Novel D-seco paclitaxel analogues: Synthesis, biological evaluation, and model testing

Article abstract of DOI:10.1021/jo0015467

Four new D-secopaclitaxel analogues were synthesized from paclitaxel. The key step of the synthesis involved the opening of the D-ring by Jones oxidation. Two of the compounds had been predicted to be nearly as active as paclitaxel in a minireceptor model of the binding site on tubulin, but all were biologically inactive in an in vitro cytotoxic assay and a tubulin assembly assay. The biological results identify a weakness in our predictive minireceptor model and suggest a corrective remedy in which additional amino acids are needed to accommodate ligand-protein steric effects around the oxetane ring. These changes to the model lead to correct predictions of the bioactivity. Conformational analysis and dynamics simulations of the compounds showed that the 4-acetyl substituent is as important as the oxetane in determining the A ring conformation.

Full text of DOI:10.1021/jo0015467

J . Org. Chem. 2001, 66, 3321-3329
3321
Novel D-Seco P a clita xel An a logu es: Syn th esis, Biologica l
Eva lu a tion , a n d Mod el Testin g
Luciano Barboni,† Apurba Datta, Dinah Dutta, Gunda I. Georg,* and David G. Vander Velde
Department of Medicinal Chemistry and Drug Discovery Program, Higuchi Biosciences Center,
University of Kansas, Lawrence, Kansas 66045
Richard H. Himes
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045
Minmin Wang and J ames P. Snyder
Department of Chemistry, Emory University, Atlanta, Georgia 30322
georg@ku.edu
Received November 1, 2000
Four new D-secopaclitaxel analogues were synthesized from paclitaxel. The key step of the synthesis
involved the opening of the D-ring by J ones oxidation. Two of the compounds had been predicted
to be nearly as active as paclitaxel in a minireceptor model of the binding site on tubulin, but all
were biologically inactive in an in vitro cytotoxic assay and a tubulin assembly assay. The biological
results identify a weakness in our predictive minireceptor model and suggest a corrective remedy
in which additional amino acids are needed to accommodate ligand-protein steric effects around
the oxetane ring. These changes to the model lead to correct predictions of the bioactivity.
Conformational analysis and dynamics simulations of the compounds showed that the 4-acetyl
substituent is as important as the oxetane in determining the A ring conformation.
Paclitaxel (1), first isolated from the bark of Taxus
brevifolia,1 and its semisynthetic analogue docetaxel (2),
They are believed to share the same or overlapping
binding sites because they are competitive inhibitors of
[3H]paclitaxel binding to microtubules.7 Clearly, the
complete understanding of the minimal structural re-
quirements to maintain microtubule binding and the
identification of a three-dimensional pharmacophore
common to these drugs are essential for the development
of new compounds with increased biological activity. SAR
studies on paclitaxel have been extensive and have led
to the conclusion that the C13 side chain and the ester
groups at C2 and C4 are essential for biological activity.8
A remarkable exception is the recent report that a taxane
lacking the C13 side chain but incorporating a 2-m-azide
in the C2 benzoyl ring blocks cancer cell proliferation at
nM concentrations.9
In the past few years special attention has been paid
to the oxetane ring. On the basis of the inactivity of the
D-seco paclitaxel 3,10 the oxetane ring has been consid-
ered essential for biological activity, but this hypothesis
cannot be considered conclusive because 3 lacks the C4
ester group known to be crucial for activity and, com-
pared to paclitaxel, an inverted configuration at C5. The
finding that the substitution of the oxygen atom in ring
D by nitrogen,11 sulfur, or selenium atoms12 causes a
marked reduction in activity is consistent with the
obtained from 10-deacetylbaccatin III, are important
therapeutic agents clinically used for the treatment of a
variety of cancers.2-5 Their mechanism of action involves
disruption of microtubule dynamics, thereby blocking cell
division.6 Recently, other structurally diverse products,
epothilones A and B, eleutherobin, and discodermolide,
have been found to have the same mechanism of action.7
† Visiting scientist from Dipartimento di Scienze Chimiche, Uni-
versita` di Camerino, via S. Agostino 1, 62032 Camerino (MC), Italy.
(1) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A.
T. J . Am. Chem. Soc. 1971, 93, 2325-2327.
(2) Eisenhauer, E. A.; Vermorken, J . B. Drugs 1998, 55, 5-30.
(3) Holmes, F. A.; Kudelka, A. P.; Kavanagh, J . J .; Huber, M. H.;
Ajani, J . A.; Valero, V. In Taxane Anticancer Agents; Georg, G. I., Chen,
T. T., Ojima, I., Vyas, D. M., Eds.; ACS Symposium Series 583;
American Chemical Society: Washington, DC, 1995; pp 31-57.
(4) Paclitaxel in Cancer Treatment; McGuire, W. P., Rowinsky, E.
K., Eds.; Marcel Dekker: New York, 1995; Vol. 8, pp 1-349.
(5) Kingston, D. G. I. J . Nat. Prod. 2000, 63, 726-734.
(6) Landino, L. M.; Macdonald, T. L. In The Chemistry and Phar-
macology of Taxol and Its Derivatives; Farina, V., Ed. Elsevier
Science: New York, 1995; pp 301-335.
(7) J ordan, A.; Hadfield, J . A.; Lawrence, N. J .; McGown, A. T. Med.
Res. Rev. 1998, 18, 259-296.
(8) Georg, G. I.; Boge, T. C.; Cheruvallath, Z. S.; Clowers, J . S.;
Harriman, G. C. B.; Hepperle, M.; Park, H. In Taxol Science and
Applications; Suffness, M., Ed.; CRC: New York, 1995; pp 317-375.
(9) He, L.; J agtap, P. G.; Kingston, D. G. I.; Shen, H.-J .; Orr, G. A.;
Horwitz, S. B. Biochemistry 2000, 39, 3972-3978.
(10) Samaranayake, G.; Magri, N. F.; J itraugsri, C.; Kingston, D.
G. I. J . Org. Chem. 1991, 56, 5114-5119.
(11) Marder-Karsenti, R.; Dubois, J .; Bricard, L.; Gue´nard, D.;
Gue´ritte-Voegelein, F. J . Org. Chem. 1997, 62, 6631-6637.
10.1021/jo0015467 CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/18/2001

3322 J . Org. Chem., Vol. 66, No. 10, 2001
Barboni et al.
Sch em e 1a
hypothesis that the C5 oxygen acts as hydrogen bond
acceptor (electronic role). The D ring might also operate
to rigidify the diterpenoid core, causing the side chains
at C2, C4, and C13 to adopt a specific orientation for
binding (conformational role).13 Very recently14 the role
of the oxetane ring has been revised on the basis of a
unified receptor model for the microtubule binding of
paclitaxel and epothilone15 and hypothesized to play both
conformational and electronic roles but not to be essential
for bioactivity. This proposition seems to be confirmed
by the synthesis of the 5(20)deoxydocetaxel (4),16 which
has a cyclopropyl ring instead of the oxetane ring and
lacks the C5 oxygen atom. Compound 4 is about half as
a
(i) TBSCl, DMAP, rt, 14h; (ii) J ones reagent, acetone, rt, 30
min.
of nonataxel, Ojima and co-workers17 proposed a phar-
macophore that incorporates the hydrophobically col-
lapsed polar conformation of paclitaxel.19 A followup
photoaffinity labeling study by the same group is ratio-
nalized in terms of the latter.20 The same conformation
in a different binding mode has been put forward by Li
et al. based on fluorescence spectroscopy and REDOR
NMR studies.18 On the other hand, a pharmacophore
model based on the hydrophobically collapsed nonpolar
conformation of paclitaxel accounts for a wide range of
taxoid and epothilone SAR.14 More recently, a computa-
tionally refined â-tubulin-paclitaxel complex has identi-
fied an extended conformation of the drug with a C13
side chain orientation related to the nonpolar conforma-
tion, the T-conformation, as most consistent with the
electron crystallographic density of the Râ tubulin dimer.21
The latter complex reinforces the notion that the oxetane
ring is a rather weak hydrogen bond acceptor.
active as docetaxel on microtubules. It is reasonable that
the cyclopropyl ring, as the oxetane ring, acts to rigidify
the C-ring, giving the side chains a favorable orientation
for binding. The absence of the C5 oxygen atom might
be responsible for the slightly decreased biological activ-
ity, suggesting that the C5 oxygen could be directly
involved in the interaction with microtubules. Alterna-
tively, differential taxane solvation can also account for
the activity of 4.14 The receptor model has also been used
to predict the activity of analogues not yet synthesized14
by employing the so-called nonpolar paclitaxel structure
as a pharmacophore template for the â-tubulin/paclitaxel
binding conformation. At present, the bioactive confor-
mation is a matter of debate15,17-19 since insufficient data
have emerged to assign it unambiguously. Resolution of
this problem would make an important contribution to
the thinking of those who seek the next generation of
paclitaxel-like drugs. Three hypothesis have emerged. On
the basis of solution NMR data and molecular modeling
D-Secop a clita xel Syn th esis. To understand the role
of the oxetane ring better and to provide probes for the
evaluation of the different models for the paclitaxel
binding conformation and the paclitaxel-tubulin binding
site, the synthesis of D-secopaclitaxel analogues having
the C4 acetate has been undertaken. Our initial target
was the synthesis of D-secopaclitaxel analogues bearing
an acetate group at C4 and a free hydroxy group at C20.
