A new stereo- and enantioselective approach to the N-terminal amino acid moiety of nikkomycins B and BX

Full text of DOI:10.1021/jo9514533

1122
J . Org. Chem. 1996, 61, 1122-1124
Notes
A New Ster eo- a n d En a n tioselective
Ap p r oa ch to th e N-Ter m in a l Am in o Acid
Moiety of Nik k om ycin s B a n d BX
Ge´rard Mandville, Mohammed Ahmar, and
Robert Bloch*
Laboratoire des Carbocycles, URA CNRS 478,
Institut de Chimie Mole´culaire d’Orsay, Baˆt. 420,
Universite´ de Paris-Sud, 91405 Orsay, France
Received August 7, 1995
Nikkomycins are peptidyl nucleoside antibiotics, struc-
turally related to polyoxins, which have been isolated
from fermentation broths of both streptomyces tendae and
streptomyces cacaoi ssp. asoensis. The purification of
these substances, as well as the determination of their
chemical structures and pharmacological activities, have
been described by Ko¨nig and Isono.1,2 These compounds
are potent competitive inhibitors of chitin synthetase and
exhibit antifungal, acaricidal, and insecticidal activities.
Due to their interesting pharmaceutical properties, much
effort has been devoted to the total synthesis of nikko-
mycins B 13 and BX 2,4 mainly by coupling the C-terminal
nucleoside amino acid with the N-terminal amino acid
component 3. The presence of three consecutive stereo-
genic centers in 3 is a synthetic challenge, and several
approaches to this amino acid 3 or its lactone derivative
4, in either racemic5 or optically active form,6 have been
recently reported.
In the course of our work directed toward the develop-
ment of stereoselective reactions induced by a thermo-
labile group, we have described the conditions under
which very high steric control is achieved in the addition
of alkyl organometallic reagents to Diels-Alder adducts
of furan.7 This method is useful for the synthesis of
enantiomerically pure 4-alkyl-but-2-enolides. However,
its scope of application has been limited by the modest
yields in the oxidation step.8 We report here an improved
procedure for the preparation of 4-arylbut-2-enolides and
the incorporation of this methodology into a short and
highly stereoselective synthesis of (2S,3S,4S)-2-amino-
4-(p-methoxyphenyl)-3-methyl-4-butanolide (4), a poten-
tial precursor of the N-terminal amino acid unit of
nikkomycins B and BX. The synthetic pathway is out-
lined in Scheme 1.
Chelation-controlled addition of (p-methoxyphenyl)-
magnesium bromide to the enantiomerically pure lactol
59 led to the corresponding diols as a 20:1 mixture of two
diastereomers. The product ratio was estimated by
integration of the 1H NMR signals for the bridgehead
protons which appear as singlets at δ ) 4.99 and 5.08
ppm for 6 and at δ ) 4.82 and 4.94 ppm for its stereomer.
The major component 6 was obtained pure as a colorless
solid in 73% yield by simple trituration of the crude
product mixture with ether. Oxidation of diol 6 [4-meth-
ylmorpholine N-oxide (NMO), tetrapropylammonium per-
ruthenate (TPAP)]10 gave the tricyclic lactone 7 which,
by cycloreversion in refluxing toluene, afforded the enan-
tiomerically pure butenolide 8 [ee > 95% determined by
1H NMR spectroscopy in the presence of Eu(hfc)3]. The
stereoselective introduction of the methyl group via
Michael addition proved to be quite critical, since both
the selectivity and the chemical yield were greatly
dependent on the reaction conditions (temperature, na-
ture of the cuprate and of the solvent). We found that
excellent yield (85%) and stereoselectivity (a single
product was detected) could be attained if the reaction
was carried out at -78 °C in ether using 3 equiv of
lithium dimethyl cuprate in the presence of chlorotri-
methylsilane. Electrophilic amination of the lithium
enolate of the disubstituted lactone 9 was then tried with
several reagents. In our hands, di-tert-butylazodicar-
boxylate11 or 1-chloro-1-nitrosocyclohexane12 did not give
(1) (a) Da¨hn U.; Hagenmaier, H.; Ho¨hne, M.; Ko¨nig, W. A.; Wolf,
G.; Za¨hner, H. Arch. Microbiol. 1976, 107, 249. (b) Hagenmaier, H.;
Keckeisen, A.; Za¨hner, H.; Ko¨nig, W. A. Liebigs Ann. Chem. 1979, 1494.
