Synthesis of novel Chlorin e6-curcumin conjugates as photosensitizers for photodynamic therapy against pancreatic carcinoma

Article abstract of DOI:10.1016/j.ejmech.2018.01.099

Curcumin (cur) has been comprehensively studied for its various biological properties, more precisely for its antitumor potential and it has shown the promising results as well. On the other hand, Chlorin e6 (Ce6) has mostly been used as a photosensitizer in photodynamic therapy (PDT) against a variety of carcinomas. In the present study, we have synthesized a series of Chlorin e6-curcumin (Ce6-cur) conjugates and investigated their photosensitizing potential against pancreatic cancer cell lines. All the synthesized compounds were characterized by UV, ¹H NMR, ¹³C NMR and LC-MS. These Ce6-cur conjugates showed better physicochemical properties and higher singlet oxygen generation capability. The cellular uptake was studied in AsPC-1 cells using fluorescence-activated cell sorting (FACS). Compound 17 was rapidly internalized within 30 min and sustained for 24 h. Compound 17 showed excellent PDT efficacy with IC₅₀ of 40, 35 and 41 nM against AsPC-1, MIA PaCa-2 and PANC-1 respectively with exceptional dark/phototoxicity ratio in the range of 2371–7500. Moreover, the treatment of compound 17 upregulated the expression of BAX, Cytochrome-C and cleaved caspase 9 while downregulating the Bcl-2 expression an anti-apoptotic protein marker. These results demonstrate outstanding capability of compound 17 as a potent photosensitizer which could improve the PDT efficacy in pancreatic cancer patients.

Full text of DOI:10.1016/j.ejmech.2018.01.099

Accepted Manuscript
Synthesis of novel Chlorin e6-curcumin conjugates as photosensitizers for
photodynamic therapy against pancreatic carcinoma
Shivakumar S. Jalde, Anil Kumar Chauhan, Ji Hoon Lee, Pankaj Kumar Chaturvedi,
Joon-Suk Park, Yong-Wan Kim
PII:
S0223-5234(18)30121-1
10.1016/j.ejmech.2018.01.099
EJMECH 10176
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 December 2017
Revised Date: 30 January 2018
Accepted Date: 31 January 2018
Please cite this article as: S.S. Jalde, A.K. Chauhan, J.H. Lee, P.K. Chaturvedi, J.-S. Park, Y.-
W. Kim, Synthesis of novel Chlorin e6-curcumin conjugates as photosensitizers for photodynamic
therapy against pancreatic carcinoma, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.01.099.
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Synthesis of Novel Chlorin e6-Curcumin Conjugates as Photosensitizers for Photodynamic
Therapy against Pancreatic Carcinoma
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Shivakumar S Jalde , Anil Kumar Chauhan , Ji Hoon Lee , Pankaj Kumar Chaturvedi , Joon-
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Suk Park , Yong-Wan Kim *.

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Synthesis of Novel Chlorin e6-Curcumin Conjugates as Photosensitizers for Photodynamic
Therapy against Pancreatic Carcinoma
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Shivakumar S Jalde , Anil Kumar Chauhan , Ji Hoon Lee , Pankaj Kumar Chaturvedi , Joon-
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Suk Park , Yong-Wan Kim *.
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1. Daegu Cancer Center, Research and Development Unit of Dongsung Pharmaceuticals
Daegu 41061, Republic of Korea.
2. New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation
Daegu 41061, Republic of Korea.
3. Department of Radiation Oncology, Chungbuk National University, College of Medicine,
Cheongju, 361-763, Republic of Korea.
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4. Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu
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*Corresponding author: Yong-Wan Kim thomas06@hanmail.net

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Abstract: Curcumin (cur) has been comprehensively studied for its various biological
properties, more precisely for its antitumor potential and it has shown the promising
results as well. On the other hand, Chlorin e6 (Ce6) has mostly been used as a
photosensitizer in photodynamic therapy (PDT) against a variety of carcinomas. In the
present study, we have synthesized a series of Chlorin e6-curcumin (Ce6-cur) conjugates
and investigated their photosensitizing potential against pancreatic cancer cell lines. All
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the synthesized compounds were characterized by UV, H NMR, C NMR and LC-MS.
These Ce6-cur conjugates showed better physicochemical properties and higher singlet
oxygen generation capability. The cellular uptake was studied in AsPC-1 cells using
fluorescence-activated cell sorting (FACS). Compound 17 was rapidly internalized within
30 min and sustained for 24h. Compound 17 showed excellent PDT efficacy with IC of
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40, 35 and 41 nM against AsPC-1, MIA PaCa-2 and PANC-1 respectively with
exceptional dark / phototoxicity ratio in the range of 2371-7500. Moreover, the treatment
of compound 17 upregulated the expression of BAX, Cytochrome-C and cleaved caspase
9 while downregulating the Bcl-2 expression an anti-apoptotic protein marker. These
results demonstrate outstanding capability of compound 17 as a potent photosensitizer
which could improve the PDT efficacy in pancreatic cancer patients.
Key Words; Photodynamic therapy, Photosensitizer, Ce6, Curcumin, Pancreatic Cancer.
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Introduction
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Photodynamic therapy (PDT) relies on the accumulation of photosensitizer in tumors, which
upon irradiation of light of appropriate wavelength generates singlet oxygen and other cytotoxic
reactive oxygen species (ROSs) that results in cell membrane damage and subsequent cell death

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[1]. PDT uniquely stimulates cell death by directly activating apoptosis, and therefore bypasses
many cell death signaling pathways. PDT has several advantages over other conventional cancer
treatment modalities. It is relatively non-invasive because irradiation is limited to the tumor site,
shows lower systemic toxicity and relatively selective destruction of tumors, partly due to
preferential localization of photosensitizer within the tumor [2]. An ideal photosensitizing agent
should be a single pure compound to allow quality control analysis with low manufacturing costs
and good stability in storage. It should have a high absorption peak between 600 and 800 nm (red
to deep red) as absorption of photons with wavelengths longer than 800 nm does not provide
enough energy to excite oxygen to its singlet state, thus there is substantially reduced ROS
generation upon irradiation [3].
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Chlorins are reduced porphyrins, they absorb light in the 660 nm region, fluoresce at 670
nm and have high quantum yield of singlet oxygen generation. Ce6 possess three different
carboxylic acids that can be derivatized in multiple ways. Remarkable clinical benefits have been
obtained with Ce6 mediated PDT in the treatment of various cancers, including melanoma,
bladder, and nasopharyngeal cancers [4]. Ce6 was conjugated to peptides [5], sugars [6],
polyamines [7], mono amino acids [8] and diamino acids [9].
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The Indian spice curcumin (also known as diferuloylmethane), extracted from the
turmeric plant, has long held a role in Indian / Hindu rituals, traditions, customs, and cuisines
[10]. It is an orange-yellow, crystalline powder remarkably insoluble in water; however, it is
exceedingly soluble in ethanol and DMSO. In contrast with conventional cytotoxic drugs which
often have side effects such as nausea, vomiting or fatigue curcumin has minimal toxicity. The
safety of curcumin has been approved by the Food and Drug Administration and World Health
Organization; In addition, its safety is strongly supported by the fact that this agent has been used

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in traditional Hindu and Chinese medicine for thousands of years. Curcumin can modulate the
activity of a variety of molecules that play important roles in cancer progression, with more than
30 molecular targets identified to date [11]. Of these molecules, NF-κB appears to be one of the
primary targets of curcumin. Curcumin may induce apoptosis of cancer cells through blocking of
NF-κB survival pathway, generation of reactive oxygen species (ROS), down-regulation of Bcl-
XL, or activation of caspase-8 pathways. Curcumin has shown good anti-cancer activity against
pancreatic cancer [12] and it is used as sensitizing agents in combination with Cisplatin,
Doxorubicin, 5-Fluorouracil and Gemcitabine [13]. Recently, curcumin is being used as a
photosensitizer for anti-microbial photodynamic therapy [14].
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Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer (PC), is
a kind of digestive-tract malignant tumor with highly invasive and metastatic features and has
become the fourth most lethal malignancies in the US [15]. The patients with pancreatic cancer
have poor survival rate with less than 5% of patients surviving 5 years after diagnosis.
Chemotherapy is the most important adjuvant treatment for recurrent pancreatic cancer patients
who are not indicated for resection. However, PDAC is also notoriously resistant to Gemcitabine
(GEM), which is the first-line chemotherapeutic agent [16]. Therefore, it is of utmost
importance to identify new molecular targets for the effective treatment of pancreatic cancer.
Ce6 is an effective PS given its several advantages for clinical use, such as activation by near-
infrared wavelengths, relatively deep penetration through layers of tissues and potency against a
broad spectrum of cancers. Curcumin is well known for anti-cancer activity, it acts as a sensitizer
for resistant cells and used as a photosensitizer in anti-microbial PDT. Herein we have
conjugated Ce6 and curcumin with linkers to improve the Ce6 PDT efficacy.

