Note: Descriptions are shown in the official language in which they were submitted.
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BENZYLLACTOBIONAMIDES AS INHIBITORS OF SMOOTH
MUSCLE CELL PROLIFERATION
BACKGROUND OF THE INVENTION
This invention relates to the use of substituted benzyliactobionamides as
smooth muscle cell proliferation inhibitors and as therapeutic compositions
for
treating diseases and conditions which are characterized by excessive smooth
muscle
proliferation such as restenosis.
All forms of vascular reconstruction such as angioplasty and vein bypass
procedures effect a response to injury that ultimately leads to smooth muscle
cell
(SMC) proliferation and subsequently, deposition of profuse amounts of
I S extracellular matrix (Clowes, A. W.; Reidy, M. A. J. Vasc. Surg 1991, 13,
885).
These events are also central processes in the pathogenesis of atherosclerosis
(Raines
E.W.; Ross R. Br. Heart J. 1993, 69 (Supplement), S. 30) as well as transplant
arteriosclerosis (Isik, F.F.; McDonald, T. O.; Ferguson, M.; Yamanaka, E.;
Gordon
Am. J. Pathol. 1992, 141, 1139). In the case of restenosis following
angioplasty,
clinically relevant solutions for controlling SMC proliferation through
pharmacological intervention have remained elusive to date (Herrman, J. P. R.;
Hermans, W.R.M.; Vos, J.; Serruys P. W. Drugs 1993, 4, 18 and 249). Any
successful approach to selective SMC proliferation inhibition must not
interfere with
endothelial cell repair or the normal proliferation and function of other
cells
(Weissberg, P.L.; Grainger, D.J.; Shanahan C.M.; Metcalfe, J.C. Cardiovascular
Res. 1993, 27, 1191 ).
The glycosaminoglycans heparin and heparan sulfate are endogenous
inhibitors of SMC proliferation, yet are able to promote endothelial cell
growth
(Castellot, J.J. Jr.; Wright, T. C.; Karnovsky, M.J. Seminars in Thrombosis
and
Hemostasis 1987, 13, 489). However, the full clinical benefits of heparin,
heparin
fragments, chemically modified heparin, low molecular weight heparins, and
other
heparin mimicking anionic polysaccharides may be compromised due to other
pharmacological liabilities (excessive bleeding arising from anticoagulation
effects, in
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particular) coupled with heterogeneity of the various preparations (Borman, S.
Chemical and Engineering News, 1993, June 28, 27).
US 5,296,588, US 5,336,765, and EP 550106A1 describe an improved
process of preparing N substituted aldonamides. US 5,310,542 and EP 551675-A1
also describe glycosides (specifically aldobionamides) being used in oral
hygiene
compositions to act as antimicrobial agents and inhibit formation and/or
growth of
bacteria responsible for dental plaque. US 2,752,334 describes a process of
preparing
N substituted lactobionamides and their use as emulsifying agents (specially
for
cheese) and antimycotic agents. The compounds of the present invention differ
in that
the compounds of this invention (a) are acetylated or sulfated
benzyllactobionamides,
(b) have substituents on the aromatic core that are different, and (c) are
being used as
smooth muscle cell antiproliferatives.
EP 312086 A2 and EP 312087 A2 describe polysulfate esters) of bis-aldonic
acid amide derivatives as antiinflammatory and antithrombotic agents. The
compounds of the present invention differ in that the compounds (a) are being
used as
smooth muscle cell antiproliferatives, (b) are not dimeric in nature, and (c)
contain no
more than two contiguous sugar residues (disaccharide).
(Klein, U.; Mohrs, K.; Wild, H.; Steglich, W. Liebigs Ann. Chem. 19$7, 485-
489.) describes the use of peracetylated aldonamides to prepare 3-substituted
pyrazoles. The compounds of the present invention differ in that the compounds
{a)
have substituents on the aromatic core that are different, (b) are not
substituted at the
benzylic position, (c) are being used as smooth muscle cell
antiproliferatives, and (d)
are not used as precursors to pyrazoles.
US 5,498,775, WO 96/14324, and US 5,464,827 describe polyanionic
benzylglycosides or cylcodextrins as smooth muscle cell proliferation
inhibitors for
treating diseases and conditions that are characterized by excessive smooth
muscle
proliferation. p-Cyclodextrin tetradecasulfate has been described as a smooth
muscle
cell proliferation inhibitor and as an effective inhibitor of restenosis
(Reilly, C. F.;
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Fujita, T.; McFall, R. C.; Stabilito, I. L; Wai-si E.; Johnson, R. G. Drug
Development
Research 1993, 29, 137). US 5019562 discloses anionic derivatives of
cyclodextrins
for treating pathological conditions associated with undesirable cell or
tissue growth.
WO 93/09790 discloses antiproliferative polyanionic derivatives of
cyclodextrins
bearing at least 2 anionic residues per carbohydrate residues. Meinetsberger
(EP
312087 A2 and EP 312086 A2) describes the antithrombotic and anticoagulant
properties of sulfated bis-aldonic acid amides. US 4431637 discloses
polysulfated
phenolic glycosides as modulators of the complement system. The compounds of
the
present invention differ from all of the prior art in that the compounds (a)
are
benzyllactobionamides which bear no structural resemblance to heparin or
sulfated
cyclodextrins, (b) are compounds which are not dimeric in nature, (c) contain
no
more than two contiguous sugar residues (disaccharide), and (d) are of defined
structure.
