Khac Minh

Huy Nguyen

Leslie Schwendimann

Pierre Gressens

Martine Largeron

Regiospecific synthesis of neuroprotective 1,4-benzoxazine derivatives through a tandem oxidation-Diels-Alder reaction

The tandem oxidation-inverse electron demand Diels-Alder reaction of o-aminophenol derivatives and enamines has been accomplished at room temperature using a stoichiometric amount of manganese dioxide as the oxidant to furnish highly substituted 1,4-benzoxazine cycloadducts with complete regiochemical control. Because of its efficiency for introducing diversity elements in both cycloadditions partners, this one-pot process should allow the assembly of libraries of biologically relevant 1,4benzoxazine derivatives. In this respect, 3,3-diphenyl-substituted-1,4-benzoxazine derivative 3n was found to be a potent neuroprotective agent in an animal model of excitotoxic lesions in newborn mice.

Introduction

1,4-Benzoxazine derivatives are an important class of heterocyclic compounds because of their natural occurrence and important biological activities. 1 They are potential drugs for treating infections, diabetes, asthma, depression as well as neurodegenerative, autoimmune, inflammatory and cardiovascular disorders (Figure 1). 2 Traditional syntheses of 1,4benzoxazine derivatives involve multistep processes, using 2nitrophenols or 2-aminophenols as the starting materials. 1,3 However, the increasing interest in 1,4-benzoxazine derivatives has prompted to develop new concise methodologies that follow diverse one-pot strategies including metal-catalyzed reactions, 4 cascade (or tandem) reactions 5 and microwave-assisted reactions. 6 A few years ago, we reported an electrochemically induced tandem oxidation-Diels-Alder reaction for the synthesis of highly functionalized 1,4-benzoxazine and 1,4-benzodioxin derivatives. 7 In particular, the anodic oxidation of o-aminophenol derivatives produced unstable o-quinone-monoimine heterodienes, which were trapped in situ by enamine dienophiles through regiospecific inverse-electron-demand Diels-Alder (IEDDA) reactions to give diverse 1,4-benzoxazine derivatives. Interestingly, some of these compounds proved to be effective neuroprotective agents both in vitro and in vivo. 8 Nevertheless, it seemed doubtful that this electrochemically induced process will ever be able to compete with more conventional chemical approaches at a preparative scale. However, the instability of o-quinone-monoimines prohibited their efficient use as heterodienes in chemically induced IEDDA reactions. For a long time, such reactions have been mainly restricted to stable o-quinone-monooximes 9 and oquinone-monoimides. 5a-d,5f,10 Recently, the synthesis of 1,4benzoxazine derivatives has been reported for the first time from in situ chemically generated unstable o-quinone-monoimine heterodienes, using diacetoxy iodobenzene as the oxidizing reagent, in the presence of vinyl ether dienophiles. 11 Inspired by this protocol, we looked for a chemical extrapolation of our previous electrochemical procedure.

Activated manganese dioxide is known as a mild oxidant in 50 organic chemistry. Especially, it has been successfully used in combination with a nucleophilic trapping agent in tandem oxidation processes (TOP) for the synthesis of diverse heterocycles. 12 As an extension of this chemistry, we report herein that a stoichiometric amount of activated MnO 2 , in 55 deaerated methanol, is sufficient to convert various oaminophenol derivatives, in the presence of enamine dienophiles, to highly functionalized 1,4-benzoxazine derivatives through a tandem oxidation-IEDDA reaction under mild conditions. The synthetic potential of this reaction has been further explored with 60 the ultimate goal of developing libraries of biologically relevant 1,4-benzoxazine derivatives.

Results and Discussion

Choice of the optimal reaction conditions

Our studies began by examining the oxidation of o-aminophenol 1a in deaerated methanol, in the presence of enamine 2a. One equivalent of 1a and 1.5 equiv of enamine 2a, together with a stoichiometric amount of MnO 2 as the oxidant, were a good reagent combination for the reaction. However, the reaction progressed slowly (entry 1, Table 1), leading after 6h to 46% of 1,4-benzoxazine 3a as a single regioisomer. As previously observed from electrochemical studies, 7 the basicity of the enamine dienophile 2a was not sufficient to produce significant amounts of the phenolate anion of 1a, that is the sole form that can be oxidized to the o-quinone-monoimine species. Accordingly, when morpholine was added to the bulk solution as the ancillary base (entries 2 and 3), the reaction could go to completion by acid-base equilibrium displacement. Using 1.5 equiv of morpholine, the yield of 3a reached 75% in only 3h (entry 3). Various temperatures and solvents were further screened (entries 4-7), but none of the changes led to an improvement of the yield of the cycloadduct 3a. As already observed, 7 strong solvation of methanol to the o-quinonemonoimine species may be required to enhance the electrophilicity of its quinonoid moiety, making it able to participate in IEDDA reaction. a Reactions were performed with 1a (0.5 mmol), 2a (0.75 mmol, 1.5 equiv) and MnO2 (0.5 mmol, 1 equiv), in 25 mL of deaerated solvent. b Yields refer to chromatographically pure isolated products.

Scope of the tandem oxidation-IEDDA process.

The optimized reaction conditions (Table 1, entry 3) were first applied to a series of aminophenol derivatives 1a-i, in the presence of alkylenamine 2a. Table 2 shows some examples of the molecular diversity that can be obtained with this reaction sequence that includes a regiospecific IEDDA reaction between the in situ generated electron-poor o-quinone-monoimine heterodiene and the electron-rich tertiary alkylenamine dienophile 2a.

40 a Reactions were performed with 1a-i (0.5 mmol), 2a (0.75 mmol, 1.5 equiv) and MnO2 (0.5 mmol, 1 equiv) in 25 mL of deaerated MeOH, at 45 room temperature, for 3h. 1.5 Equiv of morpholine was added to the bulk solution for generating the phenolate ion of 1a-i. b Yields refer to chromatographically pure isolated products.

