Impact of Substituents in Tumor Uptake and Fluorescence Imaging Ability of Near-Infrared Cyanine-like Dyes

Article abstract of DOI:10.1111/php.12482

This report presents a simple strategy to introduce various functionalities in a cyanine dye (bis-indole-N-butylsulfonate-polymethine bearing a fused cyclic chloro-cyclohexene ring structure), and assess the impact of these substitutions in tumor uptake, retention and imaging. The results obtained from the structural activity relationship (SAR) study demonstrate that certain structural features introduced in the cyanine dye moiety make a remarkable difference in tumor avidity. Among the compounds investigated, the symmetrical CDs containing an amino-phenyl thioether group attached to a cyclohexene ring system and the two N-butyl linkers with terminal sulfonate groups in benzoindole moieties exhibited excellent tumor imaging ability in BALB/c mice bearing Colon26 tumors. Compared to indocyanine green (ICG), approved by FDA as a blood pooling agent, which has also been investigated for the use in tumor imaging, the modified CD selected on the basis of SAR study produced enhanced uptake and longer retention in tumor(s). A facile approach reported herein for introducing a variety of functionalities in tumor-avid CD provides an opportunity to create multi-imaging modality agent(s). Using a combination of mass spectrometry and absorbance techniques, the photobleaching of one of the CDs was analyzed and significant regioselective photooxidation was observed.

Full text of DOI:10.1111/php.12482

Photochemistry and Photobiology, 2015, 91: 1219–1230
Impact of Substituents in Tumor Uptake and Fluorescence Imaging Ability
of Near-Infrared Cyanine-like Dyes
Nayan J. Patel1,2, Ethirajan Manivannan2, Penny Joshi2, Tymish J. Ohulchanskyy3, Roger R. Nani4,
Martin J. Schnermann4 and Ravindra K. Pandey1,2
*
1Department of Molecular Pharmacology and Cancer Therapeutics, Cell Stress Biology Roswell Park Cancer Institute,
Buffalo, NY
2PDT Center, Cell Stress Biology Roswell Park Cancer Institute, Buffalo, NY
3Institute for Lasers, Photonics and Biophotonics SUNY at Buffalo, Buffalo, NY
4Chemical Biology Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD
Received 18 January 2015, accepted 9 June 2015, DOI: 10.1111/php.12482
ABSTRACT
using a light source to excite the fluorophore. Light of a longer
wavelength is then emitted by the fluorophore and this emitted
light can provide diagnostic information based on its localization
or state. Unfortunately, limited penetration of light into tissue as
a result of it attenuation due to scattering and absorption has
been the major hindrance in the translation of this imaging tech-
nique into the clinic (3).
This report presents a simple strategy to introduce various
functionalities in a cyanine dye (bis-indole-N-butylsulfonate-
polymethine bearing a fused cyclic chloro-cyclohexene ring
structure), and assess the impact of these substitutions in
tumor uptake, retention and imaging. The results obtained
from the structural activity relationship (SAR) study demon-
strate that certain structural features introduced in the cya-
nine dye moiety make a remarkable difference in tumor
avidity. Among the compounds investigated, the symmetrical
CDs containing an amino-phenyl thioether group attached to
a cyclohexene ring system and the two N-butyl linkers with
terminal sulfonate groups in benzoindole moieties exhibited
excellent tumor imaging ability in BALB/c mice bearing
Colon26 tumors. Compared to indocyanine green (ICG),
approved by FDA as a blood pooling agent, which has also
been investigated for the use in tumor imaging, the modified
CD selected on the basis of SAR study produced enhanced
uptake and longer retention in tumor(s). A facile approach
reported herein for introducing a variety of functionalities in
tumor-avid CD provides an opportunity to create multi-imag-
ing modality agent(s). Using a combination of mass spectrom-
etry and absorbance techniques, the photobleaching of one of
the CDs was analyzed and significant regioselective photooxi-
dation was observed.
Imaging modalities such as magnetic resonance imaging
(MRI), single photon emission computed tomography (SPECT)
and positron emission tomography (PET), computed tomography
(CT) are considered as far better techniques for providing deep
tissue information (4) than fluorescence imaging. However, these
imaging techniques lack real-time imaging capabilities which
would be extremely beneficial during surgery in differentiating
malignant from normal tissues and, thereby ensure complete
tumor removal (5,6). Intraoperative imaging is not performed
with most imaging modalities due to the time of acquisition and
data processing/interpretation. A major benefit of fluorescence
optical imaging offers the ability for intraoperative imaging or
image-guided surgery as image acquisition does not interfere
with the treatment process and acquisition/data processing are
instantaneous (7). Improved fluorophores for fluorescence optical
imaging are now needed and are currently under development.
The sensitivity of fluorescence detection is often limited by
autofluorescence of biologic samples. As the extinction wave-
length becomes longer the autofluorescence decreases, and hence
detectability over background increases. For fluorescence based
image-guided surgery development, an ideal fluorophore should
be nontoxic, target selective and possess near-infrared (NIR)
absorption and fluorescence to easily distinguish the compound
from tissue and other endogenous absorbers of light such as
water, lipids and hemoglobin (4). These required NIR character-
istic limits the use of many dyes that are often used in research
such as BODIPY, DAPI, FITC, etc. The two most commonly
used classes of NIR fluorophores are the organic dyes and
nanoparticles. Organic dyes tend to be more biocompatible and
do not use potentially toxic heavy metals such as cadmium, tel-
lurium and selenium that fluorescent quantum dots use (8).
Among various NIR organic dye fluorophores, the heterocyclic
polymethine dyes (cyanine dyes or CDs) have created enormous
interest due to their desirable photophysical properties (long
wavelength absorption and fluorescence, large Stokes shift and
INTRODUCTION
Fluorescence optical imaging is a clinically underutilized imaging
modality which in the last decade has seen rapid adoption in the
field of oncology (1). Among the imaging modalities currently
being used in tumor detection, none offer the versatility and pro-
mises of optical imaging. Historically, fluorescence imaging has
almost exclusively been used in academic research for tracing
cellular movements along with understanding expression levels
and reaction rates of proteins (2). Fluorescence optical imaging
works by first administering an exogenous biologically compati-
ble fluorescent molecule (fluorophore) to the system and then
*Corresponding author email: ravindra.pandey@roswellpark.org (Ravindra K.
Pandey)
© 2015 The American Society of Photobiology
1219

1220 Nayan J. Patel et al.
measurable dimensions of approximately 4 mm 9 4 mm, the mice were
injected intravenously with the fluorophore(s) and imaged at regular inter-
vals, and the whole body images were (including tumors) were acquired.
high extinction coefficient) (9). The FDA approved heptamethine
indocyanine green (ICG) has an extremely high binding affinity
to albumin and therefore is used primarily for its diagnostic
capability in blood flow and determination of cardiac and hepatic
function (10). However, ICG lacks tumor selectivity and there-
fore has limited utility in clinical oncology (11). Several reports
have successfully demonstrated methods to improve the tumor-
avid ability of ICG analogs by conjugating them with certain tar-
geting groups such as monoclonal antibodies (12,13), peptides
(14,15) and folic acid (16) or formulated into nanoparticles
(17,18). These approaches have been effective but it is our belief
that a rational drug design and structural activity relationship
studies (SAR) could be extremely useful in developing tumor
selective fluorophores.
