Synthetic Studies of Tamandarin B Side Chain Analogues

Article abstract of DOI:10.1021/jo100457w

The syntheses of three tamandarin B analogues are described. The goal of these studies was to prepare material to determine their relative therapeutic index and to gain an oversight as to their potential for clinical applications.

Full text of DOI:10.1021/jo100457w

pubs.acs.org/joc
Synthetic Studies of Tamandarin B Side Chain Analogues
ꢀ
Kenneth M. Lassen, Jisun Lee, and Madeleine M. Joullie*
Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia,
Pennsylvania 19104
mjoullie@sas.upenn.edu
Received February 19, 2010
The syntheses of three tamandarin B analogues are described. The goal of these studies was to prepare
material to determine their relative therapeutic index and to gain an oversight as to their potential for
clinical applications.
Introduction
B (4) was shown to be equipotent with didemnin B (3) against
L1210 cells indicating for the first time that a minor modi-
fication in the alkyl side chain of Ist1 was tolerated.6
The discovery of the didemnin family of marine depsipep-
tides (Figure 1) represented an exciting and intriguing chap-
ter in natural product chemistry.1 The unusual structure of
the macrocycle2 and the impressive in vivo biological activity
of the congeners3 have led to many studies by research
groups from all over the world.4 Clinical studies of a second-
generation didemnin, dehydrodidemnin B (aplidine), con-
tinue to stimulate active research in medicinal chemistry.5
Congeners having norstatine instead of isostatine in the
macrocycle were also isolated (Figure 1). These natural
products may be viewed as having an isopropyl side chain
attached to residue 1 instead of a sec-butyl group and were
found to retain the same biological properties. Nordidemnin
In 2000, Vervoort and Fenical reported the isolation of
two novel cytotoxic cyclic depsipeptides from a Brazilian
marine ascidian of the family Didemnidae.7 They named the
major metabolite tamandarin A (1) and the corresponding
minor metabolite tamandarin B (2). Interpretation of
FABMS data and extensive 2D NMR studies supported
the structures of these new metabolites. Acid and alkaline
hydrolysis allowed the assignment of the absolute config-
urations of the various amino acid components. Tamandar-
ins A and B are structurally similar to didemnin B (3)8,9 and
nordidemnin B (4),10 differing from these natural products
only in the macrocylic core. While the didemnins possess a
23-membered macrocycle containing an R-(R-hydroxyiso-
valeryl)propionic acid (Hip2) unit, the tamandarins contain
(1) Rinehart, K. L., Jr.; Holt, T. G.; Fregeau, N. L.; Keifer, P. A.; Wilson,
G. R.; Perun, T. J., Jr.; Sakai, R.; Thompson, A. G.; Stroh, J. G.; Shield,
L. S.; Seigler, D. S.; Li, L. H.; Martin, D. G.; Grimmelikhuijzen, C. J. P.;
€
Gade, G. J. Nat. Prod. 1990, 53, 771.
(6) Jouin, P.; Poncet, J.; Dufour, M.-N.; Aumelas, A.; Pantaloni, A.
J. Med. Chem. 1991, 34, 486.
(7) Vervoort, H.; Fenical, W.; de A. Epifanio, R. J. Org. Chem. 2000, 65,
782.
(8) Rinehart, K. L., Jr.; Gloer, J. B.; Hughes, R. G., Jr.; Renis, H. E.;
McGovren, J. P.; Swynenberg, E. B.; Stringfellow, D. A.; Kuentzel, S. L.; Li,
L. H. Science 1981, 212, 933.
(2) Hossain, M. B.; Van der Helm, D.; Antel, J.; Sheldrick, G. M.;
Weinheimer, A. J.; Sanduka, S. K. Int. J. Peptide Protein Res. 1996, 47, 20.
(3) Sakai, R.; Rinehart, K. L.; Kishore, V.; Kundu, B.; Faircloth, G.;
Gloer, J. B.; Carney, J. R.; Namikoshi, M.; Sun, F.; Hughes, R. G. J.;
Gravalos, G.; de Quesada, T. G.; Wilson, G. R.; Heid, R. M. J. Med. Chem.
1996, 39, 2819.
ꢀ
(4) Vera, M. D.; Joullie, M. M. Med. Res. Rev. 2002, 22, 102.
(9) Rinehart, K. L., Jr.; Gloer, J. B.; Cook, J. C., Jr.; Mizsak, S. A.;
Scahill, T. A. J. Am. Chem. Soc. 1981, 103, 1857.
(10) Jouin, P.; Poncet, J.; Dufour, M.-N.; Pantaloni, A.; Castro, B. J. Org.
Chem. 1989, 54, 617.
(5) Rodriguez, I.; Polanco, C.; Cuevas, F.; Mendez, P.; Cuevas, C.;
Gallego, P.; Munt, S.; Manzanares, I. (Pharma Mar S.A.). U.S. patent US
7348310, 2008.
DOI: 10.1021/jo100457w
r
Published on Web 03/26/2010
J. Org. Chem. 2010, 75, 3027–3036 3027
2010 American Chemical Society

Lassen et al.
JOCArticle
FIGURE 1. Structures of tamandarins A (1) and B (2) and didemnin B (3), nordidemnin B (4), aplidine (5), and didemnin M (6).
FIGURE 2. Structures of tamandarin A side chain analogues (8, 9, and 10).
the simpler hydroxyisovaleric acid (Hiv2) unit and as such,
consist of a 21-membered macrocycle. Tamandarin A (1)
contains an isostatine (Ist1) moiety while tamandarin B (2)
contains a norstatine (Nst1) residue (Figure 1).
the only congeners that have a norstatine instead of an
isostastine unit in the macrocycle. The first total synthesis
12
ꢀ
of 1 was reported by Joullie and co-workers in 1999,
followed by that of 2 in 2000.13
The initial structural studies of the solution conformation
of 1 indicated that the lack of the chiral propionic unit
resulted in only minor conformational differences.7 The
backbone configuration of 1 is stabilized by the same three
hydrogen bonds present in 3: Ist1 NH-Leu3 CO, Leu3 NH-
MeLeu7 CO, and Lac9 CO-Thr6 NH, providing the same
“bent figure eight” shape of the cyclic peptide ring with the
side chain folding back over the cyclic portion of the
molecule.2 Tamandarin B (2)7 and nordidemnin B (4)11 are
To further explore not only the biological activity profile
of 1 and 2 but also the possibility that the tamandarins could
serve as mimics of the didemnins, a series of tamandarin A
analogues with modifications in the side chain region were pre-
pared and exhibited impressive biological activity.14,15 These
new compounds contained the tamandarin A macrocycle
ꢀ
(12) Liang, B.; Portonovo, P.; Vera, M. D.; Xiao, D.; Joullie, M. M. Org.
Lett. 1999, 1, 1319.
ꢀ
(13) Joullie, M. M.; Portonovo, P.; Liang, B.; Richard, D. J. Tetrahedron
Lett. 2000, 41, 9373.
ꢀ
(14) Liang, B.; Richard, D. J.; Portonovo, P.; Joullie, M. M. J. Am. Chem.
(11) Banaigs, B.; Jeanty, G.; Francisco, C.; Jouin, P.; Poncet, J.; Heitz, A.;
Cave, A.; Prome, J. C.; Wahl, M.; Lafargue, F. Tetrahedron 1989, 45, 181.
Soc. 2001, 123, 4469.
ꢀ
(15) Liang, B.; Vera, M. D.; Joullie, M. M. J. Org. Chem. 2000, 65, 4762.
