ALA-Porphyrin Science
Enhancing Effect of Novel Schiff Base Derivatives, UTX-134 and
UTX-135, on 5-Aminolevulinic Acid-based Photodynamic Therapy
Hirari Yamahanaa, Yusei Shinoharaa, Yoshio Endob, Tohru Obatac, Hisatsugu Yamadaa andYoshihiro Utoa*aGraduate School of Technology, Industrial and Social Science, Tokushima University, Tokushima, Japan
Central Research Resource Branch, Cancer Research Institute, Kanazawa University, Kanazawa, Japanc
School of Pharmaceutical Sciences, Aichi Gakuin University, Nagoya, Japan.
*
Corresponding Author, Fax/Tel: +81-88-656-7514; E-mail: uto.yoshihiro@tokushima-u.ac.jpbReceived in 16th August 2021, Accepted 31st August 2021

Summary
5-Aminolevulinic acid (ALA) is a precursor of protoporphyrin IX (PpIX), which is a potent
intracellular photosensitizer for photodynamic therapy (PDT). PpIX is biosynthesized from ALA by
enzymatic modifications through the heme synthesis pathway. We have previously shown that
TX-816 clearly increased the effect of PDT using ALA (ALA-PDT). We found that TX-816
derivatives in which 2-chloro-4-nitroaniline (CNA) was substituted with 4-pentyl or 4-hexyl aniline(UTX-127 and UTX-128) were more stable than TX-816 in an aqueous solution. We examined the
ALA-PDT-enhancing effects of the novel 4-alkyl aniline derivatives, which were synthesized from
4-methoxysalicylaldehyde (MeOSA), and 4-alkyl anilines with various alkyl chains from C=0 to
C=6(UTX-128
to
UTX-135).
MeOSA-based
4-alkylanilinederivatives
or
3,5-dichlorosalicylaldehyde (DCSA)-based 4-alkyl aniline derivatives, specifically MeOSA-based
4-pentyl and 4-hexyl aniline derivatives (UTX-134 and UTX-135), showed a strong
ALA-PDT-enhancing effect. These results indicated that MeOSA-based 4-pentyl and 4-hexyl
aniline derivatives are potent lead compounds for the development of an ALA-PDT sensitizer.
Keywords:
5-Aminolevulinic acid (ALA), Protoporphyrin IX (PpIX), Photodynamic therapy (PDT), Schiffbase
Introduction
Photodynamic therapy (PDT) is a therapeutic method in which cells are denatured and necrotizedby irradiating the tissue with laser after the administration of a photosensitizer [1]. The denaturationand necrosis are due to the strong oxidative effect of reactive oxygen species generated by thereaction between the laser light and the photosensitive agent.
5-Aminolevulinic acid (ALA) is biosynthesized by 5-aminolevulinate synthetase fromsuccinyl-CoA and glycine [2]. As ALA is an intermediate precursor in the heme biosynthesis
pathway, the exogenous administration of ALA in patients leads to the selective accumulation of
protoporphyrin IX (PpIX) within malignant tissues [3,4]. Intracellular PpIX accumulated in cancer
cells emits red fluorescence (635 nm) upon excitation with blue light ( = 400–410 nm). In contrast,

