AU6109600A - Apicidin-derived cyclic tetrapeptides - Google Patents

Description

WO 01/07042 PCT/US00/19627 TITLE OF THE INVENTION APICIDIN-DERIVED CYCLIC TETRAPEPTIDES 5 BACKGROUND OF THE INVENTION Field of the invention The present invention relates to anti-protozoal agents. In particular, the present invention relates to cyclic tetrapeptide compounds derived from apicidin that therapeutically inhibit histone deacetylase activity by protozoa. 10 Related background Parasitic protozoa are responsible for a wide variety of infections in man and animals. Many of the diseases are life threatening to the host and cause considerable economic loss in animal husbandry. Malaria remains a significant health 15 threat to humans despite massive international attempts to eradicate the disease. Trypanosomiasis such as i) Chagas disease caused by Trypanosoma cruzi and ii) African sleeping sickness caused by T. brucei are not uncommon in Africa and South America. Furthermore; opportunistic infections, caused by Pneumocystis carinii, Toxoplasma gondii, and Cryptosporidium sp., in immunocompromised hosts are 20 becoming increasingly significant in developed countries. A protozoal infection of great economic importance is coccidiosis, a widespread disease of domesticated animals produced by infections by protozoa of the genus Eimeria. Some of the most significant of Eimeria species are those in poultry, namely E. tenella, E. acervulina, E. necatrix, E. praecox, E. mitis, E. brunetti and E. 25 maxima. Coccidiosis can cause high levels of morbidity and mortality in poultry, resulting in extreme economic losses. In some protozoal diseases, such as Chagas disease, there is no satisfactory treatment. In other protozoal diseases, drug-resistant strains of the protozoa may develop or have developed. Accordingly, there exists a continued need 30 to identify new and effective anti-protozoal drugs. However, antiparasitic drug discovery has been, for the most part, a random and laborious process - through biological screening of natural products and synthetic compounds against a panel of parasites. Drug discovery can be greatly facilitated and made more directed if a specific target of antiprotozoal drugs can be identified, and incorporated into the 35 screening process. -1- WO 01/07042 PCT/US00/19627 Histone deacetylase ("HDA") and histone acetyltransferase ("HAT") together control the net level of acetylation of histones. Inhibition of the action of HDA results in the accumulation of hyperacetylated histones, which in turn is implicated in a variety of cellular responses, including altered gene expression, cell 5 differentiation and cell-cycle arrest. Recently, trichostatin A and trapoxin A have been reported as reversible and irreversible inhibitors, respectively, of mammalian HDA (see e.g., Yoshida et al., BioAssays, 17(5), 423-430 (1995)). Trichostatin A has also been reported to inhibit partially purified yeast HDA (Sanchez del Pino et al., Biochem. J., 303, 723-729 (1994)). Trichostatin A is an antifungal antibiotic and has 10 been shown i) to have anti-trichomonal activity as well as cell differentiating activity in murine erythroleukemia cells, and ii) the ability to induce phenotypic reversion in sis-transformed fibroblast cells (see e.g., U.S. Patent No. 4,218,478; Yoshida et al., BioAssays, 17(5), 423-430 (1995); and references cited therein). Trapoxin A, a cyclic tetrapeptide, induces morphological reversion of v-sis-transformed NIH3T3 cells 15 (Yoshida and Sugita, Jap. J. Cancer Res., 83(4), 324-328 (1992). HDA inhibition as a target for cancer research is described in Saito et al., Proc. Natl Acad. Sci. USA, 96, 4592-4597(1999); Bernardi et al., Amino Acids 6, 315-318 (1994); and R.E. Shute et al., J. Med. Chem. 30, 71-78 (1987). U.S. Patent No. 5,620,953 describes novel cyclic tetrapeptides, 20 including apicidin. Apicidin [cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8 oxo-decanoyl)] is a broad-spectrum antiprotozoal, antifungal and antineoplastic agent isolated from the fermentation culture of Fusarium fungus. The structure of apicidin is shown below: 0 a N H N H N 0 OMe O 0 4 6 8 0 25 Nevertheless, there remains a need to develop novel antiprotozoic compounds. The present inventors have found that a number of cyclic tetrapeptides -2- WO 01/07042 PCT/US00/19627 derived from apicidin, structurally related to trapoxin A, are inhibitors of histone deacetylase and possess antiprotozoal activity. SUMMARY OF THE INVENTION 5 The present invention relates to novel cyclic tetrapeptides and pharmaceutical compositions containing the tetrapeptides. The invention also concerns a method for treating protozoal infections by administering to a host suffering from protozoal infection a therapeutically effective amount of a compound that inhibits histone deacetylase. Additionally, the invention relates to the use of 10 known cyclic tetrapeptides to inhibit histone deacetylase activity and effective as antiprotozoal agents. This invention relates i) to new antiprotozoal, antifungal and antineoplastic agents related to apicidin, ii) to processes for preparation of such novel agents, iii) to compositions containing such novel agents, iv) to the use of such novel 15 agents in the treatment of parasitic infections, including malaria, in human and animals and v) the use of such novel agents in treating cancer. In treating cancer the compounds of this invention can be used as cytostatic compounds, as agents in treating abnormal cell differentiation or proliferation, as agents against tumor growth, or as antimitotic agents for cancer 20 chemotherapy. DETAILED DESCRIPTION OF THE INVENTION In one aspect, according to one embodiment, the present invention relates to a novel cyclic tetrapeptide represented by Formula I shown below: 25

R

5

R

6 0 O , X-R,

(CH

2 ) N N-R3

R

3 NR N R6

R

6 R 2 I -3- WO 01/07042 PCT/US00/19627 or a pharmaceutically acceptable salt thereof wherein X is (1) -CH2-, (2) -C(O)-, 5 (3) -CH(ORa)-, (4) =CH-, or (5) not present; n is (1) one, or 10 (2) two; R1 is (1) R7, (2) C(O)R7, (3) CN, 15 (4) CO 2 Rb, (5) C(O)N(ORb)Rc, (6) C(O)NRcRd, (7) NHCO 2 Rb, (8) NHC(O)NRcRd, 20 (9) (CO-C4alkyl)ORa, (10) (CO-C4alkyl)OCO2Rb, (11) (CO-C4alkyl)OC(O)NRcRd, (12) C(O)NRcNRcRd, (13) C(O)NRcSO 2 Rb, 25 (14) OS(O)niR7, (15) NRbS(O)niR7, wherein ni is from 0 to 2, (16) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2-C5alkenyl, Cj 30 C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or -4- WO 01/07042 PCT/US00/19627 nitrogen, in which the nitrogen optionally has an RC substituent, (17) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 5 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or 10 nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring is attached at any site to X or to the tetrapeptide, or (18) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered 15 heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, Cl-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each 20 heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent; 25 R2 is (1) optionally substituted C2-C12alkyl, (2) optionally substituted C2-C12alkenyl, (3) optionally substituted C2-C12alkynyl, or (4) (CH2)nii-O-(CH2)mii wherein nii, mii = 0 to 7, wherein the optional substituents on the alkyl, alkenyl, and alkynyl are 30 1 to 8 groups and each group independently is -5- WO 01/07042 PCT/US00/19627 (a) CO 2 Ra, (b) C(O)Rb, (c) C(O)N(ORb)Rc, (d) C(O)NRCRd, 5 (e) C(O)NRCNRCRd, (f) C(O)NRcSO2R 7 , (g) C3-C8cycloalkyl, (h) C2-C5alkenyl, (i) cyano, 10 (j) =NORa, (k) =NNRbR c , (1) =NNRbS(O)niR7, (m) N(ORb)C(O)NRbRc, (n) N(ORb)C(O)R 7 , 15 (o) NHC(O)N(ORb)Rc, (p) NRcCO 2 Rb, (q) NRcC(O)NRCRd, (r) NRcC(S)NRCRd, (s) NRcC(O)R7, 20 (t) NRbS(O)niR7, (u) NRcCH2CO 2 Ra, (v) NRcC(S)R7, (x) NRcC(O)CH2OH, (y) NRcC(O)CH2SH, 25 (z) NRcCH 2

CO

2 Ra, (aa) NRcCH2CH(OH)R7, (bb) NRcP(O)(ORa)R 7 , (cc) NY 1

Y

2 , wherein Y 1 and Y 2 are independently H or C1-C1Oalkyl, 30 (dd) NO2, (ee) N(ORb)C(O)Rb, (ff) C1-Co10alkanoylamino, (gg) OR a, (hh) OS(O)niR7, -6- WO 01/07042 PCT/US00/19627 (ii) oxo, (jj) OCO 2 Rb, (kk) OC(O)NRCRd, (11) P(O)(ORa)2, 5 (mm) P(O)(ORa)R 7 , (nn) SC(O)R7, (oo) S(O)niR7, (pp) SR7, (qq) S(O)niNRCRd, 10 (rr) NRCCH 2

CO

2 Ra, (ss) diazo, (tt) C1-C5 perfluoroalkyl, (uu) B(O)(ORa)ORa, (vv) halogen, 15 (ww) aryl(CO-C5alkyl), wherein the aryl is optionally substituted with 1 to 3 groups, wherein each group independently is R f , or (xx) a 3- to 8-membered heterocycle containing from 1 to 4 heteroatoms, each heteroatom independently is oxygen, sulfur or 20 nitrogen, wherein the heterocycle is optionally substituted by 1 to 3 groups, wherein each group independently is R f , and the heterocycle may be saturated or partly unsaturated; R3 each independently is 25 (1) hydrogen, (2) halogen, (3) OR a , (4) C1-C4alkyl, or (5) CI-C4aryl; 30 R5 is (1) isopropyl, or (2) sec-butyl; R6 each independently is -7- WO 01/07042 PCT/US00/19627 (1) O, (2) S, or (3) H; 5 R7 is (1) hydrogen, (2) optionally substituted C2-Co10alkyl, (3) optionally substituted C2-Co10alkenyl, (4) optionally substituted C2-Co10alkynyl, (5) optionally substituted C3-C8cycloalkyl, 10 (6) optionally substituted C5-C8cycloalkenyl, (7) optionally substituted aryl, wherein the optional substituents on the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and aryl are 1 to 4 groups, and each group independently is 15 (a) C1-C5alkyl, (b) X 1 -C1-Co10alkyl, wherein X 1 is O or S(O)ni, (c) C3-C8cycloalkyl, (d) hydroxy, (e) halogen, 20 (f) cyano, (g) carboxy, (h) NY 1

Y

2 , wherein Y 1 and Y 2 are independently H or C1-C10alkyl, (i) nitro, 25 (j) C1-Co10alkanoylamino, (k) aroyl amino wherein the aroyl is optionally substituted with 1 to 3 groups wherein each group independently is

R

f l 1 , wherein R f l is defined by any of the definitions below for R f except for (14), (26), (27), and (32), 30 (1) oxo, (m) aryl CO-C5alkyl wherein the aryl is optionally substituted with 1 to 3 groups, wherein each group independently is Rfl, (n) C1-C5perfluoroalkyl, -8- WO 01/07042 PCT/US00/19627 (o) N(ORb)C(O)R 7

'

, wherein R7' is any of the above definitions of R7 from (1) to (7)(n), and below of R7 from (8) to (12), or (p) NRcC(O)R7

'

, 5 (8) a 5- to 10-membered heterocycle containing from 1 to 4 heteroatoms, each heteroatom independently is oxygen, sulfur or nitrogen and the heterocycle is optionally substituted by 1 to 3 groups, each group independently is R f l 1 , and the heterocycle may be saturated or partly unsaturated, 10 (9) a benzene ring fused to a 5- to 10-membered heterocyclic ring containing from 1 to 4 heteroatoms, each heteroatom independently is oxygen, sulfur or nitrogen and the heterocycle is optionally substituted by 1 to 3 groups, each group independently is

R

f l 1 , and the heterocycle may be saturated or partly unsaturated, 15 (10) a 5- to 10-membered heterocyclic ring containing from 1 to 4 heteroatoms fused to a second 5- to 10-membered heterocyclic ring containing from 1 to 4 heteroatoms, each heteroatom in either heterocyclic ring independently is oxygen, sulfur or nitrogen and the second heterocyclic ring is optionally substituted by 1 to 3 groups, each 20 group independently is R f l 1 , and each heterocycle independently may be saturated or partly unsaturated, (11) a benzene ring fused to a C3-C8cycloalkyl ring, wherein the cycloalkyl is optionally substituted by 1 to 3 groups each independently being R f l 1 , and the cycloalkyl ring may be saturated or partly 25 unsaturated, or (12) a 5- to 10-membered heterocyclic ring containing from 1 to 4 heteroatoms, each heteroatom independently is oxygen, sulfur or nitrogen, the heterocyclic ring is fused to a C3-C8cycloalkyl ring, wherein the cycloalkyl ring is optionally substituted by 1 to 3 groups 30 each independently being R f l 1 , and the cycloalkyl ring may be saturated or partly unsaturated, Ra is (1) hydrogen, (2) optionally substituted Cl1-C 10alkyl, -9- WO 01/07042 PCT/US00/19627 (3) optionally substituted C3-C10alkenyl, (4) optionally substituted C3-Co10alkynyl, (5) optionally substituted Cl1-C10alkanoyl, (6) optionally substituted C3-C10oalkenoyl, 5 (7) optionally substituted C3-Co10alkynoyl, (8) optionally substituted aroyl, (9) optionally substituted aryl, (10) optionally substituted C3-C7cycloalkanoyl, (11) optionally substituted C5-C7cycloalkenoyl, 10 (12) optionally substituted C1-Cloalkylsulfonyl, (13) optionally substituted C3-C8cycloalkyl, (14) optionally substituted C5-C8cycloalkenyl, wherein the optional substituents on the C1-Cloalkyl, C3-Cloalkenyl, C3-C 1oalkynyl, Cl1-C 1oalkanoyl, C3-C 1oalkenoyl, C3-C1 oalkynoyl, 15 aroyl, aryl, C3-C7cycloalkanoyl, C5-C7cycloalkenoyl, Cl C10oalkylsulfonyl, C3-C8cycloalkyl and C5-C8cycloalkenyl are from 1 to 10 groups, wherein each group independently is hydroxy, C1 C6alkoxy, C3-C7cycloalkyl, aryl C1-C3alkoxy, NRXRX, CO 2 Rb, CONRcRd, or halogen, 20 (15) C1-C5perfluoroalkyl, (16) arylsulfonyl optionally substituted with 1 to 3 groups, wherein each group independently is Cl1-C5alkyl, C1 C5perfluoroalkyl, nitro, halogen or cyano, (17) a 5- or 6-membered heterocycle containing 1 to 4 25 heteroatoms, wherein each heteroatom is oxygen, sulfur or nitrogen, wherein the heterocycle is optionally substituted by 1 to 4 groups, wherein each group independently is CI1-C5alkyl, Cl1-C5alkenyl, Cl C5perfluoroalkyl, amino, C(O)NRcRd, cyano, CO 2 Rb or halogen, and wherein the heterocycle may be saturated or partly unsaturated, or 30 (18) OP(O)(ORb) 2 ; Rb is (1) H, (2) optionally substituted aryl, (3) optionally substituted C1-C 10alkyl, -10- WO 01/07042 PCT/US00/19627 (4) optionally substituted C3-C10oalkenyl, (5) optionally substituted C3-C10Oalkynyl, (6) optionally substituted C3-C15cycloalkyl, (7) optionally substituted C5-C10ocycloalkenyl, or 5 (8) optionally substituted 5- to 10-membered heterocycle containing 1 to 4 heteroatoms, wherein each heteroatom independently is oxygen, sulfur, or nitrogen, wherein the optional substituents on the aryl, C1-Co10alkyl, C3 C 10alkenyl, C3-C o10alkynyl 1, C3-C 15cycloalkyl, C5-C o10cycloalkenyl, 10 or 5- to 10-membered heterocycle are from 1 to 10 groups, wherein each group independently is (a) hydroxy, (b) C1-C6alkyl, (c) oxo, 15 (d) SO 2 NRxR x , (e) aryl C1-C6alkoxy, (f) hydroxy C1-C6alkyl, (g) C1-Cl12alkoxy, (h) hydroxy C1-C6alkoxy, 20 (I) amino C1-C6alkoxy, (j) cyano, (k) mercapto, (1) (C1-C6alkyl)-S(O)ni-(CO-C6alkyl), (m) C3-C7cycloalkyl optionally substituted with 1 to 25 4 groups, wherein each group independently is Re, (n) C5-C7cycloalkenyl, (o) halogen, (p) C1-C5alkanoyloxy, (q) C(O)NRxRx, 30 (r) CO 2 Ri, (s) . formyl, (t) -NRXRx, (u) 5 to 9-membered heterocycle, which may be saturated or partially unsaturated, containing from 1 to 4 heteroatoms, -11- WO 01/07042 PCT/US00/19627 wherein each heteroatom independently is oxygen, sulfur or nitrogen, and the heterocycle is optionally substituted with 1 to 5 groups, wherein each group independently is Re, (v) optionally substituted aryl, wherein the optional 5 substituents are 1,2-methylenedioxy or 1 to 5 groups, wherein each group independently is Re, (x) optionally substituted aryl C1-C3alkoxy, wherein the optional substituents are 1,2-methylenedioxy or 1 to 5 groups, wherein each group independently is Re, or 10 (y) C1 -C5perfluoroalkyl; Rc and Rd are independently selected from Rb; or R c and Rd together with the N to which they are attached form a 3- to 10-membered ring containing 0 to 2 additional heteroatoms, each additional heteroatom independently 15 being oxygen, nitrogen, or (O)ni substituted sulfur, wherein the ring is optionally substituted with 1 to 3 groups, wherein each group independently is Rg, hydroxy, thioxo, or oxo; Re is (1) halogen, 20 (2) C1-C7alkyl, (3) C 1-C3perfluoroalkyl, (4) -S(O)mR i , (5) cyano, (6) nitro, 25 (7) RiO(CH2)v - , (8) RiCO2(CH2)v - , (9) RiOCO(CH2)v, (10) optionally substituted aryl wherein the optional substituents are from 1 to 3 groups, wherein each group independently is halogen, 30 C1-C6alkyl, C1-C6alkoxy, or hydroxy, (11) SO 2 NRXRx, (12) CO2Rx, or (13) NRXRX; - 12- WO 01/07042 PCT/US00/19627

R

f is (1) C1-C4alkyl, (2) X 1 -C1-C4alkyl, wherein X 1 is O or S(O)mi, (3) C2-C4alkenyl, (4) C2-C4 alkynyl, 5 (5) C1-C3perfluoroalkyl, (6) NY 3 y 4 , wherein Y 3 and Y 4 are each independently hydrogen, CI-C5alkyl, or SO 2 Rb, (7) hydroxy, (8) halogen, 10 (9) C1-C5alkanoyl amino, (10) (CO-C4alkyl)CO2Ra, (11) (CO-C4alkyl)C(O)NRbR c , (12) (CO-C4alkyl)NY 5

Y

6 wherein Y 5 and Y 6 together with the N to which they are attached form a 3- to 7-membered ring 15 containing 0 to 2 additional heteroatoms, wherein the additional heteroatoms independently are oxygen, nitrogen, or (O)mi substituted sulfur, wherein the ring is optionally substituted with 1 to 3 groups, wherein each group independently is Re or oxo, 20 (13) (CO-C4alkyl)NO2, (14) (CO-C4alkyl)C(0)R7, (15) (CO-C4alkyl)CN, (16) oxo, (17) (CO-C4alkyl)C(O)N(ORb)Rc, 25 (18) (CO-C 4 alkyl)C(0)NRcRd, (19) (CO-C4alkyl)NHC(0)ORb, (20) (CO-C4alkyl)NHC(0)NRcRd, (21) (CO-C4alkyl)ORa, (22) (CO-C4alkyl)OCO2Rb, 30 (23) (CO-C 4 alkyl)OC(0)NRcRd, (24) (CO-C 4 alkyl)C(0)NRcNRcRd, (25) (CO-C4alkyl)C(0)NRcSO2Rb, (26) (CO-C4alkyl)OS(0)niR7, (27) (CO-C4alkyl)NRbS(0)niR7, - 13- WO 01/07042 PCT/US00/19627 (28) CO-C4alkyl halogen, (29) (C0-C4alkyl) SR a , (30) P(O)(ORa)2, (31) CO-C4alkyl azide, 5 (32) CO-C4aryl substituted with from 1 to 4 groups, wherein each group independently is S(O)2R7, or (33) CO-C4aryl where the aryl group is optionally substituted from 1 to 4 groups, wherein each group independently is CO 2 Rb, C(O)NRCRd, NO2, halogen, OC(O)Ra, OR a or CI1-C4alkyl; 10 Rg and Rh together with the N to which they are attached form a 3- to 7-membered ring containing 0 to 2 additional heteroatoms, wherein each additional heteroatom independently is oxygen, nitrogen, or (O)mi substituted sulfur, and the ring is optionally substituted with 1 to 3 groups, wherein 15 each group independently is Re or oxo; or Rg and Rh are each independently (1) hydrogen, (2) C1-C6alkyl optionally substituted with hydroxy, amino, or 20 CO 2 Ri, (3) aryl optionally substituted with halogen, 1,2 methylenedioxy, C1-C7alkoxy, C1-C7alkyl, or C -C3perfluoroalkyl, (4) aryl C1-C6alkyl, wherein the aryl is optionally substituted with C1-C3perfluoroalkyl or 1,2-methylenedioxy, 25 (5) C1-C5alkoxycarbonyl, (6) C 1-C5alkanoyl, (7) C1-C5alkanoyl C1-C6alkyl, (8) arylC1-C5 alkoxycarbonyl, (9) aminocarbonyl, 30 (10) (CI1-C5monoalkyl)aminocarbonyl, (11) (Ci1-C5dialkyl)aminocarbonyl, or (12) CO 2 Rb;

R

i is (1) hydrogen, - 14- WO 01/07042 PCT/US00/19627 (2) C1-C3perfluoroalkyl, (3) C I-C6alkyl, or (4) optionally substituted aryl CO-C6alkyl, wherein the aryl optional substituents are from 1 to 3 groups, wherein each group 5 independently is halogen, C1-C6alkyl, C1-C6alkoxy, or hydroxy; Rx is a C1-C4alkyl; mis 0 to 2; 10 mi is 0 to 2; ni is 0 to 2; 15 mii is 0 to 7; nii is 0 to 7; v is 0 to 3; and 20 excluding apicidin, N-desmethoxy apicidin and compounds represented by the following chemical Formula HA and Formula lIB:

CH

3

CH

2

CH

3 0 S 0 N N OCH3 H NH H N 0 0 0 25 IHA - 15- WO 01/07042 PCT/US00/19627

CH

3

CH

2

CH

3 - O N H H N NH H 0 O 0 IIB 5 Within this embodiment, the novel cyclic tetrapeptide of this invention includes a genus of compounds represented by Formula I or a pharmaceutically acceptable salt thereof wherein: X is (1) -CH2-, 10 (2) -C(0)-, (3) -CH(ORay (4) =CH-, or (5) not present; and R1 is (1) R7, 15 (2) C(0)R7, (3) CN, (4) CO 2 Rb, (5) C(O)N(ORb)Rc, (6) C(0)NRcRd, 20 (7) NHCO 2 Rb, (8) NHC(O)NRcRd, (9) (CO-C4alkyl)ORa, (10) (CO-C 4 alkyl)OCO2Rb, - 16 - WO 01/07042 PCT/US00/19627 (11) (CO-C 4 alkyl)OC(O)NRCRd, (12) C(O)NRCNRCRd, (13) C(O)NRcSO 2 Rb, (13) OS(O)niR7, 5 (14) NRbS(O)niR7, wherein ni is from 0 to 2, (15) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2-C5alkenyl, Cl C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa 10 (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc 15 substituent, (16) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, 20 C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring 25 is attached at any site to X or to the tetrapeptide, or (17) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 30 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, Cl1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is - 17- WO 01/07042 PCT/US00/19627 oxygen, sulfur, or nitrogen, and the nitrogen optionally has an RC substituent. Within this genus there is a class of compounds represented by 5 Formula I or a pharmaceutically acceptable salt thereof wherein: X is (1) -CH2-, (2) -C(O)-, (3) -CH(ORa) - , (4) =CH-, or 10 (5) not present; R1 is (1) R7, (2) C(O)R7, (3) CN, (4) CO 2 Rb, 15 (5) C(O)N(ORb)Rc, (6) C(O)NRcRd, (7) NHCO 2 Rb, (8) NHC(O)NRcRd, (9) (CO-C4alkyl)ORa, 20 (10) (CO-C4alkyl)OCO2Rb, (11) (CO-C 4 alkyl)OC(O)NRcRd, (12) C(O)NRcNRcRd, (13) C(O)NRcSO 2 Rb, (14) OS(O)niR7, 25 (15) NRbS(O)niR7, wherein ni is from 0 to 2, (16) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each group independently is CI-C5alkyl, C2-C5alkenyl, C1 C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa 30 (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or -18- WO 01/07042 PCT/US00/19627 nitrogen, in which the nitrogen optionally has an RC substituent, (17) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 5 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, CI-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or 10 nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring is attached at any site to X or to the tetrapeptide, or (18) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered 15 heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each 20 heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent; and R2 is (1) optionally substituted C2-C12alkyl, 25 (2) optionally substituted C2-C12alkenyl, (3) optionally substituted C2-C12alkynyl, or (4) (CH2)nii-O-(CH2)mii wherein nii, mii = 0 to 7, wherein the optional substituents on the C2-C12alkyl, C2-C12alkenyl, and C2-C12alkynyl are 1 to 8 groups and each group independently is 30 (a) CO 2 Ra, (b) C(O)Rb, (c) C(O)N(ORb)Rc, (d) C(O)NRcRd, (e) C(O)NRcNRcRd, -19- WO 01/07042 PCT/US00/19627 (f) C(O)NRcSO2R 7 , (g) C3-C8cycloalkyl, (h) C2-C5alkenyl, (i) cyano, 5(j) =NORa, (k) =NNRbRc, (1) =NNRbS(O)niR7, (m) N(ORb)C(O)NRbRc, (n) N(ORb)C(O)R 7 , 10 (o) NHC(O)N(ORb)Rc, (p) NRcCO 2 Rb, (q) NRcC(O)NRCRd, (r) NRcC(S)NRCRd, (s) NRcC(O)R7, 15 (t) NRbS(O)niR7, (u) NRcCH 2

CO

2 Ra, (v) NRcC(S)R7, (x) NRcC(O)CH 2 OH, (y) NRcC(O)CH2SH, 20 (z) NRcCH 2

CO

2 Ra, (aa) NRcCH2CH(OH)R7, (bb) NRcP(O)(ORa)R 7 , (cc) NY 1

Y

2 , wherein Y1 and Y 2 are independently H or C1-C10alkyl, 25 (dd) NO2, (ee) N(ORb)C(O)Rb, (if) C1-C10alkanoylamino, (gg) OR a , (hh) OS(O)niR7, 30 (ii) oxo, (jj) OCO 2 Rb, (kk) OC(O)NRCRd, (11) P(O)(ORa)2, (mm) P(O)(ORa)R 7 , - 20 - WO 01/07042 PCT/US00/19627 (nn) SC(O)R7, (oo) S(O)niR7, (pp) SR7, (qq) S(O)niNRCRd, 5 (rr) NRcCH 2

CO

2 Ra, (ss) diazo, (tt) C1-C5 perfluoroalkyl, (uu) B(O)(ORa)ORa, (vv) halogen, 10 (ww) aryl(CO-C5alkyl), wherein the aryl is optionally substituted with 1 to 3 groups, wherein each group independently is R f , or (xx) a 3- to 8-membered heterocycle containing from 1 to 4 heteroatoms, each heteroatom 15 independently is oxygen, sulfur or nitrogen, wherein the heterocycle is optionally substituted by 1 to 3 groups, wherein each group independently is R f , and the heterocycle may be saturated or partly unsaturated. 20 Within the above class of compounds, there is a subclass of compounds represented by Formula I or a pharmaceutically acceptable salt thereof wherein n is 1 or 2. 25 Within this genus there is another class of compounds represented by Formula I or a pharmaceutically acceptable salt thereof wherein: Xis (1) -CH2-, (2) -C(O)-, or (3) not present; and 30 R1 is (1) R7, (2) C(O)R7, (3) CN, (4) CO 2 Rb, (5) C(O)N(ORb)Rc, -21 - WO 01/07042 PCT/US00/19627 (6) C(O)NRCRd, (7) NHCO 2 Rb, (8) NHC(O)NRCRd, (9) (CO-C4alkyl)ORa, 5 (10) (CO-C4alkyl)OCO 2 Rb, (11) (CO-C4alkyl)OC(O)NRCRd, (12) C(O)NRCNRCRd, (13) C(O)NRcSO 2 Rb, (14) OS(O)niR7, 10 (15) NRbS(O)niR7, wherein ni is from 0 to 2, (16) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2-C5alkenyl, Cl C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa 15 (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc 20 substituent, (17) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, Cl-C5perfluoroalkyl, amino, oxo, thiono, 25 C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring 30 is attached at any site to X or to the tetrapeptide, or (18) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 - 22 - WO 01/07042 PCT/US00/19627 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, Cl-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each heterocycle may be saturated, partly unsaturated or fully 5 unsaturated, and wherein each heteroatom independently is oxygen, sulfur, or nitrogen, and the nitrogen optionally has an RC substituent. Within the above class of compounds, there is a subclass of 10 compounds represented by Formula I or a pharmaceutically acceptable salt thereof wherein n is 1 or 2. Within this genus there is yet another class of compounds represented by Formula I or a pharmaceutically acceptable salt thereof wherein: 15 X is (1) -CH2-, (2) -C(O)-, or (3) not present; and RI is (1) R7, (2) C(O)R7, 20 (3) CO 2 Rb, (4) C(O)N(ORb)Rc, (5) C(O)NRcRd, (6) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each 25 group independently is C1-C5alkyl, C2-C5alkenyl, C l C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein 30 the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, (7) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by - 23 - WO 01/07042 PCT/US00/19627 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein 5 the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring is attached at any site to X or to the tetrapeptide, or (8) a 4- to 8-membered heterocyclic ring with from 1 to 4 10 heteroatoms fused to a second 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, 15 C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent. 20 Within the above class of compounds, there is a subclass of compounds represented by Formula I or a pharmaceutically acceptable salt thereof wherein n is 1 or 2. 25 Within this genus there is yet another class of compounds represented by Formula I or a pharmaceutically acceptable salt thereof wherein: X is (1) -CH2-, (2) -C(O)-, or (3) not present; 30 R1 is (1) R7, (2) C(O)R7, (3) CO 2 Rb, (4) C(O)N(ORb)Rc, (5) C(O)NRcRd, - 24 - WO 01/07042 PCT/US00/19627 (6) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2-C5alkenyl, C1 C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa 5 (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc 10 substituent, (7) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, 15 C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring 20 is attached at any site to X or to the tetrapeptide, or (8) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 25 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is 30 oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent;and R2 is (1) optionally substituted C2-C12alkyl, (2) optionally substituted C2-C12alkenyl, (3) optionally substituted C2-C12alkynyl, or 35 (4) (CH2)nii-O-(CH2)mii wherein nii, mii = 0 to 7, - 25 - WO 01/07042 PCT/US00/19627 wherein the optional substituents on the C2-C12alkyl, C2-C12alkenyl, and C2-C12alkynyl are 1 to 5 groups and each group independently is (a) CO 2 Ra, (b) C(O)Rb, 5 (c) C(O)N(ORb)Rc, (d) C(O)NRCRd, (e) C(O)NRCNRCRd, (f) C(O)NRcSO2R7, (g) C3-C8cycloalkyl, 10 (h) C2-C5alkenyl, (i) cyano, (j) =NORa, (k) =NNRbR c , (1) =NNRbS(O)niR7, 15 (m) N(ORb)C(O)NRbRc, (n) N(ORb)C(O)R 7 , (o) NHC(O)N(ORb)Rc, (p) NRcCO 2 Rb, (q) NRcC(O)NRCRd, 20 (r) NRcC(S)NRCRd, (s) NRcC(O)R 7 , (t) NRbS(O)niR7, (u) NRCCH 2

CO

2 Ra, (v) NRcC(S)R7, 25 (x) NRcC(O)CH 2 OH, (y) NRcC(O)CH2SH, (z) NRcCH 2

CO

2 Ra, (aa) NRCCH2CH(OH)R 7 , (bb) NRcP(O)(ORa)R 7 , 30 (cc) NY 1

Y

2 , wherein Y 1 and Y 2 are independently H or methyl, (dd) NO2, (ee) N(ORb)C(O)Rb, (ff) C1-C3alkanoylamino, - 26 - WO 01/07042 PCT/US00/19627 (gg) OR a , (hh) OS(O)niR7, (ii) oxo, (jj) OCO 2 Rb, 5 (kk) OC(O)NRCRd, (11) P(O)(ORa)2, (mm) P(O)(ORa)R7, (nn) SC(O)R7, (oo) S(O)niR7, 10 (pp) SR7, (qq) S(O)niNRCRd, (rr) NRcCH 2