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SCHEME 1. Original Retrosynthetic Approach to Tamandarins A (1) and B (2)
with some of the same side chains found in naturally occurr-
ing didemnins (Figure 2).16
the tamandarin A analogues in comparison with the natural
product (1) mirrored the behavior of the analogous didemnin
analogues in comparison with the parent structure didemnin
B (3). These results indicated that tamandarins may share
the same molecular targets and mechanism of action as
the didemnins. A more definitive study using fluorescence
microscopy and synthetic fluorescent didemnin and taman-
darin analogues produced more data supporting the theory
of a common mechanism of action for these two families of
compounds.25
Our current research efforts are focused on determining
the effects of side chain structural changes on the biological
activity of tamandarin B (2). To this end, we selected three
side chains that exhibited improved or selective biological
activity when attached to the didemnin macrocycle.3 Two of
these side chains are shown in Figure 2. A side chain, a
ψ[CH2NH] surrogate, was designed in our laboratory to
investigate the importance of the prolyl amide in didemnins
and appeared to maintain the biological activity while
decreasing toxicity.16 Side chain 9 is the side chain of
aplidine, now in clinical testing.5 Analogues 21 and 22 could
therefore produce compounds of therapeutic value. Side
chain 10 present in didemnin M3,26 was chosen for testing
the immunosuppressant activity of 20. According to recent
unpublished results, 20 is believed to be one of the most
potent immunosuppressive agents known. It is currently
under investigation.
Several analogues of tamandarin B involving modifications
in the macrocycle were also prepared and evaluated for anti-
cancer activity providing further insight into the structure-
activity relationships of the tamandarins.17 On the basis of
these promising results, we decided to continue these studies
by preparing novel tamandarin B analogues with the most
active side chains found in the didemnins.
Though the SAR profile and mechanism(s) of action18-20
of these marine metabolites are still evolving, it is well estab-
lished that the biological activities of didemnins are strongly
dependent upon the structure of the side chain peptide
residues.4 The importance of the N-Me-D-Leu7 residue has
been extensively investigated.21-23 Changes including short-
ening of the leucine alkyl chain, removal of the N-Me group,
or replacing D-leucine for L-leucine all caused a significant
decrease in the cytotoxic activity. Interestingly, the two
nordidemin epimers were equipotent as protein synthesis
inhibitors and provided early evidence for separate mecha-
nisms mediating the cytotoxicity and the protein synthesis
inhibition.24 The change in biological activity observed with
ꢀ
(16) Ding, X.; Vera, M. D.; Liang, B.; Zhao, Y.; Leonard, M. S.; Joullie,
M. M. Bioorg. Med. Chem. Lett. 2001, 11, 231.
(17) Adrio, J.; Cuevas, C.; Manzanares, I.; Joullie, M. M. J. Org. Chem.
ꢀ
2007, 72, 5129.
(18) Ahuja, D.; Geiger, A.; Ramanjulu, J. M.; Vera, M. D.; SirDeshpande,
Although synthetic studies of didemnins and tamandarins
are known, synthetic approaches to their structures still
present challenging problems. The ability to selectively
couple the side chain to the macrocycle as a final step was
the strategy behind our original retrosynthetic approach
ꢀ
B. V.; Pfizenmayer, A.; Abazecd, M.; Krosky, D. J.; Beidler, D.; Joullie,
M. M.; Toogood, P. L. J. Med. Chem. 2000, 43, 4212.
(19) Ahuja, D.; Vera, M. D.; SirDeshpande, B. V.; Morimoto, H.;
ꢀ
Williams, P. G.; Joullie, M. M.; Toogood, P. L. Biochemistry 2000, 39, 4339.
(20) Vera, M. D.; Pfizenmayer, A. J.; Ding, X.; Ahuja, D.; Toogood,
ꢀ
P. L.; Joullie, M. M. Bioorg. Med. Chem. Lett. 2001, 11, 1871.
ꢀ
(21) Mayer, S. C.; Pfizenmayer, A. J.; Joullie, M. M. J. Org. Chem. 1996,
61, 1655.
(22) Pfizenmayer, A. J.; Ramanjulu, J.; Vera, M. D.; Ding, X.; Xiao, D.;
ꢀ
ꢀ
Chen, W.-C.; Joullie, M. M. Tetrahedron 1999, 55, 313.
(25) Joullie, M. M.; Leonard, M. S.; Portonovo, P.; Liang, B.; Ding, X.;
(23) Pfizenmayer, A. J. Ph.D. Thesis, University of Pennsylvania, 1998.
(24) Dominice, C.; Dufour, M.-N.; Patino, N.; Maanzoni, O.; Grazzini,
E.; Jouin, P.; Guillon, G. J. Pharmacol. Exp. Ther. 1994, 271, 107.
LaClair, J. J. Bioconjugate Chem. 2003, 14, 30.
(26) Sakai, R.; Stroh, J. G.; Sullins, D. W.; Rinehart, K. L. J. Am. Chem.
Soc. 1995, 117, 3734.
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FIGURE 3. Structures of tamandarin B side chain analogues 20, 21, and 22.
SCHEME 2. Revised Retrosynthetic Approach to the Tamandarin B Macrocycle (23)
ꢀ
as it would facilitate future side chain analogue production
(Scheme 1).
B macrocyle (19) was needed. The approach used by Joullie
and co-workers to prepare tamandarin B analogues modi-
fied in the macrocycle afforded a much improved yield in
comparison to the original syntheses of 1 and 2 and therefore
was adapted to prepare the tamandarin B macrocycle (19).28
The revised retrosynthetic approach is shown in Scheme 2.
Rather than closing the macrocycle at the Nst1-Thr6 posi-
tion, the new approach selected the Pro4-N,O-Me2Tyr5
junction as the site of macrolactamization. Several didemnin
syntheses also have used this site very successfully.29,30 With
a reliable, higher yielding route to the macrocycle,31 our
focus shifted to prepare a variety of side chains which could
be coupled to the tamandarin B scaffold and subsequently
screened for biological activity.
Results and Discussion
Naturally occurring tamandarin B (2) was reported to
possess more potent antitumor activity than didemnin B.16
In an effort to further explore the structure-activity rela-
tionship of 2 as well as the relationship between the taman-
darins and the didemnins, we now report the preparation of
three tamandarin B side chain analogues (Figure 3). The
selected side chains are those found in the naturally occur-
ring aplidine (dehydrodidemnin B)3,27 and didemnin M.3
The third side chain is a novel didemnin B analogue,16
a
ψ[CH2NH]amide bond surrogate between N-Me-D-Leu7
and Pro8 that exhibited enhanced activity in the NCI-60
tumor cell screen.
ꢀ
(28) Adrio, J.; Cuevas, C.; Manzanares, I.; Joullie, M. M. Org. Lett. 2006,
8, 511.
(29) Hamada, Y.; Kondo, Y.; Shibata, M.; Shioiri, T. J. Am. Chem. Soc.
1989, 111, 669.
(30) Jou, G.; Gonzales, I.; Albericio, F.; Lloyd-Williams, P.; Giralt, E.
J. Org. Chem. 1997, 62, 354.
To efficiently prepare these side chain tamandarin B
analogues, an improved synthetic route to the tamandarin
ꢀ
(31) Lassen, K. M.; Lee, J.; Joullie, M. M. Tetrahedron Lett. 2010, 51,
1635.
