Photoresponsive prodrugs of butyric acid based on amino naphthopyranones

In order to evaluate the application of new 7-amino-naphtho[1,2-b]pyran-2-ones as photoactive groups in the preparation of carboxylic acid derivatives, model butyric ester conjugates were synthesised by reaction with the chloromethylated heterocyclic precursor. Photocleavage studies of the ester conjugates in methanol/HEPES buffer (80:20) solution at different wavelengths of irradiation (254, 300, 350 and 419 nm) confirmed the quantitative release of the model acid in short irradiation times.


Introduction
The use of pharmacologically inert drug derivatives (the so called prodrugs) are useful to overcome common drawbacks associated with the intake of drugs (i.e. low oral absorption, chemical instability or toxicity) and have gained increasing importance in the development of improved prodrugs [1]. Photoresponsive prodrugs contain a photoactive group that can be cleaved upon irradiation with a suitable wavelength thus controlling its reactivity and such derivatives have been proposed recently for the optimisation of drug delivery with light as triggering agent also allowing spatial and temporal control of the release process [2]. Given the promising results obtained so far, this strategy could allow for a more patient-friendly drug delivery and availability scheme, involving less complex dosing schedules and with a lower potential for side effects.
There is an interest in developing prodrugs of butyric acid, which is a short chain fatty acid involved in the regulatory mechanisms for gene expression known to promote markers of cell differentiation, apoptosis and cell growth control [3].
We have been engaged for some years in the design of novel photolabile protecting groups based on oxygen and nitrogen heterocycles for temporary protection of carboxylic acids and amines for 2 organic synthesis and caging applications [4], and the present communication intends to give an account of the use of such heterocycles in the area of photoresponsive prodrugs.
Thus, in order to evaluate the differences in the photolytic release of butyric acid from the heterocyclic cage, several ester conjugates of butyric acid were prepared by reaction with an amino naphthopyranone, which was further N-alkylated. Photolysis studies were carried out under irradiation at different wavelengths (250, 300, 350 and 419 nm) in a Rayonet RPR-100 photochemical reactor in a mixture of methanol and HEPES buffer solution in a 80:20 proportion.

Results and Discussion
7-Amino-4-(chloromethyl)-naphtho [1,2-b]pyran-2-one 1 was obtained by a Pechmann reaction between 5-aminonaphthalen-1-ol and ethyl chloroacetoacetate, catalised by acid. This compound bearing a reactive chloromethyl group was used in the derivatization at the carboxylic acid function of butyric acid in the presence of potassium fluoride. The resulting ester conjugate 2a was obtained in moderate yield (Scheme 1, Table 1). Compound 2a was N-alkylated with methyl iodide yielding the monoalkylated 2b and dialkylated 2c derivatives.  (Table 1). determine the most favourable cleavage conditions. The course of the photocleavage reaction was followed by reverse phase HPLC with UV detection. The determined irradiation time represents the time necessary for the consumption of the starting materials until less than 5% of the initial area was detected (Table 2). Considering the data in Table 2, N-alkylation can be considered advantageous for promoting a faster cleavage as the alkylated derivatives generally displayed shorter irradiation times at all wavelengths of irradiation. Between the mono and dialkyl conjugates, it was found that the monoalkylated derivative 2b was always the fastest.
Considering the present compounds for practical applications, although they cleaved readily at 254 nm (the fastest being 2b with 6 min), photolysis at this wavelength can be damaging in biological 6 media. Therefore, photolysis at 300 nm and longer wavelengths is always preferable. A compromise can be achieved either at 300 (25 min for 2b) or 350 nm (68 min for 2b), depending on the media where it will be applied. At 419 nm, although possessing much lower sensitivity to irradiation at this wavelength, the irradiation time is long but still feasible (259 min for 2b).
For naphtho [1,2-b]pyranone conjugate 2b, photolysis was also attempted in a different solvent in order to see if it was possible to further improve the above result at 300 nm. Instead of a protic solvent like methanol, acetonitrile was used in an acetonitrile/HEPES buffer (80:20) solution and it was found that the irradiation time (27 min) was very similar to the previously reported for the methanol containing mixture.
Furthermore, monitorisation of the photolysis process at 300 nm was also carried out by 1 H NMR in a methanol-d 4 /D 2 O (80:20) solution for all conjugates in a concentration of 9.0 × 10 -3 M, which is several times larger than the concentration used in the experiments followed by HPLC, leading to an increase in the photolysis time for the complete release of the model acid (see Figure 1 for 2b as a representative example). It was observed that the irradiation process lead to progressively decreased of the the signals related to the linked acid, with simultaneous increase of its signals in the released form, as well as signals due to aromatic by-products related to the heterocyclic group.

Conclusions
The results of the evaluation of naphtho [1,2-b]pyranones as photoactive moieties for the preparation of butyric acid prodrugs revealed that quantitative release of the acid from the corresponding conjugates was possible under irradiation at 254, 300 and 350 nm in short times as well as at 419 nm but with longer values. Overall, this preliminary study suggests the feasibility of using an Nalkylated aminonaphtho [1,2-b]pyranone as a photocleavable unit for the carboxylic acid function.