Beyond the affinity for protein kinase C: Exploring 2-phenyl-3-hydroxypropyl pivalate analogues as C1 domain-targeting ligands

Article abstract of DOI:10.1039/c4md00564c

Over the past fifteen years, we reported the design and synthesis of different series of compounds targeting the C1 domain of protein kinase C (PKC) that were based on various templates. Out of the pivalate templates, 2-[4-(benzyloxy)phenyl]-3-hydroxypropyl pivalate (compound 1) emerged as the most potent and promising PKCα ligand, showing a K_i value of 0.7 μM. In the present contribution our efforts are aimed at better understanding which structural modifications of the pivalate template are allowed for its affinity to the C1 domain of PKC to be preserved or increased. To this aim, thirteen novel analogues of 1 were designed and their interaction with the target was evaluated in silico. Designed compounds were then prepared and fully characterized as well as their affinity for the α and δ isoforms of PKC evaluated. Additionally, in order to investigate the role of chirality in the ligand-target interaction, the pure enantiomers of the most interesting PKC ligands were prepared and their affinity for PKC isoforms was determined. Results from our study revealed that: i) the presence of the ester function seems to be essential for the ligand-target interaction; ii) only a few structural modifications at the ester group are allowed for the C1 domain affinity to be preserved; and iii) the [³H]PDBu replacement experiments showed that the C1 domain of PKC does not exhibit enantiopreference for the pure stereoisomers of tested compounds. Altogether our observations provide further insights into the ligand-target interactions of the PKC C1 domain and represent a step-forward in future development of more specific and effective PKC ligands. This journal is

Full text of DOI:10.1039/c4md00564c

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CONCISE ARTICLE
Simona Collina et al.
Beyond the affinity for protein kinase C: exploring 2-phenyl-3-hydroxypropyl
pivalate analogues as C1 domain-targeting ligands

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CONCISE ARTICLE
Beyond the affinity for protein kinase C: exploring
2-phenyl-3-hydroxypropyl pivalate analogues as
C1 domain-targeting ligands†
Cite this: Med. Chem. Commun.,
2015, 6, 547
Daniela Rossi,‡a Virpi Talman,‡b Gustav Boije Af Gennäs,‡c Annamaria Marra,‡a
Pietro Picconi,‡a Rita Nasti,‡a Massimo Serra,‡a Jihyae Ann,‡d Marialaura Amadio,‡e
Alessia Pascale,‡e Raimo K. Tuominen,‡b Jari Yli-Kauhaluoma,‡c Jeewoo Lee‡d
a
*
and Simona Collina‡
Over the past fifteen years, we reported the design and synthesis of different series of compounds
targeting the C1 domain of protein kinase C (PKC) that were based on various templates. Out of the
pivalate templates, 2-ij4-IJbenzyloxy)phenyl]-3-hydroxypropyl pivalate (compound 1) emerged as the most
potent and promising PKCα ligand, showing a Ki value of 0.7 μM. In the present contribution our efforts are
aimed at better understanding which structural modifications of the pivalate template are allowed for its
affinity to the C1 domain of PKC to be preserved or increased. To this aim, thirteen novel analogues of
1 were designed and their interaction with the target was evaluated in silico. Designed compounds were
then prepared and fully characterized as well as their affinity for the α and δ isoforms of PKC evaluated.
Additionally, in order to investigate the role of chirality in the ligand–target interaction, the pure
enantiomers of the most interesting PKC ligands were prepared and their affinity for PKC isoforms was
determined. Results from our study revealed that: i) the presence of the ester function seems to be
essential for the ligand–target interaction; ii) only a few structural modifications at the ester group are
allowed for the C1 domain affinity to be preserved; and iii) the [3H]PDBu replacement experiments showed
that the C1 domain of PKC does not exhibit enantiopreference for the pure stereoisomers of tested
compounds. Altogether our observations provide further insights into the ligand–target interactions of the
PKC C1 domain and represent a step-forward in future development of more specific and effective
PKC ligands.
