The design, synthesis, and evaluation of organic dithienopyrrole-based D-π-A dyes for use as sensitizers in photodynamic therapy

Article abstract of DOI:10.1016/j.bmcl.2018.07.025

Dithienopyrrole-based organic dyes that combine an electron-donating moiety (D), a π-conjugated bridge moiety (π), and an electron-accepting moiety (A) were designed and synthesized in short steps by previously developed one-pot Suzuki-Miyaura coupling approach. Absorption wavelengths of the dyes were readily tuned by altering the D and A moieties. The use of a strongly electron-withdrawing cyanopyridone acceptor enabled NIR absorption. A synthesized sensitizer, 2j, exerted potent phototoxicity mainly via a Type I mechanism in cells. A nitrogen atom in the dithienopyrrole ring serves as a connecting point for the introduction of functional building blocks that can improve the properties of sensitizers, which makes this D-π-A sensitizer a valuable template for the further development of sensitizers.

Full text of DOI:10.1016/j.bmcl.2018.07.025

Accepted Manuscript
The design, synthesis, and evaluation of organic dithienopyrrole-based D-π-A
dyes for use as sensitizers in photodynamic therapy
Shinichiro Fuse, Keisuke Matsumura, Miori Takizawa, Shinichi Sato, Hiroyuki
Nakamura
PII:
S0960-894X(18)30588-2
https://doi.org/10.1016/j.bmcl.2018.07.025
BMCL 25956
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
11 June 2018
13 July 2018
14 July 2018
Please cite this article as: Fuse, S., Matsumura, K., Takizawa, M., Sato, S., Nakamura, H., The design, synthesis,
and evaluation of organic dithienopyrrole-based D-π-A dyes for use as sensitizers in photodynamic therapy,
Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.07.025
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The design, synthesis, and evaluation of organic dithienopyrrole-based D--A dyes for use as
sensitizers in photodynamic therapy
,
a
a
a,b
a
Shinichiro Fuse,* Keisuke Matsumura, Miori Takizawa, Shinichi Sato, and Hiroyuki
,
a
Nakamura,*
a
Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of
Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan.
b
School of Life Science and Technology, Tokyo Institute of Technology
4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan.
*Corresponding author e-mail address: sfuse@res.titech.ac.jp; hiro@res.titech.ac.jp
Abstract
Dithienopyrrole-based organic dyes that combine an electron-donating moiety (D), a -conjugated
bridge moiety (), and an electron-accepting moiety (A) were designed and synthesized in short
steps by previously developed one-pot Suzuki-Miyaura coupling approach. Absorption wavelengths
of the dyes were readily tuned by altering the D and A moieties. The use of a strongly
electron-withdrawing cyanopyridone acceptor enabled NIR absorption. A synthesized sensitizer, 2j,
exerted potent phototoxicity mainly via a Type I mechanism in cells. A nitrogen atom in the
dithienopyrrole ring serves as a connecting point for the introduction of functional building blocks
that can improve the properties of sensitizers, which makes this D--A sensitizer a valuable template
for the further development of sensitizers.
Keywords: photodynamic therapy, Suzuki-Miyaura coupling, one-pot, dithienopyrrole
Photodynamic therapy (PDT) has garnered much attention as a non-invasive, selective, and
[
1]
cost-effective cancer therapy. In PDT, a sensitizer is administered to cancer patients and damages
cancer cells under photo irradiation via Type I (hydrogen/electron transfer) and/or Type II (singlet
oxygen generation) pathways, which leads to cell death. Several sensitizers have been approved and
[2]
clinically used for PDT. Many porphyrin-based sensitizers have been developed in ongoing efforts
[
3]
to improve PDT. Although tremendous effort has been extended to develop new sensitizers, the
number of available sensitizers remains somewhat limited due to the many requirements: proper
absorption wavelength (NIR absorption is desirable), high absorptivity, high stability under photo
irradiation and physiological conditions, low levels of dark toxicity, high levels of tumor
[
3a]
accumulation, and rapid excretion from the body following PDT.
In addition, sensitizers that
damage cancer cells involving Type I mechanisms are considered advantageous under hypoxic

