(12) United States Patent
`Naicker et al.
`
`USOO6503921B2
`(10) Patent No.:
`US 6,503,921 B2
`(45) Date of Patent:
`*Jan. 7, 2003
`
`DEUTERATED RAPAMYCIN COMPOUNDS,
`METHODS AND USES THEREOF
`
`(56)
`
`References Cited
`
`PUBLICATIONS
`
`Dennis P. Curran, et al., Intramolecular Hydrogen Transfer
`Reaction of o-(Bromophenyl)dialklsilyl Ethers. Preparation
`of Rapamycin-d, Tetrahedron Letters, vol. 33, No. 17, pp.
`2295-2298.
`Don Sticker, Senior Technical Information Specialist of
`Chemical Abstract Service, C.A.S., Commericial Database
`Search of Deuterated Rapamycin, search conducted Mar. 9,
`2OOO.
`Park et al., Jr. Bio Chem., vol. 267, No. 5 (15) pp
`3316–3324, 1992.
`Connelly et al, Biochemistry, vol. 32, pp 5583–5590 (1993).
`Primary Examiner Bruck Kifle
`(74) Attorney, Agent, or Firm-Burns Doane Swecker &
`Mathis LLP
`ABSTRACT
`(57)
`The Synthesis of deuterated analogues of rapamycin is
`disclosed together with a method for use for inducing
`immunoSupression and in the treatment of transplantation
`rejection, graft VS host disease, autoimmune diseases, dis
`eases of inflammation leukemia/lymphoma, Solid tumors,
`fungal infections, hyperproliferative vascular disorders.
`Also described is a method for the synthesis of water soluble
`deuteratred rapamycin compounds and their use as described
`above.
`
`21 Claims, 2 Drawing Sheets
`
`(54)
`
`(75)
`
`(73)
`(*)
`
`Inventors: Selvaraj Naicker, Edmonton (CA);
`Randall W. Yatscoff, Edmonton (CA);
`Robert T. Foster, Edmonton (CA)
`Assignee: Isotechnika, Inc., Edmonton (CA)
`Notice:
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`This patent is Subject to a terminal dis
`claimer.
`
`(21)
`(22)
`(65)
`
`Appl. No.: 09/987,812
`Filed:
`Nov. 16, 2001
`Prior Publication Data
`
`US 2002/0028827 A1 Mar. 7, 2002
`Related U.S. Application Data
`
`(63)
`
`(60)
`
`(51)
`(52)
`(58)
`
`Continuation of application No. 09/348,015, filed on Jul. 6,
`1999, which is a continuation-in-part of application No.
`09/148,623, filed on Sep. 4, 1998, now abandoned.
`Provisional application No. 60/057,632, filed on Sep. 5,
`1997.
`Int. Cl." ..................... C07D 491/16; A61K 31/445
`U.S. Cl. ........................................ 514/291; 540/456
`Field of Search ........................... 540/456; 514/291
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`1
`DEUTERATED RAPAMYCIN COMPOUNDS,
`METHODS AND USES THEREOF
`
`REFERENCE TO RELATED APPLICATIONS
`This application is a continuation of Ser. No. 09/348,015
`filed Jul. 6, 1999 which is a continuation-in-part of U.S.
`patent application Ser. No. 09/148,623, filed Sep. 4, 1998,
`now abandoned which is based on provisional patent appli
`cation No. 60/057,632, filed Sep. 5, 1997 both of which are
`relied on and incorporated herein by reference.
`BACKGROUND OF THE INVENTION
`This invention relates to deuterated derivatives of rapa
`mycin and a method for using them in the treatment of
`transplantation rejection, host VS. graft disease, graft VS. host
`disease, leukemia/lymphoma, hyperproliferative vascular
`disorders, autoimmune diseases, diseases of inflammation,
`Solid tumors, and fungal infections.
