A highly efficient method for the N-alkylation of aminopyrazines: Synthesis of hydrophilic red fluorescent dyes

Article abstract of DOI:10.1055/s-0029-1220009

A robust and scalable method was developed for the synthesis of N,N-dialkylated aminopyrazines by a reductive amination route. These new fluorescent pyrazine analogues were shown to absorb and emit light at relatively long wavelengths (～50 nm) compared to the corresponding diaminopyrazines. The utility of these compounds was demonstrated by the synthesis of hydrophilic fluorescent probes with potential diagnostic applications. Georg Thieme Verlag Stuttgart · New York.

Full text of DOI:10.1055/s-0029-1220009

PAPER
2383
A Highly Efficient Method for the N-Alkylation of Aminopyrazines: Synthesis
of Hydrophilic Red Fluorescent Dyes
Synthesis of
H
ydro
m
philic Re
d
Fluoresc
r
e
nt Dye
u
s
ta R. Poreddy,* Bethel Asmelash, William L. Neumann,1 Richard B. Dorshow
Covidien Pharmaceuticals, 675 McDonnell Boulevard, Hazelwood, MO 63042, USA
Fax +1(314)6548900; E-mail: amruta.poreddy@covidien.com
Received 12 February 2010; revised 5 April 2010
function as GFR agents. These new conjugates retain the
Abstract: A robust and scalable method was developed for the syn-
thesis of N,N¢-dialkylated aminopyrazines by a reductive amination
route. These new fluorescent pyrazine analogues were shown to ab-
sorb and emit light at relatively long wavelengths (~50 nm) com-
photophysical properties of the parent pyrazine 1b, with
typical absorption and emission maxima around 450 and
560 nm, and Stokes’ shifts generally greater than 100
pared to the corresponding diaminopyrazines. The utility of these nm.10 Dyes that absorb and emit at longer wavelengths
compounds was demonstrated by the synthesis of hydrophilic fluo-
rescent probes with potential diagnostic applications.
should further enhance the optical detection method due
to their increased tissue penetration resulting from the
minimal absorption of hemoglobin, water, and lipids.11
Key words: GFR, renal function, pyrazine, fluorescence, reductive
amination
Consequently, an efficient method was developed for the
synthesis of N,N¢-dialkylaminopyrazines via a reductive
amination route, the details of which are presented in this
Current medical guidelines are gravitating toward the use paper.12
of glomerular filtration rate (GFR) as the most appropriate
measure of renal function in the determination of kidney
health or illness. Interventions based on GFR values are
then applied to patients. Typically, GFR is not measured
directly; the most common method of assessing renal
function in the clinic involves determination of serum
concentration of an endogenous blood marker such as cre-
atinine. Serum creatinine has an inverse relationship to
GFR,2 and is highly dependent on age, gender, muscle
mass, and many other anthropometric variables, thus
making it a poor surrogate for a renal function assay.3
The best methodology for measurement of GFR is by
clearance of exogeneous tracer agents. Several such
agents have been employed such as inulin,4 iothalamate,5
Gd-DTPA,6 and 99mTc-DTPA.7 However, all suffer from
drawbacks such as the use and disposal of radioactivity,
and the laborious ex-vivo handling of blood and urine
samples. A rapid, dynamic (i.e., continuous, real-time),
and accurate procedure for measuring renal excretion rate
is long overdue and, in fact, considerable effort is now be-
ing directed towards developing exogenous GFR agents
for rapid real-time assessment of renal function using non-
radioactive methods.8
O
X
N
N
NH2
X
N
N
NH2
XR
RX
H2N
H2N
O
1a: X = CN
1b: X = CO2H
2a: X = NH
2b: X = NH(CH2)2NHCO
Figure 1 Diaminopyrazine scaffold and its dicarboxamide deriva-
tives
Alkyl substitution on the amino groups of pyrazine 1 was
shown to significantly increase the wavelength of both ab-
sorption and emission maxima.9,13 Whereas alkylation of
nitrile 1a with alkyl halides in the presence of sodium hy-
dride or powdered sodium hydroxide in N,N-dimethyl-
acetamide (DMA) produced N,N,N¢,N¢-tetraalkyl products
(6–28%), alkylation of esters of 1b resulted only in the
formation of the corresponding N,N¢-dialkyl compounds.
For example, esterification of 1b with alkyl/arylalkyl ha-
lides in the presence of 1,8-diazabicyclo[5.4.0]undec-7-
ene in N,N-dimethylformamide or N,N-dimethylacet-
amide at ambient temperature has been reported to give
variable yields (50% average) of the diester
3
In continuation of our efforts to develop exogenous GFR
markers that absorb and emit light in the visible region,
we have identified simple derivatives of 2,5-diaminopyra-
zine 1 as low molecular weight scaffolds with surprisingly
bright emission in the yellow-to-red region of the electro-
magnetic spectrum (Figure 1).9 A wide variety of hydro-
philic pyrazine dyes, which include amino acid and
poly(ethylene glycol) (PEG) functionalized analogues of
the type 2, have been developed as new optical tracers that
(Scheme 1).9,13a Further treatment of 3 with alkyl/aryl-
alkyl halides at elevated temperatures (70–120 °C) gave
rather low yields of N,N¢-dialkylaminopyrazines 4 (3–
14%). Exhaustive alkylation of 1b with alkyl or arylalkyl
halides in 1,8-diazabicyclo[5.4.0]undec-7-ene and N,N-
dimethylacetamide at 70–120 °C was also found to be
poor, with 7–28% overall yield of 4.13a Even with simple
2-aminopyrazines, base-induced alkylations with alkyl
halides were found to give only moderate yields (~50%)
of N-alkyl-2-aminopyrazine derivatives.14 Thus, alkyla-
tion of aminopyrazines in the presence of a strong base is
not a synthetically useful reaction because it often leads to
SYNTHESIS 2010, No. 14, pp 2383–2392
x
x.
x
x
.
2
0
1
0
Advanced online publication: 20.05.2010
DOI: 10.1055/s-0029-1220009; Art ID: M01210SS
© Georg Thieme Verlag Stuttgart · New York

2384
A. R. Poreddy et al.
PAPER
complex mixtures. The reaction is further complicated by Encouraged by this early success, we examined the scope
over-alkylation and decomposition of the pyrazine core of the reductive amination reaction with the key interme-
under harsh reaction conditions employed in the proce- diate 6a, which was prepared in 87% yield from 1b by
dure.13a Thus, there is a need to develop a simple and effi- standard coupling reaction with N-Boc-ethylenediamine
cient process for alkylating aminopyrazines. In an effort using N-(3-dimethylaminopropyl)-N¢-ethylcarbodiimide
to improve the N,N¢-dialkylation of pyrazine amines, a re- and 1-hydroxybenzotriazole (Scheme 2).17 Thus, com-
ductive amination route was explored.15 Though a myriad pound 6a was reductively alkylated with propionaldehyde
of reagents are available for direct and indirect reductive in the presence of acetic acid and sodium triacetoxyboro-
amination, the former approach, which involves sodium hydride in 1,2-dichloroethane to give 7a in 80% yield. No
triacetoxyborohydride as the reducing agent, proved to be trace of the over-alkylated tetrapropyl derivatives were
the most convenient method.16 Consequently, diethyl es- formed in the reaction. Since the reaction was found to be
ter 3a (R1 = Et), which was prepared in 68% yield from very clean and robust, the methodology was extended to a
1b, was reacted with benzaldehyde in the presence of gal- variety of aldehydes, leading to the corresponding prod-
cial acetic acid and sodium triacetoxyborohydride in 1,2- ucts 7b–h in excellent yields (Table 1). The sole excep-
dichloroethane overnight at room temperature, to yield the tion was 7f (29%), wherein the reaction was not very
corresponding bisbenzyl derivative 5 in 80% yield. The clean and never went to completion because the highly re-
overall yield of 54% from 1b is clearly superior to ~3% re- active methoxy acetaldehyde was prone to undergo aldol
alized earlier using base-induced alkylation.13a
condensation reactions. Similarly, compound 6b, which
was prepared in 79% yield from 1b and N-carbobenzoxy-
1,2-diaminoethane using N-(3-dimethylaminopropyl)-N¢-
ethylcarbodiimide/1-hydroxybenzotriazole coupling, was
smoothly transformed into 7i (70%) and 7j (77%) by re-
action with the appropriate aldehyde under reductive am-
ination conditions. The orthogonal protecting groups on
the side chains of 7g and 7i introduce the flexibility to
conjugate different moieties as desired later on. Com-
pound 7k, a four carbon variant of 7h, was synthesized in
51% overall yield by initial coupling of 1b with N-1-Boc-
1,4-diaminobutane, followed by reductive alkylation of
the bisamide intermediate 6c with 4-tert-butoxycarbonyl-
aminobutanal.18 Towards this end, reductive alkylation of
6a with propionaldehyde under Borch conditions
(NaBH3CN, MeOH)19 was attempted, however, the reac-
tion could not be driven to completion even with a large
excess of reagent over a prolonged period of time (5 d).
