Diastereo- and enantioselective synthesis of a conagenin skeletal amide moiety

Article abstract of DOI:10.1016/j.tetlet.2004.03.062

A synthetic study was carried out to obtain epimers of a protected 2,4-dihydroxy-3-methylpentanoic ester 9, which is a central building block of the immunomodulator (+)-conagenin 1. The configuration of the three contiguous stereogenic centers was determined by NMR measurements and comparison with the stereogenic centers of lactone 10.

Full text of DOI:10.1016/j.tetlet.2004.03.062

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 3579–3582
Diastereo- and enantioselective synthesis of a conagenin skeletal
amide moiety
*
ꢀ
J. Augusto R. Rodrigues, Paulo J. S. Moran, Cıntia D. F. Milagre and Cleber V. Ursini
Universidade Estadual de Campinas, Instituto de Quꢀımica, CP 6154, 13084-971 Campinas SP, Brazil
Received 12 January 2004; revised 27 January 2004; accepted 10 March 2004
Abstract—A synthetic study was carried out to obtain epimers of a protected 2,4-dihydroxy-3-methylpentanoic ester 9, which is a
central building block of the immunomodulator (þ)-conagenin 1. The configuration of the three contiguous stereogenic centers was
determined by NMR measurements and comparison with the stereogenic centers of lactone 10.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
OH OH
H
N
CO2H
OH
(þ)-Conagenin 1 was isolated from fermentation broths
of Streptomyces roseosporus by Ishizuka and co-workers
after screening for substances that exhibit specific action
on T cells by producing lymphokines as a low-molecular
mass immunomodulator.1 It was reported that 1
improved the antitumor efficacy of adriamycin and
mitomycin C against murine leukemias, which suggest
its potential utility for cancer chemotherapy.2 The bio-
logical activity and the unique highly functionalized
structure of (þ)-conagenin has stimulated several efforts
directed toward the synthesis of 1. A first study was
attempted by Sztaricskai and co-workers who obtained
conagenin diastereoisomers using as starting material a
Me
O
Me
Figure 1. (þ)-conagenin 1.
the synthesis of amide moiety of conagenin 1 and its
epimers (Fig. 1).
2. Results and discussion
Our strategy is based on the easy and inexpensive access
to poly-[(R)-3-hydroxybutyric] acid (PHB)7 from the
sugar cane industry, which is depolymerized to give
ethyl (R)-(ꢀ)-3-hydroxybutanoate 2 after esterification
of the corresponding acid in high yield and pure enan-
tiomeric form (Scheme 1).8 Protection of the hydroxyl
with TBDMSCl in the presence of imidazole/DMF gave
3 in high yield. Saponification with LiOH/H2O in
methanol and further acidification with HCl gave the
corresponding acid 4 in 60% yield after two steps from 2.
Many methods are known for the conversion of car-
boxylic acids to 2-oxo esters.9 However, we required a
strategy which would maintain the stereochemical
integrity of the molecule and we favored a method
involving ozonolysis of b-ketocyanophosphoranes,
originally described by Wasserman and Ho.10 Treatment
of the carboxylic acid 4 with (cyanomethylene)triphen-
ylphosphorane in the presence of 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide (EDCI) and 4-dimethyl-
aminopyridine (DMAP) followed by ozonolysis of 5 in
functionalized
D
-xylose.3 The first total synthesis of (þ)-
conagenin was achieved by Irie et al. starting with the
asymmetric aldol reaction of propiophenone with acet-
aldehyde followed by in situ syn-selective NaBH4
reduction.4 A formal synthesis was achieved by Enders
et al. in a 12-step sequence via asymmetric [2,3]-Wittig
rearrangement of crotyloxyacetaldehyde-SAEP-hydra-
zone and the diastereoselective reduction of a methyl-
ketone.5 A second total synthesis by Sano et al., was
accomplished based on enantioselective enzymatic
reaction followed by chemoselective reduction.6 The
combination of its biological activity and its unique
structure of encouraged us to develop a new approach to
Keywords: Conagenin; PHB-biopolymer; b-methylene-a-keto ester;
Hydrogenation.
