Tailored near-infrared contrast agents for image guided surgery

Article abstract of DOI:10.1021/acs.jmedchem.5b00253

The success of near-infrared (NIR) fluorescence to be employed for intraoperative imaging relies on the ability to develop a highly stable, NIR fluorescent, nontoxic, biocompatible, and highly excreted compound that retains a reactive functionality for co

Full text of DOI:10.1021/acs.jmedchem.5b00253

Article
pubs.acs.org/jmc
Tailored Near-Infrared Contrast Agents for Image Guided Surgery
Costyl N. Njiojob,†,§ Eric A. Owens,†,§ Lakshminarayana Narayana,†,§ Hoon Hyun,‡ Hak Soo Choi,*,‡
and Maged Henary*,†
†Department of Chemistry, Center for Diagnostics and Therapeutics and The Center for Biotechnology and Drug Design, Georgia
State University, 100 Piedmont Road SE, Atlanta, Georgia 30303, United States
‡Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School,
Boston, Massachusetts 02215, United States
S
* Supporting Information
ABSTRACT: The success of near-infrared (NIR) fluorescence to be
employed for intraoperative imaging relies on the ability to develop a highly
stable, NIR fluorescent, nontoxic, biocompatible, and highly excreted
compound that retains a reactive functionality for conjugation to a cancer-
recognizing peptide. Herein, systematic modifications to previously detailed
fluorophore ZW800-1 are explored. Specific modifications, including the
isosteric replacement of the O atom of ZW800-1, include nucleophilic
amine and sulfur species attached to the heptamethine core. These novel
compounds have shown similar satisfactory results in biodistribution and
clearance while also expressing increased stability in serum. Most
importantly, all of the synthesized and evaluated compounds display a
reactive functionality (either a free amino group or carboxylic acid moiety)
for further bioconjugation. The results obtained from the newly prepared
derivatives demonstrate that the central substitution with the studied
linking agents retains the ultralow background in vivo performance of the fluorophores regardless of the total net charge.
surface charge balanced with the meso-carboxylate balancing the
delocalized cation across the polymethine chain. Our previous
hypothesis suggests that disturbing this zwitterionic charge-
balanced system would sacrifice the optimum biological
clearance which we define as high renal excretion to the
urinary bladder within 4 h. To test the validity of this argument,
we must alter the net charge of the molecule and observe the
corresponding change in biodistribution properties. To fully
elucidate the structural characteristics of fluorophores that are
responsible for biodistribution and renal excretion, we modified
the nonresonant structure attached to the meso-carbon on the
fluorophore from the carboxylate seen in ZW800-1 (net zero
charge) to a primary amine that would be protonated at
physiological pH to yield an overall dicationic compound. The
Stokes shifts of heptamethine cyanines are very dependent on
the particular substituents that alter the stability of the excited
state; similarly, added structural stability to overcome photo-
bleaching of these compounds can be achieved using electron
donating groups, especially on the central carbon of the
polymethine chain. Increased Stokes shifts and structural
stability in solution are very desirable, and through the
incorporation of amino and sulfur moieties, a cyanine
fluorophore can become increasingly stable even in solution
when irradiated with light. Toward systematically probing this
INTRODUCTION
■
Near-infrared (NIR) based molecular imaging has emerged as
an effective technique for in vivo diagnostics. The numerous
advantages of this technique compared to other traditional
imaging techniques such as positron emission tomography
(PET) or single photon emission computed tomography
(SPECT) include the ability for real-time monitoring,1−3 the
use of nonionizing long wavelength deep tissue-penetrating
light, the increased signal-to-background ratios, cost effective-
ness, and tumor-targeting.4,5
Previously synthesized heptamethine NIR fluorophores,
including indocyanine green 1 (ICG, Figure 1), are nonideal
imaging agents because they exhibit elevated liver uptake, show
background signal in the gastrointestinal (GI) tract, demon-
strate inferior optical properties (low quantum yield/extinction
coefficient), are unstable or nonsoluble in aqueous media, and
are unable to conjugate covalently to targeting ligands.6−8 Our
previous efforts toward developing a NIR fluorophore for image
guided surgery yielded a novel zwitterionic heptamethine dye 2
(ZW800-1, Figure 1) with enhanced photophysical character-
istics (high extinction coefficient and quantum yield in serum)
and physiological properties such as fast systemic circulation,
reduced nonspecific binding, and rapid renal clearance.6
Through the development of ZW800-1, we concluded that
the balanced net zwitterionic charge directly influenced the
improved in vivo performance. The use of phenylsulfonato
(anionic) and alkylammonium (cationic) groups kept the
Received: February 12, 2015
© XXXX American Chemical Society
A
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Figure 1. Chemical structures of ICG and ZW800-1.
Scheme 1. Synthetic Route to Novel Zwitterionic NIR Fluorophores with Various meso-Substitutions
conjecture of charge-dependent biodistribution, we varied the
conjugating moiety attached to the meso-carbon for perfecting
synthetic, photophysical, and biological efficacy. We have
synthesized a number of complementary analogs (Scheme 1)
for structurally modifying the conjugating moiety of ZW800-1
(2, Figure 1) and evaluated them to ascertain the
biodistribution pattern with respect to the deviation from the
net charge which was initially zero. Furthermore, by replacing
B
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the carboxylic acid in ZW800-1 with an amine moiety, we now
have the option to conjugate other biologically relevant
molecules that possess a reactive carboxylic acid moiety in
lieu of a primary amine (i.e., folic acid, etc.). The results of this
study will provide more details about how to improve the
optical properties and in vivo biodistribution for future
implementation in the clinic.
