Total synthesis of HUN-7293

Article abstract of DOI:10.1021/ja990918u

The first total synthesis of the cyclic heptadepsipeptide HUN-7293 (1), a potent inhibitor of cell adhesion molecule expression exhibiting anti-inflammatory properties, is detailed. The most effective approach relied on an unusually efficient macrocyclization with the formation of the MLEU³ - LEU⁴ secondary amide that potentially benefits from intramolecular H-bonding preorganization of the acyclic substrate. The requisite linear depsipeptide was convergently assembled with the late stage introduction of the linking ester enlisting a Mitsunobu esterification that occurs with inversion of the DGCN α-center permitting the utilization of a readily available L-amino acid precursor to the D α-hydroxy carboxylic acid residue. An alternative and similarly attractive approach of direct macrolactonization of a substrate necessarily incorporating a D-DGCN subunit proved viable albeit less effective. Biological evaluation in cellular assays for vascular adhesion molecule expression confirmed that synthetic HUN-7923 (1) is essentially indistinguishable from the naturally occurring cyclodepsipeptide.

Full text of DOI:10.1021/ja990918u

J. Am. Chem. Soc. 1999, 121, 6197-6205
6197
Total Synthesis of HUN-7293
Dale L. Boger,*,† Holger Keim,† Berndt Oberhauser,‡ Erwin P. Schreiner,‡ and
Carolyn A. Foster‡
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037, and NoVartis Forschungsinstitut,
Brunner Strasse 59, A-1235, Vienna, Austria
ReceiVed March 22, 1999
Abstract: The first total synthesis of the cyclic heptadepsipeptide HUN-7293 (1), a potent inhibitor of cell
adhesion molecule expression exhibiting anti-inflammatory properties, is detailed. The most effective approach
relied on an unusually efficient macrocyclization with the formation of the MLEU3-LEU4 secondary amide
that potentially benefits from intramolecular H-bonding preorganization of the acyclic substrate. The requisite
linear depsipeptide was convergently assembled with the late stage introduction of the linking ester enlisting
a Mitsunobu esterification that occurs with inversion of the DGCN R-center permitting the utilization of a
readily available L-amino acid precursor to the D R-hydroxy carboxylic acid residue. An alternative and similarly
attractive approach of direct macrolactonization of a substrate necessarily incorporating a D-DGCN subunit
proved viable albeit less effective. Biological evaluation in cellular assays for vascular adhesion molecule
expression confirmed that synthetic HUN-7923 (1) is essentially indistinguishable from the naturally occurring
cyclodepsipeptide.
The cyclic heptadepsipeptide HUN-7293 (1, Figure 1) was
first isolated in 1992 from a fungal broth during a screen for
potent inhibitors of inducible cell adhesion molecule expression.
On the basis of its activity in a cell-based enzyme-linked
immunosorbant assay (ELISA), HUN-7293 (1) was purified by
assay-guided fractionation and the two- and three-dimensional
structure was subsequently determined.1 Independently, the same
cyclodepsipeptide was isolated by a Japanese group from a
different fungal species based on a screen for anti-HIV
compounds.2
Macrocyclic natural products often exhibit unique biological
properties and thus are attractive candidates for drug develop-
ment in many disease indications.3 HUN-7293 (1) represents
such a compound class with novel anti-inflammatory actions,
due to its potent inhibition of the vascular cell adhesion molecule
1 (VCAM-1).4 Endothelial cell-associated molecules such as
intercellular adhesion molecule 1 (ICAM-1), VCAM-1, and
E-selectin play a critical role in the immune response by
regulating leukocyte migration and cell-to-cell interactions.5
Modulation of these interactions in vivo by appropriate treatment
with a compound like HUN-7293 (1) could have therapeutic
potential in a variety of inflammatory disorders and autoimmune
diseases.6
† The Scripps Research Institute.
‡ Novartis Forschungsinstitut.
(1) Hommel, U.; Weber, H.-P.; Oberer, L.; Naegeli, H. U.; Oberhauser,
B.; Foster, C. A. FEBS Lett. 1996, 379, 69.
(2) Itazaki, H.; Fujiwara, T.; Sato, A.; Kawamura, Y.; Matsumoto, K.
Jpn Patent, Kokai Tokkyo Koho 07109299, 1995, Appl. 93/255592, 13 Oct.
1993; Chem. Abstr. 1995, 123, 110270.
(3) Schreiber, S. L.; Crabtree, G. R. Immunol. Today 1992, 13, 136.
(4) Foster, C. A.; Dreyfuss, M.; Mandak, B.; Meingassner, J. G.; Naegeli,
H. U.; Nussbaumer, A.; Oberer, L.; Scheel, G.; Swoboda, E.-M. J. Dermatol.
1994, 21, 847.
(5) (a) Springer, T. A. Cell 1994, 76, 301. (b) Ley, K. CardioVasc. Res.
1996, 32, 733. (c) Campbell, J. J.; Hedrick, J.; Zlotnik, A.; Siani, M. A.;
Thompson, D. A.; Butcher, E. C. Science 1998, 279, 381.
Figure 1.
HUN-7293 (1) is a cyclodepsipeptide containing six L-amino
acid residues and a D R-hydroxy carboxylic acid residue
(DGCN) coupled to form a 21-membered ring. The structure
and stereochemistry of HUN-7293 (1) has been determined by
10.1021/ja990918u CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/19/1999

6198 J. Am. Chem. Soc., Vol. 121, No. 26, 1999
Boger et al.
1H NMR spectroscopy and X-ray crystallography.3 Only one
of the six L-amino acid residues is incorporated in its unmodified
naturally occurring form, three are N-methylated, and four of
the component residues are novel: (R)-2-hydroxy-4-cyanobu-
tyric acid (DGCN), two (4R)-5-propyl-L-leucine (PrLEU)
residues, and N1′-methoxy-N-methyl-L-tryptophan (MTO). Al-
though N-methoxyindoles have been isolated as early as the
1970s,4 an N1′-methoxytryptophan derivative has only recently
been observed in one other natural product.5
Scheme 1
The conformation of HUN-7293 (1) incorporates two cis
peptide bonds in both the solution and crystalline states and
two transannular H-bonds adding stability and rigidity to the
backbone structure.3 The cis peptide bonds occur at two of the
three N-methyl amide sites and are found between PrLEU2 and
MLEU3 and between LEU4 and MTO5. The transannular
H-bonds are observed between PrLEU6-CO/PrLEU2-NH and
MLEU3-CO/PrLEU6-NH stabilizing the compact conformation.
The overall shape of the cyclopeptolide is bent or cup-shaped,
allowing the DGCN, LEU, and MTO residues to reside in close
proximity to one another on the concave side of the peptide.
The two PrLEU side chains are located on the convex face of
the peptide and extend in the same direction. Thus, the
N-methyls may be regarded as conformationally significant
structural features as well as contributing to improved protease
resistance analogous to cyclosporin A.6
Herein we report the first total synthesis of 1 and the initial
results from degradation/resynthesis studies of the natural
product conducted as part of a systematic investigation of the
structure-activity relationships of HUN-7293. The approach
relied on a key macrocyclization reaction with formation of the
MLEU3-LEU4 secondary amide, a reaction that proved to be
unusually effective potentially benefitting from intramolecular
H-bonding preorganization of the acyclic substrate. In turn, the
requisite linear depsipeptide was assembled convergently with
the late stage introduction of the linking ester enlisting a
Mitsunobu esterification. This occurs with the inversion of the
stereochemistry of the DGCN R-center which in turn permitted
the utilization of a readily available L-amino acid precursor to
the D R-hydroxy carboxylic acid residue. The requisite tetrapep-
tide 43 and tripeptide 34 were assembled convergently from
the appropriate amino acid constituents (Figure 1).
Preparation of the Amino Acid Constituents. PrLEU (10,
(4R)-5-propyl-L-leucine) was synthesized as shown in Scheme
1 starting with commercially available (R)-(+)-citronellol (2).
Ozonolysis and Wittig reaction of the resulting aldehyde 3 with
methylenetriphenylphosphorane gave alcohol 4 in 63% yield
for the two steps. Hydrogenation of 4 provided the saturated
alcohol 57,8 (94%) which was oxidized to the corresponding
aldehyde 69 by treatment with the Dess-Martin reagent10 and
directly used in the next step without further purification.
Asymmetric Strecker reaction utilizing (R)-phenylglycinol and
trimethylsilyl cyanide11 afforded 7 as an 8.6:1 mixture of
diastereomers in favor of the expected and desired (2S)-
diastereomer. This reaction proved to be sensitive to the reaction
conditions, and both kinetic and thermodynamic control could
be used to selectively generate the (2S)-diastereomer. Thus, low-
temperature condensation (-25 °C, CHCl3, 16 h) conducted
under kinetically controlled reaction conditions provided 7 (74%,
10:1), and thermal equilibration (CH3OH, 80 °C, 2-14 h) of a
mixture of C2 diastereomers provided an equilibrium ratio of
4-4.8:1 with the (2S)-diastereomer predominating. Although
separation of the diastereomers by crystallization or chroma-
tography was not successful at this stage, the free amine 9,
derived by oxidative cleavage of 7 with lead tetraacetate,12 could
easily be separated from its C2 diastereomer 8 by column
chromatography (SiO2, 40% EtOAc-hexane, R ) 1.17). The
absolute stereochemistry of the diastereomers was determined
1
by synthesis and comparison of the H NMR spectra of their
Mosher13 derivatives and ultimately confirmed upon incorpora-
tion into 1. Hydrolysis of the nitrile to the free acid 10 and Boc
protection of the amine completed of the synthesis of the PrLEU
precursor 11.