This could be partially due to relative insolubility of 6a in
the methanol solvent, and it seems that sodium triacetoxy-
O
O
N
NH2
OH
N
NH2
OR1
R1X, DBU
HO
H2N
R1O
H2N
DMF or DMA
N
N
O
O
1b
3
R
1 = alkyl, arylalkyl
R2X, DBU
DMA
70–120 °C
Ph
O
O
N
NH
N
NHR2
EtO
HN
R1O
OEt
OR1
N
R2HN
N
O
O
Ph
5
4
R
R
1 = alkyl, R2 = arylalkyl
1 = R2 = alkyl, arylalkyl
Table 1 Synthesis of Compounds 7a–k
Scheme 1 Alkylation of aminopyrazines
Product
7a
R1
R2
Yield (%)a
O
O
(CH2)2NHBoc
(CH2)2NHBoc
(CH2)2NHBoc
(CH2)2NHBoc
(CH2)2NHBoc
(CH2)2NHBoc
(CH2)2NHBoc
(CH2)2NHBoc
(CH2)2NHCbz
(CH2)2NHCbz
(CH2)4NHBoc
Et
80
62
72
85
82
29
95
92
70
77
75
N
NH2
OH
N
N
NH2
NHR1
R1NH2, DMF
R1HN
HO
H2N
7b
7c
n-Pr
EDC, HOBt
68–87%
N
H2N
Ph
O
O
6a: R1 = (CH2)2NHBoc
6b: R1 = (CH2)2NHCbz
6c: R1 = (CH2)4NHBoc
1b
7d
7e
4-MeOC6H4
4-O2NC6H4
CH2OMe
(CH2)2CO2Me
CH2NHBoc
CH2NHBoc
(CH2)2NHCbz
(CH2)3NHBoc
7f
R2
NH
O
R1HN
HN
7g
N
N
R2CHO, Na(OAc)3BH
AcOH, DCE, 0 °C, r.t.
7h
7i
NHR1
O
R2
7a–k
7j
Scheme 2 Synthesis of N,N¢-dialkylaminopyrazines 7a–k by re-
ductive alkylation of aminopyrazines
7k
a Isolated yield.
Synthesis 2010, No. 14, 2383–2392 © Thieme Stuttgart · New York

PAPER
Synthesis of Hydrophilic Red Fluorescent Dyes
2385
borohydride in an aprotic solvent such as 1,2-dichloroeth- The small molecule pyrazines described above are all li-
ane seems to be the reagent of choice. It should be pophilic and possess handles for further chemical modifi-
mentioned here that, even under optimized conditions, the cation. A few examples highlighting the utility of these
nitrile 1a failed to undergo reductive amination with pro- compounds as intermediates in the synthesis of long
pionaldehyde due to its extreme insolubility in common wavelength hydrophilic conjugates are shown in the
organic solvents that were suitable for the reaction, such Scheme 4. Thus, initial deprotection of Boc groups from
as 1,2-dichloroethane and tetrahydrofuran.
the intermediate 7a with trifluoroacetic acid, followed by
acylation of the resulting amine-trifluoroacetate salt 11
with N-hydroxysuccinimide ester of the 12-mer m-dPEG-
acid 1221 in the presence of 4-methylmorpholine and puri-
fication by reverse phase preparative HPLC, furnished
pyrazine–PEG conjugate 13 in 42% yield. Similar Boc-
deprotection of 7h gave the corresponding trifluoroacetic
acid salt 14, which, upon acylation with 12 and 15, afford-
ed the tetra-PEG conjugates 16a (29%) and 16b (10%),
respectively. Compound 16c, which contains different-
sized PEG moieties on the amino and carboxyl groups,
was prepared from orthogonally bis-protected intermedi-
ate 7i in a stepwise manner. Initial Boc-deprotection with
trifluoroacetic acid, followed by reaction of the intermedi-
ate 17 with N-hydroxysuccinimide ester 12, gave the com-
pound 18. Subsequent transfer hydrogenation with
ammonium formate in the presence of 10% Pd/C to re-
move Cbz groups furnished 19, which, upon treatment
with N-hydroxysuccinimide ester 15, afforded 16c in 20%
overall yield.
O
O
BocHN
N
N
NH2
N
H
H
+
N
H2N
NHBoc
O
6a
Na(OAc)3BH
AcOH, DCE
46%
O
R2
BocHN
N
NH
N
H
H
N
HN
R1
N
NHBoc
O
10a: R1 = R2 = c-Hex
10b: R1 = c-Hex, R2 = Et
10c: R1 = R2 = Et
The photophysical properties of some of the representa-
tive small molecule pyrazines and the PEG-conjugates are
summarized in Table 2. All the N-alkyl compounds 7b,
7d, and 7h exhibited very similar photophysical patterns,
with significant (35–45 nm) enhancements in both ab-
sorption and emission maxima over those of aminopyra-
zine 6a. The PEG conjugate 13 exhibited a bathochromic
shift of about 50 nm for both absorption and emission
maxima compared to the corresponding des-propylpyra-
zine analogue (labs = 449 nm, lem = 559 nm).10 The ab-
sorption and emission maxima for the relatively
hydrophilic tetra-PEG conjugates 16a–c were found to be
very similar, with slightly lower emission maxima com-
pared to 13, probably due to the dimished ability to donate
Scheme 3 Reductive alkylation of 6a with cyclohexanone
Reductive alkylation of 6a with ketones such as cyclohex-
anone was found to be very sluggish, and multiple addi-
tions of reagents over a period of time were needed to
complete the reaction. The product 10a was isolated in
only moderate yield (46%) due to undesired N-ethylation
that led to both 10b and 10c as byproducts (34% com-
bined; Scheme 3). The formation of these byproducts can
be rationalized by generation of acetaldehyde in situ, and
subsequent competing reductive amination under the pro-
longed reaction conditions. In fact, N-ethylation was
shown to be a major process in reductions with sodium
borohydride in neat acetic acid, and is believed to proceed
through acetaldehdye formation.20
Table 2 Absorption and Fluorescence Spectra of Small Molecule
Pyrazinesa and Pyrazine-PEG Conjugatesb
As mentioned above, the base-induced alkylation of the
weakly basic and poorly nucleophilic 2-aminopyrazines is
hampered by low yields and also by the formation of qua-
ternary salts at the ring nitrogen atoms. Moreover, de-
pending on the base strength, elimination from alkyl
halides could occur. The reductive amination reported
herein employs acid catalysis to: (i) enhance the electro-
philicity of the carbonyl component and to compensate for
the poor nucleophilicity of the amino groups; (ii) drive
formation of the intermediate imine by enhancing the rate
of the dehydration step, and (iii) increase the electrophi-
licity of the imino group by reduction through the iminum
ion intermediate. These factors provide an alkylation
methodology with far greater synthetic utility than the
base-mediated alkylation. This chemistry has become a
‘work-horse’ technology for the systematic synthesis of
mono-N-functionalized pyrazine dyes.
Entry
Compound Absorption lmax (nm) Emission lmax (nm)
1
2
3
4
5
6
7
8
6a
458
504
497
497
498
497
496
495
548
593
587
584
613
595
590
590
7b
7d
7h
13
16a
16b
16c
a Measured as DMSO solutions.
b Measured as phosphate-buffered saline (PBS) solutions.
Synthesis 2010, No. 14, 2383–2392 © Thieme Stuttgart · New York

2386
A. R. Poreddy et al.
PAPER
O
O
H
N
BocHN
N
N
NH
O
O
N
N
NH
O
(i) TFA–CH2Cl2
N
MeO
N
O
H
H
H
H
11
N
(ii) m-dPEG12-NHS (12)
NMM, DMF–CH2Cl2
O
N
OMe
HN
NHBoc
HN
N
O
H
11
42%
7a
13
O
NHBoc
O
NH2
RHN
N
N
NH
H2N
N
N
NH
O
TFA–CH2Cl2
R = Boc
N
H
N
H
H
N
H
N
HN
NHR
HN
NH2
BocHN
O
H2N
7h: R = Boc
7i: R = Cbz
14 (TFA salt)
(i) TFA–CH2Cl2
(ii) m-dPEG12-NHS (12)
NMM, DMF–CH2Cl2
m-dPEG12-NHS (12) or
m-dPEG24-NHS (15)
NMM, DMF–CH2Cl2
R = Cbz
O
O
OMe
OMe
O
N
H
O
O
N
O
H
H
11
n
CbzHN
N
N
NH
O
N
N
N
NH
O
N
H
MeO
N
O
H
H
N
H
m
O
N
OMe
HN
NHCbz
HN
N
O
H
H
H
N
m
O
N
O
O
MeO
MeO
11
n
O
18
O
16a: m = n = 11
16b: m = n = 23
16c: m = 23, n = 11
(i) 10% Pd/C, HCO2NH4, MeOH–H2O
(ii) m-dPEG24-NHS (15), NMM, DMF–CH2Cl2
Scheme 4 Synthesis of hydrophilic pyrazine-PEG conjugates 13 and 16
carried out on an Agilent 1200 series system using a Phenomenex
Luna 5 mm C18(2) 100 Å 250 × 4.6 mm column [detection: UV;
flow: 1 mL/min; gradient: 10/20% B to 80/90% B/20 min; mobile
phase A: 0.1% TFA in H2O; mobile phase B: 0.1% TFA in MeCN].
