Applied Biological and Physicochemical Activity of Isoquinoline Alkaloids: Oxoisoaporphine and Boldine

Molecules 2012, 17, 10958-10970; doi:10.3390/molecules170910958 molecules ISSN 1420-3049 www.mdpi.com/jou nal/molecules A icle Applied Biological and Physicochemical Ac i i y o Isoquinoline Alkaloids: Oxoisoapo phine and Boldine Edua do Soba zo-Sánchez 1,*, Pa icio González So o 2, C is óbal Valdés Ri e a 2, Geo gina Sánchez 3 and Ma ía Eliana Hidalgo 2,* 1 Depa men o Pha macy and Pha maceu ical Technology, Facul y o Pha macy, Uni e si y o San iago de Compos ela, San iago de Compos ela 15782, Spain 2 Facul y o Sciences, Uni e si y o Valpa aíso, Valpa aíso 33449, Chile 3 Facul y o Pha macy, Uni e si y o Valpa aíso, Valpa aíso 33449, Chile * Au ho s o whom co espondence should be add essed; E-Mails: [email p o ec ed] (E.S.-S.); [email p o ec ed] (M.E.H.); Tel.: +34-881-814-887 (E.S.-S.); Fax: +34-981-547-148 (E.S.-S.); Tel.: +56-322-508-067 (M.E.H.). Recei ed: 6 June 2012; in e ised o m: 27 Augus 2012 / Accep ed: 6 Sep embe 2012 / Published: 12 Sep embe 2012 Abs ac : The aim o his s udy was o de e mine he elec onic in luence o subs i uen g oups and annela ed ings such as oxazole-oxazinone on he physicochemical and pho op o ec ion, an ioxidan capaci y, oxici y and single oxygen pho osensi iza ion biological p ope ies o isoquinoline alkaloid amewo ks. Thus, oxoisoapo phine de i a i es 1–5 and 3-azaoxoisoapo phine (6), some o hem wi h phenolic s uc u es, did no p esen any an ioxidan capaci y, possibly ei he by o ma ion o ke o-enol au ome ism species o he o ma ion o uns able ee adicals. Due o he single oxygen quan um yields () nea o uni y, and g ea e pho os abili y han phenalenone, oxoisoapo phines 4–6 may be conside ed as pho osensi ize s o single oxygen p oduc ion and can be used as new uni e sal s udy ools. The biological applica ion as an ibac e ial agen s is an impo an and possible ool in he s udy o compounds wi h low cy o oxici y and high eac i i y in an ineoplas ic chemo he apy. On he o he hand, when boldine and i s annela ed de i a i es B1–4 a e i adia ed, a pho op o ec o e ec is obse ed (SPF = 2.35), e en a e 30 minu es o i adia ion. They also ac as pho op o ec o s in cell ib oblas cul u es. No hemolysis was de ec ed o boldine hyd ochlo ide and i s sal s wi hou i adia ion. In solu ions i adia ed be o e incuba ion (a concen a ions o e 200 ppm) pho op oduc s we e oxic o he nauplii o A emia salina. OPEN ACCESS Molecules 2012, 17 10959 Keywo ds: an ioxidan capaci y; oxoisoapo phines; pho op o ec ion; oxici y; single oxygen 1. In oduc ion The isoquinoline alkaloids e e ed o as oxoisoapo phines (7H-dibenzo[de,h]quinolin-7-one) a e isola ed om Menispe mum dau icum DC oo s, which is he only known na u al sou ce o hese alkaloids. This plan is known in China as Bei-Dou-Gen [1], and in Japan as Kohmo i-Kazu a [2]. The hizomes o hese plan s a e used in adi ional Chinese medicine as analgesics and an ipy e ics o he ea men o so e h oa s, coli is, dysen e y and heuma ic a h algia [3], easons why he plan is o icially included in he China Pha macopeia [4]. S udies and epo s ela ed o hese compounds a e sca ce, bu since he 1980s in e es has been g owing; i use in a ious pa hological condi ions has been s udied and i s an i umo ac i i y is ecognized [5] oge he wi h a bene icial ac i i y in he ca dio ascula sys em, ac ing as an an ia hy hmic-d ug [6] and wi h dopamine gic ac i i y in he D1 and D2 ecep o s in he cen al ne ous sys em. Ano he s udy ound ha phenolic compounds dec eased lipid pe oxida ion and enhanced he ac i i y o SOD (supe oxide dismu