Wassiliki Argyrakis

Christoph Köppl

Hans-Joachim Werner

Wolfgang Frey

Angelika Baro

Sabine Laschat
email: sabine.laschat@oc.uni-stuttgart.de

A combined quantum mechanical and experimental approach towards chiral diketopiperazine hydroperoxides

Keywords: Diketopiperazines, Hydroperoxides, DFT, RMP2, bond dissociation enthalpy Diketopiperazines / Hydroperoxides / DFT / RMP2 / bond dissociation enthalpy (BDE)

A combined quantum mechanical and experimental approach towards chiral diketopiperazine

Introduction

Chiral hydroperoxides have been utilized successfully in a variety of asymmetric oxidations, e.g. alkene epoxidations, [1,2] Weitz-Scheffer epoxidations of enones [3][4][5] and sulfoxidations. [5][6][7][8][9][10] Therefore several synthetic routes have been explored towards chiral hydroperoxides, e.g. kinetic resolution using peroxidases, [7,11] Sharpless epoxidation, [12] separation by column chromatography on a chiral stationary phase, [13] via diastereomeric perketals, [14] chiral flavine-derived cyclophanes, [15] nucleophilic attack to in situ formed N-(menthylcarbamoyl)-isoquinolinium ions, [16] and nor-camphor derivatives [17] utilizing hydrogen peroxide to generate the hydroperoxide. [18] In contrast, molecular oxygen has been rarely used for such purpose.

Oda reported the aerobic Weitz-Scheffer epoxidation of enones under chiral phase transfer catalysis with in situ formed 9-hexylfluorenone-9-hydroperoxide as a chiral oxidant. [19] Chiral hydroperoxides can be also obtained by enzyme catalysis, e.g. by employing lipoxygenases. [20] However, in this work we focussed on non-enzymatic hydroperoxide formation.

Scheme 1. The autoxidation of diketopiperazine (-)-1a to hydroperoxide (-)-1b with molecular oxygen. The BDE of the radical formation and the reaction enthalpy of the hydroperoxide formation are calculated in this work.

We have recently prepared the known hydroperoxide (-)-1b via aerobic oxidation of diketopiperazine (-)-1a according to a procedure by Schmidt. [21] This reaction has already been investigated by Schmidt who also described the radical mechanism of the autoxidation of (-)-1a with molecular oxygen in the presence of catalytic amounts of benzophenone whilst being irradiated with UV radiation. [21] Within the proposed We applied the chiral organic hydroperoxide (-)-1b successfully for the chemoselective sulfoxidation of sulfide 2 to sulfoxide 3 without overoxidation to sulfone and the enantioselective epoxidation of 4 to (-)-5 (Scheme 2). [5] Scheme 2. Application of the chiral organic hydroperoxide (-)-1b in chemoselective sulfoxidations and enantioselective epoxidations.

Liebscher further extended this work towards Weitz-Scheffer epoxidations of naphthoquinones and enantioselective sulfoxidations in the presence of (-)-1b and Ti(OiPr) 4 albeit with accompanying overoxidation. [16] However, with regard to our results we found that further optimization was hampered by the low reactivity of diketopiperazine (-)-1a towards autoxidation and the poor yield despite extended reaction times. Thus we had to deal first with the reactivity of the diketopiperazine prior to improvement of the oxidation reactions. Diketopiperazines were originally chosen because the steric bulkiness of these cyclic dipeptides can be properly adjusted by suitable choice of amino acid precursors. [22,[START_REF]For applications of diketopiperazine scaffolds[END_REF] In order to limit the number of diketopiperazine analogues and thus the experimental effort with regard to synthesis and autoxidations we planned to do a computational screening of potentially interesting candidates prior to experimental studies. Although several theoretical studies on diketopiperazines exist in the literature, most of them deal with calculation of spectroscopic properties, such as fragmentation patterns in ESI MS spectra, [24] vibrational and electronic spectra, [25] circular dichroic spectra, [26] confor-F o r P e e r R e v i e w 5 mational analysis [27] and crystal packing. [28] Only a couple of references treated reaction pathways such as radical formation of methionine-derived diketopiperazines as calmodulin mimics, [29] formation of imidazo[1,2-a]pyrazine-3,6-diones from diketopiperazines [30] and Pictet-Spengler cyclization of N-2-bromoacetyltetrahydroisoquinoline methyl ester in the presence of primary or secondary amines to the corresponding diketopiperazines. [31] Rauk used DFT methods to calculate kinetics and bond dissociation enthalpies of H-abstraction from symmetrical diketopiperazines by methyl or thiyl radicals. [32][33][34] For example, for glycine-and alanine-derived diketopiperazines bond dissociation enthalpies of 351 kJ mol -1 and 335 kJ mol -1 respectively were determined, which agreed well with experimental values of 340±15 kJ mol -1 and 325±15 kJ mol -1 by Rauk [32] and Schöneich. [35] Due to the captodative radical stabilization these bond dissociation enthalpies are lower than those observed for α-hydroxy-substituted C-H bonds (370 -390 kJ mol -1 ). [36] A related field of study where H-abstraction, including H-tunneling, plays an important role as the reaction rate limiting step are chemical reactions catalysed by lipoxygenase. [37][38][39][40][41][42] In light of the results obtained by Rauk and Schöneich, our aim was to use computational chemistry to identify suitable precursors with enhanced reactivity towards molecular oxygen which would allow further optimization of stereoselectivity.

The results are reported below.

Experimental

Computational details: All quantum chemical calculations presented here were performed at the DFT level of theory using the Becke3-Lee-Yang-Parr (B3LYP) [43][44][45][46][47][48] hybrid functional together with a valence triple ζ (TZVP) basis set [49][50][51] for all atoms, or using spin-restricted open-shell Møller-Plesset perturbation theory (RMP2) [52][53][54] with restricted Hartree-Fock (RHF) reference functions in combination with a correlation consistent augmented triple ζ (aug-cc-pVTZ) basis set. [55,56] Density fitting was used to compute the integrals in RHF and RMP2, employing the cc-pVTZ/JKFIT and aug-cc-pVTZ/MP2FIT basis sets of Weigend. [57,58] All DFT calculations, geometry optimizations and harmonic frequency calculations were performed at the B3LYP level of theory using TURBOMOLE [START_REF]a development of the University of Karlsruhe and Forschungszentrum Karlsruhe GmbH[END_REF][START_REF] Treutler | [END_REF][61][62][63][64][65] , whereas for all single point RMP2 calculations the MOLPRO [START_REF] Molpro | 1, a package of ab initio programs[END_REF] package of ab initio programs was used. 170 °C for 6 h. The resulting solid was isolated by filtration and the desired diketopiperazines 19 [73] (1.66 g, 56 %) and 20 [74,75] (3.16 g, 84 %) were obtained as colourless crystals after recrystallization from ethanol.

General procedure for the synthesis of diketopiperazine lactim ethers 21-26 (1S,4S)-3,6-Diisopropyl-5-ethoxy-1,6-dihydropyrazine-2(3H)-one (24)

To a solution of diketopiperazine 20 (0.792 g, 5 mmol) in dry dichloromethane (30 ml) triethyloxonium tetrafluoroborate (1.141 g, 6 mmol) was added. The reaction mixture was refluxed and after 12 h additional triethyloxonium tetrafluoroborate (1.140 g, 6 mmol) was added and stirred for further 12 h. The mixture was slowly poured into ice-cold saturated NaHCO 3 -solution (pH>8) (50 ml), the organic layer was separated and the aqueous layer was extracted with dichloromethane (3 x 20 ml). The combined organic layers were dried (MgSO 4 ), evaporated and isolated after silica gel flash chromatography (hexanes/ethyl acetate) to afford the diketopiperazine lactim ether 24. Compound 24 was obtained as a pale yellow oil (1.074 g, 95 %). 1 H-NMR (300 MHz, CDCl 3 ): δ = 0.78 (d, 3 J = 6.8 Hz, 3H, iProp-CH 3 ), 0.86 (d, 3 J = 6.8 Hz, 3H, iProp-CH 3 ), 1.05 (d, 3 J = 6.9 Hz, 3H, iProp-CH 3 ), 1.08 (t, 3 J = 6.9 Hz, 3H, OCH 2 CH 3 ), 1.28 (d, 3 J = 6.9 Hz, 3H, iProp-CH 3 ), 2.24 -2.39 (m, 1H, CH-(CH 3 ) 2 ), 2. 48 -2.58 (m, 1H, CH-(CH 3 ) 2 ), 3.90 -3.98 (m, 2H, CH-iProp), 4.08 -4.23 (m, 2H, -OCH 2 CH 3 ) ppm. The diketopiperazine lactim ethers 21, [72] 22, [72] 23, [73] 25, [73] and 26 [76] were prepared following the procedure above for lactim ether 24 and all were obtained as pale yellow oils. The spectroscopic data was according to the literature. Compounds 8a, 9a, 10a and 13a were prepared following the procedure of 12a. The pyrazinoquinazolines 9a, [77] 10a, [78d] and 13a [78d] were obtained as colourless solids and the spectroscopic data were according to the literature.

General

General procedure for autoxidation of pyrazinoquinazolines 8a, 9a, 10a, 12a to the corresponding hydroperoxides 8b, 9b, 10b, 12b

