Design, synthesis, and biological activity of isophthalic acid derivatives targeted to the C1 domain of protein kinase C

Article abstract of DOI:10.1021/jm900229p

Protein kinase C (PKC) is a widely studied molecular target for the treatment of cancer and other diseases. We have approached the issue of modifying PKC function by targeting the C1 domain in the regulatory region of the enzyme. Using the X-ray crystal structure of the PKC δ C1b domain, we have discovered conveniently synthesizable derivatives of dialkyl 5-(hydroxymethyl)isophthalate that can act as potential C1 domain ligands. Structure-activity studies confirmed that the important functional groups predicted by modeling were indispensable for binding to the C1 domain and that the modifications of these groups diminished binding. The most promising compounds were able to displace radiolabeled phorbol ester ([³H]PDBu) from PKC α and δ at K_i values in the range of 200-900 nM. Furthermore, the active isophthalate derivatives could modify PKC activation in living cells either by inducing PKC-dependent ERK phosphorylation or by inhibiting phorbol-induced ERK phosphorylation. In conclusion, we report here, for the first time, that derivatives of isophthalic acid represent an attractive novel group of C1 domain ligands that can be used as research tools or further modified for potential drug development.

Full text of DOI:10.1021/jm900229p

J. Med. Chem. 2009, 52, 3969–3981
3969
Design, Synthesis, and Biological Activity of Isophthalic Acid Derivatives Targeted to the C1
Domain of Protein Kinase C
Gustav Boije af Genna¨s,†,‡ Virpi Talman,†,§ Olli Aitio,†,| Elina Ekokoski,§ Moshe Finel,⊥ Raimo K. Tuominen,§ and
Jari Yli-Kauhaluoma*,‡
DiVision of Pharmaceutical Chemistry, Faculty of Pharmacy, UniVersity of Helsinki, FI-00014 Helsinki, Finland, DiVision of Pharmacology
and Toxicology, Faculty of Pharmacy, UniVersity of Helsinki, FI-00014 Helsinki, Finland, Finnish Biological NMR Center, Institute of
Biotechnology, UniVersity of Helsinki, FI-00014 Helsinki, Finland, Centre for Drug Research, Faculty of Pharmacy,
UniVersity of Helsinki, FI-00014 Helsinki, Finland
ReceiVed February 20, 2009
Protein kinase C (PKC) is a widely studied molecular target for the treatment of cancer and other diseases.
We have approached the issue of modifying PKC function by targeting the C1 domain in the regulatory
region of the enzyme. Using the X-ray crystal structure of the PKC δ C1b domain, we have discovered
conveniently synthesizable derivatives of dialkyl 5-(hydroxymethyl)isophthalate that can act as potential
C1 domain ligands. Structure-activity studies confirmed that the important functional groups predicted by
modeling were indispensable for binding to the C1 domain and that the modifications of these groups
diminished binding. The most promising compounds were able to displace radiolabeled phorbol ester
([3H]PDBu) from PKC R and δ at Ki values in the range of 200-900 nM. Furthermore, the active isophthalate
derivatives could modify PKC activation in living cells either by inducing PKC-dependent ERK
phosphorylation or by inhibiting phorbol-induced ERK phosphorylation. In conclusion, we report here, for
the first time, that derivatives of isophthalic acid represent an attractive novel group of C1 domain ligands
that can be used as research tools or further modified for potential drug development.
Introduction
which can be divided into three subfamilies according to their
regulatory domain structure and cofactor requirements.10,11 All
PKCs are dependent on anionic phospholipids for activation,
with phosphatidyl-L-serine being the most important. In addition,
classical PKCs (R, ꢀI, ꢀII, and γ) require calcium and
diacylglycerol (DAG) for activation, whereas novel PKCs (δ,
ε, η, and θ) require DAG but not calcium. Atypical PKCs (ꢁ
and ι/λ) require neither calcium nor DAG for activation.
DAG is a second messenger that is generated by the phos-
pholipase C-catalyzed hydrolysis of membrane phosphatidyli-
nositol-4,5-bisphosphate (PIP2).12 DAG selectively interacts with
proteins containing a C1 domain and induces their translocation
to discrete subcellular compartments. For some C1 domain-
containing proteins, such a translocation leads to their activa-
tion.13 The C1 domain is a cysteine-rich sequence that forms a
zinc finger structure.10 In the classical and novel PKC isoen-
zymes, the DAG-sensitive C1 domain is duplicated into a
tandem C1 domain consisting of C1a and C1b subdomains.
Atypical PKCs contain a single C1 domain that is not able to
bind DAG. Along with the PKC family, there are six additional
families of proteins that contain a DAG-responsive C1 domain:
the protein kinase D (PKD), the chimaerins, the guanyl
nucleotide-releasing proteins RasGRPs, the Unc-13 scaffolding
proteins, myotonic dystrophy kinase-related Cdc42-binding
kinases (MRCK), and the DAG kinases ꢀ and γ.14,15
Protein kinase C (PKCa) is a family of serine/threonine
protein kinases that is involved in the regulation of various
aspects of cell functions, including cell growth, differentiation,
metabolism, and apoptosis.1 During the past 30 years, it has
become clear that PKC isoenzymes play an important role in
the pathology of several diseases such as cancer, diabetes, stroke,
heart failure, and Alzheimer’s disease.2-8 Therefore, PKC,
particularly in the cancer field, has been a subject of intensive
research and drug development.9
PKC isoenzymes are activated by lipid-derived second
messengers and transmit their signal by phosphorylating specific
protein substrates. The PKC family consists of 10 isoenzymes,
* To whom correspondence should be addressed. Phone: +358 9
19159170. Fax: +358 9 19159556. E-mail: jari.yli-kauhaluoma@helsinki.fi.
†
These authors contributed equally to this work.
Division of Pharmaceutical Chemistry, Faculty of Pharmacy.
Division of Pharmacology and Toxicology, Faculty of Pharmacy.
Finnish Biological NMR Center, Institute of Biotechnology.
‡
§
|
⊥
Centre for Drug Research, Faculty of Pharmacy.
a Abbreviations: [3H]PDBu, [20-3H]phorbol-12,13-dibutyrate; ATP, ad-
enosine-5′-triphosphate; CDI, 1,1-carbonyldiimidazole; DAG, diacylglycerol;
DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCE, 1,2-dichloroethane; DHP,
3,4-dihydro-2H-pyran; DIPEA, diisopropylethylamine; DMEM, Dulbecco’s
modified Eagle’s medium; DMF, N,N-dimethylformamide; DMAP, 4-(dim-
ethylamino)pyridine; DMSO, dimethylsulfoxide; DOG, 1,2-dioctanoyl-sn-
glycerol; ECL, enhanced chemiluminescence; EDC, N-(3-dimethylamino-
propyl)-N′-ethylcarbodiimide; EDTA, ethylenediaminetetraacetic acid; EGTA,
ethylene glycol tetraacetic acid; GAPDH, glyceraldehyde 3-phosphate
dehydrogenase; HBA, hydrogen bond acceptor; HBD, hydrogen bond donor;
HOBt, 1-hydroxybenzotriazole; HRP, horseradish peroxidase; MRCK,
myotonic dystrophy kinase-related Cdc42-binding kinase; IgG, immuno-
globulin G; PBS, phosphate buffered saline; PDBu, phorbol-12,13-dibu-
tyrate; PIP2 phosphatidylinositol-4,5-bisphosphate; PKC, protein kinase C;
PKD, protein kinase D; PMA, phorbol 12-myristate-13-acetate; PPTS,
pyridinium p-toluenesulfonate; PS, phosphatidyl-L-serine; THF, tetrahy-
drofuran; THP, tetrahydropyran-2-yl; TBS, Tris-buffered saline; Tris,
tris(hydroxymethyl)aminomethane; TTBS, 0.1% Tween-20 in TBS; SAR,
structure-activity relationship.
Because the catalytic domain and, in particular, the ATP
binding site of PKC are substantially homologous among protein
kinases16 and the number of C1 domain containing proteins is
considerably smaller than the number of kinases17 the C1
domain provides an attractive target for designing selective PKC
modulators (see the review by Blumberg et al.18). While the
structure of the binding cleft in the C1 domain is highly
conserved, selectivity can be achieved by various strategies.18
Moreover, several studies have already shown that the regulatory
domain may have a biological function independent of the
10.1021/jm900229p CCC: $40.75  2009 American Chemical Society
Published on Web 05/13/2009