* Corresponding author. Tel.: +55-1937883141; fax: +55-1937883023;
e-mail: jaugusto@iqm.unicamp.br
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.03.062

3580
J. A. R. Rodrigues et al. / Tetrahedron Letters 45 (2004) 3579–3582
methanol and dichloromethane11 gave the required
2-oxo ester (R)-(ꢀ)-6 in 66% yield.12
Me
H
OH
H
Me
H
O
O
It was then necessary to prepare the a-keto-b-methylene
ester 7 that was obtained via direct Mannich methyl-
enation, using a methodology recently optimized in our
laboratory,13 by heating (70 °C) a mixture containing the
keto ester 6 in anhydrous acetic acid with a catalytic
amount of morpholine and an excess of paraformalde-
hyde for 3 h. Compound 7 was obtained in 35% yield as
a pale yellow oil.14 Efforts to improve the yield of 7 were
unproductive. Hydrogenation of the a-keto-b-methylene
ester 7 over 5% Pd/C were performed in ethyl acetate
under atmospheric pressure to give syn and anti keto
ester 8a/8b in 70% yield in a ratio 93:7, respectively.
Structural assignments of the isomers were determined
Figure 2. Selected 1H–1H NOE enhancements (500 MHz, 1H NMR,
CDCl3) for 10a.
Purification by silica gel flash chromatography eluted
with hexane (95%):ethyl acetate isolated pure 9a24 in
53% yield and 9c:9b25 in a 9/1 ratio, as determined by
NMR. Considering that 9c has the correct syn stereo-
chemistry for the three contiguous chiral centers of the
amide moiety of conagenin 1, we decided to search for
other reagents to reduce 8a/8b to improve its amount in
the product ratio. Reduction with LiBH4 in ethyl ether
at ꢀ90 °C increased the quantity of 9c to 41% (Scheme
2). Deprotection of 9a with tetrabutylammonium fluo-
ride in THF gave the lactone 10a26 in 70% yield. The 1H
NMR of 10a shows coupling constants J3;4 ¼ 7:3 Hz and
J4;5 ¼ 4:6 Hz that are in agreement with previous cou-
pling constant analyses and conformational studies by
MM2 calculations of substituted c-lactones with vicinal
hydrogen atoms.27 The stereochemistry of 10a was also
revealed by NOE analysis (500 MHz, CDCl3, Fig. 2).
Under the same conditions, 9c was deprotected and
cyclized to lactone 10c in high yield. The 1H NMR
coupling constant of 9c for the vicinal J2;3 (2.4 Hz) and
J3;4 (5.8 Hz) are close to those for (þ)-conagenin 1.6
Lactone 10c28 shows J3;4 ¼ 10:3 Hz and J4;5 ¼ 7:0 Hz,
also in agreement with the calculated coupling con-
stants: J3;4 ¼ 9:8 Hz and J4;5 ¼ 7:1 Hz.27 The magnitude
of J3;4 (10.3 Hz) is characteristic for hydrogens 3-H and
4-H with trans geometry.16;27
1
by H and 13C NMR spectroscopy. The magnitudes of
the 3-H and 4-H vicinal coupling constants of syn
(5.1 Hz) and anti (7.5 Hz) isomers are consistent with
those observed for b-hydroxy (or b-silyloxy)-a-methyl
esters,15 thioesters,15a aldehydes,16 acids,17 amides,18 and
ketones.19 The 13C NMR resonances of 3-C, 4-C and
methyl-3-C of syn isomer 8a appeared at higher fields
than the respective carbons of anti 8b. This behavior is
in accordance with empirical observations that allow the
assignment of the stereostructure of b-hydroxycarbonyl
compounds possessing diastereoisomerism.20 Similar b-
silyloxycarbonyl compounds show the same pattern for
13C NMR spectra.21;15f The stereochemical outcome of
this hydrogenation can be rationalized on the basis of
the Felkin22 and the Cram23 models. The a-ketoesters
mixture 8a and 8b was reduced with tetrabutylammo-
nium borohydride in methanol at ꢀ78 °C to give a
diastereomeric mixture of a-hydroxyesters 9a–c in 67%
yield, having a ratio of 85:6:9, respectively (Scheme 2).
