Per
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Oassification 5 :
`A61K 31/445, C07D 498/18
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(11) International Publication Number:
`
`WO 92/21341
`
`A1
`
`(43) International Publication Date:
`
`10 December 1992 (10.12.92)
`
`(21) International Application Number:
`
`PCT/US92/02504
`
`(22) International Filing Date:
`
`3 April1992 (03.04.92)
`
`(72) Inventor; and
`(75) Inventor/ Applicant (for US only) : SCHULTE, Gary, R.
`[US/US]; 6 Williams Street, Stonington, CT 06378 (US).
`
`(30) Priority data:
`708,412
`
`31 May 1991 (31.05.91)
`
`us
`
`(60) Parent Application or Grant
`(63) Related by Continuation
`us
`Filed on
`
`708,412 (CIP)
`31 May 1991 (31.05.91)
`
`(71) Applicant (for all designated States except US): PFIZER
`INC. [US/US]; 235 East 42nd Street, New York, NY
`10017 (US).
`
`(74)Agents: RICHARDSON, Peter, C. et al.; Pfizer Inc., 235
`East 42nd Street, New York, NY 10017 (US).
`
`(81) Designated States: AT (European patent), BE (European
`patent), CA, CH (European patent), DE (European pa(cid:173)
`tent), DK (European patent), ES (European patent), FI,
`FR (European patent), GB (European patent), GR (Eu-
`ropean patent), IT (European patent), JP, LU (European
`patent), MC (European patent), NL (European patent),
`SE (European patent), US.
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(54) Title: USE OF RAPAMYCIN PRO DRUGS AS IMMUNOSUPPRESSANT AGENTS
`
`(I)
`
`..
`
`(57) Abstract
`
`The use of rapamycin prodrugs of formula (I) as immunosuppressant agents, intermediates formed in the preparation of
`its prodrugs as well as the prodrugs themselves.
`
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`FOR 1'HE PURPOSES OF INFORMATION ONLY
`
`Codes used tu identify States pany to the Per on tht: fJOnt pages of pamphlets publishing international
`am)lications under the PCT.
`
`AT
`AU
`HB
`BE
`BF
`BG
`BJ
`BR
`CA
`CF
`('G
`CH
`Cl
`CM
`cs
`OK
`UK
`ES
`
`Au~lria
`Au)lr;tlia
`H.trbiklO)
`HciJ,:ium
`Burkin;1 l-a>t1
`Uulgo~ria
`Benin
`SriUil
`(';mada
`Central African Rt:publi!:
`Congo
`Swi!Jcrland
`Cote d'lvuirc
`('amcroun
`C!ccho>luvulht
`Germany
`Denmark
`S[lain
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`....
`FR
`GA
`GB
`GN
`GR
`HU
`IE
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`MG
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`Hnlaml
`!:'ranee
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`United Kint;dum
`Guinea
`Orecc&.:
`Hungary
`Ireland
`I tilly
`Japan
`Dcmocr .Jlk Pcu11l&.: ') Kepublic
`ofKon.:a
`Republic of Korea
`I icchtcn>t&.:in
`!iri l.<~nl.a
`l.u~cmbourg
`Muno~cu
`MaJaga.co~r
`
`Ml
`MN
`MR
`MW
`Nl.
`NO
`PL
`RO
`RU
`SD
`SE
`SN
`su
`TO
`TG
`us
`
`Mali
`Mongolia
`Mauritania
`Mo~lawi
`Nctherlnmb
`Norway
`Pul.uuJ
`Rumi.lnia
`Russian Federation
`Sudan
`Sweden
`St:ncgal
`Soviet Union
`Chad
`Togo
`United States of America
`
`..
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`
`USE OF RAPAMYCIN PRODRUGS AS IMMUNOSUPPRESSANT AGENTS
`Background of the Invention
`This invention relates to the use of rapamycin prodrugs
`as immunosuppressant agents, e.g. for use in a human host in
`the treatmen·:: of autoimmune diseases and/ or prevention of
`10 organ transplant rejections,
`intermediates formed in the
`preparation of
`the prodrugs as well as
`the prodrugs
`themselves.
`In 1983, the United States Food and Drug Administration
`licensed
`cyclosporin A,
`an anti-rejection drug
`that
`revolutionized the field of organ transplant surgery. The
`drug acts by
`inhibiting the body's
`immune system from
`mobilizing its vast arsenal of natural protecting agents to
`reject
`the
`transplant's
`foreign protein.