WO 9614325 discloses acylated benzylglycosides as smooth muscle cell
proliferation inhibitors. The compounds of the present invention differ in
that (a) the
saccharide backbone is different, (b) the open chain core has preparation
advantages
over the cyclic array, and (c) the substituents on the carbohydrate backbone
are
different.
(Zehavi, U.; Herchman, M. Carbohyd Res. 1986, 151, 371) discloses 4-
carboxy-2-nitrobenzyl 4-O-«-D-glucopyranosyl-p-D-glucopyranoside which is
attached to a polymer for study as an acceptor in the glycogen synthase
reaction. The
compounds of the present invention differ from those of the Zehavi disclosure
in that
(a) the substituents on the benzyl groups are different and (b) the use
(smooth muscle
antiproliferation) is different.
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DESCRIPTION OF THE INVENTION
This invention provides benzyllactobionamides of formula I
RIO ERs
R7 Rs
OR3 R
I R~ ~ Rto
wherein
R', RZ, R', R', R5, R6, R', and R8 are each, independently, acyl of 2-7 carbon
atoms,
haloacyl of 2-7 carbon atoms, nitroacyl of 2-7 carbon atoms, cyanoacyl of 3-
7 carbon atoms, trifluoromethylacyl of 3-8 carbon atoms, benzoyl, or -SOgH;
R9 is hydrogen, CN, NOZ, halo, CF3, alkyl of 1-6 carbon atoms, or alkoxy of 1-
6
carbon atoms;
R1~ is hydrogen, -NO~, -NHR", -NHR", -N(R")2, -NCH,R'3, -NHC02alkyl, wherein
the alkyl moiety contains 1-6 carbon atoms, alkylsulfonamide of 1 to 4 carbon
atoms,
O O O
NH ( \ R12 -~-NH ~ ~ N R12 -~-NH ~R12
~ , or ~ ;
ZisOorS;
Rl t is an a-amino acid in which the a carboxyl group forms an amide with the
nitrogen of Rto, wherein if said amino acid is glutamic acid or aspartic acid,
the non-a carboxylic acid is an alkyl ester in which the alkyl moiety contains
from 1-6 carbon atoms;
R12 is hydrogen. CN, NO" halo, CF3, alkyl of 1-6 carbon atoms, alkoxy of 1-6
carbon
atoms, acyl of 2-7 carbon atoms, or benzoyl;
R13 is hydrogen, acyl of 2-7 carbon atoms, haloacyl of 2-7 carbon atoms,
nitroacyl
of 2-7 carbon atoms, cyanoacyl of 3-7 carbon atoms, trifluoromethylacyl of 3
8 carbon atoms, or benzoyl;
or a pharmaceutically acceptable salt thereof.
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Alkyl includes both straight chain as well as branched moieties. Halogen
means bromine, chlorine, fluorine, and iodine. When R11 is an a-amino acid,
the
carboxyl moiety exists as an amide with the amide nitrogen being bonded to the
phenyl ring of the compound of formula I. The following exemplifies the
resulting
structure when R11 is alanine:
9
-~-N ~R .NH2
~ N CH3
O
When the amino acid contains a second carboxyl moiety, the moiety is an
alkyl ester of the free acid. The following example shows aspartic acid methyl
ester.
H R9
-~- ~ ~H2
NH OCH3
O
Preferred amino acids include alanine, arginine, aspartic acid, cystine,
glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
The
amino acids defined by R11 include both the D and L amino acids.
Pharmaceutically acceptable salts can be formed from organic and inorganic
acids, for example, acetic, propionic, lactic, citric, tartaric, succinic,
fumaric, malefic,
malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic, phosphoric,
nitric,
sulfuric, methanesulfonic, napthalenesulfonic, benzenesulfonic,
toluenesulfonic,
camphorsulfonic, and similarly known acceptable acids. Salts may also be
formed
from organic and inorganic bases, preferably alkali metal salts, for example,
sodium,
lithium, or potassium. Acid addition salts can be prepared when a compound of
this
invention contains a basic nitrogen, and base addition salts can typically be
prepared
when the compound of formula I contains a -S03H moiety.
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The compounds of this invention may contain an asymmetric carbon atom or
sulfoxide moiety and some of the compounds of this invention may contain one
or
more asymmetric centers and may thus give rise to optical isomers and
diastereomers.
While shown without respect to stereochemistry in Formula I, the present
invention
includes such optical isomers and diastereomers; as well as the racemic and
resolved,
enantiomerically pure R and S stereoisomers; as well as other mixtures of the
R and S
stereoisomers and pharmaceutically acceptable salts thereof.
