United States Patent [19]
`W1tzel
`
`.
`
`[11] Patent Number:
`[45] Date of Patent:
`
`4,885,276
`Dec. 5, 1989
`
`*
`
`[54] CY CLOSPORIN ANALOGS WITH
`MODIFIED C" AMINO ACIDS
`
`[56]
`
`’ References Cited
`us. PATENT DOCUMENTS
`
`[75] Invenmn Bruce E- Witzel, west?eld’ N'J-
`[73] Assignee: Merck & Co” Inc.’ Rahway’ NJ_
`
`4,396,542 8/1983 Wenger ............................. .. 530/321
`4,771,122 9/ 1988 Seebach ............................ .. 530/ 317
`
`[ "‘ ] Notice:
`
`The portion of the term of this patent
`gqbsfqumg to Jan- 17’ 2006 has been
`
`OTHER PUBLICATIONS
`Sandorama (1984) 5-11 III Wenger.
`
`15C aune .
`
`.: 261 868
`A l.
`pp No
`’
`[21]
`[22] Filed:
`Oct. 24, 1988
`
`Primary Examiner-Delbert R. Phillips
`Attorney, Agent, or Firm-Robert J. North; Joseph F.
`DiPrima
`
`Related US. Application Data
`Division of Ser. No. 57,196, Jun. 3, 1987.
`[62]
`[51] Int. 01.4 ...................... .. A61K 37/07; C07K 5/12
`[52] US. Cl. .................................... .. 514/11; 530/517;
`
`ABSTRACT
`[57]
`New cyclosporin analogs with modi?ed “C-9 amino
`f‘c‘ds have be“? made ‘tmd are f°und t° be effect“
`unm‘lmuppresswe age“ 5
`
`~
`530/321
`[58] Field of Search ................. .. 530/317, 321; 514/11
`
`8 Claims, No Drawings
`
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`
`1
`
`4,885,276 ’
`
`' 2
`
`(II)
`
`10
`
`ll
`
`1
`
`2
`
`3
`
`MeLe‘u-MeVa1-MeBmt'-Abu—-Sar
`9 MeLeu
`I
`
`D-Ala — Ala — MeLeu — Val — MeLeu
`8
`7
`6
`5
`4
`Bmt = (4R)-4-[(E)—Z-butenyl]-4-methyl-L-threonine
`Me = Methyl
`Abu = a-Aminobutyric acid
`Val = Valine
`Ala = Alanine
`MeLeu = N-methyl Leucinc
`MeVal = N-Methyl valine
`Sar = Sarcosine
`
`Generally a cyclosporin such as cyclosporin A is not
`cytotoxic nor myelotoxic. It does not inhibit migration
`of monocytes nor does it inhibit granulocytes and mac
`rophage action. Its action is speci?c and leaves most
`established immune responses intact. However, it is
`nephrotoxic and is known to cause the following unde
`sirable side effects:
`(1) abnormal liver function;
`(2) hirsutism;
`(3) gum hypertrophy;
`(4) tremor;
`(5) neurotoxicity;
`(6) hyperaesthesia; and
`(7) gastrointestinal discomfort.
`Accordingly, an object of the present invention is to
`provide new cyclosporin analogs which will (1) restore
`the balance of the help-and-suppression mechanism of
`the immune system by acting at an earlier point than the
`anti-in?ammatory agents and (2) inducespeci?c long
`term transplantation tolerance through a suppressor cell '
`circuit without increasing the body's susceptibility to
`infection.
`Another object of the present invention is to provide
`pharmaceutical compositions for administering to a
`patient in need of the treatment one or more of the
`active immunosuppressive agents of the present inven
`tion.
`’
`Still a further object of this invention is to provide a
`method of controlling graft rejection, autoimmune and
`chronic in?ammatory diseases by administering a suffi
`cient amount of one or more of the novel immunosup
`pressive agents in a mammalian species in need of such
`treatment.
`Finally, it is the object of this invention to provide
`processes for the preparation of the active compounds
`of the present invention.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`A. Scope of the Invention
`This invention relates to cyclosporins of formula (I)
`
`CY CLOSPORIN ANALOGS WITH MODIFIED “C-9
`AMINO ACIDS”
`
`5
`
`10
`
`15
`
`CROSS-REFERENCE TO RELATED
`APPLICATION
`This Application is a continuation of US. Ser. No.
`057,196, ?led by B.E. Witzel on June 3, 1987 (now
`allowed).
