Design, synthesis, and biological evaluation of novel C14-C3′BzN- linked macrocyclic taxoids

Article abstract of DOI:10.1021/jo801713q

(Figure Presented) Novel macrocyclic paclitaxel congeners were designed to mimic the bioactive conformation of paclitaxel. Computational analysis of the REDOR-Taxol structure revealed that this structure could be rigidified by connecting the C14 position of the baccatin moiety and the ortho position of C3′N-benzoyl group (C3′BzN), which are ca. 7.5 A apart, with a short linker (4-6 atoms). 7-TES-14β-allyloxybaccatin III and (3R,4S)-1-(2-alkenylbenzoyl)-β-lactams were selected as key components, and the Ojima-Holton coupling afforded the corresponding paclitaxel-dienes. The Ru-catalyzed ring-closing metathesis (RCM) of paclitaxel-dienes gave the designed 15- and 16-membered macrocyclic taxoids. However, the RCM reaction to form the designed 14-membered macrocyclic taxoid did not proceed as planned. Instead, the attempted RCM reaction led to the occurrence of an unprecedented novel Ru-catalyzed diene-coupling process, giving the corresponding 15-membered macrocyclic taxoid (SB-T-2054). The biological activities of the novel macrocyclic taxoids were evaluated by tumor cell growth inhibition (i.e., cytotoxicity) and tubulin-polymerization assays. Those assays revealed high sensitivity of cytotoxicity to subtle conformational changes. Among the novel macrocyclic taxoids evaluated, SB-T-2054 is the most active compound, which possesses virtually the same potency as that of paclitaxel. The result may also indicate that SB-T-2054 structure is an excellent mimic of the bioactive conformation of paclitaxel. Computational analysis for the observed structure-activity relationships is also performed and discussed.

Full text of DOI:10.1021/jo801713q

Design, Synthesis, and Biological Evaluation of Novel
C14-C3′BzN-Linked Macrocyclic Taxoids†
Liang Sun,‡ Xudong Geng,‡ Raphae¨l Geney,‡ Yuan Li,‡ Carlos Simmerling,‡,§ Zhong Li,‡
Joseph W. Lauher,‡ Shujun Xia,| Susan B. Horwitz,| Jean M. Veith, Paula Pera,
Ralph J. Bernacki, and Iwao Ojima*,‡,§
Department of Chemistry, State UniVersity of New York at Stony Brook, Stony Brook,
New York 11794-3400, Institute of Chemical Biology & Drug DiscoVery, State UniVersity of New York at
Stony Brook, Stony Brook, New York 11794-3400, Department of Molecular Pharmacology, Albert Einstein
College of Medicine, Bronx, New York 10461, and Department of Pharmacology and Therapeutics,
Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, New York 14263
iojima@notes.cc.sunysb.edu
ReceiVed August 1, 2008
Novel macrocyclic paclitaxel congeners were designed to mimic the bioactive conformation of paclitaxel.
Computational analysis of the “REDOR-Taxol” structure revealed that this structure could be rigidified by
connecting the C14 position of the baccatin moiety and the ortho position of C3′N-benzoyl group (C3′BzN),
which are ca. 7.5 Å apart, with a short linker (4-6 atoms). 7-TES-14ꢀ-allyloxybaccatin III and (3R,4S)-1-(2-
alkenylbenzoyl)-ꢀ-lactams were selected as key components, and the Ojima-Holton coupling afforded the
corresponding paclitaxel-dienes. The Ru-catalyzed ring-closing metathesis (RCM) of paclitaxel-dienes gave the
designed 15- and 16-membered macrocyclic taxoids. However, the RCM reaction to form the designed 14-
membered macrocyclic taxoid did not proceed as planned. Instead, the attempted RCM reaction led to the
occurrence of an unprecedented novel Ru-catalyzed diene-coupling process, giving the corresponding 15-membered
macrocyclic taxoid (SB-T-2054). The biological activities of the novel macrocyclic taxoids were evaluated by
tumor cell growth inhibition (i.e., cytotoxicity) and tubulin-polymerization assays. Those assays revealed high
sensitivity of cytotoxicity to subtle conformational changes. Among the novel macrocyclic taxoids evaluated,
SB-T-2054 is the most active compound, which possesses virtually the same potency as that of paclitaxel. The
result may also indicate that SB-T-2054 structure is an excellent mimic of the bioactive conformation of paclitaxel.
Computational analysis for the observed structure-activity relationships is also performed and discussed.
Introduction
the most widely used drugs in the fight against cancer among
a variety of chemotherapeutic agents.3,4 Paclitaxel binds to the
ꢀ-tubulin subunit, accelerates the polymerization of tubulin, and
stabilizes the resultant microtubules, which leads to apoptosis
through cell-signaling cascade.5,6 Investigation into the bioactive
conformation of paclitaxel7,8 would lead to the development of
novel drugs with much simpler structures and the same or even
higher activity.
Cancer has become the leading cause of death for people
under the age of 85 in the US.1,2 Paclitaxel is currently one of
* To whom correspondence should be addressed: Phone: 631-632-1339. Fax:
631-632-7942.
† This paper is dedicated to late Professor Albert I. Meyers for his life-long
outstanding contributions to the advancement of synthetic organic chemistry,
his exceptional leadership and mentorship in the field, as well as for numerous
fond memories of him and his warm and caring personality with big heart.
‡ Department of Chemistry, State University of New York at Stony Brook.
§ Institute of Chemical Biology & Drug Discovery.
The polar and nonpolar conformations of paclitaxel were first
proposed on the basis of the structural analysis in solution and
in the solid state using NMR and X-ray crystallography.9-13
| Albert Einstein College of Medicine.
Roswell Park Cancer Institute.
9584 J. Org. Chem. 2008, 73, 9584–9593
10.1021/jo801713q CCC: $40.75 2008 American Chemical Society
Published on Web 11/01/2008

C14-C3′BzN-Linked Macrocyclic Taxoids
Organic and medicinal chemists designed a series of structurally
restrained taxoids to mimic these two conformations, but all of
the synthesized taxoids were substantially less active than
paclitaxel.7,14-16 The structural biology study of paclitaxel did
not start until the first cryo-electron microscopy (cryo-EM)
structure of paclitaxel-bound Zn2+-stabilized R,ꢀ-tubulin dimer
was reported in 1998 at 3.7 Å resolution (1TUB structure),17
which used the docetaxel crystal structure for display. The 1TUB
structure was later refined to 3.5 Å resolution with a paclitaxel
molecule (1JFF structure) in 2001.18 However, the resolution
of these cryo-EM structures was not high enough to solve the
binding conformation of paclitaxel. Thus, a computational study
of the electron-density map was performed, which led to the
proposal of the “T-Taxol” conformation.19 To prove the validity
of the “T-Taxol” structure, rigidified paclitaxel congeners were
designed, synthesized, and assayed for their tubulin polymeri-
zation ability and cytotoxicity.7,20-23 Among those “T-Taxol”
mimcs, C4-C3′-linked macrocyclic taxoids showed higher
activities than paclitaxel in the cytotoxicity and tubulin-
polymerization assays.20,22
FIGURE 1. REDOR-Taxol-1JFF and the atom-atom distance between
C14 and the ortho carbon of the C3′N-benzoyl group.
In the meantime, two 13C-19F intramolecular distances of
the microtubule-bound 2-(4-fluorobenzoyl)paclitaxel were ex-
perimentally obtained by means of the REDOR NMR study in
2000.24 On the basis of the REDOR distances, MD analysis of
paclitaxel conformers, photoaffinity labeling,25 and molecular
modeling studies using the 1TUB coordinate,17 we proposed
the “REDOR-Taxol” as a valid microtubule-bound paclitaxel
structure in 2005.8 We have further refined the “REDOR-Taxol”
using the 1JFF coordinate and found that the “REDOR-Taxol”
is not only fully consistent with the new REDOR-experiments26
but also accommodates highly active macrocyclic paclitaxel
analogues designed on the basis of the “T-Taxol” structure (see
the Supporting Information). The critical difference between
these two structures is the orientation of the C2′-OH group. In
the “REDOR-Taxol”, the C2′-OH group interacts with His227
as the hydrogen bond donor,8 while the H-bonding is between
the C2′-OH and the backbone carbonyl oxygen of Arg369 in the
“T-Taxol”.19 Accordingly, we have designed novel macrocyclic
taxoids by linking the C14 and C3′BzN groups, which mimic
the “REDOR-Taxol” structure to examine whether these con-
formationally restricted taxoids exhibit the same level of
biological activity as that of paclitaxel.
