WO2014082286A1 - Rafamycin analogs and methods for making same - Google Patents

Abstract

A semi-synthetic rapamycin analog with a triazole moiety or a pharmaceutically acceptable salt or prodrug thereof, is a broad-spectrum cytostatic agent and a m TOR inhibitor, and is useful in the treatment of various cancers, or tumors in organs such as kidney, liver, breast, head and neck, lung, prostate, and restenosis in coronary arteries, peripheral arteries, and arteries in the brain, immune and autoimmune diseases. Also disclosed are fungal growth-, restenosis-, post- transplant tissue rejection- and immune- and autoimmune disease- inhibiting compositions and a method of inhibiting cancer, fungal growth, restenosois, post-transplant tissue rejection, and immune and autoimmune disease in a mammal. One particular preferred application of such triazole-moiety containing rapamycin analog is in treating renal carcinoma, lung cancer, colon cancer, and breast cancers wherein potency of the drug, its half-life, tissue distribution properties, and its pharmacokinetic properties including bioavailability through oral and intravenous routes are essential to the clinical outcomes.

Description

RAFAMYCIN ANALOGS AND METHODS FOR MAKING SAME

BACKGROUND OF THE INVENTION

[01] The compound cyclosporine (cyclosporin A) has found wide use since its introduction in the fields of organ transplantation and immunomodulation, and has brought about a significant increase in the success rate for transplantation procedures. Recently, several classes of macrocyclic compounds having potent immunomodulatory activity have been discovered. Okuhara et al.. in European Patent Application No. 184. 162, published Jun. 1 I. 1986, discloses a number of macrocyclic compounds isolated from the genus Streptomyces, including the immunosuppressant FK-506. a 23-membcred macrocyclic lactone, which was isolated from a strain of S. tsukubaensis.

(02 J Other related natural products, such as FR-900520 and FR-900523. which differ from FK-506 in their alkyl substituent at C-21. have been isolated from S. hygroscopicus yakushimnaensis. Another analog. FR-900525, produced by S. tsukubaensis, differs from FK- 506 in the replacement of a pipecolic acid moiety with a proline group. Unsatisfactory side- effects associated with cyclosporine and FK-506. such as nephrotoxicity, have led to a continued search for immunosuppressant compounds having improved efficacy and safety, including an immunosupressive agent which is effective topically, but ineffective systemically (U.S. Pat. 5.457.1 I I).

[03] Rapamycin. as illustrated below, is a macrocyclic triene antibiotic produced by Streptomyces hygroscopicus. which was found to have antifungal activity, particularly against Candida albicans, both in vitro and in vivo (US 3.929.992 and US 3.993.749).

[04] Rapamycin alone (US 4.885.171) or in combination with picibanil (US 4,401.653) has been shown to have antitumor activity. In 1977. rapamycin was also shown to be effective as an immunosuppressant in the experimental allergic encephalomyelitis model, a model for multiple sclerosis: in the adjuvant arthritis model, a model for rheumatoid arthritis: and was shown to effectively inhibit the formation oflgE-like antibodies.

I OS ] The immunosuppressive effects of rapamycin have also been disclosed in FASEB in 1989. as has its ability to prolong survival time of organ grafts in histoincompatible rodents. These and other biological effects of rapamycin are review ed in Transplantation Reviews. 1992. 6.39-87. Mono-ester and di-ester derivatives of rapamycin (csterification at positions 31 and 42) have been shown to be useful as antifungal agents (US 4.316.885) and as water soluble prodrugs of rapamycin (US 4,650.803).

[06] Mono-ester and di-ester derivatives of rapamycin (esterification at positions 31 and 42) have been shown to be useful as antifungal agents (US 4,316,885) and as water soluble prodrugs of rapamycin (US 4.650.803).

[07] Numerous chemical modifications of rapamycin have been attempted. These include the preparation of mono- and di-ester derivatives of rapamycin (WO 92/05179). 27-oximes of rapamycin (EPO 467606): 42-oxo analog of rapamycin (US 5.023.262); bicyclic rapamycins (US 5.120.725): rapamycin dim rs (US 5.120.727): silyl ethers of rapamycin (US 5.120.842); and arylsulfonates and sulfamates (US 5.177.203). Rapamycin was recently synthesized in its naturally occurring enantiomeric form (K. C. Nicolaou et al.. J. Am. Chem. Soc, 1993. 115. 441 -4420: S. L. Schrciber..1. Am. Chem. Soc. 1 93. 115.7906-7907; S. J. Danishefsky. J. Am. Chem. Soc. 1993. I 15. 9345-9346). One recent example of a rapamycin analog is a tetrazole containing rapamycin analog (US 6,015.815). The tetrazole heterocyclic ring is used to replace the hydroxyl group to effect the analog.

[08| Although some of these modified compounds exhibit immunosuppressive activity, anti- restenotic activities in suppressing the migration and growth of vascular smooth muscles, especially when used in a stent coating, the need remains for rapamycin analogs which possess potentially enhanced efficacy against broad spectrum of cancers such as renal cell carcinoma, breast cancers, head and neck cancers, and potentially better lipophilicity. longer half live in the blood or in local tissues, or resistance to oxidative forces and better stability in a formulation. One way to achieve these goals is through introduction of a tria/.ole moiety to the side chain of a rapamycin which may impart a better lipophilicity. better stability, better bioavailability, better tissue and cellular uptake, better efficacy compared to the known and existing modified rapamycin analogs or derivatives. The efficacy of the modified rapamycin may also have better potency against a variety of cancers, and potentially reduced toxicities.

SUMMARY OF THE INVENTION

[09] Accordingly, one object of the present invention is to provide novel semi-synthetic rapamycin analogs which possess a desired tria/.ole moiety attached to either or both to 31C-. and or 42C-position of a rapamycin molecule. [10] In accordance with one aspect, the present invention is directed to compounds represented by the structural formula illustrated below.

[111 In accordance with one aspect, the present invention is directed to compounds represented by the structural formula illustrated below.

|12| In accordance w ith yet another aspect, a compound of the present invention may contain two such substitutes at both the 42C and 31 C-positions of a rapamycin.

[ 13] The triazole moiety of the present invention may be introduced via a variety of reaction schemes, the typical ones are illustrated below:

I o I ■ °

I H OHi

O O N

ί ^{H '}

O^{' '} o

O OH I

o

Scries Λ

wherein A' is one of the following structures:

, '"' ^0H

^0H

K . o .-OH

^{^ ■"'} N -N

N- N O

N^'-N "-^"^,

\11 ΛΙ2 Λ13 Λ14

Series B: B2 113 B5

B6 B7 By B 1 U

0 o

J ^{' ■ ■} /

B l l B 1 2 B I 4

[14] Another object of the present invention is to provide a synthetic processes for the preparation of such compounds from starting materials obtained by fermentation, as well as chemical intermediates useful in such synthetic processes.

[ 15] A further object of the present invention is to provide pharmaceutical compositions containing, as an active ingredient, at least one of the above compounds.

J 16] Yet another object of the present invention is to provide a method of treating a variety of disease states, including restenosis, post-transplant tissue rejection, immune and autoimmune dysfunction, fungal growth, and cancer.

117] In addition, the compounds of the present invention may be employed as an oral tablet, oral solid or oral liquid, oral immediate or sustained release dosage, intravenous injection dosages, parenteral dosages, cream or solutions by formulation w ith pharmaceutically acceptable vehicles.

[18] Also within the scope of this invention includes pharmaceutical compositions for immediate release or sustained release of its active ingredient, each comprising a compound of this invention and pharmaceutical ly acceptable excepient.

| 19| Sti ll further related to this invention are medical devices, each comprising a compound of this invention. Examples of the medical deices include drug-eluting coronary or peripheral, esophageal, urinary. ovar\ . or neurovascu lar stent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Graph for Renal cell carcinoma tumor cell inhibition studies; FIG. 2 Graph for Renal cell carcinoma tumor cell inhibition studies; FIG. 3 Graph for Lung Cancer A549 cell inhibition studies;

FIG. 4 Graph for Lung Cancer A549 cell inhibition studies;

FIG. 5 Graph for Lung Cancer A549 cell inhibition studies;

FIG. 6 Graph for Melanoma SK-MEL-28 cell inhibition studies: FIG. 7 Graph for Melanoma SK-MEL-28 cel l inhibition studies: FIG. 8 Graph for Melanoma SK-MEL-28 cell inhibition studies: FIG. 9 Graph for Epidermal cancer A43 I tumor cell model:

FIG. 10 Graph for Epidermal cancer A43 I tumor cel l model:

FIG. 1 1 Graph for Epidermal cancer Λ43 I tumor cel l model :

FIG. 12 Graph for Glioblastoma U87 MG Tumor model studies: FIG. 13 Graph for Glioblastoma U87 MG Tumor model studies; FIG. 14 Graph for Glioblastoma U87 MG Tumor model studies: FIG. 15 Graph for Human colorectal tumor HCT l I 6 model studies; FIG. 16 Graph for H uman colorectal tumor HCT l 16 model studies; FIG. 17 C]raph for Human colorectal tumor HCT I 1 6 model studies: FIG. 18 Graph for Breast cancer MDA-MB-23 I tumor model;

FIG. 19 Graph for Breast cancer M DA-MB-23 l tumor model: FIG. 20 Graph for Breast cancer M DA-MB-23 I tumor model; FIG. 21 Graph for Breast cancer MCF-7 tumor model:

FIG. 22 Graph for Breast cancer MCF-7 tumor model;

FIG. 23 Graph for Breast cancer MCF-7 tumor model:

FIG. 24 Cjraph for Prostate cancer PC-3 tumor studies:

FIG. 25 Graph for Prostate cancer PC-3 tumor studies;

FIG. 26 Graph for Prostate cancer PC-3 tumor studies: FIG.27 Efficacy of rapamycin analog of the present invention in treating HCT 116.

I)l Ί ΛΙΙ I.I) DESCRIPTION OF THE PREFERRED EMBODIMENTS Definition of Terms

[ 20 J The term "prodrug.^" as used herein, refers to compounds which are rapidly transformed in vivo to the parent compound of the above formula, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems.^" Vol. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed., "Bioreversible Carriers in Drug Design.^" American Pharmaceutical Association and Pergamon Press. 1987. both of which are hereby incorporated by reference.

[211 The term "pharmaceutically acceptable prodrugs.^" as used herein, refers to those prodrugs of the compounds of the present invention which are. within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower mammals without undue toxicity, irritation, and allergic response, are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use. as well as the zwitterionic forms, where possible, of the compounds of the present invention. Particularly preferred pharmaceutically acceptable prodrugs of the present invention are prodrug esters of the C-31 hydroxyl group of compounds of the present invention.

[22] The term "prodrug esters.^" as used herein, refers to any of several ester-forming groups that are hydro lyzed under physiological conditions. Examples of prodrug ester groups include acetyl, ethanoy!. pivaloyl. pivaloyloxymethyl. acetoxymethyl. phthalidyl. methoxymethyl. indanyl. and the like, as well as ester groups derived from the coupling of naturally or unnaturalh-occurring amino acids to the C- 1 hydroxyl group of compounds of the present invention.

i 23 J The term "isomer^" as used herein, refers to a compound having the identical chemical formula but different structural or optical configurations.