Scheme 2. Synthesis of Aliphatic Amine Linker 12 and
Aromatic Amine Linker 14
RESULTS AND DISCUSSION
■
The chloro-substituted fluorophore 3 exhibits symmetric
quaternary ammonium cations, and sulfonate substituents
were prepared as already reported through salt condensation
with Vilsmeier−Haack reagent to build the heptamethine
carbocyanine core.6 The desired dyes shown in Scheme 1 were
synthesized by reacting the meso chlorine atom of the
fluorophore 3 with appropriate nucleophiles, through the
S
RN1 displacement pathway, in order to introduce a linking
to the increased nucleophilic nature of the alkylamine caused by
the available lone pair of electrons as compared to the aromatic
amine whose electrons are delocalized across the aromatic ring.
It has been reported that a substantial blue shift is observed in
the absorption wavelength when an aliphatic amine is attached
to the central carbon; however, this phenomenon is not
observed if the nucleophilic amine is aromatic. In order to
achieve substitution with the arylamine, we proceeded using
Boc protection of the aliphatic amine,12 followed by reduction
of the nitro group using ammonium formate in the presence of
Pd/C at reflux to afford the Boc protected amine 14 as depicted
in Scheme 2. This Boc protected amine 14 was used to replace
the chlorine fluorophore 3 followed by deprotection in
trifluoroacetic acid as elaborated in Scheme 1 to form
compound 7. The carboxylic acid derivative fluorophore 5
was prepared using similar protocol with commercially available
linker 3-(4-aminophenyl)propanoic acid.
Four new dyes containing the thiol linker were synthesized
by application of the SRN1 reaction. As highlighted in Scheme 1,
compounds 8 and 9 containing a carboxylic acid group were
synthesized from the reaction between commercially available
mercaptobenzoic acid and 2-(4-mercaptophenyl)acetic acid
respectively with the dye 3. These reactions were performed
using DMSO and water as an effective cosolvent system.
Purification of the crude was achieved by precipitation using
DMSO in ethyl acetate and by reverse phase column
chromatography using acetonitrile and water as the eluting
solvent.
The dyes 10 and 11 containing the thiol linker but with a
terminal amine group were obtained using the same procedure
as described above for dyes 8 and 9. After optimizing the
reaction conditions, we achieved more efficient synthesis with 6
molar equivalents of the 4-aminobenzenethiol being allowed to
react with 1 equiv of the dye 3 to provide compound 10. In
order to synthesize the final analog 11, the novel linker 18 had
to be prepared.
The synthesis of linker 18, highlighted in Scheme 3, was
initiated from the commercially available 4-hydroxybenzonitrile
which was treated with dimethylcarbamothioic chloride in the
presence of DABCO as base at 65 °C to provide O-(4-
cyanophenyl) N,N-dimethylcarbamothioate 15. Intermediate
15 was subjected to the Newman−Kwart rearrangement,13,14
which prompted the observed intramolecular aryl migration of
the O-thiocarbamate at elevated temperature to yield the
corresponding S-(4-cyanophenyl) dimethylcarbamothioate 16
in quantitative yield which was converted to the free thiol 17 by
treatment with potassium hydroxide in methanol.15−17 The
moiety (either carboxylic acid or amine) for subsequent
bioconjugation.9,10 Precursor 3 displays limited solubility in
dimethyl sulfoxide (DMSO) or dimethylformamide (DMF),
which has served as an ideal solvent for these reactions in the
past;6,11 however, because of the limited solubility of the
fluorophore 3 in these solvents, water was used to improve the
solubility of this fluorophore in DMSO, and this therefore led
to improved yields for these SRN1 reactions. Three nucleophilic
atoms (O, N, and S) were utilized to incorporate new linking
components containing either a terminal amine or carboxylic
acid to our highly appealing zwitterionic framework. Aromatic
amines and carboxylic acids are more difficult to couple via
conventional amide-coupling strategies because of electron
delocalization across the ring; therefore, we prepared both
compounds with the direct aromatic attachment and also with
carbon spacers between the aromatic system and the coupling
component. As a direct comparison to ZW800-1, we began the
synthesis by using boc-protected tyramine to link the phenolic
moiety to the central carbon of the methine chain, the final
ZW800-1 analog 4 displaying a free primary amine for
bioconjugated amide bond formation. The selective reactivity
at the oxygen atom was ensured by protection of the primary
aliphatic amine group prior to the reaction with the chloro dye.
The protonated oxygen exhibits limited nucleophilicity, and a
base is required to generate the phenoxide ion which is more
nucleophilic and is used in situ to react with the fluorophore 3
to provide the Boc protected product which is then converted
to the free amine product by treatment with trifluoroacetic acid
(TFA) in H2O at room temperature. Purification by
precipitation using DMSO in ethyl acetate followed by reverse
phase column chromatography provided the pure product 4.
Amino-substituted heptamethine cyanines were then pre-
pared to observe a change in biological and photophysical
properties; furthermore, we chose to study both an aliphatic
and aromatic amine attached to the meso-carbon. We
synthesized two substituted ZW800-1 analogs using the
replacement of the meso-chlorine by the diamine compound
shown in Scheme 2. The commercially available starting
material, 2-(4-nitrophenyl)ethanamine is reduced to the
diamino compound 12 using ammonium formate in Pd/C.
The diamino precursor 12 displays two nucleophilic positions,
and when the reaction is carried out with the chloro compound,
we observed a large blue shift coming from the attachment of
the aliphatic portion of the diamino precursor which afforded
compound 6. The selective nucleophilic reaction of the
aliphatic amine in the presence of the aromatic amine is due
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Scheme 3. Synthesis of Thiol Linker 18 Containing Aliphatic Amine
Figure 2. Absorbance and fluorescence spectra of the novel ZW800 analogs 4−11 recorded in 100% FBS, pH 7.4, at a 1 μM concentration (top).