To this end we chose the reaction reported by Magri and
Kingston22 for the oxetane ring opening, which was
reported to give the 5,6-dehydro-7-oxo-5-O-secopaclitaxel,
bearing an acetoxy group at C4 and a hydroxy group at
C20. The reaction of 2′-tert-butyldimethysilylpaclitaxel
(5) with the J ones reagent followed by column chroma-
tography gave compound 7 (Scheme 1), instead of 6,
revealing a facile migration of the C4 acetate to the C20
hydroxyl, as reported for the 13-oxobaccatin III deriva-
(12) Gunatilaka, A. A. L.; Ramdayal, F. D.; Sarragiotto, M.; King-
ston, D. G. I.; Sackett, D. L.; Hamel, E. J . Org. Chem. 1999, 64, 2694-
2703.
(13) Boge, T. C.; Hepperle, M.; Vander Velde, D.; Gunn, C. W.;
Grunewald, G. L.; Georg, G. I. Bioorg. Med. Chem. Lett. 1999, 9, 3041-
3046.
(14) Wang, M.; Cornett, B.; Nettles, J .; Liotta, D. C.; Snyder, J . P.
J . Org. Chem. 2000, 65, 1059-1068.
(15) Wang, M.; Xia, X.; Kim, Y.; Hwang, D.; J ansen, J . M.; Botta,
M.; Liotta, D. C.; Snyder, J . P. Org. Lett. 1999, 1, 43-46.
(16) Dubois, J .; Thoret, S.; Gue´ritte, F.; Gue´nard, D. Tetrahedron
Lett. 2000, 41, 3331-3334.
(17) Ojima, I.; Chakravarty, S.; Inoue, T.; Lin, S.; He, L.; Horwitz,
S. B.; Kuduk, S. D.; Danishefsky, S. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 4256-4261.
(18) Li, Y.; Poliks, B.; Cegelski, L.; Poliks, M.; Gryczynski, Z.;
Piszczek, G.; J agtap, P. G.; Studelska, D. R.; Kingston, D. G. I.;
Schaefer, J .; Bane, S. Biochemistry 2000, 39, 281-291.
(19) Vander Velde, D. G.; Georg, G. I.; Grunewald, G. L.; Gunn, C.
W.; Mitscher, L. A. J . Am. Chem. Soc. 1993, 115, 11650-11651.
(20) Rao, S.; He, L.; Chakravarty, S.; Ojima, I.; Orr, G. A.; Horwitz,
S. B. J . Biol. Chem. 1999, 274, 37990-37994.
(21) Snyder, J . P.; Nettles, J . H.; Cornett, B.; Downing, K. H.;
Nogales, E. PNAS 2001, in press.
(22) Magri, N. F.; Kingston, D. G. I. J . Org. Chem. 1986, 51, 797-
802.

Novel D-Seco Paclitaxel Analogues
J . Org. Chem., Vol. 66, No. 10, 2001 3323
Sch em e 2a
Sch em e 3a
a
(i) J ones reagent, acetone, rt, 30 min; (ii) DBU, CH2Cl2, rt, 1
h; (iii) HF/pyridine, pyridine, rt, 3 h.
tive.23 The acetoxy group at C20 was detected by its
HMBC correlation with H20.
A different approach to the D-seco analogue 7 has been
reported and consists of the J ones oxidation of 2′-
protected paclitaxel followed by treatment with DBU in
CH2Cl2.22 In our case this reaction gave rearranged
compound 8 (Scheme 2).
The same skeletal rearrangement was reported for 13-
oxobaccatin III in different conditions,23 but in that case
the C9-C10 acetoxy ketone transposition was not ob-
served.
1
The H NMR spectrum of 8 showed the presence of a
proton at C8 coupled with the C3 and the C19 protons.
The migration of the benzoyl group from C2 to C1 was
confirmed by the upfield shift of the C2 proton from 5.57
to 5.24 ppm and the downfield shift of the C1 carbon from
73.0 to 90.6 ppm. The 13C NMR of 8 lacked the C7
carbonyl signal but had a lactone carbonyl at 174.8 ppm,
which showed an HMBC correlation with the C5 and the
C6 protons. The presence of an acetoxy group on C20 was
also confirmed in the HMBC; it additionally showed a
correlation between H5 and C2. The absence of the
characteristic H10 singlet and the presence of a doublet
coupled with H8 indicated the acetoxy ketone transposi-
tion, confirmed by the HMBC correlation between H9 and
C19. The same transposition under similar experimental
conditions was previously reported for a compound hav-
ing a C-seco taxoid-like skeleton.26 It probably takes place
after the skeletal rearrangement of 7. Finally, a ROESY
spectrum confirmed that H3, H5, H8, H9, and one of the
H20 are on the same face of the molecule. Silyl depro-
tection of 8 with HF/pyridine gave compound 9.
To prepare analogues of 7 bearing the C4 acetate
group, we acetylated its C4 hydroxyl group with acetic
anhydride and DMAP in toluene at 80 °C (Scheme 3).27
The acetylation gave the desired compound 10 in accept-
able yield (46%). Interestingly, during the acetylation
step, new compounds 12 and 14 were formed in 13% and
2% yields, respectively. Because compound 12 gave broad
NMR spectra, the structure was confirmed on the depro-
tected compound 13. The most relevant spectroscopic
features of 13 are the absence of the H10 signal in the
1H NMR spectrum and the presence of two new non
a
(i) Ac2O, DMAP, toluene, 80 °C, 14 h; (ii) HF/pyridine, rt, 3 h.
protonated sp2 carbons in the 13C NMR spectrum (129.57
and 136.46 ppm), one of which shows a long-range
correlation with H19 in the HMBC spectrum. Compound
12 must be formed after enolization of the 9-keto group
followed by acetylation of the enol. We have observed a
similar reaction before, when the enol of 7,10-di-Troc-
baccatin III was acylated intramolecularly to yield a 9,10
cyclic carbonate.28 The benzoyl migration in compound
14 is indicated by the downfield shift af the C1 carbon
(90.10 ppm), whereas the HMBC correlation between H2
and an acetate carbonyl confirms the presence of an
acetoxy group at C2. Compound 14 is probably formed
via 2-benzoyl migration and subsequent acetylation of the
C2 hydroxy group. 1,2 Acyl migration of the paclitaxel
2-benzoyl group has been observed before in the reaction
with CS2.29 Attempts to increase the yield of compound
10 by increasing the reaction time or concentration of
reagents gave more complex mixtures of products.
Silyl deprotection of 10 with HF/pyridine gave com-
pound 11. Reduction of 10 with NaBH4 in 1:1 MeOH/
THF at 0 °C provided the 7R-hydroxy derivatives 15 and
17 (reduced at the ∆8 double bond) in a 3:4 ratio (Scheme
4). The stereoselectivity of the reduction is due to hydride
delivery to the ketone from the more accessible â-face.
Subsequent silyl deprotection afforded compounds 16 and
18, respectively.
(23) Marder-Karsenti, R.; Dubois, J .; Chiaroni, A.; Riche, C.; Gue´-
nard, D.; Gue´ritte, F.; Potier, P. Tetrahedron 1998, 54, 15833-15844.
(24) Sengupta, S.; Boge, T. C.; Georg, G. I.; Himes, R. H. Biochem-
istry 1995, 37, 11889-11894.
(25) Ali S. M.; Hoemann, M. Z.; Aube, J .; Mitscher, L. A.; Georg, G.
I.; McCall, R.; J ayasinghe, L. R. J . Med. Chem. 1995, 38, 3821-3828.
(26) Wender, P. A.; Badham, N. F.; Conway, S. P.; Floreancig, P.
E.; Glass, T. E.; Houze, J . B.; Krauss, N. E.; Lee, D.; Marquess, D. G.;
McGrane, P. L.; Meng, W.; Natchus, M. G.; Shuker, A. J .; Sutton, J .
C.; Taylor, R. E. J . Am. Chem. Soc. 1997, 119, 2757-2758.
(27) Hoemann, M. Z.; Vander Velde, D.; Aube´, J .; Georg, G. I.;
J ayasinghe, L. R. J . Org. Chem. 1995, 60, 2918-2921.
To invert the C7 stereochemistry to that of natural
taxanes, we followed the procedure reported by Chaudhary
(28) Datta, A.; AubJ , J .; Georg, G. I.; Mitscher, L. A.; J ayasinghe,
L. R. Bioorg. Med. Chem. Lett. 1994, 4, 1831-1834.
(29) Chaudhary, A. G.; Chordia, M. D.; Kingston, D. G. I. J . Org.
Chem. 1995, 60, 3260-3262.

3324 J . Org. Chem., Vol. 66, No. 10, 2001
Barboni et al.
active analogues19,31,32 and of the inactive 3.13 In the latter
case, conformational changes are relayed across the
molecule by relaxation of the A, B, and C rings and
adoption of a different preferred conformation of the C13
side chain with respect to the baccatin core.13 Examina-
tion of the compounds reported in this paper with a
saturated C ring establishes that those effects in 3 are
not solely due to the opening of the D ring but also
depend on the migration of the 4-acetyl out to C20. Table
1 summarizes the important J coupling constants in
paclitaxel and three analogues. As was the case with 3,
the C ring relaxes from a flattened, strained chair to a
more normal chair in 18 and 21, as shown by the
couplings involving H5, 6, and 7, but the effects on the
A and B rings are much less pronounced. The origin of
the A ring conformational change was investigated
computationally through molecular dynamics simulations
using Sybyl 6.5. The A ring conformation was character-
ized by the value of the torsion angle H13-C13-C14-
H14â. In the crystal structure of paclitaxel, this angle
has a value of 29° and 32° in the two inequivalent
molecules in the unit cell.33 In the simulation, the average
value was 30°, with a bell-shaped distribution of con-
formers about 50° wide. This is in agreement with the
approximately equal values of J 13-14 observed in solution.
