Annonaceous acetogenin mimics bearing a terminal lactam and their cytotoxicity against cancer cells

Article abstract of DOI:10.1016/j.bmcl.2007.03.084

Annonaceous acetogenins are a large class of naturally occurring polyketides exhibiting potent anticancer activities. Based on our previous discovery of AA005, a multi-ether mimic of natural acetogenins having potent antitumor activities and significant selectivity between normal cells and cancer cells, a new series of mimics containing a terminal lactam were designed, synthesized and evaluated. Bioactivity study against cancer cells shows that the N-methylated lactam-containing compounds 3, 4, and 5 exhibit comparable potencies to that of AA005, as well as the similar selectivity to cancer cells. Hydrocarbon-length effects of N-alkyl were further explored through synthesizing derivatives 24-26, and application of this derivation protocol to the fluorescent labeling was also investigated.

Full text of DOI:10.1016/j.bmcl.2007.03.084

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3426–3430
Annonaceous acetogenin mimics bearing a terminal lactam
and their cytotoxicity against cancer cells
Hai-Xia Liu,a Guo-Rui Huang,b Huan-Ming Zhang,a Jia-Rui Wub,* and Zhu-Jun Yaoa,*
aState Key Laboratory of Bioorganic and Natural Product Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China
bKey Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China
Received 1 December 2006; revised 8 March 2007; accepted 27 March 2007
Available online 30 March 2007
Abstract—Annonaceous acetogenins are a large class of naturally occurring polyketides exhibiting potent anticancer activities.
Based on our previous discovery of AA005, a multi-ether mimic of natural acetogenins having potent antitumor activities and sig-
nificant selectivity between normal cells and cancer cells, a new series of mimics containing a terminal lactam were designed, syn-
thesized and evaluated. Bioactivity study against cancer cells shows that the N-methylated lactam-containing compounds 3, 4, and 5
exhibit comparable potencies to that of AA005, as well as the similar selectivity to cancer cells. Hydrocarbon-length effects of
N-alkyl were further explored through synthesizing derivatives 24–26, and application of this derivation protocol to the fluorescent
labeling was also investigated.
Ó 2007 Elsevier Ltd. All rights reserved.
Annonaceous acetogenins are a large family of fatty
acid-derived natural products with unique structures.
Many diverse members in this family display a broad
spectrum of biological activities, among which the most
impressive is the antitumor activity.1,2 It is believed that
acetogenins directly act at the terminal electron transfer
step in complex I of mitochondria. However, studies to-
ward determining the accurate binding sites of acetoge-
nins still remain unclear due to lack of detail structural
information about complex I.3,4 Previous studies in our
group showed that a mimic of natural acetogenins
(AA005, 1) containing most potential functionalities of
natural acetogenins exhibited potent antitumor activity
in 50–100 nM range against a variety of human cancer
cell lines. Furthermore, this compound presented gen-
eral selectivity between human normal and cancer cells.5
It would be of great significance to reveal the detailed
action mechanism which is presented by AA005. In or-
der to carry out such studies, a fluorescent derivative
of AA005 that can be visualized in the living cells by
microscopy is required in most priority. The second,
more chemical space in the acetogenin mimicry is still
urgent to be explored for understanding essential SAR
knowledge through creative chemistry.6 As part of our
continuing efforts on AA005-initiated studies, we report
herein the synthesis of a new series of actogenin mimics
bearing a terminal lactam functionality instead of the
original lactone unit, and the evaluation of their biolog-
ical activities, as well as its application to the fluorescent
labeling.
