Benzoic acid salt of Otamixaban
FIELD OF THE INVENTION
The present invention relates to a benzoic acid salt of methyl (2R,3R)-2-{3-
[amino(imino)methyl]benzyl}-3-{[4-(1-oxidopyridin-4-yl)benzoyl]amino}butanoate, and
to a benzoic acid salt of methyl (2R,3R)-2-{3-[amino(imino)methyl]benzyl}-3-{[4-(1-
oxidopyridin-4-yl)benzoyl]amino}butanoate which is in a crystalline form or in at least
partially crystalline form, as well as a process for the preparation of the same, methods
of using such salt to treat subjects suffering from conditions which can be ameliorated
by the administration of an inhibitor of Factor Xa and shows the structure illustrated in
Formula I :
Formula I
BACKROUND OF THE INVENTION
Methyl (2R,3R)-2-{3-[amino(imino)methyl]benzyl}-3-{[4-(1 -oxidopyridin-4-
yl)benzoyl]amino}butanoate, (CAS number 193153-04-7) has the International
Nonproprietary Name Otamixaban and shows the structure illustrated in Formula II:
Formula I I
Methyl (2R,3R)-2-{3-[amino(imino)methyl]benzyl}-3-{[4-(1 -oxidopyridin-4-
yl)benzoyl]amino}butanoate use in the preparation of a medicament for treating a
patient suffering from, or subject to, conditions which can be ameliorated by the
administration of an inhibitor of Factor Xa has been disclosed in W097/241 18 .
Factor Xa is the penultimate enzyme in the coagulation cascade. Factor Xa
(fXa) is a critical serine protease situated at the confluence of the intrinsic and extrinsic
pathways of the blood coagulation cascade. FXa catalyses the conversion of
prothrombin to thrombin via the prothrombinase complex. Its singular role in thrombin
generation, coupled with its potentiating effects on clot formation render it an attractive
target for therapeutic intervention.
Both free factor Xa and factor Xa assembled in the prothrombinase complex (Factor
Xa, Factor Va, calcium and phospholipid) are inhibited by Otamixaban. Factor Xa
inhibition is obtained by direct complex formation between the inhibitor and the enzyme
and is therefore independent of the plasma co-factor antithrombin III. Effective factor
Xa inhibition is achieved by administering the compound either by continuous
intravenous infusion, bolus intravenous administration or any other parenteral route
such that it achieves the desired effect of preventing the factor Xa induced formation of
thrombin from prothrombin. In vivo experiments have demonstrated that Otamixaban is
highly efficacious in rodent, canine and porcine models of thrombosis. In addition,
recent clinical findings indicate that Otamixaban is efficacious, safe and well tolerated
in humans and therefore has considerable potential for the treatment of acute coronary
syndrome (K.R. Guertin and Yong-Mi Choi; 2007; Current Medicinal Chemistry, Vol.14,
No. 23; p. 2471-2481 ) . Clinical findings in a dose-ranging clinical trial indicate that
Otamixaban reduced prothrombin fragments 1 + 2 significantly more than
unfractionated heparin at the highest dose regimen (Cohen et al., Circulation, Vol. 115,
No. 20, May 2007, pages 2642-2651 ) , but said clinical findings do not show data in
comparison of age or renal impairment. Further clinical trials demonstrated that
Otamixaban induces dose-dependent, rapid direct factor Xa inhibition in patients with
stable coronary artery disease who are taking their usual comedication, some of whom
have mild renal impairment (Hinder et al., Clinical Pharmacology and Therapeutics,
Vol. 80, No. 6 , 2006, pages 691-702).
A crystalline form of methyl (2R,3R)-2-{3-[amino(imino)methyl]benzyl}-3-{[4-(1-
oxidopyridin-4-yl)benzoyl]amino}butanoate hydrochloride, 2-butanol hemisolvate is
disclosed in US 7,034,160. The crystalline form of (methyl (2R,3R)-2-{3-
[amino(imino)methyl]benzyl}-3-{[4-(1-oxidopyridin-4-yl)benzoyl]amino}butanoate
hydrochloride, 2-butanol hemisolvate contains 2-butanol as a solvate which is not
favourable for use in solid pharmaceutical compositions and is a hygroscopic
compound.
Hygroscopicity is the ability of a substance to attract and hold water molecules from
the surrounding environment through either absorption or adsorption with the
adsorbing or absorbing material becoming physically 'changed,' somewhat, increase in
volume, stickiness, or other physical characteristic changes of the material as water
molecules become 'suspended' between the material's molecules in the process.
Therefore hygroscopic compounds are generally very unfavorable for use in solid
pharmaceutical compositions.
