Practical Semisynthesis and Antimitotic Activity of Docetaxel and Side

Chapter 17

Practical Semisynthesis and Antimitotic Activity of Docetaxel and Side-Chain Analogues

Downloaded by UCSF LIB CKM RSCS MGMT on August 24, 2014 | http://pubs.acs.org Publication Date: December 7, 1994 | doi: 10.1021/bk-1995-0583.ch017

A. Commerçon, J. D. Bourzat, E. Didier, and François Lavelle Rhône-Poulenc Rorer, Centre de Recherches de Vitry Alfortville, 13 Quai Jules Guesde, 94403 Vitry sur Seine, France

Docetaxel (Taxotere®) and a variety of semisynthetic side-chain analogs have been prepared and evaluated for their potency in inhibiting microtubules disassembly and for their antitumor activity in in vitro and in vivo experimental models. Their partial syntheses were achieved using stereoselective approaches. Different protection/deprotection strategies have been investigated. Structure-activity relationship studies demonstrate that biological activity is very dependent on the position and nature of substituents on the aromatic ring of 3'-modified-phenyl analogs. New carbamates have also been synthesized. Yettert-butoxycarbonylremains the substituent of choice for the 3'-nitrogen atom. Among all the new taxoids reported here, 3'-para-fluoro-docetaxel was identified as one of the most powerful analogs of docetaxel.

The structure of the natural antitumor agent paclitaxel (Taxol®, 1) wasfirstestablished by Wani, Wall et al. in 1971 (7). This diterpene, extractedfromthe bark of the Western Yew, Taxus Brevifolia Nutt (Taxaceae) (2), has proved highly cytotoxic against a wide number of cancer cell lines in in vitro and in vivo experimental models (5). In the early 1980's, as the supply of paclitaxel for clinical evaluation was becoming scarce, Potier's group, i.e. Guéritte-Voegelein, Guénard et al., at Gif-sur-Yvette started the partial synthesis of new taxane diterpenoids (taxoids) from 10-deacetyl-baccatin III 2, an abundant constituent of the needles of the European yew species Taxus baccata L. (4). Using conveniently O-protected derivatives of baccatin III and 10-deacetylbaccatin III (such as 3) as key precursors (5), their semisynthetic work led to the discovery of the new and biologically potent taxoid docetaxel (Taxotere®, 4) (6). The renewable source of 10-deacetyl-baccatin III 2 makes docetaxel easily available whereas the paclitaxel used in clinical trials has been harvested so farfromthe bark of yew trees, a process that unfortunately is fatal to the tree. The unprecedented mechanism of action of docetaxel and paclitaxel has been extensively studied by Horwitz et al. (7) and Andreu et al. (8) who observed that both 0097-6156/95/0583-0233$08.00/0 © 1995 American Chemical Society

In Taxane Anticancer Agents; Georg, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


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compounds act by promoting tubulin assembly into stable microtubules. Clinical trials of both taxoids are currently underway utilizing Cremophor EL-ethanol for paclitaxel and polysorbate for docetaxel as formulation solvents. Paclitaxel and docetaxel clinical activities have been reported against many types of tumors such as advanced breast, ovarian and non-small cell lung cancers (3,9). Paclitaxel has received FDA registration approval for the treatment of metastatic ovarian cancer and breast cancer after failure of first line therapy. A wide number of analogs of docetaxel and paclitaxel have already been prepared by different pharmaceutical and academic groups (JO). Furthermore, two total syntheses of paclitaxel by Nicolaou et al. (11) and by Holton et al. (12) have been very recently reported. These major achievements from these two groups not only illustrate the present know-how of organic chemists in synthesizing complex structures but also may open the way to new and thus far inaccessible analogs. Structure-activity relationships The structural differences between paclitaxel and docetaxel are a tert-butoxycarbonyl (Boc) group instead of a benzoyl group on the nitrogen atom at C-3' on the side chain and an hydroxyl function instead of an acetate at the 10-position of the diterpene moiety (figure 1). These structural modifications lead to an increase of cytotoxicity in certain experimental models (73). These results suggested the possibility of further improvement by introducing, for instance, new side-chain modifications. Structure-activity relationships of taxoids have already been reviewed (10) and our present knowledge in this area can be outlined as depicted in figure 2. The C-13 phenylisoserine side-chain and the diterpene moiety of paclitaxel are both crucial for biological activity. Thus baccatin III and its derivatives without the phenylisoserine side-chain at C-13 are neither active in the tubulin assay nor cytotoxic (14). Studies on the diterpene moiety showed that the oxetane ring is essential for biological activity (75). Structural and molecular modelling studies show that this 4-membered ring is involved in a conformational lock of the diterpene skeleton and the C-13 sidechain through a pseudo chair conformation of ring C (6, 16). Paclitaxel and docetaxel rearrangement products possessing contracted (77) or cleaved (18) Α-rings are significantly less bioactive products. B-ring contracted analogs have been reported as maintaining antitumor activity (79). A wide number of modifications can be introduced at the 7-position without significant loss of activity as noted with epimerization and acylation (acetyl, glutaryl, phenylalanyl, alanyl, Ν,Ν-dimethyl-glycyl, etc.) products Figure 1. Structures of docetaxel, paclitaxel and baccatin ΙΠ derivatives.

