Design, synthesis, and structure-activity relationship of new isobenzofuranone ligands of protein kinase C

Article abstract of DOI:10.1016/j.bmcl.2004.02.097

Protein kinase C (PKC) is a family of enzymes, which play important roles in intracellular signal transduction. We have designed novel PKC ligands having an isobenzofuranone template, based on the proposed interaction of DAG (1,2-diacyl-sn-glycerol) with

Full text of DOI:10.1016/j.bmcl.2004.02.097

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2963–2967
Design, synthesis, and structure–activity relationship of new
isobenzofuranone ligands of protein kinase C
Yoshiyasu Baba,a,b Yosuke Ogoshi,a Go Hirai,a Takeshi Yanagisawa,b
Kumiko Nagamatsu,b,d Satoshi Mayumi,c Yuichi Hashimotoc and
Mikiko Sodeokaa,b,c,d,
*
aInstitute of Multidisciplinary Research for Advanced Materials (IMRAM), Tohoku University, 2-1-1 Katahira, Aoba,
Sendai, Miyagi 980-8577, Japan
bSagami Chemical Research Center, Kanagawa, Japan
cInstitute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
dPRESTO, Japan Science and Technology Agency, Japan
Received 20 January 2004; accepted 28 February 2004
Abstract—Protein kinase C (PKC) is a family of enzymes, which play important roles in intracellular signal transduction. We have
designed novel PKC ligands having an isobenzofuranone template, based on the proposed interaction of DAG (1,2-diacyl-sn-
glycerol) with the PKCd C1B ligand-binding domain. Several isobenzofuranone derivatives were synthesized and their PKCa
binding activities were evaluated. The pivaloyl derivative 1f was found to be a strong PKCa ligand, and the structure–activity
relationship is well explained by our proposed binding model.
Ó 2004 Elsevier Ltd. All rights reserved.
Protein kinase C (PKC) isozymes play important roles
in intracellular signal transduction of a variety of cell
events, such as proliferation, differentiation, and apop-
tosis.1 The isozymes are divided into three classes, con-
ventional PKCs (a, bI/bII, c), novel PKCs ðd; e; g; hÞ,
and atypical PKCs ðf; i=kÞ. DAG (1,2-diacyl-sn-glyc-
erol) is a physiological ligand for the former two classes
of isozymes, and several complex natural products, such
as phorbol esters, bryostatins, aplysiatoxins, and teleo-
cidins, are strong activators of these isozymes.2 It is now
believed that each isozyme participates in different sig-
naling pathways, and, in some cases, the same enzyme
triggers different cell responses depending on the stim-
ulus, though the precise molecular mechanisms of acti-
vation and the biological roles of each isozyme remain
to be clarified. The conventional PKCs have the regul-
atory domain, which includes the activator-binding do-
main (C1) and the Ca2þ-binding domain (C2). The C1
domain contains a repeat of cysteine-rich domains C1A
and C1B, each of which can potentially bind the
endogenous ligand, DAG, or an exogenous ligand such
as phorbol ester. Although many analogs of these nat-
ural products have been synthesized and tested,3 an
isozyme-specific ligand has not been found. Therefore,
development of a novel PKC ligand template, which has
distinct structural features and can be a platform for
the synthesis of variety of analogs, is of particular
interest. Here we would like to report the design,
synthesis, and evaluation of new DAG-type PKC
ligands.
DAG is a nontumor-promoting physiological ligand of
PKCs, and shows different isozyme selectivity from that
of tumor-promoting phorbol esters.4 DAG itself, how-
ever, is metabolically unstable and conformationally too
flexible to be useful for further rational structural
modifications. Marquez and co-workers have reported
thorough studies on conformationally constrained ana-
logs of DAG.5 Some of their derivatives showed strong
binding affinity to PKC; however, they suggested that
their monocyclic templates can bind PKC in two dif-
ferent binding modes and this would make further
rational structural modification difficult. As a part of
our project on clarifying the molecular mechanisms of
PKC activation,6 we decided to design a new DAG-type
template based on the crystal structure of the PKCd
C1B domain in the complex with phorbol 13-acetate
(Fig. 1).7 Hydrogen bondings between the C3-carbonyl
* Corresponding author. Tel./fax: +81-22-217-5601; e-mail: sodeoka@
tagen.tohoku.ac.jp
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.097

2964
Y. Baba et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2963–2967
bond with Gly253.9 If this is the case, there is much
extra room in the binding pocket in the direction of the
sn-2 side chain. We designed the novel isobenzofuranone
template 1, which is expected to fit into the binding
pocket. According to our binding model, the (R)-ste-
reochemistry of the quaternary carbon center should be
preferable, and the side chain at the C7 position can be
oriented to a direction similar to that of the C12 or C13
side chain of phorbol ester. Furthermore, this iso-
benzofuranone template has considerable scope for
modification on the benzene ring, which may be effective
for the development of an isozyme-selective analog.
