(12) United States Patent
`Zhu et al.
`
`I lllll llllllll Ill lllll lllll lllll lllll lllll 111111111111111111111111111111111
`US00633154 7Bl
`US 6,331,547 Bl
`Dec.18,2001
`
`(10) Patent No.:
`(45) Date of Patent:
`
`(54) WATER SOLUBLE SDZ RAD ESTERS
`
`(75)
`
`Inventors: Tianmin Zhu, Monroe, NY (US); Syed
`M. Shah, East Hanover, NJ (US);
`Richard W. Saunders, Palisades, NY
`(US)
`
`(73) Assignee: American Home Products
`Corporation, Madison, NJ (US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 09/639,610
`
`(22)
`
`Filed:
`
`Aug. 16, 2000
`
`(60)
`
`(51)
`
`(52)
`(58)
`
`(56)
`
`Related U.S. Application Data
`Provisional application No. 60/183,035, filed on Aug. 18,
`1999.
`Int. Cl.7 ...................... C07D 498/16; C07D 498/18;
`C07D 491/06; A61K 31/395; A61K 31/445
`U.S. Cl. ............................................. 514/291; 540/456
`Field of Search .............................. 514/291; 540/456
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
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`5,023,263
`5,023,264
`5,078,999
`5,080,899
`5,100,883
`5,100,899
`5,118,677
`5,118,678
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`5,130,307
`5,151,413
`5,162,333
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`5,233,036
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`5,260,300
`5,262,423
`5,302,584
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`5,385,908
`5,385,909
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`5,389,639
`5,391,730
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`5,434,260
`5,463,048
`
`12/1975 Sehgal et al. ........................ 424/122
`11/1976 Sehgal et al. ........................ 424/122
`2/1982 Rakhit .................................. 424/122
`8/1983 Eng ...................................... 424/114
`3/1987 Stella et al. .......................... 514/291
`8/1987 Mohring et al. ..................... 544/182
`12/1989 Surendra et al.
`.................... 424/122
`6/1991 Von Burg ............................. 514/291
`6/1991 Caufield et al. ..................... 514/291
`1/1992 Warner et al. ....................... 424/122
`1/1992 Sturm et al. ......................... 424/122
`3/1992 Schiehser ............................. 514/183
`3/1992 Caine ................................... 514/291
`6/1992 Caufield ............................... 514/183
`6/1992 Kao et al. ............................ 514/183
`6/1992 Failli et al.
`.......................... 540/452
`7/1992 Failli et al.
`.......................... 514/321
`9 /1992 Caufield et al. .. ... ... .... ... ... ... .. 514/63
`11/1992 Failli et al.
`.......................... 514/291
`1/1993 Failli et al.
`.......................... 540/456
`6/1993 Caufield ............................... 514/183
`8/1993 Hughes ................................ 540/455
`11/1993 Goulet et al. ........................ 514/291
`11/1993 Hu ........................................ 514/291
`11/1993 Kao ...................................... 514/291
`4/1994 Kao et al. ... ... ... ... ... .... ... ... ... .. 514/80
`11/1994 Skotnicki et al.
`... ... .... ... ... ... .. 514/63
`1/1995 Nelson et al.
`....................... 514/291
`1/1995 Nelson et al.
`....................... 514/291
`1/1995 Ocain et al.
`... ... ... ... .... ... ... ... 514/291
`2/1995 Nelson ................................. 540/456
`2/1995 Failli et al.
`.......................... 514/291
`2/1995 Skotnicki et al.
`................... 540/456
`5/1995 Kao et al. ............................ 514/291
`7/1995 Skotnicki et al.
`................... 514/291
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`................... 540/456
`
`5,480,988
`5,480,989
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`5,665,772
`5,780,462
`5,985,890
`6,015,809
`
`.......................... 540/456
`1/1996 Failli et al.
`1/1996 Kao et al. ............................ 540/456
`2/1996 Failli et al.
`.......................... 540/456
`2/1996 Nelson et al.
`....................... 540/456
`4/1996 Molnar-Kimber et al.
`......... 514/291
`10/1996 Failli et al.
`.......................... 514/291
`9 /1997 Cottens et al. . .... ... ... ... ... ... ... 514/514
`7/1998 Lee et al. ............................. 514/183
`11/1999 Cottens et al. ....................... 514/291
`1/2000 Zhu et al.
