Near infrared active heptacyanine dyes with unique cancer-imaging and cytotoxic properties

Article abstract of DOI:10.1016/j.bmcl.2011.11.070

Three near-infrared fluorescent heptacarbocyanine dyes have been synthesized using a facile one-pot synthetic approach. The reaction methodology afforded a mixture of three symmetric and unsymmetric heptacyanines containing various N-indolenine substituents, a dicarbocyclic acid (DA), a monoester (ME), and a diester (DE). These compounds were isolated, purified, characterized and biologically investigated for tumor cell cytotoxicity and uptake selectivity. Using cell viability and in vitro proliferation assays, we found that the esterified dyes (monoester, ME and diester, DE) were selectively cytotoxic to cancer cells and spared normal fibroblast cells. Additionally, confocal fluorescence imaging confirmed selective uptake of these dyes in cancer cells, thus suggesting tumor cell targeting.

Full text of DOI:10.1016/j.bmcl.2011.11.070

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1242–1246
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Near infrared active heptacyanine dyes with unique cancer-imaging
and cytotoxic properties
Maged Henary a, , Vaishali Pannu b, Eric A. Owens a, Ritu Aneja b,
⇑
⇑
a Georgia State University, Department of Chemistry, Petit Science Center, 100 Piedmont Ave., Atlanta, GA 30303, USA
b Georgia State University, Department of Biology, Petit Science Center, 100 Piedmont Ave., Atlanta, GA 30303, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Three near-infrared fluorescent heptacarbocyanine dyes have been synthesized using a facile one-pot
synthetic approach. The reaction methodology afforded a mixture of three symmetric and unsymmetric
heptacyanines containing various N-indolenine substituents, a dicarbocyclic acid (DA), a monoester (ME),
and a diester (DE). These compounds were isolated, purified, characterized and biologically investigated
for tumor cell cytotoxicity and uptake selectivity. Using cell viability and in vitro proliferation assays, we
found that the esterified dyes (monoester, ME and diester, DE) were selectively cytotoxic to cancer cells
and spared normal fibroblast cells. Additionally, confocal fluorescence imaging confirmed selective
uptake of these dyes in cancer cells, thus suggesting tumor cell targeting.
Received 23 September 2011
Revised 15 November 2011
Accepted 18 November 2011
Available online 28 November 2011
Keywords:
Fluorescent
NIR
Heptacyanine
Cancer
Ó 2011 Elsevier Ltd. All rights reserved.
Imaging
Cytotoxicity
Cellular uptake
Cl
N
R1
N
R2
X
R
1 = R2 = -(CH2)5COOH; X = Br
: R1 = -(CH2)5COOH, R2 = -(CH2)5COOMe; X = Br
DE:
DA:
ME
R
1 = R2 = -(CH2)5COOMe; X = Br
MHI-25:
R
1 = R2 = -(CH2)4SO3; X = N/A
Cyanine dyes display distinguishable characteristics and pos-
sess two nitrogen-containing heterocyclic groups connected by
an electron deficient conjugated methine chain, as shown by their
general structure in Figure 1.
Essentially, cyanine nomenclature directly corresponds to the
number of methine groups located in the bridge connecting the
two heterocycles. Dyes containing n = 0, 1, 2, and 3, are classified
as mono-, tri-, penta-, and heptacarbocyanines, respectively.1 Elec-
tron delocalization provides cyanine dyes with their characteristi-
cally wide range of absorption spectra in the visible and infrared
regions; additionally, they also exhibit narrow absorption bands
and high extinction coefficients.2 These properties combined with
low tissue auto-fluorescence, the ability to deeply penetrate tissue,
and an imaging window of 650–1000 nm have made cyanine dyes
promising for use in cancer-imaging in vivo.3 Fluorescence imaging
N
R
N
R
n
⇑
Corresponding authors. Tel.: +1 404 413 5566; fax: +1 404 413 5505.
E-mail address: mhenary1@gsu.edu (M. Henary).
