Design and campaign synthesis of piperidine- and thiazole-based histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.03.041

A lead benzamide, 3, was identified as a potent and low molecular weight histone deacetylase (HDAC) inhibitor. Optimization led to 16d, demonstrating an excellent balance of efficacy and non-efficacy properties, along with very desirable in vivo DMPK. The

Full text of DOI:10.1016/j.bmcl.2008.03.041

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2580–2584
Design and campaign synthesis of piperidine- and thiazole-based
histone deacetylase inhibitors
David M. Andrews,* Elaine S. E. Stokes,* Greg R. Carr, Zbigniew S. Matusiak,
Craig A. Roberts, Michael J. Waring, Madeleine C. Brady,
Christine M. Chresta and Simon J. East
Cancer and Infection Research, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, UK
Received 21 February 2008; revised 13 March 2008; accepted 14 March 2008
Available online 16 March 2008
Abstract—A lead benzamide, 3, was identified as a potent and low molecular weight histone deacetylase (HDAC) inhibitor. Opti-
mization led to 16d, demonstrating an excellent balance of efficacy and non-efficacy properties, along with very desirable in vivo
DMPK. The final compounds presented are >1000-fold more potent than the initial screen hit, an improvement in potency which
was achieved with a concomitant significant improvement in all the main non-efficacy properties.
ꢀ 2008 Elsevier Ltd. All rights reserved.
In the eukaryotic cell, DNA is routinely compacted to
prevent transcription factor accessibility. When the cell
is activated this compacted DNA is made available to
DNA-binding proteins, thereby allowing the induction
of gene transcription.1 Nuclear DNA associates with
histones to form chromatin and the N-terminal tails of
the core histones contain lysine residues that are sites
for acetylation.2,3 This reversible process is important
in transcriptional regulation and cell-cycle progression.4
Histone deacetylases (HDACs) are zinc-containing en-
zymes which catalyze the removal of acetyl groups from
the e-amino termini of lysine residues clustered near the
amino terminus of nucleosomal histones and inhibition
of this process is intimately linked to the induction of
gene transcription.5 In addition, HDAC disregulation
has been associated with several cancers and HDAC
inhibitors such as the commercially launched Zolinza
(1)2 and MS-275 (2)6 are undergoing study for the po-
tential treatment of cutaneous T-cell lymphoma and
various hematological malignancies (Fig. 1).7–9 Recent
data highlight the impressive response rates in the treat-
ment of Hodgkin lymphoma.10
O
O
O
N
H
NH2
H
N
HN
2
N
N
1
OH
O
O
O
C
N
NH2
NH2
H
N
H
N
B
A
O
3
4
Figure 1. Zolinza (1), MS-275 (2) and pyridine lead 3.
low molecular weight, but moderately soluble lead,
capable of being elaborated into drug-like compounds.
Although this compound demonstrated activity in a
pooled HDAC enzyme assay,12 cellular activity in an
HCT116 proliferation assay13 could not be reproducibly
demonstrated. We hypothesized that the relatively mod-
est enzyme potency of this compound leads to variable
cellular activity, but this point was not conclusively pro-
ven. However, knowledge of postulated HDAC binding
modes led us to suggest that elaboration of the ‘B’ ring
of 3 with a ‘C’ ring, would provide a template for mak-
ing compounds (4) of greater potency by being capable
of making hydrophobic contacts in the cap group region
of the HDAC protein (Fig. 2).
As part of an ongoing effort to identify novel HDAC
inhibitors, compound 311 was identified as an active,
Compounds 7a–e were therefore synthesized by
coupling the amine 6 with substituted benzoic acid
derivatives 5a–e using N-(4,6-dimethoxy-1,3,5-triazin-
2-yl)-N-methylmorpholinium chloride (DMTMM) in
Keywords: HDAC; Histone; Deacetylase; Anticancer; Benzamide.
*
Corresponding authors. Tel.: +44 1625 582828 (D.M.A.); e-mail
addresses: david.andrews@astrazeneca.com; elaine.stokes@
astrazeneca.com
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.041

D. M. Andrews et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2580–2584
2581
ally further subjected to SCX cartridge ion-exchange
chromatography to obtain the free base (Fig. 3).
