Title of Invention

A METHOD OF PREPARING BIGUANIDE DERIVATIVE

Abstract A biguanide derivative compound with N1-N5 substitution which is represented by Formula 1 or a pharmaceutically acceptable salt thereof a method of preparing the same and a pharmaceutical composition containing the same as an active ingredient are provided. The biguanide derivative may exhibit excellent effect on activation of AMPKa and inhibition of cancer cell proliferation in a low dose compared to conventional drugs and thus may be useful to treat diabetes mellitus obesity hyperlipidemia hypercholesterolemia fatty liver coronary artery disease osteoporosis polycystic ovarian syndrome metabolic syndrome cancer etc.
Full Text FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules  2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)



1. BIGUANIDE DERIVATIVE  A PREPARATION METHOD THEREOF AND A PHARMACEUTICAL COMPOSITION CONTAINING THE BIGUANIDE DERIVATIVE AS AN ACTIVE INGREDIENT

2.

1. (A) HANALL BIOPHARMA CO.  LTD.
(B) Republic of Korea
(C) 400-1 Sangseo-dong  Daeduk-gu Daejeon 306-120 Republic of Korea


The following specification particularly describes the invention and the manner in which it is to be performed.


?DESCRIPTION?
?Invention Title?
BIGUANIDE DERIVATIVE  A PREPARATION METHOD THEREOF  AND A PHARMACEUTICAL COMPOSITION CONTAINING THE BIGUANIDE DERIVATIVE AS AN ACTIVE INGREDIENT

?Technical Field?
The present invention relates to a biguanide derivative exhibiting excellent effects on activation of 5""-AMP-activated protein kinase(AMPK) and inhibition of cancer cell proliferation in a low dose compared to conventional drugs  a method of preparing the same  and a pharmaceutical composition containing the same as an active ingredient.

?Background Art?
Diabetes mellitus  a disease characterized by continuous hyperglycemia  is a disorder that affects the metabolization of carbohydrates and lipids. It is a disease aggravated by bloodstream disorders caused by hyperglycemia and systemic complications caused by decreased utilization of sugar. Diabetes mellitus is induced by insulin deficiency or insulin resistance  and diabetes mellitus that occurs due to insulin resistance is called type 2 diabetes mellitus.
Type 2 diabetes mellitus is caused by a malfunctioning of insulin in delivering sugar into cells due to the reduction in the number of insulin receptors or defects in the signal transduction system through a receptor  a condition known as insulin resistance and Type 2 diabetes mellitus directly destroys blood vessels due to hyperinsulinemia and aggravates metabolic syndrome.
Many kinds of drugs have been used to treat type 2 diabetes mellitus However  except for biguanide metformin  drugs are only partly effective in lowering blood sugar and are not sufficient in effectively preventing serious complications such as loss of sight  paralysis  apoplexy  renal failure  peripheral neuropathy  foot ulcer  etc. For example  a sulfonylurea-based drug forces insulin to be secreted from the pancreas to lower blood sugar. The medicinal effects of the sulfonylurea-based drug disappear immediately. Also  the sulfonylurea-based drug induces an abnormal lipid metabolism  thereby resulting in arteriosclerosis  weight gain  and brain damage caused by hypoglycemia. In addition  a glitazone-based drug is used in combination with metformin because it resolves the problem of insulin resistance in adipose tissues but has a side effect of destroying the retinal vessels. For these reasons  use of the glitazone-based drug requires special attention.
Metformin does not induce hypoglycemia  but it overcomes the problem of insulin resistance in adipose tissues  liver tissues and muscle tissues  and it functions to drastically lower blood sugar and decrease the level of glycosylated hemoglobin.
In addition  metformin is known to activate an AMP-activated protein kinase that physiologically controls carbohydrate and lipid metabolism and is also reported to decrease blood sugar level  improve lipid condition  and normalize menstrual irregularity  ovulation and pregnancy. Moreover  it has been proven that when metformin is used to treat p53 gene-deficient cancer cells  metformin activates an AMPK enzyme of the cancer cells and changes the metabolic energy pathway  and therefore  the cancer cells finally die [Monica Buzzai et al.  Systemic Treatment with the Antidiabetic drug Metformin Selectively Impairs p53-Deficient Tumor Cell Growth  Cancer Res 2007; 67:(14)] since they cannot adjust to the changed metabolic pathway.
In addition  Josie MM Evans reported a study concluding that a type 2 diabetes mellitus subjectpatient treated with metformin has a lower risk of cancer than a subject who has not been treated with metformin [Josie MM  Evans et al. BMJ. 2005  330  1304-1305]. Moreover  Samantha L. Browker reported that subjects with type 2 diabetes mellitus who take metformin orally have a lower cancer mortality rate than subjects who take sulfonylurea orally or are administered with insulin [Samantha L et al.  Diabetes mellitus Care. 2006  29  254-258].
There is an increasing amount of clinical evidence indicating that a cancer stem cell is involved in the recurrence and metastasis of cancer. The content of cancer stem cells in a tumor tissue is 0.2% or less  but the cancer stem cells may not be removed by conventional anticancer chemotherapy. Metformin has an anticancer effect on cancer stem cells and excellent tolerability. In recent research relating to metformin  it has been reported that when doxorubicin  which is an anticancer drug  is administered alone  there is little change in cancer stem cells  but when administered together with metformin  it removes cancer stem cells [Heather A. Hirsch et al.  Metformin Selectively Targets Cancer Stem Cells  and Acts Together with Chemotherapy to Block Tumor Growth and Prolong Remission  Cancer Res 2009; 69: (19) October 1  2009].
However  metformin is generally administered three times a day  and a single dose is approximately 500 mg or more. Thus  to prepare metformin as a sustained- released tablet to be administered once a day  the tablet should contain approximately 1 500 mg or more of metformin  but such a tablet is too large for most subjects to take. In addition  since extended release formulation available in the current market contains only approximately 750 mg of metformin  at least two tablets should be taken. For these reasons  a metformin-based substance exhibiting better pharmacological action than conventional metformin and having improved physiochemical characteristics is needed.

?Disclosure?
?Technical Problem?
The present invention is directed to provide a novel biguanide derivative or a pharmaceutically acceptable salt thereof  which exhibits excellent effects on activation AMPK activation and inhibition of cancer cell proliferation in a small amount  compared to conventional drugs  and a method of preparing the same.
The present invention is also directed to provide a pharmaceutical composition containing the above-mentioned compound as an active ingredient to treat diabetes mellitus  obesity  hyperlipidemia  hypercholesterolemia  fatty liver  coronary artery disease  osteoporosis  polycystic ovarian syndrome  metabolic syndrome  cancer  etc.

?Technical Solution?
One aspect of the present invention provides a biguanide derivative compound with N1-N5 substitution  represented by Formula 1  or a pharmaceutically acceptable salt thereof.
[Formula 1]