UV/Vis spectra were measured either with an Agilent 8453 or a
Shimadzu UV-3101 PC spectrophotometer. Fluorescence spectra
were measured either with a PTI Quantamaster or a Jobin Yvon
Fluorolog®-3 spectrofluorimeter. NMR spectra were recorded us-
ing either a Varian Gemini-300 or a VNMRS-500 spectrometer. 1H
chemical shifts are expressed in parts per million (d) relative to
TMS (d = 0 ppm) as an internal standard. 13C chemical shifts are ref-
erenced to either TMS (d = 0 ppm) or to the residual solvent peaks
in the spectra. Coupling constants (J) are reported in Hz. HRMS
(ESI) data was obtained with a ThermoFisher LTQ-Orbitrap mass
spectrometer equipped with an IonMax electrospray ionization
source operating in the FTMS mode with resolution 30 K. Elemen-
tal analyses were carried out by Atlantic Microlab, Inc., Norcross,
GA.
electrons to the pyrazine core. This is our entry into red
fluorescent hydrophilic pyrazines and the detailed photo-
physical, plasma–protein binding, and renal clearance
properties of this class of compounds will be described
elsewhere.
In summary, an efficient and improved method has been
developed for the synthesis of N,N¢-dialkylated aminopy-
razines of the type 7 by a general reductive amination
route. The utility of these new pyrazine analogues, which
absorb and emit at longer wavelengths, was demonstrated
by the synthesis of new hydrophilic compounds of the
type 13 and 16.
Unless otherwise noted, all reagents were used as supplied. Organic
extracts were dried over anhyd Na2SO4 and filtered using a fluted
filter paper (P8). Solvents were removed on a rotary evaporator un-
der reduced pressure. Analytical TLC was performed on Analtech
silica gel GF plates (250 mm) and flash chromatography was carried
out using silica gel 60 (40–63 mm). RP-LC/MS (ESI, positive ion
mode) analyses were carried out on a Waters Micromass ZQ system
equipped with a PDA detector using either a BDS Hypersil C18 (3
mm, 50 × 4.6 mm) or a ThermoElectron Hypersil Gold C18 (3 mm,
4.6 × 50 mm) column. Compounds were injected under gradient
conditions (5 to 50–95% B/6 min) with a flow rate of 1 mL/min
(mobile phase A: 0.05% TFA in H2O; mobile phase B: 0.05% TFA
in MeCN). Preparative RP-HPLC was carried out with a Waters
Dual Pump system equipped with a Liquid Handler using a Waters
XBridge™ Prep C18 5 mm OBD™ 30 × 150 mm or a 19 × 250 mm
column [lmax: PDA (200–800 nm); flow: 50 mL/min; gradient: 20–
30% to 50–95% B/10–17 min; mobile phase A: 0.1% TFA in H2O;
mobile phase B: 0.1% TFA in MeCN]. RP-HPLC analyses were
Diethyl 3,6-Diaminopyrazine-2,5-dicarboxylate (3a)
Compound 3a was prepared from 3,6-diaminopyrazine-2,5-dicar-
boxylic acid (1b)22 in 68% yield according to a reported procedure.9
Diethyl 3,6-Bis(benzylamino)pyrazine-2,5-dicarboxylate (5)
To a well-stirred, red suspension of diester 3a (0.127 g, 0.500
mmol) in anhyd DCE (20 mL), benzaldehyde (0.202 mL, 2.00
mmol) was added, and the reaction flask was immersed in an ice
bath. AcOH (0.115 mL, 2.00 mmol) was added, followed by the ad-
dition of Na(OAc)3BH (0.424 g, 2.00 mmol) in small portions over
a 15 min period. The resulting suspension was slowly allowed to
warm to r.t. and stirred overnight (ca. 16 h; RP-LC/MS analysis in-
dicated the presence of some substrate) in an atmosphere of argon.
At this stage, the reaction mixture was treated with more benzalde-
hyde (0.202 mL, 2.00 mmol), AcOH (0.115 mL, 2.00 mmol), and
Synthesis 2010, No. 14, 2383–2392 © Thieme Stuttgart · New York

PAPER
Synthesis of Hydrophilic Red Fluorescent Dyes
2387
Na(OAc)3BH (0.424 g, 2.00 mmol) as described above, and the re-
action was allowed to continue overnight (ca. 24 h). The reaction
was quenched by slow addition of sat. NaHCO3 (20 mL) while stir-
ring at 0 °C. The biphasic mixture was stirred for 30 min and ex-
tracted with CHCl3 (3 × 25 mL). The combined organic extracts
were successively washed with sat. NaHCO3, H2O, and brine (30
mL each). Removal of the solvent gave a red solid (0.200 g), which
upon flash chromatography over silica gel (CHCl3) afforded 5.13a
13C NMR (DMSO-d6): d = 165.8, 156.7, 146.7, 137.6, 128.8,
128.22, 128.20, 126.8, 65.7, 40.4 (overlaps with solvent), 39.2
(overlaps with solvent).
RP-LC/MS (ESI): m/z = 551.2 [M + H]+, 573.2 [M + Na]+ (tR = 3.86
min, 25–95% B).
Anal. Calcd for C26H30N8O6: C, 56.72; H, 5.49; N, 20.35. Found: C,
56.79; H, 5.49; N, 20.30.
Yield: 0.174 g (80%); dark-red powder; Rf = 0.49 (CHCl3).
3,6-Diamino-N2,N5-bis[4-(tert-butoxycarbonyl)amino-
butyl]pyrazine-2,5-dicarboxamide (6c)
1H NMR (DMSO-d6): d = 7.60 (t, J = 5.9 Hz, 2 H), 7.42 (dd, J = 7.7,
1.7 Hz, 4 H), 7.28–7.18 (m, 6 H), 4.51 (d, J = 5.9 Hz, 4 H), 4.32 (q,
J = 7.1 Hz, 4 H), 1.30 (t, J = 7.1 Hz, 6 H).
13C NMR (DMSO-d6): d = 165.4, 146.3, 140.1, 128.1, 128.0, 126.7,
124.8, 61.4, 44.4, 44.3, 14.1.
RP-LC/MS (ESI): m/z = 435.3 [M+H]+, 457.2 [M + Na]+ (tR = 5.53
min, 5–95% B).
The reaction of 1b (1.85 g, 11.8 mmol) and N-Boc-1,4-diaminobu-
tane hydrochloride (2.72 g, 12.1 mmol) in the presence of DIPEA
(10.3 mL, 59.2 mmol), HOBt·H2O (1.85 g, 11.8 mmol), and
EDC·HCl (2.56 g, 13.4 mmol) in anhyd DMF (40 mL) was carried
out overnight as described for the preparation of 6b. Most of the
DMF was removed under high vacuum and the viscous residue was
dissolved in CHCl3 (200 mL), and successively washed with 0.50 M
KHSO4, sat. NaHCO3, and brine (60 mL portions). Solvent was re-
moved and the crude product was subjected to flash chromatogra-
phy over silica gel (CHCl3–MeOH, 19:1, v/v) to afford the bisamide
6c.
Anal. Calcd for C24H26N4O4: C, 66.34; H, 6.03; N, 12.89. Found: C,
66.10; H, 5.98; N, 12.67.
3,6-Diamino-N2,N5-bis[2-(tert-butoxycarbonyl)amino-
ethyl]pyrazine-2,5-dicarboxamide (6a)
Yield: 1.82 g (68%); yellow powder; Rf = 0.48 (CHCl3–MeOH, 9:1,
To a well-stirred suspension of diacid 1b (0.991 g, 5.00 mmol) in
anhyd DMF (50 mL), N-(2-aminoethyl)carbamic acid tert-butyl es-
ter (1.76 g, 11.0 mmol) and HOBt·H2O (1.84 g, 12.0 mmol) were
added and the mixture was stirred for 10 min in an atmosphere of
Ar. The reaction flask was cooled in an ice bath, EDC·HCl (2.30 g,
12.0 mmol) was introduced, and the reaction mixture was slowly
warmed to r.t. and then stirred overnight (ca. 17 h). Most of the
DMF was removed under high vacuum and the residue was taken
up in CHCl3 (700 mL) and transferred to a separating funnel. The
solution was successively washed with H2O, sat. NaHCO3, H2O,
and brine (250 mL portions). The solvents were removed in vacuo
and the residue was dried under high vacuum to give spectroscopi-
cally pure bisamide 6a.
v/v).
1H NMR (CDCl3): d = 7.83 (t, J = 5.8 Hz, 2 H), 6.03 (s, 4 H), 4.61
(br s, 2 H), 3.42 (q, J = 6.7 Hz, 4 H), 3.18 (br q, 4 H), 1.68–1.54 (m,
8 H), 1.44 (s, 18 H).
13C NMR (CDCl3): d = 165.2, 156.0, 146.5, 127.0, 79.2, 40.2, 38.9,
28.4, 27.6, 26.9.
RP-LC/MS (ESI): m/z = 539.3 [M + H]+, 561.3 [M + Na]+ (tR = 4.47
min, 5–95% B).
Anal. Calcd for C24H42N8O6: C, 53.52; H, 7.86; N, 20.80. Found: C,
53.39; H, 8.01; N, 20.66.
N2,N5-Bis[2-(tert-butoxycarbonyl)aminoethyl]-3,6-bis(propyl-
amino)pyrazine-2,5-dicarboxamide (7a)
Yield: 2.10 g (87%); yellow powder.
1H NMR (DMSO-d6): d = 8.46 (t, J = 5.9 Hz, 2 H), 6.92 (t, J =
5.5 Hz, 2 H), 6.49 (s, 4 H), 3.34–3.28 (q, 4 H, overlaps with H2O in
solvent), 3.10 (q, J = 6.0 Hz, 4 H), 1.36 (s, 18 H).
