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New Fluorescent Water-Soluble Taxol Derivatives.

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M Murray. ! M ~ ~ t / t o r l U
i v ti g
Uwrtri
(Houben-Weyl), 4th ed.. Vol. V,'Za, (Ed.:
E . Muller). 1977, p. 967; P. Cadiot. W. Chodkiewicr. J. Rauss-Godineau, Bull.
Sot. Ch/nr.Fr. 1961. 2176-21Yi. H. klopfin The C/ic~mrslr:l~o/'K~rencs,
Allettrs
rind Rclotrd ~ ' o n t p o ~ ~ rVt d~.l2. ~( E
, d S . P(I/(JIJ,Wile).. Chichester 1980. p. 719
R. W Saalfi-;ink, F. SchutL. U. Moenius. Sjrtthe.si.\ 1985, 1062-1067: R W.
Saalfrank. W. Rost. F. Schutz. U ROB. Anpew Chern. 1984, Y6, 597-599;
A n ~ e i t .Chent. I n / . 0 1 . Enpl. 1984. 73. 637 638.
R. W. Saalfrank. IJ. Bauer. K . Hilbtg. A. Welch. Chrrtt. B w . 1993. /.?6, 823~
83.5.
R. LV. Saalfrank. K. Hilbig. F. SchutL. K . Peters. H. C . von Schnering. C I I P ~ J ~ .
Bw. 1988, f21. 1291-1297.
R. W. Saalfrank. E Schutz. H -U Hummel, Z. X~u/rr,-for.sd~.
1987. 4 3 . 97 100.
F. Strauss. L. Kollek, W. Heyn. Chrni. Ber. 1930, 63. 1868-1885.
R. W. Saalfrank. A. Welch, U. Bauer. M. Haubner. Licd~ixsAtin. Chpnt. 1996.
in press.
See also M. Parinantier. J. Galloy. M. van Meersbche. H. G. Viehe. Angeii,.
C'/ietit. 1975, 87. 33-34; Angeir. C'hetit. I n / . Ed. Enxl. 1975. 14. 53.
According to the spectra the compound was pure. Chromatography or distillation led to decomposition.
dansyl group linked through a 8-alanyl amino spacer to the
7-position; the bioactivity of this compound has, however, yet
to be demonstrated. Another emitting taxoid with a mrta-amino
group at the 2-benzoyl substituent, but less active than the parent drug, has also been briefly reported.["] Somewhat related to
our purpose is the synthesis of photoactivatable taxoids with an
azido substituent at the 3'-benzoylamino group," ' I which are
useful as photoaffinity labels in the mapping of the taxoid binding site in tubulin.
Here we report the synthesis and the characterization of two
new bioactive fluorescent taxol derivatives, one of which is
much more water-soluble than taxol itself, and provide an example of their use in the first direct visualization of the taxol-microtubule system in cultured cells. As it has been established that
modifications at the 7-hydroxyl group do not appreciably affect
the biological activity of t a ~ o l , [ ~ ~'I . ' we
, selected this position
for the linking of two fluorescent dyes through a spacer containing a free amino group. The synthesis of the 7-ester of taxol with
L-alanine (2) has been previously described by Mathew et al.['3]
New Fluorescent Water-Soluble Taxol
Derivatives**
Andre A. Souto, A. Ulises Acufia, Jose M. Andreu,
Isabel Barasoain, Miguel Abal, and
Francisco Amat-Guerri*
Paclitaxel (taxol, l), a natural diterpenoid found in the stem
bark of the pacific yew TU,YUS
hrevifolia,"] is one of the most
promising anticancer agents for a variety of tumors,['' due to its
unique ability to inhibit cell division as well as other interphase
processes by stabilizing micro tubule^.[^] This has stimulated an
intense and detailed study in many laboratories of the chemistry
and the biochemistry of this exciting antitumor agent. Reports
describing the total synthesis of t a ~ o I , [its
~ ]chemical modification and structural analysis,[51and the search for new taxoids
with improved pharmacological propertiesL6]have been published. The basic mechanism of the biological action of taxol is
also a research topic of relevance for the development of practical therapies with this drug."] We have described the low-resolution solution structures of taxoid-induced microtubules,[" the
assembly of purified GDP-tubulin in the presence of taxoids.