`BACKGROUND OF THE INVENTION
`Immunoregulatory abnormalities have been shown to
`exist in a wide variety of “autoimmune” and chronic
`in?ammatory diseases, including systemic lupus ery
`thematosis, chronic rheumatoid arthritis, type 1 diabetes
`mellitus, in?ammatory bowel disease, biliary cirrhosis,
`uveitis, multiple sclerosis and other disorders such as
`Crohns disease, ulcerative colitis, bullous pemphigoid,
`sarcoidosis, psoriasis, ichthyosis, and Graves ophthal
`20
`mopathy. Although the underlying pathogenesis of
`each of these conditions may be quite different, they
`have in common the appearance of a variety of autoan
`tibodies and self-reactive lymphocytes. Such self-reac
`tivity may be due, in part, to a loss of the homeostatic
`controls under which the normal immune system oper
`ates.
`Similarly, following a bone-marrow or an organ
`transplantation, the host lymphocytes recognize the
`foreign tissue antigens and begin to produce antibodies
`which lead to graft rejection.
`One end result of an autoimmune or a rejection pro
`cess is tissue destruction caused by in?ammatory cells
`and the mediators they release. Antiin?ammatory
`agents such as NSAID’s and corticosteroids act princi
`35
`pally by blocking the effect or secretion of these media
`tors but do nothing to modify the immunologic basis of
`the disease. On the other hand-cytotoxic agents such as
`cyclophosphamide, act in such a nonspeci?c fashion
`that both the normal and autoimmune responses are
`‘ shut off. Indeed, patients treated with such nonspeci?c
`immunosuppressive agents are as likely to succoumb
`from infection as they are from their autoimmune dis
`ease.
`The cyclosporins are a family of immunospressive
`compounds isolated from fermentation broths of vari
`ous fungal species including Tolvoocladium in?atum and
`Cylindrocaroon Iucidum.
`'
`The generic structure of the class of cyclosporins has
`been established as a cyclic peptide of formula (I) which
`contains 11 amino acids.
`
`30
`
`45
`
`55
`
`For example, cyclosporin A of formula (II) contains
`several N-methylated amino acids and one novel amino
`acid “MeBMT” designated as the 1- “C-9 amino acid”.
`This novel amino acid is located in position 1 and has
`been found to be important for the biological activity of
`cyclosporin. We have found that replacing the double
`bond of the “09 amino acid” (MeBM'I) with a hetero
`atom such as S and 0 decreases the toxicity of the par
`ent cyclosporin. Substantial activity in the various as
`says in which cyclosporin A expresses immunosuppres
`sive activity is also exhibited.
`
`60
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`4,885,276
`
`4
`the aryl or heteroaryl group above can be substituted
`with one or more functional groups e.g.,
`(a) cl-salkyl,
`
`where X, R, Ra, and Re are as de?ned below;
`R2 is L-2-aminobutyryl; norvalyl; L-threonyl; or R1;
`R3 is sarcosyl or a-(methylmercapto)sarcosyl; N
`methyl-D-alanyl or N-methyl-L-alanyl; or D-prolyl;
`R4 is N-methyl-L-leucyl;
`R5 is L-valyl; or norvalyl;
`R6 is N-methyl-L-leucyl;
`R7 is L-alanyl; L-Z-aminobutyryl; or L-phenylalanyl;
`R3 is D-alanyl or L-alanyl;
`R9 is N methyl~L-leucyl; or N-methyl-L-valyl;
`R10 is N-methyl-L-leucyl; or L-leucyl;
`R11 is N-methyl-L-valYl; L-valyl; or L-Z-aminobuty
`ryl;
`Preferably, this invention relates to a cyclosporin A
`derivative having modi?ed l-“C-9 amino'acid”:
`30
`
`(c) C1_5 haloalkyl;
`(d) halo;
`(6) cyano;
`(t) hydroxy C1_3 alkyl;
`(g) 01-6 alkoxy;
`(h)
`
`where n is O, 1 or 2;
`(i) —NR;,COR¢ Rb and R¢ independently are H or
`C1_6 alkyl;
`(i) —NO2;
`(k) —NRbRd;
`
`(m) -—CONRbRC;
`(n) -COR1,;
`(O) _NRbCONRbRc;
`
`RS
`
`Re
`
`-OI-l
`
`CH3
`I
`Me,Leu'— Me.Val — N’
`
`CO—Abu-Sar
`
`Me.Leu
`
`Me.Leu
`
`D—Ala_Ala——Me.Leu—-Val
`
`(q) —OCOR1,;
`(r) —SCORb; or
`
`III
`
`R, is loweralkyl;
`Re is loweralkyl; loweralkylphenyl especially benzyl
`or aryl especially phenyl; and
`X is S, SO, S02, 0, or NR5.