Received 17th December 2014,
Accepted 25th January 2015
DOI: 10.1039/c4md00564c
www.rsc.org/medchemcomm
Introduction
a Department of Drug Sciences, Medicinal Chemistry and Pharmaceutical
Technology Section, University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy.
E-mail: simona.collina@unipv.it; Fax: (+39)0382 422975; Tel: (+39)0382 987379
b Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy,
University of Helsinki, Viikinkaari 5 E (P.O. Box 56), FI-00014, Finland
c Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy,
University of Helsinki, Viikinkaari 5 E (P.O. Box 56), FI-00014, Finland
d Laboratory of Medicinal Chemistry, College of Pharmacy, Seoul National
University, Seoul 151-742, Korea
Protein kinase C (PKC) is a family of ten serine/threonine
kinases which can phosphorylate a large number of sub-
strates and is thus involved in several cellular functions.
Based on both sequence homology and sensitivity to activa-
tors, the isoforms are classified into: (1) calcium-dependent
conventional isoforms (cPKCs; α, βI, βII and γ); (2) calcium-
independent novel isoforms (nPKCs; δ ε, η and θ); and (3)
atypical isoforms (aPKCs; ζ and ι/λ).1,2 From a structural
standpoint, PKCs are generally characterized by a primary
amino acid sequence composed of a single polypeptide chain
which includes conserved domains (named C1–C4) separated
by variable sequences (Fig. 1). The N-terminal regulatory
region hosts C1 and C2 domains that control the kinase activ-
ity of the enzyme and also its subcellular localization. The C1
domain presents the binding motif for the physiological
activator diacylglycerol (DAG, Fig. 2) as well as for exogenous
activators, such as phorbol esters (i.e. phorbol-13-O-acetate,
Fig. 2). The C2 domain coordinates the binding to anionic
e Department of Drug Sciences, Pharmacology Section, University of Pavia,
Viale Taramelli 14, 27100 Pavia, Italy
† Electronic supplementary information (ESI) available: Details of chemical
synthesis, chiral resolution and all details of the molecular modeling study and
biological investigation. See DOI: 10.1039/c4md00564c
‡ Author contribution: S. C, J. Y. K., R. K. T. & J. L. conceived the work; S. C. was
responsible for the correctness of the whole study. D. R. designed the
experimental work and wrote the paper. G. B. designed, carried out and wrote
the molecular modeling part. V. T. designed and carried out biological testing
and wrote the biological part. A. M., P. P. & J. A. were responsible for the
synthesis and M. S. for the characterization of the PKC ligands. R. N. prepared
and characterized the enantiomeric compounds. A. P. & M. A. contributed to the
planning of the biological experiments. G. B., R. K. T., V. T., S. C. & J. Y. K.
revised the paper.
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Fig. 1 Schematic structures of PKC isoforms. aC1: atypical C1 domain of aPKCs; nC2: C2 domain of nPKCs; PS: pseudosubstrate sequence; PB1:
Phox and Bem 1 domain.
show either increased or decreased activity and, conse-
quently, both PKC inhibitors and activators may possess
strong therapeutic potential in various pathological contexts.
Particularly, the C1 domain of PKC is an intriguing pharma-
cological target for developing new PKC activators, which
may be useful in the treatment of several diseases, including
AD. Indeed, in vivo experiments with AD double-transgenic
mice have shown that activation of PKC with C1-targeting
compounds is a highly promising therapeutic strategy for AD
treatment.14
Fig.
