Alkylidene macrolides having immunosuppressive activity

Alkylidene macrolides of the general structural Formula I: ##STR1## have been prepared from suitable precursors by derivitization at C-9 or C-22. These macrolide immunosuppressants are useful in a mammalian host for the treatment of autoimmune diseases, infectious diseases and/or the prevention of rejection of foreign organ transplants. In addition, these macrolide immunosuppressants are useful in the topical treatment of inflammatory and hyperproliferative skin diseases and cutaneous manifestations of immunologically-mediated illnesses. Also, these macrolides are useful in the treatment of reversible obstructive airways disease, particularly asthma; as hair revitalizing agents, especially in the treatment of male pattern alopecia oralopecia senilis; in the reversal of multidrug resistance of tumor cells; in treatment of inflammation of mucosa and blood vessels, gastric ulcers, vascular damage, ischemic bowel disease, inflammatory bowel disease, necrotizing entercolitis, intestinal lesions associated with thermal burns; in the treatment of cytomegalovirus infection; and in the treatment of idiopathic thrombocytopenic purpura and Basedow's disease.

SUMMARY OF THE INVENTION 
The present invention is related to alkylidene macrolides which are useful 
in a mammalian host for the treatment of autoimmune diseases (such as 
juvenile-onset diabetes mellitus, multiple sclerosis and rheumatoid 
arthritis), immunodepression, infectious diseases and/or the prevention of 
rejection of foreign organ transplants, e.g. bone marrow and heart 
transplants and are also useful in the topical treatment of inflammatory 
and hyperproliferative skin diseases and cutaneous manifestations of 
immunologically-mediated illnesses (such as: psoriasis, atopical 
dermatitis, contact dermatitis and further eczematous dermatitises, 
seborrhoeic dermatitis, Lichen planus, Pemphigus, bullous Pemphigoid, 
Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, 
cutaneous eosinophilias, Lupus erythematosus, Alopecia areata), male 
pattern alopecia, alopecia senilis, reversible obstructive airways 
disease, particularly asthma, alopecia, inflammation of mucosa and blood 
vessels, cytomegalovirus infection, multidrug resistance, idiopathic 
thrombocytopenic purpura, and/or hepatic injury associated with ischemia. 
In addition, some of the compounds of this invention may have antagonistic 
properties and so have utility in the reversal of immunosuppressive 
activity and/or diminishing the toxicity of other immunosuppressive 
agents. 
More particularly, this invention relates to compounds of the general 
structural formula I: 
##STR2## 
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, 
R.sup.8, W, X and n are hereinafter defined. 
This invention also relates to pharmaceutical compositions containing the 
compounds and to a method of use of the present compounds and other agents 
for the treatment of and prevention of certain afflictions, diseases and 
illnesses. 
BRIEF DESCRIPTION OF DISCLOSURES IN THE ART 
Fujisawa United States European and Japanese patents and applications (U.S. 
Pat. No. 4,894,366, issued Jan. 16, 1990, EPO Publication No. 0,184,162 
and PBJ Disclosure 63-17884) and publications (J. Am. Chem. Soc., 1987, 
109, 5031 and J. Antibiotics 1987, 40, 1249) disclose 
17-allyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[2 
2.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone (FR-900506), (FK-506), 
(L-679,934), 
17-ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22 
.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone (FR-900520) and related 
compounds which are the starting materials for the preparation of the 
compounds described. The synthetic preparation of the aforementioned 
starting material (FR-900506) has recently been reported (J. Am. Chem. 
Soc., 1989, 111, 1157). A Sandoz European patent application (EPO 
Publication No. 0,356,399) discloses stereoisomers of FR-900506 and 
derivatives at the 17-position. Fisons European and WIPO patent 
applications (EPO Publication No. 0,323,042 and PCT Publication No. WO 
89/05304) discloses various derivatives of FR-900506, FR-900520 and 
related compounds. A Sandoz European Patent application (EPO Publication 
No. 0,437,680) discloses chloro, bromo, iodo and azido derivatives of 
FR-900506, FR-900520 and related compounds. A Merck European Patent 
application (EPO Publication No. 0,428,365) discloses various amino 
derivatives of FR-900506, FR-900520 and related compounds. A Fujisawa 
patent application (UK Publication No. GB 2,245,891-A) discloses various 
derivatives of FR-900506 bearing a heterocyclic group. Fujisawa patent 
applications (PCT Publication Nos. WO 92/00313 and WO 92/00980) disclose 
various derivatives of FR-900506 at the 17-position. 
Fujisawa United States patents (U.S. Pat. No. 4,929,611, issued May 29, 
1990 and U.S. Pat. No. 4,956,352, issued Sep. 11, 1990) discloses the use 
of FK-506-type compounds in treating resistance to transplantation. A 
Sandoz European patent application (EPO Publication No. 0,315,978) 
discloses the use of FR-900506 and related compounds in the topical 
treatment of inflammatory and hyperproliferative skin diseases and of 
cutaneous manifestations of immunologically-mediated illness. A Fisons 
WIPO patent application (PCT Publication No. WO 91/04025) discloses the 
use of various derivatives of FR-900506 in the treatment of 
immunodepression. A Fisons WIPO patent application (PCT Publication WO 
90/14826) discloses the use of FR-900506 and related compounds in the 
treatment of reversible obstructive airways disease, particularly asthma. 
A Fujisawa European patent application (EPO Publication No. 0,423,714) 
discloses the use of FK-506 and derivatives as hair revitalizing agents. 
Various studies have suggested the efficacy of FK-506 in the treatment of 
a number of ailments, including rheumatoid arthitis (C. Arita, et al., 
Clinical exp. Immunol., 1990, 82, 456-461; N. Inamura, et al., Clin. 
Immunol. Immunopathol. 1988, 46, 82-90), recent-onset diabetes (N. Murase, 
et al., Diabetes, 1990, 39, 1584-86; N. Murase, et al., Lancet, 1990, 336, 
373-74), posterior uveitis (H. Kawashima, Invest. Ophthalmol. Vis. Sci., 
1988, 29, 1265-71), hepatic injury associated with ischemia (M. Sakr, et 
al., Life Sci., 1990, 47, 687-91) allergic encephalomyelitis (K, Deguchi, 
et al., Brain Nerve, 1990, 42, 391-97), glomeralonephritis (J. McCauley, 
et al., Lancet, 1990, 335, 674) and systemic lupus erythematosus (K. 
Takabayashi, et al., Clin. Immunol. Immunopathol., 1989, 51, 110-117) 
multidrug resistance (M. Naito, et al., Cancer Chemother. Pharmacol., 
1992, 29, 195-200), inflammation of mucosa and blood vessels (PCT 
Publication WO 91/17754), cytomegalovirus infection (UK Publication GB 
2,247,620A), and idiopathic thrombocytophenic purpura and Basedow' s 
disease (PCT Publication WO 91/19495). 
BACKGROUND OF THE INVENTION 
Immunoregulatory abnormalities have been shown to exist in a wide variety 
of "autoimmune" and chronic inflammatory diseases, including systemic 
lupus erythematosis, chronic rheumatoid arthritis, type 1 diabetes 
mellitus, inflammatory bowel disease, biliary cirrhosis, uveitis, multiple 
sclerosis and other disorders such as Chrons disease, ulcerative colitis, 
bullous pemphigoid, sarcoidosis, psoriasis, ichthyosis, and Graves 
ophthalmophathy. Although the underlying pathogenesis of each of these 
conditions may be quite different, they have in common the appearance of a 
variety of autoantibodies and self-reactive lymphocytes. Such 
self-reactivity may be due, in part, to a loss of the homeostatic controls 
under which the normal immune system operates. 
Similarly, following a bone-marrow or an organ transplantation, the host 
lymphocytes recognize the foreign tissue antigens and begin to produce 
antibodies which lead to graft rejection. 
One end result of an autoimmune or a rejection process is tissue 
destruction caused by inflammatory cells and the mediators they release. 
Antiinflammatory agents such as NSAID's and corticosteroids act 
principally by blocking the effect or secretion of these mediators but do 
nothing to modify the immunologic basis of the disease. On the other hand, 
cytotoxic agents such as cyclophosphamide, act in such a nonspecific 
fashion that both the normal and autoimmune responses are shut off. 
Indeed, patients treated with such nonspecific immunosuppressive agents 
are as likely to succumb from infection as they are from their autoimmune 
disease. 
Cyclosporin A which was approved by the US FDA in 1983 is currently the 
leading drug used to prevent rejection of transplanted organs. 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. Though cyclosporin A is effective in fighting transplant 
rejection, it is nephrotoxic and is known to cause several undesirable 
side effects including kidney failure, abnormal liver function and 
gastrointestinal discomfort. 
Newer, safer drugs exhibiting less side effects are constantly being 
searched for in the field. 
The 23-membered tricyclo-macrolide immunosuppressant, FR-900506, FK-506, 
##STR3## 
(17-allky-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methy 
l 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[2 
2.3.1.0.sup.4,9 ]-octacos-18-ene-2,3,10,16-tetraone) and related compounds 
which were isolated and characterized by Tanaka, Kuroda, and co-workers at 
Fujisawa Pharmaceutical Co. in Japan, see J. Am. Chem. Soc., 1987, 109, 
5031, and U.S. Pat. No. 4,894,366, issued Jan. 16, 1990) have been shown 
to possess exceptional immunosupressive activity. A Fujisawa U.S. patents 
(U.S. Pat. No. 4,929,611, issued May 29, 1990 U.S. Pat. No. 4,956,352, 
issued Sep. 11, 1990) disclose the use of FK-506-type compounds in 
treating resistance to transplantation. In particular, the compound 
FR-900506 has been reported to be 100 times more effective than 
cyclosporin in the supression of in vitro immune systems (J. Antibiotics 
1987, 40, 1256). In addition, these compounds are reputed to possess 
topical activity in the treatment of inflammatory and hyperproliferative 
skin diseases and cutaneous manifestations of immunologically-mediated 
illnesses (EPO Pub. No. 0,315,978). 
The compound FK-506 and related compounds further have been suggested to be 
useful in the treatment of obstructive airways disease, particularly 
asthma (PCT Publication WO 90/14826), male pattern alopecia or alopecia 
senilis (EPO Publication No. 0,423,714), rheumatoid arthitis (C. Arita, et 
al., Clincial exp. Immunol., 1990, 82, 456-461; N. Inamura, et al., Clin. 
Immunol. Immunopathol. 1988, 46, 82-90), recent-onset diabetes (N. Murase, 
et al., Diabetes, 1990, 39, 1584-86; N. Murase, et al., Lancet, 1990, 336, 
373-74), posterior uveitis (H. Kawashima, Invest. Ophthalmol. Vis. Sci., 
1988, 29, 1265-71), hepatic injury associated with ischemia (M. Sakr, et 
al., Life Sci., 1990, 47, 687-91) allergic encephalomyelitis (K, Deguchi, 
et al., Brain Nerve, 1990, 42, 391-97), glomerulonephritis (J. McCauley, 
et al., Lancet, 1990, 335, 674), systemic lupus erythematosus (K. 
Takabayashi, et al., Clin. Immunol. Immunopathol., 1989, 51, 110-117) 
multidrug resistance (M. Naito, et al., Cancer Chemother, Pharmacol., 
1992, 29, 195-200), inflammation of mucosa and blood vessels (PCT 
Publication WO 92/17754), cytomegalovirus infection (UK Publication GB 
2,247,620A), and idiopathic thrombocytophenic purpura and Basedow's 
disease (PCT Publication WO 91/19495).

