Title of Invention

N-[ 2-HYDROXY-3-(1-PIPERIDINYL) -PROPOXY ] -PYRIDINE-1-OXIDE-3-CARBOXIMIDOYL CHLORIDE

Abstract The invention relates to N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1- oxide-3-carboximidoyl chloride, its stereoisomers, and the acid addition salts thereof, pharmaceutical composition containing the same, the use of these compounds in the treatment of pathological insulin resistance, and for the treatment of pathological conditions associated therewith, by simultaneous treatment and prevention of diabetes-induced chronic complications, especially retinopathy, neuropathy, nephropathy and/or pathologically decreased peripheral neuroregeneration caused by diabetes and methods of treatment.
Full Text N-[2-HYDROXY-3-(1-PIPERIDINYL)-PROPOXY]-PYRIDINE-1-
OXIDE-3-CARBOXIMDOYL CHLORIDE
Technical field of the invention
The invention relates to an O-(3-piperidino-2-hydroxy-1 -propyl)
hydroxymic acid halide derivative, the pharmaceutical use thereof and the
pharmaceutical products containing this derivative as active ingredient.
Namely, the invention relates to N-[2-hydroxy-3-(1-piperidirv/!)-propoxyj-
pyridine-1-oxide-3-carboximidoyl chloride, its stereoisomers, as well as their
acid addition salts. Furthermore, the invention also islates to the use of these
compounds in the treatment of insulin resistance and the pharmaceutical
products containing these derivatives as active ingredient.
Background of the invention
"O-(3-piperidino-2-hydroxy-1-propyl) hydroxymic acid halide derivatives
are already known from European Patent Specification No. 0 417 210 B1.
According to this patent specification, these compounds exhibit a selective beta
blocking effect and are thus suitable for the treatment of diabetic angiopathy,
more specifically, of diabetic retinopathy and nephropathy.
According to PCT Publication WO 98/06400, the 0-(3-piperidino-2-
hydroxy-1-propyl) hydroxymic acid halide derivatives and other compounds of
similar structure are effective in protecting and regenerating vascular
endothelial cells, and are thus suitable active agents for the treatment of
diseases caused by the'dysfunction of the endothelium.
The chaperon-expression increasing effect of several hydroxylamine

derivatives, among them of the 0-(3-piperidino-2-hydroxy-1-propyl) hydroxymic
acid halides, and the use of these compounds in the treatment of diseases
connected with the functioning of the chaperon system are known from
WO 97/16439. In this patent application, 0-(3-piperidino-2-hydroxy-1^ppyl)-3,
pyridyl hydroxymic acid chloride N-oxide derivatives (among others) are defined
and claimed as new compounds, however, the production procedure is only
described for piperidine-N-oxide and for the compound containing N-oxide
groups both in the piperidine and pyridine rings. The compound of the present
invention is not mentioned in the above application.
Insulin resistance is a pathological condition that blocks the effects of
insulin. It is generally associated with diabetes, although its formation is also
possible independently. Due to insulin resistance, the body needs higher and
higher concentrations of insulin for carbohydrate, lipid and protein metabolism,
which leads to an extremely high concentration of insulin. A long-lasting high
insulin concentration has been proven to be an independent cardio-cerebro-
vascular risk factor.
The reduction of insulin resistance is essential in both types of diabetes:
in case of diabetes type 2, it is present as a major etiological factor, while in
case of diabetes type 1, insulin resistance is caused by glucose toxicity as well
as excessive amounts of insulin applied exogenously for therapeutical
purposes.
Several active agents have been provided for the reduction of insulin
resistance. Among these, the most significant ones are the insulin sensitizer
products, the best known agent therefrom being troglitazone, a member of the
thiazolidine-dione group. (A.R.Saltiel et a!., Diabetes 45/12/1996 pp. 1661-
1669, and S. Kumar et al., Diabetologia 1996/39/6 pp. 701-709). The main
effect of this compound is the reduction of insulin resistance by lowering
peripheral insulin concentrations both in basal state and after glucose
stimulation. As a result, it improves carbohydrate metabolism as well as
corrects a number of pathological deviations arising as the secondary effect of
high insulin level, such as hyperlipidaemia and pathological hemostasis. Its

ultimate positive effect is the reduction of the cardiovascular risk. A
disadvantage is, however, that it may cause serious, mainly hepatotoxic side
effects therefore its application is limited and requires due caution.
Summary of the invention
During studies in the area of 0-(3-piperidino-2-hydroxy-1-propyl)
hydroxymic acid halides, detailed examination of the maleate of 0-(3-
piperidino-2-hydroxy-1-propyl)-3-pyridyne hydroxymic acid-chloride, known as
bimoclomol has been performed and found that its most significant effect is on
the pathological consequences of chronic neuropathy: it significantly improves
motoric and sensory nerve conduction velocity deficits in diabetes, and also
favorably effects pathological deviations resulting from autonomous
neuropathy. Furthermore, both in animal experiments and in phase II tests on
humans, it reduces pathological diabetic urinary albumin excretion, and in the
animal tests it reduces pathological histological and electrophysiological
alterations resulting from diabetic retinopathy. However, in the reduction of
insulin resistance bimoclomol was not effective.
At present, no medicinal products are available which could reduce
insulin resistance and at the same time effectively cure deviations resulting
from all three chronic diabetic complications.
In a search for suitable active materials, N-oxide derivatives of
bimoclomol were tested for biological acitvity. In a preliminary test the
effectiveness of the three N-oxide derivatives of bimoclomol on motor and
sensor neuropathy in STZ diabetic Wistar rats were studied. The effectiveness
of the three bimoclomol N-oxide derivatives in improving the peripheral motor
and sensor nerve conduction velocity deficit caused by streptozotocine-induced
diabetes was determined with use of the method described in detail in
Experiment 2. The results are summarized in the following table.


