Results And Discussion

3.1 The Lowest Excited States

The absorption spectra of the three drugs in neutral phosphate buffer are reported in

Figure 1. The spectrum of KPF is similar to that of benzophenone. It is characterised by a band with ?imax = 260 nm attributed to the S0 —» S2 transition of n,n* nature, and by a lower energy shoulder in the 300-350 nm region corresponding to the S0 Si forbidden transition of n,7i* parentage. The lowest singlet (calculated at 360 nm, f = 0.0108) participates of the K,n* nature. Two very close triplets are located at 71.0 and 72.6 kcal/mol in vacuo. The solvent effect, investigated through the method of the virtual charges,22 shows that they are almost unaffected in energy by solvation, their electronic distribution being very close to that of S0. The lowest one, if compared to the lowest n,7t* triplet state in benzophenone, participates more of the K,n* character.10 The absorption spectrum of SPF is characterised by a broad band with maximum at 300 nm, corresponding to the S0 —> S2 (k,k*) transition, a shoulder at 270 nm, attributed to S0 -» S3, the latter also of k,k* nature, and a weak tail extending beyond 360 nm, identified as the transition to Si (n,7t*) state. This latter was calculated at 74.4 kcal/mol. As far as the triplet states are concerned, T( is of n,n* nature, while T2 (n,7C*) is higher in energy by ca. 11 kcal/mol. T3 (n,n*) lies close to Si (n,7t*) (-2.5 kcal/mol below).

Figure 1 Absorption spectra ofKPF (—), SPF (-—) and TP A (•—) in phosphate bufferlO'2 moll'1, pH 7.4.

The absorption spectrum of TP A is characterised by intense bands at 314 and 266 nm and a weak shoulder extending over 350 nm. The assignment of these bands parallels that given for the corresponding ones in SPF. Si was calculated at 71.3 kcal/mol. The lowest state of the triplet manifold, Ti was calculated at 49 kcal/mol and is of n,n* character, while T2 (n,7t*) is located ca. 15 kcal/mol higher than Ti. T3 (n,n*) lies close to Si (less than 1 kcal/mol higher). The existence of a large energy gap between Ti and T2 is confirmed to be a characteristic feature of the benzoylthiophene moiety. The actual value of the energy separation in TPA is likely lower than that calculated, being the energy of Ti (k,k*) rather underestimated with respect to the experimental value (ca. 58-60 kcal/mol), and is thought to be similar to that in SPF.11

3.2 The Photoreactivity

3.2.1 Aqueous Environment. In neutral aqueous medium KPF, SPF and TPA are present as the carboxylate anions (pKa <5). The photodegradation of the drugs in these conditions is almost exclusively due to decarboxylation. For KPF in deoxygenated phosphate buffer at pH

1A the quantum yield of the photoprocess (Or) is very high, 0.75,1 and does not depend on the concentration,* the presence of oxygen and the temperature (in the range 0-50 °C). For SPF <I>r is considerably lower, 0.07, for a solution 5 x 10'5 mol H at room temperature.4 In this case the quantum yield increases by decreasing the concentration, and is lower in aerobic conditions. Moreover, it is remarkably temperature dependent between 10 and 50 °C. An activation energy AE* ~ ca. 9 kcal/mol is derived from an Arrhenius-like plot.12 Similar features were observed in the photodegradation of TPA: the reaction is affected by oxygen3 and the quantum yield, ca. 0.25 at 25 °C in anaerobic conditions, depends on the temperature with AE* ~ 8-10 kcal/mol.11 The photodegradation quantum yields of TPA and SPF at different temperatures are shown in Figure 2.

Figure 2 Quantum yield of photodegradation (@r) in deoxygenated phosphate buffer 10'2 mol l'1 pH 7.4: [SPF] = 5.0 x 10 s mol l'1; [TPA] = 6.5 x Iff4 mol V1.

Temperature / K

Figure 2 Quantum yield of photodegradation (@r) in deoxygenated phosphate buffer 10'2 mol l'1 pH 7.4: [SPF] = 5.0 x 10 s mol l'1; [TPA] = 6.5 x Iff4 mol V1.

3.2.2 fi-Cyclodextrin Cavity. The three drugs form inclusion complexes with (3-cyclodextrin characterised by 1:1 stoichiometry: the association constants were determined by studying the induced circular dichroism (i.c.d.) of the complexes and found to be 2700,8

# At high concentration (10"2 mol l"1) additional photoproducts are obtained in which the benzophenone chromophore has participated. However, this photodegradation pathway is minor.

190012 and 3000 1 mol"1 for KPF, SPF and TP A, respectively. The KPF molecule deeply includes in the host cavity with the carbonyl and the aromatic part, while the propionic group remains outside the torus.13 In the case of SPF, *H NMR spectra are in agreement with an inclusion geometry with both the thienyl and the phenyl rings located inside the cavity.

