Synthesis and biological evaluation of new paclitaxel analogs and discovery of potent antitumor agents

Article abstract of DOI:10.1039/c3ob40654g

Reaction of 10-deacetylbaccatin III (III) and its 7-TES derivative (IV) with DAST under various conditions resulted in the formation of an array of new fluorinated and non-fluorinated 13-keto taxoid compounds (2a-4a) through a vinylogous pinacol-pinacolone rearrangement. Further fluorination of some of these products (2a, 3a) with NFSi or Selectfluor gave additional derivatives. Sodium borohydride reduction of the 13-keto group of these products (2a, 2b, 3a, 3b, 4a, 8, 9, 11-14) led to a series of 9α-hydroxy taxoid derivatives, which were esterified using the docetaxel side chain employing the corresponding protected β-lactam, followed by deprotection to furnish a library of docetaxel analogs and related compounds. A selected number of synthesized compounds (7, 10, 19a, 19b, 21a, 21b, 23, 27, 29, 34-36) were submitted to the National Cancer Institute (NCI) 60 cell line screening program and tested for cytotoxic properties. Taxoids 19a, 19b, 21a, 21b, 23, 27, 29, 34 and 35 were found to exhibit significant anticancer activity against various cancerous cell lines with 23, 27, and 29 being the most potent compounds, demonstrating GI ₅₀ values of ≤5 nM in several assays.

Full text of DOI:10.1039/c3ob40654g

Organic&
BiomolecularChemistry
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Volume 11 | Number 25 | 7 July 2013 | Pages 4125–4272
ISSN 1477-0520
PAPER
Kyriacos C. Nicolaou and Roman A. Valiulin
Synthesis and biological evaluation of new paclitaxel analogs and discovery of
potent antitumor agents
1477-0520(2013)11:25;1-8

Organic &
Biomolecular Chemistry
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PAPER
Synthesis and biological evaluation of new paclitaxel
analogs and discovery of potent antitumor agents†‡
Kyriacos C. Nicolaou*a,b,c,d and Roman A. Valiulina
Cite this: Org. Biomol. Chem., 2013, 11,
4154
Reaction of 10-deacetylbaccatin III (III) and its 7-TES derivative (IV) with DAST under various conditions
resulted in the formation of an array of new fluorinated and non-fluorinated 13-keto taxoid compounds
(2a–4a) through a vinylogous pinacol–pinacolone rearrangement. Further fluorination of some of these
products (2a, 3a) with NFSi or Selectfluor gave additional derivatives. Sodium borohydride reduction of
the 13-keto group of these products (2a, 2b, 3a, 3b, 4a, 8, 9, 11–14) led to a series of 9α-hydroxy taxoid
derivatives, which were esterified using the docetaxel side chain employing the corresponding protected
β-lactam, followed by deprotection to furnish a library of docetaxel analogs and related compounds. A
selected number of synthesized compounds (7, 10, 19a, 19b, 21a, 21b, 23, 27, 29, 34–36) were sub-
mitted to the National Cancer Institute (NCI) 60 cell line screening program and tested for cytotoxic pro-
perties. Taxoids 19a, 19b, 21a, 21b, 23, 27, 29, 34 and 35 were found to exhibit significant anticancer
activity against various cancerous cell lines with 23, 27, and 29 being the most potent compounds,
demonstrating GI50 values of ≤5 nM in several assays.