`............................ 514/210
`
`FOREIGN PATENT DOCUMENTS
`
`0525960
`0532862
`9409010
`
`2/1993 (EP) .
`3/1993 (EP) .
`4/1994 (WO) .
`
`OTHER PUBLICATIONS
`
`Zalipsky et al., Eur. Polymer Journal, 19(12), 1983,
`1177-1183.
`Crowe and Lemaire, Pharmaceutical Research, 1998,
`15(11), 1666-1672.
`Sehgal et al., J. Antibiot., 1975, 28, 727.
`Baker et al., J. Antibiot., 1978, 31, 539.
`Vezina et al., J. Antibiot., 1975, 28, 721.
`Morris, R. J. Heart Lung Transplant, 1992, 11(1) pt. 2, 197.
`Baeder et al., S'h Intl' Conference of Inflammation Res.
`Assoc. 121 (Abstract), 1990.
`Martel et al., Can. J. Physiol. Pharmacol., 1997, 55, 48.
`Staruch et al. FASEB, 1989, 3(3), 3411.
`Dumont et al., FASEB, 1989, 3(4), 5256.
`Calne et al., Lancet, 1978, 1183.
`
`Primary Examiner-Bruck Kifle
`(74) Attorney, Agent, or Firm-Arnold S. Milowsky
`ABSTRACT
`
`(57)
`
`This invention provides novel water soluble pegylated esters
`of rapamycin, having the general structure:
`
`wherein n is an integer from about 5 to about 450, as well
`as pharmaceutical compositions containing these com(cid:173)
`pounds and methods for their use as immunosuppressive,
`anti-inflammatory, antifungal, antiproliferative and antitu(cid:173)
`mor agents.
`
`18 Claims, No Drawings
`
`Breckenridge Exhibit 1022
`US 6,331,547 Zhu
`Page 001
`
`

`

`1
`WATER SOLUBLE SDZ RAD ESTERS
`
`US 6,331,547 Bl
`
`2
`ful in preventing transplant rejection [FASEB 3, 3411
`(1989); FASEB 3, 5256 (1989); R. Y. Calne et al., Lancet
`1183 (1978); and U.S. Pat. No. 5,100,899].
`
`This application claims the benefit of U.S. Provisional
`Application No. 60/183,035, which was converted from
`U.S. patent application Ser. No. 09/376,685, filed Aug. 18, 5
`1999, pursuant to a petition filed under 37 C.F.R. l.53(c)
`(2)(i).
`
`BACKGROUND OF THE INVENTION
`
`Rapamycin has also been shown to be useful in prevent(cid:173)
`ing or treating systemic lupus erythematosus [U.S. Pat. No.
`5,078,999], pulmonary inflammation [U.S. Pat. No. 5,080,
`899], insulin dependent diabetes mellitus [Fifth Int. Conf.
`10 Inflamm. Res. Assoc. 121 (Abstract), (1990)], smooth
`muscle cell proliferation and intimal thickening following
`vascular injury [Morris, R. J. Heart Lung Transplant 11 (pt.
`2): 197 (1992)], adult T-cell leukemia/lymphoma [European
`15 Patent Application 525,960 Al], and ocular inflammation
`[European Patent Application 532,862 Al].
`
`This invention relates to water soluble SDZ-RAD esters,
`methods for its preparation and methods for its use for
`inducing immunosuppression and in the treatment of trans(cid:173)
`plantation rejection, autoimmune diseases, solid tumors.
`More particularly, this invention concerns pegylated esters
`of SDZ-RAD and methods for using them for inducing
`immunosuppression, and in the treatment of transplantation
`rejection, graft vs. host disease, autoimmune diseases, dis(cid:173)
`Mono- and diacylated derivatives of rapamycin ( esterified
`eases of inflammation, adult T-cell leukemia/lymphoma,
`solid tumors, fungal infections, and hyperproliferative vas-
`at the 28 and 43 positions) have been shown to be useful as
`cular disorders.
`antifungal agents (U.S. Pat. No. 4,316,885) and used to
`make water soluble aminoacyl prodrugs of rapamycin (U.S.
`SDZ-RAD is 40-0-(2-hydroxy)ethyl-rapamycin, the
`structure and synthesis of which is disclosed in WO
`Pat. No. 4,650,803). Recently, the numbering convention for
`94/09010 (Cottens et al.).
