Intrinsic sensing fluorescent probe for the solid phase synthesis of 1,4-benzodiazepine-2,5-dione

The synthesis of 4-N -naphthylethyl-1,4-benzodiazepine-2,5-dione ( 1 ) supported on solid phase is described. The fluorescent naphthyl group is used as internal sensor for monitoring various stages on the synthetic reaction by spectral differences in fluorescence. Our results show the efficacy of fluorescence as a direct method for the evaluation of reaction progress. This method is fast, sensitive and non-destructive. A target molecule was synthesized which could be used in the synthesis of a variety of benzodiazepines.


Introduction
The synthesis of chemical libraries through combinatorial chemistry has been of great importance during the last decade due to the large number of compounds that can be prepared in a fast and efficient manner.Solid phase organic synthesis (SPOS) has become to be one of the most useful synthetic technique in combinatorial chemistry.
SPOS is an important tool in the discovery of new substances biologically activite with a suitable pharmacological profile to be used as drugs.This is demonstrated by the wide range of existing reports on diverse lines of research in organic synthesis.Some of these reports describe the synthesis of quinazolinediones 1 , pyridine analogues 2 and benzodiazepines. 3SPOS was originally developed by R. B. Merrifield 4 and is based on the use of a polystyrene resin in the form of spheres which are insoluble in most common solvents.
In the development of combinatorial chemistry to produce a great diversity of substances, the analytical evaluation and structure determination is a critical step.The finding of efficient and fast techniques that allow monitoring reaction progress and chemical changes on the solid phase supported compounds has become of great importance these days. 5Proper structural analysis of these libraries is fundamental for the complete characterization of compounds and determination of reaction yields and purity of final products.Evaluation techniques for SPOS are one of the drawbacks for this process, since various analytical methods require the cleavage of the bond linking the organic compounds to the polymer before analysis.In this regard, most common analytical methods such as IR, MS, etc. are destructive.On the other hand, Gel-phase 6 in 13 C NMR has been used in some commercial resins as Wang and Merrifield.However, since this technique is slow and limited it could not be applied to combinatorial chemistry.Therefore, it is fundamental to establish new methods with high sensitivity, which allows measuring the different stages of the reaction as well as the degree of conversion during the process.
In this report, we show the use of the naphthyl group as an internal probe.This group allows direct monitoring by fluorescence spectroscopy of the several stages on a global reaction.

Results and Discussion
Polymer bound 4-N-naphthylethyl-1,4-benzodiazepine-2,5-dione (1) with the naphthyl group as the probe was synthesized according with Scheme 1.In order to evaluate the effect of the spacer length on the fluorescence, a comparison among 4-N-naphthylethyl-1,4-benzodiazepine-2,5dione supported on Merrifield resin, Wang resin, and Merrifield resin with a condensed spacer of different aliphatic chains length ( 4, 10, and 12 methylenes), was performed.

N-(R)-(+)-1-(1-Naphthyl
)ethane-2-aminobenzamide (4) was prepared from R-(+)-1,1ethylnaphthylamine and isatoic anhydride and then supported on Merrifield resin and the modified resins 3a, 3c-d.Chemistry in parallel using four different methods was applied for the coupling of o-aminobenzamide 4 with the solid support.The Best yields were achieved with method B, which is a modification of method A in which KI is added.The highest yield was achieved for Merrifield resin (100%, Method B, see Experimental), while the lowest was obtained with the resin Merrifield-1-oxy-12-chlorodecane (3e), (23%, Method A).Methods C and D showed a lower degree of coupling (92-34%).In these methods tetramethyl guanidine is used as a base with 3 and 6 equivalents, respectively.No significant effect on the yields was found.Of four methods examined, method B showed the highest degree of coupling for oaminobenzamide 4 (100-32%).Additionally, it was established that increasing the length of the spacer chain up to 12 carbons leads to a decrease in the degree of coupling (32%).
Ph 3 P/TPA or