1,4

OCOPh

1, Ri=PhCO, R2=Ac (paclitaxel) 4, Ri=tBuOCO, R =H (docetaxel) 2

2,3

OCOPh

2, R =H, R4=H (10-deacetyl-baccatin III) 3, R =R4=Troc (Cl CCH OCO) 3

3

3

2


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(20). All of these C-7-modified derivatives showed activity comparable to paclitaxel in the microtubule disassembly assay but have generally slightly reduced cytotoxicity. Oxidation at C-7 is known to reduce bioactivity (21). Interestingly enough, 7-deoxypaclitaxel exhibited in vitro activity similar to paclitaxel while 7,10-dideoxy-paclitaxel proved to be slightly less cytotoxic (22, 23). Figure 2. Modifications influencing the cytotoxicity of paclitaxel.


flexible: Boc > PhCO

flexible:

OH, H

crucial: removal => 120-fold loss

As reported by Klein with 9a-hydroxy-paclitaxel (24) and researchers at RhônePoulenc Rorer with 9a-and 9β-hydroxy-docetaxel (25), cytotoxicity of these reduced compounds is similar to paclitaxel and docetaxel respectively. Kingston et al (26), Chen et al (27), Holton et al (28) as well as our own group (25) have all reported different approaches for preparing 10-deoxy-paclitaxel and 10-deoxy-docetaxel which were found to have comparable antitumor activity with respect to paclitaxel and docetaxel. Recently our group noted that 19-hydroxy-docetaxel, obtained from natural 10-deacetyl-19-hydroxy-baccatin III, has activity similar to docetaxel (29). Furthermore Chen et al observed in the paclitaxel series an unprecedented rearrangement product possessing a cyclopropane moiety involving carbons C-7, C-8 and C-l9 (30). This constrained analog of paclitaxel exhibits cytotoxicity equivalent to paclitaxel. Thus the top part of the diterpene moiety, that is positions 7, 9, 10 and 19, tolerate a wide variety of substituents. This allows us to assume that this region of taxoids may not play a crucial role in microtubule binding or, to some extent, to cytotoxicity. 2-Debenzoyloxypaclitaxel as well as 2-debenzoyl-paclitaxel showed little in vitro cytotoxicity (37), while, very recently, different groups observed that modified benzoyl groups at C-2 have to be appropriately substituted to retain activity (32). These results indicate that the C-2 benzoate moiety plays an important role in the binding of paclitaxel to its receptor and, as a matter of fact, to cytotoxicity. Recently, it has been found that new taxoids derived from Ηβ-hydroxy-10-deacetylbaccatin III have activity close to paclitaxel and docetaxel (33). Optimum activity in the series was achieved with 14-β hydroxydocetaxel-1,14-carbonate.


