Total synthesis and stereochemistry of uncialamycin

Article abstract of DOI:10.1002/anie.200700917

Unseal the secrets of uncialamycin: The total synthesis of the C26 epimers of the newly discovered enediyne natural product allows its stereochemical assignment and further biological investigations of this potent antibiotic. Key steps involve the additio

Full text of DOI:10.1002/anie.200700917

Communications
DOI: 10.1002/anie.200700917
Uncialamycin
Total Synthesis and Stereochemistry of Uncialamycin**
K. C. Nicolaou,* Hongjun Zhang, Jason S. Chen, James J. Crawford, and Laxman Pasunoori
In memoryof Yoshihiko Ito
Uncialamycin (1a or 1b, Scheme 1) is a newly discovered
stereochemistry of the C26 hydroxy group remained unas-
signed) and the biological investigation. In view of this state
of affairs and our earlier experience with enediyne antitumor
enediyne antibiotic that possesses an intriguing molecular
[
1]
architecture and extremely potent biological properties.
[
2]
antibiotics, we set out to synthesize the two C26 epimers of
uncialamycin (1a and 1b) to address the above issues. Herein
we report the first total synthesis of both C26 epimers of
racemic uncialamycin and assign the 26R* structure (1b) as
that of the natural substance through spectroscopic studies
and X-ray analysis.
Our strategy for the construction of uncialamycin cen-
tered around three key reactions: addition of an acetylide to a
pyridinium species, an intramolecular acetylide addition to
form the enediyne ring system, and Hauser annulation to
[
3]
form the anthraquinone moiety. Fragments 2–4 were thus
identified as the key building blocks for the projected
synthesis.
Scheme 2 summarizes the synthesis of the required
[
6]
building block 2. Subjecting 5-methoxyisatin (5)
and
Scheme 1. Structures and retrosynthetic analysis of (26S*)- and
(
26R*)-uncialamycins (1a and 1b). DMB=3,4-dimethoxybenzyl,
TES=triethylsilyl, TMS=trimethylsilyl.
Uncialamycin was isolated from an unreported strain of
Streptomycete, related to Streptomyces cyanogenus, and
exhibits potent plasmid DNA cleavage and phenomenal
in vitro activity against Staphylococcus aureus (MIC
À1
0
.0000064 mgmL ), as well as activity against Escherichia
À1
coli (MIC 0.002 mgmL ) and Burkholderia cepacia (MIC
À1 [1]
0
.001 mgmL ). The scarcity of uncialamycin (only 300 mg
were isolated) limited both the structural elucidation (the
[
*] Prof. Dr. K. C. Nicolaou, Dr. H. Zhang, J. S. Chen, Dr. J. J. Crawford,
L. Pasunoori
Department of Chemistry and
The Skaggs Institute for Chemical Biology
The Scripps Research Institute
Scheme 2. Synthesis of quinoline system 2: a) KOH (1.0 equiv), H O,
2
2
58C, 5 min; then 6 (1.5 equiv), 258C, 30 min; b) 10% aq Na CO ,
2 3
8
08C, 30 min; then NaBH (4.0 equiv), 808C, 5 min, 86% over 2 steps;
4
c) aq HBr, 1208C, 18 h, then DMBBr (2.5 equiv), K CO (10 equiv),
2
3
[18]crown-6 (0.1 equiv), DMF, 258C, 4 h, 50%; d) DIBAL-H (1.1 equiv),
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
CH Cl , À78 to 258C over 1 h; e) TESCl (1.3 equiv), imidazole
2
2
Fax: (+1)858-784-2469
E-mail: kcn@scripps.edu
and
(2.6 equiv), DMF, 258C, 20 min, 86% over 2 steps, ca. 1:1 mixture of
diastereoisomers. DIBAL-H=diisobutylaluminum hydride.
Department of Chemistry and Biochemistry
University of California, San Diego
9
500 Gilman Drive, La Jolla, CA 92093 (USA)
methoxy enone 6 to a two-step Friedlander quinoline syn-
thesis via intermediate 7 afforded the keto carboxylate 8,
[
**] We thank Dr. D. H. Huang and Dr. G. Siuzdak for assistance with
NMR spectroscopy and mass spectrometry, respectively, and Dr.
