Binding of isoxazole and pyrazole derivatives of curcumin with the activator binding domain of novel protein kinase C

Article abstract of DOI:10.1016/j.bmc.2011.09.011

The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target because of its involvement in the regulation of various cellular functions, including cell growth, differentiation, metabolism, and apoptosis. The endogenous PKC ac

Full text of DOI:10.1016/j.bmc.2011.09.011

Bioorganic & Medicinal Chemistry 19 (2011) 6196–6202
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Binding of isoxazole and pyrazole derivatives of curcumin
with the activator binding domain of novel protein kinase C
⇑
Joydip Das , Satyabrata Pany, Shyam Panchal, Anjoy Majhi, Ghazi M. Rahman
Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77204, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target because of its
involvement in the regulation of various cellular functions, including cell growth, differentiation, metab-
olism, and apoptosis. The endogenous PKC activator diacylglycerol contains two long carbon chains,
which are attached to the glycerol moiety via ester linkage. Natural product curcumin (1), the active con-
stituent of Curcuma L., contains two carbonyl and two hydroxyl groups. It modulates PKC activity and
binds to the activator binding site (Majhi et al., Bioorg. Med. Chem. 2010, 18, 1591). To investigate the role
of the carbonyl and hydroxyl groups of curcumin in PKC binding and to develop curcumin derivatives as
effective PKC modulators, we synthesized several isoxazole and pyrazole derivatives of curcumin (2–6),
characterized their absorption and fluorescence properties, and studied their interaction with the activa-
Received 29 August 2011
Accepted 8 September 2011
Available online 10 September 2011
Keywords:
Curcumin
Isoxazole
Pyrazole
Fluorescence
Molecular docking
Protein kinase C
Activator
tor-binding second cysteine-rich C1B subdomain of PKCd, PKC
derivatives for protein fluorescence quenching varied in the range of 3–25
higher binding with the PKChC1B compared with PKCdC1B and PKC C1B. Fluorescence emission maxima
e
and PKCh. The EC50s of the curcumin
l
M. All the derivatives showed
e
EC50
of 2–5 were blue shifted in the presence of the C1B domains, confirming their binding to the protein.
Molecular docking revealed that hydroxyl, carbonyl and pyrazole ring of curcumin (1), pyrazole (2),
and isoxazole (4) derivatives form hydrogen bonds with the protein residues. The present result shows
that isoxazole and pyrazole derivatives bind to the activator binding site of novel PKCs and both carbonyl
and hydroxy groups of curcumin play roles in the binding process, depending on the nature of curcumin
derivative and the PKC isotype used.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Numerous analogs of curcumin have been developed for thera-
peutic purposes. The advantage of developing molecules around
Curcumin (1) is a b-diketone constituent of turmeric, which is
obtained from the powdered root of Curcuma L.1,2 Not only it is
used as a spice to give a specific flavor and yellow color to curry,
which is consumed in trace quantities daily by millions of people,
curcumin has also been used as a traditional medicine for liver
disease (jaundice), indigestion, urinary tract diseases, rheumatoid
arthritis, and insect bites.1–3 It possesses both anti-tumor and
anti-angiogenic properties. Its anti-tumor properties include
growth inhibition and apoptosis induction in a variety of cancer
cell lines in vitro, as well as the ability to inhibit tumorigenesis
in vivo.4–10 Curcumin also shows potent anti-inflammatory,11,12
antioxidant,13,14 and antimicrobial15–18 activity. It can decrease
oxidative damage, inflammation, amyloid accumulation, and is
under development as a chemotherapeutic agent.19 Curcumin is a
much stronger free-radical scavenger than vitamin E,20 protects
the brain from lipid peroxidation, and scavenges nitric oxide
(NO)-based radicals.21
the curcumin scaffold is the lack of its toxicity. Large quantities
of curcumin can be consumed without toxicity. Three phase I clin-
ical trials have demonstrated tolerances as high as 12 g per
day.22,23 These distinctive properties make curcumin a valuable
lead compound for drug development, and it remains the focus
of several clinical trials.24,25
There are about one hundred previously reported biological
targets for curcumin24 including protein kinases,26,27 membrane
ATPases,28–31 and transcription factors.32,33 The positive interfer-
ence of curcumin with the tumor promoting effects of phorbol
esters has been attributed to its effect on the phorbol ester
receptor, protein kinase C (PKC).34 There are also well documented
studies on the modulation of PKC activity by curcumin in vivo26
and in vitro35 using membrane-free systems.
