Chapter 4

Photochemical Studies on Khellin and Oleic

Acid Methyl Ester (OAME)

134

Introduction

Khellin (1), a natural occurring furochromone family, in combination with ultraviolet

light (UVA; 320≈400 nm), is some of the most effective agent available for the

photochemotherapy of a number of skin diseases such as psoriasis and vitiligo.1

OO CH3

O

OCH3

OCH3

1

The therapeutic benefit of this treatment is thought to be due to the inhibition of

hyperproliferation of skin keratinocytes. Model studies, for the most part carried out

with nucleoside or pyrimidine bases have suggested that the mono and diadducts

result from the photocycloaddition between the 5,6-bond of the pyrimidine and the 2,

3 (chromone) or 4’, 5’ (furan) bond of the khellin. For diadducts to be formed with

365 nm irradiation, monoaddition to the 4’, 5’ double bond would be the anticipated

primary photochemical act. Thymine is known to be the main target of the

photocycloaddition of excited khellin, giving rise mostly to the cis-syn cyclobutane

type adducts.2-4

Khellin forms in the dark a molecular complex with DNA. By subsequent irradiation

(365 nm) the drug photoconjugates covalently with the macromomlecules, although

the rate of photobinding is rather low. In photoaddition with the macromolecule the

drug forms inter-strand cross-links, although again in small amounts.

135

The cross-linking between the separated strands of DNA, which is caused by the

formation of cyclobutane adducts, occurs through the following mechanism.5,6

Khellin (K) + Nucleic Acid (N)Intercalated

Molecular Complex (KN)

Khv

350 nmPhotodimer, Photooxidation Products

KN350 nm

1(KN)*3(KN)*

3(KN)*energy transfer a. to epidermal cell component

b. to epidermal cells

1(KN)*3(KN)*or 2, 3-monofunctional adduct

4', 5'-monofunctional adductbifunctional adduct (cross-linked adduct)

The molecular orbital calculations predict that the triplet excited state is better

candidate for the cycloaddition reaction than the singlet excited state. The prediction

is consistent with the experimental finding that the oxygen and paramagnetic ions

quench the photoreaction between khellin and thymine.

The natural furochromones, khellin (1) and visnagin (2), two photobiologically active

compounds isolated from Ammi visnaga,7 closely resemble psoralen in structure and

have recently been used as photochemotherapeutic agents.8-11

136

OO

O

CH3

R

OH3C

1, R = OCH32, R = H

Khellin (4,9-dimethyoxy-7-methyl-5H-furo[3,2-g]-[1]-benzo-pyran-5-one) has

sometimes been used in the past as a coronary vasodilator. However, its

phototherapeutic effectiveness has also been recognized in the last few years, leading

some dermatologists to test it in association with ultraviolet light for the treatment of

skin diseases, such as psoriasis and vitiligo. The usual khellin photochemotherapy

recommended for these diseases is associated with severe side effects, such as

erythema and oedema, genotoxicity, risk of skin cancer, and cataracts.8 The various

efforts made to overcome these side effects have mainly been devoted to study of the

structural analogs of khellin, is one of the most promising agent against vitiligo.

KUVA (khellin + UV-A) therapy can, in fact, restore pigmentation in affected areas

of the skin without severe phototoxicity. Conversely, the efficacy of khellin in

clearing psoriasis is very low,12

suggesting that different mechanisms are involved in

the two therapeutic actions. While the treatment of psoriasis may mainly rely on the

antiproliferative activity of the compound, it is still an open question how these

sensitizers stimulate melanogenesis. Much work has been done to correlate

physicochemical properties with pigmentogenic activity, but no conclusive results

have been obtained.13

On the basis of the structural analogy of khellin and psoralen, some photobiological

studies have been carried out on this compound. For instance, a [2+2]-

137

photocycloadduct between khellin and thymine was isolated14

in liquid crystalline

media and the cis-syn stereochemistry similar to the photoadducts between psoralen

and pyrimidine bases has been determined. In an another study it was demonstrated

that khellin photoreacts with DNA of phage λ exposed to 360 nm UV light forming

interstrand cross-links.15 From a photobiological point of view, furochromones show

valuable phototoxicity toward various kinds of microorganisms and also valuable

genotoxic activity on various biological substrates. Among the significant aspects of

the studies required to unveil the underlying molecular mechanism are the photo-

interaction of photosensitizer with important biological molecules and the photolysis

of photosensitizers16

themselves, both to identify the products and to study their

particular biological relevance and to understand whether the intermediate by which

these products are formed interfere in some way with biological processes.