R. K. Chadha for X-ray analysis. Financial support for this work was
provided by The Skaggs Institute for Chemical Biology and a
National Defense Science and Engineering Graduate fellowship
[7]
which was reduced in situ (NaBH ) to furnish the tricyclic
4
lactone 9 in 86% overall yield. Exchange of the phenolic
methyl group for a DMB group led to 10 (50% overall yield
for the two steps). The lactone moiety of 10 was reduced
(DIBAL-H) and protected as a TES lactol to afford the
(
J.S.C.). J.J.C. acknowledges a Fullbright–AstraZeneca Research
Fellowship.
4
704
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 4704 –4707

Angewandte
Chemie
Scheme 3. Synthesis of (26S*)-uncialamycin (1a): a) 2 (1.0 equiv), 3 (1.8 equiv), EtMgBr (1.9 equiv), 258C, 30 min; then AllocCl (1.6 equiv), 258C,
min, 92% (based on 80% conversion); b) MeCN/H O/AcOH (4:1:2), 258C, 5 h, 91%; c) NaBH (1.0 equiv), MeOH, 258C, 10 min; d) mCPBA
5
2
4
(
1.4 equiv), CH Cl , 08C, 3 h, 80% over 2 steps; e) AcCl (1.1 equiv), collidine (3.0 equiv), 08C, 30 min, then 258C, 12 h, 82%; f) TESCl (1.5 equiv),
2 2
imidazole (3.0 equiv), DMF, 08C, 10 min; g) K CO (2.0 equiv), THF/MeOH (2:1), 08C, 20 min, 78% over 2 steps; h) DMP (2.0 equiv), CH Cl ,
2
3
2
2
2
58C, 3 h, 87%; i) DDQ (4.0 equiv), CH Cl /H O (10:1), 258C, 12 h, 87%; j) CeCl (4.0 equiv), 258C, THF, 30 min; then KHMDS (5.0 equiv), À78
2
2
2
3
to À408C over 1 h, 61% (based on 80% conversion), plus 30% C17 epimer; k) PhI(OAc) (1.1 equiv), MeOH, 258C, 10 min, 80%; l) nBu SnH
2
3
(
1.1 equiv), H O (4.0 equiv), [Pd(PPh ) Cl ] (0.1 equiv), CH Cl , 258C, 20 min, 74% (based on 70% conversion); m) 4 (3.0 equiv), LiHMDS
2 3 2 2 2 2
(
3.0 equiv), THF, À788C, 20 min; then 16 (1.0 equiv), À78 to 258C over 1 h, 63%; n) 3HF·Et N (100 equiv), THF, 258C, 1 h, 92%. DDQ=2,3-
3
dichloro-5,6-dicyano-1,4-benzoquinone, DMP=Dess-Martin periodinane; HMDS=hexamethyldisilazide, mCPBA=meta-chloroperoxybenzoic acid.
1
3
quinoline system 2 (86% overall yield for the two steps of
approximately a 1:1 inconsequential mixture of diastereoiso-
mers).
and C NMR spectroscopic data (Table 1) were consistent
with its structure but differed from those reported for natural
[
1]
uncialamycin.
Activation of the pyridine moiety in 2 with allyloxycar-
bonyl chloride (AllocCl) and trapping of the generated
pyridinium species with the acetylide derived from enediyne
Having proven that the structure of uncialamycin was not
1a, we then set out to synthesize the 26R* epimer 1b to
confirm the expectation that the latter was the true structure
of the natural product. The task would prove rather simple,
for it was soon discovered that an oxidation/reduction
sequence using the hydroxy acetate 12 afforded clean
inversion of the stereochemistry at the C26 position
(Scheme 4). Thus, oxidation of 12 to the ketone 17 through
[
8]
3
and EtMgBr furnished the intermediate 11 in 92% yield
[
2d,e,5,9]
(
based on 80% conversion, Scheme 3).
Removal of the
TES group from 11 (AcOH, 91% yield) followed by
reduction (NaBH ) of the resulting lactol yielded a diol,
4
which underwent selective epoxidation with mCPBA to give,
after monoacetylation (AcCl, collidine), the hydroxy epoxide
1
2 (66% overall yield for the three steps). Protection of the
free hydroxy group of 12 as a TES ether followed by K CO -
2
3
induced cleavage of both the acetate and TMS groups from
the product led to the hydroxy enediyne 13 (78% overall yield
for two steps). Oxidation of the hydroxy group in 13 (DMP,
8
8
7% yield) followed by removal of the DMB group (DDQ,
7% yield) afforded the cyclization precursor, acetylenic
aldehyde 14.