Drug formulation.
1% Tween 80/D5W. All dyes from this study
(with the exception of ICG) were formulated in 1% Tween 80/D5W.
Amount of compound and volume of solution were calculated for desired
concentration. Final solution was made to be a 1% Tween 80/D5W solu-
tion. Compound and necessary volume of tween 80 were added to a mor-
tar and mulled to a paste. The paste was allowed to sit overnight and the
next day the calculated amount of D5W was added to the paste and
mixed. Solution was filtered through a 0.2 lm syringe filter and concen-
tration was measured spectrophotometrically. Drug formulations were
stored at 4°C, when not in use.
Indocyanine green formulation. Stock solution of ICG was made by
dissolving ICG powder (Sigma Aldrich, St. Louis, MO) in ddH2O. The
formulation was filtered through a 0.2 lm syringe filter and stored at
4°C. The desired concentration(s) of the dye was obtained by diluting the
stock solution with PBS.
The objective of our study was to prepare a series of CDs and
investigate the impact of various substituents in tumor retention,
imaging ability and to select a dye with desired multifunctional
groups for developing cancer targeted and dual imaging (fluores-
cence-PET, fluorescence-MRI) agents. For our initial study, the
commercially available IR820 was synthesized via the
Vilsmeier–Haack reaction. The key feature of this particular CD
is the presence of a centrally located six member carbocyclic
system within the heptamethine chain, which improves the rigid-
ity and the stability of the dye (Fig. 1) (19). In addition, the
presence of chloro-, bromo- or iodo groups (halogens) at the
meso (or “position-a”) position of the dyes allows for several
established chemical methods of functionalization, which could
lead to improved solubility, target specificity and extended
conjugation.
Photophysical characterization.
UV-visible spectroscopy. UV-Vis
absorption spectra of the fluorophores were obtained using a Shimadzu
UV-3600 spectrophotometer by diluting drug solutions in methanol to a
final concentration of 5 lM. For in vitro photobleaching studies, the dyes
(5 lM) in methanol were irradiated with 532 nm laser light at 125 mW
over time and the fluorescence spectra were recorded using a Fluorolog-3
spectrofluorometer (532 nm excitation, 800 nm and longer emission with
2 nm slit).
Cell culture. All cells were maintained in a humidified incubator at
37°C in atmosphere of 5% CO2 at 37°C and harvested in log phase of
growth at 70–90% confluence.
Murine colon 26 carcinoma cells. Cells were cultured and maintained
in sterile RPMI-1640, containing 19 L-glutamine, supplemented with
10% Fetal Calf Serum (FCS) (Atlanta Biologicals, triple 0.1 lm filtered,
Lawrenceville, GA) and 1% Penicillin/Steptomycin/L-glutamine (P/S/l-G
10 000
I U mLꢀ1
penicillin,
10 000 lg mLꢀ1
streptomycin,
29.2 mg mLꢀ1 L-glutamine).
ABCG2 expressing HEK-293 482R. Cells were derived in the lab of
(late) Dr. Janet Morgan at Roswell Park Cancer Institute, Buffalo, NY
and cultured in DMEM supplemented with 10% FCS, 2 mM l-glutamine,
penicillin (10 IU mLꢀ1), streptomycin (10 lg mLꢀ1), 2 mg mLꢀ1 G418
sulfate (Mediatech, Inc. Manassas VA).
ABCG2 efflux activity assay. HEK-293 cells cultured in selection
media were harvested with trypsin, filtered through 30 lm mesh and
seeded at low density (400 000 cells) in 5 mL sterile polystyrene tubes.
Samples were either blocked with the 10 lM ABCG2 inhibitor Imatinib
mesylate (Novartis Pharmaceuticals, Basel Switzerland) or vehicle for
30 min. All samples were performed in triplicate. Samples were then
incubated with 0.5 lM cyanine dye for 4 h at 37°C. Previously validated
ABCG2 substrate dye N-butyl-O-butyl-bacteriopurpurin was used a posi-
tive control. Tubes were placed on ice to inhibit any further 9 dependent
efflux activity and immediately analyzed by flow cytometry (Ex. 785
Em. 830 LP).
The new compounds were characterized by 1H-NMR and
mass spectrometry analyses, and their tumor uptake and imaging
abilities were determined using BALB/c mice bearing Colon 26
tumor. To the best of our knowledge, these studies are the first
to examine how relatively simple changes to the chemical struc-
ture of heptamethine cyanines alters tumor avidity. The SAR that
emerged from these studies clearly demonstrate that tumor
avidity can be dramatically improved with modest structural
modifications.
MATERIALS AND METHODS
Animals. All animals used in experiments were housed and cared for
under the strict guidelines of the Institutional Animal Care and Use Com-
mittee (IACUC) in the Department of Laboratory and Animal Resources
(DLAR) core facility at Roswell Park Cancer Institute (Buffalo, NY).
Animals were fed with a standard laboratory diet and water ad libitum
and monitored daily.
Animals used in this study were BALB/cAnNCr mice obtained from
Fredrick National Laboratory (Fredrick, MD). Eight to twelve-weeks old
animals were inoculated subcutaneously with 1 9 106 colon 26 cells on
the right shoulder where the fur was removed. When tumors reach
In vivo fluorescence optical imaging. For all imaging experiments,
three mice per drug were injected i.v. with final concentration of each
dye was 0.3 lmol kꢀ1 body weight of the mice.
Maestro GNIR FLEX. For the CDs 9–10 and 14–19, in vivo imaging
was carried out using the Maestro GNIR FLEX (Cri Inc. Woburn, MA)
spectral imaging system. Mice were anesthetized by inhalation of isoflu-
rane (2% in oxygen) and fluorescence spectral cubes were acquired using
NIR (illumination light from 780 nm to 950 nm in 10 nm steps at 2 s
exposure for each step Ex. 710 nm to 760 nm, Emission 800 nm long-
pass) preset filter combinations. Unmixed images, in which background
signals were subtracted and quantified by using, built in Maestro soft-
ware. ROIs (region of interests) were manually selected over various sites
over mice and average signal was analyzed.
Nuance camera. Imaging for CDs 17, 21, 23 and 25 was performed
using a 12 bit Nuance camera (CRI, Worburn, MA) imaging system.
Mice were anesthetized by i.p. injection of ketamine/xylene mixture
(100/10 mg kgꢀ1) and images were acquired for 5 s with excitation
782 nm emission 800/830 nm long-pass filters over various time points.
IVIS spectrum. Epi-fluorescence imaging of CD 16 and ICG was car-
ried out using the IVIS Spectrum (Perkin-Elmer Waltham, MA) imaging
system. Mice were anesthetized by inhalation of isoflurane (2% in oxy-
gen) and fluorescence images were obtained using the Ex. 745 ꢁ 15 nm,
Em. Of 840 ꢁ 10 nm filter combination. The Living Image software
Figure 1. General structure of a NIR cyanine dye.