2 Structure of the physiological PKC activator diacylglycerol
(DAG) and the exogenous PKC activator phorbol-13-O-acetate, both
Over the past fifteen years, we and others have reported
the design and synthesis of different series of C1-targeting
PKC ligands (templates I–IV, Fig. 3). First, conformationally
constrained DAG analogues embedded in a variety of lactone
templates (i.e. templates I and II) were designed to reduce
part of the entropic penalty associated with the binding of
the flexible glycerol backbone of DAG.15–17 As an alternative
approach, we also studied a novel group of synthetic C1 domain
ligands based on the dialkyl 5-IJhydroxymethyl)isophthalate
template III,18 as well as a compound series built on 2-phenyl-
and 2-benzyl-3-hydroxypropyl pivalate template IV.19
For compounds based on template IV, both benzyl (n = 1)
and phenyl (n = 0) derivatives bearing hydrogen bonding
groups, such as hydroxy, benzyloxy and hexanoyl groups at
different positions of the aromatic moiety, were investigated
as novel PKCα ligands. Among this compound series,
2-ij4-IJbenzyloxy)phenyl]-3-hydroxypropyl pivalate (compound 1,
Fig. 4) emerged as the most potent PKCα ligand, showing a Ki
value of 0.7 μM.
targeting the C1 domain of PKC.
phospholipids on the cell membrane and in the case of
cPKCs includes the binding site for Ca2+.3–5 The regulatory
region also contains an auto-inhibitory pseudosubstrate
sequence (PS, Fig. 1) that clamps the catalytic site in the
inactive conformation and inhibits substrate binding thus
preventing the activation of the enzyme in the absence of
external stimuli. The C-terminal kinase domain is responsible
for the phosphotransfer activity and thus contains ATP and
substrate binding sites located in C3 and C4 domains, respec-
tively (Fig. 1).
Signaling pathways mediated by DAG/phorbol ester-
responsive PKCs have a pivotal role in various cellular func-
tions, such as apoptosis, proliferation and differentiation, as
well as in cognitive processes.6,7 Accordingly, the aberrant
function of PKC-mediated pathways underlies several patho-
logical conditions, ranging from cancer to neurodegenerative
diseases, especially Alzheimer's disease (AD), passing through
diabetes, heart disease and autoimmune diseases.8–13
Depending on the pathological condition, PKC isoforms may
As a part of our on-going research in the PKC field, the
present contribution is aimed at better understanding which
structural modifications of template IV are allowed for the
affinity for the C1 domain of PKC to be preserved or
Fig. 3 Structures of the previously investigated PKC ligands.
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found in the hydrophobic “top” third of the C1 domain,
where a deep, narrow and polar groove can be found between
two β-sheets. To investigate if compounds 2–14 would bind
to the binding groove of the C1 domain of PKC, we
performed docking experiments using the crystal structure
(Fig. 5). Compound 1 was also included in this study as the
reference compound. Results suggested that e.g. compound
(S)-2 can bind to the bottom of the binding groove of the C1
domain and has the same interactions as phorbol-13-O-acetate
in the crystal structure (Fig. 5A). These interactions include
hydrogen bonds between the hydroxy group and the back-
bone amide proton of Thr 242 and the carbonyl of Leu
251. In addition to these interactions, the carbonyl of com-
pound (S)-2 forms a hydrogen bond with the amide proton of
Gly 253. A similar interaction can be seen between the C3 car-
bonyl of phorbol-13-O-acetate and the C1 domain in the crys-
tal structure. Even though the C13 ester carbonyl of phorbol-
13-O-acetate is essential for the binding activity, no interac-
tion between the carbonyl and the protein can be seen in the
crystal structure, as in the case with the other carbonyl of
DAG.21 Interestingly, the docking results of the retro-esters
(compounds 3–5) and retro-amides (compounds 6–14) sug-
gest that these compounds can bind to the deep groove of
the C1 domain of PKC similarly to 1 and 2 [e.g. compound
(S)-3, Fig. 5B]. The docking results also suggest that some of
the compounds could adapt another binding mode, similarly
to the DAG-lactones.16 In this binding mode Gly 253 does not
act as a hydrogen donor to the carbonyl of e.g. compound 4
but instead to the oxygen of the 3-benzyloxy group. Given the
considerations above, the designed compounds were consid-
ered to be suitable for the purpose of this study.
Syntheses
Fig. 4 Structures of compounds 1–14.