In the series 11, 16, and 21, removal of the D ring
constraint relaxes the rigidity of the C ring and leads to
a decrease in the average value of the angle in the
simulations (from 30° to 18°) and an increase in the width
of the distribution. In accord with the experimental
finding, the two J 13-14 values become more unequal as
the 13-14â torsion approaches 0° and the 13-14R torsion
moves closer to 90°. These effects are further amplified
under the influence of 4-deacetylation. In simulations of
4-deacetylpaclitaxel and 4-deacetyl 21, the average tor-
sion angle shifted to negative values. Compound 3
displays a bimodal distribution of torsion angles, with
maximum populations at small positive and small nega-
tive values. In general, the simulations indicate that the
4-acetyl moiety is actually a much more important
determining factor for the A ring conformation than the
D ring. This is consistent with the observation that in
10-acetyl-4-deacetyltaxotere the H13 signal is a broad
doublet34 with J 13-14R having a very small value.
Sch em e 4a
a
(i) NaBH4, MeOH/THF (1:1), 0 °C, 30 min; (ii) HF/pyridine,
pyridine, rt, 3 h.
Sch em e 5a
D-seco compounds 11, 16, 18, and 21 sustain three
major orientations of the C20 side chain. As shown in
Figure 1 for the saturated C ring, each arises from
rotation about the axial C4/C20 bond, i.e., conformations
22a -22c. In structure 22a , the C20 C-O bond orienta-
tion is very similar to that of paclitaxel’s oxetane ring,
with the acetate methyl being directed away from the
bulk of the molecule (Figure 2). All three conformers are
internally strained as a result of 1,3-diaxial interactions.
An important geometric consequence of the latter is that
the C4-C20 bond is predicted by four separate force
fields (MM2*, MM3*, MMFF, AMBER*)35-37 to be par-
tially eclipsed in each of the forms. Thus, the OC4-C20O
a
(i) NaH, CS2, CH3I, THF, rt, 15 min; (ii) Bu3SnH, AIBN, H2O,
toluene, 75 °C, 1 h; (iii) HF/pyridine, pyridine, rt, 3 h.
et al.30 Treatment of compound 17 with NaH, CS2, and
MeI converted it into the S-methylthiocarboxy derivative
19 with the â stereochemistry at C7 (Scheme 5). Subse-
quent hydrolysis of 19, achieved by treatment with Bu3-
SnH and AIBN in wet toluene at 75 °C, gave compound
20 in 71% yield. Silyl deprotection yielded compound 21.
Compounds 9, 11, 13, 16, 18, and 21 were inactive both
in cytotoxicity (MCF-7 and MCF-7/ADR) and a tubulin
assembly assay.24,25
(31) Georg, G. I.; Harriman, G. C. B.; Hepperle, M.; Clowers, J . S.;
Vander Velde, D. G. J . Org. Chem. 1996, 61, 2664-2676.
(32) Boge, T. C.; Himes, R. H.; Vander Velde, D. G.; Georg, G. I. J .
Med. Chem. 1994, 37, 3337-3343.
(33) Mastropaolo, D.; Camerman, A.; Luo, Y.; Brayer, G. D.; Cam-
erman, M. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6920-6924.
(34) Datta, A.; J ayasinghe, L. R.; Georg, G. I. J . Med. Chem. 1994,
37, 4258-4260.
(35) Mohamadi, F.; Richards, N. G. J .; Guida, W. C.; Liscamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J .
Comput. Chem. 1990, 11, 440-449.
NMR a n d Con for m a tion a l An a lysis. We have pre-
viously published solution conformational analyses of
(30) Chaudhary, A. G.; Rimoldi, J . M.; Kingston, D. G. I. J . Org.
Chem. 1993, 58, 3798-3799.

Novel D-Seco Paclitaxel Analogues
J . Org. Chem., Vol. 66, No. 10, 2001 3325
Ta ble 1. Selected 1H NMR Cou p lin g Con sta n tsa of P a clita xel (1), D-Secop a clita xel (3), 18, a n d 21
J 2,3
J 5R,6R
J 5R,6â
J 5â,6R
J 5â,6â
J 6R,7R
J 6â,7R
J 6R,7â
J 6â,7â
J 13,14R
J 13,14â
1
3
18
21
7.1
5.3
6.6
5.8
9.6
2.3
6.7
4.4
11.0
11.0
9.0
4.4
8.1
7.2
9.0
10.3
9.4
smb
smb
smb
smb
3.1
sm
4.0
∼13.5
∼13.5
smb
smb
smb
4.4
11.2
9.9
a
b
CDCl3, 400 MHz. Small but not correctly measurable.
NMR coupling constants and ROESY cross-peak volumes
taken in DMSO-d6 were thus combined with 5126 con-
formations obtained from conformational searches with
four separate force fields using an aqueous continuum
model (see Experimental Section). Unlike previous NAM-
FIS analyses,38,39 the present one must be taken as more
qualitative than quantitative, since the cross-peaks were
characterized only as strong, medium, and weak. To the
latter, we have assigned separation values of 2.7, 3.5, and
4.0 Å, respectively. Using the corresponding 29 ROE-
derived distances and six coupling constants (Table 1),
the 5126 conformer collection was deconvoluted to a set
of seven conformations with estimated populations rang-
ing from 6 to 46% and with a sum of square differences
SSD ) 44.38 Of the seven, three are in the 22a family
and four in the 22b family. No representatives of 22c
are compatible with the NMR data, although the confor-
mational data set contains many such representatives.
Of the three most populated forms 18a , 18b, and 18c
(46, 13, and 11%, respectively), the first and third
incorporate the features of 22b, the second, 22a . Not
unexpectedly, 18a is very similar to the polar conforma-
tion deduced from NMR studies in DMSO-d6/D2O show-
ing hydrophobic collapse between the C2 and C3′ phenyl
groups.19 The 169° HC2′-C3′H dihedral angle corre-
sponds to J H2′H3′ ) 10.6 Hz.42 Of the six remaining
conformations, three also adopt a variation of the polar
conformer leading to an estimated total of 71% polar
character in the conformational profile of 18. Evidence
has been presented both for and against the “hydrophobic
collapse” as the bioactive conformation of taxanes. How-
ever, from both active32 compounds that do not adopt this
conformation, and inactive compounds that do (this
work), it is clear that the population of this conformation
in solution does not correlate with bioactivity.
Interestingly, the second and third most populated
structures 18b and 18c are extended conformers. In this
respect, the D-seco analogue 18 resembles paclitaxel in
both nonpolar39 and polar40 solvents. That is, while the
dominant conformation represents the family character
with respect to hydrophobic collapse, a not insignificant
fraction of the perceived conformers are extended forms.
More importantly, the latter structures bear a close
resemblance to an emerging model regarding the binding
conformation of paclitaxel at the â-tubulin binding site,
namely, the T conformation.21 Figure 3 illustrates the
nonpolar, polar, and T conformations. Superposition of
the A and B rings in 18b and T-paclitaxel results in a
remarkably similar placement of the four terminal hy-
drophobes emanating from C2, C4, and C3′. A key
difference between paclitaxel and the inactives 18 and
F igu r e 1. Rotamers around the C4-C20 bond.
F igu r e 2. Partial view of compound 18b illustrating how
D-ring opening to a type 22a conformation can mimic the
oxetane ring. The C20 acetate methyl is directed away from
the body of the molecule.
torsion angles are in the vicinity of 113, -95, and 30°
for conformers 22a , 22b, and 22c, respectively. This is
not the case for the diaxial cyclohexanes that are unfused
to the taxane core. For diaxial monocyclic rings, regard-
less of the remaining substitution pattern, the C4 and
C8 diaxial groups in 22 are nicely staggered. In sharp
contrast, (A)B-C ring fusion forces the axial C8 methyl
in the direction of C4. As a result, there is a pronounced
gearing effect between the axial centers and the C2
benzoyl group that causes a less than ideal local orienta-
tion of the C4-C20 bond. As described above, conforma-
tional changes are relayed between rings A and D in
taxol. These are relaxed to some extent when ring D is
severed. At the same time, relaxation is not complete.
The bicyclic A-B architecture still appears to exert a
forceful influence on the C ring (e.g., the 19-Me tilt angle)
as measured by molecular mechanics.
To examine the conformational profile of the C ring in
the context of the full C13 side chain bearing structure,
we performed a NAMFIS analysis38-40 on compound 18.
(40) Nevins, N. N.; Cicero, D. O.; J ansen, J .; J iminez-Barbero, J .;
Snyder, J . P. (unpublished results).
(41) Four of the conformations sustain gauche interactions for the
HC2′-C3′H torsional angle; two, 175-180°. As a result, the final
weighted conformational average yields J H2′H3′ ) 7.9 Hz as the fit to
the experimental value of 6.4 Hz.
(36) Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T. J .
Am. Chem. Soc. 1990, 112, 6127-6129.
(37) Cf. http://www.schrodinger.com/macromodel2.html.