Summary of the physicochemical and optical properties of the various novel synthesized NIR dyes in serum at pH 7.4 (bottom). Note: The peak
sizes of fluorescence spectra are arranged by the peak values of absorbance spectra to show them together as a single pair for clear observation of the
Stokes shift. Abbreviations used are the following: Ar designates that the moiety is attached to an aromatic system, while Alk indicates attachment to
an alkyl group.
resulting 4-mercaptobenzonitrile 17 was conveniently reduced
to 4-(aminomethyl)benzenethiol 18 by reaction with lithium
aluminum hydride.18 After preparation of the linker, it was
attached to the chloro compound 3 using similar reaction
conditions previously described. The difference between
compound 11 and the other compounds described was that
it took a shorter time to achieve complete conversion of the
product. It is important to note that because of the very high
nucleophilicity of the sulfur atom compared to the nitrogen
atom, no protecting group was required for the complete and
selective reaction of the sulfur with the nucleofugal carbon of
chloro compound 3 to afford the final ZW800-1 analogs 10 and
11.
Physicochemical Characteristics, Optical Properties,
and Stability Measurements. Figure 2 summarizes the
physicochemical and optical properties of the various
heptamethine ZW800-1 NIR analogs synthesized. These
measurements were either theoretically calculated using the
JChem plugin of ChemAxon or experimentally determined in
fetal bovine serum (FBS). It can be observed from Figure 2 that
the molecular weight and log D values are quite similar;
however, there are great differences concerning the pKa of the
D
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linker units and the functional moieties. Indeed, the net charge
of the compounds and the resulting biodistribution properties
are dependent on the pKa of each of these ionizable units and
the synthesized compounds span the breadth from a net charge
of 0 to +2 depending on the placement of carboxylic acids and
alkyl- or arylamines. From the optical measurements
enumerated in Figure 2, it can be observed that the molar
extinction coefficient of compound 4 is the highest, and this is
due to the fact that the oxygen atom linked to the polymethine
system has less effect on the polymethine chain compared to
the compounds possessing the S linker such as compounds 8−
11. Compounds 5−7 containing the amine linkers have the
lowest molar extinction coefficient probably because of the
competing resonance pathways leading to a broad absorption
spectrum.
As expected, the dyes containing the amine linkers have the
largest Stokes shift compared to the dyes possessing the oxygen
and sulfur linkers, as the nitrogen atom participates in an
intermolecular charge transfer which affords the large Stokes
shift.19 Also, as expected we observe from the table in Figure 2
that the quantum yield of compound 4 is the highest while the
dyes containing the amine linkers are the lowest. The stability
studies, highlighted in Figure 3, were also performed in serum
the urines from bladder were collected after 4 h of imaging and
the volumes and optical absorbance were measured. In Figure
5, compounds 4−7 containing the oxygen and amine linkers
showed higher excretion amounts over 80−90% ID during 4 h
postinjection, while compounds 8−11 containing sulfur linker
showed similar excretion amounts with ZW800-1 over 70−
80% ID. All of ZW800-1 analogs exhibited rapid and excellent
renal excretion for 4 h compared to ICG showing only 3% ID
in urine. In terms of the clinical use for safety and toxicity, we
demonstrated that the zwitterionic NIR fluorophores excreted
rapidly through renal filtration in certain time and showed
ultralow nonspecific backgrounds. Therefore, ZW800-1 analogs
may be good candidates for future in vivo applications such as
ligand−receptor specific targeting; accordingly, we plan to
exploit 9 and 11 with their increased stability in conjugated
imaging using various targeting ligands.
The increased stability is very important during surgical
resection of diseased tissue because the fluorophores must be
retained and demonstrate optimum imaging capabilities for
long periods in the body. These new sulfur-based fluorophores
have the potential to greatly assist the medical community in
these long-duration surgical resections, and we will begin
assessing their efficacy postconjugation, which will be discussed
in a separate manuscript.
CONCLUSIONS
■
We have demonstrated that the systematic modification of
zwitterionic heptamethine fluorophore ZW800-1 with various
charged linking functionalities does not have a significant
impact on the renal excretion yet is facilitated by only the
strong surface charge of the core. We can now utilize the amine
functionalized zwitterionic compounds for bioconjugation
without jeopardizing the effective clearance properties. The
sulfur atom bound to the cyanine core has increased the overall
stability of the fluorophore while allowing excellent optical and
clearance properties.
MATERIALS AND METHODS
Figure 3. Optical stability of ZW800-1 derivatives in 100% FBS, pH
7.4, at 37 °C for 24 h.
■
All chemicals and solvents were of American Chemical Society grade
or HPLC purity and were used as received. HPLC grade acetonitrile
(CH3CN) and water were purchased from VWR International (West
Chester, PA) and American Bioanalytic (Natick, MA), respectively. All
other chemicals were purchased from Fisher Scientific (Pittsburgh, PA,
USA), Sigma-Aldrich (Saint Louis, MO), and Acros Organics. The
reactions were followed using silica gel 60 F254 thin layer
chromatography plates (Merck EMD Millipore, Darmstadt, Germany).
Open column chromatography was utilized for the purification of all
hydrophobic final compounds using 60−200 μm, 60A classic column
silica gel (Dynamic Adsorbents, Norcross, GA). Highly charged final
products were isolated using revered phase C18 column chromatog-
at 37 °C for 24 h, and the results revealed that the least stable
was compound 4 because of the ether linker which can easily be
degraded, while the linkers containing sulfur atoms are larger
and more highly polarizable which leads to increased stability
without much influence on the polymethine resonance which is
confirmed in the optical spectra. Also, as expected, the final
compounds containing aliphatic amines expressed a better
stability than the aromatic amines which can be explained by
the better leaving group character in the aromatic anilide
leaving group supplied by the aromatic character versus the
primary amine.
