Structurally modified indocyanine green dyes. Modification of the polyene linker

Article abstract of DOI:10.1016/j.dyepig.2013.05.001

We have prepared a series of indocyanine green dicarboxylic acid derivatives (9a,b, 4, 10, 13) with modified polyene linkers in an attempt to increase the structural rigidity of the polyene linker and thereby the fluorescent yield. Incorporation of five- and six-membered rings into the polyene system led to lower florescent yield for 9a,b, 4 and 10, but shortening the chain by two carbon atoms led to an increase in fluorescent yield for 13.

Full text of DOI:10.1016/j.dyepig.2013.05.001

Dyes and Pigments 99 (2013) 275e283
Contents lists available at SciVerse ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Structurally modified indocyanine green dyes. Modification of the
polyene linker
Innus Mohammad a, Courtney Stanford d, Martha D. Morton c, Quing Zhu b,
,
Michael B. Smith a
*
a Department of Chemistry, University of Connecticut, 55 N. Eagleville Road, Storrs, CT 06269-3060, United States
b Department of Electrical and Computer Engineering, University of Connecticut, 371 Fairfield Way, Storrs, CT 06269-2157, United States
c Department of Chemistry, University of Nebraska, Hamilton Hall 838, Lincoln, NE 68588, United States
d The University of Iowa, E331 Chemistry Building, Iowa City, IA 52242-1294, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We have prepared a series of indocyanine green dicarboxylic acid derivatives (9a,b, 4, 10, 13) with
modified polyene linkers in an attempt to increase the structural rigidity of the polyene linker and
thereby the fluorescent yield. Incorporation of five- and six-membered rings into the polyene system led
to lower florescent yield for 9a,b, 4 and 10, but shortening the chain by two carbon atoms led to an
increase in fluorescent yield for 13.
Received 31 October 2012
Received in revised form
25 April 2013
Accepted 1 May 2013
Available online 10 May 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Indocyanine
Fluorescence
Near infrared
Dye
Synthesis
Absorbance
1. Introduction
leakage in blood vessels. The use of ICG in molecular imaging
probes may be limited due to loss of fluorescence after protein
Fluorescence imaging is an optical imaging technique proven to
be a useful tool for diagnostic imaging without the complications
that often accompany the use of nuclear contrast agents [1]. In
fluorescence imaging, a near infrared (NIR) fluorescent probe
(>700 nm) is administered, and irradiation with NIR light allows
collection of the remitted fluorescence for imaging. Fluorescence
imaging has great potential for probing tumor molecular markers
associated with tumor proliferation, growth, and metastasis.
Indocyanine dyes are important abiotic molecules [2e5], and useful
NIR probes. An important member of this family is the clinically
approved indocyanine green (ICG, 1; Scheme 1), which has many
applications for fluorescence imaging [6,7]. ICG is the only FDA
approved agent that can be used for human subjects, due to low
toxicity and high absorbance in the NIR spectrum. However, ap-
plications of ICG are mitigated by several limitations, including
optical instability in the body, low quantum yield, and promiscuous
binding. Despite these problems, the NIR probe remains available
after cell binding and internalization, due to the fact that ICG may
be dissociated from the targeting antibody, thus activating fluo-
rescence [8]. It is known, for example, that ICG binds to plasma
proteins, and protein-bound ICG emits light with a peak wave-
length of about 830 nm in the NIR. [9]. An attempt to improve the
targeting capability of the dye, as it affects NIR imaging of
cancerous tumors, led us to attach of a cancer targeting 2-
nitroimidazole moiety to the fluorescent dye in order to improve
detection of the tumor.
We prepared bis(carboxylic acid) derivative 2 using literature
procedures, and used it to synthesize dye-conjugates 3 by linking
two nitroimidazoacetate units to the dye via ethanolamine linkers
[10,11]. The purpose of this research was and is to develop a non-
invasive probe for the detection of cancerous tumors using NIR
fluorescent dyes that contain a nitroimidazole moiety known to
target hypoxic tumors [10,12]. The quantum yield for the dyes used
in the study were about 0.06, which was sufficient for the NIR
detection of hypoxic cancerous tumors in vivo [10]. To further
develop this work, we targeted the synthesis of dyes with a larger
* Corresponding author.
E-mail address: michael.smith@uconn.edu (M.B. Smith).
0143-7208/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2013.05.001

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I. Mohammad et al. / Dyes and Pigments 99 (2013) 275e283
Scheme 1. ICG, 1, ICG-dicarboxylic acid derivative 2, imidazole-ICG conjugate 3, dye 4, and indolium 5.
quantum yield with the goal of increasing the sensitivity of our
tumor detection system. However, any structural changes should
retain the emission/absorption wavelengths in the NIR region.
Once the synthetic work was completed, the next step was to
examine parameters that influence the fluorescent yield of the dye,
without significant perturbation of the emission and absorption
wavelengths.
Similarly, reaction with amines followed by conversion to the cor-
responding amide produced NIR amide tricarbocyanine dyes that
were further modified to include lipoic acid residues used for cancer
imaging due their ability to serve as highly sensitive NIR SERS re-
porter molecules [19].