`Although
`cyclosporin A is effective
`in fighting
`transplantation
`rejection, it suffers drawbacks in causing kidney failure,
`liver damage, and ulcers which in many cases can be very
`severe. Newer, safer drugs exhibiting less side effects are
`constantly being searched for.
`Rapamycin has been found to be useful as an antifungal
`25 agent, United States Patent 3,929,992, as well as capable of
`inhibition of the immune response, Martel, et al., can. J.
`Physiol. Pharmacal. 55, 48-51 (1977).
`summary of the Invention
`for
`The present
`invention
`relates
`to a method
`suppressing the immune system, for example,
`in treating
`autoimmune disease or preventing or ameliorating organ or
`tissue transplant rejection comprising administering to a
`mammal
`in
`need
`of
`such
`treatment
`an
`effective
`immunosuppressive amount of a compound of the formula
`
`15
`
`20
`
`30
`
`35
`
`40
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`
`5
`
`10
`
`I
`
`R3
`
`I I
`
`wherein R1 and R2 are independently selected from hydrogen,
`and a group of the formula
`0
`I
`II
`-C-<CH 2>.-N
`"'R4
`20 wherein m is 1-6, R3 and R4 are each hydrogen; branch or
`to C8 alkyl; phenyl;
`to C8 alkyl; cyclic C3
`straight c1
`benzyl; or R3 and R4 taken together with the nitrogen to
`which they are attached form a saturated heterocyclic ring
`having four or five carbon atoms, with the proviso that R1
`In a preferred embodiment
`and R2 can not both be hydrogen.
`of the present invention, at least one of R1 and R2 is a
`group of the formula II, more preferred R3 and ~ are C1 to C8
`alkyl.
`The present invention also relates to intermediates for
`forming prodrugs of rapamycin of formula I wherein R1 or R2
`are each independently
`0
`
`15
`
`25
`
`30
`
`35
`
`A<CH2l•
`l
`X
`
`1 I I
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`X is a suitable leaving group, and m is 1 to 6. Preferred
`leaving groups include, Br, Cl, I, -OS02CH3 , and p-toluene(cid:173)
`sulfonate.
`The present invention also relates to prodrugs of
`rapamycin of formula I wherein R2 is hydrogen and R1 is
`
`5
`
`10
`
`independently
`wherein n is 1 to 6; R3 and R4 are each
`hydrogen; branch or straight C1 to C8 alkyl; cyclic C3 to c8
`alkyl; phenyl; benzyl; or R3 and R4 taken together with the
`nitrogen to which they are attached to form a saturated
`15 heterocyclic ring having four or five carbons atoms, or
`thereof, as well as
`pharmaceutically acceptable salts
`pharmaceutical compositions including the prodrugs.
`Detailed Description of the Invention
`The compounds of formula I are rapamycin prodrugs.
`20 Rapamycin and certain prodrugs thereof are described in
`United States Patents 3,929,992; 3,993,749; 4,316,885; and
`4,650,803, the disclosure of which is hereby incorporated
`herein by reference.
`The prodrug compounds of the present invention are
`25 produced by first forming the acetate ester of rapamycin.
`This is accomplished by reacting rapamycin, the compound of
`formula I where R1 and R2 are both hydrogen with an acylating
`(V) where m is as defined
`agent of the formula YCO{CH2 )mX
`above in the presence of an alkyl amine base and a non-polar
`30 solvent. For the acylating agent of formula V, Y is, for
`example, halogen, N3, -o-cocH2-X,
`
`35
`
`or
`
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`
`and X is a suitable leaving group such as, for example,
`
`Preferably the acylating
`
`agent is where X and Y are each a bromine group. Suitable
`amine bases include the following trialkylamine bases:
`R9
`
`5
`
`R
`
`10
`
`R7 = C1 to C4 alkyl, C5-C6 cycloalkyl
`Z = -(CH2)m-, -o-
`m = 0,1
`R9 = H, C1 to C4 alkyl, N(CH3h
`15 Rs, R10 = H, C1 to C4 alkyl
`n = H, 1,3.
`the
`and dialkylamine bases will react with
`Mono-
`acylating substrate and alkali hydroxides and alkali
`hydrides will cause reaction to the macrolide substrate.
`20 Preferably a trialkylamine base is used, the preferred is
`the following
`Single or mixtures of
`2,4,6-collidine.