Compounds 8a–e validated our hypothesis by showing a
significant increase in potency compared to the starting
hit 3, albeit with a commensurate increase in logD
(data not shown). We therefore sought to maintain or
increase potency and to improve drug-like properties
by appending a solubility-enhancing group to the ‘C’
ring (Table 1).
Our synthetic strategy relied upon the ability to access
the key boronate ester 916 in good yield and couple it
in a Suzuki reaction with benzyl 4-{[(trifluoromethyl)sul-
fonyl]oxy}-3,6-dihydropyridine-1(2H)-carboxylate 1017
or commercially available 2-chloro-1,3-thiazole-5-carb-
aldehyde. Both reactions proved amenable to scale-up
on multi-gramme scale, yielding 82% and 68% of the
late-stage, divergent intermediates 11 and 12, respec-
tively. Compound 12 could be used in a series of reduc-
tive aminations in multiple parallel synthesis format
with a large set of amines; as an alternative, reduction
to 13, mesylation and amine displacement were also
highly effective. Compound 11 was simultaneously re-
duced and deprotected using H2/10% Pd/C to yield the
free amine 14 which could be alkylated or subjected to
reductive amination, again in multiple parallel synthesis
format. The final test compounds were liberated in 70–
95% yields by Boc deprotection using TFA/DCM and
were characterized by LC–MS and proton NMR.
Approximately 300 compounds were made and tested
using this synthetic strategy (Figs. 4 and 5).
Figure 2. Crystal structure of Zolinza bound to HDAC-like protein.14
acetonitrile at room temperature, followed by overnight
deprotection using HCl in dioxane. Compounds 8a–e
generally precipitated from the reaction mixture, and
were isolated by filtration and washing with a non-polar
solvent. Where free bases were required, they were usu-
NHBoc
R
X
H2N
+
a)
The synthetic library of thiazole and piperidine phenyl
benzamides were subjected to testing in the primary as-
say cascade. Gratifyingly, a large proportion of the
compounds synthesized met the potency criteria and
were therefore selected for progression into other dis-
criminating in vitro cascade assays. Acquired long
QT syndrome causes significant cardiac side effects
and represents a major problem in clinical studies of
drug candidates. One of the reasons for development
of arrhythmias related to long QT is inhibition of the
human ether-a-go-go-related-gene (hERG) potassium
5a-e
CO2H
6
R
R
X
NHBoc
X
NH2
H
H
N
N
b)
7a-e
O
O
8a-e
Figure 3. Synthesis of benzamide derivatives. (a) DMTMM, acetoni-
trile, rt, 50–82%; (b) 4 M HCl/dioxane, rt, 20 h, 42–82%. For the
identity of R, see Table 1.
Table 1. Enzyme and cell potency of benzamide HDAC inhibitors
15
Compound
R
H
X
N
Mean logD
HDAC enzyme pIC50
4.29
HDAC1 enzyme pIC50
HCT116 proliferation pIC50
—
3
1.0
5.61
S
8a
C
2.3
5.02
6.91
5.74
N
*
O
8b
8c
C
C
1.2
4.78
5.23
NT
5.13
6.08
N
*
N
<0.5
6.86
*
N
8d
8e
C
C
2.51
NT
4.57
4.44
7.35
NT
6.36
5.63
*
*
N
N

2582
D. M. Andrews et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2580–2584
R1
6.0
N
HDAC Piperidine
Potent HDAC
& Potent hERG
S
NH2
15a-c
HDAC Thiazole
H
5.8
5.6
5.4
N
Weak HDAC &
Potent hERG
O
d), i)
e), i)
O
N
HO
N
S
HNBoc
S
HNBoc
H
N
H
N
12
13
5.2
5.0
O
O
c)
b)
O
B
O
Cbz
HNBoc
HNBoc
N
10
OTf
N
9
4.8
4.6
O
a)
Cbz
N
HN
14
HNBoc
H
N
H
N
11
5.4
5.6
5.8
6.0
6.2
6.4
6.6
6.8
HCT116 Proliferation Mean pIC50
O
N
O
f)
R2
Figure 5. HCT116 cell potency versus hERG pIC50 for a series of
thiazole and piperidine-based benzamide HDAC inhibitors.
