Field of Invention
The present invention is directed to improved methods
for reductively alkylating glycopeptide antibiotics, the
improvement residing in the use of pyridine.borane complex
as the reducing agent.
The present invention relates to reductive alkylation
of glycopeptide antibiotics.
Background of the Invention
The glycopeptide antibiotics are a large class of
substances either produced by microorganisms, or produced
by microorganisms and thereafter subsequently modified in
part. 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 A82846A (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:
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-;
R6 and R7 are independently R5, R5-(Ci-C5-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-Cio-
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 nisi.
EPO 435 503 Al is directed to alkylated and acylated
glycopeptides of the formula:
X is hydrogen or chloro;
Y is hydrogen or chloro;
Rl, R2, and R3 are independently hydrogen; C1-C12 alkyl;
C2-C9 alkanoyl; or a group of formula
n i s 1 t o 3 ;
R4 is hydrogen, halo, Ci-Cg 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-qlucosvl group, Ri, R2, and R3 are
not all hydrogen, and where R is hydrogen or a glucosyl
group, Ri and R3 are not both hydrogen.
Where R is (4-epi-vancosaminvl)-0-glucosyl, the
glycopeptides so defined are
X = H, Y = CI, A82846A
X = Y = CI, A82846B
X = Y = H, A82846C
X = CI, Y = H, orienticin A.
wherein:
X and Y are each independently hydrogen or chloro;
R is hydrogen, 4-epi-vancosaminyl, actinosaminyl, or
ristosaminyl;
R^ is hydrogen, or mannose;
r2 is -NH2, -NHCH3, or-N(CH3)2;
R^ is -CH2CH (CH3)2, [p-OH, /n-Cl]phenyl, p-rhamnose-phenyl,
or [p-rhamnose-galactose]phenyl, [p-galactose-
galactose]phenyl, [p-CH30-rhamnose]phenyl;
R-^ is -CH2{CO)NH2, benzyl, [p-OH]phenyl, or [p-OH, m-
Cl1 phenyl;
R^ IS hydrogen, or mannose;
R^ is vancosaminyl, 4-epi-vancosaminyl, L-acosaminyl, L-
ristosaminyl, or L-actinosaminyl;
r"^ is {C2-Ci6)alkenyl, (C2-C12) alkynyl, (C1-C12 alkyD-Rg,
(C1-C12 alkyl)-halo, (C2-C6 alkenyD-Rs, (C2-C6 alkynyl)-Rg,
(C1-C12 alkyl)-0-R8, 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,
(li) 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-Ce)alkoxy,
(xi) carbobenzyloxy,
(xii) carbobenzyloxy substituted with (Ci-Ce)alkyl,
(Ci-Ce)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-Ce)alkoxy, halo, or nitro, and
(xiv) a group of the formula -C (0) N (rI*^) 2 wherein each
R^^ substituent is independently hydrogen, (Ci-Cs)-alkyl,
(C1-C6)-alkoxy, phenyl, or phenyl substituted with (Ci-Ce)-
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-Ce)alkyl,
(v) halo-(Ci-Ce)alkoxy,
(vi) phenyl,
(vii) thiophenyl,
(viii) phenyl substituted with halo, (Ci-Ce)alkyl, (C2-
C6)alkenyl, (C2-C6)alkynyl, (Ci-Ce)alkoxy, or nitro,
(ix) carbo-(Ci-Cg)alkoxy,
(x) carbobenzyloxy,
(xi) carbobenzyloxy substituted with (Ci-Cg)alkyl, (Ci-
Ce) 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 (R^'^) 2 as defined
above, and
(xiv) thienyi;
c) a group of the formula:
wherein A^ is -OC(a2)2-C(a2)2-O-, -0-C(a2)2-O-,-C(A^)2-
0-, or -C (a2) 2-C (a2) 2-C ia2)2-C {a2)2-, and each A^ substituent
is independently selected from hydrogen, (Ci-Ce)-alkyl, (Ci-
Cg)alkoxy, and {C4-C10)cycloalkyl;
d) a group of the formula:
wherein p is from 1 to 5; and
R^l is independently selected from the group consisting
of:
(1) 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-Ci3)alkynyloxy
(xiii) halo-{Ci-C6)alkyl,
(xiv) halo-(Ci-Ce)alkoxy,
(xv) (C2-C6)alkylthio,
(xvi) (C2-C10)alkanoyloxy,
(xvii) carboxy-(C2-C4)alkenyl,
(xviii) {C1-C3)alkylsulfonyloxy,
(xix) carboxy-(C1-C3)alkyl,
(xx) N-[di (C1-C3)-alkyl]amino-(C1-C3)alkoxy,
(xxi) cyano-(Ci-Cg)alkoxy, and
(xxii) diphenyl-(Ci-Ce)alkyl,
with the proviso that when R^^ is (Ci-Cg) alkyl, (Ci-
Cs)alkoxy, or halo, p must be greater or equal to 2, or when