A solution of the pyrazinoquinazoline derivative 8a, 9a, 10a or 12a (0.3 mmol) and benzophenone (0.01 eq.) in chloroform (300 ml) was irradiated with UV radiation (UV LED) under oxygen atmosphere at room temperature for 24 hours. The solvent was removed under reduced pressure and the residue was purified by silica gel flash chromatography (hexanes/ethyl acetate) to afford the desired products (8b, 9b, 10b Pyrazinoquinazoline hydroperoxide 9b was obtained as a colourless solid (39 mg, 27 % yield). 1 H-NMR (500 MHz, CDCl 3 ): δ = 1.04 (d, 3 J = 6.8 Hz, 3H, CH 3 ), 1.17 (d, 3 J = 6.8 Hz, 3H, CH 3 ), 2.03 -2.13 (m, 2H, 2-H), 2.27 (dqq, 3 J = 7.8 Hz, 3 J = 6.8 Hz, 3 J = 6.8 Hz, 1H, CH-(CH 3 ) 2 ) 2. 35 -2.44 (m, 1H, 1-H a ), 2. 69 -2.73 (m, 1H, 1-H b ), 3.64 -3.70 (m, 2H, 3-H), 5.33 (d, 3 J = 9.0, 1H, 3-H), 7.50 (ddd, 3 J = 8.0 Hz, 4 J = 7.0 Hz, 5 J = 1.2 Hz, 1H, 10-H), 7.67 (ddd, 3 J = 8.3 Hz, 4 J = 1.6 Hz, 5 J = 0.5 Hz, 1H, 12-H), 7.77 (ddd, 3 J = 8.3 Hz, 4 J = 7.0 Hz, 5 J = 1.6 Hz, 1 H, 11-H), 8.29 (ddd, 3 J = 8.0 Hz, 4 J = 1.5 Hz, 5 J = 0.5 Hz, 1H, 9-H), 11.95 (s, 1 H, OOH) ppm. 13 Pyrazinoquinazoline hydroperoxide 10b was obtained as a colourless solid (11 mg, 12 % yield). 1 H-NMR (500 MHz, d6-DMSO): δ = 0.89 (d, 3 J = 6.8 Hz, 3H, CH 3 ), 1.04 (d, 3 J = 6.8 Hz, 3H, CH 3 ), 2. 56-2.68 (m, 1H, CH-(CH 3 ) 2 ), 4.32 (d, 2 J = 8.8 Hz, 1H, 1-H a ), 4.79 (dd, 2 J = 8.8 Hz, 3 J = 1.1 Hz, 1H, 1-H b ), 5.89 (d, 3 J = 5.5 Hz, 1H, 2-H), 7.56 (ddd, 3 J = 8.0 Hz, 3 J = 7.2 Hz, 5 J = 1.2 Hz, 1H, 8-H), 7.70 (ddd, 3 J = 8.2 Hz, 4 J = 1.2 Hz, 5 J = 0.6 Hz, 1H, 10-H), 7.82 (ddd, 3 J = 8.2 Hz, 4 J = 7.2 Hz, 5 J = 1.5 Hz, 1H, 9-H), 8.32 (ddd, 3 J = 8.0 Hz, 4 J = 1.5 Hz, 5 J = 0.6 Hz, 1H, 7-H), 8.63 (d, 3 J = 1.1 Hz, 1H, NH), 10.48 (s, 1H, OOH) ppm. Pyrazinoquinazoline hydroperoxide 12b was obtained as a colourless solid (71 mg, 67 % yield). 1 H-NMR (500 MHz, CDCl 3 ): δ = 0.98 (d, 3 J = 6.9 Hz, 3H, CH 3 ), 1.05 (d, 3 J = 6.9 Hz, 3H, CH 3 ), 1.17 (d, 3 J = 6.9 Hz, 3H, CH 3 ), 1.34 (d, 3 J = 6.9 Hz, 3H, CH 3 ), 2. 42 -2.64 (m, 1H, CH-(CH 3 ) 2 ), 2. 64 -2.75 (m, 1H, CH-(CH 3 ) 2 ), 5.57 (d, 3 J = 6.5 Hz, 1H, 4-H), 7.69 (ddd, 3 J = 8.3 Hz, 3 J = 7.9 Hz, 4 J = 1.3 Hz 1H, 8-H), 7.91 (ddd, 3 J = 7.9 Hz, 3 J = 7.0 Hz, 4 J = 1.3 Hz, 1H, 9-H), 8.06 (dd, 3 J = 8.3 Hz, 3

3-[2-Oxo-2(1-pyrrolidine-2-onyl)ethyl]-2,4-(1H,3H)-quinazolinedione (28)

A solution of 8a (0.3 mmol) and benzophenone (0.01 eq.) in chloroform (300 ml) was irradiated with UV radiation (UV LED) under oxygen atmosphere at room temperature for 48 hours. The solvent was removed under reduced pressure and the residue was purified by silica gel flash chromatography to afford 8b in 13 % yield and quinazolinedione 28 was isolated as a crystalline, colourless solid (12 mg, 14 % yield). 1 H-NMR (500 MHz, CDCl 3 ): δ = 2.02 (tt, 3 J = 8.0 Hz, 3 J = 6.5 Hz, 2H, 14-H), 2.63 (t, 3 J = 8.0 Hz, 2H, 13-H), 3.70 (t, 3 J = 7.1, 2H, 15-H), 5.11 (s, 2H, 9-H), 7.23 (ddd, 3 J = 8.1 Hz, 4 J = 1.0 Hz, 5 J = 0.7, 1H, 8-H), 7.24 (ddd, 3 J = 7.9 Hz, 3 J = 7.2 Hz, 4 J = 1.0, 1H, 6-H), 7.70 (ddd, 3 J = 8.1 Hz , 3 J = 7.2 Hz, 4 J = 1.5 Hz, 1H, 7-H), 7.93 (ddd, 3 J = 7.9 Hz, 4 J = 1.5, 5 J = 0.7 Hz, 1H, 5-H), 11.58 (s, 1H, -NH) ppm.

Results

Theoretical studies

The following set of diketopiperazines 1a, 1b, 6a, 7a and pyrazinoquinazolines 8a-13a (Scheme 3) was chosen for computational investigation. Due to the large system sizes and the fact that electronically excited states are involved (cf. Scheme 1), it is currently impossible to obtain reliable quantitative results for the barrier heights of the investigated reactions. Therefore only a qualitative model was used. According to the mechanism discussed in the introduction (Scheme 1), we expect that the first reaction step, the abstraction of a hydrogen atom from a C-H bond is the most crucial and therefore reaction rate determining step for the reactions of all considered structures. Hence the investigation and comparison of these atom abstraction reactions within the set of chosen compounds should be an informative basis to judge the reactivity of the substrates towards hydroperoxide formation.

We based our investigations on the ideas of Evans and Polanyi. [79] For our set of similar atom abstraction reactions, the Evans-Polanyi relation suggests a linear relation between activation energy and exothermicity, i.e., changes in activation energy should be linearly related to changes in exothermicity. However, since the changes in exothermicity are directly related to the C-H bond strengths, it seems reasonable to use BDEs as a direct measure for the reactivity of the substrates.

Increasing reactivity (higher rate constants and lower activation barriers) can then be expected with decreasing BDEs. A generalization of the concept that the activation energy relates to the exothermicity of a reaction in a set of similar reactions is also known as LFER (Linear Free Energy Relation). [START_REF] Warshel | Computer Modeling of Chemical Reactions in Enzymes and Solutions[END_REF] The validity of the Evans-Polanyi relation was for example demonstrated for hydrogen abstractions from haloethers by OH radicals in a study by Chandra and Uchimaru, [START_REF] Chandra | [END_REF] although, as shown by Finn et al., this relation is only valid as long as the reactions are not entropy controlled. [82] For an understanding of the substrate reactivity and to support the experiment with promising structures prior to synthesis, BDEs for tertiary α-C-H bonds were therefore calculated. In this investigation, only tertiary C-H positions were considered for the calculations, since they should be more prone towards hydroperoxide formation as compared to the secondary C-H positions.

The diketopiperazine derivative 7a was selected for the calculations because we anticipated electron withdrawing sulfonamide groups should decrease the capto- dative stabilization characteristic for peptide radicals. [32] The pyrazino-quinazolines 8a-13a were selected, due to the easy synthetic conversion of diketopiperazines to pyrazinoquinazolines which has been well established by Söllhuber and Avendano. [78] The presence of an N-aryl-imino moiety in 8a-13a should further improve captodative stabilization via conjugation. In addition to the BDEs, the overall reaction enthalpies for the autoxidation of the heterocycles 1a, 6a-13a with molecular oxygen to the corresponding hydroperoxides 1b,c, 6b-13b, 9d and12d were evaluated. The calculated α-C-H bond dissociation and corresponding reaction enthalpies are summarized in Table 1. Whereas symmetric tricyclic diketopiperazine 1a and the unsymmetric bicyclic derivative 6a gave similar BDEs of 326.6 kJ mol -1 and 320.7 kJ mol -1 at the B3LYP level of theory, respectively (Table 1,Entries 1,3), the presence of electron withdrawing sulfonamide groups in tricyclic diketopiperazine 7a decreases the α-C-H BDE to 318.8 kJ mol -1 (Table 1, Entry 4). Furthermore, the second tertiary α-C-H bond at C-10a in tricyclic diketopiperazine hydroperoxide 1b

shows a BDE of 327.0 kJ mol -1 (Table 1, Entry 2), a value similar to the one observed for the first autoxidation of 1a. Next a series of pyrazinoquinazolines 8a-13a was studied, differing in the steric and electronic environment of the α-C-H bonds. Within this set of compounds, the lowest BDE was found for the α-C-H bond at C-1 of compound 12a (248.4 kJ mol -1 , Table 1, Entry 10) whereas the highest BDE was found for the α-C-H bond at C-4 of compound 10a (368.4 kJ mol -1 , Table 1, Entry 8).

According to the results obtained at the B3LYP and RMP2 level of theory, the molecules can be ordered according to decreasing BDEs as:

10a > 13a > 9a (C-6) > 12a (C-4) > 9a (C-13b) > 8a > 11a > 12a (C-1) (Table 1, Entries 5-12).
Some general trends can be deduced form the B3LYP calculations. An N-aryl-imino moiety in the vicinity of the α-C-H bond reduces the BDE to a much larger extent as compared to an amido moiety, see for example compounds 9a (C-13b) versus 9a (C-6) (Table 1,Entries 6,7),10a versus 11a (Table 1,Entries 8,9) or 12a (C-1) versus 12a (C-4) (Table 1,Entries 10,11). These results suggest that electronic parameters are much more important than sterical parameters with regard to BDEs. Surprisingly, pentacyclic pyrazinoquinazoline 13a exhibits a relatively high BDE of 323.7 kJ mol -1 (Table 1, Entry 12).

All autoxidation reactions leading to the corresponding hydroperoxides 1b,c, 6b-13b, 9d and 12d were found to be exothermic within a range of -6.9 to -74.9 kJ mol -1 at the B3LYP level of theory. While for the diketopiperazines 1a, 6a and 7a, the reaction enthalpies are all similar and range between -53.3 kJ mol -1 and -55.1 kJ mol - 1 (Table 1, Entries 1, 3, 4), with the enthalpy for the second oxidation of the tricyclic peroxide 1b being -36.8 kJ mol -1 (Table 1, Entry 2), the reaction enthalpies for the pyrazinoquinazolines 8a -13a spread between -6.9 kJ mol -1 to -74.9 kJ mol -1 (Table 1, Entries 5-12). The most exothermic reactions within this set of compounds were found to be the oxidations of pyrazinoquinazolines 12a (C-1) and 9a (C-13b) with reaction enthalpies of -74.9 kJ mol -1 and -74.7 kJ mol -1 , respectively (Table 1,Entry 6,10). The exothermicities of the reactions decrease in the following order for the hydroperoxides: 12b > 9b > 8b > 11b > 9d > 12d > 13b > 10b (Table 1, Entries 5-12). Thus with regard to BDEs and reaction enthalpies one could expect that the autoxidation of tetracyclic pyrazinoquinazoline 9a should preferently yield the hydroperoxide 9b in favour of regioisomer 9d. In the same manner the autoxidation of tricyclic pyrazinoquinazoline 12a should give hydroperoxide 12b rather than the regioisomer hydroperoxide 12d.

