Synthesis and biological evaluation of methoxylated analogs of the newer generation taxoids IDN5109 and IDN5390

Article abstract of DOI:10.1016/j.bmcl.2005.08.066

Starting from 10-deacetylbaccatin III (7), the 2-debenzoyl-2-m- methoxybenzoyl analogs of the newer generation taxoids IDN5109 (3) and IDN5390 (4) were synthesized. The biological evaluation of these compounds (5 and 6, respectively) showed a general incr

Full text of DOI:10.1016/j.bmcl.2005.08.066

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5182–5186
Synthesis and biological evaluation of methoxylated analogs
of the newer generation taxoids IDN5109 and IDN5390
Luciano Barboni,a,* Roberto Ballini,a Guido Giarlo,a Giovanni Appendino,b
Gabriele Fontanac and Ezio Bombardellic
a
`
Dipartimento di Scienze Chimiche, Universita di Camerino, Via S. Agostino 1, 62032 Camerino (MC), Italy
b
`
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita del Piemonte Orientale,
Viale Ferrucci 33, 28100 Novara, Italy
cIndena S.p.A. Viale Ortles 12, 20139 Milano, Italy
Received 7 July 2005; revised 11 August 2005; accepted 22 August 2005
Available online 23 September 2005
Abstract—Starting from 10-deacetylbaccatin III (7), the 2-debenzoyl-2-m-methoxybenzoyl analogs of the newer generation taxoids
IDN5109 (3) and IDN5390 (4) were synthesized. The biological evaluation of these compounds (5 and 6, respectively) showed a
general increase of cytotoxicity, as observed in first-generation anticancer taxanes.
Ó 2005 Elsevier Ltd. All rights reserved.
Paclitaxel (1), a natural product first isolated from the
bark of Taxus brevifolia, and its semi-synthetic deriva-
tive docetaxel (2), are currently used in the treatment
of a large number of human tumors such as ovarian can-
cer, breast cancer, melanoma, and nonsmall cell lung
cancer. Their mechanism of action involves stabilization
of microtubules and inhibition of tubulin depolymeriza-
tion.1 Paclitaxel and docetaxel suffer from a series of dis-
advantages, including a poor water solubility and the
quick development of resistance,2 that have fuelled the
search of analogs endowed with a better clinical profile.3
The norstatin esters IDN5109 (3)3 and IDN5390 (4)4
have recently emerged as interesting clinical candidates
to overcome resistance to paclitaxel and to allow oral
administration. When assayed against paclitaxel-respon-
sive tumors, the activity of 3 and 4 was basically in the
range of the first-generation taxoids.5 Since the intro-
duction of an azido- or a methoxy meta-substituent on
the 2-benzoate has been reported to magnify the cyto-
toxicity of taxoids,6 we have investigated this effect in
the newer generation taxoids 3 and 4, hoping to improve
their cytotoxic potency while substantially retaining the
assets of the lead structures in terms of solubility and
spectrum of activity.
Keywords: Paclitaxel; IDN5109; IDN5390; Methoxylated; Newer
generation taxoids.
Synthesis of the IDN5109 analog 5 started from the
enone 8, in turn available from 10-deacetylbaccatin III
(7) in five steps (Scheme 1).7
*
Correspondig author. Tel.: +390737402240; fax: +390737637345;
e-mail: luciano.barboni@unicam.it
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.066

L. Barboni et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5182–5186
5183
Reduction of the 13-keto group of 9 with Et4N(BH4)8
(Scheme 2) gave a mixture the a- and b-epimers (10a
and 10b, respectively), that could be isolated by column
chromatography.10 The configuration of C13 and C14
of compounds 10a and 10b was assigned on the basis
of the NOESY1D spectra. Compound 10a showed an
enhancement of the H-17 signal upon selective satura-
tion of H-13, whereas irradiation of H-14 showed a
NOE correlation with H-3. Selective saturation of H-
14 in 10b showed an enhancement of the H-13 and H-
3 signals, which indicates that both H-14 and H-13 lie
on the a-face of the molecule. With 10a in hand, the
completion of the synthesis was easily accomplished by
coupling with N-Boc (4S,5R)-2-(2,4-dimethoxyphenyl)-
4-phenyl-5-oxazolidine carboxylic acid4 and global
deprotection of the crude esterification product (Scheme
2).11
Scheme 1.