`rapamycin has been changed; therefore according to Chemi-
`Authors Crowe and Lemaire describe the in vitro and in 25 cal Abstracts nomenclature, the esters described above
`situ absorption of SDZ-RAD, an analog of rapamycin hav(cid:173)
`would be at the 31- and 42-positions. U.S. Pat. No. 5,023,
`ing the structure:
`263 describes the preparation and use of 42-oxorapamycin
`and U.S. Pat. No. 5,023,264 describes the preparation and
`use of 27-oximes of rapamycin.
`
`20
`
`30
`
`35
`
`Polyethylene glycol (PEG) is a linear or branched, neutral
`polymer available in a variety of molecular weights and is
`soluble in water and most organic solvents. At molecular
`weights less than 1000 PEGs are the viscous, colorless
`liquids; higher molecular weight PEGs are waxy, white
`solids. The melting point of the solid is proportional to the
`molecular weight, approaching a plateau at 67° C. Molecular
`40 weights range from a few hundred to approximately 20,000
`are commonly used in biological and biotechnological appli(cid:173)
`cations. Of much interest in the biomedical areas is the fact
`that PEG is nontoxic and was approved by FDA for internal
`consumption.
`
`45
`
`SDZ-RAD is a rapamycin derivative which has immuno(cid:173)
`suppressive activity in animal model (U.S. Pat. No. 5,665,
`772). U.S. Pat. No. 5,665,772 (Cottens et al.) teaches
`50 0-alkylated rapamycin derivatives, particularly
`9-deoxorapamycins, 26-dihydro-rapamycins, 40-0-
`substituted rapamycins and 28,40-0,0-disubstituted rapa(cid:173)
`mycins. It is known that pegylated rapamycin is an ester
`derivative which shows immunosuppressive activity with
`55 improved aqueous solubility (U.S. Pat. No. 5,780,462).
`Using the similar synthetic method, water soluble deriva(cid:173)
`tives of SDZ-RAD has been prepared.
`
`DESCRIPTION OF THE INVENTION
`
`This invention provides novel polyethylene glycol esters
`of SDZ-RAD, an analog of rapamycin, which are com(cid:173)
`pounds of the formula:
`
`in their article in Pharmaceutical Research, Vol. 15, No. 11,
`1998
`Rapamycin is a macrocyclic triene antibiotic produced by
`Streptomyces hygroscopicus, which was found to have anti(cid:173)
`fungal 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. No.
`3,929,992; and U.S. Pat. No. 3,993,749].
`Rapamycin alone (U.S. Pat. No. 4,885,171) or in combi(cid:173)
`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 60
`model, a model for multiple sclerosis; in the adjuvant
`arthritis model, a model for rheumatoid arthritis; and effec(cid:173)
`tively inhibited the formation of IgE-like antibodies.
`The immunosuppressive effects of rapamycin have been
`disclosed in FASEB 3, 3411 (1989). Cyclosporin A and 65
`FK-506, other macrocyclic molecules, also have been shown
`to be effective as immunosuppressive agents, therefore use-
`
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`US 6,331,547 Zhu
`Page 002
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`

`

`US 6,331,547 Bl
`
`3
`
`wherein n is an integer from about 5 to about 450.
`The compounds of this invention are water soluble ana(cid:173)
`logs of SDZ-RAD and rapamycin which are useful as
`immunosuppressive, antiinflammatory, antifungal,
`antiproliferative, and antitumor agents. Of the compounds of
`this invention, it is preferred that n=5-200; more preferred
`that n=S-135. Most preferred members are those in which
`n=S-20 and those in which n=90-120. The compounds of
`this invention may also be described and understood based
`upon the average molecular weight of the polyethylene
`glycol chains used to produce their ester chains. For
`instance, an SDZ-RAD-PEG-5000 conjugate ester refers to
`a compound of the general formula above in which the
`42-0-(2-Hydroxy)-ethyl position ester is formed utilizing a
`polyethylene glycol derivative having an average molecular
`weight range at or near 5,000.