ALA-Porphyrin Science, Vol. 10 No 1 2021, 1-14

PpIX exposed to red light ( = 630–700 nm) produces reactive oxygen species that results in cell
death of various cancer cells [5–7].
The former characteristic of ALA can be used to diagnose tumors (intraoperative photodynamic
diagnosis, ALA-PDD), and its latter property can be applied to the treatment of various diseases as aless invasive therapy (ALA-PDT). ALA-PDD is now widely used for malignant glioma and bladdercancer [8,9]. Recently, clinical applications of ALA-PDD have expanded to a wide variety of cancer
types [10–13]. ALA-PDT has been successfully used to treat patients with various neoplastic andnon-neoplastic diseases [14,15]. However, it has been reported that the cytotoxic effect of
ALA-PDT is attenuated in cells with low expression of PEPT1, a transporter that allows ALA into
cells, cells with overexpression of ABCG2, a transporter that exports PpIX, and cells expressing
mutant p53 [4,16,17]. Therefore, it is essential to develop a novel strategy to enhance the cytotoxic
effect of ALA-PDT.
We
recently
found
that
the
Schiffbasederivative
N-3’,5’-dichloro-2’-hydroxybenzylidene-2-chloro-4-nitroaniline, TX-816, could significantly
increase the effect of ALA-PDT by accelerating intracellular PpIX accumulation [18]. However,TX-816 was unstable in the aqueous solution and was quickly hydrolyzed into
3,5-dichlorosalicylaldehyde (DCSA) and 2-chloro-4-nitroaniline (CAN). Therefore, we searched fora method to enhance the hydrolysis resistance of TX-816 derivatives. In this study, we reported theenhanced cytocidal activity of ALA-PDT against human cancer cells using novel derivatives of
TX-816.
Experimentals
Materials
All solvents and chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan),Tokyo Chemical Industries (Tokyo, Japan), and Sigma-Aldrich (St. Louis, MO, USA). ALA waspurchased from Tokyo Chemical Industries. During chemical synthesis, the progress of thereactions was monitored using Merck silica gel 60 F254 TLC plates (Merck, Darmstadt, Germany)with ethyl acetate (EtOAc)/n-hexane. Column chromatography was performed using silica-gel 60N
purchased from Kanto Chemical (Tokyo, Japan). 1H-NMR spectra were recorded using
JNM-EX-400 (JEOL, Tokyo, Japan) in a deuterated solvent. Data were reported as follows:
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = double doublet, m= multiplet, brs = broad singlet), coupling constants (Hz), and integration. Broad singlet peaks,
speculated to arise from OH protons, were also noted. The purity of all final compounds was
confirmed at >95% using high performance liquid chromatography (JASCO PU-2089 Plus: Tokyo,