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Results and Discussion
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Synthesis of Ce6-curcumin
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The synthetic route of the Ce6-cur conjugates is depicted in Scheme 1 & 2. Four Ce6-cur
conjugates were synthesized by incorporating hydrophobic and hydrophilic linkers. The linkers
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for
conjugating
curcumin
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Ce6
are
Propane,
Hexane,
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(Ethylenedioxy)bis(ethylamine) (mono PEG) and 4,9-Dioxa-1,12-dodecanediamine (diPEG). N-
Boc-monoprotected Propane 1, Hexane 2, 2′-(Ethylenedioxy)bis(ethylamine) (mono PEG) 3, and
4,9-Dioxa-1,12-dodecanediamine (diPEG) 4 (Scheme 1) were prepared with minor
modifications to the preparations described in the literature [17]. The mono-carboxylic acid
derivative of curcumin 6 was synthesized by reacting curcumin 5 with glutaric anhydride in the
presence of DMAP and TEA [18]. Dimethyl ester of Ce6 8 (DME Ce6) was obtained by
selective esterification of Chlorin e6 7 using 5 % of H SO in methanol [8]. The DME Ce6 8 was
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activated using 1-hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3-(dimethylamino)propyl)-
carbodiimide hydrochloride (EDCI) and coupled individually with 1, 2, 3 and 4 to give tert-butyl
protected Ce6-propane 9, Ce6-hexane 12, Ce6-monoPEG 15, Ce6-diPEG 18 derivatives
(Scheme 2), which were followed by deprotection with trifluoroacetic acid (TFA) to afford Ce6-
2 2 2 2
propane-NH 10, Ce6-hexane-NH 13, Ce6-monoPEG-NH 16, Ce6-diPEG-NH 19 derivatives.
The final coupling of curcumin monocarboxylic acid 6 with 10, 13, 16, and 19 using HOBt and
EDCI to give Ce6-cur conjugates 11, 14, 17 and 20.
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Determination of absorbance
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We recorded the absorption of compounds in DMSO in which all compounds and reference
compounds are soluble as shown in Fig. 1a. The absorption spectra of all compounds were

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typical to Ce6 derivatives with one soret band and Q-band. All the photosensitizers 7, 11, 14, 17,
and 20 effectively absorbed the red light with the major soret peak at λmax = 405-408 nm. The
soret peak of Ce6 7 was sharp while Ce6-cur conjugates have a broad peak, the broad peak was
due to curcumin conjugation. All the compounds exhibit a bathochromic shift as compared to
starting material 7 by 10 nm. Conjugation of curcumin to Ce6 increases the absorption to a
longer wavelength.
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Fluorescence
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Curcumin and Ce6 are two fluorophores, herein we conjugated them together with linkers, and
evaluated their fluorescence. All the compounds show fluorescence at 650-700 nm (Fig. 1b).
Compound 17 and 11 show higher fluorescence than Ce6 7, while compound 14 & 20 have same
fluorescence intensity as that of Ce6 7.
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Measurement of singlet oxygen generation
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Singlet oxygen generation is the key event in the photodynamic killing of cancer cells. The
ability of Ce6-cur conjugates and Ce6 to produce singlet oxygen under photosensitizing
condition was studied in DMSO using 1, 3-diphenylisobenzofuran (DPBF) as a singlet oxygen
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trap. DPBF reacts irreversibly with O
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and the reaction can be monitored by measuring the
decrease in DPBF absorption intensity at 418 nm. Irradiation of a solution of Ce6-cur conjugates,
Ce6, methylene blue (MB) and DPBF with 660 nm laser led to quenching of DPBF absorption
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band at 418 nm as shown in Fig. 1c. All the compounds show higher O generation than MB.
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The O generation of all the compounds is in the order 11 (7.39 %), 7 (9.88 %), 14 (10.05%), 17
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(12.13%), 20 (12.32 %) and MB (33.93 %).
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Cellular Uptake Assay

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There is a direct correlation of cellular uptake of PS to PDT, higher the cellular internalization,
stronger the PDT efficacy of PS [19]. The cellular uptake of 5 µM Ce6-cur conjugates and Ce6
in AsPC-1 cells at various incubation time was measured by Fluorescence-activated cell sorting
(FACS). All the compounds have shown time dependent uptake into the cells as shown in Fig.
2a-e. Interestingly compound 17 uptake was higher at 3h. The fluorescence intensity of
compound 17 was nearly 47 and 29 fold higher than free Ce6 7 after 3h and 24h respectively
(Fig. 2c). In order to understand the uptake of compound 17, we again studied the time
dependent uptake at 30 min, 1h and 2h as shown in Fig. 2d & e. The compound 17 was uptaken
by the cells within 30 min of incubation, reached maximum within one hour and thereafter
sustained within the cells for 24h. The Compound 11 and 20 have also shown higher uptake
compare to free Ce6. The difference in uptake might be due to linkers as hydrophilic linkers (17
& 20) have higher uptake than hydrophobic (11 & 14), with exception 11.
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Cytotoxicity Assessment
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An ideal PS should possess minimal dark toxicity, be cytotoxic in the presence of light at a
defined wavelength and have higher singlet oxygen quantum yield [20]. The dark toxicity of
Ce6-cur conjugates and Ce6 was evaluated in AsPC-1, MIA PaCa-2 and PANC-1. The cells
were exposed to increasing concentrations (0-300 µM) of Ce6-cur conjugate and Ce6 after 72h
and then cell viability was determined by classical MTT assay (Fig. 3a-c). All the compounds
under study have shown less dark toxicity than the starting material Ce6 7 except compound 17
against MIA PaCa-2, increased dark toxicity in the presence of Ce6-cur conjugate can be
attributed to curcumin. However, we couldn’t understand clearly why the dark toxicity was
observed only in one of the cancer cell line. Interestingly, after exposure to light (660 nm, 50
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mW, 9 J/cm ), all the conjugates were found to be highly toxic to AsPC-1, MIA PaCa2 and

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PANC-1 (Fig. 4 a-d). From the Table-1, it is clear that Ce6-cur conjugates are highly effective
and have lower IC values. The most active compound of the series was compound 17 (Fig. 4d)
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its IC values were in the range of 40, 35, 41 nM against AsPC-1, MIA PaCa-2 and PANC-1
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respectively while maintaining a higher ratio of dark/phototoxicity in the range of 2371-7500
(Table-1). AsPC-1 and PANC-1 cells are Gemcitabine resistant cells, thus the anti-cancer
activity on these cell lines at nanomolar concentrations of conjugates might be due to the
sensitization facilitated by curcumin [16]. The IC50 of compound 17 was nearly 488, 452, 247
folds lower than the free Ce6 7(19.53, 15.84, 10.14 µM against AsPC-1, MIA PaCa-2 and
PANC-1 respectively). Compound 11 and 20 also shown good PDT efficacy with IC50 in the
range of 4.2-5.74 µM and 4.64 to 15.74 µM respectively. We speculate that the higher
cytotoxicity of the compound 17 might be attributed to its higher cellular uptake, higher singlet
oxygen generation potential as evidenced by FACS and DPBF assay and synergistic activity of
curcumin and Ce6.
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Apoptosis
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There are varieties of mechanism by which a photosensitizer (PS) execute the cell death process;
however, apoptosis and necrosis are the most common mechanisms among them. We therefore,
examined the mechanism of cell death induced by compound 17, by using flow cytometer with
the help of Annexin V-FITC and Propidium iodide staining. Annexin V dye binds with
phosphatidylserine residues which are released in the outer membrane of the apoptotic cells and
produces a florescence due to FITC labeling allowing the differentiation of apoptotic and
necrotic cells [21]. As shown in Fig. 5, increasing dose of compound 17 (20 to 60 nM) induced a
massive apoptotic population in AsPC-1 cells (21 to 40 %, respectively) suggesting the intense
apoptotic potential of compound 17 in AsPC-1 pancreatic cells.