3970 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Boije af Genna¨s et al.
catalytic activity of the enzyme,19,20 thus confirming the validity
of targeting the regulatory domain as a therapeutic strategy.
Several classes of high-affinity ligands that target the DAG-
binding C1 domain have been previously described. Naturally
occurring tumor promoters, phorbol esters, were the first ligands
that were found to bind to the C1 domain of PKC.21 In addition
to phorbol esters, naturally occurring C1 domain ligands include
bryostatins,22 teleocidins,23 aplysiatoxins,24 ingenols,25 and
iridals.26 As most of these C1 domain ligands from natural
sources are highly complex in their chemical structure, we and
other groups have been interested in designing a simple C1
domain binding template that is easily amenable to chemical
modification. Blumberg and Marquez groups have described a
large number of conformationally constrained DAG-lactones that
act as potent C1 domain ligands,27,28 while the Irie group has
studied indolactam and benzolactam derivatives as selective
activators of novel PKC isoenzymes.29,30 A number of “first
generation” C1 domain ligands (including calphostin C, bry-
ostatin 1, PEP005, and phorbol esters) are already in clinical
trials for the treatment of different types of cancer.18
In this study, we describe the design, synthesis, biological
activity, and structure-activity relationship of a novel class of
C1 domain ligands: small hydrophobic isophthalic acid deriva-
tives. At low micromolar concentrations, these isophthalic acid
derivatives can compete with phorbol ester binding to PKC and
modulate ERK1/2 phosphorylation in living cells in a PKC-
dependent manner.
Design and Synthesis
Figure 1. Crystal structure of PKC δ C1b domain complexed with
phorbol-13-O-acetate.31 The pivotal polar functional groups of the
phorbol ester involved in C1 domain recognition include the hydroxyl
groups attached to carbon C4 and C20 and the carbonyl group on C3.
The carbonyls of Gly 253 and Leu 251 are HBAs (green arrows), and
the amide proton of Gly 253 and Thr 242 are HBDs (red arrows). The
figure was created using VMD (v.1.8.6., University of Illinois).
We used the crystal structure of the PKC δ C1b domain
complexed with phorbol-13-O-acetate31 as a starting point in
the design of novel C1 domain ligands that bind to the same
site as phorbol esters and DAGs. The X-ray structure of the
complex shows that the pivotal polar functional groups of the
phorbol ester that participate in the recognition of the C1 domain
include the hydroxyl groups attached to carbons C20 and C4
and the carbonyl group on C3 (Figure 1). According to
structure-activity studies,32,33 the C13 carbonyl group is also
important for binding of phorbol-13-O-acetate to the C1 domain.
We selected diethyl 5-(hydroxymethyl)isophthalate as a template
for our SAR studies because it is commercially available and
easily derivatizable. The C2 symmetry of diethyl 5-(hydroxy-
methyl)isophthalate enables two binding orientations, similar
to DAG-lactones;34 it also contains two of the phorbol ester
pharmacophores, namely the hydroxyl and the carbonyl func-
tionalities with the same interatomic distance as that found in
phorbol.35 Furthermore, dialkyl 5-(hydroxymethyl)isophthalates
contain two carbonyl groups, a prerequisite for high affinity
binding of DAG-lactones.34 We conducted docking experiments
to verify that the phorbol-like binding mode, with the correct
positioning of the hydrogen bonding network, was feasible for
dialkyl 5-(hydroxymethyl)isophthalate structures. Indeed, our
docked model (I) (Figure 2) showed that dipentyl 5-(hydroxy-
methyl)isophthalate was anchored to the bottom of the binding
site via the same interactions as the phorbol ester, i.e., the
ligand’s hydroxyl group is hydrogen bonded to the backbone
amide proton of Thr 242 and the carbonyls of Thr 242 and Leu
251. The model also showed that one of the carbonyl groups
of I forms a hydrogen bond with the backbone amide proton of
Gly 253 in a similar fashion as the C3 carbonyl of phorbol-
13-O-acetate. The second carbonyl group of I seemed to make
no contact with the C1 domain; however, it has been proposed
that the complex might be stabilized by either a bridging water
molecule between this carbonyl and the backbone carbonyl of
Met 239 or by interactions with charged lipid head groups.36
Hydrophobic interactions are crucial for high affinity binding
to the C1 domain. The hydrophobic side chains of phorbol
esters, DAGs, and their analogues are thought to interact with
either the hydrophobic amino acids (Met 239, Pro 241, Phe 243,
Leu 250, Trp 252, and Leu 254) surrounding the ligand binding
cleft or with the hydrophobic moiety in the lipid bilayer.31,37
Because it is not known how the C1 domain interacts with the
membrane, hydrophobic interactions are difficult to model. We
therefore prepared dialkyl 5-(hydroxymethyl)isophthalate deri-
vates with different side chains to analyze their effect on binding
affinity and activity.
We initially prepared simple derivatives of the template (I)
that differed in the composition of the ester groups in order to
investigate the length and shape of the alkyl chains required
for activity. As starting material for the esters, we chose
compounds containing primary and secondary alkyl chains as
well as compounds containing aromatic groups. (see Table 1
for the structures). To investigate the hydrophobicity of the
compounds that was necessary for activity, we also prepared
three additional hydrophilic derivatives.
The dialkyl 5-(hydroxymethyl)isophthalates (1) were conve-
niently synthesized in only four steps using the commercially
available diethyl 5-(hydroxymethyl)isophthalate as the starting
material (2) (Scheme 1). Protection of the hydroxyl group of 2
as a THP ether (3), which was followed by hydrolysis of the
ester groups, gave the dicarboxylic acid (4). This was subse-
quently treated with an alcohol, CDI, DMAP, and DBU, or with
an alkyl halide, KI, and K2CO3, to give the diesters (5a1-3
and 5b1-19). Some of the alcohols used in these acylation and