Figure 1. General structure of cyanine dyes.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.070

M. Henary et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1242–1246
1243
techniques with near-infrared dyes generally display excellent
sensitivity and selectivity. An increase in the fluorophores’ rigidity
upon binding directly induces a significant increase in fluores-
cence. Commonly, near-infrared emission agents are conjugated
with ligands to target specific tumors and to be used in optical
imaging applications.
anti-proliferative activity of these dyes were evaluated and com-
pared with MHI-25 using the trypan blue and MTT assays, respec-
tively (Figs. 3 and 4).12–15 As shown in Scheme 1, the indolenine 1
was reacted with 6-bromohexanoic acid in boiling acetonitrile for
18 h under a nitrogen atmosphere to afford quaternary salt 2.
Compound 2 was allowed to react with Vilsmeier–Haack reagent16
in acetic anhydride for 3 h in the presence of sodium acetate.
Subsequently, when the reaction mixture was quenched with
methanol, it furnished a mixture of three heptamethine cyanine
dyes 4, 5, and 6 which were isolated by column chromatography.17
It should be noted that the reaction of salt 2 with Vilsmeier–Haack
reagent in boiling ethanol under basic conditions yielded only
dye 6.
We have previously reported the unique tumor imaging and tar-
geting properties of the heptamethine cyanine dyes MHI-25 and
MHI-148. It has been shown that these NIR dyes are actively taken
up by and accumulate within cancer cells but not normal cells.11
They were found to be superior for cancer detection compared to
other cyanine dyes, such as indocyanine green7 and noncyanine
dyes, such as rhodamine 123. This was particularly significant
because imaging with NIR dyes can yield much higher signal/noise
ratios with minimal interfering background fluorescence.
Since MHI-148 was found to be a mixture, we optimized the
purification process that yielded the diacid (DA), diester (DE),
monoester (ME).17 Although these heptamethine dyes show selec-
tive tumor targeting attributes, some of them have been reported
to be cytotoxic with specificity to cancer cells without affecting
normal cells. Thus, we first asked if these dyes killed cancer cells
while sparing normal cells. We employed two representative can-
cer cell lines viz., HeLa (cervical) and prostate (PC-3).18 Primary hu-
man dermal fibroblast (HDF) cells were used as non-cancerous
normal cells. Our trypan blue exclusion data demonstrated that
the esterified dyes (both monoester, ME and diester, DE) were
cytotoxic to cancer cells but spared normal cells (Fig. 3).13,14 The
Optical imaging is becoming increasingly recognizable as an
imaging technique that produces high-resolution imaging of fluo-
rophores in cancerous tissue.4 Employing near-infrared (NIR) dyes
as contrast agents in optical imaging is becoming popular.5 This
non-radiative technique4 is safer than current radiological tech-
niques. In addition, this imaging method is favorable due to its
low tissue absorption and minimal auto-fluorescence of NIR light.6
Conversely, NIR fluorescence may be able to provide a fast, inex-
pensive screening for breast cancer as well as other cancers.4–7
Although indocyanine green (ICG)8 has been clinically approved
for testing in fluorescence angiography and ophthalmology and
has been shown to be a potent contrast agent for the detection of
tumors in animal models and humans, the application of cyanine
dyes in cancer diagnosis and detection is yet to be fully ex-
plored.9,10 The long range goals are to synthesize cyanine dyes that
will natively target specific tumors without requiring molecular
conjugation.3,11
Two heptamethine cyanine dyes MHI-148 and MHI-25 (IR-783;
Fig. 2) containing a rigid cyclohexenyl ring within the methine
bridge were synthesized in our laboratory, and were shown to
selectively target cancer cells.11 We had reported cancer detection
properties of these dyes that were found to accumulate specifically
and selectively in cancer cells while sparing normal cells.11
Recently, our laboratory discovered that MHI-148 is a mixture
of three analogous compounds. The three dyes were carefully iso-
lated using flash chromatography and a gradient elution of dichlo-
romethane/methanol. The structure of each dye was identified, and
they were characterized as containing various N-substituted func-
tionalities including a monoester, a diester, and a diacid. Subse-
quent testing concluded that the monoester and the diester
specifically target and induce cellular death of human cervical
(HeLa) and prostate (PC-3) cancer cells. However, our trypan blue
exclusion data show that the diacid derivative and MHI-25 were
found to have no cytotoxic activity in these cells. In light of these
interesting data, further in vitro and preclinical research is war-
ranted to evaluate the antiproliferative and proapoptotic activity
of similar dyes with modified esters in order to evaluate their po-
tential usefulness as anti-cancer agents.