(c) Ko¨nig, W. A.; Hass, W.; Dehler, W.; Fiedler, H. P.; Za¨hner, H. Liebigs
Ann. Chem. 1980, 622. (d) Hagenmaier, H.; Keckeisen, A.; Dehler, W.;
Fiedler, H. P.; Za¨hner, H.; Ko¨nig, W. A. Liebigs Ann. Chem. 1981, 1018.
(2) (a) Kobinata, K.; Uramoto, M.; Nishii, M.; Kusakabe, H.; Naka-
mura, G.; Isono, K. Agric. Biol. Chem. 1980, 44, 1709. (b) Uramoto,
M.; Kobinata, K.; Isono, K.; Higashijima, T.; Miyazawa, T.; J enkins,
E. E.; McCloskey, J . A. Tetrahedron Lett. 1980, 21, 3395. (c) Uramoto,
M.; Kobinata, K.; Isono, K.; Higashijima, T.; Miyazawa, T.; J enkins,
E. E. McCloskey, J . A. Tetrahedron 1982, 38, 1599.
(3) Barrett, A. G. M.; Lebold, S. A. J . Org. Chem. 1991, 56, 4875.
(4) Hahn, H.; Heitsch, H.; Rathmann, R.; Zimmerman, G.; Bormann,
C.; Za¨hner, H.; Ko¨nig, W. A. Liebigs Ann. Chem. 1987, 803.
(5) (a) Hass, W.; Ko¨nig, W. A. Liebigs Ann. Chem. 1982, 1615. (b)
J a¨ger, W.; Grund, H.; Buss, V.; Schwab, W.; Mu¨ller, I.; Schohe, R.;
Franz, R.; Ehrler, R. Bull. Soc. Chim. Belg. 1983, 92, 1039. (c) Banks,
B.; Barrett, A. G. M.; Russell, M. A.; Williams, D. J . J . Chem. Soc.,
Chem. Commun. 1983, 873. (d) Melnick, M. J .; Weinreb, S. M. J . Org.
Chem. 1988, 53, 850. (e) Barrett, A. G. M.; Dhanak, D.; Lebold, S. A.;
Russell, M. A. J . Org. Chem. 1991, 56, 1894.
(6) (a) Zimmerman, G.; Hass, W.; Faash, H.; Schmalle, H.; Ko¨nig,
W. A. Liebigs Ann. Chem. 1985, 2165. (b) Barrett, A. G. M.; Lebold, S.
A. J . Org. Chem. 1990, 55, 5818. (c) Barluenga, J .; Viado, A. L.; Aguilar,
E.; Fustero, S.; Olano, B. J . Org. Chem. 1993, 58, 5972. (d) Mukai, C.;
Miyakawa, M.; Hanaoka, M. Synlett 1994, 165.
(7) (a) Bloch, R.; Gilbert, L. Tetrahedron Lett. 1987, 28, 423. (b)
Bloch, R.; Brillet, C. Tetrahedron: Asymmetry, 1992, 3, 333.
(8) Bloch, R.; Gilbert, L. J . Org. Chem. 1987, 52, 4603.
(9) Lactol 5 is readily available from the Diels-Alder adduct of furan
and maleic anhydride either via an enzymatic desymmetrization
(Bloch, R.; Guibe´-J ampel, E.; Girard; C. Tetrahedron Lett. 1985, 26,
4087) or by an asymmetric reduction: Matsuki, K.; Inoue, H.; Takeda,
M.Tetrahedron Lett. 1993, 34, 1167.
(10) (a) Ley, S. V.; Norman, J .; Griffith, W. P.; Marsden, S. P.
Synthesis 1994, 639. (b) Bloch, R.; Brillet, C. Synlett 1991, 829.
(11) Duthaler, R. O. Tetrahedron 1994, 50, 1539 and references
therein.
0022-3263/96/1961-1122$12.00/0 © 1996 American Chemical Society

Notes
J . Org. Chem., Vol. 61, No. 3, 1996 1123
Sch em e 1
course of this reaction to obtain stereoselectively the
desired azide 11 via the corresponding bromide 10.
