Tetrahedron Vol. 42. No. l6. pp. 445] to 4460. I986
`Printed in Great Britain.
`
`0040-4020/86 :3.oo+.oo
`(0 I986 Pagamon Journals Ltd.
`
`CHEMICAL STUDIES OF 10—DEACETYL BACCATIN III.
`HEMISYNTHESIS 0F TAXOL DERIVATIVES.
`
`F. GUéRITTE-VOEGELEIN,+ v. séNILH,
`B. DAVID. D. GUéNARD and P. POTIER
`
`Institut de Chimie des Substances Naturelles, CNRS,
`91190 Gif—sur-Yvette, France
`
`(Received in France 6 June I986)
`
`Abstract - The chemical reactivities of 10—deacetyl baccatin
`III and of baccatin III,
`two natural products extracted from
`Taxus baccata L., were studied with the aim of synthesizing
`taxol analogues having a modified side-chain at 0-13,
`thereby
`restoring good binding to tubulin.
`
`In 1971 taxol
`
`1 was isolated from Taxus brevifolia Nutt and was
`
`the
`
`taxol has
`first taxane diterpene shown to exhibit cytotoxic activity.1 In give,
`antileukemic and tumor inhibiting propertieg’énuiit is currently in clinical
`trials in France
`and in the USA.
`The biological activity has been related to
`the in gitgg interaction with microtubule proteinsg'QIn contrast with other
`spindle poisons such as vinblastine and colchicine which prevent the assembly of
`tubulin5'6taxol l promotesthe assembly of microtubules and inhibits the depoly-
`merisation process of tubulin.
`In addition to taxol, other taxane derivatives
`
`showing similar biological activity have been isolated from various species of
`yew tree7_gfiecause of its unique mode of action taxol may be the prototype of a
`new class of chemotherapeutic drugs. However, one of the disadvantages of taxol
`is associated with its limited availability from natural sources : it is ex-
`To
`tracted in low yield from the stem bark of the very slow—growing yew tree.
`circumvent this major problem seVeral attempts to synthesize the unusual
`taxane
`skeleton have been described10 but
`to date no total synthesis of taxol has been
`reported. One other way to prepare this compound is to use simpler taxane deri-
`vatives which could be used as precursorsin a taxol hemisynthesis.
`and
`Baccatin III E has been isolated from an alcohol extract of heart wood
`10-deacetyl baccatin III 2 was easily extracted from the annual cut of the yew
`leaves.9 These two compounds are not as active as taxol 1 both in vitro and in
`__-_.
`vivo
`but
`they can be used as raw materials for the preparation of taxol and
`derivatives. In this paper we wish to report some chemical properties of these
`compounds and the preparation of new taxane diterpenes which could be used
`as
`
`8
`
`intermediates in the hemisynthesis of taxol itself.. The compounds obtained in
`this study haVe been submitted for in vitrg antitubulin evaluation which will
`allow establishment of structure-activity relationships in this series.
`‘)Some of this work has been the subject of the PhD Tbgsis of one of us12 and
`has been partly described in a short communication.1 We
`thank Professor D.
`Kingston for a personal communication concerning his study of baccatin III.
`
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`

`4452
`
`F. GUERITrE-VOEGELHN er a].
`
`taxane skeleton of 3 and 2 has a very folded structure (figure 1)
`The unusual
`in which the a hydroxyl group at C-13 is in a hindered position and furthermore,
`it can form a hydrogen bond with the “a acetyl group. It is also important
`to remember that the 7B hydroxy1 can easily epimerize into the 7m isomer
`via a retro aldol mechanism. The presence of a hydrogen bond with the ho acetyl
`group stabilizes the a isomer during the aldol condensation.11
`
`Figure 1.
`
`The other functionalities of tetraol 3 seem stable enough to our experimental
`conditions, except for the 1-hydroxyl group and the oxetan ring which can be
`rearranged in acidic media-12'13
`
`I. Reactivity cg 10—dgacetyl baccatin III 2_with acxlating agents.
`
`Acetylation of 2 with acetic anhydride yielded three acetylation products
`depending on the experimental conditions (Table 1). Structure elucidation of
`these compounds was obtained by considering the chemical shifts of the three
`protons at C-7. C-10 and C-13 in their proton NMR spectra (see Experimental PartL
`These data thus show that there is no selectivity between C-7 and 0-10 hydroxyl
`
`groups toward acylating agents and that the C—13 hydroxyl group is the least
`reactive. as expected.
`
`Considering these results, We next undertook to protect the two most reactive
`hydroxyl groups.
`
` 3 or 2 m.- Raom
`
`cu co,cu,ccu3 in
`Dy.
`
`R2=Ac
`1 R1=H
`g R1=R2=R3=H
`2 R1=R3=H R2=Ac
`
`R3=C0CHOHCHPhNHCOPh
`
`Figure 2.
`
`g R1=Ac R2=R3=H
`2 R1=R2=Ac
`R3=H
`g R1=R2=R3=Ac
`Z R1=R2=C0~O-CH2- c-c13, R3=H
`g R1=coocnzcc13, R2=Ac, 123:}!
`2 R1=R2=R3=COOCH2CC1 3
`
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`Chemical studies of lo-deaoetyl baccatin III
`
`4453
`
`2. Protection and Deprotgction.
`Taking into consideration the instability of taxol in basic medium1 we
`thought that 2,2,2-trichloroethy1 chloroformete
`could be a good protective group,
`since it can be removed under very mild conditions. We thus obtained compound 1
`in good yield from 10-deacetyl baccatin III 2. With an excess of the acid chlo-
`ride compound 2 was also prepared. Alkyl 2.2,2-trichloroethy1 carbonate can
`be
`cleaved by B-elimination with zinc dust in methanol or acetic acid.15 Compound 1
`was cleaved as expected with zinc dust in acetic acid to give the starting mate-
`rial in quantitative yield.