CO

2 Ra, (ss) diazo, (tt) C1-C5 perfluoroalkyl, 15 (uu) B(O)(ORa)ORa, (vv) halogen, (ww) aryl(C0-C5alkyl), wherein the aryl is optionally substituted with 1 to 3 groups, wherein each group independently is R f , or 20 (xx) a 3- to 6-membered heterocycle containing from 1 to 4 heteroatoms, each heteroatom independently is oxygen, sulfur or nitrogen, wherein the heterocycle is optionally substituted by 1 to 3 groups, wherein each group 25 independently is R f , and the heterocycle may be saturated or partly unsaturated. Within the above class of compounds, there is a subclass of compounds represented by Formula I or a pharmaceutically acceptable salt thereof 30 wherein n is 1 or 2. Within this embodiment there is a second genus of compounds represented by Formula I or a pharmaceutically acceptable salt thereof wherein: R3 each independently is - 27 - WO 01/07042 PCT/US00/19627 (1) hydrogen, (2) halogen, (3) OR a, (4) C1-C4alkyl, or 5 (5) C1-C4aryl; and Ra is (1) hydrogen, (2) optionally substituted C1-C6alkyl, (3) optionally substituted C3-C6alkenyl, (4) optionally substituted C2-C4alkanoyl, 10 (5) optionally substituted C3-C4alkenoyl, (6) optionally substituted aroyl, (7) optionally substituted aryl, (8) optionally substituted C5-C6cycloalkanoyl, (9) optionally substituted C1-C4alkylsulfonyl, 15 (10) optionally substituted C5-C6cycloalkyl, (11) optionally substituted C5-C6cycloalkenyl, wherein the optional substituents on the CI-C6alkyl, C3-C6alkenyl, C2-C4alkanoyl, C3-C4alkenoyl, aroyl, aryl, C5-C6cycloalkanoyl, C C4alkylsulfonyl, C5-C6cycloalkyl and C5-C6cycloalkenyl are from 1 20 to 10 groups, wherein each group independently is hydroxy, methoxy, aryl methoxy, NRXRX, CO 2 Rb, CONRCRd, or halogen, (12) CF3, (13) arylsulfonyl optionally substituted with 1 to 3 groups, wherein each group independently is methyl, CF3, nitro, 25 halogen or cyano, or (14) a 5- or 6-membered heterocycle containing 1 to 3 heteroatoms, wherein each heteroatom is oxygen, sulfur or nitrogen, wherein the heterocycle is optionally substituted by 1 to 3 groups, wherein each group independently is 30 methyl, CF3, NMe2, C(O)NRCRd, cyano, CO 2 Rb or halogen, and wherein the heterocycle may be saturated or partly unsaturated. - 28 - WO 01/07042 PCT/US00/19627 Within this second genus is a class of compounds represented by Formula I or a pharmaceutically acceptable salt thereof wherein: R3 each independently is (1) hydrogen, 5 (2) halogen, (3) ORa, (4) C1-C4alkyl, or (5) C1-C4aryl; Ra is (1) hydrogen, 10 (2) optionally substituted C1-C6alkyl, (6) optionally substituted C3-C6alkenyl, (7) optionally substituted C2-C4alkanoyl, (5) optionally substituted C3-C4alkenoyl, (6) optionally substituted aroyl, 15 (7) optionally substituted aryl, (8) optionally substituted C5-C6cycloalkanoyl, (9) optionally substituted C1-C4alkylsulfonyl, (10) optionally substituted C5-C6cycloalkyl, (11) optionally substituted C5-C6cycloalkenyl, 20 wherein the optional substituents on the C1-C6alkyl, C3-C6alkenyl, C2-C4alkanoyl, C3-C4alkenoyl, aroyl, aryl, C5-C6cycloalkanoyl, C1 C4alkylsulfonyl, C5-C6cycloalkyl and C5-C6cycloalkenyl are from 1 to 10 groups, wherein each group independently is hydroxy, methoxy, aryl methoxy, NRXRX, CO 2 Rb, CONRcRd, or halogen, 25 (12) CF3, (13) arylsulfonyl optionally substituted with 1 to 3 groups, wherein each group independently is methyl, CF3, nitro, halogen or cyano, or (14) a 5- or 6-membered heterocycle containing 1 to 3 30 heteroatoms, wherein each heteroatom is oxygen, sulfur or nitrogen, wherein the heterocycle is optionally substituted by 1 to 3 groups, wherein each group independently is methyl, CF3, NMe2, C(O)NRcRd, cyano, CO 2 Rb or - 29 - WO 01/07042 PCT/US00/19627 halogen, and wherein the heterocycle may be saturated or partly unsaturated. X is (1) -CH2-, (2) -C(O)-, 5 (3) =CH-, or (4) not present; and RI is (1) R7, (2) C(O)R7, (3) CN, 10 (4) CO 2 Rb, (5) C(O)N(ORb)Rc, (6) C(O)NRCRd, (7) NHCO 2 Rb, (8) NHC(O)NRCRd, 15 (9) (CO-C4alkyl)ORa, (10) (CO-C4alkyl)OCO2Rb, (11) (CO-C4alkyl)OC(O)NRCRd, (12) C(O)NRCNRCRd, (13) C(O)NRcSO 2 Rb, 20 (14) OS(O)niR7, (15) NRbS(O)niR7, wherein ni is from 0 to 2, (16) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each group independently is CI1-C5alkyl, C2-C5alkenyl, C1 25 C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or 30 nitrogen, in which the nitrogen optionally has an Rc substituent, (17) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups each independently is C1-C5alkyl, C2 - 30 - WO 01/07042 PCT/US00/19627 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or 5 nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring is attached at any site to X or to the tetrapeptide, or (18) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered 10 heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each 15 heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent. 20 Within the above class of compounds, there is a subclass of compounds represented by Formula I or a pharmaceutically acceptable salt thereof wherein n is 1 or 2. Within this second genus is a class of compounds represented by 25 Formula I or a pharmaceutically acceptable salt thereof wherein: R3 each independently is (1) hydrogen, (2) halogen, (3) OR a, 30 (4) C1-C4alkyl, or (5) C1 -C4aryl;); Ra is (1) hydrogen, (2) optionally substituted C 1-C6alkyl, (3) optionally substituted C3-C6alkenyl, -31 - WO 01/07042 PCT/US00/19627 (4) optionally substituted C2-C4alkanoyl, (5) optionally substituted C3-C4alkenoyl, (6) optionally substituted aroyl, (7) optionally substituted aryl, 5 (8) optionally substituted C5-C6cycloalkanoyl, (9) optionally substituted C 1-C4alkylsulfonyl, (10) optionally substituted C5-C6cycloalkyl, (11) optionally substituted C5-C6cycloalkenyl, wherein the optional substituents on the C1-C6alkyl, C3-C6alkenyl, 10 C2-C4alkanoyl, C3-C4alkenoyl, aroyl, aryl, C5-C6cycloalkanoyl, C1 C4alkylsulfonyl, C5-C6cycloalkyl and C5-C6cycloalkenyl are from 1 to 10 groups, wherein each group independently is hydroxy, methoxy, aryl methoxy, NRXRX, CO 2 Rb, CONRcRd, or halogen, (12) CF3, 15 (13) arylsulfonyl optionally substituted with 1 to 3 groups, wherein each group independently is methyl, CF3, nitro, halogen or cyano, or (14) a 5- or 6-membered heterocycle containing 1 to 3 heteroatoms, wherein each heteroatom is oxygen, sulfur or 20 nitrogen, wherein the heterocycle is optionally substituted by 1 to 3 groups, wherein each group independently is methyl, CF3, NMe2, C(O)NRcRd, cyano, CO 2 Rb or halogen, and wherein the heterocycle may be saturated or partly unsaturated; 25 X is (1) -CH2-, (2) -C(O)-, (3) =CH-, or (4) not present; and R1 is (1) R7, 30 (2) C(O)R7, (9) CO 2 Rb, (10) C(O)N(ORb)Rc, (11) C(O)NRcRd, - 32 - WO 01/07042 PCT/US00/19627 (12) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2-C5alkenyl, C1 C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa 5 (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc 10 substituent, (13) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C -C5perfluoroalkyl, amino, oxo, thiono, 15 C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring 20 is attached at any site to X or to the tetrapeptide, or (14) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 25 to 4 groups, each group independently is CI1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is 30 oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent. Within the above class of compounds, there is a subclass of compounds represented by Formula I or a pharmaceutically acceptable salt thereof 35 wherein n is 1 or 2. - 33 - WO 01/07042 PCT/US00/19627 Within this embodiment there is a third genus of compounds represented by Formula I or a pharmaceutically acceptable salt thereof wherein: R6 each independently is 5 (1) 0, (2) S, or (3) H; X is (1) -CH2-, (2) -C(O)-, 10 (3) =CH-, or (4) not present; and R1 is (1) R7, (2) C(0)R7, (3) CN, 15 (4) CO 2 Rb, (5) C(O)N(ORb)Rc, (6) C(0)NRcRd, (7) NHCO2Rb, (8) NHC(O)NRcRd, 20 (9) (CO-C4alkyl)ORa, (10) (CO-C4alkyl)OCO2Rb, (11) (CO-C4alkyl)OC(O)NRcRd, (12) C(O)NRcNRcRd, (13) C(O)NRcSO 2 Rb, 25 (14) OS(0)niR7, (15) NRbS(O)niR7, wherein ni is from 0 to 2, (16) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2-C5alkenyl, C1 30 C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(0)niRa (where ni = 0, 1 or 2), C(0)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or - 34- WO 01/07042 PCT/US00/19627 nitrogen, in which the nitrogen optionally has an RC substituent, (17) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 5 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or 10 nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring is attached at any site to X or to the tetrapeptide, or (18) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered 15 heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 to 4 groups, each group independently is CI1-C5alkyl, C2 C5alkenyl, C1 -C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each 20 heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent. 25 Within this third genus is a class of compounds represented by Formula I or a pharmaceutically acceptable salt thereof wherein n is 1 or 2. Within this third genus is a class of compounds represented by Formula I or a pharmaceutically acceptable salt thereof wherein: 30 R3 each independently is (1) hydrogen, (2) halogen, (3) OR a, (4) C1-C4alkyl, or - 35 - WO 01/07042 PCT/US00/19627 (5) C1-C4aryl; R6 each independently is (1) 0, (2) S, or 5 (3) H; X is (1) -CH2-, (2) -C(O)-, (3) =CH-, or (4) not present; and 10 R1 is (1) R7, (2) C(0)R7, (3) CN, (4) CO 2 Rb, (5) C(0)N(ORb)Rc, 15 (6) C(O)NRCRd, (7) NHCO 2 Rb, (8) NHC(0)NRCRd, (9) (CO-C4alkyl)ORa, (10) (CO-C 4 alkyl)OCO2Rb, 20 (11) (CO-C4alkyl)OC(0)NRCRd, (12) C(0)NRCNRCRd, (13) C(0)NRcSO 2 Rb, (14) OS(O)niR7, (15) NRbS(0)niR7, wherein ni is from 0 to 2, 25 (16) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2-C5alkenyl, C1 C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa (where ni = 0, 1 or 2), C(O)Ra, C(0)NRcRd, cyano, (CO 30 C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, - 36 - WO 01/07042 PCT/US00/19627 (17) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, 5 C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring 10 is attached at any site to X or to the tetrapeptide, or (18) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 15 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is 20 oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent. Within the above class of compounds, there is a subclass of compounds represented by Formula I or a pharmaceutically acceptable salt thereof 25 wherein n is 1 or 2. In one aspect, the present invention provides a novel cyclic tetrapeptide represented by Formula I, wherein X is preferably -CH2-. In another aspect, the present invention provides a novel cyclic 30 tetrapeptide represented by Formula I, wherein X is preferably -C(O)-. In still another aspect, the present invention provides a novel cyclic tetrapeptide represented by Formula I, wherein X is preferably not present. In yet another aspect, the present invention provides a novel cyclic tetrapeptide represented by Formula I, wherein R1 is preferably a 3- to 8-membered - 37 - WO 01/07042 PCT/US00/19627 heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2-C5alkenyl, C1-C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO-C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly 5 unsaturated or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent. In another aspect, the present invention provides a novel cyclic tetrapeptide represented by Formula I, wherein R1 is preferably a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally 10 substituted by 1 to 4 groups each independently is C1-C5alkyl, C2-C5alkenyl, CI C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring is 15 attached at any site to X or to the tetrapeptide In another aspect, the present invention provides a novel cyclic tetrapeptide represented by Formula I, wherein R1 is preferably a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently 20 optionally substituted by 1 to 4 groups, each group independently is CI-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent. 25 In another aspect, the present invention provides a novel cyclic tetrapeptide represented by Formula I, wherein the compound is Example 2, 3a, 3b, 3d, 10, 11, 12d, 12e, 17, or 18: -38- WO 01/07042 PCT/US00/19627 o 0 0N O NHNH H;~~r [[N N OMe 0 OMe 0 E NOH (4-Me)PhSO 2 HNN 00 N~ OHH H N PhCH20N Na+C 2 C OSO2N Ph(4-Me) OAc o 0 N N ' O e N.,M OH N NH N NH [,t - N(yH N O e- oM SO2MeSO2Ph(4-NO2) No I : ,or O O H O OMe OMe S0O

OSO

2 Ph( 4 -Me) OAc 0 -0 H I 3 N N 4 H NH I H 0 O- e N0 M 8 0 OMe

SO

2 M SO 2 Ph( 4 -N0 2 ) 0 00 HNH H N H N__ 1M or N NH oHOIe HO 0 8_0 OMe 08 0 SH - 39 - WO 01/07042 PCT/US00/19627 In another aspect, the present invention provides a novel cyclic tetrapeptide represented by Formula I, wherein the compound is Example 22a, 22b, 23a, 23b, 145, 146c, 146d, 146e, 146f, or 147: 00 O H NO 0 H NNM'OMe N NH N NH H 0 OH H O 0 80 8 0 0 O O' ) N oH O" H N HO H NH O O O O 5 oo a. 0 0 0N NH 0 ii~S N, N NH H NH HJ~N OO 8 0 0 0 0a -40 - WO 01/07042 PCT/US00/19627 N .ON N L NnPr

CH

2 CO2CH 3 H2CO2H N NHH N HNH H Hor 0- 00 0- 0 0 o H NNO N NHH 0 N H aY 0 0 O0 00 In yet another aspect, the present invention provides a novel cyclic tetrapeptide represented by Formula I, wherein the compound is Example 21a, 21b, 24a, 24b, 26, 27, 28, 29, 30, 32, 37, 39, 43, 44, 46, 51, 56a, 63, 64, or 67: o' 0 OY N H NH N~~ 0' C N NH N NHNI NHI H ~ 0MN N A 5o Ha H None m. Oe 8 0 8N(OMe)Me 5 O0 0 Y 0Y0 0 0' 0' NH NI H N. -- N NH U OOH ~ O0 NHOCH 2 Ph 0 -41- WO 01/07042 PCT/USOO/19627 0 0 0y0 N NHNN NON He N N H I N H ' 0 (0-LI. 0 NHOH 8NHOH 0 0 0 0 0 0) N NN o o H I NH N ~ N H NI ~~OMe N>N NH 1" 0 oMe HUt N o80 0~ 0 OMe 0 0 7O0 e 0 7 y0 0 ")-N NIN II " 0' y 0 PIN HI NHJ ~ ~H N iN 0 ~ OMe N NH N 0 0- - 0 J~ I"~0 ome S11 6 '/'o 0SM 5 0N~Y N NHIIHJ N I N N HN N H N HI N 0 , 8O Y 0 OMe 0 OMe o /O 0 S H 0 'o. K SH 07 -42 - WO 01/07042 PCT/USOO/19627 0 'NO~ N H HI II ' N NH r HI NNN N IHI N 0 OMe I 0 0 8 -' 0 0 In another aspect, the present invention provides a novel cyclic 5 tetrapeptide represented by Formula I, wherein the compound is Example 69, 70, 72, 73, 74a. 74b, 74c, 74d, 74e, 74f, 74g, 74h, 74i, 74j, 75, 79, 91, 93, 97, 98, 129a, or 129b: Y 0 0 0 Y N H H I aNNH H N] N NH H NA ~ I HI N NK . H N I N--0 P(0)(OPh), 0 0 0 0 0 0 0 0 ' N H' O -43 H N NNHI 1 02Me NH, H-I -k IP 0 8 Er 0 0 0 10 0 - 43 - WO 01/07042 PCT/USOO/19627 a- 0 0 NH0IH H I NH N H N N X I H ' NN/ OY CH CO tBu Nck 0i CH-CO 2 Me 0o CH CH OSI(tmu)Me 2 0Me 0 0 0 000 I' I 0 N N H NH N NH 0 C(O)Ph( 4 -Oc HI, NIY , 0o ; : C(O)Ph 0 CO Ph( 4 -N0 2 ) 0 0 0 00 0 N-0 0 1 0 N H NHo ~ I I N H NHN N3 t 0 CO2CH2Ph NI k--K O 0SO Ph( -Me) 0 O)OCH 2

CH

2 NMe 2 00 5 0 0 y 00 ) N NH N N NH N,, IN H NK CH CO H H NNI1 oY 8 8 Ii

O

0 a C - 44 - WO 01/07042 PCT/US00/19627 OO OH0 or I HCHNHSO Me CH2N Me)SO In yet another aspect, the present invention provides a novel cyclic 5 tetrapeptide represented by Formula I, wherein the compound is Example 132a, 133, 135, 138, 139a, 139b, 139c, 139d, 139e, 139f, 139g, 139h, 139i, 139j, 140, 141, 142, 144b, 144d, 144f, 158, 159, 160, 162a, or 162b. In still another aspect, the present invention provides a novel cyclic tetrapeptide represented by Formula I, wherein the compound is Example 102, 103, N NH 10 108a, or 108b. In another aspect, the present invention provides a novel cyclic tetrapeptide represented by Formula I, wherein the compound is Example 109 or 110. In another aspect, the present invention provides a novel cyclic tetrapeptide represented by Formula I, wherein the compound is Example 168. 0 0H 2 HCNH20H 2 M 0 8 tetrapeptide represented by Formula I, wherein the compound is Example 156, 157a, 157b, 157c, or 157d. In another aspect, the present invention provides a novel cyclic tetrapeptide represented by Formula I, wherein the compound is 20 -45 - WO 01/07042 PCT/US00/19627 O0 0 NN N NH H N OMe 0 0 In another aspect, the present invention provides a novel cyclic tetrapeptide represented by Formula I, wherein the compound is 5 0 0N H N NH H N NI O OMe 0 0 In another aspect, the present invention provides a novel cyclic tetrapeptide represented by Formula I, wherein the compound is Example 153 or 154. 10 In another aspect, the present invention provides a method for the treatment of protozoal infections comprising the step of administering to a host suffering from a protozoal infection a therapeutically effective amount of the novel compounds of the invention which inhibits histone deacetylase. A therapeutically effective amount is that safe amount sufficient to inhibit histone deacetylase activity 15 of the causative protozoa to control and overcome the infection. The present invention also provides a method for the prevention of protozoal infections comprising the step of administering to a host an effective preventative amount of the -46 - WO 01/07042 PCT/US00/19627 novel compounds of the invention, which inhibits histone deacetylase. An effective preventative amount is that safe amount sufficient to inhibit the infection of the host. In yet another aspect, the present invention provides a composition useful for the treatment or prevention of protozoal diseases which comprises an inert 5 carrier and an effective amount of a compound of formula I. As used herein, "alkyl" as well as other groups having the prefix "alk" such as, for example, alkoxy, alkanoyl, alkenyl, alkynyl and the like, means carbon chains which may be linear or branched or combinations thereof. Examples of alkyl 10 groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl and the like. "Alkenyl", "alkynyl" and other like terms include carbon chains containing at least one unsaturated C-C bond. The term "cycloalkyl" means carbocycles containing no heteroatoms, and includes mono-, bi- and tricyclic saturated carbocycles, as well as fused ring 15 systems. Such fused ring systems can include one ring that is partially or fully unsaturated such as a benzene ring to form fused ring systems such as benzofused carbocycles. Cycloalkyl includes such fused ring systems as spirofused ring systems. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, decahydronaphthalene, adamantane, indanyl, indenyl, fluorenyl, 1,2,3,4 20 tetrahydronaphalene and the like. Similarly, "cycloalkenyl" means carbocycles containing no heteroatoms and at least one non-aromatic C-C double bond, and include mono-, bi- and tricyclic partially saturated carbocycles, as well as benzofused cycloalkenes. Examples of cycloalkenyl include cyclohexenyl, indenyl, and the like. The term "halogen" includes fluorine, chlorine, bromine and iodine 25 atoms. The term "heterocycle", unless otherwise specified, means cyclic systems such as those described above for cycloalkyl and cycloalkenyl in which at least one atom is a sulfur, oxygen or nitrogen atom in a group of atoms that form the backbone of a ring. Such heterocycles include mono- or bicyclic compounds that are 30 saturated or partly unsaturated, as well as benzo- or heteroaromatic ring fused saturated heterocycles or partly unsaturated heterocycles, and containing from 1 to 4 heteroatoms independently selected from oxygen, sulfur and nitrogen. Examples of saturated heterocycles include morpholine, thiomorpholine, piperidine, piperazine, tetrahydropyran, tetrahydrofuran, dioxane, tetrahydrothiophene, oxazolidine, 35 pyrrolidine; examples of partly unsaturated heterocycles include dihydropyran, -47 - WO 01/07042 PCT/US00/19627 dihydropyridazine, dihydrofuran, dihydrooxazole, dihydropyrazole, dihydropyridine, dihydropyridazine and the like. Examples of benzo- or heteroaromatic ring fused heterocycle include 2,3-dihydrobenzofuranyl, benzopyranyl, tetrahydroquinoline, tetrahydroisoquinoline, benzomorpholinyl, 1,4-benzodioxanyl, 2,3-dihydrofuro(2,3 5 b)pyridyl and the like. The term "aryl" is intended to include mono- and bicyclic aromatic and heteroaromatic rings containing from 0 to 5 heteroatoms independently selected from nitrogen, oxygen and sulfur. The term "aryl" is also meant to include benzofused cycloalkyl, benzofused cycloalkenyl, and benzofused heterocyclic groups. Examples 10 of "aryl" groups include phenyl, pyrrolyl, isoxazolyl, pyrazinyl, pyridinyl, oxazolyl, thiazolyl, imidazolyl, triazolyl, tetrazolyl, furanyl, triazinyl, thienyl, pyrimidinyl, pyridazinyl, pyrazinyl, naphthyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, furo(2,3-B)pyridyl, 2,3-dihydrofuro(2,3-b)pyridyl, benzoxazinyl, benzothiophenyl, quinolinyl, indolyl, 2,3-dihydrobenzofuranyl, benzopyranyl, 1,4 15 benzodioxanyl, indanyl, indenyl, fluorenyl, 1,2,3,4-tetrahydronaphthalene and the like. Aroyl means arylcarbonyl in which aryl is as defined above. Examples of NRcRd or NRgR h forming a 3- to 10- membered ring containing 0 to 2 additional heteroatoms selected from O, S(O)m and N are aziridine, azetidine, pyrrolidine, piperidine, thiomorpholine, morpholine, piperazine, 20 octahydroindole, tetrahydroisoquinoline and the like. The term "CO" means that the carbon is not present. Thus, "C0-C5" means that there are from none to five carbons present - that is, five, four, three, two, one, or no carbons present. The term "optionally substituted" is intended to include both 25 substituted and unsubstituted. Thus, for example, optionally substituted aryl could represent a pentafluorophenyl or a phenyl ring. Compounds described herein contain one or more asymmetric centers and may thus give rise to diastereomers and optical isomers. The present invention includes all such possible diastereomers as well as their racemic mixtures, their 30 substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. The above Formula I is shown without a definitive stereochemistry at certain positions. The present invention includes all stereoisomers of Formula I. Further, mixtures of stereoisomers as well as isolated specific stereoisomers are also included. During the the course of the synthetic 35 procedures used to prepare such compounds, or in using racemization or -48 - WO 01/07042 PCT/US00/19627 epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. When the compound of 5 the present invention is acidic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from such inorganic bases include aluminum, ammonium, calcium, copper (ic and ous), ferric, ferrous, lithium, magnesium, manganese (ic and ous), potassium, sodium, zinc and the like salts. Particularly 10 preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, as well as cyclic amines and substituted amines such as naturally occurring and synthesized substituted amines. Other pharmaceutically acceptable organic non-toxic bases from which salts can be formed 15 include ion exchange resins such as, for example, arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2 dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, 20 procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like. When the compound of the present invention is basic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include, for 25 example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids. 30 The concept of the inhibition of histone deacetylase as a target for antiprotozoal compounds is described in pending U.S. Patent Applications 09/296,834, filed April 22, 1999, and 08/716,978, filed September 20, 1996. Known compounds that may be histone deacetylase inhibitors and therefore useful in the treatment of protozoal diseases include, for example, trichostatin A, trapoxin A and B, 35 HC-toxin, chlamydocin, Cly-2, WF-3161, Tan-1746, apicidin, and analogs thereof. -49 - WO 01/07042 PCT/US00/19627 Trapoxin A is described in Itazaki et al., J. Antibiot. 43, 1524-1532(1990); HC-Toxin is described in Liesch et al., Tetrahedron 38, 45-48(1982); chlamydocin is described in Closse et al., Helv. Chim. Acta 57, 533-545(1974); Cly-2 is described in Hirota et al., Agri. Biol. Chem 37, 955-56(1973); WF-3161 is described in Umehana et al., J. 5 Antibiot. 36, 478-483(1983); and Tan-1746 is described in Japanese Patent No. 7196686. Unlike the ethyl ketone sidechain found in apicidin, HC toxin, chlamydocin, trapoxin A and trapoxin B contain a C8 c-ketoepoxide functionality. Apicidin and analogs thereof referred to herein are described by the following chemical formula: 10

CH

3

CH

2

-CH

3 NH 2 2

(CH

2 O . R 3 00 0 Apicidin 15 Examples include Compound n RI R 2

R

3 Apicidin Ia 1 H OMe H Ib 0 H OMe H Ic 1 H OMe OH IIA 1 =0 OMe H i11B 1 =0 H H These compounds are described in pending U.S. Patent Application Nos. 08/281,325, filed July 27, 1994 and 08/447,664, filed May 23, 1995. The 20 compounds are produced from a strain of Fusarium as disclosed in the applications. - 50- WO 01/07042 PCT/US00/19627 The compounds of the present invention have been found to be histone deacetylase inhibitors. Accordingly, they can be useful in the treatment and prevention of protozoal diseases in human and animals, including poultry. Examples of protozoal diseases against which histone deacetylase inhibitors may be used, and 5 their respective causative pathogens, include: 1) amoebiasis (Dientamoeba sp., Entamoeba histolytica); 2) giardiasis (Giardia lamblia); 3) malaria (Plasmodium species including P. vivax, P. falciparum, P. malariae and P. ovale); 4) leishmaniasis (Leishmania species including L. donovani, L. tropica, L. mexicana, and L. braziliensis); 5) trypanosomiasis and Chagas disease (Trypanosoma species including 10 T. brucei, T. theileri, T. rhodesiense, T. gambiense, T. evansi, T. equiperdum, T. equinum, T. congolense, T. vivax and T. cruzi); 6) toxoplasmosis (Toxoplasma gondii); 7) neosporosis (Neospora caninum); 8) babesiosis (Babesia sp.); 9) cryptosporidiosis (Cryptosporidium sp.); 10) dysentary (Balantidium coli); 11) vaginitis (Trichomonas species including T.vaginitis, and T. foetus); 12) coccidiosis 15 (Eimeria species including E. tenella, E. necatrix, E. acervulina, E. maxima and E. brunetti, E. mitis, E. bovis, E. melagramatis, and Isospora sp.); 13) enterohepatitis (Histomonas gallinarum); and 14) infections caused by Anaplasma sp., Besnoitia sp., Leucocytozoan sp., Microsporidia sp., Sarcocystis sp., Theileria sp., and Pneumocystis carinii. 20 The histone deacetylase inhibiting compounds and compositions of the present invention are preferably used in the treatment or prevention of protozoal infections caused by a member of the sub-phylum Apicomplexans. More preferably the compounds and compositions are used i) in the treatment or prevention of malaria, toxoplasmosis, cryptosporidiosis and trypanosomiasis in humans and animals, and ii) 25 in the management of coccidiosis, particularly in poultry, either to treat coccidial infection or to prevent the occurrence of such infection. When the histone deacetylase inhibiting compounds and compositions of this invention are administered on a chronic basis, such as in the prevention of coccidiosis in poultry, the histone deacetylase inhibitor preferably is selective for 30 protozoal histone deacetylase over the host histone deacetylase. Such a selective inhibitor would minimize adverse histone deacetylase inhibition effects to the host over the long term. Two specific examples of the method of this invention of administering an effective preventative amount of an histone deacetylase inhibitor to 35 prevent the establishment of parasitic infections in humans and animals are 1) the -51 - WO 01/07042 PCT/US00/19627 prevention of Plasmodium (malaria) infection in humans in endemic areas and 2) the prevention of coccidiosis in poultry. The histone deacetylase-inhibiting compound can be conveniently administered continually in the feed or drinking water, or regularly by oral or parenteral dosing. 5 Malaria is the number one cause of death in the world. The disease is transmitted by mosquitoes in endemic areas and can very rapidly progress to a life threatening infection. Therefore, individuals living in or visiting areas where malaria carrying mosquitoes are present routinely take prophylactic drugs to prevent infection. Thus, according to an embodiment of the present invention, a histone deacetylase 10 inhibitor is administered orally or parenterally one or more time(s) a day, preferably each dose ranges from about 0.01mg/kg to about 100mg/kg. The compound may be administered for the entire period during which the patient or animal is at risk of acquiring a parasitic infection. Coccidiosis is a disease that can occur in humans and animals and is 15 caused by several genera of coccidia. The most economically important occurrence of coccidiosis is the disease in poultry. Coccidiosis in poultry is caused by protozoan parasites of the genus Eimeria. The disease can spread quite rapidly throughout flocks of birds via contaminated feces. The parasites destroy gut tissue and damage the gut lining, thereby impairing nutrient absorption. An outbreak of coccidiosis in a poultry 20 house can cause such dramatic economic losses for poultry producers that it has become standard practice to use anticoccidial agents prophylactically in the poultry feed. Thus, according to another embodiment of this invention, a histone deacetylase inhibitor is administered in the feed or drinking water for the entire or a portion of the lifetime of domestic birds with a dose that ranges between about 0.1 ppm to about 25 500ppm in the feed or water. For treatment of established parasitic infections in humans or animals, the histone deacetylase inhibitor is conveniently administered orally or parenterally when the infection is suspected or diagnosed. The treatment period varies according to the specific parasitic disease and the severity of the infection. In general the 30 treatment is continued until the parasites are effectively eradicated and/or the symptoms of the disease are resolved. Two specific examples of the method of this invention for the treatment of protozoal infections by administering a therapeutically effective amount of a histone deacetylase inhibitor are 1) the treatment of a Cryptosporidium parvum infection in an animal or human and 2) the treatment of 35 acute Plasmodiumfalciparum malaria in humans. - 52 - WO 01/07042 PCT/US00/19627 Cryptosporidium parvum is a protozoan parasite that infects and destroys cells lining the intestinal tract of humans and animals. The infection establishes quite rapidly and has acute effects on the patient. In the case of humans, patients get severe dysentery for a period of 5-7 days. In immune compromised 5 patients C. parvum infections can persist and can be life threatening. In animals C. parvum infection is the leading cause of death in young dairy calves. A C. parvum infection can be easily diagnosed by symptoms and examination of a stool sample. When the disease is suspected and/or diagnosed, treatment with a histone deacetylase inhibitor according to the method of this invention can be initiated. The dose 10 preferably ranges from about 0.01mg/kg to about 500mg/kg. The histone deacetylase is administered one or more time(s) a day, orally or parenterally until the infection is eliminated. The dosing period typically is in the range of about 1-3 weeks. P. falciparum causes acute life threatening malarial infections in humans. The infection if left untreated can often result in the death of the patient. A 15 malaria infection can be easily diagnosed by symptoms and examination of a blood sample from the patient. Treatment would be initiated following diagnosis. According to an embodiment of this invention, a histone deacetylase inhibitor is administered one or more time(s) a day, orally or parenterally, until the infection is eliminated. The dose preferably ranges from about 0.01mg/kg to about 200 mg/kg. 20 The histone deacetylase inhibiting compositions of this invention may be administered to a host in need of treatment in a manner similar to that used for other known antiprotozoal agents. For example, the compositions may be administered parenterally, orally, topically, or rectally. The dosage to be administered will vary according to the particular compound used, the infectious organism 25 involved, the particular host, the severity of the disease, the physical condition of the host, and the selected route of administration; the appropriate dosage can be readily determined by a person skilled in the art. For the treatment of protozoal diseases in human and animals, the dosage preferably ranges from about 0.01mg/kg to about 500mg/kg. For prophylactic use in human and animals, the dosage preferably ranges 30 from about 0.01mg/kg to about 100mg/kg. For use as an anticoccidial agent, particularly in poultry, the compound is preferably administered in the animals' feed or drinking water. The dosage preferably ranges from about 0. lppm to about 500ppm. In one aspect, the composition of the present invention comprises a histone deacetylase inhibitor and an inert carrier. The compositions include - 53 - WO 01/07042 PCT/US00/19627 pharmaceutical compositions for human and veterinary usage, and feed compositions for the control of coccidiosis in poultry. The pharmaceutical compositions of the present invention comprise a histone deacetylase inhibitor as an active ingredient, a pharmaceutically acceptable 5 carrier and optionally other therapeutic ingredients or adjuvants. The compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The 10 pharmaceutical compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. In practice, the histone deacetylase inhibitor of this invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may 15 take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). Thus, the pharmaceutical compositions of the present invention can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions 20 can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the histone deacetylase inhibitors may also be administered by controlled release means and/or delivery devices. The compositions may be prepared by any of the methods of 25 pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation. 30 In preparing the compositions for oral dosage form, any convenient pharmaceutical media may be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like may be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, 35 binders, disintegrating agents, and the like may be used to form oral solid preparations - 54 - WO 01/07042 PCT/US00/19627 such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets may be coated by standard aqueous or nonaqueous techniques 5 A tablet containing the composition of this invention may be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing 10 agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Each tablet preferably contains from about 1mg to about 500mg of the active ingredient and each cachet or capsule preferably containing from about 1 to about 500mg of the active ingredient. 15 Pharmaceutical compositions of the present invention suitable for parenteral administration may be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be 20 included to prevent the detrimental growth of microorganisms. Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final 25 injectable form must be sterile and must be effectively fluid for easy syringability. The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene 30 glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof. Pharmaceutical compositions of the present invention can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, or the like. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations may be prepared, utilizing the histone 35 deacetylase inhibiting compounds of this invention, via conventional processing - 55 - WO 01/07042 PCT/US00/19627 methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt% to about 10 wt% of the compound, to produce a cream or ointment having a desired consistency. Pharmaceutical compositions of this invention can be in a form 5 suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in moulds. 10 In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above may include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can be included to render the formulation 15 isotonic with the blood of the intended recipient. As described above, to manage coccidiosis in poultry, the histone deacetylase inhibitor of this invention can be conveniently administered as a component of a feed composition. The poultry feed preferably contains from about lppm to about 1000ppm, more preferably from about 10ppm to about 150ppm of the 20 histone deacetylase inhibitor of this invention. The optimum levels will vary with the species of Eimeria involved, and can be readily determined by one skilled in the art. It is preferred that the histone deacetylase inhibitor of this invention be added to poultry feed in the amount of from about 0.01% to about 0.1% by weight of the diet. The compositions of this invention are especially useful in controlling the pathology 25 associated with E. tenella. The preferred concentration for similar control of intestinal-dwelling species is from about 0.01% to about 0.1% by weight of the diet. Amounts of about 0.01% to about 0.1% percent by weight are advantageous in reducing the pathogenic effects of both fecal coccidiosis and intestinal coccidiosis. In the preparation of poultry feed incorporating the compositions of the 30 invention, the histone deacetylase inhibitor can be conveniently dispersed, for example, by i) being mechanically mixed in a finely ground form with the poultry feedstuff, or ii) being first mixed with an intermediate formulation (to form a premix) that is subsequently blended with other poultry feedstuff components. Typical components of poultry feedstuffs include molasses, fermentation residues, corn meal, 35 ground and rolled oats, wheat shorts and middlings, alfalfa, clover and meat scraps, - 56 - WO 01/07042 PCT/US00/19627 together with mineral supplements such as bone meal, calcium carbonate and vitamins. Compositions containing a compound described by formula I may also be prepared in powder or liquid concentrate form. In accordance with standard 5 veterinary formulation practice, conventional water-soluble excipients, such as lactose or sucrose, may be incorporated in the powders to improve their physical properties. It is preferable that the powder compositions of this invention comprise from about 50wt% to about 100wt%, and more preferably about 60wt% to about 80wt% of the compound. These powders may either be added to animal feedstuffs, for example, by 10 way of an intermediate premix, or added to the animal drinking water by dilution. Liquid concentrates of this invention suitably contain a water-soluble compound combination and may optionally further include a veterinary acceptable water miscible solvent. For example, a solvent such as polyethylene glycol, propylene glycol, glycerol, or glycerol formal can be mixed with up to 30% v/v of ethanol. It is 15 preferable that the liquid concentrates of this invention comprise from about 50wt% to about 100wt%, and more preferably about 60wt% to about 80wt% of the compound. The liquid concentrates may be administered to the drinking water of animals, particularly poultry. 20 The following examples are provided to more fully illustrate the present invention, and are not to be construed as limiting the scope of the claims in any manner. Preparation of Side Chain-Modified Apicidin Analogs 25 Referring to Scheme I below, apicidin can be converted into alpha substituted analog compounds 4 and 5. - 57 - WO 01/07042 PCT/US00/19627 Scheme I 1: Apicidin 1) Base . 2) RX 3 + ~ 3-I 5 7-19 OSiR 3 OSiR 3 2 3 1) Base 1) Oxidize 2) RX 2) Deprotect R8 = R10 = 4a USIMe3 5aUSI-e3 - Rio1 4b SePh 5b SePh 4c SPh 5c SPh 3 5 3 4d OH 5d OH 4e =0 5e =0 O O 4f F 5f F 4 5 S Oxidative Cleavage 0 O H3 7 6 5 Apicidin is first enolized with an appropriate amine base including, but not limited to, LiN(iPr)2, NaN(SiMe3)2, KN(SiMe3)2, and the like at temperatures ranging from -78 0 C to 0OC to form an enolate. The amine base is preferably KN(SiMe3)2. Appropriate solvents for this reaction include, but are not limited to, Et20, dioxane, tetrahydrofuran (THF), dimethoxyethane, and the like. The solvent is 10 preferably THF. The enolate is reacted with an appropriate electrophile RX including, but not limited to, Mel, EtI, allyl bromide, benzyl bromide, PhSeCl, PhSC1, PhSSPh, (MeO)2P(O)Cl, (CF2SO2)20, Et3SiC1, tBu(Me)2SiCl, (nPr)3SiCl, Me3SiCl, Ph(Me)2SiCI, and the like to form a silyl enol ether. The electrophile is preferably Me3SiCl. 15 Treatment of the thus prepared silyl enol ethers with an oxidant, including but not limited to, H202, tBuOOH, Me3SiOOH, AcOOH, dimethydioxirane and the like, or preferably MCPBA (meta-chloroperbenzoic acid), at temperatures from -78 0 C to RT (room temperature) but preferably 0OC to RT will produce the corresponding alpha-silyloxyketones, compounds 4a/5a. The silyl -58 - WO 01/07042 PCT/US00/19627 protecting groups can be then removed using a variety of acid or fluoride sources including, but not restricted to, HC1, H2SO4, HBF4, acetic acid, PPTS (pyridinium p toluenesulfonate), TsOH (p-toluenesulfonyl hydroxide), HF, HFspyridine, or nBu4NF and the like in protic or aprotic solvents including, but not limited to, CH2Cl2, 5 CHCI3, MeOH, EtOH, iPrOH, THF, Et20 and dioxane and the like at temperatures from 0 0 C to 50 0 C to generate the alpha-hydroxyketones, compounds 4d/5d. The alpha-hydroxyketone compounds 4d/5d may be separated or used with no further separation, as desired. Compounds 4d/5d can be oxidized to the corresponding diketones, compounds 4e/5e, by treatment including, but not limited to, 10 Swern oxidation, Dess-Martin oxidation, PCC (pyridinium chlorochromate), PDC (pyridinium dichromate), Moffat-oxidation, and the like, or most preferably TPAP/NMO (tetrapropylammonium perruthenate(VII)/4-methylmorpholine N-oxide) in solvents including, but not limited to, toluene, CH2Cl2, CHCl3 and the like at temperatures ranging from -78 0 C to RT. 15 The alpha-hydroxyketone compounds 4d/5d can be converted into the corresponding alpha-haloketone compounds such as 4f/5f by treatment with Ph3P/CBr4, Ph3P/12, PH3P/CC14, Ph3P/CHC12CHCl2, DAST (diethylaminosulfur trifluoride), morpholinyl sulfur trifluoride, and the like in solvents such as CH2Cl2, CHCl3, benzene, toluene and the like at temperatures from -78 0 C to RT. 20 The alpha-hydroxyketone compounds 4d/5d can be treated with an oxidizing agent including, but not restricted to, NalO4, HIO4, MnO2, Amberlite® IRA-904 ion-exchange resin available from Aldrich Chemical Company, Milwaukee, Wisconsin, NalO4, KIO4, and nBu4NIO4, or most preferably Pb(OAc)4 to yield a C7-aldehyde compound 6, and a C8-methyl ester compound 7, by an oxidative 25 cleavage reaction. The oxidative cleavage reaction may be performed in a variety of solvents or mixtures of solvents, including water, EtOH, iPrOH (isopropanol), tBuOH (tert-butanol), acetone, ether, THF, benzene, toluene, CH2Cl2, CHCl3, and the like, or most preferably MeOH. Generally, the oxidative cleavage reaction is performed at temperatures from about -78 0 C to about 80oC. When utilizing MeOH, the reaction 30 should be performed at temperatures from -20 0 C to RT. The oxidative cleavage reaction may be improved by the addition of a base, including but not restricted to NaHCO3, Et3N, EtN(iPr)2, lutidine and the like, or most preferably pyridine. The oxidative cleavage reaction is generally complete in from about 5 minutes to about 24 hours. - 59 - WO 01/07042 PCT/US00/19627 Referring to Scheme II below, the phenylsulfide compounds 4c/5c or phenylselenide compounds 4b/5b, analogs of apicidin, are oxidized to the corresponding sulfoxide or selenoxide compounds (not shown) using reagents which include, but not limited to, Oxone, MCPBA, tBuOOH, AcOOH, NaIO4, 5 dimethyldioxirane, and the like, or most preferably H202, in solvents or mixtures of solvents, including, but not limited to toluene, CHCI3, MeOH, water, or most preferably CH2Cl2 and at temperatures ranging from -20 0 C to 50 0 C. Scheme HI 7 9 7 9 4b/5b + Oxidation 0 0 8 9 1) Dihydroxylate 2) Oxidative cleavage OMe 3H- 3 7 0 0 10 10 7 Although the Scheme II shows only compounds 4b/5b as the starting compounds, the same scheme applies just as well to using compounds 4c/5c as 15 starting compounds. The sulfoxides and selenoxides are thermally eliminated to generate the corresponding enone compounds 8 and 9 in solvents including, but not limited to, CH2CI2, CHCl3, MeOH, or most preferably toluene, at temperatures ranging from RT to 110oC. Enone compounds 8 and 9 can be epoxidized (not shown) with 20 appropriate epoxidizing agents including, but not limited to, dimethyldioxirane, H202, tBuOOH, AcOOH, and the like, or most preferably MCPBA, in solvents or - 60 - WO 01/07042 PCT/US00/19627 mixtures of solvents including, but not limited to, toluene, CHCl3, MeOH, or most preferably CH2CI2, at temperatures ranging from -20 0 C to RT. Enone compounds 8 and 9 also may be dihydroxylated with OsO4 under conditions known to those skilled in the art to form the corresponding diols. 5 Osmium tetroxide may be used either stoichiometrically or catalytically in the presence of an oxidant including, but not restricted to, morpholine N-oxide, trimethylamine N-oxide, hydrogen peroxide, tert-butyl hydroperoxide and the like. The dihydroxylation reactions are performed in a variety of solvents or mixtures of solvents. The solvents include both protic and aprotic solvents such as water, MeOH, 10 EtOH, tert-butanol, ether, THF, benzene, pyridine, acetone, and the like. The dihydroxylation reactions are performed at from -78'C to 80'C and are complete in from 5 minutes to 24 hours. The diol products thus obtained can be oxidatively cleaved as described previously for compounds 6 and 7 to yield a C6-aldehyde compound 10 and a C8 methyl ester compound 7 from compounds 8 and 9, 15 respectively. Referring to Scheme I below, apicidin's sidechain C8-ketone group can be a starting point for analog synthesis. Scheme III 1: Apicidin 3 5 7 9 R1 = 11 11a: OH 11b: OH, R11b 11c: OC(O)Rli c or OC(S)R 11c 11d: OSO 2 Rlld 11e: H 11f: NR 11 f 1