`In a more preferred embodiment of this invention,
`R is
`(1) hydrogen;
`(2) C1-6 alkyl;
`
`45
`
`60
`
`wherein
`R is
`(1) hydrogen; ‘(2) loweralkyl especially C1_6 alkyl
`such as methyl, ethyl, Propyl, isopropyl, t-butyl,
`pentyl, benzyl, cyclopropyl, cyclopentyl or cyclo
`hexyl;
`(3) loweralkenyl especially C2_6 alkenyl, for or ex
`ample, vinyl, allyl, and buten-Z-yl:
`(4) haloloweralkyl especially C1_6 haloalkyl such as
`tri?uoromethyl;
`(5) aryl especially phenyl or substituted phenyl;
`(6) oxyloweralkyl especially alkoxy C14; alkyl such as
`——CHzORb where Rb is H or C1..6 alkyl;
`(7) thioloweralkyl especially alkylthio C1_6 alkyl
`such as -—CH2SRa wherein R, is C1_6 alkyl; or
`mercapto C1_6 alkyl;
`(8) heteroaryl especially pyridyl, pyrryl, furyl or
`thienyl;
`
`(4) phenyl;
`(5) CHZORb; 01'
`(6) CHZSRb;
`Ra is CH3;
`X is S or O.
`In a even more preferred embodiment of this inven
`tion,
`R is C14; alkyl or phenyl;
`Rd is CH3; and
`X is S.
`B. Preparation of the compounds within the scope of
`the present invention
`The cyclosporins of this invention are prepared via
`cyclization of appropriate linear undecapeptide follow
`ing well-established procedures which were slightly
`modi?ed for better results. The procedure most used is
`published by R.W. Wenger et al. in Helv. Chim. Acta,
`67. 502(1984). The following scheme illustrates the ap
`plication of this procedure to the cyclosporins of this
`invention.
`
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`

`
`4,885,276
`
`SCHEME I
`
`H
`CH3N\ COOH W
`-
`
`A
`
`-o1-r
`
`CH3
`
`RS
`
`(1)
`
`9”
`
`CH3 N
`
`CH
`3
`
`0
`
`II1
`CH3N \
`
`\
`
`coon
`
`(l) H-Abu-Sar—MeLeu—Val—MeLeu-Ala—O—Benzyl (3) ;
`(2) 11+
`
`CH3
`
`sn
`
`(2)
`
`CO-Abu-Sar-MeLeu-Val-MeLeu-Ala-O-Bemyl Bm-D-Ala-MCLCu-MaLer-MeV?-CH 5
`
`-0H
`
`CH3
`
`RS
`
`(4)
`
`CH3
`Bo<:—D-Ala-MeLeu-MeLeu-MeVal-N\ '\
`
`CO-Abu—Sar-MeLeu—Val-MeLw-Ala-O-Benzyl 117%
`(2) TFA
`
`CH3
`
`RS
`
`(6)
`
`CH3
`
`CH3
`
`CO-Abu-Sar-MeLeu-Vd-MeLeu-Ala-OH ——>III
`
`RS
`
`(7)
`
`According to the scheme, the threom'ne derivatives 60
`of formula (1) were converted to various cyclic un
`decapeptides of formula (8) utilizing, for the most part,
`published procedures. Notably, the methods described
`in Helv. Chim. Acta. 67, 502 (1984) are the preferred
`procedures.
`Generally, the key starting material, (1) was heated in
`acetone to form the intermediate oxazolidinecarboxylic
`acid (2). Condensation of compound (2) with the hex
`
`65
`
`apeptide ester (3) in the Presence of DCC, N
`hydroybenzotriazole and N-methylmorpholine yielded
`an intermediate which upon treatment with an acid, for
`example, HCl in methanol afforded the heptapeptide
`(4). Further condensation with the tetrapeptide (5) fol
`lowed by treatment with a strong base (e.g. NaOH or
`KOH) and then an acid such as TFA_ yielded the linear
`undecapeptide (7). Cyclization of compound (7) at high
`
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`

`
`4,885,276
`7
`8
`yl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl-)
`dilution and in the presence of a condensation agent
`such as l-proPanephosphonic acid cyclic anhydride and
`with excess lithium diisopropylamide followed by ex
`dimethylaminopyridine led to the cyclosporin deriva
`cess methyl iodide via published procedures yields
`cyclo-((2S,3R,4R)-N,4-dimethyl-4-(methYlthiome
`tive, (8).
`,
`thyl)-threonyl)-L-2-aminobutyryl-N-methyl-D-alanyl
`Table I below lists the representative compounds
`prepared by following essentially the same procedures
`N-methyl-L—leucyl-L-valyl-N-methyl-L-leucyl-L- ala
`nyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N
`described in Scheme 1.
`Alternatively, cyclosporins of this invention may be
`methyl-L-valyl) .
