Design, synthesis, and biological evaluation of a highly water-soluble psoralen-based photosensitizer

Article abstract of DOI:10.1016/j.bmc.2017.02.050

In recent years, photodynamic therapy (PDT) has been approved for treating various medical conditions, including cancer. PDT is a treatment that employs a particular drugs, called photosensitizers which work along with specific light source. The growth of this medical industry is expanding as it is another promising alternative to treat cancer which lessen the burden of treatments in patients. This includes the benefits of minimally invasive procedures and delivering high accuracy in targeting mutations. In recent two decades, cancer researchers have produced remarkable studies on developing photosensitizers that enhance understanding of biology and genetics of cancer. It is unfortunate that not all PDT can work as well as other profound treatment because PDT has various limitations like PDT leads photosensitivity reaction that arises when the photosensitizer remains in the body for a long period of time. In this paper, our studies centers on synthesizing a highly soluble photosensitizing agent with improved effectiveness on detecting cancer cells.

Full text of DOI:10.1016/j.bmc.2017.02.050

Accepted Manuscript
Design, synthesis, and biological evaluation of a highly water-soluble psoralen-
based photosensitizer
Yoshiyuki Uruma, Takuya Nonomura, Priscilla Yoong Mei Yen, Marie Edatani,
Ryotaro Yamamoto, Kunishige Onuma, Futoshi Okada
PII:
S0968-0896(16)30832-X
http://dx.doi.org/10.1016/j.bmc.2017.02.050
BMC 13580
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 September 2016
24 February 2017
24 February 2017
Please cite this article as: Uruma, Y., Nonomura, T., Yen, P.Y.M., Edatani, M., Yamamoto, R., Onuma, K., Okada,
F., Design, synthesis, and biological evaluation of a highly water-soluble psoralen-based photosensitizer, Bioorganic
& Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.02.050
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<Manuscript for Journal of Bioorganic Medicinal Chemistry>
<Title>
Design, synthesis, and biological evaluation of a highly water-soluble
psoralen-based photosensitizer
<Author and Affiliation>
Yoshiyuki Uruma,*1 Takuya Nonomura,1 Priscilla Yoong Mei Yen,1 Marie
Edatani,1 Ryotaro Yamamoto,1 Kunishige Onuma,2 and Futoshi Okada2
1Department of Materials Science, National Institute of Technology, Yonago College
Yonago City, Tottori 683-8502
2Department of Biomedical Sciences, Tottori University, Yonago City, Tottori
683-8503
<Abstract>
In recent years, photodynamic therapy (PDT) has been approved for treating various
medical conditions, including cancer. PDT is a treatment that employs a particular drugs,
called photosensitizers which work along with specific light source. The growth of
this medical industry is expanding as it is another promising alternative to treat cancer
which lessen the burden of treatments in patients. This includes the benefits of
minimally invasive procedures and delivering high accuracy in targeting mutations. In
recent two decades, cancer researchers have produced remarkable studies on developing
photosensitizers that enhance understanding of biology and genetics of cancer. It is
unfortunate that not all PDT can work as well as other profound treatment because PDT
has various limitations like PDT leads photosensitivity reaction that arises when the
photosensitizer remains in the body for a long period of time. In this paper, our studies

centers on synthesizing a highly soluble photosensitizing agent with improved
effectiveness on detecting cancer cells.
Introduction
In recent years, the mortality rate due to cancer has increased at an alarming rate,
prompting the global scientific community to seek new treatments to control the disease.
One of the more promising new treatments is photodynamic therapy (PDT).1,2 PDT
employs a type of drug called a photosensitizer, which is activated by light of a specific
wavelength. The photosensitizer is injected into the bloodstream and absorbed by cells in
the body. Then, the cancer cells are exposed to monochromatic light, causing the
photosensitizer to produce singlet oxygen, which induces apoptosis in the cancer cells.3
The light sources used for PDT can be coherent-wavelength lasers, which enable the
treatment of organs and tissues in the body, or light-emitting diodes, that may be used for
treating exterior tumors such as skin cancer.
Unfortunately, most photosensitizers approved for PDT medication have drawbacks
that limit their application in treating cancer, such as poor absorption, which leads to
inefficient singlet-oxygen generation, and poor excretion from healthy cells. For
instances, photofrin and psoralen are the approved and commonly used photodynamic
therapy drugs as photosensitizers. Photofrin, also known as porfimer sodium is a