g) or h), i)
NH2
16a-e
H
N
chrome P450 isoform. A concern arising from P450
inhibition by an HDAC inhibitor is possible drug
interactions that can be the result of abrogation of
the P450 pathway(s) of metabolism causing toxicity
due to elevated exposures of other drugs metabolized
by these pathways. Compound 16a shows pIC50 be-
tween 5 and 6 for 3A4, 2D6, 2C9 and 2C19, whereas
16b–e demonstrate pIC50 < 5 against all isoforms
tested. The most likely explanation of the increased
P450 inhibition by 16a is its higher flexibility, provid-
ing greater opportunity for it to adopt a conformation
for cytochrome binding, coupled with its increased
logD.
O
Figure 4. Synthesis of thiazole and piperidine-based benzamide
derivatives. (a) PdCl2(dppf), NaHCO3, DME, H2O, 60 ꢁC, 5–9 h,
82%; (b) 2-chloro-1,3-thiazole-5-carbaldehyde, PdCl2(dppf), NaHCO3,
DME, H2O, 60 ꢁC, 5–9 h, 68%; (c) NaBH4, MeOH, water, 10 ꢁC to rt,
18 h, 97%; (d) piperidine, NaBH(OAc)3, DCM, rt, 3 h, H2O 80%; (e)
MsCl, DCM, Et3N, 30 ꢁC, cool to 10 ꢁC, add ethylamine or propyl-
amine in THF, rt, 22 h 49–60%; (f) 10% Pd/C, MeOH, H2, 5 bar,
50 ꢁC, 2 h 81%; (g) R2CHO, NaBH(OAc)3, DCM, rt, 3 h, H2O 50–
80%; (h) R2 alkyl halide, DIPEA, IPA, reflux, 4 h 50–82%; (i) DCM,
TFA, rt, 30 min, SCX2 cartridge elution–DCM, MeOH, 2 M NH3/
MeOH.
channel.18 Therefore, early identification of hERG
affinity is becoming increasingly important. We there-
fore chose to profile the compounds early in the cas-
cade using a high-throughput patch-clamp hERG
assay19; plotting the HCT116 proliferation pIC50
against hERG pIC50 allowed rapid identification of
compounds worthy of progression to more detailed
in vitro and in vivo studies. Compounds of high po-
tency and low hERG liability are boxed in blue in Fig-
ure 5; the results of detailed testing for several of these
compounds are described in Tables 2–4.
The thiazole 15b shows good in vivo properties, clear-
ance being higher than desirable, but quite reasonable
in light of its very high plasma free levels. Of the three
compounds, however, it demonstrates the lowest expo-
sure after oral dosing to the nude mouse.
Compound 16d demonstrates superior exposure in the
nude mouse and was therefore progressed to a nude
mouse xenograft study, in which a statistically signifi-
cant difference in tumor weight can be demonstrated,
by comparison to sham-dosed control animals (Fig. 6).
An excellent dose–response profile was observed, with
significant growth inhibition observed at 12.5 mg/kg
orally.
Compounds 15b, 16b and 16d offer optimal in vitro effi-
cacy and non-efficacy characteristics and were therefore
selected for further in vivo study.
In summary, 16d20 therefore emerged as the compound
demonstrating the most favorable balance of efficacy
and non-efficacy properties, along with very desirable
in vivo DMPK properties across the species tested.
The final leading series of compounds are >1000-fold
more potent than the initial screen hit, an improvement
in potency which was achieved with a concomitant sig-
nificant improvement in all the main non-efficacy prop-
erties. Finally, 16d can be assembled in a highly
convergent manner from readily available precursors,
enabling further detailed studies, which will be reported
in due course.
The thiazole 15a demonstrates an encouraging increase
in potency; however, its solubility is still modest
(37 lM at pH 7.4). The secondary amines 15b and 15c
were marginally less potent in the cellular proliferation
assay, but showed solubility greater than 500 lM with
minimal hERG inhibition (data not shown).
