Case 1:15-cv-00697-RGA Document 39-11 Filed 01/29/16 Page 1 of 5 PageID #: 531
`Case 1:15-cv—00697-RGA Document 39-11 Filed 01/29/16 Page 1 of 5 PagelD #: 531
`
`EXHIBIT 10
`
`EXHIBIT 10
`
`

`

`Case 1:15-cv-00697-RGA Document 39-11 Filed 01/29/16 Page 2 of 5 PageID #: 532
`
`United States Patent [191
`Karjalainen et a1.
`
`[11] Patent Number:
`[45] Date of Patent:
`
`4,910,214
`Mar. 20, 1990
`
`[54] OPTICAL ISOMER OF AN IMIDAZOLE
`DERIVATIVE MEDETOMIDINE AS AN
`ALPHA-Z-RECEPTOR AGONIST
`[75] Inventors: Arto J. Karjalainen, Oulu; Raimo E.
`Virtanen, Rusko; Eino J. Savolainen,
`Oulu, all of Finland
`[73] Assignee: Farmos Yhtyma 0y, Turku, Finland
`[21] Appl. N0.: 219,637
`[22] Filed:
`Jul. 15, 1988
`[30]
`Foreign Application Priority Data
`Jul. 16, 1987 [GB] United Kingdom ............... .. 8716803
`
`[51] Int. Cl.‘ .................. ..A61K 31/415; C07D 233/58
`[52] US. Cl. ................................... .. 514/396; 548/335
`[58] Field of Search ....................... .. 548/335; 514/396
`[56]
`References Cited
`U.S. PATENT DOCUMENTS
`
`*
`
`FOREIGN PATENT DOCUMENTS
`0024829 3/ 1981 European Pat. Off. .
`0058047 8/1982 European Pat. Off. .
`0072615 2/1983 European Pat. Off. .
`02114528 3/ 1987 European Pat. Off. .
`
`OTHER PUBLICATIONS
`Chemical Abstracts, 105: 1839770 (1986) [JPN. Kokai 61,
`134, 314, Farrnos, 6/21/86].
`Noller, C., Chemistry of Carbon Compounds, 2nd Ed.,
`W. B. Saunders, Philadelphia, 1957, pp. 341-344.
`Primary Examiner-Richard A. Schwartz
`Attorney, Agent, or Firm--Armstrong, Nikaido,
`Marmelstein, Kubovcik & Murray
`[57]
`ABSTRACT
`The separated d and l enantiomers of medetomidine and
`their salts are selective and potent dz-I'CCCPIOI' agonists.
`
`4,544,664 10/1985 Karjalainen et a1. ............. .. 514/400
`
`4 Claims, N0 Drawings
`
`