Photochemistry and Photobiology, 2015, 91 1221
(Perkin-Elmer Waltham, MA) was used to process data. Circular regions
of interest (ROI) were defined around the tumor and fluorescent intensity
was recorded as the average radiant efficiency ((p sꢀ1 cmꢀ2 srꢀ1)/
(lW cmꢀ2)).
Postacquisition image processing. Maestro and Nuance Camera
unmixed images were processed using ImageJ 1.44p (NIH) software.
Grayscale images were enhanced using Lookup Tables setting Royal and
brightness was adjusted using Image Adjust setting of Brightness/Con-
trast. All images shown were set to different scales.
Statistical analysis. All data were analyzed using Graph Pad Prism 5
software (GraphPad Software, San Diego, CA). To test for differences
between two groups with normal distribution, a two-tailed Student’s t-test
(unpaired) was used, error bars represent ꢁ standard deviation of mean,
the confidence intervals set at 95%.
Chemistry. All reactions were carried under an inert atmosphere using
heat gun dried glassware. The reaction mixture(s) was stirred using a
magnetic stirrer. Thin-layer chromatography (TLC) was done on pre-
coated silica gel sheets (layer thickness: 0.25 mm) or aluminum oxide
sheets. Column chromatography was performed either over silica gel 60
(70–230 mesh) or neutral alumina grade III. In some cases preparative
TLC was used for the purification of compounds. Solvents were dried
following the standard methodology. 1H NMR spectra were recorded at
room temperature in CDCl3 solution using a Varian VNMRS-400 spec-
trometer. All chemicals shifts are reported in parts per million (d). 1H
NMR (400 MHz) spectra were referenced to residual CHCl3 (7.26 ppm)
or TMS (0.00 ppm). Mass spectrometry analyses were performed at the
Mass Spectrometry Facility, Michigan State University, East Lansing.
UV-visible spectra were recorded on FT UV-visible spectrophotometer
using dichloromethane/THF as solvent.
General procedure for the synthesis of NIR cyanine dyes. Commer-
cially available CD 9 (IR820) was purchased from Sigma Aldrich, St.
Louis, MO, while the CDs 10, 15 and 16 were prepared according to the
published procedure (20–22). In general, the overall synthesis of cyanine
dyes involves three main steps (2.9.1–2.9.3) and was carried out using
modified reported procedure (23).
starting chloro cyanine disappears. The reaction mixture was then
cooled to room temperature. The reaction product obtained after
removing the water (under vacuum) was suspended in ether, and the
product was filtered and was further purified by silica gel column
chromatography (10% MeOH-DCM) to afford the desired CD 21.
Synthesis of the cyanine dye 11. The synthetic steps 1 and 2 as dis-
cussed above were followed by reacting compound 5 (obtained from
cyclohexanone 3) with benzoindolium salt 8, and the title compound was
isolated in 46% yield after following the standard procedure. UV-vis kmax
(in MeOH): 820 nm; 1HNMR (400 MHz, CD3OD): d 8.60 (d, 2H,
J = 14 Hz), 8.26 (d, 2H, J = 10 Hz), 7.99 (m, 4H), 7.62–7.69 (m, 5H),
7.49 (t, 2H, J = 7.2 Hz), 7.32 (t, 2H, J = 7.2 Hz), 7.10 (m, 1H), 6.43 (d,
2H, J = 14 Hz), 4.35 (t, 4H, J = 7.6 Hz), 3.22 (dd, 1H, J = 4, 12 Hz),
2.82–3.00 (m, 8H), 1.87–2.15 (m, 22H),. EIMS (m/z): 991 (M+ +2Na).
HRMS: Calcd. For C53H56ClN3O7S: 945.3248. Found: 945.3276.
Synthesis of cyanine dye 12. The cyclohexanone 3 was converted to
the corresponding intermediate 7, by following the general procedure
(step 1). It was dissolved in N, N-dimethyl formamide and reacted with
benzoindolinium salt 8 to afford the title compound in 42% yield. UV-
vis kmax (in MeOH): 820 nm, 1HNMR (400 MHz, CD3OD): d 8.55 (d,
2H, J = 14 Hz), 8.26 (d, 2H, J = 8.4 Hz), 8.02 (d, 2H, J = 10.8 Hz),
7.98 (d, 2H, J = 8.4 Hz), 7.79 (d, 2H, J = 9.2 Hz), 7.63 (dt, 2H,
J = 8.4, 1.2 Hz), 7.48 (dt, 2H, J = 7.6, 0.8 Hz), 6.41 (d, 2H,
J = 14 Hz), 4.37 (t, 4H, J = 7.2 Hz), 3.15–3.18 (m, 2H), 2.90 (t, 4H,
J = 7.6 Hz), 2.67–2.76 (m, 3H), 2.08–2.12 (m, 4H), 2.03 (s, 12H), 1.96–
2.00 (m, 4H),. EIMS (m/z): 915 (M+ +2Na); HRMS: Calcd. For
C47H50N2O8S2Cl: 869.2697. Found: 869.2659.
Synthesis of cyanine dye 14. It was prepared by reacting 9 with thio-
phenol 13 following the general procedure discussed above (step 3) in
78% yield. UV-vis kmax (in MeOH): 831 nm (2 = 157 000 cmꢀ1);
1HNMR (400 MHz, CD3OD): d 8.90 (d, 2H, J = 14 Hz), 8.14 (d, 2H,
J = 10 Hz), 7.95 (t, 4H, J = 10 Hz), 7.56–7.64 (m, 5H), 7.44 (t, 2H,
J = 7.2 Hz), 7.33 (m, 3H), 7.10 (m, 1H), 6.36 (d, 2H, J = 14 Hz), 4.29
(t, 4H, J = 7.6 Hz), 2.84–2.94 (m, 8H), 1.97–2.15 (m, 10H), 1.79 (s,
12H). EIMS (m/z): 946 (M+ +2Na); HRMS: Calcd. For C52H56N2O6S3:
901.3379. Found: 900.3362.
Step-1: A solution of N, N-Dimethyl formamide (1 mmole) in
dichloromethane (10 mL) was stirred at 0°C and was added phospho-
rus oxychloride (1 mmole) drop wise over a period of 15 min. After
stirring for an additional hour at 0°C, cyclohexanone (1–3) derivative
(2 mmole) was added to the reaction vessel. The entire mixture was
heated under reflux for 1 h, then cooled and treated with a mixture of
aniline and ethanol (1:1). For 7, N,N-Dimethyl formamide was used
instead of the combination of aniline and ethanol. Stirring was contin-
ued for an additional 30 min, and then the resulting solution was
poured onto a mixture of crushed ice and concentrated hydrochloric
acid. The crude mixture was kept at 4°C for overnight; the resulting
crystalline product was filtered. The precipitate was washed with cold
water and ether, and finally dried under a reduced pressure; yielded
the corresponding cyclohexene analogs 4–7 in >60% yield.
