Case 1:16-cv-00954-SLR Document 1-1 Filed 10/17/16 Page 2 of 100 PageID #: 19
`
`(12) United States Patent
`Maruyama et al.
`
`US006346532B1
`US 6,346,532 B1
`(10) Patent No.:
`Feb. 12, 2002
`(45) Date of Patent:
`
`(*) Notice:
`
`(54) AMIDE DERIVATIVES OR SALTS THEREOF
`(75) Inventors: Tatsuya Maruyama; Takayuki Suzuki;
`Kenichi Onda; Masahiko Hayakawa;
`Hiroyuki Moritomo; Tetsuya
`Kimizuka; Tetsuo Matsui, all of
`Tsukuba (JP)
`(73) Assignee: Yamanouchi Pharmaceutical Co.,
`Ltd., Tokyo (JP)
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`(21) Appl. No.:
`09/529,096
`(22) PCT Filed:
`Oct. 15, 1998
`(86) PCT No.:
`PCT/JP98/04671
`§ 371 Date:
`Apr. 7, 2000
`§ 102(e) Date: Apr. 7, 2000
`(87) PCT Pub. No.: WO99/20607
`PCT Pub. Date: Apr. 29, 1999
`Foreign Application Priority Data
`(30)
`Oct. 17, 1997 (JP) ............................................. 9-285778
`(51) Int. Cl." .................... A61K 31/495; A61K 31/505;
`CO7D 239/02; CO7D 213/00; CO7D 249/00
`(52) U.S. Cl. .................... 514/252.1; 514/256; 544/330;
`544/332; 546/1; 546/152; 548/190; 548/214;
`548/186; 548/252, 548/260
`(58) Field of Search ................................. 544/330, 332;
`546/1, 152; 548/190, 214, 186, 252, 260:
`514/252.1, 256
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`5,223,614 A * 6/1993 Schromm et al. ........... 544/105
`5,541,197 A * 7/1996 Fisher et al. .........
`... 514/311
`5,553,475 A
`9/1996 Hayashi et al. ............... 72/225
`5,614,544 A 3/1997 Sohda et al. ................ 514/376
`6,048,884 A 4/2000 Maruyama et al. ......... 514/370
`6,177,454 B1
`1/2001 Maruyama et al. ......... 514/394
`
`
`
`FOREIGN PATENT DOCUMENTS
`
`DE
`JP
`WO
`
`* 6/1989
`3743265
`* 6/1989
`1021.8861
`* 11/1995
`95291.59
`OTHER PUBLICATIONS
`Konosu T. et al. “Triazole antif.”, Chem.Pharm. Bull., 39/10,
`2581–9, Oct. 1991.*
`* cited by examiner
`Primary Examiner—Richard L. Raymond
`Assistant Examiner—Sudhaker B. Patel
`(74) Attorney, Agent, or Firm—Finnegan, Henderson,
`Farabow, Garrett & Dunner, L.L.P.
`(57)
`ABSTRACT
`
`OH
`
`(I)
`
`2–1–
`
`R S2 JK, S2 J. G.)
`
`S.
`
`Amide derivatives represented by general formula (I) or
`salts thereof wherein each symbol has the following mean
`ing: ring B: an optionally substituted heteroaryl optionally
`fused with a benzene ring; X: a bond, lower alkylene or
`lower alkenylene optionally substituted by hydroxy or lower
`alkyl, carbonyl, or a group represented by —NH–(when X
`is lower alkylene optionally substituted by lower alkyl
`which may be bonded to the hydrogen atom bonded to a
`constituent carbon atom of ring B to form lower alkylene to
`thereby form a ring); A: a lower alkylene or a group
`represented by -(lower alkylene)—O—; R* and R'": the
`same or different and each hydrogen or lower alkyl; Rº:
`hydrogen or halogeno; and Z. nitrogen or a group repre
`sented by =CH-. The compounds are useful as a diabetes
`remedy which not only functions to both accelerate the
`secretion of insulin and enhance insulin sensitivity but has
`an antiobestic action and an antihyperlipemic action based
`on its selective stimulative action on a £3 receptor.