`Rapamycin, known as Sirolimusis, is a 31-membered
`macrollide lactone, C5H7NO, with a molecular mass of
`913.6 Da. In solution, Sirolimus forms two conformational
`trans-, cis-isomers with a ratio of 4:1 (chloroform) due to
`hindered rotation around the pipecolic acid amide bond. It is
`sparingly Soluble in water, aliphatic hydrocarbons and
`diethyl ether, whereas it is Soluble in alcohols, halogenated
`hydrocarbons and dimethyl Sulfoxide. Rapamycin is
`unstable in Solution and degrades in plasma and low-, and
`neuteral-pH buffers at 37° C. with half-life of <10 h. the
`Structures of the degradation products have recently been
`characterized. Rapamycin is a macrocyclic triene antibiotic
`produced by Streptomyces hygroscopicus, which was found
`to have antifungal activity, particularly against Candida
`albicans, both in vitro and in vivo C. Vezina et al., J.
`Antibiot. 28, 721 (1975); S. N. Sehgal et al., J. Antibiot. 28,
`727 (1975); H. A. Baker et al., J. Antibiot. 31, 539 (1978);
`U.S. Pat. Nos. 3,929,992; and 3,993,749).
`Rapamycin alone (U.S. Pat. No. 4.885,171) or in combi
`nation with picibanil (U.S. Pat. No. 4,401,653) has been
`shown to have antitumor activity. R. Martel et al. Can. J.
`Physiol. Pharmacol. 55, 48 (1977) disclosed that rapamycin
`is effective in the experimental allergic encephalomyelitis
`model, a model for multiple Sclerosis, in the adjuvant
`arthritis model, a model for rheumatoid arthritis; and effec
`tively inhibited the formation of IgE-like antibodies.
`The immunosuppressive effects of rapamycin have been
`disclosed in FASEB 3, 3411 (1989). Cyclosporin A and
`FK-506, other macrocyclic molecules, also have been shown
`to be effective as immunosuppressive agents, therefore use
`ful in preventing transplant rejection FASEB 3, 3411
`(1989); FASEB 3, 5256 (1989); and R. Y. Calne et al.,
`Lancet 1183 (1978)). Although it shares structural homology
`with the immunosuppressant tacrolimus and binds to the
`Same intracellular binding protein in lymphocytes, rapamy
`cin inhibits S6p70-kinase and therefore has a mechanism of
`immunosuppressive action distinct from that of tacrolimus.
`Rapamycin was found to prolong graft Survival of different
`transplants in Several Species alone or in combination with
`other immunoSupreSSants. In animal models its spectrum of
`toxic effects is different from that of cyclosporin or FK-506.,
`comprising impairment of glucose homeostasis, Stomach,
`ulceration, weight loSS and thrombocytopenia, although no
`nephrotoxicity has been detected.
`Mono- and diacylated derivatives of rapamycin (esterified
`at the 28 and 43 positions) have been shown to be useful as
`antifungal agents (U.S. Pat. No. 4,316,885) and used to
`make water soluble prodrugs of rapamycin (U.S. Pat. No.
`
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`4,650,803). Recently, the numbering convention for rapa
`mycin has been changed; therefore according to Chemical
`Abstracts nomenclature, the esters described above would
`be at the 31- and 42-positions. Carboxylic acid esters (PCT
`application No. WO92/05179), carbamates (U.S. Pat. No.
`5,118,678), amide esters (U.S. Pat. No. 5,118,678), (U.S.
`Pat. No. 5,118,678) fluorinated esters (U.S. Pat. No. 5,100,
`883), acetals (U.S. Pat. No. 5,151,413), silyl ethers (U.S.
`Pat. No. 5,120,842), bicyclic derivatives (U.S. Pat. No.
`5,120,725), rapamycin dimers (U.S. Pat. No. 5,120,727) and
`O-aryl, O-alkyl, O-alkyenyl and O-alkynyl derivatives (U.S.
`Pat. No. 5,258,389) have been described.
`Rapamycin is metabolized by cytochrome P-4503A to at
`least Six metabolites. During incubation with human liver
`and Small intestinal microSomes, Sirolimus was hydroxy
`lated and demethylated and the structure of 39-O-demethyl
`Sirolimus was identified. In bile of Sirolimus-treated rats >16
`hydroxylated and demethylated metabolites were detected.
`In rapamycin, demethylation of methoxy group at C-7
`Carbon will lead to the change in the conformation of the
`Rapamycin due to the interaction of the released C-7
`hydroxyl group with the neighbouring pyran ring System
`which is in equilibrium with the open form of the ring
`system. The C-7 hydroxyl group will also interact with the
`triene System and possibly alter the immunoSupressive activ
`ity of rapamycin. This accounts for the degradation of
`rapamycin molecule and its altered activity.
