TECHNICAL FIELD

The present invention is related to intermediates for the preparation of new bafilomycin and concanamycin macrolides, which bafilomycin and concanamycin macrolides are useful for the inhibition of bone resorption and the treatment of bone affecting diseases.

BACKGROUND ART

Bafilomycins are a class of so-called unusual macrolide antibiotics with a 16-membered lactone ring, isolated from the organism Streptomyces griseus (Werner, G. et al. (1984) J. Antibiotics 37, 1 10- 1 17). Closely related to the bafilomycins are the 16-membered hygrolidins, which is produced by Streptomyces hygroscopicus (Seto, H. et al. ( 1982) Tetrahedron Lett. 23, 2667-2670), and the 18-membered concanamycins, originally isolated from Streptomyces diastatochromogenes (Westley, J.W. et al. ( 1984) J. Antibiotics 37, 1738-1740) and recently isolated from unidentified Streptomyces sp. (Bindseil, K.U. et al. ( 1993) J. Org. Chem. 58, 5487-5492).

Macrolide compounds of the above-mentioned type, useful as antibiotics, parasiticides and pesticides are disclosed in EP-B-0050964 and US 4,385,065 (both Merck & Co.); in US 4,558,139 (Bayer AG); and in JP 3,191 ,788-A (Nippon Kayaku KK). Use of 16-membered macrolide-type compounds as antitumor agents is disclosed in WO 93/18652 and WO 93/18653 (both Merck & Co.) and in JP 1 ,216,927-A (Ajino oto KK).

Studies directed toward the Cι -C]g aldol bond construction in bafilomycin A| by condensation are described in Evans, D.A. and Calter, M.A. ( 1993) Tetrahedron Letters 34, 6871 -6874.

Bafilomycin Ai has been shown to be a specific inhibitor of vacuolar-type H ⁺ - ATPase (Bowman, E.J. et al. ( 1988) Proc. Natl. Acad. Sci. USA 85, 7972-7976). Also concanamycin A has been identified as a valuable tool for distinguishing among different types of ATPases (Drose, S. et al. ( 1993) Biochemistry 32, 3902-3906).

WO 91/06296 (Aktiebolaget Astra) describes the use of bafilomycin macrolides for the inhibition of bone resorption and the treatment of bone affecting diseases. The finding that bafilomycin A i inhibits bone resorption suggests an important role of vacuolar H ⁺ - ATPases in osteoclast acid secretion (Sundquist, K. et al. (1990) Biochem. Biophys. Res. Comm. 168, 309-313).

Use of bafilomycins in the inhibition of vacuolation induced by Helicobacter pylori is described in Papini et al. (1993) Mol. Microbiol. 7, 323-327; and in WO 94/04161 (Biocine Sclavo S.P.A.).

PURPOSE OF THE INVENTION

There is a need for new, in particular less toxic, compounds which inhibit osteoclast acid secretion and thereby are useful for the inhibition of bone resorption and the treatment of bone affecting diseases. The purpose of the present invention is to provide useful intermediates for the preparation of new bafilomycin and concanamycin macrolides with the said activity.

DISCLOSURE OF THE INVENTION

The invention relates to a compound of formula I

wherein

R ¹ is COOH, CH ₂ OH, CHO or CH ₂ NHR ⁸ ;

R is methyl or OMe;

O

R »3 i ^• s OH, ORio , o __r θ R

R ⁴ is H; or R and R" are together =O or =N-OH

R ,5 is H, methyl or ethyl;

R is H, alkyl or alkenyl, which alkyl and alkenyl groups may be optionally substituted with NR ⁸ R ⁹ , SR ⁷ , OR ^{1 1} , halogen, COR ¹² or aryl, which aryl group may be substituted with alkyl,

NR R > 9, SR , OR , halogen, COR , 12 ; or R >6 is aryl which may be optionally substituted with alkyl , alkenyl, NR ⁶ R ^V , SR', OR , 1"1, halogen, COR , 1 ¹ 2.