and the link between the taxoid binding and microtubule assembly reactions.['] In the course of these investigations we realized
that a fluorescent and water-soluble taxol derivative would be
an enormous help, because a) it would allow the direct in vivo
study of the target microtubules in tumour cell cultures by fluorescence microscopy. and b) it would provide an extremely useful tool for the study of the molecular mechanisms of taxoidinduced microtubule assembly, as well as for the discovery of
other putative taxol targets and clinical diagnostic assays.
The synthesis of fluorescent taxol analogues has been attempted before but with limited success. Thus, Kingston et al.[5a1
mentioned a fluorescent taxol derivative with a low-absorbance
[*) Dr. F. Amat-Guerri. A. A. Souto
lnstituto de Quimica Orginica. C. S I. C.
Juan de la Cierva 3. E-28006 Madrid (Spain)
Telefax. Int. code +(1)564-48.53
Dr. A. U . Acufia
Instituto de Quimica Fisica Rocasolano. C. S. I . C.. Madrid
Dr. J. M. Andreu, Dr. I. Bardsoain, M. Abal
Centro de Iiivestigdciones Biologicas. C. S. 1. C., Madrid
[**I This work was supported by the Direccihn General de Investigacion Cientifica
y Tecnica of Spain (Projects PBY3-0126 and PB92-0007) and by Accion Especial C . S. I . C . Taxol was provided by Bristol-Myers Squibb, the owner of the
registered trade name. A. A. S. acknowledges a fellowship from CNPq
(Brazil). We thank J. Evangello for the HPLC analysis.
271 0
((-'
VC'H Vrrlu~sjiesell.sthufi
m h H , D-6Y4.iI Weinhein?,IYY.5
I
2
3, 4
4. R = CO
5F
3, R = COCHz
during their search for water-soluble taxoids. Starting from tax01 ( I ) , this synthesis comprises three steps: 1) esterification of
both the 7- and 2'-OH groups of the taxol molecule with the
NH,-protected amino acid N-(tert-butyloxycarbony1)-L-alanine
(BOC-alanine) , yielding 2',7-di(BOC-~-alanyl)taxol;2) deprotection with formic acid of both amino groups, producing 2',7di(L-alany1)taxol ; and 3) selective aqueous hydrolysis of the 2'ester group, eventually yielding 7-(~-alanyl)taxol(2)
Amino
acid esters of taxol deprotected at the NH, group are difficult to
purify. because they degrade easily through epimerization and
side-chain cleavage during the workup.[' 31 However, we have
found that the crude product 2 can readily react through the free
amino group with amine-reactive fluorescent dyes such as the
succinimidyl esters of 7-dimethylaminocoumarin-4-acetic acid"
or of 4'-fluoresceincarboxylic acid, to produce the corresponding conjugates 3 and 4 in good yields. The proper selection of
solvent for this reaction is crucial, particularly for compound 4.
The structure of each compound was confirmed by MS-FAB+
and H N M R spectroscopy."'] These two fluorophors were
preferred, because they show spectroscopic and photophysical
'
0~70-O833,1Y5:3473-,7710Y 10.00+ .ZS:O
Angrii,. Cheni. In/. Ed. Engl. 1995. 34.
No. 23/24
~
--
~
COMMUNICATIONS
-
properties that are optimal for our purposes: large absorption
coefficients at wavelengths well separated from the protein's
intrinsic absorption and matching available laser excitation
sources, a s well as large emission yield and anisotropy. The
synthetic method reported here is ofgeneral applicability for the
introduction of fluorophors into the taxol molecule and can be
used to genei-ate a family of new fluorescent taxol derivatives.
