Rf value of 0.-32 on TLC in solvent
system C.
31
.
- 43 -
~,
'` '` ` - ~ ~ . :
~:
'
- ~28~S7
.
I Example 19. Preparation of 4,2'-di-N-(2-aminoethxl)fortimicin 8
2 (Compound No. 5):
3 In this example, 170 mg (0.37 millimole) of 2'-N-
~ glycylfortimicin A obtained as in Example 14 is suspended in 10
ml of tetrahydrofuran and 5 ml (5 millimoles) of lM diborane-
6 tetrahydrofuran solution is added thereto. The reaction mixture
7 is heated by reflux with'stirring for one hour. After the reac-
8 tion mixture is cooled to room temperature, 0.5 ml of water is
9 added thereto to decompose excess diborane and the resulting
o solution is then concentrated to dryness under reduced pressuLe.
Il To the resulting residue 10 ml of 80~ aqueous hydrazine is added
12 and the mixture is heated by reflux for 3 hours and then concen-
13 trated to dryness. The thus obtained residue is dissolved in
1~ about 10 ml of water and the solution is ad]usted to a pH of 6
IS with lN hydrochloric acid. The solution is then passed through a
B 'column packed with 10 ml of Amberlite~CG-50 (NH4 form). After
l7 the column is washed with 50 ml of water, elution is carried out
18 with 0.6~l aqueous ammonia and t~.e eluate is taken in 5 ml frac-
19 tions. ~raction Nos. 22 to 46 containing a compound having a
Rf value of 0.35 in solvent system A are combined ancl the solvent
21 is distilled off to obtain 139 mg of a white solid. ~roperties
2z of the product are as follows:
23 Mass spectra: m/e 435 (M+), 417, 404, 387, 375', 3G7, 361,
2~ ' 344, 325, 301, 278, 262, 250, 233, 219,
207, 198, 136, 169, 155, 142, 129
26 P~R (deuterium oxide): c~ 1.06 (3~, d), 1.2-1~'(4~, m),
27 3.43 (3H, s), 5.10- (1~1, d)
28 On the basis of the data, it is confirmed that the
29 product is 4,2'-di-N-(2-aminoethyl)fortimicin B. Yield 85~.
Then" 110 mg (0.25 millimole) of 4,2'-di-N-(2-
31 aminoethyl)fortimicin B obtained above is dissolved in 2 ml of
- 44 -
~ . ' .
,
.
1~28~57
1 water and the solution is adjusted to a pH of 4 with 5N sulfuric
2 acid. The solution is then added dropwise to 20 ml of ethanol
3 and the resulting precipitate is collected. The precipitate is
4 dried to obtain 180 mg (0.23 millimole) of the sulfate of the
compound as a white powder. Yield 92~. Properties of the product
6 are as follows:
7 ~]23 = +61 0 (c=0 5 water)
8 Elementary analysis
9 Calculated as ClgH42N6s-3H2S4 C2H5H~2H2
o C, 31.10%: H, 7.21%: N, 10.36%
~1 Found
12 C, 31.01%: H, 7.50g: N, 10.40%
13
14 Example 20. Preparation of 4-N-t(S)-4-amino-2-h~droxybutyl]-
2'-N-glycylfortimicin ~ (Compound No. 2a) and 4-N-~(S)-4-
6 amino-2-hydrox~butx~-2'-N-(2-am noethyl)fortimicin
17 (Compound No. 7):
.
a In this ex~lple, 50 ml of lM diborane-tetrahydrofuran
19 solution is added to 3.46 g (3.5 millimoles) of the trifluoro-
acetate of 4-N-[(S)-4-amino-2-hydroxybutyryl]-2'-N-glycyl-
21 ~ortimicin B obtained in Example 15 and the mixture is stirred
22 at room temperature for 30 minutes. ~fter about lO ml of water
23 is added to the reaction mixture under ice coolin~ to decompose
24 - excess diborane, the solvent is distilled off to obtain a
solid residue. To the resldue 150 ml of 0.2N hydrochloric acid-
26 methanol solution is added and the mlxture is heated at a tempera-
27 ture of 50C for 4 hours. The solvent in the mi.YtUre is distilled
28 off to obtain a solid residue. The residue is dissolved in about
29 50 ml of water and the solution is adjusted to a pH of 7 wi.th lN
sodium hydro~ide. The solution is passed through a column packed
31 uith lO0 ml of hmberlite1cG-so (NH4 form). After th- column is
- 45 -
~ . . ,
' ~'
'
~ ~2~3~)57
I washed with about 500 ml of water, elution is carried out with
2 0.4N aqueous ammonia and the eluate is taken in 20 ml fractions.