The more electron-rich carbon atom of enamine dienophile 2a added to the nitrogen atom of the o-quinone-monoimine heterodiene. In all cases (Table 2, entries 1-9), the method was successful leading to the 1,4-benzoxazine derivatives in acceptable yields ranging from 58 to 75%. Unexpectedly, oaminophenol 1i (entry 9) proved to be as reactive as electron-poor aminophenol derivatives 1a-f. This result could indicate that the electron-rich enamine 2a was the more active participant in the IEDDA reaction. However, when electron-rich o-aminophenol bearing methyl or methoxy groups were oxidized in the presence of enamine 2a no identifiable products were obtained under the same experimental conditions. In a second series of experiments, diverse enamine dienophiles 2a-i were screened in the presence of aminophenol 1a (Table 3). As earlier shown, the use of 3,3-disubstituted enamines 2 was required for the preparation of 1,4-benzoxazine derivatives in acceptable yields. 13 As expected, tertiary alkylenamines 2a-c with a pronounced electron-rich character produced the desired cycloadduct in high yield (entries 1-3), whereas introduction of phenyl substituents resulted in lower yields (entries 4 and 5). The cycloaddition reaction could also be extended to less stable secondary enamines 2f-h and afforded the 1,4-benzoxazine derivatives 3n-p (entries 6-8) without further elimination of the 2-alkylamino chain, in contrast to what had been previously reported for similar cycloaddition reactions of enamines with heterodienes. 14 Despite of its aromaticity, commercially available 2,3-dihydro-1H-cyclopent [b]indole 2i with latent enamine functionality could also participate in the IEDDA reaction giving the indolobenzoxazine ring system in 30% yield (entry 9).

In vivo biological evaluation of compound 3n.

In a previous work, we had shown that introduction of 3-phenyl substituents was crucial for efficient neuroprotective activity without the manifestation of intrinsic toxicity using in vitro assays on murine HT-22 hippocampal cell cultures. From these results, we had identified a potential candidate A (Figure 2) that proved to be active in vivo in newborn mice. 8 Brain lesions induced in newborn mice by the glutamatergic agonist Sbromowillardiine acting on kainate receptors mimic some aspects of white matter cysts and transcortical necrosis observed in human perinatal brain damage, especially in the case of cerebral palsy. 15 Our present investigation into the substrate scope led us to select another candidate 3n. The neuroprotective effects of benzoxazine 3n were assessed and compared with those of compound A, together with reference benzoxazines B and C, two established neuroprotective agents both in vitro and in vivo. 16 Cotreatment with 3n protected both the white matter and the cortical plate against the insult (Figure 2). Furthermore, both doses (1 and 10 mg/kg) of A, B and 3n were neuroprotective, while only the highest dose (10 mg/kg) of C yielded a significant protection in this model. Finally, in newborn mice, 3,3diphenylsubstituted 1,4-benzoxazine 3n proved to be more potent than benzoxazines A-C, making it a promising candidate as neuroprotective agent. PBS: control animals injected with S-bromo-willardiine into the cortex and white matter and i.p. with physiological saline; all the other experimental groups were cotreated with S-bromo-willardiine and the selected drug at the doses indicated in mg/kg, i.p. 10

Conclusions

We have reported a successful use of in situ generated monoimine heterodienes for the regiospecific IEDDA reaction with enamine dienophiles. The possibility of introd variations in both cycloaddition partners led to highly substituted 15 1,4-benzoxazine cycloadducts with up to five elements of diversity. The reactions proceeded under mild conditions temperature, with a good efficiency. The low toxicity, low and ease of handling stoichiometric amount of activated MnO combined with its commercial availability, are more attractive 20 than the previously reported electrochemical conditions for the synthesis of derivatives at a preparative scale. By means of the current protocol, 3,3-diphenyl-substituted-1,4-benzoxazine was identified as an effective neuroprotective agent in newborn mice, suggesting that it could be a potential candidate for the willardiine into the cortex and white matter and i.p. with physiological saline; all the other ardiine and the generated o-quinonemonoimine heterodienes for the regiospecific IEDDA reaction with enamine dienophiles. The possibility of introducing variations in both cycloaddition partners led to highly substituted benzoxazine cycloadducts with up to five elements of diversity. The reactions proceeded under mild conditions, at room with a good efficiency. The low toxicity, low cost, chiometric amount of activated MnO 2 , more attractive than the previously reported electrochemical conditions for the synthesis of derivatives at a preparative scale. By means of the benzoxazine 3n gent in newborn mice, suggesting that it could be a potential candidate for the treatment and prevention of cerebral palsy, together with probably other neurodegenerative disorders involving High-resolution mass spectra (HRMS) were Bruker maXis mass spectrometer operating in positive ion mode 50 by the "Fédération de Recherche" ICOA/CBM (FR2708) platform.

Chemicals were commercial products of the highest available purity and were used as supplied. 2-Aminophenol 60 dihydro-1H-cyclopent [b]indole 2i were commercially available compounds. Substituted o-aminophenol starting materials were synthesized through previously reported procedures. Enamine dienophiles 2a-h were prepared by standard methods from a mixture of aldehyde, appropriate amine (slight excess) and 65 toluene-p-sulfonic acid (catalytic amount) heated at reflux, in benzene, for 18h, the water being continuously removed by means of a Dean-Stark separator.

General procedure for the tandem oxidation of o-aminophenol derivatives 1a-i with enamines 2a

A solution of enamine (0.75 mmol, 1.5 equiv) in deaerated methanol (5 mL) was added to a solution derivative (0.5 mmol, 1 equiv) in deaerated methanol (20 mL) containing activated MnO 2 (43.5 mg, 0.5 mmol, 1 equiv) and morpholine (66 µL, 0.75 mmol, 1.5 equiv). The resulting mixture 75 was stirred at room temperature for 3h, filtered off, and the filtrate was evaporated under reduced pressure at 30°C. The residue was purified by column chromatography (toluene/acetone 95/5 v/v) on silica gel to afford the desired 1,4 derivative.