`(1) hydrocarbons (i.e., pentane,
`solvents can be used:
`hexane, and cyclohexane), (2) halocarbons (i.e., methylene
`carbon
`and
`dichloroethane,
`chloroform,
`chloride,
`tetrachloride), (3) aromatic hydrocarbons (i.e., benzene or
`ether,
`diethyl
`(i.e.,
`ethers
`(4)
`and
`toluene),
`tetrahydrofuran, dimethoxyethane, and dioxane), preferably,
`dichloromethane. Reaction conditions range from about -78°C
`-43°C.
`temperature preferably about
`in
`to about +50°C
`30 Reactions are carried out under an inert atmosphere using,
`times
`Reaction
`for example, nitrogen or argon gas.
`typically range from 5 minutes to 24 hours, preferably about
`5 hours.
`
`25
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`
`After the first step, the reaction mixture includes
`rapamycin substituted at the 28 position, the 43 position,
`Standard flash chromatography which
`and both positions.
`separates based on polarity is used to isolate each of the
`three substituted rapamycins in order to proceed with each
`separately.
`The second step involves reacting the a~etate ester of
`rapamycin with a nucleophile of the formula HNR3R.. to form
`the corresponding glycinate ester. For the nucleophile, R3
`10 and ~ are each independently hydrogen; branch or straight c1
`to C8 alkyl; phenyl; or benzyl,
`to C8 alkyl; cyclic C3
`For non-polar solvent choices
`preferably each is methyl.
`the following are used in mixtures or independently and
`(2)
`(1) halocarbons,
`the previously described
`include
`15 aromatic hydrocarbons, and (3) ethers as well as (4) polar
`aprotic solvents (i.e., N,N-dimethylformamide, dimethyl(cid:173)
`sulfoxide, and acetonitrile), preferably dimethylformamide.
`The reaction is run in a range of temperatures from about
`(about 27°C),
`temperature
`-78°C to approximately room
`20 preferably about -45°C. The reaction is run under an inert
`atmosphere of, for example, nitrogen or argon.
`The prodrug salts of the glycinate ester are formed by
`reacting the amine of the ester with mineral and organic
`acids (H+·x) where X is, for example halogen, HS04 , H2P04 ,
`25 HC03 , R7coo, and R7S03 where R7 is C1-C24 alkyl, C1-~ alkenyl,
`or
`Preferably X = Cl, MeS03 , MeCOO,
`or phenyl.
`The solvents for salt
`(oleate).
`CH3 (CH2),CH=CH(CH2),COO
`(3)
`{2) halocarbons,
`(1) hydrocarbons,
`formation are:
`( 4) ethers, described above,
`aromatic hydrocarbons, and
`30 preferably diethyl ether. All salt formations are carried
`out at temperatures ranging from -78°C to room temperature,
`preferably 0°C.
`Rapamycin and its prodrugs exhibit immunosuppressive
`activity, i.e., positive inhibition of T-cell activation.
`35 The immunosuppressant effect of rapamycin and its prodrugs
`may be demonstrated by the following assays.
`
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`Immunosuppression can be measured by measuring 3H(cid:173)
`thymidine uptake in human lymphocytes. The procedure is a
`lymphocyte reaction using frozen
`two-way mixed
`modified
`donor cells as a stimulator population and freshly isolated
`5 cells as a responder population.
`The responder population is prepared by drawing human
`blood into a sterile syringe containing 0.8 ml (preservative
`free heparin (1,000 USP unitsfml.)). Twenty-five ml of the
`blood and 20 ml of RPMI-1640 lymphocyte medium are added and
`10 mixed in a centrifuge tube. The mixture is then underlay
`(SIGMA) and centrifuged at room
`with 10 ml HISTOPAQUE-1083
`temperature for about 3 0 minutes at approximately 925 g.
`After centrifugation is complete, the mononuclear cells in
`the layer of HISTOPAQUE-1083, the third layer from the top,
`These cells are then reconstituted with
`15 are drawn off.
`rpms,
`for 5 minutes at 1800
`centrifuged
`RPMI-1640,
`This
`supernatant removed, and resuspended in RPMI-1640.
