Total synthesis and structural confirmation of chlorodysinosin A

Article abstract of DOI:10.1021/ja0625834

The first enantiocontrolled total synthesis of the marine sponge metabolite chlorodysinosin A is described. The structure and absolute configuration are identical to those of dysinosin A except for the presence of a novel 2S,3R-3-chloroleucine residue in

Full text of DOI:10.1021/ja0625834

Published on Web 07/21/2006
Total Synthesis and Structural Confirmation of
Chlorodysinosin A
Stephen Hanessian,*,† Juan R. Del Valle,† Yafeng Xue,‡ and Niklas Blomberg§
Contribution from the Department of Chemistry, UniVersite´ de Montre´al, C.P. 6128,
Station Centre-Ville, Montre´al, P.Q., H3C 3J7 Canada, and Structural Chemistry Laboratory
and Global Compound Sciences, AstraZeneca, R&D Mo¨lndal S-431 83, Mo¨lndal, Sweden
Received April 13, 2006; E-mail: stephen.hanessian@umontreal.ca
Abstract: The first enantiocontrolled total synthesis of the marine sponge metabolite chlorodysinosin A is
described. The structure and absolute configuration are identical to those of dysinosin A except for the
presence of a novel 2S,3R-3-chloroleucine residue in the former. A concise stereocontrolled synthesis of
the new chlorine-containing amino acid fragment was developed. An X-ray cocrystal structure of synthetic
chlorodysinosin A with the enzyme thrombin confirms the structure and configuration assignment achieved
through total synthesis. Within the aeruginosin family of natural products, chlorodysinosin A is the most
potent inhibitor of the serine proteases thrombin, factor VIIa, and factor Xa, which are critical enzymes in
the process leading to platelet aggregation and fibrin mesh formation in humans.
The aeruginosins are a family of peptide natural products of
aquatic origin characterized structurally by the presence of a
substituted 2-carboxyperhydroindole core residue.1 Most of the
aeruginosins exhibit inhibitory activity against the serine pro-
tease thrombin, which is the penultimate enzyme in the blood
coagulation cascade that converts fibrinogen into soluble fibrin
monomers and activates the process of platelet aggregation.2 A
number of aeruginosins also display inhibitory activity against
other coagulation enzyme factors vital for hemostasis.3 The
medicinal implications of identifying novel, potent, and selective
serine protease inhibitors have fostered the development of
efficient methodologies for the synthesis of the aeruginosin
subunits as well as analogues for biological evaluation in our
laboratories4 and elsewhere.5 To date, 21 members of the
aeruginosin family have been isolated from freshwater algae6
and marine sponges.7 The total syntheses of six aeruginosins
have been completed,8-12 four of these involving revisions of
the originally proposed structures.13
Within the aeruginosin family, the dysinosins represent a
subset of structures isolated from the Dysideidae family of
(4) (a) Hanessian, S.; Therrien, E.; van Otterlo, W. A. L.; Bayrakdarian, M.
A.; Nilsson, I.; Fjellstro¨m, O.; Xue, Y. Bioorg. Med. Chem. Lett. 2006,
16, 1032. (b) Hanessian, S.; Tremblay, M.; Marzi, M.; Del Valle, J. R. J.
Org. Chem. 2005, 70, 5070. (c) Hanessian, S.; Sailes, H.; Therrien, E.
Tetrahedron 2003, 59, 7047. (d) Hanessian, S.; Balaux, E.; Musil, D.;
Olsson, L.-L.; Nilsson, I. Bioorg. Med. Chem. Lett. 2000, 10, 243. (e)
Hanessian, S.; Therrien, E.; Granberg, K.; Nilsson, I. Bioorg. Med. Chem.
Lett. 2000, 12, 2907.
(5) (a) Liu, Z.; Ranier, J. D. Org. Lett. 2006, 8, 459. (b) Nie, X.; Wang, G. J.
Org. Chem. 2005, 70, 8687. (c) Fukuta, Y.; Ohshima, T.; Gnanadesikan,
V.; Shibuguchi, T.; Nemoto, T.; Kisugi, T.; Okino, T.; Shibasaki, M. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 5433. (d) Toyooka, N.; Nakazawa, A.;
Himiyama, T.; Nemoto, H. Heterocycles 2003, 59, 75. (e) Toyooka, N.;
Okumura, M.; Himiyama, T.; Nakazawa, A.; Nemoto, H. Synlett 2003,
55. (f) Valls, N.; Vallribera, M.; Font-Bard`ıa, M.; Solans, X.; Bonjoch, J.
Tetrahedron: Asymmetry 2003, 14, 1241. (g) Radau, G.; Stu¨rzebecher, J.
Pharmazie 2002, 57, 11. (h) Radau, G.; Rauh, D. Bioorg. Med. Chem.
