ES2364696T3

Scooping device for container having an electromagnetic surveillance device

Abstract

A compound having the formula: ** Formula ** in which A is a saturated C1-6 or unsaturated C2-6 hydrocarbon skeleton, said skeleton being unsubstituted or having between 1 and 10 substituents, both inclusive, independently selected from cyano, halogen, azido, oxo and Q1; Each Q1 is independently selected from OR1, SR1, SO2R1, OSO2R1, NR2R1, NR2 (CO) R1, NR2 (CO) (CO) R1, NR4 (CO) NR2R1, NR2 (CO) OR1, (CO) OR1, O ( CO) R1, (CO) NR2R1 and O (CO) NR2R1; each of R1, R2 and R4 is independently selected from H, C1-6 alkyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 aminoalkyl, C6-10 aryl, C6-10 haloaryl, C6-10 hydroxyaryl, alkoxy C1-3-C6 aryl, C6-10 aryl-C1-6 alkyl, C1-6 alkyl-C6-10 aryl, C6-10 haloaryl, C1-6 alkyl, C1-6 alkyl-C6-10 haloaryl, (C1 alkoxy -3-C6 aryl) -C1-3alkyl, C2-9 heterocyclic radical, C2-9 heterocyclic radical, C1-6 alkyl, C2-9 heteroaryl and C2-9 heteroaryl-C1-6 alkyl; each of D and D 'is independently selected from R3 and OR3 in which R3 is H, C1-3 alkyl or C13 haloalkyl; n is 0 or 1; E is R5 or OR5 in which R5 is independently selected from H, C1-6 alkyl, C1-6 haloalkyl, C1-6 hydroxyalkyl and C1-6 aminoalkyl; G is O; each of J and J 'is independently H, C1-6 alkoxy or C1-6 alkyl; or J and J 'taken together are = CH2; Q is C1-3 alkyl; T is ethylene or ethenylene; each of U and U 'is independently H, C1-6 alkoxy or C1-6 alkyl; or U and U 'taken together are = CH2; X is H or C1-6 alkoxy; each of Y and Y 'is independently H or C1-6 alkoxy; or Y and Y 'taken together are = O; and each of Z and Z 'is independently H or C1-6 alkoxy; or Z and Z 'taken together are = O; or a pharmaceutically acceptable salt thereof.

ES2364696T3, drawing sheet 1 — Sheet 1 of 296

Term

Term ended

Projected expiry passed 16 June 2019, 7.3 years ago.

38 claims: 15 independent, 23 dependent

1
ES 2 364 696 T3 REIVINDICACIONES 1. Un compuesto que tiene la fórmula:en la que A es un esqueleto de hidrocarburo Ci_ 6 saturado o C 2 .6 insaturado, estando dicho esqueleto sin sustituir o teniendo entre 1 y 10 sustituyentes, ambos inclusive, seleccionados independientemente de ciano, halógeno, azido, oxo y Qi;cada Qi se selecciona independientemente de OR-ι, SR-i, SO2R1, OSO2R1, NR2R1, NR2(CO)Ri, NR2(CO)(CO)Ri, NR 4 (CO)NR 2 R1, NR 2 (CO)ORi, (CO)ORi, O(CO)R1, (CO)NR 2 Ri yO(CO)NR 2 Ri;cada uno de R1, R 2 y R4 se selecciona independientemente de H, alquilo Ci_e, haloalquilo Ci_e, hidroxialquilo Ci_e, aminoalquilo Ci_e, arilo Ce-io, haloarilo Ce-io, hidroxiarilo Ce-io, alcoxi Ci_ 3 -arilo Ce, aril Ce-io-alquilo Ci_e, alquil Ci.e-arilo Ce-io, haloaril Ce-io-alquilo Ci_e, alquil Ci.e-haloarilo Ce-io, (alcoxi C-i_3-aril Ce)-alquilo C1.3, radical heterocíclico C2-9, radical heterocíclico C 2 .9-alquilo Ci_ 6 , heteroarilo C2.9 y heteroaril C 2 .9-alquilo Ci. 6 ;cada uno de D y D' se selecciona independientemente de R 3 y OR 3 en las que R 3 es H, alquilo Ci_ 3 o haloalquilo C-i. 3¡ n es 0 ó 1;Ees R50 OR5 en las que R5 se selecciona independientemente de H, alquilo Ci_e, haloalquilo Ci_e, hidroxialquilo Ci_e y aminoalquilo Ci_e;G es O;cada uno de J y J' es independientemente H, alcoxi Ci_e o alquilo Ci_e;o J y J' tomados conjuntamente son =ΟΗ 2 ;Q es alquilo C-|. 3 ;T es etileno o etenileno;cada uno de U y U' es independientemente H, alcoxi Ci_e o alquilo Ci_e;o U y U' tomados conjuntamente son =ΟΗ 2 ;X es H o alcoxi Ci_e;cada uno de Y e Y' es independientemente H o alcoxi Ci_e;o Y e Y' tomados conjuntamente son =O;y cada uno de Z y Z' es independientemente H o alcoxi Ci_e;o Z y Z' tomados conjuntamente son =O;o una sal farmacéuticamente aceptable del mismo.
2
Un compuesto según la reivindicación 1, en el que n es 0;o una sal farmacéuticamente aceptable del mismo.
3
Un compuesto según la reivindicación 1 o la reivindicación 2, en el que cada uno de D y D' se selecciona independientemente de R 3 , alcoxi Ci_ 3 y haloalquiloxi Ci_ 3 ;o una sal farmacéuticamente aceptable del mismo.
4
Un compuesto según una cualquiera de las reivindicaciones precedentes, en el que A comprende un esqueleto de hidrocarburo Ci_e saturado o C2-6 insaturado, tendiendo dicho esqueleto al menos un sustituyente seleccionado de ciano, halógeno, azido, oxo y Q cada Q1 se selecciona independientemente de OR-ι, SR-i, SO2R1, OSO2R1, NR2R1, NR2(CO)Ri y O(CO)NR 2 Ri;n es 0;J y J' tomados conjuntamente son =CH 2 ;Q es metilo;T es etileno;U y U' tomados conjuntamente son =ΟΗ 2 ;Xes H;cada uno de Y e Y' es H;y Z y Z' tomados conjuntamente son =O;o una sal farmacéuticamente aceptable del mismo.
5
Un compuesto según una cualquiera de las reivindicaciones precedentes, en el que A tiene al menos un ES 2 364 696 T3 sustituyente seleccionado de hidroxilo, amino, azido, halógeno y oxo;o una sal farmacéuticamente aceptable del mismo.
6
Un compuesto según una cualquiera de las reivindicaciones precedentes, en el que A comprende un esqueleto de hidrocarburo saturado que tiene al menos un sustituyente seleccionado de hidroxilo, amino y azido;o una sal farmacéuticamente aceptable del mismo.
7
Un compuesto según una cualquiera de las reivindicaciones precedentes, en el que A tiene al menos dos sustituyentes seleccionados independientemente de hidroxilo, amino y azido;o una sal farmacéuticamente aceptable del mismo.
8
Un compuesto según una cualquiera de las reivindicaciones precedentes, en el que A tiene al menos dos sustituyentes seleccionados independientemente de hidroxilo y amino;o una sal farmacéuticamente aceptable del mismo.
9
Un compuesto según una cualquiera de las reivindicaciones precedentes, en el que A tiene al menos un sustituyente hidroxi y al menos un sustituyente amino;o una sal farmacéuticamente aceptable del mismo.
10
Un compuesto según una cualquiera de las reivindicaciones 1 a 8, en el que A tiene al menos dos sustituyentes hidroxilo;o una sal farmacéuticamente aceptable del mismo.
11
Un compuesto según una cualquiera de las reivindicaciones precedentes, en el que A comprende un esqueleto de hidrocarburo C2-4;o una sal farmacéuticamente aceptable del mismo.
12
Un compuesto según una cualquiera de las reivindicaciones precedentes, en el que A comprende un esqueleto de hidrocarburo C3;o una sal farmacéuticamente aceptable del mismo.
13
Un compuesto según una cualquiera de las reivindicaciones precedentes, en el que A tiene un (S)-hidroxilo en el átomo de carbono alfa con respecto al enlace A del átomo de carbono al anillo que contiene G;o una sal farmacéuticamente aceptable del mismo.
14
Un compuesto según una cualquiera de las reivindicaciones 1 a 4, en el que A comprende un esqueleto de hidrocarburo C1-6 saturado que tiene al menos un sustituyente seleccionado de hidroxilo y ciano;o una sal farmacéuticamente aceptable del mismo.
15
Un compuesto según una cualquiera de las reivindicaciones 1 a 3, en el que cada Q1 se selecciona independientemente de OR-ι, SR1, SO2R1, OSO2R1, NH(CO)R-i, NH(CO)(CO)R1 y O(CO)NHR-i;cada R 1 se selecciona independientemente de alquilo C 1-6 , haloalquilo C 1-6 , arilo C 6 , haloarilo C 6 , alcoxi C 1-3 -arilo Ce, aril C6-alquilo C1-3, alquil C1-3-arilo Ce, haloaril C6-alquilo C1-3, alquil C1-3- haloarilo Ce, (alcoxi C1-3-aril Ce)alquilo C1-3, radical heterocíclico C2-9, heteroarilo C2-9 y heteroaril C2-9-alquilo C-i-e;uno de D y D' es metilo o metoxi, y el otro es H;n es 0;J y J' tomados conjuntamente son =CH2;Q es metilo;T es etileno;U y U' tomados conjuntamente son =CH2;X es H;cada uno de Y e Y' es H;y Z y Z' tomados conjuntamente son =O;o una sal farmacéuticamente aceptable del mismo.
16
Un compuesto según la reivindicación 15, en el que Q1 se selecciona independientemente de OR1, SR1, SO2R1 y OSO2R1 en las que cada R1 se selecciona independientemente de alquilo C1-6, haloalquilo C1-6, arilo Ce, haloarilo Ce, alcoxi C 1-3 -arilo C e , aril C e -alquilo C 1-3 , alquil C 1-3 -arilo C e , haloaril C e -alquilo C 1-3 , alquil C 1-3 -haloarilo C 6 y (alcoxi C 1-3 aril C^-alquilo C 1-3 ;o una sal farmacéuticamente aceptable del mismo.
17
Un compuesto de la siguiente estructura ES 2 364 696 T3
18
Un compuesto de la siguiente estructura o una sal farmacéuticamente aceptable del mismo.
19
Un compuesto según una cualquiera de las reivindicaciones precedentes o una sal farmacéuticamente aceptable del mismo para su uso como un medicamento.
20
Un compuesto según una cualquiera de las reivindicaciones 1 a 18 o una sal farmacéuticamente aceptable del mismo para su uso en la inhibición del crecimiento tumoral o cáncer.
21
Un compuesto según una cualquiera de las reivindicaciones 1 a 18 o una sal farmacéuticamente aceptable del mismo para su uso en el tratamiento de cáncer.
22
Un compuesto según la reivindicación 20 o la reivindicación 21 o una sal farmacéuticamente aceptable del mismo, en el que el tumor o el cáncer es melanoma, fibrosarcoma, leucemia monocítica, carcinoma de colon, carcinoma de ovario, carcinoma de mama, osteosarcoma, carcinoma de próstata o carcinoma de pulmón.
23
Un compuesto según una cualquiera de las reivindicaciones 20 a 22 o una sal farmacéuticamente aceptable del mismo, en el que el tumor o el cáncer es carcinoma de mama.
24
Un compuesto según una cualquiera de las reivindicaciones 20 a 22 o una sal farmacéuticamente aceptable del mismo, en el que el tumor o el cáncer es carcinoma de pulmón.
25
Un compuesto según una cualquiera de las reivindicaciones 20 a 22 o una sal farmacéuticamente aceptable del mismo, en el que el tumor o el cáncer es carcinoma de ovario.
26
Un compuesto según una cualquiera de las reivindicaciones 20 a 22 o una sal farmacéuticamente aceptable del mismo, en el que el tumor o el cáncer es carcinoma de próstata.
27
Un compuesto según una cualquiera de las reivindicaciones 1 a 18 o una sal farmacéuticamente aceptable del mismo para su uso en la inhibición de mitosis en una célula.
28
Uso de un compuesto según una cualquiera de las reivindicaciones 1 a 18 o una sal farmacéuticamente aceptable del mismo para la preparación de un medicamento para uso uso en la inhibición del crecimiento tumoral o cáncer.
29
Uso de un compuesto según una cualquiera de las reivindicaciones 1 a 18 o una sal farmacéuticamente aceptable del mismo para la preparación de un medicamento para su uso en el tratamiento de cáncer. ES 2 364 696 T3
30
Uso según la reivindicación 28 o la reivindicación 29, en el que el tumor o el cáncer es melanoma, fibrosarcoma, leucemia monocítica, carcinoma de colon, carcinoma de ovario, carcinoma de mama, osteosarcoma, carcinoma de próstata o carcinoma de pulmón.
31
Uso según una cualquiera de las reivindicaciones 28 a 30, en el que el tumor o el cáncer es carcinoma de mama. 5
32
Uso según una cualquiera de las reivindicaciones 28 a 30, en el que el tumor o el cáncer es carcinoma de pulmón.
33
Uso según una cualquiera de las reivindicaciones 28 a 30, en el que el tumor o el cáncer es carcinoma de ovario.
34
Uso según una cualquiera de las reivindicaciones 28 a 30, en el que el tumor o el cáncer es carcinoma de próstata. 10
35
Uso de un compuesto según una cualquiera de las reivindicaciones 1 a 18 o una sal farmacéuticamente aceptable del mismo para la preparación de un medicamento para su uso en la inhibición de mitosis en una célula.
36
Una composición farmacéutica que comprende un compuesto según una cualquiera de las reivindicaciones 1 a 18 o una sal farmacéuticamente aceptable del mismo y un vehículo farmacéuticamente aceptable.
37
Un compuesto que tiene la fórmula:
38
Un compuesto que tiene la fórmula:

Independent claims38

982 paragraphs in 226 sections, as filed

ES 2 364 696 T3

DESCRIPTION

Macrocyclic analogs and procedures for their use and preparation

The invention relates to pharmaceutically active macrolides. Halichondrine B is a potent anticancer agent originally isolated from the marine sponge Halichondria okadai, and later found in Axinella sp., Phakellia caríen, and Lissondendryx sp.

A total synthesis of halichondrine B was published in 1992 (Aicher, TD et al., J. Am. Chem. Soc. 114: 3162-3164). Halichondrine B has shown in vitro inhibition of tubulin polymerization, microtubule assembly, beta crosslinkings-tubulin, GTP and vinblastine binding to tubulin and tubulin-dependent GTP hydrolysis and has shown anticancer properties in vitro and in vivo.

EP-A-0572 109 discloses halichondrine derivatives of the formula:

in which R1 and R2 together they form certain fused ring systems containing bi or tricyclic oxygen substituted with a hydroxylated alkyl chain. These halichondrine derivatives have been found to have cytotoxic activity.

WO 93/17690 discloses certain halichondrine derivatives that can be used to synthesize halichondrine B and norhalichondrine B. Some of these derivatives also exhibit antitumor activity.

The invention provides analogues of halichondrine which have pharmaceutical activity such as anticancer or antimitotic activity (blocking of mitosis). These compounds are substantially smaller than Halichondrine B. The invention features a compound having the formula (I):

Formula (I)

In formula (I), A is a hydrocarbon backbone Ci_6 saturated or C2.6 unsaturated, the backbone being unsubstituted or having between 1 and 10 substituents, for example, at least one substituent selected from cyano, halogen, azido, Qi and oxo. Each Qi is independently selected from OR-ι, SR-i, SO2Ri, BEAR2Ri, NR2Ri, NR2(CO) Ri, NR2(CO) (CO) Ri, NR4(CO) NR2Ri, NR2(CO) ORi, (CO) ORi, O (CO) Ri, (CO) NR2Ri and O (CO) NR2Ri. The number of substituents may be, for example, between 1, and 6, 1 and 8, 2 and 5, or 1 and 4. Throughout the disclosure numerical ranges are understood to be inclusive.

ES 2 364 696 T3

Ri, R2 and R4 are each independently selected from H, C1-5 alkyl, C1-6 haloalkyl, C1-5 hydroxyalkyl, C aminoalkyl1-5, aryl C6-10, haloaryl C6-10 (for example, p-fluorophenyl or p-chlorophenyl), hydroxyaryl C6-10, Cíñanlo C alkoxy6 (for example, p-methoxyphenyl, 3,4,5-trimethoxyphenyl, p-ethoxyphenyl or 3,5-diethoxyphenyl), aryl C6-10-C alkyl1-5 (e.g. benzyl or phenethyl), C 1-6 alkyl-C6-10 aryl, haloaryl C6-10-C 1-6 alkyl, C 1-6 alkyl-C6-10 haloaryl, (C 1-3 alkoxy-C6 aryl) -alkyl C1-3, C2-9 heterocyclic radical, C2-9 heterocyclic radical-C1-6 alkyl, C2-9 heteroaryl and C2-9 heteroaryl C1-6alkyl. There may be more than one R1, for example, if A is substituted with two different alkoxy groups (OR1) such as butoxy and 2-aminoethoxy.

Examples of A include 2,3-dihydroxypropyl, 2-hydroxyethyl, 3-hydroxy-4-perfluorobutyl, 2,4,5-trihydroxypentyl, 3-amino2-hydroxypropyl, 1,2-dihydroxyethyl, 2,3-dihydroxy-4- perfluorobutyl, 3-cyano-2-hydroxypropyl, 2-amino-1-hydroxyethyl, 3-azido-2-hydroxypropyl, 3,3-difluoro-2,4-dihydroxybutyl, 2,4-dihydroxybutyl, 2-hydroxy-2 (p- fluorophenyl) -ethyl, CH2 (CO) (substituted or unsubstituted aryl), -CH2 (CO) (alkyl or substituted alkyl such as haloalkyl or hydroxyalkyl) and 3,3-difluoro-2-hydroxypent-4-enyl.

Examples of Q1 include -NH (CO) (CO) - (heterocyclic or heteroaryl radical), -OSO2- (aryl or substituted aryl), O (CO) NH- (aryl or substituted aryl), aminoalkyl, hydroxyalkyl, -NH ( CO) (CO) - (aryl or substituted aryl), NH (CO) (alkyl) (heteroaryl or heterocyclic radical), O (substituted or unsubstituted alkyl) (substituted or unsubstituted aryl) and NH (CO) (alkyl) (aryl or substituted aryl).

Each of D and D 'is independently selected from R3 and OR3 where R3 is H, C1-3 alkyl or C1 haloalkyl. 3. Examples of D and D 'are methoxy, methyl, ethoxy, and ethyl. In some embodiments, one of D and D 'is H.

The value for n is 1 or preferably 0, thus forming both a six-membered ring and a five-membered ring. The ring may be unsubstituted or substituted, for example where E is R5 or OR5 where R5 is independently selected from H, C1-6 alkyl, C1-6 haloalkyl, C1-6 hydroxyalkyl and C1-6 aminoalkyl, y is a heterocyclic radical in which G is O.

J and J 'are each independently H, C1-6 alkoxy, or C1-6 alkyl; or J and J 'taken together are = CH2, Q is C1-3 alkyl, and is preferably methyl. T is ethylene or ethenylene. Each of U and U 'is independently H, C1-6 alkoxy, or C1-6 alkyl; or U and U 'taken together are = CH2. X is H or C1-6 alkoxy. Each of Y and Y 'is independently H or C1-6 alkoxy; or Y and Y 'taken together are = 0. Each of Z and Z 'is independently H or C alkoxy1-6; or Z and Z 'taken together are = O.