80

The above procedure is generally representative for all the products shown in Tables 2 and3. Any deviations from this 75 protocol are specified in the footnotes of the tables.

[(R,S)-3,3-diethyl-5-hydroxy-2-morpholino 1,4-benzoxazin-6-yl](methyl) methanone (3a

Pale yellow solid (125 mg, 75%): mp 138 85 (toluene/acetone 98/2 v/v); 1 H NMR (300 MHz, CDCl (t, J = 7.4 Hz, 3H), 0.97 (t, J=7.4 Hz, 3H), 1.40 2.59 (m, 5H), 3.41 (m, 2H), 3.70 (m, 4H), 4.13 (s, 1H), 4.57 (s, 1H), 6.45 (d, J= 8.8 Hz, 1H), 7.16 (d, J= 8.8 Hz, 1H), 12.83 (s, 1H); 13 C{ 1 H} NMR (75 MHz, CDCl 3 ) δ 6. 7, 7.7, 25.8, 26.2, 27.1, 90 49.1, 55. 4, 67.2, 95.7, 107.1, 113.3, 120.2, 120.8, 150.2, 151.3, 203.3 C NMR spectra were performed on a Bruker 300 spectrometer operating at 300 and 75 MHz, respectively.

units (part per million) downfield from TMS (tetramethylsilane). The measurements ied out using the standard pulse sequences. The carbon type (methyl, methylene, methine, or quaternary) was determined C NMR spectra of all compounds are included in the supporting information as a proof resolution mass spectra (HRMS) were performed on a operating in positive ion mode by the "Fédération de Recherche" ICOA/CBM (FR2708) Chemicals were commercial products of the highest available Aminophenol 1i and 2,3were commercially available aminophenol starting materials 1a-h d through previously reported procedures. 7c, 17 were prepared by standard methods from a mixture of aldehyde, appropriate amine (slight excess) and sulfonic acid (catalytic amount) heated at reflux, in he water being continuously removed by

andem oxidation-IEDDA reaction i with enamines 2a-i

A solution of enamine (0.75 mmol, 1.5 equiv) in deaerated was added to a solution of o-aminophenol derivative (0.5 mmol, 1 equiv) in deaerated methanol (20 mL) (43.5 mg, 0.5 mmol, 1 equiv) and L, 0.75 mmol, 1.5 equiv). The resulting mixture e for 3h, filtered off, and the filtrate was evaporated under reduced pressure at 30°C. The residue was purified by column chromatography (toluene/acetone 95/5 v/v) on silica gel to afford the desired 1,4-benzoxazine nerally representative for all the products shown in Tables 2 and3. Any deviations from this protocol are specified in the footnotes of the tables.

morpholino-3,4-dihydro-2H-3a)

mp 138-142°C; R f = 0.35 H NMR (300 MHz, CDCl 3 ) δ 0.82 Hz, 3H), 1.40-2.00 (m, 4H), 2.59 (m, 5H), 3.41 (m, 2H), 3.70 (m, 4H), 4.13 (s, 1H), 4.57 (s, 8.8 Hz, 1H), 12.83 (s, 6.7, 7.7, 25.8, 26.2, 27.1, 49.1, 55.4, 67.2, 95.7, 107.1, 113.3, 120.2, 120.8, 150.2, 151.3 7, 7.7, 25.87, 25.89, 25.92, 27.1, 29.7, 29.8, 44.9, 49.1, 55.4, 67.1, 95.6, 106.9, 111.9, 112.3, 119.9, 120.4, 149.8, 152.0, 209 7,7.7,22.8,25.83,25.88,27.1,46.8,49.1,55.4,67.2,95.7,106.9,113.3,120.26,120.33,150.0,151.6,205.5;Anal. calcd CDCl 3 ) δ 6.7, 7. 5,26.18,26.22,27.0,49.2,55.7,67.2,95.1,114.5,114.7,121.0,130.4,131.9,149.3,197.1;Anal. calcd 7.5, 26.4, 26.9, 49.1, 55.8, 67.1, 95.1, 103.5, 115.8, 117.7, 119.7 CDCl 3 ) δ 6.7, 7. 5, 26.1, 27.0, 49.2, 51.8, 55.7, 67.2, 95.0, 114.9, 116.2, 121.3, 122.5, 131.6, 148.9, 167.2 55.7,67.2,95.1,114.4,116.5,123.3,128.0,129.7,130.2,131.6,132.0,138.6,149.1,195.8;Anal. calcd 7, 7.6, 20.2, 26.2, 27.0, 28.6, 47.3, 49.2, 55.6, 67.2, 94.7, 114.56, 114.63, 116.8, 127.3, 132.1, 147.3, 167.6 8, 7.6, 26.2, 27.1, 49.3, 55.6, 67.3, 94.1, 115.0, 115.2, 118.9, 120.6, 131.9, 144.6 1,26.2,28.3,48.8,50.9,67.2,96.9,107.2,113.3,120.5,120.9,150.0,151.5,203.3;Anal. calcd 4H), 4.27 (s,1H),4.58 (s,1H),6.42 (d,J= 8.8 Hz,1H),7.13 (d,J= 8.8 Hz,1H),12.81 (s,1H); 13 C{ 1 H} NMR (75 MHz, CDCl 3 ) δ .3,21.5,25.4,26.1,34.2,34.7,48.9,52.0,67.1,96.3,107.0,113.2,120.0,120.7,150.0,151.4,203.1;Anal. calcd 1, 49.1, 63.3, 67.0, 94.7, 107.2, 113.4, 120.0, 121.8, 126.7, 126.8, 126.98, 127.02, 128.0, 128.4, 144.0, 145.8, 150.0, 151.9, 203.2 37.6,57.8,63.3,64.5,64.8,94.2,104.6,107.2,113.2,120.0,121.7,126.7,126.9,127.0,127.9,128.3,143.8,146.1,150.2,151.8,203.1;Anal. calcd 44.9, 58.6, 62.4, 72.6, 89.5, 109.0, 113.5, 120.6, 121.4, 126.6, 127.0, 127.1, 128.2, 128.4, 143.0, 144.3, 146.9, 151.5, 203.5 2, 43.7, 62.3, 87.5, 109.0, 113.6, 120.6, 121.5, 124.7, 124.9, 126.56, 126.62, 127.0, 127.1, 127.2, 128.3, 128.4, 142.8, 143.6, 144.1, 146.7, 151.6, 203.4 4,54.2,62.4,89.3,104.2,109.0,113.5,120.6,121.4,126.6,127.0,127.1,128.2,128.5,143.0,144.2,146.8,151.6,203.5;Anal. calcd 41.5,70.5,107.2,113.6,120.6,121.5,124.7,124.9,126.56,126.62,127.0,127.1,127.2,128.3,128.4,142.8,143.6,144.1,146.7,151.6,203.4;Anal. calcd

