Design, synthesis and biological activity of novel C2-C3′ N-Linked macrocyclic taxoids

Article abstract of DOI:10.1016/S0960-894X(01)00747-8

A series of novel macrocyclic taxoids was designed and synthesized by connecting the C-2 and C-3′ N positions of the taxoid framework with various tethers. Cytotoxicity of these macrocyclic taxoids was evaluated against a human breast cancer cell line LCC

Full text of DOI:10.1016/S0960-894X(01)00747-8

Bioorganic & Medicinal Chemistry Letters 12 (2002) 349–352
Design, Synthesis and Biological Activity of Novel
C2-C30 N-Linked Macrocyclic Taxoids
Iwao Ojima,a,* Xudong Geng,a Songnian Lin,a Paula Perab and Ralph J. Bernackib
aDepartment of Chemistry, State University of New York at Stony Brook, NY 11794-3400, USA
bDepartment of Experimental Therapeutics, Rosewell Park Cancer Institute, Elm and Carton Streets, Buffalo, NY 14263, USA
Received 26 September 2001; accepted 5 November 2001
Abstract—A series of novel macrocyclic taxoids was designed and synthesized by connecting the C-2 and C-30 N positions of the
taxoid framework with various tethers. Cytotoxicity of these macrocyclic taxoids was evaluated against a human breast cancer cell
line LCC6-WT, and a couple of the taxoids exhibited 0.09–0.3 mM IC50 values. # 2002 Elsevier Science Ltd. All rights reserved.
Taxol1 (paclitaxel)1 and its semi-synthetic analogue
Taxotere1 (docetaxel)2 are two extremely important
chemotherapeutic drugs widely used today. These drugs
act by facilitating tubulin polymerization and stabilizing
the resulting microtubules, thereby inhibiting the nor-
mal dynamic reorganization of microtubular network
required for mitosis, which eventually induces apopto-
sis.3 This mechanism of action remained unique until
several structurally dissimilar natural products (epothi-
lones,4,5 eleutherobin,6 discodermolide,7 and laulima-
close to that found in its X-ray crystallographic analy-
sis.13 Accordingly, we became interested in designing
and synthesizing a new series of macrocyclic taxoids,
mimicking the tubulin-bound docetaxel structure pro-
posed by Nogales et al.12 Thus, we designed novel N-
linked macrocyclic taxoids 1 connecting the C-2 car-
boxylate with the C-30 N acyl moiety with various lin-
kers (Fig. 1). We report here the synthesis and biological
activity of these novel ‘N-linked’ macrocyclic taxoids 1.
lide8,9
)
were found recently to share the same
In designing the synthetic routes to the designed
N-linked macrocyclic taxoids, the ring-closing meta-
thesis (RCM)14 was chosen for the key step based on
our successful use of this method in the syntheses of the
C-linked series taxoids.10,11 The retrosynthetic analysis
for the construction of N-linked macrocyclic taxoids is
shown in Scheme 1. As Scheme 1 illustrates, N-linked
macrocyclic taxoids 1 can be obtained from diene pre-
cursor 2 by RCM. Using our b-lactam ring-opening
coupling protocol,15À20 the diene precursor 2 can be
synthesized by coupling C-2 modified baccatin 4 with
b-lactam 3 bearing a terminal alkenyl tether. Baccatin 4
can be easily prepared from 2-debenzoyl-7,10,13-tri-
TES-baccatin 5 that should be readily derived from the
mechanism of action. In order to account for this
intriguing finding, a possible common pharmacophore
for those microtubule-stabilizing agents was proposed
by these laboratories.10 Based on this common phar-
macophore model, a series of novel macrocyclic taxoids
bearing a linker connecting the C-2 and C-30 positions
was synthesized10,11 as potential hybrids of nonataxel
and epothilone B. These ‘C-linked’ macrocyclic taxoids
were found to be moderately cytotoxic against a human
breast cancer cell line LCC6.10,11
In the first common pharmacophore proposal, a
‘hydrophobic clustering’ conformation of nonataxel was
employed, which was the prevalent conformation in
aqueous media.10 However, the electron crystal-
lographic structure of paclitaxel-bound Zn2+-stabilized
a,b-tubulin dimer (3.7 A resolution) by Nogales et al.12
incorporated a docetaxel molecule with a conformation
*Corresponding author. Tel.:+1-631-632-7947; fax:+1-631-632-7942;
e-mail: iojima@notes.cc.sunysb.edu
Figure 1. ‘N-Linked’ macrocyclic taxoids 1.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00747-8

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I. Ojima et al. / Bioorg. Med. Chem. Lett. 12 (2002) 349–352
naturally occurring 10-deacetylbaccatin III (DAB). The
enantiopure b-lactam 3 can be prepared through highly
efficient chiral enolate-imine cyclocondensation,15,19À21
followed by N-acylation.
