Synthesis and biological evaluation of new boron-containing chlorin derivatives as agents for both photodynamic therapy and boron neutron capture therapy of cancer.

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1339–1343

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Synthesis and biological evaluation of new boron-containing chlorin derivatives as agents for both photodynamic therapy and boron neutron capture therapy of cancer Ryuji Asano a, Amon Nagami b, Yuki f*ckumoto b, Kaori Miura b, Futoshi Yazama b, Hideyuki Ito c, Isao Sakata d, Akihiro Tai b,⇑ a

Fushimi Pharmaceutical Co., Ltd, 1676 Nakazu-cho, Marugame, Kagawa 763-8605, Japan Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, 562 Nanatsuka-cho, Shobara, Hiroshima 727-0023, Japan c Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja, Okayama 719-1197, Japan d Porphyrin Lab., 1-2-35 Tsushimanishizaka, Kita-ku, Okayama 700-0086, Japan b

a r t i c l e

i n f o

Article history: Received 24 September 2013 Revised 10 January 2014 Accepted 18 January 2014 Available online 29 January 2014 Keywords: Photodynamic therapy Boron neutron capture therapy Porphyrin Chlorin derivatives

a b s t r a c t New boron-containing chlorin derivatives 9 and 13 as agents for both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT) of cancer were synthesized from photoprotoporphyrin IX dimethyl ester (2) and L-4-boronophenylalanine-related compounds. The in vivo biodistribution and clearance of 9 and 13 were investigated in tumor-bearing mice. The time to maximum accumulation of compound 13 in tumor tissue was one-fourth of that of compound 9, and compound 13 showed rapid clearance from normal tissues within 24 h after injection. The in vivo therapeutic efﬁcacy of PDT using 13 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3 h after injection of 13. Tumor growth was signiﬁcantly inhibited by PDT using 13. These results suggested that 13 might be a good candidate for both PDT and BNCT of cancer. Ó 2014 Elsevier Ltd. All rights reserved.

Photodynamic therapy (PDT) is a medical treatment that utilizes a cancer-selective photosensitizing agent and visible light in the presence of oxygen to selectively destroy cancer cells by generating singlet oxygen (1O2) and free radicals that have a strong cytocidal effect through photochemical reactions.1–3 Porphyrin derivatives such as PhotofrinÒ4,5 have been most frequently used as cancer-selective photosensitizing agents in PDT. Recently, chlorin derivatives represented by LaserphyrinÒ (mono-L-aspartyl chlorin e6, Npe6, Talaporﬁn sodium),6,7 which was approved in Japan in 2004 for treatment of lung cancer, have been applied to PDT as cancer-selective photosensitizing agents, since the excitation efﬁciency of chlorin derivatives is higher than that of porphyrin derivatives. On the other hand, boron neutron capture therapy (BNCT) has attracted attention as a bimodal therapy for cancer as is PDT. BNCT is a cancer treatment that selectively destroys cancer cells by producing high linear energy transfer a particles and lithium-7 nuclei through nuclear reaction between boron-10 (10B)-containing agents selectively incorporated into cancer cells and low-energy thermal neutrons.8–11 In this treatment, accumulation in and selec-

tivity to cancer cells of boron-containing agents are the most important factors for reducing damage to normal cells. Although 12,13 L-4-boronophenylalanine (BPA) and disodium mercapto-closo-dodecaborate (BSH)14,15 (Fig. 1), which are boron-containing agents used in current clinical trials for BNCT, are very safe, accumulation in and selectivity to cancer cells of BPA and BSH are insufﬁcient, and improvement of these drugs is required. A number of candidate boron-containing agents for BNCT, such as boron-containing nucleic acids,16–18 amino acids,19,20 peptides,21 carbohydrates22,23 and liposomes,24 have been synthesized and evaluated over the past few decades. Boron-containing porphyrin25–28 and chlorin29 derivatives have also been synthesized, and dual applications with boron-containing porphyrin and chlorin derivatives in PDT and BNCT have been attempted.

Figure 1. Chemical structures of BPA and BSH.

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OH 10

H N

A HO

NOEt

O N

NaO

ONa

O O

NaO

O N

O O

ONa

NH N N

NH N

NaO

ONa HO

ONa

NOEt

NH N

Chlorin derivative 1 HN

ONa

NaO

10 HO B

B OH

Figure 2. Molecular design strategy for efﬁcient PDT/BNCT agents.