[
4]
conditions in cancers. A sensitizer that could fulfill all requirements awaits development.
We have developed an efficient synthetic approach to thiophene-based, organic dyes that
combines an electron-donating moiety (D), a -conjugated bridge moiety (), and an
[
5]
electron-accepting moiety (A). We recently reported that the D--A sensitizer 1 exerted potent
[6]
phototoxicity via both Type I and Type II mechanisms (Figure 1). However, this did not
demonstrate absorption in the NIR region. Herein, we report the design and synthesis of
dithienopyrrole-based D--A sensitizer 2. Use of strongly electron-withdrawing cyanopyridone
acceptor enabled NIR absorption. Evaluation revealed that one of the synthesized sensitizers, 2j,
exerted potent phototoxicity mainly via a Type I mechanism.
Fig. 1 Chemical structures of our previously reported thiophene-based organic D--A sensitizer 1,
and of the new dithienopyrrole-based organic D--A sensitizer 2.
We designed the dithienopyrrole-based D--A sensitizer 2 for four reasons: 1) dithienopyrrole
consists of a fused ring system with extended -conjugation that enables a red shift of absorption; 2)
dithienopyrrole consists of 5-membered heteroaromatic rings (pyrrole and thiophene), and when
linked to a benzene ring, both dithienopyrrole and benzene rings reside on a common plane that
[
7]
allows an extension of the -conjugated system, which results in a red-shift of absorption; 3) a
nitrogen atom in the dithienopyrrole ring serves as a connecting point for the introduction of a
functional group; and, 4) dithienopyrrole is readily available.
Initially, we prepared dithienopyrrole-based 12 D--A sensitizers, 2a-2l from a combination of 3
donors and 4 acceptors with different levels of electron-donating and -withdrawing abilities (Scheme
1). In order to improve the solubility of the sensitizers, a tert-butyl group was attached to the
[8]
dithienopyrrole ring via the nitrogen atom. A one-pot Suzuki-Miyaura coupling of dibromo
[9]
dithienopyrrole 3 with 3 donors, 4a-4c, and thiophene boronic acid 5 was based on our previously

[5f]
reported procedure, and afforded the desired compounds 2a, 2e, and 2i in moderate yields. A
subsequent Knoevenagel condensation of three aldehydes with 3 acceptors, 6a-6c, afforded the
desired D--A sensitizers 2b-2d, 2f-2h, and 2j-2l in moderate to excellent yields. Knoevenagel
condensations of 2e with 6a did not afford the desired product 2f under basic conditions, and,
therefore, acidic conditions were used for the synthesis of 6a. In the cases of the syntheses of 2c and
[5f]
2k, Knoevenagel condensation was performed in a one-pot fashion.
Scheme 1 Preparation of dithienopyrrole-based 12 D--A sensitizers, 2a-2l.
The photo absorption spectra of 2a-2l in DMSO were measured (Figure 2 and Table 1). The
-1
-1
D--A sensitizers exerted good absorption intensity ( = 20,000-53,000 L mol cm ) as shown in
Table 1. As expected, the introduction of a dithienopyrrole moiety to sensitizer 1 resulted in a ca 40
nm red shift (Table 1, entry 1 vs. 2). When a sensitizer had a more electron-donating donor (Me
Ph N- > MeO-), it tended to have longer wavelength absorption (2e with Me N-: λmax = 488 nm, 2a
with Ph N-: λmax = 477 nm, 2i with MeO-: λmax = 469 nm), as shown in entries 2, 7, and 11. On the
2
N- >
2
2
2