Compounds 1 and 2 were essentially prepared according to
the methodology described in our previous work,19 with suit-
able modifications (Scheme 1). Briefly, starting from the
commercially available 4-IJbenzyloxy)benzaldehyde, the alco-
hol 15 was easily obtained using lithium aluminium hydride
and then transformed into the corresponding benzyl chloride
16 with thionyl chloride. Compound 16, in turn, was
converted into the nitrile 17 by using sodium cyanide. Inter-
mediate 17 was hydrolysed by refluxing it in an aqueous solu-
tion of 30% NaOH, and the resulting carboxylic acid 18 was
converted into the methyl ester 19, which in turn was acyl-
ated with dimethyl carbonate to give compound 20. The dies-
ter groups of 20 were then reduced to the corresponding
alcohols by using LiAlH4, yielding 21. Finally, one of the
hydroxy groups of 21 was selectively acylated by pivaloyl chlo-
ride or 2-phenylacetyl chloride, giving the target products 1
and 2, respectively.
increased. To this purpose, thirteen novel analogues of com-
pound 1 (Fig. 4) were prepared and fully characterized as well
as their affinity for the α and δ isoforms of PKC evaluated.
Additionally, since enantiomers of a biologically active com-
pound may show different interactions with the target pro-
tein, the pure enantiomers of the most interesting PKC
ligands were prepared and their affinity for PKC isoforms
was determined.
Results and discussion
Compound design
We designed compounds 2–14 which are characterized by
different functionalities at the carbonyl group as well as dif-
ferent aromatic substitutions (Fig. 4).
The crystal structure of the C1b domain of PKCδ (PDB ID:
1PTR)20 complexed with phorbol 13-O-acetate reveals an
approximately 50 residue-long sequence with the “bottom”
third of the domain containing negatively charged amino
acids. The “central” part of the domain is hydrophilic,
whereas the binding site of DAG and phorbol esters can be
The key step for the synthesis of compounds 3–14 was
the preparation of 3-unsaturated γ-butyrolactones 28–30
(Scheme 2), which was easily achieved through a two-step
reaction sequence.
First, the Heck reaction, performed according to our
previously optimized phosphine-free protocol, gave the
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Fig. 5 (A) Compounds (S)-2 and (B) (S)-3 docked to the C1b domain of PKCδ (PDB ID: 1PTR).20 Hydrogen bonds are represented as green spheres
and the surface of the binding pocket of the protein is colored as follows: hydrogen bond acceptor area (red), hydrogen bond donor area (blue)
and hydrophobic area (green). The figure was created using ICM-Browser (version 3.7-2a, Molsoft L.L.C.).
Scheme 1 Synthesis of 1 and 2. Reagents and conditions: (a) LiAlH4, diethyl ether, 0 °C, then reflux, 30 min; (b) SOCl2, DCM, 0 °C, 1 h; (c) NaCN,
DMF, 100 °C, 2 h; (d) 30% NaOH (aq), reflux, overnight; (e) H2SO4, MeOH, reflux, 2 h; (f) NaH, 1,4-dioxane, 0 °C, then rt, 30 min; g) (MeO)2CO,
1,4-dioxane, 0 °C then reflux, 2 d; (h) LiAlH4, THF, 0 °C, then rt, 2 h; (i) IJMe3)3CCOCl, pyridine, DCM, 0 °C, then rt, 2 h; (l) PhCH2COCl, pyridine,
DCM, 0 °C, then rt, 2 h.
Scheme 2 Synthesis of compounds 3–14. Reagents and conditions: (a) TBAC, AcONa, PdIJOAc)2, DMF, reflux, overnight; (b) SeO2, tBuOOH, DCE,
reflux, overnight; (c) HCOONH4, 10% Pd/C, MW conditions: 100 °C, 100 W, 180 s; (d) Cs2CO3, benzyl bromide, MeCN, MW conditions: 100 °C,
60 W, 40 min; (e) 10% NaOH (aq), MeOH, reflux, 3 h, then K2CO3, benzyl bromide, H2O/DMF (1 : 10), rt, overnight; (f) 10% NaOH (aq), MeOH, reflux,
3 h, then AlCl3, amine, DCE, MW conditions: 100 °C, 60 W, 25 min.