DE at concentrations ranging from 5-20 lM killed >90% of Hela
and PC-3 cells at 24 h whereas HDF cells were unaffected at the
same dose levels. This demonstrated remarkably significant selec-
tivity of DE for cancer cells while normal cells showed 100% cell
viability. Along similar lines, ME was cytotoxic to cancer cells, with
HeLa cells showing enhanced sensitivity compared to PC-3 at the
concentrations studied. ME treatment however compromised cell
viability of HDFs. Lower concentrations (5–10
10–15% cell death, whereas 20
M affected viability of ꢀ80% HDF
cells, indicating cytotoxicity at this concentration. On the other
hand, ME at 5 and 10 M concentrations displayed significant tu-
lM) resulted in
l
In this Letter, we describe a simple methodology for synthesiz-
ing symmetrical and unsymmetrical heptamethine cyanine dyes as
potential precursors for developing functionalized near-infrared
labels for proteins and peptides. We discovered that MHI-148 con-
tains three separate dyes. The dyes 4, 5, and 6 were isolated from
the previously tested MHI-148 sample.11 They were identified
and characterized as the diester (4, DE), monoester (5, ME) and
diacid (6, DA) as depicted in Scheme 1. The cell viability and
l
mor cell selectivity with only ꢀ20% cell survival in HeLa and PC-
3 cells. This was in stark contrast to HDF cells which were ꢀ85%
viable at the same concentration. The diacid (DA) and the disulph-
onate MHI-25, did not exert any cytotoxic effects in cancer or nor-
mal cells under the concentration conditions used in our
experiments.
We next performed an in vitro cellular proliferation assay to
determine the IC50 of these dye forms in cancer (HeLa and PC-3)
and normal (HDF) cells as shown in Figure 4.15 Essentially, the
IC50 value represents the concentration at which 50% of cell prolif-
eration is inhibited. Our MTT data show that the IC50 of DE and ME
were 0.8 and 1.0
values for DE and ME were higher in PC-3 cells (3 and 2
l
M, respectively, in HeLa cells (Fig. 4). The IC50
Cl
lM,
respectively) indicating lower sensitivity of prostate cancer cells
N
N
compared to cervical cancer cells (Fig. 4). The IC50 of DE in normal
HDF was 10 l
M, indicating a wide ‘therapeutic window’ (Fig. 4).15
However, the therapeutic window for ME was much narrower
compared to DE. Since DA and MHI-25 did not affect the prolifera-
tion of cancer or normal cells, we were unable to deduce the IC50 of
these two dye forms over a wide range of concentration in the cell
lines included in our study (data not shown).
O3S
SO3Na
Figure 2. Structure of MHI-25 (IR-783).

1244
M. Henary et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1242–1246
Cl
Cl
1.
N
H
N
H
Cl
3
Br
COOH
N
N
(CH3CO)2O,reflux 3h
N
Br
Br
N
MeCN,
reflux 18 hrs
2. MeOH
1
R1OOC
R2OOC
HOOC
4 [DE]: R1 = R2 = Me (15%)
5 [ME]: R1 = H, R2 = Me (30%)
6 [DA]: R1 = R2 = H (20%)
2
Scheme 1. Synthetic route for the one pot preparation of symmetrical and unsymmetrical heptacyanine dyes 4–6.17
Figure 3. HeLa, PC-3 and HDF cells were treated with three different dose-levels (5, 10 and 20
lM) each of DA, DE, ME and MHI-25 for 24 h. The percentage of dead cells upon
various treatments was measured using trypan blue staining. The corresponding bar-graphs show percentage cell viability determined by cell counting upon each treatment.