[ 41 The term "epimer^" as used herein, refers to a compound having the identical chemical formula but a different optical configuration at a particular position. In the case of a rapamycin, a 42-Epi rapamycin refers to the compound that has the opposite optical rotation compared to the rapamycin obtained by a fermentation process. [25 j The term "15-isomer^" as used herein, refers to the analog of rapamycin that contains a 7- member ring at the 15-position as opposed to a regular rapamycin obtained from a fermentation process which contains a six-member ring. This kind of conversion is also called "tautomcrization^". The 15-isomer^"' as used herein, may also be referred to as a 15 tautomer of a rapamycin.

Preparation of Compounds

[26] The compounds and processes of the present invention will be better understood in connection with the following synthetic schemes which illustrate the methods by which the compounds of the present invention may be prepared.

[27] The compounds of the present invention may be prepared by a variety of synthetic routes. Most of the common conjugation reactions of rapamycin at 42- and/or 31-hydroxyl positions are found in the rapamycin patents mentioned above, the contents of which are incorporated herein by reference in their entireties.

EXAMPIJ-S

Synthesis of Rapamycin Derivatives. The parent rapamycin structure is shown below .

R p a

The synthetic scheme of series Λ of rapamycin analogs of the present invention is shown below:

Rapann in Λ I Scries Λ [2 1 Shown below are additional rapamvcin analogs of this invention that were synthesized similarly:

W herein A' - "

Example 1: Synthesis of Compound Λ1

[29| To a stirred solution of Rapamvcin (3 g. 3.2 mmol) and Cs₂C0 (3.2 g. 9.6 mmol) in dried D F (90 ml.) was added Nal ( 1.5 g.9.6 mmol) and 3-bromoprop- 1 -yne ( 1.2 g.9.6 mmol). The reaction mixture was stirred at rl for 30 hours. Upon the completion of reaction. 300 ml. water was added in and extracted with ethyl acetate (200 ml. x 3). The combined organic layer was washed by brine (300 ml.) and dried over anhydrous Na:S0. After concentration, the residue was purified with silica gel chromatography (50% to 100%) of ethyl acetate in petroleum ether as eluent) to give the compound Λ I (2.1 g, 68%) as a light green oil.

LCMS (m/z.) F.S- 950 ( -D^".

Example 2: Synthesis of Compound A3

|3()| To a solution of 40-O-(prop-2-ynyloxy) rapamvcin A I (200 mg.0.2 mmol) and 1-azido- Admantane (100 mg. 0.6 mmol) in anhydrous THF (9 ml.) was added DIPF.A (100 _,uL. 0.6 mmol) and Cul (20mg.0.1 mmol) under N;. The solution was stirred at rt overnight. Then.20 ml. water was added and extracted w ith ethyl acetate (20 ml, x 3). The combined organic layer was washed by brine and dried over anhydrous Na:S()_t. After concentration, the residue was purified w ith silica gel chromatography (25% to 50% of ethyl acetate in petroleum ether as eluet) to give white solid which was further purified by prep-HPLC to give Compound A3 (26 mg. 10%) as a white solid. Ή NMR (300 MHz. CDC ) δ 7.71 (s. 1H).6.74 (m. 1H).6.39-6.02 (m. 5H).5.62-5.36 (m.511): I.CMS (m//)I^:,S- I I28(M-1 ) .

Example 3: Synthesis of Compound A4 [31] To a solution of

rapamycin ΛΙ (200 mg. 0.2 mmol) and 4- azidobenzoic acid (100 mg.0.6 mmol) in anhydrous THF (9 ml.) was added DIPEA (100 μL·. 0.6 mmol) and Cul (20 mg.0.1 mmol) under N;. The solution was stirred at rt for 3 hours. Then. 20 ml. water was added and extracted with ethyl acetate (20 ml. x 3). The combined organic layer was washed b brine and dried over anhydrous a^S .). After concentration, the residue was purified with silica gel chromatography (5% to 10% of methanol in dichloromethane as eluent) to give white solid which was further purified by PREP-I1PI.C to give Compound A4 (29 mg. 12%) as a white solid. Ή NMR (300 Mil/. CDCE,) δ 8.27 (m. 111).7.90 (m. IFI).7.73 (m. 1H). 7.56 (m. IH). 6.74 (m. 111). 6.55-6.00 (m. 5H). 5.60-5.36 (m. 5H); LCMS (m/z) ES- 1 I I4(M-I)^'.

Example 4: Synthesis of Compound A5

Preparation of Intermediate 2

2

|32| To a stirred suspension of NaN;, (2.0 g. 30.8 mmol) in acetonitrile (20 ml) was added Tf;0 (7.3 g.25.8 mmol) via syringe slowly at 0 °C. The mixture was stirred for another 2 h at this temperature. The insoluble solids were removed through filtration. At 0 °C. the filtrate was added dropwise into the mixture of Compound I (2.0 g. 13 mmol). CuS0₄ (160 mg. 1 mmol). IEO (6 ml.) and Et3N (3.6 ml..25.8 mmol). The reaction mixture was stirred for 6 h at room temperature. The mixture was diluted with EtOAc and washed with brine. The organic layer was dried over Na;SO_t and evaporated to yield brown solid which was purified with silica gel chromatography (30% to 50% of EtOAc in petroleum ether as eluent) to give Intermediate 2 (1.1 g.48%) as a white solid. Ή NMR (300 MHz. CDCE) 69.93 (s. III).7.63 (m.211).7.03 (m.2H). 2.02 (s.311): ECMS (m/z) ES + 177 (MH)\

|33] To a solution of 40-O-(prop-2-ynylox ) rapamycin Al (200 mg. 0.2 mmol) and N-(4- a/ido- phenyl) acetamide. Intermediate 2 (100 mg. 0.6 mmol) in anhydrous THF (9 ml.) was added DI EA (100 μΕ, 0.6 mmol) and Cul (20 mg.0.1 mmol) under Nk The solution was stirred at rt for 4 hours. Then.20 inE water was added and the mixture was extracted with EtOAc (20 ml. x 3). The combined organic layer was washed by brine and dried over anhydrous Na^SOa. A tier concentration, the residue was purified ith silica gel chromatography (30% to 100% of EtOAc in petroleum as eluent) to give white solid which was further purified by prep-HPLC to give Compound A5 (56 mg.25%) as a white solid. Ή NMR (300 Hz, CDC1₃) δ 8.13 (m, 1H). 7.73 (m.411).6.74 (m. 111).6.49-6.00 (m.51-1).5.65-5.37 (m.5H): LCMS (m/z) ES- 1127 (M-l)\

Example 5: Synthesis of Compound A6

|34| To a solution of 40-O-(prop-2-ynyloxy) rapamycin A I (200 mg.0.2 mmol) and TMS-NL (!OO mg.0.9 mmol) in t-BuOII (6 ml.) and I EO (6 ml.) was added Na_C0₃ (I00 mg. I mmol). CuS04 (20 mg. 0.13 mmol) and sodium ascorbate (40 mg, 0.2 mmol) under M₂. The solution was stirred at rt for 3 hours. Then.20 mL water was added and extracted with EtOAc (20 mL x 3). The combined organic layer was washed by brine and dried over anhydrous Na:S0₄. After concentration, the residue was purified with silica gel chromatography (25% of EtOAc in petroleum ether as eluent) to give compound A2 (189 mg. 82%) as a white solid which was dissolved in TBAE in THE (10 ml.) at 0 °C and stirred at rt for 7 hours. Then the reaction mixture was partitioned between EtOAc and water. The aqueous phase was extracted with EtOAc (25 ml. x 3). The combined organic lax cr was washed with brine and dried over anhydrous Na;S⁽ ).. After concentration, the residue was purified with silica gel chromatography (5% to 10% of methanol in diehloromethane as eluent) to give white solid which was further purified by prep-HPLC to give compound A6 (38 mg.24%) as a white solid. Ή NMR (300 MHz. CDCIj) δ 7.75-7.55 (m. 111).6.76 (m. III).6.49-6.08 (m.5H).5.53-5.35 (m, 3H); LCMS (m/z) ES- I0I2 (M-LH8) .

Example 6: Synthesis of Compound A7

Preparation of intermediates 5 and 6:

_Qr NaN₃, H₂0 TsCI, TEA _N Cs₂C0₃. morpholine ^N^-^³

HO - ^► HO" " ³ - ^► TsO

refluxmg, DCM rt, 4h DMF, rt, overnight

³ overnight 4 5 6 |35| To a solution of 3 ⁽5 g.41 mmol) in 100 mL of water was added NaNU (5g.83 mmol) and was relluxed overnight. Then 100 ml. DCM was added in alter reaction mixture was cooled to rt. The organic phase separated was dried over Na^SO filtered. To the solution was added Eb,N (5.05 g.50 mmol) and TsCl (9.55 g.50 mmol) at 0 deg. The reaction mixture was stirred at rt for 4 hours. 100 ml. water was added. The organic phase was separated and dried over Na:SO._t. Filtration and concentration in vacuo gave the crude product. Purification by column chromatography (10% of EtOAc in petroleum ether as eluent) gave intermediate 5 (5.7 g, 58%) as a colorless oil. Ί ΐ NMR (300 Hz, CDCI₃) δ 7.81 (d.2H).7.39 (d.2FI).4.14 (m.2H), 3.48 (m. 2FI).2.43(s.3H): I.CMS (m/z) ES+ 242(Μ+1Γ\

|36| Fo a solution of intermediate 5 (1 g.4.1 mmol) and Cs;CO¾ (2.8 g.8.2 mmol) in 30 ml. of anhydrous DMF was added morpho!ine (0.71 g.8.2 mmol) at 0°C. ^'I^'hen it was stirred at rt overnight. The reaction mixture was partitioned between 50 ml. of EtOAc and 60 ml. of water. The organic phase was dried over Na^SO Filtration and concentration in vacuo gave the crude product. Purification by column chromatography (50% of EtOAc in petroleum ether as eluent) gave intermediate 6 (0.4 g.72%) as a colorless oil. LCMS (m/z) ES + 157 (M+l )\

|37] To a solution of

rapamycin A I (200 mg. 0.2 mmol) and 4-(2- azido-

ne. Intermediate 6 (100 mg, 0.6 mmol) in anhydrous THF (9 mL) was added DIPEA (100 μ_^.0.6 mmol) and Cul (20 mg, 0.1 mmol) under N₂. The solution was stirred at rt for 3 hours. ^'I^'hen. 20 ml. water was added and extracted with EtOAc (20 mL x 3). The combined organic layer was washed by brine and dried over anhydrous Na;SO After concentration, the residue was purified with silica gel chromatography (30% of EtOAc in petroleum ether as eluent) to give white solid which was further purified by prep-HPLC to give compound A7 (45 mg.20%) as a white solid. Ή NMR (300 MHz. CDCh) δ 7.89 (m. IH).6.72 (m. III).6.44-6.05 (in.511).5.60-5.37 (m.511): I CMS (m/z) ES- I 1 7 (M-l )^".