Japan) with detection at 330 nm. Gradient elution was performed using mixtures of solvent
n-hexane and chloroform (CHCl3) on a TSKgel Amide-80 3 μm column (4.6 mm I.D.×15 cm) at aflow rate of 0.5 mL/min. The column temperature was not controlled. High-resolution mass

spectroscopy (HRMS) was performed using Waters LCT Premier XE (Waters, Milford, MA, USA)and Waters ACQUITY UPLC (Waters, Milford, MA, USA).
Synthesis of Schiff base derivatives
TX-816DCSA (504 mg, 2.64 mmol) was dissolved in dry toluene (30 mL). CNA (683 mg, 3.96 mmol, 1.5eq.) was added to the solution on the ice bath. The reaction mixture was warmed up to room
temperature and refluxed for 24 h using Dean-Stark apparatus. After the reaction, the solvent wasevaporated. The residue was purified by flash silica-gel column chromatography using
EtOAc/n-hexane. TX-816 was afforded as a red solid (266 mg, 0.77 mmol, 29%). 1H-NMR (400
MHz, DMSO-d6): δ 13.5 (brs, 1H), 9.14 (s, 1H), 8.48 (d, J = 2.4 Hz, 1H), 8.38 (dd, J = 8.8, 2.4 Hz,
1H), 7.87 (d, J = 2.4 Hz, 1H), 7.84 (s, 1H), 7.82 (d, J = 2.8 Hz, 1H); HRMS (ESI+) m/z calcd. for
C13H8N2O3Cl3 [M + H]+ 344.9601, found 344.9617.
Compound 1 (UTX-122)
Aniline (220 μL, 2.40 mmol, 1.5 eq.) and dried 3A molecular sieves (975 mg) were mixed with
DCSA (305 mg, 1.60 mmol) dissolved in dry toluene (15 mL) on the ice bath. The reaction mixture
was warmed up to room temperature and refluxed for 3 h. After evaporation, the residue waspurified by flash silica-gel column chromatography using EtOAc/n-Hexane. Compound 1(UTX-122) was afforded as an orange solid (415 mg, 1.56 mmol, 98%). 1H-NMR (400 MHz,DMSO-D6): δ 9.01 (d, J = 3.6 Hz, 1H), 7.69–7.73 (m, 2H), 7.46-7.48 (m, 4H), 7.33–7.36 (m, 1H);
HRMS (ESI-TOF) m/z calcd. for C13H10NOCl2 [M + H]+ 266.0139, found 266.0152.
Compound 2 (UTX-123)
Compound 2 (UTX-123) was synthesized from p-toluidine (186 mg, 1.74 mmol) in 94% yieldusing the method described for the preparation of UTX-122. 1H-NMR (400 MHz, DMSO-d6): δ
14.7 (brs, 1H), 9.02 (s, 1H), 7.72 (d, J = 2.4 Hz, 1H), 7.70 (d, J = 2.8 Hz, 1H), 7.40 (d, J = 8.8 Hz,
2H), 7.30 (d, J = 8.0 Hz, 2H), 2.34 (s, 3H); HRMS (ESI-TOF) m/z calcd. for C14H12NOCl2 [M +H]+ 280.0296, found 280.0298.
Compound 3 (UTX-124)
Compound 3 (UTX-124) was synthesized from 4-ethylaniline (400 μL, 3.20 mmol) in 95% yieldusing the method described for the preparation of UTX-122. 1H-NMR (400 MHz, DMSO-d6): δ
9.01 (s, 1H), 7.71 (d, J = 2.8 Hz, 1H), 7.69 (d, J = 2.0 Hz, 1H), 7.40 (d, J = 8.8 Hz, 2H), 7.32 (d, J =8.4 Hz, 2H), 2.63 (dd, J = 7.6 Hz, 2H), 1.18 (t, J = 7.6 Hz, 3H); HRMS (ESI-TOF) m/z calcd. for
C15H14NOCl2 [M + H]+ 294.0452, found 294.0439.
Compound 4 (UTX-125)

