Molecular simplification in bioactive molecules: Formal synthesis of (+)-muconin

Article abstract of DOI:10.1021/jo0524674

The concept of molecular simplification as a drug design strategy to shorten synthetic routes, while keeping or enhancing the biological activity of the lead drug, has been applied to (+)-muconin, an acetogenin with remarkable cytotoxicity. A novel approa

Full text of DOI:10.1021/jo0524674

Molecular Simplification in Bioactive Molecules:
Formal Synthesis of (+)-Muconin
Fernando R. Pinacho Cris o´ stomo, Romen Carrillo, Leticia G. Le o´ n, Tom a´ s Mart ´ı n,§,|
†
†
†,‡
Jos e´ M. Padr o´ n,† and V ´ı ctor S. Mart ´ı n*
,‡,|
,†,|
Instituto UniVersitario de Bio-Org a´ nica “Antonio Gonz a´ lez”, UniVersidad de La Laguna, Astrof ´ı sico
Astrof ´ı sico Francisco S a´ nchez 2, 38206 La Laguna, Tenerife, Islas Canarias, Spain,
Instituto Canario de InVestigaci o´ n Biom e´ dica, Hospital UniVersitario NS de Candelaria,
Ctra. del Rosario s/n, 38010 S/C de Tenerife, Spain, Instituto Canario de InVestigaci o´ n del C a´ ncer,
Red Tem a´ tica de InVestigaci o´ n CooperatiVa de Centros de C a´ ncer, Ctra. del Rosario s/n, 38010 S/C de
Tenerife, Spain, and Instituto de Productos Naturales y Agrobiolog ´ı a, Consejo Superior de InVestigaciones
Cient ´ı ficas, Astrof ´ı sico Francisco S a´ nchez 3, 38206 La Laguna, Tenerife, Islas Canarias, Spain
Vmartin@ull.es
ReceiVed NoVember 30, 2005
The concept of molecular simplification as a drug design strategy to shorten synthetic routes, while
keeping or enhancing the biological activity of the lead drug, has been applied to (+)-muconin, an
acetogenin with remarkable cytotoxicity. A novel approach that enables the stereoselective synthesis of
such a natural compound or its enantiomer from a common precursor is described. An additional advantage
of the method is complete stereochemical control and the decrease in the number of chemical steps
required, thus providing an enhancement of the overall yield. Antiproliferative studies against the human
solid tumor cell lines showed that the aliphatic chain-THF/THP fragment of (+)-muconin has modest
cytotoxic activity. The strategy opens the way to preparing novel bioactive acetogenin analogues by
shorter synthetic routes.
Introduction
mono-tetrahydrofuran, adjacent bis-tetrahydrofuran, nonadjacent
bis-tetrahydrofuran, and nonclassical acetogenins.
1
Acetogenins constitute a broad family of compounds with
interesting biological properties. From the plant family Annon-
aceae, a large number of natural acetogenins have been isolated.
These compounds have attracted the attention of many research
groups as a result of their interesting structural complexity and
the important biological activities (anticancer, pesticidal, im-
(
+)-Muconin (1), a nonclassical acetogenin, was isolated for
2
the first time by McLaughlin et al. in 1996. This natural product
can be split up into three parts: a long carbon chain, sequential
THF and THP rings, and a 2,4-disubstituted R,â-unsaturated
γ-lactone (Figure 1). Furthermore, this compound has eight
stereogenic centers, six of which are found in the middle part
(THF/THP rings). Focusing on the biological activity, (+)-
muconin (1) showed a potent in vitro cytotoxicity against human
1
munosuppressive, and antifeedant) they exert. The four main
classes of structural features of annonaceous acetogenins are
2
pancreatic and breast tumor cells. In light of the interesting
†
‡
§
|
biological results and its backbone novelty, some groups
Universidad de La Laguna.
dedicated their efforts to achieve its total synthesis.3
Instituto Canario de Investigaci o´ n Biom e´ dica.
Consejo Superior de Investigaciones Cient ´ı ficas.
Instituto Canario de Investigaci o´ n del C a´ ncer.
1) For reviews, see: (a) Alali, F. Q.; Liu, X.-X.; McLaughlin, J. L. J.
The critical issue encountered in the diverse reported total
syntheses has been to introduce in a straightforward manner
(
Nat. Prod. 1999, 62, 504-540. (b) Bermejo, A.; Figadere, B.; Zafra-Polo,
M.-C.; Barrachina, I.; Estornell, E.; Cortes, D. Nat. Prod. Rep. 2005, 22,
(2) Shi, G.; Kozlowski, J. F.; Schwedler, J. T., Wood, K. V.; MacDougal,
2
69-303.
J. M.; MacLaughlin, J. L. J. Org. Chem. 1996, 61, 7988-7989.
1
0.1021/jo0524674 CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/11/2006
J. Org. Chem. 2006, 71, 2339-2345
2339

Pinacho Cris o´ stomo et al.
SCHEME 2. Retrosynthetic Analysis Developed by
Takahashi’s Group for the Synthesis of (+)-Muconin (1)
FIGURE 1. Main structural blocs in (+)-muconin.
SCHEME 1. Common Step in Several Synthetic
Approaches to (+)-Muconin (1)
SCHEME 3. Epoxides as Nucleophiles in the
Intramolecular Nicholas Reaction; Strategy for the Key THP
Ring
the six stereogenic carbons present in the sequential THF/THP
3
moiety. In most reported syntheses, a common strategy is the
oxidation and subsequent stereoselective reduction of a second-
ary alcohol (Scheme 1). Although the sequence of oxidation/
reduction reactions works well and with moderate stereocontrol,
it represents an increase in the total number of synthetic steps.
Structural simplification represents a drug design strategy to
shorten synthetic routes while keeping or enhancing the biologi-
cal activity of the natural template. The concept has been applied
successfully in naturally occurring acetogenins, leading to
analogues with remarkable cytotoxicity against human solid
synthetic intermediates against the human ovarian cancer cell
line A2780 and the human non-small cell lung cancer (NSCLC)
cell line SW1573 is described. The application of molecular
simplification in this class of compounds is also discussed.
Recently, we have developed a reliable strategy to synthesize
polysubstituted oxanes and oxepanes with a high degree of
stereocontrol by a Nicholas reaction, based on the intramolecular
nucleophilic attack of an epoxide to a carbocation generated
4
tumor cells. In acetogenins in general, the 2,4-disubstituted R,â-
unsaturated γ-lactone fragment is claimed to be essential for
1a
the biological activity. However, the contribution of the other
structural fragments of these molecules to the cytotoxic activity
has not been studied in detail. We addressed our attention toward
7
by acid treatment of exo-Co2(CO)6 propargylic alcohols. This
3
e
the synthetic strategy developed by Takahashi’s group, since
such studies should permit analysis of the influence of the
aliphatic chain and THF/THP fragments in the cytotoxic activity
of muconin. Their strategy relies on the Sonogashira coupling
between the building blocks 2 and 3 (Scheme 2). The building
block 2 was synthesized from compound 4, which represents a
very advanced intermediate in the synthesis and in which the
six stereogenic centers are installed. Therefore, this molecular
block represents an interesting synthetic target inasmuch as its
synthesis achieved in some alternative manner represents a new
formal synthesis of the natural compound.
synthetic tool allows us to introduce a new stereocenter at the
propargylic position with total control of the stereochemistry
(Scheme 3).
