United States Patent [191
`Karjalainen et al.
`
`4,910,214
`[111 Patent Number:
`[45] Date of Patent: Mar. 20, 1990
`
`[75]
`
`[54] OPTICAL ISOMER OF AN IMIDAZOLE
`DERIVATIVE MEDETOMIDINE AS AN
`ALPHA·2·RECEPTOR AGONIST
`Inventors: Arto J. Karjalainen, Oulu; Raimo E.
`Vi.rtanen, Rusko; Eino J. SaTolainen,
`Oulu, all of Finland
`(73] Assignee: Farmos Yhtyma Oy, Turku, Finland
`[21] Appl. No.: 219,637
`Jul. 15, 1988
`(22] Filed:
`Foreign Application Priority Data
`[30]
`Jul. 16, 1987 [GB] United Kingdom ................. 8716803
`Int. a ....................... A61K 31/415; com 233/58
`[51]
`(52] U.S. CJ •..................................... 514/396; 548/335
`[58] Field of Search ......................... 548/335; 514/396
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`4,544,664 10/1985 Karjalaincn ct al . ............... 514/400
`
`FOREIGN PATENT DOCUMENTS
`0024829 3/1981 European PaL Oft .
`0058047 8/1982 European Pat. Off ..
`0072615 2/1983 European Pat. Off ..
`021 14528 3/1987 European Pat. Off. .
`
`OTHER PUBLICATIONS
`Chemical Abstracts, 105: 183977c (1986) [JPN. Kokai 61,
`134, 314, Farmos, 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 I enantiomers of medetomidine and
`their salts are selective and potent az-receptor agonists.
`
`4 Claims, No Drawini1
`
`Petition for Inter Partes Review of US 8,338,470
`Amneal Pharmaceuticals LLC – Exhibit 1005 – Page 1
`
`

`
`1
`
`OPTICAL ISOMER OF AN INIIDAZOLE
`DERIVATIVE MEDETOMIDINE AS AN
`ALPHA-2-RECEPTOR AGONIST
`
`This invention relates to optical isomers of imidazole
`derivatives and to their preparation
`Medetomidine which has the formula:
`
`i
`
`N
`
`ll“
`H
`
`ll
`
`CH3
`lcm
`
`cu;
`
`CH3
`
`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 antihypenensive agent and in the
`European Patent Publication No. 18747] 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 fonnulae:
`
`5
`
`l0
`
`l5
`
`20
`
`25
`
`H
`
`N CH3‘c
`< J”
`['4
`H
`
`CH3
`
`CH1
`
`H
`
`‘CH3
`
`C
`
`N
`
`ll
`
`30
`
`35
`
`III
`
`@( W >
`
`CH3
`
`CH;
`
`N
`I!‘
`
`50
`
`SS
`
`According to a feature of the invention, racemic
`medetomidine is separated into the enantiomers IX and 45
`Ill by conversion of the raeemate into a mixture of
`diastereoisomers and separating the latter by fractional
`crystallization. Since medetomidine is a base. it may be
`converted into a ditstereoisomer 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 1-enantiomers of medetomidine react with
`organic and inorganic acids to form the corresponding
`acid addition salts, which have the same therapeutic
`activities as the bmes. They can thus form many phar-
`maceutically usable acid addition salts. as, for instance.
`chlorides. bromides. sulfates, nitrates. phosphates, sulfa-
`
`65
`
`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 a;-receptor agonists.
`Adrenergic receptors are physiologically important
`binding sites which are specific to noradrenaline and
`adrenaline and located on the surface of the cell mem-
`brane. The adrenoceptors of the sympathetic nervous
`system have been classified into two different subtypes,
`alpha-(a) and beta-(fl) receptors, which can be further
`divided into two subgroups, viz at and azand B1 and B2.
`Of these receptor types, B1, B2 and (11 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 az 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-baclt 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 0.2-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:
`
`l. ALPHA-2 AGONISM IN VITRO
`
`az-agonisin 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 ct:-agonist is able to block electrically
`induced muscular contractions by activating the presy-
`naptic az-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 pD2-
`value (negative logarith of the molar concentration of
`the compound producing 50 percent of maximal inhibi-
`tion.)
