Synthesis of Protein Kinase C (PKC)-C1 Domain Targeted Alkyl Cinnamates
5.1 Background and Focus of the Present Work
The antioxidant cinnamic acids such as ferulic, caffeic, sinapic, chlorogenic, coumaric acids and their conjugates are aromatic secondary plant metabolites exist ubiquitously in nature.
They are also very important intermediates in the bio-synthetic pathways of most of the natural products. Most of the cinnamic acids and their derivatives exhibit a broad spectrum of bio-activities such as antibacterial, antiviral, anti-inflammatory, antiatherosclerotic, anti-HIV, antitumor and others mainly due to their antioxidant properties. Numerous studies highlighted the effects of natural and synthetic cinnamates in a variety of human disease models. They are also good scavenger of a number of reactive species, including 2,2-diphenyl-1- picrylhydrazyl (DPPH) radicals, and peroxyl and hydroxyl radical.169 The caffeic acid phenethyl ester (CAPE) induces apoptosis on transformed cell lines, inhibit lipoxygenases and cyclooxygenase (COX)-2 enzymes from free radical generation and lipid hydroperoxidation. CAPE is also known to play an important role in PKC regulation.170 Long chain alkyl cinnamates have been reported to be free radical scavengers. The antioxidant activity of radish sprout (Raphanus sativus L.) has been attributed to be related mainly with the presence of sinapic acid long chain esters. The esters of long chain alcohols with caffeic acid have been shown to inhibit COX-1 enzymes. Recently, the relation-ship between the redox potentials and the antioxidant activity of hydroxy cinnamic acids and derivatives has been also demon-strated.171 Esters of sinapic and ferulic acids with alkanols have been also used in commercial applications as sunscreens.172-174
Caffeic acid and ferulic acid are reported to be weak inhibitors of 12-O- Tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion and ornithine decarboxylase activity in mouse skin. The long chain esters of both caffeic and ferulic acid are also inhibitor of tumor cell proliferation, COX enzyme, and lipid peroxidation.175,176 In this context, the present chapter describes the synthesis and binding properties to the C1b subdomains of PKCδ and PKCθ using fluorescence spectroscopy and molecular modeling studies. The alkyl cinnamates interact with the C1b subdomains of PKC isoforms through the DAG or phorbol ester binding site and forms hydrogen bonds with the residues at the activator binding site. The results also show that the hydroxyl groups and methoxy groups of curcumin plays an important role in recognizing the C1 domain of PKC isoforms.
Design and Synthesis of Alkyl Cinnamates Chapter 5 Design and synthesis─ Curcumin is one of the thoroughly studied natural compounds. It has potential uses against several diseases including, HIV-infections, cancer, cystic fibrosis and others.It is reported to be implicated in altering the activity of membrane ATPases, protein kinases and transcription factors. It has also been reported to inhibit phorbol esters induced tumor promotion and some of the phorbol esters-responsive markers in cancer.10,38,177 Recent studies showed that both curcumin and phorbol ester modulate PKC activity through the common C1 domains. Curcumin is a symmetric molecule and it is reported that long chain derivatives of curcumin interact with the C1 domain more efficiently than natural curcumin in membrane-free systems.138,177 The reported binding affinity and molecular docking analysis of curcumin and long chain curcumin derivatives show that the two halves of the symmetric curcumin molecule might act as two separate units under the experimental conditions and specifically direct the hydrophilic pharmacophores of the ligand for their interaction with C1 domains of PKC isoforms. To understand the role of both sides of the symmetric curcumin molecule and to develop cinnamic acid derivatives for improved PKC binding and activity, we prepared alkyl esters of selected cinnamic acids related to the curcumin structure (Scheme 5.1.1). Most of the compound also contains two of the phorbol ester pharmacophores, the hydroxyl and the carbonyl functionalities within the same molecule.
Scheme 5.1.1: p-TsOH catalyzed esterification of cinnamic acid derivatives (1-4, 6 and 7)
Design and Synthesis of Alkyl Cinnamates Chapter 5 The compounds also contain hydrophobic alkyl chainsfor their possible interaction either with the hydrophobic residues of the C1 domain or with the membrane lipids. To investigate the role of hydroxyl and methoxy groups of curcumin for their interaction with PKC C1b subdomains, we prepared alkyl cinnamates according to the reported and modified procedures. The vanillin derivatives closely resemble to the one half of the natural curcumin molecule. To determine the effect of both the functional groups for PKC binding, we prepared isovanillin, where only the positions of the functional groups were switched.