The RMP2 bond dissociation and reaction enthalpies are generally somewhat larger than the B3LYP ones. However, as can be seen in Table 1, the trends are the same in both cases.

Table 1. Bond dissociation and reaction enthalpies for diketopiperazine derivatives 1a, b, 6a, 7a and pyrazinoquinazolines 8a-13a. Experimental studies. With the above mentioned results in hand, the diketopiperazines 1a, 6a and pyrazinoisoquinolines 8a, 9a, 10a, 12a were prepared (Schemes 4, 5, Table 2).

BDEs [kJ mol -1 ] ∆H R [kJ mol -1 ] Entry Compound Heterocycle Product B3LYP a RMP2 a,b B3LYP a RMP2 a,
The known diketopiperazine 16 was synthesized in 3 steps following a procedure by de Costa starting from Boc-protected L-proline 14 and L-valine methyl ester hydrochloride 15 (Scheme 4). [71] The compounds 1a and 6a were also prepared according to this procedure starting from Boc-protected L-proline 14 and using L-proline methyl ester hydrochloride or glycine methyl ester hydrochloride 18, respectively, instead of 15.

The diketopiperazines 19 and 20 were prepared in 3 steps whereas the first 2 steps were following a procedure by Balaram [83] and the cyclization to the diketopiperazines was achieved by heating in mono ethylene glycol (MEG) at 170° C (Scheme 4).

Coupling 2). [78] reflux The diketopiperazines 1a, b, 6a as well as the pyrazinoquinazolines 8a, 9a, 10a, 12a

(68 %) (95 %) + N NH O OEt R + N N OEt OEt R R = H 25 (14 %) R = H 26 (38 %) R = H R = iPr
were stirred at room temperature under oxygen atmosphere with a catalytic amount 3). The absolute configuration of the diketopiperazine hydroperoxides 1b, c and 6b was determined by X-ray crystallographic data.

A longer reaction time for the autoxidation of the pyrazinoquinazoline 8a leads to decomposition of the desired hydroperoxide 8b. The proline glycine diketopiperazine 6a as well as anthranilic acid 27 and the achiral side product 28 could be isolated after 48 hours (Scheme 6). It should be noted that the stereochemical assignment for pyrazinoquinazoline hydroperoxides was based on NMR-assignments and comparison with earlier work in our group [72] on diketopiperazine hydroperoxides where we obtained NMR-and X-Ray data for both cis-and trans-isomers. The configuration of the diketopiperazine hydroperoxides 1b, c could be determined by X-Ray crystal structure analysis (see supporting information). Identical optical rotation of single crystals and bulk material were taken as evidence that both had the same configuration. Obviously the pseudoequatorial cis-configuration of the five-membered proline rings on the diketopiperazine unit form a stronger stabilization than other conformations. [22b,84] For this reason the pseudoequatorial cis-configuration of the three rings is maintained even in this radical autoxidation. In case of pyrazinoquinazolines the configuration of 8a, 9a, 10a and 12a was determined by optical rotation and comparison with data in the literature. [77,78d] Unfortunately, we could not get suitable single crystals of the pyrazinoquinazolines 8b, 9b, 10b and 12b. Therefore assignment was based on the NMR data in analogy with the diketopiperazine series due to the close steric and electronic relationship between the two series. Furthermore in the cases of 9b and 12b an isomerisation would lead to different diastereomers which would differ in NMR data. This was not observed for the main products.

Discussion

Only a subset of the computationally investigated compounds have been experi- 3, Entry 1), subsequent oxidation of 1b to the hydroperoxide 1c was achieved after 48 hours in 32 % yield (Table 3, Entry 4). For the autoxidation of 1a

ethyl acetate was chosen as solvent, because there it precipitates as a colourless solid due to its low solubility. This increases the yield of 1b in comparison to chloroform. For a direct comparison of autoxidation of diketopiperazines and pyrazinoquinazolines the autoxidation of 1a was prepared in chloroform, but after 24 hours the desired hydroperoxide 1b could not be detected (Table 3, Entry 2).

Prolonging the reaction time in chloroform up to 5 days lead to a yield of 23 % which is significantly lower than the yield obtained in ethyl acetate (Table 3, Entries 1, 3). To increase the yield for the autoxidation of 1b to dihydroperoxide 1c, a change of solvent to chloroform was necessary due to the solubility of 1b. The bicyclic diketopiperazine 6a required an even longer reaction time (7 d) and gave the hydroperoxide 6b in only 14 % yield (Table 3, Entry 5) despite its slightly decreased BDE and similar reaction enthalpy as compared to 1a. The oxidation of 6a as well as the oxidation of 1a for 24 hours in chloroform did not lead to a noteworthy amount of the corresponding hydroperoxides (Table 3,Entries 2,6).

The autoxidation of the tetracyclic pyrazinoquinazoline 8a yielded the corresponding hydroperoxide 8b after 24 hours in 44 % (Table 3,Entry 7). Although the calculated reaction enthalpy is similar to the one calculated for the autoxidation of diketopiperazine 1a to 1b (-54.1 versus -55.1 kJ mol -1 ), the decreased BDE (299.2 versus 326.6 kJ mol -1 ) reflects the much faster reaction (Table 1, Entries 1, 5).

A prolonged reaction time of 48 hours for the autoxidation of 8a decreases the yield of the desired hydroperoxide 8b to 13 % while raising the yield of the side product 28

to 14 % (Table 3, Entry 8, Scheme 6). For this reason the autoxidations of the pyrazinoquinazolines 9a, 10a and 12a were discontinued after 24 hours.

The autoxidation of the tetracyclic pyrazinoquinazoline 9a which differs in the isopropyl group on C 6 from 8a yielded the hydroperoxide 9b in 27 % (Table 3, Entry 9). Although the reaction from 9a to 9b was calculated to be more exothermic than the autoxidation of 8a (-74.7 versus 54.1 kJ mol -1 ), the obtained yield is significantly lower. This agrees with the calculated results of the BDEs of 8a and 9a

(299.2 versus 304.1 kJ mol -1 ) (Table 1, Entries 5, 6). In good agreement with the calculated BDE (368.4 kJ mol -1 ), tricyclic pyrazinoquinazoline 10a yielded hydroperoxide 10b after 24 hours in only 12 % yield ( ) and the smallest exothermic reaction enthalpy (-6.9 kJ mol -1 ) (Table 1, Entry 8), thus reflecting the poor yield.

When tricyclic pyrazinoquinazoline 12a was submitted to the autoxidation, hydroperoxide 12b could be isolated after 24 hours in 67 % yield (Table 3,Entry 11).

It should be noted that the autoxidation of 12a has to be much more exothermic than the reaction of 8a (-74.9 versus -54.1 kJ mol -1 ) and that the BDE of 12a

(248.4 kJ mol -1 ) is the lowest in the whole series indicating the improved yield of 12b

(Table 1, Entry 10).

Although 13a has a lower BDE than 10a (323.7 versus 368.4 kJ mol -1 ) and even a similar reaction enthalpy (-10.9 versus -6.9 kJ mol -1 ), neither autoxidation to the corresponding hydroperoxide nor decomposition could be observed and compound 13a could be reisolated completely ( Thus the theoretical forecast of regioisomeric hydroperoxide formation via comparison of calculated BDEs and reaction enthalpies seem to be highly valuable for the experimentalist.

Conclusion

Upon comparison of the results from calculations of BDEs and reaction enthalpies with the experimentally determined yields and reaction times of organic hydroperoxides derived from diketopiperazines and pyrazinoquinazolines, several trends can be concluded. Pyrazinoquinazolines are much faster oxidised with molecular oxygen to the corresponding hydroperoxides than diketopiperazines.

Formation of the hydroperoxide occurs preferently at a tertiary α-C-H bond next to an

  , triplet benzophenone abstracts a hydrogen atom from the diketopiperazine producing a diketopiperazine radical. This diketopiperazine radical then adds molecular oxygen to form the corresponding peroxy radical, which dehydrogenated an additional diketopiperazine or the diphenylhydroxymethyl radical to close the catalytic cycle (Scheme 1).

  13 C-NMR (75 MHz, CDCl 3 ): δ = 14.2 (iProp-CH 3 ), 14.3 (iProp-CH 3 ), 15.9 (iProp-CH 3 ), 18.3 (OCH 2 CH 3 ), 19.5 (iProp-CH 3 ), 31.8 (CH-(CH 3 ) 2 ), 58.1 (CH-(CH 3 ) 2 ), 61.2 (CH-iProp), 62.4 (CH-iProp), 62.6 (OCH 2 CH 3 ), 173.4 (C=O), 173.8 (N=C-O) ppm. HRMS (ESI): Anal. calcd. for C 12 H 22 N 2 O 2 Na + : 249.1579, found: 249.1577. MS (ESI): m/z = 249.15. FT-IR (ATR): 3232 w, 2961 m, 2873 w, 1646 s, 1140 s, 1327 w

