United States Patent c191
`Hu
`
`[54) RAPAMYCIN CARBONATE ESTERS AS
`IMMUNO-SUPPRESSANT AGENTS
`Inventor: David C. Hu, Highland Park, N.J.
`[75)
`[73] Assignee: American Home Products
`Corporation, New York, N.Y.
`[21] Appl. No.: 979,072
`[22] Filed:
`Nov. 19, 1992
`Int. Cl.5 ................... A61K 31/395; C07D 491/06
`[51)
`[52] U.S. Cl ....................................... 514/291; 546/90;
`540/456; 540/452
`[58) Field of Search ........................... 546/90; 540/456
`References Cited
`[56]
`U.S. PATENT DOCUMENTS
`3,929,992 12/1975 Sehgal et al. ....................... 424/122
`3,993,749 ll/1976 Sehgal et al. ....................... 424/122
`4,316,885 2/1982 Rakhit ................................... 546/90
`4,401,653 8/1983 Eng ..................................... 424/114
`4,650,803 3/1987 Stella et al. ........................... 546/90
`4,885,171 12/1989 Surendra et al. ................... 424/122
`5,078,999 1/1992 Warner et al. ...................... 424/122
`5,080,899 1/1992 Sturm et al. ........................ 424/122
`5,091,389 2/1992 Ondeyka et al. .................... 514/291
`5,100,899 3/1992 Caine ................................... 514/291
`5,118,678 6/1992 Kao et al ............................. 514/183
`
`OTHER PUBLICATIONS
`Martel, et al., Can. J. Physiol. Pharm. 55, 48-51, (1977).
`Calne, et al., The Lancet, Jun. 3, 1978, pp.
`Dumont et al., FASEB 3(4), 5256 (1989).
`Staruch et al., FASEB 3(3), 3411 (1989).
`Vezina et al., Journal of Antibiotics 28(10), 721-26
`(1975).
`Sehgal et al., Journal of Antibiotics 28(10), 727-32
`(1975).
`Baker et al., Journal of Antibiotics 31(6), 539-545
`(1978).
`Fieser and Fieser, Reagents for Organic Synthesis, vol.
`Ip. 364, 366 Wiley 1967.
`Primary Examiner-Mukund J. Shah
`
`I lllll llllllll Ill lllll lllll lllll lllll lllll lllll lllll lllll llllll Ill lllll llll
`US005260300A
`S,260,300
`[11) Patent Number:
`[45) Date of Patent:
`Nov. 9, 1993
`
`Assistant Examiner-Pavanaram K. Sripada
`Attorney, Agent, or Firm-R. F. Boswell, Jr.
`
`ABSTRACT
`[57]
`Carbonate esters with rapamycin at position 42 or posi(cid:173)
`tions 31 and 42 have been shown to have immunosup(cid:173)
`pressant properties and are useful in the treatment of
`transplant rejections and autoimmune diseases. These
`esters are represented by the formula below:
`
`wherein:
`R 1 and R 2 are independently H or -COOR 3 but both
`RI and R 2 cannot be H, and
`R3 is C1-C6 alkyl where 1 to 3 hydrogens may be
`replaced by fluorine, chlorine, bromine or iodine,
`C3-Cgcycloalkyl, C2-C6alkenyl, or Ar-(CH2)n(cid:173)
`where n is 0 to 6 and Ar is phenyl, phenyl substi(cid:173)
`tuted by fluorine, chlorine, bromine, iodine, trifluo(cid:173)
`romethyl, nitro, cyano, C1-C6 alkyl or C1-C6 alk(cid:173)
`oxy; pyridinyl, indolyl, quinolyl or furanyl;
`or a pharmaceutically acceptable salt thereof.
`
`13 Claims, No Drawings
`
`Breckenridge Exhibit 1065
`Hu '300 patent
`Page 001
`
`

`

`1
`
`2
`
`RAP AMYCIN CARBONATE ESTERS AS
`IMMUNO-SUPPRESSANT AGENTS
`
`FIELD OF THE INVENTION
`
`s
`
`This invention relates to novel carbonate esters of
`rapamycin, their use in methods of treatment, and phar-
`maceutical compositions thereof.
`
`10
`
`BACKGROUND OF THE INVENTION
`
`Under Formula I, RI and R2 are independently Hor
`-COOR3 but both RI and R2 cannot be Hat the same
`time. R3 is selected from C1-C6 alkyl where 1 to 3 hy(cid:173)
`drogens may be replaced by fluorine, chlorine, bromine
`or
`iodine, C3-Cg cycloalkyl, C2-C6 alkenyl or
`Ar-(CH2)n-where n is 0-6 and Ar is phenyl, phenyl
`substituted by fluorine, chlorine, bromine, iodine, triflu(cid:173)
`oromethyl, nitro, cyano, C1-C6 alkyl or C1-C6 alkoxy;
`pyridinyl, indolyl, quinolyl or furanyl. Formula I also
`encompasses the pharmaceutically acceptable salts
`which includes hydrates, solvates, and acid addition
`salts when they can be formed.
