Development of a near infrared fluorescence labeling reagent: Synthesis of indole-functionalized indocyanine green derivatives

Article abstract of DOI:10.1055/s-0031-1290139

We have demonstrated a facile synthesis of functionalized indocyanine green (ICG) derivatives. Heteroatom-substituted indolenine was synthesized via S _NAr reaction of 5-chloro-2,4-di-nitroanisole with 1,2-dimethyl-1-propenyl trimethylsilyl ether followed by reduction of the nitro groups. After the introduction of hydrophilic butanesulfonate moieties, homo- and heterocondensations with glutaconaldehyde dianilide provided symmetrical and unsymmetrical ICG derivatives, which exhibit near infrared (NIR) absorption and fluorescence emission similar to those of ICG. NIR fluorescence labeling reagent was synthesized using the amino group in the ICG derivative. The 1,3-dipolar cycloaddition with benzyl azide was performed utilizing copper nanoparticles toward a versatile method for the synthesis of NIR molecular imaging probes. Georg Thieme Verlag Stuttgart . New York.

Full text of DOI:10.1055/s-0031-1290139

306
LETTER
Development of a Near Infrared Fluorescence Labeling Reagent: Synthesis of
Indole-Functionalized Indocyanine Green Derivatives
I
ndole-Function
a
alized Indo
k
c
yanine
G
re
a
e
n
D
erivativ
y
e
s
uki Doi,*a Koya Oikawa,a Jun Suzuki,a Masahito Yoshida,a Nobuhiko Ikib
a
Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aza-aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan
Fax +81(22)7956864; E-mail: doi_taka@mail.pharm.tohoku.ac.jp
b
Graduate School of Environmental Studies, Tohoku University, 6-6-07 Aramaki-Aoba, Aoba-ku, Sendai 980-8579, Japan
Received 9 September 2011
Abstract: We have demonstrated a facile synthesis of functional-
ized indocyanine green (ICG) derivatives. Heteroatom-substituted
indolenine was synthesized via SNAr reaction of 5-chloro-2,4-di-
nitroanisole with 1,2-dimethyl-1-propenyl trimethylsilyl ether fol-
lowed by reduction of the nitro groups. After the introduction of
hydrophilic butanesulfonate moieties, homo- and heterocondensa-
tions with glutaconaldehyde dianilide provided symmetrical and
unsymmetrical ICG derivatives, which exhibit near infrared (NIR)
absorption and fluorescence emission similar to those of ICG. NIR
fluorescence labeling reagent was synthesized using the amino
group in the ICG derivative. The 1,3-dipolar cycloaddition with
benzyl azide was performed utilizing copper nanoparticles toward a
versatile method for the synthesis of NIR molecular imaging
probes.
N
N
SO3
SO3Na
indocyanin green (ICG) (1)
Figure 1
Z
Key words: cycloaddition, indoles, nucleophilic aromatic substitu-
tion, indocyanine green, near infrared fluorescence
Y
N
N
Indocyanine green (ICG, 1, Figure 1) is a near infrared
(NIR) fluorescent dye having large molar extinction coef-
ficients. It has been used as a noninvasive optical imaging
agent in clinical diagnosis.1 To design NIR-absorbing fluo-
rescence probes for in vivo imaging, various ICG deriva-
tives have been studied to identify structural components
that improve fluorescence efficiency, photostability,
quantum yield, solubility, molecular targeting, and so
on.2–9 Because ICG shows reversible binding to serum
proteins by both hydrophobic and hydrophilic properties,
we planned to displace the naphthalene ring of ICG to a
less hydrophobic heteroatom-substituted benzene ring
and expected to decrease nonspecific interaction with the
proteins.10 In this paper, we report an efficient synthetic
method for the preparation of functionalized symmetrical
and unsymmetrical ICG derivatives 2 with heteroatom
modifications at the indole moiety.
SO3
SO3Na
2
W
N
+
NHPh
PhN
•HCl
4
SO3
3
MeO
H2N
N
We set up indolenine 5 as a key synthetic intermediate, as
illustrated in Scheme 1. Because the amino group in 5 can
be easily converted into amide11 and five- or six-mem-
bered heterocycles can be newly formed on the basis of o-
aminophenol after demethylation of the anisidine moiety.
After introducing a hydrophilic sulfonate group, stepwise
condensation of 3 and 4 would yield symmetrical and un-
symmetrical ICG derivatives 2.
5
Scheme 1 Synthetic strategy for various ICG derivatives 2
Although Fischer indole synthesis is a conventional meth-
od for the synthesis of 2,3,3-trimethylindolenine,12 it is
not applicable to functionalized indolenine derivatives
owing to a lack of functional compatibility under harsh re-
action conditions and poor regioselectivity. After our in-
tensive studies for the preparation of 5, it was found that
aromatic nucleophilic substitution of 5-chloro-2,4-dini-
SYNLETT 2012, 23, 306–310
x
x.
x
x.
2
0
1
2
Advanced online publication: 03.01.2012
DOI: 10.1055/s-0031-1290139; Art ID: U06711ST
© Georg Thieme Verlag Stuttgart · New York