`
` Raoun
`
`HO
`
`2 R1=R2=R3=C00CH2CC13
`
`ll R=Me
`1_0 R4=Me
`l2. Rq=CH2-CH20H L R=CH2CH20H
`
`Figure 3.
`
`The same experiments have been done with the 7,10,13—tri(2,2,2-trichloro-
`ethyloxycarbonyl)—10-deacety1 baccatin III 2. Treatment of this compound with
`zinc dust in methanol yielded a new derivative 19. Deprotection of the 7 and 10-
`
`positions by reductive cleavage in acetic acid gave a quantitative yield of 13-
`methyloxycarbonyl-10—deacety1 baccatin III 11. This product was also obtained by
`direct methanolysis of 2 after deprotection of the 7 and 10-positions.
`These
`results led us to try other nucleophilic agents. Thus preparation of compounds
`
`1% and 12 was achieved by treatment of 2 with ethylene glycol. Unfortunately the
`in vitrg activities on microtubule assembly of $1 and 12 were less than that of
`taxol 1.
`
`Baccatin III 2 was protected in the same way to give g.
`
`3. Hemisynthesis of taxol derivatives from Z and fi-
`
`16
`
`Various Taxus species contain a mixture of alkaloids named "taxine".
`basic
`property of these compounds is due to the Winterstein's acid (3-
`dimethyl amino-3-phenyl propanoic acid). Biosynthetic study of this acid has
`shown that it arises from phenylalanine by a B—amination of cinnamic acid}7 It
`is also well known that cinnamic esters of taxane diterpenes have been isolated
`
`The
`
`from different species of yew trees. Cinnamic acid is thus an attractive candi-
`date for esterification of the free 0-13 hydroxyl group of compound 1.
`
`H
`
`O
`
`OH
`
`7
`'
`
`R. cocu
`AgCN /To|u'ono o
`00°C
`
`Ow'"
`
`In I «on
`
`0'"
`
`R.
`
`H0
`
`1_l: R9: CH3
`_1_6 Rh=-CH=CH-Ph
`Figure 4.
`
`‘
`
`OH
`
`a".
`:
`551°“
`
`1; RFCHB
`1_7_ Rq=-CH=CH-Ph
`
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`

`4454
`
`F. GUEerz-Voscauzm er al.
`
`In contrast to the relatively easy acetylation of 1 leading to $2 and 12
`after deprotection, acylation with cinnamoyl chloride has proved to be a diffi-
`cult reaction under the usual experimental conditions. Preparation of 13-
`
`cinnamoyl-lO-deacetyl baccatin III 11 was finally achieved by the coupling of
`cinnamoyl chloride and 1 in the presence of siIVer cyanide at 110°C in toluene.
`followed by the deprotection of the 10 and 7 positions of l§ by treatment with
`zinc in acetic acid. The use of more complex acid chlorides such as B phenyl
`
`to prepare taxol l was not successful in our-hands.
`isoserine (Three and Erythro)
`Therefore, our next approach was to investigate some addition reactions on the
`
`double bond of the cinnamoyl ester 12.
`
`0...... .3
`
`Cum
`
`
`
`0| 0.
`1 6
`— Toluene
`
`H
`
`OH
`
`Pb(0A5)
`Ac OH
`
`ZnIAcOH
`
`0
`
`Ph
`
`1_8
`
`Zn / A<OH
`
`.22
`
`a
`
`OH
`
`OH
`
`you.
`
`
`
`:
`
`0"
`.
`OAc
`081
`
`(
`Pb OAc).
`
`_—>Acon
`
`0
`
`HO
`
`Ph
`
`12.
`
`an-
`
`o
`
`H
`
`a
`
`NH on
`’
`
`0""
`
`NH-OH
`
`H
`
`22
`
`Figure 5.
`
`The relative configuration of the 2' and 3' carbons in taxol and the trans
`
`configuration of the cinnamate ester 16 require a gig addition on the 2'3'
`double bond. The reaction of lg with osmium tetroxide in pyridine led to the
`rapid formation of the 2'v3'—dihydroxy derivative lg as a mixture of diastereo-
`isomers (2's, 3'R and 2'R, 3'5) which could be purified by HPLC of the deprotec-
`ted mixture 12 (123 and 32b). The 1H NMR spectrum of $22 and 122 showed two new
`doublets (J = 2) corresponding to the C-3' and C-2' protons. The FAB mass spec-
`
`trum gave peaks at m/z 709 (MH+) corresponding to the addition of two hydroxyl
`groups.
`
`Oxidation of Ag with lead tetraacetate followed by deprotection of the resul-
`ting aldehyde 32 with zinc in acetic acid gave 13-hydroxyacetyl-10-deacety1
`baccatin III 31. In a similar manner oxidation of the mixture 12 gave the alde-
`hyde intermediate 33 which was characterized as its oxime 32.
`We also tried acylation of baccatin III with crotonyl chloride in order to
`
`Treatment of 7-(2,2,2-trichloroethyloxycarbonyl) baccatin III-é with crotonyl
`chloride gaVe the C-13 ester 32 which yielded 22 after deprotection of the C—7
`position.
`
`Hydroxylation of 24 with osmium tetroxide followed by deprotection of the
`
`resulting dihydroxy derivative Eé with zinc in acetic acid gave the ester 27
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`

`Chemical studies of lO—deacctyl baccatin III
`
`4455
`
`resulting dihydroxy derivative fig with zinc in acetic acid gave the ester 21
`which is less active on tubulin than the esters 12 containing a phenyl group at
`C—3'.
`
`__ a 1 -
`8
`CHrCHICH—COCI CY“
`Py.
`o
`
`/ 25
`z""‘°”
`OIO‘/Py
`
`Zn/fl HO
`
`24
`
`22
`
`Figure 6.
`Structure-activity relationships of these new taxol derivatives will be dis-
`
`cussed in a subsequent publication but it is already interesting to note that,in
`
`contrast to the phenyl group, a methyl group at C-3‘ 22 destroys the in yitrg
`activity and that hydroxyl groups at the 2' and 3' positions 12 increase the
`activity in comparison to the cinnamate ester AZ.