The crucial cyclization of 14 to afford the desired 10-
[
2d,e,5a,b]
membered ring enediyne 15
was achieved in 61% yield
(
based on 80% conversion) by exposure to KHMDS in the
presence of CeCl . The undesired C17 epimer of 15 was also
3
[
10]
formed in 30% yield. Oxidation of 15 to the corresponding
methoxy hemiquinone system (PhI(OAc) , MeOH, 80%
2
yield) and subsequent removal of the Alloc group from the
resulting product (cat. [Pd(PPh ) Cl ], 74% yield based on
3
2
2
7
1
0% conversion) furnished the rather labile iminoquinone
[
5]
[11]
6. Finally, Hauser annulation
of 16 with nitrile 4
Scheme 4. Synthesis of (26R*)-uncialamycin (1b): a) DMP (2.0 equiv),
(
9
LiHMDS, 63% yield) followed by desilylation (3HF·Et N,
2% yield) furnished (26S*)-uncialamycin (1a), whose H
3
1
CH Cl , 258C, 90 min, 92%; b) NaBH (1.0 equiv), MeOH, 258C,
2
2
4
10 min, 98%, >96% stereoselectivity.
Angew. Chem. Int. Ed. 2007, 46, 4704 –4707
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 4705

Communications
the use of DMP, followed by reduction (NaBH ) furnished the
Table 1: Selected data for compounds 1a, 1b, and 19.
4
hydroxy acetate 18, which was epimeric at C26, in 90%
1
1
a: R =0.13 (silica gel, EtOAc/hexanes 2:3); H NMR (600 MHz,
f
overall yield and greater than 96% stereoselectivity. The
[D ]DMSO): d=13.18 (s, 1H), 10.01 (d, J=4.4 Hz, 1H), 8.56 (s, 1H),
6
previously developed sequence that produced (26S*)-uncial-
8.25 (overlapping doublets, 2H), 7.95 (t, J=7.6 Hz, 1H), 7.90 (t,
J=7.3 Hz, 1H), 6.68 (d, J=5.0 Hz, 1H), 6.10 (d, J=9.9 Hz, 1H), 5.99
[
12]
amycin (1a) from intermediate 12 also served well
to
(d, J=9.8 Hz, 1H), 5.62 (d, J=5.6 Hz, 1H), 5.51 (d, J=4.9 Hz, 1H),
deliver, from intermediate 18, (26R*)-uncialamycin (1b),
1
13
5.03 (d, J=3.4 Hz, 1H), 4.20 (quint, J=6.4 Hz, 1H), 1.34 ppm(d,
whose H and C NMR spectroscopic data (Table 1) were
1
3
J=6.7 Hz, 3H); C NMR (150 MHz, [D ]DMSO): d=186.9, 182.2,
[
1]
6
consistent with those reported for the naturally occurring
substance. Synthetic 1b formed deep-purple crystals (1758C
decomp) from ethyl acetate/hexanes that yielded to X-ray
1
1
54.7, 143.5, 135.7, 134.8, 134.3, 133.5, 132.1, 129.9, 126.5, 126.0, 124.0,
23.3, 112.6, 110.4, 101.2, 98.2, 89.7, 88.5, 76.2, 66.0, 65.2, 62.7, 42.3,
+
+
21.8 ppm; HRMS (ES): calcd for C H NO : 440.1129 [M+H] , found
2
5
18
6
[
13]
analysis (Figure 1).
These results provided unambiguous
440.1133.
proof of the structure of uncialamycin as being that of 1b.