Biological Studies and Comparison to ZW800-1. In
order to confirm the in vivo performances of the various
zwitterionic NIR fluorophores, the ZW800-1 analogs were
administered intravenously into CD-1 mice, and their
biodistribution and clearance were investigated in real time
over 4 h. As shown in Figure 4, all of ZW800-1 analogs were
eliminated from the body into urine by renal clearance with
almost no significant nonspecific uptake in other organs and
tissues, the same as the in vivo performance of ZW800-1,
although compounds 8 and 9 showed weak uptake in liver. To
compare the excretion amount of injected fluorophores in mice,
1
raphy (Fluka). The H NMR and 13C NMR spectra were obtained
using high quality Kontes NMR tubes (Kimble Chase, Vineland, NJ)
rated to 500 MHz and were recorded on a Bruker Avance (400 MHz)
spectrometer using D2O, DMSO-d6, or MeOD-d4 containing
tetramethylsilane (TMS) as an internal calibration standard set to
0.0 ppm. NMR abbreviations used throughout the experimental
section are as follows: s = singlet, d = doublet, t = triplet, q = quartet, p
= pentet, m = multiplet, dd = doublet doublets, and bs = broad singlet.
UV−vis/NIR absorption spectra were recorded on a Varian Cary 50
spectrophotometer. High-resolution accurate mass spectra (HRMS)
were obtained either at the Georgia State University Mass
Spectrometry Facility using a Waters Q-TOF micro (ESI-Q-TOF)
mass spectrometer or utilizing a Waters Micromass LCT TOF ES+
Premier mass spectrometer. The purity of each compound tested was
determined by using LC−MS instrument possessing a Waters 2487
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Figure 4. In vivo biodistribution and clearance of ZW800-1 derivatives. In silico calculations of minimized structures are calculated using density
functional theory at the B3LYP level: red, negative charge; blue, positive charge; green, hydrophobicity. 10 nmol of each NIR fluorophore was
injected intravenously into CD-1 mice 4 h prior to imaging. NIR fluorescence images for in vivo biodistribution (top) and resected organs (bottom)
have identical exposure and normalizations. Abbreviations used are the following: Bl, bladder; Du, duodenum; He, heart; In, intestine; Ki, kidneys; Li,
liver; Lu, lungs; Pa, pancreas; Sp, spleen; St, stomach. Scale bars are 1 cm.
single wavelength absorption detector with wavelengths set between
640 and 700 nm depending on the particular photophysical properties.
The column used in LC was a Waters Delta-Pak 5 μm 100A 3.9 mm ×
150 mm reversed phase C18 column, with a flow rate of 1 mL/min
employing a 5−100% acetonitrile/water/0.1 formic acid gradient. A
SEDEX 75 evaporative light scattering detection (ELSD) was also
utilized in tandem with liquid chromatography to confirm purity. All
compounds tested were >95% pure.
Synthesis. Mono(2-((E)-2-((E)-2-(4-(2-aminoethyl)phenoxy)-3-
((E)-2-(3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)-
F
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Mono(2-((E)-2-((E)-2-((4-aminophenethyl)amino)-3-((E)-2-(3,3-di-
methyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-
ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-
(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate) Tribro-
mide (6). To a solution of the chloro dye 3 (0.3 g, 0.273 mmol)
under N2, in a mixture of DMSO and water (5 mL/5 mL) was added
4- aminophenethylamine (0.11 g, 0.82 mmol), and the reaction
mixture was stirred at 90 °C for 1 h while monitoring by vis/NIR
spectrophotometry. Water was evaporated from the reaction mixture
on rotavapor. The residue was cooled to rt and then poured into ethyl
acetate (200 mL) while stirring. The precipitated product was filtered
and dried under vacuum. The obtained crude product was then
purified by reverse phase column chromatography to obtain the pure
target dye (0.21 g, 85%). Mp: 243−245 °C. Vis/NIR abs, λmax: 580
nm. 1H NMR (D2O): 7.96 (d, J = 6.8 Hz, 2H), 7.91 (s, 2H), 7.57 (d, J
= 11.6 Hz, 2H), 7.19 (br s, 4H), 6.99 (d, J = 8.0 Hz, 2H), 5.85 (d, J =
13.2 Hz, 2H), 4.23 (br s, 2H), 4.03 (br s, 4H), 3.59 (br s, 4H), 3.29 (s,
18H), 3.12 (br s, 2H), 2.56 (br s, 4H), 2.39 (br s, 4H), 1.82 (br s, 2H),
1.67 (s, 12H). 13C NMR (D2O): 166.52, 145.04, 140.18, 137.66,
136.82, 130.03, 126.86, 122.99, 119.88, 108.49, 95.13, 63.89, 53.34,
47.08, 39.57, 28.25, 25.28, 20.40. HRMS (TOF MS ES+) calcd for
C50H70N6O6S2: m/z 914.4798 ([M + H]+). Found: m/z 457.2389 [M
+ H/2]+.
Precursor to Dye 7. The pure Boc protected dye mono(2-((E)-2-
((E)-2-((4-(2-((tert-butoxycarbonyl)amino)ethyl)phenyl)amino)-3-
((E)-2-(3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)-
indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-
(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate) tribro-
mide was then subjected to deprotection using TFA and water (10
mL:10 mL) while stirring at rt and monitoring the reaction by vis/NIR
spectrophotometry. At the end of 2 h, solvents were evaporated from
the reaction mixture and the obtained solid product was dried under
vacuum to afford the target dye (1.0 g, 83%). Mp: 260−263 °C. Vis/
NIR abs, λmax: 705 nm. 1H NMR (D2O): 8.21 (br s, 2H), 7.98 (d, J =
6.4 Hz, 2H), 7.92 (s, 2H), 7.45 (d, J = 7.2 Hz, 2H), 7.39 (br s, 2H),
7.34 (br s, 2H), 6.19 (d, J = 8.0 Hz, 2H), 4.31 (br s, 4H), 3.64 (br s,
4H), 3.31 (s, 18H), 3.18 (br s, 2H), 2.72 (br s, 4H), 2.48 (br s, 4H),
2.02 (br s, 2H), 1.53 (s, 12H), 1.52 (s, 2H). 13C NMR (DMSO-d6):
170.66, 160.26, 145.72, 145.08, 142.86, 142.58, 140.22, 130.48, 129.97,
126.54, 124.99, 120.07, 118.37, 109.72, 98.81, 63.17, 53.00, 48.45,
32.85, 28.16, 24.71, 21.83, 20.98. HRMS (TOF MS ES+) calcd for
C50H70N6O6S2: m/z 914.4798 ([M + H]+). Found: m/z 457.2393 [M
+ H/2]+.