For the preparation of compound 10 we capitalized on
the recently reported diastereoselective 14b-hydroxyl-
ation of 13-oxotaxoids.8 Thus, treatment of 8 with
2-benzenesulfonyl-3-(3-nitrophenyl)oxaziridine, in the
presence of tBuOK, followed by in situ carbonyl-
ation with carbonyldiimidazole gave compound 9
(Scheme 2).9
IDN5390 (4) is a ring C-seco taxoid, and the synthesis of
its methoxylated analog was carried out according to a
general retro-aldol entry for this type of compound.12
Thus, compound 5 was first protected at C20 with
TBDMSCl13 to afford 11,14 next converted to the C-seco
derivative 6 in four steps (Scheme 3)15 that could be car-
ried out without purification of any intermediate. Thus,
11 was deacetylated at C10 with hydrazine, and the ketol
function obtained was oxidized with Cu(OAc)2.12
Reductive trapping of the ring C-seco tautomer was ob-
tained by treatment with L-Selectride.12 Final deprotec-
tion with TBAF gave 6 in a 48% overall yield.
The cytotoxicity of 5 and 6 was investigated on MCF7
and MCF7/R cell lines (Table 1).16 As expected, the
meta-methoxy group had a general boosting effect on
cytotoxicity, both on MCF7 and MCF7/R cell lines,
showing that the ꢀmeta-effectꢁ (enhanced interaction be-
tween the C-2 ring and asp224 in the b-tubulin binding
pocket)17 has broad generality in anticancer taxoids.
Scheme 2. Reagents and conditions: (a) i—tBuOK, 2-benzenesulfonyl-
3-(3-nitrophenyl)oxaziridine, THF, DMPU, ꢀ65 °C to ꢀ55 °C, 1.5 h;
ii—carbonyldiimidazole, imidazole, DCM, 40 °C, 1.5 h, 30%; (b)
Et4N(BH4), MeOH, ꢀ40 °C to 0 °C, 2.5 h, 60%; (c) i—N-Boc
(4S,5R)-2-(2,4-dimethoxyphenyl)-4-phenyl-5-oxazolidine
carboxylic
In conclusion, we have demonstrated that the insertion
of a meta methoxy group on the C-2 benzoate leads to
acid, DMAP, EDC, DCM, rt, 1.5 h; ii—HF, pyridine, CH3CN, 0 °C
to rt, 2 h; iii—HCl 0.6 N (MeOH), DCM, 0 °C, 5 h, 55%.
Scheme 3. Reagents and conditions: (a) TBDMSCl, DMAP, DCM, rt, overnight, 90%; (b) N2H4ÆH2O, EtOH 96%, ꢀ15 °C, 45 min; (c)
Cu(OAc)2ÆH2O, MeOH, rt, overnight; (d) L-Selectride, THF, ꢀ5 °C, instantaneous; (e) TBAF, THF, 0 °C, 20 min; overall yield (b + c + d + e) 48%.

5184
L. Barboni et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5182–5186
Table 1. Cytotoxic potency of 5 and 6 in comparison with 1, 3, and 4
IC50a
9. Synthetic procedure for 9: a suspension of t-BuOK
(450 mg, 4 mmol) in THF (2.5 mL) was stirred at rt for
10 min, cooled at ꢀ65 °C, and then a solution of 8
(729 mg, 1.0 mmol) in THF/DMPU (8:3, 11 mL) was
added in 2 min. After 15 min, a solution of 2-benze-
nesulfonyl-3-(3-nitrophenyl)oxaziridine (0.86 g, 2.8 mmol)
in THF/DMPU (9:1, 10 mL) was added in 3 min. and the
temperature was slowly raised (about 1 h) to ꢀ55 °C. At
this point, acetic acid (0.6 mL) and aqueous NH4Cl (10%,
25.0 mL) were sequentially added, and the temperature of
the mixture was allowed to rise to rt under stirring. The
organic layer was extracted with water, dried, and
evaporated. The crude product was dissolved in anhy-
drous DCM (20 mL), and carbonyldiimidazole (2.3 g, 22
mmol) and imidazole (734 mg, 3.2 mmol) were sequential-
ly added. The solution was stirred at 40 °C for 1.5 h and
then diluted with DCM (200 mL), washed with HCl 5%,
NaHCO3 5%, water, and brine, dried, and the solvent was
evaporated. Chromatography of the residue (SiO2, hex-
ane/EtOAc, 8:2) gave compound 9 (230 mg, 0.3 mmol,
Compound
72 h cell drug exposure SE (SRB test)b
MCF7c
MCF7/Rc
RId
1
3
4
5
6
0.6 0.08
1.6 0.1
585 62
32 2.4
1044 93
975
20
11.2 0.81
0.24 0.04
0.5 0.05
93
17
4 0.8
120 30
240
a IC50: concentration inhibiting the 50% of cellular growth compared
to untreated cells in nanomoles.