`
`gonclusion.
`The results obtained in our work show that it is possible to carry out este-
`rification of the extremely hindered C 13-hydroxy group of baccatin III and 10-
`
`deacetyl baccatin III. In particular, hemisynthesis of cinnamate ester lg allowal
`us to prepare some taxol derivatives. This compound would seem to be a good pre-
`.
`.
`.
`8
`.
`cursor of taxol itself using Sharpless hydroxyamination1
`as described pre—
`viously.14
`
`EXPERIMENTAL SECTION
`
`Due to the complexity of the molecules and the small size of the samples
`available, no elemental analysis is given. Purity of the samples was determined
`by chromatographic homogeneity and careful analysis of NMR spectra (200 MHz or
`400 MHz). Preparative T.L.C. was performed on Merck Silica Gel PF—ZSQ plates.
`Melting points were observed on a Kofler apparatus. optical rotations measured
`(c, g/100 ml) on a Perkin-Elmer 141 MC,
`infrared spectra (ch‘l, CHCl
`) on a
`Perkin-Elmer 257, ultraviolet spectra EtOH, Amax nm (t)] on a Jobin-evon duospac
`203.
`1H NMR spectra were obtained at 2 0 MHz or at 400 MHz (Bruker AM 200 or AM
`400) using TMS as internal standard (coupling constants (J) are given in Hertz
`(Hz)
`; s, d,
`t, dd and m indicates singlet. doublet, triplet, doublet of doublets
`and multiplet. respectively). Mass spectra were measured on an AEI MS 9 (CI) or
`a Kratos MS 80 (FAB). C13 NMR Spectra of taxol derivatives will be presented and
`discussed in a further publication.
`
`Acetylation of 10-Deacetyl bacgggin III 2
`in
`Exp.
`1
`: Acetic anhydride (0.1 ml) was added to a solution of 2 (17 mg)
`pyridine (1 ml) with stirring at room temperature for 21 h. Work Eb by standard
`methods and purification by preparative TLC (solvent
`: CH2C12-MeQH, 93-7) yielded
`7-acetyl-10-deacetyl baccatin III_& (10 mg) and 7-acetyl baccatin III 2 (8.4 mg).
`Exp.
`2
`: Acetic anhydride (0.18 ml) was added to a solution of 2 (30 mg)
`in
`pyridine (1 ml) with stirring at 60°C for 08 h. Usual work—up and isolation of
`the products by preparative TLC (solvent
`: CH2C12-Me0H, 97-3) gave 5 (17 mg) and
`7.13—diacetyl baccatin E (18 mg).
`_
`Exp.
`3
`: Acetic anhydride (0.25 ml) was added to a solution of E (25.5 mg)
`pyridine (1 ml) with stirring at 80°C for 24 h. Usual work up yielded 7,13-
`diacetyl baccatin III 2 (30 mg).
`
`7-Acetyl-10—deacetzl baccatip III 2
`; UV : 231(16100).
`(c = 0.53, cuc13)
`Mp 265—266°c (neon—H20);
`«333 = -56°
`6 :1.04 (3H, s, c17u9
`275(1090). 280(920) ;IR :
`34 o, 1730, 1710 ;
`1H NMR(CDC13)
`1.09 (3H, s, c15H3), 1.83 (3H, s, C19H3), 1.97 (3H. s, C18H3), 2.08 and 2.32
`(2 x 3H, 23.
`2 x OAc), 4.06 (1H, d, J = 7, C3—H), 4.20 and 4.30 (2H, 2d, J = 9,
`CZOHZ), 4.80 (1H, t, J = 9, c1 -H). 4.95 (1H, d,
`J = 9, c -H), 5.33 (1H, s,
`c 0-H), 5.59 (1H, m, C7-H), 5.81 (1H, d, J = 7, c -H . 7.25, 7.58 and 8.08 (5H,
`Oéz)
`; MS(C.I.
`, m/z : 587(MH+), 569. 551. 509, 307
`327, 309, 123.
`
`in
`
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`

`4456
`
`F. Gulmm-Vomamn er a1.
`
`Z;Acetx1 baccatin III 2.
`
`; uv : 230(19500),
`)
`; @033 = .790 (c = 0.6, CHCl
`Mp 249-251°c (MeOH-Hzo)
`276(1150), 282(1040)
`;
`IR : 3400, 1730, 1710 ;
`1H NMR(c0313)
`6
`: 1.08 (3H, s,
`01 H3), 1.13 (3H, s, C15H3), 1.79 (3H, s. 01 H ), 2.03 (3H, s, Cé8H3), 2.10,
`2.16 and 2.29 (3 x 3H, 35,
`3 0A6), 2.60 and 1.30 (2H, 05H2), 2.2
`(m, Clgflz),
`4.00 (1H, d, J = 7, C3—H), 4.14 and 4.31 (2H, 2d, J = 9, conz), 4.86 (1H, t,
`J = 9, C. -H), 4.97 (1H, d, J = 9, c -H), 5.59 (1H, m, c -H), 5.62 (1H, d,
`J =7,
`Cz-H), 6.26 (1H, 6, C1o-H), 7.46, 7.58 and 8.08 (5H, OBz , MS(CI), m/z : 629
`(1411+), 611. 569. 551, 509. 491. 387. 327. 309. 123.
`7,12—Diacetyl baccatin III E.