`Stable isotopes (e.g., deuterium, C, N, 'O) are non
`radioactive isotopes which contain one additional neutron
`than the normally abundant isotope of the atom in question.
`Deuterated compounds have been used in pharmaceutical
`research to investigate the in Vivo metabolic fate of the
`compounds by evaluation of the mechanism of action and
`metabolic pathway of the non deuterated parent compound.
`(Blake et al. J. Pharm. Sci. 64, 3, 367-391, 1975). Such
`metabolic Studies are important in the design of Safe, effec
`tive therapeutic drugs, either because the in Vivo active
`compound administered to the patient or because the
`metaboliteS produced from the parent compound prove to be
`toxic or carcinogenic (Foster et al., Advances in drug
`Research Vol. 14, pp. 2-36, Academic press, London, 1985).
`Incorporation of a heavy atom particularly Substitution of
`deuterium for hydrogen, can give rise to an isotope effect
`that can alter the pharmacokinetics of the drug. This effect
`is usually insignificant if the label is placed in a molecule at
`the metabolically inert position of the molecule.
`Stable isotope labeling of a drug can alter its physico
`chemical properties Such as pKa and lipid Solubility. These
`changes may influence the fate of the drug at different Steps
`along its passage through the body. Absorption, distribution,
`metabolism or excretion can be changed. Absorption and
`distribution are processes that depend primarily on the
`molecular size and the lipophilicity of the Substance.
`Drug metabolism can give rise to large isotopic effect if
`the breaking of a chemical bond to a deuterium atom is the
`rate limiting Step in the process. While Some of the physical
`properties of a stable isotope-labeled molecule are different
`from those of the unlabeled one, the chemical and biological
`properties are the Same, with one important exception:
`because of the increased mass of the heavy isotope, any
`bond involving the heavy isotope and another atom will be
`Stronger than the same bond between the light isotope and
`that atom. In any reaction in which the breaking of this bond
`is the rate limiting Step, the reaction will proceed slower for
`the molecule with the heavy isotope due to kinetic isotope
`effect. A reaction involving breaking a C-D bond can be up
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`to 700 per cent slower than a similar reaction involving
`breaking a C-H bond.
`More caution has to be observed when using deuterium
`labeled drugs. If the C-D bond is not involved in any of the
`Steps leading to the metabolite, there may not be any effect
`to alter the behavior of the drug. If a deuterium is placed at
`a site involved in the metabolism of a drug, an isotope effect
`will be observed only if breaking of the C-D bond is the
`rate limiting Step. There are evidences to Suggest that
`whenever cleavage of an aliphatic C-H bond occurs,
`usually by oxidation catalyzed by a mixed-function oxidase,
`replacement of the hydrogen by deuterium will lead to
`observable isotope effect. It is also important to understand
`that the incorporation of deuterium at the Site of metabolism
`Slows its rate to the point where another metabolite produced
`by attack at a carbon atom not Substituted by deuterium
`becomes the major pathway by a process called “metabolic
`Switching”.
`It is also observed that one of the most important meta
`bolic pathways of compounds containing aromatic Systems
`is hydroxylation leading to a phenolic group in the 3 or 4
`position to carbon Substituents. Although this pathway
`involves cleavage of the C-H bond, it is often not accom
`panied by an isotope effect, because the cleavage of this
`bond is mostly not involved in the rate-limiting step. The
`Substitution of hydrogen by deuterium at the Stereo center
`will induce a greater effect on the activity of the drug.
`Clinically relevant questions include the toxicity of the
`drug and its metabolite derivatives, the changes in distribu
`tion or elimination (enzyme induction), lipophilicity which
`will have an effect on absorption of the drug. Replacement
`of hydrogen by deuterium at the Site involving the metabolic
`reaction will lead to increased toxicity of the drug. Replace
`ment of hydrogen by deuterium at the aliphatic carbons will
`have an isotopic effect to a larger extent. Deuterium placed
`at an aromatic carbon atom, which will be the Site of
`hydroxylation, may lead to an observable isotope effect,
`although this is less often the case than with aliphatic
`carbons. But in few cases Such as in penicillin, the Substi
`tution on the aromatic ring will induce the restriction of
`rotation of the ring around the C-C bond leading to a
`favorable Stereo-Specific Situation to enhance the activity of
`the drug.