The invention features compounds of sufficient stability to be suitable for pharmaceutical development. The invention also features pharmaceutically acceptable salts of disclosed compounds, disclosed novel synthetic intermediates, pharmaceutical compositions containing one or more disclosed compounds, methods of preparing the disclosed compounds or intermediates, and uses of the disclosed compounds or compositions. Uses include the use of the disclosed compounds for the preparation of a medicament for reversibly or irreversibly inhibiting mitosis in a cell, and for inhibiting cancer or tumor growth in vitro, in vivo, or in a patient.

A. Definitions

B. Halichondrine analogs

C. Synthesis of Halichondrine Analogs

D. Pharmacological activity

E. Uses

A. Definitions

The following terms are defined in part below and by their use herein.

Hydrocarbon skeletons contain carbon and hydrogen atoms and can be linear, branched, or cyclic. Unsaturated hydrocarbons include one, two, three, or more CC double bonds (sp2) or triple CC (sp) bonds. Examples of unsaturated hydrocarbon radicals include ethynyl, 2-propynyl, 1-propenyl, 2-butenyl, 1,3-butadienyl, 2-pentenyl, vinyl (ethenyl), allyl, and isopropenyl. Examples of divalent unsaturated hydrocarbon radicals include alkenylenes and alkylidenes such as methylidino, ethylidene, ethylidino, vinylidene, and isopropylidene. In general, the compounds of the invention have hydrocarbon skeletons ("A" in formula (I)) that are substituted, for example, with hydroxy, amino, cyano, azido, heteroaryl, aryl, and other moieties described herein . The hydrocarbon skeletons may have two oxo-substituted geminal hydrogen atoms, a divalent carbonyl oxygen atom (= O), or a ring-forming substituent such as -O- (linear or branched alkylene or alkylidene) -O- for form an acetal or ketal.

Alkyl C1-and includes linear, branched and cyclic hydrocarbons such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, sec-pentyl, neopentyl, tert-pentyl, cyclopentyl, hexyl, isohexyl, sec-hexyl, cyclohexyl, 2-methylpentyl, tert-hexyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1,3-dimethylbutyl and 2,3-dimethylbut2-yl. Alkoxy (-OR), alkylthio (-SR), and other alkyl-derived moieties (substituted, unsaturated, or bivalent) are analogous to alkyl (R) groups. Representative alkyl groups and groups derived from alkyl such as alkoxy, haloalkyl, hydroxyalkyl, alkenyl, alkylidene and alkylene groups may be C2-6, C3-6, C1-3 or C2-4.

ES 2 364 696 T3

Halogen, hydroxy, amino, cyano, azido, etc. substituted alkyls may have 1, 2, 3, 4, 5 or more substituents that are independently selected (may or may not be the same) and may or may not be in the same carbon atom. For example, haloalkyls are alkyl groups with at least one substituent selected from fluorine, chlorine, bromine, and iodine. Haloalkyls may have two or more halo substituents which may or may not be the same halogen and may or may not be on the same carbon atom. Examples include chloromethyl, periodomethyl, 3,3-dichloropropyl, 1,3-difluorobutyl, and 1-bromo-2-chloropropyl.

Heterocyclic and heteroaryl radicals include furyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathienyl, 2H-pyrrolyl, pyrrolyl, imidazolyl (eg 1-, 2- or 4-imidazolyl), pyrazolyl, isothiazolyl, isoxazolyl, pyridyl (eg, 1-, 2- or 3-pyridyl), pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl (for example, 1-, 2- or 3-indolyl), indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl , quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinolinyl, pteridinyl, pyrrolinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, indolinyl, isoindolinyl, and morpholinyl. Heterocyclic and heteroaryl radicals can be attached to the rest of the molecule at any position along the ring. Heterocyclic and heteroaryl radicals can be C2-9, or smaller, such as C3-6, C2-5, or C3-7.

Aryl groups include phenyl, benzyl, naphthyl, tolyl, mesityl, xylyl, and cumenyl.

"Heterocyclic radical", "aryl" and "heteroaryl" are understood to include those having 1, 2, 3, 4 or more substituents independently selected from lower alkyl, lower alkoxy, amino, halogen, cyano, nitro, azido and hydroxyl. Heterocyclic, heteroaryl and aryl radicals can also be bivalent substituents on the hydrocarbon skeleton "A" in formula (I).

B. Halichondrine analogs

With reference to formula (I) above, embodiments of the invention include compounds wherein n is 0; wherein each of D and D 'is independently selected from R3, C1-3 alkoxy, and C1-3 haloalkyloxy; and combinations thereof.

Other embodiments include compounds having one or more of the following characteristics: (a) wherein A is a C1-4 saturated or C2-6 unsaturated hydrocarbon skeleton, the skeleton having at least one substituent selected from cyano, halo, azido , Q1 and oxo; (b) each Q1 is independently selected from OR1, SR1, SO1R1, OSO2R1, NR2R1, NR2 (CO) R1, and O (CO) NR2R1; (c) Z and Z 'taken together are = O; (d) wherein each Q1 is independently selected from OR1, SR1, SO2R1, OSO2R1, NH (CO) R1, NH (CO) (CO) R1, and O (CO) NHR1; (e) each R1 is independently selected from C1-6 alkyl, C1-6 haloalkyl, C6 aryl, C6 haloaryl, C1-3 alkoxy-C6 aryl, C6 aryl-C1-3 alkyl, C1-3 alkyl-C6 aryl, haloaryl C6-C1-3 alkyl, C1-3 alkyl-C6 haloaryl, (C1-3 alkoxy-C6 aryl) -C 1-3 alkyl, C2-9 heterocyclic radical, C2-9 heteroaryl and C3-9 heteroaryl-C1-6 alkyl ; (f) one of D and D 'is methyl or methoxy and the other is H; (g) n is 0; (i) J and J 'taken together are = CH2; (j) Q is methyl; (k) T is ethylene; (I) U and U 'taken together are = CH2; (m) X is H; (n) each of Y and Y 'is H; and (o) Z and Z 'taken together are = O. Examples of combinations are the combination of (i) - (m) and the combination of (a) and (b). Two particularly preferred compounds are B1793 and B1939.

Another embodiment includes compounds wherein Q is independently selected from OR1, SR1, SO2R1, and OSO2R1; and each R1 is independently selected from C1-6 alkyl, C1-6 haloalkyl, C6 aryl, C6 haloaryl, C1-3 alkoxy-C6 aryl, C6 aryl-C1-3 alkyl, C1-3 alkyl-C6 aryl, C6 haloaryl- C1-3 alkyl, C1-3 alkyl-C6 haloaryl and (C1-3 alkoxy-C6 aryl) -C 1-3 alkyl. Other embodiments include compounds where: one of D and D 'is alkyl or alkoxy, where n is 1: (f) as before, where n is 1; E is alkoxy where n is 1; n is 0 where one of D and D 'is hydroxy and the other is H; and (f) as before, where n is 1 and E is ethyl.

The invention also features compounds in which: (1) A has at least one substituent selected from hydroxyl, amino, azido, halogen and oxo; (2) A is a saturated hydrocarbon backbone having at least one substituent selected from hydroxyl, amino, and azido (eg, B1793, B1939, B2042, B1794, and B1922); (3) A has at least two substituents independently selected from hydroxyl, amino, and azido (eg, B2090 and B2136); (4) A has at least two substituents independently selected from hydroxyl and amino (eg, B2042 and B2090); (5) A has at least one hydroxyl substituent and at least one amino substituent (eg, B1939 and B2136); (6) A has at least two hydroxyl substituents (eg, B1793 and B1794); (7) A is a C1-4 hydrocarbon backbone that is substituted (eg, B2004, B2037, B1920, B2039, B2070, B2090, and B2043); (8) A is a C hydrocarbon backbone that is substituted (eg, B1793, B1920, B1984, B1988, B1939, B1940, B2014); (9) A has an (S) -hydroxyl-alpha to the carbon atom that binds A to the ring containing G (for example, B1793, B1939 or B1920) or a (R) -hydroxyl (for example, B2102, B2013, B2042); and (10) A is a C1-6 saturated hydrocarbon backbone having at least one substituent selected from hydroxyl and cyano (eg, B2013, B2037, B2102, B2086, and B2091). By (S) -hydroxyl it is meant that the configuration of the carbon atom having the hydroxyl group is (S). Embodiments of the invention also include compounds having at least two substituents on the carbon atoms (1) alpha and gamma, (2) beta and gamma, or preferably (3) alpha and beta to the carbon atom that binds A to the ring that contains G. Alpha, beta, and gamma carbon atoms can have an (R) or (S) configuration

ES 2 364 696 T3

The invention further provides preferred compounds having the formula (1) -A, shown below, in which the substituents are identical to those defined above.

The following monosaccharide intermediate having the formula (II) is also disclosed:

wherein R is methyl or methoxy, and each of P1, P2, and P3 is independently selected from H and primary alcohol protecting groups. Preferably, the diol side chain is below the plane of the page and OP2 is above the plane of the page. Primary alcohol protecting groups include esters, ethers, silyl ethers, alkyl ethers, and alkoxyalkyl ethers.

Examples of esters include formates, acetates, carbonates, and sulfonates. Specific examples include formate, benzoyl formate, chloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate, 4,4- (ethylenedithio) pentanoate, pivaloonate, crotonate, 4-methoxy-benzochronate, 4-methoxy-crotonate. 2,4,6-trimethylbenzoate, carbonates such as methyl, 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2 (trimethylsilyl) ethyl, 2- (phenylsulfonyl) ethyl, vinyl, allyl, and p-nitrobenzyl.

Examples of silyl ethers include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl, and other trialkylsilyl ethers. Alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl, allylic, and allyloxycarbonyl ethers or derivatives. Alkoxyalkyl ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy) methyl, benzyloxymethyl, beta (trimethylsilyl) ethoxymethyl and tetrahydropyranyl ethers. Examples of benzyl ethers include p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl. Preferably, each of P1 and P2 is TBS and P3 is MPM (see alcohol 19 below). In one aspect, formula (II) can be modified so that the hydroxyethyl side chain can also be a protected hydroxyl, CH2CH2O-P4, where P4 is independently selected from values for P1. A related intermediate is alcohol 17, in which the hydroxyethyl side chain is a hydroxymethyl side chain. Similarly, a corresponding hydroxypropyl side chain, or an aminoalkyl side chain, can be prepared.

P1 and P2, taken together, can be a diol protecting group such as acetals and cyclic ketals (methylene, ethylidene, benzylidenes, isopropylidene, cyclohexylidene, and cyclopentylidene), silylene derivatives such as di-t-butylsilylene derivatives, and 1,1 , 3,3-tetra-isopropyldisiloxanylidene, cyclic carbonates and cyclic boronates. Procedures for adding and removing such hydroxyl protecting groups, and additional protecting groups, are well known in the art and are available, for example, in PJ Kocienski, Protecting Groups, Thieme, 1994, and in TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 2to edition, John Wiley & Sons, 1992.

ES 2 364 696 T3

The following section provides representative syntheses of intermediates of formula (II) and halichondrine analogs of formula (I).

C. Synthesis of Halichondrine Analogs

An overview is provided below, followed by Synthesis Schemes 1-16, and several detailed protocols.

Compounds of general formula 4 can be prepared by the route outlined in Scheme 1. The key F-2 fragment exemplified by vinyl iodide compound X2 can be prepared according to the procedure of Kishi et al. (Total synthesis of halichondrin B and norhalichondrin B. Aicher, TD: Buszek, KR; Fang, FG; Forsyth. CJ; Jung. SH; Kishi, Y .; Matelich, MC; Scola, PM; Spero, DM; Yoon, SKJ Am. Chem. Soc. 1992, 114. 3162-4).

Vinyl Iodide X2

XF3

The key F-3 fragment can be obtained by DIBALH reduction of the corresponding methyl ester, XF3, prepared according to the procedure of Stamos et al. (Scheme 2).

[Synthetic studies on halichondrins: a practical synthesis of the C.1-C.13 segment. Stamos, DP; Kishi, Y. Tetrahedron Lett. 1996, 37, 8643-8646], The synthesis of key F-1 fragment exemplified by compound 20 can be synthesized as described in Scheme 3 or Scheme 4.

Using B1793 as a representative example, the coupling of the three key fragments proceeded as outlined in Scheme 5: Nozaki-Hiyama-Kishi coupling of fragments 20 and X2 followed by formation of the intramolecular Williamson ether provided the tetrahydropyran B2318. Modification of the protecting group as described in Scheme 5 or alternatively Scheme 6 provided the primary iodide B2313. The halogen-metal exchange reaction and coupling with the key fragment F-3 provided a mixture of diastereomeric alcohols B2308. Manipulation of the protecting group and further oxidation followed by an intramolecular Nozaki-Hiyama-Kishi reaction provided an intermediate product which when oxidized and treated with TBAF underwent intramolecular hetero-Michael ring closure. Acetal formation mediated by PPT yielded B1793.

Aryl groups can be incorporated into the C32 side chain (eg, B2043) as exemplified in Scheme 7. Intermediate B2318 was deprotected and the resulting diol was oxidatively cleaved to the corresponding aldehyde. Treatment with a Grignard reagent (eg, pF-PhMgBr), removal of the resulting diastereomers, and silylation provided 204, which became the final product in a manner similar to that described in Scheme 6.

Ether analogs can be prepared from B1793 by treatment with an appropriate alkylating agent (eg, Scheme 8). Similarly, sulfonates, esters, carbamates, etc., can be prepared from B1793 by treatment with an activated carbonyl component. Cleavage and oxidative reduction of the diol or selective oxidation of the hydroxyl group could provide derivatives such as B2037 and B1934, respectively.

Alternatively, one or more hydroxyl groups could be converted to the corresponding amino groups with

ES 2 364 696 T3 subsequent coupling with an activated carbonite component (Scheme 9). Displacement of the sulfonyl intermediate (eg, B1920) by carbon or heteroatom nucleophiles could also easily be provided (Scheme 10).

C31 methyl analogs can be prepared as outlined in Scheme 11. Indium-mediated coupling of an allyl bromide ester with 2,3-0- (3-isopropylidine) -D-glyceraldehyde provided lactone 103 Hetero-Michael addition, lactone reduction, Wittig coupling, and intramolecular Michael addition provided tetrahydrofuran 107. Pummerer rearrangement, protective group adjustment, and DIBALH reduction provided the F1 key fragment (eg, 114), which was converted to the final compound in a manner analogous to that described in Scheme 6.

Fluorine atoms could be introduced as described in Schemes 12-14. Starting with the appropriate tetrahydrofuran intermediate the fluorinated F1 key fragment was obtained and carried to the final compound in a manner analogous to that illustrated in Scheme 6.

The triol derivatives could be similarly prepared from the tetrahydrofuran intermediate. For example, as outlined in Scheme 15, the addition of allyltributylstannane to the X32 aldehyde 15 provided the homoallyl alcohol 33a which was carried into the final compound in a manner similar to that described in Scheme 6. These triols could be further modified as exemplified in Scheme 6.

The 1,3-diol derivatives could be prepared from the previously described intermediates. For example, B2086 could be oxidatively cleaved and reduced to provide 1,3-diol B2091 (Scheme 16).

Scheme 1

ES 2 364 696 T3

Scheme 2

1) separate isomers

4a, 4b

2) TBSCI

3) Bad

4) HCI

l) PvCI

2) BnBr

t) AD '' -x ^^ * 2) separate isomers

3) TBSCI

υ hj

2) 1PM »

3) Teübe

4) 9-BBN

5) Swem

6) EtaM

7) NaBH4

1) MPMOTCI 2JLAH 3) Swem

4) Wittlg

5) 9-BBN

6j Swem

ES 2 364 696 T3

Scheme 4

L-arabinose

1) EtSH, ZnC (2

2) TIPSCI

3) l2

4) Ac2OR

XXI4

1) AliITMS

BFj ^ EtíÓ

2) KjCO3

XX15

1) separate isomers

2) TBSCI

3) Met

4) TBAF

XX16

1) PvCl

2) MPMOTCI

MPM

XX17

1) AD v

2) separate isomers

3) TBsAOTf

1JLAH

2) Swem

3) WRtífl xxie

4) 9-BBN 5} PvCI 6) DDQ

l) Swem 2> wmi0 3) 9-8BN

4) Swem

5) EUN

6) NaBH4

7) MPMOTCI

8) LAH

9) Oess * Marttn

ES 2 364 696 T3

Scheme 5

X2

1) NlCIj / CrClj

2) KHMDS

B2318

4) LAH

5) MMTrCI

6) Nal

1) DDQ

2) separate isomers

3) TsCI

1) ΡΡΤβ

2) Oess-Martfri

3) NlCfeCiCh

4) Deae-Martín

5) 7BAF

6) PPTe

01793

ES 2 364 696 T3

Scheme 6

W βΧ231β

DLAH

2) MMTíCt

3) 00Q

t-BuU. F3

4) MsCI

5) separate isomers

6) Nal

MMTr

8X2313

BX 2307, 8X2308 tZ

1) PPTe

2) Dess-Martln

3) N1CljA> Cl2

-------- 81794

4) Dess-Martln fijTBAF β) ΡΡΤβ

ES 2 364 696 T3

Scheme 7

B2318

ΟΡν

ES 2 364 696 T3

Scheme 8 pF-PhCHjBr

PUNCO

B1793

i) NaiO4

2) NaBH «

1) TBDPSCI

2) Dess-Marttn

3) TBAF

B2014

B1984

B2037

H

B1934

ES 2 364 696 T3

Scheme 9

ES 2 364 696 T3

Scheme 10

B192O

ES 2 364 696 T3

Scheme 11

Scheme 12

3) CFjTMS

4) TBSOTf

B9-BBN

2) Swem

1) LAH

2) Swem

3) wmig

4) 9-BBN

5) PvCI

1)000

2) Swem 31Tebbe

314

1) 9-6BN

2) Swem

3) 6t / l

4) NaBHx

316

1) MPM0TCI

2) LAH

3) 10)

319

ES 2 364 696 T3

Scheme 13

MeO OH

1) MMTrCt

2} NaH. MPMCI

MeO OMPM

X399 n hci

2) PvCI

3) AD

.OMPM

1) NalO4

2) CHj = CHCFjBr

X400

3) Bear4

4) NalO4 5> NaBH «

402

1) TBSOTT

2) LAH

3) Swem

4) W1ttig

406

<td>S) Tebbe</td><td></td><td></td>

1J9-BBN

2) Swem

3) Et3N

4) NaBH4

DMPMOTCI

2) LAH

3) separate isomers

4) Dess-Martin

X412

ES 2 364 696 T3

Scheme 14

n gnaw

2) CFtoTMS

3) TBSOTf

4) LAH

3) WMIg

4) 9-BBN

1) Swem

<td>MeQ OBn τηςη</td><td>1) PvCI 2) H2. Pd (OH)23) Dess «Martin</td>

<td>EX12</td><td>IGDDG 5) 9-8BN</td>

EX 13

<td>1) Dess-Martín</td><td>TBS (</td><td>MeQ z-OH * \ j</td><td>1) MPMOTCI</td>

EX1S

ES 2 364 696 T3

Scheme 15

3) TBSCI

4) H2

1) NalO «

2) NaBH4

1JTPAP

2) Tebbe

3) S-BBN

1) Swem

2) EÍ3N

3) NaBH4

3) Swem

4) Wittíg 5J9-BBN

IJMPMOTCI

2) LAH

1) PvQ

2) TBAF

3) Dess-Martin

MeO and — OMPM

X32

2> separate isomeric

1) TBSOTf

3) Dess-Martin

35a, higher major C36 isomer

35b, lower secondary C36 isomer

1JTBSOTÍ

2) LAH

2) AD

3) separate isomers

ES 2 364 696 T3

Scheme 16

EXPERIMENTAL SECTION

Synthesis of the key fragment F-3:

DIBALH

Key fragment F-3. DIBALH (1M in toluene, 3.86 mL) was added to a solution of XF-3 (1.46 g, 1.93 mmol) in toluene (37 mL) at -78 ° C. After stirring for 10 min, the reaction was quenched by the careful addition of MeOH (0.46 mL) and H2O (0.21 ml), warmed to rt and stirred for 15 min. The white suspension was filtered at

ES 2 364 696 T3 through Celite with 1: 1 CH2Cl2 / Et2O. The filtrate was concentrated and purified by column chromatography (10% EtOAc-hexanes) to give the key fragment F-3 (1.34 g, 96%) as an oil.