`made from existing analogs. For example, treatment of
`Also, treatment of the same substrate with a variety
`cyclo(2S,3R,4S)-N,4-dimethyl-4-(methylthiomethyl)~
`of oxidants, e.g., sodium metaperiodate or m-chloroper
`threonyl)-L-Z-aminobutyrylsarcosyl-N-methyl-L-level
`benzoic acid produces the corresponding sulfoxide or
`L-valyl-N-methyl-L-leucy-L-alanyl-D-alanyl-N-meth
`sulfone.
`
`10
`
`20
`
`25
`
`30
`
`35
`
`45
`
`55
`
`65
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`4,885,276
`13
`14
`Starting Materials of the process described in Scheme
`hard or soft capsules, or syrups or elixirs. Compositions
`I are mostly known and available commercially except
`intended for oral use may be prepared according to any
`the key starting material, the threonine derivative (1).
`method known to the art for the manufacture of phar
`maceutical compositions and such compositions may
`Generally the threonine derivatives of formula (1) can
`be prepared by known methods. For example:
`contain one or more agents selected from the group
`consisting of sweetening agents, ?avoring agents, color
`(a) amination of an epoxide derived from a suitable
`unsaturated carboxylic acid derivative (J. Chem.
`ing agents and preserving agents in order to provide
`pharmaceutically elegant and palatable preparation.
`Soc.(l962) 1116); (b) condensation of a substituted-thio
`Tablets containing the active ingredient in admixture
`propioualdehyde with glycine (Bull. Chem. . Soc. Japan
`with non-toxic pharmaceutically acceptable excipients
`44 (1979) 3967); (c) condensation of an aldehyde with
`isonitriles (Angew. Chem, Int. Ed. Engl. 13 (1974) 789;
`may also be manufactured by known methods. The
`(d) preparation from chiral glycine synthon enolates (J.
`excipients used may be for example, (1) inert diluents
`Am. Chem. Soc. 108 (1986) 6757); and (e) preparation
`such as calcium carbonate, lactose, calcium phosphate
`or sodium phosphate; (2) granulating and disintegrating
`from diethyl tartrate (Helv. Chim. Acta 66 (1983) 2308).
`agents such as corn starch, or alginic acid; (3) binding
`Various threonine derivaties as illustrated below in
`Scheme 11 were prepared, for the most part, via modi?
`agents such as starch, gelatin or acacia, and (4) lubricat
`cations of procedures described in (e).
`ing agents such as magnesium stearate, stearic acid or
`
`10
`
`15
`
`5
`
`o
`
`CH3’
`
`0
`
`CH3
`
`Scheme II
`
`OH
`
`on
`X
`X
`fri
`0 CH3 9 0 CH3 9 CH3 (l)-P'h_COCl/pyridine 9 o
`CH3
`CH3
`(2) ethylvinyl
`CH3
`0 Tosyl
`SR
`SR
`ether
`SI
`
`,
`oco
`
`CH2CH3
`
`H
`
`CH3—N
`.
`
`I
`
`O
`CN (1) H+
`0
`0H (2) R1NH2.HX,KCN
`CH3<—-—————-— CH3
`SR
`SR
`
`\Lcm
`
`r
`N ,ICN
`o=<
`0
`
`CH3
`
`(1)1(gCO3/EtOH
`(2)1-ICl/EtOl-I
`
`r
`N
`o=<
`0
`
`Deni-CH3 SO3/Pyridine
`DMSO
`
`on
`
`0
`cm
`sa
`
`°CH1CH3
`
`.
`Iii
`Rr-Ns COOH
`‘
`
`V-OH
`
`,COOCHgCH;
`
`ca;
`1 on
`-¢"& ‘3%,,
`(2)01‘!
`
`SR
`
`RS
`
`.
`
`‘From Diethyl L(+)-tartrate according to the procedures of R. M. Wenger, Helv. Chim. Acta, 66,2317 (1983) or Kenji Morl et al.,
`Tetrahedron, 36Y 87 (1980).
`
`C. Utility of the compounds within the scope of the .
`invention
`This invention also relates to a method of treatment
`for patients suffering from'immunoregulatory abnor
`malities involving the administration of a compound of
`formula (I) as the active constituent.
`55
`For the treatment of these conditions and diseases
`caused by immunoirregularity a compound of formula
`(I) may be administered orally, topically, parenterally,
`by inhalation spray or rectally in dosage unit formula-_
`tions containing conventional non-toxic pharmaceuti
`60
`cally acceptable carriers, adjuvants and vehicles. The
`term parenteral as used herein includes subcutaneous
`injections, intravenous, intramuscular, intrastemal in
`jection or infusion techniques.
`The pharmaceutical compositions containing the ac
`tive ingredient may be in a form suitable for oral use, for
`example, as tablets, troches, lozenges, aqueous or oily
`suspensions, dispersible powders or granules, emulsions,
`
`65
`
`tale. The tablets may be uncoated or they may be coated
`by known techniques to delay disintegration and ab
`sorption in the gastrointestinal tract and thereby pro
`vide a sustained action over a longer period. For exam
`ple, a time delay material such as glyceryl monostearate
`or glyceryl distearate may be employed. They may also
`be coated by the techniques described in the U.S. Pat.