To see if this novel isobenzofuranone template can act as
a PKC ligand, we first synthesized optically active iso-
benzofuranones 1a and 1b having a hydrophobic side
chain at the C7 position. Scheme 1 shows the synthetic
route to both enantiomers of 1a and 1b. The tert-
butyldimethylsilyl (TBS) ether of salicylamide 3 was
lithiated and then reacted with the ketone 5 prepared
from the dihydroxyacetone dimer 4. Treatment of the
resulting adduct with sodium hydride afforded the five-
membered lactone. The TBS group of the aromatic
alcohol was selectively removed by treatment with
potassium carbonate in methanol to give 6. After pro-
tection of the phenol moiety with an allyloxycarbonyl
(alloc) group, two TBS groups were removed, and
monoacetylation was carried out using acetyl chloride in
the presence of silica gel. Separation of enantiomers of
the monoacetate 7 was achieved by preparative HPLC
using a chiral phase column. The optically pure (R)- and
(S)-7 was then converted to (S)- and (R)-8, respectively,
via two-step sequences. After introduction of a hydro-
Figure 1. (A) Hydrogen bondings in the PKCd-phorbol 13-acetate
complex, and design of novel isobenzofuranone ligand, based on the
DAG structure. (B) Superposition of (R)-1 (R ¼ R0 ¼ Me) on phorbol
13-acetate.
group of phorbol and the NH group of Gly253 and
between the C20-hydroxyl group of phorbol and the
NH, and carbonyl groups of Thr242 and Leu251 were
found to be critical for the ligand binding, and these
interactions should be conserved throughout the iso-
zymes. Though DAG has two carbonyl groups, SAR
studies indicated that the sn-1 carbonyl group is more
important than the sn-2 carbonyl group.8 A molecular
modeling study also suggested that the sn-1 carbonyl
group of DAG would preferentially form a hydrogen
OR1
OR1
OR1
OH
OCOCH2CH=CH2
j, k
O
O
O
O
O
f, g, h, i
Et2N
a
m
c, d, e
O
O
O
O
O
*
*
*
2: R1 = H
TBSO
AcO
3: R1 = TBS
AcO
AcO
OTBS
n, o
OH
OTBS
OH
6
(R)-(-)-1a: R1 = (CH2)9CH3
(R)-(-)-1b: R1 = (CH2)11CH3
(S)-(+)-1a: R1 = (CH2)9CH3
(S)-(+)-1b: R1 = (CH2)11CH3
(S)-(+)-9a: R1 = (CH2)9CH3
(S)-(+)-9b: R1 = (CH2)11CH3
(R)-(-)-9a: R1 = (CH2)9CH3
(R)-(-)-9b: R1 = (CH2)11CH3
(R)-(-)-7
TBSO
OTBS
(S)-(+)-7
l
(S)-(+)-8: R1 = H
5
OH
OBn
O
(R)-(-)-8: R1 = H
p, q, r
O
O
b
HO
O
OH
O(CH2)11CH3
OBn
O
O
O
OBn
u, v
w, x, y, z
4
OH
OH
OH