Lett. 2000, 10, 779. (i) Bonjoch, J.; Catena, J.; Isa´bal, E.; Lo´pez-Canet,
M.; Valls, N. Tetrahedron: Asymmetry 1996, 7, 1899.
(6) (a) Aeruginosin 298A: Murakami, M.; Okita, Y.; Matsuda, H.; Okino, T.;
Yamaguchi, K. Tetrahedron Lett. 1994, 35, 3129. (b) Aeruginosins 98A
and 98B: Murakami, M.; Ishida, K.; Okino, T.; Okita, Y.; Matsuda, H.;
Yamaguchi, K. Tetrahedron Lett. 1995, 36, 2785. (c) Aeruginosins 102A
and 102B: Matsuda, H.; Okino, T.; Murakami, M.; Yamaguchi, K.
Tetrahedron 1996, 52, 14501. (d) Aeruginosins 205A and 205B: Shin, H.
J.; Matsuda, H.; Murakami, M.; Yamaguchi, K. J. Org. Chem. 1997, 62,
1810. (e) Oscillarin: Engh, R.; Konetschny-Rapp, S.; Krell, H.-W.; Martin,
U.; Tsaklakidis, C. PCT WO97/21725; Chem Abst. 1997, 127, 122002. (f)
Aeruginosin 103A: Kodani, S.; Ishida, K.; Murakami, M. J. Nat. Prod.
1998, 61, 1046. (g) Microcin SF608: Banker, R.; Carmelli, S. Tetrahedron
1999, 55, 10835. (h) Aeruginosins 98C, 101, 298B, 89A, and 89B: Ishida,
K.; Okita, Y.; Matsuda, H.; Okino, T.; Murakami, M. Tetrahedron 1999,
55, 10971. (i) Aeruginosin EI461: Pluotno, A.; Shoshan, M.; Carmelli, S.
J. Nat. Prod. 2002, 65, 973.
(7) (a) Dysinosin A: Carroll, A. R.; Pierens, G.; Fechner, G.; de Almeida Leone,
P.; Ngo, A.; Simpson, M.; Hooper, J. N. A.; Bostro¨m, S.-L.; Musil, D.;
Quinn, R. J. J. Am. Chem. Soc. 2002, 124, 13340. (b) Chlorodysinosin A:
Goetz, G. H.; Harrigan, G. G.; Likos, J. J.; Kasten, T. P. PCT WO03/
051831; Chem. Abst. 2003, 139, 47155. (c) Dysinosins B, C, and D: Carroll,
A. R.; Buchanan, M. S.; Edser, A.; Hyde, E.; Simpson, M.; Quinn, R. J. J.
Nat. Prod. 2004, 67, 1291.
† Department of Chemistry, Universite´ de Montre´al.
‡ Structural Chemistry Laboratory, AstraZeneca.
§ Global Compound Sciences, AstraZeneca.
(1) For recent reviews on cyanobacterial metabolites, see: (a) Burja, A. M.;
Banaigs, B.; Abou-Mansour, E.; Burgess, L. G.; Wright, P. C. Tetrahedron
2001, 57, 9347. (b) Leusch, H.; Harrigan, G. G.; Goetz, G.; Horgen, F. D.
Curr. Med. Chem. 2002, 9, 179. (c) Shiori, T.; Hamada, Y. Synlett 2001,
184. (d) Lewis, J. R. Nat. Prod. Rep. 1998, 417. (e) Namikoshi, M.;
Rinehart, K. L. J. Indust. Microbiol. 1996, 17, 373. (f) Rinehart, K. L.;
Namikoshi, M.; Choi, B. W. J. Appl. Phycol. 1994, 6, 159.
(2) (a) Colman, R. W.; Clowes, A. W.; George, J. N.; Hirsch, J. L.; Marder,
J. V. In OVerView of Homeostasis, 4th ed.; Colman, R. W., Hirsh, J. L.,
Marder, J. V., Clowes, A. W., George, J. N., Eds.; Hemostasis and
Thrombosis. Basic Principles and Clinical Practice; Lippincott Williams
& Wilkins: Philadelphia, 2001; p 3. (b) Dahlba¨ck, B. Lancet 2000, 355,
1627. (c) Steinmetz, T.; Hauptman, J.; Stu¨rzbecher, J. Exp. Opin. InVest
Drugs 2000, 10, 845. (d) Kimball, S. D. Blood Coagul. Fibrinol. 1995, 6,
511.
(8) Total syntheses of aeruginosin 298A: (a) Methot, J.-L.; Wipf, P. Org. Lett.
2000, 2, 4213. (b) Valls, N.; Lo´pez-Canet, M.; Vallribera, M.; Bonjoch, J.
J. Am. Chem. Soc. 2000, 122, 11248. (c) Ohshima, T.; Gnanadesikan, V.;
Shibuguchi, T.; Fukuta, Y.; Nemoto, T.; Shibasaki, M. J. Am. Chem. Soc.
