United States Patent c191
`Stella et al.
`
`4,650,803
`[11] Patent Number:
`[45] Date of Patent: Mar. 17, 1987
`
`[54] PRODUCTS OF RAPAMYCIN
`
`[75]
`
`Inventors: Valentino J. Stella; Paul E. Kennedy,
`both of Lawrence, Kans.
`
`[73] Assignee: University of Kansas, Lawrence,
`Kans.
`
`[21] Appl. No.: 806,152
`
`[22] Filed:
`
`Dec. 6, 1985
`
`Int. Cl,4 ................... A61K 31/395; C07D 491/06
`[51]
`[52] U.S. Cl ........................................ 514/291; 546/90
`[58] Field of Search ........................... 546190; 514/291
`
`[56]
`
`References Cited
`U.S. PA TENT DOCUMENTS
`4,316,885 2/1982 Rakhit ................................. 514/291
`
`FOREIGN PATENT DOCUMENTS
`877700 1/1980 Belgium .............................. 514/291
`EP41795 12/1981 European Pat. Off ............. 514/291
`Primary Examiner-Robert T. Bond
`Attorney, Agent, or Firm-Arthur G. Seifert
`[57]
`ABSTRACT
`Water soluble prodrugs of rapamycin are disclosed
`which are useful as components in injectable pharma(cid:173)
`ceutical formulations for the treatment of tumors in
`mammals.
`
`5 Claims, 1 Drawing Figure
`
`Rapamycin
`
`Breckenridge Exhibit 1010
`Breckenridge v. Novartis AG
`
`

`
`U.S. Patent
`
`Mar. 17, 1987
`
`4,650,803
`
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`
`1
`
`4,650,803
`
`PRODUCTS OF RAPAMYCIN
`
`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 of its 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 20
`injectable formulations have been developed for admin(cid:173)
`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(cid:173)
`cluding toxicity of the carrier. Accordingly, there is a 25
`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.
`
`30
`
`2
`DETAILED DESCRIPTION OF THE
`INVENTION
`The preparation of typical water soluble prodrugs of
`5 rapamycin of this invention is illustrated in the examples
`which were carried out using the following procedures.
`In the examples, chemical stability studies for rapa(cid:173)
`mycin and the prodrugs were done at 20 µ.g/ml with an
`ionic strength of0.5. Stabilities at pH 3.3 (0.05M acetate
`10 bu·"fer) and pH 7.4 (0.05M phosphate buffer) were stud(cid:173)
`ied at 25° and 37.5° C. No antioxidants were added and
`the buffers were not deoxygenated.
`The plasma studies were conducted at 37.5° C. for rat
`and human plasma. Rat plasma was obtained from
`15 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 µ.g/ml of prodrug. The experi(cid:173)
`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 µ.l were re-
`moved at predetermined times and added to 200 µ.l of
`10% metaphosphoric acid to quench the reaction. Be(cid:173)
`fore centrifugation 200 µ.l 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(cid:173)
`SUMMARY OF THE INVENTION
`lumn (50 mm). The mobile phase was 87:13 methanol:(cid:173)
`phosphate buffer (0.025M, pH 3.4). The detector was
`It has how been found that water soluble prodrugs of
`set at 254 nm and the flow rate was 1 ml/min for rapa-
`rapamycin can be synthesized which decompose into
`mycin studies and 1.5 ml/min for the prodrug studies.
`products including rapamycin in the presence of human 35
`Chart speed was 1 inch/10 minutes.
`plasma and animal tissue homogenates. Such prodrugs
`The liver homogenate studies were done using livers
`of rapamycin provide a component of a valuable phar-
`freshly obtained from male albino Sprague-Dawley
`rats. A 20% liver homogenate was prepared in Soren(cid:173)
`maceutical injectable composition for the treatment of
`40 sen's buffer at pH 7.4. Chemical stability studies ofrapa-
`tumor in humans.
`mycin and the two prodrugs of Examples 2 and 3 were
`The water soluble prodrugs of this invention com-
`carried out at concentrations of 20, 50 and 50 µ.g/ml
`prise mono-substituted derivatives at position 28 and
`respectively, at 37.5° C.
`. disubstituted derivatives at positions 28 and 43 of the
`Rapamycin hydrolysis data in buffers, plasm and in
`rapamycin structure. The assignments are based on a
`structural elucidation published by Findlay et al in Can. 45 rat liver homogenate are shown in the following table:
`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- 50
`ture having the following configuration.