Effectively, electrophilic bromination of the trimethylsilyl
enol ether of lactone 9 with the bulky reagent phenyl-
trimethylammonium tribromide (PTT) generated stereo-
selectively bromide 10 as the major component of a
mixture containing also a small amount of its diaster-
eomer (ratio 92/8). After purification by crystallization,
the bromo lactone 10 was treated with sodium azide in
dimethyl sulfoxide at 0 °C to give, in excellent yield and
without any epimerization during the course of azide
displacement, the desired compound 11. Finally, trans-
formation of the azido group to the primary amino group
was achieved by reduction with triphenylphosphine/
water to give the target compound, butanolide 4.15
In conclusion, we have accomplished a short and
efficient synthesis (seven steps, 23% from lactol 5) of
(2S,3S,4S)-2-amino-4-(p-methoxyphenyl)-3-methyl-4-bu-
tanolide, a potential precursor of the N-terminal amino
acid moiety of nikkomycins B and BX.
Exp er im en ta l Section 16
(1R,2R,3S,4S,1′S)-2-[1′-(p-Meth oxyph enyl)h ydr oxym ethyl]-
3-(h yd r oxym eth yl)-7-oxa bicyclo[2.2.1]-5-h ep ten e (6). To a
solution of the Grignard reagent prepared from 4-bromoanisole
(8.5 g, 45.4 mmol) and Mg (1.1 g, 0.042 g‚atoms) in THF (10
mL) at 0 °C was added anhydrous ZnBr2 (2.05 g, 9.09 mmol).
The suspension was stirred for 10 min, and a solution of lactol
5 (1.4 g, 9.09 mmol) in THF (50 mL) was added dropwise at 0
°C. The reaction mixture was allowed to warm to rt and stirred
overnight. The solution was cooled to 0 °C, diluted with
saturated aqueous NH4Cl (50 mL), extracted with ether (2 × 50
mL) and CH2Cl2 (2 × 50 mL), dried over MgSO4, and concen-
trated in vacuo. The residue was triturated with hexane (10
mL). The solid formed was decanted, washed with ether (25
mL), and isolated by filtration. The filtrate was concentrated
in vacuo and the residue triturated with a small amount of ether
(5 mL) to give a second crop of solid after filtration: total yield
good results, mainly due to the conditions necessary for
the regeneration of the free amine after alkylation. In
contrast, excellent yields of azides 11 and 11′ were
obtained when the lithium enolate of 9 was treated with
arenesulfonyl azides in the presence of a small amount
of HMPA. The use of a sterically demanding sulfonyl-
azide such as 2,4,6-triisopropylbenzenesulfonyl azide (or
trisyl azide) developed by Evans13 led to a fairly stereo-
selective reaction as shown in eq 1.
1.75 g (73%) of diol 6 as a white solid; mp 155 °C; [R]20 +5.7°
D
1
(c 1.06, CHCl3); IR (KBr) 3440, 1615, 1540 cm-1; H NMR (250
MHz, CDCl3) δ 1.88 (m, 1H), 2.11 (m, 1H), 2.3 (bs, 1H), 3.15 (bs,
1H), 3.7-3.9 (m, 2H), 3.81 (s, 3H), 4.9 (d, J ) 5.3 Hz, 1H), 4.99
(s, 1H), 5.08 (s, 1H), 6.33 (m, 1H), 6.49 (m, 1H), 6.9 (m, 2H),
7.32 (m, 2H); 13C NMR (50 MHz, CDCl3) δ 42.2, 46.3, 55.3, 62.1,
71.2, 79.2, 80.9, 113.9, 127.2, 135.8, 136.2, 136.4, 158.9; CIMS
(NH3) m/ z (relative intensity) 262 (M+, 2), 196 (30), 180 (60),
179 (100), 177 (65). Anal. Calcd for C15H18O4: C, 68.68; H, 6.92.
Found: C, 68.46; H, 7.07.