`

`Case 1:15-cv-00697-RGA Document 39-11 Filed 01/29/16 Page 3 of 5 PageID #: 533
`
`1
`
`OPTICAL ISOMER OF AN IMIDAZOLE
`DERIVATIVE MEDETOMIDINE AS AN
`ALPHA-Z-RECEPTOR AGONIST
`
`This invention relates to optical isomers of imidazole
`derivatives and to their preparation.
`Medetomidine which has the formula:
`
`cu
`i
`3
`cu
`
`CH3
`
`cm
`
`II
`
`N
`
`<’
`If
`H
`
`is known as a selective and potent az-receptor agonist.
`It has been described, e.g. in European Patent Publica
`tion No. 72615, as an antihypertensive agent and in the
`European Patent Publication No. 187471 as a veterinary
`sedative-analgesic agent.
`The present invention provides, as new compounds,
`the optically active d- and l-enantiomers of medetomi
`dine, and their non-toxic pharmaceutically acceptable
`acid addition salts. These compounds may be repre
`sented by the formulae:
`
`CH3
`N ‘ C
`
`H
`
`< T N
`
`CH3
`
`l!‘
`
`H
`
`CH3
`‘CH3
`c
`
`N
`
`T >
`
`CH3
`
`CH3
`
`N
`XL
`
`According to a feature of the invention, racemic
`medetomidine is separated into the enantiomers II and
`45
`III by conversion of the racemate into a mixture of
`diastereoisomers and separating the latter by fractional
`crystallization. Since medetomidine is a base, it may be
`converted into a diastereoisomer salt mixture by reac
`tion with an optically active acid such as (+)-tartaric
`acid. Other useful optically active acids are, e.g., (-)
`malic acid, (—)-mandelic acid and (+)-camphor-l0-sul
`fonic acid. (+)-Tartaric acid is especially useful for the
`resolution. The separation of the diastereisomers is per
`formed by repeated crystallizing from an alcohol such
`as methanol or ethanol or a mixture of them.
`Once the diastereoisomers have been separated the
`acid addition salts can be converted back to the free
`bases by making their aqueous solutions alkaline with
`sodium hydroxide and by extracting the liberated base
`in an appropriate organic solvent such as methylene
`chloride.
`‘
`The d- and l-enantiomers of medetomidine react with
`organic and inorganic acids to form the corresponding
`acid addition salts, which have the same therapeutic
`activities as the bases. They can thus form many phar
`maceutically usable acid addition salts, as, for instance,
`chlorides, bromides, sulfates, nitrates, phosphates, sulfo
`
`65
`
`55
`
`4,910,214
`2
`nates, formates, tartrates, maleates, citrates, benzoates,
`salicylates, ascorbates and the like.
`The d- and l-enantiomers of medetomidine are selec
`tive and potent az-receptor agonists.
`Adrenergic receptors are physiologically important
`binding sites which are speci?c to noradrenaline and
`adrenaline and located on the surface of the cell mem
`brane. The adrenoceptors of the sympathetic nervous
`system have been classi?ed into two different subtypes,
`alpha-(a) and beta-(B) receptors, which can be further
`divided into two subgroups, viz a1 and a2 and B1 and 3;.
`Of these receptor types, B1, B2 and 01.1 are mainly lo
`cated post-synaptically on the surface of, e.g., smooth
`muscle and thus mediate, e.g., smooth muscle contrac
`tion or relaxation; whereas (12 receptors are mainly
`located presynaptically on the terminals of noradrener
`gic nerves. If az-receptors are stimulated by noradrena
`line under physiological conditions noradrenaline re
`lease is blocked, i.e. there is a negative feed-back phe
`nomenon. This negative feed-back phenomenon may
`also be induced by certain synthetic az-agonists like
`medetomidine and some of its near derivatives.
`In animal experiments, the d- and l- enantiomers of
`the present invention and especially the d-enantiomer,
`have proved to possess highly enhanced az-selectivity
`and potency compared to the racemic mixture (i.e.
`medetomidine). The d-enantiomer can be expected to be
`of value, e.g., as a sedative-analgesic, anxiolytic or anti
`hypertensive agent. Furthermore, it can be used as a
`pharmacological tool in the study of the physiology and
`pharmacology of az-adrenoceptors.
`The pharmacological activity of the compounds of
`the invention was determined as follows:
`
`15
`
`20
`
`30
`
`35
`
`1. ALPHA-2 AGONISM IN VITRO
`az-agonism was determined by means of isolated,
`electrically stimulated mouse was deferens preparation
`(Marshall et al., Br. J. Pharmac. 62, 147-151, 1978). In
`this model, an az-agonist is able to block electrically
`induced muscular contractions by activating the presy
`naptic (lg-adrenoceptors and thus diminishing the secre
`tion on the motor transmitter. Known az-agonists like
`detomidine, medetomidine and clonidine were used as
`reference substances. Results are shown in Table 1,
`where the az-agonist effect is presented as the pDZ
`value (negative logarith of the molar concentration of
`the compound producing 50 percent of maximal inhibi
`tion.)
`
`TABLE 1
`az-agonism in' vitro (mouse
`vas deferens). pDg
`9.3
`6.0 (partial agonist)
`9.0
`8.5
`8.5
`
`Compound
`d-enantiomer
`laenantiomer
`medetomidine
`detomidine
`clonidine
`
`These results show that the az-agonist activity of
`medetomidine is limited to the d-enantiomer. The d
`enantiomer shows an enhanced az-agonist activity com
`pared to the outer agents studied.
`
`2. aZ/cq-SELECTIVITY IN VITRO
`The selectivity of the d-enantiomer as an az-agonist
`was studied in receptor binding experiments using rat
`brain membranes. The ability of the d-isomer and the
`reference compounds to compete with 3l-I-clonidine
`
`