Received 2nd April 2013,
Accepted 3rd May 2013
DOI: 10.1039/c3ob40654g
www.rsc.org/obc
their potency, selectivity and novel mechanism of action.9–11
Paclitaxel is clinically used in the treatment of breast cancer,
Introduction
Cancer remains as one of the most devastating diseases of our advanced ovarian cancer, non-small cell lung cancer, and
times with millions of patients suffering from it around the advanced forms of Kaposi’s sarcoma,12 whereas docetaxel is
world.1–6 Moreover, the disease is on the rise and no organ of employed against breast cancer, non-small cell lung cancer,
the human body is immune to its intrusion. Despite the gastric cancer, prostate cancer, and squamous cell carcinoma
plethora of anticancer drugs currently available for combating of the head and neck.13–15 Cabazitaxel (Jevtana, II′), a recently
cancer, serious deficiencies still prevail, particularly with
regards to their efficacy and safety. These chemotherapeutic
agents range from small organic molecules and natural pro-
ducts and their analogs, to antibodies and antibody–drug
conjugates.7,8
Among the natural products and their semi-synthetic
analogs, paclitaxel (I, Fig. 1) and docetaxel (II, Fig. 1) are
perhaps the most well-known and frequently employed due to
aDepartment of Chemistry, The Scripps Research Institute, 10550 North Torrey
Pines Road, La Jolla, California 92037, USA. E-mail: kcn@scripps.edu, kcn@rice.edu
bSkaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037, USA
cDepartment of Chemistry and Biochemistry, University of California, San Diego,
9500 Gilman Drive, La Jolla, California 92093, USA
dDepartment of Chemistry, Rice University, BioScience Research Collaborative,
6100 Main Street, Houston, Texas 77005, USA
†Electronic supplementary information (ESI) available: Includes experimental
procedures, selected physical data for the synthesized compounds, and further
details of biological assays. CCDC 931453–931456. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c3ob40654g
‡Dedicated to Professor Teruaki Mukaiyama on the occasion of his 85th
Fig. 1 Structures of paclitaxel (I), docetaxel (II), cabazitaxel (II’), and 10-deacetyl-
baccatin III (III). Bz = benzoyl; Ac = acetyl; Ph = phenyl.
birthday.
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introduced derivative of docetaxel (docetaxel methylated at C7 fluorine residues into small molecules,39 we initiated a
and C10 hydroxyl groups), is used to treat hormone-refractory research program directed towards the synthesis of fluorinated
prostate cancer. The latter success underscores the importance and other novel taxoids starting from 10-deacetylbaccatin III
of fine-tuning the structures of these agents in order to (III, Scheme 1) and its 7-OTES derivative (IV, Scheme 1). The
amplify their selectivity towards specific cancers, a theme that virtues of introducing fluorine atoms into organic molecules
is currently attracting special attention in view of personalized have been widely recognized and reported;39–44 they include
medicine.
increased metabolic stability (resistance to biochemical degra-
A large number of paclitaxel and docetaxel analogs became dation), enhanced lipophilicity (logP), and stronger binding
available over the last three decades leading to a useful body of affinities, often leading to improved pharmacological pro-
structure–activity relationships (SARs).16–38 Enabled by syn- perties. It is notable that the current number of clinically used
thesis, these studies were facilitated by the ready availability of drugs containing one or more fluorine atoms stands at
paclitaxel and its precursor 10-deacetylbaccatin III (III, Fig. 1). 20–30% and is steadily increasing.40,42
Motivated by the approval of cabazitaxel and the successes in
In this article, we report the results of our investigations on
medicinal chemistry resulting from the introduction of the synthesis and biological evaluation of new taxoids, includ-
ing a number of fluorinated docetaxel analogs.45
Results and discussion
Reaction of 10-deacetylbaccatin III (III) with DAST
The present study began with 10-deacetylbaccatin III (III) and
its reaction with the common fluorinating agent (diethyl-
amino)sulfur trifluoride (DAST).46 Our intention was to obtain
as many fluorinated species as possible for the purposes of
converting them, through side chain attachment, to full doce-
taxel structures for biological evaluation. The top priority at
the initial stage was, therefore, structural elucidation of pro-
ducts rather than yield optimization, the latter being post-
poned until the properties of the molecule would dictate it.
Thus, and as shown in Scheme 1, reaction of III with 2.0
equiv. of DAST in THF in the presence of 4 Å MS at −78 °C
over 3 h [conditions (a), Scheme 1] furnished enone 1a in 93%
yield.47 Product 1a was found to be labile and easily trans-
formed to its apparently more stable isomeric paclitaxel-
related form 1b (red ellipsoid, Scheme 1) on silica gel. This
conversion could also be induced by 4-DMAP in THF at
ambient temperature in 98% yield [conditions (d)]. For the
purposes of further manipulations, compound 1a was also
converted to its 7-OTES derivative 2a under conditions
(c) [Et3SiCl (5.0 equiv.), Et3N (5.0 equiv.), 4-DMAP (0.5 equiv.),
THF, 0 °C, 1.5 h; in 74% yield] and thence to the isomerized
taxoid 2b under basic conditions [conditions (d), 96% yield].