LETTER
Indole-Functionalized Indocyanine Green Derivatives
307
Table 1 SNAr Reaction of 6 with 7
AcONa, Ac2O, EtOH
R1
R2
50 °C for 3a
70 °C for 3b
+
NHPh
4 (0.5 equiv)
PhN
•HCl
N
OTMS
7
MeO
O2N
Cl
MeO
O2N
O
SO3
NO2
NO2
3a R1 = OMe, R2 = NHBoc
3b R1, R2 = OC(=O)NMe
6
8
R1
R1
R2
Entry
Additivea Solvent
Temp (°C) Time (h) Yield (%)
R2
N
N
1
2
3
KF
DMSO
DMSO
DMSO
60
r.t.
60
6
5
6
83
CsF
none
92
n.r.b
SO3
SO3Na
a The reagent was heated at 120 °C for 1 h in vacuo prior to use.
b No reaction was carried out and the starting material was recovered.
2a R1 = OMe, R2 = NHBoc (45%)
2b R1, R2 = OC(=O)NMe (41%)
2c R1 = OMe, R2 = NH2
TFA, CH2Cl2
r.t.
93%
H2, Pd/C
THF–MeOH
Scheme 3 Synthesis of symmetrical ICG derivatives 2a–c
MeO
r.t.
8
N
temperature (Table 1, entries 1 and 2). It is essential to use
the reagents dried at 120 °C in vacuo. In the absence of
fluoride, no reaction occurred (Table 1, entry 3). Note that
the reaction mixture turned blue as the reaction proceed-
ed. It is likely that the addition of the enolate of 7 to 6
produces a Meisenheimer-type complex, a known blue in-
termediate, which can undergo elimination of the chloride
ion, resulting in 8.16b,18
H2N
O
9
PBr3
MeO
THF, 0 °C
73% (2 steps)
N
RHN
5 R = H
Boc2O, NaHCO3
CH2Cl2–H2O
r.t.
10 R = Boc (90%)
O
SO2
NaHCO3
100 °C
MeO
11
AcONa, Ac2O
EtOH, r.t.
52%
N
BocHN
N
+
NHPh
PhN
•HCl
3a
4 (2 equiv)
SO3
1) AlCl3, toluene, reflux
2) carbonyl diimidazole
THF, r.t.
SO3
3c
3) MeI, K2CO3
11
120 °C
O
acetone, r.t.
3a or 3b
5
O
AcONa, Ac2O
EtOH, 50 °C
36% (3 steps)
95%
N
N
Me
Ph
N
N
12
Ac
O
N
O
O
O
+
N
N
N
Me
SO3
Me
13
SO3H
SO3
R1
R2
3b
3b'
Scheme 2 Preparations of 3a,b from 8
N
N
troanisole (6)13,14 with trisubstituted trimethylsilyl enol
ether 715 proceeded smoothly in DMSO using KF or CsF
to provide the desired 8.16,17 The results are shown in
Table 1. Addition of KF enhanced the reaction when heat-
ed at 60 °C (Table 1, entry 1). CsF was more effective
than KF, and the reaction was allowed to proceed at room
SO3
SO3Na
2d R1 = OMe, R2 = NHBoc (93%)
2e R1, R2 = OC(=O)NMe (75%)
Scheme 4 Synthesis of unsymmetrical ICG derivatives 2d and 2e
Synlett 2012, 23, 306–310
© Thieme Stuttgart · New York