OH
O
OP
O
OP
O
b
d
a
PHB
CN
PPh3
In conclusion, we have developed a new short route for
the synthesis of the amide moiety of conagenin and its
epimers. The b-methylene-a-keto ester 7 proved its
versatility for obtaining the conagenin skeleton through
hydrogenation reduction of the enone moiety with
asymmetric induction. The 1H NMR coupling constants
and formation of the lactones allow characterization of
all the isolated diastereoisomers.
OEt
OR
3 R = Et
(R)-2
5
c
4 R = H
OP
OP
O
e
P = TBDMSCl
OMe
OMe
OP
O
OP
O
O
g
f
OMe
8a
OMe
O
O
6
O
7
O
8b
Scheme 1. Reagents and conditions: (a) 1,2-dichloroetane, EtOH, cat.
H2SO4, two days (60% yield); (b) TBDMSCl, imidazole, DMF; (c)
LiOH, MeOH, H2O/HCl (60% yield, two steps); (e) Ph3PCHCN,
EDCI, DMAP, CH2Cl2 ; (e) O3, MeOH, CH2Cl2, ꢀ78 °C (66% yield,
two steps); (f) (CH2O)n, morpholine, HOAc, 70 °C (35% yield); (g) H2,
Pd–C 5%, EtOAc (70% yield, 8a:8b 93:7).
Acknowledgements
We thank FAPESP for financial support and for a
scholarship for C.V.U., and Dr. Gilson H. M. Dias for
the use of his lab facilities.
OP
O
OP OH
OP OH
OP OH
OMe
OMe
OMe
OMe
O
O
O
O
8a/b
9a
9b
9c
Scheme 2. Reagents and conditions: (1) Bu4NBH4, MeOH, ꢀ78 °C (67% yield, 9a:b:c 85:6:9); (2) LiBH4, Et2O, ꢀ90 °C (53% yield, 9a:b:c 54:5:41).

J. A. R. Rodrigues et al. / Tetrahedron Letters 45 (2004) 3579–3582
3581
18. Evans, D. A.; Ratz, A. M.; Huff, B. E.; Sheppard, G. S. J.
Am. Chem. Soc. 1995, 117, 3448–3467.
References and notes
19. Enders, D.; Bartsch, M.; Runsink, J. Synthesis 1999, 243–
248.
20. Heathcock, C. H.; Pirrung, M. C.; Sohn, J. E. J. Org.
Chem. 1979, 44, 4294–4299.
21. Hoffmann, R. W.; Weidmann, U. Chem. Ber. 1985, 118,
3966–3979.
22. Jackson, R. F. W.; Standen, S. P.; Clegg, W.; McCamley,
A. Tetrahedron Lett. 1992, 33, 6197–6200.
23. Augustine, R. L. Catal. Today 1997, 37, 419–440.
24. Methyl (2S,3S,4R)-4-(t-butyldimethylsilyloxy)-2-hydroxy-
1. (a) Yamashita, T.; Iijima, M.; Nakamura, H.; Isshiki, K.;
Naganawa, H.; Hattori, S.; Hamada, M.; Ishizuka, M.;
Takeuchi, T. J. Antibiot. 1991, 44, 557–559; (b) Kawatsu,
M.; Yamashita, T.; Osono, M.; Ishizuka, M.; Takeuchi, T.
J. Antibiot. 1993, 46, 1687–1691; (c) Ishizzuka, M.;
Kawatsu, M.; Yamashita, T.; Ueno, M.; Takeuchi, T.
Int. J. Immunopharmacol. 1995, 17, 133–139.
2. (a) Kawatsu, M.; Yamashita, T.; Ishizuka, M.; Takeuchi,
T. J. Antibiot. 1995, 48, 222–225; (b) Hamada, M.;
Yamamoto, S.; Moriguchi, S.; Kishino, Y. J. Antibiot.