2003, 125, 11206. (d) For the total synthesis of aeruginosins 298B, see:
Valls, N.; Lo´pez-Canet, M.; Vallribera, M.; Bonjoch, J. Chem.sEur. J.
2001, 7, 3446.
(3) For selected reviews on thrombin inhibitors, see: (a) Sanderson, P. E.;
Nayler-Olson, A. M. Curr. Med. Chem. 1998, 5, 289. (b) Ripka, W. E.
Curr. Opin. Chem. Biol. 1997, 1, 242. (c) Pfau, R. Curr. Opin. Drug
DiscoVery 2003, 6, 437.
9
10.1021/ja0625834 CCC: $33.50 © 2006 American Chemical Society
J. AM. CHEM. SOC. 2006, 128, 10491-10495
10491

A R T I C L E S
Hanessian et al.
Synthesis
Despite various reports describing the preparation of halo-
genated R-amino acids,19 the synthetic utility of â-halogenated
residues is limited by their susceptibility to base-promoted
elimination across the R,â-carbons.20 In our synthesis plan
toward 2, we made an intuitive assumption that the amino acid
component was 3R-chloro-D-leucine. A number of challenges
were clearly apparent, not the least of which was the propensity
of the amino acid or its derivatives to undergo â-elimination.
To circumvent this problem, we chose 3R-chloro-D-leucinol to
be our initial subtarget for synthesis. Toward this objective, we
considered a regioselective opening of an appropriately N-
substituted aziridine precursor with a source of chloride anion.21
The synthesis of 3-chloroleucinol commenced with the known
epoxy alcohol 4 (Scheme 1).22 The N-substituted aziridines
7a-d were obtained in good yield over four efficient steps by
way of intermediates 5a,b and 6. Our initial attempts to open
Figure 1.
marine sponges.7 We recently reported the total synthesis and
structural confirmation of dysinosin A (1) (Figure 1) which
revealed a unique 5,6-dihydroxyoctahydroindole-2-carboxylic
acid core structure and an unprecedented amidinopyrroline P1
basic subunit.12 Oscillarin (3), isolated from the freshwater algae
Oscillatoria aghardii,6e is the third member of the aeruginosin
family which was found to contain an amidinopyrroline subunit
as a naturally occurring arginine mimetic residue. The originally
misassigned structure for this unique heterocyclic entity was
corrected in conjunction with the total synthesis of oscillarin,
which was also found to be the most active aeruginosin against
thrombin (IC50 ) 28 nM).11
Soon after, researchers at Pharmacia Corp. isolated a chlo-
rinated analogue of dysinosin A (2, here referred to as
chlorodysinosin A) and reported its potent inhibitory activity
against thrombin, trypsin, and factor VIIa.7b Structural studies
of 2 revealed that it differed from 1 only in the presence of a
3-chloroleucine residue14 instead of D-leucine. Although the
stereochemistry of 2 was not assigned, its structural similarity
to 1 suggested identical absolute configurations for the individual
subunits. This assumption could not, however, be made to
predict the configuration of the novel 3-chloroleucine residue,
although we assumed the D-leucine backbone to be the same as
that in 1.
19e
the N-Boc (7a) and N-Ts (7b) aziridines with MgCl2 were
unsuccessful (Table 1, entries 1 and 3). A recent report on the
use of CeCl3‚7H2O to cleave unhindered N-Ts aziridines23
prompted us to apply these conditions to subtrates 7a and 7b.
While the N-Boc-aziridine proved to be unreactive (Table 1,
entry 2), a regioisomeric mixture of chlorosulfonamides was
obtained in 69% yield from 7b (Table 1, entry 4). The desired
3-chloro N-tosyl derivative could be isolated as the major
(15) (a) For an insightful report on the possible link between metabolites from
Oscillatoria cyanobacteria and Lamellodysidea marine sponges, see: Ridley,
C. P.; Bergquist, P. R.; Harper, M. K.; Faulkner, D. J.; Hooper, J. N. A.;
Haygood, M. G. Chem. Biol. 2005, 12, 397. (b) Vaillancourt, F.; Yeh, E.;
Vosburg, D. A.; Garneau-Tsodikova, S.; Walsh, C. T. Chem. ReV. 2006,
ASAP. (c) van Pee´, K.-H. Annu. ReV. Microbiol. 1996, 50, 375. (d) Gribble,
G. J. Nat. Prod. 1992, 55, 1353. (e) Bewley, C. A.; Faulkner, D. J. Angew.
Chem., Int. Ed. 1998, 37, 2162. (f) Fusetani, N.; Matsunaga, S. Chem. ReV.
1993, 93, 1783. (g) Fukuchi, N.; Isogai, A.; Yamashita, S.; Suyama, K.;
Takemoto, J. Y.; Suzuki, A. Tetrahedron Lett. 1990, 31, 1589. (h) Moore,
R. E.; Bornemann, V.; Niemczura, W. P.; Gregson, J. M.; Chen, J.-L.;
Norton, T. R.; Patterson, G. M. L.; Helms, G. L. J. Am. Chem. Soc. 1989,
111, 6128.