Compound 4 (UTX-125) was synthesized from 4-propylaniline (231 μL, 1.57 mmol) in 100%
yield using the method described for the preparation of UTX-122. 1H-NMR (400 MHz, DMSO-d6):
δ 9.01 (s, 1H), 7.71 (d, J = 1.6 Hz, 1H), 7.69 (d, J = 1.6 Hz, 1H), 7.40 (d, J = 8.4 Hz, 2H), 7.30 (d, J= 8.0 Hz, 2H), 2.58 (t, J = 7.6 Hz, 2H), 1.54–1.63 (m, 2H), 0.88 (t, J = 7.4 Hz, 3H); HRMS
(ESI-TOF) m/z calcd. for C16H16NOCl2 [M + H]+ 308.0609, found 308.0613.
Compound 5 (UTX-126)
Compound 5 (UTX-126) was synthesized from 4-butylaniline (415 μL, 2.62 mmol) in 95% yieldusing the method described for the preparation of UTX-122. 1H-NMR (400 MHz, DMSO-d6): δ
14.7 (brs, 1H), 9.02 (s, 1H), 7.72 (d, J = 2.8 Hz, 1H), 7.70 (d, J = 2.4 Hz, 1H), 7.41 (d, J = 8.0 Hz,2H), 7.31 (d, J = 8.0 Hz, 2H), 2.63 (t, J = 9.2 Hz, 2H), 1.53–1.60 (m, 2H), 1.26–1.35 (m, 2H), 0.90(t, J = 7.6 Hz, 3H); HRMS (ESI-TOF) m/z calcd. for C17H18NOCl2 [M + H]+ 322.0765, found322.0750.
Compound 6 (UTX-127)
Compound 6 (UTX-127) was synthesized from 4-pentylaniline (185 μL, 1.05 mmol) in 97% yieldusing the method described for the preparation of UTX-122. 1H-NMR (400 MHz, DMSO-d6): δ
9.01 (s, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 6.8 Hz, 2H), 7.30 (d, J = 7.6 Hz, 2H), 2.59 (t, J =7.6 Hz, 2H), 1.53–1.60 (m, 2H), 1.27–1.30 (m, 4H), 0.84 (t, J = 6.2 Hz, 3H); HRMS (ESI-TOF) m/z
calcd. for C18H20NOCl2 [M + H]+ 336.0922, found 336.0938.
Compound 7 (UTX-128)
Compound 7 (UTX-128) was synthesized from p-hexylaniline (400 μL, 2.09 mmol) in 47% yieldusing the method described for the preparation of UTX-122. 1H-NMR (400 MHz, DMSO-d6): δ
14.7 (brs, 1H), 9.01 (s, 1H), 7.71 (d, J =2.8 Hz, 1H), 7.69 (d, J = 2.8 Hz, 1H), 7.39 (d, J = 8.4 Hz,
2H), 7.29 (d, J = 8.4 Hz, 2H), 2.59 (t, J = 7.8 Hz, 2H), 1.52–1.57 (m, 2H), 1.23–1.29 (m, 6H), 0.83
(t, J = 7.0 Hz, 3H); HRMS (ESI-TOF) m/z calcd. for C19H22NOCl2 [M + H]+ 350.1078, found350.1067.
Compound 8 (UTX-129)
2-Hydroxy-4-methoxybenzaldehyde (MeOSA) (300 mg, 1.97 mmol) was dissolved in dry
1,2-dichloroethane (10 mL). Aniline (275 μL, 3.00 mmol, 1.5 eq.) and dried 3A molecular sieves(905 mg) were added to the mixture on the ice bath. The reaction mixture was treated as describedabove. Compound 8 (UTX-129) was afforded as a yellow solid (259 mg, 1.14 mmol, 58%).
1
H-NMR (400 MHz, DMSO-D6): δ 13.6 (brs, 1H), 8.84 (s, 1H), 7.50 (d, J =8.8 Hz, 1H), 7.41 (t, J =8.2 Hz, 2H), 7.34 (d, J = 7.2 Hz, 2H), 7.24 (t, J = 7.2 Hz, 1H), 6.52 (d, J = 8.4 Hz, 1H), 6.46 (d, J =2.0 Hz, 1H), 3.77 (s, 3H); HRMS (ESI-TOF) m/z calcd. for C14H14NO2 [M + H]+ 228.1025, found228.1028.