For this study, we prepared 4 as well as several related indoc-
yanine dyes, first with a six- or a five-membered ring incorporated
into the polyene system (9a,b and 10), and another derivative with
a shorter polyene chain (13). Our primary interest was the synthesis
of these compounds and determination of changes in florescent
yield as a function of structure, if any. In Tung and coworker’s work,
4 was used for protein labeling experiments and 4 was coupled to a
peptide as an amino acid residue by initial conversion to the NHS
activated ester [15,20]. Human transferrin (Tf) was used as a model
protein and coupling of activated 4 with Tf gave a conjugate that
facilitated florescence measurements in PBS solution as well as the
protein binding studies.
Our long term goal for any new dye is to eventually prepare
derivatives of 3 for targeting hypoxia in cancerous tumors rather
than protein labeling experiments. The first step toward that goal
requires synthesis of the dye and a study of the chemical and
in vitro physical properties of the dye. Therefore, work from this
laboratory reports the chemical synthesis, chemical properties, and
in vitro stability studies in PBS and sucrose solution, as well as
emission/absorbance data of the dyes. Dye-conjugates such as 3 are
required for our in vivo studies and they were not prepared as part
of this work, which focuses on identifying a new dye with improved
fluorescent properties. It is important to point out that protein
binding studies such as those published by Tung and co-workers
It is known that conversion of a nonplanar molecule to a similar
but more planar and rigid molecule often leads to an increase in the
quantum yield of fluorescence [13]. Decreases in fluorescence may
be associated with a higher collisional probability observed with
molecules that have a high degree of flexibility. In other words,
molecules with more rigid structures have a lower probability of
collision, which leads to a higher fluorescence potential, because the
energy absorbed is not lost due to molecular vibration or collision. It
has been shown, however, that rigidity (maintenance of a planar or
near planar configuration) in the first excited S1 excited state is more
important than rigidity in the S0 state [14]. One possible structural
change that might lead to rigidity is incorporation of a ring into the
polyene chain. Tung and co-workers reported just such a molecule
as a near infrared fluorochrome, in their synthesis of compound 4
(reported as compound NIR820) [15]. In earlier work, Reynolds and
Drexhage had prepared heptamethine pyrylium dyes that con-
tained a ring in the polyene moiety [16]. These dyes were relatively
unstable and exhibited a bathochromic shift. Slominskii et al. pre-
pared a series of dyes containing a polyene moiety (methine dyes),
including examples with a cyclohexane ring [17]. It is noted that the
reaction of 4 with amines produced a new class of NIR amine
tricarbocyanine dyes with excellent photostability properties [18].

I. Mohammad et al. / Dyes and Pigments 99 (2013) 275e283
277
are beyond the scope of our current studies would take our
research in a different direction.
d6) d 165.9, 147.7, 139.8, 129.6, 129.5, 129.3, 125.6, 118.8, 117.5, 115.1,
29.1, 23.5, 19.8, 14.3.
Indeed, for this study we were primarily interested in the effects
of a more rigid ring versus incorporation of more atoms into the
structure, and also how the length of the conjugated chain would
influence fluorescence. We found that incorporation of a ring
diminished the fluorescence yield, but shortening the polyene
chain increased the fluorescence yield.
2.2. N-((E)-(2-phenyl-3-((E)-(phenylimino)methyl)cyclohex-2-
enylidene)methyl)aniline, 7b
A stirred solution of N-formyl-N-methylaniline (6, 1.28 g,
9.5 mmol) in chloroform (2 mL) at ꢁ5 ꢀC was treated with phos-
phorous oxychloride (0.9 mL, 9.5 mmol), dropwise, and stirred for
1 h at 10 ꢀC. Phenylcyclohexene (0.5 g, 3.2 mmol) was added
dropwise and then stirred at 45 ꢀC for 20 h. The reaction mixture
was poured into a beaker of vigorous stirred water (20 mL), and
potassium carbonate (2 g, 14.5 mmol) was carefully added. A so-
lution of aniline hydrochloride salt (0.92 g, 7.11 mmol) in water
(3 mL) was added and the mixture stirred at ambient temperature
for 30 min. After addition of potassium carbonate (2 g, 14.5 mmol)
portionwise, the mixture was cooled and the resulting precipitate
was filtered, washed several times with cold water, stirred vigor-
ously with acetone (2 ꢂ 3 mL), filtered and dried in vacuo to afford
7b as a dark red powder (0.85 g, 2.12 mmol, 67.5%) [22]. Mp ¼ 246e
250 ꢀC; 1H NMR (400 MHz, DMSO-d6) d 11.01 (s, 2 H), 7.57e7.55 (m,
3 H), 7.42e7.40 (m, 2 H), 7.35 (t, J ¼ 8 Hz, 4 H), 7.25 (bs, 2 H), 7.15 (t,
J ¼ 8 Hz, 2 H), 7.07 (d, J ¼ 8 Hz, 4 H); 13C (100 MHz, DMSO-d6)
d 157.8, 150.5, 142.0, 139.8, 136.2, 135.3, 134.7, 130.6, 130.2, 129.8,
129.5, 128.3, 128.0, 125.8, 121.2, 118.7, 118.2, 114.6, 113.6, 23.9, 23.3,
21.9, 20.5, 19.8.
2. Materials and methods
All glassware was oven-dried under vacuum, and all reactions in
organic solvents were performed under a nitrogen atmosphere,
unless otherwise noted. 3-Methyl-2-butanone, butane sultone,
methylcyclohexene, phenylcyclohexene, cyclohexanone, and 11
were obtained from Aldrich Chemical Co. Indole derivative 5 and
dye 2 were prepared using our previously reported procedure [10].