R

1 1 f 2 11g: =NOR,g 11h: =NNHSO 2 R11h 11i: =CR 11 i 1

R

11 i2 11j: epoxide 20 11k: SR11 k -61 - WO 01/07042 PCT/US00/19627 R11b, Riic, Rl1d, RIlfl, Rllf2, Rlg, RIlh, R11il, Rlli2, and R11lk are each independently an alkyl or aryl group which optionally is substituted. By Scheme IB, the sidechain C8-ketone group can be reduced using reagents known to those skilled in the art, including, but not limited to LiBH4, 5 LiA1H4, DIBAL-H (diisobutylaluminum hydride), K-Selectride® (potassium tri-sec butylborohydride) available from Aldrich Chemical Company, Milwaukee, Wisconsin, L-Selectride® (lithium tri-sec-butylborohydride) available from Aldrich, Alpine-Borane® (B-isopinocampheyl-9-borabicyclo[3.3.1]-nonane) available from Aldrich, and the like or most preferably NaBH4 to yield the C8 alcohol compound 10 1la. These reduction reactions may be performed in protic or aprotic solvents including, but not limited to, THF, ether, dimethyl ether, dioxane, EtOH, CH2Cl2, EtOAc, CHCl3, benzene, toluene, or most preferably MeOH, and at temperatures from -78 0 C to RT. Apicidin's sidechain C8-ketone group can also be treated with RMgBr, 15 RMgC1, RMgI, RLi, R2CuLi, RCeCl2Li and the like to generate substituted alcohol compounds lIb. In these RLi, RLiX, or RMgX type reactants, R is an alkyl or aryl group, and the alkyl and aryl groups are optionally substituted. These substitution reactions may be performed in solvents or mixtures of solvents, including but not limited to, Et20, dioxane, HMPA (hexamethylphosphoramide), DMSO, NMP (1 20 methyl-2-pyrrolidinone), dimethoxyethane, and the like, or most preferably TH , at temperatures from -78 'C to RT, and are complete in from 5 minutes to 12 hours. The C8-alcohol compound 1la generated above can be alkylated, acylated or sulfonylated using known methods for acylation, sulfonylation and alkylation of alcohols to generate apicidin derivative compounds 11c or lid. Thus, 25 acylation may be accomplished using reagents such as acid anhydrides, acid chlorides, chloroformates, carbamoyl chlorides, CIC(S)OPh(F5), thiocarbonyldimidazole, isocyanates, and the like, and amine bases according to general procedures known to those skilled in the art. Sulfonylations may be carried out using sulfonyl chlorides or sulfonic anhydrides. Alkylations may be carried out using alkyl halides or 30 trichloroacetimidates. Suitable solvents for these reactions include benzene, toluene, CHCl3, CH2CICH2C1, and the like, or most preferably CH2Cl2, and may be performed from temperatures of -40 0 C to 80 0 C. The hydroxyl group at C8 of compound 1la can be eliminated using Burgess reagent, Martin's sulfurane reagent or by treating compound lid with a base - 62 - WO 01/07042 PCT/US00/19627 to generate a mixture of C6, C7- and C7, C8-olefin isomers. Suitable bases include, but are not limited to, Et3N, EtN(iPr)2, NaOMe, KOtBu, and the like or most preferably DBU in solvents such as CH2Cl2, CHCl3, toluene, benzene, MeOH, EtOH, pyridine and the like and at temperatures from 0 0 C to 110 0 C. The C8-hydroxyl group 5 of compound 11a can also be eliminated by reduction via the intermediary compound 11c wherein R is OPh, OPh(F5), Set, and the like, or most preferably N-1-imidazolyl. Intermediary compound 11c is treated with i) a radical initiator such as oxygen/Et3B, AIBN (2,2'-azobisisobutyronitrile), benzoyl peroxide and the like, and ii) a hydride source, including, but not limited to, Et3SiH, Me3SnH, Ph3SnH, Ph3AsH, 10 nBu3SnCl/NaBH4, and the like, or most preferably nBu3SnH in solvents including but not limited to CH2Cl2, CHCI3, benzene, MeOH, EtOH, or most preferably toluene, and the like, at temperatures from -78 0 C to 110 0 C, to form compound 11e. Apicidin can be treated with mono- or disubstituted amines, a hydride 15 source, and a proton source to generate compound llf. Suitable solvents include, but are not restricted to, benzene, toluene, EtOH, iPrOH and the like, or more preferably, MeOH. Suitable proton sources include, but are not limited to, TsOH, HC1, HCO2H, PPTS and the like, or most preferably HOAc. The intermediate imine may be reduced in situ as it is formed or after azeotropic removal of water using a Dean-Stark trap. 20 Suitable reducing agents include, but are not limited to, LiAlH4, NaBH4, LiBH4, H2/(10% Pd/C) and the like, or most preferably NaBH3CN. Oxime compound lg and hydrazone compound llh are prepared by treating apicidin with hydrazine in a solvent with a proton source. For example, apicidin can be treated with mono- or disubstituted amines, and a proton source. 25 Suitable solvents include, but are not restricted to, benzene, toluene, EtOH, iPrOH and the like, or more preferably, MeOH. Suitable proton sources include, but are not limited to, TsOH, HC1, HCO2H, PPTS and the like, or most preferably HOAc. Apicidin is treated with stabilized Wittig reagents, unstabilized Wittig reagents or Horner-Emmons reagents to generate the unsaturated product, compound 30 1li. Suitable reagents include, but are not limited to, Ph3P=CH2, Ph3P=CHMe, Ph3P=CH(nPr), (MeO)2P(O)CH2CO2Me, Ph3P=CH2C(O)Me and the like. These olefination reactions may be performed in solvents including, but not limited to, DMF (N,N-dimethylformamide), MeOH, CH2Cl2, toluene, Et20, MeCN, THF and the like and may be performed at from -78 0 C to 110C. The C8 ketone of apicidin may be 35 converted into an epoxide (compound llj) by treated with CH2=N2 or Me3SiCH=N2 - 63 - WO 01/07042 PCT/US00/19627 in MeOH, or Me3S(O)I in a solvent such as tBuOH, dimethoxyethane, THF, DMF, DMSO, or more preferably HMPA and a strong base such a tBuOK, nBuLi, or more preferably NaH at temperatures from -78 0 C to 50 0 C. Treatment of compound lld with an appropriate sulfur containing 5 nucleophile permitted the introduction of sulfur at C8 to form compound 11k. Suitable nucleophiles include NaSMe, KSAc, HSPh/Et3N, HSCH2CH2OH/EtN(iPr)2 and the like. These reactions proceed readily in polar solvents such as MeOH, EtOH, DMF, DMSO, HMPA, NMP and the like at temperatures from 0 0 C to 50 0 C. Referring to Scheme IV below, a Beckmann rearrangement to form 10 compounds 12a and 12b can be induced by treatment of compound 11g with an acylating agent, including but not limited to, POCI3, SOC12, MeSO2Cl and the like or more preferably TsCI and an amine base at temperatures from 0 0 C to 50 0 C. Suitable amine bases include Et3N, EtN(iPr)2, lutidine, DBU (1,8-diazabicyclo[5.4.0]undec-7 ene) and the like, or most preferably pyridine. Pyridine also may serve as a solvent for 15 this reaction or alternatively MeCN, benzene, toluene, dioxane and the like may be used. Scheme IV 3'-- 7 9 Beckmann Rearrangement N-OH 11g 0 N' + -N,_, 3-- 5 7 H 0 20 12a 12b Referring to Scheme V below, the C7-aldehyde compound 6 could be oxidized to the corresponding C7 methyl ester compound 13 by treating with suitable oxidants including NaOCI/HOAc/MeOH, tBuOCl/MeOH/pyridine, and the like, or - 64 - WO 01/07042 PCT/US00/19627 most preferably PDC/DMF/MeOH under conditions known in the art. The C7 methyl ester compound 13 can further serve as the starting material for additional derivatives. Similarly, the C6-aldehyde compound 10 can be oxidized to its corresponding C6 methyl ester (not shown). 5 Scheme V 0 0 3'--' 5 7 H -- ' --- W 3 3 5 7OMe 3 5 H 6 13 10 Referring to Scheme VI below, the methyl ester compounds 7 and 13 can be converted into a series of esters, amides and ketones. Scheme VI 1) Saponify 3 5 (CH2)nCO 2 Me 2) Amide or ester formation 7: n = 2 3) RMgBr (optional) 13: n=l

R

1 9~ 14a: OR14a 3 (CH2)nC(O)R12 14: O bR14 14b2 14b: NR1 4 bIl a 4 b 2 14c: H 14 (n = 1 or 2) 14d: alkyl group 14e: aryl group 15 R14a, R14bl, and R14b2, is each independently an alkyl or aryl group, which optionally is substituted. Saponification could be accomplished by treating compound 7 with reagents including, but not limited to, NaOH, KOH, Me3SiOOK, LiOOH and the like, or more preferably LiOH. Solvents, or mixtures of solvents, include MeOH, EtOH, - 65 - WO 01/07042 PCT/US00/19627 tBuOH, DMF, DMSO, HMPA, Et20, THF, water and the like. The reaction proceeds at temperatures from 0 0 C to 100 0 C. Amide and ester formation may be accomplished by reacting the C8-carboxylic acid (compound 14a) thus prepared using standard ester- and amide-forming reagents known to those skilled in the art. The esterification 5 reaction is carried out using at least one equivalent of an alcohol, HOR. Although preferably ten to one hundred equivalents of alcohol are used, the esterification also may be carried out using the alcohol as solvent. Esterification reagents include, but are not restricted to, dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3 ethylcarbodiimide hydrochloride (EDC*HC1), diisopropylcarbodiimide, benzotriazol 10 1-yloxy-tris(dimethylamino)phosphonium hexafluorphosphate (BOP), bis(2-oxo-3 oxazolidinyl)phosphinic chloride (BOP-C1), benzotriazole-1-yl-oxy-tris-pyrrolidino phosphonium hexafluorophosphate (PyBOP), chloro-tris-pyrrolidino-phosphonium hexafluorophosphate (PyCIOP), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBrOP), diphenylphosphoryl azide (DPPA), 2-(1H 15 benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O benzotriazol-1-yl-N,N,N',N'-bis(pentamethylene)uronium hexafluorophosphate and 2 chloro-1-methylpyridinium iodide. The ester-forming reactions may be facilitated by the optional addition of N-hydroxybenzotriazole, N-hydroxy-7-aza-benzotriazole, 4 (N,N-dimethylamino)pyridine or 4-pyrrolidinopyridine. The ester-forming reaction is 20 generally performed using at least one equivalent (although several equivalents may be employed) of amine bases such as triethylamine, diisopropylethylamine, pyridine and the like. The carboxyl group may be activated for ester bond formation via its corresponding acid chloride or mixed anhydride, using conditions known to those skilled in the art. The ester-forming reaction is carried out in an aprotic solvent such 25 as, for example, methylene chloride, tetrahydrofuran, diethyl ether, dimethylformamide, N-methylpyrrolidine, and the like, at temperatures ranging from 20'C to 60 0 C, and is complete in about 15 minutes to about 24 hours. Amides (where R12 is NR14b1R14b2) are prepared as described for esters (vida supra) from the corresponding carboxylic acids using and an appropriate amine, HNR14blR14b2. 30 The amide compound 14b (in which NR14blR14b2 is N(OMe)Me) can be treated with nucleophilic agents to yield the corresponding aldehyde (compound 14c) and ketones (compounds 14d and 14e). Suitable nucleophiles include, but are not limited to, hydride reagents, RLi or RMgX and the like as described above for the preparation of compounds 11a and 11b. In addition, the - 66 - WO 01/07042 PCT/US00/19627 aldehyde and ketone products 14c, 14b and 14e can be further reacted with hydride reagents, RLi or RMgX, to generate the corresponding alcohol adducts as described previously. Referring to Scheme VII below, the aldehyde compounds 6, 10 and 14c 5 serve as starting material for the preparation of a variety of derivatives. Scheme VII

-

1) Reduce 3 5 (CH 2 )nCHO 2) Sulfonylate 14c: n = 2 3) Nucleophile 6: n=1 14 : n = 0 ~R15= 7 15a: OR15a 3'- (CH2)nR15 15b: S(O)nR1 5 b 15c: N3 15d: NR1 5 dlR1 5 d 2 15 (n = 1, 2 or 3) 15e: P(O)(ORi1 5 e) 2 R15a, R15b, R15d1, R15d2, and R15e, are each independently an alkyl 10 or aryl group which optionally is substituted. Reduction of the side chain aldehyde group in compounds 6, 10 and 14c with hydride reagents produced compound 15a (where R15a = H). The side chain alcohol thus obtained can then be sulfonylated, as described above in Scheme III. The sulfonyl group can then be displaced with an appropriate sulfur, nitrogen or 15 phosphorous nucleophile to form compounds 15b, 15c and 15e respectively. Suitable nucleophiles include NaSMe, KSAc, NaN3, (PhCH20)2P(O)H, (P(OCH2Ph)3, (MeO)2P(O)H, P(OMe)3 and the like. Further, the side chain azide compound 15c can be reduced using conditions known to those skilled in the art including, but not restricted to, H2/10% 20 Pd/C, HSAc/MeOH, SnCl2, Ph3P/H20 and the like to form a side chain amine compound (not shown). The amine compound thus obtained canbe acylated, alkylated or sulfonylated as described above. Alternatively, reductive amination of - 67 - WO 01/07042 PCT/US00/19627 the aldehyde compounds 6, 10 and 14c with a suitable amine as described above will generate the amine compound 15d. Referring to Scheme VUI below, the side chain of compounds 6, 10 or 14c can be extended by reacting the aldehyde with stabilized Wittig reagents, 5 unstabilized Wittig reagents or Homer-Emmons reagents to form compound 16a. Scheme VIII 1) Wittig 3 i 5_(CH 2 )nCHO 2) Reduction 3) Saponification 14c: n=2 9: n =1 10: n= 0

R

1 6 16a: OR16a - 16b: S(O)nR1 6 b 3' (CH 2 )n R16 16c: N 3 16d: NR16dlR16d 2 16 (n = 1, 2 or 3) 16e: P(O)(OR1 6 e) 2 R16a, R16b, R16d1, R16d2, and R16e are each independently an alkyl 10 or aryl group, which optionally is substituted. The side chain unsaturation of compound 16a can be reduced by catalytic hydrogenation using conditions known to those skilled in the art. Suitable catalysts include 5% Pd/C, 10% Pd/C, Pd(OH)2, PtO2, RhC13, RuCl2(PPh3)3, and the like. The hydrogenation reactions may be performed in solvents or mixtures of 15 solvents including CH2Cl2, CHCl3, toluene, MeOH, EtOH, EtOAc, acetone, THF, Et20, dimethoxyethane, DMF, DMSO, and the like. The reductions may be run at from one to 10 atmospheres of hydrogen pressure and the reactions are complete in from 5min to 24h. For apicidin analog compounds 16a or 16b in which Rl6a or R16b represents an ester moiety, the ester may be saponified and the carboxylic acid thus 20 obtained may be converted into other esters or amides as described previously. Referring to Scheme IX below, the N-methoxy group of apicidin may be removed by hydrogenation as described previously and the liberated indole nitrogen compound thus generated may be N-alkylated, acylated or sulfonylated using -68- WO 01/07042 PCT/US00/19627 known methods for acylation, sulfonylation and alkylation of indoles to generate apicidin derivative compound 17. Scheme IX - 0 Apicidin N H NH 1) Hydrogenation H N 2) Alkylation NR O -8 17 5 t R17 is an alkyl or aryl group, which optionally is substituted. Thus, acylation may be accomplished using reagents such as acid anhydrides, acid chlorides, chloroformates, carbamoyl chlorides, isocyanates and the like according to general procedures known to those skilled in the art. Sulfonylations 10 may be carried out using sulfonyl chlorides or sulfonic anhydrides. Alkylations may be carried out using alkyl halides. Suitable bases for these acylation, sulfonylation and alkylation reactions include KH, nBuLi, tBuLi, LiN(iPr)2, NaN(SiMe3)2, KN(SiMe3)2 and the like or more preferably Nail. Suitable solvents, or mixtures of solvents for these reactions include benzene, toluene, CHCI3, CH2CICH2C1, CH2Cl2, 15 DMSO, HMPA, NMP and the like or most preferably DMF and may be performed from temperatures of -40 0 C to 80 0 C. When the newly incorporated R17 group contains an ester moiety, the apicidin derivative compound 17 can be saponified to the corresponding carboxylic acid and converted into a series of amides using conditions described previously. 20 When the newly incorporated R17 group contains an alcohol function, the apicidin derivative compound 17, can be acylated, alkylated, phosphorylated or sulfonylated as described previously. Alternatively, this alcohol function may be converted into a leaving group such as a sulfonate or halide and displaced with appropriate sulfur, nitrogen or phosphorus nucleophiles as described previously 25 Referring to Scheme X below, apicidin's tryptophan may be allylically - 69 - WO 01/07042 PCT/US00/19627 oxidized to generate beta-oxo apicidin analog compound 18 using conditions known to those skilled in the art. . (What is R18?) Scheme X 0 0 ,OO O N IH Apicidin N NH 1) Hydrogenation H N 2) Oxidation N ' 0 3) Alkylation o - 8 5 18 R18 is an alkyl or aryl group, which optionally is substituted. Suitable oxidants include but are not restricted to tBuOOH, SeO2, CrO3, Na2CrO4, PCC, and the like, or more preferably DDQ (2,3-dichloro-5,6 dicyano-1,4-benzoquinone). Appropriate solvents, or mixtures of solvents, include 10 DMF, toluene, benzene, CH2Cl2, CHC13, HOAc, pyridine, THF, MeOH, EtOH, water, and the like, or more preferably MeCN. These reactions are performed at from -20oC to 50oC and are complete in from 5min to 24h. The stereochemistry of the beta-oxo-tryptophan attachment of compound 18 may be changed by treatment with bases such as pyridine, EtN(iPr)2, NaH, KH, DBU, lutidine, or most preferably Et3N. 15 The epimerization reaction proceeds at from 0OC to 50 0 C in solvents including CHCl3, CH2CICH2C1, MeOH, EtOH, DMF, DMSO, NMP, and the like, or most preferably CH2Cl2. The nitrogen of the beta-oxo-tryptophan may be alkylated, acylated, sulfonylated or phosphorylated as described previously. The beta-oxo carbonyl of compound 18 may be selectively reduced 20 using a hydride source under radical conditions. Suitable hydride sources include Me3SnH, nBu3SnCl/NaBH4, Ph3SnH, Ph3AsH, and the like, or most preferably nBu3SnH, in the presence of radical initiators. Suitable radical initiators include, for example, benzoyl peroxide, Et3B/02, and the like, or most preferably AIBN. Suitable solvents for the carbonyl reduction include MeOH, EtOH, water, benzene, or most 25 preferably toluene. The reaction proceeds at temperatures from 0OC to 110'C. - 70 - WO 01/07042 PCT/US00/19627 Referring to Scheme XI below, the indole of apicidin may be subjected to oxidative degradation to prepare carboxylic acid compound 19a (where R2 = OH) using conditions known to those skilled in the art. 5 Scheme XI 00 O N R19 Apicidin , N H NH 1) Oxidative degradation N 2) Esterify 0 3) Amide formation O 8 4) Carbonyl reduction 5) Nucleophilic attack O 6) Carbonyl oxidation 19 RI = 19a: CH2C(O)R1 9 a 19b: C(O)R19 b 19c: CH 2

CH

2

OR

19c 19d: CH 2 OR1 9 d Rl9a, R19b, R19c, and R19d are each independently an alkyl or aryl group, which optionally is substituted. Suitable oxidants include, but are not restricted to, KMnO4, 10 KMnO4/NaIO4, NaIO4/RuO4, and the like, or most preferably NaIO4/RuCI3. Suitable solvents, or mixtures of solvents include CHCl3, CH2CICH2C1, MeCN, MeOH, EtOH, tBuOH, and the like, or most preferably CH2Cl2. The reaction proceeds at temperatures from 0 0 C to 50oC. This carboxylic acid may be converted into esters or amides as described previously. 15 Alternatively, a methyl ester may be prepared first (eg. compound 19a, wherein R19a is OMe) and reacted with LiN(OMe)Me, Me2AIN(OMe)Me, or most preferably BrMgN(OMe)Me, to produce a Weinreb amide compound 19a, in which R19a is N(OMe)Me. Suitable solvents for this reaction include Et20, dimethoxyethane, dioxane, and the like, or most preferably THF. The reactions are 20 performed at from -78 0 C to 50oC and are complete in from 30min to 12h. Reduction of the sidechain C8-ketone group of compound 19a to the corresponding alcohol proceeds as described previously. The Weinreb amide thus -71- WO 01/07042 PCT/US00/19627 directly generated can then be reacted with hydride reagents, RLi, or RMgX as described previously to prepare the corresponding aldehyde or ketones (eg. 19a where R19a is H, alkyl or aryl group). At this point, the side chain C8-alcohol may be oxidized back to regenerate the C8-ketone as described previously. 5 When R19a is OH in compound 19a, the carboxylic acid may be reduced using BH3 to form an alcohol compound 19c (where R19c is H). This alcohol may be acylated, sulfonylated or phosphorylated as described previously. Treatment of the alcohol compound 19c with Ar3Bi reagents will generate the corresponding aryl ether compound 19c in which R19c is an aryl group. Both the 10 alpha- and beta-stereoisomers at the tetrapeptide are accessible as described previously. Substitution of beta-oxo apicidin derivative compound 18 for apicidin in Scheme XI above results in the formation of the truncated apicidin analog compounds 19b and 19d. 15 Referring to Scheme XII, the 2,3-indole bond in Apicidin can be cleaved oxidatively to form compound 20 using conditions known to those skilled in the art. - 72 - WO 01/07042 PCT/US00/19627 Scheme XII = 0 Nl"' Apicidin N N 0) Oxidative N H NH 0 N(R20)CHO cleavage N o O0 0 8 20 - 0 00 N NHr N H N ' 21 Cyclize N O 0 0 0 21 R20 and R21 are each independently an alkyl or aryl group which optionally is substituted. 5 Suitable oxidants include KMnO4, NaIO4, Pb(OAc)4, and the like, or more preferably ozone. This reaction may be run in solvents such as CHCl3, CH2CICH2C1, and the like, or more preferably CH2Cl2, at temperatures from -78 0 C to RT and the reaction is complete in from Imin to 2h. Treatment of compound 20 with a base induces Aldol cyclization to form a quinolone compound 21. Suitable 10 bases for this reaction include Et3N, EtN(iPr)2, pyridine, DBU, NaOMe, NaOEt, NaHCO3, and the like, or more preferably KOtBu. The Aldol cyclization may be performed in solvents, or mixtures of solvents including CH2Cl2, CHC13, MeOH, EtOH, DMF, THF, Et20, DMSO, water, and the like, or more preferably tBuOH. The reaction is complete in from 10min to 12h at 0OC to RT. Substitution of N -73- WO 01/07042 PCT/US00/19627 substituted-N-desmethoxy-apicidin derivatives (Compound 17) for apicidin in Scheme XII leads to the formation of N-substituted quinolone derivatives. Referring to Scheme XIII below, the quinolone compound 21 can be treated with sulfonylating agents as described previously to form compound 22 5 wherein R22 is a sulfonate moiety Scheme XIII

R

22 -0 N N IH 21 _,,IN H NH 1) Sulfonylation N 2) Nucleophile O 3) Reduction O HO N - 0 O N NR 23 H 21 N H NH 1) Reduction N 0 2) Nucleophile 3) Oxidation 0 8 O . 23 R22 and R23 are each independently an alkyl or aryl group, which 10 optionally is substituted. During this reaction, some inversion of stereochemistry at the tetrapeptide ring juncture occurs. When R22 of compound 22 is OSO2CF3, the triflate can be displaced with suitable nucleophiles, such as halogen, sulfur nucleophiles or nitrogen nucleophiles including, but not limited to, NaBr, NaC1, KI, 15 NaN3, NaSMe, KSAc, pyridine and the like. The resulting compounds are not shown. Suitable solvents for the displacement reaction include, but are not limited to, CH2CI2, CHC13, DMF, DMSO, HMPA, NMP, and the like. The reactions proceed at temperatures from O'C to 80 0 C. - 74 - WO 01/07042 PCT/US00/19627 For apicidin derivative compound 22 in which R22 is N-1-pyridinium, the pyridinium group may be reduced using catalytic hydrogenation as described previously. Further, the C8-ketone group of apicidin derivative compound 21 may 5 be reduced first. The thus formed quinolone carbonyl can then be reacted with nucleophiles such as hydride reagents, RLi or RMgX as described previously. The apicidin derivative compound 23 can be prepared by reoxidation of the C8-alcohol as described previously. Referring to Scheme XIV below, apicidin may be brominated at the 10 indole C2 position following removal of the N-methoxy group using conditions known to those skilled in the art to form compound 24 where R24 is Br. Scheme XIV - 0 0 N Apicidin __ __ HO N H N Apicidin 1) Hydrogenation H N 2) Bromination N IH 3) Palladium coupling O 0 24 15 Suitable brominating agents include, but are not limited to, Br2, Hg(OAc)2/Br2, CBr4, CuBr2, HOBr, Br2/HOAc/NaOAc, and the like, or most preferably N-bromosuccinamide. The bromination reaction can be facilitated by a radical initiator such as benzoyl peroxide, Et3B/02 or AIBN. The 2-bromo-indole thus obtained can be further reacted with a 20 palladium catalyst, a base and ArX to induce an aryl coupling reaction. Suitable palladium catalysts include, but are not limited to, Pd(OAc)2, Pd(OAc)/PPh3, PdCI2(PPh3)2, Pd(dba)2/PPh3, and the like, or most preferably Pd(PPh3)4. Suitable bases for this reaction include, but are not limited to, KOtBu, CsCO3, or most preferably NaHCO3. Suitable solvents, or mixtures of solvent for this coupling 25 reaction include toluene, DMF, MeCN, NMP, DMSO, H20, EtOH, or most preferably dioxane/water. Suitable ArX groups include, but are not limited to, -75- WO 01/07042 PCT/US00/19627 PhB(OH)2, 2-napthylboronic acid, (4-Me)PhB(OH)2, (4-F)PhOTf, and the like. The reactions are complete in from 30min to 48h at temperatures from RT to 110 0 C. Synthesis of Side Chain Modified Apicidin Derivatives 5 In the Examples, and elsewhere herein, all percentages are by weight unless specifically stated otherwise. Further, all ratios of compounds are by volume unless specifically stated otherwise. Room temperature (RT) means a temperature from about 18 0 C to about 25 0 C. If no temperature is specified, then the conditions are 10 understood to be room temperature. In certain steps that describe using an ingredient without specifying an amount, one of ordinary skill would understand the desired result and can determine the amount without difficulty. In general, the purities of the pure Examples were better than about 95% pure. 15 EXAMPLE 1 0 N N NH H I N O-~ o -8 Example 1 was prepared by the following procedure. At room temperature, 27mg of Me3S(O)I was added to a mixture of i) 5.6mg of 60% NaH and ii) 0.35mnL HMPA. The resulting solution was allowed to stand for 5min. Then, a 20 mixture of 12mg apicidin in 96tL DMF was added to form a reacting mixture. After 48 hours, the reaction was quenched with water, extracted with EtOAc and dried in Na2SO4 to produce 8mg Example 1. Example 1 was thus obtained without requiring further purification and was characterized by 1H NMR and MS [m/z: 638 (M ++1)]. - 76 - WO 01/07042 PCT/US00/19627 EXAMPLE 2 - 0 N N NN OMe 0 OH N . OH Example 2 was prepared by the following procedure. At room temperature, 60mg HCl*H2NOH and 181 tL Et3N was added to 20mg apicidin in 5 10mL CH2Cl2. The resulting solution was aged for 12h. The volatiles were then removed under reduced pressure. Example 2 was obtained following preparative RP HPLC (reversed phase high performance liquid chromatography), without workup, using a gradient elution characterized by 1:3 MeCN:H20 to 100% MeCN, with a 60min linear ramp. The pure Example 2 thus obtained was characterized by 1H NMR 10 and MS [m/z: 639.3 (M ++1)]. EXAMPLES 3A-3M Examples 3a-3m were prepared following the general procedure 15 described in Scheme E for compound 1 If, I Ig, and 1 1h, and for Ex.2. Examples 3a 3m are described by the following chemical formula and were characterized by NMR and mass spectroscopy: 0 N H H N N O OMe o 0 X -77 - WO 01/07042 PCT/US00/19627 Table 1 Example X Group Mass Spec Ex. 3a NNHSO2Ph(4-Me) -- Ex. 3b NOCH2Ph 729.2 (M+1) Ex. 3c NNH-Dansyl 871.2 (M +1) Ex. 3d NOCH2CO2-Na+

-

Ex. 3e NOCH2CO2H 697.2 (M+1) Ex. 3f NOMe 653.2 (M++1) Ex. 3g NNH-Texas Red 1227.2 (M+1) Ex. 3h NOCH2C(O)NHCH2CH2OH

---

Ex. 3i NOCH2C(O)(N-1-pyrrolindinyl) Ex. 3j NOCH2CO2Me

-

Ex. 3k NOC(O)Ph Ex. 31 NOC(O)Me

----

Ex. 3m NOC(O)tBu 5 EXAMPLES 4A AND 4B - 00 N N ' O N O NN O H Ex. 4a Ex. 4b Examples 4a and 4b were prepared by the following procedure. At 10 0OC, 4.5mg of p-toluenesulfonyl chloride was added to 3mg of Example 2 (the C8 ketoxime of apicidin) in 0.5mL pyridine to form a solution. The solution was -78- WO 01/07042 PCT/US00/19627 maintained at 0 0 C for 10min, then warmed to RT and aged for 50min. Then lmL each of saturated brine and saturated NaHCO3(aq) were added. Next, the solution was extracted with EtOAc and dried with Na2SO4. A mixture of pure Examples 4a and 4b was obtained following preparative RP-HPLC using gradient elution (1:3 5 MeCN:H20 isocratic for 10min, then a 75min linear ramp to 100% MeCN). The pure mixture thus obtained was characterized by 1H NMR and MS [m/z: 639.2 (M ++1)]. EXAMPLE 5 0 N ONOH NH OMe 0