`washing procedure is repeated twice more. After the final
`washing, the medium in which cells are suspended (RPMI-1640)
`is enriched with 0.5% MEM non-essential amino acids (100x}
`solution, 1% L-glutamine (200 mM), 1% MEM vitamins (100x),
`1% penicillin streptomycin solution (10,000 Unitsfml), and
`15% heat-inactivated human AB serum (NABI). The cells are
`adjusted in concentration to about 5 x 105/ml and 100~1/well
`25 of cell stock added to round bottom 96 well plates.
`The stimulator pool is prepared by collecting blood
`from seven different donors blood, separating, and washing
`mononuclear cells as described above with the responder
`population up to and including the washing steps. After the
`final wash, the mononuclear cells from the donors are pooled
`and suspended at 2 x 107 cellsfml in 90% human AB serum and
`10% DMSO and stored in liquid nitrogen. When ready to
`perform the assay, the frozen cells are placed in a water
`bath at a temperature of about 37°C until thawing begins.
`35 The cells are removed from the bath at that point and room
`temperature RPMI-1640 is added to the pellet. After the
`cells have thawed completely, they are washed once in RPMI-
`
`30
`
`20
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`10
`
`1640, resuspended in a minimum volume, and viability is
`determined using,
`i.e •. ,
`trypan blue exclusion stain
`(viability should be in excess of 80%}. Those cells meeting
`the viability standard are diluted with RPMI-1640 to a
`5 concentration of 5 x 105 cells/ml and 100~1/well of
`stimulator cells are added to the wells containing the
`responder cells.
`The test compounds are solubilized in either ethanol or
`DMSO (10%) and sterile water {90%). The concentrations of
`the solubilized compounds is adjusted such that the addition
`of 50~1 of test compound solution will result in final
`concentrations in each well of 10, 1, or 0.1 mg/ml. The
`assay for each dose is run in triplicate wells and the
`plates are incubated at about 37°C in 5% C02, humidified for
`15 5 days. One ~Cifwell of 3H-thymidine is then added to each
`well and the plates are incubated for another 18 hours,
`after which the cells are harvested and tritium counted
`using the LKB BETA PLATE counter system.
`Controls for the assay included triplicate well of
`responder
`and
`stimulatory
`cells
`run
`separately
`(unstimulated) as well as the above referred to 1:1 mixture
`of those cells without the addition of drug (stimulated).
`The uptake of
`3H-thymidine for each of
`the wells
`is
`determined and the average cpm value for each set of
`triplicates is calculated. The % inhibition is calculated
`using the following formula:
`%inhibition=[1-(avg. cpm with drug)f{avg. cpm stimulated
`control)] X 100.
`Inhibition of 50% or better at any compound concentration is
`30 considered active.
`interleukin-2
`involves evaluating
`A second assay
`biosynthesis inhibition. A rat spleen cell suspension is
`first prepared by sacrificing rats. The spleens from the
`rats are minced and gently pushed through a fine meshed
`screen with frequent washings with RPMI-1640,
`5% heat(cid:173)
`inactivated fetal calf serum (FCS), 100~g streptomycin/ml,
`and 2 mM 1-glutamine (RPMI/5). Approximately 5x108 spleen
`
`20
`
`25
`
`35
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`
`cells are recovered from each 200-250 g rat. The rat spleen
`cells are resuspended in RPMI/5 at a concentration of 5xlo6
`cells/ml. 2 #g/ml of Con A is added to the cell suspension.
`CTLL-2 cells are also prepared using known methods and
`5 are maintained prior to the assay in 60% RPMI 1640, 10% FCS,
`penfstrep, !-glutamine {RPMI/10) with 40% rat growth factor.
`Rat growth factor is a mixture of cytokines prepared by
`incubating 5x106 rat spleen cells/ml with 2 #g/ml Con A for
`48 hours in RPMI/5. Cultures are then centrifuged and the
`supernatants sterile filtered and stored at -20°C until
`used.
`Approximately 1 mg of a test compound is dissolved in
`1.0 ml of approximately 10% DMSO or ethanol and 90%
`phosphate buffered saline (PBS) or sterile water. Compounds
`15 are then further diluted in RPMI/5 to a final concentration
`of 30.0, 3.0, and 0.3 #g/ml. 0.1 ml of each concentration
`of test compound is added in triplicate to each well of a
`tissue culture plate which already contains 0.1 ml of the
`above spleen cell suspension. 0.1 ml of media is also added
`to each well, giving a final test compound concentration of
`The cells are incubated with
`10.0, 1.0, and 0.1 #gfml.