,7

R is H, alkyl, aryl orarylalkyl;

R is H, alkyl, aryl or arylalkyl;

R ⁹ is H, alkyl, aryl or arylalkyl;

R ⁸ and R ⁹ may form a heterocycle including the nitrogen;

R ¹⁰ is a hydroxy protecting group such as methoxymethyl (MOM), tetramethylsilyl (TMS) or t-butyldimethylsilyl (TBDMS);

R ^{1 1} is R ¹⁰ , H, alkyl, alkenyl, aryl or arylalkyl, alkylcarbonyl, arylcarbonyl or arylalkylcarbonyl ;

R ¹² is H, OR ⁷ ; alkyl, alkenyl, aryl or arylalkyl;

A is a single bond, provided that the compound of the formula I is a bafilomycin-type compound,

or a radical of the formula , provided that the compound of the formula I is a concanamycin-type compound;

R ^π is R or R ⁵ and

R ¹⁴ is H; or R ¹³ and R ¹⁴ are together =O or =N-OH.

Throughout the specification and the appended claims, a given chemical formula or name shall encompass all stereo and optical isomers and racemates thereof where such isomers exist, as well as pharmaceutically acceptable acid addition salts thereof and solvates thereof such as for instance hydrates.

The term "alkyl" denotes a straight, branched or cyclic alkyl group having from 1 to 10 carbon atoms. Examples of said alkyl include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl and straight- and branched-chain pentyl and hexyl A cyclic alkyl group can have a ring size from C3 to C I Q, optionally further substituted by alkyl. Examples of said cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclohexyl and cycloheptyl.

The term "alkenyl" denotes a straight, branched or cyclic alkenyl group having from l to 10 carbon atoms, containing from 1 to 4 double bonds.

The term "aryl" denotes an aromatic system with one or two aromatic rings, containing 0-4 heteroatoms, selected from N, O or S. Preferred is phenyl;

The term "arylalkyl" denotes an alkyl chain as descπbed above, substituted with an aromatic system as defined above. Preferred is benzyl.

Preferred compounds of the invention are compounds of formula I wherein:

R ¹ is COOH, R ² is OMe, R* is OH, R ⁴ is H, R ^s is Me, and A is a single bond (Compound lb; Example 1 );

R ¹ is CH2OH, R ² is OMe, R ³ is OH, R ⁴ is H, R ⁵ is Me, and A is a single bond (Compound 2; Example 2),

R ¹ is CHO, R ² is OMe, R ³ is OH, R ⁴ is H, R^ is Me, and A is a single bond (Compound 3b; Example 3),

R ¹ is CH ₂ NH , R ² is OMe, R ³ is OH, R ⁴ is H, R ^s is Me, and A is a single bond (Compound 4c ; Example 4);

In a further aspect, the invention is related to a process for production of the compounds of the formula 1. The compounds represented by the formula I can be obtained from the natural product bafilomycin Ai as described below.

Compounds of the formula I can be obtained by selective oxidation of bafilomycin A| using dimetyl sulfoxide (DMSO) based oxidation agents or agents such as tetraalkyl ammonium perruthenate, gives 21-keto-bafilomycin A, (la), which by treatment with a primary amine hydrochloride under neutral conditions results in the formation of carboxylic acid lb (Example 1 ). Hydride reduction of the carbonic-carboxylic mixed anhydride, obtained by the reaction of lb with alkyl chloroformate in the presence of base, gives the alcohol 2 (Example 2). Treatment of lb with l-chloro-N,N-2-trimethylprop- l - enyl amine followed by N-methoxy-N-methyl amine hydrochloride under basic conditions results in the formation of the amide chloride 3a, which is reduced to the aldehyde 3b (Example 3) with a reducing agent such as lithium aluminum hydride. From the alcohol 2, the corresponding primary amine (4c) is obtained via formation of the p- nitrobenzenesulphonate of 2 and nucleophilic displacement with azide followed by reduction.

Bafilomycin A) can be obtained according to known methods by culturing of

Streptomyces. See further Werner, G. et al. ( 1984) J. Antibiotics 37, 1 10-1 17, and Huang, L. et al. ( 1984) J. Antibiotics 37,970-975.

The compounds of the formula I thus obtained are useful intermediates for the preparation of new bafilomycin- and concanamycin-like macrolides. An intermediate compound according to the invention can be converted to a bafilomycin- or concanamycin-like macrolide according to methods known in the art. The desired activity, i.e. the inhibition of bone resorption, can be tested according to methods well known in the art. A suitable test method is disclosed in e.g. WO 91/06296.