As it appears from the above results, the pyridine N-oxide derivative of
bimoclomol is equivalent with bimoclomol while the two other N-oxide
derivatives have significantly weaker effect on the motor and sensor
neuropathy. Based on this experience, investigations were continued with the
pyridine N-oxide derivative of bimoclomol, namely N-[2-hydroxy-3-(1-
piperidinyl)-1-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride.
Our investigations yielded the unexpected result that N-[2-hydroxy-3-(1-
piparidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride reduces
peripheral insulin resistance in addition to exhibiting an effect equal to or in
some cases greater than that of bimoclomol in the treatment of the above
mentioned three main diabetic complications. Due to this characteristic, the
compound is suitable for the treatment of chronic diabetic complications,
especially of retinopathy, neuropathy and nephropathy, and for the
simultaneous reduction of peripheral insulin resistance, but it is also suitable for
the treatment of non-diabetic pathological insulin resistance and any
pathological conditions related to it.

The favorable biological properties of N-[2-hydroxy-3-(1-piperidinyl)-
propoxy]-pyridine-1-oxide-3-carboximidoyl chloride were proven by the following
experiments. For these tests, the maleate of N-[2-hydroxy-3-(1-piperidinyl)-
propoxy]-pyridine-1-oxide-3-carboximidoyl chloride or the maleate of the
suitable optically active compound were used as test compounds. In the
description of the experiments, the maleate of the racemic compound is
referred to as Compound A, while the maleate of the optically active
stereoisomer is always specifically indicated.
Experiment 1
Effect of Treatment by Compound A and Bimoclomol on Carbohydrate
Metabolism in Obese, Insulin Resistant, Hyperinsulinaemic Zucker fa/fa Rats
with Impaired Glucose Tolerance, Following a 2-Month Treatment
Materials and methods:
In the experiments so-called Zucker fa/fa rats (Charles River Laboratories Inc.)
were used. In monozygotic animals, obesity, insulin resistance, high blood
insulin level, impaired glucose tolerance and hypertriglyceridaemia result from
hypothalamic leptin receptor mutation. Due to the above characteristics, this is
an accepted model of early type 2 diabetes.
The animals were placed in individual metabolic cages for 24 hours at
the beginning of the study, and after 1 or 2 months of treatment for 24 hours of
urine collection.
The animals were given either the test substances or physiological saline
solution by gavage for control once a day between weeks 14-22.
Biochemical parameters of the blood and the urine were measured by a
Kodak Ectachem 700 automatic analyzer. The total protein level of the urine
was determined spectrophotometrically with the use of Bradford staining
(Hitachi U-3200) at 595 nm. The insulin concentration of the serum was
determined by the RIA method, using rat anti-insulin antibodies.

Systolic, diastolic blood pressure and heart rate were measured weekly
on the tails of the rats (so-called tail cuff method) by a Letica 200 automatic
analyzer. After two months of treatment, the level of glucose tolerance was
determined by the intraperitoneal glucose tolerance test (2 g/kg ip.).
Results:
Bimoclomol administered in daily doses of 20 mg/kg p.os significantly reduced
the level of fasting blood glucose, however, it did not effect the fasting insulin
concentration.
In comparison with previous data, the unexpected outcome was that
Compound A administered in daily doses of 20 mg/kg p.os significantly reduced
both fasting blood glucose and insulin concentrations, the latter by approx.
50%. The results are given in Table 1 and Figure 1.

In the course of the intraperitoneal glucose tolerance test, neither bimoclomol,
nor Compound A effected the areas under the blood glucose curve (AUC).
However, in case of the AUC of insulin there is a difference between the two
compounds: bimoclomol had- no effect, while Compound A reduced it

significantly, to the same level as the Lean control. The results are given in
Table 2 and Figure 2.
The table contains the values of the area under the curve (AUC) between 0-60
minutes.

