AU2006252266A1 - Tamandarin and didemnin analogs and methods of making and using them - Google Patents

Description

I Regulation 3.2

AUSTRALIA

Patents Act 1990 DIVISIONAL

APPLICATION

Name of Applicant(s): The Trustees of the University of Pennsylvania, of 3700 Market Street, Suite 300, Philadelphia, Pennsylvania 19104-3147, United States of America Actual Inventor(s): Address for Service: Invention Title: JOULLIE, Madeleine LIANG, Bo; DING, Ziaobin DAVIES COLLISON CAVE, Patent Trademark Attorneys, Level 3, 303 Coronation Drive, Milton, 4064, Queensland, Australia Ph: 07 3368 2255 Fax 07 3368 2262 "Tamandarin and didemnin analogs and methods of making and using them" is a full description of this invention, including the best method of The following statement performing it known to us:- Q:\ApcWp&2oo6\Dcc0\4 23 870 AU P0 file new dcisional tulee uni of pennsyluania 356 do 22112/06 WO 01/76616 PCT/USO1/11607 \0

TITLE

Tamandarin and Didemnin Analogs i and Methods of Making and Using Them BACKGROUND OF THE INVENTION s Didemnin B is a macrocyclic depsipeptide isolated from a species of marine CN tunicate. Didemnin B exhibits potent anti-viral, immunosuppressive, and anti-tumor activities 't in vitro and in vivo, and was the first marine natural product to enter clinical testing against human cancers (Li et al., 1992, Studies in Natural Products Chemistry, 10:241-302; Sakai et C al., 1996, J. Med. Chem. 39:2819-2834; Wipf, 1995, Chem. Rev. 95:2115-2134). Didemnin B is a didemnin, a family of compounds which potently inhibit protein synthesis and cell cycle progression, and induce more rapid apoptosis than any other natural products that has been isolated to date (Grubb et al., 1995, Biochem. Biophys, Res. Commun. 215:1130-1136; Johnson et al., 1996, FEBS Lett. 383:1-5; Johnson et al., 1999, Immunol. Cell Biol. 77:242- 248; Johnson et al., 1999, J. Cell. Biochem. 72:269-278). Other members of this family of compounds, including didemnin M and dehydrodidemnin B, exhibit cytotoxic and cytostatic effects as well.

2 Tamandarin A (also designated {(2S)Hiv }didemnin B) is a naturally occurring didemnin congener which has recently been isolated from a marine tunicate. Tamandarin A exhibits biological activity which is analogous to the activities exhibited didemnin B. For example, tamandarin A is a potent inhibitor of protein synthesis, cell growth, and tumorigenesis. Tamandarin A exhibits greater in vitro activity against pancreatic carcinoma than does didemnin B (Liang et al., 1999, Org. Lett. 1: 1319-1322). A significant limitation on use of tamandarin A, either for research or for practical applications, is the limited supply of tamandarin A that is available from natural sources and the difficulty and expense of isolating this product A need exists for a method of synthesizing tamandarin A and other didemnin analogs (including dehydrodidemnin analogs).

Despite the potency of didemnin B in isolated studies, its clinical effectiveness is hampered by side effects associated with therapeutic doses of the compound. As with many anti-proliferative agents, didemnin B exhibits a relatively narrow therapeutic window.

Although didemnin M and dehydrodidemnin B exhibit improved therapeutic potential, relative -1- WO 01/76616 PCT/USO1/11607 'C to didemnin B, a need still exists for anti-proliferative agents which exhibit less toxicity at a therapeutic dose didenin analogs having a greater therapeutic index).

The present invention satisfies the needs set forth above.

N BRIEF SUMMARY OF THE INVENTION The invention relates to tarnandarin and didemnin analogs that have a deoxoproline residue or a dehydro-proline residue in their structure. In one embodiment, the C invention relates to a composition comprising a tamandarin analog having the structure of C formula I

R

1

R

22 HN 0 N 0 '1 1 N-R1

HN

0 0 OY 0).

In formula I, R is selected from the group consisting of

-H,

-(tert-butyloxycarbonyl), -leucine, -{N-methyl)leucine, -{N-methyl)leucine-(a first fluorophore), -(N-methyl)eucineproline, -(N-methyl)leucine-proline-lactate, -(N-methyl)leucine-proline-pyruvate, -(N-methyl)leucine-proline-lactate-(a first fluorophore), -(N-metyl)leucine-proline-lactate-glutamine-pyroglutamate, -(N-methyl)leucne-prohne-lactat-glutamme-cyclopentanoate, -(N-methyl)leucine-proline-alane-leune-pyroglutamate, -(N-methyl)leucine-proline-(N-methyl-alanine)-leucine-pyroglutamate, -(N-methyl)leucine-deoxo-proline, -2- WO 01/76616 WO 0176616PCT[USOI1/11607 .O -(N-methyl)leucine-deoxo-proline-lactate, S -{N-methyl)leucine-deoxo-proline-pyruvate, U-(N-methyl)Ieucine-deoxo-proline-Iactate-(a first fluorophore), -(N-methyl)leucine-eoxo-proline-lactate-glutamine-pyroglutainate, ci -(N-methyl)leucine-deoxo-proline-latate-glutanine-ylopentanoate, -(N-methyl)Ieucine-deoxo-proline-alanine-leucine-pyroglutamate, and -(N-methyl)Ieucine-deoxo-.proline-{N-methyl-alanine)-leucine-pyroglutamate, N- -(N-methyil)eucine-dehydro-proline, cI -(N-methyl)leucine-dehydro-proline-lactate, S -(N-methyl)leucine-dehydro-proie-pyruvate, -(N-methyl)leucine-dehydro-proline-lactate-(a first fluorophore), -(N-methyl)eucine-dehydro-proline-actat--lutamine-pyroglutamate, (N-methyl)leucine-dehydro-prome-lactate-glutamine--cyclopentanoate, -{Nmehy~lucnedeydo-roin-aannelecie-yrgltaiaean -{N-methyl)Ieucine-ehydro-proline-alN-meh-laine-luine, yoa te -(-ehl~ecn-dhdoprln-Nmehlaa2n) 3cn-yoguaae R 2and R 3in formula 1, can be separate moieties or they can, together, be a single moiety. When R 2and R 3are separate moieties, R 3is either a methyl group or a hydride radical and R 2is selected from the group consisting of an isoleucine side chain, a valine side chain, an alanine side chain, a norleucine side chain, a norvaline side chain, leucine side chain, a histidine side chain, a tryptophan side chain, an arginine side chain, a lysine side chain, a second fluorophore, and a substituent having the structure of formula'Il R

R

RR

6 11) When R 2and R 3are, together, a single substituent, this substituent has the structure of formula

IV

-3- WO 01/76616 PCT/US01/11607 0 R C R 8

R

R

x

(IV).

cI SIn formulas II and IV, each of R 5

R

6

R

7 R8, and R is independently N selected from the group consisting of-H, -OH, -OCH 3

-CO(C

6

H

5 -Br, -Cl, -CH 3

O

and -C 2

H

5 c- 4 R in formula I is either an isoleucine side chain or a valine side chain. Also, in formula I, X is either or Y is either a hydride radical or a hydroxyl protecting group, and R0 is either a leucine side chain or a lysine side chain. The didemnin analog is an analog other than tamandarin A {(2S)Hiv2}didemnin In one embodiment, every proline or lactate moiety that is present in RI exhibits stereochemistry. In another, every moiety capable of exhibiting stereochemistry in R 1 is present in its naturally occurring form the form for amino acid residues and lactate. It is believed that cyclopentanoate occurs naturally in an stereochemistry.

In another embodiment, the invention relates to a composition comprising a didemnin analog having the structure of formula XXI

R

3 N N R2

ON-

O

-4- O OY N-R

H

NH

R

4

(XXI).

In formula XXI, each of R

RR

3

R

4 R R, R R and RI has the same meaning as in formula I.

-4- WO 01/76616 PCT/US01/11607 SIn preferred classes of deoxo-proline tamandarin and didemnin analogs having the formula of I and XXI, respectively, R has the structure of formula III, R is methyl, R is S an isoleucine side chain, each of R 5 R R and R is a hydride radical, R is methoxy, R 0 is a leucine side chain, X is and Y is a hydride radical. Examples of tamandarin and

C-I

CN didemnin analogs that are included in the invention are compounds 103, 104, 105, 106, 107, 108, 109, 110, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 133, 134, 136, 137, 139, 141, 142, 143, 201,202, 203, and 204, which are shown in the CN Figures.

C -In one embodiment, the tamandarin or didemnin analog has a photoreactive substituent, such as an R moiety having the structure 0 The photoreactive substituent can be directly attached to the analog, or it can be attached by way of a linker having a chain that comprises 1 to about 13 or more carbon atoms, and optionally having secondary amine or amide moieties in the chain.

In another embodiment, the tamandarin or didemnin analog has a fluorophore attached, such as an analog in which a fluorophore is attached at the omega amino moiety of a 2 10 lysine side chain at R or at R 10 An example of the structure of such a fluorescent didemnin analog is show in Figure 29. Alternatively, the didemnin analog can be attached covalently) with a support. In most embodiments, Y in formulas I and XXI is preferably a hydride radical.

WO 01/76616 PCT/US01/11607 SThe invention includes an embodiment of a tamandarin or didemnin analog which can be activated (or the activity of which can be enhanced) by enzymatic cleavage of a U moiety bound with the analog. For example, the invention includes compositions which comprise an analog having a structure selected from the group consisting of formulas -q as follows.

(a) ND R3

R

13 R N N HN O HN 4 1 2 R 4 (c) (c) WO 01/76616 PCT/US01/11607 and (d)

ON

0 NH OR 13 0 OR 13 0 OY

N

IX NH 0 0 D 2 3 4 SIn formulas R R R R X, and Y have the same identities 13 CI described above for formula I. R is an enzyme-cleavable moiety that is cleavable by an enzyme, such as one selected from the group consisting of a carboxypeptidase, a betalactamase, a beta-galactosidase, a penicillin V-amidase, a cytosine deaminase, a nitroreductase, a alkaline phosphatase, a beta-glucuronidase, and a catalytic antibody. By way of example,

R

13 can have the structure of either of formulas V and VI SI H S N S

O

0 ,H 0 COOH N HO OH

(VI).

Examples of such analogs include compound 131 and compound 132.

The invention also relates to compositions which comprise a didemnin fragment having the structure of formula VII -7- WO 01/76616 PCT/US01/11607 OY 0 R4X R" HN

O

"APG (vn.

In formula VII, Y is either a hydride radical or a hydroxyl protecting group, X 4 O is either or R is either an isoleucine side chain or a valine side chain, and APG S is a amine protecting group. R 1 can be any of-OH, -NH2, -O(allyl), -O(pentafluorophenyl), Ci and a substituent having the structure of formula VIII IR 2 R I O O NH 0 0 NH Ox 0 S12 x nVm).

In formula VIII, R R 2

R

3 and R10 have the same identities described above for formula I, and R 2 can be either a hydride radical or a -2-(trimethylsilyl) ethoxycarbonyl moiety.

The tamandarin and didemnin analogs described herein can be formulated,.

together with one or more pharmaceutically acceptable carriers, to make pharmaceutical preparations. These preparations can be administered to a mammalian human) cell either in vitro or in vivo) in order to inhibit protein synthesis, inhibit growth, inhibit proliferation, inhibit tumorigenesis, or enhance apoptosis in the cell or in one or more tissues of the mammal.

The invention further includes a method of making a didemnin fragment. This method comprises coupling a first reactant having the structure 0O

II

x

H

and a second reactant having the structure WO 01/76616 PCT/US01/11607 OY 0 R4,,OH

HN

APG

to yield a first didemnin fragment having the structure

APG

In this structure, X is either or APG is an amine protecting group; Y is a hydroxyl 4 protecting group a -triisopropylsilyl group), and R can be either an isoleucine side chain or a valine side chain. The first didemnin fragment can be hydrolyzed to yield a second didemnin fragment having the structure

'APG

An activator (ACT) can be added to the carbonyl moiety of the second didemnin fragment to yield a third didemnin fragment having the structure

OYO

R4,A X )O-ACT V C h n The third didemnin fragment can be coupled with a third reactant which has the structure

SEM

-0 to yield a fourth didemnin fragment having the structure WO 01/76616 PCT/US01/11607 \0

R"

I R2 /SEM C, N 0 U R 10 y 0 1pG R O O O N-APG NH

H

SO HN-APG X H 0 Y In this structure, R 2 and R 3 have the identities described above for formula I, APG is an amine protecting group, SEM is a 2-(trimethylsilyl)ethoxycarbonyl group, and R 1 is either a leucine side chain or a lysine side chain.

j The invention also relates to a method of making a didemnin analog from the fourth didemnin fragment. This method comprises removing the SEM and CBZ moieties from the fourth didemnin fragment and cyclizing the fragment to yield a first didemnin analog having the following structure.

3 SR 2 X R4 The APG group (which can, for example, be a carbobenzyloxy moiety or a tertbutyloxycarbonyl moiety) can be removed from the first didemnin analog to yield a second didemnin analog having the structure WO 01/76616 WO 0176616PCTJUS01II 1607 CN R NH 2 0 0 c-N HN This second didemnin analog can be coupled with a fourth reagent having the structure c-I 0 HO R 14 to yield a third. didemnin analog having the structure

HNN

141 In these structures, R 14is can be one of (tert-butyloxycarbonyl), -leucine, -(N-methyl)leucine, -{N-methyl)leucine-(a first fluorophore), {N-methyl)leucine-proline, -(N-methyl)leucine-proline-lactate, -{(N-inethyl)leucine-proline-pyruvate, -(N-methyl)eucine-proline-lactate-(a first fluorophore), (N-methyl)leucine-proline-lactate-glutamine-pyroglutamate, -11- WO 01/76616 WO 0176616PCT/USO 1/11607 IC. -(N-methyl)leucine-proline-lactate.-glutaniine--cyclopentanoate, S-(N-methy)leucine-proline-alanine-eucine-~pyroglutamate, U -(N-methyl)leucine-proline-(N-methyl-alanine)-leucine-pyroglutamate, *(N-methyl)leucine'-eoxo-proline, -(N-methyl)leucine-deoxo-proline-lactate, -{N-methyl)leucine-deoxo-proline-pyruvate, IN -(N-methyl)leucine-deoxo-proline-lactate-(a first fluorophore), ICi (Nmtylecndeoprlnlaaeglamepyoltnae NI -(N-methyl)leucine-deoxo-proline-lactate-glutamine-cycolopetanate, N -(N-methyl)leucine-deoxo-proline-laninte-lucine-youtaoate,d I -(N-methyl)leucine-deoxo-proline-al(nimeh-laine-yluine, yoaa e -(N-methyl)leucine-dehydo-proline--actlae-luie-yoluaa -{N-methyl)leucine-deydro-proline-pruae -(N-methyl)leucine-dehydro-proline-lactate-(, is loohr) -(N-methyl)leucine-dehydro-proline-lactate-(guaiste---rpru e), -(N-methyl)leucine-dehydro-proline-lactate-glutamine-pycolopetanate, (N-methyl)leucine-dehydro-proline-laninte-lucine-youtaate,n -{N-methyl)leucine-ehydro-proline(N hy-alanine)-leucine-pyroglutamate, oritcadb o(n-efthesemoiee copd in eNyme-laabele oieyrgue thr is caac an em suc ase onhee oeti ca opethse an benzy-acaae ietthatidsceaabl eniilln en- m auchdas e a csine eiase, a itroredctase, ake-al poshaase, a betguonicillne, and a catalytic antibody. If Y is a hydroxyl protecting group, then that can be removed from the third didemnin analog (either before or after addition of R.I: to yield a fourth didemnin analog having the structure -12- WO 01/76616 PCT/US01/11607 O R2 HN HN O SHN X R4 0 O OH The invention further relates to a method of making deoxo-proline-containing \O tamandarin and didemnin analogs. These methods employ known methods for making 0 tamandarin and didemnin analogs, and are modified to incorporate a deoxo-proline residue in Splace of a proline residue of the analog.

The invention still further relates to a method of making dehydro-prolinecontaining tamandarin and didemnin analogs. These methods employ known methods for making tamandarin and didemnin analogs, and are modified to incorporate a dehydro-proline residue in place of a proline residue of the analog.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1, comprising Figures lA and 1B, depicts the structure of tamandarin A {(2S)HIV2 didemnin Figure IA is the structure tamandarin A (compound 101).

Figure 1B is the structure of a diastereomer (compound 102) tamandarin A. The chiral center at which these two molecules differ is indicated with an arrow.

Figure 2, comprising Figures 2A and 2B, depicts the structure of tamandarin M {(2S)HIV 2didemnin Figure 2A is the structure tamandarin M (compound 103). Figure 2B is the structure of a diastereomer (compound 104) of tamandarin M. The chiral center at which these two molecules differ is indicated with an arrow.

Figure 3, comprising Figures 3A and 3B, depicts the structure of tamandarin B {(2S)HTI }didemnin Figure 3A is the structure of tamandarin B (compound 105).

Figure 3B is the structure of a diastereomer (compound 106) of tamandarin B. The chiral center at which these two molecules differ is indicated with an arrow.

Figure 4, comprising Figures 4A, 4B, 4C, and 4D, depicts the structure of several fluorescent tamandarin-type didemnin analogs. Figure 4A is the structure of compound -13- WO 01/76616 PCTIUS01/11607 N 107. Figure 4B is the structure of compound 108. The chiral center at which compounds 107 S and 108 differ is indicated with an arrow. Figure 4C is the structure of compound 109. Figure 4D is the structure of compound 110. The chiral center at which compounds 109 and 110 differ is indicated with an arrow.

C1 Figure 5, comprising Figures 5A and 5B, depicts a class of immobilizable tamandarin-type didemnin analogs. Figure 5A is the structure of a didemnin analog of formula ^0 \0 I herein, wherein R is a lysine side chain. Figure 5B is the structure of the didemnin analog NC of Figure 5A bound with a solid support (SS).

O Figure 6, comprising Figures 6A and 6B, depicts another class of immobilizable tamandarin-type didemnin analogs. Figure 6A is the structure of didemnin CN 1 analog of formula I herein, wherein R is -leucine. Figure 6B is the structure of the didemnin analog of Figure 6A bound with a solid support (SS).

Figure 7 is the structure of compound 115.

Figure 8 is the structure of compound 116.

Figure 9 is the structure of compound 117.

Figure 10 is the structure of compound 118.

Figure 11 is the structure of compound 119.

Figure 12 is the structure of compound 120.

Figure 13 is the structure of compound 121.

Figure 14 is the structure of compound 122.

Figure 15 is the structure of compound 123.

Figure 16 is the structure of compound 124.

Figure 17 is the structure of compound 125.

Figure 18 is the structure of compound 126.

Figure 19 is the structure of compound 127.

Figure 20 is the structure of compound 128.

Figure 21 is the structure of compound 129.

Figure 22 is the structure of compound 130.

Figure 23 depicts enzymatic cleavage of the cephalosporin moiety of didemnin analog 131 by beta-lactamase to yield compound 101.

Figure 24 depicts enzymatic cleavage of the glucoside moiety of didemnin analog 132 by beta-glucuronidase to yield compound 101.

-14- WO 01/76616 PCT/US01/11607 N Figure 25, comprising Figures 25A and 25B, is a pair of structures which S illustrates the structural difference between tamandarin A (101; Figure 25A, and didemnin B (201; Figure 25B. The macrocyclic core of 101 differs from that of 201 in that 101 contains an alpha-hydroxyisovaleryl (Hiv) moiety, and 201 contains an alpha-(alpha-hydroxyisovaleryl)- C-i propionyl (Hip) moiety at the analogous position, as indicated by the brackets and dotted lines in each of the figures.

0 Figure 26, comprising Figures 26A-26E, depicts a synthetic method for C-i CI generating didemnin analogs described herein.

N Figure 27, is the structure Tamandarin A {(2S)HIV 2 didemnin B), illustrating the numbering convention used herein and in Sakai et al. (1996, J. Med. Chem.

39:2819-2834) for didemnin analogs.

Figure 28, comprising Figures 28A and 28B, depicts the structure of a dehydrotamandarin-type didemnin analog {(2S)HIV 2dehydrodidemnin Figure 28A is the structure of dehydrotamandarin (compound 133). Figure 28B is the structure of a diastereomer of dehydrotamandarin (compound 134). The chiral center at which these two molecules differ is indicated with an arrow. The position at which these dehydrotamandarintype didemnin analogs differ from tamandarin-type didemnin analogs is indicated with an asterisk.

Figure 29 is the structure of a fluorescent dehydrotamandarin-type didemnin analog. "FL" is a fluorophore.

Figure 30 is the structure of compound 136.

Figure 31 is the structure of compound 137.

Figure 32 depicts a dehydrotamandarin-type didemnin analog bound with a solid support (SS).

Figure 33 is the structure of compound 139.

Figure 34 is the structure of compound 140 Figure 35, comprising Figures 35A and 35B, depicts the structure of 2 1 dehydrotamandarin B, also designated {(2S)Hiv 2 Norsta }didemnin B. Figure 35A is the structure dehydrotamandarin B (compound 141). Figure 35B is the structure of a diastereomer dehydrotamandarin B (compound 142). The chiral center at which these two molecules differ is indicated with an arrow.

Figure 36 is the structure of compound 143 WO 01/76616 PCT/USO1/11607 NO Figure 37, comprising Figures 37A and 37B, depicts a synthetic method for O2 generating dehydrotamandarin {(2S)HIV2 dehydrodidemnin B, compound 133).

O Figure 38, comprising Figures 38A, 38B, and 38C, depicts a synthetic method for generating fluorescent didemnin analogs described herein.

C Figure 39 is the structure of a preferred deoxo-proline tamandarin analog designated compound 201.

\NO Figure 40 is the structure of a preferred deoxo-proline didemnin analog CN designated compound 202.

CI Each of Figure 41, Figure 42, and Figure 43 depicts a method of making S) deoxo-proline-containing side chain moieties for tamandarin or didemnin analogs.

Figure 44 depicts a method of making dehydro-proline-containing side chain moieties for tamandarin or didemnin analogs.

Figure 45 is the structure of a preferred dehydro-proline tamandarin analog designated compound 203.

Figure 46 is the structure of a preferred dehydro-proline didemnin analog designated compound 204.

Each of Figure 47, Figure 48, Figure 49, and Figure 50 depicts a method of making didemnin analogs having photoreactive side chain moieties.

DETAILED DESCRIPTION OF THE INVENTION The invention relates to tamandarin and didemnin analogs, including analogs which have a deoxo-proline residue or a dehydro-proline residue in their structure. The invention includes compositions comprising such analogs, and methods for making and using these analogs. These analogs are useful for, among other things, inhibiting protein synthesis, cell growth, cell proliferation, and tumorigenesis. The analogs of the invention can also exhibit anti-viral, anti-tumor, apoptosis-inducing, and immunosuppressive activities in animals, including in humans.

The invention includes compositions comprising a tamandarin analog having the structure -16- WO 01/76616 WO 0176616PCT/USO 1/11607 1-1 HN

H

wherein R ,R X, and Y have the identities described herein. Examples of analogs according to this formula are shown in the figures.

The invention also includes compositions comprising a didemnin analog having the structure wherein R R X, and Y have the identities described herein.

Definitions As used herein, each of the following terms has the meaning associated with it in this section.

The articles and "an" are used herein to refer to one or to more than one to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

-17- WO 01/76616 PCT/US01/11607 N As used herein, amino acid residues are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as 0 indicated by the following: Full Name Three-Letter Code One-Letter Code cN 5 Aspartic Acid Asp D Glutamic Acid Glu E SLysine Lys K CN Arginine Arg R SHistidine His H Tyrosine Tyr Y Cysteine Cys C Asparagine Asn N Glutamine Gin

Q

Serine Ser S Threonine Thr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L Isoleucine lie I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan Trp W As used herein, the term "amino acid side chain" refers to a moiety comprising all of the atoms of an amino acid excluding the alpha-carbon atom, a hydrogen atom bound with the alpha-carbon, the atoms of the alpha-carboxyl moiety and the alpha-amine moiety. By way of example, an "alanine side chain" refers to a methyl group, and a "valine side chain" refers to a 2-propyl group.

"Inhibition" of a process in a cell inhibition of protein synthesis, inhibition of cell growth, inhibition of cell cycle progression, inhibition of cell proliferation, or inhibition of tumorigenesis) means reduction by at least 10%, 25%, 50%, 75%, 90%, 95%, or even -18- WO 01/76616 PCT/USOI/11607 100%) of the rate at which the process proceeds, reduction by at least 10%, 25%, N 75%, 90%, 95%, or even 100%) of the rate at which the process is initiated, or both.

a"Enhancement" of a process in a cell enhancement of apoptosis) means increasing by at least 10%, 25%, 50%, 75%, 90%, 95%, or even 100%) the rate at which C1 the process proceeds, increasing by at least 10%, 25%, 50%, 75%, 90%, 95%, or even 100%) the rate at which the process is initiated, or both.

IDO As used herein, the term "pharmaceutically acceptable carrier" means a 0C chemical composition with which a didemnin analog or fragment, as described herein, can be S combined and which, following the combination, can be administered to a subject a 0 human or other animal).

As used herein, the term "physiologically acceptable" ester or salt means an ester or salt form of a didemnin analog or fragment, as described herein, which is compatible with other ingredients of a pharmaceutical composition and which is not deleterious to a subject to which the composition is to be administered.

As used herein, "parenteral administration" of a pharmaceutical composition.

includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue.

Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating nonsurgical wound, and the like. In particular, parenteral administration can include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.

As used herein, the term "anti-viral activity" means preventing replication of a virus in the cell, preventing infection of the cell by a virus, or reversing a physiological effect of infection of the cell by a virus. An anti-viral agent a composition of matter which, when delivered to a cell, exhibits anti-viral activities. Anti-viral agents are well known and described in the literature. By way of example, AZT (zidovudine, Retrovir® Glaxo Wellcome Inc., Research Triangle Park, NC) is an anti-viral agent which is thought to prevent replication of HIV in human cells.

As used herein, a "deoxo-proline" moiety or residue is a chemical moiety which has the following structure.

-19- WO 01/76616 PCT/US01/11607 As used herein, a "dehydro-proline" moiety or residue is a chemical moiety IN which has the following structure.

Q

O INN Description The present invention relates to tamandarin and didemnin analogs, including those having a deoxo-proline moiety or a dehydro-proline in their structure (and, of course, those which do not have a deoxo-proline moiety or a dehydro-proline in their structure). These analogs exhibit potent pharmacological properties when administered to humans and other mammals. By way of example, these compounds can inhibit protein synthesis and cell growth and proliferation. These compounds can also enhance apoptosis in cells. These properties render the compounds useful for treating a variety of disorders which are characterized by one or more of aberrant protein synthesis, aberrant cell growth, aberrant proliferation of cells, and aberrant apoptosis. Examples of such disorders include tumorigenesis, tumor growth, tumor metastasis, infection of a cell by a virus, and replication of a virus within a cell.

Among the compositions of the inventions are those which comprise a tamandarin analog having the structure of formula I or a didemnin analog having the structure of formula XXI.

WO 01/76616 WO 9176616PCTUSO 1/11607 KN

HH

N NR 2

R

0 0 0 1*4 NH 0 o 0TI

INNH

IND 3

(RI)

12 TeR sustt0 n of fomua I dX cnhaeadoopoln oeyi it tutradc NHo xmlb yrgnao ra mn rtciggopsial for~ ~O prtcino mn cd.Sc rtciggop r n n hrt n eerdt thouhotths iclsue Eamle f uialeprtcn ruscnb onnrfrne sbstiuentcane an am in ci r u a eg. an eu c an id eo a polypeptoie oprisin o0sbsne n moe amino acid residue xmpesg. of sucn residue and polypeptide icudesin -(N-methyl)leucine, -(N-methyl)leucine-(a first fluorophore), -(N-methyl)leucine-proline, -{N-methyl)leucine-proline-lactate, -21- WO 01/76616 WO 0176616PCT/USO 1/11607 ID -(N-methyl)Ieucine-proine-pyruvate, -(N-methyl)leucine-proline-lactate-glutamine-pyroglutamate,' U -(N-methyl)leucine-proine-lactate-glutamine-cyclopentanoate, -(N-methyl)leucine-proline-lactate-leucine-pyroglutanate, N- 5 -(N-methyl)leucine-proline-alanine-leucine-pyroglutamate, -(N-methyl)leucine-proline--(N-methyl)alanine-leucine-pyroglutamate, ID -(N-niethyl)Ieucine-deoxo-proline, Ni -(N-methyl)leucine-deoxo-proline-lactate, N- -(N-methyl)leucine-deoxo-proline-pyruvate, -(N-methyl)leucine-deoxo-proline-Iactate-gutainine-pyroglutamate, -{N-methyl)leucine--eoxo-proline-lactate--glutamine-cyclopentanoate, -(N-methyl)leucine-deoxo-poline-lactate-leucine-pyroglutamate, -(N-inethyl)leucine-deoxo-proline-alanine-leucine-yroglutainate,.