The latter was also obtained from the previously prepared
taxoid 1b upon silylation in 80% yield [conditions (c)]. Impor-
tantly, enone 2a could also be prepared directly from 7-tri-
methylsilyl-10-deacetylbaccatin III48 (IV) (Scheme 1) in 70%
yield by exposure of the latter to DAST [2.0–4.0 equiv., THF, 4 Å
MS, −78 °C, 1–3 h, conditions (a)].
Interestingly, 10-deacetylbaccatin III (III) reacted differently
Scheme 1 Reaction of 10-deacetylbaccatin III (III) with DAST. Synthesis of
with DAST under modified conditions (b) (Scheme 1). Thus,
compounds 1a–4a and 1b–3b. Reagents and conditions: (a) DAST (2.0 equiv.),
THF, 4 Å MS, −78 °C, 3 h, III → 1a (93%); IV → 2a (70%); (b) DAST (4.0 equiv.), treatment of III with 4.0 equiv. of DAST in THF–CH2Cl2 (5 : 2),
THF–CH2Cl2 (5 : 2), 4 Å MS, −78→ 25 °C, 24 h, III → 3a (63%), plus 4a (≤20%);
(c) Et3SiCl (5.0 equiv.), Et3N (5.0 equiv.), 4-DMAP (0.5 equiv.), THF, 0 °C, 1.5 h,
in the presence of 4 Å MS, at −78 to 25 °C, over 24 h led to
fluorinated enone 3a in 63% yield accompanied by varying
amounts (≤20%) of elimination product 4a, inseparable by
1a → 2a (74%); 1b → 2b (80%); (d) 4-DMAP (2.5 equiv.), THF, 4 Å MS, 25 °C,
20–24 h, 1a → 1b (98%); 2a → 2b (96%); 3a → 3b (78%). DAST = (diethyl-
silica gel chromatography. The formation of product 4a could
amino)sulfurtrifluoride; TES = triethylsilyl; MS = molecular sieves; THF = tetra-
hydrofuran; 4-DMAP = 4-(dimethylamino)pyridine.
be suppressed by diluting the reaction mixture with further
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Scheme 2 Preparation of compounds 3a, 4a, and 6 and proposed mechanism for their formation. Reagents and conditions: see Scheme 1 for (a) and (b).
amounts of CH2Cl2 after the addition of the first 2.0 equiv. of THF in the presence of 4 Å MS at −78 °C [conditions (a),
DAST and carefully controlling the temperature during the Scheme 3] led to a complex mixture of products from which
reaction (see ESI† for experimental details). Similarly to 1a, rather unexpected sulfonate 7 was isolated as a major product
enone 3a isomerizes to taxoid 3b in 78% yield under con- (33% yield), while the expected 14β-fluorotaxoid
9 was
ditions (d) (Scheme 1). obtained as a minor product (≤5% yield). In contrast to these
Plausible mechanistic explanations for the reactions men- results, the reaction of 2a with 1-chloromethyl-4-fluoro-1,4-di-
tioned in Scheme 1 are summarized in Scheme 2. Thus, initial azoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (F-TEDA-BF4,
reaction of III with DAST may produce intermediate 5a with or Selectfluor)57 under conditions (b) (Scheme 3) [KHMDS (2.0
elimination of HF. This intermediate may then undergo con- equiv.), Selectfluor (10.0 equiv.), THF–DMF (10 : 1), 4 Å MS,
certed 1,2-H-shift and elimination of Et2NS(O)F and F− to −78 °C, 1–2 h and 25 °C, 1 h] furnished 12β,14β-bis-fluorinated
afford protonated species 1a′, from which observed product 1a product 8 (24% yield) and varying amounts of 14β-fluorinated
may be formed through the loss of an H+. A stepwise process compound 9 (≤20%) depending on stoichiometry and reaction
involving the formation of an allylic carbocation for this viny- time.58 The latter product is presumed to arise from 2a via
logous pinacol–pinacolone rearrangement (5a → 1a) may also mono-fluorination and enone transposition.