PKC is a family of serine/threonine protein kinases involved in
the regulation of various aspects of cell functions, including cell
growth, differentiation, metabolism, and apoptosis.36 PKC isoforms
play an important role in the pathology of several diseases such as
cancer, diabetes, stroke, heart failure, and Alzheimer’s disease.37–43
Therefore, PKC has been a subject of intensive research and drug
development.44
⇑
Corresponding author. Tel.: +1 713 743 1708; fax: +1 713 743 1884.
E-mail address: jdas@uh.edu (J. Das).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.011

J. Das et al. / Bioorg. Med. Chem. 19 (2011) 6196–6202
6197
The PKC family has been divided into three main groups: con-
ventional isoforms ( , bI, bII, and
) that require Ca2+ and diacyl-
glycerol (DAG) for activation; novel isoforms (d, , h, and
, and k) that re-
high affinity phorbol ester 12-O-tetradecanoylphorbol-13-acetate
(TPA) contain long aliphatic chain that facilitate the ligand for its
binding to the membrane. The compounds used in this study are
shown in Figure 1. Pyrazole derivative (2) of curcumin was pre-
pared in good yield by heating curcumin (1) for 8 h with hydrazine
hydrate in acetic acid, whereas isoxazole derivative (4) of curcumin
was synthesized by treatment of curcumin with hydroxylamine
hydrochloride in acetic acid at 85 °C for 6 h. Long chain derivative
of curcumin-pyrazole was synthesized by refluxing curcumin-
pyrazole in the presence of 1-bromobutane and K2CO3 in dry
acetone for 24 h. Long chain derivatives of curcumin-isoxazole
were prepared at refluxing condition in the presence of
1-bromooctane/1-bromobutane and K2CO3 in dry acetone. All the
synthesized derivatives were characterized by 1H and 13C NMR
and mass spectrometry.
a
c
e
,
g
l)
that require only DAG and atypical isoforms (f,
i
quire neither Ca2+ nor DAG.45 DAG is a second messenger that is
generated by the phospholipase C-catalyzed hydrolysis of mem-
brane phosphatidylinositol-4,5-bisphosphate (PIP2).46 DAG selec-
tively interacts with proteins containing
a C1 domain and
induces their translocation to discrete sub cellular compartments.
In the classical and novel PKC isoenzymes, the DAG-sensitive C1
domain is duplicated into a tandem C1 domain consisting of C1A
and C1B sub domains. Atypical PKCs contain a single C1 domain
that does not bind DAG. Along with the PKC family, there are six
additional families of proteins that contain a DAG-responsive C1
domain.47,48 The C1 domains have become an attractive target in
designing the PKC based drugs. Recently it has been found that
alcohol and anesthetics also bind to the PKC C1 domains.49–51
In the present paper, we describe the design, synthesis, spectral
characteristics and binding of several isoxazole and pyrazole deriv-
atives of curcumin with the activator binding C1B subdomain of
novel PKCs. Our results show that modifications of the carbonyl
group to isoxazole or pyrazole change the spectral characteristics
of curcumin, but still retain binding properties with the PKCs.
The addition of aliphatic chain in the isoxazole or pyrazole com-
pounds reduced the binding affinity. Molecular docking studies
suggest that hydrogen bonds are formed between the hydroxyl,
carbonyl or nitrogen in the heterocyclic ring of these derivatives
and the protein residues.
3. Results and discussion
A large number of curcumin derivatives have been synthesized
and studied for their binding and activity on receptors and
proteins.52 However, compounds designed around the scaffold of
curcumin have not been explored systematically for binding and
modulation of PKC activity. In our previous papers,53,54 we showed
that dietary polyphenols curcumin, resveratrol and their long chain
derivatives bind to the activator-binding site in the PKCs by form-
ing hydrogen bonds with the residues at the activator-binding site.
The present investigation is a continuation of our previous study
with an aim of developing subtype specific modulator of PKCs. In
this study, we highlighted the role of carbonyl groups of curcumin
by derivatizing them to isoxazole and pyrazole rings and studying
their binding affinities with the activator-binding site of three no-
2. Chemistry
2.1. Curcumin and its cyclic derivatives
vel PKCs, PKCd, PKCe, and PKCh. In order to compare the binding
affinities of these derivatives with the other curcumin derivatives
described earlier,53 we used the same PKC subtypes. The isoxazole
and pyrazole derivatives were further modified with the C4 ali-
phatic chains at the hydroxyl groups and their effects on the pro-
tein binding were also measured to determine the role of
hydroxyl group in the protein–ligand interactions.
The isoxazole and pyrazole derivatives of curcumin were pre-
pared in order to study the role of carbonyl group of curcumin in
the PKC binding. Further, these derivatives were modified with
aliphatic chains to study the effect of long chain in the PKC binding.
