Spectral and Kinetic Study of 3-Styrylquinoxalin-2(1H)-ones

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Spectral and Kinetic Study of 3-Styryl-quinoxalin-2(1H)ones Photoreduced by N-Phenylglycine and Amines Dafne Díaz-Hernández, Álvaro Cañete, Lynda Pavez, Alberto PérezSanhueza, Germán Günther, Tomasz Szreder, and Julio Ramón De la Fuente J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b01950 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

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The Journal of Physical Chemistry

Spectral and Kinetic Study of 3-Styryl-quinoxalin-2(1H)-ones Photoreduced by N-Phenylglycine and Amines

Dafne Díaz-Hernández,1 Álvaro Cañete,2 Lynda Pavez,2 Alberto Pérez-Sanhueza,1 Germán Gunther,1 Tomasz Szreder,3 Julio R. De la Fuente1,*

1Departamento

de Química Orgánica y Fisicoquímica, Facultad de Ciencias Químicas y

Farmacéuticas, Universidad de Chile, Casilla 223, Santiago 1, Santiago, Chile. 2

Departamento de Química Orgánica, Facultad de Química y de Farmacia, Pontificia Universidad Católica de Chile, Casilla 306, Correo 22, Santiago, Chile.

3 Institute

of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland.

*Corresponding Author: J. R. De la Fuente. E-mail: [email protected] 1 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract. The photoreduction by amines and N-phenylglycine, NPG, of six styryl-quinoxalin2(1H)-ones derivatives substituted in the styryl moiety, R-SQ, was studied by using flash photolysis. The photoreaction is initiated via a single electron transfer from the electron donor (amines or NPG) to R-SQ excited triplet state, 3R-SQ*, with the formation of a triplet state radical ion pair or a charge transfer exciplex, 3[CRIP/CTE]. These species live longer than the respective 3R-SQ* and have very similar transient spectra. In the presence of NPG these 3[CRIP/CTE] evolve on s time scale to the respective hydrogenated radicals, RSQH, whose transient spectra and reaction rate constants with NPG are reported. The identity of these hydrogenated radicals was supported by the spectra obtained with the -H donor triethylamine and previous pulse radiolysis studies in 2-propanol. Our findings allow proposing a radical chain reaction mechanism that explains the observed spectral behavior and rationalizes formation of the main product formed by binding of four PhNHCH2 derived from NPG decarboxylation.

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Introduction. Quinoxalin-2-one derivatives have numerous pharmacological properties showing activity as bacteriostatic,1-3 anticarcinogenic effects,4-6 and many biological effects (e.g. inhibition of certain enzymes related to HIV-1,7. Almost all of the quinoxalin-2-one derivatives that have biological activity bear substituent in the position 3 of the pyrazine ring of the quinoxalin-2-one. Some attention has been put in the study of 3-styryl derivatives as aldose reductase inhibitors8,9 and anti-parasite against Toxoplasma Gondii that may affect the fetal development.10 Some styryl derivatives have been tested as staining for amyloid fibers useful for diagnosis of Parkinson’s, Huntington’s and Alzheimer’s diseases.11 Although the broad range of pharmacological applications of quinoxalin-2-ones derivatives there is a relatively scarce literature concerning their radical processes initiated photochemically or by pulse radiolysis. In the last years our research has been focused on the identification of radical transients species derived from quinoxalin-2-one either photochemically or by pulse radiolysis.12-16 The studies of quinoxalin-2-one derivatives photoreduction by amines or amino acids have revealed a common reaction mechanism that comprises a single electron transfer, SET, from the electron donor to the quinoxalin-2-one derivative generating a radical ion pair, followed by a proton transfer from the donor radical cation to the N4 of the quinoxalin-2-one radical anion which leads to the expected stabilized captodative hydrogenated radical located over C3 that finally yields the observed products.17,18 In the photoreduction by amino acids among the stepwise electron-proton transfer the decarboxylation of an amino acid should be considered in order to explain the product formation, leading to imidazoquinoxalinone products for the reaction with N3 ACS Paragon Plus Environment


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phenylglycine, NPG.14 This decarboxylation occurs spontaneously for the radical cation NPG•+, with rates around 108 s-1 avoiding back electron transfer to the donor.19,20 For Nacetyl tryptophan, NAT, we reported the formation of an adduct with the quinoxalin-2-one toghether with NAT dimer.15 The study of amphiphilic 3-styryl-quinoxalin-2-ones with different length alkyl chains at the position 4 of the styryl moiety, reveals that the main photoreaction path is the trans-cis isomerization in solution, while a [2, 2] photodimerization is the preferred path in Langmuir-Blodgett films.21 This photodimerization process is similar to the early reported by Quina et al. for styrylpyridinium salts in solid state.22 Actually, our interest deals with the photoreactivity of substituted 3-styrylquinoxalin-2-one derivatives (see Chart 1) and the spectroscopic and kinetic characterization of the transient species generated in the presence of NPG and amines. We selected NPG because it decarboxylates in less than 200 ns, giving rise to the strongly reducing -amino-alkyl radical PhNHCH2• that shows weak absorption at 400 and a stronger band around 320 nm.23 On the other hand, the fast decarboxylation of the radical cation of N-phenylglycine, NPG•, should prevent a back electron transfer in the initial radical-ion pair generated by the SET. Moreover, the presence of substituents in position 4 of the styryl should modify the electronic distribution of the hydrogentaed radical. Recently, we reported characterization of radical ions and hydrogenated radicals of compounds presented in Chart 1 generated by pulse radiolysis in acetonitrile and 2-propanol.16 Pulse radiolysis allows by choice of solvent and saturating gas exercise control over the nature of generated species and thus


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selectively form the anion radical or hydrogenated radical of interest substrates without spectral interference of counter ions and other species as occur by flash photolysis. Therefore, we can compare our laser flash photolysis spectra with those obtained by pulse radiolysis.

