Synthesis and photochemical activity of designed enediynes [19]

Full text of DOI:10.1021/ja000766z

9872
J. Am. Chem. Soc. 2000, 122, 9872-9873
Scheme 1. Photochemical Bergman Cycloaromatization
Synthesis and Photochemical Activity of Designed
Enediynes
Graham B. Jones,* Justin M. Wright, Gary Plourde, II,
Ajay D. Purohit, Justin K. Wyatt, George Hynd, and
Farid Fouad
Bioorganic and Medicinal Chemistry Laboratories
Department of Chemistry, Northeastern UniVersity
Boston, Massachusetts, 02115
Scheme 2. Synthesis of Alicyclic Photo-Bergman Candidates
ReceiVed March 2, 2000
ReVised Manuscript ReceiVed August 23, 2000
The enediynes are a growing class of antitumor agents, with
spectacular biological profiles.1 Nearly 20 discrete enediynes have
been discovered, and clinical trials of a number of these are
ongoing.2 While the in vitro and in vivo effectiveness of enediynes
against certain cancers is unquestioned, the exact mechanism(s)
of biological activity remains to be fully resolved. Enediynes per
se are biologically inactive, but undergo cycloaromatization
reactions which give rise to cytotoxic diyl radicals, which are
capable of inducing DNA strand scission at low concentration.1
Depending on local environment, however, it is also possible for
other events to occur. One potential class of targets is proteins.3
Excepting water, proteins are the most abundant constituents of
cells and extracellular fluids by weight. We recently demonstrated
that simple amino acids are viable targets for diyl radicals,
resulting in both degradation and dimerization via intermediate
carbon centered amino acyl radicals.4 Conventionally, the con-
trolled degradation of proteins is often accomplished through
affinity cleavage systems, typically containing a metal-redox
center tethered to an entity recognized by the protein of interest.5
Application of enediynes for this purpose could be an attractive
proposition but would require two conditions be met: (i) the
system must have affinity for a protein of interest and (ii) the
enediyne needs to be thermally stable until activated “on demand”.
An attractive method for activation lies in the photochemical
triggering of an otherwise unreactive enediyne. Indeed, examples
of the direct “photochemical Bergman” cyclization have been
reported.6-9 In most examples studied to date the vinyl moiety
of the enediyne is embedded in an arene, and mindful of this
restriction, we wished to design a family of alicyclic enediynes
1, and investigate their photochemical activation to diyls 2, as a
function of ring strain and electronic effects.10
Commencing from commercially available acyl chlorides 3,
the corresponding diketones 4 were produced via the intermediate
Weinreb amides (Scheme 2).11 Low-valent Ti-mediated coupling
gave 6 directly, but the yields were variable, in part due to
problems recovering product from complex mixtures. Alterna-
tively, conversion to bromides 5 followed by carbenoid coupling
gave 6 (n ) 1-5) in good yield on a preparative scale.12 Though
stable at room temperature, photochemical Bergman cyclo-
aromatization gave adducts 8 in moderate yield, presumably via
diyls 7 (Table 1). As had been found previously, the nature of
the hydrogen donor plays an important role in the conversion to
arene adduct.7 Thus, although consumption of enediyne was often
rapid using 1,4-cyclohexadiene, optimal yields of cycloaromati-
zation products were obtained using 2-propanol, the balance of
material typically composed of uncharacterized polymeric byprod-
ucts. Evidently ring strain effects play a role in the cycloaroma-
tization, with lower conversion efficiency observed with the C-7
and C-8 analogues despite the appreciable reduction in intra-
molecular “c-d” distances relative to the six-membered analogue
(Table 1).1 Though photo-Bergman cycloaromatization yields are
modest, the synthesis of this class of enediynes [4- through
8-membered] is noteworthy and may lead to many new applica-
tions in materials and polymer chemistry.13
With a route to photoactivated enediynes secure, we wished
to investigate interaction of the intermediate diyl radicals 2 with
protein targets, and accordingly sought to prepare a hydrophilic
variant, which was capable of recognizing protein architecture.
Our design was influenced by the work of Kumar,14 who reported
that an alkyl pyrenyl derivative of phenylalanine (Figure 1)
recognizes the proteins bovine serum albumin (BSA) and
lysozyme, inducing photocleavage following irradiation in the
presence of an electron acceptor.14 Molecular modeling studies
indicated that arene 14, the expected photocycloaromatization
product of enediyne 13 (Scheme 3), bears a close structural
resemblance to the reference probe, providing us with a logical
candidate for proof-of-principle studies.
* Address correspondence to this author.
(1) Smith, A. L.; Nicolaou, K. C. J. Med. Chem. 1996, 39, 2103.
(2) Xi, Z.; Goldberg, I. DNA Damaging enediyne compounds. In Com-
prehensiVe Natural Products Chemistry; Barton, D. H. R., Nakanishi, K., Eds.;
Pergamon: Oxford, 1999; Vol. 7, p 553.
