Synthesis of APTRA derivatives as building blocks for low-affinity fluorescent Ca2+ indicators

Article abstract of DOI:10.1055/s-2005-869900

A new method for the synthesis of trialkyl aminophenoltriacetates (APTRA triesters) and the functionalization of these into suitable building blocks for potential fully conjugated low-affinity fluorescent Ca²⁺ indicators are described. Georg Th

Full text of DOI:10.1055/s-2005-869900

1838
PAPER
Synthesis of APTRA Derivatives as Building Blocks for Low-Affinity
Fluorescent Ca2+ Indicators
APTRA
Derivatie
t
Ca Indicator
M
s
etten, Mario Smet, Noël Boens, Wim Dehaen*
Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium
Fax +32(16)327990; E-mail: Wim.Dehaen@chem.kuleuven.ac.be
Received 17 November 2004; revised 28 February 2005
NH2
NaOOC
ClCH2COOH
N
COONa
Abstract: A new method for the synthesis of trialkyl aminophenol-
triacetates (APTRA triesters) and the functionalization of these into
suitable building blocks for potential fully conjugated low-affinity
fluorescent Ca2+ indicators are described.
OH
O
COONa
NaOH
pH > 10
1
2
Key words: heterocycles, supramolecular chemistry, aminophe-
noltriacetate, intracellular calcium, fluorescence, indicator
H2SO4
ROH
ROOC
N
COOR
COOR
O
From a functional point of view, Ca2+ is probably the most
important intracellular cation. Measurements of (changes
in) cytosolic free Ca2+ concentrations with fluorescent
probes have enabled scientists to investigate the role of
Ca2+ in numerous physiological processes. The develop-
ment of fluorescent indicators, with APTRA1 (o-ami-
nophenol-N,N,O-triacetic acid) and BAPTA [1,2-bis(o-
aminophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid]2 moi-
eties as Ca2+ chelators, showing shifts in their excitation
and/or emission spectra upon binding Ca2+, entailed a ma-
jor breakthrough in the elucidation of intracellular Ca2+
dynamics.3 Cells at rest have a cytosolic free Ca2+ concen-
tration of around 100 nM; upon activation of the cells, this
level can rise to well above 1 mM. These changes play a
pivotal role in many cellular processes, ranging from mus-
cle contraction, neuronal signaling, secretion, fertiliza-
tion, cell division, to metabolism and cell death.4 New
fluorescent Ca2+ indicators with improved properties can
supplement the list of commercially available Ca2+ indica-
tors.5 Therefore, in this respect, we designed and synthe-
sized a number of new APTRA derivatives allowing
coupling with previously used fluorophores as well as di-
rect transformation into more novel conjugates.
3a
3b
R = Me 49%
R = Et 40%
Scheme 1
between 10 and 12 by addition of NaOH (Scheme 1). The
yield of 3 was found to drop when an equivalent amount
of NaOH was added in one portion. Because of the lack of
nucleophilicity of the carboxylate anions, cyclization was
avoided. Subsequent evaporation of water and Fischer es-
terification of 2 (using 1.1 equiv of H2SO4 with respect to
the amount of NaOH used in the previous step) gave the
triester of APTRA in fair yields and good purity. Attempts
to prepare the tribenzyl APTRA by Fischer esterification
of APTRA 2 with benzyl alcohol or benzylation in basic
media with benzyl bromide failed. The prepared trialkyl
o-aminophenol-N,N,O-triacetates 3a and 3b were used as
substrates for the synthesis of APTRA building blocks.
The synthesis of a previously described7 derivative, the 5-
bromo APTRA triester 4, was optimized. The use of bro-
mine and CH2Cl2 as solvent at room temperature instead
of NBS in AcOH reduced the reaction time and gave a
higher yield (Scheme 2). Several attempts to iodinate 3 al-
ways gave small amounts of persubstituted side-products
that were difficult to remove.
To prepare the APTRA-based fluorescent Ca2+ indicators
we needed to put the appropriate substituents on the
APTRA moiety. We also looked for an easier and cheaper
method to synthesize the APTRA triester 3 on a large
scale (10–100 g). For the reported synthesis, expensive
bases (e.g. proton sponge) and purification by column
chromatography were necessary.7 Yields were suppressed
due to the formation of benzoxazinone side-products after
cyclization of the intermediates.
To prepare the 5-pinacolatoboronic ester derivative 5
starting from 4 (Scheme 2), it was necessary to use micro-
wave irradiation because of dehalogenation occurring un-
der conventional conditions. To this end we adapted our
published conditions for the microwave assisted synthesis
of electron-rich aromatic boronic esters.8 Both 4 and 5
have potential as reagents for coupling with other arenes
in order to extend the conjugated system. This could result
in APTRA derivatives that show fluorescence at an appro-
priate wavelength.
The new method for synthesizing APTRA consists of an
adaptation of a known reaction6 between o-aminophenol
(1) and chloroacetic acid in water while keeping the pH
SYNTHESIS 2005, No. 11, pp 1838–1844
x
x.
x
x
.
2
0
0
5
Advanced online publication: 18.05.2005
DOI: 10.1055/s-2005-869900; Art ID: T13704SS
© Georg Thieme Verlag Stuttgart · New York