With the aforementioned methodology, we planned the
retrosynthetic analysis of 4 outlined in Scheme 4, where the
1
THP ring 5 (R ) H) would act as a scaffold. We first
disconnected the saturated chain from the terminal epoxide 6,
bearing in mind the possibility of introducing such an alkyl chain
by a nucleophilic opening of the terminal epoxide ring. The
THF ring was envisioned through a regio- and stereoselective
exo-cyclization in situ of the suitable epoxy alcohol, which in
turn could be obtained through a Katsuki-Sharpless asymmetric
Within our research program directed toward the synthesis
5
6
of cyclic ethers and biologically active molecules, we report
herein on the synthesis of intermediate 4. In addition, a
preliminary structure-activity relationship (SAR) study of the
(5) (a) Mart ´ı n, T.; Soler, M. A.; Betancort, J. M.; Mart ´ı n, V. S. J. Org.
Chem. 1997, 62, 1570-1571. (b) Betancort, J. M.; Mart ´ı n, V. S.; Padr o´ n,
J. M.; Palaz o´ n, J. M.; Ram ´ı rez, M. A.; Soler, M. A. J. Org. Chem. 1997,
6
2, 4570-4583. (c) Ram ´ı rez, M. A.; Padr o´ n, J. M.; Palaz o´ n, J. M.; Mart ´ı n,
(
3) (a) Schaus, S. E.; Branalt, J.; Jacobsen, E. N. J. Org. Chem. 1998,
V. S. J. Org. Chem. 1997, 62, 4584-4590. (d) Garc ´ı a, C.; Soler, M. A.;
Mart ´ı n, V. S. Tetrahedron Lett. 2000, 41, 4127-4130. (e) Garc ´ı a, C.; Mart ´ı n,
T.; Mart ´ı n, V. S. J. Org. Chem. 2001, 66, 1420-1428. (f) Betancort, J. M.;
Mart ´ı n, T.; Palaz o´ n, J. M.; Mart ´ı n, V. S. J. Org. Chem. 2003, 68, 3216-
3224.
(6) (a) Padr o´ n, J. M.; Tejedor, D.; Santos-Exp o´ sito, A.; Garc ´ı a-Tellado,
F.; Mart ´ı n V. S.; Villar, J. Bioorg. Med. Chem. Lett. 2005, 15, 2487-2490.
(b) Donadel, O. J.; Mart ´ı n, T.; Mart ´ı n, V. S.; Villar, J.; Padr o´ n, J. M. Bioorg.
Med. Chem. Lett. 2005, 15, 3536-3539.
(7) Cris o´ stomo, F. R. P.; Mart ´ı n, T.; Mart ´ı n, V. S. Org. Lett. 2004, 66,
565-568.
6
7
1
4
5
2
3, 4876-4877. (b) Yang, W.-Q.; Kitahara, T. Tetrahedron Lett. 1999, 40,
827-7830. (c) Yang, W.-Q.; Kitahara, T. Tetrahedron 2000, 56, 1451-
461. (d) Takahashi, S.; Kubota, A.; Nakata, T. Tetrahedron Lett. 2002,
3, 8661-8664. (e) Takahashi, S.; Kubota, A.; Nakata, T. Tetrahedron 2003,
9, 1627-1638. (f) Yoshimitsu, T.; Makino, T.; Nagaoka, H. J. Org. Chem.
004, 69, 1993-1998.
(4) (a) Zeng, B. B.; Wu, Y.; Jiang, S.; Yu, Q.; Yao, Z. J.; Liu, Z. H.; Li,
H. Y.; Li, Y.; Chen, X. G.; Wu, Y. L. Chem. Eur. J. 2003, 9, 282-290. (b)
Fujita, D.; Ichimaru, N.; Abe, M.; Murai, M.; Hamada, T.; Nishioka, T.;
Miyoshi, H. Tetrahedron Lett. 2005, 46, 5775-5779.
2340 J. Org. Chem., Vol. 71, No. 6, 2006

Formal Synthesis of (+)-Muconin
SCHEME 4. Retrosynthetic Analysis of Compound 4
SCHEME 5. Improved Synthesis of the Linear Precursor
a
13
a
Reagents and conditions: (a) SO3‚Py, DMSO, TEA, Ph3PdCHCO2Et,
CH2Cl2; (b) DIBAL-H, toluene, 0 °C; (c) (i) (+)-DET, Ti(OPr-i)4, TBHP,
CH2Cl2, -20 °C (ii) toluene, DMAP, Boc2O; (d) (i) OsO4 cat., NaIO4, NMO,
THF/H2O (1:1) (ii) TMS-acetylene, n-BuLi, THF, -78 °C.
8
epoxidation (KSAE) of the allylic alcohol 7. Compound 7 could
be synthesized by an allyl Grignard regioselective opening of
SCHEME 6. Synthesis of the Allylic Alcohol 7 Precursor of
epoxide 8 with further homologation and reduction. Compound
the THF/THP Ringsa
1
8
would be derived from 5 (R ) H) after a simple four-step
sequence: hydrogenation of the triple bond, Sharpless asym-
metric dihydroxylation (SAD) of the olefin, selective tosylation
9
of the primary alcohol, and terminal epoxide formation under
basic conditions. Three major advantages derived from this
strategy: first, the synthesis of the THF and THP stereoisomers
would be modulated by simply changing the chiral auxiliary at
the KSAE and SAD steps; second, the application of three
consecutive enantioselective reactions ensures the high enan-
tiomeric purity of the final products; third, the building block 8
could be homologated in both directions opening the possibility
to synthesize the enantiomer of compound muconin (1) starting
from the common intermediate 5.
Results and Discussions
1
The synthesis of 5 (R ) H) began with the commercially
available alcohol 5-hexen-1-ol (9), which was homologated by
a protocol recently developed in our group.10 Reduction of the
R,â-unsaturated ester 10 with DIBAL-H generated the allylic
alcohol 11. With 11 in our hands, we performed a KSAE using
a
Reagents and conditions: (a) see ref 6; (b) (i) K2CO3, MeOH (ii) 2,2-
dimethoxypropane, CSA, CH Cl ; (c) Lindlar catalyst, quinoleine, EtOAc;
(d) AD-mix-R, t-BuOH/H2O (1:1); (e) (i) TsCl, Py, 0 °C (ii) NaH, THF;
(f) AllylMgBr, CuI, THF; (g) (i) OsO4 cat., NaIO4, NMO, THF/H2O (1:1)
2
2
11
as chiral auxiliary (+)-diethyl tartrate, followed by alcohol
protection as tert-butyl carbonate (Boc). The double bond
present in compound 12 was cleaved with OsO4/NaIO4. Thus,
the obtained aldehyde was submitted to the next step without
further purification. The coupling of this aldehyde with the
lithium salt of trimethylsilylacetylene furnished the diastereo-
meric propargylic alcohols 13. This synthetic route to 13
represents an improvement in the overall yields and in the
number of chemical steps (47% in 6 steps) on an earlier strategy
(
ii) Ph3P(O)dCHCO2Et, CH2Cl2; (h) DIBAL-H, toluene, 0 °C.
mix R affording diol 16 as an 8:1 inseparable epimeric mixture.
Fortunately, when the primary alcohol was selectively tosylated
and reacted with NaH, pure terminal epoxide 8 was obtained.
The key epoxide 8 was then opened with allyl Grignard, in the
presence of CuI, furnishing the allylic alcohol 17. Application
of the known OsO4/NaIO4 protocol to cleave double bonds,
followed by Wittig reaction, led to the R,â-unsaturated ester
8 in good yields. Reduction with DIBAL-H allowed us to
obtain intermediate 7 (Scheme 6).