b Cells were exposed for 72 h to the drugs and the IC50 was determined
by SRB metabolic test. Values of IC50 (means of at least three
independent experiments) are reported standard error.
c Cell lines: MCF7 human mammary carcinoma; MCF7/R: subline
with acquired resistance to doxorubicin (DX).
d RI, resistance index; IC50 resistant line/IC50 sensitive line.
1
30%). H NMR (CDCl3, 300 MHz): d 0.56 (q, J = 7.8 Hz,
6H), 0.90 (t, J = 7.8 Hz, 9H), 1.16 (s, 3H), 1.34 (s, 3H),
1.70 (s, 3H), 1.89 (m, 1H), 2.17 (s, 3H), 2.21 (s, 6H), 2.52
(m, 1H), 3.77 (d, J = 6.9 Hz, 1H), 3.83 (s, 3H), 4.18 (d,
J = 8.7 Hz, 1H), 4.35 (d, J = 8.7 Hz, 1H), 4.44 (m, 1H),
4.75 (s, 1H), 4.89 (br d, J = 7.8 Hz, 1H), 6.08 (d,
J = 6.9 Hz, 1H), 6.49 (s, 1H), 7.14 (br d, J = 8.7 Hz, 1H),
7.37 (t, J = 8.0 Hz, 1H), 7.49 (br s, 1H), 7.54 (d,
J = 7.8 Hz, 1H); 13C NMR (CDCl3, 75 MHz): d 5.2, 6.7,
9.8, 13.9, 19.3, 20.7, 21.7, 32.7, 36.9, 41.6, 45.4, 55.4, 59.2,
68.3, 72.0, 74.6, 75.8, 77.1, 80.5, 83.8, 86.4, 114.7, 120.7,
122.0, 128.9, 130.0, 139.3, 151.1, 151.3, 159.8, 164.3, 168.7,
170.2, 190.8, 199.0; MS (ESI) m/z 793 (M+Na+).
a general increase of cytotoxicity also in newer genera-
tion taxoids, including those of the ring C-seco type that
differ substantially from taxanes in terms of topology
and conformational properties. While having minimal
effect on water solubility, the introduction of a methoxyl
can in principle have dramatic effects on drug metabo-
lism, and pharmacokinetics data are clearly needed to
assess the overall effect of this maneuver on the clinical
profile of newer generation taxoids.
10. Synthetic procedure for 10a and 10b: To a solution of 9
(771 mg, 1 mmol) in MeOH (15 mL) at ꢀ40 °C,
Et4N(BH4) (1.74 g, 12 mmol) in MeOH (5 mL) was added,
and the reaction mixture was stirred at ꢀ40 °C for 30 min.
The temperature was then raised to 0 °C and, after stirring
for 2 h, citric acid (1.5 g) and a saturated solution of citric
acid in water (7 mL) were sequentially added. The
temperature was allowed to raise to rt and the reaction
mixture was extracted with DCM, dried, and evaporated.