Example 7: Synthesis of Compound A :

[38] To a solution of 40-O-(prop-2-ynyloxy) rapamycin A I (200 mg. 0.2 mmol) and 2- azidoethanol (100 mg. 1.2 mmol) in anhydrous TIIF (9 mL) was added DIPEA (100 μΕ. 0.6 mmol) and Cul (20 mg.0.1 mmol) under Nk The solution was stirred at rt overnight. Then.20 ml. water was added and extracted with EtOAc (20 mL x 3). The combined organic layer was washed by brine and dried over anhydrous Na S ._t. After concentration, the residue was purified with silica gel chromatography (5% to 10% of methanol in dichloromethane as eluent) to give white solid which was further purified by prep-HPLC to give compound A9 (26 mg, 11%) as a white solid. Ή NMR (300 MHz.. CDCl_.,) δ 8.03-7.78 (m. 1 H).6.70 (m. 1 H), 6.46-6.00 (m.5H). 5.61-5.39 (m.5H): I. CMS (nvz.) LS- 1038 (M-l)\

Example 8: Synthesis of Compound A10:

Preparation oflntermediate 8

|39| A solution of compound 7 ( 1.3 g. 12.7 mmol) in 40% HBr (10 mL) was stirred at rt for 1 hour. Then. 20 mL water was added and extracted with LtOAc (20 mL x 3). The combined organic layer was washed by brine, dried over anhydrous Na:S0₄. After concentration, the residue was purified with silica gel chromatography (50% of LtOAc in petroleum ether as eluent) to give intermediate 8 (0.7 g.31%) as a white solid.

I.CMS (in /) I^'s- 183 (M l I .

Preparation of Intermediate 9

|4()| A solution of intermediate 8 (0.7 g.3.9 mmol) and NaN; ( 1.13 g. 15 mmol) in DMSO (16 mL) was stirred at 80 deg for 2 days. Then.20 ml. water was added and extracted with EtOAc (20 ml. x 3). The combined organic layer was washed by brine, dried over anhydrous Na^SO.;. After concentration, the residue was purified with silica gel chromatography (50% to 100% of LtOAc in petroleum ether as eluent) to give intermediate 9 (0.25 g.45%) as a white solid.

LCMS (m/z) F.S+ 146 (M M)'.

Preparation of Compound A 10

[41| To a solution of

rapamycin A I (200 mg. 0.2 mmol) and 2- (azidomethyl) 2-mcthylpropane- 1.3-diol Intermediate 9 (100 mg.0.7 mmol) in t-BuOH (6 mL) and fLO (6 ml.) was added a^ O;, (100 mg. 1 mmol). CuS0₄ (20 mg.0.13 mmol) and sodium ascorbate (40 mg.0.2 mmol) under NL. The solution was stirred at rt for 6 hours. Then.20 mL water was added and extracted with LtOAc (20 ml. x 3). The combined organic layer was washed by brine and dried over anhydrous Na;SO.₍. A ter concentration, the residue as purified with silica gel chromatography (5% to 10% of methanol in dichloromethane as eluent) to give white solid which was further purified prep- 1 IPl to give compound ΛΙ0 (15 mg.7%) as a white solid. Ή NMK (300 Mil/. CDC; j 67.76 (m. 111).6.69 (m. Ill), 6.55-6.00 (m.511).5.63- 5.33 (m.511). I. CMS (m/z) HS- 1096 (M-l )^".

Example 9: Synthesis of Compound Λ12

Preparation of Intermediate I I

11

10

|42| To a mixture of compound 10 (1 g. 7.8 mmol) in of MeOH/TI IF (10 mL/10 ml) was added a solution ofl.iOI I (0.9 g.39 mmol) in 10 ml. of water. The resulting solution is stirred at room temperature for .3 hours. The mixture was acidified by 2N Ι Ί to PI I 4. and extracted w ith LtOAc (25 ml. 2). The combine organic layer was concentrated under vacuum to give intermediate 1 I (0.7 g. 91%) as a colorless oil. Ή NMR (300 MHz. CDCI₃) δ 2.34 (s,2H): LCMS (m/z) ES+ 102 (M+l)\

Preparation of Compound Λ 12

I -43 J To a solution of 40-O-(prop-2-ynyloxy) rapamycin Λ1 (200 mg. 0.2 mmol) and 2- azidoacetic acid Intermediate I I ( 100 mg. 1 mmol) in t-BuOH (6 ml.) and [TO (6 ml) was added Na^CO;, (100 mg. I mmol). CuS0 (20 mg.0.13 mmol) and sodium ascorbate (40 mg.0.2 mmol) under . The solution was stirred at rt lor 2 hours. Then.20 ml. water was added and extracted ith LtOAc (20 ml. x 3). The combined organic layer was washed by brine and dried over anhydrous Na SO.₍. A ter concentration, the residue was purified with silica gel chromatography (5% to 20° of methanol in dichloromethane as eluent) to give white solid which was further purified by prep-I IPI.C to give compound A 12 (15 mg.7%) as a white solid. Ή NMR (300 MHz. C CI3) 67.89 (m. 1 H).6.72 (m. 1H).6.49-6.08 (m.5H).5.60-5.35 (m.5H): LCMS (m/z) ES- 1052 (M-l )^".

Example 10: Synthesis of Compound ΛΙ3

Preparation of intermediate 13

12

13

[44] To a stirred suspension of NaNR (2.0 g. 30.8 mmol) in acetonitrile (20 ml.) was added TRO (7.3 g.25.8 mmol) b>

slo ly at 0 deg. The mixture was stirred for another 2 h at this temperature. The insoluble solids were removed through nitration. At 0 deg. the filtrate was added dropwise into the mixture of compound 12 (2,0 g. 10 mmol), uSO (l60 mg. I mmol). PRO (6 ml.) and Ft3N (3.6 ml..25.8 mmol). The reaction mixture was stirred for 6 h at room temperature. The mixture was diluted with RtOAc and washed with brine. The organic layer was dried over Na S0₄ and evaporated to yield brown solid which was purified with silica gel chromatography (30% to 50%> of RtOAc in petroleum ether as eluent) to give brown solid which was further purified by prep-IIPI.C to give intermediate 13 (0.4 g. 17%) as a brown solid. Ή NMR (300 Mil/. CDCR) 6 6.94 (m. 411). 3.19 (m. 411). 2.60 (m. 411). 2.36 (s. 311): I. CMS (m /)l S- 218 ( M)'.

Preparation of Compound A 13

[45] To a solution of 40-(⁾-( prop-2-yn_\ lo.xy ) rapamycin A I (200 mg. 0.2 mmol) and l-(4- a/ido- phcnyl)-4-melhylpipera/ine Intermediate 13 (100 mg.0.5 mmol) in l-BuOII (6 ml) and H;0 (6 ml.) was added Na₂CO;, (100 mg. I mmol). CuS04 (20 mg. 0.13 mmol) and sodium ascorbate (40 mg.0.2 mmol) under N₂. The solution was stirred at rt for 3 hours. Then.20 ml water was added and extracted with RtOAc (20 ml. x .3). The combined organic layer was washed by brine and dried over anhydrous Na S0 . Alter concentration, the residue was purified with silica gel chromatography (5% to 20% of methanol in dichloromethane as eluent) to give white solid which was further purified by prep-IIPI.C to give compound A 13 (33 mg. 14%) as a white solid, 'll NMR (300 MM/. CDCR) 8.11 (m. III).7.71 (m.211).7.06 (m.211).6.70 (m. III).6.45-6.00 (m.511).5.66-5.36 (m.511): I. CMS (m/z) RS- I 168 (M-l ) .

Example 11: Synthesis of Compound A14

[4 j To a solution of

rapamycin A I (200 mg.0.2 mmol) and l-(azido- mcthyl)-4-fiuorobeir/cne (100 mg.0.6 mmol) in anhydrous 11 IF (9 inL) was added DIPRA (100 μί,.0.6 mmol) and Cul (20 mg.0.1 mmol) under Nk The solution was stirred at rt for 3 hours. Then.20 ml. water was added and extracted with LtOAc (20 ml. x 3). The combined organic layer was washed by brine and dried over anhydrous aiSOj. After concentration, the residue was purified with silica gel chromatography (30% to 100% of EtOAc in petroleum ether as eluent) to give white solid which was further purified by prep-l IPLC to give compound A 14 (32 mg. 14%) as a white solid. Ή N (300 MHz. CDCP,) δ 7.58 (m. III).7.26 (m.211).7.06 (m. 211).6.75 (m. III).6.50-6.00 (m.511).5.60-5.36 (m.511): I ,CMS(m/z)l^:. S-= 1 I02(M-1).

Example 12: Synthesis of Compound A15

BDPSCI, TTEEAA,, DDMMAAPP DIEA, Tf-,Ο, DCM

.OH TBDPSCI,

HO^' ,OTBDPS OTBDPS

" h HOO^' TfO'

₁₄ DC , rt, overnight rt, overnight ₁₆

Preparation of Intermediate 1

[ 471 To a solution of compound 14 (3 g.48 mmol) and HhN (5 g.50 mmol) in DCM ( 100 ml.) was added DMAP (0.6 g. 5 mmol) and dropwised TBDPS-CI (4.4 g. 16 mmol) at 0 cleg and stirred at rt overnight. Then. 100 ml. water was added and extracted with DCM (80 ml, x 3). The combined organic layer was washed by brine and dried over anhydrous a;S0. After concentration, the residue was purified with silica gel chromatography (30% of LtOAc in petroleum ether as eluent) to give intermediate 15 ( 1.7 g. 12%)) as a colorless oil. Ή NMR (300 MHz. C C I_.,) δ 7.6.3 (m.411).7.36 (m.6H).3.74 (m.2H).3.66 (m.2H). 1.04 (s.9H); LCMS (m/z) ES+ 301 (Μ+Ι .

Preparation oflntermediate 16

[48] To a solution of intermediate 15 (1.7 g. 5.7 mmol) and DIPHA (1.5 g. 11.4 mmol) in DCM (40 ml.) was added ( LO ( 1.7 g.6 mmol) at 0 cleg and stirred at rt overnight. Then.50 ml. water was added and extracted with DCM (40 ml. x 3). The combined organic layer was washed by brine and dried over anhydrous \a-S^{( )}.. After concentration, the residue was purified with silica gel chromatograph^y ( 10% of KtOAc in petroleum ether as eluent) to give intermediate 16 (1.5 g.63%) as a colorless oil. II NMR (300 Mil/.. CIX^'U) δ 7.63 (m.411).7.36 (m.611).4.58 (m.2H).3.95 (m.2ll). 1.04 (s.QII): LCMS (m//.) LS+ 433 (M+l )^'.

Preparation oflntermediate 17

|49| To a solution of Rapamycin (400 mg. 0.43 inmol) and DIPBA (278 mg.2.15 mmol) in toluene (30 ml.) was added intermediate 16 (0.93 g.2.15 mmol) at rt and stirred at 80 deg for 2 hours. Then. 50 ml, water was added and extracted with F.tOAc (30 inL ,\ 3). The combined organic layer was washed by 0.5 N HCI. saturated NaHC0₃ and brine, dried over anhydrous Na;S0₄. After concentration, the residue was purified with silica gel chromatography (25% to 40% of EtOAc in petroleum ether as cluent) to give intermediate 17 (280 mg. 53%)) give as a white solid. I.CMS (m/z) I S- I 1 4 (M-l )^'.