Summary:
Compound A significantly reduces peripheral insulin resistance, while
bimoclomol does not.
Experiment 2
The Effect of Treatment by Compound A and Bimoclomol on the Pathological
Deviations Resulting from Peripheral Neuropathy in STZ-Diabetic Wistar Rats,
Following a 1-Month Treatment.
Materials and methods:
In the experiments, male Wistar rats were used (Charles River Laboratories

Inc.). At the start of the experiment, their weights were 340-370 g. Diabetes was
induced by the intravenous administration of a single 45 mg/kg dose of
streptozotocine (STZ, Sigma, St. Louis, MO) dissolved in physiological saline
solution. The development of diabetes was checked after 1 day by a blood
glucose test, accepting a value over 15 mmol/l.
The test and reference substances were administered p.os to the
animals once a day.
For the determination of nerve conduction velocity (NCV), the method of
Stanley, modified by De Konig and Gispen was used. The animals were
anesthetized by the simultaneous administration of Hyponorm (1 mg/kg ip.,
Janssen, Tilburg, Denmark), fluanisone (10 mg/ml), and phentanyl citrate (0.2
mg/ml). Afterwards, the left ischiadicus and tibial nerves were stimulated at
standard points. Supramaximal stimulus (square impulse, 0.03 ms) was used
with a platinum needle electrode, through a Nihon-Kohden (model SEN-1104,
Japan) stimulator. The electromyogram (EMG) transferred from the sole
muscles and intensified by a myograph (Elema-SchCnander, Stockholm,
Sweden) was analyzed further by the Matlab for Windows (Mathwork Inc. UK)
program. The extent of NCV damage caused by diabetes was expressed in
m/s. The effectiveness of the treatment was compared to this in terms of
percentage (%). The statistical calculations were done by unpaired t-test or
single-criterion ANOVA test (along with the Newman-Keuls post hoc test).
(Graphpad Instat, San Diego, CA)
Results:
Bimoclomol administered once a day in a dose of 20 mg/kg and
Compound A administered once a day in a dose of 5 mg/kg significantly
improved motor (MNCV) and sensor (SNCV) nerve conduction velocities to the
same significant extent in diabetic animals. An increase in the dose of
Compound A over 10 mg/kg did not increase the effect. The results are given in
Table 3.


Experiment 3
The Effect of Treatment by Compound A and Bimoclomol on the Pathological
Deviations Resulting from Diabetic Autonomous Neuropathy in STZ-Diabetic
Wistar Rats, after 1 Month of Treatment
Materials and methods:
In the experiments, male Wistar rats were used (Charles River
Laboratories Inc.), at the start of the experiment their weights were 340-370 g.
Diabetes was induced by the intravenous administration of a single 45 mg/kg
dose of streptozotocine (STZ, Sigma, St. Louis, MO) dissolved in physiological
saline solution. The development of diabetes was checked after 1 day by a
blood glucose test, accepting a value over 15 mmol/l.

The test and reference substances were administered p.os_to the
animals once a day.
The experiments were performed under anesthesia achieved by
administering 60 mg/kg ip. pentobarbital sodium (Nembutal, Sanofi, Phylaxia).
After this, an intratracheal tube or polyethylene canula was inserted into the
fermoral artery and vein. The arterial catheter was connected to a pressure
transducer for the simultaneous measurement of systolic and diastolic blood
pressure (online automatic measuring and forwarding system, with a Haemosys
computer program). After 20 minutes of equilibration period, the following
substances were administered intravenously: Noradrenalin, 5 ug/kg iv. -
Isoproterenol 0.4 yg/kg iv. - N.vagus stimulation (2 V, duration: 500 jasec,
delay: 1 msec). The effects of the substances were monitored for 10 minutes.
Results:
Autonomous neuropathy is one of the leading causes of sudden cardiac
death both in the case of diabetes and of other diseases (e.g. liver diseases).
Therefore, all products that can effectively reduce pathological deviations
resulting from autonomous neuropathy are very important.
In the experiments, a daily 20 mg/kg single dose of either bimoclomol or
Compound A significantly reduced several pathological deviations resulting
from autonomous neuropathy.
A summary and comparison of our results are shown in Table 4.
The double arrow in the Table indicates that the test substance is
statistically more effective than the other one.


Experiment 4
The Effect of Treatment by Compound A and Bimoclomol on the Pathological
Histological Alterations Caused by Early Diabetic Retinopathy in STZ-Diabetic
Wistar Rats, Following 1 Month of Treatment

Materials and methods:
In the experiments, male Wistar rats were used (Charles River Laboratories
Inc.), at the start of the experiment their weights were 340-370 g. Diabetes was
induced by the intravenous administration of a single 45 mg/kg dose of
streptozotocine (STZ, Sigma, St. Louis, MO) dissolved in physiological saline
solution. The development of diabetes was checked after 1 day by a blood
glucose test, accepting a value over 15 mmol/l.
The test and reference substances were administered p.os_to the
animals once a day.
After anesthesia (Calypsovet, 125 mg/kg. Ip., Richter Rt., Hungary), the
eyes were enucleated and fixed in 4% formaldehyde dissolved in a phosphate
buffer (pH:7.4).
Afterwards, they were embedded in paraffin (Medim DDM P800.
embedding center: Lignifer L-120-92-014, stainer: Shandon Eliott, Microtome:
Leica SM 2000 R, Microscope: Jenaval Karl Zeiss Jena). Several 6 micron
sections of the eyes were prepared, and hematoxilyn/eosine (Fluka) and PAS
(periodic acid-Schiff, Fluka) staining were used. The light-microscopic
evaluation was performed at a magnification of 40x and 100x. Photographs and
slide positives were prepared of representative samples.
The histological evaluation was performed on coded samples, the group
division was unknown for the examiner. Pathological deviations of the retina
were graded on a scale of 0-20, while those of the lens on a scale of 0-3.
The statistical calculations were done with the Statistica 4.5 (SatSoft,
USA) program. The given value for negative cases was 0.1. A Box and Whisker
plot graph was also prepared.
For each group in the experiment, the mean±SE (Standard Error) values
were calculated, and the comparison was done with the help of a non-
parametric Mann-Whitney U-test (Graphpad Instat, San Diego, CA).
Results:
A daily single 5 mg/kg dose of Compound A and a daily single 20 mg/kg dose
of bimoclomol significantly improved the diabetic retinopathy induced

pathological histological alterations after 1 month of treatment. Of these two
compounds, Compound A was statistically more effective in comparison with
the diabetic, non-treated animals. The results are given in Table 5.

