The present invention is directed to improved methods
for reductively alkylating glycopeptide antibiotics. The
invention provides increased regioselectivity of reaction
among multiple sites and thereby results in increased yields
of the preferred product. In particular, the invention is
directed to methods for preferentially conducting a
reductive alkylation reaction on an amine on the saccharide
of a glycopeptide antibiotic having one or more additional
amines.
The essence of the invention is the discovery that
conducting the reaction in the presence of soluble copper
favors preferential reaction with the amine on the
saccharide position, and thereby improves the yields of the
reductive alkylation at this site. The initial step is the
formation of a copper complex of the glycopeptide
antibiotic, which subsequently undergoes the reductive
alkylation. This invention is also directed to these copper
complexes of the starting glycopeptide antibiotics. The
alkylated glycopeptide antibiotic products are obtained as
copper complexes, which are another embodiment of the
present invention.
The present invention relates to reductive alkylation
of glycopeptide antibiotics.
The glycopeptide antibiotics are a large class of
substances either produced by microorganisms, or produced by
microorganisms and thereafter subsequently modified in part.
3
Two of these, vancomycin and teicoplanin, are sold as
antibacterial products, but many others have been discovered
and are being considered for development, especially since
the emergence in the late 1980s of resistance to various
antibiotics, including the glycopeptides themselves. The
entire class of glycopeptide antibiotics is well described
in "Glycopeptide Antibiotics", edited by Ramakrishnan
Nagarajan (Marcel Dekker, Inc., New York, 1994). Among the
more recently discovered glycopeptides are those known as
A82845A (also called ereomomycin), A82846B (also known as
chloroorienticin A), A82846C (also known as orienticin C),
and orienticin A. The present invention is preferred for
use with vancomycin type glycopeptide antibiotics, including
vancomycin, A82846A, A82846B, A82846C, and orienticin A; the
invention is especially preferred for use with A82846B.
Many modifications of naturally-occurring glycopeptides
have been made. Among the modifications are reductive
alkylations of reactive amine(s) in glycopeptides. See, for
example, U.S. 4,698,327 describing reductive alkylations of
vancomycin, and EPO 435 503 Al and EPO 667 353 Al, both of
which describe reductive alkylations of a variety of
glycopeptides including vancomycin, A82846A, A82846B,
A82846C, and orienticin A. These references describe
reductive alkylations which introduce into the parent
glycopeptides a great variety of alkyl groups.
4,698,327 describes alkylated vancomycin compounds of
the formula:
wherein
R is hydrogen or methyl;
n is 1 or 2; and
Rl is hydrogen or methyl;
R2 and R3, independently, are hydrogen or a group of
the formula: R6R7CH-;
R5 and R7 are independently R5, R5-(Ci-Cs-alkyl) or R5-
(C2-C5-alkenyl) ;
R5 is hydrogen, Ci-Cio-alkyl, C2-Cio-alkenyl, C1-C4
alkoxy, C3-Cio-cycloalkyl, C5-Ci2-cycloalkenyl, phenyl,
naphthyl, indenyl, tetralinyl, decalinyl, adamantyl, a
monocyclic heterocyclic ring system comprising 3 to 8 atoms
in the ring or a bicyclic heterocyclic ring system
comprising 6 to 11 atoms, provided that at least one atom of
the ring system is carbon and at least one atom of the ring
system is a heteroatom selected from O, N, and S, and R5 may
be substituted with one or more hydroxy, nitro, Ci-Ciq-
alkoxy, Ci-Cio-alkyl, phenyl, Ci-Cg-alkylthio, nitrile,
halo, C2-C4-acylamino, amino, Ci-C4-dialkylamino groups; and
R4 is hydrogen, provided that: (1) at least one of R2 and
R3 must be other than hydrogen; (2) when n is 2, R must be
hydrogen; (3) when R is methyl and R3 is hydrogen, R2 cannot
be methyl and (4) when R and Ri are both methyl, then R2 is
hydrogen or methyl and n is 1.
EPO 435 503 Al is directed to alkylated and acylated
glycopeptides of the formula:
p,

wherein:
R is hydrogen or a (4-epi-vancosaminyl)-O-glucosyl
group of formula
or the glucosyl group of formula

X is hydrogen or chloro;
Y is hydrogen or chloro;
Rl, R2, and R3 are independently hydrogen; C1-C12 alkyl;
C2-CQ alkanoyl; or a group of formula

n is 1 to 3;
R4 is hydrogen, halo, Ci-Cs alkyl, Ci-Cg alkoxy, or a
group of formula
R5 and Re are independently hydrogen or C1-C3 alkyl;
p is 0 to 2;
m is 2 or 3, and r = 3 - m; provided that, where R is a
(4-epi-vancosaminvl)-0-glucosvl group, Ri, R2, and R3 are
not all hydrogen, and where R is hydrogen or a glucosyl
group, Rl and R3 are not both hydrogen.
Where R is (4-epi-vancosaminvl)-O-glucosvl, the
glycopeptides so defined are
X = H, Y = CI, A82846A
X = Y = CI, A82846B
X = Y = H, A82846C
X = CI, Y = H, orienticin A.
Thus, EPO 435 503 Al describes alkyl derivatives of
A82846A, A82846B, A82846C, and orienticin A wherein the
alkyl group is
Preferred groups are C8-C12 alkyl and groups of the formula

wherein R4 is hydrogen, halo, Ci-Cg alkyl, or Ci-Cg alkoxy.