ase) [7], as well as ha ing an i-in lamma o y ac ion. This sca old has a g ea po en ial wi h se e al pha macological p ope ies, howe e , i s pho ochemical ac i i y linked o he biological applica ion as an ineoplas ic d ugs has no been s udied. In his con ex , phenalenone (1H-phenalen-1-one) is used as a sensi ize in pho ochemis y and pho obiology. This compound is soluble in a a ie y o sol en s and he quan um yield, measu ed expe imen ally, is close o uni y in he sol en s used. Acco ding o hese cha ac e is ics, i was p oposed ha phenalenone could be a uni e sal e e ence single oxygen sensi ize [8]. In ecen yea s, a new ype o phy oalexins he e ha e been isola ed and cha ac e ized, whose phy oan icipyne s uc u e is based on he skele on o phenalenone. These a e he 4- and 9-phenylphenalenone de i a i es whose o ma ion in esponse o ungal in ec ion o banana plan s has ecen ly been desc ibed [9]. Compounds wi h a s uc u e based on he phenalenone skele on a e no mal me aboli es in a ious plan s and mic oo ganisms [9,10]. These au ho s demons a ed ha he p esence o he na u al phenalenone skele on o hese de i a i es ga e hem single oxygen pho osensi iza ion capaci y, and ha i s an i ungal ac i i y is inc eased signi ican ly by he p esence o ligh . Mo eo e , hey ound ha phenylphenalenone de i a i es ha ing highe quan um yield de eloped highe an i ungal ac i i y unde illumina ion, allowing hem o es ablish a ela ionship be ween he 1O2 gene a ion and he plan de ence ac i i y agains a pa hogen. Gi en he la ge numbe o po en ial pa hogens ha could p o oke a a ie y o diseases, plan s ha e de eloped de ense s a egies ha can be classi ied as physical o biochemical. Thus, among biochemis y s a egies he e a e se e al: he gene a ion o eac i e species de i ed om molecula oxygen, he supe oxide ion [O−•], he hyd ogen pe oxide [H2O2], he hyd oxyl adical [OH•] and he elec onic s a e o molecula single oxygen [O21] [11]. The single oxygen sensi iza ion by he isoquinoline alkaloids de i ed om glaucine, namely oxoglaucine, co unine, pon e ed ine has been s udied [12,13] (Figu e 1). Based on hese easons, i is necessa y o s udy and p o ide na u al compounds ha a e s uc u ally ela ed o phenalenone, ha a e e icien and able o sensi ize single oxygen wi h pho oquan um yields close o uni y, g ea e Molecules 2012, 17 10960 pho os abili y han phenalenone and possibly pho o oxic o pa hogens like he oxoapo phines [11]. The e o e, compounds such as oxoisoapo phine de i a i es ha include a phenalenone moie y, could be excellen candida es as possible single oxygen gene a o s and o conside a ion ei he as e ec i e an i ungals o agains o he pa hogens. Figu e 1. Chemical s uc u es o phenalenone and oxoapo phines. On he o he hand, boldine [(S)-2,9-dihyd oxy-1,10-dime hoxyapo phine], he majo alkaloid p esen in he lea es and ba k o he Chilean boldo ee (Peumus boldus Molina, Monimiaceae), has been cha ac e ized in he pas ew yea s as an an ioxidan ha e ec i ely p o ec s di e en sys ems agains ee- adical-induced lipid pe oxida ion and enzyme inac i a ion [14]. Boldine and o he ela ed apo phine alkaloids ha e been shown o beha e as po en an ioxidan s in a numbe o expe imen al models. Pha macological ac i i ies such as cy o-p o ec i e, an i- umo al p omo ing, an i-in lamma o y, an ipy e ic and an ipla ele ac i i ies ha e been associa ed wi h he abili y o boldine o sca enge highly eac i e ee adicals [15]. In a p e ious pape [16], he pho os abili y and pho op o ec i e ac i i y o boldine and glaucine we e s udied; bo h a e pho ouns able unde i adia ion. Howe e , he apo phine alkaloid s uc u e emained unchanged