(38) Nevins, N.; Cicero, D. O.; Snyder, J . P. J . Org. Chem. 1999, 65,
3979-3986.
(39) Snyder, J . P.; Nevins, N. N.; Cicero, D. O.; J ansen, J . J . Am.
Chem. Soc. 2000, 122, 724-725.
(42) Nogales, E.; Wolf, S. G.; Downing, K. H. Nature 1998, 391, 199-
202.

3326 J . Org. Chem., Vol. 66, No. 10, 2001
Barboni et al.
F igu r e 3. Graphical representations of the three conformations of paclitaxel. The depicted distances (C) are between the centroids
of the phenyl rings at C2 and C3′. Only the closest of the two rings at C3′ are labeled: (a) nonpolar, (b) T-paclitaxel, (c) polar
conformation.
Ta ble 2. P r ed iction of th e Bin d in g Affin ities of D-Seco
P a clita xel An a logu es by th e Secon d -Gen er a tion
P a clita xel-Ep oth ilon e Min ir ecep tor Mod el
of the acetate at C20 in the D-seco compounds is sterically
blocked by Leu217. Likewise, when the less stable
conformations 22b are docked into the site, an equally
unproductive interaction arises from close contact be-
tween the C20 acetate methyl and Leu275. These model-
ing observations are fully consistent with the results of
the conformational analysis described in the previous
section. Not surprisingly, the enhanced minireceptor
model predicts that 11, 16, 18, and 21 all bind to
â-tubulin with an affinity at least 100 times less than
that of paclitaxel.
Kpred (M)
Kpred/KTX
∆Gsolv (kcal/mol)
paclitaxel
2.9e-5
2.3e-3
1.5e-3
1.0e-4
2.5e-5
1
79
52
3.5
0.9
-13.2
-23.0
-20.6
-15.7
-16.13
11
16
18
21
21 is the steric bulk at C20 in the latter. Given the
availability of the T-rotational form to the D-seco ana-
logues, it implies that the disrupted D-ring and the
orientation of the pendant substitutents are responsible
for the lack of activity (see below).
Con clu sion
In summary, four novel D-seco paclitaxel analogues
bearing acetate groups at C4 and C20 were prepared. All
compounds were inactive in cytotoxicity and tubulin
assembly assays. Conformational analysis and dynamics
simulations strongly suggest that the tetracyclic taxane
core is capable of a hierarchy of inter-ring transmission
effects. On one hand, removal of the D ring has a decisive
influence on the conformations of the A and C rings.
Flattened in the four-ring core, the latter assumes a more
relaxed shape in the D-seco analogues. Nonetheless, the
intact AB pair still appears to exert a gearing effect on
the axial substitutents of the C ring. Surprisingly, as
judged by J 13-14 changes and dynamics simulation, the
largest determining factor for the A ring conformation
is the presence of the 4-acetyl moiety. As to prediction of
biological activity, the present results illuminate an
important weakness in our second-generation minire-
ceptor model and a strategy for correcting it. Clearly,
bulky groups at C5 and C20 will impair the activity of
paclitaxel analogues even though the molecule can adopt
what we believe to be the bioactive form (T-paclitaxel)
at the â-tubulin binding site.
Min ir ecep tor P r ed iction s. Prior to synthesis, we
predicted the 22a analogues of D-seco analogues 11 and
16 to bind â-tubulin only weakly relative to paclitaxel
but 18 and 21 to be similar to the drug in their binding
affinities (Table 2). Unfortunately, in light of the biologi-
cal data described above, the second-generation model15
overpredicts binding of the compounds by a factor of 10-
60.
The basis for the lack of agreement between experi-
ment and prediction lies in the constitution of the
minireceptor in the vicinity of the oxetane ring. In this
model, a conservative set of â-tubulin protein residues
taken from photoaffinity labeling experiments was em-
ployed. In particular, we selected an Arg to form a strong
hydrogen bond with the oxetane oxygen but did not
supplement this region of the receptor with other side
chains. With the availability of the electron crystal-
lography structure of Râ tubulin42 and complementary
modeling studies,14 it has become clear that the H-bond
to the oxetane is rather weak and supplied by the neutral
residue Thr instead of cationic Arg. For oxetane ring
replacements such as thietane and cyclopropane, the
arginine model is sufficient to account for bioactivity.14
However, the present synthetic and biological results add
another critical requirement to the receptor model.
Namely, additional residues are necessary to accom-
modate local ligand-protein steric effects around the
opened oxetane ring. In an evolving third-generation
minireceptor construct, a threonine and two leucines
were positioned in close contact with the C4-C5 four-
membered ring in accord with the complementary loca-
tions of Thr276, Leu217, and Leu275 in the tubulin
protein.43 When conformations corresponding to 22a are
docked into this receptor cavity, the terminal CH3 group
Exp er im en ta l Section
Gen er a l Meth od s. Cytotoxicity and tubulin binding were
carried out according to established literature protocols24,25
except that the MCF-7 breast cancer cell line was used in the
cytotoxicity assays. Commercially available reagents and
solvents were used without further purification. CH2Cl2 was
dried by distillation from CaH2, and THF by distillation from
Na-benzophenone. Flash column chromatography was carried
out with silica gel as the stationary phase. J ones reagent was
prepared as follows: CrO3 (1.67 g, 0.017 mmol) was dissolved
(43) Wang, M.; Snyder, J . P. (unpublished results).

Novel D-Seco Paclitaxel Analogues
J . Org. Chem., Vol. 66, No. 10, 2001 3327
in water (2.5 mL). The solution was cooled at 0 °C and
concentrated H2SO4 (1.44 mL) was added. Then water was
added up to 7 mL.
J ) 9.7, 1H), 7.32 (bt, J ) 7.4, 1H), 7.36-7.44 (m, 7H), 7.49
(bt, J ) 7.6, 2H), 7.54 (bt, J ) 7.3, 1H), 7.79 (bd, J ) 7.7, 2H),
7.90 (bd, J ) 7.9, 2H); 13C NMR (100 MHz, CDCl3) δ 12.72,
18.21, 20.98, 21.07, 22.32, 27.56, 34.05, 35.08, 37.78, 42.61,
50.40, 54.69, 67.00, 72.22, 73.93, 79.57, 82.65, 82.85, 90.17,
96.25, 127.44, 127.55, 128.51, 128.78, 129.15, 129.25, 130.00,
132.01, 132.25, 133.17, 134.52, 138.73, 139.69, 141.28, 165.36,
167.56, 170.60, 170.74, 172.56, 174.77, 199.43; IR (CHCl3)
3470, 1810, 1760, 1730, 1675, 1540, 1380, 1250 cm-1; FAB
HRMS m/z calcd for (M + H)+ C47H50NO14 852.3231, found
20-Acetyl-2′-O-(ter t-bu tyld im eth ylsilyl)-4-d ea cetyl-5,6-
d eh yd r o-7-oxo-5-O-secop a clita xel (7). Compound 544 (785
mg, 0.81 mmol) was dissolved in acetone (15 mL, freshly
distilled from KMnO4), and freshly prepared J ones reagent (1.6
mL) was added. After stirring for 30 min at room temperature
(TLC monitoring), 2-propanol was added as the solution turned
green. Then brine was added and the solution extracted with
EtOAc (4 × 15 mL). The combined extract was dried over Na2-
SO4, filtered, and evaporated under vacuum. After flash
column chromatography on silica gel (9:1 to 4:1 hexane/EtOAc),
520 mg of compound 7 (0.54 mmol, 66%) was collected as an
amorphous solid. 1H NMR (400 MHz, CDCl3): δ -0.30 (s, 3H),
-0.11 (s, 3H), 0.81 (s, 9H), 1.06 (s, 3H), 1.20 (s, 3H), 1.54 (s,
3H), 1.73 (s, 3H), 1.92 (s, 3H), 2.22 (s, 3H), 2.41 (dd, J ) 15.7,
10.6, 1H), 2.94 (dd, J ) 15.7, 4.4 Hz, 1H), 4.13 (d, J ) 12.0,
1H), 4.15 (d, J ) 6.0, 1H), 4.32 (d, J ) 12.0, 1H), 4.45 (bs,
1H), 5.57 (d, J ) 6.0, 1H), 5.86 (bd, J ) 9.8, 1H), 6.03 (d, J )
10.3, 1H), 6.05 (bd, J ) 8.3, 1H), 6.38 (s, 1H), 7.02 (d, J )
10.3, 1H), 7.18 (t, J ) 7.7), 7.26-7.53 (m, 9H), 7.57 (bt, J )
7.2, 1H), 7.81 (bd, J ) 7.7, 2H), 8.13 (bd, J ) 7.9, 2H). 13C
NMR (100 MHz, CDCl3): δ -5.53, -5.16, 16.33, 18.66, 19.07,
20.63, 21.34, 25.95, 27.82, 30.10, 36.24, 43.00, 54.40, 55.54,
63.71, 67.32, 72.16, 73.03, 74.75, 75.90, 77.92, 123.46, 126.92,
127.42, 128.24, 128.76, 128.92, 129.27, 129.34, 130.92, 132.27,
133.89, 134.80, 135.37, 140.08, 140.19, 155.75, 167.47, 168.05,
169.64, 170.62, 170.65, 198.53, 199.73. MS (ES+) m/z 988 (M
+ Na)+, 966 (M + H)+, 567, 549, 507, 400.