308
T. Doi et al.
LETTER
Subsequently, the reduction of the nitro groups in 8 was separable mixture (30:70). Thus, the mixture was used
investigated (Scheme 2). Typical reduction conditions, directly for the coupling reaction with glutaconaldehyde
such as H2 and Pd/C, did not render complete reduction of dianilide (4).
the nitro groups to generate indolenine 5 and instead af-
According to a previous study, symmetrical 2a and 2b
forded nitrone 9. Further reduction of 9 using PBr3/THF19
were synthesized by the coupling of 3a and 3b, respec-
provided 5 in 73% overall yield. Reduction of 9 using
tively, with 0.5 equivalents of 4 using NaOAc–Ac2O on
TiCl3/aqueous HCl–THF also gave 5 in 65% yield, where-
as overreduction to indoline occurred when either TiCl4/
NaBH4/THF or SnCl2/CH2Cl2 was used. After the protec-
tion of amine 5 with a Boc group, N-alkylation of 10 with
1,4-butane sultone (11) was investigated. The desired
reaction did not proceed under conventional conditions
such as refluxing 1,2-dichlorobenzene, which resulted in
the removal of the Boc group.20 However, it was accom-
plished in the presence of sodium bicarbonate under sol-
ventless conditions11a to yield indolium salt 3a (52%).
Similarly, the formation of N-methylcarbamate 12 from 5
followed by N-alkylation with 11 afforded indolium 3b
along with its tautomer, 2-methyleneindoline 3b¢, as an in-
heating in EtOH (Scheme 3).11 Removal of the Boc group
in 2a using 50% TFA in CH2Cl2 provided 2c without de-
composition of the polyene moiety. Unsymmetrical 2d
and 2e were synthesized as follows (Scheme 4): Coupling
of 3c21 with two equivalents of 4 was performed at room
temperature. The resulting product 13 was unstable; there-
fore, the product was immediately passed through a short
pad of silica gel and used directly for the second coupling
reaction with 3a and 3b on heating in EtOH to yield 2d
and 2e, respectively.11 Similarly, 2f was synthesized by
the coupling of 3b and 4 at room temperature followed by
treatment with 3a at 50 °C (69% overall yield).
Table 2 Absorbance and Fluorescence Data of ICG Derivativesa
O
MeO
O
N
BocHN
FG1
Me
FG2
FG
C
B
A
D
=
MeO
MeO
N
N
MeO
H2N
HN
HN
O
O
SO3
SO3Na
E
F
2
N
N
N
Ph
Entry
Compd
ICG (1)
2a
FG1
FG2
Absorbance maximum (nm) eb (·105 M–1 cm–1) Fluorescence maximum (nm) fc
1
2
A
B
C
D
A
A
B
A
A
A
A
B
C
D
B
C
C
D
E
F
794
1.97
1.74
2.04
1.28
1.78
2.01
1.90
0.89
1.27
1.67
826
831
815
822
829
821
825
822
828
828
0.13d
0.032
0.12
792.5
780
3
2b
4
2c
814.5
793
0.001
0.017
0.052
0.012
0.003
0.015
0.012
5
2d
6
2e
787
7
2f
783.5
810.5
792
8
14
9
15
10
16
792
a All samples were measured in a 1 mM DMSO solution.
b Molar absorption coefficients.
c Fluorescence quantum yield based on ICG as a standard.
d Reported in ref. 23.
Synlett 2012, 23, 306–310
© Thieme Stuttgart · New York