3 J 3 J 5 J

 335 4S)-1,4-Diisopropyl-1H-pyrazino[2,1-b]quinazoline-3,6(2H,4H)-dione (12a)Sublimated anthranilic acid (1.324 g, 9.66 mmol) was added to a solution of (3S,6S)-3,6-diisopropyl-5-ethoxy-1,6-dihydropyrazin-2(3H)-one 24 (1.713 g, 7.57 mmol) in ). The reaction mixture was heated in microwave for 15 min at 135°C. The solvent was removed under reduced pressure and the residue was purified by silica gel flash chromatography (hexanes/ethyl acetate) affording pyrazinoquinazoline 12a as a colourless solid (1.841 g, 81 % yield). 1 H-NMR (500 MHz, CDCl 3 ): δ = 0.93 (d, 3 J = 6.8 Hz, 3H, CH 3 ), 1.15 (d, 3 J = 6.8 Hz, 3H, CH 3 ), 1.16 (d, 3 J = 6.7 Hz, 3H, CH 3 ), 1.30 (d, 3 J = 6.7 Hz, 3H, CH 3 ), 2.07 -2.15 (m, 2H, CH-(CH 3 ) 2, CH-(CH 3 ) 2 ), 4.02 (dd, 3 J = 8.9Hz, 3 J = 5.2 Hz, 1H, 1-H), 5.05 (d, 3 J = 9.3 Hz, 1H, 4-H), 7.50 (ddd, 3 J = 8.1 Hz, 3 J = 7.1 Hz, 4 J = 1.2 Hz, 1H, 8-H), 7.66 (dd, = 8.3 Hz, 4 J = 1.2 Hz, 1H, 10-H), 7.79 (ddd, 3 J = 8.3 Hz, 3 J = 7.1 Hz, 4 J = 1.5 Hz, 1H, 9-H), 8.22 (dd, 3 J = 8.1 Hz, 4 J = 1.5 Hz, 1H, 7-H), 8.74 (d, 3 J = 5.2 Hz, 1H, -NH) ppm. 13 C-NMR (125 MHz, CDCl 3 ): δ = 24.0 (CH 3 ), 24.8 (CH 3 ), 25.1 (CH 3 ), 25.8 (CH 3 ), 38.6 (CH-(CH 3 ) 2 ), 40.0 (CH-(CH 3 ) 2 ), 65.0 (C-1), 67.2 (C-6), 124.6 (C-10a), 131.5 (C-7), 131.6 (C-10), 131.9 (C-8), 139.4 (C-9), 151.6 (C-6a), 155.5 (C-11a), 166.3 (C-6), 173.0 (C-3) ppm. HRMS (ESI): Anal. calcd. for C 17 H 21 N 3 O 2 Na + : 322.1526, found: 322.1517. MS (ESI): m/z = 322.1. FT-IR (ATR): 2960 (m), 2925 (s), 2873 (s), 2853 (m), 1741 (m), 1669 (s), 1615 (s), 1517 (m), 1377 (m), 1231 (s), 1024 (s), 753 (sPyrazinoquinazoline 8a was obtained as colourless crystals (0.089 g, 73 %). 1 H-NMR (500 MHz, CDCl 3 ): δ = 2.06 -2.10 (m, 1H, 2-H a ), 2.13 -2.15 (m, 1H, 2-H b ), 2.56 -2.65 (m, 1H, 1-H a ), 2.65 -2.68 (m, 1H, 1-H b ), 3.58 -3.61 (m, 1H, 3-H a ), 3.63 -3.73 (m, 1H, 3-H b ), 4.15 (d, 2 J = 17.1 Hz, 1H, 6-H a ), 4.69 (dd, 3 J = 8.4 Hz, 3 J = 7.1 Hz, 1H, 13b-H), 5.42 (d, 2 J = 17.1 Hz, 1H, 6-H b ), 7.50 (ddd, 3 J = 8.0 Hz, 3 J = 7.0 Hz, 4 J = 1.2 Hz, 1H, 10-H), 7.68 (ddd, 3 J = 8.2 Hz, 4 J = 1.2 Hz, 5 J = 0.6 Hz, 1H, 12-H), 7.76 (ddd, = 8.2 Hz, 3 J = 7.0 Hz, 4 J = 1.6 Hz, 1H, 11-H), 8.28 (ddd, 3 J = 8.0 Hz, 4 J = 1.6 Hz, = 0.6 Hz, 1H, 9-H) ppm. 13 C-NMR (125 MHz, CDCl 3 ): δ = 22.9 (C-2), 28.5 (C-1), 44.7 (C-3), 46.0 (C-6), 58.5 (C-13b), 119.9 (C-12a), 126.2 (C-9), 127.0 (C-12), 127.1 (C-11), 134.6 (C-8a), 146.9 (C-10), 152.7 (C-13a), 159.7 (C-8), 162.4 (C-5) ppm. HRMS (ESI): Anal. calcd. for C 14 H 13 N 3 O 2 Na + 278.0900, found: 278.0893. MS (EI): m/z = 255.1 (100), 226.1 (21), 199.1 (24), 185.1 (5), 170.1 (4), 130.0 (5), 102.8 (4), 76.0 (4). FT-IR (ATR): 2949 (w), 2896 (w), 2367 (w), 2182 (w), 1982 (w), 1680 (s), 1658 (s), 1606 (s), 1439 (m), 1390 (m), 1164 (m), 769 (s), 694 (s) cm -1 . 20 D ] [α = + 172° (c 1.0; CHCl 3 ).

3 J 3 J

 33 Pyrazinoquinazoline hydroperoxide 8b was obtained as a colourless solid(41 mg, 44 % yield). 1 H-NMR (500 MHz, DMSO-d 6 ): δ = 1.95 (m, 1H, 2-H a ), 2.15 (m, 1H, 2-H b ), 2.45 (ddd, 2 J = 12.6 Hz, 3 J = 7.2 Hz, 3 J = 2.1 Hz, 1H, 1-H a ), 2.58 (dd, 2 J = 12.6 Hz, 3 J = 7.2 Hz, 1H, 1-H b ), 3.41 (m, 1H, 3-H a ), 3.67 (m, 1H, 3-H b ), 4.45 (d, 2 J = 17.2 Hz, 1H, 6-H a ), 4.90 (d, 2 J = 17.2 Hz, 1H, 6-H b ), 7.59 (ddd, 3 J = 8.0 Hz, = 7.0 Hz, 4 J = 1.2 Hz, 1H, 10-H), 7.73 (ddd, 3 J = 8.3 Hz, 4 J = 1.2 Hz, 5 J = 0.6 Hz, 1H, 12-H), 7.87 (ddd, 3 J = 8.3 Hz, 3 J = 7.0 Hz, 4 J = 1.5 Hz, 1H, 11-H), 8.17 (ddd, = 8.0 Hz, 4 J = 1.5 Hz, 5 J = 0.6 Hz, 1H, 9-H), 11.57 (s, 1H, OOH) ppm. 13 C-NMR (125 MHz, DMSO-d6):δ = 20.1 (C-2), 36.9 (C-1), 44.5 (C-3), 45.7 (C-6), 88.2 (C-13b), 120.0 (C-12a), 126.2 (C-9), 127.3 (C-12), 127.3 (C-11), 134.7 (C-10), 139.4 (C-8a), 149.8 (C-13a), 161.6 (C-8), 167.4 (C-5) ppm. HRMS (ESI): Anal. calcd. for C 14 H 13 N 3 O 4 +Na + : 310.0798, found: 310.0785. MS (ESI): m/z = 310.1, 288.1, 272.1, 254.1, 203.0, 175.1. FT-IR (ATR): 2921 (m), 2851 (m), 1749 (m), 1731 (m), 1663 (s), 1607 (m), 1169 (s), 1124 (s), 1024 (s), 800 (s), 696 (s) cm -1

9 (

 9 13 C-NMR (125 MHz, d6-DMSO): δ = 18.ppm. HRMS (ESI): Anal. calcd. for C 14 H 15 N 3 O 4 Na + : 312.0955, found: 312.0959. MS (ESI): m/z = 312.1. FT-IR (ATR): 2966 (w), 1664 (s), 1604 (s), 1471 (m), 1401 (m), 1332 (m), 1171 (m), 1022 (s), 771 (m), 727 (s), 695 (m) cm -1 . 20 D ] [α = -6°(c 1.0; CHCl 3 ).

  4), 167.4 (C-10), 176.6 (C-12) ppm. HRMS (EI): Anal. calcd. for C 14 H 13 N 3 O 4 , M-H + 286.0822, found 286.0824. MS (EI): m/z = 286.1, 228.0, 201.0, 157.0, 113.0, 87.0. FT-IR (ATR): 2915 (m), 1719 (s), 1691 (s), 1661 (s), 1620 (s), 1262 (s), 1024 (s), 801 (m), 753 (m), 692 (m) cm -1 .

Scheme 3 .

 3 Scheme 3. Diketopiperazines, pyrazinoquinazolines and their corresponding hydroperoxides. (Conditions for the autoxidation: 1 atmosphere O 2 , UV radiation, benzophenone (0.01 eq.), room temperature.)

  Scheme 4. Synthesis of the diketopiperazines.

Scheme 5 .

 5 Scheme 5. Synthesis of lactim ethers from the corresponding diketopiperazines.

  being irradiated with UV radiation (UV LED) to obtain the corresponding hydroperoxides (Table

19 Scheme 6 :

 196 Scheme 6: Side product formation during prolonged autoxidation.

  mentally oxidized; namely diketopiperazines 1a,b and 6a and pyrazinoquinazolines 8a, 9a, 10a and 12a. Whereas autoxidation of symmetric tricyclic diketopiperazine 1a for maximum conversion and gave hydroperoxide 1b in only 39 % yield (Table

Table 2 :

 2 Condensation of lactim ethers with anthranilic acid to the corresponding

	Lactimether Equiv. 27	Product	Yield [%]
	21	1.1	8a	73
	22	1.1	9a	59
	23	1.3	10a	84
	25	1.3	11a	0
	24	1.3	12a	81
	26	2.4	13a	12

pyrazinoquinazolines. Reaction conditions: 1.1-2.4 equivalents of anthranilic acid, solvent: acetonitrile, microwave irradiation 300 W, 135°C, 15 min.

Table 3 :

 3 Aerobic oxidation of diketopiperazines 1a, b, 6a and pyrazinoquinazolines

	8a, 9a, 10a, 12a to the corresponding hydroperoxides. Reaction conditions: 1 atmos-
	phere O 2 , room temperature, UV-radiation (UV LED), benzophenone (0.01 eq.).
	Entry Heterocycle Solvent Time [h]	Hydroperoxide	Yield [%]
	1	1a	EtOAc	120 h	1b	39
	2	1a	CHCl 3	24 h	1b	0 a
	3	1a	CHCl 3	120 h	1b	23
	4	1b	CHCl 3	48 h	1c	32
	5	6a	EtOAc	168 h	6b	14
	6	6a	CHCl 3	24 h	6b	0 a
	7	8a	CHCl 3	24 h	8b	44
	8	8a	CHCl 3	48 h	8b	13
	9	9a	CHCl 3	24 h	9b	27
	10	10a	CHCl 3	24 h	10b	12
	11	12a	CHCl 3	24 h	12b	67
	12	13a	CHCl 3	24 h	13b	0 a
	13	13a	CHCl 3	48h	13b	0 a

a No conversion was observed; starting material was completely reisolated.

Table 3 ,

 3 Entry 10). This further

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 1 ,

 1 Entry 12, Table 3, Entries 12, 13).

With regard to the formation of regioisomeric hydroperoxides 9b, d and 12b, d it should be pointed out, that autoxidation experiments did not give any trace of regioisomers 9d and 12d, respectively. Only those hydroperoxides 9b, 12b were detected experimentally (Table

3

, Entries 9, 11) which had the lower reaction and bond dissociation enthalpy (Table

1

,

Entries 6, 7, 10, 11, Table 3, Entries 9, 11)

.

Table 1 .

 1 Calculated bond dissociation and reaction enthalpies for diketopiperazine derivatives 1a, b, 6a, 7a and pyrazinoquinazolines 8a -13a. Thermal and ZPE corrections were calculated with harmonic frequencies scaled by a factor of 0.99 at the B3LYP level of theory.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

N-aryl-imino moiety not next to a tertiary α-C-H bond next to an amido moiety. If both regioisomeric hydroperoxides are possible in a particular heterocycle, the N-arylimino α-hydroperoxide is exclusively formed in agreement with the theoretical calculations which reveal a much smaller BDE and a more exothermic reaction enthalpy as compared to the amido moiety. Thus theoretical calculations can be used a

http://mc.manuscriptcentral.com/poc Journal of Physical Organic Chemistry

Journal of Physical Organic Chemistry

Acknowledgments

Generous financial support by the Deutsche Forschungsgemeinschaft (SFB 706), the Fonds der Chemischen Industrie, the Ministerium für Wissenschaft, Forschung und Kunst des Landes Baden-Württemberg is gratefully acknowledged. We would like to thank Katharina Kress, Raphael Marx and Dominik Dengler for the skilful technical assistance.

chiral diketopiperazine hydroperoxides

Wassiliki Argyrakis, [a] Christoph Köppl, [b] Hans-Joachim Werner, [b] Wolfgang Frey, [a] Angelika Baro, [a] Sabine Laschat* [a] [a] Institut für Organische Chemie der Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany, Fax: (internat.) +49 (0)711/685 64285, E-mail: sabine.laschat@oc.uni-stuttgart.de [b] Institut für Theoretische Chemie der Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany

Supplementary Material Table of content

1. A summary of B3LYP, RMP2, thermal and ZPE results for all calculated reactions. The results were obtained as described in the article.