Scheme 1
The amidation step of the o-aminobenzamide supported on solid phase (5a-5e) was performed with bromoacetyl bromide in the presence of TEA and DMAP to give the resins oamidonaphthylbenzamide (6a-e).The resin-o-aminobenzamide with the highest degree of amidation was Wang-o-aminonaphthylbenzamide (5a) (100 %), while with Merrifield resin the yields decrease upon increasing the length of the aliphatic chain (14 % for the spacer with 12 carbons).Also, chemistry in parallel for the ring formation was performed using four different methods.The best results were obtained with cesium carbonate in DMF (Method B) and the resin o-amidonaphthylbenzamide (6b), without the aliphatic spacer group (90%).The use of sodium methoxide in refluxing methanol yielded 30% for the hydrolysis of naphthylamide (Method D).It was also observed that increasing the length of the spacer chain to 12 carbons, the degree of ring formation increased, in contrast with the previous stage.Increasing the length of the aliphatic chain results in an increment of lipophylicity of the support, which in turn makes it more easily solvated by organic solvents.The yields of this stage depend on the type of base.Using either bases of low solubility in organic solvents like K 2 CO 3 (Method A) or tetramethyl guanidine (Method C), lowers the yield of benzodiazepines 7. Sodium methoxide, which is soluble in methanol, considerably increases the yields of ring formation as well as of hydrolysis.The last reaction involved ia the detachment of benzodiazepine from the polymeric support by treatment with THF/TFA (1:1) for 30 min to obtain N-naphthylethylamine-1,4-benzodiazepine-2,5-dione (1).The yields in this stage were quantitative for the Wang resin.
The resins synthesized in each stage were analyzed in a 0.2 mL fluorescence cell.The emission and excitation spectra of all the intermediates supported in Merrifield resin without spacer are shown in Figure 1.Fluorescence spectroscopy analysis for Merrifield resin (Bands A) shows, after supporting o-aminonaphthylbenzamide (4) an increment on the intensities of the excitation and emission bands (Bands C).These intensities are greater than those of the oaminonaphthylbenzamide (4) (Bands B), and they appear at longer wavelength leading to considerably accurate measurements.Amidation of the o-aminobenzamide supported on solid phase (5a) with bromoacetyl bromide showed a quenching on the excitation and emission bands.This decrement of intensities is attributable to the incorporation of bromine, a heavy atom that deactivates fluorescence in the resin. 7pon ring closing, the resin benzodiazepine (7a) is obtained.This product shows a wavelength shift on the emission band from λ max 430 nm to λ max 637 nm, and the excitation band from λ max 382 nm to λ max 560 nm.These notable shifts are due to the formation of a new ring, and therefore, increasing rigidity in the molecule. 7luorescence spectroscopy analysis of the other resins shows the same tendency as in Merrifield resins.The only difference observed was the decreasing of the intensities when increasing the number of carbons on the spacer group.This could be explained since molecules acquire greater mobility, and part of the energy is lost by intermolecular collisions.This tendency is lineal and allows identification of the maximum number of carbons in a spacer necessary to maintain accurate measurements in fluorescence, allowing the evaluation of reaction progress (Figure 2).In summary, the use of fluorescent probes anchored to the resin provided evidence for the progress of the ongoing structural changes.Fluorescence spectroscopy is a very sensitive technique that provides valuable information involving changes in quantum yields, which in turn induces changes in the degree of conversion in a given reaction.The shifts in the signals are indicative of structural changes related to rigidity and planarity in the molecule.The naphthyl group acts as a sensor allowing the use of fluorescence spectroscopy as an analytical technique for the analysis of each step and determination of the reaction kinetics on solid phase.
H and 13 C NMR spectra were measured on a Varian Mercury 200 Spectrometer in CDCl 3 with TMS as internal standard (200 and 50.289MHz, respectively).EIMS spectra were obtained on a Hewlett Packard 5989 MS Spectrometer at 70 eV by direct insertion.Combinatorial chemistry procedures were carried out in a Reactor Quest Argonaut model SLN-210.Fluorescence spectra were recorded on a Shimadzu RF-5301 PC.