mixture of oligomers structured by eight units of porphyrin via ether and ester linkages.
Nonetheless, photofrin exhibits absorption at low wavelength, poor excretion from
healthy cells and low excitation yields in photocytotoxicity, it was approved to be
practiced for the treatment in esophageal cancer. On the other hand, psoralen suppress
apoptosis under ultraviolet (UVA) exposure, psoralen plus UVA (PUVA) therapy has
indicated to be a considerable photosensitizer in treating skin cancer. Yet, similar in sound
to what was intended, psoralen has poor solubility and selectivity for cancer cells, due to
these adverse reactions, it is only used to treat early stage of skin cancer.4 To overcome
these drawbacks, we have developed glycoconjugate psoralen as photosensitizer in PDT
therapy.4 Since increasing glycolytic capacity in cancer cells activates Warburg effect, it
is recognized that glycoconjugated psoralen has a better molecular design as
photosensitizer because it may help develop the cancer cells selectivity.5 Yano et al.,
among others also suggests that highly water-soluble photosensitizers exhibited
improved rate of absorption and excretion therefore reduces the probability of drug side
effects.6,7,8 This paper presents the synthesis strategies of glycoconjugated psoralen,
quantitation in terms of solubility via Q-NMR method and detailed analytic report on
how increase in solubility effects singlet-oxygen production and photocytotoxicity.

Results and Discussion
Chemical Synthesis
The retrosynthetic analysis, shown in Scheme 1 envisaged that the target molecule 1
could be accessed via a triazole ring formation between 2 and 3 under Huisgen reaction
conditions, in which a dipolarophile reacts with a 1,3-dipolar compound to afford a
5-membered cycle.
O
OCH3
OH
R2
O
N
O
R1
HO
O
N
O
N
OH
O
1
OAc
R1
OCH3
O
R2
AcO
O
O
OAc
N3
O
3
2
O
OAc
R1
OCH3
O
R2
AcO
OAc
O
OAc
Cl
O
4
5
Scheme 1. Retrosynthetic analysis for glycoconjugated psoralen
To perform this reaction here, we employed the acetylene and azide functional groups of
2 and 3, respectively. These key intermediates were obtained via the protected
carbohydrate 4 and the chlorinated molecule 5, respectively.9

We synthesized the acetylene derivatives 2a and 2b based on a previously reported
method,10 obtaining the galactose acetylene 2a in 21% yield. Preparation of the glucose
acetylene 2b was accomplished with the crude product as 2b was difficult to purify with
silica gel column chromatography.
<Insert Scheme 2>
OAc
OAc
OH
OAc
OAc
OH
Propargyl alcohol
BF3・Et2O
O
O
O
Pyridine
Ac2O
OAc
OH
AcO
HO
O
AcO
CH2Cl2, 21%
OAc
OH
OAc
4a
2a
6a
OAc
OAc
OH
Propargyl alcohol
BF3・Et2O
CH2Cl2
O
O
O
Pyridine
Ac2O
AcO
AcO
AcO
AcO
HO
HO
O
OAc
OH
OAc
OAc
OH
4b
6b
2b
Scheme 2. Synthesis of acetylene derivatives
As direct introduction of the azide group into psoralen is an unconcreted pathway,
compound 7 was first converted to a chlorinated compound 8 by using chloromethyl
methyl ether11.

<Insert Scheme 3>
Cl
N3
ClCH2OCH3
NaN3
CH3COOH, 81%
DMF, 65%
O
O
O
O
O
O
O
O
O
OCH3
OCH3
OCH3
8
3
7
Scheme 3. Synthesis of psoralen derivatives
The chlorinated substituent was utilized in the synthesis of the psoralen azide derivative
through nucleophilic substitution where chlorinated substituent 8 was reacted with
sodium azide to afford 3 as presented in scheme 3.