(27) Depenbrock, H.; Peter, R.; Faircloth, G. T.; Manzanares, I.; Jimeno,
J.; Hanauske, A. R. Br. J. Cancer 1998, 78, 739.
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SCHEME 3. Synthesis of Aplidine Side Chain 32
SCHEME 4. Installation of the Aplidine Side Chain 32
Synthesis of Dehydrotamandarin B. Dehydrodidemnin B
(aplidine)3,32,33 features the same macrocyclic core structure
as didemnin B, with the point of differentiation lying in the
side chain. Aplidine features a pyruvyl group at position 9
whereas didemnin B possesses a lactyl group. Aplidine was
found to be approximately 10 times as active as didemnin B
against L1210 murine leukemia cells and P388 murine
leukemia. Aplidine is also strikingly active against two more
difficult cancers, B16 melanoma and Lewis lung. Addition-
ally, an increased life span was observed with leukemia,
melanoma, and ovarian cancer, and a reduction in tumor
size observed with nonsmall cell lung, breast, prostate, and
gastric tumors. Researchers were impressed by the enhanced
antitumor activity and the reduced toxicity, in particular the
lack of cardiac toxicity, which had been an issue with
didemnin B clinical trials.3,32,33 Aplidine was in phase II
clinical trials for renal, head and neck, pancreas, lung
(NSCLC), and colorectal cancer.5,34-37 Aplidin is used
clinically in anticancer treatments in combination with
Gemcitabine or Carboplatin. In addition to its cytotoxicity
activity it also exhibits strong antiangiogenic activity that
supports its use in multiple myeloma. It appears to be well
tolerated in vivo.
In an effort to examine the influence of the pyruvyl group
on the biological activity of tamandarin B, the synthesis of
dehydrotamandarin B (21) was undertaken.
The first step in the synthesis was Boc protection of D-
leucine followed by benzyl ester formation (Scheme 3).38
Treatment of the Boc protected amine with NaHMDS and
methyl iodide resulted in N-methylation in good yield (29).
Removal of the Boc group afforded the hydrochloride salt 29
in quantitative yield. This salt was then coupled to Boc-Pro
with BOPCl in high yield (30). Removal of the Boc group and
coupling to L-lactic acid with BOP provided alcohol 31.
Oxidation followed by benzyl group removal under hydro-
genolysis conditions led to the fully functionalized side chain
(32). The synthesis of this side chain is similar to our
previously published route. The only minor difference is
the Me-D-Leu acid portion is protected as a benzyl ester
instead of a methyl ester. This minor change allows inter-
mediate 31 to be used as a common intermediate in the
synthesis of 20 without protecting group manipulation
(Scheme 3).14 The synthesis of the novel analogue (-)-dehydro-
tamandarin B was completed by the BOP- mediated coupling
between acid 32 and the amine salt of macrocycle 23 in 73%
yield (Scheme 4). This coupling was significantly better than
previous efforts on the tamandarin A macrocycle which
proceeded in 43% yield.14
(32) Lobo, C. G.; GarciaPozo, S. G.; deCastro, I. N.; Alonso, F. J.
Anticancer Res. 1997, 17, 333.
(33) Urdiales, J.; Morata, P.; Nunes de Castro, I.; Sanchez-Jimenez, F.
Cancer Lett. 1996, 102, 31.
(34) Armand, J. P. A. V.; Ady-Vago, N.; Faivre, S. Proc. Am. Soc. Clin.
Oncol. 2001, 20, 120a (Abstract).
(35) Broggini, M.; Marchini, S. V.; Galliera, E.; Borsotti, P.; Taraboletti,
G.; Erba, E.; Sironi, G.; Jimeno, J.; Faircloth, G. T.; Giavazzi, R.; D’Incalci,
M. Leukemia 2003, 17, 52.
(36) Faivre, S.; Chieze, S.; Delbaldo, C.; Ady-Vago, N.; Guzman, C.;
Lopez-Lazaro, L.; Lozahic, S.; Jimeno, J.; Pico, F.; Armand, J. P.; Martin,
J. A.; Raymond, E. J. Clin. Oncol. 2005, 23, 7871.
Synthesis of Tamandarin M. The isolation of didemnin M,
along with six other didemnins, was reported in 1995 by
Rinehart and co-workers.26 Didemnin M had previously
been reported by another group, but named didemnin H.39
(37) Gutierrez-Rodriguez, M.; Martin-Martinez, M.; Garcia-Lopez,
M. T.; Herranz, R.; Cuevas, F.; Polanco, C.; Rodriguez-Campos, I.; Man-
zanares, I.; Cardenas, F.; Feliz, M.; Lloyd-Williams, P.; Giralt, E. J. Med.
Chem. 2004, 47, 5700.
(38) Dutton, F. E.; Lee, B. H.; Johnson, S. S.; Coscarelli, E. M.; Lee, P. H.
J. Med. Chem. 2003, 46, 2057.
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SCHEME 5. Synthesis of Tamandarin Side Chain M (35)
SCHEME 6. Installation of the Tamandarin M Side Chain (35)
In an extensive study of the biological properties of several
didemnin congeners, Rinehart and co-workers reported that
didemnin M showed the strongest inhibition of the immune
response, with an IC50 value in the picomolar range in a two-
way mixed lymphocyte response (MLR) assay.3 In an effort
to further explore the structure-activity relationships of the
tamandarins, we sought to prepare a hybrid analogue,
tamandarin M (20). The synthesis of the tamandarin M side
chain began with 31, an intermediate in the synthesis of the
aplidine side chain (Scheme 3). Esterification of 31 with
BocGlu(Xan)OH using EDCI led to compound 33
(Scheme 5). Simultaneous removal of the Boc and xanthyl
group with HCl gas led to the corresponding salt, which was
condensed with the pentafluorophenyl ester of Cbz-
pGluOPfP to yield 34. The remaining protecting groups were
removed under hydrogenolysis conditions to yield side chain
35. This second-generation synthesis of this side chain is
shorter than its original synthesis. Compound 31 was where
the first- and second-generation synthesis converged. The
original synthesis takes eight steps and six of these are
protection/deprotection steps. In this more efficient second-
generation synthesis compound 31 is made in seven steps,
but only four reactions are protections/deprotections. Also
no protecting groups need to be exchanged in this second-
generation synthesis. In the original synthesis compound
31 is first formed as a methyl ester and has to be con-
verted to a benzyl ester through a hydrolysis/reprotection
sequence.15
The preparation of 20 was achieved via the coupling of the
amine salt of the tamandarin B macrocycle (23) and fragment
35, using BOP to afford the final product in 81% yield
(Scheme 6). This yield was significantly increased with the
tamandarin B macrocycle. Coupling of this side chain to the
tamandarin A macrocycle only proceeded in 32% yield.15
Synthesis of ψ[CH2NH] Amide Surrogate Analogue. In an
effort to explore the structure-activity relationship of the
tamandarins, in particular, the influence of the side chain
portion, and perhaps improve the biological potency of these
compounds, a side chain with an amide bond surrogate,
previously tested in the didemnins,16 was prepared. In this
analogue (22), the amide bond between the leucine and
proline residues of the side chain was reduced. Commercially
available N-Boc-L-proline (36) was converted to aldehyde 37
with use of a known reduction/oxidation sequence.40 Re-
ductive amination of 36 with the free amine of salt 29 led to
38.41 Removal of the Boc protecting group, followed by
coupling to L-lactic acid provided the protected side chain 39
in 61% yield over two steps. Benzyl group removal afforded
side chain acid 40 (Scheme 7).42 This synthesis is one step
(40) Reed, P. E.; Katzenellenbogen, J. A. J. Org. Chem. 1991, 56, 2624.