EPO 667 353 Al describes alkylated glycopeptide
antibiotics of the formula
wherein:
X and Y are each independently hydrogen or chloro;
R is hydrogen, 4-epi-vancosaminyl, actinosaminyl, or
ristosaminyl;
r1 is hydrogen, or mannose;
r2 is -NH2, -NHCH3, or-N(CH3)2;
r3 is -CH2CH(CH3)2, [p-OH, m-Cl]phenyl, p-rhamnose-phenyl,
or [p-rhamnose-galactose]phenyl, [p-galactose-
galactose]phenyl, [p-CHsO-rhamnose]phenyl;
R^ is -CH2(CO)NH2, benzyl, [p-OH]phenyl, or [p-OH, m-
Cl]phenyl;
R^ is hydrogen, or mannose;
R^ is vancosaminyl, 4-epi-vancosaminyl, L-acosaminyl, L-
ristosaminyl, or L-actinosaminyl;
r7 is (C2-Ci6)alkenyl, (C2-C12)alkynyl, (C1-C12 alkyl)-R8,
(C1-C12 alkyl)-halo, (C2-C6 alkenyl)-R8, (C2-C6 alkynyl)-Rg,
(C1-C12 alkyD-O-Rs, and is attached to the amino group of
r6;
R^ is selected from the group consisting of:
a) multicyclic aryl unsubstituted or substituted with
one or more substituents independently selected from the
group consisting of:
(i) hydroxy,
(ii) halo,
(iii) nitro,
(iv) (Ci-C6)alkyl,
(v) (C2-C6)alkenyl,
(vi) (C2-C6)alkynyl,
(vii) (Ci-Ce)alkoxy,
(viii) halo-(Ci-C6)alkyl,
(ix) halo-(Ci-Ce)alkoxy,
(x) carbo-(Ci-Cg)alkoxy,
(xi) carbobenzyloxy,
(xii) carbobenzyloxy substituted with (Ci-Cg)alkyl,
(Ci-Cg)alkoxy, halo, or nitro,
(xiii) a group of the formula -S(0)n'-R^/ wherein n' is
0-2 and R^ is (Ci-Ce) alkyl, phenyl, or phenyl substituted
with (Ci-Ce)alkyl, (Ci-Cs)alkoxy, halo, or nitro, and
(xiv) a group of the formula -C(0)N(r1^)2 wherein each
rI*^ substituent is independently hydrogen, (Ci-Ce)-alkyl,
(Ci-Ce)-alkoxy, phenyl, or phenyl substituted with (Ci-Cg)-
alkyl, (Ci-Ce)-alkoxy, halo, or nitro;
b) heteroaryl unsubstituted or substituted with one or
more substituents independently selected from the group
consisting of:
(i) halo,
(ii) (Ci-C6)alkYl,
(iii) (Ci-Ce)alkoxy,
(iv) halo-(Ci-C6)alkYl,
(v) halo-(Ci-Cg)alkoxy,
(vi) phenyl,
(vii) thiophenyl,
(viii) phenyl substituted with halo, (Ci-Ce)alkyl, (C2-
C6)alkenyl, (C2-C6)alkynyl, (Ci-Cg)alkoxy, or nitro,
(ix) carbo-(Ci-Cg)alkoxy,
(x) carbobenzyloxy,
(xi) carbobenzyloxy substituted with (Ci-Ce)alkyl, (Ci-
Cg) alkoxy, halo, or nitro,
(xii) a group of the formula -S(0)n'-R^/ as defined
above,
(xiii) a group of the formula -C (0) N(r1'^) 2 as defined
above, and
(xiv) thienyl;
c) a group of the formula:
wherein A^ is -OC(a2)2-C(A^)2-O-, -0-C(a2)2-O-,-C(A^)2-
0-, or -C(a2)2-C(a2)2-C(a2)2-C(a2)2-, and each a2 substituent
-1/ 'I
is independently selected from hydrogen, (Ci-Cg)-alkyl, (Ci-
C6)alkoxy, and (C4-C10)cycloalkyl;
d) a group of the formula:

wherein p is from 1 to 5; and
r11 is independently selected from the group consisting
of:
(i) hydrogen,
(ii) nitro,
(iii) hydroxy,
(iv) halo,
(v) (Ci-C8)alkyl,
(vi) (Ci-C8)alkoxy,
(vii) (C9-Ci2)alkyl,
(viii)(C2-C9)alkynyl,
(ix) (C9-Ci2)alkoxy,
(x) (C1-C3)alkoxy substituted with (C1-C3)alkoxy,
hydroxy, halo(C1-C3)alkoxy, or (C1-C4)alkylthio,
(xi) (C2-C5)alkenyloxy,
(xii) (C2-C13)alkynyloxy
(xiii) halo-(Ci-C6)alkyl,
(xiv) halo-(Ci-Ce)alkoxy,
(xv) (C2-C6)alkylthio,
(xvi) (C2-C10)alkanoyloxy,
(xvii) carboxy-(C2-C4)alkenyl.