13C NMR (DMSO-d6): d = 165.8, 156.2, 146.6, 126.8, 78.2, 28.7.
RP-LC/MS (ESI): m/z = 483.3 [M + H]+, 505.4 [M + Na]+ (tR = 3.62
min, 25–95% B).
To a partially dissolved, yellow suspension of bisamide 6a (0.483 g,
1.00 mmol) in anhyd DCE (20 mL), propionaldehyde (0.290 mL,
4.02 mmol) and AcOH (0.290 mL, 5.03 mmol) were added with
stirring at 0 °C under an argon atmosphere. The resulting, some-
what lighter suspension was allowed to stir for 5 min before the ad-
dition of Na(OAc)3BH (0.848 g, 4.00 mmol) in small portions over
a 10 min period. The reddish suspension was slowly allowed to
warm to r.t. and stirred overnight (ca. 19 h) in an atmosphere of ar-
gon. The reaction was quenched by the slow addition of sat.
NaHCO3 (20 mL) at 0 °C. The biphasic mixture was stirred for 30
min and extracted with CHCl3 (3 × 25 mL). The combined organic
extracts were successively washed with H2O and brine (50 mL
each). Removal of the solvent gave a red solid (0.680 g), which
upon flash chromatography over silica gel (CH2Cl2–EtOAc, 17:3 to
3:1, v/v) afforded 7a.
Anal. Calcd for C20H34N8O6: C, 49.78; H, 7.10; N, 23.22. Found: C,
49.66; H, 7.25; N, 22.98.23
3,6-Diamino-N2,N5-bis[2-(benzyloxycarbonyl)amino-
ethyl]pyrazine-2,5-dicarboxamide (6b)
A suspension of N-carbobenzoxy-1,2-diaminoethane hydrochloride
(4.61 g, 20.0 mmol) in anhyd DMF (100 mL) was stirred with N,N-
disopropylethylamine (DIPEA; 3.50 mL, 20.1 mmol) for 30 min in
an atmosphere of N2. Compound 1b (1.98 g, 10.0 mmol) was added
and, after 15 min, HOBt·H2O (3.37 g, 22.0 mmol) and EDC·HCl
(4.22 g, 22.0 mmol) were added. The resulting dark suspension was
stirred at r.t. overnight (ca. 16 h). Most of the DMF was removed
under high vacuum and the slurry was stirred with anhyd Et2O–
MeOH (1:3, v/v; 200 mL) for about 30 min. The precipitate was col-
lected by filtration, thoroughly washed with MeOH and anhyd Et2O
and dried under high vacuum to give the bisamide 6b.
Yield: 0.454 g (80%); crimson red solid; Rf = 0.44 (CH2Cl2–EtOAc,
7:3, v/v).
1H NMR (CDCl3): d = 8.13 (br s, 2 H), 7.78 (t, J = 5.4 Hz, 2 H), 4.87
(br s, 2 H), 3.53 (q, J = 5.9 Hz, 4 H), 3.39–3.34 (quint, J = 7.8 Hz,
8 H), 1.70–1.63 (sext, J = 7.2 Hz, 4 H), 1.42 (s, 18 H), 1.01
(t, J = 7.4 Hz, 6 H).
13C NMR (CDCl3): d = 166.8, 156.3, 146.0, 126.1, 79.6, 42.9, 40.4,
39.8, 28.3, 22.8, 11.8.
RP-LC/MS (ESI): m/z = 567.4 [M + H]+, 589.4 [M + Na]+ (tR = 5.17
min, 5–95% B).
Yield: 4.37 g (79%); yellow powder.
1H NMR (DMSO-d6): d = 8.50 (t, J = 5.5 Hz, 2 H), 7.39–7.31 (m,
10 H), 6.50 (br s, 4 H), 5.02 (s, 4 H), 3.37–3.34 (br q, 4 H), 3.20–
3.17 (br q, 4 H).
Anal. Calcd for C26H46N8O6: C, 55.11; H, 8.18; N, 19.77. Found: C,
55.17; H, 8.31; N, 19.53.
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N2,N5-Bis[2-(tert-butoxycarbonyl)aminoethyl]-3,6-bis(butyl-
amino)pyrazine-2,5-dicarboxamide (7b)
RP-LC/MS (ESI): m/z = 723.3 [M + H]+, 745.3 [M + Na]+ (tR = 4.08
min, 50–95% B).
The reaction of 6a (0.483 g, 1.00 mmol) with butyraldehyde (0.358
mL, 4.00 mmol) in the presence of AcOH (0.230 mL, 4.00 mmol)
and Na(OAc)3BH (0.848 g, 4.00 mmol) in DCE (25 mL) was car-
ried out overnight (ca. 18 h) as described for the preparation of 7a.
After a similar workup, the brick-red crude product (0.610 g) was
subjected to flash chromatography over silica gel (CHCl3–EtOAc,
3:1, v/v), and the residue was triturated with anhyd Et2O to give 7b.
Anal. Calcd for C36H50N8O8: C, 59.82; H, 6.97; N, 15.50. Found: C,
60.01; H, 7.05; N, 15.43.
N2,N5-Bis[2-(tert-butoxycarbonyl)aminoethyl]-3,6-bis(4-nitro-
benzylamino)pyrazine-2,5-dicarboxamide (7e)
The reaction of 6a (0.121 g, 0.250 mmol) with 4-nitrobenzaldehyde
(0.151 mL, 1.00 mmol) in the presence of AcOH (0.058 mL, 1.00
mmol) and Na(OAc)3BH (0.212 g, 1.00 mmol) in DCE (10 mL) was
carried out overnight (ca. 18 h) as described for the preparation of
7a. After a similar workup, the brick-red crude product (0.260 g)
was subjected to flash chromatography over silica gel (CHCl3–
EtOAc, 7:3, v/v), and the residue was recrystallized from EtOAc–
Et2O to give 7e.
Yield: 0.366 g (62%); red powder; Rf = 0.48 (CHCl3–EtOAc, 7:3,
v/v).
1H NMR (CDCl3): d = 8.13 (br s, 2 H), 7.76 (t, J = 5.1 Hz, 2 H), 4.86
(br s, 2 H), 3.53 (q, J = 6.0 H, 4 H), 3.43–3.34 (2 × q, overlapping,
8 H), 1.66–1.60 (m, 4 H), 1.49–1.38 (m, 22 H, includes Boc singlet
at d = 1.42 ppm), 0.96 (t, J = 7.3 Hz, 6 H).
13C NMR (CDCl3): d = 166.9, 156.3, 146.0, 126.1, 79.6, 40.7, 40.5,
39.8, 31.7, 28.3, 20.4, 14.0.
RP-LC/MS (ESI): m/z = 595.4 [M + H]+, 617.3 [M + Na]+ (tR = 4.45
min, 50–95% B).
Yield: 0.155 g (82%); orange microcrystalline solid; Rf = 0.33
(CHCl3–EtOAc, 1:1, v/v).
1H NMR (CDCl3): d = 8.44 (br t, 2 H), 8.18 (d, J = 8.7 Hz, 4 H),
8.03 (br s, 2 H), 7.57 (d, J = 8.5 Hz, 4 H), 4.78 (br m, 6 H), 3.46–
3.42 (br q, 4 H), 3.36–3.30 (br m, 4 H), 1.39 (s, 18 H).
13C NMR (CDCl3): d = 166.2, 156.7, 148.2, 147.0, 145.6, 127.8,
126.5, 123.8, 79.8, 44.6, 40.7, 40.0, 28.3.
Anal. Calcd for C28H50N8O6: C, 56.55; H, 8.47; N, 18.84. Found: C,
56.39; H, 8.57; N, 18.73.
3,6-Bis(benzylamino)-N2,N5-bis[2-(tert-butoxycarbonyl)amino-
ethyl]pyrazine-2,5-dicarboxamide (7c)
RP-LC/MS (ESI): m/z = 753.2 [M + H]+, 775.1 [M + Na]+ (tR = 4.02
min, 50–95% B).
The reaction of 6a (0.121 g, 0.250 mmol) with benzaldehyde (0.101
mL, 1.00 mmol) in the presence of AcOH (0.058 mL, 1.00 mmol)
and Na(OAc)3BH (0.212 g, 1.00 mmol) in DCE (10 mL) was car-
ried out overnight (ca. 16 h) as described for the preparation of 7a.
After a similar workup, the brick-red crude product (0.240 g) was
subjected to flash chromatography over silica gel (CHCl3–EtOAc,
4:1, v/v), and the residue triturated with anhyd Et2O to give 7c.
Anal. Calcd for C34H44N10O10: C, 54.25; H, 5.89; N, 18.61. Found:
C, 54.20; H, 5.97; N, 18.32.
N2,N5-Bis[2-(tert-butoxycarbonyl)aminoethyl]-3,6-bis(2-meth-
oxyethylamino)pyrazine-2,5-dicarboxamide (7f)
The reaction of 6a (0.121 g, 0.25 mmol) with methoxyacetaldehyde
(0.074 g, 1.00 mmol) in the presence of AcOH (0.058 mL, 1.00
mmol) and Na(OAc)3BH (0.212 g, 1.00 mmol) in DCE (10 mL) was
carried out overnight (ca. 15 h) as described for the preparation of
7a. After a similar workup, the red crude product (0.146 g) was sub-
jected to flash chromatography over silica gel [CHCl3–EtOAc, 1:1
(v/v) to 2% MeOH in CHCl3–EtOAc, 1:1 (v/v)] to afford 7f.