R N

N H

O

R 1a : H 1b : 4-CH3 1c : 4-OCH3 1d : 4-OCF3 1e : 3-OCH3 ; 4-OCH3 1f : 2-OCH3 ; 5-OCH3

Chart 1. 3-Styryl-quinoxalin-2-one derivatives. Experimental. Materials. Acetonitrile and 2-propanol Merck, HPLC grade were used as received. N-phenylglycine, Aldrich 97%, was crystallized twice from water before use; 1,4diazabicyclo[2.2.2]octane, DABCO, Aldrich 98%, was purified by sublimation before use and triethylamine Sigma-Aldrich > 99,5 % was distilled trap to trap at 10-4 mm Hg before use. Laser flash photolysis experiments were performed on flash photolysis set up described previously.14,15 The flash photolysis setup is now provided with a Continuum Surelite I 10 Hz Q-switched Nd-YAG laser with the second and third harmonic generators. The signals are captured by a Hamamatsu 928 photomultiplier into a WaveSurfer 600 MHz



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LeCroy oscilloscope. Software written in National Instrument LabViews 8.0 controls the laser, monochromator and shutters. The captured data are fed into a program, written in Igor Pro 6.3 for treatment and display. The system is provided with a peristaltic pump and a 0.5 mL flow cell to assure the continuous renovation of solutions. Optimal results were obtained with solutions with absorbance between 0.4 - 0.6 at the 355 nm excitation wavelength. The laser power impinging the cell was attenuated to ≈ 15 mJ/pulse by using glass plates. However, for some of the derivatives samples were irradiated without attenuation due the low triplet quantum yields. Quenching by DABCO experiments were typically made with solutions (3 mL) of substrates and the appropriate concentration of quenchers. Solutions were bubbled with Ar for 20 min in 10 mm square quartz cells sealed with a septum. After purging the lifetimes  at

selected

wavelengths

were

measured.

Our

flow

system

was

used

when

photoconsumption was evident as occurred with N-phenylglycine, NPG, and triethylamine, TEA. The lifetime of the short-lived component of biexponential decay was used for the rate constant calculations. Quenching rate constants were obtained from the slopes of SternVolmer type plots of 1/ vs. [quencher] which were linear with r > 0.97 in all of the experiments. Transient spectra in Ar-saturated acetonitrile and 2-propanol containing 3styrylquinoxalin-2-ones (3-SQ) ≈ 0.1 mM (with absorbance at  = 355 nm between 0.4 and 0.6) in the presence of amines and NPG were measured after laser pulse. Variable amounts of the quenchers at the appropriate concentrations were added to the solutions. These solutions were monitored in a 0.5 mL flow cell with 10 mm light path at a flow rate of 1 mL/min and taking an average of 3 laser shots for each monitored wavelengths at each 6 ACS Paragon Plus Environment

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quencher concentration used. The solutions were bubbled with Ar for 1 hour before the experiments and continuously during them Triplet quantum yields for 3-styrylquinoxalin-2-ones were measured by energy transfer to -carotene with benzophenone in benzene as a standard. The measurements were made by monitoring OD at  = 520 nm, the wavelength which corresponds to max of -carotene triplets24 in solutions of styrylquinoxalin-2-ones and benzophenone in acetonitrile, whose absorbances at  = 355 nm were matched approximately to 0.2. Aliquots of -carotene in benzene were added to the Ar saturated benzophenone solution to reach a plateau in the absorption signal at  = 520 nm, to ensure complete energy transfer to -carotene. The same quantity of -carotene was added to acetonitrile solutions containing 3-styryl-quinoxalinones, and OD at  = 520 nm was measured attenuating the laser power with glass slides. The impinging power was measured with a pyroelectric power meter Gentec-e model S-Link 2. The triplet quantum yields were calculated from the slope of the plots of OD520 versus laser power, measured with the styrylquinoxalin-2-one and benzophenone by using equation 1: T = (mSQ/mbzph)(nbz/nan)2 Tbzph

(1)

where msq and mbzph were the slopes of the absorbance of sensitized -carotene triplet absorption at  = 520 nm, , by the styrylquinoxalin-2-one and benzophenone, respectively and Tbzph is the triplet quantum yield of benzophenone taken as 1.0025. Measurements were corrected by the respective refraction indexes of benzene (nbz) and acetonitrile (nan). The stability of the solutions was monitored by UV spectrophotometry, and no more than



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four laser pulses were used at each laser power. This procedure assumes an energy transfer yield of 100%. Phasors calculations. Each absorption spectrum at different elapsed times was Fourier transformed, as described in literature.26,27 Briefly, the absorption spectrum obtained at each elapsed time is transformed in one point by using the following equations:

(

Σ𝐴(𝜆)𝑐𝑜𝑠

𝑥 𝑐𝑜𝑜𝑟𝑑 (𝐺) =

)

𝐿

Σ𝐴(𝜆)

(

Σ𝐴(𝜆)𝑠𝑖𝑛

𝑦 𝑐𝑜𝑜𝑟𝑑 (𝑆) =

2𝜋𝑛(𝜆 ― 𝜆𝑖

(2)

2𝜋𝑛(𝜆 ― 𝜆𝑖 𝐿

)

Σ𝐴(𝜆)