`14 Claims, No Drawings
`
`Sawai Ex. 1001
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`Case 1:16-cv-00954-SLR Document 1-1 Filed 10/17/16 Page 3 of 100 PageID #: 20
`
`US 6,346,532 B1
`
`1
`AMIDE DERIVATIVES OR SALTS THEREOF
`
`TECHNICAL FIELD
`The present invention relates to pharmaceuticals and,
`more particularly, it relates to novel amide derivatives or
`salts thereof and also to therapeutic agents for diabetes
`mellitus containing them as effective components.
`
`2
`example, WO 95/29159 describes substituted sulfonamide
`derivatives represented by the formula set forth below and
`discloses that due to their selective stimulating action to
`f{s-receptors in human being, they are useful against obesity,
`hyperglycemia, etc. However, this patent does not specifi
`cally disclose an insulin secretion promoting action and an
`insulin sensitivity potentiating action of those compounds.
`
`10
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`35
`
`BACKGROUND OF THE INVENTION
`Diabetes mellitus is a disease accompanied by continuous
`hyperglycemic state and is said to be resulted by action of
`many environmental factors and genetic factors. The main
`controlling factor for blood sugar is insulin, and it has been
`known that hyperglycemia is resulted by deficiency of
`insulin or by excess of factors which inhibit its action (such
`as genetic cause, lack of exercise, obesity and stress).
`Diabetes mellitus is classified into two main types. One is
`insulin-dependent diabetes mellitus (IDDM) caused by a
`lowering of insulin-secreting function of pancreas due to
`autoimmune diseases, and another is non-insulin-dependent
`diabetes mellitus (NIDDM), caused by a lowering of
`insulin-secreting function of pancrease due to pancreatic
`fatigue accompanied by continuous high insulin secretion.
`95% or more of diabetic patients in Japan are said to suffer
`from NIDDM, and an increase in the patients due to a
`change in daily life style is becoming a problem.
`As to the therapy of diabetes mellitus, dietetic treatment,
`therapeutic exercise and remedy of obesity are mainly
`conducted in mild cases while, when the disease progresses,
`oral antidiabetic drugs (for example, insulin secretion pro
`moters such as sulfonylurea compounds and insulin sensi
`tivity potentiators which potentiate the sensitivity of insulin)
`are administered. In severe cases, an insulin preparation is
`administered. However, there has been a brisk demand for
`creation of the drugs whereby higher control for blood sugar
`is possible, and development of antidiabetic drugs having a
`new mechanism and having high usefulness has been
`demanded.
`U.S. Pat. Nos. 4,396,627 and 4,478,849 describe phenyle
`thanolamine derivatives and disclose that those compounds
`are useful as drugs for obesity and for hyperglycemia.
`Action of those compounds is reported to be due to a
`stimulating action to fºs-receptors. Incidentally, it has been
`known that fl-adrenaline receptors are classified into fl 1, 52
`and 53 subtypes, that stimulation of 51-receptor causes an
`increase in heart rate, that stimulation of £2-receptor stimu
`lates decomposition of glycogen in muscles, whereby syn
`thesis of glycogen is inhibited, causing an action such as
`muscular tremor, and that stimulation of 53-receptor shows
`an anti-obesity and an anti-hyperglycemia action (such as
`decrease in triglyceride, decrease in cholesterol and increase
`in HDL-cholesterol).
`However, those fºs-agonists also have actions caused by
`stimulation of £1- and £2-receptors such as increase in heart
`rate and muscular tremor, and they have a problem in terms
`of side effects.
`Recently, it was ascertained that fl-receptors have differ
`ences to species, and it has been reported that even com
`60
`pounds having been confirmed to have a ?º-receptor selec
`tivity in rodential animals such as rats show an action due to
`stimulating action to £1- and £2-receptors in human being.
`In view of the above, investigations for compounds having
`a stimulating action which is selective to £3-receptor in
`human being have been conducted recently using human
`cells or cells where human receptors are expressed. For
`
`40
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`45
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`50
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`55
`
`65
`
`OH H R
`
`(R').