-(N-nethyl)leucine-deoxo-proline--(N-methyl)alanine--Ieucine-pyroglutamate, -{N-methyl)leucine-dehydro-proline, -(N-methyl)leucine-dehydro-proline-lactate, -(N-methyl)leucine-dehydro-proline-pyruvate, -(N-methyl)leucine-dehydro-proline-lactate-(a first fluorophore), -(N-methyl)leucine-dehydro-proline-lactate-gutamine-pyroglutanate, -(N-methyl)leucine-dehydro-proline-lactate-glutanine--cyclopentanoate, -(N-methyl)leucine-dehydro-proline-alanine-leucine-pyroglutamate, and -(N-methyl)leucine-dehydro-proline-(N-methyl-alanine)-leucine-pyroglutamate.

Additional examples of alternative R Isubstitue nts include peptides which comprise a fluorophore rhodamine or coumarin), an amino acid residue, a polypeptide, an enzymnatically-cleavable group, or another chemical moiety bound covalently attached) with a support a glass or silica plate, an agarose or other polymeric bead, etc.). The fluorophore or enzymatically-cleavable group can be directly linked with the analog, or it can be bound thereto with a linker having I toabout 13 or more carbon atoms (optionally including one or more secondary amine or amide moieties) therein. When R comprises an N-methylleucine residue, the alpha-carbon atom of that residue can have either or (S) stereochemistry. Other amino acid residues within R Ican have either or (S) stereochemistry, but they preferably have stereochemistry at their alpha-carbon atom.

-22- WO 01/76616 PCT/US01/11607 O When R comprises a lactate residue, the lactate residue is preferably an (S)lactate residue. In a preferable embodiment, every amino acid residue within R other than the leucine (or Nmethyl-leucine) residue (if present) attached directly to the nitrogen atom of the ring of formula S I or XXI has stereochemistry.

CO 3 3 2 C 5R can be either of-CH 3 and Alternatively, R can, together with R be a single substituent.

IND 2 0 The R substituent can be an amino acid side chain such as an isoleucine side C1 chain a 2-butyl moiety, preferably having stereochemistry), a valine side chain a S 2-propyl moiety), an alanine side chain a methyl moiety), a norleucine side chain a 1-butyl moiety), a norvaline side chain a 1-propyl moiety), a leucine side chain an isobutyl moiety, preferably having stereochemistry), a phenylalanine side chain a phenylmethyl moiety), a histidine side chain a 4-methyl-imidazole moiety), a tryptophan side chain a 3-methyl-indole moiety), a tyrosine side chain a 4-hydroxyphenylmethyl moiety), an arginine side chain a 4-guanidinyl-butyl moiety), and a lysine side chain a 4-aminobutyl moiety).

An R 2 substituent can comprise a fluorophore a fluorophore linked with one of the amino acid side chains described above). In addition, R 2 substituent can have the structure of formula III

R

7 R8 R 6 R q R In an alternative embodiment, R 2 and R 3 together are a substituent having the structure of formula IV -23- WO 01/76616 PCT/US01/11607 0

R

7 SR R 6 0(IV).

In formulas I and IV, each of R 5

R

6

R

7 R and R independently, can be a c- substituent selected from the group consisting of-H, -OH, -OCH3, O -CO(C 6

H

5 -Br, -Cl, -CH 3 and -CH 2

CH

3 4 C 5 R can be an isoleucine side chain or a valine side chain.

X can be or Y can be -H or a hydroxyl protecting group. Examples of hydroxyl protecting groups which can be present at Y include an alkyl-substituted silyl moiety, an aryl- substituted silyI moiety, or a silane substituted with both alkyl- and aryl- moieties. An example of a useful hydroxyl protecting group is a triisopropylsilyl moiety. Other hydroxyl protecting groups which can be used at Y in formula I are described in references such as Green and Wutz (1999, Protecting Groups in Organic Synthesis, Wiley, New York).

R can be an amino acid side chain such as a leucine side chain or a lysine side chain. Alternatively, R 10 can be an amino acid or other chemical moiety which is bound with covalently attached to) a support a solid support). An example of a support with an analog having the structure of formula I bound therewith is depicted in Figure Another group of compositions included within the invention are those which comprise a tamandarin analog having a structure selected from the group consisting of formulas set forth above.

Each of R2, R 3 R, R 10 X, and Y has the same meaning in formulas that it has in formulas I and XXI.

In formulas R 13 can be hydrogen or a chemical moiety which can be enzymatically cleavable an enzyme-cleavable moiety) or photoreactive. As used herein, an enzyme-cleavable moiety can include any chemical moiety which can be cleaved chemically detached from) in the presence of a specific enzyme. Examples of enzymes capable of chemically detaching an enzyme-cleavable moiety include carboxypeptidases, beta- -24- WO 01/76616 PCTIUS01/11607 lactamase, beta-galactosidase, penicillin V-amidase, cytosine deaminase, nitroreductase, alkaline phosphatase, beta-glucuronidase, and catalytic antibodies. Examples of enzyme- S cleavable moieties which can be incorporated in a compound described herein include cephalosporins, beta-glucosides, phosphate, pyrophosphate, beta-D-galactosides, c-I 13 CI nitrobenzamidine, cytosine, carbamates, peptides, and amino acids. Alternatively, R can be an enzyme-cleavable moiety such as a di-peptide linked with glutamine-pyroglutamate, or a O moiety having the structure of formula V or formula VI cNq O -IN n H tfN 00 N

O

COOH

0 HO "OH N/ HO COOH

(VI).

By way of illustration, in compound 131, depicted in Figure 23, R 13 is an enzyme-cleavable moiety having the structure of formula V a cephalosporin moiety). The cephalosporin moiety of compound 131 can be cleaved by contact with the enzyme, betalactamase, to generate compound 101. An R 13 substituent having the structure of formula VI can, for example, be in the form of a sodium or potassium salt.

After cleavage of an enzyme-cleavable moiety by an enzyme, the resulting didemnin analog can exhibit one or more of the physiological activities described herein. A tamandarin or didemnin analog having the structure of one of formulas wherein R 13 is an enzyme-cleavable moiety, can, optionally, exhibit these activities before the cleavage of the enzyme-cleavable moiety. However, in a preferred embodiment, the analog exhibits therapeutic activity only following cleavage of the enzyme-cleavable moiety therefrom.

As described above, a tamandarin or didemnin analog having the structure of one of formulas I, XXI, and can be bound with a support. The identity of the support is WO 01/76616 PCT/US1/11607 not critical. The support can be substantially any material with which such an analog can be 2 3 1 C bound by covalent attachment through one of the R R R or R moieties).

a Examples of support materials include bonded silicates, cross-linked agarose, polyacrylamide, dextran, and allyl dextran. Such support materials can be chemically modified using reactive C chemical moieties in order to facilitate covalent attachment of the analog with the support.

Chemical modifications of this type are known in the art, and can, for example, include 'O modification of a support with cyanogen bromide groups, epoxide groups, mesyl groups, and carboxyhexyl groups. Protocols for preparation of a support and subsequent attachment of a compound to the support are available in the art, and can be modified by one skilled in the art I for use with a didemnin analog described herein.

Examples of didemnin analogs having the structure of formula I or formula XXI, include, compound 21, and compounds 101-143, some of which are depicted in one or more of Figures 1-39.

1 2 In compound 21, R is -(tert-butyloxycarbonyl), R is an O-methyl-tyrosine 568 9 7 3 4 side chain R R R and R are each and R is -OCH 3 R is -CH 3 R is an isoleucine side chain, R1 0 is a leucine side chain, X is and Y is -(triisopropylsilyl).

Preferred tamandarin analogs are based on the structure of tamandarin A. In tamandarin A ({(2S)Hiv2}didemnin B; compound 101), R is -(N-methyl-R-leucine)-proline- 2 5 6 8 9 7 lactate, R is an O-methyl-tyrosine side chain R R R and R are each and R is

-OCH

3

R

3 is -CH 3

R

4 is an isoleucine side chain, R10 is a leucine side chain, X is and Y is -H.

In tamandarin M ({(2S)Hiv2}didemnin M; compound 103), R 1 is -(N-methyl- R-leucine)-proline-actate--glutamine-pyroglutamate, R is an O-methyl-tyrosine side chain 6 8 9 7 3 4 R R 6 R and R are each and R is -OCH3), R is -CH 3 R is an isoleucine side chain, R1 0 is a leucine side chain, X is and Y is -H.

Preferred didemnin analogs are based on the structure of didemnin B. In 2 1 1 tamandarin B ({(2S)Hiv 2 Norsta }didemnin B; compound 105), R is -(N-methyl-Rleucine)-proline-lactate, R 2 is an -methyl-tyrosine side chain R 5 R6, R and R are each and R is -OCH3), R is -CH 3 R is a valine side chain, R 1 is a leucine side chain, X is and Y is -H.

-26- WO 01/76616 WO 0176616PCT/USO1/1 1607 IDIn compound 107, R Iis -(N-niethyl-R-leucine)-glyin-(7-dimethylcouam 2 568, 9 -,adR7 S 4-acetate), R is an 0-methyl-tyrosine side chain R R Rand R are each-HanR 0 is -OCHA) R' is -CH 3 R 4is an isoleucine side chain, R 10is a leucine side chain, X .is and Y is -H.

c-I 12 c-I In compound 109, R is -(N-methyl-R-leucine)-proline-lactate-rhodamine, R is an 0-methyl-tyrosine side chain R 5, R 6, R 8, and R 9are each -HK and R 7is -OCH 3 3 4 .CR is -CH 3 R is an isoleucine side chain, R is a leucine side chain, X is and Y is -H.

c-I1 2 c-I In compound 111, R is -{N-methyl-R-leucine)-proline-lactate, R is an 0- 68 9 7 3.

cI methyl-tyrosine side chain R R R and R are each and R is -OCH 3 R is S CH 3 R 4is an isoleucine side chain, R 10is a lysine side chain, X is and Y is cI -H.

In compound 113, R Iis -(N-methyl-R-Ieucine), R 2is an 0-methyl-tyrosine side chain R 5,R 6,R 8,and R 9are each and R 7is -OCH 3 R 3is -CH 3 R 4is an isoleucine side chain, R 10is a leucine side chain, X is and Y is -H.

In compound 115, R Iis -{N-methyl-R-leucine)-proline-actate, R 2is a lysine side chain, R is -CH 3 R 4is an isoleucine side chain, R 10is a. leucine side chain, X is and Yis -H.

In compound 123, R Iis -{N-methyl-R-Ieucine)-proline-lactate, R 2and R3 together are a tetrahydroisoquinoline substituent having the structure of formula IV, R 5, R6 8 7 4 and R are each R is -OCH3, R is a valine side chain, R is a leucine side chain, X is and Yis -H.

In compound 124, R Iis -(N-methyl-R-leucine)-proline-lactate, R 2is an 0methyl-tyrosine side chain R 5, R 6, R 8, and R 9are each -H and R 7is -OCH 3 R 3is-

CH

3 R 4is an isoleucine side chain, R 10is a leucine side chain X is and Y is -H.

In compound 128, R Iis -(N-methyl-R-leucine)-proline-lactate-glutaminecyclopentanoate, R 2is an 0-methyl-tyrosine side chain R 5, R 6, R 8, and R 9are each -H 7 3 4 and R is -OCHA) R is -CH3, R is an isoleucine side chain, R is a leucine side chain, X is and Yis -H.

In compound 129, R Iis -(N-methyl-R-leucine)-proline-(N-methyl-S-alanine)- 2 5 68 .9 leucine-pyroglutamate, R is an 0-methyl-tyrosine side chain R R R and R are each -H and R 7is -OCHA) R 3is -Cl! 3 R 4is an isoleucine side chain, R 10is a leucine side chain, X is and Y is -H.

-27- WO 01/76616 WO 0176616PCTIUSO 1/1 1607 IND In compound 131, R Iis -(N-methyl-R-leucine)-proline-lactate, R 2is an 0- 56 8 9 7 3 methyl-tyrosine side chain R R R and R are each -H and R is -OCH 3 R is 4 10 13

CH

3 R is an isoleucine side chain, R is a leucine side chain, R is a cephalosporin moiety cleavable by the enzyme, beta-lactamase, X is and Y is

-H.

In compound 132, R Iis -(N-methyl-R-leucine)-(S)proline-(S)lactate, R 2is an ID56 8 9 7 ID 0-methyl-tyrosine side chain R R R and R are each -H and R is 3 4. 10 13

-OCH

3 R is -CH 3 R is an isoleucine side chain, R is a leucine side chain, R is a beta- N glucoside moiety cleavable by the enzyme, beta-glucuronidase, X is and Y is -H.

01 2.

0 10 In compound 134, R is -(N-methyl-S-leucine)-(S)proline-pyruvate, R is an 0-methyl-tyrosine side chain R 5, R 6, R 8, and R 9are each -H and R 7is -OCH3), R3is -CH 3 R 4is an isoleucine side chain, R 10is a leucine side chain, X is 0--,and Yis -H.

In compound 137, R Iis (N-methyl-R-leucine)-(S)proline-pyruvate, R 2and 3 R together are a tetrahydroisoquinoline substituent having the structure of formula IV, R R 6, and R 8are each R 4is an isoleucine side chain, R 10is a leucine side chain, X is and Yis -H.I 1 2.

In compound 138, R is -(N-methyl-R-leucine)-(S)proline-pyruvate, R is an 0-methyl-tyrosmne side chain R 5, R 6, R 8, and R 9are each -H and R 7is 3 4. Z0 -OCH 3 R is -CH 3 R is an isoleucine side chain, R is a lysine side chain, covalently attached to a support, X is and Y is -H.

In compound 142, R Iis -(N-methyl-S-leucine)-(S)proline-pyruvate, R 2is an 0-methyl-tyrosine side chain R 5, R 6, R 8, and R 9are each -H and R 7is -0CU 3 R 3is -CH 3 R 4is a valine side chain, R 10is a leucine side chain, X is and Y is

-H.

In compound 143, R Iis -(N-methyl-R-leucine)-(S)prolie-pyruvate, R 2is an 0-methyl-tyrosine side chain R 5, R 6, R 8, and R 9are each -H and R 7is

-OCH

3 R 3is 0H3, R 4is an isoleucine side chain, R 10is a leucine side chain, X is -Nil-, and Y is -H.

The structural similarity of didemnin B and taniandarin A ((2S)Hiv 2}dideinnin B; compound 101), is ilustrated in Figure 25. The primary structural difference, indicated by brackets and dotted lines, is in the macrocycle portion of these 28 WO 01/76616 PCT/US01/11607 o compounds. Tamandarin A, shown in Figure 25A, contains an alpha-hydroxyisovaleryl (Hiv) moiety, and didemnin B, shown in Figure 25B, contains an alpha-(alpha-hydroxyisovaleryl)propionyl (Hip) moiety. The simpler macrocycle structure of tamandarin A, and of any compound having the structure of formula I or formula XXI, can be synthesized more readily Cl than the macrocyclic structure of didemnin B. Compounds which have the structure of either of formula I and formula XXI can be more easily (and generally more inexpensively) prepared \0 than compounds which are identical but for the presence of an Hip moiety in place of the Hiv NC moiety.

Another group of compounds that can exhibit the physiological activities described herein and which are included in the invention are compounds which correspond to fragments of didemnin analogs having the structure of formula I or formula XXI. Fragments which exhibit this activity include those which have the structure of formula VII OY 0 R4~ X R11 x r HN O APG

(V)

In formula VII, X, Y, and R 4 have the identities described for formulas I and XXI, and APG is an amine protecting group. Examples of amine protecting groups which can be present in the active fragments include carbobenzyloxy (CBZ) and a tert-butyloxycarbonyl (BOC) moieties. Other useful amine protecting groups are described in references such as Green and Wutz (1999, Protecting Groups in Organic Synthesis, Wiley, New York) and Bodansky (1993, Principles of Peptide Synthesis, Springer, Berlin).

R in formula VII can be any of-OH, -NH2, -(O-allyl), and pentafluorophenyl). Alternatively R 11 can be a substituent having the structure shown in formula VII

R

3 I R2 N 0

R

1 0 NH-APG

NH

OR1

(VHM).

-29- WO 01/76616 PCT/US01/11607 N In formula VII, R2, R 3 and RI have the identities described for formulas I

O

S and XXI, and APG is an amine protecting group as described for formula VII (although it need not be the same APG as in formula VII). R 12 can be either-H or a hydroxyl protecting group, as described herein. Compounds having the structure of formula VII that can be made and N 5 used as described herein, and include the compounds designated 6, 17, 19, and 20 in Figure 26.

,0 Methods of Using Compounds Described Herein r C The didemnin and tamandarin analogs disclosed herein can be used to affect a O variety of physiological processes. Each of these types of compounds can be used to inhibit C 0 protein synthesis. Furthermore, the compounds can be. used to inhibit progression of a cell through the cell cycle. While not being bound by any particular theory of operation, it is believed that the cell cycle-inhibiting activity of the compounds can be attributed to inhibition of protein synthesis, to inhibition of other cellular activities associated with DNA replication or cell division, or to some combination of these activities. These tamandarin and didemnin analogs also induce apoptosis in cells. The physiological activities attributable to these tamandarin and didemnin analogs make these compounds useful for alleviating a variety of disorders in which one or more of cell growth, proliferation, and survival are aberrant.

Examples of such disorders include cancers at various stages tumorigenesis, tumor growth, and metastasis) and viral infections at various stages infection of cells with virus 0 particles, production of virus particles within a cell, and survival of virus-infected cells).

While still not being bound by any particular theory of operation, it is believed that the physiological activities attributable to the tamandarin and didemnin analogs described herein result from one or more interactions between such analogs and at least one cellular component. This interaction(s) leads, directly or indirectly, to the observed cellular response.

Accordingly, the invention encompasses use of these compounds to identify one or more cellular components which contributes to a disorder phenotype in an individual. Identification of such a cellular component can indicate an effective course of treatment for alleviating the disorder. Examples of compounds useful for this purpose include analogs which comprise a 1 2 fluorescent substituent at R or R a photoreactive chemical moiety, such as a moiety having the structure WO 01/76616 PCT/US01/11607 0 O0

O

or a moiety bound with a support.

IO

NO Fluorescent and other detectably labeled tamandarin and didemnin analogs CN described herein (as well as their physiologically active fragments) can be used to identify cells C c in which those analogs and fragments can exert their physiological effects. For example, cells l which absorb or bind with a fluorescent analog can be identified or isolated. Identification or isolation of such cells can be used to diagnose a disorder associated with the presence of such cells. Identification or isolation of these cells can also indicate which of the tamandarin or didemnin analogs are efficacious for treating a disorder involving the cells.

The tamandarin and didemnin analogs described herein can be used for antiproliferative, anti-tumor, anti-viral, and immunosuppressive purposes. For example, these compounds can be used in a pharmaceutical preparation or medicament to be administered to a patient afflicted with a disorder in which one or more of protein synthesis, cell growth, proliferation, and survival are aberrant. Such medicaments can be used to treat disorders such as cancers breast cancer), viral, fungal, parasitic, and bacterial infections, auto-immune disorders, allergies, other hyper-immune disorders, and atherosclerosis.

Examples of anti-tumor activities that can be exhibited by the compounds described herein include inhibition of tumorigenesis, inhibition of metastasis, inhibition of tumor cell growth, inhibition of tumor cell proliferation, and enhancement of tumor cell apoptosis. Dehydrodidemnin exhibits activity against cell lines derived from several human solid tumor types, including non-small cell lung cancer and colon tumor cell lines, and exhibits selective anti-tumor activity against non-small cell lung cancer,;melanomas, ovarian cancer, and colorectal cancer (Depenbrock et al., 1998, Brit. J. of Cancer 78(6): 739-744). The tamandarin and didemnin analogs described herein exhibit anti-tumor activities in cells of one or more of these lines, as well as in cells of the corresponding tumor type in vivo.

Determination of the effectiveness of any particular analog against any particular tumor type can be made using standard methods involving, for example, one or more of the 60 standard tumor cell lines maintained in the U.S. National Cancer Institute drug screening program.

-31- WO 01/76616 PCTUS01/11607 I, Examples of anti-viral activities that can be exhibited by the tamandarin and didemnin analogs described herein include inhibition of binding of a virus with a cellular target, inhibition of infection of a cell by a virus, inhibition of cellular synthesis of virus components, inhibition of intracellular assembly of virus particles, inhibition of release of virus N 5 particles from an infected cell, inhibition of growth of a cell infected by a virus, inhibition of proliferation of a cell infected by a virus, and induction of death apoptosis) of a cell \0 \0 infected by a virus. The anti-viral activity of the compounds described herein can, for CI example, be used to treat or prevent viral infections of mammals and associated symptoms. By way of illustration, the didemnin and tamandarin analogs can be used to treat or prevent infections by viruses such as Rift Valley Fever virus, Dengue virus, or any of the equine encephalitis viruses.

Examples of immunosuppressive activities that can be exhibited by the tamandarin and didemnin analogs described herein include inhibition of a cellular immune response to an immunogen an infectious agent, or a transplanted cell or tissue) and inhibition of a humoral immune response to an immunogen. Examples of disorders in which immunosupression can be desirable include autoimmune disorders, transplant rejection disorders rejection of a solid tissue or bone marrow transplant), development of an immune response to an implanted device a stent or a heart valve), immune hypersensitivity, and anaphylaxis.

The tamandarin and didemnin analogs described herein can be administered in vitro to a cell or tissue a cultured cell or tissue, or a cell or tissue harvested from one animal prior to introduction into the same or a different animal). Alternatively, the analogs can be administered to the cell or tissue in vivo by administering the analog or a pharmaceutical composition comprising the analog to an animal a mammal such as a human) that comprises the cell or tissue.

In one embodiment of the treatment methods described herein, a tamandarin or didemnin analog described herein and having an enzyme-cleavable group attached thereto a compound having the structure of formula XXI) is administered to an animal. Upon cleavage of the enzyme-cleavable group, the compound is transformed from an inactive (or less active) form to an active (or more active) form, as shown in Figures 23 and 24. Thus, a tamandarin or didemnin analog can be selectively activated at a body location at which the enzyme activity occurs.

-32- WO 01/76616 PCT/US01/11607 u The enzyme which is used to cleave a tamandarin or didemnin analog having an enzyme-cleavable moiety attached can be an enzyme which naturally occurs at a body location in an animal. Alternatively, the enzyme can be provided to the animal, for example as a composition comprising the enzyme or a nucleic acid which encodes the enzyme. As another CI example, the enzyme can be coupled covalently, using a cross-linking agent or by expression as an enzyme-antibody fusion protein) with an antibody that specifically binds with

INO

'0 a tissue cancerous cells such as leukemic cells or cells of a solid tumor) at a body location CI in the animal, and the antibody-enzyme complex can be administered to an animal.

Administration of a tamandarin or didemnin analog having an attached enzyme-cleavable group to the same animal results in preferential activation of the compound at the tissue or body location. The physiological effect of the compound can thereby be localized at the tissue or body location, and any side effect attributable to the activated compound can thereby be reduced or minimized.

A support-bound tamandarin or didemnin analog can be used to identify cells which comprise, on their surfaces or elsewhere, receptor proteins, glycoproteins, and the like, which are capable of interacting or binding with the analog. As an example, a tamandarin or didemnin analog having the structure of formula I or XXI and attached to a support can, by virtue of its interaction with a particular cellular receptor, be used to identify or physically isolate cells of a particular type tumor cells) which are characterized by the presence of Sthe particular receptor.

Methods of Making Compounds Described Herein The present invention include methods of making didemnin analogs and fragments described herein. Methods of making tamandarin and didemnin analogs have been described Harris et al., 1987, Tetrahedron Lett. 28:2837-2840; Harris et al., 1988, Tetrahedron 44:3489-3500; Ewing et al., 1986, Tetrahedron 42:5863-5868; Ewing, W.R., 1988, Ph.D. Dissertation, University of Pennsylvania, Philadelphia PA; Ewing et al., 1989, Tetrahedron Lett. 30:3757-3760; Li et al., 1990, J. Am. Chem. Soc. 112:7659-7672; Mayer et al., 1994, J. Org. Chem. 59:5192-5205; Mayer et al., 1994, Tetrahedron: Asymmetry 5:519- 522; Xiao et al., 1997, Tetrahedron: Asymmetry 9:47-53; Pfizenmayer et al., 1998, 8:3653- 3656; U.S. Patent Application serial number 09/545,848, filed April 7, 2000). The contents of -33- WO 01/76616 PCT/US01/11607 S each of these references and patent application are incorporated herein by reference. The

O

CN precise method used to make a tamandarin or didemnin macrocycle or analog is not critical.

SPreferably, the method that is used results in the stereoselective synthesis of a compound described herein. For example, synthesis of 2 iC tamandarin A ({(2S)Hiv }didemnin B; compound 101) is exemplified herein at Example 1.

In reference to methods of making the analogs and fragments described herein, N 2 3 4 5 6 7 8 9 IO the substituents R 2

R

3

R

4

R

5

R

6

R

7 R R R and Y, have the same meanings as S used above.

As used in the present disclosure, a protection reaction can include any reaction 0 0 whereby one or more chemical moieties are covalently (but reversibly) attached to one of a

(N

nitrogen atom, an oxygen atom, and a sulfur atom of a molecule. Such attachment prevents the atom or atoms from participating in non-desired chemical reactions, becoming covalently attached to other chemical moieties, and donating or accepting either of protons and electrons to other chemical moieties. A chemical moiety thus attached is referred to as "a protecting group." By way of example, the nitrogen atom of a compound having the structure of formula IX, such as D-allo-isoleucine, can be protected using a reagent such as carbobenzyloxysuccinimide(CBZ-succinimide). Use of this reagent in a standard protocol yields a protected D-allo-isoleucine, N(alpha)-CBZ-D-allo-isoleucine, having the structure of compound 8 in Figure 26A. In compound 8, the CBZ moiety acts as an amine protecting group, and the nitrogen atom to which it is attached cannot readily undergo additional chemical reactions.

Further by way of example, when X is a protected amine group can be used in the reactions described herein. As an alternative example, the hydroxyl moiety of compound 11 in Figure 26A, can be protected using a reagent such as triisopropylsilyltriflate (TIPSOTf) to yield compound 12 in Figure 26A. In this compound, Y is a triisopropylsilyl (TIPS) moiety and acts as a hydroxyl protecting group, preventing chemical reactions with the oxygen atom to which this moiety is attached.

Protocols for performing protection reactions and comprehensive information about chemical moieties can be used as protecting groups is found in references such as Green and Wutz (1999, Protecting Groups in Organic Synthesis. Wiley, New York) or Bodansky (1993, Principles of Peptide Synthesis. Springer, Berlin).

Didemnin analogs and fragments can be made by converting a compound having the structure of formula IX -34- WO 01/76616 PCT/US01/11607 I 0 R4

OH

o NH 2 2

(TX)

to a compound having the structure of formula X OY O SNR4

OH

(C HN-APG CN Such a series of reactions can include, but is not limited to, a protection reaction, an activation O reaction, an esterification reaction, and an ester hydrolysis reaction. The amine group of S formula IX is preferably protected prior to performing the esterification and hydrolysis reactions. A specific example of making a compound having the structure of formula X is given in Example 1. In formulas X-XVII, "APG" refers to an amine protecting group such as carbobenzyloxy (CBZ) moiety or tert-butyloxycarbonyl (BOC) moiety. Alternative amine protecting groups can also be used, as described herein and in the art.