Synthesis of B1793:

TBDPSCI

Triol 1 A solution of TBDPSCI (444 ml, 1.7 mol) in DMF (0.5 I) was added in three portions to a suspension of L-arabinose (250.0 g, 1.66 mol), imidazole (231 , 4 g, 3.40 mol) and DMF (2.5 I). The addition of each portion took 1.5 h with an interval of 30 min and one of 15 h separating the second and third portions, respectively. The resulting solution was stirred for 3 h, concentrated, and purified by flash chromatography (5% to 33% EtOAchexanes) to provide triol 1 (394 g, 61%).

ACjO, pyridine

Triacetate 2 Acetic anhydride (6.06 mol) was added over 1.5 h to triol 1 (1.01 mol) in pyridine (1.0 I) at 15 ° C. The solution was stirred for 1 hr, concentrated, and purified by flash chromatography (15% to 25% EtOAchexanes) to provide triacetate 2 (518 g, 97%).

Diacetates 3 Allyltrimethylsilane (1.11 mol) was added followed by BF3OEt2 (1.11 mmol) for 1.5 h to triacetate 2 (164 g, 0.32 mol) in toluene (1.5 I) at 0 ° C. The orange solution was stirred for 1 h at 0 ° C and for 2 h at rt. The mixture was slowly poured into NaHCO3 aqueous saturated (1.7 I) at 0 ° C and stirred for 30 min. The separated aqueous phase was extracted with EtOAc (3-600 mL) and the combined organic phases were dried over Na2SO4, concentrated and purified by flash chromatography (5% to 10% EtOAc-hexanes) to provide a mixture of diacetates 3 (108 g, 69%).

MeOH

K2CO3

4th

Diol 4a K was added2CO3 solid (72 mmol) to diacetates 3 (108 g, 218 mmol) in MeOH (0.5 I) at rt. The suspension was stirred for 2.5 h and then concentrated. The orange residue was suspended in saturated aqueous NH4CI (150 ml), extracted with EtOAc (3 χ 150 ml) and the combined organic phases were dried over Na2SO4, concentrated and purified by flash chromatography (15% to 50% EtOAc-hexanes) to provide alpha-isomer 4a (33.86 g, 37%) and beta-isomer 4b (58 g, 63%).

ES 2 364 696 T3

4th

Η,> DTBDPS

TBSCI

HQ OTBS ^ X ..... (^ kzOTBDPS

Alcohol 5 Imidazole (16.75 g, 246 mmol) and TBSCI (16.08 g, 107 mmol) were added to a solution of diol 4a (33.86 g, 82 mmol) in CH2CI2 (250 ml) at 0 ° C . After 18 h at 0 ° C and 5 h at rt, the reaction mixture was diluted with NaHCO3 aqueous saturated (250 ml), stirred for 30 min and the phases were allowed to separate. The aqueous phase was extracted with EtOAc (3 * 250 mL) and the combined organic phases were dried over Na2SO4, concentrated and purified by flash chromatography (2% to 50% EtOAc-hexanes) to provide alcohol 5 (36.0 g, 83%).

Met, NaH

Methyl ether 6 Iodomethane (16.5 ml, 265 mmol) and NaH (60% in mineral oil, 5.28 g, 132 mmol) were added to a solution of alcohol 5 (34.93 g, 66 mmol), THF ( 320 ml) and DMF (80 ml) at 0 ° C. After 19 h at 0 ° C, the reaction was quenched with saturated aqueous NH4CI and Na2S2O3 saturated aqueous. The resulting mixture was stirred for 20 min and the phases were allowed to separate. The aqueous phase was extracted with EtOAc (3 * 200 ml) and the combined organic phases were dried over Na2SW4, concentrated and purified by flash chromatography (3% EtOAc-hexanes) to provide methyl ether 6 (34.23 g, 96%).

HCl

Diol 7 HCl (37% aqueous solution, 12.75 mL, 153 mmol) was added to a solution of methyl ether 6 (32.93 g, 61 mmol) in MeOH (110 mL) at rt. After 17 h NaHCO was added3 (17 g) to the reaction mixture. The mixture was stirred for 30 min, concentrated, suspended in EtOAc, and filtered. The filtrate was concentrated and purified by flash chromatography (50% EtOAc-hexanes to EtOAc) to give diol 7 (10.0 g, 87%).

Alcohol 8 A solution of pivaloyl chloride (8.4 ml, 67 mmol) in pyridine (50 ml) was added over 1.5 h to a solution of diol 7 (12.24 g, 65 mmol) in pyridine (100 ml) at 0 ° C. After 1 h at 0 ° C and 18 h at rt, the mixture was diluted with NH4Aqueous CI saturated and extracted with EtOAc (3-800 mL). The combined organic phases were dried over Na2SW4, concentrated and purified by flash chromatography (50% EtOAc-hexanes) to provide alcohol 8 (16.9 g, 96%).

ES 2 364 696 T3

BnBr

Olefin 9 Benzyl bromide (62 ml, 521 mmol) and BU4NHSO4 (10.6 g, 31 mmol) were added to a solution of alcohol 8 (16.9 g, 62 mmol) in CH2CI2 (100 ml) at 0 ° C . A solution of NaOH (9.95 g, 248 mmol) in H2O (10 mL) was added to the reaction mixture over 15 min. After 30 min at 0 ° C and 18 h at rt, the reaction mixture was diluted with NH4Aqueous CI saturated and extracted with CH2Cl2 (3 * 100 mL). The combined organic phases were dried over Na2SO4, concentrated and purified by flash chromatography (30% EtOAc-hexanes hexanes) to provide olefin 9 (22.1 g, 98%).

10a

Diol 10a Added OsO4 (0.1 M solution in toluene, 7.3 ml, 0.73 mmol) and a solution of olefin 9 (24.9 g, 69 mmol) in t-BuOH (165 ml) to a solution of K2CO3 (31.3 g, 161 mmol), K3Faith (CN)6 (74.4 g, 161 mmol), (DHQ)2PYR (1.33 g, 1.50 mmol), H2O (500 ml) and i-BuOH (330 ml) at 0 ° C. After 3 h at 0 ° C Na2S2O5'5 H2O (37.3 g, 150 mmol) was added. The reaction mixture was warmed to rt, stirred for 1 hr, and extracted with EtOAc (3-300 mL). The combined organic phases were dried over Na2SW4, concentrated and purified by flash chromatography (5% isopropanol-C ^ Ch) to provide diol 10a (17.98 g, 75%).

Silyl ether 11 Imidazole (21 g, 308 mmol) and TBSCI (26.5 g, 176 mmol) were added to a solution of diol 10a (17.4 g, 44 mmol) in DMF (90 ml) at rt. After 18 h, the reaction mixture was diluted with saturated aqueous NaHCO (250 mL), stirred for 1 h, and extracted with CH2Cl2 (3 * 100 mL). The combined organic phases were dried over Na2SO4, concentrated and purified by flash chromatography (5% EtOAc-hexanes) to provide silyl ether 11 (25.7 g, 94%).

Alcohol 12 A mixture of silyl ether 11 (21.2 g, 33.8 mmol), Pd (OH) 2 (20%, 4.7 g, 33.8 mmol) and EtOAc (200 ml) was stirred at low rt 1 atm of H2 for 3 h. The mixture was filtered through Celite, concentrated, and purified by flash chromatography (10% to 20% EtOAc-hexanes) to provide alcohol 12 (17.4 g, 96%).

ES 2 364 696 T3

2) Tebbe

1) TPAP

Olefin 13 4-Methylmorpholine N-oxide (7.66 g, 65 mmol) and TPAP (1.15 g, 3.26 mmol) were added in four portions over 20 min to a solution of alcohol 12 (17.4 g , 32.5 mmol) in CH2Cl2 (145 mL) at 0 ° C. After 20 min, the reaction mixture was diluted with Et2O (50 ml) and Na2S2OR5 aqueous saturated (50 ml) and filtered through Celite. The organic phase was separated, washed sequentially with CuSO4 saturated aqueous-brine (1: 1) and brine, dried over Na2SO4, filtered through Celite and concentrated to provide the desired crude ketone.

The Tebbe reagent was prepared by stirring bis (cyclopentadienyl) titanium (11.36 g, 45.6 mmol) and Me3AI (2.0 M in toluene, 45.6 mL, 91.2 mmol) for 4 days at rt. This material was cooled to -25 ° C and a crude ketone solution in THF (150 ml) was added. The reaction mixture was warmed to 0 ° C, stirred for 30 min, quenched by slow addition of 0.1N NaOH (3.5 mL), and then stirred for an additional 20 min at rt. The mixture was diluted with Et2Or, it was filtered through Celite and concentrated. The residue was dissolved in CH2CI2, leaked via AI2OR3 Basic, concentrated and purified by flash chromatography (5% EtOAc-hexanes) to give olefin 13 (12.8 g, 74% for both steps).

Alcohol 14 9-BBN (0.5 M in THF, 165 ml, 83 mmol) was added to a solution of olefin 13 (12.78 g, 24 mmol) in THF (280 ml) at 0 ° C. After stirring for 5 h at rt, the reaction mixture was recooled to 0 ° C while adding H2O (200 ml), THF (100 ml) and NaBO3- 4H2Or (75 g). The mixture was warmed to rt, stirred for 16 h, and then concentrated. The aqueous residue was extracted with EtOAc (4 * 300 mL) and the combined organic phases were dried over Na2SO4. Concentration and purification by flash chromatography (20% to 35% EtOAchexanes) provided alcohol 14 (12.05 g, 91%).

MeQ OH

1) Swem

2) Et3N

3) NaBH4

Alcohol 15 DMSO (9 ml, 127 mmol) was added to a solution of oxalyl chloride (5.6 ml, 64 mmol) in CH2Cl2 (350 ml) at -78 ° C. After stirring for 15 min a solution of alcohol 14 (11.7 g, 0.021 mmol) in CH2CI2 (50 ml) was added and stirring continued for 1 h, after which Et3N (26.7 ml, 192 mmol). The reaction mixture was heated to 0 ° C, stirred for 15 min, diluted with NH4Aqueous CI saturated and extracted with CH2Cl2 (3 * 200 mL). The combined organic phases were dried over Na2SO4 and concentrated to provide the desired crude aldehyde.

This material was dissolved in CH2Cl2 (200 ml) and treated with Et3N (20 ml) at rt. After stirring overnight, the reaction mixture was diluted with NH4Aqueous CI saturated and extracted with CH2Cl2 (3 * 200 mL). The combined organic phases were dried over Na2SW4, concentrated and filtered through a column of SiO2 short (20% EtOAc-hexanes) providing the crude epimerized product.

The aldehyde was dissolved in EIO2-EtOH (1: 1, 100 mL), cooled to 0 ° C, and treated with sodium borohydride (1.21 g, 32 mmol). The mixture was stirred for 20 min, carefully diluted with saturated aqueous NH4Cl, stirred for 30 min at rt, and extracted with CH2Cl2 (3 * 150 mL). The combined extracts were dried over Na2SO4, concentrated, and purified by flash chromatography (20% EtOAc-hexanes) to provide alcohol 15 (9.95 g, 85% for all three steps).

ES 2 364 696 T3

MPMOTCI

Ether of MPM 16 BF was added3OEI2 (0.1 M in CH2Cl2, 1.8 ml, 0.18 mmol) to a solution of alcohol 15 (9.87 g, 18 mmol), MPM trichloroimidate (4.9 ml, 27 mmol) and CH2Cl2 (175 ml) at 0 ° C. After 40 min a second portion of ΒΡ3· ΟΕί2 (0.1 M in CH2CI2, 0.9 ml, 0.09 mmol) to the reaction mixture. After 20 min, the reaction was quenched with saturated aqueous NH4CI, stirred 1 h at rt, and diluted with Et2Or (600 ml). The organic phase was separated and the aqueous phase was extracted with EIO2 (150 ml). The combined organic extracts were washed sequentially with 0.1N aqueous NaOH, NaHCO3 aqueous saturated brine, dried over Na2SO4, concentrated and purified by flash chromatography (20% EtOAc-hexanes) to give the ether of MPM 16 (10.20 g, 85%).

Alcohol 17 LAH (1M in THF, 22.5 mL, 22.5 mmol) was added to a solution of the ether of MPM 16 (10.05 g, 15 mmol) in Et2O (1.0 I) at 0 ° C. After 30 min, the reaction was cautiously quenched with H2O (1.3 ml) and 1N aqueous NaOH (1.3 ml). After stirring for 1 hr, the suspension was filtered through Celite, concentrated, and purified by flash chromatography (20% EtOAc-hexanes) to provide alcohol 17 (8.18 g, 93%).

1) Swem

2) Wittig

Olefin 18 DMSO (5.8 mL, 82.4 mmol) was added to a solution of oxalyl chloride (3.6 mL, 41.2 mmol) in ΟΗ2ΟΙ2 (100 ml) at -78 ° C. After 15 min a solution of alcohol 17 (7.94 g, 13.5 mmol) in ΟΗ2ΟΙ2 (35 ml) to the reaction mixture. After stirring for 1 h, Et3N (17 ml, 122 mmol), the mixture was heated to 0 ° C, stirred for 20 min, diluted with NH4Aqueous CI saturated and then extracted with CH2Cl2, (3 * 100 mL). The combined organic extracts were dried over Na2SO4, concentrated and filtered through a short S102 column (20% EtOAc-hexanes) to provide the desired crude aldehyde.

N-BuLi (1.6 M, 20 ml, 30 mmol) was added dropwise to a solution of CH3PPh3Br (10.1 g, 30 mmol) in THF (350 ml) and DMSO (100 ml) at 0 ° C. After 1 h a solution of the crude aldehyde in THF (50 ml) was added. The reaction mixture was warmed to rt and stirred for 3 h. NH was added4Saturated aqueous CI and the mixture was extracted with EtOAc (3 ~ 500 mL). The combined extracts were washed with brine, dried over Na2SO4, concentrated and purified by flash chromatography (7% EtOAc-hexanes) to provide olefin 18 (5.57 g, 71% yield for the 2 steps).

ES 2 364 696 T3

Alcohol 19 9-BBN (0.5 M in THF, 65 mL, 33 mmol) was added to a solution of olefin 18 (5.56 g, 9.6 mmol) in THF (85 mL) at 0 ° C. The mixture was stirred for 5 h at rt and then cooled to 0 ° C. H2O (200 ml), THF (100 ml) and NaBO3and4 H2Or (30 g). After stirring overnight at rt, the volatile organic compounds were removed under reduced pressure. The aqueous residue was extracted with EtOAc (3 * 200 ml) and the combined organic phases were dried over Na2SO4. Concentration and purification by flash chromatography (30% EtOAc-hexanes) provided alcohol 19 (12.05 g, 92%).

TBS

OMPM

Aldehyde 20. DMSO (1.36 mL, 19.2 mmol) was added dropwise over 4 min to a solution of oxalyl chloride (1.26 mL, 14.4 mmol) in CH2CI2 (120 ml) at -78 ° C. After stirring for 10 min a solution of alcohol 19 (5.76 g, 9.61 mmol) in CH2CI2 (20 ml) through a cannula. The transfer was completed by rinsing with CH2CI2 additional (2 »5 ml). After stirring for 20 min, the mixture was treated with Et3N (5.36 ml, 38.4 mmol) and stirred for 10 min at -78 ° C, 30 min at 0 ° C and 10 min at rt. The reaction mixture was poured into saturated aqueous NaHCO3 (200 ml) and the separated aqueous phase was extracted with CH2CI2 (3 *) followed by EtOAc (100 ml). The combined organic phases were dried over Na2SO4, concentrated and purified by column chromatography (10% to 20% EtOAc-hexanes) to provide aldehyde compound 20 (5.28 g, 92%) as an oil.

B2318. 0.1% NiCI added2/ CrCI2, (w / w, 3.21 g) and 1% NiCI2/ CrCI2 (w / w, 4.31 g) to a solution of aldehyde 20 (3.73 g, 6.25 mmol), the key F-2 fragment exemplified by vinyl iodide X2 (5.10 g, 9.16 mmol), THF (85 ml) and DMF (21 ml) at rt in a glove-sealed chamber. The reaction mixture was stirred for 24 h, removed from the glove box, cooled to 0 ° C, diluted with EtOAc (100 mL), quenched with NH4CI saturated (200 ml) and stirred for 30 min. The separated aqueous phase was extracted with EtOAc (6 *) and the combined organic phases were dried over Na2SO4, concentrated and purified by column chromatography (20% to 30%) giving B2318 (-3 g) contaminated with nearby running impurities and uncyclized intermediate (4.61 g). The latter (4.51 g, 4.48 mmol) was dissolved in THF (150 ml), cooled to 0 ° C and treated with KHMDS (0.5 M in toluene, 14 ml, 7.0 mmol) for a period of 2 min. After stirring at 0 ° C for 15 min, the reaction was quenched with saturated aqueous NH4Cl (150 mL) and warmed to rt. The separated aqueous phase was extracted with EtOAc (3 *) and the combined organic phases were dried over Na2SO4, concentrated and combined with the partially purified product obtained above. Column chromatography (10% EtOAc-hexanes) provided B2318 (3.17 g, 55%) as an inseparable -3: 1 mixture of C27 diastereomers.

ES 2 364 696 T3

B2318

B2317

B2317. DDQ (1.45 g, 6.42 mmol) was added portionwise over 30 min to a stirred solution of B2318 (3.12 g, 3.35 mmol) in CH2Cl2 (50 mL) and pH 7 phosphate buffer ( 5 ml) at rt. The reaction was quenched with NaHCO3 aqueous saturated (50 ml), stirred for 5 min, diluted with NaHCO3 additional saturated aqueous (100 ml), H2O 5 (200 ml) and extracted with EIO2 (5χ). The combined organic phases were dried over Na2SO4, concentrated and purified by column chromatography (15% to 30% EtOAc-hexanes) to give recovered B2318 (1.40 g) and a mixture of the C27 isomeric products. The recovered B2318 was re-subjected to the reaction conditions described above providing additional product. The recovered starting material was cyclized again under the deprotection conditions. All desired material was combined and separated by MPLC 10 to provide B2317 (1.65 g, 61%).

B2317 B2316

B2316. TsCl (0.63 g, 3.30 mmol) was added to a solution of B2317 (1.60 g, 1.97 mmol) in CH2Cl2 (8 ml) and pyridine (2 ml) at rt. After stirring for 29 h, the reaction was quenched with NaHCO3 saturated aqueous (30 ml) and H2O (10 ml). The separated aqueous phase was extracted with EIO2 and the combined organic phases were dried over Na2SO.4, concentrated and purified by column chromatography (15% to 30% EtOAc-hexanes) to give B2316 (2.01 g, 92%) as an oil along with recovered B2317 (92 mg, 5.8%).