N-Formyl-N-methylaniline (6) was prepared in 68% yield by the
reaction of commercially available N-methylaniline with formic
acid and sodium formate [21]. All solvents were dried according to
standard procedures. THF was distilled from sodium benzophenone
ketyl, methylene chloride was distilled from calcium hydride, and
dimethylformamide was vacuum distilled from calcium hydride.
Thin-layer chromatography was done on Sorbent Technologies
aluminum-backed TLC plates with fluorescent indicator and 0.2 mm
silica gel layer thickness, and p-anisaldehyde or phosphomolybdic
acid were used as developing agents. Column chromatography was
done using 60 A porosity, 32e63
m
m silica gel. 1H and 13C NMR were
2.3. N-((E)-(2-chloro-3-((E)-(phenylimino)methyl)cyclohex-2-
enylidene)methyl)aniline, 7c
ꢀ
collected on a Brüker Avance 300 (300.13 MHz for 1H, 75.48 MHz for
13C), Brüker DRX-400 (400.144 MHZ 1H,100.65 MHz 13C) or a Brüker
Avance 500 (500.13 MHz for 1H,125.65 MHz for 13C). Chemical shifts
are given in ppm downfield from TMS in the following format
chemical shift, multiplicity (s ¼ singlet, d ¼ doublet, t ¼ triplet,
q ¼ quartet, m ¼ multiplet) coupling constants J in Hz. Mass spec-
trometry data was collected on a HP 5870B GC/MSD mass spec-
trometer with an HP-1 column, and high resolution mass
spectrometry was done on a Micromass VB-QTOF tandem mass
spectrometer. IR spectra were taken on FT/IR-410/C031560585
JASCO and Nexus 670 FT-IR E.S.P., neat, unless otherwise stated.
UVeVis spectra were recorded on Cary 50 spectrometer and fluo-
rescence spectra on a Cary Eclipse. Melting points were taken on a
Uni-melt capillary melting point apparatus and Digimelt MPA160
and recorded to a maximum of 270 ꢀC. For products described as
waxy solid, melting points could not be obtained.
Phosphorus oxychloride (22 mL, 0.12 mol) was added dropwise
to dimethylformamide (6, 26 mL, 0.17 mol) at 0 ꢀC, and the resulting
solution was stirred for 30 min. Cyclohexanone (11 mL, 0.053 mol),
was added and the reaction mixture was heated at reflux for 1 h.
The solution was cooled to ambient temperature, 36 mL aniline/
ethanol (1:1 (v/v)) was added dropwise and the solution stirred for
30 min. The reaction mixture was poured into 220 mL of ice cold
water/concentrated HCl (10:1) and cooled for 2 h in an ice bath.
Subsequent filtration gave a solid that was washed with cold water,
diethyl ether and then acetone to give 7c. (15.3 g, 42.7 mmol, 40.1%)
[23]. Mp ¼ 190e193 ꢀC; 1H NMR (400 MHz, DMSO-d6) d 8.29 (s,
2 H), 7.44e7.42 (m, 4 H), 7.25e7.18 (m, 6 H), 7.05e7.02 (m, 2 H); 13C
NMR (100 MHz, MeOD-d4) d 173.7, 168.6, 151.0, 146.0, 145.1, 141.5,
131.2, 128.8, 123.5, 110.9, 102.6, 50.5, 49.3, 44.2, 27.3, 27.1, 26.2, 26.1,
22.4, 20.9.
2.1. N-((E)-(2-Methyl-3-((E)-(phenylimino)methyl)cyclohex-2-
enylidene)methyl)aniline, 7a
2.4. N-((E)-(2-chloro-3-((E)-(phenylimino)methyl)cyclopent-2-
enylidene)methyl)aniline, 8
A stirred solution of N-formyl-N-methylaniline (6, 2.11 g,
15.6 mmol) in chloroform (2 mL) at ꢁ5 ꢀC was treated with phos-
phorous oxychloride (1.5 mL, 15.6 mmol), dropwise, and stirred for
1 h at 10 ꢀC. Methylcyclohexene (0.6 mL, 5.2 mmol) was added
dropwise and the solution stirred at 45 ꢀC for 20 h. The reaction
mixture was poured into a beaker containing vigorously stirred
water (20 mL). Solid potassium carbonate (2 g, 14.5 mmol) was
added carefully added. A solution of aniline hydrochloride salt
(1.52 g, 11.7 mmol) in water (3 mL) was added and the mixture
stirred at ambient temperature for 30 min. At this time, potassium
carbonate (2 g, 14.5 mmol) was added portionwise and the
resulting solution cooled to give a precipitate that was filtered,
washed several times with cold water and stirred vigorously with
acetone (2 ꢂ 3 mL) filtered and dried in vacuo to afford 7a (0.68 g,
2.0 mmol, 38.6%) [22]. Mp ¼ 207e210 ꢀC; 1H NMR (400 MHz,
DMSO-d6) d 8.47 (s, 2 H), 7.48e7.43 (m, 8 H), 7.28e7.24 (m, 2 H),
2.60e2.57(m, 7 H), 1.98e1.91 (m, 2 H); 13C NMR (100 MHz, DMSO-
A stirred solution of N-methylformanilide (6, 13.5 g, 99.9 mmol)
in chloroform (13 mL) was treated with phosphorous oxychloride
(14 mL, 153 mmol) at 10 ꢀC and the stirred at ambient temperature
for 1 h. Cyclopentanone (3.36 g, 39.9 mmol) was added and the
solution stirred at 50 ꢀC for 4 h, cooled to ambient temperature and
potassium carbonate (10 g, 72.5 mmol) was added, followed by
aniline (8.4 g, 90.2 mmol), concentrated HCl (7.5 mL) and water
(50 mL) in that order. Subsequent stirring for 1 h at ambient tem-
perature was followed by addition of CH2Cl2, A solid precipitated
formed, which was filtered and washed with acetone (5 ꢂ 10 mL)
and water (5 ꢂ 20 mL) to afford 8 (10 g, 29.0 mmol, 72.7%) [23].