21

In vivo biological assay. Neonatal mice

At postnatal day 5, Swiss mouse pups were anesthetized with isoflurane for intracerebral (ic) and intraperitoneal (ip) injections. 15,18 Intracerebral injections were performed using a 26-80 gauge needle mounted on a calibrated microdispenser. The needle was inserted 2mm under the external surface of scalp skin in the frontoparietal area of the right hemisphere 2 mm from the midline in the lateral-medial plane and 3 mm (in the rostro-caudal plane) from the junction between sagittal and lambdoid sutures. Two 1 85 µl boluses were injected at a 30-s interval.

Fifteen µg S-bromo-willardiine (Tocris, Bristol, UK), diluted in phosphate buffer saline (PBS), were injected ic. Immediately following ic injection, 1 or 10 mg/kg compound C (n = 8 and 6, respectively), 1 or 10 mg/kg compound B (n = 6 and 15, 90 respectively), 0.1, 1 or 10 mg/kg compound A (n = 8, 7 and 9, respectively), and 1 or 10 mg/kg 3n (n = 10 in both groups) diluted in PBS containing 10% dimethylsulfoxide (DMSO), were administered ip. Controls received ip PBS containing 10% DMSO alone (n = 19).

95

Five days later, the surviving pups were sacrificed and brains fixed in formalin. Coronal serial sections, 15 µm thick, were cut and every third section was stained with cresyl-violet. Brain was completely and serially sectioned from the frontal pole to the occipital lobes. In theory, neocortical and white matter lesions can 100 be defined by the maximal length of three orthogonal axes: the lateral-medial axis (in a coronal plane), the radial axis (also in a coronal plane, from the pial surface to the lateral ventricle) and the fronto-occipital axis (in a sagittal plane). Due to the difficulty of accurately evaluating the degree of damage to neurons in neocortical 105 layers in the epicenter of the lesion focus, the radial axis did not appear as an objective measure of the lesion size. In previous studies, 15,18 we had shown an excellent correlation between the maximal size of the radial and fronto-occipital diameters of the excitotoxic lesions. Based on these observations, we serially 110 sectioned the entire brain in the coronal plane. This permitted an accurate and reproducible determination of the maximal sagittal

65 70Fig. 1

 651 Fig.1Example of important pharmaceutically active compounds possessing the 1,4-benzoxazine core.

a 4 |Fig. 2

 42 Fig. 2 In vivo biological evaluation in newborn mice. The histograms represent the mean length of the neocortical lesion in the sagittal fronto occipital axis ± S.E.M. Asterisks indicate difference from control (* 5

  25 treatment and prevention of cerebral palsy, together with probably other neurodegenerative disorders involving excitotoxicity as a pathomechanism. This journal is © The Royal Society of Chemistry [year] newborn mice. The histograms represent the mean length of the neocortical lesion in the sagittal fronto-S.E.M. Asterisks indicate difference from control (* 0.05, ** P<0.01 in ANOVA with Dunnet's multiple comparison test).

  thin-layer chromatography was carried out on silica 35 gel Macherey-Nagel Polygram SIL G/UV Column chromatography was performed on Macherey 60 M silica gel (40-63 µm). Melting points were measured on a Köfler 1 H NMR and 13 C NMR spectra were performed on a Br 45 AC-300 spectrometer operating at 300 and 75 MHz, respectively. Chemical shifts are expressed as δ units (part per million) downfield from TMS (tetramethylsilane). The measurements were carried out using the standard pulse sequences. The carbon type (methyl, methylene, methine, or quaternary) was determined 50 by DEPT experiments. 1 H and 13 C NMR spectra of all compounds are included in the supporting information as a proof of their identity.

  layer chromatography was carried out on silicaPolygram SIL G/UV 254 (0.25 mm). Column chromatography was performed on Macherey-Nagel Si Melting points were measured on a Köfler apparatus.

105[

  (R,S)-5-hydroxy-2-morpholino-3-spiro-1'-cyclohexyl-3,4dihydro-2H-1,4-benzoxazin-6-yl](methyl)methanone (3k) Pale yellow solid (144 mg, 83%): mp 68-72°C; R f = 0.45 (toluene/acetone 95/5 v/v); 1 H NMR (300 MHz, CDCl 3 ) δ 1.30-1.90 (m, 10H), 2.56 (s, 3H), 2.58 (m, 2H), 3.39 (m, 2H), 3.66 (m, 110 6 | Journal Name, [year], [vol], 00-00 This journal is © The Royal Society of Chemistry [year]

Table 1

 1 Optimization of the reaction conditions a

Table 2

 2 Variation of the diene part: tandem oxidation-IEDDA reaction of diverse aminophenol derivatives

1a-i with enamine 2a a

	Entry	Substrate 1	Product 3	Yield
				(%) b
	1			75
	2			75
	3			74
	4			68
	5			68
	6			62
	7			60
	8			58
	9			62

Table 3

 3 Variation of the dienophile part: tandem oxidation-IEDDA reaction of aminophenol 1a with different enamines 2a-i a

  for C 21 H 32 N 2 O

		81
		(t, J = 7.4 Hz, 3H), 0.93 (t, J=7.4 Hz, 3H), 1.51-1.61 (m, 3H),
	30	1.70-1.90 (m, 1H), 2.53 (s, 3H), 2.58 (m, 2H), 3.35 (m, 2H), 3.65
		(m, 4H), 3.83 (s, 1H), 4.52 (s, 1H), 6.83 (d, J= 8.3 Hz, 1H), 7.22
		(s, 1H), 7.33 (d, J= 8.3 Hz, 1H); 13 C{ 1 H} NMR (75 MHz,

4 : C, 67.02; H, 8.51,N, 7.45. Found: C, 66.90; H, 8.54,N, 7.40

.