`The esters of the present invention may he produced
`utilizing the polyethylene glycols known in the art, such as
`those described on pages 355 to 361 of the Handbook of
`Pharmaceutical Excipients, Second Edition, 1994 (Library
`of Congress Catalog Card No. 94-79492), which are incor(cid:173)
`porated herein by reference. The preferred compounds of
`this invention may also be described as those of the formula
`esterified using polyethylene glycols having an average
`molecular weight of from about 200 to about 200,000. A
`preferred range of the PEG esters of this invention includes
`those in which the molecular weight of the polyethylene
`glycol portion of the ester chain has a molecular weight in
`the range of from about 300 to about 20,000, more prefer(cid:173)
`ably between about 350 and about 6,000. Non-limiting
`specific examples of compounds of this invention include
`compounds of the formula above prepared using each of the
`PEGs listed in the Handbook of Pharmaceutical Excipients,
`Second Edition, 1994.
`The compounds are useful in treating, preventing or
`inhibiting conditions for which rapamycin, its analogs and
`prodrugs may be used. These methods include the treatment
`or inhibition of transplantation rejection such as kidney,
`heart, liver, lung, bone marrow, pancreas (islet cells), cornea,
`small bowel, and skin allografts, and heart valve xenografts;
`in the treatment or inhibition of graft vs. host disease; in the
`treatment or inhibition of autoimmune diseases such as
`lupus, rheumatoid arthritis, diabetes mellitus, myasthenia
`gravis, and multiple sclerosis; and diseases of inflammation
`such as psoriasis, dermatitis, eczema, seborrhea, inflamma(cid:173)
`tory bowel disease, pulmonary inflammation (including
`asthma, chronic obstructive pulmonary disease,
`emphysema, acute respiratory distress syndrome, bronchitis,
`and the like), and eye uveitis.
`Because of the activity profile obtained, the compounds of
`this invention also are considered to have antitumor, anti(cid:173)
`fungal activities, and antiproliferative activities. The com(cid:173)
`pounds of this invention therefore also useful in treating
`
`5
`
`20
`
`4
`solid tumors, including sarcomas and carcinomas, such as
`astrocytomas, prostate cancer, breast cancer, small cell lung
`cancer, and ovarian cancer; adult T-cell leukemia/
`lymphoma; fungal infections; and hyperproliferative vascu-
`lar diseases such as restenosis and atherosclerosis. When
`used for restenosis, it is preferred that the compounds of this
`invention are used to treat restenosis that occurs following
`an angioplasty procedure. When used for this purpose, the
`compounds of this invention can be administered prior to the
`procedure, during the procedure, subsequent to the
`10 procedure, or any combination of the above.
`When administered for the treatment or inhibition of the
`above disease states, the compounds of this invention can be
`administered to a mammal orally, parenterally, intranasally,
`intrabronchially, transdermally, topically, intravaginally, or
`15 rectally.
`The compounds of this invention are particularly advan(cid:173)
`tageous as immunosuppressive, antiinflammatory,
`antifungal, antiproliferative, and antitumor agents because
`of their water solubility.
`It is contemplated that when the compounds of this
`invention are used as an immunosuppressive or antiinflam(cid:173)
`matory agent, they can be administered in conjunction with
`one or more other immunoregulatory agents. Such other
`immunoregulatory agents include, but are not limited to
`azathioprine, corticosteroids, such as prednisone and
`25 methylprednisolone, cyclophosphamide, rapamycin,
`cyclosporinA, FK-506, OKT-3, andATG. By combining the
`compounds of this invention with such other drugs or agents
`for inducing immunosuppression or treating inflammatory
`conditions, the lesser amounts of each of the agents are
`30 required to achieve the desired effect. The basis for such
`combination therapy was established by Stepkowski whose
`results showed that the use of a combination of rapamycin
`and cyclosporin A at subtherapeutic doses significantly
`prolonged heart allograft survival time. [Transplantation
`35 Proc. 23: 507 (1991)].
`This invention also comprises pharmaceutical composi(cid:173)
`tions comprising one or more of the PEG esters of this
`invention and one or more pharmaceutically acceptable
`carriers or excipients. The pharmaceutical carriers or excipi(cid:173)
`ents 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(cid:173)
`ers or tablet-disintegrating agents; it can also be an encap(cid:173)
`sulating material. In powders, the carrier is a finely divided
`45 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(cid:173)
`able proportions and compacted in the shape and size
`desired. The powders and tablets preferably contain up to
`50 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 carboxymethyl cellulose,
`polyvinylpyrrolidine, low melting waxes and ion exchange
`55 resms.