With compound 7 in hand we proceeded to the THF ring
formation. Interestingly, the KSAE of 7 using (+)-diethyl tartrate
as chiral auxiliary provided by concomitant epoxide opening
the anti-tetrahydrofuran 19 as the sole detected stereoisomer
(
25% in 11 steps) (Scheme 5).7
Compound 13 was then transformed into the THP 5 (R )
1
1
TMS) by a regio- and stereoselective intramolecular Nicholas
reaction as described earlier. K2CO3 treatment and diol protec-
6
tion with 2,2-dimethoxypropane led to the key compound 14.
The next step was the introduction of a secondary alcohol
adjacent to the THP ring, a process that proceeded with total
stereocontrol. To carry out this transformation a two-step
sequence was applied: partial hydrogenation with Lindlar
catalyst to generate the double bond in 15 and a SAD with AD-
(
Scheme 7). The sequential THF/THP rings were then obtained
as a single diastereoisomer. In addition, the six stereocenters
were installed with complete stereocontrol. Compound 19 was
subjected to selective benzoylation of the primary alcohol and
further mesylation of the secondary alcohol. Basic hydrolysis
of the benzoyl ester group provided the terminal epoxide 6 by
intramolecular displacement of the secondary mesylate group
with the primary alkoxide. Finally, the regioselective opening
of the terminal epoxide with the Grignard reagent derived from
commercially available 1-bromoundecane, in the presence of
CuI, furnished the desired compound 4 achieving a formal
(
8) (a) Katsuki, T.; Sharpless, K. B. J. Am. Chem. Soc. 1980, 102, 5974-
976. (b) Kasutki, T.; Mart ´ı n, V. S. Organic Reactions; Paquette, L. A.,
Ed.; John Wiley & Sons: New York, New York, 1996; Vol. 48, pp 1-299.
9) Kolb, H. C.; Van Nieuwenhze, M. S.; Sharpless, K. B. Chem. ReV.
994, 94, 2483-2547.
10) Cris o´ stomo, F. R. P.; Carrillo, R.; Mart ´ı n, T.; Garc ´ı a-Tellado, F.;
Mart ´ı n, V. S. J. Org. Chem. 2005, 70, 10099-10101.
11) Rossiter, B. E.; Katsuki, T.; Sharpless, K. B. J. Am. Chem. Soc.
981, 103, 464-465.
5
(
1
(
(
3
e
1
synthesis of (+)-muconin (1).
J. Org. Chem, Vol. 71, No. 6, 2006 2341

Pinacho Cris o´ stomo et al.
SCHEME 7. Completion of the Formal Synthesis of
(
stereocenters present in the THF/THP moiety is ensured by
means of three enantioselective reactions, and the yield in each
step is over 70%. In addition, intermediate 5 can serve as a
scaffold to obtain both enantiomers of muconin (1). Moreover,
the described methodology can be applied to other natural
products with a similar structure. In addition, the developed
methodology opens the way to synthesize other diastereoiso-
mers/enantiomers by simply changing the chiral auxiliary used
in the enantioselective reactions. Biological observations suggest
that our data could lead to the development of a new class of
structurally simplified anticancer agents. Further studies along
these lines are currently under way in our laboratories, and the
results will be reported elsewhere.
+)-Muconin (1)a
a
Reagents and conditions: (a) (+)-DET, Ti(OPr-i)4, TBHP, CH2Cl2,
-
20 °C; (b) (i) TEA, benzoyl chloride, then MsCl, CH2Cl2 (ii) MeOH,
NaH, CH2Cl2, 0 °C; (c) bromoundecane, Mg, CuI, THF, -78 °C.
TABLE 1. In Vitro Cytotoxic Activity (GI50) against Human Solid
a
Experimental Section
Tumor Cells
cell line
Preparation of (E)-Ethyl Octa-2,7-dienoate (10). To a solution
of 5-hexen-1-ol (9) (12 mL, 99.8 mmol) in dry CH
under N atmosphere were added DMSO (80 mL) and triethylamine
83 mL, 0.6 mol) at room temperature. Then, to the stirred solution
was added Ph PdCHCO Et (70 g, 0.2 mol), and after it was
completely dissolved, SO ‚Py (48 g, 0.3 mol) was added. The
2 2
Cl (400 mL)
compound
A2780
SW1573
2
4
7
8
90 ( 18
>100
85 ( 20
>100
>100
24 ( 5
>100
>100
>100
>100
(
3
2
>100
1
1
1
1
1
5
6
7
8
9
18 ( 11
>100
3
reaction was monitored by TLC until the starting material could
not be detected. Subsequently, 5% HCl solution was added until
>100
>100
acidic pH. Extraction with CH
phases were washed with water and brine, dried over MgSO
2 2
Cl was done. The combined organic
>100
4
,
a
filtered, concentrated, and purified by column flash chromatography
to yield 13 g (80% yield) of 10. H NMR (300 MHz) δ 1.28 (t, J
Values are given in µM and are means of at least three experiments;
1
standard deviation is given.
)
7.1 Hz, 3H), 1.56 (m, 2H), 2.08 (m,2H), 2.20 (m, 2H), 4.17 (q,
J ) 7.1 Hz, 2 H), 4.99 (m, 2H), 5.79 (m, 2H), 6.95 (dt, J ) 15.5,
A series of the aforementioned intermediates was then
screened for growth inhibition and cytotoxicity against the
human solid tumor cell lines A2780 and SW1573 using the SRB
assay of the NCI.12 The sensitivities expressed as 50% growth
inhibition (GI50) are listed in Table 1. Interestingly, compounds
13
6
.9 Hz, 1H); C NMR (75 MHz) δ 14.0 (q), 26.9 (t), 31.2 (t),
32.8 (t), 59.9 (t), 114.8 (t), 121.3 (d), 137.8 (d), 148.7 (d), 166.5
-
1
(s); IR (cm ) 1367, 1443, 1654, 1721, 2933, 2980, 3078; MS (EI)
m/z (relative intensity) 54 (91), 81 (66), 95 (100), 123 (54), 140
+
(
39), 168 (M) (8); HRMS (EI) calcd for C10
H O
16 2
168.1150, found
1
7
68.1152. Anal. Calcd for C10
1.38, H 10.02.
H O C 71.39, H 9.59. Found: C
16 2
4
and 15 were the only products that reached a GI50 value within
the experimental range. Compound 4, which represents the left
half of muconin (1), showed modest GI50 values of 90 and 85
µM against A2780 and SW1573 cells, respectively. Despite the
absence of the 2,4-disubstituted R,â-unsaturated γ-lactone
fragment, which is essential for the activity of acetogenins,
intermediate 4 is able to inhibit cell growth. This effect is
masked in muconin (1) by the extraordinarily potency of the
butenolide fragment. Interestingly, compound 15 proved to be
a more potent derivative with GI50 values in the low micromolar
range, which are comparable to those of the anticancer drugs
cisplatin and 5-fluorouracil currently used in NSCLC and
ovarian cancer chemotherapy. Furthermore, compound 15 may
be considered a molecular or structural simplification of the
more complex intermediate 4, where the long aliphatic chain
and the THF ring have been replaced by a vinyl group.