`
`(In the formula, the symbols should be referred to in the
`specification of this patent.)
`As such, there has been still a demand for creation of
`therapeutic agents for diabetes mellitus of a new type which
`have a highly clinical usefulness.
`DISCLOSURE OF THE INVENTION
`The present inventors have conducted an intensive inves
`tigation on compounds having both an insulin secretion
`promoting action and an insulin sensitivity potentiating
`action and found that novel amide derivatives show both a
`good insulin secretion promoting action and a good insulin
`sensitivity potentiating action and furthermore show a selec
`tive stimulating action to fºs-receptors, leading to accom
`plishment of the present invention.
`That is, the present invention relates to an amide deriva
`tive represented by the general formula (I) set forth below or
`a salt thereof that is useful for the therapy of diabetes
`mellitus, having both an insulin secretion promoting action
`and an insulin sensitivity potentiating action and further
`having anti-obesity and anti-hyperlipemia actions due to a
`selective stimulating action to fºs-receptors. The present
`invention also relates to a pharmaceutical agent, particularly
`to a therapeutic agent for diabetes mellitus containing the
`amide derivative or the salt thereof as an effective ingredi
`ent.
`
`(I)
`
`ºv Agºs O
`R US 2 J. CJºo
`
`Z
`
`(In the formula, each of the symbols means as follows:
`ring B: a heteroaryl group which may be substituted and
`may be fused with a benzene ring;
`X: a bond, lower alkylene or alkenylene which may be
`substituted with hydroxy or a lower alkyl group,
`carbonyl, or a group represented by —NH– (when X
`is a lower alkylene group which may be substituted
`with a lower alkyl group, the hydrogen atoms bonded
`to the carbon atom constituting the ring B may form a
`lower alkylene group together with the lower alkyl
`group so that a ring is formed);
`A: lower alkylene or a group represented by -lower
`alkylene-O-;
`R*, R* they may be the same or different and each is a
`hydrogen atom or a lower alkyl group;
`R*: a hydrogen atom or a halogen atom; and
`
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`
`US 6,346,532 B1
`
`6
`Second Manufacturing Method
`
`5
`(DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
`(EDCI), 1,1'-carbonyldiimidazole (CDI), diphenylphospho
`ryl azide (DPPA), diethylphosphoryl cyanide (DEPC), etc.
`When Y" is lower alkoxy, a method where the reaction is
`conducted under heating or refluxing as it is or in the
`above-mentioned inert solvent may be applied.
`When Y" is halide, a method where the reaction is
`conducted in the above-mentioned inert solvent in the pres
`ence of a base may be applied.
`Examples of the inert solvent are dimethylformamide
`(DMF), dimethylacetamide, tetrachloroethane,
`dichloromethane, dichloroethane, chloroform, carbon
`tetrachloride, tetrahydrofuran, dioxane, dimethoxyethane,
`ethyl acetate, benzene, toluene, xylene, acetonitrile, dim
`ethyl sulfoxide, etc., and mixed solvents thereof, and they
`may be appropriately selected depending upon each reaction
`condition. Examples of the base are inorganic bases such as
`sodium hydroxide, potassium hydroxide, sodium carbonate,
`potassium carbonate, etc.; and organic bases such as
`N-methylmorpholine, triethylamine, diisopropylethylamine,
`pyridine, etc.
`The protective group of the amino represented by Rº
`means a protective group which is commonly used for
`amino by those skilled in the art, and its representative
`examples are acyl such as formyl, acetyl, propionyl,
`methoxyacetyl, methoxypropionyl, benzoyl, thienylacetyl,
`thiazolylacetyl, tetrazolylacetyl, thiazolylglyoxyloyl,
`thienylglyoxyloyl, etc.; lower alkoxycarbonyl such as
`methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl,
`etc.; aralkyloxy-carbonyl such as benzyloxycarbonyl,
`p-nitrobenzyloxycarbonyl, etc.; lower alkanesulfonyl such
`as methanesulfonyl, ethanesulfonyl, etc.; aralkyl such as
`benzyl, p-nitrobenzyl, benzhydryl, trityl, etc.; tri-(lower
`alkyl)silyl such as trimethylsilyl, etc.; and the like.