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Perhaps more than anyone else, Potier, Guéritte-Voegelin, Guénard et al have focused on the study of structure-activity relationships of side-chain modified taxoids (34). They observed that the regio- and stereochemistry of each hetero atom at the 2 - and 3'positions are crucial for retaining biological activity (34). The hydroxyl group at C-2' is critical since 2-deoxy-paclitaxel is nearly 70-fold less cytotoxic than paclitaxel while isosteric 2'-deoxy-2-fluoro-paclitaxel and 2-O-methyl-paclitaxel proved to be nearly 100-fold and 200-fold less active respectively than the natural product (35). Introduction of an acyl group at 2' such as acetyl or hydrophilic acyl groups reduced activity in the tubulin assay but many of these derivatives retain activity in vivo or in cell-based assays since they are very likely to act as prodrugs (36). Replacement of the 3-phenyl group of paclitaxel with methyl or hydrogen drastically lowers cytotoxicity (34, 37). The importance of an aryl group at C-3' was emphasized in Rhône-Poulenc Rorer (38) and Florida State University (39) patents as well as by the work of other groups (40) with the preparation of a wide number of very active analogs. Klein et al (19a) observed that 3'-dephenyl-3'-isobutyl-9-dihydro-paclitaxel is highly cytotoxic, while Holton et al patented 3'-dephenyl-3'-isobutenyl-paclitaxel (41) and 3-dephenyl3'-cyclohexyl-paclitaxel (42) as highly cytotoxic new taxoids. Our group (43) as well as Holton et al (39) and more recently Georg et al (44) reported that heteroaromatic groups at C-3' such as thienyl, furyl or pyridyl retain cytotoxicity. Regarding modifications of the nitrogen atom at C-3', a free amino group as well as deletion of the amine function lead to less active analogs (34). Replacement of the 3benzoyl group of paclitaxel with other acyl groups such as tigloyl (cephalomanine), tosyl, butyryl and substituted benzoyl gives access to equally or less active compounds (34, 45) whereas introduction of a 3'-ter/-butyloxycarbonyl substituent (docetaxel) leads to a more active compound in experimental models (73). These preliminary results suggested that other modifications at C-3' might further improve the antitumor efficacy. We report herein some of our results regarding the stereoselective semisynthesis of docetaxel and new taxoids with either a 3-modifiedphenyl ring or a 3-N-modified-carbamate moiety, along with their biological activity.


,

Chemistry. Semisynthetic taxoid work has generated a demand for new stereoselective preparations of isoserine-type structures. This demand has been met during the last three years by a wide number of papers describing new approaches to phenylisoserinates or structurally related β-lactams (46). Our own efforts in this area have provided different stereoselective approaches. f

Stereoselective approaches to 3-modified-phenyI taxoids. Ourfirstmethod used an aldol reaction as the key step (Scheme 1) (47). Asymmetric aldol reaction of the boron enolate of the chiral compound 5, with differently substituted benzaldehydes led to the expected bromohydrins 6 as single isomers in moderate to good yields. Treatment of 6 with lithium ethoxide concomitantly cleaved the chiral auxiliary and formed epoxides 7. Sodium azide ring opening gave, after hydrogenolysis and acylation, the corresponding ethyl phenylisoserinates 8. At this point, an oxazolidine-type protection was introduced to increase the reactivity of the corresponding acid and minimize the risk of epimerization in the esterification process (47). Cyclic protection was achieved using 2methoxy-propene. Subsequent alkaline hydrolysis afforded acids 9 in nearly quantitative


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overall yields. Esteriflcation with O-diprotected baccatin III derivative 3 gave esters 10. Deprotection under acidic conditions, N-acylation with (Boc) 0 and reductive Odeprotection with zinc in acetic acid led to compounds 12 in satisfactory yields. This methodology was used to prepare a wide number of analogs 12 modified in the ortho, meta and para positions (R = Me, F, CI, etc.). 2


Scheme 1. Approach based on a stereoselective aldol reaction

OCOPh

12

Reagents: i) 5 (1 equiv.), E t N (1.4 equiv.), Bu BOTf (1.2 equiv.), C H C 1 , -70°C to 20°C, 2h then RC H C H O (1 equiv.), -78°C to 0°C, lh. ii) EtOLi, THF, -75°C to 15°C, 15min. iii) N a N , EtOH, NH C1, 60°C, 8h. iv) H (1 atm.), Pd/C (10%), AcOEt. v) (Boc) 0, N a C 0 , C H C 1 , 20°C. vi) C H = C ( C H ) O C H (8 equiv.), PPTS, toluene, 80°C, (dist). vii) LiOH, EtOH, H 0 , 20°C then H 0 . viii) 3 (1 equiv.), 9 (1.5 equiv.), DCC (1.6 equiv.), DMAP (0.5 equiv.), toluene, 80°C, 2h. ix) HCOOH, 20°C, 4h. x) (Boc) 0, C H C 1 , NaHC0 , 20°C. xi) Zn, AcOH, MeOH, 60°C, lh. 3