Within the context we have investigated:

[A] Photocycloaddition of khellin and thymine in liquid crystalline media.

[B] Photocycloaddition of khellin and oleic acid methyl ester in liquid crystalline

media.

138

[A] Photocycloaddition of Khellin and thymine in liquid crystalline

media

Khellin is a naturally occurring furochromone which, when combined with artificial

ultraviolet (UV) A or solar irradiation (KUVA), is reported to repigment vitiligo skin

as effectively as PUVA photochemotherapy.17

The exact mechanism of KUVA-

induced repigmentation is unknown. With the aim of elucidating its mechanism of

action, the interaction both in ground and excited states between the drug and DNA

were studied in vitro.12b Its therapeutic effects in the presence of near UV light (365

nm) are believed to be mediated, at least in part, by two successive [2 +2]

photoadditions to pyrimidine residues in duplex DNA, resulting in cross links which

appear to inhibit the overproduction of DNA associated with vitiligo.The

photoreactive sites in khellin (1) are 2,3 (chromone) and 4’,5’ (furan) bonds. As the

2,3 monoadduct (3) does not absorb near UV light, the 4’,5’-adduct (4) is the

intermediate involved in the formation of cross links.18

O O

O

H3C

OCH3

OCH3

(1)

OO

HN

N

O

O

HCH3

H HR

(4)

OCH3

OOCH3

O O

O OCH3

OCH3

HN

N

O

O

HCH3

H CH3R

CH3

(3)

139

Further investigations of the biochemistry of the khellin are needed and this will

continue to be benefited by the understanding of underlying chemistry of khellin-

DNA cycloaddition. A number of studies on structural, regiochemical and

stereochemical aspects of khellin-DNA photoadducts and their models have been

conducted.19 It was shown that the regio- and stereochemical possibilities permit the

formation of number of isomeric photocycloadducts.19a,d A model for the reaction of

khellin with DNA in the phototherapy of vitiligo was presented,19c in the study of UV

irradiation of a mixture of khellin and thymine in ice-methanol matrix in the presence

of benzophenone, with selective formation of a 4’,5’-cis-[2 + 2] photo-adduct.

The [2+2] photocycloadduct formation is well recognized to be solvent dependent,

however, a systematic study on the control of isomer formation is lacking. The regio-

and stereoselectivity of these cycloadditions depend upon the nature of reaction

medium and it is therefore, desirable to study such reactions in organized medium

which could mimic biological systems.

In biological systems enzymes conduct chemical synthesis with remarkable efficiency

and specificity, whereas, organic chemist’s efforts have been much less successful

than nature’s synthetic activity. Chemists have long recognized the important role, the

reaction media play in controlling rates, product distributions and stereochemistry.

Recently, much effort20

has been directed towards the use of organized media to

modify reactivity (especially photochemical), as compared to that in isotropic liquids.

A major goal of such studies is to utilize the order of the medium so as to increase the

rate and selectivity of the chemical process involved, in much the same way that

enzymes modify the reactivity of the substrates to which they are bound.

140

Among the many ordered or constrained systems utilized to organize the reactants, the

notable ones are micelles, micro-emulsions, liquid crystals, inclusion complexes,

monolayers and solid phases such as adsorbed surfaces and crystals, because of their

analogies to some biological systems.

In an extension to the model reports,19a,c we chose to study the photoreaction of

clinically important khellin (1) with thymine (5) in the liquids crystalline solvent.

Experimental

All chemicals used were of analytical grade. Khellin and cholestryl oleyl carbonate

were purchased from Sigma Chemical Co. and Aldrich Chemical Co., respectively.

UV spectra were recorded on a Shimadzu 160 A instrument. 1H-NMR spectra were

recorded on a Bruker Avance DRX-300 spectrometer using TMS as internal standard

and (CD3)2CO as solvent. High resolution mass spectra were recorded on VG-ZAB-

BEQ9 mass spectrometer.