An example of an activation reaction included in the method of making a compound having the structure of formula X is depicted in Figure 26A, reaction B. Activation of a compound such as compound 8 can involve a reagent such as pentafluorophenol (PFPOH) to yield compound 9. Compound 9 is an example of an activated intermediate that more readily undergoes subsequent reactions, such as esterification, at the carbonyl carbon of its PFP ester moiety. Esterification reactions which do not require an activated intermediate can also be employed to make a compound having the structure of formula X.

Any method of ester hydrolysis known in the art that does not comprise harsh conditions which favor racemization and can be used to make a compound having the structure of formula X. By way of example, a compound having the structure OY o R4_OCH 3

HN-APG

can be hydrolyzed using a strong base in a solvent mixture, as exemplified in Figure 26A, reaction F. Reagents and conditions suitable for ester hydrolysis under milder conditions WO 01/76616 PCTUSO1/11607 including conditions which do not favor racemization) can be readily selected by one skilled in the art.

A compound having the structure Zlyo COH

X-H

I can be esterified with, for example, allyl bromide as described in Example to yield a compound having the structure of formula XI c-i 0

I'D

c-iH

(XI).

A compound having the structure of formula IX and a compound having the structure of formula XI can be coupled esterified) to yield a compound having the structure of formula XII 0Y 0 HN 0 APG (xi).

reaction I of Figure 26B). Optionally, such a reaction can be performed using a catalyst, a coupling reagent, or an esterification reagent. Reagents and conditions useful for this type of reaction are known in the art and exemplified in Example 1. Didemnin fragments having the structure of formula XII exhibit one or more of the pharmacological activities described herein.

A compound having the structure of formula XII can be hydrolyzed to yield a compound having the structure of formula XIII

OYO

HNRXII OH "N APG0 As described elsewhere herein, reaction conditions and reagents suitable for ester hydrolysis are known in the art, and can be readily applied by a skilled artisan. An example of this type of -36- WO 01/76616 PCT/US01/11607 hydrolysis is depicted in Figure 26B, reaction J. Didemnin fragments having the structure of N formula XIII exhibit one or more of the pharmacological activities described herein.

The carboxyl group of a compound having the structure of formula XIII can be activated to yield a compound having the structure of formula XIV N OYo R4-A XO-ACT SHN 0 APG (XIV).

CI In formula XIV, "ACT" refers to an activating group, such as a pentafluorophenyl (PFP) moiety. Another example of an activating group is an N-hydroxysuccinimide moiety.

Chemical activation can be performed using reagents such as an activating reagent, a catalyst, an activating group donor, or the like. By way of example, compound 6, depicted in Figure 26C, is activated by covalent attachment of a PFP group to yield compound 19. Protocols for activating a compound in the manner disclosed herein are known in the art. Didemnin fragments having the structure of formula XIV exhibit one or more of the pharmacological activities described herein.

The activated compound having the structure of formula XIV can be coupled with a third reactant having the structure of formula XV

R

3 I R 2 ~Oy 0 NoO

APG

°'^NH

NH

2 o=KN 0 SEM (xV) to yield a compound having the structure of formula XVI R 3 I N 3

R

2 0 SEM R1OI N 0 0 H 0

AP

O NH HN-APG

H

0 0 Y (XVI).

-37- WO 01/76616 PCT/US01/11607 IND In formulas XV and XVI, SEM refers to 2-(trimethylsilyl)ethoxycarbonyl. An example of this N reaction is depicted in reaction M of Figure 26C, in which compound 19 is coupled with S compound 18 to yield compound 20. The reagents and conditions necessary for preparation of a protected peptide such as compound 18 are described, for example, in Li et al. (1990, J. Am.

c"i Ci Chem. Soc. 112: 7659-7672). Didemnin analogs having the structure of formula XVI exhibit one or more of the pharmacological activities described herein.

A didemnin analog having one or more of the pharmacological activities S described herein can be made by removing one of the amino protecting groups and the c-i 0 carbonyl hydroxyl protecting group of a compound having the structure of formula XVI and Q cyclizing the compound to yield a PSI having the structure of formula XVII N

H

R N' O

N-APG

HNH "YY H< X R 4 0 0 OY (xv An example of reactions of this type is shown in step N of Figure 26D.

Chemically de-protecting a compound such as one having the structure of formula XVI can be accomplished by reacting the compound with one or more reagents to remove a protecting group of the compound. Exemplary de-protection reactions are disclosed herein, for example for compound 20, as shown in Figure 26D. Other protocols for de-protecting a compound are known in the art, and can be readily applied by a skilled artisan to de-protection of a compound having the structure of formula XVI. Cyclization of a de-protected compound otherwise having the structure of the formula XVI can be accomplished using any method known in the art for macrocyclization ofpeptides. For example, the macrocyclization conditions can be similar or identical to those used in the cyclization that yields compound 21, depicted in Figure 26D and described in Example 1. Didemnin analogs having the structure of formula XVII exhibit one or more of the therapeutic activities described herein.

One or more of the protecting groups of a compound having the structure of the formula XVII can be removed to yield a compound having the structure of formula XVIII -38- WO 01/76616 PCT/US01/11607 R2

R

C O R2 NH 2 RO

O

0 X R 4 O

O

a described herein.

Yet another active compound can be made by coupling a compound having the structure of formula XVIII and a reagent having the structure of formula X 0 O HOA R14(XX) to yield a PSI having the structure of formula XX

R

3 1N R 2 R o OH N

O

N ^0 OH SR14

(XX).

This reaction is exemplified in reaction N of Figure 26E. R 14 can, for example, be any 1 14 of the moieties described above as R The R 4 substituent group can comprise an enzyme cleavable moiety, preferably at or near the distal end thereof (relative to the macrocycle). Such a moiety can be cleavable by an enzyme, for example, a carboxypeptidase, a beta-lactamase, a beta-galactosidase, a penicillin V-amidase, a cytosine deaminase, a nitroreductase, an alkaline phosphatase, a beta-glucuronidase, and a catalytic antibody. An example of an R 14 moiety which comprises an enzyme-cleavable moiety is -(N-methyl)leucine-(S)proline-(S)lactate- (S)glutamine-(S)pyroglutamate. Other examples of enzyme-cleavable moieties are described -39- WO 01/76616 PCT/US01/11607 S herein. By way of illustration, compounds 131 and 132, depicted in Figures 23 and 24,

O

CN respectively, can be prepared using the methods described herein. Didemnin analogs and fragments which have an enzyme cleavable moiety attached thereto and which otherwise have the structure of one of formulas XI-XVII upon cleavage of the enzyme-cleavable group C ;i therefrom exhibit one or more of the therapeutic activities described herein.

Variation of the substituents of the didemnin analogs and fragments can require I slight modifications in the general methods described herein. It is understood that the invention includes such modifications, as they could be readily designed by one of ordinary S skill in the art of synthetic chemistry.

O

0 Tamandarin and didemnin analogs can also be made by a method that includes incorporation of a deoxo-proline residue or a dehydro-proline residue in the side chain of a tamandarin or didemnin analog. The deoxo-proline residue or dehydro-proline residue can be used in place of a proline residue in any known tamandarin or didemnin analog. Particularly contemplated tamandarin and didemnin analogs are those in which the deoxo-proline residue or i dehydro-proline residue is linked to the macrocycle by way of a leucine residue having a methylated amine moiety an -(N-methyl)leucine-(deoxo or dehydro)-proline-containing tamandarin or didemnin analog).

Of course, the (deoxo or dehydro)-proline residue can be further substituted, for example by lactate, by pyruvate, by lactate-fluorophore, by lactate-glutamine-pyroglutamate, by lactateglutamine-cyclopentanoate, by -alanine-leucine-pyroglutamate, or by -(N-methyl-alanine)leucine-pyroglutamate. One or more of the (deoxo or dehydro)-proline, lactate, glutamine, pyroglutamate, cyclopentanoate, and alanine residues is preferably the enantiomer.

Incorporation of a deoxo-proline residue can be achieved by any method known in the art. Examples of methods of incorporating a deoxo-proline residue are included in this disclosure in Example 5 and in Figures 41-43.

In one embodiment, the method of incorporating a deoxo-proline residue comprises protecting the hydroxyl and amine moieties of leucine, methylating the leucine amine moiety, and de-protecting the leucine amine moiety. The amine group of proline is protected, and the ester function of the proline is reduced to an aldehyde using a strong base such as LiBH4 coupled with oxidation with an oxidizing agent such as pyridine-S03).

Reductive amination in the presence of a non-aqueous solvent, a strong base, and a carboxylic acid catalyst; in the presence ofNa(AcO) 3 BH, AcOH, and CH 2 C1 2 can be WO 01/76616 PCT/US01/11607 used to couple the hydroxyl-protected leucine with the amine protected proline to form N compound 43 in Figure 43, in one embodiment). The reductive amination can, for example, be performed as described by Abdel-Magid and co-workers Abdel-Magid et al., 1990, Tetrahedron Lett. 31:5595-5598; Abdel-Magid et al., 1990, Synlett. 537-539).

cN The resulting leucine-deoxo-proline dipeptide can be further substituted with other moieties with -lactate, -pyruvate, -lactate-fluorophore, -lactate-glutamine- \0 pyroglutamate, -lactate-glutamine-cyclopentanoate, -alanine-leucine-pyroglutamate, or NC methyl-alanine)-leucine-pyroglutamate), with (preferably) or without first removing the 0 protecting groups. The leucine-deoxo-proline dipeptide (optionally further substituted) can be attached to a tamandarin or didemnin macrocycle in the position identified as R in formulas I and XXI.

Incorporation of a dehydro-proline residue can be achieved by any method known in the art. Examples of methods of incorporating a dehydro-proline residue are included in this disclosure in Example 6 and in Figure 44.

In one embodiment, the method of incorporating a dehydro-proline residue comprises protecting the carboxyl and amino groups of 4-hydroxyprolinate, mesylating the 4hydroxyl moiety, displacing the mesylate moiety with an aryl-selenyl moiety, oxidatively eliminating the aryl-selenyl moiety, and de-protecting the carboxyl moiety. The resulting dehydro-proline moiety can be coupled with one or more additional amino acid residues or organic acids those identified herein, removing the amino-protecting group if necessary or desired) and coupled with a tamandarin or didemnin macrocycle made or obtained by conventional means.

Pharmaceutical Compositions The invention encompasses pharmaceutical compositions comprising at least one of the tamandarin and didemnin analogs and the physiologically active fragments described herein. Such compositions can comprise the analog/fragment and a pharmaceutically acceptable carrier. By way of example, a pharmaceutical composition can comprise a pharmaceutically acceptable carrier and a tamandarin or didemnin analog having the structure of any of formulas I, XXI, and as an active agent. As a further example, a pharmaceutical composition can comprise a pharmaceutically-acceptable carrier and one or more of the compounds depicted in the figures in this disclosure.

-41- WO 01176616 WO 0176616PCT[US0 1/11607 IND Such pharmaceutical compositions can be used,. for example, in the methods described herein for and for inhibiting one or more of protein synthesis, cell cycle progression, 0 tumorigenesis, growth, and proliferation in a cell. In addition, such compositions can be used in the methods described herein for enhancing apoptosis in a cell.

N- 5 Pharmaceutical compositions that are useful in the methods of the invention can be administered systemically in oral solid formulations, ophthalmic, suppository, aerosol, ID topical or other similar formulations. In addition to the active agent, such pharmaceutical

N-

N- compositions can contain pharmaceutically-acceptable carriers and other ingredients known to ci enhance and facilitate drug administration. Other possible formulations, such as nanoparticles, 3) liposomes, resealed erythrocytes, and immunologically based systems can also be used to ci administer the active agent according to the methods of the inVention.

The invention encompasses pharmaceutical compositions which consist of the active agent, in a form suitable for administration to a subject, or the pharmaceutical composition can comprise the active agent and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active agent can be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active agent into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humnans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, and -42- WO 01/76616 PCT/US01/11607 IN mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

Pharmaceutical compositions that are useful in the methods of the invention can be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, C i topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed N0 erythrocytes containing the active agent, and immunologically-based formulations.

CI A pharmaceutical composition of the invention can be prepared, packaged, or o sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit 8 dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active agent. The amount of the active agent is generally equal to the dosage of the active agent which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

In addition to the active agent, a pharmaceutical composition of the invention S can further comprise one or more additional pharmaceutically active agents such as, other tumor therapy agents, other anti-infective agents, and the like.

Controlled- or sustained-release formulations of a pharmaceutical composition of the invention can be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitable for oral administration can be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active agent. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion.

As used herein, an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.

A tablet comprising the active agent may, for example, be made by compressing or molding the active agent, optionally with one or more additional ingredients. Compressed tablets can be prepared by compressing, in a suitable device, the active agent in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets can be made by molding, in a suitable device, a mixture of the active agent, a pharmaceutically -43- WO 01/76616 PCT/US01/11607 S acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically N, acceptable excipients used in the manufacture of tablets include, but are not limited to, inert a diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycollate.

c 5 Known surface active agents include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, IND microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginate. Known binding agents include, but are not limited to, gelatin, acacia, pre-

O

S gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearate, silica, and talc.

Tablets can be non-coated or they can be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active agent. By way of example, a material such as glyceryl monostearate or glyceryl distearate can be used to coat tablets. Further by way of example, tablets can be coated using methods described in U.S. Patents numbers 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled release tablets. Tablets can further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation.

Hard capsules comprising the active agent can be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active agent, and can further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active agent can be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active agent, which can be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration can be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.

-44- WO 01/76616 PCT/US01/11607 I Liquid suspensions can be prepared using conventional methods to achieve

O

C suspension of the active agent in an aqueous or oily vehicle. Aqueous vehicles include, for OO example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated C0 vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions can further comprise one or more additional ingredients including, but not limited to, suspending agents,

ID

O dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, S flavorings, coloring agents, and sweetening agents. Oily suspensions can further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, 0 hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan mono-oleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para- hydroxybenzoates, ascorbate, and sorbate. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

Liquid solutions of the active agent in aqueous or oily solvents can be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active agent is dissolved, rather than suspended in the solvent. Liquid solutions of the pharmaceutical composition of the invention can comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active agent in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

WO 01/76616 PCT/US01/11607 Powdered and granular formulations of a pharmaceutical preparation of the invention can be prepared using known methods. Such formulations can be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each CN 5 of these formulations can further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, IND flavoring, or coloring agents, can also be included in these formulations.

N1 A pharmaceutical composition of the invention can also be prepared, packaged, cI or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase can be a g vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions can further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions can also contain additional ingredients including, for example, sweetening or flavoring agents.

A pharmaceutical composition of the invention can be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition can be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation.

Suppository formulations can be made by combining the active agent with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature about 20 0 C) and which is liquid at the rectal temperature of the subject about 37°C in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations can further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.

Retention enema preparations or solutions for rectal or colonic irrigation can be made by combining the active agent with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations can be administered using, and can be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations can -46- WO 01/76616 PCT/US01/11607 further comprise various additional ingredients including, but not limited to, antioxidants and 04 preservatives.

SA pharmaceutical composition of the invention can be prepared, packaged, or sold in a formulation suitable for vaginal administration. Such a composition can be in the CN form of, for example, a suppository, an impregnated or coated vaginally-insertable material such as a tampon, a douche preparation, or a solution for vaginal irrigation.

\0 O Methods for impregnating or coating a material with a chemical composition are 0c known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying.

Douche preparations or solutions for vaginal irrigation can be made by combining the active agent with a pharmaceutically acceptable liquid carrier. As is well known in the art, douche preparations can be administered using, and can be packaged within, a delivery device adapted to the vaginal anatomy of the subject. Douche preparations can further comprise various additional ingredients including, but not limited to, antioxidants, antibiotics, anti-fungal agents, and preservatives.

Formulations of a pharmaceutical composition suitable for parenteral administration can comprise the active agent combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations can be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.

Injectable formulations can be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.

Such formulations can further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active agent is provided in dry powder or granular) form for reconstitution with a suitable vehicle sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

-47- WO 01/76616 PCT/US01/11607 The pharmaceutical compositions can be prepared, packaged, or sold in the C form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution can be formulated according to the known art, and can comprise, in addition to the active agent, additional ingredients such as the dispersing agents, wetting agents, or suspending Ci 5 agents described herein. Such sterile injectable formulations can be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other I acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active 0 0 10 agent in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained:release or implantation can comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topicallyadministrable formulations may, for example, comprise from about 1% to about 10% (w/w) active agent, although the concentration of the active agent can be as high as the solubility limit of the active agent in the solvent. Formulations for topical administration can further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation can comprise dry particles which comprise the active agent and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active agent dissolved or suspended in a low-boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater -48- WO 01/76616 PCT/US01/11607 D than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 S nanometers. Dry powder compositions preferably include a solid fine powder diluent such as U sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having a boiling C 5 point of below 65°F at atmospheric pressure. Generally the propellant can constitute 50 to 99.9% of the composition, and the active agent can constitute 0.1 to 20% of the I composition. The propellant can further comprise additional ingredients such as a liquid non- S ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same S order as particles comprising the active agent).

0 Pharmaceutical compositions of the invention formulated for pulmonary delivery can also provide the active agent in the form of droplets of a solution or suspension.

Such formulations can be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active agent, and can conveniently be administered using any nebulization or atomization device. Such formulations can further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers.

0 The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of a pharmaceutical composition of the invention.

Another formulation suitable for intranasal administration is a coarse powder comprising the active agent and having an average particle from about 0.2 to 500 micrometers.

Such a formulation is administered in the manner in which snuff is taken by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% and as much as 100% of the active agent, and can further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% active agent, the balance comprising an orally dissolvable or degradable -49- WO 01/76616 PCT/US01/11607 composition and, optionally, one or more of the additional ingredients described herein.

O

Alternately, formulations suitable for buccal administration can comprise a powder or an S)aerosolized or atomized solution or suspension comprising the active agent. Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle cI 5 or droplet size in the range from about 0.1 to about 200 nanometers, and can further comprise one or more of the additional ingredients described herein.

IND A pharmaceutical composition of the invention can be prepared, packaged, or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% solution or

O

0 10 suspension of the active agent in an aqueous or oily liquid carrier. Such drops can further comprise buffering agents, salts, or one or more other of the additional ingredients described herein. Other ophthalmalmically-administrable formulations which are useful include those which comprise the active agent in microcrystalline form or in a liposomal preparation.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; anti-fungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" which can be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, which is incorporated herein by reference.

The relative amounts of the active agent, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and the type and severity of condition of the subject treated and further depending upon the route by which the composition is to be administered.

By way of example, the composition can comprise between 0.1% and 100% active agent.

Typically dosages of the active agent which can be administered to an animal, preferably a human, range in amount from 1 microgram to about 100 grams per kilogram of WO 01/76616 PCTUS01/11607 body weight of the animal. While the precise dosage administered will vary depending upon C any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration. Preferably, the dosage of the active agent will vary from about 1 milligram to about 10 g per kilogram of body weight of Cl the animal. More preferably, the dosage will vary from about 10 milligram to about 1 gram per kilogram of body weight of the animal. Alternatively, the dosage can be determined in units of DO square meters of the body surface of an animal milligrams or kilograms per square meter, C2 2 C mg/m or kg/m Preferably, this dosage will vary from about 0.1 milligram to about 5 grams per square meter of body surface of the animal. More preferably, the dosage will vary from about 1 milligram to about 1 gram per square meter of body surface of the animal.

The active agent can be administered to an animal as frequently as several times daily, or it can be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even lees frequently, such as once every several months or even once a year or less. The frequency of the dose is determinable by the skilled artisan and depends upon various factors including, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

The invention is now described with reference to the following Examples.

These Examples are provided for the purpose of illustration only and the invention is not limited to these Examples, but rather encompasses all variations which are evident as a result of the teaching provided herein.

Examples The reagents and procedures which were used in the Examples are now presented.

Unless otherwise stated, all reactions were conducted in the presence of an inert atmosphere argon or nitrogen). All solvents were reagent grade distilled solvents, chromatography solvents, and reaction work-up solvents) or HPLC grade reaction solvents). Anhydrous diethyl ether and tetrahydrofuran (THF) were distilled from sodium and benzophenone. The boiling point range of the hexane used was 38-55 0 C. Methylene chloride

(CH

2 C1 2 benzene, toluene, and N,N-dimethyl formamide (DMF) were distilled from calcium hydride (CaH2). Organic acids and bases were reagent grade. Triethylamine (Et 3

N),

diisopropylethylamine (DIPEA), morpholine, and N-methylmorpholine (NMM) were distilled -51- WO 01/76616 PCTIUS01/11607 from calcium hydride (CaH2). All other reagents, including dimethylaminophenol and diethyl N- 1,3-acetonedicarboxylate, were the highest purity commercial available. Analytical thin-layer a chromatography (TLC) was performed using EM Separations Tech./Merck silica gel (60-F254) plates (0.25 millimeter) pre-coated with a fluorescent indicator. Visualization was effected C 5 using ultraviolet light (254 nanometers), phosphomolybdic acid w/v) in 95% ethanol.

Melting points (mp) were determined using a Thomas-Hoover capillary melting point Sapparatus and are reported without correction. Proton and carbon magnetic resonance spectra N 1 13 S (1H- and 1C-NMR, respectively) were recorded on a Bruker AM-500 (500MHz) Fourier transform spectrometer, and chemical shifts were expressed in parts per million (ppm) relative 1 13 to CHC1 3 as an internal reference (7.24 ppm for H and 77.0 for Multiplicities are designated as singlet doublet doublet of doublets doublet of triplets triplet quartet multiplet and broad singlet Infrared spectra (IR) were obtained using a Perkin-Elmer Model 1600 FT-IR spectrophotometer. Absorptions are reported in wave number Optical rotations (in degrees) were measured using a Perkin-Elmer Model 341 polarimeter. High resolution mass spectra (HRMS) were obtained using either a VG 70-70HS, or a Micromass AutoSpect. Elemental Analyses were performed using a Perkin-Elmer 2400 Series II CHNS/O Analyzer. Flash column chromatography was performed using Merck silica gel 60 (240-400 mesh) using the solvent systems indicated for individual experiments.

Example 1 Total Synthesis Tamandarin A A method of synthesizing Tamandarin A is described in this example. The method is illustrated in Figure 26. The method is initiated with the synthesis of compound 13, depicted in Figure 26A.

Reaction A of Figure 26A: Synthesis of Compound 8 A solution comprising 5.13 milliliters (36.9 millimoles) of Et 3 N was added drop-wise to a solution comprising 1.56 grams (11.9 millimoles) of compound 7, D-alloisoleucine, and 50 milliliters of freshly distilled CH2Cl 2 at 0°C. To the resulting mixture was added 3.114 grams (12.5 millimoles) of carbobenzyloxy succinimide (Cbz-succinimide). This reaction was stirred at 0°C for 1 hour, and maintained with stirring at room temperature overnight The reaction mixture was concentrated, diluted with 20 milliliters of a saturated -52- WO 01/76616 PCT/US01/11607 S solution of NaHCO 3 and washed twice with 10 milliliters aliquots of ether. The combined CN ether layers were extracted with 10 milliliters of a saturated solution of sodium bicarbonate O) (NaHCO 3 The combined aqueous layers were cooled to o0C, acidified to pH 2 by drop-wise addition of 1 normal KHS04, and extracted three times with 20 milliliters of ethyl acetate (EtOAc). The EtOAc layers were combined, washed with 20 milliliters of a saturated solution of NaCI, and dried in the presence of anhydrous sodium sulfate (Na2SO4). The resulting solution was filtered and concentrated under reduced pressure to yield 3.13 grams of compound t% 8 (99% yield). Compound 8 was bbtained as a colorless oil and used directly in the next step 0 without purification. Analytical data for compound 8 were as follows: Rf 0.08 (20:80-ethyl 1 0 10 acetate:hexane); H NMR (500 MHz, CDCl 3 5 0.86-0.90 3H), 0.93-0.97 3H), 1.20- 1.27 1H) and 1.42-1.47 1H), 1.98-2.09 1H), 4.47-4.50 (dd, Ji=9.1 Hz, J 2 =3.4 Hz, 1H), 5.10 2H), 5.17-5.19 J 9.1 Hz, 1H), 7.29-7.35 5H); 13C NMR (250 MHz,

CDCI

3 8 11.69, 14.35, 26.20, 37.42, 56.9,6, 67.20, 128.14, 128.24, 128.55, 135.06, 156.42, 177.41; IR (CHCI 3 2470-3540, 3440, 2980, 2950, 2890, 1720, 1510, 1455, 1405, 1385, 1325- -1 1355, 1230-1280, 1165, 1095, 1040, 1005, 910 cm Reactions B and C of Figure 26A: Synthesis of Compound A solution comprising 3.534 grams (13.3 millimoles) of compound 8 crude Cbz-D-allo-isoleucine) 50 milliliters of anhydrous CH2C12 was cooled in an ice bath to 0°C.

!0 Each of the following was added in solid form to the cooled solution: 2.574 grams (14.0 millimoles) pentafluorophenol (PFPOH), 3.064 grams (16.0 millimoles) of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDAC-HC1), and 0.325 gram (2.7 millimoles) 4-dimethylaminopyridine (DMAP). The resulting mixture was maintained at 0 C for half an hour with stirring, and at room temperature for an additional 4 hours. The mixture was diluted with 50 milliliters of CH 2 Cl 2 The CH 2 C1 2 layer was washed once using milliliters of a 10% solution of hydrochloric acid (HC1), once using 25 milliliters of a solution of NaHCO 3 and once using 25 milliliters of a saturated solution of NaCl. The washed CH2CI 2 layer was dried in the presence ofNa2SO4, filtered, and concentrated under reduced pressure. The resulting PFP ester, compound 9 (5.70 grams), was obtained as a colorless oil and used in the next step without purification. The following analytical data were obtained for compound 9: Rf 0.52 (20:80 EtOAc:hexane); H NMR (500 MHz, CDCI 3 0.90-1.12 6H), 1.29-1.39 1H) and 1.45-1.52 1H), 2.05-2.15 1H), 4.79-4.84 (m, -53 WO 01/76616 PCT/US01/111607 ID13 0 1H), 5.10 2H), 5.12-5.14 J= 9.1 Hz, 1H), 7.29-7.37 5H); C NMR(250 MHz, IN CDC13) 8 11.68, 14.25, 26.24 37.64, 57.09 67.45, 128.20, 128.35, 128.59, and 135.95, 136.9, a 139.0, 140.0 and 142.0, 156.13, 168.87.

A solution comprising 5.70 grams of compound 9 and 25 milliliters of 5 anhydrous THF was cooled to -78 0 C. An enolate solution comprising the lithium enolate of methyl acetate was prepared by cooling a solution comprising 49.2 millimoles of lithium N dialdehyde and 25 milliliters anhydrous THF in a dry ice bath at -78 0 C, adding to the solution it 3.92 milliliters (49.2 millimoles) of methyl acetate by syringe, and maintaining the resulting IN solution with stirring at -78 0 C for 1 hour. The resulting enolate solution was added drop-wise 0 10 to the solution comprising compound 9, and the resulting mixture was maintained with stirring for 0.75 hours at -78 0 C. The reaction was quenched at -78 0 C by adding 50 milliliters of a saturated solution of aqueous ammonium chloride (NH 4 CI). The quenched reaction was brought to room temperature, and THF was removed under reduced pressure. The resulting aqueous solution was extracted 3 times with 25 milliliters aliquots of CH 2 Cl 2 The combined

CH

2 C1 2 layers were washed once using 25 milliliters of a 10% solution of hydrochloric acid (HC1), once using 25 milliliters of a 5% solution of NaHCO3, and once using 25 milliliters of a saturated solution of NaCl. The washed CH 2 C1 2 layer was dried in the presence of Na2SO4, filtered, and concentrated under reduced pressure. This reaction yielded a yellow oil which was purified by flash column chromatography. The yellow oil was applied to a column of silica gel and eluted with a solution comprising EtOAc and hexane in a ratio of 10 to respectively. The product obtained from chromatography was 3.42 grams of a colorless oil corresponding to the beta-keto ester, compound 10. The yield of compound 10 was 80%, as calculated for both Reactions B and C. The following analytical data were obtained for compound 10: Rf 0.42 (35:65, EtOAc:hexane); H NMR (500 MHz, CDCl 3 5 0.75-0.79 (m, 3H), 0.90-0.98 3H), 1.26-1.30 1H) and 1.42-1.46 1H), 1.97-1.99 1H), 3.53 (s, 2H), 3.72 3H), 4.56-4.58 J 7.6 Hz, 1H), 5.10 2H), 5.26-5.28 J 6.4 Hz, 1H), 7.30-7.36 5H); 13C NMR (250 MHz, CDCI 3 8 11.83, 13.79, 26.83, 36.12, 46.56, 52.46, 63.00, 67.19, 128.10, 128.24, 128.56, 136.16, 156.42, 166.96, 201.68; IR (CHC1 3 3349.1, 2964.4, 1748.3, 1712.9, 1520.6, 1454.8, 1328.2, 1232.1 cm-1; HRMS m/z calculated forC 17

H

2 3

NO

5 Na (M Na 344.1498, found 344.1490; [a]D 2 0 -27.85 (c 0.53, CHCl 3 Anal. Calculated forC 17

H

2 3 NO5: C, 63.52; H, 7.22, N, 4.36. Found: C, 63.32; H, 7.15, N, 4.24.