<Insert Scheme 4>
O
OCH3
OAc
OAc
Sodium Ascorbate
CuSO4
O
O
N
O
3
N
2a
O
AcO
N
tBuOH, H2O, 72%
OAc
O
9a
OCH3
OH
OH
CH3ONa
O
O
N
O
CH3OH, 31%
N
O
HO
N
OH
1a
O
OCH3
Sodium Ascorbate
CuSO4
OAc
O
O
N
3
O
2b
tBuOH, H2O, 79%
AcO
AcO
N
O
N
OAc
O
OCH3
9b
OH
O
O
CH3ONa
N
O
HO
HO
N
CH3OH, 38%
O
N
OH
1b
Scheme 4. Synthesis of glycoconjugated psoralen derivatives
Then, to prepare glycoconjugate psoralen derivative, heterocoupling between compound

3 and acetylene derivatives azide 2a and 2b by Huisgen reaction12 is performed to
obtain acetyl protected glyconjugate psoralen derivatives 9a and 9b shown in scheme 4.
Finally, deprotection of the obtained acetyl protected coupling compound proceeded
smoothly with sodium methoxide in methanol, affording 1a and 1b in 31% and 38%
yields, respectively.

Evaluation of Synthetic Compounds
Assessment of singlet-oxygen production
The efficiency of converting ground state oxygen to singlet oxygen is an ultimate
determinant used to evaluate the new PDT agents because the singlet oxygen has a
unique chemistry that induces irreversible destruction of cancer cells within irradiated
domain. In this examination, the samples, psoralen and glycoconjugated psoralen were
dissolved in DMSO respectively and 1,3-diphenylisobenzofuran (DPBF), singlet
oxygen acceptor which has an absorption maximum ꢀ(ꢁ3 around 416 nm, was
employed to monitor the production of singlet oxygen. The sample mixture was
exposed under a constant light source of 50 W halogen lamp and the disappearance of
DPBF was monitored spectroscopically.13 The experimental results indicate that
psoralen exhibits quantum yield of 17% and glycoconjugated psoralen shows
improvement in singlet oxygen production with a quantum yield of 24%. From the
obtained data, it is clearly implied that a glycoconjugated photosensitizer performs
productively in PDT.
Water solubility test

The characterization of solubility of psoralen 7 and glycoconjugate psoralen 1b were
determined by using quantitative nuclear magnetic resonance method (Q-NMR), which
is a relatively new spectroscopic method in this field14 to assess the solubility of the
drugs. Both samples were prepared using 3-(trimethylsilyl)-1-propanesulfonic acid
sodium salt (DSS-d6) as an internal standard and dissolved in deuterated solvent D2O.
<Insert Figure1>

D2O
Figure 1 Q-NMR spectra of psoralen 7 and glucose-conjugated psoralen 1b; (down) full
spectrum (ꢂ 8.50~0.00 ppm) and (up) magnification of aromatic region (ꢂ 8.30~6.40
ppm). The aromatic peaks were indicated as A~D and were assigned based on the 2D
NMR spectroscopy (HMBC and HMQC). Samples were prepared in D2O and
quantitation was evaluated using internal reference, DSS-d6.
The Q-NMR analysis of psoralen and glucose-conjugated psoralen were shown in
Figure 1. The singlet peak at δ 0.00 with integral value of 1.00 encodes the internal

reference, DSS-d6 and was assigned as an analytic peak in calculating the solubility of
dissolved compound. Several areas of the peaks of hydrogen atoms originated from the
samples, without any overlapping were selected and labeled as A~D. The peaks A~D
are signals aroused from aromatic ring of both samples, for psoralen are A: δ 6.47 (d,
1H, J = 9.6 Hz); B: δ 8.13 (d, 1H, J = 9.6 Hz); C: δ 7.01 (d, 1H, J = 2.4 Hz); D: δ
7.88 (d, 1H, J = 2.4 Hz) and for glucose-conjugated psoralen are A: δ 7.82 (br, 1H);
B: δ 6.56 (br, 1H); C: δ 7.04 (br, 1H); D: δ 7.47 (d, 1H, J = 16 Hz). The irregular
chemical shift of the aromatic protons of 1b was detected; peak A was shifted to
downfield and peak D was shifted to upfield. The reason of the unexpected
displacement was ambiguous. The average integral values of 4 peaks for psoralen and
glucose-conjugated psoralen are 0.01 and 0.28 respectively. From the relationship of the
known amount of DSS-d6 and its integral area, the number of moles of dissolved
psoralen and glucose-conjugated psoralen in deuterated solvent are 1.4 × 10−8 and 1.7 ×
10−7 mol and therefore, the measurement indicates that glucose-conjugated psoralen is
12 times more soluble than psoralen .
Photocytotoxicity assay