An important limitation in the clinical use of taxol is its low
solubility in water ( 5 pM),['" and therefore it is not surprising
that much effort is aimed at the synthesis of soluble taxol derivatives. This has been achieved by introducing polar or ionic substituents at the 2' and/or 7-hydroxyl groups, in many cases
linked through spacers.[t81 In this way water solubilities as
high as 0.01 M
~ and,~ in the
~ case~ of a -derivative
~
with
~
a
7-(polyethylene glycol) chain, 0.1 M [ have
~ been
~
~demonstrat~
ed, although in many cases the biological activity of these drugs.
when tested in whole cells, was seriously impaired relative to
that of the parent compound 1 . In the case of the fluorescent
derivatives 3 and 4.although the water solubility of the former
(approximately 1 p ~is )less than that of taxol. that of the bioactive fluorescein taxoid 4 is about two orders of magnitude higher
(0.3 mu)than that of the natural drug.
The biochemical and cell biological properties of compound
4 are being compared with those of taxol. Interestingly, preliminary results indicate that compound 4 retains essentially the
activity of the parent drug, and that it is able to induce the
assembly of otherwise inactive GDP-tubulin into microtubules.
a unique characteristic of active taxoids.['] When this probe is
tested in epithelial PtK2 cultured cells, the microtubule system
can readily be seen under the fluorescence microscope (Fig. 1 ) .
(C-4column. 20 to 80% acetonitrile in water ;is eluciil) };/\I3
\IS IIII-YUHAI. W I :
1 1 7 6 . 5 0 ( M + N a t ) . 1184 SO(?MH'.calculatedIbr(',,H,,,U,O ,1153 -13).U V \ i s
(MeOH): in,,,
( i : ) = 376 nm (20200 M - I cm - I ) . ciiiissti)n ic\citrition ;it 376 i n i n ) :
i,,, 462 n m ; ' H NMR (500 MHz. CDCI,. 3lJ'C. TMSj: 0 = \ I 0 (d. .I = 7 . 3 Hr.
2H, n-H of Ph(a)). 7.75 (d. J = 7 . 3 Hz. 2H. (1-H o f P h ( c l ) .
p-H ofPh(a)). 7.80 (m,
6H. H-5C. ni-H o f P h ( a ) . (1-H o f Phihi p-lJ ofPh(c)). 7 41
(m. 4H. n - H of Ph(b). ni-H of Ph(c)). 7.35 (t. .I = 7.3 Hz. I H . p-H o f P l i ( b ) ) .6.98
(d.J=9.0H7.1H.3'-NH).6.67(d.J=8.9H~,IH.NHofA1;1).~~.59(dd.J=2.-1.
8.9 Hz. I H . H-6C), 6.48 (d. J = 2 4 Hz. 1H. H-XC). 6 17(t. I = X.5 H7. 1 H . H-13).
6.17(s. l H . H - 3 C ) . 6 . 0 9 ( ~I,H . H-lO).5.78(dd. J = 2.4.9.0
ti/. IH, H-3'). 5.65(d,
J = 6 7 Hz, IH, H-2),5.60(dd, J = 7 . 3 . 10.4 H7, 1H. H-7),4.S9(d..J = 8 9 Hz, I H ,
H-5). 4 78 (dd, .I = 2.4. 5.1 Hz. IH. H-7'). 4.68 (m,IH, C11 of A h ) , 4.30 (d, J
= 8.3 Hz. I H . H-202). 4.16 (d. J = 8.3 Hr. IH. H-70b). i.XS i d . J = 6.7 Hz. IH,
H-3). 3.65 (s, 2H. CH,CONH). 3.56 (d. J = 5.1 Hr, l H , ? - O H ) . 3.03 (5. 6H,
N(CH3)J.7.47(ni. lH,H-6a).236(s.3H.4-CH,CO).1_.32(in,2H.H-14).2.10(s
3H. 10-CH,CO). 1.84(in, lH, H-6P). 1.79 (s. 3H. H-18). 1
(s. 3H. H-19). 1 73
:IT
(s,1H.1-OH),1.19(~.3H.H-l7),1.13(overlappedd.iH.CH,ofi\la).1.12(s.