In Formula 1  R1  R2  R3 and R4 are independently hydrogen or a non-hydrogen substituent selected from the group consisting of C1-12 alkyl unsubstituted or substituted with at least one non-hydrogen substituent selected from the group consisting of C3-10 cycloalkyl  C5-12 aryl  C5-12 heteroaryl  hydroxyl  halogen and C1-4 alkoxycarbonyl; C3-10 cycloalkyl; C1-12 alkoxy; C5-12 aryl; C5-12 heteroaryl; hydroxyl and halogen  and the aryl and heteroaryl are unsubstituted or substituted with at least one non-hydrogen substituent selected from the group consisting of C1-4 alkyl  C1-4 alkoxy  hydroxyl and halogen.
A “substituted” group used herein is a group in which at least one hydrogen atom is replaced with at least one non-hydrogen atom group  provided that the group has to satisfy a requirement of valence and generate a chemically stable compound from the substitution. In the specification  unless explicitly described as “unsubstituted ” it should be understood that all of substituents may be substituted or unsubstituted. R1 to R4 substituents on the biguanide according to the present invention may each be substituted again with at least one of the above-defined substituents.
“Alkyl” refers to a linear and branched saturated hydrocarbon group  generally having a specified number of carbon atoms (for example  1 to 12 carbon atoms). Examples of the alkyl group include  without limitation  methyl  ethyl  n-propyl  isopropyl  n-butyl  sec-butyl  isobutyl  tert-butyl  n-pentyl  n-hexyl and n-heptyl. The alkyl may be attached to a parent group or a substrate at any ring atom  unless such attachment would violate valence requirements. Likewise  the alkyl or alkenyl group may include at least one non-hydrogen substituent unless such substitution would violate valence requirements.
“Cycloalkyl” refers to saturated monocyclic and polycyclic hydrocarbon rings  generally having a specified number of carbon atoms that include the ring (for example  C3-10 cycloalkyl refers to a cycloalkyl group having 3  4  5  6  7  8  9  or 10 carbon atoms as ring members). The cycloalkyl may be attached to a parent or substrate at any ring atom  unless such attachment would violate valence requirements. Likewise  the cycloalkyl group may include at least one non-hydrogen substituent unless such substitution would violate valence requirements.
“Aryl” refers to monovalent and bivalent aromatic groups  respectively including 5- and 6-membered monocyclic aromatic groups and “heteroaryl” refers to monovalent and bivalent aromatic groups  respectively including 5- and 6-membered monocyclic aromatic groups that contain 1 to 4 heteroatoms independently selected from nitrogen  oxygen and sulfur. Examples of the monocyclic aryl group and heteroaryl group include  without limitation  phenyl  pyridinyl  furanyl  pyrrolyl  thiopheneyl  thiazolyl  isothiazolyl  imidazolyl  triazolyl  tetrazolyl  pyrazolyl  oxazolyl  isoxazolyl  pyrazinyl  pyridazinyl  pyrimidinyl  etc. The aryl and heteroaryl groups also include bicyclic groups  tricyclic groups  etc.  including fused 5- and 6-membered rings as described above. Examples of the polycyclic aryl and heteroaryl groups include  without limitation  isoquinolinyl  naphthyl  biphenyl  anthracenyl  pyrenyl  carbazolyl  benzoxazolyl  benzodioxazolyl  benzothiazolyl  benzoimidazolyl  benzothiopheneyl  quinolinyl  indolyl  benzofuranyl  purinyl  indolizinyl  etc. The aryl and heteroaryl groups may be attached to a parent group or to a substrate at any ring atom  unless such attachment would violate valence requirements. Likewise  the aryl and heteroaryl groups may include at least one non-hydrogen substituent unless such substitution would violate valence requirements. Non-hydrogen substituents of the acryl and heteroaryl groups may also be substituted with additional non-hydrogen substituents.
“Carbonyl” refers to –C(O)R’. “(O)” used herein refers to an oxygen binding to an atom such as carbon or sulfur by a double bond. Here  “R’” refers to non-hydrogen substituents such as lower alkyl  lower alkoxy  etc. Examples of the carbonyl group include  without limitation  2-methoxyoxoethyl  3-methoxyoxopropyl  etc. The carbonyl group may be attached to a parent group or to a substrate at any ring atom  unless such attachment would violate valence requirements. Likewise  the carbonyl group may include at least one non-hydrogen substituent unless such substitution would violate valence requirements.
“Alkoxy” refers to alkyl-O-. Here  the alkyl is defined above. Examples of the alkoxy group include  without limitation  methoxy  ethoxy  etc. The alkoxy may be attached to a parent group or to a substrate at any ring atom  unless such attachment would violate valence requirements. Likewise  the alkoxy group may include at least one non-hydrogen substituent unless such substitution would violate valence requirements.
“Hydroxyl” refers to –OH  “halogen” refers to fluoro  chloro  bromo  and iodo  and “oxo” refers to =O.
In the compound of Formula 1 of the present invention  R1  R2  R3 and R4 are independently hydrogen or a non-hydrogen substituent selected from the group consisting of C1-12 alkyl; C3-10 cycloalkyl; C1-12 alkoxy; C5-12 aryl; C5-12 heteroaryl; hydroxyl and halogen.
Here  the C1-12 alkyl may be a linear or branched C1-12 alkyl unsubstituted or substituted with at least one non-hydrogen substituent. When the alkyl is substituted with a non-hydrogen substituent  the alkyl may be  but is not limited to  a linear or branched alkyl having 1 to 12 carbon atoms. Here  non-hydrogen substituents for the alkyl may be selected from the group consisting of C3-10 cycloalkyl  C5-12 aryl  C5-12 heteroaryl  hydroxyl  halogen and C1-4 alkoxycarbonyl  but the present invention is not limited thereto. The non-hydrogen substituents may also be further substituted or unsubstituted. For example  the aryl and heteroaryl may be unsubstituted or substituted with at least one non-hydrogen substituent selected from the group consisting of C1-4 alkyl  C1-4 alkoxy  hydroxyl and halogen.
In one embodiment  R1  R2  R3 and R4 are independently hydrogen or a non-hydrogen substituent selected from the group consisting of unsubstituted C1-7 alkyl; C1-6 alkyl substituted with at least one non-hydrogen substituent selected from the group consisting of unsubstituted C3-7 cycloalkyl  C5-12 aryl  C5-12 heteroaryl  hydroxyl  halogen and C1-4 alkoxycarbonyl; unsubstituted C3-7 cycloalkyl; C1-6 alkoxy; C5-12 aryl; C5-12 heteroaryl; hydroxyl; and halogen. The aryl and heteroaryl may be unsubstituted or substituted with at least one non-hydrogen substituent selected from the group consisting of C1-4 alkyl  C1-4 alkoxy  hydroxyl and halogen.
In one embodiment  R1 and R4 are independently non-hydrogen substituents selected from the group consisting of unsubstituted C1-7 alkyl; C1-6 alkyl substituted with at least one non-hydrogen substituent selected from the group consisting of unsubstituted C3-7 cycloalkyl  C5-12 aryl  C5-12 heteroaryl and C1-4 alkoxycarbonyl; unsubstituted C3-7 cycloalkyl; C5-12 aryl; and C5-12 heteroaryl  R2 and R3 are hydrogen; unsubstituted C1-7 alkyl; C5-12 aryl; or C5-12 heteroaryl  and the aryl and heteroaryl may be unsubstituted or substituted with at least one non-hydrogen substituent selected from the group consisting of C1-4 alkyl  C1-4 alkoxy  hydroxyl and halogen.