PKC-Targeted Compounds
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3971
To increase the hydrophilicity of the compounds, we
prepared several amide derivatives of the diesters (Schemes
4 and 5; Table 2). Synthesis of the 5-(hydroxymethyl)isoph-
thalamides (24a-b) was initiated by coupling the amines with
4. Subsequent deprotection of the THP groups in 23a-b
produced compounds 24a-b. The reverse diamide (27) was
prepared from the 3,5-diaminobenzyl alcohol (25) by acy-
lation and the subsequent chemoselective hydrolysis of 26.
The second reverse diamide was synthesized by catalytic
hydrogenation of the nitro groups in 28, followed by acylation
of the resultant phenylenediamine (29) to give the diamide
(30), which was used as a negative control compound for
binding to the C1 domain of PKC R and δ.
In the following set of synthesis reactions, we wanted to
investigate the effect of having one ester and one reverse
amide group in the compound. Synthesis of the 3-(hydroxym-
ethyl)-5-(alkanoylamino)benzoates (36a-e) was initiated
from the commercially available 5-nitroisophthalic acid
monomethyl ester (31) (Scheme 6 and Table 2). The
hydroxymethyl group, which is important for binding, was
obtained in the first step by chemoselective reduction of the
carboxylic acid (31) using borane dimethyl sulfide complex.
This gave a better yield (84%) than using the borane
tetrahydrofuran complex (70%).39 The alcohol (32) was
protected as a THP ether (33), and the subsequent catalytic
reduction of the nitro group gave the amine product (34).
The subsequent N-acylation with a set of carboxylic acids
yielded the corresponding amides (35a-e), while the final
deprotection of the THP-groups by PPTS gave the products
36a-e.
We also prepared one monoester that contained an amide
group (39). This synthesis commenced with monohydrolysis
of the THP-protected diester (3) (Scheme 7) and the
subsequent N-acylation of 3-(trifluoromethyl)benzylamine
with 37 to give 38. Deprotection of the THP ether using PPTS
and purification by SiO2 column chromatography yielded the
amide product (39).
Figure 2. Dipentyl (5-hydroxymethyl)isophthalate (I) docked in the
PKC δ C1b domain. The hydroxyl group of the ligand is hydrogen
bonded to the backbone amide proton of Thr 242 (red arrow) and the
carbonyls of Thr 242 and Leu 251 (green arrows). The model also
shows that one of the carbonyl groups of the template (I) forms a
hydrogen bond with the backbone amide proton of Gly 253 (red arrow).
A bridging water molecule between the backbone carbonyl of Met 239
and the other carbonyl (black arrows) could stabilize the complex. The
figure was created using VMD (v.1.8.6., University of Illinois).
substitution reactions were not commercially available and were
synthesized in our laboratory (see Supporting Information). The
hydroxymethyl groups of 5a1-3 and 5b1-19 were deprotected
to give the targeted diesters (1a1-3 and 1b1-19) (Table 1).
This procedure was used to prepare all the required diesters
with the need for only a single chromatographic purification
step. In addition to the diesters, the dicarboxylic acid (1b20)
was prepared by the hydrolysis of (2) (Table 1, see Supporting
Information).
The ester groups of 1a3 and 1b2 were selected to further
SAR studies. We focused on the hydroxymethyl group of
the template (I) and synthesized a group of compounds
(15a-e) that lacked this group (Scheme 2, Table 2). These
were prepared from the corresponding carboxylic acids, and
one unsymmetrical diester (15c) was synthesized among these
esters.
To investigate the significance of having two ester groups
in the template molecule (I), we prepared three monoesters
(22a-c) (Scheme 3 and Table 2). Treatment of 16 with NaOH
gave monomethyl isophthalate (17). Reduction of the car-
boxylic acid was performed with borane dimethyl sulfide
complex (BH3 · SMe2),38 and the resultant alcohol (18) was
protected as a THP-ether (19). Hydrolysis of the methyl ester
(19) gave the carboxylic acid (20), which was converted to
esters (21a-c). Finally, the deprotection of the THP group
by PPTS yielded the ester products (22a-c).
Biological Data
Screening and Structure-Activity Analysis for Binding
to PKC C1 Domain. We first screened the synthesized
compounds for binding to the C1 domain of recombinant human
PKC R and δ using a filtration method on 96-well plate format.40
We used crude cell lysates from baculovirus-infected Sf 9 cells
as the source for PKC in the assay; the lysate from uninfected
Sf 9 cells did not exhibit any phorbol ester binding (not shown).
The compounds were used at a concentration range of 0.1-20
µM, with the highest concentration being based on the estimated
maximal solubility in aqueous buffer. The results revealed that
the first set of compounds with different ester side chains, with
the exception of compounds 1b17-1b21 (Table 1), displaced
[20-3H]phorbol-12,13-dibutyrate ([3H]PDBu) from PKC R and
δ in a concentration-dependent manner. Compounds 1b17, 1b18,
and 1b19 have more hydrophilic side chains and were unable
to displace [3H]PDBu at the concentration range used. In
addition, diethyl 5-(hydroxymethyl)isophthalate (1b21, Table
1), which contains significantly shorter side chains, and 5-(hy-
droxymethyl)isophthalic acid (1b20, Table 1), which lacks the
alkyl side chains, were unable to bind to the PKC C1 domain
under the conditions employed in this study. Compounds 1b10,
1b11, and 1b13, all with aliphatic carbon side chains and high
hydrophobicity, appeared to be the most effective in displacing
[3H]PDBu from PKC R and δ. Total binding was the only factor

3972 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Boije af Genna¨s et al.
Table 1. 5-(Hydroxymethyl)isophthalates 1a1-3 and 1b1-21, Their clogP Values, and Binding Affinity to PKC R and δa
a The binding affinity was screened as described in the experimental section and is expressed as mean ( sem (n ) 3-8) of residual [3H]PDBu binding
(% of control) with 20 µM ligand concentration.
Scheme 1a
a Conditions: (a) DHP, PPTS, DCE, rt, 6 h, 97%; (b) KOH (10%, aq), MeOH, reflux, 1 h, 85%; (c) alkyl halide, K2CO3, KI, DMF, 110 °C, 2 h (5a1-3)
or alcohol, CDI, DMAP, DBU, DMF, rt f 40 °C, 22 h (5b1-19); (d) Dowex 50W × 8, MeOH, 40 °C, overnight, 10-95% (two steps).
that was measured in these initial assays, and therefore no
binding constants or IC50 values were calculated based on these
results.
We next examined the contribution of the hydroxymethyl
group and the ester groups to the binding affinity of isophthalic
acid derivatives. All compounds lacking the hydroxymethyl

PKC-Targeted Compounds
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3973
Scheme 2a
a Conditions: (a) 2-methyl-1-pentanol, CDI, DMAP, DBU, DMF, rt f 40 °C, 19-21 h (14a-d) or 3-(trifluoromethyl)benzyl chloride, K2CO3, KI, DMF,
80 °C, 2 h, (14e), 28-76%.
Table 2. Synthesized Derivatives, Their clogP Values, and Binding Affinity to PKC R and δa
a The binding affinity was screened as described in the Experimental Section and is expressed as the mean ( SEM (n ) 3-8) of residual [3H]PDBu
binding (% of control) with 20 µM ligand concentration.
group (replaced with hydrogen, or a methyl, or a nitro group)
exhibited a significantly diminished binding at the concentration
range used in this study (Table 2, compounds 15a-e). Ad-
ditionally, the unsymmetrical ligands that lacked one of the ester
groups (Table 2, compounds 22a-c) were not able to displace
[3H]PDBu from PKC (Table 2). Thus, the hydroxymethyl group
and both of the ester groups are essential for creating sufficient
binding affinity to the C1 domain.
We also investigated the effect of replacing one or both of
the ester bonds with amide bonds, both to test derivatives with