It is an object of the present invention to find a salt of methyl (2R,3R)-2-{3-
[amino(imino)methyl]benzyl}-3-{[4-(1-oxidopyridin-4-yl)benzoyl]amino}butanoate with
reduced absorption or adsorption of water molecules from the surrounding
environment. It has been found that a benzoic acid salt of methyl (2H,3R)-2-{3-
[amino(imino)methyl]benzyl}-3-{[4-(1-oxidopyridin-4-yl)benzoyl]amino}butanoate has
favorable reduced absorption or adsorption of water molecules from the surrounding
environment.
SUMMARY OF THE PRESENT INVENTION
In one embodiment the present invention relates to a benzoic acid salt of methyl
(2R,3R)-2-{3-[amino(imino)methyl]benzyl}-3-{[4-(1-oxidopyridin-4-
yl)benzoyl]amino}butanoate and shows the structure illustrated in Formula I:
Formula I
In another embodiment the invention relates to a benzoic acid salt of methyl
(2R,3R)-2-{3-[amino(imino)methyl]benzyl}-3-{[4-(1-oxidopyridin-4-yl)benzoyl]amino}
butanoate, which is in a crystalline form or in at least partially crystalline form.
DETAILED DESCRIPTION OF THE INVENTION
In one embodiment the present invention relates to a benzoic acid salt of methyl
(2R,3R)-2-{3-[amino(imino)methyl]benzyl}-3-{[4-(1-oxidopyridin-4-yl)benzoyl]amino}
butanoate and shows the structure illustrated in Formula I .
In another embodiment the invention relates to a benzoic acid salt of methyl
(2R,3f?)-2-{3-[amino(imino)methyl]benzyl}-3-{[4-(1-oxidopyridin-4-yl)benzoyl]amino}
butanoate, which is in a crystalline form or in at least partially crystalline form.
Polymorphism is the ability of a single compound to exist in more than one form
or crystal structure. Different polymorphs represent distinct solids sharing the same
molecular formula, yet each polymorph may have distinct physical properties. A single
compound may give rise to a variety of polymorphic forms wherein each form may
have different and distinct physical properties, such as different solubility profiles,
different thermodynamic stability, different crystallization behavior, different filterability,
different melting point temperatures and/or different X-ray diffraction peaks. The
difference in the physical properties of different polymorphic forms results from
different orientation and intermolecular interactions of adjacent molecules in the solid.
Polymorphic forms of a compound can be distinguished by X-ray diffraction and by
other methods such as, infrared spectroscopy or Raman spectroscopy.
"Amorphous" means a solid that exhibits in an X-ray powder diffraction pattern
measured in transmission with CUKOH radiation at room temperature no characteristic
reflections at degrees 2 theta which can be separated from each other by their
diffraction angle oder specific degree 2 theta.
In another embodiment the invention relates to a crystalline benzoic acid salt of
Formula I wherein the crystalline salt exhibits in an X-ray powder diffraction pattern
measured in transmission with CUKOH radiation at room temperature a characteristic
reflection at degrees 2 theta of 19.8, 18.8 and 7.9, each time ± 0.2 degrees 2 theta.
In another embodiment the invention relates to a crystalline benzoic acid salt of
Formula I wherein the crystalline salt exhibits in an X-ray powder diffraction pattern
measured in transmission with CuKoh radiation at room temperature a characteristic
reflection at degrees 2 theta of 22.0, 19.8, 8.8, 17.9, 15.7 and 13.6 each time ± 0.2
degrees 2 theta.
The selection of characteristic reflections was determined by the number of reflections
at a specified degree 2 theta.
In another embodiment the crystalline benzoic acid salt of Formula I may also be
characterized by its X-ray powder diffraction pattern substantially by the one shown in
Figure 1, which has been obtained using CUKOH radiation in transmission mode,
wherein the intensities of the reflections depicted in the Figure as well as those of the
reflections specified above are not a prerequisite, but may vary.
The crystalline benzoic acid salt of formula I may also be characterized by its crystal
lattice parameters which have been determined by indexing its powder pattern. The
crystalline salt of formula I crystallizes in the orthorhombic crystal system with
a = 33.524 A, b = 7.928 A, c = 9.896 A, volume = 5947 A3.
Moreover, crystalline benzoic acid salt of Form ula I may also be characterized by its
dynam ic vapor sorption (DVS) water vapor sorption and desorption isotherms
measured at 25°C. Before starting the sorption cycle the crystall ine salt of Formula I
sample is treated with dry nitrogen gas. As shown in the examples the sorption and
desorption isotherms are almost the same wherein a moderate water uptake of 1.1%
at 80% room hum idity (RH) and 2.0% at 95% RH takes place.