DETAILED DESCRIPTION OF THE INVENTION 
A. Scope of the Invention 
The novel compound of this invention has structural Formula I: 
##STR4## 
or a pharmaceutically acceptable salt thereof, wherein: R.sup.1 is 
selected from: 
(1) hydrogen; and 
(2) C.sub.1-4 alkanoyl; 
R.sup.2 is selected from: 
(1) methyl; and 
(2) hydrogen; 
R.sup.3 is hydrogen, hydroxy, or C.sub.1-6 alkoxy; 
R.sup.4 is hydrogen, or R.sup.3 and R.sup.4 taken together form a double 
bond; 
R.sup.5 is selected from: 
(1) methyl, 
(2) ethyl, 
(3) propyl, and 
(4) allyl; 
R.sup.6 is hydroxy or hydrogen; 
R.sup.7 is hydrogen, or R.sup.6 and R.sup.7 taken together form a double 
bond; 
R.sup.8 is hydrogen, hydroxy, or fluoro; 
W and X are independently selected from: 
(1) oxo, 
(2) (H, OH), 
(3) (OH, R.sup.9), wherein R.sup.9 is selected from: 
(a) C.sub.1-6 alkyl, 
(b) C.sub.2-6 alkenyl, 
(c) --CH.sub.2 --SO.sub.m --C.sub.1-6 alkyl, wherein m is 0, 1 or 2, 
(d) --CH.sub.2 S(C.sub.1-6 alkyl).sub.2.sup.+ M.sup.-, wherein M.sup.- is a 
negative counterion selected from: chloro, bromo, iodo, 
(e) phenyl, 
(f) furanyl, 
(4) .dbd.CR.sup.10 R.sup.11, wherein R.sup.10 and R.sup.11 are 
independently selected from: 
(a) hydrogen, 
(b) C.sub.1-6 alkyl, 
(c) C.sub.2-6 alkenyl, 
(d) --SO.sub.m --C.sub.1-6 alkyl, wherein m is as defined above; 
with the proviso that W and X are not simultaneously oxo, (H, OH), or 
combinations thereof; 
n is 1 or 2. 
The compounds of the present invention have asymmetric centers and this 
invention includes all of the optical isomers and mixtures thereof. 
In addition compounds with carbon-carbon double bonds may occur in Z- and 
E- forms with all isomeric forms of the compounds being included in the 
present invention. 
When any variable (e.g., alkyl, aryl, R.sup.9, R.sup.10, R.sup.11, etc.) 
occurs more than one time in any variable or in Formula I, its definition 
on each ocurrence is independent of its definition at every other 
occurrence. 
As used herein, the term "alkyl" includes those alkyl groups of a 
designated number of carbon atoms of either a straight, branched, or 
cyclic configuration. Examples of "alkyl" include methyl, ethyl, propyl, 
isopropyl, butyl, sec-and tert-butyl, pentyl, hexyl, heptyl, cyclopropyl, 
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, and the like. 
"Alkoxy" represents an alkyl group of indicated number of carbon atoms 
attached through an oxygen bridge, such as methoxy, ethoxy, propoxy, 
butoxy and pentoxy. "Alkenyl" is intended to include hydrocarbon chains of 
a specified number of carbon atoms of either a straight- or 
branched-configuration and at least one unsaturation, which may occur at 
any point along the chain, such as ethenyl, propenyl, butenyl, pentenyl, 
dimethylpentyl, and the like, and includes E and Z forms, where 
applicable. "Halo", as used herein, means fluoro, chloro, bromo and iodo. 
As will be understood by those skilled in the art, pharmaceutically 
acceptable salts include, but are not limited to salts with inorganic 
acids such as hydrochloride, sulfate, phosphate, diphosphate, 
hydrobromide, and nitrate or salts with an organic acid such as malate, 
maleate, fumarate, tartrate, succinate, citrate, acetate, lactate, 
methanesulfonate, p-toluenesulfonate or palmoate, salicylate and stearate. 
Similarly pharmaceutically acceptable cations include, but are not limited 
to sodium, potassium, calcium, aluminum, lithium and ammonium. 
Preferred compounds of the present invention are the compounds identified 
as follows: 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-methyl-13,19,21,27-tetramethyl-11,28-dioxa-4-a 
zatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione; (1) 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-methyl-13,19,21,27-tetramethyl-11,28-dioxa-4-a 
zatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione; (2) 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-butyl-13,19,21,27-tetramethyl-11,28-dioxa-4-az 
atricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione; (3) 
17-Ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylv 
inyl]-23,25-dimethoxy-16-butyl-13,19,21,27-tetramethyl-16-methenyl-11,28-di 
oxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione; (4) 
17-Ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylv 
inyl]-23,25-dimethoxy-16-butyl-13,19,21,27-tetramethyl-16-(2-butenyl)-11,28 
-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione; (5) 
17-Ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylv 
inyl]-23,25-dimethoxy-16-propenyl-13,19,21,27-tetramethyl-11,28-dioxa-4-aza 
tricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione; (6) 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-(methylthiomethyl)-13,19,21,27-tetramethyl-11, 
28-dioxa-a-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3, 10-trione; 
(7) 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-(methylsulfinylmethyl)-13,19,21,27-tetramethyl 
-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione; 
(8) 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-(methylsulfinylmethenyl)-13,19,21,27-tetrameth 
yl-11, 28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3, 
10-trione; (9) 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-phenyl-13,19,21,27-tetramethyl-11,28-dioxa-4-a 
zatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione; (10) 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-furan-2-yl-13,19,21,27-tetramethyl-11,28-dioxa 
-4-azatricyclo[2-2.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione; (11) 
17-Ethyl-14-hydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylvinyl 
]-23,25-dimethoxy-13,19,21, 
-27-tetramethyl-11,28-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 
]-octacos-1,18-diene-2,3,10,16-tetraone; (12) 
17-Ethyl-14-hydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylvinyl 
]-23,25-dimethoxy-13,19,21, 
27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 
]-octacos-18-ene-2,3,10,16-tetraone; (13) 
17-Ethyl-14-hydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylvinyl 
]-23,25-dimethoxy-13,19,21, 
epi-27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10,16-tetraone (14). 
B. Preparation of Compounds Within the Scope of the Present Invention 
The starting materials for the preparation of the compounds of this 
invention are represented by Formula II: 
##STR5## 
wherein: E is hydrogen or methyl; 
W is O or (H, OH); 
R.sup.3 is hydrogen, hydroxy, or C.sub.1-6 alkoxy; 
R.sup.4 is hydrogen, or R.sup.3 and R.sup.4 taken together form a double 
bond; 
R.sup.5 is methyl, ethyl, propyl or allyl; and 
n is 1 or 2. 
The production and characterization of compounds of Formula II is well 
known in the literature (see U.S. Pat. No. 4,894,366 issued Jan. 16, 1990; 
U.S. Pat. No. 4,929,611 issued May 29, 1990; U.S. Pat. No. 3,244,592 
issued Apr. 15, 1966; EPO Publication No. 0,323,042,; EPO Publication No. 
0,356,399; PBJ Disclosure 63-17884; J. Am. Chem. Soc., 1987, 109, 5031; J. 
Antibiotics, 1987, 40, 1249; J. Antibiotics, 1988, 41 (11), 1592; and J. 
Antibiotics, 1992, 45 (1), 118). Both biological fermentation and 
synthetic processes may be found. A synthetic route to compounds of 
Formula II can involve modifications of a route described in J. Am. Chem. 
Soc., 1989, 111, 1157. 
Biological fermentation followed by synthetic modification is presently 
favored in the art as the method to produce compounds of Formula II. 
Organisms belonging to the genus Streptomyces such as Streptomyces 
tsukubaensis, No. 9993 and Streptomyces hygroscopicus, var. ascomycetis, 
No. 14891 placed in an aqueous nutrient medium will produce desired 
compounds in isolable amounts. The nutrient medium contains sources of 
assimilable carbon and nitrogen, preferably under aerobic conditions. 
Produced in fermentation are four compounds of Formula II, (A) where E is 
methyl, W is O, R.sup.3 is hydroxyl, R.sup.4 is hydrogen, R.sup.5 is allyl 
and n is 2; (B) where E is methyl, W is O, R.sup.3 is hydroxyl, R.sup.4 is 
hydrogen, R.sup.5 is ethyl and n is 2; (C) where E is methyl, W is O, 
R.sup.3 is hydroxyl, R.sup.4 is hydrogen, R.sup.5 is methyl and n is 2; 
and (D) where E is methyl W is O, R.sup.3 is hydroxyl, R.sup.4 is 
hydrogen, R.sup.5 is allyl and n is 1. 
A lyophilized sample of the isolated Streptomyces tsukubaensis, No. 9993 
was deposited with the Fermentation Research Institute, Agency of 
Industrial Science and Technology (No. 1-3, Higashi 1-chome, Yatabemachi 
Tsukuba-gun, Ibaraki Prefecture, Japan) under the deposit number of FERM 
P-7886 (deposit date: Oct. 5th, 1984), and then converted to Budapest 
Treaty route of the same depository on Oct. 19, 1985 under the new deposit 
number of FERM BP-927. 
Using the four compounds produced in fermentation above, the remaining 
compounds of Formula II may be easily produced. The allyl of R.sup.5 may 
be conveniently reduced to propyl by well known methods, for example as 
described in U.S. Pat. No. 4,894,366. The hydroxy of R.sup.3 may be 
protected by well known methods, for example as disclosed in EPO 
Publication No. 0,323,042. Likewise, the hydroxyl at C-4" may also be 
protected. In addition, the hydroxy of R.sup.3 may be reduced to a 
hydrogen or eliminated to form a double bond with R.sup.4 (by methods 
disclosed in U.S. Pat. No. 4,894,366, EPO Publication No. 0,323,042 or EPO 
Publication No. 0,413,532). The carbonyl of W may be reduced to the 
alcohol by methods disclosed in EPO Publication No. 0,323,042 or by 
methods disclosed in EPO Publication No. 0,445,975. The hydroxy of R.sup.8 
may be introduced by methods disclosed in EPO Publication No. 0,463,690. 
The methyl of E as produced may be replaced with hydrogen or demethylated 
and subsequently protected as desired, if necessary. This demethylation of 
compounds wherein E is methyl may be carried out in a fermentation 
reaction using the compounds of Formula II as a feedstock. For instance, 
compound A named under Formula II above may be demethylated at E above by 
using the microorganism Actinomycetales ATCC No. 53771 (described in U.S. 
Pat. No. 4,981,792) or by using the microorganism Streptomyces 
tsukubaensis, No. 9993 (described in EPO Publication No. 0,353,678). 
Similarly, compound B named under Formula II above may be demethylated at 
E above using the microorganism Actinoplanacete sp. ATCC No. 53771 
(described in EPO Publication No. 0,349,061). In addition the compound of 
Formula II wherein E is H, W is O, R.sup.3 is hydroxy, R.sup.4 is 
hydrogen, R.sup.5 is ethyl and n is 2 may be produced directly by 
fermentation using the mutant microorganism Streptomyces hygroscopicus 
sup. ascomyceticus, No. 53855 (being a blocked mutant of Streptomyces 
hygroscopicus sup. ascomyceticus, No. 14891) (as described in EPO 
Publication No. 0,388,152). Similarly, the compound of Formula II wherein 
E is hydrogen, W is O, R.sup.3 is hydroxy, R.sup.4 is hydrogen, R.sup.5 is 
methyl and n is 2 may be produced directly by fermentation using the 
mutant microorganism Streptomyces hygroscopicus sup. ascomyceticus, No. 
