Total Synthesis of [(2S)-Hiv2]Didemnin M

Full text of DOI:10.1021/jo000253a

4762
J . Org. Chem. 2000, 65, 4762-4765
Tota l Syn th esis of [(2S)-Hiv2]Did em n in M
potency enhancement caused by the glutamine-contain-
ing side chain of 3. We also wish to establish the
consequences of the HIP-to-Hiv modification present in
tamandarin-type congeners for the wide spectrum of
biological effects exerted by this family of natural prod-
ucts. As part of this study, we now disclose the synthesis
of [(2S)-Hiv2]didemnin M (1). This analogue, which has
not been reported as a natural product, contains both the
simplified tamandarin-type macrocycle and the more
complex side chain of didemnin M (3). Biological testing
of this compound and comparison with 3 and 4 will
establish the relevance of both the Hiv2 residue and the
glutamine-containing side chain for biological activities.
Bo Liang, Matthew D. Vera, and Madeleine M. J oullie´*
Department of Chemistry, University of Pennsylvania,
Philadelphia, Pennsylvania 19104-6323
mjoullie@sas.upenn.edu
Received February 23, 2000
In tr od u ction
The didemnins are cyclic depsipeptides isolated from
the tunicate Trididemnum solidum by Rinehart and co-
workers.1 Several reviews discussing their syntheses and
biological activities have been published.2,3 Their struc-
ture-activity relationships have been reviewed.4 Didem-
nin B (2) is the best-studied member of this group of
natural products, and it exhibits antitumor, antiviral,
immunosuppressive, and protein biosynthesis inhibition
activities. Didemnin H,5 also known as didemnin M6(3),
which contains glutamine and pyroglutamic acid residues
in the side chain, displayed subpicomolar potency in an
in vitro immunosuppressive assay,4 roughly 1000-fold
more potent than didemnin B.
Tamandarin A (4), a novel cyclodepsipeptide first
isolated, identified and reported by Vervoort et al.,7 was
recently synthesized in our laboratory.8 This natural
product could be formally viewed as [(2S)-Hiv2]didemnin
B,9 since it contains a hydroxyisovaleryl (Hiv) residue,
rather than the more complex R-(R-hydroxyisovaleryl)-
propionyl (HIP) unit at position 2 of the didemnin
macrocycle. The structures of compounds 1-4 are shown
in Figure 1. Tamandarin A has shown levels of cytotoxic
and protein biosynthesis inhibition activities that are
comparable to those of didemnin B (2),7 indicating that
the macrocylic HIP subunit may not be required for
bioactivity. However, the immunosuppressive activity of
tamandarin-type congeners has not been examined. Our
current research effort is focused on the nature of the
Resu lts
Two semisynthetic approaches to 3 have been exam-
ined. Katauskas and co-workers10,11 synthesized didem-
nin M (3) by derivatization of didemnin A. Wen and co-
workers12 reported methods for the synthesis of a
depsipeptide containing residues 8-11. Deprotection
operations and ester formation between Lac9 and Gln10
were cited as the primary difficulties. We report here an
efficient total synthesis of the side chain containing
residues 7-11 and its coupling to the tamandarin-type
macrocycle to furnish analogue 1. This method should
also permit access to 3 by total synthesis. The retrosyn-
thetic analysis of 1 showing the macrocycle (6) and the
side chain (5) are shown in Figure 2.
The synthesis of the side chain started with the
coupling of O-Bn-Lac-Proline13 (7, Scheme 1) with the
methyl ester of N-Me-D-Leu to afford compound 8,
followed by removal of the benzyl group, and then
saponification of the ester to provide compound 10, which
is the didemnin B side chain.14 Protection of the carboxyl
functionality as its benzyl ester (11), followed by DCC
coupling with BocGln(Xan)OH, afforded a high yield of
compound 12. Removal of the xanthyl group proved to
be nontrivial. Although a 4.0 M HCl solution in dioxane
could not accomplish deprotection, the cleavage of both
the xanthyl and Boc groups was effected using HCl gas
in EtOAc and anisole as a scavenger. The resulting free
amine was coupled with the activated PFP ester of
Z-pGlu to give compound 13, followed by hydrogenolysis
of both the benzyl and benzyloxycarbonyl protecting
groups, to afford the free acid 5. The coupling of the side
chain 5 with the macrocycle 6, synthesized according to
our previous strategy,8 using BOP and NMM in CH2Cl2
at 0 °C, afforded the desired product 1 in 32% yield.