10
O
O
*
O
10
*
*
(R)-(-)-1c: R3 = c-Hex
(R)-(-)-1d: R3 = C6H5
(R)-(-)-1e: R3 = 3,5-t-Bu2-C6H3
(R)-(-)-1f: R3 = t-Bu
R3COO
OH
OH
OTBS
OR2
AcO
(S)-(+)-14
(R)-(-)-14
aa
(S)-(+)-11: R2 = TBS
(R)-(-)-12: R2 = H
(S)-(+)-12: R2 = H
t
s
(S)-(+)-8
(R)-(-)-8
(S)-(+)-11
(R)-(-)-11
(R)-(-)-11: R2 = TBS
(S)-(+)-1f: R3 = t-Bu
Scheme 1. Synthesis of the optically active isobenzofuranone derivatives. Reagents and conditions: (a) TBSCl, Et3N, DMAP, CH2Cl2, rt, 15 h
(quant); (b) TBSCl, DMAP, pyridine, rt, 5 d (92%); (c) s-BuLi, TMEDA, THF, )40 °C, 1.5 h, then 5, )40–)30 °C, 17 h; (d) NaH, THF, 70 °C, 3 h; (e)
K2CO3, MeOH, 70 °C, 3 h (46% from 5, three steps); (f) CH2@CHCH2OCOCl, Et3N, DMAP, THF, rt, 3 h (92%); (g) HFÆPy, pyridine–CH2Cl2, rt,
12 h (95%); (h) AcCl, SiO2, THF–Et2O, 0 °C, 21 h (79%); (i) separation by HPLC (CHIRALPAK AD, hexane:i-PrOH ¼ 3:1); (j) TBSCl, imidazole,
DMF, 0 °C, 3 h (72%); (k) Pd(PPh3)4, PPh3, HCOOH, BnNH2, THF, rt, 15 h (96%); (l) R1OH, DEAD, PPh3, THF, rt, 10 h (R1 ¼ (CH2)9CH3: 9a,
92%; R1 ¼ (CH2)11CH3: 9b, 79%); (m) HFÆPy, pyridine–CH2Cl2, rt, 8 h (R1 ¼ (CH2)9CH3: 1a, 90%; R1 ¼ (CH2)11CH3: 1b, 89%); (n) BnBr, K2CO3,
acetone, rt, 19 h (85%); (o) HFÆPy, pyridine–CH2Cl2, rt (94%); (p) AcCl, SiO2, THF, 0 °C–rt, 5 d (69%); (q) TBSCl, imidazole, DMF, rt, 3 h (94%); (r)
separation by HPLC (CHIRALPAK AD, hexane:i-PrOH ¼ 9:1); (s) HFÆPy, pyridine–CH2Cl2, rt, 113 h (98%); (t) H2, Pd/C, EtOH, rt (97%); (u)
TBSCl, imidazole, DMF, 0 °C, 2 h (51%); (v) separation by HPLC (CHIRALPAK AD-H, hexane:i-PrOH ¼ 3:1); (w) PvCl, Et3N, THF, rt, 5.5 h or
R2COCl, pyridine, rt; (x) H2, Pd/C, EtOH or MeOH, rt (R2 ¼ c-Hex: 84%, R2 ¼ C6H5: 30%, R2 ¼ 3,5-t-Bu2-C6H3: 75%, R2 ¼ t-Bu: 60%, two steps);
(y) CH3(CH2)11OH, DEAD, PPh3, THF, rt; (z) HFÆPy, pyridine–CH2Cl2, 0 °C–rt (R2 ¼ c-Hex: 1c, 96%, R2 ¼ C6H5: 1d, quant, R2 ¼ 3,5-t-Bu2–C6H3:
1e, 96%, R2 ¼ t-Bu: 1f, 98%, two steps); (aa) AcCl, Et3N, DMAP,CH2Cl2 (64%).

Y. Baba et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2963–2967
2965
phobic side chain (C10 or C12) by means of the Mitsun-
obu reaction, the TBS group was removed using
HFÆpyridine complex to give (R)- or (S)-1a,1b. Optical
purity of these final products was confirmed by HPLC
analysis. When tetrabutylammonium fluoride was used
for deprotection, significant racemization was observed.