Experiment 5
The Effect of Treatment by Compound A and Bimoclomol on Pathological
Urinary Protein Loss Caused by Diabetic Nephropathy in STZ-Diabetic Wistar
Rats, After a 1-Month Treatment
Materials and methods:
In the experiments, male Wistar rats were used (Charles River Laboratories
Inc.), at the start of the experiment their weights were 340-370 g. Diabetes was
induced by the intravenous administration of a single 45 mg/kg dose of
streptozotocine (STZ, Sigma, St. Louis, MO) dissolved in physiological saline

solution. The development of diabetes was checked after 1 day by a blood
glucose test, accepting a value over 15 mmol/l.
The test and reference substances were administered p.os_to the
animals once a day.
For the 24-hour period of urine collection, the animals were placed in
individual metabolic cages. During this period they were given water ad libitum
but no food. The latter measure was necessary to prevent possible
contamination by the protein content of the food. Urine was collected in
calibrated glass containers, in which Thymol crystal (Reanal 3135-1-08-38) was
placed to prevent bacterial contamination.
Before measurement, the urine samples were centrifuged (2500 rpm)
and filtered through a paper filter (Whatmann 1). If necessary, they were stored
at -20 "C until measurement.
The total protein content of the urine was determined by the Bradford staining
method (Sigma B-6916, St. Louis, MO), and color intensity was detected by
spectrophotometry (Hitachi-U-3200).
Results:
Bimoclomol, administered in a daily single dose of 20 mg/kg, significantly
reduced STZ-diabetes-induced elevated urinary protein loss. Compound-A,
administered in a daily single dose of 10 mg/kg, non-significantly reduced
protein loss. However, the (+) enantiomer of Compound A, administered in a
daily single dose of 5 mg/kg significantly reduced diabetic protein loss. The
results are given in Table 6.


Experiment6
The effect of Compound-A and its (+) and (-) enantiomers on pathological
alterations of peripheral neuropathy in STZ-diabetic Wistar rats after 1 month
treatment
Materials and methods: experimental animals and all the methods applied are
the same as described in Experiment 2.

Results:
Compound-A in a single daily dose of 10 mg/kg and Compound A(+) in a single
daily dose of 5 mg/kg were equiactive and significantly improved both defective
motor (MNCV) and sensory (SNCV) nerve conduction velocity deficits in
diabetic animals. On the contrary, Compond A(-) did not have significant
improving effect on either parameter. Results are shown in Table 7.

Experiment 7
The effects of Compound-A and its A(+) and A(-) enantiomers on pathological
histological alterations of early diabetic retinopathy in STZ-diabetic rats after 2
month treatment

Materials and methods: the experimental animals and all the methods applied
are the same as described in Experiment 4.
Results:
The A(+) enantiomer in a single daily dose of 5 mg/kg significantly improved
both lenticular and retinal pathologic histological alterations caused by diabetic
retinopathy after 2 month treatment, while the effect of Compound-A in a single
daily dose of 10 mg/kg was not significant and A(-) enantiomer in a single daily
dose of 5 mg/kg was not effective. Regarding retinal histological alterations
only, both Compound-A and its A(+) enantiomer were effective while the effect
of the A(-) enantiomer was not significant. The results are shown in Table 8.


Experiment 8
The effects of A(+) and A(-) enantiomers on in vivo insulin-dependent glucose
uptake in dietary induced insulin resistant animal model
Materials and methods:
Male Wistar rats (Charles River Laboratories Inc.) with an initial body weight of
300-350 g were used in the experiments.
Insulin resistance was induced by dietary manipulation: animals were given a
high fat (HF) diet for 3 weeks. In the HF diet the proportion of saturated fats
were dominant and gave 70 % of total daily caloric intake. The A(+) and A(-)
enantiomers were given once a day in preventive application in a dose of 20
mg/kg/day.
At the end of 3 weeks treatment the following parameters were investigated: 1.
carbohydrate and lipid parameters from serum and 2. in vivo insulin-mediated
glucose uptake by the euglycaemic glucose clamp method being currently the
most accurate method for the quantitative determination of glucose uptake (
DeFronzo et al., American Journal of Physiology, 1979/237/ E214-223 pages).
Briefly: the fasting blood glucose concentration in different animal groups must
be identical. Experiments were carried out in conscious, freely moving,
chronicly canulated rats: first an insulin infusion (6.4 mU/kg/min) was started,
followed by a parallel-run continuous glucose infusion to maintain blood glucose
concentrations in the euglycaemic range. After stabilisation the quantity of
infused glucose was measured for a 90 min. period (glucose infusion rate=
GIR, mg/kg/min) which is the quantitative parameter of insulin sensitivity.
Results:
The A(+) and A(-) enantiomers in a daily dose of 20 mg/kg/min did not affect
the body weight and food consumption and the fasting blood glucose levels of
the rats.
On the contrary both compounds normalized HF diet induced elevated fasting
insulin and triglyceride concentrations and significantly decreased elevated
muscle triglyceride content as well. The euglycaemic glucose clamp test