be envisioned. Reaction of hydroxyenone 1a with further
7α-Fluoroenone 3a also entered a number of interesting
amounts of DAST may then lead, initially to 5b, and thence to reactions with NFSi and Selectfluor (Scheme 4). Thus, treat-
intermediate 5c, from which all observed products (i.e. 3a, 4a, ment of 3a with NFSi under conditions (a) [KHMDS (2.2
and 6) may be generated49–52 as shown in Scheme 2.
equiv.), NFSi (3.0 equiv.), THF, 4 Å MS, −78 °C, 1 h and 25 °C,
1 h] furnished 7α-fluoro vinyl sulfonate 10 (24% yield) mirror-
ing the reaction of its 7-OTES counterpart 2a (Scheme 3). On
the other hand, reaction of 3a with Selectfluor under con-
ditions (b) [KHMDS (2.0 equiv.), Selectfluor (10.0 equiv.), THF–
DMF (10 : 1), 4 Å MS, −78 °C, 1–2 h and 25 °C, 1 h] led to
7α,14β-difluoroenone 11 (≤5% yield) as a minor product (plus
trace amounts of compound 12 (≤2%), presumably arising
from contaminant 4a present in starting material 3a, see
Reaction of enones 2a and 3a with NFSi and Selectfluor
Having gained access to the new taxoid scaffolds mentioned
above, we proceeded to explore their chemistry with additional
fluorinating agents. Enones 2a and 3a proved to be the most
fertile in these pursuits as shown in Schemes 3 and 4, respect-
ively. Thus, treatment of compound 2a with N-fluoro-N-(phe-
nylsulfonyl)benzenesulfonimide (NFSi)53–56 and KHMDS in
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Scheme 3 Fluorination of 2a with NFSi and Selectfluor. Synthesis of compounds
7–9. Reagents and conditions: (a) KHMDS (2.2 equiv.), NFSi (3.0 equiv.), THF,
4 Å MS, −78 °C, 1 h and 25 °C, 1 h, 2a → 7 (33%), plus 9 (≤5%); (b) KHMDS
(2.0 equiv.), Selectfluor (10.0 equiv.), THF–DMF (10 : 1), 4 Å MS, −78 °C, 1–2 h
and 25 °C, 1 h, 2a → 8 (24%), plus 9 (≤20%). KHMDS = potassium bis(trimethyl-
silyl)amide; NFSi = N-fluorobenzenesulfonimide; Selectfluor = 1-chloromethyl-
4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate); DMF
= N,N-
dimethylformamide.
Scheme 1), and 7α,10α-difluoroenone 13 (17% yield). Further
treatment of the latter with excess of Selectfluor under the
same set of conditions led to trifluoro enone 14 (mp =
229–230 °C, CH3Cl–hexanes–EtOAc 10 : 5 : 1) in 97% yield. The
structure of 14 was unambiguously established by 2D NMR
spectroscopic techniques and X-ray crystallographic analysis
(see ORTEP drawing, Scheme 4).
Scheme 4 Fluorination of 3a with NFSi and Selectfluor. Synthesis of compounds
10–14. Reagents and conditions: (a) KHMDS (2.2 equiv.), NFSi (3.0 equiv.),
THF, 4 Å MS, −78 °C, 1 h and 25 °C, 1 h, 3a → 10 (24%); (b) KHMDS (2.0 equiv.),
Selectfluor (10.0 equiv.), THF–DMF (10 : 1), 4 Å MS, −78 °C, 1–2 h and 25 °C,
1 h, 3a → 11 (≤5%), plus 12 (≤2%), plus 13 (17%); 13 → 14 (97%).
Having a variety of rearranged (2a, 2b, 4a) and fluorinated
(3a, 3b, 8, 9, 11–14) taxoids in hand, we pursued their conver-
sion to docetaxel analogs through reduction of their C-13 car-
bonyl moieties and attachment of the side chain.
