Syntheses and Cellular Investigations of Di(aspartate) and Aspartate-lysine Chlorin e₆ Conjugates

Abstract

Chlorin e₆ is a tricarboxylic acid degradation product of chlorophyll a. Four chlorin e₆ bis(amino acid) conjugates were regioselectively synthesized bearing two aspartate conjugates in the 13¹,17³- and 15²,17³-positions, or at the 13¹,15² via an ethylene diamine linker. One additional conjugate bearing two different amino acids, lysine at 13¹ via an ethylene diamine linker and an aspartate at 15² via a β-alanine linker was also synthesized. The cytotoxicity and uptake of four di(amino acid) chlorin e₆ conjugates were investigated in human HEp2 cells, and compared with chlorin e₆. The most cytotoxic and most taken up conjugates were the zwitterionic 13¹,15²-disubstituted conjugates 28 and 33; these also localized in multiple organelles. In contrast, the tetra-anionic 13¹,17³- and 15²,17³-di-aspartyl chlorin e₆ conjugates 12 and 13 showed low dark cytoxicity and lower phototoxicity compared with chlorin e₆.

Graphical Abstract

Regioselective syntheses of chlorin e₆ bis(amino acid) conjugates bearing aspartates in the 13¹,17³-, 15²,17³- and 13¹,15²-positions, and their cell studies are reported.

Introduction

Photodynamic therapy (PDT) is a special form of cancer photochemotherapy involving a photosensitizer and light. Selective uptake of a photosensitizer into tumor cells followed by irradiation with laser light within the “therapeutic window” (λ_max 600-800 nm) produces singlet oxygen and other reactive oxygen species (ROS).^1-5 Singlet oxygen, in particular, readily reacts with nearby local biomolecules (unsaturated lipids, amino acids and DNA) to cause destruction of apparatus within the cell. Because of limited diffusion of singlet oxygen through tissue, the destructive PDT effects are mainly localized to the photosensitizer-containing cells, thus minimizing the possibility of damage to normal cells near the tumor. Two essentials for the success of PDT treatment are: (i) the tumor-targeting ability of the photosensitizer, and (ii) the wavelength of the light required to activate the photosensitizing drug. Researchers aim for minimal “dark” toxicity, a high quantum yield of triplet state formation upon excitation, high selectivity for tumor cells compared with normal cells, reasonably rapid clearance from normal tissues, and a strong absorption peak between 600 and 800 nm to facilitate maximum light penetration through tumor and normal tissues.

Photofrin is a porphyrin-based photosensitizer mixture that was developed and approved in several countries for the PDT of melanoma, lung, digestive tract, genitourinary tract and for treatment of Barrett’s esophagus.^3-6 It suffers from the fact that it is ineffective at wavelengths above 630 nm, and that it clears from tissues so slowly that patients can suffer from residual skin photosensitivity for weeks after treatment. Thus, a number of so-called second generation tetrapyrrole photosensitizers have been invented, these usually possessing strong red-shifted absorption maxima at the red end of the therapeutic window. Examples are tetra(meta-hydroxyphenyl)chlorin, (mTHPC or Temoporfin), 2-(1-hexyloxyethyl)-2-devinyl-pyropheophorbide a (HPPH or Photochlor), mono-L-aspartylchlorin e₆ (LS-11, or Talaporfin) and a phthalocyanine called “Pc4”.^3-6 Of these, chlorins (dihydroporphyrins) have received special attention for PDT because of their intense absorptions above 640 nm. Indeed, chlorophyll a derivatives are amphiphilic tetrapyrroles that have been extensively investigated and shown to exhibit low dark toxicities while at the same time able to generate cytotoxic ROS upon irradiation with light.^7-9 Both HPPH and Talaporfin are in advanced-stage clinical trials for oncologic PDT treatment;^10-13 they exhibit superior PDT activity, and are rapidly cleared from tissue such that residual photosensitivity in patients is minimized compared with Photofrin.

Chlorin e₆ (1) is a particularly interesting chlorophyll a derivative because it possesses no less than three chemically different carboxylic acid functions (that could potentially be differentially functionalized with biomolecules): a nuclear aromatic acid in position 13¹, a nuclear acetic acid in position 15², and a propionic side-chain acid that is truly aliphatic, in position 17³. Conjugations of peptides, sugars, lipoproteins, polyethylene glycols, and polyamines with chlorin e₆ have been reported.^14-22 Amino acid conjugations, in particular, have been shown to improve the PDT effects of tetrapyrrole compounds, and the type and position on the macrocyclic core can have a major impact upon efficacy in PDT. Talaporfin (2) was originally formulated in 1987 as the 17³-aspartyl derivative (3) of chlorin e₆. Gomi et al.²³ used spectroscopic techniques (mostly) to propose that Talaporfin was in fact (2), not (3), but this work was almost universally ignored until our own group confirmed by total synthesis of the three possible isomers (2-4) (and comparison with authentic material), that (2) was indeed the correct structure for Talaporfin.²⁴ A definitive X-ray structure of the tetramethyl ester (5) of Talaporfin finally ended all conjecture.²⁴ It was proposed that the anhydride (6) is an intermediate in the formation of (2) whereby the aspartic acid is not involved in the first step of the reaction. The anhydride (6) is formed by dehydration using only DCC or EDC, and then the aspartic acid nucleophile opens the anhydride ring by attacking the more electrophilic aliphatic carbonyl at position 15².²⁴ This point of view was solidified by isolation of the anhydride, a single crystal X-ray structure of the anhydride methyl ester (7), and the demonstration that ring-opening of the anhydride with eight different nucleophiles resulted only in formation of the 15²-conjugate (8).²⁵ Bis-conjugation was shown to be possible (at 17³ and 15²) in one case,²⁵ or when two equivalents of DCC or EDC were used,²⁶ but at no time was the 17³-conjugate the sole product of the conjugation.

Making use of the differential conjugation methods developed in our earlier work,²⁴ regioselective amino acid conjugates at the 17³, 15² and 13¹ positions of chlorin e₆ were synthesized²⁷ to evaluate the effect in vitro of the conjugation site and structure of the amino acid on the PDT efficacy of the conjugates, using human carcinoma HEp2 cells. Syntheses of the 17³ and 13¹ aspartyl regioisomers of mono-L-aspartylchlorin e₆ were developed, along with the corresponding cationic lysine derivatives (which would be expected to show strong interactions with anionic biomolecules and membranes, resulting in enhanced PDT effectiveness.²⁸ In addition, molecules with an ethylene diamine or β-alanine spacer group between the macrocycle and the amino acid were also synthesized. Based on in vitro biological evaluations, it was concluded that the site of amino acid conjugation, rather than the nature of the amino acid conjugated, was the major determinant of phototoxicity.²⁷ The most phototoxic compounds were found to be the 13¹-regioisomers, bearing either an aspartic acid or a lysine residue directly conjugated to position 13 of the chlorin macrocycle, or connected via a β-alanine or ethylene diamine spacer. The most phototoxic compound of this series, and the most promising for PDT applications, was 13¹-aspartylchlorin e₆. Unfortunately, poor solubility of these molecules was one of the major drawbacks in the cellular studies, and some of the sensitizer molecules tended to self-aggregate easily, resulting in lower cell uptake. In the present work, in order to overcome these shortcomings, we report the syntheses and biological evaluations of a new series of chlorin e₆ derivatives that possess two amino acids in a single chlorin e₆ molecule (Figure 1).

Figure 1.

All possible bis-amino acid regioisomers of chlorin e₆

Kimani et al.¹⁹ recently reported the synthesis of mono-, di- and tri-PEGylated chorin e₆ photosensitizers with tri(ethylene glycol) attached at the three carboxylic acid positions in chlorin e₆.¹⁹ These were tested for solubility and hydrolytic stability, as well as phototoxicity, cell uptake and localization in ovarian OVCAR-5 cancer cells. Their results showed that increasing the number of PEG groups increased the water solubility and cellular uptake of the compounds. Computational studies also indicated that the PEG groups prevent formation of aggregates by π-π stacking, by wrapping onto the chlorin ring. Most significantly an increased number of PEG groups increased the phototoxicity, and cellular uptake. We therefore anticipated that an increase in the overall charge of the molecule and resulting amphiphilicity, might increase the water solubility^29,30 and concomitantly decrease the self-aggregation in biological media.³¹ Our hypothesis was that a higher number of charged substituents would increase both the phototoxicity and the cellular uptake. To test this hypothesis all three possible regioisomers of diamino acid chlorin e₆ derivatives were synthesized (Figure 1). It was found from our previous study, that the position of the amino acid plays a vital role in determining the conformation of the molecule.²⁷ Therefore new synthetic routes were developed to prepare all three regioisomers. Both the 15²,17³- and 13¹,17³-di(amino acid) derivatives were synthesized starting from chlorin e₆ (1) and the 13¹,15²-diamino derivatives were synthesized from pheophytin a. Thus, the aims of the present work were to determine: (i) the degree to which di-(amino acid)-chlorin e₆ derivatives were taken up and localized in cancer cells, (ii) the main site(s) of intracellular accumulation, and (iii) whether the position and charge of the amino acid substituents influences the dark and photo-toxicity.

Results and Discussion

Synthesis of 15²,17³-diaspartylchlorin e₆ derivative

A major side product in the synthesis of 15²-mono-aspartylchlorin e₆ derivative (2) was the 15²,17³-diaspartylchlorin e₆ derivative 8,²⁶ the yield of which can be increased by increasing the number of equivalents of coupling reagent and aspartic acid. According to our proposed mechanism,²⁴ DCC and DMAP activated the acetic side chain in 1 and formed the anhydride intermediate 6. The excess coupling reagent can further activate the propionic acid side chain to form the O-acylisourea anhydride intermediate 9 (Scheme 1).²⁵

Scheme 1.