The crude material was purified by gravity chromatogra-
phy (SiO2, hexane/EtOAc, 3:2) to yield 10a (464 mg,
0.60 mmol, 76%) and 10b (147 mg, 0.19 mmol, 19%). 10a:
1H NMR (CDCl3, 300 MHz): d 0.55 (q, J = 7.8 Hz, 6H),
0.89 (t, J = 7.8 Hz, 9H), 1.12 (s, 3H), 1.29 (s, 3H), 1.67 (s,
3H), 1.86 (m, 1H), 2.17 (s, 3H), 2.18 (s, 3H), 2.28 (s, 3H),
2.51 (m, 1H), 3.69 (d, J = 7.5 Hz, 1H), 3.83 (s, 3H), 4.16
(d, J = 8.0 Hz, 1H), 4.31 (d, J = 8.0 Hz, 1H), 4.44 (m, 1H),
4.77 (d, J = 5.7 Hz, 1H), 4.93 (br d, J = 7.5 Hz, 1H), 4.98
(br d, J = 5.7 Hz, 1H), 6.04 (d, J = 7.2 Hz, 1H), 6.41 (s,
1H), 7.13 (br d, J = 7.2 Hz, 1H), 7.35 (t, J = 8.1 Hz, 1H),
7.53 (br s, 1H), 7.59 (d, J = 7.8 Hz, 1H); 13C NMR
(CDCl3, 100 MHz): d 5.2, 6.7, 10.0, 14.8, 20.8, 21.6, 22.3,
25.9, 37.0, 41.4, 46.6, 55.4, 58.7, 69.4, 71.9, 72.0, 75.0, 76.0,
80.3, 83.9, 84.1, 88.4, 114.5, 120.5, 122.1, 129.4, 129.9,
132.8, 142.2, 152.8, 159.7, 164.7, 169.2, 170.4, 200.8; MS
(ESI) m/z 795 (M+Na+). 10b: 1H NMR (CDCl3,
Acknowledgments
We gratefully acknowledge Arturo Battaglia and Maria
Luisa Gelmi for their helpful suggestions. We also thank
Ralph J. Bernacki and Paula Pera for the cytotoxicity
tests.
References and notes
1. Horwitz, S. B.; Cohen, D.; Rao, S.; Ringel, I.; Shen, H.-J.
Monogr. Natl. Cancer Inst. 1993, 15, 55.
2. Rowinsky, E. K.; Donehower, R. C. N. Engl. J. Med.
1995, 332, 1004.
3. (a) Eckstein, J. W. IDrugs 2004, 7, 575; (b) Miller, M. L.;
Ojima, I. Chem. Rec. 2001, 1, 195.
4. Appendino, G.; Danieli, B.; Jakupovic, J.; Belloro, E.;
Scambia, E.; Bombardelli, E. Tetrahedron Lett. 1997, 38,
4273.
5. (a) Nicoletti, M. I.; Colombo, T.; Rossi, C.; Monardo, C.;
Stura, S.; Zucchetti, M.; Riva, A.; Morazzoni, P.; Donati,
M. B.; Bombardelli, E.; DꢁIncalci, M.; Gavazzi, R. Cancer
Res. 2000, 60, 842; (b) Ferlini, C.; Raspaglio, G.; Mozzetti,
S.; Cicchillitti, L.; Filippetti, F.; Gallo, D.; Fattorusso, C.;
Campiani, G.; Scambia, G. Cancer Res. 2005, 65, 2397.
6. Chaudhary, A. G.; Gharpure, M.; Rimoldi, J. M.;
Chordia, M. D.; Gunatilaka, A. A. L.; Kingston, D. G.
I. J. Am. Chem. Soc. 1994, 116, 4097.
7. (a) Chordia, M. D.; Kingston, D. G. I. Tetrahedron 1997,
53, 5699; (b) Appendino, G. J.; Jakupovic, J.; Cravotto,
G.; Enriu`, R.; Varese, M.; Bombardelli, E. Tetrahedron
Lett. 1995, 36, 3233.
300 MHz):
d 0.58 (q, J = 7.8 Hz, 6H), 0.892 (t,
J = 7.8 Hz, 9H), 1.29 (s, 3H), 1.42 (s, 3H), 1.71 (s, 3H),
1.90 (m, 1H), 2.20 (s, 3H), 2.25 (s, 3H), 2.31 (s, 3H), 2.51
(m, 1H), 3.67 (d, J = 7.3 Hz, 1H), 3.85 (s, 3H), 4.20 (d,
J = 8.8 Hz, 1H), 4.34 (d, J = 8.8 Hz, 1H), 4.42 (m, 2H),
4.80 (d, J = 7.8 Hz, 1H), 4.90 (br d, J = 8.8 Hz, 1H), 6.10
(d, J = 7.3 Hz, 1H), 6.43 (s, 1H), 7.15 (br d, J = 7.8 Hz,
1H), 7.37 (t, J = 7.8 Hz, 1H), 7.53 (br s, 1H), 7.58 (d,
J = 7.8 Hz, 1H); 13C NMR (CDCl3, 100 MHz): d 5.2, 6.7,
9.9, 19.9, 20.8, 21.3, 22.0, 30.4, 37.0, 40.7, 46.1, 55.4, 59.1,
8. Baldelli, E.; Battaglia, A.; Bombardelli, E.; Carenzi, G.;
Fontana, G.; Gambini, A.; Gelmi, M. L.; Guerrini, A.;
Pocar, D. J. Org. Chem. 2003, 68, 9773.