and he pho op oduc s o glaucine exhibi ed a highe pho op o ec ion ac o (SPF) han he non-i adia ed compounds. We also ound [17] ha boldine de i a i es bea ing subs i uen g oups and annela ed he e ocycles wi h he apo phine amewo k ha e a g ea e pho os abili y agains UVB adia ion. Thus, he compounds de i ed om boldine bea ing oxazole and oxazinone annela ed ings ha e p omising pho ophysical and pho ochemical p ope ies ha could allow hei possible use as sunsc eens. In his sense, he e is e idence ha oxoisoapo phines, some o which possess phenolic g oups in hei s uc u es, inhibi lipid pe oxida ion; o his eason he an ioxidan capaci y o oxoisoapo phine de i a i es wi h phenolic s uc u e, migh gi e us excellen a p io i in o ma ion abou his p ope y when no ye s udied. In his s udy, we e alua ed he in luence o subs i uen g oups and annela ed a oma ic sys ems buil on he boldine apo phine alkaloid amewo k: 8-ni oso-boldine hyd ochlo ide (B1), 8-aminoboldine Molecules 2012, 17 10961 hyd ochlo ide (B2), 9H-benzo[de]oxazoloboldine hyd ochlo ide (B3), [1,4]oxazino[3',2':3,4]benzo- [1,2-g]benzo[de]boldine hyd ochlo ide (B4), and se e al oxoisoapo phine de i a i es and 3- azaoxoisoapo phine; 2,3-dihyd o-5-me hoxy-6-hyd oxy-7H-dibenzo[de,h]quinolin-7-one (1), 5-me hoxy- 6-hyd oxy-7H-dibenzo[de,h]quinolin-7-one (2), 5-hyd oxy-7H-dibenzo[de,h]quinolin-7-one (3), 7H- dibenzo[de,h]quinolin-7-one (4), 5-me hoxy-7H-dibenzo[de,h]quinolin-7-one (5), and 7H-benzo[e] pe imidin-7-one (6) on se e al biological and physicochemical p ope ies: pho op o ec ion, an ioxidan capaci y, oxici y and single oxygen pho osensi iza ion, in o de o explain he possibili y o such isoquinoline compounds o ac ei he as an imic obial, an i ungal o an ineoplas ic agen s. 2. Resul s and Discussion 2.1. SPF in Vi o Tes Pho op o ec o capaci y was calcula ed as Sun P o ec ion Fac o (SPF) wi h espec o homome hyl salicyla e (SPF = 4). The SPF o he un-i adia ed ni oso, amino, phenyloxazinone and oxazole boldine de i a i es was simila o he SPF a e age alue o un-i adia ed boldine hyd ochlo ide. A e 30 min o i adia ion he a e age alue SPF o boldine hyd ochlo ide and i s de i a i es was 2.35. No signi ican di e ences we e obse ed a ibu able o he subs i uen s (Table 1). Table 1. Sun p o ec ion ac o (SPF) e sus i adia ion ime o boldine and i s de i a i es B1–4. I adia ion ime (min) Boldine B1 B2 B3 B4 0 6.30 6.33 6.51 6.39 6.43 5 4.12 4.08 4.20 4.00 4.02 10 3.71 3.93 4.07 3.95 3.97 15 3.70 3.90 4.00 3.80 3.86 20 3.23 3.25 2.68 2.71 2.77 25 2.96 2.80 2.58 2.68 2.70 30 2.33 2.35 2.41 2.31 2.36 The SPF o boldine hyd ochlo ide and i s de i a i es in he sal o m did no di e in any ime ange o he expe imen s, om ime ze o o 30 min o i adia ion. The e o e boldine hyd ochlo ide and i s de i a i es ha e a simila e ec , exp essed in he SPF alue, as de e mined by he me hod o pho op o ec ion in i o. F om ze o o hi y minu es o i adia ion, he SPF o boldine and i s de i a i es dec eased om 6 o 2.35. In acco dance wi h he Food and D ug Adminis a ion (FDA), sunsc eens wi h SPF = 2 a e accep able as comme cial p oduc s. Boldine and i s de i a i es, e en a e exposu e o inc easing i adiance doses, main ained hei pho op o ec i e p ope ies. 2.2. Pho ohemolysis The pho op o ec o e ec o boldine hyd ochlo ide was simila o ha o he es o i s de i a i es. Molecules con aining ni oso g oups we e mo e oxic when i adia ed be o e incuba ion due o he p esence o he subs i uen g oup (NO), which may ac by a ee adical pho odecomposi ion mechanism [18,19] (Table 2); howe e , he gene a ion o pho op oduc s wi h a s uc u e simila o Molecules 2012, 