-54- WO 01/76616 PCT/US01/11607

IO

O

c Reaction D of Figure 26A: Synthesis of Compound 11 SA solution comprising 2.797 grams (8.7 millimoles) of compound 10 and milliliters of HPLC-grade methanol (MeOH) was cooled to -78 0 C, and 1.644 grams (30.5 millimoles) of potassium borohydride (KBH 4 was added in portions. The resulting mixture Swas initially maintained with stirring at -78 0 C for 10 minutes. The reaction vessel was next C, warmed to -20 0 C and maintained with stirring for 30 minutes, following which, the reaction tt vessel was warmed to 0 C and maintained with stirring for 10 minutes. The resulting mixture 0 was quenched at 0 OC by adding a solution comprising glacial acetic acid in a drop-wise 0 10 manner until the pH of the aqueous layer was not less pH 6 when tested with litmus paper. The resulting neutralized, bilayer solution was concentrated under reduced pressure, and milliliters of a solution comprising EtOAc and H20 in a ratio of 1 to 1, was added. The organic layer was separated from the aqueous layer and washed with 10 milliliters of a saturated solution of NaC1. The washed organic layer was dried in the presence of Na 2 SO4, filtered, and concentrated under reduced pressure. The crude product thus obtained was 2.786 grams of a colorless oil comprising an 11:1 ratio of compound 11 and its stereoisomer, respectively. Crystallization of the colorless oil in a solution comprising ether and hexane afforded pure compound 11 as a white crystalline solid in 99% yield. The following analytical data were obtained for compound 11: Rf 0.29 (35:65, EtOAc:hexane); 1H NMR (500 MHz, CDC1 3 8 0.83-0.85 3H), 0.89-0.92 3H), 1.19-1.23 1H) and 1.32-1.34 1H), 1.91-1.93 1H), 2.45-2.51 (dd, J 1 =16.7 Hz, J 2 =9.1 Hz, 1H) and 2.58-2.62 (dd, J 1 =16.7 Hz,

J

2 =2.7 Hz, 1H), 3.12-3.14 J= 4.5 Hz, 1H), 3.68 3H), 3.90-3.91 1H), 4.62-4.65 J 10.0 Hz, 1H), 5.07-5.08 J 5.1 Hz, 2H), 7.29-7.35 5H); 13C NMR (250 MHz,

CDCI

3 6 12.10, 13.64, 27.52, 34.28, 38.74, 52.25, 57.57, 67.39, 69.43, 128.50, 128.61, 128.97 and 136.82, 157.08, 174.09; IR (CHCI 3 3421, 3316, 2951, 1709,1537, 1443, 1229 -1 cm HRMS m/z calculated forC 17

H

2 5

NO

5 Na (M Na 346.1630, found 346.1645; [a]D 2 0 -10.9 (c 0,595, CHC1 3 Anal. Calculated for C 17

H

2 5 N0 5 C, 63.12; H, 7.80, N, 4.33. Found: C, 63.23; H, 7.85, N, 4.06.

Reaction E of Figure 26A: Synthesis of Compound 12 A solution comprising 0.8636 gram (2.67 millimoles) of compound 11 as a colorless oil and 10 milliliters of CH 2

CI

2 was placed under an argon atmosphere and cooled to WO 01/76616 PCT/US01/11607 0°C. A solution comprising 0.778 milliliters (6.68 millimoles) of 2,6-lutidine was added,

O

followed by addition of a solution comprising 1.08 milliliters (4.01 millimoles) of S triisopropylsilyl triflate (i-Pr3SiOTf). The reaction mixture was initially maintained with stirring at 0 0 C for 30 minutes, following which, the reaction mixture was maintained at room temperature for 2 hours. The resulting mixture was diluted with 20 milliliters of CH 2

CI

2 The

CH

2 C1 2 layer was washed once using 15 milliliters of a 10% solution of hydrochloric acid S(HCI), once using 15 milliliters of a 5% solution ofNaHCO3, and once using 15 milliliters of a S saturated solution of NaCl. The resulting washed CH 2

CI

2 layer was dried in the presence of C anhydrous Na 2 SO4, filtered, and concentrated under pressure. The concentrated residue was S purified by flash column chromatography, eluting with solutions comprising ether and hexane C1 in a ratio of from 2 to 98, respectively, to 15 to 85, respectively. Chromatography yielded 1.204 grams (94% yield) of compound 12 as the major isomer in the form of a colorless oil.

The following analytical data were obtained for compound 12: Rf 0.65 (35:65, EtOAc:hexane); IHNMR (500 MHz, CDCI 3 8 0.84-0.91 6H), 0.99-1.05 21H), 1.12- 1.34 2H), 1.81-1.84 1H), 2.54-2.62 2H), 3.54 3H), 3.73-3.75 1H), 4.34-4.36 1H), 4.69-4.71 J 10.5 Hz, 1H), 5.01-5.04 J 12.3 Hz, 1H) and 5.09-5.11 J 12.3 Hz, 1H), 7.28-7.34 5H); 13C NMR (250 MHz, CDC13) 12.50, 12.74, 13.97, 18.08, 27.44, 34.40,40.45, 51.54, 58.62,66.67,70.49, 128.03, 128.08, 128.46 and 136.67, 156.51, 172.00; IR (CHC13) 3450, 3359, 2944,2863, 1728, 1510,1459, 1434, 1384, 1308, 1232, 1171, -1 D 1090 cm HRMS m/z calculated forC 2 6

H

4 5 NSiO 5 Na (M Na 480.3145, found 480.3128; [a]D 2 0 +15.88 (c 0.57, CHCl 3 Anal. Calculated forC1 7

H

2 5

NO

5 C, 65.09; H, 9.46, N, 2.92.

Found: C, 64.80; H, 9.41, N, 2.69.

Reaction F of Figure 26A: Synthesis of Compound 13 A solution comprising 1 normal NaOH (20 milliliters, 11 millimoles) was added to a solution comprising 0.84 gram (1.753 millimoles) of compound 12, 10 milliliters of THF, and 10 milliliters of MeOH, which was cooled to 0°C. This reaction mixture was maintained with stirring at 0°C for 2 hours. The reaction mixture was then maintained with stirring at room temperature overnight. The reaction mixture was concentrated under reduced pressure and diluted with 10 milliliters of H 2 0. The resulting mixture was cooled to 0oC in an ice bath, acidified to pH 2 by adding a solution comprising 1 normal KHSO4, and extracted 3 times with milliliters aliquots of EtOAc. The EtOAc layers were combined, washed with 10 milliliters -56- WO 01/76616 PCT/US01/11607 Sof a saturated solution of NaCI, dried in the presence of anhydrous Na 2

SO

4 filtered, and

O

concentrated. This reaction yielded 0.7709 gram (95%yield) of compound 13 in the form of a S white foam. Compound 13 was used in the subsequent reaction without purification.

Q Analytical data for compound 13 were as follows: Rf 0.08 (35:65-ethyl acetate:hexane); H CN NMR (500 MHz, CDCl 3 8 0.86-1.00 1.06-1.08 21H), 1.19-1.24 1H) and 1.64-1.72 1H), 1.85-1.92 1H), 2.50-2.80 2H), 3.60-3.80 1H), 4.28-4.33 (m, O 1H), 4.86-4.88 J 10.4 Hz, 1H), 5.06-5.22 2H), 7.29-7.45 Reaction G of Figure 26B: Synthesis of Compound SSynthesis of a protected form of (2S)-a-hydroxyisovaleryl-isostatine is depicted in Figure 26B. In reaction G of Figure 26B, L-valine was dissolved in a solution comprising sodium nitrite (NaNO 2 and 1 normal sulfuric acid (H2S0 4 The reaction was carried out using a standard method as described, for example, in Green and Wutz (1999, Protecting Groups in Organic Synthesis, Wiley, New York) or Bodansky (1993, Principles of Peptide Synthesis; Springer, Berlin). This reaction yielded alpha-hydroxy valine Reaction H of Figure 26B: Synthesis of Compound 16 In reaction H,'2.46 grams (17.78 millimoles) of anhydrous potassium carbonate

(K

2 C0 3 and 1.25 grams (3.4 millimoles) of tetrabutyammonium iodide (Bu 4 NI) were added to a solution comprising 2 grams (16.93 millimoles) of alpha-hydroxy valine (15) and milliliters of re-distilled DMF, followed by drop-wise addition of 5.86 milliliters (67.72 millimoles) of allyl bromide. The resulting solution was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, diluted with milliliters of H 2 0, and extracted three times with 20 milliliters aliquots of ether. The combined organic layers were washed once using 15 milliliters of a 10% solution of hydrochloric acid (HCI), once using 15 milliliters of a 5% solution of NaHCO 3 and once using milliliters of a satiirated solution of NaC1. The resulting washed organic layers were dried in the presence ofNa 2

SO

4 filtered, and concentrated under reduced pressure. This reaction yielded 2.53 grams (96%yield) of compound 16 in the form of an orange oil. Compound 16 was used in the subsequent reaction without purification. The following analytical data were obtained for compound 16: Rf 0.50 (25:75, EtOAc:hexane); 1H NMR (500 MHz, CDC1 3 8 0.71-0.86 J 6.8 Hz, 3H), 0.92-1.00 J 7.0 Hz, 3H), 2.04-2.10 1H), 2.65-2.66 (d, -57- WO 01/76616 PCT/US01/11607 I J 6.1 Hz, 1H), 4.03-4.05 (dd, Ji=5.9 Hz, J 2 =3.5 Hz, 1H), 4.62-4.70 2H), 5.24-5.27 (dd,

O

0 Jl=10.4 Hz, J2=1.1 Hz, 1H) and 5.31-5.35 (dd, J 1 =17.2 Hz, J2=3.5 Hz, 1H), 5.86-5.94 (m, I 13 1H); C NMR (250 MHz, CDC1 3 8 15.93, 18.76, 32.17, 66.04, 75.03, 119.12, 131.48, 174.62; IR (CHC1 3 3521-3458, 2964, 2880, 1735, 1646, 1462, 1367, 1257, 1204, 1136, 1073, -1 C 1026,983,931cm 1 I Reaction I of Figure 26B: Synthesis of Compound 17 C A solution comprising 0.6827 gram (1.47 millimoles) of compound 13 and C milliliters of freshly distilled toluene, was placed under an inert atmosphere and cooled to 0°C.

S) A solution comprising 0.232 gram (1.47 millimoles) of compound 16 and 2.5 milliliters of CN toluene was added drop-wise to the cooled solution of compound 13, followed by the addition of 0.333 gram (1.61 millimoles) of dicyclohexylcarbodiimide (DCC) and 0.036 gram (0.29 millimole) of DMAP. The reaction mixture was stirred at 0°C for 2 hours and maintained at room temperature overnight. The reaction was quenched by adding 2 milliliters of a solution comprising MeOH and acetic acid (AcOH) in a ratio of 1:2, and EtOAc, and stirred for minutes at room temperature. The resulting mixture was concentrated under reduced pressure.

The residue which remained after this procedure was dissolved in 10 milliliters of ether, which resulted in the formation of a solid, which was removed by filtration. The filtrate was washed once using 10 milliliters of a 10% solution of citric acid, once using 10 milliliters of a solution of NaHCO3, and once using 10 milliliters of brine solution a saturated NaCI solution). The organic layer was dried in the presence of anhydrous Na2SO4, filtered, and concentrated. The resulting residue was purified by flash column chromatography, eluting with a solvent mixture comprising ether and hexane in a ratio of from 2 to 98, respectively, to 12 to 88, respectively. Concentration of the eluant under reduced pressure yielded 0.5774 gram (65% yield) of compound 17 in the form of a colorless oil. The following analytical data were obtained for compound 17: Rf 0.55 (20:80-ethyl acetate:hexane); IH NMR (500 MHz, CDCI 3 8 0.85-1.08 33H), 1.16-1.19 1H), 1.34-1.36 1H), 1.80-1.82 1H), 2.18-2.21 1H), 2.68-2.73 2H), 3.78-3.82 1H), 4.37-4.42 1H), 4.55-4.64 (m, 2H), 4.77-4.78 J 4.4 Hz, 1H), 4.86-4.88 J 10.7 Hz, 1H), 5.07 2H), 5.21-5.23 (dd,

J

1 =9.4 Hz, J 2 =0.9 Hz, 1H), 5.28-5.32 (dd, J 1 =17.0 Hz, J 2 =1.2 Hz, 1H), 5.83-5.87 1H), 7.27-7.34 5H); 13C NMR (250 MHz, CDCl 3 8 12.69, 12.93, 14.14, 17.25, 18.12, 18.78, 26.33, 29.98, 34.48, 40.28, 58.15, 66.69, 68.67, 70.66, 76.74, 118.82, 128.01, 128.40, 128.47,.

-58- WO 01/76616 PCT/US01/11607 ND 136.75, 131.63, 156.48, 169.13, 170.78; IR (CHCI 3 3380, 2964, 2867, 1743, 1508, 1463, O -1 8 1374, 1201, 1129, 994, 882 cm HRMS m/z calculated forC33H 5 5NSiO 7 Na (M Na

C-I

628.364552, found 628.365878; Anal. Calculated forC 3 3

H

5 5 NSiO 7 C, 65.41; H, 9.16.

Found: C, 65.09; H, 9.05.

CN Reaction J of Figure 26B: Synthesis of Compound 6 I0 In reaction J, tetrakis-(triphenylphosphine)palladium (Pd(PPh 3 4 0.044 grams, N 0.038 millimole) was added to a solution comprising 0.2315 gram (0.38 millimole) of the isostatine-Hiv-allyl ester, compound 17, and 3 milliliters of freshly distilled THF. Freshly distilled morpholine (0.33 milliliters, 3.8 millimoles) was added drop-wise to the resulting CI mixture. The addition of reagents was performed in a dark hood, and the reaction mixture was maintained with stirring at room temperature and in a dark hood for at least 8 hours. The reaction mixture was concentrated under reduced pressure and diluted with 5 milliliters of

CH

2 C1 2 The solution obtained from this procedure was washed once with 5 milliliters of a solution comprising 1 normal HCI and once using 5 milliliters of H20. The washed organic layer was dried in the presence of anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was dissolved in 5 milliliters of ether, filtered, and concentrated again under reduced pressure. The residue which remained after these procedure was a white foam corresponding to compound 6 (0.218 grams, quantitative yield). Compound 6 was used in the subsequent reaction without purification.

The synthesis of a linear hexapeptide compound (20) is depicted in Figure 26C.

Compound 5 was prepared as described (Li et al., 1990, J. Am. Chem. Soc. 112:7659-7672).

Reaction K of Figure 26C: Synthesis of Compound 18 A solution comprising 0.6653 gram (0.74 millimole) of compound 5 and milliliters of MeOH was added to a suspension comprising 0.1996 gram of 10% Palladium on carbon 10 milliliters of MeOH, and 10 milliliters of EtOAc. The reaction mixture was agitated using a Parr apparatus for 5 hours at room temperature. The resulting slurry was filtered through Celite® and the Celite® was washed with an excess of a solvent mixture comprising MeOH and EtOAc in a ratio of 1 to 1. The filtrate was dried in the presence of anhydrous Na 2

SO

4 filtered, and concentrated under reduced pressure to yield compound 18 (0.552 grams, 98% yield). Compound 18 Leucylprolyl-N,O-dimethyltyrosine-N-Boc-O- -59- WO 01/76616 PCT/US01/11607 ID SEM-threonine) was obtained in the form of a white solid using this procedure, and was used Sin the subsequent reaction without purification. The following analytical data were obtained for compound 18: Rf 0.03 (40:60, acetone:hexane); IH NMR (500 MHz, CDCI 3 8 -0.001 (s, 9H), 0.64-0.86 2H), 0.88-0.96 6H), 1.19-1.21 J 6.3 Hz), 1.33-1.34 J 6.3 Hz, 5 3H, RI), 1.44 9H), 1.63-1.92 3H), 1.92-2.01, 2.08-2.24 4H), 2.73 3H), 2.86-2.96 3.10-3.19 2H), 3.45-3.72 4H), 3.75 3H), 4.34-4.69 3H), 4.78-4.81 (dd, ND Ji=8.0 Hz, J 2 =3.3 Hz, 1H), 5.01-5.10 1H), 5.17-5.52 7.58-7.60 J= 9.7 Hz, 3H), IND 13 6.77-6.83 2H), 7.00-7.10 2H); C NMR (250 MHz, CDC1 3 5 -1.46, 16.87, 17.96, S 22.00, 23.19, 23.76, 25.14, 28.16, 29.31, 33.65, 39.31, 47.34, 50.54, 55.37, 55.46, 58.66, 62.25, 68.16, 72.36, 81.15, 89.83, 114.39, 128.75, 128.75, 130.47, 157.04, 158.84, 168.15, 168.95, N 169.57, 173.13; IR(CHCl 3 )3285,2951, 1 7 4 0,170 9 1 6 4 1,1511,14 4 8 ,1 3 65,1250,1161 cm I HRMS m/z calculated forC 3 7H 6 3

N

4 SiOl 0 (M 751.4314, found 751.4343; [aD 2 44.68 (c 1.03, CHC13); Anal. Calculated forC 37

H

6 2

N

4 SiO 10 C, 59.17; H, 8.33;N, 7.46.

Found: C, 59.17; H, 8.35; N, 7.26.

Reaction L of Figure 26C: Synthesis of Compound 19 In reaction L, the entire yield of compound 6 from reaction J was dissolved in 1 milliliter of freshly distilled CH 2 C1 2 and cooled to o0C. To this solution was added 0.074 gram (0.40 millimole) of PFPOH, (0.088 grams, 0.46 millimole) of EDACHCI, and 0.0093 gram of DMAP (0.076 millimole). The resulting mixture was stirred at o0C for 30 minutes.

The reaction mixture was maintained at room temperature for an additional 4 hours and diluted with 10 milliliters of CH 2 C1 2 The organic layer was washed once using 5 milliliters of a solution of HCI, once using 5 milliliters of a 5% solution of NaHCO 3 and once using milliliters of a saturated solution of NaCl. The CH 2 C1 2 layer was dried in the presence of anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography, eluting with a solvent mixture comprising ether and hexane in a ratio of from 3 to 97, respectively, to 7 to 93, respectively. Concentration of the eluate under reduced pressure yielded 0.2315 gram of a colorless oil corresponding to the PFP ester, compound 19 (83% yield for reaction J of Figure 26B and reaction L of Figure 26C). The following analytical data were obtained for compound 19: Rf 0.57 (20:80, EtOAc:hexane); H NMR (500 MHz, CDC1 3 8 0.86-1.09 33H), 1.18-1.20 1H), 1.27- 1.29 1H), 1.84-1.86 1H), 2.31-2.36 1H), 2.69-2.72 1H), 2.80-2.85 1H), WO 01/76616 PCT/US01/11607 I 3.79-3.82 1H), 4.38-4.42 1H), 4.78-4.80 J 10.7 Hz, 1H), 4.97-4.98 J 4.4 Hz, 1H), 5.00-5.06 2H), 7.25-7.29 5H); C NMR (250 MHz, CDCI 3 8 12.72, 12.95, o 14.07, 17.20, 18.09, 18.45, 27.52, 30.19, 34.40, 40.12, 58.17, 65.82, 66.74, 70.47, 127.92, 128.18, 128.33, 136.57, 138.83, 140.06, 140.65, 141.97, 156.56, 165.68, 170.76; IR (CHC1 3 C1 5 3480,2962,2868, 1793, 1730, 1516, 1464, 1381, 1214,1094, 995,880 cm-; HRMS m/z N, calculated forC3 6

H

5 0

NF

5 SiO 7 Na (M Na 754.3174, found 754.3191.

ID

CI Reaction M of Figure 26C: Synthesis of Compound l t A solution comprising 0.2855 gram (0.39 millimole) of compound 19 and milliliters of CH2C1 2 was cooled to 0°C in an ice bath. To this solution 0.17 milliliters (0.98 NC millimole) of DIEA was added drop-wise, and the resulting mixture was maintained with stirring at o0C for 20 minutes. A solution comprising 0.522 gram of compound 18, 0.0095 gram (0.078 millimole) of DMAP, and 1.5 milliliters of CH 2 C1 2 was added to the solution comprising compound 19 using a syringe. The resulting mixture was stirred at 0°C for 1 hour and maintained at room temperature for an additional 1 hour. The reaction was quenched at o0C by adding 3 milliliters of a saturated solution of NH4CI and diluting the reaction with milliliters of CH 2 Cl 2 The resulting mixture was separated at room temperature. The aqueous layer was extracted 3 times using 10 milliliters aliquots of CH 2 C1 2 and the combined organic layers were washed once using 10 milliliters of a 10% solution of HC1, once using milliliters of a 5% solution of NaHCO 3 and once using 10 milliliters of a saturated NaCl solution. The washed organic layer was dried in the presence of anhydrous Na 2

SO

4 filtered, and concentrated under reduced pressure. This reaction yielded 0.4861 gram (96% yield) of the fully protected hexapeptide precursor, compound 20. Compound 20 were obtained in the form of a white foam using this procedure, and was used in the subsequent reaction without further purification. Analytical data for hexapeptide 20 was as follows: Rf0.47 (05:95acetone:CH2Cl 2 H NMR (500 MHz, CDCl 3 5 -0.0008 9H), 0.73-0.83 2H), 0.85- 0.92 9H), 0.92-1.08 21H), 1.45 9H), 1.13-2.19 11H), 2.43-2.46 1H) and 2.54-2.58 1H), 2.64 and 2.88 3H, RI), 3.09-3.17 2H), 3.44-3.73 4H), 3.75 (s, 3H), 3.79-3.89 1H), 4.38-4.45 1H), 4.21-4.35 3H), 4.70-4.81 1H), 4.96-5.06 3H), 5.18-5.43 3H) and 8.32-8.34 J 9.0 Hz, 3H), 5.46-5.48 J 6.1 Hz, 1H), 6.74-6.83 2H), 6.95-7.11 2H), 7.25-7.38 5H), 7.75-7.77 J 8.5 Hz) and 8.85- 13 8.87 J= 10.1 Hz, 2H); 3C NMR (250 MHz, CDC1 3 -1.45, 11.73, 12.73, 14.47, 16.54, -61- WO 01/76616 PCT/US01/11607 S17.73, 17.99, 18.14, 18.92, 21.40, 23.54, 24.42, 25.13, 28.21, 28.27, 29.20, 29.67, 30.13, 33.59, 34.86, 39.58, 39.73, 46.87, 49.04, 55.13, 55.39, 56.90, 58.94, 62.24, 66.42, 68.12, 70.96, 72.25, S 78.71, 80.39, 89.85, 113.92, 114.37, 127.70, 127.76, 127.86, 128.35, 128.45, 128.81, 128.91, 137.01, 130.44, 130.86, 156.39, 158.40, 158.82, 169.20, 169.75, 169.97, 170.58, 171.77, C 5 173.85; IR (CHCI 3 3275, 2952, 2868, 1735, 1704, 1636, 1511, 1454, 1380, 1365, 1250, 1167, 1110, 1047 cm HRMS m/z calculated forC67H N5Si2Ol 6 Na (M Na 1320.7462, found 1320.7520; [aD 2 0 -44.56 (c 1.13, CHCI 3 Anal. Calculated forC6 7

H

1 iN 5 Si 2 0 16

C,

N 61.95; H, 8.62; N, 5.40. Found: C, 61.75; H, 8.59; N, 5.15.

c, Cyclization of hexapeptide 20 to yield compound 21 is depicted in Figure 26D.

C

N Reaction N of Figure 26D: Synthesis of Compound 21 A solution comprising 0.3505 gram (0.27 millimole) of compound 20 and milliliters of CH 2 C1 2 was cooled to 0°C, and 0.21 gram (0.81 millimole) of magnesium bromide etherate (MgBr 2 .Et2O) was added. The resulting mixture was stirred at 0 C for 2 Shours and maintained at room temperature for an additional 4 hours. The reaction mixture was diluted by adding 10 milliliters of CH 2 C1 2 and washed once using 10 milliliters of a solution of HCl, and once using 10 milliliters of a saturated NaCI solution. The CH 2 C12 layer was dried in the presence of anhydrous Na 2

SO

4 filtered, and concentrated under reduced pressure. The white foam obtained as a product of this de-protection reaction was used in the subsequent hydrogenolysis reaction without purification. The entire yield of white foam (0.3195 grams) was dissolved in 10 milliliters of MeOH and subjected to hydrogenolysis as described for preparation of compound 18. This hydrogenolysis reaction yielded 0.296 gram of a white foam which was used in the subsequent coupling reaction without purification. The hydrogenolysis product was dissolved in 27 milliliters of freshly-distilled DMF and cooled to 0°C. To the cooled solution was added 0.123 gram (0.32 millimole) of 2-(1H-9azobenzotriazole-l-yl)-1,1, 3 3 -tetramethyluronium hexafluorophosphate (HATU), followed by drop-wise addition of 0.141 milliliters (0.81 millimole) of DIEA. The resulting mixture was stirred at 0 C for 1 hour and maintained with stirring at room temperature for at least 8 hours.

The reaction mixture was concentrated under reduced pressure, diluted with 10 milliliters of EtOAc, and washed once using 10 milliliters of a 10% solution of HC1, once using milliliters of a 5% solution of NaHCO 3 and once using 10 milliliters of a saturated NaCI solution. The CH 2 C1 2 layer was dried in the presence of anhydrous Na 2

SO

4 filtered, and -62- WO 01/76616 PCT/US01/11607 \O concentrated under reduced pressure. The crude residue obtained from this procedure was

O

Spurified by flash column chromatography using a solvent mixture comprising acetone and Shexane in a ratio of from 5 to 95, respectively, to 15 to 85, respectively. The resulting eluate was concentrated under reduced pressure to yield 0.173 gram of the protected macrocycle, compound 21, in the form of a white foam. The yield of compound 21 represents a 63% yield for the three reactions starting with compound 20 the de-protection, hydrogenolysis, and IO coupling reactions). The following analytical data were obtained for compound 21: Rf0.55 (30:70, acetone:hexane); H NMR (500 MHz, CDCI 3 8 0.78-1.07 39H), 1.21-1.48 (m, 17H), 1.56-1.92 4H), 1.95-2.11 2H), 2.43-2.44 1H), 3.11-3.17 1H), 2.53 (s, O 10 3H), 2.89-3.02 1H), 3.30-3.34 1H), 3.49-3.52 1H), 3.60-3.62 1H), 3.66-3.70 CN 1H), 3.77 3H), 4.14-4.19 1H), 4.37-4.43 1H), 4.46-4.48 J 7.6 Hz, 1H), 4.55-4.86 1H), 4.88-4.91 7.60-7.66 4H), 6.82-6.83 J 8.5 Hz, 2H), 7.06-7.07 J 8.5 Hz, 2H), 7.41-7.48 2H); 13C NMR (250 MHz, CDC13) 8 12.24, 12.66, 14.21, 15.11, 15.61,17.98, 18.60, 18.74, 19.31, 21.10, 23.92, 25.24, 27.21, 28.42, 30.48, 34.87, 37.92, 39.00,41.65, 47.10,48.52, 55.67, 56.16, 57.25, 63.26, 66.33, 68.65, 71.78, 80.46, 81.66, 114.50, 130.31, 130.82, 156.40, 159.01, 169.13, 170.90, 171.34, 172.78, 176.75; IR (CHC1 3 -I 3330, 2952, 2876, 1735, 1629, 1508, 1447, 1243, 1168, 1024, 843 cm HRMS m/z calculated forC 5 3 Hg 9

N

5 SiO 12 Na (M Na 1038.6175, found 1038.6166.

The final reactions in the total synthesis Tamandarin A, 101, are depicted in Figure 26E. Compound 4 was prepared in the manner previously reported in Li et al. (1990, J. Am. Chem. Soc. 112:7659-7672).