Mono(2-((E)-2-((E)-2-((4-(2-aminoethyl)phenyl)amino)-3-((E)-2-
(3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-
2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-
(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate) Tribro-
mide (7). To a solution of 4-nitrophenethylamine hydrochloride
(1.0 g, 4.94 mmol) in ethyl acetate (15 mL) under N2 was added
triethylamine (0.75 g, 7.41 mmol) followed by Boc anhydride (1.18 g,
5.43 mmol), and the reaction mixture was stirred at rt for 17 h. The
reaction mixture was then diluted with ethyl acetate (25 mL) and was
washed with water (2 × 30 mL), dried over Na2SO4 and concentrated
to afford 1.3 g of tert-butyl 4-nitrophenethylcarbamate as a pale yellow
solid (quantitative). To a solution of the above tert-butyl 4-
nitrophenethylcarbamate (1.3 g, 4.94 mmol) in methanol (25 mL)
was added ammonium formate (1.56 g, 24.7 mmol) followed by 10%
palladium on carbon (130 mg), and the reaction mixture was refluxed
for 24 h. The reaction mixture was filtered through Celite and washed
with methanol. The filtrate and washings were combined, and the
solvent was then evaporated on rotavapor, diluted with ethyl acetate
(50 mL), and washed with water (3 × 40 mL). The organic layer was
then dried over Na2SO4 and concentrated to afford 1.04 g of tert-butyl
4-aminophenethylcarbamate (89%) which was used in the next step
without further purification. To a solution of the above, tert-butyl 4-
aminophenethylcarbamate (1.0 g, 4.24 mmol) in a mixture of DMSO
and water (20 mL:20 mL) under N2 was added the chloro dye 3 (1.55
g, 1.41 mmol), and the reaction mixture was stirred at 90 °C for 2 h
while monitoring by vis/NIR spectrophotometry. Water was
evaporated from the reaction mixture on rotavapor. The residue was
cooled to rt and poured into ethyl acetate (300 mL) while stirring. The
Figure 5. Renal excretion (% ID) of ZW800-1 derivatives. ZW800-1
and ICG are used as controls (N = 5).
indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-
1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate) Tri-
bromide (4). To a solution of tyramine (1.25 g, 9.112 mmol) in
DMF (20 mL) under N2, was added triethylamine (1.38 g, 13.67
mmol) followed by Boc anhydride (2.98 g, 13.67 mmol), and the
reaction mixture was stirred at rt for 2 h. To the above solution under
N2 was then added sodium hydride (0.22 g, 9.112 mmol), and the
mixture was stirred at rt for 1 h. To the above was then added the
chloro dye 3 (0.66 g, 0.6 mmol) under N2, and the reaction mixture
was stirred at rt for 17 h while monitoring by vis/NIR
spectrophotometry. To the above solution was then added a mixture
of TFA and water (10 mL/10 mL), and the reaction mixture was
stirred at rt for 2 h. The solvents were then evaporated from the
reaction mixture on rotavapor at 60 °C, and the obtained residue was
stirred with ethyl acetate (200 mL), filtered, and dried under vacuum.
The obtained crude product was stirred in methanol (100 mL),
filtered, and dried under vacuum to afford the pure dye as a green solid
(0.44 g, 80%). Mp: 230−232 °C. Vis/NIR abs, λmax: 770 nm. 1H NMR
(D2O): 7.85 (d, J = 14.0 Hz, 2H), 7.72 (d, J = 8.4 Hz, 2H), 7.69 (s,
2H), 7.26 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 8.4
Hz, 2H), 6.07 (d, J = 14.0 Hz, 2H), 4.10 (br s, 4H), 3.41 (br s, 4H),
3.04 (s, 18H), 2.88 (br s, 2H), 2.55 (br s, 4H), 2.22 (br s, 4H), 1.84
(br s, 2H), 1.23 (s, 12H). 13C NMR (D2O): 172.58, 164.51, 158.77,
143.56, 142.61, 141.17, 139.81, 131.31, 130.87, 126.83, 124.33, 119.72,
115.10, 110.72, 100.90, 63.09, 53.03, 48.81, 40.62, 32.13, 27.18, 23.78,
20.73. HRMS (TOF MS ES+) calcd for C50H69N5O7S2: m/z 915.4638
([M + H]+). Found: m/z 457.7321 [M + H/2]+.
Mono(2-((E)-2-((E)-2-((4-(2-carboxyethyl)phenyl)amino)-3-((E)-2-
(3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-
2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-
(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate) Tribro-
mide (5). To a solution of the chloro dye 3 (1.0 g, 0.91 mmol) in a
mixture of DMSO and water (15 mL/15 mL) under N2, was added 3-
(4-aminophenyl)propionic acid (0.45 g, 2.73 mmol), and the reaction
mixture was stirred at 90 °C for 2h while monitoring by vis/NIR
spectrophotometry. Water was then evaporated from the reaction
mixture. The residue was cooled to room temperature and poured into
ethyl acetate (300 mL) while stirring. The precipitated product was
filtered, washed with ethyl acetate, and dried under vacuum. The crude
material was dissolved in DMSO (70 mL) and methanol (20 mL) and
poured into ethyl acetate (400 mL). The precipitated product was
filtered and dried under vacuum. This precipitation procedure was
repeated once more and the obtained pure product was dried under
vacuum (0.67 g, 78%). Mp: 263−265 °C. Vis/NIR abs, λmax: 700 nm.
