2-Trifluoroacetylthiophene oxadiazoles as potent and selective class II human histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.09.076

Trifluoroacetylthiophene carboxamides have recently been reported to be class II HDAC inhibitors, with moderate selectivity. Exploration of replacements for the carboxamide with bioisosteric pentatomic heteroaromatic like 1,3,4-oxadiazoles, 1,2,4-oxadiazo

Full text of DOI:10.1016/j.bmcl.2008.09.076

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6083–6087
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Trifluoroacetylthiophene oxadiazoles as potent and selective class II
human histone deacetylase inhibitors
*
Ester Muraglia , Sergio Altamura, Danila Branca, Ottavia Cecchetti, Federica Ferrigno, Maria Vittoria Orsale,
Maria Cecilia Palumbi, Michael Rowley, Rita Scarpelli, 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:
Trifluoroacetylthiophene carboxamides have recently been reported to be class II HDAC inhibitors, with
moderate selectivity. Exploration of replacements for the carboxamide with bioisosteric pentatomic het-
eroaromatic like 1,3,4-oxadiazoles, 1,2,4-oxadiazoles and 1,3-thiazoles, led to the discovery that 2-triflu-
oroacetylthiophene 1,3,4-oxadiazole derivatives are very potent low nanomolar HDAC4 inhibitors, highly
selective over class I HDACs (HDAC 1 and 3), and moderately stable in HCT116 cell culture.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 29 August 2008
Revised 17 September 2008
Accepted 18 September 2008
Available online 24 September 2008
Keywords:
HDAC
Inhibitors
Trifluoroacetyl
Oxadiazoles
Histone deacetylase (HDAC) and histone acetyltransferase
(HAT) enzymes regulate the acetyl removal/addition to the N-ter-
minal Lys residues on histones. The acetylation status of histones
is directly related to gene expression, with hyperacetylation being
associated with a less condensed chromatin state, a conformation
that induces activation of gene transcription.
single isoforms is still not completely deciphered. Notwith-
standing, the urgency for alternative therapies for cancer has
allowed a rapid development of broad spectrum HDAC inhibi-
tors. Indeed, most of the HDAC inhibitors that are known or
in clinical trials are unselective, or are just partially selective
for one class of HDACs2,3 (e.g. Vorinostat, formerly SAHA, ap-
proved by the FDA in 2006, is an inhibitor of HDAC1, 2, 3
and 6).4 Elucidation of the function of each HDAC subtype
would potentially address toxicity issues and clinical adverse
HDACs are also involved in the deacetylation of other non-his-
tone proteins (HSP90,
scription factors and nuclear receptors (p53, Er
a
-tubuline), and in interactions with tran-
a
).1
The involvement of HDACs in regulating cell proliferation, to-
gether with the experimental evidence that inhibition of HDACs
induces growth arrest or apoptosis in various tumor cell lines,2
has highlighted HDACs as an appealing target for cancer
therapy.
The 18 known human HDACs can be divided into four classes:
class I (HDAC1, 2, 3 and 8), class II, which can be further divided
into the two subclasses HDAC IIa (HDAC 4, 5, 7, 9) and IIb (HDAC
6 and 10) and class IV (HDAC11) are Zn-dependent HDACs, while
class III, or Sirtuins (SIRT1-7), are structurally unrelated NAD-
dependent deacetylases.
effects of HDAC inhibitors. Consequently, there remains a
widely recognized need to identify selective HDAC inhibitors.5
Selective HDAC inhibitors would also provide more focused
cancer therapies and new clinical opportunities. For instance,
it has been demonstrated for HDAC inhibitors to be additive
or synergistic with conventional anticancer chemotherapeutics
and to produce cellular sensitization to ionizing radiation.6
The former seems to be correlated to the inhibition of class
I7 HDACs, while studies on modulation of cellular response
to radiation provide evidence of class II HDAC involvement,
implying a role for the HDAC4 isoform.8
Class I, II and IV HDACs are sensitive to the classical HDAC
inhibitor trichostatin A (TSA), whereas those of class III are insen-
sitive to this inhibitor.1b
The cellular function of each HDAC class is not fully under-
stood and also the specific role in the antitumor activity of the
In our company a research program was initiated aiming at
developing a portfolio of different chemotypes, each with a dif-
ferent HDAC selectivity profile. This effort led to the discovery
of potent and selective class I HDAC inhibitors, belonging to
various chemical classes, including methyl ketone A (Fig. 1)9
and aminobenzamides B10 and C,11 which have been demon-
strated to cause tumor growth inhibition in a HCT-116 xeno-
graft model.