LETTER
Indole-Functionalized Indocyanine Green Derivatives
309
For the synthesized compounds, absorption maxima, mo- ter. In fact, the acylation proceeded in MeOH at room
lar absorption coefficients (e), fluorescence emission temperature in the dark, providing 15 in 84% yield. Then,
maxima, and fluorescence quantum yields (f)22 are listed copper(I)-catalyzed 1,3-dipolar cycloaddition of 15 with
in Table 2. The absorption spectra of 2a,b, and 2d–f con- benzyl azide was investigated in t-BuOH–H2O (1:1) in the
tain intense maxima at 780–793 nm, which are similar to dark. We attempted two methods as follows: (A) CuSO4,
that of ICG (1, 794 nm). Their fluorescence spectra exhib- sodium ascorbate, room temperature25,27 and (B) Cu,
it fluorescence emission at 815–831 nm upon excitation at AlO(OH), room temperature.28 Both methods afforded
700 nm. All the dyes except for diamine 2c have a strong 1,4-disubstituted triazole 16 in 84% and 87% yields, re-
NIR absorbance and a 34–41.5 nm Stokes shift of the flu- spectively. Note that in method B copper nanoparticles in
orescence emission maxima. Note that the naphthalene the aluminum oxyhydroxide fiber catalyzed the reaction
rings in ICG can be replaced by heteroatom-substituted in t-BuOH–H2O without additives. As the catalyst was re-
benzene rings, such as structure units B and C shown in covered simply by filtration, the workup and purification
Table 2, in both symmetrical and unsymmetrical deriva- process was considerably simplified in comparison with
tives.
standard protocols. The absorption and fluorescence max-
ima of 15 and 16 are 792 and 828 nm, respectively, similar
to those of ICG (Table 2, entries 9 and 10 vs. entry 1). Al-
though the fluorescence quantum yield of 16 (f = 0.012 in
DMSO) was not as good as that of ICG (f = 0.13 in
DMSO),23 15 was found to be an NIR fluorescence label-
ing agent.29
The incorporation of a terminal alkyne into the dyes was
expected to permit fluorescence labeling by copper-cata-
lyzed 1,3-dipolar cycloaddition with an azide.24,25 There-
fore, we explored the introduction of a 5-hexynoyl group
onto the amino substituent of 2d (Scheme 5). Removal of
the Boc group in 2d with 50% TFA in CH2Cl2 at 0 °C af-
forded amine 14. Because 14 is highly hydrophilic and is In summary, we have demonstrated a facile synthetic
only soluble in an alcohol, we investigated the acylation method for the preparation of functionalized ICG deri-
of 14 with 5-hexynoic acid utilizing 4-(4,6-dimethoxy- vatives 2a–f. Furthermore, we have determined that a
1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride heteroatom-substituted benzene ring is a suitable replace-
(DMTMM),26 which has been utilized for the condensa- ment for the naphthalene ring of ICG. Utilizing an amino
tion of carboxylic acids and amines in an alcohol or in wa- group of 2d, NIR fluorescence labeling reagent 15 was
CO2H
Na2CO3
OMe
MeOH, r.t.
84%
N
N
NHR
MeO
N
Cl
N
N
N
O
Me
SO3
SO3Na
MeO
DMT-MM
TFA, CH2Cl2
0 °C, 99%
2d R = Boc
14 R = H
OMe
NH
Ph
N3
t-BuOH–H2O (1:1)
N
N
A: CuSO4, Na ascorbate,
r.t. (84%)
O
B: Cu/AlO(OH), r.t. (87%)
SO3
SO3Na
15
OMe
NH
N
N
O
SO3
SO3Na
N
N
N
Ph
16
Scheme 5 Synthesis of NIR fluorescence labeling reagent 15 and copper-catalyzed 1,3-dipolar cycloaddition of 15 with benzyl azide
© Thieme Stuttgart · New York
Synlett 2012, 23, 306–310

310
T. Doi et al.
LETTER
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synthesized. The 1,3-dipolar cycloaddition of 15 with
benzyl azide was achieved using copper nanoparticles.
The process developed herein can become a versatile
method for the synthesis of NIR molecular imaging
probes.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.
Acknowledgment
We thank Prof. Jaiwook Park (POSTEC, Korea) for a generous gift
of copper nanoparticles in aluminum oxyhydroxide fiber. We also
thank Prof. Hideo Takeuchi (Tohoku University) for his kind help
in the measurement of NIR absorption. This study was supported by
the Asahi Glass Foundation.
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