`Approaching half a century of Stable-isotope usage in
`human metabolic Studies has been without documented
`Significant adverse effect. Side-effects with acute D dosing
`are transitory with no demonstrated evidence of permanent
`deleterious action. The threshold of D toxicity has been
`defined in animals and is far in exceSS of concentrations
`conceivably used in human studies (Jones PJ, Leatherdale
`ST Clin Sci (Colch) April 1991; 80(4):277–280). The pos
`Sibility that D may have additional beneficial pharmacologi
`cal applications cannot be excluded. For isotopes other than
`D, evidence of observed toxicity remains to be produced
`even at dosages far in excess of the range used in metabolic
`studies. Absence of adverse effect may be attributable to
`Small mass differences and the Similar properties of tracer
`and predominantly abundant isotopes. The precision of
`extrapolating toxicity thresholds from animal Studies
`remains unknown. However, should perturbation of the
`delicate homoeostatic characteristic of living organisms
`occur with use of Stable isotopes, it is almost undoubtedly at
`Some level of administration greatly in excess of those
`administered currently in biomedical research.
`In the prior art, no details are described regarding deu
`terated derivatives to improve the Stability of rapamycin
`
`4
`molecule and also about glycosylated deuterated rapamycin
`to improve the stability and also the solubility of the
`molecule in order to increase the bio-availability of the drug.
`We therefore defined the global objective of preparing a
`rapamycin derivative which is more Stable, leSS prone to
`degradation, and more water Soluble to improve the bio
`availability.
`
`SUMMARY OF THE INVENTION
`Deuteration of the rapamycin molecule results in altered
`physicochemical and pharmacokinetic properties which
`enhance its usefulneSS in the treatment of transplantation
`rejection, host VS. graft disease, graft VS. host disease,
`leukemia/lymphoma, hyperproliferative vascular disorders,
`autoimmune diseases, diseases of inflammation, Solid
`tumors, and fungal infections.
`Deuterium isotope is selected based on the fact that if 'C,
`'N or another heavy isotope differing from the light one by
`less than 10% in mass is incorporated at the site of
`metabolism, there may be a Small isotope effect. In addition
`to this, there are Secondary isotope effects away from the Site
`of isotope Substitution due to changes in electronic environ
`ment.
`Substitution of deuterium in methyl groups of rapamycin
`will result in a slower rate of oxidation of the C-D bond
`relative to the rate of oxidation of a non deuterium Substi
`tuted C-H bond. The isotopic effect acts to reduce forma
`tion of demethylated metabolites and thereby alters the
`pharmacokinetic parameters of the drug. Lower rates of
`oxidation, metabolism and clearance result in greater and
`more Sustained biological activity. Deuteration is targeted at
`various sites of the rapamycin molecule to increase the
`potency of drug, reduce toxicity of the drug, reduce the
`clearance of the pharmacologically active moiety and
`improve the stability of the molecule.
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 is the chemical structure of 7-deuteromethyl rapa
`mycin showing sites of deuteration.
`FIG. 2 is the chemical structure of epi-7 deuteromethyl
`rapamycin showing sites of deuteration.
`FIG. 3 is the chemical structure of 7,43-de-rapamycin
`showing sites of deuteration.
`FIG. 4 is the chemical structure of 31,42-d showing sites
`of deuteration.
`FIG. 5 illustrates the preparation of glycosylated deuter
`orapamycin.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`Substitution of deuterium for ordinary hydrogen and
`deuterated Substrates for protio metabolites can produce
`profound changes in biosystems. Isotopically altered drugs
`have shown widely divergent pharmacological effects. Pet
`tersen et al., found increased anti-cancer effect with deuter
`ated 5,6-benzylidene-dl-L-ascorbic acid (Zilascorb)
`Anticancer Res. 12, 33 (1992)).