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Walsh, J. J. J. Am. Chem. Soc. 2000, 122, 5343–5353.
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J. Med. Chem. 2004, 47, 5937–5944.
Results and Discussion
Design of Novel C14-C3′BzN-Linked Macrocyclic Tax-
oids. Computational analysis of the “REDOR-Taxol” revealed
that this structure could be rigidified by connecting the C14
position of the baccatin moiety and the ortho position of C3′N-
benzoyl group (C3′BzN), which are ca. 7.5 Å apart, with a short
linker (4-6 atoms) (Figure 1). Also, the same analysis indicated
that the use of such linkers would not interfere with the amino
acid residues in the paclitaxel binding site. Since the naturally
occurring 14ꢀ-OH-10-deacetylbaccatin (14-OH-DAB) is readily
available to us and we have performed substantial SAR studies
on the taxoids derived from this material, we chose 14-OH-
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G.; Jagtap, P. G.; Studelska, D. R.; Kingston, D. G. I.; Schaefer, J.; Bane, S.
Biochemistry 2000, 39, 281–291.
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361–370.
J. Org. Chem. Vol. 73, No. 24, 2008 9585

Sun et al.
just by using (3R,4S)-4-(2-methylpropen-2-yl)-ꢀ-lactam 3d in
place of 3a-c.
As Scheme 2 shows, (3R,4S)-ꢀ-lactams 3a-d were prepared
in excellent yields through the acylation of enantiopure ꢀ-lac-
tams 1a,b with the corresponding 2-alkenylbenzoyl chlorides
2a-c34-36 in the presence of triethylamine and DMAP. 7-TES-
14ꢀ-allyloxybaccatin 6 was prepared following the method
previously reported by us from 14-OH-DAB through selective
acetylation of the C10-hydroxyl group37 and subsequent TES
protection of the C7-hydroxyl group (83% for two steps, Scheme
3).8 The Ojima-Holton coupling of ꢀ-lactams 3a-d with
baccatin 6 was carried out under the standard conditions
(LiHMDS in THF at -30 °C) to give the corresponding
paclitaxel-dienes 7a-d bearing olefinic groups at the C14
position as well as the ortho position of the C3′BzN moiety
(Scheme 3).
FIGURE 2. Designed novel C14-C3′BzN-linked macrocyclic taxoids.
As Scheme 4 shows, the RCM reactions of paclitaxel-dienes
7a and 7d catalyzed by the “1st-generation Grubbs catalyst”,
Cl2Ru()CHPh)(PCy3)2, proceeded smoothly at room temper-
ature to give the corresponding macrocyclic taxoids 8a and 8d,
respectively, which were isolated in high yields by passing
through a short silica gel column to remove the Ru catalyst.
The crude 8a and 8d, thus obtained, were subjected to the
deprotection of all silyl groups with HF-pyridine to give the
designed macrocyclic taxoids 1a and 1d, respectively, in high
yields. In both products, E-isomer was formed exclusively. We
reported the synthesis of 1a (SB-T-2053) in 2005,8 but we
needed to resynthesize this macrocyclic taxoid for additional
cytotoxicity assay for comparison purposes. We confirmed that
the reported synthesis is perfectly reproducible.
The RCM reaction of 7c proceeded slowly at room temper-
ature. Thus, the reaction mixture was heated at reflux for 3 days
until 7c had disappeared. The reaction gave E and Z isomers at
the alkene moiety formed, i.e., 8c-E and 8c-Z, in 40% and 42%
yields, respectively. The observed low reactivity of 7c and the
lack of stereoselectivity in the alkene formation can be attributed
to the larger ring size of the product(s). The silyl groups of
8c-E and 8c-Z were removed by HF-pyridine to give 1c-E
and 1c-Z, respectively, in high yields (Scheme 5).
In contrast to the RCM reactions described above, the reaction
of paclitaxel-diene 7b did not proceed as anticipated. The
attempted RCM reaction of 7b was sluggish. Thus, the reaction
was run in refluxing dichloromethane for 5 days. As often
observed in RCM reactions, deactivation of the Ru catalyst
appeared to have occurred. Accordingly, more Ru catalyst was
added during the reaction (0.25 equiv × 3). The reaction stopped
at 75% conversion (i.e., 25% of the unreacted 7c was recovered),
giving ring-closed product 8b in 50% yield. Then, the silyl
groups were removed by HF-pyridine to give the corresponding
macrocyclic taxoid 9b in high yield. However, the LCMS
analysis of 9b showed its molecular weight higher than the
anticipated RCM product 1b by 14 mass units, which was quite
puzzling. The 1H and 13C NMR and 2D NMR analyses
FIGURE 3. Overlays of REDOR-Taxol (green) with 1a (cyan), 1b
(red), and 1c (pink).
DAB as a key starting material for the synthesis of novel
macrocyclic taxoids. Thus, the C14 position of the designed
novel taxoids was fixed to ꢀ-ether functionality. The structures
of the designed macrocyclic taxoids are shown in Figure 2. As
the overlays in Figure 3 illustrate, the designed macrocyclic
taxoids 1a and 1c appear to mimic the REDOR-Taxol structure
very well, while the C3′N-benzoyl group of 1b deviates from
the rest.
In addition, we included a C3′-(2-methylpropen-2-yl) ana-
logue of 1a, i.e., 1d, for comparison, since the replacement of
the 3′-phenyl moiety with a 3′-(2-methylpropen-2-yl) group in
paclitaxel and docetaxel produced the corresponding taxoids
with higher potency.27,28
Synthesis of Macrocyclic Taxoids. As the retrosynthetic
analysis in Scheme 1 shows, the designed C14-C3′BzN-linked
macrocyclic taxoids 1a-c can be synthesized through ring-
closing metathesis (RCM)29 of diene key intermediates 7a-c,
which can be obtained from the corresponding (3R,4S)-1-(2-
alkenylbenzoyl)-ꢀ-lactam 3a-c and 14ꢀ-allyloxybaccatin 6
through the Ojima-Holton coupling.14,15,27,30,31 A similar
strategy worked very well previously for the synthesis of the
C2-C3′-linked14,32 and C2-C3′N-linked33 macrocyclic taxoids
in our laboratory. Macrocyclic taxoid 1d can be synthesized
(27) Ojima, I.; Slater, J. C.; Michaud, E.; Kuduk, S. D.; Bounaud, P.-Y.;
Vrignaud, P.; Bissery, M.-C.; Veith, J.; Pera, P.; Bernacki, R. J. J. Med. Chem.
1996, 39, 3889–3896.
(28) Ojima, I.; Chen, J.; Sun, L.; Borella, C. P.; Wang, T.; Miller, M. L.;
Lin, S.; Geng, X.; Kuznetsova, L. V.; Qu, C.; Gallager, D.; Zhao, X.; Zanardi,
I.; Xia, S.; Horwtiz, S. B.; Mallen-St. Clair, J.; Guerriero, J. L.; Bar-Sagi, D.;
Veith, J. M.; Pera, P.; Bernacki, R. J. J. Med. Chem. 2008, 51, 3203–3221.
(29) Grubbs, R. H.; Chang, S. Tetrahedron 1998, 54, 4413–4450.
(30) Ojima, I.; Habus, I.; Zhao, M.; Zucco, M.; Park, Y. H.; Sun, C. M.;
Brigaud, T. Tetrahedron 1992, 48, 6985–7012.
(32) Ojima, I.; Chakravarty, S.; Inoue, T.; Lin, S.; He, L.; Horwitz, S. B.;
Kuduk, S. D.; Danishefsky, S. J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4256–
4261.
(33) Ojima, I.; Geng, X.; Lin, S.; Pera, P.; Bernacki, R. J. Bioorg. Med.
Chem. Lett. 2002, 12, 349–352.
(34) Reich, S. H.; Melnick, M.; Pino, M. J.; Fuhry, M. A. M.; Trippe, A. J.;
Appelt, K.; Davies, J. F.; Wu, B. W.; Musick, L. J. Med. Chem. 1996, 39, 2781–
2794.
(35) Chang, S.; Grubbs, R. H. J. Org. Chem. 1998, 63, 864–866.
(36) Kim, B. M.; Park, J. K. Bull. Korean Chem. Soc. 1999, 20, 744–746.
(37) Holton, R. A.; Zhang, Z.; Clarke, P. A.; Nadizadeh, H.; Procter, D. J.
Tetrahedron Lett. 1998, 39, 2883–2886.