Preferred compounds of this invention are benzyllactobionamides of formula I
Ri0 ORs
O OR7 Rs
R20 O ORe '
OR3 R4O N
R5O O Rio
wherein
R', R2, R', R°, R5, R6, R', and Rg are each, independently, acyl of 2-7
carbon atoms or
-S03H;
R9 is hydrogen, CN, NOZ, halo, CF3, alkyl of 1-6 carbon atoms, or alkoxy of 1-
6
carbon atoms;
Rl~ is hydrogen, -NOZ, -NHR", -NHR'3, -N(R'3)Z, -NCH3R'3, -NHC02alkyl, wherein
the alkyl moiety contains 1-6 carbon atoms, alkylsulfonamide of 1 to 4 carbon
atoms,
O
NH ( ~ R12 '~-NH ( ~ ~N R12 -~-NH jRl2
~ , or "~.~ ;
Z is O;
R11 is an a-amino acid in which the a carboxyl group forms an amide with the
nitrogen of R1~, wherein if said amino acid is glutamic acid or aspartic acid,
the non-a carboxylic acid is an alkyl ester in which the alkyl moiety contains
from 1-6 carbon atoms;
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R12 is hydrogen, CN, NO2, halo, CF3, alkyl of 1-b carbon atoms, alkoxy of 1-6
carbon
atoms, acyl of 2-7 carbon atoms, or benzoyl;
Rt3 is hydrogen, acyl of 2-7 carbon atoms, haloacyl of 2-7 carbon atoms,
nitroacyl
of 2-7 carbon atoms, cyanoacyl of 3-7 carbon atoms, trifluoromethylacyl of 3-
S 8 carbon atoms, or benzoyl;
or a pharmaceutically acceptable salt thereof.
More preferred compounds of this invention are benzyllactobionamides of
formula I
R10 ORs
O O R7 R9
R20 O OR8
OR3 R40 NH
R5p O Rio
wherein
R', R2, R', R°, RS, R6, R', and R8 are each, independently, acetyl or
-S03H;
R9 is hydrogen, CN, NOZ, halo, CF3, alkyl of 1-6 carbon atoms, or alkoxy of 1-
6
carbon atoms;
Rl~ is hydrogen, -NO~, -NHR'3, -N(R'3)2,
R13 is hydrogen, or acyl of 2-7 carbon atoms;
or a pharmaceutically acceptable salt thereof.
Specifically preferred compounds of this invention are:
N-Benzyl-octa-O-acetyl-lactobionamide or a pharmaceutically acceptable salt
thereof;
N-Benzyl-octa-O-sulfo-lactobionamide or a pharmaceutically acceptable salt
thereof;
N-(4-Nitro-benzyl)-octa-O-acetyl-lactobionamide or a pharmaceutically
acceptable
salt thereof;
N (4-Amino-benzyl)-octa-O-acetyl-lactobionamide or a pharmaceutically
acceptable
salt thereof;
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N (3-Amino-benzyl)-octa-O-acetyl-lactobionamide or a pharmaceutically
acceptable
salt thereof;
N [3-(Acetylamino)-benzyl]-octa-O-acetyl-lactobionamide or a pharmaceutically
acceptable salt thereof; and
N [3-(Acetylamino)-benzyl]-octa-O-sulfo-lactobionamide or a pharmaceutically
acceptable salt thereof;
The compounds of this invention were prepared according to the following
scheme from commercially available starting materials or starting materials
which
can be prepared using literature procedures. This scheme shows the preparation
of
representative compounds of this invention.
Lactobiono-1,5-lactone (1, *H. S. Isbell; H. L. Frush. Methods Carbohyd.
Chem. 1963, 2, 16-18.) is coupled with a benzyl amine 2 (in the presence of
sodium
carbonate when using amine salt) in a protic solvent such as methanol at
temperatures
ranging from 0 to 60 °C to yield glycoside 3 (Scheme 1). Reduction of
the nitro
group of 3 can be accomplished with a reducing agent such as stannous chloride
in a
polar aprotic solvent such as ethyl acetate at ambient temperature to reflux
to afford
an anilino compound 4. Coupling of 4 with an acid chloride can be completed in
the
presence of an amine base such as triethylamine or diisopropylethylamine or
using a
stronger base such as sodium hydride (for sterically hindered systems) in an
aprotic
solvent such as dichloromethane or tetrahydrofuran at temperatures ranging
from 0°C
to ambient temperature to yield the target compound 5. The peracetylated
compound
5 can be converted to its octahydroxy intermediate with catalytic sodium
methoxide
in methanol or aqueous sodium hydroxide in methanol at temperatures ranging
from
ambient temperature to reflux. This intermediate can be further converted to
the
octasulfo compound 6 with sulfur trioxide trimethylamine complex in a polar
solvent
such as N,N dimethylformamide at reflux. Many salts of the sulfate group can
be
prepared by using different ion exchange columns such as Dowex Na' or K'.
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Scheme 1
H
H
HO O + H2N ~ N02
HO HO
HO O 2'
1
1 ) lactobionamide formation
2) acetylation
Ac Ac
Ac
O Ac
AcOAcO Ac0 N ~"N02
A c0 O
3
reduction
Ac Ac
Ac
O Ac
AcOAcO Ac0 N ~NH2
Ac0 O
4
acylation
Ac Ac 1
Ac
O Ac
AcOAcO Ac0 N ~ NHAc
Ac0
5
1 ) hydrolysis
2) sulfation
R R
R
O R
RO RO RO N ~ NHAc
RO
6
R = SO aNa
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The compounds of this invention are useful as antiproliferative agents. The
following procedures show the evaluation of representative compounds of this
invention in standard pharmacological test procedure which measured ability of
the
evaluated compound to inhibit smooth muscle cell proliferation
Effects of Compounds on Cell Proliferation Usin 'g-H Thvmidine Incorporation
Human and porcine smooth muscle cells were tested in early passage (generally
passage 3 - 7) at sub-confluent conditions. Cultures were grown in 16 mm (24
well)
mufti-well culture dishes in medium 199 supplemented with 10% fetal bovine
serum
and 2% antibiotic / antimycotic. At sub-confluence, the cells were placed in a
defined serum free medium (AIM-V; Gibco) for 24 - 48 h prior to initiating the
experimental protocol.