* Corresponding author. Tel.: +39 06 91093276; fax: +39 06 91093654.
E-mail address: ester_muraglia@merck.com (E. Muraglia).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.076

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E. Muraglia et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6083–6087
Table 1
N
(S)
HN
Inhibitory activity of trifluoroacetylthiophene carboxamides against HDAC 4, 1, 3 and
6
N
H
O
O
F3C
O
O
S
O
N
R2
R1
R
N
H
a
A
Compound
R
R1
R2
HDAC IC50 (nM)
4WTb
1
3
6
3
4
5
6
7
Ph
H
H
H
Me
H
H
243
76
170
510
1700
467
320
5300
1800
7100
520
230
9400
1200
>10000
370
360
210
230
310
Ph(R)
Ph(S)
Ph
Me
Me
H
S
Me
H
O
a
Values are means of >2 experiments.
His-tagged HDAC4 catalytic domain (653–1084) from Escherichia coli (Ref. 15).
b
N
H
NH2
R
Methylation of the amide nitrogen as in 6 was also tolerated,
NH
suggesting that the NH was not essential for HDAC4 activity. The
simple ethylamide 7 proved to be only a weak inhibitor of HDAC4
and a submicromolar HDAC6 inhibitor.
Based on the above observations, we reasoned that it should be
possible to substitute the amide bond in 8 (Fig. 3) with pentatomic
heteroaromatic bioisosteres such as 1,2,4-oxadiazoles 9a, 1,3,4-
oxadiazoles 9b and 1,3-thiazole 9c.
H
O
N
O
B: R =
C: R =
O
H2N
Figure 1. Class I selective HDAC inhibitors.
Research directed at the identification of selective class II HDAC
inhibitors, targeting in particular HDAC4, led to the discovery that
5-(trifluoroacetyl)thiophene-2-carboxamides of general structure
1 (Fig. 2) 12 are active site class II HDAC inhibitors,13 with moderate
selectivity over class I HDACs. SAR studies on the amide moiety of
1 showed that various substitutions are tolerated.
We were pleased to see that 1,2,4-oxadiazole 10a retained
HDAC4 potency, and displayed appreciable selectivity over HDAC1
and 3. Submicromolar activity on HDAC6 was also detected. The
isomeric 1,3,4-oxadiazole 10b proved to be 4-fold less active than
10a on HDAC4 WT and less selective, and thiazole 10c was only a
weak class II HDAC inhibitor.
The lack of cellular activity displayed by these inhibitors can be
rationalized in terms of the metabolic instability of the trifluoro-
acetyl ketone moiety of 1. This functionality is readily reduced in
the presence of HCT116 cells to the corresponding alcohol, which
is inactive against HDAC4.12,14 The half-life of the amide inhibitors
1 was shown to vary depending on the nature of the amide substit-
uents, and to be in the range of 20 min to 2 h.
The modest selectivity and the instability in cells prompted us
to seek alternative structures, aiming to obtain more potent and
selective HDAC4 inhibitors. Structural modification could also pos-
sibly address the metabolic instability issue by reducing the mole-
cule recognition by the intracellular carbonyl reductases, thus
decreasing the susceptibility of the trifluoroacetyl group towards
reduction.
