United States Patent
`Stella et al.
`
`[191
`
`[11]
`
`[45]
`
`Patent Number:
`
`4,650,803
`
`Date of Patent:
`
`Mar. 17, 1987
`
`[54]
`
`[75]
`
`PRODUCTS OF RAPAMYCIN
`
`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
`
`[5 1]
`[52]
`[5 8]
`
`Int. Cl.‘ ................. .. A61K 31/395; CO7D 491/06
`U.S. Cl. .............................. .. 514/291; 546/90
`Field of Search ........................... 546/90; 514/291
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`4,316,885
`
`2/1982 Rakhit ................................. 514/291
`
`FOREIGN PATENT DOCUMENTS
`
`1/1980 Belgium .............
`877700
`. EP4l795 12/1981 European Pat. Off.
`
`..... 514/291
`............ 514/291
`
`Primary Exam1‘ner~——Robert T. Bond
`Attorney, Agent, or Firm-——Arthur G. Seifert
`
`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
`mammals.
`
`5 Claims, 1 Drawing Figure
`
`Rapamycin
`
`Par Pharm., Inc.
`Exhibit 1041
`Page 001
`
`

`
`U. s. Patent
`
`Mar. 17,1987
`
`4,650,803
`
`.Eo
`>u
`E0
`<1
`
`00
`
`:
`
`Par Pharm., Inc.
`Exhibit 1041
`Page 002
`
`

`
`1
`
`4,650,803
`
`2
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`PRODUCTS or RAPAMYCIN
`
`5
`
`10
`
`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
`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.
`
`SUMMARY OF THE INVENTION
`
`35
`
`40
`
`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 Findlay et 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.
`’
`
`ii“C‘(CH2)m"N
`
`/1“
`
`R2
`
`wherein m is an integer from 1 to 3, wherein R1 and R2
`are each hydrogen or an alkyl radical having from one
`to three carbon atoms or wherein R1 and R2 together
`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
`substituents at both positions 28 and 43 of the rapamycin
`structure having the same configuration as the substitu-
`ent for the mono-substituted derivative.
`
`The preparation of typical water soluble prodrugs of
`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-
`mycin and the prodrugs were done at 20 ug/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 conducted at 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 p.l were re-
`moved at predetermined times and added to 200 pl of
`10% metaphosphoric acid to quench the reaction. Be-
`fore centrifugation 200 id 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 done using 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 p.g/ml
`respectively, at 37.5‘ C.
`Rapamycin hydrolysis data in buffers, plasm and in
`rat liver homogenate are shown in the following table:
`
`TABLE 1
`Chemical Stability Study
`
`A.
`
`25' C.
`
`- B.
`
`37.5’ C.
`
`pH
`3.3
`7.4-
`3.3
`7.4
`
`V
`Plasma Stability Study g37.s" C.)
`conc (pg/ml)
`50
`50
`50
`
`Human plasma
`Rat plasma
`Liver homogenate
`
`A.
`B.
`C.
`
`t; (hrs)
`35.8
`47.6
`9.9
`10.2
`
`tg (hrs)
`3
`2.83
`5.5
`
`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.
`
`Par Pharm., Inc.
`Exhibit 1041
`Page 003
`
`