B3LYP optimised geometries of the investigated compounds in angstrom (Å).

The geometries were obtained as described in the article and follow the same numbering. Structure names with a prime (e.g. 9a') are derived from the corresponding parent structures (e.g. 9a) by the abstraction of a hydrogen atom and therefore represent radicals; on doubly primed structures (e.g. 9a'') a hydrogen atom located on an alternative position was removed.


Malgorzata Farnik

Wladyslaw Pierzchala

Grzegorz Brożek

Jan E Zejda

Michal Skrzypek

Małgorzata Farnik

M D Phd¹

Władysław Pierzchała

Grzegorz Broŝek

M D Phd²

MS Michał Skrzypek

MD Małgorzata Farnik

Małgorzata Farnik

MS Michał Skrzypek

MD Małgorzata Farnik

Quality of Life Protocol in the Early Asthma Diagnosis in Children

Keywords: asthma, quality of life, early diagnosis, child asthma, pediatrics, health-related quality of life, early diagnosis asthma, pediatrics, health-related quality of life, early diagnosis

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The results of International Study of Asthma and Allergies in Childhood (ISAAC) have shown that childhood asthma represents an important public health problem worldwide (1). Previously increasing prevalence of childhood asthma, well documented in many countries (2,3,4) can be observed in Eastern Europe over the years 1994-2007 (1,5,6,7). The magnitude of the problem could be underestimated because of underdiagnosis (8). The resulting public health burden justifies the development and implementation of population-based measures. Asthma not only has a profound impact on public health, but also on the everyday lives of individual patients.

A patients' burden of chronic illness cannot be assessed by disease severity; it requires measurement reflecting personal perception. There is an increasing interest in activity limitation, emotional functioning, impact on every-day life due to disease and the implementation of health-related quality of life (HRQOL) questionnaires.

Deterioration of HRQOL has been described in children and adults with allergic diseases, such as eczema (9) and rhinitis (10). HRQOL assessment highlights important and troublesome problems experienced by patients with asthma (11,12,13). Children with asthma suffer from activity limitation, emotional problems such frustration, fear and irritation (14). Although HRQOL does not correlate closely with asthma control, it is known that children with very severe asthma tend to have a poorer quality of life than those suffering from mild or moderate condition (15).

Parents of children with asthma could be limited in normal daily activities and experience emotional distress (16).

Materials and Methods

A large population-based respiratory health survey resulted in identification of new pediatric asthma patients. All newly identified children with asthma aged 7-11 years and their parents were involved in HRQOL study. A disease-specific quality-oflife questionnaire was used for the children and a generic questionnaire for parents.

Clinical status data was obtained prior to an HRQOL assessment.

Participants

Children were recruited using cluster sampling during a respiratory health survey study in primary schools in Katowice (Southern Poland). 2254 children grades I-IV, in 7 out of 51 schools in the town were invited (17.0% of the town population in the defined age group) for respiratory questionnaire assessment. This was a validated questionnaire, used in The Central European Study of Air Pollution and Respiratory Health (CESAR) [Table 1] and other surveys (22,23,24). The response rate was 80.8% (1822 children) . Study design is shown in Fig. 1] -Respiratory Questionnaire (examples of questions) Clinical evaluation was proposed to all children in "Asthma"-group and 60% samples from groups "Respiratory Symptoms" and "No Respiratory Symptoms", with the response rates: 41.4%, 70.8% and 25.6%, respectively. A certified pulmonologist (Department of Pneumonology, Medical University of Silesia) examined 415 children.

The physician was blind to the results of the questionnaire. Diagnostic procedures were based on GINA guidelines for childhood asthma (25). Forced expiratory volumes were measured according to ERS/ATS recommendations (26). Spirometry was considered normal if FEV 1, FVC and PEF values exceeded 80% predicted and if FEV 1 %FVC ratio was greater than 75%. If actual values presented predicted values, a further challenge test of bronchial hyperresponsiveness, described by Gerald et al (27), was used. A sub-maximal exercise test (stepping up and down on a single step with metronome rhythm) was followed by spirometry obtained at minutes 3, 5 and 10 after exercise. An FEV 1 decrease greater than 15% was considered positive for asthma. Medical records were provided; additional skin allergy tests and a chest radiograph were carried out, where needed.

Clinical assessment of 415 children confirmed 33 previously known and revealed 29 new cases of asthma. All newly identified children with asthma and their parents were invited to HRQOL assessment.

HRQOL measurements

Quality of life questionnaires were completed by children with asthma at school in the patient's normal psychosocial environment. Parents of newly diagnosed children The questionnaire required circling a choice from seven responses devised for each question, from 1 (most bothered or limited) to 7 (not bothered or limited). All questions concerned parental HRQOL of previous two-week period.

Psychometric data

PAQLQ

The instrument is reproducible (ICC =0.95) and has shown ability to detect HRQOL changes as a result of treatment (p<0.001). Estimation of reliability in stable asthma has shown that it was consistent throughout all domains (ICC: 0.84-0.93).

QLCCDQ

Reproducibility based on the weighted kappa coefficients showed an expected level of agreement. Values of weighed kappa coefficients suggested almost perfect agreement (0.81-1.0) in case of 8 questions, substantial (0.61-0.8) for 5 questions and moderate (0.41-0.6) for 2 questions. The reliability of the defined subscales was evaluated by means of Cronbach's α . The values of α ranged between 0.74 and 0.94: symptoms: 0.93, emotions: 0.91, family roles: 0.77, social roles: 0.79, occupational roles: 0.92 and activity limitations in roles function: 0.91. Supplementary data such as school grades and absenteeism (missed school days due to asthma symptoms) were provided to determine if there were relationships between reported HRQOL and child's functioning at school. It could be expected that untreated asthma has an impact on school functioning resulting in children lower school grades and missed school days. Polish school grades assessment is based on 6-point scale: from 1 (Fail) to 6 (Excellent).

Child's school grades and absenteeism

The study protocol was approved by the ethics committee (Medical University of Silesia). Each child admitted to the study had the written consent of a parent.

Statistical analyses

The statistical analysis was performed using standard procedures available in the Statistica 7. Correlations between child's and parental HRQOL scores were analyzed. Relations between clinical status (symptoms, lung function tests) and HRQOL were examined, as well as links between educational markers (school grades and absenteeism) and patient HRQOL scores. Gender and age effects on HRQOL were also analyzed.

Results

Participating children

In children with newly diagnosed asthma (mean age = 8.6 years; SD=1.4 years), classified according to GINA protocol, the disease was either episodic (20 children: 6 boys and 14 girls) or chronic mild (9 children: 4 boys and 5 girls). New diagnoses of asthma were made in 10.9% of children from the "Respiratory Symptoms" group and in 3.6% of children from the "No Respiratory Symptoms" group. All 29 newly diagnosed children with asthma and 21 parents responded to the subsequent HRQOL questionnaire.

[Table 2 ] -Demographic and asthma severity Patient and parental HRQOL HRQOL was assessed for each domain, the subscale mean ranging from 1.0 to 7.0. Lower HRQOL scores reflected greater impact of disease on every-day functioning.

Children's most affected HRQOL domain was limitation of activity. Parents' impairment was most severe in emotional functioning and perceived symptoms, while less impaired domains included social functioning. Domains and overall HRQOL scores for children and parents appear in the table 3.

[Table 3]. HRQOL -domains and overall scores Statistically important positive correlations were observed between all PAQLQ subscales, as well as most QLCCDQ subscales [ Table 4]. QLCCDQ symptom perception subscale correlated only with parental emotional functioning, while Bonferroni correction suggested significance level was p < 0.0001.

[Table 4]. PAQLQ and QLCCDLQ subscales Spearman's correlations

HRQOL measures and clinical assessment Symptoms

The differences of mean QLCCDQ and PAQLQ subscales scores between groups of symptomatic and nonsymptomatic children over in the past 12 months were analyzed. Both for QLCCDQ and PAQLQ the differences in scores did not reach statistical significance. Bonferroni correction suggested significance level was p < 0.01.

Lung function tests

Statistically important correlations were found between HRQOL and spirometry. Challenge tests were done in 23 of the 29 children -3 children did not obtain reproducible spirometry after exercise, 2 children were wheezing before exercise commenced, 1 child refused to cooperate. Baseline FEV 1 %FVC and symptom domain in children showed a correlation (r=0.42), while exercise value FEV 1 %FVC correlated with emotional domain (r=0.7). MEF25-75% correlated with QLCCDQ family roles (r=0.81) and social functioning (r=0.81). PAQLQ activity limitation was correlated with MEF 25-75% (r=0.44) and PEF (r=0.55), both obtained during the exercise test. All exercise test correlations were based on spirometry results collected 10 minutes after the challenge test [Table 5].

[ Table 5]. HRQOL scores and lung function -Spearmans' correlations No correlations were observed in parents' HRQOL and missed school days or school marks.

Patient gender and age

Females showed a significantly lower symptom score in comparison to males (p=0.01), however this effect was not translated into total PAQLQ scores nor into parents' HRQOL.

The relationship between HRQOL scores and age were not statistically significant.

Discussion

Our study focused on the implementation of the HRQOL protocol in early diagnosis of childhood asthma. The analysis involved data obtained in children with newly diagnosed asthma and in their parents. Since asthma cases were identified through population-based screening, it is not surprising that all previously undiagnosed cases represented either episodic or mild asthma -children with more advanced disease had been diagnosed on regular basis, via pediatric health care.

An important finding of our study is that the profile of HRQOL impairment is different in children with asthma and in their parents. Not only did PAQLQ and QLCCDQ scores not correlate but -in addition -the highest impaired domains differed between children and parents. Symptom perception and emotional functioning appeared to be the most affected HRQOL domains in parents, while activity limitation was the most impaired domain in children. Some parents might limit their child's activity, due to emotional distress caused by anticipation of symptoms, and such behavior could explain the lack of correlation between emotion domain QLCCDQ and symptom or activity limitation domains in PAQLQ.