Step-2: Indolium salt 8 (2 mmol), Vilsmeier–Haack reagent (1 mmol)
and anhydrous sodium acetate (4 mmol) were dissolved in absolute etha-
nol (25 mL) and the entire mixture was stirred at room temperature for
12 h under a nitrogen atmosphere. The intermediate 7 showed limited
solubility in ethanol, and therefore, N, N-dimethylformamide was used as
a solvent. The residue obtained after removing the solvent on a rotary
evaporator, was treated with ether (drop by drop). The product so
obtained was filtered, and the crude dye was further purified by Silica Gel
column chromatography, eluting with dichloromethane/methanol
(90:10) to afford the pure cyanine dye(s) 9–12 in modest yield (40–55%).
Step-3: A solution of cyanine dye (1 mmol) and thiophenol deriva-
tive (10 mmol) in anhydrous N,N-dimethylformamide (20 mL) under
a nitrogen atmosphere was stirred at room temperature for 12 h. The
solvent was removed on a rotary evaporator and the crude obtained
was triturated with diethyl ether to precipitate the thiol substituted
cyanine dye, which was further purified by silica gel chromatography,
eluting with dichloromethane/methanol (90:10) to obtain the pure dye
(s) 14–19 in the range 60–80% yield.
Synthesis of cyanine dye 17. Compounds 10 with 13 (4-aminophenylth-
iol) were reacted by following the methodology as discussed above (step 3)
and the title compound was obtained in 73% yield; UV-vis kmax (in
MeOH): 829 nm, 1HNMR (400 MHz, CD3OD):
d 9.00 (d, 2H,
J = 14 Hz), 8.22 (d, 2H, J = 14 Hz), 7.91–8.02 (m, 4H), 7.58–7.63 (m,
4H), 7.44 (t, 2H, J = 7.2 Hz), 7.10 (d, 2H, J = 8.4 Hz), 6.62 7.10 (d, 2H,
J = 8.4 Hz), 6.38 (d, 2H, J = 14 Hz), 4.34 (t, 4H, J = 7.6 Hz), 4.22 (q,
2H, J = 8.0 Hz), 2.82–2.94 (m, 2H), 1.95–2.08 (m, 10H), 1.85 & 1.82 (s,
12H), 1.24 (t, 3H, J = 7.2 Hz). EIMS (m/z): 1032 (M+ +2Na); HRMS:
Calcd. For C55H61N3O8S3: 987.3621. Found: 987.3570.
Synthesis of cyanine dye 18. Synthesized by following the procedure
discussed above (step 3) by reacting dye 11 with 13 (4-aminophenylthiol)
in 77% yield; UV-vis kmax (in MeOH): 825 nm, 1HNMR (400 MHz,
CD3OD): d 9.02 (d, 2H, J = 14 Hz), 8.18 (d, 2H, J = 8.4 Hz), 8.02 (d,
2H, J = 10.8 Hz), 7.97 (t, 2H, J = 8.4 Hz), 7.67 (d, 2H, J = 9.2 Hz),
7.60–7.63 (m, 4H), 7.48 (dt, 2H, J = 7.6, 0.8 Hz), 7.32 (t, 2H,
J = 7.6 Hz), 7.14 (d, 2H, J = 8.8 Hz), 6.68 (d, 2H, J = 8.8 Hz), 6.39 (d,
2H, J = 14 Hz), 4.29 (t, 4H, J = 7.2 Hz), 3.15–3.21 (m, 2H), 2.80–2.96
(m, 3H), 2.85 (t, 4H, J = 7.6 Hz), 1.92–2.12 (m, 4H), 1.98 (s, 6H), 1.83
(s, 6H).. EIMS (m/z): 1079 (M+ +2Na); HRMS: Calcd. For
C59H62N4O7S3: 1034.3781. Found: 1034.3770.
Synthesis of cyanine dye 19. The desired dye was obtained by reacting
12 with 13 (4-aminophenylthiol) in 80% yield (for a general procedure,
see step 3). UV-vis kmax (in MeOH): 825 nm, 1HNMR (400 MHz,
CD3OD): d 8.43 (d, 2H, J = 14 Hz), 8.15 (d, 2H, J = 8.4 Hz), 7.92 (d,
2H, J = 8.8 Hz), 7.88 (d, 2H, J = 10.8 Hz), 7.59 (d, 2H, J = 8.8 Hz),
7.53 (dt, 2H, J = 8.4, 1.2 Hz), 7.37 (dt, 2H, J = 7.6, 0.8 Hz), 6.91 (d,
2H, J = 8.4 Hz), 6.55 (d, 2H, J = 9.2 Hz), 6.31 (d, 2H, J = 14 Hz), 4.26
(t, 4H, J = 7.0 Hz), 3.13–3.17 (m, 2H), 2.82 (t, 4H, J = 7.6 Hz), 2.55–
2.63 (m, 3H), 1.86–2.02 (m, 8H), 1.92 (s, 12H).. EIMS (m/z): 915 (M+
+2Na); HRMS: Calcd. For C53H58N3O8S3: 960.3381. Found: 960.3410.
Synthesis of cyanine dye 21. Following the methodology presented
above (step 4), the CD 10 and 4-aminoboronic acid 20 were reacted and
the title CD was obtained in 48% yield; UV-vis kmax (in MeOH):
787 nm, 1HNMR (400 MHz, CDCl3): 8.07 (d, 2H, J = 8.0 Hz), 7.94 (t,
4H, J = 8.0 Hz), 7.53–7.61 (m, 6H), 7.40–7.44 (m, 2H), 6.98–7.01 (m,
4H), 6.23 (d, 2H, J = 12 Hz), 4.21–4.27 (t, 6H), 3.02–3.05 (m, 4H),
Step 4: (Used only for the preparation of CD 21): To a dry flask, the
dye 10 (1.0 mmol), 4-carboxyphenylboric acid (20) (1.8 mmol) and
degassed water (10 mL) was added. The reaction mixture was heated
under reflux in the presence of Pd(PPh3)4 (0.065 mmol) for 12 h.
The reaction progress was monitored by visible/near-infrared spec-
troscopy for aliquots diluted with methanol until absorption of the

1222 Nayan J. Patel et al.
2.86–2.92 (m, 6H), 1.90–2.03 (m, 10H),1.59 (s, 12H), 1.27–1.32 (m,
3H). EIMS (m/z): 1000 (M+ +2Na); HRMS: Calcd. For C55H62N3O8S2
(M+ 2): 956.3973. Found: 956.4007.
Table 1. Photophysical properties of cyanine dyes in equimolar concen-
trations (solvent: methanol). Extinction coefficients values (molar absorp-
tivity) are presented at the longest wavelength absorption of each dye.