`
`3
`EXAMPLE 1
`
`4,650,803
`
`4
`TABLE 2-continued
`
`Synthesis of Mono—(28)-N,N-Dimethylglycinate Ester
`of Rapamycin
`In a dry 100 mL round bottom flask was placed 2.80
`(3.07>< lO'*3 moles)
`of
`rapamycin,
`0.616
`g
`g
`(5.98>< l0"3 moles) of N,N-dimethyl glycine and 1.40 g
`(6.80X l0*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-dimethylarninopyri-
`dine. The reaction was stirred overnight at room tem-
`perature. A thin layer chromatogram (TLC) of the
`reaction (solvent system 1:1 acetonezmethylene chlo-
`ride) was taken and indicated the reaction to be com-
`plete. The Rf of the monoglycinate prodrug was 0.32.
`Some bisglycinate was also present at a Rfof 0.09. The
`reaction was worked-up by first filtering off the dicy-
`clohexylurea (DCU). The solvent was removed on the
`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-
`zacetone to give 1.67 g of product, yield 55%. This
`material was found difficult to recrystallize. NMR (300
`MHZ, solvent CDCI3) 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
`at 32.32. The methylene group of the glycinate was
`found at 33.16 as a singlet.
`EXAMPLE 2
`
`Synthesis of Methanesulfonic Acid Salt of
`Mono-(28)-N,N Dimethylglycinate Ester of Rapamycin
`In a dry 100 mL round bottom flask was placed 3.00
`g (3.l0><10—3 moles) of mono N,N-dimethylglycinate
`prodrug of rapamycin. This was dissolved in 15 mL of
`anhydrous methylene chloride (distilled from P205). To
`this was added 2.71 X l0‘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
`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-
`nate methanesulfonic acid salt-prodrug of rapamycin
`are shown in the following table:
`TABLE 2
`
`20
`
`40
`
`Physical Properties
`MW
`MP
`Solubility in water
`HPLC Ograting Conditions
`Column
`
`Precolumn
`Mobile phase
`
`Detector
`
`Flow rate
`Retention
`
`Chemical Stability. 25“ C.
`Conditions
`
`1095
`93-99° C.
`>50 mg/mL
`
`RP—l8, 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'
`
`tg (hrs)
`
`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
`50 ug prodrug/niL liver homogenate
`Plasma/Tissue Stability Study (37.5“ C.)
`conc (pg/ml)
`t; (hrs)
`5.6
`200
`4.8
`100
`50
`5.0
`2.5
`200
`1.8
`100
`1.75
`50
`4.5
`50
`C.
`Liver liomogenate
`‘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.
`
`A.
`
`B.
`
`Human plasma
`
`Rat plasma
`
`Reconstitution Procedure
`
`The prodrug can be reconstituted with either water
`for injection on distilled water containing 5% by weight
`dextrose (DSW). 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.
`EXAMPLE 3
`
`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 (l.09X l0‘3 moles) of rapamycin, 0.34 g (2.l6>< 10-3
`moles) N,N-diethylaminopropionic acid hydrochloride
`salt and 0.50 g (2.43X 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 of the reac-
`tion (solvent system: ethyl acetate) on silanized silica gel
`plate was taken and indicated the reaction to be com-
`plete. The Rfof the monopropionate hydrochloride salt
`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 of silan-
`ized silica gel. The column was first developed with 200
`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-
`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 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.
`
`Par Pharm., Inc.
`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:
`
`Physical Properties
`M.W.
`M.P.
`Solubility
`
`1088
`94-98‘ C.
`--15 mg/mL in water
`
`20
`
`30
`
`Reconstitution Procedure
`
`The prodrug can be reconstituted with either water
`for injection or DSW. The solutions 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.
`EXAMPLE 5
`
`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.
`of N,N-dimethylglycine, 3.3 eq. of dicyclohexy1carbo-
`diirnide and 0.16 eq. of 4-N,N-dimethylaminopyridine.
`After purification on silica gel, 64% ofbis-glycinate was
`obtained. NMR confirmed the product with two 6 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
`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 half life of the prodrug
`of Example 3 was the shortest, i.e. that half of the pro-
`drug decomposed into products including mainly rapa-
`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-dipropyl-4—aminobutyric acid, 2-(N-pyrrolidino)a-
`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:
`‘
`
`R1
`
`/
`
`R2
`
`0I
`
`I
`—c-(CH2),,,N
`
`wherein m is an integer from 1 to 3,
`wherein R1 and R2 is each hydrogen or an alkyl radi-
`cal having from one to three carbon atoms or
`wherein R1 and R2 together with the nitrogen to
`which they are attached form a saturated heterocy-
`
`5
`\
`Data with respect to mono—(28)-N,N-diethylamino-
`propionate hydrochloride salt-prodrug of rapamycin
`are shown in the following table:
`.
`TABLE 3
`
`Physical Properties
`M.W.
`M.P.
`Solubility
`HPLC Opgrating Conditions
`‘Column
`
`Precolumn
`Mobile phase
`
`Detector
`
`Flow rate
`Retention volume
`
`1077
`99-106“ C.
`>50 mg/mL in water
`
`RP-18, 150 mm length,
`4.6 mm id
`50 mm length, 4.6 mm id
`87 parts methanol:l3
`parts phosphate buffer
`(0.025 M. pH 3.4)
`Kratos 783
`UV 254 nm
`1.5 mL/min
`9.75 ml."
`
`Chemical Stability
`
`Conditions tg (hrs)
`pH 3.3, 25' C.
`33
`pH 7.4, 25' C.
`17
`pH 3.3, 37.5‘ C.
`7.9
`pH 7.4, 37.5’ C.
`6.3
`Plasma/Tissue Stability, 37.5” C.
`t; (hrs)
`Conditions
`2.5
`50 ug prodrug/mL human plasma
`1
`50 ug prodrug/mL rat plasma
`50 ug prodrug/mL liver homogenate 3.7
`
`Plasmaffissue Stability Study §37.5° C.)
`conc (pg/ml)
`ti (hrs)
`3.25
`200
`2.15
`100
`50
`2.50
`60
`200
`58
`100
`58
`50
`3.7
`50
`C.
`Liver homogenate
`‘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.
`
`A.
`
`Human plasma
`
`Rat plasma
`
`Reconstitution Procedure
`
`The prodrug can be reconstituted with either water
`for injection or D5W. The solutions 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.
`EXAMPLE 4
`
`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 l0‘3 moles) of rapamycin, 1.48 g (7.66)< l0*3
`moles) of 4-pyrrolidino-butyric acid hydrochloride salt
`and 50 mL of anhydrous methylene chloride (distilled
`from P205). The reaction was placed under a nitrogen
`atmosphere and 2.50 g (1.21 X 10-2 moles) of dicyclo-
`hexylcarbodiimide
`and
`15 mg of 4-N,N-dime-
`thylaminopyridine. The reaction was stirred overnight
`at room temperature. The following day the dicy-
`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
`developed with 75:25 ethyl acetatezhexane to remove
`the starting material. The product was eluted with ethy-
`lacetate to give 3.24 g of a white solid, yield 78%.
`
`Par Pharm., Inc.
`Exhibit 1041
`Page 005
`
`