-ll/'-
(xviii) (C1-C3)alkylsulfonyloxy,
(xix) carboxy-(C1-C3)alkyl,
(xx) N-[di(C1-C3)-alkyl]amino-(C1-C3)alkoxy,
(xxi) cyano-(Ci-C6)alkoxy, and
(xxii) diphenyl-(Ci-Cg)alkyl,
with the proviso that when R^^ is (Ci-Cg)alkyl, (Ci-
Cg)alkoxy, or halo, p must be greater or equal to 2, or when
r"^ is {C1-C3 alkyl)-r8 then R^^ is not hydrogen, (Ci-
Cg)alkyl, (Ci-Cs)alkoxy, or halo;
e) a group of the formula:
wherein q is 0 to 4;
r12 is independently selected from the group consisting
of:
(i) halo,
(ii) nitro,
(iii) (Ci-C6)alkyl,
(iv) (Ci-Cg)alkoxy,
(v) halo-(Ci-Ce)alkyl,
(vi) halo-(C1-C6)alkoxy, and
(vii) hydroxy, and
(vii) (Ci-Ce)thioalkyl;
r is 1 to 5; provided that the sum of q and r is no
greater than 5;
Z is selected from the group consisting of:
(i) a single bond,
(ii) divalent (Ci-C6)alkYl unsubstituted or
substituted with hydroxy, (Ci-Cg)alkyl, or (Ci-Cg)alkoxy,
(iii) divalent (C2-C6)alkenyl,
(iv) divalent (C2-C6)alkynyl, or
(v) a group of the formula - (C (R^'^) 2) s-R''"^- or -
r15-(C(r1^) 2) s-/ wherein s is 0-6; wherein each R^^
substituent is independently selected from hydrogen, (Ci-
C6)-alkyl, or (C4-C10) cycloalkyl; and R^^ is selected from -
0-, -S-, -SO-, -SO2-, -SO2-O-, -C(0)-, -0C(0)-, -C(0)0-, -
NH-, -N(Ci-C6 alkyl)-, and -C(0)NH-, -NHC(O)-, N=N;
R^-^ is independently selected from the group consisting
of:
(i) (C4-C10)heterocyclyl,
(ii) heteroaryl,
(iii) (C4-C10) cycloalkyl unsubstituted or
substituted with (Ci-Ce)alkyl, or
(iv) phenyl unsubstituted or substituted with 1 to
5 substituents independently selected from: halo, hydroxy,
nitro, (Ci-Cio) alkyl, (Ci-Cio)alkoxy, halo-(C1-C3)alkoxy,
halo-(C1-C3)alkyl, (C1-C3)alkoxyphenyl, phenyl, phenyl-(Ci-
C3)alkyl, (Ci-Cg)alkoxyphenyl, phenyl-(C2-C3)alkynyl, and
(Ci-C6)alkylphenyl;
f) (C4-C10) cycloalkyl unsubstituted or substituted with
one or more substituents independently selected from the
group consisting of:
(i) (Ci-C6)alkyl,
(ii) {Ci-C6)alkoxy,
(iii) (C2-C6)alkenYl,
(iv) (C2-C6)alkYnYl,
(v) (C4-Cio)cYcloalkyl,
(vi) phenYl,
(vii) phenYlthio,
(viii) phenYl substituted by nitro, halo, (Ci-
Cg)alkanoyloxY, or carbocYcloalkoxY, and
(ix) a group represented by the formula -Z-R^^ wherein
Z and R^2 are as defined above; and
g) a group of the formula:
wherein
A^ and A'^ are each independently selected from
(i) a bond,
(ii) -0-,
(iii) -S(0)t-, wherein t is 0 to 2,
(iv) -C(r1'^)2-/ wherein each R^"^ substituent is
independently selected from hydrogen, (Ci-Ce)alkyl, hydroxy,
(Ci-Ce) alkyl, (Ci-Cg) alkoxy, or both R^"^ substituents taken
together are 0,
(v) -N(R^®)2-/ wherein each R^^ substituent is
independently selected from hydrogen; (Ci-Cg)alkyl; (C2-
C6)alkenyl; (C2-C6)alkynyl; (C4-C10) cycloalkyl; phenyl;
/
phenyl substituted by nitro, halo, (Ci-Ce)alkanoyloxy; or
both r1^ substituents taken together are (C4-C10) cycloalkyl
r1^ is r12 or R^^ as defined above; and
u is 0-4.
In this reference, preferred glycopeptide antibiotics are
A82846A, A82846B, A82846C, and orienticin A; preferred
alkyls are those wherein r"^ is CH2-R8; and preferred R^
moieties are those defined as groups "(d)" and "(e)".
The present invention can be utilized to make the
alkylated glycopeptides described in these references.
Preferred alkylated glycopeptides which can be prepared by
the present process include the following:
N4-n-octylA82 846B
N^-n-decylA82 84 6B
N4-benzylA82846B
N^-(p-chlorobenzyl)A82846B
N'i- (p-bromobenzyl) A82846B
N"^- (p-propylbenzyl) A82846B
N^-(p-isopropylbenzyl)A82846B
N^-(p-butylbenzyl)A82846B
N"^- (p-isobutylbenzyl) A82846B
N^-(p-pentylbenzyl)A82846B
N^-(p-isohexylbenzyl)A82846B
N^-(p-octylbenzyl)A82846B
N^-(p-propoxybenzyl)A82 846B
N'^- (p-isopropoxybenzyl) A82846B
N^-(p-butoxybenzyl)A82846B
N^-(p-tert-butoxybenzyl)A82846B
N'^- (p-pentYloxYbenzYl)A82846B
N^-(p-hexyloxybenzyl)A82846B
N^-{o-hexyloxYbenzYl)A82846B
N^-(p-heptyloxybenzyl)A82846B
N'^- (p-octyloxybenzyl) A82846B
N4-phenethylA82 846B
N^-(4-phenylbenzyl)A82846B
N^-(4-(4-chlorophenyl)benzylA82846B
n4_ (4_ (4-inethylbenzyloxy)benzyl) A82 84 6B
n4_(4_(4-ethylbenzyloxy)benzyl)A82846B
n4_(4-(4-chlorophenethyl)benzyl)A82846B
N'*- (4- (2- (4-inethoxyphenyl) ethynyl) benzyl) A82846B.