Compound 16a emerged as one of the most potent
piperidine-based inhibitors in the cellular assay (Table
3), however, unusually for this series, the compound
shows significant inhibition of more than one cyto-

D. M. Andrews et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2580–2584
Table 2. Enzyme and cell potency of thiazole benzamide HDAC inhibitors
2583
Compound
R1=
MW
393
HDAC1 enzyme pIC50
7.66
HCT116 proliferation pIC50
6.84
ACD logD
*
N
15a
2.94
15b
15c
CH3CH2NH–
CH3(CH2)2NH–
352
366
7.42
7.56
6.69
6.73
1.79
2.4
Use R1 definition in conjunction with Figure 4.
Table 3. Enzyme and cell potency of piperidine benzamide HDAC inhibitors
Compound
R2=
Method
HDAC1 enzyme pIC50
HCT116 proliferation pIC50
6.52
Mean logD
F
O
16a
h
7.17
2.76
N
H
*
N
O
16b
g
7.07
6.32
1.53
*
O
16c
16d
h
h
7.24
7.46
6.38
6.44
2.31
2.28
N
*
NH
O
*
NH
16e
h
7.51
6.36
1.99
O
*
Use R2 definition in conjunction with Figure 4.
Table 4. DMPK properties of the HDAC inhibitors 15b and 16d in Han Wistar rat, nude mouse and beagle dog
Compound/species Dose IV CL t1/2 IV Vss
AUC(0Àt)
[lmol/kg] [ml/min/kg] [h] [l/kg] IV norm [h kg/L] [lmol/kg]
Dose oral t1/2 oral [h] AUC(0Àt) oral Plasma % free
norm [h kg/L]
15b/rat (HW)
15b/mouse (nude)
16b/rat (HW)
5.0
—
38
—
49
—
16
36
7
5.2
7.3
—
0.44
—
70.9
70.9
10.0
54.8
54.8
54.8
3.0
2.1
1.1
2.1
3.8
3.7
3.2
7.2
0.31
0.06
0.07
0.23
0.74
0.59
8.4
54
—
28
40
21
—
33
—
5.0
—
1.6
3.1
—
0.34
—
16b/mouse (nude)
16d/rat (HW)
—
2.5
5.0
0.8
1.7
4.8
3.5
5.0
4.2
1.08
0.46
4.9
16d/mouse (nude)
16d/dog (beagle)
10.2
Acknowledgments
Vehicle
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
We thank Andrew Mortlock and Keith Gibson for sup-
port, encouragement and advice. We thank Graham
Sproat & Heather Haye for cloning of HDAC1 and pro-
vision of HDAC enzyme; Helen Pointon, Greg Carr,
Graham Duncan, Jon Eden, Neil Findlay, Euan Ford-
yce, Mark Graham, Galith Karoutchi, Mark Maybury,
Steven Raw, and Andy Turner for provision of interme-
diates and final test compounds; Graham Sproat, Helen
Cotterill, Natalie Byrne and Neil Hewitt for biochemical
test data; Ross Chawner, Alison Hunter, Clare King,
Janet Smallwood and Rebecca Watson for physical
measurements and rat, mouse and dog DMPK data.
Compound 16d 62.5 mg/kg
Compound 16d 50 mg/kg
Compound 16d 25 mg/kg
Compound 16d 12.5 mg/kg
0
2
4
6
8
10
12
14
16
18
References and notes
Days of dosing
1. (a) Beato, M. J. Mol. Med. 1996, 74, 711; (b) Wolffe, A. P.
Nature 1997, 387, 16.
Figure 6. Nude mouse A549a xenograft efficacy for 16d when
administered orally once daily (5 d/week) for 18 days.

2584
D. M. Andrews et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2580–2584
2. Grant, S.; Easley, C.; Kirkpatrick, P. Nat. Rev. Drug Disc.
2007, 6, 21.
3. Marchion, D. C.; Bicaku, E.; Daud, A. I.; Sullivan, D. M.;
Munster, P. N. Cancer Res. 2005, 65, 3815.
4. Lindemann, R. K.; Gabrielli, B.; Johnstone, R. W. Cell
Cycle 2004, 3, 779.