Both the endogenous PKC activator diacylglycerol (DAG) and the
N
NH
N
NH
MeO
HO
OMe
MeO
HO
OMe
OH
b
3
2
OC4H9
a
N
O
O
O
MeO
HO
OMe
OH
c
MeO
HO
OMe
OH
4
1
b
d
N
O
N
O
MeO
OMe
Me O
C8H17
OMe
5
C4H9O
OC4H9
6
O
OC8H17
a, NH2NH2.H2O/CH3COOH, 8 h reflux; b, Br-C4H9, K2CO3/Acetone, reflux; c, NH2OH.HCl/CH3COOH, 85 oC, 6 h; d, Br-
C8H17, K2CO3/Acetone, reflux.
Figure 1. Scheme outlining the synthesis of the isoxazole and pyrazole derivatives of curcumin (1).

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J. Das et al. / Bioorg. Med. Chem. 19 (2011) 6196–6202
Table 1
Absorptiona and fluorescenceb properties of curcumin and its derivatives in different solvent at 25 °C
Compound
Absorbance maximum (kmax), nm
Emission maximum, nm
EtOH
427
CH3CN
417
Hexane
404
Water
425
EtOH
560
CH3CN
Hexane
Water
572
1
2
3
4
5
6
518
432
433
413
425
420
474
449
383
362
384
367
378
367
382
370
384
368
329
328
337
335
335
327
328
332
335
335
325
331
332
334
333
320
328
332
326
328
389
387
418
418
416
395
392
428
418
419
a
concn, 2 ꢀ 10ꢁ6 M.
b
concn, 5 ꢀ 10ꢁ6 M in water and 2 ꢀ 10ꢁ6 M in other solvents.
3.1. Absorption and emission characteristics of the curcumin
derivatives
forbidden n–p⁄ transition might have been buried under the strong
p⁄ transition. Because curcumin can exist as an equilibrium
p–
mixture of keto and enol form, both this forms contribute to this
absorption maximum. For compounds 2–6, in which the carbonyl
groups curcumin were derivatized to either cyclic isoxazole or
pyrazole rings, the absorption maximum values decreased
drastically in the range of 320–337 nm and showed reduced
solvent sensitivity (Table 1). Figure 2A shows the absorption
spectra of isoxazole derivative 4 recorded in different solvents.
No significant solvent-induced wavelength shift was observed for
4. The maximum difference of 9 nm in the absorption values was
observed for 2 and 5 in water and ethanol. Although, among all
the compounds examined here, the oxazole derivative showed
higher absorption maximum values than the pyrazole derivative,
modification of the phenolic OH group of the curcumin moiety
with C4 or C8 aliphatic chain did not alter the values significantly.
The presence of the two C8 aliphatic chains, however, made com-
pound 6 poorly soluble in all the solvents tested here.
The absorption maximum of curcumin is dependent on the
polarity of the solvent.55,56 In a nonpolar solvent such as hexane,
its absorption maximum is 404 nm, while in polar solvent such
as ethanol, the absorption maximum is 427 nm. This strong band
is assigned to be of
spectral shift from non-polar to polar organic solvent and the
p–
p⁄ nature57 based on the spectral position,
intensity of the band. It was also proposed that the dipole
The emission maximum of curcumin is also solvent dependent,
showing a single band at 572 nm in water and two maxima at 449
and 474 nm in hexane.53,56 These emissions are believed to have
characteristics of intramolecular charge transfer states.58,59 In prot-
ic solvent such as ethanol, emission characteristics are influenced
by intermolecular proton transfer, and in the aprotic solvent like
0.8
0.6
0.4
0.2
0.0
10
30
00
05
15
20
25
35
[Ligands], µM
Figure 3. Binding of curcumin and its derivatives with PKCh C1B. Plot of
fluorescence intensity of PKCh C1B (2 M) in buffer (50 mM Tris, 150 mM NaCl,
2 mM DTT, 50 M ZnSO4, pH 7.2) in the presence of varying concentration of 2 (j),
Figure 2. (A) Effect of solvent polarity on the absorption properties of curcumin
and its derivatives. Normalized absorption spectra of 4,40-((1E,10E)-isoxazole-3,5-
diylbis (ethene-2,1-diyl))bis(2-methoxyphenol) (4), 2 ꢀ 10ꢁ6 M in (a) water, (b)
ethanol, (c) acetonitrile, and (d) hexane. (B) Effect of solvent on the emission
properties of curcumin and its derivatives. Normalized fluorescence emission
spectra of 4,40-((1E,10E)-isoxazole-3,5-diylbis(ethene-2,1-diyl))bis(2-methoxyphe-
nol) (4) in (a) water, (b) acetonitrile, (c) ethanol, and (d) hexane. The concentration
of 4 was 10 ꢀ 10ꢁ6 M in water and 5 ꢀ 10ꢁ6 M in other solvents.