Preparation of (E)-Octa-2,7-dien-1-ol (11). To a solution of
0 (13 g, 77.3 mmol) in dry toluene (400 mL) under N atmosphere
1
2
at 0 °C was added dropwise DIBAL-H (154 mL, 0.15 mol). When
TLC indicated the complete reaction of the starting material, the
2
reaction was quenched by the addition of H O. To the obtained
slurry was added MgSO , and then the mixture was filtered through
4
a pad of Celite. The solvent was evaporated, and the residue was
purified by column chromatography to give 9 g (93% yield) of 11.
1
H NMR (300 MHz) δ 1.48 (m, 2H), 2.04 (m, 4H), 4.07 (d, J )
1
3
4.6 Hz, 2H), 4.97 (m, 2H), 5.64 (m, 2H), 5.79 (m, 2H); C NMR
1
3
14
(75 MHz) δ 28.0 (t), 31.3 (t), 32.9 (t), 63.5 (t), 114.4 (t), 129.0 (d),
-1
1
32.7 (d), 138.4 (d); IR (cm ) 1670, 1721, 2857, 2928, 3077, 3354;
MS (EI) m/z (relative intensity) 55 (75), 67 (100), 79 (51), 93 (35),
+
1
1
1
8 14
07 (12), 125 (M - 1) (4); HRMS (EI) calcd for C H O
26.1045, found 126.1047. Anal. Calcd for C
1.18. Found: C 76.16, H 11.71.
8
H14O C 76.14, H
Preparation of tert-Butyl[(2S,3S)-3-(pent-4-enyl)]oxidan-2-yl]-
Conclusions
methyl Carbonate (12). To a solution of crushed molecular sieves
(MS 4 Å) and (+)-DET (17 mL, 99 mmol) in dry CH Cl under
In summary, we have accomplished a new formal and
practical synthesis of (+)-muconin (1). Total control of the six
2
2
N
2
4
atmosphere at -20 °C was added Ti(OPr-i) (25 mL, 85 mmol).
After 15 min, compound 11 was added (8.4 g, 71 mmol,) and 30
min later a 6.1 M solution of TBHP in isooctane (21 mL, 127 mmol)
was added. The reaction was stirred overnight. The reaction was
quenched by the addition of tartaric acid solution (15% in water),
(12) Skehan, P.; Storeng, P.; Scudeiro, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl.
Cancer Inst. 1990, 82, 1107-1112.
(13) (a) Van Moorsel, C. J.; Smid, K.; Voorn, D. A.; Bergman, A. M.;
and further extraction was done with CH
dried over MgSO , and the solvent was evaporated under reduced
2 2
Cl . The extracts were
Pinedo, H. M.; Peters, G. J. Int. J. Oncol. 2003, 22, 201-207. (b) Fokkema,
E.; Groen, H. J.; Helder, M. N.; de Vries, E. G.; Meijer, C. Biochem.
Pharmacol. 2002, 63, 1989-1996.
4
pressure. The residue obtained was dissolved in ether and washed
with NaOH solution (15% in water). The organic phase was dried
(14) Bergman, A. M.; Giaccone, G.; van Moorsel, C. J.; Mauritz, R.;
4
with MgSO and filtered. Following solvent evaporation the residue
Noordhuis, P.; Pinedo, H. M.; Peters, G. J. Eur. J. Cancer 2000, 36, 1974-
1
983.
was employed in the next step without further purification.
2342 J. Org. Chem., Vol. 71, No. 6, 2006

Formal Synthesis of (+)-Muconin
To a solution of the aforementioned crude epoxy alcohol in dry
(cm-1) 1063, 1250, 1715, 2179, 2954, 3384; MS (EI) m/z (relative
+
toluene (280 mL) under N
was added Boc anhydride (19 g, 85 mmol) followed by DMAP
867 mg, 7.1 mmol). The reaction was monitored by TLC and was
complete after 2 h. The reaction mixture was treated with CHCl
and HCl solution (5% in water) and extracted with more CHCl
The organic phase was dried and concentrated. Chromatography
2
atmosphere and at room temperature
intensity) 73 (100), 97 (29), 109 (15), 125 (84), 181 (34), 242 (M)
+
(3); HRMS (EI) calcd for C12
H O
22 3
Si (M) 242.1338, found
(
242.1331. Anal. Calcd for C12
59.44, H 9.01.
22 3
H O Si C 59.46, H 9.15. Found: C
3
3
.
Preparation of (2S,6R)-2-[(R)-2,2-Dimethyl-1,3-dioxolan-4-yl]-
6-ethynyl-tetrahydro-2H-pyran (14). To a solution of 5 (3.52 g,
14.5 mmol) in methanol (50 mL) at room temperature was added
K CO (1.0 g, 7.2 mmol). After 6 h the reaction was completed.
2
5
on silica gel afforded 14.5 g (85% yield) of 12. [R]
D
) -19.9 (c
1
1
4
1
(
5
.1, CHCl ); H NMR (300 MHz) δ 1.48 (s, 9H), 1.47-1.57 (m,
3
2
3
H), 2.08 (m, 2H), 2.85 (m, 1H), 2.97 (m, 1H), 4.00 (dd, J ) 5.8,
2.0 Hz, 1H), 4.22 (dd, J ) 3.8, 12.0 Hz, 1H), 4.97 (m, 2H), 5.76
2 2
To the reaction mixture was added CH Cl , and the mixture was
filtered through a pad of Celite. The solvent was evaporated under
reduced pressure, and the resulting residue was employed in the
next step without purification.
13
m, 1H); C NMR (75 MHz) δ 24.8 (t), 27.5 (q), 30.6 (t), 33.0 (t),
4.8 (d), 56.3 (d), 66.8 (t), 82.3 (s), 114.7 (t), 137.9 (d), 153.0 (s);
-
1
IR (cm ) 1370, 1641, 1744, 2862, 2980, 3077; MS (FAB) m/z
This crude was dissolved in dry CH Cl (50 mL) under N
2
2
2
(
relative intensity) 57 (100), 107 (41), 137 (14), 154 (9), 187 (33),
atmosphere and at room temperature. To the solution were added
2,2-dimethoxypropane (2.7 mL, 21.7 mmol) and camphor-10-
sulfonic acid (1.68 g, 7.2 mmol). The reaction was monitored by
TLC, and after 3 h starting material was not observed. A saturated
+
+
2
2
9
43 (M + 1) (8); HRMS (FAB) calcd for C13
H
23
O
4
(M + 1)
43.1596, found 243.1595. Anal. Calcd for C13
.15. Found: C 64.44, H 9.53.