1H NMR (D2O): 8.14 (d, J = 12.8 Hz, 2H), 7.94 (d, J = 8.0 Hz, 2H),
7.84 (s, 2H), 7.35−7.25 (m, 4H), 7.19 (d, J = 7.6 Hz, 2H), 6.12 (d, J =
13.6 Hz, 2H), 4.17 (br s, 4), 3.61 (br s, 4H), 3.29 (s, 18H), 2.91 (br s,
2H), 2.75−2.65 (br s, 6H), 2.41 (br s, 4H), 1.99 (br s, 2H), 1.29 (s,
12H). 13C NMR (DMSO-d6): 173.98, 170.45, 160.78, 144.96, 142.67,
140.19, 133.98, 130.05, 126.49, 124.87, 120.06, 118.52, 109.69, 98.59,
63.12, 53.00, 48.44, 35.69, 30.07, 28.21, 24.83, 21.90, 21.02. HRMS
(TOF MS ES+) calcd for C51H69N5O8S2: m/z 943.4588 ([M + H]+).
Found: m/z 471.7291 [M + H/2]+.
G
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Journal of Medicinal Chemistry
Article
precipitated crude product was then filtered and dried under vacuum.
Final purification was achieved by reverse phase column chromatog-
raphy to obtain the Boc protected dye (1.14 g, 1.128 mmol, 80%). Mp:
250−252 °C. Vis/NIR abs, λmax: 705 nm. 1H NMR (D2O): 8.14 (d, J
= 12.8 Hz, 2H), 7.95 (d, J = 7.6 Hz, 2H), 7.84 (br s, 2H), 7.34 (d, J =
8.0 Hz, 2H), 7.29 (br s, 2H), 7.22 (br s, 2H), 6.15 (d, J = 13.2 Hz,
2H), 4.23 (br s, 4H), 3.65 (br s, 4H), 3.31 (s, 18H), 2.79 (br s, 2H),
2.68 (br s, 4H), 2.45 (br s, 6H), 1.96 (br s, 2H), 1.41 (s, 12H), 1.36 (s,
9H). 13C NMR (DMSO-d6): 170.43, 160.84, 145.07, 144.97, 142.73,
142.64, 140.16, 132.56, 130.36, 126.50, 124.73, 120.07, 118.68, 109.64,
98.53, 78.01, 63.14, 53.00, 48.42, 28.73, 28.23, 24.83, 21.91, 21.01.
Mono(2-((E)-2-((E)-2-((4-carboxyphenyl)thio)-3-((E)-2-(3,3-di-
methyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-
ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-
(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate) Tribro-
mide (8). To a solution of the chloro dye 3 (0.5 g, 0.613 mmol)
under N2 in a mixture of DMSO and water (10 mL/10 mL) was added
4-mercaptobenzoic acid (0.24 g, 1.55 mmol), and the reaction was
stirred at rt for 30 min while monitoring by vis/NIR spectropho-
tometry. After complete conversion of the starting material, water was
evaporated from the reaction mixture on rotavapor and the residue was
cooled to rt and then poured into ethyl acetate (200 mL) while
stirring. The precipitated product was filtered and dried under vacuum
to obtain the pure target dye as a green solid (0.52 g, 91%). Mp: 250−
255 °C. Vis/NIR abs, λmax: 791 nm. 1H NMR (D2O): 8.62 (d, J = 13.6
Hz, 2H), 7.96 (d, J = 7.2 Hz, 4H), 7.79 (s, 2H), 7.51(d, J = 8.4 Hz,
2H), 7.38 (d, J = 8.4 Hz, 2H), 6.46 (d, J = 14 Hz, 2H), 4.37 (s, 4H),
3.71 (br s, 4H), 3.30 (s, 18 H), 2.77 (br s, 4H), 2.45 (br s, 4H), 1.93
(s, 2H), 1.43 (s, 12H). 13C NMR (DMSO): 172.87, 167.12, 148.61,
146.11, 143.12, 142.29, 140.97, 134.49, 130.95, 128.58, 126.65, 126.21,
120.27, 111.07, 102.89, 62.83, 52.90, 49.37, 40.89, 27.59, 26.46, 21.34.
evaporated to dryness to obtain the pure target dye as a green solid
(0.63 g, 77%). Mp: 210−212 °C. Vis/NIR abs, λmax: 790 nm. 1H NMR
(D2O, 50 °C): 8.80 (d, J = 14.0 Hz, 2H), 8.00 (d, J = 8.0 Hz, 2H), 7.95
(s, 2H), 7.55−7.45 (m, 4H), 7.31 (d, J = 7.2 Hz, 2H), 6.47 (d, J = 14.4
Hz, 2H), 4.42 (br s, 4H), 3.69 (br s, 4H), 3.30 (s, 18H), 2.84 (br s,
4H), 2.50 (br s, 4H), 2.05 (br s, 2H), 1.60 (s, 12H). 13C NMR
(DMSO-d6, 50 °C): 172.86, 146.16, 145.80, 142.41, 142.33, 140.96,
134.71, 128.13, 126.71, 121.97, 120.18, 111.04, 102.77, 63.01, 53.06,
52.91, 49.34, 41.58, 27.83, 27.73, 26.57, 21.39, 21.00. HRMS (TOF
MS ES+) calcd for C48H65N5O6S3: m/z 903.4097 ([M + H]+). Found:
m/z 451.7050 [M + H/2]+.
Mono(2-((E)-2-((E)-2-((4-(aminomethyl)phenyl)thio)-3-((E)-2-(3,3-
dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-
ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-
(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate) Tribro-
mide (11). To a solution of the chloro dye 3 (1.2 g, 1.47 mmol)
under N2 in a mixture of DMSO and water (10 mL/10 mL) was added
4-(aminomethyl)benzenethiol (1.23 g, 8.83 mmol), and the reaction
was stirred at rt for 3 h while monitored by vis/NIR spectropho-
tometry. After complete conversion of the starting material, water was
evaporated from the reaction mixture on rotavapor and the residue was
cooled to rt and then poured into ethyl acetate (300 mL) while
stirring. The precipitated product was filtered and dried under vacuum.