CH

2 10 Example 5 was prepared by the following procedure. At room temperature (RT), 114mg of Ph3PCH3 +Br - was added to i) 16.8mg of a 60% dispersion of NaH in oil, ii) 2mL DMF, and iii) 0.2mL HMvPA to form a mixture. After the mixture ceased foaming, a solution of 20mg apicidin in lmL DMF was 15 added. The resulting solution was aged for 4 hours. Preparative RP-HPLC without workup using gradient elution (1:3 MeCN:H20 isocratic for 10min, then a 75min linear ramp to 100% MeCN yielded 14mg of pure Example 5 which was characterized by 1 H NMR and MS [m/z: 622.3 (M +1)]. - 79 - WO 01/07042 PCT/US00/19627 EXAMPLES 6A-6D o 0 iil N N HO CI Ex. 6a 5 Example 6a was prepared by the following procedure. At 0oC, 0.12mL of 1.0M (4-C1)PhMgBr in Et20 was dropwise added to 15mg apicidin in a mixture of 1.75mL THF and 0.25mL pyridine. After lb at 0C, an additional 0.12mL of 1.0M (4-C1)PhMgBr in Et20 was added. The resulting solution was aged for lh at 0 0 C and then lh at RT. The reaction was quenched by the addition of saturated NH4Cl(aq) to 10 the solution. The quenched mixture was extracted with EtOAc and dried with Na2SO4. Preparative RP-HPLC using gradient elution (1:3 MeCN:H20 isocratic for 10min, then a 75min linear ramp to 100% MeCN) yielded 8mg of pure Example 6a, IH which was characterized by 1H NMR and MS [m/z: 736.3 (M++1)]. Examples 6b, 6c, and 6d are described by the chemical structure shown 15 below. The specific substituents are tabulated in Table 2. Examples 6b, 6c, and 6d were prepared following the general procedure described in Scheme 11I for compound lb under conditions similar to those described above for Ex. 6a - 80 - WO 01/07042 PCT/US00/19627 0 H NH N N 0 OMe O 8 HO R Table 2 Example R Group Mass Spec Ex. 6b CH2Ph 716.4 (M++1) Ex. 6c C6H11 708.4 (M++1) Ex. 6d CH2CH3 654.4 (M+1) 5 EXAMPLE 7 cyclo(N-O-Methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-hydroxy-decanoyl) 0 O-e OH Example 7 was made by first adding 18mg NaBH4 to 300mg apicidin 10 in 12mL MeOH at 0OC. Next, the ice bath was removed immediately and the solution was stirred at RT for 4 hours. Acetone was added to quench the reaction and the solvents were removed under reduced pressure at ambient temperature. The residue was dissolved in CH2C12, poured into saturated NaHCO3, extracted with 1:9 iPrOH:CH2Cl2 and dried with Na2SO4. The pure product was obtained following -81 - WO 01/07042 PCT/US00/19627 flash chromatography on silica gel using 1:1 acetone:hexanes as eluant. The pure Example 7 was characterized by 1H NMR. TLC: Rf = 0.32 (1:1 acetone:hexanes). EXAMPLE 8 - 0 NN N NH .N 5 OH Example 8 was prepared following the general procedure of Example 7 but using N-desmethoxy-apicidin as the starting material. Example 8 was characterized by H NMR and MS [m/z: 596 (M ++1)]. 10 EXAMPLE 9 0 N NH H N O Example 9 was prepared by the following process. At room temperature, 57mg of thiocarbonylimidazole was added to 40mg of cyclo(N-O methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-hydroxy-decanoyl) in 1.6mL CH2C12. The 15 resulting solution was heated to 75 0 C for 2 hours. Next, 1mg of DMAP (4 dimethylaminopyridine) was added and the solution aged for lh at 75oC and 48h at RT. The solvent then was removed under reduced pressure. 59mg of the pure intermediary product 8-OC(S)imidazolyl-apicidin (also known as cyclo(N-O-methyl L-Trp-L-Ile-D-Pip-L-2-amino-8-imidazoylylthionooxy-decanoyl) was obtained by - 82- WO 01/07042 PCT/US00/19627 PTLC (2 x 15001tm plate) using 1:3:96 NH4OH:MeOH:CHCl3 as eluant and was characterized by 1H NMR and MS [m/z: 736 (M ++1)]. To the above prepared 59mg of intermediary product 8 OC(S)imidazolyl-apicidin in 1.6mL toluene was added 2.6mg AIBN and 53[tL 5 nBu3SnH. The solution was then degassed and heated to 80oC for lh, concentrated under reduced pressure, and partitioned between MeCN and hexanes. The hexanes layer was discarded. The volatiles were removed under reduced pressure and pure Example 9 product was obtained following RP-HPLC using gradient elution (4:6 to 1:0 MeCN:H20). Example 9 was characterized by 1H NMR and MS [m/z: 610 10 (M ++1)]. EXAMPLE 10 0 N NH H I NI SOMe o -8

OSO

2 Ph(4-Me) Example 10 was made by adding 10mg DMAP to 100mg cyclo(N-O 15 methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-hydroxy-decanoyl) in 2mL pyridine at RT. Next, 94mg tosic anhydride was added. After 3d at RT, the solution was poured into saturated NaHCO3, extracted with CH2Cl2 and dried with Na2SO4. Pure Example 10 was obtained following flash chromatography on silica gel using gradient elution (1:1:98 then 1:2:97 then 1:3:96 NH4OH:MeOH:CHC13 as eluant). Example 10 was 20 characterized by 1H NMR. TLC: Rf = 0.36 (1:3:96 NH4OH:MeOH:CHC13). - 83 - WO 01/07042 PCT/US00/19627 EXAMPLE 11 0 H "H NN N OMe O o -8 OAc The procedure to form Example 11 was as follows. At room temperature, 50mg NaBH4 was added to 100mg apicidin in 10mL 1:1 THF:MeOH. 5 After 30min at RT, the solution was poured into brine, extracted with CH2Cl2 and dried with Na2SO4. To the residue thus obtained was added 2mL pyridine, followed by addition of 10mg DMAP and 10 drops of Ac20. After another 15min at RT, the solution was heated to 80 0 C for 10min. without noting any reaction. An additional 5 drops of fresh Ac20 (from an unopened bottle) were added and the solution stirred at 10 RT for 24 hours. The solvents were removed under reduced pressure and the residue was lyophilized from dioxane. Preparative RP-HPLC using gradient elution (3:7 to 6:4 MeCN:H20) yielded 69mg of pure Example 11 product, which was characterized by 1H NMR and MS [m/z: 668.6 (M +1)]. HPLC: tR = 4.95min (6:4 MeCN:H20, 1.5mL/min, Zorbax T M RX-8 available from Rainin Co. 15 EXAMPLES 12A-12E Following the general procedure described in Scheme III, compounds I ic and 1 1d, and similarly to the procedure for Examples 10 and 11, the following Examples 12a-12f were prepared and characterized by NMR and mass spectroscopy: 0 "N NNH H N OMe 0 -8 20 OR - 84 - WO 01/07042 PCT/US00/19627 Table 3 Example R Group 12a C(O)Ph 12b C(O)tBu 12c C(O)Ph(F5) 12d SO2Me 12e SO2Ph(4-NO2) 10 SO2Ph(4-Me) EXAMPLE 13 0 N NNHH H N OMe 0 -8

NH

2 5 To form Example 13, 0.16mL (Me3Si)2NH and 235mg ZnCl2 was added to 100mg apicidin in 5mL EtOAc at RT. The solution was heated to 55oC for 12 hours. The solution was then cooled to 0 0 C and 12mg NaBH4 was added. After lh, the solution was warmed to RT and aged an additional 2h. The solution was poured into 1:1 brine:saturated NaHCO3, extracted with CH2Cl2 and dried with 10 Na2SO4. Pure Example 13 product was obtained following preparative RP-HPLC using gradient elution (3:7 to 6:4 MeCN:H20) and was characterized by 1H NMR and MS [m/z: 625.3 (M +1)]. TLC: Rf= 0.22min (1:9:90 NH4OH:MeOH:CHCl3). - 85 - WO 01/07042 PCT/US00/19627 EXAMPLE 14 0 NN N. O OMe H 0 8 NHAc To form Example 14, 2 drops Ac20 and a catalytic amount of DMAP was added to 14mg 8-amino-8-desoxo apicidin in 2mL pyridine at 0 0 C. The solution 5 was stirred at 0OC for 30min and at RT for another 30min. Next, lmL methanol was added and the solution was then concentrated under reduced pressure. Pure Example 14 was obtained following preparative RP-HPLC purification (gradient elution using 25:75 MeCN:H20 for 10min, then a 70min ramp to 100% MeCN) and was characterized by 1H NMR and MS [m/z: 667.4 (M +1)]. TLC: Rf = 0.67 (1:9:90 10 NH4OH:MeOH:CHCl3). HPLC: tR = 4.60min, 1:1 MeCN:H20, 1.5mL/min, Zorbax T M RX-8). EXAMPLE 15 - 0 N NHt N I OOMe 0 8

NHCH

2

CH

2 0H 15 Example 15 was made by first adding to 60mg apicidin in 0.5mL MeOH at RT i) 1mL pyridine, ii) 40gL ethanolamine, iii) 60gL glacial HOAc (pH 5.0), and iv) powdered 4A sieves. The solution was cooled to 0 0 C and 7.9mg NaCNBH3 was added. After 2h, the solution was warmed to RT and aged for 12h. The solution was then filtered through Celite filter agent (available from Aldrich - 86 - WO 01/07042 PCT/US00/19627 Chemical Company, Milwaukee, Wisconsin) using 1:1 CH2Cl2:MeOH as eluant, reduced in volume in vacuo, poured into saturated NaHCO3, extracted with CH2Cl2 and dried with Na2SO4. Following preparative RP-HPLC using 1:1 MeCN:H20 to 100% MeCN gradient elution, 4.2mg pure Example 15 was obtained. The product 5 thus obtained was characterized by 1H NMR and MS [m/z: 669 (M +1)]. EXAMPLE 16 0 "oe N NH N O~ OMe ON 0 8

NHCH

2

CH

2

CH

3 Example 16 was prepared similarly to Example 15. At room 10 temperature, to 60mg apicidin in 0.5mL MeOH was added i) 2mL pyridine, ii) 0.5rmL propylamine, iii) lmL glacial HOAc (pH - 4.5), and iv) powdered 4A sieves. The solution was cooled to O'C and 60mg NaCNBH3 was added. After 2h, the solution was warmed to RT and aged for 12h. The solution was filtered through Celite using 1:1 CH2CI2:MeOH as eluant, reduced in volume in vacuo, poured into saturated 15 NaHCO3, extracted with CH2C12 and dried with Na2SO4. Pure Example 16 was obtained following PTLC on silica gel (1 x 1500gm plate) using 2:18:80 NH4OH:MeOH:CHCl3 as eluant. The pure Example 16 product thus obtained was characterized by 1H NMR and MS [m/z: 667 (M +1)]. - 87 - WO 01/07042 PCT/US00/19627 EXAMPLE 17 0 N H NH NLN o -8 Y 0 To form Example 17, 32mg KSAc was added to 18.1mg of the Example 10 C8-tosylate compound in 3mL 95% EtOH. The solution was heated to 5 70 0 C for 3 hours. The solution was then cooled to RT and saturated NH4Cl(aq) was added. Next, the solution was extracted with EtOAc and dried with Na2SO4. The solution then was filtered, evaporated to dryness. PTLC on silica gel (1 x 1000pm plate) using 3:7 acetone:hexanes as eluant yielded 3.4mg of pure Example 17 product that was characterized by H NMR. 10 EXAMPLE 18 0 H

NH

NO M 0O ~ SH To form Example 18, 3.4mg of the Example 17 C8-thioacetate compound was placed at RT in 0.2mL NaOMe 2M solution in MeOH) and aged for 15 3h. The solution was poured into saturated NH4CI(aq), extracted with CH2CI2, and dried with Na2SO4. The solution was filtered, concentrated to dryness, and pure - 88 - WO 01/07042 PCT/US00/19627 Example 18 was obtained following RP-HPLC. Example 18 thus obtained was characterized by 1H NMR. EXAMPLES 19A AND 19B 0 0 Ex. 19a Ex. 19b Examples 19a and 19b were prepared by the following procedure. 50mag apicidin was heated in 5mL THF at 500C until the resulting solution 10 became homogenous. The solution was then cooled to -78C and immediately 800pL 0.5M potassium hexamethyldisilazane in toluene was added. After NHH NH HNH N 5min, 40L TMSCl as a solution in 1mL THF was added. After 10main at 78C the reaction was stopped by the addition of 5mL saturated NaHCO3. Next, the solution was extracted, first with EtOAc, followed by CH2Cl2 and 15 dried with Na2SO4. The crude mixture of Example 19a and Example 19b was used with no further purification in the next reaction. The crude yield was 74mg (145%). Thoe mixture was characterized by 1H NMR. TLC: Rf = 0.52Oe (1:2 acetone:hexanes). -89 88 5 OSiMe 3

O&W~

3 Ex. 19a Ex. 19b Examples 19a and 19b were prepared by the following procedure. 50mg apicidin was heated in 5mL THE at 50'C until the resulting solution 10 became homogenous. The solution was then cooled to -78'C and immediately 800gL 0.5M potassium hexamethyldisilazane in toluene was added. After 5min, 40L TMSCI as a solution in lmL TILE was added. After 10min at 78'C the reaction was stopped by the addition of 5mL saturated NaHCO3. Next, the solution was extracted, first with EtOAc, followed by CH2Cl2 and 15 dried with Na2SO4. The crude mixture of Example 19a and Example 19b was used with no further purification in the next reaction. The crude yield was 74mg (145%). The mixture was characterized by I H NMR. TLC: Rf = 0.52 (1:2 acetone: hexanes). - 89 - WO 01/07042 PCT/US00/19627 EXAMPLES 20A AND 20B O OSiMe 3 O OSiMe 3 0 o Ex. 20a Ex. 20b 5 To form Examples 20a and 20b, 74mg of the crude -1:1 mixture Example 19a, cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-trimethylsiloxy-7 ene- decanoyl), and Example 19b, cyclo(N-O-Methyl-L-Trp-L-Ile-D-Pip-L-2-amino 8-trimethylsiloxy-8-ene-decanoyl), was placed in 5mL CH2Cl2 at RT to which was added 200mg solid NaHCO3. To this solution was added 20mg 85% MCPBA. After 10 5min, the reaction was quenched with 1:1 saturated Na2S203:saturated NaHCO3, extracted with CH2Cl2, and dried with Na2SO4. This yielded a 43mg pure mixture of Example 20a, cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-7 trimethylsiloxy-decanoyl), and Example 20b, cyclo(N-O-Methyl-L-Trp-L-Ile-D-Pip L-2-amino-8-oxo-9-trimethylsiloxy-decanoyl) following flash chromatography on 15 silica gel using 4:1 hexanes:acetone as eluant. The mixture was characterized by 1H NMR. TLC: Rf = 0.33 (1:2 acetone:hexanes). - 90 - WO 01/07042 PCT/US00/19627 EXAMPLES 21A AND 21B - 0 - 0 O O 0- ., ' I H NH N - N H N N OMe N OMe O OH 0 OH O 0 Ex. 21a Ex. 21b 5 Example 21a and Example 21b were prepared by the following procedure. To 43mg of a 1:1 mixture of Example 20a, cyclo(N-O-methyl-L-Trp-L Ile-D-Pip-L-2-amino-8-oxo-7-trimethylsiloxy-decanoyl), and Example 20b, cyclo(N O-Methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-9-trimethylsiloxy-decanoyl), in 4mL THF at RT was added 120gL 1M nBu4NF in THF. After 20min at RT, the solvent 10 was evaporated under reduced pressure and the crude mixture purified by RP-HPLC without workup using 6:4 MeCN:H20. The resulting pure mixture of Examples 21a and 21b was characterized by 1H NMR and MS [m/z: 657.2 (M ++NH4)]. TLC: Rf = 0.14 (1:2 acetone:hexanes). 15 EXAMPLES 22A AND 22B +0 SN,. N N,~~ O' W.NOe O NA NOMe N NH + N H NH N N O OH 0 OH O 0 O 0 Ex. 22a Ex. 22b -91 - WO 01/07042 PCT/US00/19627 Following the general procedure of Examples 19-21, a 95% pure mixture of Examples 22a and 22b was prepared and characterized by H NMR. EXAMPLES 23A AND 23B O -0 NHN -N H N NH + N NH OH OH O - O 8 0 5 o o Ex. 23a Ex. 23b Following the general procedure of Examples 19-21, a 95% pure mixture of Example 23a and 23b was prepared and characterized by 1 H NMR. 10 EXAMPLES 24A AND 24B 0 O 0 ~NcfN N H 'H N H I H N H~ N O OMe 0 OMe O 0 0 8 WO e 7 H0 Ex. 24a Ex. 24b 15 Examples 24a and 24b were prepared by adding 10mL pyridine to 10mg -1:1 mixture of Examples 21a and 21b in 3mL MeOH at 0 oC, followed by the addition of 10mg Pb(OAc)4. After 10min, the solution was quenched with 2mL Na2S203, diluted with about 2mL brine, extracted with CH2Cl2 and dried with - 92 - WO 01/07042 PCT/US00/19627 Na2SO4. Following preparative TLC on silica gel (500ptm plate) using 1:2 acetone:hexanes as eluant, separated pure products were obtained. Example 24a, cyclo(N-O-Methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-oxo heptanoyl) (5.5mg) was characterized by IH NMR and MS [m/z: 582.2 (M+1)]. TLC: 5 Rf = 0.16 (1:2 acetone:hexanes). Example 24b, cyclo(N-O-Methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7 carboxymethyl-heptanoyl) (6.5mg) was characterized by 1H NMR and MS [m/z: 626.3 (M++I)]. TLC: Rf = 0.23 (1:2 acetone:hexanes). 10 EXAMPLES 25A-25D Examples 25a-25d were prepared by following the general procedure of Example 24b. Starting with Examples 21a and 21b, and using an appropriate alcohol as solvent, the following derivatives were prepared and analyzed by NMR and 15 mass spectroscopy: 0 N N H O OR Oe 0 Table 4 Example R Group Mass Spec 25a Et 640.5 (M +1) 25b nPr 654.4 (M++I) 25c nBu 668.3 (M +1) 25d iPr 654.4 (M +1) - 93 - WO 01/07042 PCT/US00/19627 EXAMPLE 26 0 N H H NH N OMe O 0 O" Li 0 Example 26 was prepared by the following procedure. To 41mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-carbomethoxy-heptanoyl) in 4mL 5 3:1:1 THF:MeOH:H20 at 0 0 C was added 100pL IM LiOH. The solution was stirred for lh and then additional 300L IM LiOH was added. After 12h, 33mg pure Example 26 product was obtained following preparative RP-HPLC without workup using gradient elution (column equilibrated in 5:95 MeCN:H20, using 25:75 MeCN:H20 for 40min followed by a 20min ramp to 100% MeCN, flow rate 10 lOmlJmin). Example 26 was characterized by 1H NMR and MS [m/z: 629.2 (M++NH4)]. HPLC: tR = 1.98min 45:55 MeCN:H20, 1.5mL/min, Zorbax T M RX-8). EXAMPLE 27 H N N o OMe 0 NHOH O 0 15 Example 27 was prepared by the following procedure. To 15mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-carboxy-heptanoyl), lithium salt in 3mL DMF at RT was added 5.4mg H2NOSi(Me)2tBu and 7mg EDC*HC1. After 2h at RT, 15mg additional H2NOSi(Me)2tBu (15mg) and 14mg EDC*HCl were added and the solution allowed to stir overnight. The reaction was quenched by the addition - 94 - WO 01/07042 PCT/US00/19627 of 5 drops glacial HOAc and lmL MeOH. The solution was poured into brine, extracted with CH2C12, and dried with Na2SO4. The crude product was chromatographed on silica gel using gradient elution (1:3:96 NH4OH:MeOH:CHCl3 to 1:4:95 NH4OH:MeOH:CHCl3, to 1:9:90 NH4OH:MeOH:CHCl3). To remove 5 some contaminating EDU present in the chromatographed material, the product was dissolved in 2mL CHC13 and 2mL 10% aq. HOAc. After 5min, the aqueous layer was decanted and the washing repeated twice more to yield 5.5mg pure Example 27 product. The pure Example 27 stained positive (purple-orange) for a hydroxamic acid using Fe(II)Cl 3 stain. The product was characterized by 1 H NMR and MS [m/z: 10 627.3 (M++1)]. TLC: Rf= 0.26 (then 1:9:90 NH4OH:MeOH:CHCl3). EXAMPLE 28 0 N H NH OMe O 8 N(OMe)Me O 0 Example 28 was prepared by the following procedure. To 30mg 15 cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-carboxy-heptanoyl) lithium salt in lmL DMF at RT was added 47mg HCl*HN(OMe)Me, 2mg DMAP, 7mg HOBT (1 hydroxybenzotriazole hydrate) and 90tL DIEA (Et2NiPr) followed by 12mg EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride). After 36h, the solution was poured into brine, acidified to pH-4.0 with 2N HC1, extracted with 20 CH2Cl2 and dried with Na2SO4. Following flash chromatography on silica gel using 1:3:96 NH4OH:MeOH:CHCl3 as eluant, 29.6mg purified Example 28 was obtained and was characterized by 1H NMR and MS [m/z: 655.3 (M ++1)]. TLC: Rf = 0.39 (1:3:96 NH40OH:MeOH:CHCl3). HPLC: tR = 3.90min (62:38 MeCN:H20, 1.5mL/min, Zorbax T M RX-8). 25 - 95 - WO 01/07042 PCT/US00/19627 EXAMPLE 29 O N H N ,H N O OMe 0 8 NHOCH2Ph O Example 29 was prepared by the following procedure. To 150mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-carboxy-heptanoyl) in 14mL 5 CH2Cl2 at 0OC was added 78mg HCIH2NOCH2Ph, 0.13mL DIEA, 33mg HOBT, 2mg DMAP, and 108mg BOP. After lh at 0 0 C and 12h at RT, the solution was poured into saturated NaHCO3, extracted with CH2Cl2 and dried with Na2SO4. Following preparative TLC on silica gel (5 x 1000pm plates) using 5:95 MeOH:CHC13 as eluant, 137mg pure Example 29 was obtained and was characterized 10 by IH NMR. TLC: Rf = 0.62 (5:95 MeOH:CHCI3). HPLC: tR = 7.46min (45:55 MeCN:H20, 1.5mL/min, Zorbax T M RX-8). EXAMPLE 30 0 SHNHOH HO N 0 8 NHOH 0 15 Example 30 was prepared by the following procedure. To 130mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-(N-benzyloxy-carboxamido) heptanoyl) in 5mL MeOH at RT was added 5% Pd/C and an H2 atmosphere (balloon pressure) was established. After 12h, 10mg Pd(OH)2 was added and the reaction continued for an additional 2h. The catalyst was removed by filtration through Celite - 96 - WO 01/07042 PCT/US00/19627 using MeOH as eluant and the solution concentrated under reduced pressure. Pure Example 30 product was obtained following RP-HPLC purification using gradient elution (5:95 MeCN:H20 for 5min then 55min ramp to 50:50 MeCN:H20). The pure Example 30 was characterized by IH NMR and MS [m/z: 597.5 (M +1)]. TLC: Rf 5 = 0.11 (1:9:90 NH4OH:MeOH:CHC13). HPLC: tR = 10.65min (2min ramp from 5:95 MeCN:H20 to 1:1 MeCN:H20, 1.0mL/min, Zorbax T M RX-8). EXAMPLE 31 0 N O HN OMe o - 8H 0 10 Example 31 was prepared by the following procedure. To 10mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-(N-O-methyl-N-methyl carboxamido)-heptanoyl) in 2mL THF at 0OC was added 150pL IM CH2=CHMgBr in Et20. After 15min at 0OC, the solution was cooled to -78oC and quenched by addition of ImL saturated NH4C1. The solution was poured into brine and extracted 15 with CH2C12 and dried with Na2SO4. The product was partially purified on a silica gel pipette plug using 1:2 acetone:hexanes as eluant. Following preparative TLC on silica gel (1 x 250Am plate) using 1:3:96 NH4OH:MeOH:CHCl3 as eluant, 2.1mg pure Example 31 was obtained and was characterized by 1H NMR and MS [m/z: 596.3 (M +1I)]. TLC: Rf = 0.57 (1:3:96 NH40H:MeOH:CHCl3). 20 - 97 - WO 01/07042 PCT/US00/19627 EXAMPLE 32 O 0 H NH H OMe o -8 0 Example 32 was prepared by the following procedure. To 7mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-(N-methoxy-N-methyl 5 carboxamido)-heptanoyl) in lmL THF at 0 0 C was added 551tL IM MeMgBr in Et20. After 10min, an additional 55gL IM MeMgBr in Et20 was added. The solution was poured into saturated NH4C1, extracted with CH2Cl2 and dried with Na2SO4. 4.3mg pure Example 32 product was obtained following preparative TLC on silica gel (1 x 500pm plate) using 4:6 acetone:hexanes as eluant. The pure Example 32 was 10 characterized by 1H NMR and MS [m/z: 610.3 (M +1)]. TLC: Rf = 0.22 (1:2 acetone:hexanes). HPLC: tR = 4.51 min (1:1 MeCN:H20, 1.5mL/min, Zorbax T M RX-8). EXAMPLES 33A-33C 15 Following the general procedure illustrated in Example 26-34, the following derivatives were prepared: 0 N H H H N N U Olve O8 R Oe 0 - 98 - WO 01/07042 PCT/US00/19627 Table 5 Example R Group Mass Spec 32 Me 610.3 (M+1) 33a nPr 638.5 (M++1) 33b iPr 638.5 (M+1) 33c Ph 672.5 (M+1) 5 EXAMPLE 34 - 0 N N H N OMe 0 O OMe Example 34 was prepared by the following procedure. To 25mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-oxo-heptanoyl), 11 mg anhydrous LiCl, and 21mL (MeO)2P(O)CH2CO2Me in 2.5mL MeCN at RT, was added 42mL 10 DIEA. After 2h the solution was poured into saturated NaHCO3, extracted with CH2Cl2 and dried with Na2SO4. Pure Example 34 product was obtained following flash chromatography on silica gel using 1:2 acetone:hexanes as eluant. The pure Example 34 was characterized by 1H NMR and MS [m/z: 638.2 (M +1)]. TLC: Rf = 0.38 (1:2 acetone:hexanes). HPLC: tR = 5.09min, (1:1 MeCN:H20, 1.5mL/min, 15 Zorbax T M RX-8). - 99 - WO 01/07042 PCT/US00/19627 EXAMPLE 35 0 N NH H N NI N 0 H 0 0 O 9Me Example 35 was prepared by the following procedure. To 35mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7,8-dehydro-8-carbomethoxy 5 octanoyl) in 4mL 1:1 THF:MeOH was added 20mg Pd(OH)2 and an H2 atmosphere (balloon pressure) was established. After 12h, the catalyst was filtered off and 11.7mg pure Example 35 product was obtained following flash chromatography on silica gel using 1:2 acetone:hexanes as eluant. The pure Example 35 was characterized by IH NMR. TLC: Rf = 0.21 (1:2 acetone:hexanes). HPLC: tR = 3.84min (55:45 10 MeCN:H20, 1.5mL/min, Zorbax T M RX-8). EXAMPLE 36 O N HI N NH 0 H 0 0 O9 OH Example 36 was prepared by the following procedure. To 10.6mg 15 cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-carbomethoxy-octanoyl) in 1mL 3:1:1 THF:MeOH:H20 at 0 0 C was added 15mL 1M LiOH. The solution was stirred for lh at 0 0 C, 6h at RT, 3 days at 4oC and then an additional 30mL IM LiOH was added. After 8h longer, the solvents were removed using a vigorous stream of N2 and pure Example 36 product was obtained by purification without workup using 20 preparative RP-HPLC (gradient elution using 2:8 MeCN:H20 for 10min followed by - 100 - WO 01/07042 PCT/US00/19627 a 60min ramp to 100% MeCN). Pure product was characterized by 1H NMR and MS [m/z: 596.3 (M++1)]. HPLC: tR = 2.89min (3:7 MeCN:H20, 1.5mL/min, Zorbax T M RX-8). 5 EXAMPLE 37 0 N NH H N N I 0O OMe o 0 7 OMe Example 37 was prepared by the following procedure. To 25mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-oxo-heptanoyl) in 1.25mL DMF at RT was added 0.25nmL MeOH followed by 67.5mg PDC. The solution was stirred for 10 3.5h and then filtered through 1" silica gel, with 0.5" Celite on top of it, using MeOH as eluant. The solvents were removed under reduced pressure. Pure 9mg Example 37 product was obtained following preparative TLC on silica gel (2 x 1000gm plates) using 5:95 MeOH:CHCl3 as eluant. The pure Example 37 was characterized by 1H NMR and MS [m/z: 612.3 (M ++1)]. TLC: Rf = 0.24 (1:2 acetone:hexanes). HPLC: 15 tR = 9.41min (45:55 MeCN:H20, 1.5mL/min, Zorbax T M RX-8). - 101 - WO 01/07042 PCT/US00/19627 EXAMPLE 38 - 0 N O N O OMe O O " OMe 7 Example 38 was prepared by following the general procedure of Example 15, and Scheme fI, utilizing methyl glycinate in place of ethylamine, and 5 was characterized by H NMR and MS [m/z: 655.0 (M+ +1)]. EXAMPLE 39 0 N S O OMe O 0 H O -I O-Li+ 7 Example 39 was prepared by following the general procedure of 10 Example 36 and starting with the methyl ester of Example 38, and was characterized by 1H NMR and MS [m/z: 641.4 (M ++1)]. - 102- WO 01/07042 PCT/US00/19627 EXAMPLE 40 0 N H NH N H N N OMe O OH 7 Example 40 was prepared by following the general procedure of Example 7, utilizing Example 32 as the starting material, and was characterized by H 5 NMR and MS [m/z: 598.3 (M ++1)]. EXAMPLE 41 N~o OMe 0 N OH Example 41 was prepared by following the general procedure of 10 Example 7 to convert the C7-aldehyde of Example 23 and was characterized by 1H H N NMR and MS [m/z: 584.2 (M +1)]. - 103 O H Example 41 was prepared by following the genera] procedure of 10 Example 7 to convert the C7-aldehyde of Example 23 and was characterized by I H NMR and MIS [mlz: 584.2 (M ++1)]. - 103 - WO 01/07042 PCT/US00/19627 EXAMPLE 42 S0 0 N NH O H 6 Example 42 was prepared by the following two methods. 5 Method A Following the general procedure of Example 7, the C6-aldehyde of Example 58a was converted into Example 42 by adding 2.1mg NaBH4 to 64mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-6-oxo-hexanoyl) in lmL 1:1 THF:EtOH at 0 0 C. After lh, the resulting solution was poured into saturated NH4C1, 10 extracted exhaustively with CH2Cl2 and 3:7 iPrOH:CHCl3 (lx). The organic layer was dried with Na2SO4. Pure Example 42 was obtained following PTLC on silica gel (1 x 500m plate) using 1:1 acetone:hexanes as eluant. Example 42 was characterized by 1H NMR and MS [m/z: 570 (M ++1)]. 15 Method B 7.3mg of a -1:1 mixture of 6,7- and 9,10-enones of apicidin, Example 55a and 55b, was placed in lmL CH2Cl2 at -78oC. Ozone was bubbled through the solution until a blue color persisted. A vigorous stream of nitrogen was then used to 20 remove the excess ozone. To this solution was added 3.6mg NaBH4 in 120L 1:1 EtOH:H20, the cooling bath was removed and the solution was aged overnight. The solution was poured into saturated NH4CI(aq), extracted with CH2C]2 and dried with Na2SO4. Pure Example 42 was obtained following PTLC purification on silica gel (1 x 500tm plate) using 1:1 acetone:hexanes as eluant. 25 - 104 - WO 01/07042 PCT/US00/19627 EXAMPLE 43 0 N~ N H NH ,H N 0 OMe 7 0 0 O Example 43 was prepared by the following procedure. To 32mg Example 41, cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-hydroxy-heptanoyl), 5 in 2.5mL CH2Cl2 at 0 0 C, was added 27tL DIEA, a catalytic amount of DMAP, and 36mg toluene sulfonic anhydride. After lh at 0 0 C and 12h at RT, the solution was poured into saturated NaHCO3, extracted with CH2CI2 and dried with Na2SO4. Following preparative TLC on silica gel (2 x 1000pm plates) using 1:3:96 NH4OH:MeOH:CHC13 as eluant. 20mg pure Example 43 product was obtained and 10 was characterized by 1H NMR and MS [m/z: 755.5 (M ++NH4)]. TLC: Rf= 0.58 (1:3:96 NH4OH:MeOH:CHCl3). EXAMPLE 44 60 O 15 Example 44 was prepared from Example 42 cyclo(N-O-methyl-L-Trp L-Ile-D-Pip-L-2-amino-6-hydroxy-hexanoyl) by following the general procedure of Example 43, and was characterized by 1H NMR and MS [m/z: ??? (M++NH4)]. -105 N N H NH H N 0 00 15 Example 44 was prepared from Example 42 cyclo(N-O-methyl-L-Trp L-le-D-Pip-L-2-amino-6-hydroxy-hexanoyl) by following the general procedure of Example 43, and was characterized by IH NMR and MS [mlz: ??? (M ++NH4)]. - 105 - WO 01/07042 PCT/US00/19627 EXAMPLE 45 0 H NH ,H 0 O OMe o . 8,0Me II OMe O Example 45 was prepared by the following procedure. To 9pL (MeO)2P(O)H in 350gL THF was added 2.5mg 95% NaH at RT via syringe and the 5 solution heated to reflux for 20min. The solution was then cooled to RT and 25mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-(para-toluenesulfonyl)-heptanoyl) was added as a solution in 350[tL THF, heated to reflux for 2h, cooled to RT and stirred for 12h. The solution was poured into saturated NaHCO3, extracted CH2Cl2 and dried with Na2SO4. Pure 4.1 lmg Example 45 product was obtained following 10 PTLC (1 x 1000am plate) on silica gel using 1:3:96 NH4OH:MeOH:CHC13 as eluant. The pure product was characterized by 1H NMR and MS [m/z: 676 (M ++1)]. EXAMPLE 46 - 0 N ' H S OMe O SMe 15 Example 46 was prepared by the following procedure. To 5mg cyclo(N-0-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-(para-toluenesulfonyl)-heptanoyl) in lmL DMF at RT was added 5mg NaSMe. After 2h, the solution was poured into brine, extracted with CH2Cl2 and dried with Na2SO4. The pure Example 46 product was obtained following preparative TLC on silica gel (1 x 500pm plate) using 1:2 - 106 - WO 01/07042 PCT/US00/19627 acetone:hexanes as eluant. The pure product was characterized by H NMR and MS [m/z: 614.5 (M ++1)]. TLC: Rf = 0.33 (1:2 acetone:hexanes). EXAMPLE 47 0 N NH HJ N SOMe O 5 0 Example 47 was prepared by the following procedure. To 5mg cyclo(N-0-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-(para-toluenesulfonyl)-heptanoyl) in lmL DMF at RT was added 5mg NaSAc. After 2h, the solution was poured into brine, extracted with CH2Cl2 and dried with Na2SO4. Pure Example 47 product was 10 obtained following preparative TLC on silica gel (1 x 500gm plate) using 1:2 acetone:hexanes as eluant. The pure product was characterized by 1H NMR and MS [m/z: 642.5 (M +1I)]. TLC: Rf= 0.22 (1:2 acetone:hexanes). EXAMPLE 48 N O O OH 15 OH Example 48 was prepared by starting with Example 22b and following the general procedure described for Example 7. Example 22b's C8 ketone group was converted to a hydroxyl to form Example 48, which was characterized by 1H NMR. - 107 - WO 01/07042 PCT/US00/19627 EXAMPLE 49 - 0 N H NH N I U OMe o 8O0CH 2 Ph O 1/ IP OCH 2 Ph 0 Example 49 was prepared by the following procedure. A solution of 5 634L dibenzyl phosphonate in lmL THF was added via syringe to 7mg 95% NaH and the solution heated to reflux for 20min. The mixture was cooled to RT and 70mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-(para-toluenesulfonyl)-octanoyl) was added as a solution in lmL THF. The resultant white, heterogeneous solution was heated to reflux for 2h followed by 12h at RT. The solution was added to water, 10 extracted with CH2Cl2 and dried with Na2SO4. Pure 26mg Example 49 was obtained following PTLC on silica gel (1 x 1500pm plate) using 1:3:96 NH4OH:MeOH:CHCl3 as eluant. The product was characterized by IH NMR and MS [m/z: 828 (M ++1)]. 15 EXAMPLE 50 0 N N H N H H N NI 0 OMe o U Om /OCH 2 Ph O P/ IIO K + O 0 Example 50 was prepared by the following procedure. To 11 mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-dibenzylphosphono-octanoyl), in 2mL iPrOH containing 44gL H20 and 1.3mg KHCO3 at RT was added lmg 10% - 108- WO 01/07042 PCT/US00/19627 Pd/C. An atmosphere of H2 was established (balloon pressure). After 12h, the catalyst was removed by filtration through Celite using 1:1 MeOH:H20 as eluant. The solution was concentrated in vacuo and the residue was washed with CHCl3 followed by EtOAc. The remaining glassy material was lyophilized from water to 5 yield 3mg product. The product was characterized by H NMR and MS [m/z: 738 (M+1)]. EXAMPLE 51 0 NH H NH N O OMe om 0 8 8/OH II o- K + 0 10 Example 51 was prepared by the following procedure. To 2mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-dibenzylphosphono-octanoyl) in 0.35mL iPrOH was added 8gL water, 0.25mg KHCO3 and 0.5mg 10% Pd/C and a balloon atmosphere of hydrogen was established. After 7h at RT, the catalyst was removed via filtration through Celite and washed with water. 3mg of pure Example 15 51 product was characterized by 1H NMR and MS [m/z: 648 (M +1)]. - 109 - WO 01/07042 PCT/US00/19627 EXAMPLES 52A AND 52B O F O F 0 0 Ex. 52a Ex. 52b 5 Examples 52a and 52b were prepared by the following procedure. To 3mg -1:1 cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-hydroxy-8-oxo decanoyl) and cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-9- hydroxy -8-oxo decanoyl) 3mg) in 0.25mL CH2Cl2 at -78'C was added powdered, activated 4A sieves followed by 1.5pL Et2NSF3. The solution was warmed to -10 0 C over lh and 10 then quenched by the addition of saturated NaHCO3. The solution was extracted with CH2CI2 and dried with Na2SO4. A pure mixture of approximately 1:1 Example 52a and 52b was obtained following PTLC on silica gel (1 x 500pm plate) using 1:3:96 NH4OH:MeOH:CHCl3 as eluant. 2.5mg of the mixture were characterized by 1H NMR and MS [m/z: 641 (M ++1)]. 15 - 110- WO 01/07042 PCT/US00/19627 EXAMPLES 53A AND 53B 0 0 NH H NOje + 0 W~e 0 0o 0 0 0 Ex. 53a Ex. 53b 5 Examples 53a and 53b were prepared by the following procedure. To 6mg -1:1 cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7-hydroxy-8-oxo decanoyl) and cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-9-hydroxy-8-oxo decanoyl), Examples 20a and 20b, at RT was added powdered, activated 4A sieves followed by 3mg N-methylmorpholine-N-oxide and 0.3mg TPAP. After lh, the 10 mixture was diluted with CH2Cl2 and filtered through Celite using CH2Cl2 as eluant. The filtrate was extracted with 10% NaHSO3(aq), washed with water, and dried with Na2SO4. Pure products were obtained following PTLC (1 x 500 tm plate) separation using 1:3:96 NH4OH:MeOH:CHCl3 as eluant. 3.5mg of each pure Example 53a and 53b were characterized by 1H NMR and MS [m/z: 637 (M +1)]. 15 -111- WO 01/07042 PCT/US00/19627 EXAMPLES 54A AND 54B -oO o 0 0 NH NHIK NH NH'< H N H N OYOMe +OMe 0 SePh 0SePh 0 0 Ex. 54a Ex. 54b 5 Examples 54a and 54b were prepared by the following procedure. To 2g apicidin in 32mL THF at 0oC was added 14mL 0.5M potassium hexamethyldisilazane solution in toluene. The solution was aged at 0 0 C for 30min. Next, 25.g solid PhSeCI was added and the solution was warmed to RT for 2h. The reaction was quenched by the addition of saturated NaHCO3(aq), was extracted with 10 CH2Cl2, dried with Na2SO4 and filtered through 8 x 14 cm plug of silica gel using gradient elution (hexanes to 1:1 EtOAc:hexanes to 1:1 acetone:hexanes). The product was used in the preparation of Examples 55a and 55b with no further purification. The 2.3g mixture thus obtained was characterized by 1H NMR and MS [m/z: 780.3 (M++1)]. TLC: Rf = 0.60 (1:3:96 NH4OH:MeOH:CHCl3). 15 - 112- WO 01/07042 PCT/US00/19627 EXAMPLES 55A AND 55B 0 - 0 -O O O O 0 0 Ex. 55a Ex. 55b 5 Examples 55a and 55b were prepared by the following procedure. To 2.2g -1:1 cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-7-phenylselenyl decanoyl) and cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-9 phenylselenyl-decanoyl) in 40mL THF at 0OC was added 7.3mL 30% H202. The solution was warmed to 50oC and after 10min was cooled to 0OC, quenched with 10 saturated Na2S203, extracted with CH2Cl2 and dried with Na2SO4 Following purification on silica gel using 4:6 acetone:hexanes as eluant, a 230mg pure mixture of Examples 55a and 55b was characterized by 1H NMR and MS [m/z: 622.3 (M++1)]. TLC:. Rf = 0.38 (1:3:96 NH40H:MeOH:CHCI3). 15 EXAMPLES 56A AND 56B O OMe O OMe O O Ex. 56a Ex. 56b - 1130 N H NH N H NH H IJ N *H N 0 OMe 0Y OMe 0 0 8 0 -8 0 0 0 Ex. 56a Ex. 56b - 113 - WO 01/07042 PCT/US00/19627 Examples 56a and 56b were prepared by the following procedure. To 5.6mg -1:1 cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-6,7-dehydro decanoyl) and cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-9,10-dehydro decanoyl) in 0.225mL THF at RT was added 0.8mL PhCH2N(Me)3 (40% solution in 5 MeOH) followed by 1.5mL 70% t-BuOOH(aq). After 4.5h, EtOAc and minimal water were added and the aqueous phase thoroughly extracted with EtOAc. The organic layer was washed with cold 1N HCI (lx), quickly washed again with saturated NaHCO3, and then dried with Na2SO4. Pure products were separated by PTLC (1 x 500gm plate) using 4:6 acetone:hexanes as eluant. The pure examples 56a and 56b 10 were characterized by 1H NMR. The procedure yielded 1.4mg cyclo(N-O-Methyl-L Trp-L-Ile-D-Pip-L-2-amino-6,7-oxiranyl-decanoyl); MS [m/z: 638 (M+1)]; TLC: Rf = 0.4 (4:6 acetone:hexanes). The procedure yielded 2mg cyclo(N-O-Methyl-L-Trp-L Ile-D-Pip-L-2-amino-9,10-oxiranyl-decanoyl); MS [m/z: 638 (M+I1)]; TLC: Rf = 0.3 (4:6 acetone:hexanes). 15 EXAMPLES 57A AND 57B 0 0 N NH N HN H NH H N HN N I +f N 0 OMe N 0O OMe O OH O OH O OH OH O Ex. 57a Ex. 57b 20 Example 57a and 57b were prepared by the following procedure. To 115mg -1:1 cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-6,7-dehydro decanoyl) and cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-9,10-dehydro decanoyl) in 4mL 8:1 acetone:water at 0 0 C was added 45mg trimethylamine-N-oxide followed by 0.77mL 0.024M OsO4(aq). The solution was warmed to RT for 3h and 25 then aged at 4 0 C for 12h. The brown homogenous solution was quenched at 0oC by the addition of 2mL 10% NaHSO3(aq). After 10min, brine was added and the -114- WO 01/07042 PCT/US00/19627 solution thoroughly extracted with 3:7 iPrOH:CHCl3 (9x) and dried with Na2SO4. The solvent was removed in vacuo to yield 230mg crude product (121mg theoretical) which was used with no additional purification. A small aliquot of the regioisomeric diols were separated by PTLC on silica gel (1 x 1000tm plate) using 1:1 5 acetone:hexanes as eluant and the products were characterized by 1H NMR and MS. cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-6,7-dihydroxy-decanoyl): MS [m/z: 656 (M ++1)]; TLC: Rf = 0.5 (4:6 acetone:hexanes). cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-9,10-dihydroxy-decanoyl): MS [m/z: 656 (M +1I)]; TLC: Rf = 0.25 (4:6 acetone:hexanes). 10 EXAMPLES 58A AND 58B 0 0 Ex. 58a Ex. 58b 15 Examples 58a and 58b were prepared by the following procedure. To 121mng ~1:1 cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-6,7-dihydroxy decanoyl) and cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-9,10 NH NH N H NH H ~ N H N dihydroxy-decanoyl) in 6mL MeOH at C was added 75mL pyridine followed by 20 184 0g Pb(OAc)4. After 40in, the solution was poured into saturated Na2S203,e 0 0 Ex.extracted with CH2C2 and dried with Na2SO4. Pure separated Examples 58a and 15 58bExamples 58a and 58b were prepared by theobtained following PTLC (3 x 1500m plate) on silica gel using 1:3:96procedure. To 121mg -1:1 cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-6,7-dihydroxy decanoyl) and cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-9, 10 dihydroxy-decanoyl) in 6mL MeGH at 0 0 C was added 75rnL pyridine followed by 20 184mg Pb(OAc)4. After 40min, the solution was poured into saturated Na2S2O3, extracted with CH2Cl2 and dried with Na2SO4. Pure separated Examples 58a and 58b were obtained following PTLC (3 x 15001im plate) on silica gel using 1:3:96 NH4OH:MeOH:CHCI3 as eluant. The pure products were characterized by 1H NMR and MS. - 115- WO 01/07042 PCT/US00/19627 Example 58a, cyclo(N-O-Methyl-L-Trp-L-Ile-D-Pip-L-2-amino-6-oxo hexanoyl): Yield: 30mg. MS [m/z: 568 (M ++1)]; TLC: Rf = 0.45 (1:1 acetone:hexanes). Example 58b, cyclo(N-O-Methyl-L-Trp-L-Ile-D-Pip-L-2-amino-7 5 carboxymethyl-heptanoyl): Yield: 20mg. EXAMPLE 59 0 N NH H N N U o O 8 OCH 2