(41) Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.;
Shah, R. D. J. Org. Chem. 1996, 61, 3849.
(39) Boulanger, A.; Abou-Mansour, E.; Badre, A.; Banaigs, B.;
Combaut, G.; Francisco, C. Tetrahedron Lett. 1994, 35, 4345.
(42) Ding, A. X. Ph.D. Thesis, University of Pennsylvania, 2000.
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SCHEME 7. Synthesis of Reduced Side Chain 40
SCHEME 8. Installation of the ψ[CH2NH] Amide Surrogate Side Chain 40
shorter than our previous synthesis and uses amine salt 29,
which is a common intermediate in the synthesis of the two
previously discussed side chains.16 Finally, coupling side
chain 40 to the macrocyclic salt led to the desired compound
(22) in 80% yield (Scheme 8). This coupling yield is higher
than the previous example of coupling this side chain to the
didemnin B macrocycle (72%).16
analogues were tested at the National Cancer Institute
against various cell lines in vitro and all new compounds
exhibited promising bioactivity. The immunosuppressant
activity of 20 is under investigation and appears to be more
potent than that of didemnin M.
Experimental Section
Biological Activity. The three analogues were submitted to
the NCI for screening in the NCI-60 tumor cell screen.
Tamandarin M (20) delivered remarkable activity with
GI50 values for all cell lines that were e10 nM while main-
taining TGI values that were less than 200 nM in 50 of the cell
lines and LC50 values that were greater than 1 μM in 45 of the
cell lines examined. Dehydrotamandarin B (21) had GI50
values that were e10 nM in 23 of the 60 cell lines with TGI
values that were less than 700 nM in 29 of the cell lines and
LC50 values of greater than 1 μM in 55 of the cell lines. Amide
surrogate (22) had 28 GI50 values below 10 nM, 44 TGI
values below 700 nM, and 48 LC50 values greater than 1 μM.
Of these three analogues tamandarin M (20) has the most
potent activity across the cell lines tested.
General Methods. All reactions were carried out under an
argon atmosphere. Tetrahydrofuran was distilled over sodium
benzophenone, and dichloromethane, methanol, and 1,2-di-
chloroethane were distilled over calcium hydride prior to use.
Column chromatography was conducted with silica gel 60
(240-400 mesh) and solvent systems are listed under individual
experiments. Analytical thin-layer chromatography (TLC) was
performed on silica gel (60F-254) plates (0.25 mm). Plates were
visualized using ceric ammonium molybdate, or ninhydrin.
Proton and carbon magnetic resonance spectra were recorded
on a 500 MHz spectrometer at 500 and 125 MHz, respectively.
General Procedure for Removal of Benzyl and Cbz Groups.
The desired substrate was dissolved in a 1:1 mixture of anhy-
drous MeOH/EtOAc (0.1 M). To this solution was added 10%
Pd/C (10 wt %). The reaction vessel was evacuated and purged
with hydrogen gas. The reaction was monitored by TLC and was
filtered through a pad of Celite when complete. Removal of
solvent under reduced pressure provided the product, which was
used without further purification.
Procedure for the Preparation of Tamandarin B Macrocyclic
Salt. Macrocycle 23 (14 mg, 0.017 mmol) was dissolved in
EtOAc (4 mL) and cooled to -30 °C. HCl gas was bubbled
over this solution while keeping the temperature at -30 °C for
10 min. The flow of HCl gas was stopped and stirring con-
tinued at -30 °C for an additional 30 min. The temperature was
raised to 0 °C for 1 h. At this point TLC showed that the start-
ing material had been consumed. The solvent was removed
Conclusion
In view of the promising bioactivity of some of the
members of the tamandarin family, we have been actively
investigating their synthetic routes, many of which have
proven challenging and have required extensive experimen-
tation. Three novel tamandarin B side chain analogues (20,
21, and 22) have been prepared by using an alternate synth-
esis of the tamandarin B macrocycle. Two of the compounds
(20 and 21) contain side chains found in naturally occurring
didemnin compounds with enhanced bioactivity. All three
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JOCArticle
under reduced pressure to yield the macrocyclic salt (13 mg,
quantitative) as a yellow solid: 1H NMR(500MHz, CDCl3) δ0.94
(m, 18H), 1.19-1.52 (m, 5H), 1.66 (s, 2H), 1.80 (s, 2H), 1.92-2.15
(m, 5H), 2.25 (m, 2H), 2.37 (m, 1H), 2.65 (s, 3H), 2.99 (m, 1H),
3.16-3.36 (m, 2H), 3.43-3.66 (m, 2H), 3.78 (s, 3H), 3.96-4.06
(m, 2H), 4.53-4.76 (m, 2H), 4.83 (d, J = 4.16 Hz, 1H), 5.46 (s,
1H), 6.82 (m, 1H), 7.05 (m, 2H), 7.53 (s, 1H), 7.92-8.42 (m, 3H);
13C NMR (125 MHz, CDCl3) δ 16.6, 17.8, 18.7, 20.6, 23.4, 24.9,
28.2, 30.2, 33.7, 37.6, 40.4, 47.0, 49.8, 55.3, 57.8, 59.3, 66.4, 67.4,
69.4, 80.2, 114.0, 129.5, 130.3, 158.6, 166.2, 168.7, 170.6, 171.1,
172.2, 175.1; IR (neat, cm-1) 3333, 2956, 2925, 2873, 1739, 1636,
1512, 1246; HRMS (ESI) m/z calcd for C38H60N5O10 (M - Cl)þ
746.4340, found 746.4342; [R]19D -83.03 (c 0.67, CH2Cl2).
in 4.0 M HCl in dioxane (20 mL) at room temperature. The
reaction was complete after 2 h. The solvent was evaporated to
yield N-Me-D-leucine benzyl ester hydrochloride (29), (3.78 g,
quantitative) as a white solid, which was used without further
purification. Boc-proline (2.99 g, 13.9 mmol) was dissolved in
anhydrous CH2Cl2 (46 mL) and cooled to -15 °C. To this
solution was added BOP-Cl (3.72 g, 14.6 mmol) and NMM
(1. 64 mL, 14.8 mmol). The resulting mixture was stirred for
30 min, then 29 (3.78 g, 13.9 mmol) and NMM (5 mL, 45.9 mmol)
were added. The reaction was allowed to warm to room tempera-
ture and stirred for 24 h. The mixture was diluted with EtOAc
(100 mL), washed with 10% HCl (30 mL), saturated NaHCO3
(30 mL), and brine(30mL), dried over MgSO4, and filtered. After
solvent evaporation the crude product was purified by column
chromatography(10f20%acetone/hexanes) toyieldthe product
(4.75 g, 92%) as a white solid: Rf 0.5 (30% acetone/hexanes); 1H
NMR (500 MHz, CDCl3) δ 0.91 (m, 6H) 1.40 (m, 9H), 1.63
(s, 1H), 1.70-1.78 (m, 1H), 1.79-2.20 (m, 3H), 3.02 (m, 3H),
3.37-3.48 (m, 1H), 3.53-3.69 (m, 1H), 4.60 (dd, J = 8.59, 3.39
Hz), 5.14 (m, 2H), 7.32 (m, 5H); 13C NMR (125 MHz, CDCl3) δ
21.2, 23.3, 25.1, 28.2, 30.4, 31.7, 32.6, 37.8, 46.4, 55.2, 57.1, 66.7,
Boc-D-leucine Benzyl Ester. D-Leucine (5 g, 38.1 mmol) was
suspended in 1 M NaOH (76 mL) and dioxane (23 mL) and the
mixture was cooled to 0 °C. To this solution was added Boc
anhydride (9.15 g, 41.9 mmol) in dioxane (30 mL). The reaction
was allowed to warm to room temperature and stirred over-
night. The reaction mixture was diluted with water (100 mL) and
extracted with hexanes (3ꢀ100 mL). The remaining aqueous
layer was acidified with solid citric acid and extracted with ethyl
acetate (3 ꢀ 100 mL). The organic extracts were washed with
water (50 mL) and brine (50 mL) and dried over MgSO4.