Description of the Figures
Fig. 1 - X-ray powder diffraction pattern of crystalline benzoic acid salt of methyl
(2 ,3R)-2-{3-[amino(imino)methyl]benzyl}-3-{[4-(1 -oxidopyridin-4-yl)benzoyl]amino}
butanoate, measured in transm ission mode with CUKCH radiation at room temperature
(x-axis: diffraction angle 2theta (2Q) [°]; y-axis: relative intensity
The benzoic acid salt of methyl (2R,3R)-2-{3-[am ino(im ino)methyl]benzyl}-3-{[4-
( 1-oxidopyridin-4-yl)benzoyl]amino}butanoate may also be prepared by dissolving the
methyl (2R, 3R)-2-{3-[amino(im ino)methyl]benzyl}-3-{[4-(1 -oxidopyridin-4-
yl)benzoyl]am ino}butanoate in aqueous or aqueous-alcohol solution or other suitable
solvents to which the benzoic acid or a salt of benzoic acid is added. Under stirring, the
mixture can be heated to 65°C yielding a clear solution and subsequently cooling
overnight yields a precipitate. The precipitate obtained can be filtered, washed with
water and dried under reduced pressure.
In general, the crystalline benzoic acid salt of methyl (2R,3R)-2-{3-
[amino(im ino)methyl] benzyl}-3-{[4-(1 -oxidopyridin-4-yl)benzoyl]am ino}butanoate of the
present invention (in the following benzoic salt of formula I) can be obtained by
crystallizing or recrystallizing compound of Formula I , starting from a solution of
compound of Formula I or from a suspension of compound of Formula I or from solid
compound of Formula I. A solution of com pound of Form ula I, or a suspension of
compound of Formula I, may have been obtained at the end of the chem ical synthesis
of com pound of Formula I, or it may have been obtained by dissolving or suspending
previously synthesized crude compound of Form ula I . The term "crude compound of
Form ula I" comprises any form of compound of Form ula I , e.g . the material directly
obtained from chemical synthesis, a distinct crystalline form or amorphous material of
the compound of Formula I .
More specifically, the crystalline salt of Formula I of the invention can be obtained by
(a) providing a solution or suspension of compound of Formula I , for example by
dissolving or suspending crude compound Formula I in a suitable solvent such as an
alcohol, e.g. methanol, ethanol, 2-propanol; wherein a solution of compound of
Formula I generally is a clear solution and may optionally have been filtered,
(b) maintaining, heating, cooling and/or concentrating the solution or suspension
and/or adding one or more further solvents, with or without agitation such as stirring, to
form crystals of a desired distinct crystalline form or solvate or to allow the formation of
a desired distinct crystalline form or solvate, and
(c) isolating the distinct crystalline salt of Formula I .
The processes for preparing crystalline forms and solvates of compound of Formula I
can be performed with conventional equipment and according to standard procedures.
For example, concentrating of a solution or suspension in step (b) may be done by
distilling off solvent partially or totally at atmospheric pressure or at reduced pressure.
Isolating of a crystalline form or solvate in step (c) may be done by any conventional
technique such as filtration or vacuum filtration or centrifugation. Isolating may also
comprise drying, e.g. by applying elevated temperatures and/or reduced pressure, for
example at moderately reduced pressure at about room temperature, i.e. a
temperature of about 18 °C to about 65 °C, for example about 20 °C, or at about 65 °C.
In a preferred embodiment, the solution or suspension may be seeded in step (a) or
step (b) to promote crystallization. Seeding is preferably done with a small amount of
the crystalline salt of Formula I already prepared.
The benzoic acid salt of methyl (2F?,3R)-2-{3-[amino(imino)methyl]benzyl}-3-{[4-
( 1-oxidopyridin-4-yl)benzoyl]amino}butanoate of the present invention (in the following
benzoic salt of formula I) may be useful in inhibiting Factor Xa. Accordingly, the
present invention provides methods for the treatment or prevention of a pathological
condition that may be capable of being modulated by inhibiting production of Factor
Xa.
Examples of pathological conditions that may be capable of being treated with
the benzoic salt of formula I of the present invention include, for example, acute
myocardial infarction (AMI), non-ST elevation myocardial infarction, unstable angina,
thromboembolism, acute vessel closure associated with thrombolytic therapy,
percutaneous transluminal coronary angioplasty (PTCA), transient ischemic attacks,
stroke, intermittent claudication, and restenosis.
The benzoic salt of formula I described herein thus may be useful for, inter alia,
inhibiting blood coagulation by virtue of their general ability to inhibit the penultimate
enzyme in the coagulation cascade, Factor Xa, rather than thrombin. Benzoic salt of
formula I within the scope of the present invention may exhibit marked
pharmacological activities according to tests described in the literature, including in
vivo tests and in vitro tests, the latter of which are believed to correlate to
pharmacological activity in humans and other mammals. For example, both free
Factor Xa and Factor Xa assembled in the prothrombinase complex (Factor Xa, Factor
Va, calcium and phospholipid) may be inhibited. Factor Xa inhibition may be obtained
by direct complex formation between the inhibitor and the enzyme and is therefore
independent of the plasma co-factor antithrombin III. Effective Factor Xa inhibition
may be achieved by administering the benzoic salt of formula I according to the
invention by continuous intravenous infusion, bolus intravenous administration or any
other suitable route such that it may achieve the desired effect of preventing the Factor
Xa induced formation of thrombin from prothrombin.