Mp ¼ 195e198 ꢀC; 1H NMR (400 MHz, DMSO-d6) d 11.40 (bs, 2 H),
8.30 (s, 2 H), 7.62e7.60 (m, 4 H), 7.46 (t, J ¼ 8 Hz, 4 H), 7.27e7.24 (m,
2 H), 3.02 (s, 4 H); 13C NMR (100 MHz, DMSO-d6) d 142.8, 140.5,
139.6, 139.3, 138.1, 130.2, 129.7, 129.5, 126.0, 122.9, 118.5, 116.3,
114.9, 110.6, 26.1.

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I. Mohammad et al. / Dyes and Pigments 99 (2013) 275e283
2.5. Sodium 2-((E)-2-((E)-3-((Z)-2-(5-carboxy-3,3-dimethyl-3-(4-
sulfonatobutyl)lndolin-2-ylidene)ethylidene)-2-methylcyclohex-1-
enyl)vinyl)-3,3-dimethylindole-1-sulfonate-5-carboxylic acid, 9a
2.8. Sodium 2-((E)-2-((E)-3-((Z)-2-(5-carboxy-3,3-dimethyl-3-(4-
sulfonatobutyl)lndolin-2-ylidene)ethylidene)-2-chlorocyclopent-1-
enyl)vinyl)-3,3-dimethylindole-1-sulfonate-5-carboxylic acid, 10
A vigorously stirred solution of 3-(5-carboxy-2,3,3-trimethyl-
3H-indolium-1-yl)propane-1-sulfonate (5, 0.107 g, 0.32 mmol) and
7a (0.05 g, 0.15 mmol) in acetic anhydride (1 mL) and acetic acid
(0.5 mL) was treated with sodium acetate (0.041 g, 0.5 mmol) and
heated at reflux (120 ꢀC) for 45 min. The reaction mixture was
cooled to ambient temperature and anhydrous diethyl ether
(5 mL) was added. The resulting precipitate was isolated by vac-
uum filtration to give a crude solid that was recrystallized
(methanol: water) to give 9a as a green solid (0.030 g, 0.04 mmol,
25%). Mp ¼ charring at 255e260 ꢀC; 1H NMR (400 MHz, MeOD)
d 8.20 (d, J ¼ 16 Hz, 2 H), 8.09e8.06 (m, 2 H), 7.34 (d, J ¼ 8 Hz,
2 H), 6.34 (d, J ¼ 12 Hz, 2 H), 4.21e4.18 (m, 4 H), 2.91e2.88 (m,
4 H), 2.66e2.63 (m, 4 H), 2.49 (s, 3 H), 2.05e1.90 (m, 10 H), 1.76 (s,
12 H); 13C NMR (100 MHz, MeOD) d 173.4, 158.4, 146.8, 142.3,
134.1, 124.6, 111.4, 102.6, 52.0, 45.2, 28.7, 27.4, 26.6, 23.8, 15.4;
HRMS (TOF). Calcd for C41H51N2O10S2 (Mþ þ H) 795.2985 Obs.
795.2964.
A vigorously stirred solution of 3-(5-carboxy-2,3,3-trimethyl-
3H-indolium-1-yl)propane-1-sulfonate (5, 0.197 g, 0.58 mmol) and
8 (0.1 g, 2.9 mmol) in acetic anhydride (1.5 mL) and acetic acid
(1 mL) was treated with sodium acetate (0.084 g, 1.02 mmol) and
heated at reflux (120 ꢀC) for 45 min. The reaction mixture was
cooled to ambient temperature and anhydrous diethyl ether
(10 mL) was added. The resulting filtrate was isolated by vacuum
filtration to give a crude solid that was recrystallized (methanol:
water) to give 10 as a green solid (0.06 g, 0.07 mmol, 25%).
Mp ¼ charring at 145e150 ꢀC; 1H NMR (400 MHz, MeOD) d 8.10e
8.08 (m, 4 H), 7.97 (d, J ¼ 16 Hz, 2 H), 7.40 (d, J ¼ 8 Hz, 2 H), 6.25 (d,
J ¼ 16 Hz, 2 H), 4.23 (t, J ¼ 8 Hz, 4 H), 2.90e2.87 (m, 4 H), 2.02e1.94
(m, 8 H), 1.75 (s, 12 H); 13C NMR (100 MHz, DMSO-d6) d 167.8, 167.0,
153.9, 148.6, 145.9, 141.2, 138.4, 137.3, 130.8, 123.4, 122.6, 103.9, 99.4,
53.8, 51.3, 51.1, 50.7, 48.8, 44.5, 28.2, 27.7, 27.4, 26.1, 23.0, 22.7, 22.5,
20.1; HRMS (TOF). Calcd for C39H45ClN2O10S2 (Mþ þ H) 801.2282
Obs. 801.2367.