r"^ is (C1-C3 alkyl)-R^ then R^^ is not hydrogen, (Ci-
Cg)alkyl, (Ci-Cg)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-C6)alkoxy,
(v) halo-(Ci-C6)alkyl,
(vi) halo-(Ci-Cg)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-Ce)alkoxy,
(iii) divalent (C2-C6)alkenyl,
(iv) divalent (C2-C6)alkynyl, or
(v) a group of the formula - (C (R^'^) 2) s-R-"-^- or -
r1^-(C (rI"*) 2) s-/ wherein s is 0-6; wherein each R^^
substituent is independently selected from hydrogen, (Cj-
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;
r13 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-Ce)alkoxyphenyl, phenyl-(C2-C3)alkynyl, and
(Ci-Cg)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-Ce)alkoxy,
(iii) (C2-C6)alkenyl,
(iv) (C2-C6)alkynyl,
(v) (C4-C10) 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^^ 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-Cg) alkyl, (Ci-Ce) alkoxy, or both R^"^ substituents taken
together are 0,
(v) -N(r1^)2-/ wherein each R^^ substituent is
independently selected from hydrogen; (Ci-Ce)alkyl; (C2-
C6)alkenyl; (C2-C6)alkynyl; {C4-C10) cycloalkyl; phenyl;
phenyl substituted by nitro, halo, (Ci-Cg)alkanoyloxy; or
both r1^ substituents taken together are (C4-C10) cycloalkyl;
r1^ is R^2 or R^3 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)".
9^35!5it_of the Invention .^.^^^^
The pre53ent, invention can be ur.iiLzed to make the
alkylated glycopeptides described in these references.
Preferred alkylated glycopeptides which can be prepared by
the present process include the following:
N^-n -octylA82 84 6B
N'^-n-decylA82846B
N'^-benzylA82846B
N^-(p-chlorobenzyl)A82846B
N^-- (p-bromobcnzyl) A82 84 6B
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) A82846B
;p-isopropoxybenzyl)A82846B
N^-(p-butoxybenzyl)A82846B
W^-(p-tert-butoxybenzyl)A82846B
N'^- (p-pentyloxybenzyl) A82846B
N'^-(p-hexyloxybenzyl) A82846B
N^-(o-hexyloxybenzyl)A82846B
N^-(p-heptyloxybenzyl)A82846B
N'i- (p-octyloxybenzyl) A82846B
N'^-phenethylA82 84 6B
N^-(4-phenylbenzyl)A82846B
n4_ (4.. (4-chlorophenyl)benzylA82846B
n4_(4_(4-methylbenzyloxy)benzyl)A82846B
n4_(4_(4-ethylbenzyloxy)benzyl)A82846B
n4-(4-{4-chlorophenethyl)benzyl)A82846B
N^-(4-(2-(4-methoxyphenyl)ethynyl)benzyl)A82 84 6B.
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 suggest a strong preference for sodium
cyanoborohydride as reducing agent. While sodium
cyanoborohydride is a successful reagent for small scale
use, its use in large scale production is less satisfactory.
This is due to safety and environmental issues posed by the
cyanide ion. Accordingly, sodium cyanoborohydride is less
than an ideal reducing agent for larger scale reactions.
Reducing agents are legion, but many are unsatisfactory
for the glycopeptides. One of the many reducing agents
known for use in reductive alkylations is pyridine•borane
(see J. Chem Soc. Perkin Trans. 1 (1984), pages 717-720,
which is incorporated herein by reference). It has now been
discovered that pyridine•borane is a uniquely acceptable
reagent for alkylative reductions on glycopeptides, while
presenting no safety or environmental hazards as is the case
with sodium cyanoborohydride. Furthermore, in a preferred
embodiment, it has been discovered that portionwise addition of
the pyridine.borane increases yields.
Statement of the Invention
The present invention is directed to an improved process
for reductively alkylating an amine-containing glycopeptide
antibiotic, which process comprises reacting the glycopeptide
antibiotic with an aldehyde or ketone in the presence of a
reducing agent, wherein the improvement comprises employing
pyridine.borane as reducing agent. In a preferred embodiment,
the glycopeptide antibiotic, aldehyde or ketone, and a portion of
the reducing agent are mixed together at the same time, and one
or more additional portions of reducing agent are added
thereafter.