Preparation of Compound A I 5

[50] To a solution of intermediate 17 (280 mg.0.23 mmol) in THF (10 ml..) was added 2 ml. I II in pyridine at 0 deg and stirred at rt for 4 hours. Then.20 ml. water was added and extracted with PtOAc (20 ml. x 3). The combined organic lay cr was washed by 0.5N HCI. saturated NallCO, and brine, dried over anhydrous Na?SO._t. After concentration, the residue was purified with silica gel chromatography (30% to 100 % of !itOAc in petroleum ether as eluent) to give white solid which was further purified by prep-IIPLC to give compound A 15 (34 mg. 1 %) as a white solid. Ή NMR (300 Mil/.. CDC ) 66.40-6.00 (in.511).5.53-5.25 (m.4H).4.83 (s. 111). 4.13 (m. Ill): l.CMS (m//) F.S- 957 (M-l)^".

Synthesis of series ol^'j'apamycin derivatives of the present invention

|51| B series of rapamycin

were prepared according to the following reaction scheme:

^R -- > Ti\⁽⁾ NaNn ^ ^M ^

OTf ₃ 'Click Reaction' ^|i

Rapamycin Conipiuind \i Compound B 1 Series P>

[52| In the formula of [he schemes shown above. R and B^" have the following structures in some examples of the B series of rapamycin derivatives.: HO

-OH

OH

OH

152 B3 B4 155

-N

-C0₂H

/ COpH

156 157 158 159 1510

BH BI2 1514

Example 13: Synthesis of Compound 131

compound B compound B1

I S 1 To a solution of rapann ein (5 g.5.5 mmol) and 2.6-di-terl-but\ l-4-methylpyridine (3.4 g. 3.4 mmol) in dried DCM (I 50 ml.). iritluoromethanesullOnic anhydride (1.55 g.5.5 mmol) was added at 0 oC. After the mixture was stirred for 2 h at room temperature. NaNU (3.6 g.55 mmol) was added and D.MSO (60 ml.) s added at 40 oC. the mixture was stirred for at 40 oC for 5 h. The mixture was quenched

addition of water, extracted with DCM (200 ml. ^x 2). and the combined extracts were washed with water, dried over Na^SO,. and evaporated to dryness in vacuo. The crude product was purified by column chromatography (25% of LTOAc in petroleum ether as cluent) to give compound B ( 1.5 g.30%)) as a white solid. LCMS (m/z) ES- 937 (M-H)^'.

Exam le 14: Synthesis of Compound B2

|54] To a solution of compound Bl (I 50 mg.0.16 mmol) and S I (27 mg.0.48 mmol) in MeOII/ILO (4 ml. '2 ml.) was added vitamine C sodium salt (6.3 mg.0.32 mmol). followed with the addition of CuSO, (5I mg. 0.32 mmol) and a;C()_; (5I mg. 0.48 mmol). After stirred overnight, the mixture was filtered, the filtrate was concentrated and purified by column chromatography (0 to 2% of methanol in dichloromethane as eluent) to give Compound B2 (33.2 mg.21%) as yellow solid. Ί I W1R (300 Mil/. ODCI_.O 67.83 (m. III).6.37-6.01 (m.411).5.40- 5.31 (m.411): I. CMS (m/x) |-S- 993 (M-l I) .

Example 15: Synthesis of Compound B3

salts

|55| ΊΌ a solution of compound Bl (I 50 mg.0.1 mmol) and SMI (40 mg. 0.48 mmol) in MeOII/ll-O (4 ml./2 ml.) was added vitamine C sodium salt (63 mg.0.32 mmol) followed with the addition of CuS0. (5 I mg. 0.32 mmol) and a^ O, (5 I mg. 0.48 mmol). After stirred overnight, the mixture was filtered, the filtrate was concentrated and was purified by column chromatography (0 to 2% of methanol in dichloromethane as eluent) to give Compound B3 (20.7 mg. I3%> as white solid. ^! 11 NM (300 Ml I/. CDCh) 8.55 (s. 111).7.89 (m. III).6.39-6.02 (m. 4II).5.46-4.83 (m.4II): I CMS (m/z) f-S- 1020 (M-l I) .

Example 16: Synthesis of Compound B4

S 1

|56| To a solution of l (2 g.32.8 mmol) in DOM ( I00 ml.) was added SM l (2 g. I6.4 mmol) at 0 oC dropwise over I hour. I lie mixture was concentrated and the residue was purified

column chromatography (0 to 2% of methanol in dichloromethane as eluent) to give intermediate 2 (0.9 g.54%) as yellow oil. I.CMS (m/ ) HS 100 (M i II)^'.

|57| To a solution of compound Bl (150 mg. 0.16 mmol) and intermediate 2 (48 mg. 0.48 mmol) in MeOII/I ) (4 ml.2 ml.) was added vitamine C sodium salt (63 mg. 0.32 mmol) followed with the addition of C11SO (51 mg. 0.32 mmol) and Na^CO^ (51 mg. 0.48 mmol). Alter stirred overnight, the mixture was filtered, the filtrate was concentrated and purified by column chromatography' (0 to 2% of methanol in dichloromethane as eluent) to give Compound 1*4 (17.1 mg. 11%) as white solid. Ή NMR (300 Mil/.01)01_.008.40 (s. 111).6.39-6.02 (in.411). 5.37-4.94 (m.411): I .CMS (m./) KS- 1036 (M-l I) . Example 17: Synthesis of Compound B5

SM1

|58] To a solution of l (2 g. 19.0 mmol) in I MF (40 ml.) was added K ^'O (5.2 g.38 mmol) and SMI (2.2 g. I 9 mmol) at 0°C. Alter stirred for 4 h at r.t. the mixture was filtered, the filtrate was concentrated and purified by column chromatography (0 to 3% of methanol in diehloromethane as eluent) to give intermediate 2 (1.2 g.44%) as yellow oil. Ή NMR (300 MHz. CDCh) δ 3.65 (m.4I I)..3.49 (m.2I I).2.75 (m.4H).2.22 (m. 111).

|59| To a solution of compound Bl (I50 mg. 0.16 mmol) and intermediate 2 (69 mg. 0.48 mmol) in MeOH/H₂0 (4 ml./2 ml.) was added vitamine C sodium salt (63 mg. 0.32 mmol) followed w ith the addition of CuSCTi (5 I mg. 0.32 mmol) and Na₂CO_.i (51 mg. 0.48 mmol). After stirred overnight, the mixture was tlltered. the llltrate was concentrated and purilled by column chromatography (0 to 2% of methanol in diehloromethane as eluent) to give Compound B5 (26.7 mg. I 1%) as white solid, 'l l NMR (300 MHz. ΠΧΊ3) δ 7.92 (s. 111). 6.39-5.99 (m. 411).5.45-4.84 (m.411); 1 CMS (m.'z) 1 IS- 1080 (M-l If.

Example 18: Synthesis of Compound B6

S 1 1 2 compound B6

|6()| To a solution of I (0.5 g. 5.8 mmol) in I I If (40 ml.) was added _:CO_:, ( 1.6 g. I 1.6 mmol) and S I (0.69 g. 5.8 mmol) at 0 oC. After stirred for 4 h at r.t. the mixture was filtered, the llltrate was concentrated and purified by column chromatography (3% of methanol in diehloromethane as eluent) to give 2 (0.3 g.42%) as yellow oil. I.CMS (m/z) I-S+ 125 (M+Hf . |61] To a solution of compound Bl ( 150 mg. 0.I6 mmol) and 2 (60 mg. 0.48 mmol) in MeOII/HT) (4 ml /2 ml.) was added vitamine (^' sodium salt (63 mg. 0.32 mmol) followed with the addition of uSO, (5 I mg. 0.32 mmol) and : O, ( I mg- 0.48 mmol). After stirred overnight, the mixture was filtered, the llltrate was concentrated and purified by column chromatography (0 to 2% of methanol in diehloromethane as eluent) to give Compound B6 (25.8 mg. 15%) as white solid. Ή NMR (300 MHz. CDCb,) ό 7.81 (s. 111).6.40-6.02 (m, 411).5.45- 4.81 (m.4ll): I. CMS (m/z) CS- 1062 (M-ll)^'.

Example 19: Synthesis of Compound B7

compound B7

|62| To a solution of l (0.5 g.5 mmol) in Till (40 ml.) was added Κ._:(Ό¾ ( 1.4 g. I0 mmol) and SMI (0.6 g.5 mmol) al 0°C. After stirred for 4 h at r.t. the mixture was filtered, the filtrate was coneentrated and purified by column chromatography (3% of methanol in dichloromethane as eluent) to give intermediate 2 (0.5 g.72"») as \ el low oil. I .CMS (m/z) i.S · 139 (M+H)^'.

[63| To a solution of compound Bl ( 150 mg. 0.16 mmol) and 2 (60 mg. 0.48 mmol) in Me011/ll:() (4 mI./2 ml.) was added vitamine C sodium salt (63 mg.0.32 mmol) followed with the addition of CuS0 (51 mg. 0.32 mmol) and Na^CO;, (51 mg. 0.48 mmol). After stirred overnight, the mixture was filtered, the filtrate was concentrated and purified by column chromatography (2% of methanol in dichloromethane as eluent) to give Compound B7 (12 mg. 7%) as white solid. Ί 1 NMR (300 MHz. CDCU) δ 8.39 (s. 111).7.76 (s. 111).6.37-6.01 (m.411). 5.41-4.78 (m.411): I. CMS (m'z) l!S- 1075 (M-l l) .

SM1

[64 j To a solution of compound Bl (150 mg.0.16 mmol) and SMI (50 mg.0.48 mmol) in Me011/ll_:0 (4 mL/2 ml.) was added vitamine C sodium salt (63 mg.0.32 mmol) followed with the addition of CuSO.j (51 mg. 0.32 mmol) and \a.⁽. O. (51 mg. 0.48 mmol). After stirred overnight, the mixture was filtered, the filtrate was concentrated and purified by column chromatography (2% of methanol in dichloromelhane as eluent) to give Compound [39 (37.1 mg. 22%) as white solid. Ή NMR (300 MHz. CDCI3)89.37-7.89 (m.511).6.39-6.01 (m.411).5.42- 4,99 (m.411): I .CMS (m/z.) LS- 1040 (M-ll)^".

Exam le 21: Synthesis of Compound HI 1

Step 1

[65 j To a solution of SMI (2.1 g. I0 mmol) in Dioxane (20 ml.) was added

(730 mg. I mmol) under Nk then LhN (10 g. 100 mmol) was added dropwise. After stirred at 100 oC overnight, the mixture was quenched by water, extracted with LtOAc (50 ml., x 2). The combine organic layer was dried over anhydrous Na:$0₄. concentrated to give crude intermediate I .

|66| The crude intermediate I was dissolved treated with TBAF in THF (20 ml...20 mmol) at r.t. for 2 hours, then quenched by water and extracted with LtOAc (50 ml. x 2). The combine organic laser as dried over anhxdrous a S0. concentrated to give crude intermediate 2 which was purified by column chromatography to give 2 (0.9 g.58%) as yellow solid. Ή NMR (300 MHz. DMSO-d6)o 7.96 (d.2II).7.62 (d.21 i).4.48 (s. III).3.87 (s.311).

|67| To a solution of2 (0.5 g.3.13 mmol) in MeOH (10 ml.) was added LiOH (0.312 . 12.52 mmol) in water (10 ml.). The mixture was stirred for 2 h. then quenched by NCI solution (2N). extracted with LtOAc (30 ml.*3). The combined organic was dried over anhydrous Na;SO.|. concentrated to give the desired intermediate 3 (0.35 g.77%) as yellow solid.

|68| To a solution of compound Bl (150 mg. 0.16 mmol) and intermediate 3 (70 mg. 0.48 mmol) in MeOI l/fFO (6 ml./3 ml.) was added vitamine C sodium salt (63 mg. 0.32 mmol) followed with the addition of OiSO., (51 mg. 0.32 mmol) and

(51 mg. 0.48 mmol). After stirred overnight, the mixture was adjusted to pi I about 3-4. filtered, the filtrate was concentrated and purified by column chromatography (1.5% of methanol in dichloromethane as eluent) to give Compound Bl I (15.5 mg.9%) as white solid. Ή NMR (300 MHz. CDCI ) 8.I (m.3H).7.98 (d.211).6.77-6.1 I (m.411).5.49-4. 1 (m.411): I. CMS (m/z) KS- 108.3 (M-H)^".