H
22
O
4
C 64.44, H
Preparation of tert-Butyl{(2R,3R)-3-[4-hydroxy-6-(trimeth-
ylsilyl)hex-5-ynyl]oxiran-2-yl}methyl Carbonate (13). Compound
2 (14 g, 57.8 mmol) was dissolved in THF/H O 1:1 (350 mL) at
room temperature. To this solution were added NMO (17 g, 0.14
mol) and a catalytic amount of OsO . The reaction mixture was
stirred overnight, quenched with a saturated solution of Na
and extracted with EtOAc. The organic phase was dried, and the
solvent was evaporated under reduced pressure. The crude aldehyde
obtained was used in the next step without purification.
solution of NaHCO was added to the reaction mixture, and the
3
mixture was extracted with CH Cl . The organic phase was dried
2
2
1
2
and the solvent evaporated. Column chromatography afforded 2.35
g (78% yield) of compound 14. [R]25 ) -44.1 (c 1.1, CHCl ); H
1
D
3
4
NMR (300 MHz) δ 1.33 (s, 3H), 1.39 (s, 3H), 1.26-1.68 (m, 3H),
1.80-1.87 (m, 3H), 2.45 (d, J ) 2.1 Hz, 1H), 3.24 (m, 1H), 3.91-
3.97 (m, 2H), 4.04-4.13 (m, 2H); 13C NMR (75 MHz) δ 22.4 (t),
25.0 (q), 26.5 (q), 27.2 (t), 32.3 (t), 67.1 (t), 67.7 (d), 72.3 (d),
2 2 3
S O ,
-
1
77.7 (d), 79.0 (d), 82.8 (s), 109.0 (s); IR (cm ) 1371, 1719, 2120,
To a solution of ethynyltrimethylsilane (9 mL, 63.5 mmol) in
2860, 2942, 3284; MS (FAB) m/z (relative intensity) 55 (100), 69
+
dry THF under N
2
atmosphere at -78 °C was added a solution of
(65), 81 (73), 101 (75), 154 (52), 210 (M) (4); HRMS (FAB)
+
n-BuLi (2.1 M, 30 mL). After 30 min at the same temperature the
calcd for C12H
18
O
3
(M) 210.1256, found 210.1258. Anal. Calcd
crude aldehyde was added. The reaction was quenched by addition
for C12
H
18
O
3
C 68.54, H 8.63. Found: C 68.64, H 8.85.
of a saturated solution of NH
4
Cl, and the extraction was done with
, and the solvent
Preparation of (2S,6R)-2-[(R)-2,2-Dimethyl-1,3-dioxolan-4-yl]-
6-vinyl-tetrahydro-2H-pyran (15). A mixture of compound 14
(1.79 g, 8.5 mmol), Lindlar catalyst (89 mg, 5% w), and quinoline
(4 µL, 0.2% w) in dry EtOAc (50 mL) was stirred at room
Et O. The organic phase was dried over MgSO
2
4
was evaporated under reduced pressure. Purification by chromato-
graphic column afforded 14.8 g (75% yield) of 13. H NMR (300
MHz) δ 0.17 (s, 9H), 1.49 (s, 9H), 1.57-1.86 (m, 6H), 2.88 (m,
1
2
temperature under H atmosphere (1 atm) for 1 h. After this time,
1
3
2
6
1
5
H), 2.99 (m, 1H), 4.01 (dd, J ) 5.8, 12.0 Hz, 1H), 4.24 (dd, J )
.7, 12.0 Hz, 1H), 4.36 (m, 1H); 13C NMR (75 MHz) δ -0.4 (q),
1.4 (t), 27.5 (q), 30.8 (t), 36.9 (t), 57.7 (d), 56.2 (d), 62.4 (d),
6.7 (t), 82.3 (s), 89.5 (s), 106.2 (s), 153.0 (s); IR (cm-1) 1281,
370, 1745, 2170, 2957, 3459; MS (FAB) m/z (relative intensity)
TLC showed complete conversion. The solution was filtered through
a pad of Celite, and the filter cake was washed with EtOAc. After
purification by chromatographic column, 1.79 g (99% yield) of
25
1
compound 15 was obtained. [R]
D
3
) -38.1 (c 1.0, CHCl ); H
NMR (300 MHz) δ 1.35 (s, 3H), 1.40 (s, 3H), 1.23-1.67 (m, 4H),
1.79-1.92 (m, 2H), 3.32 (m, 1H), 3.84 (m, 1H), 3.95 (m, 2H),
4.05 (m, 1H), 5.06 (dt, J ) 10.6, 1.4 Hz, 1H), 5.19 (dt, J ) 17.3,
1.6 Hz, 1H), 5.83 (ddd, J ) 17.3, 10.6, 5.3 Hz, 1H); 1 C NMR (75
MHz) δ 22.6 (t), 25.0 (q), 26.4 (q), 27.5 (t), 31.0 (t), 66.8 (t), 77.9
7 (95), 73 (100), 137 (25), 154 (23), 181 (9), 269 (30); HRMS
+
(FAB) calcd for C17
H
30
O
5
SiNa (M + Na) 365.1760, found
3
3
5
65.1768. Anal. Calcd for C17
9.61, H 8.89.
30 5
H O Si C 59.61, H 8.83. Found: C
-
1
Preparation of (R)-1-{(2S,6R)-6-[2-(Trimethylsilyl)ethynyl]-
tetrahydro-2H-pyra-2-yl}ethane-1,2-diol (5). To a solution of 13
8.52 g, 24.9 mmol) in dry CH Cl under N atmosphere and at
room temperature was added Co (CO) (9.36 g, 27.3 mmol). The
(d), 78.0 (d), 78.2 (d), 108.8 (s), 114.1 (t), 138.8 (d); IR (cm )
1075, 1212, 1370, 2859, 2936, 2986; MS (EI) m/z (relative intensity)
+
(
2
2
2
67 (100), 93 (95), 101 (52), 111 (79), 197 (54), 212 (M) (1);
+
HRMS (EI) calcd for C12
211.1342. Anal. Calcd for C12
H
O
20 3
(M - 1) 211.1334, found
2
8
reaction was monitored by TLC until the starting material was
completely consumed. To the solution of the hexacarbonyldicobalt
20 3
H O C 67.89, H 9.50. Found: C
67.87, H 9.67.
complex of 13 was added BF
temperature. The reaction mixture was stirred for 3 h. The mixture
was poured with vigorous stirring into a saturated solution of
3
‚OEt
2
(3.15 mL, 24.9 mmol) at room
Preparation of (R)-1-{(2R,6S)-6-[(R)-2,2-Dimethyl-1,3-diox-
olan-4-yl]-tetrahydro-2H-pyran-2-yl}ethane-1,2-diol (16). Com-
pound 15 was added to a mixture of t-BuOH (43 mL), H O (43
2
NaHCO
3
at 0 °C and extracted with CH
2
Cl
2
. The combined organic
), and concentrated.
mL), and AD-mix-R (11.83 g, 1.4 g/mmol of 15) at 0 °C. The
solution was stirred for 12 h at 0 °C. After addition of a saturated
phases were washed with brine, dried (MgSO
4
The crude mixture was dissolved in acetone (2.1 mL) at 0 °C. Ceric
ammonium nitrate (41.0 g, 74.7 mmol) was added in portions with
stirring until evolution of CO ceased and the CAN color persisted
Na
2 3
SO solution (50 mL) the mixture was extracted with EtOAc.