6

2

2

2

4

3

4

2

2

2

3

2

2

+

2

3

3

2

2

2

2

3

3

As the approach via the aldol reaction has several limitations (aldehydes bearing a basic nitrogen atom sometimes presented difficulties in the initial condensation reaction and,



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in the third step, regioselectivity of the oxirane opening is controlled by the substituents of the phenyl ring), we looked for other stereoselective approaches to phenylisoserines. Another way to prepare phenylisoserine derivatives is via opening of a corresponding βlactam (48). Furthermore Holton (49) and Ojima (50) have shown that N-acyl βlactams are very helpful in the esterification of baccatin derivatives at C-13. Extensive efforts at generating new stereoselective methods for the preparation of β-lactams quickly followed (46). Our own efforts at utilizing β-lactam chemistry as an expeditious way to generate isoserines after hydrolytic opening are outlined below (Scheme 2) (57). Staudinger [2+2] cycloaddition of readily available benzylidene-imines 13 with acetoxyketene generated in situ led to β-lactams 14a and 14b as a mixture of diastereomers, in approximately 50% d.e. After acetate hydrolysis the corresponding hydroxy-lactams were obtained. At this step (or in some cases at the previous one), we were generally able to separate the major diastereomer 15 by a single crystallization. After ring opening and hydrogenolysis, the desired phenylisoserine derivatives 16 were isolated in good overall yield. This latter compound was taken on to acid 9 as described in scheme 2. Our approach has proven general enough to access a series of analogs modified on the phenylringat C-3' particularly at the para-position (R = F, N(Me>2, etc.). Scheme 2. Preparation of phenylisoserines using the Staudinger reaction.

14a (major)

1

4

b

( j m

n 0

r)

COOH OMe

v, vi, vu

R=H,

R=H,

67%

91%

BocN

χ 9

Reagents: i) A c O C H C O C l , CHC1 , E t N , 0°C, 1.5h then 20°C, 3h. ii) K O H / H 0 (IM), THF, 0°C, lh or N H , 0°C, lh then crystallisation from AcOEt. iii) HC1 (6N)/MeOH (5/1), reflux 20h or HC1 gas, MeOH, 40°C, 2.5h. iv) H (345 psi), Pd/C (3%), MeOH/AcOH (3/1), 65°C, 4h or H (15 psi), Pd(OH) (20%), MeOH/AcOH (20/1), 20°C, 18h. v) (Boc) 0, N a C 0 , C H C 1 , 20°C. vi) C H = C ( C H ) O C H (8 equiv.), PPTS, toluene, 80°C, (dist.). vii) LiOH, EtOH, H 0 , 20°C then H 0 . 2

3

3

2

3

2

2

2

2

2

3

2

2

+

2

3

3

2

3

In the early stage of our studies on structure-activity relationships, it was very important to know if the distance of the phenyl ring to the isoserine chain could be modified. Thus the corresponding compound, i.e. 3'-dephenyl-3'-benzyl-docetaxel 21, was prepared by yet another route, as depicted in scheme 3, but still utilizing the same protection/deprotection sequence (Bourzat, J.D.; Commerçon, Α., unpublished data). Benzylisoserinate 18 was obtained in several stepsfromN-Boc-phenylalaninol 17. After


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isopropylidene protection and alkaline hydrolysis the derived acid 19 was esterified with the 7,10-diTroc baccatin derivative 3 to give the 13-O-esterified baccatin derivative 20. Final deprotection, reacylation and cleavage of the Troc groups afforded the desired analog 21. 3-Dephenyl-3'-benzyl-docetaxel 21 proved to be 150-fold less active than docetaxel in the tubulin assay (4) and inactive in the different in vitro cytotoxicity assays (IC50 (P388): 8 μg/ml) showing that the distance of the phenyl group on the isoserine-chain was quite important. ,

v


Scheme 3. Preparation of 3-dephenyl 3'-benzyldocetaxel

Reagents: i) C5H5NSO3 (3 equiv.), E t N , DMSO, CH C1 , -70°C. ii) Me SiCN, MgBr , CH C1 , 26h, -20°C, then chromatogr. iii) EtOH, HCI, H 0 , 20°C. iv) (Boc) 0, CH C1 , N a H C 0 , 20°C, 16h. v) C H = C ( C H ) O C H (8 equiv.), PPTS, toluene, 80°C (dist.). vi) LiOH, EtOH, H 0 , 20°C, 3h then H 0 . vii) 3 (1 equiv.), 19 (1.5 equiv.), DCC (1.6 equiv;), DMAP (0.5 equiv.), toluene, 80°C, 2h. viii) HCOOH, 20°C, 4h. ix) (Boc) 0, C H C 1 , N a H C 0 , 20°C, 20h. x) Zn, AcOH, MeOH, 60°C, 35 min. 3