As mentioned as above, the acetogenin mimic AA005
(1) was found to inhibit complex I of the mitochondrial
electron transport chain.7 Considering the proposed
model by McLaughlin’s group8 for the interaction
between natural acetogenins with complex I, introduc-
tion of a labeling group into the hydrocarbon skeleton
between terminal lactone unit and middle ethylene gly-
col moiety of AA005 might alter the configuration of
the whole skeleton and weaken the binding of AA005
to the target. Based on our previous results,6 we suppose
that introduction of a fluorescent label at one terminal
of the molecule (lactone moiety) may affect less on bind-
ing of AA005 to the target. It is known the a,b-unsatu-
rated c-butenolide is an essential functionality for the
bioactivities of these acetogenins. If this lactone moiety
could be replaced by an unsaturated lactam, the newly
introduced nitrogen atom may offer us the opportunities
Keywords: Annonaceous acetogenin; Mimicry; Anticancer; Fluores-
cent labeling; Cytotoxicity.
*
Corresponding authors. Fax: +86 21 64166128; e-mail: yaoz@
mail.sioc.ac.cn
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.084

H.-X. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3426–3430
3427
d
to introduce a fluorescent label directly through a prop-
a - c
OH
OMs
OMOM
17
7
7
er spacer. Also, such new AA005-like molecules with a
terminal lactam are of value to be explored. Guided
by the above idea, a series of new acetogenin mimics
2–5 with a terminal lactam moiety were designed
(Fig. 1).
OH
13
OH
I
e
O
O
OMOM
11
7
7
OMOM
18
Retrosynthetically, the terminal a,b-unsaturated lactam
could be constructed by a sequential aldol condensation,
lactamization, and b-elimination (Fig. 2). Two direc-
tional successive O-alkylations of ethylene glycol could
introduce both required long-hydrocarbon chains. This
protocol allows us to rapidly assemble the lactam moiety
in considerable diversity from a common intermediate 6
and a variety of easily prepared aminoaldehydes 7–10
from corresponding amino acids.
Scheme 1. Reagents and conditions: (a) HC(OCH3)3, D-10-CSA,
CH2Cl2; (b) DIBAL-H (1.0 M in toluene), CH2Cl2, 0 °C, 90% (two
steps); (c) MsCl, Et3N, CH2Cl2, 100%; (d) ethylene glycol, NaH,
DMF, 130 °C, 80%; (e) I2, Ph3P, imidazole, CH2Cl2, 90%.
and followed by the mesylation10 of the primary hydro-
xyl group afforded mesylate 17 in 90% yield (2 steps).
Mono O-alkylation of ethylene glycol with freshly pre-
pared mesylate 17 gave alcohol 18, which was further
converted to the corresponding iodide 11 in 72% yield
(2 steps).
As shown in Scheme 1, the synthesis started from diol
13.5b Selective protection of the secondary alcohol9
Right-hand part of the common skeleton 6 was prepared
from the commercially available cis-eruic acid (Scheme
2). Ozonization of the double bond of cis-eruic acid gave
the desired aldehyde, which was reduced immediately by
KBH4. Bromination of the resulting alcohol with HBr
followed by esterification afforded the methyl ester 16
in a satisfactory overall yield. Subsequently, Wittig olef-
ination, hydrogenation, and removal of the acetonide
protecting group gave the expected diol 12.
2 R3
R
O
R1
N
O
O
O
O
O
O
OH
OH
OH
OH
2 R1 = H, R2 = Me, R 3= H
3 R1 = Me, R2 = Me, R3 = H
4 R1 = Me, R2 = Me, R3 = Me
5 R1 = Me, R2 = H, R3 = H
AA005 (1)
Cross-coupling of both fragments 11 and 12 was accom-
plished through an etherification in the presence of
Bu2SnO and CsF (Scheme 3). Protection of exposed sec-
ondary hydroxyl group with MOMCl gave the linear
skeleton 6 in good yield. Final construction of the termi-
nal lactam moiety was achieved by the following three-
step sequential procedure. Aldol reactions of the enolate
derived from ester 6 with various adehydes 7–10,11 re-
moval of N-Cbz-protection by hydrogenation and
in situ cyclization (to form the lactams), and b-elimina-
tion gave compounds 20–23 in reasonable yields, respec-
tively. Finally, global deprotection of the MOM ethers
by treatment with BF3ÆEt2O in Me2S12 afforded target
mimics 2–5.
