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PACLITAXEL ANALOGUES FROM TAXUS BACCATA

BRUNOGARETTA,* PAOLOORSINI,FEDERICOPETERLONGO and GIOVANNIAPPENDINO~

Indena S.p.A.. Via Don Minzoni 6. I-20090 Settala (Milano), Italy; i- Dipartimento di Scienra e Tecnologia del Farmaco. Via Giuria 9. I-10125 Torino, Italy

(Rrceiwd in rvr~i.wd,fbrm I I July 1997)

Key Word Index-Tu.rus haccntcr; Taxaceae; N-debenzoyl-N-butanoyl-IO-deacetylpaclitaxel; N-debenzoyl-N-propanoyl-IO-deacetylpaclitaxel.

Abstract-N-Debenzoyl-N-butanoyl- and N-debenzoyl-N-propanoyl-IO-deacetylpaclitaxel were identified as minor constituents of T. haccutn by HPLC-MS. The structure of these compounds was confirmed by com- parison with synthetic samples prepared from 7-triethylsilylbaccatin III. J; 1998 Elsevier Science Ltd. All rights reserved

INTRODUCTION

Over the past few years, the needles of the yew tree (Turus spp. vv.) have become an important com- modity for the pharmaceutical industry, serving as a renewable source of IO-deacetylbaccatin III (1) [I]. Thanks to improvements in synthetic methodologies [2]. commercially viable methods for the conversion of 1 to the anticancer drug paclitaxel ( =Taxol”) (2a) have been developed [2]. As a result. semisynthetic paclitaxel obtained from 1 was approved for clinical use in December 1994 [3], effectively solving the eco- logical issues raised by its isolation from the bark of the Pacific yew (T. hrec#iA Nutt.), a non renewable source [4]. Compound 1 has also been extensively used in studies aimed at unravelling the structure-activity relationship within antitumor taxoids [5], serving as a starting material for the synthesis of first generation [6] and second generation [7] chemical analogues of paclitaxel.

IO-Deacetylbaccatin III can be isolated from yew more easi!y than paclitaxel, owing to a higher con- centration and a very poor solubility in a variety of organic solvents. As part of an investigation aimed at RI R2 the identification of minor paclitaxel analogues in yew tissues, we examined the mother liquors of a batch of 1 prepared from T. haccutu L.

RESL'LTSANDDlSCtiSSlON

In previous studies, the coupling of high per- formance liquid chromatography with mass spec- trometry using a thermospray (TSP) interface proved

2a 2b 2c 2d 2e

AC 1-i H AC AC

Ph n-Pr Et iz-Pr Et

*Author to whom correspondence should be addressed.

very useful for the on-line analysis of taxanes in crude plant extracts [8]. Paclitaxel and its analogues give positive thermospray mass spectra showing, beside the protonated molecule ion MH+, an extensive and characteristic fragmentation pattern [8]. The main

1325

1326 B. GABI,TTA ~‘7 trl

- (4a, R= n-Pr: 4b, R=Et)

* DMA!?, Toluene, h

3

HC1, MeOH

cl

NH2-YH:

* 2d (2e) (overall 40-50% from3)

Scheme I, Synthesis of 2b and 2c from 7.Tesbaccatin III (3)

ions correspond to the phenylisoserine chain, ([ScH + HI+, base peak), and to the terpcnoid moiety (ions [MH-ScH]+ and [MH~ScH~PhCOOH] ). The presence of these fragments allows a straightfol-Mard identification of the terpenoid core and the N-acyl- ation pattern of the side chain. The HPLC TSP-MS analysis of the mother liquors of a methanol crys- tallization of 1 revealed two minor constituents 2b (< 1X, [MH + at fl?/s 7781) and 2c ([MH + at nz/: 7641). The presence of an ion at r?,/: 527 in both compounds showed that they were esters of IO-deacetylbaccatin III, whereas the ions at nz,l= 252 and 777:: 238 (100% relative intensity) suggested the presence of side chains where the N-benzoyl moiety of paclitaxel was replaced by a N-butanoyl and a N-propanoyl residue. respec- tively. Mass spectra thus suggested that 2b was N- debenzoyl-N-butanoyl-IO-deacetylpachtaxel and 2c was N-debenzoyl-N-propanoyl-IO-deacetylpaclitaxel. The very low concentration of these metabohtes pre- cluded their isolation and thus an examination of their stereochemical features, which cannot be deduced by mass spectral analysis alone. Therefore, compounds corresponding to these structures were synthesized, in order to compare their chromatographic and mass spectral properties with those of the natural products. The synthesis of 2b and 2c starts from 7-tri- ethylsilylbaccatin 111 (3) [9] (Scheme 1). The baccatin 111 derivative 3 was preferred over diprotected IO- deacetylbaccatin III derivatives (7,10-bistriethylsilyI- 7, IO-bistrichloroethoxycarbonyl-derivatives [5]) since also the corresponding compounds of the pachtaxel series (IO-acetyl) would have been available in this way. The oxazolidine carboxylic acids 4a and 4b were used as compact synthons of the phenylisoserine side chains of 2b and 2c, respectively, and were prepared as shown in Scheme 2. Both syntheses proceeded uneventfully, giving the paclitaxel analogues 2d and 2e, which were then deacetylated with hydrarine in ethanol [IO]. The synthetic products showed HPLC