Synthesis of cyanine dye 23. By following the procedure discussed in
step 1, compound 2 was first converted to 6, which on reacting with 22
(step 2), gave the title compound in 51% yield; UV-vis kmax (in MeOH):
816 nm; 1HNMR (400 MHz, CDCl3): d 8.52 (d, 2H, J = 14 Hz), 8.23
(d, 2H, J = 8.8 Hz), 7.95–8.0 (m, 4H), 7.68 (d, 2H, J = 8.8 Hz), 7.61 (t,
2H, J = 7.2 Hz), 7.46 (t, 2H, J = 7.2 Hz), 6.41 (d, 2H, J = 14 Hz), 4.52
(t, 4H, J = 7.2 Hz), 4.19 (q, 2H, J = 7.2 Hz), 3.28–3.30 (m, 3H), 3.05–
3.30 (m,2H), 2.90–2.94 (m, 2H), 2.69 (t, 4H, J = 7.6 Hz),1.99 (s, 12H),
1.27 (t, 3H, J = 7.2 Hz). EIMS (m/z): 816 (M+ 2Na). HRMS: Calcd. For
C47H48N2O6Cl (M+ 1): 771.3201. Found: 771.3218.
Synthesis of cyanine dye 25. The cyanine dye 23 was reacted with
thiol 24 by following the general procedure discussed above (step 3) and
the title compound was isolated in 55% yield; UV-vis kmax (in MeOH):
827 nm; 1HNMR (400 MHz, CDCl3): 8.95 (d, 2H, J = 14.4 Hz), 8.16
(d, 2H, J = 8 Hz), 7.95 (t, 4H, J = 8.0 Hz), 7.56–7.64 (m, 4H), 7.43 (t,
2H, J = 7.2 Hz), 7.08 (d, 2H, J = 8.0 Hz), 6.65 (d, 2H, J = 8.0 Hz),
6.44 (d, 2H, J = 16 Hz), 4.49 (t, 4H), 4.17 (q, 2H, J = 8.0 Hz), 2.97–
3.09 (m, 5H), 2.65–2.74 (m, 4H),1.87 & 1.81 (s, 12H), 1.27 (t, 3H,
J = 7.2 Hz). EIMS (m/z): 905 (M+ 2Na). HRMS: Calcd. For
C53H54N3O6S (M+ 1): 860.3733. Found: 860.3748.
General plate irradiation procedure. A stock solution of 21a (5 mM
DMSO) was diluted in water to a final concentration of 20 lM. Samples
in 96-well plates (Corning UV-transparent acrylic copolymer) were irradi-
ated using a 780 nm LED ꢁ 20 nm (L690-66-60, Marubeni America
Co.) at a light intensity of 15 mW cmꢀ2 as measured using a power
meter. Wells containing 300 lL of the 20 lM solution were irradiated
and analyzed at 5 min intervals by single point absorption (825 nm).
Experiments were run in duplicate and plotted with error bars derived
from the standard deviation (<5% in all cases).
Procedure for LC/MS relative Ion analysis of photolysis of 21a. Leav-
ing the solution of compound 19 containing a –COOH functionality in
the central cyclohexene ring in methanol converted it the corresponding
methyl ester 21a. A stock solution of 21a (5 mM DMSO) was diluted in
water to a final concentration of 40 lM. Phenylalanine (40 lM) was
used as an internal standard. The photolysis was run for 60 min at rt with
20 mW cmꢀ2 780 nm (ꢁ20 nm) light in a 1.5 mL HPLC vial. At
time = 0 (prior to irradiation) and 60 min the samples were analyzed by
a direct loop injection method with a Shimadzu LCMS-2020 Single
Quadrupole instrument (normal resolution). The relative ion counts
were calculated by integrating the extracted ion chromatogram (EIC) of
the m/z of 21a, oxindole 26, analogue 28, and corresponding carbonyl 27
and 29, and then dividing by the ion count of the phenylalanine internal
standard.
Molar
Abs.
Fluor.
absorptivity
Compound (kmax, nm) (kmax, nm)
Stokes shift
(Mꢀ1 cmꢀ1)
9
820
816
831
837
830
829
825
835
788
816
827
854
854
872
876
878
866
864
870
813
852
859
34 nm or 485 cmꢀ1 184 000
38 nm or 545 cmꢀ1 194 300
41 nm or 565 cmꢀ1 157 000
39 nm or 532 cmꢀ1 197 000
38 nm or 659 cmꢀ1 207 500
37 nm or 515 cmꢀ1 217 800
39 nm or 547 cmꢀ1 226 000
35 nm or 482 cmꢀ1 203 000
25 nm or 390 cmꢀ1 190 000
36 nm or 518 cmꢀ1 175 000
32 nm or 450 cmꢀ1 180 000
10
14
15
16
17
18
19
21
23
25
were performed. The main objectives of this study were (1) to
determine the optimal time of accumulation of the dye within the
tumor; (2) to determine clearance of the dye from the tumor with
time; (3) to evaluate tumor specificity (e.g. uptake in tumor vs
surrounding tissues tissue, and finally; and (4) to monitor the
clearance of the dye from the liver and other organs. For imag-
ing studies, a single filter set was used to capture images. How-
ever, due to significant differences in the absorption maxima of
the dyes synthesized, and the instrumentation limitations to
excite the wavelength at the optimal maxima of each CD, direct
comparison of one dye over the other could not be performed.
Therefore, analysis for selectivity was based on the relative
intensities of the fluorescence signal through the body and more
specifically, to those measured in tumor, liver and distal skin.
This selectivity was determined by injecting a single dose of
0.3 lmol kgꢀ1 compound in groups of 3 BALB/c mice bearing
colon 26 tumors and observing uptake at various time points post
injection.
To determine the impact of various substituents on the tumor
imaging ability of heterocyclic heptamethine dyes, a variety of
functionalities were introduced at position-a, position-b (cyclo-
hexene moiety) and position-c (Fig. 1). A known methodology
was followed for the preparation of CDs 9 and 10. Attempts to
convert the ethyl ester functionality at position-b of the CD
under acidic conditions (under basic conditions, the CD was not
stable) produced decomposition products, and the desired analog
was isolated in a poor yield. Therefore, we used another strategy
and the substituted cyclohexane 2 was reacted with aniline and
phosphorous oxychloride (POCl3) in dimethylformamide (DMF),
which gave the corresponding Schiff base intermediates
(Scheme 1), which on reacting with indolium salt 8, afforded the
desired cyanine dyes 10 and 12 in modest yields. To avoid the
formation of side products associated with reacting cyclohexane
3 with aniline, cyclohexane 3 was reacted with POCl3 in DMF
to yield the intermediate 7, which on subsequent reaction with
NaOAC in DMF gave CD 12. To investigate the impact of
chloro vs phenyl or substituted (-NH2, -COOH, -H) phenyl
groups in tumor uptake, CDs 10–12 were reacted with various
aromatic thiols 13 (X = various groups) and a series of substi-
tuted CDs were synthesized (Scheme 1).
RESULTS
Photophysical properties of NIR cyanine dyes
The photophysical characterizations [absorption, fluorescence and
extinction coefficient values (molar absorptivity)] of all the cya-
nine dyes were performed in methanol. All synthetic dyes
reported herein exhibited similar photophysical properties and
the results are summarized in Table 1.
The longest wavelength absorptions of all dyes were in a
range 788–837 nm. The Stokes shifts (shifts between the absorp-
tion and emission wavelengths) were in
a range 25 nm
(390 cmꢀ1) to 41 nm (565 cmꢀ1) and the extinction coefficient
values (molar absorptivity) in methanol were in the range
157 000–226 000 Mꢀ1 cmꢀ1
.