(1S,2R,5S,6S,7R)-4,10-Dioxa-5-(p-m eth oxyph en yl)tr icyclo-
[5.2.1.02,6]d ec-8-en -3-on e (7). To a solution of diol 6 (985 mg,
3.75 mmol) in CH2Cl2 (20 mL) were added activated 4 Å
molecular sieve powder (1 g) and 4-methylmorpholine N-oxide
(1.32 g, 11.25 mmol). After the mixture was stirred for 10 min,
tetrapropylammonium perruthenate (39 mg, 0.11 mmol) was
added. The mixture was stirred at rt for 1 h and was then
filtered through a pad of silica gel. The solid was washed with
CH2Cl2 (20 mL) and EtOAc (100 mL). The solvents were
removed under reduced pressure, and the residue was purified
by flash chromatography on silica gel (hexane/CH2Cl2/CH3CO2Et
1/1/1) to give 757 mg (78%) of lactone 7 as a white solid: mp
In contrast to reported results involving similar trans-
formations of trans disubstituted butyrolactones,14 the
major diastereomer formed was the undesired azide 11′.
This indicates that during the approach of the electro-
phile 1,3-interactions with the aryl group were stronger
than 1,2-interactions with the methyl group. The respec-
tive stereochemistry of the stereomers 11 and 11′ was
1
easily assigned by comparison of their H NMR spectra
with the corresponding data for known lactone 11.15 We
then decided to take advantage of the stereochemical
135-136 °C; [R]20 +4.3° (c 1.2, CHCl3); IR (KBr) 1770, 1620,
D
1590 cm-1; H NMR (200 MHz, CDCl3) δ 2.78 (dd, J ) 7.8, 7.8
1
(12) Oppolzer, W.; Tamura, O.; Deerberg, J . Helv. Chim. Acta 1992,
75, 1965.
(13) Evans, D. A.; Britton, T. C.; Ellman, J . A.; Dorow, R. L. J . Am.
Chem. Soc. 1990, 112, 4011.
Hz, 1H), 3.03 (d, J ) 7.8 Hz, 1H), 3.83 (s, 3H), 4.43 (bs, 1H),
5.36 (bs, 1H), 5.69 (d, J ) 7.8 Hz, 1H), 6.34 (m, 1H), 6.47 (m,
1H), 6.94 (m, 2H), 7.28 (m, 2H); 13C NMR (50 MHz, CDCl3) δ
(14) Hanessian, S.; Murray, P. J . J . Org. Chem. 1987, 52, 1170.
(15) 1H NMR spectra of 11 and 4 were in excellent agreement with
those of the products already described in ref 6d. We thank Professor
C. Mukai for sending us these documents. However, we found much
higher values of optical rotations for these products than the one
reported in this reference: 11, [R]20 -141° (c 1.0, CHCl3) (lit.6d [R]19
46.3, 49.1, 55.3, 80.4, 81.6, 113.9, 127.4, 128.0, 136.9, 137.7,
+
159.6, 175.5; CIMS (NH3) m/ z (relative intensity) 276 (MNH4
,
71), 258 (M+, 3), 208 (49), 191 (100). Anal. Calcd for C15H14O4:
C, 69.76; H, 5.46. Found: C, 69.71; H, 5.49.
D
D
D
-90.4° (c 0.50, CHCl3)); 4, [R]20 -32° (c 0.57, CHCl3) (lit.6d [R]19
D
-27.3° (c 0.50, CHCl3)). In order to confirm the enantiomeric purity of
our product, 4 was chemically transformed in its N-Boc derivative:
[R]20 +20.7° (c 0.24, CHCl3) (lit.6c [R]23 +20.1° (c 0.82, CHCl3)).
(16) For a general description of experimental parameters see:
Bloch, R.; Bortolussi, M.; Girard, C.; Seck, M. Tetrahedron 1992, 48,
453.