3974 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Boije af Genna¨s et al.
Scheme 3a
a Conditions: (a) NaOH (1.05 equiv), MeOH, acetone, rt, 21 h then NaOH (0.1 equiv), rt, 4 h, 98%; (b) BH3 ·SMe2, THF, 0 °C f rt, 5 h, 29%; (c) DHP,
PPTS, DCE, rt, 16 h, 99%; (d) KOH (10%, aq), MeOH, 90 °C, 17 h, 67%; (e) alcohol, CDI, DMAP, DBU, DMF, rt f 40 °C, 21 h; (f) Dowex 50W × 8,
MeOH, 40 °C, 17 h, 65-75% (two steps).
Scheme 4a
a Conditions: (a) DIPEA, EDC, HOBt, amine, rt f 40 °C, 4-23 h; (b) Dowex 50W × 8, MeOH, 40 °C, 23-24 h, 28-39% (two steps).
Scheme 5a
a Conditions: (a) heptanoyl chloride (R ) (CH2)5CH3), pyridine, DCM, rt, 20 h; (b) KOH (10%, aq), MeOH, rt, 4 h, 95%; (c) H2, Pd/ C (10%), EtOH:
EtOAc (2:1), rt, 22 h, 99%; (d) Heptanoyl chloride, DIPEA, DCM, rt, 21 h, 48%.
different groups around the aromatic backbone and to increase
the hydrophilicity of the compounds. The C2 symmetric amides,
where both of the ester groups of the template I were replaced
with amide groups (Table 2, compounds 24a-b, 27, and the
negative control 30), were unable to bind to PKC with a
comparable affinity compared to the original compounds. This
was true irrespective of the orientation of the amide group
(compound 24b versus the reverse amide 27). Additionally, most
of the monoamide derivatives (Table 2, compounds 36a-e and
39) were unable to displace [3H]PDBu from the PKC C1 domain
as effectively as the original compounds. Only compound 36a,
which had a long hydrophobic side chain, showed a slight
concentration-dependent binding affinity to PKC R and δ.
In summary, our results show that the hydroxymethyl group
and the ester groups of the template (I) are indispensable
for binding to the PKC C1b domain. Modifying these regions
of the molecule leads to diminished binding. In addition to
these functional groups, the amount of hydrophobicity of the
side chains appears to play an important role in achieving
binding affinity in the micromolar range.
Binding Affinity Constants of Selected Compounds to
Purified PKC r and δ. Following the initial screening for
binding against PKC R and δ, we determined the binding
constants for three diesters (1a3, 1b10, and 1b11). Because
the screening data suggested that the affinities of most of
the ligands were within the same concentration range, we
did not determine binding constants for all of the ligands.
The Kd values determined with [3H]PDBu saturation binding
and Scatchard analysis to purified commercial recombinant
human PKC R and δ were 1.29 and 2.35 nM, respectively
(data not shown). We therefore used a concentration of 10
nM for the radioligand in the subsequent competition binding
experiments. To validate the assay, we determined the Ki
values for 1,2-dioctanoyl-sn-glycerol (DOG) and bryostatin
1. DOG inhibited the binding of [3H]PDBu to PKC R with a
Ki of 40.1 ((6.0) nM, and bryostatin 1 inhibited the binding
of [3H]PDBu to PKC δ with a Ki of 0.44 ((0.08) nM (Table
3). These values are in accordance with published data.41,42
The inhibitory binding constants for compounds 1a3, 1b10,
and 1b11 are shown in Table 3. All three ligands were

PKC-Targeted Compounds
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3975
Scheme 6a
a Conditions: (a) BH3 ·SMe2, THF, 0 °C f 60 °C, 21 h, 86%; (b) DHP, PPTS, DCE, rt, 25 h, 99%; (c) H2, Pd/C (10%), EtOH:THF (2:1), rt, 23 h, 100%;
(d) RCO2H, DIPEA, EDC, HOBt, DCM, 40 °C, 21-23 h, 13-58%; (e) Dowex 50W × 8, MeOH, 40 °C, 19-24 h, 46-89%.
Scheme 7a
a Conditions: (a) KOH (1 equiv), MeOH, 40 °C, 20 h, 65%; (b) DIPEA, EDC, HOBt, amine, rt f 40 °C, 4 h; (c) Dowex 50W × 8, MeOH, 40 °C, 23 h,
41% (two steps).
Table 3. Binding Affinities of Selected Isophthalate Derivativesa
ERK1/2 phosphorylation in living cells. ERK1/2 is member
of the mitogen activated protein (MAP) kinase signaling
cascade that controls a broad range of cellular activities.43
It has been shown that multiple PKC isoforms can induce
ERK1/2 phosphorylation via the Raf-MEK pathway; ad-
ditionally, PMA-induced ERK1/2 phosphorylation is medi-
ated by PKC.44
Ki (nM) PKC R
Ki (nM) PKC δ
1a3
1b10
1b11
205 ( 14 (4)
661 ( 90 (3)
319 ( 12 (3)
590 ( 195 (4)
915 ( 132 (3)
529 ( 61 (3)
DOG
bryostatin 1
40.1 ( 6.0 (4)
nd
nd
0.44 ( 0.08 (5)
a Data represent the means ( SEM. The number of independent
experiments is shown in parentheses (nd ) not determined).
Human cervical cancer (HeLa) cells were treated with a
20 µM concentration of compounds 1a3, 1b10, and 1b11
for 1-20 min. Compounds 1a3 and 1b10, but not 1b11,
induced ERK1/2 phosphorylation within 3 min (Figure 4A).
The phosphorylation induced by 1a3 and 1b10 was blocked
by pretreatment with the MEK inhibitor U0126 (Figure 4A).
The PKC inhibitor Go¨6983 (final conc 1 µM) inhibited the
1a3 and the 1b10-induced ERK1/2 phosphorylation by 38%
( 7% and 36% ( 9%, respectively (Figure 4A), as
determined by the quantification of Western blots from 3-4
independent experiments. Because 1b11 was able to displace
[3H]PDBu from PKC in vitro but could not induce ERK1/2
phosphorylation by itself, we examined whether it could still
compete with phorbol esters binding to PKC in living cells.
HeLa cells were treated with various concentrations of 1b11
for 30 min before stimulating the cells for 5 min with 10
nM PMA. PMA alone induced a pronounced phosphorylation
of ERK1/2 (Figure 4B). Pretreatment of the cells with 1b11
capable of displacing the radioactively labeled PDBu with a
comparable submicromolar affinity. The ligands bound to
both PKC R and δ at nearly identical affinities and with no
significant selectivity toward either of the isoenzymes. In
general, the Ki values were somewhat lower for PKC R than
for δ. However, compound 1a3 was able to displace a
substantially smaller proportion of the radioligand from PKC
δ than from R, while 1b10 and 1b11 exhibited similar
efficacy toward both isoenzymes (Figure 3).
The Effect of Isophthalic Acid Derivatives on ERK1/2
Phosphorylation in HeLa Cells. Because the compounds
could compete with PDBu in vitro, we wanted to examine
whether they were able to penetrate the cell membrane and
modulate PKC activity within the cellular environment. To
investigate this issue, we studied their ability to modulate