`Substitution of deuterium in methyl groups of rapamycin
`will result in a slower rate of oxidation of the C-D bond
`relative to the rate of oxidation of a non deuterium Substi
`tuted C-H bond. The isotopic effect acts to reduce forma
`tion of demethylated metabolites and thereby alters the
`pharmacokinetic parameters of the drug. Lower rates of
`oxidation, metabolism and clearance result in greater and
`more Sustained biological activity. Deuteration is targeted at
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`various Sites of the rapamycin molecule to increase the
`potency of drug, reduce toxicity of the drug, reduce the
`clearance of the pharmacologically active moiety and
`improve the stability of the molecule.
`Determination of the physicochemical, toxicological and
`pharmacokinetic properties can be made using Standard
`chemical and biological assays and through the use of
`mathematical modeling techniques which are known in the
`chemical and pharmacological/toxicological arts. The thera
`peutic utility and dosing regimen can be extrapolated from
`the results of Such techniques and through the use of
`appropriate pharmacokinetic and/or pharmacodynamic
`models.
`The compounds of this invention may be administered
`neat or with a pharmaceutical carrier to an animal, Such as
`a warm blooded mammal, and especially humans, in need
`thereof. The pharmaceutically effective carrier may be solid
`or liquid.
`A Solid carrier can include one or more Substances which
`may also act as flavoring agents, lubricants, Solubilizers,
`Suspending agents, fillers, glidants, compression aids, bind
`erS or tablet-disintegrating agents, it can also be an encap
`Sulating material. In powders, the carrier is a finely divided
`solid which is in admixture with the finely divided active
`ingredient. In tablets, the active ingredient is mixed with a
`carrier having the necessary compression properties in Suit
`able proportions and compacted in the shape and size
`desired. The powders and tablets may contain up to 99% of
`the active ingredient. Suitable Solid carriers include, for
`example, calcium phosphate, magnesium Stearate, talc,
`Sugars, lactose, dextrin, Starch, gelatin, cellulose, methyl
`cellulose, Sodium carboxy methyl cellulose,
`polyvinylpyrrolidine, low melting waxes and ion eXchange
`CSS.
`35
`Liquid carriers are used in preparing Solutions,
`Suspensions, emulsions, Syrups, elixirs and pressurized com
`positions. The active ingredient can be dissolved or SuS
`pended in a pharmaceutically acceptable liquid carrier Such
`as water, an organic Solvent, a mixture of both or pharma
`ceutically acceptable oils or fats. The liquid carrier can
`contain other Suitable pharmaceutical additives Such as
`Solubilizers, emulsifiers, buffers, preservatives, Sweeteners,
`flavoring agents, Suspending agents, thickening agents,
`colors, Viscosity regulators, Stabilizers or oSmo-regulators.
`Suitable examples of liquid carriers for oral and parenteral
`administration include water (partially containing additives
`as above, e.g. cellulose derivatives, possibly Sodium car
`boxymethyl cellulose Solution), alcohols (including mono
`hydric alcohols and polyhydric alcohols, e.g. glycols) and
`50
`their derivatives, and oils (e.g. fractionated coconut oil and
`arachis oil). For parenteral administration, the carrier can
`also be an oily ester Such as ethyl oleate and isopropyl
`myristate. Sterile liquid carriers are useful in Sterile liquid
`form compositions for parenteral administration. The liquid
`carrier for pressurized compositions can be halogenated
`hydrocarbon or other pharmaceutically acceptable propel
`lent.
`Liquid pharmaceutical compositions which are Sterile
`Solutions or Suspensions can be utilized by, for example,
`intramuscular, intraperitoneal or Subcutaneous injection.
`Sterile solutions can also be administered intravenously. The
`compound can also be administered orally either in liquid or
`Solid composition form.
`The pharmaceutical composition can be in unit dosage
`form, e.g. as tablets or capsules. In Such form, the compo
`Sition is Sub-divided in unit dose containing appropriate
`
`6
`quantities of the active ingredient; the unit dosage forms can
`be packaged compositions, for example, packeted powders,
`Vials, ampoules, prefilled Syringes or Sachets containing
`liquids. The unit dosage form can be, for example, a capsule
`or tablet itself, or it can be the appropriate number of any
`Such compositions in package form. The dosage to be used
`in the treatment must be subjectively determined by the
`attending physician.