Each of these hydroxy compounds was acylated using pro-
cedure (b) (Scheme 5): NaHMDS (2.5 equiv.) and β-lactam side
chain equivalent (3R,4S)-3-triethylsilanyloxy-4-phenyl-N-Boc-
2-azetidinone (V) (5.0–10.0 equiv.) to afford 7,2′-bis-silylated-4-
acetoxy products 18a (53% yield), 20a (53% yield), 2′-silylated-
4-acetoxy-Δ7,8 product 22 (31% yield) or 7,2′-bis-silylated-4-dea-
cetyl products 18b (60% yield), and 20b (52% yield), depending
on the conditions employed (see ESI†). These compounds were
then desilylated by exposure to conditions (c) (Scheme 5):
aqueous HCl, THF–MeOH (1 : 2), 0 °C, leading to docetaxel
analogs 19a (83% yield), 19b (89% yield), 21a (96% yield), 21b
(73% yield), and 23 (94% yield). The absolute stereochemical
configurations of 19a (mp = 178–181 °C, CH3CN) and 21a (mp =
233–235 °C, CH3CN–hexanes–EtOH 10 : 2 : 5) were confirmed by
X-ray crystallographic analysis (see ORTEP representations of
19a and 21a, Scheme 5).
Reduction of diketones 2a, 3a, and 4, and preparation of
docetaxel analogs 19a, 19b, 21a, 21b and 23
As a prelude to the preparation of docetaxel analogs from the
dicarbonyl compounds 2a, 3a, and 4a, we subjected the latter
to NaBH4 reduction in the hope that the less hindered 13-car-
bonyl moiety would be preferentially and selectively reduced to
the corresponding 13-hydroxy precursors. Indeed, exposure of
2a, 3a and 4a (as a pure compound or a ca. 2 : 1 of 3a : 4a in-
separable mixture) to NaBH4 [2.5 equiv., THF–MeOH (1 : 2), 0 °C,
procedure (a), Scheme 5] led to alcohols 15 (94%), 16 (71%),
and 17 [30% yield after chromatographic separation from the
concomitantly formed 16 (66% yield)], respectively, all posses-
sing the desired 13α configuration as shown in Scheme 5.
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Scheme 6 Stereo- and regioselectivity of the reduction of ketones 2b and 3b.
Synthesis of compounds 24–29. Reagents and conditions: (a) NaBH4 (2.5 equiv.→
large excess), THF–MeOH (1 : 2),
0 °C, 1–3 h; (b) NaHMDS (2.5 equiv.),
V (5.0–10.0 equiv.), THF, 4 Å MS, 0 °C, 40 min, 24 (mixture)→ 26 (24%);
25 (mixture)→ 28 (30%); (c) aq. HCl (1.0 M), THF–MeOH (1 : 2), 0 °C, 1–3 h,
26 → 27 (58%); 28 → 29 (89%).
was detected as the major compound. The same complications
arose in the case of fluorinated diketone 3b (Scheme 6b)
which led, upon treatment with excess of NaBH4, to a complex
mixture, in which the desired alcohol 25 (note the retention at
the Δ11,12 olefinic bond as contrasted with 24) was determined
to be the predominant product. Unfortunately, neither product
(i.e. 24 and 25) could be isolated in pure form by flash chro-
matography (silica gel), forcing us to employ the crude mixtures
in the side chain attachment step. Thus, treatment of the mix-
tures containing 24 or 25 (see Scheme 6) with NaHMDS and
β-lactam V at 0 °C as described above [conditions (b)] fur-
nished pure coupling products 26 (24% yield) or 28 (30% yield),
respectively, in pure form after chromatographic separation.
Desilylation of products 26 and 28 under conditions (c),
(Scheme 6) [aq. HCl, THF–MeOH (1 : 2), 0 °C] led to docetaxel
analogs 27 (58% yield) and 29 (89% yield), respectively.
Scheme 5 Synthesis of compounds 15–17, 18a–21a, 18b–21b, 22 and 23.