Both enantiomers of the 7-benzyloxy derivative 12 were
prepared from 6 in six steps, including the separation of
the enantiomers of 11 with preparative HPLC. The
absolute stereochemistry of (þ)-12 was unequivocally
determined to be (S) by X-ray analysis of its (1S)-
camphanic acid ester 13 (Fig. 2). Since the stereochem-
istry of ())-11 was automatically determined to be (R) by
the correlation with (S)-(þ)-12, the stereochemistry of all
the other compounds, 7, 8, 9a,b, and 1a,b, was deter-
mined by the conversion of (R)-())-11 to (R)-())-8.10
sequences shown in Scheme 1. Dose-dependent inhibi-
tion curves of these compounds for [3H]PDBu binding
to PKCa are shown in Figure 4. As expected, when the
acetyl group was changed to a cyclohexanoyl group
((R)-1c), 10-fold improvement of the binding affinity
was observed.12 The benzoyl derivative (R)-1d was not
as potent as (R)-1c. Further introduction of a hydro-
phobic substituent on the benzene ring rather decreased
the binding to PKCa. Finally, the pivaloyl derivative
(R)-1f showed the strongest binding to PKCa among
these compounds. The Ki value of (R)-1f was 122 nM,
which is much smaller than that of 1-oleoyl-2-acetyl-sn-
glycerol (OAG, Ki ¼ 540 nM). The (S)-enantiomer of 1f
was, again, less effective than the (R)-enantiomer.
Figure 5 shows the results of molecular modeling of the
complex of the PKCd C1B domain with the pivaloyl
derivative. Calculation was carried out for the com-
pound with the shorter C3 methylene chain at the C7
position instead of the C12 methylene chain of (R)-1f.13
The model suggests that positive hydrophobic interac-
tion between the tert-butyl group and Leu250 and
Leu254 contributes to the increase of the affinity.
Next, the binding affinity of isobenzofuranone deriva-
tives to PKCa was preliminarily examined by measuring
competitive inhibition of the binding of tritium-labeled
phorbol dibutyrate ([3H]PDBu) to PKCa.11 As expected,
compounds (R)-1a and 1b exhibited significant com-
petitive binding to PKCa (Fig. 3). The (S)-enantiomers
of these compounds were less potent than the
(R)-enantiomers, supporting our design concept. Com-
pounds having a less hydrophobic side chain, such as
alloc ((R)-7) or benzyl ((R)-12), were less effective, and at
least a C12 methylene chain seemed to be required for
strong binding to PKC (Fig. 3).
100
(R)-1b
(
R
)-1c
)-1d
)-1e
)-1f
80
60
40
20
0
(R
(R
To further improve the affinity of the isobenzofuranone
derivatives to PKC, we next synthesized several acyl
chain analogs (R)-1c–f according to the reaction
(R
(S
)-1f
TPA
OAG
0.01
0.1
1
10
100
Concentration [µM]
Figure 4. Dose-dependent inhibition curves of 1b–f for [3H]PDBu
binding to PKCa.
Figure 2. Determination of absolute stereochemistry. The crystal
structural information has been deposited in the Cambridge Crystal-
lographic Data Center (deposition no CCDC 223959).
100
80
60
40
20
0
(
R
)-7
(R
)-12
(R
)-1a
(
R
)-1b
(S
)-1a
(S)-1b
Figure 3. Inhibition of binding of [3H]PDBu to PKCa by the iso-
benzofuranone derivatives at 100 lM.
Figure 5. Binding model of (R)-1f with PKCd C1B. Modeling was
carried out on the compound with the shorter (C3) side chain.13

2966
Y. Baba et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2963–2967
Koehler, M. F. T.; Lippa, B.; Park, C.-M.; Shiozaki, M.
J. Am. Chem. Soc. 1998, 120, 4534–4535; (e) Wender,
P. A.; Baryza, J. L.; Bennett, C. E.; Bi, F. C.; Brenner,
Although the data shown in Figure 4 were obtained
using PKCa, these two leucines are conserved in the
PKCa C1B domain (Leu121 and Leu125). As a result,
the tert-butyl group fills the gap between the two leu-
cines and forms a continuous hydrophobic surface with
Leu250 and Leu254, which may be favorable for inter-
action with the membrane.
^
S. E.; Clarke, M. O.; Horan, J. C.; Kan, C.; Lacote, E.;
Lippa, B.; Nell, P. G.; Turner, T. M. J. Am. Chem. Soc.
2002, 124, 13648–13649; (f) Wender, P. A.; Mayweg,
A. V. W.; VanDeusen, C. L. Org. Lett. 2003, 5, 277–279;
For aplysiatoxin analogs: (g) Nakamura, H.; Kishi, Y.;
Pajares, M.; Rando, R. R. Proc. Natl. Acad. Sci. 1989, 86,
9672–9676; For teleocidin analogs: (h) Ohno, M.; Endo,
Y.; Hirano, M.; Itai, A.; Shudo, K. Tetrahedron Lett.