Reaction 0 of Figure 26E: Synthesis of Compound 22 A solution comprising 167 milligrams (0.165 millimole) of protected macrocycle (compound 21) and 20 milliliters of EtOAc was cooled to -30 0 C. Gaseous HC1 was introduced into the solution, and the temperature of the reaction mixture was maintained between -10°C to -20 0 C during introduction of HC1. The reaction mixture was stirred and maintained at a temperature of between -10 0 C to -20 0 C for an additional 30 minutes. The reaction mixture was warmed to 0 C and maintained at o0C for 1 hour. The reaction vessel was purged with N2 gas for at least 30 minutes while the temperature of the vessel was maintained at 0°C. The purged solution was warmed to room temperature and concentrated under reduced pressure. The residue remaining after concentration of the reaction was -63- WO 01/76616 PCTUS01/11607 I triturated and washed by decanting using three 5 milliliter aliquots of a solvent mixture

O

Scomprising tert-butylmethyl ether and hexane in a ratio of 1 to 4, respectively. The solid which O was produced by this procedure was collected by filtration and dried under reduced pressure to provide 127.5 milligrams (quantitative yield) of the hydrochloride salt of compound 22.

Compound 22 was obtained as a white solid, and was used in the final coupling reaction without purification.

ID

S Reaction P of Figure 26E: Synthesis Tamandarin A (Compound 101) A solution comprising 63.3 milligrams (0.082 millimole) of compound 22, 37.7 0 10 milligrams (0.12 millimole) of compound 4, and 0.50 milliliters of CH 2 Cl 2 was cooled to 0 C c-I in an ice bath. To this cooled solution was added 53.1 milligrams (0.12 millimole) of benzotriazole-l-yl-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP) and 0.035 milliliters (0.32 millimole) of NMM. The reaction mixture was stirred for 30 minutes at 0°C and allowed to warm to room temperature. The reaction mixture was maintained with stirring at room temperature for at least an additional 8 hours. The resulting solution was diluted with 2 milliliters of a saturated solution of NaCl and extracted with 10 milliliters of EtOAc. The extracted organic layer was washed once using 10 milliliters of a 10% solution of HCI, once using 10 milliliters of a 5% solution of NaHCO 3 and once using 10 milliliters of a saturated solution of NaC1. The washed organic layer was dried in the presence of anhydrous Na 2

SO

4 filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography using a solvent mixture comprising MeOH and CH 2 C1 2 in a ratio of from 2 to 98, respectively, to 5 to 95, respectively. This procedure yielded 0.0471 gram of tamandarin A (compound 101) in the form of a yellow-greenish solid. The yield of compound 101 was 56%, as calculated for both reactions O and P, and 12% as calculated for reactions A-F, I, J, and L-P starting with D-allo-isoleucine). The following analytical data were obtained for tamandarin A (101): Rf 0.57 (10:90-MeOH:CH 2

C

2 1H NMR (500 MHz, CDC1 3 8 0.86-1.08 24H), 1.19-2.30 (dd, J 1 =17.1 Hz, J 2 =7.9 Hz, 1H), 3.29-3.33 (d, J 17.0 Hz, 1H), 2.62 3H), 3.14 3H), 3.16-3.21 (dd, Ji=14.4 Hz, J2=1 1.1 Hz, 1H), 3.41- 3.45 1H), 3.59-3.80 5H) 3.82 3H), 3.90-3.95 1H), 4.03-4.08 1H), 4.29-4.30 1H), 4.37-4.42 1H), 4.67-4.69 1H), 4.74-4.77 1H), 4.89-4.93 1H), 5.06 (d, J 4.3 Hz, 1H), 5.31-5.35 J1=l 1.6 Hz, J 2 =3.3 Hz, 1H), 5.44-5.46 1H), 6.86-6.88 J= 8.3 Hz, 2H), 7.09-7.11 J 8.3 Hz, 2H), 7.37-7.39 J= 9.1 Hz, 1H), 7.48-7.50 J 5.1 -64- WO 01/76616 PCT/US01/11607 I Hz, 1H1), 7.78-7.80 J 9.7 Hz, 1H); 13C NMR (250 MHz, CDC1 3 5 11.78,14.07, 16.52, S 17.55, 18.93, 20.28, 20.90, 21.32, 23.47, 23.79, 24.83, 24.90,, 25.98, 27.33, 27.96, 28.40,

(N

30.11, 31.26, 33.55, 33.93, 35.73, 38.70, 39.41, 39.65, 46.66,47.02,48.28, 54.91, 55.27, 56.71, 56.97, 57.90, 66.07, 66.22, 68.98, 70.70, 78.87, 114.08, 130.07, 130.33, 156.62, 168.52, I 5 169.57, 170.11, 170.35,170.59,171.09,172.67,173.84,174.53; IR (CHC1 3 3330,2952,2876, 1735, 1629, 1508, 1447, 1243, 1168, 1024, 843 cm HRMS m/z calculated IN forC54H 8 5

N

7 0 1 4 Na (M Na 1078.6052, found 1078.6044; [a]D 2 0 -43.93 (c 1.05, CHCI 3 N The inventors believe that the series of reactions disclosed above represent the S first stereoselective synthesis of Tamandarin A.

0 0 (Ni Example 2 Biological Activity of Tamandarin A The experiments described in this example demonstrate that tamandarin A is an effective protein synthesis inhibitor and anti-tumoragent. Initial results are shown in Table 1 for inhibition of protein biosynthesis (column cytotoxicity (column and anti-tumor activity (column 3) of tamandarin A, as compared to the related anti-tumor compound didemnin B. The results in Table 1 are given in units of concentration of the selected compound.

1 Compound Protein Cytotoxicity Anti-tumor Synthesis (NCI-60 Mean) activity (Assay Mean) Tamandarin A 1.3 tiM GI 5 0 10.4 U.M LC50 4.8 jM 1.31 nM Didemnin B 4 jiM G1 5 0 1.8 pM LC50 7.4 nM 1.58 nM As used herein, "GI50" refers to the dose of a compound which is capable of producing 50% inhibition of cell growth. G1 5 0 is assessed by comparing growth of cells to which a compound has been administered with growth of the same cells to which the compound has not been administered.

As used herein, "LC 5 0" refers to the dose of a compound which is capable of producing 50% lethality in cells. LC 50 is assessed by comparing death of cells in a population WO 01/76616 PCT/US01/11607 I of cells to which a compound has been administered with the death of cells in a population of Sthe same cells to which the compound has not been administered.

refers to a 60 tumor cell line panel which from the National Cancer Institute (NCI, Frederick, MD). "NCI-60 Mean" is the average GI50 or LC5 0 for the panel 0C 5 treated with the selected compound.

These in vitro findings indicate that tamandarin A exhibits comparable potency INO to didemnin B, a known anti-cancer agent. Tamandarin A is significantly less cytotoxic in c these assays than is didemnin B. The comparison indicate the usefulness of tamandarin A and 0C other didemnin analogs as pharmacological agents having anti-cancer and other activities

ID

0 10 characteristic of didemnin B.

O

Example 3 Synthesis of (-)Dehvdrotamandarin A method of synthesizing dehydrotamandarin is described in this example.

The method involves the macrocyclic compound 22, which can be generated as described in Example 1. The method described in this Example is illustrated in Figure 37, and begins with synthesis of compound 27, depicted in Figure 37A.

Reaction 0 of Figure 37A: Synthesis of Compound 26 To a solution comprising 0.1084 gram (0.33 millimole) of compound 25 and 1 milliliter of freshly-distilled CH 2 Cl2 was added 0.182 g (0.43 millimole) of Dess-martin periodinane. The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with 10 milliliters of ether and poured into 6 milliliters of a saturated NaHCO 3 solution comprising 5% Na2S20 3 Another 10 milliliter aliquot of ether was added to the bi-phasic mixture, and the layers were separated. The organic layer was washed once using 10 milliliters of a saturated NaHCO 3 solution, and once using 10 milliliters of water.

The washed organic layer was dried in the presence of anhydrous Na2SO 4 filtered, and concentrated under reduced pressure. The residue obtained by this procedure was purified by flash column chromatography, eluting with a solvent mixture comprising MeOH and CH 2

CI

2 in a ratio of 5 to 95, respectively, to yield 0.083 gram of purified compound 26 (77% yield) in the form of a white foam. The following analytical data were obtained for compound 26: Rf 0.61 (10:90-MeOH:CH2Cl2); H NMR (500 MHz, CDC1 3 8 0.86-1.05 6H), 1.41-1.54 (m, -66- WO 01/76616 PCT/US01/11607 IO 1H), 1.70-1.74 2H), 1.87-1.89 3H) and 2.05-2.19 I 2.33 3H), 2.97 2H), 0 3.59-3.63 2H), 3.70 3H), 4.95-4.98 J=7.97Hz, 1H), 5.11-5.13 1H); HRMS m/z

C

O calculated forC 16

H

2 6

N

2 0 5 (M H 327.1920, found 327.1912; D +1.14 (c 0.49, Q CHCI 3 Reaction R of Figure 37A: Synthesis of Compound 27 IN A solution comprising 0.0678 gram (0.21 millimole) of compound 26, 4 S milliliters of distilled THF, and 4 milliliters of MeOH was cooled to 0°C. To this solution, 8 "1 milliliters of a solution comprising 0.2 molar LiOH was added. The reaction mixture was 0 initially maintained with stirring at 0°C for 1 hour, after which the reaction mixture was NC warmed to room temperature and maintained with stirring for at least an additional 8 hours.

The resulting mixture was concentrated under reduced pressure, cooled to 0°C, and acidified to pH 3 by adding 1 normal KHSO4. The acidified mixture was extracted three times with milliliters aliquots of EtOAc. The combined EtOAc layers were dried in the presence of anhydrous Na2SO 4 filtered, and concentrated under reduced pressure to yield 0.042 gram (64% yield) of compound 27 in the form of a white solid. The following analytical data were obtained for compound 27: H NMR (500 MHz, CDC13) 8 0.86-1.03 6H), 1.35-1.50 (m, 1H), 1.70-1.77 2H), 1.85-1.93 2H) and 2.07-2.18 2H), 2.33 3H), 3.02 3H), 3.58-3.86 2H), 4.78-4.81 1H), 5.08-5.12 1H); 13C NMR (250 MHz, CDCI 3 8 21.26 and 22.55, 23.21, 24.64, 26.36 and 28.17, 31.16, 37.09, 45.70, 56.79, 58.66, 163.32, 173.12, 174.59, 199.07.

Reaction S of Figure 37B: Synthesis of (-)Dehvdrotamandarin (Compound 133) A solution comprising 19.7 milligrams (0.063 millimole) of compound 27, 33.4 milligrams (0.042 millimole) of compound 22, and 0.50 milliliters of CH 2

C

2 was cooled to 0°C in an ice bath. To this cooled solution was added 28 milligrams (0.063 millimole) of BOP and 0.0185 milliliters (0.17 millimole) of NMM. The reaction mixture was stirred for minutes at 0°C and allowed to warm to room temperature. The reaction mixture was maintained with stirring at room temperature for at least an additional 8 hours. The resulting solution was diluted with 2 milliliters of a saturated solution of NaCl and extracted with milliliters of EtOAc. The extracted organic layer was washed once using 5 milliliters of a solution of HC1, once using 5 milliliters of a 5% solution of NaHCO 3 and once using -67- WO 01/76616 PCT/US01/11607 milliliters of a saturated solution of NaCl. The washed organic layer was dried in the presence 0 of anhydrous Na 2

SO

4 filtered, and concentrated under reduced pressure. The crude residue 0 was purified by flash column chromatography, eluting with a combination of solvent mixtures comprising MeOH and CH 2 Cl 2 in ratios from 2 to 98, respectively, to 10 to 90, respectively, to S yield 18.1 milligrams of dehydrotamandarin (compound 133) in the form of a yellow-white solid. The yield of compound 133 was 41%, as calculated for both reaction O the de- IND protection of compound 21 to yield compound 22 in Example 1) and reaction S. The following S analytical data were obtained for dehydrotamandarin A: Rf 0.48 (10:90-MeOH:CH 2

C

2

IH

NMR (500 MHz, CDCI 3 8 0.80-1.00 24H), 1.16-1.45 (m 11H), 1.51-2.25 10H), 2.38- 0 2.48 and 3.19-3.30 2H), 2.52-2.53 J=6.2Hz, 3H), 2.57 3H), 3.04 and 3.08 3H, CN rotomers), 3.12-3.16 1H) and 3.31-3.35 1H), 3.53-3.72 5H), 3.77 3H), 3.81-4.03 1H), 4.05-4.10 1H), 4.25-4.26 1H), 4.61-4.65 1H), 4.68-4.71 1H), 4.85- 4.88 1H), 5.00-5.01 J=4.6 Hz, 1H), 5.15-5.20 1H), 5.28-5.31 1H), 6.81-6.83 (d, Hz, 2H), 7.05-7.07 J=8.2 Hz, 2H), 7.28-7.30 J-9.8 Hz, 1H) and 7.33-7.35 (d, J=10.2 Hz, 1H), 7.39-7.40 J=5.5 Hz, 1H), 7.72-7.74 J-9.7 Hz, 1H) and 7.78-7.80 (d, J=9.7 Hz, 1H); 13C NMR (500 MHz, CDC1 3 8 11.80, 14.11, 16.52, 17.63, 18.91,20.87, 21.33, 22.32, 23.53, 24.89 (overlap), 24.83, 27.08, 27.35, 27.99, 29.62, 30.13, 31.00, 33.56, 34.04, 35.10, 35.84, 38.90,39.65, 46.72, 48.29, 48.81, 54.78, 55.27 (overlap), 56.91, 57.46, 58.91, 66.10, 68.92, 70.90, 78.94, 114.12, 130.00, 130.36, 158.68, 161.50, 168.43, 169.59, 170.61, 171.00, 172.26, 173.00, 174.52, 197.36; IR (KBr) 3339, 2960, 2927, 2872, 1736, 1715, -1 1655, 1633, 1508, 1460, 1438, 1248, 1177, 1085, 1031, 830 cm HRMS m/z calculated forC54H83N 7 01 4 Na (M+Na 1076.5896, found 1076.5946; D 35.29 (c 0.35, CHCl 3 Example 4 Synthesis of Fluorescent Tamandarin analogs A method of synthesizing a fluorescent tamandarin, compound 108, is described in this example. As in Example 3, the method illustrated in this Example involves the macrocyclic compound 22, which can be generated as described in Example 1. The method described in this Example is illustrated in Figure 38, and is initiated with synthesis of compound 206a, depicted in Figure 38A.

-68- WO 01/76616 PCT/US01/11607 NO Reaction T of Figure 38A: Synthesis of Compound 205a.

0A solution comprising 10.00 grams (49.5 millimoles) ofdiethyl 1,3- O acetonedicarboxylate, 7.12 grams (51.9 millimoles) of m-dimethylaminophenol, 8.56 grams S (62.8 millimoles) of zinc chloride, and 25 milliliters of absolute ethanol was maintained at C reflux for 13 hours. The reaction mixture was diluted with 50 milliliters of EtOAc and washed using 25 milliliters of H20. The aqueous layer obtained from this procedure was extracted ^O three times with 50 milliliters aliquots of EtOAc. The combined EtOAc layers were dried in the presence of anhydrous magnesium sulfate (MgSO4), filtered, and concentrated under l reduced pressure. The resulting crude solid was re-crystallized from absolute ethanol to yield

\O

C 2.64 grams (20% yield) of compound 205a in the form of orange crystals. The following CN analytical data were obtained for compound 205a: mp 131-132°C; Rf 0.20 (acetone/hexane 1 30:70); H NMR (500 MHz, CDCI3) 8 1.22 (3H, t, .J=7.1 Hz), 3.01 (6H, 3.64 (2H, 4.15 (2H, q, J=7.2 Hz), 6.02 (1H, 6.48 (1H, d, J=2.6 Hz), 6.58 (1H, dd, J 2.6 Hz, 8.9 Hz), 7.37 (1H, d, J 13C NMR (125 MHz, CDCI 3 8 14.02, 38.16, 40.01, 61.49, 98.28, 108.45, 108.93, 110.63, 125.22, 148.39, 152.91, 155.89, 161.65, 169.01; IR (KBr) 2907 1720 1600 1534 1484 1427 1405 1371 1330 1232 cm 1; HRMS Calculated forC 1 5

H

1

NO

4 2.76.1236, found 276.1244. Anal. Calculated forC 1 5H 1 7NO 4 C, 65.43; H, 6.23; N, 5.09. Found: C, 65.26; H. 6.36; N, 5.03.

Reaction U of Figure 38A: Synthesis of Compound 205b.

A solution comprising 0.46 gram (11.0 millimoles) of lithium hydroxide hydrate (LiOH-H 2 0) and 25 milliliters of H20 was added to a solution comprising 1.50 grams millimoles) of compound 20Sa and 10 milliliters of THF. The reaction mixture was maintained with stirring at room temperature for 2.5 hours. The resulting solution was washed with 10 milliliters of ether and acidified to pH 2. A precipitate formed upon acidification and was collected by filtration. The precipitate comprised 0.364 gram of compound 205b in the form of yellow crystals. The following analytical data were obtained for compound 205b: mp 166-167 OC, 1H NMR (500 MHz, CDCI 3 /DMSO) 8 3.01 (6H, 3.62 (2H, 6.00 (1H, s), 6.44 (1H, d, J= 2.3 Hz), 6.58 (1H, dd, J 1 2.4 Hz, 8.9 Hz), 7.40 (1H, d, J 9.0 Hz); 13C NMR (125 MHz, CDCI 3 /DMSO) 8 38.42, 40.25, 98.34, 108.89, 109.35, 110.67, 125.84, 149.59, 153.22, 156.15, 162.00, 171.33; IR (KBr) 2923 1690 1619 1534 1404 -69- WO 01/76616 PCT/US01/11607 D 1248 1145 1055 cm HRMS Calculated forCi 3 Hi 4

NO

4 (M H):

O

248.0922, found 248.0929.

Reaction V of Figure 38A: Synthesis of Compound 206a.

A solution comprising 0.364 gram (1.47 millimoles) of compound 205b and milliliters of freshly-distilled CH 2 Cl 2 was placed under an argon atmosphere and cooled to OC.

O0 To this solution was added 0.282 gram (1.47 millimole) of EDAC-HCI and 0.035 gram (0.29 c, millimole) of DMAP. The resulting mixture was stirred for 10 minutes, and 0.246 gram (1.47 'C millimoles) of glycine tert-butyl ester was added. The reaction mixture was maintained with

IND

C stirring at 0°C for 2 hours, warmed to room temperature, and maintained at room temperature C1 for at least 8 additional hours. The reaction mixture was diluted with CH 2 C12 and washed once using 10 milliliters of a 10% solution of HCl and once using 10 milliliters of a saturated NaCI solution. The combined, washed CH 2 C1 2 layers were dried in the presence of anhydrous MgSO 4 filtered, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography, eluting with a solvent mixture comprising equal amounts of EtOAc and CH 2 C1 2 to yield 0.250 gram (42% yield) of compound 206a in the form of a yellow solid. The following analytical data were obtained for compound 206a: 1H NMR (500 MHz, CDC1 3 6 1.42 9H), 3.03 6H), 3.64 2H), 3.88 J 5.12 Hz, 2H), 6.05 1H), 6.48 (brs, 1H), 6.58 J 2.5 Hz, 1H), 6.60 (dd, J 1 2.6 Hz, 2 8.9 Hz, 1H), 7.45 J 8.9 Hz, 1H); 13C NMR (125 MHz, CDCI 3 8 168.5, 167.8, 161.6, 156.0, 153.1,149.2, 125.6, 110.5, 109.2, 108.3, 98.3, 82.5, 42.4, 40.3, 40.1, 27.9; IR (CHCI 3 3450, 1679, 1618, 1530, -1 1405, 1370, 1230, 1157 cm HRMS (CI) m/z calculated forCl9H24N205 (M 360.1685, Found 360.1686. Anal. Calculated forC 1 9H 2 4

N

2 0 5 C, 63.30; H, 6.72; N, 7.78. Found: C, 62.99; H, 6.60; N, 7.52.

Reaction W of Figure 38B: Synthesis of Compound 207a.

A solution comprising 0.250 gram (0.69 millimole) of Gly-DACA-tert-butyl ester, compound 206a, and CH 2 C1 2 was cooled to o0C, and maintained for 10 minutes. To the cooled solution a solution comprising 10% trifluoroacetic acid (TFA) was added drop-wise.

This addition was completed in 10 minutes. The resulting mixture was maintained with stirring at room temperature for at least 8 hours. The reaction mixture was evaporated to dryness using a rotary evaporator, and the resulting residue, comprising 0.033 gram (0.108 WO 01/76616 PCT/USO 1/11607 millimole) of compound 206b, was used in the subsequent reaction without further 0 purification.

A solution comprising 0.033 gram (0.108 millimole) of compound 206b and

CH

2 C1 2 was cooled to 0°C. To the cooled solution was added 0.027 gram (0.108 millimole) of C i1 BOP and 0.012 gram (0.108 millimole) of NMM. The resulting mixture was maintained for minutes with stirring. An excess of the methyl ester of N-methyl-D-leucine was added to the 0 stirred mixture, after which the reaction was maintained with stirring at o°C for at least 8 hours.

C1 The reaction mixture was diluted with CH 2 C1 2 and washed once using 10 milliliters of a Ssolution of HCI and once using 10 milliliters of a saturated NaCI solution. The combined, I washed CH 2

CI

2 layers were dried in the presence of anhydrous MgSO4, filtered, and

O

CI concentrated under reduced pressure. The resulting residue was purified by flash column chromatography, eluting with a solvent mixture comprising equal amounts of EtOAc and

CH

2 C12. Evaporation of the eluate yielded 0.030 gram (63% yield) of compound 207a in the form of a yellow solid. The following analytical data were obtained for compound 207a: 1 S [a]D +19.08 (c 0.865, CHCI 3 H NMR (500 MHz, CDC1 3 8 0.87 J= 6.52 Hz, 3H), 0.91 J 6.79 Hz, 3H), 1.40 1H), 1.69 2H), 2.85 3H), 3.01 6H), 3.63 2H), 3.67 3H), 4.06 2H), 5.22 (dd, J1 10.6 Hz, 5.2 Hz, 1H), 6.04 1H), 6.47 J Hz, 1H), 6.57 (dd, J1 2.5 Hz, 2 8.9 Hz, 1H), 6.76 (brs, 1H), 7.42 J 8.9 Hz, 1H); 13C NMR (125 MHz, CDCI3) 5 171.7, 168.6 167.8, 161.6, 155.9, 152.9, 149.1,125.4, 110.5, -1 109.1, 108.4, 98.3, 54.7, 52.3, 41.8, 40.1, 39.9, 37.1, 30.0, 24.8, 23.1, 21.3 cm 1 HRMS m/z calculated forC 2 3

H

3 1

N

3 0 6 (M Na 468.2111, Found 468.2133.

Reaction X of Figure 38B: Synthesis of Compound 207b A solution comprising 0.135 gram (0.304 millimole) of compound 207a and THF was cooled to 0 C, and a solution comprising 0.025 gram (0.606 millimole) of LiOH-H 2 0 in 2 milliliters of water was added. This addition was completed over a period of 5 minutes.

The resulting mixture was maintained with stirring at 0°C for 30 minutes and then warmed to room temperature. The reaction mixture was maintained at room temperature for 1.5 hours, and then washed twice using 4 milliliter aliquots of ether. The washed aqueous layer was evaporated to dryness under reduced pressure. The resulting residue, comprising compound 207b, was dissolved in a solution comprising 2 milliliters of water and 4 milliliters of EtOAc, cooled to 0°C, and acidified to pH 2 by adding a solution comprising 1 normal KHSO 4 The -71- WO 01/76616 PCT/US01/11607 ND separated aqueous layer formed by this procedure was washed twice with 4 milliliters aliquots of EtOAc. The combined organic layers were dried in the presence of anhydrous MgSO 4 Sfiltered, and concentrated to dryness under reduced pressure. The residue resulting from this procedure comprised 0.010 gram (0.023 millimole) of compound 207b, and was used in the c 5 subsequent reaction without purification.

INO Reaction Y of Figure 38C: Synthesis of Compound 107 c- A solution comprising 0.010 gram (0.023 millimole) of compound 207b and

CH

2 C1 2 was cooled to 0°C. To this cooled solution were added 0.010 gram (0.023 millimole) of BOP and 0.010 milliliter (0.092 millimole) of NMM. The resulting mixture was maintained C- with stirring at 0°C for 10 minutes, and the hydrochloride salt of compound 22 (0.018 gram, 0.023 millimole) was added. The reaction was maintained at 0°C for 1 hour and at room temperature for at least an additional 8 hours. The resulting solution was diluted with 1 milliliter of brine and extracted twice using 2 milliliter aliquots of EtOAc. The organic layer formed by extraction was washed once using 1 milliliter of a 10% solution of HCI and twice using 1 milliliter aliquots of water. The washed organic layer was dried in the presence of anhydrous MgSO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash chromatography, eluting with a solvent mixture comprising acetone and hexane in a ratio of 30 to 70, respectively. Evaporation of the eluate yielded 0.008 gram yield) of fluorescent compound 107. The following analytical data were obtained for 1 compound 107: [a]D -160.1 (c 0.3, CHCl 3 H NMR (500 MHz, CDC13) 8 0.73-0.93 (m, 24H), 1.07-1.74 15H), 2.04 2H), 2.14 3H), 2.15 2H), 2.31 2H), 2.53 4H), 2.84 2H), 3.03 6H), 3.17 (brd, J 10.4 Hz, 2H), 3.35 1H), 3.57 2H), 3.68 2H), 3.78 3H), 3.99 (brd, 2H), 4.084.12 3H), 4.55-4.56 1H), 4.78-4.80 1H), 4.82- 5.01 2H), 5.13-5.14 (brd, 1H), 6.08 1H), 6.51 1H), 6.61 J= 9.1 Hz, 1H), 6.83 J 8.54 Hz, 2H), 7.07 J 8.35 Hz, 2H), 7.24-7.41 3H), 7.51 J 8.90 Hz, 1H), 7.81 -1 J 8.65 Hz, 1H); IR (KBr) 3462, 2953, 2358, 1732, 1658, 1632, 1555, 1538, 1456, cm-1 MS z calculated forC64H92N 6 (M +Na):1251,6529,Found 1251.6528.

HRMS m/z calculated forC64H9 2 NgOl 6 (M Na 1251, 6529, Found 1251.6528.

-72- WO 01/76616 PCT/US01/11607 IND O Example SSynthesis of a Deoxo-Proline Side Chain Moiety and Coupling to a Didemnin Macrocycle N 5 An amino methylene single bond was used to replace the amide bond between D-leucine and L-proline in the side chain ofdidemnin B. Synthetically, the amino methylene 0 bond was prepared by reductive amination, as described (Abdel-Magid et al., 1990, CN Tetrahedron Lett. 31:5595-5598; Abdel-Magid et al., 1990, Synlett. 537-539).