(1) Rinehart, K. L., J r.; Gloer, J . B.; Hughes, R. G., J r.; Renis, H.
E.; McGovren, J . P.; Swynenberg, E. B.; Stringfellow, D. A.; Kuentzel,
S. L.; Li, L. H. Science 1981, 212, 933-935.
(2) Li, W.-R.; J oullie´, M. M. In Studies in Natural Products
Chemistry, Vol. 10, Stereoselective Synthesis (Part F); Atta-ur-Rahman,
Ed.; Elsevier: Amsterdam, 1992; pp 241-302.
(3) Wipf, P. Chem. Rev. 1995, 95, 2115-2134.
(4) 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 r.;
Gravalos, G.; de Quesada, T. G.; Wilson, G. R.; Heid, R. M. J . Med.
Chem. 1996, 39, 2819-2834.
(5) Boulanger, A.; Abou-Mansour, E.; Badre, A.; Banaigs, B.; Com-
baut, G.; Francisco, C. Tetrahedron Lett. 1994, 35, 4345-4348.
(6) Sakai, R.; Stroh, J . G.; Sullins, D. W.; Rinehart, K. L. J . Am.
Chem. Soc. 1995, 117, 3734-3748.
(7) Vervoort, H.; Fenical, W.; de Epifanio, R. J . Org. Chem. 2000,
65, 782-792.
(8) Liang, B.; Portonovo, P.; Vera, M. D.; Xiao, D.; J oullie´, M. M.
Org. Lett. 1999, 1, 1319-1322.
(9) Because of the similarities among the didemnins, nordidemnins
and tamandarins, we have adopted a systematic nomenclature for
these natural products. To this end, we use the numbering convention
as proposed by Sakai et al.6 This system numbers isostatine as residue
1, HIP as residue 2, etc. Using this nomenclature, nordidemnin B (see
J ouin et al. J . Org. Chem. 1989, 54, 617-627) would be named
[Norsta1]didemnin B and tamandarin A would be [(2S)-Hiv2]didemnin
B. This convention provides a convenient and unambiguous description
of the natural products and their synthetic analogues and highlights
the sites of the structural changes made.
The synthetic analogue (1) was tested by the National
Cancer Institute against various cancer cell lines in
vitro.15 A representative sample of cytotoxic activity data,
(10) Katauskas, A. J .; Rinehart, K. L. 213th National Meeting of
the American Chemical Society, San Francisco, CA, 1997; American
Chemical Society: Washington, DC, 1997; pp ORG-408.
(11) Rinehart, K. L.; Katauskas, A. J . PCT Int. Appl. WO 9817275,
April 30, 1998.
(12) Wen, J . J .; Crews, C. M. Tetrahedron Lett. 1998, 39, 779-782.
(13) Xiao, D. Ph.D. Thesis, University of Pennsylvania, Philadelphia,
PA, 2000.
(14) Li, W.-R.; Ewing, W. R.; Harris, B. D.; J oullie´, M. M. J . Am.
Chem. Soc. 1990, 112, 7659-7672.
(15) Boyd, M. R.; Paul, K. D. Drug Dev. Res. 1995, 34, 91-109.
10.1021/jo000253a CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/23/2000

Notes
J . Org. Chem., Vol. 65, No. 15, 2000 4763
F igu r e 1. Structures of [(2S)-Hiv2]didemnin M (1), didemnin B (2), didemnin M (3), and tamandarin A (4).
F igu r e 2. Retrosynthesis of [(2S)-Hiv2]didemnin M (1).
derived from 48 h continuous drug exposure of at least
five concentrations at 10-fold dilutions, is shown in Table
1. Growth inhibition concentratios (GI50) were below 2.50
nM for 32 of 57 cell lines tested; the other 25 cell lines
test data are all in the range of nanomolar concentra-
tions. Interestingly, compound 1 is about 1000-fold more
sensitive against colon cancer/HT29 cell line than didem-
nin B (2).
underway. The results and the comparison with didem-
nin M (3) and tamandarin A (4) will be reported in due
course.