N1′-Methoxytryptophan derivative 23 was initially prepared
from N-methoxyindole (12)14 as shown in Scheme 2. Pheny-
lacetic acid was condensed with methoxyamine hydrochloride15
(98%) and subsequent ring closure was achieved following a
(6) (a) Lobb, R. R.; Hemler, M. E. J. Clin. InVest. 1994, 94, 1722. (b)
Foster, C. A. J. Allergy Clin. Immunol. 1996, 98, S270. (c) Cornejo, C. J.;
Winn, R. K.; Harlan, J. M. AdV. Pharmacol. 1997, 39, 999.
(11) (a) Inaba, T.; Kozono, I.; Fujita, M.; Ogura, K. Bull. Chem. Soc.
Jpn. 1992, 65, 2359. (b) Chakraborty, T. K.; Reddy, G. V.; Hussain, K. A.
Tetrahedron Lett. 1991, 32, 7597.
(12) Gawley, R. E.; Rein, K.; Chemburkar, S. J. Org. Chem. 1989, 54,
3002.
(7) (a) Bergdahl, M.; Nilsson, M.; Olsson, T.; Stern, K. Tetrahedron
1991, 47, 9691. (b) Fang, C.; Ogawa, T.; Seumune, H.; Sakai, K.
Tetrahedron: Asymmetry 1991, 2, 389. (c) Bezuidenhoudt, B. C. B.; Castle,
G. H.; Innes, J. E.; Ley, S. V. Recl. TraV. Chim. Pays-Bas 1995, 114, 184.
(d) Mori, K.; Kato, M. Liebigs Ann. Chem. 1985, 10, 2083. (e) Norsikian,
S.; Marek, I.; Normant, J.-F. Tetrahedron Lett. 1997, 38, 7523. (f) Orsini,
F.; Rinaldi, S. Tetrahedron: Asymmetry 1997, 8, 1039. (g) Marek, I.;
Alexakis, A.; Mangeney, P.; Normant, J. F. Bull. Soc. Chim. Fr. 1992, 129,
171. (h) Somfai, P.; Tanner, D.; Olsson, T. Tetrahedron 1985, 41, 5973.
(8) Breitenbach, R.; Chiu, C. K.-F.; Masset, S. S.; Meltz, M.; Murtiashaw,
C. W.; Pezzullo, S. L.; Staigers, T. Tetrahedron: Asymmetry 1996, 7, 435.
(9) Naemura, K.; Kittaka, K.; Murata, M.; Ida, H.; Hirose, H. Enantiomer
1996, 1, 219.
(13) (a) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512. (b)
Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34, 2543. (c)
The stereochemistry of the undesired (R,R)-diastereomer was established
1
by the synthesis of both Mosher derivatives and the comparison of the H
NMR spectra. The difference in the chemical shift of C3-proton δ (C3-H
(R-derivative)-C3-H (S-derivative)) was found to be -0.05. According
to the correlations established by Mosher,13a the configuration at C2 was
established to be R. Details are provided in the Supporting Information.
(14) (a) Acheson, R. M.; Hunt, P. G.; Littlewood, D. M.; Murrer, B. A.;
Rosenberg, H. E. J. Chem. Soc., Perkin Trans. I 1978, 1117. (b) Hanke, F.
J.; Kubo, I. J. Nat. Prod. 1989, 52, 1237.
(10) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.

Total Synthesis of HUN-7293
J. Am. Chem. Soc., Vol. 121, No. 26, 1999 6199
Scheme 2
Scheme 3
to the N1-oxidation of a N-methyltryptophan derivative which
itself was not successful. Thus, reduction of the indole 21 to
the corresponding indoline 22 (83%) followed by N-oxidation
(Na2WO4-H2O2) with in situ reoxidation to the indole and
subsequent O-methylation provided the protected MTO deriva-
tive 23 (Scheme 2). Fmoc deprotection afforded 20 and provided
a much more concise route to this key amino acid subunit.
The precursor 29 to the (R)-2-hydroxy-4-cyanobutyric acid
(DGCN) constituent was synthesized according to Scheme 3
starting with the N-BCbz-L-GLN (24). Formation of the methyl
ester 25 by treatment with TMSCHN2 and subsequent dehydra-
tion utilizing cyanurchloride21 gave the desired nitrile 26 (85%,
2 steps).22 Hydrolysis of the methyl ester with LiOH and Cbz
hydrogenolysis (H2, Pd-C) gave the free amino acid 28 (>90%,
2 steps), which provided (S)-2-hydroxy-4-cyanobutyric acid (29)
upon treatment with NaNO2 and H2SO4 (>90%). Alternatively,
direct amide dehydration of N-Cbz-GLN followed by transfer
hydrogenolysis removal of the N-Cbz protecting group also
provided 28 (72% overall).22 Alcohol esterification of (S)-29
under Mitsunobu conditions would proceed with inversion of
the R-stereochemistry and, as such, permitted the use of a readily
available L-amino acid precursor.23
sequence developed by Kikugawa,16 which involved N-chlorina-
tion17 followed immediately by Ag(I)-catalyzed ring closure to
give 12 (86%). The amide was reduced cleanly to the hemi-
aminal 13 using LiAlH4 (88%), which could be converted
directly to the racemic tryptophan derivative 15 by heating with
R-acetamidoacrylic acid (14) in HOAc-Ac2O presumably
proceeding through the N-methoxyindole.18 Resolution of 15
by enantioselective enzymatic hydrolysis of the N-acetate using
Acylase I19 from Aspergillus sp. afforded N1′-methoxy-L-
tryptophan (16). The crude reaction mixture containing N1′-
methoxy-L-tryptophan and N-acetyl-N1′-methoxy-D-tryptophan
was submitted to standard N-Boc protection conditions which,
upon chromatographic separation, gave pure 17 in 86% (43%)
yield for the 2 steps. N-Methylation of 17 utilizing MeI-NaH
followed by conversion of 18 to the methyl ester 19 upon
reaction with TMSCHN220 and removal of the Boc group (HCl-
EtOAc) provided the MTO derivative 20 in 75% yield over the
last 3 steps. The optical purity of intermediate 19 was established
to be 98.6% ee by chiral phase HPLC (Chiralcel OD, 0.45 ×
25 cm, 3% i-PrOH/hexane 1 mL/min flow rate, R ) 1.19, tR )
9.86 (D) and 11.73 (L) min), indicating that the enzymatic
resolution was highly effective and, importantly, that the
subsequent N-methylation step proceeded without detectable
racemization.
Synthesis of Tripeptide 34. N-Boc-MLEU (30) was treated
with isobutene and H2SO4 in CH2Cl2 to generate the tert-butyl
ester 31 (89%) with simultaneous removal of the Boc group
(Scheme 4). Following conversion of the amine to the HCl salt
(HCl-EtOAc), 31 was coupled with N-Boc-PrLEU (11) utiliz-
ing EDCI-HOAt24 and NaHCO3 as base to provide 32 (54%).
Removal of the Boc group was accomplished by treatment with
HCO2H (30 min, 22 °C),25 and the tert-butyl ester was stable
under these reaction conditions. Without purification, coupling
of 33 with the DGCN residue precursor 29 utilizing EDCI-
HOAt and NaHCO3 as base provided the desired tripeptide 34
1
(65%, 2 steps) as a single diastereomer as determined by H
NMR.
Synthesis of Tetrapeptide 41. MALA-OCH3 (35) was
coupled with N-Boc-PrLEU (11, EDCI-HOAt, NaHCO3, 74%)
(20) Hashimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm. Bull. 1981,
29, 1475.
(21) Maetz, P.; Rodriguez, M. Tetrahedron Lett. 1997, 38, 4221.
(22) Yoneta, T.; Shibahara, S.; Fukatsu, S.; Seki, S. Bull. Chem. Soc.
Jpn. 1978, 51, 3296. Boehlein, S. K.; Rosa-Rodriguez, J. G.; Schuster, S.
M.; Richards, N. G. J. J. Am. Chem. Soc. 1997, 119, 5785. 26 has been
prepared previously by esterification of Cbz-Glu (SO2Cl2, CH3OH) and
dehydration with BnSO2Cl or Burgess reagent; see the following: Van, T.
T.; Kojro, E.; Grzonka, Z. Tetrahedron 1977, 33, 2299. Claremon, D. A.;
Phillips, B. T. Tetrahedron Lett. 1988, 29, 2155. For 28, see the following:
Boehlein, S. K.; Rosa-Rodriguez, J. G.; Schuster, S. M.; Richards, N. G. J.
J. Am. Chem. Soc. 1997, 119, 5785.
Upon completion of the synthesis a shorter and more effective
preparation of 20 was developed employing an indirect approach
(15) (a) De Rosa, M.; Alonso, J. L. T. J. Org. Chem. 1978, 43, 2639.