5. Brittain, D.; Weinmann, H.; Ottow, E. Annu. Rep. Med.
Chem. 2007, 42, 337.
was added to each well and the plates were reincubated for
3 h. Absorbance was then measured on a 96-well plate
reader at 490 nm. The IC50 values for HDAC inhibitors
were determined by performing dose–response curves with
individual compounds and determining the concentration
of inhibitor producing 50% decrease in maximal signal
(diluent control). For other examples of the use of
HCT116 cells in the evaluation of HDAC inhibitors, see
also: (a) Nagaoka, Y.; Maeda, T.; Kawai, Y.; Nakashima,
D.; Oikawa, T.; Shimoke, K.; Ikeuchi, T.; Kuwajima, H.;
Uesato, S. Eur. J. Med. Chem. 2006, 41, 697; (b)
Remiszewski, S. W.; Sambucetti, L. C.; Bair, K. W.;
Bontempo, J.; Cesarz, D.; Chandramouli, N.; Chen, R.;
Cheung, M.; Cornell-Kennon, S.; Dean, K.; France, G.
D.; Green, M. A.; Howell, K. L.; Kashi, R.; Kwon, P.;
Lassota, P.; Martin, M. S.; Mou, Y.; Perez, L. B.; Sharma,
S.; Smith, T.; Sorensen, E.; Taplin, F.; Trogani, N.;
Versace, R.; Walker, H.; Weltchek-Engler, S.; Wood, A.;
Wu, A.; Atadja, P. J. Med. Chem. 2003, 46, 4609; (c) Kim,
D.-K.; Lee, J. Y.; Kim, J.-S.; Ryu, J.-H.; Choi, J.-Y.; Lee,
J. W.; Im, G.-J.; Kim, T.-K.; Seo, J. W.; Park, H.-J.; Yoo,
J.; Park, J.-H.; Kim, T.-Y.; Bang, Y.-J. J. Med. Chem.
2003, 46, 5745; (d) Moradei, O. M.; Mallais, T. C.;
Frechette, S.; Paquin, I.; Tessier, P. E.; Leit, S. M.;
Fournel, M.; Bonfils, C.; Trachy-Bourget, M.-C.; Liu, J.;
Yan, T. P.; Lu, A.-H.; Rahil, J.; Wang, J.; Lefebvre, S.; Li,
Z.; Vaisburg, A. F.; Besterman, J. M. J. Med. Chem. 2007,
50, 5543.
6. Suzuki, T.; Ando, T.; Tsuchiya, K.; Fukazawa, N.; Saito,
A.; Mariko, Y.; Yamashita, T.; Nakanishi, O. J. Med.
Chem. 1999, 42, 3001.
7. (a) Olsen, E. A.; Kim, Y. H.; Kuzel, T. M.; Pacheco, T. R.;
Foss, F. M.; Parker, S.; Frankel, S. R.; Chen, C.; Ricker,
J. L.; Arduino, J. M.; Duvic, M. J. Clin. Oncol. 2007, 25,
3109; (b) O’Connor, O. A.; Heaney, M. L.; Schwartz, L.;
Richardson, S.; Willim, R.; MacGregor-Cortelli, B.;
Curly, T.; Moskowitz, C.; Portlock, C.; Horwitz, S.;
Zelenetz, A. D.; Frankel, S.; Richon, V.; Marks, P.; Kelly,
W. K. J. Clin. Oncol. 2006, 24, 166; (c) Kelly, W. K.;
Richon, V. M.; O’Connor, O. Clin. Cancer Res. 2003, 9,
3578.
8. Kummar, S.; Gutierrez, M.; Gardner, E. R.; Donovan, E.;
Hwang, K.; Chung, E. J.; Lee, M. J.; Maynard, K.;
Kalnitskiy, M.; Chen, A.; Melillo, G.; Ryan, Q. C.;
Conley, B.; Figg, W. D.; Trepel, J. B.; Zwiebel, J.;
Doroshow, J. H.; Murgo, A. J. Clin. Cancer Res. 2007, 13,
5411.
9. Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. J.
Med. Chem., 2008. doi:10.1021/jm7011408.
10. Younes, A.; Fanale, M.; Pro, B.; McLaughlin, P.;
Neelapu, S.; Fayad, L.; Wedgwood, A.; Dubay, M.;
Backstrom, J.; Martell, R. E. J. Clin. Oncol. (Meeting
Abstracts) 2007, 25, 8000.
14. Finnin, M. S.; Donigian, J. R.; Cohen, A.; Richon, V. M.;
Rifkind, R. A.; Marks, P. A.; Breslow, R.; Pavletich, N. P.