Yield: 0.119 g (72%); orange powder; Rf = 0.40 (CHCl3–EtOAc,
7:3, v/v).
1H NMR (CDCl3): d = 8.20 (br t, J = 5.0 Hz, 2 H), 7.76 (br t, 2 H),
7.37–7.30 (m, 8 H), 7.25–7.21 (m, 2 H), 4.77 (br s, 2 H), 4.58 (d,
J = 5.4 Hz, 4 H), 3.44–3.40 (br q, 4 H), 3.31–3.25 (br q, 4 H), 1.43
(s, 18 H).
Yield: 0.043 g (29%); orange-red solid; Rf = 0.34 [2% MeOH in
CHCl3–EtOAc, 1:1 (v/v)].
13C NMR (CDCl3): d = 166.5, 156.2, 145.6, 140.3, 128.5, 127.0,
126.8, 126.4, 79.6, 45.6, 40.4, 39.8, 28.4.
1H NMR (CDCl3): d = 8.20 (br, 2 H), 7.96 (t, J = 4.9 Hz, 2 H), 4.93
(br, 2 H), 3.66–3.59 (m, 8 H), 3.52 (q, J = 5.9 Hz, 4 H), 3.41 (s,
6 H), 3.36 (br q, J = 5.3 Hz, 4 H), 1.42 (s, 18 H).
13C NMR (CDCl3): d = 166.6, 156.5, 145.9, 126.4, 79.6, 71.6, 59.0,
41.0, 40.4, 40.1, 28.4.
RP-LC/MS (ESI): m/z = 599.4 [M + H]+, 621.4 [M + Na]+ (tR = 4.47
min, 5–95% B).
HRMS (ESI): m/z [M + H]+ calcd for C26H47N8O8: 599.3511; found:
599.3511; [M + Na]+ calcd for C26H46N8O8Na: 621.3331; found:
621.3329.
RP-LC/MS (ESI): m/z = 663.2 [M + H]+, 685.2 [M + Na]+ (tR = 4.30
min, 50–95% B).
Anal. Calcd for C34H46N8O6: C, 61.61; H, 7.00; N, 16.91. Found: C,
61.72; H, 7.07; N, 16.89.
N2,N5-Bis[2-(tert-butoxycarbonyl)aminoethyl]-3,6-bis(4-meth-
oxybenzylamino)pyrazine-2,5-dicarboxamide (7d)
The reaction of 6a (0.483 g, 1.00 mmol) with 4-methoxybenzalde-
hyde (0.485 mL, 4.00 mmol) in the presence of AcOH (0.230 mL,
4.00 mmol) and Na(OAc)3BH (0.848 g, 4.00 mmol) in DCE (25
mL) was carried out overnight as described for the preparation of
7a. After a similar workup, the brick-red crude product (1.14 g) was
subjected to flash chromatography over silica gel (CHCl3–EtOAc,
3:1, v/v), and the material was recrystallized from EtOAc–Et2O to
give 7d.
Dimethyl 4,4¢-[3,6-Bis{2-(tert-butoxycarbonylamino)ethyl-
carbamoyl}pyrazine-2,5-diyl]bis(azanediyl)dibutanoate (7g)
The reaction of 6a (0.965 g, 2.00 mmol) with methyl 4-oxobu-
tanoate (0.838 mL, 8.00 mmol) in the presence of AcOH (0.460 mL,
7.98 mmol) and Na(OAc)3BH (1.70 g, 8.00 mmol) in DCE (40 mL)
was carried out overnight (ca. 20 h) as described for the preparation
of 7a. After a similar workup, the orange crude product (1.74 g) was
subjected to flash chromatography over silica gel (CHCl3–EtOAc,
7:3, v/v) to give 7g.
Yield: 0.615 g (85%); orange-red microcrystalline solid; Rf = 0.30
(CHCl3–EtOAc, 7:3, v/v).
1H NMR (CDCl3): d = 8.14 (br t, J = 5.0 Hz, 2 H), 7.90 (br t, 2 H),
7.28 (d, J = 8.5 Hz, 4 H), 6.86 (d, J = 8.5 Hz, 4 H), 4.82 (br t, 2 H),
4.52 (d, J = 5.4 Hz, 4 H), 3.78 (s, 6 H), 3.46–3.43 (br q, 4 H), 3.33–
3.28 (br q, 4 H), 1.42 (s, 18 H).
Yield: 1.30 g (95%); orange-red powder; Rf = 0.33 (CHCl3–EtOAc,
1:1, v/v).
13C NMR (CDCl3): d = 166.6, 158.6, 156.3, 145.6, 132.2, 128.3,
126.4, 113.9, 79.6, 55.3, 45.0, 40.5, 39.8, 28.4.
1H NMR (CDCl3): d = 8.66 (t, J = 5.9 Hz, 2 H), 7.93 (t, J = 6.0 Hz,
2 H), 5.21 (br t, 2 H), 3.67 (s, 6 H), 3.56 (q, J = 5.8 Hz, 4 H), 3.46–
Synthesis 2010, No. 14, 2383–2392 © Thieme Stuttgart · New York

PAPER
Synthesis of Hydrophilic Red Fluorescent Dyes
2389
3.30 (m, 8 H), 2.42 (t, J = 6.5 Hz, 4 H), 1.99–1.89 (quint, J = 6.9 Hz,
4 H), 1.41 (s, 18 H).
13C NMR (CDCl3): d = 174.4, 166.7, 156.0, 145.6, 126.1, 79.2, 51.8,
40.8, 40.4, 39.5, 30.9, 28.4, 24.4.
RP-LC/MS (ESI): m/z = 683.3 [M + H]+, 705.3 [M + Na]+ (tR = 4.75
min, 15–95% B).
HRMS (ESI): m/z [M + H]+ calcd for C30H51N8O10: 683.3723;
found: 683.3719.
N2,N5-Bis[2-(benzyloxycarbonyl)aminoethyl]-3,6-bis[3-(benzyl-
oxycarbonylamino)propylamino]pyrazine-2,5-dicarboxamide
(7j)
The reaction of 6b (1.10 g, 2.00 mmol) with 3-[(benzyloxycarbon-
yl)amino]propionaldehyde (1.24 g, 6.00 mmol) in the presence of
AcOH (0.340 mL, 5.90 mmol) and sodium triacetoxyborohydride
(1.27 g, 6.00 mmol) in anhyd DCE (50 mL) was carried out over-
night (ca. 40 h) as described above for the preparation of 7a. After
a similar workup, the crude product was suspended in MeCN–
anhyd Et2O (1:1, v/v; 100 mL) and stirred at r.t. for 30 min. The pre-
cipitate was collected by filtration, washed with MeCN–anhyd
Et2O, and dried under high vacuum to give 7j (1.35 g). The filtrate
was concentrated and subjected to flash chromatography over silica
gel (CHCl3–MeOH, 49:1, v/v)] to obtain an additional 0.09 g of the
product.
Anal. Calcd for C30H50N8O10·1/6CHCl3: C, 51.56; H, 7.20; N,
15.95. Found: C, 51.97; H, 7.25; N, 15.59.
N2,N5-Bis[2-(tert-butoxycarbonyl)aminoethyl]-3,6-bis[2-(tert-
butoxycarbonylamino)ethylamino]pyrazine-2,5-dicarbox-
amide (7h)
The reaction of 6a (2.00 g, 4.15 mmol) with N-Boc-2-aminoacetal-
dehyde (2.60 g, 16.3 mmol) in the presence of AcOH (0.960 mL,
16.6 mmol) and Na(OAc)3BH (3.52 g, 16.6 mmol) in DCE (25 mL)
was carried out overnight (ca. 20 h) as described for the preparation
of 7a. After a similar workup, the red crude product obtained was
subjected to flash chromatography over silica gel (CHCl3–EtOAc,
1:1, v/v) to afford 7h.
Yield: 1.44 g (77%); orange powder; Rf = 0.42 (CHCl3–MeOH,
19:1, v/v).
1H NMR (DMSO-d6): d = 8.53 (t, J = 5.5 Hz, 2 H), 7.86 (br t, 2 H),
7.42 (t, J = 5.5 Hz, 2 H), 7.36–7.21 (m, 20 H), 4.99 (s, 4 H), 4.98 (s,
4 H), 3.50–3.30 (m, 10 H), 3.18 (q, J = 6.1 Hz, 4 H), 3.07 (q, J = 6.4
Hz, 4 H), 1.66 (quint, J = 6.6 Hz, 4 H).
13C NMR (DMSO-d6): d = 165.7, 156.4, 156.1, 145.3, 137.2, 137.0,
128.22, 128.18, 127.64, 127.60, 125.7, 65.2, 65.1, 38.1, 37.6, 29.6.
Yield: 2.94 g (92%); brick-red solid; Rf = 0.27.
1H NMR (DMSO-d6): d = 8.81 (t, J = 5.9 Hz, 2 H), 7.95 (t, J = 5.9
Hz, 2 H), 6.96 (t, J = 5.6 Hz, 2 H), 6.86 (br t, J = 5.1 Hz, 2 H), 3.41
(q, J = 6.4 Hz, 4 H), 3.35 (q, J = 6.2 Hz, 4 H), 3.15–3.08 (quint,
J = 6.3 Hz, 8 H), 1.38 (s, 18 H), 1.35 (s, 18 H).
