Studies of the metabolic stability in cells of 5-(trifluoroacetyl)thiophene-2-carboxamides and identification of more stable class II histone deacetylase (HDAC) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.10.041

5-(Trifluoroacetyl)thiophene-2-carboxamides were found to be potent and selective class II HDAC inhibitors. This paper describes their further development and the investigation on the cause for the lack of cell-based activity. A rapid screening assay was set up which enabled the identification of more metabolic stable compounds as potent and selective class II HDAC inhibitors.

Full text of DOI:10.1016/j.bmcl.2008.10.041

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6078–6082
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Studies of the metabolic stability in cells of 5-(trifluoroacetyl)thiophene-2-
carboxamides and identification of more stable class II histone deacetylase
(HDAC) inhibitors
*
Rita Scarpelli , Annalise Di Marco, Federica Ferrigno, Ralph Laufer, Isabella Marcucci, Ester Muraglia, Jesus
M. Ontoria, Michael Rowley, Sergio Serafini, Christian Steinkühler, Philip Jones
IRBM—Merck Research Laboratories Rome, Via Pontina km 30,600, Pomezia 00040, Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
5-(Trifluoroacetyl)thiophene-2-carboxamides were found to be potent and selective class II HDAC inhib-
itors. This paper describes their further development and the investigation on the cause for the lack of
cell-based activity. A rapid screening assay was set up which enabled the identification of more metabolic
stable compounds as potent and selective class II HDAC inhibitors.
Received 29 August 2008
Revised 7 October 2008
Accepted 7 October 2008
Available online 12 October 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Histone deacetylase
HDAC
Trifluoroacetyl ketones
Histone deacetylases (HDACs) are a class of Zn2+ metalloen-
zymes involved in the deacetylation of histones, transcription fac-
tors, and other proteins in eukaryotic cells.1 HDAC inhibition
causes hyperacetylation of histones which leads to a more open
chromatin structure, thereby facilitating gene transcription and
ultimately leading to cell-growth arrest, differentiation, and apop-
tosis. The development of specific HDAC inhibitors (HDACi) repre-
sents a new strategy in cancer therapy.2
In an effort to develop a second generation HDACi with en-
hanced efficacy and larger therapeutic window, we were interested
in targeting a more restricted set of HDAC isoforms. In particular,
RNAi knock-down experiments were performed against all 11 class
I and II HDACs in three different cancer cell lines (HeLa, HCT116,
and A549)8 where it was observed that knock-down of HDAC 4
was sufficient to inhibit cell proliferation. Consequently a program
was initiated to develop HDAC 4 or selective class II HDAC inhibi-
tors which would be expected to show growth inhibition in the
same cancer cell lines.
As previously reported, a series of 5-(trifluoroacetyl)thiophene-
2-carboxamides was identified as a novel class of potent and selec-
tive class II HDAC inhibitors.9 These compounds typically display
around 10-fold selectivity for class II HDACs (HDAC 4+6) over their
class I HDACs (HDAC 1+3) counterparts (Table 1).10,11
In human, Zn2+ dependent HDACs are divided in two major sub-
classes (class I and class II), based on sequence homology to the
yeast HDACs. Class I HDACs include HDAC 1, 2, 3, and 8 which
are related to yeast RPD3 deacetylase. Class II HDACs are related
to yeast Hda1 and include HDAC 4, 5, 6, 7, 9, and 10. All these
HDACs possess
a highly conserved zinc-dependent catalytic
domain.3
Both hydroxamic acid-based HDACi, for example, vorinostat
(SAHA)4 and PDX1015 and non-hydroxamic acid-based HDACi,
for example, the 2-aminophenylamide MS-2756 and the dithiol
FK2287 are currently in the clinic. Most of them hit a subset of both
class I and class II or, as in the case of the 2-aminophenylamides,
show selectivity versus class I HDACs. All of them elicit similar ad-
verse effects, mainly fatigue, nausea, vomiting, and diarrhea that
become dose-limiting in clinical trials.