3 Fraction Nos. 13 to 26 containing a compound having RE value 0.33
~ in solvent system A are combined and the solvent is distilled off
to obtain 494 mg of a white solid. Properties of the product are
6 as follows: `~
7 Mass spectra: m/e 493 (M + 1), 492 (M ), 474, 444, 418,
8 387, 322, 294, 245, 219, 200
9 PMR (deuterium oxide): ~ 1.06 (3H, d),
1.2-1.9 (6H, m), 2.40 (3H, s),
11 3.29 (2H, s), 3.43 (3H, s),
12 5.10 (lH, d)
13 One the basis of the data, it is confirmed that the
product is 4-N-[(S)-4-amino-2-hydro~ybutyl]-2'-N-glycylfortimicin
B. The sulfate of the compound is prepared as in E~ample 19.
6 ~]23 = +70 oo (c=0 2 water)
Elementary analysis
8 Calculated as C21H44N6O7-2-5112SO4 2 5 2
C, 34.45%: H, 7.17%: N, 10.48
zo ~'ound
21 C, 34.29~: H, 7.45%: N, 10.61~
22 Then, elution is carried out with 0.5N aqueous ammonla
2a and the eluate is taken in 20 ml fractions. Fraction Nos. 11 to
2~ - 90 containin~ a compound havin~ a Rf value of 0.30 in solvent
system A are combined and the solvent is'distilled ofE to obta.in
26 638 m~ of a white powder. Properties of the product are as
27 follows: -
2a Mass spectra: m/e 479 (M+ + 1), 478 (M+), 460, 448, 443,
29 ' 430, 40~1, 375, 373, 361, 3~r4, 338, 322, 311,
306, 294, 272, 245, 219, 207, 202, 186, 173,
169, 155, 142, 126
4~ -
-
,
1128~57
PMR ~deuterium oxide): ~ 1.04 (-3H, d), 1.2-1.9 (4H, m),
2.43 (3H, s), 3.43 (3H, s), 5.13
~lH, d).
On the basis of the data, it is confirmed that the
product is 4-M-[(S)-4-amino-2-hydroxybutyl]-2'-~l-(2-aminoethyl)
fortimicin B. Yield 38%.
The sulfate of the compound is prepared as in Example
19. 2
[alD = +67.0 (c=0.2, water)
Elementary analysis
Calculated as C2lH46N606.3H2SO4.C2HsOH.H2O
C, 33.00~: H, 7.23%: N, 9.98%
Found
C, 32.76%: H, 7.18%: N, 9.70~
Example 21._ Preparation of 4-N-[(S)-4-amino-2-hydroxybutyl]-
2'-N-[(S)-4-amino-2-hydroxybut~ryl]fortimicin B (Compound ~o. 8)
and 4,2'-di-N-[(S)-4-amino-2-hydroxybutyl]fortimicin B
~Compound No. 9):
In this example, 1.06 q ~0.99 millimole) of the
trifluoroacetate of 4,2'-di-N-[(S)-4-amino-2-hydroxybutyryl]
fortimicin B is dissolved in 30 ml of tetrahydrofuran. To the
solution, 15 ml of lM diborane-tetrahydrofuran is added and the
mixture is stirred at room temperature for 30 minutes. After
about 5 ml of water is added to the reaction mixture under ice
coolin~ to decompose excess diborane, the solvent is distilled
off to obtain a residue. To the residue 50 ml of 0.2N hydro-
chloric acid-methanol solution is added. The solution is heated
at a temperature of 50C for 4 hours and the solvent is dis-
solved in about 20 ml of water. The solution is then adjusted
to a pH of 7 with sodium hydroxide and passed throu~h a colu~n packed
~,. b dm ~t~ - ~7 -
- ~
'
' 1 1 2 8 ~15 7
.