(3)

where A(λ) is the absorption at each wavelength, (λ - λi) corresponds to the difference of wavelengths of the actual absorption and the wavelength where spectrum (or conversion to phasor) starts (in nm), L is the window of spectrum used for the conversion excluding negative OD, n is the number of the harmonic (we only considered the first harmonic, n = 1). Summation over the selected wavelength range, the spectrum yields one phasor point, with G and S coordinates that should take values between 1 to −1, excluding negative values of OD. Preparative photolysis. Preparative photolysis was done with the unsubstituted 3styrylquinoxalin-2-one (1a) in solutions containing an excess of NPG (1.6 mmol) and 40 mg (0.16 mmol) of 1a in HPLC quality acetonitrile that was bubbled with N2 for 1 hour. The solution was photolyzed, at room temperature, for 24 h under continuous N2 bubbling and stirring. The light source was a light emitting diode LED of 365 nm M365LP1 with the power source DC4100 and DC4014 four-channel LED driver from Thorlabs. The post8 ACS Paragon Plus Environment

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irradiation reaction mixture was filtered and separated from the NPG excess, and the filtrate was evaporated to dryness and the solid separated by flash column chromatography on silica gel using CH2Cl2 : acetonitrile 5:1 as the eluent. The main fraction isolated was dried out and dissolved in CDCl3 for NMR and mass spectroscopy. HRMS-ESI were done by using a Thermo ScientificExactive Plus Orbitrap spectrometer with a constant nebulizer temperature of 250 °C. The experiments were carried out in the positive and negative ion mode, with a scan range of m/z 100-700 with a resolution of 140000. The samples were infused directly into the ESI source via a syringe pump at flow rates of 5 μL min-1. NMR Spectroscopy. 1H NMR, COSY, HMQC and

13C

NMR DEPT 135º were

done in a Bruker Advance DRX-400, 400 MHz spectrometer, using TMS as the internal standard. Due to the low concentration of the samples, the experiments lasted for several hours.



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Results and Discussion. Photophysical behavior. The substituted 3-styryl-quinoxalin-2-ones, R-SQ, (Chart 1) have strong absorptions in the near UV and visible region and show emission with low fluorescence quantum yields f (of the order of 10-2) in acetonitrile while the triplet quantum yields in the same solvent, T, are in the range from 0.18 to 0.82 for the 4-CH3Oderivative (1c) and the 4-CF3O- derivative (1d), respectively, (see Table 1). Table 1. Photophysical parameters for substituted 3-styryl-quinoxalin-2-ones (RSQ) measured in acetonitrile SQ derivative  M-1cm-1 (max nm) f 10-2 (max nm) T

H1a 9250 (382) 2.32 (453) 0.55

4-CH31b 16260 (382) 1.41 (454) 0.33

4-CH3O1c 13980 (397) 3.88 (477) 0.18

4-CF3O1d 6400 (382) 3.07 (458) 0.82

3,4-CH3O- 2,5-CH3O1e 1f 19810 16490 (403) (400) not not measured measured 0.17 0.19

Transient absorption spectra and interaction with DABCO and TEA. All RSQ derivatives in Ar-saturated acetonitrile solution have very similar transient absorption spectra over 400 nm and a strong ground depletion centered at  = 350 nm which is shifted to the red for the methoxylated derivatives. Since the absorption bands over 400 nm and the ground depletion disappear completely under air or O2 for all the derivatives, these absorption bands were attributed to the triplet-triplet absorptions of the excited triplet states of the 3-styryl-quinoxalin-2-ones, 3R-SQ*.


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0.03

0.05 0.04

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B

0.02

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OD

OD

0.01

0.00 -0.01 -0.01

0.010

D

C

0.01

0.00 0.000

OD

0.005

OD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-0.01

-0.005

-0.02

-0.010 300

350

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Wavelength / nm

550

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Figure 1 Triplet-triplet transient absorption spectra recorded in Ar-saturated acetonitrile solutions for derivatives: (A) SQ, 1a; (B) 4-CH3-SQ, 1b; (C) 2,5-(CH3O)2SQ, 1f and (D) 3,4-(CH3O)2SQ, 1e at elapsed times after the laser pulse of: 0.5 (); 5 (); () 10 and 40 s ().

Addition of DABCO. As for other quinoxalin-2-one derivatives, previously studied by us, we expected quenching of triplet-triplet absorption bands by DABCO which is known as a good electron donor that is unable to further react.17,18 For all the derivatives studied we observed the typical Stern-Volmer behavior with the shortening of lifetimes of the excited triplet 3-styryl-quinoxalinones at low DABCO concentrations but a lengthening of them at higher concentrations without significant spectral changes. The quenching rate constants of the excited triplet 3-styryl-quinoxalinones, 3R-SQ* by DABCO in acetonitrile were evaluated by measuring lifetimes in independent cells with the appropriate amount of quencher (low concentration). These rate constants, kET, attributed to a single electron 11 ACS Paragon Plus Environment


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transfer from DABCO to the excited triplet states of 3-styryl-quinoxalinones, 3R-SQ*, (equation 4), are in the range of 107 M-1s-1, well below the diffusional limit, (see Table 2 for the kET values). Table 2 Rate constants of 3R-SQ* quenching by DABCO R-SQ H4-CH3- 4-CH3O- 4-CF3O3,4-CH3O- 2,5-CH3Oderivative 1a 1b 1c 1d 1e 1f 7 2.4+/-0.7 2.3+/-0.7 2.4+/-0.1 2.0+/-0.6 not measured 3.7+/-0.3 kET 10 M-1s-1

However, these values are higher than those measured for the quenching of excited triplet states of 3-phenylquinoxalin-2-ones by DABCO and other amines.17 3R-SQ*

+ DABCO  R-SQ− + DABCO+

(kET)

(4)