Unfortunately, most of these derivatives lacked cell-based
activity as they showed no antiproliferative effect against HeLa
and A549 cell lines, and failed to show inhibition of either histone
H3 or
a-tubulin deacetylation in HCT116 cells, the cellular sub-
strates of HDAC 1 and 6, respectively.12
In the light of these results, it was necessary to establish the
cause for the lack of cellular activity. Several possibilities were con-
sidered: (a) the lack of cell penetration and/or efflux of these com-
pounds from cells13; (b) high binding to plasma protein of the cell
incubation medium; (c) metabolic degradation of compounds dur-
ing incubation with cells; or (d) lack of activity on HDAC 1+3,
assuming that these compounds can enter into cells and are stable,
* Corresponding author. Tel.: +39 0691093248; fax: +39 0691093654.
E-mail address: rita_scarpelli@merck.com (R. Scarpelli).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.041

R. Scarpelli et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6078–6082
6079
Table 1
the whole cell system would suggest these class II HDACi suffer
from poor cell permeability.
Activity of 5-(trifluoroacetyl)thiophene-2-carboxamides on the various HDAC
isoforms.
All five analogs were shown to be low micromolar inhibitors of
CYP2C9 in microsomes, as determined by the release of tritium as
tritiated water that occurs upon CYP2C9-mediated 40-hydroxyl-
ation of diclofenac labeled with tritium in the 40 position.15
When those compounds were tested in human hepatocytes
R
N
CF3
R'
S
O
O
similar IC50 were observed, for example, 5
in hepatocytes for compound 5 and 1.8 M in both HLM and hepa-
tocytes for compound 6. The one compound that showed a modest
shift was compound 7 with IC50 of 1.4 M in HLM and 11 M in
lM in HLM and 8 lM
H
N
Ph
(S)
Ph
N
N
N
Ph
N
l
N
4
1
2
3
l
l
–NRR0
HDAC IC50 (nM)a
hepatocytes. In order to exclude that the lack of cellular activity
of these compounds was due to high plasma protein binding, the
inhibition of CYP2C9 activity in hepatocytes was also determined
in the presence of 10% fetal bovine serum (FBS), a component of
the cell culture medium used for proliferation assays. The addition
of serum to the cells caused no significant effect on IC50 (Table 2).
Together these results suggested that the lack of activity in cell-
based assays is not due to a cell permeability issue and/or high
plasma protein binding, with the caveat that these data were gen-
erated in hepatocytes, whereas deacetylation and proliferation as-
says were carried out in HeLa, A549, and HCT116 cell lines.
We next focused our attention on the stability of these HDACi in
cell culture. It has been previously documented that electrophilic
1
3
4WT14
6
1
2
3
4
890
560
510
170
150
300
230
150
130
310
5000
1300
4700
9400
1400
>5000
a
IC50 is the concentration of drug required for 50% inhibition in vitro; values are
means of >2 experiments with standard deviations <30%.
and that class II HDACs are not involved in the control of cell
proliferation.
Consequently, a focused research was initiated to understand
the causes for the lack of antiproliferative effects and remedy
any untoward observations.
ketones, such as trifluoromethyl ketones,
a
a-keto heterocycles and
-keto esters, can be rapidly reduced by carbonyl reductases.16
As a part of any lead optimization program routine counter
screening for cytochrome P (CYP)450 inhibition is performed on
all compounds synthesized. Five 5-(trifluoroacetyl)thiophene-2-
carboxamides were identified that were relatively potent class II
This phenomenon seems to be especially pronounced in straight-
chain alkyl-linked compounds, both peptidic and non-peptidic.17
Representative
5-(trifluoroacetyl)thiophene-2-carboxamides
M in cell culture
were incubated with HCT116 cells at 1 and 5
l
HDACi, but displayed little or no inhibition of
tion in cells (Table 2). These five compounds showed modest
CYP2C9 inhibition in human liver microsomes (HLM) at 10 M. Gi-
ven this observation, the level of CYP2C9 inhibition in HLM was
compared to that seen in human hepatocytes as any shifts in the
IC50 between the sub-cellular microsome fractions compared to
a
-tubulin deacetyla-
medium containing 10% FBS for 24 h and aliquots were taken at
different times and analyzed by LCMS/MS; control incubation
was also performed in cell culture medium without cells. All tested
compounds were stable in culture medium, but in the presence of
HCT116 cells, rapid degradation of compounds was observed, typ-
ically with t1/2 < 2 h. Degradation of tested compounds occurred in
l
Table 2
Effect on the CYP2C9 activity in human liver microsomes (HLM) versus human hepatocytes of 5-(trifluoroacetyl)thiophene-2-carboxamides.