(b) Gassman, P. G.; Campbell, G. A.; Metha, G. Tetrahedron 1972, 28,
2749.
(16) Kikugawa, Y.; Kawase, M. J. Am. Chem. Soc. 1984, 106, 5728.
(17) Mintz, M. J.; Walling, C. Org. Synth. 1969, 49, 9.
(18) Snyder, H. R.; Smith, C. W. J. Am. Chem. Soc. 1944, 66, 350.
(19) (a) Cho, H.-Y.; Tanizawa, K.; Tanaka, H.; Soda, K. Agric. Biol.
Chem. 1987, 51, 2793. (b) Chenault, H. K.; Dahmer, J.; Whitesides, G. M.
J. Am. Chem. Soc. 1989, 111, 6354.
(23) The corresponding (R)-29 was prepared from D-24 in an analogous
fashion and utilized for the preparation of 55 as detailed in Scheme 9.
(24) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
(25) Bodanszky, M.; Bodanszky, A. Int. J. Pept. Res. 1984, 23, 565.

6200 J. Am. Chem. Soc., Vol. 121, No. 26, 1999
Boger et al.
Scheme 4
Scheme 6
Scheme 5
Coupling of the dipeptides 37 and 40 (HATU, NaHCO3) gave
the desired tetrapeptide 41 together with small amounts of an
undesired diastereomer 42 in 95% yield. Alternative coupling
reagents including EDCI-HOAt provided larger amounts of
42. Although attempts to separate the isomers by SiO2 chro-
matography were unsuccessful, separation by semipreparative
reversed-phase HPLC was easily accomplished to give clean
samples of both isomers (13:1).
Final Steps: Esterification and Macrocyclic Ring Closure.
Tetrapeptide 41 was hydrolyzed to the free acid 43 by treatment
with LiOH, and the crude product was subjected to the following
esterification step without further purification (Scheme 6).
Reaction of 43 with alcohol 34 under modified Mitsunobu
conditions27 (Ph3P-DIAD) proceeded smoothly to give the
desired linear heptadepsipeptide 44 in good conversion. In
optimizing this esterification, we found that the selection of the
reaction conditions was critical. The combination of Ph3P-
DIAD in toluene gave the desired product in 67% yield. The
use of Bu3P instead of Ph3P resulted in poor yields and various
byproducts, and toluene was found to be a superior solvent over
benzene and THF. The less hindered DEAD gave results
comparable with DIAD although the yields were generally
lower. Alternative attempts to generate 44 enlisting the corre-
sponding (2R) versus (2S) DGCN precursor and direct esteri-
fication with carboxylate activation were not nearly as success-
ful. Thus, coupling promoted by DCC required the presence of
DMAP (0.1-2 equiv) to effect reasonable conversions at useful
rates (-20 to 22 °C, 2-6 h, 18-69%) but provided a 1-2:1
ratio of diastereomers derived from racemization of the inter-
mediate activated carboxylate together with significant amounts
(∼20%) of the N-acyl urea. Epimerization is problematic
because of the requirement for activation of a N-methyl
C-terminus carboxylate, and this is strategically avoided through
use of the Mitsunobu esterification.
to give the desired dipeptide 36 in which the extent of
racemization during the coupling was less than 10% as
determined by 1H NMR (Scheme 5). Removal of the Boc group
was accomplished by treatment with HCl-EtOAc to give 37
as the HCl salt which was used directly in the next coupling
reacting without further purification. The dipeptide 40 was
prepared from the tryptophan derivative 20 and N-Boc-LEU
(38) in a coupling reaction that proceeded smoothly to give 39
as a single isomer in 91% yield. Hydrolysis of the methyl ester
by treatment with LiOH in t-BuOH-H2O gave the free acid
40 without evidence that racemization occurred under the
reaction conditions26 and in a reaction that was sufficiently
clean that crude product could be used directly in the next
reaction.
Removal of the Boc group and the tert-butyl ester was
accomplished in one step by treatment with CF3CO2H at 22
°C. The use of HCl-EtOAc or HCl-dioxane resulted in the
formation of a byproduct derived from hydrolysis of the nitrile
to the corresponding amide. Cyclization of the crude 45 utilizing
EDCI-HOAt, NaHCO3 proceeded smoothly within 6 h at 0
°C to give 1 in 71% overall yield identical in all respects with
(26) Treatment of 40 with TMSCHN2 gave the methyl ester 39 which
was indistinguishable from the H NMR of the authentic starting material
sample, indicating that no racemization occurred during the hydrolysis.
(27) (a) Mitsunobo, O.; Yamada, M. Bull. Chem. Soc. Jpn. 1967, 40,
2380. (b) Mitsunobo, O. Synthesis 1981, 1. (c) Hughes, D. L.; Reamer, R.
A.; Bergan, J. J.; Grabowski, E. J. J. J. Am. Chem. Soc. 1988, 110, 6487.
1

Total Synthesis of HUN-7293
J. Am. Chem. Soc., Vol. 121, No. 26, 1999 6201
Scheme 7
Scheme 8
authentic HUN-729328 (1H NMR, 13C NMR, HPLC, TLC, [R]D,
IR). Although not examined in detail, the use of DMF as solvent
in initial trials was superior to CH2Cl2 or a mixture of CH2-
Cl2-DMF (5:1), which gave only traces of the desired product.
Similarly, closure of crude 45 effected by treatment with HATU
(3 equiv, 9 equiv of collidine, CH2Cl2, 25 °C, 10 h) provided 1
in superb conversion (72%), and in this case substantial
racemization was observed in CH3CN but not CH2Cl2. PyBroP
(3 equiv, 6 equiv of iPr2NEt, CH2Cl2 0-25 °C, 3-4 h, ∼70-
80%) also promoted ring closure, but difficulty was encountered
in removing reaction byproducts in the final purification of 1.
Closures conducted with DPPA (5 equiv, 4 equiv of NaHCO3,
DMF, 0 °C, 24 h, trace of 1), BOP-Cl (3 equiv, DMAP, CH3-
CN, 25 °C), or HATU (2 equiv, 5 equiv of NaHCO3, DMF, 0
°C, 4 h, 20% 1) were less effective in their initial examinations.
Additional strategically attractive attempts that reverse the
order of the last amide coupling and esterification reaction such
that the final macrocyclization step entailed formation of the
sensitive ester were not nearly as successful (Scheme 7).
Enlisting the substrate with the (2R)-DGCN subunit and the
corresponding direct esterification (DCC, DMAP) was only
modestly successful as detailed in the next section, and
Mitsunobu displacement enlisting 49 with inversion of the
DGCN R-center accompanying esterification was unsuccessful.29
Degradation and Resynthesis of HUN-7293. The most
successful of the ring closures was independently shown to be
effective on the corresponding seco-derivative derived from the
natural product via a selective ring-opening procedure at the
MLEU39LEU4 amide bond. Upon treatment of the natural
product with 0.5 equiv of Lawesson’s reagent, the amide
bond between MLEU and LEU was primarily attacked and
converted into the corresponding thioamide 50 (Scheme 8). This
preference most likely reflects the accessibility of this amide,
which in the crystal structure is the only amide to form a H-bond
to a solvent molecule. As the only side product, a double
thioamide with the additional introduction of sulfur at the
MTO5-PrLEU6 amide bond was formed. Benzylation of the
mono-thioamide was achieved in a two-phase system with
benzyl bromide in CH2Cl2 and aqueous NaOH. The resulting
benzylthioamidate 52 could quantitatively be cleaved by short
treatment with aqueous acid (aqueous HCl/t-BuOH) at 55 °C
without affecting the lactone. In a second step, the benzyl-
thioester 53 at the newly formed C-terminus was selectively
cleaved to the unprotected depsipeptide 45 by silver-promoted
hydrolysis in aqueous t-BuOH. The crude material after gel
filtration was subjected directly to ring closure in acetonitrile
using BOP as condensing agent and DMAP as base. Using this
complement to the ring closure detailed in Scheme 6, HUN-
7293 was formed in a clean reaction (80%), most probably
facilitated by a conformational preorganization of the precursor,
due to a preformed H-bond of the activated ester C-terminus
and the PrLEU2 NH and partial cis-geometry of the methylated
amide bonds.
(28) Dreyfuss, M. M.; Foster, C. A.; Naegeli, H.-U.; Oberhauser, B. PCT
Int. Appl. 9603430, 1996; Chem. Abstr. 1996, 125, 34177.
(29) For 55: 1H NMR (CDCl3, 500 MHz) δ 7.74 (d, J ) 7.5 Hz, 2H),
7.52-7.56 (m, 2H), 7.37-7.41 (m, 2H), 7.28-7.33 (m, 2H), 7.04 (d, J )
8.0 Hz, 1H), 5.23 (dd, J ) 10.7, 5.2 Hz, 1H), 4.88 (m, 1H), 4.54 (m, 2H),
4.22 (dd, J ) 7.5, 4.0 Hz, 1H), 4.18 (t, J ) 5.9 Hz, 1H), 2.77 (s, 3H), 2.50
(dd, J ) 16.5, 7.7 Hz, 1H), 2.40 (ddd, J ) 16.9, 8.3, 5.5 Hz, 1H), 2.09-
2.15 (m, 1H), 1.90-1.97 (m, 1H), 1.17-1.59 (m, 12H), 0.94 (d, J ) 6.0
Hz, 3H), 0.86 (d, J ) 6.5 Hz, 3H), 0.84 (t, J ) 7.0 Hz, 3H), 0.81 (d, J )
6.5 Hz, 1H); FABHRMS (NBA-CsI) m/z 722.2541 (M + Cs+, C35H47N3O5
requires 722.2570). For 58: FABHRMS (NBA-CsI) m/z 1141.5740 (M
+ Cs+, C54H88N8O10 requires 1141.5678).