EXAMPLES

General: All 1H NMR spectra were recorded at 500 MHz, ^{l 3} C NMR spectra were recorded at 75 MHz. Tetramethylsilane was used as internal reference. Solvents were used as purchased from the manufacturer without further purification.

EXAMPLE 1

Synthesis of 21-keto-bafilomycin A | (compound la):

To a solution of oxalyl chloride (4.9 g, 38 mmol) in CH C1 ₂ (100 mL) at -70 °C (dry ice bath), DMSO (8 g, 100 mmol) dissolved in CH ₂ C1 (20 mL) was added dropwise during 5 min. The solution was stirred for 10 min and bafilomycin A\ (13 g, 20 mmol) dissolved in CH ₂ C1 ₂ (50 mL) was added dropwise (5 min) at -70 °C to the oxidation mixture. Stirring was continued for additional 10 min at -70 °C whereupon triethylamine (10 g, 100 mmol) dissolved in CH ₂ Cl2 (20 mL) was added dropwise (5 min). The cooling was interrupted and the reaction mixture was left at ambient temperature 15 min whereupon it was transferred to a separatory funnel and diluted with diethyl ether (500 mL). Brine (100 mL) was added and the mixture was shaken to equilibrium. After separation the organic phase was washed with further portions of brine (3 100 mL), dried over Na ₂ SO4, filtered and concentrated in vacuo. The residue was treated with hexane / ethyl acetate (4: 1 ) whereby the title compound crystallized. Yield 7.4 g (12 mmol). Mp: 157-159 °C. (Reference: Drose et al. (1993) Biochemistry 32, 3902)

Η NMR (CDCI3): δ 6.69 (s, 1 H), 6.51 (dd, 1 H), 5.81 (d, 1 H), 5.78 (d, 1 H), 5.69 (s, 1 H), 5.16 (dd, 1 H), 4.94 (d, 1 H), 4.74 (d, 1 H), 4.14 (m, 1 H), 3.89 (dd, 1 H), 3.80 (m, 1 H), 3.65 (s, 3 H), 3.30 (m, 1 H), 3.25 (s, 3 H), 2.73 (d, 1 H), 2.54 (m, 1 H), 2.33 (m, 1 H), 2.26 (m, 1 H), 2.15 (m, 2 H), 1.99 (s, 1 H), 1.98 (m, 2 H), 1.94 (m, 3 H), 1.93 (s, 1 H), 1.91 (s, 3 H), 1.57 (d, 1 H), 1.07 (d, 3 H), 1.02 (d, 3 H), 1.97 (d, 6 H), 1.93 (d, 3 H), 1.86 (2xd, 2x3 H).

^I3 C NMR (CDCl3) :δ 208.9, 167.4, 143.2, 143.0, 141.2, 133.8, 133.1 , 133.0, 127.1 , 125.2, 101.2, 82.2, 81.1 , 77.2, 76.8, 70.7, 60.0, 55.5, 50.9, 47.3, 41.8, 41.2, 40.0, 37.1 , 36.7, 28.8, 21.6, 21.0, 20.2, 17.3, 14.2, 14.0, 9.8, 8.8, 6.9.

Synthesis of the compound of the formula I wherein R ¹ is COOH, R ² is OMe, R ³ is OH, R ⁴ is H, R ^" is Me, and A is a single bond (compound lb):

To a solution of methylamine hydrochloride (7.57 g, 1 13 mmol) in methanol ( 100 mL) 4 A molecular sieves (7 g) was added and the flask was cooled in an ice bath. Sodium hydroxide powder (2.98 g, 74.6 mmol) was added in portions and the resulting white suspension was allowed to stir at ambient temperature for ten minutes before la (7.4 g, 1 1.9 mmol) was added in portions (c:a 10 min). The stirring was continued for six hours and the solid was filtered off and washed with methanol until no product could be detected in the eluate. The filtrate was concentrated in vacuo to give a yellow oil which was dissolved in water (200 mL). The solution was cooled and stirred in an ice bath and was acidified to pH 2-3 by adding cold IM hydrochloric acid. The aqueous solution was extracted three times with ethyl acetate (100 mL each) and the combined extracts were washed twice with water, once with brine. The organic layer was dried (MgSO4), filtered

and evaporated to dryness. The residue was chromatgraphed on silica gel using ethyl acetate / hexane (2:3) followed by 1 % acetic acid in the eluent, to give 4.6 g (78 %) of the desired product.