RP-LC/MS (ESI): m/z = 933.4 [M + H]+ (tR = 4.96 min, 15–95% B).
Anal. Calcd for C48H56N10O10: C, 61.79; H, 6.05; N, 15.01. Found:
C, 61.53; H, 5.92; N, 14.96.
13C NMR (DMSO-d6): d = 165.4, 155.8, 155.5, 145.0, 125.7, 77.7,
N2,N5-Bis[4-(tert-butoxycarbonyl)aminobutyl]-3,6-bis[4-(tert-
butoxycarbonylamino)butylamino]pyrazine-2,5-dicarbox-
amide (7k)
77.5, 40.2 (overlaps with solvent), 39.1 (overlaps with solvent).
RP-LC/MS (ESI): m/z = 769.3 [M + H]+, 791.3 [M + Na]+ (tR = 5.10
The reaction of 6c (0.250 g, 0.464 mmol) with N-Boc-4-
aminobutyraldehyde18 (0.695, 3.71 mmol) in the presence of AcOH
(0.212 mL, 3.68 mmol) and sodium triacetoxyborohydride (0.787 g,
3.71 mmol) in DCE (15 mL) was carried out overnight as described
for the preparation of 7a. After a similar workup, the crude product
(0.678 g) was subjected to flash chromatography over silica gel
(CHCl3–EtOAc, 3:2, v/v) to give 7k.
min, 15–95% B).
HRMS (ESI): m/z [M + H]+ calcd for C34H61N10O10: 769.4567;
found: 769.4567.
Anal. Calcd for C34H60N10O10·2/3EtOAc: C, 53.21; H, 7.96; N,
16.92. Found: C, 53.01; H, 8.03; N, 16.58.
N2,N5-Bis[2-(benzyloxycarbonyl)aminoethyl]-3,6-bis[2-(tert-
butoxycarbonyl)aminoethylamino]pyrazine-2,5-dicarbox-
amide (7i)
The reaction of 6b (0.500 g, 0.908 mmol) with N-Boc-2-aminoacet-
aldehyde (0.578 g, 3.63 mmol) in the presence of AcOH (0.208 mL,
3.61 mmol) and Na(OAc)3BH (0.770 g, 3.63 mmol) in anhyd DCE
(40 mL) was carried out overnight (ca. 19 h) as described above for
the preparation of 7a. After a similar workup, the crude product
(0.900 g) was subjected to flash chromatography over silica gel
(CH2Cl2–MeOH, 97:3, v/v) to afford 7i.
Yield: 0.307 g (75%); brick-red powder; Rf = 0.45.
1H NMR (CDCl3): d = 7.92 (br s, 2 H), 7.85 (t, J = 5.5 Hz, 2 H), 4.73
(br s, 2 H), 4.61 (br s, 2 H), 3.44–3.39 (m, 8 H), 3.17 (br q, 8 H),
1.68–1.54 (m, 16 H), 1.43 (s, 36 H).
13C NMR (CDCl3): d = 166.1, 156.02, 156.0, 145.9, 126.2, 79.1,
40.4, 40.2, 38.9, 28.4, 27.7, 27.6, 27.1, 26.8.
RP-LC/MS (ESI): m/z = 881.4 [M + H]+, 903.4 [M + Na]+ (tR = 5.32
min, 5–95% B).
Anal. Calcd for C42H76N10O10: C, 57.25; H, 8.69; N, 15.90. Found:
C, 57.53; H, 8.83; N, 15.70.
Yield: 0.530 g (70%); brick-red powder; Rf = 0.77 (CH2Cl2–MeOH,
19:1, v/v).
1H NMR (CDCl3): d = 9.03 (br t, 2 H), 7.93 (t, J = 5.8 Hz, 2 H),
7.33–7.26 (m, 10 H), 5.73 (br t, 2 H), 5.07 (s, 4 H), 4.85 (t, J = 6.2
Hz, 2 H), 3.61 (br q, 4 H), 3.48–3.25 (m, 8 H), 3.30–3.25 (m, 4 H),
1.42 (s, 18 H).
13C NMR (CDCl3): d = 167.1, 156.7, 156.5, 145.8, 136.8, 128.4,
128.0, 127.9, 126.4, 79.5, 66.5, 41.7, 41.1, 39.3, 39.2, 28.5.
N2,N5-Bis[2-(tert-butoxycarbonyl)aminoethyl]-3,6-bis(cyclo-
hexylamino)pyrazine-2,5-dicarboxamide (10a)
To a partially dissolved yellow suspension of 6a (0.121 g, 0.250
mmol) in anhyd DCE (10 mL), cyclohexanone (0.104 mL, 1.00
mmol) was added, and the reaction flask was immersed in an ice
bath. AcOH (0.058 mL, 1.00 mmol) was added followed by the ad-
dition of sodium triacetoxyborohydride (0.212 g, 1.00 mmol) in
small portions over a 10 min period. The resulting suspension
was slowly allowed to warm to r.t. and stirred overnight (ca. 17 h;
RP-LC/MS analysis indicated intact substrate) in an atmosphere of
N2. At this stage, the reaction mixture was treated with further cy-
clohexanone (0.104 mL, 1.00 mmol), AcOH (0.058 mL, 1.00
mmol), and sodium triacetoxyborohydride (0.212 g, 1.00 mmol) as
described above, and the reaction was continued for 48 h (RP-LC/
MS analysis indicated some substrate remaining). Similar quantities
RP-LC/MS (ESI): m/z = 837.4 [M + H]+, 859.3 [M + Na]+ (tR = 5.15
min, 5–95% B).
Anal. Calcd for C40H56N10O10: C, 57.40; H, 6.74; N, 16.74. Found:
C, 57.33; H, 6.78; N, 16.48.
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2390
A. R. Poreddy et al.
PAPER
of the reagents were added once again and the reaction was contin-
ued over the weekend. After the usual workup described for the
preparation of 7a, the crude red product (0.456 g) was subjected to
flash chromatography over silica gel (CHCl3 to CHCl3–EtOAc,
17:3, v/v) to afford the desired product 10a along with the byprod-
ucts 10b and 10c.
RP-LC/MS (ESI): m/z = 367.3 [M + H]+ (tR = 3.36 min).
N2,N5-Bis(38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-
39-azahentetracontan-41-yl)-3,6-bis(propylamino)pyrazine-
2,5-dicarboxamide (13)
To a red solution of the above TFA salt 11 (0.759 mmol) in anhyd
DMF (7 mL), 4-methylmorpholine (NMM; 0.835 mL, 7.59 mmol)
was added at 0 °C, and the reaction was stirred for 30 min in an at-
mosphere of argon. A solution of m-dPEG12-NHS (12; 1.25 g, 1.82
mmol) in anhyd CH2Cl2 (3 mL) was added and the reaction mixture
was stirred overnight at ambient temperature. Most of the solvent
was removed under high vacuum and the crude product (1.82 g) was
subjected to purification by preparative RP-HPLC (30 × 150 mm,
20–50% B/10 min). The pure fractions were concentrated in vacuo,
the residue was partitioned between CHCl3 and sat. NaHCO3, and
the CHCl3 layer was washed with H2O and brine. After removal of
the solvent, the residue was dried under high vacuum to give 13.
Yield: 0.075 g (46%); crimson red powder; Rf = 0.58 (CHCl3–
EtOAc, 7:3, v/v).
1H NMR (CDCl3): d = 8.02 (br t, 2 H), 7.75 (d, J = 7.7 Hz, 2 H),
4.83 (br t, 2 H), 3.90–3.76 (br m, 2 H), 3.52 (q, J = 5.9 Hz, 4 H),
3.34 (q, J = 5.9 Hz, 4 H), 2.02–1.20 (m, 38 H, includes Boc singlet
at d = 1.42 ppm).
13C NMR (CDCl3): d = 166.5, 156.4, 144.8, 125.8, 79.4, 48.9, 40.4,
39.5, 32.8, 28.3, 25.9, 24.6.
RP-LC/MS (ESI): m/z = 647.5 [M + H]+ (tR = 5.36 min, 30–95% B).
HRMS (ESI): m/z [M + H]+ calcd for C32H55N8O6: 647.4239; found:
Yield: 0.481 g (42%); brick-red solid.
647.4238.
1H NMR (DMSO-d6): d = 8.58 (t, J = 5.7 Hz, 2 H), 8.02 (t, J = 5.6
Hz, 2 H), 7.87 (t, J = 5.6 Hz, 2 H), 3.60–3.24 (m, 110 H, includes
characteristic m-dPEG signals at d = 3.50 and 3.24 ppm), 2.32 (t,
J = 6.5 Hz, 4 H), 1.60–1.52 (sext, J = 7.2 Hz, 4 H), 0.95 (t, J = 7.5
Hz, 6 H).
13C NMR (DMSO-d6): d = 171.2, 166.4, 146.0, 126.2, 71.7, 70.3,
70.09, 70.05, 70.0, 67.2, 58.5, 42.4, 38.5, 36.6, 23.0, 12.1.
Anal. Calcd for C32H54N8O6·1/3EtOAc·1/3H2O: C, 58.69; H, 8.47;
N, 16.43. Found: C, 58.31; H, 8.39; N, 16.08.