CH

3 0 10 Example 59 was prepared by the following procedure. To 4mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-6-hydroxy-hexanoyl) in 0.14mg CH2Cl2 and lmL pyridine at 0OC was added lmL ethyl chloroformate. The solution was warmed to RT, aged for 3h and the solvents removed in vacuo. 1.3mg pure Example 59 was obtained following PTLC (1 x 500tm plate) on silica gel using 4:6 15 acetone:hexanes as eluant. The pure product was characterized by 1H NMR and MS [m/z: 659 (M +NH4)]. - 116- WO 01/07042 PCT/US00/19627 EXAMPLE 60 0 N H NH Hl- I N0 OMe O O..8 OPh(4-NO 2 ) O 0 Example 60 was prepared by the following procedure. 7.5mg of the Example 64 C6-alcohol was placed in about lmL CH2Cl2 at 0OC to which was added 5 3.2mg (4-NO2)PhOC(O)Cl followed by 1.3tL pyridine. After 2h at 0OC, the volatiles were removed under reduced pressure without workup and 9mg pure Example 60 product was obtained following PTLC on silica gel (1 x 5001tm plate) using 1.1 acetone:hexanes as eluant. The pure Example 60 thus prepared was characterized by 1H NMR and MS [m/z: 735 (M ++1)]. 10 EXAMPLE 61 S0 S N H N NH N O OMe 0 O NH 2 Of 0 Example 61 was prepared by the following procedure. Anhydrous ammonia was bubbled into 2mL dioxane at 0OC to generate a -0.5 M solution. This 15 solution was added to 6mg solid cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-anmino-6 para-nitrophenoxycarbonyloxy-hexanoyl) at 0OC. The ice bath was removed and the solution aged at RT for 2h. The solution was concentrated under reduced pressure and 1.7mg pure Example 61 was obtained following PTLC (1 x 500pm plate) using 1:9:90 - 117- WO 01/07042 PCT/US00/19627 NH4OH:MeOH:CHC13 as eluant, and was characterized by 1H NMR and MS [m/z: 613 (M ++1)]. EXAMPLE 62 0 N N NH H N NI o O OMe 0 S 5 0o Example 62 was prepared by the following procedure. To 4mg Ph3P in 0.2mL THF at 0OC was added 2.4mL DEAD (diethyl azodicarboxylate) and aged for 30min. To this resulting solution was added about 4mg solid cyclo(N-O-methyl L-Trp-L-Ile-D-Pip-L-2-amino-6-hydroxy-hexanoyl) at 0OC. After lh at 0OC, the 10 solution was warmed to RT for lh. Solvent was removed under reduced pressure. 2mg of pure Example 62 product was obtained following PTLC (1 x 250/m plate) using 1:1 acetone:hexanes as eluant and was characterized by H NMR and MS [m/z: 628 (M++I)]. 15 EXAMPLE 63 - 0 0 N N. NH H N NI 0 OMe 0 SH Example 63 was prepared by the following procedure. To 1.5mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-6-acetylthio-hexanoyl) in 0.2mL MeOH at 0OC was added 0.3mL 25 wt % NaOMe in MeOH. After 5h, water was -118- WO 01/07042 PCT/US00/19627 added to quench the reaction. The solution was extracted with CH2Cl2 and dried with Na2SO4. 0.5mg pure Example 63 product was obtained following PTLC (1 x 250Am plate) using 1:1 acetone:hexanes as eluant. Example 63 was characterized by H NMR and MS [m/z: 588 (M ++1)]. 5 EXAMPLE 64 O 'NN N___O OMe O-~ SH Following the general procedure of Example 63, the C7 thiol was prepared from the corresponding thioacetate of Example 47. Example 64 was 10 characterized by 1H NMR and MS [m/z: 599 (M ++1)]. EXAMPLE 65 0 H N NI 0 OMe 0 - OSO2Ph(4-Me) Example 65 was prepared by the following procedure. To 1.6mg 15 cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-6-hydroxy-hexanoyl) in 0.28mL CH2Cl2 at 0OC was added 0.2mg DMAP followed by 2mg TsC1. After 16h, the solution was aged at RT for 16h. The solvent was removed under reduced pressure. Following PTLC (1 x 2501m plate) using 1:3:96 NH40H:MeOH:CHC13 as eluant, 0.3mg pure Example 65 was obtained. The pure product was characterized by 1H 20 NMR and MS [m/z: 724 (M ++1)]. - 119- WO 01/07042 PCT/US00/19627 EXAMPLE 66 0 N N NH H e' OMe 0

-

N

3 Example 66 was prepared by the following procedure. To 4mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-6-hydroxy-hexanoyl) in 0.35mL 5 CH2Cl2 at 0 0 C was added i) 3.7mg PPh3, ii) lmg imidazole and iii) 3.2mg Zn(N3)2*(pyridine)2 followed by iv) 2.2pL DEAD. The solution was warmed to RT for 12h. Following PTLC (1 x 500Am plate) using 1:3:96 NH4OH:MeOH:CHCI3 as eluant, 2mg pure Example 66 was obtained. Example 66 was characterized by 1H NMR and MS [m/z: 595 (M ++1)]. 10 EXAMPLE 67 0 N .NH H N NI o O Example 67 was prepared by the following procedure. To 1mg cyclo(N-O-methyl-L-Trp-L-Ile-D-Pip-L-2-amino-6-azido-hexanoyl) at 0oC in 0.1mL 15 THF was added 0.1mL thiolacetic acid. After lh, the solution was warmed to RT for lh. The solvents were then removed with a vigorous stream of nitrogen. The residue was dissolved in 0.2mL neat thiolacetic acid, aged for 4h and then concentrated with a vigorous stream of nitrogen. Pure Example 67 product was obtained following PTLC - 120 - WO 01/07042 PCT/US00/19627 (1 x 250pm plate) using 1:3:96 NH4OH:MeOH:CHC13 as eluant. The pure product (0.7mg) was characterized by 1 H NMR and MS [m/z: 611 (M ++1)]. 5 EXAMPLE 68 0 O O 0 0 Example 68 was prepared by the following procedure. 40mg Pd(OH)2 was added to 500mg apicidin in 40mL 1:1 THF:MeOH. An H2 atmosphere was established (balloon pressure). After 12h, the palladium catalyst was removed by 10 filtration through Celite using MeOH as eluant. Following flash chromatography on silica gel using 4:6 acetone:hexanes as eluant, 467mg pure Example 68 product was obtained and was characterized by 1H NMR. TLC: Rf = 0.18 (1:2 acetone:hexanes). HPLC: tR = 7.54min (1:1 MeCN:H20, 1.5mlJmin, Zorbax T M RX-8). 15 EXAMPLE 69 Q N N H ON - ~ O 0 0 Example 69 was prepared by the following methods: - 121 - WO 01/07042 PCT/US00/19627 Method C To 30mg N-desmethoxy apicidin in 500[tL DMF at RT was added 4 drops Mel followed by the addition of 11 mg tBuOK. The solution was stirred for 2h at RT, 12h at 4oC and then for another 4h at RT. The solution was then heated at 5 60 0 C for 1.5h and cooled back to RT. An additional 20mg tBuOK was added and the solution stirred for lh. The solution was then poured into 3mL of 1:2 saturated NaHCO3:saturated brine, extracted with CH2Cl2 and dried with Na2SO4. Following preparative TLC on silica gel (2 x 1500pm plates) using 1:2 acetone:hexanes as eluant, 19mg pure Example 69 was obtained and was characterized by 1H NMR and 10 MS [m/z: 625.3 (M ++NH4)]. TLC: Rf = 0.31 (1:2 acetone:hexanes). HPLC: tR = 3.90min (62:38 MeCN:H20, 1.5mL/min, Zorbax T M RX-8). Method D 1.3mg 60% NaH was added to 20mg N-desmethoxy apicidin in 15 0.35mL DMF at RT). After 30min, 4p.L Mel was added and the solution stirred for 10h. The solution was then poured into saturated NH4C1, extracted with CH2Cl2 and dried with Na2SO4. Following preparative TLC on silica gel (1 x 500 pm plate) using 1:1 acetone:hexanes as eluant, and further purification by preparative RP-HPLC using a linear gradient (1:1 to 1:0 MeCN:H20), 5mg pure Example 69 was obtained 20 which was characterized by 1H NMR and MS [m/z: 608.5 (M +1)]. EXAMPLE 70 - 0 H N H H N 0O CH 2

CO

2 Me O O Example 70 was prepared by the following procedure. At RT, 467mg 25 N-Desmethoxy apicidin was placed in 16mL DMF to which was added 63mg 60% NaH. After 10min, 206[tL BrCH2CO2Me and 871mg nBu4NI were added and the - 122 - WO 01/07042 PCT/US00/19627 solution heated to 80'C. After 15min, the solution was poured into water, extracted with CH2Cl2 and dried with Na2SO4. Following flash chromatography on silica gel using 1:1 acetone:hexanes as eluant, 401mg pure Example 70 was obtained which was characterized by 1H NMR and MS [m/z: 666 (M +1)]. TLC: Rf = 0.46 (1:1 5 acetone:hexanes). HPLC: tR = 7.21min 1:1 MeCN:H20, 1.0mL/min, Zorbax T M

RX

8). EXAMPLE 71 0 o ON NH O -8 NH 0 NN 0 Example 71 was prepared by the following procedure. At 0 oC, 0.65mg 10 HOBT, 1.6mg NaHCO3, 0.5mg 5-aminotetrazole and Img EDCI was added to 3.5mg N-desmethoxy-N-(para-carboxyphenylmethyl) apicidin in DMF. After 12h, the solution was poured into saturated NHCl4, extracted with CH2Cl2 and dried with Na2SO4 Following RP-HPLC using gradient elution (4:6 to 1:0 MeCN:water), 1.6mg pure Example 71 was obtained which was characterized by 1H NMR and MS [m/z: 15 795 (M1+I)]. -123 - WO 01/07042 PCT/US00/19627 EXAMPLE 72 0 HH O P(0)(OPh) 2 O 8 0 Example 72 was prepared by the following procedure. At RT, 3.4mg 60% NaH was added to 50mg N-desmethoxy apicidin in 0.2mL DMF and 0.2mL 5 HMPA. After gas evolution ceased, 35pL (PhO)2P(O)CI was added. After 24h, the solution was poured into water, extracted with EtOAc and dried with Na2SO4. Following preparative chromatotron TLC (1000pm plate) using 1:2 acetone:hexanes as eluant, 16mg pure Example 72 was obtained which was characterized by 1H NMR and MS [m/z: 826 (M ++1)]. 10 EXAMPLE 73 o-0 NO Example 73 was prepared by the following procedure. At RT, 7mL Et3N, and 1mg DMAP was added to 10mg N-desmethoxy apicidin in 0.17mL 15 CH2Cl2. Then 3.9pL MeSO2Cl was added. After 20h, the solution was poured into water, extracted with EtOAc and dried with Na2SO4. Following preparative RP HPLC using a linear gradient (4:6 to 1:0 MeCN:H20), 0.6mg pure Example 73 was - 124 - WO 01/07042 PCT/US00/19627 obtained (Rf= 0.4, 4:6 acetone:hexanes) which was characterized by 1H NMR and MS [m/z: 672 (M +1)]. EXAMPLES 74A-74J 5 Following the general procedure of Examples 69-72, utilizing an appropriate electrophile (R-X) readily determined by one in the art, the following compounds were prepared: 0 N NH N 0 0 - 125 O -125 - WO 01/07042 PCT/US00/19627 Table 6 Example R Group Mass Spec 69 Me 608.5 (M+1) 70 CH2CO2Me 666 (M +1) 71 CH2Ph[4-C(O)NH(5-tetrazolyl)] 795 (M +1) 72 P(O)(OPh)2 826 (M +1) 73 SO2Me 672 (M +1) 74a Et 639.4 (M +NH4) 74b nPr 653.3 (M++NH4) 74c CH2CO2tBu 708 (M+1) 74d CH2CH2OSi(tBu)Me2 752 (M+1) 74e CH2Ph(4-CO2Me) 742 (M++1) 74f C(O)Ph(4-Oac) 756 (M +1) 74g C(O)Ph 698 (M+1) 74h CO2Ph(4-NO2) 759 (M +1) 74i CO2CH2Ph 728 (M +1) 74j SO2Ph(4-Me) 748 (M'+1) 75 CO2CH2CH2NMe2 709 (M +1) EXAMPLE 75 O NO N H NH H N 0 0 OCH 2

CH

2 NMe 2 0O 8 0 Example 75 was prepared by the following procedure. At RT, 0.lmL 5 pyridine was added to 9mg N-desmethoxy-N-(para-aminophenoxycarbonyl) apicidin in 0.22mL DMF, followed by the addition of 22RL HOCH2CH2NMe2. After 15h, - 126 - WO 01/07042 PCT/US00/19627 the solution was poured into saturated NaHCO3, extracted with CH2Cl2 and dried with Na2SO4. Following preparative chromatotron TLC (1000am plate) using 1:2 acetone:hexanes as eluant, pure Example 75 was obtained which was characterized by 1H NMR and MS [m/z: 709 (M ++1)]. 5 EXAMPLE 76 O - 0 NH NH N I O CH 2 Ph(4-CO 2 H) o 8 0 Example 76 was prepared by the following procedure. At 0OC, 7.8[L IN LiOH was added to 3.8mg N-desmethoxy-N-(para-carboxymethylphenylmethyl) 10 apicidin in 0.13mL of a 3:1:1 mixture of THF:MeOH:H20. After 2h at 0 0 C and 17h at RT, the volatiles were then removed with a vigorous stream of nitrogen. The aqueous layer was then extracted with EtOAc and the aqueous layer acidified to pH-4 with 2N HC1. The aqueous layer was further extracted with 5 aliquots of a 3:7 mixture of iPrOH:CHCl3 and finally dried with Na2SO4. Following RP-HPLC using 15 a linear gradient (2:8 to 1:0 MeCN:H20), 2.5mg pure Example 76 was obtained which was characterized by 1H NMR and MS [m/z: 728 (M ++1)]. - 127 - WO 01/07042 PCT/US00/19627 EXAMPLE 77 O N O NH ~ S0 Ph(4-OH) 0 - 8 0 Example 77 was prepared by the following procedure. At -10 0 C, 6.5pL IM LiOH was added to a solution of 3.3mg N-desmethoxy-N-(para 5 acetoxyphenylcarbonyl) apicidin in 0.1 lmL of a 3:1:1 mixture of THF:MeOH:H20. After lh, the volatiles were removed with nitrogen. Then, about 2mL each of water and EtOAc was added. The resulting solution was carefully neutralized to pH-7 with 2N HC1. The solution was extracted with EtOAc and dried with Na2SO4. Following PTLC (1 x 500[tm plate) using 6:4 acetone:hexanes as eluant, 1.7mg pure Example 77 10 was obtained which was characterized by 1H NMR and MS [m/z: 714 (M ++1)]. EXAMPLE 78 0 N H N N N OO OPh(4-NH 2 ) - 8 O 0 Example 78 was prepared by the following procedure. At RT, 0.5mg 15 10% Pd/C catalyst was added to 2mg N-desmethoxy-N-(para-nitrophenoxycarbonyl) apicidin in 0.2mL CH2Cl2 and an atmosphere of hydrogen established (balloon pressure). After 6.5h, the catalyst was removed by filtration through Celite using 1:1 - 128- WO 01/07042 PCT/US00/19627 MeOH:CH2Cl2 as eluant. Without any further purification, the resulting 1.8mg Example 78 was characterized by H NMR and MS [m/z: 729 (M ++1)]. EXAMPLE 79 0 N NH H H O CH 2

CO

2 H OO 5 0 Example 79 was prepared by the following procedure. At O'C, 200gL IM LiOH was added to 89mg N-desmethoxy-N-carbomethoxymethyl apicidin in 3.5mL of a 1:1:1 mixture of THF:MeOH:H20. After 45min at 0 0 C, the slightly cloudy solution was warmed to RT and became homogenous. After an additional 10 20min, the MeOH and THF were removed using a vigorous stream of N2. Then, 2mL Ethyl acetate was added to the solution and removed to dispose of residual organic soluble material. The solution was acidified to pH-4.0 using 2N HC1, 3mL brine was added to the aqueous layer, and then extracted with a 1:4 mixture of iPrOH:CHCl3. The organic layer was dried with Na2SO4 to yield 51mg pure Example 79, which was 15 characterized by 1H NMR and MS [m/z: 652.5 (M +1)]. HPLC: tR = 1.21 min (1:1 MeCN:H20, 1.5mL/min, Zorbax T M RX-8). - 129 - WO 01/07042 PCT/US00/19627 EXAMPLE 80 N NH N

NO

2 O 00 0 Example 80 was prepared by the following procedure. At RT, 2.6mL TEA was added to 6mg N-desmethoxy-N-(6-amino-hexylaminocarbonylmethyl) 5 apicidin in lmL CH2Cl2. Next, 4mg NBD-Cl was added and the vial was wrapped with foil. After 3h at RT, pure Example 80 was obtained by flash chromatography on silica gel without workup using 1:1 hexanes:acetone as eluant. The pure product was characterized by H NMR. TLC: Rf = 0.19 (1:1 acetone:hexanes). 10 EXAMPLE 81 0 N NH H NII O CH 2 C(0)NH(CH 2

)

6 NH-CBZ 0 0 Example 81 was prepared by the following procedure. At 0 'C, 19mg EDCI was added to 50mg N-desmethoxy-N-carboxymethyl apicidin, 29mg CBZ 15 HN(CH2)6NH2, 10mg HOBT and 19jtL DIEA in 5mL CH2Cl2. After 15min at OoC and 1h at RT, 3mg DMAP was added. After an additional 2 hours, the CH2Cl2 was removed using a vigorous stream of N2 and 2mL DMF was added. After 2h, the solution was poured into 20jL 2:1 H20:brine, acidified to pH-3.0 with 2N HCI and -130- WO 01/07042 PCT/US00/19627 extracted with 5 15mL aliquots of CH2CI2. The organic layer was dried with Na2SO4. Without further purification, 54mg pure Example 81 was obtained which was characterized by 1H NMR and MS [m/z: 884.6 (M ++1)]. TLC: Rf = 0.72 (1:9:90 NH4OH:MeOH:CHC13). HPLC: tR = 5.38min (6:4 MeCN:H20, 1.5mL/min, 5 Zorbax T M RX-8). EXAMPLE 82 0 N I 0 CH 2 C(0)NHCH 2

CH

2

SCH

2

CH

3 8 0 Example 82 was prepared by the following procedure. At O'C, 1.2mg 10 HOBT was added to 5.7mg N-desmethoxy-N-carboxymethyl apicidin in 0. lmL DMF, 2.9mg NaHCO3, and 1.2mg EtSCH2CH2NH2*HC1. This was followed by the addition of 1.8mg EDCI. The solution was warmed to RT and aged for 16h. The aged solution was poured into saturated NaHCO3, extracted with EtOAc and dried with Na2SO4. Following RP-HPLC using gradient elution (4:6 to 1:0 MeCN:H20), 15 3.3mg pure Example 82 was obtained which was characterized by 1H NMR and MS [m/z: 739 (M +1)]. - 131 - WO 01/07042 PCT/US00/19627 EXAMPLE 83 0 ,N N H H~y O CH 2 C(0)NH(CH 2

)

6

NH

2 0 o0 Example 83 was prepared by the following procedure. At RT, 10mg 5% Pd/C catalyst was added to 54mg N-desmethoxy-N-[6-(benzyloxycarbonylamino) 5 hexylaminocarbonylmethyl]-apicidin in 3mL DMF and a H2 atmosphere (balloon pressure) was established. After 2h, an additional 40mg 5% Pd/C catalyst was added and the solution stirred overnight. The catalyst was then filtered off and the solvents were removed under reduced pressure. Following flash chromatography on silica gel using gradient elution (using first neat CHCl3, then three subsequent elutions of 10 1:3:96, then 1:4:95 and then 1:9:90 NH4OH:MeOH:CHCl3 as eluant), pure Example 83 was obtained which was characterized by 1H NMR and MS [m/z: 750.4 (M +1)]. TLC: Rf = 0.12 (1:9:90 NH4OH:MeOH:CHCl3). EXAMPLE 84 015 o Example 84 was prepared by the following procedure. At RT, 3.2mag NHS-SS-Biotin was added to 4mg N-desmethoxy-N-(6 aminohexylaminocarbonylmethyl)-apicidin in 0.5mL CH2Cl2 followed by 2pL DIEA. The solution was stirred for 1h at RT, followed by 12h at 4oC and 2h at RT. N' 0 H H NH N H0 0 Hr 0 H N N 0 H H N 0 S 15 0 Example 84 was prepared by the following procedure. At RT, 3.2mg NHS-SS-Biotin was added to 4mg N-desmethoxy-N-(6 aminohexylaminocarbonylmethyl)-apicidin in 0.5mL CH2Cl2 followed by 2ptL DIEA. The solution was stirred for lh at RT, followed by 12h at V 0 C and 2h at RT. 20 Additional 3.2mg NHS-SS-Biotin and 2tL DIEA were added followed by 100pL - 132 - WO 01/07042 PCT/US00/19627 DMF. After an additional 1 hour, the solution was loaded directly onto a silica gel pipette column using gradient elution (1:3:96 to 1:9:90 NH40H:MeOH:CHCI3 as eluant) to yield 4mg pure Example 84 which was characterized by 1H NMR. TLC: Rf = 0.26 (1:9:90 NH4OH:MeOH:CHCl3). 5 EXAMPLE 85 0 H I N 0 N H ON NH-Fmoc O H 0 O\ I O 0 Example 85 was prepared by the following procedure. At RT, 0.5mg HOBT, 2.6mg Fmoc-Phe(4-Bz)-OH (Fmoc = 9-fluorenylmethyl oxycarbonyl) and 10 1mg EDCI was added to 2mg N-desmethoxy-N-(6-aminohexylaminocarbonylmethyl) apicidin in 0.5mL CH2Cl2. Then, 3tL DIEA was added. After 2h at RT, the crude was purified without workup on a pipette flash column with silica gel using gradient elution (1:1 acetone:hexanes followed by 5:95 MeOH:CHCl3). The partially purified Example 85 was characterized by 1H NMR. TLC: Rf = 0.26 (1:9:90 15 NH4OH:MeOH:CHCl3). TLC: Rf = 0.53 (5:95 MeOH:CHCl3). - 133 - WO 01/07042 PCT/US00/19627 EXAMPLE 86 0 N N HHO OH N NH O H NNA0 NN-~-- N oO\H O 0 Example 86 was prepared by the following procedure. At RT, 0.2mL piperidine was added to 15mg of the Fmoc-protected Example 85 compound in 2mL 5 CH2CI2. After 3h at RT, the volatiles were removed under reduced pressure to produce Example 86. This material was used with no additional purification in Example 87. EXAMPLE 87 OO oo\H 10 o Example 87 was prepared by adding 5pL Et3N to 2mg of the crude product of Example 86 in 0.2mL CH2Cl2 at 0 oC followed by 2pL MeSO2C1. After 30min, the reaction was quenched by the addition of 3 drops of a 1:9:90 mixture of NH4OH:MeOH:CHCl3. Following flash chromatography on silica gel using 1:3:96 -134 - WO 01/07042 PCT/US00/19627 NH4OH:MeOH:CHCI3 as eluant, pure Example 87 was obtained without workup which was characterized by H NMR. EXAMPLE 88 - 0 NNH NH H N 0 N H O N N O H 5 o o Example 88 was prepared by the following procedure. First, 6mg HOBT, 10mg (4-Bz)PhCO2H, 23pL DIEA, and 19.6mg BOP were added to 25OtL CH2C12 at RT to generate (4-Bz)PhCO(OBT). Then, 20[tL of freshly prepared (4 Bz)PhCO(OBT) solution was added to 1mg N-desmethoxy-N-(6 10 aminohexylaminocarbonylmethyl)-apicidin in 200pL CH2Cl2 in a vial. The vial was wrapped in foil and allowed to stir at RT overnight. Partially purified product was obtained following preparative TLC on silica gel (1 x 250Rm plate) using 1:9:90 NH4OH:MeOH:CHC13 as eluant. Following preparative TLC on silica gel (1 x 250pm plate) using 1:3:96 NH4OH:MeOH:CHCl3 as eluant, pure Example 88 was 15 obtained which was characterized by 1H NMR. TLC: Rf = 0.27 (1:3:96 NH4OH:MeOH:CHC13). - 135- WO 01/07042 PCT/US00/19627 EXAMPLE 89 O H H N N H O0 N N O H o O o o 0 Example 89 was prepared by the following procedure. At RT, 3mg HOBT, 6LL Et3N, and 4.1mg (4-Bz)PhCH=CHCO2H was added to 9mg N 5 desmethoxy-N-(6-aminohexylaminocarbonylmethyl)-apicidin in 1mL CH2Cl2 followed by 13mg BOP. After 4h, the crude was purified without workup by flash chromatography on silica gel using 1:3:96 NH4OH:MeOH:CHCl3. This yielded 13.4mg pure Example 89, which was characterized by 1H NMR. TLC: Rf = 0.29 (1:3:96 NH4OH:MeOH:CHCl3). HPLC: TR = 4.90min (7:3 MeCN:H20, 10 1.5mL/min, Zorbax T M RX-8). EXAMPLE 90 0 H 'N 0 2 H N H 0 N N N N H I o 2 H 0 0 Example 90 was prepared by the following procedure. At RT, 3mg 15 5% Pd/C catalyst was added to 4mg Example 89 in 1:1 MeOH:CH2Cl2 and a deuterium gas atmosphere was established (balloon pressure). After 1h, the solution was purified on a silica gel pipette column using 1:9:90 NH4OH:MeOH:CHC13 as eluant to yield 2.9mg pure Example 90, which was characterized by 1H NMR. TLC: - 136- WO 01/07042 PCT/US00/19627 Rf = 0.34 (1:3:96 NH4OH:MeOH:CHCl3). HPLC: tR = 4.66min (7:3 MeCN:H20, 1.5mL/min, Zorbax T M RX-8). EXAMPLE 91 0 N I H CH 2