H-16).
4: To a solution ofcrude 2 (10 mg. 10.8 minol) i n DMF (1 in1 ) a n d aqueous pH 9
carbonate:bicarbonate buffer ( 1 mL) was added another solution of' succinimidyl
fluorescein-4'-carboxylate (7.7 mg, 16.2 mmol) in the same solvent mixtiire ( I mL).
After stirring for 2 11, the solvent was removed. and the crude product 4 was purified
by preparati\e TLC (silica gel. 1 :4ethyl acetate-dichloromcth~~iie).
Yield 8.0 mg
(60%). M.p >300 C. HPLC puritq > 9 2 % (C-I8 column. 20 to X0'%, acetonitrile
inwatrraseluent). FAB'-MS(fn-NBA).iii:_ 1305 5 0 [ M + N ~ i ' ] .1283 5 0 ( M H + .
calculated for C7,H,,N,02, 1282.42); UV'Vis (water):
493 nm; (basic MeOH)
i,,, 499 nm (e 70000 M-'c m - I ) : emission (in water. excitii[ion at 494 iim) i,,,
520 nm: ' H N M R (500 MHr. D,O, 40 C, ref. H,O at (i= 4 61): ij = 8 20 (s. 1H.
H-3'F). 8.00 (d. J = 8 0 Hz. -3H. o-H ofPh(a). H-5.F). 7.81 (d. .I = 7 Y Hr. 2H. 0-H
of Ph(c)). 7.68 (t. J = 8 0 HZ. IH. p-H ofPh(a)). 7.63 ( t , J =7.9 HL. I H . /I-H of
Ph(c)). 7.58 ( t , J = X.O H;.. 2H, 111-H of Ph(a)). 7.54 (t. J - 7 9 Hz, 2H. riz-13 of
Pii(c)),7 . 5 0 ( o ~ ~ i a p p e d2H.
d . H-1F.H-8F). 7.4Y(s,ZH. H-4t. H-5Fj.7.31 (m.IH.
p-HofPh(b)).7.13(m,2H.in-HofPh(b)).h.69(d..I
= 8.0 H7.4tt. H-?F, tl-7F.o-H
of Ph(b)). 6.62 (d. J = 8.4 Hr. I H , H - 6 F ) . 6.34 (a. I H . H-10). 6 10 (t. J = 9.0 H7.
1H. H-13). 5.64 (d. J = 7 7 Hz. 1 H. H-2). 5.52 (in. IH. H-7). 5 46 (d. .J =7.1 Hz,
I H . H-3'). 8.04(m,
I H . H-5).4.85(d, J = 7 1 Hz, I H , H - 2 ' j . J 73(q. J = 7 . 5 Hz, I H ,
CHofAla).4.24(m.2H.H-20).3.79(d.J=7.7H~.lH.H-i).2.63(m.lH,H-6a
2 29 (s. 3H, 4-CH3CO). 2.19
15. 3H. 10-CH,CO).
1.91 (ni.ZH, H-6b. H-14a). 1 Xh(,, .?H, H-19). I 78
(s. 3H, H-lX). 1 7 4 (m,1H. H-14b). 1.45 (d. J
=7.5 Hz. 3H. CH, of A h ) . 1 16 ( s . 6H. H-16.
H-17).
Receihed: Jul! 18. 1995 [Z82241E]
German version: Aii,y01. ( 3 m i . 1995. 107.