The values and error bars shown in all the graphs represent average and standard deviations, respectively, of three independent experiments (p <0.05). Note: Nearly 100% of
cell death was observed with 10 and 20 l
M ME in PC-3 cells. Thus, the bars are not visible for percent cell viability at indicated concentrations of ME.13,14
Figure 4. HeLa, PC-3 and HDF cells were treated for 48 h with increasing concentrations (0, 0.75, 1, 2, 5, 10, 15, 20 lM) of DA, DE, ME, MHI25 and cell proliferation was
measured using MTT assay. A (i, ii, iii) Line-graph depicting percentage cell survival versus concentration representing the sensitivity profile of corresponding cell lines to
each dye. B (i, ii, iii) Bar-graphical representation of IC50 values of DE and ME in the corresponding cell lines. The values and error bars shown in all the graphs represent
average and standard deviations, respectively, of three independent experiments (p <0.05).15
The inability of DA and MHI-25 to inhibit cell proliferation
could be attributed to their limited cellular uptake, thus rendering
them ineffective. It is also likely that they are taken up by cancer
cells but are not cytotoxic. To confirm the intracellular localization
of these dyes, we performed fluorescence confocal imaging in PC-3
and HDF cells upon 8 h of dye exposure (Fig. 5).19,20 Both the
esterified dyes (DE and ME) showed considerable cytoplasmic
localization in PC-3 cells; however, neither DE nor ME showed
accumulation in HDF cells. PC-3 cells demonstrated enhanced up-
take of ME as compared to DE, perhaps explaining its lower IC50
.
DA and MHI-25 however, exhibited negligible intracellular locali-
zation in both PC-3 and HDF cells. It is reported that esters are

M. Henary et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1242–1246
1245
Figure 5. (A) Confocal micrographs showing PC-3 cells treated with DE, ME, DA and MHI-25 (20
localization of the corresponding dyes is visible in red. (B) Merged micrographs showing HDF cells treated with the corresponding dyes (20
(green) and DNA with DAPI (blue). Any intracellular dye localization should appear in red. Scale bar = 10
m.19,20
l
M) for 8 h and stained for actin (green) and DNA with DAPI (blue). Cellular
lM) for 8 h, stained with actin
l
more readily transported across cellular membranes.21 This might
explain the enhanced cellular uptake of the esterified dyes (ME and
DE) compared to the diacid and MHI-25. It is also likely that the
partition coefficient (logP) values of the mono- and diesterified
dyes is optimal to facilitate cellular uptake. Our data thus under-
score the usefulness of DE and ME as potential tumor-specific
imaging as well as tumor targeting dye candidates. Although the
accumulation properties of MHI-25 have previously reported11 in
different cancer cell lines including prostate (LNCaP, C4-2, C4-2B,
ARCaPE, ARCaPM, and PC-3), lung (H358), breast (MCF-7), cervical
(HeLa), leukemia (K562), renal (SN12C, ACHN), bladder (T24), and
pancreatic (MIA PaCa-2), our data show minimal cellular uptake of
MHI-25 in HeLa and PC-3 cells under our experimental conditions.
In conclusion, a unique synthesis of asymmetric carbocyanine
dyes has been presented that is important for the preparation of
NIR dyes containing monofunctional groups for biomolecule conju-
gation. Furthermore, this one-pot synthesis appears feasible with a
variety of carboxylic acid alkylated salts. Interestingly, the synthe-
sized mono- and diester dyes were selectivity taken up and accu-
mulated in tumor cells but not normal cells, providing the
advantage of tumor-specific targeting that does not require recep-
tor conjugation of the imaging dyes.
4. Nitziachristos, V.; Bremer, C.; Weissleder, R. Eur. Radiol. 2003, 13, 195.
5. Choi, H. S.; Nasr, K.; Alyabyev, S.; Feith, D.; Lee, J. H.; Kim, S. H.; Ashitate, Y.;
Hyun, H.; Patonay, G.; Strekowski, L.; Henary, M.; Frangioni, J. V. Angew. Chem.,
Int. Ed. 2011, 50, 6258.
6. Bhushan, K. R.; Misra, P.; Liu, F.; Mathur, S.; Lenkinski, R. E.; Frangioni, J. V. J.
Am. Chem. Soc. 2008, 130, 17648.
7. Alacam, B.; Yazici, B.; Intes, X.; Nioka, S.; Chance, B. Phys. Med. Biol. 2008, 53,
837.
8. Ntziachristos, V.; Yodh, A. G.; Schnall, M.; Chance, B. Proc. Natl. Acad. Sci. 2000,
97, 2767.
9. Armitage, B. Top. Heterocycl. Chem. 2008, 14, 11.
10. Wuestenfeld, J.; Herold, J.; Niese, U.; Kappert, U.; Schmeisser, A.; Strasser, R.;
Braun-Dullaeus, R. C. J. Vasc. Surg. 2008, 48, 1315.