Proposed intermediates in the formation of 15²,17³ di-aspartic acid derivative 8.

Thereafter, the free amine group of the amino acid can attack both carbonyl groups in intermediate 9, one in the anhydride ring and the other one in the O-acylisourea group. Mechanistic studies revealed the nucleophile first attacks the more reactive 15² position of the anhydride ring instead of the 13¹ position, regardless of the size or nucleophilicity of the molecule. Then the second equivalent can react with the O-acylisourea group to form diaspartylchlorin e₆ derivative 8.

The detailed synthetic route to 15²,17³-diaspartylchlorin e₆ methyl ester (12) is shown in Scheme 2. It was possible to obtain diaspartyl methyl ester 11 in 33% overall yield from chlorin e₆ (1). The optimal yield of diaspartyl chlorin e₆ 12 (26%) was obtained upon activation of chlorin e₆ with three equivalents of DCC and DMAP in CH₂Cl₂ at room temperature for two hours followed by coupling with 2.5 equivalents of di-tert-butyl protected aspartic acid for 24 hours. Less than three equivalents of DCC and aspartic acid tended to form significant amounts of the 15²-monoaspartic acid conjugate as a side product. When the number of equivalents of either DCC or aspartic acid was increased, the yield of diaspartyl chlorin e₆ 10 improved, but it was not possible to prevent the formation of mono-aspartyl conjugate as a side product. Longer reaction times also helped to increase the yield of diaspartyl derivative 10. After confirming the formation of tert-butyl protected diaspartyl chorin e₆ 10 by TLC and mass spectrometry, freshly prepared diazomethane gas was bubbled through the mixture for five minutes to convert the remaining free acid groups into methyl esters. This reaction was monitored by TLC. It was possible to see two new spots in the TLC plate. A brighter spot with lower R_F value belonged to the desired product 15²,17³-diaspartylchlorin e₆ 11 and the second spot had a higher R_F value similar to that of 15²-monoaspartlychlorin e₆ methyl ester. Purification was achieved via silica gel column chromatography and the identity of the esterified product 11 was confirmed using ¹H NMR and mass spectrometry. ¹H NMR spectroscopy showed four singlets between 3.0 and 4.5 ppm each integrating to three protons. Of the four peaks, three belong to the methyl groups directly connected to the macrocycle and the remaining peak belongs to the 13¹ methyl ester group. The most downfield singlet at δ 4.25 confirmed the formation of the 13¹ methyl ester because this peak is unique to the methyl group on the formic (13-) side chain of the trimethyl ester of chlorin e₆ (1). This observation confirmed that aspartic acid coupled with the acetic (15-) and propionic (17-) side chains of the molecule.

Scheme 2.

Synthesis of 15²,17³-diaspartylchlorin e₆ methyl ester (12).(Reagents: i. 3 equiv. DCC, DMAP, CH₂Cl₂, 2 h; ii. 3 equiv. Asp di-t-Bu.HCl, DIEA, rt, 24 h; iii. CH₂N₂, CH₂Cl₂, 5 min, 30% (3 steps); iv. TFA/CH₂Cl₂ (1:3), thioanisole, 6 h, rt, 88%.)

In the final step, all four tert-butyl esters in molecule 11 were removed to give 12 in a TFA/CH₂Cl₂ mixture for six hours at room temperature. TFA and CH₂Cl₂ were first removed and the product was freeze-dried by dissolving it in a water/acetonitrile mixture. Once the sample was neutralized the color changed from purple to dark green. The identity of product 12 was confirmed by ¹H NMR, ¹³C NMR and mass spectrometry.

Synthesis of 13¹,17³-diaspartylchlorin e₆ derivative

There are two possible routes to synthesize the 13¹,17³-chlorin e₆ conjugate 13: 1) coupling of the first amino acid to the propionic acid chain of pheophorbide a (14) and subsequent isocyclic ring opening of pheophorbide 15 with ethylene diamine to give 16, followed by coupling of the carboxylate of a second amino acid to the free amine group of the resulting chlorin derivative, to give 17 (Scheme 3), or 2) selective esterification of the acetic acid side chain (15² ester) of chlorin e₆ (1) followed by coupling with two equivalents of an identical amino acid to afford the 13¹,17³-di(amino acid) derivative (Scheme 4).

Scheme 3.

Retrosynthetic route to 13¹,17³-di(amino acid) derivatives, Method 1.

Scheme 4.

Retrosynthetic route to 13¹,17³-di(amino acid) derivatives, Method 2

In the first method, pheophorbide a (14) can be obtained by selective hydrolysis of the phytyl ester group of pheophytin a (obtained from Spirulina maxima or S. pacifica alga³²) using a procedure developed by Wasielewski and Svec.³³ Then the amino acid can be coupled to the propionic side chain to obtain the pheophorbide derivative 15. The pheophorbide isocyclic ring in 15 can be cleaved, using ethylene diamine,³⁴ to provide 16 bearing a tethered free amine group able to couple with the carboxylic group of a second (potentially different) amino acid.

The key step in the second method (Scheme 4) is the selective protection of the acetic acid side chain of chlorin e₆ (1) in the presence of the formic and propionic carboxylic side chains. From the previous work, it was found that the acetic acid group of chlorin e₆ can be activated selectively by forming a cyclic anhydride. Chlorin e₆ was activated using one equivalent of DCC and DMAP to form the anhydride intermediate (6).²⁴ Formation of cyclic anhydride 6 can be confirmed by using UV-Vis spectroscopy. Then freshly prepared 0.5 M sodium methoxide was added until the color of the reaction mixture changed from brown-purple to dark green. Mass spectroscopy confirmed the formation of desired product 18. Purification of di-acid 18 was challenging due to its high polarity. After work-up it was possible to obtain a ¹H NMR spectrum of the crude product and this was clear enough to identify the unique peak for the 15² methyl ester, and indicated it was sufficiently pure for the next step. Appearance of a new peak at 3.7 ppm for 3 protons provided evidence that esterification took place uniquely at the acetic acid side chain. Then tert-butyl-protected aspartic acid was coupled with crude 15²-chlorin e₆ monomethyl ester (18). The optimal yield was obtained with HOBt and TBTU as coupling reagents at room temperature for 48 hours.³⁵ This reaction was monitored by TLC. The product was purified via silica gel column chromatography and the first moving band with 5% methanol/DCM was collected. The structure of product 20 was confirmed by mass and ¹H NMR spectroscopy. Purified tert-butyl protected diaspartyl chlorin e₆ 20 was treated with TFA/CH₂Cl₂ mixture for six hours at room temperature to deprotect all four carboxylic acid groups. Pure compound 13 was obtained after removal of TFA. The final product, 13¹,17³-diaspartyl chlorin e₆ 13, was obtained in 28% yield over four steps (Scheme 5).

Scheme 5.

Synthesis of 13¹,17³-diaspartyl chlorin e₆ methyl ester (13). (Reagents: i. DCC, DMAP, CH₂Cl₂, 1 h; ii. 0.5 M NaOMe/MeOH, 0 °C, 99% (2 steps); iii. HOBt, TBTU, DMF, L-Asp di-t-Bu.HCl, DIEA, CH₂Cl₂, rt, 48 h, 48%; iv. TFA/CH₂Cl₂, thioanisole, 6 h, rt, 53%.).

Synthesis of 13¹,15²-di(amino acid) chlorin e₆ derivatives

Previous studies have revealed that the formic and acetic acid derivatives of chlorin e₆ are more potent photosensitizers than the propionic acid conjugates.²⁷ Thus it was assumed that the 13¹,15²-di(amino acid) derivatives may show more potent photosensitizing ability in this di(amino acid) series compared to the two other regioisomers (Figure 1). Therefore two different amino acid conjugates of the 13¹,15² regioisomer were synthesized.

The main challenge was to protect the propionic side chain in the presence of the two other carboxyl groups. There is no known chemical method to selectively methylate the propionic side chain of chlorin e₆ without methylating the acetic side chain. Esterification with methanol under acidic conditions will form the 15²,17³-chlorin e₆ dimethyl ester.³⁶ Diazomethane will methylate all three carboxylic groups to form chlorin e₆ trimethyl ester.³⁷ But in the recent literature there is a reported chemo-selective aminolysis of the β-keto ester of methyl pheophorbide a (21).^38-40 This opens up a new route to synthesize 13¹,15² chlorin e₆ derivative from methyl pheophorbide a (21).

Previously, Shinoda and Osuka reported a facile transesterification of the β-keto ester of methyl pheophorbide a.⁴¹ These authors were able to introduce various alcohols and steroid groups in the presence of 2-chloro-1-methylpyridinium iodide (CMPI) and of 4-(N,N-dimethylamino)pyridine (DMAP) to the 13²-position of pheophorbide. Haavikko and coworkers also reported a selective aminolysis of the β-keto ester of the methyl pheophorbide a (21).³⁸ They used various primary, secondary and aromatic amines for the aminolysis of the β-keto ester. It is known that aminolysis of the β-keto ester is usually facile compared to inactivated esters.^42,43 In the previous literature the formation of β-enaminoesters by reaction between secondary amines and β-ketoesters at low temperatures is described.⁴⁴ It was noticed that at higher temperatures they tend to form substantial amounts of the corresponding β-ketoamide along with the expected enamino-ester.

