2-Anilino-4-aryl-1,3-thiazole inhibitors of valosin-containing protein (VCP or p97)

Article abstract of DOI:10.1016/j.bmcl.2010.01.058

Valosin-containing protein (VCP; also known as p97) is a member of the AAA ATPase family with a central role in the ubiquitin-degradation of misfolded proteins. VCP also exhibits antiapoptotic function and metastasis via activation of nuclear factor kappa-B signaling pathway. We have discovered that 2-anilino-4-aryl-1,3-thiazoles are potent drug-like inhibitors of this enzyme. The identified compounds show low nanomolar VCP potency, demonstrate SAR trends, and show activity in a mechanism based cellular assay. This series of compounds represents the first steps towards a novel, small molecule VCP inhibitor as a cancer therapeutic.

Full text of DOI:10.1016/j.bmcl.2010.01.058

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1677–1679
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Anilino-4-aryl-1,3-thiazole inhibitors of valosin-containing
protein (VCP or p97)
a
a
b
b
Matthew G. Bursavich a, , Daniel P. Parker , J. Adam Willardsen , Zhong-Hua Gao , Thaylon Davis ,
*
Kirill Ostanin b, Rosann Robinson b, Ashley Peterson b, Daniel M. Cimbora b, Ju-Fen Zhu b, Burt Richards b
a Medicinal Chemistry, Myriad Pharmaceuticals, Inc., 305 South Chipeta Way, Salt Lake City, UT 84108, United States
b In Vitro Pharmacology, Myriad Pharmaceuticals, Inc., 305 South Chipeta Way, Salt Lake City, UT 84108, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Valosin-containing protein (VCP; also known as p97) is a member of the AAA ATPase family with a central
role in the ubiquitin-degradation of misfolded proteins. VCP also exhibits antiapoptotic function and
metastasis via activation of nuclear factor kappa-B signaling pathway. We have discovered that 2-anili-
no-4-aryl-1,3-thiazoles are potent drug-like inhibitors of this enzyme. The identified compounds show
low nanomolar VCP potency, demonstrate SAR trends, and show activity in a mechanism based cellular
assay. This series of compounds represents the first steps towards a novel, small molecule VCP inhibitor
as a cancer therapeutic.
Received 23 November 2009
Revised 7 January 2010
Accepted 11 January 2010
Available online 21 January 2010
Keywords:
ATPase
VCP
Ó 2010 Elsevier Ltd. All rights reserved.
P97
Oncology
Thiazole
Oxazole
Hit to lead
Valosin-containing protein (VCP; also known as p97) is a mem-
ber of the AAA ATPase family that plays a role in several biological
processes. These include ERAD (endoplasmic reticulum associated
degradation), nuclear membrane fusion after completion of mito-
sis, golgi reassembly, apoptosis repression, and activation of tran-
scription factors.1 VCP also plays a central role in removing
ubiquitin-tagged, misfolded proteins from the endoplasmic reticu-
lum and escorting them to the 26S proteasome for degradation.2
VCP protein is over expressed in gastric, colon, pancreatic, and
hepatocellular cancers.1 Overabundance of VCP has been shown
Here we report the discovery and structure–activity relation-
ships (SAR) of thiazole-based VCP inhibitor 1 found via HTS. To
our knowledge, this class of compounds represents the first disclo-
sure of small, drug-like VCP inhibitors with potential utility as a
cancer therapeutic.
2
R
1
R
N
N
S
to target IjB for degradation, thereby activating the NFjB pathway.
H
Activation of the NF
j
B pathway is commonly observed during
1
tumorigenesis.3 Disruption of VCP function, via RNA interference
or over expression of ATPase deficient protein in tumor cell lines,
has been shown to cause cell death.4,5 Thus, a VCP inhibitor has
the potential as a novel small molecule cancer therapeutic.
A high throughput screening (HTS) assay for VCP inhibitors was
therefore developed to identify an entry point for lead identifica-
tion/optimization. Inhibition of ATPase activity was determined
by a glucokinase coupled enzyme assay.6–8 Conversion of ATP to
ADP was detected by resorufin signal and VCP inhibition confirmed
by an ATPase assay.