53855 (being a blocked mutant of Streptomyces hygroscopicus sup. 
ascomyceticus, No. 14891) (EPO Publication No. 0,388,153). The hydroxy of 
C-3" may be protected by methods similar to those known for the protection 
of the hydroxyl groups of R.sup.3 and/or C-4", for example as disclosed in 
U.S. Pat. No. 4,894,366. 
Suitable protecting groups for hydroxyl include those groups well known in 
the art such as: methylthiomethyl, ethylthiomethyl; trisubstituted silyl 
such as trimethylsilyl, triethylsilyl, tributylsilyl, tri-i-propylsilyl, 
t-butyldimethylsilyl, tri-t-butylsilyl, methyl-diphenylsilyl, 
ethyldiphenylsilyl, t-butyldiphenylsilyl, and the like; acyl such as 
acetyl, pivaloyl benzoyl, 4-methoxybenzoyl, 4-nitrobenzoyl and aliphatic 
acyl substituted with aromatic group, which are derived from carboxylic 
acids; and the like. 
Compounds A, B, C and D of Formula II, organisms to produce the same, 
conditions of fermentation, separation techniques, and chemical 
modification of the products are fully described in U.S. Pat. No. 
4,894,366, dated Jan. 16, 1990 and U.S. Pat. No. 4,929,611, issued May 29, 
1990. 
The novel processes for preparing the novel compounds of the present 
invention are illustrated as follows, wherein R.sup.1, R.sup.2, R.sup.3, 
R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, E, W and 
n are as defined above unless otherwise indicated. It will be readily 
apparent to one of ordinary skill in the art reviewing the synthetic route 
depicted below that other compounds within Formula I can be synthesized by 
substitution of appropriate reactants and agents in the synthesis shown 
below. 
##STR6## 
Reaction Scheme A 
As shown in Reaction Scheme A, when the reaction mixture is maintained at 
low temperature (-78.degree. C.) throughout the reaction sequence and the 
nucleophile contains a non-chelating carbanion [e.g. a lithium carbanion 
(either alkyl or aryl], the nucleophile adds selectively to the 
22-carbonyl group of a 24,32-protected analog. When the nucleophile 
(R.sup.9 Li) is a primary carbanion (RCH.sub.2 Li or ArLi), the addition 
results in a 22-alkyl-22-hydroxy product [Examples 3, 4 (methyl), 5 
(butyl), 10 (methylthiomethyl), 16 (phenyl), 17 (furan2-yl)]. These anions 
tend to add to the less-hindered face of the carbonyl and form the 
.alpha.-hydroxy group as indicated in the Scheme. 
Reaction Scheme B 
As shown in Reaction Scheme B, when the nucleophile (R.sup.9 Li) is a 
secondary carbanion (R.sup.10 R.sup.11 CHLi) or when the nucleophile is an 
.alpha.-silyl-.alpha.-lithio carbanion (Examples 6,7,8,9), the addition is 
followed by elimination, resulting in an olefination sequence (R.sup.10 
R.sup.11 CHLi) and affording a 22-alkylidene product. 
Reaction Scheme C 
As shown in Reaction Scheme C, when the nucleophile contains a sulfur 
heteroatom, that sulfur may be oxidized to afford the corresponding 
sulfoxide as a mixture of S-stereoisomers (Example 11). When the 
22-hydroxy group is .beta. to the newly formed sulfoxide, that hydroxy 
group may eliminate, forming the corresponding vinyl sulfone (Example 12). 
Reaction Scheme D 
As shown in Reaction Scheme D, when the nucleophile is a sulfur ylid, it 
adds to either face of the 22-carbonyl, forming a mixture of stereoisomers 
at that position (Example 13). The new sulfonium species may be 
dealkylated to afford the corresponding 22 alkyl-22-hydroxy analogs with 
either configuration at that position (Examples 14 and 15). 
Reaction Scheme E 
As shown in Reaction Scheme E, when the reaction is initiated at low 
temperature (-78.degree. C.) but allowed to warm to 0.degree. C. and when 
a chelating carbanion (either alkyl or aryl) is used as the nucleophile, 
the nucleophile will preferentially add to the 9-carbonyl group of a 
24,32-protected analog (Examples 19, 20). 
Reaction Scheme F 
As shown in Reaction Scheme F, when the carbanion contains an .alpha.-silyl 
substituent, the addition is followed by elimination, affording a 
9-alkylidene product (Example 18). This 9-alkylidene may rearrange to 
afford the intramolecular Michael adduct (Example 21). 
Reaction Scheme G 
As shown in Reaction Scheme G, this Michael rearrangement might be 
prevented by utilizing the 10-deoxy analog or the .delta..sup.10,11 
analogs as substrates (Examples 22, 23, and 24). 
The object compounds of Formula I obtained according to the reactions as 
explained above can be isolated and purified in a conventional manner, for 
example, extraction, precipitation, fractional crystallization, 
recrystallization, chromatography, and the like. 
It is to be noted that in the aforementioned reactions and the 
post-treatment of the reaction mixture therein, the stereoisomer(s) of 
starting and object compounds due to asymmetric carbon atom(s) or double 
bond(s) of the object compounds of Formula I may occasionally be 
transformed into the other stereo isomer(s), and such cases are also 
included within the scope of the present invention. 
In the present invention, compounds with asymmetric centers may occur as 
racemates, as diastereomeric mixtures and as individual diastereomers, 
with all isomeric forms of the compounds being included in the present 
invention. These may be prepared by methods such as those disclosed in 
publications which describe synthetic routes to fragments of the macrolide 
FR-900506 and the total synthesis of the macrolide FR-900506 itself (J. 
Am. Chem. Soc. 1989, 111, 1157; J. Am. Chem. Soc. 1990, 112, 2998; J. Org. 
Chem. 1990, 55, 2786; J. Am. Chem. Soc. 1990, 112, 5583. Tetrahedron Lett. 
1988, 29, 277; Tetrahedron Lett. 1988, 29, 281; Tetrahedron Lett. 1988, 
29, 3895; J. Org. Chem. 1988, 53, 4643; Tetrahedron Lett. 1988, 29, 4245; 
Tetrahedron Lett. 1988, 29, 4481; J. Org. Chem. 1989, 54, 9; J. Org. Chem. 
1989, 54, 11; J. Org. Chem. 1989, 54, 12; J. Org. Chem. 1989, 54, 15; J. 
Org. Chem. 1989, 54, 17; Tetrahedron Lett. 1989, 30, 919; Tetrahedron 
Lett. 1989, 30, 1037; J. Org. Chem. 1989, 54, 2785; J. Org. Chem. 1989, 
54, 4267; Tetrahedron Lett. 1989, 30, 5235; Tetrahedron Lett. 1989, 30, 
6611; Tetrahedron Lett. 1989, 30, 6963; Synlett 1990, 38; J. Org. Chem. 
1990, 55, 2284; J. Org. Chem. 1990, 55, 2771; J. Org. Chem. 1990, 55, 
2776; Tetrahedron Lett. 1990, 31, 1439; Tetrahedron Lett. 1990, 31, 1443; 
Tetrahedron Lett. 1990, 31, 3007; Tetrahedron Lett. 1990, 31, 3283, 3287). 
The compounds of the present invention are capable of forming salts with 
various inorganic and organic acids and bases and such salts are also 
within the scope of this invention. Examples of such acid addition salts 
include acetate, adipate, benzoate, benzenesulfonate, bisulfate, butyrate, 
citrate, camphorate, camphorsulfonate, ethanesulfonate, fumarate, 
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, 
hydroiodide, methanesulfonate, lactate, maleate, methanesulfonate, 
2-naphthalenesulfonate, oxalate, pamoate, persulfate, picrate, pivalate, 
propionate, succinate, tartrate, tosylate, and undecanoate. Base salts 
include ammonium salts, alkali metal salts such as sodium, lithium and 
potassium salts, alkaline earth metal salts such as calcium and magnesium 
salts, salts with organic bases such as dicyclohexylamine salts, 
N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine 
and so forth. Also, the basic nitrogen-containing groups may be 
quaternized with such agents as: lower alkyl halides, such as methyl, 
ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates 
like dimethyl, diethyl, dibutyl; diamyl suflfates; long chain halides such 
as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; 
aralkyl halides like benzyl bromide and others. The non-toxic 
physiologically acceptable salts are preferred, although other salts are 
also useful, such as in isolating or purifying the product. 
The salts may be formed by conventional means, such as by reacting the free 
base form of the product with one or more equivalents of the appropriate 
acid in a solvent or medium in which the salt is insoluble, or in a 
solvent such as water which is removed in vacuo or by freeze drying or by 
exchanging the anions of an existing salt for another anion on a suitable 
ion exchange resin. 
C. Utility of the Compounds Within the Scope of the Invention 
The compounds of Formula I may be employed as immunosuppressants or 
antimicrobial compounds by methods and in dosages known in the prior art 
for compounds of Formula II. These compounds possess pharmacological 
activity such as immunosuppressive activity, antimicrobial activity, and 
the like, and therefore are useful for the treatment and prevention of the 
resistance to transplantation or transplantation rejection of organs or 
tissues such as heart, kidney, liver, duodenum, small-bowel, medulla 
ossium, skin, pancreatic islet-cell, etc., graft-versus-host diseases by 
medulla ossium transplantation, autoimmune diseases such as rheumatoid 
arthritis, systemic lupus erythematosis, Hashimoto's thyroiditis, multiple 
sclerosis, myasthenia gravis, type I diabetes, uveitis, allergic 
encephalomyelitis, glomerulonephritis, etc., and infectious diseases 
caused by pathogenic microorganisms. 
The compounds of Formula I are also useful for treating inflammatory and 
hyperproliferative skin diseases and cutaneous manifestations of 
immunologically-mediated illnesses such as: psoriasis, atopical 
dermatitiis, contact dermatitis and further eczematous dermatitises, 
seborrhoeic dermatitis, Lichen planus, Pemphigus, bullous Pemphigoid, 
Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, 
cutaneous eosinophilias or Alopecia areata. More particularly, the 
compounds of Formula I are useful in hair revitalizing, such as in the 
treatment of male pattern alopecia or alopecia senilis, by providing 
epilation prevention, hair germination, and/or a promotion of hair 
generation and hair growth. 
The compounds of Formula I are further useful for treating reversible 
obstructive airways disease, including conditions such as asthma, 
including bronchial asthma, allergic asthma, intrinsic asthma, extrinsic 
asthma and dust asthma, particularly chronic or inveterate asthma (for 
example late asthma and airway hyper-reponsiveness), bronchitis and the 
like. The compounds of Formula I may also be useful for treating hepatic 
injury associated with ischemia. 