17 10962 ni osoboldine hyd ochlo ide (B1) is possible, which can be co obo a ed wi h he kine ics o pho odecomposi ion in ai and pho o-consump ion quan um yield [16]. Table 2. Hemolysis and pho ohemolysis (%) wi h p ei adia ed boldine hyd ochlo ide and i s de i a i es h ough ed blood cell model. Samples % Hemolysis in da kness % Hemolysis p ei adia ed % Pho ohemolysis Boldine 0.00 ± 0.00 0.00 ± 0.00 2.31 ± 2.35 × 10−3 B1 0.00 ± 0.00 19.45 ± 3.68 × 10−30.00 ± 0.00 B2 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 B3 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 B4 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 Signi ican di e ences (p < 0.05, Mann-Whi ney es ) exis ed be ween he solu ions o ni oso-boldine hyd ochlo ide i adia ed be o e incuba ion and hose i adia ed oge he wi h he ed blood cell suspension. 2.3. Toxici y Tes : Eggs o A emia salina The esul s (Table 3) indica e ha LD50 a e age was 880 ppm o non-i adia ed solu ions, while o i adia ed solu ions he LD50 a e age was 620 ppm. UVB exe ed a di ec dele e ious e ec on nauplii (11% mo ali y) as was p o en in expe imen s wi hou oxoisoapo phines. Solu ions i adia ed be o e incuba ion p oduced he same e ec , h ough pho odecomposi ion by UVB adia ion o boldine hyd ochlo ide and de i a i es in oxic pho op oduc s, on A emia salina. Solu ions i adia ed be o e incuba ion p oduced a g ea e numbe o dead nauplii han i adia ed solu ions. The i adia ed solu ions he e o e appea o exe a pho op o ec i e e ec up o a concen a ion o 200 ppm. A highe concen a ions he e was inc eased mo ali y, p obably due o a high accumula ion o oxic pho op oduc s. Table 3. Median le hal dose (LD50, ppm) o boldine hyd ochlo ide and i s de i a i es (B1–4) wi h he A emia salina model. Samples Le hal dose 50 (LD50) Le hal Dose 50 (LD50) P ei adia ed Le hal dose 50 (LD50) I adia ed Boldine 900 400 685 B1 860 335 602 B2 900 305 580 B3 900 360 662 B4 850 366 560 2.4. Me hod o Fib oblas Cell Su i al The comme cial o mula ions Biode ma Pho ode m Spo ® Labo a oi e De ma ologique (SPF 1) and An helios XL ® La Roche-Posay (SPF 2) ha we e used in his s udy p oduced ib oblas su i al a es a ound 96%. Molecules 2012, 17 10963 In ou expe imen s, p epa a ions con aining boldine and i s de i a i es da kened when i adia ed. P e ious s udies showed ha he pho op o ec ion obse ed in i o o boldine hyd ochlo ide could be a ibu able in pa o physical e ec s on he skin o guinea pigs due o pho op oduc s ha showed a da k colo a ion, p o iding addi ional p o ec ion agains UV adia ion [20]. The ehicle used o he s udy was he No obase c eam, which is made o a mix u e o alcohols such as ce yl alcohol and s ea yl alcohol and had a s ong e ec on cell su i al, which was a ound 50%. Oxazole boldine hyd ochlo ide (B3) and phenyloxazinone boldine hyd ochlo ide (B4) had a highe pho op o ec o capaci y han ib oblas cells, al hough no s a is ically signi ican ; possibly due o he e ec o subs i uen - used he e ocyclic con aining s able unc ional g oups wi h a highe esonance e ec (Figu e 2). P e ious s udies demons a ed ha boldine wi h oxazine and phenyloxazinone moie ies had g ea e s abili y han boldine agains UVB adia ion. Figu e 2. Pho op o ec o capaci y (% ali e ib oblas s) o SPFs comme cial o mula ions compa ed wi h boldine de i a i es B1–4. 