The Stern-Volmer plots (at low concentration range) for derivatives 4-CF3O-SQ, 1d and 4-CH3O-SQ, 1c are shown in the inset of Figure 2A. As it was mentioned earlier, at higher [DABCO] the Stern-Volmer behavior reverts showing an increase of absorption lifetime with concentration of DABCO. The monoexponential decays recorded at the absorption maximum of 1a at 0; 18 and 71 mM DABCO with lifetimes of: 4.5; 6.9 and; 10.5 s respectively, are shown in the inset in Figure 2B. The same lifetimes behavior was observed for the other derivatives in the presence of high DABCO concentrations: for derivative 4-CH3O-SQ, 1c, the lifetime increases from 11.1 s in absence of DABCO to 12.2 s at 20 mM DABCO and for 4-CF3O-SQ, 1d, the lifetime increases from 9.5 s in absence of DABCO to 16.5 s at 42 mM DABCO. This behavior strongly suggests that after the SET a contact radical ion pair, CRIP, or a charge 12 ACS Paragon Plus Environment

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transfer exciplex, CTE, is formed, probably with a triplet character and hence decaying slowly and mono-exponentially inside of the solvent cage due to the spin forbidden back electron transfer, equation 5. 3[R-SQ−

 DABCO+] / 3[R-SQ−  DABCO+]  R-SQ + DABCO

250

300

350

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450

500

550

600

650

(5)

700

5

3.6x10

A

5

OD

0.015 0.010

5

2.8x10

-1

.5 s 1 2 5 10 20 40

0.020

-1

3.2x10  /s

0.025

5

2.4x10

5

2.0x10

0

1

2

3

4

5

6

[DABCO] / mM

0.005 0.000 -0.005

B

0.020 0.015 0.010

.5 s 1 2 5 10 20 40

0.02 OD

0.025

OD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.01 0.00 -0.01

0

5

10

15

t / s

0.005 0.000 -0.005 -0.010 250

300

350

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650

700

Wavelength / nm



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Figure 2. (A) Transient absorption spectra recorded for unsubstituted derivative (1a) in the absence of quencher. Inset: The Stern-Volmer plots for the 3SQ* quenching by DABCO for derivatives: 1d CF3O-SQ (black squares); and CH3O-SQ 1c (red circles). (B) Transient absorption spectra recorded for (1a) in the presence of 36 mM DABCO. Inset: The decays of 3SQ* at various DABCO concentrations: 0 (black), 18 (red) and 71 (green) mM.

The transient spectra obtained by laser flash photolysis of all the derivatives studied in the presence of high DABCO concentrations and the respective transient absorption spectra of radical anions obtained by pulse radiolysis are collected in Supp. Inf. Figure S1. Almost all the spectra at high [DABCO] differ notably with those obtained by pulse radiolysis in Ar-saturated acetonitrile and attributed to styrylquinoxalin-2-ones radical anions R-SQ.16 The only two radical anion derivatives, R-SQ−, whose pulse radiolysis spectra resemble those obtained by flash photolysis at high [DABCO] are 1c and 1e with CH3O- substituent in 4- and 3,4- positions, respectively. Addition of TEA. The same lifetime behavior in the presence of high concentration of trimethylamine (TEA) was observed for two derivatives studied by us: 4-CH3-SQ and 4CF3O-SQ. This is shown for 4-CF3O-SQ, 1d, where the lifetimes at high [TEA] increase with respect to those observed for 34-CF3O-SQ* absorption ( 7.8 s) changing to 17.5 s and 18.2 s at [TEA] 24 and 120 mM respectively (inset in Figure 3A). Moreover, a decrease in transient absorption intensity was observed as TEA concentration increases. The observed transient spectra at short-times, in the presence of high concentration of TEA, (Figure 3B) are very similar to those attributed to the triplet-triplet absorption, (Figure 3A). However, decrease in a transient absorption intensity was observed as [TEA] increases and


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at long elapsed time (40 s) after the laser pulse a new transient with absorption maxima at  = 430 and 480 nm was formed (Figure 3B). Since, the radical cation of TEA (TEA+) is able to donate H+ from the -C to the radical anion 4-CF3O-SQ, this spectrum might be tentatively assigned to the hydrogenated radical, 4-CF3O-SQH generated after TEA+deprotonation, (Eq 6). 3[4-CF

3O-SQ

−

 TEA+]/3[4-CF3O-SQ−  TEA+]  4-CF3O-SQH + TEA(-H) (6)

The spectrum resembles a spectrum obtained by pulse radiolysis in Ar-saturated 2propanol and attributed to 4-CF3O-3-styrylquinoxalin-2-one hydrogenated radical, 4-CF3OSQH,16 (see Figure 3B). The spectrum of 4-CF3O-SQH obtained by pulse radiolysis is shown in Figure 3B, for comparison. The same behavior was observed for 4-CH3-SQ derivative 1b showing a shoulder located at  = 480 nm at long elapsed time (data not shown) attributed to 4-CH3-3-styrylquinoxalin-2-one hydrogenated radical, 4-CH3-SQH . No attempts to identify the photoproducts of both these reactions were done. Interestingly, the absorption spectrum observed at short times in the presence of high concentration of TEA does not resemble absorption spectrum attributed to 4-CF3OSQ obtained by pulse radiolysis in Ar-saturated acetonitrile.16 It is reasonable to claim that absorption spectrum can be attributed to a triplet state CRIP (3[R-SQ−  Amine+]) or CTE (3[R-SQ−  Amine+]), where the back electron transfer, forbidden by spin, explain the long life of their absorptions as compared with the respective triplet-triplet absorption. Similar spectral behavior was observed in the presence of high concentration of DABCO (vide supra). From the above results it is clear that SET quenching by amines took place with the formation of a long lived CRIP or CTE with triplet character. Moreover, by 15 ACS Paragon Plus Environment


considering the spectral similarity of these absorptions with the triplet-triplet absorption, these species are probably locally excited in the styrylquinoxalin-2-one moiety. Similar processes considering a CTE for diaza-fluorenones and phenols, where the radical anion of diaza-fluorenones cannot be detected, were invoked to explain the formation of ketyl/phenoxyl radical pair for these compounds.28,29