In one embodiment  R1 and R4 are independently non-hydrogen substituents selected from the group consisting of unsubstituted C1-7 alkyl; C1-4 alkyl substituted with at least one non-hydrogen substituent selected from the group consisting of unsubstituted C3-7 cycloalkyl  C5-12 aryl  C5-12 heteroaryl and C1-4 alkoxycarbonyl; unsubstituted C3-7 cycloalkyl; C5-12 aryl; and C5-12 heteroaryl  R2 and R3 are hydrogen; unsubstituted C1-7 alkyl; C5-12 aryl; or C5-12 heteroaryl  and the aryl and heteroaryl may be unsubstituted or substituted with at least one non-hydrogen substituent selected from the group consisting of C1-4 alkoxy  hydroxyl and halogen  and the aryl and heteroaryl may be selected from the group consisting of phenyl  pyridinyl  furanyl and isoquinolinyl.
In one embodiment  R1 is unsubstituted C1-7 alkyl; C1-4 alkyl substituted with C5-12 aryl or C5-12 heteroaryl; C5-12 aryl; or C5-12 heteroaryl  R2 is hydrogen; or unsubstituted C1-7 alkyl  R3 is hydrogen; unsubstituted C1-7 alkyl; C5-12 aryl; or C5-12 heteroaryl  and R4 is a non-hydrogen substituent selected from the group consisting of unsubstituted C1-7 alkyl; C1-4 alkyl substituted with at least one non-hydrogen substituent selected from the group consisting of unsubstituted C3-7 cycloalkyl  C5-12 aryl  C5-12 heteroaryl and C1-4 alkoxycarbonyl; unsubstituted C3-7 cycloalkyl; C5-12 aryl; and C5-12 heteroaryl. The aryl and heteroaryl may be unsubstituted or substituted with at least one non-hydrogen substituent selected from the group consisting of C1-4 alkoxy  hydroxyl and halogen  and the aryl and heteroaryl may be selected from the group consisting of phenyl  pyridinyl  furanyl and isoquinolinyl.
In one embodiment  the compound of Formula 1 may be N1-hexyl-N5-propyl biguanide; N1-hexyl-N5-cyclopropylmethyl biguanide; N1-hexyl-N5-cyclohexylmethyl biguanide; N1-hexyl-N5-benzyl biguanide; N1 N5-bis(4-chlorophenyl) biguanide; N1 N5-bis(3-chlorophenyl) biguanide; N1-(4-chloro)phenyl-N5-(4-methoxy)phenyl biguanide; N1 N5-bis(3-chloro-4-methoxyphenyl) biguanide; N1 N5-bis(3 4-dichlorophenyl) biguanide; N1 N5-bis(3 5-dichlorophenyl) biguanide; N1 N5-bis(4-bromophenyl) biguanide; N1-benzyl-N5-(pyridine-3-yl)methyl biguanide; N1-(phenethyl)-N5-propyl biguanide; N1-(phenethyl)-N5-cyclopropylmethyl biguanide; N1-(phenethyl)-N5-cycloheptyl biguanide; N1 N5-bis(phenethyl) biguanide; N1 N1 N5-trimethyl biguanide; N1 N1-dimethyl-N5-butyl biguanide; N1 N1-dimethyl-N5-(butane-2-yl) biguanide; N1 N1-dimethyl-N5-t-butyl biguanide; N1 N1-dimethyl-N5-pentyl biguanide; N1 N1-dimethyl-N5-methoxycarbonylethyl biguanide; N1 N1-dimethyl-N5-cycloheptyl biguanide; N1 N1-dimethyl-N5-cyclopropylmethyl biguanide; N1 N1-dimethyl-N5-(4-bromo)phenyl biguanide; N1 N1-dimethyl-N5-(furan-2-yl)methyl biguanide; N1 N1-dimethyl-N5-(pyridine-3-yl)methyl biguanide; N1 N1-dimethyl-N5-benzyl biguanide; N1 N1-dimethyl-N5-(phenethyl) biguanide; N1 N1-diethyl-N5-(3-chloro)phenyl biguanide; N1 N1-dipropyl-N5-(3-chloro)phenyl biguanide; N1 N1-(ethyl)(propyl)-N5-(4-chloro)phenyl biguanide; N1 N1-dipropyl-N5-(isoquionline-5-yl) biguanide; N1 N1-dihexyl-N5-(3-chloro)phenyl biguanide; N1 N1 N5 N5-tetraethyl biguanide; N1 N1-diethyl-N5 N5-(cyclohexyl)(methyl) biguanide; N1 N1-dipropyl-N5 N5-diethyl biguanide; N1 N1-dipropyl-N5 N5-(methyl)(phenethyl) biguanide; N1 N1-dipropyl-N5 N5-(4-hydroxylphenyl)(phenyl) biguanide; or N1 N1 N5 N5-bis((benzyl)(methyl)) biguanide.
Meanwhile  the pharmaceutically acceptable salt of the compound of Formula 1 according to the present invention may be an acid addition salt formed using an organic acid or inorganic acid. Examples of the organic acid include formic acid  acetic acid  propionic acid  lactic acid  butyric acid  isobutyic acid  trifluoroacetic acid  malic acid  maleic acid  malonic acid  fumaric acid  succinic acid  succinic acid monoamide  glutamic acid  tartaric acid  oxalic acid  citric acid  glycolic acid  glucuronic acid  ascorbic acid  benzoic acid  phthalic acid  salicylic acid  anthranyl acid  dichloroacetic acid  aminooxy acetic acid  benzensulfonic acid  p-toluenesulfonic acid and methanesulfonic acid  and examples of the inorganic acid include hydrochloric acid  bromic acid  sulfuric acid  phosphoric acid  nitric acid  carbonic acid and boric acid. The above-mentioned acid addition salt may be prepared by a general method of preparing a salt  including a) directly mixing the compound of Formula 1 and an acid  b) dissolving one of the compound and an acid in a solvent or a hydrated solvent and mixing the resulting solution with the other element  or c) dissolving the compound of Formula 1 and an acid in a solvent or a hydrated solvent  respectively  and mixing them.
When the compound of Formula 1 has an acid group such as a carboxyl group and a sulfonic group  the compound may become an zwitterionic salt  and examples of the salt may include alkali metal salts (i.e.  a sodium salt  a potassium salt  etc.)  alkali earth metal salts (i.e.  a calcium salt  a magnesium salt  etc.)  inorganic acid-based salts (i.e.  an aluminum salt  an ammonium salt  etc.)  and basic addition salts (i.e.  trimethyl amine  triethyl amine  pyridine  picoline  ethanolamine  diethanolamine  triethanolamine  dicyclohexyl amine  N  N’-dibenzylethylenediamine-based salt  etc.). In addition  the salt of the compound of Formula 1 may be a basic amino acid-based salt (i.e.  an arginine  lysine  or ornitine-based salt) or an acidic amino acid-based salt (i.e.  an aspartame-based salt).
In one embodiment  the pharmaceutically acceptable salt of the compound of Formula 1 may be a salt with an acid selected from the group consisting of formic acid  acetic acid  propionic acid  lactic acid  butyric acid  isobutyic acid  trifluoroacetic acid  malic acid  maleic acid  malonic acid  fumaric acid  succinic acid  succinic acid monoamide  glutamic acid  tartaric acid  oxalic acid  citric acid  glycolic acid  glucuronic acid  ascorbic acid  benzoic acid  phthalic acid  salicylic acid  anthranyl acid  benzensulfonic acid  p-toluenesulfonic acid  methanesulfonic acid  dichloroacetic acid  aminooxy acetic acid  hydrochloric acid  bromic acid  sulfuric acid  phosphoric acid  nitric acid  carbonic acid and boric acid.
The compound of Formula 1 according to the present invention may be prepared by multiple methods.
In one embodiment  a method of preparing the compound of Formula 1 includes reacting a compound of Formula 2 with a dicyanoamide in at least one organic solvent to obtain a compound of Formula 3; and reacting the compound of Formula 3 with a compound of Formula 4 in at least one organic solvent to obtain the compound of Formula 1.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