`
`TABLE 1
`Chemical Stability Study
`
`ti (hrs)
`pH
`--A-.--2-5·-c-.--------3.-3-----3-5.-8--
`7.4
`47.6
`3.3
`9.9
`7.4
`t0.2
`
`B.
`
`37.5' C.
`
`55
`
`Plasma Stability Study (37.5' C.)
`cone (µ.g/ml)
`
`w
`/R1
`-C-(CH2)m-N'\.
`
`R2
`
`wherein m is an integer from 1 to 3, wherein Rt and R1
`are each hydrogen or an alkyl radical having from one
`to three carbon atoms or wherein Rt and R1 together 60
`with the nitrogen atom to which they are attached form
`a saturated heterocyclic ring having four to five carbon
`atoms.
`The di-substituted derivatives include those having 65
`substituents at both positions 28 and 43 of the rapamycin
`structure having the same configuration as the substitu(cid:173)
`ent for the mono-substituted derivative.
`
`A.
`B.
`C.
`
`Human plasma
`Rat plasma
`Liver homogenate
`
`50
`50
`so
`
`3
`2.83
`s.s
`
`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.
`
`

`
`4,650,803
`
`4
`TABLE 2-continued
`
`pH 3.3
`pH 7.4
`
`Plasma/Tissue Stability, 37.5° C.
`Conditions
`50 ug prodrug/mL human plasma
`50 ug prodrug/mL rat plasma
`SO ug prodrug/mL liver homogenate
`
`73
`45
`
`ti (hrs)
`5
`1.8
`4.5
`
`Plasma/Tissue Stability Study (37.S' C.)
`cone (µg/ml)
`
`11 (hrs)
`
`10
`
`A.
`
`Human plasma
`
`B.
`
`Rat plasma
`
`S.6
`200
`4.8
`100
`so
`5.0
`2.5
`200
`100
`1.8
`so
`1.75
`so
`4.5
`C.
`Liver homogenate
`•With a new RP C-18 column two peaks were observed which are believed to be
`cis-trans isomers about the amide bond in the macrocyclic lactone ring.
`
`Reconstitution Procedure
`The prodrug can be reconstituted with either water
`for injection o~ distilled water containing 5% by weight
`dextrose (D5W). The solutions should be freshly pre(cid:173)
`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.
`
`3
`EXAMPLE 1
`Synthesis of Mono-(28)-N,N-Dimethylglycinate Ester
`of Rapamycin
`In a dry 100 mL round bottom flask was placed 2.80 5
`(3.07X lQ-3 moles) of
`g
`rapamycin, 0.616 g
`(5.98 X lQ-3 moles) of N,N-dimethyl glycine and 1.40 g
`(6.80X lQ-3 moles) of dicyclohexylcarbodiimide. The
`flask was placed under a nitrogen atmosphere and 60
`mL of anhydrous methylene chloride (dried over P205)
`was added followed by 60 mg of 4-dimethylaminopyri(cid:173)
`dine. The reaction was stirred overnight at room tem(cid:173)
`perature. A thin layer chromatogram (TLC) of the
`reaction (solvent system 1:1 acetone:methylene chlo- 15
`ride) was taken and indicated the reaction to be com(cid:173)
`plete. The Rf of the monoglycinate prodrug was 0.32.
`Some bisglycinate was also present at a Rtof 0.09. The
`reaction was worked-up by first filtering off the dicy(cid:173)
`clohexylurea (DCU). The solvent was removed on the 20
`rotovapor to give a white solid. The crude product was
`chromatographed on 18 gm of silica gel using 300 mL of
`ethyl acetate to elute rapamycin plus residual DCU.
`The product was eluted with 1:1 methylene chloride(cid:173)
`:acetone to give 1.67 g of product, yield 55%. This 25
`material was found difficult to recrystallize. NMR (300
`MHZ, solvent CDCb) indicated the spectrum of the
`prodrug to be practically identical to that of rapamycin
`except for the two singlets arising from the glycinate
`group. The N,N dimethyl protons appeared as a singlet 30
`at a2.32. The methylene group of the glycinate was
`found at a3.16 as a singlet.
`
`35
`
`EXAMPLE2
`Synthesis of Methanesulfonic Acid Salt of
`Mono-(28)-N,N Dimethylglycinate Ester ofRapamycin
`In a dry 100 mL round bottom flask was placed 3.00
`g (3.lOX lQ-3 moles) of mono N,N-dimethylglycinate
`prodrug of rapamycin. This was dissolved in 15 mL of
`anhydrous methylene chloride (distilled from P205). To 40
`this was added 2.71 X lQ-3 moles) of a stock solution of
`methanesulfonic acid dissolved in diethyl ether. The
`solvent was immediately removed to give a white solid,
`wt. 3.25 g, yield 99%. This compound was also found
`difficult to recrystallize. The salt form of this compound 45
`was found to be unstable to long stirring times. Even in
`the crystalline form long exposures to light resulted in a
`slow discoloration of the material.