Compound 9 (UTX-130)
Compound 9 (UTX-130) was synthesized from p-toluidine (194 mg, 1.81 mmol) in 61% yieldusing the method described for the preparation of UTX-129. 1H-NMR (400 MHz, DMSO-D6): δ
8.82 (s, 1H), 7.48 (d, J =8.8 Hz, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 6.51 (d, J =8.8 Hz, 1H), 6.44 (d, J = 2.8 Hz, 1H), 3.77 (s, 3H), 2.29 (s, 3H); HRMS (ESI-TOF) m/z calcd. for
C15H16NO2 [M + H]+ 242.1181, found 242.1177.
Compound 10 (UTX-131)
Compound 10 (UTX-131) was synthesized from 4-ethylaniline (150 μL, 1.20 mmol) in 82% yieldusing the method described for the preparation of UTX-129. 1H-NMR (400 MHz, DMSO-D6): δ
13.8 (brs, 1H), 8.83 (s, 1H), 7.48 (d, J =8.8 Hz, 1H), 7.25 (q, J = 15.8, 7.9 Hz, 4H), 6.51 (d, J = 8.8
Hz, 1H), 6.44 (d, J = 2.4 Hz, 1H), 3.76 (s, 3H), 2.59 (q, J = 15.8, 7.9 Hz, 2H), 1.15 (t, J = 7.4 Hz,3H); HRMS (ESI-TOF) m/z calcd. for C16H18NO2 [M + H]+ 256.1338, found 256.1326.
Compound 11 (UTX-132)
Compound 11 (UTX-132) was synthesized from 4-propylaniline (200 μL, 1.36 mmol) in 84%
yield using the method described for the preparation of UTX-129. 1H-NMR (400 MHz,DMSO-D6): δ 13.8 (brs, 1H), 8.83 (s, 1H), 7.48 (d, J =8.4 Hz, 1H), 7.26 (d, J = 8.8 Hz, 2H), 7.21 (d,J = 8.8 Hz, 2H), 6.51 (d, J = 8.8 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 3.76 (s, 3H), 2.53 (t, J = 7.4 Hz,
2H), 1.62–1.51 (m, 2H), 0.86 (t, J = 7.6 Hz, 3H); HRMS (ESI-TOF) m/z calcd. for C17H20NO2 [M +H]+ 270.1494, found 270.1481.
Compound 12 (UTX-133)
Compound 12 (UTX-133) was synthesized from 4-butylaniline (420 μL, 2.63 mmol) in 44% yieldusing the method described for the preparation of UTX-129. 1H-NMR (400 MHz, DMSO-D6): δ
8.82 (s, 1H), 7.48 (d, J =8.8 Hz, 1H), 7.26 (d, J = 6.4 Hz, 2H), 7.21 (d, J = 8.8 Hz, 2H), 6.51 (d, J =8.4 Hz, 1H), 6.44 (d, J = 2.8 Hz, 1H), 3.76 (s, 3H), 2.55 (t, J = 7.8 Hz, 2H), 1.55–1.48 (m, 2H),
1.31–1.19 (m, 2H), 0.86 (t, J = 7.4 Hz, 3H); HRMS (ESI-TOF) m/z calcd. for C18H22NO2 [M + H]+
284.1651, found 284.1662.
Compound 13 (UTX-134)
Compound 13 (UTX-134) was synthesized from 4-pentylaniline (310 μL, 1.75 mmol) in 63%
yield using the method described for the preparation of UTX-129. 1H-NMR (400 MHz,DMSO-D6): δ 8.82 (s, 1H), 7.48 (d, J =8.8 Hz, 1H), 7.26 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.4 Hz,
2H), 6.51 (d, J = 8.4 Hz, 1H), 6.44 (d, J = 2.4 Hz, 1H), 3.76 (s, 3H), 2.55 (t, J = 7.8 Hz, 2H),
1.58–1.50 (m, 2H), 1.32–1.21 (m, 4H), 0.82 (t, J = 7.0 Hz, 3H); HRMS (ESI-TOF) m/z calcd. for
C19H24NO2 [M + H]+ 298.1807, found 298.1795.