Exp er im en ta l Section
Gen er a l Meth od s. All reactions were performed under
nitrogen. THF and dichloromethane were distilled over sodium-
benzophenone and calcium hydride, respectively. Proton and
carbon magnetic resonance spectra were recorded (500 MHz for
1H, 250 MHz for 13C) on a Bruker AM-500 Fourier transform
spectrometer. Flash column chromatography was carried out on
E. Merck silica gel 60 (240-400 mesh) using the solvent systems
listed under individual experiments.
Compound 1 has shown comparable or even better
antitumor activity than didemnin B (2). In conclusion,
we have achieved the first total synthesis of 1, which also
provides a feasible synthetic route to 3. The synthesis of
3 and the further biological characterization of 1 are
O-Ben zyl-L-lactyl-L-pr olyl-N-m eth yl-D-leu cin e Meth yl Es-
ter (8). To a solution of O-Bn-Lac-proline (7) (1.37 g, 4.95 mmol)

4764 J . Org. Chem., Vol. 65, No. 15, 2000
Notes
Sch em e 1a
7.29-7.35 (m, 5H); HRMS (EI) m/z calcd for C23H34N2O5 (M +
Na+) 441.2365, found 441.2359.
L-La ctyl-L-p r olyl-N-m eth yl-D-leu cin e Meth yl Ester (9).
The protected dipeptide 8 (0.51 g,1.2 mmol) was dissolved in
MeOH/EtOAc (1:1)(10 mL), followed by the addition of 10% Pd/C
(0.152 g), and the suspension was placed under an atmosphere
of H2 for 3 h. The catalyst was collected by filtration, the residue
was washed with HPLC MeOH, and the filtrate was concen-
trated to give a white foam, which was recrystallized in ether/
hexane to afford white crystals (9, 0.4246 g, 98% yield): Rf 0.68
(10:90 methanol/methylene chloride); mp 95.5-96.0 °C; 1H NMR
δ 0.84-1.01 (m, 6H), 1.34-1.36 (d, J ) 6.60 Hz, 3H), 1.39-1.45
(m, 1H), 1.68-1.71 (m, 2H), 1.72-1.96 (m, 2H), 2.07-2.20 (m,
2H), 3.04 (s, 3H), 3.54-3.56 (t, d ) 6.7 Hz, 2H), 3.67 (s, 3H),
4.28-4.32 (m, 1H), 4.90-4.93 (m, 1H), 5.06-5.09 (m, 1H); 13C
NMR δ 20.3, 21.1, 23.2, 25.0, 28.3, 31.9, 37.5, 46.6, 52.2, 55.5,
57.0, 65.4, 171.9, 172.1, 173.3; IR (KBr) 3409, 1736, 1642 cm-1
;
HRMS (EI) m/z calcd for C16H29N2O5 (M + H+) 329.2076, found
329.2081; Anal. Calcd for C16H28N2O5: C, 58.50; H, 8.60; N, 8.53.
Found: C, 58.34; H, 8.67; N, 8.53.
L-La ctyl-L-p r olyl-N-m eth yl-D-leu cin e (10). The white crys-
tals (9, 0.31 g, 0.94 mmol) were dissolved in distilled THF/HPLC
MeOH (1:1) (20 mL), 0.2 M LiOH solution (20 mL) was added,
and the reaction mixture was stirred at room temperature
overnight. The mixture was concentrated in vacuo, and the
aqueous solution was cooled to 0 °C, then acidified to pH 3 with
1 N KHSO4, and extracted with EtOAc. The organic layers were
combined and dried (Na2SO4), filtered and concentrated to afford
a white solid (0.23 g, 77% yield): Rf 0.08 (10:90-methanol:
1
methylene chloride); H NMR δ 0.87-0.88 (d, J ) 6.6 Hz, 3H)
and 0.91-0.92 (d, J ) 6.7 Hz, 3H), 1.40-1.42 (d, J ) 6.7 Hz,
3H), 1.65-1.68 (m, 1H), 1.80-1.85 (m, 2H), 1.98-2.02 (m, 2H),
2.07-2.18 (m, 2H), 2.99 (s, 3H), 3.61-3.63 (m, 2H), 4.42-4.43
(m, 1H), 4.86-4.87 (m, 1H), 5.35-5.38 (dd, J 1)11.3 Hz, J 2)4.4
Hz, 1H),; 13C NMR δ 20.2, 21.3, 23.4, 25.1, 28.5, 31.2, 36.8, 47.0,
54.5, 57.2, 66.2, 171.8, 175.0, 175.1; IR (KBr) 3384, 1703, 1651,
1604 cm-1; HRMS (EI) m/z calcd for C15H27N2O5Na (M + H+)
315.1922, found 315.1924; [R]20D - 24 (c 1.05, CHCl3) [lit.14 [R]25
-23.9 (c 0.85, EtOH)].