`humidification under 5% C02 at 37°C for about 24 hours.
`After the incubation, 0.1 ml of supernatant is removed and
`tissue culture plate
`the wells of another
`to
`added
`25 containing the CTLL-2 cells. The spleen cells are saved for
`viability determinations.
`they are
`The day before the CTLL-2 cells are used,
`washed and resuspended in RPMI/10 without rat growth factor
`at 105 cellsjml, and 0.1 ml plated into the wells of a 96
`30 well tissue culture plate to which 0.1 ml of the above
`After
`spleen cell suspension has already been added.
`addition of test supernatant, the plates are incubated for
`48 hours with humidification under 5% C02 at 37°C. six hours
`before harvesting, 1.0 #Ci tritiated thymidine (spec. act.
`The cells are then
`is added to each well.
`35 2. o Ci/mM)
`harvested and tritium counted (using the LKB BETA PLATE
`system).
`
`20
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`
`to
`relative
`calculated
`is
`inhibition
`Percent
`introduction of only con A as control supernatant and is
`determined for each concentration. Viability of the spleen
`Compounds
`cells and the CTLL-2 cells is also assessed.
`5 which inhibit IL-2 production 8 0% or more, which do not
`than 80%
`the spleen cells more
`decrease viability of
`compared to Con A controls, and which are not toxic to the
`CTLL-2 cells are considered active.
`A third assay for delayed-type hypersensitivity (DTH)
`10 can be used to determine the ability of compounds to inhibit
`the delayed response of sensitized mice to a challenge of
`sheep red blood cells. The assay uses 20 g C57BL/6 male
`mice (Charles River Breeding Laboratories), typically five
`per group. Defibrinated sheep blood can be purchased from,
`On the first day
`for example, Scott Laboratories, Inc.
`sheep red blood cells are prepared by centrifuging about
`4 ml of sheep red blood cells {srbcs) for 10 min. at 3000
`rpms. The supernatant is poured off and the red cells are
`resuspended in 9 mls of sterile saline. This washing step
`The red blood cells are then
`is performed twice more.
`adjusted to a concentration of 5x106/ml.
`Animals are treated with 0.2 ml of a test compound in
`10% ETOH, OMSO, or similar vehicle. Control groups include
`a vehicle control, and non-treated immune control, and a
`25 normal, i.e., non-sensitized, group. Also, on the first day
`treatment
`the above
`and approximately 1 hour after
`(depending on which group a given animal is in), animals are
`sensitized by injecting 106 srbcs in 0.2 ml i.v. Animals are
`treated daily for four additional days. On day 4, srbcs are
`30 prepared as before, except that the final concentration is
`adjusted to 3x108/ml. Approximately 1 hour after treatment,
`implanting 108 srbcs into the
`animals are ·challenged by
`planar surface of 1 hind footpad in a volume of 0.03 ml.
`Approximately 24 hours after challenge, the thickness
`35 of both hind footpads is measured with dial gauge calipers.
`increase in footpad size of the challenged footpad
`The
`
`20
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`versus the control footpad is a measure of DTH, and is
`typically in the range of o.s to 1.2 mm.
`Results are reported as % inhibition on DTH in drug
`treated groups versus control.
`Rapamycin prodrugs of the present application possess
`immunosuppressive activity and antimicrobial activity, and
`are therefore useful for the treatment, prevention of the
`rejection of transplanted organs and tissues (such as heart,
`liver, medulla ossium, skin, etc.),
`heart-lung, kidney,
`autoimmune diseases such as rheumatoid arthritis, systematic
`thyroiditis, multiple
`lupus erythematosus, Hashimoto's
`type I diabetes, uveitis,
`sclerosis, myasthenia gravis,
`obstructive airway diseases, and psoriasis), as well as
`infectious diseases caused by pathogenic microorganisms.
`the present
`compositions of
`pharmaceutical
`The
`in the form of a pharmaceutical
`invention can be used
`in solid, semi-solid or liquid
`preparation, for example,
`form, which contains the rapamycin prodrugs as an active
`inorganic
`in admixture with an organic or
`ingredient,
`20 carrier or excipient suitable for external, enteral or
`ingredient may be
`The active
`parenteral applications.