29 10- 791 2
Keywords: drugs . fluorescence markers . taxol
M. C. Wan]. H. L. T a ~ l o r .M . E Wall. P
Coggon, A T. McPhail. J. A n i . Clrrwr Soc.
1971, Y3, 2325-2327.
Recent reviews on the taxol therap) : a ) K.
Gelmon, Tiir Laricc,r 1Y94, 344. 1267- 1272;
b) E. K. Rowinsky. R C. Donehower. .Vm
Engi. J Med 1995. 332. I004 1014.
a) P. B. Schrff. J. Faint. S. B Horwitx. Nurrira
1979. 277. 665 667; b) P. B. Schiff. S. 8.
Horwitz, Pro(.. ,Vurl . 4 c d Sci. L S A 1980. 77,
1561 -1565.
The total synthesis of t'iwl has been reported
independent11 by the groups of Nicolaou and
Holton: a) K . C. Nicolaou, Z. Xing. J. J. Lin,
H. Ueno. P. G . Nanteiinet. R K. Guy. C. F.
Clairborne, J. Renaud. E. 4 Couladouros.
K . Paulvannan. E. J. Sorensen. Nurnre 1994.
367.630 634: K . C Nicolaou. P. G . NanterFig 1 Image ol'inicrotubules in epithelial PtK2 cultured cells with the fluoresceinated taxol derivative 4 [19]. A : A
met, H. Ueno, R K Guy, E . A. Courepresentnti\e hie\% of interphase cell5 B: Enlargement of a bright mitotic spindle in a dividing cell. The bar
ladouros, E J. Sorenscn. J. 4in. C I i ~ i n Sot
corrcspr>ndsto 10 !irn
1995. 117.624 633; K ('. Nicolaou, J.-J. Liu.
2 Xing. H. Ueno. E J Yorenaen. C. F. Clairborne, R. K Guy, C.-K Huanc, M . Nakdda,
P. G. Nantermet. h i d . 1995, 117, 634-644; K. C. Nicolaoii, Z. Yang. J.-J. Liu,
E.up~i-inwnt i i l Proc.c&rr
P. G. Nantermet. C. F. Clairborne. J. Renaud, R. K. <hi>.
K Shibayama. h i d .
3: To 'I coIutroin ofcrude 7-(~-alanyl)taxol2 (10 mg, 10.8 minol) and Et,N (15 pL,
1995. 117. 648-682: K. C Nicolaou. H. Ueno, J.-J Liu. P G Nantermet. Z.
108 rnimol) in dioxanc ( I inL) was added another solution of succinrmidyl 7Yang. J Renaud. K. Paulvannan. R . Chadha. ihrrl. 1995. 117.683 659. see also
dimrtliqlarninocouiiiarin-4-acetate (8.6 mg, 16.2 pmol) in dioxane (0.5 m L ) . After
K . C. Nicolaou. R. K. Gu), Angeiv. Clim? 1995, 107. 2247. Airpen ('licw. Inr
stirring foi- 74 h 211 room temperature in the dark. the solvent h a s removed. The
Ed. Eizgl. 1995, 34. 2079-2090: b) R. A Holton. C Sornoia. H:B. Kim. F.
crude product .I was purified by preparative TLC (silicagel, 1 : 1 ethyl acetateLiang. R. J. Biediger. P. D. Boatman. M . Shindo, C < Smith. S. Kim, H.
dichlo~~iinetlnanc).
Yield 9.5 mg ( 7 7 % ) . M.p IX2-18S"C. HPLC punt) > 9 6 %
Nadizadeh. Y. Suruki. C. Tao, P. Vu. s. Tang. P. Zhang. K K . Murtiu, i N.
COMMUNICATIONS
Gentile. J. H Liu. J. h i . Cheni. So<. 1994, 116, 1597-1598: R. A. Holton.
H.-B. Kim. C. Somoza. F. Liang. R. J. Biediger. P. D. Boatman. M. Shindo.
