Solid state physicochemical study of chlorophyll a derivatives and their glycol conjugates Текст научной статьи по специальности «Химические науки»

Porphyrins

Порфирины

Макрогэтероцмклы

http://macroheterocycles.isuct.ru

Paper

Статья

DOI: 10.6060/mhc160963c

Solid State Physicochemical Study of Chlorophyll a Derivatives and Their Glycol Conjugates

Dmitry B. Berezin,a@ Dmitry V. Belykh,b Olga M. Startseva,b |Nikolay G. Manin|,c Mikhail A. Krest'yanmov,c and Andrey V. Kustovc

aIvanovo State University of Chemistry and Technology, Institute ofMacroheterocyclic Compounds, 153000 Ivanovo, Russia hInstitute of Chemistry of Komi Scientific Center of Ural Division of Russian Academy of Sciences, 167982 Syktyvkar, Russia Krestov Institute of Solution Chemistry of Russian Academy of Sciences, 153045 Ivanovo, Russia @Corresponding author E-mail: [email protected]

This study focuses on solid state physico-chemical properties of recently synthesized potential sensitizers for photodynamic therapy developed on a chlorophyll a platform. Pheophorbide a and chlorin e6 derivatives as well as two their glycol conjugates have been synthesized and identified via UV-Vis, NMR and mass spectra. The behavior of photosensitizers (PSs) in a solid state has been studied. They were found to be stable in inert atmosphere at least up to 520 K, the thermal stability decreases from phlorin to chlorin molecules and, especially, after their glycol substitution. The thermodestruction of macroheterocycles takes place after their melting. Both glycol conjugates form intramolecular H-bonds in two different conformations, what is confirmed by phase transitions on DSC curves and quantum-chemical analysis data.

Keywords: Chlorophyll a, photosensitizers, phase transitions, thermal stability, intramolecular H-bonding.

Физико-химические свойства производных хлорофилла а и их диэтиленгликолевых конъюгатов в твердой фазе

Д. Б. Березин,а@ Д. В. Белых,ь О. М. Старцева,ь |Н. Г. Манин],с М. А. Крестьянинов,с А. В. Кустовс

аИвановский государственный химико-технологический университет, Институт макрогетероциклических соединений, 153000 Иваново, Россия

ьИнститут химии Коми Научного центра Уральского отделения Российской академии наук, 167982 Сыктывкар, Россия

сИнститут химии растворов им. Г.А. Крестова Российской академии наук, 153045 Иваново, Россия @Е-таИ: [email protected]

Производные феофорбида а и хлорина в& а также их коньюгаты с диэтиленгликолем были синтезированы и идентифицированы спектральными (ЭСП, ЯМР 'И, МС) методами. Изучено поведение фотосенсибилизаторов (ФС) в твердой фазе. Соединения стабильны в инертной атмосфере до 520 К и выше, их термическая устойчивость снижается при переходе от флоринов к хлоринам, особенно после введения в молекулу гликоль-ных фрагментов, а само разрушение всегда следует за процессом плавления. Из данных ДСК и квантово-хими-ческого анализа следует, что оба гликольных производных образуют внутримолекулярные И-связи, формируя два типа конформаций.

Ключевые слова: Хлорофилл а, фотосенсибилизаторы, фазовые переходы, термическая устойчивость, внутримолекулярные ^связи.

Introduction

H2C=CH

H3C

H2C=CH

2I

H3COOC

H H ROOC

C2H5 CH3

O

CH3

H3C—<k 1 1 >— C2H5

3 Vnh n^ C2H5

H3C

H3COOC

CH3

NHCH3

1, 2

CO

Aor

3, 4

1, 4. R=CH3; 2, 3. R=-(CH2-CH2O)2H

Chlorophyll a derivatives are perspective photosen-sitizers (PSs) for different medical purposes like diagnostics, anti-tumor, antifungial and antimicrobial therapy, etc. [1-7] During last years much attention is paid to the state of PSs in solutions mimicking bioliquids.[8,9] It is stipulated by great importance of photophysical, associative and other solution properties of PSs for an effective photodynamic therapy (PDT) application.[1011] Meantime, objective conclusions drawn from the analysis of quantitative characteristics of solubility, solvation, interphase redistribution and other solution processes of chlorin macroheterocycles can be made only after taking under consideration of some knowledge about their solid phase statement, particularly, phase transitions which could significantly change the solubility of a photosensitizer. These data are useful, for instance, for preparation of dosage forms of PSs.[5] Therefore here we discuss the solid state properties of some chlorins and their recently synthesized glycol conjugates (compounds 1-4). Chlorins 3-4 are found[9] to generate singlet oxygen intensively, effectively penetrate into cell membranes due to their moderate hydrophilicity and therefore can be considered as potential PDT sensitizers.