Figure 1. Annonaceous mimics AA005 (1) and lactam-containing
derivatives 2–5.
R3
O
R2
R1
N
( )7
( )7
O
O
OH
OH
2 - 5
R1
CbzN
O
( )7
O
+
H
( )7
OMOM
O
O
The results of biological evaluation of compounds 2–5
are shown in Table 1. All five lactam-based mimics of
R3 R2
OMOM
OMe
6
7 R1 = H, R2 = Me, R3 = H
8 R1 = Me, R2 = Me, R3 = H
9 R1 = Me, R2 = Me, R3 = Me
10 R1 = Me, R2 = H, R3 = H
a - c
d
COOH
11
10
Br
COOMe
6
15
cis-Eruic acid
16
OMe
( )7
HO
+
( )7
O
I
O
OH
MOMO
12
11
O
COOMe
e
HO
COOMe
11
11
O
OH
12
O
Br
19
( )7
O
H
+
OH
OMe
HO OH
+
Scheme 2. Reagents and conditions: (a) i—O3, 0–5 °C, EtOH-cyclo-
hexane (1:5), ii—KBH4; (b) HBr, HOAc; (c) MeOH, SOCl2 61% in 3
steps; (d) i—PPh3, ii—t-BuOK, then (R)-glyceraldehyde acetonide 15,
iii—H2 (1 atm), EtOH, 10% Pd-C, 80% over three steps; (e) 50%
HOAc, THF, 94%.
( )7
O
OH
13
14
15
O
16
Figure 2. Retrosynthetic analysis of terminal lactam-containing mim-
ics of acetogenin.

3428
H.-X. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3426–3430
nal lactam were designed. Such a protocol extended to
the fluorescent labeling is illustrated in Figure 3.
a, b
O
OMe
11
+
( )7
( )7
OMOM
12
O
O
OMOM
O
6
R1
CbzN
R3 R2
7 - 10
Derivatives 24–26 were prepared using a similar proce-
dure as described above (Scheme 4). Reductive alkyl-
ation14 of L-alanine methyl ester 28 with aldehyde 29
followed by N-Cbz protection of the secondary amine
afforded ester 30, which was further reduced by Dibal-
H at ꢀ78 °C to give aldehyde 31.15 By a similar three-
step procedure, lactam 32 was prepared in a satisfactory
overall yield. After removal of two MOM ethers and the
N-Boc protecting group, further branch extension was
achieved by coupling(s) with the linear amino acid 33
under standard conditions (EDCI/HOBt), affording
compounds 24 (a by-product), 25, and 26, respectively.
c
H
R3
R2
N R1
( )7
( )7
O
O
OMOM
OMOM
O
20 - 23
R3
d
R2
N R1
( )7
( )7
O
O
OH
OH
O
2 - 5
Scheme 3. Reagents and conditions: (a) i—Bu2SnO, CHCl3/MeOH,
reflux, ii—CsF, then 11, DMF, 79%; (b) MOMCl, i-Pr2NEt, CH2Cl2,
90%; (c) i—LDA, then 7–10 (see Fig. 2), THF, ii—H2, cat. Pd(OH)2,
EtOH, 40 °C, ii—TFAA, Et3N (R1 = H), or MsCl, Et3N, then DBU
(R1 = Me), 77% for 7, 62% for 8, 55% for 9, and 58% for 10 over three
steps; (d) BF3ÆEt2O, Me2S, 83% for 2, 70% for 3, 72% for 4 and 67%
for 5.
spacer
O
N
7
O
O
Branch extension
OH
OH
Labeling group
Table 1. Cytotoxicity screenings of acetogenin mimics 2–5a,b
c
Compound
IC50 (lM)
Figure 3. Fluorescent labeling protocol through the N-alkylation.