L. Barboni et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5182–5186
5185
67.7, 68.7, 71.8, 74.8, 75.0, 76.0, 80.9, 83.9, 92.3, 114.8,
120.3, 122.0, 129.3, 129.9, 138.7, 139.9, 152.7, 159.8, 164.5,
169.0, 170.1, 200.5; MS (ESI) m/z 795 (M+Na+).
(25 mg, 0.5 mmol) was added at ꢀ15 °C. After stirring
for 45 min, the reaction mixture was worked up by
dilution with EtOAC, washing with saturated NH4Cl
and brine, and concentration under reduced pressure to
give crude 12a, which was used for the next step. Thus, 12a
was dissolved in MeOH (3 mL), Cu(OAc)2ÆH2O (974 mg)
was added, and the suspension was magnetically stirred
for 24 h. The reaction mixture was worked up by
concentration under reduced pressure, filtration through
Celite, and washing of the cake with EtOAc. The organic
phase was washed with 2% NH3 and brine, and dried,
giving the epimeric mixture 12b that was directly employed
for the reductive fragmentation step. 12b was then
dissolved in dry THF (ca. 2 mL), cooled to ꢀ15 °C, and
a solution of L-selectride (1.0 M, 0.2 mL, and 0.2 mmol)
was added dropwise. At the end of the addition, the yellow
solution was diluted with EtOAc and washed with 2 N
H2SO4 and brine. After evaporation of the solvent, the
residue was dissolved in dry THF (2 mL), cooled to 0 °C,
and treated with tetrabutylammonium fluoride (TBAF,
1 M in THF, 0.2 mL, and 0.2 mmol). After stirring for
20 min, the reaction mixture was diluted with EtOAC,
washed with 2N H2SO4 and brine, dried, and evaporated.
The residue was purified by chromatography (SiO2,
hexane/EtOAc, 1:1) to give 6 (40 mg, 48%). Amorphous
11. Synthetic procedure for 5: to a solution of N-Boc (4S,5R)-
2-(2,4-dimethoxyphenyl)-4-phenyl-5-oxazolidine carbox-
ylic acid (obtained by acidification of 0.75 g of its
corresponding sodium salt, 1.75 mmol) in dry DCM
(5 mL), a solution of 10a (386 mg, 0.5 mmol) in dry
DCM, EDCI (288 mg, 1.5 mmol), and DMAP (183 mg,
1.5 mmol) was added. After stirring at rt for 1.5 h, the
reaction was worked up by dilution with EtOAc and then
addition of sat. NaHCO3. The organic phase was washed
with brine, dried, and evaporated. The residue was
dissolved in CH3CN/pyridine, (5:4, 5 mL), cooled to
0 °C, and HF (70% pyridine solution, 3.5 mL) was added.
After stirring at rt for 2 h, the solution was diluted with
EtOAc and washed with KHSO4 (10%), NaHCO3 5%,
water, and brine. The organic phase was evaporated, the
residue was dissolved in dry DCM (8 mL), cooled to 0 °C,
and treated with 0.25 mL of methanolic HCl (obtained
from the reaction of 9 lL AcCl in 0.25 mL MeOH). After
stirring overnight at room temperature, the reaction was
worked up by dilution with DCM and washing with sat.
NaHCO3. The organic phase was washed with water and
brine, dried, and evaporated. The residue was purified by
gravity column chromatography (SiO2, hexane/EtOAc,
20
solid; ½aꢁD ꢀ 20:5ꢂ (c 1.1, CHCl3); 1H NMR (CDCl3,
1:1) to afford
5
(248 mg, 55%). Amorphous solid;
300 MHz, 50 °C): d 0.98 (d,J = 6.0 Hz, 6H), 1.27 (s, 3H),
1.34 (s, 9H), 1.37 (s, 3H), 1.49 (m, 1H), 1.58 (m, 1H), 1.70
(m, 1H), 1.91 (s, 3H), 1.94 (m, 1H), 1.99 (br s, 6H), 2.37
(m, 1H), 3.71 (m, 1H), 3.85 (s, 3H), 3.87 (m, 1H), 4.20 (m,
2H), 4.29 (d, J = 8.1 Hz, 1H), 4.33 (d, J = 3.6 Hz, 1H),
4.78 (d, J = 9.9 Hz, 1H), 5.14 (m, 1H), 5.24 (br d,
J = 10.8 Hz, 1H), 5.42 (br s, 1H), 6.05 (d, J = 9.3 Hz,
1H), 6.49 (br d, J = 3.3 Hz, 1H), 7.15 (dd, J = 8.1, 2.4 Hz,
1H), 7.39 (t, J = 8.1 Hz, 1H), 7.48 (br s, 1H), 7.57 (d,
J = 8.1 Hz, 1H); MS (ESI) m/z 882 (M + Na+). HRMS
m/z calcd for C43H57NO17Na+ 882.3524, found 882.3531.