The compounds of Formula I are also useful for treating multidrug 
resistance of tumor cells, (i.e. enhancing the activity and/or sensitivity 
of chemotherapeutic agents), preventing or treating inflammation of mucosa 
or blood vessels, LTB.sub.4 -mediated diseases, gastric ulcers, vascular 
damage caused by ischemic diseases and thrombosis, ischemic bowel disease, 
inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis) 
necrotizing enterocolitis, or intestinal lesions associated with thermal 
burns, cytomegalovirus infection, particularly HCMV infection, idiopathic 
thrombocytopenic purpura and Basedow's disease. 
Further, the compounds of Formula I are also useful for treating or 
preventing renal diseases selected from interstitial nephritis, 
Goodpasture's syndrome, hemolytic-uremic syndrome and diabetic 
nephropathy; nervous diseases selected from multiple myositis, 
Guillain-Barre syndrome, Meniere's disease and radiculopathy; endocrine 
diseases selected from hyperthyroidism; hematic diseases selected from 
pure red cell aplasia, aplastic anemia, hypoplastic anemia, autoimmune 
hemolytic anemia, agranulocytosis and anerythroplasia; bone diseases such 
as osteoporosis; respiratory diseases selected from sarcoidosis, fibroid 
lung and idiopathic interstitial pneumonia; eye diseases selected from 
herpetic keratitis, conical cornea, dystrophia epithelialis corneae, 
corneal leukmas, ocular pemphigus, Mooren's ulcer, scleritis and Grave's 
ophthalmopathy; skin diseases selected from dermatomyositis, leukoderma 
vulgaris, ichthyosis vulgaris, photoallergic sensitivity and cutaneous T 
cell lymphoma; circulatory diseases selected from arteriosclerosis, 
aortitis syndrome, polyarteritis nodosa and myocardosis; collagen diseases 
selected from scleroderma, Wegener's granuloma and Sjogren's syndrome; 
adiposis; eosinophilic fasciitis; periodontal disease; and muscular 
dystrophy. 
The compounds of Formula I may also act as antagonists of macrocyclic 
immunosuppressive compounds, including derivatives of 
12-(2'-cyclohexyl-1'-methylvinyl)-13,19,21,27-tetramethyl-11,28-dioxa-4-az 
atricyclo[22.3.1.0.sup.4,9] octacos-18-ene, and so be useful in the 
treatment of immunodepression (such as AIDS, cancer, senile dementia, 
trauma (including wound healing, surgery and shock), chronic bacterial 
infection and certain central nervous system disorders), overdosages or 
toxicity of such immunosuppressive compounds, and as an adjunct to the 
administration of an antigen in vaccination. 
The pharmaceutical compositions of this invention can be used in the form 
of a pharmaceutical preparation, for example, in solid, semisolid or 
liquid form, which contains one or more of the compounds of the present 
invention, as an active ingredient, in admixture with an organic or 
inorganic carrier or excipient suitable for external, enteral or 
parenteral applications. The active ingredient may be compounded, for 
example, with the usual non-toxic, pharmaceutically acceptable carriers 
for tablets, pellets, capsules, suppositories, solutions, emulsions, 
suspensions, 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 other carriers suitable for use in 
manufacturing preparations, in solid, semisolid, or liquid form, and in 
addition auxiliary, stabilizing, thickening and coloring agents and 
perfumes may be used. For example, the compounds of Formula I may be 
utilized with hydroxypropyl methylcellulose essentially as described in 
U.S. Pat. No. 4,916,138, issued Apr. 10, 1990, or with a surfactant 
essentially as described in EPO Publication 0,428,169. Oral dosage forms 
may be prepared essentially as described by T. Hondo, et al., 
Transplantation Proceedings, 1987, XIX, Supp. 6, 17-22. Dosage forms for 
external application may be prepared essentially as described in EPO 
Publication 0,423,714. The active object compound is included in the 
pharmaceutical composition in an amount sufficient to produce the desired 
effect upon the process or condition of diseases. 
For the treatment of these conditions and diseases caused by 
immunoirregularity a compound of Formula I may be administered orally, 
topically, parenterally, by inhalation spray or rectally in dosage unit 
formulations containing conventional non-toxic pharmaceutically acceptable 
carriers, adjuvants and vehicles. The term parenteral as used herein 
includes subcutaneous injections, intravenous, intramuscular, intrasternal 
injection or infusion techniques. 
For modifying the activity and/or toxicity of FK-506-type 
immunosuppressants, a compound of Formula I may be administered prior to, 
in conjunction with or subsequent to the administration of an FK-506-type 
of a compound. 
Dosage levels of the compounds of the present invention are of the order 
from about 0.005 mg to about 50 mg per kilogram of body weight per day, 
preferably from about 0.1 mg to about 10 mg per kilogram of body weight 
per day, are useful in the treatment of the above-indicated conditions 
(from about 0.7 mg to about 3.5 mg per patient per day, assuming a 70 kg 
patient). In addition, the compounds of the present invention may be 
administered on an intermittent basis; i.e. at semiweekly, weekly, 
semi-monthly or monthly intervals. 
The amount of active ingredient that may be combined with the carrier 
materials to produce a single dosage form will vary depending upon the 
host treated and the particular mode of administration. For example, a 
formulation intended for the oral administration of humans may contain 
from 0.5 mg to 5 gm of active agent compounded with an appropriate and 
convenient amount of carrier material which may vary from about 5 to about 
95 percent of the total composition. Dosage unit forms will generally 
comprise from about 0.01 mg to about 500 mg, and preferably about 0.5 mg 
to about 100 mg of active ingredient. For external administration the 
compound of Formula I may be formulated within the range of, for example, 
0.0001% to 60% by weight, preferably from 0.001 to 10% by weight, and most 
preferably from about 0.005 to 0.8% by weight. 
It will be understood, however, that the specific dose level for any 
particular patient will depend on a variety of factors including the 
activity of the specific compound employed, the age, body weight, general 
health, sex, diet, time of administration, route of administration, rate 
of excretion, drug combination and the severity of the particular disease 
undergoing therapy. 
The following examples are given for the purpose of illustrating the 
present invention and shall not be construed as being limitations on the 
scope or spirit of the instant invention. 
PREATION OF STARTING INTERMEDIATES 
17-Ethyl-1-hydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylvinyl] 
-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1. 
0.sup.4,9 ]-octacos-18-ene-2,3,10,16-tetraone 
A solution of 500 mg of 
17-ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22 
.3.1.0.sup.4,9] octacos-18-ene-2,3,10,16-tetraone in 7 ml of benzene was 
treated with 10 mg of p-toluenesulfonic acid and the solution was heated 
at 60.degree. C. for two hours. The reaction mixture was quenched into 
saturated sodium bicarbonate solution and extracted with ethyl acetate. 
The combined organic layers were washed with water and saturated sodium 
chloride solution, dried with anhydrous magnesium sulfate and 
concentrated. The residue was chromatographed on silica gel (66% ethyl 
acetate: 33% hexane: 1% methanol) to give 350 mg of product. This material 
was dissolved in 10 ml of ethyl acetate and treated with 15 mg of 5% Rh/C. 
A balloon containing hydrogen was placed over the reaction mixture and the 
mixture stirred until the reaction was complete. The mixture was filtered 
through diatomaceous earth, concentrated and the residue subjected to 
chromatography (75% CH.sub.2 Cl.sub.2 : 5% MeOH: 20% Hexane) to give 294 
mg of product. 
17-Ethyl-1-hydroxy-12-[2'-(4",3"-dihydroxyoxycyclohexyl)-1'-methylvinyl]-23 
,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[22.3.1.0.s 
up.4,9 ]octacos-18-ene-2,3,10,16-tetraone 
A solution of 
17-ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-hydroxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo-[2 
2.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone (210 mg) and a catalytic 
amount of p-toluenesulfonic acid in 40 ml of benzene was refluxed for 4 
hours under a nitrogen atmosphere. The solvent was removed under reduced 
pressure and the dark residue was purified by chromatography (silica gel, 
7% i-propanol/CH.sub.2 Cl.sub.2) to give 
17-ethyl-1-hydroxy-12-[2'-(4"-hydroxy-3"-isopropyloxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,-27-tetramethyl-11,28-dioxa-4-azatricyclo-[ 
22.3.1.0.sup.4,9 ]octacos-14,18-diene-2,3,10,16-tetraone (180 mg) as a 
white solid. This material was dissolved in ethanol (20 ml) and treated 
with 5% Rh/C (40 mg). Hydrogen was introduced via balloon for 30 min. and 
the mixture was filtered through celite. Removal of solvent followed by 
chromatography (silica gel) gave 172 mg of the title compound. Mass, 
.sup.1 H and .sup.13 C NMR data were consistant with the title structure. 
17-Ethyl-1-hydroxy-12-[2'-(4"-triisopropylsilyloxy-3"-methoxycyclohexyl)-1' 
-methylvinyl]-14-triisopropylsilyloxy-23,25-dimethoxy-13,19,21,27-tetrameth 
yl-11,28-dioxa-4-azatricyclo-[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10,16-tetraone 
To a cooled solution (0.degree. C.) of 
17-ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22 
.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone (120 mg) in dry methylene 
chloride (15 ml) was added 2,6-lutidine (64.3 mg) followed by 
triisopropylsilyl trifluoromethanesulfonate (184 mg). Reaction temperature 
was raised to r.t. and stirred overnight under nitrogen atmosphere. The 
reaction was quenched with 10 ml of water and extracted with ethyl 
acetate. Organic layer was washed (water, sat'd NaHCO.sub.3, sat'd NaCl) 
and dried (anhydrous MgSO.sub.4). Removal of solvent followed by 
chromatography on silica gel (70% hexane/ethyl acetate) gave 150 mg of 
product. MASS: (FAB) 1110 (M.sup.+ +Li). 
17-Ethyl-1-hydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylvinyl] 
-14-triisopropylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-diox 
a-4-azatricyclo[22.3.1.0.sup.4,9 ]-octacos-18-ene-2,3,10,16-tetraone 
The title compound from the previous preparation (680 mg) was dissolved in 
methylene chloride (45 ml) and 10% solution of p-toluenesulfonic acid in 
methanol (45 ml) was added with stirring. The mixture was stirred at room 
temperature and the progress was followed by tlc analysis. After 4 hr, 
reaction was quenched with sat'd sodium bicarbonate and extracted with 
ethyl acetate three times. Normal work-up and removal of solvent followed 
by purification on silica gel column (80% ethyl acetate/hexane) gave 560 
mg of the product (2a) as a white solid. MASS: (FAB) 954 (M.sup.+ +Li). 
17-Ethyl-1-hydroxy-12-[2'-(4"-t-butyldimethylsilyloxy-3"-methoxycyclohexyl) 
-1'-methylvinyl]-14-t-butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tet 
ramethyl-11,28-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10,16-tetraone 
To a cooled solution (0.degree. C.) of 
17-ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetra-methyl-11,28-dioxa-4-azatricyclo[2 
2.3.1.0.sup.4,9 [octacos-18-ene-2,3,10,16-tetraone (1a) (395 mg) in dry 
methylene chloride (15 ml) was added 2,6-lutidine (160 mg) followed by 
t-butyldimethylsilyl triflouromethanesulfonate (250 mg). Reaction 
temperature was raised to r.t. and stirred under nitrogen atmosphere. 
After 6 hr, the reaction was quenched with 10 ml of water and extracted 
with ethyl acetate. Organic layer was washed (water, saturated 
NaHCO.sub.3, saturated NaCl) and dried (anhydrous MgSO.sub.4). Removal of 
solvent under reduced pressure gave 500 mg of crude product. MASS: (FAB) 
1026 (M.sup.+ +Li). 