`Removal of the protective group in this manufacturing
`method may be conducted by customary manners. For
`example, the protective group for amino represented by Rº
`may be easily removed, for example, by i) a method where
`in case that the protective group is benzhydryl,
`p-methoxybenzyl, trityl, tert-butoxycarbonyl, formyl, etc.,
`treatment with an acid such as formic acid, trifluoroacetic
`acid, a trifluoroacetic acid-anisole mixed solution, a hydro
`bromic acid-acetic acid mixed solution, a hydrochloric acid
`dioxane mixed solution, etc. is conducted; ii) a method
`where in case that the protective group is benzyl,
`p-nitrobenzyl, benzhydryl, trityl, etc., a catalytic reduction
`method using palladium-carbon or palladium hydroxide
`carbon is conducted; and iii) a method where in case that the
`protective group is a tri-(lower alkyl) silyl or the like,
`treatment with water, fluoride anion (e.g., tetra-n
`butylammonium fluoride, sodium fluoride, potassium
`fluoride, hydrofluoric acid), etc. is conducted.
`
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`50
`
`OH
`
`(I)
`
`rº Cl O
`R2 S2 JK, S2 *_C)
`
`(In the formulae, Rºº, Rºº, Rº, A, B, X and Z have the same
`meanings as defined already.)
`In this manufacturing method, the compound (IV) is
`reacted with the compound (V) to give the compound (I) of
`the present invention.
`The amine compound (IV) and the compound (V) are
`reacted under heating or refluxing for 1 to 24 hours as they
`are or in an inert solvent, to give the compound (I) of the
`present invention.
`Examples of the inert solvent are acetonitrile,
`tetrahydrofuran, 2-butanone, dimethyl sulfoxide and
`N-methylpyrrolidone. In the reaction, a base such as sodium
`bicarbonate, potassium carbonate or diisopropylethylamine
`may be added to the reaction mixture.
`Incidentally, in the above manufacturing methods, it is
`possible to purify the resulting substance by removing
`undesired by-products by means of recrystallization,
`pulverization, preparative thin layer chromatography, silica
`gel flash chromatography (as described in W. C. Still, et al.,
`J. Org. Chem., 43, 2923 (1978)), medium-pressure liquid
`chromatography and HPLC. The compound produced
`through HPLC can be isolated as a corresponding salt.
`The starting material used in the above-mentioned manu
`facturing methods may be easily manufactured by the meth
`ods which are known to those skilled in the art. One of the
`representative methods is shown as hereunder.
`
`Manufacturing Method for the
`Starting Compound (II)
`
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`US 6,346,532 B1
`
`-continued
`
`Z
`
`(II)
`
`3.
`
`(VIIIb)
`
`(In the formulae, Rºº, R*, R*, R*, A and Z have the same
`meanings as defined already; Rº is a hydrogen atom or an
`aralkyl-based protective group for amino; and Rº is epoxy,
`2-haloacetyl or 1-carboxymethan-1-ol.)
`This manufacturing method is composed of from step (a)
`to step (c) in which the step (a) is a step where the compound
`(VI) is reacted with the compound (VII), followed by
`reduction reaction to give the compound (VIIIa) depending
`upon the type of Rº, the step (b) is a step where protection
`is conducted when R* of the compound (VIIIa) is a hydrogen
`atom; and the step (c) is a step where nitro is reduced to
`amino to give the compound (II).
`Examples of the aralkyl-based protective group for amino
`used in this manufacturing method are benzyl,
`p-nitrobenzyl, benzhydryl, etc.
`Step (a)
`Illustration is made for the following three cases.
`1) When R* is epoxy, the compound (VI) may be reacted
`with the compound (VII) by the same manner as in the
`above-mentioned second manufacturing method. Reac
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`3) When R* is 1-carboxymethan-1-ol, the compound (VI)
`is reacted with the compound (VII) in the presence of
`a condensing agent, followed by reduction reaction in
`the same manner as in 2) to prepare the compound
`(VIIIa). The condensing agent is the same as that
`mentioned in the first manufacturing method.