Example 22: Synthesis of Compound B12

SM1 Step 2

|6 | To a solution of SM I (4 g.23 mmol) in propan-2-ol (20 ml,) was added NaOH (2.8 g.69 mmol) in water and 3-biomoprop- 1 -ync (2.4 g. 20 mmol). After stirred lor 4li at 70 °C. the mixture was concentrated, tillered, the filter cake was washed by water, dried to give intermediate 1 (2 g..37%) as

solid. I. CMS (m/z) HS 234 (M+Na)^'.

|7 | To a solution of intermediate I ( I g. 4.3 mmol) in DMF (8 ml.) was added oxalyl dichloride (I.I g.8.6 mmol) in IX^'M (4 ml.) dropwise at 0°C. After stirred overnight at r.t. the mixture was quenched by water, extracted with DCM (30 ml. x 3). The combined organic was dried over anhydrous Na:S()4. concentrated, purified by column chromatography (0 to 10% of F.tOAc in petroleum ether as eluent) to give the desired intermediate 2 (0.35 g.77%) as yellow solid.

Intermediate 2 (0.35 g. 1.5 mmol) was added into ammonia water (5 ml.), the mixture was stirred for I h al r.t.. quenched by addition of water, extracted with ITOAc (20 ml. x 2). The combined organic was dried over anlndrous Na;S().|. concentrated, purilled by column chromatography (0 to 50% of HtOAc in petroleum ether as eluent) to give intermediate 3 (0.2 g.20% for two steps) as yello solid.¹11 NMR (300 MHz. MS()-d6) ό 7.77 (d.211).7.24 (s.211).7.14 (d.211).4.90 (d.211).3.63 (m. III).

|71| To a solution of compound B! ( 1 0 mg.0.16 mmol) and intermediate 3 (101 mg.0.48 mmol) in VleOl ll ⁽) (6 ml.3 ml,) was added viiaminc C sodium salt (6.3 mn. 0.32 mmol) followed with the addition of C^'uSO.i (51 mg. 0.32 mmol) and a^C (51 mg. 0.48 mmol). After stirred overnight, the mixture was liltered. and the filtrate was concentrated and purified by column chromatography (0 to 2.5% of methanol in dichloromethane as eluent) to give Compound B 12 (13.4 mg.7.3%) as white solid. ' H M R (300 MHz. CDCI₃)67.88 (m.311).7.12 (m.211).6.39-6.01 (m.4H).5.42-4.63 (m.4H): I.CMS (m/z) ES- 1148 (M-H)^".

Example 23: Synthesis of Compound B13

Step 1 1

SM1

compound B13

[72] To a solution of S I (2.3 g. 10 mmol) in dioxane (20 mL) was added ethynyltrimethylsilane (2 g. 20 mmol). Cul (191 mg. I mmol) and Pd(PPh3);CN (730 mg. 1 mmol) under \ . then EhN ( 10 g. 100 mmol) was added dropwise. After stirred overnight at 100 °C. the mixture was quenched b\ addition of water, the mixture was extracted with HtOAc (50 ml. x 2). The combine organic layer was dried over anhydrous

concentrated to give crude intermediate I .

[73) fhe crude intermediate 1 was treated with THAI-^' in TUP (20 ml..20 mmol) and stirred for 2h at r.t.. then quenched by water and extracted with KtOAc (50 ml. x 2). fhe combine organic laver was dried over anhydrous N'a SO|. concentrated to give crude intermediate 2. which was purified by column chromatography to give pure intermediate 2 (1.5 g. 84%) as Ncllow solid. Ή NMR (300 MHz. DMSO-d6) δ 7.82 (d.211).7.68 (d.2H).7.46 (s.2H).4.45 (s.

[741 To a solution of compound Bl (150 mg. 0.16 mmol) and intermediate 2 (87 mg. 0.48 mmol) in MeOI l/I \₂ (6 ml./3 ml.) was added vitamine C sodium salt (6.3 mg. 0.32 mmol) followed with the addition of CuS0 (51 mg.0.32 mmol). Na;CO¾ (51 mg.0.48 mmol). After stirred at r.t. overnight, the mixture was liltered. The llltrate was concentrated and purilled b\ column chromatograph) (f) to 1.5% of methanol in dichloromethane as eluent) to give Compound I3 (48.7 mg.27%) as white solid. ^!ll NMR (300 Mil/. (ΊΧ.Ί,) 8.21 (m. I II).7.93 (m, 411).6.37-6.00 (m.411).5.44-5.30 (m.511). I. CMS (m/z) I^'.S- I I 18 (M-l I)^'.

Ex mple 24: Synthesis of Compound B14

|7Sj To a solution of S I (2.3 g. 20 mmol) in l/tOII (20 ml.) was added aOI I (1.6 g. 40 mmol) in water, then 3-bromoprop- 1 -yne (2.4 g. 20 mmol) was added at 0°C. After stirred for 4h at r.l. the mixture was quenched by I Id solution (2N) and pi I was adjusted lo 3-4. extracted with 1/tOAc (50 ml.*5). I he combined organic was dried over anhydrous Na₂SO.|. concentrated, purified column chromatograph) (0-3% of methanol in dichloromethane as eluent) to give intermediate 1 ( 1.2 g.40%) as colorless crystal. I CMS ϋη /' I S I54 (M I I)^'.

|76) To a solution of compound Bl ( 150 mg. 0.16 mmol) and intermediate 1 (73 mg. 0.48 mmol) in MeOI I/ICO (4 ml. -2 ml.) was added vitamine C sodium salt (63 mg. 0.32 mmol) followed with the addition of CuSO., (5lmg.0.32 mmol) and Na_.-CO, (51 mg.0.48 mmol). After stirred overnight, the mixture was filtered, the filtrate was concentrated and purified by column chromatography (2% of methanol in dichloromethane as cluent) to give Compound BI4 (12.6 mg. 7.2%) as white solid. Ή NMR (300 MHz. CDCh) δ 8.17 (m. 114).6.39-5.99 (m.411).5.56- 4.87 (m.411): I CMS (m 7) l/S- 1090 (M-l I) .

I n/ymalic activities of

analogs of the present invention

|77| I^'he m TOR is a serine/threonine protein kinase that has been shown to regulate multiple cellular responses including cell growth, proliferation, motility, survival and protein synthesis, m TOR kinase activity is regulated by several upstream signaling pathwa s and its dysreg lation has been implicated in several forms of cancer. Now we use a Terbium labeled ant i- phosphorv lated 4I/-BPI antibodv to detect phosphorylation of the Of ['-labeled substrate b\ m TOR. This Κ-Ι RITI based assay can be used to screen inhibitors of mTOR in vitro.

|78| Materials: Assay buffer components: I 111 PI. S pi 17.5. GIBCO. Cat// 15630 : IM MgCk Sigma. Cut// M l 028: 0.5M 1/1) I A. GIBCO. Cat;; 15575: DTI. Sigma Cat// 43819: I ( 1 Λ. Sigma Cat// F.3889; Triton XI 00. Sigma. Cat// Ί 8787: BSA CAFBIOCPIF. I Cat/f 126575.

179 ] Fnzyme. substrate and detection reagents: mTOR: Invitrogen. Cat// PV4753: GFP-4H- BP I : Invitrogen. Cat// PV4759: F BPI2: SinoBiologicak Cat// I0268-H08F: ATP: Sigma Cat// A26209: Tb-anti-p4P:-BP 1 : Invitrogen. Cat// PV4755: TR-FRFT Dilution buffer Invitrogen. Cat# PV3574.

[ 80 j Plate: Compounds preparation plate: 384-well. Corning cat/ 3657: Assa\ plate: black low volume microliter plate (Creiner Bio-One. Cat// 784076).

|81| Procedure: Compounds dosage gradient solution preparation:

Compounds were 3-fold serial diluted in 1 0% DMSO in a microliter plate (Corning 3674) at I I different concentrations in the range of lOOu.M to 1.7nM ( 100 μ\1.33 μ,Μ. 1 ΙμΜ.3.7μΜ.

Ι.2μΜ.4Ι Ι ιιΜ. 137 ηΜ.46 ηΜ. 15 ηΜ.5 ηΜ. 1.7 ηΜ). Then the diluted compounds in 100%

DMSO was 10-fold diluted with ddH20. so the compounds were in 10% DMSO.

|82| A typical assay protocol of measuring the mTOR inhibitory ability of the rapamvcin derivatives of the invention is as follows:

Assay protocol:

0.5 μ| diluted compounds in 10% DMSO was pipetted into a black low volume 384well microliter plate (Greiner Bio-One. Frickenhausen. Germany, cat/' 784076):

2μΙ of a solution of mTOR in aqueous assa> buffer |50 mVI I II PFS'NaOI I pi I 7.5.5 niM MgCI2. 1.0 mM dilhiothreitol. I mM P.GTA.0.01% (v/v) Triton-X 100 (Sigma).0.01 % (w/v| bovine serum albumine (BSA)| (mTOR.0. 1 5 ng i.l > final cone, in the 5 μ| assay volume is 0.125 ng μΙ) were added to the assay plate and the compound-enzyme mixture was incubated for 15 ιηίη at 22^JC to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction:

|83] The kinase reaction was started by the addition of 2.5 μΙ of a solution of ATP (ATP.200 μΜ > inal cone, in the 5 μΙ

volume is 10 μΜ) and substrate (0.8μΜ > final cone, in the 5 μΙ assa> volume is 0.4 μΜ) in assay buffer and the resulting mixture was incubated for 18 min at 22°C.