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Figure 3. (A) Transient absorption spectra recorded for derivative 1d, 4-CF3O-SQ in the absence of TEA at elapsed times from 0.5 to 40 s. (B) Transient absorption spectra recorded in the presence of 120 mM TEA for derivative 1d, 4-CF3O-SQ at elapsed time of 0.5 to 40 s. Spectrum at 40 s () shows maxima at 430 and 480 nm, see text. Inset: the decays of 34-CF3SQ* at various [TEA]: 0; 24 and; 120 mM, (black), (red) and,(green), respectively Red solid line shows the absorption spectrum obtained by pulse radiolysis in Ar-saturated 2-propanol and attributed to hydrogenated radical 4-CF3O-SQH, 1dH.16

Addition of NPG. Results of other experiments performed with 7-substituted-3phenylquinoxalin-2-ones in the presence of NPG are also compatible with the generation of a CRIP or CTE after the laser pulse. As an example, the transient spectra change only in intensity on addition of NPG showing an increase of the absorption band with max = 320 nm attributed to PhNHCH2 radical as shown in Supp. Inf., Figure S2 for the 7-CF3-3phenylquinoxalin-2-one. Moreover, the stable photoproducts of exhaustive photolysis in excess of NPG are analogous to those found for 3-methylquinoxalin-2-ones as shown in Supp. Inf. Figure S3 for the unsubstituted 3-phenylquinoxalin-2-one. Transient species in the presence of NPG. Flash photolysis experiments for all of the 3-styryl-quinoxalin-2-one derivatives in the presence of NPG show similar features to those observed with amines, i.e. at low concentrations of NPG and at short elapsed time after the laser pulse, spectra stayed nearly the same as those of excited triplet state, but the decays become biexponential at very low NPG concentration showing the presence of at least two species. At higher concentrations of NPG a long-lived shoulder around 470-480 nm appears for all the derivatives and is accompanied by the absorption band with max = 440 nm. The quenching rate constant by



NPG were obtained from the short-lived component of biexponential decay at the absorption maximum of the respective excited triplet state (see below). Transient absorption spectra recorded in acetonitrile solutions containing 4-CH3-SQ, 1b and 4-CH3O-SQ, 1c, in the presence of high NPG concentration are shown in Figure 4. High concentration of NPG assures an almost complete quenching of excited triplet states. A strong absorption at  = 320 nm attributable to PhNHCH214,15,23 was observed in both spectra.

.5 s 1 2 5 20 80

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Figure 4 (A) Transient absorption spectra recorded in acetonitrile solutions containing 4CH3-SQ, 1b, in the presence of 19 mM of NPG at various times after the laser pulse. Inset: kinetic traces recorded at selected wavelengths: 480 nm (green), 445 nm (red), 520 nm (blue) and 320 nm (black). (B) Transient absorption spectra in acetonitrile solutions containing 4-CH3O-SQ, 1c, in the presence of 22.2 mM of NPG at various times after the laser pulse. Inset: kinetic traces recorded at selected wavelengths: 475 nm (green); 445 nm (red) and 320 nm (black). This absorption at  = 320 nm grows initially within the laser pulse with a secondary delayed growth reaching maxima at times < 1 μs, depending on the derivative and NPG concentration. For the 4-CH3-SQ derivative (1b) and 19 mM NPG, an absorption band with max = 445 nm and shoulders located at  = 480 nm and 520 nm were observed at short elapsed times (Figure 4A). For this derivative, it is interesting to note that the initial decay of absorption at  = 445 nm, k = (1.1 +/- 0.04)  106 s1 perfectly matches, within the experimental error, the delayed growth of absorption attributed to PhNHCH2 at  = 320 nm with k = (1.1 +/- 0.1)  106 s1. Therefore, these former absorptions should be attributed to a precursor of PhNHCH2, the CRIP or CTE. These bands evolve at longer times to a spectrum with max = 480 nm and a broad shoulder centered at   445 nm. A close analysis of spectra reveals that the ratio of absorbances at  = 445 nm and  = 480 nm (R445/480) starts from 1.34 at 0.5 μs and further changes to 0.73 at 20 μs and remains nearly constant up to 80 μs. This result allows us to conclude that the observed spectrum with two maxima located at  = 445 and   480 nm after 20 μs can be attributed mainly to the presence of one species, plausibly, the hydrogenated styryl-quinoxalin-2-one radical 4-CH3-SQH. On the other hand, the absorption spectra recorded at short elapsed time after the laser pulses resemble the spectra obtained at high DABCO concentration. Therefore, they might be mainly attributed to the contact radical ion pair (CRIP) or charge transfer exciplex 19 ACS Paragon Plus Environment


Page 20 of 37

(CTE) in triplet state 3[SQ−NPG+] / 3[SQ−NPG+] with a small contribution of the long-lived hydrogenated radical SQH, 1bH. The latter radical is generated within the laser pulse and persists up to 80 μs. Similar spectral features were observed for the 4-CH3O-SQ, derivative, 1c, in the presence of 22 mM NPG, at short elapsed times (Figure 4B). They are characterized by the strong absorption band with max = 320 nm assigned earlier to PhNHCH2- radical and the second absorption band with max = 480 nm and a shoulder at  = 445 nm. These absorptions evolve with similar kinetics; though the max shifts slightly to 475 nm at longer elapsed times (see in Figure 4B). A close examination of the ratio of absorbances at  = 445 and 475 nm, (R445/475) shows that it changes from 0.82 at 0.5 μs to 0.76 at 20 μs and then remains constant at longer elapsed times. This behavior reveals again that the absorption spectra observed at long times, can be attributed to a single species, plausibly to the hydrogenated radical 4-CH3O-SQH, 1cH. Summarizing these data one can conclude that there are at least 3 species absorbing between 400 and 550 nm including PhNHCH2 which is also characterized by the strong absorption band with max = 320 nm. These three species can be tentatively identified as the remnant of excited triplet states 3R-SQ*; the CRIPs 3[R-SQ−NPG+] or CTEs 3[RSQ−NPG+] (they are similar for all the derivatives to those observed with DABCO) and to the hydrogenated radicals R-SQH with absorption maxima located around 445 and 480 nm for all the derivatives studied. However, the strong overlap of absorption bands made difficult the unequivocal spectral assignments.