17-Ethyl-1-hydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylvinyl] 
-14-t-butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-d 
ioxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone 
The product from the previous example (500 mg) was dissolved in 
acetonitrile (20 ml) and 100 ml of hydrogen fluoride (48%) was added. 
Reaction was stirred for 20 minutes at room temperature, quenched with 
saturated sodium bicarbonate, then extracted with ethyl acetate. Removal 
of solvent in vacuo followed by chromatography on silica gel (80% ethyl 
acetate/hexane) gave 300 mg of product (Mass, .sup.1 H and .sup.13 C NMR 
data consistent with the title compound. 
17-Ethyl-1-hydroxy-12-[2'-(4"-(tert-butyldimethylsiloxy)-3"-hydroxycyclohex 
yl)-1'-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-a 
zatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone 
To a solution of 
17-ethyl-1-hydroxy-12-[2'-(3",4"-dihydroxycyclohexyl)-1'-methylvinyl]-23,2 
5-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.04,9]o 
ctacos-18-ene-2,3,10,16-tetraone (3.01 g) in dry methylene chloride (70 ml) 
was added an excess of imidazole (809 mg) followed by 
tert-butyldimethylsilyl chloride (716 mg). After 3 days of stirring at 
room temperature, the mixture was diluted with ethyl acetate which in turn 
was washed with 1N HCl, saturated sodium bicarbonate and brine, dried over 
magnesium sulfate and purified by flash chromatography (ethyl 
acetae:hexane (1:3)) to give the title compound (941 mg). .sup.1 H NMR 
consistent with the desired structure. 
17-Ethyl-1-hydroxy-12-[2'-(4"-(tert-butyldimethylsilyloxy)-3"-methoxycycloh 
exyl)-1'-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4 
-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone 
To a solution of 
17-ethyl-1-hydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylvinyl 
]-23,25-dimethoxy-13,19,21,-27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1 
.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone (200 mg) in dry methylene 
chloride (3 ml) was added an excess of 2,6-lutidine (45 .mu.l) and the 
mixture was stirred at room temperature. After 10 minutes, 
tert-butyldimethylsilyl trifluoromethanesulfonate (64 .mu.l) was added by 
syringe. After 15 minutes the reaction mixture was diluted with ethyl 
acetate, extracted from saturated bicarbonate, washed with brine and the 
organic phase dried over magnesium sulfate. Removal of solvent in vacuo 
and flash chromatography on silica gel (ethyl acetate:hexane (1:2)+1% 
methanol) gave the title compound (235 mg). 
(.sup.1 H NMR consistent with the desired structure). 
17-Ethyl-1,20-dihydroxy-12-[2'-(4"-tert-butyldimethylsilyloxy)-3"-methoxycy 
clohexyl)-4"1'-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-d 
ioxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone 
To a stirred solution of 
17-ethyl-1-hydroxy-12-[2'-(4"-(tert-butyldimethylsilyloxy)-3"-methoxycyclo 
hexyl)-1'-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa- 
4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone (235 mg) 
in 95% ethanol (2.2 ml) was added 53 .mu.l of pyridine followed by 
selenium dioxide (58 mg). The flask was fitted with a water condenser and 
heated to 70.degree. C. on a mantle. After 20 hours the mixture was cooled 
to room temperature filtered through diatomaceous earth and the filtrate 
poured into a saturated sodium bicarbonate solution. This was extracted 
with ethyl acetate, washed with brine and dried over magnesium sulfate. 
The solution was concentrated and purified by flash chromatography on 
silica gel (ethyl acetate:hexane (1:2)+1% methanol) to give the title 
compound (89 mg). 
(.sup.1 H NMR consistent with the desired structure). 
17-Ethyl-20-fluoro-1-hydroxy-12-[2'-(4"-(tert-butyldimethylsiloxy)-3"-metho 
xycyclohexyl)-1'-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28 
-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10.16-tetraone 
A solution of 
17-ethyl-20-dihydroxy-12-[2'-(4"-(tert-butyldimethylsiloxy)-3"-methoxycycl 
ohexyl)-1'-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa 
-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10.16-tetraone (30.5 
mg) in methylene chloride (0.5 ml) was cooled to -78.degree. C. in a dry 
ice/isopropanol bath. To this stirred solution, diethylaminosulfur 
trifluoride (4.5 .mu.l) was added. After 3 minutes, saturated sodium 
bicarbonate (500 .mu.l) was added followed by ethyl acetate (2 ml) and the 
mixture was warmed to room temperature. Extraction from ethyl acetate, 
drying over magnesium sulfate and purification by flash chromatography on 
silica gel (ethyl acetate:hexane (1:2)+1% MeOH) gave the title compound 
(22 mg). 
(.sup.1 H NMR consistent with the desired structure). 
17-Ethyl-1,20-dihydroxy-12-[2'-(4"-(hydroxy-3"-methoxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22 
.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10.16-tetraone 
To a solution of 
17-ethyl-1,20-dihydroxy-12-[2'-(4"-(tert-butyldimethylsiloxy)-3"-methoxycy 
clohexyl)-1'-methylvinyl]23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-diox 
a-4-azatricyclo[22.3.1.0.sup.4,9 ]-octacos-18-ene-2,3,10.16-tetraone (7 mg) 
in acetonitrile (0.3 ml) was added a solution of 2% hydrogen fluoride in 
aqueous acetonitrile (100 .mu.l), and the mixture stirred at room 
temperature. After 28 hours the solution was diluted with ethyl acetate, 
extracted with saturated sodium bicarbonate and the organic phase dried by 
passage through a magnesium sulfate column. Purification of the 
concentrate by flash chromatography on silica gel (ethyl acetate:hexane 
(2:1)+1% methanol) gave the title compound. 
17-Ethyl-20-fluoro-1-hydroxy-12-[2'-(4"-(hydroxy-3"-methoxycyclohexyl)-1'-m 
ethylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyc 
lo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10.16-tetraone 
To a solution of 
17-ethyl-20-fluoro-1-hydroxy-12-[2'-(4"-(tert-butyldimethylsiloxy)-3"-meth 
oxycyclohexyl)-1'-methylvinyl]23,25-dimethoxy-13,19,21,27-tetramethyl-11,28 
-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10.16-tetraone (7 
mg) in acetonitrile (0.3 ml) was added a solution of 2% hydrogen fluoride 
in aqueous acetonitrile (100 .mu.l), and the mixture stirred at room 
temperature. After 2 hours the solution was diluted with ethyl acetate, 
extracted with saturated sodium bicarbonate and the organic phase dried by 
passage through a magnesium sulfate column. Purification of the 
concentrate by flash chromatography on silica gel (ethyl acetate:hexane 
(1:1)+1% methanol) gave the title compound. MASS: (FAB) 816 (M+Na). 
partial .sup.13 C NMR .delta.: 211.5 (C-16); 196.1 (2) 169.3 (10); 165.0 
(3); 138.1 (C-19); 135.8 (C-1'); 121.0 (C-18' major); 84.1 (C-3"); 43.1 
(C-15); 26.0 (C-21). 
17-Ethyl-1,14,20-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo 
[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone 
Alternate Route 
To a solution of 
17-ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22 
.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone (5.15 gm, 0.065 mol) in 
glacial acetic acid (500 ml) at room temperature, was added a solution of 
selenium dioxide (9.27 gm, 0.083 mol) in H.sub.2 O (90 ml). The reaction 
mixture was stirred at room temperature for 41 hours whereupon, it was 
poured into a stirred mixture of H.sub.2 O (3 L) and celite. After 
stirring for 15 minutes, the mixture was filtered through a pad of celite 
and extracted with diethyl ether (1.times.2 L, 2.times.1 L). The organic 
fractions were washed with saturated sodium bicarbonate and brine, dried 
over magnesium sulfate, filtrated and evaporated in vacuo. The product was 
purified by chromatography (silica, acetone:hexanes 2:5) to give the title 
compound MASS and .sup.1 H NMR were consistent with the structure. 
EXAMPLE 1 
17-Ethyl-1-hydroxy-12-[2'-(4"-t-butyldimethylsilyloxy-3"-methoxycyclohexyl) 
-1'-methylvinyl]-14-t-butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tet 
ramethyl-11,28-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10,16-tetraone 
To a cooled solution (0.degree. C.) of 
17-ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22 
.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone (1a) (4.00, 5 mmole) in 
dry methylene chloride (50 ml) was added 2,6-lutidine (4 mL, 33 mmole) 
followed by t-butyldimethylsilyl trifluoromethanesulfonate (5 mL, 21 
mmole). Reaction temperature was raised to r.t. and stirred under nitrogen 
atmosphere. After 2 hr, the reaction was quenched with 10 ml of methanol 
and partitioned between ether and water. The ether layer was washed 
sequentially with 2 portions of 2M H.sub.2 SO.sub.4, brine, NaHCO.sub.3, 
and brine, then dried over MgSO.sub.4. Removal of solvent under reduced 
pressure gave 5.4 g of crude product. MASS SPECTRUM (FAB) m/e 1026(M+Li). 
EXAMPLE 2 
17-Ethyl-1-hydroxy-12-[2'-(4"-triethylsilyloxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-14-triethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28 
-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone 
To a cooled solution (0.degree. C.) of 
17-ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22 
.3.1.0.sup.4,9 ]-octacos-18-ene-2,3,10,16-tetraone (4.00, 5 mmole) in dry 
methylene chloride (50 ml) was added 2,6-lutidine (4 mL, 33 mmole) 
followed by triethylsilyl trifluoromethanesulfonate (5 mL, 22 mmole). 
Reaction temperature was raised to r.t. and stirred under nitrogen 
atmosphere. After 1 hr, the reaction was quenched with 10 ml of methanol 
and partitioned between ether and water. The ether layer was washed 
sequentially with 2 portions of 2M H.sub.2 SO.sub.4, brine, NaHCO.sub.3, 
and brine, then dried over MgSO.sub.4. Removal of solvent under reduced 
pressure gave 5.3 g of crude product. MASS SPECTRUM (FAB) m/e 1026(M+Li). 
EXAMPLE 3 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-methyl-13,19,21,27-tetramethyl-11,28-dioxa-4-a 
zatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione 
Step 1: 
17-Ethyl-14-t-butyldimethylsilyloxy-1,16-dihydroxy-12-[2'-(4"-t-butyldimet 
hylsilyloxy-3"-methoxycyclohexyl)-1'-methylvinyl]-23,25-dimethoxy-16-methyl 
-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10-trione 
A solution of 0.101 g (0.1 mmole) of 
17-ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22 
.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetrone in 5 mL of dry THF was 
cooled to 0.degree. C. under nitrogen. Then 1 mL of a 0.5M solution of 
methyllithium was added dropwise and the solution was stirred at 
-78.degree. C. for 24 h. The solution was quenched at -78.degree. C. by 
addition of 0.2 mL of glacial acetic acid and then diluted with 20 mL of 
ether and partitioned between ether and 1M KHCO.sub.3 solution. The ether 
layer was washed with 1M KHCO.sub.3 and brine, dried over MgSO4, and 
concentrated. The residue was purified by flash chromatography (2.times.20 
cm silica gel) using 20% ethyl acetate-hexane to afford the 0.091 g (88%) 
of title compound as a colorless oil. 