CH

2 OH 0 8 5 0 Example 91 was prepared by the following procedure. To 9mg of the silyl ether Example 74d in 0.2mL pyridine at 0OC was added 0.2mL HF*pyridine solution (prepared from 25g IIFpyridine, 10mL pyridine and 25mL THF). After 1.5h, the reaction was quenched by the addition of saturated NaHCO3, extracted with 10 CH2Cl2 and the combined organic layers were dried with Na2SO4. The 7.4mg of alcohol thus obtained was used in Example 92 below with no additional purification and was characterized by 1H NMR and MS [m/z: 638 (M ++1)]. EXAMPLE 92 - 0 N NH H N N O O OCH 2 Ph 0 I- OCH 2 Ph 8 0 15 o Example 92 was prepared by adding to 7.4mg of the Example 91 alcohol in 4mL CH2Cl2 at RT 422mg 1,2,4-triazolyle followed by 610pL (PhCH20)2PNEt2. After aging the solution for 3h, the volatiles were removed in - 137 - WO 01/07042 PCT/US00/19627 vacuo to form a yellow residue. Then, 7mL THF was added to the yellow residue to form a solution, which was cooled to -40 0 C. To this solution was added 4.6mL 30% H202 and warmed to RT. After aging for 30min, the reaction was quenched by the addition of 10% Na2S203(aq), diluted with saturated NaHCO3(aq) and water, 5 extracted with CH2CI2 and dried with Na2SO4. Following chromatotron purification (1000 m plate) using 1:2 acetone:hexanes as eluant, 255mg pure Example 92 was obtained which was characterized by 1H NMR and MS [m/z: 898 (M +1)]. EXAMPLE 93 0 N NH H_ N No0 II 0 O1 IO- K+ 8 0 10 0 Example 93 was prepared by adding 27mg KHCO3 and 25mg 10% Pd/C catalyst at RT to 245mg of Example 92 in 40mL iPrOH and lmL water. An atmosphere of hydrogen (balloon pressure) was established for 12h. After the catalyst was removed by filtration through Celite using 1:1 MeOH:H20 as eluant, the volatiles 15 were removed under reduced pressure. No further purification was required and yielded 214mg Example 93, which was characterized by 1H NMR and MS [m/z: 718 (M++1)]. - 138 - WO 01/07042 PCT/US00/19627 EXAMPLE 94 0 N I O CH 2

CH

2

OSO

2 Ph(4-Me) o 8 0 Example 94 was prepared by the following procedure. At 0oC, 2mg DMAP was added to 20mg apicidin alcohol in 2mL CH2Cl2 followed by the addition 5 of 26mg Ts20. After 10min the solution was warmed to RT for 3h. Then, 10mg TsCl was added and the solution aged for 16h. The solvent was removed under reduced pressure and 1mg pure Example 94 was obtained following centrifugal TLC (4:6 acetone:hexanes to 1:9:90 NH4OH:MeOH:CHCl3) as eluant. The product was characterized by H NMR and MS [m/z: 792 (M +1)]. 10 EXAMPLE 95 0 NI H N N 0 CH 2

CH

2

N

3 0 8 0 Example 95 was prepared by the following procedure. At 0 0 C, 247mg Ph3P and 217mg Zn(N3)3*pyridine was added to 300mg N-desmethoxy-N-(2 15 hydroxyethyl)-apicidin in 25mL CH2Cl2, followed by the addition of 150gL DEAD. The solution was then warmed to RT. After aging for 12h, the volatiles were removed under reduced pressure. Following chromatotron TLC on silica gel (2mm plate) using 1:3:96 NH4OH:MeOH:CHC13 as eluant, 311 lmg pure Example 95 (Rf= 0.32, 1:9:90 -139- WO 01/07042 PCT/US00/19627 NH4OH:MeOH:CHC13) was obtained which was characterized by 1H NMR and MS [m/z: 663 (M ++1)]. EXAMPLE 96 0 N I O CH 2

CH

2

NH

2 0 8 5 0o Example 96 was prepared by the following procedure. At RT, 60mg 10% Pd/C catalyst was added to 311mg N-desmethoxy-N-(2-azidoethyl) apicidin in CH2Cl2 and an atmosphere of hydrogen was established (balloon pressure). After 8h, the catalyst was filtered through Celite using 3:7 iPrOH:CHCl3 as eluant to yield the 10 desired product. Following chromatotron PTLC (1 x 2000pm plate) using 1:3:96 NH4OH:MeOH:CHCl3 as eluant, pure Example 96 (200mg, Rf= 0.21 (1:3:96 NH4OH:MeOH:CHCl3) was obtained which was characterized by 1 H NMR and MS [m/z: 637 (M++1)]. 15 EXAMPLE 97 N NH H" N N I 0 CH 2

CH

2

NHSO

2 Me o 8 O 0 Example 97 was prepared by the following procedure. At 0OC, 9ptL Et3N was added to 10mg N-desmethoxy-N-(2-aminoethyl) apicidin in 0.5mL CH2Cl2 - 140 - WO 01/07042 PCT/US00/19627 followed by the addition of 3.6jiL MeSO2C1. The solution was warmed to RT and stirred for 30min. The solution was quenched by the addition of saturated NaHCO3, extracted with CH2Cl2 and dried with Na2SO4. Following PTLC (1 x 250pm plate) on silica gel using 1:1 acetone:hexanes as eluant, 9mg pure Example 97 was obtained 5 which was characterized by 1H NMR and MS [m/z: 732.7 (M+ NH4)]. TLC: Rf = 0.26 (1:1 acetone:hexanes). HPLC: tR = 4.7 min (1:1 MeCN:H20, 1.5 ml/min, Zorbax T M RX-C8). EXAMPLE 98 0 N NH N I O K ACH 2

CH

2 N(Me)SO 2 Me o 8 10 0 Example 98 was prepared by the following procedure. At RT, 7 pL NaN(TMS)2 (IM in THF) was added to 4mg N-desmethoxy-N-2 methanesulfonamidoethyl apicidin in 0.28mL THF followed by the addition of 1.5pL Mel. After 16h, the solution was quenched by the addition of water, extracted with 15 CH2Cl2 and dried with Na2SO4. Following PTLC (1 x 250gm plate) on silica gel using 1:3:96 NH40H:MeOH:CHCl3 as eluant, 2.2mg pure Example 98 was obtained which was characterized by 1H NMR and MS [m/z; 746.6 (M ++NH4)]. TLC: Rf = 0.42 (1:3:96 NH4OH:MeOH:CHC13). - 141 - WO 01/07042 PCT/US00/19627 EXAMPLE 99 0 0 N H N -N NH O 0 NH-Fmoc O 0 Example 99 was prepared by the following procedure. At RT, 5mg HOBT, 7pL TEA and 18.4mg Fmoc-Phe(4-Bz)-OH was added to 16mg N 5 desmethoxy-N-(2-aminoethyl)-apicidin in lmL CH2Cl2 followed by the addition of 16mg BOP. After 3h at RT, the solution was purified by flash chromatography on silica gel using 1:3:96 NH4OH:MeOH:CHCl3 as eluant to yield pure Example 99, which was characterized by 1H NMR. TLC: Rf = 0.50 (1:3:96 NH4OH:MeOH:CHCl3). 10 EXAMPLE 100 0 0 N N NHNHS HO N N H o 0,I, N....N

NHSO

2 Me 00 0 Example 100 was prepared by adding 50pL piperidine to 15mg of the Fmoc-protected amine of Example 99 at RT in 2mL CH2Cl2. After 2h at RT, the 15 solution was concentrated under reduced pressure and lyophilized from dioxane to remove residual piperidine. The crude deprotected amine product was dissolved in 2mL CH2C12 at O'C and 5.6gL Et3N was added followed by 62pL MeSO2Cl (0.26M in CH2Cl2). After lh, the reaction was quenched by the addition of saturated - 142- WO 01/07042 PCT/US00/19627 NaHCO3(aq), extracted with CH2Cl2 and dried with Na2SO4. Following PTLC on silica gel (1 x 1000pm plate) using 1:4:95 NH4OH:MeOH:CHCl3 as eluant, pure Example 100 was obtained which was characterized by 1H NMR and MS [m/z: 1080 (M ++1)]. 5 EXAMPLE 101 0 NI N H NH O:v U CH 2

CH

2

NH

2 0 OH Example 101 was prepared by the following procedure. At RT, 8mg NaBH4 was added to 20mg N-desmethoxy-N-(2-aminoethyl) apicidin in 2mL MeOH. 10 After 2h at RT, acetone was added to the solution to quench the reaction and the solution was poured into saturated NaHCO3, extracted with CH2Cl2 and dried with Na2SO4. Following flash chromatography on silica gel using 1:9:90 NH4OH:MeOH:CHCI3 as eluant, pure Example 101 was obtained which was characterized by 1 H NMR. TLC: Rf= 0.28 (1:9:90 NH4OH:MeOH:CHC13). 15 - 143- WO 01/07042 PCT/US00/19627 EXAMPLE 102 0 0 NH NH H H 0 8 0 Example 102 was prepared by the following procedure. At RT, 16.1 mg DDQ was added to 20mg N-desmethoxy apicidin in 1. lmL 9:1 MeCN:H20 5 to form a dark purple solution, which became blood-red over 30 min. The solution was aged at 0 0 C for 12h. The solution was purified without workup by RP-HPLC using 4:6 MeCN:H20 as eluant. This yielded 15mg of Example 102 which was characterized by 1H NMR and MS [m/z: 608 (M ++1)]. 10 EXAMPLE 103 0 0 oY o 8 O Example 103 was prepared by the following procedure. At RT, 1.5RL Et3N was added to 6mg cyclo(beta-oxo-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo decanoyl) in 0.5mL CH2Cl2. After 10min, the solution was purified without workup 15 by RP-HPLC using 1:1 MeCN:H20 as eluant. This yielded 3mg pure Example 103, which was characterized by 1 H NMR and MS [m/z: 608 (M +1)]. - 144 - WO 01/07042 PCT/US00/19627 EXAMPLES 104A AND 104B - 0 0 - 0 0 NH N NH 0 m H& N HJ N N _ I N I O CH 2

CH

2

CH

2

CH

2 CI O CH 2

CH

2

CH

2

CH

2 CI O 0 0 0 Ex. 104a Ex. 104b 5 Examples 104a and 104b were prepared by the following procedure. At RT, 0.14mL BrCH2CH2CH2CH2C1, 0.5g nBu4NI and 25mg 95% NaH were added to 300mg beta-oxo-N-desmethoxy apicidin in 0.5mL DMF containing 0.25mL HMPA. The solution was degassed with bubbling N2 for 4min and then heated to 10 100 0 C for 90min. The solution was then cooled to RT, poured into saturated brine/saturated NaHCO3, extracted with CH2Cl2 and dried with Na2SO4. Following PTLC (2 x 1500tm plates) on silica gel using 1:3:96 NH3:MeOH:CHCl3 as eluant, a pure mixture of Example 104a and Example 104b was obtained. The pure products were characterized by 1H NMR and MS [m/z: 698.5 (M+1) for each isomer]. The 15 yield was 150mg D-Trp isomer and 120mg L-Trp isomer. TLC: Rf = 0.42 for D-Trp isomer and 0.25 for L-Trp isomer (2:3 acetone:hexanes). - 145 - WO 01/07042 PCT/US00/19627 EXAMPLES105A AND 105B - 0 0 - 0 0 N N o H H, NN E N 0 CH 2

CH

2

CH

2 CI 0

CH

2

CH

2

CH

2 CI 0 O 0 0 Ex. 105a Ex. 105b 5 Examples 105a and 105b were prepared by adding 0.12mL BrCH2CH2CH2C1, 0.5g nBu4NI and 25mg 95% NaH at RT to 300mg beta-oxo-N desmethoxy apicidin in 0.5mL DMF containing 0.25mL HMPA. The solution was degassed with bubbling N2 for 4min and then heated to 100oC for 90min. The 10 solution was cooled to RT, poured into 1:1 saturated brine:saturated NaHCO3, extracted with CH2Cl2 and dried with Na2SO4. Following PTLC (2 x 1500/Lm plates) on silica gel using 1:3:96 NH3:MeOH:CHC13 as eluant, a pure mixture of Examples 105a and 105b were obtained which were characterized by 1H NMR and MS [m/z: 684.5 (M +1) for each isomer]. Yield: 120mg D-Trp isomer and 80mg L 15 Trp isomer. TLC: Rf = 0.55 for D-Trp isomer and 0.27 for L-Trp isomer (2:3 acetone:hexanes). -146- WO 01/07042 PCT/US00/19627 EXAMPLES 106A AND 106B - 0 0 - 0 0 0 CH 2

CH

2

CH

2

CH

2 1 O CH 2

CH

2

CH

2

CH

2 1 O o Ex. 106a Ex. 106b 5 Examples 106a and 106b were prepared by adding 516mg Nal to 120mg cyclo(N-(4-chloro-n-butyl)-beta-oxo-D-Trp-L-Ile-D-Pip-L-2-amino-8-oxo decanoyl) in 2.2mL anhydrous MeCN. The resulting solution was heated to 60 0 C for 12h. The solution was cooled to RT and diluted with 1:1 brine:saturated NaHCO3, extracted with CH2Cl2 and dried with Na2SO4. This yielded 100mg of a mixture of 10 Example 106a and 106b which was characterized by 1H NMR and MS [m/z: 790.5 (M++1) for each isomer] without purification. TLC: Rf = 0.58 for D-Trp isomer and 0.41 for L-Trp isomer (1:3:96 NH4:MeOH:CHCI3) EXAMPLES 107A AND 107B OI . _1

N

H N H HN H N O C 2

H

2

CH

2 CH21 0 CH 2

CH

2

CH

2 o O0 15 o o Ex. 107a Ex. 107b - 147 - WO 01/07042 PCT/US00/19627 Examples 107a and 107b were prepared by adding 350mg Nal to 80mg cyclo(N-(4-chloro-n-propyl)-beta-oxo-D-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-decanoyl) in 1.5mL anhydrous MeCN. The resulting solution was heated to 60 0 C for 12h. The solution was cooled to RT, diluted with 1:1 brine:saturated NaHCO3, extracted with 5 CH2Cl2 and dried with Na2SO4. This yielded 70mg of a mixture of Example 107a and Example 107b which were characterized by 1H NMR and MS [m/z: 776.5 (M++1) for each isomer] without purification. TLC: Rf = 0.53 for D-Trp isomer and 0.42 for L-Trp isomer (1:3:96 NH4OH:MeOH:CHC13). 10 EXAMPLES 108A AND 108B - 0 0 - 0 0 N H H NH N H NH 0 CH 2

CH

2

CH

3

CH

2 2

CH

3 CH o o0 0 0 Ex. 108a Ex.108b 15 Examples 108a and 108b were prepared by adding 30mg MgBr2*Et20, and 30gL nBu3SnH to 40mg of an - 1:1 mixture of cyclo(N-(3-iodo-n-propyl)-beta oxo-D-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-decanoyl) and cyclo(N-(3-iodo-n-propyl) beta-oxo-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-decanoyl) in 0.5mL CH2Cl2. The resulting solution was cooled to -78 0 C. Next, 100tL Et3B was added followed 20 by 500gL oxygen gas via syringe over 2h. The reaction was quenched by the addition of 1:1 brine:saturated NaHCO3 at -78'C. The solution was then warmed to RT, partitioned with CH2Cl2 and the organic layer dried with Na2SO4. The solution was concentrated under reduced pressure and the residue partitioned between hexanes:MeCN (1:3). The MeCN layer was washed (3x) with hexanes and the MeCN 25 layer concentrated under reduced pressure. Pure products were obtained following PTLC (1 x 1000m plate) on silica gel using 1:3:96 NH4OH:MeOH:CHCl3 as eluant. Pure products were characterized by 1H NMR and MS [m/z: 650.6 (M ++1) for each - 148- WO 01/07042 PCT/US00/19627 isomer]. Yield: 14mg D-Trp isomer and 14mg L-Trp isomer. TLC: Rf = 0.69 for D Trp isomer and 0.51 for L-Trp isomer (1:3:96 NH4OH:MeOH:CHC13). EXAMPLE 109 - 0 OH N N H N O CH 2

CH

2

CH

3 0 5 0o Example 109 was prepared by the following procedure. At RT, 5mg 2,2'-azobisisobutyronitrile and 38gL nBu3SnH were added to a 22mg mixture of cyclo(N-(3-iodo-n-propyl)-beta-oxo-D-(and L, - 1:1)-Trp-L-Ile-D-Pip-L-2-amino-8 oxo-decanoyl) in 0.6mL toluene. Nitrogen was bubbled through the solution for 5min 10 and it was then heated to 100 0 C for 2h. The solution was cooled to RT, concentrated under reduced pressure and the residue partitioned between hexanes:MeCN (1:3). The MeCN layer was washed (3x) with hexanes and the MeCN layer concentrated under reduced pressure. Following PTLC on silica gel using 4:6 acetone:hexanes as eluant, 10mg pure Example 109 was obtained which was characterized by 1H NMR 15 and MS [m/z: 652.7 (M ++1)]. TLC: Rf = 0.50 and 0.43 (mixture of beta-hydroxy isomers) (1:3:96 NH4OH:MeOH:CHCl3). -149- WO 01/07042 PCT/US00/19627 EXAMPLE 110 0 OH S NH NH O CH 2

CH

2

CH

2

CH

3 0 0 Example 110 was prepared by the following procedure. At RT, 6mg 2,2'-azobisisobutyronitrile and 52gL nBu3SnH was added to 31mg of a -1:1 mixture 5 of cyclo(N-(4-iodo-n-butyl)-beta-oxo-D-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-decanoyl) and cyclo(N-(4-iodo-n-butyl)-beta-oxo-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo decanoyl) in 0.8mL toluene. Nitrogen was bubbled through the solution for 5min and it was then heated to 100 0 C for 2h. The solution was cooled to RT, concentrated under reduced pressure and the residue partitioned between hexanes:MeCN (1:3). 10 The MeCN layer was washed (3x) with hexanes and the MeCN layer concentrated under reduced pressure. Example 110 was characterized by 1H NMR without purification. TLC: Rf = 0.66 (1:3:96 NH4OH:MeOH:CHCl3). EXAMPLES 111A AND 111B L,, O O H -', O O H 15 OH OH OH Ex. 111a Ex. 111b N NHExamples 11a and 11b were prepared by adding mg NaBH4 to 3mg beta-oxo-N-desmethoxy apicidin in 0.25mL EtOH at C. After 2.5h at RT and 10h N- 150 N I 0 H 0 H 0 8 08 15 OH OH Ex. lila Ex. 111b Examples Il1a and 11lib were prepared by adding 1 mg NaBH4 to 3mg beta-oxo-N-desmethoxy apicidin in 0.25rnL EtOH at 0 0 C. After 2.5h at RT and 10h -150- WO 01/07042 PCT/US00/19627 at 0 0 C, the solution was poured into saturated NH4CI, extracted with 3:1 EtOAc:iPrOH and dried with Na2SO4. Following RP-HPLC using gradient elution (2:3 to 1:1 MeCN:H20), a mixture of pure Example 11 la and 11 lb were obtained which was characterized by 1H NMR and MS [m/z: 594 (M +-H20) for both isomers]. 5 TLC: Rf = 0.50 for D-Trp isomer and 0.28 for L-Trp isomer (1:9:90 NH4OH:MeOH:CHCI3). HPLC: tR = 3.9min for D-Trp isomer and 3.5min for L-Trp isomer (1:1 MeCH:H20, 1.5mL/min, Zorbax T M RX-C8). EXAMPLES 112A AND 112B -0 0 0 0 N" H N H~L N; 0 H 0 H O8 0 8 10 OH OH Ex. 112a Ex.112b Examples 112a and 112b were prepared by adding 5.8mg CeC13*6H20 to 10mg cyclo(beta-oxo-L-Trp-L-Ile-D-Pip-L-2-amino-8-oxo-decanoyl) at RT in 15 0.2mL MeOH. After 5min, the solution was cooled to 0 0 C and 0.6mg NaBH4 was added. The solution was poured into saturated NH4C1, extracted with EtOAc and dried with Na2SO4. Following RP-HPLC using 1:1 MeCN:H20 as eluant, a pure mixture of 0.7mg Example 112a and 1.3mg Example 112b was obtained which was characterized by 1 H NMR and MS [m/z: 609 (M ++1) for each isomer]. 20 - 151 - WO 01/07042 PCT/US00/19627 EXAMPLES 113A AND 113B - 0 0 - 0 0 Ioo. OHO HN H N 0 Ac 0 Ac o 0 OAc OAc Ex. 113a Ex. 113b 5 Examples 113a and 113b were prepared by adding 14mg DMAP and 0.533mL Ac20 to 700mg cyclo(beta-oxo-L-Trp-L-Ile-D-Pip-L-2-amino-8-hydroxy decanoyl) in 115mL dichloroethane at RT. After 8h, the mixture was poured into saturated NH4C1, extracted with CH2CI2 and dried with Na2SO4. Following 10 preparative chromatotron (4pm plate) on silica gel using 2:8 to 4:6 acetone:hexanes gradient elution as eluant, 8mg of a mixture of Examples 113a and 113b was obtained. Pure epimeric products were characterized by 1H NMR and MS. D-Trp isomer: yield: 140mg; TLC: Rf= 0.71 (1:1 acetone:hexanes); MS [m/z: 694.4 (M ++1)]. L Trp isomer: yield: 110mg; TLC: Rf = 0.57 (1:1 acetone:hexanes); MS [m/z: 694.5 15 (M ++1)]. - 152- WO 01/07042 PCT/US00/19627 EXAMPLE 114 O N H N-r N N O OMe O-m 0 8 0 Example 114 was prepared by the following procedure. At RT, 28.5mg N-bromosuccinamide and 1.2mg benzoyl peroxide was added to 100mg 5 apicidin in 5.3mL CCl4. Nitrogen then was bubbled through the solution for 5min. The solution was refluxed for 15min and then cooled to RT. Following PTLC on silica gel (3 x 1000tm plates) using 1:3:96 NH4OH:MeOH:CHCl3 (one development) followed by 4:6 acetone:hexanes (two developments) as eluant, 62mg pure Example 114 was obtained which was characterized by 1H NMR and MS [m/z: 10 704 (M++1)]. RP-HPLC: tR = 5.02 min (apicidin: tR = 4.82min), 6:4 MeCN:H20, 1.5mL/min. EXAMPLES 115A AND 115B N 0 H O Example 115a (mobile product A) and Example 115b (polar product Q8 08 0 0 15 Ex. 115a Ex. 115b Example 115~a (mobile product A) and Example 1 15b (polar product B) were prepared by the following methods E and F. - 153 - WO 01/07042 PCT/US00/19627 Method E At 0OC, 10mg Example 114 was added to 4mg AgBF4 in 250pL 3:1 DMSO:CH2Cl2. After aging for 10min (at this point, TLC showed the disappearance 5 of the starting bromide), 10L Et3N was added and the solution aged for an additional hour. The reaction was quenched by the addition of water. The mixture was then extracted with CH2CI2 and dried with Na2SO4. Following PTLC on silica gel (1 x 250am plate) using 1:1 acetone:hexanes as eluant, a pure mixture of Examples 115a and 115b was obtained which were characterized by 1H NMR and MS [m/z: 640 10 (M++1) for both isomers]. TLC: Rf= 0.48 Example 115a (mobile product A) and 0.41 Example 115b (polar product B), 1:1 acetone:hexanes. Method F At RT, 12mg NaHCO3 was added to 43mg apicidin in CH2Cl2, 15 followed by 18mg 85% MCPBA. The resulting solution was vigorously stirred for 12h. The solution was then poured into saturated NaHCO3(aq), extracted with CH2Cl2 and dried with Na2SO4. Following PTLC on silica gel (1 x 250[tm plate) using 1:1 acetone:hexanes, pure Example 115a was obtained which was identical in all respects to Example 115a, mobile product A, from Method E above. 20 EXAMPLE 116 0 N Br N-OH 1 11 H"J N N OMe O 8 0 Example 116 was prepared by following the general procedure of Example 115a, method E. Starting with 10mg of Example 114, 4mg of Example 116 25 was prepared which was characterized by 1H NMR and MS [m/z: 718.6 (M ++1)]. - 154 - WO 01/07042 PCT/US00/19627 EXAMPLE 117 O-0 SBr -OA H CN SOMe 0 8 0 Example 117 was prepared by adding 4pL Ac20 to 5.4mg of Example 115b at RT in 375pL CICH2CH2C1, followed by the addition of 0.3mg DMAP. After 5 1.5h, the volatiles were removed under a stream of nitrogen. Following PTLC on silica gel (1 x 250m plate) using 1:1 acetone:hexanes as eluant, 6mg pure Example 117 which was characterized by 1H NMR and MS [m/z: 762 (M ++1)]. 10 EXAMPLE 118 0 N N-OAc' NO O - A o 8 0 Example 118 was prepared by the following procedure. To 5mg of Example 117 at RT in lmL CH2CI2 was added lmg Pd(OH)2 and a hydrogen atmosphere was established (balloon pressure). After aging for 2h, the solution was 15 filtered and concentrated under reduced pressure. Following flash chromatography on silica gel using 1:9:90 NH4:MeOH:CHCl3, 3.5mg pure Example 118 was obtained which was characterized by 1 H NMR and MS [m/z: 652 (M +1I)]. - 155- WO 01/07042 PCT/US00/19627 EXAMPLE 119 - O 3/1 naifepi 0 N N N-OM H N N O oO o 8 0 Example 119 was prepared by the following procedure. To 25mg beta 5 oxo-N-desmethoxy-apicidin at RT in 1.5mL MeOH was added 15[tL pyridine followed by the addition of 126mg Pb(OAc)4. After aging for 48h, the solution was cooled to 0OC and saturated Na2S203(aq) was added. The solution was poured into saturated NH4Cl(aq):brine (1:1), extracted with iPrOH:CHCl3 (3:7) and dried with Na2SO4. The solution was filtered and concentrated under reduced pressure. 10 Following flash chromatography on silica gel using 1:9:90 NH4:MeOH:CHC13, 36mg pure Example 119 was obtained which was characterized by H NMR and MS [m/z: 638 (M++1)]. EXAMPLE 120 o-0 N HH N HXO N 0 W~e 0 8 15 o Example 120 was prepared by the following procedure. To 81mg of Example 114 in 6mL THF:H20 at RT was added 141mg basic A1203 and 191mg Ag2CO3. The solution was warmed to 50 0 C for 5h, then cooled to RT. The mixture - 156 - WO 01/07042 PCT/US00/19627 was partitioned between water and CH2Cl2, the layers separated, the organic layer dried with Na2SO4 and then filtered through Celite. Following PTLC (1 x 500tm plate) using 1:3:96 NH40H:MeOH:CHCl3 as eluant, pure Example 120 was obtained which was characterized by 1H NMR and MS [m/z: 640.5 (M +1)]. 5 EXAMPLE 121 O-0 Br O N 8 0 Example 121 was prepared by following the general procedure of Example 120, and utilizing the dibromide Example 126 as the starting material. The 10 product thus obtained was characterized by 1H NMR and MS [m/z: 720 (M+ +1)]. EXAMPLE 122 0

O

NNH H Ji N U OMe o 8 0 Example 122 was prepared by the following procedure. To 200[L 15 CH2Cl2 at -78 0 C was added 6AL oxallyl chloride (2M solution in CH2Cl2) followed by the addition of 2pL DMSO. After 5min, 3.3mg of Example 120 (as a solution in 50pL CH2Cl2) was added to the DMSO/oxallyl chloride solution. After aging for 15min, 14pL Et3N was added and the solution was warmed to 0 0 C. The reaction was - 157 - WO 01/07042 PCT/US00/19627 then quenched by the addition of water, extracted with CH2Cl2 and dried with Na2SO4. Following PTLC on silica gel (1 x 500m plate) using 1:3:96 NH4OH:MeOH:CHCl3 as eluant, pure Example 122 was obtained which was characterized by 1H NMR and MS [m/z: 658 (M ++1)]. 5 EXAMPLE 123 -0 Br N NH H jyeS N Oe 0 8 0 Example 123 was prepared by mixing 28mg of Example 126 in 1.5mL DMF at RT with 13mg NaSMe. The mixture was then warmed to 50oC. After lh, the 10 solution was poured into water, extracted with CH2Cl2 and dried with Na2SO4. Following PTLC purification on silica gel (1 x 250m plate) using 4:6 acetone:hexanes as eluant (two developments). Pure Example 123 was obtained which was characterized by 1H NMR and MS [m/z: 748 (M+ +I)]. 15 EXAMPLE 124 o0 N H N O Me OO 0 8 0 Example 124 was prepared by adding 5.4mg KSAc to 11mg of Example 114 in 260pL DMF at 0OC. After aging the solution for 48h, it was warmed - 158- WO 01/07042 PCT/US00/19627 to RT and aged an additional 20h. The solution was poured into water, extracted with CH2Cl2 and dried with Na2SO4. Following PTLC on silica gel using 4:6 acetone:hexanes as eluant, the product thus obtained was characterized by H NMR and MS [m/z: 622 (M ++1)]. 5 EXAMPLE 125 O N

O

H 0 N OMe o 8 0 Example 125 was prepared by adding 5mg DDQ to 5mg of Example 115a (mobile product A) at RT in 200L THF. The resulting solution was warmed to 10 65 0 C. After aging for 20h, an additional 5mg DDQ was added. After an additional 6h, the volatiles were removed at ambient temperature under reduced pressure. Methylene chloride was added, the solution was filtered and the filtrate was loaded onto a preparative TLC plate (1 x 250Am plate, silica gel). Following PTLC purification using 4:6 acetone:hexanes as eluant, the pure Example 125 thus obtained 15 was characterized by 1H NMR and MS [m/z: 608.6 (M ++1)]. - 159 - WO 01/07042 PCT/US00/19627 EXAMPLE 126 O 0 cy~ .. < ~ Br N N-Br NN OMe 0 8 0 Example 126 was prepared by the following procedure. At RT, 86mg N-bromosuccinamide was added to 100mg apicidin in 5.3mL CC14, followed by the 5 addition of 1.2mg benzoyl peroxide. The resulting solution was purged with vigorous nitrogen bubbling for 5min. The solution was heated to reflux for 45min and then cooled to RT. The volatiles were removed under reduced pressure and pure Example 126 was obtained following PTLC purification on silica gel (1500tm plate) using 1:3:96 NH4OH:MeOH:CHCl3 as eluant. The dibromide Example 126 product thus 10 obtained was characterized by 1H NMR and MS [m/z: 780 (M ++1)]. TLC: Rf= 0.49 (1:3:96 NH40H:MeOH:CHCl3). HPLC: t R = 10.02min, ImL/min, 6:4 MeCN:H20, Zorbax T M RX-8). EXAMPLES 127A AND 127B 0 0 - H Br N Br N N Br H- H H N N NI NI H OMe O OMe 0 8 0 8 0 0 15 Mobile product A Polar product B Ex. 127a Ex. 127b -160- WO 01/07042 PCT/US00/19627 Examples 127a and 127b were prepared by adding 0.32mL DMF, and 0.32mL 1:1 saturated NaHCO3:H20 to 10mg of Example 126, followed by the addition of 4.5mg Na2S204. The milky white solution thus formed was aged at RT for 24h. Then, 2mL Acetonitrile was added, and the solids were removed by 5 filtration. This yielded 1mg pure Example 127a (mobile product A) and 4mg pure Example 127b (polar product B) following RP-HPLC using 1:1 MeCN:H20 as eluant. Both products were characterized by 1H NMR and MS. Example 127a, mobile product A: MS: [m/z: 704 (M ++1)]; TLC: Rf = 0.75 (1:1 acetone:hexanes); HPLC: tR = 8min, 2mL/min, 1:1 MeCN:H20, 10 Zorbax T M RX-8). Example 127b, polar product B: MS: [m/z: 702 (M ++1)]; TLC: Rf= 0.60 (1:1 acetone:hexanes); HPLC: tR = 7min, 2mi/min, 1:1 MeCN:H20, Zorbax T M RX-8). 15 EXAMPLE 128 O-0 Oe Example 128 was prepared by the following procedure. At 0oC, 6mg N-bromosuccinamide was added to 13mg apicidin in 1mL CH2Cl2 and 0.5mL MeOH. After 4min, 1mL saturated Na2SO3(aq) was added, followed by 1mL brine. 20 The solution was extracted with EtOAc and dried with Na2SO4. Partially purified product was obtained following PTLC on silica gel (1 x 1500ptm plate) using 1:2 acetone:hexanes as eluant. Pure Example 128 was subsequently obtained following flash chromatography on silica gel using 1:2 acetone:hexanes as eluant. The Example 128 thus obtained was characterized by 1H NMR and MS [m/z: 670.4 (M +1)]. 25 -161 N NH H 0~ N NI 0 OMe 0 8 0 Example 128 was prepared by the following procedure. At 0 0 C, 6mg N-bromosuccinamide was added to 13mg apicidin in lmL CH2Cl2 and 0.5mL MeGH. After 4mmn, lmL saturated Na2SO3(aq) was added, followed by lmL brine. 20 The solution was extracted with EtOAc and dried with Na2SO4. Partially purified product was obtained following PTLC on silica gel (1 x 1500/.km plate) using 1:2 acetone:hexanes as eluant. Pure Example 128 was subsequently obtained following flash chromatography on silica gel using 1:2 acetone:hexanes as eluant. The Example 128 thus obtained was characterized by I H NMR and MIS [mlz: 670.4 (M ++1)]. 25 - 161 - WO 01/07042 PCT/US00/19627 EXAMPLES 129A AND 129B 0 0 NN O O" -,,0 0CH 2 OH 0 8 0 8 o 0 Ex. 129a Ex. 129b 5 Examples 129a and 129b were prepared by the following procedure. To 10mg N-desmethoxy apicidin at RT in 0.1mL DMF was added 3RL 37% formaldehyde(aq) and 3RL pyrrolidine. After 48h, the reaction was quenched with saturated NaHCO3, extracted with EtOAc and dried with Na2SO4. Pure 2mg of the pyrrolidino Example 129a (Rf= 0.2) and 2mg of the hydroxymethyl Example 129b 10 (Rf= 0.1) was obtained following PTLC (1:3:96 NH4OH:MeOH:CHC13, Rf= 0.2) as eluant. The pure products were characterized by 1H NMR and MS [m/z: 624 (M +1) for the pyrrolidino Example 129a and 677 (M+1) for the hydroxymethyl Example 129b]. 15 EXAMPLE 130 - 0 N I O

CH

2 0Ac 0 8 0 Example 130 was prepared by the following procedure. To 5mg N desmethoxy-N-hydroxymethyl apicidin in 0.16mL pyridine at RT was added 0.63mL - 162 - WO 01/07042 PCT/US00/19627 acetyl chloride and one crystal of DMAP. After 12h, the reaction was quenched with saturated NH4CI, extracted with EtOAc and dried with Na2SO4. Following RP HPLC using a linear gradient of 4:6 to 1:0 MeCN:H20 as eluant, pure Example 130 was obtained which was characterized by 1H NMR and MS [m/z: 666 (M ++1)]. 5 EXAMPLE 131 0 '.s N NH' N I O CH 2

OP(O)(OCH

2 Ph) 2 o 8 O 0 Example 131 was prepared by the following procedure. To 9mg N desmethoxy-N-hydroxymethyl apicidin in CH2Cl2 at 0 0 C was added 13pL EtN(iPr)2 10 followed by the addition of 43gL (PhCH20)2P(O)C1. After 30min at 0 0 C, 0.4mg DMAP was added and the solution was aged for 1.5h at O'C, followed by 2.5h at RT. The reaction was quenched by the addition of water, extracted with CH2Cl2 and dried with Na2SO4. Following preparative RP-HPLC using a linear gradient of 4:6 AE 1:0 MeCN:H20 as eluant, 0.3mg pure Example 131 was obtained which was 15 characterized by 1H NMR and MS [m/z: 884 (M+1)]. - 163- WO 01/07042 PCT/US00/19627 EXAMPLES 132A AND 132B 0 Q N 0. OH O N OH HN N NH O + N H NH O NO 2 0 - 0 0 0 Ex. 132a Ex. 132b 5 Examples 132a and 132b were prepared by the following methods G, H and I. Method G 10 To 100mg apicidin in 4mL MeCN and 3mL CH2Cl2 at RT was added 800mg NaIO4 in 10mL water, followed by the addition of 10mg RuCI3. The solution was then aged overnight. The solution was poured into brine, acidified with glacial acetic acid and filtered to remove particulates. The solids were rinsed with CH2Cl2 15 and the solution was extracted with CH2Cl2. The combined organic layers were dried with Na2SO4, filtered, and concentrated under reduced pressure. Pure 52mg carboxylic acid Example 132a was obtained following preparative RP-HIIPLC using gradient elution (1:4 to 1:1 MeCN:H20, 50min linear ramp). Example 132a obtained was characterized by 1H NMR and MS [m/z: 523.2 (M ++1)]. Also obtained from this 20 reaction was the nitrophenylketone apicidin analog, Example 132b, which was characterized by 1 H NMR and MS [m/z: 628.2 (M ++1)]. Method H To a solution containing 0.3mg RuCl3*xH20 and 50mg N-desmethoxy 25 apicidin in 2mL 1:1 MeCN:CCl4 was added 324mg NaIO4 (as a solution in lmL H20). After 45h, the resulting green solution was partitioned between 1:1 brine:saturated NH4Cl and 3:7 iPrOH:CHCl3. The organic layer then was dried with -164- WO 01/07042 PCT/US00/19627 Na2SO4. The solution:was concentrated under reduced pressure to yield 60mg crude product. Method I 5 To 9mg cyclo(L-Asp-L-Ile-D-Pip-L-2-amino-8-oxo-decanoyl), methyl ester in lmL 3:1:1 THF:MeOH:H20 at 0 oC was added 50gL IM LiOH. After lh at 0 0 C, followed by 2 days at RT, the solution was filtered through a reversed-phase plug (0.5 g C-18) with MeOH as eluant, concentrated under reduced pressure, and purified without workup by RP-HPLC using gradient elution (10min ramp from 5:95 10 MeCN:H20 to 25:75 MeCN:H20, then 60min ramp to 100% MeCN). EXAMPLE 133 0 yO