In addition to their use in anticoagulant therapy, Factor Xa inhibitors may be
useful in the treatment or prevention of other diseases in which the generation of
thrombin may play a pathologic role. For example, thrombin has been proposed to
contribute to the morbidity and mortality of such chronic and degenerative diseases as
arthritis, cancer, atherosclerosis and Alzheimer's disease by virtue of its ability to
regulate many different cell types through specific cleavage and activation of a cell
surface thrombin receptor. Inhibition of Factor Xa may effectively block thrombin
generation and therefore neutralize any pathologic effects of thrombin on various cell
types.
The methods preferably comprise administering to a patient a pharmaceutically
effective amount of the benzoic salt of formula I of the present invention, preferably in
combination with one or more pharmaceutically acceptable carriers or excipients. The
relative proportions of pharmaceutical compositions and carrier and/or excipient may
be determined, for example, by the solubility and chemical nature of the materials,
chosen route of administration and standard pharmaceutical practice.
The dosage of the benzoic salt of formula I that will be most suitable for
prophylaxis or treatment may vary with the form of administration, the particular novel
form of the compound chosen and the physiological characteristics of the particular
patient under treatment. Broadly, small dosages may be used initially and, if
necessary, increased by small increments until the desired effect under the
circumstances is reached.
Generally speaking, in the adult, suitable doses may range from about 0.01 to
about 100 mg/Kg body weight, and all combinations and subcombinations of ranges
and specific doses therein. Preferred doses may be from about 0.01 to about 10
mg/kg body weight per day by inhalation, from about 0.01 to about 100, preferably 0.1
to 70, more preferably 0.5 to 10 mg/Kg body weight per day by oral administration, and
from about 0.01 to about 50, preferably 0.01 to 10 mg/Kg body weight per day by
intravenous administration. In each particular case, the doses may be determined in
accordance with the factors distinctive to the subject to be treated, such as age,
weight, general state of health and other characteristics which can influence the
efficacy of the medicinal product.
The benzoic salt of formula I according to the invention may be administered as
frequently as necessary to obtain the desired therapeutic effect. Some patients may
respond rapidly to a higher or lower dose and may find much weaker maintenance
doses adequate. For other patients, it may be necessary to have long-term treatments
at the rate of about 1 to about 4 doses per day, in accordance with the physiological
requirements of each particular patient. Generally, the active product may be
administered orally about 1 to about 4 times per day. It goes without saying that, for
other patients, it may be necessary to prescribe not more than one or two doses per
day.
The benzoic salt of formula I of this invention may be administered in oral
dosage forms such as tablets, capsules (each of which includes sustained release or
timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions,
syrups, and emulsions. Solid dosage forms (pharmaceutical compositions) suitable for
administration may generally contain from about 1 mg to about 1000 mg of the benzoic
salt of formula I per dosage unit.
For oral administration in solid form such as a tablet or capsule, the benzoic salt
of formula I can be combined with a non-toxic, pharmaceutically acceptable inert
carrier, such as lactose, starch, sucrose, glucose, methylcellulose, magnesium
stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like.
Preferably, in addition to the active ingredient, solid dosage forms may contain a
number of additional ingredients referred to herein as "excipients." These excipients
include, among others, diluents, binders, lubricants, glidants and disintegrants.
Coloring agents may also be incorporated. "Diluents" as used herein, refers to agents
which may impart bulk to the formulation to make a tablet a practical size for
compression. Examples of diluents are lactose and cellulose. "Binders" as used
herein, refers to agents that may be used to impart cohesive qualities to the powered
material to help ensure the tablet remains intact after compression, as well as to
improve the free-flowing qualities of the powder. Examples of typical binders include
lactose, starch and various sugars. "Lubricants", as used herein, have several
functions including preventing the adhesion of the tablets to the compression
equipment and improving the flow of the granulation prior to compression or
encapsulation. Lubricants are in most cases hydrophobic materials. Excessive use of
lubricants is undesired, however, as it may can result in a formulation with reduced
disintegration and/or delayed dissolution of the drug substance. "Glidants", as used
herein, refers to substances which may improve the flow characteristics of the
granulation material. Examples of glidants include talc and colloidal silicon dioxide.
"Disintegrants", as used herein, refer to substances or mixtures of substances added
to a formulation to facilitate the breakup or disintegration of the solid dosage form after
administration. Materials that may serve as disintegrants include starches, clays,
celluloses, algins, gums and cross-linked polymers. A group of disintegrants referred
to as "super-disintegrants" generally are used at a low level in the solid dosage form,
typically 1% to 10% by weight relative to the total weight of the dosage unit.