Compound 14 (UTX-135)
Compound 14 (UTX-135) was synthesized from p-hexylaniline (335 μL, 1.74 mmol) in 68% yieldusing the method described for the preparation of UTX-129. 1H-NMR (400 MHz, DMSO-D6): δ
8.82 (s, 1H), 7.48 (d, J =8.8 Hz, 1H), 7.26 (d, J = 8.8 Hz, 2H), 7.21 (d, J = 8.8 Hz, 2H), 6.51 (d, J =8.4 Hz, 1H), 6.44 (d, J = 2.8 Hz, 1H), 3.77 (s, 3H), 2.55 (t, J = 7.4 Hz, 2H), 1.55–1.49 (m, 2H),
1.27–1.21 (m, 6H), 0.82 (t, J = 6.8 Hz, 3H); HRMS (ESI-TOF) m/z calcd. for C20H26NO2 [M + H]+
312.1964, found 312.1950.
Cells and cell culture
The poorly differentiated gastric cancer cell line MKN-45 was provided by Dr. Suzuki (Fukushima
Medical College, Fukushima, Japan). As per our previous report, the MKN-45 cell lines resistant to
ALA-PDT were established by repeated treatment with 5 min PDT, after exposure with 200 µM ALA
and sub-cloning [18]. Briefly, MKN-45 cells were seeded into a 35-mm dish and incubated with 200µM ALA for 4 h. The cells were exposed to red LED light (630 nm, 80 mW/cm2) for 5 min. The
surviving and re-growing cells, which were resistant to ALA, were collected and seeded into a newdish, treated again with ALA-PDT (R and R400), and re-seeded into a new 15-cm dish after eachtreatment. Colonies of re-growing resistant cells were picked up and cultured (R13). R400 is a cellline resistant to 5 min PDT using 400 µM ALA. Both R and R400 were repeatedly treated with
ALA-PDT over 3 months.
Cells were maintained in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal
bovine serum and 50 μg/mL kanamycin at 37 °C in a humidified atmosphere containing 5% CO2.
ALA-PDT sensitivity assay by LED irradiation
Cells were seeded at a density of 5 × 103 cells/well in a 96-well plate and cultured at 37 °C in 5%
CO2 for 24 h. Test compounds such as DCSA, MeOSA, DCSA-based 4-alkyl aniline derivatives,and MeOSA-based 4-alkyl aniline were dissolved in DMSO and added to the culture medium at 10µM. The final concentration of DMSO in the culture medium was 0.5%. Thereafter, serial dilutionsof ALA were added to the culture medium at concentrations between 7.8 and 1,000 µM. After 4 hof incubation, the medium was replaced by a completely fresh medium. Then, the 96-well plate was
exposed to LED irradiation (630 nm, 80 mW/cm2) emitted by an LED irradiation unit provided by
SBI ALA PROMO (Tokyo, Japan) for 5 min. The LED light spot was an equally illuminatedrectangular spot measuring 128 × 86 mm covering the whole area of the 96-well culture plate. For
each concentration, triplicate wells were used and all assays were performed at least three times.
After LED irradiation, the cells were further incubated for 48–72 h, and the cell viability wasmeasured by a colorimetric assay using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto,Japan) according to the manufacturer’s instructions. After 72 h of incubation, the medium was
replaced by a fresh medium containing the WST-8 reagent. After 3 h, the absorbance in each well wasmeasured at 450 nm, using a microplate spectrophotometer, ImmunoMini NJ-2300 (BioTec, Tokyo,
Japan, Tokyo, Japan).
The percentage of cell growth inhibition was calculated by applying the following formula: cell
growth inhibition (%) = (1-[T/C]) × 100, where C and T are the mean absorbance values of thecontrol and treated groups, respectively. The IC50 value was determined graphically from thedose-response curve with at least three drug concentration points.
Measurement of intracellular PpIX
The cells (2 × 106 cells) were seeded in 6-cm dishes and incubated for 24 h. DCSA, MeOSA,
DCSA-based 4-alkyl aniline derivatives, and MeOSA-based 4-alkyl aniline derivatives were added
to the medium at the stated concentrations before ALA addition. All treatments were performed intriplicate. After 4 h of incubation, the cells were harvested using 2 mL of 0.25% trypsin-1 mM
EDTA. A cell suspension was collected in a 5-mL tube and centrifuged at 800 rpm for 5 min. The
supernatant was aspirated and the cells were washed with phosphate-buffered saline. Finally, the cellpellet was suspended in 500 µL of 2.5% Triton X-100 solution and the cell suspension was transferred
into a 0.5-mL micro tube, incubated for 5 min at room temperature, homogenized using Physcotron
NS36D micro homogenizer (Microtec, Chiba, Japan), and centrifuged at 15,000 rpm for 10 min. The
fluorescence intensity of PpIX in the supernatant was measured using a SEC2000-UV/VIS
spectrophotometer (BAS, Tokyo, Japan: excitation, 410 nm; emission, 634 nm).
Evaluation of chemical stability by gas chromatography-mass spectrometry
The stability of 4-alkyl aniline derivatives and TX-816 was evaluated by gas
chromatography-mass spectrometry (GC/MS) using the Agilent 7820A GC system coupled to theAgilent 5977E MSD system (Agilent Technologies, CA, USA). 1,5-Diphenyl-1,4-pentadien-3-one
as an internal control was dissolved in 10% water/90% DMSO at 1.2 mM. The samples tested weredissolved at concentrations from 5 to 10 mM with the solvent including an internal control.
Subsequently, the samples were immediately sonicated for 5 min. Then, the sample solutions were
filtrated with a syringe filter (Minisart Syringe Filter, 0.2 µm, Sartorius AG, Göttingen, Germany)and settled at room temperature until measurement by GC/MS analysis. After appropriate
incubation time, an aliquot of the sample solution was separated by GC system and all peaks
corresponding to each compound including an internal control were identified by ion-fragment
pattern based on a single quadrupole mass spectrometer with an electrospray ionization source.
Separation was achieved with Ultra Inert Columns HP-5ms capillary GC column (30 m × 0.25 mm,
0.25 µm) (Agilent Technologies). Chromatographic conditions were as follows: the initial
temperature was 50 °C or 120 °C for 0 min; it increased at a rate of 15 °C/min to 250 °C (15 min).
The gas flow rate was 1.0 mL/min with helium used as a carrier gas. The amount of originalcompound and degraded materials were corrected with an internal control.