The references noted above describe the reductive
alkylation as comprising a first step, in which the
glycopeptide is reacted with the respective aldehyde or
ketone to form a Schiff's base, which in a second step is
reduced to the desired alkylated product. In one variation
of this procedure, EPO 667 353 Al describes a process in
which the reducing agent is added simultaneously with the
glycopeptide and aldehyde or ketone. The references say
that any chemical reducing agent can be employed, but the
references also suggest a preference for sodium
cyanoborohydride.
Essentially all glycopeptides contain multiple reactive
sites. Manipulation of these multiple sites is not
uniformly advantageous. It is sometimes desired to react
the glycopeptide regioselectivity, to have the reaction
occur at only one of multiple sites. This is equally true
in the case of reductive alkylations of glycopeptides. An
example of this is A82846B. While derivatives alkylated on
the leucine amine (N^) and/or the monosaccharide (N^) are
active as antibacterials, alkylation of the N'^
(disaccharide) amine appears to be preferred.
Pharmaceutical practices require a relatively pure form, and
therefore preferential reaction of the N'^ site is desirable
in order to achieve a highly pure N^-alkylated product.
The present invention provides a technique for
obtaining reaction preferentially on the amine on a
saccharide at the N'^ position in the glycopeptide
antibiotic. In the case of vancomycin, A82846A, A82846B,
A82846C, and orienticin A, the present process reduces
reactivity at sites N^ and N^ and thereby increases reaction
selectivity for the N'^ (disaccharide) site. The invention
requires the initial preparation of a soluble copper complex
of the glycopeptide, which is then reductively alkylated.
The soluble copper complex is achieved by reacting the
glycopeptide antibiotic with copper, typically by adding a
source of soluble copper to a reaction mixture containing
the glycopeptide antibiotic. The identity of the copper
source is not critical, so long as it is at least partially
soluble and does not negatively impact the pH. Such a
copper salt can be used in anhydrous or hydrated form. A
preferred source of copper is copper (II) acetate, most
conveniently employed as the hydrate.
Supplying copper to the reaction mixture results in the
initial production of a copper complex with the glycopeptide
antibiotic starting material, typically in a 1:1 ratio.
This copper complex of the glycopeptide antibiotic starting
material is one of the features of the present invention.
The reducing agent to be employed in the present
invention is sodium cyanoborohydride or pyridine-borane
complex.
The identity of the solvent is important. Straight
methanol has given high yields, and it is expected that
methanol somewhat diluted as with DMF or DMSO would provide
acceptable yields. Other solvents have not produced
satisfactory results. Therefore, the reaction solvent is at
least predominantly methanol.
The reaction should be conducted at a pH of 6-8, and
preferably at a pH of 6.3-7.0.
The amounts of reactants and reagents to be employed
are not critical; amounts to maximize the yield of product
will vary somewhat with the identity of the reactants. The
reaction consumes the glycopeptide antibiotic and the
aldehyde or ketone in equimolar amounts. A slight excess of
the aldehyde or ketone, e.g., 1.3 to 1.7:1, is preferred.
The amount of the glycopeptide antibiotic to be used must be
corrected for its purity. The reaction consumes an
equimolar amount of the reducing agent. At least that
amount should be employed, and a slight excess is preferred.
The amount of soluble copper is not critical when employing
sodium cyanoborohydride as reducing agent. When employing
pyridine-borane as reducing agent, the amount of soluble
copper to be employed is more important, since excess copper
will react with the pyridine-borane. Regardless of the
identity of the reducing agent, the present process first
results in the formation of a 1:1 complex with the
glycopeptide antibiotic; therefore, the copper is preferably
present in an amount approximately equimolar with the
glycopeptide antibiotic. Amounts exceeding one molar
equivalent (in the case of pyridine-borane) or two molar
equivalents (in the case of sodium cyanoborohydride) are
undesirable.
Summarizing the foregoing, the ideal amounts to be
employed are a ratio of:
glycopeptide:aldehyde or ketone:reducing agent:copper salt
of:
1:1.3 to 1.5:1.3:1
with the exception that when using pyridine-borane complex
as reducing agent, the preferred ratio is:
1:1.3 to 1.7:1.5:0.9 to 1.0.
The concentration of the reactants in the solvent has
some bearing on the process. Methanol volume relative to
mass of glycopeptide antibiotic can vary from 50:1 to 500:1;
a 100:1 dilution appears to be a useful, practical ratio,
although higher dilutions may give slightly higher yields.
The temperature at which the process is carried out is
not critical. Reaction mixtures in methanol boil at about
67°C., thereby setting the maximum temperature when
employing straight methanol as the solvent. Higher
temperatures are of course possible when employing mixtures
of methanol or when operating under pressure. Lower
temperatures can be tolerated, but preferably not lower than
about 45°C. The ideal condition for sodium cyanoborohydride
as reducing agent is the use of straight methanol and
conducting the reaction at reflux; the ideal condition for
pyridine-borane as reducing agent is also the use of
straight methanol but at temperatures of about 58-63°C.
Some product is produced with even short reaction
times. Longer reaction times, such as from 6 hours to 48
hours, are preferred. However, the ideal reaction time
appears to be approximately 2 0 to 2 5 hours. Longer times
may increase the yield of products alkylated at undesired
sites in the glycopeptide antibiotic.