Khellin (100 mg, 0.385mM) thymine (121 mg, 0.96 mM) and benzophenone (72 mg,

0.6 mM) were mixed with cholesteryl oleyl carbonate (3 gm), and mesomorphic range

of this mixture was then determined with a Reichert polarizing microscope equipped

with a heating stage. The values of the transition temperatures of cholesteryl oleyl

carbonate (mesomorphic range: –10º smectic 19º cholesteric37ºC) in the mixture were not

altered appreciably at this low concentration of khellin, thymine and benzophenone.

The mixture was thermostated at different temperatures of the mesomorphic range of

cholesteryl oleyl carbonate and was irradiated in a layer (few millimeters) in a cell for

≈30 minutes with near UV light (ca. 365 nm) from Philips HPW 125 lamp. The

irradiated mixture was collected and most of the excess of reactants and sensitizer

141

removed by fractional crystallization from methanol followed by extraction with

benzene. Column chromatography (silica gel 60, 70-230 mesh; methanol-chloroform:

80: 20) afforded the adduct 6 (ca. 13 % yields) as colourless needle, which

decompose, without melting at 285-310º C.

UV λmax (MeOH) 320 nm; HRMS calcd. For (M+) C19H18N2O7 386.1112 found

386.1105; IR (KBr): 3200-3600, 1718, 1715, 1700 cm-1

; 1H-NMR ((CD3)2CO): δ

1.36 (3H,s, Me), 1.73 (3H, s, Me), 3.82 (3H, s, -OMe), 3.85 (3H,s,-OMe), 4.13 (1H,

ddd, J= 6.5, 2.5 and 0.7 Hz, H-8) 4.26 (1H, quintet, J=2.6 Hz, collapsed to qd, J = 2.6,

1.0 Hz by irradiation at δ 6.80, and also after shaking with D2O, H-14), 5.62 (1H, dd,

J = 6.5, 5.5 Hz, H-15), 6.26 (1H, d, J=9.6 Hz, H-3), 6.80 (1H, brs, H-13), 6.92 (1H,

brs, H-11); MS m/z: 386, 260, 245, 231, 216, 126.

Result and Discussion

A highly regio- and stereoselective [2+2] photocycloaddition occurred on irradiation

of a mixture of 1,5 and benzophenone (triplet sensitizer) in the liquid crystalline

solution at temperatures such as to preserve the mesomorphic states. Thus, a racemic

cis-syn-[2+2] photoadduct (6) was nearly exclusively formed (Scheme 4.1), when the

medium was maintained anisotropic, regardless to the smectic or cholesteric phases of

the used liquid crystal: cholesteryl oleyl carbonate (mesomorphic range: –10º smectic

19º cholesteric37ºC). The adduct was assigned structure 6 by the comparison of its

spectral values with the reported19c

one.

142

O O

O

H3C

OCH3

OCH3

(1)

NH

HN

O

O

CH3

(5)

hvBenzophenonecholesteryl oleylcarbonate

OO

HN

N

O

O

HCH3

H HH OCH3

OOCH3

CH3

(6)

+

Scheme 4.1

143

The adduct 1:1 was indicated by chemical ionization mass spectrometry, which

showed (M+) at m/z 386 and its fragments, corresponding to the adduct. This was

confirmed as a furan-side monoadduct by its characteristic UV absorption spectra

(λmax 320 nm) for a 4’,5’-dihydrokhellin-type chromophore.

The IR spectrum shows almost no shift in the α-β unsaturated carbonyl absorption of

khellin with an absorption at 1700 cm-1 while a new carbonyl absorption at 1718 cm-1

is very close to that of the thymine cyclodimer at 1715 cm-1. A braod absorption

between 3200-3600 cm-1 can be attributed to the N-H bonds of the photoadduct.

The 1H-NMR spectrum notably showed resonance for cyclobutane ring protons at H-8

at δ 4.13 (1H, ddd, J = 6.5, 2.5 and 0.7 Hz), H-14 at δ 4.23 (1H, Quintet, J = 2.6 Hz,

collapsed to qd, J= 2.6, 1.0 Hz by irradiation at δ 6.80, and also after shaking with

D2O) and H-15 at δ 5.62 (1H, dd, J = 6.5, 5.5 Hz). In addition the two different NH

protons at position 11 and 13 appeared as D2O exchangeable resonance at δ 6.92 (1H,

brs) and 6.80 (1H, brs). The magnitude of the couplings between cyclobutane ring

protons (H-8, H-14 and H-15) suggested that the adduct has regio- and stereo-

chemistry depicted in 6.