`Step (b):
`When R* in the compound (VIIIa) is a hydrogen atom, the
`amino group is protected by customary manners using
`di-tert-butyl dicarbonate, etc., to prepare the compound
`(VIIIa).
`Step (c):
`A method for the reduction of nitro to amino may be
`conducted by customary manners such as metallic reduction
`using iron, zinc, etc. and catalytic reduction using a catalyst
`such as palladium-carbon, palladium hydroxide-carbon,
`Raney nickel, etc. R* becomes a hydrogen atom depending
`upon the reduction conditions, but it may be protected again
`by customary manners.
`
`Manufacturing Method for Starting Compound (IV)
`
`A)
`
`|
`R1b 2 NH2
`
`R1a
`(IX)
`
`(III)
`
`(IVa)
`
`55
`
`tion conditions such as reaction temperature, solvent,
`etc. are the same as well.
`2) When R* is 2-haloacetyl, the compound (VI) is reacted
`with the compound (VII) in the presence of a base,
`followed by reduction reaction to prepare the com
`pound (VIIIa). The base is the same as that mentioned
`60
`in the first manufacturing method. The reduction reac
`tion may be conducted in the above-mentioned inert
`solvent or in a solvent of an alcohol type with stirring
`in the presence of a reducing agent. Examples of the
`reducing agent are sodium borohydride, sodium
`cyanoborohydride, lithium aluminum hydride, borane,
`etc.
`
`65
`
`A.
`
`"ºx SU S O
`|
`R1a
`R1b 2 * @ (IV)
`
`(In the formulae, Rºº, Rºº, Rº, A, B, X and Yº have the same
`meanings as defined already.)
`
`This reaction is a reaction where the compound (IX) and
`the compound (III) are subjected to amidation reaction to
`give a compound (IVa) and, when R* is a protective group
`for amino, the protective group is removed to give a com
`pound (IV). The amidation reaction can be conducted by the
`same manner as in the above-mentioned first manufacturing
`method, and the reaction conditions such as reaction
`temperature, solvent, etc. are the same as well.
`
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`US 6,346,532 B1
`
`B)
`
`sº
`2 NH2
`
`O º, O
`(III)
`
`(X)
`
`">“CU 2.sºs ()
`
`H
`
`10
`
`(IVb)
`
`15
`
`10
`sitivity potentiating action, so that its usefulness in diabetes
`mellitus is expected. Although the fºs-receptor stimulating
`action may have a possibility of participating in expression
`of the insulin secretion promoting action and the insulin
`sensitivity potentiating action, other mechanism might also
`possibly participate therein, and the details thereof have
`been still unknown yet. The ?ys-receptor stimulating action
`of the compound of the present invention is selective to
`??s-receptors in human being. It has been known that the
`stimulation of fºs-receptor stimulates decomposition of fat
`(decomposition of the fat tissue triglyceride into glycerol
`and free fatty acid), whereby a disappearance of fat mass is
`promoted. Therefore, the compound of the present invention
`has an anti-obesity action and an anti-hyperlipemia action
`(such as triglyceride lowering action, cholesterol lowering
`action and HDL cholesterol increasing action) and is useful
`as a preventive and therapeutic agent for obesity and hyper
`lipemia (such as hypertriglyceridemia, hypercholester
`olemia and hypo-HDL-lipoproteinemia). Those diseases
`have been known as animus factors in diabetes mellitus, and
`amelioration of those diseases is useful for prevention and
`therapy of diabetes mellitus as well.
`The compound of the present invention is also useful as
`a preventive and therapeutic agent for other diseases where
`the improvement of symptom can be achieved by reducing
`the symptoms of obesity and hyperlipemia such as ischemic
`coronary diseases such as arteriosclerosis, myocardial
`infarction, angina pectoris, etc. cerebral arteriosclerosis such
`as cerebral infarction, etc., or aneurysm, etc.