N2,N5-Bis[2-(tert-butoxycarbonyl)aminoethyl]-3-(cyclohexyl-
amino)-6-(ethylamino)pyrazine-2,5-dicarboxamide (10b)
Yield: 0.040 g (27%); red solid; Rf = 0.38 (CHCl3–EtOAc, 7:3, v/v).
1H NMR (CDCl3): d = 8.16 (br t, 1 H), 8.01 (br t, 1 H), 7.79 (d,
J = 7.7 Hz, 1 H), 7.63 (t, J = 5.1 Hz, 1 H), 4.83 (br s, 2 H), 3.83 (br
m, 1 H), 3.55–3.34 (m, 10 H), 1.99–1.21 (m, 31 H, include Boc sin-
glet at d = 1.42 and Me triplet at d = 1.27 ppm).
RP-HPLC (264 nm): 90.3% (tR = 17.63 min, 20–80% B).
RP-LC/MS (ESI): m/z = 1507.9 [M + H]+ (tR = 3.44 min, 25–95%
B).
13C NMR (CDCl3): d = 166.9, 166.8, 156.4, 156.3, 145.7, 145.3,
126.1, 79.6, 49.0, 40.5, 40.4, 39.9, 39.6, 35.8, 32.9, 28.3, 28.2, 26.0,
24.6, 14.9.
RP-LC/MS (ESI): m/z = 593.4 [M + H]+ (tR = 4.88 min, 30–95% B).
HRMS (ESI): m/z [M + H]+ calcd for C28H49N8O6: 593.3770; found:
HRMS (ESI): m/z [M + 2H]2+ calcd for C68H132N8O28: 754.4570;
found: 754.4568; [M + H]+ calcd for C68H131N8O28: 1507.9067;
found: 1507.9055.
N2,N5-Bis(2-aminoethyl)-3,6-bis[2-(amino)ethylamino]-
pyrazine-2,5-dicarboxamide TFA Salt (14)
The reaction of 7h (0.93 g, 1.21 mmol) with TFA (15 mL) in anhyd
CH2Cl2 (15 mL) was carried out as described above for the prepara-
tion of 11 to give TFA salt 14 as a reddish brown solid that was used
as such in the next reaction.
1H NMR (DMSO-d6): d =8.75 (t, J = 6.1 Hz, 2 H), 8.06 (br t, 2 H),
7.97 (br s, 4 H), 7.86 (br s, 4 H), 3.73 (br q, 4 H), 3.55 (q, J = 6.3
Hz, 4 H), 3.04–2.95 (m, 8 H).
593.3760.
N2,N5-Bis[2-(tert-butoxycarbonyl)aminoethyl]-3,6-bis(ethyl-
amino)pyrazine-2,5-dicarboxamide (10c)
Yield: 0.010 g (7%); orange solid; Rf = 0.25 (CHCl3–EtOAc, 7:3,
v/v).
1H NMR (CDCl3): d = 8.17 (br t, 2 H), 7.67 (t, J = 5.0 Hz, 2 H), 4.86
(br t, 2 H), 3.55–3.33 (m, 12 H), 1.42 (s, 18 H), 1.27 (t, J = 7.2 Hz,
6 H).
RP-LC/MS (ESI): m/z = 369.3 [M + H]+ (tR = 1.22 min, 5–95% B).
13C NMR (CDCl3): d = 166.8, 156.4, 145.9, 126.1, 79.6, 40.4, 39.9,
3,6-Bis(38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-
azahentetracontan-41-ylamino)-N2,N5-bis(38-oxo-
2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azahentetra-
contan-41-yl)pyrazine-2,5-dicarboxamide (16a)
35.9, 28.3, 14.9.
RP-LC/MS (ESI): m/z = 539.3 [M + H]+, 561.5 [M + Na]+ (tR = 4.34
min, 30–95% B).
The reaction of the above TFA salt 14 (1.21 mmol) with NHS ester
12 (3.89 g, 5.67 mmol) in the presence of NMM (2.66 mL, 24.2
mmol) in anhyd DMF (20 mL) and anhyd CH2Cl2 (5 mL) was car-
ried out overnight (ca. 15 h) as described above for the preparation
of 13. A similar purification of the crude product by preparative RP-
HPLC (30 × 150 mm, 20–50% B/15 min) afforded 16a.
HRMS (ESI): m/z [M + Na]+ calcd for C24H42N8O6Na: 561.3120;
found: 561.3117.
N2,N5-Bis(2-aminoethyl)-3,6-bis(propylamino)pyrazine-2,5-di-
carboxamide TFA Salt (11)
To a solution of 7a (0.430 g, 0.759 mmol) in anhyd CH2Cl2 (5 mL),
was carefully added TFA (5 mL) while stirring at ice-bath temper-
ature. After a few minutes, the reaction mixture was slowly allowed
to warm to r.t. and stirred for 1 h in an atmosphere of argon. The re-
action mixture was concentrated in vacuo, and the viscous residue
was co-evaporated with CH2Cl2 (4 × 20 mL), and then dried over-
night under high vacuum to give TFA salt 11, which was used as
such in the next reaction.
1H NMR (DMSO-d6): d = 8.68 (t, J = 6.2 Hz, 2 H), 7.90 (br s, 6 H),
3.54 (q, J = 6.2 Hz, 4 H), 3.44 (t, J = 6.7 Hz, 4 H), 3.03–2.97 (sext,
J = 5.8 Hz, 4 H), 1.61–1.55 (sext, J = 7.2 Hz, 4 H), 0.96 (t, J = 7.2
Hz, 6 H).
Yield: 0.94 g (29%); brick-red solid.
1H NMR (DMSO-d6): d = 9.03 (t, J = 5.9 Hz, 2 H), 8.12 (t, J = 6.0
Hz, 2 H), 7.96, 7.95 (2 × t, 4 H), 3.62 (t, J = 6.5 Hz, 4 H), 3.58 (t,
J = 6.6 Hz, 4 H), 3.51–3.22 (m, 204 H, includes characteristic m-
dPEG signals at d = 3.50 and 3.24 ppm), 2.33 (t, J = 6.5 Hz, 4 H),
2.30 (t, J = 6.6 Hz, 4 H).
13C NMR (DMSO-d6): d = 171.3, 170.8, 166.1, 145.7, 126.4, 71.7,
70.3, 70.1, 70.05, 70.0, 69.98, 67.3, 67.2, 58.5, 39.3, 38.7, 38.6,
36.6.
RP-HPLC (264 nm): 95.5% (tR = 16.35 min, 20–80% B).
Synthesis 2010, No. 14, 2383–2392 © Thieme Stuttgart · New York

PAPER
Synthesis of Hydrophilic Red Fluorescent Dyes
2391
RP-LC/MS (ESI): m/z = 884.3 [M + 3H]3+, 1325.4 [M + 2H]2+
(tR = 3.81 min, 5–95% B).
70.58, 70.55, 70.5, 70.2, 70.1, 66.9, 66.4, 65.8, 59.0, 41.9, 41.0,
40.0, 39.1, 38.8, 36.5, 32.2, 25.6.
HRMS (ESI): m/z [M + 3H]3+ calcd for C118H231N10O54: 884.1874;
found: 884.1872; [M + 2H]2+ calcd for C118H230N10O54: 1325.7774;
found: 1325.7769.
RP-LC/MS (ESI): m/z = 1778.3 [M + H]+, 889.9 [M + 2H]2+
(tR = 4.24 min, 5–95% B).
HRMS (ESI): m/z [M + 2Na]2+ calcd for C82H140N10O32Na2:
911.4710; found: 911.4713; [M + Na]+ calcd for C82H140N10O32Na:
1799.9527; found: 1799.9534.
3,6-Bis(74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,
53,56,59,62,65,68,71-tetracosaoxa-75-azaheptaheptacontan-77-
ylamino)-N2,N5-bis(74-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,
41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75-azaheptahep-
tacontan-77-yl)pyrazine-2,5-dicarboxamide (16b)
The reaction of the above TFA salt 14 (0.221 g, 107% mass balance,
0.250 mmol) with NHS ester 15 (1.40 g, 1.15 mmol) in the presence
of NMM (0.55 mL, 5.00 mmol) in anhyd DMF (9 mL) and anhyd
CH2Cl2 (1 mL) was carried out overnight (ca. 24 h) as described
above for the preparation of 13. A similar purification of the crude
product by preparative RP-HPLC (19 × 250 mm, 25–95% B/17
min) afforded 16b.
N2,N5-Bis(2-aminoethyl)-3,6-bis(38-oxo-
2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azahentetra-
contan-41-ylamino)pyrazine-2,5-dicarboxamide (19)
To a suspension of 18 (0.714 g, 0.400 mmol) in MeOH (25 mL), a
slurry of 10% Pd/C (0.143 g) in H2O (5 mL) was added, followed
by ammonium formate (0.103 g, 1.64 mmol). The resulting mixture
was heated at 70 °C for 1 h, allowed to cool to r.t., and filtered
through a pad of Celite to remove the catalyst. The filtrate was con-
centrated and the residue was dissolved in CHCl3 (100 mL), and
washed with brine–H2O (4:1, v/v). Removal of the solvent afforded
19, which was used as such in the next reaction.
Yield: 0.118 g (10%); brick-red semi-solid.