We have observed the same behavior with pheophorbide a.²⁷ In our previous work,²⁵ it was possible to open the isocylic ring of pheophorbide a (by cleaving the β-keto ester) using ethylene diamine at room temperature. It was noticed that at higher temperatures (90 °C in toluene) the reaction with ethylene diamine tended to form two major products (two new very close spots in TLC). Both products were isolated using preparative TLC and characterized by ¹H NMR and mass spectroscopy. One of the products still clearly shows the unique peak for the pheophorbide isocylic ring: the proton appears as a sharp singlet at δ 6.26 (Figure 2). The disappearance of the peak for the methyl group of the β-ketoester and new peaks for the ethylene diamine methylene groups confirmed the formation of β-ketoamide 22. The second product was identified as the expected chlorin e₆ derivative 23. As reported in the literature, temperature and the concentration of the amine play vital roles in deciding the major product.³⁸ A large excess of amine and mild heating always produced the classical ring-opened product 23, and a stoichiometric amount of amine in refluxing toluene yielded β-ketoamide 22 as the major product.

Figure 2.

¹H NMR spectral comparison of: A) methyl pheoporbide a (21); B) β-ketoamide 22 (*contains 10% of ring-opened product 24); and C) 13¹-ethylene-diaminyl-chlorin e₆ dimethyl ester 24. Assignments: a, 13² CH; b, 13³ CO₂CH₃; c, 15¹ CH₂; d, 15² CO₂CH₃.

The first attempt to introduce the ethylene diamine to both ester and keto groups of methyl pheophorbide 21 to form the 13¹,15²-disubstituted chlorin e₆ derivative 23 was unsuccessful. Both β-keto-ester aminolysis and ring-opening reactions were attempted in one pot. First methyl pheophorbide 21 and ethylene diamine were refluxed in toluene until disappearance of the starting material, as monitored by TLC. Then excess amine was added and the mixture was stirred at 40 °C for 12 hours to complete the ring cleavage reaction. Unfortunately, this process resulted in a mixture of compounds, including the desired product 23. Mass spectrometry revealed three major compounds (22-24) in this mixture, as shown in Scheme 6. Purification of these compounds was challenging due to the high polarity of the free amine groups.

Scheme 6.

Attempted synthesis of 13¹,15²-bis-ethylenediamine substituted chlorin e₆ derivative 23

It was decided to use half-protected ethylene diamine to simplify the purification process. As indicated by TLC, aminolysis of the β-keto-ester of methyl pheophorbide a (21) with protected ethylene diamine was completed after 12 hours. But the second step ring-cleavage was unsuccessful with Boc-protected ethylene diamine, possibly due to steric bulkiness of the amine. Thus, product 25 was isolated and purified before going on to the ring cleavage reaction. As it was possible to achieve the isocyclic ring cleavage using free ethylene diamine, unprotected ethylene diamine was used for the second step and the diethylene diamine substituted product 26 was obtained. Product 26 was purified using a short silica gel column and its identity was confirmed using mass and ¹H NMR spectroscopy. Boc deprotection was achieved using TFA to obtain the free amines (23) for the subsequent coupling reaction. Coupling with protected aspartic acid, to give 27 followed by deprotection produced the desired product 28, in 15% yield over 5 steps from methyl pheophorbide a (21) (Scheme 7).

Scheme 7.

Synthesis of 13¹,15²-di(ethylenediaminylaspartyl)chlorin e₆ 28, (Reagents: i. 1.5 equiv (Boc)NHCH₂CH₂NH₂, toluene, 100 °C, 12-16 h, 65%; ii. 10 equiv NH₂CH₂CH₂NH₂, toluene, 45 °C, 12 h, 64%; iii. TFA, CH₂Cl₂ 1:3, 6 h, rt, 88%; iv. Boc-Asp(O-tBu)OH, DIEA, CH₂Cl₂, TBTU, DMF, 40%; iv. TFA/CH₂Cl₂ 1:3, 6 h, rt, 70%.)

Advantage was taken of a previous route to introduce two types of amino acids to the 13¹ and 15² positions. Protected β-alanine was used for the aminolysis reaction of 21 as it greatly reduces the unwanted ring-opened product and creates a carboxylic end to couple aspartic acid through its amino group. Subsequent aminolysis with protected β-alanine, to give 29, followed by deprotection of the tert-butyl ester provided free acid 30. Its identity was confirmed by mass spectrometry. Free acid 30 was activated and coupled with protected aspartic acid to obtain pheophorbide derivative 31. This was purified and characterized by ¹H NMR and mass spectroscopy. A classical ring-opening reaction was performed with excess ethylene diamine in toluene at 40 °C to provide the chlorin e₆ derivative 32. The reaction was monitored using UV-Vis spectroscopy, and the color changed from dark green to bright green during isocyclic ring-opening. ¹H NMR spectroscopy shows the new peaks for ethylene diamine and mass spectroscopy confirmed the identity of the product. Then Boc-protected lysine was coupled with the free amine group of chlorin e₆ derivative 32 to produce the desired 13¹-ethylenediaminyl-lysyl-15²-β-alanylaspartylchlorin e₆ tert-butyl methyl ester. This product was purified using silica gel chromatography. Finally, global deprotection with TFA in CH₂Cl₂ produced the final product 33 in 9% yield over six steps starting from methyl pheophorbide a (21) (Scheme 8).

Scheme 8.

Synthesis of 13¹,15²-di(amino acid) derivative 33. (Reagents: i. 1.5 equiv NH₂CH₂CH₂CO₂t-Bu, toluene, 100 °C, 12-16 h, 54%; ii. TFA, CH₂Cl₂ 1:3, 6 h, rt, 99%; iii. L-Asp di-t-Bu.HCl, DIEA, CH₂Cl₂, HOBt, TBTU, DMF, 59%; iv. 10 equiv NH₂CH₂CH₂NH₂, toluene, 45 °C, 12 h, 58%; v. Boc-Lys(Boc)-OH, DCHA, DIEA, CH₂Cl₂, HOBt, TBTU, DMF; vi. TFA, CH₂Cl₂ 1:3, 6 h, rt, 88%; iv. Boc-Asp(O-t-Bu)OH, DIEA, CH₂Cl₂, TBTU, DMF, 40%; iv. TFA/CH₂Cl₂ 1:3, 12 h, rt, 48% (2 steps).

Cell Culture Studies

Cytotoxicity

The dark cytotoxicity and phototoxicity (~1.5 J/cm²) of chlorin e₆ (1) and its di(amino acid) conjugated derivatives 12, 13, 28 and 33 were evaluated in HEp2 cells using a Cell Titer Blue assay. Significant differences were observed in the dark and phototoxicity of the di(amino acid) conjugates, as shown in Table 1. These differences are attributed to both the sites of conjugation (13¹,15² or 13¹,17³ or 15²,17³) and the overall charge and amphiphilic character of the chlorin e₆ derivatives. While conjugation of two aspartate groups via their α-amine groups gave tetra-anionic conjugates 12 and 13, the conjugation of two aspartates via the carboxylate groups gave zwitterionic 28. On the other hand compound 33 bearing an aspartate conjugated via the α-amine group and a lysine conjugated via the carboxylate group, is also zwitterionic.

Table 1.

Dark and phototoxicity (~ 1.5 J/cm²) for chlorin e₆ and its di-amino acid derivatives in HEp2 cells, using a Cell Titer Blue assay

Compound	Dark toxicity (IC50, μM)	Phototoxicity (IC50, μM)	Ratio
Chlorin e6 (1)	350	14.5	24.1
152,173-di(Asp)Ce6 MME (12)	>400	35	>11
131,173-di(Asp)Ce6 MME (13)	305	26	11.7
131,152-di(EDAsp)Ce6 MME (28)	70	11	6.4
131-EDLys-152-β-AlaAspCe6 MME (33)	65	9.1	7.2

In comparison with chlorin e₆ (1), the zwitterionic 13¹,15²-disubstituted conjugates 28 and 33 showed higher phototoxicity while the tetra-anionic 12 and 13 showed lower phototoxicity. These results are in agreement with our previous observations that 13¹- and 15²-mono(amino acid) chlorin e₆ derivatives tend to show higher phototoxicities than the corresponding 17³ derivatives.²⁷ This might in part be due to the different molecular conformations conferred by 13¹, 15² or 17³ substitutions; while the 17³ derivatives tend to assume L-shape conformations with the side group positioned perpendicular, or folded over the macrocycle, the 15² and in particular the 13¹ derivatives tend to assume linear conformations with the side groups extending away from the macrocycle.²⁷ Such linear conformations might favor binding to specific intracellular components, inducing higher phototoxicity. However, the zwitterionic 13¹,15² conjugates also showed about 5-fold higher dark cytotoxicity than chlorin e₆ and the tetra-anionic conjugates, and lower dark/photo cytotoxicity ratio. The observed higher dark and photo cytotoxicities of conjugates 28 and 33 might in part be due to their higher cellular uptake (see below).

Time-dependent Cellular Uptake

The results obtained for the time-dependent uptake of chlorin e₆ and its di(amino acid) derivatives at a concentration of 10 μM in human carcinoma HEp2 cells are shown in Figure 3. Conjugates 12 and 13 showed similar uptake to chlorin e₆, probably due to their low interaction and low permeability across the cell membrane as a result of their negative charge. In addition to phosphate groups, the plasma membranes of tumor cells generally contain higher net negative charge compared with normal cells because of over-expression of polysialic acid residues.⁴⁵ On the other hand, the zwitterionic conjugates 28 and 33, showed much higher cellular uptake at all time points investigated, and after 24 h about 4-fold and 6-fold higher cellular uptake were observed for 28 and 33 than chlorin e₆, respectively. Although the 13¹,15²-substituted conjugates 28 and 33 were found to have similar cytotoxicities (see Table 1), conjugate 33 bearing two different amino acid residues, lysine and aspartate, had significant higher uptake than 28 at all times investigated, maybe due to its higher amphiphilicity conferred by substitution with two different amino acids.

Figure 3.