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The mechanism of apoptosis is much complicated having two major pathways i.e. intrinsic
apoptosis which is associated with mitochondrial dysfunctioning and extrinsic pathway regulated
by death domain receptors [22]. We therefore, elucidated the particular mechanism of apoptosis
induced by compound 17 through the immunoblot examination. The results obtained from the
examination suggest that the treatment of compound 17 triggered the intrinsic apoptotic pathway
as the downregulation of Bcl-2 and up regulation of Bax was observed (Fig. 6). Also, the
Cytochrom-C release a defining feature of mitochondrial stress was observed to be over
expressed along with the upregulation of cleaved caspase 9 (Fig. 6). Thus, compound 17 have
potential to trigger the mitochondria associated intrinsic apoptotic pathway.
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Conclusion
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In summary, we have designed and synthesized the novel Ce6-cur conjugates and characterized
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by UV, H NMR, C NMR and LC-MS. These conjugates exhibit excellent physicochemical
properties and higher singlet oxygen generation. On cellular level, the uptake of these conjugates
was far better as compared to free Ce6. Compound 17 has shown outstanding cytotoxicity
against three pancreatic cancer cells AsPC-1, MIA PaCa2 and PANC-1 with IC values ranging
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from 35-41 nM. Additionally it had highest dark/photo toxicity ratio in the range of 2371-7500.
Moreover, Compound 17 has potential to trigger the intrinsic apoptotic pathway in AsPC-1 a
pancreatic cancer cell line. So, in the premise of our data we propose that compound 17, a novel
Ce6-cur conjugate with highest dark/ phototoxicity ratio is a potent photosensitizer with
enhanced PDT efficacy in vitro. We further plan to validate its promising potential as a
photosensitizer in vivo.
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Acknowledgements

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Financial support of this research was provided by a grant of the Daegu-Gyeongbuk Medical
Innovation Foundation Medical Device Development Center R & D Project (DG15D001).
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Materials and Methods
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All air and moisture sensitive reactions were performed in dried and distilled solvents under
argon atmosphere. All solvents and reagents were purchased from commercial sources, unless
otherwise stated. Silica gel 60 (230 X 400 mesh, Zeochem) was used for column
chromatography. Analytical thin-layer chromatography (TLC) was carried out on silica gel plates
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G254 (Merck). H and C spectra were recorded on Bruker 400 spectrometers, with TMS as
internal standard and CDCl as the solvents. Chemical shifts (δ) and coupling constant (J) are
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given in ppm and Hz, respectively. ESI mass spectrometric data were obtained on Shimadzu LC-
MS. UV absorption spectra were measured on Thermo Scientific UV spectrophotometer.
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Measurement of Singlet Oxygen Photogeneration
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1, 3-Diphenylisobenzofuran (DPBF) was used as a selective O acceptor, which was bleached
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upon reaction with O [23]. Five sample solutions of DPBF in DMSO (50 µM) containing,
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respectively, DPBF only (50 µM, a control sample), DPBF + methylene blue (MB) (1 µM),
DPBF + 7 (1 µM), DPBF + 11 (1 µM), DPBF + 14 (1 µM), DPBF + 17 (1 µM), DPBF + 20 (1
µM), were prepared in the dark. All the samples were placed in a 96-well plate and the container
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was covered with aluminum foil. The samples were irradiated (660 nm, 50 mW, 2 J/cm ) for 40
seconds. After irradiation, visible spectra of the sample solutions were measured
spectrophotometrically. The normalized absorbance of DPBF at 418 nm in these samples was
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reported. The O photo generation activities of MB, 7, 11, 14, 17, and 20 can be compared with
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the different absorbance decay of each sample relative to the DPBF control sample.

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Synthesis of 5-(4-((1E, 6E)-7-(4-hydroxy-3-methoxyphenyl)-3, 5-dioxohepta-1, 6-dien-1-yl)-
2-methoxyphenoxy)-5-oxopentanoic acid (6)
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To a solution of curcumin (2.01 g, 5.46 mmol), and DMAP (112 mg, 0.92 mmol) in THF (100
mL) was added (1.33 mL, 9.55 mmol) of Et N. Glutaric anhydride (95%) (0.685 g, 6 mmol) in
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THF (5 mL) was added slowly drop wise to the curcumin solution. The mixture was stirred and
refluxed under argon overnight. THF was removed under vacuum, EtOAc (55 mL) was added,
followed by the addition of 1M HCl (15 mL); the mixture was stirred for 10 minutes. The
organic phase was separated and extracted with EtOAc three times; the solvent was removed and
dried. The product was purified via column chromatography, and eluted with CH Cl : MeOH,
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95: 5. Yield: 69 %. UV-Vis (DMSO): λmax (ɛ / M cm ) 370 (4.7 X 10 ), 414 (4.4 X 10 ) nm.
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HNMR (CDCl 400 MHz): δ 7.65 (d, J = 16 Hz, 2H), 7.20-6.95 (m, 5H), 6.96 (d, 1H), 6.48-
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6.57 (m, 2H), 5.85 (s, 2H), 3.98 (s, 3H), 3.90 (s, 3H), 2.75-2.71 (t, J = 8 Hz, 2H), 2.61-2.57 (t, J
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= 8 Hz, 2H), 2.15-2.12 (t, J = 8 Hz, 2H). C NMR (CDCl , 100 MHz): δ 184.56, 181.80, 178.26,
170.84, 151.28, 148.03, 146.84, 141.09, 139.40, 134.12, 127.53, 124.25, 123.07, 121.73, 120.99,
114.89, 111.37, 109.69, 101.58, 55.96, 32.82, 19.92. LC-MS: 483 [M+H].
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Synthesis of Dimethylester of chlorin e6 (DME Ce6) (8)
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Chlorin e6 (7) (3 g, 5.02 mmol) was dissolved in 5% sulfuric acid in methanol and allowed to stir
in the dark, under argon overnight. The reaction was poured into cold saturated aqueous
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NaHCO and extracted twice with CH Cl . The extract was washed twice with brine, dried over
Na SO and filtered. The solvent was evaporated. It was then purified on a silica gel column
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afford 2.8 g, Yield: 88 %. UV-Vis (DMSO): λmax (ɛ / M cm ) 656 (9.2 X 10 ), 501 (2.9 X 10 ),
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399 (3.6 X 10 ) nm. H NMR(CDCl , 400 MHz ): δ 9.62 (s, 1H), 9.49 (s, 1H), 8.73 (s, 1H), 8.03
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(m, 1H), 6.32 (dd, J = 17.8, 1.2 Hz, 1H), 6.13 (dd, J = 11.5, 1.2 Hz, 1H), 5.50 (d, J = 18.6 Hz,
1H), 5.23 (d, J = 18.6 Hz, 1H), 4.45 (m, 2H), 3.82 (s, 3H), 3.76 (q, J = 7.6 Hz, 2H), 3.62 (s, 6H),
3.46 (s, 3H), 3.25 (s, 3H), 1.69 and 2.12 (m, 2H), 2.19 and 2.56 (m, 2H), 1.81 (d, J = 7.1 Hz,
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3H), 1.64 (t, J = 7.6 Hz, 3H), -1.71 (s, 1H), -1.92 (s, 1H). C NMR (CDCl , 100 MHz): δ
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173.58, 169.89, 167.31, 155.01, 148.84, 145.12, 139.77, 137.17, 136.17,135.85, 135.58, 134.84,
130.67, 129.33, 121.80, 102.47, 98.63, 93.60, 52.84, 51.65, 49.53, 39.22, 30.98, 29.34, 22.75,
19.59, 17.66, 12.69, 12.15, 11.29. LC-MS: 625 [M+H].
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Synthesis of tert-butyl (3-aminopropyl) carbamate (1)
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To a stirred and cooled solution (0 ºC) of 1, 3-diaminopropane (3.64 mL, 43.5 mmol) in CHCl3
(45 mL) was added a solution of di-tert-butyl bicarbonate (0.95 g, 4.35 mmol) in CHCl (22 mL)
3
drop wise over a period of 3h. The reaction mixture was allowed to warm to room temperature
and stirred for additional 20h. The precipitated white solid was filtered and the CHCl
3
was
washed with water (2 x 20 mL). The organic layer was dried over Na SO and concentrated in
2
4
vacuo to give compound tert-butyl (3-aminopropyl)carbamate (1) 475 mg, Yield: 63% as a clear
1
oil which was used for the next reaction without any further purification. H NMR (CDCl 400
3
,
MHz): δ 4.91 (bs, 1H), 3.16 (dq, J = 12.7, 6.4 Hz, 2H), 2.73 (t, J = 6.6 Hz, 2H), 1.58 (p, J = 6.6
Hz, 2H), 1.41 (s, 9H).
2
65
Synthesis of Ce6-Propane-NHBoc (9)
2
2
2
2
66
67
68
69
DME Ce6 8 (1 g, 1.60 mmol) was dissolved in anhydrous CH Cl (30 mL). EDCI (368 mg, 1.92
2
2
mmol) and HOBt (260 mg, 1.92 mmol) were then added and allowed to stir until completely
dissolved under nitrogen. After 30 min, tert-butyl (3-aminopropyl)carbamate (1) (836 mg, 4.80
mmol) and DIEA (413 mg, 3.2 mmol) were mixed in CH Cl (20 mL) and added to the reaction
2
2