2

2

2

3

2

3

2

3

2

2

2

2

2

3

2

+

3

2

2

2

3

Protective groups improvements. One of the problems that we constantly faced in all of the above synthetic schemes involved the side-chain protection: cleavage of the isopropylidene protection of compounds 10 occurs under acidic conditions but with removal of the Boc moiety. Such a cleavage is desired when Boc replacement analogs are to be prepared (vide infra) but is of course less desirable when the Boc group is to remain on the molecule. In order to retain the Boc moiety, we investigated a large number of other acidic conditions but none afforded a chemoselective opening of the oxazolidine ring. These results led us to examine other 2-modifled oxazolidines as protecting groups. Our idea was to modify the 2-position with a group such as trichloromethyl, which might give, under reducing conditions, both cleavage of the cycle and removal of the 7,10-diTroc groups on the baccatin moiety. Although trichloroacetaldehyde (chloral) is known to react with β-aminoalcohols to prepare 2-trichloromethyl-oxazolidines (52), to our knowledge, the use of such derivatives as protective groups of β-aminoalcohols has never been reported. In the event (scheme 4)(53), cyclization of N-Boc phenylisoserine 22 with chloral in the presence of pyridinium /?ara-toluenesulfonate (PPTS) in refluxing toluene yielded the expected oxazolidine derivatives as a mixture of diastereomers (d.e. 30%) but unfortunately with removal of the Boc group. We subsequently found that we could



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directly obtain these trichloromethylene-protected isoserinates 24 by treating phenylisoserinate 23 with chloral under the same conditions. The corresponding acids 25 were then prepared by alkaline hydrolysis. The nitrogen atom of these oxazolidines proved to be weakly basic. Thus we were able to directly esterify 25 with the Odiprotected baccatin derivative 3 affording the corresponding esters in good yield. Simultaneous cleavage of the trichloroethylidene protection and the Troc protective groups was performed with zinc in acetic acid at room temperature to give arninotaxoid 27 which was acylated to give docetaxel 4. Although in this case the initial goal offindinga selective protection with respect to the Boc was not fulfilled, this new approach represents a very expeditious semisynthetic access to docetaxel and side-chain modified analogs. Scheme 4. 2-Trichloromethyl-oxazolidine-type protection cci

.' P

h

CCI3

3

RNH O H

êooMe

22 : R = tB0C

23 : R = H

—L_> 9

i

-

9

6

H N ^ O

"

y-i^

%

P

h

9

COOMe

o

> %

Ο

JiL 3

M Ph

COOH

24

25

Reagents: i) from 22: CCI3CHO (20 equiv.), PPTS, toluene (dist), flash chromatography in AcOEt/cyclohexane 1/3, 91% (d.e.=30% by NMR); from 23: CCI3CHO (4 equiv.), PPTS, toluene (dist.), 96% (d.e.=35%by HPLC); ii) KOH or L i O H H 0 (1.1 equiv.), MeOH, H 0 , HC1 IN, >90%; iii) 3 (1 equiv.), 25 (1.8 equiv.), DCC (1.03 equiv.), DMAP (0.2 equiv.), toluene, 25°C (3h); iv) 26, Zn (10 equiv.), AcOH (40 equiv.), AcOEt, 25°C (16h), 65% by HPLC from 3; v) 27, Oi-tertbutyldicarbonate (1.2 equiv.), MeOH, 25°C (15h), 70% by HPLC. 2

2

Another idea was to introduce at the 2-position of the oxazolidine a substituent, such as an alkoxy, a phenyl or a mono- or dimethoxy-phenyl group, which might favor the hydrolytic cleavage of the ring without removal of the Boc group. We tested different aryl and alkoxy groups at the 2-position of the oxazolidine-protection (54). In considering the resulting yields, products stability and ability to give crystallized compounds, we selected the mono- or dimethoxy-phenyl groups. As an example, we report in scheme 5 the preparation of docetaxel with para-methoxy-phenyl as the activating group. N-Boc phenylisoserine methyl ester reacts under kinetic control with /?ara-methoxybenzaldehyde dimethylacetal to give the corresponding 2-monosubstituted-l,3oxazolidine-carboxylates 28a and 28b in 70% de. allowing the isolation of the major diastereomer 28b after a single crystallization. Alkaline hydrolysis of 28b followed by coupling reaction with the 7,10-O-diprotected baccatin derivative 3 afforded the corresponding esterification product 30 in nearly quantitative overall yield and without detectable epimerization at the 5-position of the oxazolidine ring. Acid-mediated