The photocytotoxicity of psoralen 7 and its glycol-derivates, 1a and 1b were evaluated
against BALB/3T3 cells after incubation for 24h at 37℃ in controlled environmental
condition of humidified atmosphere containing 5% CO2. Figure 2 shows the
photoactivity of photosensitizers against percentage cell survival evaluated at light dose
of 3.3 J/cm2 upon incubation with photosensitizers for 48 h.
<Insert Figure2>
Figure 2 Percentage of cell survival Ə S.D. towards different concentration of
photosensitizers. Photocytotoxicity was determined against cancer cells, BALB/3T3
under light fluences rate of 3.3 J/cm2 from the source of 365 nm UV light. The

incubation time with photosensitizers and irradiation time were kept constant, 48 h and
500 s respectively. Mean Ə S.D of percentage of cell survival was analysed vs non
treated (The bar denotes standard deviation (P<0.05) of mean, n=3). ‘Non treated’
indicates no addition of photosensitizer agents and ‘DMSO’ refers to solvent used.
Above result implied that at high concentration of photosensitizers (42 μM >), galactose
conjugated psoralen exhibited a degree of enhancement in PDT activity compared to
psoralen, but psoralen showed slightly higher apoptosis than glucose conjugated
psoralen in spite of its high water solubility. At concentration between 7 and 23 μM,
there was no major difference observed between two examined glycoconjugated
photosensitizers, yet psoralen produced slightly better PDT effects. As reported
previously by Hirohara et. al., 15 in general, increased molecular size and high
concentration of photosensitizers suppress cellular uptake due to aggregation and
subsequently lead to poor PDT effects. Hence, this could be the possible explanation for
the unexpected results obtained at concentration 7~23 μM. However, at low
concentration range from 2.1 to 5.3 μM, the data interprets that glycoconjugated
psoralens showed improvement in photocytotoxicity. Particularly at concentration of 2.9

μM, glucose conjugated psoralen exhibited a significant contribution to PDT effect,
inducing 15% cell death. It can be safely concluded that glycopyranosyl groups not only
influence the amount of cellular uptake but also PDT effect16, which support the
hypothesis that high solubility photosensitizers promote photocytotoxicity.
Conclusion
The main conclusion of our experiments is that increased solubility of photosensitizers,
evaluated based on the Q-NMR quantitation, promote singlet oxygen production and
photocytotoxicity in low presence of drug. Further work on the photocytotoxicity
assessment of analogues against several of cancer cells is underway to provide concrete
guidelines and fundamentals for implementing in clinical practices.

Experimental
1H and 13C NMR spectra were recorded on a BRUKER 400 at 400 MHz and 100 MHz,
respectively. The chemical shifts are reported as δ values using tetramethylsilane (0 ppm)
and CHCl3 (77.0 ppm) as internal standards for proton and carbon spectra, respectively.
Infrared (IR) spectra were recorded on a JASCO FT/IR 420 spectrometer as liquid films
on NaCl plates. Low- and high-resolution mass spectra (LRMS and HRMS, respectively)
were obtained on JMS-AX 500 and JMS-700 T spectrometers at the Analytical Center of
Osaka City University. Silica gel (silica gel 60, 230–400 mesh) was used for flash
chromatography. Melting point was measured by MP-21 (Yamato Kagaku). Precoated
silica gel plates (Merck 5715, 60F254) were used for thin-layer chromatography. All
air-sensitive reactions were conducted in flame-dried glassware under Ar.
Tetrahydrofuran, methylene chloride, dimethyl formamide (DMF), methanol, ethanol and
ether were purchased from Wako (Japan).
Chemical syntheses
See Figures 1–2 for compound labels.