2001, 54, 349–353; (c) Hamada, M.; Sonotake, E.;
Yamamoto, S. Morigushi, S. J. Antibiot. 1999, 52,
3-methylpentanoate 9a: IR (thin film) 3476 (br) and
1738 cmꢀ1
23
D
;
½aꢁ ꢀ 4:0 (c 1.03, CHCl3); 1H NMR
(500 MHz, CDCl3) dH 4.16 (dd, 1H, J2;3 ¼ 7:0 Hz,
J2;2-OHÞ ¼ 6:4 Hz, 2-H), 4.09 (dq, 1H, J4;5 ¼ 6:4 Hz,
J3;4 ¼ 3:0 Hz, 4-H), 3.88 (d, 1H, J2;2-OH ¼ 6:1 Hz, OH),
3.77 (s, 3H, CO2CH3), 1.92 (dquint, 1H, J3;3-methyl ¼ 7:0 Hz,
548–551.
ꢀ
3. Kovacs-Kulyassa, A.; Herczegh, P.; Sztaricskai, F. J.
ꢀ
Tetrahedron Lett. 1996, 37, 2499–2502.
4. Hatakeyama, S.; Fukuyama, H.; Mukugi, Y.; Irie, H.
Tetrahedron Lett. 1996, 37, 4047–4050.
5. Enders, D.; Bartsch, M.; Runsink, J. Synthesis 1999, 243–
248.
6. Sano, S.; Miwa, T.; Hayashi, K.; Nozaki, K.; Ozaki, Y.;
Nagao, Y. Tetrahedron Lett. 2001, 42, 4029–4031.
7. Bachmann, B. M.; Seebach, D. Helv. Chim. Acta 1998, 81,
2430–2461.
8. Seebach, D.; Beck, A. K.; Breitschuh, R.; Job K. Org.
Synth. 1993, 71, 39–47.
J2;3 ¼ 7:0 Hz, J3;4 ¼ 3:0 Hz, 3-H), 1.18 (d, 3H, J4;5
¼
6:4 Hz, 5-H3), 0.93 (d, 3H, J3;3-methyl ¼ 7:0 Hz 3-C–CH3),
0.90 [s, 9H, SiC(CH3)3], 0.11 (s, 3H, SiCH3), 0.09 (s, 3H,
SiCH3); 13C (1H) NMR, (125.7 MHz, CDCl3) dC 174.59
(1-C), 74.73 (2-C or 4-C), 70.42 (4-C or 2-C), 51.92 (O–
CH3), 42.74 (3-C), 25.78 [SiC(CH3)3], 19.99 (SiC), 17.93
(SiC), 11.22 (3-CCH3), ꢀ4.24 (SiCH3), ꢀ5.03 (SiCH3).
Anal. Calcd for C13H28O4Si (276.44): C, 56.48; H, 10.21.
Found: C, 56.44; H, 10.47.
25. 9c/9b: 9:1 ratio, IR (thin film): m (OH) 3500 (br s), m (C@O)
9. For reviews of the synthesis of 2-oxo esters see: (a)
Kovacs, L. Recl. Trav. Chim. Pays-Bas 1993, 112,
471–496; (b) Copper, A. J. L.; Ginos, J. Z.; Meister, A.
Chem. Rev. 1983, 83, 321–358.
23
1738 cmꢀ1 (s) ½aꢁ ꢀ 70 (c 0.22, CHCl3)Methyl (2R,3S,
D
4R)-4-(tert-butyldimethylsilyloxy)-2-hydroxy-3-methyl-
1
pentanoate 9c: H NMR (500 MHz, CDCl3) dH 4.45 (dd,
1H, J2;3 ¼ 2:4 Hz, J2;2-OH ¼ 4:3 Hz, 2-H) 3.93 (apparent
quint, 1H, J4;5 ¼ 6:1 Hz, J3;4 ¼ 5:8 Hz, 4-H), 3.79 (s, 3H,
CO2CH3), 3.05 (d, 1H, J2;2-OH ¼ 4:3 Hz, OH), 1.95 (ddq,
1H, J3;3-methyl ¼ 7:0 Hz, J2;3 ¼ 2:4 Hz, J3;4 ¼ 5:8 Hz, 3-H),
1.22 (d, 3H, J4;5 ¼ 6:1 Hz, 5-H3), 0.90 [s, 9H, SiC(CH3)3],
0.88 (d, 3H, J3;3-methyl ¼ 7:0 Hz, 3-C–CH3), 0.087 (s, 3H,
SiCH3), 0.081 (s, 3H, SiCH3); 13C{1H} NMR.