Experimental

Compounds investigated were synthesized, purified and identified according to the procedures described earlier.112"141

Pheophorbide a 17(3)-methyl ester (1). Pheophytin a was extracted from the Spirulina platensis alga (Germany).1121 Methylpheophorbide a 1 was prepared according to the well-established procedures described in[1213]. Spectral characteristics were in agreement with the literature data. Mass spectrum (ESI) m/z: for MH+ (C36H38N4O5), calculated value - 607.3, experimental value - 607.2. 1H NMR (500 MHz, CDCl3, 298 K) SH ppm: 9.42 s (1H, H10), 9.30 s (1H, H5), 8.55 s (1H, H20), 7.92 dd (1H, 3-CH=CH2, J 18.0 and 12.0 Hz), 6.27 s (1H, H13(2)), 6.20 dd (1H, 3-CH=CHHtrans, J 18.0 and 2.0 Hz), 6.12 dd (1H, 3-CH=CHHcis, J 12.0 and 2.0 Hz), 4.50-4.10 m (2H, H18, H17), 3.80-3.40 m (2H, 8-CH2CH3), 3.91 s (3H, 13(2)-COOCH3), 3.63 s (3H, 17(3)-CH2CH2COOCH3), 3.61 s (3H, 12-CH3), 3.32 s (3H, 2-CH3), 3.00 s (3H, 7-CH3), 2.80-2.17 m (4H, 17-CH2CH2COOCH3), 1.84 d (3H, 18-CH3, J7.2 Hz), 1.58 t (3H, 8-CH2CH3, J 7.0 Hz), 0.51 br.s (1H, I-NH), -1.52 br.s (1H,

III-NH). 2 3

Pheophorbide a 13(2)-diethylene glycol ester 17(3)-methyl ester (2). Glycol derivatives of pheophorbide a (2) and chlorin e6 (3) were synthesized from 17(3)-methyl ester of pheophorbide a

according to the previously reported schemes.[14] trans-Esterification of the ester group of the substituent at the 13(2)-position of methylpheophorbide a (1) was initiated by its reaction with the 50-fold excess of diethylene glycol at refluxing in toluene under the action of 4-dimethylaminopyridine and 2-chloro-N-methylpyridinium iodide added as activators. After 3 hours the final product 2 existing as a diastereomers mixture (the 13(2)-R/13(2)-S ratio is 7:1) was isolated, purified and dried as described above. The total yield of glycol 2 equals to 67 %. Mass spectrum (ESI) m/z: for MH+ (C39H44N4O7, calculated value - 680.3, experimental value - 681.4. 1H NMR (300 MHz, CDCl3, 298 K) SH ppm: 13(2)-R diastereomer: 9.55 (1H, s, H10), 9.42 (1H, s, H5), 8.59 (1H, s, H20), 8.03 (1H, dd, J=18.5 and 11.6 Hz, 3-CH=CH2), 6.33 (1H, dd, J=17.4 and 1.4 Hz, 3-CH=CHHtrans), 6.32 (1H, s, H13(2)), 6.22 (1H, dd, J=11.4 and 1.5 Hz, 3-CH=CHHcis), 4.61-4.45 (3H, m, 13(2)-COOCH2CH2OCH2CH2OH, H18), 4.28 (1H, br.d, J=8.5 Hz, H17), 3.85-3.50 (8H, 8-CH2CH3, 13(2)-COOCH2CH2OCH2CH2OH), 3.72 (3H, s, 12-CH3), 3.61 (3H, s, 17(3)-CH2CH2COOCH3), 3.44 (3H, s, 2-CH3), 3.28 (3H, s, 7-CH3), 2.77-2.15 (4H, m,

