Synthesis and structure-activity relationships of benzothienothiazepinone inhibitors of protein kinase D

Article abstract of DOI:10.1021/ml100230n

Protein kinase D (PKD) is a member of a novel family of serine/threonine kinases that regulate fundamental cellular processes. PKD is implicated in the pathogenesis of several diseases, including cancer. Progress in understanding the biological functions and therapeutic potential of PKD has been hampered by the lack of specific inhibitors. The benzoxoloazepinolone CID755673 was recently identified as the first potent and selective PKD inhibitor. The study of structure-activity relationships (SAR) of this lead compound led to further improvements in PKD1 potency. We describe herein the synthesis and biological evaluation of novel benzothienothiazepinone analogues. We achieved a 10-fold increase in the in vitro PKD1 inhibitory potency for the second generation lead kb-NB142-70 and accomplished a transition to an almost equally potent novel pyrimidine scaffold, while maintaining excellent target selectivity. These promising results will guide the design of pharmacological tools to dissect PKD function and pave the way for the development of potential anticancer agents.

Full text of DOI:10.1021/ml100230n

pubs.acs.org/acsmedchemlett
Synthesis and Structure-Activity Relationships of
Benzothienothiazepinone Inhibitors of Protein Kinase D
†
†
†
‡
ꢀ
Karla Bravo-Altamirano, Kara M. George, Marie-Celine Frantz, Courtney R. LaValle,
Manuj Tandon,‡ Stephanie Leimgruber,‡,§ Elizabeth R. Sharlow,‡,§
John S. Lazo,‡,§ Q. Jane Wang,‡,§ and Peter Wipf*,†,§
†Department of Chemistry, ‡Department of Pharmacology and Chemical Biology, and §Drug Discovery Institute,
University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States
ABSTRACT Protein kinase D (PKD) is a member of a novel family of serine/
threonine kinases that regulate fundamental cellular processes. PKD is implicated
in the pathogenesis of several diseases, including cancer. Progress in understanding
the biological functions and therapeutic potential of PKD has been hampered by
the lack of specific inhibitors. The benzoxoloazepinolone CID755673 was recently
identified as the first potent and selective PKD inhibitor. The study of structure-
activity relationships (SAR) of this lead compound led to further improvements in
PKD1 potency. We describe herein the synthesis and biological evaluation of novel
benzothienothiazepinone analogues. We achieved a 10-fold increase in the in vitro
PKD1 inhibitory potency for the second generation lead kb-NB142-70 and accom-
plished a transition to an almost equally potent novel pyrimidine scaffold, while
maintaining excellent target selectivity. These promising results will guide the
design of pharmacological tools to dissect PKD function and pave the way for the
development of potential anticancer agents.
KEYWORDS Protein kinase D, small molecule inhibitor, benzothienothiazepinone,
pyrimidines, CID755673
iacylglycerol (DAG) is a key lipid secondary messenger
that primarily targets protein kinase C (PKC) in cells.1 In
sites, including Ser
cellular localization of PKD by binding to its C1 domain.4-6
.
916 13 Furthermore, DAG regulates the intra-
Daddition to PKC, an increasing number of DAG receptors Indeed, activated PKD seems to be mobile and able to shuttle
between different subcellular compartments. Accordingly,
the DAG-PKC-PKD signaling cascade represents a key mech-
anism that controls PKD function in cells. Recent evidence
also indicates that PKD can be activated independently of
PKC, adding to the complexity of the signaling network.5
PKD contributes to a range of cellular responses, including
cell proliferation, cell survival through oxidative stress-
induced activation of nuclear factor-kappaB (NF-κB) signal-
ing, gene expression by regulation of class IIa histone
deacetylases, protein trafficking from the Golgi to the plasma
membrane, cell motility, and immune responses.4-6,14,15
This ubiquitous role of PKD at the cellular level accounts for
its implication in pathological processes such as cardiac
hypertrophy,16,17 angiogenesis,18,19 as well as tumor cell
proliferation and metastasis,20-23 therefore constituting a
potential therapeutic target for cardiovascular diseases and
oncology.14,15
have been discovered: protein kinase D (PKD), the chimaerin
Rac GTPase-activating proteins, the Ras guanyl nucleotide-
releasing proteins (RasGRPs), Munc13s, and the DAG kinases
(DGKs).2 All of these receptors are thought to be partially
responsible for the diverse biological actions of DAG signaling.
