Efficient synthesis of 3-trifluoromethylphenyldiazirinyl oleic acid derivatives and their biological activity for protein kinase C

Article abstract of DOI:10.1016/S0960-894X(03)00200-2

3-Trifluoromethylphenyldiazirine based oleic acids derivatives are synthesized to elucidate the functions of specific activation of protein kinase C (PKC) with oleic acid. The synthetic route is based on the alkylation of phenolic derivative with oleic acid equivalent and the post-functionalization of the compound to achieve radiolabeling. Several compounds have biological activity for PKC with similar efficacy with that of oleic acid. The results indicated that the diaizinyl oleic acid derivatives should be useful to study the specific functions of oleic acid for PKC.

Full text of DOI:10.1016/S0960-894X(03)00200-2

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1531–1533
Efficient Synthesis of 3-Trifluoromethylphenyldiazirinyl Oleic Acid
Derivatives and Their Biological Activity for Protein Kinase C
Makoto Hashimoto,a,* Kensuke Nabetaa and Kentaro Murakamib,*
aDepartment of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine,
Inada-cho, Obihiro 080-8555, Hokkaido, Japan
bDepartment of Biology, University of Vermont, 307 Marsh Life Sciences Building,
Burlington, Vermont 05405-0086, USA
Received 15 January 2003; accepted 21 February 2003
Abstract—3-Trifluoromethylphenyldiazirine based oleic acids derivatives are synthesized to elucidate the functions of specific acti-
vation of protein kinase C (PKC) with oleic acid. The synthetic route is based on the alkylation of phenolic derivative with oleic
acid equivalent and the post-functionalization of the compound to achieve radiolabeling. Several compounds have biological
activity for PKC with similar efficacy with that of oleic acid. The results indicated that the diaizinyl oleic acid derivatives should be
useful to study the specific functions of oleic acid for PKC.
# 2003 Elsevier Science Ltd. All rights reserved.
Protein kinase C (PKC) plays a pivotal role in trans-
membrane signaling in response to various hormones
and neurotransmitters.1,2 It has been established that
PKC is regulated by diacylglycerol (DAG) generated by
receptor mediated phospholipid hydrolysis. The binding
site of DAG in PKC has been determined using tumor
promoting phorbol esters that bind to PKC with high
affinity in a competitive manner with DAG.3 In addi-
tion to DAG, PKC can be activated by free unsaturated
fatty acid such as arachidonic acid and oleic acid.1
However, the binding site of free fatty acid within PKC has
not been determined. This is partly because PKC
requires relatively high concentrations of free fatty acid
for its activation, and there is no agent available that
mimics free fatty acid with high affinity binding to PKC.
Recently, it was demonstrated that PKC translocation
occurs at remarkably low concentrations of arachidonic
acid; a brief exposure of low concentrations (10–30 nM)
of arachidonic acid translocates PKC from the cytosol
to the membrane.4 Other free unsaturated fatty acids
have the similar effect on PKC translocation but ara-
chidonic acid metabolites, such as leukotriene B4 and
5-hydroxyicosatetraenoate (5-HETE), do not have such
an effect. This indicates that free fatty acid itself, but not
arachidonic acid metabolites, mediates the translocation
of PKC. Furthermore, arachidonic acid at similar low
concentrations stimulated the binding of PKC to tumor
promoting phorbol esters.4 These findings that free
unsaturated fatty acid is able to bind to PKC with high
affinity and translocate it in vivo at low concentrations
prompted us to develop photoreactive fatty acid deri-
vatives for studying the fatty acid regulation of PKC
photoaffinity labeling in vivo. Oleic acid was chosen
over arachidonic acid since oleic acid is not metabolized
through cyclooxygenase/lipoxygenase pathways and
therefore the effect on PKC can be directly attributed to
fatty acid itself but not to its metabolites.
Photoaffinity labeling is a powerful method in the study
of the structure and function of biomolecules.5À10 The
technique has been widely used for analysis of inter-
actions in vivo based on the affinity of the ligand moiety
with its target macromolecules. Various photophors
such as phenyldiazirine, arylazide and benzophenone,
have been used. Comparative irradiation studies of
these photophors for living cells suggested that a car-
bene precursor 3-trifluoromethylphenyldiazirine is most
promising.11 However, the complicated synthesis of the
3-trifluoromethylphenyldiazirinyl three membered ring
has resulted in fewer applications of it in biomolecular
studies than those of other photophors. The synthesis of
*Corresponding author. Tel.: +81-155-49-5542; fax: +81-155-49-
5577; e-mail: hasimoto@obihiro.ac.jp(M. Hashimoto); tel.: +1-802-
656-0455; fax: +1-802-656-2914; e-mail: kentaro.murakami@uvm.edu
(K. Murakami)
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00200-2