17-CH2CH2COOCH3), 1.86 (3H, d, J=7.3 Hz, 18-CH3), 1.74 (3H, t, 8-CH2CH3, J=7.4 Hz), 0.62 (1H, br. s, 21-NH), -1.55 (1H, br.s, 23-NH); 13(2)-S diastereomer: 9.53 (1H, s, H10), 9.39 (1H, s, H5), 8.53 (1H, s, H20), 8.07-7.97 (1H, m, 3(1)-CH=CH2), 6.33 (1H, dd, J=17.4 and 1.4 Hz, 3(2)-CH=CHHtrans), 6.21 (1H, s, H13(2)), 6.22 (1H, dd, J=11.4 and 1.5 Hz, 3(2)-CH=CHHcis), 4.61-4.45 (3H, m, 13(2)-COOCH2CH2OCH2CH2OH, H18), 4.28 (1H, br.d, J=8.5 Hz, H17), 3.85-3.50 [8H, m, 8-CH2CH3, 13(2)-COOCH2CH2OCH2CH2OH], 3.69 (3H, s, 12-CH3), 3.48 (3H, s, 17-CH2CH2COOCH3), 3.42 (3H, s, 2-CH3), 3.27 (3H, s, 7-CH3), 2.77-2.15 (4H, m, 17-CH2CH2COOCH3), 1.86 (3H, d, J=7.3 Hz,

18-CH3), 1.74 (3H, t, 8-CH2CH3, J=7.4 Hz), 0.72 (1H, br.s, 21-NH), -1.37 (1H, br.s, 23-NH).

Chlorin e6 13(1)-N-methylamide, 15(2)-diethelene glycol, 17(3)-methyl ester (3). This compound was obtained through disclosure of an exocycle in compound 2 by addition of a 33 % aqueous solution of methylamine to the solution of 2 in tetra-hydrofuran at room temperature. The duration of this reaction was found to be of 20 minutes, the yield of the final product is 60 %. Mass spectrum (ESI) m/z: MH+ (C40H49N5O7), calculated value - 711.4, experimental value 712.3. 1H N MR (300 MHz, CDCl3, 298 K) SH ppm: 9.74 (1H, s, H10), 9.69 (1H, s, H5), 8.86 (1H, s, H20), 8.13 (1H, dd, J=18.3 and 11.7 Hz, 3-CH=CH2), 7.31-7.22 (1H, m, 13-CONHCH3), 6.40 d (1H, d, J=18.3 Hz, 3-CH=CHHt ), 6.18 (1H, d, J= 11.7 Hz, 3-CH=CHH), 5.59

trans^' v 5 5 5 cis-"

(1H, d, J=19.4 Hz, 15-CHAHBCOOCH2CH2OCH2CH2OH), 5.40 (1H, br.d, J=19.4 Hz, 15-CHAHBCOOCH2CH2OCH2CH2OH), 4.52 (1H, q, J=7.0 Hz, H18), 4.45 (1H, br.d, J= 8.8 Hz, H17), 4.374.11 (2H, m, 15-CH2COOCH2CH2OCH2CH2OH), 3.84 (2H, q., J=7.7 Hz, 8-CH2CH3), 3.63 (3H, s, 17(3)-CH2CH2COOCH3), 3.60 (3H, s, 12-CH3), 3.54 (3H, s, 2-CH3), 3.36 (3H, s, 7-CH3), 3.28 (3H, d, J=4.4 Hz, 13(2)-CONHCH3), 3.19-2.79 (6H, m, 15-CH2COOCH2CH2OCH2CH2OH), 2.60-2.46 and 2.35-2.22 (both m 1H, 17-CH2CH2CO OCH3), 2.16-2.01 and 1.98-1.83 (both m 1H, 17-CH2CH2C OOCH3), 1.76 (3H, t, J=7.3 Hz, 8-CH2CH3), 1.72 (3H, d, J=7.0 Hz, 18-CH3), -1.63 (1H, br. s, 21-NH), -1.83 (1H, br.s, 23-NH).

Chlorin e6 13(1)-N-methylamide, 15(2)-,17(3)-dimethyl ester (4). Compound 4 was synthesized from methylpheophorbide a 1 in accordance with the same procedure as for compound 3.[14] The yield of chlorin e6 derivative was 72 %. Mass spectrum (ESI) m/z: MH+ (C37H43N5O5), calculated value - 637.3, experimental value 637.8 BNMR (300 MHz, CDCl3 298 K) SH ppm: 9.70 (1H, s, H10), 9.64 (1H, s, H5), 8.81 (1H, s, H20), 8.10 (1H, dd, J=23.8 and 15.4 Hz, 3-CH=CH2), 6.37 (1H, dd, J=23.8 and 1.8 Hz, 3-CH=CHHtrans), 6.15 (1H, dd, J=15.8 and 1.8 Hz, 3-CH=CHHcis), 6.40 (1H, In, 13(1)-CONHCH3), 5.54 (1H, d, J=25.0 Hz, 15-CHAHBCOOCH2), 5.26 (1H, d, J=25.0 Hz, 15-CHAHBCOOCH2), 4.36-4.49