Time-dependent uptake of chlorin e₆ (1, green) and its derivatives 15²,17³-di(Asp)Ce₆ MME (12, blue), 13¹,17³-di(Asp)Ce₆ MME (13, red), 13¹,15²-di(EDAsp)Ce₆ MME (28, purple) and 13¹-EDLys-15²-β-AlaAspCe₆ MME (33, black), at 10 μM by HEp2 cells.

The preferential sites of subcellular localization of the di(amino acid) conjugates of chlorin e₆ were evaluated by fluorescence microscopy, and the results are shown in Table 2 and Figures 4-7. The organelle specific probes BODIPY Ceramide (Golgi), LysoSensor Green (Lysosomes), MitoTracker Green (mitochondria), and ER tracker Blue/White fluorescence (ER) were used in the overlay experiments. All conjugates localized in the lysosomes and the Golgi apparatus, and to a smaller extent in the ER. In addition, conjugate 33 bearing lysine and aspartate residues, was also observed in mitochondria, and this might in part be responsible for its higher dark and phototoxicities. These results are in agreement with the known preferential localization of LS-11 (15²-aspartyl chlorin e₆) in the lysosomes,⁴⁶ and with our previous observations of 13¹, 15² and 17³ mono(amino acid) conjugates of chlorin e₆ localizing in lysosomes, ER and Golgi.²⁷

Table 2.

Major and minor subcellular sites of localization for chlorin e₆ and its di-amino acid derivatives in HEp2 cells

Compound	Lyso	ER	Golgi	Mito
Chlorin e6 (1)	+	++	−	−
152,173-di(Asp)Ce6 MME (12)	+	+	+++	−
131,173-di(Asp)Ce6 MME (13)	+++	+	+++	−
131,152-di(EDAsp)Ce6 MME (28)	+++	+	+++	−
131-EDLys-152-β-AlaAspCe6 MME (33)	+++	+	+++	+

Figure 4.

Subcellular localization of 15²,17³-di(aspartate) chlorin e₆ 12 in HEp2 cells at 10 μM for 6h, (a) phase contrast, (b) overlay of 12 and phase contrast, (c) ER Tracker Blue, (d) overlay of 12 and ER Tracker Blue, (e) BODIPY Ceramide, (f) overlay of 12 and BODIPY Ceramide, (g) MitoTracker Green, (h) overlay of 12 and MitoTracker Green, (i) LysoSensor Green, (j) overlay of 12 and LysoSensor Green. Scale bar: 10 μm.

Figure 7.

Subcellular localization of 15²,17³-di(aspartate) chlorin e₆ 33 in HEp2 cells at 10 μM for 6h, (a) phase contrast, (b) overlay of 33 and phase contrast, (c) ER Tracker Blue, (d) overlay of 33 and ER Tracker Blue, (e) BODIPY Ceramide, (f) overlay of 33 and BODIPY Ceramide, (g) MitoTracker Green, (h) overlay of 33 and MitoTracker Green, (i) LysoSensor Green, (j) overlay of 33 and LysoSensor Green. Scale bar: 10 μm.

Experimental

General

All air and moisture sensitive reactions were performed in dried and distilled solvents under an argon atmosphere. All solvents and reagents were purchased from commercial sources, unless otherwise stated. Silica gel 60 (230×400 mesh, Sorbent Technologies) was used for column chromatography. Analytical thin-layer chromatography (TLC) was carried out using polyester backed TLC plates 254 (precoated, 200 μm) from Sorbent Technologies. NMR spectra were recorded on an AV-400 spectrometer (400 MHz for ¹H, 100 MHz for ¹³C). The chemical shifts are reported in δ ppm using the following partially deuterated solvents as internal references: CD₂Cl₂ 5.32 ppm (¹H), 54 ppm (¹³C); d-DMSO 2.49 ppm (¹H), 39.5 ppm (¹³C); d-CH₃OH 4.78 ppm (¹H), 49.0 ppm (¹³C); CDCl₃ 7.26 ppm (¹H), 77.16 ppm (¹³C); (CH₃)₂CO 2.50 ppm (¹H), 29.84 ppm (¹³C). Electronic absorption spectra were measured on an Agilent 8453 UV/Vis spectrophotometer. Mass spectra were obtained on a Bruker Omniflex MALDI Time-of-Flight Mass Spectrometer. Spirulina pacifica alga was purchased as a spray-dried powder from Cyanotech, Hawaii. Pheophytin a (1) was extracted from Spirulina pacifica alga as previously published and its spectroscopic characterization agreed with the published data.³⁴ All compounds synthesized were purified and isolated in ≥ 95% purity, as evidenced by analytical TLC in at least two solvent systems, and confirmed by the absence of extraneous tetrapyrrole resonances in ¹H- and ¹³C-NMR spectra.

Synthetic procedures and characterization

15²,17³-Diaspartylchlorin e₆ tetra(tert-butyl)-monomethyl ester (11)

Chlorin e₆ (1, 100 mg, 0.168 mmol) was dissolved in dry CH₂Cl₂ (7 mL) and DIEA (0.06 mL, 0.34 mmol) was added. A mixture of DCC (105 mg, 0.51 mmol) and DMAP (163 mg, 0.51 mmol) in CH₂Cl₂ (8 mL) was added and the mixture was allowed to stir for 2 h. Then aspartic acid di(tert)butyl ester hydrochloride (125 mg, 0.445 mmol) and DIEA (0.075 ml) were mixed in CH₂Cl₂ (2 ml) and added to the reaction mixture. The solution was allowed to stir overnight at room temperature and after 12 h it was treated with excess ethereal diazomethane. Then the mixture was diluted with CH₂Cl₂ and washed with 5% aqueous citric acid, followed by a wash with brine and finally water. It was dried over anhydrous Na₂SO₄ and the solvent was evaporated. The residue was dissolved in 5% methanol/CH₂Cl₂ and purified via silica gel column chromatography with the same mobile phase to afford 15²,17³-diaspartylchlorin e₆ tera(tert-butyl)-monomethyl ester (11 , C₅₉H₈₀N₆O₁₂, 54 mg, 0.051 mmol, 30% yield); UV-Vis (acetone): λ_max (ε/mM⁻¹cm⁻¹) 664 nm (52), 608 (3.9), 528 (3.6), 500 (13), 400 (170); ¹H NMR (acetone-d₆, 400 MHz): δ 9.80 (s, 1H), 9.63 (s, 1H), 9.06 (s, 1H), 8.15 (dd, J = 17.9, 11.6 Hz, 1H), 7.37 (d, J = 7.9 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 6.37 (dd, J = 17.9, 1.5 Hz, 1H), 6.10 (dd, J = 11.6, 1.4 Hz, 1H), 5.37 (d, J = 12.0 Hz, 2H), 4.67 (s, 2H), 4.26 (s, 3H), 3.77 (d, J = 7.6 Hz, 2H), 3.57 (s, 3H), 3.48 (s, 4H), 3.25 (s, 3H), 2.83 – 2.63 (m, 3H), 2.52 – 2.28 (m, 2H), 2.14 (s, 2H), 1.89 – 1.79 (m, 2H), 1.78 (d, J = 7.1 Hz, 3H), 1.69 (t, J = 7.6 Hz, 3H), 1.43 (s, 9H), 1.35 (s, 9H), 1.26 (s, 9H), 1.16 (s, 9H), −1.36 (s, 1H), −1.57 (s, 1H); MS (MALDI-TOF) m/z 1065.591 [M+H]⁺, calcd. for C₅₉H₈₁N₆O₁₂ 1065.591.

15²,17³-Diaspartylchlorin e₆ methyl ester (12)

15²,17³-Diaspartylchlorin e₆ tetra(tert-butyl)-monomethyl ester (11, 54 mg, 0.051 mmol) was dissolved in 2 mL of dry CH₂Cl₂ in an ice bath under argon and TFA (1 mL) was added and the reaction mixture was allowed to stir overnight. The solvent was rotevaporated several times with diethyl ether to remove residual TFA. The residue was washed with CH₂Cl₂ several times. The final product was re-dissolved in a water/acetonitrile mixture and freeze dried to afford 15²,17³-diaspartylchlorin e₆ methyl ester (12, C₄₃H₄₈N₆O₁₂, 38 mg, 0.045 mmol, 88% yield); UV-Vis (MeOH): λ_max (ε/mM⁻¹cm⁻¹) 661 nm (71.6), 607 (11), 527 (9.3), 499 (22.4), 399 (172.3); ¹H NMR (acetone-d₆, 400 MHz): δ 9.88 (s, 1H), 9.66 (s, 1H), 9.21 (s, 1H), 8.11 (dd, J = 17.8, 11.6 Hz, 1H), 6.35 (d, J = 17.8 Hz, 1H), 6.14 (d, J = 11.6 Hz, 1H), 5.35 (t, J = 22.7 Hz, 2H), 4.81 (br. s, 1H), 4.65 (br. s, 1H), 4.63 (br. S, 1H), 4.25 (s, 3H), 3.70 (d, J = 7.9 Hz, 2H), 3.54 (s, 3H), 3.46 (s, 3H), 3.18 (s, 3H), 3.06 – 2.60 (m, 5H), 2.27 (m, 2H), 1.81 (d, J = 7.1 Hz, 3H), 1.55 (t, J = 7.4 Hz, 3H). 2.00 – 1.50 (m, 4H) 1.25 (m, 2H); ¹³C NMR (acetone-d₆, 101 MHz) δ 193.12, 192.77, 191.95, 191.88, 191.84, 191.04, 188.72, 168.55, 164.14, 163.25, 160.42, 158.10, 157.50, 155.68, 155.26, 155.20, 151.92, 150.02, 149.04, 145.83, 142.64, 137.66, 134.79, 124.88, 121.07, 117.59, 116.00, 73.49, 73.03, 69.43, 69.24, 68.92, 59.56, 55.70, 53.44, 52.59, 50.75, 42.86, 39.10, 36.79, 31.75, 31.58, 30.41. MS (MALDI-TOF) m/z 840.451 [M]⁺, calcd. for C₄₃H₄₉N₆O₁₂ 840.333

