119)
`United States Patent
`4,650,803
`[11] Patent Number:
`
` Stella et al. [45] Date of Patent: Mar. 17, 1987
`
`[54] PRODUCTS OF RAPAMYCIN
`.
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`[75]
`
`Inventors: Valentino J. Stella; Paul E. Kennedy,
`both of Lawrence, Kans.
`
`:
`.
`:
`.
`[73] Assignee: Kaversity of Kansas, Lawrence,
`
`[21] Appl. No.: 806,152
`
`“1.
`[22] Filed:
`
`Dec.6, 1985
`
`ABSTRACT
`[57]
`Water. soluble prodrugs of rapamycin are disclosed
`which are useful as components in injectable pharma-
`ceutical formulations for the treatment of tumors in
`[51] Tint. C14 cece A61K 31/395; CO7D 491/06|mammals.
`
`[52] US, CL. cssesessesssseesceccensessessensasenes 514/291; 546/90
`[58] Field of Search ..........cssscereeers 546/90; 514/291
`
`4,316,885
`
`2/1982 Rakhi........sssssssssseeeseseees 514/291
`
`FOREIGN PATENT DOCUMENTS
`
`877700
`1/1980 Belgium .........sscsscesseseees 514/291
`_EP41795 12/1981 European Pat. Off. ......... 514/291
`Primary Examiner—Robert T. Bond
`Attorney, Agent, or Firm-—Arthur G. Seifert
`
`5 Claims, 1 Drawing Figure
`
`
`
`Rapamycin
`
`West-Ward Pharm.
`Exhibit 1041
`Page 001
`
`West-Ward Pharm.
`Exhibit 1041
`Page 001
`
`

`

`U.S. Patent
`
`Mar. 17, 1987
`
`4,650,803
`
`5
`
`ro
`
`_ oO
`
`Rapamycin
`
`West-Ward Pharm.
`Exhibit 1041
`Page 002
`
`West-Ward Pharm.
`Exhibit 1041
`Page 002
`
`