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
2
2
70
71
72
73
74
75
76
77
78
79
80
81
82
mixture. The mixture was allowed to stir at room temperature for 12h under nitrogen. The
reaction mixture was diluted with CH Cl (200 mL) and then washed with brine and water,
2
2
respectively. The organic layer was dried over anhydrous Na SO and then evaporated. The
2
4
product was purified via column chromatography to afford 520 mg of 9, Yield: 41 %. UV-Vis
-1
-1
5
5
5
1
(DMSO): λmax (ɛ / M cm ) 670 (4.3 X 10 ), 504 (2.0 X 10 ), 408 (7.6 X 10 ) nm. H
NMR(CDCl , 400 MHz ): δ 9.62 (s, 1H), 9.57 (s, 1H), 8.73 (s, 1H), 8.03 (m, 1H), 6.31 (dd, J =
3
17.8, 1.2 Hz, 1H), 6.08 (dd, J = 11.5, 1.2 Hz, 1H), 5.48 (d, J = 18.6 Hz, 1H), 5.20 (d, J = 18.6
Hz, 1H), 4.38 and 4.27 (m, 2H), 3.76 (m, 5H), 3.58 (m, 8H), 3.40 (s, 6H), 3.25 (s, 4H), 1.69 and
2.12 (m, 2H), 1.90 and 2.49 (m, 2H), 1.90 (m, 3H), 1.68 (m, 3H) 1.50 (s, 11 H), -1.67 (s, 1H), -
1
3
1.88 (s, 1H). C NMR (CDCl , 100 MHz): δ 173.87, 168.78, 166.70, 156.56, 154.17, 149.07,
3
144.74, 138.86, 136.10, 13503, 134.77, 134.50, 130.14, 129.90, 129.45, 128.20, 121.61, 102.15,
101.40, 98.83, 93.67, 79.35, 53.08, 52.17, 51.65, 49.26, 37.62, 31.14, 30.37, 29.64, 28.38, 23.05,
19.69, 17.76, 12.19, 11.35. LC-MS: 781 [M+H].
2
83
Synthesis of Ce6-Propane amine (10)
2
2
2
2
2
2
2
2
2
84
85
86
87
88
89
90
91
92
The compound 9 (500 mg, 0.64 mmol) was dissolved in dry CH Cl (20 mL) in an ice bath under
2
2
argon. TFA (2 mL) was added, and the reaction mixture was stirred overnight. The reaction
mixture was evaporated several times with diethyl ether to remove residual TFA. Then the
precipitate was dissolved in CH Cl and washed three times with H O and once with 10%
2
2
2
3 2 4
NaHCO to remove TFA. The organic layer was dried over anhydrous Na SO and then
evaporated to give a crude compound, purified by silica gel chromatography to give 350 mg of
-1
-1
5
5
10, Yield: 80 %. UV-Vis (DMSO): λmax (ɛ / M cm ) 658 (6.4 X 10 ), 501 (2.0 X 10 ), 400 (2.5
6
1
X 10 ) nm. H NMR(CDCl , 400 MHz ): δ 9.62 (s, 1H), 9.56 (s, 1H), 8.73 (s, 1H), 8.01 (m, 1H),
3
6.29 (dd, J = 16 Hz, 1H), 6.07 (dd, J = 16 Hz, 1H), 5.50 (d, J = 20 Hz, 1H), 5.20 (d, J = 20 Hz,

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
93
94
95
96
97
98
1H), 4.40 and 4.27 (m, 2H), 3.86 and 3.61 (m, 2H), 3.71 (m, 5H), 3.53 (s, 3H), 3.48 (s, 4H), 3.41
(s, 3H), 3.26 (s, 3H), 2.89 (t, J = 8 Hz, 2H), 2.49 (m, 2H), 1.69 and 2.12 (m, 2H), 1.85 (t, J = 4 &
1
3
8 Hz, 2H), 1.77 (m, 6H), -1.71 (s, 1H), -1.92 (s, 1H); C NMR (CDCl , 100 MHz): δ 173.87,
3
168.78, 166.70, 154.17, 149.07, 144.74, 138.86, 136.10, 135.03, 134.77, 134.50, 130.14, 129.90,
129.45, 128.20, 121.61, 102.15, 101.40, 98.83, 93.67, 53.08, 52.17, 51.65, 49.26, 37.62, 31.14,
30.37, 29.64, 28.38, 23.05, 19.69, 17.76, 12.19, 11.35. LC-MS: 681 [M+H ].
2
99
Synthesis of Chlorin e6-curcumin conjugate (11)
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
Compound 6 (250 mg, 0.51 mmol) was dissolved in dry CH Cl . A mixture of HOBt (83 mg,
2
2
0.62 mmol), EDCI (120 mg, 0.62 mmol), and DIEA (66 mg, 0.51 mmol) in CH Cl was added,
2
2
the mixture was then allowed to stir for 30 min. Compound 10 (352 mg, 0.51 mmol) and DIEA
(66 mg, 0.51 mmol) were mixed in CH Cl and added to this reaction mixture. The mixture was
2
2
stirred overnight. It was diluted with CH
followed by a wash with brine and water. It was dried over anhydrous Na
evaporated. The residue was purified by silica gel column chromatography to afford 280 mg of
2
Cl
2
and then washed with 5% aqueous citric acid,
2
SO and then
4
-1
-1
5
5
11, Yield: 47 %. UV-Vis (DMSO): λmax (ɛ / M cm ) 668 (3.3 X 10 ), 504 (1.6 X 10 ), 403 (7.6
5
1
X 10 ) nm. H NMR (CDCl , 400MHz): δ 9.59 (s, 1H), 9.55 (s, 1H), 8.72 (s, 1H), 8.00 (m, 1H),
3
7.49 (d, J = 16 Hz, 1H), 7.40 (d, J = 16 Hz, 1H), 7.01 (m, 2H), 6.93 (m, 4H), 6.85-6.81 (m, 2H),
6.33-6.25 (m, 2H), 6.07 (dd, J = 4 Hz, 1H), 5.56 (s, 1H), 5.44 (d, J = 16 Hz, 1H), 5.19 (d, J = 20
Hz, 1H), 4.40 and 4.26 (m, 2H), 3.85 (m, 5H), 3.75-3.69 (m, 8H), 3.57-3.53 (m, 6H), 3.46-3.40
(m, 8H), 3.23 (s, 3H), 2.58 (t, J = 8 Hz, 2H), 2.50 (m, 1H), 2.34 (t, J = 8 & 4 Hz, 2H), 2.15-2.09
1
3
3
(m, 2H), 2.05 (t, J = 8 Hz, 2H), 1.67 (m, 6H), -1.71 (s, 1H), -1.92 (s, 1H). C NMR (CDCl , 100
MHz): δ 184.43, 181.53, 173.59, 172.69, 171.19, 170.04, 168.90, 166.73, 151.17, 149.03,
147.92, 146.75, 144.81, 141.01, 140.94, 139.13, 136.17, 134.92, 134.85, 134.60, 134.56, 133.94,