[(R,S)-3,3-diethyl-2-morpholino-3,4-dihydro-2H-1,4-

benzoxazin-6-yl](methyl)methanone (3d)

White solid (108 mg, 68%): mp 134-138°C; R f = 0.25 (toluene/acetone 95/5 v/v); 1 H NMR (300 MHz, CDCl 3 ) δ 0.

,S)-3,3-diethyl-2-morpholino-3,4-dihydro-2H-1,4- benzoxazin-6-yl]carbonitrile (3e)

		for
	35	C 18 H 26 N 2 O 3 : C, 67.92; H, 8.18, N, 8.80. Found: C, 67.81; H,
		8.21, N, 8.77.
	40	[(RWhite solid (103 mg, 68%): mp 156-160°C; R f = 0.35 (toluene/acetone 95/5 v/v); 1 H NMR (300 MHz, CDCl 3 ) δ 0.81
		(t, J = 7.4 Hz, 3H), 0.94 (t, J=7.4 Hz, 3H), 1.50-1.70 (m, 3H),
		1.70-1.90 (m, 1H), 2.59 (m, 2H), 3.31 (m, 2H), 3.66 (m, 4H),
		3.85 (s, 1H), 4.53 (s, 1H), 6.78 (s, 1H), 6.83 (d, J= 8.3 Hz, 1H), 6.99 (d, J= 8.3 Hz, 1H); 13 C{ 1 H} NMR (75 MHz, CDCl 3 ) δ 6.7,

  ; Anal. calcd for C 18 H 26 N 2 O 4 : C, 64.67;H, 7.78,N, 8.38. Found: C, 64.70; H, 7.80,N, 8.38.

	60	[(R,S)-3,3-diethyl-2-morpholino-3,4-dihydro-2H-1,4-
		benzoxazin-6-yl](phenyl)methanone (3g)
		Yellow solid (114 mg, 60%): mp 150-154°C; R f = 0.35 (toluene/acetone 95/5 v/v); 1 H NMR (300 MHz, CDCl 3 ) δ 0.83
		(t, J = 7.4 Hz, 3H), 0.94 (t, J=7.4 Hz, 3H), 1.50-1.7 (m, 3H),
	65	1.82 (m, 1H), 2.61 (m, 2H), 3.38 (m, 2H), 3.68 (m, 4H), 3.83 (s,
		1H), 4.54 (s, 1H), 6.83 (d, J= 8.1 Hz, 1H), 7.15 (d, J= 8.2 Hz,
		1H), 7.17 (s, 1H), 7.48 (m, 2H), 7.57 (m, 1H), 7.77 (d, J= 7.5 Hz, 2H); 13 C{ 1 H} NMR (75 MHz, CDCl 3 ) δ 6.7, 7.6, 26.3, 27.0, 49.2,

  for C 16 H 22 N 2 O

4 : C, 62.74; H, 7.19,N, 9.15. Found: C, 62.47; H, 7.21,N, 9.11.

  ; Anal. calcd for C 27 H 24 N 2 O

		(toluene/acetone 95/5 v/v); 1 H NMR (300 MHz, CDCl 3 ) δ 2.40
		(broad s, 1H), 2.53 (s, 3H), 2.85-3.10 (m, 2H), 3.22 (s, 3H), 3.24
		(s, 3H), 4.32 (t, J= 5.7 Hz, 1H), 5.15 (s, 1H), 5.77 (s, 1H), 6.37
	60	(d, J= 8.8 Hz, 1H), 7.11 (d, J= 8.8 Hz, 1H), 7.15-7.55 (m, 10H), 12.98 (s, 1H); 13 C{ 1 H} NMR (75 MHz, CDCl 3 ) δ 26.2, 46.9,
		53.
	55

3 S: C, 71.05; H,

5.26,N, 6.14. Found: C, 50

70.83; H, 5.27, N, 6.12.

[(R,S)-2-[(2,2-dimethoxy)ethylamino]-3,3-diphenyl-5hydroxy-3,4-dihydro-2H-1,4-benzoxazin-6-yl](methyl) methanone (3p)

Pale yellow solid (117 mg, 52%): mp 64-68°C; R f = 0.3

  fronto-occipital diameter (which is equal to the number of sections where the lesion was present multiplied by 15 µm) and was as an index of the volume of the lesion. Statistical analyses were performed by analysis of variance (ANOVA) with Dunnet's multiple comparison of means test. Results were expressed as means ± S.E.M. Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie de Paris, 4 avenue de l'Observatoire, 75270 Paris cedex 06, France. Fax: +33144073588; Tel: +33153739646; E-mail: martine.largeron@parisdescartes.fr b Inserm, U 1141, F-75019 Paris, France c Université Paris Diderot, Sorbonne Paris Cité,UMRS 1141, F-75019 Paris, France †Electronic Supplementary Information (ESI) available: 1 H and 13 C NMR spectra for benzoxazine derivatives 3a-q. See DOI: 10.1039/b000000x/ 1 For recent reviews on biologically active 1,4-benzoxazine 20 derivatives, see: (a) B. Achari, S. B. Mandal, P. K. Dutta, C. Chowdhury, Synlett, 2004, 14, 2449 and references therein; (b) F. A. Macias, D. Marin, A. Oliveros-Bastidas, J. M. G. Molinillo, Nat. Prod. Rep. 2009, 26, 478. 2 For selected recent examples, see: (a) P. Stefanic Anderluh, M.