In carrying out the present invention, the glycopeptide
antibiotic and copper are preferably mixed in a solvent,
creating the soluble copper complex of the glycopeptide
antibiotic, and the aldehyde and reducing agents are then
added. However, the precise order of addition is not
critical. Portionwise addition of the reducing agent is
preferred, and is required for good results when employing
pyridine-borane complex as reducing agent. The reaction is
continued for a period of time, after which the product is
produced and can be separated from the reaction mixture.
Upon the completion of the reaction period, the
reaction mixture is preferably quenched, as by the addition
of sodium borohydride. This reagent consumes residual
-2/- ^
aldehyde or ketone and thereby prevents further undesired
reactions.
The product is isolated from the reaction mixture as a
copper complex of the alkylated glycopeptide. Isolation is
achieved by concentration of the reaction mixture and
precipitation of the complex by addition of an antisolvent
such as ethyl acetate, acetone, 1-propanol, isopropyl
alcohol, or preferably acetonitrile. The complex can be
broken by aqueous treatment at pH 24^ freeing the simple
alkylated glycopeptide product, which can, if desired, be
purified in conventional manner.
The following examples illustrate the present invention
and will enable those skilled in the art to practice the
same.
Reference Example A (no copper)
A82846B (6.0 g, 76.5% potency, 4.59 bg, 2.88 mmol), 4'-
chloro-4-biphenylcarboxaldehyde (0.86 g, 3.97 mmol), and
sodium cyanoborohydride (84 mg, 1.34 mmol) were added to 600
mL methanol and the solution was heated at reflux for 3
hours. An additional portion of sodium cyanoborohydride (84
mg, 1.34 mmol) was added and the mixture was heated 3 hours
longer at reflux. A final portion of sodium
cyanoborohydride (84 mg, 1.34 mmol) was added and heating at
reflux was continued an additional 17 hours. The clear
colorless solution was cooled to ambient temperature and
concentrated to 13 0 mL on a rotary evaporator. The product
was precipitated by addition of 200 mL of isopropyl alcohol
over 2 hours. After cooling to 0° C and stirring 1 hour,
filtration afforded N^-(4-(4-chlorophenYl)benzyl)A82846B as
a white solid (5.61 g, 49.3% potency, 2.77 bg, 53.7%).
EXAMPLE 1
A82846B(0.50 g, 76.3% potency, 0.38 bg, 0.24 iranol), 4'-
chloro-4-biphenylcarboxaldehyde (70 mg, 0.32 iranol), and
cupric acetate monohydrate (51 mg, 0.26 mmol) were stirred
in 50 mL methanol. Sodium cyanoborohydride (2 0 mg, 0.32
mmol) was added and the solution was heated at reflux for 23
hours. The clear purple solution was cooled to ambient
temperature and 12% sodium borohydride in aqueous 14 M
sodium hydroxide (0.03 mL, 0.14 mmol) was added. One drop
of acetic acid was added to pH adjust the solution to 7.3.
An additional portion of 12% sodium borohydride in aqueous
14 M sodium hydroxide (0.23 mL, 0.10 mmol) was added
followed by one drop of acetic acid to maintain the solution
pH at 7.3. The mixture was stirred at ambient temperature
for 1 hour and concentrated to 12 mL on a rotary evaporator.
The product was precipitated by addition of 25 mL of
acetonitrile over 20 min. After stirring 20 min at ambient
temperature, filtration afforded the copper complex of N'^-
(4-(4-chlorophenyl)benzyl)A82846B as a purple solid (0.58 g,
potency 59.5%, 0.35 bg, 80.3 %).
EXAMPLE 2
A82846B (6.0 g, 78.4% potency, 4.7 bg, 2.95 iranol), was
stirred in 600 mL methanol and cupric acetate (0.66 g, 3.6
mmol) was added. After stirring at ambient temperature for
15 min, 4'-chloro-4-biphenylcarboxaldehyde (0.95 g, 4.4
mmol), and sodium cyanoborohydride (0.27 g, 4.3 mmol) were
added and the mixture was heated at reflux for 24 hours.
After cooling to ambient temperature, HPLC analysis of a
reaction aliquot afforded a yield of 4.52 g (85.4%) of N"^-
(4-(4-chlorophenyl)benzyl)A82 84 6B.
EXAMPLE 3
A82846B (2.5 g, 78.5% potency, 1.96 bg, 1.23 mmol), was
stirred in 250 mL methanol and cupric acetate monohydrate
(0.26 g, 1.32 mmol) was added. After stirring at ambient
temperature for 10 min, 4'-chloro-4-biphenylcarboxaldehyde
(0.35 g, 1.6 mmol), and sodium cyanoborohydride (34 mg, 0.54
mmol) were added and the mixture was heated at reflux for 3
hours. An additional portion of sodium cyanoborohydride (34
mg, 0.54 mmol) was added and the mixture was heated 3 hours
longer at reflux. A final portion of sodium
cyanoborohydride (34 mg, 0.54 mmol) was added and heating at
reflux continued an additional 17 hours. The mixture was
cooled to ambient temperature and 12% sodium borohydride in
aqueous 14 M sodium hydroxide (0.14 mL, 0.63 mmol) was
added. A few drops of acetic acid were added to pH adjust
the solution to 7.3. A second portion of 12% sodium
borohydride in aqueous 14 M sodium hydroxide (0.13 mL, 0.6
mmol) was added and a few drops of acetic acid were added to
adjust the solution pH to 8.1. After stirring at ambient
temperature for 2 hours, the reaction mixture was
concentrated to 60 mL on a rotary evaporator. Isopropyl
alcohol (17 5 mL) was added dropwise over a period of 1 hour
to precipitate the copper complex of N'^-(4-(4-
chlorophenyl)benzyl)A82846B. Filtration afforded the
complex as a purple solid (6.50 g, 26.9% potency as wet
cake, 1.75 bg, 79.1%) .