other hand, the electron-withdrawing ability of the acceptors (rhodanine > cyanoacrylic acid >
cyanoacrylic amide > aldehyde) was not consistent with absorption wavelength. In detail, the
sensitizer 2b with more electron-withdrawing cyanoacrylic acid acceptor had similar or shorter
wavelength absorption comparing with those of the sensitizers 2c and 2a with less
electron-withdrawing cyanoacrylic amide and aldehyde acceptors, respectively (2d with rhodanine:
λmax = 557 nm, 2b with cyanoacrylic acid: λmax = 477 nm, 2c with cyanoacrylic amide: λmax = 526 nm,
2a with aldehyde: λmax = 477 nm), as shown in entries 2, 3, 5, and 6. We speculated that the
deprotonation of cyanoacrylic acid generated less electron-withdrawing cyanoacrylate, which
shortened the absorption wavelength. In order to confirm this speculation, a photo absorption
spectrum of sensitizer 2b in DMSO with 0.5% acetic acid was measured. As expected, longer
wavelength absorption was observed (Table 1, entry 3 vs. 4).
Fig. 2 Absorption spectra of D--A sensitizers (a) 1-2d and (b) 2e-2l in DMSO.
Table 1 Absorption maxima (λmax), molar absorption coefficients (ε), and optical edges of the
sensitizers 1-2l.
entry sensitizer
λ
max
ε
optical
-
1
-1
1
[
nm]
[Lmol cm ] edge*
nm]
33,000 507
[
1
1
438

2
3
4
5
6
7
8
9
1
2a
2b
2b
2c
2d
2e
2f
477
477
510
526
557
488
487
540
574
469
471
503
553
51,000
47,000
28,000
53,000
30,000
35,000
33,000
35,000
36,000
45,000
35,000
46,000
20,000
548
552
669
618
656
566
567
642
684
534
540
597
642
2
*
2g
2h
2i
0
11
1
1
1
2
3
4
2j
2k
2l
*1 Optical absorption edge was defined by the wavelength for which the absorbance revealed 1/10
of the peak top.
*2 D--A sensitizer 2b with 0.5% acetic acid.
The antitumor effects of sensitizers 2a-2l and that of the positive controls, sensitizer 1,
[10]
[2b]
merocyanine 540 (MC540),
and protoporphyrin IX (PPIX)
on HeLa cells under photo
[11]
irradiation are shown in Table 2.
Although our previously reported D--A sensitizer with a
[
6]
triphenyl amine donor exerted potent phototoxicity, the triphenyl amine-containing D--A
sensitizers 2a-2d showed weak phototoxicity (entries 1-4). These results indicate the importance of
the systematic preparation and evaluation of sensitizers by combining various building blocks for the
development of highly potent sensitizers. Sensitizers containing dimethyl phenyl amine, 2e, 2f-2h
(
entries 5-8), or methoxy phenyl donor, 2i-2l (entries 9-13), exerted a higher level of phototoxicity.
With respect to an acceptor moiety, cyanoacrylic acid or cyanoacrylic amide acceptor contributed to
a higher level of phototoxicity. In fact, highly phototoxic compounds, 2f, 2j, and 2k (IC50 < 1 M),
are found in combinations of dimethyl phenyl amine or methoxy phenyl donor and cyanoacrylic acid
or cyanoacrylic amide acceptor (entries 6, 10, and 12). These sensitizers exerted a level of
phototoxicity that was higher than that of the positive controls MC540 and PPIX, and the same level
of phototoxicity as that of sensitizer 1. Since much stronger photo irradiation conditions are used in
2
clinical PDT (laserphyrin: 150 mW/cm , 3-12 min), the antitumor activity of the most potent
sensitizer, 2j, was evaluated over an extended amount of photo irradiation time (60 min, entry 11),
and exerted a significantly higher level of phototoxicity (IC50 of 2j: 0.063 M). In addition, 2j was
less toxic (IC50 = 43 M) without irradiation by comparison with 2f, 2k, and 1 (IC50 = 15-22 M).