D
a
Reagents and conditions: (a) N,O-dimethyl-D-Leu, BOP-Cl,
NMM, CH2Cl2, -15 °C to room temperature (83%); (b) H2, 10%
Pd/C, MeOH/EtOAc, (98%); (c) 0.2 M LiOH solution, MeOH/THF/
H2O (1:1:1) (77%); (d) BnBr, K2CO3, DMF, Bu4NI, (94%); (e) DCC,
DMAP, BocGln(Xan)OH, CH2Cl2, rt (87%); (f) HCl (g), EtOAc/
anisole (20:1); (g) Z-p-GluOPFP, DIEA, CH2Cl2, (67%, two steps
overall); (h) H2, 10% Pd/C, MeOH/EtOAc (1:1), (99%).
L-La ctyl-L-p r olyl-N-m eth yl-D-leu cin e Ben zyl Ester (11).
A solution of compound 10 (0.163 g, 0.52 mmol) in redistilled
DMF (5 mL) was added to anhydrous K2CO3 (0.075 g, 0.54
mmol), Bu4NI (0.038 g, 0.10 mmol), and BnBr (0.25 mL, 2.08
mmol) sequentially. The reaction mixture was stirred at room
temperature for 1 h, and the solvent was removed in vacuo. The
residue was diluted with water and extracted with EtOAc. The
organic layers were combined and washed with 10% HCl, 5%
NaHCO3, and brine and then dried (Na2SO4), filtered, and
concentrated to afford a white solid (11, 0.197 g, 94% yield): Rf
Ta ble 1. Cytotoxic Activities of Did em n in B (2) a n d
[(2S)-Hiv2]d id em n in M (1)a
didemnin B [(2S)-Hiv2]didemnin M
1
0.31 (5:95 methanol/methylene chloride); H NMR δ 0.87-1.02
(2)
(1)
(m, 6H), 1.36-1.37 (d, J ) 6.64 Hz, 3H), 1.38-1.46 (m, 1H),
1.70-1.75 (m, 2H), 1.76-1.90 (m, 2H), 2.06-2.18 (m, 2H), 3.03
(s, 3H), 3.51-3.57 (m, 2H), 4.29-4.47 (m, 1H), 4.91-4.94 (m,
1H), 5.08-5.14 (m, 3H), 7.27-7.36 (m, 5H); 13C NMR δ 20.4,
21.4, 23.2, 25.1, 28.4, 29.4, 37.4, 46.6, 55.7, 57.0, 65.6, 66.8, 128.1,
128.5, 128.7 and 135.8, 171.2, 172.2, 173.3; IR (KBr, CHCl3)
3426, 1738 cm-1; HRMS (EI) m/z calcd for C22H32N2O5Na (M +
Na+) 427.2209, found 427.2213.
GI50
TGI
LC50
13 nM
66 nM
3.8 µM
38.2 µM
4 nM
126 nM
7.6 µM
colon cancer/HT29 (LC50)
29.5 nM
a
Preliminary results from the NCI-60 tumor cell screen.
Concentrations for 50% growth inhibition (GI50), total growth
inhibition (TGI), and 50% cell kill (LC50) are based upon the mean-
graph midpoint across all cell lines.