Monohydroxy, dihydroxy, monomethoxy and dimethoxy alkyl cinnamates were synthesized to understand the importance of hydroxyl and methoxy groups separately. To extend our findings for better PKC binding cinnamic acid derivatives, we also prepared 3,4-dihydroxy-5- methoxy alkyl cinnamates. The alkyl cinnamates were prepared from the respective benzaldehydes through Knoevenagel condensation and Verley–Doebner modification of Knoevenagel condensation in two steps.172,178,179
The alkyl cinnamates 1–4, 6 and 7 were prepared from the respective benzaldehydes using malonic acid, pyridine and catalytic amount of piperidine, followed by refluxing with octanol or 1-hexadecanol in presence of p- toluenesulfonic acid (Scheme 5.1.1). The other alkyl cinnamates 5 and 8 were prepared from the corresponding half esters of malonic acids and benzaldehydes.
Scheme 5.1.2: Synthesis of long chain alkyl cinnamates (5 and 8) from monomalonates
Design and Synthesis of Alkyl Cinnamates Chapter 5 The monomalonates were obtained in high yields by refluxing Meldrum’s acid (2,2- dimethyl- 1,3-dioxane-4,6-dione) with octanol and 1-hexadecanol in toluene. Condensation of monomalonates with protocatechualdehyde and 5-hydroxyvanillin in presence of pyridine and piperidine produced the respective alkyl cinnamates in moderate to good yields (Scheme 5.1.2). All the compounds were fully characterized on the basis of their spectral data (1H and
13C NMR and HRMS). The alkyl cinnamates were prepared both with long (1-hexadecanol) and short chain (octanol) alcohols to study the impact of hydrophobicity on the binding affinity.
Spectral Properties
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Cinnamic acid derivatives of our interest show a broad absorbance peaks in the range of 304-330 nm. The absorption maxima show significant solvent dependency both in polar and non-polar environment. The compounds show one major absorption peak in polar, non-polar and liposome environments (Table 5.1.1 and Table 5.1.2).The absorption spectra of the compounds in ethanol are shown in Figure 5.1.1. The fluorescence emission maximums of the compounds are in the range of 341- 444 nm (Table 5.1.1 and Table 5.1.2). For all the compounds one major broad emission peak were observed in polar, non-polar and liposome environment. The emission maxima were blue shifted in the non-polar solvent such as hexane as compared with ethanol or water. This could be due to the effect of polarity in intermolecular hydrogen bond formation.
Table 5.1.1: Absorption and emission propertiesa of alkyl cinnamates and curcumin in different solvents at room temperature.
compound Hexane EtOH Water Hexane EtOH Water
1a 313 325 324 360 410 418
1b 314 323 323 360 410 417
2a 313 324 322 358 410 416
2b 312 323 323 357 409 417
3a 311 313 319 345 400 404
3b 310 315 320 346 400 408
5a 322 330 328 358 412 425
5b 323 329 329 358 413 424
6a 305 309 317 342 400 402
6b 304 310 319 341 400 402
8a 315 324 324 373 426 437
8b 313 325 326 372 425 444
Curcumin 404 427 425 474 560 572
a Compound, 2 µM. Absorbance maximum was used for recording emission spectra.
Design and Synthesis of Alkyl Cinnamates Chapter 5 In non-polar solvents like hexane the intramolecular hydrogen bond formation and in polar solvents like ethanol the intermolecular hydrogen bond formation may affect the position of fluorescence emission maxima.
Figure 5.1.1: (A) Normalized absorption spectra and (B) fluorescence emission spectra of cinnamic acid derivatives 1b, 3b, 5b and 8b in ethanol.
In water, the compounds showed much lower and broader fluorescence intensity compared to the organic solvent indicating lower quantum yields in water. The emission spectra of the compounds in ethanol are shown in Figure 5.1.1. We also measured the spectral properties of liposomes containing long chain alkyl cinnamates in buffer solution (20 mM Tris, 150 mM NaCl, 50 µM ZnSO4, pH 7.4). The results show that the absorption and fluorescence properties of the liposomes containing alkyl cinnamates are very similar to that of compounds in polar medium (Table 5.1.2).
Table 5.1.2: Absorption and emission propertiesa of alkyl cinnamates embedded in liposome at room temperature.
Compound Absorbance maximum (λmax), (nm)
Emission maximum (λem), (nm)
1a 331 412
3a 316 405
5a 332 413
6a 312 411
8a 334 441
a) Compound, 2 µM. Absorbance maximum was used for recording emission spectra
Design and Synthesis of Alkyl Cinnamates Chapter 5 The alkyl chain length did not show any significant effect in the absorption and emission maxima.