Croscarmelose, crospovidone and sodium starch glycolate represent examples of a
cross-linked cellulose, a cross-linked polymer and a cross-linked starch, respectively.
Sodium starch glycolate swells seven- to twelve-fold in less than 30 seconds
effectively disintegrating the granulations that contain it.
As would be apparent to a person of ordinary skill in the art, once armed with
the teachings of the present disclosure, when dissolved, crystalline benzoic salt of
formula I loses its crystalline structure, and is therefore considered to be a solution of
benzoic salt of Formula I . All forms of the present invention, however, may be used for
the preparation of liquid formulations in which crystalline benzoic salt of formula I may
be, for example, dissolved or suspended. In addition, the crystalline benzoic salt of
formula I may be incorporated into solid formulations.
The following non-limiting examples illustrate the inventors' preferred methods for
preparing and using the benzoic acid salt of formula I of the present invention.
EXAMPLES
Exam le 1 Preparation of Compound (III)
3 C H C0 H ( I
water/toluene
TsOH is p-Toluenesulfonic acid with the formula CH3C6H4SO3H. TsOH refers to the
monohydrate. To a reactor were charged Compound (Ma) (100.0 g) and anhydrous
tetrahydrofuran (THF) (320 g). The resulting suspension was cooled down to -20 ±
3 °C and lithium hexamethyldisilazide (LiHMDS) (475.6 grams, 1.3 M solution in THF)
was added over 55 minutes and stirred for 20 minutes at -20 ± 3 °C. A solution of abromo-
m-tolunitrile in THF (65.1 g in 181 g of THF) was then charged into the reactor
over 40 minutes while maintaining the temperature at -20 ± 3 °C and stirred for another
30 minutes. Benzoic acid (126.6 grams) was charged as a solid to the reactor. Water
(1000 g) was then added and mixture distilled at a 65 ± 3 °C jacket temperature and
200-233 mbar vacuum. After distilling to a constant pot temperature of 57 °C and
constant head temperature of 45 °C, the distillation was stopped. Toluene (432 g) was
added to the hot solution and stirred while cooling down to 10 + 2 °C. The resulting
suspension was then filtered and the filter cake washed with water (250 grams) and
toluene (432 grams). Compound (III) was dried at 45-50 °C at -350 mbar vacuum
under a nitrogen stream for 24 hours until constant weight. The isolated solid weighed
76.0 grams (62.0 % yield).
Example 2 Preparation of Compound (V)
Compound (III) was partitioned between dichloromethane and aqueous sodium
carbonate. The organic phase (containing the free base of (III)) was washed with
additional aqueous sodium carbonate and was distilled under reduced pressure and
solvent exchanged with dimethylformamide (DMF). This solution was assayed for
wt/wt content of (III). To a suspension of (IV) ( .0 equivalent vs. (Ill)) in DMF were
added 2 equivalents of 4-methylmorpholine and 1. 1 equivalents of O-Benztriazol-1-yl-
L, ,L ' , '-tetramethyluronium tetrafluoroborate (TBTU). This mixture was stirred at
ambient temperature until ester activation was complete (about 90 minutes). The DMF
solution of Compound (III) ( 1 equivalent) was added and the resulting solution stirred
overnight after which HPLC indicated that the reaction was complete. Water was
added at 75 °C and the mixture was cooled to crystallize the product. The mixture was
cooled to 5 °C, filtered, and the filter cake was washed with water. The product was
dried under reduced pressure at 70 °C.
Example 3 Preparation of Compound (VI)
In a well-stirred reactor, 45g of Compound (V) in 450 mL dichloromethane was
reacted for at least 5 hours with 6 1 g of magnesium monoperoxyphthalate (66.4%
based on available oxygen, 1.5 eq.) in 450 g of water until the reaction was complete.
The phases were separated and the organic phase was washed successively with
equal volumes of water, a 5% aqueous sodium bicarbonate solution, and water. The
resulting solution was concentrated to an approximately 40 wt% solution and diluted
with 180 g of methyl isobutyl ketone (MIBK). Further distillation to remove residual
dichloromethane, seeding with appropriate crystals, and cooling gave the product as a
crystalline solid. The crystals were filtered, rinsed with 30 g of MIBK, and dried at 50
°C under reduced pressure to give 4 1.8 g of Compound (VI) (89.3% yield).