(31) Ojima, I. Acc. Chem. Res. 1995, 28, 383–389, and references cited
therein.
9586 J. Org. Chem. Vol. 73, No. 24, 2008

C14-C3′BzN-Linked Macrocyclic Taxoids
SCHEME 1. Retrosynthetic Analysis of the Designed Macrocyclic Taxoids 1a-c
SCHEME 2a
SCHEME 3a
a Reagents and conditions: (i) acid chloride (2.0 equiv), Et3N (4 equiv),
CH2Cl2, DMAP, overnight.
suggested that 9b should possess a butenylene unit between the
C14-O and the ortho position of the C3′N-benzoyl moiety and
the newly formed double bond should be conjugated to the
phenyl group. This proposed structure was confirmed by the
X-ray crystallographic analysis of 9b (SB-T-2054) as shown
in Figure 4. This unexpected reaction is shown in Scheme 6.
A plausible mechanism for this unique reaction is proposed
in Scheme 7. The first step of this process would be the
formation of a Ru-carbene intermediate M-1 through a normal
Ru-carbene exchange reaction. In the next step, however, a
normal metalacyclobutane intermediate M-2 is not formed,
which is very likely due to the macrocyclic ring strain for the
formation of a 14-membered ring. Since M-2 is the precursor
of the RCM product diTES-1b, the expected RCM reaction is
blocked at this stage. Instead, regioisomeric metalacyclobutane
intermediate M-3 is generated, forming a 15-membered ring.
There are two possible pathways from M-3, i.e., (i) reductive
elimination to form a cyclopropane 10 or (ii) ring-opening
through ꢀ-hydride elimination to form a σ-allylic Ru intermedi-
ate M-5a and/or its regioisomer M-5b, which may well be
a Reagents and conditions: (i) (a) Ac2O (10 equiv), CeCl3 ·7H2O (0.1
equiv), THF, rt, 2 h, (b) TESCl (3.0 equiv), imidazole (4.0 equiv), DMF,
rt, 5 h (83% in 2 steps), (ii) allyl iodide (1.1 equiv), NaHMDS (1.1 equiv),
DMF, -40 °C, 1 h (82%), (iii) 3a-d (3.0 equiv), LiHMDS (1.5 equiv),
THF, -30 to 0 °C, 2.5 h.
equilibrated via π-allylic Ru intermediate M-5. Apparently, the
first pathway did not happen since the formation of cyclopropane
product 10 was not observed. Thus, the reaction should have
proceeded through the possible pathway (ii). Since M-5a has a
clear stabilization through double-bond conjugation to the
benzene ring, the reaction gives thermodynamically favorable
product 8b through reductive elimination. To the best of our
knowledge, this is the first example of a unique ring-closing
olefin-olefin coupling reaction promoted by the Grubbs RCM
catalyst.
J. Org. Chem. Vol. 73, No. 24, 2008 9587

Sun et al.
SCHEME 4a
FIGURE 4. X-ray crystal structure of 9b (SB-T-2054).
SCHEME 6a
a Reagents and conditions: (i) (a) Cl2Ru(dCHPh)(PCy3)2 (0.2 equiv),
CH2Cl2, overnight; (ii) HF/Py, Py, CH3CN, rt, overnight.
SCHEME 5a
a Reagents and conditions: (i) (a) Cl2Ru(dCHPh)(PCy3)2 (0.25 equiv ×
3), CH2Cl2, reflux, 5d; (ii) HF/Py, Py, CH3CN, rt, overnight.
sensitive and drug-resistant human breast cancer cell lines, drug-
resistant human ovarian cancer cell line, as well as drug-sensitive
human colon and overian cancer cell lines. The results are
summarized in Table 1.
As Table 1 shows, 9b (SB-T-2054) is the most potent
compound among the macrocyclic taxoids synthesized and
evaluated. It is worthy of note that the potency of 9b is almost
equivalent to that of paclitaxel against all six cell lines; i.e., 9b
is more potent than paclitaxel against NCI/ADR, LCC6-WT,
and A2780 cell lines but less potent than paclitaxel against
MCF7, LCC6-MDR, and HT-29. The results may suggest that
9b is very closely mimicking paclitaxel’s bioactive conforma-
tion. This is also confirmed by the tubulin polymerization assay
described below. Macrocyclic taxoid 1a (SB-T-2053) is an olefin
regioisomer of 9b, which is 2-3 times less potent than 9b. The
results appear to indicate high sensitivity of the potency to the
subtle difference in the position of C3′N-benzoyl group and the
rigidity of the macrocyclic structure. Larger ring analogues 1c-E
and 1c-Z exhibit substantial loss of potency. Also, there is a
marked difference between the E isomer and the Z isomer,
wherein the Z isomer (1c-Z) is 6-8 times more potent than the
E isomer, except for the LCC6-MDR cell line. The result further
indicates the highly delicate effects of ring size and rigidity of
the macrocyclic taxoids on their potency. Macrocyclic taxoid
1d, which is a 3′-(2-methylpropen-2-yl) analogue of 1a, shows
2-30 times less potency than 1a. This result is a surprise for
a Reagents and conditions: (i) (a) Cl2Ru(dCHPh)(PCy3)2 (0.2 equiv),
CH2Cl2, overnight, flash chromatography; (ii) HF/Py, Py, CH3CN, rt,
overnight.
It should be noted that this Ru-carbene complex promoted
reaction does not regenerate the living Ru-carbene complex,
different from RCM reactions, since the final step in this process
is the reductive elimination of the alkene product 8b and Ru(II)
species without a carbene bond. The fact that 75% conversion
was attained by using 0.75 equiv (total) of Cl2Ru(dCHPh)-
(PCy3)2 may indicate that the reaction proceeded very well as
it should, although we did not recognize it at the time of the
experiment.
Evaluation of Biological Activities. Cytotoxicity of Mac-
rocyclic Taxoids against Human Breast, Ovarian, And
Colon Cancer Cell Lines. Novel macrocyclic taxoids, thus
obtained, were evaluated for their cytotoxicity against drug-
9588 J. Org. Chem. Vol. 73, No. 24, 2008

C14-C3′BzN-Linked Macrocyclic Taxoids
SCHEME 7. Plausible Mechanism for the Formation of 8d via Novel Ring-Closing Coupling
same assay.8 This result also supports our observation that 9b
is closely mimicking paclitaxel’s bioactive conformation, as
TABLE 1. Cytotoxicity of C14-C3′BzN-Linked Macrocyclic
Taxoids (IC50 nM)a
NCI/
LCC6- LCC6-
mentioned above.
taxoids
MCF-7b
A2780f HT-29g
ADRc
WTd
2.45
12.2
2.09
MDRe
Electron Microscopy Analysis. The microtubules formed
with 9b were analyzed further by electron microscopy for their
morphology and structure in comparison with those formed by
using GTP and paclitaxel. As Figure 6 shows, the microtubules
formed with 9b and paclitaxel are very similar, while those
formed with GTP are longer and more uniform.
paclitaxel
1a
9b
1c-E
1c-Z
1d
1.85
12.3
3.49
1,650
196
398
395
592
183
110
300
129
36.1
114
31.0
7.28
29.2
17.0
523
62.0
10,010 2,067
1,135
1,000
2,285 1,475
348
130
1,924
618
198
Molecular Modeling Analysis. The microtubule-bound
structure of 9b (SB-T-2054) was energy minimized in the 1JFF
coordinate and overlaid with the “REDOR-Taxol” structure in
the 1JFF ꢀ-tubulin. The overlay of 9b with the “REDOR-Taxol”
is shown in Figure 7. The H-bond between the C2′-OH of 9b
and His227 is 2.7 Å.
Figure 8 shows the overlay of 9b with 1a (SB-T-2053). Those
two structures are very close, but there is a slight difference in
the position of the C3′N-benzoyl group as well as the rigidity
of the macrocyclic structure; i.e., 9b is more rigid than 1a, due
to the conjugation of the ethenyl group to the benzoyl group.
a Concentration of compound which inhibits 50% (IC50, nM) of the
growth of human tumor cell line after 72 h drug exposure. b MCF7:
human breast carcinoma cell line. c NCI/ADR: multidrug resistant
human ovarian carcinoma cell line. d LCC6-WT: human breast
carcinoma cell line (Pgp-). e LCC6-MDR: mdr1-transduced cell line
(Pgp+). f A2780 human ovarian carcinoma cell line. g HT-29 human
colon carcinoma cell line.
us since the replacement of the 3′-phenyl moiety of paclitaxel
and docetaxel with a 2-methylpropen-2-yl group has been shown
to increase the potency.27,28 Thus, the result may suggest that
the second-generation taxoids bearing 3′-(2-methylpropen-2-yl)
moiety have a slightly different binding site from that of
paclitaxel or these taxoids have a different bioactive conforma-
tion that that of paclitaxel.