ACCEPTED MANUSCRIPT
3
3
3
16
17
18
130.27, 129.77, 129.34, 127.86, 127.40, 124.06, 123.20, 122.92, 121.67, 120.90, 114.79, 111.27,
109.53, 102.15, 101.43, 98.84, 93.72, 55.82, 53.07, 51.67, 49.25, 37.91, 36.32, 33.01, 31.11,
29.62, 23.05, 21.02, 19.68, 17.76, 12.17, 11.35. LC-MS: 1145 [M+H].
3
19
Synthesis of tert-butyl (6-aminohexyl)carbamate (2)
3
3
3
3
3
3
3
3
3
20
21
22
23
24
25
26
27
28
Di-tert-butyl dicarbonate (4.0 g, 18.4 mmol) was dissolved in CHCl
solution of hexamethylenediamine (10.6 g, 91.6 mmol) in CHCl
3
and added drop-wise to a
3
at 0 °C. The mixture was
allowed to warm to room temperature. After stirring for 12h, the reaction crude was filtered and
washed with CHCl . The filtrates were collected and solvent was evaporated. The residue was re-
3
dissolved in EtOAc and washed with water and then brine. The organic solution was dried over
anhydrous Na SO , filtered and concentrated under reduced pressure to afford 1.68 g, Yield: 42
2
4
1
% of tert-butyl (6-aminohexyl)carbamate (2). H NMR (CDCl , 400 MHz): δ 4.52 (bs, 1H), 3.10
3
1
3
(q, J = 6.6 Hz, 2H), 2.68 (t, J = 7.0 Hz, 2H), 1.49-1.30 (m, 17H), 1.25 (t, J = 7.2 Hz, 2H). C
NMR (100 MHz, CDCl ): δ 156.1, 79.1, 42.2, 40.5, 33.8, 30.2, 28.4, 26.7, 26.6.
3
3
29
Synthesis of Ce6-Hexane-NHBoc (12)
3
3
3
3
3
3
3
3
30
31
32
33
34
35
36
37
DME Ce6 8 (1 g, 1.60 mmol) was dissolved in anhydrous CH Cl (30 mL). EDCI (614 mg, 3.2
2
2
mmol) and HOBt (432 mg, 3.2 mmol) were then added and allowed to stir until completely
dissolved under nitrogen. After 30 min, tert-butyl (6-aminohexyl)carbamate (2) (1.73 g, 8.0
mmol) and DIEA (620 mg, 4.8 mmol) were mixed in CH Cl (20 mL) and added to the reaction
2
2
mixture. The mixture was allowed to stir at room temperature for 12h under nitrogen. The
reaction mixture was diluted with CH Cl (200 mL) and then washed with brine and water,
2
2
respectively. The organic layer was dried over anhydrous Na SO and then evaporated. The
2
4
product was purified via column chromatography to afford 700 mg of 12, Yield: 53 %. UV-Vis

ACCEPTED MANUSCRIPT
-
1
-1
5
5
5
1
3
3
3
3
3
3
3
3
3
38
39
40
41
42
43
44
45
46
(DMSO): λmax (ɛ / M cm ) 670 (3.57 X 10 ), 504(9.8 X 10 ), 408(9.82 X 10 ) nm. H
NMR(CDCl , 400 MHz ): δ 9.63 (s, 1H), 9.58 (s, 1H), 8.74 (s, 1H), 8.03 (m, 1H), 6.31 (dd, J =
3
16 Hz, 1H), 6.09 (dd, J = 12 Hz, 1H), 5.49 (d, J = 20 Hz, 1H), 5.21 (d, J = 20 Hz, 1H), 4.40 and
4.26 (m, 2H), 3.85 (m, 5H), 3.58 (m, 8H), 3.42 (s, 3H), 3.25 (s, 3H), 3.10 (m, 2H), 1.69 and 2.12
1
3
(m, 2H), 1.74 (m, 2H), 1.67 (m, 6H), 1.50 (s, 17H), 1.36 (s, 2H), -1.71 (s, 1H), -1.92 (s, 1H). C
NMR (CDCl , 100 MHz): δ 173.56, 169.39, 168.74, 166.67, 156.05, 154.15, 149.10, 144.74,
3
138.82, 136.12, 134.99, 134.75, 134.49, 130.14, 129.47, 128.43, 121.63, 102.15, 101.35, 98.85,
93.68, 79.08, 52.14, 51.65, 49.25, 40.49, 37.81, 31.14, 30.95, 29.48, 28.45, 26.76, 23.07, 19.70,
17.78, 14.22, 12.20, 11.37. LC-MS: 823 [M+H].
3
47
Synthesis of Ce6-Hexane amine (13)
3
3
3
3
3
3
3
3
3
3
3
3
3
48
49
50
51
52
53
54
55
56
57
58
59
60
The compound 12 (700 mg, 0.85 mmol) was dissolved in of dry CH Cl (30 mL) in an ice bath
2
2
under argon. TFA (3 mL) was added, and the reaction mixture was stirred overnight. The
reaction mixture was evaporated several times with diethyl ether to remove residual TFA. Then
the precipitate was dissolved in CH Cl and washed three times with H O and once with 10%
2
2
2
NaHCO to remove TFA. The organic layer was dried over anhydrous Na SO and then
3
2
4
evaporated to give a crude compound, purified by silica gel chromatography to give 350 mg of
-1
-1
5
5
13, Yield: 73 %. UV-Vis (DMSO): λmax (ɛ / M cm ) 658 (4.7 X 10 ), 501 (1.4 X 10 ), 400 (1.9
6
1
X 10 ) nm. H NMR(CDCl , 400 MHz ): δ 9.63 (s, 1H), 9.58 (s, 1H), 8.73 (s, 1H), 8.01 (m, 1H),
3
6.31 (dd, J = 16, 4 Hz, 1H), 6.09 (dd, J = 12 Hz, 1H), 5.50 (d, J = 20 Hz, 1H), 5.21 (d, J = 20
Hz, 1H), 4.40 and 4.27 (m, 2H), 3.75 (m, 5H), 3.53 (s, 3H), 3.49 (s, 4H), 3.42 (s, 3H), 3.25 (s,
3H), 2.62 (t, J = 4 & 8 Hz, 2H), 2.46 (m, 2H), 1.69 and 2.12 (m, 2H), 1.76-1.63 (m, 10H), 1.43
1
3
(m, 8H), -1.71 (s, 1H), -1.92 (s, 1H). C NMR (CDCl , 100 MHz): δ 173.57, 169.40, 168.75,
3
166.68, 154.15, 149.10, 144.74, 138.83, 136.14, 134.98, 134.80, 134.75, 134.49, 130.14, 129.45,