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oxazolidine cleavage of the baccatin ester 30 was conducted with /?ara-toluenesulfonic acid (PTSA) in methanol without removal of the Boc to give compound 31. Thus, this methodology fulfilled our original goal of maintaining the Boc group during the oxazolidine cleavage. It has also proved general and easily applicable to the preparation of docetaxel and side-chain modified analogs. These oxazolidine-type protections have different advantages: compared to their open forms, the cyclic acids are very stable and reactive in the esterification reaction. Furthermore, in the cyclic-protected phenylisoserine, phenyl and carboxylic functions enjoy a trans disposition, which limits the risk of isomerization at C-2' during the esterification reaction. This 2-epimerization possibility has also recently been reported by Greene et al using trichloromethoxymethyl as protecting group at C-2* under the same esterification conditions (55). Scheme 5. 4-MethoxyphenyI-oxazolidine-type protection

OMe

Reagents: i) 22, 4-Me0-PhCH(0CH ) (1.1 equiv.), PPTS, toluene (dist.), 0.5-2h. ii) cryst. toluene/cyclohexane. iii) KOH or LiOH H 0 (1.1 equiv.), MeOH, H 0 then HCI IN. iv) 3 (1 equiv.), 29 (1.7equiv.), D C C (1.06 equiv.), DMAP (0.2 equiv.), toluene, 25°C, 2h. v) PTSA (1 eq.), MeOH, 25°C, (2h). vi) Zn (11 equiv.), AcOH (40 equiv.), AcOEt, 30°C, 3h. 3

2

2

2

Surprisingly enough, we observed limitations of such cyclic protections in the coupling of the corresponding enantiomeric or diastereomeric forms of acids 9 or 29 with the baccatin derivative 3: partial to complete epimerization at 2' occurred in the esterification process (Bourzat, J.D.; Commerçon, Α., unpublished data). This was also independently observed by Greene et al (55). Preparation of 3'-N-modified taxoids. Partial synthesis of new carbamate and amide derivatives of the amino group at C-3' have been easily realized using the free amino intermediate 32 (Scheme 6). Acylation was achieved with a wide range of acylating


242


agents. For instance new carbamates were prepared from the free amino derivative with halocarbonates (when available or stable), or /?ara-nitrophenyl carbonates. We also applied Senet's method which makes use of very reactive 1,2,2,2-tetrachloroethylcarbonates (56). Application to our case proved to be general since we rarely faced difficulties in introducing bulky substituents at the N-3* position.


Scheme 6. General access to new carbamate analogs of docetaxel.

Reagents: i) method A: C1COR', N a H C 0 , AcOEt, H 0 , 20°C, 15min; method B: (R'CO) 0, N a H C 0 , C H C 1 , 20°C, 24h; method C: R'C00CHC1CC1 , pyridine, C H C 1 , 20°C, 16h; method D: R'COOC H -(p-N0 ), pyridine, C H C 1 , 20°C. ii) Zn (11 equiv.), AcOH (40 equiv.), MeOH, 60°C, lh.. 3

3

2

6

2

2

2

3

4

2

2

2

2

2

Biological results and discussion. Results of inhibition of the disassembly process of microtubules at 4°C (4) for docetaxel and its side-chain analogs as well as their in vitro cytotoxicity (P388 leukemia cell line) (75) and in vivo antitumor activity (B16 melanoma grafted in mice) (57) are reported in Tables I and II. Table I : Biological activities of 3'-modified-phenyl analogs of docetaxel Entry Substituent Tubulin assay P388 IC50 Β16 Melanoma R mg/Kg/inj %T/C NCI Score IC50/IC50 (1) μg/ml ++d 1 13.4 0 0.64 0.04 H (4) + 2 o-CH 35 2.2 30 0.8 3 /w-CH 2.8 0.7 30 42 ++ 4 p-CH a 8 1.3 15.5 0.04 ++ 5 o-F 0.04 10.8 0 1.1 6 /w-F 42 1 30 0.1 ++e 7 /7-F 6 20 0.9 0.03 8 m-OMe 3 18 88 0.7 ++ 9 0 0.7 23 p-OMe 0.05 ++ 10 p-Cl 1 18 0 0.05 11 p-I 2.2 35 88 0.3 + 12 39 p-NMe 3.5 20 0.65 Prepared using scheme 1. ^Prepared using scheme 2. Obtained by another method (Bourzat, J-D. etal., in preparation). ^Log cell kill: 3.3. Log cell kill: 3.2 a