This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00-00 | 7 a UMR 8638 CNRS-

Organic & Biomolecular Chemistry Accepted Manuscript Published on 03 February 2015. Downloaded by BIBLIOTHEQUE INTERUNIVERSITAIRE DE PHARMACIE on 03/02/2015 10:39:46. View Article Online DOI: 10.1039/C5OB00049A

Organic & Biomolecular Chemistry Organic & Biomolecular Chemistry Accepted Manuscript Published on 03 February 2015. Downloaded by BIBLIOTHEQUE INTERUNIVERSITAIRE DE PHARMACIE on 03/02/2015 10:39:46. View Article Online DOI: 10.1039/C5OB00049A

Acknowledgements

We thank CNRS, Inserm together with Paris Descartes and Paris Diderot Universities for financial support.

Notes and references


M Kepaptsoglou

Q Lazzeri

Ramasse

F S Hage

G Radtke

D M Kepaptsoglou

M Lazzeri

Q M Ramasse
email: qmramasse@superstem.org

Single Atom Vibrational Spectroscopy in the Scanning Transmission Electron Microscope

Main Text

Changes in the normal mode frequencies of dynamical systems arising from the presence of impurities have been studied as far back as the 19 th century, resulting in the set of classical theorems now referred to as the Rayleigh Theorems (1,[START_REF] Strutt | The Theory of Sound[END_REF]. However, the modern theory of defect modes in crystals was established in the 1940s with the pioneering work of Lifschitz [START_REF] Lifšic | [END_REF]. A wealth of studies followed, mainly based on optical spectroscopies (4), which identified two types of non-trivial defect-induced modes known as localized and resonant modes. Resonant modes are also called quasi-or pseudo-localized modes because, despite being spatially extended, they involve a large amplitude vibration of the impurity itself. In turn, defect modes can control materials' properties such as electric and heat transport or more generally processes that are affected by the scattering of electrons or phonons. This can be exploited for instance to suppress heat propagation in thermoelectrics using rattler modes (5), to tune the superconductivity in 2-dimensional films (6) or the optoelectronic properties of conducting polymers (7). Although the existence of an atomically localized spectroscopic signature of single-atom defects has long been discussed [START_REF] Wallis | Localized Excitations in Solids[END_REF] conventional vibrational spectroscopies typically average information over much larger lengthscales.

Vibrational electron energy loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) has recently emerged as a powerful means of probing the vibrational response of materials at a spatial resolution superior to other experimental techniques [START_REF] Krivanek | [END_REF]10). Tip-enhanced Raman spectroscopy (TERS) (11) or inelastic electron tunneling spectroscopy (IETS) (12,13) provide high spatial and energy resolution alternatives, but they are strictly limited to surface experiments and therefore present challenges for a range of applications. Vibrational STEM-EELS on the other hand takes advantage of versatile probe-forming optics to offer ground-breaking capabilities: nanometer-scale thermometry (14), mapping of bulk and surface-phonon-polariton modes (15), establishing phonon dispersion diagrams from nanoobjects (16), site-specific isotopic labeling in molecular aggregates (17). These reports highlight the complementarity of STEM-EELS with conventional vibrational spectroscopies whose energy resolution remains unmatched. However, the ultimate promise of vibrational STEM-EELS is to reach the single atom or molecular level, in the same way that modern microscopes enabled electronic structure analysis (18), plasmonic (19) and UV-optical response fingerprinting (20), or energy-dispersive X-ray spectroscopy (21) from single atoms. Atomically resolved phonon maps of bulk systems are preliminary steps in this direction (22).

Here, we use STEM-EELS to measure the localized vibrational signature of a single trivalent substitutional Si atom in single-layer graphene (Si@Gr). From ab initio simulations we attribute the measured atomic-scale spectroscopic response to scattering by pseudo-localized vibrational modes arising from a resonance between the Si impurity-specific modes and the bulk continuum.

Figure 1A illustrates how electron beam deflectors are adjusted to displace the EEL spectrometer entrance aperture by 69 mrad (or a 8.87 Å -1 momentum transfer) with respect to the bright field (BF) disc, so that these no longer overlap. Further details of the experimental geometry are provided in the Supplementary Materials (SM), fig. S1. Compared to a conventional on-axis geometry where the EELS aperture is centered on the BF disc, this off-axis or dark-field EELS geometry significantly suppresses the relative contributions of electrons having undergone elastic and delocalized phonon scattering, favoring instead highly localized impact phonon scattering (23). This approach makes it possible to record atomic resolution phonon scattering maps of nmthick flakes of hexagonal boron nitride (22) or of single-layer graphene (fig. S2 in the SM), where the off-axis geometry is key since the on-axis EELS phonon response of graphene is vanishingly small (24). Note that the large beam convergence necessary for an atomic-size probe results in spectral integration over a range of momentum transfer in the sample plane. To achieve a signal-to-noise ratio sufficient for resolving the phonon loss spectrum fine structure, the electron beam is scanned repeatedly over a small window tightly defined around the impurity of interest while the spectrum intensity is accumulated (25).

Figure 1B shows a dark-field EEL spectrum from a single Si atom impurity in graphene ("Si", red) alongside that acquired from a comparably sized region of pristine graphene ("C", blue), located a mere few atoms away from the Si impurity. The relative positions of the two scanned regions are indicated by red (Si) and blue (C) boxes on the high-angle annular-dark-field (HAADF) image in fig. 1C. A close-up of the probed Si atom (fig. 1D) and the corresponding fine structure of the Si L 2,3 ionization edge (fig. S3C) confirm that the brighter contrast Si atom is trivalently substituted into the graphene lattice. Asymmetric annular-dark-field movies (aADF, thus denoted due to the off-axis geometry) were recorded during spectrum acquisition to monitor possible beam-induced structure modifications, while ensuring the probed atom remained centered within the scanned region. Averaged aADF movies are shown as insets in fig. 1B, with individual frames shown in the SM.