`lmmunosuppressive activity was determined in an in
`vitro standard pharmacological test to measure lympho(cid:173)
`cyte proliferation (LAF). In this in vitro test, rapamycin
`itself has an ICsoof 1.7-6.3 nM and the carbonate esters
`of this invention have ICso's ranging from 0.32 to 2.5
`nM. Thus in this assay, the invention compounds have
`LAF activity equal or greater than rapamycin and
`would therefore be expected to posses the immunologi(cid:173)
`cal properties of rapamycin and be useful in the tre3t(cid:173)
`ment of transplantation rejection and autoimmune dis(cid:173)
`eases such as lupus, rheumatoid arthritis, diabetes melli(cid:173)
`tus, multiple sclerosis, psoriasis and the like. Because of
`the structural similarity of the invention compounds
`with rapamycin and other analogs having antifungal
`activity against several strains of Candida a/bicans, the
`compounds of this invention would also be expected to
`have antifungal properties. Similarly, the compounds of
`this invention would be expected to have antiinflamma(cid:173)
`tory activity.
`
`45
`
`so
`
`Rapamycin is a macrocyclic triene antibiotic pro(cid:173)
`duced by Streptomyces hygroscopicus, which was found IS
`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. Seghal et al, J. Antibiot.
`28, 727 (1975); H. A. Baker et al., J. Antibiot. 31, 539 20
`(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 com(cid:173)
`bination with picibanil (U.S. Pat. No. 4,401,653) has 2s
`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 en(cid:173)
`cephalomyelitis model, a model for multiple sclerosis; in 30
`the adjuvant arthritis model, a model for rheumatoid
`arthritis; and effectively inhibited the formation of lgE(cid:173)
`like antibodies.
`The immunosuppressive effects of rapamycin have JS
`been disclosed in FASEB 3, 3411 (1989). Rapamycin
`has been shown to be effective in inhibiting transplant
`rejection (U.S. Pat. No. 5,100,899). Cyclosporin A and
`FK-506, other macrocyclic molecules, also have been 40
`shown to be effective as immunosuppressive agents,
`therefore useful in preventing transplant rejection
`[FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); and R.
`Y. Calne et al., Lancet 1183 (1978)].
`U.S. Pat. No. 5,080,899 discloses a method of treating
`pulmonary inflammation with rapamycin. U.S. Pat. No.
`5,078,999 discloses a method of treating systemic lupus
`erythematosus with rapamycin.
`Mono- and diacylated derivatives of rapamycin (es-
`DETAILED DESCRIPTION OF THE
`terified at the 28 and 43 positions) have been shown to
`INVENTION
`be useful as antifungal agents (U.S. Pat. No. 4,316,885)
`The invention compounds are prepared by dissolving
`and used to make water soluble prodrugs of rapamycin ss rapamycin in a mixture of ether and tetrahydrofuran
`(U.S. Pat. No. 4,650,803). Recently, the numbering
`(THF) and treating with 3 equivalents of pyridine. The
`mixture is cooled to o• C. and treated with a chlorofor-
`convention for rapamycin has been changed; therefore
`according to Chemical Abstracts nomenclature
`the
`mate (3 equivalents) of the formula ClC02R 3 where R 3
`is as defined under Formula I. After a suitable period of
`.
`'
`.
`esters descnbed above would be at the 31- and 42- post- 60 time, the reaction is quenched with dilute acid and the
`isolated mixture of carbonate esters and starting materi-
`tions. lmmunosuppressive, antifungal, and antitumor
`activities of rapamycin 31 and/or 42 carbamate esters
`als subjected to flash chromatography on silica gel to
`are disclosed in U.S. Pat. No. 5,118,678.
`obtain the desired carbonate esters. The position 42
`carbonate esters are the major reaction product. The 31,
`6S 42-dicarbonate esters, and 31-carbonate esters, when
`isolated, are obtained as a minor product.
`Specific examples of the synthetic procedures follow.
`These examples are given for ilhlstrative purposes only
`
`SUMMARY OF THE INVENTION
`
`The novel carbonate derivatives of rapamycin have
`the structure shown in Formula I below.
`
`Breckenridge Exhibit 1065
`Hu '300 patent
`Page 002
`
`

`

`3
`and are not to be construed as limiting to this disclosure
`which is limited only by the scope of the appended
`claims.