D
D

1124 J . Org. Chem., Vol. 61, No. 3, 1996
Notes
(4S)-4-(p-Meth oxyp h en yl)bu t-2-en olid e (8). A solution of
tricyclic adduct 7 (740 mg, 2.85 mmol) in toluene (10 mL) was
heated under reflux for 2 h. The toluene was removed under
reduced pressure, and the residue was purified by flash chro-
matography on silica gel (hexane/CH2Cl2/EtOAc: 50/30/20) to
layer was extracted with ether (2 × 10 mL). The combined
organic layers were washed with saturated aqueous NaHCO3
(3 mL) and water (5 mL) and dried over MgSO4. Concentration
in vacuo and purification of the residue by silica gel chroma-
tography (ether/hexane: 60/40) gave 80 mg (70%) of a mixture
of two stereomers (92/8). Recrystallization from ether/hexane
50/50 (1 mL) afforded 64 mg of pure bromo lactone 10 as a white
solid: mp 82 °C; [R]20D +46° (c 1.0, CHCl3); IR (KBr) 1805, 1785,
give 487 mg (90%) of butenolide 8 as a white solid: mp 88 °C;
1
[R]20 -266° (c 1, CHCl3); IR (KBr) 1775, 1750, 1620 cm-1; H
D
NMR (250 MHz, CDCl3) δ 3.82 (s, 3H), 5.98 (t, J ) 1.6 Hz, 1H),
6.24 (dd, J ) 5.6, 2 Hz, 1H), 6.92 (m, 2H), 7.19 (m, 2H), 7.51
(dd, J ) 5.6, 1.6 Hz, 1H); 13C NMR (63 MHz, CDCl3) δ 55.0,
84.0, 114.1, 120.6, 125.7, 128.0, 155.8, 160.1, 173.0; CIMS (NH3)
m/ z (relative intensity) 208 (MNH4+, 28), 191 (MH+, 100). Anal.
Calcd for C11H10O3: C, 69.46; H, 5.29. Found: C, 69.13; H, 5.10.
(3S,4S)-4-(p-Meth oxyp h en yl)-3-m eth yl-4-bu ta n olid e (9).
To a suspension of CuI (660 mg, 3.47 mmol) in ether (10 mL)
cooled at -20 °C was added dropwise a solution of methyllithium
1.5 M in ether (4.63 mL, 6.94 mmol). The reaction mixture was
warmed to 0 °C and stirred at this temperature until a colorless
clear solution was obtained. To this solution cooled at -78 °C
was added dropwise freshly distilled chlorotrimethylsilane (440
µL, 376 mg, 3.47 mmol) followed by a solution of butenolide 8
(220 mg, 1.16 mmol) in ether (40 mL). After addition completion,
the mixture was stirred for an additional 4 h at -78 °C and the
1620 cm-1 1H NMR (250 MHz, CDCl3) δ 1.15 (d, J ) 6.5 Hz,
;
3H), 2.42 (m, 1H), 3.83 (s, 3H), 4.56 (d, J ) 5.7 Hz, 1H), 5.07 (d,
J ) 9.5 Hz, 1H), 6.94 (m, 2H), 7.28 (m, 2H); 13C NMR (50 MHz,
CDCl3) δ 12.6, 44.6, 47.9, 55.3, 86.0, 114.2, 127.0, 128.1, 160.6,
172.0; CIMS (NH3) m/ z (relative intensity): 304 and 302
(MNH4+, 64 and 77), 287 and 285 (MH+, 99 and100). Anal.
Calcd for C12H13BrO3: C, 50.55; H, 4.60. Found: C, 50.64; H,
4.57.
(2S,3S,4S)-2-Azid o-4-(p-m eth oxyp h en yl)-3-m eth yl-4-bu -
ta n olid e (11). To a solution of bromobutanolide 10 (64 mg, 0,
22 mmol) in dimethyl sulfoxide (1 mL), cooled at 0 °C was added
sodium azide (58 mg, 0.9 mmol). The mixture was stirred for 1
h, and the reaction was quenched with water (1 mL). After
extraction with CH2Cl2 (3 × 5 mL), the organic layers were dried
over MgSO4. The solvent was removed in vacuo and the residue
was purified by chromatography on silica gel (ether/hexane: 60/
40) to give 51 mg (92%) of compound 11 as a white solid: mp 93
°C; [R]20D -141° (c 1.0, CHCl3); IR (KBr) 2140, 1805, 1795, 1620
temperature was allowed to rise gradually to -30 °C.