The PKD family has attracted particular attention because it is
not only a target of DAG but also a direct substrate of PKC,3 thus
positioning itself downstream of DAG and PKC at a central
position in the signal transduction pathway.4-6
PKDs are serine/threonine kinases that are now classified
as a subfamily of the Ca2þ/calmodulin-dependent kinase
(CaMK) superfamily.7 To date, three PKD isoforms have been
identified: PKD1 (formerly PKCμ),8,9 PKD2,10 and PKD3
(formerly PKCν).11 They share a highly homologous sequence
that comprises a catalytic domain and a regulatory domain.
The regulatory domain of each PKD isoform is mainly con-
stituted bya C1 domain, which binds DAG/phorbol esters, and a
pleckstrin homology (PH) domain, which mediates protein-
protein interactions and PKD autoinhibition.8-11
The lack of specificity of early PKD inhibitors, i.e. the
isoquinoline sulfonamide H89, staurosporine analogues, and
DAG-responsive PKC is known to activate PKD by direct
interaction with the PH domain of PKD and transphosphor-
ylation of its activation loop at the conserved Ser744 and
Received Date: September 28, 2010
Accepted Date: December 6, 2010
Published on Web Date: December 14, 2010
Ser .
748 3,12 Subsequent autophosphorylation occurs at multiple
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Table 1. Chemical Structure, SAR Analysis, and PKD Inhibitory Activity of CID755673 and Selected Synthetic Analogues
structure
Z
IC50
zone
compd
X
Y
R
n
PKD1 (nM)a
cellular PKD1 (μM)b
CID755673
CH
CH
CH
CH
CH
CH
CH
N
O
CH2
CH2
S
OH
1
1
1
1
1
2
2
1
182 ( 27 (n = 5)
130 ( 14 (n = 3)
28.3 ( 2.3 (n = 3)
82.5 ( 4.6 (n = 4)
74.9 ( 14.8 (n = 5)
111 ( 6 (n = 3)
11.8 ( 4.0 (n = 3)
>50 (n = 3)
II
kb-NB123-57
kb-NB142-70
kb-NB165-09
mcf292-08
NH
S
OH
II, III
OH
2.2 ( 0.6 (n = 3)
3.1 ( 0.5 (n = 3)
2.2 ( 0.2 (n = 3)
2.6 ( 0.7 (n = 2)
18.6 ( 2.0 (n = 3)
6.8 ( 1.3 (n = 3)
I
S
S
OMe
N3
I
S
S
III
III
I
kb-NB165-92
kb-NB184-02
kmg-NB4-23
S
S
OH
S
S
OMe
OMe
193 ( 27 (n = 3)
124 ( 31 (n = 4)
S
S
a PKD1 IC50 was determined using a radiometric kinase activity assay as previously described.24 Each IC50 was calculated as the mean ( SEM of at
least three independent experiments with triplicate determinations at each concentration in each experiment; n = number of independent
experiments. b Cellular IC50 was determined by densitometry analysis of Western blotting data for PKD1 autophosphorylation at S916 in LNCaP cells
as previously described.25 Each IC50 was calculated as the mean ( SEM of at least two independent experiments; n=number of independent
experiments.