l
l
3 (N), 4 (d), and 5 (.), where F and F0 are fluorescence intensities in the presence
and absence of the ligand, respectively. Solid lines indicate the fit using modified
Hill equation, fðxÞ ¼ min þðmax ꢁ minÞxnH=ðxnH þ EC5n0H). The corresponding EC50 for
2, 3, 4, and 5 are 7.24, 15.86, 3.16, and 7.14 lM, respectively and the corresponding
Hill coefficients (nH) are 0.74, 0.69, 1.46, and 0.51, respectively. Excitation
wavelength used was 280 nm.

J. Das et al. / Bioorg. Med. Chem. 19 (2011) 6196–6202
6199
Table 2
EC50 values (in
l
M) and corresponding Hill coefficient (n) for the binding of curcumin and its derivatives with the C1B sub domains of PKC
e
, PKCd, and PKCh measured by
fluorescence quenching. Protein concentration is 2
lM in buffer (50 mM Tris, 150 mM NaCl, 2 mM DTT, 50
lM ZnSO4, pH 7.2)
Compounds
dC1B
e
C1B
hC1B
EC50
n
EC50
8.81 0.21
18.81 0.39
16.09 0.18
11.81 0.20
23.38 0.52
n
EC50
n
1a
2
3
4
5
10.67 0.35
14.97 0.38
21.64 0.69
11.55 0.19
25.23 0.43
1.00
1.00
11.08 0.76
7.25 0.42
15.84 0.25
3.16 0.13
7.14 0.20
1.00
0.78 0.031
0.66 0.03
1.03 0.03
0.89 0.02
0.70 0.022
0.75 0.013
1.14 0.03
0.76 0.025
0.74 0.05
0.69 0.02
1.46 0.07
0.51 0.01
a
Ref. 53.
acetonitrile and hexane, it is influenced by intramolecular proton
transfer. Similar to what was observed in the absorption character-
istics, the isoxazole and pyrazole compounds showed large blue
shift in their emission maxima as compared to curcumin in all
the solvents tested in this study. In ethanol and water, the oxazole
derivatives showed higher emission maximum values as compared
to the pyrazole derivatives. On the other hand, in acetonitrile, pyr-
azole derivatives showed higher emission maximum values as
compared to the oxazole derivatives. In hexane, however, all the
derivatives showed peaks at around 370 and 380 nm. For curcumin
(1), the order of the emission maximum values was found to be
water > ethanol > acetonitrile > hexane. Same order was followed
for the isoxazole derivative 4. Fig. 2B represents the emission spec-
tra of 4 in different solvents. The isoxazole derivatives with ali-
phatic chains showed similar emission maxima in ethanol,
acetonitrile, and water. For pyrazole derivatives, the order of emis-
sion maximum values is acetonitrile > water > ethanol > hexane.
This indicates that the presence of different heteroatom can cause
different solvent sensitivity in the emission properties of the cur-
cumin derivatives.
The derivative having two C8 aliphatic chains (6) was not inves-
tigated for the binding studies because of its poor solubility in the
concentration range tested in this study.
To further investigate the binding of the isoxazole and pyrazole
derivatives of curcumin with the PKC C1B subdomains, the effect of
protein on their emission maxima was studied. In the presence of
10-fold excess of PKCC1B, the emission maxima of compounds 2–5
were blue shifted. The emission maximum of 2 shifted from
395 nm in buffer to 387 nm in the presence of PKCd C1B, and for
5, it shifted from 415 nm in buffer to 408 nm in protein. Under sim-
ilar experimental condition, curcumin showed a blue shift53 of 10–
13 nm and for a high affinity phorbol ester, sapintoxin (SAPD), the
shift was even higher (15–19 nm).53 Emission maxima of the other
derivatives also were blue shifted in the presence of protein and
the maximum shift of 18 nm was observed for compound 4 in
PKCeC1B. (Fig. 4, Table 3). These results also indicate that isoxazole
and pyrazole derivatives bind to the C1B domains of novel PKCs
and the extent of binding is different for different isotype.