`

`1
`
`4,650,803
`
`2
`
`PRODUCTS OF RAPAMYCIN
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`BACKGROUND OF THE INVENTION
`
`"
`
`This invention relates to water soluble prodrugs of
`rapamycin and in particular to certain derivatives of
`rapamycin such as, for example, the glycinate prodrugs
`of rapamycin, the propionate prodrugs of rapamycin
`and the pyrrolidino butyrate prodrugs of rapamycin.
`Rapamycin is a known compound described and
`claimed in U.S. Pat. Nos. 3,929,992, issued Dec. 30,
`1975, and 3,993,749 issued Nov. 23, 1976. Morever,
`certain ofits acyl derivatives are disclosed and claimed
`in-U.S. Pat. No. 4,316,885, issued Feb. 23, 1982.
`Rapamycin has been disclosed and claimed as useful
`in the treatment of tumors in Belgian Pat. No. 877,700.
`Rapamycin is, however, only very slightly soluble in
`water,
`i.e. 20 micrograms per milliliter, and special
`injectable formulations have been developed for admin-
`istration to patients, such as those described and claimed
`in European Pat. No. EP 41,795. These formulations are
`not altogether satisfactory for a number of reasons in-
`cluding toxicity of the carrier. Accordingly, there is a
`need in the art for a rapamycin derivative or prodrug
`which is relatively soluble in water so as to form a safe
`injectable solution and which is as-effective as rapamy-
`cin in the treatment of tumors.
`
`SUMMARYOF THE INVENTION
`
`It has how been found that water soluble prodrugs of
`rapamycin can be synthesized which decompose into
`products including rapamycin in the presence of human
`plasma and animal tissue homogenates. Such prodrugs
`of rapamycin provide a component of a valuable phar-
`maceutical injectable composition for the treatment of
`tumor in humans.
`The water soluble prodrugs of this invention com-
`prise mono-substituted derivatives:at position 28 and
`-disubstituted. derivatives at positions 28 and 43 of the
`rapamycin structure. The assignments are based on a
`structural elucidation published by Findlayet al in Can.
`J. of Chem. 58, 579 (1980). This structure is reproduced
`in FIG. 1 of the accompanying drawing.
`The mono-substituted derivatives include those hav-
`ing a substituent at position 28 of the rapamycin struc-
`ture having the following. configuration.
`
`The preparation of typical water soluble prodrugs of
`rapamycin ofthis inventionis illustrated in the examples
`which werecarried out. using the following procedures.
`In the examples, chemical stability studies for rapa-
`mycin and the prodrugs were doneat 20 wg/ml with an
`ionic strength of 0.5. Stabilities at pH 3.3 (0.05M acetate
`bu‘fer) and pH 7.4 (0.05M phosphate buffer) were stud-
`ied at 25° and 37.5° C. No antioxidants were added and
`the buffers were not deoxygenated.
`The plasma studies were conductedat 37.5° C.for rat
`and human plasma. Rat plasma was obtained from
`Sprague-Dawley male albino rats and was used within
`several days. Human plasma was obtained from the
`Lawrence Memorial Hospital in Lawrence, Kans. The
`plasma studies were done at three prodrug concentra-
`tions: 200, 100 and 50 pg/ml of prodrug. The experi-
`mental procedure was as follows: The compound to be
`tested was taken from a stock aqueous solution of 5
`mg/ml and added to the plasma to give the desired
`prodrug concentration. Samples of 200 pl were re-
`movedat predetermined times and added to 200 pl of
`10% metaphosphoric acid to quench the reaction. Be-
`fore centrifugation 200 pl of methanol was added to
`further precipitate the plasma proteins. The results are
`expressed in half-lives in hours.
`The chemical and plasma studies were followed by
`HPLC using a RP C-18 column (150 mm) and a preco-
`lumn (50 mm). The mobile phase was 87:13 methanol:-
`phosphate buffer (0.025M, pH 3.4). The detector was
`set at 254 nm and the flow rate was 1 ml/min for rapa-
`mycin studies and 1.5 ml/min for the prodrug studies.
`Chart speed was 1 inch/10 minutes.
`The liver homogenate studies were doneusing livers
`freshly obtained from male albino Sprague-Dawley
`rats. A 20% liver homogenate was prepared in Soren-
`sen’s buffer at pH 7.4. Chemical stability studies of rapa-
`mycin and the two prodrugs of Examples 2 and 3 were
`carried out at concentrations of 20, 50 and 50 pg/ml
`respectively, at 37.5° C.
`Rapamycin hydrolysis data in buffers, plasm and in
`rat liver homogenate are shown in the following table:
`
`5
`
`30
`
`45
`
`TABLE 1
`
`
`Chemical Stability Study
`
`pH
`ty (hrs)
`A.
`25°C.
`3.3
`35.8
`14
`47.6
`: B.
`37.5° C.
`3.3
`9.9
`14
`10.2
`
`
`u (CH2),
`Nn’
`~C=(CHy)m—
`PlasmaStability Study (37.5°C.)
`araN
`
`conc (4g/ml)
`ty (hrs)
`R2
`
`A.—Human plasma 30 3
`
`B.
`Rat plasma
`50
`2.83
`
`c. 5.5 Liver homogenate 50
`
`
`
`Ri
`
`55
`
`wherein m is an integer from 1 to 3, wherein R; and Ro
`are each hydrogenor an alkyl radical having from one
`to three carbon atoms or wherein R; and R2 together
`with the nitrogen atom to which they are attached form
`a saturated heterocyclic ring having fourto five carbon
`atoms.
`The di-substituted derivatives include those having
`substituents at both positions 28 and 43 ofthe rapamycin
`structure having the same configuration as the substitu-
`ent for the mono-substituted derivative.
`
`In all the prodrug studies, the disappearance of the
`prodrug peak appeared to result in the formation of a
`peak with a retention time nearly equal to rapamycin.
`Analysis of the plasma and homogenate studies by thin
`layer chromatography (TLC) tended to suggest that
`rapamycin initially formed but then it further degraded
`to other decomposition products, as does rapamycin
`itself in these studies.
`
`65
`
`West-Ward Pharm.
`Exhibit 1041
`Page 003
`
`West-Ward Pharm.
`Exhibit 1041
`Page 003
`
`