M

e NH N ', , " O M e N NH 0 NO 0 0 00 Example 133 was prepared by adding lmL Me3SiCH=N2 (0.5M 15 solution in hexanes) to 12mg of the carboxylic acid product of Example 132a in 4mL 2:1 MeOH:Et20 at RT. After 20min, the solution became homogenous and 0.25mL glacial acetic acid was added. The solution was poured into brine, extracted with CH2Cl2 and dried with Na2SO4. The solution was filtered and concentrated under reduced pressure. Pure Example 133 was obtained following PTLC on silica gel (1 x 20 1000pm plate) using 1:1 acetone:hexanes as eluant. The methyl ester Example 133 thus obtained was characterized by 1H NMR and MS [m/z: 537.5 (M +1)]. - 165 - WO 01/07042 PCT/US00/19627 EXAMPLE 134 N ',-OMe N NH 0 NO 0 o OH Example 134 was prepared by adding 9.6mg NaBH4 to 120mg of Example 133 in 7mL THF at 0 0 C. After aging for 3h, the reaction was quenched by 5 the addition of saturated NH4CI(aq), extracted with CH2Cl2 and dried with Na2SO4. Following PTLC using 4:6 acetone:hexanes (Rf= 0.53) as eluant, 117mg of pure Example 134 was obtained which was characterized by 1H NMR. EXAMPLE 135 0 N * ,-y N(OMe)Me N NH 0 H Nl tO 0 0 10 0 Example 135 was prepared by the following methods J and K. Method J To 50mg of Example 132a in 2mL CH2Cl2 at RT was added 15 sequentially 14p1L Et3N followed by 81aL MeSO2C1. After aging for 2h, 18mg solid HCl*HN(OMe)Me was added. After an additional hour, the volatiles were removed under reduced pressure. Following flash chromatography on silica gel using 1:2:97 NH4OH:MeOH:CHCI3 as eluant, 1.9mg pure Example 135 was obtained which was characterized by 1H NMR and MS. - 166 - WO 01/07042 PCT/US00/19627 Method K To 20mg of Example 132a in lmL THF at -78 0 C was added 10.6mg HCI*HN(OMe)Me followed by the dropwise addition of 112gL iPrMgBr (2M 5 solution in THF). The resulting solution was slowly allowed to warm to 4 0 C and was aged for 12h. The reaction was quenched by addition of lmL saturated NH4Cl(aq), extracted with CH2Cl2 and dried with Na2SO4. Following flash chromatography on silica gel using 1:2:97 NH4OH:MeOH:CHCl3 as eluant, 1 1mg pure Example 135 was obtained which was characterized by 1H NMR and MS. 10 EXAMPLE 136 0 N ',- N(OMe)Me H N H NH O NOOi 0 OH Example 136 was prepared from 117mg of Example 134 using Method K as described in Example 135. This resulted in 76mg of Example 136 (Rf= 0.46, 15 1:9:90 NH40H:MeOH:CHCl3) which was characterized by 1 H NMR and MS [m/z: 568 (M+1)]. - 167 - WO 01/07042 PCT/US00/19627 EXAMPLE 137 0 N NH 0 N0 OH Example 137 was prepared by the following procedure. To 11 mg cyclo(N-O-methyl-N-methyl-L-Asp-L-Ile-D-Pip-L-2-amino-8-hydroxy-decanoyl) in 5 0.39mL THF and 80L HMPA at 0 0 C was added 388 pL n-C10H21MgBr (1M in Et20). The solution was warmed immediately to RT and aged for 12h. The solution was poured into saturated NH4Cl(aq), partitioned with THIF and dried with Na2SO4. Following PTLC (1 x 500m plate) on silica gel using 1:9:90 NH40H:MeOH:CHCl3 as eluant, 2.5mg pure Example 137 was obtained which was characterized by 1H 10 NMR and MS [m/z: 649 (M ++1)]. EXAMPLE 138 0 H N 0 0 Example 138 was prepared by adding 5pL pyridine to 2mg of Example 15 137 in 0.35mL CH2Cl2 at 23 0 C, followed by the addition of 7mg Dess-Martin periodinane. After 1.5h, the solution was poured into 1:1 saturated NaHCO3:10% NaHSO3, aged for 10min, then extracted with CH2Cl2, and dried with Na2SO4. Following PTLC (1 x 250pm plate) on silica gel using 1:3:96 NH4OH:MeOH:CHCl3 - 168- WO 01/07042 PCT/US00/19627 as eluant, 1.5mg pure Example 138 was obtained which was characterized by 1H NMR and MS [m/z: 647 (M ++1)]. EXAMPLES 139A-139J 5 Following the general procedures described in Examples 137 and 138, and utilizing the appropriate starting compounds and reactants - particularly the appropriate nucleophile for the R1 group - which would be clear to one in the art, the following compounds were prepared: 0 )-- N '.,.- R, yRi N NH O NH O

K

0 0 10 R 2 Table 7 Example R1 Group R2 Group Mass Spec 139a CH2Ph H, OH 599 (M +1) 139b CH2Ph =0O 597 (M+1) 139c 1-napthyl H, OH 635 (M+1) 139d 1-napthyl =0 633 (M+1) 139g 5-(N-methyl-indolyl) H, OH 638 (M +1) 139h 5-(N-methyl-indolyl) =0 636 (M ++1) 139i tBu H, OH 565 (M+1) 139j tBu =0 563 (M +1) - 169 - WO 01/07042 PCT/US00/19627 EXAMPLE 140 - 0 0 OOH N J OH NNH O 0 0 0 o0 Example 140 was prepared by the following procedure. To 100mg cyclo(beta-oxo-L-Trp-L-lBe-D-Pip-L-2-amino-8-oxo-decanoyl) at RT in 6mL 1:1:1 5 MeCN:CCl4:H20 was added 0.7mg RuCl3*2H20 followed by the addition of 634mg NalO4 (as a sonicated solution in 2mL H20). After 30h, the resulting tan-white heterogeneous solution was partitioned between 1:1 brine:saturated NH4Cl and 3:7 iPrOH:CHCl3. The organic layer was dried with Na2SO4 and concentrated under reduced pressure to yield 100mg of Example 140. The crude product was 10 characterized by IH NMR and MS [m/z: 526 (M ++NH4)] with no additional purification. EXAMPLE 141 - 0 0 N OMW e NH H 0 0 00 15 Example 141 was prepared by the following procedure. To 80mg cyclo(D-2-amino-2-carboxy-ethanoyl-L-Ile-D-Pip-L-2-amino-8-oxo-decanoyl) in 3.3mL 2:1 MeOH:Et20 at RT was added lmL TMSCHN2 (2M in hexanes). After 1.5h, glacial HOAc was added dropwise until foaming ceased and the solution was partitioned between 1:1 brine:saturated NH4Cl and CH2Cl2. The organic layer was - 170 - WO 01/07042 PCT/US00/19627 dried with Na2SO4. Following PTLC (1 x 1500ptm plate) on silica gel using 3:97 HOAc:EtOAc as eluant, 28mg pure Example 141 was obtained which was characterized by H NMR and MS [m/z: 540 (M ++NH4)]. 5 EXAMPLE 142 - 0 0 N N(OMe)Me NNH H 0 0 O Example 142 was prepared by the following procedure. To 26.5mg of Example 140 in 2mL CH2Cl2 at RT was added 51mg HClIHN(OMe)Me and 13mg DMAP, followed by the addition of 46mg BOP. After aging for 8h at RT, the solution 10 was warmed to 40 0 C for 12h. Following removal of volatiles, 2mg pure Example 142 was obtained following PTLC on silica gel using 1:1 acetone:hexanes as eluant. The product was characterized by 1H NMR and MS [m/z: 552 (M +1)]. EXAMPLE 143 H0 0 N N(OMe)Me NNH H 0 15 OH Example 143 was prepared by starting with Example 141. First, the side chain carbonyl of Example 141 was reduced as described in Example 134. The resulting intermediate compound was then treated by the procedure described in Example 135. The pure Example 143 thus obtained was characterized by 1H NMR. - 171 - WO 01/07042 PCT/US00/19627 EXAMPLES 144A-144G Following the general procedures described above in Examples 142 5 and 143 (procedure of Example 142 was utilized for Example 144g, while the procedure of Example 143 was utilized for the other Examples 144a-144f), and using the appropriate materials which would be clear to one in the art, the following compounds were prepared: o N NH H NO 0 0

R

2 10 Table 8 Example R1 Group R2 Group Mass Spec 144a CH2Ph H, OH 602 (M++NH4) 144b CH2Ph =0 600 (M++NH4) 144c iPr H, OH 537 (M ++1) 144d iPr =0 552 (M++NH4) 144e 5-(N-methylindolyl) H, OH 624 (M +1) 144f 5-(N-methylindolyl) =0 622 (M +1) 144g CH2Ph PhCH2-, OH 689 (M+1) - 172- WO 01/07042 PCT/US00/19627 EXAMPLE 145 S NH O 0 0 Example 145 was prepared by the following procedure. To 10mg N desmethoxy-N-methyl apicidin in 2.5mL CH2Cl2 at -78 0 C was bubbled 03 until the 5 solution turned light blue. The resulting solution was stirred for 10min and then N2 was bubbled through the solution for 5min. Next, 250gL Dimethylsulfide was added, the solution then warmed slowly to RT and concentrated under reduced pressure. The resulting residue was dissolved in 1:1 THF:tBuOH at 0 0 C, followed by the addition of 3.7mg tBuOK. After 2h at 0OC, the solution was poured into 1:1 water:saturated 10 NaHCO3, extracted with CH2Cl2 and dried with Na2SO4. Following preparative TLC on silica gel (1 x 500am plate) using 1:3:96 NH4OH:MeOH:CHCl3 as eluant, pure Example 145 was obtained which was characterized by IH NMR and MS [m/z: 622.7 (M++1)]. TLC: Rf = 0.15 (1:3:96 NH4OH:MeOH:CHCl3). - 173 - WO 01/07042 PCT/US00/19627 EXAMPLES 146A-146F Following the general ozonalysis procedure described for Example 145, the following compounds were prepared: 0 Q NN R N NH N-- O 0 5 o Table 9 Example R Group Starting Compound Mass Spec 145 Me Ex. 69 622.7 (M+I1) 146a H Apicidin 608.3 (M'+1) 146b OMe Apicidin 638.3 (M+1) 146c Et Ex. 74a 636.8 (M +1) 146d nPr Ex. 74b 650.3 (M +1) 146e CH2CO2Me Ex. 70 680.7 (M +1) 146f CH2CO2H Ex. 79 666.6 (M+1) - 174 - WO 01/07042 PCT/US00/19627 EXAMPLE 147 O' N , N"- H HN N-'- O 0 0 Example 147 was prepared by the following procedure. To 10mg cyclo(L-2-amino-2-(3'-(quinol-4'-onyl))-ethanoyl-L-Ile-D-Pip-L-2-amino-8-oxo 5 decanoyl) in lmL CICH2CH2CI at RT was added 4mg DMAP and 19pL TEA, followed by the addition of 5RL MeSO2Cl. After 15min at RT, the solution was poured into saturated NaHCO3, extracted with CH2Cl2 and dried with Na2SO4. Following preparative TLC on silica gel (1 x 500m plate) using 1:1 acetone:hexanes as eluant, pure Example 147 was obtained which was characterized by 1H NMR and 10 MS [m/z: 608.5 (M ++1)]. TLC: Rf = 0.43 (1:1 acetone:hexanes). EXAMPLES 148A AND 148B 0 . 0 N OMe N H H H H H + 0 0 OH OH 15 Ex. 148a Ex. 148b Examples 148a and 148b were prepared by adding 3mg NaBH4 to 20mg cyclo(L-2-amino-2-(3'-(N-O-methyl-quinol-4'-onyl))-ethanoyl-L-Ile-D-Pip-L - 175- WO 01/07042 PCT/US00/19627 2-amino-8-oxo-decanoyl) in 5mL MeOH at 0 0 C. Then the cooling bath was removed promptly. After 20min, acetone was added to quench the reaction and the solution was poured into saturated NaHCO3, extracted with CH2Cl2 and dried with Na2SO4. Initial purification was accomplished following flash chromatography on silica gel 5 using 1:1 acetone:hexanes as eluant. At this juncture, it was noted that the resulting product was approximately 1:1 mixture of two compounds with similar TLC Rf values (product A: 0.39 and Product B: 0.28 in 1:1 acetone:hexanes). Repurification by preparative TLC on silica gel (1 x 500pim plate) yielded two pure products which were characterized by 1H NMR and MS [m/z: 640.6 (M ++1) for Example 148a and 10 610.5 (M++1) for Example 148b]. Example 148a: cyclo(L-2-amino-2-(3'-(N-O-methyl-quinol-4'-onyl)) ethanoyl-L-Ile-D-Pip-L-2-amino-8-hydroxy-decanoyl); TLC: Rf = 0.55 (1:3:96 NH4OH:MeOH:CHCl3); HPLC: tR = 7.17min (1:1 MeCN:H20, 1.0mL/min, Zorbax T M RX-8). 15 Example 148b: cyclo(L-2-amino-2-(3'-quinol-4'-onyl)-ethanoyl-L-Ile D-Pip-L-2-amino-8-hydroxy-decanoyl); TLC: Rf = 0.18 (1:3:96 NH4OH:MeOH:CHCl3); HPLC: tR = 5.86min (1:1 MeCN:H20, 1.0mL/min, Zorbax T M RX-8). 20 EXAMPLE 149 - 0 N '"r N NH O N(OMe)CHO N 0 0 o - 8 0 Example 149 was prepared by the following procedure. Ozone was bubbled through 25mg apicidin in 2.5mL CH2C12 at -78 0 C until the resulting solution remained pale blue. After 10min, the solution was purged with a vigorous stream of 25 nitrogen, followed by the addition of limL Me2S. Then the solution was warmed to RT. The volatiles were removed under reduced pressure and pure Example 149 was obtained following PTLC on silica gel (1 x 2000pm plate) using 1:2 acetone:hexanes - 176- WO 01/07042 PCT/US00/19627 as eluant. The pure Example 149 thus obtained was characterized by 1 H NMR and MS [m/z: 662.5 (M ++Li)]. EXAMPLE 150 - 0 N NH 0 HN H N O Y O 5 0 Example 150 was prepared by the following procedure. Ozone was bubbled through a solution of 470mg N-desmethoxy-apicidin in 40mL CH2Cl2 at 78 0 C for about 10min until a blue color persisted. Then the solution was purged with a vigorous stream on nitrogen, followed by the addition of lmL Dimethylsulfide. The 10 resulting solution was allowed to warm to RT and the volatiles were removed under reduced pressure. Following flash chromatography on silica gel using gradient elution (2:3 to 1:1 acetone:hexanes), 320mg pure Example 150 was obtained which was characterized by 1H NMR and MS [m/z: 626 (M ++1)]. 15 EXAMPLE 151 - 0 0 N H NNH 0 HN H O 0 0 Example 151 was prepared similarly to the procedure described for Example 150 utilizing beta-oxo-N-desmethoxy-apicidin as the starting material. - 177- WO 01/07042 PCT/US00/19627 Example 151 thus obtained was characterized by 1H NMR and MS [m/z: 640 (M +1)]. EXAMPLES 152A AND 152B

CF

3

SO

3 N H N N Ni HH o o 0 - 0 5 0 0 Ex. 152a Ex. 152b Examples 152a and 152b were prepared by adding 30tL pyridine to 43mg cyclo(L-2-amino-2-(3'-quinol-4'-onyl)-ethanoyl-L-Ile-D-Pip-L-2-amino-8-oxo 10 decanoyl) in 1.2mL CH2C12. The mixture was cooled to 0 oC. To the resulting solution was added 14gL (CF3SO2)20. After 40min, the solvent was removed in vacuo. The crude pyridinium salt thus obtained was characterized by 1H NMR and MS [m/z: 740 (M ++1)]. - 178- WO 01/07042 PCT/US00/19627 EXAMPLE 153 oN@ ,(DN 0 Ir N NH 0 0 Example 153 was prepared by the following methods L and M. 5 Method L To 52mg crude cyclo(L-2-amino-2-(3'-(4'-pyridium-quinolyl)) ethanoyl-L-Ile-D-Pip-L-2-amino-8-oxo-decanoyl) in 4mL CH2Cl2 at RT was added 1mg 20% Pd(OH)2 Degussa catalyst. A hydrogen atmosphere (balloon pressure) was 10 established. After 12h, the catalyst was removed by filtration through Celite using acetone as eluant. Following PTLC (1 x 1000 pm plate) on silica gel using 1:3:96 NH4OH:MeOH:CHCl3 as eluant, 28mg pure Example 153 was obtained which was characterized by 1H NMR and MS [m/z: 675 (M ++1)]. 15 Method M To 20mg cyclo(L-2-amino-2-(3'-quinol-4'-onyl)-ethanoyl-L-Ile-D-Pip L-2-amino-8-oxo-decanoyl) in 0.6mL CH2C12 at 0 'C was added 8mg 2,6-di-t-butyl 4-methyl-pyridine followed by 7ptL (CF3SO2)20. After 3.5h, 7tL piperidine was added, the solution was aged for 2.5h and then was warmed to RT for 12h. Following 20 PTLC without workup (1 x 500am plate) on silica gel using 1:3:96 NH4OH:MeOH:CHC13 as eluant, 6mg pure Example 153 was obtained which was characterized by 1H NMR and MS [m/z: 675 (M ++1)]. - 179 - WO 01/07042 PCT/US00/19627 EXAMPLE 154 CI N NH NO H O" 0O 0 Example 154 was prepared by the following procedure. At 23 0 C, 13mg of Example 146a was placed in 360L DMF. Then 5.3mg 2,6-di-tert-butyl-4 5 methyl-pyridine was added followed by 6.9mg 2,4-dinitrobenzenesulfonyl chloride. After aging for 6h, 2.7mg LiCl was added and the solution was warmed to 60 0 C for 12h. The reaction was cooled to RT, quenched by the addition of water, extracted with CH2Cl2 and dried with Na2SO4. Following PTLC on silica gel (1 x 500im plate) using 1:9:90 NH4OH:MeOH:CHCl3 as eluant, 5mg pure Example 154 was 10 obtained which was characterized by 1H NMR and MS [m/z: 626 (M ++1)]. EXAMPLE 155 0 N N' H~ NH Br O 0 0 Example 155 was prepared by mixing 1.2g N-Desmethoxy-apicidin, 15 360mg N-bromosuccinamide and 15mg benzoyl peroxide in 70mL CCl4. The resulting mixture was heated to 80 0 C for 15min. The solvent was then removed under reduced pressure and the crude product was purified in two batches by RP-HPLC - 180 - WO 01/07042 PCT/US00/19627 using 4:6 MeCN:H20 as eluant to yield 400mg pure Example 155 which was characterized by 1H NMR and MS [m/z: 674 (M ++1)]. EXAMPLE 156 -0 Wj H NH H - MeO OMe oe W 0 5 o Example 156 was prepared by dissolving 100mg cyclo(2-bromo-L-Trp L-Ile-D-Pip-L-2-amino-8-oxo-decanoyl) in 3mL dioxane and 3mL EtOH. Then 63mg LiC1, 270mg (3,5-diMeO)PhB(OH)2 and 1.5mL IM NaHCO3 was added. To the resulting mixture was added 17mg Pd(PPh3)4 and the resulting solution was heated in 10 sequence to 90 0 C for 90min, 100oC for 15min and 80 0 C for 12h. The solution was poured into 1:1 saturated NaHCO3:brine, extracted with CH2Cl2 and dried with Na2SO4. Following preparative TLC on silica gel (1 x 5001tm plate) using 1:3:96 NH40H:MeOH:CHCl3 as eluant (four developments), 67 pure Example 156 was obtained which was characterized by 1 H NMR and MS [m/z: 730 (M ++1)]. 15 - 181 - WO 01/07042 PCT/US00/19627 EXAMPLES 157A-157D Examples 157a-157d were prepared following the procedure described in Example 156. S NH N H o H 0 0 5 0 Table 10 Example R Group Mass Spec 156 Ph(3,5-OMe) 730 (M+1) 157a 2-napthyl 720 (M+1) 157b 5-(N-methylindolyl) 723 (M +1) 157c 1-napthyl 720 (M ++1) 157d Ph 687 (M++NH4) EXAMPLE 158 o N OH H N- O 0 10 OH Example 158 was prepared by adding 9mg NaBH4 to 100mg of Example 141 in 6mL THF at 0 0 C. After 2h, the reaction was quenched by the - 182 - WO 01/07042 PCT/US00/19627 addition of acetone followed by the addition of saturated NaHCO3(aq), extracted with CH2C12 and dried with Na2SO4. This yielded 10mg pure diol Example 158 (Rf= 0.37) following PTLC on silica gel using 3:7 acetone:hexanes as eluant. The product thus obtained was characterized by 1H NMR. 5 EXAMPLE 159 - 0 N' "s-OH NH HO 0 O OH Example 159 was prepared by the following methods N and O. 10 Method N: To 100mg of Example 133 in 3.5mL THF at 0 0 C was added 11.6mg LiBH4. After aging for 4h at 0oC, the reaction was warmed to RT. After an additional 2h, the reaction was quenched by the addition of acetone followed by the addition of saturated brine(aq), extracted with 3:7 iPrOH:CHCl3 and dried with 15 Na2SO4. Following PTLC on silica gel using 1:3:96 NH4OH:MeOH:CHCl3 as eluant, 60mg pure diol Example 159 was obtained which was characterized by IH NMR and MS [m/z: 511 (M +1)]. Method O 20 To 250mg of Example 133 in 1 lmL THF at 0 0 C was added 2.65mL DIBAL-H (IM solution in toluene). After aging for 4h at O'C, the reaction was quenched by the addition of acetone followed by the addition of saturated brine, extracted with 3:7 iPrOH:CHCl3 and dried with Na2SO4. Following flash chromatography on silica gel using 1:3:96 to 1:9:90 NH4OH:MeOH:CHCl3 gradient 25 elution, 100mg pure diol Example 159 (Rf= 0.41, 1:9:90 NH4OH:MeOH:CHCI3) was obtained which was characterized by IH NMR. - 183 - WO 01/07042 PCT/US00/19627 EXAMPLE 160 N 0 NO N H N O

-

0 OH Example 160 was prepared by the following procedure. To 27mg 5 Ph3Bi dissolved in 1mL CH2Cl2 at RT was added 0.5gL CH3CO3H. After 10min, a Ph3Bi/CH3CO3H solution resulted. To the solution, 22mg of Example 159 was added as a solution in lmL CH2Cl2, followed by the addition of 3.5mg Cu(OAc)2. The resulting solution was then warmed to 60'C for 3h. After cooling to RT, the reaction was quenched by the addition of saturated NaHCO3(aq), extracted with 3:7 10 iPrOH:CHCl3 and dried with Na2SO4. Following PTLC on silica gel using 4:6 acetone:hexanes (Rf= 0.66) as eluant, 4mg pure Example 160 was obtained which was characterized by 1H NMR and MS [m/z: 587 (M ++1)]. EXAMPLE 161 0 N NH 0 15 o Example 161 was prepared by oxidizing 3mg of Example 160 using Dess-Martin reagent similarly to the general procedure described in Example 138. This resulted in 2mg Example 161, which was characterized by 1H NMR and MS [m/z: 585 (M +1)]. - 184 - WO 01/07042 PCT/US00/19627 EXAMPLES 162A AND 162B Following the general procedure for Examples 160 and 161, the 5 following Examples 162a and 162b were prepared and characterized by NMR and MS: 0 OR oO OO 0O 0 Table 12 Example R Group Mass Spec 161 Ph 585 (M++1) 162a Ph(4-OPh) ---- 162b Ph(4-F) 10 - 185- WO 01/07042 PCT/US00/19627 EXAMPLE 163 O HO NHO H 0 0 Example 163 was prepared by the following procedure. To 68mg of Example 148a in 6mL THF at RT was added 2mL PhMgBr (2M solution in THF). 5 After aging at RT for 20h, the reaction was quenched by the addition of saturated NH4Cl(aq), extracted with CH2Cl2 and dried with Na2SO4. Following PTLC on silica gel (1 x 1500ptm plate) using 1:9:90 NH40H:MeOH:CHCl3 as eluant, 18.9 mg pure intermediate compound having a side chain alcohol (Rf= 0.49, 1:9:90 NH4OH:MeOH:CHCl3) was obtained which was characterized by 1H NMR. The 10 side chain alcohol of the intermediate was then oxidized using Dess-Martin reagent as described in Example 138. Following PTLC on silica gel (1 x 500pm plate) using 1:9:90 NH4OH:MeOH:CHCl3 as eluant, 13mg pure Example 163 was obtained (Rf= 0.66, 1:9:90 NH4OH:MeOH:CHCl3) which was characterized by H NMR and MS [m/z: 684 (M +1)]. 15 - 186 - WO 01/07042 PCT/US00/19627 EXAMPLES 164A AND 164B Following the general procedure for Example 163, the following Examples 164a and 164b were prepared: R O HO R N NH H N-,O 0 5 o Table 13 Example R Group Mass Spec 163 Ph 684 (M+I1) 164a Ph(4-tBu) 757.8 (M++NH4) 164b CH2Ph 698 (M+1) EXAMPLE 165 0 N '"" M NWe 0 OMe 0 0 10 Example 165 was prepared by the following procedure. To 20mg apicidin in 321[tL DMF at RT was added 16jiL Mel followed by the addition of 3.8mg NaH (60% suspension in mineral oil). After 20h, water was added and the solution extracted with EtOAc and dried with Na2SO4. Following PTLC on silica gel (1 x 100O~m plate) using 1:3:96 NH4OH:MeOH:CHCl3 as eluant, 9.9mg pure - 187 o o 10 Example 165 was prepared by the following procedure. To 20mg apicidin in 321pL DMF at RT was added 16pL Mel followed by the addition of 3.8mg NaHl (60% suspension in mineral oil). After 20h, water was added and the solution extracted with EtOAc and dried with Na2SO4. Following PTLC on silica gel (1 x 1000pm plate) using 1:3:96 NH40H:MeOH:CHCl3 as eluant, 9.9mg pure -187 - WO 01/07042 PCT/US00/19627 Example 165 was obtained which was characterized by 1H NMR and MS [m/z: 666 (M +1)]. EXAMPLES 166A-166C 5 Examples 166a-166c were prepared similarly to the procedure described in Example 165. Apicidin was treated with benzyl bromide, in place of the methyl iodide in Example 165, to yield a mixture of mono-, di- and tri-benzylated derivatives. The three compounds, Examples 166a-166c, thus obtained were 10 characterized by 1H NMR and MS. The regiochemistry of the mono- and di benzylated derivatives was not established. 0 N NR Nj bom 0 0 N O OMe Table 14 Example R Groups Mass Spec 166a Mono-benzylated 714 (M'+1) 166b Di-benzylated 804 (M+1) 166c Tri-benzylated 894 (M +1) 15 - 188 - WO 01/07042 PCT/US00/19627 EXAMPLES 167A-167D

X

2 OMe X3 0 Examples 167a-167d were prepared by the following procedure. To 5 10mg apicidin in 2mL toluene was added 13mg Lawesson's reagent. The resulting solution was heated at 80oC for 25min and then cooled to RT. The entire solution was loaded directly onto a silica gel flash chromatography column and purified by gradient elution (100 % CHCl3, one column, followed by 1:3:96 NH4OH:MeOH:CHCl3 elution) to yield two fractions: monothiono Example 167a (Fraction One - Product A, 10 Rf = 0.83, 1:3:96 NH4OH:MeOH:CHCl3) and impure bis- and tris-thiono Examples 167b-167d (Fraction Two - Products B, C, and D, Rf= 0.68, 1:3:96 NH40H:MeOH:CHCl3). Fraction Two was further purified by preparative RP-HPLC using gradient elution (2:3 MeCN:H20 to 100% MeCN, 70min linear gradient). The products thus obtained were characterized by 1H NMR and MS. The following 15 retention times were obtained for the four products during the preparative RP-HPLC run: tR = 34.2min (product A - Example 167a); 39.9min (product B Example 167b); 45.6min (product C - Example 167c); 48.8min (product D - Example 167d); (2:3 MeCN:H20 to 100% MeCN, 70min linear gradient). 20 -189- WO 01/07042 PCT/US00/19627 Table 15 Example Product X1 X2 X3 Mass Spec 167a Product A S O O 640.3 (M++1) 167b Product B S S O 656.3 (M+1) 167c Product C S O S 656.3 (M +1) 167d Product D S S S 672.3 (M+1) EXAMPLE 168 0 N H NH H H H ' N H I S OMe O 0 0 5 Example 168 was prepared by adding 0.160mL BH3*THF (IM solution in THF) to 10mg apicidin in 2mL THF at 0 0 C. After 30min, the resulting solution was warmed to RT and aged for 12h. At this point, after 12.5h total, the solution was heated to 60 0 C for 30min, then cooled to RT. Then, lmL methanol was 10 added, followed by the addition of 0.15mL Me 2

NCH

2

CH

2 OH, and the solution was stirred for 2h. The stirred solution was poured into saturated brine, extracted with EtOAc, and dried with Na2SO4. The volatiles were removed under reduced pressure and the crude product was filtered through a 1.5inch pad of silica gel using 1:3:96

NH

4 0H/MeOH/CHCI 3 as eluant to remove baseline contaminants. The filtered 15 solution was concentrated under reduced pressure and pure product was obtained following preparative RP-HPLC using 1/3 MeCN/H 2 0 isocratic for 20 min, followed by a 60min linear gradient to 100% MeCN. The pure Example 168 thus obtained was characterized by 1H NMR and MS [m/z: 612.4 (M++1)]. HPLC: tR = 6.69min, 1/1 MeCN:H20, 1.5mL/min, Zorbax T M RX-8 column. TLC: Rf = 0.50, 1:3:96 20 NH4OH:MeOH:CHCI3. - 190 - WO 01/07042 PCT/US00/19627 EXAMPLES 169 AND 170 0 O< N NH N H N NO IMe 0O ~ 0 0 Ex. 169 5 0o N N H N NH H_, N N I S0 Me 0 Ex. 170 - 191 -

Claims (28)