EXAMPLE 4
A82846B (2.5 g, 78.5% potency, 1.96 bg, 1.23 mmol), was
stirred in 250 mL methanol and cupric acetate monohydrate
(0.26 g, 1.32 mmol) was added. After stirring at ambient
temperature for 10 min, 4'-chloro-4-biphenylcarboxaldehyde
(0.35 g, 1.6 mmol), and sodium cyanoborohydride (34 mg, 0.54
mmol) were added and the mixture was heated at reflux for 3
hours. An additional portion of sodium cyanoborohydride (34
mg, 0.54 mmol) was added and the mixture was heated 3 hours
longer at reflux. A final portion of sodium
cyanoborohydride (34 mg, 0.54 mmol) was added and heating at
reflux continued an additional 17 hours. The mixture was
cooled to ambient temperature and 12% sodium borohydride in
aqueous 14 M sodium hydroxide (0.14 mL, 0.63 mmol) was
added. A few drops of acetic acid were added to pH adjust
the solution to 7.3. A second portion of 12% sodium
borohydride in aqueous 14 M sodium hydroxide (0.13 mL, 0.6
mmol) was added and a few drops of acetic acid were added to
adjust the solution pH to 8.2. After stirring at ambient
temperature for 1.5 hours, the reaction mixture was
concentrated to 60 mL on a rotary evaporator. Isopropyl
alcohol (175 mL) was added dropwise over a period of 1 hour
to precipitate the product. It was filtered and dried in
vacuo to afford the copper complex of N^-(4-(4-
chlorophenyl)benzyl)A82846B as a purple solid (2.56 g, 62,9%
potency, 1.61 bg, 72.9%).
EXAMPLE 5
A82846B (6.0 g, 76.1% potency, 4.56 bg, 2.9 mmol), was
stirred in 600 mL methanol and cupric acetate monohydrate
(0.63 g, 3.15 mmol) was added. After stirring at ambient
temperature for 15 min, 4'-chloro-4-biphenylcarboxaldehyde
(0.85 g, 3.9 mmol), and sodium cyanoborohydride (84 mg, 1.3
mmol) were added and the mixture was heated at reflux for 3
hours. An additional portion of sodium cyanoborohydride (84
mg, 1.3 mmol) was added and the mixture was heated 3 hours
longer at reflux. A final portion of sodium
cyanoborohydride (84 mg, 1.3 mmol) was added and heating at
reflux continued an additional 16 hours. The mixture was
cooled to ambient temperature and 50% aqueous sodium
hydroxide solution was added to adjust the pH of the
reaction mixture to 7.6. Sodium borohydride (0.11 g, 2.9
mmol) was added and the solution was stirred 3.5 hours at
ambient temperature. The reaction mixture was concentrated
to 110 mL on a rotary evaporator and isopropyl alcohol (250
mL) was added dropwise over a period of 4 hours to
precipitate the product. After cooling the purple slurry to
0°C for 1 hour, filtration afforded the purple complex of
n4-(4-(4-chlorophenyl)benzyl)A82846B (11.03 g, 36.2% potency
as wet cake, 3.99 bg, 77.6%.
Reference Example B (no copper)
A82846B (0.50 q, 84.3% potency, 0.42 bg, 0.26 mmol) was
stirred in 50 mL methanol and 4'-chloro 4-
biphenylcarboxaldehyde (72 mg, 0.3 3 mmol) and
pyridine-borane complex (0.033 mL, 0.33 mmol) were added.
The mixture was heated at reflux for 6 hours before being
cooled to ambient temperature. HPLC analysis of a reaction
aliquot afforded a yield of 0.25 g (53.2%) of N'i-(4-(4-
chlorophenyl)benzyl)A82846B.
EXAMPLE 6
A82846B (0.50 g, 84.3% potency, 0.42 bg, 0.26 mmol )
was stirred in 50 mL methanol and cupric acetate (45 mg,
0.25 mmol) was added. After stirring at ambient temperature
for 10 min, 4'-chloro-4-biphenylcarboxaldehyde (84 mg, 0.39
mmol) and pyridine-borane complex (0.039 mL, 0.39 mmol) were
added. The mixture was heated at 57° C for 24 hours before
being cooled to ambient temperature. HPLC analysis of a
reaction aliquot afforded a yield of 0.34 g (72.3%) of N"^-
(4-(4-chlorophenyl)benzyl)A82846B.
EXAMPLE 7
A82846B (0.50 g, 76.3% potency, 0.38 bg, 0.24 iranol )
and cupric acetate monohydrate (43 mg, 0.216 mmol) were
stirred in 50 mL methanol and 4'-chloro-4-biphenylcarbox-
aldehyde (84.5 mg, 0.3 9 mmol) and pyridine-borane complex
(0.011 mL, 0.11 mmol) were added. The mixture was heated at
63°C for 2 hours and an additional portion of
pyridine-borane was added (0.01 mL, 0.1 mmol). After 2
hours more at 63°C a third portion of pyridine-borane (0.005
mL, 0.05 mmol) was added. A fourth portion of
pyridine-borane (0.005 mL, 0.05 mmol) was added 2 hours
later followed by a fifth portion of pyridine-borane (0.005
mL, 0.05 mmol) after another 5 hours at 63°C. The mixture
was heated at 63°C for another 11 hours before being cooled
to ambient temperature. HPLC analysis of a reaction aliquot
afforded a yield of 0.34 g (79.2%) of N'i-(4-(4-
chlorophenyl)benzyl)A82846B.