Herein we report the first total synthesis and structural and
stereochemical confirmation of 2, including the 2S,3R config-
uration of the 3-chloroleucine subunit. In addition, it was our
aim to compare the enzymatic inhibitory activity of 2 against
representative serine proteases involved in the blood coagulation
cascade to that of 1, which lacks the chloro substituent. While
many chlorinated peptide natural products have been isolated
over the years, mostly from cyanobacteria,15 â-chloro-R-amino
acids have only been found in a few natural products which
include the astins,16 FR900148,17 and FR225659.18 To the best
of our knowledge, the isolation of chlorodysinosin A marked
the first occurrence of the 3-chloroleucine residue in a natural
product.
(16) (a) Morita, H.; Nagashima, S.; Koichi, T.; Itokawa, H. Chem. Pharm. Bull.
1993, 41, 992. For examples of other peptide natural products featuring
â-chlorinated proline residues, see: (b) Marumo, S.; Sumiki, Y. Nippon
Nogei Kagaku Kaishi 1955, 29, 395. (c) Ghosh, A. C.; Ramgopal, M. J.
Heterocycl. Chem. 1980, 17, 1809. (d) Kosemura, S.; Ogawa, T.; Kazuo,
T. Tetrahedron Lett. 1993, 34, 1291.
(17) (a) Kuroda, Y.; Okuhara, M.; Goto, T.; Yamashita, M.; Iguchi, E.; Kohsaka,
M.; Aoki, H.; Imanaka, H. J. Antibiot. 1980, 33, 259. (b) Yasuda, N.;
Sakane, K. J. Antibiot. 1991, 44, 801.
(18) Ohtsu, Y.; Sasamura, H.; Tsurumi, Y.; Yoshimura, S.; Shigematsu, N.;
Takase, S.; Hashimoto, M.; Shibata, T.; Hino, M.; Fujii, T. J. Antibiot.
2003, 56, 682.
(19) For reviews on the synthesis of fluorinated amino acids, see: (a) Kukhar,
V. P.; Soloshonok, V. A. Fluorine Containing Amino Acids: Synthesis
and Properties; Wiley: New York, 1995. (b) Qiu, X.-L.; Meng, W.-D.;
Qing, F.-L. Tetrahedron 2004, 60, 6711. (c) Sutherland, A.; Willis, C. L.
Nat. Prod. Rep. 2000, 17, 621. For relevant examples of the synthesis of
other halogenated amino acids, see: (d) Meyer, F.; Laaziri, A.; Papini, A.
M.; Uziel, J.; Juge´, S. Tetrahedron: Asymmetry 2003, 14, 2229. (e) Pansare,
S. V.; Vederas, J. C. J. Org. Chem. 1989, 54, 2311. (f) Chuang, T.-H.;
Sharpless, K. B. Org. Lett. 2000, 2, 3555. (g) Choi, D.; Kohn, H.
Tetrahedron Lett. 1995, 36, 7011. (h) Righi, G.; D’Achille, R. Tetrahedron
Lett. 1996, 37, 6893. (i) Schumacher, K. K.; Jiang, J.; Joullie´, M. M.
Tetrahedron: Asymmetry 1998, 9, 47. (j) Laszlo, S. E.; Willard, P. G. J.
Am. Chem. Soc. 1985, 107, 199.
(20) (a) Srinivasan, A.; Stephenson, R. W.; Olsen, R. K. J. Org. Chem. 1977,
42, 2253. (b) Dunn, M. J.; Gomez, S.; Jackson, R. F. W. J. Chem. Soc.,
Perkin Trans. 1 1995, 1639. (c) Gelb, M. H.; Lin, Y.; Pickard, M. A.;
Song, Y.; Vederas, J. C. J. Am. Chem. Soc. 1990, 112, 4932. (d) Murkin,
A. S.; Tanner, M. E. J. Org. Chem. 2002, 67, 8389.
(21) For the opening of N-Boc aziridines with MgBr2 and MgBr2/NaI as halide
sources, see: (a) Righi, G.; Franchini, T.; Bonini, C. Tetrahedron Lett.
1998, 39, 2385. For recent reviews on halogenolysis of activated aziridines,
see: (b) Righi, G.; Bonini, C. Recent Res. Org. Chem. 1999, 343. (c)
McCoull, W.; Davis, F. A. Synthesis 2000, 1347. (d) Hu, X. E. Tetrahedron
2004, 60, 2701.
(9) Aeruginosin EI461: Valls, N.; Vallribera, M.; Carmeli, S.; Bonjoch, J. Org.
Lett. 2003, 5, 447.
(10) Microcin SF608: Valls, N.; Vallribera, M.; Lo´pez-Canet, M.; Bonjoch, J.