ACCEPTED MANUSCRIPT
3
3
61
62
128.47, 121.63, 102.15, 101.35, 98.86, 93.69, 52.14, 51.65, 49.25, 41.97, 40.52, 37.78, 31.14,
29.45, 26.92, 23.08, 19.71, 17.78, 12.20, 11.37. LC-MS: 723 [M+H].
3
63
Synthesis of Chlorin e6-curcumin conjugate (14)
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
Compound 6 (300 mg, 0.62 mmol) was dissolved in dry CH
2
Cl
2
. A mixture of HOBt (100 mg,
Cl was added,
0.74 mmol), EDCI (143 mg, 0.74 mmol), and DIEA (66 mg, 0.51 mmol) in CH
2
2
the mixture was then allowed to stir for 30 min. Compound 13 (352 mg, 0.51 mmol) and DIEA
(160 mg, 1.24 mmol) were mixed in CH Cl and added to this reaction mixture. The mixture was
2
2
stirred overnight. It was diluted with CH Cl and then washed with 5% aqueous citric acid,
2
2
followed by a wash with brine and water. It was dried over anhydrous Na SO and then
2
4
evaporated. The residue was purified by silica gel column chromatography to afford 450 mg of
-1
-1
5
5
14, Yield: 61 %. UV-Vis (DMSO): λmax (ɛ / M cm ) 668 (2.9 X 10 ), 505 (1.4 X 10 ), 406 (6.9
5
1
3
X 10 ) nm. H NMR (CDCl , 400 MHz): δ 9.60 (s, 1H), 9.55 (s, 1H), 8.72 (s, 1H), 8.02 (m, 1H),
7.38 (m, 2H), 6.94-6.81 (m, 8H), 6.30-6.19 (m, 3H), 6.07 (dd, J = 4 Hz, 1H), 5.50-5.44 (m, 2H),
5.23-5.17 (m, 2H), 4.40 and 4.26 (m, 2H), 3.86-3.78 (m, 5H), 3.73-3.71 (m, 9H), 3.54 (s, 4H),
3.46 (s, 4H), 3.41 (s, 3H), 3.27-3.24 (m, 6H), 2.54 (t, J = 8 Hz, 2H), 2.50 (m, 1H), 2.26 (t, J = 8
1
3
Hz, 2H), 2.10-1.99 (m, 5H), 1.74-1.62 (m, 9H), -1.71 (s, 1H), -1.92 (s, 1H); C NMR (CDCl ,
3
100 MHz): δ 184.38, 181.46, 173.61, 172.21, 171.20, 169.51, 168.75, 166.68, 154.15, 151.08,
149.08, 147.91, 146.71, 144.75, 140.89, 138.99, 136.15, 134.95, 134.75, 134.72, 134.45, 133.96,
130.19, 129.51, 129.42, 128.33, 127.28, 124.11, 123.13, 122.84, 121.60, 120.90, 114.75, 111.29,
109.45, 102.10, 101.41, 98.84, 93.70, 55.84, 53.11, 52.18, 51.67, 49.23, 40.31, 39.18, 38.90,
37.78, 35.18, 32.83, 29.68, 26.52, 23.09, 21.14, 19.69, 17.78, 12.19, 11.36. LC-MS: 1187
[M+H].

ACCEPTED MANUSCRIPT
3
83
Synthesis of tert-butyl(2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (3)
3
3
3
3
3
3
3
3
3
3
84
85
86
87
88
89
90
91
92
93
Under a nitrogen atmosphere, to a solution of 2,2’-(ethylenedioxy)-bis-(ethylamine) (14.8 g, 100
0
mmol) in anhydrous CHCl3 (100 mL) cooled to 0 C was added dropwise di-tert-
butyldicarbonate (2.18 g, 10 mmol) in CHCl (50 mL). After been stirred 24h at room
3
temperature, the solvent is evaporated under vacuum. The thick oil obtained is taken up in
CH
2
Cl
2
(100 mL). The organic layer is successively washed with saturated aqueous NaCl (50
SO and concentrated in vacuo to afford 2.20 g,
mL), water (50 mL), dried over anhydrous Na
2
4
Yield: 89% of crude tert-butyl(2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (3). This material
1
was used without further purification. H NMR (CDCl , 400 MHz): δ 5.15 (br s, 1H, NH), 3.63–
3
3.51 (m, 8H), 3.31 (td, J = 5.5 Hz, 2H), 2.88 (t, J = 4.8 Hz, 2H), 1.45 (s, 9H), 1.40 (s, 2H, NH2);
1
3
C NMR (CDCl , 100 MHz): d 155.42, 78.13, 72.80, 69.63, 41.08, 39.67, 27.77.
3
3
94
Synthesis of Ce6-MonoPEG-NHBoc (15)
3
3
3
3
3
4
4
4
4
4
95
96
97
98
99
00
01
02
03
04
DME Ce6 8 (1.5 g, 2.40 mmol) was dissolved in anhydrous CH Cl (50 mL). EDCI (552 mg,
2
2
2.88 mmol) and HOBt (388 mg, 2.88 mmol) were then added and allowed to stir until
completely dissolved under nitrogen. After 30 min, tert-butyl(2-(2-(2-
aminoethoxy)ethoxy)ethyl)carbamate (3) (2 g, 8.41 mmol) and DIEA (620 mg, 4.8 mmol) were
mixed in CH Cl (20 mL) and added to the reaction mixture. The mixture was allowed to stir at
room temperature for 12h under nitrogen. The reaction mixture was diluted with CH Cl (200
2
2
2
2
mL) and then washed with brine and water, respectively. The organic layer was dried over
anhydrous Na SO and then evaporated. The product was purified via column chromatography to
2
4
-1
-1
5
afford 1.3 g of 15, Yield: 63 %. UV-Vis (DMSO): λmax (ɛ / M cm ) 667 (4.1 X 10 ), 506 (1.8 X
5
5
1
10 ), 410 (8.5 X 10 ) nm. H NMR(CDCl , 400 MHz ): δ 9.63 (s, 1H), 9.57 (s, 1H), 8.73 (s, 1H),
3

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
05
06
07
08
09
10
11
12
8.02 (m, 1H), 6.31 (dd, J = 16 Hz, 1H), 6.09 (dd, J = 16 Hz, 1H), 5.51 (d, J = 20 Hz, 1H), 5.24
(d, J = 20 Hz, 1H), 4.66 and 4.02 (m, 2H), 4.42 and 4.31 (m, 2H), 3.84 (m, 2H), 3.74-3.66 (m,
7H), 3.54-3.50 (m, 9H), 3.29-3.25 (m, 5H), 2.97 (m, 2H), 2.50 and 2.17 (m, 2H), 1.74 and 2.08
1
3
(m, 2H), 1.67 (m, 8H), 1.50 (s, 9H ) -1.71 (s, 1H), -1.92 (s, 1H). C NMR (CDCl , 100 MHz): δ
3
173.56, 169.51, 168.83, 166.69, 155.86, 154.24, 149.10, 144.75, 138.88, 136.09, 135.02, 134.78,
134.52, 130.16, 129.90, 128.26, 121.60, 102.32, 101.37, 98.83, 93.66, 79.12, 70.44, 69.86,
53.11, 52.19, 51.62, 49.22, 40.41, 37.89, 31.12, 29.70, 28.43, 23.09, 19.71, 17.76, 12.18, 11.36.
LC-MS: 855 [M+H].
4
13
Synthesis of Ce6-MonoPEGamine (16)
4
4
4
4
4
4
4
4
4
4
4
4
4
4
14
15
16
17
18
19
20
21
22
23
24
25
26
27
The compound 15 (1.3 g, 1.52 mmol) was dissolved in dry CH Cl (30 mL) in an ice bath under
2
2
argon. TFA (3 mL) was added, and the reaction mixture was stirred overnight. The reaction
mixture was evaporated several times with diethyl ether to remove residual TFA. Then the
2 2 2
precipitate was dissolved in CH Cl and washed three times with H O and once with 10%
NaHCO to remove TFA. The organic layer was dried over anhydrous Na SO and then
3
2
4
evaporated to give a crude compound, purified by silica gel chromatography to give 1 g of 16,
-1
-1
5
5
Yield: 87 %. UV-Vis (DMSO): λmax (ɛ / M cm ) 658 (5.7 X 10 ), 501 (1.8 X 10 ), 399 (2.2 X
6
1
10 ) nm. H NMR(CDCl , 400 MHz ): δ 9.63 (s, 1H), 9.58 (s, 1H), 8.74 (s, 1H), 8.03 (m, 1H),
3
6.31 (dd, J = 16, 4 Hz, 1H), 6.09 (dd, J = 12 Hz, 1H), 5.54 (d, J = 20 Hz, 1H), 5.25 (d, J = 20
Hz, 1H), 4.42 and 4.00 (m, 2H), 4.28 and 4.01 (m, 2H), 3.86 (m, 2H), 3.75 (m, 6H), 3.66 (m, 2H)
3.54-3.49 (m, 9H), 3.43 (m, 2H), 3.26 (s, 3H), 3.21 (t, J = 8 & 4 Hz, 2H), 2.49 and 2.10 (m, 2H),
1
3
3
1.69 and 2.10 (m, 2H), 1.67-1.63 (m, 6H), -1.71 (s, 1H), -1.92 (s, 1H); C NMR (CDCl , 100
MHz): δ 173.58, 169.57, 168.79, 166.78, 154.08, 149.06, 144.71, 138.80, 136.14, 135.08,
134.83, 134.73, 134.49, 130.12, 129.51, 128.60, 121.63, 102.36, 101.26, 98.84, 93.68, 72.78,