profiles and mass spectra identical to those of the natural products, confirming that 2b and 2c were indeed :V-debenzoyl-N-butanoyl and N-debenzoyl-N- propanoyl-lo-deacetylpaclitaxel, respectively. The proton and carbon resonances of 2b and 2c are reported in Tables I and 2. An examination ofextracts from various yews, showed that 2b and 2c are present as trace constituents (<O.OOOl%) in the European and the Himalayan yews (T. haccuta L. and T. real- lichirmtr Zucc.. respectively).

The identification of minor compounds structurally related to paclitaxel is interesting in the context of the biogenesis of these products. Indeed, the isolation of 2c. with a N-propanoyl group, makes it unlikely that compounds like taxol C [ 111 and taxol D [ 121, having a ,Y-hexanoyl and a N-butanoyl group, are syn- thesized by homologation of an hitherto unreported N-acetyl starter, since only acyl residues with an even number of carbon atoms would be expected. It is thus more likely that the various N-alkanoyl residues of the natural taxols are introduced as such, in what is considered the final step of their biosynthesis [ 131.

EXPERIMENTAL

Liy14itl chromcltogruphy thermosprq~ tnuss spec- tron7efr~~~. The HPLC-MS system used included a Wat- ers 600-MS pump/system controller and Waters 486 tunable UV!Vis detector. Injections were performed with a Waters 717 plus autosampler. A Zorbax SB- CN column (250 x 4.6 mm I.D., 5 pm) was used. The mobile phase composition was eluent A (H,O-MeOH 8:2), eluent B (MeCN-MeOH 8:2), in gradient conditions (eluent A from 99% to 35% in 65 min). The flow rate was 1 ml min-’ and the UV detector was set at 227 nm. The liquid chro- matography system was connected to a Finnigan- MAT TSQ triple quadrupole mass spectrometer equipped with a TSP-2 thermospray interface and a

Paclitaxel analogues from Tu.w.s hacw~cr 1327

RCO,

MeOH-HI0

R 4a n-Pr 4b Et

Scheme 2. Synthesis of the side chain synthons 4a (R = C,H,) and 4b (R = C2Hi). PPTS = pyridinium p-toluenesulphonate.

Table I. ‘H NMR spectral data of 2b and 2c (200 MHz. CDCI,. TMS as reference, J in Hz)

H 2b

2 3 5 br

6P 7 IO 13 14a.b I6 I7 I8 I9 2Oa 20b 2’ 3’

NH-COCHZR NH-COCH,CH2R NH-COCHICH2CH, 4-OAc 3’NH BZ

Ph

5.67 d(7.0) 3.86 d(7.0) 4.91 &I (9.6, I .9) 2.53 &W(l4.8.8.9,5.5) I .83 1n 4.22 m 5.21 J 6.16 &(8.9.9.0) 2.26 m I.11 J 1.22 s I .80 hr s 1.73 s 4.29 d (8.6) 4.19 d(8.6) 4.67 dd(4.5,2.7) 5.56 dd(9.0.2.7)

2.16 ((7.7) 1.59 I?! 1.20 f(7.5) 2.32 s 6.44 d(9.0) 8.14 d(8.0) 7.62 t (8.0) 7.52 r(8.0) 7.34-7.56 m

2c

5.68 d(7.2) 3.89 d(7.2) 4.92 dd (9.5.2. I) 2.56 ddd(14.5. 11.5.4.9) I .80 n, 4.22 m 5.19 .Y 6. I8 dd(8.9.9.0) 2.28 n, I.12 s I .23 s I.81 hrs 1.75 s 4.22 ,u

4.76 dd(4.5.2.5) 5.56 dd(9.1.2.5) 2.23 q(7.7)

I.10 ((7.6)

2.34 s 6.36 d(9.1) 8.10 d(8.0) 7.62 t(8.0) 7.52 t (8.0) 7.34-7.56 m

5100 DEC Station with ICIS data system. Mass spec- repeller voltage 30 V, discharge 1800 V, filament off- trometer conditions were optimized in order to ach- mode. The electron multiplier and dynode voltages ieve maximum sensitivity. Typical values were as fol- were set to 2000 V and 15 kV, respectively. Pre- lows: source block temp. 230 C, vaporizer temp. 70 C. amplifier sensitivity was IO -’ A,%‘. Positive ther-

1328 B. C;ABETTA cf cd

Table 2. “C NMR spectral data of 2b and 2c (SO MHz. CDCl,. TMS as reference)

C

4 5

8 9

IO 11 12 13 14 15 16 17 18 19 20

2’