The Si and C spectra in fig. 1B are normalized to the maximum of their respective zero-loss peaks (ZLPs). As a result the tails of the ZLPs closely overlap immediately before the first observable loss features, allowing for a straightforward visual comparison of relative changes in energy loss due to inelastic scattering by phonons. Any change in spectrum intensity above the coinciding ZLP tails should be representative of differences in relative phonon scattering probability. The fine structure in the phonon energy range of the two recorded spectra is strikingly different. While the C spectrum is consistent with that of non-doped bulk graphene (24), the Si spectrum comprises phonon loss features at different energies.

Figure 1E shows in greater detail the phonon loss region of the spectra. The C spectrum exhibits two distinct loss peaks at 85 meV (685 cm -1 ) and 170 meV (1371 cm -1 ). Following Ref. (24), we assign these peaks to scattering by transverse (T) and longitudinal (L), acoustic (A) or optical (O) modes in graphene, respectively (the graphene phonon dispersion diagram is presented for reference in fig. S10). Spectral contributions of out-of-plane phonon modes are expected to be negligible as the incident electron beam is normal to the graphene plane. In spite of stemming from a position only a few atoms away, the Si spectrum shows a remarkably different phonon fine structure comprising a prominent loss peak at about 55 meV (443 cm -1 ) and weaker structures at 125 and 150 meV (1008 and 1209 cm -1 ). To enhance the differences between the spectra, we subtracted the C from the Si spectrum: the "difference spectrum" is shown in fig. 1E-F. This process has the additional benefit of effectively subtracting the elastic scattering ZLP tail (making the reasonable assumption that the tail contribution, before any expected loss contribution, is similar between spectra) without possible errors associated with common background removal techniques, as discussed in the SM (fig. S4). The difference can thus be interpreted as a relative change in phonon scattering probability induced by the presence of the single Si atom impurity. Virtually identical results (in SM) were obtained from complementary measurements carried out in a different area of the sample. These experimental results lead to the remarkable conclusion that the single Si atom impurity in graphene possesses a characteristic vibrational signature localized at the atomic scale.

To gain insights into the physics associated with these results, we have calculated within the framework of density functional theory (DFT) (26) the vibrational spectrum of a large 96×96 supercell of graphene containing one substitutional Si atom, using periodic boundary conditions. The structure of the defect and computational details are presented in the SM. As discussed therein, the important features observed in the vibrational EEL spectra of graphene can safely be interpreted in terms of the phonon density of states (DOS) of the bulk. The local behavior of the DOS can be quantified by the projected phonon DOS (PPDOS) defined as

( ) = | | (
), where denotes a specific atom, and are the phonon angular frequency and normalized polarization, and the sum is carried over all the phonon modes of the supercell. Since in our experiments the momentum transfer occurs predominantly in the plane perpendicular to the electron beam trajectory, only the components of the phonon polarization parallel to the graphene plane are relevant. A tentative comparison to the experimental difference spectrum is then provided by combining the PPDOS projected on the Si atom , on its three carbon neighbors , and the bulk phonon DOS per atom : ( ) = ( ) + 3 ( ) 4 ( ) /4. This differential PPDOS reflects the experimental spectrum averaging over the scanning window, which is expected to include contributions from the impurity's neighboring C atoms. The resulting differential PPDOS is shown in fig. 1F, after broadening to match the experimental resolution: it predicts all the main features of the experimental difference spectrum, including: a single peak at ~55 meV (443 cm -1 ), two overlapping peaks at 125 meV (1008 cm -1 ) and 150 meV (1209 cm -1 ), and "dips" centered around 100 meV (807 cm -1 ) and 180 meV (1452 cm -1 ).

The physical origin of these spectral features can be understood by considering the individual inplane PPDOS employed to construct the differential PPDOS and those of carbon atoms at increasing distances away from the Si impurity: fig. 2A. The Si PPDOS is dominated by an intense peak at 55 meV, matching closely the low-energy experimental feature seen in fig. 1E. This peak is followed by a broad band with weaker structures at 105, 127 and 155 meV (847, 1024 and 1250 cm -1 ).

The absence of intense features in the bulk graphene DOS at 55 meV implies that the corresponding modes should possess a degree of localization. Indeed, by inspecting the PPDOS of neighboring carbon atoms in fig. 2, it is evident that whereas the first two carbon neighbor shells coordinating the impurity still display traces of the 55 meV peak, its contribution is weak and the PPDOS of subsequent neighbors rapidly tend to the bulk signature, which is fully retrieved after 6 shells. Corresponding atomically resolved experimental spectra in fig. 2C, from a full spectrum image over equivalent carbon neighbor positions (fig. S8), exhibit an identical trend: the EELS signal reproduces the main features observed in the in-plane PPDOS.

It is instructive to consider a calculation performed on a smaller 13-atom fragment of C 3v symmetry centered on the impurity (fig. S9), decoupled from the supercell by artificially setting the interatomic force constants linking the fragment to the rest of the 96×96 supercell to zero.

The fragment displays two modes with E symmetry at 52 and 124 meV (419 and 1000 cm -1 ) involving large in-plane displacements of the Si, in-phase (mode A) or out-of-phase (mode B) with the neighboring carbon atoms: fig. 3. The resonances in the full Si@Gr system, simulated by the 96×96 supercell, can thus be interpreted as a hybridization of these local impurity modes with the vibrational continuum of the graphene bulk.

The associated atomic displacements, including those arising from the in-plane vibration of the silicon atom, do not decay far from the defect: the full system presents a delocalized continuum, a concept quantified with the inverse participation ratio analysis shown in the SM. However, these delocalized phonons modes possess an enhanced component atomically localized on the impurity: the power of EELS is the technique's ability to probe this quasi-localization thus revealing the paradoxical nature of defect-induced resonant modes. It is also remarkable that the experimentally measured ~30 meV (242 cm -1 ) full-width at half-maximum (FWHM) of the impurity peak at ~55 meV (fig. 1F) matches closely the intrinsic theory-predicted width of the quasi-resonant mode: fig. 3A. The experimental energy resolution is therefore not limiting and the EEL spectra capture faithfully the fine structure of the Si@Gr system's vibrational response.