3976 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Boije af Genna¨s et al.
Figure 3. Binding of selected isophthalate derivatives to purified PKC R and δ. Binding of 10 nM [3H]PDBu was measured in the presence of
different concentrations of the isophthalate derivatives. Representative competition binding curves for (A) PKC R and 1a3, (B) PKC R and 1b10,
(C) PKC R and 1b11, (D) PKC δ and 1a3, (E) PKC δ and 1b10, and (F) PKC δ and 1b11. Data represent means from three parallel samples in
a single experiment.
inhibited the PMA-evoked phosphorylation of ERK1/2 in a
concentration-dependent manner (Figure 4B).
[3H]PDBu for binding to PKC R or δ either. The results that
highlight the importance of hydrophobicity in ligand binding
are similar to those reported for other C1 domain ligands,35
including the DAG-lactones, which have been shown to have
an estimated optimal clogP value between 5 and 6.37 However,
because the clogP value of PDBu (3.43) is significantly lower
and PDBu has been shown to have a higher affinity to the C1
domain45 than the isophthalate derivatives or the DAG-lactones,
it might be possible to modify the structure and reduce the
lipophilicity while increasing the binding affinity.
Discussion
Several laboratories have focused their studies on the C1
domain of PKC in order to discover PKC-modulating com-
pounds for drug development. Bryostatin 1, a marine-derived
compound that acts through the C1 domain, is currently in phase
II clinical trials for the treatment of cancer. However, naturally
occurring C1 domain ligands are highly complex in their
structure. Therefore, structural modification with the aim of
altering the ligand specificity is difficult.18 DAG-lactones and
indolactam derivatives, the major groups of synthetic C1 domain
ligands, are considerably simpler than the natural products,18
however, they are also laborious to synthesize and modify. We
therefore sought to find simpler template for ligand synthesis,
whose structure could be easily modified and fine-tuned in order
to achieve selectivity.
C2 symmetric diethyl 5-(hydroxymethyl)isophthalate is a
commercially available and easily derivatizable compound.
While being structurally simple, our molecular docking experi-
ments showed that its derivative, dipentyl 5-(hydroxymethyl)-
isophthalate, could bind to the C1 domain of PKC in a similar
fashion as phorbol esters. The same hydrogen bond interactions
that have been proposed to occur for other C1 domain ligands
(such as phorbol esters35 and DAG-lactones27) could be attained
with the isophthalate core structure. We synthesized a series of
isophthalic acid derivatives to confirm their binding to the C1
domain of PKC and to study the impact that different side chains
had on the binding affinity. Binding studies showed that the
compounds were able to displace [3H]PDBu from the C1
domains of PKC R and δ. Modifying the ester side chains had
a very modest effect on the binding as long as the side chains
provided sufficient hydrophobicity, which appears to be an
important determinant for the binding affinity. The best com-
pounds had clogP values higher than 5, whereas analogues with
lower clogP values, e.g., the ether analogue 1b19 of 1b1 (clogP
values of 2.35 and 6.33, respectively) did not exhibit high
binding affinity. In addition, 1b21, which carries short ester
groups and has a clogP value of 2.10, could not compete with
We also studied the effects of replacing the structural moieties
that, as predicted by the pharmacophore model, were required
for C1 domain recognition. As expected, the hydroxymethyl
group and both of the carbonyl groups were important for
binding. Surprisingly, the diamide derivatives (24a-b and 27)
were unable to bind to the PKC C1 domain. This might be due
to unfavorable interactions of the two amide protons with the
C1 domain. The monoamide derivatives 36a-e were also unable
to compete with [3H]PDBu for binding to the C1 domain of
PKC R and δ in a similar manner as the corresponding diester
analogues. However, the compound with the longest and most
hydrophobic amide side chain (36a) and therefore the highest
clogP value (4.84, compared to 0.56-4.26 for 36b-e), exhibited
some binding toward PKC. One can therefore speculate that
both the decreased hydrophobicity and the lack of the other ester
group contribute to the diminished binding affinity observed
with the monoamide derivatives. A compound with some
similarity to these monoamide derivatives (36a-e) has previ-
ously been described to inhibit phorbol ester binding to the PKC
regulatory domain.46
On the basis of the results from screening for [3H]PDBu
displacement, we selected compounds 1a3, 1b10, and 1b11 for
further testing. Using the assay developed by the Blumberg
group,47 we were able to show that the compounds bind to PKC
R and δ with comparable submicromolar affinities. Compounds
1b10 and 1b11 displaced [3H]PDBu from both PKC isoenzymes
in a similar manner. However, compound 1a3 was able to
displace only a small proportion of the radioligand from PKC
δ while exhibiting more efficient displacement of [3H]PDBu
from PKC R. The differences in the spatial geometry of the

PKC-Targeted Compounds
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3977
C1a domain of PKC R binds DAG with a higher affinity than
phorbol esters, whereas the PKC R C1b domain exhibits higher
affinity for phorbol esters than for DAG.41 On the other hand,
both the C1a and C1b domains of PKC γ bind DAG and phorbol
esters with high affinities.41 Thus, while it is tempting to
speculate about the basis for this potential selectivity of 1a3
toward classical isoenzymes, making definitive conclusions
about the specificity mechanism at this stage may be premature.
Further studies with different PKC isoenzymes and individual
C1 domain constructs are needed to clarify the selectivity profile
of these compounds.
The hydrophobicity of the compounds suggests that within a
cellular environment the compounds could easily diffuse into
the cell membranes and reach PKC in a similar manner as that
of phorbol esters and DAGs. To address both the cell membrane
penetration and the modulation of PKC activity within cellular
environment, we studied the effects of compounds 1a3, 1b10,
and 1b11 on ERK1/2 phosphorylation in HeLa cells. Com-
pounds 1a3 and 1b10 induced ERK1/2 phosphorylation, which
was inhibited by pretreatment with the PKC inhibitor Go¨6983,
thus confirming that the effect was PKC-dependent. Surprisingly,
while the structure of 1b11 differs from 1b10 by only a single
carbon in the ester side chains, 1b11 was unable to induce
ERK1/2 phosphorylation. However, 1b11 inhibited the PMA-
induced ERK1/2 phosphorylation, demonstrating that it can
penetrate the cell membrane and bind to the PKC C1 domain
in living cells. These results suggest that, under these conditions,
compounds 1a3 and 1b10, but not compound 1b11, are capable
of activating one or more PKC isoforms in HeLa cells. It is
possible that 1b11 had a weak activating effect on PKC, which
was not detected with the ERK phosphorylation assay. As a
weak activator, 1b11 could still antagonize the effect of a much
stronger activator (PMA). Such an antagonism has been reported
between the PKC activator bryostatin 1 and PMA.50 The
activation and/or inhibition profiles of these compounds need
to be studied in more detail.
Figure 4. Effects of 1a3, 1b10, and 1b11 on ERK1/2 phosphorylation
in HeLa cells. (A) Cells were treated with 20 µM concentration of
1a3, 1b10, or 1b11 for the indicated times. The PKC inhibitor Go¨6983
(1 µM) or the MEK inhibitor U0126 (10 µM) were added to the cells
5 min before the addition of the test compounds. PMA (10 nM for 5
min) was used as a positive control. Cells were harvested and ERK1/2
phosphorylation was detected as described in Experimental Section.
(B) Effect of 1b11 on PMA-induced ERK1/2 phosphorylation. A
representative blot from a single experiment (upper panel) and
quantification results (mean ( SEM) from three independent experi-
ments are shown (lower panel). The cells were treated with different
concentrations of 1b11 for 25 min and subsequently stimulated with
10 nM of PMA for 5 min. The cells were then harvested and ERK1/2
phosphorylation was detected as described in the Experimental Section.
(a) Untreated cells, (b) PMA alone, (c) 1 µM of 1b11 + PMA, (d) 5
µM of 1b11 + PMA, (e) 20 µM of 1b11 + PMA, (f) 20 µM of 1b11
alone.
On the basis of the results presented here, we are currently
working toward improving the affinity and selectivity of the
isophthalate derivatives. Because there are additional proteins
in cells that contain a phorbol-responsive C1 domain, binding
of these compounds to non-PKC phorbol ester receptors will
be studied. Selectivity, while difficult to attain because of the
high degree of conservation within the binding cleft of the C1
domain, is achievable, as demonstrated with the DAG-lac-
tones.28,51 Furthermore, preliminary studies have shown that a
number of compounds described in this report inhibit HeLa cell
proliferation (Talman et al. unpublished results); studies to
delineate the mechanism of those effects are in progress.
Additionally, preliminary pharmacokinetic evaluation of cell
membrane permeability and absorption are currently being
carried out on these compounds.52
side chains could be one possible explanation to the observed
phenomenon. The side chains of 1b10 and 1b11 are aliphatic
and therefore more flexible than the aromatic side chains of
1a3, which are more constrained as a result of the planar
conformation of the aromatic rings. In addition to the differences
in the side chains of the compounds studied, there are also
differences between individual C1 domains of different PKC
isoenzymes. Particularly, there is a marked difference in DAG
binding between the novel and the classical PKC isoenzymes.48
The novel PKC isoenzymes are more sensitive to DAG binding
than the classical isoenzymes, a phenomenon that appears to
be dependent on a single amino acid residue within the C1
domain.49 Because the PKC isoforms studied here include both
classical PKCs and novel PKCs (PKC R and δ, respectively),
it is possible that the structures of their binding sites would
differ sufficiently to contribute to the observed phenomenon.
However, differences exist also between the affinity of individual
C1a and C1b domains of PKC isoenzymes within the subgroups
of PKC isoenzymes.11 For example, in the case of cPKCs, the
Conclusions
In conclusion, we describe here a novel group of compounds,
dialkyl 5-(hydroxymethyl)isophthalates, that target the phorbol
ester binding site of PKC. The compounds displace [3H]PDBu
from the C1 domain of PKC R and δ in vitro at submicromolar
concentrations and can regulate PKC-dependent signaling in
living cells, as shown by the ERK phosphorylation studies. The
compounds are conveniently and effectively synthesized starting
from the commercially available diethyl 5-(hydroxymethyl)-
isophthalate. This makes isophthalic acid derivatives an at-