C. C. Smith. S. Kim. H. Nadizadeh. Y Suzuki. C. Tao. P. Vu. S. Tang. P.
Zhang. K . K. Murthi, L. N. Gentile. J. H. Liu. hid. 1994. 116,1599-1600. c)
For a commented history of both synthetic achievements see: L. Wessjohann.
Angrw. (%ern. 1994.106. 101 1 ; Ang~ ii.Chem. I n ! . Ed. Engl. 1994.33.959-961;
S.B. Horwitz. Nuturc, 1994,367. 593-594.
[5] Selected reviews on the chemistry and biology of taxol: a) D. G. I . Kingston,
Phurma(,ol. Thw. 1991. 52. 1-34: b) Trends Bioferhno/.1994. 12. 222-227, c)
K.C.Nicolaou. W.-M. Dai, R. K. Guy, Angew. Chew. 1994,106. 38; Angew.
Chein. In/. E d EngI. 1994.33. 15--44.The conformation of taxol in several
organic solvents has been studied: S. V. Balasubramanian, J. L. Alderfer. R. M.
StrdUblngCr. J. Pharni. Sri. 1994. 83. 1470-1476
[6]a) F. Gueritte-Voegelein. D. Guenard, F. Lavelle. M.-T. Le Goff. L. Mangatal.
P. Potier. .I Med. Chem. 1991. 34. 992-998; b) C. S.Swindell. N. E. Krauss,
S. B. Horwitz. I. Ringel, ibid. 1991. 34, 1176-1184;c) G . Appendino. H. C.
Ozen, P. Gariboldi. B. Gabetta. E. Bombardelli, Fitoterupm 1993.64,47-81;
d) A. N. Boa, P. R. Jenkins. N. J. Lawrence. Conremp. Org. Swi!h. 1994. 1,
47-75.
[7] a) W. D. Howard. S. N. Timasheff. J. B i d . Chem. 1988, 263, 1342-1346; b)
S. B. Horwitz. Trends Pharmucol. S C I .1992. 13, 134-136: c) R. B. Dye, S. P.
Fink. R. C. Williams. Jr., J. B i d Chem. 1993,268,6874-6850;d) M. A. Jordan, R. J. Toso. H. Thrower. L. Wilson. Prur. Null. Acurl. Sci. USA 1993,90.
9552-9556; e) E. Nogaks, S. G . Wolf, I. A. Khan, R. F. Luduefia. K. H.
Downing, Nurure 1995,375,424-427.
[El a ) J. M. Andreu. J. Bordas, J. F. Diaz, J. Garcia de Ancos, R. Gil, F. J. Medrano,
E. Nogales. E. Pantos, E. Towns-Andrews, J. Mol. B i d . 1992.226. 169-184;
b) J. M. Andreu, J. F. Diaz, R. Gil. J. M. de Pereda, M. Garcia de Lacoba, V.
Peyrot, C. Briand, E. Towns-Andrews, J. Bordas. J. Bid. Chem. 1994, 269.
31785-31792.
(91 a) 3. F. Diaz, J. M. Andreu, Biochrmisrr,v 1993,32.2747-2755;b) J. F.Diaz. M
Menendez, J. M. Andreu, ibid. 1993,32. 10067-10077.
[lo] Y. Han. A. G . Chaudhary. M. D. Chordia, D. L. Sackett. D. G . I. Kingston,
S. B. Hastie, Mot. B i d . Ce// 1994. 5, 28421.
[ l l ] a) G . 1. Georg. T. C. Boge, H. Park, R. H. Himes, Biourg. Mrrl. Chem. Left.
1995, 5 . 615-620; b) C. Combeau. A. Commercon. C. Mioskowski. B.
Rousseau. F. Aubert. M. Goeldner. B i o c h e m i s f r ~1994, 33. 6676-6683; c) S.
Rao. N. E. Krauss, J. M. Heerding, C. S. Swindell, I. Ringel, G . A. Orr, S. B.