Results
Enhancing effect of DCSA-based or MeOSA-based 4-alkyl aniline derivatives on ALA-PDT
The synthesized Schiff base derivatives tested in this study are summarized in Fig. 1.
We investigated the enhancement effect of newly synthesized DCSA-based and MeOSA-based
derivatives on ALA-PDT. When the IC50 values of ALA in the ALA-PDT sensitive assay were
evaluated, the combination treatment of DCSA and DCSA-based all 4-alkyl aniline (C=0 to C=6)
derivatives with ALA significantly enhanced the effect of ALA-PDT as compared with ALA alone(Fig. 2). On the other hand, the enhancing effect of MeOSA was less than that of DCSA and its
derivatives. Among the Schiff base derivatives, MeOSA-based 4-pentyl and 4-hexyl aniline
derivatives (UTX-134 and UTX-135) showed a stronger ALA-PDT enhancing effect than the otherderivatives, including DCSA-based all 4-alkyl aniline derivatives. Specifically, UTX-135 wassignificantly the most effective ALA-PDT sensitizer.

Fig. 1Synthesis of TX-816, DCSA-based derivatives and MeOSA-based derivatives

Fig. 2Comparison of IC50 values in ALA-PDT sensitivity assay

Cells were seeded at a density of 5 × 103 cells/well in a 96-well plate. After 24 h of incubation, the compounds
were added to the culture medium. Thereafter, serial dilutions of ALA (7.8–1,000 µM) were added to the culture
medium. After 4 h of incubation, the 96-well plate was exposed to LED irradiation for 5 min and further
incubated. Mean and standard deviation (error bars) of IC50 values were calculated by the data from three
independent experiments, each made in triplicate.

Stability of DCSA-based and MeOSA-based 4-alkyl aniline derivatives in aqueous solution
TX-816 is very unstable in an aqueous solution and quickly produced aldehyde and amine byhydrolysis (Fig. 3) [18]. Therefore, we examined the stability of the 4-alkyl aniline derivatives
using GC/MS system.
In 10%-water/90%-DMSO solvent (Fig. 4), TX-816 was rapidly hydrolyzed and > 50% of TX-816
was degraded within the first 5 min. After 100 min incubation, only 1.4% of the original TX-816
structure remained and almost the whole compound was converted into DCSA and CNA (55.0%
and 43.6% respectively). In contrast, DCSA-based and MeOSA-based 4-butyl, 4-pentyl or 4-hexyl
aniline derivatives (UTX-126–128 and UTX-133–135) were more stable than TX-816. Also, the
MeOSA-based derivatives showed higher stability than the DCSA-based derivatives. After >1000
min of incubation, MeOSA-based derivatives were maintained original structures (> 80%).

Fig. 3Chemical structures of Schiff base and its degradation products

Fig. 4 Stability of Schiff base derivatives in 10% water/90% DMSO solvent

DCSA-based derivatives and MeOSA-based derivatives increased an intracellular PpIX
accumulation in MKN-45 cells

Fig. 5 Increased intracellular accumulation of PpIX in MKN-45 after combination treatment with
ALA and Schiff base derivatives
MKN-45 cells were seeded at a density of 2 × 106 cells/well in a 6-cm dish. After 24 h of incubation, thecompounds were added to the medium before ALA addition. After 4 h of incubation, the fluorescence intensityof PpIX was measured.

We investigated the relationship between the intracellular PpIX accumulation and the enhancement
effect of DCSA-based and MeOSA-based derivatives on ALA-PDT. When MKN-45 cells weretreated with 10 µM each compound and 50 µM ALA, the intracellular PpIX accumulation clearly
increased in the cells treated with the 4-pentyl and 4-hexyl aniline derivatives (UTX-127, 128, 134,
and 135) as compared to those treated with the corresponding original compounds as shown in Fig.
5. Interestingly, the enhancing activity of UTX-134 and UTX-135 on the intracellular PpIX
accumulation tended to be slightly lower than that of the DCSA-based derivatives.
UTX-134 and UTX-135 enhanced treatment effects of ALA-PDT-resistant cells
Since TX-816 showed the effect of attenuating ALA-PDT resistance [18], we also examined theeffect of DCSA-based and MeOSA-based derivatives on ALA-PDT-resistant cells. UTX-127, 128,
134, and 135 could effectively recover the sensitivity to ALA-PDT in R, R13, and R400 cells,
which are MKN-45 cells that have acquired resistance (Fig. 6, Table 1). Specifically, UTX-134 and
UTX-135 were more effective ALA-PDT sensitizers than DCSA-based derivatives.