R
N
CF3
R'
S
O
O
H
N
H
N
N
X
Y
N
N
5; X = H; Y =OMe
7
8
6; X = OMe; Y = Cl
N
N
N
N
N
O
N
Ph
9
10
a
b
c
c
–NRR0
HDAC IC50 (nM)
Tub-Ac EC50
(
lM)
CYP2C9 in HLM IC50
(l
M)
CYP2C9 in hepatocytes IC50 (lM)
4WT
6
No serum
10% FBS
5
6
7
8
9
480
530
170
90
170
50% inh at 50
l
M
l
M
5( 0.9)
1.8( 0.4)
1.4( 0.2)
13( 4)
8( 1)
8( 4)
ND
17( 4)
3( 1)
5( 0.5)
175
360
260
160
NA up to 10
l
1.8( 0.3)
11( 3)
10( 2)
8( 0.8)
50% inh at 25
M
10
lM
70
NA up to 25
l
M
5( 0.3)
a
Values are means of >2 experiments with standard deviation <30%.
b
Results were calculated assuming the signal obtained treating cells with 10
treatment; NA, not active.
l
M of SAHA as the 100% activation signal. The activation of
a
-tubulin is measured after 24 h
c
MTS assay showed no toxicity of these derivatives which were stable in the cells; standard deviation is given in parentheses; ND, not determinate.

6080
R. Scarpelli et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6078–6082
a timeframe which was much shorter than the duration of prolifer-
ation and deacetylation assays, 72 h and 24 h, respectively. The
corresponding inactive alcohol was identified as a major metabo-
lite after incubation with HCT116 cells. Conversion seemed to be
quantitative as shown for compound 10, as a representative exam-
ple, which was incubated with two different cell lines HCT116 and
HeLa cells. The amount of inactive alcohol after 6 h of incubation
with HCT116 cells was about equal to the initial amount of parent
compound (Fig. 1).18
Similar results were obtained when compound 10 was incu-
bated with HeLa cells in cell culture medium containing 10% FBS.
In addition, no significant amount of the product of amide bond
hydrolysis was detected. These results suggest that the lack of cel-
lular activity of these compounds is due to metabolic instability.
Compound 9, which was the most potent and selective class II
HDAC inhibitor, unfortunately was very unstable in HCT116 cells
with a t1/2 < 2 h (Table 3). This is in agreement with the lack of inhi-
10
8
no cofactor
NADH
6
NADPH
4
2
0
0
20
40
60
80
14
12
10
8
no cofactor
NADH
6
4
bition of either histone H3 or
a-tubulin deacetylation in cell cul-
NADPH
2
ture. On the other hand, compounds 8 and 10 which were the
most stable compounds with t1/2 = 5 h and 3.5 h, respectively, re-
0
0
20
40
Time (min)
60
80
sulted to be efficient inhibitors of
a-tubulin acetylation with
IC50 = 10 M and 12 M. Inhibition of histone H3 deacetylation
l
l
was seen only at higher concentration.
Figure 2. Conversion of compound 10 into alcohol metabolite in S9 fraction of
In order to increase throughput of the HCT116 stability assay it
was shown that the S9 sub-cellular fraction of HCT116 cells was
suitable as enzyme source for a rapid screening of these trifluoro-
methyl ketones. The assay was validated using compound 10, as
representative compound, and other compounds and carried out
in presence of NADPH which is the preferred cofactor for reduction
of these trifluoromethyl ketones (Fig. 2).19 Interestingly, we ob-
HCT116 cells in the presence of NADH or NADPH.