Com p ou n d 8. To a CH2Cl2 (5 mL) solution of compound 7
(150 mg, 0.16 mmol) was added DBU (509 mg, 0.5 mL, 3.3
mmol), and the reaction mixture was stirred at room temper-
ature for 1 h. Then CH2Cl2 and HCl (1 N) were added. The
organic layer was washed with saturated NaHCO3 and brine
and dried over Na2SO4. After filtration, evaporation of the
solvent and flash column chromatography (3:2 hexane/EtOAc),
120 mg of compound 8 (0.124 mmol, 80%) was collected as an
amorphous solid: 1H NMR (400 MHz, CDCl3) δ -0.34 (s, 3H),
-0.08 (s, 3H), 0.79 (s, 9H), 1.24 (s, 3H), 1.26 (d, J ) 7.1, 3H),
1.31 (s, 3H), 2.06 (bs, 6H), 2.24 (s, 3H), 2.45 (dd, J ) 16.5, 6.1,
1H), 2.55 (d, J ) 18.6, 1H), 2.77 (m, 2H), 2.91 (m, 1H), 3.25
(dd, J ) 16.5, 9.9, 1H), 4.16 (d, J ) 11.9, 1H), 4.66 (d, J ) 1.8,
1H), 5.06 (d, J ) 11.9, 1H), 5.20 (bd, J ) 6.1, 1H), 5.26 (d, J )
8.6, 1H), 5.40 (d, J ) 6.7, 1H), 5.91 (dd, J ) 9.3, 1.7, 1H), 5.98
(bt, J ) 7.7, 1H), 7.12 (d, J ) 9.4, 1H), 7.30 (m, 1H), 7.40 (m,
8H), 7.52 (m, 2H), 7.84 (m, 4H); 13C NMR (100 MHz, CDCl3)
δ -5.64, -4.99, 12.71, 18.18, 18.61, 21.00, 21.07, 22.54, 25.91,
27.36, 35.08, 37.63, 42.63, 50.52, 56.07, 67.21, 71.10, 76.03,
79.36, 82.05, 82.92, 90.62, 96.17, 127.15, 127.51, 128.20,
128.72, 128.93, 129.17, 129.99, 132.03, 132.08, 133.06, 134.89,
138.90, 139.46, 141.69, 165.26, 167.53, 170.57, 170.67, 170.85,
174.76, 199.60; MS (FAB+) m/z 965, 845, 445, 400, 354, 210,
177, 154.
852.3261; [R]20 124 (c 1.00, CHCl3).
D
Acetyla tion of com p ou n d 7. Compound 7 (200 mg, 0.21
mmol) was dissolved in toluene (10 mL); then DMAP (122 mg,
1 mmol) and Ac2O (790 µL, 857 mg, 8.4 mmol) were added.
The reaction mixture was stirred under argon at 80 °C for 17
h (the solution turned brown, TLC check showed some starting
material but the reaction was worked up to avoid decomposi-
tion); then the solvent was concentrated under vacuum and
the residue was dissolved in CH2Cl2 and washed with satu-
rated NaHCO3. The aqueous solution was extracted with CH2-
Cl2 (3 × 15 mL), and the combined organic layers were washed
with brine and then dried over Na2SO4, filtered, and evapo-
rated. After flash column chromatography on silica gel (4:1 to
7:3 hexane/EtOAc), 80 mg of compound 10 (0.079 mmol, 38%),
28 mg of 12 (0.026 mmol, 13%), and 4 mg of 14 (0.004 mmol,
2%) were collected.
Com p ou n d 10. Amorphous solid: 1H NMR (400 MHz,
CDCl3) δ -0.35 (s, 3H), -0.06 (s, 3H), 0.76 (s, 9H), 1.18 (s,
3H), 1.20 (s, 3H), 1.63 (s, 3H), 1.85 (s, 3H), 1.89 (s, 3H), 2.18
(m, 1H, 14), 2.21 (s, 3H), 2.48 (s, 3H), 2.79 (dd, J ) 15.3, 8.6
Hz, 1H), 4.27 (d, J ) 13.1, 1H), 4.33 (d, J ) 7.0, 1H), 4.52 (d,
J ) 13.1, 1H), 4.67 (d, J ) 1.9, 1H), 5.65 (d, J ) 7.0, 1H), 5.80
(dd, J ) 9.2, 1.9, 1H), 6.07 (d, J ) 10.6, 1H), 6.27 (bt, J ) 8.7,
1H), 6.36 (s, 1H), 7.08 (d, J ) 9.2, 1H), 7.18 (d, J ) 10.6, 1H),
7.26-7.62 (m, 11H), 7.77 (bd, J ) 8.1, 2H), 8.10 (bd, J ) 7.8,
2H); 13C NMR (100 MHz, CDCl3) δ -5.48, -4.81, 15.00, 17.91,
18.52, 20.87, 21.27, 21.32, 23.30, 25.89 (x3), 26.52, 30.11, 35.89,
43.53, 51.30, 55.96, 63.79, 68.16, 70.99, 74.55, 75.72, 76.85,
79.32, 82.97, 124.77, 126.70, 127.40, 128.30, 129.13, 129.23,
129.30, 129.80, 130.48, 132.26, 132.42, 134.31, 134.57, 138.72,
141.58, 149.18, 167.31, 167.57, 169.54, 169.87, 171.65, 196.99,
199.48; IR (CHCl3) 3465, 2960, 1760, 1690, 1520, 1240 cm-1
;
MS (FAB+) m/z 1008 (M + H)+, 948, 549, 489, 400, 354.
Com p ou n d 12. Amorphous solid: 1H NMR (400 MHz,
CDCl3, 50 °C) δ -0.34 (s, 3H), -0.03 (s, 3H), 0.79 (s, 9H), 1.23
(s, 3H), 1.44 (s, 3H), 1.68 (s, 3H), 1.84 (s, 3H), 1.86 (s, 3H),
2.06 (s, 3H), 2.18 (s, 3H), 2.27 (m, 1H, 14), 2.50 (s, 3H), 2.90
(dd, J ) 15.0, 7.6 Hz, 1H), 3.94 (d, J ) 5.3, 1H), 4.14 (d, J )
12.5, 1H), 4.60 (d, J ) 12.5, 1H), 4.64 (bs, 1H), 5.79 (bd, J )
9.1, 1H), 5.98 (d, J ) 5.3, 1H), 6.07 (d, J ) 10.6, 1H), 6.21 (bt,
J ) 8.9, 1H), 7.08 (d, J ) 9.1, 1H), 7.24 (d, J ) 10.6, 1H), 7.26-
7.61 (m, 11H), 7.79 (bd, J ) 7.8, 2H), 8.06 (bd, J ) 7.8, 2H);
13C NMR (125 MHz, CDCl3) δ -5.85, -5.16, 16.48, 18.10,
20.24, 20.44, 20.51, 20.77, 21.37, 23.92, 25.50, 35.53, 43.28,
49.64, 55.14, 55.81, 66.30, 70.71, 74.67, 75.56, 78.93, 83.39,
126.21, 126.31, 126.71, 126.96, 127.28, 127.75, 128.64, 128.83,
129.66, 129.85, 130.10, 130.59, 134.50, 136.71, 138.84, 139.06,
143.90, 146.48, 166.34, 166.91, 167.01, 168.01, 169.15, 169.37,
171.00, 194.96; MS (FAB+) m/z 1051 (M + H)+, 991, 400, 354.
Com p ou n d 14. Amorphous solid: 1H NMR (400 MHz,
CDCl3) δ -0.27 (s, 3H), -0.11 (s, 3H), 0.83 (s, 9H), 1.12 (s,
3H), 1.39 (s, 3H), 1.52 (s, 3H), 1.89 (s, 3H), 1.94 (s, 3H), 1.99
(s, 3H), 2.22 (s, 3H), 2.96 (dd, J ) 16.4, 4.5 Hz, 1H), 3.78 (dd,
J ) 16.4, 10.2 Hz, 1H), 4.12 (d, J ) 11.9, 1H), 4.18 (d, J ) 6.1,
1H), 4.39 (d, J ) 11.9, 1H), 4.49 (d, J ) 1.6, 1H), 5.28 (s, 1H),
5.65 (d, J ) 6.1, 1H), 5.95 (m, 1H), 6.03 (bd, J ) 8.5, 1H), 6.06
(d, J ) 10.4, 1H), 6.33 (s, 1H), 7.17 (d, J ) 10.6, 1H), 7.25-
7.67 (m, 12H), 7.67 (bd, J ) 8.3, 2H), 7.83 (bd, J ) 7.8, 2H);
13C NMR (100 MHz, CDCl3) δ -5.46, -5.16, 16.17, 18.64,
18.68, 20.79, 20.98, 21.34, 21.56, 25.95, 28.81, 44.60, 54.45,
55.62, 63.62, 67.46, 71.49, 72.90, 73.83, 75.63, 77.03, 90.09,
123.42, 127.32, 127.80, 128.16, 128.79, 128.82, 128.92, 129.80,
131.32, 131.92, 133.22, 133.28, 135.14, 140.01, 141.19, 156.93,
164.76, 168.01, 169.53, 170.98, 171.13, 171.33, 197.98, 199.29;
MS (FAB+) m/z 1008 (M + H)+, 950, 852, 826, 705.