4-(azidomethyl)-9-methoxy-7H-furo[3,2-g]chromen-7-one (3). 17
To a solution of 8 (387.4 mg, 1.47 mmol) in DMF (55 mL) was added NaN3 (572.3 mg,
8.80 mmol) at r.t., and the reaction was stirred for 24 h. The reaction was then quenched
with H2O and extracted with EtOAc. The combined organic layers were washed with
H2O and brine and then dried over anhydrous Na2SO4 and evaporated in vacuo. The
residue was chromatographed on silica gel (CH2Cl2) to afford 3 (363 mg, 91%) as a
white solid.
Rf = 0.4 (EtOAc:n-Hexane, 2:1); m.p. 154.0–156.0 °C; 1H NMR (CDCl3, 400 MHz) δ
4.31 (3H, s), 4.71 (2H, s), 6.45 (1H, d, J = 9.9 Hz), 6.94 (1H, d, J = 2.4 Hz), 7.73 (1H, d,
J = 2.4 Hz), 8.02 (1H, d, J = 9.9 Hz); 13C NMR (CDCl3, 100 MHz) δ 47.8, 61.3, 105.0,
115.4, 117.0, 126.9, 133.1, 139.9, 143.6, 146.6, 146.7, 147.1, 159.7; IR (NaCl) νmax
(cm−1): 1714, 1421, 1362, 1221, 1092; HRMS (FAB, pos) m/z 272.0643 (calcd for
C13H10N3O4, 272.0671 [M+H]+)
Compound 9a.
To a solution of 3 (151 mg, 0.6 mmol) in H2O (2.8 mL) and tert-BuOH (2.8 mL) was

added acetylene derivative 2a (258 mg, 0.7 mmol), l-(+)-ascorbic acid sodium salt (11
mg, 0.1 mmol), and copper (II) sulfate (18 mg, 0.1 mmol) at r.t., and the reaction was
stirred for 24 h. The reaction was then quenched with H2O and extracted with EtOAc.
The combined organic layers were washed with H2O and brine and then dried over
anhydrous Na2SO4 and evaporated in vacuo. The residue was chromatographed on silica
gel (EtOAc) to afford compound 9a (81 mg, 72%) as a white solid.
Rf = 0.42 (EtOAc); m.p. 95–96.0 °C; 1H NMR (CDCl3, 400 MHz) δ 1.85 (3H, s), 1.95
(3H, s), 2.02 (3H, s), 2.11 (3H, s), 3.89 (1H, td, J = 6.5 Hz, 1.0 Hz), 4.05 (1H, m), 4.12
(1H, m), 4.32 (3H, s), 4.57 (1H, d, J = 7.9 Hz), 4.72 (1H, d, J = 12.5 Hz), 4.86 (1H, d, J
= 12.6 Hz), 4.97 (1H, dd, J = 10.4, 3.4 Hz), 5.13 (1H, dd, J = 10.5, 7.8 Hz), 5.36 (1H,
dd, J = 4.4, 1.6 Hz), 5.91 (2H, s), 6.46 (1H, d, J = 9.9 Hz), 6.95 (1H, J = 2.3 Hz), 7.33
(1H, s), 7.78 (1H, d, J = 2.3 Hz), 8.16 (1H, d, J = 9.9 Hz); 13C NMR (CDCl3, 100 MHz)
δ 20.5, 20.6(3C), 21.0, 47.3, 60.4, 63.1, 66.9, 68.7, 70.7, 70.8, 100.6, 104.6, 115.1,
115.3, 116.3, 122.12, 127.1, 133.7, 139.2, 143.6, 145.2, 146.7, 147.7, 159.3, 169.4,
170.0, 170.1, 170.4; IR (NaCl) νmax (cm−1): 1751, 1714, 1590, 1422, 1363, 1222, 1162,
1132, 1091, 1046; HRMS (FAB, pos) m/z 658.1856 (calcd for C30H32N3O14, 658.1884
[M+H]+)

Compound 9b.
The procedure was the same as that used for the preparation of 9a but proceeded from
2b instead of 2a. 9b was prepared in 79% yield from corresponding compound (2a and
3)
Rf = 0.42 (EtOAc); 1H NMR (CDCl3, 400 MHz) δ 1.87 (3H, s), 1.96 (3H, s), 2.03 (3H,
s), 2.11 (3H, s), 3.67 (1H, ddd, J = 9.7 Hz, 4.6 Hz, 2.4 Hz), 4.48 (1H, d, J = 7.9 Hz),
4.72 (1H, d, J = 12.5 Hz), 4.86 (1H, d, J = 12.6 Hz), 4.99 (1H, dd, J = 9.5 Hz, 8.0 Hz),
5.09 (1H, t, J = 12.4 Hz), 5.14 (1H, t, J = 9.3 Hz), 5.91 (2H, s), 6.46 (1H, d, J = 9.9 Hz),
6.95 (1H, d, J = 2.3 Hz), 7.78 (1H, d, J = 2.3 Hz), 8.16 (1H, d, J = 9.9 Hz); 13C NMR
(CDCl3, 100 MHz) δ 20.6(3C), 20.7, 47.2, 61.3, 61.6, 67.7, 68.2, 70.8, 71.9, 72.5, 100.5,
104.6, 114.8, 115.4, 116.3, 123.0, 127.1, 132.8, 139.1, 143.6, 146.7, 146.8, 159.3,
169.3(3C), 170.2, 170.4; IR (NaCl) νmax (cm−1): 1741, 1727, 1585, 1427, 1354, 1208,
1162, 1146, 1091; HRMS (FAB, pos) m/z 658.1886 (calcd for C30H32N3O14, 658.1884
[M+H]+)
Compound 1a.

