Synthesis of 6-acetamido-5-amino- and -5-guanidino-3, 4-dehydro-N-(2-ethylbutyryl)- 3-piperidinecarboxylic acids related to zanamivir and oseltamivir, inhibitors of influenza virus neuraminidases.

Article abstract of DOI:10.1021/ol000261d

[reaction: see text] 6-Acetamido-5-amino- and -5-guanidino-3, 4-dehydro-N-(2-ethylbutyryl)-3-piperidinecarboxylic acids (8 and 9) have been synthesized starting from natural siastatin B, a bacterial neuraminidase inhibitor isolated from Streptomyces cultu

Full text of DOI:10.1021/ol000261d

ORGANIC
LETTERS
2000
Vol. 2, No. 24
3837-3840
Synthesis of 6-Acetamido-5-amino- and
-5-guanidino-3,4-dehydro-N-(2-ethylbutyryl)-
3-piperidinecarboxylic Acids Related to
Zanamivir and Oseltamivir, Inhibitors of
Influenza Virus Neuraminidases
,†
Eiki Shitara,† Yoshio Nishimura,* Kuniaki Nerome,‡ Yasuaki Hiramoto,‡ and
Tomio Takeuchi†
Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku,
Tokyo 141-0021, Japan, and Laboratory of Respiratory Viruses, National Institute of
Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
mcrf@bikaken.or.jp
Received September 8, 2000
ABSTRACT
6-Acetamido-5-amino- and -5-guanidino-3,4-dehydro-N-(2-ethylbutyryl)-3-piperidinecarboxylic acids (8 and 9) have been synthesized starting
from natural siastatin B, a bacterial neuraminidase inhibitor isolated from Streptomyces culture in a stereospecific fashion. These compounds
are related to zanamivir and oseltamivir, inhibitors of influenza virus neuraminidases.
Two integral membrane glycoproteins, haemagglutinin (HA)
and neuraminidase (NA), on the envelope of the surface of
the influenza virus interact with receptors which contain
terminal sialic acid residues of glycoproteins on the surface
of the host cell.1 Infection by the influenza virus begins with
the attachment of HA to cellular receptors and subsequent
fusion of viral and host cellular membranes.2 NA is a
glycosidase which cleaves the R-ketosidic bond linking a
terminal neuraminic acid to the adjacent oligosaccharide
residues of glycoproteins and glycolipids.3 This cleavage is
thought to facilitate transport of the virus through the mucus
within the respiratory tract and to be involved in the elution
of progeny virions from the infected cells and the prevention
of self-aggregation of progeny virions.4 Thus, NA plays a
crucial role in the life cycle of the virus and it has been
† Institute of Microbial Chemistry.
‡ National Institute of Infectious Diseases.
(3) (a) Gottschalk, A. Bochem. Biophys. Acta 1957, 23, 645. (b) Schauer,
R. Trends Biochem. Sci. 1985, 10, 357.
(1) (a) Weis, W.; Brown, J. H.; Cusack, S.; Paulson, J. C.; Skehel, J. J.;
Wiley: D. C. Nature 1988, 333, 426. (b) Colman, P. M.; Varghese, J. N.;
Laver, W. G. Nature 1983, 303, 41. (c) Varghese, J. N.; Laver, W. G.;
Colman, P. M. Nature 1983, 303, 35. (d) Varghese, J. N.; Colman, P. M.
J. Mol. Biol. 1991, 221, 473. (e) Burmeister, W. P.; Ruigrok, R. W. H.;
Cusak, S. EMBO J. 1992, 11, 49.
(2) (a) Paulson, J. C. In The Receptors; Conned, P. M., Ed.; Academic
Press: New York, 1985; Vol. 2, pp 131-219. (b) Wiley, D. C.; Skehel, J.
J. Annu. ReV. Biochem. 1987, 56, 365.
(4) (a) Palese, P.; Tobita, K.; Ueda, M.; Compans, R. W. Virology 1974,
61, 397. (b) Palese, P.; Compans, R. W. J. Gen. Virol. 1976, 33, 159. (c)
Griffin, J. A.; Basak, S.; Compans, R. W. Virology 1983, 125, 324. (d)
Colman, P.; Ward, C. W. Curr. Top. Microbiol. Immunol. 1985, 114, 177.