[84 ) ^'fhc reaction was stopped by the addition of 5ul of 30mM FDTA (HDTA. 30 mM ^r > final cone, in the 10 μΙ assa_\ volume is I m\1) and 4nM Tb-chelate labeled anti-4F.-BPl [pT46| phosphospecitic antibod

Cat- PV4755| (^'Fb-labelecl antibod .4 nM - > final cone. in the 10 μΙ assay volume is 2 ii ) in TR-I RLf dilution buffer, the resulting mixture was incubated I hour at 22°C to allow the formation of complex of the phosphor) lated substrate and the Tb-chelate labeled antibody.

|85| I he amount of phosphor) lated substrate was evaluated

measurement of the resonance energy transfer from the Tb-chelate to the (II P. Therefore, the fluorescence emission at 495 nm and 520 nm after excitation at 340 nm was measured on envision 2104 multilabel reader (Perkin- Llmer). The ratio of the emission at 520 nm and at 495 nm was taken as the measure for the amount of phosphorylated substrate. The data were normalised (enzyme reaction without inhibitor 0 % inhibition, all other assay components but no enzyme - 100 % inhibition) and I 50 values were calculated by a 4 parameter lit (equation ( I )) using IDBS XLlit software (ID Business Solutions

)

Y-Bottom r (T()P-Botlom)/( I ί IO^A((LoglC50-X)*hillslope)) equation ( 1 )

|86] In this equation. Y was the normalized "oinhibition value. X was the log value of the test compound concentration, i 50 was the concentration of compound where half of maximal inhibition was achieved.

|87j I he testing results of m IO inhibitor) effects of the rapamycin analogs/derivatives of the invention are shown below:

Compound ID : IC50(nM) ( ^'impound I IC50(n

-i -

Rapam) cin 5.09

Compound Λ 15 6.43 B2 18.43

Compound A 4 ^' 6604.427021 B3 15.40

Compound A7 ^* > 10000 B4 28.52

Compound A 10 ^' 10000 B5 19.38 ^"

Compound A5 ; ■^■ 10000 B(> 12.96

Compound A6 19143.20 ^~'~ 7 ^~23^~7 ^"'

Compound A3 I > 10000 B9 27.16

... . .

Compound A 1 ^: > 10000 Bl 1 446.00 Compound A 12 > 10000 B12 35.19

Compound Λ 14 > 10000 BI3 29.03

Compound Λ9 > I 0000 B14 6.80

Tumor coll inhibition studios:

Cell proliferation assay

[88 j The effect of different compounds on the cellular activities was quantitated through determining the number of living cells in a culture by a homogeneous detection method for quantitative determination of cell viability by the CellTiter-Glo ¾ chemiluminesccnce detection kit for A I P. ΛΤΡ is an indicator of the metabolism of living cells. Homogeneous detection step is added directly to the single reagent (CellTiter-Glo ® Reagent) in serum-containing cultured cells, without washing the cells or removing the medium. After adding reagent and mixing in a 96-well or 384-well plates, the number ol^' cells that can be quantified hv the system within 10 minutes, is as low as 15 cells in each well,

Preparation of the reagents

|8')| Different cell types were cultured using a medium, containing 10% I BS plus 1% penicillin streptomycin double antibiotics, and the following appropriate additives: I ) \ 11 Λ I medium (Ciibco. Item No. I 1995073) for culturing colorectal cancer cells I ICTI 16. breast cancer cells MCF-7 and MDA- B-23 I melanoma cells SK-MEL-28. A549 and epidermal squamous cell carcinoma cell A43 I : RPMI-1640 medium (containing 2 mM L-glutam^'mc. 1.5 g / I. sodium bicarbonate.4.5 g / 1. glucose. 10 mM HP^'.PI-S. 1.0 mM sodium pyruvate ( Item it 72400-120 from Ciibco) for culturing U87/MG and kidney cancer786-0: P-I2K mixed medium (Item «21127 from Ciibco) for culturing prostate cancer cell line PC-3.

Instrumentation

Multi-label Micro-plate Reader I^'nvison 14 from Perk i n I^'lmer

Cell Culture conditions

|90| All 9 cell lines were cultured in the wells in the plates, at a cell density of 3000 cells/well after 9 passages.

Preparation of culture media and cell culture conditions: [91] Prepare the compounds and condition the cells the next day. each chemical compound to be assaved was diluted to 10 mM stock solution with 100% DMSO. followed by additional dilution with 1 0% DMSO diluted to 2 mM. followed

serial 5X dilution using serum-free cell culture medium to a final 10 different diluted concentrations points (2000.400.80. 16.3.2.0.64. 0.128.0.0256.0.00512.0.00102 μΜ). plus 0.5% DMSO (no compound) as a maximum control and ΙΟμΜ Rapamycin as a minimum control. A solution of 0.5μΙ of each diluted compound is added to the ΙΟΟμΙ of cell culture plate, the final compound concentration of 10 points ( 10.2.0.4. 0.08. 0.016. 0.0032. 0.00064. 0.000128. 0.0000256. .00000512 μΜ). The cells were then cultured in 37 "C incubator for 72 hours. In order to ensure the reliability of the experiments of determining the inhibition of each compound, a duplicate was used for each compound concentration gradient will do two repeated ( fable 1 ). and the determination of each compound as repeated tw ice.

Plate reading

|92| After 72-hr of cell culture.50 ul of Cell^'fiter Cilo was added to each well on the plate, and shaken for 5 min on a shaker followed by 10 mm at room temperature. The cell number was analyzed by the Micro-Plate reader.

Data analysis:

|93| Cell viability was obtained through the reading by the multi-label micro-plate reader. The effect of each dilution value on the % cell viabilitv was calculated using the follo ing fotnula: % cell inhibition- 1 0-1 0^"- (Signal-low control ) ( I ligli control-low control, in which signal . low control, and high control are the test compound, minimal value, and maximal value respectively. |94| The IC50

of each test compound in inhibiting the cells is obtained by formula 2 (below ):

Y Bottom * (T P-Bottom)4 I ¹ (( IC50/X Lhillslope)). in which X and y are known values. IC50. Ilillslope. fop and Bottom 4 parameters generated by the analysis software. Y as the % inhibition. X as the test compound concentration, and IC50 as the concentration of the compound needed to inhbit 50% of the cells. Ilillslope is the slope of curve fitting, usually around 1.

|95| All experimental data were analyzed bv IDBS X141l5 (ID Business Solutions Ltd.. UK).

Experimental results and conclusion

|96| All the potency of each lest compound is shown in one of the following graphs for each of the cancer cell models tested. The lower the curve in the graph, the more potent each compound is. from the data shown in each of the graphs, it is clear that all the series β compounds showed varying levels of high potency against the cancer cell tested. Some of the B series compounds were extremely potent, reaching a potency level of nM concentration range. Renal cell carcinoma tumor cell inhibition studies:

Renal cell carcinoma tumor cell inhibition studies: Figl and Fig2.

Lun Cancer A549 cell inhibition studies: Fig3, Fig4 and Fig5.

Melanoma SK-MFI -28 cell inhibition studies: Fig6, Fig7 and Fig8.

Fpidermal cancer Λ431 tumor cell model: Fig9, Fig 10 and Figl 1.

Glioblastoma U87 IY1G Tumor model studies: Figl2, Figl3 and Figl4.

Human colorectal tumor HCT 116 model studies: FiglS, Fig 1 and Fig 17.

Breast cancer ΜΟΛ- Β-231 tumor model: Figl8, Figl9 and Fig20.

Breast cancer IVICF-7 tumor model: Fig21, Fig22 and Fig23.

Prostate cancer C-3 tumor studies: Fig24, Fig25 and Fig26.

Fft^'icacv studies of rapamycin derivatives in Human Colon Tumor (IICTl 16) model

|97| Purpose: I lie object i

is to evaluate preclinical!) the iii vivo therapeutic efficac) of Λ 1 (positive control) and a lead compound from the B series administrated as per os (p.o.) in the slowing or eliminating tumor development in subcutaneous li(^'T-l 16 human colon cancer model.

[98] Animals: Balb/c nude mice, female.6-8 weeks, weighing approximately 18-20». A total of 70 will be needed for the study, which will be purchased from Vital River Laboratory Animal Technology Co.1.id.

|99| Tumor Inoculation: Lach mouse will be inoculated subcutaneous!) at the right Hank with IK^' f-l 16 tumor cells (3x106) in 0.1 ml of PBS for tumor development. The treatments will be started when the tumor size reaches approximately -150 miii.l The test article administration and the animal numbers in each group are shown in the follow ing experiment design table.

Groups and Treatments

Dose 1 )osing 1 )osing

Group n Treatment Schedule

(mg kg) Route volume

1 10 Vehicle - p.o. 1 u! g Ol) X 1

2 10 A 15 9 p.o. ΙΟ μΙ/g QD x 21 10 B p.o. 10 μΐ/g 01) x 1

4 10 B 9 p.o. 10 μΙ/g QD x 21

5 10 B 18 p.o. 10 μΙ/g QD x 21

Note: n: animal number: Dosing volume: adjust dosing volume based on bod weight 10 μΙ/g). Treatment schedule may be adjusted ifbodv weight loss > 15%.

11 0] Assignment to Groups: Before commencement of treatment, all animals will be weighed and the tumor volumes will be measured. Since the tumor volume can affect the effectiveness of any given treatment, mice will be assigned into groups using randomized block design based upon their tumor volumes, This ensures that all the groups arc comparable at the baseline.

[1011 l-ndpoints: The major endpoinl is to see if the tumor growth can be delayed or mice can be cured. Tumor sizes will be measured t ice w ekly in two dimensions using a caliper, and the volume will be expressed in mm3 using the formula: V - 0.5 a x b2 where a and b are the long and short diameters of the tumor, respectively. The tumor sizes are then used for the calculations of both T-C and T/C values. f-C is calculated with T as the median time (in days) required for the treatment group tumors to reach a predetermined size (e.g.. 500 mm3). and C is the median time (in days) for the control group tumors to reach the same size. The /C value (in percent) is an indication of antitumor effectiveness. T and C are the mean volume of the treated and control groups, respectively, on a given day. Tumor tissues will be collected for the tumor weight and photo at the end of (he si Lid \ .

|102| Termination: This studv will be terminated when the mean tumor size of the control group reach the volume of 600- 1000 min3. Animals that arc observed to be in a continuing deteriorating condition will be euthanized prior to death, or before reaching a comatose state. Animals showing obvious signs of severe distress and/or pain should be humanely sacrificed. In case of following situations, the animals will be euthanized:

[103| Animals have lost significant body mass (emaciated). Obvious body weight loss > 20%:

[104| Animals cannot get to adequate food or water.

|1()5| The study will be terminated with all animals in all groups being sacrificed when the mean tumor burden in the vehicle treated control group reaches a value of 2000 mm3.

|106| Statistical Analysis: I or comparison between two groups, an independent sample t-test will be used, lor comparison among three or more groups, a onc-wav ANOVA will be performed. If a significant I -statistics (a ratio of treatment variance to the error variance) is obtained, multiple comparison procedures will be applied after ANOVA. The potential synergistic effect between treatments will be analyzed by LSI) or Dunnetfs T3. All data will be anal\ zed using SPSS I 7.0 software, p^■ 0.05 is considered to be statistically .significant.

|1()7| Summary: As shown in the following figure, after 22 days of treatment, the 9 mg/kg/day of A 15 positive control compound (Afinitor from Novartis) significantly inhibited tumor growth by 63% in SubQ HCTI 16 resistant colon cancer xenograft model(* P<0.05). which is consistent with the reports from literature. After 22 days treatment. B compound suppressed the tumor grow th by 42%.57%) and 64%, (**. P<0.01 ). at 3.9. and 18 mg/kg/day. respectively, compared to the vehicle control. No obvious toxicity was observed. These data indicate that B compound is \ery potent in \ ivo and may overcome the colon cancer resistance.