The insertion of the drugs in the apolar cavity of the cyclodextrin has marked consequences on their photoreactivity. In the KPF-(3-CD inclusion complex, in the absence of oxygen, the quantum efficiency of decarboxylation is 0.42, remarkably lower than in aqueous solution; correspondingly, the photodegradation quantum yield is 0.85, i.e. somewhat higher than in water. A fraction of ca. 50% of the photoreacted compound leads to a product with an absorption A,max = 205 nm, characteristic of the reduced carbonyl group, and with NMR properties typical of an adduct containing the macrocycle as covalently linked moiety.13 The (3-CD-SPF inclusion complex exhibits similar behaviour: the photodegradation and the photodecarboxylation quantum yields are different, 0.23 and 0.11 respectively in the absence of oxygen at 20°C, and negligibly concentration dependent (up to 8xl0"5 mol l"1), the formation of addition products of the drug to the macrocycle in which the thienoyl chromophore is lost was inferred from absorption and NMR spectra.12

3.3 The Photochemical Intermediates

In these molecules photoexcitation either of the Sq-Sj or of the S0-S2 transitions leads to practically quantitative population of the lowest triplet states. This was evidenced by laser flash photolysis. This high ISC efficiency is accounted for by the results of the quantum mechanical calculations which show the presence of a n,n* triplet almost isoenergetic with the lowest n,7t* excited singlet.

In aqueous environment both the formation and the decay of the lowest triplet of the KPF carboxylate anion was observed in the subnanosecond time domain (Figure 3). The T-T absorption has a maximum at 526 nm (a) and is quite similar in shape and intensity to that of benzophenone, taken in the same conditions (c). The close spectroscopic parentage of the lowest n,rc* triplets of the two molecules is patent. However, the two excited states differ in their kinetic evolution: while the benzophenone triplet is very long-lived and rather unreactive (the lifetime is of the order of tens of microseconds), the KPF triplet has a decay time constant of only 250 ps and, with the same time constant, a transient absorption with A,max = 570 nm (b) grows up. This dynamic behaviour was ascribed to the decarboxylation reaction which was suggested to be promoted by the more delocalised and partial K,it* charge distribution of the KPF lowest triplet state. Alternatively, the presence of an upper state, very close to Ti and reactive so that it induces fast deactivation of the latter, can be hypothesised.

The study of the intermediates along the pathway to the final product (nanosecond and microsecond domains) led us to propose the mechanism reported in Scheme l.10In the triplet state (1) an intramolecular electron transfer from the carboxyl (donor) to the carbonyl (acceptor) groups occurs with a very low energy barrier and leads to the triplet biradical 2, with A^ax = 570 nm and the structure of a benzophenone-like anion. Release of carbon dioxide proceeds adiabatically from 2, with rate constant 8.3 x 106 s"1, and the biradical 3 is formed, undergoing protonation equilibrium with the biradical 4 (pKa ~ 7.6). This latter at neutral pH has a decay rate of 2.5 x 105 s"1 and is very sensitive to dissolved oxygen (kq ~ 2

x 109 M'V1). The conversion to (3-benzoylphenyl)ethane needs ISC and from 4 is rather slow, since it involves a formal intramolecular H-shift and further long-lived intermediates (~105 s"1), while from 3 is faster (~ 4 x 106 s_1 at pH 12.5) since it only requires an intramolecular electron transfer from the oxygen site to the benzylic site, followed by protonation from solvent of the formed carbanion 6,§

Wavelength / nm

Figure 3 Absorbance changes observed in KPF, 7.0 x 10'5 moll'1 in phosphate buffer 10' mol I'1 at pH 7.4, after a 35 ps laser pulse at 266 nm: (a) 99 ps; (b) 693 ps; (c) benzophenone, 1.1 xlO'4 mol I'1, at 99 ps.

In the KPF-fJ-CD inclusion complex, where two comparably efficient photochemical pathways are present, one leading to decarboxylation and the other to attack of the cyclodextrin, no subnanosecond triplet decay is observed: the triplet lifetime becomes of the order of 100 ns. This was attributed to the slowing down of the intramolecular electron

* A minor contribution to the photocleavage of KPF is also provided by photoionisation. This additional mode may become important at high light intensities due to a prevailing biphotonic nature.10

transfer process, thermodynamically disfavoured by the location of the C = O in the apolar CD interior. On this time scale, an intrinsically slower process, like H-abstraction from a CD-glucose unit, may compete. A transient with lifetime 250 ns, not observed in the absence of CD, was assigned to the triplet radical pair diarylketyl radical-CD', recombining upon ISC to form the addition product.13

'coo electron transfer ch3

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