The regio- and stereoselective formation of cis-syn-[2 + 2] adduct (6) may have

relevance to molecular mechanism in KUVA therapy of vitiligo. Since liquid crystals

are ordered fluids with structures and properties intermediate between those of

isotropic liquids and crystalline solid and hence, the reactivity of a molecule

incorporated in a liquid crystal is expected to be different from that in isotropic

solvents. The rigidity present in solids matrix is absent in liquid crystals and hence

permits molecular motion as well as conformational flexibility. At the same time, due

144

to the order in the liquid crystalline phase, the randomness in motion and flexibility of

dissolved solutes, prevalent in solutions, is restricted. The guest-host intermolecular

interactions resulting from dissolving an organic substance in thermotropic liquid

crystal induce modification in the orientational correlation of the molecules of the

mesomorphic solvents and anisotropy in both the diffusion and molecular orientations

of the solute.

The long and short range order of the liquid crystalline phase should influence solute

molecules to align themselves in the defined packing arrangement, and hence the

preferred orientations of solute-solute collisions may result in a high regio- and

stereo-selectivity as observe in the formation of 6.

145

[B] Photocycloaddition of Khellin and Oleic Acid Methyl Ester in

Liquid Crystalline Media

Unlike psoralens, khellin does not photoinduce cross-links in DNA in vitro or Chinese

hamster cells in vivo. This behaviour was associated to explain the low

photogenotoxicity in yeast and the lack of phototoxic erythemal response when

treating vitiligo with khellin.12a

Its very low DNA-photobinding, and consequently

poor antiproliferative and antipsoriasic activity, was attributed to the hindering effect

of the two bulky methoxy groups which prevent interaction into the double helix. The

furan side C4-cycloadduct between khellin and thymine was in fact produced by

irradiation in a frozen state14 when steric effect should be less important. When

irradiation is carried out in the presence of suitable substrate (e.g. pyrimidine bases,

unsaturated fatty acids) the pyrone ring is still involved. No evidence for such

behaviour was found with khellin, probably, because the 6,7-olefinic double bond not

conjugated with the benzofuran moiety, and therefore should not be involved in the

excited states of the molecule.

Khellin, a naturally occurring furochromone, used in the past as a coronary

vasodilator, has recently been used in the photochemotherapeutic treatment of vitiligo

and psoriasis. Its therapeutic effects in the presence of near UV light (350 nm) are

believed to be mediated, at least in part, by two successive [2 +2] photoadditions to

pyrimidine residues in duplex DNA, resulting in cross links which appear to inhibit

the overproduction of DNA associated with vitiligo. The photoreaction sites in

Khellin (1) are 2,3 (chromone) and 4’, 5’ (furan) bonds. As the 2,3 monoadduct (7)

does not absorb near UV light, the 4’, 5’ -adduct (8) is the intermediate involved in

the formation of cross links.18

The furan side monoadduct between khellin and oleic

146

acid methyl ester, formed in the photoreaction between the drug and oleic acid methyl

ester was isolated and characterized, and shows a cis-syn configuration.

O O

O

H3C

OCH3

OCH3

(1)

O O

O OCH3

OCH3

(7)

O O

O

H3C

OCH3

OCH3

(CH2)7CH3

(CH2)7COOCH3

HH

H H

H

H H3C

H(H2C)7CH3

(CH2)7COOCH3

(8)

Further investigations of the biochemistry of the khellin are needed and this will

continue to be benefited by the understanding of underlying chemistry of khellin-

OAME cycloaddition. A number of studies on structural, regiochemical and

stereochemical aspects of khellin-OAME photoadducts and their models have been

conducted. It was shown that the regio- and stereochemical possibilities permit the

formation of number of isomeric photocycloadducts. A model for the reaction of

khellin with DNA in the phototherapy of vitiligo was presented, in the study of UV

irradiation of a mixture of khellin and OAME in ice-methanol matrix in the presence

of benzophenone, with selective formation of a 4’, 5’-cis-[2 + 2] photo-adduct.