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Spectral phasors. The transient spectral behavior was tested by applying spectral phasors that also suggest the presence of the same three transient species. To our best knowledge phasor analysis has not been applied to absorption spectra in general including transient absorption spectra obtained by flash photolysis. Phasor based image analysis has been shown to be very valuable for frequency domain30-33 and time domain34,35 lifetime imaging data. It has been shown that phasor representation of spectral data affords rapid and reliable resolution of individual components in images. Therefore, to unveil spectral information underlying combined absorption of transient species, we applied phasor analysis to the evolution in time of transient spectra. For instance, a total phasor from the combination of two spectra should fall in a line which connects their pure phasors. Therefore, a linear trajectory indicates the evolution of spectra from one compound to another. If the total spectrum is composed of 3 pure components, the phasor will fall inside a triangle where the vertices are the phasors of pure spectra, thus, curved trajectories must involve at least three components. See Supp. Inf. Figures S4 and S5. Data were treated removing any ground state depletion (to keep the universal circle, despite the spectral range is narrowed and some information is lost). Results obtained show clearly that at least three species are involved in the absorption during the time domain monitored. Figure 5A shows the evolution transient spectra of derivative 4-CF3O-SQ (1d) in the presence of 22 mM NPG and in the absence of quencher from an independent experiment. The respective phasor plot, Figure 5A’, shows the initial evolution from one transient to another as shown by the linear trend from 0.5 to 5 s indicating the transition from the excited triplet state 34-CF3O-SQ*, (black point in the phasor plot) to the CRIP or 21 ACS Paragon Plus Environment


CTE. After 5 s, the excited triplet state disappeared completely and the formation of protonated radical started, moving the phasor trajectory over a new straight line in the phasor space. For 4-CH3-SQ (24 mM NPG), a slightly different behavior is seen (Figure 5B’. For this compound a curved trajectory was obtained indicating again that at least three components are involved: the excited triplet state, the CRIP or CTE and the hydrogenated radical. The latter radical should be generated after 15 s from the CRIP or CTE. After 15 s a linear trend which involves only two remaining species, the CRIP or CTE and the protonated radical, is seen in the plot.

0.06 0.04

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Page 22 of 37

-0.3 350

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Wavelength / nm

0.50

5 30

10 25

15

0.55

G

Figure 5. (A): In black, excited triplet state absorption of 4-CF3O-SQ, in color, transient spectra of 4-CF3O-SQ in the presence of 22mM of NPG at 0, 1, 2, 5, 10, 15, 20, 25, 30, 35 and 40 s. (A’): Phasors obtained from spectra shown in A tagged with the respective 22 ACS Paragon Plus Environment

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times. (B): Transient absorption spectra of 4-CH3-SQ in the presence of 24 mM of NPG at 0, 1, 2, 5, 10, 15, 25, 30 and 40 sec. (B’): Phasors obtained from spectra shown in B, tagged with the respective times.

The information on the evolution of transient absorption, extracted from the phasor treatment, is fully consistent with the evolution of absorption at different wavelengths discussed above. Hydrogenated radicals. The transient spectra for all the derivatives at high NPG concentrations at short and long elapsed times together with the respective spectrum of the hydrogenated styrylquinoxalin-2-one radicals (R-SQH) obtained by pulse radiolysis in Ar-saturated 2propanol (2-PrOH)16 are shown in Figure 6. These spectra match reasonably well with the spectra obtained by flash photolysis in the presence of NPG at long elapsed times. This observation supports our earlier spectral assignment to the protonated radical of the styrylquinoxalinones R-SQH.



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Page 24 of 37

2,5-(CH3O)2-SQ

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Wavelength / nm

Figure 6 Transient absorption spectra recorded in acetonitrile solutions containing styrylquinoxalin-2-one derivatives R-SQ at short () and long () elapsed times in the presence of  20 mM NPG and transient absorption spectra () recorded 10 s after the electron pulse in Ar saturated 2-PrOH and assigned to protonated radicals R-SQH.16

Quenching of triplet excited states of 3-styryl-quinoxalin-2-ones by N-phenylglycine. The respective quenching rate constants were determined by studying the effect of NPG concentration on the kinetics observed at wavelengths corresponding to max of the absorption bands assigned to the respective triplet excited state (3R-SQ*) (see Table 3). 3R-SQ*

+ NPG  3[R-SQ−  NPG+] / 3[R-SQ−  NPG+]

(7)


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Moreover, the kinetic behavior of the absorption at  = 470-480 nm attributed to the hydrogenated radical R-SQH was also analyzed. The results obtained for the unsubstituted derivative 1a are presented in Figure 7A and B. The kinetic traces at  = 440 nm (corresponds to max of the absorption band of the excited triplet state 31a*) at various [NPG] are presented in the inset in figure 7A. The 31a* decays monotonically by the first order kinetic in absence of NPG which becomes biexponential by adding NPG. As expected the absorption of the long-lived bi-exponential component increases and the lifetime of the short-lived component, attributed to the triplet excited state, decreases. By plotting the lifetime reciprocal, -1, of the short-lived component v/s [NPG] we obtained the Stern-Volmer plot of Figure 7A, were the slope is the quenching rate constant kET = (4.1 +/0.4)107 M-1s-1 for derivative 1a. Table 3. Rate constants of quenching of 3R-SQ* by NPG in acetonitrile. R-SQ derivative kET 107 M-1s-1 astrong

H1a 4.1+/-0.4

4-CH31b 0.96 +/- 0.02

4-CH3O1c 1.4 +/-0.5

4-CF3O1d 3.5 +/- 0.3

3,4-CH3O1e a not measured

2,5-CH3O1f 2.8+/-0.3

spectral overlap



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Page 26 of 37

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Figure 7. (A) Stern-Volmer like plots of the effect of NPG concentration on the excited triplet absorption kinetic traces of the unsubstituted derivative 1a at 440 nm. (B) Effect of NPG concentration on the kinetic traces of the absorption at 475 nm attributed to the hydrogenated radical 1aH. The insets in A and B: kinetic traces at various [NPG]: 0.0 (black); ; 4.90 mM (red); 9.18 mM (green) and 25.10 mM (blue) at  = 440 and 475 nm, respectively.