2.6. Sodium 2-((E)-2-((E)-3-((Z)-2-(5-carboxy-3,3-dimethyl-3-(4-
sulfonatobutyl)lndolin-2-ylidene)ethylidene)-2-phenylcyclohex-1-
enyl)vinyl)-3,3-dimethylindole-1-sulfonate-5-carboxylic acid, 9b
2.9. N-((1E,3E)-3-(phenylimino)prop-1-enyl)benzenamine
hydrochloride (12)
A solution of propanedial bis(dimethyl acetal) (11, 5.25 mL,
0.03 mol) and HCl (4.25 mL) in distilled water (90 mL) was treated
with a solution of distilled water (70 mL), HCl (5 mL) and aniline
(3.7 mL, 0.04 mol) at 50 ꢀC. The resulting solution was stirred at
50 ꢀC for 30 min, cooled, and the resulting precipitate was isolated
by vacuum filtration and dried in vacuo to yield 12 as an orange
solid (6.35 g, 24.5 mmol, 81.8%) [24]. Mp ¼ charring at 150 ꢀC; 1H
NMR (400 MHz, CDCl3) d 8.72 (d, J ¼ 12 Hz, 2 H), 7.45e7.38 (m,
9 H), 7.24 (t, J ¼ 8 Hz, 2 H), 6.28 (t, J ¼ 12 Hz, 1 H); 13C NMR
(100 MHz, DMSO-d6) d 159.6, 139.8, 131.1, 131.0, 127.6, 118.8, 118.7,
99.5, 99.4.
A vigorously stirred solution of 3-(5-carboxy-2,3,3-trimethyl-
3H-indolium-1-yl)propane-1-sulfonate (5, 0.36 g, 1.06 mmol) and
7b (0.2 g, 0.5 mmol) in acetic anhydride (1.5 mL) and acetic acid
(1 mL) was treated with sodium acetate (0.139 g, 1.7 mmol) and
heated at reflux (120 ꢀC) for 45 min. The reaction mixture was
cooled to ambient temperature and anhydrous diethyl ether
(15 mL) was added. The resulting precipitate was isolated by vac-
uum filtration to give a crude solid that was recrystallized (meth-
anol: water) to give 9b as a green solid (0.025 g, 0.03 mmol, 57%).
Mp ¼ charring at 255e260 ꢀC; 1H NMR (400 MHz, MeOD) d 8.04 (d,
J ¼ 8 Hz, 2 H), 7.91 (s, 2 H), 7.63 (t, J ¼ 8 Hz, 3 H), 7.34 (t, J ¼ 8 Hz,
4 H), 7.26 (d, J ¼ 4 Hz, 2 H), 6.32 (d, J ¼ 12 Hz, 2 H), 4.16 (m, 4 H), 2.88
(t, J ¼ 8 Hz, 4 H), 2.77 (m, 4 H), 2.07 (m, 2 H), 1.93 (m, 8 H), 1.21 (s,
12 H); 13C NMR (100 MHz, MeOD) d 175.6, 169.2, 156.8, 147.4, 142.8,
132.3, 128.7,128.6, 124.6,112.0,106.0, 51.7, 50.4, 45.1, 27.9,27.2, 23.5;
HRMS (TOF). Calcd for C46H53N2O10S2 (Mþ þ H) 857.3142 Obs.
857.3097.
2.10. Sodium 4-[2-[(1E,3E,5Z)-7-[1,1-dimethyl-3-(4-sulfonatobutyl)
indol-2-ylidene]penta-1,3-dienyl]-1,1-dimethylindol-3-ium-3-yl]
butane-1-sulfonate, 13
A vigorously stirred solution of 3-(5-carboxy-2,3,3-trimethyl-
3H-indolium-1-yl)propane-1-sulfonate (5, 0.14 g, 0.41 mmol) and
12 (0.05 g, 0.19 mmol) in acetic anhydride (1 mL) and acetic acid
(0.5 mL) was treated with sodium acetate (0.054 g, 0.66 mmol)
and heated at reflux (120 ꢀC) for 45 min. The reaction mixture was
cooled to ambient temperature and anhydrous diethyl ether
(5 mL) was added. The resulting precipitate was isolated by vac-
uum filtration to give a crude solid that was recrystallized
(methanol: water) to give 13 as a blue solid (0.09 g, 0.12 mmol,
63.4%). Mp ¼ decomposition at 287 ꢀC; 1H NMR (400 MHz, MeOD)
d 12.98e12.92 (bs, 2 H), 8.43 (t, J ¼ 12 Hz, 2 H), 8.17 (s, 2 H), 7.99
(d, J ¼ 8 Hz, 2 H), 7.53 (d, J ¼ 8 Hz, 2 H), 6.74e6.67 (m, 1 H), 6.49 (d,
J ¼ 16 Hz, 2 H), 4.15 (m, 4 H), 1.18e1.76 (m, 8 H), 1.72 (s, 12 H); 13C
NMR (100 MHz, MeOD) d 175.7, 169.2, 156.8, 147.4, 142.8, 132.3,
128.7128.6, 124.6, 112.0, 106.0, 51.7, 50.4, 45.1, 27.9, 27.2, 23.5;
HRMS (TOF). Calcd for C35H43N2O10S2 (Mþ þ H) 715.2359 Obs.