147

The [2+2] photocycloadduct formation is well recognized to be solvent dependent,

however, a systematic study on the control of isomer formation is lacking. The regio-

and stereoselectivity of these cycloadditions depend upon the nature of reaction

medium and it is therefore, desirable to study such reactions in organized medium

which could mimic biological systems.

In biological systems enzymes conduct chemical synthesis with remarkable efficiency

and specificity, whereas, organic chemist’s efforts have been much less successful

than nature’s synthetic activity. Chemists have long recognized the important role, the

reaction media play in controlling rates, product distributions and stereochemistry.

Recently, much effort

has been directed towards the use of organized media to

modify reactivity (especially photochemical), as compared to that in isotropic liquids.

A major goal of such studies is to utilize the order of the medium so as to increase the

rate and selectivity of the chemical process involved, in much the same way that

enzymes modify the reactivity of the substrates to which they are bound.

Among the many ordered or constrained systems utilized to organize the reactants, the

notable ones are micelles, micro-emulsions, liquid crystals, inclusion complexes,

monolayers and solid phases such as adsorbed surfaces and crystals, because of their

analogies to some biological systems.

In the present study, photoreaction of khellin (1) with oleic acid methyl ester

(OAME, 9) was investigated in the liquid crystalline media.

148

Experimental

All chemicals used were of analytical grade. Khellin was purchased from Sigma

Chemical Company and recrystallized from methanol. UV spectra were recorded on a

Shimadzu 160 A instrument. IR spectra were recorded as KBr discs on a Perkin Elmer

model spectrum RXI. 1H-NMR and

13C-NMR spectra were recorded on a Bruker

Avance DRX-300 spectrometer using TMS as internal standard and (CD3)2CO as

solvent. High-resolution mass spectra were determined with a VG-ZAB-BEQ9

spectrometer.

Khellin (100 mg, 0.385 mM), oleic acid methyl ester (516 mg, 1.93 mM) and

benzophenone (72 mg, 0.6 mM) were mixed with cholesteryl oleyl carbonate (3 gm),

and mesomorphic range of this mixture was then determined with a Reichert

polarizing microscope equipped with a heating stage. The values of the transition

temperatures of cholesteryl oleyl carbonate (mesomorphic range: –10º smectic

19º

cholesteric37ºC) in the mixture were not altered appreciably at this low concentration of

khellin, oleic acid methyl ester and benzophenone. The mixture was thermostated at

different temperatures of the mesomorphic range of cholesteryl oleyl carbonate and

was irradiated in a layer (few millimeters) in a cell for ≈30 minutes with near UV

light (ca. 350 nm) from Philips HPW 125 lamp. The irradiated mixture was collected

and most of the excess of reactants and sensitizer removed by fractional

crystallization from methanol followed by extraction with benzene. Column

chromatography (silica gel 60, 70-230 mesh; methanol-chloroform: 80: 20) afforded

the adduct 10 (yields 26 mg) as colourless needle, which decompose, without melting

at 178º C.

149

Yield: 26 mg; mp 178 oC; UV λmax (MeOH) 251, 348 sh. nm; IR (KBr): 830, 1655,

1741, 1240, 1205, 2962; HRMS calcd. for C33H48O7 556.3400 found 556.3410; 1H-

NMR ((CD3)2CO): δ 6.12 (s, 1H, H-3), 4.51 (dd, 1H, J=6.5, 6.1 Hz, H-11), 3.83 (s,

3H, COOCH3), 3.75 (s, 3H, -OCH3), 3.69 (dd, J=6.5, 6.2 Hz, 1H, H-8), 3.61 (s, 3H,

-OCH3), 2.46 (m, 1H, H-10), 2.35 (m, 1H, H-9), 2.25-1.25 (m, (CH2)n-OAME, CH3-

pyrone), 0.98 (t, 3H, CH3); 13C-NMR (CD3)2CO): 180.2 (C-4), 175.1 (COOCH3),

162.7 (C-2), 159.4 (C-6), 153.2 (C-13), 148.2 (C-15), 134.2 (C-14), 119.7 (C-7),

111.8 (C-3), 103.5 (C-5), 73.9 (C-11), 56.4 (C-10), 50.2 (COOCH3), 40.5 (OCH3),

38.3 (C-8), 37.2 (OCH3), 33.1 (C-9), 32.5-25.1 ((CH2)n-OAME), 21.7 (CH3), 17.1

(CH3); MS m/z: 556, 525, 541, 494, 485, 392, 260, 232, 231.