with 25 ml of Amberlite~CG-50 (NH4 form). After the column is
washed with 150 ml of water and 180 ml of aqueous ammonia, elution
3 is carried out with 0.5N aqueous ammonia. The eluate is ta~en
~ in 10 ml fractions. Fraction Nos. 6 to 100 conta-ning a compound
having a Rf value of 0.25 in solvent system A are combined and
6 the residue is concentrated to dryness to obtain 86 mg of a white
7 solid. Properties of the product are as follows:
8 Mass spectra: m/e 537 (~1+ + 1), 387, 361, 344, 322, 294,
9 226, 219, 143
o PMR (deuterium oxide): ~ 1.05 (3H, d), 1.2-1.9 (8H, m),
Il 2.43 (3H, s), 3.43 (3H, s),
l2 5.07 (lH, d)
~3 On the basis of the data, it is confirmed that the
l~ product is 4-N-[(S)-4-amino-2-hydroxybutyl]-2'-N-[(S)-4-amino-2-
hydroxybutyryl~fortimicin B. Yield 16%.
16 The sulfate of the compound is prepared as in
17 Example 19.
18 [~]25 = +87.5 (c-0.2, water)
19 Elementary analysis
Calclllated as C23H48N6o8-2-5H2so4 2 5 2
2i C, 3~.76~: H, 7.35~: N, 9.73
22 Found
23 C, 34.84~: E~, 7.61~: N, 9.78~
2~ Then, elution is carried out with 0.6N aqueous
ammonia and the eluate is taken in 10 mi fractions.
26 Fraction Nos. 82-170 containing a compound having a Rf value of
27 0.20 in solvent system A are combined and the solvent is distilled
2a off to obtain l50 mg of a white solid. Properties of the product
'29 are as follows:
30 . Mass spectra: m/e 523 tM + 1), 487, 474, 448, 401, 344,
3~ 322, 294, 230, 219, 213, 207, 175, 155
,
.
- 1128QS~
I PMR (deuterium oxide): ~ 1.05 (3H, d), 1.2-1.9 (8H, m),
2 2.44 (3H, s), 3.43 (3H, s),
3 5.20 (lH, d)
~ On the basis of the data, it is confirmed that the
s product is 4,21-di-N-[~S)-4-amino-2-hydroxybutyl]fortimicin B.
The sulfate of the compound is prepared as in
7 Example 19.
3 [~]23 = +88 0 ( 0 2
9 Elementary analysis
Calculated as C23HsoN67 3H2S4 1 5H2
1I C, 33.74%: H, 7.36%: N, 9.44%
12 Found
13 C, 33.77%: H, 7.61~: N, 9.20~
Example 22. Preparation of 4-N-[(S)-4-amino-2-hydroxvbutvl]-
6 2'-N-(2-hydroxyethyl)fortimicin B (Compound No. 10):
-
17 In this example, 15 ml of lM diborane-tetrahydrofuran
18 solution is added to the trifluoroacetate of 4-N-[(S)-4-amino-
19 2-hydroxybutyryl]-2'-N-glycolylfortimicin B obtained in
Ex~lple 18 and the mixture is heated by reflux with stirring
2i for 2 hours. After the reaction solution is cooled to room
22 temperature, about 2 ml of water is added thereto to decompose
23 excess diborane and the solvent is distilled off. To the
2s resultinq residue, 20 ml of 0.2N hydrochloric acid-methanol
2S solution is added and the solution is allowed to stand at room
26 temperature for 23 hours. Then, the solvent is distilled off.
27 ~he resulting residue is dissolved in about 10 ml of water. The
28 solution is ad~usted to a p~l o ~ 7 and passed through a column
B 29 packed with 30 ml of ~nberlite1 CG-50 (NH4 form). After the
Jo column is washed with 150 ml of water and 300 ml of 0.2N aqueous
31 ammonia, elut~on is carried out with 0.4N aqueous a~monia. The
.' . ;
.