revealed that HF diet significantly suppressed in vivo insulin mediated glucose
uptake: control: 26.7+0.68 mg/kg/min, HF diet: 15.0+0.39 mg/kg/min.
This suppressed insulin mediated glucose uptake is increased by both
enantiomers: HF+ A(+): 20.5+0.89 mg/kg/min and HF+ A(-): 19.7+1.38
mg/kg/min (a significant increase in both cases at the level of p According to these results both enantiomers increased the insulin-mediated
glucose uptake which proves from a new perspective the insulin resistance
reducing action of the compounds of the invention.
Experiment 9
The antidiabetic activity of A(+) enantiomer in Zucker Diabetic Fatty rats after
chronic administration
Materials and methods:
A genetically diabetic animal model was selected and the Zucker Diabetic Fatty
(ZDF) rats were used in the experiments. This model is the diabetic variant of
the insulin resistant, obese but non-diabetic Zucker fa/fa animal model (see
Experiment 1). In the ZDF rats diabetes developed at the age of 6-8 weeks
preceded by an insulin resistant phase.
The effect of (A+) enantiomer was investigated in a treatment with a dose of
2x20 mg/kg /day started in the non-diabetic phase, at the age of 7 weeks and
continued for 6 weeks.
Clinical chemistry parameters were measured by standard methods.
The serum insulin concentrations were measured by a recently developed
method (ELISA method, DRG International.lnc,U.S.A.).
Results:
It has been detected in this experiment as a new result that A(+) enantiomer
has a strong antidiabetic activity in diabetic animals.
Results obtained after 3 and 5 week treatment are shown in Table 9.


Though the treatment with A(+) enantiomer did not normalize blood glucose
concentrations the combined effects of strong antidiabetic activity and the
previously identified significant healing efficiency on chronic diabetic
complications gives this compound a unique character.
The therapeutic indication area of the A(+) enantiomer can be significantly
broadened on the basis of this new combined efficiency.
Further experiments have led to the conclusion that the compounds of the
invention, besides their efficacy on pathological complications of diabetes are
useful in the treatment of other damages of peripheral nerves caused by
diabetes. This conclusion is supported by the results of the following
neuroregeneration experiment.
Experiment 10
The therapeutical effect of Compound-A and A(+) enantiomer on the
neuroregeneration in STZ-diabetic Wistar rats
Materials and methods:
The experiments were accomplished on Wistar rats with a body weight of 320-
350 g. Diabetes was induced and checked as described in Experiment 2. In the

test animals having been diabetic for 3 weeks the left nervus ischidiacus was
injured by freezing and the right side one was used as non-injured control. The
regeneration was observed by monitoring the signals of the flexor reflex
provoked by irritation of the sole that is areas under curve (AUC) of the
electromyogram transferred from the forward tibial muscle. For the stimulation
and detection the system described in Experiment 2 was used.
Single daily doses of 10 mg/kg of Compound-A and 5 mg/kg of A(+) enantiomer
were administred for 5 weeks after the injury by freezing.
Results:
At the end of the 3 week period of diabetes, before the injury by freezing a
sensor neuropathy developed and caused a 23-25 % decrease in AUC on both
legs. No response was observed during 2 weeks after the injury by freezing.
The extent of neuroregeneration was 63 % on the 5. week. The regeneration
was enhanced to 73 % by the Compound-A, the A(+) enantiomer was effective
for an extent of 93 %. Neuroregenerations of 83 % and only 44 % of the non-
freezed nerves were observed as a result of treatment with A(+) enantiomer
and Compound-A, respectively. Consequently, the A(+) enantiomer has a
strong neuroregenerative effect.
N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl
chloride can be prepared by the following procedure, which is not known from
the prior.
According to the oxidation method described in WO 97/16349, a
derivative oxidized on the nitrogen atom of both rings, or with less reagent a
derivative oxidized on the alicyclic ring can be prepared from 0-(3-piperidino-2-
hydroxy-1-propy!)-3-pyridine hydroxymic acid-chloride, since the alicyclic
nitrogen atom is preferred in the oxidation reaction. The regioselectivity of the
oxidation had to be directed towards the pyridine ring for the production of
compound of the invention, therefore the procedure was modified. The main
point of the modification is that, in order to facilitate the selective oxidation of
the pyridine ring, the peracidic oxidation is performed in the presence of a
strong acid, preferably methanesulphonic acid, which protonates the alicyclic