Com p ou n d 9. To an ice cooled solution of compound 8 (50
mg, 0.052 mmol) in dry pyridine (3 mL) was added HF‚pyridine
(25 drops), and the mixture was stirred at room temperature
for 3 h (TLC check, 3:2 hexane/EtOAc). The solution was then
neutralized with saturated NaHCO3, acidified with 3 N HCl,
and extracted with CH2Cl2 (3 × 10 mL). The organic layer was
washed with brine and then dried over Na2SO4, filtered, and
evaporated. After flash column chromatography (7:3 hexane/
EtOAc), 22 mg of compound 9 (0.026 mmol, 50%) was collected
1
as a colorless powder: mp 142-144 °C; H NMR (400 MHz,
CDCl3) δ 1.23 (s, 3H), 1.23 (d, J ) 7.0, 3H), 1.30 (s, 3H), 2.00
(s, 3H), 2.02 (s, 3H), 2.21 (s, 3H), 2.53 (d, J ) 18.8, 1H), 2.59
(dd, J ) 16.7, 6.1, 1H), 2.73 (dd, J ) 18.7, 5.8 1H), 2.76 (d, J
) 8.5, 1H), 2.88 (m, 1H), 3.35 (dd, J ) 16.7, 9.8, 1H), 3.37 (bs,
1H), 4.28 (d, J ) 11.8, 1H), 4.79 (bt, J ) 2.0, 1H), 4.92 (d, J )
11.8, 1H), 5.13 (d, J ) 8.3, 1H), 5.21 (bd, J ) 5.8, 1H), 5.37 (d,
J ) 6.7, 1H), 5.93 (dd, J ) 9.7, 1.8, 1H), 5.98 (m, 1H), 6.98 (d,
(44) Magri, N. F.; Kingston, D. G. I. J . Nat. Prod. 1988, 51, 298-
306.

3328 J . Org. Chem., Vol. 66, No. 10, 2001
Barboni et al.
Com p ou n d 11. To an ice cooled solution of compound 10
(30 mg, 0.030 mmol) in dry pyridine (3 mL) was added HF‚
pyridine (20 drops), and the mixture was stirred at room
temperature for 3 h (TLC check, 3:2 hexane/EtOAc). The
solution was then neutralized with saturated NaHCO3, acidi-
fied with 3 N HCl, and extracted with CH2Cl2 (3 × 10 mL).
The organic layer washed with brine and then dried over Na2-
SO4, filtered, and evaporated. After flash column chromatog-
raphy (3:2 hexane/EtOAc), 19 mg of compound 11 (0.021 mmol,
71%) was collected as a colorless powder: mp 150-152 °C; 1H
NMR (400 MHz, CDCl3) δ 1.15 (s, 3H), 1.19 (s, 3H), 1.60 (s,
3H), 1.61 (s, 3H), 1.89 (s, 3H), 2.20 (s, 3H), 2.31 (s, 3H), 2.40
(dd, J ) 15.5, 9.7, 1H), 2.50 (dd, J ) 15.5, 7.6, 1H), 3.96 (d, J
) 4.2, 1H), 4.25 (d, J ) 13.0, 1H), 4.28 (d, J ) 6.9, 1H), 4.51
(d, J ) 13.0, 1H), 4.78 (dd, J ) 4.2, 2.6, 1H), 5.62 (d, J ) 6.9,
1H), 5.80 (dd, J ) 9.1, 2.6, 1H), 6.02 (d, J ) 10.6, 1H), 6.11
(bt, J ) 8.5, 1H), 6.30 (s, 1H), 6.97 (d, J ) 10.6, 1H), 7.19 (d,
J ) 9.1, 1H), 7.28-7.54 (m, 10H), 7.61 (bt, J ) 7.7, 1H), 7.77
(bd, J ) 7.7, 2H), 8.07 (bd, J ) 7.7, 2H); 13C NMR (100 MHz,
CDCl3) δ 14.95, 17.95, 20.64, 20.87, 21.25, 22.95, 26.71, 36.04,
43.35, 51.01, 55.15, 63.84, 68.27, 71.80, 74.06, 74.29, 76.90,
79.12, 83.42, 124.81, 127.42, 127.46, 128.66, 129.16, 129.24,
129.29, 129.68, 130.40, 132.40, 132.92, 134.11, 134.39, 138.45,
140.79, 149.14, 167.11, 167.47, 169.44, 169.92, 170.40, 172.04,
11H), 7.74 (bd, J ) 8.1, 1H), 8.18 (bd, J ) 8.5, 1H); 13C NMR
(100 MHz, CDCl3) δ -6.07, -5.33, 14.11, 15.47, 16.78, 20.23,
20.91, 23.23, 25.44, 25.91, 36.59, 42.18, 43.69, 55.49, 58.58,
68.52, 70.35, 72.89, 75.54, 75.99, 78.49, 80.09, 82.31, 126.30,
127.01, 127.78, 127.81, 128.71, 128.72, 128.80, 130.09, 130.31,
130.96, 131.28, 131.74, 133.37, 134.20, 138.45, 140.21, 166.80,
167.77, 169.65, 170.10, 170.93, 171.41, 205.96; MS (FAB+) m/z
1010 (M + H)+, 1009, 950, 768, 491, 400, 354, 210, 177.
Com p ou n d 17. Amorphous solid: 1H NMR (400 MHz,
CDCl3) δ -0.33 (s, 3H), -0.07 (s, 3H), 0.78 (s, 9H), 1.11 (s,
3H), 1.19 (s, 3H), 1.48 (s, 3H), 1.58 (m, 1H), 1.91 (s, 3H), 1.95
(s, 3H), 1.95 (m, 1H), 2.01 (m, 1H), 2.15 (m, 1H), 2.19 (s, 3H),
2.41 (s, 3H), 2.89 (m, 1H), 3.23 (dd, J ) 15.0, 8.9 Hz, 1H), 3.58
(bs, 1H), 3.76 (d, J ) 6.6, 1H), 4.16 (d, J ) 13.9, 1H), 4.44 (d,
J ) 13.9, 1H), 4.66 (d, J ) 1.9, 1H), 5.71 (d, J ) 6.6, 1H), 5.76
(dd, J ) 9.0, 1.9, 1H), 6.31 (bt, J ) 8.9, 1H), 6.89 (s, 1H), 7.12
(d, J ) 9.0, 1H), 7.25-7.58 (m, 11H), 7.79 (bd, J ) 7.7, 1H),
8.03 (bd, J ) 7.8, 1H); 13C NMR (100 MHz, CDCl3) δ -5.84,
-5.30, 15.04, 17.63, 18.15, 20.65, 20.91, 21.42, 23.08, 25.51,
26.27, 27.19, 35.48, 42.70, 47.67, 55.72, 59.26, 67.05, 71.34,
74.97, 75.41, 76.20, 79.12, 79.68, 85.46, 126.32, 126.42, 127.00,
127.15, 127.84, 128.65, 128.79, 129.89, 131.77, 132.77, 133.59,
134.26, 138.51, 140.49, 166.76, 167.61, 168.97, 169.61, 170.30,
171.38, 206.66; MS (FAB+) m/z 1012 (M + H)+, 952, 493, 400,
354, 210, 177.
196.75, 199.27; IR (CHCl3) 3490, 1750, 1670, 1540, 1230 cm-1
;
FAB HRMS m/z calcd for (M + H)+ C49H52NO15 894.3337,
Com p ou n d 16. To an ice cooled solution of compound 15
(30 mg, 0.030 mmol) in dry pyridine (3 mL) was added HF‚
pyridine (20 drops), and the mixture was stirred at room
temperature for 3 h (TLC check, 3:2 hexane/EtOAc). The
solution was then neutralized with saturated NaHCO3, acidi-
fied with 3 N HCl, and extracted with CH2Cl2 (3 × 10 mL).
The organic layer was washed with brine and then dried over
Na2SO4, filtered, and evaporated. After flash column chroma-
tography (3:2 hexane/EtOAc), 25 mg of compound 16 (0.028
mmol, 94%) was collected as a colorless powder: mp 153-155
found 894.3362; [R]20 -58 (c 0.70, CHCl3).
D
Com p ou n d 13. To an ice cooled solution of compound 12
(60 mg, 0.057 mmol) in dry pyridine (3 mL) was added HF‚
pyridine (25 drops), and the mixture was stirred at room
temperature for 3 h (TLC check, 3:2 hexane/EtOAc). The
solution was then neutralized with saturated NaHCO3, acidi-
fied with 3 N HCl, and extracted with CH2Cl2 (3 × 10 mL).