715.2321.
All UVeVis spectra were recorded on Cary 50 spectrometer and
fluorescence spectra on a Cary Eclipse. Fluorescence was measured
in a 10 mm fluorescence cuvette and sample concentrations were
maintained below 0.1 absorbance by diluting the stock solutions to
the corresponding concentrations. All UV absorbance measure-
ments were done with solvent background correction, at the con-
centrations listed in Table 1.
2.7. Sodium 2-((E)-2-((E)-3-((Z)-2-(5-carboxy-3,3-dimethyl-3-(4-
sulfonatobutyl)lndolin-2-ylidene)ethylidene)-2-chlorocyclohex-1-
enyl)vinyl)-3,3-dimethylindole-1-sulfonate-5-carboxylic acid, 4 [15]
A vigorously stirred solution of 3-(5-carboxy-2,3,3-trimethyl-
3H-indolium-1-yl)propane-1-sulfonate (5, 0.1 g, 0.29 mmol) and
7c (0.05 g, 0.14 mmol) in acetic anhydride (1 mL) and acetic acid
(0.5 mL) was treated with sodium acetate (0.039 g, 0.47 mmol)
and heated at reflux (120 ꢀC) for 45 min. The reaction mixture was
cooled to ambient temperature and anhydrous diethyl ether
(5 mL) was added. The resulting precipitate was isolated by vac-
uum filtration to give a crude solid that was recrystallized
(methanol: water) to give 4 as a green solid (0.055 g, 0.07 mmol,
47%) [15]. Mp ¼ charring at 130e140 ꢀC; 1H NMR (400 MHz,
MeOD) d 8.49 (d, J ¼ 16 Hz, 2 H), 8.13-8.10 (m, 4 H), 7.45 (d,
J ¼ 8 Hz, 2 H), 6.43 (d, J ¼ 12 Hz, 2 H), 4.26 (t, J ¼ 8 Hz, 4 H), 2.90e
2.88 (m, 4 H), 2.80e2.77 (m, 4 H), 2.05e1.99 (m, 10 H), 1.77 (s,
12 H); 13C NMR (100 MHz, MeOD) d 174.9, 169.8, 152.1, 147.2, 146.3,
142.7, 132.4, 130.0, 124.7, 112.1, 103.7, 45.3, 28.5, 28.3, 27.4, 27.2,
23.6, 22.1; HRMS (TOF). Calcd for C40H48N2O10
815.2439 Obs. 815.2451.
S
(Mþ
þ
H)

I. Mohammad et al. / Dyes and Pigments 99 (2013) 275e283
279
Table 1
[10,29]. Ozinskas and co-workers prepared 5 by a similar route, in
98% yield [28]. In this later case, treatment of 2,3,3-trimethyl-3H-
indole-5-carboxylic acid with ethyl iodide at 120 ꢀC in a sealed tube
gave a N-ethyl derivative of 5 in 26% yield, whereas heating with
butanesultone at 180 ꢀC gave 5 in 82% yield [15]. Licha et al. pre-
pared 6 by a similar route [25]. In all cases the dyes were assembled
by condensation of 5 with the commercially available glutaconic
aldehyde dianilide$HCl [25,28,30]. In our hands, 2,3,3-trimethyl-
3H-indole-5-carboxylic acid was prepared in 68% yield, 5 in 88%
yield and 2 in 85% yield [10].
Dye solutions used for UVeVis analysis.
Dye in PBS
Stock solution concentration in
m
m
m
M
M
M
9a
9b
4
10
13
542.7
428.5
354.3
734.1
420.7
Dye in Ethanol
Stock solution concentration in
9a
9b
4
10
13
636.5
417.1
728.5
307.7
144.8
Both the dye 2 and dye-conjugates 3 showed absorption at about
754 nm, and emission at about 778 nm in PBS (see Scheme 3 and
Table 1) [10]. Spectral data for 2 had been reported previously in
DMSO, with emission at 817 nm and absorption at 794 nm, molar
absorbtivity of 286,000 and a fluorescence quantum yield of 0.12
[31]. Our stated goal was to modify the structure of 2 to increase ri-
gidity and thereby increase the fluorescent quantum yield. Any
structural modification of the polyene unit must produce a dye with
absorption/emission in the NIR region. In order to introduce rigidity
into the molecule, we targeted two new structural types: one
introduced a ring intothe polyeneunit as observed previously with 4,
but with modification of the group attached to the cyclohexene ring
and we included a five-membered ring derivative, and the second
type of structurally modification shortened the polyene chain.
N-Formyl-N-methylaniline (6) was prepared by the reaction of N-
methylaniline with formic acid and sodium formate [21], and then
treated first with POCl3 and then with 1-methylcyclohexene. As
shown in Scheme 2, subsequent reaction with aniline hydrochloride
gave anilide 7a in 39% yield [22]. 1-Phenylcyclohexene was con-
verted to 7b in 68% yield usingan identicalprocedure [22]. The direct
treatment of N-formyl-N-methylaniline with POCl3 in the presence
of cyclohexanone and aniline hydrochloride led to a 79% yield of 7c
[22]. The identical reactionwith cyclopentanone led to 8 in 73% yield
[23]. With these anilides in hand, coupling with indolium sulfonate 5
was accomplished using the standard procedure from our previous
work, as shown in Scheme 3: anilide and indole heated to 120 ꢀC in
acetic acid-acetic anhydride, buffered with sodium acetate [10].