`Nos. 4,256,108; 4,160,452; and 4,265,874 to form os
`motic therapeutic tablets for controlled release.
`In some cases, formulations for oral use may be in the
`form of hard gelatin capsules wherein the active ingre
`dient is mixed with an inert solid diluent, for example,
`calcium carbonate, calcium phosphate or kaolin. They
`may also be in the form of soft gelatin capsules wherein
`the active ingredient is mixed with water or an oil me
`dium, for example peanut oil, liquid paraffin, or olive
`oil.
`
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`
`l0
`
`15
`
`20
`
`4,885,276
`15
`Aqueous suspensions normally contain the active
`materials in admixture with excipients suitable for the
`manufacture of aqueous suspensions. Such excipients
`may be
`(1) suspending agents such as sodium carboxymethyl~=
`cellulose, methylcellulose, hydroxypropylmethylcel
`lulose, sodium alginate, polyvinylpyrrolidone, gum
`tragacanth and gum acacia;
`(2) dispersing or wetting agents which may be
`(a) a naturally-occurring phosphatide such as lecithin,
`a condensation product of an alkylene oxide with a
`fatty acid, for example, polyoxyethylene stearate,
`(c) a condensation product of ethylene oxide with a
`long chain aliphatic alcohol, for example, hep
`tadecaethyleneoxycetanol,
`(d) a condensation product of ethylene oxide with a
`partial ester derived from a fatty acid and a hexitol
`such as polyoxyethylene sorbitol monooleate, or
`(e) a condensation product of ethylene oxide with a
`partial ester derived from a fatty acid and a hexitol
`anhydride, for example polyoxyethylene sorbitan
`monooleate.
`The aqueous suspensions may also contain one or
`more preservatives, for example, ethyl or n-propyl p
`hydroxybenzoate; one or more coloring agents; one or
`more ?avoring agents; and one or more sweetening
`agents such as sucrose or saccharin.
`Oily suspension may be formulated by suspending the
`active ingredient in a vegetable oil, for example arachis
`oil, olive oil, sesame oil or coconut oil, or in a mineral
`30
`oil such as liquid paraffin. The oily suspensions may
`contain a thickening agent, for example beeswax, hard
`paraffm or cetyl alcohol. Sweetening agents and ?avor
`- ing agents may be added to provide a palatable oral
`preparation. These compositions may be preserved by
`the addition of an antioxidant such as ascorbic acid.
`Dispersible powders and granules are suitable for the
`preparation of an aqueous suspension. They provide the
`active ingredient in admixture with a dispersing or wet
`ting agent, a suspending agent and one or more preser
`vatives. Suitable dispersing or wetting agents and sus~
`pending agents are exempli?ed by those already men
`tioned above. Additional excipients, for example, those
`sweetening, ?avoring and coloring agents described
`above may also be present.
`The pharmaceutical compositions of the invention
`may also be in the form of oil-in-water emulsions. The
`oily phase may be a vegetable oil such as olive oil or
`arachis oils, or a mineral oil such as liquid paraffin or a
`mixture thereof. Suitable emulsifying agents may be (1)
`50
`naturally occurring gums such as gum acacia and gum
`tragacanth, (2) naturally-occurring phosphatides such
`as soy bean and lecithin, (3) esters or partial esters de
`rived from fatty acids and hexitol anhydrides, for exam
`ple, sorbitan monooleate, (4) condensation products of 55
`said partial esters with ethylene oxide, for example,
`polyoxyethylene sorbitan monooleate. The emulsions
`may also contain sweetening and ?avoring agents.
`Syrups and elixirs may be formulated with sweeten
`ing agents, for example, glycerol, propylene glycol,
`sorbitol or sucrose. Such formulations may also contain
`a demulcent, a preservative and flavoring and coloring
`agents.
`The pharmaceutical compositions may be in the form
`of a sterile injectable aqueous or oleagenous suspension.
`65
`This suspension may be formulated according to known
`methods using those suitable dispersing or wetting
`agents and suspending agents which have been men
`
`16
`tioned above. The sterile injectable preparation may
`also be a sterile injectable solution or suspension in a
`non-toxic parenterally-acceptable diluent or solvent, for
`example as a solution in 1,3-butane diol. Among the
`acceptable vehicles and solvents that may be employed
`are water, Ringer’s solution and isotonic sodium chlo
`ride solution. In addition, sterile, ?xed oils are conven
`tionally employed as a solvent or suspending medium.