Fig. 6 The ALA-PDT enhancing effects of Schiff base derivatives in parental MKN-45 cells and its
ALA-PDT-resistant cell lines(A) DCSA-based derivatives. (B) MeOSA-based derivatives. Cells were seeded at a density of 5 × 103
cells/well in a 96-well plate. After 24 h of incubation, the compounds were added to the culture medium.
Thereafter, serial dilutions of ALA were added to the culture medium as described in the “Experimentals”
section. After 4 h of incubation, the 96-well plate was exposed to LED irradiation for 5 min and further
incubated for 72 h.

Table 1

Comparison of IC50 values between DCSA-based and MeOSA-based derivatives in

ALA-PDT sensitivity assay
IC50 (µM) of ALA at 10 µM compound
DCSA-based derivative

MeOSA-based derivative

Cells

ALA alone UTX-123

UTX-127

UTX-128

ALA alone UTX-130

UTX-134

UTX-135

MKN-45

59.8

22.8

17.7

17.3

63.5

47.3

10.0

8.20

R

>400

192.0

226.4

379.9

>400

>400

19.4

27.3

R13-2

>400

>400

378.3

>400

>400

>400

85.0

71.8

R400-1

>400

261.0

335.1

198.0

>400

>400

39.9

14.1

Discussion
In this study, we investigated the ALA-PDT enhancing activity of novel TX-816 derivatives andfound that 4-alkyl aniline (C=0 to C=6) derivatives exhibited potent ALA-PDT enhancement effects(Fig. 2). In addition, the 4-butyl, 4-pentyl, and 4-hexyl aniline derivatives (UTX-126–128 and
UTX-133–135) had higher stability than TX-816 (Fig. 4). This improved stability was probably due
to the substitution of CNA with 4-alkyl aniline containing an electron-donating group [19, 20].
Interestingly, ALA-PDT enhancement effects of DCSA-based derivatives were relatively unaffectedby the alkyl chain length, whereas those of MeOSA-based derivatives were significantly improvedin a carbon-number dependent manner. Of these, the effects of UTX-134 and UTX-135 on
ALA-PDT were better than that of the TX-816 and DCSA-based derivatives, and they also
exhibited a potent activity on ALA-PDT-resistant cells (Fig. 6). Since DCSA-based derivatives weredegraded more rapidly than MeOSA-based derivatives, the inhibition of PpIX emission by DCSA
may have contributed to the ALA-PDT enhancement effect. This is also apparent from the findingthat the intracellular PpIX levels were increased in the cells treated with DCSA-based derivatives,
regardless of the length of the alkyl side chain (Fig. 5). On the contrary, because MeOSA-based
derivatives are extremely stable, their ALA-PDT enhancement effects depend on PpIX emission
inhibitory activities of compounds themselves. Therefore, it is speculated that the affinity to
ABCG2 is increased as the alkyl side chain is extended, leading to an improvement in intracellular
PpIX accumulation and PpIX emission inhibitory efficacy. However, the intracellular PpIX
accumulation in MKN-45 cells treated with MeOSA-based derivatives tended to be lower than thatin cells treated with DCSA-based derivatives (Fig. 5). The superior effects of UTX-134 and
UTX-135 on ALA-PDT compared to DCSA-based derivatives were not reflected in the increased
intracellular accumulation of PpIX. Although the ALA-PDT enhancement mechanism of thesecompounds has not yet been clarified, it cannot be ruled out that the intact Schiff base structure of
MeOSA derivatives may directly promote the cytocidal activity of ALA-PDT [21]. Further studies
are needed to determine the mechanism of ALA-PDT enhancement effects of the MeOSA-based
derivatives.

Conclusion
UTX-134 and UTX-135 showed higher stability to an aqueous solution and a stronger
ALA-PDT-enhancing effect than the TX-816 and DCSA-based derivatives. These results suggestthat MeOSA-based derivatives are excellent lead compounds for the development of novel
ALA-PDT enhancers.
Acknowledgement
We thank Tokushima Regional Base for Industry-Academia-Government joint Research for use of
facilities. This study was partly supported by JSPS KAKENHI Grant Numbers JP26430160 (YoshioEndo). This work was partly supported by the Extramural Collaborative Research Grant of Cancer
Research Institute, Kanazawa University. Several research resources were kindly provided by the
Joint Usage/Research Program of Cancer Research Institute, Kanazawa University.
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