`In addition, the compounds of this invention may be
`employed as a Solution, cream, or lotion by formulation with
`pharmaceutically acceptable vehicles administered to a fun
`gally affected area.
`
`EXAMPLES
`FIGS. 1-4 show examples of sites for deuteration of the
`rapamycin molecule. Nonlimiting examples of deuterated
`rapamycin molecules include the compounds;
`7-deuteromethyl rapamycin (FIG. 1), epi-7-deuteromethyl
`rapamycin (FIG. 2), 7,43-de-rapamycin (FIG. 3) and 31,42
`d-rapamycin (FIG. 4) including the cis and trans isomers of
`the compounds shown in FIGS. 1-4. FIG. 5 shows the
`preparation and Structure of the compound glycosylated
`deuterorapamycin.
`
`Example 1
`Preparation of 7-Deuteromethyl Rapamycin
`(FIG. 1)
`5 mg of Rapamycin was dissolved in 2.5 ml of dichlo
`romethane. 40 mg of deuterated methanol was added. 10
`beads of NAFIONOR catalyst were added to the above
`Solution. The contents were stirred under nitrogen at room
`temperature for 14 hours. The reaction was monitored by
`mass spectrum. The Solution was filtered and concentrated.
`The residue was dissolved in dry benzene and freeze dried.
`The white Solid obtained was homogenous by mass Spec
`trum analysis and characterized by LC/MS.
`Example 2
`Preparation of 31, 42 d-7-deuterated Rapamycin
`(FIG. 3)
`Rapamycin (11 mM) was dissolved in a mixture of
`cyclohexane and dichloromethane (1:1) 10 ml. The contents
`were cooled in ice bath and poly(Vinylpyridinium)
`dichromate 0.5 grams was added. The reaction mixture was
`Stirred overnight and the reaction was followed by mass
`Spectrum. The reaction mixture was filtered, washed with
`water and dried using anhydrous magnesium Sulphate. The
`organic Solution was filtered and concentrated. The crude
`product was Subjected to purification by Silica column using
`chloroform-methanol (20:10) mixture. The pure fractions
`were collected and concentrated. The residue was dissolved
`in benzene and freeze dried. The product was characterized
`by LC/MS. M+(Na)932. This material was dissolved in dry
`ether (10 ml). 10 equivalents of lithium aluminum deuteride
`was added. The reaction mixture was stirred for 24 hours.
`After the completion of the reaction, the excess of LiAlD.
`was decomposed by the addition of acetone. The complex
`was decomposed by adding ice cooled acetic-acid. The
`mixture is filtered. The filtrate was diluted with ether and
`washed with water, dried, and concentrated. The crude
`mixture was Subjected to column chromatography and the
`required material was eluted using chloroform-methanol
`Solvent System. The pure fractions were collected and con
`centrated. The compound was tested by mass Spectrum.
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`M=(Na) 940. This compound was converted to the desired
`final compound (2) by following the procedure as described
`in Example 1.
`
`Example 3
`Preparation of Glycosylated deuteroRapamycin
`(FIG. 5)
`Referring to FIG. 5, compound 10 prepared by example 1
`(20 mg) was dissolved in 5 ml of dichloromethane. Dim
`ethylaminopyridine (2.2 mg) was added to the above Solu
`tion. The contents were cooled to -70 C.
`4-Nitrophenylchloroformate in dichloromethane was added
`to the reaction mixture. The Solution was Stirred under
`nitrogen at room temperature for 14 hours. The reaction was
`followed by mass spectrum. After the completion of the
`reaction, the reaction mixture was diluted with dichlo
`
`1O
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`3. The deuterated rapamycin compound of claim 1
`wherein the rapamycin compound is 31,42-d-rapamycin.
`4. The deuterated rapamycin compound of claim 1
`wherein the deuterated rapamycin compound is glycosylated
`at position 42.
`5. The deuterated rapamycin compound of claim 2
`wherein the deuterated rapamycin compound is glycosylated
`at position 42.
`6. The deuterated rapamycin compound of claim 3
`wherein the deuterated rapamycin compound is glycosylated
`at position 42.