J. Org. Chem. 2002, 67, 4945.
(11) Oscillarin: Hanessian, S.; Tremblay, M.; Petersen, J. F. W. J. Am. Chem.
Soc. 2004, 126, 6064.
(12) Dysinosin A: Hanessian, S.; Margarita, R.; Hall, A.; Johnstone, S.;
Tremblay, M.; Parlanti, L. J. Am. Chem. Soc. 2002, 124, 13342.
(13) The originally proposed structures of aeruginosins 298A, 298B, EI461 and
oscillarin have been revised through synthesis. The proposed structures of
aeruginosins 205A and 205B have been called into question through
synthesis and NMR analysis of the putative hydroindole core residue (see
ref 14a).
(14) (a) For a stereoselective synthesis of diastereomeric 3-chloroleucines, see:
Valls, N.; Borrega´n, M.; Bonjoch, J. Tetrahedron Lett. 2006, 47, 3701. (b)
A nondiastereoselective synthesis of racemic 3-chloroleucine has been
reported: Hayashi, K.; Skinner, C. G.; Shive, W. J. Org. Chem. 1961, 26,
1167.
(22) Caldwell, C. G.; Bondy, S. S. Synthesis 1990, 34.
(23) Sabitha, G.; Babu, R. S.; Rajkumar, M.; Reddy, Ch. S.; Yadav, J. S.
Tetrahedron Lett. 2001, 42, 3955.
9
10492 J. AM. CHEM. SOC. VOL. 128, NO. 32, 2006

Synthesis/Structural Confirmation of Chlorodysinosin A
A R T I C L E S
Table 1
entry
SMa
conditions
yieldb
3-Cl:2-Clc
R′
1
2
3
4
5
6
7
8
7a
7a
7b
7b
7c
7d
7d
7e
10 equiv of MgCl2, Et2O, 72 h, rt
1.1 equiv of CeCl3‚7H2O, MeCN, 90 °C, 24 h
10 equiv of MgCl2, Et2O, 72 h, rt
1.1 equiv of CeCl3‚7H2O, MeCN, 90 °C, 24 h
1.1 equiv of CeCl3‚7H2O, MeCN, 90 °C, 24 h
2.0 equiv of CeCl3‚7H2O, MeCN, 90 °C, 48 h
4.0 equiv of CeCl3‚7H2O, MeCN, 90 °C, 72 h
4.0 equiv of CeCl3‚7H2O, MeCN, 90 °C, 48 h
no rxn
no rxn
no rxn
69%
57%
56%
TBS
TBS
TBS
TBS
TBS
5:1 H:TBS
H
H
2:1
1:1
10:1
10:1
10:1
80%
74%
1
a For the structure of 7e and R groups, see Scheme 1. b Combined isolated yield. c Determined by H NMR.
Scheme 1 a
Scheme 2 a
a Reagents and conditions: (a) NaN3, NH4Cl, MeOEtOH/H2O (9:1),
reflux; (b) TBSCl, Et3N, DMAP, DCM, 0 °C; (c) PPh3, MeCN, 50 °C; (d)
Boc2O, Et3N, DCM; (e) TsCl, Et3N, DCM; (f) Tf2O, pyr, DCM, 0 °C; (g)
t-BuSOCl, Et3N, DCM, 0 °C then mCPBA, DCM, 0 °C; (h) 0.5 M TBAF/
THF, 2 h.
a Reagents and conditions: (a) 4.0 equiv of CeCl3‚7H2O, MeCN, 90 °C,
72 h; (b) 0.1 M TfOH/DCM, anisole; (c) PyBOP, (R)-3-tert-butyl-
diphenylsilyloxy-2-methoxypropionic acid, 2,6-lutidine, DCM; (d) 0.1 M
H2IO6/wet MeCN, cat. CrO3, 0 °C.
product in over 40% yield, but the modest regioselectivity and
harsh conditions eventually required for the deprotection of the
N-tosyl group led us to investigate alternative activating groups.
Treatment of the trifluoromethylsulfonamide 7c with CeCl3‚
7H2O in refluxing MeCN led to a 1:1 mixture of 2-chloro and
3-chloro regioisomers (Table 1, entry 5). We were pleased to find
that the tert-butylsulfonamide 7d gave significantly improved
regioselectivity in favor of the desired 3-chloro isomer (Table
1, entry 6). By employing 4.0 equiv of CeCl3‚7H2O in refluxing
MeCN for 72 h, it was possible to achieve efficient, regioselec-
tive aziridine opening and cleavage of the TBS group affording
(2S,3R)-N-Bus-3-chloroleucinol (8) as a crystalline solid in 80%
yield (Table, entry 7). A single-crystal X-ray structure of 8
confirmed the stereo- and regiochemistry of the product.