`

`3
`
`4,650,803
`
`4
`
`TABLE2-continued
`
`i
`
`EXAMPLE1
`ae *4
`Synthesis of Mono-(28)-N,N-Dimethylglycinate Ester
`aml
`of Rapamycin
`5 Plasma/T:issue Stability, 37.5° C.
`ty (hrs)
`Conditions
`In a dry 100 mL round bottom flask was placed 2.80
`;
`;
`g
`(.07X10-3 moles)
`of
`rapamycin,
`0.616
`g
`3
`20 ie oredremL vatplasPlasma
`(5.98 x 10-3 moles) of N,N-dimethyl glycine and 140g
`45
`59 ug prodrug/mLliver homogenate
`(6.80 x 10-3 moles) of dicyclohexylcarbodiimide. The
`flask was placed under a nitrogen atmosphere and 60 10 Plasma/Tissue Stability Smdv G75".
`
`
`
`mL of anhydrous methylene chloride (dried over P205)Plasma/TissueStabilityStudy(375°C)conc (wg/ml) ty (hrs)
`
`was added followed by 60 mg of 4-dimethylaminopyri-
`A.
`Human plasma
`200
`5.6
`dine. The reaction wasstirred overnight at room tem-
`100
`4.8
`50
`5.0
`perature. A thin layer chromatogram (TLC) of the
`reaction (solvent system 1:1 acetone:methylene chlo-
`200
`2.5
`100
`1.8
`ride) was taken and indicated the reaction to be com-
`50
`1.75
`plete. The Rf of the monoglycinate prodrug was 0.32.
`50
`4.5
`Liver homogenate
`C.
`Somebisglycinate was also present at a Ryof 0.09. The
`*With a new RP C-18 column two peaks were observed which are believed to be
`reaction was worked-upbyfirst filtering off the dicy-
`cis-trans isomers about the amide bond in the macrocyclic lactonering.
`clohexylurea (DCU). The solvent was removed on the
`rotovaporto give a white solid. The crude product was
`chromatographed on 18 gm ofsilica gel using 300 mL of
`ethyl acetate to elute rapamycin plus residual DCU.
`The product was eluted with 1:1 methylene chloride-
`sacetone to give 1.67 g of product, yield 55%. This
`material was founddifficult to recrystallize. NMR (300
`MHZ,solvent CDCl3) indicated the spectrum of the
`prodrugto be practically identical to that of rapamycin
`except for the twosinglets arising from the glycinate
`- group. The N,N dimethyl protons appearedas a singlet
`at 22.32. The methylene group of the glycinate was
`found at 03.16 as a singlet.
`EXAMPLE2
`
`B.
`
`Rat plasma
`
`20
`
`Reconstitution Procedure
`
`The prodrug can be reconstituted with either water
`for injection or distilled water containing 5% by weight
`dextrose (D5W). The solutions should be freshly pre-
`pared and used immediately (<1 hr if possible). The
`prodrug appears to discolor upon prolonged exposure
`to light. Precaution should be taken to prevent this.
`EXAMPLE3
`
`Synthesis of
`Mono-(28)-3-(N,N-Diethylamino)propionate
`Hydrochloride Salt Ester of Rapamycin
`
`Synthesis of Methanesulfonic Acid Salt of
`Mono-(28)-N,N Dimethylglycinate Ester of Rapamycin
`Ina dry 100 mL round bottom flask was placed 3.00
`g (3.10X 10-3 moles) of mono N,N-dimethylglycinate
`prodrug of rapamycin. This was dissolved in 15 mL of
`anhydrous methylene chloride(distilled from P20s). To