Fig 27. In this figure, the lop line (diamond) is for Vehicle, second line (triangle) is for B7 at 3 mg/kg dose: third line (purple cross) represents B7 at 9gm kg dose: fourth line from the top (pink squared represent Afinitor at 9 mg/kg dose), the bottom line (blue cross) represents B7 a I 8 mg kg dose.

Methods of Treatment

|1()8| The compounds of the present invention, including but not limited to those specified in the examples, possess immunomodulatory and anti-tumor activity in mammals (especially humans). As immunosuppressants, the compounds of the present invention are useful for the treatment and prevention of immune-mediated diseases such as the resistance by transplantation of organs or tissue such as heart, kidney, liver, medulla ossium. skin, cornea, lung, pancreas, intestinum tonne, limb, muscle, nerves, duodenum, small-bowel, pancrcatic-islet-cell. and the like:

diseases brought about by medulla ossium transplantation: autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus. Hashimoto's th oiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, allergic encephalomyelitis, glomerulonephritis, and the like, further uses include the treatment and prophylaxis of inilammatory and by perproliferative skin diseases and cutaneous manifestations of immunologically-mediated illnesses, such as psoriasis, atopic dermatitis, contact dermatitis and further eczematous dermatitises, is scborrhocis dermatitis, lichen planus, pemphigus, bullous pemphigoid, epidermolysis bullosa, urticaria, angioedemas. vasculitides. erythemas, cutaneous eosinophijias. lupus erythematosus, acne and alopecia areata: various eye diseases (autoimmune and otherwise) such as keratoconjunctivitis, vernal conjunctivitis, uveitis associated with Behcet's disease, keratitis, herpetic keratitis, conical cornea, dystrophia epithelialts corneae. corneal leukoma, and ocular pemphigus. In addition, reversible obstructive airway disease, which includes conditions such as asthma (for example, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma), particularly chronic or inveterate asthma (for example, late asthma and airway hyper-responsiveness), bronchitis, allergic rhinitis, and the like are targeted by compounds of the present invention. Inllammalion of mucosa and blood vessels such as gastric ulcers, vascular damage caused by ischemic diseases and thrombosis. Moreover.

lerativ e vascular diseases such as intimal smooth muscle cell hyperplasia, restenosis and vascular occlusion, particularly following biologically- or mechanically-mediated vascular injury, may be treated or prevented by the compounds of the present invention. Other treatable conditions include ischemic bowel diseases, inflammatory bowel diseases, necrotizing enterocolitis, intestinal inflammations/allergies such as Coeliac diseases, proctitis, eosinophilic gastroenteritis, mastocytosis. Crohn's disease and ulcerative colitis: nervous diseases such as multiple myositis. (iuillain-Barre syndrome. Meniere's disease, polyneuritis, multiple neuritis, mononeuritis and radiculopathy: endocrine diseases such as hyperthyroidism and Basedow's disease: hematic diseases such as pure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia and anerythroplasia: bone diseases such as osteoporosis: respiratory diseases such as sarcoidosis, fibroid lung and idiopathic interstitial pneumonia: skin disease such as dermalomyositis. leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity and cutaneous T cell lymphoma: circulatory diseases such as arteriosclerosis, atherosclerosis, aortitis syndrome, polyarteritis nodosa and myocardosis: collagen diseases such as scleroderma. Wegener's granuloma and Sjogren's syndrome: adiposis: eosinophilic fasciitis: periodontal disease such as lesions of gingiv a, periodontium, alveolar bone and substantia ossea dentis: nephrotic syndrome such as glomerulonephritis: male pattern aleopecia or alopecia senilis by preventing epilation or prov iding hair germination and'or promoting hair generation and hair growth: muscular dystrophy: Pyoderma and Sevan^'s syndrome: Addison's disease: active oxygen-mediated diseases, as for example organ injury such as ischemia-reperl^'usion injury of organs (such as heart, liver, kidney and digestive tract) which occurs upon pres rvation, transplantation or ischemic disease (lor example, thrombosis and cardiac infarction): intestinal diseases such as endotoxin-shock. pseudomembranous colitis and colitis caused by drug or radiation: renal diseases such as ischemic acute renal insufficiency and chronic renal insufficiency: pulmonary diseases such as toxinosis caused by lung-oxygen or drug (for example, paracort and bleomycins), lung cancer and pulmonary emphysema: ocular diseases such as cataracta. sidcrosis. retinitis, pigmentosa, senile macular degeneration, vitreal scarring and corneal alkali burn: dermatitis such as erythema multiforme, linear IgA ballous dermatitis and cement dermatitis: and others such as gingivitis, periodontitis, sepsis, pancreatitis, diseases caused b_\ environmental pollution (for example, air pollution), aging, carcinogenesis, metastasis of carcinoma and hypobaropalhy : diseases caused by histamine or lcukotricne-C^'.siib.4 release: Behcet's disease such as intestinal-, vasculo- or neuro-Behcet's disease, and also Behcet's which affects the oral cav ity, skin. eye. vulva, articulation, epididymis, lung, kidney and so on. furthermore, the compounds of the present invention may be useful for the treatment and prevention of hepatic disease such as immunogenic diseases (for example, chronic autoimmune liver diseases such as autoimmnune hepatitis, primary biliary cirrhosis and sclerosing cholangitis), partial liver resection, acute liver necrosis (e.g. necrosis caused by toxin, viral hepatitis, shock or anoxia). B-viriis hepatitis. non-A/non-B hepatitis, cirrhosis (such as alcoholic cirrhosis) and hepatic failure such as fulminant hepatic failure, late-onset hepatic failure and ^"acute-on-chronic^" liver failure (acute liver failure on chronic liver diseases), and moreover are useful for various diseases because of their useful activity such as augmention of chemotherapeutic effect, cytomegalovirus infection, particularly I IC V infection, antiinflammatory activity, sclerosing and llbrotic diseases such as nephrosis, scleroderma, pulmonary fibrosis, arteriosclerosis, congestive heart failure, ventricular hypertrophy, postsurgical adhesions and scarring, stroke, myocardial infarction and injur associated with ischemia and rcperfusion. and the like.

|1()9| Additionally, compounds of the present invention possess Γ -506 antagonistic properties. The compounds of the prcsenl invention may thus be used in the treatment of immunodepression or a disorder involving immunodepression. Rxamplcs of disorders involving immunodepression include AIDS, cancer, fungal infections, senile dementia, trauma (including wound healing, surgery and shock) chronic bacterial infection, and certain central nervous system disorders. The immunodepression to be treated ma be caused by an overdose of an immunosuppressive macroe die compound, for example derivatives ol^' 12-(2-cyclohcxyl- 1 -methyl vinyl )- 1 .19.21 ,27-tetramethyl- 11.28-dioxa4-azatricyclo|22.3.1.0.sup.4.9|octacos- 18-cnc such as \^' - 506 or rapamvein. The overdosing of such medicants by patients is quite common upon their realizing that the) have forgotten to take their medication at the prescribed time and may lead to serious side effects.

I he compounds of the present invention, including but not limited to those specified in the examples, possess anti-tumor activity in mammals (especially humans). .As an anti-cancer drug, the compounds of the invention can be used to treat brain and neurovascular tumors, head and neck cancers, breast cancer, lung cancer, mesothelioma, lymphoid cancer, stomach cancer, kidney cancer, renal carcinoma, liver cancer and liver cirrhosis, ovarian cancer, ovary endometriosis, testicular cancer, skin cancer, melanoma, neuro and all endocrine cancers, spleen cancers, pancreatic cancers, blood proliferative disorders such as I lodgkin^'s cancer, lymphoma, leukemia, and anv cancer disorders that result from uncontrolled cellular proliferations.

[Ill] The compounds of the present invention, may be mixed with commonly known pharmaceutical excipients such as udragit. sodium carboxymelhv lcellulosc (N'a CM), sodium carboxypropv (cellulose, anv oilier natural Ix derived or svnthetic excipients to effect an efficacious pharmaceutical formulation. The formulation comprising the compounds of the invention mav be made as a immediate release formulation, or a sustained release formulation, or site injection depot formulation, depending on the medical needs. The compound of the present invention ma\ also be combined with a medical device, such as a stent, a balloon, a balloon catheter, an orthopedic dev ice, lo further enhance the efficacy of the medical device. The compound of the present invention may be the main lunction component of a medical treatment regime, such as a local injection formulation, or an ancillary function, such as a coaling on a medical device, or in combination with a low -molecular weight or polymer excipient. and used as a coaling or lillcr of a medical device.

|112| When used to treat restenosis following a balloon angioplasty or stent placement, the compounds of the present invention, and the native rapamycin. are thought to exhibit their therapeutic functions through the inhibition of the mammalian target of rapamycin or m^'l OR. I hex mav ;II > bind to 1 K i i I ' receptors.

[11 { When used in the above or other treatments, a therapeutically effective amount of one of the compounds of the present invention mav be employed in pure form or. where such forms exist, in pharmaceutical!) acceptable salt, ester or prodrug form. Alternatel , the compound may be administered as a pharmaceutical composition containing the compound of interest in combination with one or more pharmaceutically acceptable excipients. The phrase "therapeutically effective amount^" of the compound of the present invention means a sufficient amount of the compound to treat disorders, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention w ill be decided by the attending ph sician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder: activity of the spec i tic compound employed: the specific composition employed: the age. body weight, general health, sex and diet oflhe patient: the time of administration, route of administration, and rate of excretion of the specific compound employed: the duration o lhe treatment: drugs used in combination or coincidental with the specific compound employed: and like factors well known in the medical arts. For example, it is well w ithin the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect

|114] The total daily dose of the compounds oflhe present invention administered to a human or lower mammal may range from about 0.01 to about 20 mg/kg/day. l or purposes of oral administration, more preferable doses may be in the range of from about 0.001 to about 3 mg/kg/day. If desired, the effective daily dose may be divided into multiple doses for purposes of administration: consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. I opical administralion may involve doses ranging from 0.001 to 10 percent mg/kg/day. depending on the site of application. When administered locally to treat restenosis and vulnerable plaque, the dose may range from about I microgram ^''mm stent length to about 100 microgram/mm stent length.

Pharmaceutical Compositions

[115| The pharmaceutical compositions of the present invention comprise a compound and a pharmaceutically acceptable carrier or excipient. which may be administered orally, recta 11 \ . parenteral ly . intracisternall . intrav aginally . intraperiionealry . topically (as by powders, ointments, drops or transdermal patch), hucally. or as an oral or nasal spray. The phrase ^"pharmaceutical!) acceptable earner^" means a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term ^"'parenteral.^" as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal. subcutaneous and intraarticular injection and infusion.

|116| Pharmaceutical compositions of the present invention for parenteral inieetion comprise pharmaceutical!) acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. P.xamples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol. polyols (such as glycerol, propylene glycol, polyethylene gl eol. and the like), carboxv methylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper lluiditv ma) be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle si/e in the case of dispersions, and by the use of surfactants. |117| I hese compositions mav also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms mav be ensured bv the inclusion of various antibacterial and antifungal agents, for example, paraben. chlorobutanol. phenol sorbic acid, and the like. It mav also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical fo m mav be brought about by the inclusion of agents w hich delay absorption such as aluminum monostearate and gelatin.

|118| In some cases, in order to prolong the effect of the drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This mav be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution w hich, in turn, mav depend upon crvstal size and crv sialline form. Alternately, delayed absorption of a parenteral l> administered drug form is accomplished bv dissoK ing or suspending the drug in an oil vehicle.