B2316 B2315

B2315. LAH (1M in THF, 2.61 mL, 2.61 mmol) was added over 1 min to a solution of B2316 (1.68 g, 1.74 27

ES 2 364 696 T3 mmol) in Et2O (80 ml) at 0 ° C. After stirring for 7 min, the reaction was quenched by the careful addition of MeOH (0.42 mL, 10.4 mmol) and H2O (0.19 mL, 10 mmol), warmed to rt and stirred for 20 min. Filtration through Celite with 1: 1 of 0Η20Ι2-Εί2*, Concentration and purification by column chromatography (30% to 40% EtOAc-hexanes) gave B2315 (1.38 g, 90%) as an oil.

B2314. MMTrCI (0.70 g, 2.26 mmol) was added to a solution of B2315 (1.33 g, 1.51 mmol) in CH2CI2 (25 ml) and iPr2NEt (0.79 ml, 4.53 mmol) at rt. The resulting mixture was stirred for 1 h and then poured into a mixture of saturated aqueous NaHCO3 (20 ml), H2O (10 ml) and Et2Or (50 ml). The separated aqueous phase was extracted with Et2Or (3χ). The combined organic phases were dried over Na2SO4, concentrated and purified by column chromatography (CH2CI2 followed by 15% to 30% EtOAc-hexanes) giving B2314 (1.65 g, 95%) as a solid foam.

B2314 B2313

B2313. A mixture of B2314 (1.60 g, 1.39 mmol) and Nal (3.10 g, 20.8 mmol) in acetone (50 ml) was refluxed for 13 h. After cooling to rt, the reaction mixture was diluted with EtOAc and concentrated. H2O (5 ml), brine (20 ml) and Na2S2O3 (200 mg) and the resulting mixture was extracted with Et2Or (4 *). The 15 combined extracts were dried over Na2SO4, concentrated and purified by column chromatography (10% EtOAc-hexanes) to give B2313 (1.50 g, 97%) as an oil.

ES 2 364 696 T3

B2307 and B2308. Tert-BuLi (1.7 M in pentane, 1.00 ml, 1.7 mmol) was added over 1 min to a solution of B2313 (0.90 g, 0.81 mmol) in Et2O (14 ml) at -78 ° C. After stirring for 9 min, the mixture was transferred by cannula for 4 min into a solution of the key fragment F-3 (0.83 g, 1.14 mmol) in Et2O (4 ml) at -78 ° C. The transfer was completed by rinsing with Et2Or additional (2 ml). The resulting mixture was stirred at -78 ° C for 5 min 5 and then at 0 ° C for 10 min, quenched with saturated aqueous NaHCOs (30 mL) and warmed to rt. The separated aqueous phase was extracted with Et2O (3 *) and the combined organic phases were dried over Na2SO4 and concentrated. The residue was combined with those of two other batches (corresponding to 0.11 g and 0.44 g of B2313) and purified by column chromatography (10% to 20% EtOAc-hexanes) giving a mixture of B2307 and B2308 ( 1.86 g, 83%) as a solid foam. Although the isomers could be separated by prep CCL (20% EtOAc-hexanes), 10 were transferred as a mixture.

B2305 and B2306. The B2307-B2308 (1.80 g, 1.05 mmol) mixture was dissolved in EtOH (20 mL), treated with PPTS (10.0 mg, 0.04 mmol), stirred at for 11 h and then quenched with NaHCOs (20.0 mg, 0.24 mmol). After stirring for 15 min, the mixture was concentrated, azeotroped with toluene (15 ml) and purified by column chromatography (20% to 30% EtOAc-hexanes) to give a mixture of B2305 and B2306 (1.22 g, 81%) as a solid foam. Although the isomers could be separated by prep TLC (30% EtOAc-hexanes), they were transferred as a mixture.

ES 2 364 696 T3

B2305, B2306. B2304

B2304. A mixture of B2305 / B2306 (1.16 g, 0.68 mmol) and Dess-Martin periodinane (0.61 g, 1.44 mmol) in CH2Cl2 (35 mL) was stirred at rt for 1 h. Additional Dess-Martin periodinane (0.54 g, 1.27 mmol) was added to the mixture and stirring continued for an additional 1 hr. The mixture was diluted with EIO2 (100 mL), stirred for 20 min and filtered through Celite with EIO2. The colorless filtrate was washed with saturated aqueous NaHCO3 (100 ml) and the separated aqueous phase was extracted with Et2Or (3X). The combined organic phases were dried over Na2SW4, concentrated and purified by column chromatography (10% to 15% EtOAc-hexanes) to give B2304 (0.98 g, 84%) as a solid foam.

Alternatively, B2304 can be prepared as follows and in fact the synthesis described below is superior to that given above.

B2317

ER804025

To a solution of alcohol, 2.4 g, in methylene chloride, 29 ml, was added methanesulfonyl anhydride, 770 mg. The mixture was stirred for 15 minutes, extracted with saturated sodium bicarbonate, dried and chromatographed to give 2.737 g, 100%.

ES 2 364 696 T3

ER804025 ER804026

To a solution of the mesylate, 405 mg, in DMF, 0.06 ml, was added diisopropylethylamine, 0.130 ml, followed by benzenethiol, 0.061 ml. After 4 hours and after 22 hours, additional amine, 0.03 ml, and benzenethiol, 0.015 ml were added.After 24 hours, the mixture was diluted with 5% ethyl acetate / hexane, 1 ml, and 5 chromatographed to give 409 mg.

ER804026 ER804027

To a solution of the sulfide, 1.97 g, in acetonitrile, 16 ml, was added N-methylmorpholine oxide (NMO), and then a solution of 1.02 g of tetrapropylammonium perruthenate (VIL) (TPAP), 38 mg , in acetonitrile, 1 ml. After 3.5 hours at room temperature, the mixture was heated to 40 ° C for 1 hour. The mixture was cooled and 10 aqueous saturated sodium thiosulfate was added and the mixture was partitioned between water and ethyl acetate. The usual work-up gave 1.567 g of a brown oil.

ER0O4O27 ER8O4O28

To a solution of the pivaloate ester, 1.567 g, in methylene chloride, 11.2 ml, at -78 ° C was added DIBAL, 2.5 ml, of a 1 M solution in toluene. After 15 minutes additional DIBAL, 0.8 ml was added. After an additional 5 minutes, methanol, 0.46 ml, was added slowly, followed by water, 0.2 ml. The mixture was filtered through Celite and chromatographed to give 1.386 g of an oil.

ES 2 364 696 T3

To a solution of the sulfone, 36 mg, in DME, 1 ml, at -40 ° C was added n-butyllithium, 2.8 equivalents. After 35 minutes a solution of aldehyde, 42 mg, in DMB, 0.5 ml was added. After 40 minutes saturated aqueous ammonium chloride was added and the mixture was extracted with chromatography to give 52 mg of an oil.

ethyl acetate.

The usual final treatment, followed by

ER804029 ER804030

To a solution of alcohol, 42 mg, in methylene chloride, 2 ml, was added the Dess-Martin reagent, 36.4 mg. The mixture was stirred for 30 minutes and ether was added. The mixture was filtered through Celite, washed with saturated sodium bicarbonate, with saturated sodium thiosulfate, worked up in the usual way, and chromatographed to give 38 mg of an oil.

ER804030 B2304

ES 2 364 696 T3

Preparation of the SmÍ2 solution

A solution of 1,2-diiodoethane in 10 ml of THF was added to a suspension of Sm, 0.16 g, in THF, 1 ml. The mixture was stirred for 1 hour.

An aliquot of this solution, 0.03 ml, was added to a solution of the sulfone in THF at -78 ° C. After 5 min. Additional SmI2 reagent, 0.05 ml, was added. After a few additional minutes more reagent was added,

0.25 ml. The cooling bath was removed and saturated aqueous sodium bicarbonate, 3 ml, was added. The mixture was partitioned between ether and water and the usual work-up gave 9.1 mg, 81%, of an oil.

B2304 B2302, B2303

B2302 and B2303. NiCfe / CrCb (1% w / w, 1.09 g, 8.86 mmol) was added to a solution of B2304 (1.01 g, 0.70 mmol) in THF (600 ml ) and DMF (150 ml) at rt. After stirring for 2 days, the reaction mixture was removed from the glove box, cooled to 0 ° C, quenched with saturated aqueous NH4Cl (300 mL), and stirred at 0 ° C for 20 min. After the addition of H2O (100 ml), the two phases were separated and the aqueous phase was extracted with EtOAc (5 *). The combined organic phases were washed with brine, dried over Na2SO4, concentrated and purified by column chromatography (15% EtOAc-hexanes) to provide a mixture of B2302 and B2303 (0.84 g, 92%) as a solid foam. Although the isomers could be separated by prep TLC (20% EtOAc-hexanes), they were transferred as a mixture.

ES 2 364 696 T3

B2301. A mixture of B2302 / B2303 (0.79 g, 0.60 mmol) and Dess-Martin periodinane (0.26 g, 0.60 mmol) in

CH2Cl2 (30 mL) at rt was stirred for 30 min. Additional Dess-Martin periodinane (0.26 g, 0.60 mmol) was added to the mixture and stirring continued for an additional 1.5 h. The mixture was then diluted with Et2O (100 ml), stirred for 15 min and filtered through Celite. The filtrate was washed with saturated aqueous NaHCO3 (100 mL) and the separated aqueous phase was extracted with Et2 <D (3χ). The combined organic phases were dried over Na2SO4, concentrated, and purified by column chromatography (10% to 155% EtOAc-hexanes) to give B2301 (0.67 g, 85%) as an oil.

B1793. TBAF (1 M in THF containing 0.5 M imidazole HCl, 4.60 mL, 4.60 mmol) was added over 2 min to a solution of B2301 (0.62 g, 0.48 mmol) in THF ( 29 ml) at rt and the resulting mixture was stirred for 18 h. After

ES 2 364 696 T3 the dilution with hexanes (10 ml), the reaction mixture was directly loaded onto a column of S1O2 packed with 50% EtOAc-hexanes and eluted with 50% EtOAc-hexanes (1 I) followed by 10% MeOH / EtOAc to collect a mixture of intermediates. After removal of the solvent, the residue was dissolved in CH2Cl2 (15 ml) and treated with PPTS (645 mg). After stirring for 1 hr at rt additional PPTS (414 mg) was added and the resulting white suspension was stirred for 4.5 hr. The reaction mixture was then directly loaded onto a S102 column packed with 70% EtOAc-hexanes and eluted with 70% EtOAc / hexanes (0.5 I), EtOAc (1 I). Elution with 5% to 10% MeOH / EtOAc provided pure B1793 (181 mg) and elution with 15% MeOH-EtOAc gave additional semi-pure product, which after purification by preparative TLC (10% MeOH-EtOAc) provided additional pure B1793 (42 mg). B1793 (total 223 mg, 64%) was obtained as a white solid. HRMS: cale, for C40H58O12 + Na 753.3826. Found: 753.3808.

Synthesis of B1794:

Arabinose

Arabinose

B1794. Except for stereochemical and protecting group differences (Schemes 4 and 6), arabinose was converted to B1794 in a manner similar to that described for B1793 (see Schemes 3 and 5). HRMS: cale, for C40H58O12 + Na 753.3826. Found: 753.3856.

Synthesis of Representative Analogs of B1793:

B1793

B1920 and B1921 TsCI (9.9 mg, 0.052 mmol) was added to a solution of the diol B1793 (7.6 mg, 0.010 mmol) in CH2CI2 (1 ml) and pyridine (0.1 ml) at rt. After 48 h, the reaction was quenched with a 1: 4 mixture of saturated aqueous NaHCO3-brine and extracted with CH2CI2 (4*). The combined extracts were dried over Na2SW4 and

ES 2 364 696 T3 concentrated. Purification by preparative TLC (80% EtOAc-hexanes) provided monotosylate B1920 (6.0 mg, 67%) and ditosylate B1921 (1.8 mg, 18%).

B1793 B2294

B2294 MsCI (0.3 M in CH2Cl2, 98 µl, 0.030 mmol) was added dropwise over 40 min to a mixture of collidine (75 µl, 0.054 mmol), B1793 (20.8 mg, 0.028 mmol) and CH2Cl2 (1 ml) at 0 ° C. After 76 h at 4 ° C, the reaction was quenched with a 1: 4 mixture of saturated aqueous NaHCO3-brine and extracted with CH2Cl2 (4 *). The combined extracts were dried over Na2SO4 and concentrated. The crude product was dissolved in toluene (3 ml), concentrated and purified by preparative TLC (1.5% MeOH-EtOAc) to provide mesylate B2294 (21.4 mg, 95%).

B2014 B20I5

B2014 and B2015 A 0.016 M solution of 4-fluorobenzyl bromide in EÍ2O (800 μΙ, 13 μιτιοΙ) and Ag2 <D (10 mg, 43 μιτιοΙ) were each added in three portions at intervals of 1 h to a solution at rt of B1793 (1.7 mg, 2.3 pmol) in EIO2 (1.2 ml). The mixture was protected from light, stirred for 7 h, then filtered through Celite. Concentration and purification by preparative TLC (EtOAc) provided primary ether B2014 (1.1 mg, 56%) and secondary ether B2015 (0.6 mg, 31%). HRMS (FAB): cal, for C47H63FO12 + Na 861.4201. Found: for B2014

ES 2 364 696 T3

861.4178, for B2015 861.4160.

General. A mixture of B1793 (1 mg, 1.37 micromole), Et3N (10 microL, 72 micromole) and ArNCO (2 to 4 equiv.) In CH2CI2 (0.2 ml) was stirred at rt for 4 hrs to overnight until the reaction was judged to be complete by TLC. The reaction mixture was diluted with NaHCO3 saturated (3 ml), extracted with CH2CI2 (3 *) and EtOAc (2χ), dried over Na2SO4 and purified by preparative TLC (5% MeOH-CH2CH2CI2) providing the products:

B1984. (1.0 mg, 86%) HRMS (FAB): warm, for 047Η63ΝΟι3 + Na 872.4197. Found: 872.4214.

B1990. (1.1 mg, 92%) HRMS (FAB): heat, for C47H62CINOi3 + Na 906.3807. Found: 906.3826.

B1992. (1.0 mg, 83%) HRMS (FAB): heat, for C48H65NOi4 + Na 902.4303. Found: 902.4269.

B2042 DEAD (0.4 M in ether, 50 μΙ, 19 μιτιοΙ) was added to a solution of B1793 (2.0 mg, 2.7 μιτιοΙ), triphenylphosphine (5 mg, 19 μιτιοΙ), 4-nitrobenzoic acid (3 , 2 mg, 19 μιτιοΙ) and Et2O (500 μΙ) at rt. After 22 h, the reaction mixture was directly loaded onto a preparative TLC plate and eluted with 60% EtOAc-hexanes to give the intermediate diester (3.0 mg). This material was taken up in MeOH (300 μΙ) and treated with K2CO3 (approximately 1 mg).

After stirring at rt for 30 min, the reaction mixture was diluted with brine and extracted with CH2CI2 (5*).

The combined extracts were dried over Na2SW4, concentrated and purified by preparative TLC (5%

ES 2 364 696 T3

MeOH-EtOAc) providing B2042 (1.2 mg, 60% for both steps). HRMS (FAB): cale, for C40H58O12 + Na 753.3826. Found: 753.3810.

B1793

B1896 and B1897. NaBH was added4 (3 mg, 0.08 mmol) to a solution of B1793 (2.30 mg, 3.15 pmol) in 1: 1 of 5 CH2Cl2-MeOH (0.2 mL) at rt. The concentration of the reaction mixture and purification by preparative TLC (8%

MeOH-EtOAc) provided B1896 (0.80 mg, 35%) and B1897 (2: 1 mixture, 0.15 mg, 6.5%). HRMS (FAB) for B1896: cale, for C4oHeoOi2 + Na 755.3983. Found: 753.3969.

B1793 B1918

B1918. A mixture of B1793 (2.0 mg, 2.74 pmol), NalO4 (35 mg, 0.16 mmol), MeOH (0.8 ml) and H2O (0.2 ml) were stirred at rt for 40 min. The reaction mixture was diluted with H2O (1 ml), extracted with CH2Cl2 (6 *), dried over

Na2SO4, concentrated and purified by column chromatography (5% MeOH-CH2Cl2) to give B1918 (1.9 mg, 100%).

ES 2 364 696 T3

B1918 B2037

B2037. A 0.034 M solution of NaBhL (0.1 ml, 3.4 pinol) in EtOH was added portionwise to a solution of B1918 (1.9 mg, 2.72 μmol) in MeOH (0.8 ml) and CH2CI2 (0.2 ml) at -78 ° C to rt until the reaction was judged to be complete by TLC. The reaction was quenched with saturated aqueous NH4CI (2 mL) at -78 ° C, warmed to 5 rt, extracted with CH2Cl2 (6χ), dried over Na2SO4, and purified by preparative TLC (5% MeOH-CH2CÍ2) yielding B2037 (1.7mg, 89%). HRMS (FAB): cale, for C39H56Oi5 + Na 723.3720. Found: 723.3749.

B1793 B2035

B2035

NaICU (35 mg, 0.16 mmol) was added to a solution of B1793 (1.7 mg, 0.0023 mmol), MeOH (800 μΙ) and H2O (200 μΙ) 10 and after 15 min the mixture was diluted with H2O and extracted with CH2Cl2 (5 *). The organic extracts were dried over Na2SW4, were concentrated and the intermediate aldehyde was immediately dissolved in DMF (300 µΙ). 3-Bromo-3,3-difluoropropene (3 μΙ, 0.023 mmol) and indium powder (3 mg, 0.23 mmol) were added and after 24 h additional 3-bromo-3,3-difluoropropene (1 μΙ, 0.008 mmol). After 18 h H 2 O was added, the mixture was extracted with EtOAc (3 *). The combined organic extracts were washed successively with H2O and brine, dried over 15 Na2SW4, concentrated and purified by preparative TLC (80% EtOAc-hexanes) to provide B2035

ES 2 364 696 T3 (0.74 mg, 41% for the 2 steps) as a mixture of isomers. HRMS (FAB): cale, for C42H58F2O11 + Na 799.3845. Found: 799.3883.

B1793 B2011: X = H

B2008: X = F

B2008, B2011

NaBH4 (2 mg, 0.05 mmol) was added to a solution of B1793 (2.2 mg, 0.003 mmol) in 1: 1 CH2Cl2-MeOH (200 µΙ) at rt. After 15 min saturated aqueous NH4Cl and H2O were added and the mixture was extracted with CH2Cl2 (6x) and EtOAc (2χ). The combined organic extracts were dried over Na2SW4, concentrated and purified by column chromatography (10% MeOH-EtOAc) providing an intermediate triol that was dissolved in MeOH (800 µ 800) and H2O (200 µΙ). NaICU (35 mg, 0.16 mmol) was added and after 20 min the mixture was diluted with H2O and extracted with CH2Cl2 (6x). The organic extracts were dried over Na2SC> 4, concentrated and the intermediate aldehyde was immediately dissolved in THF (500 µΙ). 4-Fluorophenylmagnesium bromide (2M in EIO, 12 µΙ, 0.024 mmol) was added and after 20 min the reaction was quenched with saturated aqueous NH4Cl. The mixture was extracted with CH2Cl2 (6χ) and the combined organic extracts were dried over Na2SC> 4 and concentrated. Purification by preparative TLC (EtOAc) provided the desired product as a mixture of 4 isomers (1.32 mg, 55% for all 3 steps).