In these formulas  R1  R2  R3 and R4 are the same as defined in Formula 1. The method may be illustrated in Reaction Scheme 1  and will be described in steps.
[Reaction Scheme 1]

In the method of preparing the compound of Formula 1  the cyanoguanidine compound of Formula 3 used as an intermediate may be prepared by reacting substituted amine of Formula 2 with a dicyanoamide such as sodium or potassium dicyanoamide in at least one organic solvent to convert the substituted amine into the cyanoguanidine compound of Formula 3 and then refluxing the cyanoguanidine compound of Formula 3 with the compound of Formula 4 in at least one organic solvent.
An amount of the sodium cyanoamide used in the preparation of the cyanoguanidine compound of Formula 3 is equivalent to approximately 1 to 2 moles with respect to the compound of Formula 2  and examples of the organic solvent used herein may be ethanol  isopropanol  n-butanol  t-butanol  etc. The reaction temperature is in the range of 60 to 150 °C.
After the cyanoguanidine compound of Formula 3 obtained above is dissolved in at least one organic solvent (i.e.  ethanol  isopropanol  n-butanol or 1 4-dioxane)  the compound of Formula 4 is added and then refluxed with stirring. Here  an amount of the compound of Formula 4 is equivalent to approximately 1 to 2 moles with respect to the compound of Formula 3  and the reaction temperature is in the range of the reflux temperature of the solvent used (i.e.  room temperature to 140 °C for butanol). When the reaction is completed  the resulting product is filtered  and the pH of the reaction solution is controlled to approximately 4 to 5 using an acid  such as hydrochloric acid. A solution produced as such is concentrated and purified  thereby yielding the compound of Formula 1 or a pharmaceutically acceptable salt thereof.
Compared to conventional drugs  only a small amount of the compound of Formula 1 or the pharmaceutically acceptable salt thereof produced as such may exhibit effects on activation of AMPK and inhibition of cancer cell proliferation  which can be confirmed in the following example. It is known that AMPK activation inhibits cancer  decreases blood sugar  and lowers lipid concentration  as described above. Therefore  the compound of Formula 1 or the pharmaceutically acceptable salt thereof may be useful to treat diabetes mellitus  obesity  hyperlipidemia  hypercholesterolemia  fatty liver  coronary artery disease  osteoporosis  polycystic ovarian syndrome  metabolic syndrome  etc.  as well as cancer.
Another aspect of the present invention provides a drug comprising the compound of Formula 1 or the pharmaceutically acceptable salt thereof as an active ingredient.
Still another aspect of the present invention provides a pharmaceutical composition comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient to treat a disease selected from the group consisting of diabetes mellitus  obesity  hyperlipidemia  hypercholesterolemia  fatty liver  coronary artery disease  osteoporosis  polycystic ovarian syndrome  metabolic syndrome  cancer  muscle pain  myocyte damage and rhabdomyolysis  a use of the compound of Formula 1 or the pharmaceutically acceptable salt thereof to treat the above-mentioned disease  and a method of treating the disease including administering a therapeutically effective amount of the compound of Formula 1 or the pharmaceutically acceptable salt thereof to a subject.
In one embodiment  diabetes mellitus may be non-insulin dependent diabetes mellitus.
In one embodiment  the cancer may be breast cancer  colorectal cancer  gastric cancer  liver cancer  lung cancer  blood cancer  prostate cancer  brain cancer  pancreatic cancer  ovarian cancer  or endometrial cancer.
The pharmaceutical composition of the present invention comprises at least one pharmaceutically acceptable carrier in addition to an active ingredient. As used in the present invention  “pharmaceutically acceptable carrier” refers to a known pharmaceutically acceptable excipient  which is useful to formulate a pharmaceutically active compound for administration to a subject  and is substantially non-toxic and non-sensitive under the conditions it is used. An exact ratio of the excipient is determined by standard pharmaceutical practice  as well as solubility  chemical characteristics and selected route of administration of the active compound.
The pharmaceutical composition of the present invention may be formulated in a suitable type for a desired administration method using adjuvants such as a excipient  a disintegrating agent  a sweetening agent  a binder  a coating agent  a swelling agent  a lubricating agent  a glossing agent  a flavoring agent  etc  which are suitable and physiologically acceptable.
The pharmaceutical composition may be formulated as a tablet  a capsule  a pill  a granule  powder  an injection or a liquid  but is not limited thereto.
Meanwhile  in the present invention  “subject” refers to warm-blooded animals such as mammals with a specific disease  disorder or illness  for example  including humans  orangutans  mice  rats  dogs  cows  chickens  pigs  goats  sheep  etc.  but the present invention is not limited thereto.
In addition  “treating” includes relieving a symptom temporarily or permanently  eliminating a cause of the symptom  and preventing or lowing occurrence of the symptom  progression of the disease  disorder or illness  but the present invention is not limited thereto.
An effective amount of the active ingredient of the pharmaceutical composition of the present invention refers to an amount required for treating a disease. Therefore  the effective amount may be controlled by various factors such as type and severity of a disease  kinds and contents of an active ingredient and other ingredients contained in the composition  a type of formulation  age  body weight  general medical conditions  sex and diet of a subject  duration and route for administration  release rate of the composition  treatment regime  and drugs simultaneously used. For example  to a male adult having a body weight of 60 kg  the compound of Formula 1 may be administered once to several times a day in a dosage range from 0.5 to 100 mg/kg of body weight. However  the dosage may vary depending on various factors listed above  and in some cases  a smaller or larger amount than the above-mentioned dosage of the composition may be administered.

?Advantageous Effects?
A biguanide derivative of Formula 1 according to the present invention can exhibit excellent effects on activation of AMPK and inhibition of cancer cell proliferation in a low dose compared to conventional drugs  and thus  can be useful to treat diabetes mellitus  obesity  hyperlipidemia  hypercholesterolemia  fatty liver  coronary artery disease  osteoporosis  polycystic ovarian syndrome  metabolic syndrome  cancer  etc.

?Best Mode?
The advantages and features of the present invention and the method of revealing them will be explicit from the following examples described in detail. However  it is to be distinctly understood that the present invention is not limited thereto but may be otherwise variously embodied and practiced. It is obvious that the following examples are to complete the disclosure of the invention and to indicate the scope of the present invention to a skilled artisan completely  and the present invention will be defined only by the scope of the claims.

[Examples]
Example 1: Preparation of N1-hexyl-N5-propyl biguanide hydrochloride

(1-1) Synthesis of 1-hexyl-3-cyanoguanidine
While a solution prepared by dissolving 1-hexyl amine (3.6 g  35.9 mmol) in n-butanol (30 ml) was stirred  sodium dictanoamide (3.5 g  39.5 mmol) and concentrated hydrochloric acid (3.4 ml  39.5 mmol) were added thereto at room temperature. The mixed solution was refluxed with stirring for 24 hours. After confirming the completion of the reaction  the generated sodium chloride was removed by filtering the reaction mixture  and then the filtered solution was concentrated under reduced pressure. After the concentrate was filtered  the filter-cake was washed with distilled water (30 ml). The filter-cake was vacuum-dried  thereby obtaining a target compound as a white solid (3.4 g  58%). The compound was used in a subsequent reaction without another step of purification.
(1-2) Preparation of N1-hexyl-N5-propyl biguanide hydrochloride
Concentrated hydrochloric acid (0.47 ml  5.31 mmol) was added to a solution prepared by dissolving 1-propyl amine (0.58 g  5.84 mmol) in n-butanol (10ml)  and the mixed solution was stirred for 30 minutes at room temperature. The compound obtained in the previous step (1-1) (0.89 g  5.31 mmol) was added to the reaction mixture and refluxed with stirring for 24 hours. The mixture was concentrated under reduced pressure  and the concentrate was purified using flash column chromatography (dichloromethane:methanol = 9:1). A 6N methanol hydrochloride solution (1 ml) was added to the compound to dissolve  and the mixture was concentrated under reduced pressure  thereby obtaining a target compound as a white solid (0.92 g  65%).
1H NMR (600 MHz  DMSO-d6) d 7.46(br s  2H)  6.81(br s  2H)  3.05(m  4H)  1.44(m  4H) 1.25 (m  6H)  0.85 (t  3H  J = 7.2 Hz); mp 148-149 °C
Target compounds of the following Examples 2 to 40 were prepared by the same method as described in Example 1  except that an amine compound corresponding to the target compound was used instead of 1-hexyl amine and 1-propyl amine respectively used in steps (1-1) and (1-2) of Example 1.
Example 2: N1-hexyl-N5-cyclopropylmethyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 7.50(br s  2H)  6.83(br s  3H)  3.07(m  2H)  2.97(m  2H)  1.42(m  2H)  1.26(m  6H)  0.95(m  1H)  0.85(t  3H  J = 6.6 Hz)  0.42(m  2H)  0.17(d  2H  J = 4.8 Hz); mp 162-163 °C
Example 3: N1-hexyl-N5-cyclohexylmethyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 8.22(br s  2H)  3.12(m  2H)  2.73(dd  2H  J = 7.5  7.2 Hz)  1.96 (m  2H)  1.68(m  2H)  1.50-1.59 (m  7H)  1.46(m  2H)  1.38(m  2H)  1.23-1.36(m  4H)  0.87(t  3H  J = 7.2 Hz)
Example 4: N1-hexyl-N5-benzyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 8.45(br s  1H)  7.24-7.36(m  5H)  6.92(br s  2H)  4.33(d  2H  J = 6.0 Hz)  3.08(m  2H)  1.21-1.44 (m  8H)  0.86(m  3H) : mp 121-123 °C
Example 5: N1 N5-bis(4-chlorophenyl) biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 10.02(br s  2H)  7.54(br s  2H)  7.38(d  4H  J = 9.0 Hz)  7.32(d  4H  J = 9.0 Hz); mp 263-264 °C
Example 6: N1 N5-bis(3-chlorophenyl) biguanide hydrochloride