We knew that, among amide substitutions, the benzyl and het-
erobenzyl substitutions produced the most active compounds
when screened on class II HDACs (2, Fig. 2).12 The simplest benzyl
derivative 3 (Table 1) showed basically no selectivity over class I
HDACs (HDAC1 and 3). The presence of a methyl in the benzylic
position was tolerated (4 and 5), showing a marked difference be-
tween the two enantiomers, the (S)-enantiomer displaying a high-
er selectivity for HDAC4 over HDAC1 and 3.
The trend of 1,2,4-oxadiazole being a more potent HDAC4
inhibitor than 1,3,4-oxadiazole was confirmed for other pairs of
isomers, such as 11a–b and 12a–b. This evidence prompted us to
concentrate our efforts on a wider investigation of the 3-position
of the oxadiazole nucleus in the 1,2,4-oxadiazole series (Table 3),
with the aim to improve activity and selectivity.
Aromatic substitutions proved to be tolerated both in terms of
the position and the nature of the substituents, as demonstrated
by compounds 13-18 (Table 3). 4-t-Bu derivative (19) showed
micromolar activity on all tested HDAC isoforms.
The introduction of heteroaryl moieties as R, (such as in 20 and
21, R = 2-pyridyl and 3-pyridyl, respectively), essentially retained
the HDAC4 activity of phenyl analog 10a, with somewhat reduced
selectivity over HDAC1. This is in line with what was observed pre-
viously for thiophene derivative 12a. Isomeric 4-pyridyl derivative
22 displayed a marked drop in HDAC4 potency, though maintain-
ing the same level of activity on HDAC6.
F3C
O
F3C
X
S
X
S
O
N
O
X
Me,H
R
R
8
9a, b, c
F3C
O
F3C
O
S
S
O
N
R1
O
N
R''
N
R'
R
O
N
N
N
1
2
S
N
O
Aryl
c
a
b
R, R1 = H, Me
Figure 3. Isosteres of the alkylamide moiety of trifluoroacetylthiophene carbox-
Figure 2. Trifluoroacetylthiophene 2-carboxamides class II HDAC inhibitors.
amides: 1,2,4-oxadiazole, 1,3,4-oxadiazole, 1,3-thiazole derivatives.

E. Muraglia et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6083–6087
6085
Table 3
Interestingly, a methylene spacer between the oxadiazole core
HDAC activity of 1,3,4-oxadiazole derivatives
and thiophene (23) proved very favorable (compare 23 with
12a). 23 displayed 15 nM activity on HDAC4, and a 70-fold selec-
tivity with respect to HDAC1, maintaining high HDAC6 activity.
This result prompted us to further explore alkyl substitutions,
and some of the most relevant results are reported in Table 3. A
variety of different functionalities provided HDAC4 active com-
pounds, such as alkylamide 24 and benzoxazolone 25, which
showed excellent activity on HDAC4 (IC50 = 15 nM), high selectiv-
ity over HDAC1 (>75-fold), and a >20-fold class IIa selectivity.
Phenoxy derivative 26 proved to be the most potent HDAC4 inhib-
itor and displayed the highest selectivity in the series (>600-fold
and >100-fold, respectively, HDAC1/HDAC4 and HDAC6/HDAC4
selectivity). Alkyl and aryl sulfones were also favorable substitu-
tions, providing potent and selective HDAC4 inhibitors, such as
27 and 28 (HDAC4 IC50 = 30 nM, >70-fold HDAC4 vs HDAC1 selec-
tivity), sulfonylthiophene derivative 29 (>100-fold HDAC4/HDAC1
selectivity) and benzyl sulfone 30. Surprisingly, we discovered that
the simple methyl oxadiazole 31 was a very potent HDAC4 inhib-
itor and highly selective over HDAC1. This result, in a head-to-head
comparison with the corresponding carboxamide 7 (Table 1), con-
firmed the high potential of the oxadiazole series as potent and
selective HDAC4 inhibitors.