The obtained crude product was then purified by reverse phase
column chromatography using water and acetonitrile as the mobile
phase to obtain the pure target dye as a green solid. (0.7 g, 52%). Mp:
240−245 °C. Vis/NIR abs, λmax: 790 nm. 1H NMR (DMSO): 8.63 (d,
J = 13.6 Hz, 2H), 8.36 (s, 2H), 7.72 (d, 2H), 7.62 (d, J = 8.0 Hz, 2H),
7.47 (m, J = 8.0 Hz, 4H), 7.32 (d, J = 8.0 Hz, 2H), 6.41(d, J = 14.0 Hz,
2H), 4.24 (s, 4H), 3.92 (br s, 2H), 3.58 (t, 4 H), 3.10 (s, 18H), 2.82
(br s, 4H), 2.15 (br s, 4H), 1.92 (s, 2H), 1.44 (s, 12H)). 13C NMR
(DMSO): 172.79, 149.88, 145.96, 145.66, 142.33, 140.95, 137.51,
134.62, 132.20, 130.89, 126.60, 120.25, 111.07, 102.87, 62.83, 52.90,
49.33, 41.98, 40.88, 27.74, 26.49, 21.34. LCMS ESI TOF calcd for
+
LCMS ESI TOF calcd for C49H63N4O8S3 : m/z 932.2483 ([M + H]+).
Found: m/z 466.1337 [M + H/2]+.
Mono(2-((E)-2-((E)-2-((4-(carboxymethyl)phenyl)thio)-3-((E)-2-
(3,3-dimethyl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-
2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-
(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate) Tribro-
mide (9). To a solution of the chloro dye 3 (0.5 g, 0.61 mmol)
under N2 in a mixture of DMSO and water (10 mL/10 mL) was added
2-(4-mercaptophenyl)acetic acid (0.26g, 1.52 mmol), and the reaction
was stirred at rt for 1 h while monitoring by vis/NIR
spectrophotometry. After complete conversion of the starting material,
water was evaporated from the reaction mixture on rotavapor and the
residue was cooled to rt and then poured into ethyl acetate (300 mL)
while stirring. The precipitated product was filtered and dried under
vacuum. The obtained crude product was then purified by reverse
phase column chromatography using water and methanol as the
mobile phase to obtain the pure target dye as a green solid (0.42 g,
71%). Mp: 248−250 °C. Vis/NIR abs, λmax: 791 nm. 1H NMR
(DMSO): 8.64 (d, J = 14 Hz, 2H), 7.70 (s, 4H), 7.59 (d, J = 8.4, 2H),
7.41(d, J = 8.4 Hz, 2H), 7.22 (s, 4H), 6.37 (d, J = 14 Hz, 2H), 4.21 (s,
4H), 3.51 (br s, 4H), 3.08 (s, 18 H), 2.79 (br s, 4H), 2.14 (br s, 4H),
1.92 (s, 2H), 1.38 (s, 12H)). 13C NMR (DMSO): 172.88, 172.65,
150.64, 146.18, 145.80, 142.30, 140.98, 135.27, 134.62, 133.49, 131.19,
126.69, 126.55, 120.19, 111.02, 102.76, 63.03, 53.06, 49.35, 41.63,
+
C49H66N5O6S3 : m/z 917.4242 ([M + H]+). Found: m/z 458.1678 [M
+ H/2]+.
Synthesis of Linkers. O-(4-Cyanophenyl) Dimethylcarbamo-
thioate (15). A 250 mL flask equipped with a magnetic stirring bar was
charged with 4-hydroxybenzonitrile (5.0 g, 41.9 mmol), DABCO (9.41
g, 83.8 mmol), and 10 mL of DMF. To this well stirred mixture was
added N,N-dimethylthiocarbamoyl chloride (6.21 g, 50.28 mmol). The
resulting mixture was heated to 65 °C and monitored by TLC until
complete disappearance of the starting material. The reaction mixture
was poured into a beaker containing ice, and the pH of the solution is
reduced to 2 by adding HCl. The crystals formed are filtered, and
further purification is accomplished by recrystallization in ethanol to
afford the pure compound 15 as a cream white solid (8.0 g, 93%). 1H
NMR (CDCl3): 7.67 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H),
3.42 (s, 3H), 3.33 (s, 3H). 13C NMR (CDCl3): 186.30, 157.10, 133.36,
124.22, 118.38, 109.62, 43.38, 39.01.
S-(4-Cyanophenyl) Dimethylcarbamothioate (16). To a 250 mL
round-bottom flask equipped with a magnetic stir bar and a reflux
condenser was charged with O-(4-cyanophenyl) dimethylcarbamo-
thioate (4.80 g, 23.27 mmol). The contents of the flask were
maintained under N2 and immersed in a preheated oil bath maintained
at 200 °C while stirring. The reaction was monitored by TLC, and
complete disappearance of the starting material was observed after 4 h
of reaction. After completion of the reaction, the content of the flask
solidified when cooled to rt. This provided the pure product 16 which
was used in the next step without further purification ((4.50 g, 94%).
1H NMR (CDCl3): 7.64−7.58 (m, J = 8.0 Hz, 4H), 3.08−3.03 (d,
+
27.81, 26.56, 21.39. LCMS ESI TOF calcd for C50H65N4O8S3 : m/z
946.4031([M + H]+). Found: m/z 473.1717 [M + H/2]+.
Mono(2-((E)-2-((E)-2-((4-aminophenyl)thio)-3-((E)-2-(3,3-dimeth-
yl-5-sulfonato-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)-
ethylidene)cyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-1-(3-
(trimethylammonio)propyl)-3H-indol-1-ium-5-sulfonate) Tribro-
mide (10). To a solution of the chloro dye 3 (1.0 g, 0.91 mmol) in
a mixture of DMSO and water (15 mL/15 mL) under N2, was added
4-aminothiophenol (0.68 g, 5.45 mmol), and the reaction mixture was
stirred at rt for 17h while monitoring by vis/NIR spectrophotometry.