`For this purpose any bland fixed oil may be employed
`including synthetic mono~ or diglycerides. In addition,
`fatty acids such as oleic acid ?nd use in the preparation
`of injectables.
`A compound of (I) may also be administered in the
`form of suppositories for rectal administration of the
`drug. These compositions can be prepared by mixing
`the drug with a suitable non-irritating excipient which is
`solid at ordinary temperatures but liquid at the rectal
`temperature and will therefore melt in the rectum to
`release the drug. Such materials are cocoa butter and
`polyethylene glycols.
`For topical use, creams, ointments, jellies, solutions
`or suspensions, etc., containing the immunoregulants
`are employed.
`Dosage levels of the order from about 0.5 mg to
`about 100 mg per kilogram of body weight per day are
`useful in the treatment of the above-indicated condi
`tions (from about 25 mg to about 5 gms. per patient per
`day).
`>
`The amount of active ingredient that may be com
`bined with the carrier materials to produce a single
`dosage form will vary depending upon the host treated
`and the particular mode of administration. For example,
`a formulation intended for the oral administration of
`humans may contain from 5 mg to 5 gm of active agent
`compounded with an appropriate and convenient
`amount of carrier material which may vary from about
`5 to about 95 percent of the total composition. Dosage
`unit forms will generally contain between from about 25
`mg to about 500 mg of active ingredient.
`It will be understood, however, that the specific dose
`level for any particular patient will depend upon a vari
`ety of factors including the activity of the speci?c com
`pound employed, the age, body weight, general health,
`sex, diet, time of administration, route of administration,
`rate of excretion, drug combination and the severity of
`the particular disease undergoing therapy.
`D. Biological evidence in support of utility of the com
`pounds within the scope of the invention
`It has been found that the compounds of formula (I)
`have immunosuppressive activities and are thereby use
`ful in the treatment of various “autoimmune” and
`chronic inflammatory diseases. They may also be useful
`in the prevention of graft rejection or rejection of “do
`nor” organs in transplantation operations. The follow
`ing table illustrates and supports the utility of the com
`pounds of the present invention:
`
`25
`
`35
`
`40
`
`45
`
`NOVARTIS EXHIBIT 2007
`Par v Novartis, IPR 2016-00084
`Page 9 of 13
`
`

`
`17
`TABLE 2
`CYCLOPHILIN BINDING ASSAY“ '
`RS
`
`4,885,276
`
`18
`(B). N.m.r. spectrum was consistent with the structure;
`Mass spectrum, M+=l50; [a]p=30 l4.5° (c=0.9,
`CDCl3).
`
`CH3
`
`-Ol-l
`
`CH3 ..
`I
`MeLeu-MeVal-Nl
`
`CO—R2—Sar
`
`MeLeu
`
`MeLeu
`
`D—Ala—Ala-MeLeu—Val
`
`Step C: Preparation of
`(2R,3R]-l-Benzoyloxy-3-methyl-4-(methylthio)-2
`butanol (C)
`To a stirred, ice-cooled solution of (B) (3.7g.,0.025m.)
`in dried pyridine (30 ml.) was added dropwise over ca.
`5 minutes under a nitrogen atmosphere a solution of
`benzoyl chloride (3.0 ml., 0.026m.) in dry ether (5 ml.).
`The mixture was stirred for an additional 5 minutes, the
`ice-bath removed, and the mixture allowed to stir at
`ambient temperatures. After ca.l.5 hr., tlc analysis indi
`cated that the reaction was complete and the mixture
`was transferred to a separatory funnel with ether (300
`ml.). The mixture was washed IX with water (100 ml.),
`4 X with saturated copper sulfate solution, 1X with
`saturated sodium chloride solution and dried over so
`dium sulfate. Concentration of the filtered solution fol
`lowed by chromatography on silica gel (200g; 1%
`methanol/methylene chloride as eluant) yielded 5.4g
`(C) as an oil. N.m.r. was consistent with the structure
`[a]p=30 9.1 (c=0.75, CDClg).
`Step D: Preparation of
`(2R,3S)-l-Benzoyloxy-Z-(l’-ethoxyethoxy)-3-methyl-4
`(methylthio)-butane (D)
`To a stirred solution of (C) (5.4g., 0.021 m.).in dry
`methylene chloride (60 ml.) was added ethyl vinyl ether
`(8 ml., 0.084 m.) followed by one small drop of anhy
`drous tri?uoroacetic acid. The mixture was allowed to
`stir in a closed container at ambient temperatures until
`tlc analysis indicated that the reaction was complete.
`Anhydrous sodium hydrogen carbonate (0.1g.) was then
`added, the mixture allowed to stir for ca. one hour, and
`the volatiles removed in vacuo. The crude (D) was used
`immediately in the next step. Step B: Preparation of
`(2R,3.R)-2-(l’-Ethoxyethoxy)-3methyl 4-(methylthio)
`l-butanol (E)
`A solution of the crude (D) in cold ethanol (60 ml.)