1993, 34, 8119–8122; (i) Endo, Y.; Ohno, M.; Hirano, M.;
Itai, A.; Shudo, K. J. Am. Chem. Soc. 1996, 118, 1841–
1855; (j) Irie, K.; Isaka, T.; Iwata, Y.; Yanai, Y.;
Nakamura, Y.; Koizumi, F.; Ohigashi, H.; Wender, P.
A.; Satomi, Y.; Nishino, H. J. Am. Chem. Soc. 1996, 118,
10733–10743; (k) Nakagawa, Y.; Irie, K.; Yamanaka, N.;
Ohigashi, H.; Tsuda, K. Bioorg. Med. Chem. Lett. 2003,
13, 3015–3019; (l) Kozikowski, A. P.; Ma, D.; Pang, Y.-P.;
Shum, P.; Likic, V.; Mishra, P. K.; Macura, S.; Basu, A.;
Lazo, J. S.; Ball, R. G. J. Am. Chem. Soc. 1993, 115, 3957–
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A. P. Org. Lett. 2002, 4, 2169–2172, and references cited
therein.
In conclusion, we have designed a novel isobenzofura-
none template for PKC ligands based on the proposed
interaction of DAG with PKC. Several derivatives were
synthesized and their PKCa binding activities were
evaluated. The pivaloyl derivative (R)-1f was found to
be a strong PKCa ligand, and the structure–activity
relationship is well explained by our binding model.
Design and synthesis of novel isobenzofuranone analogs
based on this binding model should be useful for re-
search to clarify the molecular mechanisms of PKC
activation and also for the development of isozyme
selective ligands.
4. Kazaniets, M. G.; Areces, L.; Bahador, A.; Mischak, H.;
Goodnight, J.; Mushinski, J. F.; Blumberg, P. M. Mol.
Pharmacol. 1993, 44, 298–307.
Acknowledgements
5. (a) Teng, K.; Marquez, V. E.; Milne, G. W. A.; Barchi,
J. J., Jr.; Kazanietz, M. G.; Lewin, N. E.; Blumberg,
P. M.; Abushanab, E. J. Am. Chem. Soc. 1992, 114, 1059–
1070; (b) Sharma, R.; Lee, J.; Wang, S.; Milne, G. W. A.;
Lewin, N. E.; Blumberg, P. M.; Marquez, V. E. J. Med.
Chem. 1996, 39, 19–28; (c) Lee, J.; Wang, S.; Milne, G. W.
A.; Sharma, R.; Lewin, N. E.; Blumberg, P. M.; Marquez,
V. E. J. Med. Chem. 1996, 39, 29–35; (d) Lee, J.; Sharma,
R.; Wang, S.; Milne, G. W. A.; Lewin, N. E.; Szallasi, Z.;
Blumberg, P. M.; George, C.; Marquez, V. E. J. Med.
Chem. 1996, 39, 36–45; (e) Nacro, K.; Bienfait, B.; Lee, J.;
Han, K.-C.; Kang, J.-H.; Benzaria, S.; Lewin, N. E.;
Bhattacharyya, D. K.; Blumberg, P. M.; Marquez, V. E.
J. Med. Chem. 2000, 43, 921–944; (f) Marquez, V. E.;
Nacro, K.; Benzaria, S.; Lee, J.; Sharma, R.; Teng, K.;
Milne, G. W. A.; Bienfait, B.; Wang, S.; Lewin, N. E.;
Blumberg, P. M. Pharmacol. Ther. 1999, 82, 251–261, and
references cited therein.
We thank Mr. Yasuharu Ijuin (Sagami Chemical Re-
search Center) for X-ray analysis. This work was sup-
ported in part by grants from Uehara Memorial
Foundation and Hoh-ansha Foundation, Kanagawa
Academy of Science and Technology Research Grants, a
Grant-in-Aid for Creative Scientific Research (no
13NP0401) and a Grant-in-Aid for Scientific Research
from the Japan Society for the Promotion of Science,
and a Grant-in-Aid for the COE project, Giant Mole-
cules and Complex Systems, from The Ministry of
Education, Culture, Sports, Science and Technology.
References and notes
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Horwood: New York, 1992.