Microtubule Polymerization Assay. The activity of 9b (SB-
T-2054) was evaluated in in vitro tubulin polymerization assay.
Paclitaxel was used as the standard for comparison. Changes
in absorbance in this spectrophotometric assay provide the direct
measure of turbidity, hence indicating the extent of tubulin
polymerization. As Figure 5 shows, 9b induced tubulin polym-
erization in the absence of GTP in a manner similar to that of
paclitaxel. The microtubules formed with 9b as well as paclitaxel
were stable against Ca2+-induced depolymerization. We have
already reported that 1a (SB-T-2053) also exhibited virtually
the same or slightly better activity than that of paclitaxel in the
FIGURE 5. Tubulin polymerization with 9b and paclitaxel: microtubule
protein 1 mg/mL, 37 °C, GTP 1 mM, drug 10 µM.
J. Org. Chem. Vol. 73, No. 24, 2008 9589

Sun et al.
FIGURE 6. Electromicrographs of microtubule: (a) GTP, (b) paclitaxel, and (c) 9b (SB-T-2054).
FIGURE 7. Overlays of REDOR-Taxol (green) with 9b (SB-T-2054)
(purple).
FIGURE 9. Overlay of REDOR-Taxol (green) with 1c-E (pink) and
1c-Z (yellow).
(see the Supporting Information). The results indicate that the
X-ray crystal structure of 9b (see Figure 5) is just one of the
preferred conformations in a crystalline state. Also, the dihedral
angle of C13-C1′-C2′-O2′ of 9b does not match those of
the “REDOR-Taxol” and the “T-Taxol”, which may suggest
that both proposed bioactive conformations of paclitaxel need
further refinements.
In summary, novel C14-C3′BzN-linked macrocyclic taxoids
1a (SB-T-2053), 1c-E, 1c-Z, 1d, and 9b (SB-T-2054) were
synthesized based on the computational design, mimicking the
“REDOR-Taxol” structure. The Ru-catalyzed RCM reaction was
employed in the key macrocyclization step of the paclitaxel-
dienes 7a-d. The formation of 15- and 16-membered macro-
cyclic taxoids (1a, 1c, and 1d) proceeded as anticipated, while
that of 14-membered macrocyclic taxoid 1b did not go through.
However, a novel Ru-catalyzed intramolecular coupling of two
ethenyl moieties was discovered instead, which gave 15-
membered macrocyclic taxoid 9b (SB-T-2054). This new
process is likely to involve a metalacyclobutane intermediate,
regioisomeric to the RCM intermediate, followed by ꢀ-hydride
elimination and reductive elimination. Evaluation of the biologi-
cal activities of these novel macrocyclic taxoids has revealed
that their potency is highly sensitive to the subtle conformational
differences as well as the rigidity of the compounds. Compu-
tational analysis for the structure-activity relationships of these
novel macrocyclic taxoids is consistent with this observation.
Macrocyclic taxoid 9b is found to be the most potent compound
among those synthesized and assayed. It is noteworthy that 9b
exhibits virtually the same potency as that of paclitaxel in the
cytotoxicity and tubulin-polymerization assays as well as
morphology analysis of microtubules by electron microscopy,
which may indicate that 9b structure almost perfectly mimics
the bioactive conformation of paclitaxel.
FIGURE 8. Overlay of 9b (purple) and 1a (SB-T-2053) (cyan).
These subtle differences appear to be correlated to the 2-3 times
difference in their potency,
Figure 9 shows the overlay of 1c-E, 1c-Z, and the “REDOR-
Taxol”. As the cytotoxicity assay revealed (see Table 1), there
is a 6-8 times difference in the potency of these two
stereoisomers; i.e., 1c-Z is substantially more potent than 1c-
E. It is clear that 1c-Z has a C3′N-benzoyl group orientation
closer to that of 1a and 9b as compared to that of 1c-E and
1c-Z is more rigid than 1c-E. Monte Carlo conformational
analysis of 1c-E and 1c-Z in a simulated aqueous environment
supports the substantial difference in the rigidity of these two
stereoisomes (see the Supporting Information).
We also performed the Monte Carlo conformational analysis
of 9b and 1c under the same conditions. We started from their
“REDOR-Taxol” conformation as well as “T-Taxol” conforma-
tion, but both converged to the same distribution of conformers
9590 J. Org. Chem. Vol. 73, No. 24, 2008

C14-C3′BzN-Linked Macrocyclic Taxoids
(3R,4S)-1-(2-Allyloxybenzoyl)-3-triethylsiloxy-4-phenylazeti-
din-2-one (3c): colorless oil; 1H NMR (300 MHz, CDCl3) δ
0.40-0.50 (m, 6 H), 0.75-0.81 (m, 9 H), 4.62 (dd, J ) 5.7, 1.2
Hz, 2 H), 5.10 (d, J ) 6.0 Hz, 1 H), 5.22 (dd, J ) 10.5, 0.9 Hz, 1
H), 5.36 (d, J ) 6.0 Hz, 1 H), 5.45 (dd, J ) 17.4, 1.8 Hz, 1 H),
6.04-6.20 (m, 1 H), 6.96-7.03 (m, 2 H), 7.32-7.45 (m, 7 H);
13C NMR (75 Hz, CDCl3) δ 4.34, 6.15, 61.5, 69.7, 70.1, 112.2,
118.3, 120.5, 124.2, 127.8, 127.9, 128.0, 129.4, 132.8, 132.9, 133.6,
156.9, 164.6, 165.0; HRMS (FAB/DCM/NaCl) m/z calcd for
C25H31NO4SiH+ 438.2100, found 438.2088 (∆ ) -2.7 ppm).
14ꢀ-Allyloxy-7-triethylsilylbaccatin III (6). To a solution of 5
(427 mg, 0.596 mmol) in DMF (20 mL) was added 1.0 M sodium
bis(trimethylsilyl)amide (NaHMDS) in THF (0.656 mL, 0.656
mmol) at -40 °C, the reaction mixture was stirred for 5 min, and
then allyl iodide (0.060 mL, 0.656 mmol) was added. The reaction
was quenched after 10 min with saturated aqueous NH4Cl (5 mL).
The reaction mixture was extracted with ethyl acetate (100 mL)
and washed with water (10 mL × 3) and brine (10 mL). The organic
layer was dried over anhydrous MgSO4 and filtered, and the solvent
was removed under reduced pressure. The residue was purified on
a silica gel column using hexanes/EtOAc (6/1) as the eluant to
afford 6 as a white solid (361 mg, 80%): mp 133-135 °C; 1H NMR
(300 MHz, CDCl3) δ 0.55 (m, 6 H), 0.90 (m, 9 H), 1.19 (s, 3 H),
1.60 (m, 2 H), 1.66 (s, 3 H), 1.84 (m, 1 H), 2.00 (m, 3 H), 2.12 (s,
3 H), 2.29 (s, 3 H), 2.50 (m, 1 H), 3.22 (d, J ) 4.2 Hz, 1 H), 3.69
(d, J ) 5.7 Hz, 1 H), 3.77 (m, 2 H), 4.06 (m, 1 H), 4.18 (m, 1 H),
4.29-4.47 (m, 3 H), 4.69 (bs, 1 H), 4.87-4.98 (m, 3 H), 5.61-5.72
(m, 1 H), 5.83 (d, J ) 6.9 Hz, 1 H), 7.42 (m, 2 H), 7.55 (m, 1 H),
8.06 (m, 2 H); 13C NMR (62.9 MHz, CDCl3) δ 5.1, 5.4, 6.6, 6.7,
9.7, 10.0, 14.7, 20.8, 21.4, 22.4, 26.2, 37.1, 42.7, 46.4, 58.7, 72.1,
73.0, 74.5, 75.6, 75.7, 76.2, 76.4, 80.9, 81.5, 82.0, 84.0, 117.4,
128.4, 129.7, 129.8, 133.3, 133.6, 133.7, 141.2, 165.6, 169.3, 170.1,
201.9; HRMS (FAB/DCM/NaCl) m/z calcd for C40H56O12SiH+
757.3619, found 757.3643 (∆ ) -3.1 ppm).