of the resulting macrocyclic taxoids 1d,e were separated
by column chromatography on silica gel to give 1d-E,
1d-Z, 1e-E, and 1e-E (Scheme 5). Hydrogenation of 1d
and 1e on Pd/C gave 1(H)d and 1(H)e, respectively, in
excellent yield (Scheme 5).
The synthesis started with 2-debenzoyl-10-deacetyl-
7,10,13-tri-TES-baccatin 5, which was prepared from
10-DAB by the previously reported method.11,22 Ester-
ification of 5 with 3-vinylbenzoic acid (6a) or (3R)-3-
methylhept-6-enoic acid (6b) in the presence of DIC and
DMAP gave desired C-2-modified baccatins 7a and 7b,
respectively. It should be noted that the formation of a
considerable amount of D-ring-opened byproduct11,23
(36–50%) was observed when using acid 6a. Optimiza-
tion for this particular reaction is in progress. Removal
of all silyl protecting groups of 7a,b by HF-pyridine,
followed by selective protection of C-7 hydroxyl group
with TES gave 2-modified-7-TES-10-deacetylbaccatin
(8a,b) in excellent yield. Subsequent selective C10
acetylation at À40 ꢀC using LiHMDS as base gave C-2-
modified baccatin 4a,b in good yield (Scheme 2).
Enantiopure b-lactams 3a–c were prepared in high
yields by acylation of N-H-b-lactam 924 with allyl
chlorofomate (10a), pent-4-enoyl chloride (10b), and
but-3-enyl chloroformate (10c) (Scheme 3).
Scheme 2. Synthesis of C-2-modified baccatin III 4. (i) DIC (3.5
equiv), DMAP, CH2Cl2, overnight: 7a, 54%; 7b: 89%; (ii) HF-pyri-
dine, pyridine, CH3CN, overnight; (iii) TESC1 (3.0 equiv), imidazole,
(4.0 equiv), DMF, 0 ꢀC to rt, 1 h; 8a, 95% for two steps; 8b, 96% for
two steps; (iv) LiHMDS (1.1 equiv), AcCl (1.1 equiv), THF, À40 ꢀC,
4a, 85%; 4b, 90%.
Ring-opening coupling of b-lactams 3a–c with C-2-
modified baccatin 4a using LiHMDS in THF at À40 ꢀC
gave dienes 2a–c in 70–80% yield. The diene precursors
2a–c were subjected to RCM using the Grubbs cata-
lyst14 in CH2Cl2 to afford the macrocyclic taxoids 11a–c
in 70–96% yield. It is worthy of note that the formation
of only E-isomers of 11a–c was observed in these RCM
reactions. Removal of silyl protecting groups gave
macrocyclic taxoids 1a–c in 74–80% yields (Scheme 4).
Hydrogenation of macrocyclic taxoids 1a–c, thus
obtained, on Pd/C afforded the corresponding macro-
cyclic taxoids 1(H)a–c bearing a saturated linker and 2-
methylpropyl group at C-30 (Scheme 4).