N2-Boc-N5-xa n t h yl-L-glu t a m in yl-O-L-la ct yl-L-p r olyl-N-
m eth yl-D-leu cin e Ben zyl Ester (12). The alcohol 11 (0.1972
g, 0.49 mmol) was dissolved in CH2Cl2 (2 mL) under argon and
cooled to 0 °C. To this solution were added BocGln(Xan)OH
(0.3121 g, 0.73 mmol), DCC (0.151 g, 0.73 mmol), and DMAP
(0.1192 g, 0.98 mmol) sequentially. The reaction mixture was
stirred at 0 °C for 30 min and then at room temperature
overnight, at which time it was quenched with AcOH/MeOH in
EtOAc (2 mL). The solvent was evaporated in vacuo and the
residue was dissolved in ether (10 mL) and the solid formed
collected by filtration. The ether layer was washed with 10%
citric acid, 5% NaHCO3 and brine. The organic layer was then
dried (Na2SO4), filtered, and concentrated. The resulting residue
was purified by flash column chromatography eluting with
acetone:hexane (10:90 to 30:70) to obtain 12 as a white solid
(0.3461 g, 87% yield): Rf 0.25 (40:60 acetone/hexane); 1H NMR
δ 0.71-0.95 (m, 6H), 1.22-1.30 (m, 1H), 1.34-1.35 (d, J ) 6.2
Hz, 3H), 1.43 (s, 9H), 1.62-1.67 (m, 2H), 1.77-2.05 (m, 4H),
2.15-2.39 (m, 4H), 2.70 (s, 3H), 3.43-3.68(m, 2H), 4.36-4.39
in 15 mL of freshly distilled CH2Cl2 at 0 °C was added BOP-Cl
(1.33 g, 5.2 mmol), followed by the dropwise addition of NMM
(0.6 mL, 5.5 mmol), and the reaction was stirred at -15 °C for
30 min. To this mixture was added N,O-diMe-D-Leu (0.97 g, 4.95
mmol) and another portion of NMM (1.8 mL, 16.4 mmol)
dropwise. The reaction was stirred at -15 °C for 10 min and
then warmed to 0 °C for overnight stirring. The reaction mixture
was diluted with EtOAc, washed with 10% HCl, 5% NaHCO3,
and saturated NaCl solution sequentially, and then dried (Na2-
SO4), filtered, and concentrated. The crude mixture was purified
using flash column chromatography by eluting with MeOH/CH2-
Cl2 (5:95) to obtain the protected dipeptide 8 (1.73 g, 83% yield)
as a yellow oil: Rf 0.64 (10:90 methanol/methylene chloride); 1H
NMR δ 0.86-1.04 (m, 6H), 1.40-1.44 (d, J ) 6.6 Hz, 3H), 1.56-
1.58 (m, 1H), 1.70-1.73 (m, 2H), 1.78-1.88 (m, 2H), 2.05-2.15
(m, 2H), 3.08 (s, 3H), 3.60-3.68 (m, 2H), 3.70 (s, 3H), 4.19-
4.22 (m, 1H), 4.90-4.93 (m, 1H), 5.01-5.04 (t, 1H), 5.29 (s, 2H),

Notes
J . Org. Chem., Vol. 65, No. 15, 2000 4765
(m, 1H), 4.51 (m, 1H), 4.60-4.63 (m, 1H), 4.86-4.94 (m, 2H),
5.09-5.16 (m, 2H), 5.40-5.42 and 7.41-7.48 (m, 1H), 6.48-6.50
(m, 1H), 7.02-7.40 (m, 13H); 13C NMR δ 15.9, 21.3, 21.7, 23.1,
24.9, 28.2 (overlap), 32.0, 37.3, 43.5, 46.6, 52.7, 55.7, 56.7, 66.5,
69.2, 79.8, 116.2, 121.3, 123.3 and 123.5, 127.9, 128.4, 128.8 and
135.7, 151.1, 168.7 (overlap), 171.0, 171.5, 172.0; IR (KBr) 3298,
(0.0534 g, 99%) as a white solid: Rf 0.05 (10:90 MeOH/CH2Cl2);
1H NMR δ 0.82-0.98 (m, 6H), 1.26-1.41 (m, 1H), 1.44-1.45 (d,
J ) 6.68 Hz, 3H), 1.61-1.77 (m, 2H), 1.96-2.18 (m, 8H), 2.19-
2.27 (m, 2H), 2.31-2.45 (m, 2H), 2.94 (s, 3H), 3.550-3.71 (m,
2H), 4.21-4.27 (m, 1H), 4.37-4.43 (m, 1H), 4.86-4.88 (m, 1H),
5.07-5.10 (m, 1H), 5.14-5.22 (m, 1H), 6.68(s, 1H) and 7.29 (s,
1H), 7.53 (s, 1H), 8.02 (s, 1H); 13C NMR δ 15.7, 21.1, 23.2, 25.0,
25.1, 27.0, 28.4, 29.4, 31.0, 31.3, 37.1, 46.9, 52.1, 54.6, 56.8, 57.6,
69.3, 169.1, 171.3, 172.4, 173.0, 174.0, 176.9, 179.8; IR (KBr)
3300, 1734, 1654 cm-1; HRMS (EI) m/z calcd for C25H39N5O9Na
(M + Na+) 576.2645, found 576.2645.