Experimental Section
General Methods. 1H and 13C NMR spectra were measured on
a Varian 300, 400, 500, or 600 MHz NMR spectrometer or a Bruker
AC-250 NMR spectrometer. Melting points were measured on a
Thomas-Hoover Capillary melting point apparatus and are uncor-
rected. Specific optical rotations were measured on a Perkin-Elmer
Model 241 polarimeter. IR spectra were recorded on a Perkin-Elmer
Model 1600 FT-IR spectrophotometer. TLC was performed on
Merck DC-alufolien with Kieselgel 60F-254, and column chroma-
tography was carried out on silica gel 60 (Merck; 230-400 mesh
ASTM). Purity was determined with a Waters HPLC assembly
consisting of dual Waters 515 HPLC pumps, a PC workstation
running Millennium 32, and a Waters 996 PDA detector, using a
Phenomenex Curosil-B column, employing CH3CN/water (2/3) as
the solvent system with a flow rate of 1 mL/min. High-resolution
mass spectra were obtained at the Mass Spectrometry Laboratory,
University of Illinois at Urbana-Champaign, Urbana, IL, or the
Mass Spectrometry Facility, University of California, Riverside,
CA.
Materials. The chemicals were purchased from a commercial
supplier and used as received or purified before use by standard
methods. Tetrahydrofuran was freshly distilled from sodium metal
and benzophenone. Dichloromethane was also distilled immediately
prior to use under nitrogen from calcium hydride. N,N-Dimethyl-
formamide (DMF) was distilled over 4A molecular sieves under
reduced pressure. 4-(N,N-Dimethylamino)pyridine (DMAP) was
used as received. 14ꢀ-Hydroxyl-deacetylbaccatin III (DAB, 4) was
obtained from a commercial supplier. (3R,4S)-3-Triethylsiloxy-4-
phenylazetidin-2-one (1a)15 and (3R,4S)-3-tert-butyldimethylsiloxy-
4-(2-methylpropen-2-yl)azetidin-2-one (1b)27 were prepared by the
methods we reported. (3R,4S)-1-(2-Allylbenzoyl)-3-triethylsiloxy-
4-phenylazetidin-2-one (3a), 7-triethylsilyl-14ꢀ-hydroxybaccatin III
(5), 14ꢀ-allyloxy-3′N-debenzoyl-3′N-(2-allylbenzoyl)-7,2′-triethyl-
silylpaclitaxel (7a), and macrocyclic taxoid 1a (SB-T-2053) were
synthesized using our methods previously reported.8
1-(2-Alkenylbenzoyl)-ꢀ-lactams (3a-d). The procedure for the
synthesis of ꢀ-lactam 3d is described as a typical example. To a
solution of (3R,4S)-3-triisopropylsiloxy-4-phenylazetidin-2-one (1b)
(106 mg, 0.365 mmol), triethylamine (0.2 mL, 1.46 mmol), and
DMAP (8 mg, 0.073 mmol) in CH2Cl2 (2 mL) was added
2-allylbenzoyl chloride (2a) (2 equiv) in a solution of CH2Cl2 (2
mL) dropwise at room temperature. The mixture was stirred
overnight at room temperature, and the reaction was quenched with
saturated aqueous NH4Cl solution (2 mL). The mixture was then
extracted with CH2Cl2 (10 mL × 3). The combined extracts were
dried over anhydrous MgSO4 and concentrated in vacuo. The crude
product was purified on a silica gel column using hexanes/EtOAc
(9/1) as the eluent to afford (3R,4S)-1-(2-allylbenzoyl)-3-triisopro-
pylsiloxy-4-(2-methylprop-1-enyl)azetidin-2-one (3d) as a colorless
7,2′-Disilylpaclitaxel-diene Analogues 7a-d, Substrates for
RCM Reactions. The procedure for the synthesis of 7d is described
as a typical example (for 7a, see Materials, vide supra). To a
solution of 6 (100 mg, 0.13 mmol) and ꢀ-lactam 3d (3 equiv) in
THF (15 mL) was added 1 M LiHMDS in THF (0.2 mL, 0.20
mmol) at -40 °C. The reaction mixture was warmed to -20 °C
over 1 h and then quenched with saturated aqueous NH4Cl solution
(10 mL) and extracted with EtOAc (30 mL × 3). The organic layers
were combined, and solvent was removed by reduced pressure
vacuum. The residue was purified by using chromatography column
on silica gel using hexanes/EtOAc (9/1) to afford 7-triethylsilyl-
14ꢀ-allyloxy-2′-triisopropyl-3′-dephenyl-3′-(2-methylprop-1-enyl)-
3′N-debenzoyl-3′N-(2-allylbenzoyl)paclitaxel (7d) as a white solid
1
(115 mg, 73%): H NMR (300 MHz, CDCl3) δ 0.60-0.65 (m, 6
H), 0.93-1.00 (m, 9 H), 1.18 (s, 21 H), 1.32 (s, 3 H), 1.77 (s, 3
H), 1.83 (s, 3 H), 1.91-2.03 (m, 3 H), 1.92 (s, 3 H), 2.05 (s, 3 H),
2.20 (s, 3 H), 2.43 (s, 3 H), 2.50-2.54 (m, 1 H), 3.45-3.61 (m, 2
H), 3.74 (s, 1 H), 3.84 (m, 2 H), 3.92-3.98 (m, 1 H), 4.30-4.32
(d, J ) 8.4 Hz, 1 H), 4.43-4.58 (m, 3 H), 4.69 (s, 1 H), 4.79 (d,
J ) 9.9 Hz, 1 H), 4.94-5.09 (m, 3 H), 5.14-5.20 (m, 1 H), 5.45
(d, J ) 9.0 Hz, 1 H), 5.67-5.81 (m, 1 H), 5.91-6.11 (m, 3 H),
6.48-6.51 (m, 2 H), 7.22-7.26 (m, 2 H), 7.30-7.54 (m, 5),
8.14-8.16 (m, 2 H); 13C NMR (62.9 MHz, CDCl3) δ 5.3, 6.7, 9.8,
12.6, 14.5, 17.9, 18.1, 18.7, 20.1, 22.7, 25.7, 26.1, 37.2, 43.3, 46.0,
50.8, 58.7, 72.2, 72.5, 74.5, 75.0, 75.1, 76.4, 78.7, 78.9, 81.6, 84.1,
115.8, 118.2, 122.2, 126.2, 127.3, 128.5, 129.6, 129.9, 130.1, 133.0,
133.4, 135.4, 136.0, 136.2, 136.4, 137.4, 137.9, 165.5, 168.5, 169.3,
171.3, 172.0, 201.1; HRMS calcd. for C66H95NO15Si2H+ 1198.6318,
found 1198.6320 (∆) -0.1 ppm).
1
oil (126 mg, 80% yield): H NMR (300 MHz, CDCl3) δ 0.99 (m,
21 H), 1.81 (br s, 6 H), 3.42-3.61 (m, 2 H), 4.98-5.04 (m, 4 H),
5.36 (d, J ) 8.2 Hz, 1 H), 5.87-6.01 (m, 1 H), 7.27 (m, 2 H), 7.37
(m, 1 H), 7.50 (m, 1 H); 13C NMR (75.0 MHz, CDCl3) δ 12.2,
17.4, 18.3, 26.0, 37.3, 56.3, 116.0, 117.7, 125.8, 128.5, 129.9, 131.4,
133.6, 136.8, 138.4, 140.4, 165.6, 166.7. HRMS (EI) m/z calcd for
C26H39NO3SiH+ 442.2777, found 442.2776 (∆ ) 0.1 ppm).
In a similar manner, ꢀ-lactams 3a-c were prepared from (3R,4S)-
3-triethylsiloxy-4-phenylazetidin-2-one (1a) (for 3a, see Materials,
vide supra).
(3R,4S)-1-(2-Ethenylbenzoyl)-3-triethylsiloxy-4-phenylazeti-
din-2-one (3b): colorless oil; 1H NMR (600 MHz, CDCl3) δ 0.44
(m, 6 H), 0.77 (m, 9 H), 5.15 (d, J ) 6.6 Hz, 1 H), 5.33 (m, 2 H),
5.69 (d, J ) 17.4 Hz, 1 H), 6.94 (dd, J ) 17.4, 10.8 Hz, 1 H), 7.33
(m, 6 H), 7.47 (m, 1 H), 7.52 (m, 1 H), 7.59 (d, J ) 6.8 Hz, 1 H);
13C NMR (100.5 MHz, CDCl3) δ 4.3, 6.16, 61.4, 76.9, 117.3, 126.1,
127.2, 128.1, 128.1, 128.3, 128.5, 131.3, 132.1, 133.5, 133.8, 136.9,
165.3, 166.4; HRMS (FAB/DCM/NaCl) m/z calcd for
C24H29NO3SiH+ 408.1995, found 408.1995 (∆ ) 0 ppm).