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The kinetic traces at  = 475 nm, attributed to the hydrogenated radical are shown in the inset of Figure 7B. By increasing NPG concentration, the kinetic traces at  = 475 nm become bi-exponential with a concomitant increase in the absorption intensity within duration of the laser pulse. Moreover, a secondary growth that depends on the NPG concentration at larger concentrations of NPG appears in the first 5 s, see inset of Figure 7B. This secondary growth strongly suggests some chain radical reaction as observed earlier for 3-methylquinoxalin-2-ones.15 The first-order rate constants obtained by a mono-exponential fitting of this secondary growth at  = 475 nm at various concentrations of NPG yield fairly linear SternVolmer plot (Figure 7B) From the slope, the second order rate constant of reaction 8, attributed to the chain propagation reaction, was determined for the unsubstituted derivative 1a: kSQH/NPG = (5.7 +/- 0.3)107 M-1s-1. This pseudo 2nd order rate constant (NPG and RSQ are in a large excess with respect to R-SQH) of the reaction between the hydrogenated radical R-SQH, ground state R-SQ and NPG, giving origin to more R-SQH might explain the increase of the absorption attributed to R-SQH. On the other hand, the intercept of this plot might be interpreted as the rate constant for the decay of R-SQH in the media, kdSQH = 2.6105 s-1 leading to products, Equation 9. [R-SQH + R-SQ] + NPG   2 R-SQH + PhNHCH2 + CO2 (kSQH/NPG)

(8)

R-SQH   Products

(9)

(kdSQH)

By applying the same treatment to 4-CF3O-SQ 1d derivative, from the secondary growth, the pseudo-first-order rate constants were determined at each [NPG] and from the respective Stern-Volmer plot the rate constant of the radical chain propagation reaction 27 ACS Paragon Plus Environment


Page 28 of 37

between the hydrogenated radical R-SQH and NPG was determined from the slope: kSQH/NPG = (5.2 +/- 0.3)107 M-1s-1 with the pseudo first-order with an intercept, measuring the decay reaction leading to products of R-SQH, kdSQH = 2.4105 s-1. These decay constant rates, kdSQH, are 2 magnitude order larger than the decay rates of the same radicals determined by pulse radiolysis for the same derivatives16 because kdSQH were measured in the presence of the reactive phenylaminoalkyl radical PhNHCH2. These decay rate constants, kPhNHCH2, are 10 times larger than the decay rates estimated before for the PhNHCH2 intermediate radicals in the reaction of 3-methylquinoxalin-2-ones with NPG.15 If reaction 8 occurred, an increase in [PhNHCH2] should be observed which will be reflected by an increase of the absorption at 320 nm. However the absorption at this wavelength remains almost independent of concentration of NPG as is shown for derivative 4-CH3O-SQ, 1c, (Figure S6 Supp. Inf), likely due to the spectral overlap between this absorption and ground depletion of R-SQ.

Moreover, the

styrylquinoxalin-2-one hydrogenated radical R-SQH might be able to oxidize NPG to NPG or abstract the  hydrogen from NPG leading to PhNHCHCOOH, both leading to PhNHCH2 by decarboxylation.14,15 This latter species, the -aminoalkyl radical is known as a strong one-electron reductant likely able to reduce the 3-styryl-quinoxalin-2-one as we proposed earlier for the 3-methyl derivatives.14,15 Therefore, we can propose the following radical chain reaction mechanism.


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3

[CRIP / CTE]

3

[R-SQNPG] / 3[R-SQNPG]

NPG

3

R-SQ* + NPG

kSQH/NPG

PhNHCH2

kET

R-SQH

kPhNHCH2 R-SQ Products

Scheme 1. Proposed radical chain reaction mechanism for the photoreaction between styrylquinoxalin-2-ones and N-phenylglycine. Transient spectra in 2-propanol. To check for a possible photoinduced hydrogen transfer, some experiments were done using 2-propanol, 2-PrOH, as hydrogen donor solvent to form the respective ketyl radical and the styrylquinoxalin-2-one hydrogenated radical, equation 9. 2-PrOH + 3R-SQ*  (CH3)2-C-OH + R-SQH

(11)

However, transient spectra of styrylquinixalin-2-one derivatives in neat 2-PrOH were almost identical to those observed in acetonitrile and their triplet quantum yields, T, resulted, in all the measured cases, lower than those in acetonitrile, see Table 4. Table 4. Triplet quantum yields for Styrylquinoxalin-2-one derivatives in 2-PrOH. R-SQ derivative T

H1a 0.22

4-CH31b not measured

4-CH3O1c 0.10

4-CF3O1d 0.23

3,4-CH3O- 2,5-CH3O1e 1f 0.09 0.11



Page 30 of 37

The behavior of transient spectra in the presence of NPG was very similar to those observed in acetonitrile, with decays becoming biexponential at very low NPG concentrations and the appearance of long lived absorptions at 440-450 and 480 nm attributable to the protonated radicals, Figures S7 and S8 in Supp. Inf. show this behavior for derivatives 4-CF3O-SQ and 4-CH3-SQ. From the decays of the short lived component and Stern-Volmer plots, the quenching rate constants in 2-propanol, kET_2PrOH, were determined resulting in (2.3 +/- 0.1)107 and (5.4 +/- 0.8)106 M-1s-1 for 4-CH3-SQ, 1b, and 4-CF3O-SQ, 1d, respectively.