Reagents and conditions: (a) NaBH4 (2.5 equiv.), THF–MeOH (1 : 2),
0 °C,
0.5–3 h, 2a → 15 (94%); 3a → 16 (71%); 3a : 4a (ca. 2 : 1 by 1H NMR)→
16 (66%), plus 17 (30%); (b) NaHMDS (2.5 equiv.), V (5.0–10.0 equiv.), THF,
4 Å MS, 0 °C, 40 min (or −78→ 0 °C, 40 min see ESI† for detailed procedure B1,
B2, and B3), 15 → 18a (53%); 15 → 18b (60%); 16 → 20a (53%); 16 →
20b (52%); 17 → 22 (31%); (c) aq. HCl (1.0 M) (excess), THF–MeOH (1 : 2), 0 °C,
1–3 h, 18a → 19a (83%); 18b → 19b (89%); 20a → 21a (96%); 20b →
21b (73%); 22
→ 23 (94%). NaHMDS = sodium bis(trimethylsilyl)amide;
V = (3R,4S)-3-triethylsilanyloxy-4-phenyl-N-Boc-2-azetidinone.
Reduction of diketones 2b and 3b and preparation of
docetaxel analogs 27 and 29
In contrast to the smooth reduction of diketones 2a, 3a, and
4a discussed above, the reaction of compounds 2b and 3b with
NaBH4 proved to be complicated and low yielding with regards
to the desired 13α-hydroxy products. Thus, and as shown in
Scheme 6a, treatment of 2b with a large excess of NaBH4
Reduction of diketones 8, 13, and 14
resulted, through a rather sluggish reaction, in the formation Fluorinated diketones 8, 13, and 14 exhibited interesting reac-
of a complex mixture, in which the over-reduced product 24 tivity towards NaBH4 reduction as shown in Scheme 7. Thus,
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which led partially to reversal of stereoselectivity (see 8 → 30a
+ 30b, Scheme 7a).
While these results were interesting on their own right, they
precluded the attachment of the docetaxel side chain at the
proper position of the products in order to obtain useful
analogs for biological evaluation.
Preparation of unusual taxoids 34–36 from 15 and 16
While optimizing the conditions for the preparation of doce-
taxel analogs from hydroxy compounds 15 and 16, we encoun-
tered
a
number of unusual side-products, including
compounds 34, 35, and 36 (Scheme 8). These compounds were
of interest not only from the biological point of view, but also
for mechanistic considerations of their formation. Speculative
pathways for their generation are outlined in Scheme 8. Thus,
under the basic conditions (a) employed for the side chain
attachment (i.e. NaHMDS), the C-13-alkoxide (33a) formed
from 15 or 16 may undergo intramolecular rearrangement
through C-4-acetyl migration as shown in the case of 15 (R1 =
β-OTES), or intramolecular enolate formation as shown in the
case of 16 (R1 = F) to afford acetate 33b or enolate 33d, respect-
ively. The former may also rearrange to enolate 33c. All three
reactive species (i.e. 33c, 33b, and 33d) may then react with
β-lactam V to afford the corresponding esters, which upon
acid-induced desilylation lead to compounds 34 (39% overall
yield from 15), 35 (12% overall yield from 15), and 36 (16%
overall yield from 16), respectively as shown in Scheme 8. The
absolute configuration of 34 (mp = 161–163 °C, hexanes–EtOAc
10 : 1) was confirmed by X-ray crystallographic analysis (see
ORTEP representation, Scheme 8).
Scheme 7 Stereo- and regioselectivity of the reduction of ketones 8, 13, and
14. Synthesis of compounds 30a, 30b, 31, and 32. Reagents and conditions:
(a) NaBH4 (2.5 equiv.→ large excess), THF–MeOH (1 : 2), 0 °C, 0.5–2 h (or NaBH4
(large excess), THF–MeOH (1 : 2), 0 °C, CeCl3·7H2O (cat.), see ESI†), 8 → 30a
(44%), plus 30b (39%); 13 → 31 (58%); 14 → 32 (95%).
Biological evaluation of selected compounds
Selected compounds from those synthesized and described
above, including sulfonates 7, 10, docetaxel analogs 19a, 19b,
21a, 21b, 23, 27, 29 and side-products 34–36 were submitted to
the NCI 60 cell line screening program.45 Initial one-dose
screening (at 10 μM conc.) revealed that sulfonates 7, 10, and
7α-fluorinated compound 36 possessed weak cytotoxicity
(Fig. 2). Specifically, sulfonate 7 demonstrated moderate
results, however, not sufficient for the five-dose screening
[98% growth inhibition (GI) for HOP-92 (non-small cell lung
cancer) and 8% lethality for A498 (renal cancer)]. 7α-Fluori-
nated sulfonate 10 was less active [76% growth inhibition (GI)
for MDA-MB-435 (melanoma), 66% growth inhibition for HT29
(colon cancer), and RPMI-8226 (leukemia)]. Compound 36 was
the least active, suppressing the growth of CNS cancer cell line
(SNB-75) by only 33%. While these activities did not warrant
further investigation of the compounds mentioned above,
those of the remaining did.