`Further, the selective fºs-receptor stimulating action of the
`compound of the present invention is useful for prevention
`and therapy of s several diseases which have been reported
`to be improved by the stimulation of £3-receptor. Examples
`of those diseases are shown as follows.
`It has been mentioned that the fºs-receptor mediates the
`motility of non-sphincteral smooth muscle contraction, and
`because it is believed that the selective ?ys-receptor stimu
`lating action assists the pharmacological control of intestinal
`motility without being accompanied by cardiovascular
`action, the compound of the present invention has a possi
`bility of being useful in therapy of the diseases caused by
`abnormal intestinal motility such as various gastrointestinal
`diseases including irritable colon syndrome. It is also useful
`as the therapy for peptic ulcer, esophagitis, gastritis and
`duodenitis (including that induced by H. pylori), enterelcosis
`(such as inflammatory intestinal diseases, ulcerative colitis,
`clonal disease and proctitis).
`It is further shown that the fis-receptor affects the inhibi
`tion of release of neuropeptide of some sensory fibers in
`lung. The sensory nerve plays an important role in neuro
`genic inflammation of respiratory tract including cough, and
`therefore, the specific f{3-agonist of the present invention is
`useful in the therapy of neurogenic inflammation and in
`addition, has little action to cariopulmonary system.
`Moreover, the [3-adrenaline receptor is capable of result
`ing in a selective antidepressant action due to stimulation of
`the fºs-receptor in brain, and accordingly, the compound of
`the present invention has a possibility of being useful as an
`antidepressant.
`The action of the compound of the present invention has
`been ascertained to be selective to 53-receptors as a result of
`experiments using cells expressing human type receptors,
`and the adverse action caused by other fºs-receptor stimu
`lation is low or none.
`Effects of the compound of the present invention have
`been ascertained by the following tests.
`
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`
`This reaction is a reaction where the compound (X) and
`the compound (III) are subjected to amidation reaction and
`then to reduction reaction to give a compound (IVb). The
`amidation reaction can be conducted by the same manner as
`in the above-mentioned first manufacturing method, and the
`reaction conditions such as reaction temperature, solvent,
`etc. are the same as well. In the reduction reaction, the
`above-mentioned catalytic reduction, or a method where
`reduction is conducted using sodium borohydride in the
`presence of cobalt chloride, may be applied.
`With regard to other compounds such as the compound
`(III), the compound (V), the compound (VI), and the com
`pound (VII), those which are available in the market or are
`30
`appropriately synthesized by known methods (such as
`N-alkylation reaction, cyclization reaction, hydrolysis
`reaction, etc.) from the commercially available compounds
`may be used.
`The compound (I) of the present invention which is
`manufactured as such is isolated and purified as a free
`compound, a salt thereof obtained by means of salt forma
`tion by customary manners, a hydrate, a solvate with various
`solvents such as ethanol, etc., or polymorphic crystals, etc.
`The isolation and purification may be conducted by applying
`common chemical operations such as extraction,
`concentration, evaporation, crystallization, filtration,
`recrystallization, various chromatographic methods, etc.
`Various isomers may be isolated by customary manners
`utilizing the physico-chemical differences between the iso
`mers. For example, the racemate can be converted to stere
`ochemically pure isomers by common racemic resolution
`(such as a method where the racemate is changed to dias
`tereomer salts with usual optically active acid (for example,
`tartaric acid), followed by optical resolution, and the like).
`Incidentally, a mixture of diastereomers may be separated by
`customary method such as fractional crystallizaiton or
`chromatography, etc. In the case of an optically active
`compound, it may be manufactured starting from an appro
`priate optically active material.
`Industrial Applicability
`The phenethanol derivative of the present invention rep
`resented by the general formula (I) or the salt thereof has
`both an insulin secretion promoting action and an insulin
`sensitivity potentiating action and also has a selective
`??s-receptor stimulating action, so that it is useful as a
`therapeutic agent for diabetes mellitus.