The most active/selective compounds were then analyzed for
metabolic stability in cellular S9 fraction,14 which has been dem-
onstrated to be a fast and reliable test to rank stability towards
reduction of the trifluoroacetyl moiety to the alcohol. Pleasingly,
some of the oxadiazoles tested (Table 4) showed a half-life signif-
icantly higher than that observed for amide derivatives (amide 6 as
reference),15,16 ranging from 18–20 min (25, 26) to more than 2 h
(17, 27, 31). Compounds 27–30, which showed half-lives ranging
from 35 to >120 min in the S9 preliminary test, and high potency
on HDAC4 (IC50: 30–60 nM), were then tested in the standard
HCT116 cell based assay conditions (in the presence of 10% FBS, Ta-
ble 4). The data obtained confirmed the stability trend observed in
F3C
O
N
S
N
O
R
a
Compound
R
HDAC IC50 (nM)
4WT
1
3
6
13
14
15
16
17
18
19
3-Me-Ph
3-CF3-Ph
4-CF3-Ph
4-F-Ph
2,4-diF-Ph
2,4-diCl-Ph
4-tBu-Ph
280
320
200
220
120
240
6540
>1000
>1000
nd
>1000
>1000
nd
680
17500
>10000
5900
4200
>5000
16600
2450
11000
270
580
3000
3540
>2000
>1000
20
21
22
23
24
90
2800
2200
>10000
990
nd
nd
nd
630
nd
500
840
570
170
710
N
70
N
590
15
N
S
N
60
5900
O
O
N
25
26
15
1170
4800
>1000
>1000
410
760
O
Table 2
Comparison of 1,3,4-oxadiazoles, 1,2,4-oxadiazoles and 1,3-thiazole activities on
HDACs
O
F3C
X
X
S
7
X
O
R
N
N
O
N
O
N
N
N
O
S
S
N
O
O
O
27
28
30
30
2470
2220
>1000
>1000
320
240
c
a
b
O
S
Compound
het
R
HDAC IC50 (nM)a
3
4WT
1
6
10
10
10
11
11
a
b
c
a
b
Ph
Ph
Ph
4-Me-Ph
4-Me-Ph
110
420
1500
110
7400
2970
>10000
6800
>1000
500
nd
nd
>1000
580
330
1350
860
O
S
O
29
30
35
3890
>1000
520
S
270
1800
600
12
a
95
2900
2600
nd
670
160
O
S
O
S
60
75
6550
7600
>1000
7100
700
650
F
12
b
450
500
S
31
Me
See notes a–b in Table 1.
a
a
See notes a–b in Table 1.

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E. Muraglia et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6083–6087
the S9 preliminary test, leading to the identification of compounds
with half-life longer than 10 h, such as 27 (half-life 13 h).
As the cellular substrate for HDAC4 is unknown, no cellular tar-
get engagement assay was available. HDAC6 has been demon-
and 28, were tested for the inhibition of a-tubulin deacetylation
and showed values (27: IC50 = 640 nM, 28: IC50 = 500 nM) that
were compatible with the corresponding values for HDAC6 inhibi-
tion reported in Table 3, thus confirming the ability of these inhib-
itors to inhibit class II HDACs in cells.
strated to be a specific
a a-tubulin
-tubulin deacetylase,17 so that
hyperacetylation in HCT116 cells was used as surrogate activity
All the derivatives described in Tables 2 and 3 were prepared as
shown in Scheme 1 from the common intermediate 5-(trifluoro-
acetyl)thiophene-2-carboxylic acid 32.18 To obtain the 1,2,4-oxadi-
azoles 9a19 a modification of a literature procedure was used.20
Acid 32, upon activation with 1,1’-carbonyldiimidazole, followed
by treatment with the appropriate amidoxime, provided O-acyl
amidoxime 33, which was dehydrated in one pot by treatment
with a further equivalent of CDI and microwave irradiation for
2 min to give 9a.
marker.12 Two of the most stable compounds from Table 4, 27
Table 4
Stability of 1,3,4-oxadiazole derivatives in the presence of S9 fraction and HCT116
cells
Compound
Half life
S9a (min)
HCT116b (h)
When 32 was treated with the appropriate acyl hydrazide and
polymer-supported carbodiimide in amide coupling conditions,
intermediate 34 was obtained, which was cyclized to the corre-
sponding 1,3,4-oxadiazoles 9b21 by treatment with thionyl chlo-
ride under microwave irradiation conditions. Thiazole 10c22 was
prepared reacting amide 35 with Lawesson’s reagent in toluene
under microwave irradiation.