Chang
B16
BEL-7404
SK-Hep1
AA005 (1)
NA
NA
NA
NA
NA
0.035
0.87
0.041
2.20
0.065
NA
O
O
Ala-OMe-HCl
2
3
4
5
b
c
a
28
0.013
0.478
0.168
0.234
0.845
0.168
0.589
0.583
0.104
H
OMe
+
6
CbzN
31
OHC
CbzN
NHBoc
NHBoc
29
30
NHBoc
a AA005 was used as a positive control.
b Exponentially growing cells were treated with compounds 1–5 for
48 h, respectively. Cell viability was determined by MTT assay.
c NA, not active.
N
( )7
( )9
O
O
NHBoc
OMOM
OMOM
O
32
NHBoc
d) TFA, CH2Cl2
AA005 retain the selectivity between the human cells
(Chang) and tumor cells (B16, BEL-7404, and SK-
Hep1), while they are 5–10-fold less potent than
AA005. Compound 2 (without an N-methyl in the lac-
tam) exhibits weaker activities as compared to com-
pound 3 (with an N-methyl in the lactam). Such kind
of difference was often found in some known N-methyl-
ated peptides with unique physicochemical properties.13
Compound 3 also shows some kind of selectivity among
different tumor cells, which is a defection of AA005, and
other three N-methylated compounds 3–5 only show
slight difference. In addition, elimination of the stereo-
genic center on the lactam by replacement of the (S)-
methyl with a hydrogen atom or replacement of the
hydrogen there with a methyl (compounds 4 and 5) does
not decrease the bioactivity dramatically.
HO2C
e) EDCI, HOBt, iPr2NEt, TFA trace
33
N
( )7
( )9
O
O
24
NHCOCF3
OH
OH
O
+
H
N
N
HN
( )9
( )7
O
O
O
Boc
OH
OH
O
25
f) TFA, CH2Cl2
g) 33, EDCI, HOBt, iPr2NEt
H
N
O
( )4
N
( )7
( )9
O
O
HN
O
OH
OH
O
NHBoc
26
( )4
According to the above biological results, N-methyla-
tion on the terminal lactam increases the activity, and
those mimics retain the cell selectivity. It mentions that
the chemical space to introduce other longer alkyls car-
rying on more functional groups, including a fluorescent
group, is possible. Therefore, compounds 24–26 with
different lengths of alkyls at the nitrogen atom of termi-
Scheme 4. Reagents and conditions: (a) NaBH(OAc)3, then aq.
NaHCO3, CbzCl, 42%; (b) DIBAL-H, CH2Cl2, ꢀ78 °C, 90%; (c)
i—LDA, THF, then 31, ii—H2, cat. Pd(OH)2, EtOH, 40 °C, ii—MsCl,
Et3N, then DBU, 62% over three steps; (d) TFA, CH2Cl2; (e) EDCI,
HOBt, 33, i-Pr2NEt, TFA trace CH2Cl2, 17% for 24 and 41% for 25; (f)
TFA, CH2Cl2; (g) 33, EDCI, HOBt, i-Pr2NEt, CH2Cl2, 60%.

H.-X. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3426–3430
3429
A fluorescent probe 27 was synthesized from intermedi-
ate 25 ( Scheme 5). Deprotection of the N-Boc group of
25 followed by coupling with a commonly used fluores-
cent labeling reagent 34 gave the designed fluorescein-la-
beled compound 27 in 59% yield.
work opens an entrance to those new AA005-like ace-
togenin mimics containing nitrogen functionalities. Fur-
ther optimization of the structures and selective labeling
of the acetogenin mimics are currently underway in our
laboratory.
Unfortunately, these compounds bearing the longer
branches decrease the antitumor activity dramatically
as compared to the N-methyl compound 3 ( Table 2),
and no activity was measured for the fluorescent label-
ing compound 27. According to these data, it mentions
that the longer the N-terminal alkyl chain is, the less ac-
tive it is.
