A new monofunctional psoralen, pyrazinopsoralen (PzPs), was efficiently synthesized by Suzuki coupling reaction as a key step starting from 5-bromo-6-methoxybenzofuran and methyl 3-iodopyrazine 2-carboxylate. Demethylative lactonization of coupling product with $BBr_3$ gave PzPs.
PzPs has UV absorption spectra similar to other psoralens except that it absorbs more strongly in the long-UVA than 8-methoxypsoralen. The solvent effects on the UV absorption and fluorescence emission spectra indicate that the lowest excited singlet state is the $π,π^*$ state like other psoralens. It shows much lower fluorescence quantum yield (0.0008 in ethanol at room temperature) than other psoralens as expected by increased proximity effects (vibronic perturbation) due to close $^1(n,π^*)$ to $^1(π,π^*)$ states. The fluorescence lifetime was 1.05 ns in methylcyclohexane with single exponential decay, while more than two components were observed in other solvents with the short-lived component being the major (>95%). The triplet state of PzPs could not be detected by phosphorescence, laser flash excitation (T-T absorption) and singlet oxygen formation probably due to very low intersystem crossing quantum yield ($Φ_ISC, S_1$ to $T_1$), or short lifetime of the triplet state ($τ_T$) caused by the fast $T_1$ to $S_0$ intersystem crossing. Pyrazinocoumarin (PzC), 7-methoxypyrazinocoumarin (7-MOPzC) and 5,7-dimethoxypyrazinocoumarin (5,7-DMPzC) show the similar UV absorption spectra as the corresponding coumarins except for more distinct shoulders in the near UV absorption band (300 ~ 350 nm), probably due to $n → π^*$ transition. Fluorescence excitation spectra of PzC derivatives in nonpolar solvents have the lowest energy maxima in the region of shoulders of UV absorption, indicating the lowest fluorescent state to have the same energy as these shoulders. Fluorescence quantum yields of PzC derivatives are on the order of $10^{-2} ~ 10^{-4}$, much lower than the corresponding coumarins. The activation energies (Ea(nr)) for non-radiative processes (internal conversion, intersystem crossing, etc.) determined by the temperature effect on the fluorescence are 10.2, 6.35, 6.57 kJ/mol for PzC, 7-MOPzC and 5,7-DMPzC, respectively. Phosphorescence emission spectra of PzC and 7-MOPzC are structureless with $Φ_P$ of 0.12 and 0.17, respectively.
Direct photolysis of PzPs with excess olefins such as dimethyl fumarate (DMFu), dimethyl maleate (DMMa) and dimethyl ethylidenemalonate (DMEM) gave C4-photocycloadducts. The photoadducts were determined to be 1:1 $C_4$-cycloaddition products formed through the addition of 4',5'-furan double bond of excited PzPs to the olefins. The fluorescence of PzPs was quenched by olefins with rate constants on the order of $10^9 ~ 10^10 M^{-1}s^{-1}$. The appearance of the long-lived fluorescence component implies a singlet exciplex mechanism for the photocycloaddition reaction of PzPs with excess olefins.
Limited solubility of PzPs to water could be an important disadvantage in the application into clinics. Hydroxymethyl substituent was introduced at 5'-position of PzPs to increase water solubility. 5'-Hydroxymethylpyrazinopsoralen (HMPzPs) was synthesized by the similar method as PzPs. Candida albicans growth inhibition, which is useful to measure the phototoxicity involving DNA damage, was checked for 8-MOP, 5-MOP, PzPs and HMPzPs. PzPs derivatives did not inhibit the growth of Candida albicans whereas excited 8-MOP and 5-MOP killed Candida albicans strain very efficiently. PzPs and HMPzPs do not seems to be good candidate to treat psoriasis which is characterized by excessive DNA proliferation of melanocyte cells. However, possibility for the treatment of vitiligo remains because of different mechanism of occurrence of diseases and the treatment of those diseases. Vitiligo is known to be cured by psoralens because psoralens activate the formation of melanocyte and keratinocyte, resulting in pigmentation.
Psoralens show remarkable DNA base sequence selectivity in the photoreaction with DNA or RNA. Psoralens prefer the AT-rich sequence of nucleic acids. In addition to the biological mechanism (AT-rich region is reported to be highly flexible and stable compared with GC-rich region), the series of photo-induced electron transfer (PIET) reactions were proposed to account for the DNA base sequence selectivity. The absorption of 8-MOP radical anion was observed in laser flash photolysis after addition of adenine into 8-MOP solution. Psoralens and DNA bases are good electron donors and acceptors, respectively. In addition to 8-MOP radical anion, Other spectral peaks were observed in the laser excitation of 8-MOP in the presence of both adenine and thymine. Ps-thy adduct radical anion was suspected for those peaks.
The addition of adenine to 8-MOP solution results in the appearance of new fluorescence emission peak centered at 80 nm in addition to 8-MOP fluorescence emission peak. The addition of thymine to 8-MOP plus adenine solution has an evident effect on the relative intensity of new peak, indicating that thymine has a certain role in the interaction of 8-MOP with adenine. Thymine may react with the species formed by prior PIET reaction between 8-MOP and adenine as proposed.