In a first synthetic method, exhaustive methylation of Cbz-D-leucine was performed using dimethylsulfate, as illustrated in Figure 41. The Cbz group was removed

O

C using hydrogenolysis to yield dimethylated D-leucine free amine (Compound 40). The amine was used in reductive amination without purification. Commercially available L-proline was first esterified using SOC1 2 in MeOH, and its amino group was subsequently protected using Boc 2 0. After purification, the ester function was reduced to an aldehyde in two steps. NaBH4 was combined with LiCl to generate LiBH 4 in situ, and this compound was used to reduce the ester to the corresponding alcohol. Oxidation with S03*pyridine reagent gave the aldehyde, designated compound 42, in good yield. Reduction of the ester to the aldehyde in one step using DIBAL could be performed, but depended on the freshness of the reducing reagent and was not very reproducible. Reductive amination between the aldehyde designated compound 0 40 and the free amine (compound 42) yielded compound 43. The Boc group of compound 43 was removed using TFA/CH 2 Cl 2 The resulting free amine was coupled with pyruvic acid using BOP to yield the protected Y(CH 2 NH) side chain designated compound 44.

The next step was hydrolysis of the methyl ester of compound 44. But this step was proved to be non-trivial. Perhaps owing to steric hindrance, the methyl ester was difficult to cleave using 2 equivalents of LiOH*H20 in THF/H20. When the amount of was increased to 10 equivalents, the methyl ester appeared to hydrolyze, since the mass spectrum of the reaction mixture did show the acid peak. But the acid was so hydrophilic and buried inside the excess inorganic salts, that it could not be extracted by any organic solvent.

We also tried to precipitate the acid using ether but only precipitated the inorganic salts. On account of this purification problem, we decided not to use the methyl ester to protect the acid.

We needed a protective group which could survive all the synthetic steps but did not require aqueous conditions for its removal. The benzyl group was found to serve this purpose well.

-73- WO 0 1/76616 PCTIUSOI11 6 o 7 IND The Synthetic Procedure was modified as indicated in Figure 42. In order to be comatile it th bezy ester, teleucine aiogroup wsprotected by DUCinta of Cbz. Selective luethylation of the amino group gave the acid designated compound 46. The U free acid was beuzylated to yield the desired benzyl ester, compound 47. The Boc protecting N- 5 group was removed using TFA. The resulting amine (compound 48) was condensed with protected prohinal by reductive ainination. Removal of the Boc protecting group and ID subsequent coupling with pyruvic acid yielded compound 50. [lydrogenolysis was used to N- remove the benzyl group and yielded the desired

'P(CH

2 NIH) side chain, in the form of the free V) acid (compound 51). Coupling of the free acid side chain with a didemnin macrocycle gave the desired deoxo-proline didemnin analog designated compound 12.

A second deoxo-proline dideninin analog (designated compound 72a) was synthesized using a similar synthetic strategy, as illustrated in Figure 43 and described in detail as follows.

g 0 t~ r O k dN b h

~C

Benzyl N-Boc D-leucmnate (Compound 46).

A solution of N-Boc-D-Ieucine (1.0 gramn, 4.1 inillinioles) in 20 iiliters of DMF was cooled to 0* C. Finely powdered Li2CO 3 (1.5 grains, 20.5 inillimoles) was added, followed by the addition of benzyl bromide (2.43 milliliters, 20.5 mfillimoles). The reaction mixture was stirred for 6 hours and monitored by TLC. When the reaction was complete, the reaction mixture was diluted with H20 and extracted with EtOAc three times. The EtOAc extracts were combined and washed with brine. DMF was removed in vacuo. The crude mixture was purified with coluimn chromatography eluting with 20% acetone/liexane to afford compound 46 in 78% yield. The following analytical data were obtained for compound 46: Rf 0.60 (40% actn/hxn) [affl +16

CHCI

3 lHj NMR (500 MH7, ODC1 3 8 Ha) 0.92 (in, 611), 111) 1.48 914), Hc) 1.50-1.59 (in, 114), Hd) 1.60-1.69 (dd, 211), He) 4.36 (in, IIIM, Hf) 4.90 11W), Hg) 5.11-5.20 (mn, 2H1), Hh) 7.3 3 (in, 511; 13 C NIMR (125 MH7,

CDCI

3 8 Ca) 21.8, Ca') 22.7, Cc) 24.7, Oh) 28.2, Cd) 41.6, Ce) 52.1, Cg) 66.7, CO) 79.6, Cb) 74- WO 01/76616 PCT/USO /11607 IN 128.0, 128.2, 128.4, Cl) 135.5, Cj) 155.3, Ck) 173.2; IR (neat) 3367 2958 1732 0 1715 1500 1455 1366 1120 cm-1; HRMS m/z calculated for O C11H23N402 322.2017, found 322.2018.

0U e SMe INIf 0 'b I N a d O b (N h c

C

in

(N

SN-Boc N-Methyl benzyl D-leucinate (compound 47).

C, A solution of compound 46 (1.2 grams, 3.74 millimoles) in 200 milliliters of THF was cooled to 0' C. NaHMDS (1 molar in THF; 5.6milliliters, 5.6 millimoles) was added, followed by the addition of methyl iodide (1.0 milliliter, 18.7 millimoles). The reaction mixture was stirred overnight and monitored by TLC. When the reaction was complete, the reaction mixture was diluted with ether. The organic layer was washed with 5% HC1, NaHCO3, and brine. The resulting solution was dried over Na2SO4 and concentrated. The crude mixture was purified by column chromatography, eluting with 20% acetone/hexane to afford compound 47 in 71% yield. The following analytical data were obtained for compound 47: Rf 0.55 (30% Acetone/Hexane); [a]B +20.4 CHCI3); 1 H NMR (500 MHz, CDC13) 8 Ha) 0.85-0.90 6H), He) Hd) 1.35-1.65 3H), Hb) 1.45 9H), He) 2.75 (d, 3H), Hf) 4.53-4.58 4.81-4.88 (rm, 1H), Hg) 5.10 2H), Hh) 7.15-7.28 5H); 13 C NMR (125 MHz, CDC13) 8 Ca) 22.0 and 23.5, Cc) 25.8, Cb) 29.0, Cd) 31.0, Ce) 38.0, Cf) 57.0, Cg) 66.1, Ci) 80.2, Ch) 128.0, 128.2, 128.4, Cl) 136.5, Cj) 156.3, Ck) 173.2; IR (neat) 2958.3 1742.7 1696.8 1455.6 1390.7 1366.6 1323.5 1151.3 cm- 1

HRMS

m/z calculated for C11H23N402 336.2174, found 336.2178.

Sd y e

NH*HCI

i e h 'a c g b 75 WO 01/76616 PCT/US01/11607 I Benzyl N-Methyl D-leucinate hydrochloride salt (compound 48).

Compound 47 (0.1 gram) was dissolved in HCl-dioxane (5 milliliters) and O stirred at room temperature. When the reaction was completed, the solvent was removed in Svacuo. Toluene was added twice and concentrated. The residue was dried under reduced CN 5 pressure overnight to afford the desired HCI salt (compound 48) in 98%. The following analytical data were obtained for compound 48: Rf baseline (10% MeOH/CH2Cl2); [a]1 IN +48.5 CHCI 3 1 H NMR (500 MHz, CDC13) 8 Ha) 0.91 6H), Hb) 1.75 1H) He) 1.90-1.95 (dd, 2H), Hd) 2.71 3H), He) 3.83 1H), Hf) 5.20-5.30 (rm, 2H), Hg) 7.35-7.40 5H), Hh) 9.85 10.15 (br, 2H); 13 C NMR (125 MHz, CDC13) 6 Ca) 21.8, and 23.4, Cc) O 10 25.8, Cb) 31.9, Cd) 38.2, Ce) 60.1, Cf) 68.8, Cg) 128.4, 128.6, 128.8, Cj) 134.6, Ci) 168.2; IR CN (neat) 2958.3 1742 1696 1455 1390 1366 1323 1151 cm- 1 HRMS m/z calculated for Cl 1H23N402 336.2174, found 336.2178.

c b MeO d e N 0O O Methyl N-Boc L-Prolinate (compound 41).

Commercially available N-Boc-L-proline (5.0 grams, 23.2 millimoles) was dissolved in acetone (200 milliliters). K2C03 (3.8 grams, 27.84 millimoles) was added at 0* C, followed by the addition of Mel (2.9 milliliters, 46.4 millimoles). The reaction mixture was stirred overnight. The reaction was quenched with 5% NaHCO3 solution and extracted with ether. The combined ether layers were washed with 5% HCI and brine, and concentrated to afford the crude product (compound 41) in 91% yield. This compound was purified by column chromatography eluting with 20% acetone/hexane. The following analytical data were obtained for compound 41: Rf 0.4 (30% acetone/hexane); -61.2 CHC13); 1

H

NMR (500 MHz, CDC13) 8 Ha) 1.45 9H), Hb) 1.98 (dd, 2H) He) 1.88 2.22 rm, 2H), Hd) 3.42-3.58 2H), He) 3.74 3H), Hf) 4.24 4.35 (rm, 1H); 13 C NMR (125 MHz, CDC13) 8 Cb) 24.2, and 24.4, Ca) 28.3, Cc) 31.6 and 31.8, Cd) 47.3, Cf) 52.1, Ce) 59.7, Cg) 80.1, Ch) 153.8, Ci) 174.0; IR (neat) 2975 1749 1700 1396 1365 1200 1161 1151 HRMS m/z calculated for Cl H23N402.(M+H) 230.1391, found 230.1389.

-76- WO 01/76616 PCT/US01/11607 O b c

I

HO a SO a N-Boc L-Prolinol (Compound 41a).

Compound 41 was dissolved in 200 milliliters THF/EtOH LiCI (1.8 grams, 32.7 millimoles) and NaBH4 (1.2 grams, 32.7 millimoles) was added in portions at 0°C.

k0 The reaction mixture was stirred overnight and monitored by TLC; more LiCI and NaBH4

O

0 were added during the operation. When the reaction was complete, the white solid was collected and washed with ether. The solvent was removed using a rotary evaporator. The residue was neutralized to pH 4 and then extracted twice with EtOAc. The EtOAc extracts 0 were combined, washed with brine, dried, and concentrated. The crude product was purified by column chromatography eluting with 10% acetone/hexane to afford the desired alcohol (compound 41a) in 83% yield. The following analytical data were obtained for compound 41a: Rf 0.30 (30% Acetone/Hexane); [a]U -60.0 CHC13); 1 H NMR (500 MHz, CDC13) 5 Ha) 1.48 9H), Hb) 1.60 2.05 (rm, 2H) Hc) 1.83-1.98 (dd, 2H), Hd) 3.35 3.45 (rm, 2H), He) 3.61-3.70 2H), Hi) 4.03 1H); 13C NMR (125 MHz, CDC13) 5 Cc) 23.8, Ca) 28.4, Cb) 28.9, Cf) 47.3, Ce) 59.9, Cd) 67.3, Cg) 80.0, Ch) 156.8; IR (neat) 3424 (br), 2973 2877 1695 1670 1477 1406 1366.2 HRMS m/z calculated for C 11H23N402 202.1443, found 202.1449.

b c 0O hO a N-Boc L-prolinal (compound 42).

A solution of compound 41a (2.0 grams, 9.9 millimoles) and Et3N in 200 milliliters of CH2CI2 was cooled to -78' C. S03*pyridine complex (4.7 grams, 29.7 millimoles) in DMSO (30 milliliters) was added to the previous solution. The reaction mixture was warmed to room temperature, stirred overnight, and monitored by TLC. When the 77 WO 01/76616 PCT/US01/11607 N0 reaction was complete, the reaction mixture was diluted with ether. The organic layer was

O

0 washed with 5% HCI, 5% NaHCO3, and brine. The resulting solution was dried over Na2SO4 0 and concentrated. The crude mixture was purified with a short column to afford the desired Q aldehyde (compound 42) in 81% yield. The following analytical data were obtained for compound 42: Rf 0.55 (30% Acetone/Hexane); [a]21 +20.40 CHC13); IH NMR (500 MHz, CDC13) 5 Ha) 1.45 9H), Hb) 1.75-1.90 2H), Hc) 1.90-2.15 2H), Hd) 3.20- IN 3.50 2H), He) 3.90-4.20 1H), Hf) 9.72 1H); 13 C NMR (125 MHz, CDC13) 8 Cb) 24.0, Cc) 27.3, Ca) 28.9, Cd) 47.5, Ce) 65.5, Cg) 80.1, Ch) 154.2, Cf) 200.1.

INh SO g d S I Me I O p N 1 N f e OjOn b a N-Boc-pro y(NHCH2) N-Methyl-Benzyl-D-Leucinate (compound 43).

Compound 42 (0.1 gram, 0.5 millimoles) was dissolved in CH2C12 (8 milliliters), and free amine compound 48 (0.15 gram, 0.55 millimoles) was added with efficient stirring. At 0 0 C, AcOH (0.02 milliliters, 0.3 millimoles) was added as a catalyst and stirred for 10 minutes before NaBH(OAc)3 (0.13 gram, 0.6 millimoles) was added to the reaction mixture. The reaction mixture was stirred at room temperature and monitored by TLC. When the reaction was complete, the mixture was diluted with CH2C12. The excess reagent was quenched by dropwise addition of saturated NH4Cl solution. The organic layer was washed with 5% HCI, 5% NaHCO3, and brine, dried, and concentrated. The crude product was purified by flash chromatography, eluting with 20% EtOAc/petroleum ether to afford the desired amine (compound 43). The following analytical data were obtained for compound 43: Rf 0.50 (30% Acetone/Hexane);.[a]?l -44.0 CHC13); 1 H NMR (500 MHz, CDCI3) 8 Ha) 0.92 6H), Hb) 1.45 9H), He), Hd) He) 1.50-1.90 5H), Hf) Hg)2.3- 2 4 5 (m, Hhl) 2.22-2.90 1H), Hh2) Hi) 3.25-3.4 3H), Hj) Hk) 3.70-3.95 2H), HI) 5.35 5H), Hm) 7.30 5H); 13 C NMR (125 MHz, CDC13) 6 Ca) 22.2, Cd) 23.4, Cc) 25.6, Cb) 28.4, Ce) 28.8, Cg) 36.6, Cf) 38.4, Ch) 46.2, Ci) 56.1, Cj) 58.5, C) 56.2, Ck) 66.0, Cn) 79.6, Cm) 128.2, Cq) 136.2, Co) 154.1, Cp) 174.2; IR (neat) 2955 2868 1730 -78 WO 01/76616 WO 01/66 16PCTLJSOI/1 1607 IND 1693 1455 1392 1364 1170 cm- 1 FIRMS m/z calculated for C24H138N204 419.2910 found 419.2897.

0 f b IND e g H*H.CI IND d a L-Pro TI(NHCH2) N-Methyl-Benzyl-D-Leuci'nate Hydrochloride salt (compound 44).

Compound 43 (0.1 gram) was dissolved in HCl-dioxane (5 millilters), and the solution was stirred at room temperature. When the reaction was completed, al solvent was removed in vacuo. Toluene was added twice and concentrated. The residue was dried under reduced pressure overnight to afford the desired HCI salt (compound 44) in 98% yield. The following analytical data were obtained for compound 44: Rf baseline (60% EAIPE); [a]U +15.3 CHCI3); 1 H NMR (500 MHz, CDC13) 8 Ha) 0.91-1. 10 6H1), Fib), Hc) Fid) 1.68-2.30 (in, S5H), He) 2.83 (dd, 2H), Hf) 3.12 3H), Hg) Hh) 3.3 1-3.65 (mn, 4H), Hi) Hj) 4.11-4.30 (in, 2H), Hk) 5.28 2H), Hk) 7.40 (in, 5H1); 1 3 C NMR (125 MHz, CDCl3) Cc) 20.8, Cb) 23.5, Ca) 25.9, Cd) 26.1, Ce) 36.2, Ct) 46.2, Cg) 55.8, Ch) 61.6, CO) 64.2, Cj) 67.0, Ck) 68.2, Cl) 125.0, 128.0, 128.8, 128.9 134. 1, Cn) 13 7.9, Cm,) 167.0; JR (neat) 2954 2360 2338 1740 1455 1389 1197 1141 cm-1;HR.MS m/z calculated for C19H30N202 (M±H4) 319.2386 found 319.2392.

a OHh L-Lactyl-Pro 1 P(NHCH2) N-Methyl-Benzyl-D-Leucinate (compound -79- WO 01/76616 PCT/US01/11607 IN Compound 44 (20 milligrams, 0.063 millimoles) was dissolved in CH2C12 milliliters), and lactic acid (5.55 milligrams, 0.063 millimoles) was added at o0C, followed by 0 the addition of BOP (28 milligrams, 0.063 millimoles and NMM (0.035 milliliters, 0.31 millimoles). The reaction mixture was stirred at 0°C and monitored by TLC. When the CN 5 reaction was completed, the reaction mixture was diluted with ether. The organic layer was washed with 5% HC1, 5% NaHCO3, and brine. The resulting solution was dried over Na2SO4 ND and concentrated. The crude mixture was purified by column chromatography, eluting with FC 30% acetone/hexane to afford compound 50a in 61% yield. The following analytical data were c, obtained for compound 50a: Rf 0.50 (50% Acetone/Hlexane); [a]2f +21.0 CHC13); 1 HNMR (500 MHz, CDC13) 8 Ha) 0.82-0.92 6H), Hb) 1.20 3H), He) 1.45-1.66 (m, cN 2H), Hd) 1.66-1.72 1H), He) Hf) 1.76-1.90 4H), Hg) Hh) 2.25-2.45 5H), Hi), Hj) Hk) 3.19-3.85 4H), HI) 4.10-4.21 (rm, 1H), Hm) 5.05-5.18 2H), Hn) 7.12-7.38 5H); 1 3 C NMR (125 MHz, CDC13) 8 Ca) 22.0, Cc) 22.8, Cd) 24.2, Ce) 24.8, Cb) 25.4, CO 28.1, Cg) 37.8, Ch) 38.6, Cj) 45.8, Ci) 46.2, Ck) 56.0, CO 56.8, Cm) 66.0, Cn) 128.8, Co) 136.1, Cp) 173.8, Cq) 174.1; IR (neat), 3415 2955 2869 1729 1638 1455 1379 1366 1147 1126 HRMS m/z calculated for C22H34N204 (M+Na) 413.2416 found 413.2423.

Se f Me HO n h d b

C

a

OH

N-Methyl-leu '(NHCH 2 lac-pro acid (compound 51a).

Compound 50a (20 milligrams, 0.063 millimoles) was dissolved in milliliters MeOH/EtOAc The mixture was added to a solution of MeOH and EtOH (1:1) containing Pd/C catalyst (10 milligrams). The reaction mixture was shaken in a Parr hydrogenator under H2 (40 pounds per square inch, gauge) and monitored by TLC. When the reaction was completed, the catalyst was removed by filtration. The remaining solution was concentrated in vacuo. The crude product (compound 51a) was used directly in the next step.

The following analytical data were obtained for compound 51a: Rf 0.20 WO 01/76616 PCT/USO1/11607 I MeOH/CH2Cl2); [a]S +65.0 CHCI3); 1 H NMR (500 MH-z, CDCI3) 6 Ha) 0.82-0.92 6H), Hb) 1.34 3H), He) 1.45-1.55 1H), Hd) 1.78-1.91 2H1), He) 1.91-2.0 (m, 2H11), Hf) 2.0-2.18 2H), Hg) 2.81 3H), Hh) 3.03-3.18 2H1), Hi) 3.45-3.54 1H), Hj) 3.56-3.68 2H), Hk) 4.25-4.35 1H), HI) 4.40-4.49 1H); 1 3 C NMR (125 MHz, CDC13) 6 Ca) 21.0, Cb) 22.8, Cc) 23.4, Cd) 24.8, Ce) 25.8, Cf) 29.4, Cg) 36.5, Ch) 38.6, Ci) 47.2, Cj) 55.2, Ck) 66.4, C) 68.2, Cm) 172.4, Cn) 176.2; IR (neat), 3336 2957 2870 D 1718 1627 1466 1368 1250 1197 1128 HRMS m/z

IND

calculated for C15H27N204 (M+Na) 323.1947, found 323.1939.

S,,jJOMe r a O i e dd a Ue N 0 fwit Fl 'Me tk d C) MMe k NHff k O p1 e 0 n cc bb so kk kk( 0 .0 l o H kk N z N'b Ng b j k m g a

OH

a a a Cyclo[N-(N-3,4-didehydro-L-prolyl-N-methyl-D-leucyl)-O[[N-[(2S,3S,4S)-4- [(3S,4R,5S)-4-amino-3-hydroxy-5-methyl-heptanoyl]oxy-3-oxo-2,5-dimethylhexanoyl] -Lleucyl]-L-prolyl-N,O-dimethyl-L-tyrosyl]-L-threonyl] (compound 72a).

The crude acid (12.5 milligrams, 0.038 millimoles) was combined with the macrocycle HCI salt (15.0 milligrams, 0.019 millimole) in CH2CI2 (0.25 milliliter) at 0* C.

HATU (8.2 milligrams, 0.020 millimole) and DIEA (0.026 milliliter, 4 equivalents) was added.

The reaction was stirred at O'C overnight and monitored by TLC. When the reaction was complete, the mixture was diluted with Et20 and the organic layer was washed with 5% HCI, NaHC03, and brine. The resulting solution was dried over Na2SO4 and concentrated.

The crude residue was purified by column chromatography eluting with 5% MeOJI/CH2Cl2 to yield the desired product (compound 72a) in 72% yield. The following analytical data were obtained for compound 72a: Rf 0.40 (10% MeOHICH2CI2); [alj +81.2 (c=0.15, CHC13); -81- WO 01/76616 WO 0176616PCTJUSOI/I 1607 IND HNMR (500 M~z, CDC13) 8 Ha) 0.85-0.97 (in, 24ff), Hb) 1. 17-1.27 (in, 2ff), Hc) 1.28-1.39 Hci) 1.40 3H), He) 1.43 3H), Hf) 1.46-1.50 (mn, 111) and 1.61 1H), Hg) 1.68- 1.73 (mn, 1H), Hh) 1.74-1.79 (nm, 1ff), Hi) 1.82-1.88 (in, 2ff), 2.02-2.08 (in, 1ff) and 2.11-2.17 (in, 2ff), Hj) 2.34-2.37 (in, 1ff), Hk&) 2.57 3H1), HI) 2.38 2ff), Hm) 3.18 and 3.39 (dd, 1ff), Hn) 2.83 3ff, H 0 3.37-.3.57 (mn, 2ff), Hp) 3.60 (di, 111), Hq) 3.70-3.73 (mn, I Hr) 3.80 3ff), Hs) 4.05-4.14 (in, 3H1) and 4.36-4.48 (mn, 2ff), Ht) 4.27 11H), Hu) 4.56 (cli, IND1I1), Hv) 4.65 (dd, 1ff), Hw) 4.81 1ff), Hx) 5.19 (di, 1ff), Hy) 5.3 5 (dd, IHM, H 7 5.42 (dd, cIN 111), Haa) 5.61 (dci, 1ff), Hbb) 5.73-5.75 (mn, IM1, Hcc) 6.11 (dci, 1IM, Hdd) 6.85 (ci, 2ff), Hee) 7.08 (di, 211), Hif) 7.20 (di, 1I1), Hgg) 7.54 11H), Hhh) 7.78 1ff); 13 C NMR (125 MHz, C) 10 CDCI3) 5 Ca), Cc)l 11.7, 14.7, 15.2, 16.3, 16.9, 18.6, 20.1 and 20.9, Cd) 23.3, Ce) 23.7, Cb) Cl 24.8 and 27.2, Co) 24.9,25.0 and 33.9, Cf) 27.9, Cg) 31.2, Ch~) 31.3, Cq) 33.9, Co) 36.2, Cm~) 38.7, Ck) 38.8, Cn) 41.3, C 0 46.9, Cq) 49.46, Cs) 49.52, 53.0, and 67.9, Cr) 55.3, Cv) 55.4, Cw) 55.5, Cu) 57.2, Cy) 57.6, Ct) 63.9, Caa) 65.9, Cs) 66.4, Cp) 66.5, Cx) 70.4, Cz) 81.5, Cdd) 114.1, Ccc) 124.0, Cbb) 129.1, Cii) 130.0, Cee) 130.3, Cjj) 158.6, Ckk) 168.6, 169.3, 170.5, 170.6, 171.3, 171.5, 172.4, 173.9, CIi) 204.9; IR (neat) 3331 2956 2871 1732 1638.3 (br, overlap), 1543 1513 1448 1379 1247 1167 cmr 1 fIRMS m/z calculated for C57H91IN70*14 (M+ff) 1098.6702, found 1098.6726.

Example 6 Synthesis of a 3,4-Dehydro-Proline Side Chain Moiety and Coupling to a Dideinnin Macrocycle Synthesis of the 3,4-dehydroproline unit began with trans-4-hydroxy-L-proline (compound 52), which was produced as described (Rueger et al., 1982, Can. J. Chem.

60:2918; see Figure 44). The acid was first protected as its ethyl ester. The amino group was further protected using Boc20 to yield compound 53. The hydroxyl group was inesylated using MsC1 and pyridine. The mesylate methyl sulfonate moiety of compound 53) was displaced by sodium benzene selenide with inversion of stereochemistry to yield compound 54.

Oxidative elimination of the phenyl selenium group afforded the corresponding alkene (compound 55) in 73% yield.

If compound 54 was directly exposed to the basic elimination condition, two regioisomers would be generated. But oxidlative elimination of compound 54 through phenyl selenium as the intermediate gave only the desired regioisoiner. This regioselectivity may be 82 WO 01/76616 PCT/US01/11607 due to the fact that the transition state leading to the undesired isomer has larger dipole moment 0 0 with higher energy.

SHydrolysis of the ethyl ester (compound 55) yielded the free dehydroproline acid (compound 56), which was coupled with D-leucine ester to yield compound 57.

c Hydrolysis of the methyl ester afforded the free acid, which was coupled to the didemnin macrocycle salt using HATU to afford compound 58. The Boc protecting group was removed using HCI gas, and the HCI salt was neutralized using saturated NaHCO3 solution to afford the final analog, compound 59.

C The steps in this synthesis are now described in greater detail.

e

OH

a f c CH 3CH 20 d 0 A b Ethyl N-Boc-trans-4-Hydroxyprolinate (compound 53). Ethyl trans-4-hydroxy prolinate hydrochloride salt (1.0 gram, 0.005 mole) was dissolved in saturated CH2C12 milliliter). Et3N (2.09 milliliter, 0.015 mole) was added at 0 C, followed by addition of (2.23 g, 0.01 mole). The reaction mixture was stirred overnight. When the reaction was complete, the pH was measured, and was then 8. The reaction mixture was washed with ether, and the ether layer was discarded. The aqueous layer was acidified with 1 normal KHSO 4 to pH 4, followed by extraction three times with ethyl acetate. The organic extracts were combined, washed with brine, dried, and concentrated. The crude mixture was purified by column chromatography, eluting with 20% acetone/hexane to afford the desired product (compound 53) in 71% yield. The following analytical data were obtained for compound 53: Rf 0.50 (40% Acetone/Hexane); [a]j +71.3 CHCl 3 1 HNMR (500 MHz, CDC1 3 8 Ha) 1.07-1.13 6H), Hb) 1.32-1.36 9H), He) 1.84-2.15 dr, 2H), Hd) 3.23-3.49 dr, 2H), He) 3.78-3.95 (br, 1H), Hf) 3.96-4.09 2H), Hg) 4.17-4.26 1H), Hh) 4.27-4.32 (t, 1H); 1 3 C NMR (125 MHz, CDCI 3 8 Ca) 14.0, Cb) 28.1, Cc) 38.2, Cd) 54.2, Cf) 57.7, Cg) 61.0, Ch) 69.0, Ci) 80.0, Cj) 153.9, Ck) 172.6; IR (neat) 3448 2978 2935 1746 -83- WO 01/76616 PCT/US01/11607 1702 1676 1477 1402(s), 1367 1339 cm- 1 HRMS m/z calculated for

O

S C 1

H

2 3

N

4 0 2 260.1497, found 260.1503.