resveratrol, has significantly impeded the further analysis of the
regulation and biology of this kinase, but more recently, several
more selective inhibitors have been reported.14,15,17,23 A sig-
nificant accomplishment in this area was recently achieved by
the identification and characterization of CID755673, the first
potent and selective PKD inhibitor (Tabl e 1 ).24 CID755673
inhibits PKDs with an in vitro IC50 of 182 nM for PKD1, 280 nM
for PKD2, and 227 nM for PKD3. In addition to its potency,
CID755673 was unique because it was not an ATP-competitive
inhibitor, implying that it may bind to an alternative site on PKD
and, consequently, offer a greater selectivity for PKD vs other
protein kinases. CID755673 effectively blocked PKD-mediated
cellular functions and revealed novel tumor-promoting func-
tions of PKD isoforms in prostate cancer cells.24
Herein, we report our structure-activity relationship
(SAR) efforts based on CID755673. The search for further
information of the binding interaction and the identification
of more potent analogues was guided by the dissection of the
parent compound CID755673 into four major structural
zones (Table 1): zone I (aryl moiety), zone II (furan), zone
III (azepine), and zone IV (amide function).25 The com-
pounds resulting from the structural modifications of each
of these zones were evaluated in vitro and in cells for their
PKD1 inhibitory activity. Among the ca. 50 analogues that
were prepared, data for the most representative in each
series are summarized in Table 1.
Interestingly, replacement of the oxygen with a sulfur atom
significantly improved the activity and led to the discovery of
the potent benzothienothiazepinone kb-NB142-70, exhibit-
ing an in vitro IC50 of 28.3 nM.
Zone I modifications included functionalization across the
aryl moiety and substitutions on the phenolic hydroxyl
group. Most of these zone I derivatives were less active than
CID755673. Specifically, carbon substituents ortho to the
phenol and O-benzylations were detrimental, whereas
ortho-halogenation25 had a minor effect on the in vitro
activity (data not shown). Furthermore, phenol O-methyla-
tion was well tolerated, as illustrated by an IC50 of 82.5 nM
for the methoxy analogue, kb-NB165-09. This remarkable
potency of the methyl ether compared to the phenol
kb-NB142-70 may indicate that a hydrogen bond donor at
this position is not crucial for activity. In contrast, the
dramatic loss in activity of the benzyl analogue suggests
that the zone I pocket in the protein is sterically limiting.
Notably, the azide analogue mcf292-08 maintained a high
inhibitory activity with an IC50 of 74.9 nM. This result
confirms the limited significance of a hydrogen bond donor
at this position and also suggests that an azide group can fit
quite well inside the sterically restricted binding pocket.
In zone III, the ring size was altered by addition or removal
of methylene groups and substitution at the benzylic position.
Concomitant to zone II modification by a thiophene, thioether
insertion exo to the five-membered heterocycle was not
detrimental to the activity, as shown for kb-NB142-70 and
kb-NB165-09, although no conclusion could be drawn at this
point regarding the relative contribution of each modification.
Moreover, increasing the ring size from 7 to 8 atoms by
inserting an additional methylene group in zone III was
also well tolerated, as suggested by the potency of the
All modifications to zone IV, which included functional group
interconversions and replacement of the amide moiety, failed
to enhance the inhibitory activity (data not shown).
In zone II, replacement of the oxygen in the parent
compound CID755673 with a nitrogen atom resulted in
equivalent or slightly enhanced potency, as exemplified by
pyrrole kb-NB123-57, which showed an IC50 of 130 nM.
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Scheme 1. Synthesis of the β-Carboline Analogue kb-NB123-57a
Scheme 3. Synthesis of the Benzothienothiazocinones kb-NB165-92
and kb-NB184-02a
a Reagents and conditions: (a) H2SO4, EtOH, reflux, 5 h. (b) Pd/C,
NH4HCO3, MeOH, reflux, 2 h.
Scheme 2. Synthesis of the Benzothienothiazepinone Analogues
kb-NB142-70 and kb-NB165-09a
a Reagents and conditions: (a) (i) ClSO3H, CCl4, rt, overnight; (ii) CS2, NaOH,
EtOH/H2O, 0 °C to reflux, 40 min. (b) conc HCl, reflux, N2, 14 days. (c) 5,
DBU, DMF, rt, 2 h; then 70 °C, 18 h. (d) BBr3, DCM, -20 °C to rt, 2 h. (e) MeI,
K2CO3, DMF, rt, overnight.