A
saturated NH4Cl aqueous solution (20 mL) was then added at
this temperature, and the reaction mixture was warmed to rt,
extracted with CH2Cl2 (3 × 30 mL), dried over MgSO4, and
concentrated in vacuo. The residue was purified by flash
chromatography on silica gel (ether/hexane: 70/30) to afford 203
cm-1 1H NMR (250 MHz, CDCl3) δ 1.21 (d, J ) 6.7 Hz, 3H),
;
2.32 (m, 1H), 3.84 (s, 3H), 4.04 (d, J ) 11.5 Hz, 1H), 4.86 (d, J
) 10.1 Hz, 1H), 6.94 (m, 2H), 7.28 (m, 2H); 13C NMR (63 MHz,
CDCl3) δ 13.5, 45.8, 55.3, 64.4, 84.7, 114.2, 127.4, 128.1, 160.4,
172.3; CIMS (NH3) m/ z (relative intensity) 265 (MNH4+, 66),
248 (MH+, 71), 222 (57), 220 (100). Anal. Calcd for
mg (85%) of butanolide 9 as a white solid: mp 109 °C; [R]20
D
+11.8° (c 1.0, CHCl3); IR (KBr) 1780, 1770, 1730, 1610 cm-1; 1H
NMR (250 MHz, CDCl3) δ 1.17 (d, J ) 6.4 Hz, 3H), 2.34 (dd, J
) 16.3, 10.7 Hz, 1H), 2.5 (m, 1H), 2.80 (dd, J ) 16.3, 7.1 Hz,
1H), 3.83 (s, 3H), 4.90 (d, J ) 8.4 Hz, 1H), 6.92 (m, 2H), 7.28
(m, 2H); 13C NMR (63 MHz, CDCl3) δ 16.2, 37.4, 39.6, 55.3, 88.1,
114.0, 127.5, 129.6, 159.9, 176.1; CIMS (NH3) m/ z (relative
intensity) 224 (MNH4+, 96), 207 (MH+, 100), 206 (M+, 10). Anal.
Calcd for C12H14O3: C, 69.89; H, 6.84. Found: C, 69.82; H, 6.90.
(2R,3R,4S)-2-Br om o-4-(p -m et h oxyp h en yl)-3-m et h yl-4-
bu ta n olid e (10). To a solution of diisopropylamine (63 µL, 0,
48 mmol) in THF (1 mL) cooled at 0 °C was added dropwise a
solution of butyllithium 1.6 M in hexane (302 µL, 0.48 mmol).
To this mixture, cooled at -78 °C was added dropwise a solution
of butanolide 9 (83 mg, 0.40 mmol) in THF (2 mL). The solution
was stirred for an additional 30 min at -78 °C, and the
temperature was allowed to rise gradually to -40 °C. To the
solution cooled again at -78 °C were added successively freshly
distilled chlorotrimethylsilane (102 µL, 0.80 mmol) and a solu-
tion of phenyltrimethylammonium tribromide (227 mg, 0.6
mmol) in THF (2 mL). The solution was stirred for an additional
3 h at -78 °C, quenched at this temperature by the addition of
saturated aqueous NH4Cl (5 mL), and warmed to ambient
temperature. The organic layer was separated and the aqueous
C
12H13N3O3: C, 58.29; H, 5.30. Found: C, 58.17; H, 5.45.
(2S,3S,4S)-2-Am in o-4-(p-m eth oxyp h en yl)-3-m eth yl-4-bu -
ta n olid e (4). To a solution of azide 11 (44 mg, 0.18 mmol) in
THF (1 mL) was added triphenylphosphine (58 mg, 0.22 mmol),
and this solution was stirred at room temperature until no more
nitrogen was released (∼15 min). After addition of water (200
µL), the mixture was heated at 60 °C for 4 h (monitored by TLC).
The solution was cooled, diluted with water (5 mL), extracted
with ethyl acetate (5 × 5 mL), and dried over MgSO4. The
solvent was removed in vacuo, and the residue was purified by
chromatography on silica gel (EtOAc/CH3OH: 95/5, aqueous
NH4OH 1/1000) to give 32 mg (80%) of amino lactone 4 as an
oil: [R]20 -32.2° (c 0.57, CHCl3); IR (CDCl3) 3450, 1790, 1650,
D
1620 cm-1
;
1H NMR (200 MHz, CDCl3) δ 1.19 (d, J ) 6.5 Hz,
3H), 1.66 (bs, 2H), 2.12 (m, 1H), 3.41 (d, J ) 11.4 Hz, 1H), 3.83
(s, 3H), 4.80 (d, J ) 10.1 Hz, 1H), 6.93 (m, 2H), 7.28 (m, 2H);
13C NMR (63 MHz, CDCl3) δ 13.6, 48.3, 55.2, 58.8, 84.6, 114.0,
128.1, 128.5, 160.1, 177. Anal. Calcd for C12H15NO3: C, 65.14;
H, 6.83. Found: C, 65.03; H, 6.97.
J O9514533