In conclusion, substitution of the carboxamide moiety of triflu-
oroacetylthiophene carboxamides HDAC4 inhibitors with bioisos-
teric pentatomic heteroaromatic 1,2,4-oxadiazole, 1,3,4-
oxadiazole and 1,3-thiazole led to the discovery of 1,2,4-oxadiazole
derivatives as potent low nanomolar inhibitors of HDAC4, highly
selective with respect to class I HDAC1 and 3 isoforms and with
appreciable subclass (class IIa/IIb) selectivity. Some of the oxadiaz-
ole inhibitors displayed significant stability in the presence of
HCT116 cells and oxadiazole 27 demonstrated good inhibition of
class II HDAC, HDAC6, in cells.
6
17
23
25
26
27
28
29
30
31
15c
>120
35
25
18
>120
45
44
35
>120
13
12
6
5.5
a
S9 fraction of HCT116 cells, in the presence of NADPH and regenerating system
(Ref. 14).
b
Compounds (5 lM) were incubated with HCT116 cells in cell culture medium
containing 10% FBS (Fetal Bovine Serum) (Ref. 14).
c
Ref. 14.
These compounds represent an important tool to elucidate the
role and possible therapeutic implications of HDAC4 as a target
in cancer therapy.
c
F3C
O
F3C
O
N
N
S
S
O
O
N
O
Acknowledgments
H2N
R
R
9a
33
The authors thank Annalise Di Marco for stability experiment in
HCT116 cells and Sergio Serafini for routine biological screening.
a, b
References and notes
H
N
d
F3C
OH
F3C
NH
1. (a) Glozak, M. A.; Sengupta, N.; Zhang, X.; Seto, E. Gene 2005, 363, 15; (b)
Ropero, S.; Esteller, M. Mol. Oncol. 2007, 1, 19.
2. Minucci, S.; Pelicci, P. G. Nat. Rev. Cancer 2006, 6, 38.
S
S
O
O
O
O
O
R
32
34
3. Karagiannis, T. C.; El-Osta, A. Leukemia 2007, 21, 61.
4. Grant, S.; Easley, C.; Kirkpatrick, P. Nat. Rev. Drug Discov. 2007, 6, 21.
5. For a review on isoform-selective HDAC inhibitors, see Itoh, Y.; Suzuki, T.;
Miyata, N. Curr. Pharm. Design 2008, 14, 529.
e
f
6. Karagiannis, T. C.; El-Osta, A. Oncogene 2006, 25, 3885.
7. Inoue, S.; Mai, A.; Dyer, M. J.; Cohen, G. M. Cancer Res. 2006, 66, 6785.
8. Karagiannis, T. C.; El-Osta, A. Cell Cycle 2006, 5, 288.
9. Jones, P.; Altamura, S.; De Francesco, R.; Gonzalez Paz, O.; Kinzel, O.; Mesiti, G.;
Monteagudo, E.; Pescatore, G.; Rowley, M.; Verdirame, M.; Steinkühler, C. J.
Med. Chem. 2008, 51, 2350.
10. Wilson, K. J.; Witter, D. J.; Grimm, J. B.; Siliphaivanh, P.; Otte, K. M.; Kral, A. M.;
Fleming, J. C.; Harsch, A.; Hamill, J. E.; Cruz, J. C.; Chenard, M.; Szewczak, A. A.;
Middleton, R. E.; Hughes, B. L.; Dahlberg, W. K.; Secrist, J. P.; Miller, T. A. Bioorg.
Med. Chem. Lett. 2008, 18, 1859.