` ~ "
11280~7
eluate is taken ln 20 ml fractions. Fraction Nos. 14 to 21
containing a compound having a Rf value of 0.50 in solvent system
C are combined and the solvent is distilled off to obtain 228 mg
of a white solid. Properties of the product are as follows:
Mass spectra: m/e 480 (M+ + 1), 444, 431, 405, 322, 294,
261, 219, 203, 187
PMR (dPuterium oxide): ~ 1.02 (3H, d), 1.2-1.9 (6H, m),
2.40 (3H, s), 3.42 (3H, s), 5.15
(lH, d).
On the basis of the data, it is confirmed that the
product is 4-N-[(S)-4-amino-2-hydroxybutyl]-2'-N-(2-hydroxyethyl)
fortimicin B. Yield 61~.
The sulfate of the compound is prepared as in Example
19, Properties of the product are as follows:
23
[a]D = ~59 5 (c=0.2, water)
Elementary analysis
Calculated as C2lH4sNsO7.2.5H2SO4.C2HsOH.H2O
C, 35.01%: H, 7.41%: N, 8.89%
Found
C, 34.92~: H, 7.56%: N, 8.67%
Example 23. Preparation of 1!6~-di-N-benzyloxycarbonyl-4-N
[(S)-4-benzyloxycarbonylamino-2-hydroxybutyll-2l-N-(N-ben
carbonylglycyl)fortimicin B (Compound No. 27) and 1,6'-di-N-
benzv~g ~ )-4-benzyloxycarbonyl-amino-2-
hydroxybutvl]-2'-N-(2-benzyloxvcarbonylaminoethyl)fortimicin B
~ComPound No. 28):
In this example, 324 mg (0.30 millimole) of 1,6'-
di-M-benzyloxycarbonyl-4-N-[(S)-4-benzyloxycarbonyl-amino-2
hydroxybutyryl]-2'-N-~N-benzyloxycarbonylglycyl)fortimicin B
obtained in Example 10 is dissolved in 10 ml of tetrahydrofuran.
dm:c~ ~ 50
~ ~128~
I To the solution S ml of lM diborane-tetrahydrofuran solution is
added and the mixture is stirred at room temperature for 5 minutes.
3 About 1 ml of water is added to the reaction mixture to decompose
~ excess diborane and the solvent is distilled off. To the result-
ing solid residue 24 ml of 0.2N hydrochloric acid-methanol solu-
6 tion is added. The solution is allowed to stand at room tempera-
7 ture for 46 hours and the solvent is then distilled off. To the
8 resulting solid residue 20 ml each of chloroform and 5~ sodium
9 bicarbonate is added and the mixture well stirred is then sub-
jected to separation. The water layer is washed twice with 10 ml
of chloroform. After the chloroform layer and the washed chloro-
12 form are combined and washed with 10 ml of water, the combined
13 chloroform solution is dried with anhydrous sodium sulfite. The
1~ chloroform solution is concentrated to dryness to obtain a resi-
due. The residue is dissolved in a small amount of chloroform
16 and passed through a column packed with 15 g of silica gel.
17 Elution is carried out with a solvent system of chloroform-
18 methanol (96:4 by volume) and the eluate is taken in 7 ml frac-
19 tions. Fraction Nos. 21-26 containing a compound having a Rf
val~Ie of 0.51 in solvent system D are combined and the solvent
ZI is distilled off to obtain 51 mg of a white solid. PMR (methanol-
22 d4) spectra of the product is as follows:
23 ~ 1.03 (3H, d), 1.2-1.9 ~6H, m), 2.46 (31~, s),
2~ 3.40 (3H, s), 7.30 (201~, 9)
On the basis of the data, it is confirmed that the
26 product is 1,6'-di-N-benzyloxycarbonyl-4-N-[(S)-4-benzyloxy-
27 carbonylamino-2-hydroxybutyl]-2'-N-(N-benzyloxycarbonylglycyl-
28 )fortimicin B. Yield 16~.
Fraction Nos. 36 to 66 containing a compound having a
Rf value of 0.15 in solvent system D are combined and the solvent
3I is distilled off to obtain 121 mg of a white solid. P~IR
-- 51 -
" ~ 28(3 S7
; .