To a solution of 9a (73 mg, 0.1 mmol) in CH3OH (1.1 mL) was added NaOCH3 (12 mg,
0.2 mmol) at r.t., and the reaction was stirred for 30 min. The reaction was then
evaporated in vacuo, and the residue was chromatographed on silica gel (EtOAc) to
afford compound 1a (17 mg, 31%) as a white solid.
Rf = 0.12 (EtOAc); m.p. 127.0–129.0 °C; 1H NMR (CDCl3 + CD3OD, 400 MHz) δ 3.38
(1H, d, J = 3.3 Hz), 3.42 (1H, d, J = 3.2 Hz), 3.48 (1H, dd, J = 9.0, 7.3 Hz), 3.79 (1H,
dd, J = 3.1, 0.8 Hz), 4.27 (3H, s), 4.71 (1H, d, J = 12.5 Hz), 4.88 (1H, d, J = 12.6 Hz),
6.06 (2H, s), 6.47 (1H, d, J = 9.9 Hz), 7.17 (1H, d, J = 2.4 Hz), 7.87 (1H, s), 7.91 (1H, d,
J = 2.2 Hz), 8.46 (1H, d, J = 10.0 Hz); 13C NMR (CDCl3 + CD3OD, 100 MHz) δ 47.8,
61.8, 62.3, 62.9, 70.0, 72.2, 74.7, 76.6, 104.0, 106.1, 115.9, 116.5, 117.9, 124.8, 128.7,
134.5, 142.0, 144.5, 146.2, 148.1, 149.0, 161.7; IR (NaCl) νmax (cm−1): 2045, 1449,
1422, 1115, 1031; HRMS (FAB, pos) m/z 490.1469 (calcd for C22H24N3O10, 490.1462
[M+H]+)
Compound 1b.
The procedure was the same as that used for the preparation of 1a but proceeded from
9b. 1b was prepared in 38% yield from 9b.

Rf = 0.12 (EtOAc); 1H NMR (CD3OD + CDCl3, 400 MHz) δ 3.17 (1H, dd, J = 9.8, 8.0
Hz), 3.23 (1H, d, J = 1.8 Hz), 3.26 (1H, t, J = 9.5 Hz), 3.31 (1H, t, J = 8.5 Hz), 3.62 (1H,
dd, J = 11.9, 5.4 Hz), 3.82 (1H, dd, J = 11.8, 1.9 Hz), 4.27 (3H, s), 4.31 (1H, d, J = 7.8
Hz), 4.71 (1H, d, J = 12.5 Hz), 4.88 (1H, d, J = 12.5 Hz), 6.06 (2H, s), 6.47 (1H, d, J =
10.0 Hz), 7.17 (1H, d, J = 2.1 Hz), 7.9 (1H, d, J = 2.2 Hz), 8.65 (1H, d, J = 10.0 Hz);
13C NMR (CDCl3 + CD3OD, 100 MHz) δ 48.2, 61.4, 62.3, 62.4, 70.8, 72.2, 74.7, 76.6,
103.5, 106.7, 115.5, 116.3, 118.2, 124.8, 128.2, 133.5, 142.5, 144.5, 146.8, 148.7, 148.4,
162.42; IR (NaCl) νmax (cm−1): 2057, 1433, 1428, 1100; HRMS (FAB, pos) m/z
490.1472 (calcd for C22H24N3O10, 490.1462 [M+H]+)
Biological Evaluation
Cell culture
BALB/3T3 cells were grown in Dulbecco's modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), and were cultured at 37 °C in a
humidified atmosphere containing 5% CO2.