`Liquid carriers are used in preparing solutions,
`suspensions, emulsions, syrups, elixirs and pressurized com(cid:173)
`positions. The active ingredient can be dissolved or sus(cid:173)
`pended in a pharmaceutically acceptable liquid carrier such
`as water, an organic solvent, a mixture of both or pharma-
`60 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.
`65 Suitable examples of liquid carriers for oral and parenteral
`administration include water (partially containing additives
`as above, e.g. cellulose derivatives, preferably sodium car-
`
`40
`
`Breckenridge Exhibit 1022
`US 6,331,547 Zhu
`Page 003
`
`

`

`US 6,331,547 Bl
`
`15
`
`5
`boxymethyl cellulose solution), alcohols (including mono(cid:173)
`hydric alcohols and polyhydric alcohols, e.g. glycols) and
`their derivatives, lethicins, and oils (e.g. fractionated coco(cid:173)
`nut oil and arachis oil). For parenteral administration, the
`carrier can also be an oily ester such as ethyl oleate and 5
`isopropyl myristate. Sterile liquid carriers are useful in
`sterile liquid form compositions for parenteral administra(cid:173)
`tion. The liquid carrier for pressurized compositions can be
`halogenated hydrocarbon or other pharmaceutically accept(cid:173)
`able propellant.
`Liquid pharmaceutical compositions which are sterile 10
`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 compounds of this invention may be administered
`rectally in the form of a conventional suppository. For
`administration by intranasal or intrabronchial inhalation or
`insufflation, the compounds of this invention may be for(cid:173)
`mulated into an aqueous or partially aqueous solution, which
`can then be utilized in the form of an aerosol. The com- 20
`pounds of this invention may also be administered transder(cid:173)
`mally through the use of a transdermal patch containing the
`active compound and a carrier that is inert to the active
`compound, is non toxic to the skin, and allows delivery of
`the agent for systemic absorption into the blood stream via 25
`the skin. The carrier may take any number of forms such as
`creams and ointments, pastes, gels, and occlusive devices.
`The creams and ointments may be viscous liquid or semi(cid:173)
`solid emulsions of either the oil-in-water or water-in-oil
`type. Pastes comprised of absorptive powders dispersed in 30
`petroleum or hydrophilic petroleum containing the active
`ingredient may also be suitable. A variety of occlusive
`devices may be used to release the active ingredient into the
`blood stream such as a semi-permeable membrane covering
`a reservoir containing the active ingredient with or without 35
`a carrier, or a matrix containing the active ingredient. Other
`occlusive devices are known in the literature.
`In addition, the compounds of this invention may be
`employed as a solution, cream, or lotion by formulation with
`pharmaceutically acceptable vehicles containing 0.1-5
`percent, preferably 2%, of active compound which may be 40
`administered to a fungally affected area.
`The dosage requirements vary with the particular com(cid:173)
`positions employed, the route of administration, the severity
`of the symptoms presented and the particular subject being
`treated. Based on the results obtained in the standard phar- 45
`macological test procedures, projected daily dosages of
`active compound would be 0.1 µg/kg-100 mg/kg, preferably
`between 0.001-25 mg/kg, and more preferably between
`0.01-5 mg/kg. Treatment will generally be initiated with
`small dosages less than the optimum dose of the compound. 50
`Thereafter the dosage is increased until the optimum effect
`under the circumstances is reached; precise dosages for oral,
`parenteral, nasal, or intrabronchial administration will be
`determined by the administering physician based on expe(cid:173)
`rience with the individual subject treated. Preferably, the 55
`pharmaceutical composition is in unit dosage form, e.g. as
`tablets or capsules. In such form, the composition is sub(cid:173)
`divided in unit dose containing appropriate 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. 60
`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 42-0-(2-Hydroxy)ethyl on SDZ-RAD esterified
`compound of this invention can be prepared by initially 65
`acylating the hydroxyl group on 42-0-(2-Hydroxy)ethyl or
`31-position or both position of SDZ-RAD with an acylating
`
`6
`agent having the general structure X-CH2 C02H, where X
`is a suitable leaving group such as iodine or bromine, in the
`presence of a coupling reagent, such as dicyclohexlcarbo(cid:173)
`diimide (DCC) and a base catalyst such as dimethylami(cid:173)
`nopyridine (DMAP) to provide either a 42-0-(2-Hydroxy)
`ethyl and/or 31-hydroxy acylated SDZ-RAD having the
`following structure where R1 and R2 are independently from
`-H and -COCH2X.