Injectable depot forms arc made bv forming mieroencapsulc matrices of the drug in biodegradable polymers such as po laetide-polv gh colide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release may be controlled. I^'xamples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or niicroemulsions which arc compatible with body tissues.

[12()| The injectable formulations may be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which may be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

[121] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutical I \ acceptable excipient or carrier such as sodium citrate or dicaleium phosphate and/or a) tillers or extenders such as starches, lactose, sucrose, glucose, mannitol. and silicic acid, b) binders such as carboxy melliy lcellulose. alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cctyl alcohol and glycerol monoslearate. h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate. magnesium stearate. solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise b ffering agents.

|122| Solid composilions of a similar type may also be employed as fillers in soft, semi-solid and hard-filled gelatin capsules or liquid-lllled capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

|123| The solid dosage forms of tablets, dragees. capsules, pills, and granules may be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient(s) only, or preferentially , in a certain part of the intestinal tract, optionally, in a delayed manner, bxamples of embedding compositions which may be used include polymeric substances and waxes.

|124| The active compounds may also be in a micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[125| Liquid do>agc forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art. for example, water or other solvents, solubilizing agents and emulsi!lers such as ethyl alcohol, isopropvl alcohol, ethyl carbonate. ethyl acetate, benzyl alcohol, benzv I benzoale. propylene glycol. 1.3-butylene glycol, dimethyl I rmamide. oils (in particular, cottonseed, groundnut, corn. germ, olive, castor, and sesame oils), glycerol, tetrah drofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan. and mixtures thereof.

[126J Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, s eetening, flavoring, and perfuming agents.

[127) Suspensions, in addition to the active compounds, may contain suspending agents, for example, cthoxy lated isostcary I alcohols. polyoxy ethylene sorbitol and sorbitan esters, microcry stalline cellulose, aluminum metahydroxide. bentonite. agar--agar. and tragacanth. and mixtures thereof.

|128| Topical administration includes administration to the skin or mucosa, including surfaces of the lung and eye. Compositions for topical administration, including those for inhalation. may be prepared as a dry powder which may be pressurized or non-pressurized. In non-pressurized powder compositions, the active ingredient in finely divided form may be used in admixture with a larger-sized pharmaceutically acceptable inert carrier comprising particles having a size, for example, of up to 100 micrometers in diameter. Suitable inert carriers include sugars such as lactose. Desirably at least > percent by weight of the particles of the active ingredient have an effective particle size in the range of 0. 1 to 10 micrometers. Compositions for topical use on the skin also include ointments, creams, lotions, and gels.

|12⁽)| Alternately the composition may lie pressurized and contain a compressed gas. such as nitrogen or a liquefied gas propel lant. The liquelled propel lanl medium and indeed the total composition is preferably such that the active ingredient does not dissolve therein to any substantial extent. The pressurized composition may also contain a surface active agent. The surface active agent ma> be a liquid or solid non-ionic surface active agent or may be a solid anionic surface active agent. It is preferred to use the solid anionic surface active agent in the form of a sodium salt.

[ 1 0 j A further form of topical administration is to the eye. as for the treatment of immune- mediated conditions of the eye such as autoimmune diseases, allergic or inflammatory conditions, and corneal transplants. I he compound of the present invention is delivered in a pharmaceutically acceptable ophthalmic vehicle, such that the compound is maintained in contact with the ocular surface for a sufficient time period to allow the compound to penetrate the corneal and internal regions of the eye. as for example the anterior chamber, posterior chamber, vitreous bod). ac|ueous humor, vitreous humor, cornea, iris/eilary. lens, choroid/retina and sclera. The pharmaceutical!) acceptable ophthalmic vehicle may. for example, be an ointment, vegetable oil or an encapsulating material.

11311 Compositions for rectal or vaginal administration are preferably suppositories or retention enemas which may be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at bod) temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

J 1 21 Compounds of the present invention ma ' also be administered in the form of liposomes. As is known in the art. liposomes are general!) derived from phospholipids or other lipid substances, l iposomes are formed

mono- or multi-lamellar hvdralcd liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologicall) acceptable and metaboli/able lipid capable of forming liposomes can be used. The present compositions in liposome form ma\ contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients. and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and s nihetic. Methods to orm liposomes are known in the art. See. for example. Prescott. I d.. Methods in Cell Biology. Volume XIV. Academic Press. Ne York. N.Y. (1976). p.33 el seq.

J 13 ] Compounds of the present invention may also be coadministered ith one or more immunosuppressant agents. The immunosuppressant agents within the scope of the present invention include IMURAN. RTM. a/athioprine sodium, brequinar sodium. SPANIDIN.RTM. gusperimus trih) drochloride (also known as dcoxyspergualiii). mizoribine (also known as bredinin). CI I I (Ί PT.R I^'M.

mofetil. NUORAI ..RTM. Cylosporin A (also marketed as different formulation of Cyclosporin Λ under the trademark SANDIMMl ^:NI ..R^'l AT). PROCRAI .RTM. tacrolimus (also known as l^'k ()6). sirolimus and RAPAMl NI^'.R I^'M., lellunomide (also known as IIW A-1 6). glucocorticoids, such as prednisolone and its derivatives, antibody therapies such as orthoclone (OK 1 ) and /cnapax.R I M.. and antith) mvocvie globulins, such as ihymoglobulins. |134| The local deliver} of drug/drug combinations from a stent or other implantable device has the following advantages: namely, the prevention of vessel recoil and remodeling through the scaffolding action of the stent and the prevention of multiple components of neointimal hyperplasia or restenosis as well as a reduction in inflammation and thrombosis. This local administration of drugs, agents or compounds to stented coronary arteries may also have additional therapeutic benefit. l or example, higher tissue concentrations of the drugs, agents or compounds mav be achieved utilizing local delivery, rather than systemic administration. In addition, reduced systemic toxicity may be achieved utilizing local deliver} rather than systemic administration while maintaining higher tissue concentrations. Also in utilizing local deliver}' from a stent rather than systemic administration, a single procedure may suffice with better patient compliance. An additional benefit of combination drug, agent, and. or compound therapv mav be to reduce the dose of each of the therapeutic drugs, agents or compounds, thereby limiting their to icity . while still achieving a reduction in restenosis, inflammation and thrombosis. Local stent-based therapy is therefore a means of improving the therapeutic ratio (cHlcaey< toxicity) of anti-restenosis. anti-inllammalor} . antithrombotic drugs, agents or compounds.

|135| It is understood that the foregoing detailed description and accompanying examples are merel} illustrativ e and are not to be taken as limitations upon the scope of the invention, which is defined solelv b_v the appended claims and their equivalents. Various changes and modi ications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including ithout limitation those relating to the chemical structures, substiluents. derivativ es, intermediates, s ntheses, formulations and/or methods of use of the invention, mav be made without departing from the spirit and scope thereof.

Claims

What Is Claimed Is:

1. A compound of Formula I or a pharmaceutically acceptable salt or prodrug thereof:

Formula

wherein A is selected from the group consisting of:

a) hydrogen, alkyl and substituted alkyl, alkenyl and substituted alkenyl, alkynyl and substituted alkynyl, cycloalkyl and substituted cycloalkyl, heterocycloalkyl and substituted heterocycloalkyl; the substitution group including hydroxyl, sulfonyl, carbonyl, amino, cyano, halogen, alkoxy, aryl, and heteroaryl, and

b) aryl and substituted aryl, heteroaryl and substituted heteroaryl; the substitution group including hydroxyl, halogen, amino, carbonyl, cyano, nitro, sulfonyl, alkyl, alkoxy, cycloalkyl, heterocycloalkyl.

2. The compound from claim 1 , wherein the compound is selected from the group consisting of:

Formula II

wherein B is selected from the group consisting of:

a) hydrogen, alkyl and substituted alkyl, alkenyl and substituted alkenyl, alkynyl and substituted alkynyl, cycloalkyl and substituted cycloalkyl, heterocycloalkyl and substituted heterocycloalkyl; wherein each substituent is independently hydroxyl, sulfonyl, carbonyl, amino, cyano, halogen, alkoxy, aryl, or heteroaryl, and

b) aryl and substituted aryl, heteroaryl and substituted heteroaryl; wherein each substituent is hydroxyl, halogen, amino, carbonyl,cyano, nitro, sulfonyl, alkyl, alkoxy, cycloalkyl, or heterocycloalkyl.

4. The compound from claim 3, wherein the compound is selected from the group consisting of:

5. A pharmaceutical composition comprising a compound of any of claims 1-4 and a pharmaceutical excipient.

6. The pharmaceutical formulation of claim 5, wherein the formulation is suitable for administration to a mammal via a route selected from the group consisting of oral, nasal, intravenous, transdermal, parenteral, subcutaneous, intramuscular, intra-ocular, and peritoneal routes.

7. The pharmaceutical formulation of claim 6, wherein the mammal is a human.

8. A method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any of claims 1-4.

9. The method of claim 8, wherein the cancer is selected from the group consisting of brain and neurovascular tumors, head and neck cancers, breast cancer, lung cancer, mesothelioma, lymphoid cancer, stomach cancer, kidney cancer, renal carcinoma, liver cancer and liver cirrhosis, ovarian cancer, ovary endometriosis, testicular cancer, skin cancer, melanoma, neuro and all endocrine cancers, spleen cancers, pancreatic cancers, blood proliferative disorders such as Hodgkin's cancer, lymphoma, leukemia, and any cancer disorders that result from

uncontrolled cellular proliferations

10. A method for treating or preventing an immune-mediated disease in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound of any of claims 1-4.

11. The method of claim 10, wherein the immune-mediated disease is selected from the group consisting of resistance by transplantation of heart, kidney, liver, medulla ossium, skin, cornea, lung, pancreas, intestinum tenue, limb, muscle, nerves, duodenum, small-bowel, or pancreatic-islet-cell; graft- versus-host diseases brought about by medulla ossium transplantation.

12. The method of claim 10, wherein the immune-mediated disease is rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, allergic encephalomyelitis, or glomerulonephritis.

13. The method of claim 10, wherein the immune-mediated disease is a graft-versus-host disease brought about by medulla ossium transplantation.

14. Use of a compound of any of claims 1-4 for manufacturing of a medicament for the treatment of a cancer or an immune-mediated disease.

15. The use of claim 14, wherein the cancer is selected from the group consisting of brain and neurovascular tumors, head and neck cancers, breast cancer, lung cancer, mesothelioma, lymphoid cancer, stomach cancer, kidney cancer, renal carcinoma, liver cancer and liver cirrhosis, ovarian cancer, ovary endometriosis, testicular cancer, skin cancer, melanoma, neuro and all endocrine cancers, spleen cancers, pancreatic cancers, blood proliferative disorders such as Hodgkin's cancer, lymphoma, leukemia, and any cancer disorders that result from uncontrolled cellular proliferations; and the immune-mediated disease is selected from the group consisting of resistance by transplantation of heart, kidney, liver, medulla ossium, skin, cornea, lung, pancreas, intestinum tenue, limb, muscle, nerves, duodenum, small-bowel, or pancreatic- islet-cell; graft-versus-host diseases brought about by medulla ossium transplantation.