Using this procedure, 7a was converted to 9a in 41% yield, 7b to 9b in
37% yield, 7c to 4 in 22% yield, and 8 to 10 in 25% yield. As noted
earlier, Tung and co-workers prepared 4 in 67% yield by heating
commercially available 7c, obtained from Aldrich, with 5 in ethanol
at reflux for two hours in the presence of sodium acetate [15].
We prepared a single example of the second target type (13, see
Scheme 4), with two carbons less in the polyene linker. Reaction of
the bis(dimethyl acetal) of propanedial (11) with aniline, in aqueous
HCl led to anilide 12 in 90% yield [24]. Subsequent reaction with
indole 5 under the standard conditions gave a 30% yield of dye 13.
Dye in 9.25% sucrose
Stock solution concentration in
9a
9b
4
10
13
681.4
731.9
700.2
1202.4
986.2
3. Results and discussion
3.1. Synthesis
The ultimate goal of our work is to develop nitroimidazole de-
rivatives covalently linked to indocyanine green derivatives for use
as NIR florescent dyes that target hypoxia in cancerous tumors. For
this purpose, we chose a bis(carboxylic acid) derivative of ICG as the
prototype dye, specifically 2. The choice was dictated by the need to
attach nitroimidazole units, and suggested by our previous work
that used functionalized indocyanine carboxylic acid derivatives
[10]. The fluorescence quantum yield of 2 (0.066) is slightly greater
than the commercial ICG (0.012) [10], probably due to the obser-
vation that formation of fluorescence-quenched aggregates is
suppressed because dyeedye interaction and aggregation is
reduced by hydrophilic, non-charged but sterically demanding
substituents [25,26]. It is noted that the synthesis of 2 may lead to a
mixture of stereoisomers due to rotation about each of the four CeC
single bonds of the polyene unit. As seen in Fig. 1 (a), which is the
HPLC trace for 1, there is a clear indication that this compound is a
mixture of stereoisomers. We can find no instance in the literature
where a similar stereochemical analysis was reported in connection
for a study. Our analysis of synthetic 2 in Fig. 1(b), on the other
hand, shows two peaks, indicating there are fewer stereoisomers
when compared with 1. Many polyenes exist as the lower energy
all-E isomer, but solvent effects as well as structural features of the
molecule can stabilize other isomers, leading to mixtures [27]. The
major stereoisomer of 2 may be the all-E isomer, but we have no
evidence to absolutely rule out other isomers. It is clear, however,
that 2 is relatively pure when compared to 1. The dyes used in our
study are dicarboxylic acid derivatives, were prepared in a manner
identical to 2, and we believe the stereochemistry of all dyes are
similar to 2. However, the compounds we have prepared, as well as
those similar compounds reported in the literature, are probably
not a single isomer and isomers would arise from bond rotation
associated with the polyene linker. The interactions of the polar
substituents incorporated into these dyes may lead to stabilization
and the relatively small mixture of observed isomers.
3.2. Absorption and fluorescence properties
All derivatives were characterized using an UVeVis spectro-
photometer and
a fluorescence spectrophotometer (Varian
Analytical Instruments, Walnut Creek, CA). The wavelength range
of both spectrophotometers is 250 nme1100 nm. The dyes were
suspended in either Phosphate Buffer Saline (PBS) or a 9.25%
aqueous sucrose solution, and the absorption and fluorescence
spectra were recorded. These two solvent systems were chosen to
be compatible with our previously reported in vivo studies with 3
and related compounds.
A straightforward synthesis of 2 was reported by Lindsey and
co-workers [28], in which the reaction of p-hydrazinobenzoic acid
with 3-methyl-2-butanone gave a 71% yield of 2,3,3-trimethyl-3H-
indole-5-carboxylic acid via a Fischer indole synthesis [10]. Sub-
sequent treatment with butanesultone gave a 32% yield of 3-(5-
Carboxy-2,3,3-trimethyl-3H-indolium-1-yl)propane-1-sulfonate, 5
Quantum yields were calculated relative to the standard sam-
ples that have a fixed fluorescence quantum yield value, according
to the following equation [32]:
ꢀ
ꢁ
FX
¼
FSTðGradX=GradSTÞ h2X=hS2T

280
I. Mohammad et al. / Dyes and Pigments 99 (2013) 275e283
Fig. 1. HPLC of commercial ICG dye 1 (a) and synthetic dye conjugate 2 (b).
Where ST ¼ standard and X ¼ unknown, respectively,
F
is the
enhancement in the quantum yield, with quantum yields ranging
from a low of 0.028 to a high of 0.34.
fluorescence quantum yield, Grad is the gradient from the plot of
integrated fluorescence intensity versus absorbance, and h the
refractive index of the solvent. Our results are shown in Table 2.