The tert-butylsulfonyl (Bus) protecting group, originally
introduced by Weinreb and co-workers,24 and later utilized in
the opening of aziridines with secondary amines by Sharpless
and co-workers,25 has received scant attention as an N-protecting
group. The high regioselectivity of CeCl3-mediated opening of
the Bus-protected 7d may be due to a steric clash between the
tert-butyl and isopropyl groups, thus providing an open path
for SN2 attack at C3. The less sterically demanding N-tosyl and
N-trifluoromethylsulfonyl groups of 7b and 7c may allow for a
competitive reaction at C2. This hypothesis is supported by the
fact that cleavage of the aziridine bearing the smallest N-sulfonyl
group studied (7c) resulted in the lowest regioselectivity at C3.
Furthermore, CeCl3-mediated opening of hydroxymethyl aziri-
dine 7e (Table 1, entry 8) provided the corresponding 3-chloro-
sulfonamide with the same regioselectivity and yield as those
of 7d, despite diminished steric bulk at C2.
With a concise and selective synthesis of (2S,3R)-N-tert-
butylsulfonyl-3-chloroleucinol (8), we proceeded with the elab-
oration of amide 10. Treatment of 8 with 0.1 M TfOH smoothly
cleaved the N-Bus group, and the resulting amine salt was coup-
led with (R)-3-tert-butyldiphenylsilyloxy-2-methoxypropionic
acid12 in the presence of PyBOP (Scheme 2). Oxidation of 9
under a variety of well-known conditions resulted in complex
mixtures of products. Gratifyingly, oxidation with H5IO6 and
catalytic CrO3, developed by Reider, Grabowski, and co-
workers,26 provided the desired N-terminal acid 10 in 78% yield.
We have recently described an efficient intramolecular
N-acyliminium ion halocarbocyclization route to the 2-carboxy-
5-hydroxyoctahydroindole (Choi) core found in oscillarin and
various other members of the aeruginosin family.4b,11 As shown
in Scheme 3, treatment of the mixture of hemiaminal acetates
12 with SnBr4 in CH2Cl2 at -78 °C afforded, within a few
minutes, the brominated intermediate 13 in good yield. This
method was exploited toward a new synthesis of the dihydroxy-
hexahydroindole core previously obtained via tin-mediated
C-allylation and subsequent ring closing metathesis of a diene
intermediate.12 Thus, dehydrohalogenation of 13 by heating in
neat DBU gave the previously obtained RCM product 14 in
(24) Sun, P.; Weinreb, S. J. Org. Chem. 1997, 62, 8604
(25) Gontcharov, A. V.; Liu, H.; Sharpless, K. B. Org. Lett. 1999, 1, 783.
(26) Zhao, M.; Li, J.; Song, Z.; Desmond, R.; Tschaen, D. M.; Grabowski, E.
J. J.; Reider, P. J. Tetrahedron Lett. 1998, 39, 5323.
9
J. AM. CHEM. SOC. VOL. 128, NO. 32, 2006 10493

A R T I C L E S
Scheme 3 a
Hanessian et al.
a Reagents and conditions: (a) SnBr4, DCM, -78 °C, 5 min; (b) neat DBU, 80 °C, 4 h; (c) DEPBT, 2,6-lutidine, DCM, 0 °C, 4 h; (d) 20 equiv of
MeSnOH, 1,2-DCE, 75 °C, 48 h; (e) PyBOP, 2,6-lutidine, 2.0 equiv, DCM, 0 °C, 4 h; (f) 0.3 M TBAF/THF, 15 min, 0 °C; (g) SO3‚pyr, cat. Bu2SnO, DCM,
18 h, then repeat; (h) 10% TFA/DCM, rt, 6 h; (i) preparative RP-HPLC.
70% yield. Following the four-step procedure described for
dysinosin A,12 14 was converted to 15 in preparation for
coupling to the chloro amino acid fragment 10.
in 78% yield.32 Coupling of carboxylic acid intermediate 16b
to amine 17 in the presence of PyBOP and 2,6-lutidine
proceeded in high yield to give 18a.
The installation of the sulfate ester and final deprotection was
the last challenge to face en route to the intact intended target
2. This was achieved by treatment of 18a with TBAF for 15
min to cleave the TBDPS ether. Longer reaction times resulted
in the formation of byproducts, including elimination. Reaction
of 18b with SO3‚pyr and catalytic Bu2SnO (2 times),33 followed
by global deprotection with 10% TFA/DCM and purification
of the crude peptide by RP-HPLC, gave 2 in 38% overall yield.
The spectral data obtained from synthetic 2 matched those
reported for the natural product7b in every respect, thus
confirming the structure of chlorodysinosin A as well as the
configuration of the (2S,3R)-3-chloroleucine residue.