.sup.1 H NMR (CDCl.sub.3, 300 MHz) .delta.0.02-0.04 (s, 12H), 0.6-1.0 (m, 
16H), 0.90 (s, 18H), 1.2 (s, 3H, 16-CH.sub.3), 1.3-2.4 (m), 2.9 (m, 2H), 
3.2-3.6 (m), 3.8 (s, 1H), 4.05 (m, 1H), 4.25 (m, 1H), 4.39 (d, 1H), 4.78 
(d, 1H), 4.95 (m, 2H), 5.25 (d, 1H); .sup.13 C NMR (CDCl.sub.3, 75 MHz) 
.delta.83.8, 97.5, 128.0, 128.7, 133.4, 136.9, 164.8, 168.9, 195.8; MASS 
SPECTRUM (FAB) 1042(M+Li). 
Step 2: 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-me 
thylvinyl]-23,25-dimethoxy-16-methyl-13,19,21,27-tetramethyl-11,28-dioxa-4- 
azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione 
A solution of 0.086 g (0.082 mmole) of 
17-ethyl-14-t-butyldimethylsilyloxy-1,16-dihydroxy-12-[2'-(4"-t-butyldimet 
hylsilyloxy-3"-methoxycyclohexyl)-1'-methylvinyl]-23,25-dimethoxy-16-methyl 
-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10-trione in 2 mL of a solution of 1:9 48% aqueous 
HF-acetonitrile was stirred at room temperature for 8 h. The reaction was 
quenched by addition of 2 mL of ethoxytrimethylsilane and the solution was 
concentrated to dryness under vacuum. The residue was purified by flash 
chromatography (2.times.20 cm silica gel) using 25% acetone-hexane to 
afford 0.051 g (77%) of the title compound as a white solid. 
.sup.1 H NMR (CDCl.sub.3, 300 MHz) .delta.0.7-1.1 (m, 16H), 1.22 (s, 3H, 
16CH.sub.3), 1.25-2.5 (m), 2.71 (m, 1H), 2.90 (t, 1H), 2.95 (m, 1H), 
3.2-3.6 (m), 3.76 (s, 1H), 4.05 (m, 1H), 4.29 (m, 1H), 4.43 (d, 1H), 4.80 
(d, 1H), 4.95 (m, 2HO, 5.24 (m, 1H); .sup.13 C NMR (CDCl.sub.3, 300 MHz) 
.delta.84.1, 97.7, 127.8, 128.2, 133.6, 136.9, 164.9, 168.6, 195.9; MASS 
SPECTRUM (FAB) 814 (M+Li). 
EXAMPLE 4 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-methyl-13,19,21,27-tetramethyl-11,28-dioxa-4-a 
zatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione 
A solution of 0.101 g (0.1 mmole) of 
17-ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22 
.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetrone in 5 mL of dry THF was 
cooled to 0.degree. C. under nitrogen. Then 1 mL of a 0.5M solution of 
methyllithium was added dropwise and the solution was stirred at 
-78.degree. C. for 24 h. The solution was quenched at -78.degree. C. by 
addition of 0.2 mL of glacial acetic acid and then diluted with 20 mL of 
ether and partitioned between ether and 1M KHCO.sub.3 solution. The ether 
layer was washed with 1M KHCO.sub.3 and brine, dried over MgSO4, and 
concentrated. The residue was dissolved in 2 mL of 1:9 48% aqueous 
HF-acetonitrile was stirred at room temperature for 8 h. The reaction was 
quenched by addition of 2 mL of ethoxytrimethylsilane and the solution was 
concentrated to dryness under vacuum. The residue was purified by flash 
chromatography (2.times.20 cm silica gel) using 25% acetone-hexane to 
afford 0.053 g (68%) of the title compound as a white solid. 
.sup.1 H NMR (CDCl.sub.3, 300 MHz) .delta.0.7-1.1 (m, 16H), 1.22 (s, 3H, 
16CH.sub.3), 1.25-2.5 (m), 2.71 (m, 1H), 2.90 (t, 1H), 2.95 (m, 1H), 
3.2-3.6 (m), 3.76 (s, 1H), 4.05 (m, 1H), 4.29 (m, 1H), 4.43 (d, 1H), 4.80 
(d, 1H), 4.95 (m, 2H), 5.24 (m, 1H); .sup.13 C NMR (CDCl.sub.3, 300 MHz) d 
84.1, 97.7, 127.8, 128.2, 133.6, 136.9, 164.9, 168.6, 195.9; MASS SPECTRUM 
(FAB) 814 (M+Li). 
EXAMPLE 5 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-butyl-13,19,21,27-tetramethyl-11,28-dioxa-4-az 
atricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione 
Prepared as in Example 4 except that a 2.0M solution of butyllithium was 
used in place of methyllithium to afford 0.062 g (72%) of the title 
compound as a white solid; MASS SPECTRUM (FAB) m/e 856 (M+Li). 
EXAMPLE 6 
17-Ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylv 
inyl]-23,25-dimethoxy-16-butyl-13,19,21,27-tetramethyl-16-methenyl-11,28-di 
oxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione 
A solution of 0.201 g (0.2 mmole) of 
17-ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22 
.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetrone in 5 mL of dry THF was 
cooled to 0.degree. C. under nitrogen. Then 1 mL of a 0.5M solution of 
trimethylsilylmethyllithium was added dropwise and the solution was 
stirred at -78.degree. C. for 24 h. The solution was quenched at 
-78.degree. C. by addition of 0.2 mL of glacial acetic acid and then 
diluted with 20 mL of ether and partitioned between ether and 1M 
KHCO.sub.3 solution. The ether layer was washed with 1M KHCO.sub.3 and 
brine, dried over MgSO4, and concentrated. The residue was dissolved in 2 
mL of 1:9 48% aqueous HF-acetonitrile was stirred at room temperature for 
8 h. The reaction was quenched by addition of 2 mL of 
ethoxytrimethylsilane and the solution was concentrated to dryness under 
vacuum. The residue was purified by flash chromatography (2.times.20 cm 
silica gel) using 25% acetone-hexane to afford 0.137 g (86%) of the title 
compound as a white solid. 
.sup.1 H NMR (CDCl.sub.3, 300 MHz) .delta.0.7-1.1 (m, 16H), 1.25-2.5 (m), 
2.59 (s, 1H), 2.7-3.1 (m, 3H), 3.2-3.6 (m), 3.63 (d, 1H), 3.76 (s, 1H), 
4.35 (d, 1H), 4.45 (d, 1H), 4.80 (d, 1H), 5.95 (m, 3H), 5.16 (s, 1H), 5.29 
(m, 1H); .sup.13 C NMR (CDCl.sub.3, 300 MHz) d 84.1, 97.7, 117.8, 127.9, 
128.2, 133.6, 136.9, 147.4, 164.9, 168.6, 195.9; MASS SPECTRUM (FAB) 796 
(M+Li). 
EXAMPLE 7 
17-Ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylv 
inyl]-23,25-dimethoxy-16-butyl-13,19,21,27-tetramethyl-16-(2-butenyl)-11,28 
-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione 
Prepared as in Example 4 except that a 2.0M solution of sec-butyllithium 
was used in place of methyllithium to afford 0.068 g (71%) of the title 
compound as a white solid; MASS SPECTRUM (FAB) m/e 838 (M+Li). 
EXAMPLE 8 
17-Ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylv 
inyl]-23,25-dimethoxy-16-propenyl-13,19,21,27-tetramethyl-11,28-dioxa-4-aza 
tricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione (fast isomer) 
Prepared as in Example 6 except that a solution of 
1-trimethylsilylpropyllithium was used in place of methyllithium to afford 
0.042 g (51%) of the title compound as a white solid; MASS SPECTRUM (FAB) 
m/e 824 (M+Li). 
EXAMPLE 9 
17-Ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylv 
inyl]-23,25-dimethoxy-16-propenyl-13,19,21,27-tetramethyl-11,28-dioxa-4-aza 
tricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione (slow isomer) 
Further elution of the column from Example 8 with 25% acetone-hexane 
afforded 0.032 g (39%) of the title compound as a white solid; MASS 
SPECTRUM (FAB) m/e 824 (M+Li). 
EXAMPLE 10 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-(methylthiomethyl)-13,19,21,27-tetramethyl-11, 
28-dioxa-a-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione 
Prepared as in Example 4 except that a solution of methylthiomethyllithium 
was used in place of methyllithium to afford 0.079 g (92%) of the title 
compound as a white solid; MASS SPECTRUM (FAB) m/e 860 (M+Li). 
EXAMPLE 11 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-(methylsulfinylmethyl)-13,19,21,27-tetramethyl 
-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione 
A solution of 0.050 g (0.059 mmole) of 
17-ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-me 
thylvinyl]-23,25-dimethoxy-16-(methylthiomethyl)-13,19,21,27-tetramethyl-11 
,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione in 5 
mL of methanol was cooled to 0.degree. C. in an ice bath. Then 2 mL of a 
0.5M solution of NaIO.sub.4 was added and the mixture was stirred at room 
temperature for 1 h. The solution was partitioned between ether and water 
and the ether layer was washed with KHCO.sub.3 and brine, then dried over 
MgSO.sub.4 and concentrated. The residue was purified by flash 
chromatography (2.times.20 cm silica gel) using 20% acetone-hexane to 
afford 0.022 g (44%) of the title compound as a white solid; MASS SPECTRUM 
(FAB) m/e 876 (M+Li). 
EXAMPLE 12 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-(methylsulfinylmethenyl)-13,19,21,27-tetrameth 
yl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione 
Further elution of the column in Example 10 with 30% acetone-hexane 
afforded 0.018 g (35%) of the title compound as a white solid; MASS 
SPECTRUM (FAB) m/e 858 (M+Li). 
EXAMPLE 13 
17-Ethyl-16-(triethylsilyloxy) 
1,14-dihydroxy-12-[2'-(4"-triethylsilyloxy-3"-methoxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22 
.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione-16-(methyldimethylsulfonium) 
iodide 
To a stirred solution of 612 mg trimethylsulfonium iodide (3.0 mmole) in 
3.9 mL of THF at 0.degree. C. was added methyllithium (1.4M in ether, 2.1 
mL, 2.94 mmole). The mixture was stirred for 1 h at 0.degree. C. To 2 mL 
of the resultant solution at -78.degree. C. was added 200 mg of 
17-ethyl-1-hydroxy-12-[2'-(4"-triethylsilyloxy-3"-methoxycyclohexyl)-1'-me 
thylvinyl]-14-triethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,2 
8-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone 
(0.2 mmole) and the mixture was stirred at -78.degree. C. for 2 h. The 
reaction was quenched with two drops of glacial acetic acid, diluted with 
ether and washed with sat aq. KHCO.sub.3, water and brine. The residue was 
purified by flash chromatography (2.times.20 cm silica gel) using 20% 
ethyl acetate-hexane remove starting material, followed by 5% CH.sub.3 
OH--CH.sub.2 Cl.sub.2 to afford 0.034 g (17%) of the title compound as a 
white solid; MASS SPECTRUM (FAB) m/e 1096 (M+) 
EXAMPLE 14 
17-Ethyl-16-triethylsilyloxy-1,14-diihydroxy-12-[2'-(4"-triethylsilyloxy-3" 
-methoxycyclohexyl)-1'-methylvinyl]-23,25-dimethoxy-16-(methylthiomethyl)-1 
3,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10-trione (faster isomer) 
Step 1: 
17-Ethyl-1,16-dihydroxy-12-[2'-(4"-triethylsilyloxy-3"-methoxycyclohexyl)- 
1'-methylvinyl]-14-triethylsilyloxy-23,25-dimethoxy-16-(methylthiomethyl)-1 
3,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10-trione (faster isomer) 
A sample of 34 mg (0.034 mmole) of 17-ethyl-16-(triethylsilyloxy) 
1,14-dihydroxy-12-[2'-(4"-triethylsilyloxy-3"-methoxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22 
.3.1.0.sup.4,9 ]one-16-(methyldimethylsulfonium) iodide was dissolved in 3 
mL of CH.sub.2 Cl.sub.2 and treated with four drops of triethylamine. The 
solution was concentrated under vacuum and the residue was purified by 
flash chromatography (1.5 cm.times.15 cm) using 10% ethyl acetate-hexane 
to afford 0.12 g (32%) the title compound as a white solid; MASS SPECTRUM 
(FAB) m/e 1088 (M+Li). 