Although compounds were found to be more effective with longer pre-
incubations,
in general, the procedures were initiated with the addition of compound, 'H
thymidine
and serum / growth factor to serum deprived synchronized cells and results are
reported accordingly.
Compounds were added to each well at SO fold dilution (20 pL / well) and the
plates
were incubated for 24 - 36 h at 37 °C in 5% CO2. Compounds were
initially
dissolved in 50% ethanol and serially diluted into media. Compounds were
routinely
evaluated at concentrations from 1 to 100 E.~M. As a control, grade II porcine
intestinal mucosal heparin (sodium salt} was routinely evaluated in all cell
preparations at concentrations from 0.1 to 100 pg/mL.
At the completion of the test procedure, plates were placed on ice, washed
three
times with ice cold phosphate buffered saline {PBS) and incubated in ice cold
10%
trichloroacetic acid (TCA) got 30 min to remove acid soluble proteins.
Solution was
transferred to scintillation vials containing 0.4 N HCI (500 ~tL,/ vial to
neutralize
NaOH) and each well was rinsed two times with water (500 pL) for a total
volume of
2 mL / vial.
Data was obtained, in triplicate, for both control and experimental samples.
Control
(100%) data was obtained from maximally stimulated cells, as the result of
growth
factor or serum stimulation. Experimental data was obtained from cells
maximally
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stimulated with growth factor or serum and treated with compound. Data are
expressed as an ICSp or percent inhibition in Table I below.
Table 1.
Porcine Smooth Muscle
Com ound of Exam Cell
le _Antiproliferation IC50
1 118 M
2 45% @ 500 M
30% @ 500
12% @ S00 M
122
The compounds of this invention are useful in treating or inhibiting diseases
which are characterized by excessive smooth muscle cell proliferation (smooth
muscle cell hyperproliferation). The compounds are particularly useful in
treating
hyperproliferative vascular diseases which are characterized by smooth muscle
cell
hyperproliferation, such as restenosis, which most frequently arises from
vascular
reconstructive surgery and transplantation, for example, balloon angioplasty,
vascular
graft surgery, coronary artery bypass surgery, and heart transplantation.
Other
disease states in which there is unwanted "cellular" vascular proliferation
include
hypertension, asthma, and congestive heart failure. The compounds of this
invention
are also useful as inhibitors of angiogenesis. Angiogenesis
(neovascularization), the
process by which new capillaries are formed, is of principal importance for a
number
of pathological events including chronic inflammation and malignant processes.
The
compounds of this invention are therefore useful as antineoplastic agents.
The compounds of this invention can be formulated neat or with a
pharmaceutical carrier for administration, the proportion of which is
determined by
the solubility and chemical nature of the compound, chosen route of
administration
and standard pharmacological practice. The pharmaceutical carrier may be solid
or
liquid.
A solid carrier can include one or more substances which may also act as
flavoring agents, lubricants, solubilizers, suspending agents, fillers,
glidants,
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compression aids, binders or tablet-disintegrating agents; it can also be an
encapsulating material. In powders, the carrier is a finely divided solid
which is in
admixture with the finely divided active ingredient. In tablets, the active
ingredient is
mixed with a carrier having the necessary compression properties in suitable
proportions and compacted in the shape and size desired. The powders and
tablets
preferably contain up to 99% of the active ingredient. Suitable solid carriers
include,
for example, calcium phosphate, magnesium stearate, talc, sugars, lactose,
dextrin,
starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose,
polyvinylpyrrolidine, low melting waxes and ion exchange resins.
Liquid Garners are used in preparing solutions, suspensions, emulsions,
syrups, elixirs and pressurized compositions. The active ingredient can be
dissolved
or suspended in a pharmaceutically acceptable liquid carrier such as water, an
organic
solvent, a mixture of both or pharmaceutically acceptable oils or fats. The
liquid
carrier can contain other suitable pharmaceutical additives such as
solubilizers,
emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending
agents,
thickening agents, colors, viscosity regulators, stabilizers or osmo-
regulators.
Suitable examples of liquid carriers for oral and parenteral administration
include
water (partially containing additives as above, e.g. cellulose derivatives,
preferably
sodium carboxymethyl cellulose solution), alcohols (including monohydric
alcohols
and polyhydric alcohols, e.g. glycols) and their derivatives, lethicins, and
oils (e.g.
fractionated coconut oil and arachis oil). For parenteral administration, the
carrier
can also be an oily ester such as ethyl oleate and isopropyl myristate.
Sterile liquid
carriers are useful in sterile liquid form compositions for parenteral
administration.
The liquid carrier for pressurized compositions can be halogenated hydrocarbon
or
other pharmaceutically acceptable propellant.
Liquid pharmaceutical compositions which are sterile solutions or suspensions
can be utilized by, for example, intramuscular, intraperitoneal or
subcutaneous
injection. Sterile solutions can also be administered intravenously. The
compounds
of this invention can also be administered orally either in liquid or solid
composition
form.