Filtration and concentration under reduced pressure led to the
product as a clear oil (8.51 g, 97%) that was used directly in the
next step. The Boc protected product (8.51 g, 36.8 mmol) was
dissolved in anhydrous DMF (75 mL) and cooled to 0 °C. To
this solution was added cesium carbonate (12.0 g, 36.8 mmol)
and the reaction was allowed to stir for 20 min. Benzyl bromide
(4.37 mL, 36.8 mmol) was added to the reaction mixture via
syringe and the mixture was allowed to warm to room tempera-
ture and stirred overnight. Water (300 mL) was added to the
reaction and was then extracted with hexanes (3 ꢀ 150 mL). The
combined organic extracts were washed with water and brine
and dried over MgSO4. Concentration under reduced pressure
led to the product as a clear oil (10.76 g, 91%): Rf 0.41 (10%
79.5, 128.1, 128.5, 135.6, 153.9, 171.6, 173.3; IR (neat, cm-1
)
2958, 2931, 2872, 1741, 1699, 1661, 1396, 1169; HRMS (ESI) m/z
calcd for C24H36N2O5Na (M þ Na)þ 455.2522, found 455.2515;
[R]26D -1.54 (c 1.0, CH2Cl2).
L-Lactyl-L-prolyl-N-methyl-D-leucine Benzyl Ester (31). Ben-
zyl ester 30 (300 mg, 0.69 mmol) was dissolved in 4.0 M HCl in
dioxane (3 mL) and the mixture was allowed to stir at room
temperature for 2 h. The solvent was removed and the resulting
salt was redissolved in anhydrous CH2Cl2 (3 mL) and cooled to
0 °C. To this solution was added L-lactic acid (52 μL, 0.69 mmol),
BOP (0.368 g, 0.83 mmol), and NMM (0.31 mL, 2.78 mmol).
The mixture was allowed to warm to room temperature and
stirred overnight. The reaction was diluted with CH2Cl2 (25 mL)
and washed with 10% HCl (10 mL), saturated NaHCO3
(10 mL), and brine (10 mL). The organic phase was dried over
Na2SO4, filtered, and concentrated. The crude product was
purified by column chromatography (25% acetone/hexanes)
to yield the product (0.172 g, 61%) as a clear oil: Rf 0.48 (50%
acetone/hexanes); 1H NMR (500 MHz, CDCl3) δ 0.78-1.01 (m,
6H), 1.33 (m, 6H), 1.43 (m, 2H), 1.74 (m, 2H), 1.91 (m, 2H), 2.09
(m, 2H), 3.01 (s, 3H), 3.52 (t, J = 6.75 Hz, 2H), 4.29 (q, J =
5.98 Hz, 1H), 5.15 (m, 3H), 7.31 (m, 5H); 13C NMR (125 MHz,
CDCl3) δ 20.4, 21.3, 23.1, 24.8, 28.3, 32.1, 37.4, 46.5, 55.6, 568,
65.5, 66.6, 128.0, 128.4, 128.6, 135.6, 171.1, 172.1, 173.2; IR
(neat, cm-1) 3415, 2957, 2873, 1739, 1651, 1456, 1129, 843;
HRMS (ESI) m/z calcd for C22H32N2O5Na (M þ Na)þ
427.2209, found 427.2194; [R]26D -14.07 (c 0.76, CHCl3).
Pyruvyl-prolyl-N-methyl-D-leucine Benzyl Ester (32). Lactyl-
prolyl-N-methyl-D-leucine benzyl ester (120 mg, 0.296 mmol)
was dissolved in anhydrous CH2Cl2 (1.00 mL) and dimethyl
sulfoxide (0.30 mL). To this solution was added Dess-Martin
periodinane (629 mg, 1.48 mmol). The reaction was allowed to
stir for 24 h at which time it was quenched with 5% Na2S2O3
(5 mL) and diluted with Et2O (25 mL). The organic layer was
separated and washed with saturated NaHCO3 (5 mL) and
water (5 mL), dried over Na2SO4, filtered, and concentrated.
The crude product was purified by column chromatography
(20% acetone/hexanes) to yield the product (87 mg, 73%) as a
1
EtOAc/hexanes); H NMR (500 MHz, CDCl3) δ 0.92 (d, J =
4.10 Hz, 3H), 0.93 (d, J = 4.10 Hz), 1.46 (s, 9H), 1.49 (m, 1H),
1.65 (m, 2H), 4.49 (s, 1H), 4.89 (s, 1H), 5.16 (q, J = 13.4 Hz, 2H),
7.32 (m, 5H); 13C NMR (125 MHz, CDCl3) δ 21.9, 22.8, 24.8,
28.3, 41.7, 52.1, 66.9, 79.8, 128.1, 128.3, 128.5, 135.7, 155.5,
173.5; IR (neat, cm-1) 3365, 2958, 1718, 1508, 1366, 1251, 1164;
HRMS (ESI) m/z calcd for C18H27NO4Na (M þ Na)þ 344.1838,
found 344.1850; [R]23D 32.56 (c 1.12, MeOH).
N-Me-N-Boc-D-leucine Benzyl Ester. N-Boc-leucine benzyl
ester (3.61 g, 11.2 mmol) was dissolved in anhydrous THF
(56 mL) and cooled to 0 °C. Methyl iodide (3.54 mL, 56.2 mmol)
was added to this solution followed by 1 M NaHMDS (16.8 mL,
16.8 mmol). The reaction mixture was allowed to warm to room
temperature and stirred overnight. The reaction was quenched
by addition of saturated ammonium chloride (50 mL). The
reaction mixture was extracted with diethyl ether (2 ꢀ 50 mL).
The combined organic phases were washed with 10% HCl
(30 mL), saturated NaHCO3 (30 mL), and brine (30 mL), and
dried over MgSO4. The solvent was evaporated to yield the
crude product as a brown oil. The oil was purified by column
chromatography (2f4% EtOAc/hexanes) to yield the product
as a clear oil (2.76 g, 73%): Rf 0.40 (10% EtOAc/hexanes); 1H
NMR (500 MHz, CDCl3) δ 0.92 (d, J = 6.55 Hz, 3H), 0.94 (d,
J = 5.83 Hz, 3H), 1.43 (s, 9H), 1.56 (m, 1H), 1.68 (m, 2H), 2.79
(m, 3H), 4.6-4.8 (m, 1H), 5.15 (m, 2H), 7.33 (m, 5H); 13C NMR
(125 MHz, CDCl3) δ 21.2, 23.7, 24.6, 28.2, 32.4, 37.4, 56.0, 57.2,
66.5, 80.1, 127.3, 128.2, 128.5, 135.7, 155.4, 156.2, 172.26; IR
(neat, cm-1) 2958, 1740, 1702, 1455, 1391, 1367, 1323, 1156, 970;
HRMS (ESI) m/z calcd for C19H29NO4Na (M þ Na)þ 358.1995,
found 358.1990; [R]25D 23.96 (c 1.0, MeOH).