`
`7
`
`4,650,803
`
`clic ring having four carbon atoms and the pharma-
`
`ceutically acceptable salts of such derivatives.
`_
`_
`_
`'
`2. The mono—subst1tuted derivative of claim 1
`
`5
`
`0
`ll
`‘C‘CH2CH2N
`
`CHZCHS
`
`/
`
`CH2CH3
`
`Wherem the subsmuem 15
`
`4. The mono-substituted derivative of claim 1
`wherein the substituent is
`
`0I
`
`I
`‘- C""CHzCH2CHzN
`
`10
`
`/
`
`CH3
`
`3. The mono-substituted prodrug derivative 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.
`‘
`"
`"
`‘
`*
`
`Par Pharm., Inc.
`Exhibit 1041
`Page 006
`
`

`
` ——-j——
`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`
`CERTIFICATE OF CORRECTION
`
`PATENT N0.
`
`1
`
`4,650,303
`
`DATED
`INVENTOR(S)
`
`1 March 17, 1987
`2 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 Column 1, line 1 after "[54]", amend
`
`the title to read:
`
`"PRODRUGS OF RAPAMYCIN"
`
`Signed and Sealed this
`
`Eighth Day of September, 1987
`
`DONALD J. QUIGG
`
`Atlesting Ofiicer
`
`Commisximror of Pammx and Trrr(/I'Iv1nr/(J
`
`Par Pharm., Inc.
`Exhibit 1041
`Page 007