`As confirmed by a glucose tolerance test and a hypogly
`cemic test in insulin-resisting model animals as described
`later, the compound of the present invention has both a good
`insulin secretion promoting action and a good insulin sen
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`US 6,346,532 Bl
`
`11
`1. Hypoglycemic Test in kk Mice (insulin-resisting model;
`Obesity and Hyperglycemia)
`Male kk mice (blood sugar level: not lower than 200
`mg/dl) were subjected to a measurement of blood sugar level
`under feeding and then randomly classified into groups. The 5
`drug to be tested was compulsorily administered orally or
`subcutaneously once daily for four days, and the blood sugar
`level after 15 to 18 hours from the final administration was
`compared with that before the administration (n=6). The
`blood was collected from a tail vein of the mice using a glass 10
`capillary (previously treated with heparin), the protein was
`removed therefrom, and the amount of glucose in the
`supernatant liquid (mg/dl) was measured by calorimetric
`determination by means of a glucose oxidase method.
`Further, a dose at which the blood sugar level was lowered 15
`by 30% as compared with that before the administration
`with the drug to be tested was expressed as an ED 30 value.
`As a result, the compound of the present invention sig(cid:173)
`nificantly lowered the blood sugar level as compared with
`that before the administration with the drug to be tested in 20
`both cases of oral and subcutaneous administrations. In
`particular, some of the compounds of the present invention
`exhibited a strong activity so that the ED 30 value in the oral
`administration was 3 mg/kg/day or less. On the other hand,
`in the above-referenced WO 95/29159, the compound of
`Example 90 had an ED 30 value of 30 mg/kg/day or more,
`and the compound of Example 92 had an ED 30 value of 30
`mg/kg/day. From this fact, it has become clear that the
`compounds of the present invention have a superior poten(cid:173)
`tiating action to insulin sensitivity as compared with those of 30
`the above-referenced WO 95/29159.
`2. Glucose Tolerance Test in Normal Rats
`Male rats of SD strain of seven weeks age were fasted for
`a whole day and night, then randomly classified into groups
`and subjected to an oral glucose tolerance test (OGTT)
`(n+4). The compound to be tested was administered orally or
`subcutaneously at 30 minutes before administration of glu(cid:173)
`cose (2 g/kg by oral administration). The blood was col(cid:173)
`lected from an abdominal aorta using a heparin-treated glass
`syringe from the rats which were anesthetized with pento(cid:173)
`barbital (65 mg/kg), the protein was removed therefrom, and
`the amount of glucose in the supernatant liquid (mg/dl) was
`measured by colorimetric determination by means of a
`glucose oxidase method. The insulin value in blood was
`determined by measuring the amount of insulin in plasma
`(ng/ml) by means of radioimmunoassay (RIA).
`As a result, in a group where the compound of the present
`invention was administered orally or subcutaneously, a
`significant increase in the insulin value in blood was
`observed as compared with the group to which no drug was
`given. An increase in the sugar blood level after adminis(cid:173)
`tration of glucose was significantly inhibited as well. From
`those results, it is apparent that the compound of the present
`invention has a good insulin secretion promoting action and
`a good hyperglycemia inhibiting action.
`3. Stimulating Test to Human ~3 -, ~2- and ~ 1 -receptors
`Human ~3-stimulating action was investigated using an
`SK-N-MC cell system (cells in which human ~3-receptor
`and human ~1 -receptor were permanently expressed were
`purchased) while human ~2- and ~1 -stimulating actions were
`investigated using a CHO cell system (cells in which each of
`human ~2- and ~ 1 -receptors was compulsorily expressed
`were purchased). Stimulating action of the compound (10- 10
`to 10-4 M) were investigated by incubating 105 cells/well of
`each of the cells on a 24-well plate and checking under a
`subconfiuent state after two days using a producing activity
`of cyclic AMP ( cAMP) as an index. Incidentally, the human
`
`12
`~3 -stimulating action was investigated in the presence of a
`~1 -receptor blocker (CGP20712A, 10- 6 M). Amount of
`production of cAMP in each cell (pmol/ml) was measured
`by an RIA method using 125I-cAMP. Intensity of action of
`each compound was compared by calculating the pD2 value
`and the maximum activity (I.A (%) where the maximum
`reaction of 10- 6 M isoproterenol was defined as 100%) from
`the resulting dose-reaction curve.