`
`0
`
`Every possible compound can be separated by chroma(cid:173)
`tography. Reaction of the acylated SDZ-RAD with
`monomethoxypoly(ethylene glycol) thiol in the presence of
`base such as sodium bicarbonate provides the desired ester
`on 42-0-(2-Hydroxy)ethyl of SDZ-RAD of this invention.
`This novel method can be applied to any hydroxyalkyl,
`dihydroxyalkyl, hydroalkylarylalkyl, dihydroalkylarylalkyl,
`dihydroxyalkylally derivative of rapamycin in U.S. Pat, No.
`5,665,772.
`The preparation of rapamycin 42-silyl ethers is described
`in U.S. Pat. No. Bl 5,120,842, which is hereby incorporated
`by reference. In the case of the tert-butyl dimethylsilyl
`protecting group, deprotection can be accomplished under
`mildly acidic conditions, such as acetic acid/water/THF. The
`deprotection procedure is described in Example 15 of U.S.
`Pat. No. 5,118,678, which is hereby incorporated by refer(cid:173)
`ence.
`This invention also covers analogous esters of other
`rapamycins known in the art such as, but not limited to,
`29-demethoxyrapamycin, [U.S. Pat. No. 4,375,464,
`32-demethoxyrapamycin under C.A. nomenclature]; rapa(cid:173)
`mycin derivatives in which the double bonds in the 1-, 3-,
`and/or 5-positions have been reduced [U.S. Pat. No. 5,023,
`262]; 29-desmethylrapamycin [U.S. Pat. No. 5,093,339,
`32-desmethylrapamycin under C.A. nomenclature]; 7,29-
`bisdesmethylrapamycin [U.S. Pat. No. 5,093,338, 7,32-
`desmethylrapamycin under C.A. nomenclature];
`27-hydroxyrapamycin [U.S. Pat. No. 5,256,790] and
`15-hydroxyrapamycin [U.S. Pat. No. 5,102,876]. This
`invention also covers esters at the 31-position of
`42-oxorapamycin [U.S. Pat. No. 5,023,263]. The disclosures
`in the above cited U.S. Patents are hereby incorporated by
`reference.
`The reagents used in the preparation of the compounds of
`this invention can be either commercially obtained or can be
`prepared by standard procedures described in the literature.
`The following examples illustrate the preparation and
`biological activities of representative compounds of this
`invention.
`
`EXPERIMENTAL SECTION
`Material and Instruments
`1,3-Dicyclohexylcarbodiimide (DCC) and
`4-dimethylaminopyridine were purchased from Aldrich
`
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`US 6,331,547 Zhu
`Page 004
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`US 6,331,547 Bl
`
`7
`Chemical Co. (Milwaukee, Wis.). Methoxy-PEG-SH of
`average molecular weight 5000 (mPEG-SH 5000) was pur(cid:173)
`chased from Shearwater Polymers, Inc. (Huntsville, Ala.).
`SDZ-RAD was obtained from Chemical Development
`Wyeth-Ayerst Research, Pearl River, N.Y. All solvents were 5
`IIPLC grade and all other chemicals were analytical reagent
`or equivalent. The preparative HPLC consisted of two
`Dynamax solvent delivery systems (Model SD-1) and one
`Dynamax absorbance detector (Model UV-1) from Rainin
`Instrument Inc.(Woburn, Mass.). An automatic speed-vac
`concentrator (Savant, Model AS 160) was from Savant
`Instruments, Inc. (Holbrook, N.Y.) and a BUCHI rotary
`evaporation system (RE 260 and R 124) was from Buchi
`(Flawil, Switzerland). 1H-NMR spectra were recorded on
`400 MHz NMR spectrophotometer using CDC13 as solvents.
`Mass spectra were obtained from API 365 mass spectropho- 15
`tometer or MALDI(fOF mass spectrophotometer from PE
`Sciex. All samples were prepared and run at ambient tem(cid:173)
`perature.