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α,β-unsaturated esters 25–27 as mixtures of (E,Z)-isomers.22,23
Subsequently, crude 25–27 underwent allylic oxidation with
selenium dioxide and tert-butyl hydroperoxide without
further purification,24 furnishing the desired oxidation prod-
ucts 28–30. γ-Butyrolactones 28–30 were then subjected to
microwave-assisted catalytic hydrogenation under phase
transfer conditions,25 yielding intermediates 31–33. It has to
be noted that catalytic hydrogenation of 28 and 29 resulted
in the simultaneous reduction of the double bond and cleav-
age of the benzyloxy group, thus furnishing compounds 31
and 32, respectively. These, in turn, were fully O-benzylated26
to yield the desired γ-butyrolactones 34 and 35.
Target products 3–5 were synthesized starting from
γ-butyrolactones 33–35 by a tandem hydrolysis–esterification
process.27 First, γ-butyrolactones 33–35 were converted into the
corresponding sodium 3-aryl-4-hydroxybutanoates, which in
turn were treated with benzyl bromide in the presence of K2CO3
to yield the corresponding benzyl esters 3–5. The target prod-
ucts 6–14 were synthesized from the same γ-butyrolactones
33–35 using the appropriate secondary amine and AlCl3,
according to the protocol developed by Lesimple et al.,28 with
suitable modifications. Specifically, to speed up the synthetic
procedure, we decided to take advantage of microwave irradia-
tion instead of using conventional heating, thus obtaining the
desired products in a very short reaction time.
length, 5 μm) columns (for details see Table S2, ESI†). As
presented in Fig. 6, for both compounds, baseline resolution
was achieved on the Chiralpak IC column eluted with
n-hexane/2-propanol IJ90/10, v/v) at a flow rate of 1 mL min−1
[tr1 19.9 min, tr2 22.5 min, α 1.16 and Rs 2.00 for 1, Fig. 6A;
tr1 36.7 min, tr2 47.1 min, α 1.31 and Rs 4.16 for 2, Fig. 6B].
Hence, these experimental conditions were selected to be
properly scaled-up to the semipreparative scale. Briefly,
48 mg of 1 was processed in 24 cycles according to the condi-
tions reported in Table 1, yielding 20.0 mg of the first eluted
20
405
enantiomer 1A (ee 99.9%, [α]
= + 37.7, c 0.6 in methanol)
and 19.3 mg of the second eluted enantiomer 1B (ee 99.9%,
20
405
[α]
= − 37.2, c 0.6 in methanol), together with 7.5 mg of
an intermediate fraction which was a mixture of the two
enantiomers. As regards 2, 30 mg of the racemate was
processed in 5 cycles following the conditions summarized in
Table 1, furnishing 14.2 mg of the first eluted enantiomer 2A
(ee 99.9%, [α]24005 = + 37.5, c 0.4 in methanol) and 13.1 mg of the
second eluted enantiomer 2B (ee 99.9%, [α]24005 = − 37.4, c 0.4 in
methanol), together with 1.1 mg of the intermediate fraction.