To gain finer details of the possible binding mode of the isoxaz-
ole and pyrazole derivatives and the interacting residues of the C1B
subdomains, we performed molecular docking experiments. The
molecules were docked into the activator-binding site using the
phorbol ester binding residues of PKCd and the corresponding res-
idues of epsilon and theta at the homologous positions and the re-
sults are shown in Table 4. All the molecules showed positive total
score values and most of them formed hydrogen bonds (up to
ꢂ3 Å) with the protein residues. The mode of interactions of curcu-
min (1), pyrazole (2) and pyrazole containing a C4 aliphatic chain
(3) with PKChC1B is depicted in Figure 5. Compounds 1, 2, and 3
interacted with the PKChC1B by forming 3, 5, and 4 hydrogen
bonds, respectively. For curcumin (1), all the three hydrogen bonds
were formed with the 4-OH group and Gly-254. In 2, however,
three out of five hydrogen bonds, were formed with the pyrazole
ring nitrogen (two with Gln-258 and one with Gly-254), and one
3.2. Binding of curcumin derivatives to the PKC C1B domains
Following our previous study,53 the binding of the isoxazole and
pyrazole derivatives has been determined by fluorescence quench-
ing methods. The C1B subdomain of the PKCd, PKCe, and PKCh con-
tains a tryptophan residue, and 2, 1, and 2 tyrosine residues,
respectively, which account for their intrinsic fluorescence. Com-
pounds 2–5 quenched protein fluorescence in a concentration
dependent manner and a plateau was reached at around 25–
30 lM (Fig. 3). The EC50 values and the corresponding Hill coeffi-
cients of the quenching were determined using the modified Hill
equation49 and are shown in Table 2. In general, both isoxazole
and the pyrazole derivatives showed higher binding affinity for
PKChC1B as compared with PKCdC1B and PKCeC1B. While for isox-
azole (4), the affinity for theta was about three times higher than
delta and epsilon, for pyrazole (2), the affinity was about two times
higher. Similar higher affinity for theta was also observed earlier in
the case of curcumin and its long chain derivatives.53 Between
isoxazole and pyrazole derivatives, the former showed higher
affinity with all the three C1B subdomains. For example, the isox-
0.8
a
0.6
0.4
0.2
b
azole (4) binds to theta with an EC50 of 3.16
lM, whereas the pyr-
azole (2) binds with an EC50 of 7.25 M. It is also evident from
l
Table 2 that the modification of isoxazole and pyrazole derivative
of curcumin with aliphatic chain reduced their affinities for delta
and theta. Addition of two C4 aliphatic chains in compound 5 also
reduced its affinity for theta as compared with the nonchain deriv-
ative 4, although with a lesser extent. Similarly, addition of a C4
aliphatic chain to pyrazole reduced the affinity of 3 for theta to
550
400
500
450
Wavelength (nm)
15.84 lM from 7.25 lM for 2. However, different observations
were made when curcumin was modified with aliphatic chains.
The long chain derivatives of curcumin showed increased affinity
for all the three PKC subtypes as compared with curcumin.53
Figure 4. Blue shift in emission maxima of 4 (5 ꢀ 10ꢁ6 M) in the presence of PKC
e
C1B (50 ꢀ 10ꢁ6 M). Emission spectrum of 4 in (a) buffer and in (b) protein. The
buffer used was 50 mM Tris, 150 mM NaCl, 2 mM DTT, 50 lM ZnSO4, pH 7.2.

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J. Das et al. / Bioorg. Med. Chem. 19 (2011) 6196–6202
each with 4-OH (with Leu-252) and 40-OH (with Lys-240).
Compound 3, with a C4 aliphatic chain, adopted a conformation
in which its 4-OH formed four hydrogen bonds with Gly-254. Dif-
ferent types of interactions were observed in the isoxazole deriva-
tives. Compound 4, in which the carbonyl groups of curcumin were
derivatized to an isoxazole ring, formed one hydrogen bond be-
tween the 4-OH and Gly-254. When both the hydroxyl groups
were derivatized with alkyl chains resulting in 5, no hydrogen
bond formation was observed. Interestingly, the carbonyl group
of curcumin formed hydrogen bonds with delta and epsilon,
although no hydrogen bonds were observed with the theta. This
however does not rule out the possibility of water mediated hydro-
gen bond between the carbonyl and PKChC1B, which was not taken
into account in the docking methods used in this study. Therefore,
in general, both hydroxyl and carbonyl (or pyrazole and isoxazole
rings) groups of these cyclic derivatives of curcumin are important
for their interaction with PKCs and the nature of these interactions
depend on the curcumin derivative and the subtype of PKC used.
Using the same docking methods, it was found that one of the
high-affinity activator of PKC, phorbol-13-OAc formed three hydro-
gen bonds with PKChC1B (Table 4). Although we did not find a
quantitative correlation between the EC50 values and the total
score, or between the EC50 and the number of hydrogen bonds,
which could be due to the altered conformation of both the protein
and the small molecule in solution, the docking results revealed
that all the derivatives interact with the protein and their possible
mode of interactions.