`was treated with l0N KOH (12 ml.), and the ice-cooled
`mixture allowed to gradually warm to room tempera
`ture. After ca. 40 minutes, tlc analysis indicated that the
`reaction was complete. The mixture was diluted with
`methylene chloride (300 ml.), washed with water (200
`ml.), the aqueous layer re-extracted 2X with methylene
`chloride and the combined organic layers dried (sodium
`sulfate), ?ltered, and concentrated to 4.6g. of (E) which
`was used in the following step without further puri?ca
`tion. Nmr was consistent with the structure [a]D=4.O
`(c=0.8, CDCl3).
`Step F: Preparation of
`(2R,3S)-2-(1'Ethoxyethoxy)-3-methyl-4-(methylthio)-l
`butanal (F).
`To a well stirred solution of (F) (4.lg., 0.0l85m.) in
`dried DMSO (50 ml.) was added dry triethylamine (17
`ml.) and the resulting mixture cooled under a nitrogen
`atmosphere to ca. 16° C. A solution of sulfur dioxide
`pyridine complex (8.84g., 0.055m) in dry DMSO (50
`ml.) was then added dropwise over ca. 8 minutes while
`the temperature was kept below 30° C. The resulting
`mixture was stirred at ambient temperatures until tlc
`analysis indicated complete reaction. The mixture was
`diluted with ether (200 ml.), the ether layer washed IX
`with water (200 ml.), the aqueous layer re-extracted 2X
`
`Compound a
`(3)
`CH3
`(b)
`CH3
`(c)
`CH3CH2
`
`(d)
`
`CH3
`
`R2
`Abu
`nVal
`Abu
`
`Abu
`
`CH3
`
`15
`
`CYCLOPHILIN BINDING
`(% OF CsA Activity)b
`179
`58
`96
`
`184
`
`20
`
`18
`Abu
`Phenyl
`(e)
`"This assay is described in detail by R. Handschumacher et. al., Science, 226, 544
`1984)
`-
`gAverage of multiple assays
`
`25
`
`35
`
`EXAMPLE 1
`Preparation of Starting Threonine Derivatives
`Step A: Prepartion of (2R.3S)-3,4‘Isopropylidene
`dioxy-Z-methyl-l-(methylthio)-butane (A)
`To a stirred, ice-cooled solution of sodium methyl
`mercaptide (from 3.0g., 0.l3g.atm. of sodium and excess
`anhydrous methyl mercaptan) in anhydrous methanol
`(250 ml.) was added dropwise a solution of p-toluenesul
`fornate (10.0 g., 0.0318 111) (prepared according to the
`procedures described by Helv. Chim. Acta.. 66 (1983),
`2317 or Tetrahedron 36, (1980), 89) in anhydrous ether
`(50 ml.). The resultant mixture was stirred cold for an
`additional one hour, and then allowed to stir at ambient
`temperatures until thin-layer (tlc) analysis indicated
`the absence of tosylate. Nitrogen gas was used to dis
`place the excess methyl mercaptan, the reaction mixture
`concentrated in vacuo to a residue, the residue distrib
`uted between methylene chloride and water, the aque
`ous layer re-extracted one time with methylene chlo
`ride, and the combined organic layers dried over so
`dium sulfate. The concentrated residue was then chro=
`matographed on a 300g. silica gel column using 10%
`ether in hexane as eluant to yield 3.7g.(A) as a volatile
`oil. N.m.r. and Mass spectrum were consistent with the
`structure. [a] D= + 15.0° (c= l,CDCl3).
`Step B: Prepartion of
`(2R,3S)3-Methyl-4-(methylthio)-1,2-butanediol (B)
`To a stirred solution of (A) (5.6g.,0.0294m.) in perox
`ide free tetrahydrofuran (250 ml.) was added dropwise
`a solution of dilute HCl (from 17 ml. Fisher 2N HCl
`60
`diluted to 68 ml. with water). The resultant mixture was
`evacuated 4 times under a nitrogen atmosphere and
`allowed to stir at room temperature until tlc analysis
`(10% ether in hexane) indicated the absence of (A). The
`pH was adjusted to ca.7 with fresh saturated sodium
`65
`hydrogen carbonate solution and the THF removed in
`vacuo. Repeated extraction of the resulting aqueous
`layer with methylene chloride yields 3.7g(84%) of oily
`
`50
`
`55
`
`NOVARTIS EXHIBIT 2007
`Par v Novartis, IPR 2016-00084
`Page 10 of 13
`
`

`
`4,885,276
`19
`with ether, and the combined ether layers washed 2X
`with water and IX with saturated sodium chloride solt
`uion. The ether solution was then concentrated in
`vacuo (temperature <23° C.), and the oily residue used
`immediately in the next step.