Although we had achieved our first objectives with the
synthesis of fragments 10 and 15, we were cognizant that the
assembly of 2 by previously employed methods for dysinosin
A12 had to be extensively modified due to the sensitive nature
of the 3-chloroleucine moiety. The first of the anticipated
challenges was encountered during attempted amide bond
formation. Use of triethylamine or diisopropylethylamine in
combination with EDC/HOBt, HBTU/HOBt, or PyBOP gave
only low (<15%) yields of 16a (Scheme 3). By employing 2,6-
lutidine as a base in combination with various coupling
reagents,27 yields improved slightly, although mixtures of
eliminated products still predominated. After extensive optimi-
zation, we found that 16a could be obtained reproducibly in
55-60% yield when coupling was mediated by 1.5 equiv of
recrystallized DEPBT.28
The next major hurdle to overcome was the hydrolysis of
the methyl ester in 16a. As expected, the various hydroxide
bases effected the desired ester cleavage, but at the expense of
unwanted elimination. Unsuccessful attempts with both sodium
hydroperoxide29 and enzymatic methods such as pig liver
esterase30 led us to explore the MeTeAlMe2 complex, recently
reported by Corey and co-workers.31 Although we observed
efficient ester cleavage without elimination of HCl under the
described conditions, the reaction was attended by rapid
deprotection of the MOM ethers. However, we were gratified
to find that 16b could be obtained without any detectable
elimination by employing an excess of Me3SnOH with heating
Biological and Structural Data
Although our “hunch” in choosing the (2S,3R)-3-chloroleu-
cine diastereomer (rather than the equally possible 3S) proved
correct, we had not yet appreciated the subtleties of replacing
a pro-R methine hydrogen in 1 by a chlorine atom in 2, until
their enzyme inhibitory activities were compared. Chlorodysi-
nosin A proved to be the most potent inhibitor to date of throm-
bin and factor VIIa among the known aeruginosins (IC50 values
for 2: thrombin ) 5.7 nM, trypsin ) 37 nM, and factor VIIa
) 39 nM; for 1: thrombin ) 46 nM, factor VIIa ) 326 nM).34
A cocrystal structure of 2 with thrombin (at 2 Å resolution)
confirms the structure and configurational assignments through
synthesis and provides insights into the enhanced binding.35
While the overall conformation of 2 bound to the thrombin
active site is very similar to the binding mode of 1, there are
some interesting differences (Figure 2, left). For instance, both
Glu192 and Arg173 differ in the orientation of their respective
side chains in the cocrystal structures of 1 and 2 bound to
(27) For a review of peptide coupling methods as applied to the synthesis of
complex natural products, see: Humphrey, J. M.; Chamberlin, A. R. Chem.
ReV. 1997, 97, 2243.
(28) (a) Li, H.-T.; Jiang, X.-H.; Ye, Y.-H.; Fan, C.-X.; Romoff, T.; Goodman,
M. Org. Lett. 1999, 1, 91. (b) Fan, C.-X.; Hao, X.-L.; Ye, Y.-H. Synth.
Commun. 1996, 26, 1455. For discussions on the effectiveness of DEPBT
in difficult coupling reactions en route to complex natural products, see:
(c) Jiang, W.; Wanner, J.; Lee, R. J.; Bounaud, P.-Y.; Boger, D. L. J. Am.
Chem. Soc. 2003, 125, 1877. (d) Ye, Y.-H.; Li, H.-T.; Jiang, X.-H.
Biopolymers 2005, 80, 172.
(29) See: Hanessian, S.; Kagotani, M. Carbohydr. Res. 1990, 202, 67 and
referenced cited therein.
(30) Ohno, M.; Otsuka, M. Org. React. 1989, 37, 1.
(31) (a) Reddy, L. R.; Fournier, J.-F.; Reddy, B. V. S.; Corey, E. J. J. Am.
Chem. Soc. 2005, 125, 8974. (b) Reddy, B. V. S.; Rajender, Reddy, L. J.;
Corey, E. J. Tetrahedron Lett. 2005, 46, 4589.
(32) For a recent application, see: (a) Nicolaou, K. C.; Estrada, A. A.; Zak,
M.; Lee, S. H.; Safina, B. S. Angew. Chem., Int. Ed. 2005, 44, 1378. See
also: (b) David, S.; Hanessian, S. Tetrahedron 1985, 4, 643.
(33) (a) Lubineau, A.; Lemoine, R. Tetrahedron Lett. 1994, 35, 8795. (b)
Sanders, W. J.; Manning, D. D.; Koeller, K. M.; Keissling, L. L.
Tetrahedron 1997, 53, 16391.
(34) Nilsson, T.; Sjoling-Ericksson, A.; Deinum, J. J. Enzyme Inhibition 1998,
13, 11.
(35) Data for cocrystal structure of 2 bound to thrombin has been registered
with the protein data bank under PDB ID: 2GDE.