Table 2 Antitumor activity of D--A sensitizers 2a-2l against HeLa cells.
*
1
entry
Sensitizer
IC50 (mM)
dark
Irradiation (15 min)
45.0 ± 6.4
> 100
1
2
3
4
5
6
7
8
9
2a
67.0 ± 13.8
> 100
2b
2c
> 100
> 100
2d
2e
60.2 ± 11.2
> 10
17.1 ± 1.8
2.5 ± 0.1
2f
15.5 ± 2.7
54.0 ± 7.6
>100
0.6 ± 0.1
2g
5.0 ± 0.4
2h
2i
12.1 ± 1.9
3.8 ± 0.2
68.6 ± 10.6
43.4 ± 3.3
43.4 ± 3.3
15.7 ± 1.7
63.9 ± 8.8
21.9 ± 2.4
>100
1
0
2j
0.19 ± 0.01
*
2
11
2j
0.063 ± 0.005
1
1
1
1
1
*
2
3
4
5
6
2k
2l
0.56 ± 0.02
5.7 ± 0.3
1
0.21 ± 0.03
16 ± 3.0
MC540
PPIX
12.5 ± 4.3
0.75 ± 0.02
1 The sensitizer concentration required to reduce cell viability by 50% (IC50) was determined from
semi-logarithmic, dose-response plots. *2 Irradiation time was 60 min.
The oxidative damage to human serum albumin (HSA) caused by the photo irradiation of the
sensitizers 2a-2l and positive control (MC540) was monitored. HSA has only one tryptophan residue
(
Trp), and the intrinsic fluorescence of HSA originates almost entirely from it. This tryptophan
residue is easily oxidized by photo-irradiated sensitizers. It results in a decrease in the characteristic
fluorescence of tryptophan at around 350 nm, which makes HSA useful for evaluating the
[
4, 12]
protein-damaging activity of photosensitizers.
A sample solution containing 10 M of sensitizer
and 20 M of HSA in a PBS buffer was irradiated via LED (ISL-150×150 H3WH4R, white light,
2
4
20-750 nm, 8.0 ± 0.5 mW/cm , CCS Inc.). MC 540 was used as a positive control. The observed
protein-damaging activity of the sensitizers was almost consistent with their phototoxicity (Figure 3).
The highly phototoxic sensitizers 2f, 2j, and 2k obviously damaged HSA, whereas the other
sensitizers showed no obvious damage to HSA. The most phototoxic sensitizer 2j (Table 2, entry 10),
showed the most significant oxidative damage to HSA (Figure 3).

Fig. 3 Time-dependent damage of human serum albumin (HSA). A sample solution containing 10
M of sensitizer and 20 M of HSA in a PBS buffer was irradiated via LED.
In order to achieve NIR absorption, the acceptor moiety of the most potent sensitizer, 2j was
changed to strongly electron-withdrawing cyanopyridone. Knoevenagel condensation of aldehyde 2i
[
13]
with the acceptor 6d afforded the desired sensitizer 2m in a moderate yield. As expected, 2m
demonstrated excellent absorption in the NIR region (> 650 nm), as shown in Scheme 2. These
results clearly demonstrated the ready tunability of the absorption wavelength of the D--A
sensitizer. However, the antitumor activity of sensitizer 2m on HeLa cells under photo irradiation
was low. This result again indicated the importance of cyanoacrylic acid or cyanoacrylic amide
acceptor for antitumor activity of sensitizers.

Scheme 2 Synthesis of a D--A sensitizer, 2m, containing strongly electron-withdrawing
cyanopyridone acceptor and its antitumor activity and photo absorption spectrum.
To further elucidate the mode of action for the most potent sensitizer, 2j, damage to Trp was
measured in the presence of L-ascorbic acid (ROS quencher), and NaN
3
(Type II singlet oxygen
quencher) (Figure 4). Damage to the protein was obviously decreased in the presence of L-ascorbic
acid and slightly decreased in the presence of NaN
HSA mainly through Type I mechanisms.
3
. These results indicate that sensitizer 2j oxidizes
Fig. 4 Time-dependent damage of human serum albumin by sensitizer 2j. A sample solution
containing 10 M of sensitizer and 20 M of HSA in a PBS buffer was irradiated via LED.
In order to confirm the cellular uptake of sensitizer 2j, HeLa cells that were treated with 2j and
Hoechst 33342 were observed via fluorescence confocal microscopy (Figure 5). The sensitizer 2j
obviously went into the HeLa cells and was distributed over the cytoplasm. In particular, 2j seemed
to localize near the nuclei.