The reactions reported in Reference Examples A and B
and Examples 1-7 were also evaluated (1) for the amount of
the remaining starting glycopeptide, (2) for the amount of
products alkylated on amine sites other than the N'^-
position, and (3) for the amount of multiply-alkylated
products. The results are set forth in the following table
and are expressed as a percentage relative to the intended
product monoalkylated on the N^-amine; yields of the
intended product are actual yields as recited in the
foregoing examples.
These data show that the present invention provides several
advantages. First, the yield of the product alkylated on N'^
is increased. Second, the yields of products alkylated on
N^ and/or N^ are decreased. Therefore, the present
invention provides significant improvement in reaction
regieselectivity.
EXAMPLE 8
N^-(4-(4-chlorophenyl)benzyl)A82846B Copper Complex
A82846B (0.50 g, 75.6 - 78.8 % potency, 0.24 - 0.25
mmol) was stirred in 50 mL methanol and cupric acetate (53 -
56 mg, 0.29 - 0.31 mmol) was added followed by 4'-chloro-4-
biphenylcarboxaldehyde (70 - 73 mg, 0.32 - 0.34 mmol) and
sodium cyanoborohydride (20 - 22 mg, 0.3 2 - 0.3 5 mmol). The
reaction mixture was heated at reflux for 24 hours and
cooled to ambient temperature. The pH was adjusted to 9.0 -
9.3 by addition of 1 M NaOH solution. The reaction mixture
was concentrated to 10 - 2 0 mL on a rotary evaporator and
isopropyl alcohol (13 - 2 0 mL) was added dropwise to
precipitate the purple glycopeptide copper complex which was
isolated by suction filtration. Drying in vacuo at 60°C
afforded the glycopeptide copper complex as a purple powder.
After four repetitions of the process the combined
glycopeptide complex was assayed for copper content and was
found to contain 3.0% copper, confirming a 1:1 copper
complex with N"^-(4-(4-chlorophenyl) benzyl) A82846B.
Reference Example C
k
EXAMPLES 9-19
Various copper salts were evaluated
in a standardized procedure.
A82846B (1 equivalent as potency adjusted free base)
was stirred in 50 mL methanol and a divalent metal salt
(MX2, 0.63 equivalent) or a monovalent metal salt (MX, 1.25
equivalent) was added followed by 4'-chloro-4-
biphenylcarboxaldehyde (1.25 equivalent) and sodium
cyanoborohydride (1.25 equivalent). The mixture was heated
at reflux for 24 hours. After cooling to ambient
temperature, an aliquot was removed for HPLC analysis.
The following HPLC System was used for in situ reaction
monitoring and yield calculation: HPLC system Waters 600E
with HP3395 integrator and Applied Biosystems 757 detector
set at 230 nm, sensitivity 0.1 absorption units, 1 sec.
filter rise time. Column: DuPont Zorbax SB-Phenyl, 4.6 mm x
25 cm. Eluant A: 10% acetonitrile, 90% buffer (0.2%
triethylamine, 0.25% H3PO4). Eluant B: 60% acetonitrile,
40% buffer (0.2% triethylamine, 0.25% H3PO4). Gradient
profile at 1 mL/min: initialize 100% A, gradient to 80% A,
20% B over 5 minutes, hold 5 minutes, gradient to 100% B
over 20 minutes, gradient to 100% A over 5 minutes, hold 20
minutes. Sample preparation: 0.5 - 1.0 g of reaction
mixture diluted to 25 mL in acetonitrile - buffer. Hold at
ambient temperature about 3 0 minutes until the purple color
of the copper complex is discharged. The desired
glycopeptide alkylation product elutes at 16-18 minutes, the
starting glycopeptide nucleus at 3-4 minutes, the site N^
(monosugar) alkylation product at 18-19 minutes, the site N^
(methyl leucine) alkylation product at 19-21 minutes,
dialkylated impurities at 24-26 minutes, and aldehyde at 35-
36 minutes. In situ yield is determined by correlation to
standards prepared with a reference sample of the product.
The results are shown in the following table. Results
for alkylated byproducts are expressed as percentage
relative to the desired N^ alkylation product.
The same copper salts were further evaluated for their
solubility in methanol and for the solubility of the
starting glycopeptide antibiotic in their presence. The
procedure was as follows: the copper salt (0.165 mmol) was
added to 50 mL methanol and stirred at ambient temperature
for 15 min. Solubility data was recorded as well as the pH.
Glycopeptide nucleus (0.55 g, 74.7% potency, 0.41 bg, 0.26
mmol) was added and stirring continued 15 min. Solubility
and pH data was recorded.
The foregoing examples illustrate several facets of the
present invention. First, copper must be supplied to the
reaction mixture in a form which is at least partially
soluble. Copper salts such as CuF2 and Cu(0H)2, which are
nearly insoluble in methanol, are not effective. Further,
the copper salt preferably should allow full solubility of
the starting glycopeptide antibiotic, and ideally at the
preferred pH. The salts which work the best (Cu(0Ac)2/
Cu(cyclohexanebutyrate)2/ and Cu(2-ethylhexanoate)2) afford
complete dissolution of nucleus and afford nucleus solutions
-3/- ^
at about pH 6.7. The salts which afford improvements over
no additive but are not optimal (Cu(O2CCF3 ) 2, CUCI2, CuBr2)
either afford solubility of nucleus but at less than optimal
pH (CuBr2 and Cu(02CCF3)2) or are at optimal pH but do not
afford complete nucleus solubility (CUCI2)•
In summary, the copper must be in a form which is at
least partially soluble, and should allow or maintain full
solubility of the starting glycopeptide antibiotic at an
acceptable pH, typically 6.3-7. Also, these experiments
were conducted with suboptimal amounts of the copper;
further advantage from the present invention is obtained at
higher copper concentration.