`
`5,260,300
`
`4
`by analytical TLC indicated at least three compounds
`are present in the reaction mixture.
`The reaction mixture is dissolved in EtOAc (15 mL)
`and the compounds separated by flash chromatography
`EXAMPLE 1
`5 (silica gel on a 50 mm column, 50% EtOAc-hexane
`gradient to 100% EtOAc ). Two compounds (A and B)
`(A) 31, 42-0-Bis(methoxycarbonyl)rapamycin
`are separated and the fractions concentrated in vacuo.
`(B) 42-0-Methoxycarbonylrapamycin
`Analysis of compound A (0.011 g, 1.8% overall yield)
`indicated that it is 31, 42-0-bis(ethoxycarbonyl)-rapa-
`To a solution of rapamycin (0.83 g, 0.908 mmol) in
`ether/THF (30 mL, ether:THF=2:1) in a 250 mL flask to mycin.
`equipped with a magnetic stirrer under nitrogen at
`IH NMR (400 MHz, CDCh): 85.38 (d, lH), 4.5-4.58
`room temperature is added pyridine (0.25 mL, 3.09
`(m, IH), 4.2 (q, 2H), 4.15 (q, 2H), 1.3 (t, 3H), 1.25 (t,
`mmol) dropwise. The solution is cooled to o• C. and
`3H).
`stirred for 30 minutes. To the solution is added methyl MS (neg. ion FAB) m/e: 1057 (M-), 590, 393.
`chloroformate (0.22 mL, 2.85 mmol) dropwise over a 15 IR (KBr, cm-1): 3400, 2910, 1735, 1640, 1450.
`period of ten minutes. The reaction mixture is stirred at
`Anal. calcd. for Cs7H 87N0 17: C 64.69%, H 8.29%, N
`0° C. for thirty minutes, warmed to room temperature,
`1.32%; Found: C 64.63%, H 8.19%, N 1.20%.
`and stirred for 24 hours.
`Compound B (0.3054 g, 53% overall yield) is 42-0-
`The reaction is quenched with approximately 50 mL
`ethoxycarbonyl-rapamycin.
`of IN HCl. The organic layer is separated, and the 20 IH NMR: 84.55-4.6 (m, IH), 4.2 (q, 2H), 1.2 (t, 3H).
`aqueous layer is extracted three times with ether fol-
`MS (neg. ion F AB) m/e: 985 (M-), 590, 393.
`lowed by three times with ethyl acetate. The combined
`IR (KBr, cm-I): 3430, 2910, 1740, 1640, 1450.
`organic layers are washed with brine and dried over
`Na2S04. The solution is filtered and concentrated in
`vacuo to afford a pale yellow powder (0.88 g). Analysis 25
`by analytical TLC indicated at least three compounds
`are present in the reaction mixture.
`The reaction mixture is dissolved in EtOAc (20 mL)
`and the compounds separated by flash chromatography
`(silica gel on a 50 mm column, 50% EtOAc-hexane 30
`gradient to 100% EtOAc). Two compounds (A and B)
`are separated and the fractions concentrated in vacuo.
`Analysis of compound A (0.0143 g, 15% overall yield)
`indicated that it is 31, 42-0-bis(methoxycarbonyl)-rapa-
`mycin.
`IH NMR (400 MHz, CDCIJ): 85.2 (d, lH), 4.5-4.55 (m,
`lH), 3.8 (s, 3H), 3.7 (s, 3H).
`MS (neg. ion FAB) m/e: 1029 (M-), 997, 952, 590, 379.
`Compound B (0.5725 g, 65% overall yield) is 42-0-
`methoxycarbonyl-rapamycin.
`IH NMR (400 MHz, CDCl3): 84.5-4.58 (m, lH), 3.79 (s,
`3H).
`MS (neg. ion FAB) m/e: 971 (M-), 590, 379.
`IR (KBr, cm-1): 3400, 2950, 1750, 1645, 1440.
`Anal. calcd. for Cs3Hs1N01s.H20: C 64.30%, H 45
`8.29%, N 1.42%; Found: C 64.08%, H 8.13%, N 1.35%.
`
`EXAMPLE 3
`42-0-(Phenyloxycarbonyl)rapamycin.
`To a solution of rapamycin (1.05 g, 1.148 mmol) in
`ether/THF (30 mL, ether:THF= 1:1) in a 250 mL flask
`equipped with a magnetic stirrer under nitrogen at
`room temperature is added pyridine (0.30 mL, 3.71
`mmol) dropwise. The solution is cooled to o• C. and
`stirred for 30 minutes. To the solution is added phenyl
`chloroformate (0.50 mL, 3.99 mmol) dropwise over a
`period of twelve minutes. The reaction mixture is
`stirred at o· C. for thirty minutes, warmed to room
`temperature, and stirred for three hours.