Fig. 5 Colocalization study (a) Hoechst 33342; (b) sensitizer 2j; and, (c) a merging of two images,
(
a) and (b).
We designed dithienopyrrole-based D--A sensitizers and rapidly prepared them via our
developed one-pot coupling approach. As expected, the D--A structure enabled a ready tuning of
the absorption wavelength. The highly phototoxic sensitizer 2j was developed, and it exerted
phototoxicity mainly via a Type I mechanism following its absorption into cells. A nitrogen atom in a
dithienopyrrole ring served as a connecting point to introduce functional building blocks that could
improve sensitizer properties such as solubility and/or tumor targeting ability. Therefore, a D--A
sensitizer with a dithienopyrrole structure could be a valuable template for further development of
sensitizers that could fulfil the requirements for PDT.
A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at
https://doi.org/10.1016/j.bmcl.xxxxx.

Acknowledgement
We thank Mr. Yiming Zhao, University of Birmingham, for assistance in the synthesis of 2m.
References
[
1]
a) W. Fan, P. Huang, X. Chen, Chem. Soc. Rev. 2016, 45, 6488-6519; b) P. Agostinis, K. Berg, K.
A. Cengel, T. H. Foster, A. W. Girotti, S. O. Gollnick, S. M. Hahn, M. R. Hamblin, A. Juzeniene,
D. Kessel, M. Korbelik, J. Moan, P. Mroz, D. Nowis, J. Piette, B. C. Wilson, J. Golab, CA
Cancer J. Clin. 2011, 61, 250-281; c) Z. Huang, Technol. Cancer Res. Treat. 2005, 4, 283-293;
d) D. E. J. G. J. Dolmans, D. Fukumura, R. K. Jain, Nat. Rev. Cancer 2003, 3, 380-387.
a) J. Akimoto, Neurol. Med. Chir. (Tokyo) 2016, 56, 151-157; b) C. Cantisani, G. Paolino, V.
Faina, F. Frascani, F. Cantoresi, D. Bianchini, G. Fazia, S. Calvieri, Int. J. Photoenergy 2014, 7;
c) M. O. Senge, J. C. Brandt, Photochem. Photobiol. 2011, 87, 1240-1296; d) J. Moan, Q. Peng,
in Photodynamic Therapy, Vol. 2 (Ed.: T. Patrice), RSC, Nantes France, 2003; e) T. J. Dougherty,
J. Clin. Laser Med. Surg. 1996, 14, 219-221; f) T. J. Dougherty, Photochem. Photobiol. 1983, 38,
[
2]
3
77-379.
[
3]
a) A. B. Ormond, H. S. Freeman, Materials 2013, 6, 817-840; b) M. Sakai, N. Fujimoto, K. Ishii,
H. Nakamura, Y. Kaneda, K. Awazu, J. Biomed. Opt. 2012, 17, 1083-3668; c) M. Yamauchi, N.
Honda, H. Hazama, S. Tachikawa, H. Nakamura, Y. Kaneda, K. Awazu, Photodiagnosis
Photodyn. Ther. 2014, 11, 48-54; d) S. Tachikawa, M. E. El-Zaria, R. Inomata, S. Sato, H.
Nakamura, Bioorg. Med. Chem. 2014, 22, 4745-4751; e) S. Tachikawa, S. Sato, H. Hazama, Y.
Kaneda, K. Awazu, H. Nakamura, Bioorg. Med. Chem. 2015, 23, 7578-7584.
[
[
4]
5]
D. Ouyang, S. Inoue, S. Okazaki, K. Hirakawa, J. Porphyrins Phthalocyanines 2016, 20,
8
13-821.
a) S. Irie, S. Fuse, M. M. Maitani, Y. Wada, Y. Ogomi, S. Hayase, T. Kaiho, H. Masui, H. Tanaka,
T. Takahashi, Chem. Eur. J. 2016, 22, 2507-2514; b) S. Fuse, R. Takahashi, M. M. Maitani, Y.
Wada, T. Kaiho, H. Tanaka, T. Takahashi, Eur. J. Org. Chem. 2016, 508-517; c) K. Matsumura, S.
Yoshizaki, M. M. Maitani, Y. Wada, Y. Ogomi, S. Hayase, T. Kaiho, S. Fuse, H. Tanaka, T.
Takahashi, Chem. Eur. J. 2015, 21, 9742-9747; d) S. Fuse, S. Sugiyama, M. M. Maitani, Y. Wada,
Y. Ogomi, S. Hayase, R. Katoh, T. Kaiho, T. Takahashi, Chem. Eur. J. 2014, 20, 10685-10694; e)
S. Fuse, K. Matsumura, A. Wakamiya, H. Masui, H. Tanaka, S. Yoshikawa, T. Takahashi, ACS
Comb. Sci. 2014, 16, 494-499; f) S. Fuse, K. Matsumura, Y. Fujita, H. Sugimoto, T. Takahashi,
Eur. J. Med. Chem. 2014, 85, 228-234; g) S. Fuse, Y. Asai, S. Sugiyama, K. Matsumura, M. M.
Maitani, Y. Wada, Y. Ogomi, S. Hayase, T. Kaiho, T. Takahashi, Tetrahedron 2014, 70,
8
690-8695; h) S. Fuse, H. Yoshida, T. Takahashi, Tetrahedron Lett. 2012, 53, 3288-3291.