I (methanol-d4) spectra of the product is as follows:
2 ~ 1.02 (3H, d), 1.2-1.9 (6H, m), 2.40 (3H, s),
3 3-39 13H, s), 7.33 (20H, s)
~ On the basis of the data, it is confirmed that the
product is 1,6'-di-N-benzyloxycarbonyl-4-N-[(S)-benzyloxy-
6 carbonylamino-2-hydroxybutyl]-2'-N-(2-benzyloxycaxbonylamino-
7 ethyl)fortimicin ~. Yield 38%.
9 Example 24. Preparation of 4-N-[(S)-4-amino-2-hydroxyb-utyl]
2'-N-glycylfortimicin B (Compound Mo. 6):
Il In this example, 51 mg of 1,6'-di-N-benzyloxycarbonyl-
4-N-[(S)-4-benzyloxycarbonylamino-2-hydroxybutyl]-2'-N-(N-
l3 benzyloxycarbonylglycyl)fortimicin B obtained in Example 23 is
14 dissolved in 10 ml of 0.2N hydrochloric acid-methanol solution.
1~ About 5 mg of 10~ palladium-carbon is added to the solution and
- 16 then hydrogen gas at room temperature and atmospheric pressure
17 is bubbled therethrough. The catalyst in the reaction mixture is
8 removed by filtration and the filtrate is concentrated to dryness
19 to obtain a residue. The residue is then dissolved in about 2 ml
of water. The solution is adju5ted to a pH of 7 with lN sodium
2I hydr ~ide and passed through a column packed with 5 ml of Amber-
B 22 litelCG-50 (NH4 form). After the column is washed with about 25
2~ m~ of water, elution is carried out with 0.6N aqueous ammonia and
24 the eluate is taken in 2 ml fractions. Fraction Nos. 11 to 16
containinq a compound h~?vinq a Rf value of 0.33 in solvent system
26 A are combined and the solvent is distilled off to obtain 16 mg
27 of a white solid. Since the product is coincident with the
28 compound obtained in Example 20 in Rf values on TLC and mass
29 spectrum, it is confirmed that the product is 4-N-[(S)-4-amino-2-
hydroxybutyl]-2'-N-glycylfortimicin B. Yield 40%.
31
- 52 -
.
:: ~
:
;
:
28Q57-
I Example 25. Preparation of 4-N-[(S)-4-amino-2-hydroxybutyl]-
2 2'-N-(2-a inoethyl)fortimicin B (Compound No. ?)
3 In this example, 121 mg (0.12 millimole) of 1,6'-
4 di-N--benzyloxycarbonyl-4-N-[(S)-4-benzyloxycarbonylamino-2-
hydroxybutyl]-2'-W-(2-benzyloxycarbonylaminoethyl)fortLmicin B
6 obtained in Example 23 is dissolved in 15 ml of 0.2N hydrochloric
7 acid-methanol solution. To the solution about 10 mg of 10%
8 palladium-carbon is added and then hydrosen gas at room tempera-
9 ture and atmospheric pressure is bubbled therethrough. After the
IO catalyst in the reaction mixture is removed by filtration, the
11 filtrate is concentrated to dryness under reduced pressure to
12 obtain a residue. The residue is ~issolved in about 2 ml of
Il water. The solution is adjusted to a pH of 7 with lN sodium
1~ hydr ~ide and passed through a column packed with 5 ml of Amber-
lite1CG-S0 (NH4 form). After the column is washed with about 25
16 ml of water, elution is carried out with 0.6N aqueous ammonia and
17 the eluate is ta~en in 2.5 ml fractions. Fraction Nos. 20 to 40
8 containing a compound having a Rf value of 0.30 in solvent system
9 A are combined and the solvent is distilled off to obtain 27 mg
o of a white solid.- Since the product is coincident with tho
2I compound obtained in Example 20 in the Rf values on TLC and mass
22 spectrum, it is confirmed that the product .is 4-~-t(S)-d-amino-2-
~1 hydroxybutyl]-2'-N-(2-aminoethyl)fortimicin B. Yield 47~.
26
t7
28
-29
3l
- -
- 53 -
~:
:',
:. ~:
