Optimized S-trityl-l-cysteine-based inhibitors of kinesin spindle protein with potent in vivo antitumor activity in lung cancer xenograft models

Article abstract of DOI:10.1021/jm3014597

The mitotic kinesin Eg5 is critical for the assembly of the mitotic spindle and is a promising chemotherapy target. Previously, we identified S-trityl-l-cysteine as a selective inhibitor of Eg5 and developed triphenylbutanamine analogues with improved pot

Full text of DOI:10.1021/jm3014597

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Article
Optimized S-Trityl-L-Cysteine Based Inhibitors of Kinesin Spindle Protein
with potent in Vivo Antitumor Activity in Lung Cancer Xenograft Models
James A.D. Good, Fang Wang, Oliver Rath, Hung Yi Kristal Kaan, Sandeep
K Talapatra, Dawid Podgorski, Simon Paul Mackay, and Frank Kozielski
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm3014597 • Publication Date (Web): 11 Feb 2013
Downloaded from http://pubs.acs.org on February 19, 2013
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Optimized S-Trityl-L-Cysteine Based Inhibitors of Kinesin Spindle Protein with
potent in Vivo Antitumor Activity in Lung Cancer Xenograft Models
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*,†, ǀ, ǁ
†, ǁ
†,&
†,#
⊥
,
James A. D. Good
Fang Wang, Oliver Rath , Hung Yi Kristal Kaan ,
†
†,‡
‡
*,†
Sandeep S. Talapatra , Dawid Podgórski , Simon P. MacKay and Frank Kozielski
†
Molecular Motors Laboratory, The Beatson Institute for Cancer Research, Garscube
Estate, Switchback Road, Glasgow G61 1BD, Scotland, U.K.
‡
Strathclyde Institute of Pharmacy and Biomedical Sciences, 161 Cathedral Street,
University of Strathclyde, Glasgow G4 0RE, Scotland, U.K.
*
Corresponding authors: James Good & Frank Kozielski, The Beatson Institute for
Cancer Research, Molecular Motor Laboratory, Garscube Estate, Switchback Road,
Glasgow G61 1BD, Scotland, UK, Fax: +44-141-9426521; Email:
james.good@chem.umu.se; f.kozielski@beatson.gla.ac.uk.
Present Address
ǀ
Umeå University, Department of Chemistry, SE 901 87 Umeå, Sweden.
⊥
Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang
District, Beijing, 100101, China.
&
Redx Oncology Ltd, Duncan Building, Royal Liverpool University Hospital,
Liverpool, L69 3GA.
#
Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore
1
38673.
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Author Contributions
ǁ
These authors contributed equally.
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Abbreviations
c/f, confer; dia-, diastereomeric mixture; DMP, Dess-Martin periodinane; GI50, the
app
concentration required to achieve 50 % growth inhibition; K
i
i
, apparent K value;
KSP, kinesin spindle protein; LE, ligand efficiency; MTs, microtubules; rac-,
racemate; RTV, relative tumor volume, STDC, S-trityl-D-cysteine; STLC, S-trityl-L-
cysteine; n.d.; not determined; n.i., no inhibition; T/C, relative test tumor versus
control value.
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Abstract
The mitotic kinesin Eg5 is critical for the assembly of the mitotic spindle and is a
promising chemotherapy target. Previously, we identified S-trityl-L-cysteine as a
selective inhibitor of Eg5 and developed triphenylbutanamine analogues with
improved potency, favorable drug-like properties, but moderate in vivo activity. We
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report here their further optimisation to produce extremely potent inhibitors of Eg5
app
(
K
i
> 10 nM) with broad-spectrum activity against cancer cell lines comparable to
or surpassing the Phase II drug candidates ispinesib and SB-743921. They have good
oral bioavailability and pharmacokinetics and induced complete tumor regression in
nude mice explanted with lung cancer patient xenografts. Furthermore, they display
fewer liabilities with CYP metabolizing enzymes and hERG compared with ispinesib
and SB-743921, which is important given the likely application of Eg5 inhibitors in
combination therapies. We present the case for this preclinical series to be
investigated in single and combination chemotherapies, especially targeting
hematological malignancies.
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Introduction
1
Mitotic kinesins represent an exciting class of oncology targets. The members of this
structurally divergent superfamily of microtubule (MT) based molecular motor
proteins have extensive involvement throughout mitosis, with functions ranging from
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the organization of the mitotic architecture to the transport of regulatory proteins.
One of the most studied is the kinesin spindle protein (KSP, also known as Eg5,
Kif11, KNSL1; kinesin-5 family), whose primary role is to establish the bipolar
4, 5
spindle in early prometaphase.
Inhibition of Eg5 by small molecule inhibitors
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results in monopolar spindles and mitotic arrest which can lead to cell death. This
has proved highly effective across a variety of in vitro and in vivo models of cancer,
7-9
and total regression has been demonstrated in a number of xenograft models.
A
3
, 10, 11
great variety of inhibitor scaffolds have been described in the literature,
the
majority of which are allosteric inhibitors that bind to an induced fit pocket formed by
1
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loop L5, approximately 10 Å from the nucleotide binding site.
These allosteric
inhibitors exhibit excellent selectivity for Eg5 amongst kinesins due to the unique
1
3
length of loop L5 amongst the kinesin superfamily. A number of compounds have
progressed to clinical trials, with the most advanced in phase II (e.g. 1-3, Figure 1);
however, to date the best outcome in the majority of studies against advanced solid
1, 11
tumors has been limited to disease stabilization.
Better responses were achieved
1
4, 15
targeting hematological malignancies with Eg5 inhibitors.
In a phase II study
8
with the thiadiazole based candidate ARRY-520 (3), a 19% objective response rate
15
was recorded in patients with relapsed/refractory multiple myleomas. Synergistic
responses were recorded on treatment with 3 and the currently approved proteasome
inhibitor bortezomib in xenograft models of multiple myeloma, and combination
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clinical trials are now planned.
Targeting hematological malignancies with Eg5
inhibitors as single agents or in combination with existing therapies therefore
represents an exciting area of investigation which may offer clinical advantages over
current regimens.
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Our group previously identified S-trityl L-cysteine (STLC, 4) as a tight-binding and
1
7-19
selective allosteric inhibitor of kinesin Eg5 (Figure 2).
In common with other
loop L5 Eg5 inhibitors, 4 blocks bipolar spindle formation causing mitotic arrest,
1
8
which ultimately leads to apoptotic cell death across a variety of tumor cell lines.
This primarily occurs through activation of the spindle checkpoint and the intrinsic
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0
apoptotic pathway.
Antitumor activity has been reported for 4 and the p-
2
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methoxyphenyl analogue 7 across leukemia, bladder and prostate xenograft models.
23
In initial SAR investigations, analogues with improved cellular potency were
identified which incorporated a phenyl containing lipophilic para substituents (e.g. 5-
2
4-26
7
, Figure 2).
The subsequent elucidation of the crystal structure of 4 in complex
2
7, 28
with Eg5
enabled us to pursue the rational SAR-based optimization of this
scaffold, which led to the development of butanamine analogues with improved
2
9
activity against Eg5 (11-14, Figure 2).
The lead triphenylbutanamine from our
previous study (11) had improved potency, favorable drug-like properties and in vivo
antitumor activity in a lung cancer xenograft model. We report here on the next phase
of development, which has produced analogues which significantly improve on both
the potency and in vivo efficacy of the previous leads. Across multiple cancer cell
lines, these new leads demonstrate comparable or superior in vitro activity to the
phase II candidate ispinesib (1), and its more potent second generation analogue SB-
1
, 9, 30
743921 (3) which is also undergoing clinical studies (Figure 1).
Favorable drug-
like properties are evident across a selection of the most potent analogues, in addition
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to compelling in vivo pharmacokinetics. Finally, the new lead analogue induces
complete tumor regression in a lung cancer xenograft model. In addition to
surpassing the activity of all previously described S-trityl L-cysteine analogues, this in
vivo efficacy is comparable with that of the drug candidates 1-3, and strongly
warrants further development.
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Results and discussion
Synthesis
S-Trityl thioethanamine and cysteine analogues were prepared by dehydration of trityl
alcohols in trifluoroacetic acid and thioetherification with L-cysteine or cysteamine
31
hydrochloride (Scheme 1).
Intermediate trityl alcohols were synthesized by the
reduction of benzophenone analogues with lithiated aryl bromides; key
thioethanamines were therefore accessible in two steps. The trityl alcohol 59
containing a disubstituted m-ethyl, p-methyl phenyl was prepared from the 5-
bromobenzoic acid derivative 55 by converting to the Weinreb amide 56 to enable
32
controlled access to ketone 57, which was subsequently reduced with hydrazine-
hydrate to afford the dialkyl aryl bromide 58 (Scheme 2). Subsequently, lithium
bromine exchange and reduction of benzophenone and thioetherification of 59
afforded the target thioether 36.
2
Triphenylbutanamine (CH -trityl) analogues with the carboxylic acid were prepared
by the route reported previously from common trityl alcohol intermediates, as
29
illustrated for rac-42 (Scheme 3).
Allylation of trityl alcohol 60 with iron
3
3
trichloride mediating carbon-oxygen bond cleavage afforded the carbon framework,
prior to successive oxidations by hydroboration-oxidation to the primary alcohol 62
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then Dess-Martin periodinane (DMP) to aldehyde 63.
This was converted to α-
aminonitrile rac-64 by a modified version of the Strecker synthesis employing
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Montmorillonite KSF clay. Hydrolysis of rac-64 and subsequent hydrogenation of
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benzylamine rac-65 using ammonium formate gave the racemic amino acid 45.
Attempts to resolve rac-42 by chiral HPLC using a ChiralPak IC column as described
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previously were not successful. Triphenylbutanamines without the carboxylic acid
were prepared by functional group interconversion from primary alcohol
intermediates (e.g. 62) to the primary amine via the mesylate and azide (Supporting
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Information, Schemes S6-S8).
The β-fluorinated triphenylbutanamine rac-52 was prepared by the enamine-based
organocatalysed α-fluorination of aldehyde 66 with L-proline and N-
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fluorobenzenesulfonimide, followed by immediate reduction to afford β-fluorinated
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alcohol rac-67 (Scheme 4).
Expedient access to rac-28 was then achieved by
stepwise conversion via the tosylate and azide. The β,β-difluorinated amine 53 was
®
synthesized by a comparable methodology with SelectFluor as the electrophilic
3
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source of fluorine. Direct access to azide 69 was possible from β,β-difluorinated
alcohol 68 under microwave conditions, and subsequent reduction of 69 yielded the
β,β-difluorinated-amine 53. The α-trifluoromethyl amine rac-54 was prepared from
40
aldehyde 66 by trifluoromethylation with TMS-CF3; subsequent functional group
interconversion of trifluoromethyl alcohol rac-70 via the triflate and azide gave the
4
1
desired product rac-54.
Structure activity relationship (SAR) studies: monosubstituted phenyl rings
The most potent analogues identified previously contained a lipophilic substituent in
24, 29
the meta or para position of one phenyl ring.
Crystallographic studies with p-
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chlorophenyl and m-tolyl containing analogues 5 and 11 revealed these substituents to
be preferentially positioned in the predominantly hydrophobic environment of the P3
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pocket (Figure 3B).
In the meta position, the optimal substituents were typically
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small alkyl groups that were electron-donating and hydrophobic,
which we
rationalized as being due to strengthening the C-H···π interaction between the i-
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propyl side chain of Leu 214 and the phenyl ring (Figure 3A). A small number of
thioethanamines was prepared to determine whether this was also applicable for
substituents to the para position. (15-21, Table 1). The p-methyl and p-methoxy
analogues 17 and 19 demonstrated comparable basal inhibition activity, that was
comparable to 16 and improved ~ 4-fold over 4 , and while a drop in potency was
apparent upon increasing from methyl to ethyl (17 and 18), this was not seen with the
ether analogues 19-21. We evaluated all inhibitors of Eg5 in the basal assay for
growth inhibition against K562 human leukemia cells. This cell-line has previously
4
2, 43
been shown to be susceptible to Eg5 inhibition and to overexpress Eg5.
Improved activity confer (c/f) 4 was exhibited in the cellular assay by p-alkyl and p-
methoxy containing substituents 17, 18 and 19, with 18 and 19 matching that of the
2
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potent meta-substituted analogue 16. Ethoxy and triofluoromethoxy derivatives 20
and 21 proved less active, which may be due to less appropriate physicochemical
properties such as reduced aqueous solubility (e.g. 20: turbidimetric solubility at pH
7
.4 = 3.75 µM). To determine whether the optimal thioethanamine trityl substituents
were translatable to the butanamine-based inhibitors, several triphenylbutanamine
analogues containing a single substituent in the meta or para position were prepared
(
22-27, Table 1). Triphenylbutanamines 22 and 23 incorporating m-ethyl and m-
isopropyl substituents respectively proved very potent in both assays, with 22 ~3-fold
more active against cells than the comparable thioether 16. The p-methoxyphenyl
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analogues rac-25 and 26 with and without the α-carboxylate both displayed GI50
<
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00 nM, which improved significantly over the equivalent S-trityl pair 7 and 19.
While these findings confirmed the systematically improved activity of the
butanamines versus analogous thioethanamines, in general these new compounds
exhibited equivalent in vitro activity to the previously described leads (e.g. rac-11 and
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3). The activity of the p-methoxy analogue 26 was however improved (~2-fold)
versus other analogues without the carboxylate, and now equivalent in the cell-based
assay to the zwitterionic rac-11. Interestingly, while rac-25 and 26 exhibited
comparably strong activity across both assays, the zwitterionic thiother 7 was clearly
superior in the cellular assay to 19.
Disubstituted phenyl rings
Having identified the optimal substituents to improve affinity, we next investigated
disubstituted phenyl rings. By broadening the scope of our SAR data, we hoped to
utilize this knowledge to allow for more permutations to modulate the metabolic and
pharmacokinetic properties of the ultimate lead candidates by unearthing multiple
modifications capable of imbuing Eg5 affinity. A 2,5-difluorophenyl ring in the P3
pocket produced impressive increases in Eg5 inhibitory activity during optimization
4
4
of the clinical candidate MK-0731.
In our own series, although a m-fluoro
29
substituent had proved detrimental to Eg5 affinity, we had not examined the
influence of fluoro substituents in alternative positions in combination with the
optimal alkyl and methoxy substituents. An o-fluoro substituent diminished
inhibitory activity against Eg5 substantially when combined with a methyl substituent
in either meta or para (28 and 29, Table 2). However, when combined with a p-
methoxy substituent (31), the o-fluorine produced a ~ 2-fold improvement in potency
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against Eg5 c/f the p-methoxyphenyl 19. Retaining the p-methoxy substituent and
repositioning the fluoro substituent in the meta position was not tolerated (32). In the
cellular assay, 28, 29, 32 and 33 proved weak inhibitors, but the improvements
evident in the basal assay for 31 translated to a modest improvement in growth
inhibition c/f the p-methoxyphenyl containing 19. On introduction of the carboxylic
acid (30), a further 3-fold improvement in growth inhibitory activity was realized c/f
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, equivalent to the butanamine lead rac-11. Interestingly the butanamine analogue of
30, compound 40 proved less potent in both the enzymatic and cellular assays.
Next, we investigated combinations of dialkyl substitutuents following from the
observation that a β-napthyl analogue of 4 demonstrated good potency against both
Eg5 and HeLa cells, with docking studies suggesting the bicyclic ring occupied the P3
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pocket. Compounds 34-39 were prepared to investigate whether more metabolically
amenable and favorable physiochemically combinations of dialkyl substituents could
4
5
be implemented in place of the napthyl moiety (Table 2). The dialkylphenyl and
tetralene analogues (35, 37 and 39) all proved comparably effective inhibitors of Eg5
basal activity to singly substituted analogues (e.g. 16-27). When the terminal
carboxylic acid was introduced, incremental improvements in activity were apparent:
the dimethylphenyl compound 34 displayed the highest affinity for Eg5 of any S-trityl
app
L-cysteine analogue, with an estimated K
i
≈ 1.2 nM. However in the cell-based
assay, 35, 37 and 39 lacking the carboxylate proved disappointingly weak inhibitors,
with poor solubility probably the principle cause of their reduced cellular efficacy
(
e.g. turbidimetric solubility at pH 7.4: 35 = 3.75 µM, Supporting Information, Table
S3). The presence of the terminal α-carboxylic acid in the tail ameliorated this: in
app
addition to K
i
< 5 nM against Eg5 in the basal assay, the L-cysteine analogues 34,
3
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based assay. The most potent growth inhibition activity was exhibited by 36 (GI50
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the importance of achieving the correct physicochemical balance. S-Alkylated
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56
57
58
59
60
derivatives of cysteine were first designed as anticancer agents following the
observation that radiolabelled cysteine was incorporated by leukemic white blood
46, 47
cells,
so active uptake of the amino acid zwitterion also cannot be discounted as
contributing to the improved activity of 36 c/f 37. Next, we transferred the optimal
dialkyl substituent pattern to the butanamine scaffold. The poor aqueous solubility of
tetralene 38 even when the carboxylic acid was present precluded continuing with this
modification (turbidimetric solubility at pH 7.4 = 20 µM). Although 36 was more
potent in the cellular assay, the superior Eg5 affinity and physicochemical properties
of the dimethylphenyl analogue 34 marked this as the optimal modification from this
selection. The prepared compound rac-42 was less potent in the basal assay, but
importantly proved 3-fold more active in the cellular growth inhibition assay (GI50
≈
2
3 nM), thereby improving on the activity of both the previously reported butanamine
2
9
leads 11 and 12 (Figure 2), and the L-cysteine analogues 34, 36 and 38 in this assay.
Analogues with modifications on two phenyl rings
All the modifications to the trityl group described so far increased its hydrophobicity,
which in certain cases compromised cellular efficacy due to poor aqueous solubility
(e.g. 38). In addition to this immediately apparent penalty, excessively lipophilic (i.e.
log P/log D7.4 > 3) compounds are more likely to possess unfavorable ADME
properties, and bind more promiscuously, which is likely to result in greater side
4
8
effects and general toxicity.
We were interested in developing hydrophilic
modifications to the trityl group to produce a more physicochemically balanced
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scaffold to serve as a backup to the lead amino acid based compounds. One way to
achieve this was by replacing a phenyl ring with a heterocycle, however none of the
investigated analogues displayed noteworthy activity (S1-S5, Supporting Information,
0
1
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8
9
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26, 49
Table S1), in agreement with other reports on thiophene and pyridyl analogues.
Previously we identified certain hydrophilic phenyl substituents that reduced the
lipophilicity of the trityl head group and improved cellular efficacy (e.g. 8 and 9,
2
9
Figure 2). The most effective of these modifications was the m-hydroxy 8, designed
to form hydrogen-bonding interactions with the peptide backbone of the P2 pocket
12, 29
(Figure 3A).
Modest improvements in cellular efficacy were also noted in
analogues containing primary and secondary amide phenyl substituents (e.g. 9, Figure
2
). These amides were proposed to occupy the solvent exposed P1 region due to the
44, 50, 51
steric restrictions of the P2 pocket observed in other SAR studies (Figure 3B).
We combined each of these modifications in turn, with a lipophilic substituent on a
different phenyl in the trityl group, to investigate if synergistic increments in activity
could be achieved (44-51, Table 3). While no improvement was apparent for
thioethanamine rac-45 containing a m-phenol and m-chlorophenyl, the related m-
phenol analogues rac-46 and rac-47 with m-ethyl and p-methyl phenyl substituents
were 5-fold and 4-fold more potent inhibitors of Eg5 basal activity respectively than
comparable analogues 16 and 17. These analogues also exhibited improved inhibition
over their respective benchmarks in the K562 assay: e.g. rac-46 improved ≥ 2-fold
over the benchmarks 8 and 16 and rac-47 improved ≥ 5-fold over 8 and 17. These
data implied a synergistic binding motif was being realized, with hydrogen bonding
interactions from the m-hydroxyl in the P2 pocket augmented by hydrophobic
interactions with the alkyl substituted phenyl rings situated in the P3 pocket. While
no increase in potency was evident in the basal assay when a p-tolyl was combined
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with a m-primary amide containing phenyl, this combination afforded a ~ 2-fold
improvement in the cellular assay (rac-49 c/f 9 and 17) which also supported the
binding conformation proposed. With GI50 ≈ 100 nM, rac-47 is the most active
thioethanamine without the terminal α-carboxylic acid in the cellular assay reported to
date, and comparable to the lead butanamine 11. These modifications did not prove
advantageous when applied in the comparable butanamine rac-51, which was less
active in both assays than rac-47 or 11. Better activity was evident on addition of the
terminal carboxylate in the basal assay, but not in the cellular assay (dia-50). We
previously noted that passive cellular diffusion was adversely affected by the presence
of both the α-carboxylate in the tail and amide or hydroxyl phenyl substituents, which
may offer a partial explanation for the comparatively weak cellular activity of the
zwitterionic butanamine dia-50.
10
11
12
13
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52
53
54
55
56
57
58
59
60
Crystal structure of the Eg5-46 complex.
The binding mode proposed for analogues with two orthogonal phenyl substituents
was confirmed when the crystal structure of 46 in complex with Eg5 was solved to a
resolution of 2.65 Å. This showed the m-phenol in P2 and the m-ethyl in P3 (Figure
3
C). Data collection and refinement statistics are presented in Table 4. The Eg5-46
complex crystallised in space group I2 3 with 1 molecule per asymmetric unit.
1
Although the racemic mixture of 46 was used for crystallization, only the R-
enantiomer was observed in the inhibitor-binding pocket. The three phenyl rings of
(R)-46 are positioned in the same overall conformation as 4, and consequently the
hydrophobic and aromatic interactions between the three phenyl rings and the
2
7, 28
residues in the inhibitor-binding pocket are conserved.
In the thioethanamine tail,
the primary amine maintains the crucial hydrogen bonds exhibited by all analogues of
2
6, 27,
4
with the carboxylate of Glu116 and the main chain amide carbonyl of Gly117.
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29
The well-defined electron density affords accurate positioning of the two phenyl
substituents and provides a clear structural rationale for the exhibited improvements
in binding affinity. The m-ethylphenyl is situated in the predominantly hydrophobic
P3 pocket bounded by Leu160, Gly217, Ala218, Arg221, mimicking the m-tolyl and
0
1
2
3
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6, 29
p-chlorophenyl rings in previous crystal structures.
3
However, the terminal CH of
the ethyl extends into previously unutilized space between the methyl of Ala218 and
the aliphatic chain of Arg221. The m-phenol of 46 occupies the P2 pocket formed by
Glu118, Arg119, Trp127, Asp130, Leu132, Ala133 and Pro137, with the hydroxyl
pointing towards the core of the protein instead of outwards towards the solvent.
Hydrogen-bonding interactions are evident in this position from the hydroxyl to the
main chain carbonyl of Glu118, the main chain nitrogen of Ala133 and additional
weak electrostatic interactions with the main chain nitrogen of Arg119, one of the
side chain oxygen of Asp130, and the carbonyl oxygen of Leu132 (distance cut-off =
4
.0 Å), similar to those observed in the Eg5-monastrol complex and structurally
related dihydropyrimidines, thereby stabilising the inhibitor in the allosteric site and
1
2, 52
increasing the affinity for the protein.
Inhibition of MT-stimulated ATPase activity and kinesin selectivity.
The inhibitory activity of the most potent analogues from the initial in vitro screens
was next examined in a MT-stimulated Eg5 ATPase assay, together with selected
control compounds (4, 7, rac-11 and 13) and external benchmarks (1 and 2, Table 5).
In cells, Eg5 uses MT filaments as tracks to push anti-parallel MTs apart, which
makes this particular assay closer to physiological conditions. Three new analogues
app
(36, rac-42 and rac-47) possessed K
i
estimates of < 10 nM. Recently, the inhibitory
activity of monastrol against Eg5 was shown to be dependent on the ionic strength of
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the buffer (Luo 2004, Biochemistry). To investigate whether this was the case for our
scaffold, we assessed MT-stimulated ATPase activity of compounds 1 and rac-42 at
different salt concentrations. For both compounds, the IC50 values systematically
decrease with increasing salt concentrations indicating that activity is optimal under
more physiological conditions represented by 150 mM NaCl (Supporting Information,
Figure S2).
10
11
12
13
14
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16
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19
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46
47
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50
51
52
53
54
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60
Analogues 4, 7 and 11 were all previously tested against multiple kinesins and found
to specifically inhibit Eg5. Against a panel of seven human kinesins containing
Kif3B
(kinesin-2;
organelle
transport),
neuron-specific
Kif5A
and
Kif5B/conventional kinesin (both kinesin-1; cargo transport), Kif7 (kinesin-4,
involved in Hedgehog signaling), Kif9 (kinesin-9; regulation of matrix degradation)
2, 3
and Kif20A/MKLP-2 and Kif20B/MPP1 (both kinesin-6; required for cytokinesis),
rac-42 displayed no activity at a maximum inhibitor concentration of 200 µM
(
Supporting Information, Table S4 & Figure S1), which confirms the proposed high
1
7, 18, 25, 29
selectivity of this class of inhibitors within the kinesin superfamily.
Our
analogues have not yet been tested on ATPases beyond the kinesins, although other S-
trityl L-cysteine analogues were recently shown to be weak inhibitors of Hepatitis C
5
3
Virus NS5B Polymerase.
Evaluation of inhibitors across multiple cell lines.
A selection of the new lead compounds was evaluated in five additional cell lines
derived from colon (HCT116), pancreas (BxPC-3), prostate (LNCaP and PC3) and
lung (NCI-H1299) tumors, to determine in vitro efficacy against a histologically
broader panel of cancer cell lines (Table 5). The lead compounds from our previous
study with and without the carboxylic acid (11 and 13 respectively) were included to
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assess progress, along with the original hit 4 and the Phase II drug candidates 1 and
2
. In general, HCT116 cells were the most responsive to the Eg5 inhibitors tested,
whereas NCI-H1299 cells were the least affected. Three of the new analogues (34, 36
0
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29
and rac-42) displayed higher activity than the original lead (rac-11); both 36 and
rac-42 were furthermore more potent than the clinical candidate 1, and equal its more
potent second generation analogue 2. For those compounds without the carboxylic
acid group, three (26, rac-47, rac-51) were more active than the previous lead 13, but
less active than rac-11. We have shown previously that for 4, 11 and 12, the absolute
configuration of the stereocentre in the amino acid tail has very little effect on
1
8, 29
efficacy.
The phenol compounds rac-47 and rac-51 were prepared racemically
and contain a stereocentre at the trityl carbon more likely to impact on activity,
especially given that only the (R)-enantiomer was observed in the crystal structure of
the Eg5-46 complex. Compounds prepared via the TFA mediated dehydration of
4
9
chiral tertiary alcohols form a 1:1 ratio of enantiomers (Scheme 1). However, we
did not investigate the resolution of rac-47 and rac-51 further, as rac-47 displayed
-
1
½
very high clearance in human hepatocytes (Clint = 50.1 ± 4.2 µL min ; t = 27.7
min), and thus was not suitable for progression as a drug candidate.
Profiling of drug-like properties of lead compounds
We subsequently subjected the new lead compounds from this study (34, 36 and rac-
42) to a series of in vitro and in vivo physicochemical and ADMET assays to evaluate
if in addition to pharmacodynamics improvements, they exhibited favorable drug-like
properties (Table 6). The Phase II candidate 2 was included as an external
benchmark; the profile for the first generation candidate 1 has been reported
29
previously. The p-methoxy analogue 7 was also profiled, as this compound was
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Luc
reported to inhibit the rate of tumor growth in subcutaneous prostate (PC3 cells)
2
2, 23
and bladder xenograft tumor models.