16. Human tumor cell lines: the MCF7-S and MCF7-R
(multidrug resistant) human mammary carcinoma cell
lines were purchased from the American Type Culture
Collection (ATCC), and the MDA435/LCC6-WT and
MDR1 cell lines were provided by Dr. R. Clarke,
Lombardi Cancer Center, Georgetown University School
of Medicine. Cell lines are propagated as monolayers in
RPMI-1640 containing 5% FCS, 5% NuSerum IV, 20 mM
Hepes, and 2 mM L-glutamine at 37 °C in a 5% CO2
humidified atmosphere. The doubling times for the cell
lines ranged between 20 and 30 h. Growth inhibition assay
in 96-well microtiter plates. Assessment of cell growth
inhibition was determined according to the methods of
Skehan.et al. (Skehan, P.; Streng, R.; Scudierok, D.;
Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.;
Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst.
1990, 82, 1107). Briefly, cells were plated between 800 and
1500 cells/well in 96-well plates and incubated at 37 °C 15–
18 h prior to drug addition to allow cell attachment.
Compounds to be tested were solubilized in 100% DMSO
and further diluted in RPMI-1640 containing 10 mM
Hepes. Each cell line was treated with 10 concentrations of
compound (5 log range). After a 72 h incubation, 100 lL
of ice-cold 50% TCA was added to each well and
incubated for 1 h at 4 °C. Plates were then washed five
times with tap water to remove TCA, low-molecular-
weight metabolites, and serum proteins. Then, 50 lL of
0.4% sulforhodamine B (SRB), an anionic protein stain,
was added to each well. At cell densities ranging from very
sparse to supraconfluent, SRB staining changed linearly
with increases or decreases in the number of cells and
protein concentrations. These staining characteristics
20
½aꢁD ꢀ 39:6ꢂ (c 1.0, CHCl3); 1H NMR (CDCl3,
300 MHz):
d
0.94 (d, J = 6.7 Hz, 3H), 0.97 (d,
J = 6.7 Hz, 3H), 1.26 (s, 3H), 1.30–1.34 (m, 1H), 1.33 (s,
3H), 1.34 (s, 9H), 1.69 (m, 2H), 1.70 (s, 3H), 1.87 (m, 1H),
1.88 (s, 3H), 2.23 (s, 3H), 2.46 (s, 3H), 2.54 (m, 1H), 3.68
(d, J = 7.3 Hz, 1H), 3.84 (s, 3H), 4.08 (m, 1H), 4.20 (d,
J = 8.3 Hz, 1H), 4.30 (m, 1H), 4.32 (d, J = 8.3 Hz, 1H),
4.37 (m, 1H), 4.73 (d, J = 9.8 Hz, 1H), 4.83 (d, J = 6.7 Hz,
1H), 4.93 (br d, J = 9.8 Hz, 1H), 6.08 (d, J = 7.3 Hz, 1H),
6.25 (s, 1H), 6.44 (br d, J = 6.7 Hz, 1H), 7.13 (dd, J = 7.9,
3.0 Hz, 1H), 7.36 (t, J = 7.9 Hz, 1H), 7.54 (br s, 1H), 7.61
(d, J = 7.9 Hz, 1H); 13C NMR (CDCl3, 100 MHz): d 9.7,
15.0, 20.8, 22.1, 22.5, 23.1, 23.2, 24.8, 26.0, 28.2, 35.4, 40.5,
41.8, 45.0, 51.6, 55.4, 58.7, 69.5, 71.7, 73.9, 74.6, 74.8, 76.0,
79.6, 80.4, 80.5, 84.2, 88.1, 114.0, 121.0, 122.3, 129.1,
130.0, 133.5, 139.8, 151.8, 156.2, 159.8, 164.7, 170.6, 170.9,
173.0, 202.2; MS (ESI) m/z 924 (M+Na+); HRMS m/z
calcd for C45H59NO18Na+ 924.3630, found 924.3636.