Table 3
Emission maxima (nm)a of 2–5 in the presence of PKC C1B domains
Compound
Buffer
PKCd
PKCe
PKCh
The isoxazole and pyrazole derivatives of curcumin were syn-
thesized earlier to prevent tautomerization of the keto groups
and to study if the electron rich isoxazole and pyrazole rings could
lessen the potential for nucleophilic addition with receptor.60,61
These analogs exhibited increased antitumor activity against hepa-
tocellular carcinoma HA22T/VGH cell line.60 They were also evalu-
ated for lipoxygenase inhibitory activity,62 cytotoxic activity,63,64
and anti-oxidant activity,65 anti-tumor activity against breast can-
cer cell lines (MCF-7, SKBR3)66 and anti-malarial activity.18 In the
present investigation, we observed that the EC50 values of the isox-
azole and pyrazole derivatives did show improved binding with
the PKCh as compared with curcumin, but, did not show much
1b
2
3
4
5
572
395
396
428
415
559
387
393
420
408
562
390
392
410
405
561
389
393
419
410
2–5, 5 ꢀ 10ꢁ6 M and protein, 50 ꢀ 10ꢁ6 M. Spectra were recorded after incu-
a
bating the compound for 40 min at 25 °C.
b
Ref. 53.
Table 4
Summary of the results obtained from molecular docking. The compounds were
docked into the activator binding sites of the C1B domains using Surflex dock module
of Sybyl 8.0. Hydrogen bonds those are formed within the distance of 3.0 Å are
reported here. Higher total score indicates higher binding probability
improvement for the PKCd and PKCe.
The PKC C1B domains are highly conserved in their sequences
and have similar overall structure. However, there is less conserva-
tion among the residues exterior of the domain that influences
their interaction and affinity for the phospholipid bilayer. In fact,
the lipid selectivity was found to be quite different for PKCs
belonging to the same novel class.67–69 In the present study, the
binding assay was performed in the absence of any lipid, thereby
ruling out the effects of lipid environment on the binding affinity
of these ligands with the novel PKCs observed here. Therefore,
these affinity differences are solely on the basis of the differences
in the residues and surface area of the activator binding pockets.
Compound
PKCdC1B
PKCe
C1B
Total
PKChC1B
No. of
H-bond
Total
score
No. of
H-bond
No. of
H-bond
Total
score
score
1
2
3
4
4
3
1
1
1
4
6.39
5.90
4.07
5.66
6.13
5.56
6
7
5
3
4
2
4.13
4.83
3.61
4.16
5.54
3.02
3
5
4
1
0
3
4.18
4.66
3.85
4.33
4.87
3.76
5
Phorbol-13-OAc
Figure 5. Docking of the isoxazole derivatives into PKChC1B. Structure of (A) curcumin (1), (B) 4,40-((1E,10E)-(1H-pyrazole-3,5-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol
(2), and (C) 4-((E)-2-(5-((E)-4-butoxy-3-methoxystyryl)-1H-pyrazol-3-yl)vinyl)-2-methoxyphenol (3) docked into PKChC1B. Only the region of the protein that interacts with
the ligand is shown here. The docked structures are generated using the Surflex dock module of Sybyl 8.0 and visualized using Chimera package from the Computer Gpaphics
Laboratory, University of California San Francisco, San Francisco, CA, USA. The NMR structure of PKChC1B (PDB code: 2ENZ) has been used as the receptor template.

J. Das et al. / Bioorg. Med. Chem. 19 (2011) 6196–6202
6201
solvent mixture (hexane:ethyl acetate:methanol = 60:27:13). Yield
57%; 1H NMR (CD3OD, 300 MHz): d 7.13 (2H, d, J = 15.0 Hz), 7.10–
7.0 (4H, m), 6.91–6.89 (2H, m), 6.75 (2H, d, J = 14.5 Hz), 6.64 (1H,
s), 3.99 (2H, t), 3.89 (3H, s), 3.87 (3H, s), 1.77 (2H, m), 1.46 (2H,
m), 0.89 (3H, t); 13C NMR (CD3OD, 300 MHz): d 149.2, 147.2,
131.0, 130.4, 129.3, 122.3, 118.0, 116.6, 109.0,105.2, 68.7, 56.2,
31.6, 19.2, 14.1; MALDI: 421.3 [M+H].