13¹,17³-Diaspartylchlorin e₆ tetra(tert-butyl)-monomethyl ester (20)

Chlorin e₆ (1, 100 mg, 0.168 mmol) was dissolved in methanol (5 mL). DCC (35 mg, 0.17 mmol) and DMAP (21 mg, 0.17 mmol) were added and the mixture was stirred until the anhydride intermediate was observed in the TLC. After 1 h, freshly prepared sodium methoxide (0.34 mL of a 0.5 M solution) was added into the reaction mixture dropwise until the color changed from brown to light green. The reaction was monitored by UV-Vis spectroscopy. The solution changed from brown to light green as the anhydride ring opened. The mixture was diluted with ethyl acetate and then washed with 5% aqueous citric acid, followed by a wash with brine and finally with water. It was dried over anhydrous Na₂SO₄ to afford chlorin e₆ 15²-methyl ester (18, C₃₅H₃₉N₄O₆, 101 mg, 0.165 mmol, 100% yield). Purification was challenging due to the polarity induced by the two free acid groups; thus, the crude product was subjected to the next reaction without purification. A ¹H NMR spectrum of the crude product confirmed the methylated acetic side chain. [MS (MALDI-TOF) m/z 633.311 [M+Na]⁺, calcd. for C₃₅H₃₉N₄O₆ 633.269]. Chlorin e₆ monomethyl ester (18, 101 mg, 0.165 mmol) was dissolved in dry DMF (5 mL). A mixture of HOBt (46 mg, 0.34 mmol), TBTU (109 mg, 0.34 mmol) and DIEA (0.06 ml, 0.34 mmol) in DMF (5 ml) was added and the mixture was allowed to stir for 30 min at room temperature. Then aspartic acid di-tert-butyl ester hydrochloride (125 mg, 0.445 mmol) and DIEA (0.08 ml, 0.445 mmol) were mixed in CH₂Cl₂ (2 mL) and added to the reaction mixture. The solution was allowed to stir for 48-72 h until formation of the desired product was confirmed by TLC. Then the reaction mixture was washed with 5% aqueous citric acid, followed by a wash with brine and then water. The organic phase was dried over anhydrous Na₂SO₄ and solvent was evaporated. The residue was dissolved in 5% methanol/CH₂Cl₂ and purified via chromatography on a short silica gel column with the same mobile phase to afford 15²,17³-diaspartylchlorin e₆ tetra(tert-butyl)-monomethyl ester (20, C₅₉H₈₀N₆O₁₂, 85 mg, 0.079 mmol, 48% yield); UV-Vis (acetone): λ_max (ε/mM⁻¹cm⁻¹) 663 nm (54), 607 (3.8), 528 (3.1), 500 (13), 399 (165); ¹H NMR (acetone-d₆, 400 MHz): δ 9.79 (d, J = 6.1 Hz, 1H), 9.65 (s, 1H), 9.10 (s, 1H), 8.42 (d, J = 7.9 Hz, 1H), 8.12 (dd, J = 17.8, 11.6 Hz, 1H), 7.29 (d, J = 8.2 Hz, 1H), 6.32 (d, J = 17.8 Hz, 1H), 6.06 (d, J = 11.4 Hz, 1H), 5.73 (d, J = 18.9 Hz, 1H), 5.50 – 5.21 (m, 2H), 4.76 – 4.61 (m, 2H), 4.61 – 4.48 (m, 1H), 3.76 (s, 3H), 3.74 – 3.68 (m, 1H), 3.65 (s, 3H), 3.47 (s, 3H), 3.42 (s, 1H), 3.22 (s, 3H), 3.20 – 3.11 (m, 2H), 2.66 (d, J = 5.2 Hz, 2H), 2.46 – 2.27 (m, 1H), 2.24 – 2.14 (m, 1H), 1.83 – 1.72 (m, 2H), 1.66 (s, 9H), 1.55 (s, 9H), 1.41 (s, 9H), 1.35 (s, 9H), 1.72-1.2 (m, 6H), −1.66 (1H), −1.85 (1H); MS (MALDI-TOF) m/z 1065.681 [M+H]⁺, calcd. for C₅₉H₈₁N₆O₁₂ 1065.591.

13¹,17³-Diaspartylchlorin e₆ methyl ester (13)

15²,17³-Diaspartylchlorin e₆ tetra(tert)butyl methyl ester (20, 50 mg, 0.047 mmol) was dissolved in 2 mL of dry CH₂Cl₂ in an ice bath under argon. TFA (1 mL) was added and the reaction mixture was allowed to stir overnight. The mixture was rotavaporated several times with diethyl ether to remove residual TFA. The residue was dissolved in water/acetonitrile mixture and freeze dried to obtain 13¹,17³-diaspartylchlorin e₆ methyl ester (13, C₄₃H₄₈N₆O₁₂, 21 mg, 0.025 mmol, 53%). UV-Vis (MeOH): λ_max (ε/mM⁻¹cm⁻¹) 662 nm (75.3), 607 (23), 529 (10), 500 (8.3), 400 (165.2); ¹H NMR (MeOD, 400 MHz) δ 10.07 (s, 1H), 9.86 (s, 1H), 9.45 (s, 1H), 8.06 (dd, J = 17.3, 11.9 Hz, 1H), 6.30 (d, J = 17.8 Hz, 1H), 6.24 (d, J = 11.4 Hz, 1H), 5.62 (d, J = 18.9 Hz, 1H), 5.28 (d, J = 18.3 Hz, 2H), 4.78 – 4.76 (m, 2H), 4.76 - 4.56 (m, 3H), 3.80 (s, 3H), 3.59 (s, 3H), 3.52 (s, 3H), 3.21 (s, 3H), 2.87 – 2.69 (m, 2H), 2.68 – 2.47 (m, 1H), 2.21 (d, J = 19.9 Hz, 2H), 1.94 – 1.72 (m, 4H), 1.59 (d, J = 6.9 Hz, 4H), 1.30 – 1.01 (m, 4H). ¹³C NMR (MeOD, 101 MHz) δ 175.33, 174.90, 174.72, 174.02, 173.88, 173.83, 173.66, 169.35, 143.94, 142.95, 142.47, 140.57, 140.42, 138.22, 138.01, 136.12, 135.46, 135.25, 133.96, 132.61, 131.29, 129.47, 124.69, 107.02, 100.12, 98.81, 97.77, 54.94, 53.05, 51.25, 50.55, 38.89, 36.86, 34.68, 33.77, 26.68, 26.02, 23.70, 20.00, 16.98, 12.22, 10.91, 9.11. MS (MALDI-TOF) m/z 840.532 [M]⁺, calcd. for C₄₃H₄₉N₆O₁₂ 840.333.

Ethylenediaminyl(boc) pheophorbide a (25)

Methyl pheophorbide a (21, 100 mg, 0.165 mmol) was dissolved in dry toluene (20 ml) and the mixture was heated to 100 °C under nitrogen. Then, mono-boc protected ethylene diamine (32 mg, 0.20 mmol) was added. The reaction mixture was allowed to stir overnight at 100 °C while monitoring by TLC. Then the solvent was removed and the residue was dissolved in CH₂Cl₂ and washed with 5% aqueous citric acid, followed by water and brine. It was dried over anhydrous Na₂SO₄ and the solvent was evaporated. The residue was dissolved in 3% methanol/CH₂Cl₂ and purified via silica gel column chromatography with the same mobile phase to afford ethylenediaminyl(boc) pheophorbide a (25, C₄₂H₅₀N₆O₆, 80 mg, 0.109 mmol, 65%); UV-Vis (DCM): λ_max (ε/mM⁻¹cm⁻¹) 667 nm (46), 609 (6.5), 535 (8), 505 (10), 413 (99); ¹H NMR (acetone-d₆, 400 MHz) δ 9.58 (s, 1H), 9.30 (s, 1H), 8.87 (s, 1H), 8.13 – 7.92 (m, 2H), 6.27 (dd, J = 17.9, 1.5 Hz, 1H), 6.18 (s, 1H), 6.12 (dd, J = 11.5, 1.4 Hz, 1H), 6.16 (m, 1H), 4.77 – 4.55 (m, 1H), 4.37 (dt, J = 9.3, 2.5 Hz, 1H), 3.59 (s, 3H), 3.52 (s, 3H), 3.42 – 3.36 (m, 2H), 3.39 (s, 3H), 3.35 (d, J = 6.5 Hz, 3H), 3.08 (s, 3H), 2.74 – 2.57 (m, 2H), 2.46 – 2.33 (m, 1H), 2.31 – 2.19 (m, 1H), 1.84 (d, J = 7.3 Hz, 3H), 1.76 – 1.64 (m, 1H), 1.58 (t, J = 7.6 Hz, 3H), 1.35 (s, 9H), −1.77 – −2.11 (m, 2H); MS (MALDI-TOF) m/z 735.561 [M+H]⁺, calcd. for C₄₂H₅₁N₆O₆ 735.387

15²-Ethylenediaminyl(boc)-13¹-ethylenediaminylchlorin e₆ methyl ester (26)