1H NMR (CDCI3) δ 6.65 (s, 1 H), 5.80 (d, 1 H), 5.75 (d, 1 H), 5.15 (dd, 1 H), 5.03 (d, 1 H), 3.87 (dd, 1 H), 3.74 (dd, 1 H), 3.66 (s, 3 H ), 3.28 (dd, I H), 3.24 (s, 3 H), 2.72 (m, 1 H), 2.51 (m, 1 H), 2.13 (m, 2 H), 1.98 (s, 3 H), 1.92 (s, 3 H), 1.9-2.0 ( , 2 H), 1.25-0.90 (4 x d, 4 x 3 H). MS : (M +Na)+ = 517

EXAMPLE 2

Synthesis of the compound of the formula I wherein R ¹ is CH ₂ OH, R ² is OMe, R ³ is OH, R ⁴ is H, R ^s is Me, and A is a single bond (compound 2):

To a solution of compound lb from above (3.95 g, 8 mmol) and triethylamine ( 1.26 mL, 9 mmol) in THF (16 mL) at -25°C, ethyl chloroformate (0.86 mL, 9 mmol) was added dropwise during 10 min under nitrogen. The resulting white suspension was stirred at this temperature for 15 min and then filtered and washed with THF (40 mL). The filtrate was added dropwise to a suspension of sodium borohydride (0.76 g, 20 mmol) in water (5 mL) at 0°C. The ice bath was removed and the stirring was continued for 30 min at ambient temperature, satd. ammonium chloride was added and the phases were separated. The aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with water and brine, dried over MgSO4, filtered and the solvent removed in vacuo. The

residue was chromatographed on silica gel using ethyl acetate / hexane (2:3) to give 2.4 g (62 %) of the desired product.

Η NMR (CDCl3) δ 6.16 (s, 1 H), 6.50 (dd, 1 H), 5.81 (d, 1 H), 5.75 (d, 1 H), 5.15 (dd, 1 II), 5.01 (d, 1 H), 4.30 (d, 1 H), 3.88 (dd, 1 H), 3.78 (m, 1 H), 3.70 (m, 1 H), 3.68 (s, 3 H), 3.46 (m, 1 H ), 3.30 (m, 1 H), 3.25 (s, 3 H), 2.90 (m, 1 H), 2.60 (m, 1 H), 2.20-2.10 (m, 2 H), 1.95 (s, 3 H), 1.90 (s, 3 H), 2.0- 1 .8 (m, 3 H), 1.60 (d, 1 H), 1.20-0.90 (4 x d, 4 x 3 H). MS: (M+Na)+ = 503.

EXAMPLE 3

Synthesis of compound 3a:

To compound lb (540 mg, 1.2 mmol) dissolved in CH C1 ₂ ( 15 mL) was added 1 -chloro- N,N-2-trimethyl-l-propenyl amine at ambient temperature. After 15 min N-methoxy-N- methylammonium chloride (300 mg; 3 mmol) was added to the rection mixture whereupon it was neutralized by dropwise addition of triethyl amine (600 mg, 6 mmol) dissolved in CH ₂ CI ₂ (5 mL), while stirring efficiently. The mixture was left at ambient temperture for 15 min whereupon it was hydrolyzed with brine ( 10 mL ) and transferred to a separatory funnel. The solvent phases were separated and the organic phase was washed with brine (2x 10 mL ), dried over Na ₂ SO4, filtered and concentrated under in vacuo. The residue was chromatographed on silica gel using hexane / ethyl acetate (1 : 1 ) to give the title compound contaminated with N,N-dimethylisobutyramide. Compound 3a was crystallized from this mixture by treatment with hexane/ethyl acetate (9:1). Yield 300 mg (0.55 mmol). Mp: 164- 167 °C.