It is of interest that parental HRQOL pattern is in line with the published qualitative evidence that parents tend to perceive symptoms of asthma in a child as intermittent acute episodes rather than a chronic condition (28). Such interpretation could explain the lack of diagnosis of episodic or mild asthma in our study. It cannot be excluded that such a presentation is not entirely neglected by parents, but treated by them as an annoying condition and not overt disease. Our finding could have some implication as far as screening is concerned -occurrence of symptoms is necessary The relationships between clinical indices and HRQOL were analyzed in many studies as an important observation for clinicians (29,30,31,32). Our study has shown that there were no statistically significant differences in both patient and parental HRQOL scores between symptomatic and asymptomatic groups. However some of analyzed relationships were suggested as significant; before the significance level was lowered by applying Bonferroni correction to p < 0.01 . Our findings were obtained in newly diagnosed and mild cases of asthma. Studies addressing established childhood asthma showed that the correlations between the clinical status and HRQOL scores are weak to moderate, even for disease-specific measures ( 29,30,31). A perception of dyspnea, although a crucial asthma symptom, might show interpersonal and intrapersonal variations [START_REF] Juniper | Byrne PM Relationship between quality of life and measures of clinical status in asthma: a factor analysis[END_REF]. Although children with severe asthma tend to have a poorer quality of life than children with milder disease [START_REF] Martinez -Moragon | Asthma patients' perception of dyspnea during acute bronchoconstriction[END_REF], parental and children's perception can be influenced by a number of factors. Consistent with findings in the G. Guyatt's study, in children aged 7-10, clinicians cannot rely on parental reports to provide insight into the child's HRQOL. This study also showed that the patient's assessment of change in symptoms were more closely related to HRQOL scores than parental assessment (20). A different effect provided by our study could be discussed in terms of early society [START_REF] Trute | Family adjustment to Childhood Developmental Disability : a measure of parent appraisal of family impacts[END_REF]. In contrast an increasing parental stress could diminish compliance and adjustments to illness [START_REF] Trute | Family adjustment to Childhood Developmental Disability : a measure of parent appraisal of family impacts[END_REF][START_REF] Streisand | Childhood Illness -Related Parenting Stress: The Pediatric Inventory for Parents[END_REF].

The limitation of the study is its relatively small sample, although over 2000 randomly selected children were involved in the study. Despite the response rate being 80.8% (1822 children) during the first stage of study, fewer children responded to invitation for clinical evaluation (the response rates ranged from 25.6 to 70.8% depending the clinical group). Decreasing response rates should be considered as a limitations if longitudinal assessment would be considered. Final data collection (HRQOL) was completed on 29 newly diagnosed asthmatic children and 21 parents. Thus not all parents responded to HRQOL assessment and this should be also considered as the limitation of this study.

More complete data could be received if a multicenter study was conducted or if rural areas were included. Further studies are necessary to determine whether similar results could be found in other areas.

Other aspects should be considered, for example parental education or financial situation. Such conditions could be considered as possible mediators in a future research. Further studies could explain the role of social factors in the underdiagnosis of asthma in children.

Nevertheless the method used in our study provides pertinent data concerning the parental perception of patient disease and the questionnaire is a useful method to describe the impact of disease on parental quality of life. Pre and post-intervention HRQOL as an outcome measure could be implemented for evaluation education or coaching programs, and provide important information if strategies satisfy individual needs and health expectations. (usually mild cases) has an important impact on every-day family functioning. What seems to be of importance is that asthma-related emotional changes were visible both in parents and in affected children. This observation could translate into a practically important result. The added value of the assessment of HRQOL extends beyond the diagnostic phase. It offers a better means of asthma management, such as for example family-tailored education and coaching. Therefore it is necessary to consider a multidisciplinary approach in the childhood asthma, even in mild cases, as ill children and their parents are subjects of care.

In conclusion, HRQOL newly recognized children with asthma and their parents doesn't correlate closely with clinical status. Patient and parental HRQOL should be considered as independent dimensions. A patients' burden of chronic illness cannot be assessed by disease severity; it requires measurement reflecting personal perception. There is an increasing interest in activity limitation, emotional functioning, impact on every-day life due to disease and the implementation of health-related quality of life (HRQOL) questionnaires.

Deterioration of HRQOL has been described in children and adults with allergic diseases, such as eczema (9) and rhinitis (10). HRQOL assessment highlights important and troublesome problems experienced by patients with asthma (11,12,13). Children with asthma suffer from activity limitation, emotional problems such frustration, fear and irritation (14). Although HRQOL does not correlate closely with asthma control, it is known that children with very severe asthma tend to have a poorer quality of life than those suffering from mild or moderate condition (15).

Parents of children with asthma could be limited in normal daily activities and experience emotional distress (16). Clinical experience shows that a caregiver's HRQOL and their perception of a child's symptoms are important in diagnosis and control of established asthma (17).

Less is known about the potential impact of disease on HRQOL in newly recognized children with asthma which were found as the result of screening programs. It remains unknown to what extent such a procedure could be helpful in both early assessment of asthma and its impact on patients' and parental quality of life.

We implemented the HRQOL protocol in examination of newly identified pediatric asthma patients and their families, participants of a large population-based respiratory health survey. The goal of the study was to determine the impact of asthma on the health related quality of life of newly recognized pediatric asthma patients and their parents. 1] and other surveys (22,23,24). The response rate was 80.8% (1822 children) . Study design is shown in Fig. 1 1] -Respiratory Questionnaire (examples of questions) Clinical evaluation was proposed to all children in "Asthma"-group and 60% samples from groups "Respiratory Symptoms" and "No Respiratory Symptoms", with the response rates: 41.4%, 70.8% and 25.6%, respectively. A certified pulmonologist (Department of Pneumonology, Medical University of Silesia) examined 415 children.

The physician was blind to the results of the questionnaire. Diagnostic procedures were based on GINA guidelines for childhood asthma (25). Forced expiratory volumes were measured according to ERS/ATS recommendations (26). Spirometry was considered normal if FEV 1, FVC and PEF values exceeded 80% predicted and if FEV 1 %FVC ratio was greater than 75%. If actual values presented predicted values, a further challenge test of bronchial hyperresponsiveness, described by Gerald et al (27), was used. A sub-maximal exercise test (stepping up and down on a single step with metronome rhythm) was followed by spirometry obtained at minutes 3, 5 and 10 after exercise. An FEV 1 decrease greater than 15% was considered positive for asthma. Medical records were provided; additional skin allergy tests and a chest radiograph were carried out, where needed.

Clinical assessment of 415 children confirmed 33 previously known and revealed 29 new cases of asthma. All newly identified children with asthma and their parents were invited to HRQOL assessment.

HRQOL measurements

Quality of life questionnaires were completed by children with asthma at school in the patient's normal psychosocial environment. Parents of newly diagnosed children Supplementary data such as school grades and absenteeism (missed school days due to asthma symptoms) were provided to determine if there were relationships between reported HRQOL and child's functioning at school. It could be expected that untreated asthma has an impact on school functioning resulting in children lower school grades and missed school days. Polish school grades assessment is based on 6-point scale: from 1 (Fail) to 6 (Excellent).

The study protocol was approved by the ethics committee (Medical University of Silesia). Each child admitted to the study had the written consent of a parent.

Statistical analyses

The statistical analysis was performed using standard procedures available in the Statistica 7.1 package and SAS, version 9.2 (SAS Institute Inc., Gary, NC). Correlations between child's and parental HRQOL scores were analyzed. Relations between clinical status (symptoms, lung function tests) and HRQOL were examined, as well as links between educational markers (school grades and absenteeism) and patient HRQOL scores. Gender and age effects on HRQOL were also analyzed.

Normality of distributions of continuous variables was analyzed using the

Results

Participating children

In children with newly diagnosed asthma (mean age = 8.6 years; SD=1.4 years), classified according to GINA protocol, the disease was either episodic (20 children: 6 boys and 14 girls) or chronic mild (9 children: 4 boys and 5 girls). New diagnoses of asthma were made in 10.9% of children from the "Respiratory Symptoms" group and in 3.6% of children from the "No Respiratory Symptoms" group. All 29 newly diagnosed children with asthma and 21 parents responded to the subsequent HRQOL questionnaire.

[Table 2 ] -Demographic and asthma severity Patient and parental HRQOL HRQOL was assessed for each domain, the subscale mean ranging from 1.0 to 7.0. Lower HRQOL scores reflected greater impact of disease on every-day functioning.

Children's most affected HRQOL domain was limitation of activity. Parents' impairment was most severe in emotional functioning and perceived symptoms, while less impaired domains included social functioning. Domains and overall HRQOL scores for children and parents appear in the table 3.

[Table 3]. HRQOL -domains and overall scores Statistically important positive correlations were observed between all PAQLQ subscales, as well as most QLCCDQ subscales [ Table 4]. QLCCDQ symptom perception subscale correlated only with parental emotional functioning, while Bonferroni correction suggested significance level was p < 0.0001.

[Table 4]. PAQLQ and QLCCDLQ subscales Spearman's correlations

HRQOL measures and clinical assessment Symptoms

The differences of mean QLCCDQ and PAQLQ subscales scores between groups of symptomatic and nonsymptomatic children over in the past 12 months were analyzed. Both for QLCCDQ and PAQLQ the differences in scores did not reach statistical significance. Bonferroni correction suggested significance level was p < 0.01.

Lung function tests

Statistically important correlations were found between HRQOL and spirometry. Challenge tests were done in 23 of the 29 children -3 children did not obtain reproducible spirometry after exercise, 2 children were wheezing before exercise commenced, 1 child refused to cooperate. Baseline FEV 1 %FVC and symptom domain in children showed a correlation (r=0.42), while exercise value FEV 1 %FVC correlated with emotional domain (r=0.7). MEF25-75% correlated with QLCCDQ family roles (r=0.81) and social functioning (r=0.81). PAQLQ activity limitation was correlated with MEF 25-75% (r=0.44) and PEF (r=0.55), both obtained during the exercise test. All exercise test correlations were based on spirometry results collected 10 minutes after the challenge test [Table 5].

[ Table 5]. HRQOL scores and lung function -Spearmans' correlations

School grades and absenteeism

Higher scores on the symptoms subscale of PAQLQ correlated with better school grades (r= 0.64; p=0.02) and fewer missed school days (r= -0.53; p=0.02).

No correlations were observed in parents' HRQOL and missed school days or school marks.

Patient gender and age

Females showed a significantly lower symptom score in comparison to males (p=0.01), however this effect was not translated into total PAQLQ scores nor into parents' HRQOL.

The relationship between HRQOL scores and age were not statistically significant.

Discussion

Our study focused on the implementation of the HRQOL protocol in early diagnosis of childhood asthma. The analysis involved data obtained in children with newly diagnosed asthma and in their parents. Since asthma cases were identified through population-based screening, it is not surprising that all previously undiagnosed cases represented either episodic or mild asthma -children with more advanced disease had been diagnosed on regular basis, via pediatric health care.