11. Yang, X.; Shi, C.; Tong, R.; Qian, W.; Zhau, H. E.; Wang, R.; Zhu, G.; Cheng, J.;
Yang, V. W.; Cheng, T.; Henary, M.; Strekowski, L.; Chung, L. W. Clin. Cancer Res.
2010, 16, 2833.
12. Mishra, R. C.; Karna, P.; Gundala, S. R.; Pannu, V.; Stanton, R. A.; Gupta, K. K.;
Robinson, M. H.; Lopus, M.; Wilson, L.; Henary, M.; Aneja, R. Biochem.
Pharmacol. 2011, 82, 110.
13. Cell viability by trypan blue-exclusion assay: The loss of membrane integrity in
dead and dying cells allows preferential uptake of labels like trypan blue. We
used trypan blue assay to determine cell viability upon treatment with dyes.14
At the end of incubation times with the dyes, PC-3, HeLa or HDF cells were
pelleted and washed with PBS. Well-suspended cells were mixed with equal
volume of 0.4% trypan blue in 1Â PBS, pH 7.4, followed by incubation at room
temperature for 5 min. Cells were examined under the microscope and blue-
stained cells were considered non-viable.
14. Zhou, J.; Liu, M.; Luthra, R.; Jones, J.; Aneja, R.; Chandra, R.; Tekmal, R. R.; Joshi,
H. C. Cancer Chemother. Pharmacol. 2005, 55, 461.
15. Cytotoxicity assay: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay was employed to evaluate the proliferative capacity of cells as
described previously.12 Essentially, MTT is a colorimetric assay, which utilizes
the colorless tetrazolium dye and converts it into a colored formazan salt,
which can be quantified by measuring absorbance at 570 nm. Briefly, a 96-well
format was used to seed 100 mL medium containing cells at a density of
5 Â 103 cells per well. After incubation with dyes, cells were treated with
gradient concentration of the test compounds, which were dissolved in DMSO.
The final concentration of DMSO in the culture medium was maintained at
0.1%. After 48 h of incubation with dye, the spent medium was removed and
the wells were washed twice with PBS. Hundred milliliter of fresh medium and
10 mL of MTT (5 mg/mL in PBS) were added to the wells and cells were
incubated at 37 °C in the dark for 4 h. The formazan product was dissolved by
adding 100 mL of 100% DMSO after removing the medium from each well. The
absorbance was measured at 570 nm using a Spectra Max Plus multi-well plate
Supplementary data
Supplementary data associated with this Letter can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.070.
References and notes
1. Mojzych, M.; Henary, M. In Topics in Heterocyclic; Strekowski, Ed.; Springer:
Berlin, 2008; Vol. 14, p 1. Verlag, Heidelberg.
2. Patonay, G.; Kim, J. S.; Kodagahally, R. K.; Strekowski, L. http://
www.ncbi.nlm.nih.gov/pubmed/15969815 Appl. Spec. 2005, 59, 682.
3. Shi, C.; Zhang, C.; Su, Y.; Cheng, T. The Lancet 2011, 11, 815.

1246
M. Henary et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1242–1246
reader (Molecular Devices, USA). We first performed MTT with a broader
concentration range (0.1–100 M with a 10-fold increment) to determine the
IC50 of the dyes. Since the IC50 range was between 1 and 10 M for DE and ME,
respectively, in PC-3 and below 1 M in HeLa, we decided to take a narrower
range to precisely determine the IC50 values. The IC50 table for HeLa, PC-3 and
HDF is shown in Table 1 in Supplementary data.
6.26 (d, JHH = 14.0 Hz, 2H), 7.18 (d, JHH = 7.6 Hz, 2H), 7.27 (m, 2H), 7.40 (m, 4H),