Biological activity
After synthesis and chemical characterization, compounds
1–14, 1A–B and 2A–B were subjected to biological assays to
determine their binding affinity for PKCα and PKCδ. Com-
pounds 1–14 were used in the concentration range of 0.1–100 μM
to displace [3H]-phorbol 12,13-dibutyrate ([3H]PDBu) from
the C1 domain of PKCα and PKCδ using a 96-well plate filtra-
tion method as previously described.18 The percentages of
residual [3H]PDBu binding after exposure to 1–14 at 100 μM
concentration are presented in Table 2. As regards the affin-
ity to PKCα, the novel compound 2 displaced [3H]PDBu in a
concentration-dependent manner with an IC50 value of 73.9 μM
Isolation of 1 and 2 enantiomers
The resolution of racemic 1 and 2 by semi-preparative HPLC
using chiral stationary phases was carried out. In order
to identify the optimal experimental conditions for the
enantioresolution of 1 and 2, a standard screening protocol29
was applied to both Chiralcel OJ-H (0.46 cm diameter × 15 cm
length, 5 μm) and Chiralpak IC (0.46 cm diameter × 25 cm
Fig. 6 Analytical separation of (A) compound 1 (detection at 274 nm) and (B) compound 2 (detection at 254 nm) on a Chiralpak IC column
(0.46 cm diameter × 25 cm length, 5 μm), mobile phase: n-hexane/2-propanol IJ90/10, v/v), flow rate: 1 mL min−1
.
Table 1 Semi-preparative resolution of 1 and 2 on a Chiralpak IC column (10 mm diameter × 250 mm length, 5 μm), eluted with n-hexane/2-propanol
IJ90/10, v/v)
Cmpd
n-Hexane/2-propanol
Flow rate [mL min−1
]
tr1 [min]
tr2 [min]
Inj. vol [mL]
Conc. [mg mL−1
]
λ [nm]
1
2
90/10
90/10
3
2
29.5
48.1
33.5
63.5
0.2
1
10
6
274
254
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Table 2 Binding affinity of compounds 1–14 to PKCα and PKCδa
(Fig. 7A), showing a binding profile very similar to that of the
reference compound 1 (IC50 = 37.5 μM). The retro-ester deriv-
atives 3–5 also exhibited clear concentration-dependent bind-
ing to the C1 domain of PKCα (Fig. 7B), although they were
less potent than compounds 1 and 2 in displacing [3H]PDBu.
However, no significant displacement of [3H]PDBu was
observed for the retro-amides 6–14, not even at the highest
concentration (100 μM) tested (Table 2). Only compounds
10–11 displaced 27.6% and 23.9% of [3H]PDBu at 100 μM
concentration, respectively, but exhibited no clear concentra-
tion–response (data not shown). The poor affinities of the
retro-amides were contradictory to our molecular modeling
predictions, in which their affinities were expected to be sim-
ilar to those of the ester derivatives. However, this is in line
with our observations with the isophthalate derivatives, in
which the substitution of the esters with the respective
amides diminished the binding affinity.19
Residual [3H]PDBu binding
Residual [3H]PDBu binding
Cmpd
to PKCα (% of control)
to PKCδ (% of control)
1
2
3
4
5
6
7
8
41.6 2.7 (4)
51.3 5.8 (4)
73.9 8.0 (2)
67.4 2.1 (2)
64.4 11.5 (2)
100.5 12.2 (2)
90.0 11.6 (2)
84.4 4.9 (3)
80.6 10.3 (3)
72.4 10.7 (3)
76.1 7.7 (3)
106.7 7.8 (3)
99.7 0.9 (2)
86.6 11.9 (3)
29.3 2.1 (4)
34.8 2.2 (4)
61.6 0.4 (2)
52.5 1.3 (2)
56.7 3.5 (2)
n.d.
n.d.
n.d.
n.d.
n.d.
9
10
11
12
13
14
n.d.
n.d.
n.d.
n.d.
a
The binding affinities were studied as described in the ESI1 and
are expressed as mean SEM of residual [3H]PDBu binding (% of
control) with 100 μM ligand concentration. The number of
independent experiments is presented in brackets for each value.
n.d. – not determined.
For PKCδ, the results were consistent with those obtained
from the PKCα binding assays: compounds 1 and 2 exhibited
the highest affinity (IC50 values of 33.2 μM and 39.9 μM,
respectively; Fig. 8A), while compounds 3–5 displaced
Fig. 7 Binding of compounds 1–5 to PKCα. Affinities of 1, 2 and 3–5 for PKCα were determined as described in the ESI† and are presented as
percentage of control (DMSO). (A) Results are expressed as mean SEM of 4 independent experiments. IC50 values were calculated with
GraphPad Prism 5 software. (B) Results are expressed as mean SEM of 2 independent experiments.