`this was added 2.71 x 10-3 moles) ofa stock solution of
`methanesulfonic acid dissolved in diethyl ether. The
`solvent was immediately removed to give a whitesolid,
`wt. 3.25 g, yield 99%. This compound was also found
`difficult to recrystallize. The salt form of this compound
`was foundto be unstable to longstirring times. Even in
`the crystalline form long exposuresto light resulted in a
`slow discoloration of the material.
`Data with respect to mono-(28)-N,N-dimethylglyci-
`nate methanesulfonic acid salt-prodrug of rapamycin
`are shown in the following table:
`TABLE 2
`
`Physical Properties
`MW
`MP
`Solubility in water
`HPLC Operating Conditions
`Column
`
`Precolumn
`Mobile phase
`
`Detector
`
`Flow rate
`Retention
`
`1095
`93-99° C.
`>50 mg/mL
`
`RP-18, 150 mm length,
`4.6 mm id
`50 mm length, 4.6 mm id
`87 parts methanol:13
`parts phosphate buffer
`(0.025 M, pH 3.4)
`Kratos 783
`UV 254 nm
`1.5 mL/min
`9.5 mL*
`
`Chemical Stability, 25° C.
`Conditions
`
`ty (hrs)
`
`In a dry 100 mL round bottom flask was placed 1.00
`g (1.09 x 10~3 moles) of rapamycin, 0.34 g (2.16 10-3
`moles) N,N-diethylaminopropionic acid hydrochloride
`salt and 0.50 g (2.43 x 10-3 moles) of dicyclohexylcar-
`bodiimide.
`The vessel was placed under a nitrogen atmosphere
`and 25 mL of anhydrous methylene chloride (dried
`over P205) was added followed by 15 mg of 4-dime-
`thylaminopyridine. The reaction was stirred overnight
`at room temperature. The next day a TLC ofthe reac-
`tion (solvent system: ethyl acetate) on silanizedsilica gel
`plate was taken and indicated the reaction to be com-
`plete. The Ryof the monopropionate hydrochloridesalt
`of rapamycin was 0.34 and 0.01 for the bispropionate
`hydrochloride salt which was also formed in the reac-
`tion. The dicyclohexylurea was filtered from the reac-
`tion and the solvent removed on the rotovapor. The
`crude product was chromatographed on 12 g ofsilan-
`ized silica gel. The column was first developed with 200
`mLofethyl acetate to remove any rapamycin and also
`residual dicyclohexylurea. The product was eluted with
`ethyl acetate to give 0.61 g of product, yield 53%. This
`compound was found difficult to recrystallize and un-
`stable to prolonged exposure to light. NMR (300 MHz,
`solvent CDCL3) indicated the spectrum of the prodrug
`to be practically identical with that of rapamycin. The
`propionate group did not give sharp easily interpreted
`resonances as was the case with the glycinate prodrug.
`This is the result of the resonances being multiplets
`resulting from the ethyl groups whichare notas easily
`seen among the other resonances from rapamycin.
`Broad peaks did appear around 1.2 and 1.5 which were
`not found in rapamycin.
`
`40
`
`45
`
`50
`
`355
`
`65
`
`West-Ward Pharm.
`Exhibit 1041
`Page 004
`
`West-Ward Pharm.
`Exhibit 1041
`Page 004
`
`