(3) Zein, N.; Solomon, W.; Casazza, A. M.; Kadow, J. F.; Krishnan, B.
S.; Tun, M. M.; Vyas, D. M.; Doyle, T. W. Bioorg. Med. Chem. Lett. 1993,
3, 1351. Jones, G. B.; Kilgore, M. W.; Pollenz, R. S.; Li, A.; Mathews, J. E.;
Wright, J. M.; Huber, R. S.; Tate, P. L.; Price, T. L.; Sticca, R. P. Bioorg.
Med. Chem. Lett. 1996, 6, 1971.
(4) Jones, G. B.; Plourde, G.; Wright, J. W. Org. Lett. 2000, 2, 811. Jones,
G. B.; Wright, J. M.; Hynd, G.; Wyatt, J. K.; Yancisin, M.; Brown, M. A.
Org. Lett. 2000, 2, 1863.
(5) Rana, T. M.; Meares, C. F. J. Am. Chem. Soc. 1991, 113, 1859. Hoyer,
D.; Cho, H.; Schultz, P. G. J. Am. Chem. Soc. 1990, 112, 3249. Cuenoud, B.;
Tarasow, T. M.; Schepartz, A. Tetrahedron Lett. 1992, 33, 895. Hegg, E. L.;
Burstyn, J. N. J. Am. Chem. Soc. 1995, 117, 7015. Wu, J.; Perrin, D. M.;
Sigman, D. S.; Kaback, H. R. Proc. Nat. Acad. Sci. 1995, 92, 9186. Parac, T.
N.; Kostic, N. M. J. Am. Chem. Soc. 1996, 118, 5946. Kumar, C. V.;
Buranaprapuk, A. J. Am. Chem. Soc. 1999, 121, 4262. Miyake, R.; Owens, J.
T.; Xu, D.; Jackson, W. M.; Meares, C. F. J. Am. Chem. Soc. 1999, 121,
7453.
(10) Schmittel, M.; Kiau, S. Chem. Lett. 1995, 953. Schmittel, M.; Kiau,
S. Liebigs Ann./Recl. 1997, 1391. Jones, G. B.; Plourde, G. W. Org. Lett.
2000, 2, 1757.
(6) Funk, R. L.; Young, E. R. R.; Williams, R. M.; Flanagan, M. F.; Cecil,
T. L. J. Am. Chem. Soc. 1996, 118, 3291.
(7) Turro, N. J.; Evenzahav, A.; Nicolaou, K. C. Tetrahedron Lett. 1994,
35, 8089. Evenzahav, A.; Turro, N. J. J. Am. Chem. Soc. 1998, 120, 1835.
(8) Ramkumar, D.; Kalpana, M.; Varghese, B.; Sankararaman, S.; Ja-
gadeesh, M. N.; Chandrasekhar, J. J. Org. Chem. 1996, 61, 2247.
(9) Kaneko, T.; Takahashi, M.; Hirama, M. Angew. Chem., Int. Ed. 1999,
38, 1267.
(11) Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815.
(12) Jones, G. B.; Wright,J. M.; Plourde, G. W., II; Hynd, G.; Huber, R.
S.; Mathews, J. E. J. Am. Chem. Soc. 2000, 122, 1937.
(13) John, J. A.; Tour, J. M. J. Am. Chem. Soc. 1994, 116, 5011.
(14) Kumar, C. V.; Buranaprapuk, A.; Opiteck, G. J.; Moyer, M. B.;
Jockusch, S.; Turro, N. J. Proc. Natl. Acad. Sci. 1998, 95, 10361.
10.1021/ja000766z CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/23/2000

Communications to the Editor
J. Am. Chem. Soc., Vol. 122, No. 40, 2000 9873
Table 1. Synthesis and Cycloaromatization of Enediyne 6
c-d
irradiation
(h)b
% 8d
(based on 6)
entry
n
(Å)a
conversionc
1
2
3
4
5
6
2
3
4
4
5
6
4.941
4.284
4.018
4.018
3.947
3.893
3
3
3
12
12
12
100e
97
49
95
70
0 (0)
13 (13)
15 (31)
21 (22)
11 (16)
9 (14)
66
a Equilibrium geometry calculated using PM3 (PC Spartan Pro).
b Reactions conducted at 0.4 g/L enediyne in 2-propanol in a quartz
vessel, irradiated using a 450 W (Hanovia) lamp. c Based on recovered
starting material. d Isolated yields. e Decompostion and formation of
polymeric adducts ensues in <1 h.
Figure 2. 10% SDS polyacrylamide gel of the reaction of bovine serum
albumin (BSA) with 13 and 14. All reactions were conducted in 50 mM
Tris-HCl, pH 7.0 at 37 °C. From right to left; lane 1, molecular weight
markers (KDa); lane 2, BSA control (1.5 µM); lane 3, BSA (1.5 µM),
13 (150 µM), no irradiation; lane 4, BSA (1.5 µM), 14 (150 µM), 3h
irradiation; lane 5, BSA (1.5 µM), 13 (7.5 µM), 3h irradiation. The running
gel was overlaid with a 4% stacking gel and samples were run top to
bottom. Analogous reactions with 6 (n ) 3) showed no change from
control (data not shown).