3

a

3

3

a

a

b

a

a

a

c

b

2

a

c

e

Regarding the 3-modified-phenyl analogs (Table I), one observes a rather good correlation between the microtubule disassembly inhibitions and both the cellular and the in vivo antitumor activities. The results obtained in this study demonstrate the influence of the substituent position on biological activity. We detected that introduction of a substituent at the weta-position (entries 3, 6, 8,) significantly



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decreases the level of cytotoxicity in experimental models. This lowering of activity is not apparently dependent on the electronic properties of the substituent (electronwithdrawing groups such as fluorine, or electron-donating groups such as methyl and methoxy). The presence of a substituent in the ortho-position looks acceptable as long as it is quite small (F versus Me, entries 2, 5). Modifications at the /?ara-position are better tolerated although with modulated activity. Thus electronic effects are not detectable at para (entries 4, 9, 10) while bulky and basic substituents cause a loss in activity (entries 11, 12). Based on the in vivo results (log cell kill: 3.2), 3'-/?am-fluorodocetaxel (entry 7) is a very powerful analog of docetaxel. Concerning 3-N-modified analogs, biological results (Table II) clearly demonstrate the superiority of the Boc group (entry 1) over other primary (entries 3, 4), secondary (entries 5, 6) or other tertiary (entries 7, 8, 9) alkyl carbamates. Isosteric forms of tertbutyl carbamate were prepared such as the C-isostere obtainedfromtert-butylacetyl chloride (entry 2). None of these derivatives exhibits higher cytotoxicity than docetaxel. t

Table Π : Biological activities of 3-modified-carbamate analogs of docetaxel Β16 Melanoma Entry Tubulin assay P388 IC50 Substituent mg/Kg/inj %T/C NCI Score R' IC50/IC50(1) μg/ml 0 13.4 0.04 1 0.64 0-ter/-Bu (4) 0.5 38 107 2 1.3 CH -tert-Bu 6 50 68 0.7 3 OMe 0.3 50 64 4 0-fl-Bu 1.1 0.06 22 4 ++f 5 0-/so-Pr 1.2 6 0.08 20 49 0.5 OCH(Et) + 7 OC(Me) Et 0.6 0.04 38 18 + 8 OC(Me) CH Cl 0.6 0.07 20 30 + 9 OC(Me) CH CN 0.65 0.45 20 16 Prepared using method A. ^Prepared using method B. Prepared using method C. ^Prepared using method D. Log cell kill: 3.3; *Log cell kill: 1.4. a

2

a

a

a

c

2

b

2

c

2

2

d

2

2

a

c

e

Concluding remarks In conclusion, our work has contributed to the ongoing search for facile and practical preparations of phenylisoserinates and phenyl-modified phenylisoserinates. These methodologies led to the synthesis of docetaxel and docetaxel analogs modified at the 3-position (phenyl ring or substituent of the nitrogen atom). Preliminary results clearly demonstrate that the para-position of the phenylringcan be modified with retention of activity whilefert-butylcarbamateremains thus far one of the best substituents for antitumor efficacy. Among all the taxoids disclosed, 3'-/?ara-fluoro-docetaxel proved to be the most powerful analog of docetaxel. Acknowledgements We would like to thank Drs. H. Bouchard, E. Fouque, T. Hart, P. Ni and I. Taillepied as well as M . Alasia, D. Bézard, F. Bernard, E. Bouley, M-F. Marzin, C. Massey, P. Nemecek and C. Souder for their chemical contributions. We are also grateful to Dr. M. Vuilhorgne and his collaborators for structural analyses, Drs. M-C. Bissery, C. Combeau, J-F. Riou, P. Vrignaud and their collaborators for biological evaluation and Dr. C.J. Burns for critical reading of this manuscript. We also gratefully acknowledge


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