Photocytotoxicity
The photocytotoxicities of the galactose- and glucose-conjugated photosensitizers
against BALB/3T3 cells were investigated as follows: The cells (2.5 × 103 cells) in 100
µL DMEM containing 10% FBS were plated in a 96-well plate and incubated for 24 h
(37 °C, 5% CO2), and the required amount of photosensitizer in 100 µL of DMEM
containing 10% FBS and 0.2–13.8 mM DMSO was added to each well. The
photosensitizer concentration was varied from 1 to 500 μM, with the final DMSO
content being 1% in all cases. The plate was then incubated for 48 h under normal
oxygen condition. The cells were washed twice with brine, and then 100 µL of DMEM
containing 10% FBS was added. The cells were exposed to 365 nm UV light for 500 s.
The irradiation time was adjusted to obtain the desired light dose of 3.3 J cm−2. The
mitochondrial activity of nicotinamide adenine dinucleotide (NADH) dehydrogenase in
the cells in each well was measured at 18 h after photoirradiation using Alamar Blue
reagent (10 µL, DAL1025, Biosource, Camarillo, CA, USA) according to the
manufacturer’s instructions. The absorbance at 570 nm was measured using a plate
reader. The percentage cell survival was calculated by normalization with respect to a
control experiment containing no drug.

Acknowledgements
We gratefully acknowledge Prof. Yuzo Fujii for his valuable suggestions and
discussions. This work was performed within the framework of the KOSEN-GIKADAI
project, and partly sponsored by NIT, Yonago College financial support.
References
1. M, Tim. Mini-Rev. Med. Chem., 2009, 9, 974.
2. R. Bonnett. Chem. Soc. Rev. 1995, 24, 19.
3. Lang, K.; Mosinger, J.; Wagnerovia, D. M. Coord. Chem. Rev., 2004, 248, 321.
4. Mikata, Y.; Takagi, S.; Tanahashi, M.; Ishii, S.; Obata, M.; Miyamoto, Y.; Wakita,
K.; Nishisaka, T.; Hirano, T.; Ito, T.; Hoshino, M.; Ohtsuki, C.; Tanihara, M.; Yano,
S. Bioorg. Med. Chem. Lett., 2003, 13, 3289.
5. Warburg, O. Science, 1956, 123, 309.
6. Sol, V.; Blais, J. C.; Bolbach, G.; Carre, V.; Granet, R.; Guilloton, M.; Spiro, M.;
Krausz, P. Tetrahedron Lett., 1997, 38, 6391.
7. Maillard, P.; Hery, C.; Momenteau, M. Tetrahedron Lett., 1997, 38, 3731.
8. Oulmi, D.; Maillard, P.; Guerquin-Kern, J-L.; Huel, C.; Momenteau, M. J. Org.

Chem., 1995, 60, 1554.
9. Hansen, J. B.; Bjerring, P.; Buchardt, O.; Ebbesen, P.; Kanstup, A.; Karup, G.;
Knudsen, P. H.; Nielsen, P. E.; Norden, B.; Ygge, B. J. Med. Chem., 1985, 28, 1001.
10. Mereyala, H. B.; Gurrala, S. R. Carbohydr. Res., 1998, 307, 351.
11. Aboulezz, F.; EI-Attar, A. A.; EI-Sockary, M. A. Acta chimica Academiae
Scientiarum Hungaricae 1973, 77, 205.
12. Hartmuth, C.; kolb, M.; G. Finn, and K. Barry Sharpless, Angew. Chem. Int. Ed.
2001, 40, 2004.
13. Spiller, W.; Kliesch, H.; Wöhrle, D.; Hackbarth, S.; Röder, B.; Schnurpfeil, G. J.
Porphyrins Phthalocyanines 1998, 2, 145.
14. Kuchta, K.; Ortwein, J.; L. Hennig, HW. Rauwald, Fitoterapia 2014, 96, 8.
15. Hirohara, S.; Obata, M.; Alitomo, H.; Sharyo, K.; Ando, T.; Tanihara, M.; Yano, S. J.
Photochem. Photobiol. B: Biol. 2009, 97, 22.
16. Obata, M.; Hirohara, S.; Sharyo, K.; Alitomo, H.; Kajiwara, K.; Ogata, S.; Tanihara,
M.; Ohtsuki, C.; Yano, S. Biochim. Biophys. Acta. 2007, 1770, 1204.
17. Hansen, J. B.; Buchardt, O. Tetrahedron Lett. 1981, 22, 1847.