Synthesis and fluorescence of new 3-biphenylpyrrolo[1,2-c]pyrimidines

2.1 General

Melting points were determined on a Boëtius hot plate microscope. The elemental analysis was carried out on a COSTECH Instruments EAS32 apparatus. The IR spectra were recorded on a Bruker Vertex 70 ATR spectrometer or Nicolet Impact 410 spectrometer, in KBr pellets. The NMR spectra were recorded on a Varian Gemini 300 BB instrument, operating at 300 MHz for ¹H NMR and 75 MHz for ¹³C NMR. UV–VIS spectra were recorded with a JASCO V550 spectrophotometer. Fluorescent excitation and emission spectra were measured with a Jasco FP6500 spectrofluorimeter, with reading at an angle of 90°. The refractive indexes were measured at room temperature using a refractometer (Abbe from CETI Belgium).

2.2 Synthesis of the starting 4-Biphenylpyrimidine

4-Biphenylpyrimidine was obtained from 4-acetylbiphenyl and trisformylaminomethane according to the reported method for the synthesis of 4-phenylpyrimidine (Brederek et al., 1965). 2-Bromo-3′,4′-dimethoxyacetophenone was obtained by the brominating of corresponding acetophenone with bromine in diethyl ether. Other reagents used for synthesis were commercially available products. Acetonitrile and chloroform (Fluka) were used as received. Quinine sulphate (Buchler) was used as reference to calculate the quantum yield.

2.3 General procedure for syntheses of 3-biphenylpyrrolo[1,2-c]pyrimidines 4a–i

A mixture of 4-biphenylpyrimidine 1 (2 mmol), bromoacetyl derivative 2 (2 mmol) and a non-symmetrical electron-deficient alkyne 3 (2 mmol) in 30 mL of 1,2-epoxybutane was heated at reflux temperature for 24 h. The solvent was partly removed under vacuum, 3 mL of MeOH was added under a gentle stirring, and the mixture was left overnight in the refrigerator. The solid formed was filtered off and recrystallized from CHCl₃/MeOH giving 3-biphenyl-pyrrolo[1,2-c]pyrimidines 4a–i. In the case of symmetrical dipolarophiles, the procedure is similar with the small modification that the dipolarophile was added to the reaction mixture after an hour of reflux.

3.1 Synthesis of 3-biphenylpyrrolo[1,2-c]pyrimidines

The new 3-biphenylpyrrolo[1,2-c]pyrimidines 4a–i have been obtained by 1,3-dipolar cycloaddition reactions of the corresponding 4-biphenylpyrimidinium-N-ylides (generated in situ from the corresponding salts) with electron-deficient alkynes, in the 1,2-epoxybutane at reflux (Georgescu et al., 2013; Georgescu et al., 2012; Popa et al., 2015). The advantage of performing the reaction in a one-pot three-component approach is the direct formation of the final aromatic compounds, avoiding the formation of dipyrimidino-pyrazinic inactivated products. The 3-biphenylpyrrolo[1,2-c]pyrimidines have been synthesized according to Scheme 1 with medium to good yields (Table 1).

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Scheme 1

Synthesis of 3-biphenylpyrrolo[1,2-c]pyrimidines.

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Table 1 Structures, melting points and synthesis yields for the new pyrrolo[1,2-c]pyrimidine 4a–i.

Entry	R	R1	R2	Mp (°C)	Yield (%)
4a	4-Cl	H	Me	234–235	53
4b	3-NO2	H	Me	286–288	49
4c	4-F	H	OEt	222–224	58
4d	4-Br	H	OEt	234–235	53
4e	4-NO2	H	OEt	267–269	47
4f	3,4-diMeO	H	OEt	216–218	55
4g	4-NO2	CO2Me	OMe	286–288	62
4h	4-C6H5	CO2Et	OEt	216–218	51
4i	3,4-benzo	CO2Et	OEt	215–217	44

The reaction mechanism (Scheme 2) for obtaining the new 3-biphenylpyrrolo[1,2-c]pyrimidines implies the attack of the bromide ion of the pyrimidinium bromide of type 5 on the oxirane ring of 1,2-epoxybutane with formation of an alkoxide (Popa et al., 2015). This reactive alkoxide extracts one of the methylene protons in the bromide salt, generating the pyrimidinium-N-ylide 6 which reacts in the form of the mesomeric 1,3-dipole 7. In the next step, 1,3-dipolar cycloaddition between the pyrimidinium-N-ylide 7 and the activated acetylenic dipolarophile leads to the formation of the primary cycloadduct 8 which gives the final aromatic pyrrolo[1,2-c]pyrimidine 9 by oxidation in the reaction conditions.