Dess-Martin periodinane (-3 mg, 0.007 mmol) was added to a solution of the above product (0.95 mg, 0.0012 mmol) in CH2CI2 (300 µΙ) and the mixture was stirred at rt for 20 min. Additional Dess-Martin periodinane (-3 mg, 0.007 mmol) and CH2Cl2 (300 μΙ) were added and after another 40 min ΕΪ2 <3, saturated aqueous NaHCC> 3 (4 mL) and saturated aqueous Na2S2C> 3 ( 1 ml). The mixture was extracted with Et2 <0 (3χ) and the combined extracts were washed with brine, dried over NasSCU, concentrated, and purified by column chromatography (20% BtOAc-hexanes) to provide B2008 (0.58 mg, 61%). HRMS (FAB): cale, for C45H57FO11 + Na 815.3783. Found: 815.3758.

B2011. In an analogous fashion, B1793 (1.9 mg, 0.003 mmol) was converted to B2011 (0.87 mg, 42% for all 4 steps). HRMS (FAB): cale, for C45H58O11 + Na 797.3877. Found: 797.3877.

ES 2 364 696 T3

B1920

B2013

A solution of B1920 (1.4 mg, 0.0016 mmol), KCN (1 mg, 0.016 mmol), and DMSO (500 μΙ) was heated at 60 ° C for

h. After cooling to rt, H2O and the mixture was extracted with EtOAc (3 *). The combined organic extracts were washed successively with H2O and brine, dried over Na2SO4, concentrated and purified by preparative TLC (80% EtOAc-hexanes) to provide B2013 (0.78 mg, 67%). HRMS (FAB): cale, for C41H57NO11 + Na 762.3829. Found: 762.3848.

B1920

X1920

A mixture of B1920 (1.3 mg, 1.47 pmol), Nal (30 mg, excess) and acetone (1 ml) was stirred at 60 ° C for 3.5 h. After cooling to rt, the reaction mixture was diluted with NaHCO3 aqueous saturated (3 ml), extracted with CH2CI2 (5 *) and EtOAc, dried over Na2SW4 and purified by column chromatography (50% EtOAc-CH2CI2 at 80% EtOAc-hexanes) to give the iodide X1920 (1.3 mg, 100%).

ES 2 364 696 T3

ArSH

X1920

B1998; Ar = p-Cl-Ph

B2010: Ar = / j-MeO-Ph

B2019: Ar - 2-imidazole

General. A mixture of X1920 iodide (1.0 equiv.), IP ^ EtN (11 to 22 equiv.), ArSH (9 to 46 equiv.) And DMF (0.3 ml) was stirred at rt until TLC judged that the reaction was complete (usually 24 to 48 hrs). The reaction mixture was diluted with saturated aqueous NaHCOs (2 mL), extracted with CH2CI2 and EtOAc, dried over Na2SO4 and purified by preparative TLC (80% EtOAc-hexanes or 5% MeOH-CH2CI2) providing the sulfur products:

B1998. (1.3 mg gave 1.1 mg, 85%) HRMS (FAB): warm, for C46H6iCIOiiS + Na 897.3521. Found: 897.3533.

B2010. (1.1 mg gave 0.7 mg, 59%). HRMS (FAB): calo, for C47H64Oi2S + Na 875.4016. Found: 875.4057.

B2019. (1.1 mg gave 0.7 mg, 61%) MS (FAB): M + Na

mCPBA

B1998: Ar = p-Cl-Ph

B2010: Ar = p-MeO-Ph

B2016: Ar = p-Cl-Ph

B2030: Ar = / j-MeO-Ph

General. A 0.01 M solution of mCPHA (1.2 equiv.) In CH2Cl2 was added to a solution of a sulfide (1.0 equiv.) In CH2Cl2 (0.5 ml) at 0 ° C over 30 min. The reaction mixture was diluted with saturated NaHCOs (2 ml), extracted with CH2Cl2 and EtOAc, dried over Na2SO4 and purified by preparative TLC (80% EtOAc-hexanes or EtOAc) to provide the products:

ES 2 364 696 T3

B2016. (0.9 mg gave 0.7 mg, 74%)

B2030. (1.0 mg gave 0.6 mg, 61%) HRMS (FAB): lime, for C47H64Oi4S + Na 907.3914. Found: 907.3950.

B1934. TBDPSCI (3.0 µl, 12 pmol) was added to a solution of B1793 (1.3 mg, 1.78 pmol), imidazole (10 mg, 166 5 pmol) and DMF (0.10 ml) at rt. After stirring for 1 h, the reaction mixture was diluted with NaHCO3 aqueous saturated (2 ml), extracted with CH2Cl2 (3χ) and EtOAc (2χ), dried over Na2SO4 and purified by preparative TLC (5% MeOH-CH2CI2) to give the intermediate silyl ether (1.3 mg, 77%).

This material was dissolved in CH2Cl2 (0.5 ml) and treated with Dess-Martin periodinane (10 mg, 24 pmol) for 1.5 h at rt, diluted with Et2Or and leaked through Celite. The filtrate was concentrated and purified by preparative TLC (50% EtOAc-hexanes) to provide the diketone intermediate (1.0mg, 77%) which was dissolved in THF (0.5ml) and treated with TBAF 0.02 M containing 0.01 M imidazole hydrochloride (THF solution, 75 µl, 1.5 pmol) at rt for 15 min. The reaction mixture was eluted through a column of SiO2 (50% EtOAc-hexanes to 5% MeOH-CH2CI2) and the desired product was further purified by preparative TLC (5% MeOH-CH2CI2) to provide B1934 (0.75 mg, 100%). HRMS (FAB): calo, for C4qH36Oi2 + Na 751.3669. Found: 751.3690.

Synthesis of B1939:

B2294 B1922

ES 2 364 696 T3

B1922 Tetra-n-butylaminonium azide (0.2 M in DMF, 0.5 mL, 0.10 mmol) was added to a solution of B2294 mesylate (21.4 mg, 0.026 mmol) in DMF (2 mL) to ta. After stirring at 83 ° C for 3.5 h, the reaction mixture was cooled to rt, diluted with toluene, concentrated, and purified by preparative TLC (80% ethyl acetate-hexanes) to provide B1922 (18 mg, 92%).

I3P

B1922

B1939 Me was added sequentially3P (1 M in THF) and H2O (0.8 ml) to a solution of azide B1922 (24.6 mg, 0.032 mmol) in THF (3.2 ml) at rt. The mixture was stirred for 22 h, diluted with toluene, concentrated, and purified by flash chromatography [step gradient, 10% MeOH-EtOAc followed by MeOH-EtOAc-30% aqueous NH4OH (9: 86: 5) ] yielding the desired primary amine (23.3 mg), which by NMR1H contained 1% trimethylphosphine oxide. Lyophilization from benzene and standing in high vacuum for 2 d provided B1939 (20.3 mg, 87%).

Synthesis of Representative B1939 Analogs: B1930, B1940, B1973, B1987, B1988, B1991, B2003, B2004

ES 2 364 696 T3

B1930 Added Me3P (1 M in THF, 13 µl, 0.013 mmol) to a solution of B1922 (1.6 mg, 2.1 pmol), THF (400 µl) and H2O (100 pl) at rt. The mixture was stirred for 22 h, diluted with toluene, concentrated and azeotroped with toluene (2 *) to give the crude amine which was used directly in the next step.

EDC (0.06 M in CH2CI2, 100 µl, 11 pmol) to a solution of the crude amine, benzoylformic acid (0.8 mg, 5.3 pmol) and CH2CI2 (200 pl) at rt. After 30 min, the reaction was quenched with a 1: 4 mixture of saturated aqueous NaHCO3-brine and extracted with CH2CI2 (5*). The combined extracts were dried over Na2SW4 and concentrated and purified by preparative TLC (EtOAc) to provide B1930 (1.5 mg, 83% for both steps). HRMS (FAB): cale, for C48H63NOi3 + Na 884.4197. Found: 884.4166.

B1940 Using the procedure described above for B1930, B1922 was reduced, coupled with 3-pyridylacetic acid hydrochloride, and purified by preparative TLC [(MeOH-EtOAc-30% NH4Aqueous OH (9: 86: 5)] affording B1940 (0.8 mg, 67% for both steps). HRMS (FAB): cale, for ΰ47Ηβ4Ν2Οι2 + Na 871.4357. Found: 871.4362.

B1973 Using the procedure described above, B1922 (0.9 mg, 1.2 pmol) was reduced, coupled with phenylacetic acid, and purified by preparative TLC (5% MeOH-EtOAc) to provide B1973 (0.44 mg, 44 % for both stages). HRMS (FAB): cale, for C48H65NOi2 + Na 870.4404. Found: 870.4447.

B1987 Using the procedure described above, B1922 (0.9 mg, 1.2 pmol) was reduced, coupled with 3-indolglyoxylic acid, and purified by preparative TLC (3% MeOH-EtOAc) to provide B1987 (0.8 mg, 75 % for both stages). HRMS (FAB): cale, for C5oH84N2I heard3 + Na 923.4306. Found: 923.4338.

B1991 Using the procedure described above, B1922 (1.0 mg, 1.3 pmol) was reduced, coupled with 4-chlorobenzoic acid, and purified by preparative TLC (3% MeOH-EtOAc) to provide B1991 (0.8 mg, 70 % for both stages). HRMS (FAB): cale, for C47H62CINOi2 + Na 890.3858. Found: 890.3843.

B2003 Using the procedure described above, B1922 (1.0 mg, 1.3 pmol) was reduced, acid coupled

3.4.5- trimethoxybenzoylformic and purified by preparative TLC (EtOAc) yielding B2003 (0.7 mg, 56% for the two steps). HRMS (FAB): cale, for C5iH69NOi6 + Na 974.4514. Found: 974.4525.

B2004 Using the procedure described above, B1922 (1.0 mg, 1.3 pmol) was reduced, acid coupled

3.4.5- trimethoxybenzoic and purified by preparative TLC (5% MeOH-EtOAc) yielding B2004 (0.7 mg, 58

ES 2 364 696 T3% for the two stages). HRMS (FAB): cale. CsoHegNO-is + Na 946.4565. Found: 946.4599.

B1930 B1988

B1988 Dess-Martin periodinane (1 mg, 2.3 pmol) was added to a solution of B1930 (0.80 mg, 0.93 pmol) in

CH2CI2 (500 µl) at rt. After 1 hr, the reaction was diluted with Et2Or and leaked through Celite. The filtrate was washed sequentially with a 1: 9 mixture of NaHCO3 aqueous saturated-Na2S20 and brine, dried over Na2SO4, concentrated and purified by preparative TLC (80% EtOAc-hexanes) to provide B1938 (0.45 mg, 56%). HRMS (FAB): cale, for Ci8H6iNOi3 + Na 882.4041. Found: 884.4012.

Synthesis of B2090:

Compound 103. Indium powder (1.35 g, 11.8 mmol) was added to a solution of 102 (3.38 g, 17.6 mmol) in DMF (20 mL) at rt. After stirring for 30 min, the reaction mixture was cooled to 0 ° C. Then the pure aldehyde 101 (3.72 g, 28.6 mmol) was added and the mixture was stirred overnight while allowing the temperature to warm to rt. The reaction mixture was recooled to 0 ° C and then carefully quenched with saturated aqueous NH4Cl (100 mL). After stirring for 30 min, the resulting mixture was extracted with EIO2 (3χ), dried over Na2SO4, concentrated and purified by column chromatography (10% to 20% EtOAc-hexanes) to give 103 pure crystalline (2 , 20 g, 59%).

ES 2 364 696 T3

HSPh

103

104

105

Compound 104. Et was added3N (12 μΙ, 0.51 μιτιοΙ) to a solution of 103 (1.09 g, 5.13 mmol) and thiophenol (0.63 ml, 7.16 mmol) in CH2CI2 and the resulting mixture was stirred at 0 ° C for 1 hr. Filtration through SiO2 gave a mixture of 104 and 105 which after MPLC (15% to 20% EtOAc-hexanes) gave 104 (0.53 g, 32%) and 105 (0.92 g, 56%).

DIBALH

Compound 106. DIBALH (1 M in toluene, 3.28 mL, 3.28 mmol) was added to a solution of 104 (0.53 g, 1.64 mmol) in toluene (10 mL) at -78 ° C and The mixture was stirred at -78 ° C for 10 min. The reaction was quenched by the careful addition of MeOH (0.40 mL, 9.84 mmol) and H2O (0.17 mL, 9.84 mmol), warmed to rt and stirred for 10 min. The white suspension was filtered through a mixture of Celite and SiO2 with 1: 1 CH2CI2-Et2O and concentrated to give 106 (0.53 g, 100%) as an oil.

2) DBU

1) Ph3P = CHCO2Et

106

107

Compound 107. A mixture of 106 (0.53 g, 1.64 mmol) and ethyl (triphenylphosphoranylidene) acetate (1.15 g, 3.29 mmol) in toluene (10 mL) was heated at 80 ° C for 15 h. The mixture was cooled to rt and DBU (25 µΙ, 0.16 mmol) was introduced. The mixture was heated to 80 ° C for 1.5 h, cooled to rt, concentrated and purified by column chromatography (10% to 20% EtOAc-hexanes) to give 107 (0.54 g, 83%) as an oil (3: ratio of α: β isomers).

mCPBA

Compound 108. A solution of mCPBA (-55%, 450 mg in 4.5 ml CH2CI2, 1.44 mmol) to a solution of 107 (0.54 g, 1.36 mmol) in CH2CF2 (10 ml) at -78 ° C. The reaction mixture was diluted with NaHCO3 saturated aqueous

ES 2 364 696 T3 (50 ml), H2O (10 ml) and Et2O (60 ml) and then warmed to rt. The separated aqueous phase was extracted with EtOAc (4χ) and the combined organic phases were dried over Na2SW4, concentrated and purified by column chromatography (50% EtOAc-hexanes) to give 108 (0.51 g, 92%) as an oil.

SOPh

I

Ac¿0

108

Compound 109. A mixture of 108 (0.51 g, 1.24 mmol) and NaOAc (1.00 g, 12.4 mmol) in Ac2O (10 mL) was stirred at 140 ° C for 12 h, cooled to rt and then concentrated. The residue was partitioned between saturated aqueous NaHCO3 (20 mL) and Et2O (30 ml) and stirred vigorously at rt for 30 min. The separated aqueous phase was extracted with Et2O (2 *) and the combined organic phases were dried over Na2SW4, concentrated and purified by column chromatography (5% to 15% EtOAc-hexanes) to give 109 (0.41 g, 73%) as an oil.

1) K2CO3

2) NaBHí

110

Compound 110. A mixture of 109 (0.41 g, 0.91 mmol) and K2CO3 (44.3 mg, 0.32 mmol) in EtOH (5 mL) was heated at 60-70 ° C for 1 d. After cooling to rt, the reaction mixture was concentrated and eluted through a column of SiO2 (10% to 20% EtOAc-hexanes) giving the partially purified aldehyde intermediate. This material was dissolved in EtOH (2.5 ml), treated with NaBH4 (50 mg, 1.33 mmol) and stirred at rt for 30 min. The mixture was concentrated and purified by column chromatography (40% EtOAc-hexanes) to give 110 (181 mg, 66%).

MPMOTCI

110

111

Compound 111. BF was added3OEt2 (0.05 M in CH2CI2, 175 µl, 8.75 pmol) to a solution of 110 (181 mg, 0.60 mmol) and p-methoxybenzyl 2,2,2-trichloroacetimidate (0.50 ml, 1.80 mmol) in CH2CI2 (5 ml) at 0 ° C. The resulting mixture was stirred for 1.5 h at 0 ° C and for 2 h at rt until the reaction was complete. The mixture was quenched with NaHCO3 aqueous saturated (25 ml) and extracted with Et2Or (5 *). The combined organic phases were dried over Na2SW4, concentrated and purified by column chromatography (CH2CI2 and then 20% EtOAc-hexanes) to give 111 semi-pure (0.37 g,> 100%) as an oil.

ES 2 364 696 T3

TsOH

111

112

Compound 112. A mixture of 111 (0.37 g, max = 0.60 mmol) and TSOH H2O (36 mg) in EtOH (5 mL) was stirred initially at rt overnight and then at 60 ° C for 1 h. TsOH H was added2Additional O (31 mg) at rt and the reaction mixture was stirred for 1 h at rt. The mixture was then concentrated, quenched with saturated aqueous NaHCO3, and extracted with EtOAc (5 *). The combined organic phases were dried over Na2SO4, concentrated and purified by column chromatography (20% to 50% EtOAc-hexanes and then 5% MeOH-ChCl) to give 112 (121 mg, 53%) as an oil along with recovered 111 (49 mg , twenty-one%).

OMPM

OH

TBSOTI

TBSO> · '

TBSÓ

112

113

Compound 113. TBSOTf (250 µl, 1.09 mmol) was added to a solution of 112 (121 mg, 0.32 mmol) and EtsN (176 µl, 10 1.26 mmol) in CH2Cl2 at 0 ° C and the mixture resulting was stirred for 25 min. The reaction was quenched with saturated aqueous NaHCO3 (15 mL) and the separated aqueous phase was extracted with ether (3 *). The combined organic phases were dried over Na2SO4, concentrated and purified by column chromatography (5% to 10% EtOAc / hexanes) to give 113 (165 mg, 85%) as an oil.

TBSOx'xand * TBSÓ

I

DIBALH

TBSO '' ~ V TBSÓ

113

114

Compound 114. DIBALH (1 M in toluene, 0.54 ml, 0.54 mmol) was added to a solution of 113 (165 mg, 0.27 mmol) in toluene (5 ml) at -78 ° C and the mixture The resulting mixture was stirred at -78 ° C for 10 min. The reaction was quenched by the careful addition of MeOH (65 µΙ, 0.81 mmol) and H2O (29 µΙ, 0.81 mmol), warmed to rt and stirred for 25 min. The white suspension was filtered through Celite with 1: 1 CH2Cl2-EÍ2O. Concentration and purification by column chromatography (10% to 20% EtOAc-hexanes) gave 114 (153 mg, 100%) as an oil.

ES 2 364 696 T3

TBSO ^> TBSÓ

OMPM

114

B2090. In a manner similar to that described in Scheme 6 for the synthesis of B1794, intermediate 114 was converted to B2090. HRMS (FAB): cale, for C39H56O11 + Na 723.3720. Found: 723.3731.

B2136. In a manner analogous to B1939, B2090 became B2136. HRMS (FAB): cale, for C39H57NO10 + Na

722.3880. Found: 722.3907.

Synthesis of B2039 / B2043:

ES 2 364 696 T3

Diol 201 TBAF (1 M in THF, 383 μΙ, 0.383 mmol) was added to a solution of X2318 (350-LS-218) (80.8 mg, 0.0765 mmol) in THF (7 ml) and stirred at ta for 16 h. After partial concentration, the residue was directly loaded onto a S1O2 column packed using 30% EtOAc-hexanes. Gradient elution 5 (30% EtOAc-hexanes to EtOAc) provided diol 201 (49.7 mg, 92%).

Aldehyde 202 A mixture of Diol 201 (49.7 mg, 0.0707 mmol), NalO4 (100mg, 0.47mmol), MeOH (10ml) and H2O (2.5 ml) was stirred at rt for 30 min. H2O was added and the mixture was extracted with CH2Cl2 (4 *). The combined organic extracts were dried over Na2SO4, concentrated and purified by column chromatography (30% EtOAc-hexanes) to provide aldehyde 202 (41.7 mg, 88%).