Effects of the presence and position of substituents in in vivo
efficacy of cyanine dyes: A structure–activity relationship
(SAR) study
To determine the in vivo tumor selectivity of the series of dyes,
time course studies using in vivo fluorescence optical imaging
Effect of substituents at position-a. To understand the importance
of chloro (-Cl) group at position-a of CD 9, it was reacted with

Photochemistry and Photobiology, 2015, 91 1223
Scheme 1. Synthesis of substituted cyanine dyes bearing N-alkyl sulfonate groups.
phenyl thiol, thio-benzoic acid and p-thio-aniline and the respec-
tive CDs 14, 15 and 16 were obtained in modest yields.
appeared to maintain high levels of uptake in the liver at 72 h
post injection and therefore, did not provide the same level of
tumor to whole body contrast as CD 16. Attempts to hydrolyze
the ethyl ester functionality at position-b of CD 10 to a car-
boxylic acid had limited success and resulted in decomposition.
Under similar reaction conditions, the CD 17 resulted in a loss
of the 4-aminophenylthiolether functionality. Therefore, CD 19
(position-a bearing a 4-aminophenylthiol, and at position-b
bearing a carboxylic acid) was prepared via a stepwise synthesis
(Scheme 1).
The newly synthesized CD analogs were evaluated for tumor–
uptake/imaging potential at variable time points under similar
imaging dose (0.3 lmol kgꢀ1) in BALB/c mice bearing colon 26
tumors, and the results are illustrated in Fig. 3. In brief, com-
pared to CD 17, the dye 19 produced similar tumor uptake, but
showed faster clearance from the liver. This could possibly be
attributed due to the change in pharmacokinetic profile of the
molecule on introduction a carboxylic acid functionality. Again,
the presence of an amino-phenyl group at position-b (CD 18)
showed slightly enhanced tumor uptake.
As seen in Fig. 2, CD 9 displayed limited selectivity toward
tumor at 4-96 h post injection with significantly high uptake in
liver. Among the three CD 9 derivatives, compound 16 in which
the chloro group was replaced by a 4-aminophenylthioether
group displayed high tumor selectivity, and retained in tumor at
24, 48, 72 and 96 h post injection. In contrast to tumor, a rapid
liver clearance especially at 72 and 96 h post injection was
observed. Compound 16 exhibited highest liver accumulation at
4 h post injection and dropped to almost undetectable level in
liver at 72 h post injection resulting in excellent contrast between
the tumor and the rest of the body.
Replacing the amino (-NH2) group in CD 16 with a car-
boxylic acid (-COOH) group to yield CD 15 drastically changed
the pharmacokinetic profile of the dye, and under similar imag-
ing parameters, displayed extremely low tumor avidity and a fas-
ter clearance from the system.
CD 14 obtained by removing the amino group in CD 16 at
position-a showed limited accumulation in tumor and, in contrast
produced high uptake and retention in liver for a long time, even
at 96 h post injection. Effect of substituents at position-b: Posi-
tion-b modification started with the synthesis of CD 10 (posi-
tion-a -Cl, position-b ethyl ester) following the method described
by Strekowski et al. (23). After introducing anethyl ester func-
tionality at position-b of IR820, the compound was reacted with
4-aminophenylthiol and CD 17 was obtained in good yield.
Interestingly, compared to CD 16, the addition of the ethyl ester
functionality to it (CD 17) reduced its tumor uptake/specificity
(Fig. 3). However, compared to the parent CD 10 bearing a Clꢀ
group at position-a, the 4-aminophenylthioetrher analog 17 did
display more favorable tumor localizing ability. The CD 17
Importance of a thioether linker at position-a of the CDs investi-
gated. To determine the importance of thioether linker between
the 4-aminophenyl and the cyanine dye, CD 10 (position-a -Cl,
position-b ethyl ester) was reacted with p-aminophenyl boronic
acid 20 under Suzuki–Miyaura reaction conditions and the
desired compound 21 was isolated in modest yield (Scheme 2).
In contrast to other position-a substituted CDs, which resulted in
a bathochromic shift, the CD 21, where the chloro group was
substituted with an aminophenyl group (linked with C–C bond)
exhibited a hypsochromic shift exhibiting a strong absorption at
788 nm (e = 190 000).

1224 Nayan J. Patel et al.
A
24 h
72 h
96 h
4 h
8 h
12 h
48 h
9
16
15
14
High Signal
Low Signal
B
CD 9
CD 16
CD 15
CD 14
Figure 2. Unscaled whole body imaging of 0.3 lmol kgꢀ1 CDs 9, 14, 15 and 16 over 96 h in a representative BALB/c mouse with colon 26 tumors.
Images acquired using Maestro GNIR Flex NIR filter set (Ex. 710–760 nm, Em. 800 nm long-pass, 2 s exposure) (A) One representative mouse per dye
across all time points is depicted. Tumor location is indicated with red arrows and while liver is located just right of tumor. Arbitrary scale bar included
for signal reference (B) Average signals from tumor and liver of three mice per group per drug at only the 24 h time point. Maestro software was used
to acquire signal level. Error bars represent standard deviation; Student’s t-test P values are shown.
The in vivo results summarized in Fig. 4 indicate that similar
to CD 17 (containing a sulfur linker), compound 21 (no sulfur
linkage) produced higher uptake at 24 to 48 h post injection.
The position-c carboxylic acid derivatives were synthesized by
following a similar synthetic approach as discussed for the prepa-
ration of CD 10, in which the benzoindole 22 (instead of 8) was
reacted with Schiff’s base 6 (Scheme 3). The resulting intermedi-
ate CD 23 was reacted with 4-aminophenylthiol (24) to give the
desired cyanine dye 25 in 55% yield.
To investigate the difference in tumor specificity of the CDs
containing the sulfonic acid vs carboxylic acid functionalities,
tumor uptake of CD 17 (4-aminophenylthioether group at posi-
tion-a ethyl ester functionality at position-b, and N-alkyl
sulfonate group at position-c), and CD 25 (4-aminophenylth-
ioether group at position-a ethyl ester functionality at position-b,
and N-alkyl carboxylic acid group at position-c), was compared
in tumored mice (BALB/c mice bearing Colon26 tumors). To
Further validate the importance of 4-aminophenylthiol, CD 23
(position-a –Cl, position-b ethyl ester, position-c N-alkyl
Effect of substituents at position-c. In the most of the symmetri-
cal and nonsymmetrical cyanine dyes, including ICG, in which
the sulfonic acid functionalities are attached either at position-c
or in aromatic ring system(s) and the dyes generally show
enhanced tumor uptake/retention and faster clearance from the
rest of the organs. Presence of sulfonic acid groups in CDs
seems to improve their water solubility, and also prevents the
aggregation of the dyes (24).
To investigate the impact of such substitutions, the tumor
uptake and fluorescence imaging potential of CD 17 was
compared with CD 25, in which the sulfonic acid functionalities
(-SO3H) were replaced with carboxylic acid (-COOH) groups.