(e) Klenk, H.-D.; Rott, R. AdV. Virus Res. 1988, 34, 247.
(5) Palese, P.; Schulman, J. L. In Chemoprophylaxis of Virus Infection
of the Respiratory Tract; Oxford, J. S., Ed.; CRC: West Palm Beach, FL,
1977; Vol. 1, pp 189-205.
10.1021/ol000261d CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/28/2000

postulated that inhibitors of this enzyme should have antiviral
properties.5 Zanamivir (1) and oseltamivir (2) (Figure 1) are
In the course of our studies on the relationships between
the structure and biological activity of siastatin B (6)9 (Figure
2) isolated from a Streptomyces culture as an inhibitor of
Figure 2. Siastatin B and its analogues.
bacterial neuraminidase, we demonstrated, in 1993, that
3-episiastatin B (7) (Figure 2) shows potent inhibitory
activities against influenza virus neuraminidases and the
influenza virus in vitro infectivities10 while several analogues
having the same equatorial carboxyl group as 6 act as
Figure 1. Representative inhibitors of influenze virus neuramini-
dases and N-acetylneuraminic acid.
1
inhibitors only for bacterial neuraminidases.11 From the H
NMR spectrum, it was assumed that 7 exists in a boat
conformation in an aqueous solution and also in a nonsolution
by molecular modeling using PM3/MOPAC.10,12 The lowest
energy boat conformer of 7 was also superimposed onto the
R-boat conformer of 5 (Figure 2) in a pocket of the active
site residue of the crystal structure of influenza virus
B/Beijing/1/87 neuraminidase complexed with 5 by a dock-
ing experiment using BIOCES/AMBER.1e,10,12 Under these
circumstances, we are interested in the synthesis of similarly
functionalized analogues of 6 to 1 and 2 for development of
a new class of influenza virus neuraminidase inhibitors. Here,
we describe the synthesis of 6-acetamido-5-amino- and -5-
guanidino-3,4-dehydro-N-(2-ethylbutyryl)-3-piperidinecar-
boxylic acids (8 and 9) together with 3,4-dehydro-4-deoxy-
N-(2-ethylbutyryl)siastatin B (27) (Figure 2). First, we
potent inhibitors of neuraminidases from both influenza A
and B viruses and are currently used for treatment of
influenza viral infection.6 This postulation was conceived
through a rational drug design based on the crystal structure
of influenza virus neuraminidase complexed with sialic
acid.6,7 These results revealed the presence of a large
hydrophobic pocket as well as a hydrophilic pocket in the
region corresponding to the glycerol substituent of N-
acetylneuraminic acid (5). Several analogues based on 1 and
2 have been synthesized in the search for effective anti-
influenza agents, and it appears that 4-amino- and 4-guani-
dino-4H-pyran-6-carboxamides (3 and 4) (Figure 1) also
exhibit strong inhibition particularly against influenza virus
A neuraminidases and show both in vitro and in vivo antiviral
efficacy.8
(11) (a) Nishimura, Y.; Kudo, T.; Umezawa, Y.; Kondo, S.; Takeuchi,
T. Nat. Prod. Lett. 1992, 1, 33; 1992, 1, 39. (b) Kudo, T.; Nishimura, Y.;
Kondo, S.; Takeuchi, T. J. Antibiot. 1992, 45, 1662; 1993, 46, 300.
(12) Nishimura, Y. In Stuides in Natural Products Chemistry; Atta-ur-
Rahman, Ed.; Elsevier: Amsterdam, 1995; Vol. 16, pp 75-121.
(6) (a) von Itzstein, M.; Wu, W.-Y.; Kok, G. B.; Pegg, M. S.; Dyason,
J. C.; Jin, B.; Phan, T. V.; Smythe, M. L.; Whitem, H. F.; Oliver, S. W.;
Colman, P. M.; Varghese, J. N.; Ryan, D. M.; Woods, J. M.; Bethell, R.