`Step G: Preparation of
`(2R,3S)-2-Hydroxy-3-methyl-4-(methylthio)-l-butanal
`(G)
`A stirred solution of (F) (from above) in fresh THIF
`(75ml) was treated with 1N HCl (15 ml.) The‘ mixture
`was deaerated under a nitrogen atmosphere and stirred
`at ambient temperatures until tlc analysis indicated no
`compound (F) remained. The mixture was transferred
`to a separatory funnel with methylene chloride (300 ml
`). washed 1X with water (300 ml.), and the aqueous
`layer re-extracted 2X with methylene chloride. The
`organic layers were combined and washed lX with
`water and concentrated in vacuo to compound (G) as
`an oil which was used immediately in the following
`step.
`
`20
`
`25
`
`30
`
`Step H: Preparation of
`(2RS,3R,4S)-3-Hydroxy-4-methyl-2-(methylamino)-5
`(methylthio)pentanenitrile (H)
`Compound (G) was dissolved in methanol (120 ml.)
`and the stirred solution treated sequentially with potas
`sium cyanide (l.82g., 0.028m), methylamine hydrochlo
`ride (2.03g., 0.03m.), and water (20 ml.). The sides of the
`?ask were rinsed down with an additional 5 ml of meth
`anol, the mixture covered with a nitrogen atmosphere
`and allowed to stir overnight at room temperature. The
`mixture was then concentrated in vacuo to a slurry.
`This was distributed between methylene chloride (300
`ml.) and water (400 ml), separated, and the aqueous
`35
`layer re-extracted 2x with methylene chloride. The
`combined organic layers were dried over sodium sulfate
`and concentrated to yield compound (H) as a waxy
`solid (3.lg.). It was used directly in the next step.
`Step I: Preparation of
`3-Methyl-5-((l’-methyl-2'-(methylthio))ethyl)-2-0xoox
`azolidine-4-carbonitrile (I).
`To a stirred solution of (H) (3.lg., 0.0165m.) in dried
`methylene chloride (120ml) at room temperature was
`45
`added l,l’-carbonyldiimidazole (5.63g.,0.0347m.) and
`the mixture covered with a nitrogen atmosphere and
`allowed to stir overnight. After transferring to a separa
`tory funnel with methylene chloride (200ml.), the resul
`tant solution was washed with water, the aqueous layer
`re~extracted 2X with methylene chloride and the com
`bined organic layers dried over sodium sulfate. Concen
`tration of the dried solution yielded 4.0g. of an oil which
`after chromatography (silica gel, 0.5% methanol/
`methylene chloride as eluant) gave 2.4g. of compound
`(I) as an oil which crystallized on standing overnight.
`[a]p= +37.5° (c=0.6, CDC13).
`Step J: Preparation of Ethyl (4S,
`5R)~3-methyl-5-(((1'S-1‘methyl-2.-(methylthio))ethyl)2
`oxooxazolidineA-carboximidate (I)
`To a stirred solution of Compound (I) (0.8g.,
`0.0037m) in 95% ethanol (35 ml.) was added crushed,
`anhydrous potassium carbonate (1.05g.). The sides of
`the flask was rinsed down with an additional 5 ml. of
`65
`ethanol, and the mixture allowed to stir at room temper
`ature. After 6 hours, the mixture was transferred
`quickly to a separatory funnel, and methylene chloride
`
`50
`
`20
`(500 ml.) followed by water (150ml) was added. The
`mixture was shaken, separated, the aqueous layer re
`extracted 2X with methylene chloride, and the com
`bined organic layer dried over sodium sulfate. Concen
`tration of the dried organic layer yielded 0.85 g of com
`pound (J) (Mass spectrum, M+=2l4) which was used
`directly in the next step.
`
`Step K: Preparation of Ethyl
`(4S,5R)~3-methyl-5-(((1'R)~1'-methyl-2-(methylthio))e
`thyl)-2-oxooxazolidine-4~carboxylate (K)
`To a stirred solution of Compound (J) (0.85g.,
`0.00327m.) in 95% ethanol (90 ml.) was immediately
`added lN HCL (4.5 ml., 0.0045m.), and the mixture
`allowed to stir until tlc analysis indicated no compound
`(J) remained. The pH of the solution was brought to ca.
`7 with lN sodium hydrogen carbonate solution and the
`mixture distributed between methylene chloride
`(300ml.) and water (150ml). The aqueous layer was
`re-extracted with 100ml. methylene chloride and the
`combined organic layers dried over sodium sulfate.
`Concentration of the dried organic layer in vacuo
`yielded 0.8g. compound (K) as an oil. [a]D=+33.7