3978 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Boije af Genna¨s et al.
can Type Culture Collection (ATCC, Manassas, VA) and cultured
at 37 °C in a humidified, 5% CO2 atmosphere with DMEM (Sigma-
Aldrich, Steinheim, Germany) supplemented with 10% fetal bovine
serum, 100 U/mL penicillin, and 100 µg/mL streptomycin.
Molecular Modeling. Molecular docking modeling was per-
formed using the crystal structure of PKC δ C1b domain complexed
with phorbol-13-O-acetate (Protein Data Bank code: 1PTR).31 First,
the phorbol-13-O-acetate was removed from the structure. The
hydrogens, as well as the side chains of Lys 234, Arg 273, and
Glu 274 not visible in the X-ray structure, were subsequently added
to the protein and the structure was energy minimized in SYBYL
7.0 (Tripos, St Louis, MO) using the TRIPOS force field. A hundred
iterations using the conjugate gradient method were performed.
Superimposition of the experimental and minimized structures
showed no significant deviations. The minimized structure was used
for ligand docking. Ligand geometry was optimized using the
semiempirical AM153 method in Spartan 5.1.3 (Wave function Inc.,
Irvine CA). The docking experiments were performed using
AUTODOCK3.0.54 Gasteiger-Hu¨ckel partial charges were calcu-
lated in SYBYL7.0 for both protein and ligand. Grid maps with
60 × 60 × 60 points and a grid-point spacing of 0.375 Å centered
in the phorbol binding pocket were calculated. All ligand torsion
angles were considered as flexible. Fifty separate docking simula-
tions were performed using the Lamarckian genetic algorithm. A
validation of the docking protocol was performed using phorbol-
13-O-acetate as ligand. The X-ray structure was well reproduced
as described previously.37
tractive candidate for further SAR studies and for further develop-
ment as research tools or lead compounds in drug development.
Experimental Section
Materials and General Procedures. All reagents were com-
mercially available and were acquired from Fluka (Buchs, Swit-
zerland) and Sigma-Aldrich (Schnelldorf, Germany). THF and Et2O
were distilled from sodium/benzophenone ketyl. CHCl3 was distilled
from CaH2. Anhydrous DMF was from Fluka (Buchs, Switzerland)
and was stored over molecular sieves (4 Å) under an inert
atmosphere of dry argon. All reactions in anhydrous solvents were
performed in flame-dried glassware under an inert atmosphere of
dry argon. The progress of chemical reactions was monitored by
thin-layer chromatography on silica gel 60-F254 plates acquired from
Merck (Darmstadt, Germany) using phosphomolybdic acid stain
(10% by weight in EtOH) or ninhydrin stain (1.5% by weight in
EtOH). Flash SiO2 column chromatography was performed with a
Merck silica gel 60 (230-400 mesh) or with a Biotage high-
performance flash chromatography Sp4-system (Uppsala, Sweden)
using a 0.1 mm path length flow cell UV-detector/recorder module
(fixed wavelength: 254 nm), 12 mm or 25 mm flash cartridges,
and the indicated mobile phase.
1H NMR, 13C NMR and DEPT spectra were recorded on a Varian
Mercury 300 MHz or a Varian Unity 500 MHz spectrometer
(Varian, Palo Alto, CA) as solutions in CDCl3, DMSO-d6, CD3OD,
or CD2Cl2. Deuterated solvents were purchased from Aldrich.
Chemical shifts (δ) are given in parts per million (ppm) relative to
the NMR solvent signals (CDCl3 7.26 and 77.21 ppm, DMSO-d6
2.50 and 39.52 ppm, CD3OD 3.31 and 49.00 ppm, CD2Cl2 5.32
Synthesis. Procedures for the synthesis and analysis of the
remaining compounds are available in the Supporting Information.
Diethyl 5-(Tetrahydropyran-2-yloxymethyl)isophthalate (3). A
mixture of diethyl (5-hydroxymethyl)isophthalate (2) (5.72 g, 22.7
mmol), 3,4-dihydro-2H-pyran (5.54 mL, 61.3 mmol, 2.7 equiv),
pyridinium p-toluenesulfonate (285 mg, 1.14 mmol, 0.05 equiv),
and 1,2-dichloroethane (50 mL) was stirred at room temperature
for 6 h. The reaction mixture was quenched by the addition of cold
water (50 mL), extracted with DCM (50 mL) and washed with a
saturated solution of NaHCO3 (3 × 20 mL) and brine (3 × 20
mL). The organic phases were dried over Na2SO4, filtered, and
evaporated in vacuo to give 3 as a pale-yellow oil (7.41 g, 97%
yield). This oil was used without further purification, and full
product analysis was carried out for the following product.
5-(Tetrahydropyran-2-yloxymethyl)isophthalic Acid (4). A mix-
ture of 3 (3.46 g, 10.3 mmol), a 10% solution of KOH (46.2 mL,
82.4 mmol, 8 equiv), and MeOH (50 mL) was refluxed for 1 h.
The solvents were evaporated in vacuo and pH was adjusted with
a 25% solution of KHSO4 to 4.0. The white precipitate was filtered,
washed with water (10 mL), dissolved into a solution of EtOAc
and THF (1:1, 3 × 50 mL), dried over Na2SO4, filtered, and
evaporated in vacuo to give 4 as a white solid (2.45 g, 85% yield).
1
and 53.80 ppm for H and 13C NMR, respectively). Multiplicities
are indicated by s (singlet), d (doublet), t (triplet), q (quartet), quintet
(qn), and m (multiplet). The synthesized compounds were analyzed
by HPLC-MS. HPLC-MS analyses were performed to determine
the purity of all tested compounds by a HP1100 instrument with
UV detector (λ 210 nm) and a Perkin-Elmer Sciex API3000
triple-quadrupole LC/MS/MS mass spectrometer (Applied Bio-
systems/MDS Sciex, Concord, Canada) with a turbo ESI source.
Signal separation was carried out by a XTerra MS RP18 column
(4.6 mm × 30 mm, 2.5 µm). High resolution mass spectra (HRMS)
were run on a Q-TOF Micro (quadrupole time-of-flight) mass
spectrometer (Waters/Micromass, Manchester, UK) with an ESI
source in positive ion mode. The instrument was calibrated with
an internal standard. The elemental analyses were conducted by
Robertson-Microlit Laboratories (Madison, NJ). Melting points were
measured with an Electrothermal IA 9100 apparatus. Purity of all
tested compounds was >95%. logP Values for the compounds were
calculated using ChemBioDraw Ultra 11.0 (CambridgeSoft, Cam-
bridge, MA).
1
[20-3H]Phorbol-12,13-dibutyrate ([3H]PDBu) was custom labeled
by Amersham Radiolabeling Service (GE Healthcare, Little Chal-
font, UK). Unlabeled PDBu, phorbol 12-myristate-13-acetate
(PMA), phosphatidyl-L-serine (PS), and bovine immunoglobulin
G (IgG) were purchased from Sigma-Aldrich (Steinheim, Germany).
Purified recombinant human PKC R and δ were from Invitrogen
(Carlsbad, CA). Protease inhibitors (Complete Protease Inhibitor
Cocktail Tablets) and phosphatase inhibitors (PhosSTOP Phos-
phatase Inhibitor Cocktail Tablets) were from Roche (Mannheim,
Germany). Anti-ACTIVE MAPK polyclonal antibody was from
Promega (Madison, WI), anti-GAPDH monoclonal antibody, and
HRP-conjugated goat antimouse IgG were from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA), and the HRP-conjugated goat
antirabbit IgG was from Bio-Rad Laboratories (Hercules, CA).
Precision Plus Protein Kaleidoscope standard ladder (Bio-Rad
Laboratories, Hercules, CA) was used as the SDS-PAGE marker.
ECL reagents (SuperSignal-West-Pico-Chemiluminescent-Substrate-
Kit) were from Pierce (Thermo Fisher Scientific Inc., Rockford,
IL).
This solid was used without further purification. H NMR (300
MHz, DMSO-d6): δppm 13.3 (bs, 2H), 8.39 (t, 1H, J 1.5 Hz), 8.13
(d, 2H, J 1.5 Hz), 4.80 (d, 1H, J 12.6 Hz), 4.72 (t, 1H, J 3.0 Hz),
4.59 (d, 1H, J 12.6 Hz), 3.82-3.75 (m, 1H), 3.52-3.45 (m, 1H),
1.80-1.62 (m, 2H), 1.58-1.48 (m, 4H). 13C NMR (75.4 MHz,
DMSO-d6): δppm 166.5, 139.8, 132.1, 131.3, 129.0, 97.7, 67.3, 61.4,
30.1, 25.0, 19.1.
Bis(3-trifluoromethylbenzyl) 5-(Tetrahydropyran-2-yloxymeth-
yl)isophthalate (5a3). A mixture of 4 (1.00 g, 3.57 mmol),
3-(trifluoromethyl)benzyl chloride (2.08 mL, 10.7 mmol, 3 equiv),
K2CO3 (2.47 g, 17.8 mmol, 5 equiv), and KI (650 mg, 3.92 mmol,
1.1 equiv) in dry DMF (18 mL) was heated at 80 °C for 150 min.
The reaction mixture was cooled to rt, quenched by the addition of
an ice-water mixture (20 mL), extracted with EtOAc (3 × 20 mL),
dried over Na2SO4, filtered, and evaporated in vacuo to give 5a3
as a brown oil. This and the following crude diesters (5b10-11)
were used in the subsequent deprotection step without further
purification, and full product analysis was carried out for the
products 1a3 and 1b10-11.
Cell culture solutions and reagents were purchased from Invit-
rogen (Carlsbad, CA) unless otherwise stated. Sf9 cells were
purchased from Invitrogen. HeLa cells were acquired from Ameri-
Bis(1-ethylbutyl) 5-(Tetrahydropyran-2-yloxymethyl)isophtha-
late (5b10). A mixture of 4 (250 mg, 0.89 mmol) and 1,1-
carbonyldiimidazole (318 mg, 1.96 mmol, 2.2 equiv) in dry DMF