Scheme 3. Preparation of b-lactam 3. (i) 10 (2–5 equiv), TEA, (4–6
equiv), DMAP, CH2Cl2, 1-2 d, 3a, 89%; 3b, 80%; 3c, 88%.
In a similar manner, C-2-modified baccatin 4b was cou-
pled with b-lactam 3a,b to give dienes 2d and 2e in 93
and 77% yields, respectively. The RCM reactions of 2d
and 2e afforded 11d (E/Z=3.5, 91%) and 11e (E/Z=2,
93%) in high yields. The silyl protecting groups of 11d,e
were removed by HF-pyridine and the E and Z isomers
Scheme 4. Synthesis of macrocyclic taxoids 1a–c and 1(H)a–c. (i)
LiHMDS (1.5 equiv), 3 (1.5 equiv), THF, À40 ꢀC, 40 min; 2a, 70%;
2b, 80%; 2c, 70%; (ii) (Cy3P)2Ru(=CHPh)Cl2 (0.2 equiv), CH2Cl2,
2–3 d; 11a, 87%; 11b, 70%; 11c, 96%; (iii) HF-pyridine, pyridine,
CH3CN, overnight; 1a, 80%; 1b, 77%; 1c, 74%; (iv) H2, Pd/C, EtOAc,
overnight, 100%.
Scheme 1. Retrosynthetic analysis of macrocyclic taxoid 1.

I. Ojima et al. / Bioorg. Med. Chem. Lett. 12 (2002) 349–352
351
Table 1. Cytotoxicity (IC50, mM)a of 30-N-linked macrocyclic taxoids
Entry
Taxoid
LCC6-WTb
LCC6-MDRc
1
2
3
1a
1b
1c
0.30
0.09
1.9
3.5
3.0
18
4
5
6
7
8
9
10
11
12
1(H)a
1(H)b
1(H)c
1d-E
1d-Z
1e-E
1e-Z
1(H)d
1(H)e
0.10
2.1
7.4
2.7
0.70
2.2
0.30
9.6
1.5
21
10
19
4.0
29
9.4
19
2.0
16
aThe concentration of compound which inhibits 50% of the growth of
human tumor cell line after 72 h drug exposure.28
bLCC6-WT, human breast carcinoma.
cLCC6-MDR, MDR1 transduced line.
Scheme 5. Synthesis of macrocyclic taxoids 1d,e and 1(H)d,e. (i)
LiHMDS (1.5 equiv), 3 (1.5 equiv), THF, À40 ꢀC, 40 min; 2d, 93%;
2e, 77%; (ii) (Cy3P)2Ru(=CHPh)Cl2 (0.2 equiv), CH2Cl2, 2–3 d; 11d,
91%, E/Z=3.5/1; 11e, 93%, E/Z=2/1; (iii) HF-pyridine, pyridine,
CH3CN, overnight; 1d-E, 65%; 1d-Z, 18%; 1e-E, 56%; 1e-Z, 28%;
(iv) H2, Pd/C EtOAc, overnight; 1(H)d, 95%; 1(H)e, 99%.
identification of tubulin-bound structures of micro-
tubule-stabilizing agents will form a solid basis for the
development of the next generation de nove anticancer
agents. Further studies along this line are actively
underway in these laboratories.
Novel macrocylic taxoids 1a–e and 1(H)a–e, thus
obtained, were tested for their cytotoxicity against
human breast cancer cell lines LCC6-WT and LCC6-
MDR (drug-resistant). As Table 1 shows, most of these
N-linked series of macrocyclic taxoids exhibit IC50
values ranging from micromolar to submicromolar
against LCC6-WT cell line. (Note: IC50 of paclitaxel-
LCC6-WT, 0.0031 mM; LCC6-MDR, 0.346 mM. In
particular, 1b25 and 1(H)a26 exhibit IC50 values of 0.09
and 0.1 mM, respectively (entries 2 and 4), which show
substantial increase in cytotoxicity as compared to that
of the C-linked macrocyclic taxoids.11,27 Naturally, the
cytotoxicity of these N-linked macrocyclic taxoids
against a drug-resistant LCC6-MDR cell line is one
order of magnitude weaker than that against LCC6-
WT. It should be noted, however, the level of resistance
observed for these taxoids, 1.35–33 times, is much
smaller than that observed for paclitaxel (115 times)
against the same pair of LCC6 cell lines. Also, the fact
that the Z-isomers of 1d and 1e possess much better
potency than their E-isomers (entries 7,8 and 9,10) pro-
vides valuable information for further SAR studies.