`7. The deuterated rapamycin compound of claim 4
`wherein the glycosylated deuterorapamycin has the Struc
`ture:
`
`OH
`
`HO
`HO
`
`OH
`
`O
`
`
`
`romethane and the organic Solution was washed with water,
`0.2M ice cold HCl solution. The organic layer was dried
`over anhydrous magnesium Sulphate. After filtration, the
`organic Solution was filtered and concentrated. The crude
`product was purified by LC/MS to provide the pure com
`pound 30 (Yield 10 mg.) Compound 30 (0.9 m.mol)was
`dissolved in dry DMF(0.5 ml) To this mixture, a solution of
`2-aminoethyl-a-D-glucopyranoside (7.2 m.mol) was added.
`The reaction mixture was stirred for 14 hours at room
`temperature. After the completion of the reaction, the mix
`ture was diluted with dichloromethane. The organic Solution
`was concentrated in vacuum. The residue was extracted with
`water and the aqueous Solution was Subjected to biogel
`column to get the required pure compound 50. This material
`was characterized by LC/MS. M+(Na)1185.
`Further variations and modifications of the present inven
`tion will be apparent to those skilled in the art from the
`foregoing and are intended to be encompassed by the claims
`appended hereto.
`We claim:
`1. A deuterated rapamycin compound that is Selected from
`the group consisting of 7-deuteromethyl rapamycin and
`31,42-d-rapamycin and isomers thereof.
`2. The deuterated rapamycin compound of claim 1
`wherein the rapamycin compound is 7-deuteromethyl rapa
`mycin.
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`50
`
`55
`
`60
`
`65
`
`8. A pharmaceutical composition comprising deuterated
`rapamycin or a pharmaceutically acceptable Salt thereof and
`a pharmaceutically acceptable carrier, wherein the deuter
`ated rapamycin is Selected from the group consisting of
`7-deuteromethyl rapamycin and 31,42-d-rapamycin and
`isomers thereof.
`9. The pharmaceutical composition of claim 8 that is in
`tablet form.
`10. The pharmaceutical composition of claim 8 wherein
`the deuterated rapamycin or a pharmaceutically acceptable
`Salt thereof is 7-deuteromethyl rapamycin.
`11. The pharmaceutical composition of claim 8 wherein
`the deuterated rapamycin or a pharmaceutically acceptable
`Salt thereof is 31,42-d-rapamycin.
`12. The pharmaceutical composition of claim 8 wherein
`the deuterated rapamycin or a pharmaceutically acceptable
`Salt thereof is glycosylated at position 42.
`13. The pharmaceutical composition of claim 10 wherein
`the deuterated rapamycin or a pharmaceutically acceptable
`Salt thereof is glycosylated at position 42.
`14. The pharmaceutical composition of claim 11 wherein
`the deuterated rapamycin or a pharmaceutically acceptable
`Salt thereof is glycosylated at position 42.
`15. The pharmaceutical composition of claim 12 wherein
`the glycosylated deuterated rapamycin has the Structure:
`
`Apotex Ex. 1004
`
`

`

`US 6,503,921 B2
`
`10
`
`OH
`
`HO
`HO
`
`
`
`OH
`
`16. The deuterated rapamycin compound which is epi-7-
`deuteromethyl rapamycin and which is glycosylated at posi
`tion 42.
`17. The deuterated rapamycin compound which is 7,43
`de-rapamycin and which is glycosylated at position 42.
`18. A pharmaceutical composition comprising deuterated
`rapamycin or a pharmaceutically acceptable Salt thereof and
`a pharmaceutically acceptable carrier, wherein the deuter
`ated rapamycin is epi-7-deuteromethyl rapamycin and
`which is glycosylated at position 42 and isomers thereof.
`
`25
`
`19. The pharmaceutical composition of claim 18 that is in
`tablet form.
`20. A pharmaceutical composition comprising deuterated
`rapamycin or a pharmaceutically acceptable Salt thereof and
`a pharmaceutically acceptable carrier, wherein the deuter
`ated rapamycin is 7,43-de-rapamycin and which is glycosy
`lated at position 42 and isomers thereof.
`21. The pharmaceutical composition of claim 20 that is in
`tablet form.
`
`Apotex Ex. 1004
`
`