4. Conclusion
We described here the synthesis, spectral properties and bind-
ing of isoxazole and pyrazole derivatives of curcumin to the C1B
sub domains of novel protein kinase C. Our results showed that
isoxazole, pyrazole and their long chain derivatives bind to the
activator-binding domain of PKC by forming hydrogen bonds with
the residues at the activator binding site. Of the three novel PKCs
studied here, PKCh showed higher binding and the addition of ali-
phatic chain did not show any improvement in binding. Our results
also indicated that both carbonyl and hydroxyl group of curcumin
are important for its binding to the protein. The results presented
here laid a foundation for future detailed study involving various
curcumin derivatives and PKCs. On the basis of the results pre-
sented here, we are currently working towards improving the
affinity and selectivity of the curcumin derivatives for the C1 do-
main containing proteins and also studying the effects of curcumin
derivatives in membrane translocation properties of various PKC
subtypes.
5.2.3. Synthesis of the isoxazole derivative of curcumin,
4,40-((1E,10E)-isoxazole-3,5-diyl bis(ethene-2,1-diyl))bis(2-
methoxyphenol) (4)
Hydroxylamine hydrochloride (1.1 equiv) is refluxed with
1 equiv of curcumin in the presence of acetic acid as the solvent
for 6 h. The reaction mixture was evaporated to dryness; residue
was dissolved in dichloromethane and washed with water. The
crude product was subjected to chromatographic purification on
a silica gel column. The compound was eluted with the solvent
mixture (ethyl acetate/hexane/DCM, 11:7:82).
5.2.4. Synthesis of the isoxazole analog, 3,5-bis((E)-4-butoxy-3-
methoxystyryl)isoxazole (5)
5. Experimental protocols
5.1. General
Di-alkylation of the isoxazole derivative (1 equiv) was per-
formed by addition of bromobutane (2.2 equiv) and K2CO3
(2.2 equiv) in acetone. The mixture was refluxed for 24 h. After
cooling the mixture to room temperature and filtering, solvent
was removed under vacuum. The pure product was obtained by sil-
ica gel column chromatography (ethyl acetate:hexanes = 1:10).
Yield 68%; 1H NMR (CD3OD, 300 MHz): d 7.30 (d, J = 16.0 Hz, 2H),
7.10 (d, J = 16.0 Hz, 2H), 6.96 (d, J = 8.0 Hz, 2H), 6.91 (s, 2H), 6.83
(d, J = 8.0 Hz, 2H), 6.43 (s, 1H), 4.05 (t, 4H), 3.96 (s, 6H), 1.82 (m,
4H), 1.5 (m, 4H), 0.98 (t,6H); 13C NMR (CD3OD, 300 MHz): d
167.7, 161.8, 149.7, 149.4, 135.2, 134.0, 128.9, 128.4, 121.0,
120.5, 113.9, 112.2, 110.9, 109.1, 108.9, 97.7, 68.1, 56.0, 19.9,
14.2; ES-MS: 478.2 [M+H].
Curcumin was purchased from Sigma and used without further
purification. Glutathione-sepharose 4B was from GE healthcare
biosciences. Solvents were purchased from VWR and Fisher. All
other reagents were from Sigma. Progress of chemical reaction
was monitored through thin layer chromatography (TLC) on
pre-coated glass plates (silica gel 60 F254, 0.25 mm thickness)
purchased from EMD chemicals. 1H and 13C NMR spectra were re-
corded on a QE-300 spectrometer. Unless otherwise specified, all
NMR spectra were obtained in deuterated chloroform (CDCl3)
and referenced to the residual solvent peak; chemical shifts are re-
ported in parts per million, and coupling constants in hertz (Hz).
Multiplicities are reported as follows: s (singlet), d (doublet), t
(triplet), m (multiplet) and br (broadened). Mass spectra were
obtained on either a VG 70-S Nier Johnson or JEOL Mass Spectrom-
eter. Absorption spectra were recorded on Hitachi U-2910
spectrophotometer.
5.2.5. Synthesis of the isoxazole analog, 3,5-bis((E)-3-methoxy-
4-(octyloxy)styryl)isoxazole (6)
Di-alkylation of the isoxazole derivative (1 equiv) was per-
formed by addition of bromo-octane (2.2 equiv) and K2CO3
(2.2 equiv) in acetone. The mixture was refluxed for 24 h. After
cooling the mixture to room temperature and filtering, solvent
was removed under vacuum. The pure product was obtained by sil-
ica gel column chromatography (ethyl acetate:hexanes = 1:10).