Η NMR (CDCI3): δ 6.63 (s, 1 H), 6.46 (dd, 1 H), 5.81 (d, 1 H), 5 74 (d, 1 H), 5.21 (dd, 1 H), 5.17 (d, 1 H), 3.81 (m, 2 H), 3.69 (s, 3 H), 3.68 (s, 3 H), 3.31 (m, 1 H), 3.23 (s, 3 H), 3.18 (s, 3 H), 3.10 ( , 1 H), 2.51 (m, 1 H), 2.05 (m, 3 H), 1.97 (s, 3 H), 1.89 (m, 1 H), 1.88 (s, 3 H), 1.58 (d, 1 H), 1.22 (d, 3 H), 1.06 (d, 3 H), 0.94 (d, 6 H). ^π C NMR (CDCI3): δ 177.2, 165.9, 142.3, 141.8, 141.6, 132.7, 132.5, 132.2, 127.2, 125.3, 83.7, 81.3, 76.1 , 73.0, 61.3, 60.0, 55 7, 41.3, 39.4, 38.6, 37.2, 36.9, 32.2, 22.1 , 19.7, 17.5, 14.0, 1 1.3, 10.8.

Synthesis of the compound of the formula I wherein R ¹ is CHO, R ² is OMe, R ³ is OH, R ⁴ is H, R is Me, and A is a single bond (compund 3b).

Lithium aluminum hydride ( 100 mg; 2.63 mmol) was dissolved in dry diethyl ether (25 L) and cooled to -70 °C (dry ice bath) under an argon atmosphere. To this efficiently stirred mixture 3a (770 mg; 1.43 mmol) THF ( 15 mL) was added dropwise with a syringe The reaction mixture was left at -70 °C for 10 min whereupon it was quenched with ammonium chloride (0.2 g dissolved in 2 mL water). The cooling was interrupted and the reaction mxture was allowed to reach ambient temperature whereupon 2 M hydrochloric acid (2 mL) was added. The mixture was transferred to a separatory funnel and the phases were separated. The organic phase was washed with brine (2 x 15 mL), dried over Na ₂ SO4, filtered and concentrated under in vacuo. The residue was chromatographed on silica gel with hexane / ethyl acetate (2: 1 ) to give the title compound as an oil, yield: 620 mg ( 1.29 mmol). Η NMR (CDCI3): δ 9.74 (s, 1 H), 6.67 (s, 1 H), 6.51 (dd, 1 H), 5.81 (d, 1 H), 5.77 (d, 1 H), 5.17 (dd, 1 H), 5.03 (d, 1 H), 4.08 (d, 1 H), 3.86 (m, 2 H), 3.69 (s, 3 H), 3.29 (m, 1 H),

3.25 (s, 3 H), 2.53 ( , 1 H), 2.46 (m, 1 H), 2.13 (m, 2 H), 1.99 (s, 3 H), 1.93 (s, 3 H), 1.90 (m, 2 H), 1.55 (d, 1 H), 1.17 (d, 3 H), 1.07 (d, 3 H), 0.94 (d, 3 H), 0.90 (d, 3 H). ¹ C NMR (CDCl3): δ 205.2, 167.0, 143.2, 142.8, 141.2, 133.5, 133.0, 132.9, 127.0, 125.2, 82.5, 81.2, 76.5, 70.7, 60.2, 55.6, 48.1, 41.3, 40.0, 37.8, 36.7, 21.7, 20.1 , 17.3, 13.9, 10. 1 , 6.1.

EXAMPLE 4

Synthesis of compound 4a.

Compound 2 (4.50 g, 9.37 mmol), triethylamine (4.77 g, 46.8 mmol) and 4- dimethylaminopyridine (1.14 g, 9.37 mmol) were dissolved in 20 mL of dichloromethane. The resulting solution was stirred at ambient temperature and 4-nitrobenzenesulphonyl chloride (3.00 g, 13.5 mmol) was carefully added in portions. The reaction mixture was stirred 20 min. and quenched by the addition of 25 mL water and 20 L dichloromethane. The organic layer was washed with 1 M HCl ( 1 x 30 + 1 x 10 mL), and finally with 20 mL brine. The organic layer was dried over MgSO4, filtered and evaporated in vacuo. The residue was purified by chromatography on silica gel with heptane/ethyl acetate (4: 1, 2: 1) and after collection of homogeneous fractions and removal of solvents in vacuo compound 4a as a pale yellow glass. Yield 5.45 g, 87 %.