PAPER
APTRA Derivatives for Fluorescent Ca2+ Indicators
1839
ROOC
N
COOR
O
ROOC
N
COOR
O
COOR
COOR
Br2,
HNO3
AcOH
3
3
CH2Cl2
Br
4
NO2
13a
13b
R = CH3
84%
Pd(dppf)Cl2
DME, MW
150 °C, 200 W
O
O
R = CH2CH3 51%
B B
H2, Pd/C,
Et2O
O
O
ROOC
N
B
COOR
O
4a
4b
R = CH3
90%
COOR
ROOC
N
COOR
R = CH2CH3 77%
R = CH2CH3 63%
O
COOR
5
12a
12b
R = CH3
96%
O
O
R = CH2CH3 63%
5
NH2
Scheme 4
Scheme 2
The very useful 4-amino-APTRA triesters 12 were pre-
pared by subsequent nitration of 3 to 13 and reduction of
13 to 12 (Scheme 4). These products constitute the key in-
termediates in the synthesis of fused APTRA analogues
(Scheme 5).
Formylation of 3a,b under Vilsmeier–Haack conditions
gave 5-formyl APTRA triesters 6a,b in acceptable yields
(Scheme 3).
The [2+1]-condensation reaction of aldehyde 6b with 5-
amino-3-methyl-1-phenylpyrazole (8) gave tricyclic com-
pound 9 in low yield (Scheme 3). The low yield may be
due to intramolecular Claisen-type condensation between
the acetate functions. Analogs of 9 were described by us
to be highly fluorescent.9 Condensation of aldehyde 6a
with 9,10-phenanthrenequinone (10) in the presence of
ammonium acetate gave 11 in moderate yield (Scheme 3).
As a model compound for the APTRA triester 12 we have
used the commercial 4-aminoveratrol (14) to tune the re-
action conditions leading to benzothiazole and quinoline
derivatives (Table 1).
Model compound 14 and 4-amino-APTRA triester 12b
were easily transformed into isothiocyanates 15 and 16
with thiophosgene. An earlier attempt to prepare 2-chlo-
robenzothiazole 17 from 4-isothiocyanatoveratrol with
PCl5 had failed,10 probably due to the sensitivity of the
electron-rich aromatic nucleus to the harsh reaction con-
ditions. As an alternative, we prepared the 2-aminoben-
zothiazole derivatives 18 and 19 by reaction of the
anilines 12 or 14 with freshly prepared thiocyanogen11
followed by Sandmeyer reaction under anhydrous condi-
tions to afford the 2-halobenzothiazoles 17, 20, 21 and 22.
ROOC
N
COOR
O
COOR
POCl3
DMF
3
6a
6b
R = CH3
63%
R = CH2CH3
CHO
O
O
Quinolines can be prepared from the acetanilides 23 and
24 by a Vilsmeier-type methodology reported by Meth–
Cohn.12 The reaction with model compound 23 gave the
expected quinoline 25 in good yield. Unfortunately, start-
ing from anilide 24 and 8 equivalents of DMF, beside the
expected quinoline 26, substantial amounts of diformylat-
ed product 27 were found. Optimal results were obtained
using 2 equivalents of DMF, affording 26 in moderate
yield. The halogen substituents on the benzothiazole and
quinoline are easily substituted with aryl groups using the
Suzuki13 or Stille14 coupling reactions. We are currently
investigating the scope of these coupling reactions.
N
N
2
∆
H2N
NH4OAc,
AcOH
8
10
ROOC
N
N
COOR
ROOC
N
COOR
O
COOR
O
COOR
N
NH
N
N
N
Condensation reactions of anilines with o-chloroalde-
hydes can give fully conjugated aromatic heterocycles.
The solventless condensation reaction at 190–200 °C of 4-
aminoveratrol (14) and 5-chloro-4-formyl-3-methyl-1-
phenylpyrazole (28) gave the expected pyrazolo[3,4-
b]quinoline 29 in acceptable yield (Scheme 5). An
APTRA triester functionalized pyrazolo[3,4-b]quinoline
N
9
R = CH2CH3
17%
11
R = CH3
35%
Scheme 3
Synthesis 2005, No. 11, 1838–1844 © Thieme Stuttgart · New York