Initial efforts to explore the 2-anilino-4-aryl-1,3-thiazole VCP
inhibitor scaffold began with the Hantzsch thiazole synthesis out-
lined in Scheme 1.9,10 Various commercial arylthioureas 2 and
readily or commercially available a-bromo ketones 3 were cyclized
in refluxing EtOH to afford the desired 2-anilino-4-aryl-thiazole 1.
Based on our work with another scaffold (data not shown), we
first investigated R1 substitution while holding R2 constant (R2 = p-
Cl). VCP inhibition data for this initial set of 2-anilino-4-aryl-1,3-
thiazoles (4–18) is shown in Table 1. Compounds 4–18 demon-
strate that the R1 aniline substitution plays a key role in VCP activ-
ity. When R1 = H or a meta substituent, minimal VCP inhibition is
seen (R1 = H 4: 14% inhibition; R1 = 3-OH, 3-CN, or 3-CF3 5–7:
* Corresponding author. Tel.: +1 801 214 7839; fax: +1 801 214 7994.
E-mail address: Matthew.Bursavich@myriadpharma.com (M.G. Bursavich).
26% inhibition, 26% inhibition, and IC50 = 8.3 lM, respectively). At-
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.058

1678
M. G. Bursavich et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1677–1679
2
Table 2
R
VCP inhibition for various 2-(para-hydroxy)anilino 4-aryl/heteroaryl-1,3-thiazoles
O
1
1
R
S
Br
a
HO
R
N
2
Aryl/HetAryl
R
N
H
NH2
N
N
H
S
N
S
2
3
1
H
VCP% inhibb
a
Compd
Aryl/hetaryl
VCP IC50
(lM)
Scheme 1. Reagents and conditions: (a) EtOH, reflux.
19
20
21
22
23
24
25
26
27
28
29
30
31
33
34
35
36
37
38
41
42
Phenyl
2-Thiophene
2-Pyridyl
3-Pyridyl
4-Pyridyl
0.19
0.11
0.23
0.30
0.29
1.30
0.36
0.67
2.7
Table 1
VCP inhibition for various R1 substituents of 2-anilino-4-(para-chloro)phenyl-1,3-
2-Naphthyl
(2-F) Phenyl
thiazoles
(2-OMe) Phenyl
(2-CF3) Phenyl
(2-CO2H) Phenyl
(3-Cl) Phenyl
(3-CN) Phenyl
(3-OMe) Phenyl
(3-NO2) Phenyl
(3-OH) Phenyl
(4-CN) Phenyl
(4-CF3) Phenyl
(4-Me) Phenyl
(4-OMe) Phenyl
(4-Morpholine) Phenyl
(3,4-di-Cl) Phenyl
Cl
44
53
26
27
R1
N
N
S
0.76
0.12
0.18
H
R1
VCP IC50
(lM)
VCP% inhibb
a
Compd
9
55
4
5
6
7
8
H
3-OH
3-CN
14
26
25
2
13
3-CF3
8.3
0.43
3-CO2H
3-NHAc
4-CN
4-NO2
4-OH
4-NH2
4-OMe
4-NHAc
4-CO2H
4-CONH2
4-CONMe
35
25
10
15
a
9
Values are means of two experiments, standard deviations are 10%.
Values are VCP percent inhibition at 10 lM inhibitor concentration.
b
10
11
12
13
14
15
16
17
18
0.07
0.08
7
methoxy ortho-substituted phenyl analogs 25 (IC50 = 0.36
and 26 (IC50 = 0.67 M) showed sub-micromolar VCP activity. Con-
versely, the electron withdrawing trifluoromethyl and carboxylic
acid phenyl analogs 27 and 28 showed reduced VCP activity
lM)
>10
>20
l
40
25
a
(IC50 = 2.7 lM and 44% inhibition, respectively). Various meta-sub-
Values are means of two experiments, standard deviations are 10%.