`TABLE I
`a3-agonism in vitro (mouse
`
` Compound vas defereus). pD-_v
`d-enantiomer
`9.3
`I-enantiomer
`6.0 (partial agonist)
`medetornidine
`9.0
`detomidine
`8.5
`
`clonidine 8.5
`
`the a.z-agonist activity of
`These results show that
`medetomidine is limited to the d-enantiomer. The d-
`enantiomer shows an enhanced a2-agonist activity com-
`pared to the outer agents studied
`2. a1/at-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 3H-clonidine
`
`Petition for Inter Partes Review of US 8,338,470
`Amneal Pharmaceuticals LLC — Exhibit 1005 — Page 2
`
`

`
`3
`(for a1-receptors) and 3H-prazosin (for a1-receptors)
`was studied essentially as described by Virtanen and
`Nyman in Eur. J. Pharmac. 108, 163-9, 1985. Results of
`the test are presented in Table 2, where the ability of the
`studied agents to compete with lH-clonidine and 3H. 5
`prazosin binding is expressed as the IC50-value (molar
`concentration of the competing ligand needed to dis(cid:173)
`place 50 percent of the radioactive ligand).
`TABLE2
`lH-clonidine
`JH-prazosin
`displacement
`displacement
`ICso..nM
`ICso. nM
`1.2
`55019
`189975
`46
`3.3
`16700
`242
`3.7
`6.4
`6200
`
`4,910,214
`
`4
`center of the t-maze and the number of open and en(cid:173)
`closed entries is recorded during 5 minutes. Results
`obtained are shown in Table 5.
`TABLE 5
`Mean number of entries (n = §2
`open/total
`open
`closed
`total
`3.4
`8.6
`12.0
`0.28
`
`Drug/dose. mg/kg
`NaCl
`d-enantiomer
`0.0003
`0.001
`0.003
`0.01
`0.03
`diaupam
`I
`
`4.8
`3.2
`4.0
`5.8
`2.5
`
`S.2
`
`10.6
`10.6
`9.5
`8.8
`3.0
`
`10.5
`
`14.0
`13.8
`13.5
`14.6
`5.5
`
`15.7
`
`0.20
`0.23
`0.29
`0.39
`0.45
`
`0.33
`
`10
`
`15
`
`20
`
`The results show that the d-enantiomer has an anxi(cid:173)
`olytic profile in the elevated t-maze test.
`It is well known that anxiety states connected to
`withdrawal symptoms are due to noradrenergic hyper·
`activity. Therefore such symptoms can be successfully
`treated with drugs reducing the level of noradrenaline,
`e.g. clonidine. Experiments in the rat indicate that the
`d-enantiomer is able to reduce noradrenaline release and
`25 thus sympathetic tone both in the central and peripheral
`nervous systems. This has clearly been demonstrated by
`measuring CSP-concentrations of MHPG-S04 (the
`principal metabolite of central noradrenaline) in the rat
`after d-enantiomer administration. The results are
`30 shown in Table 6.
`
`d-enantiomer dose
`J.Lg/kg
`0
`3
`10
`30
`100
`
`TABLE 6
`CSF MHPG-S04 (% of control)
`(4 h after d-enantiomer adm.)
`too
`-10
`- 20
`- 30
`- 65
`
`35
`
`40
`
`5. ANTIHYPERT ENSIVE EFFECT S
`The antihypertensive properties of the compounds of
`the invention have been studied as follows: Sprague-
`45 Dawley rats of normal weight were first anesthetized
`with urethane. After this, the femoral artery was con(cid:173)
`nected by a polyethylene tube to a blood pressure trans(cid:173)
`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
`shown in Table 7.
`
`Compound
`d-enantiomer
`1-enantiomer
`medetomidine
`detomidine
`clonidine
`
`ava1-
`selectivity
`45849
`4129
`5060
`65
`969
`
`The results show that the d-enantiomer is an extremely
`selective a1-agonist compared to medetomidine and the
`other reference compounds.
`3. SEDATIVE ANAL YGESIC EFFECTS
`The sedative-analgesic properties of the compounds
`were studied in the spontaneous motility and writhing(cid:173)
`test in the mouse. Spontaneous motility of mice and rats
`was measured using the Animex-activity meter. The
`test compounds were administered i.p. 30 minutes be(cid:173)
`fore the measuring periods of two minutes. In the writh(cid:173)
`ing test the compounds studied and saline were adminis(cid:173)
`tered s.c. to rats, and 45 min. later l ml of 1 % acetic acid
`was administered i.p. The number of writhes was re(cid:173)
`corded in the following 25 min. period (Koster et al.,
`Fred. Proc. 18: 412, 1959). Results are shown in Tables
`3 and 4.
`
`TABLE 3
`EDso -values of the studied compounds
`in reducing spontaneous motility in mice
`ED so (mg/kg s.c.)