Thus, because of their significant potencies against numer-
ous tumor cell lines, docetaxel analogs 19a, 19b, 21a, 21b, 23,
27, 29, 34, and 35 were advanced to the five-dose screening
stage, revealing more details about their cytotoxicities as
depicted in Fig. 2 [the most potent compounds are shown in
boxes (e.g., 23, 27, and 29); the highest potencies are empha-
sized in red (less than 5 nM) and bold black (from 10 to 100
treatment of 12β,14β-difluorodiketone 9 with a large excess of
NaBH4 [THF–MeOH = 1 : 2, conditions (a)] resulted in the for-
mation of a mixture of 13α- and 13β-hydroxyenones 30a (44%
yield) and 30b (39% yield) (Scheme 7a). Apparently the replace-
ment of the hydrogen atoms at the C-12 and C-14 positions
with fluorines influences the stereoselectivity of this reduction
(compare 2a → 15, Scheme 5). Even more interesting was the
NaBH4-mediated reduction of 7α,10α-difluorodiketone 13
(Scheme 7b) which led to 9α-hydroxyenone 31 (58% yield). In
contrast, the reduction of the trifluoro diketone 14
(Scheme 7c) under the same conditions (a) proved sluggish
and required Luche conditions (NaBH4–CeCl3·7H2O), leading
to 9β-hydroxyenone 32 (95% yield). The observed reactivity
enhancement of the C-9 carbonyl moiety over the C-13 carb-
onyl group within diketones 13 and 14 may be attributed to the
electron-withdrawing properties of the fluorine substituents at
C-7 and C-10 carbons adjacent to the former functional group.
The opposite stereochemical outcome of this reduction in the
case of 14, as compared to that of diketone 13 (Scheme 7b), is
presumably due to the steric effect of the 10β-fluorine residue
in the latter, an outcome observed also in the reduction of 8,
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Scheme 8 Generation of compounds 34–36 and postulated mechanism for their formation from 15, 16, and V. Reagents and conditions: (a) NaHMDS (2.0–3.0
equiv.), −78 °C, 1 h, then (b) V (5.0–10.0 equiv.), THF, −78 °C, 0.5 h and 0 °C, 0.5 h, see ESI† for detailed procedures B2 and B3; then PTLC, followed by (c) aq.
HCl (1.0 M) (excess), THF–MeOH (1 : 2), 0 °C, 1–3 h (yields reported over 3 steps), 15 → 34 (39%), plus 35 (12%); 16 → 36 (16%). PTLC = preparative thin-layer
chromatography.
nM)]. Specifically, analogs 23, 27, and 29 demonstrated very for 21a [CNS cancer (SNB-75) GI50 ≤ 10 nM; melanoma
low GI50 values (less than 5 nM) against most of the 60 cell (MDA-MB-435) GI50 = 13 nM; renal cancer (RXF 393) GI50 = 24
lines tested: for 23 [for most of the leukemia, non-small cell nM; breast cancer (HS 578T) GI50 = 21 nM and (MDA-MB-468)]
lung cancer, colon cancer, CNS cancer, melanoma, ovarian GI50 = 18 nM]; and for 21b [non-small cell lung cancer
cancer, renal cancer, prostate cancer, and breast cancer cell (NCI-H460) GI50 = 210 nM; colon cancer (HT29) GI50 = 259 nM;
lines GI50 ≤ 5 nM]; for 27 [for many of the leukemia, non- CNS cancer (SNB-75) GI50 = 87 nM; melanoma (MDA-MB-435)
small cell lung cancer, colon cancer, CNS cancer, melanoma, GI50 = 144 nM; breast cancer (MCF7) GI50 = 295 nM]. Analogs
ovarian cancer, prostate cancer, and breast cancer cell lines 34 and 35 exhibited low cytotoxicities (GI50 ≥ 500 nM for most
GI50 ≤ 5 nM]; for 29 [CNS cancer (SNB-75) GI50 ≤ 5 nM; mela- of the 60 cell lines) as shown in Fig. 2. For further details of
noma (MDA-MB-435) GI50 ≤ 5 nM and (SK-MEL-5) GI50
=
the cytotoxicity data see http://dtp.nci.nih.gov/.