Example 4 Preparation of Compound (VII)
To a 200-mL jacketed reaction flask were charged Compound (VI) (50.0 g , 116
mmol) and methanol (50 mL). This mixture was cooled to -5 °C and sealed after
establishing a partial vacuum (about 100 torr). Anhydrous HCI (52.2 g , .43 mol) was
added while maintaining the reaction temperature at less than 0 °C. The reaction was
stirred at 0 ± 1 °C under closed conditions. After 16 hours, the reaction was complete
(less than 2 A% (VI) by HPLC). To the intermediate product solution was added
anhydrous methanol ( 00 mL) while maintaining the temperature at less than
5 °C. The solution was treated with NH3 (27.7 g , 1.62 mol) keeping the temperature
less than 0 °C. Before allowing the mixture to warm to room temperature, a pH check
was made of an aliquot dissolved in distilled water (a pH of 8-10 indicates a sufficient
charge of ammonia). The reaction was stirred at 20 °C overnight at which point the
reaction was complete.
Example 5 Preparation of Compound (VIII) by solvent addition
To the ammonium chloride slurry from Example 4 was added 2-butanol
(840 mL), and the resulting mixture was stirred for 1 hour while warming to 70 °C. The
ammonium chloride was removed by hot filtration and the cake was washed with a
solution of 20 ml_ methanol in 160 ml_ 2-butanol. The filtrates were combined and 0.5
g of seed crystals were added. The mixture was allowed to stir overnight at ambient
temperature. The slurry was cooled to - 5 °C and held for 2 hours to ensure complete
crystallization. The solid was filtered and the reactor and cake were washed with 165
mL of 2-butanol. The solid was dried under reduced pressure at 45°C to 50 C with a
nitrogen bleed giving 44.3 g (73.2%) of Compound (VIII) as an off-white crystalline
solid.
Example 6 Preparation of benzoic acid salt
The material was prepared by dissolving 4 g of the compound prepared in Example 5
(the monohydrochloride - hemi-2-butanol-solvate) in 80 ml of hot water and adding
1. 1 1 g of solid sodium benzoate. Under stirring, the mixture was cooled overnight. The
precipitate obtained was filtered, washed with water and dried under reduced pressure
at 40°C for 6 hours. The yield amounted to 79.4 %.
NMR data confirmed the received salt as a :1 ratio of benzoic acid and methyl
(2f?,3R)-2-{3-[amino(imino) methyl]benzyl}-3-{[4-(1-oxidopyridin-4-yl)benzoyl]amino}
butanoate and shows the structure illustrated in the following schema:
Calibration H
DMSO = 2.50 ppm DMSO = 39.476 ppm
DMSO means dimethyl sulfoxide
For a complete assignment of NMR chemical shifts see Tab. 1.
Tab. 1: NMR chemical shifts of the benzoic acid salt of methyl (2ft,3R)-2-{3-
[amino(imino) methyl]benzyl}-3-{[4-(1-oxidopyridin-4-yl)benzoyl]amino} butanoate,
c = 4.5 mg in 600 m I DMSO at 300 °K.
Alternatively to a solution of 5.0 g of the compound prepared in Example 4 in 100 mL
of water was added 2.2 g of sodium benzoate. The mixture was heated on a steam
bath until homogeneous. Charcoal (approximately 2 g) was added and this mixture
was filtered through celite® washing with 20 mL of water. Crystallization began
immediately. After cooling for 2 h , the solids were collected and washed with water.
After drying the solids for 3 days in a vacuum oven at 50 °C, 3.9 g (67%) was
collected. H NMR confirmed the salt as a 1:1 ratio of benzoic acid and methyl
(2fi,3R)-2-{3-[amino(imino)methyl]benzy
butanoate.
Example 7 Differential scanning calorimetry (DSC)
The DSC measurement was performed with a Mettler DSC822e (module
DSC822e/700/1 09/414935/0025). 40m I_Aluminum-crucibles with sealed lid and pinhole
were used. All measurements were carried out in a nitrogen gas flow of 50mL/min and
a heating rate of 107min. The measured data was evaluated via the software STARe
V8.10.
The crystalline form used was prepared as in Example 6 and showed during heating
from 25 °C to 300 °C an onset peak at 216.01 °C and a peak at 221 .15 °C.
Example 8 Preparation of a crystalline form
A crystalline form was prepared by rapid cooling of a solution of 0.190 g benzoic acid
salt of formula I as prepared in Example 6 in 2 mL ethanol from 65°C to 0°C. Since no
precipitation occurred, the sample was left over night at 0 C. The precipitate was
isolated by vacuum filtration and then dried under reduced pressure at 25°C.
Alternatively a crystalline form was prepared by controlled cooling of a stirred solution
of 0.200 g benzoic acid salt of formula I as prepared in Example 6 in 5.0 mL ethanol
from 65°C to 10°C in 18 h . The precipitate was isolated by vacuum filtration.
Alternatively, a crystalline form was prepared by controlled cooling of a stirred solution
of 0.201 g benzoic acid salt of formula I as prepared in Example 6 in 15 mL 2-propanol
and 1.0 mL water from 65°C to 10°C in 18 h . Since no precipitation occurred, the
solvent was allowed to evaporate at 65°C.