ACCEPTED MANUSCRIPT
4
4
28
29
70.47, 69.98, 53.10, 52.19, 51.66, 49.22, 41.05, 40.32, 37.75, 31.11, 29.68, 23.12, 19.73, 17.78,
12.21, 11.39. LC-MS: 755 [M+H].
4
30
Synthesis of chlorin e6-curcumin conjugate (17)
4
4
4
4
4
4
4
4
4
4
4
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4
4
4
4
4
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35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Compound 6 (700 mg, 1.45 mmol) was dissolved in dry CH
2
Cl
2
. A mixture of HOBt (235 mg,
Cl was added,
1.74 mmol), EDCI (333 mg, 1.74 mmol), and DIEA (187 mg, 1.45 mmol) in CH
2
2
the mixture was then allowed to stir for 30 min. Compound 16 (1.09 g, 1.45 mmol) and DIPEA
(187 mg, 1.45 mmol) were mixed in CH Cl and added to this reaction mixture. The mixture was
2
2
stirred overnight. It was diluted with CH Cl and then washed with 5% aqueous citric acid,
2
2
followed by a wash with brine and water. It was dried over anhydrous Na SO and then
2
4
evaporated. The residue was purified by silica gel column chromatography to afford 770 mg of
-1
-1
5
5
17, Yield: 44 %. UV-Vis (DMSO): λmax (ɛ / M cm ) 668 (2.9 X 10 ), 502 (1.5 X 10 ), 406 (7.3
5
1
3
X 10 ) nm. H NMR (CDCl , 400 MHz): δ 9.62 (s, 1H), 9.56 (s, 1H), 8.73 (s, 1H), 8.01 (m, 1H),
7.48 (d, J = 16 H, 1H), 7.37 (d, J = 16 H, 1H), 7.01-6.99 (m, 2H), 6.93-6.83 (m, 4H), 6.71 (d, J
= 8 Hz, 1H), 6.31-6.24 (m, 2H), 6.08 (d, J = 12 Hz, 1H), 5.56-5.48 (m, 2H), 5.25-5.12 (m, 2H),
4.40 and 4.26 (m, 2H), 4.01 (m, 1H), 3.83 (m, 6H), 3.73 (m, 6H),3.64 (m, 2H), 3.59 (s, 3H),
3.49-3.47 (m, 6H), 3.41 (s, 3H), 3.25 (s, 3H), 3.07 (m, 2H), 2.70 (m, 2H), 2.50 (m, 2H), 2.14-
1
3
2.06 (m, 6H), 1.94 (t, J = 4 & 8 Hz, 2H), 1.71-1.58 (m, 8H), -1.71 (s, 1H), -1.92 (s, 1H).
C
NMR (CDCl , 100 MHz): δ 184.36, 181.56, 173.58, 172.01, 170.82, 169.56, 169.00, 166.67,
3
151.03, 147.96, 146.76, 144.78, 140.90, 140.87, 139.08, 136.21, 134.96, 134.84, 134.60, 133.79,
130.35, 129.79, 129.37, 128.27, 127.31, 124.01, 123.00, 122.87, 121.74, 120.76, 114.83, 111.16,
109.54, 102.28, 101.33, 98.84, 93.81, 70.29, 69.55, 55.76, 53.10, 52.26, 51.68, 49.22, 40.39,
38.59, 37.79, 34.26, 31.12, 29.69, 23.12, 20.47, 19.69, 17.76, 12.17, 11.36. LC-MS: 1219
[M+H].

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Synthesis of tert-butyl (3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamate (4)
4
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4
4
4
4
4
4
53
54
55
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59
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A solution of 4,7,10-trioxa-1,13-tridecanediamine (7.5 g, 34.1 mmol) in CHCl (100 mL) was
3
treated with BOC-anhydride (3.7 g, 16.9 mL). The mixture was stirred at room temperature for
12h. The solvent was removed, and the resulting yellow oil was purified by silica gel flash
chromatography
to
produce
the
oil
5.5
g
of
tert-butyl
(3-(2-(2-(3-
1
aminopropoxy)ethoxy)ethoxy)propyl)carbamate (4), Yield: 49 %. H NMR (CDCl , 400 MHz):
3
δ 5.1 (s, 1H), 3.58-3.50 (m, 12H), 3.21 (d, J = 6.9 Hz, 2H), 2.79 (t, J = 8 Hz, 2H), 1.75-1.69 (m,
1
3
4H), 1.59 (s, 2H), 1.42 (s, 9H). C NMR (CDCl , 100 MHz): δ 155.0, 69.2, 68.9, 66.7, 48.0,
3
37.6, 30.4, 28.6, 27.3.
4
61
Synthesis of Ce6-diPEG-NHBoc (18)
4
4
4
4
4
4
4
4
4
4
4
62
63
64
65
66
67
68
69
70
71
72
DME Ce6 8 (1 g, 1.60 mmol) was dissolved in anhydrous CH Cl (50 mL). EDCI (368 mg, 1.92
2
2
mmol) and HOBt (260 mg, 1.92 mmol) were then added and allowed to stir until completely
dissolved under nitrogen. After 30 min, tert-butyl (3-(2-(2-(3-
aminopropoxy)ethoxy)ethoxy)propyl)carbamate (4) (1.28 g, 4 mmol) and DIEA (414 mg, 3.20
mmol) were mixed in CH Cl (20 mL) and added to the reaction mixture. The mixture was
2
2
allowed to stir at room temperature for 12h under nitrogen. The reaction mixture was diluted
with CH Cl (200 mL) and then washed with brine and water, respectively. The organic layer
2
2
was dried over anhydrous Na SO and then evaporated. The product was purified via column
2
4
-1
-1
chromatography to afford 1.2 g of 18, Yield: 81 %. UV-Vis (DMSO): λmax (ɛ / M cm ) 667 (4.2
5
5
5
1
X 10 ), 506 (2.1 X 10 ), 410 (7.9 X 10 ) nm. H NMR(CDCl , 400 MHz ): δ 9.68 (s, 1H), 9.63
3
(s, 1H), 8.79 (s, 1H), 8.07 (m, 1H), 6.37 (dd, J = 20 Hz, 1H), 6.14 (dd, J = 14.8 Hz, 1H), 5.61 (d,

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74
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76
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78
79
J = 20 Hz, 1H), 5.27 (d, J = 16 Hz, 1H), 4.44-4.34 (m, 3H), 4.05 (m, 1H), 3.82-3.72 (m, 8H),
3.63-3.48 (m, 12H), 3.31 (s, 5H), 2.70 (s, 2H), 2.60-2.49 (m, 3H), 2.35 (m, 2H), 2.20-2.09 (m,
1
3
6H), 1.78-1.70 (m, 8H), 1.37 (s, 9H) -1.64 (s, 1H), -1.94 (s, 1H); C NMR (CDCl , 100 MHz):
3
δ 173.55, 169.44, 168.86, 166.76, 154.15, 148.99, 144.79, 138.82, 136.18, 135.01, 134.92,
134.74, 134.62, 130.25, 129.75, 129.35, 128.31, 121.84, 102.15, 101.27, 98.87, 93.69, 79.08,
70.12, 69.67, 69.05, 68.28, 53.07, 52.45, 51.68, 49.07, 39.09, 37.60, 31.06, 29.75, 29.00, 23.14,
19.56, 17.73, 12.18, 11.29. LC-MS: 927 [M+H].
4
80
Synthesis of Ce6-diPEGamine (19)
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
81
82
83
84
85
86
87
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91
92
93
94
95
The compound 18 (1.2 g, 1.52 mmol) was dissolved in of dry CH Cl (30 mL) in an ice bath
2
2
under argon. TFA (5 mL) was added, and the reaction mixture was stirred overnight. The
reaction mixture was evaporated several times with diethyl ether to remove residual TFA. Then
the precipitate was dissolved in CH
NaHCO to remove TFA. The organic layer was dried over anhydrous Na
evaporated to give a crude compound, purified by silica gel chromatography to give 1 g of 19,
2
Cl
2
and washed three times with H
2
O and once with 10%
3
2
SO and then
4
-1
-1
5
5
Yield: 93 %. UV-Vis (DMSO): λmax (ɛ / M cm ) 657 (5.0 X 10 ), 501 (1.5 X 10 ), 400 (2.0 X
6
1
10 ) nm. H NMR(CDCl , 400 MHz ): δ 9.63 (s, 1H), 9.58 (s, 1H), 8.78 (s, 1H), 8.04 (m, 1H),
3
6.33 (dd, J = 16 Hz, 1H), 6.12 (dd, J = 12 Hz, 1H), 5.58 (d, J = 20 Hz, 1H), 5.31 (d, J = 16 Hz,
1H), 4.43 and 4.29 (m, 2H), 3.82-3.76 (m, 5H), 3.63-3.56 (m, 9H), 3.45-3.39 (m, 9H) 3.26-3.22
1
3
(m, 5H), 2.76 (m, 2H), 2.18-2.13 (m, 8H), 1.73-1.61 (m, 8H), -1.76 (s, 1H), -1.96 (s, 1H);
C
NMR (CDCl , 100 MHz): δ 173.55, 169.44, 168.86, 166.75, 154.15, 148.99, 144.79, 138.82,
3
136.18, 135.01, 134.92, 134.74, 134.62, 130.25, 129.75, 129.35, 128.31, 121.84, 102.15, 101.27,
98.87, 93.69, 69.98, 69.67, 68.28, 53.07, 52.45, 51.68, 49.07, 39.09, 37.60, 31.06, 29.75, 23.14,
19.56, 17.73, 12.18, 11.29. LC-MS: 827 [M+H].