Parent
14
12
10
8
Metabolite
6
N
N
N
N
in cells
4
N
CF3
N
CF3
OH
S
S
2
O
O
O
10
inactive alcohol metabolite
0
Parent HCT116
Parent HeLa
8
7
6
5
4
3
2
1
0
Alcohol HCT116
Alcohol HeLa
Figure 3. Effect of carbonyl reductase inhibitors on the reduction of compound 10
(10
M) in S9 fraction of HCT116 cells.20
l
R
N
1) 2N KOH (2 eq)/MeOH,
H2O, 40°C, 12h
EtO
CF3
CF3
R'
S
S
O
O
O
O
2) PS-DCC (4 eq), DCM,
R'RNH, rt, 12h,
0
5
10
15
20
25
30
Time (hours)
then MP-CO3 (2 eq),
rt, 48h
Figure 1. Stability of compound 10 and formation of the corresponding inactive
alcohol in HCT116 and HeLa cells.
Scheme 1. Synthesis of 5-(trifluoroacetyl)thiophene-2-carboxamides.
Table 3
Stability of 5-(trifluoroacetyl)thiophene-2-carboxamides in HCT116 cells.
a
–NRR0
HDAC IC50 (nM)
HCT116 Stability t1/2 (h)
Tub-Ac EC50
NA up to 10
(
l
M)
H3 EC50 (lM)
1
3
4WT
6
6
7
8
9
4400
830
1600
1300
580
>10
840
1500
2800
670
l
M
530
170
90
175
360
260
160
90
2
3
5
2
l
M
lM
NA up to 10
NA up to 25
l
l
M
M
50% inh at 25
10
lM
>35
lM
70
NA up to 25
12
l
M
NA up to 25
l
M
10
100
3.5
40%inh at 50 lM
a
Values are means of >2 experiments with standard deviations <30%; NA, not active.

R. Scarpelli et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6078–6082
6081
Table 4
Metabolic stability in S9 fraction of HCT116 cells.
R
N
CF3
N
R'
S
O
O
NH
Cl
Y
CF3
X
O
O
11
12; X = S, Y = N
13; X = N, Y = CH
O
N
N
CF3
N
H
CF3
N
S
O
O
O
14
15
a
–NRR0
HDAC IC50 (nM)
S9 Stability t1/2 (min)
Tub-Ac EC50 (lM)
1
4WT
6
1
890
580
550
1600
NA up to 10
NA up to 10
7600
510
100
100
13
NA up to 10
NA up to 10
75
230
90
190
410
580
15
20
15
20
15
>120
>120
ND
12
NA up to 10
NA up to 10
ND
ND
6.5
10
11
12
13
14
15
lM
lM
l
l
M
M
l
M
lM
3320
650
a
Values are means of >2 experiments with standard deviations <30%; NA, not active; ND, not determinate.
served that in general the rank order with the selected compounds
was the same with a good correlation between the two assays.
Given that selective class II selective inhibitors had been
developed, a rapid way to assess the importance of class II HDACs
for cell proliferation could be to stabilize those compounds using
a reductase inhibitor. Studies were carried out on the effect of
drugs that inhibit various classes of carbonyl reductase on the
reduction of compound 10 in HCT116 S9 fractions. In particular,
flufenamic acid and phenolphthalein were used as aldo-ketone
reductase (ADK) inhibitors, while quercetin, menadione, rutin,
and ethacrynic acid were used as short-chain dehydrogenase/
reductase (SDR) inhibitors.20 The choice of carbonyl reductase
inhibitors was somewhat limited to avoid drugs known to have
antiproliferative effects.
Attempts to modulate this rapid reduction via heterocycle var-
iation such as thiazoles and pyrroles offered modest but not suffi-
cient improvements in stability (Table 4, compounds 12 and 13). In
general, those compounds that showed an improved stability suf-
fered from poor class II HDAC activity. We concluded that we were
unable to identify suitable compounds in the amide series.
A more substantial structural change was made by the modifi-
cation of the amide bond to an oxadiazole ring which led to the
identification of compound 15 which, although a weaker HDAC 4
and 6 inhibitor, showed improved metabolic stability.