The extracts were dried, and the solvent was removed. Flash
25
chromatography yielded 16 (1.70 g, 83% yield). [R]
D
) -6.6 (c
); H NMR (300 MHz) δ 1.32 (s, 3H), 1.40 (s, 3H),
1.17-1.53 (m, 3H), 1.72 (m, 2H), 1.93 (m, 1H), 3.35 (m, 1H),
1
(30 min). The solvent was removed under vacuum, and the pink
1.1, CHCl
3
solid residue was then partitioned between Et
aqueous phase was extracted additionally twice with Et
2
O and H
2
O. The
O. The
1
3
2
3.43 (m, 1H), 3.56 (m, 1H), 3.68 (m, 2H), 3.85-4.02 (m, 3H); C
combined organic extracts were dried, filtered, concentrated, and
NMR (75 MHz) δ 22.2 (t), 25.0 (q), 26.3 (q), 27.0 (t), 27.6 (t),
subjected to silica gel flash chromatography yielding compound 5
63.2 (t), 66.2 (t), 73.5 (d), 77.9 (d), 78.2 (d), 79.3 (d), 109.1 (s); IR
2
5
1
-1
in 70% yield (4.2 g). [R]
300 MHz) δ 0.15 (s, 9H), 1.48-1.65 (m, 5H), 1.80-1.92 (m, 3H),
.45 (m, 1H), 3.65 (m, 1H), 3.75 (m, 2H), 4.10 (dd, J ) 2.3, 11.1
D
) -26.6 (c 0.7, CHCl
3
); H NMR
(cm ) 1212, 1371, 2861, 2936, 2985, 3417; MS (FAB) m/z (relative
+
(
3
intensity) 81 (55), 132 (78), 189 (54), 231 (60), 247 (M + 1)
+
+
(100), 269 (M + Na) (64); HRMS (FAB) calcd for C12
H O
22 5
(M)
13
Hz, 1H); C NMR (75 MHz) δ -0.4 (q), 22.6 (t), 26.0 (t), 32.3
t), 63.1 (t), 68.7 (d), 73.3 (d), 79.8 (d), 89.0 (s), 104.2 (s); IR
246.1467, found 246.1464. Anal. Calcd for C12
9.00. Found: C 58.51, H 9.10.
22 5
H O C 58.52, H
(
J. Org. Chem, Vol. 71, No. 6, 2006 2343

Pinacho Cris o´ stomo et al.
Preparation of (2S,6R)-2-[(R)-2,2-Dimethyl-1,3-dioxolan-4-yl]-
-[(R)-oxiran-2-yl]-tetrahydro-2H-pyran (8). To a stirred solution
of 16 (1.5 g, 6.1 mmol) in dry pyridine (30.5 mL, 0.2 M) was
added TsCl (1.39 g, 7.3 mmol) at 0 °C. The reaction mixture was
stirred for 6 h, after which time TLC showed the end, and then
3H), 1.38 (s, 3H), 1.16-1.59 (m, 5H), 1.79 (m, 1H), 1.91 (m, 1H),
2.09-2.40 (m, 3H), 3.29 (m, 2H), 3.58 (m, 1H), 3.87 (m, 2H),
4.02 (m, 1H), 4.16 (q, J ) 7.1 Hz, 2H), 5.82 (d, J ) 15.6 Hz, 1H),
6.96 (dt, J ) 6.9, 15.4 Hz, 1H); 13C NMR (75 MHz) δ 14.0 (q),
22.2 (t), 24.8 (t), 25.1 (q), 26.4 (q), 27.9 (t), 28.3 (t), 30.2 (t), 59.9
(t), 66.7 (t), 72.5 (d), 78.0 (d), 78.6 (d), 80.0 (d), 109.0 (s), 121.4
6
diluted with H
2
O, and the aqueous layer was extracted with Et
2
O.
-
1
The ethereal extracts were washed with H
2
O, saturated CuSO
4
(d), 148.5 (d), 166.4 (s); IR (cm ) 1370, 1652, 1719, 2860, 2938,
solution, and saturated NaCl solution (60 mL), dried, and concen-
trated. The residue monotosylated alcohol was used in the next step
without purification.
3487; MS (FAB) m/z (relative intensity) 55 (83), 67 (65), 83 (58),
+
101 (50), 327 (65), 343 (M + 1) (100); HRMS (FAB) calcd for
+
C
C
18
H
H
31
O
O
6
(M + 1) 343.2121, found 343.2120. Anal. Calcd for
The crude was dissolved in dry THF (61 mL) under N
2
18
30
6
C 63.14, H 8.83. Found: C 63.14, H 9.67.
atmosphere at room temperature, and NaH (60%, 175 mg, 12.2
mmol) was added. After 4 h, TLC showed that starting material
was completely consumed. The reaction was quenched by addition
of a saturated solution of NaCl, and the mixture was extracted with
Preparation of (2E,6R)-6-{(2R,6S)-6-[(R)-2,2-Dimethyl-1,3-
dioxolan-4-yl]-tetrahydro-2H-pyran-2-yl}hex-2-ene-1,6-diol (7).
To a solution of 18 (900 mg, 2.6 mmol) in toluene (26 mL) under
N
2
atmosphere at 0 °C was added DIBAL-H (7.9 mL, 7.9 mmol).
When TLC indicated the complete conversion of the starting
material, the reaction was quenched by addition 0.25 mL of H O.
To the slurrish solution obtained was added MgSO , and it was
2
Et O. The organic phase was dried, and the solvent was evaporated
under reduced pressure. Purification by chromatographic column
2
2
5
gave 1.01 g (73% yield) of epoxide 8. [R]
D
) -6.0 (c 0.7, CHCl
H NMR (300 MHz) δ 1.35 (s, 3H), 1.41 (s, 3H), 1.24-1.91 (m,
H), 2.67 (dd, J ) 2.6, 5.2 Hz, 1H), 2.75 (dd, J ) 4.1, 5.2 Hz,
3
);
4
1
filtered through a pad of Celite. The solvent was evaporated, and
the residue was purified by chromatographic column to give 630
6
1
(
5
13
25
1
H), 2.94 (m, 1H), 3.25 (m, 2H), 3.88-4.05 (m, 3H); C NMR
mg of 7 (80% yield). [R]
D
3
) -6.4 (c 1.9, CHCl ); H NMR (300
75 MHz) δ 22.1 (t), 25.1 (q), 26.4 (q), 27.5 (t), 44.9 (t), 53.2 (t),
MHz) δ 1.34 (s, 3H), 1.39 (s, 3H), 1.22-1.59 (m, 6H), 1.76-2.24
(m, 6H), 3.29 (m, 2H), 3.60 (m, 1H), 3.86-4.08 (m, 5H), 5.67 (m,
2H); 13C NMR (75 MHz) δ 22.3 (t), 24.6 (t), 25.1 (q), 26.4 (q),
27.9 (t), 28.3 (t), 31.2 (t), 63.3 (t), 66.6 (t), 72.7 (d), 78.0 (d), 78.5
-
1
3.5 (d), 66.7 (t), 77.2 (d), 77.9 (d), 78.4 (d), 109.3 (s); IR (cm
)
1092, 1370, 1456, 2861, 2937, 2986; MS (FAB) m/z (relative
intensity) 55 (100), 69 (90), 83 (61), 95 (50), 213 (16), 229 (M +
+
+
-1
1)
(16); HRMS (FAB) calcd for C12
H
H
21
O
4
(M + 1) 229.1440,
C 63.14, H 8.83.
(d), 80.1 (d), 109.0 (s), 129.2 (d), 132.3 (d); IR (cm ) 1074, 1371,
found 229.1441. Anal. Calcd for C12
20
O
4
1669, 2858, 2936, 3415; MS (FAB) m/z (relative intensity) 55 (100),
+
Found: C 63.14, H 9.19.
73 (98), 147 (43), 221 (16), 281 (13), 300 (M) (1); HRMS (FAB)
+
Preparation of (1R)-1-{(2R,6S)-6-[(R)-2,2-Dimethyl-1,3-diox-
olan-4-yl]-tetrahydro-2H-pyran-2-yl}penten-4-en-1-ol (17). To
a mixture of CuI (75 mg, 0.4 mmol) in dry THF (40 mL) under
argon atmosphere was added allylmagnesium bromide (11.8 mL,
calcd for C16H
28
O
5
(M) 300.1937, found 300.1926. Anal. Calcd
for C16
H
28
O
5
C 63.97, H 9.40. Found: C 63.84, H 9.08.