(2H, m, H17, H18), 3.84 (3H, s, 15-CH2COOCH3), 3.62 [3H, s, 17-CH2CH2COOCH3], 3.56 (3H, s, 12-CH3), 3.50 (3H, s, 2-CH3), 3.33 (3H, s, 7-CH3), 3.81 (2H, q, J=10.0 Hz, 8-CH2CH3), 1.90-2.60 [4H, m, 17-(CH2CH2COOCH3)], 1.73 (6H, m, 18-CH3, 8-CH2CH3) -1.60 (br.s, 21NH), -1.80 (br.s, 23NH).

MS-spectra were recorded on Thermo Finnigan LCQ spectrometer. 1H NMR spectra were registered in CDCl3 using spectrometers Bruker AVANCE-II-300 (300 MHz) and Bruker Avance 500 (500 MHz).

Differential Scanning Calorimetry (DSC) and Thermogravimetry Studies

Calorimetric studies were carried out with the differential heat flow calorimeter of DSC 204 F1 Phoenix "NETZSCH". All samples with mass of 10 mg were run from 263 K to 473 K in an argon atmosphere with sapphire as a standard. The heating rate of a hermetic aluminum crucible with a dried macroheterocycle was equal to 5 K/min and reproducibility of the results obtained was estimated to be within 2 %. The experimental DSC signals plotted versus T are shown in Figure 1.

Thermogravimetric measurements were performed in an argon atmosphere using a Netzsch TG 209 F thermobalances. A weighted crystalline sample (3-7 mg) was placed into a platinum crucible and heated in a static atmosphere of Ar at a rate of 5 K/min in the temperature range of 298-1173 K (25-950 °C). The samples were pre-dried to constant weight in a Fisher vacuum apparatus at room temperature.

Quantum-Chemical Calculations

The geometry optimization of macrocycles 1-4 was performed using the GAUSSIAN 09 program package,[15] the density functional method, the B3LYP hybrid functional,»61 and the CC-pVDZ basis set.[17] The stabilization energy of the hydrogen bonds (Estab, kJ/mol) and the value of the transferred charge (qstab, charge units) were calculated by means of NBO analysis.118191 A full description of the calculation procedure is given in reference.™

Results and Discussion

Differential Scanning Calorimetry

intramolecular ones. Meantime, compounds with several H-donor and H-acceptor centers like macrocycles 2 or 3 can form some sort of intermolecular H-bonded and supramolec-ular structures destroyed then at about 300 K in a solid state. Namely breaking of these bonds give signals on DSC curves at low temperatures about 298 K (Figure 1). Low energy of these phase transitions is explained by weakness of H-bonds which are only about 10 kcal/mol (Table 1). Two stable conformations are found for macrocycles 2 and 3 (Table 1, Figure 2). Formation of H-bonds between oxygen atom of ester group and OH-group of glycol residue is found to be about 5 kcal/mol more favorable in the case of phorbine derivative 2 if compare to chlorin 3 (Table 1) because of better conditions for spatial preorganization of reaction centers in former macrocycle.

0,350,30-f 0,25-^ 0,20-

I" 0,15-|

X ■

0,050,00250 300 350 400 450 500 T, K

Figure 1. DSC traces for solid compounds 1-3.

Curves are shifted from each other on 0.02 mW for better

visualization. Dash lines show calculated curves for estimating the

heat effects of processes occurring in a solid state.

For 1 no any phase transitions have been detected;

2 reveals two peaks at T=293 K and T=456 K; 3 gives one sharp

peak at T=466 K and smaller effect at about 303 K.

Figure 1 compares the DSC curves for methylpheophorbide a 1 and its two glycol derivatives 2 and 3 at 263-473 K. The compound 1 is rather stiff and does not reveal any phase transitions in the temperature range studied. In contrast, two peaks have been detected for compounds 2 and 3. It could be assumed the first one is associated with conformational changes of the glycol fragment which is absent in macrocycles 1 and 4. The relatively low temperature of transition and its small heat effect (7 kJ/mol and lower, Figure 1) testifies that molecular packing is not too dense. The most probable reason is H-bond formation between the lone pair of oxygen atom of carbonyl group and hydrogen of glycol fragment of the neighbour molecules. These H-bonds strongly restrict the number of possible conformations of glycol fragments.