`As a result, it has been ascertained that the compound of
`the present invention has a selective stimulating action to
`human ~3-receptor.
`A pharmaceutical composition containing one or more of
`the compound of the present invention or the salt thereof as
`an effective ingredient is prepared using common pharma(cid:173)
`ceutically acceptable vehicles. Administration of the phar(cid:173)
`maceutical composition according to the present invention
`may be either by oral administration or by parenteral admin(cid:173)
`istration by, for example, injection, suppository, subcutane(cid:173)
`ous agent, inhaling agent or intracystic infusion.
`The dose may be appropriately decided depending upon
`each particular case while taking into consideration
`symptom, age, sex, etc. of the patient but usually, is around
`0.01 mg/kg to 100 mg/kg per day for adults in the case of
`oral administration, and that is administered at a time or by
`25 dividing into 2 to 4 times a day. When intravenous injection
`is conducted depending upon the symptom, the dose is
`usually around 0.001 mg/kg to 10 mg/kg per day for adults,
`and that is administered at a time or by dividing into two or
`more times a day.
`With regard to a vehicle for the preparation, nontoxic
`solid or liquid substances for pharmaceuticals may be used.
`Examples of the solid composition for use by means of
`oral administration according to the present invention are
`tablets, pills, capsules, diluted powder and granules. In such
`35 a solid composition, one or more active substances are
`mixed with at least one inert excipient such as lactose,
`mannitol, glucose, hydroxypropyl cellulose, microcrystal(cid:173)
`line cellulose, starch, polyvinylpyrrolidone, agar, pectin,
`magnesium metasilicate aluminate and magnesium alumi-
`40 nate. The composition may also contain additives other than
`the inert excipient such as lubricants such as magnesium
`stearate; disintegrants such as calcium cellulose glycolate;
`stabilizers such as lactose; and auxiliary solubilizers such as
`glutamic acid or aspartic acid by customary manners. Tab-
`45 lets and pills may, if necessary, be coated with sugar coat
`such as sucrose, gelatin, hydroxypropyl cellulose, hydrox(cid:173)
`ypropylmethyl cellulose phthalate, etc., or with film of
`gastric or enteric coating substances.
`The liquid composition for oral administration includes
`50 pharmaceutically acceptable emulsions, solutions,
`suspensions, syrups and elixirs and contains commonly used
`inert excipients such as purified water or ethanol. In addition
`to the inert excipient, the composition may further contain
`auxiliary agents such as moisturizing or suspending agents,
`55 sweeteners, tasting agents, aromatic agents and antiseptic
`agents. The injection for parenteral administration includes
`aseptic aqueous or non-aqueous solutions, suspensions and
`emulsions. The non-aqueous solutions and suspensions
`include, for example, distilled water for injection and a
`60 physiological saline solution. Examples of the solvent for
`non-aqueous solution and suspension are propylene glycol;
`polyethylene glycol; plant oils such as cacao butter, olive oil
`and sesame oil; alcohols such as ethanol; gum arabic; and
`Polysolvate 80 (trade name). Such a composition may
`65 further contain auxiliary agents such as isotonizing agents;
`antiseptic agents; moisturizing agents; emulsifiers; dispers(cid:173)
`ing agents; stabilizers such as lactose; and auxiliary solubi-
`
`Sawai Ex. 1001
`Page 7 of 30
`
`

`

`Case 1:16-cv-00954-SLR Document 1-1 Filed 10/17/16 Page 9 of 100 PageID #: 26
`
`US 6,346,532 B1
`
`13
`lizers such as glutamic acid and aspartic acid). These may be
`sterilized, for example, by filtration passing through a
`bacteria-preserving filter or by compounding of or irradia
`tion with a bactericide. These may also be used by manu
`facturing a sterile solid composition, followed b