In summary, we have identified the cause for the lack of cellular
activity of the 5-(trifluoroacetyl)thiophene-2-carboxamides as
being rapid metabolism in cells by carbonyl reductases. A rapid
screening assay was set up in S9 sub-cellular fraction of HCT116
cells in order to identify rapidly more stable compounds. Com-
pound 15 was discovered as a valid lead. Further development of
this finding will be the topic of the accompanying paper.21
However, partial inhibition of reduction of compound 10 was
observed with only flufenamic acid at 10
ethacrynic acid at high concentration 500
l
l
M or menadione and
M (Fig. 3).20 Unfortu-
nately, the partial effects of these drugs was insufficient to test
the effects of our HDACi in long term cellular assays and we needed
a way to further stabilize this series of compounds.
In the light of these results, being unable to stabilize the trifluo-
romethyl ketones with carbonyl reductase inhibitors, we needed to
further explore this series and design new compounds that could
show improved stability in order to demonstrate if inhibitors of
class II HDACs have any antiproliferative effects.
An extensive SAR exploration was performed on the carboxam-
ide moiety of our inhibitors using solid phase supported coupling
reagents, which allowed for the parallel synthesis of a large num-
ber of amides in a short period of time, avoiding the need of HPLC
purification.
The optimized procedure used PS-carbodiimide and excess of
carboxylic acid followed by treatment with MP-carbonate as scav-
enger resin enabled the preparation of compounds with more than
90% purity by LC/MS analysis (Scheme 1).
Acknowledgments
The authors thank Vincenzo Pucci for performing LCMS/MS
analysis and Ottavia Cecchetti for biological screening.
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0.1
lM (limit of quantification).
19. HCT116 cells were homogenized on ice at 4 °C in 20 mM Tris–HCl, pH 7.5,
150 mM KCl, 250 mM sucrose, 1 mM EDTA, and 1 mM DTT. Cell homogenate
was centrifuged at 10,000g, for 30 min at 4 °C and the supernatant (S9 fraction)
was frozen in liquid nitrogen and used for stability experiments. Similar results
were obtained in fresh S9 fraction (data not shown). The reaction was carried
out using S9 fraction (1 mg/ml) in the presence of 1 mM NADPH and a NADPH
regenerating system. The experiment was run in 96-well conical plates, in a
final assay volume of 200
l
l, at 37 °C, under low shaking. The reaction was
l 1N HCl, followed by centrifugation for 10 min at
1690g, and aliquots of the supernatant were diluted with 1 volume of
stopped by addition of 20
l
13. The antiproliferative activity of selected compounds in HCT116 cells was not
affected by inhibitors of MDR1 or MRP (data not shown), suggesting that lack
of cellular activity is not due to active efflux mechanisms.
acetonitrile containing an analytical internal standard and analyzed by LCMS/
MS.
14. Compounds were incubated for 10 min with His-tagged HDAC4 CD (653-1084)
from Escherichia coli in assay buffer (25 mM Tris/HCl, pH 8, 137 mM NaCl,
2.7 mM KCl, 1 mM MgCl2, 0.1 mg/ml BSA), trifluoacetamide substrate solution
was added and left for 1 h at 37 °C and the reaction stopped by adding
developer/TSA solution. The fluorescence was measured at ex.360 nM/
em.460 nM.
20. Compound 10 (5
HCT116 cells, in the presence or absence of the indicated reductase inhibitors:
10 M flufenamic acid (FLU), 50 M phenolphthalein (Phen) (AKR family),
500 M menadione (Menad), quercetin (Querc), rutin and ethacrynic acid
(Ethacry) (SDR family).
21. Muraglia, E.; Branca, D.; Cecchetti, O.; Ferrigno, F.; Orsale, M. V.; Palumbi, M. C.;
Rowley, M.; Scarpelli, R.; Steinkühler, C.; Jones, P. Bioorg. Med. Chem. Lett.,
accompanying paper.
lM) was incubated for 30 min with 1 mg/ml of S9 fraction of
l
l
l
15. Di Marco, A.; Marcucci, I.; Chaudhary, A.; Taliani, M.; Laufer, R. Drug Metab.
Disp. 2005, 33, 359.