(125.7 MHz, CDCl3) dC 175.27 (1-C), 72.29 (2-C or 4-C),
71.26 (4-C or 2-C), 52.45 (O-CH3), 43.44 (3-C), 25.81
[SiC(CH3)3], 21.06 (5-C), 17.98 (SiC), 8.94 (3-CCH3),
ꢀ4.11 (Si–CH3), ꢀ4.90 (Si–CH3).
10. Wasserman, H. H.; Ho, W.-B. J. Org. Chem. 1994, 59,
4364–4366.
11. This methodology was also employed by Bhalay, G.;
Clough, S.; McLaren, L.; Sutherland, A.; Willis, C. L.
J. Chem. Soc., Perkin Trans. 1 2000, 901–910.
20
12. (R)-6: ½aꢁ ꢀ 22:4 (c 1.21 CHCl3); IR (thin film) m (C@O)
D
1755, 1733 cmꢀ1; 1H NMR (500 MHz, CDCl3) dH 4.38 (m,
1H, 4-H), 3.85 (s, 3H, CO2CH3), 3.04 (dd, 1H, J3;3 ¼ 15:6,
J3;4 ¼ 7:3, 3-HH), 2.87 (dd, 1H, J3;3 ¼ 15:6, J3;4 ¼ 5:2, 3-
HH), 1.21 (d, 3H, J4;5 ¼ 6:1, 5-H3), 0.84 [s, 9H,
SiC(CH3)3], 0.053 (s, 3H, SiCH3), 0.017 (s, 3H, SiCH3).
13. Rodrigues, J. A. R.; Siqueira-Filho, E. P.; de Mancilha,
Methyl
(2R,3R,4R)-4-(tert-butyldimethylsilyloxy)-2-hy-
droxy-3-methylpentanoate 9b: 1H NMR dH (500 MHz,
CDCl3): 4.21 (dd, 1H, J2;3 ¼ 4:3 Hz, J2;2-OH ¼ 5:5 Hz, 2-H)
3.89 (apparent quint, 1H, J ¼ 6:4 Hz, 4-H), 3.78 (s, 3H,
CO2CH3), 3.37 (d, 1H, J2;2-OH ¼ 5:9 Hz, OH), 2.00 (m, 1H,
J ¼ 7:0 Hz, J ¼ 4:6 Hz, 3-H), 1.16 (d, 3H, J4;5 ¼ 6:1 Hz, 5-
H3), 0.97 (d, 3H, J3;3-methyl ¼ 7:0 Hz, 3-C–CH3), 0.89 [s, 9H,
SiC(CH3)3], 0.077 (s, 3H, SiCH3), 0.071 (s, 3H, SiCH3);
13C{1H} NMR. dC (125.7 MHz, CDCl3): 175.04 (1-C),
73.57 (2-C or 4-C), 70.84 (4-C or 2-C), 52.08 (O-CH3),
44.31 (3-C), 25.86 [SiC(CH3)3], 21.55 (5-C), 17.98 (SiC),
13.52 (3-CCH3), ꢀ4.34 (Si–CH3), ꢀ4.73 (Si–CH3).
M.; Moran, P. J. S. Synth. Commun. 2003, 33, 331–340.
20
D
14. (R)-7: ½aꢁ ꢀ 27:6 (c 1.66, CHCl3); IR (thin film) m (C@O)
1
1746, 1682 cmꢀ1; H NMR (300 MHz, CDCl3) dH 6.54 (d,
1H, J ¼ 1:1 Hz, ¼ CHH), 6.24 (s, 1H, ¼ CHH), 4.76 (q,
1H, J4;5 ¼ 6:2 Hz, 4-H), 3.91 (s, 3H, CO2CH3), 1.28 (d, 3H,
J4;5 ¼ 6:2 Hz, 5-H3), 0.91 [s, 9H, SiC(CH3)3], 0.084 (s, 3H,
SiCH3), 0.039 (s, 3H, SiCH3). Anal. Calcd for C13H24O4Si:
C, 57.32; H, 8.88. Found: C, 57.07; H, 9.0.