Analogue 7 has a balanced profile, with
favorable physiochemical characteristics and reasonable metabolic stability in both
human microsomes and hepatocytes. Moderate affinity is displayed for the 2C19
CYP isoform. In comparison to the other lead compounds however, 7 is
systematically less potent in vitro (Table 5) so was not investigated further. The most
potent analogue lacking the carboxylic acid 26, which also contains a p-
methoxyphenyl, was profiled and interestingly proved metabolically more resilient in
human microsomal and hepatocyte assays than 7. Several liabilities were however
evident, including low turbidimetric solubility at physiological pH (> 30 µM), hERG
10
11
12
13
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60
inhibition and moderate to significant inhibition of CYP isoforms from four of the
54
main families.
This agreed with the trend previously observed that compounds
lacking the carboxylic acid group have generally more liabilities than those
2
9
possessing it. At this stage, we investigated whether modulating the basicity of the
2
9
primary amine would be an effective strategy to ameliorate these.
Although
5
5
alkylation of primary amines can overcome hERG inhibition, the hydrogen bonds
formed by the primary amine in 4 are crucial to maintaining activity (Figure 3A), with
2
6, 27
secondary amines only weakly active.
We therefore applied the subtler strategy
5
6
of modulating the pK by β-fluorination to produce 52-54 (Table 7). In the basal
a
assay, whilst inhibitory activity comparable to the unmodified 26 was displayed by β-
fluorinated rac-52, the binding affinity was substantially reduced for β,β-difluorinated
5
3 and lowered to micromolar levels for α-trifluoromethyl containing rac-54. Despite
good potency against Eg5, in cells rac-52 proved ~ 8-fold less active than the
unmodified 26, and 53 only exhibited micromolar levels of growth inhibition. Whilst
these results reinforce the importance of the protonation state of the primary amine at
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physiological pH, the reasonable basal activity suggests multiple factors contributed
to the reduced cellular efficacy. As this strategy did not appear useful in this series,
we did not investigate further.
0
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0
Consequently, we profiled our two most potent cysteine based analogues, 34 and 36,
which differ only by an extra methylene in the meta position that imparts 3 to 4-fold
higher potency across cellular assays (Table 5). This gain in potency comes at a cost:
3
6 exhibits higher clearance in human hepatocytes (24.4 ± 4.7 versus 7.9 ± 3.4
µL/min/million cells) and is less available in human plasma (1.55% versus 4.51%,
Table 6). A distinct profile is also evident towards CYP enzymes: like the p-
methoxy analogue 7, 34 inhibits CYP 2C19 (IC50 = 7.9 ± 1.3 µM), whereas 36 does
not, but instead weakly inhibits CYP 2C9 (IC50 = 14.0 ± 4.9 µM). Compound 36
displays high bioavailability (62%) when compared to that determined previously for
2
9
1
(45%), 13 (4%) and rac-11 (51%) (Table 8). The pharmacokinetic data is in good
agreement with the in vitro mouse metabolic profiling: 36 has a shorter half-life and
higher clearance than the clinical candidate 1, with rac-11 displaying low clearance in
2
9
comparison.
The butanamine analogue of 34, and most active from the in vitro profiling rac-42,
was investigated next. Direct comparison with 34 illustrates that rac-42 displays
higher turbidimetric solubility (> 100 µM versus 65 µM), increased metabolic
resilience in human hepatocytes (5.91 ± 1.89 versus 7.9 ± 3.4 µL/min/million cells)
and does not inhibit any of the common CYP isoforms examined. In common with
the profiled zwitterionic S-trityl analogues, no affinity is evident towards the hERG
channel. This attractive profile is analogous to that reported for our previous lead
2
9
rac-11.
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Examining clinical candidate 2 in the same assays, improved solubility was displayed
2
9
at the three pHs tested compared to 1 (> 100 µM versus 65 µM).
The reduced log
P of 2 also translates to a higher proportion of unbound compound in human plasma
10
11
12
13
14
15
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46
47
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(
22% versus 8%). However, 2 retains the potentially physiologically significant
2
9
hERG channel interactions and affinity for the CYP isoform 3A4 evident for 1.
Therefore the butanamines 11 and rac-42 possess a more balanced ADMET profile
which may offer distinct clinical advantages.
In vivo xenograft studies
We advanced the three most active and balanced of the new analogues (34, 36, and
rac-42) into in vivo xenograft experiments with lung cancer patient explants (LXFS
5
38) passaged as subcutaneous xenografts in nude mice. As controls, our previous
lead analogue rac-11 and the clinical candidate 1 were selected. We adopted lung
cancer xenograft models as this is a key area for improving therapy due to the poor
5
7
prognosis for most patients; the indications that Eg5 inhibitors may achieve better
clinical outcomes for hematological malignancies rather than in solid tumors came
after this work had commenced. We used explanted tumors as these are a more
realistic model than solid tumors derived from in vitro cell lines. The transplanted
tumors retain the histological complexity from the patient and reflect prior treatment,
58, 59
and have been shown to be better predictors of clinical response.
Administering
our previous lead rac-11 by an improved dosing schedule resulted in complete tumor
regression at day 44 (Figure 4A), in comparison to partial remission recorded
2
9
previously. When treatment stopped, tumor growth relapsed around day 76 (data
not shown). Xenograft experiments for 1 have previously been reported at a dose of
60
8
-10 mg/kg in breast tumor models. In mice implanted with LXFS 538 tumors,
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5
6
treatment at this dose proved to be lethal (data not shown), and similar toxicity issues
7
have been observed previously. When we repeated at a lower dose of 6 mg/kg, total
tumor regression was evident around day 13 and the mice remained tumor free over
the entire observation time (Figure 4A). For the cysteine containing 36, although
significantly more potent than rac-11 in cell-based assays, only a reduced tumor
growth rate was observed (Figure 4B), although the difference was still statistically
significant (p = 0.0124). No tumor growth inhibition was evident for 34 (Figure 4C).
Metabolic profiling of 34 and 36 explained this poor in vivo activity versus the strong
in vitro efficacy: although stable in mouse microsomes, 36 showed high clearance in
mouse hepatocytes (58.7 ± 5.64 µL/min/million cells; t1/2 = 23.6 min). For 34, a
similar explanation applied: while stable in human microsomes with only low
clearance in human hepatocytes (7.9 ± 3.4 µL/min/million cells), medium clearance
was evident in mouse microsomes (17.8 ± 3.7 µL/min/mg protein) and extremely high
clearance by mouse hepatocytes (170 ± 9.4 µL/min/million cells; t1/2 = 8.2 min).