Water was evaporated from the reaction mixture on rotavapor, the
residue was cooled to rt and was then poured into ethyl acetate (200
mL) while stirring. The precipitated product was filtered and dried
under vacuum. The obtained crude product was then dissolved in
water (30 mL) and was washed with dichloromethane (3 × 40 mL)
followed by ethyl acetate (3 × 40 mL) and the aqueous layer was
6H). 13C NMR (CDCl3): 164.83, 136.36, 133.24, 118.40, 112.45,
37.05
4-Mercaptobenzonitrile (17). To a 250 mL round-bottom flask
equipped with a magnetic stir bar was added S-(4-cyanophenyl)
dimethylcarbamothioate 13 (4.0 g, 19.4 mmol) and dissolved in THF.
To this solution was added KOH (2.40 g, 16.27 mmol) dissolved in
MeOH, and this was stirred at rt for 3 h while monitoring with TLC.
After completion of the hydrolysis, the mixture was poured into
crushed ice, acidified with HCl to attain a pH value of 2, and stirred
H
DOI: 10.1021/acs.jmedchem.5b00253
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
well until the oil solidified. The precipitate formed was collected,
ACKNOWLEDGMENTS
■
washed with cold water, and dried to yield the desired product as a
1
The research was supported through the Brains and Behavior
grant to M.H. and through the National Institutes of Health
Grant NIBIB R01-EB-011523 to H.S.C. E.A.O. was supported
through the Center for Diagnostics and Therapeutics at
Georgia State University.
light brown solid (2.20 g, 84%). H NMR (CDCl3): 7.49 (d, J = 8.0
Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 3.72 (s, 1H). 13C NMR (CDCl3):
139.30, 132.52, 128.70, 118.66, 108.73.
4-(Aminomethyl)benzenethiol (18). 4-Mercaptobenzonitrile (2.0 g,
14.8 mmol) was dissolved in anhydrous THF. The solution was added
dropwise to a stirred suspension of lithium aluminum hydride (0.67 g,
17.7 mmol) in THF under N2 atmosphere. This suspension was slowly
heated to reflux. After 4 h, the excess hydride was removed by adding
ethyl acetate followed by methanol and filtered. The filtrate was
poured into crushed ice and the precipitate filtered, collected, and
dried to yield the desired product as a white solid (1.82 g, 89%). Mp:
ABBREVIATIONS USED
■
NIR, near-infrared; SPECT, single photon emission computed
tomography; ICG, indocyanine green; ZW800-1, zwitterionic
heptamethine dye; SRN1, unimolecular radical nucleophilic
substitution; DMSO, dimethyl sulfoxide; DMF, dimethylfor-
mamide; TFA, trifluoroacetic acid; DABCO, 1,4-diazabicyclo-
[2.2.2]octane; FBS, fetal bovine serum; % ID, percent injected
dose
1
215−218 °C. H NMR (CDCl3): 8.75 (br s, 2H), 7.56−7.51 (m, J =
8.0 Hz, 4H), 3.95 (s, 2H), 3.47 (s, 1H). 13C NMR (CDCl3): 136.10,
134.11, 130.73, 127.47, 41.95.
Determination of Optical Properties and Prediction of
Physicochemical Descriptors. All optical measurements were
performed at 37 °C in 100% fetal bovine serum (FBS) buffered
with 50 mM HEPES, pH 7.4. Absorbance and fluorescence emission
spectra of the series of NIR fluorophores were measured using fiber
optic HR2000 absorbance (200−1100 nm) and USB2000FL
fluorescence (350−1000 nm) spectrometers (Ocean Optics, Dunedin,
FL). NIR excitation was provided by 8 mW of either 655 or 765 nm
NIR laser diode light source (Electro Optical Components, Santa
Rosa, CA) (depending on the exact chemical structure) coupled
through a 300 μm core diameter, NA 0.22 fiber (Fiberguide Industries,
Stirling, NJ). For fluorescence quantum yield (QY) measurements,
oxazine 725 in ethylene glycol (QY = 19%) and ICG in DMSO (QY =
13%) were used as a calibration standards, under conditions of
matched absorbance at 655 and 765 nm. In silico calculations of the
partition coefficient (log D at pH 7.4) and pKa were done using
Marvin and JChem calculator plugins (ChemAxon, Budapest,
Hungary). Molecular skeleton (2D) and hydrophobicity (3D)
structures were minimized and calculated using Spartan density
function theory calculations at the B3LYP level using the 6-311+
+G(2df,2pd) basis set.
Biological Fluorescence Imaging. Animals were housed in an
AAALAC-certified facility and were studied under the supervision of
BIDMC IACUC in accordance with approved institutional protocol
(no. 101-2011 for rodents). Male CD-1 mice weighing ∼25 g (Charles
River Laboratories, Wilmington, MA) were anesthetized with 100 mg/
kg ketamine and 10 mg/kg xylazine intraperitoneally (Webster
Veterinary, Fort Devens, MA). For in vivo fluorescence imaging in
real-time, the FLARE imaging system has been described in detail
previously.20−22 In this study, 11 mW/cm2 of 760 nm excitation light
was used with white light (400−650 nm) at 40 000 lx. Color and NIR
fluorescence images were acquired simultaneously with custom
software at rates up to 15 Hz over a 15 cm diameter field of view.
The imaging system was positioned at a distance of 18 in. from the
surgical field. For each experiment, camera exposure time and image
normalization were held constant.
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ASSOCIATED CONTENT
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S
* Supporting Information
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AUTHOR INFORMATION
Corresponding Authors
*H.S.C.: phone, 617-667-6024; e-mail, hchoi@bidmc.harvard.
■
edu.
*M.H.: phone, 404-413-5566; e-mail, mhenary1@gsu.edu.
Author Contributions
§C.N.N., E.A.O., and L.N. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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