15. (a) Evans, D. A.; Kozlowski, M. C.; Murry, J. A.; Burgey,
C. S.; Campos, K. R.; Connell, B. T.; Staples, R. J. J. Am.
Chem. Soc. 1989, 121, 669–685; (b) Slough, G. A.;
Bergman, R. G.; Heathcock, C. H. J. Am. Chem. Soc.
1999, 111, 938–949; (c) Hoffmann, R. W.; Dresely, S.;
26. (3S,4S,5R)-3-hydroxy-4,5-dimethyl-tetrahydrofuran-2-
23
one 10a: ½aꢁ þ17 (c 0.080, CHCl3); IR (nujol): m (OH)
D
3440 cmꢀ1 (br, s), m (C@O) 1766 cmꢀ1 (s); 1H NMR dH
(500 MHz, CDCl3): 4.58 (dq, 1H, J4;5 ¼ 4:6 Hz,
J5;5-methyl ¼ 6:4 Hz, 5-H), 4.57 (d, 1H, J3;4 ¼ 7:3 Hz, 3-H),
2.70 (apparent dquint and br s, 2H, J ¼ 7:3 Hz,
ꢀ
Lanz, J. W. Chem. Ber. 1988, 121, 1501–1507; (d) Frater,
€
€
G.; Muller, U.; Gunther, W. Tetrahedron 1984, 40, 1269–
1277; (e) Scheidt, K. A.; Bannister, T. D.; Tasaka, A.;
Wendt, M. D.; Savall, B. M.; Fegley, G. J.; Roush, W. R.
J. Am. Chem. Soc. 2002, 124, 6981–6990; (f) Mateus, C.
R.; Feltrin, M. P.; Costa, A. M.; Coelho, F.; Almeida, W.
P. Tetrahedron 2001, 57, 6901–6908; (g) Keck, G. E.;
Kachensky, D. F.; Enholm, E. J. J. Org. Chem. 1985, 50,
4317–4325.
J4;5 ¼ 4:6 Hz, 4-H and –OH), 1.38 (d, 3H, J5;5-methyl
¼
6:4 Hz, 5-CH3), 0.95 (d, 3H, J4;4-methyl ¼ 7:3 Hz, 4-CH3);
13C{1H} NMR. dC (125.7 MHz, CDCl3): 177.32 (2-C),
76.20 (3-C or 5-C), 71.80 (5-C or 3-C), 39.24 (4-C), 15.45
(5-CCH3),6.37 (4-CCH3).
ꢀ
27. Jaime, C.; Segura, C.; Dinares, I.; Font, J. J. Org. Chem.
16. Barrett, A. G. M.; Lebold, S. A. J. Org. Chem. 1991, 56,
4875–4884.
17. Renauld, P.; Seebach, D. Helv. Chim. Acta 1986, 69, 1704–
1710.
1993, 58, 154–158.
28. (3R,4S,5R)-3-hydroxy-4,5-dimethyl-tetrahydrofuran-2-
23
D
one 10c: mp 69–72 °C; ½aꢁ þ 60 (c 0.027, CHCl3); IR

3582
J. A. R. Rodrigues et al. / Tetrahedron Letters 45 (2004) 3579–3582
(nujol): m (OH) 3400 cmꢀ1 (br, s), m (C@O) 1760 cmꢀ1 (s);
1H NMR dH (300 MHz, CDCl3): 4.72 (quint, 1H,
J4;5 ¼ 7:0 Hz, J5;5-methyl ¼ 7:0 Hz, 5-H), 4.11 (d, 1H, J3;4
10:3 Hz, 3-H), 2.58 (m, 2H, 4-H and –OH), 1.31 (d, 3H,
J5;5-methyl ¼ 6:6 Hz, 5-CH3), 1.21 (d, 3H, J4;4-methyl ¼ 6:9 Hz,
4-CH3).
¼