The less successful ring closure via macrolactonization was
also examined both with synthetic material as well as with that
derived from the natural product (Scheme 9). The lactone bond
in HUN-7293 was selectively cleaved under alkaline conditions
(LiOH, THF/H2O), producing the lithium salt of the corre-

6202 J. Am. Chem. Soc., Vol. 121, No. 26, 1999
Boger et al.
Scheme 9
Figure 2. A representative cell-ELISA comparing the inhibitory effect
of HUN-7293 natural (1) versus synthetic (1) on VCAM-1 expression
in primary human endothelial cells (HUVEC). Optical density (OD
550 nm) values ( standard deviation indicate VCAM-1 protein levels
on the left y-axis and relative cell density on the right axis, using
triplicate wells/experimental group. Briefly, cells in 96-well microtiter
plates were incubated with serial dilutions of HUN-7293 for 4 h and
then cytokine-stimulated with TNFR (tumor necrosis factor alpha) +
IL-1 (interleukin 1) (100 units/mL each) for 16 h, as previously
described.4 Possible mitogenic, cytostatic, or cytotoxic effects were
analyzed in the same plate by subsequently quantifying the cell number
(absorbance of Giemsa nuclear dye), relative to control wells.
sponding hydroxy acid 54. Due to 15-20% racemization in this
saponification at the R-carbon of MALA,7 we obtained 54 as
an inseparable mixture together with its D-MALA7 isomer.
Recyclization to HUN-7293 was achieved applying a modified
protocol (acetonitrile, DCC/DMAP/DMAP‚TFA, 55 °C) of the
Keck method30 which gave the natural product in 29% yield.
For the cyclization, acetonitrile was found to be as efficient a
solvent as chloroform which is usually used for this reaction.
Efforts to form the (1S) analogue of HUN-7293 by a Mitsunobu
reaction with inversion of the configuration were not successful.
This same hydroxy acid 54 incorporating the (R)-DGCN
subunit was also prepared from tetrapeptide 41 and the corre-
sponding tripeptide 5529 constituting a second, albeit less
effective, total synthesis of HUN-7293 (Scheme 9). Notably,
hydrolysis of 58 to provide 54 also proceeded with extensive
racemization (20-30%), further detracting from this approach.
Biological Activity of HUN-7293. To compare the biological
activity of the naturally occurring HUN-7293 [natural 1] with
the cyclodepsipeptide achieved by total synthesis [synthetic 1],
we evaluated both compounds in parallel using a cell-based
ELISA for inducible adhesion molecules in human endothelial
cells. The vascular proteins VCAM-1, ICAM-1, and E-selectin,
due to their prominent roles in regulating leukocyte extravasa-
tion,5,6 were investigated in primary cells derived from umbilical
vein (HUVEC) and a microvascular cell line (HMEC-1). Figure
2 shows that natural and synthetic 1 potently and dose-
dependently inhibited VCAM-1 expression in HUVEC to a
similar extent, with IC50 (concentration resulting in 50%
inhibition) values of 3 and 6 nM, respectively (Table 1).
Complete down-regulation of VCAM-1 occurred by 37 nM,
essentially equal to background levels in the unstimulated group,
whereas the cell density was not altered under these experimental
conditions (Figure 2). Natural and synthetic 1 also showed
similar effects on VCAM-1 in the cell line HMEC-1 (Table 1),
confirming earlier results with the naturally occurring HUN-
7293.4
Table 1. Comparison of HUN-7293 Natural (1) Versus Synthetic
(1) Biological Activity (nM IC50) in Cell ELISA for VCAM-1,
ICAM-1, and E-selectin Expression Using Primary Endothelial Cells
(HUVEC) and a Human Micro-Vascular Cell Line (HMEC-1)
VCAM-1 IC50
ICAM-1 IC50
E-sel
HUN-7293
HUVEC HMEC HUVEC HMEC HUVEC
natural (1)
synthetic (1)
3
6
1
2
26
49
24
42
44
69
1). In addition, approximately 13-fold higher concentrations of
both compounds were needed to inhibit E-selectin expression
in HUVEC, relative to their effect on VCAM-1 (Table 1). The
present results with natural 1 are in agreement with previous
studies, such as in HMEC-1 cells where the mean IC50 for
VCAM-1 was 2 nM (range 0.3-4 nM in 12 experiments) and
that for ICAM-1 was 39 nM (range 11-89 nM in 7 experi-
ments). Therefore, considering the usual variablility seen in such
biological assays, our data indicate that the synthesis of HUN-
7293 (1) has provided a compound that is indistinguishable from
the naturally occurring cyclodepsipeptide.
Experimental Section
N-[(2S,4R)-2-[N-(tert-Butyloxycarbonyl)amino]-4-methylheptanoyl]-
N-methyl-L-leucine tert-Butyl Ester (32). A solution of 1131 (43.7
mg, 160 µmol) and 3131 (56.8 mg, 1.5 equiv) in 0.4 mL of CH2Cl2-
DMF (5:1) was treated with NaHCO3 (20.1 mg, 1.5 equiv), HOAt (26.0
mg, 1.2 equiv), and EDCI (60.9 mg, 2 equiv) at 0 °C. The mixture
was stirred for 32 h at 22 °C and diluted with 2 mL of aqueous 1 M
HCl. The mixture was extracted with EtOAc (2 × 2 mL), and the
combined organic phase was washed with 1 mL of H2O and dried over
MgSO4. The mixture was filtered, and the solvent was removed under
reduced pressure. Chromatography (SiO2, 25% EtOAc-hexane) af-
forded 32 (39.5 mg, 54%) as a white solid: [R]2D2 -31 (c 0.50,
1
Compared to VCAM-1 inhibition, natural and synthetic 1
were both 8- to 20-fold less effective in suppressing ICAM-1
up-regulation in HUVEC and HMEC-1 cells, respectively (Table
CHCl3); H NMR (CDCl3, 500 MHz) δ 5.19 (dd, J ) 5.2, 10.7 Hz),
5.15 (d, J ) 9.2 Hz), 4.61 (m, 1H, C2-H), 2.92 (s, 3H), 1.40 (s, 9H),
(31) Details of the preparation may be found in the Supporting Informa-
tion.
(30) Boden, E. P.; Keck, G. E. J. Org. Chem. 1985, 50, 2394.

Total Synthesis of HUN-7293
J. Am. Chem. Soc., Vol. 121, No. 26, 1999 6203
1.39 (s, 9H), 1.20-1.70 (m, 12H), 0.96 (d, J ) 6.3 Hz, 3H), 0.87-
0.92 (m, 6H), 0.86 (t, J ) 5.9 Hz, 3H); 13C NMR (CDCl3, 125 MHz)
δ 173.8, 170.8, 81.5, 79.4, 55.1, 48.8, 40.4, 37.3, 37.0, 30.8, 29.2, 29.1,
28.3, 28.0, 24.8, 23.3, 22.8, 21.4, 19.2, 14.0; IR (neat) Vmax 2958, 2928,
1732, 1716, 1652, 1368, 1272, 1254, 1170 cm-1; FABHRMS (NBA)
m/z 457.3629 (M + H+, C25H48N2O5 requires 457.3641).
1 mL of t-BuOH-H2O (2:1) was treated with LiOH (10.1 mg, 422
µmol) at 0 °C. After being stirred for 2 h at 0 °C, the mixture was
diluted with aqueous 1 N HCl (5 mL) and extracted with EtOAc (3 ×
8 mL). The combined organic phase was dried over MgSO4 and
evaporated under reduced pressure to give 40 (101 mg, quantitative)
as a pale yellow solid: [R]2D2 -60 (c 1.1, CHCl3); H NMR (CDCl3,
1
500 MHz, both rotamers) δ 7.55 (d, J ) 7.9 Hz, 1H), 7.38 (t, J ) 8.2
Hz, 1H), 7.22 (m, 1H), 7.02-7.13 (m, 2H), 5.23-5.28 (m, 1.66H),
4.87 (dd, J ) 3.7, 10.7 Hz, 0.33H), 4.54 (m, 0.66H), 4.09 (m, 0.33H),
4.01 and 4.00 (s, 3H), 3.42-3.50 (m, 1H), 3.25 (dd, J ) 10.7, 15.8
Hz, 0.66H), 3.13 (dd, J ) 10.7, 15.1 Hz, 0.33H), 2.98 and 2.91 (s,
3H), 1.70 (m, 0.66H), 1.16-1.52 (m, 2.33H), 1.39 (s, 9H), 0.92 (d, J
) 6.6 Hz, 2H), 0.89 (d, J ) 6.6 Hz, 2H), 0.44 (d, J ) 6.3 Hz, 1H),
0.14 (d, J ) 6.3 Hz, 1H), -0.17 (m, 0.33H), 13C NMR (CDCl3, 125
MHz, both rotamers) δ 174.5, 174.2, 173.5, 170.6, 156.8, 155.8, 132.3,
123.6, 123.2, 123.0, 122.6, 122.0, 121.5, 120.4, 119.8, 118.6, 118.4,
108.6, 108.4, 106.7, 106.0, 81.4, 79.7, 65.9, 65.7, 61.2, 58.3, 49.2, 47.6,
41.7, 39.3, 33.0, 29.5, 28.25, 28.17, 24.6, 24.1, 23.9, 23.7, 23.4, 22.8,
21.6, 19.7; IR (neat) Vmax 3317, 2959, 1716, 1652, 1616, 1506, 1456,
1367, 1252, 1168, 1100, 1024, 955, 739 cm-1; FABHRMS (NBA-
NaI) m/z 484.2407 (M + Na+, C24H35N3O6 requires 484.2424).