Photochemistry and Photobiology, 2015, 91 1225
A
24 h
72 h
96 h
4 h
8 h
12 h
48 h
High
Low
B
CD 10
CD 17
CD 18
CD 19
Figure 3. Whole body imaging of BALB/C mice (three mice/group, the image of only 1 mouse/CD is shown) bearing colon 26 tumors injected with
CDs 10, 17, 18 or 19 (dose: 0. 3 lmol kgꢀ1) over 96 h. Images acquired using Maestro GNIR Flex NIR filter set (Ex. 710–760 nm, Em. 800 nm long-
pass, 2 s exposure) (A) One representative mouse per dye across all time points is depicted. Tumor location is indicated with red arrows and while liver
is located just right of tumor. Arbitrary scale bar included for signal reference (B). Maestro software was used to acquire average concentration of the
dye from tumor vs liver (using three mice/group/drug at 24 h post injection). More specifically, using the built in Maestro software, regions of interest
(ROI) were drawn over the areas where the tumor and other organs of interest are located. The software subtracted out signals from skin and tissue from
a control mouse, and displayed the signal of only the drug within the desired ROI. Error bars represent standard deviation and the Student’s t-test P val-
ues are shown.
Scheme 2. Synthesis of 40-aminophenylcyanine dye without thioether linkage.

1226 Nayan J. Patel et al.
therapeutic applications. Cyanine fluorophores have been
reported to undergo oxidative C–C cleavage reactions upon irra-
diation (26–28). These reactions have been proposed to occur
through a mechanism comprising photosensitization to form sin-
glet oxygen, regioselective dioxetane formation and final dioxe-
tane cleavage to form carbonyl products. Using a combination of
spectrometry and absorption techniques, it was observed that
irradiation of 21a at 780 nm reduces the near-IR absorbance and
leads to the formation of four photooxidative products, 26, 27,
28 and 29 (Fig. 6).
24 h
48 h
72 h
High
21
17
Signal
Low
Signal
Figure 4. Whole body images of tumor mice showing difference in
tumor uptake of CD 17 and CD 21 at 24, 48 and 72 h post injection.
One representative BALB/c mouse with colon 26 tumor is shown across
multiple time points injected with 0.3 lmol kgꢀ1. Images acquired using
Nuance Camera (Ex. 782 nm, Em. 800/830 nm long-pass filters, 5 s
exposure). Tumor location is indicated with red arrows. Arbitrary scale
bar included for signal reference. Based on the scale from the Image J
software, higher signal intensity was observed for compound 17 over 21.
Cyanine dyes specificity toward ABC transport proteins. One of
the factors that have been reported to play an important role in
the cellular accumulation of many agents including the cyanine
dyes is their specificity toward ABC (ATP-binding cassette)
transport proteins (29). ABCG2 or breast cancer resistance pro-
tein has been found to efflux many classes of compounds used
for cancer imaging and therapy. Tyrosine kinase inhibitors
(TKI), including imatinib mesylate (Gleevec), are a part of a
novel class in cancer treatment which have been found to reverse
resistance to chemotherapy drugs and certain photosensitizers by
blocking their efflux by ABCG2 (30).
Therefore, our interest was to investigate if the increase in
tumor accumulation and retention that occurs by introducing the
aminophenylthioether functionality to the CD was the result of
pump efflux evasion. Using HEK-293 cells transfected with
ABCG2 and NIR flow cytometry we compared the structurally
carboxylic acid) was also included in this study. From the results
summarized in Fig. 5, it can be seen that the tumor and liver dis-
tribution of CD17 was far more favorable than CD 25. CD 17
was rapidly cleared from nontumor sites while CD 25 appeared
only to slightly favor tumor accumulation to liver at 24 and
48 h. However, CD 25 displayed much more favorable distribu-
tion than CD 23, again confirming the importance
4-aminophenylthioether group at position-a of the cyanine dye.
Photooxidative reactivity of 21a. Besides the use of CDs in
tumor imaging, certain respective targeted or nontargeted CD
analogs have also been investigated for their application as pho-
tosensitizers for photodynamic therapy (PDT) (25). In our hands,
the newly synthesized cyanine dyes, which showed significant
potential for tumor imaging produced limited PDT efficacy (not
shown) in colon 26 cell lines under the normal PDT treatment
parameters (MTT assay), where the required time for light expo-
sure was much longer than used for tumor imaging.
similar CDs
9
(position-a -Cl) and 16 (position-a
4-aminophenylthioether). The in vitro results summarized in
Fig. 7 indicate that neither dye was a substrate for the ABCG2
dye efflux pump.
Comparative tumor imaging ability of symmetrical cyanine dyes
bearing heptamethine linker with and without a fused six mem-
ber ring in IVIS system. Indocyanine green has extremely high
binding affinity to albumin and does not show preferential uptake
in tumor cells versus normal cells. This partially explains why
tumor sites accumulate ICG only for a short time after intra-
Therefore, determining the stability of cyanine fluorophores
under tumor imaging and treatment parameters is extremely
important as these methods proceed toward diagnostic and
Scheme 3. Synthesis of N-alkyl carboxylic acid containing cyanine dyes.

Photochemistry and Photobiology, 2015, 91 1227
of this six member ring, we compared the in vivo tumor accumu-
lation of ICG and one of the top candidates (CD 16) under simi-
lar imaging parameters. The in vivo results summarized in Fig. 8
illustrate the difference the two compounds in terms of accumu-
lation and pharmacokinetic profile. As can be seen, ICG shows
optimal uptake in the tumor at 30 min after injection. However,
even at this optimal time of tumor uptake, actual drug localiza-
tion is limited and ICG is primarily distributed in the digestive
system. ICG was found to rapidly clear from the system within
24 h and very little signal above background can be detected.
Unlike ICG, CD 16 has very little accumulation in the tumor at
30 min after injection. However, over the course of the experi-
ment this changes greatly. At 48 h post-injection, most of the
dye was localized in the tumor, and comparatively low fluores-
cence throughout the body provided excellent contrast.
24 h
23
48 h
72 h
High
Signal
25
17
Low
Signal
Figure 5. Whole body images of tumored mice showing uptake of CDs
17, 23 and 25 at 24, 48 and 72 h post injection. One representative
BALB/c mouse with colon 26 tumor is shown across multiple time points
injected with 0.3 lmol kgꢀ1. Images acquired using Nuance Camera (Ex.
782 nm, Em. 800/830 nm long-pass filters, 5 s exposure).Tumor location
is indicated with red arrows. Arbitrary scale bar included for signal
reference.