14ꢀ-Allyloxy-3′N-debenzoyl-3′N-(2-ethenylbenzoyl)-7,2′-tri-
ethylsilylpaclitaxel (7b): white solid; mp 99-102 °C; [R]20D -42
1
(c 0.6, CHCl3); H NMR (600 MHz, CDCl3) δ 0.34-0.47 (m, 6
H), 0.55-0.63 (m, 6 H), 0.78 (m, 9 H), 0.93 (m, 9 H), 1.15 (s, 3
H), 1.31 (s, 3 H), 1.74 (s, 3 H), 1.95 (m, 1 H), 2.03 (s, 3 H), 2.19
(s, 3 H), 2.56 (m, 1 H), 2.60 (s, 3 H), 3.67 (s, 1 H), 3.82 (d, J )
J. Org. Chem. Vol. 73, No. 24, 2008 9591

Sun et al.
7.2 Hz, 1 H), 3.84 (d, J ) 8.0 Hz, 1 H), 3.93 (dd, J ) 10.8, 4.2
Hz, 1 H), 4.28 (d, J ) 9.0 Hz, 1 H), 4.32 (dd, J ) 10.5, 4.0 Hz, 1
H), 4.47 (m, 3 H), 4.62 (d, J ) 10.5 Hz, 1 H), 4.71 (d, J ) 1.2 Hz,
1 H), 5.00 (d, J ) 9.0 Hz, 1 H), 5.32 (d, J ) 7.6 Hz, 1 H), 5.36
(m, 1 H), 5.61 (d, J ) 8.4 Hz, 1 H), 5.70 (d, J ) 11.4 Hz, 1 H),
5.99 (d, J ) 7.8 Hz, 1 H), 6.21 (d, J ) 7.0 Hz, 1 H), 6.47 (s, 1 H),
6.99 (m, 2 H), 7.26-7.48 (m, 8 H), 7.50-7.58 (m, 3 H), 8.10 (d,
J ) 8.4 Hz, 2 H); 13C NMR (75.5 MHz, CDCl3) δ 4.3, 5.3, 6.4,
6.7, 9.9, 14.4, 20.8, 23.3, 26.1, 37.3, 43.5, 45.9, 56.1, 58.8, 72.3,
72.5, 74.8, 74.9, 76.5, 76.6, 78.6, 78.6, 81.7, 84.1, 117.2, 117.8,
126.3, 126.5, 127.7, 128.0, 128.7, 129.5, 129.9, 130.6, 132.7, 133.5,
134.2, 134.8, 135.6, 136.2, 136.5, 138.6, 165.6, 168.3, 169.4, 169.7,
171.4, 201.0; HRMS (FAB/DCM/NaCl) m/z calcd for
C64H85NO15Si2H+ 1164.5536, found 1164.5535 (∆ ) 0.1 ppm).
14ꢀ-Allyloxy-3′N-debenzoyl-3′N-(2-allyloxybenzoyl)-7,2′-tri-
ethylsilylpaclitaxel (7c): white solid; 1H NMR (400 MHz, CDCl3)
δ 0.28-0.48 (m, 6 H), 0.52-0.66 (m, 6 H), 0.76-0.86 (m, 9 H),
0.88-0.98 (m, 9 H), 1.11 (s, 3 H), 1.25 (s, 3 H), 1.73 (s, 3 H),
1.88-1.98 (m, 1 H), 2.01 (s, 3 H), 2.17 (s, 3 H), 2.50-2.60 (m, 1
H), 2.66 (s, 3 H), 3.74 (s, 1 H), 3.76-3.88 (m, 3 H), 4.20 (dd, J )
10.8, 4.4 Hz, 1 H), 4.28 (d, J ) 8.4 Hz, 1 H), 4.42-4.55 (m, 4 H),
4.70 (d, J ) 1.6 Hz, 1 H), 4.74-4.83 (dd, J ) 13.2, 5.2 Hz, 1 H),
4.83-4.89 (dd, J ) 12.8, 5.6 Hz, 1 H), 5.0 (d, J ) 8.4 Hz, 1 H),
5.20-5.30 (m, 1 H), 5.34 (d, J ) 10.4 Hz, 1 H), 5.44 (d, J ) 16.4
Hz, 1 H), 5.64 (d, J ) 6.4 Hz, 1 H), 5.96 (d, J ) 7.2 Hz, 1 H),
6.16-6.30 (m, 2 H), 6.45 (s, 1 H), 6.92-7.0 (m, 2 H), 7.26-7.42
(m, 6 H), 7.48 (t, J ) 7.2 Hz, 2 H), 7.59 (t, J ) 7.6 Hz, 1 H), 8.02
(dd, J ) 1.6, 8 Hz, 1 H), 8.10 (d, J ) 7.2 Hz, 2 H), 9.09 (d, J )
7.6 Hz, 1 H); 13C NMR (100 MHz, CDCl3) δ 4.3, 5.3, 6.5, 6.7,
9.9, 14.3, 20.9, 22.4, 23.4, 26.0, 29.7, 37.3, 43.4, 45.9, 56.6, 58.8,
70.3, 72.2, 72.7, 74.7, 74.9, 75.1, 76.6, 78.2, 78.5, 81.6, 84.2, 113.0,
117.2, 118.6, 121.1, 126.6, 127.6, 128.5, 128.6, 129.6, 129.9, 132.5,
132.8, 132.9, 133.4, 135.4, 136.4, 139.0, 157.1, 164.6, 165.6, 169.4,
170.0, 171.2, 201.0; HRMS (FAB/DCM/NaCl) m/z calcd for
C65H87NO16Si2H+ 1194.5462, found 1194.5509 (∆ ) 3.9 ppm).
Macrocyclic Taxoid 1c (E and Z). To a solution of 7c (108 mg,
0.09 mmol) in dry CH2Cl2 (25 mL) was added Cl2Ru(dCHPh)-
(PCy3)2 (17 mg, 0.022 mmol) in dry CH2Cl2 (1 mL) under a nitrogen
atmosphere. The reaction was stirred for 3 days at reflux to convert
all of the starting materials. Solvent was removed in vacuo. Two
products were separated by flash chromatography on silica gel
(hexanes/EtOAc ) 4/1-3/1) to afford 8c-E as a crude yellow solid
(42 mg, 40%) and 8c-Z as a crude yellow solid (44 mg, 42%).
To a solution of 8c-E (35 mg) in CH3CN (0.7 mL) and pyridine
(0.7 mL) was added HF-pyridine (70:30, 0.35 mL) at 0 °C under
nitrogen, and the mixture was stirred overnight. Ethyl acetate (60
mL) was added to the reaction mixture, and the resulting solution
was washed with saturated aqueous NaHCO3 (10 mL × 2), aqueous
CuSO4 (8 mL × 3), water (10 mL × 3), and brine (5 mL). The
organic layer was dried over anhydrous MgSO4, and solvent was
removed in vacuo. Flash chromatography of the residue on silica
gel (hexanes/EtOAc ) 1/2) afforded 1c-E as a white solid (24 mg,
85%). In the same manner, 8c-Z (48 mg) was deprotected to give
1c-Z as a white solid (35 mg, 91%).
131.7, 132.0, 132.9, 133.6, 134.4, 136.9, 138.7, 156.4, 165.2, 165.7,
170.3, 171.0, 202.9; HRMS m/z calcd for C51H55NO16H+ 938.3599,
found 938.3590 (∆ ) -0.9 ppm).