N-[(2S,4R)-2-[[N-[N-(tert-Butyloxycarbonyl)-L-leucinyl]-N1′-meth-
oxy-N-methyl-L-tryptophanyl]amino]-4-methylheptanoyl]-N-meth-
yl-L-alanine Methyl Ester (41). A solution of 36 (29.3 mg, 79.0 µmol)
in 1.0 mL of EtOAc was saturated with HCl(g), at 0 °C and the mixture
was stirred for 70 min at 0 °C. The solvent was removed with a stream
of N2, and the residue was dried under vacuum to give 37 (27 mg) as
a white solid. A sample of 40 (33.0 mg, 71.9 µmol) in 1.0 mL of CH2-
Cl2-DMF (5:1) was added at 0 °C, followed by NaHCO3 (10.0 mg,
119 µmol) and HATU (54.6 mg, 144 µmol). The mixture was stirred
for 3 h at -30 °C and 1 h at 0 °C before being diluted with 2 mL of
aqueous 1 M HCl. The mixture was extracted with EtOAc (3 × 2 mL),
and the combined organic phase was washed with 1 mL of H2O and
dried over MgSO4. The mixture was filtered and the solvent removed
under reduced pressure. Chromatography (SiO2, 60% EtOAc-hexane)
afforded the mixture of 41 and 42 (48.8 mg, 95%) as a white solid.
Further purification by semipreparative HPLC (reverse-phase C18,
80% CH3OH-H2O) gave 45.0 mg of the desired diastereomer 41 (88%)
and 3.5 mg (7%) of the undesired diastereomer 42 as white solids. For
N-[(2S,4R)-2-[N-[(S)-2-Hydroxy-4-cyanobutanoyl]amino]-4-me-
thylheptanoyl]-N-methyl-L-leucine tert-Butyl Ester (34). A solution
of 32 (29.0 mg, 64.2 µmol) in 3 mL of HCO2H was stirred for 1 h at
22 °C. The solvent was removed with a steam of N2, and the residue
was dissolved in 0.5 mL of HCl-EtOAc. The solvent was removed
with a stream of N2, and the residue was dissolved in 0.5 mL of CH2-
Cl2-DMF (5:1). A sample of 2931 (16.8 mg, 1.5 equiv), NaHCO3 (7.3
mg, 1 equiv), HOAt (17.7 mg, 1.5 equiv), and EDCI (33.1 mg, 2 equiv)
was added at 0 °C. The mixture was stirred for 2 h at 0 °C and diluted
with 2 mL of aqueous 1 M HCl. The mixture was extracted with EtOAc
(2 × 2 mL), and the combined organic phase was washed with 1 mL
of H2O and dried over MgSO4. The mixture was filtered, and the solvent
was removed under reduced pressure. Chromatography (SiO2, 25%
EtOAc-hexane) afforded 34 (19.5 mg, 65%) as a white solid: [R]D22
-58 (c 0.65, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 7.26 (d, J ) 8.9
Hz, 1H), 5.08 (dd, J ) 10.7, 5.2 Hz, 1H), 4.92 (m, 1H), 4.17 (m, 1H),
2.97 (s, 3H), 2.39-2.51 (m, 2H), 2.13-2.25 (m, 2H), 1.94 (m, 1H),
1.20-1.71 (m, 11H), 1.41 (s, 9H), 0.83-0.96 (m, 12H); 13C NMR
(CDCl3, 125 MHz) δ 173.8, 172.4, 170.3, 119.3, 81.8, 69.8, 55.5, 47.3,
39.4, 37.2, 36.9, 31.0, 30.5, 29.5, 29.1, 24.8, 23.2, 22.8, 21.4, 18.9,
14.0, 13.0; IR (neat) Vmax 2958, 2931, 2872, 1732, 1634, 1520, 1368,
1274, 1160, 1127, 1090 cm-1; FABHRMS (NBA) m/z 468.3424 (M
+ H+, C25H45N3O5 requires 468.3437).
N-[(2S,4R)-2-[N-(tert-Butyloxycarbonyl)amino]-4-methylheptanoyl]-
N-methyl-L-alanine Methyl Ester (36). A solution of 1131 (6.5 mg,
24 µmol) and 3532 (5.5 mg, 36 µmol) in 0.25 mL of CH2Cl29DMF
(5:1) was treated with NaHCO3 (2.9 mg, 35 µmol), HOAt (6.5 mg, 48
µmol), and EDCI (9.1 mg, 48 µmol) at 0 °C. The mixture was stirred
for 35 h at 22 °C and diluted with 2 mL of aqueous 1 M HCl. The
mixture was extracted with EtOAc (3 × 2 mL), and the combined
organic phase was washed with 1 mL of H2O and dried over MgSO4.
The mixture was filtered and the solvent removed under reduced
pressure. Chromatography (SiO2, 25% EtOAc-hexane) afforded 36
(6.5 mg, 74%) as a white solid: [R]2D2 -6.8 (c 0.33, CHCl3); 1H NMR
(CDCl3, 500 MHz) δ 5.25 (q, J ) 7.0 Hz, 1H), 5.18 (d, J ) 9.0 Hz,
1H), 4.65 (m, 1H), 3.69 (s, 3H), 2.97 (s, 3H), 1.45-1.61 (m, 3H), 1.41
(s, 9H), 1.15-1.29 (m, 6H), 0.98 (d, J ) 6.5 Hz, 3H), 0.89 (d, J ) 6.5
Hz, 3H), 0.87 (t, J ) 6.5 Hz, 3H); 13C NMR (CDCl3, 125 MHz) δ
173.6, 172.1, 155.7, 79.4, 52.2, 52.0, 48.8, 40.9, 37.3, 30.9, 29.24, 29.17,
28.3, 22.8, 19.2, 14.2, 14.0; IR (neat) Vmax 2960, 2928, 1746, 1709,
1647, 1497, 1461, 1366, 1248, 1174, 1093 cm-1; FABHRMS (NBA)
m/z 373.2694 (M + H+, C19H36N2O5 requires 373.2702).