C.; Hotham, V. J.; Cameron, J. M.; Penn, C. R. Nature 1993, 363, 418. (b)
Bishofberger, N. W.; Kim, C. U.; Lew, W.; Liu, H.; Williams, M. A. PCT
Int. Appl. WO9626933; Chem. Abstr. 1996, 125, 300503. (c) Kim, C. U.;
Lew, W.; Williams, M. A.; Zhang, L.; Liu, H.; Swaminathan, S.;
Bishofberger, N.; Chen, M. S.; Tai, C. Y.; Mendel, D. B.; Lavert, W. G.;
Stevens, R. C. J. Am. Chem. Soc. 1997, 119, 681.
(7) (a) von Itzstein, M.; Dyason, J. C.; Oliver, S. W.; White, H. F.; Wu,
W.-Y.; Kok, G. B.; Pegg, M. S. J. Med. Chem. 1996, 39, 388. (b) Kim, C.
U.; Lew, W.; Williams, M. A.; Zhang, L.; Liu, H.; Swaminathan, S.;
Bishofberger, N.; Chen, M. S.; Tai, C. Y.; Mendel, D. B.; Laver, W. G.;
Stevens, R. C. J. Am. Chem. Soc. 1997, 119, 681.
(8) (a) Smith, P. W.; Sollis, S. L.; Howes, P. D.; Cherry, P. C.; Starkey,
I. D.; Cobley, K. N.; Weston, H.; Scicinski, J.; Merritt, A.; Whittington,
A.; Wyatt, P.; Taylor, N.; Green, D.; Bethell, R. C.; Madar, S.; Fenton, R.
J.; Morley, P. J.; Pateman, T.; Beresford, A. J. Med. Chem. 1998, 41, 787.
(b) Taylor, N. R.; Cleasby, A.; Singh, O.; Skarzynski, T.; Wonacott, A. J.;
Smith, P. W.; Sollis, S. L.; Howes, P. D.; Cherry, P. C.; Betnell, R.; Colman,
P.; Varghese, J. J. Med. Chem. 1998, 41, 798.
(13) The synthesis of 12 will be reported elsewhere.
1
(14) 8: [R]23 +83.7° (c 0.37, H2O); H NMR (D2O) δ 6.91 and 6.90
D
(total 1H, d each, J4,5 ) 5.9 Hz, H-4), 6.7 and 6.3 (total 1H, s each, H-6),
4.627 and 4.625 (total 1H, d each, J2,2′ ) 18.6 and 20.5, H-2), 4.2 and 4.1
(total 1H, d each, H-5), 4.1 and 3.9 (total 1H, d each, H-2′). 9: [R]23
D
+60.1° (c 0.34, H2O); 1H NMR (D2O) δ 6.6 and 6.7 (total 1H, d each, J4,5
) 5.9 Hz, H-4), 6.5 and 6.1 (total 1H, s and d, J6,2 ) 1.5 Hz, H-6), 4.8
(1H, dd, J2,2′ ) 19.5 Hz, H-2), 4.4 and 4.3 (total 1H, d each, H-5), 3.8 (1H,
d, H-2′). 20: [R]23 +136.4° (c 0.27, H2O); H NMR (D2O) δ 6.6 (1H d,
1
D
J4,5 ) 5.9 Hz, H-4), 6.3 and 5.9 (total 1H, s and d, J6,2 ) 2.0 Hz, H-6), 4.6
and 4.5 (total 1H, d and dd, J2,2′ ) 18.6 Hz, H-2), 4.2 (1H, d, H-5), 3.9 and
3.6 (total 1H, d each, H-2′). 27: [R]23 +146.8° (c 0.29, H2O); H NMR
1
D
(CD3OD) δ 6.99 and 6.98 (total 1H, dd each, J4,2′ ) 1.5 and J4,5 ) 5.9 Hz,
H-4), 6.6 and 6.1 (total 1H, d each, J6,2 ) 1.0 and 1.5 Hz, H-6), 4.81 and
4.84 (total 1H, dd each, J2,2′ ) 20 Hz, H-2), 4.2 and 4.1 (total 1H, d each,
H-5), 3.7 (1H, dt, H-2′). 12: [R]23 +3.7° (c 0.45, MeOH). 13: [R]23
D
D
D
D
+57° (c 0.45, MeOH). 14: [R]23 -12.3° (c 0.40, MeOH). 15: [R]23
D
(9) Umezawa, H.; Aoyagi, T.; Komiyama, T.; Morishima, H.; Hamada,
M.; Takeuchi, T. J. Antibiot. 1974, 27, 963.