S

h S.Seph ci a g c .e

CH

3 CH20 k/ f cI CM 0 eOOK O b 0 Ethyl N-Boc-cis-4-phenylselenyl-L-prolinate (compound 54). Sodium O borohydride (0.15 gram, 0.004 mole) was added in small portions, at room temperature, to a solution of diphenyl diselenide (0.556 gram, 0.0018 mole) in EtOH. The mixture was stirred for about 5 minutes, until the bright yellow color disappeared. The previously prepared mesylate (1.0 gram, 0.003 mole) was added, the solution was refluxed for 2 hours, and the solvent was removed in vacuo. The residue was diluted with Et 2 0 (5 milliliters), and the organic layer was washed with H 2 0 (10 milliliters) and brine. The resulting organic layer was dried and concentrated. The crude oil was purified by column chromatography, eluting with acetone/hexane to afford the product (compound 54) in 85% yield. The following analytical data were obtained for compound 54: Rf 0.53 (30% Acetone/Hexane); [a]4 -16.4 CHCI3); IH NMR (500 MHz, CDC13) 8 Ha) 1.21-1.24 3H), Hb) 1.45 9H), He) 2.05 2.68 (dr, 2H), Hd) 3.40 1H), He) 3.58 1H), Hfl) 3.95 1H), Hf2) Hg) 4.10-4.28 2H), Hh) 7.25, 7.55 5H); 13 C NMR (125 MHz, CDC1 3 8 Ca) 14.0, Cb) 28.2, Cc) 36.1, Cd) 39.8, Ce) 53.6, Cf) 58.8, Cg) 60.8, Ci 80.2, Ch) 127.0, 128.2, 134.3, Cj) 152.8, Ck) 171.8; IR (neat) 2977.0, 1747.1, 1701.9, 1477.4, 1394.4, 1190.1, 1114.1 cm- 1

HRMS

m/z calculated for ClgH 2 5

NO

4 Se 399.0948, found 399.0957.

a d f CH 3 CH 20

C

0 o.O b Ethyl N-Boc-3,4-Dehydro-L-prolinate (compound 55). A mixture of compound 54 (0.9 gram, 2.26 millimoles) and CH2C12 was initially cooled to 0°C in an ice bath. Pyridine -84- WO 01/76616 PCT/USO1/11607 NO (0.27 milliliter, 3.4 millimoles) was added dropwise to this solution. A solution of aqueous H202 (0.58 milliliter) was then gradually added over a 5 minute period. The reaction was stirred at room temperature for 1 hour, and then diluted with EtOAc. The organic layer was washed with 5% HCI, saturated Na 2

CO

3 solution, and brine. The resulting solution was C 5 dried and concentrated. The residue was purified by column chromatography to afford the desired product (compound 55) in 73% yield. The following analytical data were obtained for ND compound 55: Rf 0.63 (30% Acetone/Hexane); [a] 2 -32.3 CHC3); 1 H NMR (500 MHz, CDC13) 8 Ha) 1.15-1.30 3H), Hb) 1.45 9H), He) Hd) 4.15-4.30 4H), He) t 4.98 1H), Hf) 5.75 (dt, 1H), Hg) 5.95 (dd, 1H); 13 C NMR (125 MHz, CDCI 3 8 Ca) 14.1, Cb) 29.2, Cc) 53.8, Cd) 61.8, Ce) 67.0, Ch) 80.1, Cf) 125.0, Cg) 129.2, Ci) 154.0, Cj) 170.2; C( IR (neat) 3458 2979 1786 1750 1710 1448 1395 1369 1318 1258 1159 1896 cmrl; HRMS m/z calculated for C 12 Hi 9 N0 4

(M+H)

242.1392, found 242.1386.

e d HO b O hoj< a 0 N-Boc-3,4-Dehydro-L-proline (compound 56). Ethyl ester 55 (0.18 gram, 0.8 millimole) was dissolved in THF/H 2 0 10 milliliters). LiOH-H 2 0 (0.33 gram, 8 millimoles) was added to the solution at 0°C. The mixture was stirred for about 6 hours and monitored by TLC. When the reaction was completed, THF was removed in vacuo. Saturated NaHCO 3 solution was added and washed with ether twice. All aqueous layers were combined and acidified to pH 4 with 1 normal KHSO 4 Ethyl acetate was used to extract the acidified aqueous solution three times. The extracts were dried and concentrated to afford the desired acid (compound 56) in 92% yield. The following analytical data were obtained for compound 56: Rf 0.20 (30% acetone/hexane); [a]1 +8.4 CHC1 3 IH NMR (500 MHz, CDCI3) 6 Ha) 1.49 rm, 9H), Hb) 4.25 2H), He) 5.05 1H), Hd) 5.80 (dt, 1H), He) 6.01 (dd, 1H), Hf) 8.40 (br, 1H); 13 C NMR (125 MHz, CDCl 3 S Ca) 28:9, Cb) 53.9, Cc) 66.2, Cg) 81.8, Cd) 124.2, Ce) 129.9, Ch) 154.5, Ci) 165.3; IR (neat) 3000-3400 2957 1736 1704 1666 1400.2 1367 1177 1136 cm-l; HRMS m/z calculated for C 1 0 H1 5 N0 4 213.1079, found 242.1086.

WO 01/76616 PCT/US01/11607

IO

S e k j SMe d 0 MeO c I N-Boc-3,4-Dehydro-L-Prolyl-Methyl-N-Methyl-Leucinate (compound 57).

I-N

Compound 56 (0.1 gram, 0.469 millimole) was dissolved in CH2C12 (5 milliliters). At 0°C, CI 5 HATU (0.26 gram, 0.58 millimole) was added, followed by the addition of DIEA (0.26 0 milliliter, 1.88 millimoles). Finally, dimethyl D-leucine hydrochloride salt (0.09 gram, 0.469 c millimole) was added to the mixture. The reaction mixture was stirred for 6 hours and monitored by TLC. When the reaction was completed, the mixture was diluted with Et20 and the organic layer was washed with 5% HC1, 5% NaHCO3, and brine. The resulting solution was dried and concentrated. The crude residue was purified by column to afford the desired product (compound 57) in 76% yield. The following analytical data were obtained for compound 57: Rf 0.32 (30% Acetone/Hexane); [a] 2 -22.7 CHCI 3 1H NMR (500 MHz, CDCI 3 8 Ha) 0.90-1.05 rm, 6H), Hb) 1.48 rm, 9H), Hc) Hd) 1.65-1.80 3H), He) 3.05 3H), Hf) 3.75 3H), Hg) 4.20-4.35 2H), Hh) 5.28 1H), Hi) 5.40 1H), Hj) 5.70 (dt, 1H), Hk) 6.01 (dd, 1H); 13 C NMR (125 MHz, CDCl 3 8 Ca) 22.0,23.8, Cc) 25.2, Cb) 29.0, Cd) 37.8, Ce) 52.2, Ch) 53.8, Ci) 65.8, Cl) 80.0, Cj) 124.0, Ck) 129.2, Cm) 159.5, Cn) 171.0, Co) 172.2; IR (neat) 2956 1743 1705 1667 1400 1366 1316 1258 1177 1128 cm-; HRMS m/z calculated for C 18

H

3 0

N

2 0 5

(M+H)

355.2233, found 355.2225.

e k j

S

M e h HO o d O i b N-Boc-3,4-Dehydro-L-Prolyl-N-Methyl-Leucine (compound 57a). Compound 57 (0.13 gram, 0.37 millimole) was dissolved in THF/H 2 0 5 milliliters). LiOH*H 2 0 -86- WO 01/76616 PCT/USo01/11607 I (0.15 gram, 3.7 millimoles) was added to the solution at o 0 C. The mixture was stirred for about 6 hours and monitored by TLC. When the reaction was completed, THF was removed in vacuo. Saturated NaHCO 3 solution was added and washed with ether twice. All aqueous layers were combined and acidified to pH 4 with 1 normal K1SO 4 Ethyl acetate was used to C 5 extract the acidified aqueous solution three times. The extracts were dried and concentrated to afford the desired acid (compound 57a) in 92% yield. The following analytical data were D obtained for compound 57a: Rf 0.20 (40% Acetone/Hexane); [a]2 +52.3 CHC1 3 1

H

NMR (500 MHz, CDCl 3 8 Ha) 0.85-1.05 rm, 6H), Hb) 1.48 rm, 9H), Hc) Hd) 1.65- 1.92 3H), He) 3.12 rm, 3H), Hf) br, 1H, Hg) 4.10-4.28 2H), Hh) 5.15 1H), Hi) 5.35 IH), Hj) 5.70 (dt, 1H), Hk) 6.01 (dd, 1H); 13 C NMR (125 MHz, CDCI 3 8 Ca) 23.3, C-I 24.5, Cc) 25.0, Cb) 28.3, Cd) 31.5, Cg) 38.5, Ce) 53.6, Ci) 55.8, Ch) 65.2, C) 80.0, Cj) 124.1, Ck) 129.5, Cm) 153.8, Cn) 171.3, Co) 175.3; IR (neat) 3300 2957 1731 1783 1667 1612 1481 1400 1367 1177 1136.7 HRMS m/z calculated for C 17

H

2 8

N

2 0 5 341.2076, found 341.2063.

jij OMe i r a f I k 0 1 1dd a ee kk O fwkk 0 e tk d NH ff k 0Me n cc bb kk s s hh kkMe kk. s O 0H kk H Y 'aa Nc kk qlNH b O k z pk gg9 0 gg h 0 0 mm ga M 9h a a a a a Cyclo[N-(N-Boc-3,4-dehydro-L-prolyl-N-methyl-D-leucyl)-O[[N-[(2S,3S,4S)- 4[(3S,4R,5S)-4-amino-3-hydroxy-5-methyl-heptanoyl]oxy-3-oxo-2,5-dimethylhexanoyl]-Lleucyl]-L-prolyl-N,O-dimethyl-L-tyrosyl]-L-threonyl] (compound 58). The crude acid 57a (5.0 milligrams, 0.012 millimole) was combined with the macrocycle HCI salt (14.9 milligrams, 0.012 millimole) in CH 2

C

2 (0.1 milliliter) at 0 0 C. HATU (48 milligrams, 0.012 millimole) and DIEA (0.048 milliliter, 0.048 millimole) was added. The reaction was stirred at -87- WO 01/76616 PCT/USO 1/11607 IND 0°C overnight and monitored by TLC. When the reaction was complete, the mixture was 0 diluted with Et 2 0 and the organic layer was washed with 5% HCI, 5% NaHCO 3 and brine.

The resulting solution was dried and concentrated. The crude residue was purified by column Q chromatography, eluting with 5% MeOH/CH 2 CI2 to afford the desired product (compound 58) in 72% yield. The following analytical data were obtained for compound 58: Rf 0.40 MeOH/CH 2 Cl 2 [a] 5 -83.2 CHCI 3 IH NMR (500 MHz, CDCl 3 8 Ha) 0.85-0.97 '0 24H), Hb) 1.17-1.27 3H), Hc) 1.45 9H), Hd) 1.40 3H), He) 1.43 3H), Hf) ID 1.46-1.50 1H) and 1.61 1H), Hg) 1.68-1.73 1H), Hh) 1.74-1.79 1H), Hi) 1.82- 1.88 2H), 2.02-2.08 1H) and 2.11-2.17 2H), Hj) 2.34-2.37 1H), Hk) 2.57 (s, 0 3H), Hi) 2.63 (dd, IH) and 2.95 I1H), Hm) 3.18 and 3.39 (dd, 1H), Hn) 3.23 3H), Ho) N 3.57-3.61 2H), Hp) 3.33 1H), Hq) 3.69-3.71 11), Hr) 3.80 3H), Hs) 4.05-4.14 (m, 2H) and 4.36-4.48 2H), Ht) 4.27 1H), Hu) 4.56 (dd, 1H), Hv) 4.65 (dd, 1H), Hw) 4.81 (t, 1H), Hx) 5.19 1H), Hy) 5.35 (dd, 1H), Hz) 5.42 (dd, 1H), Haa) 5.61 (dd, 1H), Hbb) 5.73-5.75 IH), Hec) 6.11 (dd, IH), Hdd) 6.85 2H), Hee) 7.08 2H), Hff) 7.20 1H), Hgg) 7.54 1H), Hhh) 7.78 1H); 13C NMR (125 MHz, CDCl 3 8 Ca) 11.7, 14.7, 15.2, 16.3, 16.9, 18.6, 20.1 and 20.9, Cd) 23.3, Ce) 23.7, Ca) 24.8 and 27.2, Ci) 24.9, 25.0 and 33.9, C) 27.9, Cc) 28.6, Cg) 31.2, Ch) 31.3, Cj) 33.9, CO) 36.2, Cm) 38.7, Ck) 38.8, Cn) 41.3, Co) 46.9, Cq) 49.46, Cs) 49.52, 53.0, and 67.9, Cr) 55.3, Cv) 55.4, Cw) 55.5, Cu) 57.2, Cy) 57.6, Ct) 63.9, Caa) 65.9, Cp) 66.5, Cx) 70.4, Cz) 81.5, Cz) 81.7, Cdd) 114.1, Ccc) 124.0, Cbb) 129.1, Cii) 130.0, Cee) 130.3, Cjj) 158.6, Ckk) 168.6, 169.3, 170.5, 170.6, 171.3, 171.5, 172.4, 173.9, 204.9; IR (neat) 3337 2959 2870 1733 1645 1640 1543 1514 1454 1407 1368 1302 1248 1171 cm-l; HRMS m/z calculated for

C

5 9

H

9 1

N

7 0 15 (M+Na) 1160.6471, found 1160.6415.

88- WO 01/76616 PCTUS0O1/11607 a I ddr a 0 0 1 e d h fw kk tk 0 Ot d S kkMe s OO s v O kk. ss c-I DH %Ia 4

S

11 kk HihhY

N

J kk i NH b 0 H n z O P 99 h a m o a a a b Cyclo[N-(N-3,4-dehydro-L-prolyl-N-methyl-lI-leucyl)-O[[N-[(2S,3S,4S)- 4 [(3S, 4 R,5S)-4-amino-3-hydroxy-5-methyl-heptanoyl]oxy-3-oxo-2,5-dimethylhexanoyl]-Lleucyl]-L-prolyl-N,O-dimethyl-L-tyrosyl]-L-threonyl] (compound 59). Compound 58 (4 milligrams) was dissolved in HC1*dioxane (0.5 milliliter) and stirred at room temperature.

When the reaction was completed, the solvent was removed in vacuo. Toluene was added twice, and the solution was concentrated. The residue was dried in vacuo ovemrnight to afford the desired HCI salt in 98%. The HCI salt was dissolved in EtOAc, washed with saturated NaHCO 3 and the organic layer washed again with brine, dried over Na 2

SO

4 and concentrated 0 to afford the desired product (compound 59) in 75% yield. The following analytical data were obtained for compound 59: Rf 0.20 (10% MeOH/CH 2 Cl 2 [aD2f -242.3 CHCl 3 1H NMR (500 MHz, CDCl 3 8 Ha) 0.79-0.97 24H), Hb) 1.12-1.23 3H), Hd) 1.32-1.40 (d, 3H), He) 1.43 3H), Hf) 1.46-1.50 1H) and 1.61 1H), Hg) 1.68-1.73 1H), Hh) 1.74-1.79 1H), Hi) Hj) 1.82-1.88 2H), 2.02-2.08 1H) and 2.11-2.17 3H), Hk) 2.25 3H), Hi) 2.51 2H), Hm) 3.18 and 3.39 (dd, 1H), Hn) 2.95 3H), Ho) 3.57-3.61 (m, 2H), Hp) 3.33 1H), Hq) 3.69-3.71 Hr) 3.73 3H), Hs) 3.90-4.18 (mn, 2H), H) 4.49 1H), Hu) 4.56 (dd, 1H), H) 4.65 (dd, 1H), H) 4.81 1H), Hx) 5.19 3H), Hbb) Haa) 5.73-5.75 3H), Hec) Hz)6.01 (dd, 3H), Hdd) 6.85 2H), Wee) 7.08 2H), Hff) 7.20 (d, 1H), Hgg) 7.54 1H), Hhh) 7.78 1H); 1 3 C NMR (125 MHz, CDC 3 8 Ca) 11.7, 14.7, 15.2, 16.3, 16.9, 18.6, 20.1 and 20.9, Cd) 23.3, Ce) 23.7, Ca) 24.8 and 27.2, Ci) 24.9, 25.0 and 33.9, Cf) 27.9, Cc) 28.6, Cg) 31.2, Ch) 31.3, Cj) 33.9, C) 36.2, Cm) 38.7, C) 38.8, Cn) 41.3, Co) 46.9, Cq) 49.46, Cs) 49.52, 53.0, and 67.9, Cr) 55.3, Cv) 55.4, Cw) 55.5, Cu) 57.2, Cy) 57.6, Ct) -89- WO 01/76616 PCT/US01/11607 I 63.9, Caa) 65.9, Cp) 66.5, Cx) 70.4, Cz) 81.5, Cdd) 114.1, Ccc) 124.0, Cbb) 129.1, Ci) 130.0, Cee) 130.3, Cjj) 158.6, Ckk) 168.6, 169.3, 170.5,171.3, 171.5, 172.4, 173.9, Cu) 204.9; IR (neat) 3337 2958 2861 1734 1642 1638 1547 1514 1451 1385 1243 1166 cmur; HRMS m/z calculated for C 54 Hg 3

N

7 0 13

(M+H)

CN 5 1038.6127, found 1038.6103.

INO Example 7 Synthesis and Biological Evaluation of Didemnin Photoaffinitv Analogs t Two proteins which bind with didemnins have been identified, namely, 0 eukaryotic elongation factor 1 alpha (EF-la; Crews et al., 1994 J. Biol. Chem. 269:15411c0 15414) and palmitoyl protein thioesterase 1 (PPTI; Crews et al., 1996, Proc. Natl. Acad. Sci.

USA 93:4316-4319). However, the precise structural interaction between didemnin B and these two proteins are not fully understood., The experiments described in this Example were performed in order to generate photo-reactive didemnin derivatives that can be used to investigate binding of the corresponding didemnins with these and other cellular targets, and that analogous photo-reactive tamandarin derivatives can be synthesized.

Synthesis of didemnin analogs designated compounds 403, 404, 405, and 406 is described in this example. These compounds have the structure of formula XXI, wherein R2 is a substituent having the structure of formula III, R 3 is methyl, R 4 is an isoleucine side chain, each of R 5

R

6

R

8 and R 9 is a hydride radical, R 7 is a methoxy radical, R 1 0 is a leucine side chain, X is Y is a hydride radical, and R 1 has the identity shown in Figures 47A, 47B, 47C, and 47D, respectively.

Each of compounds 403, 404, 405, and 406 is a didemnin A derivative, and each of these molecules comprises a 4-benzoylbenzoic acid moiety. This type of benzophenone derivative was selected as the photo-reactive group because of its multiple advantages in photoaffinity labeling, as described (Dorman et al., 1994, Biochemistry 33:5661-5673; Fleming, 1995, Tetrahedron 51:12479-12520). Benzophenones are easily manipulated in ambient light, but are photoactivated by light having a wavelength of about 350 nanometers.

Light having this wavelength does not generally damage proteins, and was used in order to avoid light-induced changes in the target proteins used in these experiments. The activated diradical ofbenzophenone tends to avoid solvent molecules and nucleophiles, and cross-links preferentially at unreactive carbon centers. Because photo-excitation ofbenzophenones is WO 01/76616 PCT/US01/11607 reversible, a given molecule of the analog which is not proximal to the ligand binding site can S undergo several excitation-relaxation cycles without suffering non-specific side reactions. As a result, benzophenones tend to cross-link with greater site-specificity than photophores such as aryl azides, which undergo an irreversible photolysis reaction.

N 5 The side chain portions of compounds 403, 404, 405, and 406 were synthesized and then coupled to an advanced macrolide intermediate as a final step. Figure 48 shows the INO synthesis of the D-leucine moiety which is common to all four analogs. D-Leucine was N converted to its benzyl carbamate (compound 407) using benzyl chloroformate in NaHCO 3 S and water. Compound 407 was dialkylated using dimethyl sulfate in the presence of powdered O 0 KOH, THF, andBu 4

N+HSO

4 Catalytic hydrogenolysis of the carbamate over a Pd/C catalyst 0C in the presence of hydrogen and ethyl acetate:methanol, followed by trapping with HCI in the presence of methanol yielded the amine hydrochloride salt, compound 409. As shown in Figure 49, compound 409 was coupled with 4-benzoylbenzoic acid by maintaining the acid with BOP-C1, a reagent which is generally useful in coupling to N-methyl amines in the presence ofNMM and DMF at 0°C for 30 minutes, and then contacting the reaction product with compound 409. Removal of the methyl group by ester saponification yielded compound 412.

The benzophenone derivative designated compound 410 (see Figure 50) was elongated by BOP-mediated coupling with a glycine methyl ester linker in the presence of BOP, NMM, DMF, and the glycine methyl ester hydrochloride salt. Saponification of the ester compound 413 in the presence of LiOH, water, THF, and methanol, followed by coupling with amine salt 409 in the presence ofBOP-C1, NMM, and DMF, and subsequent saponification in the presence of LiOH, water, THF, and methanol yielded compound 415. Compound 417 was prepared in an analogous fashion, using a 6-aminohexanoic acid methyl ester. Compound 419 was prepared by twice coupling compound 410 with 6-aminohexanoic acid methyl ester prior to coupling with compound 409 and subsequent saponification; Compounds 412, 415, 417, and 419 were coupled with the previously reported macrocyclic salt (prepared as described, Mayer et al., 1994, J. Org. Chem. 59:5192-5205; Mayer et al., 1995, Acta Crystallogr. C51:1609-1614) using the BOP coupling reagent to obtain compounds 403, 404, 405, and 406, respectively, in 68-81% yield.

In order to evaluate their suitability for photoaffinity labeling of the EF-la binding protein, compounds 403, 404, 405, and 406 were assayed for protein biosynthesis -91- WO 01/76616 PCT/US01/11607 ,O inhibition potency. The results of this evaluation are listed in Table 2. The cell-free translation assay system was as described (SirDeshpande et al., 1995, Biochemistry 34:9177-9184; Ahuja et al., J. Med. Chem. 2000 43:4212-4218). In this assay, didemnin B exhibited a half-maximal Sinhibitory concentration (IC 5 0 of 3 micromolar. The results in Table 2 demonstrate that lN 5 protein biosynthesis inhibition potency remains intact even when a large benzophenone moiety is incorporated into the structure of didemnin B. The length of the linker seems to exert a marginal effect, with the two shortest side chains compounds 403 and 404) being nearly I equipotent with didemnin B. Increasing side chain length correlates with modest reductions in t inhibitor potency. These results are consistent with those obtained by others (Sakai et al., 1996, J. Med. Chem. 39:2819-2834; Ahuja et al., J. Med. Chem. 2000 43:4212-4218), and CI demonstrate that protein biosynthesis inhibition is broadly tolerant of side chain modification.

Table 2

IC

50 Compound Coupling Yield (micromolar) Didemnin B 403 68% 404 78% 405 74% 24 406 81% 19 Table 3 lists preliminary results obtained using the NCI-60 tumor cell screen (described in Boyd et al., 1995, Drug Dev. Res. 34:91-109). The data in Table 3 indicate that the benzophenone photoaffinity analogs can be used to study anti-tumor activity of didemnins.

Examination of the 50% growth inhibition parameter (GI 5 0 shows that all four analogs retain potency comparable to didemnin B. In contrast to the results obtained with regard to in vitro protein biosynthesis, there does not appear to be any clear-cut relationship between linker length and activity. However, total growth inhibition (TGI) by the four analogs requires significantly higher concentrations than are required by didemnin B. Interestingly, all four of the analogs exhibit three to ten-fold lower toxicity as measured by the lethal concentration parameter,

LC

5 0 92- WO 01/76616 PCT/USO1/11607 Table 3 Compound Didemnin B 403 404 405 406

GI

50 (nanomolar) 13 3.0 13 4.3 17

TGI

(micromolar) 0.066 0.35 2.0 20.5 5.0

LC

5 0 (micromolar) 3.8 23 19 48 The results of the experiments presented in this Example demonstrate that i benzophenone-containing analogs ofdidemnins can be made by total synthesis. Biological evaluation of the analogs indicates that incorporation ofbenzophenone in the side chain peptide is a feasible strategy for photoaffinity labeling of molecular targets of didemnins.

The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

While this invention has been disclosed with reference to specific embodiments, other embodiments and variations of this invention can be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims include all such embodiments and equivalent variations.