The compounds of this invention may be administered rectally or vaginally in
the form of a conventional suppository. For administration by intranasal or
intrabronchial inhalation or insufflation, the compounds of this invention may
be
formulated into an aqueous or partially aqueous solution, which can then be
utilized
in the form of an aerosol. The compounds of this invention may also be
administered
transdermally through the use of a transdermal patch containing the active
compound
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and a carrier that is inert to the active compound, is non toxic to the skin,
and allows
delivery of the agent for systemic absorption into the blood stream via the
skin. The
carrier may take any number of forms such as creams and ointments, pastes,
gels, and
occlusive devices. The creams and ointments may be viscous liquid or semisolid
emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of
absorptive powders dispersed in petroleum or hydrophilic petroleum containing
the
active ingredient may also be suitable. A variety of occlusive devices may be
used to
release the active ingredient into the blood stream such as a semipermeable
membrane covering a reservoir containing the active ingredient with or without
a
carrier, or a matrix containing the active ingredient. Other occlusive devices
are
known in the literature.
The dosage requirements vary with the particular compositions employed, the
route of administration, the severity of the symptoms presented and the
particular
subject being treated. Based on the results obtained in the standard
pharmacological
test procedures, projected daily dosages of active compound would be 0.1 to 10
mg/kg administered parenterally (intravenous preferred), with projected daily
oral
dosage being approximately ten-fold higher. Anticipated intravenous
administration
would last for approximately 5-30 days following acute vascular injury (i.e.,
balloon
angioplasty or transplantation) and for a longer duration for the treatment of
chronic
disorders. Treatment will generally be initiated with small dosages less than
the
optimum dose of the compound. Thereafter the dosage is increased until the
optimum effect under the circumstances is reached; precise dosages for oral,
parenteral, nasal, or intrabronchial administration will be determined by the
administering physician based on experience with the individual subject
treated.
Preferably, the pharmaceutical composition is in unit dosage form, e.g. as
tablets or
capsules. In such form, the composition is sub-divided in unit dose containing
appropriate quantities of the active ingredient; the unit dosage forms can be
packaged
compositions, for example, packaged powders, vials, ampoules, pre filled
syringes or
sachets containing liquids. The unit dosage form can be, for example, a
capsule or
tablet itself, or it can be the appropriate number of any such compositions in
package
form.
The following provides the preparation of representative compounds of this
invention.
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Example 1
N Benzyl-octa-O-acetyl-lactobionamide
step 1
To a stirred solution of lactobiono-1,5-lactone (0.480 g, 1.41 mmol, *H. S.
Isbell; H. L. Frush. Methods Carbohyd. Chem. 1963, 2, 16-18.) in warm MeOH (5
mL), from gentle heating, was added benzyl amine (0.177 mL, 1.62 mmol)
dropwise.
The reaction was stirred at rt for 18 h. After concentration, the oily residue
was
purified by recrystallization from i-PrOH to afford the product (0.300 g,
48%a) as a
glassy white solid; 'H NMR (DMSO-d6) s 3.26-3.36 (m, 2H), 3.36-3.41 (m, 1H),
3.46-3.55 (m, 3H), 3.55-3.64 (m, 2H), 3.66-3.74 (m, 2H), 4.02-4.07 (m, 1H),
4.12 (d,
J = 5.9 Hz, 1H), 4.17 (dd, J = 1.5, 5.1 Hz, 1H), 4.26 (d, J = 7.0 Hz, 1H),
4.30 (d, J =
6.4 Hz, 2H), 4.46-4.52 (m, 2H), 4.66 (t, J = 5.7 Hz, 1H), 4.78 (dd, J = 5.5,
9.7 Hz,
2H), 5.14 (d, J = 3.7 Hz, 1H), 5.25 (d, J = 5.7 Hz, 1H), 7.16-7.24 (m, 1H),
7.24-7.32
(m, 4H), 8.13 (t, J = 6.2 Hz, 1H); IR (KBr) 3380, 2930, 2890, 1650, 1555,
1455,
1430, 1400, 1370, 1325, 1280, 1220, 1125, 1080, 1045, 970, 880, and 710 cm-';
mass
spectrum [(-) FAB], m/z 446 (M - H)-; Anal. Calcd. for C~9HZ9NO": C, 51.00; H,
6.53; N, 3.13, Found: C, 50.87; H, 6.49; N, 3.12.
step 2
To a stirred solution of N benzyl-lactobionamide (0.150 g, 0.335 mmol) and
triethylamine (0.822 mL, 5.90 mmol) in DMF (3.4 mL) at rt was added dropwise
acetic anhydride (0.278 mL, 2.95 mmol) followed by a catalytic amount of DMAP
(0.0327 g, 0.268 mmol). After 18 h, the mixture was concentrated, and the
resulting
residue was diluted with EtOAc (100 mL). This layer was washed with 1 N HCl
(10
mL), sat. aq. NaHC03 (10 mL), and brine (10 mL) and then dried (MgSO,). After
concentration, the residue was purified by flash chromatography (10:90 to
90:10
EtOAc:petroleum ether gradient) to afford the product (0.221 g, 84%) as a
glassy
solid, mp 85-86 °C; 'H NMR (CDC13) s 1.88 (s, 3H), 1.90 (s, 3H), 1.98
(s, 3H), 2.02
(s, 6H), 2.03 (s, 3H), 2.09 (s, 3H), 2.15 (s, 3H), 3.94-4.02 (m, 2H), 4.03 (d,
J = 7.2
Hz, 1H), 4.11-4.19 (m, 2H), 4.23 (t, J = 20.4 Hz, 1H), 4.27-4.37 {m, 2H), 4.79
(d, J =
7.9 Hz, 1H), 4.84-4.95 (m, 2H), 5.14 (dd, J = 3.7, 10.1 Hz, 1H), 5.21-5.24 (m,
2H),
5.45 (dd, J = 3.1, 6.4 Hz, 1H), 7.17-7.25 (m, 3H), 7.26-7.32 (m, 2H), 8.58 (t,
J = 1.9
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Hz, 1H); IR (KBr) 3390, 2970, 1755, 1670, 1540, 1420, 1375, 1220, 1130, 1050,
950, and 610 cm~'; mass spectrum [(+) FAB], m/z 784 (M + H)+, 806 (M + Na)+;
Anal. Calcd. for C,SH,SN0~9: C, 53.64; H, 5.79; N, 1.79, Found: C, 53.51; H,
5.86;
N, 1.82.