Yield 72%; 1H NMR (CDCl3, 300 MHz): d 7.40 (2H, d, J = 16.5 Hz),
7.21–7.05 (6H, m), 6.95 (2H, d, J = 16.4 Hz), 6.42 (1H, s), 4.03 (4H,
t), 3.92 (3H, s), 3.91 (3H, s), 1.85 (4H, m), 1.46 (4H, m), 1.38–1.24
(16H, m), 0.86 (6H, t); 13C NMR (CD3OD, 300 MHz): d 168.5,
162.2, 149.9, 149.6, 135.5, 134.8, 128.8, 128.5, 121.2, 120.9,
114.1, 112.6, 111.0, 109.5, 109.2, 97.7, 69.1, 56.1, 56.0, 31.9, 29.8,
29.4, 293, 29.1, 26.0, 22.7, 14.2; ES-MS: 590.9 [M+H].
5.2. Chemical synthesis
5.2.1. Synthesis of the pyrazole derivative of curcumin,
(4,40-((1E,10E)-(1H-pyrazole-3, 5-diyl) bis (ethene-2,1-diyl))
bis(2-methoxyphenol) (2)
Curcumin (1.0 equiv) was dissolved in glacial acetic acid (5 mL),
and hydrazine hydrate (1.1 equiv) was added to the solution. The
solution was refluxed for 8 h, and then the solvent was removed
under vacuum. Residue was dissolved in ethyl acetate and washed
with water. Organic portion was collected, dried over sodium sul-
fate, and concentrated under vacuum. Crude product was purified
by column chromatography on a silica column. The solvent system
used was hexane/ethyl acetate, 65:35.
5.3. Bacterial expression and purification of the PKC eC1B, dC1B,
and hC1B sub domains
The PKCC1B subdomain fused with glutathione S-transferase
(GST) was expressed in BL21 gold Escherichia coli and purified as
described earlier.53 Binding studies were performed with the pro-
teins devoid of the GST tag.
5.2.2. Synthesis of the pyrazole analog, 4-((E)-2-(5-((E)-4-
butoxy-3-methoxystyryl)-1H-pyrazol-3-yl)vinyl)-2-
methoxyphenol (3)
Mono alkylation of the pyrazole derivative (2) (1 equiv) was
performed by addition of bromobutane (1 equiv) and K2CO3
(1 equiv) in acetone refluxed at 85 °C. The reaction was allowed
to run for 30 h, and then washed with water. Following extraction
with ethyl acetate (2 ꢀ 10 ml), the combined organic layers were
evaporated under vacuum to obtain crude products. The product
was purified through silica gel column chromatography using a
5.4. Fluorescence studies
Fluorescence spectra were recorded on PTI LPS 220B equipped
with temperature and stirring control systems. A 1.5-ml cuvette
(Hellma) with a Teflon stopper was used for fluorescence measure-
ments. For fluorescence quenching experiments, protein (2
and varying concentration of ligands (0–35 M) were incubated
lM)
l

6202
J. Das et al. / Bioorg. Med. Chem. 19 (2011) 6196–6202
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ZnSO4, pH 7.2) at 25 °C. Protein was excited at 280 nm and emis-
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intensity data, (F0 ꢁ F)/F were plotted against the ligand concentra-
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l
M) in the presence of
M protein in buffer (50 mM Tris, 150 mM NaCl, 2 mM DTT,
M ZnSO4, pH 7.2). Compounds 2–5 were incubated with the
50
50
l
l
proteins for 1 h and excited at their corresponding absorption
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5.5. Generation of 3D models of PKC C1B sub domains and
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Generation of the homology model for PKCeC1B and subsequent
energy minimization was done according to the methods described
earlier.53 Molecular docking was performed on SurflexDoc module
of Sybyl 8.0. Protomols were generated using threshold, bloat and
radius values of 0.5, 2.0, and 3 Å, respectively. Residues Tyr-239,
Lys-240, Ser-241, Pro-242, Thr-243, Phe-244, Leu-251, Leu-252,
Trp-253, Gly-254, Leu-255, and Glu-258 of PKC theta; Tyr-238,
Met-239, Ser-240, Pro-241, Thr-242, Phe-243, Leu-250, Leu-251,
Trp-252, Gly-253, Leu-254, and Gln-257 for PKC delta and Tyr-
250, Lys-251, Val-252, pro-253, Thr-254, Phe-255, Leu-262, Leu-
263, Trp-264, Gly-265, Leu-266, and Gln-269 for PKC epsilon were
used for protomol generation. These residues were selected by
comparing the PKC activator phorbol ester binding site in PKCd
C1B. For docking, max conformation and max rotation values were
20 and 100, respectively. Pre-dock and Post-dock energy minimiza-
tion methods were also applied. Docking results were compared by
the total score values. A higher total-score value represents better
docking of the ligands in the receptor site.
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