9
10494 J. AM. CHEM. SOC. VOL. 128, NO. 32, 2006

Synthesis/Structural Confirmation of Chlorodysinosin A
A R T I C L E S
Figure 2. (Left) Overlay of the cocrystal structures of 1 (grey) and 2 (pink) with thrombin. (Right) Overlay of the cocrystal structures of 1 (grey) and 2
(pink) showing changes in the thrombin residue side chain orientations in the case of 2.
thrombin (Figure 2, right). The flexibility of Glu192 has been
previously observed in substrate-bound thrombin structures.36
Although Glu192 in thrombin adopts a different side chain
orientation when bound to 2 rather than 1, there is no evidence
of a direct interaction between Glu192 and the bound ligand in
either X-ray structure. The Arg173 residue in thrombin points
toward the sulfate group of the bound ligand 1, an orientation
not frequently observed in other thrombin complexes. The
presence of the chlorine seems to cause the side chain of this
Arg173 to return to its “normal” orientation (Figure 2, right).
Other differences in the orientation of the residues in the binding
pocket include the backbone atoms of Glu217 (0.36 Å for CR)
and Gly219 (0.47 Å). Shifts of a similar scale are also observed
for some of the ligand atoms.
Perhaps most significant, the addition of the chlorine does
not seem to change the conformation of either the side chain of
the leucine or the protein binding site. We rationalize the
enhanced inhibitory activity of 2 by favorable changes in charge
distribution, better accommodation of the chlorine in the S3
subsite, and stabilization of the ø1 dihedral angle of the (2S,3R)-
chloroleucine residue. The presence of the chlorine atom also
enhances lipophilicity (log P of -1.8 for 2 vs -3.8 for 1)
contributing to increased hydrophobic interactions via a de-
creased desolvation penalty. In addition, the presence of a
chlorine atom in the S3 subsite of thrombin leads to a release
of water from the protein, which provides an entropic gain.
To test to what extent the chlorine atom stabilizes the biolog-
ically active conformation of the dysinosins, we ran a short mol-
ecular dynamics simulations starting from the bound conforma-
tions of 1 and 2. Dysinosin A was found to sample two major
conformations of the ø1 dihedral angle (approximately trans and
gauche), whereas the sampling of ø1 in chlorodysinosin A (2)
is clearly more restricted.37 These results suggest that conforma-
tional stabilization of the leucine side chain in fact plays an
important role in the enhanced potency of 2. A detailed calori-
metric study of the relative binding thermodynamics of the dy-
sinosins would provide insight into the relative importance of
the different terms and provide a basis for future refinements.38
Conclusion
We have described a concise total synthesis of chlorodysi-
nosin A (2), a highly potent inhibitor of the serine proteases
thrombin, trypsin, and factor VIIa. The synthesis also established
the absolute configuration of the natural product. A novel 3R-
chloro-D-leucine amide residue was synthesized in a stereocon-
trolled manner en route to the target compound. An X-ray
cocrystal structure of 2 bound to thrombin reveals important
differences with the des-chloro analogue, dysinosin A (1). The
importance of chlorinated amino acids in natural products is
highlighted by enhanced potency and functional novelty in these
metabolites. The incorporation of a chlorine in related aerugi-
nosins as hybrid analogues and the replacement of the 3-chloro-
leucine with other hydrophobic â-branched residues are currently
being investigated.
Acknowledgment. This paper is dedicated to Professor K. C.
Nicolaou, chemist extraordinaire, on the occasion of his 60th birthday.
We thank NSERC and AstraZeneca R&D Mo¨lndal, Sweden for gener-
ous financial support. We also thank Michel Simard for the X-ray struc-
ture analysis of 7. Thanks are due to Drs. Ingemar Nilsson and Ola
Fjellstrom of AstraZeneca for continued support and useful discussions.
Note Added after ASAP and Print Publication: Due to a
production error, the references following ref 21 were misnumbered
and structure 3 in Figure 1 was incorrect in the version of this paper
published on the Web July 24, 2006 (reflecting a change to Figure 1
after initial ASAP publication on July 21, 2006), and in the August
16, 2006 print issue. This electronic version of the paper, with the
correct Figure 1 and Scheme 1 and the references correctly numbered,
was published on August 22, 2006; an Addition and Correction appears
in the September 6, 2006 issue (Vol. 128, No. 35, pp 11727-11728).
Supporting Information Available: Experimental procedures,
1H, 13C NMR spectra for all new compounds. Molecular modeling data
and a table of comparative biological activities are provided for 1 and
2. These materials are available free of charge via the Internet at
http://pubs.acs.org.
JA0625834
(36) (a) Recacha, R.; Costanzo, M. J.; Maryanoff, B. E.; Carson, M.; DeLucas,
L.; Chattopadhyay, D. Acta Crystallogr. 2000, D56, 1395. (b) Malikowski,
M. G.; Martin, P. D.; Guzik, J. C.; Edwards, B. F. P. Protein Sci. 1997, 6,
1438.
(37) See Supporting Information for experimental details on molecular modeling
and for a comparison of ø1 angles and energy minimized conformations of
1 and 2.
(38) Ward, W. H; Holdgate, G. A. Prog. Med. Chem. 2001, 38, 309.
9
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