`

`Case 1:15-cv-00697-RGA Document 39-11 Filed 01/29/16 Page 4 of 5 PageID #: 534
`
`4,910,214
`4
`3
`(for az-receptors) and 3H-prazosin (for m-receptors)
`center of the t-maze and the number of open and en
`closed entries is recorded during 5 minutes. Results
`was studied essentially as described by Virtanen and
`Nyman in Eur. J. Pharmac. 108, 163-9, 1985. Results of
`obtained are shown in Table 5.
`the test are presented in Table 2, where the ability of the
`TABLE 5
`‘
`studied agents to compete with 3H-clonidine and 3H- 5
`prazosin binding is expressed as the IC50~value (molar WM
`concentration of the competing ligand needed to dis-
`Dwg/dwev "18/ k3
`°Pen
`“med
`‘ml
`Wen/mm‘
`place 50 percent of the radioactive ligand).
`NaCl
`3.4
`8.6
`12.0
`0.28
`d-enantiomer
`10 0.0003
`0.001
`0.003
`0.01
`3%03 am
`1 ml’
`
`4.8
`3.2
`4.0
`5.8
`2'5
`5 2
`'
`
`10.6
`10.6
`9.5
`8.8
`3‘o
`10 5
`'
`
`14.0
`13.8
`13.5
`14.6
`5'5
`l 5 7
`'
`
`0.20
`0.23
`0.29
`0.39
`0'45
`0 33
`'
`
`Compound
`d-enantiomer
`l-enantiomer
`medetomidine
`detomidine
`clomdme
`
`az/ay-
`selectivity
`45849
`4129
`5060
`65
`969
`
`15
`
`TABLE 2
`3H-clonidine
`3H-prazosin
`displacement
`displacement
`lC50,_ nM
`IC50, nM
`1.2
`55019
`46
`189975
`3.3
`16700
`3.7
`242
`The results show that the d-enantiomer has an anxi
`6'4
`6200
`'
`'
`l v
`t-
`2 test.
`.
`.
`olymi pro?le m the e e ated .ma e
`The results show that the d-enantiomer 15 an extremely
`It 15 well known that anxiety states connected to
`.
`.
`.
`.
`.
`.
`selective az-agontst compared to medetormdine and the
`20 withdrawal symptoms are due to noradrenergic hyper
`.
`.
`other reference compounds.
`activity. Therefore such symptoms can be successfully
`treated with drugs reducing the level of noradrenaline,
`3. SEDATIVE ANALYGESIC EFFECTS
`e.g. clonidine. Experiments in the rat indicate that the
`The 5edative.ana1g¢sic properties of the compounds
`d-enantiomer 1S able to reduce noradrenaline release and
`were studied in the spontaneous motility and writhing.
`test in the mouse. Spontaneous motility of mice and rats 25 thus sympathetic tone both in the central and peripheral
`was measured using the Animex-activity meter. The
`nervous systems. This has clearly been demonstrated by
`test compounds were administered i.p. 30 minutes be-
`measuring CSF-concentrations of MHPG-SO4 (the
`fore the measuring periods of two minutes. In the writh-
`principal metabolite of central noradrenaline) in the rat
`ing test the compounds studied and saline were adminis-
`after d-enantiomer administration. The results are
`tered 5.0. to rats, and 45 min. later 1 ml of 1% acetic acid 39 shown in Table 6.
`was administered i.p. The number of writhes was re-
`corded in the following 25 min. period (Koster et al.,
`Fred. Proc. 18: 412, 1959). Results are shown in Tables
`3 and 4.
`
`35
`
`_
`d'enanmmef ‘3°58
`ug/kg
`g
`m
`30
`100
`
`TABLE 6
`
`CSF MHPGSO“ (_% °f “mm”
`‘ (4 h after d-enantlomer adm.)
`“53
`:20
`_30
`—65
`
`TABLE 3
`ED50 -values of the studied compounds
`in reducinv spontaneous motility in mice
`Compound
`ED5Q (mg/kg s.c.)
`d-enantiomer
`0.02
`l-enantiomer
`> 10
`m=de“.’"."d'"e
`0'05
`detomidine
`0.3
`C‘Onidine
`Q3
`
`TABLE 4
`‘
`_ EDs0~v=1lpd§ (21f lhzswqlzfi commlmds
`c'“ am'cdac' "“ “Ce wmEgg ‘85' 'nkm'cc
`ompofm
`somgv/ g 8'6‘)
`d‘enamfmer
`O-Ol -
`l-enantiomer
`>10
`medetomidine
`0.02
`detomidine
`0.02
`clonidine
`0.03
`
`4O
`
`5. ANTIHYPERTENSIVE EFFECTS
`The antihypertensive properties of the compounds of
`the invention have been studied as follows: Sprague
`45 Dawley rats of normal weight were ?rst anesthetized
`with urethane. After this, the femoral artery was con
`nected by a polyethylene tube to a blood pressure trans
`ducer. The test substance was then injected into the
`femoral vein and the blood pressure and the pulse fre
`50 quency were registered with a recorder. Results are
`h
`- T bl 7
`5 Own m a e ‘
`
`TABLE 7
`Antihypertensive effects of
`the d-enantiomer in anesthetized rats
`Decrease in BP, % Decrease in heart rate, %
`_8
`_21
`_23
`_40
`—43
`—47
`1g
`“51g
`
`_
`
`_
`
`Dose‘ ing/kg
`0m]
`0003
`0.01
`Us)?
`
`60
`
`'
`
`These results shown that lih? d-enantiomer has an en- 55
`hanced sedative/analgesic property compared to the
`racemic mixture (medetomidine) and other reference
`compounds. The sedative/analgesic
`effects
`of
`medetomidine are con?ned to the d-enantiomer.
`4. ANXIOLYTIC EFFECTS
`The results show that the d-enantiomer possesses clear
`The anxiolytic effects of the compounds were studied
`anti-hypertensive and bradycardia effects.
`using a method described by l-landley and Mithoni:
`The d- and l-enantiomers, and their non-toxic, phar
`Naunyn-Schmiedeb, Arch. Pharmacol. 327, 1-5, 1984.
`In this test the manner of exploration of open and en- 65 maceutically acceptable acid addition salts or mixtures
`thereof may be administered parenterally, intravenously
`closed arms in an elevated t-maze by a rat is examined.
`It has been shown that anxiolytic drugs increase the
`or orally. Typically, an effective amount of the com
`relative exploration of open arms. A rat is placed in the
`pound is combined with a suitable pharmaceutical car
`
`