6. (a) Sodeoka, M.; Arai, M. A.; Adachi, K.; Uotsu, K.;
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Shibasaki, M. Bioorg. Med. Chem. 1998, 6, 1117–
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2. (a) Kishi, Y.; Rando, R. R. Acc. Chem. Res. 1998, 31, 163–
172; (b) Castagna, M.; Takai, Y.; Kaibuchi, K.; Sano, K.;
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7847–7851; (c) Fujiki, H.; Tanaka, Y.; Miyake, R.;
Kikkawa, U.; Nishizuka, Y.; Sugimura, T. Biochem.
Biophys. Res. Commun. 1984, 120, 339–343; (d) Smith,
J. B.; Smith, L.; Pettit, G. R. Biochem. Biophys. Res.
Commun. 1985, 132, 939–945; (e) Berkow, R. L.; Kraft, A.
S. Biochem. Biophys. Res. Commun. 1985, 131, 1109–1116.
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M.; Sasai, H.; Shibasaki, M. Chem. Pharm. Bull. 1996, 44,
463–465; (b) Wender, P. A.; Kirshberg, T. A.; Williams, P.
D.; Bastiaans, H. M. M.; Irie, K. Org. Lett. 1999, 1, 1009–
1012; (c) Tanaka, M.; Irie, K.; Nakagawa, Y.; Nakamura,
Y.; Ohigashi, H.; Wender, P. A. Bioorg. Med. Chem. Lett.
2001, 11, 719–722; For bryostatin analogs: (d) Wender,
P. A.; De Brabander, J.; Harran, P. G.; Jimenez, J.-M.;
7. Zhang, G.; Kazanietz, M. G.; Blumberg, P. M.; Hurley,
J. H. Cell 1995, 81, 917–924.
8. Heymans, F.; Da Silva, C.; Marrec, N.; Godfroid, J.-J.;
Castagna, M. FEBS Lett. 1987, 218, 35–40.
9. Nacro, K.; Sigano, D. M.; Yan, S.; Niklaus, M. C.;
Pearce, L. L.; Lewin, N. E.; Garfield, S. H.; Blumberg,
P. M.; Marquez, V. E. J. Med. Chem. 2001, 44, 1892–1904.
10. All compounds were characterized by spectroscopic anal-
yses. Purity of the compounds obtained was determined to
be more than 95% based on the 1H NMR spectral analysis.
Spectral data of selected compounds are as follows. (R)-
20
D
())-1b: Colorless oil; ½aꢀ )14.6 (c ¼ 0:640, CHCl3); 1H
NMR (200 MHz, CDCl3) d 0.88 (t, J ¼ 6:7 Hz, 3H), 1.1–
1.6 (m, 18H), 1.8–2.0 (m, 2H), 2.03 (s, 3H), 2.20 (br s, 1H),
3.89 (d, J ¼ 6:6 Hz, 2H), 4.14 (t, J ¼ 6:8 Hz, 2H), 4.42 (d,
J ¼ 11:9 Hz, 1H), 4.53 (d, J ¼ 11:9 Hz, 1H), 6.96 (d,
J ¼ 8:4 Hz, 1H), 7.02 (d, J ¼ 7:4 Hz, 1H), 7.60 (dd,

Y. Baba et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2963–2967
2967
J ¼ 8:4, 7.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) d
14.10, 20.63, 22.67, 25.81, 28.82, 29.33, 29.51, 29.59, 29.61,
29.65, 31.90, 64.19, 64.49, 69.14, 85.32, 112.73, 113.55,
178.04; MALDI-TOFMS (positive ion, a-cyano-4-
hydroxycinnamic acid) calcd for C27H42O6Na (M+Naþ)
485.29; found 485.24.
114.00, 136.41, 149.97, 158.49, 166.89, 170.67; LRMS (EI,
11. Assay protocol: Inhibition of [3H]PDBu binding was
measured essentially according to the reported procedure.
See: Tanaka, Y.; Miyake, R.; Kikkawa, U.; Nishizuka, Y.