Stable photoproduct. In order to identify the stable photoproducts we selected the unsubstituted derivative 1a which was photolysed, by light of 365 nm, in N2-saturated acetonitrile solution in the presence of excess of NPG until complete reactant consumption. The main photoproduct was isolated by silica gel column-chromatography as described in experimental section and analyzed by NMR and HMRS-ESI techniques. The 1H-NMR spectrum allows deducing from the 24 aromatic protons that almost 3 additional phenyl units, coming from the NPG, have been incorporated in the SQ. The 13C NMR spectrum shows 7 aliphatic carbons being one of them quaternary and 4 of them diasterotopic methylenes. Two of these appearing at 54.96 and 52.58 ppm might be identified in spite of the low signal intensity in the DEPT spectrum while the other two appearing at 56.59 and 50.39 ppm show intense signals. The quaternary C, the former C3 of quinoxalin-2-one moiety is also seen at 55.36 ppm in the same spectrum. All these 30 ACS Paragon Plus Environment

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spectra are included in Supp. Inf. Figures S8 to S15. Therefore, for the reaction of H-SQ , 1a, it can be expected the attachment of four PhNHCH2 radicals, with the cyclization reaction accompanying aniline elimination. Two of these PhNHCH2 will attack the stereogenic double bond of styryl and the other two PhNHCH2 attack at N-4 and C3 of quinoxalin-2-one core, undergoing cyclization together with elimination of aniline as we report for the 3-phenyl-quinoxalin-2-one derivatives in this work (Figure S3) and proposed earlier for 3-methyl-quinoxalin-2-ones.14 Therefore, the following structure of the main photoproduct was proposed (Chart 2).

NPG Fragment NH Styryl Fragment H N

O

Quinoxalinone fragment

H N N N

NPG Fragment

Chart 2. Main photoproduct of photoreaction (C38H37N5O). HRMS-ESI experiments. Samples of the isolated main photoproduct were analyzed by HRMS-ESI. The results obtained in positive mode show clearly the adduct with sodium, [M + Na]+, with a precision of 2.16 ppm with respect the calculated exact mass. On the other hand, results obtained in negative mode show the adduct with chloride [M + Cl]- within 1.47 ppm of calculated exact mass. These mass spectra are shown in Figures S17 and S19 in Supp. Inf. 31 ACS Paragon Plus Environment


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Among these signals, one with m/z = 579.2940 which can be attributed to molecular ion (with precision of 10.01 ppm) was identified in positive mode, (Figure S16). Furthermore, a fragment, (Figure S18), was identified as the protonated N-phenethylbenzenamine (C14H16N+, m/z = 198.1275) with a m/z just 1.00 ppm different to the calculated exact mass. Conclusions In general, photoreduction of quinoxalin-2-ones derivatives are initiated by a single electron transfer from amines or NPG to the excited triplet of the quinoxalin-2-one. For the 3-styryl-quinoxalin-2-ones and 3-phenylquinoxalin-2-ones, the SET with amines and NPG is followed by the generation of a triplet state contact ion radical pair or charge transfer exciplex, 3[CRIP/CTE], locally excited in the quinoxalin-2-one moiety. Formation of these species can explain the lengthening of lifetime and nearly no spectral changes between CRIP/CTE and triplet transient absorptions. This CRIP/CTE evolves to the respective hydrogenated radical R-SQH stabilized by a captodative effect whose absorption spectrum matches absorption spectrum attributed to it obtained by pulse radiolysis.16 Moreover, these R-SQH are likely able to react with NPG, in the radical chain propagation reaction, Eq 8, leading to more R-SQH and PhNHCH2. Furthermore, the main photoproduct might probably formed by reaction of the PhNHCH2 with the ground state excess of R-SQ. On the other hand, the experiments performed in 2-propanol allow to disregard the H transfer as a reaction path. The proposed radical chain mechanism proposed in Scheme 1 rationalizes formation of the identified main product and of the transient spectral behavior reported.


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As far as triplet quantum yields are concerned, , the largest quantum yield (T) was found for the 4-CF3O- derivative 1d containing electron withdrawing group by inductive effect but electron donating group by resonance. The T values gradually decrease for the unsubstituted SQ, 1a, (without any substituent effect) and for the 4-CH3-SQ, 1b, containing electron donating group by inductive effect. The lowest T values were observed for the 4CH3O-SQ monosubstituted, 1c, and the disubstituted derivative

3,4-(CH3O-)2-SQ, 1e,

both with a CH3O- in position 4, contributing by donating inductive and resonance effect. For the derivative 1f, 2,5-(CH3O-)2-SQ doublesubstituted in positions 2 and 5 T is slightly higher than for the 3,4-(CH3O-)2-SQ disubstituted derivative 1e. Therefore, it can be concluded that an increase in electronic density on the quinoxalin-2-one moiety, transmitted through the vinyl bridge, decrease the intersystem crossing probably due an increase of non-radiative decay path of excited singlet state. Supporting Information is available free of charge on the ACS Publication website. Transient spectra in acetonitrile and 2-propanol, HRMS-ESI mass and NMR spectra used for characterization of photoproducts. (PDF file) Acknowledgments to FONDECYT Grant N° 1150567 for the financing support, to Prof. K. Bobrowski from INCT, Warsaw, Poland for the valuable and fruitful discussion of the manuscript content.



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Spectral and Kinetic Study of 3-Styrylquinoxalin-2(1H)-ones

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