Recently, we synthesized a water-soluble chlorin derivative 1 (Fig. 2) that had tumor selectivity and rapid clearance from photoprotoporphyrin IX dimethyl ester (2),30,31 and we conﬁrmed the usefulness of PDT using the water-soluble chlorin derivative 1.32 By using chlorin derivatives based on the chemical structure of 1 as boron delivery agents, it is expected that more effective agents for both PDT and BNCT can be developed. In this study, we synthesized two kinds of new boron-containing chlorin derivatives bonded with BPA-related compounds and chlorin derivatives prepared from photoprotoporphyrin IX dimethyl ester (2). We investigated the cancer-selective accumulation and clearance from normal tissues of boron-containing chlorin derivatives, and then evaluated the therapeutic efﬁcacy of PDT using a candidate agent for both PDT and BNCT. We designed two kinds of new boron-containing chlorin derivatives that combined BPA-related compounds and chlorin derivative 1 (Fig. 2). One is a derivative 9 that combined a BPA-related compound in the A-position of the chlorin derivative 1 to leave iminodiacetic acid groups because iminodiacetic acid groups of the chlorin derivative 1 were shown in our previous study to be important for superior tumor accumulation and clearance characteristics.32 The other is a derivative 13 in which one carboxyl group of the iminodiacetic acid group (B-position) was replaced by another polar group, namely, a 4-boronophenyl group. The synthesis of boron-containing chlorin derivative 9 is shown in Scheme 1. Reaction of aldehyde 2 with hydroxylamine hydrochloride in pyridine gave oxime 3. Diamide 4 was prepared by hydrolysis of methyl esters of 3 with aqueous sodium hydroxide in tetrahydrofuran and condensation with dimethyl iminodiacetate in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride in N,N-dimethylacetamide, followed by reoxime formation of aldehyde produced by removal of the oxime group during condensation (94% yield for three steps). O-Alkylation of 4 with sodium hydride and 3-(tert-butoxycarbonylamino)propyl

bromide in N,N-dimethylformamide afforded tert-butyloxycarbonyl (BOC) derivative 5, which was then N-deprotected with triﬂuoroacetic acid in dichloromethane to give aminopropyl derivative 6 (41% yield for two steps). In addition, aminopropyl derivative 6 was synthesized directly from 4 by O-alkylation using non-protected 3-aminopropylbromide in 69% yield, because decomposition of 5 was conﬁrmed under the acidic condition to remove the BOC group. Condensation of 6 with 4-boronocinnamic acid 7 (10B-enriched) in the presence of 4-dimethylaminopyridine and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride in N,N-dimethylacetamide afforded amide 8 (60%), which was then hydrolyzed under basic conditions to give boronocinnamic acid derivative 9 in 83% yield. On the other hand, boronophenylalanine derivative 13 was synthesized by condensation of 10 with L-4-boronophenylalanine ethyl ester hydrochloride 11 (10B-enriched) in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride in N,N-dimethylacetamide, followed by hydrolysis of ethyl esters 12 with aqueous sodium hydroxide in N,N-dimethylformamide in 42% yield for two steps (Scheme 2). The newly synthesized boron-containing chlorin derivatives 9 and 13 were fully characterized by 1H NMR spectroscopy, UV–visible absorption spectroscopy and liquid chromatography mass spectrometry analysis, and they showed good water solubility (100 mg/mL). The in vivo biodistribution and clearance of synthesized boroncontaining chlorin derivatives 9 and 13 were evaluated according to our previous procedure.32 Figure 3 shows the distributions of boron-containing chlorin derivatives 9 and 13 in mouse tissues and serum for periods ranging from 1 h to 24 h after intravenous injection of 10 lmol/kg of 9 and 13. Both compounds 9 and 13 showed good tumor-selective accumulation and rapid clearance from normal tissues. These results suggest that the molecular designs of the boron-containing chlorin derivatives 9 and 13 based on the chemical structure of compound 1 are successful. The

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Scheme 1. Synthesis of boron-containing chlorin derivative 9. Reagents and conditions: (a) hydroxylamine hydrochloride, pyridine, 1 h, 94%; (b) i-NaOH, THF, 1 h; iidimethyl iminodiacetate hydrochloride, dicyclohexylamine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, DMAc, 7 h; iii-hydroxylamine hydrochloride, pyridine, 30 min, 93% for 3 steps; (c) 3-(tert-butoxycarbonylamino) propyl bromide, NaH, DMF, 1 h, 68%; (d) TFA, CH2Cl2, 20 min, 61%; (e) 3-bromopropylamine hydrobromide, NaH, DMF, 1 h, 69%; (f) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, DMAc, 2 h, 60%; (g) NaOH, DMF, 30 min, 83%.