1741, 1713, 1651 cm-1; HRMS (EI) m/z calcd for C45H56N4O10
-
Na (M + Na+) 835.3894, found 835.3921; [R]20 -23.3 (c 0.69,
D
CHCl3). Anal. Calcd for C45H56N4O10: C, 66.47; H, 6.95; N, 6.89.
Found: C, 66.13; H, 7.06; N, 6.44.
L-pGlu -L-Glu -O-L-lactyl-L-pr olyl-N-m eth yl-D-leu cin e Ben -
zyl Ester (13). A solution of compound 12 (0.4065 g, 0.5 mmol)
in EtOAc/MeOPh (20:1, 5 mL) was cooled to -20 °C, and gaseous
HCl was introduced at such a rate that the temperature of the
mixture was maintained between -10 and -20 °C at saturation.
After being stirred for 30 min at this temperature, the reaction
mixture was stirred at 0 °C for 1 h. The solution was then purged
with N2 for about 30 min, maintaining the temperature at 0 °C.
After the solution was concentrated, the residue was triturated
and washed by decantation with three 5.0 mL portions of tert-
butyl methyl ether/hexane (1:4). The product was collected by
filtration and dried in vacuo to provide the hydrochloride salt
(0.285 g, quantitative yield) as a white solid, which was used
directly in the next step. The crude hydrochloride salt was
dissolved in fresh CH2Cl2 (2 mL) at 0 °C, followed by the addition
of DIEA (0.35 mL, 2 mmol) and Z-pGluOPFP (0.215 g, 0.5 mmol).
The reaction was stirred at 0 °C for 1 h, then at room
temperature for another 1 h. The reaction mixture was quenched
with saturated NaCl solution (3 mL), and diluted with CH2Cl2
(5 mL). The mixture was separated and the aqueous layer was
extracted with CH2Cl2, the combined organic layers were washed
with 10% HCl, 5% NaHCO3 and brine, then dried (Na2SO4),
filtered and concentrated. The crude residue was purified by
flash column chromatography eluting with MeOH/CH2Cl2 (5:95)
to provide a white solid (13, 0.2614 g, 67% yield, two steps
overall): Rf 0.47 (10:90 MeOH/CH2Cl2); 1H NMR δ 0.71-0.96
(m, 6H), 1.17-1.23 (m, 1H), 1.46-1.48 (d, J ) 6.84 Hz, 3H),
1.68-1.75 (m, 2H), 1.92-2.25 (m, 10H), 2.35-2.41 (m, 2H),
2.60-2.70 (m, 1H) and 2.95 (s, 2H), 3.55-3.69 (m, 2H), 4.05-
4.10 (dd, J 1)14.27 Hz, J 2)7.13 Hz, 1H), 4.41-4.50 (m, 1H),
4.54-4.59 (m, 1H), 4.82-4.92 (m, 1H), 5.04-5.13 (m, 1H), 5.15-
5.24 (m, 4H), 5.54 (s, 1H) and 6.90 (s, 1H), 7.25-7.34 (m, 10H),
7.58-7.60 (d, J ) 6.4 Hz, 1H); 13C NMR δ 15.8, 21.1, 22.4, 23.1,
25.1, 27.2, 28.2, 31.1, 31.7, 37.4, 46.7, 52.0, 55.1, 57.0, 59.5, 66.7,
68.2, 69.2, 127.9, 128.0, 128.2, 128.4, 128.5, 128.6 and 135.1,
135.5, 151.3, 168.6, 170.7, 171.2, 171.5, 172.0, 173.5, 175.7; IR
(KBr) 3333, 3211, 1791, 1741, 1661 cm-1; HRMS (EI) m/z calcd
for C40H51N5O11Na (M + Na+) 800.3483, found 800.3513.
L-p Glu -L-Glu -O-L-la ctyl-L-p r olyl-N-m eth yl-D-leu cin e (5).