An important finding of our study is that the profile of HRQOL impairment is different in children with asthma and in their parents. Not only did PAQLQ and QLCCDQ scores not correlate but -in addition -the highest impaired domains differed between children and parents. Symptom perception and emotional functioning appeared to be the most affected HRQOL domains in parents, while activity limitation was the most impaired domain in children. Some parents might limit their child's activity, due to emotional distress caused by anticipation of symptoms, and such behavior could explain the lack of correlation between emotion domain QLCCDQ and symptom or activity limitation domains in PAQLQ.

It is of interest that parental HRQOL pattern is in line with the published qualitative evidence that parents tend to perceive symptoms of asthma in a child as intermittent acute episodes rather than a chronic condition (28). Such interpretation could explain the lack of diagnosis of episodic or mild asthma in our study. It cannot be excluded that such a presentation is not entirely neglected by parents, but treated by them as an annoying condition and not overt disease. Our finding could have some implication as far as screening is concerned -occurrence of symptoms is necessary The relationships between clinical indices and HRQOL were analyzed in many studies as an important observation for clinicians (29,30,31,32). Our study has shown that there were no statistically significant differences in both patient and parental HRQOL scores between symptomatic and asymptomatic groups. However some of analyzed relationships were suggested as significant; before the significance level was lowered by applying Bonferroni correction to p < 0.01 . Our findings were obtained in newly diagnosed and mild cases of asthma. Studies addressing established childhood asthma showed that the correlations between the clinical status and HRQOL scores are weak to moderate, even for disease-specific measures ( 29,30,31). A perception of dyspnea, although a crucial asthma symptom, might show interpersonal and intrapersonal variations [START_REF] Juniper | Byrne PM Relationship between quality of life and measures of clinical status in asthma: a factor analysis[END_REF]. Although children with severe asthma tend to have a poorer quality of life than children with milder disease [START_REF] Martinez -Moragon | Asthma patients' perception of dyspnea during acute bronchoconstriction[END_REF], parental and children's perception can be influenced by a number of factors. Consistent with findings in the G. Guyatt's study, in children aged 7-10, clinicians cannot rely on parental reports to provide insight into the child's HRQOL. This study also showed that the patient's assessment of change in symptoms were more closely related to HRQOL scores than parental assessment (20). A different effect provided by our study could be discussed in terms of early society [START_REF] Trute | Family adjustment to Childhood Developmental Disability : a measure of parent appraisal of family impacts[END_REF]. In contrast an increasing parental stress could diminish compliance and adjustments to illness [START_REF] Trute | Family adjustment to Childhood Developmental Disability : a measure of parent appraisal of family impacts[END_REF][START_REF] Streisand | Childhood Illness -Related Parenting Stress: The Pediatric Inventory for Parents[END_REF].

The limitation of the study is its relatively small sample, although over 2000 randomly selected children were involved in the study. Despite the response rate being 80.8% (1822 children) during the first stage of study, fewer children responded to invitation for clinical evaluation (the response rates ranged from 25.6 to 70.8% depending the clinical group). Decreasing response rates should be considered as a limitations if longitudinal assessment would be considered. Final data collection (HRQOL) was completed on 29 newly diagnosed asthmatic children and 21 parents. Thus not all parents responded to HRQOL assessment and this should be also considered as the limitation of this study.

More complete data could be received if a multicenter study was conducted or if rural areas were included. Further studies are necessary to determine whether similar results could be found in other areas.

Other aspects should be considered, for example parental education or financial situation. Such conditions could be considered as possible mediators in a future research. Further studies could explain the role of social factors in the underdiagnosis of asthma in children.

Nevertheless the method used in our study provides pertinent data concerning the parental perception of patient disease and the questionnaire is a useful method to describe the impact of disease on parental quality of life. Pre and post-intervention HRQOL as an outcome measure could be implemented for evaluation education or coaching programs, and provide important information if strategies satisfy individual needs and health expectations. (usually mild cases) has an important impact on every-day family functioning. What seems to be of importance is that asthma-related emotional changes were visible both in parents and in affected children. This observation could translate into a practically important result. The added value of the assessment of HRQOL extends beyond the diagnostic phase. It offers a better means of asthma management, such as for example family-tailored education and coaching. Therefore it is necessary to consider a multidisciplinary approach in the childhood asthma, even in mild cases, as ill children and their parents are subjects of care.

In conclusion, HRQOL newly recognized children with asthma and their parents doesn't correlate closely with clinical status. Patient and parental HRQOL should be considered as independent dimensions.

Figure 1 .

 1 Figure 1. Study design

  HRQOL questionnaire as well at school, before the diagnosis was communicated. The Paediatric Asthma Quality of Life Questionnaire The Paediatric Asthma Quality of Life Questionnaire (PAQLQ), dedicated to children aged 7-17 years old, was used as a well-validated and reliable measure of HRQOL in childhood asthma. The questionnaire consists of 23 questions, covering symptoms, emotional function and limitations of activity. Children were asked to recall their experiences over the previous week and responded on a 7-point scale (7 -no impairment, 1 -severe impairment). Patients completed the questionnaire with physicians experienced in HRQOL research. Interviewer-administration was used to avoid parental interference with patient responses. Quality of Life in a Child's Chronic Disease Questionnaire An accompanying questionnaire for parents was devised by the authors, based on standard procedure -starting from initial questions (identification of needs, definition of operational objectives), tool development, pretesting and final version (format, wording) preparation, pilot testing and validation of the questionnaire (19, 20). Quality of Life in a Child's Chronic Disease Questionnaire (QLCCDQ) is concerned with daily problems and limitations that concern parents of children with chronic disease. QLCCDQ is a self -report measure of parent's HRQOL, only symptoms subscale is parent-proxy measure. Most questions concern parents' functioning, only the symptom subscale reflects parents' perception of symptoms presence. QLCCDQ consisted of 15 questions and covered patient-perceived symptoms (e.g Did the child show any worrying symptoms?) , emotions (e.g. Have you experienced anxiety because of your child's health problems?) and activity limitation in roles function: social (e.g. Please indicate how much you have been limited by your child's activities in past two weeks: going to church, cinema, visiting friends), family roles (e.g. Do you struggle to find time to spend with other family members (spouse, another child) because of your child' disease?) and occupational (e.g. Please indicate how much you have been limited by your child's disease in work related activities in past two weeks).

  1 package and SAS, version 9.2 (SAS Institute Inc., Gary, NC). Normality of distributions of continuous variables was analyzed using the Shapiro-Wilk test. Statistical significance of differences between continuous variables was analyzed by t-Student test and ANOVA while for data with non-normal distribution, U Mann-Whitney and Kruskal-Wallis tests were used. Differences between categorical variables were examined by the chi-square test. Strength of associations, due to nonnormal distributions, were measured by Spearman's rank correlation coefficient. The statistical inferences were based on confidence levels p<0.05. If multiple comparisons were used statistical significance was lowered according to the Bonferroni adjustment.

10 Variables

 10 included patient and parental HRQOL assessment, symptoms over the previous 12 months, baseline and exercise spirometry, school grades and absenteeism.

  between most subscales describing roles functioning.

  Higher scores on the symptoms subscale of PAQLQ correlated with better school grades (r= 0.64; p=0.02) and fewer missed school days (r= -0.53; p=0.02).

  have to be a sufficient cause to seek medical advice. Other important factors include individual experience, perception and emotions.

  As the examplecompared to parents of asymptomatic children, parents of children complaining of wheezing had a lower (p=0.02) mean score in emotional function and symptom domain. As well the group of children possessing dyspnea showed a lower mean parental emotional function score (p=0.04) than in asymptomatic children. In children an emotional function score was lower in the group of children complaining of cough in the past 12 months (p=0.01) as compared to asymptomatic children. Correlations were observed for exercise lung function and QLCCDQ domains, such as emotional, family and social functions. Worthy to note, that the correlations between children's lung function and parental HRQOL domains were stronger than analogous correlations concerning HRQOL in children. The most impaired parent HRQOL domain -emotions, significantly correlated with FEV 1 % FVC. Activity limitation, the most impaired patient domain, correlated with PEF and MEF 25-75.Even if the correlations between clinical indices and HRQOL scores were observed only for few variables it should not determine a limitation of HRQOL protocol in early diagnosis. As HRQOL is not a diagnostic scale nor a disease or symptom severity scale index -the impact of disease is not systematically correlated with the disease severity as it is categorized (32) by health -care providers. Patient' or caregiver -complement traditional clinical or biological evaluation, which could suggest that HRQOL could function as part of an early assessment or as part of disease monitoring.

  diagnosis and mild clinical presentation of the newly diagnosed cases. Parental experience of their child's disease is very important for the diagnosis of asthma, and it would confirm the importance of screening programs addressed to parents, including emotional context of their functioning in relation to the child's symptoms. Parental HRQOL could be recommended as a valuable method of a more holistic assessment of disease impact on everyday functioning of child and their family. Both patient and parental perception are important in care strategies planning and implementation. Children are influenced by their parents and reduction of parental distress could be beneficial for child's family life and integration within

  EF, Wisniewski ME, Cox FM, Emmet AH, Nielsen KE, O'Byrne PM Relationship between quality of life and measures of clinical status in asthma: a factor analysis. Eur Respir J, 2004, 23: 287-291. 34. Martinez -Moragon E, Perpina M, Belloch A, de Diego A, Martinez-Frances ME. Asthma patients' perception of dyspnea during acute bronchoconstriction. Arch Bronchoneumol 2003; 39 (2): 67-73. 35. Trute B, Hiebert -Murphy D, Family adjustment to Childhood Developmental Disability : a measure of parent appraisal of family impacts. J Ped Psych; 2002; 27: 271-280. 36. Streisand R, Braniecki S, Tercyak KP, Childhood Illness -Related Parenting Stress: The Pediatric Inventory for Parents. J Ped Psych, 2001; 26 (3): 155-162. Asthma is considered an important problem in children and influences on their everyday functioning. This study was concerned with the impact of asthma on the health related quality of life (HRQOL) of newly recognized pediatric asthma patients and their parents. Methods. Patients were identified by a general respiratory survey. HRQOL was assessed by self-report measures: disease-specific questionnaire for children, the Pediatric Asthma Quality of Life Questionnaire (PAQLQ) and generic for parents -the Quality of Life in Child's Chronic Disease Questionnaire (QLCCDQ).Results. 29 newly diagnosed asthmatic children aged 7-11 years (mean age = 8.6 SD=1.4) were identified from a population-based respiratory health survey. The most impaired HRQOL domains differed in parents and children. In children, activity limitation was the most impaired; however in parents, emotional functioning and symptom perception domains were most impaired. There were no statistically significant differences in patient and parental HRQOL observed in either the group where symptoms were present or in the group were symptoms were not present.Baseline spirometry showed correlation for FEV 1 %FVC and PAQLQ symptom domain (r=0.42). Exercise test showed correlations between FEV 1 % FVC and QLCCDQ emotional domain (r=0.7); MEF25-75% and family roles (r=0.81), as well as social functioning (r=0.81). Activity limitation on the PAQLQ correlated with the MEF 25-75% (r=0.44) and PEF (r=0.55).Conclusions. HRQOL newly recognized children with asthma and their parents does not correlate closely with clinical status. Patient and parental HRQOL should be considered as independent dimensions.