Acknowledgments
This work was co-supported by the NSFC (30271531,
20425205, and 20621062), Ministry of Science and Tech-
nology of China (2005BA711A04), and Shanghai Muni-
cipal Commission of Science and Technology
(04DZ14901 and 06QH14016).
In summary, a new series of AA005-like acetogenin
mimics bearing a terminal lactam moiety have been syn-
thesized16 for the first time and their cytotoxicities were
measured and evaluated. Among these compounds,
N-methyl compound 3 shows comparable activities to
that of AA005 and retains similar cell selectivity. It
was also revealed that the stereogenic center on the lac-
tam is not an essential to the antitumoral activity. Utiliz-
ing the newly introduced nitrogen atom, new fluorescent
labeling through N-branching was also studied, and all
these efforts are negative. The compounds bearing long-
er chains through N-alkylation dramatically decrease
the activities, and no activity was observed for the de-
signed fluorescent probe 27. However, the presented
References and notes
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china, I.; Estornell, E.; Cortes, D. Nat. Prod. Rep. 2005,
22, 269.
2. For most recent examples on the total synthesis of
annonaceous acetogenins, see: (a) Keum, G.; Hwang, C.
H.; Kang, S. B.; Kim, Y.; Lee, E. J. Am. Chem. Soc.
2005, 127, 10396; (b) Tinsley, J. M.; Roush, W. R. J. Am.
Chem. Soc. 2005, 127, 10818; (c) Takahashi, S.; Ogawa,
N.; Koshino, H.; Nakata, T. Org. Lett. 2005, 7, 2783; (d)
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Roush, W. R. Org. Lett. 2005, 7, 4245; (e) Keaton, K.
A.; Phillips, A. J. J. Am. Chem. Soc. 2006, 128, 408; (f)
Curran, D. P.; Zhang, Q.; Richard, C.; Lu, H.; Gudipati,
V.; Wilcox, C. S. J. Am. Chem. Soc. 2006, 128, 9561; (g)
Crimmins, M. T.; Zhang, Y.; Diaz, F. A. Org. Lett. 2006,
8, 2369; (h) Hoye, T. R.; Eklov, B. M.; Jeon, J.;
Khoroosi, M. Org. Lett. 2006, 8, 3383, and references
cited therein.
25
O
OH
HO
1) TFA, CH2Cl2
2) iPr2NEt, 34, CH2Cl2
O
59%
O
34
SCN
H
N
O
( )4
N
( )7
( )9
O
O
HN
S
OH
OH
O
O
NH
O
27
HO
3. Okun, J. G.; Lummen, P.; Brandt, U. J. Biol. Chem. 1999,
274, 2625.
¨
O
OH
Scheme 5. Synthesis of fluorescent labeling compound 27.
4. Miyoshi, H. J. Bioenerg. Biomembr. 2001, 33, 223.
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X.-G. Angew. Chem., Int. Ed. Engl. 2000, 39, 1934; (b)
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H.; Li, H.-Y.; Li, Y.; Chen, X.-G.; Wu, Y.-L. Eur. J.
Chem. 2003, 9, 282.
Table 2. Biological evaluation of compounds 3 and 24–27a,b
6. Liu, H.-X.; Huang, G.-R.; Zhang, H.-M.; Jiang, S.; Wu,
J.-R.; Yao, Z.-J. Chem. Bio. Chem. 2007, 8, 172.
7. Huang, G.-R.; Jiang, S.; Wu, Y.-L.; Jin, Y.; Yao, Z.-J.;
Wu, J.-R. Chem. Bio. Chem. 2003, 4, 1216.
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65, 7618.