Alternatively, a crystalline form was prepared by dissolving 0.217 g of benzoic acid salt
of formula I as prepared in Example 6 in 5.0 mL of ethanol at about 65°C. The solvent
was then allowed to evaporate from the stirred solution at the same temperature over
night. The solid residue was dried at room temperature under reduced pressure.
Alternatively, a crystalline form was prepared by dissolving 0 .197 g of benzoic acid salt
of formula I as prepared in Example 6 in 10 ml of 2-propanol and 1.0 L water at
about 65°C. The solvent was then allowed to evaporate from the stirred solution at the
same temperature over night. The solid residue was dried at room temperature under
reduced pressure.
Example 9 Dynamic vapor sorption (DVS)
Moisture sorption/desorption isotherms were recorded on a DVS-1000 from Surface
Measurement Systems. Two cycles were run at 25 °C, in which the sample was first
treated with dry nitrogen gas and then the relative humidity was stepwise increased
from 0 to 95% and subsequently decreased again back to 0% and the weight of the
sample was measured. Typical total measurement times for both cycles were about 20
to 30 hours.
The measured data for the benzoic acid salt of methyl (2R,3R)-2-{3-[amino(imino)
methyl] benzyl}-3-{[4-(1-oxidopyridin-4-yl)benzoyl]amino}butanoate as prepared in
Example 8 are shown in the following table 2 .
Table 2 : DVS Isotherm at 24.9 °C
Target Change In Mass (%)
RH (%) Sorption Desorption Hysteresis
Cycle 1 0.0 0.000 -0.035
10.0 0.096 0.215 0.1 19
20.0 0.180 0.326 0.146
40.0 0.363 0.534 0.172
60.0 0.596 0.810 0.214
80.0 0.926 1.300 0.374
90.0 1.408 1.697 0.290
95.0 2.031 2.031
Cycle 2 0.0 -0.035 -0.056
10.0 0.109 0.1 98 0.089
20.0 0.226 0.310 0.084
40.0 0.461 0.523 0.062
60.0 0.742 0.800 0.058
80.0 1.241 1.299 0.058
90.0 1.61 6 1.687 0.071
95.0 1.999 1.999
"RH" means relative hum idity; the relative hum idity of an air-water mixture is defined as
the ratio of the partial pressure of water vapor in the mixture to the saturated vapor
pressure of water at a prescribed tem perature.
DVS shows a moderate water uptake of 1.1% at 80% RH and 2.0% at 95% RH for the
benzoic acid salt of methyl (2R,3R)-2-{3-[amino(im ino)methyl]benzyl}-3-{[4-(1 -
oxidopyridin-4-yl)benzoyl]amino}butanoate.
DVS Comparison Experiment:
Methyl (2R,3R)-2-{3-[amino(im ino)methyl]benzyl}-3-{[4-(1 -oxidopyridin-4-
yl)benzoyl]am ino}butanoate monohydrochloride - hemi-2-butanol-solvate as prepared
in Example 5 .
The measured data for methyl (2R,3f?)-2-{3-[amino(imino)methyl]benzyl}-3-{[4-(1 -
oxidopyridin-4-yl)benzoyl]amino}butanoate monohydrochloride - hemi-2-butanolsolvate
as prepared in Exam ple 5 are shown in the following table 3 .
Table 3 : DVS Isotherm at 25.5 °C
Change In Mass
Target (%)
RH (%) Sorption Desorption Hysteresis
Cycle 1 0.0 0.00 0.35
10.0 0.79 3.33 2.54
20.0 1.28 5.29 4.01
40.0 2.22 7 .14 4.91
60.0 3.69 9.78 6.09
80.0 17.79 16.92 -0.87
90.0 26.66 26.07 -0.59
95.0 34. 13 34. 13
Cycle 2 0.0 0.35 0.34
10.0 0.69 3 .17 2.48
20.0 1.32 4.97 3.65
40.0 2.67 6.78 4 .11
60.0 4.57 9.41 4.84
80.0 13.43 16.37 2.94
90.0 23.91 25.32 1.41
95.0 32.86 32.86
DVS shows a strong water uptake of 3.69% at 60% RH, 1 .79% at 80% RH, 26.66%
at 90% RH and 34. 13% at 95% RH for methyl (2R3R)-2-{3-[amino(imino)methyl]
benzyl}-3-{[4-( 1-oxidopyridin-4-yl)benzoyl]am ino}butanoate monohydrochloride - hemi-
2-butanol-solvate.
Exam ple 10 Differential scanning calorimetry (DSC)
The DSC measurement was performed with a Mettler DSC822e (module
DSC822e/700/1 09/41 4935/0025). 40m I_Aluminum-crucibles with sealed lid and pinhole
were used. All measurements were carried out in a nitrogen gas flow of 50m l_/m in and
a heating rate of 10 in. The measured data was evaluated via the software STARe
V8. 10 .