Ethylenediaminyl(boc) pheophorbide-a (25, 80 mg, 0.109 mmol) was dissolved in toluene (10 mL) and ethylene diamine (30 mg, 0.5 mmol) was added. The reaction mixture was heated at 40 °C overnight. Progress was monitored by TLC and UV-Vis spectrometry. After the reaction was complete by TLC, the solvent was removed and the residue was dissolved in CH₂Cl₂ and washed with 5% aqueous citric acid to remove excess amine, followed by a wash with brine. The organic phase was dried over anhydrous Na₂SO₄ and the solvent was evaporated. The residue was dissolved in 5% methanol/CH₂Cl₂ and eluted from a silica gel column with the same mobile phase. Then the methanol percentage of the mobile phase was gradually increased up to 20% to elute the pure product from the column. The solvent was evaporated and the residue was re-dissolved in 5% acetone/CH₂Cl₂ and filtered to remove silica from the sample. After evaporation of the solvent pure 15²-ethylenediaminyl(boc)-13¹-ethylenediaminylchlorin e₆ methyl ester was obtained (26, C₄₄H₅₈N₈O₆, 55 mg, 0.069 mmol, 64%); UV-Vis (acetone): λ_max (ε/mM⁻¹cm⁻¹) 663 nm (55), 607 (2.6), 528 (1.3), 500 (12), 399 (170); ¹H NMR (acetone-d₆, 400 MHz) δ 9.74 (s, 1H), 9.73 (s, 1H), 9.13 (s, 1H), 8.36 (br. s, 1H), 8.23 (dd, J = 17.8, 11.5 Hz, 1H), 7.46 (br. s, 1H), 6.64 (br. s, 1H), 6.40 (dd, J = 17.9, 1.5 Hz, 1H), 6.12 (dd, J = 11.7, 1.5 Hz, 1H), 5.55 (d, J = 18.0 Hz, 1H), 5.09 (d, J = 9.3 Hz, 1H), 4.76 – 4.51 (m, 2H), 4.28 (s, 1H), 4.01 – 3.83 (m, 1H), 3.80 – 3.67 (m, 4H), 3.58 (s, 3H), 3.54 (s, 3H), 3.52 (s, 3H), 3.29 (s, 3H), 3.25 – 3.16 (m, 2H), 3.08 (dd, J = 9.1, 4.9 Hz, 1H), 2.78 – 2.64 (m, 1H), 2.43 – 2.21 (m, 2H), 2.00 (d, J = 8.0 Hz, 1H), 1.76 (d, J = 7.1 Hz, 3H), 1.68 (t, J = 7.6 Hz, 3H), 1.20 (s, 9H), 0.97 (m, 3H), −1.66 (s, 1H), −2.02 (s, 1H); MS (MALDI-TOF) m/z 795.651 [M+H]⁺, calcd. for C₄₄H₅₉N₈O₆ 795.456

13¹,15²-Diethyleneaminylaspartylchlorin e₆ di(tert)butyl di(boc) methyl ester (27)

15²-Ethylenediaminyl(boc)-13¹-ethylenediaminylchlorin e₆ methyl ester (26, 55 mg, 0.069 mmol) was dissolved in 3 mL of dry CH₂Cl₂ in an ice bath under argon and 1 m of TFA was added. Then the reaction mixture was allowed to stir overnight. The mixture was rotavaporated several times with diethyl ether to remove residual TFA. The residue was dissolved in water/acetonitrile mixture and freeze dried. Without any further purification the crude product was taken to the next step. (23, C₃₉H₅₀N₈O₄, 42 mg, 0.06 mmol, 88%), [MS (MALDI-TOF) m/z 695.445 [M+H]⁺,calcd. for C₃₉H₅₁N₈O₄ 695.403]. (Boc)Asp(tBu)OH (70 mg, 0.24 mmol) was dissolved in dry DMF (5 mL). A mixture of HOBt (32 mg, 0.24 mmol), TBTU (77 mg, 0.24 mmol) and DIEA (0.05 ml, 0. mmol) in DMF (3 mL) was added and the mixture was allowed to stir for 1 h. 13¹,15²-Diethylenediaminylchlorin e₆ methyl ester (23, 42 mg, 0.060 mmol) was added to the reaction mixture and stirring was continued for 48 h. The mixture was diluted with CH₂Cl₂ and then washed with 5% aqueous citric acid, followed by washes with brine and water. The organic layer was dried over anhydrous Na₂SO₄ and the solvent was evaporated. The residue was dissolved in 6% MeOH/CH₂Cl₂ and purified via silica gel column chromatography using the same mobile phase to afford 13¹,15²-diethyleneaminyl-diaspartylchlorin e₆ di(tert)butyl di(boc) methyl ester (27, C₆₅H₉₂N₁₀O₁₄, 30 mg, 0.024 mmol, 40%); UV-Vis (acetone): λ_max (ε/mM⁻¹cm⁻¹) 663 nm (48), 607 (2.1), 528 (1.7), 500 (11), 399 (150); ¹H NMR (chloroform-d, 400 MHz) δ 9.68 (s, 1H), 9.61 (s, 1H), 8.79 (s, 1H), 8.07 (dd, J = 17.9, 11.5 Hz, 1H), 7.97 (d, J = 5.7 Hz, 1H), 7.74 (s, 1H), 6.34 (d, J = 17.8 Hz, 1H), 6.13 (d, J = 11.5 Hz, 1H), 5.81 (d, J = 8.3 Hz, 1H), 5.47 (d, J = 18.1 Hz, 1H), 5.28 – 4.88 (m, 2H), 4.67 – 4.30 (m, 3H), 4.10 – 3.68 (m, 10H), 3.65 – 3.12 (m, 5H), 3.54 (s, 3H), 3.48 (s, 3H), 3.31 (s, 3H), 2.93 – 2.69 (m, 2H), 2.67 – 2.53 (m, 1H), 2.23 (m, 3H), 2.04 (s, 1H), 1.90 – 1.55 (m, 7H), 1.31 (s, 18H), 1.14 (s, 18H), 0.94 – 0.80 (m, 1H), −1.62 (s, 1H), −1.80 (s, 1H); MS (MALDI-TOF) m/z 1237.651 [M+H]⁺, calcd. for C₆₅H₉₃N₁₀O₁₄ 1237.687.

13¹,15²-Diethyleneaminyl-diaspartylchlorin e₆ methyl ester (28)

13¹,15²-Diethyleneaminyl-diaspartylchlorin e₆ di(tert)butyl di(boc) methyl ester (27, 30 mg, 0.024 mmol) was dissolved in 2 mL of dry CH₂Cl₂ in an ice bath under argon. TFA (1 mL) was added and the reaction mixture was allowed to stir overnight. The reaction mixture was rotavaporated several times with diethyl ether to remove residual TFA. The residue was washed with CH₂Cl₂ several times. The final product was dissolved in water and freeze dried to obtain 13¹-15²-diethyleneaminyl-diaspartylchlorin e₆ methyl ester (28, C₄₇H₆₀N₁₀O₁₀, 16 mg, 0.016 mmol, 70%). UV-Vis (MeOH): λ_max (ε/mM⁻¹cm⁻¹) 661 nm (74.7), 606 (8.4), 527 (6.2), 500 (20.5), 400 (190.2); ¹H NMR (methanol-d₄, 400 MHz) δ 10.18 (s, 1H), 10.03 (s, 1H), 9.47 (s, 1H), 8.20 (dd, J = 17.7, 11.6 Hz, 1H), 6.39 (d, J = 17.8 Hz, 1H), 6.29 (d, J = 11.5 Hz, 1H), 5.59 (d, J = 18.7 Hz, 1H), 5.42 (d, J = 18.7 Hz, 1H), 4.79 (d, J = 7.2 Hz, 1H), 4.54 (d, J = 10.8 Hz, 1H), 4.44 (ddd, J = 24.1, 8.5, 4.2 Hz, 1H), 4.25 – 4.05 (m, 1H), 4.03 – 3.83 (m, 4H), 3.74 (s, 3H), 3.67 (s, 3H), 3.56 (s, 3H), 3.49 (td, J = 10.6, 9.5, 4.6 Hz, 2H), 3.41 – 3.26 (m, 6H), 3.38 (s, 3H), 3.23 – 3.00 (m, 2H), 2.99 – 2.82 (m, 2H), 2.70 (dd, J = 17.9, 9.0 Hz, 1H), 2.59 – 2.47 (m, 1H), 2.46 – 2.31 (m, 1H), 1.91 – 1.51 (m, 3H), 1.83 (d, J = 7.1 Hz, 3H), 1.68 (t, J = 7.3 Hz, 3H), 1.29 (m, 3H); ¹³C NMR (MeOD, 101 MHz) δ 174.42, 173.97, 173.75, 172.86, 171.55, 171.39, 169.05, 168.72, 168.05, 143.07, 142.13, 141.77, 139.03, 138.76, 137.66, 136.98, 135.23, 134.58, 132.81, 131.33, 130.49, 128.33, 123.54, 105.19, 99.20, 97.43, 97.06, 53.90, 51.08, 49.96, 49.13, 39.50, 39.30, 39.19, 38.70, 34.67, 34.46, 30.47, 29.65, 29.30, 22.83, 22.37, 18.95, 15.79, 11.06, 10.88, 9.85. MS (MALDI-TOF) m/z 947.559 [M+Na]⁺, calcd. for C₄₇H₆₀N₁₀NaO₁₀ 947.439.