The organic layer was washed with brine and then dried over
Na2SO4, filtered, and evaporated. After flash column chroma-
tography (3:2 hexane/EtOAc), 30 mg of compound 13 (0.032
mmol, 56%) was collected as a colorless powder: mp 148-150
°C; 1H NMR (400 MHz, CDCl3) δ 1.22 (s, 3H), 1.43 (s, 3H),
1.63 (bs, 3H), 1.66 (s, 3H), 1.88 (s, 3H), 2.08 (s, 3H), 2.20 (s,
3H), 2.38 (s, 3H), 2.43 (dd, J ) 15.2, 10.0, 1H), 2.58 (dd, J )
15.2, 7.0, 1H), 3.92 (bs, 1H), 4.04 (d, J ) 4.0 Hz), 4.10 (bd, J
) 12.6, 1H), 4.59 (d, J ) 12.6, 1H), 4.80 (dd, J ) 4.0, 2.6, 1H),
5.79 (bd, J ) 9.4, 1H), 5.95 (d, J ) 5.4, 1H), 6.02 (d, J ) 10.7,
1H), 6.11 (bt, J ) 8.5, 1H), 7.04 (d, J ) 10.7, 1H), 7.15 (d, J )
9.4, 1H), 7.30-7.55 (m, 10H), 7.62 (bt, J ) 7.3, 1H), 7.79 (bd,
J ) 7.8, 2H), 8.06 (bd, J ) 7.8, 2H); 13C NMR (100 MHz, CDCl3)
δ 16.43, 18.90, 20.36, 20.52, 20.88, 21.12, 22,66, 23.95, 35.80,
43.13, 49.50, 55.01, 55.11, 66.50, 71.51, 73.95, 74.55, 78.95,
83.68, 125.92, 127.00, 127.07, 128.13, 128.71, 128.81, 128.87,
129.57, 129.97, 130.84, 131.83, 133.73, 134.04, 136.46, 138.46,
138.90, 143.03, 146.48, 166.26, 166.91, 166.98, 168.05, 169.24,
169.82, 171.73, 194.70; IR (CHCl3) 3480, 1750, 1700, 1530,
1225 cm-1; FAB HRMS m/z calcd for (M + H)+ C51H54NO16
1
°C; H NMR (400 MHz, CDCl3) δ 1.13 (bs, 6H), 1.33 (s, 3H),
1.72 (bs, 3H), 1.75 (s, 3H), 2.18 (s, 3H), 2.31 (s, 3H), 2.33 (m,
2H), 2.96 (d, J ) 9.8 Hz), 3.60 (dd, J ) 9.8, 5.7 1H), 3.69 (d, J
) 3.4 Hz), 4.15 (d, J ) 12.6, 1H), 4.33 (d, J ) 12.6, 1H), 4.47
(d, J ) 8.4, 1H), 4.80 (bt, J ) 3.4, 1H), 5.62 (d, J ) 10.2, 1H),
5.68 (d, J ) 8.4, 1H), 5.89 (bd, J ) 9.7, 1H), 5.99 (dd, J )
10.2, 5.7 1H), 6.14 (bt, J ) 8.1, 1H), 6.94 (s, 1H), 7.15 (d, J )
9.7, 1H), 7.20-7.60 (m, 11H), 7.76 (bd, J ) 7.8, 2H), 8.16 (bd,
J ) 7.6); 13C NMR (100 MHz, CDCl3) δ 16.00, 17.09, 20.74,
21.10, 21.33, 23.38, 26.69, 36.89, 42.47, 44.17, 54.72, 59.05,
69.01, 71.99, 73.09, 74.04, 76.13, 79.04, 80.34, 83.35, 125.92,
127.31, 127.47, 128.48, 129.09, 129.17, 129.25, 130.42, 130.63,
131.00, 132.28, 132.45, 133.94, 134.23, 138.63, 139.74, 166.96,
168.15, 169.96, 170.41, 171.97, 172.21, 205.95; IR (CHCl3)
3460, 1750, 1675, 1530, 1240 cm-1; MS (FAB+) m/z 896 (M +
H)+, 836, 778, 654, 491; HRMS (FAB+) m/z calcd for (M+
-
OAc) C47H50NO13 836.3281, found 836.3275; [R]20D -24 (c 0.80,
CHCl3).
936.3443, found 936.3397; [R]20 30 (c 0.90, CHCl3).
Com p ou n d 18. To an ice cooled solution of compound 17
(30 mg, 0.030 mmol) in dry pyridine (3 mL) was added HF‚
pyridine (20 drops), and the mixture was stirred at room
temperature for 3 h (TLC check, 3:2 hexane/EtOAc). The
solution was then neutralized with saturated NaHCO3, acidi-
fied with 3 N HCl, and extracted with CH2Cl2 (3 × 10 mL).
The organic layer washed with brine and then dried over Na2-
SO4, filtered, and evaporated. After flash column chromatog-
raphy (3:2 hexane/EtOAc), 16 mg of compound 18 (0.018 mmol,
60%) was collected as a colorless powder: mp 160-162 °C; 1H
NMR (400 MHz, CDCl3) δ 1.11 (s, 3H), 1.18 (s, 3H), 1.46 (s,
3H), 1.60 (m, 1H), 1.77 (s, 3H), 1.89 (s, 3H), 1.99 (m, 1H), 2.19
(s, 3H), 2.23 (m, 1H), 2.26 (s, 3H), 2.34 (dd, J ) 15.2, 9.4, 1H),
2.69 (bd, J ) 13.3, 1H), 3.01 (dd, J ) 15.3, 8.1 Hz, 1H), 3.56
(bs, 1H), 3.63 (d, J ) 5.0, 1H), 3.85 (d, J ) 6.6, 1H), 4.16 (d, J
) 13.6, 1H), 4.40 (d, J ) 13.6, 1H), 4.78 (dd, J ) 5.0, 2.5, 1H),
5.70 (d, J ) 6.6, 1H), 5.79 (dd, J ) 9.0, 2.5, 1H), 6.17 (bt, J )
8.1, 1H), 6.85 (s, 1H), 7.09 (d, J ) 9.0, 1H), 7.28-7.60 (m, 11H),
7.79 (bd, J ) 8.0, 2H), 8.03 (bd, J ) 8.0, 2H); 13C NMR (100
MHz, CDCl3) δ 15.43, 18.10, 21.06, 21.31, 21.37, 23.22, 26.22,
26.26, 27.61, 35.90, 42.95, 47.74, 55.41, 59.84, 67.29, 72.79,
73.89, 75.20, 76.44, 79.42, 80.13, 86.30, 127.42, 127.49, 128.66,
D
Red u ction of Com p ou n d 10. To an ice cooled solution of
compound 10 (100 mg, 0.1 mmol) in 1:1 MeOH/THF (20 mL)
was added NaBH4 (19 mg, 0.5 mmol). The reaction mixture
was stirred at 0 °C for 25 min (TLC check, 3:2 hexane/EtOAc);
then 3 N HCl was added, the solvent was concentrated under
vacuum, and the residue was diluted with CH2Cl2 and washed
with water. The aqueous solution was extracted with CH2Cl2
(3 × 15 mL), and the organic layer was washed with brine
and then dried over Na2SO4. After filtration, evaporation of
the solvent, and flash column chromatography (7:3 hexane/
EtOAc), 28 mg of compound 15 (0.028 mmol, 28%) and 38 mg
of compound 17 (0.038 mmol, 38%) were collected.
Com p ou n d 15. Amorphous solid: 1H NMR (400 MHz,
CDCl3) δ -0.37 (s, 3H), -0.08 (s, 3H), 0.74 (s, 9H), 1.13 (s,
3H), 1.16 (s, 3H), 1.34 (s, 3H), 1.69 (s, 3H), 1.94 (s, 3H), 2.13
(dd, J ) 15.5, 8.8, 1H), 2.18 (s, 3H), 2.45 (s, 3H), 2.51 (dd, J )
15.5, 9.3 Hz, 1H), 3.61 (dd, J ) 10.1, 5.7 1H), 4.20 (d, J )
12.5, 1H), 4.27 (d, J ) 12.5, 1H), 4.50 (d, J ) 8.6, 1H), 4.66 (d,
J ) 1.9, 1H), 5.68 (d, J ) 8.6, 1H), 5.69 (d, J ) 10.1, 1H), 5.86
(dd, J ) 9.1, 1.9, 1H), 6.01 (dd, J ) 10.1, 5.7 1H), 6.27 (bt, J
) 9.1, 1H), 6.99 (s, 1H), 7.06 (d, J ) 9.1, 1H), 7.25-7.56 (m,

Novel D-Seco Paclitaxel Analogues
J . Org. Chem., Vol. 66, No. 10, 2001 3329
129.11, 129.13, 129.34, 130.29 (x2), 132.34, 133.52, 134.04,
134.20, 138.54, 140.22, 167.24, 168.01, 169.70, 170.11, 170.73,
tography (2:3 hexane/EtOAc), 24 mg of compound 21 (0.027
mmol, 80%) was collected as a colorless powder: mp 159-161
°C; 1H NMR (400 MHz, CDCl3) δ 1.15 (s, 3H), 1.23 (s, 3H),
1.35 (s, 3H), 1.54 (m, 1H), 1.76 (m, 1H), 1.79 (s, 3H), 1.89 (m,
1H), 1.93 (s, 3H), 2.10 (d, J ) 3.5 Hz, 1H), 2.23 (s, 3H), 2.24
(m, 1H, 5), 2.43 (dd, J ) 15.4, 9.9, 1H), 3.08 (dd, J ) 15.4, 7.2
Hz, 1H), 3.16 (m, 1H), 3.46 (d, J ) 5.8 Hz, 1H), 3.56 (d, J )
5.9, 1H), 3.86 (m, 1H), 4.08 (d, J ) 13.7, 1H), 4.36 (d, J ) 13.7,
1H), 4.79 (dd, J ) 6.0, 2.7, 1H), 5.59 (d, J ) 5.8, 1H), 5.75 (dd,
J ) 8.9, 2.7, 1H), 6.18 (bt, J ) 8.2, 1H), 6.43 (s, 1H), 7.07 (d,
J ) 8.9, 1H), 7.33-7.49 (m, 9H), 7.54 (bt, J ) 7.4, 1H), 7.59
(bt, J ) 7.4, 1H), 7.80 (bd, J ) 7.8, 2H), 7.98 (bd, J ) 8.1, 2H);
13C NMR (100 MHz, CDCl3) δ 10.97, 15.85, 20.88, 21.01, 21.31,
23.05, 26.87, 27.24, 32.55, 35.37, 43.70, 52.84, 55.88, 61.65,
67.03, 72.96, 73.94, 74.72, 75.50, 75.72, 78.90, 84.94, 127.48
(×2), 128.85, 129.12, 129.21, 129.45, 129.96, 130.17, 132.52,
133.97, 134.18, 134.24, 138.18, 141.32, 167.53, 167.64, 169.39,
169.94, 171.39, 172.83, 203.86; IR (CHCl3) 3434, 1729, 1654,
172.78, 206.98; IR (CHCl3) 3480, 1750, 1680, 1535, 1260 cm-1
;
FAB HRMS m/z calcd for (M + H)+ C49H56NO15 898.3650,
found 898.3641; [R]20 -49 (c 0.70, CHCl3).