(10) Nishimura, Y.; Umezawa, Y.; Kondo, S.; Takeuchi, T.; Mori, K.;
Kijima-Suda, I.; Tomita, K. J. Antibiot. 1993, 46, 1883.
+149.4° (c 0.55, MeOH). 19: [R]23 +182° (c 0.71, MeOH). 21: [R]23
D
+94.2° (c 0.3, MeOH), mp 149-150 °C. 23: [R]23 +44.1° (c 0.49,
D
MeOH). 24: [R]23 +186.2° (c 0.44, MeOH). 25: [R]23 +83.1° (c 1.25,
CHCl3). 26: [R]23D +73.4° (c 0.47, MeOH).
D
D
3838
Org. Lett., Vol. 2, No. 24, 2000

attempted the synthesis of analogues (10 and 11) containing
an N-alkyl side chain related to 1 and 2 (Scheme 1 and
Scheme 3
Scheme 1
Scheme 2). Hydrogenolysis of the methyl ester 1213,14
prepared from 6 gave imine 13, which was transformed into
14 by N-alkylation with 2-ethylbutyraldehyde and NaBH3-
CN in excellent yield. Elimination of the acetate group of
14 proceeded smoothly upon treatment with t-BuOK in THF
to afford the R,â-unsaturated ester 15 in 76% yield. However,
removal of the protecting tert-butoxycarbonyl group with an
acid such as HCl in ether or trifluoroacetic acid in THF did
not result in the desired amine 10 but gave the unresolved
aromatized products. To confirm whether this kind of
N-alkylation generally results in aromatization or not in the
removal of protecting groups, we examined the preparation
of 11 starting from amine 13. Unfortunately, removal of the
protecting groups of ester 17 was unsuccessful and gave the
unresolved aromatized products. Therefore, we turned our
attention to the synthesis of the imides (8 and 9) analogous
to 4H-pyran-6-carboxamides (3 and 4), the alternative potent
inhibitors particularly of influenza virus A neuraminidases
(Figure 1 and Figure 2). This strategy was improved by
shortening the number of steps in the above synthesis
(Scheme 3). The desired R,â-unsaturated ester 19 was
prepared in 84% yield by reaction of 2-ethylbutyryl chloride
in pyridine and subsequent treatment with DBU in benzene
starting from siastatin B methyl ester (18).11b Basic hydrolysis
of 19 gave the R,â-unsaturated acid 20, which was protected
with an easily removable diphenylmethyl group to afford
21 in a good yield. After several unsuccessful attempts of
direct epimerization of the hydroxy group, attention was
directed to the general two-step epimerization by oxidation
and reduction. The Dess-Martin oxidation15 of 21 yielded
enone 22 in 85% yield. Reduction of 22 with sodium
borohydride was stereoselectively carried out to furnish allyl
alcohol 23 in excellent yield. Introduction of an equatorial
amino group was best achieved by Mitsunobu reaction16 with
HN3 in benzene followed by treatment with Ph3P in CH3CN
to give 25 in a good yield. Removal of a protecting group
with CF3CO2H in CH2Cl2 resulted in the desired 4-amino
product 8 in excellent yield. Compound 8 was further
functionalized with a guanidino group upon treatment with
N,N′-bis(tert-butyloxycarbonyl)thiourea in the presence of
17
HgCl2 to afford 26 in 58% yield. Compound 26 was
straightforwardly converted into the 4-guanidino product 9
with acid. Compound 20 was also transformed into the R,â-
unsaturated methyl ester 27 by treatment with TMSCl in
MeOH in 75% yield.