Example 2
N-Benzvl-octa-O-sulfo-lactobionamide octasodium salt
To a stirred solution of N benzyl-lactobionamide (0.760 g, 1.70 mmol) in
DMF (59.4 mL) at rt was added sulfur trioxide trimethylamine complex (9.46 g,
68.0
mmol); this mixture was then heated to 70 °C. After 4 days, the mixture
was cooled
to 0 °C and quenched with H20 (60 mL). At this point, the solution was
concentrated
using no heat, and the resulting residue was slurried in H20 (~20-25 mL) and
filtered.
The filtrate was applied to a G-10 Sephedex column. The resulting compound was
collected and applied directly to a Dowex Na' ion exchange column to afford
the
product (1.47 g, 68%), after elution, as a glassy solid, mp >200 °C
(decomp.); 'H
NMR (DZO) s 3.86 (t, J = 6.2 Hz, 1H), 3.98-4.14 (m, 3H), 4.19-4.47 (m, SH),
4.52-
4.57 (m, 1H), 4.67-4.86 (m, 3H), 4.92-4.96 (m, 2H), 7.10-7.18 (m, 1H), 7.19-
7.29
(m, 4H); IR (KBr) 3490, 2950, 2320, 1680, 1570, 1500, 1450, 1270, 1190, 1070,
1025, 920, 820, 700, 620, and 580 cm-'; mass spectrum [(-) ESI], (m - zNa)/z
608.9
(M - 2Na)2-, 398.3 (M - 3Na)3-; Anal. Calcd. for C,9HZ~NO,SSBNaB' 3H20: C,
17.32;
H, 2.07; N, I.06, Found: C, 17.12; H, 2.08; N, 1.03.
Example 3
N l4-Nitro-benzvl)-octa-O-acetyl-lactobionamide
To a stirred solution of lactobiono-1,5-lactone (5.00 g, 14.7 mmol, *H. S.
Isbell; H. L. Frush. Methods Carbohyd. Chem. 1963, 2, 16-18.) in warm MeOH (53
mL), from gentle heating. was added 3-nitro-benzyl amine HCl salt (3.56 g,
19.1
mmol). The reaction was cooled to 0 °C, followed by Na2C03 (1.56 g,
14.7 mmol)
addition, and then stirred at rt for 4 days. After concentration, the oily
residue was
taken up in DMF (74 mL). To this stirred solution at rt was added dropwise
triethylamine (40.6 mL, 292 mmol) followed by acetic anhydride (13.7 mL, 146
mmol) and finally a catalytic amount of DMAP ( 1.63 g, 13.4 mmol). After 2 h,
the
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mixture was concentrated, and the resulting residue was diluted with EtOAc
(500
mL). This layer was washed with 1 N HCl (50 mL), sat. aq. NaHC03 (50 mL), and
brine (50 mL} and then dried (MgSO,). After concentration, the residue was
purified
by flash chromatography (0% to 40% acetone:CHCl3 gradient) to afford the
product
(7.23 g, 59%) as a white foam, mp 100-103 °C;'H NMR (CDC13) s 1.98 (s,
3H), 2.03
(s, 3H), 2.04 (s, 3H), 2.05 (s, 3H}, 2.08 (s, 6H), 2.16 (s, 3H), 2.18 (s, 3H),
3.91 (t, J =
7.0 Hz, 1H), 4.01 (dd, J = 5.7, 12.5 Hz, 1H), 4.07 (dd, J = 7.0, 11.2 Hz, 1H),
4.18
(dd, J = 6.4, 11.2 Hz, 1H), 4.31 (dd, J = 4.0, 6.6 Hz, 1H), 4.47 (dd, J = 5.5,
15.8 Hz,
1H), 4.54 (dd, J = 2.9, 12.5 Hz, 1H), 4.63 (d, J = 7.9 Hz, 1H}, 4.64 (dd, J =
6.8, 15.8
Hz, 1 H), 5.00 (dd, J = 3.3, 10.3 Hz, 1 H), 5.09 (td, J = 2.9, 5.7 Hz, 1 H),
5.17 (dd, J =
7.9, 10.3 Hz, 1H), 5.38 (dd, J = 1.1, 3.5 Hz, 1H), 5.59 (dd, J = 4.0, 5.7 Hz,
1H), 5.63
(d, J = 5.7 Hz, 1H), 6.66 (t, J = 6.2 Hz, 1H), 7.41 (d, J = 8.8 Hz, 2H), 8.16-
8.21 (m,
2H); IR (KBr) 3400, 2980, 1755, 1670, 1560, 1525, 1420, 1380, 1360, 1220,
1125,
1050, 950, 900, 860, and 600 cm''; mass spectrum [(-) FAB], m/z 828 (M}';
Anal.