nitrogen and thus prevents its oxidation; therefore the oxidation of the pyridine
becomes primary. As an oxidant, any type of peracid, preferably peracetic acid
may be used.
The optically active enantiomers of the compound of the invention are
prepared by using the suitable optically active 0-(3-piperidino-2-hydroxy-1-
propyl)-3-pyridine hydroxymic acidchloride enantiomer as starting material,
which can be produced for example according to EP 0 417 210 B1, by re-
solving the racemic compound. In the course of the reaction, the chirality of the
molecule is not damaged, and the resulting product has the same optical purity
as the starting substance.
If desired, the N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-
carboximidoyl chloride that obtained or one of its optically active enantiomers
can be transformed into an acid addition salt with a mineral or organic acid, by
known methods.
N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl
chloride, its optically active (+) or (-) enantiomer, a mixture of the enantiomers
of any ratio, and the racemic compound, furthermore, the acid addition salts
formed from any of the above compounds with mineral or organic acids
constitute objects of the present invention. All possible geometrical isomer
forms of N-[2-hydroxy-3-(1 -piperidinyl)-propoxyJ-pyridine-1-oxide-3-
carboximidoyl chloride belong to the scope of the invention. The term Mthe
stereoisomers of N-[2-hydroxy-3-(1-piperidinyl)-propoxyj-pyridine-1-oxide-3-
carboximidoyl chloride" refers to all possible optical and geometrical isomers of
the compound.
According to the invention, these compounds are applied for the
treatment of pathological insulin resistance and for the treatment and
prevention of pathological conditions associated with it.
A special embodiment of the invention is that these compounds are used
for simultaneous treatment or prevention of chronic diabetes-induced
complications, especially retinopathy, neuropathy and nephropathy, and of
pathological insulin resistance and the pathological conditions associated

therewith.
According to an other special embodiment of the invention, N-[2-
hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride or
its stereoismers or acid addition salts thereof are used in the treatment of
pathological insulin resistance and pathological conditions associated therewith
it and a simultaneous increasing of a diabetes-iduced pathologically decreased
peripheral neuroregeneration.
The compounds of the invention may be applied both in human and
veterinary therapy.
Therefore, the object of the invention also includes the method for the
treatment of pathological insulin resistance and treatment and prevention of
pathological conditions associated therewith, in the course of which the patients
are administered N-[2-hydroxy-3-(1-piperidiny!)-propoxy]-pyridine-1-oxide-3-
carboximidoyl chloride or one of its stereoisomers in the form of base or acid
addition salt. The preferred embodiment of the procedure of the invention is
when N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl
chloride or one of its stereoisomers, or a acid addition salt thereof is
administered to a patient suffering from diabetic retinopathy, neuropathy or
nephropathy.
According to another special embodiment of the invention N-[2-hydroxy-
3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride or one of its
stereoisomers or an acid addition salt thereof is administered to a patient in
case of pathological decrease of neuroregeneration caused by diabetes.
The dose of the compounds depends on the condition and the disease
of the patient, and the daily dose is 0.1-400 mg/kg, preferably 0.1-100 mg/kg. In
human therapy, the oral dose is preferably 10-300 mg, in the case of rectal
administration 1-15 mg. while in the case of parenteral administration 1-15 mg
for an adult patient. These doses are preferably administered in dosage unit
forms, which may be divided into 2-3 smaller doses for a day in certain cases,
especially in oral treatment. ........

Preferably, the stereoisomer of the racemic compound, most preferably
the (+) enantiomer is used. In this case, a smaller quantity of active ingredient
within the above limits is sufficient for the treatment.
Pharmaceutical preparations suitable for the treatment are also object of
the invention. These pharmaceutical compositions contain, in addition to the
usual auxiliary substances and carriers, N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-
pyridine-1-oxide-3-carboximidoyl chloride or one of its stereoisomers, or an acid
addition salt of one of the above, as active ingredients.
The pharmaceutical compositions of the invention can be prepared in the
form of a solid or fluid preparation generally used in human or veterinary
therapy. Simple or coated tablets, dragees, granulates, capsules, solutions or
syrups can be prepared for oral administration, suppositories for rectal
administration, and lyophilised or not lyophilised injection or infusion solutions
for parenteral administration. These can be produced by the usual methods.
The products for oral use can contain filling materials such as microcrystalline
cellulose, starch or lactose, lubricants such as stearic acid or magnesium
stearate, coating materials such as sugar, film forming materials such as
hydroxy-methyl-cellulose, aromas or sweeteners such as methyl-paraben or
saccharine, or coloring substances. The suppositories can contain cocoa butter
or polyethylene glycol as auxiliary. The parenteral products can contain, in
addition to the effective substance, saline solution, or in certain cases
dispersing and moistening substances such as propylene glycol.
The invention is further illustrated by the following examples:
Example 1
Preparation of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-
carboximidoyl chloride (Z)-2-butenedioate (1:1)
40,4 g (0,136 mol) of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridine-
carboximidoyl chloride was dissolved in the mixture of 238 ml of glacial acetic
acid and 13.0 g (0.136 mol) of methane sulphonic acid. 61.5 ml (0.591 mol) of
30% hydrogen peroxide solution was added at 60 °C. The reaction mixture was