Result and discussion

A highly regio- and stereoselective [2+2] photocycloaddition occurred on irradiation

of a mixture of 1,9 and benzophenone (triplet sensitizer) in the liquid crystalline

solution at temperatures such as to preserve the mesomorphic states. Thus, a racemic

cis-syn-[2+2] photoadduct (10) was nearly exclusively formed (Scheme 4.2), when

the medium was maintained anisotropic, regardless to the smectic or cholesteric

phases of the used liquid crystal: cholesteryl oleyl carbonate (mesomorphic range: –

10º smectic 19º cholesteric37ºC). The adduct was assigned structure 10 by the comparison

of its spectral values.

The structure of the photoproduct was characterized by the spectroscopic methods as

described below. The UV absorption spectra of the photoproducts, λmax (MeOH) 251,

348 sh. nm, are very similar to each other and resemble to that of 4’,5’-

dihydrokhellin, indicating that the 4’,5’-double bond of khellin is saturated in the

150

photoproducts. IR spectra also indicated the saturation of 4’,5’-double bond on

photoadduct formation. The olefinic C-H stretching bands of furan ring of khellin at

3060 and 3100 cm-1

disappear in the photoproduct and a characteristic cyclobutane

ring deformation band appeared at 830 cm-1

. Moreover, the carbonyl stretching band

of khellin at 1655 cm-1 was intact, indicating that chromone double bond of khellin

was not affected on photoreaction. Mass spectra of the photoproducts showed

molecular ion peaks of 1:1 adduct of khellin and OAME at m/z 556 and its fragments,

corresponding to the adducts. These data suggested that the photoadduct is composed

of khellin and OAME (1:1), formed by [2π+2 π] cycloaddition of 4’,5’-furyl bond of

khellin to C=C double bond of OAME.

151

O O

O

H3C

OCH3

OCH3

(CH2)7CH3

HH

H3COOC(H2C)7+

O O

O

H3C

OCH3

OCH3

(CH2)7CH3

(CH2)7COOCH3

HH

H H1

2

34

56 7 8 9

1011

12

13

1415

(1) (9)

(10)

hvBenzophenonecholesteryl oleylcarbonate

Scheme 4.2

152

The 1H-NMR spectrum notably showed resonance for cyclobutane ring protons at H-8

at δ 3.69 (1H, dd, J = 6.5, 6.2 Hz), H-9 at δ 2.35 (1H, m), H-10 at δ 2.46 (1H, m) and

H-11 at δ 4.51 (dd, 1H, J= 6.5, 6.1 Hz). The magnitude of the couplings between

cyclobutane ring protons (H-8, H-9, H-10 and H-11) suggested that the adduct has

regio- and stereo-chemistry depicted in 10.

The regio- and stereoselective formation of cis-syn-[2 + 2] adduct 10 may have

relevance to molecular mechanism in KUVA therapy of vitiligo. Since liquid crystals

are ordered fluids with structures and properties intermediate between those of

isotropic liquids and crystalline solid and hence, the reactivity of a molecule

incorporated in a liquid crystal is expected to be different from that in isotropic

solvents. The rigidity present in solids matrix is absent in liquid crystals and hence

permits molecular motion as well as conformational flexibility. At the same time, due

to the order in the liquid crystalline phase, the randomness in motion and flexibility of

dissolved solutes, prevalent in solutions, is restricted. The guest-host intermolecular

interactions resulting from dissolving an organic substance in thermotropic liquid

crystal induce modification in the orientational correlation of the molecules of the

mesomorphic solvents and anisotropy in both the diffusion and molecular orientations

of the solute.

The long and short range order of the liquid crystalline phase should influence solute

molecules to align themselves in the defined packing arrangement, and hence the

preferred orientations of solute-solute collisions may result in a high regio- and

stereo-selectivity as observe in the formation of 10.

153

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