Preparation of (S)-1-((2R,5R)-5-{(2R,6S)-6-[(R)-2,2-Dimethyl-
1,3,dioxolan-4-yl]-tetrahydro-2H-pyran-2-yl}-tetrahydrofuran-
2-yl)ethane-1,2-diol (19). Crushed, activated 4 Å molecular sieves
were added to stirred CH Cl (2.5 mL) under argon. The flask was
1
M in THF, 11.8 mmol) at -78 °C. The mixture was stirred for
10 min, and epoxide 6 (900 mg, 3.9 mmol) was added dropwise.
2
2
TLC showed almost instant conversion. The reaction was quenched
with a saturated aqueous solution of NH Cl. The layers were
partitioned, and the aqueous phase was extracted with Et O. The
cooled to -20 °C, and Ti(OPr-i) (0.16 mL, 0.5 mmol), (+)-DET
4
4
(0.11 mL, 0.6 mmol), and the allylic alcohol 7 (134 mg, 0.45 mmol)
2
in CH Cl (2.5 mL) were added sequentially with stirring. The
2
2
combined organic layers were dried, filtered, and concentrated to
yield a crude oil, which was purified by chromatographic column
mixture was stirred at the same temperature for 20 min, and TBHP
(0.15 mL, 6.1 M in isooctane, 0.80 mmol) was added slowly. After
the addition, the reaction was maintained with stirring for 2 h.
Tartaric acid aqueous solution (15% w/v) was added at room
temperature, and the stirring was continued until clear phases were
reached (ca. 1 h). The phases were separated, and the aqueous phase
was extracted with CH Cl . The combined organic phases were
25
to obtain 1.00 g (94% yield) of compound 17. [R]
D
) -8.3 (c
1
0
1
1
5
.6, CHCl ); H NMR (300 MHz) δ 1.35 (s, 3H), 1.40 (s, 3H),
3
.18-1.65 (m, 6H), 1.80 (m, 1H), 1.91-2.13 (m, 3H), 2.25 (m,
H), 3.31 (m, 2H), 3.62 (m, 1H), 3.91 (m, 2H), 4.02 (m, 1 H),
.01 (m, 2H), 5.83 (m, 1H); 13C NMR (75 MHz) δ 22.3 (t), 24.6
2
2
(t), 25.1 (q), 26.5 (q), 28.0 (t), 29.8 (t), 31.0 (t), 66.8 (t), 72.8 (d),
washed with brine, concentrated, diluted with Et O, and treated
2
-1
7
1
6
8.1(d), 78.6 (d), 80.1 (d), 109.0 (s), 114.6 (t), 138.2 (d); IR (cm )
370, 1640, 2858, 2937, 3076, 3472; MS (EI) m/z (relative intensity)
with precooled (0 °C) 15% (w/v) NaOH aqueous solution. The two-
phase mixture was stirred vigorously for 15 min at 0 °C. The organic
phase was separated, and the aqueous phase was extracted with
CH Cl . The combined organic phases were washed with brine,
+
6 (84), 81 (96), 133 (44), 169 (69), 255 (100), 270 (M + 1) (1);
+
HRMS (EI) calcd for C15
Anal. Calcd for C15
.86.
Preparation of Ethyl (2E,6R)-6-{(2R,6S)-6-[(4R)-2,2-Dimeth-
H
26
O
4
(M) 270.1831, found 270.1835.
2
2
H
26
O
4
C 66.64, H 9.69. Found: C 66.56, H
dried, filtered, concentrated, and purified by silica gel column
2
5
9
chromatography to yield 19 (106 mg, 75% yield). [R] ) -3.0
1
D
3
(c 0.7, CHCl ); H NMR (300 MHz) δ 1.21 (m, 2H), 1.34 (s, 3H),
yl-1,3-dioxolan-4-yl]-tetrahydro-2H-pyran-2-yl}-6-hydroy-2-
hexenoate (18). Compound 17 (900 mg, 3.33 mmol) was dissolved
1.39 (s, 3H), 1.46 (m, 1H), 1.68-1.99 (m, 7H), 2.32 (m, 1H), 2.46
(m, 1H), 3.25 (m, 2H), 3.67 (m, 3H), 3.89 (m, 4H), 4.00 (m, 1H);
1
3
in a 1:1 mixture of THF/H
this solution were added NMO (1.01 g, 8.32 mmol) and a catalytic
amount of OsO . The reaction mixture was stirred overnight,
quenched with a saturated solution of Na , and extracted with
2
O (35 mL) at room temperature. To
C NMR (75 MHz) δ 22.4 (t), 25.1 (q), 26.5 (q), 26.9 (q), 27.5
(t), 27.7 (t), 28.0 (t), 63.7 (t), 66.7 (t), 72.7 (d), 78.1 (d), 78.4 (d),
79.9 (d), 80.4 (d), 81.6 (d), 109.0 (s); IR (cm ) 1211, 1371, 1648,
-
1
4
S
2 2
O
3
2935, 2983, 3421; MS (FAB) m/z (relative intensity) 55 (89), 69
+
EtOAc. The organic phase was dried, and the solvent was
evaporated under reduced pressure. The crude aldehyde obtained
was used in the next step without purification.
(100), 81 (59), 109 (27), 154 (14), 339 (M + Na) (10); HRMS
+
(FAB) calcd for C16
Anal. Calcd for C16
8.67.
H
H
28
O
6
Na (M + Na) 339.1784, found 339.1798.
6
28
O
C 60.74, H 8.92. Found: C 60.82, H
To a solution of the crude aldehyde in CH
atmosphere at room temperature was added Ph
2.32 g, 6.66 mmol). After 4 h the reaction was completed. The
reaction was quenched with H O, and extraction was done with
CH Cl . The organic phase was dried, and the solvent was
evaporated under reduced pressure. Chromatographic purification
2
Cl
2
(35 mL) under
N
2
3
PdCHCO Et
2
Preparation of (2S,6R)-2-[(R)-2,2-Dimethyl-1,3-dioxolan-4-yl]-
6-{(2R,5R)-5-[(R)-oxiran-2-yl]-tetrahydrofuran-2-yl}-tetrahydro-
2H-pyran (6). To a solution of 19 (95 mg, 0.30 mmol) in dry
(
2
2
2
2 2
CH Cl (6 mL) under argon atmosphere were sequentially added
Et N (0.06 mL, 0.45 mmol) and benzoyl chloride (0.047 mL, 0.36
3
2
5
gave 1.02 g (90% yield) of the ester 18. [R]
D
) -2.8 (c 1.1,
mmol) at 0 °C. The reaction was stirred for 2 h, after which time
TLC showed that the starting material had disappeared. Then
1
CHCl
3
); H NMR (300 MHz) δ 1.26 (t, J ) 7.1 Hz, 3H), 1.33 (s,
2344 J. Org. Chem., Vol. 71, No. 6, 2006

Formal Synthesis of (+)-Muconin
saturated aqueous solution of NaCl was added, and the resulting
78.5 (d), 80.0 (d), 81.3 (d), 82.6 (d), 109.0 (s); IR (cm-1) 1371,
mixture was extracted with CH
2
Cl
2
. The combined organic phases
1460, 2856, 2926, 2983, 3495; MS (EI) m/z (relative intensity) 127
+
were dried, filtered, and evaporated under reduced pressure to yield
a residue, which was used in the next step without any further
purification. To a stirred mixture of the crude monobenzoate in
(22), 197 (49), 255 (100), 353 (13), 440 (4), 454 (M) (0.4); HRMS
+
(EI) calcd for C27
H
50
O
5
(M) 454.3658, found 454.3669.