Introduction

  The results of International Study of Asthma and Allergies in Childhood (ISAAC) have shown that childhood asthma represents an important public health problem worldwide(1). Previously increasing prevalence of childhood asthma, well documented in many countries (2, 3, 4) can be observed in Eastern Europe over the years 1994-2007(1, 5, 6, 7). The magnitude of the problem could be underestimated because of underdiagnosis(8). The resulting public health burden justifies the development and implementation of population-based measures. Asthma not only has a profound impact on public health, but also on the everyday lives of individual patients.

  A large population-based respiratory health survey resulted in identification of new pediatric asthma patients. All newly identified children with asthma aged 7-11 years and their parents were involved in HRQOL study. A disease-specific quality-oflife questionnaire was used for the children and a generic questionnaire for parents.Clinical status data was obtained prior to an HRQOL assessment.ParticipantsChildren were recruited using cluster sampling during a respiratory health survey study in primary schools in Katowice (Southern Poland). 2254 children grades I-IV, in 7 out of 51 schools in the town were invited (17.0% of the town population in the defined age group) for respiratory questionnaire assessment. This was a validated questionnaire, used in The Central European Study of Air Pollution and Respiratory Health (CESAR) [Table

Figure 1 .

 1 Figure 1. Study design

  HRQOL questionnaire as well at school, before the diagnosis was communicated. The Paediatric Asthma Quality of Life Questionnaire The Paediatric Asthma Quality of Life Questionnaire (PAQLQ), dedicated to children aged 7-17 years old, was used as a well-validated and reliable measure of HRQOL in childhood asthma. The questionnaire consists of 23 questions, covering symptoms, emotional function and limitations of activity. Children were asked to recall their experiences over the previous week and responded on a 7-point scale (7 -no impairment, 1 -severe impairment). Patients completed the questionnaire with physicians experienced in HRQOL research. Interviewer-administration was used to avoid parental interference with patient responses. Quality of Life in a Child's Chronic Disease Questionnaire An accompanying questionnaire for parents was devised by the authors, based on standard procedure -starting from initial questions (identification of needs, definition of operational objectives), tool development, pretesting and final version (format, wording) preparation, pilot testing and validation of the questionnaire (19, 20). Quality of Life in a Child's Chronic Disease Questionnaire (QLCCDQ) is concerned with daily problems and limitations that concern parents of children with chronic disease. QLCCDQ is a self -report measure of parent's HRQOL, only symptoms subscale is parent-proxy measure. Most questions concern parents' functioning, only the symptom subscale reflects parents' perception of symptoms presence. QLCCDQ consisted of 15 questions and covered patient-perceived symptoms (e.g Did the child show any worrying symptoms?) , emotions (e.g. Have you experienced anxiety because of your child's health problems?) and activity limitation in roles function: social (e.g. Please indicate how much you have been limited by your child's activities in past two weeks: going to church, cinema, visiting friends), family roles (e.g. Do you struggle to find time to spend with other family members (spouse, another child) because of your child' disease?) and occupational (e.g. Please indicate how much you have been limited by your child's disease in work related activities in past two weeks).The questionnaire required circling a choice from seven responses devised for each question, from 1 (most bothered or limited) to 7 (not bothered or limited). All questions concerned parental HRQOL of previous two-week period.Psychometric dataPAQLQThe instrument is reproducible (ICC =0.95) and has shown ability to detect HRQOL changes as a result of treatment (p<0.001). Estimation of reliability in stable asthma has shown that it was consistent throughout all domains (ICC: 0.84-0.93).QLCCDQReproducibility based on the weighted kappa coefficients showed an expected level of agreement. Values of weighed kappa coefficients suggested almost perfect agreement (0.81-1.0) in case of 8 questions, substantial (0.61-0.8) for 5 questions and moderate (0.41-0.6) for 2 questions. The reliability of the defined subscales was evaluated by means of Cronbach's α . The values of α ranged between 0.74 and 0.94: symptoms: 0.93, emotions: 0.91, family roles: 0.77, social roles: 0.79, occupational roles: 0.92 and activity limitations in roles function: 0.91.Child's school grades and absenteeism

  Shapiro-Wilk test. Statistical significance of differences between continuous variables was analyzed by t-Student test and ANOVA while for data with non-normal distribution, U Mann-Whitney and Kruskal-Wallis tests were used. Differences between categorical variables were examined by the chi-square test. Strength of associations, due to nonnormal distributions, were measured by Spearman's rank correlation coefficient. The statistical inferences were based on confidence levels p<0.05. If multiple comparisons were used statistical significance was lowered according to the Bonferroni adjustment.

10 Variables

 10 included patient and parental HRQOL assessment, symptoms over the previous 12 months, baseline and exercise spirometry, school grades and absenteeism.

  between most subscales describing roles functioning.

  have to be a sufficient cause to seek medical advice. Other important factors include individual experience, perception and emotions.

  As the examplecompared to parents of asymptomatic children, parents of children complaining of wheezing had a lower (p=0.02) mean score in emotional function and symptom domain. As well the group of children possessing dyspnea showed a lower mean parental emotional function score (p=0.04) than in asymptomatic children. In children an emotional function score was lower in the group of children complaining of cough in the past 12 months (p=0.01) as compared to asymptomatic children. Correlations were observed for exercise lung function and QLCCDQ domains, such as emotional, family and social functions. Worthy to note, that the correlations between children's lung function and parental HRQOL domains were stronger than analogous correlations concerning HRQOL in children. The most impaired parent HRQOL domain -emotions, significantly correlated with FEV 1 % FVC. Activity limitation, the most impaired patient domain, correlated with PEF and MEF 25-75.Even if the correlations between clinical indices and HRQOL scores were observed only for few variables it should not determine a limitation of HRQOL protocol in early diagnosis. As HRQOL is not a diagnostic scale nor a disease or symptom severity scale index -the impact of disease is not systematically correlated with the disease severity as it is categorized (32) by health -care providers. Patient' or caregiver -complement traditional clinical or biological evaluation, which could suggest that HRQOL could function as part of an early assessment or as part of disease monitoring.

  diagnosis and mild clinical presentation of the newly diagnosed cases. Parental experience of their child's disease is very important for the diagnosis of asthma, and it would confirm the importance of screening programs addressed to parents, including emotional context of their functioning in relation to the child's symptoms. Parental HRQOL could be recommended as a valuable method of a more holistic assessment of disease impact on everyday functioning of child and their family. Both patient and parental perception are important in care strategies planning and implementation. Children are influenced by their parents and reduction of parental distress could be beneficial for child's family life and integration within

  Figure 1. Study design

Table 2 .

 2 Demographic and asthma severity

	Page 47 of 50
		Males		Females
		Number Percent Number percent
	GINA classification
	I	6	60	14	73,68
	II	4	40	5	26,32
	Total	10	34,5	19	65,5
	Age
	Average (SD)	8,6 (1,4)	8,9 (1,0)

Table 3 .

 3 HRQOL -domains and overall scores

					Page 48 of 50
		Mean	SD	Min	Max
	QLCCDQ domain
	Role functioning
	Family	5.9	0.8	4.0	7.0
	Social	6.1	0.7	4.3	7.0
	Occupational	5.7	1.0	3.7	7.0
	Roles activity	5.8	0.8	4.2	7.0
	limitations
	Symptoms	4.0	1.1	2.0	6.3
	Emotions	4.1	0.7	3.7	6.2
	Overall points	81.7	10	66	98
	Overall mean	5.4	0.7	4.4	6.5
	PAQLQ domain
	Symptoms	5.4	1.2	2.1	7.0
	Activity limitation	4. 7	1.1	1.4	7.0
	Emotions	5.9	1.3	1.4	7.0
	Overall points	126.8	24,98	41	161
	Overall mean	5.5	1.1	1.8	7.0

Table 4 .

 4 QLCCDQ and PAQLQ subscales Spearmans' correlations

		Family roles	Social roles	Occupation al roles	Activity limitation	Symptoms perception	Emotions
	Domain 1
	QLCCDQ
	Family roles	-
	Social roles	0.7 (0.0001)	-
	Occupational Roles	0.6 (0.007)	0.6 (0.007)	-
	Roles activity limitation	0.8 (0.0001)	0.8 (0.0001)	0.9 (0.0001)	-
	Symptoms perception	0.07 (0.7)	-0.005 (0.9)	0.4 (0.08)	0.2 (0.31)	-
	Emotions	0.2 (0.4)	0.2 (0.3)	0.4 (0.05)	0.4 (0.09)	0.9 (0.0001)	-
	Domain 2
	PAQLQ
	Activity Limitation				-
	Symptoms				0.7 (0.0001)	-
	Emotions				0.6 (0.0001)	0.7 (0.0001)	-
	p-value in brackets
	1 -according to Bonferroni adjustment significance level p<0.001
	2-according to Bonferroni adjustment significance level p<0.008

Table 5 .

 5 HRQOL scores and lung function -Spearmans' correlations

			Lung function test
		(10 minutes after exercises test)
		FEV1	FEV1 %FVC	FVC	PEF	MEF 25-75%
	QLCCDQ domain
	Role functioning
	Family roles	0.5	0.3	0.1	-0.3	0.8
	Social roles	0.1	0.01	0.07	-0.1	0.8
	Occupational	0.2	0.3	0.1	0.2	0.2
	roles
	Roles activity	0.2	0.3	0.1	0.2	0.6
	limitation
	Symptoms	0.06	0.6	-0.02	0.2	0.2
	Emotions	0.02	0.7	-0.09	0.2	0.2
	PAQLQ domain
	Symptoms	0.01	0.2	0.4	0.2	0.2
	Activity limitation	0.2	0.2	0.4	0.5	0.4
	Emotions	-0.05	0.2	0.2	0.09	0.2
		p<0.01 statistically significant in bold

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John Wiley & Sons, Inc.Pediatric Pulmonology

Acknowledgements

The respiratory survey was sponsored by scientific grant from State Committee for Scientific Research (Polish Ministry of Scientific Research and Information Technology). The authors are grateful to Professor Elizabeth Juniper for her kind permission to use the PAQLQ. Acknowledgements The respiratory survey was sponsored by scientific grant from State Committee for Scientific Research (Polish Ministry of Scientific Research and Information Technology). The authors are grateful to Professor Elizabeth Juniper for her kind permission to use the PAQLQ.