`The reaction is quenched with approximately 50 mL
`of IN HCl. The organic layer is separated, and the
`aqueous layer is extracted with ether followed by ethyl
`acetate. The combined organic layers are washed with
`brine and dried over Na2S04. The solution is filtered
`and concentrated in vacuo to afford a pale yellow pow-
`der (0.84 g). Analysis by analytical TLC indicated at
`least two compounds are present in the reaction mix-
`ture.
`The reaction mixture is dissolved in EtOAc (15 mL)
`and the compounds separated by flash chromatography
`(silica gel on a 50 mm column, 75% EtOAc-hexane
`EXAMPLE2.
`gradient to 100% EtOAc). The fraction containing the
`(A) 31, 42-0-Bis(ethoxycarbonyl)rapamycin
`50 major product_ is concentrated in vacuo to afford a pale
`(B) 42-0-Ethoxycarbonylrapamycin
`yellow foam (0.53 g, 47% overall yield). Analysis indi-
`cated that the compound isolated is ~he monohydrate of
`To a solution of rapamycin (0.53 g, 0.580 mmol) in
`ether/THF (40 mL, ether:THF= 1:1) in a 250 mL flask
`42-0-(phenyloxycarbonyl)-rapamycm.
`1H NMR (400 MHz, CDCh): 87.18-7.4 ·(m, SH),
`equipped with a magnetic stirrer under nitrogen at
`4.5-4.6~ (m, lH).
`room temperature is added pyridine (0.15 mL, 1.85 55
`mmol) dropwise. The solution is cooled to o• C. and MS (neg. ion FAB) m/e: 971 (M-), 590,441.
`stirred for 30 minutes. To the solution is added ethyl
`IR (KBr, cm-I): 3450, 2930, 1720, 1640, 1460.
`chloroformate (0.17 mL, 1.78 mmol) dropwise over a
`Anal. calcd. for CssHs3N01s.H20: C 67.35%, H
`8.09%, N 1.35%;
`period of ten minutes. The reaction mixture is stirred at
`o• C. for thirty minutes, warmed to room temperature, 60
`Found: C 64.88%, H 7.76%, N 1.29%.
`and stirred for 24 hours.
`EXAMPLE 4
`The reaction is quenched with approximately 50 mL
`A. 42-0-(4-Nitrophenyloxycarbonyl)rapamycin
`of lN HCI. The organic layer is separated, and the
`aqueous layer is extracted three times with ether fol(cid:173)
`To a solution of rapamycin (1.13 g, 1.240 mmol) in
`lowed by three times with ethyl acetate. The combined 65
`ether/THF (30 mL, ether:THF= 1:1) in a 250 mL flask
`equipped with a magnetic stirrer under nitrogen at
`organic layers are washed with brine and dried over
`Na2S04. The solution is filtered and concentrated in
`room temperature is added pyridine (0.30 mL, 3.71
`mmol) dropwise. The solution is cooled to o• C. and
`vacuo to afford a pale yellow powder (0.38 g). Analysis
`
`35
`
`40
`
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`Page 003
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`

`5,260,300
`6
`o• C. for thirty minutes, warmed to room temperature,
`and stirred for 36 hours.
`The reaction is quenched with approximately SO mL
`of IN HCJ. The organic layer is separated, and the
`aqueous layer is extracted with ether followed by ethyl
`acetate. The combined organic layers arc washed with
`brine and dried over Na2S04. The solution is filtered
`and concentrated in vacuo to afford a pale yellow pow(cid:173)
`der (O.S3 g). Analysis by analytical TLC indicated at
`least two compounds present in the reaction mixture.
`The reaction mixture is dis5olvcd in EtOAc (20 mL)
`and the compounds separated by flash chromatography
`(silica gel on a SO mm column, 2S% EtOAc-hexane
`gradient to 7S% EtOAc). The fraction containing the
`major product is concentrated in vacuo to afford a pale
`yellow foam (0.18 g, 14% overall yield). Analysis indi(cid:173)
`cated that the product isolated is the trihydrate of 42-0-
`(prop-2-cnyloxycarbonyl)rapamycin.
`lff NMR (400 MHz, DMSO): BS.8S-S.9S (m, IH),
`S.2-S.3S (dd, 2H), 4.3-4.4S (m, lH).
`MS (neg. ion FAB) m/e: 971 (M-), S90, 40S.