Scheme 4. Synthesis of the Azidobenzothienothiazepinone Ana-
logue mcf292-08a
a Reagents and conditions: (a) BnBr, NaOH, EtOH, rt, 14 h. (b) (i) SOCl2,
pyr, DMF, PhCl, 120 °C, 22 h; (ii) Et3N, MeOH, reflux, 12 h. (c) DBU, DMF,
rt, 1.5 h; then 70 °C, 12 h. (d) BBr3, DCM, -20 to 0 °C, 2.5 h. (e) MeI,
K2CO3, DMF, rt, 12 h.
benzothienothiazocinones kb-NB184-02 (IC50 193 nM) and
kb-NB165-92 (IC50 111 n M ). It is also worth noting that in the
case of the 8-membered ring, the same trend applies regard-
ing the slightly decreased activity of the methoxy vs the free
phenol substituent in zone I.
The synthesis of representative analogues in each series
required the development of flexible synthetic routes.
β-Carboline kb-NB123-57 was prepared from phenylhydra-
zine 2 via a Fischer-like indole synthesis with the corre-
sponding 7-membered R-ketolactam, obtained in situ by
acid-catalyzed hydrolysis of the enamine 1 (Scheme 1).26
Debenzylation by transfer hydrogenation led to the phenol
kb-NB123-57.27
Benzothienothiazepinones kb-NB142-70 and kb-NB165-
09 were synthesized according to a literature protocol28,29
beginning with benzyl protection of commercially available
3-hydroxycinnamic acid (Scheme 2). Thionyl chloride-
mediated Higa cyclization of acid 4 provided the benzo-
[b]thiophene acid chloride, which was subsequently con-
verted to methyl ester 5. Treatment of 5 with cysteamine
hydrochloride in the presence of DBU resulted in formation
of the desired benzothienothiazepinone core.29 Deprotec-
tion of the aryl benzyl ether with boron tribromide provided
kb-NB142-70 in good yield, and O-methylation of kb-NB142-
70 with methyliodide led to kb-NB165-09.
a Reagents and conditions: (a) Et3N, MeOH, 40-50 °C, 4 h. (b) Tf2O,
Et3N, DMAP, DCM, rt, 2 h. (c) DBU, DMF, rt, 1.5 h; then 70 °C, 13 h. (d)
SnCl2, EtOH, reflux, 5 h. (e) t-BuONO, TMSN3, MeCN, rt, 1.5 h.
of thiazinanethione 7, prepared in two steps from commer-
cially available 3-amino-1-propanol (Scheme 3).30 Aminothiol
8was isolated as an approximately 1:1.3 thiol/disulfide mixture
and used directly in the cyclocondensation-deprotection se-
quence to yield the desired benzothienothiazocinones.
Replacement of the phenol group by an azide on the
benzothiophene scaffold required the development of an
alternative route (Scheme 4). Aromatic nucleophilic substi-
tution of methyl 2-chloro-5-nitrobenzoate by the methyl
thioglycolate anion followed by immediate Dieckman
cyclization31,32 afforded the benzothiophene precursor 10.
Cyclization of the corresponding triflate 11 with cysteamine
hydrochloride provided the desired tricyclic core 12 in 50%
yield (67% based on recovered starting material 10). After
reduction of the nitro group, treatment of aniline 13 with
tert-butyl nitrite and TMS-azide using Moses' method33
yielded the aryl azide mcf292-08.
Our SAR had established that, in general, benzothie-
nothiazepinones were superior for PKD inhibition compared
to benzofuroazepinones. The benzothienothiazepinone kb-
NB142-70 wasthe most potent analogue, with an in vitro IC50
In order to synthesize the thiazocinone annulated ben-
zothiophenes kb-NB165-92 and kb-NB184-02, the aminopro-
panethiol hydrochloride 8 was obtained through ring-opening
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of 28 nM for PKD1, achieving a nearly 7-fold improvement in
potency over the earlier lead structure CID755673. This
trend was further confirmed in cell-based assays, for which
the cellular IC50 was lowered 5-fold from 11.8 μM for the
inhibition of PMA-induced activation of endogenous PKD1
by CID755673 to single digit 2.2 μM by kb-NB142-70. While
this result boded well for the ability of kb-NB142-70 to inhibit
PKD1 in intact cells, in vivo studies revealed a short plasma half-
life for the benzothienothiazepinone.34 Accordingly, we further
explored a zone I modification of kb-NB142-70 to install a more
electron-deficient pyrimidine moiety in place of the phenol
ether, a known site of active phase I and II metabolism.