-93-

Claims (6)

1. A composition comprising a tamandarin analog having the structure HNN 0 Q wherein: i) RI is selected from the group consisting of -H, -(tert-butyloxycarbonyl), -leucine, 0 -(N-methyl)leucine, -(N-methyl)leucine-a first fluorophore), -(N-methyl)leucine-proline, -(N-methyl)leucine-proline-actate, -(N-methyl)leucine-proline-pyruvate, -(N-methyl)leucine-proline-lactate--{a first fluorophore), -(N-methyl)leucine-proline-lactate-glutamine-pyroglutamate, -(N-methyl)leucinie-proline-lactate--glutamine-cyclopentanoate, -(N-methyl)leucine-proline-aanine-leucine-pyroglutamate, -(N-methyl)leucine-proline-(N-methyl-alanine)-leucine-pyroglutamate, (N-methyl)Ieucine-eoxo-proline, (N-methyl)leucine-eoxo-proline-lactate, -Q4-methyl)leucine-deoxo-proline-pyruvate, -(N-methyl)leucine-deoxo-proline-lactate-(a first fluorophore), -(N-methyl)leucine-deoxo-proline-lactate-glutaiine-pyroglutamate,

94- WO 01/76616 WO 01/66 16PCT/USO 1/11607 -(N-methyl)leucine-deoxo-proline-lactate-glutanmine-cyclopentanoate, C) -(N-methyl)leucine-deoxo-proline-aanine-leucine-pyroglutamate, S -(N-methyl)leucine-deoxo-proline-(N-methyl-alanine)-leucine-pyroglutaruate, S -(N-methyl)leucine-dehydro-pro line, CI-(N-methyl)leucine-dehydro-proline-lactate, -(N-methyl)leucine-dehydro-proline-pyruvate, -{N-methyl)leucine-dehydro-proline.-lactate-(a first fluorophore), CI-(N-methyl)leucine-dehydro-proline-lactate-glutamine-pyroglutanate, S -Q4-methyl)leucine-dehydro-proline-actate-glutaiine-cyclopentanoate, IND-ehlluire-eyr-rlne-lnn-ecn-prguaae n C-i -(N-methyl)leucine--dehydro-proline(N hy-alanine)-leucine-pyroglutamate ii) either 3 2. R is selected from the group consisting of -CH 3 and and R is selected from the group consisting of an isoleucine side chain, a valine side chain, an alanine side chain, a norleucine side chain, a norvaline side chain, leucine side chain, a histidine side chain, a tryptophan side chain, an arginine side chain, a lysine side chain, a second fluorophore, and a substituent having the structure 8 1 6 or R 2and R 3together are a substituent having the structure 8 6 WO 01/76616 PCTIUSOI/11607 6 78 9 IND iii) each of R R R R and R when present, is independently selected from the group consisting of -OH, -OCR 3 -CO(C 6 H5), -Br, -Cl, -CR 3 ad iv) R 4is selected from the group consisting of an isoleucine side chain and a N- 5 valine side chain; v) X is selected from the group consisting of and IND vi) Y is selected from the group consisting of H and a hydroxyl. protecting cI group; c-I vii) R is selected from the group consisting of a leucine side chain and a [0 lysine side chain; and c-I viii) the molecule is not tamandarin A. 2. The composition of claim 1, wherein R is selected from the group consisting of -H, (tert-butyloxycarbonyl), -leucine, (N-methyl)leucine, -(N-methyl)leucine-(a first fluorophore), !O -(N-methyl)leucine--(S)proline, -(N-methyl)leucine-(S)proline-(S)lactate, -(N-methyl)leucine-(S)prolinc-pyruvate, -(N-methyl)leucine-(S)proline-(S)lactate-(a first fluorophore), -(N-methyl)leucine-{S)proline-{S)lactate-glutamine-pyroglutamate, -(N-methyl)leucine-(S)proline-(S)lactate-glutamine-cryclopentanoate, (N-methyl)leucine-(S)proline-alanin.-eucine-pyroglutamate -(N-methyl)leucine-(S)proline-(N-methyl- alanine)-leucie-pyroglutamate, -(N-methyl)eucine-deoxo-(S)proline, -(N-methyl)leucine-eoxo-(S)proline-{S)actate, -(N-methyl)leucine-deoxo-(S)proline-pyruvate, -{N-methyl)Ieucine-eoxo-(S)proline-(S)Iactate-(a first fluorophore), -(N-methyl)leucine-deoxo-(S)proline-(S)lactate--glutamine-pyroglutamate,

96- WO 01/76616 WO 0176616PCT[USO1/ 11607 IN -N-methyl)leucine-deoxo-(S)proin-aSIanineecnglutamylpate, t -(N-methyI)leucine-deoxo(S)proline-(N-methyaanine)leucipyroguaate, -(N-methyl)eucin--ehydro-(S)proline, -{N-Inethyl)leucine-dehydro-(S)proline-{S)lactate, -(N-methyl)leucine-dehydro-(S)proline-pyruvate, ID -{N-methyl)Ieucine-dehydro-(S)proline-(S)lactate-(a first fluorophore), INDe h l l u i e d h d r S p o i e a t t g u a j e p r g u a a e -(N-methyl)leucine-debydro-(S)proline-(S)lactate--glutamine-pyrolpetanate, C) 0 -(N-methyl)leucine-dehydro-(S)proline-alanine-leucine-pyroglutamate, and Nl -(N-methyl)Ieucine-dehydro-(S)proline-(N-methyl-alanine)-Aeucine-pyroglutaniate. 3. The composition of claim 1, wherein R is selected from the group consisting of -H, -(tert-butyloxycarbonyl), -leucine, -(N-methyl)leucine, -(N-methyl)leucine-(a first fluorophore), 0 -(N-methyl)leucine-(S)proline, (N-methyl)leucine-(S)proline--S)lactate, -(N-methyl)leucine-(S)proline-pyruvate, -(N-methyl)leucine-(S)proine-(S)actae-({a first fluorophore), -(N-methyl)leucine-{S)proline-(S)lactate.-(S)glutaniine-.S)pyroglutunate, (N-methyl)leucine-(S)proline-(S)lactate--(S)glutamine.-(S)cyclopentanoate, -(N-niethy)leucine-(S)proline-(S)alainine..S)Ieucine-(S)pyroglutamate, -(N-methyl)leucine-(S)proline-{N-methyl-S-alanine)-(s)leucine-{S)pyroglutamate, -(N-methyl)leucine-deoxo-(S)proline, -(N-metbyl)leucine-deoxo-(S)proline-S)actate, -(N-methyl)leucine-deoxo-(S)proline-pyruvate, -(N-methyl)leucine-deoxo-(S)proline-(S)actate-{a first fluorophore), (N-methyl)leucine-deoxo-(S)proline-(S)actate-(S)glutamine-{S)pyroglutamate, -97- WO 01/76616 WO 0176616PCT/USO 1/11607 IND -(N-methyl)Ieucine-deoxo-(S)proineS)actate(S)gutie-(S)cyclopentaoate, -(N-methyl)leucinedeoxo-(S)proine(S)aanine(S)euci-S)pyoglutmate, and -{N-xnethyl)leucine-dehydro-(S)proline, N- 5 -(N-methyl)leucinedehydro-(S)proline-(S)lactate, -(N-metbyl)eucine-deoxo-(S)proline-pyruvate, IND -(N-methyl)leucine-deoxo-(S)proline-(S)lactate-(a first fluorophore), N- -(N-methyl)eucine-deoxo-(S)proline-(S)1actate-(S)glutamine-(S)pyroglutamate, t~t--(N-methyl)leucine-deoxo-(S)proline-(S)1actate-(s)glutamne-S)cyclopentaoate, ID 10 -(N-metbyl)leucine-deoxo-(S)proline-(S)alanine--(S)leucine-(S)pyroglutamate, and ci-(N-methy)leucinedeoxo-(S)prone(N-methy-S-aanine)(S)euciS)progutmae. 2 4. The composition of claim 1, wherein R is R 6 R R6 R6 36 45 8 9. R3 is methyl,R is an isoleucine side chain, each ofR5 ,R ,R andR isa hydride radical, R7 is methoxy, RI is a leucine side chain,: X is and Y is a hydride radical. The composition of claim 1, wherein the tamandarin analog is compound

201. 6. The composition of claim 1, wherein the tamandarin analog is compound

203. 7.The composition of claim 1, wherein R Iis -(N-methyl)leucine-deoxo- (S)proline-Iactate. -98 WO 01/76616 PCT/USO1/11607 8. The composition of claim 1, wherein R is -(N-methyl-R-leucine). U 1 9. The composition of claim 1, wherein R is -(N-methyl-R-leucine)-- cl i (S)proline-(S)lactate-(S)glutamine-(S)pyroglutamate, Y is and X is IN 10. The composition of claim 9, wherein the tamandarin analog is selected from the group consisting of compound 103 and compound 104. ND 4 S11. The composition of claim 1, wherein R is a valine side chain, Y is CN and X is 12. The composition of claim 11, wherein the tamandarin analog is selected from the group consisting of compound 105, compound 106, compound 116, compound 120, compound 123, compound 141, and compound 142. 13. The composition of claim 1, wherein R 1 is -(N-methyl)leucine-(S)proline- (S)lactate-(a first fluorophore). 14. The composition of claim 13, wherein the tamandarin analog is selected from the group consisting of compound 107, compound 108, compound 109, and compound 110. The composition of claim 1, wherein R is -(N-methyl)leucine-(S)proline- pyruvate. 16. The composition of claim 15, wherein the tamandarin analog is selected from the group consisting of compound 133 and compound 134. -99- WO 01/76616 PCT/US01/11607 17. The composition of claim 1, wherein Y is and wherein R 2 has the structure 5 2 18. The composition of claim 1, wherein R is a lysine side chain and Y is -H. 19. The composition of claim 1, wherein the tamandarin analog has the following structure, wherein FL is a fluorophore The composition of claim 1, wherein X is 21. The composition of claim 1, wherein the tamandarin analog is selected from the group consisting of compound 124, compound 125, compound 126, and compound 143. 22. The composition of claim 1, wherein the tamandarin analog is selected from the group consisting of compound 115, compound 117, compound 118, compound 119, compound 121, compound 122, compound 127, compound 128, compound 129, compound 130, compound 136, compound 137, and compound 139. -100- WO 01/76616 PCT/US01/11607 IND) 23. The composition of claim 1, further comprising a pharmaceutically acceptable carrier. 24. A support having the tamandarin analog of claim 1 covalently attached N 5 thereto. IND 25. A method of inhibiting protein synthesis in a cell, the method comprising radministering the composition of claim I to the cell. INDI 26. A method of inhibiting growth of a cell, the method comprising i administering the composition of claim I to the cell. 27. A method of inhibiting proliferation of a cell, the method comprising administering the composition of claim 1 to the cell. 28. A method of inhibiting tumorigenesis in a cell, the method comprising administering the composition of claim 1 to the cell. 29. A method of enhancing apoptosis of a cell, the method comprising administering the composition of claim 1 to the cell. A composition comprising a compound having a structure selected from the group consisting of (a) IR2 R 1 2 N HN 0 0 N0N 0 OY 101 WO 01/76616 WO 0176616PCTJUSO 1/11607 and d) wherein: i) either a) R 3is selected from the group consi sting of -CH 3 and and R 2is selected from the group consisting of an isoleucine side chain, a valine side chain, an alanine side chain, a norleuicine side chain, a norvaline side chain, a proline side chain, leucine side -102- WO 01/76616 PCT/US01/11607 chain, a histidine side chain, a tryptophan side chain, an arginine side chain, a lysine side chain, 0 a second fluorophore, and a substituent having the structure a R 6 C R7 R9 R 8 n; or N R and R together are a substituent having the structure R7 R 8 R R R 5678 9 ii) each of R R 6 R R and R when present, is independently selected from the group consisting of-H, -OH, -OCH 3 -CO(C6H5), -Br, -Cl, -CH 3 and iii) R is selected from the group consisting of an isoleucine side chain and a valine side chain; iv) X is selected from the group consisting of-O- and v) Y is selected from the group consisting of-H and a hydroxyl protecting group; vi) R 0 is selected from the group consisting of a leucine side chain and a lysine side chain; and vii) R 3 is an enzyme-cleavable moiety that is cleavable by an enzyme selected from the group consisting of a carboxypeptidase, a beta-lactamase, a beta-galactosidase, a penicillin V-amidase, a cytosine deaminase, a nitroreductase, a alkaline phosphatase, a beta- glucuronidase, and a catalytic antibody. 31. The composition of claim 30, wherein R 13 has the structure -103- WO 01/76616 PCTIUS01/11607 O 11 H NO O 32. The composition of claim 30, wherein R 13 has the structure IND 33. A method of inhibiting protein synthesis in a cell, the method comprising administering the composition of claim 30 to the cell. 34. A method of inhibiting growth of a cell, the method comprising Iadministering the composition of claim 30 to the cell. A method of inhibiting proliferation of a cell, the method comprising administering the composition of claim 30 to the cell. 36. A method of inhibiting tumorigenesis in a cell, the method comprising administering the composition of claim 30 to the cell. 37. A method of enhancing apoptosis of a cell, the method comprising administering the composition of claim 30 to the cell. -104- WO 01/76616 WO 0176616PCT/USOI/I 1607 IND 38. A composition comprising a dideminin analog having the structure R 3 N N R00 0 10 "0 0 NH 0 0N0 IN 0 N-R H NH c-I x wherein: i) RI is selected from the group consisting of -H, (tert-butyloxycarbonyl), -leucine, -(N-methyl)Ieucine, (N-methyl)leucine-(a first fluorophore), -(N-methyl)leucine-proline, -{N-methyl)leucine-proline-lactate, -(N-methyl)leucine-proline-pyruvate, -(N-methyl)Ieucine-proline-lactate-(a first fluorophore), -(N-methyl)leucine-proline-lactategutamine-pyroglutamate, -(N-methyl)leucine-proline-lactate-glutamine-cyclopentanoate, -(N-metbyl)leucine-proline-alanine-leucine-pyroglutamate, -(N-methyl)leucine-proline-(N-methyl-alanine)-leucine-pyroglutamate, -(N-methyl)leucine-deoxo-proline, -(N-methyl)leucine-deoxo-proline-lactate, -(N-methyl)leucine-deoxo-proline-pyruvate, -(N-methyl)leucine-deoxo-proline-lactate-(a first fluorophore), -(N-methy1)leucine-deoxo-proine-actate-glutamine-pyroglutaniate, {N-methyl)Ieucine-deoxo-proline-lactate-glutamine-cyclopentanoate, -105- WO 01/76616 WO 0176616PCTIUS01/1 1607 ID -(N4-methyl)leucine-deoxo-proline-alanine-leucine-pyroglutam' ate, C) -(N-rnethyl)leucine-deoxo-proline-(N-methy-aline)-leucinepyroglutmate, S -4§N-methyl)Ieucine-dehydro-proline, S -(N-methyl)leucine-dehydro-proline-lactate, CI -(N-methyl)leucine-dehydro-proline-pyruvate, -(N-methyl)leucine--ehydro-proline-lactate-(a first fluorophore), INDO -(N-methyl)leucine-dehydro-proline-lactate-glutamine-pyroglutamate, CI -(N-methyl)leucin--ehydro-proline-1actate-glutamine-cyclopentanoate, S -(N-methyl)leucine-ehydro-proLine-alanine-leucine-pyroglutaniate, and I-{N-methyl)leucine-dehydro-proline-(N-methyl-alanine-ieucine-pyroglutamate; ii) either 3 2. R is selected from the group consisting of -CH 3 and and R is selected from the group consisting of an isoleucine side chain, a valine side chain, an alanine side chain, a norleucine side chain, a norvaline side chain, leucine side chain, a histidine side chain, a tryptophan side chain, an arginine side chain, a lysine side chain, a second fluorophore, and a substituent having the structure R8R R or R 2and R 3together are a substituent having the structure -106- WO 01/76616 PCTIUSOI/i 1607 5 6 78 9 IND iii) each of R R R R and R when present, is independently selected Sfrom the group consisting of -01H, -OCH 3 -CO(C 6 H 5 -Br, -Cl, -CH 3 and iv) R 4is selected from the group consisting of an isoleucine side chain and a CI valine side chain; v) X is selected from the group consisting of and IND vi) Y is selected from the group consisting of -H and a hydroxyl protecting group; vii) Ri is selected from the group consisting of a leucine side chain and a S lysine side chain; and c-i viii) the molecule is not tamandanin A. 39. The composition of claim 38, wherein R is selected from the group consisting of -H, -(tert-butyloxycarbonyl), -leucine, -(N-methyl)leucine, -(N-nlethyl)leucine-(a first fluorophore), -(N-methyl)leucine4S)proline, -{N-methyl)leucineS)proie-S)lactte, -(N-metby)leucine{(S)prolepyvate, -(N-methyl)leucine-S)proline-(S)1acttea first fluorophore), -(N-methyl)leucine-<S)proline-.{anie-hy1aline-olutaine--yol, mt N-methyl)leucine-e(Sroli j-N-ethl lnn)luieprguaa -(N-methy)leucinedeoxo(S)prolinSIat -(N-methyl)leucinedeoxo(S)prolinepnvte, -{N-methyl)leucinedeoxo(S)prolin-S)1cttea first fluorophore), -107- WO 01/76616 WO 0176616PCT[USO 1/11607 ID -(N-methyl)leucine-deoxo-(S)proline(s)Iactatgutmn-ycopentanoate, -(N-methyl)leucine-deoxo-(S)proineaaineleucin-pyoglutamate, S -(N-Inethyl)leucine~dehydro-(S)proline, NI -(N-methyl)leucine-dehydro-(S)proine-(S)actate, -(N-methyl)leucine-dehydro-(S)proline-pyruvate, -(N-methyl)leucine-dehydro-(S)proline-(S)lactate-<a first fluorophore), CI-(N-methyl)leucine-dehydro-(S)proine(s)actategutie-pyroglutamate, S -(N-methyl)leucine-dehydro-(S)proine-S)Iac-ltatetcyclopentaoate, C) -(N-methyl)leucine--dehydro-(S)proineaanineleucinepyroglutrate, and Ni -(N-methyl)leucine--dehydro-(S)proine-Nmethylalanine)-leucine-pyoglutamate. The composition of claim 38, wherein R is selected from the group consisting of -H, -(tert-butyloxycarbonyl), -leucine, -(N-methyl)leucine, -(N-methyl)leucine-(a first fluorophore), -(N-methyl)leucine-(S)proline, -(N-inethyl)leucine--(S)proline--(S)Iactate, -(N-methyl)leucine-(S)proline-pyruvate, -(N-methyl)leucine-(S)proline-(S)Iactate-{a first fluorophore), -(N-methy)leucine-{S)proline-(S)actate-S)glutamine...S)pyroglutamate, N-methyl)leucine-(S)proline-(S)1actate-(S)glutamine-..S)cyclopentaoate, -{N-methyl)leucine-(S)proine-(S)alanine(S)eucine(S)pyroglutamte, and -{N-methyl)leucine-(S)prolinei-{N-methy-S-alanine-(S)leucine-(S)pyogutamate, -(N-methyl)leucine-deoxo-(S)proline, -{N-methyl)leucine-deoxo-(S)proine.-(S)actate, -{N-methyl)leucine-deoxo-(S)proline-pyruvate, -(N-methyl)leucine-deoxo-(S)proline--{S)lactate--{a first fluorophore), -(N-methy)leucine-deoxo-(S)proline-(S)lactate--{S)glutamine...S)pyroglutamate, -108- WO 01/76616 WO 0176616PCT/IJSOI/1 1607 C-{N-methyl)leucine-deoxo-(S)proline-(S)lactate-(S)glutamine-(S)cyclopentanoate, C) -(N-methyl)leucine-deoxo-(S)proline-S)alanine-(S)eucine(S)pyroglutamate, U -{N-methyl)leucine-deoxo-(S)proline-{N-methyl-S-alanine)-(S)leucine-(S)pyroglutaniate, S -(N-methyl)leucine-dehydro-(S)proline, S -(N-methyl)leucine-dehydro-(S)proline-(S)lactate, -{N-methyl)leucinie--ehydro-(S)proline-pyruvate, S -{N-methyl)leucine-dehydro-(S)proline-(S)actate-(a first fluorophore), S-(N-methyl)leucine-dehydro-(S)proline-(S)actate-(S)glutamine-(S)pyroglutamate, ci -(N-methyl)leucine-dehydro-(S)proline-(S)actate-(S)glutamin-{S)cyclopentanoate, IND Nmty~ecn-eyr-(~rln-(~lnn-Slecn(~yoltrae n -(N-metbyl)leucine-dehydro-(S)proline-(N-manety-Slanine--(S)ecnSpyroglutamate 2 41. The composition of claim 38, wherein R is 7 106 hydride radical, R7 is meffioxy, RI is a leucine side chain, X is and Y is a hydride radical. 42. The composition of claim 38, wherein the didemnin analog is compound 202. 43. The composition of claim 38, wherein the didemnin analog is compound

204. 44. The composition of claim 3 8, wherein R is -(N-methyl)leucine-deoxo- (S)proline-lactate. -109- WO 01/76616 PCT/US01/11607 The composition of claim 38, wherein Y is and wherein R 2 has the structure 46. e composition of claim 38, wherein R 2 is a ysine side chain and Y is -H. 46. The composition of claim 38, wherein R is a lysine side chain and Y is -H. 47. The composition of claim 38, wherein X is 48. acceptable carrier. The composition of claim 38, further comprising a pharmaceutically 49. A support covalently attached with the didemnin analog of claim 38. A method of inhibiting protein synthesis in a cell, the method comprising administering the composition of claim 38 to the cell. 51. A method of inhibiting growth of a cell, the method comprising administering the composition of claim 38 to the cell. 52. A method of inhibiting proliferation of a cell, the method comprising administering the composition of claim 38 to the cell. 53. A method of inhibiting tumorigenesis in a cell, the method comprising administering the composition of claim 38 to the cell. -110- WO 01/76616 PCT/USOI/11607 I 54. A method of enhancing apoptosis of a cell, the method comprising O administering the composition of claim 38 to the cell. In a method of making a didemnin analog, the improvement comprising C incorporating a deoxo-proline residue in place of a proline residue of the analog. I 56. The improvement of claim 55, wherein the analog comprises an (N- N methyl)leucine-proline moiety and wherein the (N-methyl)leucine-proline moiety is replaced S by an (N-methyl)leucine-deoxo-proline moiety. NO S57. The improvement of claim 56, wherein the (N-methyl)leucine-deoxo- proline moiety is made by reducing the ester function of proline to an aldehyde function; and coupling the proline with the (N-methyl)leucine moiety by reductive amination to yield the (N-methyl)leucine-deoxo-proline moiety. 58. The improvement of claim 57, wherein the amine moiety of the proline is protected with an amine-protecting group prior to the reductive amination. 59. The improvement of claim 57, wherein the ester function of the proline is reduced to an aldehyde function by contacting the proline with a strong base and then contacting the proline with an oxidizing agent. The improvement of claim 57, wherein the reductive amination is performed in a non-aqueous solvent in the presence of a strong base and a carboxylic acid catalyst. 61. In a method of making a didemnin analog, the improvement comprising incorporating a dehydro-proline residue in place of a proline residue of the analog. -111- WO 01/76616 PCT/USO1/11607 I0 62. The improvement of claim 61, wherein the analog comprises an (N- O O methyl)leucine-proline moiety and wherein the (N-methyl)leucine-proline moiety is replaced 0 by an (N-methyl)leucine-dehydro-proline moiety. 63. The improvement of claim 61, wherein the dehydro-proline residue is made by protecting the carboxyl and amino moieties of 4-hydroxyprolinate, alkyl-sulfonylating the N 4-hydroxyl moiety, displacing the alkyl-sulfonate moiety with an aryl-selenyl moiety, S oxidatively eliminating the aryl-selenyl moiety to yield a dehyro-proline moiety having C- protected carboxyl and amine moieties, and coupling the dehydro-proline moiety with an 0) amine moiety of the analog. 64. The improvement of claim 61, wherein the alkyl-sulfonate moiety is a methyl-sulfonate moiety. 65. The improvement of claim 61, wherein the aryl-selenyl moiety is a phenyl- selenyl moiety. 66. The improvement of claim 61, wherein the 4-hydroxyprolinate is trans-4- hydroxyprolinate. 67. A composition comprising a didemnin fragment having the structure OY O R4 X -1 HN, 0 APG wherein: i) Y is selected from the group consisting of-H and a hydroxyl protecting group; ii) X is selected from the group consisting of-O- and 4 iii) R is selected from the group consisting of an isoleucine side chain and a valine side chain; -112- WO 01/76616 WO 01/76616 PCT/US01/11607 iv) APG is a amine protecting group; and Sv) R 1 is selected from the group consisting of -OH, -NH 2 -O(allyl), O -O(pentafluorophenyl), and a substituent having the structure b) either3 SR2 1 O ^0 wherein: a)RI is selted om group onising of-H, and an am prt g b) either: R is selected from the group consisting of-CH 3 and and R 2 is selected from the group consisting of an isoleucine side chain, a valine side chain, an alanine side chain, a norleucine side chain, a norvaline side chain, a proline side chain, leucine side chain, a histidine side chain, a tryptophan side chain, an arginine side chain, a lysine side chain, a second fluorophore, and a substituent having the structure R R6 R. Rs ;or (II) R and R together are a substituent having the structure -113- WO 01/76616 PCT/US01/11607 R I-N c 5 6 78 9 4 c) each of R 5 R 6 R Rand R 9 when present, is independently selected from N the group consisting of -OH, -OCH 3 -CO(C 6 H 5 -Br, -Cl, -CH 3 and d) R 0 is selected from the group consisting of a leucine side chain, a lysine C-i side chain, and a protected lysine side chain; and 12 e) R 2 is selected from the group consisting of-H, and -2-(trimethylsilyl) ethoxycarbonyl. 68. A method of inhibiting protein synthesis in a cell, the method comprising administering the composition of claim 67 to the cell. 69. A method of inhibiting growth of a cell, the method comprising administering the composition of claim 67 to the cell. A method of inhibiting proliferation of a cell, the method comprising administering the composition of claim 67 to the cell. 71. A method of inhibiting tumorigenesis in a cell, the method comprising administering the composition of claim 67 to the cell. 72. A method of enhancing apoptosis of a cell, the method comprising administering the composition of claim 67 to the cell. -114- WO 01/76616 PCT/US01/11607 IND 73. A method of making a didemnin fragment, the method comprising coupling a first reactant having the structure 0 II x H IND and a second reactant having the structure SOY 0 R4 O OH O HN APG to yield a first didemnin fragment having the structure OY 0 R4, X O oY o HN 0 APG wherein X is selected from the group consisting of-O-, and wherein APG is an amine protecting group; wherein Y is a hydroxyl protecting group; and wherein R 4 is selected from the group consisting of an isoleucine side chain and a valine side chain. 74. The method of claim 73, wherein Y is -triisopropylsilyl. The method of claim 74, further comprising hydrolyzing the first didemnin fragment to yield a second didemnin fragment having the structure OY 0O HN O \APG 76. The method of claim 75, further comprising adding an activator to the carbonyl moiety of the second didemnin fragment to yield a third didemnin fragment having the structure -115- WO 01/76616 PCT/USO1/11607 IO 0 IN N IN 0- APG wherein -ACT is an activator. 77. The method of claim 76, further comprising coupling the third didemnin fragment and a third reactant having the structure 3 10 2 O3 -R20 /SEM 1 G ONA NH 2 H to yield a fourth didemnin fragment having the structure .APG HN-APG 0Xo R4 O O wherein: i) either R 3 is selected from the group consisting of -CH 3 and and R 2 is selected from the group consisting of an isoleucine side chain, a valine side chain, an alanine side chain, a norleucine side chain, a norvaline side chain, a proline side chain, leucine side chain, a histidine side chain, a tryptophan side chain, an arginine side chain, a lysine side chain, a second fluorophore, and a substituent having the structure -116- WO 01/76616 WO 01/66 16PCT/JSOI/1 1607 ;or 2 3 R and R together are a substituent having the structure ii) each of R 5, R 6 R 7, R 8, and R 9, when present, is independently selected from the group consisting of -OH, -OCH3, -CO(C 6 H4 5 -Br, -Cl, -CH 3 and -C2115; iii) APQ is an amine protecting group; iv) SEM is 2-(trinaethylsilyl)ethoxycarbonyl and iii) RI is selected from the group consisting of a leucine side chain and a lysine side chain. -117- WO 01/76616 PCT/US01/11607 78. A method of making a didemnin analog, the method comprising removing the SEM and CBZ moieties from the fourth didemnin fragment of claim 46 and cyclizing the fourth didemnin fragment to yield a first didemnin analog having the structure R3 1 2 H N ^N-APG 79. The method of claim 78, further comprising removing the APG group from the first didemnin analog to yield a second didemnin analog having the structure 80. The method of claim 78, wherein the APG group is selected from the group consisting of a carbobenzyloxy moiety and a tert-butyloxycarbonyl moiety. 81. The method of claim 79, further comprising coupling the second didemnin analog and a fourth reagent having the structure H R 1 HO R14 118- WO 01/76616 WO 0176616PCTUSO 1/11607 IND to yield a third didemin analog having the structure U3 H 1 N 0 N-R 1 010L HN H 0- 0 QY c-i 14 IC wherein R is selected from the group consisting of 0 -leucine, -{N-methyl)leucine, -(N-methyl)leucine--a first fluorophore), {N-methyl)leucine-proline, -{N-methyl)leucine-proline-lactate, -{N-methyl)leucine-proline-pyruvate, -(N-methyl)Ieucine-proline-lactate-(a first fluorophore), -{N-methyl)leucine-proline--lactate--glutamine-pyroglutamate, -(N-methyl)leucine-proline-lactate-glutamine-cyclopentanoate, -{N-methyl)Ieucine-proline-alanine-leucine-pyroglutamate,. -(N-methyl)leucine-prolin-(N-methyl-alanine)-eucin--yroglutamate, -(N-methyl)leucine-deoxo-proline, (N-methyl)leucine-deoxo-proline-lactate, -(N-methyl)leucine-deoxo-proline-pyruvate, -(N-methyl)leucine-deoxo-proline-lactate-(a first fluorophore), -(N-methyl)leucine-deoxo-proline-lactate-glutamine-pyroglutamate, -(N-methyl)leucine-eoxo-proline--lactate-glutaniine-cyclopentanoate, -{N-methyl)leucine-deoxo-prolin-aanine-leucine-pyroglutanate, -(N-methyl)leucine-eoxo-proline-(N-methyl-alanine)-leucine-pyroglutaniate, {N-methyl)leucine-dehydro-proline, -(N-methyl)leucine-dehydro-proline-lactate, -(N-methyl)leucine-dehydro-proline-pyruvate, -119- WO 01/76616 PCT[USO1/11607 -(N-methyl)leucine-dehydro-proline-lactate-{a first fluorophore), -(N-methyl)leucinedehydro-proline-lactate-gutjpyroglutaate, -(N-methyl)leucine-dehydro-proline-lactate glutamine-cyclopentanoate, C -(N-methyl)Ieucine-dehydro-proline-alanineeuci oghtmate, and i -(N-methyl)leucine-dehydro-proine-{N-methy-alanine)-leucine-pyroglutamate, IND and C one of these moieties coupled with an enzyme-cleavable moiety that is cleavable by an enzyme selected from the group consisting of a carboxypeptidase, a beta-lactanase, a beta- cIN galactosidase, a penicillin V-amidase, a cytosine deaminase, a nitroreductase, an alkaline phosphatase, a beta-glucuronidase, and a catalytic antibody. 82. The method of claim 81, wherein Y is a hydroxyl protecting group, the method further comprising removing the hydroxyl protecting group from the second didemnin analog to yield a fourth didemnin analog having the structure R 3 22 R1O>O O Of HN R 4 1OH -120-