1. A compound having a Formula I: R 5 R 6 N ,X-R, (CH 2 ) -N R3 N-R3 R 3 R 5 R 6 R

2 I or a pharmaceutically acceptable salt thereof, wherein Xis (1) -CH2-, 10 (2) -C(O)-, (3) -CH(ORa) - , (4) =CH-, or (5) not present; n is (1) one, or 15 (2) two; R1 is (1) R7, (2) C(O)R7, (3) CN, (4) CO 2 Rb, 20 (5) C(O)N(ORb)Rc, (6) C(O)NRcRd, (7) NHCO 2 Rb, (8) NHC(O)NRcRd, (9) (CO-C4alkyl)ORa, 25 (10) (CO-C4alkyl)OCO 2 Rb, (11) (CO-C4alkyl)OC(O)NRcRd, (12) C(O)NRcNRcRd, (13) C(O)NRcSO 2 Rb, - 192 - WO 01/07042 PCT/US00/19627 (14) OS(O)niR7, (19) NRbS(O)niR7, wherein ni is from 0 to 2, (20) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each 5 group independently is C1-C5alkyl, C2-C5alkenyl, C1 C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein 10 the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, (21) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 15 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or 20 nitrogen, in which the nitrogen optionally has an RC substituent, and wherein the benzene/heterocycle fused ring is attached at any site to X or to the tetrapeptide, or (22) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered 25 heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each 30 heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is - 193 - WO 01/07042 PCT/US00/19627 oxygen, sulfur, or nitrogen, and the nitrogen optionally has an RC substituent; R2 is (1) optionally substituted C2-C12alkyl, (2) optionally substituted C2-C12alkenyl, 5 (3) optionally substituted C2-C12alkynyl, or (4) (CH2)nii-O-(CH2)nmii wherein nii, mii = 0 to 7, wherein the optional substituents on the C2-C12alkyl, C2-C12alkenyl, and C2-C12alkynyl are 1 to 8 groups and each group independently is (a) CO 2 Ra, 10 (b) C(O)Rb, (c) C(O)N(ORb)Rc, (d) C(O)NRCRd, (e) C(O)NRCNRCRd, (f) C(O)NRcSO2R7, 15 (g) C3-C8cycloalkyl, (h) C2-C5alkenyl, (i) cyano, (j) =NORa, (k) =NNRbRc, 20 (1) =NNRbS(O)niR7, (m) N(ORb)C(O)NRbRc, (n) N(ORb)C(O)R 7 , (o) NHC(O)N(ORb)Rc, (p) NRcCO 2 Rb, 25 (q) NRcC(O)NRCRd, (r) NRcC(S)NRCRd, (s) NRcC(O)R 7 , (t) NRbS(O)niR7, (u) NRcCH 2 CO 2 Ra, 30 (v) NRcC(S)R7, (x) NRcC(O)CH2OH, (y) NRcC(O)CH2SH, (z) NRcCH 2 CO 2 Ra, - 194 - WO 01/07042 PCT/US00/19627 (aa) NRCH2CH(OH)R7, (bb) NRcP(O)(ORa)R 7 , (cc) NY 1 y 2 , wherein Y 1 and Y 2 are independently H or C1-Co10alkyl, 5 (dd) NO2, (ee) N(ORb)C(O)Rb, (ff) Cl1-C 10alkanoylamino, (gg) OR a , (hh) OS(O)niR7, 10 (ii) oxo, (jj) OCO 2 Rb, (kk) OC(O)NRCRd, (11) P(O)(ORa)2, (mm) P(O)(ORa)R 7 , 15 (nn) SC(O)R7, (oo) S(O)niR7, (pp) SR7, (qq) S(O)niNRcRd, (rr) NRcCH 2 CO 2 Ra, 20 (ss) diazo, (tt) C1-C5 perfluoroalkyl, (uu) B(O)(ORa)ORa, (xx) halogen, (yy) aryl(C0-C5alkyl), wherein the aryl is optionally 25 substituted with 1 to 3 groups, wherein each group independently is R f , or (xx) a 3- to 8-membered heterocycle containing from 1 to 4 heteroatoms, each heteroatom independently is oxygen, sulfur or nitrogen, wherein the heterocycle is optionally substituted by 1 to 3 30 groups, wherein each group independently is R f , and the heterocycle may be saturated or partly unsaturated; R3 each independently is (1) hydrogen, (2) halogen, - 195 - WO 01/07042 PCT/US00/19627 (3) ORa, (4) CI-C4alkyl, or (5) C1-C4aryl; R5 is (1) isopropyl, or 5 (2) sec-butyl; R6 each independently is (1) O, (2) S, or (3) H; 10 R7 is (1) hydrogen, (2) optionally substituted C2-C 10 alkyl, (3) optionally substituted C2-C10oalkenyl, (4) optionally substituted C2-Co10alkynyl, (5) optionally substituted C3-C8cycloalkyl, 15 (6) optionally substituted C5-C8cycloalkenyl, (7) optionally substituted aryl, wherein the optional substituents on the C2-Co10alkyl, C2-Co10alkenyl, C2-Co10alkynyl, C3-C8cycloalkyl, C5-C8cycloalkenyl and aryl are 1 to 4 groups, and each group independently is 20 (a) C1-C5alkyl, (b) X 1 -C1-Co10alkyl, wherein X 1 is O or S(O)ni, (c) C3-C8cycloalkyl, (d) hydroxy, (e) halogen, 25 (f) cyano, (g) carboxy, (h) NY 1 Y 2 , wherein Y 1 and Y 2 are independently H or C1-Co10alkyl, (i) nitro, 30 (j) C1-Co10alkanoylamino, (k) aroyl amino wherein the aroyl is optionally substituted with 1 to 3 groups wherein each group independently is R f l 1 , wherein R f l 1 is defined by any of the definitions below for R f except for (14), (26), (27), and (32), -196- WO 01/07042 PCT/US00/19627 (1) oxo, (min) aryl C0-C5alkyl wherein the aryl is optionally substituted with 1 to 3 groups, wherein each group independently is R f l , 5 (q) C1-C5perfluoroalkyl, (r) N(ORb)C(O)R 7 ' , wherein R7' is any of the above definitions of R7 from (1) to (7)(n), and below of R7 from (8) to (12), or (s) NRcC(O)R7 ' , 10 (8) a 5- to 10-membered heterocycle containing from 1 to 4 heteroatoms, each heteroatom independently is oxygen, sulfur or nitrogen and the heterocycle is optionally substituted by 1 to 3 groups, each group independently is R f l 1 , and the heterocycle may be saturated or partly unsaturated, 15 (9) a benzene ring fused to a 5- to 10-membered heterocyclic ring containing from 1 to 4 heteroatoms, each heteroatom independently is oxygen, sulfur or nitrogen and the heterocycle is optionally substituted by 1 to 3 groups, each group independently is R f l 1 , and the heterocycle may be saturated or partly unsaturated, 20 (10) a 5- to 10-membered heterocyclic ring containing from 1 to 4 heteroatoms fused to a second 5- to 10-membered heterocyclic ring containing from 1 to 4 heteroatoms, each heteroatom in either heterocyclic ring independently is oxygen, sulfur or nitrogen and the second heterocyclic ring is optionally substituted by 1 to 3 groups, each 25 group independently is R f l 1 , and each heterocycle independently may be saturated or partly unsaturated, (11) a benzene ring fused to a C3-C8cycloalkyl ring, wherein the cycloalkyl is optionally substituted by 1 to 3 groups each independently being R f l 1 , and the cycloalkyl ring may be saturated or partly 30 unsaturated, or (12) a 5- to 10-membered heterocyclic ring containing from 1 to 4 heteroatoms, each heteroatom independently is oxygen, sulfur or nitrogen, the heterocyclic ring is fused to a C3-C8cycloalkyl ring, wherein the cycloalkyl ring is optionally substituted by 1 to 3 groups - 197 - WO 01/07042 PCT/US00/19627 each independently being R f l 1 , and the cycloalkyl ring may be saturated or partly unsaturated, Ra is (1) hydrogen, (2) optionally substituted Cl1-C 10alkyl, 5 (3) optionally substituted C3-Co10alkenyl, (4) optionally substituted C3-C 10oalkynyl, (5) optionally substituted Cl1-C10alkanoyl, (6) optionally substituted C3-C10oalkenoyl, (7) optionally substituted C3-C 10oalkynoyl, 10 (8) optionally substituted aroyl, (9) optionally substituted aryl, (10) optionally substituted C3-C7cycloalkanoyl, (11) optionally substituted C5-C7cycloalkenoyl, (12) optionally substituted C1-C10alkylsulfonyl, 15 (13) optionally substituted C3-C8cycloalkyl, (14) optionally substituted C5-C8cycloalkenyl, wherein the optional substituents on the C1-Co10alkyl, C3-Co10alkenyl, C3-C 10alkynyl, C1-C 10alkanoyl, C3-C 10oalkenoyl, C3-C 10alkynoyl, aroyl, aryl, C3-C7cycloalkanoyl, C5-C8cycloalkenyl, C l 20 Co10alkylsulfonyl, C3-C8cycloalkyl and C5-C8cycloalkenyl are from 1 to 10 groups, wherein each group independently is hydroxy, Cl C6alkoxy, C3-C7cycloalkyl, aryl C1-C3alkoxy, NRXRX, CO 2 Rb, CONRcRd, or halogen, (15) C1-C5perfluoroalkyl, 25 (16) arylsulfonyl optionally substituted with 1 to 3 groups, wherein each group independently is C1-C5alkyl, C1 C5perfluoroalkyl, nitro, halogen or cyano, (17) a 5- or 6-membered heterocycle containing 1 to 4 heteroatoms, wherein each heteroatom is oxygen, sulfur or nitrogen, 30 wherein the heterocycle is optionally substituted by 1 to 4 groups, wherein each group independently is C1-C5alkyl, C 1-C5alkenyl, C I C5perfluoroalkyl, amino, C(O)NRcRd, cyano, CO 2 Rb or halogen, and wherein the heterocycle may be saturated or partly unsaturated, or (18) OP(O)(ORb) 2 ; - 198- WO 01/07042 PCT/US00/19627 Rb is (1) H, (2) optionally substituted aryl, (3) optionally substituted C1-C10alkyl, (4) optionally substituted C3-C10alkenyl, 5 (5) optionally substituted C3-Co10alkynyl, (6) optionally substituted C3-C15cycloalkyl, (7) optionally substituted C5-C10cycloalkenyl, or (8) optionally substituted 5- to 10-membered heterocycle containing 1 to 4 heteroatoms, wherein each heteroatom independently 10 is oxygen, sulfur, or nitrogen, wherein the optional substituents on the aryl, C1-C10alkyl, C3 C o10alkenyl, C3-C o10alkynyl, C3-C 15cycloalkyl, C5-C o10cycloalkenyl, or 5- to 10-membered heterocycle are from 1 to 10 groups, wherein each group independently is 15 (a) hydroxy, (b) C1-C6alkyl, (c) oxo, (d) SO 2 NRxR x , (e) aryl C1-C6alkoxy, 20 (f) hydroxy C1-C6alkyl, (g) C1-C12alkoxy, (h) hydroxy C1-C6alkoxy, (I) amino C1-C6alkoxy, (j) cyano, 25 (k) mercapto, (1) (C 1-C6alkyl)-S (O)ni-(CO-C6alkyl), (m) C3-C7cycloalkyl optionally substituted with 1 to 4 groups, wherein each group independently is Re, (n) C5-C7cycloalkenyl, 30 (o) halogen, (p) C1-C5alkanoyloxy, (q) C(O)NRxRx, (r) CO 2 Ri, (s) formyl, - 199 - WO 01/07042 PCT/US00/19627 (t) -NRXRx, (u) 5 to 9-membered heterocycle, which may be saturated or partially unsaturated, containing from 1 to 4 heteroatoms, wherein each heteroatom independently is oxygen, sulfur or nitrogen, 5 and the heterocycle is optionally substituted with 1 to 5 groups, wherein each group independently is Re, (vi) optionally substituted aryl, wherein the optional substituents are 1,2-methylenedioxy or 1 to 5 groups, wherein each group independently is Re, 10 (x) optionally substituted aryl C1-C3alkoxy, wherein the optional substituents are 1,2-methylenedioxy or 1 to 5 groups, wherein each group independently is Re, or (y) C1-C5perfluoroalkyl; R c and Rd are independently selected from Rb; or Rc and Rd together with the N to 15 which they are attached form a 3- to 10-membered ring containing 0 to 2 additional heteroatoms, each additional heteroatom independently being oxygen, nitrogen, or (O)ni substituted sulfur, wherein the ring is optionally substituted with 1 to 3 groups, wherein each group independently is Rg, hydroxy, thioxo, or oxo; 20 Re is (1) halogen, (2) CI-C7alkyl, (3) C1-C3perfluoroalkyl, (4) -S(O)mR i , (5) cyano, 25 (6) nitro, (7) RiO(CH2)v -, (8) RiCO2(CH2)v -, (9) RiOCO(CH2)v, (10) optionally substituted aryl wherein the optional substituents 30 are from 1 to 3 groups, wherein each group independently is halogen, C 1-C6alkyl, C 1-C6alkoxy, or hydroxy, (11) SO 2 NRXRx, (12) CO 2 Rx, or (13) NRXRX; - 200 - WO 01/07042 PCT/US00/19627 R f is (1) C1-C4alkyl, (2) X 1 -C1-C4alkyl, wherein XI is O or S(O)mi, (3) C2-C4alkenyl, (4) C2-C4 alkynyl, 5 (5) C1-C3perfluoroalkyl, (6) NY 3 y 4 , wherein Y 3 and Y 4 are each independently hydrogen, C1-C5alkyl, or SO 2 Rb, (7) hydroxy, (8) halogen, 10 (9) C1-C5alkanoyl amino, (18) (CO-C4alkyl)CO2Ra, (19) (CO-C4alkyl)C(O)NRbRc, (20) (CO-C4alkyl)NY 5 Y 6 wherein Y 5 and Y 6 together with the N to which they are attached form a 3- to 7-membered ring 15 containing 0 to 2 additional heteroatoms, wherein the additional heteroatoms independently are oxygen, nitrogen, or (O)mi substituted sulfur, wherein the ring is optionally substituted with 1 to 3 groups, wherein each group independently is Re or oxo, 20 (13) (CO-C4alkyl)NO2, (14) (CO-C4alkyl)C(O)R7, (15) (CO-C4alkyl)CN, (16) oxo, (17) (CO-C4alkyl)C(0)N(ORb)Rc, 25 (18) (CO-C4alkyl)C(0)NRcRd, (19) (CO-C4alkyl)NHC(O)ORb, (20) (CO-C4alkyl)NHC(O)NRcRd, (21) (CO-C 4 alkyl)ORa, (22) (CO-C4alkyl)OCO2Rb, 30 (23) (CO-C4alkyl)OC(0)NRcRd, (24) (CO-C 4 alkyl)C(0)NRcNRcRd, (25) (CO-C4alkyl)C(0)NRcSO2Rb, (26) (CO-C4alkyl)OS(O)niR7, (27) (CO-C4alkyl)NRbS(0)niR7, - 201 - WO 01/07042 PCT/US00/19627 (28) CO-C4alkyl halogen, (29) (CO-C4alkyl) SR a , (30) P(O)(ORa)2, (33) CO-C4alkyl azide, 5 (34) CO-C4aryl substituted with from 1 to 4 groups, wherein each group independently is S(O)2R7, or (33) CO-C4aryl where the aryl group is optionally substituted from 1 to 4 groups, wherein each group independently is CO 2 Rb, C(O)NRCRd, NO2, halogen, OC(O)Ra, OR a or C 1-C4alkyl; 10 Rg and Rh together with the N to which they are attached form a 3- to 7-membered ring containing 0 to 2 additional heteroatoms, wherein each additional heteroatom independently is oxygen, nitrogen, or (O)mi substituted sulfur, and the ring is optionally substituted with 1 to 3 groups, wherein each group independently is Re or oxo; or 15 Rg and Rh are each independently (1) hydrogen, (2) C1-C6alkyl optionally substituted with hydroxy, amino, or CO 2 Ri, (3) aryl optionally substituted with halogen, 1,2 20 methylenedioxy, C1-C7alkoxy, C1-C7alkyl, or C1-C3perfluoroalkyl, (4) aryl CI-C6alkyl, wherein the aryl is optionally substituted with C1-C3perfluoroalkyl or 1,2-methylenedioxy, (5) C1-C5alkoxycarbonyl, (6) C1-C5alkanoyl, 25 (19) C1-C5alkanoyl C1-C6alkyl, (20) arylCl1-C5 alkoxycarbonyl, (21) aminocarbonyl, (22) (C1-C5monoalkyl)aminocarbonyl, (23) (CI1-C5dialkyl)aminocarbonyl, or 30 (24) CO 2 Rb; R i is (1) hydrogen, (2) C 1-C3perfluoroalkyl, (3) C1-C6alkyl, or - 202 - WO 01/07042 PCT/US00/19627 (4) optionally substituted aryl CO-C6alkyl, wherein the aryl optional substituents are from 1 to 3 groups, wherein each group independently is halogen, C1-C6alkyl, C1-C6alkoxy, or hydroxy; Rx is a C 1-C4alkyl; 5 m is 0 to 2; mi is 0 to 2; ni is 0 to 2; mii is 0 to 7; nii is 0 to 7; 10 v is 0 to 3; and excluding apicidin, N-desmethoxy apicidin and compounds represented by chemical Formula IIA and chemical Formula fIB: CH 3 CH 2 CH 3 O 0 N OCH3 H 3 N NH 0 15 IIHA - 203 - WO 01/07042 PCT/US00/19627 CH 3 CH 2 CH 3 - O N N NH H 00 OO 0 IEB 5 2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein: X is (1) -CH2-, (2) -C(O)-, (3) -CH(ORa) - , 10 (4) =CH-, or (5) not present; and R1 is (1) R7, (2) C(0)R7, (3) CN, 15 (4) CO 2 Rb, (5) C(0)N(ORb)Rc, (6) C(0)NRcRd, (7) NHCO 2 Rb, (8) NHC(0)NRcRd, 20 (9) (CO-C4alkyl)ORa, (10) (CO-C4alkyl)OCO 2 Rb, (11) (CO-C 4 alkyl)OC(0)NRcRd, (12) C(O)NRcNRcRd, (13) C(0)NRcSO 2 Rb, - 204 - WO 01/07042 PCT/US00/19627 (21) OS(O)niR7, (22) NRbS(O)niR7, wherein ni is from 0 to 2, (23) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each 5 group independently is C -C5alkyl, C2-C5alkenyl, C I C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein 10 the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, (24) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 15 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or 20 nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring is attached at any site to X or to the tetrapeptide, or (25) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered 25 heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each 30 heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent. - 205 - WO 01/07042 PCT/US00/19627

3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein: X is (1) -CH2-, (2) -C(O)-, 5 (3) -CH(ORa)-, (4) =CH-, or (5) not present; R1 is (1) R7, (2) C(O)R7, 10 (3) CN, (4) CO 2 Rb, (5) C(O)N(ORb)Rc, (6) C(O)NRcRd, (7) NHCO 2 Rb, 15 (8) NHC(O)NRcRd, (9) (CO-C4alkyl)ORa, (10) (CO-C 4 alkyl)OCO2Rb, (11) (CO-C 4 alkyl)OC(O)NRcRd, (12) C(O)NRcNRcRd, 20 (25) C(O)NRcSO 2 Rb, (26) OS(O)niR7, (27) NRbS(O)niR7, wherein ni is from 0 to 2, (28) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each 25 group independently is C 1-C5alkyl, C2-C5alkenyl, C I C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein 30 the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, (29) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by - 206 - WO 01/07042 PCT/US00/19627 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein 5 the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring is attached at any site to X or to the tetrapeptide, or (30) a 4- to 8-membered heterocyclic ring with from 1 to 4 10 heteroatoms fused to a second 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, 15 C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent; and 20 R2 is (1) optionally substituted C2-C12alkyl, (2) optionally substituted C2-C12alkenyl, (3) optionally substituted C2-C12alkynyl, or (4) (CH2)nii-O-(CH2)mii wherein nii, mii = 0 to 7, wherein the optional substituents on the C2-Cl12alkyl, C2-Cl12alkenyl, 25 and C2-C12alkynyl are 1 to 8 groups and each group independently is (a) CO 2 Ra, (b) C(O)Rb, (c) C(O)N(ORb)Rc, (d) C(O)NRcRd, 30 (e) C(O)NRcNRcRd, (f) C(O)NRcSO2R 7 , (g) C3-C8cycloalkyl, (h) C2-C5alkenyl, (i) cyano, - 207 - WO 01/07042 PCT/US00/19627 (j) =NORa, (k) =NNRbRc, (1) =NNRbS(O)niR7, (m) N(ORb)C(O)NRbRc, 5 (n) N(ORb)C(O)R 7 , (o) NHC(O)N(ORb)Rc, (p) NRcCO 2 Rb, (q) NRcC(O)NRCRd, (r) NRcC(S)NRCRd, 10 (s) NRcC(O)R 7 , (t) NRbS(O)niR7, (u) NRcCH 2 CO 2 Ra, (v) NRcC(S)R7, (x) NRcC(O)CH2OH, 15 (y) NRcC(O)CH2SH, (z) NRcCH 2 CO 2 Ra, (aa) NRCCH2CH(OH)R7, (bb) NRcP(O)(ORa)R 7 , (cc) NY 1 Y 2 , wherein Y1 and Y 2 are independently 20 H or C1-C10alkyl, (dd) NO2, (ee) N(ORb)C(O)Rb, (ff) Cl1-C 10alkanoylamino, (gg) OR a , 25 (hh) OS(O)niR7, (ii) oxo, (jj) OCO 2 Rb, (kk) OC(O)NRCRd, (11) P(O)(ORa)2, 30 (mm) P(O)(ORa)R 7 , (nn) SC(O)R7, (oo00) S(O)niR7, (pp) SR7, (qq) S(O)niNRcRd, - 208 - WO 01/07042 PCT/US00/19627 (rr) NRcCH2CO 2 Ra, (ss) diazo, (tt) C1-C5 perfluoroalkyl, (uu) B(O)(ORa)ORa, 5 (xx) halogen, (yy) aryl(CO-C5alkyl), wherein the aryl is optionally substituted with 1 to 3 groups, wherein each group independently is R f , or (xxi) a 3- to 8-membered heterocycle containing from 10 1 to 4 heteroatoms, each heteroatom independently is oxygen, sulfur or nitrogen, wherein the heterocycle is optionally substituted by 1 to 3 groups, wherein each group independently is R f , and the heterocycle may be 15 saturated or partly unsaturated.

4. The compound according to claim 3, or a pharmaceutically acceptable salt thereof, wherein n is 1 or 2. 20

5. The compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein: X is (1) -CH2-, (2) -C(O)-, or (3) not present; and 25 R1 is (1) R7, (2) C(O)R7, (3) CN, (4) CO 2 Rb, (5) C(O)N(ORb)Rc, 30 (6) C(O)NRcRd, (7) NHCO 2 Rb, (8) NHC(O)NRcRd, (9) (CO-C4alkyl)ORa, (10) (CO-C4alkyl)OCO2Rb, - 209 - WO 01/07042 PCT/US00/19627 (11) (CO-C 4 alkyl)OC(O)NRcRd, (12) C(O)NRCNRCRd, (19) C(O)NRcSO 2 Rb, (20) OS(O)niR7, 5 (21) NRbS(O)niR7, wherein ni is from 0 to 2, (22) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2-C5alkenyl, C1 C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa 10 (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc 15 substituent, (23) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, 20 C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring 25 is attached at any site to X or to the tetrapeptide, or (24) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 30 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, CI-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is -210- WO 01/07042 PCT/US00/19627 oxygen, sulfur, or nitrogen, and the nitrogen optionally has an RC substituent.

6. The compound according to claim 5, or a pharmaceutically 5 acceptable salt thereof, wherein n is 1 or 2.

7. The compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein: X is (1) -CH2-, 10 (2) -C(O)-, or (3) not present; and R1 is (1) R7, (2) C(O)R7, (15) CO 2 Rb, 15 (16) C(O)N(ORb)Rc, (17) C(O)NRcRd, (18) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each group independently is CI1-C5alkyl, C2-C5alkenyl, Ci 20 C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or 25 nitrogen, in which the nitrogen optionally has an Rc substituent, (19) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups each independently is C1-C5alkyl, C2 30 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc -211 - WO 01/07042 PCT/US00/19627 substituent, and wherein the benzene/heterocycle fused ring is attached at any site to X or to the tetrapeptide, or (20) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered 5 heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each 10 heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatomindependently is oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent. 15

8. The compound according to claim 7, or a pharmaceutically acceptable salt thereof, wherein n is 1 or 2.

9. The compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein: 20 X is (1) -CH2-, (2) -C(O)-, or (3) not present; R1 is (1) R7, (9) C(O)R7, 25 (10) CO 2 Rb, (11) C(O)N(ORb)Rc, (12) C(O)NRcRd, (13) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each 30 group independently is C 1-C5alkyl, C2-C5alkenyl, C I C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein -212- WO 01/07042 PCT/US00/19627 the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, (14) a benzene ring fused to a 4- to 8-membered heterocyclic 5 ring with from 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein 10 the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring is attached at any site to X or to the tetrapeptide, or (15) a 4- to 8-membered heterocyclic ring with from I to 4 15 heteroatoms fused to a second 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, 20 C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent;and 25 R2 is (1) optionally substituted C2-C12alkyl, (2) optionally substituted C2-C12alkenyl, (3) optionally substituted C2-Cl12alkynyl, or (4) (CH2)nii-O-(CH2)mii wherein nii, mii = 0 to 7, wherein the optional substituents on the C2-C12alkyl, C2-C12alkenyl, 30 and C2-C12alkynyl are 1 to 5 groups and each group independently is (a) CO 2 Ra, (b) C(O)Rb, (c) C(O)N(ORb)Rc, (d) C(O)NRcRd, -213 - WO 01/07042 PCT/US00/19627 (e) C(O)NRCNRCRd, (f) C(O)NRcSO2R7, (g) C3-C8cycloalkyl, (h) C2-C5alkenyl, 5(i) cyano, (j) =NORa, (k) =NNRbRc, (1) =NNRbS(O)niR 7 , (m) N(ORb)C(O)NRbRc, 10 (n) N(ORb)C(O)R 7 , (o) NHC(O)N(ORb)Rc, (p) NRcCO 2 Rb, (q) NRcC(O)NRCRd, (r) NRcC(S)NRCRd, 15 (s) NRcC(O)R 7 , (t) NRbS(O)niR7, (u) NRcCH 2 CO 2 Ra, (v) NRcC(S)R 7 , (x) NRcC(O)CH2OH, 20 (y) NRcC(O)CH2SH, (z) NRCCH 2 CO 2 Ra, (aa) NRCH2CH(OH)R7, (bb) NRcP(O)(ORa)R 7 , (cc) NY 1 Y 2 , wherein Y1 and Y 2 are independently 25 H or methyl, (dd) NO2, (ee) N(ORb)C(O)Rb, (ff) Cl1-C3alkanoylamino, (gg) OR a , 30 (hh) OS(O)niR7, (ii) oxo, (jj) OCO 2 Rb, (kk) OC(O)NRCRd, (11) P(O)(ORa)2, -214- WO 01/07042 PCT/US00/19627 (mm) P(O)(ORa)R 7 , (nn) SC(O)R7, (oo) S(O)niR7, (pp) SR7, 5 (qq) S(O)niNRCRd, (rr) NRcCH 2 CO 2 Ra, (ss) diazo, (tt) C1-C5 perfluoroalkyl, (uu) B(O)(ORa)ORa, 10 (zz) halogen, (aaa) aryl(CO-C5alkyl), wherein the aryl is optionally substituted with 1 to 3 groups, wherein each group independently is R f , or (xxii) a 3- to 6-membered heterocycle containing from 15 1 to 4 heteroatoms, each heteroatom independently is oxygen, sulfur or nitrogen, wherein the heterocycle is optionally substituted by 1 to 3 groups, wherein each group independently is R f , and the heterocycle may be 20 saturated or partly unsaturated.

10. The compound according to claim 9, or a pharmaceutically acceptable salt thereof, wherein n is 1 or 2. 25

11. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein: R3 each independently is (1) hydrogen, or (2) halogen, 30 (3) OR a , (4) C1-C4alkyl, or (5) C1-C4aryl; and Ra is (1) hydrogen, (2) optionally substituted C1-C6alkyl, -215- WO 01/07042 PCT/US00/19627 (8) optionally substituted C3-C6alkenyl, (9) optionally substituted C2-C4alkanoyl, (5) optionally substituted C3-C4alkenoyl, (6) optionally substituted aroyl, 5 (7) optionally substituted aryl, (8) optionally substituted C5-C6cycloalkanoyl, (9) optionally substituted C1-C4alkylsulfonyl, (10) optionally substituted C5-C6cycloalkyl, (15) optionally substituted C5-C6cycloalkenyl, 10 wherein the optional substituents on the C1-C6alkyl, C3-C6alkenyl, C2-C4alkanoyl, C3-C4alkenoyl, aroyl, aryl, C5-C6cycloalkanoyl, C C4alkylsulfonyl, C5-C6cycloalkyl and C5-C6cycloalkenyl are from 1 to 10 groups, wherein each group independently is hydroxy, methoxy, aryl methoxy, NRxRX, CO 2 Rb, CONRcRd, or halogen, 15 (16) CF3, (17) arylsulfonyl optionally substituted with 1 to 3 groups, wherein each group independently is methyl, CF3, nitro, halogen or cyano, or (18) a 5- or 6-membered heterocycle containing 1 to 3 20 heteroatoms, wherein each heteroatom is oxygen, sulfur or nitrogen, wherein the heterocycle is optionally substituted by 1 to 3 groups, wherein each group independently is methyl, CF3, NMe2, C(O)NRcRd, cyano, CO 2 Rb or halogen, and wherein the heterocycle may be saturated or 25 partly unsaturated.

12. The compound according to claim 11, or a pharmaceutically acceptable salt thereof, wherein: R3 each independently is 30 (1) hydrogen, (2) halogen, (3) OR a, (4) C1-C4alkyl, or (5) C1-C4aryl; ); -216- WO 01/07042 PCT/US00/19627 Ra is (1) hydrogen, (2) optionally substituted CI1-C6alkyl, (10) optionally substituted C3-C6alkenyl, (11) optionally substituted C2-C4alkanoyl, 5 (5) optionally substituted C3-C4alkenoyl, (6) optionally substituted aroyl, (7) optionally substituted aryl, (8) optionally substituted C5-C6cycloalkanoyl, (9) optionally substituted C1-C4alkylsulfonyl, 10 (10) optionally substituted C5-C6cycloalkyl, (15) optionally substituted C5-C6cycloalkenyl, wherein the optional substituents on the C1-C6alkyl, C3-C6alkenyl, C2-C4alkanoyl, C3-C4alkenoyl, aroyl, aryl, C5-C6cycloalkanoyl, C 1 C4alkylsulfonyl, C5-C6cycloalkyl and C5-C6cycloalkenyl are from 1 15 to 10 groups, wherein each group independently is hydroxy, methoxy, aryl methoxy, NRXRX, CO 2 Rb, CONRCRd, or halogen, (16) CF3, (17) arylsulfonyl optionally substituted with 1 to 3 groups, wherein each group independently is methyl, CF3, nitro, 20 halogen or cyano, or (18) a 5- or 6-membered heterocycle containing 1 to 3 heteroatoms, wherein each heteroatom is oxygen, sulfur or nitrogen, wherein the heterocycle is optionally substituted by 1 to 3 groups, wherein each group independently is 25 methyl, CF3, NMe2, C(O)NRcRd, cyano, CO 2 Rb or halogen, and wherein the heterocycle may be saturated or partly unsaturated. Xis (1) -CH2-, (2) -C(O)-, 30 (5) =CH-, or (6) not present; and R1 is (1) R7, (2) C(O)R7, (3) CN, - 217 - WO 01/07042 PCT/US00/19627 (4) CO 2 Rb, (5) C(O)N(ORb)Rc, (6) C(O)NRCRd, (7) NHCO 2 Rb, 5 (8) NHC(O)NRCRd, (9) (CO-C4alkyl)ORa, (10) (CO-C4alkyl)OCO 2 Rb, (11) (CO-C4alkyl)OC(O)NRCRd, (12) C(O)NRCNRCRd, 10 (19) C(O)NRcSO 2 Rb, (20) OS(O)niR7, (21) NRbS(O)niR7, wherein ni is from 0 to 2, (22) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each 15 group independently is C1-C5alkyl, C2-C5alkenyl, C1 C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein 20 the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, (23) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 25 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or 30 nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring is attached at any site to X or to the tetrapeptide, or (24) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered -218- WO 01/07042 PCT/US00/19627 heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 to 4 groups, each group independently is CI-C5alkyl, C2 C5alkenyl, CI-C5perfluoroalkyl, amino, oxo, thiono, 5 C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent. 10

13. The compound according to 12, or a pharmaceutically acceptable salt thereof, wherein n is 1 or 2.

14. The compound according to claim 11, or a pharmaceutically 15 acceptable salt thereof, wherein: R3 each independently is (1) hydrogen, (2) halogen, (3) ORa, 20 (4) C1-C4alkyl, or (5) C1-C4aryl;); Ra is (1) hydrogen, (5) optionally substituted C1-C6alkyl, (6) optionally substituted C3-C6alkenyl, 25 (7) optionally substituted C2-C4alkanoyl, (5) optionally substituted C3-C4alkenoyl, (6) optionally substituted aroyl, (7) optionally substituted aryl, (8) optionally substituted C5-C6cycloalkanoyl, 30 (9) optionally substituted C1-C4alkylsulfonyl, (10) optionally substituted C5-C6cycloalkyl, (15) optionally substituted C5-C6cycloalkenyl, wherein the optional substituents on the C1-C6alkyl, C3-C6alkenyl, C2-C4alkanoyl, C3-C4alkenoyl, aroyl, aryl, C5-C6cycloalkanoyl, C 35 C4alkylsulfonyl, CS-C6cycloalkyl and C5-C6cycloalkenyl are from 1 -219- WO 01/07042 PCT/US00/19627 to 10 groups, wherein each group independently is hydroxy, methoxy, aryl methoxy, NRXRX, CO 2 Rb, CONRcRd, or halogen, (16) CF3, (17) arylsulfonyl optionally substituted with 1 to 3 groups, 5 wherein each group independently is methyl, CF3, nitro, halogen or cyano, or (18) a 5- or 6-membered heterocycle containing 1 to 3 heteroatoms, wherein each heteroatom is oxygen, sulfur or nitrogen, wherein the heterocycle is optionally substituted 10 by 1 to 3 groups, wherein each group independently is methyl, CF3, NMe2, C(O)NRcRd, cyano, CO 2 Rb or halogen, and wherein the heterocycle may be saturated or partly unsaturated; X is (1) -CH2-, 15 (5) -C(O)-, (6) =CH-, or (7) not present; and R1 is (1) R7, (2) C(O)R7, 20 (21) CO 2 Rb, (22) C(O)N(ORb)Rc, (23) C(O)NRcRd, (24) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each 25 group independently is C1-C5alkyl, C2-C5alkenyl, C1 C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein 30 the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, (25) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by - 220 - WO 01/07042 PCT/US00/19627 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein 5 the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring is attached at any site to X or to the tetrapeptide, or (26) a 4- to 8-membered heterocyclic ring with from 1 to 4 10 heteroatoms fused to a second 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, C 1-C5perfluoroalkyl, amino, oxo, thiono, 15 C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent. 20

15. The compound according to claim 14, or a pharmaceutically acceptable salt thereof, wherein n is 1 or 2.

16. The compound according to claim 1, or a pharmaceutically 25 acceptable salt thereof, wherein: R6 each independently is (1) O, (2) S, or (3) H; 30 X is (1) -CH2-, (2) -C(o)-, (5) =CH-, or (6) not present; and R1 is (1) R7, - 221 - WO 01/07042 PCT/US00/19627 (2) C(O)R7, (3) CN, (4) CO 2 Rb, (5) C(O)N(ORb)Rc, 5 (6) C(O)NRCRd, (7) NHCO 2 Rb, (8) NHC(O)NRCRd, (9) (CO-C4alkyl)ORa, (10) (CO-C4alkyl)OCO2Rb, 10 (11) (CO-C 4 alkyl)OC(O)NRcRd, (12) C(O)NRCNRCRd, (13) C(O)NRcSO 2 Rb, (19) OS(O)niR7, (20) NRbS(O)niR7, wherein ni is from 0 to 2, 15 (21) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2-C5alkenyl, C1 C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO 20 C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an RC substituent, 25 (22) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be 30 saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring is attached at any site to X or to the tetrapeptide, or - 222 - WO 01/07042 PCT/US00/19627 (23) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 5 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO2Rb or halogen, wherein each heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is 10 oxygen, sulfur, or nitrogen, and the nitrogen optionally has an Rc substituent.

17. The compound according to claim 16, or a pharmaceutically acceptable salt thereof, wherein n is 1 or 2. 15

18. The compound according to claim 16, or a pharmaceutically acceptable salt thereof wherein: R3 each independently is (1) hydrogen, 20 (2) halogen, (3) ORa, (4) C1-C4alkyl, or (5) C1-C4aryl; R6 each independently is 25 (1) O, (2) S,or (3) H; X is (1) -CH2-, (2) -C(O)-, 30 (3) =CH-, or (5) not present; and R1 is (1) R7, (2) C(0)R7, (3) CN, - 223 - WO 01/07042 PCT/US00/19627 (4) CO 2 Rb, (5) C(O)N(ORb)Rc, (6) C(O)NRCRd, (7) NHCO 2 Rb, 5 (8) NHC(O)NRCRd, (9) (CO-C4alkyl)ORa, (10) (CO-C4alkyl)OCO2Rb, (11) (CO-C4alkyl)OC(O)NRcRd, (12) C(O)NRCNRCRd, 10 (19) C(O)NRcSO 2 Rb, (20) OS(O)niR7, (21) NRbS(O)niR7, wherein ni is from 0 to 2, (22) a 3- to 8-membered heterocycle containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each 15 group independently is C1-C5alkyl, C2-C5alkenyl, C1 C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein 20 the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, (23) a benzene ring fused to a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 25 1 to 4 groups each independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or 30 nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring is attached at any site to X or to the tetrapeptide, or (24) a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered - 224 - WO 01/07042 PCT/US00/19627 heterocyclic ring with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2 C5alkenyl, C1-C5perfluoroalkyl, amino, oxo, thiono, 5 C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is oxygen, sulfur, or nitrogen, and the nitrogen optionally has an RC substituent. 10

19. The compound according to claim 18, or a pharmaceutically acceptable salt thereof, wherein n is 1 or 2.

20. The compound according to claim 1, or a pharmaceutically 15 acceptable salt thereof, wherein X is preferably -CH2-.

21. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X is preferably -C(O)-. 20

22. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X is preferably not present.

23. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R1 is preferably a 3- to 8-membered heterocycle 25 containing 1 to 4 heteroatoms, optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2-C5alkenyl, C1-C5perfluoroalkyl, NRcRd, oxo, thiono, ORa, S(O)niRa (where ni = 0, 1 or 2), C(O)Ra, C(O)NRcRd, cyano, (CO C6alkyl)aryl, CO 2 Rb, or halogen, and each group may be saturated, partly unsaturated or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, 30 or nitrogen, in which the nitrogen optionally has an Rc substituent.

24. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R1 is preferably a benzene ring fused to a 4- to 8 membered heterocyclic ring with from 1 to 4 heteroatoms, optionally substituted by 1 35 to 4 groups each independently is C1-C5alkyl, C2-C5alkenyl, C1-C5perfluoroalkyl, - 225 - WO 01/07042 PCT/US00/19627 amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, each group may be saturated, partly unsaturated, or fully unsaturated, wherein the heteroatoms are each independently oxygen, sulfur, or nitrogen, in which the nitrogen optionally has an Rc substituent, and wherein the benzene/heterocycle fused ring is attached at any site to X 5 or to the tetrapeptide

25. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R1 is preferably a 4- to 8-membered heterocyclic ring with from 1 to 4 heteroatoms fused to a second 4- to 8-membered heterocyclic ring 10 with from 1 to 4 heteroatoms, each heterocyclic ring independently optionally substituted by 1 to 4 groups, each group independently is C1-C5alkyl, C2-C5alkenyl, C 1-C5perfluoroalkyl, amino, oxo, thiono, C(O)NRcRd, cyano, CO 2 Rb or halogen, wherein each heterocycle may be saturated, partly unsaturated or fully unsaturated, and wherein each heteroatom independently is oxygen, sulfur, or nitrogen, and the 15 nitrogen optionally has an Rc substituent.

26. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1 and a pharmaceutically acceptable carrier. 20

27. A method for the treatment of protozoal infections comprising the step of administering, to a host in need of such treatment, a non-toxic amount of a composition according to claim 1 effective to inhibit a histone deacetylase activity of the infecting protozoa. 25

28. A method for the prevention of protozoal infections comprising the step of administering to a host a non-toxic effective preventative amount of a composition according to claim 1. - 226 -