`the usual non-toxic,
`example, with
`for
`compounded,
`pharmaceutically acceptable carriers for tablets, pellets,
`capsules, suppositories, solutions, emulsions, suspension,
`and any other form suitable for use. The carriers which can
`be used are water, glucose, lactose, gum acacia, gelatin,
`mannitol, starch paste, magnesium trisilicate, talc, corn
`starch, keratin, colloidal silica, potato starch, urea and
`in manufacturing
`use
`for
`suitable
`carriers
`other
`30 preparations, in solid, semi-solid, or liquid form, and in
`thickening and coloring
`addition auxiliary, stabilizing,
`agents and perfumes may be used.
`For applying such pharmaceutical compositions to a
`the compositions by
`to apply
`it is preferable
`human,
`35 parenteral or oral administration. While the dosage of
`therapeutically effective amount of the rapamycin prodrug
`depend upon the age and condition of each individual patient
`
`10
`
`15
`
`25
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`
`to be treated, a daily dose of from about 1 mg/kg to about
`250 mg/kg preferably about 20 mgfkg and an average single
`dose of about 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250
`mg is generally administered.
`The following example is given for the purpose of
`invention and should not be
`the present
`illustrating
`construed as being a limitation on the scope of the instant
`invention.
`
`5
`
`10
`
`Example 1
`43-bromoacetate ester of rapamycin
`Rapamycin (5.0 gm, 5.47 mmol) was dissolved in 500 ml
`of dichloromethane and then the solution was cooled to -72°C
`2, 4, 6 - Collidine {14.46 ml,
`{acetone/dry ice bath).
`109.41 mmol) and bromoacetylchloride (4.06 ml, 49.23 mmol)
`15 were sequentially added to the cold rapamycin solution.
`After 4. 5 hours additional 2, 4, 6 - Collidine ( 7 .12 ml,
`53.86 mmol) and bromoacetylchloride (2.00 ml, 24.24 mmol)
`were added. The reaction was worked up by pouring into a 5%
`sodium bicarbonate/ice (400 ml/600 ml) mixture after 5.5
`The aqueous solution was
`time.
`total reaction
`20 hours
`extracted with ethyl acetate (800 ml). The organic solution
`was washed with 5% sodium bicarbonate (4 x 500 ml) followed
`by in 5% hydrochloric acid (4 x 500 ml), dried (anhydrous
`Evaporation followed by
`sodium sulfate) and filtered.
`25 chromatography (Si02; hexanesfethyl acetate, 1:1) gave pure
`title compound (2.0 g, 1.94 mmol, 35% yield) as a glass.
`Physical data: Fast atom bombardment mass spec.: mfz
`1057/1059 ((M+Na)+,5%), 1034/1036 {(M+H)+, 10%); partial 1H
`NMR {CH2Cl2): 6 4.68 (1H, ddd) and 3.85 {2H, brs).
`Example 2
`43-N.N-dimethylglycinate ester of rapamycin
`A sample of 43-bromoacetate ester of rapamycin (1.01 g,
`0.978 mmol) was dissolved in 20 ml of N,N-dimethylformamide
`and then cooled to -45°C (acetonitrile/dry ice). To this
`in 3 ml of N,N(cid:173)
`35 solution excess dimethylamine {1 ml)
`dimethylformamide was slowly added by addition funnel so as
`to maintain the reaction solution below -40°C. After 1 hour
`
`30
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`the reaction was complete and poured into 600 ml of brine
`The aqueous solution was extracted with ethyl
`and ice.
`acetate (2 x 250 ml). The ethyl acetate solution was washed
`filtered and
`(Na2S04 ) ,
`(3 x 100 ml) , dried
`with brine
`5 evaporated to give pure title compound (0.95 g, 0.957 mmol,
`98% yield) as a glassy solid.
`Physical data: Fast atom bombardment mass spec.: mfz
`( (M+H) + 1 100%) ; partial 1H NMR
`5%) , 999
`( (M+Na) + 1
`1021
`(CD2Cl2) : o 4.68 (1HI ddd), 3.12 (2H, brs), and 2.30 (6H,
`10 brs).
`
`Example 3
`Methanesulfonic acid salt of 43-N.N-dimethylqlycinate
`ester of rapamycin
`A sample of 43-N,N-dimethylglycinate ester of rapamycin
`of
`mL
`5
`in
`dissolved
`was
`0.952 mmol)
`(0.95 g,
`dichloromethane and cooled to ooc. A stock solution of
`in 46.7 ml of
`methanesulfonic acid (3.25 ml, 49.9 mmol)
`diethyl ether was prepared. A 1 ml aliquot of the acid
`solution was slowly added to the cooled aminoester. After
`20 addition the solution was evaporated to give the title
`compound (1.02 g, 0.986 mmol 1 98% yield) as a light yellow
`solid, m.p. 100-120°C.