`
`; UV : 232(19500),
`)
`(c = 0.52, CHCl
`; @033 = -80°
`Mp 236-237°c (MeOH-HZO)
`275(1380), 282(1250)
`;
`IR : 3400, 1730, 1710 ;
`1H NMR (c0813)
`6
`: 1.17 (3H, 5,
`c1 H3), 1.22 (3H, s, C16H3), 1.81 (3H, s, c1 H ), 1.97 (3H, s, Clgflg), 1.80 and
`2.60 (m, 2H, C6H2), 2.03, 2.17, 2.21 and 2.32 (4 x 3H, 4s,
`4 0A6),
`.25 (m, 2H,
`Clgflz), 3.97 (1H, d, J = 7, c -H), 4.16 and 4.33 (2H, 2d, J = 9, c 0H2), 4.98
`(1H, d, J = 9, Cs-H), 5.59 (1 , m, C7-H), 5.66 (1H, d, J = 7, Cz-H?,
`.18 (1H, 6
`J = 9, 013—H), 6.26 (1H, s, Clo-H),
`7.48, 7.61 and 8.08 (5H, 082)
`; MS(CI) m/z
`671 (MH+). 653. 611. 593. 551, 491. 489, 429. 369. 309. 123-
`
`7110'diS21212 Trichloroethxlogxcarbonyl)—10-dgacetxl baccatin III Z.
`2,2,2—Trichloroethylchloroformate (0.085 ml) was added to a solution of 10-
`deacetyl baccatin III a (100 mg)
`in pyridine (2 ml) with stirring at 80°C. After
`5 min the reaction mixture was treated with H20 and extracted with CH2C12. Usual
`work-up and purification by preparative TLC (solvent
`: CH2C12-MeOH, 95-5) yielded
`7 (153 mg). mp 233—234°c (MeOH-Hao)
`; @933 : —58°
`(c = 0.465, CHC1 )
`; UV : 232
`719000), 276(990), 283(810)
`;
`IR : 3420,
`1765, 1730, 1720 ;
`1H NMQ (CDC13)
`6
`1.12 (3H, s, 01 H3), 1.16 (3H, s, 016H3), 1.85 (3H, s, C19Hg), 2.16 (3H, s,
`C18H3), 2.30 (30, s, 0A6), 2.30 (m, cquz), 2.05 and 2.65 ( m, c Hz), 4.00 (1H,
`d, J = 7, C3-H), 4.18 and 4.35 (2H, 2d,
`J = 9, C20H ), 4.63 and
`.92 (2H, 2d,
`J = 12, CH2 of the protectug group),4.76 and 4.80 ( H, 2d, J = 12, CH of the
`protecfimggroup), 4.92 (1H,
`t, J = 9, C 3-H), 5.00 (1H, d, J = 9, C5-fi), 5.61
`(1H, m, C7-H), 5.66 (1H, d, J = 7, Cz-H , 6.30 (1H, s, c o—H), 7.50, 7.64 and
`8.13 (2H,
`1H and 2H, 2t and 1d, J = 7, OBz)
`; Ms(CI). m}z 893(MH+), 875. 701.
`683. 579. 337. 327. 309. 123-
`
`2,10,12—trif2,2,2-Trichlorogthxloxxcarbonxl)-10—deacetyl baccatin III 2.
`2,2,2-Trichloroethylchloroformate
`(0.13 ml) was added to a solution of 2
`(100 mg)
`in pyridine (1.4 ml) with stirring at 80°C. After 30 min, water was
`added and the reaction mixture was extracted with CH2C12. Work-up by usualmethah
`and gurification by preparatiVe TLC gave 2 (185 mg). mp 229—231°C (MeOH-H 0)
`;
`[QJE
`:
`-55°
`(0 = 0.56, CHC13)
`; UV : 232 nm (19800), 275(1600), 282(1280? ;
`IR : 3400, 1765, 1730, 1720 ;
`1H NMR (CDC13)
`6
`: 1.19 (3H, s, C17H ), 1.21 (3H,
`s, C16H3), 1.85 (3H, 5. C1 H ), 2.12 (3H, s, C18H3), 2.42 (3H, s, gAc), 2.40 (m,
`CIQH ), 2.06 and 2.65 (m, 8682), 4.00 (1H, d, J = 7, C -H), 4.15 and 4.36 (2H,
`2d, 3 = 9, conz), 4.61 and 4.92 (2d, J = 12), 4.77 6.3 4.80 (2d, J = 12) and
`4.88 (s)
`(3 x CH2 of the protecfimg groups), 4.98 (1H, d, J = 9, C -H), 5.69 (1H,
`d, J = 7, C2-H), 5.64 (1H, m, C7-H), 6.03 (1H, t, J = 9, c1 -H), 2.29 (1H, s,
`c o-H), 7.51, 7.63 and 8.10 (2H,
`1H and 2H, 2t and 1d, J =
`, OBz)
`; MS (c1),
`m}z 1067 (MH+), 875. 533. 491. 931. 309. 123-
`z,10-di(2,2,2-Trichloroethyloxxcarbonyl)-1z-methyloxxcarbonyl-10-deacety1
`baccatin III 29.
`
`(2 m1) wiflm
`in methanol
`(20 mg) was added to a solution of 2 (30 mg)
`Zinc dust
`filtered and concen-
`stirring at 60°C. After 1h 30,
`the reaction mixture was
`trated to dryness. Purification by preparative TLC (solvent = CH2C12—Me0H, 98-2)
`gave 10 (23 mg).
`1H NMR (00013)
`6
`: 1.18 (3H, s, C17H3), 1.21 (3H, s, C16H3),
`1.85 73H, 5, C19H3), 2.08 (3H, s, C18H3), 2.36 (3H, 3, GAO), 2.36 (m, 01 H2),
`2.06 and 2.65
`( 2m, c6H2), 3.88 (3H, s, CH 0), 3.96 (1H, d, J = 7, 03—0), 4.15
`and 4.35 (2H, 2d, J = 9, conz), 4.60 and 4. 0 (2H, 2d,
`J = 12), 4.75 and 4.78
`(2H, 2d, J = 12)
`(2 x CH2 of the protectinggroups), 4.98 (1H, d, J = 9, CS-H),
`5.62 (1H, m, C7-H), 5.68 (1H, d, J = 7, 02-H), 5.95 (1H,
`t, J
`9, c13_H), 6.26
`(1H, s, c —H), 7.50, 7.63, 8.08 (2H,
`1H and 2H, 2t and 1d, J
`7, 082)
`; MS (CI)
`m/z 951 (138*). 933. 759. 683, 491. 369. 309. 123.