l
8.34 (d, JHH = 14.0 Hz, 2H); 13C NMR (CDCl3, 100 MHz) dC 173.8, 172.2, 150.2,
144.2, 142.3, 141.1, 128.8, 127.8, 125.3, 122.2, 110.9, 101.7, 51.6, 49.3, 44.8,
33.7, 28.2, 27.2, 26.8, 26.4, 24.6, 20.8; HRMS calcd for C44H56N2O4Cl, m/z
711.3921; observed m/z 711.3915. kmax absorption and fluorescence
wavelengths in methanol were 780 and 801 nm, respectively. Chloro-3-[2-
[1,3-dihydro-3,3-dimethyl-1-(methylhexanoate)-2H-indol-2-ylidene]-ethylidene]-
l
l
16. Makin, S. M.; Boiko, I. I.; Shavrygina, O. A. Zh. Org. Khim. 1977, 13, 1189.
17. Synthetic methods and spectroscopic characterization: All 1H NMR (400 MHz) and
13C NMR (100 MHz) spectra were recorded on a BRUKER AVANCE-400 NMR
spectrometer at ambient temperature with CDCl3 and DMSO-d6 as solvent and
tetramethylsilane (TMS) as an internal standard. High resolution electrospray
ionization (ESI) mass spectra were obtained on a Waters Micro-mass Q-TOF
micro analyzer in the Department of Chemistry, Instrumentation Facility at
Georgia State University. Melting points (open pyrex capillary) were measured
on a Thomas Hoover apparatus and are uncorrected. Starting reagents 2,3,3-
trimethyl indolenine and 6-bromohexanoic acid were purchased from Sigma
Aldrich or Alfa Aesar and were used as received. All of the solvents were at least
reagent grade and were used without further purification. 1-(5-Carboxypentyl)-
1-cyclo-1-yl]-ethenyl]-3,3-dimethyl-1-(5-carboxypentyl)-3H-indolum
bromide
(5). Greenish-gold solid, yield 30%, 800 mg, mp 165–167 °C; 1H NMR (CDCl3,
400 MHz) dH 1.53 (m, 4H), 1.72 (s, 14H), 1.87 (t, JHH = 7.4 Hz, 4H), 2.00 (m, 2H),
2.36 (t, JHH = 7.4 Hz, 2H), 2.42 (t, JHH = 7.4 Hz, 2H), 2.71 (m, 4H), 3.66 (s, 3H),
4.13 (m, 4H), 6.14 (d, JHH = 14.0 Hz, 2H), 7.13 (d, JHH = 7.4 Hz, 1H), 7.25 (m, 3H),
7.41 (m, 4H), 8.36 (q, JHH = 14.0 Hz, 2H); 13C NMR (CDCl3, 75 MHz) dC 176.2,
173.8, 172.8, 171.9, 150.7, 144.9, 144.0, 142.2, 142.0, 141.1, 140.9, 129.0, 128.8,
128.6, 127.3, 125.6, 125.2, 122.3, 112.2, 110.7, 101.6, 100.8, 51.6, 49.5, 49.2,
44.7, 44.5, 34.5, 33.9, 33.6, 29.6, 28.5, 28.1, 27.0, 26.5, 26.4, 26.3, 25.5, 24.5,
20.7; HRMS calcd for C43H54N2O4Cl, m/z 697.3772; observed m/z 697.3763.
kmax absorption and fluorescence wavelengths in methanol were 780 and
2,3,3-trimethyl-3H indolenium bromide, 2. 2,3,3-Trimethyl-3H indolenine
1
801 nm,
respectively.
Chloro-3-[2-[1,3-dihydro-3,3-dimethyl-1-(5-
(2.0 g, 12.6 mmol) and 6-bromohexanoic acid (7.35 g, 25.1 mmol) were mixed
in acetonitrile (15 mL) and heated to reflux for 18 h under nitrogen
atmosphere. The mixture was allowed to cool to room temperature. The
reaction was concentrated under vacuum to yield an oily residue, which was
dissolved in dichloromethane (5 mL). Ether was added dropwise to the
carboxypentyl)-2H-indol-2-ylidene]-ethylidene]-1-cyclo-1-yl]-ethenyl]-3,3-
dimethyl-1-(5-carboxypentyl)-3H-indolum bromide (6). Greenish-gold solid,
yield 20%, 520 mg, mp 171–173 °C; 1H NMR (CDCl3, 400 MHz) dH 1.56 (m,
4H), 1.71 (s, 12H), 1.76 (q, JHH = 7.4 Hz, 4H), 2.00 (br t, 2H), 2.46 (t, JHH = 7.4 Hz,
4H), 2.72 (br t, 4H), 4.13 (t, JHH = 7.4 Hz, 4H), 6.20 (d, JHH = 14.0 Hz, 2H), 7.22
(m, 4H), 7.38 (m, 4H), 8.33 (d, JHH = 14.0 Hz, 2H); 13C NMR (CDCl3, 75 MHz) dC
172.2, 148.0, 143.0, 142.0, 141.1, 128.6, 126.2, 125.1, 122.5, 111.5, 101.6, 49.0,
43.7, 33.5, 27.5, 26.7, 25.8, 25.6, 24.2; HRMS calcd for C42H52N2O4Cl, m/z
683.3616; observed m/z 683.3595. kmax absorption and fluorescence
wavelengths in methanol were 780 and 801 nm, respectively.