Nature 1999, 401, 188.
15. HDAC inhibitors were screened against recombinant
human HDAC1 produced in Hi5 insect cells. The enzyme
was cloned with a FLAG tag at the C-terminal of the gene
and affinity purified using Anti-FLAG M2 agarose. The
deacetylase assays were carried out in a 50 lL reaction.
HDAC1 (75 ng of enzyme) diluted in 15 lL of reaction
buffer (25 mM Tris–HCl (pH 8), 137 mM NaCl, 2.7 mM
KCl, 1 mM MgCl2) was mixed with either buffer alone
(10 lL) or buffer containing compound (10 lL) for 30 min
at ambient temperature. The reaction was initiated by
addition of 25 lL of acetylated histone H4 peptide
(25 lM) (KI 174 Biomol) diluted in buffer and incubated
for one hour at ambient temperature. The remaining
protocol is as for the pooled enzyme (vide supra).
16. Andrews, D. M.; Gibson, K. M.; Graham, M. A.;
Matusiak, Z. M.; Roberts, C. A.; Stokes, E. S. E.; Brady,
M. C.; Chresta C. M. Bioorg. Med. Chem. Lett., in press.
17. Eastwood, P. R. Tetrahedron Lett. 2000, 41, 3705.
18. Thai, K.-M.; Ecker, G. F. Bioorg. Med. Chem. 2008, 18,
1359.
11. Kabanos, T. A.; Tsangaris, J. M. J. Coord. Chem. 1984,
13, 89.
12. In vitro enzyme assay of pooled histone deacetylases from
nuclear extracts prepared from the human cervical cancer
cell line HeLa: assays were carried out in a 40 lL reaction.
2.5 lg of nuclear extract diluted in 15 lL of reaction buffer
(25 mM Tris–HCl (pH 8), 137 mM NaCl, 2.7 mM KCl,
1 mM MgCl2) was mixed with either buffer alone (5 lL) or
buffer containing compound (5 lL) for 30 min at ambient
temperature. 25 lM of fluor de lys substrate (Biomol)
diluted in 20 lL of buffer was then added to initiate the
reaction and incubated for 1 h at ambient temperature.
The reaction was stopped by addition of an equal volume
(40 lL) fluor de lys developer (Biomol) containing
trichostatin A at 2 lM. The reaction was allowed to
develop for 30 min at ambient temperature and then
fluorescence measured at an excitation wavelength of
360 nm and an emission wavelength of 465 nm. IC50
values for HDAC enzyme inhibitors were determined by
performing dose–response curves with individual com-
pounds and determining the concentration of inhibitor
producing 50% decrease in the maximal signal (no
inhibitor control).
19. Schroeder, K.; Neagle, B.; Trezise, D. J.; Worley, J. J.
Biomol. Screen. 2003, 8, 50.
20. (DMSO-d6) 1.13 (t, 3H, CH3CH2), 1.73 (m, 4H, piperidine
3-CH2), 2.10 (t, 2H, piperidine 2-axial CH), 2.60 (m, 1H,
piperidine 4-CH), 2.94 (d, 2H, piperidine 2-equiv CH), 3.28
(m, 2H, CH3CH2) 3.55 (s, 2H, phenyl-CH2-N), 4.85 (s, 2H,
NH2), 6.59 (m, 1H, CONHCHCH), 6.77 (d, 1H, NH2CH),
6.96 (m, 1H, NH2CHCH), 7.17 (d, 1H, CONHCH), 7.39
(m, 4H, piperidine-phenyl o-CH and ethylbenzamide
m-CH), 7.80 (d, 2H, ethylbenzamide o-CH), 7.90 (d, 2H,
piperidine-phenyl m-CH), 8.38 (t, 1H, CH3CH2NH), 9.55
(s, 1H, CONH); mass spectrum: M+H+ 457.
13. HCT116 cells are adherent epithelial human colon carci-
noma cell lines. Inhibition of proliferation in whole cells
was assayed using the Promega cell titer 96 aqueous
proliferation assay (Promega #G5421). The HCT116 cells
were seeded in 96-well plates at 1 · 103 cells/well, and
allowed to adhere overnight. They were treated with
inhibitors for 72 h. The 20 lL of the tetrazolium dye MTS