Alcohol 203 4-Fluorophenylmagnesium bromide (2M in EIO, 155 µΙ, 0.31 mmol) was added to a solution of aldehyde 202 (41.7 mg, 0.062 mmol) in THF (6 mL). After 15 min at rt, the reaction was quenched with NH4Aqueous CI saturated and extracted with CH2CI2 (4*). The combined organic extracts were dried over Na2SW4, concentrated and purified by preparative TLC (40% EtOAc-hexanes) to provide alcohol 203 (32.4 mg, 68%) as a 1: 1 mixture of C34 isomers. The secondary unwanted C27 isomer was removed at this stage and was also isolated as a 1: 1 mixture of C34 isomers (8.4 mg, 18%).

ES 2 364 696 T3

203

204

Ether 204 Et added3N (18 µΙ, 0.13 mmol) and TBSOTf (15 µΙ, 0.063 mmol) to a solution of alcohol 203 (32.4 mg, 0.042 mmol) in CH2Cl2 (5 ml) at 0 ° C. After 20 min the reaction was quenched by the addition of saturated aqueous NH4CI and extracted with CH2CI2 (3*). The combined organic extracts were dried over Na2SW4, concentrated and purified by column chromatography (20% EtOAc-hexanes) to provide ether 204 (33.1 mg, 89%).

Alcohol 205 LAH (1 M in THF, 113 μΙ, 0.113 mmol) was added dropwise to a solution of ether 204 (33.1 mg, 0.0375 mmol) in Et3O (10 ml) at 0 ° C. After 20 min, H2O and 1M NaOH were added and the mixture was stirred at rt for 10 min. Filtration through Celite, concentration, and purification by column chromatography (40% EtOAc-hexanes) provided alcohol 205 (28.4 mg, 95%).

205 206

Ether 206 Diisopropylethylamine (31 μΙ, 0.18 mmol) and MMTrCI (22 mg, 0.071 mmol) were added to a solution of alcohol 205 (28.4 mg, 0.0356 mmol) in CH2Cl2 (4 ml) at 0 ° C . After 15 h at rt, H2O was added and the mixture was extracted with CH2Cl2 (3 *). The combined extracts were washed with brine, dried over Na2SO4, concentrated and purified by preparative TLC (40% EtOAc-hexanes) to provide ether 206 as a

ES 2 364 696 T3 -1.5: 1 mixture of C34 epimers (45 mg, quantitative) that contained a small amount of impurities that ran nearby.

Alcohol 207 DDQ (40 mg, 0.18 mmol) was added to a solution of ether 206 (37 mg, 0.034 mmol) in CH2Cl2 (4 mL) and a 1:10 mixture of t-BuOH: phosphate buffer at pH 7 ( 2 ml) at 0 ° C. The mixture was vigorously stirred in the dark for 15 min. Three additional portions of DDQ (40 mg, 0.18 mmol) were added at 10 min intervals, then the reaction was diluted with saturated aqueous NaHCO3 and extracted with CH2Cl2 (3χ). The combined organic extracts were washed with brine, dried over Na2SO4, concentrated, and purified by preparative TLC (30% EtOAc-hexanes) to provide alcohol 207 (19.2 mg, 59%), in addition to the recovered ether 206. (9.7 mg, 26%).

Mesylates 208A and 208B Et was added sequentially3N (19 µl, 0.13 mmol) and MS2O (10 mg, 0.056 mmol) to a solution of alcohol 207 (21.3 mg, 0.022 mmol) in CH2Cl2 (6 mL) at 0 ° C. After 30 min saturated aqueous NaHCO3 was added and the mixture was extracted with CH2Cl2 (3 *). The combined extracts were washed with brine, dried over Na2SO4, concentrated and purified by preparative TLC (30% EtOAc-hexanes) to provide mesylates 208A (11.7 mg, 51%) and 208B (6.5 mg, 28%) as individual C34 isomers.

ES 2 364 696 T3

208A and 208B B2039, 02043

B2039 and B2043. In a manner similar to that described in Scheme 6 for the synthesis of B1794, both diastereomers 208A and 208B were independently converted to B2039 and B2043. HRMS (FAB): cal, for C45H59F11 + Na 817.3939. Found: for B2039 817.3896, B2043 817.3910.

Synthesis of B2086, B2088, B2091

1) NalO4

2) NaBH4

10a, 10b

X-20 alcohol NalO was added4 (1.16 g, 5.4 mmol) to a solution of diols 10 a, b (1.19 g, 3.0 mmol) in MeOHH2O (4: 1, 75 ml) at 0 ° C. The reaction mixture was allowed to warm to rt. After stirring for 40 min, the mixture was diluted with EtOAc, filtered through Celite, concentrated, and partitioned between brine and ΟΗ2ΟΙ2. The separated aqueous phase was extracted with CH2CI2 (2*). The combined organic phases were dried over Na2SW4 and concentrated to provide the crude aldehyde intermediate.

NaBH4 (228 mg, 6.0 mmol) was added to a solution of the aldehyde in MeOH-Et2O (1: 1, 40 ml) at 0 ° C. The mixture was stirred for 30 min, carefully quenched with saturated aqueous NH4CI, stirred for 20 min at rt, and extracted with CH2CI2 (3*). The combined extracts were dried over Na2SC> 4, concentrated and purified by flash chromatography (40% to 50% EtOAc-hexanes) to provide the alcohol X-20 (1.03 g, 93% for both steps).

TBSCI

X-20

ES 2 364 696 T3

Silyl Ether 21 Imidazole (0.94 g, 13.9 mmol) and TBSCI (0.59 g, 3.89 mmol) were added sequentially to a solution of the alcohol X-20 (1.02 g, 2.78 mmol) in DMF (10 ml) at rt. After 14 h, the reaction mixture was diluted with NH4Aqueous CI saturated and extracted with EtOAc (3 *). The combined organic extracts were washed with H2Or, brine, dried over Na2SO4, concentrated and purified by flash chromatography (5% to 15% EtOAc-hexanes) to provide silyl ether 21 (1.3 g, 98%).

Alcohol 22 A mixture of Pd (OH)2 (20%, 0.8 g), silyl ether 21 (1.3 g, 2.70 mmol) and EtOAc (30 ml) were stirred for 1 h under 1 atm H2 at rt, filtered through Celite, concentrated and purified by flash chromatography (20% to 40% EtOAc-hexanes) to provide alcohol 22 (0.96 g, 91%).

<td>MeQ</td><td>Oh</td><td>1JTPAP MeQ, OH * ** *</td>

<td>TBSO <^ Ao</td><td>X> OPv</td><td>2) Tebbe TBSOz^x^ O ^ '^OPv3) S-8BN</td>

Alcohol 25 4-methylmorpholine N-oxide (980 mg, 8.4 mmol) and TPAP (131 mg, 3.26 mmol) were added sequentially to a solution of alcohol 22 (1.78 g, 4.6 mmol) in CH2CI2 (45 ml) at rt. A cold bath was necessary to control the exotherm. After 20 min, the reaction mixture was diluted with hexanes, filtered through a column of SiO2 short (15% EtOAc-hexanes) and concentrated to give the crude ketone.

Tebbe's reagent (14.9 ml, 9.0 mmol) was added over 10 min to a solution of the crude ketone in THF (60 ml) at 0 ° C. After 20 min, the reaction mixture was poured into Et2O (100 ml) which was previously cooled to -78 ° C, quenched by slow addition of H2O (30 ml), warmed to rt, stirred for 30 min and extracted with Et2Or (4 *). The combined extracts were washed with brine, dried over Na2SO4, concentrated and purified by flash chromatography (10% EtOAc-hexanes) to provide the desired olefin contaminated by the geminal dimethyl product (1.07 g). This mixture was used directly in the next stage.

9-BBN (0.5 M in THF, 11.6 ml, 5.8 mmol) was added to a solution of the olefin in THF (15 ml) at 0 ° C. The reaction mixture was allowed to warm to rt, stirred for 5 h, and then cooled to 0 ° C. H2O (60 ml), THF (60 ml) and NaBO3-4 H2O (5.7 g). After stirring for 5 h at rt, THF was removed under reduced pressure and the aqueous residue was extracted with EtOAc (4 *). The combined organic extracts were washed with brine, dried over Na2SO4, concentrated and purified by flash chromatography (20% to 40% EtOAc-hexanes) to provide alcohol 25 (605 mg, 18% for all three steps).

MeO and — OH

TBSO ^ Q1) Swem

2) Et3N

3) NaBH4

Alcohol 26 Using the previously described procedure, alcohol 25 (604 mg, 1.49 mmol) was sequentially oxidized, isomerized, and reduced. Purification by flash chromatography (20% to 40% EtOAchexanes) provided alcohol 26 (550 mg, 91% for all three steps).

ES 2 364 696 T3

MPMOTCI

MPM 27 Ether BF3OEÍ2 (0.05 M in CH2Cl2, 270 μΙ, 0.013 mmol) was added to a solution of alcohol 26 (545 mg, 1.35 mmol) and MPM trichloroimidate (1.14 g, 4.0 mmol ) in CH2CI2 (40 ml) at 0 ° C. After 1 hr, the reaction was quenched with saturated aqueous NaHCO3, extracted with CH2CI2, dried over Na2SW4, concentrated and purified by flash chromatography (10% to 15% EtOAc-hexanes) to provide the ether of MPM 27 (580 mg, 82%).

Alcohol 28 LAH (1 M in THF, 1.9 ml, 1.9 mmol) was added to a solution of the ether of MPM 27 (580 mg, 1.11 mmol) in EIO2 (100 ml) at 0 ° C. After 30 min, the reaction was carefully quenched with H2O (0.5 mL) and 1N aqueous NaOH (0.5 mL), stirred for 1 h at rt, filtered through Celite, concentrated, and purified by Flash chromatography (30% to 50% EtOAc-hexanes) to provide alcohol 28 (460 mg, 95%).

1) Swem

2) WWg

Oleflna 29 DMSO (441 µΙ, 6.23 mmol) was added to a solution of oxalyl chloride (272 µΙ, 3.12 mmol) in CH2Cl2 (30 mL) at -78 ° C. After 15 min a solution of alcohol 28 (458 mg, 1.04 mmol) in CH2Cl2 (15 ml) was added to the reaction mixture. After stirring for 1 h at -78 ° C Et3N (1.3 ml, 9.35 mmol). The reaction mixture was heated to 0 ° C, stirred for 10 min, diluted with NH4Aqueous CI saturated and extracted with CH2Cl2 (3χ). The combined organic extracts were dried over Na2SO4, concentrated and filtered through a short SIO2 column (20% to 30% EtOAc-hexanes) to provide the crude aldehyde.

N-BuL¡ (1.63 M, 1.4 ml, 2.28 mmol) was added dropwise to a solution of CHsPPhsBr (815 mg, 2.28 mmol), THF (20 ml) and DMSO (7, 5 ml) at 0 ° C. After 1 h a solution of the aldehyde in THF (10 ml) was added. The reaction mixture was warmed to rt and stirred for 3 h. NH was added4Saturated aqueous CI and the mixture was extracted with EtOAc (4 *). The combined organic extracts were washed with H2Or, brine, dried over Na2SW4] They were concentrated and purified by flash chromatography (10% to 15% EtOAc-hexanes) to provide definition 29 (380 mg, 95% yield for the 2 steps).

2) PvCI

ES 2 364 696 T3

Compound 31 9-BBN (0.5 M in THF, 6 ml, 3 mmol) was added to a solution of olefin 29 (370 mg, 0.85 mmol) in THF (7 ml) at 0 ° C. The mixture was allowed to warm to rt and stirred for 1 hr. After being cooled back to 0 ° C, H2O (30 ml), THF (20 ml) and NaBO were added3* 4 H2O (2.8 g). After stirring for 3 h at rt, THF was removed under reduced pressure. The aqueous residue was extracted with EtOAc (4 *), dried over Na2SO4, concentrated and purified by flash chromatography (25% to 50% EtOAc-hexanes) to provide alcohol 30 which was used directly in the next step.

Pivaloyl chloride (157 µl, 1.27 mmol) was added to a solution of alcohol 30 in CH2CI2-pyridine (1: 1 mixture, 10 mL) at rt. After 18 hr, additional pivaloyl chloride (100 µΙ, 0.81 mmol) was added. After 1 hr, the reaction mixture was cooled to 0 ° C, quenched with MeOH (0.5 mL), concentrated, diluted with brine, and extracted with CH2Cl2 (4 *). The combined organic extracts were dried over Na2SW4, concentrated and purified by flash chromatography (10% to 15% EtOAc-hexanes) to provide compound 31 (410 mg, 90% for both steps).

TBAF

Alcohol 32 TBAF (1M in THF, 1.14 mL, 1.14 mmol) was added to a solution of 31 (410 mg, 0.761 mmol) in THF (5 mL) at rt. After 1.5 h the reaction mixture was concentrated and purified by flash chromatography (40% EtOAc-hexanes to 100% EtOAc) to provide alcohol 32 (320 mg 100%).

33a: C34a-OH 33b: C34 0-OH

Alcohols 33a and 33b Dess-Martin periodinane (925 mg, 2.18 mmol) was added to a solution of alcohol 32 (309 mg, 0.727 mmol) in CH2Cl2 (19 mL) at rt. After 1 hr, the reaction was diluted with EIO2 and filtered through Celite. The filtrate was washed sequentially with a 1: 9 mixture of NaHCO3 saturated aqueous-Na2S203 and brine, dried over Na2SW4, concentrated and purified by flash chromatography (20% to 30% EtOAc-hexanes) to provide the desired aldehyde, which was carried out immediately to the next step.

BF2'OEt2 (135 µΙ, 1.1 mmol) was added to a solution of the crude aldehyde, tri-n-butylalyltin (337 µΙ, 1.08 mmol) and CH2Cl2 (16 ml) at -78 ° C. After 1 hr, the reaction was quenched with NaHCO3 aqueous saturated and extracted with CH2Cl2 (3 *). The combined organic extracts were dried over Na2SO4, concentrated, and purified by MPLC (25% to 30% EtOAc-hexanes) to provide the more polar major alcohol 33a (165 mg, 49% for both steps) and the minor product 33b less polar (90 mg, 27% for the two stages).

TBSOTf

Compound 34. TBSOTf (163 µΙ, 0.710 mmol) was added to a solution of alcohol 33a (165 mg, 0.355 mmol), Et3N (247 μΙ, 1.78 mmol) and ΟΗ2ΟΙ2 (5 ml) at 0 ° C. After 25 min, the reaction was quenched with NaHCO2 aqueous saturated, extracted with CH2Cl2 (3 *), dried over Na2SW4, concentrated and purified by flash chromatography (15% to

ES 2 364 696 T3

20% EtOAc-hexanes) to provide compound 34 (200 mg, 98%).

Diools 35a and 35b OsO was added4 (0.1 M solution in toluene, 32 µl, 3.2 pmol) to a solution of K2CO3 (168 mg, 1.22 mmol), K3Faith (CN)6 (400mg, 1.32mmol), (DHQ)2PYR (11 mg, 12 pmol), H2O (3.2 ml) and t-BuOH (2.2 ml) at 0 ° C. Then a solution of definition 34 (200 mg, 0.345 mmol) in t-BuOH (1 ml) was added to the reaction mixture. After 5 h at 0 ° C Na was added2S2O5'5 H2O (200 mg). The reaction mixture was warmed to rt, stirred for 30 min, and extracted with CH2CI2 (5*). The combined organic extracts were washed with brine, dried over Na2SW4, concentrated and purified by preparative TLC (70% EtOAc-hexanes) to provide the less polar major diol 35a (118 mg, 56%) and the more polar minor diastereomeric product 35b (74 mg, 35%). The individual diastereomers were each transferred separately.

Compound 36. TBSOTf (177 µl, 0.77 mmol) was added to a solution of diol 35a (118 mg, 0.192 mmol), Et3N (267 pl, 1.92 mmol) and CH2CI2 (5 ml) at 0 ° C. After 25 min, the reaction was quenched with NaHCO3 aqueous saturated, extracted with CH2CI2 (3 *), dried over Na2SW4, concentrated and purified by flash chromatography (10% to 15% EtOAc-hexanes) to provide compound 36 (161 mg, 100%).

THE H

Alcohol 37 Using the procedure previously described for the preparation of alcohol 28, compound 36 (161 mg, 0.192 mmol) provided alcohol 37 (135 mg, 93%) after purification by flash chromatography (20% to 40% EtOAc -hexanes).

Dess-Martin

Aldehyde 38 Dess-Martin periodinane (227 mg, 0.535 mmol) was added to a solution of alcohol 37 (135 mg, 0.178 mmol) in CH2CI2 (5 ml) at rt. After 1 hr, the reaction mixture was diluted with Et2Or and it leaked through Celite. The filtrate was washed sequentially with a 1: 9 mixture of NaHCO3 saturated aqueous-Na2S203 and brine, it

ES 2 364 696 T3 dried over Na2SO4, concentrated and purified by flash chromatography (10% to 20% EtOAc-hexanes) to provide aldehyde 38 (127 mg, 95%).

B2086, B2102. Each of the diastereomers obtained above was carried separately into the final product in a manner similar to that described in Scheme 6 for B1794. Diastereomer 35a provided B2086. Diastereomer 35b provided B2102.

B2088 NalO was added4 to a solution of B2086 (1 mg, 1.29 pmol) in MeOH-H2O (4: 1, 1 mL) at rt. After 30 min, the reaction mixture was diluted with H2O, extracted with CH2Cl2 (6X), dried over Na2SO4 and was concentrated to provide B2088 (1.2 mg).

ES 2 364 696 T3

NaBH4

B2088

B2091 NaBH added4 (0.013 M in EtOH, 20 μΙ, 0.27 pmol) to a solution of B2088 (1 mg, 1.29 pmol) in MeOHCH2CI2 (4: 1. 0.5 ml) at -78 ° C. NaBH was added periodically4 additional with close monitoring of the reaction by TLC (a total of 220 μΙ of the NaBH solution was required4). The reaction mixture was quenched at 0 ° C with NH4Aqueous saturated CI, stirred for 20 min at rt and extracted with CH2CI2 (6 *). The combined extracts were dried over Na2SW4, concentrated and purified by preparative TLC (7% MeOH-EtOAc) to provide B2091 (0.40 mg, 50%).

Synthesis of B1933:

9-BBN

302

303

Alcohol 303 9-BBN (0.5 M in THF, 23 ml, 0.012 mol) was added dropwise over 30 min to a solution of alkene 302 (1.51 g, 0.00386 mol) in THF (40 ml) at 0 ° C. After stirring at rt for 80 min, the mixture was cooled to 0 ° C and H was carefully added2O (80 ml) followed by NaBO3* 4 H2O (4.2 g, 0.027 mol). The mixture was vigorously stirred at rt for 2.3 h, then extracted with EtOAc (3 *). The combined organic extracts were washed with brine, dried over Na2SW4, concentrated and purified by column chromatography (50% EtOAc-hexanes) to provide alcohol 303 (1.37 g, 87%).

Swem

303

304

Aldehyde 304 Oxalyl chloride (88 µl, 1.00 mmol) was added dropwise to a solution of DMSO (142 µl, 2.00 mmol) in CH2CI2 (20 ml) at -78 ° C. After 30 min a solution of alcohol 303 (137 mg, 0.335 mmol) in CH2CI2 (5 ml) and stirred at -78 ° C for 1 hr. Et was added3N (420 µl, 3.01 mmol) and after 10 min the reaction was stirred for 10 min at 0 ° C, at which point NH was added4Saturated aqueous CI and the resulting mixture was extracted with CH2CI2 (3*). The combined organic extracts were washed with brine, dried over Na2SW4, I know

ES 2 364 696 T3 were concentrated and purified by flash chromatography (50% EtOAc-hexanes) to provide intermediate aldehyde 304 (0.114 g, 84%) which was used immediately in the next step.