`Data with respect to mono-(28)-N,N-dimethylglyci(cid:173)
`nate methanesulfonic acid salt-prodrug of rapamycin 50
`are shown in the following table:
`TABLE2
`
`Physical Properties
`MW
`MP
`Solubility in water
`HPLC Operating Conditions
`Column
`
`Precolumn
`Mobile phase
`
`Detector
`
`Flow rate
`Retention
`Chemical Stability, 25' C.
`Conditions
`
`1095
`93-99' c.
`>50mg/mL
`
`RP-18, 150 mm length,
`4.6mmid
`50 mm length, 4.6 mm id
`87 parts methanol: 13
`parts phosphate buffer
`(0.025 M, pH 3.4)
`Kratos 783
`UV 254nm
`1.5 mL/min
`9.5 mL*
`
`EXAMPLE3
`Synthesis of
`Mono-(28)-3-(N,N-Diethylamino )propionate
`Hydrochloride Salt Ester of Rapamycin
`In a dry 100 mL round bottom flask was placed 1.00
`g (1.09X lQ-3 moles) of rapamycin, 0.34 g (2.16X lQ-3
`moles) N,N-diethylaminopropionic acid hydrochloride
`salt and 0.50 g (2.43 x lQ-3 moles) of dicyclohexylcar(cid:173)
`bodiimide.
`The vessel was placed under a nitrogen atmosphere
`and 25 mL of anhydrous methylene chloride (dried
`over P20s) was added followed by 15 mg of 4-dime(cid:173)
`thylaminopyridine. The reaction was stirred overnight
`at room temperature. The next day a TLC of the reac(cid:173)
`tion (solvent system: ethyl acetate) on silanized silica gel
`plate was taken and indicated the reaction to be com-
`plete. The Rtof the monopropionate hydrochloride salt
`of rapamycin was 0.34 and 0.01 for the bispropionate
`hydrochloride salt which was also formed in the reac(cid:173)
`tion. The dicyclohexylurea was filtered from the reac(cid:173)
`tion and the solvent removed on the rotovapor. The
`crude product was chromatographed on 12 g of silan(cid:173)
`ized silica gel. The column was first developed with 200
`55 mL of ethyl 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(cid:173)
`stable to prolonged exposure to light. NMR (300 MHz,
`60 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
`65 resulting from the ethyl groups which are not as easily
`seen among the other resonances from rapamycin.
`Broad peaks did appear around 1.2 and 1.5 which were
`not found in rapamycin.
`
`

`
`5
`Data with respect to mono-(28)-N,N-diethylamino(cid:173)
`propionate hydrochloride salt-prodrug of rapamycin
`are shown in the following table:
`TABLE 3
`
`4,650,803
`
`6
`Data with respect to the mono-(28)-4' -(pyrrolidino )(cid:173)
`butyrate hydrochloride salt-prodrug of rapamycin are
`shown below:
`
`5
`
`10
`
`Physical Properties
`M.W.
`M.P.
`Solubility
`
`1088
`94-98' C.
`-15 mg/mL in water
`
`Reconstitution Procedure
`The prodrug can be reconstituted with either water
`for injection or DSW. The solutions should be freshly
`15 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.
`
`Physical Properties
`M.W.
`M.P.
`Solubility
`HPLC Operating Conditions
`·column
`
`Precolumn
`Mobile phase
`
`Detector
`
`1077
`99-106' c.
`>50 mg/mL in water
`
`RP-18, 150 mm length,
`4.6 mmid
`50 mm length, 4.6 mm id
`87 parts methanol: 13
`parts phosphate buffer
`(0.025 M, pH 3.4)
`Kratos 783
`UV 254nm
`1.5 mL/min
`9.7S mL"
`
`t1 (hrs)
`33
`17
`7.9
`6.3
`
`A.
`
`Human plasma
`
`B.
`
`Rat plasma
`
`Flow rate
`Retention volume
`Chemical Stability
`Conditions
`pH 3.3, 2S' C.
`pH 7.4, 25' C.
`pH 3.3, 37.S' C.
`pH 7.4, 37.S' C.
`Plasmaffissue Stability, 37.5" C.