`
`10
`
`8
`a sintered glass filter. The filtrate was transferred to a
`separatory funnel, washed 50 ml of sodium bicarbonate
`solution (5.5 g/100 ml) and then washed with 2x50 ml of
`water. The methylene chloride layer was dried with 5 g
`anhydrous sodium sulfate for 4 h. Then sodium sulfate was
`filtered out and methylene chloride was removed by rotary
`evaporation. A total of 0.60 g pale yellow solid was
`obtained. Isolation of pure SDZ-RAD-iodoacetate ester was
`performed by preparative HPLC on a Prep Nova-pak HR
`C18 (300x19 mm) column from Waters. SDZ-RAD(cid:173)
`iodoacetate ester eluted at 18.4 min using a gradient (30% A,
`70% B for 5 min. then to 100% B in 30 min.). A is 90%
`water, 10% acetonitrile; B is 10% water, 90% acetonitrile.
`The fraction was collected and extracted by 2x50 ml meth(cid:173)
`ylene chloride. The organic layer was combined and dried
`with anhydrous sodium sulfate for 4 h. The organic solvent
`was removed by rotary evaporation to dryness. A yellowish
`solid was obtained (0.102 g). 1 H NMR (CDCL3 , 400 MHz)
`d 3.72 (s, 2H, I-CH2-C02- ) , 4.28 (m, 2H, -C02-C
`H 2 - ) . MS m/z 1134.6 (M+NH4t; m/z 1184.6 (M+OAC)-.
`MS/MS 213.0 (ICH2 C02 CH 2 CH2t.
`20 Preparation of SDZ-RAD-PEG 5000 conjugate
`
`HPLC Method
`
`The analytical HPLC system consisted of a Hewlett(cid:173)
`Packard model 1090 LC with a 1040 diode array detector
`system. A µ-Bondapak C-18 (300x3.9 mm) column from
`Waters was used. The mobile phase A was 10% acetonitrile,
`90% 0.1 M 0.1 M tetraethylammonium acetate (TEAA) 25
`buffer at pH 4.5. The mobile phase B was 90% acetonitrile,
`10% 0.1 M TEAA buffer at pH 4.5. The gradient was from
`50% B to 100% B in 30 min then held 100% B for 5 min
`before going beck original 50% B. The column was equili(cid:173)
`brated at 50% B for 15 min before next injection. The 30
`column temperature was 40° C. and flow rate was 1 mL/min.
`The detection wavelength was set at 280 nm. The injection
`volume was 10 µl.
`Preparation of SDZ-RAD-iodoacetate ester
`
`35
`
`40
`
`45
`
`Xis --(CH20CH2)n- , with average n = 104
`
`SDZ-RAD iodoacetate ester (76 mg, 6.8x10-5 mole) was
`dissolved in 30 ml of solution containing 50% acetonitrile
`50 and 50% aqueous NaHC0 3 (0.1 M) solution. The solution
`was flushed with N2 for 10 min. The original sample 10 µL
`was taken for HPLC analysis. Then mPEG-SH 5000 (337
`mg, 6.8x10-5 mole) was added to the reaction solution and
`the reaction mixture was stirred at room temperature for
`55 another 30 min. The reaction was checked again by taking
`10 µL sample for HPLC analysis. The chromatogram
`showed that SDZ-RAD iodoacetate ester was 100% con(cid:173)
`verted to SDZ-RAD-PEG conjugate. The reaction mixture
`was extracted with 2x150 ml methylene chloride. The
`organic layer was dried with anhydrous sodium sulfate then
`60 filtered. The filtrate was concentrated to a volume of 20 ml
`by rotaty evaporation. The crude product was precipitated
`out after adding 200 mLether. A total of 340 mg pale yellow
`powder was obtained after filtered out by a sintered glass
`funnel and dried under vacuum. Isolation of pure SDZ(cid:173)
`RAD-PEG conjugate, which may also be referred to as
`pegylated SDZ-RAD, was performed by preparative HPLC
`on a Prep Nova-pak HR C18 (300x19 mm) column from
`
`5.2xl0- 4 mole),
`g
`(0.50
`SDZ-RAD
`4-Dimethylaminopyridine (3.0 mg) and 1,3-
`Dicyclohexylcarbodiimide (0.136 g, 6.6x10- 4 mole) were
`dissolved in 20 ml anhydrous methylene chloride in a 150 ml
`round-bottom flask. lodoacetic acid (0.116 g, 6.3xl0-4
`mole) was dissolved in 10 ml anhydrous methylene chloride.