Scheme 2. Synthesis of boron-containing chlorin derivative 13. Reagents and conditions: (a) dicyclohexylamine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, DMAc, 8.5 h, 45%; (b) NaOH, DMF, 10 min, 94%.

accumulation of compound 9 in tumor tissue was more than that in all other tissues except for serum at all intervals following drug injection and reached a maximum amount (0.21 pmol/mg) at 12 h after administration (Fig. 3A). Although the accumulation of compound 13 in tumor tissue was less than that in the liver and serum at all intervals following drug injection, accumulation of compound 13 in tumor tissue reached a maximum amount (0.27 pmol/mg) at 3 h after administration (Fig. 3B). At 24 h after administration, compounds 9 and 13 were only slightly detected in all other normal tissues except for serum. The time to maximum accumulation of compound 13 in tumor tissue was one-fourth of that of compound 9. In addition, the number of boron-10 atoms in the structure of compound 13 is twice that in compound 9. A requirement for porphyrin derivatives for PDT and BNCT is that they reach the amount of maximum accumulation in tumor tissue in the shortest possible time and are rapidly discharged from the body, in order to reduce side effects such as photosensitivity. Therapeutic efﬁcacy of BNCT can be improved by using an agent that has speciﬁc tumor accumulation and many boron-10 atoms contained in its chemical structure. Therefore, the results suggested that compound 13 was superior to compound 9 as a PDT and BNCT agent. On the other hand, 10B concentration of 20–30 lg per gram

of tumor is required for clinically effective BNCT,10 and the optimal tumor/blood boron concentration (T/B) ratio of BNCT agents is around 5:1 in order to avoid inducing necrosis in the vasculature.33 The 10B concentration of compound 13 was small compared with the amount required for effective BNCT in the period in which they had maximum accumulated amounts in the tumor, and T/B ratio of compound 13 was a very low at all intervals. In a future study, it is necessary for use in BNCT to investigate the doses of compound 13, because the experiments on in vivo biodistribution and clearance of 9 and 13 were performed at doses intended for use in PDT. The in vivo therapeutic efﬁcacy of PDT using boronophenylalanine derivative 13 was evaluated by measuring tumor growth rates in colon-26 tumor-bearing BALB/c mice after light-emitting diode (LED) irradiation at 660 nm for PDT. The derivative 13 PDT group showed signiﬁcantly (P < 0.05) greater inhibition of tumor growth at 3–7 days than did the light irradiation group (Fig. 4). Although a temperature rise of 2–3 °C in the skin surface at the irradiated site was conﬁrmed during LED irradiation for PDT in the present study, the inhibition of tumor growth is due to PDT, not thermotherapy (hyperthermia),34 which is used as one of the cancer treatments based on the property of tumor cells being more sensitive than normal cells to heat, as was revealed in our previous study.32

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Compound 9

Concentration (pmol/mg)

1.2 Tumor Liver

0.9

Kidney Spleen

0.6

Lung

Brain 0.3

Muscle Skin Serum

0 1

Time after injection (hours)

Compound 13

7.6

32.2

Concentration (pmol/mg)

1.2 Tumor Liver

0.9

Kidney Spleen 0.6

Lung Brain Muscle

0.3

Skin Serum

0 1

Time after injection (hours) Figure 3. In vivo biodistribution of boron-containing chlorin derivatives in colon-26 tumor-bearing BALB/c mice. Boron-containing chlorin derivatives 9 (A) and 13 (B) (10 lmol/kg) were injected intravenously into colon-26 tumor-bearing BALB/c mice. Eight tissues and serum samples from tumor-bearing mice were analyzed at the indicated times after injection. Concentrations of boron-containing chlorin derivatives 9 and 13 in the tissue and serum samples were determined using ﬂuorescence measurements. All data represent means ± SE (n = 4).

2000 light irradiation only

Tumor volume (mm3)

1600

derivative 13 PDT

* 1200

* 800

In conclusion, we synthesized boron-containing chlorin derivatives that were intended for use in both PDT and BNCT, and we evaluated their accumulation in tumor tissues and clearance from normal tissues. The results showed that boronophenylalanine derivative 13 had good tumor selectivity and rapid clearance, and we conﬁrmed the usefulness of PDT using 13. However, optimization of the dose of 13 is required for use in BNCT. In future studies, progress in the development of more effective agents for use in combination therapy of PDT and BNCT is expected by investigating the in vivo therapeutic efﬁcacy of BNCT using 13 and clarifying the usefulness of BNCT with boron-containing chlorin derivatives. Acknowledgments

400

0 0

2 3 4 5 6 Time after treatment (days)

Figure 4. Tumor growth of colon-26 tumor-bearing BALB/c mice treated with PBS with irradiation and compound 13 (10 lmol/kg) with irradiation. Tumor volumes were measured for 7 days after treatment. All data represent means ± SE (derivative 13 PDT group: n = 4, light irradiation group: n = 5). ⁄ P < 0.05 compared with the control.

We wish to thank Stella Chemifa Corporation for the supply of 4-boronocinnamic acid 7 (10B-enriched) and L-4-boronophenylalanine ethyl ester 11 (10B-enriched). The authors are grateful to the SC-NMR Laboratory of Okayama University and the MS Laboratory of Faculty of Agriculture, Okayama University. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.01. 054.

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Synthesis and biological evaluation of new boron-containing chlorin derivatives as agents for both photodynamic therapy and boron neutron capture therapy of cancer. - PDF Download Free (2024)