Reference Example D (no copper)
k
EXAMPLE 2 0
Two reactions were conducted with the glycopeptide
antibiotic A82846A, one without copper (Reference Example D)
and one with cupric acetate monohydrate. The aldehyde was
4'-chloro-4-biphenylcarboxaldehyde. The reactions were
conducted in the essentially same procedures as reported in
the foregoing examples. Results were as set forth in the
following table:
EXAMPLE 21
A82846B Copper Complex
A82846B (3.0 g, 78.7% potency, 2.4 bg, 1.5 mmol) was
stirred in 3 00 mL methanol at ambient temperature and cupric
acetate monohydrate (0.31 g, 1.6 mmol) was added. After
stirring at ambient temperature for 20 minutes, the purple
mixture was heated to 35 to 40°C and stirred an additional
3 0 minutes. the solution was concentrated to 45 mL on a
rotary evaporator and 10 0 mL or isopropyl alcohol was added
dropwise over 2 hours. The slurry was cooled to 0°C and
filtered. Drying in vacuo at 35°C afforded 2.6 g of the
A82846B copper complex as a purple solid. Mass
spectroscopic analysis showed the expected ions for the
complex, including a series of peaks around 1653, not seen
in the analysis of a reference sample of A82846B, and
indicative of the A82846B-copper complex.
Another sample of A82846B copper complex was prepared
in like manner and analyzed by UV-visible spectroscopy,
which showed an absorbance maxima at about 54 0 mm, not seen
in the spectra of a reference standard of A82846B or of
cupric acetate and indicative of the A82846B copper complex.
We Claim:
1. A method for reductively alkylating a glycopeptide antibiotic
comprising an amine-containing saccharide at N^ and one or more
other amines, wherein the reaction alkylates the amine on said N''
saccharide selectively over said one or more other amines, said
method comprises of:
a) reacting a soluble copper complex of the glycopeptide
antibiotic with a slight molar excess of a ketone or
aldehyde, in the presence of a solvent and a reducing
agent, such as herein described, and heating at reflux and
at a pH of 6-8, to obtain a reaction mixture,
b) Isolating a copper complex of alkylated glycopeptide from
the reaction mixture of step (a) by adding an antisolvent
such as herein described to obtain a copper complex of an
alkylated glycopeptide, optionally breaking the copper
complex by aqueous treatment at pH 4 to obtain an
alkylated glycopeptide product,
2. A method as claimed in claim 1, wherein the reducing agent is
selected from sodium cyanoborohydride or pyridine.borane complex.
3. A method as claimed in claim 2, wherein the soluble copper
complex of the glycopeptide antibiotic is prepared in situ from
the glycopeptide antibiotic and a soluble form of copper.
4. A method as claimed in claim 3, wherein the glycopeptide
antibiotic is a vancomycin type glycopeptide antibiotic.
5. A method as claimed in claim 4, wherein the glycopeptide
antibiotic is A82846B.
6. A method as claimed in claim 1, wherein the pH is in a range of
6.3-7.0.
7. A method as claimed in claimed 1, wherein the solvent is selected
from a group of methanol. Dimethyl Formamide and Dimethyl
sulfoxide.
8. A method as claimed in claim 1, wherein the solvent is selected
from methanol.
9. A method as claimed in claim 1, wherein the aldehyde is 4'-
chIoro-4-biphenylcarboxaldehyde.
10. A method as claimed in any of the claims 1-5, which further
comprises recovering a copper complex of the alkylating
glycopeptide antibiotic.
11. A method as claimed in any of the claims 1-7, which further
comprises recovering the alkylated glycopeptide antibiotic.
12. A method as claimed in claim 1, wherein in step (b) the
antisolvent is selected from a group of ethyl acetate,
acetonitrile, acetone, 1-propanol, isopropyl alcohol.
13. A method as claimed in claim 10, wherein the antisolvent is
acetonitrile.
14. A copper complex of an alkylated glycopeptide antibiotic wherein
the glycopeptide antibiotic is selected from A82846A and A82846B.
15. The copper complex as claimed in claim 12, said complex is a 1:1
copper complex with A82 846B.
16. The complex of copper with a reductively alkylated glycopeptide
antibiotic, which is produced by the method as claimed in the
claims 1-11.
17. The complex as claimed in claim 14, said complex is of a
reductively alkylated A82846B.
18. The complex as claimed in claim 15, in which the reductively
alkylated A82846B is N* - (4-(4-chlorophenyl) benzyl) A82846B.
19. A method as claimed in claim 1, wherein the soluble copper
complex of the glycopeptide antibiotic is in a molar ratio with
the ketone or aldehyde of 1:1.3 to 1.7.

This invention is concerned with improved processes for
reductive alkylation of glycopeptide antibiotics. The
improvement residing in providing a source of copper which
results in the initial production of a copper complex of the
glycopeptide antibiotic. Reductive alkylation of this
complex favors regioselective alkylation and increased
yields. Copper complexes of the glycopeptide antibiotic
starting materials and of the alkylated products are also
part of the invention.