41: [R]2D2 -81.6 (c 0.58, CHCl3); H NMR (CDCl3, 500 MHz, both
1
rotamers) δ 8.23 (d, J ) 8.1 Hz, 1H), 7.57 (d, J ) 8.1 Hz, 1H), 7.50
(d, J ) 8.1 Hz, 1H), 7.36 (t, J ) 9.9 Hz, 2H), 7.22 (d, J ) 8.1 Hz,
1H), 7.19 (d, J ) 7.7 Hz, 1H), 7.06-7.11 (m, 2H), 6.96 (s, 1H), 6.59
(d, J ) 8.5 Hz, 1H), 5.42 (t, J ) 7.7 Hz, 1H), 5.38 (q, J ) 7.8 Hz,
1H), 5.19 (q, J ) 7.4 Hz, 1H), 5.14 (d, J ) 8.9 Hz, 1H), 5.04 (m, 1H),
4.92 (t, J ) 8.5 Hz, 1H), 4.76 (m, 1H), 4.57 (m, 1H), 3.99 (s, 3H),
4.00 (s, 3H), 3.67 (s, 3H), 3.69 (s, 3H), 3.38 (dd, J ) 3.3, 15.5 Hz,
1H), 3.30 (dd, J ) 7.4, 15.5 Hz, 1H), 3.13 (dd, J ) 8.1, 15.5 Hz, 1H),
3.08 (dd, J ) 10.7, 15.5 Hz, 1H), 3.07 (s, 3H), 2.99 (s, 3H), 2.94 (s,
3H), 2.92 (s, 3H), 1.10-1.78 (m, 23H), 1.41 (s, 9H), 1.38 (s, 9H),
0.96 (d, J ) 6.6 Hz, 3H), 0.94 (d, J ) 6.3 Hz, 3H), 0.84-0.92 (m,
18H), 0.42 (d, J ) 6.7 Hz, 3H), 0.00 (d, J ) 6.7 Hz, 3H), -0.43 (m,
1H); 13C NMR (CDCl3, 125 MHz, two rotamers) δ 174.3, 173.9, 172.8,
172.4, 172.1, 169.7 and 168.4, 156.3 and 155.7, 132.2 and 132.1, 123.7
and 123.5, 122.8, 122.4, 122.1, 121.3, 120.2, 119.6, 118.8 and 118.7,
108.5 and 108.2, 106.7 and 106.6, 80.3 and 79.6, 65.9 and 65.6, 60.9,
56.2, 52.2, 52.1, 52.0, 51.7, 49.1, 47.7 and 47.6, 47.3, 42.2, 40.0, 39.2,
38.7, 37.4, 37.1, 31.0, 30.9, 30.8, 30.6, 29.4, 29.3, 29.2, 29.1, 28.30
and 28.25, 24.6, 23.6, 23.5, 23.4, 23.0, 22.8, 21.5, 19.3, 19.0, 18.9,
14.4, 14.2, 14.1; IR (neat) Vmax 3303, 2956, 2930, 1744, 1707, 1637,
N-[N-(tert-Butyloxycarbonyl)-L-leucinyl]-N1′-methoxy-N-methyl-
L-tryptophan Methyl Ester (39). A solution of 2031 (36.3 mg, 139
µmol) and N-Boc-LEU (38, 64 mg, 277 µmol) in 1.4 mL of CH2Cl2
was treated with HOAt (38 mg, 277 µmol) and EDCI (53 mg, 277
µmol) at 0 °C. The mixture was stirred for 20 h at 22 °C and
concentrated to dryness under reduced pressure. Chromatography (SiO2,
33% EtOAc-hexane) afforded 39 (59.8 mg, 91%) as a pale yellow
solid: [R]2D2 -53 (c 0.56, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 7.55
(d, J ) 7.9 Hz, 1H), 7.37 (d, J ) 8.2 Hz, 1H), 7.21 (t, J ) 8.1 Hz,
1H), 7.09 (t, J ) 7.9 Hz, 1H), 7.08 (s, 1H), 5.33 (dd, J ) 5.7, 10.1 Hz,
1H), 5.14 (d, J ) 8.6 Hz, 1H), 4.56 (m, 1H), 3.99 (s, 3H), 3.71 (s,
3H), 3.42 (ddd, J ) 1.0, 5.6, 15.6 Hz, 1H), 3.18 (dd, J ) 10.1, 15.8
Hz, 1H), 2.90 (s, 3H), 1.51-1.77 (m, 3H), 1.39 (s, 9H), 0.91 (d, J )
6.5 Hz, 3H), 0.90 (d, J ) 6.5 Hz, 3H); 13C NMR (CDCl3, 125 MHz)
δ 173.7, 171.3, 155.6, 132.3, 123.6, 122.5, 121.4, 119.7, 118.6, 108.3,
106.9, 79.5, 65.6, 57.4, 52.3, 49.1, 42.2, 32.3, 28.3, 24.6, 24.1, 23.4,
21.7; IR (neat) Vmax 3346, 2957, 1742, 1709, 1650, 1504, 1454, 1440,
1392, 1367, 1251, 1169, 1047, 1024, 739 cm-1; FABHRMS (NBA)
m/z 476.2772 (M + H+, C25H37N3O6 requires 476.2761).
1453, 1408, 1366, 1251, 1169, 1097, 1045, 1023, 956, 739 cm-1
;
FABHRMS (NBA-CsI) m/z 848.3548 (M + Cs+, C38H61N5O8 requires
848.3574).
N-[(2S,4R)-2-[N-[(S)-2-[N-[(2S,4R)-2-[[N-[N-(tert-Butyloxycarbo-
nyl)-L-leucinyl]-N1′-methoxy-N-methyl-L-tryptophanyl]amino]-4-
methylheptanoyl]-N-methyl-L-alanyloxy]-4-cyanobutanoyl]amino]-
4-methylheptanoyl]-N-methyl-L-leucine tert-Butyl Ester (44). A
solution of 41 (7.6 mg, 10.6 µmol) was dissolved in 0.3 mL of
t-BuOH-H2O (2:1) and treated with LiOH (0.5 mg, 21.2 µmol) at 0
°C. After being stirred for 100 min at 0 °C, the mixture was diluted
with aqueous 1 N HCl (1 mL) and extracted with EtOAc (3 × 2 mL).
N-[N-(tert-Butyloxycarbonyl)-L-leucinyl]-N1′-methoxy-N-methyl-
L-tryptophan (40). A solution of 39 (100 mg, 211 µmol) dissolved in
(32) Olsen, R. K. J. Org. Chem. 1970, 35, 1912.

6204 J. Am. Chem. Soc., Vol. 121, No. 26, 1999
Boger et al.
The combined organic phase was dried over MgSO4 and evaporated
under reduced pressure to give a pale yellow solid. A sample of 34
(4.4 mg, 9.4 µmol) and Ph3P (13.9 mg, 53.1 µmol) in 200 µL of toluene
was added, and the mixture was cooled to 0 °C. Diisopropylazodicar-
boxylate (10.4 µL, 52.9 µmol) was added and stirring was continued
for 16 h at 22 °C. Evaporation of the solvent and chromatography (SiO2,
60% EtOAc-hexane) afforded 19.5 mg of crude product which was
contaminated with large amounts of reagent. Further purification by
semipreparative HPLC (reverse-phase C18, 90% CH3OH-H2O) gave
(toluene/CH3OH 95:5), and purified by chromatography (SiO2, 0.5-
4% CH3OH-toluene, gradient) to give 393 mg (33%) of the thioamide
50 (colorless solid foam) and 110 mg (9%) of the bis-thioamide 51
(colorless solid foam) and recovered starting material. For 50: 1H NMR
(CDCl3, 500 MHz) δ 9.38 (br d, 1H), 8.02 (br d, J ) 9 Hz, 1H), 7.49
(d, J ) 9.5 Hz, 1H), 7.48 (d, J ) 8 Hz, 1H), 7.41 (d, J ) 8 Hz, 1H),
7.22 (ddd, J ) 7, 8, 1 Hz, 1H), 7.11 (s, 1H), 7.07 (ddd, J ) 7, 8, 1 Hz,
1H), 5.32 (ddd, J ) 9.5, 8.7, 5.5 Hz, 1H), 5.16 (ddd, J ) 4.6, 7.5, 8
Hz, 1H), 5.13 (dd, J ) 9.9, 2.7 Hz, 1H), 5.02 (ddd, J ) 12, 9, 2.4 Hz,
1H), 4.19 (dd, J ) 9, 7 Hz, 1H), 4.05 (s, 3H), 3.73 (dd, J ) 5.3, 14.7
Hz, 1H), 3.72 (q, J ) 7 Hz, 1H), 3.47 (s, 3H), 3.45 (m, 1H), 3.30 (dd,
J ) 8.6, 14.7 Hz, 1H), 3.29 (s, 3H), 2.37 (s, 3H), 1.50 (d, J ) 7 Hz,
3H), 1.00 (d, J ) 7 Hz, 3H); 13C NMR (CDCl3, 125 MHz) δ 197.4,
175.5, 172.8, 169.8, 168.9, 168.3, 168.0, 132.5, 123.5, 122.6, 122.0,
119.7, 118.5, 118.1, 108.8, 108.5, 75.5, 73.2, 66.7, 65.8, 59.7, 54.2,
48.9, 46.1, 40.9, 40.0, 39.0, 38.7, 38.5, 38.1, 37.4, 37.4, 37.4, 29.5,
29.2, 29.1, 28.8, 27.7, 25.5, 25.1, 23.6, 23.2, 23.0, 23.0, 22.8, 22.3,
21.8, 19.2, 19.16, 14.6, 14.2, 14.1, 13.6; ESIMS m/z 993.8 (M + H+,
C53H84N8O8S requires 993.6); Anal. calcd for C53H84N8O8S: C, 64.08;
H, 8.52; N, 11.28. Found: C, 63.79; H, 8.51; N, 11.16.
44 (7.2 mg, 67%) as a white solid: [R]2D2 -94 (c 0.11, CHCl3); H
1
NMR (CDCl3, 600 MHz, major rotamer) δ 8.08 (d, J ) 9.2 Hz, 1H),
7.63 (d, J ) 7.8 Hz, 1H), 7.54 (d, J ) 7.8 Hz, 1H), 7.35 (d, J ) 8.2
Hz, 1H), 7.19 (t, J ) 7.3 Hz, 1H), 7.06-7.12 (m, 1H), 7.08 (s, 1H),
5.46 (dd, J ) 7.0, 8.7 Hz, 1H), 5.30 (dd, J ) 4.8, 10.4 Hz, 1H), 5.25
(d, J ) 9.1 Hz, 1H), 5.12 (dd, J ) 4.3, 7.3 Hz, 1H), 4.99 (t, J ) 7.6
Hz, 1H), 4.86 (m, 1H), 4.56 (t, J ) 8.9 Hz, 1H), 3.99 (s, 3H), 3.30
(dd, J ) 6.5, 15.6 Hz, 1H), 3.12-3.22 (m, 1H), 3.08 (s, 3H), 3.04 (s,
3H), 2.93 (s, 3H), 1.92-2.31 (m, 5H), 1.10-1.70 (m, 42H), 0.81-
0.99 (m, 21H), 0.78 (d, J ) 6.6 Hz, 3H), 0.65 (d, J ) 6.4 Hz, 3H); 13
C
NMR (CDCl3, 125 MHz) δ 173.6, 173.5, 172.0, 170.8, 169.9, 168.2,
155.7, 132.1, 123.7, 122.4, 121.4, 119.7, 118.8, 108.2, 106.8, 81.4,
79.4, 73.2, 65.6, 58.0, 56.1, 55.1, 49.3, 48.1, 46.7, 42.2, 39.5, 38.1,
37.5, 36.9, 36.8, 35.8, 30.8, 30.6, 29.34, 29.30, 29.06, 29.02, 28.3, 28.1,
27.1, 24.7, 24.6, 24.1, 23.5, 23.1, 23.0, 22.9, 21.5, 21.3, 19.5, 18.9,
14.1, 13.6, 13.5; IR (neat) Vmax 3306, 2928, 2871, 2248, 1646, 1634,
1539, 1546, 1368, 1165, 1096, 1046, 955, 803, 739 cm-1; FABHRMS
(NBA-CsI) m/z 1283.6604 (M + Cs+, C62H102N8O12 requires 1283.6672).