1
yellow oil: Rf 0.27 (30% acetone/hexanes); H NMR (CDCl3,
500 MHz) δ 0.91 (m, 6H), 1.43 (m, 1H), 1.60-1.90 (m, 6H),
2.20-2.45 (m, 3H), 2.81-3.10 (m, 3H), 3.52-3.92 (m, 2H), 4.92
(m, 1H), 5.04-5.22 (m, 3H), 7.36 (m, 3H); 13C NMR (CDCl3,
125 MHz) δ 21.2, 22.3, 23.3, 24.9, 26.1, 31.1, 32.1 37.2, 47.5,
57.8, 58.5, 66.9, 128.1, 128.3, 128.5, 136.5, 163.6, 170.9 172.6,
198.7; IR (neat, cm-1) 3460, 2958, 2873, 1739, 1715, 1652, 1455,
1206; HRMS (ESI) m/z calcd for C22H30N2O5Na (M þ Na)þ
425.2052, found 425.2040; [R]21D -2.58 (c 0.96, CHCl3).
N-Boc-proline-N-Me-D-leucine Benzyl Ester (30). N-Me-N-
Boc-D-leucine benzyl ester (4.66 g, 13.9 mmol) was dissolved
3034 J. Org. Chem. Vol. 75, No. 9, 2010

Lassen et al.
JOCArticle
Dehydrotamandarin B (21). The tamandarin B macrocyclic
salt (8.0 mg, 0.010 mmol) was dissolved in anhydrous CH2Cl2
(1 mL) and cooled to 0 °C. To this solution was added side chain
32 (5.0 mg, 0.015 mmol), BOP (7.0 mg, 0.15 mmol), and NMM
(4.5 μL, 0.041 mmol). The reaction was allowed to warm to
room temperature and stirred overnight. The reaction was
quenched with brine (4 mL) and the mixture was extracted with
EtOAc (3ꢀ10 mL). The organic phase was washed with 10%
HCl (5 mL), 5% NaHCO3 aq (5 mL), and brine (5 mL), dried
over Na2SO4, filtered, and concentrated to yield the crude
product. The product was purified by reverse phase HPLC
(10% MeOH/H2Of100% MeOH gradient over 40 min) to
yield the product (8 mg, 73%) as a white solid: Rf 0.26 (30%
acetone/hexanes); 1H NMR (500 MHz, CDCl3) δ 0.78-1.07 (m,
24H), 1.25 (m, 10H), 1.40 (t, J = 7.49 Hz, 3H), 1.45 (m, 1H), 1.61
(m, 5H), 1.72-2.28 (m, 10H), 2.43-2.50 (m, 1H), 2.53-2.62 (m,
3H), 3.03-3.12 (m, 3H), 3.13-3.20 (m, 1H), 3.55-3.74 (m, 3H),
3.79 (s, 3H), 3.81-3.89 (m, 1H), 3.91-4.04 (m,1H), 4.28-4.40
(m, 1H), 4.63 (m, 1H), 4.89 (t, J = 10.17 Hz, 1H), 5.05 (d, J =
4.49 Hz, 1H), 5.16-5.27 (m, 1H), 5.28-5.36 (m, 1H), 6.84 (m,
2H), 7.08 (d, J = 7.91 Hz, 2H), 7.42-7.56 (m, 1H), 7.72-7.85
(m, 1H); 13C NMR (125 MHz, CDCl3) δ 14.1, 16.6, 17.0, 17.4,
18.9, 20.4, 20.8, 21.4, 22.7, 23.5, 23.8, 24.9, 26.3, 27.1, 28.0, 29.4,
29.7, 30.0, 30.6, 31.2, 34.0, 35.8, 38.7, 39.2, 46.7, 48.7, 48.8, 54.7,
55.2, 56.9, 57.3, 58.3, 58.9, 66.0, 70.9, 78.7, 114.1, 129.8, 130.3,
158.6, 161.3, 168.6, 169.6, 170.4, 170.5, 171.7, 172.2, 172.9,
173.9, 201.11; IR (cm-1) 3340, 2957, 2926, 2871, 1741, 1662,
1636, 1514, 1457, 1170, 1078; HRMS (ESI) m/z calcd for
C53H81N7O14Na (M þ Na)þ 1062.5739, found 1062.5760;
[R]17D -18.68 (c 0.2, CH2Cl2).
ether solution was evaporated and triturated again to produce a
second crop of the hydrochloride salt. The product was isolated
as a white solid (0.182 g, 64%) that was used in the next step
without further purification. The HCl salt (0.182 g, 32 mmol)
was dissolved in anhydrous dichloromethane (1.4 mL) and the
solution was cooled to 0 °C. To this solution was added N-Cbz-
pGlu-OPfP (0.140 g, 32 mmol) and DIPEA (225 μL, 1.28 mmol).
The mixture was allowed to warm to room temperature and
stirred overnight. The reaction was quenched with brine (3 mL)
and diluted with CH2Cl2 (5 mL). The layers were separated and
the aqueous layer was extracted with CH2Cl2 (3ꢀ5 mL). The
combined organic extracts were washed with 10% HCl (5 mL),
5% NaHCO3 aq (5 mL), and brine (5 mL). The organic layer
was dried (MgSO4), filtered, and evaporated to yield the crude
product, which was purified by column chromatography
(1-5% MeOH/CH2Cl2) to yield the product (77 mg, 31%) as
a white solid: Rf 0.41 (10% MeOH/CH2Cl2); 1H NMR (CDCl3,
500 MHz) δ 0.86 (d, J = 6.52 Hz, 3H), 0.91 (d, J = 6.67 Hz, 3H),
1.38 (m, 1H), 1.44-1.57 (m, 3H), 1.69-1.79 (m, 3H), 1.93-2.33
(m, 9H), 2.35-2.47 (m, 2H), 2.68 (t, J = 9.87 Hz, 1H), 2.71
(t, J = 9.97 Hz, 1H), 2.99 (s, 3H), 3.57 (q, J = 7.68 Hz, 1H), 3.70
(q, J = 7.75 Hz, 1H), 4.46-4.60 (m, 2H), 4.86 (dd, J=8.40,
4.76 Hz, 1H), 5.05-5.28 (m, 4H), 5.42 (m, 1H), 6.93 (s, 1H),
7.24-7.39 (m, 10H), 7.51 (d, J = 6.55 Hz, 1H); 13C NMR
(CDCl3, 125 MHz) δ 15.8, 21.1, 22.2, 23.2, 24.9, 25.1, 27.1, 28.3,
31.1, 31.3, 31.6, 37.4, 46.7, 51.9, 55.0, 57.2, 59.5, 66.8, 68.2, 69.2,
127.9, 128.2, 128.5, 128.6, 135.1, 135.5, 151.3, 168.6, 170.7,
171.1, 171.5, 171.9, 173.5, 175.6; IR (neat, cm-1) 3432, 3328,
3214, 2951, 1791, 1742, 1661, 1452, 1304, 1189; HRMS (ESI)
m/z calcd for C40H51N5O11Na (M þ Na)þ 800.3483, found
800.3495; [R]27D -57.03 (c 0.77, CHCl3).