Figure 1.
Scheme 3. Preparation and Photo-Bergman Cyclization of the
Phe-enediyne Hybrid
Figure 3. 15% SDS polyacrylamide gel of the reaction of Lysozyme
(Lyso) with 13 and 14. All reactions were conducted in 50 mM Tris-
HCl, pH 7.0 at 37 °C. From right to left; lane 1, molecular weight markers
(in KDa); lane 2, Lyso (0.15 µM); lane 3, Lyso (0.15 µM), 3h irradiation;
lane 4, Lyso (0.15 µM), 13 (0.75 µM), 3h irradiation; lane 5, Lyso (0.15
µM), 13 (0.75 µM), 6h irradiation; lane 6, Lyso (0.15 µM), 14 (15 µM),
12h irradiation. The running gel was overlaid with a 4% stacking gel
and samples were run top to bottom. Analogous reactions with 6 (n ) 3)
showed no change from control (data not shown).
mediated cross coupling.15 Though the origins of the observed
degradation and dimerization events require further study, genera-
tion of protein free radicals has precedent.16 Furthermore, it has
recently been reported that in the case of the naturally occurring
enediyne chromoprotein C-1027, the enediyne chromophore
induces proteolysis of its own apoprotein,17 suggesting that protein
targets of other enediynes may be found. The present findings
might ultimately lead to the design of specific photoproteases,
and we envision the ready accessibility and photochemical profile
of carboxylate 12 will help expedite this process.18
Accordingly, diketone 9 was prepared from adipoyl chloride
using a mixed coupling procedure. Subsequent functional group
interconversion and carbenoid coupling gave the differentially
functionalized C6-enediyne, which was unmasked to give 10.
Photo-Bergman cycloaromatization of this enediyne (or the TIPS
protected precursor) gave <10% of the corresponding arene
product, suggesting the aryl groups play a key role in the
(reversible) cyclization process.7-9 The alkyne was then coupled
with methyl-3-(3-iododphenyl)hexynoate 11, followed by hy-
drolysis to give carboxylate 12, which was then coupled with
L-phenylalanine methyl ester and subjected to saponification to
give conjugate 13 in good yield. In contrast to the model series,
photochemical activation (450 W, 3 h) of this enediyne gave arene
product 14 in appreciable yield, together with unreacted starting
material, underscoring the influence of the conjugated arene unit.
Photochemically induced modification of BSA and lysozyme
was examined by photolyzing 13 in aqueous buffer, using 14 and
enediynes 6 (n ) 4) as controls. In the case of 66 kD protein
BSA, subsequent analysis (SDS-PAGE) showed evidence of
enediyne induced degradation, with one principal fragment (∼40
kDa) visible (Figure 2). Subsequent analysis by MALDI-TOF
revealed the presence of fragments at 44.76, 11.28, and 10.22
kDa suggesting the possibility of two sites of cleavage.15 In the
case of the 14 kDa protein lysozyme, dimerization to form a
species with m/z 28.63 kDa was observed following irradiation
(Figure 3), suggesting the possibility of intermolecular diyl
Supporting Information Available: Synthetic procedures and spec-
troscopic data for the preparation of 13 (PDF). This material is available
free of charge via the Internet at http://pubs.acs.org.
JA000766Z
(15) MS analysis performed using desalted mixtures directly following
incubation using a sinapinic acid (BSA) or R-cyano-4-hydroxy-cinnamic acid
matrix (lysozyme). Binding constants of 13 for BSA (1.6 × 105 dm3 mol-2
)
and lysozyme (0.3 × 105 dm3 mol-2) were determined following Scatchard
analysis. Densitometric analysis of Coomassie stained gels (Microtek Scan-
maker 4, processed with Adobe Photoshop/NIH-image software) quantitated
the BSA 44 kDa fragment as 4% relative to parent, and the lysozyme dimer
(28 kDa) at 8% relative to parent.
(16) Sperling, J.; Elad. D. J. Am. Chem. Soc. 1971, 93, 3839. Marsh, E. N.
G. BioEssays 1995, 17, 431. Pedersen, J. Z.; Finazzi-Agro, A. FEBS 1993,
325, 53. Stubbe, J.; van der Donk, W. A. Chem. Biol. 1995, 2, 793.
(17) Hirama, M.; Akiyama, K.; Tanaka, T.; Noda, T.; Iida, K.; Sato, I.;
Hanaishi, R.; Fukuda-Ishisaka, S.; Ishiguro, M.; Otani, T.; Leet, J. E. J. Am.
Chem. Soc. 2000, 122, 720.
(18) We thank the NIH [1RO1GM57123] for generous financial support.