`(CD2Cl2) : o 11.59 (1H,
`1H NMR
`Physical data: partial
`(2H, brs) 1 3.00 (6H,
`(1H 1 ddd) 1 3.88
`exchangeable), 4.79
`25 brs), and 2.73 (3H, s).
`
`15
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`
`CLAIMS
`system
`suppressing
`immune
`the
`for
`A method
`1.
`in need of such
`comprising administering to a mammal
`immunosuppressive amount of a
`treatment an effective
`5 compound of the formula
`Rl
`
`10
`
`15
`
`20
`
`0
`
`I
`
`wherein R1 and R2 are independently selected from hydrogen,
`and a group of the formula
`
`I I
`
`25 wherein m is 1-6, R3 and R4 are each independently hydrogen;
`branch or straight C1 to C8 alkyl; cyclic C3 to c8 alkyl;
`phenyl; benzyl; or R3 and R4 taken together with the nitrogen
`to which they are attached form a saturated heterocyclic
`ring having four or five carbon atoms with the proviso that
`3 o R1 and R2 cannot both be hydrogen.
`The method of claim 1, wherein said method is used
`2.
`in treating autoimmune disease.
`The method of claim 1, wherein said method is used
`3.
`in preventing or ameliorating organ or tissue transplant
`rejection.
`
`35
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`The method according to claim 1, wherein R3 and R•
`4 •
`are each independently a straight C1 to C8 alkyl chain.
`The method according to claim 4, wherein R3 and R•
`5.
`are both methyl.
`The method according to claim 1 wherein m is 1.
`6.
`The method according to claim 1 wherein R1 is
`7.
`
`I I
`
`5
`
`10
`
`15
`
`wherein m is 1-6, R3 and R4 are each independently hydrogen;
`branch or straight C1 to C8 alkyl; cyclic C3 to Ca alkyl;
`phenyl; benzyl; or R3 and ~ taken together with the nitrogen
`to which they are attached form a saturated heterocyclic
`ring having four or five carbon atoms.
`The method according to claim 7, wherein R3 and R•
`8.
`are each independently a straight C1 to C8 alkyl chain.
`The method according to claim 8 wherein R3 and R4
`9.
`20 are both methyl.
`10. A compound of the formula
`
`I
`
`25
`
`30
`
`35
`
`wherein R1 or ~ are each independently
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`
`Ill
`
`X is a suitable leaving group, and m is 1 to 6.
`11. The compound of claim 10 1 wherein X is Br 1 Cl,
`or I.
`12. The compound of claim 11, wherein X is Br.
`13. The compound of claim 10, wherein m is 1.
`14. The compound of claim 10, wherein the leaving
`groups are Br, -oso2CH3, p-toluenesulfonate.
`15. The compound of claim 10, wherein R1 is
`
`0 A<cH2 >.
`
`I
`)(
`
`Ill
`
`X is a suitable leaving group, and m is 1 to 6.
`16. The compound of claim 15, wherein the leaving
`groups are Br, -oso2CH3, p-toluenesulfonate.
`17. The compound of claim 16, wherein the leaving
`group is a bromine group.
`18. A compound of the formula
`Rl
`
`I
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`wherein R2 ·is hydrogen and R1 is
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`5
`
`independently
`is 1 to 6; R3 and R4 are each
`wherein n
`hydrogen, branch or straight C1 to C8 alkyl; cyclic C3 to C8
`alkyl; phenyl; benzyl; R3 and R4 taken together with the
`nitrogen to which they are attached to form a saturated
`10 heterocyclic ring having four or five carbons atoms, or
`pharmaceutically acceptable salts thereof.
`19. The compound of claim 18, wherein n is 1.
`20. The compound of claim 18, wherein R3 and R4 are
`each independently a straight C1 to C8 alkyl chain.
`21. The compound of claim 18 wherein R3 and R4 are both
`methyl.
`in
`use
`for
`composition
`pharmaceutical
`22. A
`effective
`system comprising an
`immune
`the
`suppressing
`immunosuppressive amount of the compound of claim 21 and a
`20 pharmaceutically acceptable carrier or diluent.