PKC-Targeted Compounds
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3979
(1 mL) was stirred at room temperature for 1 h. 3-Hexanol (330
µL, 2.68 mmol, 3 equiv), 4-(dimethylamino)pyridine (11 mg, 0.09
mmol, 0.1 equiv), and 1,8-diazabicyclo[5.4.0]undec-7-ene (332 µL,
1.78 mmol, 2 equiv) were added to the reaction mixture that was
stirred at 40 °C for 43 h. The reaction was quenched with ice-water
(10 mL) and extracted with EtOAc (3 × 20 mL). The combined
organic phases were washed with a saturated solution of NaHCO3
(2 × 20 mL), brine (20 mL), dried over Na2SO4, filtered, and
evaporated in vacuo to give the crude 5b10, which was obtained
as a yellow oil after work up.
Bis(1-ethylpentyl) 5-(Tetrahydropyran-2-yloxymethyl)isophtha-
late (5b11). A mixture of 4 (250 mg, 0.89 mmol) and 1,1-
carbonyldiimidazole (318 mg, 1.96 mmol, 2.2 equiv) in dry DMF
(1 mL) was stirred at room temperature for 1 h. 3-Heptanol (380
µL, 2.68 mmol, 3 equiv), 4-(dimethylamino)pyridine (11 mg, 0.09
mmol, 0.1 equiv), and 1,8-diazabicyclo[5.4.0]undec-7-ene (332 µL,
1.78 mmol, 2 equiv) were added to the reaction mixture and stirred
at 40 °C for 43 h. The reaction was quenched with ice-water (10
mL) and extracted with EtOAc (3 × 20 mL). The combined organic
phases were washed with a saturated solution of NaHCO3 (2 × 20
mL), brine (20 mL), dried over Na2SO4, filtered, and evaporated
in vacuo to give the crude 5b11, which was obtained as a yellow
oil after work up.
suspending the cells in buffer containing 25 mM Tris-HCl (pH
7.5), 0.5 mM EGTA, 0.1% Triton-X 100, and protease inhibitors
according to the manufacturer’s instructions (see Materials and
General Procedures). After a 30 min incubation on ice, the lysate
was centrifuged at 16000g for 15 min at 4 °C and the supernatant
was collected. The protein content was determined according
to Bradford,57 and the supernatant was used for [3H]PDBu
binding experiments.
[3H]PDBu Binding Assay for Initial Screening of Binding
Affinity. The ability of the compounds to compete with radio-
actively labeled phorbol ester [3H]PDBu in binding to the
regulatory domain of PKC R and δ was determined according
to Gopalakrishna et al.40 First, 20 µg of protein/well from the
supernatant (see Cloning and Production of Recombinant Human
PKC R and δ in Insect Cells) was incubated with different
concentrations of each compound and [3H]PDBu for 10 min at
room temperature in a 96-well Durapore filter plate (Millipore,
cat. no. MSHVN4B50, Bedford, MA) in a total volume of 125
µL. The final concentrations in the assay were as follows: 20
mM Tris-HCl (pH 7.5), 40 µM CaCl2, 10 mM MgCl2, 400 µg/
mL bovine IgG, 25 nM [3H]PDBu, and 0.1 mg/mL phosphatidyl-
L-serine. Proteins were precipitated by the adition of 125 µL of
cold 20% poly(ethylene glycol) 6000, and after 15 min of
incubation on a plate shaker at room temperature, the filters were
washed six times using a vacuum manifold with buffer containing
20 mM Tris-HCl (pH 7.5), 100 µM CaCl2, and 5 mM MgCl2.
The plates were dried, and 25 µL of Optiphase SuperMix liquid
scintillant (PerkinElmer, Groningen, Netherlands) was added to
each well. After equilibration period of at least two hours,
radioactivity was measured using Wallac Microbeta Trilux
microplate liquid scintillation counter (PerkinElmer, Waltham,
MA). All compounds tested were diluted in DMSO to give the
same final DMSO concentration in the binding assay (4%) in
each well. Since preliminary studies showed that the nonspecific
binding was always less than 5%, only the total binding was
measured, and all results were calculated as a percentage of
control (DMSO) from the same plate. DMSO itself decreased
[3H]PDBu binding to PKC R by 20% and to PKC δ by 10%.
[3H]PDBu Binding Assay for Determination of Binding Affin-
ity Constants. To determine Ki values for selected ligands, we
used the method developed by the Blumberg group.47 Briefly,
20 ng/tube of purified human recombinant PKC R or δ was
incubated with different concentrations of the ligands for 10 min
at 37 °C in a reaction mixture consisting of 50 mM Tris-HCl
(pH 7.4), 0.1 mg/mL phosphatidyl-L-serine, 1.8 mg/mL bovine
IgG, [3H]PDBu, and 0.1 mM CaCl2 (for PKC R) or 1 mM EGTA
(for PKC δ). Samples were chilled on ice for 10 min, and 200
µL of 35% poly(ethylene glycol) 6000 in 50 mM Tris-HCl (pH
7.4) was added. The samples were mixed, incubated on ice for
15 min, and centrifuged at 4 °C (15000g, 15 min). Radioactivity
was determined from 100 µL aliquots of supernatants and from
dried pellets by scintillation counting after addition of Optiphase
HiSafe 3 liquid scintillant and 16 h equilibration. The results
were calculated according to Lewin and Blumberg.47 The
dissociation constants (Kd) for the individual PKC isoenzymes
and inhibitory dissociation constants (Ki) for the compounds were
calculated with GraphPad Prism4 software (GraphPad Software
Inc., La Jolla, CA).
A General Procedure III, Deprotection of the THP Ethers:
Bis(3-trifluoromethylbenzyl) 5-(Hydroxymethyl)isophthalate (1a3).
A mixture of the crude 5a3 and Dowex 50W × 8 (750 mg) in
MeOH (8 mL) was stirred at 40 °C for 23 h. The crude product
was purified with flash SiO2 column chromatography (n-hexane/
EtOAc, 12:1 f 1:2) to give 1a3 as a white solid (765 mg, 42%
yield for two reaction steps). Rf 0.21 (n-hexane/EtOAc, 2:1); mp
1
90.1-90.6 °C. H NMR (300 MHz, CDCl3): δppm 8.67 (s, 1H),
8.28 (s, 2H), 7.71 (s, 2H), 7.66-7.62 (m, 4H), 7.53 (t, 2H, J 7.8
Hz), 5.44 (s, 4H), 4.83 (d, 2H, J 5.5 Hz), 1.87 (t, 1H, J 5.5 Hz).13C
NMR (75.4 MHz, CDCl3): δppm 165.5, 142.3, 136.8, 132.6, 131.9,
131.3 (q, J 32.5 Hz), 130.8, 130.3, 129.4, 125.5 (q, J 3.74 Hz),
125.3 (q, J 3.85 Hz), 124.1 (q, J 273 Hz), 66.5, 64.3. Anal.
(C25H18F6O5) C, H: calcd, 3.54; found, 3.31.
Bis(1-ethylbutyl) 5-(Hydroxymethyl)isophthalate (1b10). The
general procedure III was followed except that 5b10 was used and
the reaction mixture was stirred for 20 h. The crude product was
purified with flash SiO2 column chromatography (n-hexane/EtOAc,
2:1) to give 1b10 as a colorless oil (199 mg, 61% yield for two
reaction steps). Rf 0.47 (n-hexane/EtOAc, 2:1). 1H NMR (300 MHz,
CDCl3): δppm 8.62 (app t, 1H, J 1.5 Hz), 8.22 (app d, 2H, J 1.2
Hz), 5.16-5.08 (m, 2H), 4.82 (s, 2H), 1.86 (bs, 1H), 1.76-1.56
(m, 8H), 1.48-1.30 (m, 4H), 0.98-0.91 (m, 12H). 13C NMR (75.4
MHz, CDCl3): δppm 165.8, 141.8, 132.0, 131.8, 130.1, 76.8, 64.6,
36.1, 27.3, 18.9, 14.2, 9.9; DEPT 132.0 (CH), 130.1 (CH), 76.8
(CH), 64.6 (CH2), 36.1 (CH2), 27.3 (CH2), 18.9 (CH2), 14.2 (CH3),
9.9 (CH3). HRMS-ESI (m/z): [M + HCOO-]- calcd 409.2226;
found 409.2238.
Bis(1-ethylpentyl) 5-(Hydroxymethyl)isophthalate (1b11). The
general procedure III was followed except that 5b11 was used and
the reaction mixture was stirred for 20 h. The crude product was
purified with flash SiO2 column chromatography (n-hexane/EtOAc,
2:1) to give 1b11 as a colorless oil (213 mg, 61% yield for two
reaction steps). Rf 0.41 (n-hexane/EtOAc, 4:1). 1H NMR (300 MHz,
CDCl3): δppm 8.61 (app t, 1H), 8.23 (m, 2H), 5.10 (qn, 2H, CH, J
6.0 Hz), 4.82 (s, 2H), 1.95 (bs, 1H), 1.76-1.65 (m, 8H), 1.38-1.30
(m, 8H), 0.95 (t, 6H, J 7.5 Hz), 0.89 (t, 6H, J 7.2 Hz). 13C NMR
(75.4 MHz, CDCl3): δppm 165.8, 141.8, 132.1, 131.8, 130.1, 77.1,
64.6, 33.6, 27.7, 27.3, 22.8, 14.2, 9.9. HRMS-ESI (m/z): [M +
HCOO-]- calcd 437.2539; found 437.2512.
Determination of ERK Phosphorylation by Immunoblotting. To
study the effects of the compounds on ERK1/2 phosphorylation,
HeLa cells were treated with the test compounds in the presence
or absence of PMA or inhibitors. The amount of phosphorylated
ERK1/2 was determined by Western blotting. The cells were
seeded onto 6-well plates at a density of 4.0 × 105 cells/well.
Then, 20-24 h after seeding, the medium was changed to serum-
free DMEM. After a 24 h serum starvation incubation, the cells
were treated with the test compounds for the indicated times,
washed twice with ice-cold PBS, and then harvested in ice-cold
lysis buffer containing 20 mM Tris-HCl (pH 7.4), 0.1% Triton
X-100, 2 mM EDTA, 10 mM EGTA, 2 mM Na3VO4, and
protease and phosphatase inhibitors according to manufacturer’s
Cloning and Production of Recombinant Human PKC r and
δ in Insect Cells. The cloning of human recombinant PKC R and
δ and their expression in baculovirus-infected Sf9 insect cells
was performed as described previously.55,56 For production of
the PKC proteins, Sf9 cells were infected with an optimized
amount of the recombinant baculovirus, harvested 2 days
postinfection, and washed with PBS. The resultant cell pellets
were subsequently frozen. Crude cell lysates were prepared by