1H NMR (400 MHz  DMSO-d6) d 9.23(br s  2H)  7.54(dd  2H  J = 2.0  2.0 Hz)  7.32(dd  2H  J = 8.2  8.2 Hz)  7.23(ddd  2H  J = 8.2  2.0  0.8 Hz)  7.15 (br s  2H)  7.11(ddd  2H  J = 8.2  2.0  0.8 Hz); mp 129-131 °C
Example 7: N1-(4-chloro)phenyl-N5-(4-methoxy)phenyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 9.87(br s  1H)  9.75(br s  1H)  7.49(br s  1H)  7.35(d  2H  J = 9.0 Hz)  7.33(d  2H  J = 9.0 Hz)  7.30(br s  1H)  7.20(d  2H  9.0 Hz)  6.91(d  2H  9.0 Hz)  3.72(s  3H); mp 247-249 °C
Example 8: N1 N5-bis(3-chloro-4-methoxyphenyl) biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 9.11(br s  1H)  7.46(d  2H  J = 2.4 Hz)  7.20(dd  2H  J = 9.0  2.4 Hz)  7.09(d  2H  J = 9.0 Hz)  7.05(br s  1H)  3.83(s  6H); mp 203-204 °C
Example 9: N1 N5-bis(3 4-dichlorophenyl) biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 10.3(br s  1H)  7.73(br s  1H)  7.63(s  2H)  7.59(d  2H  J = 9.0 Hz)  7.28(d  2H  J = 9.0 Hz); mp 255-257 °C
Example 10: N1 N5-bis(3 5-dichlorophenyl) biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 10.48(br s  1H)  7.85(br s  1H)  7.40(d  4H  J = 1.8 Hz)  7.35(d  2H  J = 1.8 Hz); mp 250-251 °C
Example 11: N1 N5-bis(4-bromophenyl) biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 10.17(br s  1H)  7.59(br s  1H)  7.49(d  4H  J = 7.2 Hz)  7.27(d  4H  J = 7.2 Hz); mp 242-243 °C
Example 12: N1-benzyl-N5-(pyridine-3-yl)methyl biguanide hydrochloride

1H NMR (400 MHz  DMSO-d6) d 8.67(s  1H)  8.57(dd  1H  J = 4.8  1.6 Hz)  8.45(br s  2H)  7.93(dd  1H  J = 7.6  1.6 Hz)  7.90(br a  1H)  7.45(dd  1H  J = 7.6  4.8 Hz)  7.18-7.34(m  5H)  7.21(br s  1H)  4.32(m  2H)  4.06(s  2H); mp 138-140 °C
Example 13: N1-(phenethyl)-N5-propyl biguanide hydrochloride

1H NMR (400 MHz  DMSO-d6) d 7.33(br s  2H)  7.21-7.37(m  5H)  6.87(br s  2H)  3.33(m  2H)  2.79(m  2H)  1.46(m  2H)  0.87(t  3H  J = 7.2 Hz); mp 126-128 °C
Example 14: N1-(phenethyl)-N5-cyclopropylmethyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 7.20-7.32(m  5H)  3.32(m  2H)  2.98(t  2H  J = 6.0 Hz)  2.76(m  2H)  0.96(m  1H)  0.41(dd  2H  J = 7.8  1.8 Hz)  0.19(dd  2H  J = 4.8  1.8 Hz); mp 143-146 °C
Example 15: N1-(phenethyl)-N5-cycloheptyl biguanide hydrochloride

1H NMR (400 MHz  DMSO-d6) d 8.08(br s  2H)  7.10-7.38(m  5H)  6.86(br s  2H)  3.66(m  1H)  3.33(m  4H)  3.15(m  2H)  2.77(m  2H)  1.81(m  2H)  1.36-1.57(m  6H); mp 135-137 °C
Example 16: N1 N5-bis(phenethyl) biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 8.29(br s  2H)  7.34(m  2H)  7.26(m  3H)  3.00(t  2H  J = 9.0 Hz)  2.92(t  2H  J = 9.0 Hz); mp 204-205 °C
Example 17: N1 N1 N5-trimethyl biguanide hydrochloride

1H NMR (400 MHz  DMSO-d6) d 7.98(br s  2H)  6.91(br s  1H)  2.82(s  9H); mp 175-177 °C
Example 18: N1 N1-dimethyl-N5-butyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 7.95(br s  2H)  6.93(br s  1H)  2.85(s  6H)  2.72(t  2H  J = 7.2 Hz)  1.49(m  2H)  1.31(m  2H)  0.86(t  3H  J = 7.8 Hz); mp 131-133 °C
Example 19: N1 N1-dimethyl-N5-(butane-2-yl) biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 7.92(br s  2H)  6.94(br s  1H)  3.05(m  1H)  2.87(s  6H)  1.60(m  1H)  1.43(m  1H)  1.15(d  3H  J = 5.4 Hz)  0.88(t  3H)  J = 7.2 Hz); mp 110-112 °C
Example 20: N1 N1-dimethyl-N5-t-butyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 8.05(br s  2H)  6.93(br s  1H)  2.85(s  6H)  1.23(s  9H); mp 186-187 °C
Example 21: N1 N1-dimethyl-N5-pentyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 7.94(br s  2H)  6.95(br s  1H)  2.86(s  6H)  2.73(t  2H  J = 7.2 Hz)  1.54(m  2H)  1.27(m  4H)  0.86(t  3H  J = 7.2 Hz); mp 131-133 °C
Example 22: N1 N1-dimethyl-N5-(methoxycarbonylethyl)biguanide hydrochloride

1H NMR (400 MHz  DMSO-d6) d 8.12(br s  2H)  6.93(br s  1H)  3.61(s  3H)  2.97(t  2H  J = 4.8 Hz)  2.69(t  2H  J = 4.8 Hz); mp 100-102 °C
Example 23: N1 N1-dimethyl-N5-cycloheptyl biguanide hydrochloride

1H NMR (400 MHz  DMSO-d6) d 8.01(br s  2H)  6.93(br s  1H)  3.10(m  1H)  2.86(s  6H)  1.91(m  2H)  1.63(m  2H)  1.38-1.53(m  6H)  1.34(m  2H); mp 132-133 °C
Example 24: N1 N1-dimethyl-N5-cyclopropylmethyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 8.08(br s  2H)  6.94(br s  1H)  2.86(s  6H)  2.63(d  2H  J = 4.8 Hz)  1.01(m  1H)  0.51(m  2H)  0.31(m  2H); mp 117-118 °C
Example 25: N1 N1-dimethyl-N5-(4-bromo)phenyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 10.07(br s  1H)  7.68(br s  2H)  7.40(d  2H  J = 9.0 Hz)  7.35(d  2H  J = 9.0 Hz)  2.92(s  6H); mp 272-273 °C
Example 26: N1 N1-dimethyl-N5-(furan-2-yl)methyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 7.59(m  1H)  7.38(br s  2H)  6.77(br s  1H)  6.40(dd  1H  J = 2.7  1.5 Hz)  6.31(s  1H)  4.31(d  2H  J = 6.0 Hz)  2.92(s  6H); mp 176-177 °C
Example 27: N1 N1-dimethyl-N5-(pyridine-3-yl)methyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 8.65(d  1H  J = 1.8 Hz)  8.62(br s  2H)  8.52(dd  1H  J = 4.8  1.8 Hz)  7.93(ddd  1H  J = 7.8  1.8  1.8 Hz)  7.40(dd  1H  J = 7.8  4.8 Hz)  4.01(s  2H)  3.34(s  6H); mp 112-114 °C
Example 28: N1 N1-dimethyl-N5-benzyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 8.51(br s  2H)  7.46(m  2H)  7.36(m  3H)  6.91(br s  1H)  3.95(s  2H)  2.83(s  6H); mp 151-152 °C
Example 29: N1 N1-dimethyl-N5-(phenethyl) biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 8.24(br s  2H)  7.20-7.34(m  6H)  3.00(t  2H  J = 7.8 Hz)  2.92(s  6H)  2.91(t  2H  J = 7.8 Hz); mp 155-157 °C
Example 30: N1 N1-diethyl-N5-(3-chloro)phenyl biguanide hydrochloride