`

`4,650,803
`
`6
`Data with respect to the mono-(28)-4’-(pyrrolidino)-
`butyrate hydrochloride salt-prodrug of rapamycin are
`shown below:
`
`5
`‘
`Data with respect to. mono-(28)-N,N-diethylamino-
`propionate hydrochloride salt-prodrug of rapamycin
`are shownin the following table:
`TABLE3
`
`1077
`99-106° C,
`>50 mg/mL in water
`
`Physical Properties
`MW.
`M.P.
`Solubility
`HPLC Operating Conditions
`‘Column
`
`Precolumn
`Mobile phase
`
`Detector
`
`,
`RP-18, 150 mm length,
`4.6 mm id
`50 mm length, 4.6 mm id
`87 parts methanol:13
`parts phosphate buffer
`(0.025 M, pH 3.4)
`Kratos 783
`UV 254 nm
`Flow rate
`1.5 mL/min
`Retention volume
`9.75 mL*
`
`
`5
`
`10
`
`
`
`Physical Properties
`M.W.
`M.P.
`Solubility
`
`1088
`94-98° C.
`~15 mg/mL in water
`
`Reconstitution Procedure
`
`15
`
`The prodrug can be reconstituted with either water
`- for injection or D5W. Thesolutions should be freshly
`prepared and used immediately (<1 hr if possible). The
`prodrug appears to discolor upon prolonged exposure
`to light. Precaution should be taken to preventthis.
`EXAMPLE5
`
`Chemical Stability
`Synthesis of Bis N,N-Dimethylglycinate Ester of
`ty (hrs)
`Conditions
`Rapamycin
`33
`pH 3.3, 25° C.
`17
`pH 7.4, 25°C.
`Thebis-glycinate prodrug of rapamycin substituted
`pH 3.3, 37.5° C.
`79
`at positions 28 and 43 of the rapamycin structure was
`
`pH 7.4, 37.5° C.
`6.3
`synthesized by the addition of 1 eq. of rapamycin,3 eq.
`Plasma/Tissue Stability, 37.5° C.
`of N,N-dimethylglycine, 3.3 eq. of dicyclohexylcarbo-
`
`Conditions
`ty (hrs)
`diimide and 0.16 eq. of 4-N,N-dimethylaminopyridine.
`50 ug prodrug/mL human plasma
`2.5
`After purification on silica gel, 64% ofbis-glycinate was
`50 ug prodrug/mL rat plasma
`1
`obtained. NMR confirmed the product with two 6 pro-
`50 ug prodrug/mLliver. homogenate
`3.7
`ton singlets for the methyl groups of the two glycinate
`Piasma/Tissue Stability Study (37.5° C.)
`groups.
`ty (hrs)
`conc (g/ml)
`The formation of the methanesulfonic acid salt of the
`bis-glycinate. was accomplished by the addition of 1.95
`3.25
`200
`2.15
`100
`eq. of methanesulfonic acid. The use of two equivalents
`2.50
`50
`caused ‘the decomposition of the prodrug. This gave
`60
`200
`58
`100
`92% yield ofthe bis-glycinate prodrug of. rapamycin.
`58
`50
`- The studies carried out using fresh human plasma and
`Cc. 3.7NENSRALiver homogenate 50
`
`
`
`fresh rat plasmaindicate that the halflife of the prodrug
`*Two peaks were also.observed for this prodrug when a new RP-18 column was
`of Example 3 was the shortest, i.e. that half of the pro-
`used. This was also believed to be’ cis-trans isomers as mentioned above for the
`drug decomposedinto products including mainly rapa-
`glycinate prodrug.
`mycin within two and one-half hours with rapamycin
`being the only observed product of hydrolysis.
`Similarly as in Example 1, other water soluble deriva-
`tives of rapamycin can be prepared using as a reagent
`instead of N,N-dimethyl glycine, glycine, N,N-die-
`thylglycine, N,N-diisopropylglycine, N-propylglycine,
`3-aminopropionic acid, N-ethyl-3-aminopropionic acid,
`4-aminobutyric acid, N-ethyl-4-amino butyric acid,
`N,N-dipropy]-4-aminobutyric acid, 2-(N-pyrrolidino)a-
`cetic acid, and 3-(N-piperidino)propionic acid and using
`appropriate protecting groups where necessary.
`Whatis claimedis:
`1. Derivatives of rapamycin which are water soluble
`and which are mono-substituted derivatives at position
`28 and disubstituted derivatives at positions 28 and 43 of
`rapamycin with the substituents having the configura-
`tion:
`
`20
`
`25
`
`30
`
`35
`
`A.
`
`‘Human plasma
`
`B.
`
`Rat plasma
`:
`
`Reconstitution Procedure
`
`The prodrug can be reconstituted with either water
`for injection or D5W. Thesolutions should be freshly
`prepared and used immediately (<1 hr if possible). The
`prodrug appears to discolor upon prolonged exposure
`to light. Precaution should be taken to prevent this.
`EXAMPLE4
`
`45
`
`50
`
`Synthesis of Mono-(28)-4’-(N-pyrrolidino)-butyrate
`Hydrochloride Salt Ester of Rapamycin
`In a dry 100‘mL round bottom flask was placed 3.50
`g (3.83 xX 10-3 moles) of rapamycin, 1.48 g (7.66 10-3
`moles) of 4-pyrrolidino-butyric acid hydrochloride salt
`and 50 mL of anhydrous-methylene chloride (distilled
`from P2Qs).. The reaction was placed under a nitrogen
`atmosphere and 2.50 g (1.21 10~-2 moles) of dicyclo-
`hexylcarbodiimide
`and
`15 mg of 4-N,N-dime-
`thylaminopyridine. The reaction wasstirred overnight
`at room temperature. The following day the dicy-
`clohexylurea ‘was filtered from the reaction and the
`wherein m is an integer from 1 to 3,
`filtrate adsorbed onto 5gof silanizedsilica gel. This was
`65
`wherein R) and R2 is each hydrogen or an alkyl radi-
`loaded onto a 12 g columnofsilanizedsilica gel and was
`cal having from one to. three carbon atoms or
`developed with 75:25 ethyl acetate:hexane to remove
`wherein R; and R»2 together with the nitrogen to
`the starting materia]. The product waseluted with ethy-
`which they are attached form a saturated heterocy-
`lacetate to give 3.24 g of.a white solid, yield 78%.
`
`U (CH), Nn
`—C-—-(CH2)m
`\
`
`Ri
`
`R2
`
`West-Ward Pharm.
`Exhibit 1041
`Page 005
`
`West-Ward Pharm.
`Exhibit 1041
`Page 005
`
`