stirred at 60 °C for 3.5-4 hours. The solution was cooled to 10 °C, and then 91
ml of 0.5 M Na2S205 solution was added thereto. 315 ml of water-acetic acid
mixture was distilled off from the solution, 250 ml of 4 N NaOH solution was
added to the residue (pH=10.55), and shaken with chloroform. The chloroform
phase was washed with water, dried, treated with charcoal, and then
evaporated. Water was added to the residue, and extracted with isopropyl
ether and then with chloroform. The chloroform phase was dried, treated with
charcoal, filtered and evaporated off. The residue was dissolved in acetone and
transformed into salt with maleic acid. The precipitate was filtered, washed with
acetone and dried. The product was crystallized from boiling ethanol.
Yield: 20 g (35%)
Mp.: 150.5-154.5 °C
'H-NMR (solvent: DMSO; reference: DMSO; v = 300 MHz) [ppm]: 8.55 (s, 1H,
2-pyridine); 8.35 (d, 1H, 6-pyridine); 7.68 (d, 1H, 4-pyridine); 7.55 (m, 1H. 5-
pyridine); 6.00 (s, 2H, CH=CH); 4.23-4.48 (m, 3H, CH-OH and NOCH2); 2.95-
3.50 (m, 6H, 3 x NCH2); 1.20-1.90 (m, 6H, piperidine: 3 x CH2).
13C-NMR (solvent: DMSO; reference: DMSO; v = 300 MHz) [ppm]: 167.6 (2C, 2
COOH); 141.0 (2-pyridine); 136.8 (6-pyridine); 136.4 (2C, CH=CH); 133.4 (CCI);
131.9 (3-pyridine); 127.2 (4-pyridine); 123.6 (5-pyridine); 77.9 (NOCH2); 63.6
(CH2N); 58.3 (CHOH); 52.0-55.0 (2C, piperidine: 2 x NCH2); 22.6, and 21.7 (3C,
piperidine: 3 x CH2).
Example 2
Preparation of (+)-/R/-N-[2-hydroxy-3-(1 -piperidinyl)-propoxy]-pyridine-1 -
oxide-3-carboximidoyl chloride (Z)-2-butenedioate (1:1)
The procedure described in Example 1 was repeated with the difference that
instead of the racemic N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridine-
carboximidoyl chloride, its R enantiomer was used. The pure form of the
compound was isolated from the crude base by crystallization with hexane.

Yield: 31%
Mp.: 91-93 °C
IR (KBr. cm-1): 3167 (br); 2840; 2710; 1575; 1560; 1480; 1443 (br); 1293 (s);
1279 (s); 1093; 1053; 1043; 1023 (s); 834 (s); 810; 688
If desired, a maleate salt can also be prepared from the crude base in acetone
solution as described in Example 1.
Yield: 33%
Mp.: 132.0-133.0 oC
Enantiomer ratio: 98/2 (HPLC measurement on a Chiral AGP 100 x 4 mm
column).
1H-NMR and 13C-NMR: same as the spectra of the racemic compound.
Example 3
Preparation of (-)-/S/-N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-
oxide-3-carboximidoyl chloride (Z)-2-butenedioate (1:1)
The procedure in Example 1 was followed, with the difference that
instead of the racemic N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridine-
carboximidoyl chloride, the S-enantiomer was used.
Yield: 34%
Mp.: 132.0-133.0 °C
Enantiomer ratio: 98/2 (HPLC measurement on a Chiral AGP 100 x 4 mm
column).
1H-NMR and 13C-NMR: same as the spectra of the racemic compound.



The active ingredient and the polyvinyl pyrrolidone were dissolved in
ethanol. A mixture of the lactose and the potato starch were moistened evenly
with the granulating solution of the active ingredient. After filtering, the granulate
was dried at 50 °C and screened. The magnesium stearate was added and
pressed into tablet form, which was then covered by a sugar coating and
polished by bee wax.


Example 9
Injection
(+)-N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-
pyridine-1-oxide-3-carboximidoyl chloride maleate 2 mg
Physiological saline solution, pyrogen-free, sterile q.s.ad 2.0 ml
The solution was poured into 2 ml vials and then sealed.
Example 10
Infusion solution
500 ml of infusion solution was prepared with the following composition:
N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-
pyridine-1-oxide-3-carboximidoyl chloride maleate 20.0 mg
Physiological saline solution, pyrogen-free, sterile q.s.ad 500 ml

CLAIMS:
1. N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl
chloride, its stereoisomers, such as herein described, and the acid addition salts
thereof, such as herein described,
2. A pharmaceutical composition comprising a pharmaceutically acceptable
carrier and a therapeutically effective amount of at least one compound chosen from
N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride,
its stereoisomers, and acid addition salts thereof.
3. A pharmaceutical composition as claimed in claim 2 for the treatment of
pathological insulin resistance, and the treatment and prevention of diabetes-induced
chronic complications, especially retinopathy, neuropathy, nephropathy and/or
pathologically decreased peripheral neuroregeneration caused by diabetes.
4. A pharmaceutical composition as claimed in claim 2, optionally comprising at
least one pharmaceutically acceptable auxiliary.

The invention relates to N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-
oxide-3-carboximidoyl chloride, its stereoisomers, and the acid addition salts thereof,
pharmaceutical composition containing the same, the use of these compounds in the
treatment of pathological insulin resistance, and for the treatment of pathological
conditions associated therewith, by simultaneous treatment and prevention of
diabetes-induced chronic complications, especially retinopathy, neuropathy,
nephropathy and/or pathologically decreased peripheral neuroregeneration caused by
diabetes and methods of treatment.