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M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1531–1533
photoreactive oleic acid analogues presents a similar
problem. Ruhmann and Wentrup has been reported
diazirinyl oleic acid synthesis.12 But the synthesis consist
of over ten steps, because the oleic acid equivalents
should be introduced before construction of diazirinyl
three membered ring. Furthermore, there is no report
for the biological activity for the diazirinyl oleic acid
derivative. We have been elucidating approaches for the
post-functionalization of the 3-trifluoromethylphenyl-
diazirinyl photophor.13À18 In the course, we found the
diazirinylphenoxy linked saturated fatty acid derivatives
was recognized as substrate by biomolecules.15 In this
paper, we describe effective synthesis of 3-trifluoro-
methylphenyldiazirine based oleic acid analogues and
their substituent effects for PKC activity.
The biological activity of the synthetic compounds for
the activation of PKC was tested using purified human
recombinant PKCa (Pan Vera). As shown in Figure 2,
all compounds, except compound 8, have similar effi-
cacy with oleic acid for PKC activation. The Km values
are 102Æ21, 174Æ40, 186Æ63, and 140Æ99 mM for
oleic acid, compound 5, 7 and 10, respectively. We have
found that iodine substitution makes the compound
slightly more potent for the activation of PKC at lower
concentrations (Fig. 2). This result is consistent with the
previous observation on the iodinated diazirinyl bis-
glucose makes slightly higher activity for glucose trans-
porter than unsubstituted diazirinyl one.24 Interestingly,
the ability of benzyl alcohol type compound 8 to induce
PKC activation was drastically reduced. This could be
due to the direct interaction between the introduced
alcohol moiety and PKC, although we do not know the
precise nature of the interaction at the moment.
Figure 1 shows the synthesis of diazirine-based oleic
acid derivatives. m-Hydroxydiazirine 116 was subjected
to the phenoxy alkylation condition19 with 18-bromoo-
leic acid methyl ester20 to afford 4 with moderate yield.
Then the methyl ester was hydrolyzed to yield oleic acid
derivative 5.21 To investigate the effects of substituent
groups on benzene ring for PKC activity, the compound
5 was subjected Friedel–Craft’s reaction with dichlor-
omethyl methyl ether13 and acetyl chloride18 to yield
compound 6 and acetophenone derivative, respectively.
But the double bond in the oleic acid was not stable
under those conditions. To yield 6, the 4-formyl-3-
hydroxydiazirine 222 was applied to phenoxy alkylation.
The reaction proceeded without damage to aldehyde
moiety. After ester hydrolysis, the aldehyde 7 was
reduced to alcohol 8 with sodium borohydride, which is
easy to purchase radiolabeled reagent. The compound 1
was subjected to iodination, which is one of the most
common radioisotopes, with one equivalent of NaI and
Chloramine T23 to afford compound 3. The mono-
iodinated compound also applied to phenoxy alkylation
in a manner identical with above procedure. NOE
measurement of O-linked methylene for compound 9
revealed the methylene correlated to only singlet ben-
zene proton. The result indicated the orientation of
iodine was confirmed to 4-position against diazirinyl
moiety.
Figure 2. PKC activation by photoreactive oleic acid derivatives. Bio-
logical activity of the oleic acid derivatives were tested using lysine-rich
histone as a phosphate acceptor substrate in the presence of 100 mM
free Ca2+ using a method described previously.25,26
Figure 3. Effect of compound 10 at a low concentration on diacyl-
glycerol-induced PKC activation. The PKC activity was measured in
the absence or presence 10 mM compound 10 in the presence of 10 mM
dioctanoyl-sn-glycerol and 10 mM dioleoylphosphatidylserine using
lysine rich histone as a substrate.
Figure 1. Synthesis of diazirine based oleic acid derivatives. (i) 18-
Bromo oleic acid methyl ester, K2CO3, (n-Bu)4NI, DMF, 60 ꢀC, 58–
60%, (ii) NaI, Chloramine T, CH3OH, rt, 60%, (iii) NaOH, CH3OH,
rt, 2 h, 85%, (iv) NaOH, CH3OH, rt, 71%, (v) NaBH4, C2H5OH, rt,
90%, (vi) NaOH, CH3OH, rt, 75%.