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Scheme 2

Reaction mechanism for the 1,3-dipolar cycloaddition of pyridinium N-ylides with activated acetylenes.

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The structural characterization of the new compounds was performed by FTIR, ¹H and ¹³C NMR spectroscopy and elemental analysis. All methods present evidence for the structure of the new compounds. The main features of ¹H NMR spectra are the signals of the two hydrogen atoms in pyrimidine, H-1 and H-4, which appear as two doublets with coupling constants ranging from 1.1 to 1.5 Hz. In the case of unsymmetrical dipolarophiles the signal for H-6 appears as a sharp singlet at around 7.80 ppm or 8.0 ppm (when trifluoroacetic acid (TFA) was added). The main features of ¹³C NMR spectra are given by the presence of aliphatic carbons and carbonyl carbon atoms in the expected ranges. C-1 carbon atom appears the most deshielded at around 140 ppm due to its direct coupling with two nitrogen atoms. All the other signals and the corresponding multiplicities (for the ¹H NMR spectra) are in good agreement with the proposed structures.

3.2 Absorbance studies

UV–VIS spectra of the compounds have been recorded in order to evaluate the excitation wavelength for the study of the pyrrolo[1,2-c]pyrimidine framework fluorescence. Acetonitrile:chloroform mixture (1:1) has been found as the appropriate solvent. For instance Fig. 1A shows the obtained spectra at increasing concentrations of compound 4d in acetonitrile:chloroform (1:1) and Fig. 1B shows the linear dependences on concentration of the recorded absorbances at the main maximum wavelengths λ_max1 = 268 nm and λ_max2 = 384 nm. The extinction coefficients (ε₁, ε₂) at the two wavelengths (λ_max1 and λ_max2) have been calculated from the slopes of the linear dependences of absorbance on concentration, and they are shown in Fig. 1B.

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Figure 1

A: Spectra of 4d in acetonitrile:chloroform (1:1) at increasing concentrations (μmol/L): 1 (a), 2 (b), 3 (c), 4 (d), 5 (e) B: Linear dependences of the absorbance on concentration for 4d at λ_max1 = 268 nm (solid line) and λ_max2 = 384 nm (dotted line).

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Fig. 2 shows the absorption spectra for all compounds 4a–i at the same concentration (5 μmol/L) in acetonitrile:chloroform (1:1). It can be seen that absorption maxima show big differences in intensity varying between 0.1 and 0.8 for the investigated structures. All spectra have two main absorption maxima λ_max1, in the range 262–280 nm, and λ_max2, in the range 366–398 nm. The extinction coefficients (ε₁, ε₂) at the two maximum wavelengths (λ_max1 and λ_max2) have been calculated for all compounds from the slopes of the linear dependences in a similar way as shown in Fig. 1B for 4d. The results are summarized in Table 2 where are given also the equations for the linear dependences of the absorbances at λ_max1 and λ_max2 on concentration ([I]) (A_λmax1 and A_λmax2 vs [I] respectively).

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Figure 2

Absorption spectra for compounds 4a–i in 5 μmol/L solutions acetonitrile:chloroform (1:1).

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Fig. 2 shows that the nature of substituents R connected to the pyrrolo[1,2-c]pyrimidine framework induces significant differences in their spectra. R nature has an important influence both on λ_max and ε. The replacement of fluorine atom (from compound 4c) with a bromine atom (in compound 4d) and chlorine atom (in compound 4a) induces a bathochromic shift (to higher wavelengths) of λ_max1 and λ_max2; this is the effect of electronegativity of the atoms. By comparing the compound 4h with 4i it can be concluded that the hypsochromic shift (to lower wavelengths) for compound 4h is caused by the disturbance of conjugation due to deviation from co-planarity of the aromatic rings imposed by steric impediments.