7 nM; renal cancer (RXF 393) GI50 = 11 nM; breast cancer (HS
578T) GI50 = 7 nM]. Taxoids 19a, 19b, 21a, and 21b showed
moderate cytotoxicity (GI50 values vary from 10 to 100 nM). In
particular, for 19a [for most of the leukemia, non-small cell
lung cancer, colon cancer, CNS cancer, melanoma, ovarian
cancer, renal cancer, prostate cancer, and breast cancer cell
lines GI50 ≤ 10 nM)]; for 19b [leukemia (HL-60(TB)) GI50 = 184
nM; colon cancer (HT29) GI50 = 188 nM; CNS cancer (SNB-75)
GI50 = 25 nM; melanoma (MDA-MB-435) GI50 = 76 nM; breast
cancer (MCF7) GI50 = 188 nM and (HS 578T) GI50 = 137 nM];
Conclusion
An exploration of the reactivity of the readily available taxoid
10-deacetylbaccatin III towards various fluorinating reagents
led to a number of novel baccatin III derivatives, some of
which were successfully converted to docetaxel analogs
through reduction and side chain attachment. Biological
evaluation of these analogs at NCI employing their 60 cell line
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Fig. 2 Cytotoxicities of chosen synthesized taxoids against selected cancer cell lines (NCI). For experimental details and definitions, please use the following
weblink: (http://dtp.nci.nih.gov/branches/btb/ivclsp.html). GI50 = growth inhibition of 50%, i.e. drug concentration resulting in a 50% reduction in the net protein
increase (as measured by SRB staining) in control cells during the drug incubation; TGI = drug concentration resulting in total growth inhibition; LC50 = concentration
of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning; SRB = sulforhodamine
B. Units: molar. Color code: bold red (GI50 < 5 nM) and bold black (GI50 = 10–100 nM) numbers indicate high potencies. Average GI50 for paclitaxel for most cell lines
varies from 10–35 nM. For further details on all compounds, see ESI† and http://dtp.nci.nih.gov/ (see Table S1† for compound numbering).
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screening assays led to the identification of a number of
highly potent compounds against several cancerous cell lines
(i.e. 23, 27, and 29; GI50 ≤ 5 nM). These results indicate that
oxygenation at C-7 and C-10 of the taxoid family is not necess-
ary for biological activity. They also suggest that some of the
reported compounds warrant further investigation aiming at
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Abbreviations
DAST
TES
(Diethylamino)sulfurtrifluoride
Triethylsilyl
MS
THF
4-DMAP
KHMDS
NFSi
Molecular sieves
Tetrahydrofuran
4-(Dimethylamino)pyridine
Potassium bis(trimethylsilyl)amide
N-Fluorobenzenesulfonimide
N,N-Dimethylformamide
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DMF
NaHMDS Sodium bis(trimethylsilyl)amide
PTLC Preparative thin-layer chromatography
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Acknowledgements
We thank Drs D. H. Huang and L. Pasternack for NMR spectro-
scopic, Dr G. Siuzdak for mass spectrometric, and Dr R. K. 20 Q. Cheng, T. Oritani and T. Horiguchi, Tetrahedron, 2000,
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for this work was provided by the Skaggs Institute for Research 21 S. Iimura, K. Uoto, S. Ohsuki, J. Chiba, T. Yoshino,
and the National Institutes of Health, USA (Grant AI055475).
We are grateful to Merck for a postdoctoral fellowship (to R. A. V.).
M. Iwahana, T. Jimbo, H. Terasawa and T. Soga, Bioorg.
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Co., Ltd. (Shanghai ParlingPharmTech Co., Ltd) for generous
B. H. Long, J. F. Kadow, D. R. Langley, V. Farina and
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