Calcd. for C35H4,NZOz' ~ 0.5H20: C, 50.18; H, 5.41; N, 3.34, Found: C, 50.21;
H,
5.36; N, 3.29.
Example 4
N (4-Amino-benzvl)-octa-O-acetyl-lactobionamide
A solution containing N-(4-nitro-benzyl)-octa-O-acetyl-lactobionamide (6.97
g, 8.41 mmol) and tin (II) chloride dehydrate (13.3 g, 58.9 mmol) in EtOAc
(167 mL)
was refluxed for 4 h. The reaction was cooled to rt, carefully quenched with
sat. aq.
NaHC03 (until basic), diluted with EtOAc ( 163 mL), stirred for 0.5 h and
filtered.
The biphasic filtrate was separated and the aqueous phase extracted with
EtOAc. The
combined organic extracts were dried (NazS04) and concentrated. Purification
by
flash chromatography (0 to 30% methanol/CHC13 gradient) gave 4.61 g {69%) of
the
product as a glassy white solid, mp 87-91 °C; 'H NMR (CDCl3) s 1.97 (s,
3H), 2.01
(s, 3H), 2.04 (s, 3H), 2.05 (s, 3H), 2.07 (s, 3H), 2.09 (s, 3H), 2.12 (s, 3H),
2.15 (s,
3H), 3.70 {s, 2H), 3.75 (td, J = 0.9, 6.6 Hz, 1H), 4.00-4.17 (m, 3H), 4.25-
4.38 (m,
3H), 4.50 {dd, J = 2.9, 12.3 Hz, 1H), 4.59 (d, J = 7.9 Hz, 1H), 4.95 (dd, J =
3.5, 10.3
Hz, 1H), 5.03-5.07 (m, 1H), 5.17 (dd, J = 7.9, 10.5 Hz, 1H), 5.33 (dd, J =
l.l, 3.5 Hz,
1H), 5.58 (dd, J = 3.1, 6.6 Hz, 1H), 5.61 (d, J = 6.8 Hz, 1H), 6.38 (t, J =
5.7 Hz, 1H),
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6.62-6.67 (m, 2H), 7.02-7.07 (m, 2H); IR (KBr) 3390, 2980, 1745, 1670, 1630,
1530,
1425, 1375, 1220, 1125, 1060, 950, 900, 840, 755, and 620 cm-'; mass spectrum
[(+)
FAB], m/z 799 (M + H)', 821 (M + Na)'; Anal. Calcd. for C,SH46NZO~9 ~ 2.SH20:
C,
49.82; H, 6.09; N, 3.32, Found: C, 49.84; H, 5.43; N, 3.33.
Example 5
N (3-Amino-benzvl)-octa-O-acetyl-lactobionamide
The title compound was prepared as a glassy foam (5.90 g, 63%) from N (3
nitro-benzyl)-octa-O-acetyl-lactobionamide using a procedure similar to
Example 4,
mp 90-93 °C; 'H NMR (CDCl,) s 1.97 (s, 3H), 2.01 (s, 3H), 2.04 (s, 3H),
2.05 {s,
3H), 2.07 (s, 3H), 2.09 (s, 3H), 2.14 (s, 3H), 2.15 (s, 3H), 3.76-3.83 (m,
3H), 4.03-
4.18 (m, 4H), 4.29-4.41 (m, 2H), 4.50 (dd, J = 2.9, 12.3 Hz, 1H), 4.60 (d, J =
7.9 Hz,
1 H), 4.97 (d, J = 3.5, 10.5 Hz, 1 H), 5.03-5.08 (m, 1 H), 5.17 (dd, J = 7.9,
10.5 Hz,
1H), 5.34 (dd, J = 0.9, 3.3 Hz, 1H), 5.59 (dd, J = 3.3, 6.6 Hz, 1H), 5.65 (d,
J = 6.6
Hz, 1H), 6.45 (t, J = 5.9 Hz, 1H), 6.55-6.62 (m, 3H), 7.09 (t, J = 7.9 Hz,
1H); IR
(KBr) 3460, 3390, 2980, 1745, 1670, 1630, 1610, 1530, 1500, 1465, 1435, 1375,
1225, 1170, 1130, 1055, 955, 750, 700, 630, and 610 cm~'; mass spectrum [(+)
FAB],
m/z 799 (M + H)', 821 (M + Na)'; Anal. Calcd. for C35H46N2019 ' 1H_>O: C,
51.47; H,
5.92; N, 3.43, Found: C, S 1.40; H, 5.70; N, 3.25.