Values are VCP percent inhibition at 10 lM inhibitor concentration.
b
stituents also provided a range of VCP activities. The chloro, cyano,
and methoxy meta-substituted phenyl analogs 29–31 showed re-
duced VCP activity (53%, 26%, and 27% inhibition, respectively).
tempts to display hydrogen bond donors/acceptors in the R1 meta
position yielded minimally active VCP analogs (R1 = 3-CO2H 8:
35% inhibition; R1 = 3-NHAc 9: 25% inhibition). When R1 was a para
substituent a wide range of VCP activity was observed. Electron
withdrawing substituents show minimal activity (R1 = 4-CN 10:
10% inhibition; R1 = 4-NO2 11: 15% inhibition). Rewardingly,
hydrogen bond donating substituents showed much improved po-
However, the nitro (32: IC50 = 0.76
lM) and hydroxyl (33:
IC50 = 0.12 M) meta-substituted phenyl analogs showed nanomo-
l
lar VCP activity. Para-substituted aryl analogs 35–41 also provided
a range of VCP activities. The cyano and methoxy para-substituted
phenyl analogs, 35 and 38, provided VCP active compounds
(IC50 = 0.18 and 2.0 lM, respectively). Conversely, other para-
substituted phenyl substituents led to minimally active VCP ana-
logs 36, 37, and 41 (9%, 55%, and 31% inhibition, respectively).
The meta, para-disubstituted phenyl analog 42 (3,4-dichloro) affor-
tency (R1 = 4-OH 12: IC50 = 0.07 M; R1 = NH2 13: IC50 = 0.08
l lM).
Masking these hydrogen bonding substituents led to greatly re-
duced potency (R1 = 4-OMe 14: 26% inhibition; R1 = NHAc 15:
ded a compound with sub-micromolar potency (IC50 = 0.43 lM).
IC50 > 10
acceptors in the R1 para position yielded minimally active VCP ana-
logs (R1 = 4-CO2H 16: 40% inhibition; R1 = 4-CONH2 17: IC50
20
M; R1 = 4-CONHMe 18: 25% inhibition). Other attempts to re-
lM). Other attempts at adding hydrogen bond donors/
We then turned our efforts toward exploring a replacement of
the central thiazole core with an oxazole. This work began with
the synthetic route outlined in Scheme 2.9,11 Heating commercially
>
l
available p-OH phenyl isothioisocyanate 43 with various a-azido-
place the p-OH or p-NH2 with fused heterocycles such as indoles,
indazoles, benzimidazoles, benzoxazoles, and benzothiazoles (not
shown) led to minimally active VCP analogs.
acetophenones 44 (prepared from commercially available acetoph-
enones) in the presence of resin-bound triphenyl phosphine
afforded the desired 2-anilino-5-aryl-oxazoles 45–54.
To expand upon the identification of 12 (VCP IC50 = 0.07 lM) a
VCP inhibition data for this set of oxazoles 45–54 is shown in
Table 3. In general, the oxazoles investigated showed nanomolar
series of 2-(para-hydroxy)-anilino-4-aryl thiazoles was synthe-
sized (Table 2). While much SAR was developed around the 4-
chloro phenyl, a more potent replacement was not found. The
unsubstituted phenyl 19, and isosteric 2-thiophene analog 20,
2
R
O
1
show nanomolar VCP activity (IC50 = 0.19
tively). The three regioisomeric 2-, 3-, and 4-pyridines showed re-
duced activity (21–23: IC50 = 0.23 M, 0.30 M, and 0.29 M,
respectively). The larger 2-naphthyl substituent resulted in even
greater loss of activity (24: IC50 = 1.3 M). Various substituted phe-
nyl analogs 25–42 were then investigated. Both the fluoro and
lM and 0.11 lM, respec-
a
R
N
3
1
O
2
R
R
NCS
N
H
l
l
l
N
44
43
45-54
l
Scheme 2. Reagents and conditions: (a) PPh3 resin, dioxane, reflux.

M. G. Bursavich et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1677–1679
1679
Table 3
methyl phenyl analog 52 (7% inhibition) demonstrated only mini-
mal activity. Both para-substituted amino analogs (53–54: mor-
pholine and diethyl amine, respectively) also showed nanomolar
VCP activity.