`1
`-Meth 10x carbon l-10-deacet 1 baccatin III 11.
`in acetic acid (1 ml)
`Zinc dust
`(20 mg) was added to a solution of 12 (23 mg)
`with stirring at 40°C for 2 h. The reaction mixture was filtered, concentratedand
`extracted with ethyl acetate. The
`rganic layer was washed with water, brine and
`dried (MgSOg). Filtration and concentration gave a quantitative yield of 11. mp
`208—21ovc (MeOH-Hzo)
`;
`[“323 = -70°c (c = 0.38, CHC13)
`- UV : 231(16600),‘275
`(1130), 282(955, EtOH)
`; I
`: 3450, 1740, 1720, 1705 ;
`1H NMR (c0c13)
`6
`: 1.08
`(3H, 5’ C17H3)' 1'11 (3“:
`31 C1 H )1 1070 (3H9 57 C 9H3): 1095 (3“! 5! C18H )1
`2.27 (3H, s, OAC), 2.27 (m, c1 02?, 1.94 and 2.46 ( m, C5H2), 3.81 (3H, s, 3CH3),
`3.88 (1H, d, J = 7, c —H), 4.1
`(1H, m, c -H), 4.13 and 4.26 (2H, 2d,
`J = 9,
`c2 Hz), 4.92 (1H, d. 3 = 9, Cs—H), 5.19 (1 , s, c 0-H), 5.56 (1H, d, J = 7, 02—H)
`5.84 (1H,
`t, J = 9, C 3-H)
`7.41, 7.54 and 7.98 (2H,
`1H and 2H, 2t and 1d, J = 7,
`OBz)
`;Ms(c1). m/z 60} (11.14), 585. 509. 387. 345. 327. 309. 123.
`
`NEPTUNE GENERICS EX. 1005 00006
`
`NEPTUNE GENERICS EX. 1005 00006
`
`

`

`Chemical studies of lO—deaoetyl baccatin Ill
`
`4457
`
`Compound 1&-
`in DMF (0.6 ml)
`Ethylene glycol (1.2 ml) was added to a solution of 2 (43 mg)
`with stirring at 80°C for 22 h. The reaction mixture was concentrated and puri-
`ficated by preparative TLC (solvent
`: CH Cl —MeOH, 97.5-2.5) to give 12 (27.5 mg).
`1H NMR (CDC13)
`: 1.19 (3H, s, C17H ), 1.
`1 (3H, s, C16H3), 1.84 (3H, s, C19H3),
`2.08 (3H, s, C18H3), 2.36 (3H, 5, 3Ac), 2.36 (m, Clgflz), 2.04 and 2.62 (m, C6H2),
`3.91 and 4.36
`( 2 x 2H, m, HOCH2CH20), 3.94 (1H, d, J = 7, C3-H), 4.14 and
`4.33 (2H, 2d, J = 9, Czo“2)v 4,60 and 4.90 (2H, 2d, J = 12), 4.77 and 4.79 (2H,
`2d, J = 12)
`(2 x CH2 of the protectkm groups), 4.98 (1H, d, J = 9, C5-H), 5.60
`(1H, m, C7-H), 5.66 (1H, d, J = 7, 02—H), 5.94 (1H, t, J = 9, C13-H), 6.27 (1H,
`s, C o-H), 7.50, 7.62 and 8.08 (2H,
`1H, 2H, 2t and 1d,
`J = 7, 082)
`; MS(CI). m/z
`931 (MH+). 963-
`13-(2-dero§zethyloxxcarbony1)-10-deacetyl baccatin III 12.
`Zinc dust
`(30 mg) was added to a solution of lg in acetic acid (1 m1) at 40°C
`for 4 h, with stirring. Filtration, concentration and purification of the crude
`mix are by preparative TLC (solvent
`: CH2C1 -Me0H, 90-10) gave 12 mg of 22 (68%);
`[a] 3 = —72°
`(c = 0.33, CHCl
`)
`; UV : 231(10200), 275(1620), 282(1360)
`;
`IR :
`3450, 1740, 1720, 1705 ;
`1H RMR (CDC13)
`6
`: 1.14 (3H, s, CI7H3), 1.19 (3H, s,
`01 H3), 1.76 (3H, s, c 9H3), 2.02 (3H, s, 0 8H ), 2.36 (3H, 5, OAc), 2.02 and
`2.62 (2m, C6H2), 2.34 m, C14H2), 3.93 and 4.36 (2 x 2H, m, HOCHg-CHZO), 3.99
`(1H, d, J = 7, C3-H). 4.18 and 4.30 (2H, 2d, J = 9, c 0H2), 4.30 (1H, m, C7-H),
`4.99 (1H, d, J = 9, C5-H), 5.27 (1H, s, Cio—H), 5.67
`1H, d, J = 7, C2—H), 5.93
`(1H,
`t,
`J = 9, c 3-H), 7.50, 7.63 and 8.09 (2H,
`1H and 2H, 2t and 1d, J = 7,
`082)
`; M5031), m}z 633 (mm, 615. 597. 527. 509. 405. 345, 123. 89.
`7,10-di(2,2,2-Trichloroethxloxxcarbonyl)—1Z-acetyl-lo-dgacetxl baccatin III 14.