HUN-7293 (1). A sample of 44 (2.0 mg, 1.74 µmol) was dissolved
in 50 µL of anisole, 250 µL of trifluoroacetic acid was added, and the
mixture was stirred for 60 min at 22 °C. The solvent was removed
with a stream of N2, and the residue was dissolved in 100 µL of 3.5 M
HCl-EtOAc. The solvent was removed with a stream of N2, and the
residue was dissolved in 1 mL of DMF (freshly distilled). NaHCO3
(0.3 mg, 3.5 µmol), HOAt (0.5 mg, 3.5 µmol), and EDCI (0.7 mg, 3.5
µmol) were added at 0 °C. The mixture was stirred for 3 h at 0 °C, and
additional EDCI (0.7 mg, 3.5 µmol) was added. Stirring was continued
for 3 h at 0 °C, and the solvent was removed under reduced pressure.
Chromatography (SiO2, 10% i-PrOH-toluene) afforded 1 (1.2 mg,
71%) as a white solid: [R]2D2 -221 (c 0.06, CH3OH), lit.28 [R]2D2 -234
(c 1.11, CH3OH); 1H NMR (CDCl3, 500 MHz, major rotamer) δ 8.48
(d, J ) 10.1 Hz, 1H), 8.05 (d, J ) 9.7 Hz, 1H), 7.63 (d, J ) 7.8 Hz,
1H), 7.54 (d, J ) 7.8 Hz, 1H), 7.35 (d, J ) 8.2 Hz, 1H), 7.19 (t, J )
7.3 Hz, 1H), 7.06-7.12 (m, 1H), 7.08 (s, 1H), 5.46 (dd, J ) 7.0, 8.7
Hz, 1H), 5.30 (dd, J ) 4.8, 10.4 Hz, 1H), 5.25 (d, J ) 9.1 Hz, 1H),
5.12 (dd, J ) 4.3, 7.3 Hz, 1H), 4.99 (t, J ) 7.6 Hz, 1H), 4.86 (mc,
1H), 4.56 (t, J ) 8.9 Hz, 1H), 3.99 (s, 3H), 3.30 (dd, J ) 6.5, 15.6 Hz,
1H), 3.12-3.22 (m, 1H), 3.08 (s, 3H), 3.04 (s, 3H), 2.93 (s, 3H), 1.76-
2.31 (m, 6H), 1.10-1.67 (m, 21H), 0.81-0.99 (m, 21H), 0.78 (d, J )
6.6 Hz, 3H), 0.65 (d, J ) 6.4 Hz, 3H), -0.41 (m, 1H); 13C NMR
(CDCl3, 150 MHz, major rotamer) δ 172.9, 172.6, 171.2, 170.8, 169.8,
168.1, 167.4, 132.3, 123.6, 122.9, 122.3, 120.2, 119.9, 118.8, 108.7,
106.8, 73.8, 65.9, 61.3, 59.5, 57.3, 47.3, 46.9, 39.7, 38.9, 37.7, 37.4,
37.0, 36.9, 36.7, 29.4, 29.2, 29.1, 28.8, 28.4, 26.6, 24.6, 23.9, 23.7,
23.6, 23.0, 22.9, 22.7, 21.8, 19.8, 19.0, 18.6, 14.2, 14.1, 13.7; IR (neat)
Vmax 3280, 2957, 2928, 2822, 2249, 1750, 1652, 1634, 1558, 1539,
1456, 1287, 1194, 1097, 957, 740 cm-1; FABHRMS (NBA-CsI) m/z
1109.5369 (M + Cs+, C53H84N8O9 requires 1109.5416).
Benzylthioimidate 52. A mixture of aqueous NaOH (30%, 1 mL)
and a CH2Cl2 solution containing 37.8 mg of thioamide 50 and 100
µL of benzyl bromide (5 mL) was stirred for 30 min at 25 °C with
repeated sonication. The mixture was neutralized with aqueous 4 N
HCl and extracted with EtOAc. The organic phase was dried over Na2-
SO4 and evaporated and the residue subjected to chromatography
(Sephadex LH-20, CH3OH/EtOAc 1:1) to yield 38 mg (94%) of 52 as
a colorless solid foam; 1H NMR (CDCl3, 500 MHz, 330 K) δ 7.93 (br
d, 1H), 7.87 (br d, J ) 8 Hz, 1H), 7.81 (br d, J ) 8.5 Hz, 1H), 7.35
(d, J ) 8 Hz, 1H), 7.27-7.23 (m, 5H), 7.23 (s, 1H), 7.17 (ddd, J ) 7,
8, 1 Hz, 1H), 7.08 (ddd, J ) 7, 8, 1 Hz, 1H), 5.27 (dd, J ) 6.8, 3.4 Hz,
1H), 5.23 (dd, J ) 11.1, 2.2 Hz, 1H), 5.16 (ddd, J ) 9.5, 9.3, 5.3 Hz,
1H), 4.74 (ddd, J ) 12, 8.5, 2 Hz, 1H), 4.48 (dd, J ) 9.4, 4.5 Hz, 1H),
4.26 (d, J ) 13.5 Hz, 1H), 4.02 (s, 3H), 3.83 (d, J ) 13.5 Hz, 1H),
3.59 (q, J ) 7 Hz, 1H), 3.42 (dd, J ) 14, 9 Hz, 1H), 3.12 (s, 3H), 3.03
(s, 3H), 2.94 (s, 3H), 1.46 (d, J ) 7 Hz, 3H); 13C NMR (CDCl3, 125
MHz, 330 K) δ 174.6, 171.8, 171.2, 169.5, 168.7, 138.0, 132.6, 128.8,
128.4, 127.0, 124.7, 123.2, 122.0, 119.9, 119.6, 119.4, 108.1, 108.1,
72.6, 66.0, 65.4, 59.8, 59.2, 56.6, 49.2, 47.1, 42.5, 40.1, 38.8, 37.3,
37.3, 37.2, 36.0, 35.7, 33.5, 31.1, 29.8, 29.4, 29.0, 28.9, 27.4, 25.9,
25.7, 23.7, 23.5, 23.1, 22.9, 22.8, 22.1, 21.3, 20.1, 18.6, 18.6, 14.0,
13.9, 13.5, 13.3; FABMS (NBA) m/z 1083 (M + H+), 1051 (M -
OCH3+).
Ring-opening: Benzylthioester Hydrochloride 53. A solution of
26 mg of benzyl thioimidate 52 in 2 mL of t-BuOH and 200 µL of
concentrated HCl was warmed at 55 °C for 20 min. The reaction
mixture was diluted with EtOAc and evaporated in vacuo to give 30
mg of 53 as a colorless solid: 1H NMR (CDCl3, 500 MHz, main
conformer, characteristic signals) δ 7.96 (br d, J ) 9 Hz), 7.55 (d, J )
8 Hz, 1H), 7.36 (d, J ) 8 Hz, 1H), 7.3-7.2 (m), 7.12 (s, 1H), 7.3-7.2
(m), 7.06 (ddd, J ) 8, 7, 1 Hz, 1H), 5.55 (m, 1H), 5.24 (dd, J ) 8, 4
Hz, 1H), 5.00 (m, 1H), 4.78 (ddd, J ) 11, 8, 3 Hz, 1H), 4.33 (m, 1H),
4.16 (d, J ) 13.8 Hz, 1H), 4.01 (s, 3H), 3.99 (d, J ) 13.8 Hz, 1H),
3.79 (q, J ) 7 Hz, 1H), 3.08 (s, 6H), 2.93 (s, 3H), 1.46 (d, J ) 7 Hz,
1H), 0.63 (d, J ) 6.5 Hz, 3H); 13C NMR (CDCl3, 125 MHz) δ 199.8,
174.7, 171.6, 169.9, 169.7, 169.4, 168.6, 137.2, 132.3, 128.7, 128.5,
127.4, 123.5, 122.6, 122.0, 119.8, 119.4, 119.0, 108.3, 106.5, 72.9,
65.8, 61.9, 58.9, 57.4, 50.1, 48.3, 47.4, 47.0, 39.7, 39.6, 37.3, 37.0,
36.9, 36.6, 36.4, 33.1, 31.0, 29.7, 29.3, 29.2, 29.1, 29.0, 28.9, 27.4,
24.7, 24.4, 24.2, 23.2, 23.2, 23.0, 22.9, 21.3, 21.2, 19.7, 18.8, 14.1,
13.8, 13.6; ESIMS m/z 1101.4 (M + H+, C60H92N8O9S requires 1101.7),
1135.4 (M + Cls, requires 1135.6).