This assumption is confirmed by geometry optimization of compounds 1-4 and NBO analysis data (Table 1). Though, only glycol conjugates of methylpheophorbide a

1 and chlorin e. 4 are able to the H-bonds C=O H-O for-

6

mation (Figure 2). H-bonds considered in NBO analysis are

Table 1. Parameters of intramolecular H-bonds of PSs 2 and 3 calculated from NBO-analysis.

Bond distances (Á) Comp. and angles (deg)

O H O- O O-H- O

E , kJ/mol

st'

(kcal/mol)

2(1) 1.839 2.811 172.9 63.89 (15.3) 0.028

2(2) 1.904 2.874 172.7 49.54 (11.8) 0.024

3(1) 1.888 2.805 155.2 39.03 (9.3) 0.016

3(2) 1.843 2.790 161.3 62.67 (15.0) 0.028

DSC peaks for both derivatives 2 and 3 are found at higher temperatures. The compound 2 shows gradual disordering at T>380 K. The position of the broad peak maximum at T=456 K is close to the determined experimentally temperature of PS melting (445 K). Further temperature rise appears to lead to partial PS decomposition which can be seen from thermogravimetry data (Table 2)

and dealing with the loss of glycol residue and macroring destruction. The compound 3 gives a sharp peak at T=466 K, which is also nearly identical to experimentally determined temperature of melting (463 K). Compound 1 does not show any phase transitions at both low and high temperature of 263-473 K interval. It does not form any H-bonds and melts at 519 K only (Table 2).

Such a way, phase transitions on the DSC curves are associated with a rapid H-bonds breaking, melting and then partial decomposition of PS. It is interesting to note that the sum of two heat effects for the compounds 2 and 3 are almost equal in the temperature interval studied (Figure 1).

Thermal Stability

Investigation of thermal stability of chlorins 1-4 by thermogravimetry method (Table 2) indicates that they are quite stable in inert atmosphere. Thermograms are typical for chlorophyll derivatives and represented by two high temperature stages of aromatic chromophore destruction (Figure 2).

It should be mentioned that all compounds studied are exposed to thermodestruction after their melting points. The initial temperature of the first destruction stage is higher for pheophorbide a and chlorin e6 derivatives (compounds 1 and 4; 624 and 606 K, respectively) and decreases dramatically down to 519 K after insertion of diethylene glycol fragment into the macrocyclic molecule (Table 2).

Thus, our comparative analysis indicates that the PSs studied are stable in a wide temperature range. Introduction

of the glycol fragment into the macrocycle destabilizes PS and causes phase transitions appeared in temperature range 265-475 K. H-Bonds strongly restrict the number of possible conformations of glycol fragments for 2 and 3 and influence on solubility preventing the PS transfer from a solid state to liquid phase.

Conclusions

In this paper, we have focused our attention on the behavior of recently synthesized macrocyclic compounds which are believed to have some importance for photody-namic therapy.[9] Our comparative analysis clearly indicates that solid potential PSs created on chlorophyll a platform are stable in a wide temperature range. Hydrophobic methyl-pheophorbide a 1 does not reveal any phase transitions in a solid state both at low and high temperatures. In contrast, glycol derivatives undergo phase transitions, especially, at higher temperatures which correspond to melting of the mac-roheterocycles. Intramolecular H-bonding is responsible for the phase transitions of glycol conjugates at lower temperatures. This conclusion is confirmed by quantum-chemical calculations.

Thermodestruction of PS in inert atmosphere takes place at temperatures higher 520 K and is represented by two step processes within 520-760 K.

Acknowledgements. This work was supported by the Russian Scientific Foundation (Grant 15-13-00096).

Figure 2. Geometry structure of stable conformations of compound 2: (1) - right and (2) left, optimized by DFT method (basis CC-pVDZ).

Table 2. Thermodestruction characteristics of compounds 1, 3 and 4 in inert atmosphere.

Comp. t,, K st> Stage 1 T , K max' Tfi, K fin' Am, % tst, K st> Stage 2 Tmax, K max' Tfin, K fin' Am, %

1 624.17 672.02 681.91 26.6 699.40 708.15 753.60 25.3

3 519.48 573.73 595.11 20.6 645.03 693.84 755.67 39.1

4 606.44 641.33 654.53 27.2 697.77 704.05 741.58 23.2

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Received 29.09.2016 Accepted 09.03.2017

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