`IR (KBr, cm-1): 3420, 2910, 2370, 1740, 1640, 14SO.
`Anal. calcd. for CssHs3N01s.3H20: C 62.79%, H
`8.06%, N 1.33%;
`Found: C 62.43%, H 8.04%, N 1.34%.
`
`s
`stirred for 30 minutes. To the solution is added 4-
`nitrophenyl chloroformate (0.748 g, 3.71 mmol) por(cid:173)
`tionwise over a period of five minutes. The reaction
`mixture is stirred at o· c. for thirty minutes.
`The reaction is quenched with approximately SO mL 5
`of IN HCJ. The organic layer is separated, and the
`aqueous layer is extracted with ether followed by ethyl
`acetate. The combined organic layers are washed with
`brine and dried over Na2S04. The solution is filtered
`and concentrated in vacuo to afford a pale yellow pow- 10
`dcr (I.OS g). Analysis by analytical TLC indicated at
`least two compounds are present in the reaction mix(cid:173)
`ture.
`The reaction mixture is dissolved in EtOAc (IS mL)
`and the compounds separated by flash chromatography 15
`(silica gel on a SO mm column, SO% EtOAc-hexane
`gradient to 100% EtOAc). The fraction containing the:
`major product is concentrated in vacuo to afford a pale
`yellow foam (0.S8 g, 43% overall yield). Analysis indi(cid:173)
`cated that it is 42-0-(4-nitro-phcnyloxycarbonyl)-rapa- 20
`mycin.
`lff NMR (400 MHz, DMSO): 88.2 (d, 2H), 7.4 (d, 2H),
`4.S-4.S8 (m, lH).
`MS (neg. ion FAB) m/e: 1078 (M-), S90, 138.
`IR (KBr, cm-1): 3430, 2930, 1760, 1730, 1640, 1S30, 25
`14SO.
`B. 31, 42-0-Bis(4-Nitrophenyloxycarbonyl)rapamycin
`To a solution of rapamycin (1.33 g, 1.46 mmol) in
`ether/THF (SO mL, ether:THF= 1.1) in a 2SO mL flask 30
`equipped with a magnetic stirrer under nitrogen at
`room temperature is added pyridine (0.36 mL, 4.4S2
`mmol) dropwise. The solution is cooled to o• C. and
`stirred for 30 minutes. To the solution is added 4-
`nitrophenyl chloroformate (0.896S g, 4.4S mmol) por- 35
`tionwise over a period of five minutes. The reaction
`mixture is stirred at o• C. for one hour.
`The reaction is quenched with approximately 60 mL
`of IN HCI. The organic layer is separated; and the
`aqueous layer is extracted with ether followed by ethyl 40
`acetate. The combined organic layers are washed with
`brine and dried over Na2S04. The solution is filtered
`and concentrated in vacuo to afford a pale yellow pow(cid:173)
`der (1.47 g). Analysis by analytical TLC indicated the
`presence of two components.
`The reaction mixture is dissolved in EtOAc (21 mL)
`and the compounds separated by flash chromatography
`(silica gel on a SO mm column, 60% EtOAc-hexane).
`The fraction containing the major component is con(cid:173)
`centrated in vacuo to afford a pale yellow foam (1.129 50
`g, 62% overall yield). Analysis indicated that it is 31,
`42-0-Bis(4-nitrophcnyloxycarbonyl)rapamycin.
`lff NMR (400 MHz, DMSO): 88.3S (dd, 4H), 7.S2 (d,
`2H), 7.4S (d, 2H), 4.S8 (d, lH), 4.3S-4.42 (m, lH).
`MS (neg. ion FAB)m/e: 1243(M-), 1078, S90, 446, 138. ss
`IR (KBr, cm-1): 3430, 2930, 1760, 1730, 1640, 1S30,
`14SO.
`
`45
`
`EXAMPLE6
`42-0-(Benzyloxycarbonyl)rapamycin
`To a solution of rapamycin (1.14 g, 1.2S mmol) in
`ether/THF (40 mL, cther:THF== 1:1) in a 2SO mL flask
`equipped with a magnetic stirrer under nitrogen at
`room temperature is added pyridine (0.30 mL, 3.71
`mmol) dropwise. The solution is cooled to o· C. and
`stirred for 30 minutes. To the solution is added benzyl(cid:173)
`chlorofonnate (0.53 mL, 3.71 mmol) dropwise over a
`perioo of ten minutes. The reaction mixture is stirred at
`o• C. for two hours, warmed to room temperature, and
`stirred for 30 hours.