J. Biochem. 1986, 99, 257–261; Briefly, plastic test tubes of
each assay mixture (300 lL) contained 50 mM Tris–HCl
(pH 7.5), 4 mM CaCl2, 100 lg/mL 1,2-di-(cis-9-octadece-
22
m=z) 420 (Mþ). (R)-())-1c: Colorless oil; ½aꢀ )21.8
D
(c ¼ 0:41, CHCl3); 1H NMR (400 MHz, CDCl3) d 0.88
(t, J ¼ 6:8 Hz, 3H), 1.26 (m, 20H), 1.49 (tt, J ¼ 6:6,
6.8 Hz, 2H), 1.62–1.82 (m, 6H), 1.89 (tt, J ¼ 6:6, 6.8 Hz,
2H), 2.23–2.29 (m, 2H), 3.88 (br s, 2H), 4.14 (t, J ¼ 6:6 Hz,
2H), 4.49 (br s, 2H), 6.96 (d, J ¼ 8:3 Hz, 1H), 7.02 (d,
J ¼ 7:6 Hz, 1H), 7.59 (dd, J ¼ 8:3, 7.6 Hz, 1H); 13C NMR
(100 MHz, CDCl3) d 14.11, 22.67, 25.20, 25.24, 25.57,
25.79, 28.76, 29.33, 29.51, 29.58, 29.61, 29.65, 29.69, 31.89,
42.85, 63.71, 64.45, 69.10, 85.73, 112.65, 113.65, 114.09,
136.33, 149.95, 158.40, 166.95, 175.71; MALDI-TOFMS
(positive ion, matrix a-cyano-4-hydroxycinnamic acid)
noyl)-sn-glycero-3-phospho-L-serine sodium salt (L-PS,
from SIGMA), 4 mg/mL BSA, 10 nM [3H]PDBu, 4.3–
4.8 nM protein kinase Ca (human, recombinant, Spodop-
tera frugiperda from CALBIOCHEM), and each concen-
tration of the synthetic compound or TPA. L-PS was
sonicated in 50 mM Tris–HCl at 0 °C prior to use. After
incubation at 0 °C for 2 h, the mixture was diluted with
cold 0.5 % DMSO (2.5 mL) then filtered through a glass-
fiber filter (Whatman GF/B), which had been pretreated
with 0.3% polyethyleneimine for 1 h. The filter was washed
four times with 2 mL of cold 0.5% DMSO. The radioac-
tivity of each filter was counted in a scintillation vial with
5 mL of scintillator (Clear-sol I from NACALAI TES-
QUE) using a liquid scintillation counter. The count for
the tube with 10 lM TPA was taken as the background
(100% inhibition) and subtracted from the count of each
tube. The Kd value of [3H]PDBu under these assay
conditions was determined to be 0.19 nM by Scatchard
plot analysis. Nonspecific binding was measured in the
presence of 10 lM TPA. Free [3H]PDBu was determined
by subtraction of the bound [3H]PDBu from the total
[3H]PDBu. Ki value was determined according to the
reported method. See: Sharkey, N. A.; Blumberg, P. M.
Cancer Res. 1985, 45, 19–24.
calcd for C22H44O6Na (M+Naþ) 511.30; found 511.23.
22
(R)-())-1d: Colorless oil; ½aꢀ )15.8 (c ¼ 0:10, CHCl3); 1H
NMR (400 MHz, CDCl3) dD0.88 (t, J ¼ 6:8 Hz, 3H), 1.26
(m, 16H), 1.49 (tt, J ¼ 7:32, 6.8 Hz, 2H), 1.90 (tt, J ¼ 7:3,
6.8 Hz, 2H), 2.28 (t, J ¼ 7:1 Hz, 1H), 3.98 (d, J ¼ 7:1 Hz,
2H), 4.15 (t, J ¼ 6:8 Hz, 2H), 4.66 (d, J ¼ 12:0 Hz, 1H),
4.79 (d, J ¼ 12:0 Hz, 1H), 6.97 (d, J ¼ 8:3 Hz, 1H), 7.09
(d, J ¼ 7:3 Hz, 1H), 7.44 (dd, J ¼ 7:3, 7.1, 2H), 7.57 (dd,
7.3, J ¼ 1:5 Hz, 1H), 7.59 (dd, 8.3, J ¼ 7:3 Hz, 1H), 7.95
(dd, J ¼ 7:1, 1.5 Hz, 2H); 13C NMR (100 MHz, CDCl3) d
14.12, 22.69, 25.80, 28.80, 29.35, 29.53, 29.60, 29.63, 29.66,
29.69, 31.92, 64.47, 64.68, 69.14, 85.51, 112.82, 113.67,
113.96, 128.53, 129.07, 129.79, 133.51, 136.50, 150.00,