2.5. An ioxidan Capaci y and Single Oxygen Fo ma ion 2,3-Dihyd o-5-me hoxy-6-hyd oxy-7H-dibenzo[de,h]quinolin-7-one (1) and 5-me hoxy-6-hyd oxy- 7H-dibenzo[de,h]quinolin-7-one (2) did no p esen an ioxidan capaci y a ei he high o low concen a ions in he -ca o ene assay me hod wi h T olox as e e ence (Table 4). In his case he e is a di ec in luence by he p esence o a ca bonyl g oup which gene a es an in amolecula hyd ogen bond be ween wo oxygen a oms, whose balance may esul in he p esence o ke o-enol au ome ic species. Table 4. An ioxidan capaci ies (AA) o oxoisoapo phines 1–3 e alua ed om p o ec ion o -ca o ene. Compounds Concen a ion (μM) AA 1 100 0.35 2 100 0.40 3 100 0.35 T olox 100 89.72 Fo 5-hyd oxy-7H-dibenzo[de,h]quinolin-7-one (3), he s abiliza ion o he phenyl adicals by he esonance a successi e s ages wi h he elec on loca ed in one o he adical esonance s uc u es, posi ioned on he ni ogen a om, des abilized he molecule elec onically, which does no con ibu e o he good s abiliza ion o he adical o med, esul ing in he obse ed dec ease in an ioxidan capaci y. 0 10 20 30 40 50 60 70 80 90 100 cells da k cells i . SPF 1 SPF 2 c eme base B0 B1 B2 B3 B4 % ali e ib oblas s Molecules 2012, 17 10964 Ne e heless, oxoisoapo phines 4–6 syn he ically ob ained in bes yield o u he s udies by ou g oup, showed a su p ising e ec i eness wi h a high deg ee o pho osensi iza ion in he p oduc ion o single oxygen [O2 ( 1g)]. The pho os abili y ha he es ed compounds exhibi can be ex ended o di e en de i a i es ha con ain a ce ain ype o subs i uen ha could modula e he pho osensibili y and s abili y in a ied sol en s o di e se pola i y. Thus, he use o apola sol en s such as oluene, cyclohexane, s udied among o he s, as well as o a pola sol en like me hanol, showed ha o oxoisoapo phine de i a i es 4–6, he quan um yield () o o ma ion o single oxygen wi h e e ence o phenalenone, is nea o uni y and wi h a clea ideal beha io (Table 5). In con as o he phenalenone, he quan um yield o 4 is p ac ically independen o he pola i y o he sol en , which sugges s ha his compound migh be used also as a uni e sal e e ence. Table 5. Single oxygen quan um yield () o oxoisoapo phine de i a i es (4–6) and phenalenone (Phenal) in di e en sol en s and a ied pola i y. Sol en Pola i y  4  5 6 Phenal Cyclohexane 0.006 0.95 0.92 Toluene 0.099 1.00 0.95 1.00 0.95 Te ahyd o u an 0.207 1.00 0.87 N,N'-dime hylace amide 0.377 1.00 0.93 0.87 Ace oni ile 0.460 0.98 1.00 0.98 P opylene ca bona e 0.475 1.00 1.00 Me hanol 0.762 1.00 0.97 0.93 0.98 2,2,3.3-Te a luo op opanol 0.886 1.00 1.00 3. Expe imen al 3.1. Syn hesis o Boldine De i a i es Boldine de i a i es B1–4 (Figu e 3) we e syn hesized and cha ac e ized by s anda d me hods [21,22]. Hyd ochlo ide sal s we e p epa ed by dissol ing he compounds in isop opanol and subsequen ly adding HCl, hen p ecipi a ing he hyd ochlo ide wi h e hyl e he . The eagen s B ij 35 (Ald ich Labo a o ies, Milwaukee, WI, USA), dime hylsul oxide, e hanol, me hanol, e ahyd o u an and ace oni ile (Me ck, San iago, Chile) we e used. Dis illed wa e adjus ed o pH 3 and pH 10 wi h HCl and NaOH, espec i ely (Me ck) was used. 3.2. SPF in Vi o Tes The pho op o ec o capaci y in he UVB egion was de e mined by measu ing changes in ligh ansmission o he solu ion o he compounds and homosala e ( e e ence sunsc een, SPF = 4). The ansmission spec a o he compounds (0.123 mg/5 mL) in a uni e sal sol en , p epa ed by mixing 12.5 g me hylene chlo ide, 37.5 g cyclohexane and 50 g isop opanol, we e eco ded in a spec opho ome e . The a ea unde he cu e was ela ed o ha o homosala e o SPF de e mina ion. A bank o six lamps, p o iding a mean i adiance o abou 0.50 mW/cm2, was used as UVB adia ion sou ce. I adiance measu emen s we e made wi h a adiome e . Molecules 2012, 17 10965 Figu e 3. Chemical s uc u es o boldine de i a i es hyd ochlo ide B1–4. 