10. Williams, H. R.; Mosher, H. S. J. Am. Chem. Soc. 1954,
76, 2984.
c
Compound
IC50 (lM)
Chang
SK-hep1
B16
Bel-7404
3
24
25
26
27
NA
NA
NA
NA
NA
0.437
NA
NA
NA
NA
0.075
0.995
NA
0.295
1.35
3.20
2.50
NA
NA
NA
a Compound 3 was used as a positive control.
b Exponentially growing cells were treated with compounds 3, 24–27
for 48 h, respectively. Cell viability was determined by MTT assay.
c NA, not active.

3430
H.-X. Liu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3426–3430
11. The aminoaldehydes 7–10 were freshly prepared from the
corresponding aminoester which were reduced by DIBAL-
H at ꢀ78 °C (Ref. [15]).
0.88 (3H, t, J = 7.0 Hz), 1.22 (6H, s), 1.22–1.70 (40H, m),
2.24 (2H, t, J = 8.0 Hz), 2.85 (3H, s), 3.32 (2H, dd, J = 8.2
9.7 Hz), 3.53 (2H, dd, J = 2.9, 9.8 Hz), 3.66 (4H, m), 3.78
(2H, m), 6.54 (1H, t, J = 1.4 Hz) ppm; 13C NMR
(100 MHz, CDCl3): 170.3, 145.8, 137.9, 75.9, 70.5, 70.2,
61.5, 33.0, 31.9, 29.68, 29.60, 29.5, 29.4, 29.3, 29.32, 29.30,
27.4, 25.5, 25.4, 23.5, 23.2, 22.6, 14.0 ppm; IR (KBr): 3443,
2918, 2850, 1683, 1647, 1467, 1336, 1160, 1092 cmꢀ1; MS
(ESI, m/z): 604 (M++Na); HRMS (ESI) calcd for
C35H67NO5Na 604.4911, found 604.4911. Compound 5.
12. (a) Hu, T.-S.; Yu, Q.; Wu, Y.-L.; Wu, Y. K. J. Org. Chem.
2001, 66, 853; (b) Marshall, J. A.; Chen, M. Z. J. Org.
Chem. 1997, 62, 5996.
13. Aurelio, L.; Brownlee, R. T. C.; Hughes, A. B. Chem. Rev.
2004, 104, 5823.
14. Han, Y.-L.; Chorev, M. J. Org. Chem. 1999, 64, 1972.
15. Bastiaans, H. M. M.; van der Baan, J. L.; Ottenheijm, H.
C. J. J. Org. Chem. 1997, 62, 3880.
25
½aꢁD ¼ 5:77 (c 0.40, CHCl3); 1H NMR (500 MHz, CDCl3):
25
16. Data for final compounds: Compound 2. ½aꢁD ¼ þ34:4 (c
0.88 (3H, t, J = 6.9 Hz), 1.22–1.70 (40H, m), 2.27 (2H, t,
J = 8.0 Hz), 3.04 (3H, s), 3.32 (2H, t, J = 9.7 Hz), 3.53
(2H, dd, J = 2.7, 9.8 Hz), 3.66 (4H, m), 3.78 (2 H, m), 3.82
(2H, s), 6.59 (1H, d, J = 1.6 Hz) ppm; 13C NMR
(125 MHz, CDCl3): 171.8, 140.7, 133.5, 75.9, 70.5, 70.3,
52.6, 33.0, 31.9, 29.69, 29.61, 29.5, 29.4, 29.34, 29.33, 29.2,
25.9, 25.5, 22.6, 14.1 ppm; IR (KBr): 3440, 2918, 2850,
1669, 1633, 1472, 1464, 1330, 1160, 1143, 957 cmꢀ1; MS
(ESI, m/z): 576 (M++Na); HRMS (ESI) calcd for
C33H63NO5Na 576.4598, found 576.4596. Compound 27.