The crystalline form used was prepared as in Exam ple 8 and showed during heating
from 25 °C to 300 °C an onset peak at 225. 57 °C and a peak at 229.02 °C.
Exam ple 11 X-ray powder diffraction (XRPD)
X-ray powder diffraction was performed with Stoe Stadi-P transm ission diffractometer
using CuKod radiation (wavelength is 1.54060 Angstrom) and a linear position
sensitive detectors. Unless stated otherwise, X-ray powder diffraction was performed
at room temperature. Samples were investigated in flat preparation. The measured
data were evaluated and plotted with the Software WinXPOW V2. 12 .
The observed X-ray powder diffraction pattern of benzoic acid salt of methyl (2R,3R)-2-
{3-[amino(imino)methyl]benzyl}-3-{[4-(1 -oxidopyridin-4-yl)benzoyl]amino}butanoate as
prepared in Example 8 is displayed in the Figure 1 (Fig. 1) . The X-ray powder
diffraction pattern shown in the Figure was background-subtracted.
The 2Q (2theta) angles in ° (degree) are specified as the number of characteristic
reflections. The 2theta angles in degree have the following values in Fig. 1 and the
relative intensities are shown in brackets:
5.26 (20), 5.61 (15), 7.20 (17), 9.31 (18), 10.23 (16), 10.40 (15)
10.53 (15) 11.51 (14) 11.62 (16), 12.91 (16), 13.58 (23), 14.33 (14)
15.05 ( 1 1) 15.74 (26) 16.51 (9), 17.01 ( 1 1) , 17.53 (12), 17.93 (30)
18.12 (14) 18.79 (47) 19.80 (100), 20.52 (15), 20.65 (15), 2 1.20 (13)
2 1.36 (15) 2 1.99 (21 ) 22.32 ( 1 1) , 22.86 (16), 23.16 (9), 23.51 (13)
23.95 (14) 24.44 (1 1) 24.96 (13), 25.39 (7), 25.87 (10), 25.97 (10)
26.34 (7), 26.58 (6), 26.94 (12), 27.36 (9), 27.57 (8), 28.67 (6),
28.88 (7), 29.94 (6), 30.91 (13), 3 1.39 (6), 3 1.88 (5), 32.25 (4),
32.72 (4), 33.77 (5),
10
Claims
1 Benzoic acid salt of methyl (2R, 3R)-2-{3-[am ino(im ino)methyl]benzyl}-3-{[4-( 1-
oxidopyridin-4-yl)benzoyl]am ino}butanoate.
2 Benzoic acid salt according to claim 1 which is in a crystalline form or in at least
partially crystall ine form.
3 Benzoic acid salt according to claim 2 wherein the crystalline salt exhibits in an
X-ray powder diffraction pattern measured in transmission with CuKai radiation
at room temperature a characteristic reflection at degrees 2 theta of 19.8, 18.8
and 7.9, each time ± 0.2 degrees 2 theta.
4 Benzoic acid salt according to claim 2 wherein the crystalline salt exhibits in an
X-ray powder diffraction pattern measured in transmission with CuKai radiation
at room temperature a characteristic reflection at degrees 2 theta of 22.0, 19.8,
8.8, 17.9, 15.7 and 3.6 each time ± 0.2 degrees 2 theta.
5 Benzoic acid salt according to claim 2 , which is characterized by an x-ray
powder diffraction pattern substantially in accordance with that shown in Figure
1.
6 Process for the preparation of the benzoic acid salt according to claim ,
comprising dissolving methyl (2R,3R)-2-{3-[amino(im ino)methyl]benzyl}-3-{[4-( 1-
oxidopyridin-4-yl)benzoyl]am ino}butanoate in aqueous or aqueous-alcohol
solution and adding benzoic acid or sodium benzoate.
7 Benzoic acid salt according to claims 1 to 5 for use as a medicament.
8 A pharmaceutical composition comprising a benzoic acid salt of methyl (2R,3R)-
2-{3-[amino(im ino)methyl]benzyl}-3-{[4-(1 -oxidopyridin-4-yl)benzoyl]am ino}
butanoate as claimed in claims 1 to 5 and a pharmaceutically acceptable carrier
or excipient.
The benzoic acid salt of methyl (2R,3fi)-2-{3-[amino(imino)methyl]benzyl}-3-{[4-
(1-oxidopyridin-4-yl)benzoyl]amino}butanoate according to claims 1 to 5 for use
in the treatment of acute myocardial infarction, non-ST elevation myocardial
infarction, unstable angina, thromboembolism, acute vessel closure associated
with thrombolytic therapy, percutaneous transluminal coronary angioplasty,
transient ischemic attacks, stroke, intermittent claudication, and restenosis.