H
N
F3C
F3C
N
N
S
S
O
O
O
O
O
Ph
R
35
9b
g
11. Witter, D. J.; Harrington, P.; Wilson, K. J.; Chenard, M.; Fleming, J. C.; Haines, B.;
Kral, A. M.; Secrist, J. P.; Miller, T. A. Bioorg. Med. Chem. Lett. 2008, 18, 726.
12. Jones, P.; Bottomley, M. J.; Carfì, A.; Cecchetti, O.; Ferrigno, F.; Lo Surdo, P.;
Ontoria, J. M.; Rowley, M.; Scarpelli, R.; Schultz-Fademrecht, C.; Steinkühler, C.
Bioorg. Med. Chem. Lett. 2008, 18, 3456.
13. Trifluoromethyl ketones as HDAC inhibitors have been reported in (a) Frey,
R. R.; Wada, C. K.; Garland, R. B.; Curtin, M.-L.; Michaelides, M. R.; Li, J.;
Pease, L. J.; Glaser, K. B.; Marcotte, P. A.; Bouska, J. J.; Murphy, S. S.;
Davidsen, S. K. Bioorg. Med. Chem. Lett. 2002, 12, 3443; (b) Jose, B.; Onikib,
Y.; Katob, T.; Nishino, N.; Sumida, Y.; Yoshida, M. Bioorg. Med. Chem. Lett.
2004, 14, 5343.
F3C
N
S
S
O
Ph
10c
Scheme 1. Synthesis of compounds in Tables 2 and 3. Reagents and conditions: (a)
CDI, DMF, rt, 30 min; (b) R-C(NOH)NH2, rt, 12 h; (c) CDI, DMF,
PS-carbodiimide, DCM/DMF, R-CONHNH2, rt, 12 h; (e) SOCl2,
PS-carbodiimide, DCM, 2-aminoacetophenone hydrochloride, TEA, rt, 12 h; (g)
Lawesson’s reagent, toluene, w, 100 °C, 10 s.
l
l
w, 140 °C, 2 min; (d)
w, 100 °C, 5 min; (f)
14. Scarpelli, R.; Di Marco, A.; Ferrigno, F.; Laufer, R.; Marcucci, I.; Muraglia, E.;
Ontoria, J. M.; Rowley, M.; Serafini, S.; Steinkühler C.; Jones, P., Bioorg. Med.
Chem. Lett., accepted for publication.
l

E. Muraglia et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6083–6087
6087
15. Jones, P.; Altamura, S.; De Francesco, R.; Gallinari, P.; Lahm, A.; Neddermann,
P.; Rowley, M.; Serafini, S.; Steinkühler, C. Bioorg. Med. Chem. Lett. 2008, 18,
1814.
16. Half-life measured in S9 fraction conditions was found to range from <15 min
to 20 min for amide HDAC4 inhibitors (see Ref. 14).
17. Hubbert, C.; Guardiola, A.; Shao, R.; Kawaguchi, Y.; Ito, A.; Nixon, A.; Yoshida,
M.; Wang, X. F.; Yao, T. P. Nature 2002, 417, 455.
18. Carboxylic acid 32 was obtained by LiOH/H2O/MeOH hydrolysis of the
corresponding ethyl ester (purchased from Rieke).