1
1
b: R =0.14 (silica gel, EtOAc/hexanes 2:3); H NMR (600 MHz,
f
[
D ]DMSO): d=13.19 (s, 1H), 10.01 (d, J=4.5 Hz, 1H), 8.53 (s, 1H),
6
8.24 (overlapping doublets, 2H), 7.94 (td, J=7.4, 1.1 Hz, 1H), 7.89 (td,
J=7.4, 1.1 Hz, 1H), 6.68 (d, J=5.1 Hz, 1H), 6.06 (d, J=9.8 Hz, 1H),
5
1
.98 (d, J=10.0 Hz, 1H), 5.39 (d, J=5.7 Hz, 1H), 5.16 (d, J=5.1 Hz,
H), 5.07 (d, J=4.6 Hz, 1H), 4.33 (quint, J=6.2 Hz, 1H), 1.31 ppm(d,
1
3
J=6.5 Hz, 3H); C NMR (150 MHz, [D ]DMSO): d=186.8, 182.1,
6
1
1
2
4
54.7, 143.5, 135.5, 134.8, 134.3, 133.5, 132.1, 129.8, 126.5, 126.0, 123.9,
23.2, 112.6, 110.3, 100.3, 98.8, 89.6, 87.3, 75.9, 63.5, 62.9, 59.7, 43.1,
+
+
1.9 ppm; HRMS (ES): calcd for C H NO : 440.1129 [M+H] , found
2
5
18
6
40.1123.
1
1
9: R =0.13 (silica gel, EtOAc/hexanes 2:3); H NMR (600 MHz,
f
CD CN): d=13.26 (s, 1H), 10.80 (d, J=4.7 Hz, 1H), 8.27 (d, J=7.0 Hz,
3
1
1
H), 8.24 (d, J=7.1 Hz, 1H), 7.83 (t, J=7.0 Hz, 1H), 7.77 (t, J=7.3 Hz,
H), 7.77 (s, 1H), 7.45 (d, J=8.3 Hz, 1H), 7.43 (d, J=7.6 Hz, 1H), 7.29
Figure 1. ORTEP drawing of uncialamycin (1b). Thermal ellipsoids are
set at the 30% probability level.
(t, J=6.9 Hz, 1H), 7.25 (t, J=7.0 Hz, 1H), 5.42 (d, J=7.6 Hz, 1H), 5.14
d, J=5.2 Hz, 1H), 4.25 (d, J=7.9 Hz, 1H), 4.02 (quint, J=6.5 Hz, 1H),
(
3
.70 (s, 1H), 2.92 (d, J=7.1 Hz, 1H), 1.46 ppm(d, J=6.4 Hz, 3H);
Uncialamycin (1b) proved to be quite stable in the solid
13
C NMR (150 MHz, CD CN): d=188.3, 182.8, 156.1, 143.2, 137.6,
3
phase and in a variety of solvents. In the presence of dry HCl
136.9, 136.6, 136.1, 135.4, 133.8, 133.6, 133.3, 129.7, 129.1, 128.8, 128.0,
127.5, 126.9, 114.5, 109.1, 80.0, 78.0, 73.5, 69.3, 56.8, 20.4 ppm; HRMS
in CH Cl2 at ambient temperature, however, it rapidly
2
+
+
(
ES): calcd for C H ClNO : 478.1057 [M+H] , found 478.1074.
converts into the blue hexacyclic compound 19 (90% yield,
25 21 6
Table 1), presumably as a consequence of a cascade that
[
14]
involves a Bergman cycloaromatization reaction as shown
in Scheme 5. It is assumed that uncialamycin damages DNA
and kills cells through a mechanism that involves such a
cascade sequence initiated by bioreduction in a similar
[
1–5]
manner as dynemicin A.
While the described study proves the structure of
uncialamycin and renders its racemic form readily available,
it leaves the absolute stereochemistry unverified, although its
[
4,5a,b]
structural similarity to dynemicin A
and its DNA cleav-
[
1]
age activity are highly suggestive of the shown enantiomeric
form. An asymmetric synthesis of uncialamycin, currently in
progress in these laboratories, should prove this hypothesis
and provide ample quantities of its natural form and related
analogues for biological investigations.
Received: February 28, 2007
Published online: May 11, 2007
Keywords: antibiotics · enediynes · natural products ·
.
structure elucidation · total synthesis
[
1] J. Davies, H. Wang, T. Taylor, K. Warabi, X.-H. Huang, R. J.
Andersen, Org. Lett. 2005, 7, 5233 – 5236.
[
2] a) K. C. Nicolaou, W.-M. Dai, Angew. Chem. 1991, 103, 1453 –
1481; Angew. Chem. Int. Ed. Engl. 1991, 30, 1387 – 1416; b) K. C.