Documents:

in-pct-2001-785-kol-abstract-1.1.pdf

in-pct-2001-785-kol-abstract.pdf

in-pct-2001-785-kol-assignment-1.1.pdf

in-pct-2001-785-kol-assignment-1.2.pdf

in-pct-2001-785-kol-assignment.pdf

in-pct-2001-785-kol-claims-1.1.pdf

in-pct-2001-785-kol-claims.pdf

IN-PCT-2001-785-KOL-CORRESPONDENCE 1.1.pdf

in-pct-2001-785-kol-correspondence-1.2.pdf

in-pct-2001-785-kol-correspondence-1.3.pdf

in-pct-2001-785-kol-correspondence.pdf

in-pct-2001-785-kol-description (complete)-1.1.pdf

in-pct-2001-785-kol-description (complete).pdf

in-pct-2001-785-kol-examination report-1.1.pdf

in-pct-2001-785-kol-examination report-1.3.pdf

in-pct-2001-785-kol-examination report.pdf

in-pct-2001-785-kol-form 1.pdf

in-pct-2001-785-kol-form 18-1.1.pdf

in-pct-2001-785-kol-form 18-1.2.pdf

in-pct-2001-785-kol-form 18.pdf

in-pct-2001-785-kol-form 3-1.1.pdf

in-pct-2001-785-kol-form 3-1.2.pdf

in-pct-2001-785-kol-form 3.pdf

in-pct-2001-785-kol-form 5-1.1.pdf

in-pct-2001-785-kol-form 5-1.2.pdf

in-pct-2001-785-kol-form 5.pdf

in-pct-2001-785-kol-gpa-1.1.pdf

in-pct-2001-785-kol-gpa-1.2.pdf

in-pct-2001-785-kol-gpa.pdf

in-pct-2001-785-kol-granted-abstract-1.1.pdf

in-pct-2001-785-kol-granted-abstract.pdf

in-pct-2001-785-kol-granted-claims-1.1.pdf

in-pct-2001-785-kol-granted-claims.pdf

in-pct-2001-785-kol-granted-description (complete)-1.1.pdf

in-pct-2001-785-kol-granted-description (complete).pdf

in-pct-2001-785-kol-granted-form 1-1.1.pdf

in-pct-2001-785-kol-granted-form 1.pdf

in-pct-2001-785-kol-granted-form 2.pdf

in-pct-2001-785-kol-granted-specification-1.1.pdf

in-pct-2001-785-kol-granted-specification.pdf

in-pct-2001-785-kol-others-1.1.pdf

in-pct-2001-785-kol-others-1.2.pdf

IN-PCT-2001-785-KOL-OTHERS.pdf

in-pct-2001-785-kol-reply to examination report-1.1.pdf

in-pct-2001-785-kol-reply to examination report-1.2.pdf

in-pct-2001-785-kol-reply to examination report.pdf

in-pct-2001-785-kol-specification-1.1.pdf

in-pct-2001-785-kol-specification.pdf


Patent Number 249656
Indian Patent Application Number IN/PCT/2001/785/KOL
PG Journal Number 44/2011
Publication Date 04-Nov-2011
Grant Date 01-Nov-2011
Date of Filing 31-Jul-2001
Name of Patentee BIOREX KUTATO ES FEJLESZTO RT
Applicant Address PF: 348, H-8201 VESZPREM-SZABADSAGPUSZTA
Inventors:
# Inventor's Name Inventor's Address
1 BIRO, KATALIN TOVIS U. 7/B H-1022 BUDAPEST
2 KURTHY, MARIA BALASSI B.U. 5, H-8230 BALATONFURED
3 NAGY, KAROLY SOMLOI UT 13, H-1118 BUDAPEST
4 UROGDI, LASZLO TELEKI U.80 H-1184 BUDAPEST
5 CSAKAI,ZITA PETOFI LAKOTELEP B/19 H-6090 KUNSZENTMIKLOS
6 SZILBEREKY, JENO SZAMOS U. 7. H-1122 BUDAPEST, HUNGARY
7 MOGYOROSI, TAMAS PAL LASZLO U.10.I.3. H-3700 KAZINCBARCIKA
8 TOROK, MAGDOLNA ZOLDFA U.172 H-4700 MATESZALKA
9 KOMAROMI, ANDRAS BUZAVIRAG U. 8/A H-8200 VESZPREM
10 MARVANYOS, EDE ULASZLO U. 38.VI.6 H-1114 BUDAPEST
11 BARABAS, MIHALY EGRY J.U.36 H-1111 BUDAPEST
12 KARDOS, MIHALYNE HALLE U.9/I.I.5 H-8200 VESZPREM
13 NAGY, ZOLTAN BOGDYANFY U.10 H-1117 BUDAPEST
14 KORANYI. LASZLO HERMANN O.U.10 H-1022 BUDAPEST
15 NAGY, MELINDA MUNKACSY UT 1/C VII.23. H-8200 VESZPREM
PCT International Classification Number A61K 31/445
PCT International Application Number PCT/HU2000/00015
PCT International Filing date 2000-02-24
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 P 9900475 1999-02-26 Hungary