19. As an example, synthesis of 27: To 5-(trifluoroacetyl)thiophene-2-carboxylic
acid 32 in DMF (2 mL/mmol) 1,10-carbonyldiimidazole (1.1 equiv) in DMF
(1 mL/mmol), was added. The mixture was stirred at room temperature for
30 min, then N-hydroxy-2-(methylsulfonyl)ethanimidamide (1.1 equiv) in
anhydrous DMF (2 mL/mmol) was added and the mixture stirred at room
temperature overnight. The intermediate formed was not isolated, and reacted
by addition of CDI (1.1 equiv) in DMF (1 mL/mmol) and heating (Biotage
Initiator60 microwave apparatus, sealed tube, 140 °C, 2 min). From the
reaction mixture the product was isolated (yield: 34%) by preparative RP-
HPLC, using water (0.1% TFA) and acetonitrile (0.1% TFA) as eluents (column:
C18). 1H NMR (300 MHz, DMSO-d6, 300 K): d 8.22 (s, 2 H), 4.98 (s, 1 H), 3.21 (s,
3 H), hydrate form 8.34 and 8.33 (2s, 0.18H), 7.98 (d, J = 4 Hz, 0.15 H), 7.38 (d,
J = 4 Hz, 0.16 H). 19F NMR proton decoupled (282 MHz, DMSO-d6, 300 K): d
ꢀ75.04 (s, 2.3F, keto form), ꢀ86.42 (s, 0.7F, hydrate form). MS m/z (EI+): 341
(M + H)+, 359 (M + 18 + H)+. MS m/z (ES-): 339 (M + e)-, 357 (M + 18 + e)-.
20. Deegan, T. L.; Nitz, T. J.; Cebzanov, D.; Pufko, D. E.; Porco, J. A., Jr. Bioorg. Med.
Chem. Lett. 1999, 9, 209.
(1.7 equiv), and the suspension stirred at room temperature for 30 min.
Benzoic hydrazide (1.3 equiv) dissolved in DCM/DMF 1:1 (3 ml/mmol) was
added, and the resulting suspension was stirred at room temperature
overnight. The suspension was filtered and the filtrate evaporated. Crude
carboxyhydrazide 34 was used as such. It was dissolved in thionyl chloride
(9 mL/mmol) and the solution was heated (Biotage Initiator60 microwave
apparatus, sealed tube, 100 °C, 5 min), then the thionyl chloride was
evaporated. From the reaction mixture 10b was isolated (yield: 15%) by
preparative RP-HPLC, as reported in Ref. 19 1H NMR (300 MHz, DMSO-d6,
300 K): d 8.25 (s, 1H), 8.21–8.05 (m, 3 H), 7.75–7.60 (m, 3 H). 19F NMR proton
decoupled (282 MHz, DMSO-d6, 300 K): d ꢀ74.09 (s, 2.8F, keto form), ꢀ85.56 (s,
0.2F, hydrate form). MS m/z (EI+): 325 (M + H)+, 343 (M + 18 + H)+. MS m/z
(ES-): 323 (M + e)-.
22. Synthesis of 10c: To a solution of 5-(trifluoroacetyl)thiophene-2-carboxylic
acid 32 in DCM (22 mL/mmol) was added to PS-carbodiimide (2 equiv), and the
suspension stirred at room temperature for 30 min.
A mixture of 2-
aminoacetophenone hydrochloride (1.1 equiv) in DCM (1.5 mL/mmol) and
TEA (1.1 equiv) was added and the mixture stirred at room temperature
overnight. The suspension was filtered, the filtrate evaporated and the residue
purified by flash chromatography (silica gel/dichloromethane) to isolate 35
(yield: 68%). 1H NMR (300 MHz, CD3CN, 300 K): d 8.1–8.0 (m, 3 H), 7.8–7.50 (m,
5 H), 4.85 (d, J = 6 Hz, 2 H). A mixture of the above amide in toluene (24 mL/
mmol) and Lawesson’s reagent (4 equiv) was heated (Biotage Initiator60
microwave apparatus, sealed tube, 100 °C, 10 s), then the reaction mixture was
purified by flash chromatography (silica gel, gradient of petroleum ether/
AcOEt) to provide 10c (yield: 50%). 1H NMR (300 MHz, CDCl3, 300 K): d 8.03 (s,
1 H), 7.93–7.85 (m, 1 H), 7.61–7.55 (m, 3 H); 7.48–7.34 (m, 3 H). MS m/z (EI+):
340 (M + H)+, 358 (M + 18 + H)+.
21. As an example, synthesis of 10b: A solution of 5-(trifluoroacetyl)thiophene-2-
carboxylic acid 32 in DCM (18 mL/mmol) was added to PS-carbodiimide