1H NMR (400 MHz  DMSO-d6) d 9.91(br s  1H)  7.64(br s  2H)  7.60(dd  1H  J = 2.0  2.0 Hz)  7.29(d  1H  J = 8.0 Hz)  7.26(ddd  1H  J = 8.0  1.6  1.6 Hz)  7.05(ddd  1H  J = 8.0  1.6  1.6 Hz)  6.92(br s  1H)  3.31(m  4H)  1.08(m  6H); mp 219-220 °C
Example 31: N1 N1-dipropyl-N5-(3-chloro)phenyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 10.20(br s  1H)  7.70(br s  2H)  7.63(d  1H  J = 1.8 Hz)  7.30(m  2H)  7.05(d  1H  J = 7.8 Hz)  3.27(m  4H)  1.56(m  4H)  0.85(m  6H); mp 201-202 °C
Example 32: N1 N1-(ethyl)(propyl)-N5-(4-chloro)phenyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 9.71(br s  1H)  8.69(br s  1H)  7.42(d  2H  J = 6.6 Hz)  7.35(d  2H  J = 6.6 Hz)  7.23(br s  2H)  2.90(q  2H  J = 7.2 Hz)  2.80(t  2H  J = 7.2 Hz)  1.61(m  2H)  1.18(t  3H  J = 7.2 Hz)  0.91(t  3H  J = 7.2 Hz); mp 110-112 °C
Example 33: N1 N1-dipropyl-N5-(isoquinoline-5-yl) biguanide hydrochloride

1H NMR (400 MHz  DMSO-d6) d 9.27(br s  2H)  8.64(d  1H  J = 8.4 Hz)  7.97(m  2H)  7.78(ddd  1H  J = 8.4  6.0  2.4 Hz)  7.71(d  1H  J = 7.2 Hz)  7.38(br s  1H)  7.23(d  1H  J = 7.2 Hz)  3.21(t  2H  J = 7.6 Hz)  0.85(t  3H  J = 7.2 Hz); mp 191-192 °C
Example 34: N1 N1-dihexyl-N5-(3-chloro)phenyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 10.07(br s  1H)  7.67(br s  1H)  7.62(dd  1H  J = 2.4  1.8 Hz)  7.29(dd  1H  J = 7.8  7.8 Hz)  7.25(ddd  1H  J = 7.8  1.8  1.2 Hz)  7.05(ddd  1H  J = 7.8  2.4  1.2 Hz)  3.27(t  4H  J = 7.8 Hz)  1.52(m  4H)  1.18-1.34(m  12H)  0.82(m  6H); mp 187-188 °C
Example 35: N1 N1 N5 N5-tetraethyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 8.76(br s  2H)  6.90 (br s  1H)  2.87 (q  8H  J = 7.2 Hz)  1.17 (t  12H  J = 7.2 Hz); mp 140-141 °C
Example 36: N1 N1-diethyl-N5 N5-(cyclohexyl)(methyl) biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 8.73 (br s  2H)  6.90 (br s  1H)  3.26 (q  4H  J = 7.2 Hz)  2.86(m  1H)  2.49 (s  3H)  1.99 (m  2H)  1.74 (m  2H)  1.20-1.30 (m  6H)  1.02 (t  6H  J = 7.2 Hz); mp 115-117 °C
Example 37: N1 N1-dipropyl-N5 N5-diethyl biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 6.92 (br s  1H)  6.89(br s  1H)  3.30 (m  4H)  3.21 (m  4H)  1.51 (m  4H)  1.08 (t  6H  J = 6.0 Hz)  0.84(t  6H  J = 6.6 Hz); mp 151-152 °C
Example 38: N1 N1-dipropyl-N5 N5-(methyl)(phenethyl) biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 7.16-7.30 (m  5H)  6.90 (br s  2H)  3.47 (t  2H  J = 7.8 Hz)  3.18 (t  4H  J = 7.2 Hz)  2.84 (s  3H)  2.76 (t  2H  J = 7.8 Hz)  1.49 (m  4H)  0.83 (m  6H); mp 110-111 °C
Example 39: N1 N1-dipropyl-N5 N5-(4-hydroxylphenyl)(phenyl) biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 9.86 (br s  1H)  7.20-7.38 (m  5H)  7.16 (d  2H  J = 8.4 Hz)  6.84(br s  2H)  6.81 (d  2H  J = 8.4 Hz)  3.10 (t  4H  J = 5.4 Hz)  1.54 (m  4H)  0.89 (t  6H  J = 7.2 Hz); mp 189-190 °C
Example 40: N1 N1 N5 N5-bis((benzyl)(methyl)) biguanide hydrochloride

1H NMR (600 MHz  DMSO-d6) d 7.59(m  4H)  7.41(m  6H)  7.00(br s  2H)  4.07(s  4H)  2.47(s  6H); mp 141-142 °C
[Experimental Examples]
The compounds synthesized by the method described in the examples of the present invention were treated to cancer cells  according to a method to be described in the following Experimental Examples  to measure an effect on inhibition of cancer cell proliferation. A simple experimental method is as follows:
Experimental Example 1: Measurement of effect on inhibition of cancer cell proliferation
HCT116 cells derived from human colorectal cancer were used  and an effect of a biguanide derivative on the inhibition of cancer cell proliferation was confirmed by measuring a concentration value (cell growth inhibition concentration  GIC50) at which 50% of the cell growth was inhibited using a 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenytetrazolium bromide (MTT) reagent.
First  HCT116 cells were put on a 96-well plate and cultured in a DMEM medium containing 10% bovine serum for 24 hours to each have cell count of approximately 5000. Subsequently  to obtain the GIC50 value of each compound  100 µM (or 200 µM)  25 µM  6.25 µM  1.56 µM or 0.39 µM of the compound was treated to each culture medium and then incubated for 48 hours. To confirm living cells after treatment with the compound  MTT was added to each culture medium and further incubated for 3 hours. Generated formazane crystal was dissolved using dimethyl sulfoxide (DMSO) and absorbance of the solution was measured at 560 nm. After the 48-hourincubation  a ratio of a cell count cultured on a well plate not treated with the compound to a cell count on a well plate treated with compounds synthesized in the examples was indicated as cell viability (%) according to each administered concentration. A cell viability curve was plotted using the cell viability (%)  and the calculated concentration value (GIC50) of the compound  at which 50% of the growth was inhibited was  to confirm an effect on the inhibition of cancer cell proliferation.
Results of cancer cell growth inhibition effect are shown in Table 1.
?Table 1?
Example GIC50 (uM) @ HCT116 Example GIC50 (uM) @ HCT116
Metformin HCl 2172
1 23.8 21 >200
2 20.7 22 >200
3 >100 23 >200
4 8.4 24 >200
5 3.2 25 >100
6 >100 26 >200
7 18.3 27 >200
8 17.0 28 >200
9 5.9 29 >200
10 6.3 30 28.3
11 3.1 31 7.6
12 >200 32 >200
13 47.6 33 >100
14 47.0 34 7.8
15 17.2 35 >200
16 >100 36 >200
17 >200 37 >100
18 >200 38 36.0
19 >200 39 >100
20 >200 40 >100