DISCUSSION
Recently the work revolving around the use of fluorescent dyes
for optical imaging in cancer has proven very promising and has
contributed greatly to the adoption of the technology. While cur-
rent fluorescent dye development is geared toward chemical con-
jugations of active targeting molecules to confer selectivity, this
method has its own limitations. Tumors which do not express
these targets would not be detected by these highly selective dye
conjugates. In addition, these conjugated dyes such as antibody-
dye conjugates tend to be very bulky which limits permeability
through membranes and greatly increase the cost of the drug. In
venous injection into mice. The leaky and damaged blood ves-
sels of a tumor allow ICG to enter into the tumor and it is quite
possible that the cyanine dyes synthesized and investigated in
this study are also following a similar pattern. Structurally, ICG
does not contain the central fused six member linker which the
dyes in our study possess. To gain an understanding of the role
Figure 6. (A) Conversion of 21a to 26, 27, 28 and 29. (B) Absorption (825 nm) upon exposing a 20 lM solution of 21a in H2O to 20 mW cmꢀ2 light
at rt. (C) Relative spectral ion counts of 26, 27, 28 and 29 prior to and after irradiation. Ion counts were determined at each time point relative to an
internal standard (phenylalanine).

1228 Nayan J. Patel et al.
between the various CD dyes in vivo prevents to compare their
precise optimal potential. Favorable in vivo distribution (tumor
accumulation vs all other detectable tissue) was used as the
screening criteria for the majority of the findings in this study. In
vivo distribution showed that nucleophilic substitution of the
chloro group at position-a of CD 9 has improved selectivity with
certain functionalities. Studies on substituting the chloro group
with a 40-thioether (CD 16) gave the most effective imaging
agent. Replacing the amino functionality with a carboxyl group
or removing it entirely to form CD 14 and 15, respectively,
resulted in reduced tumor selectivity. Quantification of signal at
24 h post injection showed significantly higher (P < 0.05) liver
accumulation if compared to tumor for both derivatives while the
amino substituted CD 16 was found to have no difference
between tumor and liver uptake. This favorable distribution pro-
file of the amino substituted dye was only improved over the
time course of this study where the dye was found to be retained
in the tumor yet being excreted from the liver thereby providing
excellent contrast at later time points. To demonstrate the impor-
tance of the sulfur in the 4-aminophenylthioether functionality in
CD 16, CD 21 (with no thioether linkage) was prepared by fol-
lowing Suzuki coupling (32,33). While both compound displayed
similar clearance from the liver, CD 21 was found to be less
selective as it was shown to linger in the normal tissue around
the tumor.
p=0.58
2500
2000
1500
1000
500
0
p=0.94
p=0.01
CD9
CD16
Control
CD9 + Inhibitor
CD16 + Inhibitor
Control + Inhibitor
Figure 7. ABCG2 efflux pump susceptibility of dyes with and without
pump inhibitor measured by flow cytometry (Ex. 785 Em. 830 LP).
0.5 lmol CD 9, CD 16 and positive control N-butyl-O-butyl-bacteriopur-
purin were incubated for 4 h with and without pump inhibitor Imatinib
mesylate. Error bars represent standard deviation. Student’s t-test P val-
ues are shown.
this study we have pursued an alternative SAR-driven approach
examining the effects of subtle chemical modifications on tumor
avidity.
In general, the cyanine dyes tend to have low fluorescence
quantum yields, but their inherent high extinction coefficients
and reasonable Stokes shifts along with positioning of their
absorption/fluorescence in the NIR optical region make them
excellent candidates for tumor imaging (31). The in vitro photo-
physical studies of the dyes selected for SAR study demonstrated
these dyes possess desirable photophysical properties, e.g. high
extinction coefficients of >155 000 Mꢀ1 cmꢀ1, NIR absorbance
maxima >788 nm, fluorescence peaks >813 and Stokes shifts
>25 nm.
Once the importance of the amino functionality at position-a
was validated for enhanced tumor uptake, a synthetic strategy to
introduce a variety of substituents at position-b of the amino-phe-
nyl functionalized CD was developed. Compared to position-a
modified CD, substituents introduced at position-b did not show
such a profound difference in tumor avidity. However, the avail-
ability of a facile synthetic approach for introducing desired func-
tional groups at positions-a, -b and -c provides an opportunity to
develop a wide variety of bi or multi-imaging (fluorescence/PET,
fluorescence/MRI) agents with and without tumor targeting
The lack of availability of an optimal tunable excitation light
source and adjustable optical filter sets impedes comparison
0.5 h
48 h
ICG
ICG
High
Signal
CD 16
CD 16
Low
Signal
Figure 8. Whole body images of tumored mice (BALB/c mice with colon 26 tumors, three mice/group) injected with ICG and CD16 at a dose of
0.3 lmol kgꢀ1 over 96 h (only 0.5 and 48 h images are shown). The images were acquired using IVIS Spectrum (Ex. 745 ꢁ 15 nm, Em.
840 ꢁ 10 nm) Tumor location is indicated with blue arrows. Arbitrary scale bar included for signal reference.

Photochemistry and Photobiology, 2015, 91 1229
a prospective controlled study. Photodiagnosis Photodyn Ther. 10
(4), 356–361.
moieties, and these studies are currently in progress. The tumor
imaging ability of the most effective cyanine dye (CD16) was also
compared with ICG, a clinically approved blood pooling agent,
which is also being used for fluorescence imaging to tumors, and
it clearly demonstrated enhanced tumor specificity with desirable
pharmacokinetic profile. ABCG2 efflux pump substrate studies
show that all the cyanine dye are the substrates for the pump and
no significant difference between the tumor-avid and nontumor-
avid dyes was observed. Like many other drugs, one of the reasons
for the difference in tumor specificity of some of the CDs could be
due to their differences in binding affinity to human serum albu-
min (HSA). Possibly, the dye with higher binding affinity tends to
retain in circulation for a longer period of time and passes through
the leaky tumor vessels. The hydrophobic nature of the dye helps
it to accumulate, and retain in tumor for a longer period of time
than the other organs.
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In summary, a series of heterocyclic polymethine dyes were
synthesized and characterized and their photophysical properties
and in vivo tumor localizing/retention ability with time was
investigated. Synthetic modifications were made at 3 different
positions of heptamethine chain containing a fused chloro-cyclo-
hexene ring system, which allowed us to define, for the first
time, key structural features than can improve tumor avidity. For
example: (1) replacing the sulfonate groups of N-alkyl sulfonate
side chains with carboxylic acid functionalities reduced the tumor
uptake significantly; (2) substituting the chloro group in chloro-
cyclohexene moiety of the CD with 4-aminophenyl-thioether
functionality significantly increased the tumor uptake; and (3) a
methodology to introduce carboxylic acid functionality in cyclo-
hexene ring system was established, which provides an opportu-
nity to develop multi-imaging agents. Efforts are currently
underway in our laboratory to explore the utility of this approach
in developing tumor specific fluorescence/PET, fluorescence/
SPECT and fluorescence/MRI dual imaging agents.
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Acknowledgements—The financial support from the NIH (CA55791,
CA127369 and Photolitec, LLC is gratefully acknowledged. A partial
support from the shared resources of the RPCI support grant
(P30CA16056) is also appreciated. This work was also supported in part
by the Intramural Research Program of the National Cancer Institute,
National Institutes of Health. Nayan Patel is thankful to the Department
of Molecular Pharmacology and Cancer Therapeutics for providing
fellowship from the NIH funded graduate students research training
grant.
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