Fig. 8 Binding of compounds 1–5 to PKCδ. Affinities of 1, 2 and 3–5 for PKCδ were determined as described in the ESI† and are presented as
percentage of control (DMSO). (A) Results are expressed as mean
SEM of 4 independent experiments. IC50 values were calculated with
GraphPad Prism 5 software. (B) Results are expressed as mean SEM of 2 independent experiments.
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[3H]PDBu from PKCδ in a concentration-dependent manner
(Fig. 8B), but with lower potency.
Based on the biological results discussed so far, 1 and 2
were selected to be further investigated. Since the enantio-
mers of a biologically active molecule may behave differently
under physiological conditions (i.e., they can exhibit different
pharmacodynamic and/or pharmacokinetic properties as well
represent a step-forward in future development of more spe-
cific and effective PKC ligands. Particularly, the most interest-
ing feature of the results presented herein appears to be the
observation that, at least for ligands belonging to the tem-
plate IV series, only a few structural modifications are toler-
ated by the C1 domain of PKC.
as different toxicological profiles), the investigation of Abbreviations
enantiopure forms is a key step in the drug discovery pro-
cess.30 Accordingly, we evaluated the ability of the pure
enantiomers of 1 and 2 to displace [3H]PDBu from the C1
domain of PKC, according to the methodology described
above. Results showed that, for both compounds 1 and 2,
the dextro (1A and 2A) and the levo (1B and 2B) isomers
possess almost the same binding profile as the correspond-
ing racemates (displacement at 100 μM equal to: 34.6% for
1A, 36.3% for 1B, 27.3 for 2A and 26.3 for 2B), thus
suggesting that chirality does not seem to play a significant
role in the ligand–target interaction.
AD
Bn
Alzheimer's disease
Benzyl
Compound
Diacylglycerol
1,2-Dichloroethane
Dichloromethane
N,N-Dimethylformamide
Microwave
Cmpd
DAG
DCE
DCM
DMF
MW
[3H]PDBu [3H]-Phorbol 12,13-dibutyrate
PKC
THF
TBAC
Protein kinase C
Tetrahydrofuran
Tetrabutylammonium chloride
Conclusion
In an attempt to study the impact of modifying template IV
on the affinity for the C1 domain of PKC, we designed and
prepared thirteen novel analogues of compound 1, character-
ized by different functionalities at the carbonyl group as well
as different aromatic substitutions. In contrast to our molec-
ular modeling predictions, the results of PKC binding assays
suggested that replacing the ester group with the amide one
determines the total loss of affinity. This experimental evi-
dence is consistent with the SAR consideration previously
drawn for compounds belonging to the template III series.18
Indeed, also for the isophthalate derivatives, moving from
esters to amides led to a dramatic decrease in the binding
affinity for the C1 domain of PKC,18 even though molecular
modeling suggested that the amides should interact similarly
with the target. Moreover, the results obtained in our study
clearly revealed that the presence of the ester function is
essential for the ligand–target interaction, as widely demon-
strated by numerous studies (for an overview see the reviews
in bibliography31,32). Furthermore, it can be seen from our
study that to preserve the C1 domain affinity only a few struc-
tural modifications at the ester group are allowed. Indeed,
only the normal ester derivative 2 showed affinity for the C1
domain of PKC comparable to that of the reference com-
pound 1, while the retro-ester derivatives 3–5 bound to the
target more loosely, thus suggesting that the medicinal chem-
istry impact of modifying the normal ester functional group
is very high, independently of the aromatic moiety of the
molecules.
Acknowledgements
We thank Gloria Wissel and Yuezhou Zhang for assistance
with the extraction of the results from the molecular docking
studies. This work was funded by the Academy of Finland
(project no. 257685), Jane and Aatos Erkko Foundation and
Finnish Cultural Foundation.
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