Macrocyclic taxoid 1c-Z: white solid; mp 166-168 °C; [R]20
D
-73 (c 3.7, CHCl3); 1H NMR (500 MHz, CDCl3) δ 1.22 (s, 3 H),
1.24 (s, 3 H), 1.69 (s, 3 H), 1.84 (s, 3 H), 1.87 (m, 1 H), 2.04 (s,
3 H), 2.24 (s, 3 H), 2.40 (d, J ) 4.0 Hz, 1 H), 2.50-2.51 (m, 1 H),
2.97 (d, J ) 8.5 Hz, 1 H), 3.44 (s, 1 H), 3.74 (d, J ) 7.5 Hz, 1 H),
3.85 (d, J ) 8.0 Hz, 1 H), 4.14 (d, J ) 11.0 Hz, 1 H), 4.19 (d, J
) 8.0 Hz, 1 H), 4.20 (d, J ) 8.5 Hz, 1 H), 4.34-4.44 (m, 3 H),
4.60 (dd, J ) 12.5, 8.5 Hz, 1 H), 4.91 (d, J ) 9.0 Hz, 1 H), 5.03
(dd, J ) 8.5, 3.5 Hz, 1 H), 5.49 (dd, J ) 5.5, 4.0 Hz, 1 H),
5.70-5.77 (m, 1 H), 5.82 (t, J ) 9.0 Hz, 1 H), 5.92 (d, J ) 7.5
Hz, 1 H), 6.20 (d, J ) 6.5 Hz, 1 H), 6.28 (s, 1 H), 7.00 (d, J ) 8.0
Hz, 1 H), 7.24-7.26 (m, 1 H), 7.34-7.44 (m, 7 H), 7.60-7.66
(m, 2 H), 8.03 (d, J ) 7.5 Hz, 2 H), 8.35 (dd, J ) 7.5, 1.0 Hz, 1
H), 8.49 (d, J ) 6.0 Hz, 1 H); 13C NMR (100.5 MHz, CDCl3) δ
9.4, 14.7, 20.8, 22.2, 23.4, 26.2, 35.4, 43.4, 44.9, 58.6, 59.5, 63.3,
71.1, 71.9, 72.8, 72.9, 75.2, 75.9, 77.2, 78.0, 78.6, 78.8, 81.0, 84.3,
111.8, 122.2, 123.2, 128.1, 128.6, 128.7, 129.7, 129.9, 132.8, 133.5,
133.6, 134.6, 134.7, 135.4, 138.2, 156.3, 165.1, 165.6, 169.9, 171.1,
174.1, 202.9; HRMS m/z calcd for C51H55NO16H+ 938.3599, found
938.3589 (∆ ) -1.0 ppm).
Macrocyclic Taxoid 1d (SB-T-2061). To a solution of 7d (45
mg, 0.04 mmol) in CH2Cl2 (20 mL) was added Cl2Ru(dCHPh)-
(PCy3)2 (8 mg, 0.009 mmol) in CH2Cl2 (10 mL). The reaction was
stirred overnight, and solvent was removed by reduced pressure
vacuum. The residue was passed through a short silica gel column
to remove the catalyst to afford 8d as a yellow solid (80%).
To a solution of 8d thus obtained in CH3CN (1.0 mL) and
pyridine (1.0 mL) was added HF-pyridine (70:30, 0.5 mL), and
the reaction mixture was stirred overnight. The reaction mixture
was quenched with EtOAc (100 mL) and washed with saturated
aqueous NaHCO3 solution (10 mL), CuSO4 solution (10 mL × 3),
water (10 mL × 3), and brine (3 mL). The organic layer was dried
over anhydrous MgSO4, and solvent was removed under reduced
pressure. The residue was purified by a column chromatography
on silica gel using hexanes/EtOAc (2:1) as the eluent to afford 1d
as white solid (83%): mp 198-200 °C; 1H NMR (300 MHz, CDCl3)
δ 1.19 (s, 3 H), 1.23 (s, 3 H), 1.72 (s, 3 H), 1.90 (s, 3 H), 1.91 (s,
3 H), 1.92 (s, 3 H), 2.27 (s, 3 H), 2.39 (s, 3 H), 3.23-3.28 (m, 2
H), 3.80-3.82 (m, 2 H), 3.88 (d, J ) 5.1 Hz, 1 H), 3.98-4.05 (m,
2 H), 4.25 (d, J ) 5.4 Hz, 1 H), 4.40-4.48 (m, 2 H), 4.54 (d, J )
3.6 Hz, 1 H), 5.02 (d, J ) 5.4 Hz, 1 H), 5.19-5.31 (m, 2 H), 5.49
(d, J ) 9.3 Hz, 1 H), 5.68-5.77 (dt, J ) 15.0, 6.3 Hz, 1 H), 5.99
(d, J ) 7.2 Hz, 1 H), 6.16 (dd, J ) 7.8, 1.8 Hz, 1 H), 6.31-6.35
(m, 2 H), 7.18-7.21 (m, 1 H), 7.29-7.34 (m, 1 H), 7.38-7.55
(m, 4 H), 7.64-7.66 (m, 1 H), 8.09-8.13 (m, 2 H); 13C NMR
(62.9 MHz, CDCl3) δ 9.4, 14.9, 18.8, 20.8, 22.4, 22.9, 26.1, 35.5,
36.1, 43.4, 45.0, 49.8, 58.8, 72.0, 72.5, 74.5, 75.2, 77.1, 80.3, 81.5,
84.3, 118.5, 126.4, 126.8, 127.6, 128.6, 129.4, 130.0, 130.5, 131.1,
131.7, 133.7, 134.5, 135.7, 137.4, 138.3, 140.8, 165.2, 168.9, 170.0,
171.1, 171.8, 202.9; HRMS calcd for C49H57NO15H+ 900.3806,
found 900.3805 (∆) 0.2 ppm).
Macrocyclic Taxoid 9b (SB-T-2054). To a solution of 7b (43
mg, 0.036 mmol) in CH2Cl2 (22 mL) was added Cl2Ru(dCHPh)-
(PCy3)2 (8 mg × 3, 0.027 mmol) in CH2Cl2 (0.08 mL). The reaction
was refluxed for 5 days, and solvent was removed by reduced
pressure vacuum. The residue was passed through a short column
(eluent: hexanes/EtOAc ) 3/1) to remove the catalyst to afford 8b
as a crude yellow solid (20 mg, 50%) with unreacted diene (7b, 11
mg, 25%) recovered.
To a solution of 8b (20 mg) in CH3CN (0.4 mL) and pyridine
(0.4 mL) was added HF-pyridine (70:30, 0.2 mL), and the reaction
mixture was stirred overnight. The reaction mixture was quenched
with EtOAc (50 mL) and washed with saturated aqueous NaHCO3
solution (10 mL × 2), CuSO4 solution (10 mL × 3), water (10 mL
× 3), and brine (3 mL). The organic layer was dried over anhydrous
MgSO4, and solvent was removed under reduced pressure. The
Macrocyclic taxoid 1c-E: white solid; mp 194-196 °C; [R]20
D
1
-107 (c 12.7, CHCl3); H NMR (400 MHz, CDCl3) δ 1.08 (s, 3
H), 1.12 (s, 3 H), 1.60 (s, 3 H), 1.64 (s, 3 H), 1.78-1.86 (m, 1 H),
2.13 (s, 3 H), 2.34 (s, 1 H), 2.36 (s, 3 H), 2.44-2.56 (m, 1 H),
3.27 (s, 1 H), 3.69-3.77 (m, 3 H), 4.08-4.19 (m, 3 H), 4.30-4.33
(m, 3 H), 4.60-4.68 (m, 2 H), 4.92 (d, J ) 8.4 Hz, 1 H), 5.57 (d,
J ) 15.6 Hz, 1 H), 5.82 (dd, J ) 3.2, 9.2 Hz, 1 H), 5.86 (d, J )
7.2 Hz, 1 H), 5.94 (m, 1 H), 6.16 (s, 1 H), 6.25 (d, J ) 6.0 Hz, 1
H), 6.89 (d, J ) 8.4 Hz, 1 H), 7.04 (t, J ) 7.6 Hz, 1 H), 7.17-7.24
(m, 3 H), 7.40 (t, J ) 7.6 Hz, 3 H), 7.47 (d, J ) 7.2 Hz, 2 H), 7.53
(t, J ) 7.2 Hz, 1 H), 7.92 (dd, J ) 7.6, 1.2 Hz, 1 H), 8.02 (d, J )
7.6 Hz, 2 H), 8.52 (d, J ) 9.2 Hz, 1 H); 13C NMR (100 MHz,
CDCl3) δ 9.4, 15.3, 20.7, 22.2, 22.5, 26.2, 29.7, 35.6, 43.2, 45.1,
54.8, 58.9, 69.1, 72.1, 72.9, 74.5, 75.3, 76.3, 77.1, 81.5, 83.5, 84.3,
112.3, 121.5, 122.7, 123.9, 127.5, 127.6, 128.1, 128.6, 129.6, 129.9,
9592 J. Org. Chem. Vol. 73, No. 24, 2008

C14-C3′BzN-Linked Macrocyclic Taxoids
residue was purified by a column chromatography on silica gel using
Tris base (pH 10.5) for 5 min on a gyratory shaker. Optical density
was measured at 570 nm. The IC50 values were then calculated by
fitting the concentration-effect curve data with the sigmoid-Emax
model using nonlinear regression, weighted by the reciprocal of
hexanes/EtOAc (1:1) as the eluent to afford 9b as a white solid
1
(13 mg, 80%): mp 203-205 °C; [R]22 -28 (c 0.92, CHCl3); H
D
NMR (600 MHz, CDCl3) δ 1.19 (s, 3 H), 1.23 (s, 3 H), 1.72 (s, 3
H), 1.73 (s, 3 H), 1.91 (m, 1 H), 1.94 (m, 1 H), 2.23 (s, 3 H), 2.24
(m, 1 H), 2.57 (m, 1 H), 2.63 (s, 3 H), 3.35 (s, 1 H), 3.49 (t, J )
9.6 Hz, 1 H), 3.82 (m, 3 H), 4.26 (d, J ) 8.4 Hz, 1 H), 4.38 (m,
1 H), 4.40 (d, J ) 8.4 Hz, 1 H), 4.78 (m, 1 H), 5.00 (d, J ) 6.8
Hz, 1 H), 5.62 (d, J ) 7.8 Hz, 1 H), 5.92 (ddd, J ) 16.8, 8.4, 2.8
Hz, 1 H), 6.02 (d, J ) 7.2 Hz, 1 H), 6.31 (s, 1 H), 6.36 (d, J ) 7.8
Hz, 1 H), 6.52 (d, J ) 16.2 Hz, 1 H), 6.90 (d, J ) 8.4 Hz, 1 H),
7.22 (d, J ) 7.2 Hz, 1 H), 7.34-7.63 (m, 10 H), 8.10 (d, J ) 7.8