`The reaction is quenched with approximately SO mL
`of lN HCJ. The organic layer is separated, and the
`aqueous layer is extracted with ether followed by ethyl
`acetate. The combined organic layers arc washed with
`brine and dried over Na2S04. The solution is filtered
`and concentrated in vacuo to afford a pale yellow pow(cid:173)
`der (0.47 g). Analysis by analytical TLC indicated at
`least two compounds present in the reaction mixture.
`The reaction mixture is dissolved in EtOAc (20 mL)
`and the compounds separated by flash chromatography
`(silica gel on a SO mm column, 50% EtOAc-hexane).
`The fraction containing the major product is concen(cid:173)
`trated in vacuo to afford a pale yellow foam (0.310 g,
`23% overall yield). Analysis indicated that it is 4~-0-
`(benzyloxycarbonyl)rapamycin with 2.5 moles of wa(cid:173)
`ter.
`lH NMR (400 MHz, DMSO): 8S.15 (s, lH), S.2-5.3S
`(dd, 2H), 4.35-4.42 (m, lH).
`MS (neg. ion FAB) m/c: 1047 (M-), 590, 4S5.
`IR (KBr, cm-1): 3440, 2920, 1740, 1640, 1450.
`Anal. calcd. for Cs9HssN01s.2.SH20: C 64.88%, H
`8.24%, N 1.28%;
`Found: C 64.82%, H 7.9S%, N 1.13%.
`
`EXAMPLE 7
`Following the procedures outlined in Example 6 and
`substituting the following for benzylchlorofonnate:
`a. 4-fluorophenylchloroformate
`b. vinylchlorofonnate
`
`EXAMPLES
`42-0-(Prop-2-enyloxycarbonyl)rapamycin
`To a solution of rapamycin (1.17 g, 1.28 mmol) in
`ether/THF (30 mL, ether:THF= 1.1) in a 2SO mL flask
`equipped with a magnetic stirrer under nitrogen at
`room temperature is added pyridine (0.31 mL, 3.74
`mmol) dropwise. The solution is cooled to o• C. and 65
`stirred for 30 minutes. To the solution is added allyl
`chloroformate (0.41 mL, 3.86 mmol) dropwise over a
`period of ten minutes. The reaction mixture is stirred at
`
`60
`
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`5,260,300
`
`7
`c. 2-trichloroethylchloroformate
`d. 4-methoxyphenylchloroformate
`e. allylchloroformate
`f. 4-nitrobenzylchloroformate
`g. 2-bromoethylchloroformate
`there are obtained respectively:
`a. 42-0-(4-fluorophenyloxycarbonyl)rapamycin
`b. 42-0-(vinyloxycarbonyl)rapamycin
`c. 42-0-(2-trichloroethyloxycarbonyl)rapamycin
`d. 42-0-(4-methoxyphenyloxycarbonyl)rapamycin
`e. 42-0-(allyloxycarbonyl)rapamycin
`·
`f. 42-0-(4-nitrobenzyloxycarbonyl)rapamycin
`g. 42-0-(2-bromoethyloxycarbonyl)rapamycin. ·
`The comitogen-induced thymocyte proliferation pro(cid:173)
`cedure (LAP) was used as an in vitro measure of the
`immunosupprcssive effects of representative com(cid:173)
`pounds. Briefly, cells from the thymus of normal
`BALB/c mice are cultured for 72 hours with PHA and
`IL-1 and pulsed with tritiated thymidine during the last
`six hours. Cells are cultured with and without various 20
`concentrations of rapamycin, cyclosporin A, or test
`compound. Cells are harvested and incorporated; radio(cid:173)
`activity is determined. Inhibition of lymphoprolifera(cid:173)
`tion is assessed in percent change in counts per minute
`from non-drug treated controls. The results are ex- 25
`pressed by the following ratio, or as the percent inhibi(cid:173)
`tion of lymphoproliferation at a concentration of 1 µM
`or as the ICso.
`
`3H-control thvmus cells - 3H-rapamycin-treated thymus ceils
`3H-control thymus cells - 3H-test compound-treated cells
`
`30
`
`PHARMACEUTICAL COMPOSITION
`The compounds can be formulated neat or with a
`pharmaceutical carrier to a mammal in need thereof.
`The pharmaceutical carrier may be solid or liquid.
`A solid carrier can include one or more substances
`which may also act as flavoring agents, lubricants, solu(cid:173)
`bilizers, suspending agents, fillers, glidants, compression
`aids, binders or tablet-disintegrating agents; it can also
`be an encapsulating material. In powders, the carrier is 55
`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 suitable proportions and
`compacted in the shape and size desired. The powders 60
`and tablets preferably 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 cellu(cid:173)
`lose, sodium carboxymethyl cellulose, polyvinylpyr- 65
`rolidine, low melting waxes and ion exchange resins.