3.3. Me hod o Fib oblas Cell Su i al [23] Human ib oblas cells p o ided by he Labo a o y o Physiological Chemis y and Immunology (Facul y o Pha macy, Uni e si y o Valpa aíso) we e g own in a lask seeded wi h cul u e medium [Dulbecco’s Modi ied Eagle medium (DMEM)/Ham’s F-12] supplemen ed wi h e al cal se um (PAA), penicillin 10 U/L, s ep omycin 100 µg/mL and ampho e icin B 2.5 pg/mL (PAA). O he en plaques con aining ib oblas s, one was le in da kness wi hou i adia ion and he emaining nine we e i adia ed wi h six lamps (UVB TLK 40W/12) wi h an i adiance o 0.50 mW/cm2 o 30 min, using app oxima ely 40 minimal e y hema dose. Nine o he six-well pla es we e i adia ed, one wi hou any p o ec ion, one comple ely and homogeneously co e ed on i s uppe ace wi h a sun p o ec o (SPF > 50, Biode ma ®), one comple ely and homogeneously co e ed on i s uppe ace wi h a sun p o ec o (SPF > 50, La Roche-Posay ®), one comple ely and homogeneously co e ed on i s uppe ace wi h only a c eam base ( ehicle), one comple ely and homogeneously co e ed on i s uppe ace wi h a c eam base con aining only boldine hyd ochlo ide. Fou o he six-well pla es we e comple ely and homogeneously co e ed on hei uppe aces wi h a c eam base ha con ained only one de i a i e o boldine hyd ochlo ide each. The abso bance was de e mined in a 96-well pla es pla e eade (Sensiden Scan, Me ck, Da ms ad , Ge many) a 540 nm; he pe cen age o pho op o ec ion o each boldine hyd ochlo ide de i a i e was hen calcula ed. 3.4. Pho ohemolysis Red blood cells o heal hy adul dono s we e used. Sho ly a e collec ion he hepa inized blood was cen i uged a 2,000 g and he plasma and bu y coa we e disca ded. The emaining ed cells we e washed h ee imes wi h an iso onic solu ion (0.15 M NaCl in 0.01 M sodium phospha e (PBS), pH 7.4). The ed cells we e esuspended o app oxima ely 2% / , kep a 6 °C and used in he nex Molecules 2012, 17 10966 72 h. The pe cen age o hemolysis was de e mined immedia ely a e i adia ion and exposi ion o p e-i adia ed solu ions by measu ing he hemoglobin libe a ed in he medium om solu ions con aining 0.4% ed cells [24]. The measu emen s we e ca ied ou spec opho ome ically a e cen i uga ion a 2,000 g, and he abso bance eco ded a 540, 560, 577, 630 and 700 nm. The concen a ions we e e alua ed acco ding o he Win e bou n equa ion [18]. 3.5. Toxici y Tes : Eggs o A emia saline A emia salina (Class: C us acea, Subclass: B anchiopoda; Supe o de : Anos aca, Family: A emidae, Genus: A emia) cys s we e incuba ed in il e ed (mic opo e 0.22 μm) sea wa e and oxygena ed o 45 min a 30 °C in a he mo egula ed ba h and adjus ed o pH 8 wi h NaOH 0.1 M. A e 24 h, he eclosd nauplii ( i s s age o A emia saline) a e in an app op ia e condi ion o oxici y es s [25]. 3.6. Syn hesis o Oxozhines Oxoisoapo phine de i a i es 1–6 (Figu e 4) we e syn hesized and cha ac e ized by s anda d me hods [26]. Figu e 4. Chemical s uc u es o oxoisoapo phine de i a i es 1–6. 3.7. Single Oxygen P oduc ion The single oxygen p oduc ion [O2 (1g)] was assessed using a spec opho ome e sys em based on a li e ime o luo escen 55 luo escence PicoQuan Fluo ime 200. Using an exci a ion lase Nd: YAG pumped by diode C yLas FTSS355-Q, emi ing lase pulses a 355 nm, epe i ion a e o 10 kHz, pulse wid h o 1 ns and powe 5 mW, J1J co esponding o 0.5 pe pulse. The emission is collec ed a 90°, passed h ough a monoch oma o , and de ec ed wi h a pho omul iplie Hamama su H9170 nea -IR-45. All pho ons a e coun ed wi h a mul ichannel scale PicoQuan ’s Nanoha p 250. The his og ams a e analyzed using he so wa e FluoFi , 60 also PicoQuan .  N O H3CO N O H3 CO HO HO N O HO N O N O N N O H3CO (1) (2) (3) (4) (5) (6)