`A solution of z (65 mg)
`in toluene was stirred at 80°C. Acetyl chloride
`(0.055 ml) and silver cyanide (42 mg) were added to the reaction mixture. After
`3 h, water was added and the reaction was extracted with CH2C12. Usual work-up
`and purification by preparative TLC (solvent
`: CHZCl
`-MeOH) gave 14 (46 mg)
`;
`Em]D = -48°
`(c = 0.35, CHC13)
`- UV : 232(16200), 275(1320), 282(1210)
`;
`IR :
`3450, 1765, 1730, 1715 cm-1 ;
`iH NMR (00013)
`0
`: 1.17 (3H, s, 01 H3), 1.24 (3H,
`s, C16H ), 1.84 (3H, s, 019H3), 2.03 (3H, s, c 8H ), 2.21 and 2. 6 (2 x 3H, 2s,
`2 x 0Acg, 3.94 (1H, d, J = 7, C3-H), 4.16 and 4.33 (2H, 2d. J = 9, czonz), 4.61
`and 4.91 (2H, 2d, J = 12), 4.79 (2H, s)
`(2 x CH2 of the protecfinggroups)
`; 4.99
`(1H, d,
`J = 9, c -H), 5.59 (1H, m, c -H), 5.70 (1H, d,
`J = 7, Cz-H), 6.21 (1H,
`t, J = 9, C1 -H)? 6.27 (1H, s, C10-H , 7.50, 7.62 and 8.08 (2H,
`1H and 2H, 2t
`and 1d,
`J =
`, OBz)
`: 115(01). m/z 935 (MW). 743. 683. 623. 561. 369. 309. 291.
`123.
`
`12:Acetyl-10—deacetyl baccatin Ill 12-
`in acetic acid (1.5
`Zinc dust
`(20 mg) was added to a solution of 14 (46 mg)
`ml) at 40°C with stirring. After 2h 30,
`the reaction was filtered and extracted
`with ethyl acetate. Purification by preparative TLC (solvent
`: CH Clz-MeOH,
`95-5) gaVe 20 mg of 35.
`[0123 = -69°
`(c = 0.67, CHCl
`)
`; UV : 232(16100), 276
`(1290), 282(1200)
`;
`IR :
`3 90
`1730, 1715
`;
`1H NMR (CDCl
`)
`a
`: 1.11 (3H, 3,
`01 H3), 1.21 (3H, s, C16H3), 1.74 (3H, 5, c1 H3), 1.94 (3 , s, 018H3), 1.86 and
`2.59 (2m, C6H ), 2.23 and 2.32 (2 x 3H, 25, g x OAc), 2.23 (m, c 4H2), 3.95 (1H,
`d, J = 7, ca—fi), 4.18 and 4.32 (2H, 2d, J = 9, C20H2), 4.26 (m, C7-H), 4.98 (1H,
`d, J = 9, c -H), 5.22 (1H, s, 01 -H), 5.67 (1H, d,
`J = 7, C2-H), 6.16 (1H,
`t,
`J = 9, c 3-3), 7.48. 7.60 and 8.87 (2H,
`1H and 2H, 2t and 1d, J = 7, 082)
`; MS
`(CI ). m}z : 587 (MH+). 569. 551. 527, 509. 405. 387. 345. 327. 123-
`7,10-di(2,2,2-Trichloroethyloxycarbonyl)-12 cinnamoyl-lO deacetxl baccatin IIIlé.
`Oxalyl chloride (0.057 ml) was added to a solution of cinnamic acid (50 mg)
`in
`dry toluene (1 ml) with stirring at 60°C for 30 min. After distillation of
`oxalyl chloride,
`2 m1 of dry toluene, compound Z (60 mg) and silver cyanide
`(40 mg)were added and the reaction mixture was heated at 110°C for 20h. After
`filtration, extraction with ethyl acetate and usual work—up,
`the product lg (42
`mg) was isolated by preparative TLC (solvent
`: CH2C12-Me0H, 99.5-0.5). 16 :
`EaJZB = -56°
`(c = 0.57. CHCl
`)
`; UV : 217(26800), 222(26900), 232(161ooTT 276
`24300)
`;
`IR : 3420, 1760,
`1 25, 1710, 1635 ;
`1H NMR (cnc13)
`0
`z 1.29 (3H, 5,
`C17H ). 1.29 (3H, s, C16H3), 1.88 (3H, s, 019H3), 2.16 (3H, s, C18H3), 2.31 (3H,
`020). 3.99 (1H. d.
`J = 7. C -H), 4.20 and 4.34 (2H, 2d, J = 9, C20H2), 4.62
`and 4.93 (2H, 2d,
`J = 12), 4.7
`(2H, s)
`(2 x CH of the protecthg groups). 5.02
`(1H, d, J = 9, c5-H), 5.62 (1H, m, C7-H), 5.73
`1H, d, J = 7, Cz-H), 6.21 (1H,
`t,
`J = 8, C13—H), 6.30 (1H, s, c
`-H),
`6.53 and 7.89 (2H, 2d, J = 16, C2.-H and
`C3.—H)
`;
`7.45 and 7.60 (5H, 29)
`; 7.45. 7.60 and 8.07 (SH, 082)
`; us (c.I). m/z
`1 23 (MH+). 1005. 831. 813. 683. 665. 491. 431. 369. 309. 291. 149. 131. 123-
`
`13-Cinnamoxl-1O deacetyl baccatin III 12.
`in acetic acid (1.5m1)at
`Zinc dust
`(15 mg) was added to a solution of lg (30mg)
`40°C for 2 h. Filtration, concentration, extraction with ethyl acetate and puri-
`fication by preparativg TLC (solvent
`: CH Cl
`-Me0H, 93-7) gave 14 mg of 17. mp =
`229-231°c (neon)
`:
`[@103 = .104° (C = 0.4%, 3Hc1 )
`; UV : 217(26200), 221(26100),
`232(16400), 276(24600)
`;
`IR : 3440, 1725, 1710,
`;635 ;
`1H NMR (CDC13)
`6
`: 1.15
`(s, 3H, 017H3), 1.25 (3H, s,
`€16H3),
`1.78 (3H, s, C19H3), 2.06 (3H, s, C18H3),
`
`NEPTUNE GENERICS EX. 1005 00007
`
`NEPTUNE GENERICS EX. 1005 00007
`
`

`

`4458 F. GU&W~TE-VO~GELEM et al.