D
Com p ou n d 19. To a suspension of NaH (2.3 mg, 0.095
mmol, 4 g of 60% dispersion) in dry THF (0.5 mL)was added
compound 17 (53 mg, 0.052 mmol), dissolved in dry THF (0.5
mL). The reaction mixture was stirred at room temperature
for 5 min, and CS2 (50 µL) and MeI (50 µL) were added. After
15 min at room temperature, EtOAc (5 mL) and water (5 mL)
were added. The mixture was extracted with EtOAc (3 × 10
mL) and the organic layer dried over Na2SO4 and evaporated.
After flash column chromatography (4:1 hexane/EtOAc), 40 mg
of compound 19 (0.036 mmol, 70%) was collected as an
amorphous solid: 1H NMR (400 MHz, CDCl3) δ -0.29 (s, 3H),
-0.02 (s, 3H), 0.81 (s, 9H), 1.21 (s, 3H), 1.25 (s, 3H), 1.56 (m,
1H), 1.57 (s, 3H), 1.80 (m, 1H), 1.98 (s, 3H), 2.07 (bs, 3H), 2.16
(d, J ) 3.3 Hz, 1H), 2.28 (m, 2H), 2.42 (s, 3H), 2.47 (s, 3H),
3.36 (dd, J ) 15.2, 7.9 Hz, 1H), 3.43 (bd, J ) 13.6 Hz, 1H),
3.53 (d, J ) 5.5 Hz, 1H), 4.12 (d, J ) 13.9, 1H), 4.43 (d, J )
13.9, 1H), 4.67 (d, J ) 1.8, 1H), 5.62 (d, J ) 5.5, 1H), 5.74 (bd,
J ) 9.1, 1H), 5.87 (dd, J ) 11.4, 4.6 Hz, 1H), 6.33 (bt, J ) 8.7,
1H), 6.44 (s, 1H), 7.13 (d, J ) 9.1, 1H), 7.27-7.61 (m, 11H),
7.80 (bd, J ) 7.5, 2H), 7.99 (bd, J ) 7.9, 2H); 13C NMR (100
MHz, CDCl3) δ -5.32, -4.76, 12.78, 16.13, 18.55, 18.83, 20.57,
21.04, 21.18, 23.30, 25.93, 26.03, 32.84, 35.31, 44.16, 54.71,
56.14, 58.77, 67.18, 71.96, 75.03, 75.60, 75.71, 78.34, 82.59,
84.49, 126.63, 127.40, 128.40, 129.14, 129.19, 129.28, 129.84,
130.18, 132.29, 133.77, 134.25, 134.59, 138.63, 140.70, 167.43
(x2), 169.26, 169.38, 169.74, 171.89, 201.56, 215.39; MS (FAB+)
m/z 1102, 1042, 643, 583, 535, 493, 479, 400, 354.
Com p ou n d 20. A solution of Bu3SnH (200 µL), AIBN (3
mg), and H2O (40 µL) in toluene (1 mL) was heated at 75 °C
under an argon atmosphere. A solution of compound 19 (35
mg, 0.032 mmol) in dry toluene (1 mL) was added, and the
reaction mixture was stirred at 75 °C (TLC check, 7:3 hexane/
EtOAc). After 1 h EtOAc was added and the solvent evaporated
under vacuum. The mixture was purified as follows: the
column was first washed with hexane to eliminate the excess
of Bu3SnH and then eluted with 4:1 to 3:7 hexane/EtOAc,
collecting 23 mg of compound 20 (0.023 mmol, 71%) as an
amorphous solid: 1H NMR (400 MHz, CDCl3) δ -0.27 (s, 3H),
-0.04 (s, 3H), 0.82 (s, 9H), 1.17 (s, 3H), 1.25 (s, 3H), 1.39 (s,
3H), 1.48 (m, 1H), 1.80 (m, 1H), 1.93 (m, 1H), 1.95 (s, 3H),
1.99 (bs, 3H), 2.14 (d, J ) 3.3 Hz, 1H), 2.24 (s, 3H), 2.25 (dd,
J ) 15.1, 9.7 Hz, 1H), 2.42 (s, 3H), 3.28 (dd, J ) 15.1, 8.3 Hz,
1H), 3.31 (bd, J ) 13.5 Hz, 1H), 3.43 (d, J ) 5.9 Hz, 1H), 3.88
(m, 1H), 4.12 (d, J ) 13.8, 1H), 4.40 (d, J ) 13.8, 1H), 4.67 (d,
J ) 1.9, 1H), 5.64 (d, J ) 5.9, 1H), 5.75 (bd, J ) 9.1, 1H), 6.33
(bt, J ) 8.8, 1H), 6.47 (s, 1H), 7.13 (d, J ) 9.1, 1H), 7.30-7.61
(m, 11H), 7.80 (bd, J ) 7.6, 2H), 8.01 (bd, J ) 8.0, 2H); 13C
NMR (100 MHz, CDCl3) δ -5.31, -4.85, 10.93, 15.87, 18.57,
21.01, 21.27, 23.43, 25.93, 26.80, 26.96, 32.96, 35.32, 43.93,
52.94, 56.09, 61.58, 67.07, 72.07, 74.80, 75.41, 75.73, 75.97,
79.07, 84.79, 126.71, 127.39, 128.39, 129.14, 129.17, 129.25,
130.01, 130.21, 132.29, 133.87, 134.16, 134.57, 138.67, 141.85,
167.29, 167.65, 169.22, 169.86, 171.37, 171.98, 204.02; MS
(FAB+) m/z 1011, 953, 553, 493, 451, 400, 354.
1518, 1245 cm-1; FAB HRMS m/z calcd for (M + H)+ C49H56
-
NO15 898.3650, found 898.3619; [R]20 -98 (c 0.90, CHCl3).
D
Com p u ta tion a l P r oced u r es. Structure 18 was subjected
to Monte Carlo conformational searches with the AMBER*,
MM2*, MM3*, and MMFF force fields and the GB/SA/H2O
continuum model in MacroModel6.535-37 for a total of 35 000
steps. The global minima were located 13, 5, 2, and 10 times,
respectively. The combined set of 5126 optimized conformers
was treated to a NAMFIS38 conformer deconvolution analysis
by fitting 29 rOe’s and six J couplings. The rOe cross-peaks
were classified as weak, medium, and strong. Since NAMFIS
requires explicit distances as input, the latter were assigned
average values of 4.0, 3.5, and 2.7 Å, respectively. The
experimental J values were converted to dihedral angles with
the modified Karplus equation due to Haasnoot, Leeuw, and
Altona.45 The NAMFIS fitting procedure provided seven
conformations with predicted populations ranging from 6 to
46%, i.e., 46, 13, 11, 9, 8, 7, and 6%, with a sum of square
differences (SSD) of 44. The corresponding O-C4-C20O
optimized torsional angles are -105, 116, -82, -89, 112, -87,
and 113°, respectively. All structures were visualized on SGI
O2 workstations and further manipulated in MacroModel; for
example, for evaluation of the effect of B-C ring fusion on the
axial-axial interaction between C8-Me and C4-CH2OAc and
for the nature of the hydrophobic collapse between C2 and C3′
phenyl rings.
Molecular dynamics simulations to investigate the effect of
D-ring opening and 4-deacetylation were performed in Sybyl
6.5 using the Sybyl force field and Gasteiger-Hu¨ckel partial
atomic charges.
Ack n ow led gm en t. We acknowledge the National
Cancer Institute (1R01 CA-82801) and the Drug Dis-
covery Program, Higuchi Biosciences Center, for finan-
cial support and DABUR INDIA Ltd. for a generous gift
of paclitaxel. The authors thank Rebecca Marquez for
performing the biological assays. L.B. is grateful to
Consiglio Nazionale delle Ricerche, Italy, for a Mobilita`
di breve durata fellowship.
Su p p or tin g In for m a tion Ava ila ble: 1H and 13C spectra
of compounds 11, 16, 18, and 21. This material is available
free of charge via the Internet at http://pubs.acs.org.
Com p ou n d 21. To an ice cooled solution of compound 20
(34 mg, 0.034 mmol) in dry pyridine (3 mL) was added HF‚
pyridine (20 drops), and the mixture was stirred at room
temperature for 3 h (TLC check, 1:1 hexane/EtOAc). The
solution was then neutralized with saturated NaHCO3, acidi-
fied with 3 N HCl, and extracted with CH2Cl2 (3 × 10 mL).
The organic layer was washed with brine and then dried over
Na2SO4, filtered, and evaporated. After flash column chroma-
J O0015467
(45) Haasnoot, C. G. G.; De Leeuw, F. A. A. M.; Altona, C.
Tetrahedron 1980, 36, 2783-2792.