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Synthesis of chlorin e6-curcumin conjugate (20)
4
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4
5
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5
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
Compound 6 (500 mg, 1.45 mmol) was dissolved in dry CH Cl . A mixture of HOBt (167 mg,
2 2
1.23 mmol), EDCI (236 mg, 1.23 mmol), and DIEA (133 mg, 1.03 mmol) in CH Cl was added,
2
2
the mixture was then allowed to stir for 30 min. Compound 19 (856 mg, 1.03 mmol) and DIEA
(133 mg, 1.03 mmol) were mixed in CH Cl and added to this reaction mixture. The mixture was
2
2
stirred overnight. It was diluted with CH
2
Cl
2
and then washed with 5% aqueous citric acid,
SO and then
evaporated. The residue was purified by silica gel column chromatography to afford 770 mg of
followed by a wash with brine and water. It was dried over anhydrous Na
2
4
-1
-1
5
5
20, Yield: 41 %. UV-Vis (DMSO): λmax (ɛ / M cm ) 668 (2.7 X 10 ), 502 (1.5 X 10 ), 407 (6.1
5
1
X 10 ) nm. H NMR (400MHz, CDCl ): δ 9.67 (s, 1H), 9.63 (s, 1H), 8.80 (s, 1H), 8.09 (m, 1H),
3
7.53 (d, J = 16 H, 1H), 7.46 (d, J = 16 H, 1H), 7.20 (m, 1H), 7.06-7.04 (m, 2H), 6.96-6.84 (m,
4H), 6.37-6.32 (m, 3H), 6.15 (dd, J = 12 Hz, 1H), 5.65-5.57 (m, 2H), 5.34-5.23 (m, 2H), 4.49
and 4.37 (m, 2H), 4.01 (m, 1H), 3.92-3.89 (m, 4H), 3.82-3.69 (m, 12H), 3.62 (s, 3H), 3.55-3.51
(m, 6H), 3.47 (s, 3H), 3.38-3.36 (m, 2H), 3.31 (s, 3H), 2.89 (m, 2H), 2.64-2.52 (m, 3H), 2.43-
1
3
2.33 (m, 6H), 2.25-2.09 (m, 5H), 1.84-1.69 (m, 9H), -1.66 (s, 1H), -1.89 (s, 1H). C NMR
(CDCl , 100 MHz): δ 184.35, 181.66, 173.54, 171.56, 170.96, 169.28, 168.76, 166.90, 154.04,
3
151.13, 149.07, 147.97, 146.77, 144.74, 140.99, 139.15, 138.75, 136.18, 135.09, 134.80, 134.67,
134.48, 133.87, 130.19, 129.92, 129.43, 128.74, 127.42, 124.09, 123.09, 122.96, 121.71, 121.58,
120.81, 114.81, 111.26, 109.55, 102.42, 101.44, 101.23, 98.80, 93.72, 70.30, 69.99, 69.12,
55.86, 53.10, 52.14, 51.67, 49.19, 39.11, 37.72, 34.84, 32.91, 31.16, 29.67, 29.27, 28.15, 23.09,
20.77, 19.71, 17.81, 12.20, 11.38. LC-MS: 1291 [M+H].
5
17

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Measurement of Cellular uptake
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19
20
21
22
23
24
To examine the cellular permeability of the synthesized compounds AsPC-1 cells were used.
5
Briefly, exponentially growing 2 x 10 cells were seeded in six well plates and incubated in dark
o
at 37 C in a CO incubator. After 24h, media was removed and cells were incubated with
2
various synthesized compounds for a different time intervals. Once the incubation was
completed, the cells were collected by trypsinization and washed thrice with phosphate buffered
saline (PBS). Thereafter, the cells were centrifuged and the pellets were dissolved in 500 µl of
5
5
25
26
PBS to carry out FACS.
Cell Viability Assay
5
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38
AsPC-1 pancreatic cancer cells were grown in RPMI-1640 medium (life technologies
corporation, USA) supplemented with 10% fetal bovine serum (life technologies corporation,
USA) and 1% Penicillin & Streptomycin (life technologies corporation, USA), whereas MIA-
PaCa2 and PANC-1 cells were grown in DMEM supplemented with 10% fetal bovine serum
(life technologies corporation, USA) and 1% Penicillin & Streptomycin (life technologies
3
corporation, USA). Exponentially growing 5.0 x 10 cells were seeded in 96 well plates and
o
incubated overnight at 37 C in a CO incubator. Next morning the cells were treated with
2
different concentrations of the synthesized photosensitizers for 3h. After 3h, the cells were
2
irradiated with laser light 660 nm, 50 mW, 9 J/cm . The cells were then incubated for 72h in dark
0
at 37 C in a 5% CO
2
incubator. For the evaluation of dark toxicity of the synthesized
compounds, the cells were kept under similar conditions without laser irradiation. After 72h,
MTT assay was performed for both the groups to determine cell viability.
5
39
.

ACCEPTED MANUSCRIPT
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40
Annexin V and Propidium Iodide Staining
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43
44
45
46
47
To determine the mechanism of cell death annexin v and propidium iodide staining was carried
5
o
out. Briefly, 2 x 10 cells were seeded in six well plates and incubated at 37 C in a CO
2
incubator. After 24h, media was removed and the cells were treated with compound 17. After 3h
2
o
cells were irradiated with the light (660 nm, 50 mW, 9 J/cm ) and incubated for 24h at 37 C in a
CO incubator. Thereafter, cells were collected, washed three times with PBS and dissolved in
2
the FACS buffer. Annexin V and PI staining was then carried out with help of FACS assay kit
(BD Pharmigen) as per the manufacturer’s protocol.
5
48
Western Blot
5
5
5
5
5
49
50
51
52
53
For western blot cells were seeded in six well plates and treated with compound 17. After 3h,
2
cells were irradiated with the laser light (660 nm, 50 mW, 9 J/cm ) and incubated for additional
24h. Then the cells were washed with PBS, lysed on ice in modified RIPA buffer (50 mM Tris–
HCl pH 7.4, 1% NP-40, 0.25% Sodium Deoxycholate, 150 mM NaCl, 1 mM Na VO , and 1 mM
3
4
NaF) containing protease inhibitors (100 µM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin,
5
5
5
5
5
5
54
55
56
57
58
59
10 µg/ml pepstatin, and 2 mM EDTA). The lysates were centrifuged at 10,000g for 10 min at 4
o
C, and the supernatant fractions were collected. The proteins were separated on 10 % SDS-
PAGE and transferred to Immobilon P membranes (Millipore Corp., Bedford, MA, USA). The
specific proteins were detected using an enhanced chemiluminescence (ECL) Western blotting
kit (Amersham, GE Healthcare) according to the manufacturer’s instructions.
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