1H NMR (DMSO-d6): d = 9.04 (t, J = 5.2 Hz, 2 H), 8.12 (t, J = 6.0
Hz, 2 H), 7.95 (t, J = 5.2 Hz, 4 H), 3.74–3.22 (m, 404 H, includes
characteristic m-dPEG signals at d = 3.50 and 3.23 ppm), 2.36–2.26
(m, 8 H).
Yield: 0.548 (91%).
1H NMR (CDCl3): d = 9.08 (br, 2 H), 8.03 (br, 2 H), 6.90 (br, 2 H),
3.78–3.42 (m, 104 H, includes broad peak at d = 3.50 ppm for PEG),
3.38 (s, 6 H), 2.96 (br, 4 H), 2.45 (br, 4 H), 1.76 (br, 4 H).
RP-HPLC (264 nm): 91.5% (tR = 17.31 min, 10–90% B).
RP-LC/MS (ESI): m/z = 1192.9 [M + 4H]4+ (tR = 3.87 min, 5–95%
RP-LC/MS (ESI): m/z = 755.6 [M + 2H]2+ (tR = 3.57 min, 5–95%
B).
B).
3,6-Bis(38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-
azahentetracontan-41-ylamino)-N2,N5-bis(74-oxo-2,5,8,11,14,
17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetra-
cosaoxa-75-azaheptaheptacontan-77-yl)pyrazine-2,5-dicarbox-
amide (16c)
The reaction of the diamine 19 (0.54 g, 0.36 mmol) with NHS ester
15 (1.00 g, 0.82 mmol) in the presence of NMM (0.20 mL, 1.82
mmol) in anhyd DMF (20 mL) and anhyd CH2Cl2 (4 mL) was car-
ried out over the weekend as described above for the preparation of
13. A similar purification of the crude product by preparative RP-
HPLC (19 × 250 mm, 30–70% B/14 min) afforded 16c.
HRMS (ESI): m/z [M + 6H]6+ calcd for C214H426N10O102: 794.8070;
found: 794.8063; [M + 4H]4+ calcd for C214H424N10O102: 1191.7069;
found: 1191.7022.
3,6-Bis(2-aminoethylamino)-N2,N5-bis[2-(benzyloxycarbon-
yl)aminoethyl]pyrazine-2,5-dicarboxamide TFA Salt (17)
The reaction of 7i (0.418 g, 0.500 mmol) with TFA (3.85 mL) in an-
hyd CH2Cl2 (10 mL) was carried out as described above for the
preparation of 11 to give TFA salt 17 as a reddish brown solid that
was used as such in the next reaction.
1H NMR (DMSO-d6): d = 8.60 (t, J = 6.1 Hz, 2 H), 8.12 (br, 2 H),
7.71 (br, 6 H), 7.46 (t, J = 5.8 Hz, 2 H), 7.34–7.29 (m, 8 H), 5.01 (s,
4 H), 3.70 (br, 4 H), 3.37 (q, J = 6.3 Hz, 4 H), 3.21 (q, J = 6.3 Hz,
4 H), 3.01–2.95 (m, 4 H).
Yield: 0.379 g (28%); reddish gum.
1H NMR (DMSO-d6): d = 9.03 (t, J = 5.8 Hz, 2 H), 8.11 (t, J = 5.6
Hz, 2 H), 7.94 (t, J = 5.7 Hz, 4 H), 3.62–3.36 (m, 296 H, includes
broad peak at d = 3.50 ppm for PEG), 3.233, 3.230 (2 × s, 12 H),
2.34–2.29 (2 × t, 8 H).
RP-LC/MS (ESI): m/z = 637.2 [M + H]+ (tR = 3.72 min, 5–95% B).
N2,N5-Bis[2-(benzyloxycarbonyl)aminoethyl]-3,6-bis(38-oxo-
2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azahentetra-
contan-41-ylamino)pyrazine-2,5-dicarboxamide (18)
13C NMR (DMSO-d6): d = 171.3, 170.8, 166.2, 145.7, 126.4, 71.7,
70.3, 70.1, 70.05, 70.01, 69.98, 67.3, 67.2, 58.5, 40.4, 40.3, 40.1,
36.6.
The reaction of the above TFA salt 17 (0.50 mmol) with NHS ester
12 (1.00 g, 1.46 mmol) in the presence of NMM (0.55 mL, 5.00
mmol) in anhyd DMF (25 mL) and anhyd CH2Cl2 (5 mL) was car-
ried out overnight as described above for the preparation of 13.
Most of the solvent was evaporated under high vacuum, and the res-
idue was dissolved in CHCl3 (150 mL) and then successively
washed with 0.5 M KHSO4 in brine (1:4, v/v), H2O, and brine (50
mL each). Removal of the solvent gave a reddish gum (1.21 g),
which was subjected to flash chromatography over silica gel
(CHCl3–MeOH, 19:1, v/v) to give bis-PEG derivative 18.
RP-HPLC (264 nm): 96.2% (tR = 16.99 min, 10–90% B).
RP-LC/MS (ESI): m/z = 928.2 [M + 4H]4+, 1237.7 [M + 3H]3+
(tR = 3.87 min, 5–95% B).
HRMS (ESI): m/z [M + 4H]4+ calcd for C166H328N10O78: 927.5496;
found: 927.5543; [M + 3H]3+ calcd for C166H327N10O78: 1236.3971;
found: 1236.4004; [M
1854.0920; found: 1854.0960.
+
2H]2+ calcd for C166H326N10O78:
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.
Yield: 0.714 g (80%); reddish gum.
1H NMR (CDCl3): d = 9.22 (t, J = 5.9 Hz, 2 H), 8.02 (t, J = 6.1 Hz,
2 H), 7.34–7.28 (m, 10 H), 7.20 (br t, 2 H), 7.08 (t, J = 6.1 Hz, 2 H),
5.06 (s, 4 H), 3.86–3.35 (m, 108 H, includes characteristic m-dPEG Acknowledgment
signals at d = 3.65 and 3.38 ppm), 2.90 (t, J = 6.5 Hz, 2 H), 2.62 (br,
We would like to express our thanks to Tim A. Marzan, Rana
4 H), 2.35 (t, J = 5.6 Hz, 4 H).
Kumar, Tasha M. Schoenstein, and James M. Wilcox (Covidien),
and to Prof. Kevin D. Belfield and Hyo-Yang Ahn (University of
Central Florida, Orlando, FL) for measuring photophysical proper-
13C NMR (CDCl3): d = 173.2, 168.9, 166.7, 166.6, 156.9, 146.0,
137.0, 128.4, 128.3, 128.0, 126.5, 72.0, 70.7, 70.65, 70.64, 70.62,
Synthesis 2010, No. 14, 2383–2392 © Thieme Stuttgart · New York

2392
A. R. Poreddy et al.
PAPER
ties. We also thank Dr. Bich Vu and Dr. Jingyue Yang for providing
high-resolution mass spectral data and Dr. Raghavan Rajagopalan
and Dr. John N. Freskos for their helpful comments and suggestions
in the preparation of this manuscript.
(12) Presented in part: Poreddy, A. R.; Asmelash, B.; Galen, K.
P.; Fitch, R. M.; Shieh, J.-J.; Wilcox, J. M.; Schoenstein, T.
M.; Wojdyla, J. K.; Gaston, K. R.; Freskos, J. N.; Neumann,
W. L.; Rajagopalan, R.; Ahn, H.-Y.; Kostelc, J. G.;
Debreczeny, M. P.; Belfield, K. D.; Dorshow, R. B. In
Reporters, Markers, Dyes, Nanoparticles, and Molecular
Probes for Biomedical Applications, Vol. 7190; Achilefu,
S.; Raghavachari, R., Eds.; SPIE Press: Bellingham, 2009.
(13) (a) Kim, J. H.; Shin, S. R.; Matsuoka, M.; Fukunishi, K.
Dyes Pigm. 1998, 39, 341. (b) Kim, J. H.; Shin, S. R.;
Matsuoka, M.; Fukunishi, K. Dyes Pigm. 1999, 41, 183.
(c) Sekar, N. Colourage 1999, 46, 41.
(14) Jeanjot, P.; Bruyneel, F.; Arrault, A.; Gharbi, S.; Cavalier,
J.-F.; Abels, A.; Marchand, C.; Touillaux, R.; Rees, J.-F.;
Marchand-Brynaert, J. Synthesis 2003, 513.
(15) For reviews, see: (a) Abdel-Magid, A. F.; Mehrman, S. J.
Org. Process Res. Dev. 2006, 10, 971. (b) Tripathi, R. P.;
Verma, S. S.; Pandey, J.; Tiwari, V. K. Curr. Org. Chem.
2008, 12, 1093.
(16) (a) Abdel-Magid, A. F.; Maryanoff, C. A.; Carson, K. G.
Tetrahedron Lett. 1990, 31, 5595. (b) Abdel-Magid, A. F.;
Maryanoff, C. A. Synlett 1990, 537. (c) Abdel-Magid, A.
F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R.
D. J. Org. Chem. 1996, 61, 3849.
(17) Neumann, W. L.; Rajagopalan, R.; Dorshow, R. B. PCT Int.
Appl WO2007149479, 2007; Chem. Abstr. 2007, 148, 79073.
(18) Busnel, O.; Bi, L.; Dali, H.; Cheguillaume, A.; Chevance, S.;
Bondon, A.; Muller, S.; Baudy-Floc’h, M. J. Org. Chem.
2005, 70, 10701.
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