Nicolaou, W.-M. Dai, S.-C. Tsay, V. A. Estevez, W. Wrasidlo,
Scheme 5. Bergman cycloaromatization of uncialamycin (1b):
a) 0.005m HCl in CH Cl , 258C, 5 min, 90%.
2
2
4
706 www.angewandte.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 4704 –4707

Angewandte
Chemie
Science 1992, 256, 1172-1178; c) K. C. Nicolaou, A. L. Smith,
E. W. Yue, Proc. Natl. Acad. Sci. USA 1993, 90, 5881 – 5888;
d) K. C. Nicolaou, C.-K. Hwang, A. L. Smith, S. V. Wendeborn,
J. Am. Chem. Soc. 1990, 112, 7416 – 7418; e) K. C. Nicolaou, P.
Maligres, T. Suzuki, S. V. Wendeborn, W.-M. Dai, R. K. Chadha,
J. Am. Chem. Soc. 1992, 114, 8890 – 8907.
[8] A. Ernst, L. Gobbi, A. Vasella, Tetrahedron Lett. 1996, 37, 7959 –
7962.
[9] The lactol ring led to the rigid positioning of the methyl group
which effectively blocked the formation of the alternative
diastereoisomer in this reaction. Furthermore, this rigid ring
system allowed the definitive assignment of the relative stereo-
chemistry of 11 on the basis of HMQC NMR analysis (to
unambiguously identify the H24 position) and ROESY NMR
analysis, which indicated a correlation between the H24 and H27
atoms.
10] A tentative stereochemical assignment of C17 was made on the
basis of ROESY analysis of both C17 epimers of 15. Definitive
assignment was afforded by ROESYanalysis of 1a, derived from
the major epimer 15, which indicated a correlation between the
H17 and H26 atoms.
[
[
3] For related studies that culminated in elegant total syntheses of
[
4]
dynemicin A, which is a related enediyne antitumor antibiotic,
see reference [5].
4] a) M. Konishi, H. Ohkuma, T. Tsuno, T. Oki, G. D. VanDuyne, J.
Clardy, J. Am. Chem. Soc. 1990, 112, 3715 – 3716; b) M. Konishi,
H. Ohkuma, K. Matsumoto, H. Kamei, T. Miyaki, T. Oki, H.
Kawaguchi, G. D. VanDuyne, J. Clardy, J. Antibiot. 1989, 42,
[
1449 – 1452.
[
5] a) A. G. Myers, M. E. Fraley, N. J. Tom, S. B. Cohen, D. J. Madar,
Chem. Biol. 1995, 2, 33 – 43; b) A. G. Myers, N. J. Tom, M. E.
Fraley, S. B. Cohen, D. J. Madar, J. Am. Chem. Soc. 1997, 119,
[
[
[
11] a) F. M. Hauser, R. P. Rhee, J. Am. Chem. Soc. 1979, 101, 1628 –
1629; b) G. A. Kraus, H. Cho, S. Crowley, B. Roth, H. Sugimoto,
S. Prugh, J. Org. Chem. 1983, 48, 3439 – 3444.
12] The key cyclization to form the desired 10-membered enediyne
ring system now occurred with a 5:1 diastereomeric ratio (80%
total yield).
13] CCDC-638436 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via www.ccdc.
cam.ac.uk/data_request/cif.
6072 – 6094; c) M. D. Shair, T. Y. Yoon, S. D. Danishefsky,
Angew. Chem. 1995, 107, 1883 – 1885; Angew. Chem. Int. Ed.
Eng. 1995, 34, 1721 – 1723; d) M. D. Shair, T. Y. Yoon, K. K.
Mosny, T. C. Chou, S. J. Danishefsky, J. Am. Chem. Soc. 1996,
118, 9509 – 9525.
[
6] S. E. V. Bell, R. F. C. Brown, F. W. Eastwood, J. M. Horvath,
Aust. J. Chem. 2000, 53, 183 – 190. Purchased from TCI America.
7] H. Bretschneider, K. Hohenlohe-Oehringen, A. Rhomberg, US
patent 3,311,632, 1967.
[
[
14] a) R. R. Jones, R. G. Bergman, J. Am. Chem. Soc. 1972, 94, 660 –
661; b) R. G. Bergman, Acc. Chem. Res. 1973, 6, 25 – 31.
Angew. Chem. Int. Ed. 2007, 46, 4704 –4707
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 4707