Therefore nude mice xenografts are not an appropriate model for assessing the
therapeutic potential of 34 and 36: in future alternative models such as nude rats will
need to be employed, if these compounds prove metabolically stable in rat
hepatocytes. In contrast to the thioethanamines 34 and 36, the activity of the most
potent butanamine rac-42 was translated effectively into the xenograft model, with
total tumor regression by day 17, improving on the activity recorded for rac-11 and
comparable to the response achieved on treatment with 1 (Figure 4C).
0
1
2
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Conclusions
Optimization of 4 has led to structurally relatively simple, yet extremely effective Eg5
inhibitors, which display comparable in vitro and in vivo antitumor activity to
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benchmark inhibitors further advanced in clinical trials. The only other Eg5 inhibitors
reported to induce complete tumor regression in mice xenograft models, to the best of
7
-9
our knowledge, are the three clinical candidates 1-3 in Phase II.
We propose that
10
11
12
13
14
15
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19
20
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23
24
25
26
27
28
29
30
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34
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36
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42
43
44
45
46
47
48
49
50
51
52
53
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60
the favorable drug-like properties as highlighted by in vitro profiling strengthens the
case for the progression of triphenylbutanamine analogues based on rac-42 for further
evaluation. In general, the butanamines proved systematically more potent than the
equivalent thioethanamines, but most notably their increased metabolic stability
imparted improved efficacy in xenograft studies with nude mice. Both the profiled
butanamine and thioethanamine leads rac-11 and 36 show good oral bioavailability
and pharmacokinetics, however for an accurate assessment of the potential of these S-
trityl analogues, alternative in vivo models will need to be examined. In this and our
preceding study, we have developed a range of modifications to the core scaffold
which impart excellent affinity for the target protein Eg5. The potential of clinical
candidates based on rac-42 will be determined by selecting those which provide
optimal bioavailability and robust pharmacokinetics, a process we have initiated
through the profiling described here. The wider challenge facing Eg5 inhibitors is
translating the efficacy evident in preclinical studies into the clinic. The responses to
Eg5 inhibitors in clinical trials have been limited to date, and one explanation for this
is that tumor doubling times in xenograft models are significantly faster than those in
human patients and thus not sufficiently realistic models for developing
6
1
chemotherapeutics.
This hypothesis is corroborated by the improved responses
observed against faster growing hematological cancers and the principle dose limiting
1
toxicities for most clinical candidates of neutropenia and myelosuppression. Early
clinical indications suggest a possible application for Eg5 inhibitors in advanced
1
5
multiple myeloma, which will most likely be in combination with existing therapies.
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6
In combined chemotherapies, the potential for inducing drug-drug interactions and
toxicities becomes increasingly important. For example, the hepatic metabolism of
the synthetic glucocorticoid dexamethasone currently used in the treatment of
0
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0
62
multiple myeloma is mediated primarily by CYP 3A4. Both of the trial Eg5 drugs
we profiled, 1 and 2, are CYP 3A4 inhibitors, so careful consideration of the
physiological relevance of these interactions would be required prior to undertaking
2
9
combination based therapy.
Limited information is available on the relevant
1
properties of other candidates under investigation. We have demonstrated that in
comparison to the clinical candidates 1 and 2, the lead S-trityl L-cysteine derived
analogues rac-11 and rac-42 exhibit comparable in vivo efficacy by inducing
complete tumor regression in an advanced tumor explant xenograft model, but display
fewer liabilities towards CYP enzymes and the hERG channel. Furthermore unlike
other highly advanced Eg5 inhibitors, the described compounds are not patented and
therefore are freely available for investigation in single or combined cancer
chemotherapies by the scientific and medical community, which we propose should
focus on targeting hematological malignancies.
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Material and Methods
A) Chemistry
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56
57
58
59
60
General
Compounds 4 and 2 were purchased from Nova Biochem, Sigma Aldrich and Selleck
Chemicals, respectively, and used without further purification. Compound 1 was a
gift from Sanofi-Aventis. Compounds 8, 9, 11-16 and 43 were prepared as reported
2
9
previously.
Compound 7 (NSC123528) was obtained from the NCI/DTP Open
Chemical Repository (http://dtp.cancer.gov) of the National Cancer Institute. All
reagents and solvents were of commercial quality and used without further
purification. Anhydrous reactions were carried out in oven-dried glassware under a
nitrogen atmosphere unless otherwise noted. Microwave reactions were performed
using a Biotage Initiator-8 microwave synthesizer (operating at 2.45 GHz). Thin-
2
layer chromatography (TLC) was carried out on aluminium backed SiO plates (silica
gel 60, F254), and spots were visualized using ultraviolet light (254 nm) or by staining
with phosphomolybdic acid (alcohols) or ninhydrin (amines). Flash column
chromatography was performed on silica gel [SNAP KP-Sil, 60 Å, 40−63 µm
cartridges] using a Biotage SP4 automated chromatography system (detection
wavelength, 254 nm; monitoring, 280 nm). Melting points were determined using a
1
13
Stuart Scientific SMP1 melting point apparatus and are uncorrected. H and
C
NMR spectra were recorded on a JEOL ECX-400 (400 MHz), Avance DPX400 (400
1
9
MHz), or Avance DPX500 (500 MHz) spectrometer. F NMR spectra were recorded
1
on an Avance AV400 (400 MHz) instrument equipped with a multinuclear probe. H
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5
6
chemical shifts (δ) are reported in ppm relative to the residual signal of the deuterated
3 3 6
solvent (7.26 in CDCl , 3.31 in CD OD, and 2.50 in DMSO-d ). Multiplicities are
indicated by s (singlet), d (doublet), t (triplet), q (quartet), m (unresolved multiplet),
0
1
2
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9
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3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
and br (broad signal).
C chemical shifts (δ) are reported in ppm relative to the
, 49.00 in CD OD, and
). F spectra are referenced relative to CFCl . High-resolution
carbon resonance of the deuterated solvent (77.16 in CDCl
3
3
1
9
3
9.52 in DMSO-d
6
3
mass spectra were recorded on a Thermo Electron LTQ ORBITRAP mass
spectrometer using electrospray ionization. Gas chromatography mass spectra (GC-
MS) using electron ionization (EI) were recorded on a Thermo Scientific Focus GC
with DSQ2 single quadrupole mass spectrometer. GC-MS using chemical ionization
(
CI) were recorded on an Agilent Technologies 7890A GC system and an Agilent
975C Inert XL EI/CI MSD with DSQ2 single quadrupole mass spectrometer,
equipped with an Agilent Technologies DB5-MS column (30 m x 0.25 mm x 0.25
5
-
1
µm). Helium was the carrier gas (flow rate = 1 mL/min ). Elemental analysis data
were recorded on a Perkin-Elmer 2400 series 2 CHN analyzer. LC-MS analyzes were
performed with an Agilent Quaternary 1200 series pump and an Agilent 6130 dual
R
source mass spectrometer with UV detection at 254 nM. Retention times (t ) were in
minutes, and purity was calculated as percentage of total area. The method for
determining purity consisted of the following: Zorbax Eclipse XDB-C18 reverse
phase column (15 cm x 4.3 µm, particle size 5 µm); column temperature 40 °C;
solvent A: H
acetate); gradient of A:B, 95:5 (0 – 3 min), A:B, 95:5  B, 100% (3 – 17 min), B,
00% (17- 27 min), B, 100%  A:B, 95:5 (27-33 min), A:B, 95:5 (33 – 36 min);
2
O (5 mM ammonium acetate); solvent B: MeCN (5 mM ammonium
1
-
1
flow rate 1 ml min . All tested compounds were ≥ 95% pure by elemental or LCMS
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analyses. New compounds were named according to IUPAC nomenclature by ACD
ChemSketch 12.01 (Windows, Advanced Chemistry Development, Toronto, Canada).