1H NMR (500 MHz, CDCl3/MeOD): 0.88 (3H, t,
J = 7.1 Hz), 1.22–1.75 (51H, m), 2.24 (4H, m), 3.07 (1H,
m), 3.27 (3H, m), 3.49 (2H, m), 3.62–3.80 (10H, m), 4.08
(1H, q, J = 6.5 Hz), 6.57 (2H, d, J = 8.7 Hz), 6.68 (1H, s),
6.70 (2H, s), 6.80 (2H, d, J = 8.7 Hz), 7.14 (1H, d,
J = 8.2 Hz), 7.90 (1H, d, J = 8.2 Hz), 8.07 (1H, s) ppm;
13C NMR (125 MHz, CDCl3/MeOD): 181.9, 176.9, 175.2,
173.2, 171.4, 155.0, 142.8, 141.4, 139.1, 130.6, 129.5, 126.7,
115.6, 112.5, 103.6, 76.4, 71.0, 70.9, 57.4, 50.5, 50.0, 36.9,
33.9, 32.6, 31.4, 30.4, 30.4, 30.3, 30.2, 30.0, 26.2, 23.3, 18.2,
14.6 ppm; IR (film): 3282, 3070, 2924, 2853, 1736, 1658,
1610, 1501, 1465, 1378, 1326, 1254, 1210, 1180, 1110, 993,
913, 851, 787, 730 cmꢀ1; MS (MALDI, m/z): 1135
(M++Na); HRMS (MALDI) calcd for C63H92N4O11SNa
1135.6375, found 1135.6360.
1
0.13, CHCl3); H NMR (300 MHz, CDCl3): 0.88 (3H, t,
J = 6.9 Hz), 1.20–1.60 (43H, m), 2.24 (2H, t, J = 8.0 Hz),
2.83 (2OH, br s), 3.32 (2H, dd, J = 8.2, 9.8 Hz), 3.53 (2H,
dd, J = 2.8, 9.8 Hz), 3.67 (4H, m), 3.79 (2H, m), 4.13 (1H,
q, J = 7.0 Hz), 6.51 (1H, s), 6.60 (1H, s) ppm; 13C NMR
(75 MHz, CDCl3): 174.0, 143.2, 139.0, 75.8, 70.5, 70.2,
52.7, 33.0, 31.8, 29.6, 29.59, 29.57, 29.55, 29.51, 29.3, 29.2,
27.5, 25.5, 25.2, 22.6, 19.0, 14.0 ppm; IR (KBr): 3443,
1689, 1637, 1471, 1465, 1159, 1143, 1093, 958 cmꢀ1; MS
(ESI, m/z): 576 (M++Na); HRMS (ESI) calcd for
C33H63NO5Na 576.4598, Found 576.4599. Compound 3.
25
½aꢁD ¼ þ8:4 (c 0.25, CHCl3); 1H NMR (500 MHz,
CDCl3): 0.88 (3H, t, J = 7.1 Hz), 1.22–1.55 (43 H, m),
2.25 (2H, t, J = 8.0 Hz), 2.95 (3H, s), 3.32 (2H, dd, J = 8.3,
9.7 Hz), 3.54 (2H, dd, J = 2.8, 9.8 Hz), 3.64 (4H, m), 3.78
(2H, m), 3.88 (1H, dq, J = 1.4, 6.9 Hz), 6.53 (1H, dd,
J = 1.6, 3.1 Hz) ppm; 13C NMR (125 MHz, CDCl3):
171.2, 140.1, 139.3, 75.9, 70.5, 70.3, 57.7, 33.0, 31.9,
29.7, 29.6, 29.59, 29.57, 29.4, 29.3, 27.5, 26.6, 25.6, 25.5,
22.6, 16.7, 14.1 ppm; IR (KBr): 3441, 2977, 2850, 1683,
1641, 1464, 1431, 1398, 1328, 1159, 1141, 957 cmꢀ1; MS
(ESI, m/z): 590 (M++Na); HRMS (ESI) calcd for
C34H65NO5Na 590.4754, found 590.4753. Compound 4.
25
1
½aꢁD ¼ 5:55 (c 3.5, CHCl3); H NMR (400 MHz, CDCl3):