The synthetic route to arrive at this new thiazepinothio-
phenopyrimidinone scaffold is summarized in Scheme 5.
Starting with commercially available methyl 3-aminothio-
phene-2-carboxylate, formation of the pyrimidine moiety
using potassium cyanate, followed by chlorination with
POCl3 gave dichloride 15.35 Palladium-catalyzed regioselec-
tive dechlorination of 15 in the presence of Na2CO3 occurred
exclusively at the C-4 position,36 and substitution of the
remaining C-2 chloride with methoxide provided 17 in
79% yield over the two steps. Electrophilic bromination of
17 using bromine in AcOH37 gave the desired C-7 bromi-
nated compound 18. Functionalization at C-6 under Knochel's
conditions38 gave the required cyclization precursor 19.
Formation of the thiazepinone moiety was then achieved
by one-pot nucleophilic displacement-condensation of 19
with cysteamine hydrochloride, providing the desired meth-
oxypyrimidine kmg-NB4-23 in good yield.
Gratifyingly, pyrimidine kmg-NB4-23 exhibited an IC50 of
124 nM, which is only a slight decrease in activity compared to
that of the parent lead kb-NB165-09. This encouraging result
thus confirms the validity of our design and also suggests that
decreased π-electron density is well tolerated in the aryl region.
In order to establish the selectivity profile of our analogue
series against other kinases, we also performed a variety
of counterscreens using AKT, CAK (CDK7), PLK1, PLK2,
several PKCs, and CaMKR. In contrast to the low nanomolar
activity of our analogues against PKD, none of our inhibitors
were found to be significantly active at concentrations <10
μM in these screens. Furthermore, we tested the selectivity
of our leads kmg-NB4-23, mcf292-08, and kb-NB123-57
against PKCR, PKCβI, and PKCδ, and we did not detect
significant activities in these assays. For CaMKIIR, we found
weak (<40%) inhibition at 10 μM for mcf292-08 and
kb-NB123-57. These counterscreens clearly establish a
high selectivity profile for PKD inhibition in our analogue
series.39
Scheme 5. Synthesis of the Thiazepinothiophenopyrimidinone
Analogue kmg-NB4-23a
a Reagents and conditions: (a) (i) KOCN, AcOH, H2O, rt, 20 h; (ii) 2 N
NaOH, rt, 2 h. (b) POCl3, MeCN, reflux, 2 days. (c) H2, Pd/C, EtOH,
Na2CO3, rt, 24 h. (d) NaOMe, MeOH, reflux, 6 h. (e) AcOH, Br2, 70 °C,
24 h. (f) (i) TMPMgCl LiCl, -50 °C, 2 h; (ii) MeCO2CN. (g) DBU, DMF, rt,
3
1.5 h; then 70 °C, 10 h.
In summary, we have identified a series of novel ben-
zothienothiazepinones as PKD inhibitors. A first round of
optimization from the initial lead CID755673 led to the
discovery of kb-NB142-70 and its methoxy analogue
kb-NB165-09. These new lead compounds inhibited PKD1
in vitro in the low nanomolar range, achieving a nearly one-
log IC50 enhancement compared to the HTS hit CID755673.