158.51, 166.24, 166.92; MALDI-TOFMS (positive ion,
a-cyano-4-hydroxycinnamic acid) calcd for C29H38O6Na
(M+Naþ) 505.26; found 505.19. (R)-())-1e: Colorless oil;
23
½aꢀ )28.9 (c ¼ 0:34, CHCl3); 1H NMR (400 MHz,
D
CDCl3) d 0.88 (t, J ¼ 6:8 Hz, 3H), 1.26 (m, 16H), 1.31
(s, 18H), 1.50 (tt, J ¼ 7:3, 6.8 Hz, 2H), 1.90 (tt, J ¼ 7:3,
6.8 Hz, 2H), 2.35 (br s, 1H), 3.98 (br s, 2H), 4.13 (t,
J ¼ 6:8 Hz, 2H), 4.66 (d, J ¼ 12:0 Hz, 1H), 4.78 (d,
J ¼ 12:0 Hz, 1H), 6.96 (d, J ¼ 8:3 Hz, 1H), 7.09 (d,
J ¼ 7:4 Hz, 1H), 7.57–7.62 (m, 2H), 7.73 (s, 2H); 13C
NMR (100 MHz, CDCl3) d 14.12, 22.69, 25.84, 28.84,
29.35, 29.37, 29.53, 29.60, 29.62, 29.66, 31.28, 31.91, 34.87,
64.61, 64.64, 69.04, 85.73, 112.53, 113.56, 114.09, 123.93,
127.66, 128.43, 136.42, 150.16, 151.17, 158.47, 166.79,
166.92; MALDI-TOFMS (positive ion, a-cyano-4-
12. Increase of PKC binding affinity of branched derivatives
of DAG analogs was also reported. See, Refs. 5e,9.
13. Modeling method: To construct a binding model of
isobenzofuranone and PKCd C1B domain, a computa-
tional docking study was performed based on the reported
PKCd C1B-phorbol 13-acetate complex (pdb code 1PTR).
The isobenzofuranone derivative with a shorter side chain
(C3) was used instead of 1f. The docking model was
constructed in the Affinity module of the INSIGHT II
molecular program developed by MSI (now succeeded
by Accelrys, San Diego). The first binding placement of
isobenzofuranone within the PKCd C1B domain was
made by superimposing the molecule on the critical
functional groups of phorbol 13-acetate. We constructed
a ‘grid’, which is partitioned into bulk (nonflexible) and
movable atoms of the ligand/receptor system; interactions
among bulk atoms are approximated by using the accurate
and efficient molecular mechanical/grid (MM/Grid)
method, while interactions among movable atoms are
treated using a full force field representation. The grid was
hydroxycinnamic acid) calcd for C37H54O6 Na (M+Naþ)
28
D
617.38; found 617.30. (R)-())-1f; colorless oil; ½aꢀ )29.3
1
(c ¼ 0:91, CHCl3); H NMR (400 MHz, CDCl3) d 0.88 (t,
J ¼ 7:1 Hz, 3H), 1.08 (s, 9H), 1.26 (m, 16H), 1.48 (m, 2H),
1.89 (tt, J ¼ 7:1, 6.8 Hz, 2H), 2.13 (dd, J ¼ 6:6, 7.8 Hz,
1H), 3.85 (dd, J ¼ 6:6, 12.2 Hz, 1H), 3.89 (dd, J ¼ 7:8,
12.2 Hz, 1H), 4.15 (t, J ¼ 6:8 Hz, 2H), 4.45 (d,
J ¼ 12:0 Hz, 1H), 4.54 (d, J ¼ 12:0 Hz, 1H), 6.96 (d,
J ¼ 8:3 Hz, 1H), 7.02 (d, J ¼ 7:6 Hz, 1H), 7.59 (dd,
J ¼ 8:3, 7.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) d
14.10, 22.66, 25.76, 26.94, 28.73, 29.30, 29.32, 29.51, 29.57,
29.60, 29.64, 31.89, 38.83, 63.95, 64.51, 69.10, 85.98,
112.66, 113.59, 114.22, 136.28, 149.93, 158.37, 166.99,
ꢀ
defined by the amino acid residues within 6 A around
the isobenzofuranone. The binding model was made using
a Monte Carlo type procedure to search both conforma-
tional and Cartesian space.