`23. A process for forming a compound of the formula
`
`15
`
`I
`
`25
`
`30
`
`35
`
`wherein R1 or Rz are each independently
`
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`
`III
`
`..
`
`5
`
`X is a suitable leaving group, and m is 1 to 6 comprising
`reacting a compound of formula · I where R1 and ~ are each
`hydrogen with an acylating agent of the formula YCO(CH2)mX in
`the presence of an alkylamine base, wherein X and m are as
`10 defined above and Y is a suitable leaving group.
`24. The process of claim 23, wherein X is Br, Cl,
`or I.
`25. The process of claim 24, wherein X is Br.
`26. The process of claim 23, wherein m is 1.
`27. The process of claim 23, wherein the leaving
`groups are Br, -OS02CH3 , p-toluenesulfonate.
`28. The process of claim 23, wherein R1 is
`
`15
`
`20
`
`0 A<cH2>,
`
`I
`X
`
`Ill
`
`X is a suitable leaving group, and m is 1 to 6.
`29. The process of claim 28, wherein the leaving
`25 groups are Br, -oso2CH3 , p-toluenesulfonate.
`30. The process of claim 29, wherein the leaving group
`is a bromine group.
`31. A process for forming a compound of the formula
`
`30
`
`35
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`
`•
`
`I
`
`5
`
`10
`
`15
`
`wherein R2 is hydrogen and R1 is
`
`IV
`
`independently
`wherein n is 1 to 6; R3 and R4 are each
`20 hydrogen, branch or straight C1 to C8 alkyl; cyclic C3 to C8
`taken together with the
`alkyl; phenyl; benzyl; R3 and R4
`nitrogen to which they are attached to form a saturated
`four or five carbons atoms,
`heterocyclic ring having
`comprising reacting the compound of formula I where R1 is
`25 hydrogen and R2 is
`
`III
`
`30
`
`35
`
`where X is a suitable leaving group and m is 1 ·to 6 with a
`compound of the formula HNR3R.t where R3 and R.t are as defined
`above; and if desired, converting a compound of formula I to
`a pharmaceutically acceptable salt thereof.
`32. The process of claim 31, wherein n is 1.
`
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`33. The process of claim 31, wherein R3 and R4 are each
`independently a straight C1 to C8 alkyl chain.
`34. The process of claim 31 wherein R3 and R4 are both
`methyl.
`
`5
`
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`
`INTERNATIONAL SEARCH REPORT
`International application No. PCT /US 92/0250.!\
`(if several classification symbols apply, Indicate aU)6
`' l. CLASSIFICATION OF SUBJECT MATTER
`According to International Patent Classification (JPC) or to both National Classification and IPC :-
`C 07 D 498/18
`A 61 K 31/445
`J.;ot. Cl. 5
`
`D. FIELDS SEARCHED
`
`•
`
`Classification System
`
`Int.Cl.S
`
`Minimum Documentation Searched'
`
`Classification Symbols
`
`C 07 D
`
`A 61 K
`
`Documeatatioo Searched other than Minimum Documeatation
`to the Extent that such Documents are Included in the Fields Searched a
`
`m. DOCUMENTS CONSIDERED TO BE RELEVANT9
`Citation of Document, 11 with indication, where appropriate, of the relevant passages 12
`Category 0
`
`A
`
`p ,A
`
`Journal of the American Chemical Society, vol.
`113, no. 4, 13 February 1991, American Chemical
`Society, · H. FRETZ et a 1.: 11 Rapamycin and FK506
`, pages
`binding proteins (immunophilins) 11
`1409-1411, see page 1410, scheme I' compounds
`2c,2d,2b
`---
`Tetrahedron Letters, vol. 33, no. 17, 21 April
`1992, Pergamon Press Ltd, (GB), D.P. CURRAN et
`al.: "Intramolecular hydrogen transfer reactions
`of o-(bromophenyl)dialkYlsilyl ethers.
`, pages 2295-2298,
`Preparation of rapamycin-d1 11
`see p'age 2297, lines 13-26 and eq5
`---
`-I-
`
`Relevant to Claim No.l3
`
`10-21
`
`10-21
`
`0 Special categories of cited documents : 10
`• A"' document defining the general state of the an which is not
`considered to be of particular relevance
`"E.. earlier document but published on or after the l.aternatlonal
`filing date
`'"1.."' document which may throw doubts on prio