3’-Ph

4-OAc

NH-COR NH-COCH?R NH-COCH2CH2R NH-COCH$ZH>C’H, BZ

2b

78.2s 74.1 d 46.0 d 80.6 s 83.7 d 35.3 t 71.5 d 51.2 s

210.9 s 74.4 d

135.6 s 137.X s 72.7 tl 36.4 r 42.6 s 20.2 q 26.0 q 13.8 q 9.3 q

76.1 i 172.5 .\ 71.9 d 54.0 d

137.7 s 126.5 ri 128.5 d 127.7 d 169.9 s 22.0 q

172.5 s 38.0 t 18.6 I 13.2 q

166.5 s 128.8 s 129.7 d 128.3 d 133.3 (I

2c

78.2 .< 74.1 d 45.9 (I 80.6 ., 83.7 tl 35.3 I 71.5 tl 57.2 .!

211.0 \ 74.3 il

135.7 .Y 137.X .\

72.7 d 36.5 / 47.6 .\ 20.2 r, 26.0 c, 13.x q 9.3 q

76.2 I 172.3 \ 7 I .9 (I 54.0 il

137.6 5 126.5 tl 12x.5 tl 127.x rl 169.9 .\ 22.0 q

173.2 r 29.1 I

9.3 q

166.5 .Y 12x.x .c 129.7 d 12x.3 d 133.3 II

mospray mass spectra from nz/~ 200 to 1000 (scan time I .5 set) were obtained scanning the Q3 analy;ler and acquired in centroid mode.

N-Drh~~n~o~l-N-hutun(~~~l- IO-~~u~et~lpuc.litcl.~-rl (2b). Mp 244 (dec.); IR \I,“,,“: cm-‘: 3428 (OH), 1724 (ester C=O), 1654 (amide C=O). Cl-MS 140 eV. 177;:

(rel. int.): 795 [C&H,,NO,,+NH,]+[M + NH,]’ (50), 562 [MNH&cHpH>O)]+ (55), 544 [MNH,pScH]+ (15), 269 [ScHNH,]+ (35), 105 [PhCO]+ (100). HPLC relative retention time (RR,): 1.70 (compared to IO- deacetylbaccatin III).

N- Dc,hm;(l~l-N-propatl~~l- 1 O-~~~r~et.~,lpcrc,litcl.rc’l (2~). Mp 245 (dec.). IR $I cm-‘: 3407 (OH), 1725 (ester C==O), 1655 (amide C=O). CI-MS 140 eV, u,‘: (rel. int.): 781 [C,,H,,NO,,+NH,]+[M+NH,]+ (35). 544 [MNH,-ScH]+ (15), 255 [ScHNH,]’ (100). HPLC relative retention time (RR,): 1.58 (compared to IO- deacetylbaccatin 111).

S?sn the.5 i.s of’ 2b. To a solution of 7-tri- ethylsilylbaccatin III 3 [9] (650 mg, 0.99 mmol) in toluene (60 ml), (4S,5R) N-(butanoyl)-2-(2,4-dime- thoxyphenyl)-4-phenyl-5-oxazolidine-carboxylic acid (4a) (804 mg. 2.0 mmol, 2 mol. equiv.; prep [ 151 from (4S.5R) h’-(r-butoxycarbonyl)-2,2-dimethyl-4-phenyl- 5-oxazolidine-carboxylic acid [ 141, see Scheme 2), DCC (I .5 g, 7.3 mmol, 7.4 mol. equiv.) and 4-dime- thyla~mminopyridine (250 mg, 2.0 mmol, 2.0 mol. equiv.) were added. The mixture was heated at 70 ‘C for 3 hr and then worked up by filtration and evapn. To remove the protecting groups, the residue was dissolved in a soln of dry HCI in MeOH (prepared by dissolving AcCl (350 ,~l) in 50 ml of MeOH). After stirring 2 hr at room temp., the soln was evapd. The residue was taken up in CH$Zl, and washed with sat. NaHCO,. After drying (Na$O,) and evapn, crude 2d was obtained. The latter was dissolved in EtOH (abs., 2.5 ml) and treated with 2.6 ml of a soln prepared dissolving 1 ml 85% aq. hydrazine in IO ml abs. EtOH. After stirring 2 hr at room temp., the reaction was worked up by dilution with dil. HCI and extracted with EtOAc. The residue was purified by CC (CH,CIZ-MeOH 98:2) to give 340 mg of 2b (44% from 7-triethylsilylbaccatin III).

~~wthc.vis of’2c. The same procedure used for the synthesis of 2b was employed. but (4S,5R) N-(pro- panoyl)-2-(2,4-dimethoxyphenyl)-4-phenyl-5-oxazo- lidine-carboxylic acid 4b was used for the coupling step.

I. 2.

6.

7.

8.

9.

10.

Il.

REFERENCES

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Paclitaxel analogues from Trr.ms baccura 1329

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