`Liquid carriers are used in preparing solutions, sus(cid:173)
`pensions, emulsions, syrups, elixirs and pressurized
`
`8
`compositions. The active ingredient can be dissolved or
`suspended in a pharmaceutically acceptable liquid car(cid:173)
`rier such as water, an organic solvent, a mixture of both
`or pharmaceutically acceptable oils or fats. The liquid
`5 carrier can contain other suitable pharmaceutical addi(cid:173)
`tives such as solubilizers, emulsifiers, buffers, preserva(cid:173)
`tives, !'iwceteners, flavoring agents, suspending agents,
`thickening agents, colors, viscosity regulators, stabiliz(cid:173)
`ers or osmo-regulators. Suitable examples of liquid car-
`10 riers for oral and parenteral administration include
`water (partially containing additives as above, e.g. cel(cid:173)
`lulose derivatives, preferably sodium carboxymethyl
`cellulose solution), alcohols (including monohydric
`alcohols and polyhydric alcohols, e.g. glycols) and their
`15 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 olcate and isopro(cid:173)
`pyl myristatc. Sterile liquid carriers arc useful in sterile
`liquid form compositions for parenteral administration.
`The liquid carrier for pressurized compositions can be
`halogenated hydrocarbon or other pharmaceutically
`acceptable propellant.
`Liquid pharmaceutical compositions which are sterile
`solutions or suspensions can be utilized by, for example,
`intramuscular, intraperitoneal or subcutaneous injec(cid:173)
`tion. Sterile solutions can also be administered intrave(cid:173)
`nously. The compound can also be administered orally
`either in liquid or solid composition form.
`The compounds of this invention may be adminis(cid:173)
`tered rectally in the form ofa conventional suppository.
`For administration by intranasal or intrabronchial inha(cid:173)
`lation or insuffiation, the compounds of this invention
`may be formulated into an aqueous or partially aqueous
`35 solution, which can then be utilized in the form of an
`aerosol. The compounds of this invention may also be
`administered transdermally through the use of a trans(cid:173)
`dermal patch containing the active compound and a
`carrier that is inert to the active compound, is non toxic
`40 to the skin, and allows delivery of the agent for systemic
`absorption into the blood stream via the skin. The car(cid:173)
`rier 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-
`45 solid emulsions of either the oil-in-water or water-in-oil
`type. Pastes comprised of absorptive powders dispersed
`in petroleum or hydrophilic petroleum containing the
`active ingredient may also be suitable. A variety of
`occlusive devices may be used to release the active
`so ingredient into the blood stream such as a semipermia(cid:173)
`ble membrane covering a reservoir containing the ac(cid:173)
`tive ingredient with or without a carrier, or a matrix
`containing the active ingredient. Other occlusive de(cid:173)
`vices are known in the literature.
`Preferably, the pharmaceutical composition is in unit
`dosage form, e.g. as tablets or capsules. In such form,
`the composition is sub-divided in unit dose containing
`appropriate quantities of the active ingredient; the unit
`dosage forms can be packaged compositions, for exam(cid:173)
`ple, packeted powders, vials, ampoules, prcfilled syrin(cid:173)
`ges 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 physi(cid:173)
`cian.
`In addition, the compounds of this invention may be
`employed as a solution, cream, or lotion by formulation
`
`The pharmacological data obtained in the above tests
`with the invention compounds is shown in Table I along
`with comparative results with rapamycin.
`TABLE I
`IC~o DAT A FOR LAF ASSAY
`IC~o
`Ester Rapamycin
`0.87
`2.50
`2.00
`0.60
`0.34
`
`IC<o
`Rapamycin
`
`IA
`1B
`2A
`2B
`3
`
`2.7
`3.9
`3.9
`3.9
`6.3
`
`4A
`4B
`5
`6
`
`1.00
`23
`1.60
`0.32
`
`6.3
`1.6
`3.8
`3.8
`
`Example
`
`Example
`
`Ester
`
`Breckenridge Exhibit 1065
`Hu '300 patent
`Page 005
`
`

`

`5,260,300
`
`9
`with pharmaceutically acceptable vehicles containing
`0.1-5 percent, preferably 2%, of active compound
`which may be administered to a fungally affected area.
`What is claimed is:
`1. A compound according to the formula:
`
`10
`
`IO
`
`15
`
`25
`
`20 wherein:
`RI and R2 are independently H or -COOR 3 but both
`R 1 and R 2 cannot be H, and
`R3 is C1-C6 alkyl_ where 1 to 3 hydrogens may be
`replaced by fluorine, chlorine, bromine or iodine,
`C3-Cg cycloalkyl, C2-C6 alkenyl, or Ar-(CH2.