12. Appendino, G.; Noncovich, A.; Bettoni, P.; Dambruoso,
P.; Sterner, O.; Fontana, G.; Bombardelli, E. Eur. J. Org.
Chem. 2003, 4422.
13. Gunatilaka, A. A. L.; Ramdayal, F. D.; Sarragiotto, M.
H.; Kingston, D. G. I.; Sackett, D. L.; Hamel, E. J. Org.
Chem. 1999, 64, 2694.
1
14. 11: H NMR (CDCl3, 300 MHz): d 0.11 (s, 3H), 0.15 (s,
3H), 0.86 (s, 3H), 0.91 (s, 3H), 0.95 (s, 9H), 1.27 (s, 3H),
1.30-1.34 (m, 1H), 1.36 (s, 9H), 1.45 (s, 3H), 1.66 (m, 2H),
1.71 (s, 3H), 1.90 (m, 1H), 1.91 (s, 3H), 2.25 (s, 3H), 2.50
(s, 3H), 2.56 (m, 1H), 3.71 (d, J = 7.5 Hz, 1H), 3.88 (s,
3H), 4.16 (m, 1H), 4.23 (d, J = 8.4 Hz, 1H), 4.33 (m, 2H),
4.40 (m, 1H), 4.52 (d, J = 9.9 Hz, 1H), 4.85 (d, J = 6.9 Hz,
1H), 4.95 (br d, J = 7.2 Hz, 1H), 6.10 (d, J = 7.5 Hz, 1H),
6.27 (s, 1H), 6.38 (br d, J = 6.9 Hz, 1H), 7.15 (dd, J = 8.4,
2.7 Hz, 1H), 7.37 (t, J = 8.4 Hz, 1H), 7.55 (br s, 1H), 7.61
(d, J = 8.4 Hz, 1H); 13C NMR (CDCl3, 100 MHz): d ꢀ5.1,
ꢀ4.6, 9.9, 15.4, 21.0, 22.2, 23.0, 23.6, 23.7, 24.9, 25.9, 26.1,
28.4, 35.7, 41.7, 42.0, 45.3, 51.8, 55.6, 58.8, 69.7, 71.8, 74.5,
75.0, 75.3, 76.1, 79.6, 79.9, 80.7, 84.5, 88.3, 114.3, 121.2,
122.4, 129.3, 130.2, 133.6, 140.1, 152.0, 155.4, 160.0, 164.9,
170.8, 171.1, 171.9, 202.4; MS (ESI) m/z 1038 (M+Na+).
15. Synthetic procedure for 6: To a solution of 11 (100 mg,
0.1 mmol) in EtOH (10 mL), hydrazine monohydrate

5186
L. Barboni et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5182–5186
provided an accurate assessment of cell growth.18 Follow-
Appl. Math. 1976, 11, 431) as adapted by Nash (Nash, J.
C. Compact numerical method for computers: Linear
algebra and function minimization; John Wiley & Sons:
New York, 1979) for the nonlinear regression. The
concentration of drug which resulted in 50% growth
inhibition (IC50) was calculated.
ing a 5-min incubation at room temperature, plates were
rinsed five times with 0.1% acetic acid and air-dried.
Bound dye was solubilized with 10 mM Tris Base (pH
10.5) for 5 min on a gyratory shaker. Optical density was
measured at 570 nm. Data analysis: data were fit with the
Sigmoid-Emax concentration-effect model (Holford, N.H.
G; Scheiner, L. B. Clin. Pharmocokin. 1981, 6, 429) with
nonlinear regression and weighted by the reciprocal of the
square of the predicted response. The fitting software was
developed at RPCI with MicroSoft FORTRAN and uses
the Marquardt algorithm (Marquardt, D. W. J. Soc. Ind.
17. (a) Ganesh, T.; Guza, R. C.; Bane, S.; Ravinfra, R.;
Shanker, N.; Lakdawala, A. S.; Snyder, J. P.; Kingston, D.
G. I. PNAS 2004, 101, 10006; (b) Snyder, J. P.; Nettles, J. H.;
Cornett, B.; Downing, K. H.; Nogales, E. PNAS 2001, 98,
5312; (c) He, L.; Jagtap, P. G.; Kingston, D. G. I.; Shen, H.-
J.; Orr, G. A.; Horwitz, S. B. Biochemistry 2000, 39, 3972.