Procedures:
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56
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58
59
60
5
-Bromo-N-methoxy-N,2-dimethylbenzamide (56). Oxalyl chloride (3.43 ml, 40
mmol) was added to a solution of 5-bromo-2-methylbenzoic acid 55 (4.30 g, 20
mmol) in anhydrous CH Cl (20 ml), with a catalytic amount of DMF (1 drop), and
2
2
stirred at room temperature for 2 h. The reaction mixture was concentrated in vacuo,
and the residual oil added by slow dropwise addition over 5 min to a cooled (0 °C)
solution of N,O-dimethylhydroxylamine hydrochloride (2.24 g, 23 mmol) and
2 2
triethylamine (3.67 mL, 50 mmol) in anhydrous CH Cl (20 ml). After stirring for 1
h, the reaction mixture was warmed to room temperature, stirred for 20.5 h, then
quenched with water followed by aqueous HCl (0.5 M, 25 mL), and extracted with
CH
2
Cl
2
(3 x 25 mL). The combined organic extracts were dried (MgSO
4
),
; 0-50% EtOAc in
, 500 MHz) δ
), 7.07 (d, J = 8.6 Hz,
, 125 MHz) δ 18.77, 61.33, 118.95, 129.13,
concentrated in vacuo, and purified by flash chromatography [SiO
2
1
hexane] to afford 56 as a colourless oil (3.63 g, 70%). H NMR (CDCl
3
2.26 (s, 3H, CH
3
3 3
), 3.30 (br s, 3H, CH ), 3.48 (br s, 3H, CH
13
2
1
2
5
H), 7.38-7.40 (m, 2H). C NMR (CDCl
3
+
31.95, 132.23, 134.06, 137.20. HRMS (ESI+) calcd. for C10
H
13BrNO
2
(M+H) :
58.01242; found: 258.01244. Anal. calcd. for C10H12BrNO : C, 46.53; H, 4.69; N,
2
.43. Found: C, 45.03; H, 4.78; N, 5.60.
1
-(5-Bromo-2-methylphenyl)ethanone (57). The title compound was prepared
63
using an adaptation of the procedure reported by Hirashima et al. MeMgCl (3.0 M
in THF, 8.27 mL, 24.8 mmol) was added by slow dropwise addition over 10 min to a
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cooled (0 °C) solution of 5-bromo-N-methoxy-N,2-dimethylbenzamide 56 (3.27 g,
1
2.4 mmol) in THF (12.4 mL) and stirred for 2 h. The reaction mixture was warmed
to room temperature, stirred for 2 h, before quenching with saturated aqueous NH Cl
solution (20 mL) and extracting with EtOAc (3 x 25 mL). The organics were washed
successively with water and brine (75 ml each), dried (MgSO ) and concentrated in
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
vacuo to give 57 as a clear pale brown oil (2.40 g, 91%), which was used without
1
further purification. H NMR (CDCl
3
, 500 MHz) δ 2.45 (s, 3H, CH
3
), 2.56 (s, 3H,
3
CH ), 7.11 (d, J = 8.2 Hz, 1H), 7.48 (dd, J = 2.1, 8.2 Hz, 1H), 7.77 (d, J = 2.1 Hz,
13
1
1
H). C NMR (CDCl
3
, 125 MHz) δ 22.10, 29.62, 119.24, 132.11, 133.77, 134.39,
+
37.29, 139.46. GC-MS (EI, 70eV) t
R
= 4.68 min (m/z = 211.9, M ). Anal. calcd. for
9 9
C H
BrO C, 50.73; H, 4.26. Found: C, 50.44; H, 4.30.
4-Bromo-2-ethyl-1-methylbenzene (58). The title compound was prepared using an
6
4
adaptation of the procedure reported by Chackal-Catoen et al. Hydrazine hydrate
monohydrate (1.46 mL, 30 mmol) was added to a solution of 1-(5-bromo-2-
methylphenyl)ethanone 57 (2.13 g, 10 mmol) and powdered KOH (1.68 g, 30 mmol)
in anhydrous ethylene glycol (10 mL) and refluxed for 4 h. After cooling to room
temperature, the reaction was quenched was aqueous HCl (1.0 M, 30 mL) and
extracted with EtOAc (3 x 30 mL). The organics were washed successively with
water and brine (75 mL each), dried (MgSO
4
) and concentrated in vacuo and the
; hexane] to afford alkane 58 as a
, 400 MHz) δ 1.20 (t, J = 7.5 Hz, 3H,
), 7.00 (d, J = 8.0 Hz, 1H), 7.22
residue purified by flash chromatography [SiO
2
1
colourless oil (1.10 g, 55 %). H NMR (CDCl
3
CH
3
), 2.24 (s, 3H, CH
3
), 2.59 (q, J = 7.5 Hz, 2H, CH
2
1
3
(dd, J = 2.0, 8.0 Hz, 1H), 7.28 (d, J = 1.9 Hz, 1H). C NMR (CDCl
3
, 100 MHz) δ
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4.21, 18.84, 26.18, 119.63, 128.72, 130.80, 131.71, 134.83, 144.68. GC-MS (EI, 70
+
eV) t
R
= 4.90 min (m/z = 199.8, M ).
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11
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56
57
58
59
60
(
3-Ethyl-4-methylphenyl)(diphenyl)methanol (59). n-Butyllithium (2.5 M in
hexane, 2.21 mL, 5.53 mmol) was added by slow dropwise addition over 2 min to a
cooled (-78 °C) solution of 4-bromo-2-ethyl-1-methylbenzene 58 (918 mg, 4.61
mmol) in anhydrous THF (4.61 mL) and stirred for 1 h at ≤ -70 °C. A solution of
benzophenone (966 mg, 5.30 mmol) in anhydrous THF (5.30 mL) was added by slow
dropwise addition over 5 min, and the reaction mixture stirred with the temperature
maintained ≤ -70 °C for 5 h, before allowing the reaction to warm to room
temperature and stirring for a further 20 h. The reaction was quenched with saturated
aqueous NH
combined organic layers were then washed successively with H
each), dried (MgSO ) and concentrated in vacuo. Purification by flash
chromatography [SiO
4
Cl solution (10 mL) and extracted with EtOAc (3 x 10 mL). The
2
O and brine (30 mL
4
2
; 0-10% EtOAc in hexane] afforded trityl alcohol 59 as a
1
yellow oil (1.263 g, 91%). H NMR (CDCl
3
, 500 MHz) 1.14 (t, J = 7.6 Hz, 3H,
), 2.77 (s, 1H, OH), 6.93 (dd, J
2.0, 7.9 Hz, 1H), 7.07 (d, J = 7.9 Hz, 1H), 7.12 (d, J = 1.9 Hz, 1H), 7.26-7.32 (m,
3 3 2
CH ), 2.31 (s, 3H, CH ), 2.59 (q, J = 7.6 Hz, 2H, CH
=
1
3
10H). C NMR (CDCl
3
, 125 MHz) δ 14.60, 18.92, 82.11, 125.60, 127.24, 127.66,
+
1
27.98, 128.06, 129.67, 134.96, 142.11, 144.77, 147.26. HRMS (ESI ) calcd. for
+
C
21
H21 (M-OH) : 285.16378; found: 285.16348. Anal. calcd. for C22H22O: C, 87.38;
H, 7.33. Found: C, 87.23; H, 6.81.