MeO

OHC * ^

1) CF3TMS

2) TBAF

304 305

Alcohol 305 TBAF (1 M in THF, 5 µl, 0.005 mmol) was added to a solution of aldehyde 304 (0.114 g, 0.27 mmol) in CF3TEM (0.5 M in THF, 1.1 ml, 0.54 mmol) at 0 ° C. After 20 min a second portion of TBAF (1 M in THF, 100 µl, 0.1 mmol) was added and the mixture was stirred for 10 min, at which time excess TBAF (1 M in THF, 270 µΙ, 0.27 mmol) to cleave the intermediate silyl ether. After 30 min, the mixture was diluted with H2O and extracted with EtOAc (3 *). The organic extracts were washed with H2O, brine, dried over Na2SO4, concentrated, and purified by column chromatography (50% EtOAc-hexanes) to provide alcohol 305 (123 mg, 95%) as an inseparable 1: 1 mixture. of isomers.

TBSOTf

TBSO

305

306

Silyl ether 306 TBSOTf (265 μΙ, 1.16 mmol) was added to a solution of alcohol 305 (123 mg, 0.257 mmol) and EtsN (430 μΙ, 3.08 mmol) in ΟΗ2ΟΙ2 (8 ml) at 0 ° C. After stirring at rt for 20 h NaHCO was added3 aqueous saturated and the mixture was extracted with CH3CI3 (3 *). The combined organic extracts were washed with brine, dried over Na2SO4, concentrated, and purified by column chromatography (20% EtOAc-hexanes) to provide silyl ether 306 (148 mg, 97%).

Alcohol 307 LAH (1 M in THF, 220 µΙ, 0.22 mmol) was added dropwise to a solution of silyl ether 306 (131 mg, 0.22 mmol) in EIO2 (5 mL) at 0 ° C. After 20 min, H2O and 1M NaOH were carefully added. The mixture was stirred at rt 30 min, filtered through glass wool, concentrated, and purified by column chromatography (50% EtOAc-hexanes) to provide the alcohol 307 (112 mg, quant.).

1) Swem

2) Wlttig

ES 2 364 696 T3

Alkene 309 Oxalyl chloride (58 µΙ, 0.66 mmol) was added dropwise to a solution of DMSO (94 µΙ, 1.3 mmol) in CHCl2 (10 ml) at -78 ° C. After 30 min a solution of alcohol 307 (112 mg, 0.22 mmol) in CH2CI2 (3 ml). After 1 h, Et3N (276 µΙ, 1.98 mmol) and after 10 min at -78 ° C the reaction was stirred at 0 ° C for 10 min. Saturated aqueous NH4Cl was added and the mixture was extracted with CH2Cl2 (3 *). The combined organic extracts were washed with brine, dried over Na2SO4, concentrated and purified by flash chromatography (50% EtOAc-hexanes) to provide aldehyde 308 (101 mg, 91%) which was used immediately in the next step.

N-BuLi (1.63 M in THF, 200 μΙ, 0.33 mmol) was added dropwise to a solution of CH3PPh3Br (118 mg, 0.33 mmol) in THF (3 ml) and DMSO (1.2 ml) at 0 ° C. After 70 min a solution of aldehyde 308 (101 mg, 0.20 mmol) in THF (3 ml) was added and after 10 min at 0 ° C the reaction was stirred at rt for 1 h. NH was added4Aqueous saturated CI and the mixture was extracted with EtOAc (3 *). The combined organic extracts were washed with brine, dried over Na2SO4, concentrated and purified by column chromatography (20% EtOAc-hexanes) to provide alkene 309 (90.9 mg, 90%).

9-BBN

TBSO

309

310

Alcohol 310 9-BBN (0.5 M in THF, 17 mL, 8.45 mmol) was added dropwise to a solution of alkene 309 (1.06 g, 2.11 mmol) in THF (30 mL) to 0 ° C. After stirring for 2.5 h at rt, the reaction was cooled to 0 ° C and H2O (60 ml) followed by NaBO3-4 H2O (3.25 g, 21.1 mmol). The mixture was vigorously stirred at rt for 2 h, then diluted with H2O and extracted with EtOAc (3 *). The combined organic extracts were washed with brine, dried over Na2SW4, concentrated and purified by column chromatography (20% to 30% EtOAc-hexanes) to provide alcohol 310 (0.920 g, 84%).

PvCI

310

311

Pivaloate 311 A mixture of alcohol 310 (65.8 mg, 0.0126 mmol), pyridine (61 μΙ, 0.76 mmol) and PvCI (23 μΙ, 0.189 mmol) in CH2Cl2 (3 ml) was stirred at rt for 5 h. A second reaction using alcohol 310 (0.92 g, 1.76 mmol) was run under similar conditions and both reactions were combined during workup: saturated aqueous NH4Cl was added and the mixture was extracted with CH2Cl2 (3 *). The combined organic extracts were washed with brine, dried over Na2SO4, concentrated, and purified by column chromatography (20% EtOAc-hexanes) to provide pivaloate 311 (1.08 g, quant).

312

Alcohol 312 A mixture of ether 311 (0.811 g, 1.33 mmol), DDQ (6.1 g, 27 mmol) and BuOH 10: 1: phosphate buffer pH 7 (42 ml) in ΟΗ2ΟΙ2 (84 ml) was stirred vigorously in the dark at rt for 1.5 h at which time additional DDQ (1.0 g, 4.4 mmol) was added. After 1 h NaHCO was added3 aqueous saturated and the mixture was extracted with CH2Cl2 (4 *). The combined organic extracts were washed successively with saturated aqueous NaHCO2 and

ES 2 364 696 T3 brine, dried over Na2SO4, concentrated and purified by column chromatography (20% EtOAc-hexanes) to provide alcohol 312 (0.56 g, 87%), in addition to the recovered starting material 311 (97 mg, 12%).

TBSO

Swem

312

313

Ketone 313 Oxalyl chloride (21 µl, 0.12 mmol) was added dropwise to a solution of DMSO (34 µΙ, 0.48 mmol) in CH2Cl3 (3 ml) at -78 ° C. After 1 h a solution of alcohol 312 (39.4 mg, 0.081 mmol) in ΟΗ2ΟΙ2 (1.5 ml) and the mixture was stirred for 1.5 h. EtsN (100 µΙ, 0.73 mmol) was added and after 10 min the mixture was heated to 0 ° C. NH was added4Saturated aqueous CI and the mixture was extracted with CF2CI3 (3χ). The combined organic extracts were washed with brine, dried over Na2SO4, concentrated and purified by flash chromatography (30% EtOAc-hexanes) to provide ketone 313 (36.6 mg, 93%) which was used immediately in the next step.

<td>MeQJ_</td><td>Tebbe</td>

313

314

Alkene 314 Tebbe's reagent (-0.65 M in toluene, 720 μΙ, 0.47 mmol) was added dropwise to a solution of ketone 313 (151 mg, 0.31 mmol) in THF (5 mL) to 0 ° C. After 15 min, H2O was carefully added and the mixture was extracted with EtoAc (3 *). The combined organic extracts were washed with brine, dried over Na2SO4, concentrated and purified by column chromatography (10% EtOAc-hexanes) to provide alkene 314 (139 mg, 93%).

315

Alcohol 315 9-BBN (0.5 M in THF, 6.0 mL, 2.9 mmol) was added dropwise to a solution of alkene 314 (468 mg, 0.97 mmol) in THF (10 mL) to 0 ° C. The mixture was stirred at rt for 2 h, at which time additional 9-BBN (0.5 M in THF, 500 µΙ, 0.25 mmol) was added. After 2.5 h, the mixture was cooled to 0 ° C and H2O (10 mL) was carefully added followed by NaBO3'4 H2O (1.5 g, 9.7 mmol). The mixture was vigorously stirred at rt for 5 h, diluted with H2O and extracted with EtOAc (3 *). The combined organic extracts were washed with brine, dried over Na2§O4, concentrated and purified by column chromatography (gradient 20% to 30% EtOAc-hexanes) to provide alcohol 315 (0.47 g, 97%).

ES 2 364 696 T3

—OH

TBSO

1) Swem x ^ OPv 2) Et3N

3) NaBH4

TBSO

315 316

Alcohol 316 Oxalyl chloride (246 µΙ, 2.82 mmol) was added dropwise to a solution of DMSO (400 µΙ, 5.64 mmol) in CH2Cl2 (40 mL) at -78 ° C. After 1 h a solution of alcohol 315 (0.47 g, 0.94 mmol) in CH2CI2 (10 ml) and the mixture was stirred for 1h. Et was added3N (1.2 ml, 8.5 mmol), and after 10 min the mixture was warmed to 0 ° C and stirred for 10 min. Saturated aqueous NH4Cl was added and the mixture was extracted with CH2Cl2 (3 *). The combined organic extracts were washed with brine, dried over Na2SO4, and concentrated. The crude aldehyde was stirred in CH2Cl2 (20 mL) and Et3N (2 ml) at rt overnight. Saturated aqueous NH4Cl was added and the mixture was extracted with CH2Cl2 (3 *). The combined organic extracts were washed with brine, dried over Na2SO4, concentrated, and purified by flash chromatography (30% EtOAc-hexanes) to provide the epimerized aldehyde which was immediately dissolved in 1: 1 EI2O: EtOH (10 mL) and cooled to 0 ° C. NaBH was added4 (35 mg, 0.94 mmol) and after 10 min the reaction was quenched with NH4Saturated aqueous CI. The mixture was extracted with EtOAc (3 *) and the combined organic extracts were washed with brine, dried over Na2SO4, concentrated and purified by column chromatography (30% EtOAc-hexanes) to provide alcohol 316 (0.410 g, 87% yield for all 3 steps).

MPMOTCI

317

Ether 317 Alcohol 316 (60.7 mg, 0.12 mmol) and MPMOTCI (0.10 g, 0.36 mmol) were combined, azeotroped in toluene (3 *) and dried under high vacuum overnight. CH2Cl2 (3 ml) was added and the mixture was cooled to 0 ° C. BF was added3OEI2 (approx. 1 µΙ, 0.01 mmol) and after stirring for 10 min the reaction was quenched with saturated aqueous NH4Cl. The mixture was extracted with CH2Cl2 (3 *) and the combined extracts were washed with brine, dried over Na2SW4, concentrated and purified by preparative TLC (30% EtOAc-hexanes) to provide ether 317 (55.4 mg, 74%).

317

318

Alcohol 318 LAH (1M in THF, 104 µΙ, 0.104 mmol) was added dropwise to a solution of ether 317 (54 mg, 0.087 mmol) in EIO2 (5 mL) at 0 ° C. After 30 min, H2O and 1M NaOH were carefully added. The mixture was stirred at rt for 10 min, filtered through glass wool, concentrated, and purified by column chromatography (30% -50% EtOAc- hexanes) to provide alcohol 318 (45.5 mg, 98%).

ES 2 364 696 T3

Swern

TBSO

OMPM

TBSO

318

319

Aldehyde 319 Oxalyl chloride (11 µΙ, 0.13 mmol) was added dropwise to a solution of DMSO (18 µΙ, 0.25 mmol) in CH2Cl2 (2 ml) at -78 ° C. After 1.8 h a solution of alcohol 318 (22.6 mg, 0.042 mmol) in CH2CI2 (1 ml) and the mixture was stirred for 1h. Et was added3N (53 µΙ, 0.38 mmol) and after 10 min reaction 5 was warmed to 0 ° C and stirred 10 min. Saturated aqueous NH4CI was added and the mixture was extracted with CH2CI3 (3*).

The combined organic extracts were washed with brine, dried over Na2SC> 4, concentrated and purified by flash chromatography (20% EtOAc-hexanes) to provide aldehyde 319 (21.7 mg, 97%).

319

B1933. In a manner similar to that described in Scheme 6 for the synthesis of B1794, intermediate 319 was converted to B1933. HRMS (FAB): cal, for CH41H57F3O11 + H 783.3931. Found: 783.3940.

1) NalO4

2) CF3TMS

3) TBAF

4) Dess-Martin

B1896. B1897

B1942. A mixture of B1896 / B1897 (2 mg, 2.73 pmol), NalO4 (35mg, 0.16mmol), MeOH (0.8ml) and H2O (0.2 ml) 65

ES 2 364 696 T3 was stirred at rt for 30 min. The reaction mixture was then diluted with H2O (3 mL) and extracted with CH2Cl2 (6 *) and EtOAc (2 *). The combined organic phases were dried over Na2SW4 and purified by column chromatography (5% MeOH-CH2CI2) giving the desired aldehyde.

This material was dissolved in THF (0.1 ml), cooled to 0 ° C and treated with 0.5 M CF3TMS in THF (30 µl, 15 mmol) followed by 0.05 M TBAF in THP (5 ml, 0.025 mmol). After stirring for 30 min, the reaction mixture was diluted with saturated aqueous NaHCO3 (2 mL) and H2O (1 ml), extracted with EtOAc (6χ), dried over Na2SW4, filtered and concentrated to give the crude bis-TMS ether.

This material was dissolved in THP (0.5 ml) and treated with 1 M TBAF in THF containing 0.5 M imidazole hydrochloride (8 µl, 8 pmol) at rt for 30 min. The reaction mixture was eluted through a column of SiO2 (50% EtOAchexanes to EtOAc) providing the diol intermediate.

A mixture of this product and Dess-Martin periodinane (10 mg, 24 mmol) in CH2CI2 (0.5 ml) was stirred at rt for 1h, diluted with Et2O (5 ml) and filtered through Celite. The filtrate was concentrated and purified by preparative TLC (50% EtOAc-hexanes) to provide B1942 (1.5 mg, 72% for all 5 steps). HRMS (FAB): cale, for CD ^ HssFsOn + H 767.3516. Found: 767.3542

Synthesis of B2070 / B2073:

1) NalO4

2) CH2 = CHCF2Br In

X400 401

Alcohol 401 A mixture of NalO4, (375 mg, 1.74 mmol), X400 (674 mg, 1.58 mmol), MeOH (16 ml) and H2O (4 ml) was stirred at rt for 1h. After dilution with H2Or, the mixture was extracted with CH2CI2 (4x) and the combined organic extracts were dried over Na3SW4, concentrated and purified by flash chromatography (30% EtOAc-hexanes) to provide the intermediate aldehyde (570 mg), which was immediately dissolved in DMF (15 ml). Indium (275 mg, 2.4 mmol) and 3-bromo-3,3-difluoropropene (240 µl, 2.4 mmol) were added and after stirring at rt for 17 h H2O and 0.1 M HCl. The mixture was extracted with EtOAc (3x) and the combined organic extracts were washed successively with H2O and brine, dried over Na2SW4, concentrated and purified by column chromatography (20% to 30% EtOAc-hexanes) to provide alcohol 401 as a 1: 1 mixture of C34 isomers (605 mg, 81% for the 2 steps).

ΗΟΜβ <\ —PMPM 9 Bear4

2) NalO4 FF 3) NaBH.

FF

401 402

Diol 402 A mixture of OsO4 (1 crystal), alcohol 401 (605 mg, 1.28 mmol), 4-methyl-morpholine / V-oxide (0.45 g, 3.84 mmol), acetone (30 ml) and H2O (6 ml) was stirred at rt for 29 h. OsO added4 (3 crystals) and 4-methylmorpholine dioxide (0.1 g, 0.8 mmol) and after 2 days Na was added2S2OR3 saturated aqueous. The mixture was extracted with CH2CI2 (6x) and the combined organic extracts were dried over Na2SW4 and they concentrated. The crude intermediate triol immediately dissolved in 4: 1: MeOH: H2O (25 ml) and NalO was added4 (0.41 g, 1.9 mmol). After vigorously stirring at rt for 2 h, the mixture was diluted with H2Or, it was extracted with CH2CI2 (3 *) and the combined organic extracts were dried over Na2SW4 and concentrated to provide the intermediate aldehyde which was immediately dissolved in 1: 1 EtOH-Et2O (30 ml) and cooled to 0 ° C. NaBH was added4 (48 mg, 1.3 mmol) and after 20 min the reaction was quenched with H2O and extracted with CH2CI2 (4*). The combined organic extracts were dried over Na2SW4, concentrated and purified by column chromatography (50% EtOAc-hexanes) to provide diol 402 (485 mg, 80% over 3 steps).

ES 2 364 696 T3

402

403

Silyl ether 403 TBSOTf (2.3 ml, 10 mmol) was added dropwise to a mixture of the diol 402 (485 mg, 1.0 mmol), Et3N (2.8 ml, 20 mmol) and CH2Cl2 (30 ml) at 0 ° C. After stirring for 1 h at rt, saturated aqueous NH4Cl was added and the mixture was extracted with CH2Cl2 (3 *). The combined organic extracts were washed with brine, dried over Na2SO4, concentrated and purified by column chromatography (20% EtOAc-hexanes) to provide silyl ether 403 (668 mg, 95%).

Alcohol 404 LAH (1 M in THF, 2.8 mL, 2.8 mmol) was added dropwise to a solution of silyl ether 403 (668 mg, 0.948 mmol) in EIO2 (60 mL) at 0 ° C. After 15 min, H2O and 1M NaOH were carefully added. The mixture was stirred at rt for 20 min, filtered through glass wool, concentrated, and purified by column chromatography (30% EtOAc-hexanes) to provide alcohol 404 (500mg, 85%).

404

Aldehyde 405 Oxalyl chloride (210 µl, 2.42 mmol) was added dropwise to a solution of DMSO (345 µΙ, 4.84 mmol) in CH2Cl2 (30 ml) at -78 ° C. After 1 h a solution of alcohol 404 (500 mg, 0.806 mmol) in CH2Cl2 (10 ml) was added. After 40 min Et3N (1.0 ml, 7.2 mmol). After stirring at -78 ° C for 10 min, the reaction mixture was warmed to 0 ° C and stirred for an additional 10 min. Saturated aqueous NH4Cl was added and the mixture was extracted with CH2Cl2 (3 *). The combined organic extracts were washed successively with H2O, brine, dried over Na2SO4, and concentrated. Purification by flash chromatography (30% EtOAc-hexanes) provided aldehyde 405 (486 mg, 98%) which was used immediately in the next step.

MeQ OMPM

TBStp '' ¡—ζ Wtttlg

TBso ^ Xy ^ Ar> cho

FF

405

406

Alkene 406 n-BuLi (1.63 M, 860 μΙ, 1.4 mmol) was added dropwise to a solution of CH3PPh3Br (500mg, 1.4mmol) in THF (15ml) and DMSO (6ml) at 0 ° C. After 1 hr a solution of aldehyde 405 (486 mg) in THF (15 ml) was added. The reaction mixture was warmed to rt and stirred for 30 min. Saturated aqueous NH4CI was added, the mixture was extracted with EtOAc (3x), and the combined extracts were washed successively with H2O and brine, dried over Na2SO4, concentrated and purified by column chromatography (20% EtOAc-hexanes) to provide alkene 406 (450 mg, 93%).