Localized and resonant modes arising from point defects have been widely discussed [START_REF] Wallis | Localized Excitations in Solids[END_REF]. The former are characterized by frequencies lying out of the continuum of the unperturbed crystal and atomic amplitudes dying off faster-than-exponentially with increasing distance from the defect (27). By contrast, the latter occur at frequencies lying within the allowed bands of the host. Their recognition was delayed by their peculiar characteristics whereby the vibrational amplitude does not vanish far from the defect, extending instead over the entire crystal (28). Furthermore, experimental observations of these effects have thus far been limited to indirect fingerprints, often at the macroscopic scale. Volgmann et al. (13) used SPM-IETS to detect a local energy-dependent increase in phonon DOS on a Ag (111) surface, which they attributed to a substitutional Cu atom. But the surface nature of these experiments and the lack of more direct visualization means precluded an unambiguous interpretation.

In contrast, the ability demonstrated here to measure directly at the atomic scale the localized component of the vibrational signature of a single impurity atom within a solid and to match the observed spectral fine structure to theoretically predicted modes truly realizes the potential of phonon spectroscopy in the STEM. Single-atom defect sensitivity combined with isotope selectivity (17) and the ability to operate at cryogenic temperatures (29) marks STEM-EELS as a potentially unique technique for molecular chemistry, enabling experiments where a single functionalizing isotope is fingerprinted at the atomic scale through its vibrational signature. The approach should be applicable to three-dimensional structures, although challenges will arise from the complexity of the computational work necessary to inform these experiments. Nevertheless, the path to further tantalizing applications in solid state science opens up whereby the electron beam of the STEM is used both to assemble functional devices atom by atom (30) and to probe spectroscopically the resulting lattice dynamics and their coupling with other quasi-

Fig. 1 .

 1 Fig. 1. Experimental geometry and vibrational STEM-EEL spectrum of a Si impurity in graphene. (A) Beam deflectors shift the bright-field (BF) disc away from the EEL spectrometer entrance aperture in the diffraction plane. (B) Normalized vibrational EEL spectra of a substitutional Si impurity and of defect-free graphene. Insets show aADF images of the repeatedly scanned sample regions. Smoothed spectra (thin lines) are superimposed on the raw data (transparent thick lines). (C) HAADF overview of the experimental region. A red (blue) box indicates the position of the sub-scan region from which the Si (C) spectrum was acquired. (D) HAADF close-up of the (bright) trivalent Si impurity. (E) Detail of the normalized Si and C EEL spectra shown in B and the difference spectrum. (F) Comparison of the calculated differential PPDOS (broadened to match the experimental resolution) and experimental difference spectrum. The blue and red shaded areas highlight energy ranges where the contributions of the Si impurity and its three nearest neighbors, or of bulk graphene, are comparatively stronger.

Fig. 2 .

 2 Fig. 2. Localization of the vibrational signal. (A) Black lines: calculated in-plane component of the phonon density of states projected on the Si and carbon atoms at increasing distances from the impurity. Gray lines: bulk graphene phonon density of states per atom, fully recovered from atom 6. The curves are vertically shifted and smeared by a 2 meV FWHM Lorentzian for clarity.

Fig. 3 .

 3 Fig. 3. Localized components of the Si vibrations. (A) Gray bar-histogram: square of the phonon eigenmodes of the 96×96 supercell projected on the in-plane Si atom component. Black line: in-plane component of the phonon DOS projected on the Si atom (same as the "Si" PPDOS in fig. 2) obtained by broadening the gray bar-histogram with a 2.0 meV FWHM Lorentzian. The red bar-histogram is calculated from a 13-atom fragment centered on the impurity. Two dominant modes, denoted A and B, are observed. (B) Atomic model of the 13-atom fragment (the Si atom is in red), with relative atomic displacements for modes A and B indicated as arrows whose length is proportional to the displacement amplitude.

particles.

Acknowledgements

The authors gratefully acknowledge Prof. Rik Brydson and Drs. Matthieu Bugnet and Eric Prestat for useful discussions. Funding: SuperSTEM is the U.K. National Research Facility for Advanced Electron Microscopy, supported by EPSRC. This work was granted access to the High-Performance Computing resources of the Institut du Développement et des Ressources en Informatique Scientifique (IDRIS) under allocation 2019-A0060910820 attributed by the Grand Equipement National de Calcul Intensif (GENCI). Author contributions: QMR conceived the project. FSH, QMR and DMK designed and carried out the experiments and interpreted the data. GR and ML carried out and interpreted the theoretical calculations. All authors contributed to the preparation of the manuscript. Competing interests: The authors declare no competing interests.

Data and materials availability: All data necessary for evaluating the conclusions of the paper are included in the main text and/or Supplementary Materials. Data related to that presented here is available from the authors upon reasonable request.

Supplementary Materials

Materials and Methods

Supplementary Text Figures S1-S12

References (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)


Anthony Bonnamy

Alexandre Marciniak
email: alexandre.marciniak@irap.omp.eu

Gabi Wenzel

Sébastien Zamith

Sabela Quiroga

Diego Peña

Giacomo Mulas

Christine Joblin

VUV photo processing of large cationic PAHs in astrophysical conditions: coupling a VUV source to the PIRENEA setup

Bibliography Study as a function of the VUV irradiation time

TD-DFT calculations

Models from [4] Implications in VUV photo-processing of PAHs • The absolute VUV photo-absorption cross-section of C 78 H 26 + is about 3.25 times larger than the one of C 24 H 12 + (ratio of C atom number).

• A simple model for the photo-ionization yield of large PAH has been derived from measurements in a previous study [4] : ~0.23 at 10. ➢ The retrieved dissociation yield at a VUV energy close to the threshold is consistent with the results found by Zhen and coworkers [6].

➢ This shows that a large fraction of the 10.5 eV photons absorbed by C24H12+ are relaxed by radiative cooling.