N2-Boc-N5-xanthyl-glutaminyl-O-L-lactyl-L-prolyl-N-methyl-
D-leucine Benzyl Ester (33). L-Lactyl-L-prolyl-N-methyl-D-leu-
cine benzyl ester (2.24 g, 5.5 mmol) was dissolved in anhydrous
THF (30 mL) and the solution was cooled to 0 °C. To this was
added N2-Boc-N5-glutamine (3.07 g, 7.2 mmol), followed by
EDCI (1.48 g, 7.7 mmol) and DMAP (1.34 g, 11 mmol). The
reaction was allowed to warm to room temperature and stirred
overnight. EtOAc (50 mL) was added and the solution was
washed sequentially with 10% HCl (25 mL), saturated NaHCO3
(25 mL), and brine (25 mL), dried over MgSO4, filtered, and
concentrated. The crude product was purified by column chro-
matography (15f20% acetone/hexanes) to yield the product
(3.70 g, 83%) as a white solid: Rf 0.54 (50% acetone/hexanes);
1H NMR (500 MHz, CDCl3) δ 0.73 (t, J = 6.63 Hz, 2H), 0.80 (d,
J = 6.50 Hz, 2H), 0.86 (d, J = 6.65 Hz, 3H), 0.97 (dd, J = 16.2,
6.53 Hz, 1H), 1.35 (d, J = 5.85 Hz, 3H), 1.43 (s, 9H), 1.60-1.69
(m, 2H), 1.78-1.88 (m, 2H), 1.88-2.20 (m, 3H), 2.22-2.42 (m,
2H), 2.70 (m, 3H), 2.89-2.98 (m, 1H), 3.43-3.69 (m, 2H), 3.72
(t, J = 6.25 Hz, 2H), 4.31-4.64 (m, 1H), 4.91 (m, 2H), 5.11 (m,
2H), 6.50 (m, 1H), 7.00-7.12 (m, 4H), 7.18-7.37 (m, 7H),
7.39-7.51 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 11.4, 14.1,
16.0, 21.3, 22.5, 23.1, 24.9, 25.3, 28.3, 29.0, 31.5, 32.0, 34.6, 36.0,
37.3, 43.5, 46.7, 55.8, 56.7, 66.3, 116.2, 121.2, 123.3, 123.5, 127.9,
128.4, 128.8, 129.4, 135.7, 151.1, 168.6, 170.1, 171.9, 172.1; IR
(neat, cm-1) 3305, 2959, 1743, 1712, 1652, 1481, 1456, 1258;
HRMS (ESI) m/z calcd for C45H57N4O10 (M þ Hþ) 813.4075,
found 813.4078; [R]24D -27.24 (c 0.89, CHCl3).
Tamandarin M (20). The tamandarin B macrocyclic salt
(10.0 mg, 0.013 mmol) was dissolved in anhydrous CH2Cl2
(1 mL) and the solution was cooled to 0 °C. To this solution
was added side chain 35 (11.0 mg, 0.019 mmol), BOP (9.0 mg,
0.019 mmol), and NMM (6.0 μL, 0.051 mmol). The reaction was
allowed to warm to room temperature and stirred overnight. The
reaction was quenched with brine (4 mL) and the resulting
mixture was extracted with EtOAc (3 ꢀ 10 mL). The organic
phase was washed with 10% HCl (5 mL), 5% NaHCO3 aq
(5 mL), and brine (5 mL), dried over Na2SO4, filtered, and
concentrated toyield the crude product. The product was purified
by reverse phase HPLC (10% MeOH/H2Of100% MeOH gra-
dient over 40 min) to yield the product (13 mg, 81%) as a white
solid. Rf 0.21 (50% EtOAc/CH2Cl2); 1H NMR (500 MHz,
CDCl3) δ 0.76-1.05 (m, 24H), 1.15-1.32 (m, 10H), 1.36-1.42
(m, 5H), 1.46 (d, J = 7.07 Hz, 2H), 1.52-1.66 (m, 3H), 1.72 (m,
2H), 1.89 (m, 2H), 1.96-2.31 (m, 9H), 2.34-2.46 (m, 2H), 2.54 (s,
3H), 2.61 (m, 1H), 2.92 (m, 1H), 2.97 (s, 2H), 3.06-3.16 (m, 2H),
3.32 (dd, J = 14.16, 3.95 Hz, 1H), 3.53-3.74 (m, 4H), 3.77 (s,
3H), 3.87 (m, 2H), 4.15 (t, J = 4.16 Hz, 1H), 4.49 (m, 2H), 4.57
(dd, J = 7.73, 4.68 Hz, 1H), 4.74 (t, J = 7.05 Hz, 1H), 4.87 (m,
1H), 4.91 (d, J = 5.05 Hz, 1H), 5.01(m, 1H), 5.11 (q, J = 6.70 Hz,
1H), 5.25 (dd, J = 9.45, 6.00 Hz, 1H), 5.94 (s, 1H), 6.81 (d, J =
8.30Hz, 2H), 7.05 (d, J =8.35Hz, 2H), 7.75 (d, J =9.40Hz, 1H),
7.80 (d, J = 9.80 Hz, 1H), 8.52 (d, J = 6.35 Hz, 1H); 13C NMR
(125 MHz, CDCl3) δ 14.2, 15.9, 16.1, 16.9, 17.9, 18.8, 20.3, 20.9,
21.4, 22.7, 23.6, 23.7, 24.6, 24.9, 25.7, 27.2, 27.9, 28.8, 29.3, 29.7,
30.2, 31.1, 31.8, 31.9, 34.0, 26.1, 38.6, 39.2, 39.5, 46.8, 47.1, 48.3,
51.8, 53.8, 54.3, 55.3, 56.3, 56.6, 56.8, 57.0, 58.4, 66.1, 68.7, 69.5,
71.2, 79.4, 114.1, 129.8, 130.4, 158.6, 168.8, 169.4, 169.5, 170.5,
Protected Tamandarin M Side Chain (34). N2-Boc-N5-
xanthyl-glutaminyl-O-L-lactyl-L-prolyl-N-methyl-D-leucine benzyl
ester (407 mg, 0.50 mmol) was dissolved in EtOAc (4.75 mL) and
anisole (0.25 mL) and the mixture was cooled to -20 °C.
Gaseous HCl was added over a 5 min period in which the
reaction color went from yellow to red. The reaction was stirred
at -20 °C for 1 h and at 0 °C for 1 h. Argon was then bubbled
through the solution as it was allowed to warm to room
temperature. The solvent was evaporated and the remaining
residue was triturated with diethyl ether (10 mL) to produce a
white solid, which was removed by filtration. The remaining
170.8, 171.1, 171.3, 172.6, 173.3, 173.5, 176.5, 178.8; IR (cm-1
)
3337, 2958, 1928, 2873, 1741, 1662, 1636, 1514, 1456, 1248;
HRMS (ESI) m/z calcd for C63H96N10O18Na (M þ Na)2-
1303.6802, found 1303.6832; [R]17D -35.49 (c 0.65 CH2Cl2).
N-Boc-Prolinal (37). This compound was prepared according
procedures by Reed. The product matched reported spectral
and physical characteristics.40
J. Org. Chem. Vol. 75, No. 9, 2010 3035

Lassen et al.
66.0, 128.1, 128.3, 128.4, 135.8, 172.5, 173.6; IR (neat, cm-1
3426, 2957, 2925, 2863, 1731, 1639, 1452, 1127; HRMS (ESI)
m/z calcd for C22H35N2O4 (M þ H)þ 391.2597, found 391.2585;
[R]19D -1.55 (c 0.58, CHCl3).