`)n-where n is 0 to 6 and Ar is phenyl, phenyl
`substituted by fluorine, chlorine, bromine, iodine,
`trifluoromethyl, nitro, cyano, C1-C6 alkyl or
`C1-C6 alkoxy; pyridinyl, indolyl, quinolyl or fura(cid:173)
`nyl;
`or a pharmaceutically acceptable salt thereof.
`12. A method of treating autoimmune diseases which
`comprises administration to a warm-blooded animal in
`need thereof of a therapeutically effective amount of a
`compound according to the formula:
`
`40
`
`45
`
`50
`
`wherein:
`RI and R 2 are independently H or -COOR 3 but both
`RI and R 2 cannot be H, and
`R3 is C1-C6 alkyl where 1 to 3 hydrogens may be 30
`replaced by fluorine, chlorine, bromine or iodine,
`C3-Cg cycloalkyl, C2-C6 alkenyl, or Ar-(CH2-
`)n-where n is 0 to 6 and Ar is phenyl, phenyl
`substituted by fluorine, chlorine, bromine, iodine, 35
`trifluoromethyl, nitro, cyano, C1-C6 alkyl or
`C1-C6 alkoxy; pyridinyl, indolyl, quinolyl or fura(cid:173)
`nyl;
`or a pharmaceutically acceptable salt thereof.
`2. A compound according to claim 1 which is 31,42-
`0-bis(methoxycarbonyl)-rapamycin.
`3. A compound according to claim 1 which is 41-0-
`(methoxycarbonyl)rapamycin.
`4. A compound according to claim 1 which is 31,42-
`0-bis(ethoxycarbonyl)rapamycin.
`5. A compound according to claim 1 which is 42-0-
`(ethoxycarbonyl)rapamycin.
`6. A compound according to claim 1 is 42-0-
`(phenyloxycarbonyl)rapamycin.
`7. A compound according to claim 1 which is 42-0-
`(4-nitrophenyloxycarbonyl)-rapamycin.
`8. A compound according to claim 1 which is 31,42-
`0-bis(4-nitrophenyloxycarbonyl)-rapamycin.
`9. A compound according to claim 1 which is 42-0-
`(prop-2-enyloxycarbonyl)rapamycin.
`10. A compound according to claim 1 which is 42-0-
`(benzyloxycarbonyl)rapamycin.
`11. A method of treating organ and tissue transplant
`rejection which comprises administration to a warm(cid:173)
`blooded animal in need thereof of a therapeutically
`effective amount of a compound according to the for-
`mula:
`
`55
`
`60
`
`65
`
`wherein:
`RI and R2are independently H or-COOR3but both
`RI and R 2 cannot be H, and
`R3 is C1-C6 alkyl where 1 to 3 hydrogens may be
`replaced by fluorine, chlorine, bromine or iodine,
`C3-Cg cycloalkyl, C2-4 alkenyl, or Ar-(CH2-
`)n-where n is 0 to 6 and Ar is phenyl, phenyl
`substituted by fluorine, chlorine, bromine, iodine,
`trifluoromethyl, nitro, cyano, C1-C6 alkyl or
`C1-C6 alkoxy; pyridinyl, indolyl, quinolyl or fura(cid:173)
`nyl;
`or a pharmaceutically acceptable salt thereof.
`13. A pharmaceutical composition for the treatment
`of organ and tissue transplant rejection and autoimmune
`
`Breckenridge Exhibit 1065
`Hu '300 patent
`Page 006
`
`

`

`11
`
`5,260,300
`
`5
`
`h" h
`.
`d.
`1seases w 1c compnses a pharmaceutical carrier and
`
`10
`
`an effective amount of a compound having the formula:
`
`wherein:
`RI and R2are independently H or-COOR3but both
`RI and R 2 cannot be H, and
`R3 is C1-C6 alkyl where 1 to 3 hydrogens may be
`replaced by fluorine, chlorine, bromine or iodine,
`C3-Cs cycloalkyl, C2-C6 alkenyl, or Ar-(CH2.
`)n-where n is 0 to 6 and Ar is phenyl, phenyl
`substituted by fluorine, chlorine, bromine, iodine,
`trifluoromethyl, nitro, cyano, C1-C6 alkyl or
`C1-C6 alkoxy; pyridinyl; indolyl, quinolyl or fura(cid:173)
`nyl;
`or a pharmaceutically acceptable salt thereof.
`* * * * *
`
`IS
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`SS
`
`60
`
`65
`
`Breckenridge Exhibit 1065
`Hu '300 patent
`Page 007
`
`