3980 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
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instructions (see Materials and General Procedures). After a 30
min incubation on ice, the cell lysates were centrifuged at 16000g
for 15 min at 4 °C and the protein concentrations of the
supernatants were determined using Bradford’s method.57 The
samples were diluted in Laemmli sample buffer at equal
concentrations and stored at -20 °C. Then 20 µg of protein per
lane were subjected to SDS-PAGE and then transferred to
nitrocellulose membranes. Membranes were washed for 5 min
with 0.1% Tween 20 in Tris-buffered saline (TTBS) and then
blocked for 1 h with 5% nonfat milk powder in TTBS (milk-
TTBS). The membranes were then cut just above the 37 kDa
marker band and incubated with the primary antibody (Anti-
ACTIVE MAPK pAb 1:5000 or anti-GAPDH mAb 1:10 000 in
milk-TTBS) overnight at 4 °C. Membranes were then washed
for a total of 35 min with TTBS and incubated with horseradish
peroxidase-conjugated secondary antibody (goat antirabbit IgG
or goat antimouse IgG 1:3000 in milk-TTBS) for one hour at
room temperature. After washing (total of 40 min with TTBS),
the bands were visualized with ECL. GAPDH bands were used
as controls to ensure equal loading of proteins to all wells in
the SDS-PAGE gel. Western blots were quantified by measuring
the optical density of the immunoreactive bands with Scion
Image software (http://scioncorp.com).
Acknowledgment. We thank Matti Wahlsten, Sirkku
Ja¨ntti, and Teemu Nissila¨ for conducting the LC-MS analysis
as well as Minna Baarman, Marjo Vaha, and Tarja Va¨lima¨ki
for technical assistance. This work was supported by grants
from the European Commission Research (project no.
503467), the Academy of Finland (project no. 108376), the
Finnish Cultural Foundation, and the Magnus Ehrnrooth
Foundation.
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Supporting Information Available: Synthesis procedures,
degree of purity (elemental analysis), spectroscopic data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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