(2R)-2-Amino-3-(((3-ethyl-4-methylphenyl)(diphenyl)methyl)sulfanyl)propanoic
acid (36). A solution of the tertiary alcohol 59 (254 mg, 0.84 mmol) with L-cysteine
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5
5
5
5
5
5
5
6
(
112 mg, 0.93 mmol) in TFA (1.0 mL) was stirred for 3 h at room temperature. The
volatiles were removed in vacuo, and the crude basified (circa. pH 10) with saturated
aqueous sodium carbonate solution. The aqueous mixture was extracted with CH Cl
3 x 10 mL) and the organic layer dried (MgSO ) and concentrated in vacuo.
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
4
Purification by flash chromatography [SiO
2
2 2
; 0-25% MeOH in CH Cl ] afforded the
1
thioether 36 as a white solid (242 mg, 71%). Mpt. 149-152 °C. H NMR (500 MHz,
MeOD) δ = 1.10 (t, 3H, J = 7.6 Hz), 2.27 (s, 3H), 2.56 (q, 2H, J = 7.6 Hz), 2.69 (dd,
1H, J = 9.2, 13.4 Hz), 2.82 (dd, 1H, J = 4.2, 13.4 Hz), 3.05 (dd, 1H, J = 4.2, 9.2 Hz),
7
.06 (d, 1H, J = 8.0 Hz), 7.14 (dd, 1H, J = 2.1, 8.0 Hz), 7.18-7.24 (m, 3H), 7.27-7.32
1
3
(m, 4H), 7.42-7.46 (m, 4H). C NMR (125 MHz, MeOD) δ = 14.94, 18.73, 27.28,
3
1
4.36, 55.13, 68.05, 127.92, 128.07, 129.04, 130.47, 130.70, 130.74, 135.60, 143.16,
+
43.29, 145.91, 145.95, 172.53. HRMS (ESI+) Calcd. for C25
H28NO
2
S (M+H) :
406.1835; found 406.1843. Anal. calcd. for C25
N, 3.38. Found: C, 72.18; H, 6.37; N, 3.14.
2 2
H27NO S·½H O: C, 72.43; H, 6.81;
4-(1,1-Diphenylbut-3-en-1-yl)-1,2-dimethylbenzene (61). The title compound was
3
3
prepared using an adaptation of the method reported by Kabalka et al.
Butyllithium (2.5 M in hexane, 6.12 mL, 15.3 mmol) was added cautiously by
dropwise addition to cooled (0 °C) solution of (3,4-
dimethylphenyl)(diphenyl)methanol 61 (4.00 g, 13.9 mmol) in anhydrous CH (85
n-
a
2
Cl
2
mL), the reaction mixture allowed to warm to room temperature and stirred for 30
min. Allyltrimethylsilane (6.02 mL, 36.9 mmol) and iron trichloride (5.19 g, 32.0
mmol) were then added and the reaction stirred at room temperature for 6 h. The
reaction was quenched with H
2 2 2
O (100 mL) extracted with CH Cl (3 x 100 mL). The
combined organic layers were washed successively with saturated aqueous NaHCO
3
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solution (150 mL) and brine (200 mL), dried (MgSO4) and concentrated in vacuo.
2
Purification by flash chromatography [SiO ; 0-10% EtOAc in hexane] afforded
alkene 61 as a brown oil [3.119 g, 91% (based on 79% conversion)] and recovered
10
11
12
13
14
15
16
17
18
19
20
21
22
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
starting material 60 (834 mg). H NMR (400 MHz, CDCl
3
) δ = 2.22 (s, 3H, CH
3
),
2.26 (s, 3H, CH
3
), 3.43-3.46 (m, 2H), 4.97 (ddd, J = 1.4, 3.4, 10.3 Hz, 1H), 5.06 (ddd,
J = 1.5, 3.5, 17.0 Hz, 1H), 5.70 (ddd, J = 6.6, 10.4, 17.1 Hz, 1H), 6.94-6.98 (m, 1H),
1
3
7
.03-7.08 (m, 2H), 7.19-7.34 (m, 10H).
3
C NMR (100 MHz, CDCl ) δ = 19.40,
20.24, 45.67, 56.01, 117.18, 125.97, 126.97, 127.79, 129.14, 129.58, 130.62, 134.23,
1
R
35.87, 136.35, 144.92, 147.65. GC-MS (CI, methane) t = 16.44 min (m/z = 311.2,
+
[
M-H] ). Anal. calcd. for C24
H
24: C, 92.26; H, 7.74. Found: C, 91.51; H, 7.28.
4
3
-(3,4-Dimethylphenyl)-4,4-diphenylbutan-1-ol (62). A solution of BH ⋅THF (1.0
M in THF, 20.0 mL, 20.00 mmol) was added to a cooled (0 °C) solution of alkene 61
3.11 g, 9.95 mmol) in THF (20 mL), and stirred at room temperature for 19 h. The
reaction was cooled to 0 °C and quenched cautiously with H O (5 mL) and aqueous
NaOH (3.0 M, 6.8 mL, 20.40 mmol), followed by slow dropwise addition of
hydrogen peroxide (30% in H O, 5.11 mL, 50.01 mmol) over 5 min. The mixture
was maintained at 0 °C for 30 min, and then allowed to warm to room temperature
and stirred for a further 3.5 h, diluted with H O (50 mL) and extracted with Et O (3 x
0 mL). The combined organic extracts were washed successively with saturated
aqueous NaHCO solution (200 mL) and brine (200 mL), dried (MgSO ) and
concentrated in vacuo. Purification by flash chromatography [SiO
(
2
2
2
2
5
3
4
2
; 0-22% EtOAc in
1
hexane] afforded the primary alcohol 62 as a colourless oil (1.90 g, 58%). H NMR
(400 MHz, CDCl
3
) δ = 1.31-1.40 (m, 2H, CH
2
), 2.19 (s, 3H, CH
3 3
), 2.21 (s, 3H, CH ),
2
.59-2.66 (m, 2H, CH ), 3.62 (t, J = 6.4 Hz, 2H, CH
2
2
), 6.97-7.03 (m, 2H), 7.04-7.09
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