Detailed description of the Invention
In carrying out the present invention, standard conditions
for reductive alkylations of glycopeptides are employed, other
than the identity of the reducing agent and the preference for
its portionwise addition. Thus, the glycopeptide and aldehyde or
ketone are initially dissolved in a solvent which is at least
predominantly methanol, and which is preferably only methanol.
If only these reagents are supplied, some small amount of
Schiff's base is produced, but the reaction equilibrium does not
favor complete production of the Schiff's base. Addition of
reducing agent shifts the equilibrium as Schiff's base is
converted to alkylated product. As noted, EPO 657 353 Al teaches
a preference for simultaneous addition of the reducing agent with
the glycopeptide antibiotic and aldehyde or ketone. Thus, in the
present invention, the
glycopeptide, aldehyde or ketone, and pyridine-borane are
added at essentially the same time.
Further, it has been discovered that when employing
pyridine-borane as reducing agent, even simultaneous
addition of glycopeptide antibiotic, aldehyde or ketone, and
reducing agent leads to only modest yields and that such
yields can be increased by portionwise addition of the
pyridine-borane, with no more than a portion being added
initially to the glycopeptide antibiotic and the aldehyde or
ketone.
The exact number and timing of portions is not
critical. The reaction is generally conducted over a period
of time from 4 to 48 hours and preferably from 6 to 24
hours. In the preferred practice of the present invention,
a first portion of pyridine-borane is added with the
glycopeptide antibiotic and aldehyde or ketone, and the
remainder of the pyridine-borane is added in one, two, or
more subsequent portions. The ideal sequence of
pyridine-borane addition appears to be five portions at 2 to
4 hour intervals (counting the initial addition as the first"
of the five). Devices can be employed to provide a
continuous delivery of the pyridine-borane.
In another preferred embodiment of the invention, a source of soluble copper is supplied to the reaction mixture, initially converting the glycopeptide to a copper complex, which becomes the reactive entity. The use of '
copper confers regioselectivity of reaction in those
glycopeptides having multiple reactive amines. For example,
-\8'
in A82846B, the use of copper'minimizes reaction on the N^
(leucine) site and on the N^ (monosaccharide) site, thereby
providing higher yields of the product monoalkylated on the
N'^ (disaccharide) amine.
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. Suitable copper salts are cupric
acetate, cupric trifluoroacetate, cupric
cyclohexanebutyrate, cupric 2-ethylhexanoate, cuprous
chloride, cupric chloride, and cupric bromide. A preferred
source of copper is copper (II) acetate, most conveniently U
employed as the hydrate.
The reaction should be conducted at a pH of 6-8, and
preferably at a pH of 5.3-7.0.
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 pyridine-borane. A slight excess is
preferable. The amount of soluble copper, if used, is
important. The process first results in the formation of a
1:1 copper complex with the glycopeptide antibiotic.
Therefore, the copper is preferably present in an amount
approximately equimolar with the glycopeptide antibiotic.
However, amounts exceeding one molar equivalent are
undesirable bedause excess copper decomposes the
pyridine•borane.
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 1.7:1.5:0.9 to 1.
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
important. 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 conditions depend upon whether or not
copper is employed in the reaction. When copper is not
added, it is preferred to conduct the reaction in straight
methanol at temperatures of about 58-67°C. When employing
copper, it is important to conduct the reaction at slightly
lower temperatures of about 58-63°C; again, straight
methanol is preferred.
upon the completion of the reaction, the reaction
mixture is preferably quenched, as by the addition of sodium
borohydride. This reagent consumes residual aldehyde or
ketone and thereby prevents further undesired reactions.
The product is isolated from the reaction mixture in
conventional manner. When copper has been employed, 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-propanal, isopropyl
alcohol, or preferably acetonitrile. The complex can be
broken by aqueous treatment at pH ^4, 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.
EXAMPLE 1
A82846B (0.50 g, 84.3% potency, 0.42 bg, 0.26 mmol )
was stirred in 50 mL methanol and 4'-chloro-4-
biphenylcarboxaldehyde (72 mg, 0.33 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 n4-(4-(4-
chlorophenyl)benzyl)A82846B.