Step 2: 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-me 
thylvinyl]-23,25-dimethoxy-16-(methylthiomethyl)-13,19,21,27-tetramethyl-11 
,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione 
(faster isomer) 
A sample of 10 mg (0.009 mmole) of 
17-ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-me 
thylvinyl]-23,25-dimethoxy-16-(methylthiomethyl)-13,19,21,27-tetramethyl-11 
,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione 
(faster isomer) was deprotected as in Part 2 of Example 3 to afford 6 mg 
(76%) of the title compound as a white solid whose .sup.1 H and .sup.13 C 
NMR spectra indicated that it was isomeric to the product of Example 10 at 
the 16 position; MASS SPECTRUM (FAB) m/e 860 (M+Li). 
EXAMPLE 15 
17-Ethyl-16-triethylsilyloxy-1,14-diihydroxy-12-[2'-(4"-triethylsilyloxy-3" 
-methoxycyclohexyl)-1'-methylvinyl]-23,25-dimethoxy-16-(methylthiomethyl)-1 
3,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10-trione (slower isomer) 
Step 1 
Further elution of the column in Part 1 of Example 13 using 10% ethyl 
acetate-hexane afforded 0.10 g (26%) the title compound as a white solid 
whose .sup.1 H and .sup.13 C NMR spectra were identical to those of the 
protected product from Example 10; MASS SPECTRUM (FAB) m/e 1088 (M+Li). 
Step 2: 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-me 
thylvinyl]-23,25-dimethoxy-16-(methylthiomethyl)-13,19,21,27-tetramethyl-11 
,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione 
(faster isomer) 
A sample of 10 mg (0.009 mmole) of 
17-ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-me 
thyl-vinyl]-23,25-dimethoxy-16-(methylthiomethyl)-13,19,21,27-tetramethyl-1 
1,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione 
(faster isomer) was deprotected as in Part 2 of Example 3 to afford 6 mg 
(76%) of the title compound as a white solid whose .sup.1 H and .sup.13 C 
NMR spectra were identical to those of the product of Example 10; MASS 
SPECTRUM (FAB) m/e 860 (M+Li). 
EXAMPLE 16 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-phenyl-13,19,21,27-tetramethyl-11,28-dioxa-4-a 
zatricyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione 
Prepared as in Example 4 except that a solution of phenyllithium was used 
in place of methyllithium to afford 0.029 g (33%) of the title compound as 
a white solid; MASS SPECTRUM (FAB) m/e 876 (M+Li). 
EXAMPLE 17 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-23,25-dimethoxy-16-furan-2-yl-13,19,21,27-tetramethyl-11,28-dioxa 
-4-azatricyclo[2-2.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10-trione 
Prepared as in Example 4 except that a solution of 2-lithiofuran was used 
in place of methyllithium to afford 0.021 g (24%) of the title compound as 
a white solid; MASS SPECTRUM (FAB) m/e 866 (M+Li). 
EXAMPLE 18 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-14-triethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2-(1- 
methyl)-11,28-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-3,10,16-tetraone 
A solution of 0.100 g (0.1 mmole) of 
17-ethyl-1-hydroxy-12-[2'-(4"-triethylsilyloxy-3"-methoxycyclohexyl)-1'-me 
thylvinyl]-14-triethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,2 
8-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone 
in dry THF was cooled to -78.degree. C. Then 0.1 mL of a 3.0M solution of 
methylmagnesium bromide was added and the solution was allowed to warm to 
0.degree. C. The solution was acidified by addition of 0.1 mL of glacial 
acetic acid and the mixture was partitioned between ether and KHCO.sub.3 
solution. The ether layer was washed with KHCO.sub.3 and brine, then dried 
over MgSO.sub.4 and concentrated. The residue was dissolved in 2 mL of a 
1:19 solution of 48% aqueous HF-acetonitrile and left stirring at room 
temperature for 1 h. The reaction was quenched with 1 mL of 
ethoxytrimethylsilane and concentrated under vacuum. The residue was 
purified by preparatory TLC (4 plates, 250 m) using 15% acetone-hexane to 
afford 61 Mg (73%) of the title compound as a white solid. 
.sup.1 H NMR (CDCl.sub.3, 300 MHz) .delta.0.7-1.1 (m, 16H), 1.22 (s, 3H), 
1.25-2.5 (m), 2.59 (s, 1H), 2.7-3.1 (m, 3H), 3.2-3.6 (m), 3.63 (d, 1H), 
3.76 (s, 1H), 4.35 (d, 1H), 4.45 (d, 1H), 4.80 (d, 1H), 4.95 (m, 2H), 5.29 
(m, 1H); .sup.13 C NMR (CDCl.sub.3, 300 MHz) .delta.84.1, 97.7, 127.9, 
128.2, 133.6, 136.9, 168.6, 173.2, 212.4; MASS SPECTRUM (FAB) 814 (M+Li). 
EXAMPLE 19 
17-Ethyl-1,14,16-trihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-met 
hylvinyl]-14-triethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2-(2- 
propenyl)-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-3,10,16-tetraone 
Prepared as in Example 18 except that a solution of allylmagnesium bromide 
was used in place of methylmagnesium bromide to afford 0.056 g (67%) of 
the title compound as a white solid; MASS SPECTRUM (FAB) m/e 839 (M+Li). 
EXAMPLE 20 
17-Ethyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylv 
inyl]-14-triethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2-metheny 
l-11,28-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-3,10,16-tetraone 
A solution of 0.100 g (0.1 mmole) of 
17-ethyl-1-hydroxy-12-[2'-(4"-triethylsilyloxy-3"-methoxycyclohexyl)-1'-me 
thylvinyl]-14-triethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,2 
8-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone 
in dry THF was cooled to -78.degree. C. Then 0.3 mL of a 1.0M solution of 
trimethylsilylmethylmagnesium bromide was added and the solution was 
allowed to warm to 0.degree. C. until tlc (silica gel, 20% ethyl 
acetate-hexane) indicated that no starting material remained. The solution 
was acidified by addition of 0.1 mL of glacial acetic acid and the mixture 
was partitioned between ether and KHCO.sub.3 solution. The ether layer was 
washed with KHCO.sub.3 and brine, then dried over MgSO.sub.4 and 
concentrated. The residue was dissolved in 2 mL of a 1:19 solution of 48% 
aqueous HF-acetonitrile and left stirring at room temperature for 1 h. The 
reaction was quenched with 1 mL of ethoxytrimethylsilane and concentrated 
under vacuum. The residue was purifed by preparatory TLC (4 plates, 250 m) 
using 15% acetone-hexane to afford 15 mg (18%) of the title compound as a 
white solid. 
.sup.1 H NMR (CDCl.sub.3, 300 MHz) .delta.0.7-1.1 (m, 16H), 1.25-2.5 (m), 
2.59 (s, 1H), 2.7-3.1 (m, 3H), 3.2-3.6 (m), 3.63 (d, 1H), 3.76 (s, 1H), 
4.35 (d, 1H), 4.45 (d, 1H), 4.80 (d, 1H), 4.95 (m, 2H), 5.29 (m, 1H), 
6.2-6.3 (m, 2H); .sup.13 C NMR (CDCl.sub.3, 300 MHz) d 84.1, 97.7, 126.8, 
127.9, 128.2, 133.6, 136.9, 139.4, 164.9, 168.6, 195.9; MASS SPECTRUM 
(FAB) 796 (M+Li). 
EXAMPLE 21 
Product from intramolecular rearrangement of 
17-Ethyl-1,14-hydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylvi 
nyl]-14-triethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2-methenyl 
-11,28-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-3,10,16-tetraone 
A solution of 0.025 g (0.126 mmole) of 
17-ethyl-1,14-hydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylvi 
nyl]-14-triethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl-2-methenyl 
-11,28-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-3,10,16-tetraone in dichloromethane was left at room 
temperature for 24 h. TLC of the solution in 20% acetone-hexane indicated 
that a new, more polar product had formed and that this material no longer 
gave UV end-absorption characteristic of unsaturated carbonyl compounds. 
The solution was concentrated to dryness to afford 25 mg of the title 
compound as a pale yellow glass. 
.sup.1 H NMR (CDCl.sub.3, 300 MHz) .delta.0.7-1.1 (m, 16H), 1.25-2.5 (m), 
2.59 (s, 1H), 2.7-3.1 (m, 3H), 3.2-3.6 (m), 3.63 (d, 1H), 4.20-4.50 m, 
4H), 4.80 (d, 1H), 4.95 (m, 2H), 5.29 (m, 1H); .sup.13 C NMR (CDCl.sub.3, 
300 MHz) d 84.1, 127.9, 128.2, 133.6, 136.9, 168.6, 173.5; 204 (broad), 
212.2; MASS SPECTRUM (FAB) 796 (M+Li). 
EXAMPLE 22 
Step 1: 
17-Ethyl-12-[2'-(4"-t-butyldimethylsilyloxy-3"-methoxycyclohexyl)-1'-methy 
lvinyl]-14-t-butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl- 
11,28-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 
]octacos-1,18-diene-2,3,10,16-tetraone 
A solution of DEAD-PPH.sub.3 complex was prepared by dropwise addition of 
13.8 mL (95 mmole) of diethyl azodicarboxylate to a solution of 30 g 
(114.5 mmole) of triphenylphosphine in 200 mL of dry THF. This solution 
was stirred at room temperature for 30 minutes, then cooled to 0.degree. 
C. A solution of 34.1 g (31.6 mmole) of crude 
17-ethyl-1-hydroxy-12-[2'-(4"-t-butyldimethylsilyloxy-3"-methoxycyclohexyl 
)-1'-methylvinyl]-14-t-butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-te 
tramethyl-11,28-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10,16-tetraone in 500 mL of dry THF was cooled to 
0.degree. C. and then the first solution of DEAD-PPH.sub.3 was added at 
such a rate to keep the temperature of the solution below 10.degree. C. 