Η NMR (CDCI3) δ 8.42 (d, 2 H), 8.08 (d, 2 H), 6.67 (s, 1 H), 6.50 (dd, 1 H), 5.81 (d, 1 H), 5.78 (d, 1 H), 5.15 (dd, 1 H), 4.93 (d, 1 H), 4.18 (t, 1 H), 4.07 (dd, 1 H), 3.86 (t, 1 H), 3.82 (d, 1 H), 3.71 (s, 3 H), 3.32-3.27 (m, 2 H), 3.23 (s, 3 H), 2.54 (m, 1 H), 2.14 (m, 1 H), 2.13 (d, 1 H), 2.03 ( , 1 H), 2.00 (s, 3 H), 1.98 (dd, 1 H), 1.93 (s, 3 H), 1.90 (m, 1 H), 1. 1 (d, 1 H), 1.08 (d, 3 H), 0.95 (d, 3 H), 0.88 (d, 3 H), 0.83 (d, 3 H).

Synthesis of compound 4b.

Compound 4a (7.85 g, 1 1.8 mmol), amberlite IRA-400-N ₃ (A. Hassner and M. Stern ( 1986) Angew. Chem. 98, 479) (59 g. 148 mmol N ₃ ) and 50 mL of dry acetonitrile were placed in a 250 mL 3-necked flask equipped with a large magnetic stirring bar. The flask was evacuated and filled with argon six times. The reaction was heated at 60 °C for 1 1 hours followed by 4 hours at 90 °C. The reaction mixture was cooled to room temperature and filtered through a glass filter followed by repeated washing with acetonitrile until the washings were free from UV-active material. Removal of the solvent in vacuo gave compound 4b, 5.92 g, as a pale yellow glass. Yield 99 . Η NMR (CDCI3) δ 6.65 (s, 1 H), 6.50 (dd, 1 H), 5.82 (d, 1 H), 5.76 (d, 1 H), 5.18 (dd, I H), 5.06 (dd, 1 H), 3.97 (d, 1 H), 3.89 (t, 1 H), 3.68 (s, 3 H), 3.49 (dd, 1 H), 3.32 (ddd, 1 H), 3.30 (broad t, 1 H), 3.25 (s, 3 H), 3.25 (dd, 1 H), 2.53 (m, 1 H), 2.14 (d, 1 H), 2.08 (m, 1 H), 2.00-1.85 (m, 3 H), 1.99 (d, 3 H), 1.94 (s, 3 H), 1.51 (d, 1 H), 1.07 (d, 3 H), 0.95 (d, 3 H), 0.94 (d, 3 H), 0.87 (d, 3 H).

Synthesis of the compound of the formula I wherein R ' is CH ₂ NH ₂ , R ² is OMe, R^ IS OH, R IS H, R5 is Me, and A is a single bond (compund 4c):

Compound 4b (5 90 g, 1 1.7 mmol), triethylamine (3 54 g, 35.0 mmol), thiophenol (3 86 g, 35.0 mmol) and tιn(II) thiophenolale (M. Bartra, F Upπ and J. Vilarrasa ( 1987) Tetrahedron Letters 28(47), 5941) (13.0 g, 38.5 mmol) were treated with 58 mL of dichloromethane. The reaction mixture was stirred for 2.5 hours and then placed directly on a silica gel column. The column was eluated with dichloromethane until no LIV-active material could be spotted whereupon eluation was continued with dιchloromethane/NH ₃ Homogeneous fractions were collected and after removal of solvent in vacuo 3 37 g of compound 4c was obtained as a glass. Yield 60 %. Η NMR (CDCI3) δ 6.65 (s, 1 H), 6.49 (dd, 1 H), 5.82 (d, 1 H), 5.76 (d, 1 H), 5.21 (dd, 1 H), 5.14 (dd, 1 H), 3.89 (t, 1 H), 3.69 (s, 3 H), 3 43 (dd, 1 H), 3.30 (d, 1 H), 3.25 (s, 3 H), 2.88 (dd, 1 H), 2.75 (dd, 1 H), 2.53 (m, 1 H), 2.12 (d, 1 H), 2 04 (m, 1 H), 1.99 (s, 3 H), 1.99 (dd, 1 H), 1.91 (s, 3 H), 1.90 (m, 1 H), 1.68 (m, 1 H), 1.64 (broad s, 1 H), 1.07 (d, 3 H), 0.95 (d, 3 H), 0.94 (d, 3 H), 0.86 (d, 3 H).