3.3 Fluorescence studies

The emission spectra of compounds 4a–i were recorded in acetonitrile:chloroform (1:1) solutions (Tatu et al., 2015). For each compound the spectra were recorded at the corresponding absorption maxima λ_max1 and λ_max2 (Table 2). The emission spectra for λ_max1 have fluorescence intensities smaller (less than 50%) than for λ_max2 (Figs. 3Aand B). The spectra obtained at the same concentration with excitation at λ_max2 are shown in Fig. 4.

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Figure 3A

Emission spectra of compounds 4f for excitation at λ_max1 = 290 nm (solid line) and λ_max2 = 380 nm (dot line) in solutions (10⁻⁶ mol/L) in acetonitrile:chloroform (1:1).

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Figure 3B

Emission spectra of compounds 4c-f (A), 4g–i (B), 4a–b (C) in solutions (10⁻⁶ mol/L) in acetonitrile:chloroform (1:1); each spectrum has been recorded for excitation at the specific wavelength λ_max2 (given in Table 2).

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Figure 4

Excitation spectrum (solid line, with maximum at 389 nm) and emission spectrum (dot line, with maximum at 466 nm) for the compound 4b in solution (10⁻⁶ mol/L) in acetonitrile:chloroform (1:1).

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All compounds present one single emission band in the blue range domain (430–465 nm) as seen also in Table 3. The spectra in Fig. 3B have been grouped in 3 categories (A, B, C), in agreement with the compounds structure: 4c–f (A), 4g–i (B), 4a–b (C), and the values of their fluorescence intensity are given at the end of discussion. Studies regarding their fluorescence characteristics (Stokes shift, quantum yield and fluorescence quenching) have been performed.

3.4 Stokes shifts

For practical applications, an important feature of the fluorescence is given by the Stokes shift ( $\mathrm{\Delta }\tilde{\nu }$ ) which is equal with the difference between the wavelength of excitation and emission (Yang et al., 2015).

Stokes shifts were calculated (Table 3) using Eq. (1) for all compounds 4a–i on the basis of absorption-emission properties. Eq. (1) gives errors of 5–20%. The Stokes shifts were calculated at each maximum wavelength ( $\mathrm{\Delta }{\tilde{\nu }}_1$ and $\mathrm{\Delta }{\tilde{\nu }}_2$ ) at the same concentration (10⁻⁶ mol/L). All these compounds have fluorescence in the range of 430–470 nm. The data from Table 3 show higher Stokes shifts for excitation at λ_max,exc1 in comparison with λ_max,exc2 for all the investigated compounds.

3.5 Quantum yield

The fluorescence quantum yield (QY) is among the most important characteristics of fluorescence being defined as the efficiency of a fluorophore to convert the absorbed light into fluorescence. QY has been calculated (Table 4) for all compounds 4a–i using Eq. (2) (Brower, 2011; Fery-Forgues and Lavabre, 1999; Maree et al., 2002) in order to evaluate their fluorescence intensity. QY was measured for diluted acetonitrile:chloroform (1:1) solutions (3.5 ∗ 10⁻⁶ mol/ L) using quinine sulphate as standard (Tatu et al., 2015). In (2), QY, A, I, and n are quantum yield, maximum value of the absorbance at the emission wavelength λ_max,em2, area of the emission peak and refractive index for the solution of investigated compound, and QY_ref, A_ref, I_ref, n_ref are the corresponding values for the standard solution respectively. The following values are constant: n = 1.3942, and I_ref = 16,463; n_ref = 1.339; QY_ref = 0.6 (for the quinine sulphate standard solution) in the case of diluted solutions of all compounds 4a–i in acetonitrile:chloroform (1:1).