JOCArticle
N-Boc-Pro-ψ(NHCH2)-N-Methyl-leucine Benzyl Ester (38).
Boc-prolinal (430 mg, 2.15 mmol) and N-methyl-leucine benzyl
ester (508 mg, 2.15 mmol) were dissolved in anhydrous 1,2-
dichloroethane (5 mL). The reaction was cooled to 0 °C and
acetic acid (0.5 mL) was added then the solution was stirred for
20 min. Sodium triacetoxyborohydride (637 mg, 3.01 mmol) was
added to the reaction and the mixture was allowed to warm to
room temperature and to stir for 3 h. The reaction was quenched
with saturated NH4Cl and diluted with CH2Cl2 (15 mL). The
organic layer was washed with 10% HCl (10 mL), 5% NaHCO3
aq (10 mL), and brine (10 mL), dried over Na2SO4, filtered, and
concentrated to yield the crude product. The crude product
was purified by column chromatography (10f20% EtOAc/
hexanes) to afford the product (580 mg, 65%) as a yellow oil:
Rf 0.68 (30% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ
0.89 (m, 6H), 1.42 (s, 9H), 1.56 (m, 1H), 1.68 (m, 1H), 1.80 (m,
4H), 2.30 (m, 4H), 2.51-2.80 (m, 1H), 3.31 (m, 3H), 3.70-3.95
(m, 1H), 5.13 (m, 2H), 7.35 (m, 5H); 13C NMR (125 MHz,
CDCl3) δ 22.4, 22.9, 24.7, 28.5, 36.8, 39.2, 40.3, 46.1, 55.9, 66.1,
)
ψ[CH2NH] Amide Surrogate (22). The tamandarin B macro-
cyclic salt (15.0 mg, 0.019 mmol) was dissolved in anhydrous
CH2Cl2 (1 mL) and cooled to 0 °C. To this solution was added
side chain 39 (12 mg, 0.038 mmol), HATU (12 mg, 0.031 mmol),
and DIPEA (14 μL, 0.077 mmol). The reaction was allowed to
warm to room temperature and stirred overnight. The reaction
was diluted with EtOAc (15 mL) and washed with 10% HCl
(5 mL), 5% NaHCO3 aq (5 mL), and brine (5 mL), dried over
Na2SO4, filtered, and concentrated to yield the product (15 mg,
83%) as a white solid. Rf 0.46 (10% MeOH/CH2Cl2); 1H NMR
(500 MHz, CDCl3) δ 0.0-1.05 (m, 24H), 1.19 (m, 1H), 1.25
(t, J = 7.09 Hz, 3H), 1.29-1.39 (m, 5H), 1.40-1.52 (m, 3H),
1.57-1.81 (m, 5H), 1.82-2.05 (m, 3H), 2.07-2.18 (m, 3H), 2.33
(d, J = 10.00 Hz, 3H), 2.41 (m, 1H), 2.53 (s, 2H), 2.77 (s, 3H),
2.89 (m, 1H), 3.12 (m, 2H), 3.28-3.50 (m, 3H), 3.52-3.71 (m,
3H), 3.76 (s, 3H), 3.94 (m, 1H), 4.26 (m, 2H), 4.56 (m, 2H), 4.84
(d, J = 6.05 Hz, 2H), 5.01 (m, 1H), 6.80 (d, J = 8.15 Hz, 2H),
7.05 (d, J = 7.95 Hz, 2H), 7.62 (d, J = 9.15 Hz, 1H), 7.68 (d, J =
8.34 Hz, 1H), 7.77 (d, J = 8.53 Hz, 1H), 8.00 (t, J = 8.90 Hz,
1H); 13C NMR (125 MHz, CDCl3) δ 15.5, 16.5, 18.1, 18.5, 20.3,
20.8, 21.1, 22.0, 22.6, 23.0, 23.5, 24.4, 24.9, 25.1 25.6, 27.6, 28.0,
30.3, 34.1, 36.0, 36.7, 38.6, 45.8, 46.4, 46.8, 48.3, 54.5, 56.4, 57.1,
57.2, 58.9, 63.6, 64.6, 65.1, 65.4, 65.8, 68.8, 69.3, 71.3, 79.3,
114.0, 129.8, 130.4, 158.6, 165.8, 168.8, 169.8, 170.3, 171.2,
172.5, 172.9, 173.2; IR (cm-1) 3339, 2956, 2920, 2868, 1742,
1661, 1654, 1633, 1511, 1094; HRMS (ESI) m/z calcd for
79.1, 128.2, 128.3, 128.5, 136.0, 154.4, 172.8; IR (neat, cm-1
)
2958, 2873, 1731, 1694, 1456, 1394, 1171; HRMS (ESI) m/z calcd
for C24H38N2O4Na (MþNa)þ 441.2730, found 441.2741; [R]22
D
10.7 (c 0.8 CHCl3).
L-Lactyl-Pro-ψ(NHCH2)-N-methyl-D-leucine Benzyl Ester
(39). N-Boc-Pro-ψ(NHCH2)-N-methyl-leucine benzyl ester
(0.128 g, 0.316 mmol) was dissolved in 4.0 M HCl in dioxane
(5 mL). The reaction was stirred for 2 h and the solvent was
evaporated to yield the hydrochloride salt in quantitative yield.
The resulting salt (0.122 g, 0.316 mmol) was dissolved in
anhydrous CH2Cl2 (2 mL) and cooled to 0 °C. To this solution
was added L-lactic acid (25 μL, 0.332 mmol), BOP (0.154 g,
0.348 mmol), and NMM (173 μL, 1.58 mmol). The reaction was
allowed to warm to room temperature and stirred overnight.
The reaction was diluted with EtOAc (20 mL) and washed with
10% HCl (10 mL), 5% NaHCO3 aq (10 mL), and brine (10 mL).
The organic layer was dried over Na2SO4, filtered, and con-
centrated to yield the crude product. The crude product was
purified by column chromatography (20f50% EtOAc/
hexanes) to yield the product (75 mg, 61%) as a yellow oil: Rf
0.30 (50% EtOAc/hexanes); 1H NMR (500 MHz, CDCl3) δ
0.83-0.92 (m, 6H), 1.29 (d, J = 6.55 Hz, 3H), 1.38-1.70 (m,
4H), 1.76-2.01 (m, 4H), 2.28-2.48 (m, 5H), 2.64-2.87 (m, 1H),
3.24-3.50 (m, 3H), 4.06-4.40 (m, 1H), 5.12 (d, J = 4.75 Hz,
2H), 7.33 (m, 5H); 13C NMR (125 MHz, CDCl3) δ 20.3, 20.9,
22.3, 22.7, 23.4, 23.9, 24.8, 27.7, 29.6, 37.4, 38.4, 46.0, 56.6, 65.4,
C53H86N7O13 (M þ H)þ 1028.6284, found 1028.6300; [R]18
D
-65.55 (c 0.34, CH2Cl2).
Acknowledgment. We thank the National Institutes of
Health (National Cancer Institute) through Grant CA-40081
for financial support. Financial support for the departmental
instrumentation was provided by the National Institutes of
Health (1S10RR23444-1). We thank Dr. George Furst and
Dr. Jun Gu for NMR and cryoprobe assistance.
1
Supporting Information Available: Copies of H and 13C
NMR spectra are provided for all compounds. This mate-
rial is available free of charge via the Internet at http://
pubs.acs.org.
3036 J. Org. Chem. Vol. 75, No. 9, 2010