VCP inhibition for various 4-aryl-2-(para-hydroxy) anilino-4-aryl-1,3-oxazoles
R2
HO
To further profile these VCP inhibitors several of the more po-
tent compounds were evaluated in a mechanism based cellular as-
say shown in Table 4. This assay utilized a ubiquitin-tagged
Luciferase reporter (Ub-G76 V-Luciferase) as VCP has been shown
to play a role in targeting ubiquitinated fusion constructs for deg-
radation.2,12 Several of the potent thiazole-based VCP inhibitors
cause stabilization of the reporter construct in HeLa cells after a
24 h treatment with low micromolar (12 and 21) to nanomolar
(19, 20, and 34) cellular activity.13
In conclusion, we have presented a series of 2-anilino-4-aryl-
1,3-thiazole VCP inhibitors. These thiazoles and the related oxaz-
oles have been synthesized in a parallel synthesis approach to
investigate a HTS-derived class of inhibitors. The compounds pre-
sented herein are small, drug-like lead molecules with tractable
SAR and low nanomolar potency against an emerging oncology tar-
get, VCP. Several potent VCP inhibitors also show sub-micromolar
activity in a mechanism based cellular assay. The continued devel-
opment of novel, small molecule VCP inhibitors towards a cancer
therapeutic will be reported in due course.
O
N
N
H
a
Compd
R2
VCP IC50
(lM)
VCP% inhibb
45
46
47
48
49
50
51
52
53
54
H
0.091
0.12
2-OMe
2-F
3-Cl
3-OMe
3-CO2H
4-Cl
4-Me
4-Morpholine
4-NEt2
7
0.14
0.17
0.19
0.33
16
0.13
0.23
a
Values are means of two experiments, standard deviations are 10%.
Table 4
VCP cellular inhibition for various 2-anilino-4-aryl/heteroaryl-1,3-thiazole inhibitors
of VCP
Acknowledgments
HO
Aryl/HetAryl
We thank Drs. Kraig Yager and Robert Carlson for support of
this work. We thank Dr. Vijay Kumar for useful discussions in the
preparation of this manuscript. We thank the Myriad Pharmaceu-
tical Analytical department for their help with compound purifica-
tion and characterization.
N
N
S
H
a
a,b
Compd Aryl/hetaryl
VCP IC50
M)
Luciferase stabilization EC50
(lM)
(
l
12
19
20
21
34
(4-Cl) Phenyl 0.07
<1.20
0.52
0.09
<1.20
0.20
References and notes
Phenyl
0.19
0.11
0.23
0.12
2-Thiophene
2-Pyridyl
(3-OH)
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3. Karin, M. Nature 2006, 441, 431.
a
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B. J. Biomol. Screen. 2007, 12, 881.
Values are means of two experiments, standard deviations are 10%.
EC50 = concentration that results in half-maximal stabilization of the Luciferase
b
reporter construct in HeLa cells. Values are means of two experiments, standard
deviations are 10%. More accurate values for 12 and 21 could not be calculated due
to cell killing.
9. All newly prepared compounds were characterized by reverse phase-HPLC/MS
spectroscopy, High Resolution Mass Spectroscopy, and 1H NMR.
10. Sherman, W. R.; Dickson, D. E. J. Org. Chem. 1962, 27, 1351.
11. Dhar, T. G.; Murali; Quo, J.; Shen, Z.; Pitts, W. J.; Gu, H. H.; Chen, B.-C.; Zhao, R.;
Bednarz, M. S.; Iwanowicz, E. J. Org. Lett. 2002, 4, 2091.
12. Matsuzawa, S.; Cuddy, M.; Fukushima, T.; Reed, J. C. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 14982.
13. All compounds tested were shown to be selective against the proteasome. The
VCP inhibitory activity, but the SAR showed differing trends than
the related thiazoles. The unsubstituted phenyl derivative 45
(IC50 = 0.09 lM) demonstrated low nanomolar VCP potency. While
the ortho-fluoro analog (47: 7% inhibition) showed minimal activ-
ity, the electron donating ortho-methoxy phenyl analog 46
(IC50 = 0.12 lM) showed similar low nanomolar activity. All three
meta-substituted analogs (48–50: chloro, methoxy, and carboxcyl-
ic acid, respectively) showed potent nanomolar inhibitory activity.
The para-chloro analog, which was the most potent thiazole, pos-
compounds in Table 4 exhibit IC50 values between 0.43 and 4.5 lM against
chymotrypsin, caspase, and trypsin activities of the 20S proteasome. The EC50
values for Ub-Luciferase stabilization do not correlate with proteasome
inhibition. While we have not extensively screened for potential off-target
activities, for these purposes cellular efficacy in the luciferase assay can been
ascribed to mechanism based VCP inhibitory activity.
sessed reduced VCP activity (51: IC50 = 0.33 lM) and the para-