Biological Tests. Cells, Culture, and Plating. The human solid
dry CH
2
Cl
2
(6 mL) under argon atmosphere were added Et
3
N (0.06
tumor cell lines A2780 (ovarian and SW1573 (nonsmall cell lung
cancer) were used in this study. Cells were maintained in 25 cm
culture flasks in RPMI 1640 supplemented with 5% heat-inactivated
2
fetal calf serum and 2 mM l-glutamine in a 37 °C, 5% CO , 95%
2
mL, 0.45 mmol) and methanesulfonyl chloride (0.05 mL, 0.72
mmol) at 0 °C. The reaction mixture was stirred for 1 h, after which
time TLC showed no remaining alcohol. Then, a saturated aqueous
solution of NaCl was added, and the resulting mixture was extracted
with CH Cl . The combined organic phases were dried, filtered,
2 2
and evaporated under reduced pressure.
humidified air incubator. Exponentially growing cells were trypsinized
and resuspended in antibiotic containing medium (100 units
penicillin G and 0.1 mg of streptomycin/mL). Single cell suspen-
sions displaying >97% viability by trypan blue dye exclusion were
subsequently counted. After counting, dilutions were made to give
the appropriate cell densities for inoculation onto 96-well microtiter
plates. Cells were inoculated in a volume of 100 mL per well at
densities of 7,000 (A2780) and 6,000 (SW1573) cells per well,
based on their doubling times.
Chemosensitivity Testing. Chemosensititvity tests were per-
formed using the SRB assay of the NCI.12 Pure compounds were
initially dissolved in DMSO at 400 times the desired final maximum
test concentration. Control cells were exposed to an equivalent
concentration of DMSO (negative control). Each agent was tested
in triplicates at different dilutions in the range 1-100 µM. The
drug treatment was started on day 1 after plating. Drug incubation
times were 48 h, after which time cells were precipitated with 25
µL ice-cold 50% (w/v) trichloroacetic acid and fixed for 60 min at
4 °C. Then the SRB assay was performed. The optical density (OD)
of each well was measured at 490 nm, using BioTek’s ELx800NB
absorbance microplate reader. Values were corrected for background
OD from wells only containing medium. The effect is defined as
percentage of growth (PG) and was calculated with respect to
untreated control cells (C) at each of the drug concentration levels
To a cooled suspension of NaH (36 mg, 60% in mineral oil, 0.9
mmol) in dry CH Cl (6 mL) under argon atmosphere was added
2 2
dropwise dry MeOH (0.4 mL, 0.9 mmol) at 0 °C. The mixture was
stirred for 15 min, and the residue obtained above was added slowly.
After the addition, the mixture was allowed to warm to room
temperature until the TLC showed no remaining benzoate. Then a
saturated aqueous solution of NaCl was added, the aqueous phase
was extracted with CH
dried, filtered, and concentrated. Purification was effected by flash
column chromatography to yield 6 (73 mg, 82% yield). [R]
2 2
Cl , and the combined organic phases were
25
D
)
1
-
3
9.0 (c 0.5, CHCl ); H NMR (300 MHz) δ 1.21 (m, 3H), 1.34 (s,
3
2
3
H), 1.39 (s, 3H), 1.64-1.86 (m, 6H), 2.02 (m, 2H), 2.66 (dd, J )
.7, 5.1 Hz, 1H), 2.74 (dd, J ) 4.2, 5.1 Hz, 1H), 2.95 (m, 1H),
13
.24 (m, 1H), 3.81-3.91 (m, 4H), 3.99 (m, 1H); C NMR (75
MHz) δ 22.4 (t), 25.2 (q), 26.5 (q), 27.5 (t), 28.0 (t), 28.1 (t), 28.4
(t), 43.8 (t), 53.9 (d), 66.8 (t), 78.1 (d), 78.4 (d), 78.7 (d), 79.9 (d),
-1
82.1 (d), 109.0 (s); IR (cm ) 1210, 1370, 1456, 2860, 2934, 2984;
MS (FAB) m/z (relative intensity) 55 (100), 69 (73), 81 (60), 109
+
(
29), 132 (16), 297 (M - 1) (6); HRMS (FAB) calcd for C16
H O
25 5
+
(M - 1) 297.1702, found 297.1709. Anal. Calcd for C16
H
26
O
5
C
6
4.41, H 8.78. Found: C 64.39, H 9.40.
Preparation of (1R)-1-((2R,5R)-5-{(2R,6S)-6-[(4R)-2,2-Dimethyl-
,3-dioxolan-4-yl]tetrahydro-2H-pyran-2-yl}tetrahydro-2-fura-
based on the difference in OD at the start (T
exposure (T), according to NCI formulas. Briefly, if T is greater
than or equal to T )/(C - T )].
the calculation is 100 × [(T - T
If T is less than T
denoting cell killing the calculation is 100 ×
[(T - T )/(T )]. With these calculations a PG value of 0 corresponds
to the amount of cells present at the start of drug exposure, while
negative PG values denote net cell kill. Thus, 50% growth inhibition
(GI50) represents the concentration at which PG is +50.
0
) and end of drug
1
nyl)-1-tridecanol (4). To a mixture of CuI (4 mg, 0.02 mmol) in
dry THF (2 mL) under argon atmosphere at -78 °C was added
undecanemagnesium bromide, previously formed from bromoun-
decane (195 mg, 0.83 mmol) and magnesium turnings (20 mg, 0.8
mmol) at room temperature. The mixture was stirred for 10 min,
and epoxide 6 (62 mg, 0.2 mmol) dissolved in dry THF (2 mL)
was added slowly. TLC showed almost instant conversion. The
0
0
0
0
0
0
Acknowledgment. The authors thank the MYCT (PPQ2002-
4361-C04-02) of Spain and the Canary Islands Government
reaction was quenched with a saturated aqueous solution of NH
The layers were partitioned, and the aqueous phase was extracted
with Et O. The combined organic layers were dried, filtered, and
concentrated to yield a crude oil, which was purified by chromato-
4
Cl.
0
for supporting this research. F.R.P.C. thanks CajaCanarias for
a FPI fellowship. R.C. thanks the Spanish MEC for a FPU
fellowship. J.M.P. thanks ICIC for a postdoctoral fellowship.
2
2
5
graphic column to obtain 66 mg (70% yield) of compound 4. [R]
D
) {lit. [R]25
3e
)
+16.5 (c 0.4, CHCl
3
D
3
) +17.8 (c 0.23, CHCl )} ;
Supporting Information Available: General experimental
conditions and 1H NMR and C NMR spectra for all new
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
1
H NMR (300 MHz) δ 0.87 (t, J ) 4.7 Hz, 3H), 1.34 (s, 3H), 1.39
s, 3H), 1.18-1.91 (m, 34 H), 3.28 (m, 2H), 3.79 (m, 2H), 3.93
13
(
(
13
m, 2H), 4.05 (m, 1H); C NMR δ (75 MHz) δ 13.9 (q), 22.3 (t),
2
2.4 (t), 25.1 (q), 25.4 (t), 26.5 (q), 27.9 (t), 28.0 (t), 28.3 (t), 29.1
(t), 29.4 (t), 29.5 (t), 31.7 (t), 33.3 (t), 66.8 (t), 73.8 (d), 78.1 (d),
JO0524674
J. Org. Chem, Vol. 71, No. 6, 2006 2345