`Compound
`d-enantiomer
`0.02
`> 10
`1-enantiomer
`medctomidine
`0.05
`0.3
`detomidine
`clonidine
`0.3
`
`TABLE 4
`EDso-values of the studied compounds
`in acetic acid-induced writhing test in mice
`ED so (mg/kg s.c.)
`Compound
`d-cnantiomer
`0.01 .
`1-enantiomer
`>10
`mcdetomidine
`0.02
`detomidinc
`O.G2
`clonidinc
`O.o3
`
`TABLE 7
`Antihypcrtensive effects of
`the d-enantiomer in an~thetized rats
`Do~e. mg/kg Decrease in BP, %
`Decrease in heart rate. %
`0.001
`-21
`- 8
`- ll
`Q~
`- ~
`- 43
`-47
`0.01
`- 48
`0.03
`- 45
`60 ~~~o._1 ~~~~--~45~~~~~~~---s_o~~~~
`
`These results shown that the 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 confined to the d-enantiomer.
`
`4. ANXIOL YTIC EFFECTS
`The anxiolytic effects of the compounds were studied
`using a method described by Handley and Mithani:
`Naunyn-Schmiedeb, Arch. Pharmacol. 327, 1-5, 1984.
`In this test the manner of exploration of open and en- 65
`closed arms in an elevated t-maze by a rat is examined.
`It has been shown that anxiolytic drugs increase the
`relative exploration of open arms. A rat is placed in the
`
`The results show that the d-enantiomer possesses clear
`anti-hypertensive and bradycardia effects.
`The d- and 1-enantiomers, and thei.r non-toxic, phar(cid:173)
`maceutically acceptable acid addition salts or mixtures
`thereof may be administered parenterally, intravenously
`or orally. Typically, an effective amount of the com(cid:173)
`pound is combined .with a suitable pharmaceutical car-
`
`Petition for Inter Partes Review of US 8,338,470
`Amneal Pharmaceuticals LLC – Exhibit 1005 – Page 3
`
`

`
`4,910,214
`
`6
`mixture of 20 ml abs. ethanol and 60 ml methanol by
`heating on a steam bath. After cooling, 5 g of precipitate
`(degree of rotation + 55°) was obtained. After recrystal(cid:173)
`lization from 60 ml of methanol, 4.1 g of product was
`obtained, degree of rotation +60°. Recrystallization
`was repeated until the degree of rotation did not in(cid:173)
`crease any longer. The d-enantiomer tartrate was dis(cid:173)
`solved in water, the solution was made alkaline and the
`d-enantiomer was dissolved in an organic solvent e.g.
`dichlonnethane or diethyl ether. The degree of rotatioll
`of the d-enantiomer base ·was + 75°.
`The 1-enantiomers may be isolated from the mother
`liquors.
`We claim:
`1. The d enantiomer of medetomidine or a non-toxic
`pharmaceutically acceptable acid addition salt thereof.
`2. A pharmaceutical composition suitable for use in a
`method of sedation/analgesia or treatment of anxiety or
`hypertension
`comprising
`the
`d-enantiomer
`of
`medetomidine or a non-toxic pharmaceutically accept·
`able acid addition salt thereof in an amount sufficient to
`produce the desired effect in association with a pharma(cid:173)
`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.
`* • * • •
`
`5
`rier. As used herein, the term "effective amount" en(cid:173)
`compasses those amounts which yield the desired activ·
`ity without causing adverse side-effects. The precise
`amount employed in a particular situation is dependent
`upon numerous factors such as method of administra- s
`tion, type of mammal, condition for which the deriva(cid:173)
`tive is administered, etc. and of course the structure of
`the derivative.
`The pharmaceutical carriers which are typically em(cid:173)
`ployed with the compounds of the present invention 10
`may be solid or liquid and are generally selected with
`the planned manner of administration in mind. Thus, for
`example, solid carriers include lactose, sucrose, gelatin
`and agar, while liquid carriers include water, syrup,
`peanut oil and olive oil. Other suitable carriers are well IS
`known to those skilled in the art of pharmaceutical
`formulations. The combination of the derivative and the
`carrier may be fashioned into numerous acceptable
`forms, such as tablets, capsules. suppositories, solutions,
`emulsions, and powders.
`The following Example illustrates the separation of
`the new enantiomers.
`
`20
`
`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(cid:173)
`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
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Petition for Inter Partes Review of US 8,338,470
`Amneal Pharmaceuticals LLC – Exhibit 1005 – Page 4