`
`2.28
`
`(3H,
`
`s,
`
`OAc), 2d, J = 9, C2OR2), 4.00 (lH, d, J = 7, C3-H), 4.31 (m, C7-II), 4.20 and 4.31 (2H, 4.99 (lH, d, J = 9, C5-H), 5.28 (lH, 6.51 and 7.85 (:i,I,Cd8;H,!,=5;81) ilR:Hd, 7.45, 7.58 and 8.07 (5H, OBz) ; ii8 , 527, 509, 491, 405, 387, 345, 327, 149, 131, 123. Compound 18. - Oemium tetroxide (54 mg) waa added to a solution of 16 (140 mg) in pyridine (3.6 ml) at room temperature with stirring. After lh, szium bisulfite (245 mg) in water (3.6 ml) was added. The reaction mixture was left at room temperature for 2h and extracted with ethyl acetate, Usual work-up and purification by pre- parative TLC (solvent : side chain), 13-(2,,3,-dihydroxy-3, phenyl propanoyl)-lo-deacetyl baccatin III 19a and -_-_. E (2'R, 3'S and 2'S, 3'R) : Zinc dust (20 mg) was added to a solution of 18 (51 mg) in acetic acid (2 ml) at 4O'C for 5 h. After filtration and concentratT;n, the reaction mixture was extracted with ethyl acetate. Purification by preparative TLC (solvent : CH2Clp- 1.12 (3H, s, (MH+). 13-Hydroxy acetyl-lo-deacetyl baccatin III 21. -_ Lead tetraacetate (230 mg) was added to a solution of 18 (360 mg) in acetic acid (5 ml) with stirring at room temperature for 30 min. Usual work-up and purification by preparative TLC (solvent : CH2C12-MeOH, 95-5) gave 20 (309 mg). Compound 20 (30 mg) was treated with zinc dust (15 mg) in acetic acid (1 ml) at 600~ for i-h. Filtration, usual work-up and purification by preparative TLC #‘;e”t_& fc = CH C12-MeOH, 90-10) yielded 21 (20 mg) : mp = 188-19O'C (MeOH) i CD BD)
`
`0.4, MeOH) ; IR : 3560, 3450, 1730, 1715 i H RMR (CDC13-
`
`S,
`:
`b
`C17H3), 1.19 (3H, s, C16H3), 1.72 (3H, s, Cl9H3), 1.92 (32,
`S,
`
`Cl8H3), 2.23 (2H, Cl4H2), 2.29 (3R, a, OAc), 2i51~l~~dC~:~~,(~;1882~lH~ 2, C -H) 4.18-4.28 (5H, ij,=C;;H2], 4:18 (lH, m, C2 H2 C -H and C2,H2), C -H) H7, 8.24'(2i; d J = 7.5, C2:H2) C5-H), 5.21 (lH, s, Clo- 5:63 (lH, d: J'= 7, 4.95 (IH, d, J'= 9 C2-H) 6.23'(lH, t J = 9, Cl -;I) 7.45, 7.58 and 8.01 (5H, 0~~) ; MS (C.I), m/z 603 (MH'), 585, 567,'527, 509, a 05, 387, 345, 327, 123, 105, 77. som.ound 3. Lead tetraacetate (28 mg) was added to a solution of +P. (a + b) (30 mg) in acetic acid (1 ml) with stirring at room temperature for 30 min. USuaI work-up and purification by preparative TLC (solvent : CH2CI2-MeOH, 90-10) gave 22 (19 mg). Refluxing a mixture of 22 (9 mg) and hydroxylamine hydrochloride (4 mg) in pyridine (1 ml) for 20 min. zelded 3 in quantitative yield after usual work-up; (3H, [a]$3 = -51" (c = 0,14, MeOH) ; 'H RMR (CDCl -CD OD) 6 : 1.13 s, C17H3), 1.21 (3H, s, C16H3), 1.76 (3H, e, Cl9H3), a.oa (3& 2.31 (3~,
`
`OAC),
`
`S,
`
`3.93 (lH, d, J = 7, C3-H), 4.26 (lH, m, C -H), s, C18H3), 3 4.28 (2H, d, J = 9, C2OH2)' 5.69 (lH, d, J = 7, C2-H), 5.01 (lH, d, J = 9, C5-H), 5.29 (Ui, 8, C O-H 6.28 (1H. t, J = 9, Cl -H), 7.66 (lH, 8, C2,-H (5H, OBz)
`(FAB), m/z 616 (M&), 598, 580, 527, 509. f, 7.52, 7.64 and 8.11 7-(2,2,2_Trichloroethyloxy carbonyl)-13-crotonoyl baccatin III 22. To a solution of 8 (200 mg) in dry toluene (8 ml) was added silver cyanide (455 mg) and crotonyi chloride (0.76 ml). The reaction mixture was stirred at llO°C for 24 h. Filtration, extraction with CH2C12 and concentration gave a
`
`; MS
`
`NEPTUNE GENERICS EX. 1005 00008
`
`1.12 (3H,
`

`

`Chemical studies of Io-dcaoetyl baccatin Ill
`
`4459
`
`to
`: CH2C12-Me0H. 99—1)
`mixture which was purified by preparative TLC (solvent
`give 24 (120 mg).
`1n m (c001 )
`e
`: 1.17 (3H, s, c1711),
`1. 22 (3H, 3. c1 H ),
`1.88 73H, 5, c H3), 1.98 (3H,
`d, J = 7 et J = 2, cg.-&3), 2.04 (3H, s, c1883?,
`2.18 and 2.30 (3 x 3H 2s, 2 OAC), 4.00 (1H, d, J = 7, Ca-H), 9.19 and 9.33
`(2H, 2d, J = 9, C20H2), 4.99 (1H, d,
`J = 9, 05—H), 4.65 and 5.41 (2H, 2d, J = 12
`CH2 of the protecting group). 5.61 (1H, m, C7-H). 5.69 (1H, d, J = 7, Cz-H),
`5.96 (1H, dd, J = 15 and J = 2, C2.-H), 6.13 (1H, t, J = 9, 013—H), 6.40 (1H, s,
`Cio-H), 7.18 (1H, dd, J = 15 and J = 7, C3.-H), 7.48, 7.60 and 8.06 (SH, 082).