Acknowledgements
This work has been supported by a grant from the
National Institutes of Health (NIGMS). Generous sup-
ply of 10-deacetylbaccatin III by Indena SpA, is also
gratefully acknowledged.
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20
26. 1(H)a: mp 182–184 ꢀC; ½aꢁD À33.3ꢀ (c 0.12, CHCl3); 1H
NMR (250 MHz, CDCl3) d 0.86 (m, 6H), 1.13 (s, 3H), 1.25 (m,
5H), 1.29 (s, 3H), 1.59 (s, 3H), 1.69 (s, 3H), 1.78 (s, 3H), 2.02
(m, 2H), 2.24, (s, 3H), 2.30 (m, 2H), 2.37 (s, 3H), 2.44 (s, 3H),
2.80 (m, 1H), 3.80 (d, J=7.7 Hz, 1H), 4.24 (m, 2H), 4.47 (b,
1H), 4.81 (b, 1H), 4.90 (d, J=9.3 Hz 1H), 5.14 (b, 1H), 5.35
(d, J=5.4 Hz, 1H), 5.63 (d, J=7.7 Hz, 1H), 6.26, (m, 2H),
7.38 (b, 2H), 7.89 (s, 1H), 8.17 (s, 1H). 13C NMR (62.9 MHz,
CDCl3) d 5.4, 9.6, 10.1, 14.2, 16.3, 18.2, 18.4, 21.3, 22.4, 24.6,
24.8, 25.2, 26.7, 27.4, 30.8, 30.8, 40.9, 47.2, 53.2, 53.8, 58.3,
67.4, 67.8, 70.9, 80.1, 115.0, 123.77, 124.4, 124.9, 126.0, 129.6,
135.5, 137.8, 149.2, 162.0, 165.8, 166.8, 170.2, 199.1. HRMS
(FAB, DCM/NBA/NaCl) m/z calcd for C42H55NO15Na+
832.3156, found 832.3131 (ꢀ=3.1 ppm).
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20
25. 1b: White solid, mp 160–162 ꢀC, ½aꢁD À100.0ꢀ (c 0.16,
1
CHCl3), H NMR (250 MHz, CDCl3) d 1.12 (s, 3H), 1.25 (m,
6H), 1.6 (s, 3H), 1.7 (s, 3H), 1.8 (s, 3H), 2.14–2.50 (m, 7H), 2.2
(s, 3H), 2.4 (s, 3H), 3.76 (d, J=7.4 Hz, 1H), 4.28 (dd, J=21.3,
8.4 Hz, 1H), 4.44 (b, 1H), 4.94 (m, 2H), 5.29–5.36 (m, 3H),
5.64 (d, J=7.4 Hz, 1H), 5.74 (d, J=7.2 Hz, 1H), 6.25–6.28 (m,
3H), 6.45 (d, J=15.9 Hz, 1H), 7.31–7.48 (m, 2H), 7.87 (d,
J=7.5 Hz, 1H), 8.16, (s, 1H). 13C NMR (62.9 MHz, CDCl3) d
5.3, 9.6, 10.0, 14.0, 16.3, 18.2, 18.6, 18.8, 21.3, 22.6, 23.0, 24.8,
25.2, 27.4, 30.8, 32.3, 38.5, 40.8, 45.7, 53.8, 67.4, 67.9, 68.0,
70.8, 76.0, 76.2, 80.0, 116.1, 120.3, 124.5, 124.5, 125.0, 125.1,