`Conditions
`t1 (hrs)
`2.5
`50 ug prodrug/mL human plasma
`50 ug prodrug/mL rat plasma
`I
`3.7
`50 ug prodrug/mL liver homogenate
`_...._. _ __. _____ __. ___________
`30
`Plasmaffissue Stability Study (37.5" C.)
`ti (hrs)
`cone (µ.g/ml)
`3.25
`200
`2·15
`I~
`~.so
`200
`58
`100
`so
`58
`c.
`Liver homogenate
`so
`3.7
`•Two peaks were also observed for this prodrug when a new RP-18 column was
`used. This was also believed to be cis-trans isomers as mentioned above for the
`glycinate prodrug.
`
`20
`
`40
`
`EXAMPLES
`Synthesis of Bis N,N-Dimethylglycinate Ester of
`Rapamycin
`The bis-glycinate prodrug of rapamycin substituted
`at positions 28 and 43 of the rapamycin structure was
`synthesized by the addition of 1 eq. of rapamycin, 3 eq.
`25 of N,N-dimethylglycine, 3.3 eq. of dicyclohexylcarbo-
`diimide and 0.16 eq. of 4-N,N-dimethylaminopyridine.
`After purification on silica gel, 64% ofbis-glycinate was
`b
`· d NMR
`fi
`d h
`d
`· h
`6
`o taine ·
`con rme t e pro uct wit two pro-
`ton singlets for the methyl groups of the two glycinate
`groups.
`The formation of the methane sulfonic acid salt of the
`bis-glycinate was accomplished by the addition of 1.95
`eq. of methane sulfonic acid. The use of two equivalents
`35 caused the decomposition of the prodrug. This gave
`92% yield of the bis-glycinate prodrug of rapamycin.
`The studies carried out using fresh human plasma and
`fresh rat plasma indicate that the halflife of the prodrug
`of Example 3 was the shortest, i.e. that half of the pro-
`drug decomposed into products including mainly rapa(cid:173)
`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(cid:173)
`tives of rapamycin can be prepared using as a reagent
`instead of N,N-dimethyl glycine, glycine, N,N-die(cid:173)
`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-dipropyl-4-aminobutyric acid, 2-(N-pyrrolidino)a-
`50 cetic acid, and 3-(N-piperidino)propionic acid and using
`appropriate protecting groups where necessary.
`What is claimed is:
`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:
`
`Reconstitution Procedure
`The prodrug can be reconstituted with either water
`for injection or DSW. The solutions should be freshly 45
`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
`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 X lQ-3 moles) of rapamycin, 1.48 g (7.66X lQ-3 55
`moles) of 4-pyrrolidino-butyric acid hydrochloride salt
`and 50 mL of anhydrous methylene chloride (distilled
`from P20s). The reaction was placed under a nitrogen
`atmosphere and 2.50 g (1.21X10-2 moles) of dicyclo(cid:173)
`hexylcarbodiimide and
`15 mg of 4-N,N-dime- 60
`thylaminopyridine. The reaction was stirred overnight
`at room temperature. The following day the dicy(cid:173)
`clohexylurea was filtered from the reaction and the
`filtrate adsorbed onto 5 g of silanized silica gel. This was
`loaded onto a 12 g column of silanized silica gel and was 65
`developed with 75:25 ethyl acetate:hexane to remove
`the starting material. The product was eluted with ethy(cid:173)
`lacetate to give 3.24 g of a white solid, yield 78%.
`
`wherein m is an integer from 1 to 3,
`wherein Rt and Rz is each hydrogen or an alkyl radi(cid:173)
`cal having from one to three carbon atoms or
`wherein Rt and Rz together with the nitrogen to
`which they are attached form a saturated heterocy-
`
`

`
`7
`
`4,650,803
`
`8
`
`4. The mono-substituted derivative of claim 1
`wherein the substituent is
`
`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.
`* * * * *
`
`clic ring having four carbon atoms and the pharma-
`
`ceutically acceptable salts of such derivatives.
`
`2. The mono-substituted derivative of claim 1
`
`wherein the substituent is
`
`3. The mono-substituted prodrug derivative claim 1
`
`wherein the substituent is
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`

`
`PATENT NO.
`
`DATED
`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`CERTIFICATE OF CORRECTION
`4,650,803
`March 17, 1987
`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:
`
`INVENTOR(S)
`
`On the Cover Page in Column 1, line 1 after "[54]", amend
`
`the title to read:
`
`"PRODRUGS OF RAPAMYCIN"
`
`Signed and Sealed this
`
`Eighth Day of September, 1987
`
`Attest:
`
`Attesting Officer
`
`Commi.uirmer of Patents and Trru.lermirks
`
`DONALD J. QUIGG