`

`Case 1:15-cv-00697-RGA Document 39-11 Filed 01/29/16 Page 5 of 5 PageID #: 535
`
`4,910,214
`6
`5
`mixture of 20 ml abs. ethanol and 60 ml methanol by
`rier. As used herein, the term “effective amount" en
`heating on a steam bath. After cooling, 5 g of precipitate
`compasses those amounts which yield the desired activ
`ity without causing adverse side-effects. The precise
`(degree of rotation + 55°) was obtained. After recrystal~=
`amount employed in a particular situation is dependent
`lization from 60 ml of methanol, 4.1 g of product was
`obtained, degree of rotation +60". Recrystallization
`upon numerous factors such as method of administra
`was repeated until the degree of rotation did not in
`tion, type of mammal, condition for which the deriva
`crease any longer. The d-enantiomer tartrate was dis
`tive is administered, etc. and of course the structure of
`the derivative.
`solved in water, the solution was made alkaline and the
`The pharmaceutical carriers which are typically em
`d-enantiomer was dissolved in an organic solvent e.g.
`ployed with the compounds of the present invention
`dichlormethane or diethyl ether. The degree of rotation
`of the d-enantiomer base 'was +75°.
`may be solid or liquid and are generally selected with
`The l-enantiomers may be isolated from the mother
`the planned manner of administration in mind. Thus, for
`liquors.
`example, solid carriers include lactose, sucrose, gelatin
`and agar, while liquid carriers include water, syrup,
`_
`We claim:
`peanut oil and olive oil. Other suitable carriers are well
`1. The d enantiomer of medetomidine or a non-toxic
`pharmaceutically acceptable acid addition salt thereof.
`known to those skilled in the art of pharmaceutical
`2. A pharmaceutical composition suitable for use in a
`formulations. The combination of the derivative and the
`method of sedation/analgesia or treatment of anxiety or
`carrier may be fashioned into numerous acceptable
`hypertension comprising
`the
`d-enantiomer of
`forms, such as tablets, capsules, suppositories, solutions,
`emulsions, and powders.
`medetomidine or a non~toxic pharmaceutically accept
`20
`The following Example illustrates the separation of
`able acid addition salt thereof in an amount sufficient to
`produce the desired effect in association with a pharma»
`the new enantiomers.
`ceutical carrier.
`3. A method of sedation/analgesia or treatment of
`anxiety or hypertension by administration to a subject
`of an effective amount of an enantiomer according to
`claim 1.
`‘ 4. A method of sedation/analgesia or treatment of
`anxiety or hypertension by administration to a subject
`of an effective amount of a composition according to
`claim 2.
`
`EXAMPLE
`14 g of medetomidine (base) were dissolved in 50 ml
`25
`of methanol. 10.5 g of (+)-tartaric acid were dissolved
`in 50 ml of methanol. The solutions were mixed and the
`solvent was evaporated to a volume of 50 ml. The mix
`ture was put into an ice bath and 9 g of white precipitate
`was obtained. The precipitate was suspended in 25 ml of
`30
`ethanol, the mixture was kept in ultrasonic sound for 14
`min and filtered. The precipitate was dissolved in a
`
`Ill
`
`1i
`
`1k
`
`*
`
`1k
`
`35
`
`50
`
`55
`
`65
`
`