Additionally, these inhibited PKD1 autophosphorylation at
Ser916 in LNCaP prostate cancer cells in the low micromolar
range, a 10-fold improvement compared to the case of
CID755673. In order to circumvent metabolism issues,
further core modification with a pyrimidine moiety led to
the promising new lead structure kmg-NB4-23. SAR studies
are currently in progress to improve the activity of kmg-NB4-
23 as well as its selectivity toward PKD1 vs PKD2 or PKD3,
for which the IC50 values were found in the 50-500 nM
range for all derivatives tested (data not shown). In conclu-
sion, our results to date demonstrate that modifications of
CID755673 significantly modulate its potency but have only
limited effect on isoform selectivity.25 This suggests that the
binding domain may be highly conserved among the three
isoforms. Finally, the azide analogue mcf292-08, also active
at nanomolar (in vitro) or micromolar (in cells) concentra-
tions, represents a valuable tool for future photoaffinity
labeling studies that may provide key structural information
for binding site identification.
We have also started to probe the cellular effects of kmg-
NB4-23, mcf292-08, and kb-NB123-57.39 The azidoben-
zothienothiazepinone mcf292-08 inhibited PMA-induced
PKD1 S916 autophosphorylation in LNCaP prostate cancer
cells with an IC50 of 2.2 μM. For the thiazepinothiopheno-
pyrimidinone kmg-NB4-23, an IC50 of 6.8 μM was found in
this assay. In contrast, kb-NB123-57 did not significantly
inhibit the PMA-induced PKD1 autophosphorylation at S916
(IC50 > 50 μM), possibly due to limited membrane perme-
ability, adverse protein binding, metabolic degradation, or
other compensatory pathways in cells.
SUPPORTING INFORMATION AVAILABLE Experimental
procedures, assay results, and spectral data for compounds. This
information is available free of charge via the Internet at http://pubs.
acs.org.
AUTHOR INFORMATION
Corresponding Author: *Department of Chemistry, Parkman
Ave. CSC 1301, University of Pittsburgh, Pittsburgh, PA 15260.
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Telephone: 412-624-8606. Fax: 412-624-0787. E-mail: pwipf@
pitt.edu.
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Author Contributions: Karla Bravo-Altamirano participated in
research design (chemistry) and execution (performed synthesis);
ꢀ
Marie-Celine Frantz participated in research design (chemistry) and
execution (performed synthesis) and contributed to writing of the
manuscript; Kara M. George participated in research design (chem-
istry) and execution (performed synthesis) and contributed to
writing of the manuscript; Courtney R. LaValle participated in
research execution (performed screens); John S. Lazo participated
in research design (biology), acquired funding, and contributed to
writing of the manuscript; Stephanie Leimgruber participated in
research execution (performed counterscreens); Elizabeth R. Shar-
low participated in research design (biology) and execution (per-
formed counterscreens); Manuj Tandon participated in research
execution (performed screens); Q. Jane Wang led research design
(biology), acquired funding, and contributed to writing of the
manuscript; and Peter Wipf led research design (chemistry), ac-
quired funding, contributed to writing of the manuscript, and is the
corresponding author.
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ACKNOWLEDGMENT This study was supported in part by the
National Institutes of Health (Grants R03MH082038-01, R01CA129127,
R01CA142580), the NIH Roadmap Program (Grants U54MH074411,
GM067082), the Fiske Drug Discovery Fund, and the Elsa Pardee
Foundation. The authors thank Dr. Donna Huryn (University of
Pittsburgh) for advice on the design of analogues, Ms. Rachel Byerly
(University of Pittsburgh) for technical assistance, and Dr. Julie Eiseman
(University of Pittsburgh Hillman Cancer Center) for her suggestions on
metabolism and in vivo studies.
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ABBREVIATIONS CAK, CDK activating kinase; CaMK, Ca2þ
/
calmodulin-dependent kinase; CDK, cyclin-dependent kinase;
DAG, diacylglycerol; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene;
DGK, DAG kinase; DMAP, N,N-dimethylaminopyridine; HDAC,
histone deacetylase; NF-κB, nuclear factor-kappaB; PH, pleckstrin
homology; PKC, protein kinase C; PKD, protein kinase D;
PLK, polo-like kinase; PMA, phorbol 12-myristate 13-acetate;
RasGRP, Ras guanyl-nucleotide-releasing protein; SAR, structure-
activity relationship; TMP, 2,2,6,6-tetramethylpiperidine.
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