dichloromethane solution to give
a precipitate, which was filtered and
washed with ether to furnish 2 as a pale pink powder; yield 4.4 g (67%); mp
127–129 °C; 1H NMR (DMSO-d6, 400 MHz): d 1.43 (m, 2H), 1.54 (s, 6H), 1.56
(m, 2H), 1.85 (m, 2H), 2.23 (t, J = 7.6 Hz, 2H), 2.86 (s, 3H), 4.46 (t, J = 7.6 Hz, 2H),
7.62 (m, 2H), 7.85 (m, 1H), 7.99 (m, 1H), 11.98 (br s, 1H). Heptamethine cyanine
dyes 4, 5, and 6. A solution of salt 2 (2.40 g, 6.77 mmol), Vilsmeier–Haack
reagent 3 (1.10 g, 3.39 mmol) and sodium acetate (0.833 g, 10.2 mmol) in
acetic anhydride (15 ml) was heated at 90 °C for 3 h under nitrogen
atmosphere. The mixture was cooled to room temperature and quenched
with methanol (5 ml). After 10 min, the solution began boiling and turned
black. The solvent was concentrated under vacuum and the crude solid was
dissolved in dichloromethane (20 ml) and filtered to remove sodium acetate.
The mixture of dyes was separated by column chromatography on silica gel
eluting with methanol-dichloro-methane gradient from 1:30, 1:20, to 1:10.
The fractions of each dye were collected together and concentrated under
vacuum to give heptamethine cyanine dye diester 4, heptamethine cyanine
dye monoester 5, and heptamethine cyanine dye diacid 6. Chloro-3-[2-[1,3-
dihydro-3,3-dimethyl-1-(methylhexanoate)-2H-indol-2-ylidene]-ethylidene]-1-
18. Cell culture: PC-3 were grown in RPMI medium, and HeLa and HDF in DMEM
medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/
streptomycin.
19. Immunofluorescence microscopy: The cellular localization of the NIR dyes was
imaged using Indocyanine Green (ICG-B) filter Set (Chroma Technology, VT)
and 780 nm Collimated LED source (Zeiss, Germany) on an Axio Observer. A1
microscope (Zeiss, Germany). Cells were grown on glass coverslips for
immunofluorescence microscopy as described previously.20 After treatment
with drugs, cells were fixed with cold (À20 °C) methanol for 10 min and
blocked by incubating with 2% BSA/PBS at 37 8C for 1 h. Alexa FluorÒ 555
phalloidin (Invitrogen, Carlsbad, 1:500 dilution) was used to stain for actin and
was incubated with the coverslips for 1 h at room temperature. The cells were
washed with 2% BSA/PBS for 10 min at room temperature. Cells were mounted
with Prolong-Gold antifade reagent that contained DAPI (Invitrogen, Carlsbad).
20. Karna, P.; Rida, P. C.; Pannu, V.; Gupta, K. K.; Dalton, W. B.; Joshi, H.; Yang, V.
W.; Zhou, J.; Aneja, R. Cell Death Differ. 2011, 18, 632.
cyclo-1-yl]-ethenyl]-3,3-dimethyl-1-(methylhexano-ate)-3H-indolum
bromide
(4). Greenish-gold solid, yield 15%, 400 mg, mp 161–163 °C. 1H NMR (CDCl3,
400 MHz) dH 1.54 (m, 4H), 1.73 (s, 14H), 1.86 (m, 4H), 2.00 (m, 4H), 2.36 (t,
JHH = 7.6 Hz, 4H), 2.75 (t, JHH = 7.6 Hz, 4H), 3.65 (s, 6H), 4.25 (t, JHH = 7.6 Hz, 4H),
21. Han, H. K.; Oh, D. M.; Amidon, G. L. Pharm. Res. 1998, 15, 1382.