β-Alanylpheophorbide a tert-butyl methyl ester (29)

Methyl pheophorbide a (21, 100 mg, 0.165 mmol) was dissolved in dry toluene (10 mL) and the mixture was heated to 100 °C under nitrogen. Then β-alanine(tBu).HCl (45 mg, 0.25 mmol) and DIEA (0.06 ml, 0.33 mmol) were added. The reaction mixture was allowed to stir overnight at 100 °C in an oil bath and was monitored by TLC. Then the solvent was removed and the residue was dissolved in CH₂Cl₂ and washed with 5% aqueous citric acid followed by water and with brine. The organic layer was dried over anhydrous Na₂SO₄ and the solvent was evaporated. The residue was dissolved in 5% methanol/CH₂Cl₂ and purified via silica gel chromatography with the same mobile phase and to afford β-alanylpheoporbide a tert-butyl methyl ester (29, C₄₂H₄₉N₅O₆, 65 mg, 0.090 mmol, 54%); UV-Vis (DCM): λ_max (ε/mM⁻¹cm⁻¹) 667 nm (45), 609 (6.5), 535 (7.6), 505 (10.5), 412 (107); ¹H NMR (acetone-d₆, 400 MHz) δ 9.33, 9.23* (s, 1H), 8.99, 9.93* (s, 1H), 8.79, 8.77* (s, 1H), 7.97*, 7.92 (t, J = 6.0 Hz, 1H), 7.80 (m, 1H), 6.16, 6.13* (s, 1H), 6.07 (d, J = 9.7 Hz, 1H), 5.99 (dd, J = 11.6, 1.5 Hz, 1H), 4.65*, 4.57 (qd, J = 7.4, 2.0 Hz, 1H), 4.38 (tt, J = 9.7, 2.2 Hz, 1H), 3.68 (qd, J = 6.6, 2.3 Hz, 1H), 3.61 (q, J=6.4, 1H) 3.59*, 3.53 (s, 3H), 3.49 (s, 3H), 3.41 (s, 2H), 3.28*, 3.27 (s, 3H), 2.86*, 2.84 (s, 3H), 2.79 (m, 2H), 2.71 – 2.52 (m, 2H), 2.49 – 2.12 (m, 2H), 1.85 (d, J = 7.3 Hz, 3H), 1.64 (t, J = 7.3 Hz, 3H), 1.48*, 1.49 (s, 9H), −2.00 (s, 1H), −2.16 (s 1H).(* Minor 13² epimer); MS (MALDI-TOF) m/z 720.467 [M+H]⁺, calcd. for C₄₂H₅₀N₅O₆ 720.368.

β-Alanylaspartylpheoporbide a di-tertbutyl methyl ester (31)

β-Alanylpheophorbide a tert-butyl methyl ester (29, 65 mg, 0.090 mmol) was dissolved in 2 mL of dry CH₂Cl₂ in an ice bath under argon. TFA (1 mL) was added and the reaction mixture was allowed to stir for 6 h. The reaction mixture was diluted with CH₂Cl₂ and washed with water and then with saturated sodium bicarbonate. This formed a precipitate while washing with sodium bicarbonate then citric acid solution was added until the precipitate dissolved in the organic phase. Then the solution was washed with brine and dried over anhydrous Na₂SO₄ to give β-alanylpheoporbide a methyl ester (30, C₃₈H₄₁N₅O₆, 60 mg, 0.09 mmol, 100%). [MS (MALDI-TOF) m/z 686.387 [M+H]⁺, calcd. for C₃₈H₄₁N₅NaO₆ 686.295]. β-Alanylpheoporbide a methyl ester (30, 60 mg, 0.090 mmol) was dissolved in dry DMF (5 mL). A mixture of HOBt (18 mg, 0.135 mmol), TBTU (43 mg, 0.135) and DIEA (0.03 ml, 0.18 mmol) in DMF (3 mL) was added and the mixture was allowed to stir for 30 min. Then a mixture of aspartic acid di(tert)butyl ester hydrochloride (101 mg, 0.36 mmol) and DIEA (0.06 ml, 0.36 mmol) in CH₂Cl₂ (3 mL) was added to the reaction mixture. The mixture was allowed to stir for 24 h. The mixture was diluted with CH₂Cl₂ and then washed with 5% aqueous citric acid, followed by with water and brine. The organic layer was dried over anhydrous Na₂SO₄ and the solvent was evaporated. The residue was dissolved in 5% methanol/CH₂Cl₂ and purified via silica column chromatography with the same mobile phase to afford β-alanyl aspartyl pheophorbide a di-tertbutyl methyl ester (31, C₅₀H₆₂N₆O₉, 48 mg, 0.053 mmol, 59%); UV-Vis (DCM): λ_max (ε/mM⁻¹cm⁻¹) 667 nm (41.5), 609 (5.5), 535 (7), 505 (10), 413 (103); ¹H NMR (acetone-d₆, 400 MHz) δ 9.06, 8.98* (s, 1H), 8.70, 8.59* (s, 1H), 8.69 (s, 1H), 8.2* 8.06 (t, J = 5.9 Hz, 1H), 7.70 – 7.45 (m, 2H), 6.17, 6.08* (s, 1H), 5.93 (dt, J = 17.7, 2.3 Hz, 1H), 5.85 (dd, J = 11.5, 1.4 Hz, 1H), 4.82*, 4.76 (dt, J = 8.3, 5.8 Hz, 1H), 4.64*, 4.55 (tt, J = 9.0, 4.5 Hz, 1H), 4.42*, 4.35 (dt, J = 9.7, 2.5 Hz, 1H), 3.77 (q, J = 6.5 Hz, 2H), 3.63*, 3.59 (s, 3H), 3.42, 3.31* (s, 3H), 3.14 (s, 3H), 3.05 (h, J = 6.8, 6.3 Hz, 3H), 2.78 (dd, J = 5.9, 4.7 Hz, 2H), 2.74 – 2.66 (m, 4H), 2.63 (s, 3H), 2.49 – 2.37 (m, 1H), 2.20 (td, J = 9.0, 3.3 Hz, 1H), 1.85, 1.65* (d, J = 7.3 Hz, 3H), 1.45 (s, 9H), 1.42 (s, 9H), 1.35 (t, J = 7.5 Hz, 2H), −2.17*, −2.33 (s, 2H). ).(* Minor 13² epimer); MS (MALDI-TOF) m/z 890.461 [M]⁺, calcd. for C₅₀H₆₃N₆O₉ 890.458.

13¹-Ethylenediaminyl-15²-β-alanylaspartylchlorin e₆ di-tert-butyl methyl ester (32)

β-Alanylaspartylpeoporbide a di-tert-butyl methyl ester (31, 48 mg, 0.053 mmol) was dissolved in toluene (10 ml) and ethylenediamine (15 mg, 0.26 mmol) was added. The reaction mixture was heated at 40 °C for 24-36 h. It was monitored by TLC and UV-Vis spectroscopy. After reaction was complete as monitored by TLC, the solvent was removed and the residue was dissolved in CH₂Cl₂ and washed with 5% aqueous citric acid to remove excess amine, followed by a wash with brine. The organic phase was dried over anhydrous Na₂SO₄ and the solvent was evaporated. The residue was dissolved in 5% methanol/CH₂Cl₂ and chromatographed on a silica gel column with the same mobile phase. Then the methanol percentage of the mobile phase was gradually increased up to 20% to elute the pure product from the column. The solvent was evaporated and re-dissolved in 5% acetone/CH₂Cl₂ and filtered to remove silica from the sample. After evaporation of solvent 13¹-ethylenediaminyl-15²-β-alanyl aspartylchlorin e₆ di-tert-butyl methyl ester was obtained (32, C₅₂H₇₀N₈O₉, 30 mg, 0.031 mmol, 58 %). UV-Vis (acetone): λ_max (ε/mM⁻¹cm⁻¹) 664 nm (51.5), 607 (2.5), 528 (2), 500 (11), 400 (160); It was not possible to obtain the ¹H NMR spectrum and so the crude product was subjected directly to next step. MS (MALDI-TOF) m/z 951.767 [M+H]⁺, calcd. for C₅₂H₇₁N₈O₉ 951.534.

13¹-Ethylenediaminyllysinyl-15²-β-alanylaspartylchlorin e₆ methyl ester (33) via 13²-ethylenediaminyllysinyl-15²-β-alanylaspartylchlorin e₆ di-tert-butyl di-(boc) methyl ester

Boc-Lys(Boc)OH.DCHA (61 mg, 0.116 mmol) was dissolved in dry DMF (5 mL). A mixture of HOBt (16 mg, 0.116 mmol), TBTU (37 mg, 0.116 mmol) and DIEA (0.024 ml, 0.14 mmol) in DMF (3 mL) was added and the mixture was stirred for 30 min. 13¹-Ethylenediaminyl-15²-β-alanylaspartylchlorin e₆ di-tertbutyl methyl ester (32, 30 mg, 0.031 mmol) was added to the reaction mixture and it was stirred for 72 h. After the reaction was deemed complete by TLC, the mixture was diluted with CH₂Cl₂ and then washed with 10% sodium bicarbonate, 5% aqueous citric acid, then followed by washing with brine. The organic phase was dried over anhydrous Na₂SO₄ and the solvent was evaporated. The residue was dissolved in 10% MeOH/CH₂Cl₂ and purified via silica gel column chromatography using the same mobile phase to afford 13¹-ethylenediaminyllysinyl-15²-β-alanylaspartylchlorin e₆ di-tert-butyl di-tert-butyl carbamates methyl ester (C₆₈H₉₈N₁₀O₁₄, 24 mg, 0.018 mmol, 61%); UV-Vis (acetone): λ_max (ε/mM⁻¹cm⁻¹) 663 nm (43.5), 607 (3), 528 (2.5), 500 (10), 400 (136); ¹H NMR (acetone-d₆, 400 MHz) δ 9.67 (s, 1H), 9.65 (s, 1H), 9.10 (s, 1H), 8.71 (s, 1H), 8.26 (d, J = 5.5 Hz, 1H), 8.17 (dd, J = 17.8, 11.5 Hz, 1H), 7.05 (s, 2H), 6.34 (d, J = 17.8 Hz, 1H), 6.23 (d, J = 8.0 Hz, 1H), 6.07 (d, J = 11.5 Hz, 1H), 5.95 (s, 1H), 5.56 (d, J = 18.9 Hz, 1H), 5.22 (d, J = 19.2 Hz, 1H), 4.68 (q, J = 7.1 Hz, 1H), 4.54 (d, J = 9.6 Hz, 1H), 4.29 (q, J = 3.6 Hz, 1H), 3.98 – 3.62 (m, 9H), 3.57 (s, 3H), 3.48 (s, 6H), 3.25 (s, 3H), 3.02 (d, J = 6.0 Hz, 2H), 2.92 (s, 3H), 2.56 – 2.40 (m, 2H), 2.40 – 2.26 (m, 2H), 2.26 – 2.17 (m, 1H), 1.91 (dt, J = 23.0, 8.6 Hz, 2H), 1.74 (d, J = 7.1 Hz, 3H), 1.66 (t, J = 7.2 Hz, 3H), 1.57 – 1.44 (m, 4H), 1.37 (s, 18H), 1.34 (s, 18H), −1.65 (s, 1H), −1.94 (s, 1H); MS (MALDI-TOF) m/z 1279.775 [M+H]⁺, calcd. for C₆₈H₉₉N₁₀O₁₄ 1279.734.