Horwitz. J. Biol. Chem. 1994,269,3132-3134;d ) C. S.Swindell. J. M. Heerding, N. E. Krauss. S. B. Horwitz. S. Rao. I. Ringel. J. Med. Chem. 1994. 37.
1446-1449; e) D . Dasgupta. H. Park. G . C. Harriman, G. I. Georg, R. H.
Himes, ihid. 1994,37,2976-2980.The ester 7-[(m-azido-o-nitro)benzoyl]taxol
has been also used as a photoaffinity analog: J. M. Carboni. V. Farina, S. Rao.
S. I. Hauck, S. B. Horwitz, 1. Ringel, J. Mcd. Chem. 1993,36, 513-515.
[12] W. Mellado, N. F. Magri. D. G. I. Kingston. R. Garcia Arenas. G. A. Orr,
S. B. Horwitz. Biocheni. Biuphys. Res. Commun. 1984,124. 329-336.
[I31 A. E. Mathew, M. R. Mejillano, J. P. Ndth, R. H. Himes, V. J. Stella, J. Med.
Chem. 1992,35, 145-151.
[14] Our total yield of ester 2 was about 70%.
[15] Direct esterification of the 7-hydroxyl in 2'-[(2,2,2-trichloroethyl)oxycarhonyl]taxol (N. F. Magri, D. G . I. Kingston. J. Org. Chem. 1986,5/.797-802)
with 7-dimethylaminocoumdrin-4-aceticacid under different experimental
conditions was unsuccessful. probably due to a severe steric hindrance, as
molecular models suggest.
[16] ROESY experiments showed that in CDCI, solution the coumarin group in
compound 3 must he close to protons H-3 and H-7 of the taxol moiety, because
measurable NOE effects were observed with proton H-3C in the coumarin
group. Cross peaks between the aromatic signals in the taxol moiety were not
found, which supports the assumption that the corresponding rings are not
close (absence of clustering), as expected in this nonpolar solvent (D. G. VanderVelde, G. I . Georg. G . L. Grunewald. C W. Gunn, L. A. Mitscher, J. Am.
Chem. Soc. 1993, 115, 11650-11651).
[17] K. C. Nicolaou, C. Riemer, M. A. Kerr, D. Rideout. W. Wrasidio, Nuture 1993,
364.464-466.
[18] a) H. M. Deutsch. J. A. Glinski. M. Hernandez, R. D. Haugwitz. V. L.
Narayanan, M. Suffness, L. H. Zalkow, J Med. Chem. 1989. 32,788-792; b)
Z.Zhao, D. G. I. Kingston. A. R. Crosswell. J. Nut. Prod. 1991, 54, 16071611; c) K. C. Nicolaou. R. K. Guy. E. N. Pitsinos. W. Wrasidlo, Angeir.
Chrm. 1994, 106. 1612;Angeiv. Chem. I n / . Ed. EngI. 1994,33. 1583-1587;d)
L. G. Paloma. R. K. Guy. W. Wrasidlo, K . C. Nicoiaou, Cheni. B i d . 1994. 1 .
107-112;e) K.C.Nicolaou, J. Renaud, P. G. Nantermet. E. A. Couladouros.
R. K. Guy, W. Wrasidlo, J. Am. Chern. Soc. 1995. 117. 2409-2420. f ) R. B.
Greenwald. A. Pendri. D. Bolikal. J. Org. Chern. 1995, 60, 331 -336; g) Y.
Ueda, J. D. Matiskella, A. B. Mikkilineni, V. Farina, J. 0 . Knipe, W. C . Rose,
A . M . Casazza. D. M. Vyas. Bioorg. Mrd. Cheni. Left. 1995,5. 247-252.