After 4 h, the reaction was quenched by addition of 30 mL of H.sub.2 O and 
the solution was concentrated to about 100 mL volume under vacuum. The 
solution was partitioned between ether and water and the ether layer was 
washed two times with brine, dried over MgSO.sub.4, and concentrated. The 
residue was crystallized from ether-hexane and filtered. The filtrate was 
concentrated and the oily residue was taken up in hexane and filtered a 
second time to remove residual triphenylphosphine oxide. The clear 
filtrate was concentrated to afford 35.2 g (100%) of the title compound as 
a pale yellow oil (that was contaminated with some residual 
triphenylphosphine). 
.sup.1 H NMR (CDCl.sub.3, 300 MHz) .delta.0.02-0.04 (s, 12H), 0.6-1.0 (m, 
16H), 0.90 (s, 18H), 1.3-2.4 (m), 2.15 (s, 3H, 27-CH.sub.3), 2.9 (m, 2H), 
3.2-3.8 (m), 4.45 (m, 1H), 4.95 (m, 2H), 5.25 (d, 2H); .sup.13 C NMR 
(CDCl.sub.3, 75 MHz) d 83.8, 97.5, 117, 128.0, 128.7, 133.4, 136.9, 145, 
164.8, 168.9, 188.5; MASS SPECTRUM (FAB) 1008(M+Li). 
Step 2: 
17-Ethyl-14-hydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylviny 
l]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatri-cyclo[22.3. 
1.0.sup.4,9 ]octacos-1, 18-diene-2,3,10,16-tetraone 
A sample of 2.01 g (2.02 mmole) of 
17-ethyl-12-[2'-(4"-t-butyldimethylsilyloxy-3"-methoxycyclohexyl)-1'-methy 
lvinyl]-14-t-butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl- 
11,28-dioxa-4-aza tri-cyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10,16-tetraone was deprotected as in Part 2 of Example 
3 to afford 1.46 g (94%) of the title compound as a white powder after 
lyophillization from benzene. 
.sup.1 H NMR (CDCl.sub.3, 300 MHz) .delta.0.6-1.0 (m, 16H), 1.3-2.4 (m), 
2.15 (s, 3H, 27-CH.sub.3), 2.9 (m, 2H), 3.2-3.8 (m), 4.45 (d, 1H), 4.95 
(m, 2H), 5.25 (m, 2H); .sup.13 C NMR (CDCl.sub.3, 75 MHz) d 83.8, 97.5, 
117, 128.0, 128.7, 133.4, 136.9, 145, 164.8, 168.9, 188.5; MASS SPECTRUM 
(FAB) 1008(M+Li). 
EXAMPLE 23 
17-Ethyl-14-hydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylvinyl 
]-23,25-dimethoxy-13,19, 
21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10,16-tetraone 
Step 1: 
17-Ethyl-12-[2'-(4"-t-butyldimethylsilyloxy-3"-methoxycyclohexyl)-1'-methy 
lvinyl]-14-t-butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl- 
11,28-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10,16-tetraone 
A solution of 35.2 g (31.6 mmole) of crude 
17-ethyl-12-[2'-(4"-t-butyldimethylsilyloxy-3"-methoxycyclohexyl)-1'-methy 
lvinyl]-14-t-butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl- 
11,28-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 
]octacos-1,18-diene-2,3,10,16-tetraone in 500 mL of dry THF was cooled to 
-90.degree. C. under nitrogen. Then 150 mL of a 0.5M solution of 
KHBPH.sub.3 in dry THF was added at such a rate so that the temperature 
did not rise above -78.degree. C. After addition was complete, the 
solution was left stirring under nitrogen for 72 h, then was quenched by 
dropwise addition of methanol, again keeping the temperature of the 
solution below -75.degree. C. The mixture was partitioned between ether 
and water and the ether layer was washed sequentially with KHCO.sub.3 and 
brine, dried over MgSO.sub.4, and concentrated. The residue was purified 
by flash chromatography (15 cm.times.40 cm) using 10% ethyl acetate-hexane 
to afford 11.28 g (35.7%) of recovered starting material. Further elution 
with 15% ethyl acetate-hexane afforded 14.13 g (45%) of the title compound 
as a white foam. 
NMR (CDCl.sub.3, 300 MHz) .delta.0.02-0.04 (s, 12H), 0.6-1.0 (m, 16H), 0.90 
(s, 18H), 1.3-2.4 (m), 2.9 (m, 2H), 3.2-3.8 (m), 4.39 (d, 1H), 4.85 (d, 
1H), 4.95 (m, 2H), 5.25 (d, 1H); .sup.13 C NMR (CDCl.sub.3, 75 MHz) d 
84.1, 122.5, 131.3, 132.2, 138.9, 165.9, 169.3, 200.4. 212; MASS SPECTRUM 
(FAB) 1010 (M+Li). 
Step 2: 
17-Ethyl-14-hydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylviny 
l]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1 
.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone 
A sample of 1.62 g (1.61 mmole) of 
17-ethyl-12-[2'-(4"-t-butyldimethylsilyloxy-3"-methoxycyclohexyl)-1'-methy 
lvinyl]-14-t-butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,27-tetramethyl- 
11,28-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10,16tetraone was deprotected as in Part 2 of Example 
3 to afford 0.962 g (77%) of the title compound as a white powder after 
lyophillization from benzene. 
.sup.1 H NMR (CDCl.sub.3, 300 MHz) .delta.0.6-1.0 (m, 16H), 1.3-2.4 (m), 
2.9 (m, 2H), 3.2-3.8 (m), 4.39 (d, 1H), 4.85 (d, 1H), 4.95 (m, 2H), 5.25 
(d, 1H); .sup.13 C NMR (CDCl.sub.3, 75 MHz) d 84.1, 122.5, 131.3, 132.2, 
138.9, 165.9, 169.3, 200.4. 212; MASS SPECTRUM (FAB) 782 (M+Li). 
EXAMPLE 24 
17-Ethyl-14-hydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylvinyl 
]-23,25-dimethoxy-13,19,21, 
epi-27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10,16-tetraone 
Step 1: 
17-Ethyl-12-[2'-(4"-t-butyldimethylsilyloxy-3"-methoxycyclohexyl)-1'-methy 
lvinyl]-14-t-butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,epi-27-tetramet 
hyl-11,28-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10,16-tetraone 
Further elution of the column in Step 1 of Example 21 with 25% ethyl 
acetate-hexane afforded 1.36 g (4.3%) of the title compound as a white 
foam; MASS SPECTRUM (FAB) m/e 1010. 
Step 2: 
17-Ethyl-14-hydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylviny 
l]-23,25-dimethoxy-13,19,21,epi-27-tetramethyl-11,28-dioxa-4-azatricyclo[22 
.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone 
A sample of 0.043 g (0.042 mmole) of 
17-ethyl-12-[2'-(4"-t-butyldimethylsilyloxy-3"-methoxycyclohexyl)-1'-methy 
lvinyl]-14-t-butyldimethylsilyloxy-23,25-dimethoxy-13,19,21,epi-27-tetramet 
hyl-11,28-dioxa-4-azatri-cyclo[22.3.1.0.sup.4,9 
]octacos-18-ene-2,3,10,16-tetraone was deprotected as in Step 2 of Example 
3 to afford 0.028 g (84%) of the title compound as a white powder after 
lyophillization from benzene; MASS SPECTRUM (FAB) m/e 782. 
EXAMPLE 25 
T-Cell Proliferation Assay 
1. Sample Preparation 
The compounds to be assayed were dissolved in absolute ethanol at 1 mg/ml. 
2. Assay 
Spleens from C57B1/6 mice were taken under sterile conditions and gently 
dissociated in ice-cold RPMI 1640 culture medium (GIBC), Grand Island, 
N.Y.) supplemented with 10% heat-inactivated fetal calf serum (GIBO)). 
Cells were pelleted by centrifugation at 1500 rpm for 8 minutes. 
Contaminating red cells were removed by treating the pellet with ammonium 
chloride lysing buffer (GIBO)) for 2 minutes at 4.degree. C. Cold medium 
was added and cells were again centrifuged at 1500 rpm for 8 minutes. T 
lymphocytes were then isolated by separation of the cell suspension on 
nylon wool columns as follows: Nylon wool columns were prepared by packing 
approximately 4 grams of washed and dried nylon wool into 20 ml plastic 
syringes. The columns were sterilized by autoclaving at 25.degree. F. for 
30 minutes. Nylon wool columns were wetted with warm (37.degree. C.) 
culture medium and rinsed with the same medium. Washed spleen cells 
resuspended in warm medium were slowly applied to the nylon wool. The 
columns were then incubated in an upright position at 37.degree. C. for 1 
hour. Non-adherent T lymphocytes were eluted from the columns with warm 
culture medium and the cell suspensions were spun as above. 
Purified T lymphocytes were resuspended at 2.5.times.10.sup.5 cells/ml in 
complete culture medium composed of RPMI 1640 medium with 10% 
heat-inactivated fetal calf serum, 100 mM glutamine, 1 mM sodium pyruvate, 
2.times.10.sup.-5 M 2-mercaptoethanol and 50 .mu.g/ml gentamycin. 
Ionomycin was added at 250 ng/ml and PMA at 10 ng/ml. The cell suspension 
was immediately distributed into 96 well flat-bottom microculture plates 
(Costar) at 200 .mu.l/well. The various dilutions of the compound to be 
tested were then added in triplicate wells at 20 .mu.l/well. The compound 
17-allyl-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methyl 
vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[2 
2.3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone was used as a standard. 
The culture plates were then incubated at 37.degree. C. in a humidified 
atmosphere of 5% CO.sub.2 -95% air for 44 hours. The proliferation of T 
lymphocytes was assessed by measurement of tritiated thymidine 
incorporation. After 44 hours of culturing, the cells were pulse-labelled 
with 2 .mu.Ci/well of tritiated thymidine (NEN, Cambridge, Mass). After 
another 4 hours of incubation, cultures were harvested on glass fiber 
filters using a multiple sample harvester. Radioactivity of filter discs 
corresponding to individual wells was measured by standard liquid 
scintillation counting methods (Betacounter). Mean counts per minute of 
replicate wells were calculated and the results expressed as concentration 
of compound required to inhibit tritiated thymidine uptake of T-cells by 
50%. 
A selection of compounds were tested according to the previous procedure. 
The title compounds of the following Examples had activity in inhibiting 
the proliferation of T-cells in the aforementioned assay: 3, 6, 15, 20, 
22, 23 and 24. 
For determining antagonist activity, the foregoing procedure is modified in 
that dilutions of compounds are cultured with 
17-ally-1,14-dihydroxy-12-[2'-(4"-hydroxy-3"-methoxycyclohexyl)-1'-methylv 
inyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22. 
3.1.0.sup.4,9 ]octacos-18-ene-2,3,10,16-tetraone (as a standard) at a 
concentration of 1.2 nM, a concentration which inhibits T cell 
proliferation by 100%, the concentration of compound required to reverse 
the inhibition obtained by the standard alone by 50% is measured, and the 
ED.sub.50 value is determined. 
A selection of compounds were tested according to the previous procedure. 
The title compounds of the following Examples had activity in reversing 
the inhibition of proliferation of T-cells by the standard in the 
aforementioned assay: 5, 7, 10, 11, 12, 15 and 17. 
While the foregoing specification teaches the principles of the present 
invention, with examples provided for the purpose of illustration, it will 
be understood that the practice of the invention encompasses all of the 
casual variations, adaptations, modifications, deletions, or additions of 
procedures and protocols described herein, as come within the scope of the 
following claims and its equivalents.