Scheme 2
A preliminary biological evaluation was carried out on
compounds 8, 9, 20, 27, and the general standard inhibitor,
2,3-dehydro-2-deoxy-N-acetylneuraminic acid 28. Unfortu-
nately, compounds 8, 9, 20, and 27 were found to be inactive
when inhibitory activities of neuraminidases from influenza
virus A/PR/8/34 (H1N1), A/Japan/307/56 (H2N2), A/Aichi/
2/68 (H3N2), and B/Yamagata/16/88 and also from Clostrid-
ium perfringens (0% at 2.4 × 102 µM, compound 8; 1.5 ×
102 µM, compound 9; 3.4 × 102 µM, compound 12; and
(15) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
(16) For a review, see: Mitsunobu, O. Synthesis 1981, 1.
(17) Kim, K. S.; Qian, L. Tetrahedron Lett. 1993, 34, 7677.
Org. Lett., Vol. 2, No. 24, 2000
3839

3.2 × 102 µM, compound 27) were evaluated, while
compound 28 was moderately active against these neuramini-
dases (IC50 110, 27, 24, 2.4, and 24 µM, respectively).18,19
The inactivities of 8, 9, 20, and 27 can be explained from
1
their H NMR spectra. The small coupling constant (J5,6
0 Hz) in comparison to the large coupling constant (J5,6
)
)
6.4 Hz) of 2911 clearly indicates that these compounds
preferentially adopt the half-chair conformation A (Figure
3) in solution as the conformational flip is generally seen in
the imide- or carbamate-fashioned functionalization of imino
group of 5.12,19,20 This is distinct from the normally preferred
Figure 3. Two half-chair conformers of 8, 9, and 27.
(18) Inhibitory activities of influenza neuraminidases were assayed by
the method described in memoranda Bull. Wld. Hlth. Org. 1971, 45, 119.
(19) Inhibitory activity of Clostridium perfringens was assayed by the
method described in Aoyagi, T.; Yagisawa, M.; Kumagai, M.; Hamada,
M.; Okami, Y.; Takeuchi, T.; Umezawa, H. J. Antibiot. 1971, 24, 860.
(20) (a) Nishimura, Y.; Satoh, T.; Adachi, H.; Kondo, S.; Takeuchi, T.;
Azetaka, M.; Fukuyasu, H.; Iizuka, Y. J. Am. Chem. 1996, 118, 3051; J.
Med. Chem. 1997, 40, 2626. (b) Satoh, T.; Nishimura, Y.; Kondo, S.;
Takeuchi, T.; Azetaka, M.; Fukuyasu, H.; Iizuka, Y.; Ohuchi, S.; Shibahara,
S. J. Antibiot. 1996, 49, 321. (c) Nishimura, Y.; Satoh, T.; Kudo, T.; Kondo,
S.; Takeuchi, T. Bioorg. Med. Chem. 1996, 4, 91. (d) Satoh, T.; Nishimura,
Y.; Kondo, S.; Takeuchi, T. Carbohyd. Res. 1996, 286, 173. (e) Shitara,
E.; Nishimura, Y.; Kojima, F.; Takeuchi, T. J. Antibiot. 1999, 52, 348;
Bioorg. Med. Chem. 1999, 1241.
half-chair conformation B (Figure 3) which must be adopted
upon binding of influenza virus neuraminidases as seen in
1, 2, 3, 4, and 28.6,7,8,21 Substitution around siastatin B (6) is
in progress to probe the requirements of structural binding
to neuraminidases.
Acknowledgment. The authors are grateful to Dr. Kun-
imoto Hotta, National Institute of Infectious Disease, for his
helpful discussion and encouragement. We also thank Ms.
Fukiko Kojima for the biological evaluation.
(21) (a) Varghese, J. N.; Epa, V. C.; Colman, P. Protein Sci. 1995, 4,
1081. (b) Taylor, N. R.; von Itzstein, M. J. Comput.-Aided Mol. Des. 1996,
10, 233.
OL000261D
3840
Org. Lett., Vol. 2, No. 24, 2000