[19] PtK2 cells attached to a coverslip were cultured overnight with the taxol deriva.
twice with phosphate buffered saline. mounted without
tive 4 (1 p ~ )washed
~
buffer pH 8.6. 0.20M NaCI, 70% glycerol. and obfixation in 0 . 1 3 glycine
served. The cells were cultured and photographed a s described: C. de Ines, D.
Leynadier. I. Barasoain. V. Peyrot. P. Garcia, C. Briand. G. A. Renner, C.
Temple. Cancer Res. 1994. 54. 75-84.
271 2
C
VCH Yr.rlugs~esellschafr
nihH. 0-69451 Weinheim. 1995
Saccharide-Peptide Hybrids as Novel
Oligosaccharide Mimetics""
H a n s Peter Wessel," Catherine M . Mitchell,
Cinta Maria L o b a t o , and Gerard S c h m i d
The important role of carbohydrates in physiological processes has been increasingly recognized in recent years. The establishment of "glycobiology" as a new area of research has evoked
growing interest in carbohydrate mimetics. Examples of
monosaccharide mimetics are hydroxylated piperidine derivatives, which have been investigated particularly in their role as
glycosidase inhibitors."] Oligosaccharides have been mimicked
by C-disaccharides"] or C,C-trisac~harides[~l
in which the interglycosidic oxygen atom is replaced by a methylene group. In
other disaccharide analogues, pyranose rings are coupled directI Y , [ ~ through
]
substituted methylene groups,[51or also through
more extended
We present here novel oligosaccharide
mimetics consisting of peptide-linked monomers.
Carbohydrate amino acids such as muramic acid"' are well
known in nature and have also been prepared synthetically, for
example to induce defined peptide conformations.[*] Carbohydrate amino acids of type 1 not only contain an intact carbohydrate epitope, but can also undergo reaction to form amide
linkages. This procedure has the advantage over oligomerization through glycosidic linkages that no stereoisomers are
formed; in addition, methods for the oligomerization of amino
acids in solution or on solid phase are well established. The
oligomerization of identical o r different carbohydrate amino
acids, and thus the formation of novel compound libraries,
should be feasible. The approach is exemplified1'] here by the
synthesis of a tetramer containing normuramic acid (2)""' as a
building block.
1
2
This building block is characterized by the free anomeric center, which provides the option of introducing a hydrophobic
group to increase protein binding;["' in the present example we
have employed benzyl glycosides. Normuramic acid derivatives
suited for oligomerization can be prepared in very good yield;
reaction of the known1"I glucopyranoside 3 with a bromoacetic
acid ester results in 4, which can be deblocked to furnish the free
acid 5 or, alternatively, amine 6 (Scheme 1 ) .
Coupling to give dimers has been carried out by the diester
method (Scheme 2); the known't3] benzyl glucopyranoside 7
was chosen as a terminal building block. The benzylidene protective groups were removed at the dimer level, since the cleavage from higher oligomers proved to be troublesome. The activating reagent of our choice for the highly polar acid derivative
9 was 2-chloro-4,6-dimethoxy-l,3,5-triazine
(CDMT);[14' the
coupling could be carried out in D M F as a solvent.
[*] Dr. H. P. Wessel, C. M. Mitchell."' Dr. C. M. Lohato."' Dr. G. Schmid
Pharma Division, Preclinical Research
F, Hoffmann-La Roche Ltd
CH-4002 Basel (Switzerland)
Telefax: Int. code +(61)688-6459
E-mail: hans-p.wessel(o roche.com
[ '1 Post-doctoral fellow (1993)from the University of Barcelona, Spain. Present
address: Institute of Organic Chemistry, University of Basel
['I
[**I
Summer student 1993 from the University of Basel, Switzerland. Present address: Institute of Organic Chemistry, University of Wiirzburg (Germany)
We wish to thank Roland Keller for technical assistance.
0570-0833/95/3423-2712
S 10.00+ .25/0
Angew. Chem. Int. Ed. Engl. 1995,34%No. 23/24
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