`l3-Crotonoxl baccatin III 22.
`in acetic acid with
`Zinc dust
`(50 mg) was added to a solution of 32 (50 mg)
`stirring at 40°C for 3 h. Filtration, concentration of the solution and puri£§-
`cation by preparative TLC (solvent
`: CH C1 -Me0H, 97-3) gave 22 (25 mg).
`a D
`— 62°
`(c = 0.56, EtOH)
`; UV : 226(20200§, 374(1750), 282(1510, EtOH)
`;
`IR :
`3500, 1730, 1650 ;
`1H NMR (CDC13)
`6
`: 1.13 (3H, s, c17H3), 1.25 (3H, s, C16H ),
`1.68 (3H, s, c19n3), 1.95 (3H, s, 01 H3), 1.97 (3H, dd, J = 7 and J = 2, cqva ),
`2.25 and 2.28
`(2 x 3H, 25,
`2 0Ac§. 3.83 (1H, d,
`J = 7, C3-H), 4.18 and 4.
`1
`(2H, 2d, J = 9, C20H2), 4.45 (1H, m, C7-H), 5.00 (1H, d,
`J = 9, 05-H), 5.68 (1H,
`d, J = 7, C2-H), 5.95 (1H, dd, J = 15 and J = 2, 02.—H), 6.15 (1H, t, J = 9,
`c13—H), 6.33 (1H, 5. c1 -H), 7.16 (1H, dd, J = 15 and J = 7, c3.-H), 7.50, 7.61
`and 8-07 (SH. 082). MS ?FAB) m/z 655 (MH+), 637. 595. 577, 509. 491, 449. 349.
`327. 309-
`
`=
`
`Compound fig (2'5, 3'R and 2'R, 3's).
`in pyridine
`Osmium tetroxide (47 mg) was added to a solution of 22 (100 mg)
`(3 m1) at room temperature with stirring for 1 h. Sodium bisulfite in water was
`added. Extraction with ethyl acetate and purification by preparative TLC (solvent
`Hexane—EtOAc, 40-60) gave 2Q (43 mg, mixture of diastereoisomers).
`IR : 3500,
`1765, 1730 ;
`1H NMR (cnc137 6
`: 1.18 (s. C17H ), 1.26 (s, 01 H ), 1.40 and 1.36
`(2d, J = 6, C4.H3), 1.85
`(s, C19H
`, 2.06 and 2.01 (25, c1883?, 2.17 (s, 0A0),
`2.30 (s. cm). 3.98 (d, J = 7. c343), 4.12 (m, C2.-H), 4.33 and 4.18 (a,
`J = 9,
`C20“ ), 4.65 (s, CH2 of the protected group), 4.96 (d, J = 9, C —H), 5.58 (m,
`c -H§, 5.68 (d, J = 7, Cg-H), 6.33 and 6.23 (t, J = 9, C13-H), 0.50 (s, c 0-H),
`8.06, 7.65 and 7.51 (082)
`; MS(I.C) m/z : 863 (MH+), 803, 785, 743, 683,
`21,
`123, 121 (CH3CH0H-CHOH-C00H + H+).
`Compound 31 (2'5, 3'R and 2'R, 3'5).
`Zinc dust
`(20 mg) was added to a solution of 36 in acetic acid (2 m1) at 90°C
`for 2 h. Filtration, concentration, extraction with ethyl acetate and purifica-
`tion by preparative TLC (solvent
`: CH2C12-MeOH, 97-3) gave 10 mg of £1. UV(Et0H)
`231(13360), 276(1410), 283(1420).
`IR :
`3300, 3500, 1700.
`1H NMR (CDCl
`)
`6
`:
`1.18 (5, c1 H3), 1.25 (s, C16H3), 1.41 and 1.36 (2d, J = 6, CQ.H3), 1.68 (s,
`C19H ), 1_9§ and 1.93 (25, C18 H3), 2.23 (s, OAc), 2.30 (5, OAc), 3.83 (d, J = 7,
`c —H , 4.08 (m, Cz.-H), 4.30 and
`4.16 (d, J = 9, c2 H2), 4.47 (m, C7-H), 4.95
`(3, J = 9, c -H), 5.66 (d. J = 7, Cz-H), 6.33 and 6.26 (2t
`J = 9, c, -H), 6.32
`(s, Clo-H), 3.05, 7.61, 7.48 (082). ms
`(FAB) m/z : 689 (uni), 671, 62 , 611, 569,
`551, 527, 509, 449, 121 (CH3—CH0H-CHOH—COOH + H+).
`
`Acknowledgements :
`
`This work has been supported by the "Association pour la Recherche sur 1e
`Cancer". We
`thank Mrs. Khuong for generous gift of B-phenyl isoserine (erythro
`and three).
`
`REFERENCES
`
`
`
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`NEPTUNE GENERICS EX. 1005 00009
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`NEPTUNE GENERICS EX. 1005 00009
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`

`

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`
`11.lR.McLaughlin, R.W. Miller, R.G. Powell,
`312 (1981).
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`13. D.G.I. Kingston. private communication.
`
`14. V. Sénilh, F. Guéritte, D. Guénard. M. Colin, P. Potier, c. R. Acad. Sc..
`222, série II, 1039 (1984)-
`’I‘.B. Windholz, 0.13.12. Johnston, Tetrahedron Lett., 2555 (1967).
`II
`II
`
`(LR. Smith, J. Nat. Prod., 22,
`
`15.
`
`in the Alkaloids (Manske). 12. 597 (1968).
`16. B. Lythgoe,
`17. J.W. Harrison. R.M. Scrowston, B. Lythgoe, J. Chem. Soc., 1966, 1933 (1966).
`18. E. Herranz, S.A. Biller. K.B. Sharpless. J. Amer. Chem. Soc., 100, 3596
`(1978) -
`
`NEPTUNE GENERICS EX. 1005 00010
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`NEPTUNE GENERICS EX. 1005 00010
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`