[
[
[
6]
7]
8]
S. Fuse, M. Takizawa, K. Matsumura, S. Sato, S. Okazaki, H. Nakamura, Eur. J. Org. Chem.
017, 5170-5177.
2
When 6-membered aromatic rings are connected, planes of the aromatic rings tend to twist for
avoiding steric repulsion between substituents on the aromatic rings.
a) N. Miyaura, A. Suzuki, J. Chem. Soc., Chem. Commun. 1979, 866-867; b) N. Miyaura, K.
Yamada, A. Suzuki, Tetrahedron Lett. 1979, 3437-3440; Selected recent reviews on
Suzuki-Miyaura coupling, see c) A. J. J. Lennox, G. C. Lloyd-Jones, Chem. Soc. Rev. 2014, 43,
4
12-443; d) I. Maluenda, O. Navarro, Molecules 2015, 20, 7528-7557.
[
[
[
[
9]
S. A. Odom, K. Lancaster, L. Beverina, K. M. Lefler, N. J. Thompson, V. Coropceanu, J. L.
Brédas, S. R. Marder, S. Barlow, Chem. Eur. J. 2007, 13, 9637-9646.
10]
11]
12]
a) I. Tsujino, K. Miyagi, R. W. Sampson, F. Sieber, Photochem. Photobiol. 2006, 82, 458-465; b)
F. Sieber, J. L. Spivak, A. M. Sutcliffe, Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 7584-7587.
The antitumor effects of sensitizers 1 and 2j on HEK293 cells with/without photoirradiation was
also evaluated, see supporting information.
a) D. Ouyang, K. Hirakawa, Rapid Commun. Photosci. 2016, 4, 41-44; b) K. Hirakawa, T.
Yoshioka, Chem. Phys. Lett. 2015, 634, 221-224; c) K. Hirakawa, H. Umemoto, R. Kikuchi, H.
Yamaguchi, Y. Nishimura, T. Arai, S. Okazaki, H. Segawa, Chem. Res. Toxicol. 2015, 28,
2
62-267; d) K. Hirakawa, N. Fukunaga, Y. Nishimura, T. Arai, S. Okazaki, Bioorg. Med. Chem.
Lett. 2013, 23, 2704-2707.
[
13]
M. Matsui, R. Aoki, D. Nishiwaki, Y. Kubota, K. Funabiki, J. Jin, T. Yoshida, S. Higashijima, H.
Miura, RSC Adv. 2015, 5, 57721-57724.

Highlights
・
・
・
Dithienopyrrole-based organic dye exerted potent phototoxicity.
The dye killed cancer cells mainly through Type I mechanism.
D--A structure enabled a ready tuning of absorption wavelength.