Example 6
N f 3-(Acetylamino)-benzvll-octa-O-acetyl-lactobionamide
To a stirred solution of N (3-amino-benzyl)-octa-O-acetyl-lactobionamide
(2.90 g, 3.63 mmol) and triethylamine (1.11 mL, 7.99 mmol) in THF (45 mL) at 0
°C
was added dropwise acetyl chloride (0.310 mL, 4.36 mmol). After 0.5 h at this
temperature, it was warmed to rt and stirred an additional 72 h. At this
point, the
reaction was concentrated and taken up in EtOAc (300 mL). This organic
solution
was washed with 1 N HCl (30 mL), sat. aq. NaHC03 (30 mL), and brine (30 mL)
and
then dried (MgSO,). After concentration, the residue was purified by flash
chromatography (5% to 60% acetone:CHCl3 gradient) to afford the product (2.35
g,
77%) as a glassy white foam, mp 120-123 °C; 'H NMR (CDCl3) s 1.98 (s,
3H), 2.00
(s, 3H), 2.05 (s, 3H), 2.06 (s, 3H), 2.075 (s, 3H), 2.082 (s, 3H), 2.14 (s,
3H), 2.I5 (s,
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3H), 2.17 (s, 3H), 3.84-3.90 (m, 1H), 4.00-4.08 (m, 2H), 4.21 (dd, J = 6.4,
11.2 Hz,
1H), 4.30 (dd, J = 3.3, 6.8 Hz, 1H), 4.35 (dd, J = 5.9, 14.9 Hz, 1H), 4.46-
4.53 (m,
2H), 4.62 (d, J = 7.9 Hz, 1H), 4.98 (dd, J = 3.3, 10.3 Hz, 1H), 5.05 (td, J =
2.9, 5.7
Hz, 1 H), 5.19 (dd, J = 7.9, 10.3 Hz, 1 H), 5.36 (dd, J = 1.1, 3.5 Hz, 1 H),
5.56 (dd, J =
3.1, 6.8 Hz, 1H), 5.69 (d, J = 6.8 Hz, 1H), 6.64 (t, J = 5.9 Hz, 1H), 6.96 (d,
J = 7.7
Hz, 1H), 7.21-7.30 {m, 2H), 7.66 (dd, J = l.l, 8.1 Hz, 1H), 7.81 (s, 1H); IR
(KBr)
3390, 2990, 1755, 1675, 1620, 1560, 1490, 1425, 1375, 1220, 1130, 1055, 950,
755,
and 610 cm-'; mass spectrum ((+) FAB], m/z 841 (M + H)', 863 (M + Na)'; Anal.
Calcd. for C3,HQgNZO2o ~ 3H~0 C, 49.66; H, 6.08; N, 3.13, Found: C, 49.46; H,
5.37;
N, 3.04.
Example 7
N f3-(Acetvlamino)-benzyll-octa-O-sulfo-lactobionamide octasodium salt
step 1
A solution containing N [3-(acetylamino)-benzyl]-octa-O-acetyl-
lactobionamide (2.09 g, 2.49 mmol) and 25 weight % NaOMe in MeOH (42.6 wL,,
0.746 mmol) in MeOH (62.7 ml) was refluxed for 2 h. The reaction was cooled to
room temperature and concentrated, and the resulting residue was triturated
with
MeOH:EtzO (30:70} to afford the product (1.11 g, 88%) as a glassy white solid,
mp
>170 °C {decomp.); 'H NMR (DMSO-d6) s 2.01 (s, 3H), 3.16 (d, J = 4.4
Hz, 2H),
3.31-3.42 (m, 1H), 3.47-3.63 (m, 4H), 3.68-3.73 (m, 2H), 4.01-4.14 (m, 3H),
4.14-
4.33 (m, 4H), 4.44-4.53 (m, 2H), 4.63-4.70 (m, 1 H), 4.73-4.87 (m, 2H), 5.12-
5.19
(bs, 1H), 5.19-5.25 (bs, iH), 6.95 (d, J = 7.7 Hz, 1H), 7.19 (t, J = 7.7 Hz,
1H), 7.38
(s, 1H), 7.48 (d, J = 8.1 Hz, 1H), 8.09 (t, J = 6.2 Hz, 1H), 9.86 (s, 1H); IR
(KBr)
3380, 2920, 2320, 1660, 1620, 1600, 1560, 1495, 1430, 1370, 1320, 1275, 1080,
1050, 880, 780, and 700 cm''; mass spectrum [(+) FAB], m/z 527 (M + Na)+;
Anal.
Calcd. for CZ,H32NZO,2 ~ 1.SH20: C, 47.45; H, 6.64; N, 5.27, Found: C, 47.86;
H,
6.73; N, 4.95.
step 2
The title compound was prepared as a tan solid (1.76 g, 66%) from N [3-
(acetylamino)-benzyl]-lactobionamide using a procedure similar to Example 2,
mp
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>2I 1 °C (decomp.); 'H NMR (D20) s 1.98-2.01 (m, 3H), 3.74-3.92 (m,
IH), 3.98-
4.17 (m, 3H), 4.20-4.56 (m, 6H), 4.66-4.85 (m, 3H), 4.87-4.98 (m, 2H), 6.96-
7.08
(m, 2H), 7.18-7.27 (m, 1H), 7.30-7.36 (m, 1H); IR (KBr) 3440, 2950, 1640,
1565,
1495, 1430, 1250, 1125, 1060, 1020, 920, 820, 695, 620, and 580 cm-'; mass
spectrum [(-) ESI], m/z 1297 (M - Na)', 1195 (M - S03Na + H - Na)', (m -
zNa)/z
637.4 (M - 2Na)2-; Anal. Calcd. for C,9H~N2O36S$Nag' 6Hz0: C, 17.65; H, 2.54;
N,
1.96, Found: C, 17.31; H, 2.13; N, 1.87.