3.6 Fluorescence quenching

The fluorescence quenching of the new synthesized pyrrolo[1,2-c]pyrimidines in presence of the quencher 1,4-benzoquinone (BQ) has been examined. This is a very important property of a specific chromophore which may lead to interesting applications in the investigation of supramolecular assemblies for example (Wang et al., 2002). The fluorescence quenching curves for compounds 4a–i in the presence of BQ have been recorded. For instance Fig. 5 presents the fluorescence decrease in intensity (quenching) for the compound 4f in the presence of increasing concentrations of BQ.

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Figure 5

Fluorescence quenching curves of 4f (10⁻⁵ mol/L) in acetonitrile:chloroform (1:1) upon addition of increasing concentrations mol/L of BQ: 0 (a), 0.006 (b), 0.012 (c), 0.018 (d), 0.024 (e), and 0.030 (f).

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The changes in the fluorescence intensity related to BQ concentration are expressed by Stern-Volmer (S-V) Eq. (3) (Rose, 1964; Lakowicz, 2006) where: F₀ = fluorescence intensity in the absence of quencher; F = fluorescence intensity in the presence of quencher; K_SV = Stern-Volmer constant; [BQ] = concentration of the quencher (1,4-benzoquinone).

The ratios of F₀/F were calculated and plotted (Fig. 6) against [BQ], and K_SV were determined according to (3) from their slopes (Table 4).

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Figure 6

Stern-Volmer plots for benzoquinone (BQ) quenching of the studied compounds in solutions (10⁻⁵ mol/L) in acetonitrile:chloroform (1:1); the quenching curves have been recorded at λ_max,em2.

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Looking at the values from Table 4, it can be seen that two of the compounds (4c and 4f) have higher values of QY and K_SV (their values are highlighted with bold figures in Table 4). These higher values of QY and K_SV could be the result of a π electron conjugated system expansion for this compound. This assumption is confirmed by the high values of the fluorescence quantum yield for the two compounds.

3.7 Structure-fluorescence properties relation in the series of pyrrolo[1,2-c]pyrimidines 4a–i

The fluorescence and the related properties of the pyrrolo[1,2-c]pyrimidine derivatives 4a–i certainly are influenced by the nature of the substituents and this is discussed further. When comparing the compounds 4c, 4d, 4e and 4f of the group A (in which R is a substituted phenyl in position 4 with F, Br, NO₂ and (MeO)₂, respectively), it can be noticed that their fluorescence varies in the order of 4f > 4c > 4d > 4e; this order corresponds to the influence of the following substituents: MeO > F > Br > NO₂. Compound 4f has the highest fluorescence intensity, witch can be attributed to the presence of the two repulsive methoxy groups. Compound 4c presents a lower degree of fluorescence then 4d due to the smaller volume of the fluorine atom (in compound 4c) in comparison with bromine atom (in compound 4d). This is an example of the heavy atom effect (Guilbault, 1973), which suggests that the probability of intersystem crossing increases as the size of the molecule increases. The presence of bromine atom (compound 4d), leads to a loss of excitation energy by collision between molecules due to its big volume, which is the consequence of the fluorescence intensity decrease.

In group B, 4i, 4h and 4g which have biphenyl, 2-naphthyl and 4-nitrophenyl as substituent R and R¹ = R², compound 4g presents the lowest fluorescence intensity. This might lead to the conclusion that the moieties presenting an extended degree of conjugation such as naphthyl and biphenyl induce a high degree of fluorescence. The difference between 4i and 4h could be explained by the free rotation of the two phenyl rings in 4h, which can place the two phenyls out of the plane, thus decreasing the degree of conjugation and leading to a decrease in fluorescence. The lower fluorescence of 4g compared to that of the other two structurally analogous compounds 4i and 4h can be attributed to an increased tendency to aggregate in solution, due to the presence of nitro group (the polarized structure of the nitro group favours the appearance of compact supramolecular structures).

The compounds 4a and 4b of the group C, present low fluorescence intensities due to the presence of electron withdrawing substituents such as NO₂ and Cl. The low fluorescence intensities seen in the experimental fluorescence spectra (Fig. 3) are expressed also by the calculated values of fluorescence quantum yield and K_SV (Table 4).