General Procedure for Cyclization Experiments. A sample of 44
(10.3 mg, 9 µmol) was dissolved in 0.1 mL of anisole, 0.5 mL of
trifluoroacetic acid was added, and the mixture was stirred for 60 min
at 22 °C. The solvent was removed with a stream of N2, and the residue
was dissolved in 0.2 mL of 3.5 M HCl-EtOAc. The solvent was
removed with a stream of N2, and the residue was dissolved in 10 mL
of solvent. Base (2,6-collidine, 10 µL, 9 equiv) and reagent (HATU,
10.2 mg, 3 equiv) were added at 0 °C. The mixture was stirred at the
temperature indicated (25 °C, 10 h), and the solvent was removed under
reduced pressure. Chromatography (SiO2,10% i-PrOH-toluene) af-
forded 1 (7.5 mg, 73%) as a pale yellow solid.
N-[(2S,4R)-2-[N-[(S)-2-[N-[(2S,4R)-2-[[N-L-Leucinyl-N1′-methoxy-
N-methyl-L-tryptophanyl[amino-4-methylheptanoyl]-N-methyl-L-
alanyloxy]-4-cyanobutanoyl]amino-4-methylheptanoyl]-N-methyl-
L-leucine (45). From 53: The crude benzylthioester hydrochloride 53
(30 mg) was taken up in 2 mL of t-BuOH and 100 µL of water, and
an excess of AgClO4 was added (20 mg). After 5 min at 25 °C, the
precipitated silver thiobenzylate was removed by filtration. Excess silver
was removed by extraction of the solution with saturated aqueous NaCl/
EtOAc. After removal of the AgCl by filtration over Celite, the organic
Thioamide 50. A solution of 1.17 g of natural HUN-7293 and 0.36
g of Lawesson’s reagent in 50 mL of xylene was warmed at 130 °C
for 30 min. The mixture was evaporated in vacuo, filtered over SiO2

Total Synthesis of HUN-7293
J. Am. Chem. Soc., Vol. 121, No. 26, 1999 6205
layer was dried over Na2SO4 and evaporated. The crude product was
purified by chromatography (Sephadex LH-20) to give 22.3 mg (92%,
two steps) of 45 as a colorless solid identical to that described earlier.
Macrocyclization of 45 with BOP. A solution of 11 mg of the linear
peptide 45 was dissolved in 20 mL of CH3CN, and 12.3 mg of DMAP
and 22 mg of BOP were added at 25 °C. After 20 min, the reaction
mixture was diluted with EtOAc/aqueous HCl, and the organic layer
was dried over Na2SO4 and evaporated in vacuo. The crude product
was purified by chromatography (Sephadex LH-20, CH3OH/EtOAc 1:1)
to give 11.5 mg of the cyclization product HUN-7293. The identity
(1.0 mg, 5.4 µmol) at -30 °C. The mixture was stirred for 1 h at -30
°C and for 1 h at 0 °C. The crude reaction mixture was subjected to
chromatography (SiO2, 60% EtOAc-hexane) to give 58 (1.4 mg, 52%)
as a white solid. The sample of 58 was dissolved in 100 µL of t-BuOH/
H2O (2:1) and treated with LiOH (0.1 mg, 4.2 µmol) at 0 °C. Stirring
was continued for 1 h at 0 °C. The reaction mixture was diluted with
EtOAc and washed with 1 M aqueous HCl and H2O. Drying over
MgSO4 and evaporation of the solvent gave crude 54, which was further
purified by filtration over Sephadex G-15 (CH3OH) providing the 54
(1.4 mg, 100%) as a white solid: FABHRMS (NBA-CsI) m/z
1127.5563 (M + Cs+, C53H86N8O10 requires 1127.5521).
1
with the natural product was confirmed by H NMR (80% purity of
the crude material after gel filtration), SiO2, and C-8 reverse-phase TLC.
N-[(2S,4R)-2-[[N-[N-[N-[(2S,4R)-2-[N-[(R)-2-Hydroxy-4-cyanobu-
tanoyl]amino]-4-methylheptanoyl]-N-methyl-L-leucinyl]-L-leucinyl]-
N1′-methoxy-N-methyl-L-tryptophanyl]amino]-4-methylheptanoyl]-
N-methyl-L-alanine Methyl Ester (54). (A) Saponification of HUN-
7293. A 200 mg (0.2 mmol) sample of HUN-7293 was dissolved in
THF (5 mL). Then, 0.5 M aqueous LiOH (0.6 mL) was added at 25
°C and the mixture was stirred for 36 h. The reaction mixture was
diluted with EtOAc/cyclohexane (1:2) and washed with 1 M aqueous
HCl (10 mL) and water (3 × 10 mL). Drying over Na2SO4 and
evaporation of the solvent gave the crude free acid. Final purification
was performed by size exclusion chromatography on Sephadex LH-20
(CH3OH) providing 199 mg (98%) of 54. For characterization by NMR,
the lithium salt was prepared. For this, after saponification the solvent
was evaporated and the residue obtained redissolved in CH3OH and
purified by size exclusion chromatography on Sephadex LH-20 (CH3-
OH). 1H NMR (CD3OD, 500 MHz, main conformer, characteristic
signals) δ 7.60 (d, J ) 8 Hz, 1H), 7.41 (d, J ) 8.2 Hz, 1H), 7.22 (dd,
J ) 8.2, 7 Hz, 1H), 7.20 (s, 1H), 7.09 (dd, J ) 8, 7 Hz, 1H), 5.14 (q,
J ) 7.3 Hz, 1H), 5.12 (m, 1H), 4.95 (dd, J ) 11, 3.6 Hz, 1H), 4.89
(dd, J ) 12.1, 3 Hz, 1H), 4.81 (dd, J ) 7.7, 3 Hz, 1H), 4.37 (dd, J )
12.1, 3 Hz, 1H), 4.11 (dd, J ) 7.3, 3.8 Hz, 1H), 4.04 (s, 3H), 3.41 (dd,
J ) 13, 3.6 Hz, 1H), 3.12 (s, 3H), 3.08 (dd, J ) 13, 11 Hz, 1H), 3.01
(s, 3H), 2.92 (s, 3H), 2.55 (m, 1H), 2.47 (m, 1H), 2.07 (m, 1H), 1.89
(m, 1H), 1.36 (d, J ) 7.3 Hz, 3H), 1.12 (m, 1H), 0.83 (d, J ) 6.0 Hz,
3H), 0.39 (d, J ) 6.6 Hz, 3H), 0.05 (d, J ) 6.6 Hz, 3H), -0.52 (ddd,
J ) 13.9, 11, 3 Hz, 1H); ESIMS m/z 995.6 (M + H+, C53H86N8O10
requires 995.7).
Macrolactonization of 54, Preparation of HUN-7293 (1). A
solution of DCC (62 mg, 0.30 mmol), DMAP (55 mg, 0.45 mmol),
and DMAP‚TFA (70 mg, 0.30 mmol) in anhydrous CH3CN (20 mL)
was treated with 54 (100 mg, 0.1 mmol) in CH3CN (5 mL) continuously
added at 60 °C over a period of 5 h, and the resulting solution was
stirred overnight. The solvent was evaporated, and the residue was
dissolved in EtOAc (50 mL), extracted with 1 M aqueous HCl, saturated
aqueous NaHCO3, and saturated aqueous NaCl, and dried over Na2-
SO4. Oligomers were removed by size exclusion chromatography on
Sephadex LH-20 (CH3OH), and the sample was further purified by
chromatography (SiO2, 1-5% CH3OH/toluene gradient). Traces of silica
gel were removed again by filtration over Sephadex LH-20 (CH3OH)
yielding 29 mg (29%) of 1. The identity with the natural product was
1
confirmed by H NMR, HPLC, and TLC.
Acknowledgment. We gratefully acknowledge the financial
support of the National Institutes of Health (CA41101), the
Skaggs Institute for Chemical Biology, and the award of a
Deutsche Forschungsgemeinschaft postdoctoral fellowship (H.K.,
1997-1999). We would like to thank Brigitte Grohmann,
Michael Kern, Christa Rabeck, and Eva-Marie Swoboda for their
excellent technical assistance. Barbara Wolff-Winiski kindly
provided the HUVEC cultures.
Supporting Information Available: Experimental details
for the preparation of 3-11, 13-23, 25-29, and 31 are
provided. This material is available free of charge via the
Internet at http://pubs.acs.org.
(B) From 56 and 57. A solution of 56 (1.1 mg, 2.7 µmol) and 57
(1.7 mg, 2.7 µmol) in 100 µL of CH2Cl2/DMF (5:1) was treated with
NaHCO3 (0.2 mg, 2.7 µmol), HOAt (0.7 mg, 5.4 µmol), and EDCI
JA990918U