Experimental Example 2: Measurement of effect on AMPK activation
MCF7 cells derived from human breast cancer cells were used  and an effect of a biguanide derivative on 5""-AMP-activated protein kinase(AMPK) activation was confirmed using an AMPKa immunoassay kit (Invitrogen  catalog No. KHO0651).
The MCF7 cells were put on a 6-well plate and incubated in a DMEM medium containing 10% fetal bovine serum in an incubator to which 5% CO2 was supplied to have a cell count of approximately 5?105. 50 µM of derivatives synthesized in the examples were treated to the each culture medium  and the cells were incubated for 24 hours. Subsequently  the cells were lysed by a method presented in the operation manual of the AMPKa immunoassay kit  and 20 µg of cell lysates were yielded through protein assay. A degree of phosphorylation of an AMPKa threonine 172nd residue (Thr172) from the cell lysates were confirmed according to a method presented in the operation manual of the AMPKa immunoassay kit to thereby obtain results. A degree of the AMPKa activation by a biguanide derivative was exhibited as a degree of phosphorylated AMPKa in the cells cultured in the presence of the compounds synthesized in the examples based on phosphorylated AMPKa in cells cultured without treating the biguanide derivative.
In addition  an experiment was performed in the same manner as described in Experimental Example 2 using metformin as a control group  and the results of an effect on AMPK activation were compared to the effect on AMPK activation when 1 mM metformin was treated.
The results are shown in Table 2.
?Table 2?
Example AMPK Activation
0 50 µM fold
Metformin HCl 6.8 21.5 (@ 1mM) 3.2
1 5.3 35.6 6.7
2 5.3 29.3 5.5
3 6.8 13.1 1.9
4 2.5 15.5 6.2
5 N.D
6 6.8 4.6 0.7
7 N.D
8 N.D
9 N.D
10 N.D
11 N.D
12 5.3 7.8 1.5
13 5.3 36.2 6.8
14 5.3 31.2 5.9
15 4.6 7.0 1.5
16 6.8 4.7 0.7
17 4.9 5.5 1.1
18 5.3 4.6 0.9
19 5.3 3.6 0.7
20 5.3 4.6 0.9
21 5.3 5.6 1.1
22 5.3 3.2 0.6
23 5.3 5.4 1.0
24 5.3 5.1 1.0
25 5.3 23.1 4.4
26 5.3 8.9 1.7
27 5.3 4.6 0.9
28 4.9 5.9 1.2
29 5.3 14.7 2.8
30 2.5 19.3 7.7
31 N.D
32 5.3 5.2 1.0
33 6.8 11.3 1.7
34 N.D
35 5.3 3.8 0.7
36 5.3 4.5 0.8
37 5.3 33.3 6.3
38 5.3 35.8 6.8
39 6.8 28.8 4.2
40 6.8 11.3 1.7

Consequently  it was seen that the derivatives synthesized in the examples effectively inhibited the viability of cancer cells  particularly  colorectal cancer cells in terms of the effect on inhibition of cancer cell proliferation. In addition  it could be observed that the compounds exhibiting a greater effect on AMPKa activation at a concentration 20 times lower than the control group  metformin  may have an effect at least 20 times greater than the control group.

WE CLAIM:-
?Claim 1?
A compound of Formula 1 or a pharmaceutically acceptable salt thereof:
[Formula 1]

wherein R1  R2  R3 and R4 are independently hydrogen or a non-hydrogen substituent selected from the group consisting of C1-12 alkyl unsubstituted or substituted with at least one non-hydrogen substituent selected from the group consisting of C3-10 cycloalkyl  C5-12 aryl  C5-12 heteroaryl  hydroxyl  halogen and C1-4 alkoxycarbonyl; C3-10 cycloalkyl; C1-12 alkoxy; C5-12 aryl; C5-12 heteroaryl; hydroxyl; and halogen  and
the aryl and heteroaryl are unsubstituted or substituted with at least one non-hydrogen substituent selected from the group consisting of C1-4 alkyl  C1-4 alkoxy  hydroxyl and halogen.
?Claim 2?
The compound of Formula 1 or a pharmaceutically acceptable salt thereof of Claim 1  wherein R1  R2  R3 and R4 are independently hydrogen or a non-hydrogen substituent selected from the group consisting of unsubstituted C1-7 alkyl; C1-6 alkyl substituted with at least one non-hydrogen substituent selected from the group consisting of unsubstituted C3-7 cycloalkyl  C5-12 aryl  C5-12 heteroaryl  hydroxyl  halogen and C1-4 alkoxycarbonyl; unsubstituted C3-7 cycloalkyl; C1-6 alkoxy; C5-12 aryl; C5-12 heteroaryl; hydroxyl; and halogen  and
the aryl and heteroaryl are unsubstituted or substituted with at least one non-hydrogen substituent selected from the group consisting of C1-4 alkyl  C1-4 alkoxy  hydroxyl and halogen.
?Claim 3?
The compound of Formula 1 or a pharmaceutically acceptable salt thereof of Claim 2  wherein R1 and R4 are independently non-hydrogen substituents selected from the group consisting of unsubstituted C1-7 alkyl; C1-6 alkyl substituted with at least one non-hydrogen substituent selected from the group consisting of unsubstituted C3-7 cycloalkyl  C5-12 aryl  C5-12 heteroaryl and C1-4 alkoxycarbonyl; unsubstituted C3-7 cycloalkyl; C5-12 aryl; and C5-12 heteroaryl 
R2 and R3 are hydrogen; unsubstituted C1-7 alkyl; C5-12 aryl; or C5-12 heteroaryl  and
the aryl and heteroaryl are unsubstituted or substituted with at least one non-hydrogen substituent selected from the group consisting of C1-4 alkyl  C1-4 alkoxy  hydroxyl and halogen.
?Claim 4?
The compound of Formula 1 or a pharmaceutically acceptable salt thereof of Claim 3  wherein R1 and R4 are independently non-hydrogen substituents selected from the group consisting of unsubstituted C1-7 alkyl; C1-4 alkyl substituted with at least one non-hydrogen substituent selected from the group consisting of unsubstituted C3-7 cycloalkyl  C5-12 aryl  C5-12 heteroaryl and C1-4 alkoxycarbonyl; unsubstituted C3-7 cycloalkyl; C5-12 aryl; and C5-12 heteroaryl 
R2 and R3 are hydrogen; unsubstituted C1-7 alkyl; C5-12 aryl; or C5-12 heteroaryl  and
the aryl and heteroaryl may be unsubstituted or substituted with at least one non-hydrogen substituent selected from the group consisting of C1-4 alkoxy  hydroxyl and halogen  and the aryl and heteroaryl are selected from the group consisting of phenyl  pyridinyl  furanyl and isoquinolinyl.
?Claim 5?
The compound of Formula 1 or a pharmaceutically acceptable salt thereof of Claim 4  wherein R1 is unsubstituted C1-7 alkyl; C1-4 alkyl substituted with C5-12 aryl or C5-12 heteroaryl; C5-12 aryl; or C5-12 heteroaryl 
R2 is hydrogen; or unsubstituted C1-7 alkyl 
R3 is hydrogen; unsubstituted C1-7 alkyl; C5-12 aryl; or C5-12 heteroaryl 
R4 is a non-hydrogen substituent selected from the group consisting of unsubstituted C1-7 alkyl; C1-4 alkyl substituted with at least one non-hydrogen substituent selected from the group consisting of unsubstituted C3-7 cycloalkyl  C5-12 aryl  C5-12 heteroaryl and C1-4 alkoxycarbonyl; unsubstituted C3-7 cycloalkyl; C5-12 aryl; and C5-12 heteroaryl  and
the aryl and heteroaryl may be unsubstituted or substituted with at least one non-hydrogen substituent selected from the group consisting of C1-4 alkoxy  hydroxyl and halogen  and the aryl and heteroaryl are selected from the group consisting of phenyl  pyridinyl  furanyl and isoquinolinyl .
?Claim 6?
The compound of Formula 1 or a pharmaceutically acceptable salt thereof of Claim 1  wherein the compound of Formula 1 is N1-hexyl-N5-propyl biguanide; N1-hexyl-N5-cyclopropylmethyl biguanide; N1-hexyl-N5-cyclohexylmethyl biguanide; N1-hexyl-N5-benzyl biguanide; N1 N5-bis(4-chlorophenyl) biguanide; N1 N5-bis(3-chlorophenyl) biguanide; N1-(4-chloro)phenyl-N5-(4-methoxy)phenyl biguanide; N1 N5-bis(3-chloro-4-methoxyphenyl) biguanide; N1 N5-bis(3 4-dichlorophenyl) biguanide; N1 N5-bis(3 5-dichlorophenyl) biguanide; N1 N5-bis(4-bromophenyl) biguanide; N1-benzyl-N5-(pyridine-3-yl)methyl biguanide; N1-(phenethyl)-N5-propyl biguanide; N1-(phenethyl)-N5-cyclopropylmethyl biguanide; N1-(phenethyl)-N5-cycloheptyl biguanide; N1 N5-bis(phenethyl) biguanide; N1 N1 N5-trimethyl biguanide; N1 N1-dimethyl-N5-butyl biguanide; N1 N1-dimethyl-N5-(butan-2-yl) biguanide; N1 N1-dimethyl-N5-t-butyl biguanide; N1 N1-dimethyl-N5-pentyl biguanide; N1 N1-dimethyl-N5-methoxycarbonylethyl biguanide; N1 N1-dimethyl-N5-cycloheptyl biguanide; N1 N1-dimethyl-N5-cyclopropylmethyl biguanide; N1 N1-dimethyl-N5-(4-bromo)phenyl biguanide; N1 N1-dimethyl-N5-(furan-2-yl)methyl biguanide; N1 N1-dimethyl-N5-(pyridine-3-yl)methyl biguanide; N1 N1-dimethyl-N5-benzyl biguanide; N1 N1-dimethyl-N5-(phenethyl) biguanide; N1 N1-diethyl-N5-(3-chloro)phenyl biguanide; N1 N1-dipropyl-N5-(3-chloro)phenyl biguanide; N1 N1-(ethyl)(propyl)-N5-(4-chloro)phenyl biguanide; N1 N1-dipropyl-N5-(isoquionline-5-yl) biguanide; N1 N1-dihexyl-N5-(3-chloro)phenyl biguanide; N1 N1 N5 N5-tetraethyl biguanide; N1 N1-diethyl-N5 N5-(cyclohexyl)(methyl) biguanide; N1 N1-dipropyl-N5 N5-diethyl biguanide; N1 N1-dipropyl-N5 N5-(methyl)(phenethyl) biguanide; N1 N1-dipropyl-N5 N5-(4-hydroxylphenyl)(phenyl) biguanide; or N1 N1 N5 N5-bis((benzyl)(methyl)) biguanide.
?Claim 7?
The compound of Formula 1 or a pharmaceutically acceptable salt thereof of Claim 1  wherein the pharmaceutically acceptable salt is a salt with an acid selected from the group consisting of formic acid  acetic acid  propionic acid  lactic acid  butyric acid  isobutyic acid  trifluoroacetic acid  malic acid  maleic acid  malonic acid  fumaric acid  succinic acid  succinic acid monoamide  glutamic acid  tartaric acid  oxalic acid  citric acid  glycolic acid  glucuronic acid  ascorbic acid  benzoic acid  phthalic acid  salicylic acid  anthranyl acid  benzensulfonic acid  p-toluenesulfonic acid  methanesulfonic acid  dichloroacetic acid  aminooxy acetic acid  hydrochloric acid  bromic acid  sulfuric acid  phosphoric acid  nitric acid  carbonic acid and boric acid.
?Claim 8?
A method of preparing a compound of Formula 1  comprising:
reacting a compound of Formula 2 with dicyanoamide in at least one organic solvent to obtain a compound of Formula 3; and
reacting the compound of Formula 3 with a compound of Formula 4 in at least one organic solvent to obtain the compound of Formula 1:
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