- 2.1 -
EXAMPLE 2
(portionwise addition of pyridine-borane)
A82846B (0.50 g, 83.9 % potency, 0.26 mmol) and 4'-
chloro-4-biphenylcarboxaldehyde (98 mg, 0.45 mmol) were
stirred in 50 mL methanol and pyridine-borane complex (0.015
mL, 0.15 mmol) was added. The reaction mixture was heated
at reflux for 4 hours and an additional aliquot of
pyridine-borane complex (0.015 mL, 0.15 mmol) was added.
After heating at reflux for 4 hours longer a final addition
of pyridine-borane complex (0.015 mL, 0.15 mmol) was made.
The reaction mixture was heated at reflux for another 2 0
hours. After cooling to ambient temperature HPLC analysis
of a reaction aliquot afforded a yield of 0.27 g (58.0 %) of
N^-(4-(4-chlorophenyl)benzyl)A82846B.
EXAMPLE 3
(with copper)
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)A82 84 6B.
EXAMPLE 4
(with copper + portionwise addition of pyridine-borane)
A82846B (0.50 g, 76.3% potency, 0.38 bg, 0.24 mmol )
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 Examples 1, 3, and 4 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.
-23> -
These data show that portionwise addition of
pyridine-borane, accompanied by the use of copper, maximizes
yields of the product monoalkylated on N^, while minimizing
yields of other alkylated products.
EXAMPLE 5
(with copper + portionwise addition
of pyridine-borane by syringe pump)
A82846B (0.50 g, 83.4 % potency, 0.26 mmol) and cupric
acetate (47 mg, 0.26 mmol) were stirred in 50 mL methanol
and 4'-chloro-4-biphenylcarboxaldehyde (98 mg, 0.45 mmol)
and pyridine-borane complex (0.015 mL, 0.15 mmol) were
added. The reaction mixture was heated at 63°C for 2 hours.
Additional pyridine-borane complex (0.03 mL, 0.3 0 mmol) in 2
mL methanol was added to the reaction mixture at a rate of
400 uL/hour using a syringe pump. The temperature was
maintained at 63°C during the addition. After the addition
was complete, heating was continued to afford a total
reaction time of 24 hours. After cooling to ambient
temperature, HPLC analysis of a reaction aliquot afforded a
yield of 0.35 g (74.3 %) of n4-(4-(4-
chlorophenyl)benzyl)A82846B.
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,
2 0% 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.
We Claim:
1. A process for reduclivclx alk\iating an aminc-conlaining ghcopcptide antibiotic
comprising (i) mixing the aminc-containing gl_\copcptidc antibiotic, an aldehyde or
ketone, a p_\ridine.borane reducing agent and a source of soluble copper to form a
mixture, wherein the reducing agent is added in one or more portions.
2. The process of claim 1 wherein the glycopeptide antibiotic is vancomycin, A82846B,
A82846B, A82846C. or orienticin A.
3. The process of claim 1 wherein the glycopeptide antibiotic is A82846Ii.
4. The process oi"claim 1 wherein the aldehyde is 4'-chioro-4-biphenylcarboxyaldehyde.
5. The process ol"claim 1 wherein the reduv ing agent is added in ine portions at 2 to 4
hour inter\als.
6. The process oTclaim 1 wherein the soluble .source ofcopper is selected from the
group consisting of cupric acetate, cupric irilluoroacetale. cupric cyclohexanebutyrate,
cupric 2-ethylhcxanoaie. cuprous chloride, cupric chloride, and cupric bromide.
7. The process oiclaim I wherein the source of soluble copper is a hsdrate ofcopper
(li) acetate.
8. The process of claim 1 wherein the source of soluble copper is present in an amount
approximately cquimolar with the glycopeptide antibiotic.
9. I'he process of claim 1 wherein the amounls of the glycopeptide antibiotic and the
source of soluble copper are present at a ratio of 1 :().9 to 1 glycopeptide antibiotic:
source of soluble copper.
10. fhe process of claim 1 wherein said process is carried out in methanol at .58-6.3 "C.
11. fhe process oiclaim 1 further comprising step (ii) adding sodium borohydride.
12. A process for reducti\el\ alkylating an amine-containing ghcopcptide antibiotic
comprising (i) mixing the amine-containing glscopeplide antibiotic, an aldeh>de or
ketone, and a first portion of pyridine.borane reducing agent to form a mixture: and
(ii) adding one or more subsequent portions of the reducing agent to the mixture.

This invention is concerned with improved processes for
reductive alkylation of glycopeptide antibiotics, the
improvement residing in employing pyridine-borane as
reducing agent.