M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1531–1533
1533
We have examined the effect of compound 10 at low
concentrations (1–10 mM) for DAG-induced PKC acti-
vation because oleic acid can activate PKC at much
lower concentrations in the presence of DAG.27 In this
condition, compound 10 at 10 mM increases the DAG
activity for PKC over 40% (Fig. 3). The result indicated
photoreactive compounds have synergic effects with
DAG as same as oleic acid.
10. Hatanaka, Y.; Sadakane, Y. Curr. Top. Med. Chem. 2002,
271.
11. Gillingham, A. K.; Koumanov, F.; Hashimoto, M.; Hol-
man, G. D. Detection and analysis of glucose transporters
using photolabelling techniques; In Membrane transport: a
practical approach; Baldwin, S. A., Ed.; Oxford University
Press: Oxford, 2000; pp 193.
12. Ruhmann, A.; Wentrup, C. Tetrahedron 1994, 50, 3785.
13. Hashimoto, M.; Kanaoka, Y.; Hatanaka, Y. Heterocycles
1997, 46, 119.
The synthesis route of photoreactive oleic acid deriva-
tives could be easily changed for the different carbon
length of oleic acid equivalent and applied to synthesis
of radiolabeled compounds without loss of bioactivity
for PKC. These compounds should be useful to eluci-
date not only PKC activation but also other biological
molecules that interact and are regulated by free unsa-
turated fatty acid.
14. Hashimoto, M.; Hatanaka, Y.; Nabeta, K. Bioorg. Med.
Chem. Lett. 2000, 10, 2481.
15. Hashimoto, M.; Yang, J.; Holman, G. D. Chem. Bio.
Chem. 2001, 2, 52.
16. Hashimoto, M.; Hatanaka, Y.; Nabeta, K. Bioorg. Med.
Chem. Lett. 2002, 12, 89.
17. Hashimoto, M.; Hatanaka, Y.; Sadakane, Y.; Nabeta, K.
Biorg. Med. Chem. Lett. 2002, 12, 2507.
18. Hashimoto, M.; Hatanaka, Y.; Nabeta, K. Heterocycles
2003, 59, 395.
19. Hatanaka, Y.; Hashimoto, M.; Kanaoka, Y. Bioorg. Med.
Chem. 1994, 2, 1367.
Acknowledgements
20. Seoane, E.; Arno, M. An. Quim. 1977, 73, 1336.
21. Compound 5 1H NMR (CDCl3) d 7.29 (1H, t, J=8.2 Hz),
6.92 (1H, d, J=8.2 Hz), 6.75 (1H, d, J=8.2 Hz), 6.67 (1H, s),
5.35 (2H, m), 3.93 (2H, t, J=6.6 Hz), 2.34 (2H, t, J=7.6 Hz),
2.02 (4H, m), 1.77 (2H, m), 1.63 (4H, m), 1.46–1.26 (16H, m),
This research was partly supported by the Ministry of
Education, Science, Sports and Culture, Grant-in-Aid
for Encouragement of Young Scientists, 12780433
(M.H.) and Vermont EPSCoR (K.M.). M.H. thanks the
Akiyama Foundation and Uehara Memorial Founda-
tion for a partial financial support of this work.
1
compound 7 H NMR (CDCl3) d 10.47 (1H, s), 7.84 (1H, d,
J=8.3 Hz), 6.82 (1H, d, J=8.3 Hz), 6.68 (1H, s), 5.35 (2H, m),
4.06 (2H, t, J=6.6 Hz), 2.34 (2H, t, J=7.6 Hz), 2.02 (4H, m),
1.85 (2H, m), 1.60 (4H, m), 1.48–1.30 (16H, m), compound 8 1H
NMR (CDCl3) d 7.31 (1H, d, J=7.9 Hz), 6.78 (1H, d, J=7.9
Hz), 6.61 (1H, s), 5.35 (2H, m), 4.69 (2H, s), 3.99 (2H, t, J=6.6
Hz), 2.34 (2H, t, J=7.6 Hz), 2.00 (4H, m), 1.81 (2H, m), 1.60
(4H, m), 1.43–1.25 (16H, m), compound 10 1H NMR (CDCl3) d
7.77 (1H, d, J=8.2 Hz), 6.54 (1H, d, J=8.2 Hz), 6.50 (1H, s),
5.35 (2H, m), 3.98 (2H, t, J=6.3 Hz), 2.35 (2H, t, J=7.6 Hz),
2.02 (4H, m), 1.83 (2H, m), 1.63 (4H, m), 1.52–1.31 (16H, m).
22. Hashimoto, M.; Hatanaka, Y.; Yang, J.; Dhesi, J.; Hol-
man, G. D. Carbohydr. Res. 2001, 331, 119.
23. Hatanaka, Y.; Hashimoto, M.; Kurihara, H.; Nakayama,
H.; Kanaoka, Y. J. Org. Chem. 1994, 59, 383.
24. Hashimoto, M.; Yang, J.; Hatanaka, Y.; Sadakane, Y.;
Nakagomi, K.; Holman, G. D. Chem. Pharm. Bull. 2002, 50,
1004.
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