Wherein R1  R2  R3 and R4 are independently hydrogen or a non-hydrogen substituent selected from the group consisting of C1-12 alkyl unsubstituted or substituted with at least one non-hydrogen substituent selected from the group consisting of C3-10 cycloalkyl  C5-12 aryl  C5-12 heteroaryl  hydroxyl  halogen and C1-4 alkoxycarbonyl; C3-10 cycloalkyl; C1-12 alkoxy; C5-12 aryl; C5-12 heteroaryl; hydroxyl; and halogen  and
the aryl and heteroaryl are unsubstituted or substituted with at least one non-hydrogen substituent selected from the group consisting of C1-4 alkyl  C1-4 alkoxy  hydroxyl and halogen.
?Claim 9?
A drug comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof according to Claim 1 as an active ingredient.
?Claim 10?
A pharmaceutical composition comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof according to Claim 1 as an active ingredient to treat a disease selected from the group consisting of diabetes mellitus  obesity  hyperlipidemia  hypercholesterolemia  fatty liver  coronary artery disease  osteoporosis  polycystic ovarian syndrome  metabolic syndrome  cancer  muscle pain  myocyte damage and rhabdomyolysis and pharmaceutical acceptable carrier.
?Claim 11?
The pharmaceutical composition of Claim 10  wherein the diabetes mellitus is insulin independent mellitus.
?Claim 12?
The pharmaceutical composition of Claim 10  wherein the cancer is breast cancer  colorectal cancer  gastric cancer  liver cancer  lung cancer  blood cancer  prostate cancer  brain cancer  pancreatic cancer  ovarian cancer  or endometrial cancer.
?Claim 13?
The pharmaceutical composition of Claim 10  wherein the pharmaceutical composition is formulated in the form of a tablet  a capsule  a pill  a granule  powder  an injection or a liquid.
Dated this 3rd day of August 2012

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=DeOZccqckvGtgmGX0/5P2Q==&loc=vsnutRQWHdTHa1EUofPtPQ==


Patent Number 280055
Indian Patent Application Number 1904/MUMNP/2012
PG Journal Number 06/2017
Publication Date 10-Feb-2017
Grant Date 08-Feb-2017
Date of Filing 03-Aug-2012
Name of Patentee IMMUNOMET THERAPEUTICS INC.
Applicant Address JLABS at Texas Medical Center 2450 Holcombe Blvd, Houston, TX 77021, United States Of America
Inventors:
# Inventor's Name Inventor's Address
1 KIM Sung Wuk 102-304 Samhwan Villa 250 Gumi-dong Bundang-gu Seongnam-si Gyeonggi-do 463-500 Republic of Korea
2 JUN Sung Soo 426-701 Hyundai Apt. Seohyeon-dong Bundang-gu Seongnam-si Gyeonggi-do 463-777 Republic of Korea
3 MIN Chang Hee 135-704 Daelim Apt. Haengdang-dong Seongdong-gu Seoul 133-775 Republic of Korea
4 KIM Young Woong 210-1412 Jugong 2 Danji Apt. Beop 1-dong Daedeok-gu Daejeon 306-752 Republic of Korea
5 KANG Min Seok 506-305 Gangchon Maeul 5 Danji Apt. Madu 2-dong Ilsandong-gu Goyang-si Gyeonggi-do 410-716 Republic of Korea
6 OH Byung Kyu 501 Rishiville Sky Apt. Jijok-dong Yuseong-gu Daejeon 305-330 Republic of Korea
7 PARK Se Hwan 321-51 Seokbong-dong Daedeok-gu Daejeon 306-810 Republic of Korea
8 KIM Yong Eun 298-3 Sanseong-dong Jung-gu Daejeon 301-819 Republic of Korea
9 KIM Duck 102-5 Nowon-dong 2-ga Buk-gu Daegu 702-082 Republic of Korea
10 LEE Ji Sun 119 Gwanpyeong-dong Yuseong-gu Daejeon 305-509 Republic of Korea
11 OH Ju Hoon 202-202 2Jugong Apt. 835 Noam-dong Gangneung-si Gangwon-do 210-932 Republic of Korea
PCT International Classification Number C07C 279/26
PCT International Application Number PCT/KR2011/000097
PCT International Filing date 2011-01-06
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 10-2011-0001438 2011-01-06 Republic of Korea
2 10-2010-0001021 2010-01-06 Republic of Korea