The abovementioned 13¹-ethylenediaminyllysinyl-15²-β-alanylaspartylchlorin e₆ di-tertbutyl di-(boc) methyl ester (24 mg, 0.018 mmol) was dissolved in 2 mL of dry CH₂Cl₂ in an ice bath under argon. TFA (1 mL) was added and the reaction mixture was allowed to stir overnight. The mixture was rotavaporated several times with diethyl ether to remove TFA and the residue was washed with CH₂Cl₂ several times. The final product was dissolved in water and then freeze dried to obtain 13¹-ethylenediaminyllysinyl-15²-β-alanylaspartylchlorin e₆ methyl ester (33, C₅₀H₆₆N₁₀O₁₀, 14 mg, 0.014 mmol; 79%). UV-Vis (MeOH): λ_max (ε/mM⁻¹cm⁻¹) 658 nm (18.9), 635 (67.8), 594 (14), 511 (10), 411 (150.8); ¹H NMR (400 MHz, MeOD) δ 9.96 (s, 1H), 9.70 (s, 1H), 9.42 (s, 1H), 7.98 (s, 2H), 6.39 – 6.01 (m, 3H), 5.57 (d, J = 18.5 Hz, 1H), 5.44 (d, J = 18.5 Hz, 1H), 4.80 (d, J = 6.3 Hz, 2H), 4.60 (d, J = 8.7 Hz, 1H), 4.36 (s, 1H), 4.17 (s, 1H), 4.01 (s, 1H), 3.95 – 3.79 (m, 3H), 3, 3.70 (s, 6H), 3.60 (s, 3H), 3.47 (s, 3H), 3.08 (m, 3H), 3.03 (m, 3H), 2.86 (d, J = 7.1 Hz, 1H), 2.78 (s, 1H), 2.60-2.32 (m, 5H), 2.21 – 1.98 (m, 4H), 1.83 (m, 3H), 1.67 (t, J = 6.7 Hz, 3H), 1.56 (br. s, 4H) 1.31 (m, 2H). ¹³C NMR (MeOD, 101 MHz) δ 174.30, 173.45, 172.41, 171.99, 169.49, 169.40, 169.24, 143.12, 141.31, 138.38, 138.21, 136.42, 134.93, 134.49, 132.42, 130.83, 130.21, 128.13, 123.05, 121.13, 118.21, 115.30, 112.39, 109.05, 104.86, 99.38, 96.88, 53.87, 53.19, 51.07, 49.15, 48.73, 39.86, 39.15, 38.98, 37.50, 36.18, 35.26, 34.82, 30.76, 30.52, 29.83, 26.79, 22.43, 21.66, 18.72, 15.78, 11.06, 10.83, 9.62. MS (MALDI-TOF) m/z 967.602 [M+H]⁺, calcd. for C₅₀H₆₇N₁₀O₁₀ 967.504

Cell Studies

All reagents used in these studies were purchased from Invitrogen. The human HEp2 cells were obtained from ATCC and maintained in a 50:50 mixture of DMEM:AMEM supplemented with 5% FBS and 1% penicillin/streptomycin antibiotic. The cells were sub-cultured twice weekly to maintain sub-confluent stocks.

Time-Dependent Uptake

The HEp2 cells were plated at 7500 cells per well in a Costar 96-well plate and allowed to grow for 48 h. Compound stock solutions were prepared at 32 mM in DMSO and Cremophor (10% of Cremophor in DMSO). Further dilution into the cells of the 96-well plate gave a final concentration of 400 μM with maximum DMSO concentration of 1.95% and Cremophor concentration of 0.05%. Uptake was allowed to continue for 0, 1, 2, 4, 8, 12 and 24 h. The uptake was terminated by removing the loading medium and washing the wells once with PBS. The compound concentration was measured using intrinsic fluorescence as measured with a BMG FLUOstar plate reader equipped with a 355 nm excitation and a 650 nm emission filter. The cells were measured using a CyQuant Cell proliferation assay as per manufacturer’s instructions.

Cytotoxicity

The HEp2 cells were plated as described above for the uptake experiment. The compounds were diluted into media to give 400 μM solution concentrations. Two-fold serial dilutions were then prepared and the cells were incubated overnight. Cell toxicity was measured using Promega’s Cell Titer Blue viability assay⁴⁷ as per manufacturer’s instructions, with untreated cells considered 100% viable and cells treated with 0.2% saponin as 0% viable.

The cells were prepared as described above with compound concentration range from 0-100 μM. After loading overnight, the medium was replaced with medium containing 50 mM HEPES pH 7.2. The cells were exposed to a 1000 W halogen lamp filtered through a 610 nm liquid filter to provide approximately 1.5 J/cm² light dose. The cells were kept cool by filtering the IR radiation through 10 mm of water and placing the culture in an Echotherm chilling plate (Torrey Pines Scientific, Inc.). After 20 min exposure to light, the plate was incubated overnight. Cell viability was then measured as described above.

In comparison with the previously investigated mono-amino acid derivatives of chlorin e6,²⁷ the di-amino acid conjugates appear to be less phototoxic than the 13¹- and the 15²-aspartate derivatives (IC₅₀ = 0.6 and 4.0 uM at 1 J/cm²), probably due to the higher hydrophilicity of the di(amino acid) conjugates.

Microscopy

The Hep2 cells were plated in a 6-well plate and allowed to grow overnight. The cells were exposed to 10 μM of each compound, and then kept at 37 °C for 6 h in 5% CO₂ before adding the organelle tracer. The working concentrations of organelle tracers were as follows: LysoSensor Green 50 nM, MitoTracker Green 250 nM, ER Tracker Blue/white 100 nM, and BODIPY FL C5 Ceramide 50 nM. The organelle tracers were diluted in growing medium and the cells were incubated concurrently with the compound and the tracer for 30 min. The loading medium was removed and cells were washed with PBS 3 times before imaging. The images were acquired using a Leica DMRXA2 microscope with a water immersion objective and DAPI, GFP, and TRITC filter cubes (Chroma Technologies).

Conclusions

Previous work²⁷ has shown 13¹-monoconjugates of chlorin e₆ to be more potent PDT sensitizers (with regard to dark- and photo-toxicity) than the corresponding 15²-monoconjugates. In the present work a series of chlorin e₆ di(amino acid) conjugates were regioselectively synthesized bearing two aspartates in the 13¹,17³- and 15²,17³-positions, or at the 13¹,15² via an ethylene diamine linker. One conjugate bearing two different amino acids, lysine at 13¹ via an ethylene diamine linker and an aspartate at 15² via a β-alanine linker was also synthesized. The conjugation of aspartate residues via the α-amine groups gave anionic compounds while conjugation via the carboxylate gave a zwitterionic compound. The cytotoxicity and uptake of four di(amino acid) chlorin e₆ conjugates, two anionic and two zwitterionic, were investigated in human HEp2 cells, and compared with chlorin e₆. The most cytotoxic, IC₅₀ (dark) = 65-70 μM and IC₅₀ (light) = 9-11 μM, were the zwitterionic 13¹,15²-disubstituted conjugates 28 and 33; these were also the most taken up by cells and localized in multiple organelles. On the other hand, the tetra-anionic 13¹,17³- and 15²,17³-di-aspartyl chlorin e6 12 and 13 showed low dark cytoxicity and lower phototoxicity compared with chlorin e₆. These di(amino acid) derivatives of chlorin e₆ appear to be less phototoxic than previously investigated 13¹- and 15²-mono aspartate chlorin e₆ derivatives,²⁷ maybe due to their increased hydrophilicity.

Figure 5.

Subcellular localization of 15²,17³-di(aspartate) chlorin e₆ 13 in HEp2 cells at 10 μM for 6h, (a) phase contrast, (b) overlay of 13 and phase contrast, (c) ER Tracker Blue, (d) overlay of 13 and ER Tracker Blue, (e) BODIPY Ceramide, (f) overlay of 13 and BODIPY Ceramide, (g) MitoTracker Green, (h) overlay of 13 and MitoTracker Green, (i) LysoSensor Green, (j) overlay of 13 and LysoSensor Green. Scale bar: 10 μm.

Figure 6.

Subcellular localization of 15²,17³-di(aspartate) chlorin e₆ 28 in HEp2 cells at 10 μM for 6h, (a) phase contrast, (b) overlay of 28 and phase contrast, (c) ER Tracker Blue, (d) overlay of 28 and ER Tracker Blue, (e) BODIPY Ceramide, (f) overlay of 28 and BODIPY Ceramide, (g) MitoTracker Green, (h) overlay of 28 and MitoTracker Green, (i) LysoSensor Green, (j) overlay of 28 and LysoSensor Green. Scale bar: 10 μm.