`The iodoacetic acid solution was added into the reaction
`mixture over a period of 10 min. with stirring by a magnetic 65
`bar. Then the reaction mixture was stirred at room tempera(cid:173)
`ture for another 2.5 h. The solution was then filtered through
`
`Breckenridge Exhibit 1022
`US 6,331,547 Zhu
`Page 005
`
`

`

`US 6,331,547 Bl
`
`9
`Waters. SDZ-RAD-iodoacetate ester eluted at 15 min. using
`a gradient ( 60% A, 40% B for 5 min. then at 20% A, 80%
`B in 30 min.) The fraction was collected and extracted by
`2x100 ml methylene chloride. The organic layer was com(cid:173)
`bined and dried with anhydrous sodium sulfate for 4 hr. The 5
`organic solvent was removed by rotary evaporation to dry(cid:173)
`ness. The residue was dissolved in 5 ml methylene chloride
`and was precipitated out after adding 150 ml ether. A pale
`yellow powder was obtained after filtered out by a sintered
`glass funnel and dried under vacuum. 1H NMR (CDC13 , 400 10
`MHz) ll 2.84 (t, 2H, S-CH2-CH2 ), 3.31 (s, 2H,
`CO-CH2-S), 3.38 (s, 3H, -OCH3 ), 4.26 (m, 2H,
`-C02-CH2 - ) . MS (MALD!TOF) m/z 5795.5.
`
`U87MG Human Glioblastoma (ATCC # HTB-14)
`
`3H Thymidine Incorporation Protocol
`
`Growth Medium:
`
`ERL Minimum Essential Medium with Earle Salts (500 ml)
`+5 mLBRL MEM Non-Essential Amino Acids (10 mM)
`+5 mLBRL Penicillin-Streptomycin
`(10000 u/ml,10000 ug/ml)
`+5 mLBRL Na Pyruvate Solution (100 mM)
`+5 mLBRL L-Glutamine 200 mM
`+50 mLBRL Fetal Bovine Serum (Qualified)
`
`Assay:
`
`20
`
`25
`
`30
`
`35
`
`1. Cells were trypsinized and plated at a concentration of 104
`cells/well in a final volume of 200 µl growth medium in
`96-well fiat bottom plates and allowed to adhere for 24
`hours at 37° C.
`2. The media was removed by aspiration with care to not
`disturb the cell monolayer. 200 µl of fresh growth media
`was added per well, allowing enough wells for samples to
`be run in triplicate. Compounds were added in 10 µl PBS 40
`solutions and incubated for another 48 hours at 37° C.
`3. During the last 5 hours of incubation, plates were labeled
`with 1 µCi 3H thymidine per well. (NEN thymidine,
`catalog# NET-027, 6.7 Ci/mmole). The 1 µCi was added
`in 10 µl of PBS (on the day of harvest.). The plates were
`returned to the incubator for the last 5 hours.
`4. The radioactive media was removed by aspiration, with
`care not to disturb the cell monolayer. 50 µl of ERL
`lOxTrypsin was added to each well, followed by incuba- 50
`tion at 37° C. for 10 minutes or until the monolayer was
`loosened from the well bottom. Samples were harvested
`on a glass fiber filter mat using a Skatron 96 well
`harvester. Mats were counted in a Wallac Betaplate
`counter.
`
`45
`
`10
`
`What is claimed:
`1. A compound of the structure
`
`0
`
`wherein n is an integer from 5-450.
`
`2. The compound of claim 1 wherein n=5-200.
`
`3. The compound of claim 1 wherein n=8-135.
`
`4. The compound of claim 1 wherein n=8-20.
`
`5. The compound of claim 1 wherein n=90-120.
`
`6. The compound of claim 1 which is SDZ-RAD-PEG
`5000 conjugate.
`
`7. A method of treating transplantation rejection or graft
`vs. host disease in a mammal in need thereof, which com(cid:173)
`prises administering to said mammal an antirejection effec(cid:173)
`tive amount of a compound of the structure
`
`0
`
`Compou