ES 2 364 696 T3

Ester 407 9-BBN (0.5 M in THF, 9.0 ml, 4.5 mmol) was added dropwise to a solution of alkene 406 (0.460 g, 0.746 mmol) in THF (10 ml) at 0 ° C. After warming to rt, the mixture was stirred for 3 h and two additional portions of 9-BBN (0.5 M in THF, 3.0 mL, 1.5 mmol) were added at 30 min intervals. The reaction mixture was recooled to 0 ° C, after which THF (10 mL), H2O (10 mL) and NaBO3-4 H2O (1.72 g, 11.3 mmol) were carefully added. The mixture was vigorously stirred at rt for 1.5 h and additional NaBO3-4H2O (1.0 g, 6.5 mmol) was added. After 2 h the mixture was diluted with H2O and extracted with EtOAc (3 *). The combined extracts were washed with brine, dried over Na2SO4, concentrated and purified by column chromatography (20% to 30% EtO Ac-hexanes) to provide the intermediate alcohol (509 mg) which was immediately dissolved in CH2Cl2 (10 ml) and treated with pyridine (600 μΙ, 7 , 5 mmol) and PvCI (275 µΙ, 2.2 mmol). After 6 h NH was added4Saturated aqueous CI and the mixture was extracted with ΟΗ2ΟΙ2 (3*). The combined organic extracts were washed with brine, dried over Na2SO4, concentrated and purified by column chromatography (20% to 30% EtOAc-hexanes) to provide ester 407 (423 mg, 79% for the 2 steps).

407

408

Alcohol 408 A mixture of ester 407 (11 mg, 0.015 mmol) and Pd (OH) 2 / C (10 mg) in EtOAc (500 µΙ) was vigorously stirred under an atmosphere of H2 at rt for 6 h. The mixture was filtered through Celite, concentrated, and purified by column chromatography (30% EtOAc-hexanes) to provide alcohol 408 (9.4 mg, quant).

408

Alkene 409 Oxalyl chloride (7 µΙ, 0.075 mmol) was added dropwise to a solution of DMSO (11 µΙ, 0.15 mmol) in CH2Cl2 (2 mL) at -78 ° C under N2. After 40 min a solution of alcohol 408 (15.2 mg, 0.025 mmol) in CH2CI2 (1 ml) and the reaction was stirred at -78 ° C for 1 hr. Et was added3N (31 µΙ, 0.22 mmol) and after stirring for 10 min the mixture was heated to 0 ° C. After 10 min the reaction mixture was quenched with NH4Aqueous CI saturated and extracted with CH2Cl2 (3 *) The combined extracts were washed successively with H2O and brine, dried over Na2SO4 and they concentrated. After flash chromatography (30% EtOAchexanes), the intermediate ketone (13 mg) was immediately dissolved in THF (500 μΙ) and treated with Tebbe's reagent (-0.65 M in toluene, 62 μΙ, 0.040 mmol) at 0 ° C. After 1.5 h additional Tebbe reagent (-0.65 M in toluene, 62 µΙ, 0.040 mmol) was added and after 10 min H2O and then brine were carefully added. The mixture was extracted with EtOAc (3 *) and the combined organic extracts were washed with brine, dried over Na2SW4, concentrated and purified by column chromatography (10% EtOAc-hexanes) to provide the alkene

ES 2 364 696 T3

409 (11.9 mg, 80% for the 2 stages).

409 410

Alcohol 410 9-BBN (0.5 M in THF, 1.5 ml, 0.72 mmol) was added dropwise to a solution of alkene 409 (0.144 g, 0.242 mmol) in THF (2 ml) at 0 ° C. After warming to rt, the mixture was stirred for 3 h. The reaction mixture was recooled to 0 ° C, after which THF (2 mL), H2O (2 mL) and NaBO3-4 H2O (0.38 g, 2.4 mmol) were carefully added. The mixture was vigorously stirred at rt for 4 h, diluted with H2O and extracted with EtOAc (3 *). The combined extracts were washed with brine, dried over Na2SO4, concentrated and purified by column chromatography (20% EtOAc-hexanes) to provide alcohol 410 (0.140 g, 94%).

1) Swem

2) EtsN

3) NaBHí

410

Alcohol 411 Oxalyl chloride (26 µΙ, 0.30 ml) was added dropwise to a solution of DMSO (43 µΙ, 0.60 mmol) in CH2CI2 (4 ml) at -78 ° C. After 1 hr a solution of alcohol 410 (57 mg, 0.093 mmol) in CH2Cl2 (2 ml) was added. After 45 min Et3N (125 µΙ 0.90 mmol). After stirring at -78 ° C for 10 min, the reaction mixture was warmed to 0 ° C and stirred for an additional 10 min. NH was added4Saturated aqueous CI and the mixture was extracted with CH2Cl2 (3 *). The combined organic extracts were washed with brine, dried over Na2SO4 and they concentrated. The crude product was dissolved in CH2Cl2 (4 ml), treated with EtsN (400 µΙ) and stirred at rt for 15 h. NH was added4C, aqueous saturated and the mixture was extracted with CH2Cl2 (3x). The combined organic extracts were washed with brine, dried over Na2SO4, concentrated and purified by flash chromatography (30% EtOAc-hexanes) to provide the intermediate aldehyde (48 mg), which was immediately dissolved in 1: 1 EIO-EtOH (4 ml), cooled to 0 ° C and treated with NaBH4 solid (-4 mg, 0.09 mmol). After stirring for 15 min NH was carefully added4Aqueous saturated CI and the mixture was extracted with EtOAc (3x). The combined extracts were washed with brine, dried over Na2SO4, concentrated and purified by column chromatography (20% to 30% EtOAc-hexanes) to provide alcohol 411 (45.6 mg, 80% for all 3 steps).

1) MPMOTCI v 2) LaH

3) separate isomers

412A, 412B

412A and 412B Alcohol 411 (120 mg, 0.196 mmol) and MPMOTCI (0.17 g, 0.59 mmol) were combined, azeotroped in toluene (3x) and dried under high vacuum for 1 hr. CH2Cl2 (9 ml) was added and the mixture was cooled to 0 ° C. BF3OEÍ2 (0.016 M in CH2Cl2, 125 μΙ, 0.002 mmol) was added dropwise and after stirring for 20 min the reaction was quenched with NH4Saturated aqueous CI. The mixture was extracted with CH2Cl2 (3x) and the combined extracts were washed with brine, dried over Na2SO4, concentrated and purified by preparative TLC (20% EtOAc-hexanes) to provide the intermediate MPM ether which contained some nearby running impurities. This material immediately dissolved in Et2O (10 ml) and treated with LAH (1 M in THF, 300 µΙ, 0.300 mmol) at 0 ° C. After 10 min H2O and 1 M NaOH were added, and after stirring for 10 min at rt the mixture was filtered through Celite, concentrated and purified by preparative TLC (35% EtOAc-hexanes) to provide 412A (49 mg, 39% for the 2 stages) as a single C34 isomer and 412B (46 mg, 36% for the 2 stages) as a mixture

ES 2 364 696 T3 ~ 9: 1 of C34 isomers.

412A, 412B

B2070 and B2073. In a manner similar to that described in Schemes 4 and 6 for the synthesis of B1794, intermediates 412A and 412B were converted to B2070 and B2073, respectively. For B2070: HRMS (FAB): cale, for C41H58F2O12 + Na 803.3794. Found: 803.3801. For B2073: HRMS (FAB): cale, for C41H58F2O12 + Na 803.3793. Found: 803.3781

Synthesis of B1963:

Diol 501 (64) NaHCO was added3 aqueous saturated (21 ml) and KBr (89 mg, 0.75 mmol) to a solution of the diol 10a (1.35 g, 3.4 mmol) in CH2Cl2 (34 ml). The mixture was cooled to 0 ° C and 4-methoxy-2,2,6,6-tetramethyl-1-piperidinyloxy (0.05 M in CH2Cl2, 7.45 ml, 0.37 mmol) and NaOCI (0, 07 M in H2O, 5.6 ml, 0.39 mmol). After 1 h, the reaction mixture was quenched with Na2S2OR3 saturated aqueous, diluted with NaHCO3 aqueous saturated and extracted with CH2CI2 (3x). The combined extracts were dried over Na2SW4, concentrated and dissolved in THF (21 ml).

After cooling to 0 ° C, CF was added sequentially3TEM (1.5 g, 10.5 mmol) and TBAF (0.1 M in THF, 680 µl, 0.068 mmol). After stirring for 40 min, additional TBAF (1M in THF, 8.3 mL, 8.3 mmol) was added. After 30 min the reaction was quenched with H2O and extracted with EtOAc (3 *). The combined organic extracts were washed with brine, dried over Na2SW4, concentrated and purified by flash chromatography (30%, 40%, 50% EtOAc-hexanes followed by EtOAc) to provide a 2: 1 mixture of the diols (553 mg, 35%). Separation by MPLC (1.5% MeOH-CH2CI2) gave the 501 more polar major isomer (64) (340 mg, 22%) and the less polar minor isomer (152 mg, 10%).

ES 2 364 696 T3

B1963

B1963. In a manner similar to that described in Schemes 4 and 6 for the synthesis of B1794, intermediate 501 was converted to B1963.

Synthesis of B2320 and Related Analogs

HR

These compounds are prepared by treating B2294 with an appropriate amine in a solvent such as methanol for a period of a few hours to several days. The progress of the reaction can be monitored by thin layer chromatography. A standard work-up procedure, well known to those skilled in the art, provides the desired compounds. The following procedure is to prepare ER803868: however, this procedure is general and can be used to prepare any desired analog.

Synthesis of ER803868

To a solution of B2294, 1.2 mg, in methanol, 0.5 ml, was added morpholine, 0.012 ml. The mixture was stirred for 10 days with additional morpholine, 0.012 ml, being added on days 1, 2, 3, 4 and 8. The mixture was then purified by chromatography to give 1.4 mg of the desired compound.

ES 2 364 696 T3

B2320

B2330

B2336

B2339

B2417

B2418

B2489

B2490

B2491

ER803834

ER803835

ER803836

ER803843

ER803845

ER803846

ER803851

ER803852

ER803868

ER803869

ER803870

ER803883

ER803884

R = N, N-dimethylamino

R = N-isopropylamino

R = N-methylamino

R = Nt-butylamino

R = N-2-hydroxyethylamino

R = N-piperazinyl

R = N, N-bis- (2-hydroxyethyl) amino

R = N-1,3-dihydroxy-2-propylamino

R = N-benzylamino

R = N-piperidinyl

R = N-pyrrolidinyl

R = N-3- (R) -hydroxypyrrolidinyl

R = N-homopiperidinyl

R = N-para-methoxybenzylamino

R = N-phenethylamino

R = N-2- (S-hydroxymethyl) pyrrolidinyl

R = N-2- (R-hydroxymethyl) pyrrolidinyl

R = N-morpholinyl

R = N-ethylamino

R = N-imidazoyl

R = N, N-diethylamino

R = N-para-chlorobenzylamino

D. Pharmacological activity

Many of the individually disclosed drugs were tested for in vitro and in vivo activity (see Table 1, below). Screening procedures included a standard in vivo cell growth inhibition assay using DLD-1 human colon cancer cells (ATCC accession number CCL 221) in a 96-well microtiter plate form (Finlay, GJ. Et al. . Analytical Biochemistry 139: 272-277, 1984), a U937 mitotic block reversibility assay (ATCC accession number CRL 1593) (described below) and, in some cases, an in vivo xenograft growth inhibition assay LOX human melanoma tumor (see Table 1). Chemical stability to esterase degradation was also examined.

U937 Mitotic Block Reversibility Assay

U937 human histiocytic lymphoma cells were added to 75 cm tissue culture flasks2 about 2.5 x 106 cells in 22.5 ml of RPMI 1640 medium containing 10% fetal bovine serum. Cells were allowed to adapt to culture for 36 h incubation at 37 ° C in a humidified atmosphere containing 5% CO.2. Each test drug was then added to a flask as 2.5 ml of 10x final concentration. Final concentrations achieved were 0.1-1000 nM, in half-log increments, for a total of 10 concentration steps that included a drug-free control flask that received 2.5 ml of medium. Cells were incubated with drug for a pretreatment period of 12 h at 37 ° C in a humidified atmosphere containing 5% CO2.

The contents were removed from each flask and centrifuged at 300 xg for 10 min at room temperature, after which the drug-containing medium was removed from the cell pellet. Cells were resuspended in 25 ml of warm drug-free medium and centrifuged at 300 xg for 10 min at room temperature. After removing the medium from the cell pellet, the cells were resuspended in 35 ml of warm drug-free medium, transferred to fresh flasks and a 10 ml sample of cells was immediately taken from each flask, immediately processed as described later and saved for later cell cycle analysis (0 hour drug washout).

Incubation of the remaining 25 ml of cells continued in drug-free medium for another 10 h. A 10 ml sample of cells was taken from each flask, processed immediately and waited for subsequent cell cycle analysis (10 hour drug washout) and 10 ml of fresh replacement medium was added to each incubation flask. Incubation of cells in drug-free medium continued for 5 days. On day two, 20 ml of cell medium was removed from each flask and replaced with 20 ml of fresh medium. Cell viability was quantified after 5 days by trypan blue exclusion techniques using hemocytometer counting.

Cells were processed for cell cycle analysis using modifications of the procedure published in section 1.11 of the Becton Dickinson Immunocytometry Systems reference book (Preparation of alcohol-fixed whole cells from suspensions for DNA analysis). Briefly, each 10 ml sample of cells taken from the flasks at 0 and 10 hours after the drug wash was separately centrifuged at 300 xg for 10 min. After removing the medium from the cell pellet, the cells were resuspended in 3 ml of cold saline. Seven milliliters of cold 100% ethanol was slowly added with vigorous vortexing. Samples of

ES 2 364 696 T3 ethanol treated cells from the 0 hour and 10 hour periods of the compound wash were stored overnight at 4 ° C. The ethanol-treated cells were centrifuged at 300 xg for 10 min, the ethanol was removed, and the cells were then washed in 10 ml of phosphate buffered saline (PBS). The cells were resuspended in 0.5 ml of 0.2 mg / ml ribonuclease A (Sigma # R-5503) in PBS and incubated in a 37 ° C water bath for 30 min.

Cells were transferred to appropriate flow cytometry tubes and 0.5 ml of 10 mg / ml propidium iodide (PI) (Sigma # P4170) in PBS was added to each tube. Cells were incubated with PI at room temperature in the dark for at least 15 min before analysis with a flow cytometer (Becton Dickinson FACScan flow cytometer or equivalent). Cells should be analyzed within one hour and kept in the dark at 4 ° C until ready. Cell cycle analysis was performed on 0 hour and 10 hour cells using flow cytometric measurement of cell fluorescence intensity. The intensity of propidium iodide fluorescence for each cell was measured on a linear amplification scale with duplicate events ignored using doublet discrimination. The results obtained from analyzing 15,000 cells were presented as a histogram with increasing fluorescence intensity on the x-axis and the number of cells at a particular intensity level on the y-axis.

The intensity of PI staining depends on the amount of DNA in the cell, so it is possible to identify cells in various phases of the cell cycle such as cells that have not yet synthesized DNA since the last mitosis (G1 phase), cells that are in intermediate phases of DNA synthesis (S phase) and cells that have doubled their DNA complement and are ready to divide (G phase2). Cells that are blocked in the mitosis phase of the cell cycle also have twice the amount of DNA compared to G1 phase cells. If all cells are blocked in mitosis, there are no G1 phase cells, but if the blockage is removed when the compound is removed, the cells complete mitosis and reappear in G1 phase. Therefore, the number of cells that thus reappear in the G1 or S phase is a measure of the number of cells that have recently completed mitosis. For each sample at 0 and 10 hours after elimination of the compound, the percentage of cells that completed mitosis was quantified (as the number of cells that reappeared in the G1 phase) and was represented as a function of the initial concentration of compound used during the 12 hour pretreatment period. The percentage of cells still viable 5 days after drug washout was overlaid on the same graph. A relationship can be determined between the concentration of compound required to completely block all cells in mitosis at 0 hours and the concentration required to maintain block 10 hours after elimination of the compound. This was taken as a measure of the reversibility of a compound, indicating ratios close to or equal to one, probably potent in vivo antitumor compounds (see Table 1, columns 4-6).

Table 1

<td colspan="6">In vitro inhibition and reversibility data</td>

<td></td><td>DLD-1 *</td><td></td><td colspan="2">Complete mitotic block</td><td rowspan="2">Reversibility ratio</td>

<td>Compound</td><td>Mean IC50, mn</td><td>SB</td><td>0 hours, nM **</td><td>10 hours, uMf</td>

ES 2 364 696 T3 (cont.)

<td colspan="6">In vitro inhibition and reversibility data</td>

<td></td><td>DLD-1 *</td><td></td><td colspan="2">Complete mitotic block</td><td rowspan="2">Reversibility ratio</td>

<td>Compound</td><td>Mean IC50, mn</td><td>SB</td><td>0 hours, nM **</td><td>10 hours, uMf</td>

<td colspan="6">* = inhibition of cell growth in vitro ** = before washing φ = after washing</td>

E. Use

The disclosed compounds have pharmacological activity, including antitumor and antimitotic activity, as demonstrated in section D above. Examples of tumors include melanoma, fibrosarcoma, monocytic leukemia, colon carcinoma, ovarian carcinoma, breast carcinoma, osteosarcoma, prostate carcinoma, lung carcinoma, and ras-transformed fibroblasts.

The invention features pharmaceutical compositions that include a compound of formula (I) and a pharmaceutically acceptable carrier. The compositions can also include a combination of disclosed compounds, or a combination of one or more disclosed compounds and other pharmaceutically active agents such as an antitumor agent, an immunostimulating agent, an interferon, a cytokine, an anti-MDR agent, or an agent. antiangiogenic. The compositions can be formulated for oral, topical, parenteral, intravenous or intramuscular administration, or administration by injection or inhalation. Formulations can also be prepared for controlled release, including transdermal patches.

ES 2 364 696 T3

The use of the disclosed compounds for the preparation of a medicament for inhibiting tumor growth in a patient includes formulating a medicament containing an effective antitumor amount of a disclosed compound or composition. Such use also contemplates the preparation of a medicament for use in combination therapies that include the co-administration of a compound of formula (I) before, during or after the administration of another pharmaceutically active agent. Administration procedures may be the same or different. Tumor growth inhibition includes a growth of the cell or tissue exposed to the test compound that is at least 20% lower, and preferably 30%, 50% or 75% lower than the growth of the control (absence of known inhibitor or test compound).

Other realizations

The essential features of the invention can be easily perceived from the above description and the following claims.

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15 members in 9 offices

This recordMember, by publication datePriority claim

Priority claims3

Priority claims
Document	Office	Kind	Date
33243506	United States of America	A	2006-01-13
33243506	United States of America	A	2006-01-13
US20060332435	–	–	–

Members15

Family members, oldest first
Document	Office	Kind
CA2573546A1	Canada	A1	2007-07-13
EP1808835A2	European Patent Office (EPO)	A2	2007-07-18
US2007164869A1	United States of America	A1	2007-07-19
JP2007249940A	Japan	A	2007-09-27
BRPI0700090A	Brazil	A	2007-10-16
EP1808835A3	European Patent Office (EPO)	A3	2007-11-28
MX2007000489A	Mexico	A	2008-11-14
US7646302B2	United States of America	B2	2010-01-12
EP1808835B1	European Patent Office (EPO)	B1	2011-04-27
DE602007014127D1	Germany	D1	2011-06-09
ES2364696T3This record	Spain	T3	2011-09-12
ES2364697T3	Spain	T3	2011-09-12
PL1808835T3	Poland	T3	2011-09-30
CA2573546C	Canada	C	2014-04-08
BRPI0700090B1	Brazil	B1	2019-01-22