`

`7
`
`4,650,803
`
`clic ring having four carbon atomsand the pharma-
`
`ceutically acceptable salts of such derivatives.
`
`2. The mono-substituted derivative of claim 1
`
`I
`—C—CHCH2N
`
`CH2CH3
`
`CH2CH3
`
`wherein the substituent is
`
`4. The mono-substituted derivative of claim 1
`wherein the substituent is
`
`fl
`—C—CH)CH2CH2N
`
`5. An injectable pharmaceutical composition useful in
`the treatment of tumors comprising a pharmaceutically
`acceptable carrier and an effective amount of a water
`soluble derivative of rapamycin as defined in claim 1.
`*
`*
`*
`*
`
`i
`—C—CH2N
`
`CH3
`
`CH3
`
`3. The mono-substituted prodrug derivative claim 1
`
`wherein the substituent is
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`West-Ward Pharm.
`Exhibit 1041
`Page 006
`
`West-Ward Pharm.
`Exhibit 1041
`Page 006
`
`

`

`a
`
`UNITED STATES PATENT AND TRADEMARKOFFICE
`CERTIFICATE OF CORRECTION
`
`PATENT NO.
`4,650,803
`:
`DATED
`: March 17, 1987
`INVENTOR(S)
`: Valentino J. Stella and Paul E. Kennedy
`it is certified that error appears in the above—identified patent and that said Letters Patent
`are hereby corrected as shown below:
`
`
`
`
`
`
`
`
`
`On the Cover Page in Column1, line 1 after "[54]", amend
`
`the title to read:
`
`
`"PRODRUGS OF RAPAMYCIN"
`
`
`
` Signed and Sealedthis
`
`Eighth Day of September, 1987
`
`Attest:
`
`
`
`Attesting Officer
`
`Commissioner of Patents and Trademarks
`
`DONALD J. QUIGG
`
`West-Ward Pharm.
`Exhibit 1041
`Page 007
`
`West-Ward Pharm.
`Exhibit 1041
`Page 007
`
`