The florescence yield of 9a,b, 4, 10 and 13 were measured in
ethanol using Rh101 as the standard, in PBS and in a 9.25% sucrose
solution using 2 as the standard. All results are shown in Table 2. It
is clear that incorporating a ring into the polyene moiety as in 9a,b,
4 and 10 had little or no effect, or led to a slight decrease in the
quantum yield. Although each of these derivatives should be more
rigid in terms of rotation about the CeC bonds of the polyene, the
increase in the number of atoms may mitigate any benefits. On the
other hand, the chain-shortened derivative 13 showed a significant
Using Rh101 as the standard, the dye 13 absorbed at 657 nm and
emission was measured at 676 nm. These values are in the useful
range for NIR analysis, although a different filter would be required
for a NIR analysis using any dye conjugate prepared from 13 that is
structurally related to 3. Using 2, absorbance was at 655 nm and
emission at 669 nm in PBS, and absorbance at 655 nm and emission
at 672 nm in sucrose solution. The chloro and methyl derivatives (4
and 9a) showed slight shifts in the absorption and emission peaks,
relative to 2, although 10 showed a peak past 800 nm. However, the
emission peaks shifted to past 800 nm for all three using Rh101 and
for 9a,c using 2 (see Table 2). Tung and coworkers reported data for

I. Mohammad et al. / Dyes and Pigments 99 (2013) 275e283
281
Scheme 2. Synthesis of dianilides 7 and 8.
4 in methanol, and reported that 4 absorbs maximally at 790 nm
and emits maximally at 820 nm in methanol with a molar extinc-
tion coefficient of 184,000, and a quantum yield of 0.08 [15]. We
found that 4 absorbs at 812 nm and emits at 826 nm, in ethanol,
with a quantum yield of 0.025 and a molar extinction coefficient of
188,928. Our data was reported using Rh101 as a standard. Tung
used the same method to determine relative fluorescence quantum
yield, indicating the use of a standard, but that standard was not
identified. As seen in Table 2, phenyl derivative 9b did not show the
shifts observed for 4. In all cases, the quantum yield was less than 2,
although that of phenyl derivative 9b was close to 2.
It is clear from our results that incorporation of a ring in the
polyene chain does not increase the florescence yield. While the
ring may diminish the number of stereoisomers that arise due to
Scheme 3. Synthesis of dicarboxylic acid indocyanine dyes 9 and 10.

282
I. Mohammad et al. / Dyes and Pigments 99 (2013) 275e283
Scheme 4. Synthesis of polyene shortened dicarboxylic acid indocyanine dye 13.
rotation about the CeC bonds, any benefit may be offset by the
increased number of atoms. However, our analysis of isomers in
Fig. 1(b) suggests there are a limited number of isomers in the dye.
Loss of two carbon atoms from the polyene chain led to a significant
increase in fluorescent yield for 13. It is not clear why this relatively
minor structural change leads to such an increase, but one expla-
nation is that the diminished number of CeC bonds leads to fewer
isomers due to rotation. Since our analysis of isomers in Fig. 1(b)
suggests there are a limited number of isomers in the dye it is not at
all clear this is the explanation. It is certainly possible that fewer
structural isomers can account for the increase in florescence yield.
It is also possible that brining the two indole moieties closer
together may restrict rotation and lead to an increase in rigidity. At
this time our explanation remains speculative.
3.3. Optical stability in solution
The photostability of all dyes prepared for this study were
examined in both PBS solution and in 9.25% sucrose solution. The
dyes were dissolved in PBS solution and 9.25% sucrose solution, and
the absorbance of each solution was maintained at approximately
0.82. Each sample was irradiated under a 500 W halogen lamp,
maintained at a distance of 300 mm, for 14 h. An aqueous solution
of sodium nitrite (50.0 g/L) was placed between the samples and
the lamp to filter the light to less than 400 nm, and also to mitigate
heat from the lamp.
Table 2
The initial absorbance data was measured and then measure-
ments were carried out each hour. As shown in Fig. 2 (in PBS so-
lution) and Fig. 3 (in the sucrose solution), all dyes that contained a
ring, 9aec and 10, showed loss of absorbance over the course of
three hours. This data stands in sharp contrast to dye 13, which
showed significantly improved stability in both PBS and in sucrose.
It is noted that we also examined the stability in ethanol, but un-
avoidable evaporation of ethanol over the course of the experiment
led slight changes in concentration and we deemed the data un-
reliable. This experiment was repeated with the same result, so it is
not included here.
Photophysical properties of the dyes in different solvents against against Rh101 as
standard.
Dye
labs (in nm)
Rh101 standard
In ethanol
F
lemission (in nm)
Extinction coefficient
( 3 )
) (Mꢁ1cmꢁ1
2
759
783
775
796
820
657
778
803
791
811
840
676
191.362
182.534
250.265
188.928
144.783
210.347
0.146
0.024
0.127
0.059
0.041
0.344
9a
9b
4
10
13
Dye
labs (in nm)
Diacid 2 standard
In PBS
F
lemission (in nm)
Extinction coefficient
) (Mꢁ1cmꢁ1
(
3
)
2
756
774
766
789
812
655
778
791
779
802
826
669
236.868
183.510
209.539
210.351
254.619
220.417
0.066
0.024
0.055
0.028
0.025
0.289
9a
9b
4
10
13
Dye
labs (in nm)
Diacid 2 standard
In 9.25% sucrose soln.
F
lemission (in nm)
Extinction coefficient
( 3 )
) (Mꢁ1cmꢁ1
2
755
776
767
790
813
655
779
791
787
815
833
672
211.000
207.096
240.440
230.286
113.211
261.971
0.072
0.030
0.068
0.031
0.024
0.338
9a
9b
4
10
13
Fig. 2. Photostabilty of indocyanine dyes 4, 9a,b, 10, 13 in PBS solution.

I. Mohammad et al. / Dyes and Pigments 99 (2013) 275e283
283
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