To the suspension of 10% Pd/C (0.023 g) in EtOAc/MeOH (1:1,
2 mL) was added a solution of compound 13 (0.0755 g, 0.097
mmol) in EtOAc/MeOH (1:1, 2 mL). The reaction mixture was
shaken in a Parr hydrogenator under an atmosphere of H2 for
4 h and then was filtered through a fine funnel to collect Pd/C.
The residue was washed with MeOH, and the filtrate was
concentrated in vacuo. The resulting product (5) was obtained
[(2S)-Hiv2]Did em n in M (1). To a mixture of the macrocycle
amine salt (6, 84 mg, 0.11 mmol) and side chain (5) (53.4 mg,
0.096 mmol) in CH2Cl2 (0.30 mL) at 0 °C was added BOP (51
mg, 0.12 mmol) and NMM (0.042 mL, 0.39 mmol). After 30 min
at 0 °C, the reaction mixture was allowed to warm to room
temperature and stir overnight. The solution was then treated
with saturated aqueous NaCl (2 mL) and extracted with EtOAc.
The organic layer was washed with 10% aqueous HCl, 5%
aqueous NaHCO3, and saturated aqueous NaCl, dried (Na2SO4),
filtered, and concentrated. The crude residue was purified by
flash column chromatography (the silica gel was pretreated with
ammonia washed hexane to neutral) eluting with MeOH/CHCl3
(2:98 to 10:90) to obtain 1 (0.0331 g, 32% yield) as a pale yellow
solid: Rf 0.27 (10:90 MeOH/CHCl3); 1H NMR δ 0.78-1.04 (m,
24H), 1.11-1.74 (m, 15H), 1.91-2.30 (m, 17H), 2.34-2.45 (m,
2H), 2.54 (s, 3H), 2.99 (s, 3H), 3.06-3.15 (m, 2H), 3.54-3.61 (m,
4H), 3.64-3.67 (m, 1H), 3.76 (s, 3H), 3.82-3.84 (m, 1H), 3.85-
3.91 (m, 1H), 4.13-4.16 (m, 1H), 4.22-4.24 (m, 1H), 4.46-4.50
(m, 1H), 4.56-4.60 (m, 1H), 4.70-4.74 (m, 1H), 4.84-4.88 (m,
1H), 4.93-4.94 (d, J ) 5.29 Hz, 1H), 4.98-5.03 (m, 1H), 5.11-
5.17 (m, 1H), 5.24-5.27 (m, 1H), 6.81-6.82 (d, J ) 8.57 Hz, 2H),
7.05-7.06 (d, J ) 8.51 Hz, 2H), 7.29-7.31 (m, 1H), 7.42-7.45
(m, 1H), 7.50-7.55 (m, 1H), 7.62-7.66 (m, 1H), 7.79-7.81 (d, J
) 9.83 Hz, 1H), 8.33-8.34 (d, J ) 6.66 Hz, 1H); 13C NMR δ 11.8,
14.1, 16.0, 17.9, 18.8, 20.8, 21.3, 23.5, 23.7, 24.6, 24.8 and 24.9
(overlap), 25.5, 25.9, 27.2 (overlap), 27.9 (overlap), 28.8, 29.3,
30.1 (overlap), 31.1, 33.6, 34.1, 36.0, 37.3, 38.6, 39.4, 46.8, 47.1,
48.3, 51.8, 54.5, 55.3, 56.5, 56.6, 56.7, 57.0, 66.0, 67.8, 68.7, 69.4,
71.2, 79.4, 114.1, 130.3, 132.1, 158.6, 168.5, 169.5, 170.4, 170.6,
170.7, 171.1, 171.3, 172.5 (overlap), 173.1, 174.0, 176.1, 178.6;
IR (KBr, CHCl3) 3335, 1742, 1641 cm-1; HRMS (EI) m/z calcd
for C64H98N10O18Na (M + Na+) 1317.6958, found 1317.6896;
[R]20 -47.4 (c 3.96, CHCl3).
D
Ack n ow led gm en t. We gratefully acknowledge the
financial support of the National Institutes of Health
(CA-40081), the National Science Foundation (CHE 99-
01449), and the University of Pennsylvania.
Su p p or tin g In for m a tion Ava ila ble: Copies of 1H and 13C
NMR and IR spectra for all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
J O000253A