Hz, 2 H); 13C NMR (75.5 MHz, CDCl3) δ 9.1, 15.1, 20.8, 23.0,
23.3, 26.2, 29.7, 32.7, 35.7, 43.6, 44.9, 54.6, 58.9, 72.1, 72.7, 73.3,
75.2, 76.4, 77.2, 77.9, 79.0, 81.6, 84.3, 126.5, 126.7, 126.8, 128.3,
128.4, 128.8, 128.9, 129.0, 129.2, 129.9, 130.0, 131.1, 133.7, 134.9,
135.0, 136.6, 137.9, 138.4, 165.4, 169.4, 170.1, 171.1, 173.2, 202.9;
HRMS (FAB/DCM/NaCl) m/z calcd for C51H55NO15H+ 922.3650,
found 922.3643 (∆ ) -0.8 ppm).
39
the square of the predicted effect.
Tubulin Polymerization Assay. Assembly and disassembly of
calf brain microtubule protein (MTP) was monitored spectropho-
tometrically (Beckman Coulter DU 640, Fullerton, CA) by recording
changes in turbidity at 350 nm at 37 °C.40,41 MTP was diluted to
1 mg/mL in MES buffer containing 3 M glycerol. The concentration
of tubulin in MTP is 85%, and that is taken into consideration when
the ratios of tubulin to drug are presented in Figure 6. Microtubule
assembly was carried out with 10 µM 9b (SB-T-2054). Paclitaxel
(10 µM) and GTP (1 mM) were also used for comparison purposes.
Calcium chloride (6 mM) was added to the assembly reaction after
50 min to follow the calcium-induced microtubule depolymeriza-
tion.
Electron Microscopy. Aliquots (50 µL) were taken from in vitro
polymerization assays at the end of the reaction and placed onto
300-mesh carbon-coated, formavar-treated copper grids. Samples
were then stained with 20 µL of 2% uranyl acetate and viewed
with a JEOL Model 100CX electron microscope.
X-ray Single-Crystal Analysis of 9b (SB-T-2054). A colorless
single crystal (0.2 × 0.3 × 0.5 mm3) of 9b grown in a NMR tube by
slow diffusion of hexanes (0.5 mL) into a solution of 9b (5 mg) in a
mixture of dichloromethane (0.1 mL) and ethyl actate (0.05 mL) was
selected for X-ray crystallographic analysis. Since the crystals obtained
in this way were sensitive to air, the test single crystal was sealed
along with some mother liquid in a capillary tube. Intensity data
collection was carried out with a Bruker AXS SMART diffractometer
equipped with a CCD detector using a Siemens graphite-monochro-
mated Mo radiation tube (λ ) 0.71073 Å) at room temperature. The
unit cells were determined by a least-squares analysis using the
SMART software package. The raw frame data were integrated into
SHELX-format reflection files and corrected for Lorentz and polariza-
tion effects using SAINT. Corrections for incident and diffracted beam
absorption effects were applied using SADABS. The structure was
solved by direct method and refined by full-matrix least-squares method
on F2 using the SHELXTL 97 software package. All non-hydrogen
atoms were refined anisotropically. Hydrogen atoms were placed in
calculated positions and added as fixed contributions. There are solvent
molecules trapped in the crystals. However, we were not able to fully
solved the structure due to their highly disordered behavior. Ignorance
of these solvent molecules did not terribly harm the final refinement;
thus, we simply treated this single crystal as a solvent free sample.
The CIF file is available as Supporting Information.
In Vitro Cell Growth Inhibition Assay. Tumor cell growth
inhibition was determined according to the method established by
Skehan et al.38 Human cancer cells LCC6-WT (Pgp-), MCF-7
(Pgp-), LCC6-MDR (Pgp+), NCI/ADR (Pgp+), A2780 (Pgp-), and
HT-29 (Pgp-) were plated at a density of 400-2000 cells/well in
96-well plates and allowed to attach overnight. These cell lines
were maintained in RPMI-1640 medium (Roswell Park Memorial
Institute growth medium) supplemented with 5% fetal bovine serum
and 5% Nu serum (Collaborative Biomedical Product, MA).
Macrocyclic axoids were dissolved in DMSO and further diluted
with RPMI-1640 medium. Triplicate wells were exposed to various
treatments. After 72 h incubation, 100 µL of ice-cold 50%
trichloroacetic acid (TCA) was added to each well, and the samples
were incubated for 1 h at 4 °C. Plates were then washed five times
with water to remove TCA and serum proteins, and 50 µL of 0.4%
sulforhodamine B (SRB) was added to each well. Following a 5
min incubation, plates were rinsed five times with 0.1% acetic acid
and air-dried. The dye (SRB) was then solubilized with 10 mM
Computational Methods. The structures of the C14-C3′BzN-
linked macrocyclic taxoids 1a, 1c-E, 1c-Z, and 9b in the 1JFF were
produced by directly introducing the linker into the “REDOR-
Taxol” in the 1JFF complexes using the Builder module in the
InsightII 2000 program (CVFF). Then, these structures were energy-
minimized in 5000 steps or until the maximum derivative was
<0.001 kcal/A by means of the conjugate gradients method using
the CVFF force field and the distance-dependent dielectric. The
backbone of the protein was fixed throughout the energy minimiza-
tion. After the energy minimization, the snapshots were overlaid
by superimposing the backbones of the proteins. During the energy
minimizations of the macrocyclic taxoids, the C′2-OH-N(His227
)
H-bond was very stable in all cases. Monte Carlo conformational
searches on the C14-C3′BzN-linked macrocyclic taxoids were
performed with energy minimization (5000 MC steps, minimization
for 1000 steps with Polak-Ribiere conjugated gradients) by Mac-
romodel program using a generalized born with surface area term
(GBSA) continuum solvent description of water solvation.42
Acknowledgment. This work was supported by grants from
the National Institutes of Health (CA103314 and GM42798 to
I.O.; CA083185 and CA077263 to S.B.H.; CA 73872 to R.J.B.)
and the National Science Foundation (PACI-MCA02N028 to
C.S.). Generous support from Indena, SpA is also gratefully
acknowledged. L.S. and I.O. also thank Ms. Rebecca Rowehl
for her valuable help at the Cell Culture and Hybridoma Facility
at Stony Brook.
Supporting Information Available: Refined “REDOR-
Taxol” structure; Monte Carlo conformational search and
1
analysis results for 1a, 1c-E, 1c-Z, and 9b; H and 13C NMR
spectra of 3a-d, 6, 7a-d, 1a, 1c-E, 1c-Z, 1d, and 9b; single-
crystal X-ray analysis data for 9b. This material is available
free of charge via the Internet at http://pubs.acs.org.
JO801713Q
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1990, 82, 1107–1112.
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