1840
B. Metten et al.
PAPER
R1
R1
R2
R2
t-BuONO, CuR3
CH3CN
S
S
N
N
H2N
R3
18, 19
17, 20, 21, 22
O
(SCN)2, r.t.
PCl5
H
N
N
Cl
R1
R1
R1
R2
R2
R2
28
MW
or conventional
heating
SCCl2
N
N
N
CH2Cl2, TEA
NH2
N
C
S
29, 30
12, 14
15, 16
O
TEA, CH2Cl2
Cl
R1
R1
R4
R2
R2
O
POCl3
DMF
N
HN
O
Cl
25, 26, 27
23, 24
Scheme 5
NaOH (28.0 g, 0.70 mol in 100 mL H2O). The mixture was heated
in a preheated oil bath to 100 °C. Solid NaOH was added portion-
wise when the pH dropped below 10. After the pH remained con-
stant around 10.7 the mixture was refluxed for another 30 min. After
cooling, the water was evaporated under reduced pressure. The
crude mixture of 2, excess NaOH and acetate residues was used in
further synthesis without purification.
1H NMR (300 MHz, D2O): d = 3.74 (s, 4 H, NCH2), 4.60 (s, 2 H,
OCH2), 7.03 (d, J = 8.1 Hz, 1 H, C6H4), 7.10 (t, J = 7.3 Hz, 1 H,
C6H4), 7.21 (t, J = 7.3 Hz, 1 H, C6H4), 7.28 (d, J = 8.1 Hz, 1 H,
C6H4).
30 was prepared in low yield by heating (conventional or
microwave assisted) an equimolar mixture of 12a and 28
without solvent. The low yield may again be due to in-
tramolecular Claisen-type condensation between the ace-
tate functions.
In conclusion, we have demonstrated that functionaliza-
tion of APTRA triesters 3 can give suitable building
blocks 4, 5, 6, 12, 16, 21, 22, and 26. These compounds
offer numerous possibilities for further derivatization by
condensation reactions or by transition metal catalyzed re-
actions into custom-made fluorescent systems which,
upon saponification, can serve as low-affinity fluorescent
Ca2+indicators.
13C NMR (75 MHz, D2O): d = 180.1, 177.3, 150.4, 139.2, 124.2,
122.6, 120.5, 113.5, 67.2, 58.0.
Triethyl o-Aminophenol-N,N,O-triacetate (3b)
The fluorescence data of 9, 11 and 30 and their use as a To the crude residue obtained in the preparation of 2 were added
Ca2+indicators is under investigation and will be reported
EtOH (350 mL) and H2SO4 (75.0 g, 40.8 mL, 0.77 mol) and the
mixture was stirred for 3 d at reflux temperature. After cooling the
elsewhere.
salts were filtered off and the solvent was evaporated. The residue
was dissolved in EtOAc and washed with a 10% NaOH solution and
DMF, THF and Et2O were dried over molecular sieve 4 Å. All other
reagents and solvents were purchased from Acros Organics or Ald-
rich and used without further purification. Mass spectra were re-
corded on a Kratos MSTC instrument at 70 eV or Hewlett Pacard
5989A Quadrupole. 1H NMR and 13C NMR spectra were recorded
on a Bruker Avance 300 MHz.
then with brine. The organic layer was dried over MgSO4 and evap-
orated under reduced pressure yielding compound 3b (15.8 g, 43%
calculated from o-aminophenol) as a pale brown oil.
1H NMR (300 MHz, CDCl3): d = 1.22 (t, J = 7.3 Hz, 6 H, OCH3),
1.30 (t, J = 7.3 Hz, 3 H, OCH3), 4.15 (q, J = 7.1 Hz, 4 H, OCH2),
4.20 (s, 4 H, NCH2), 4.24 (q, J = 7.1 Hz, 2 H, OCH2), 4.65 (s, 2 H,
OCH2), 6.80–6.95 (m, 4 H, C6H4).
13C NMR (75 MHz, CDCl3): d = 171.2, 169.3, 150.4, 140.2, 123.4,
122.2, 120.8, 115.2, 66.6, 61.4, 54.3, 14.7, 14.3.
LR–MS (CI): m/z = 368 [MH+].
Trisodium o-Aminophenol-N,N,O-triacetate APTRA (2)
To a three-necked flask equipped with a pH meter and a reflux con-
denser, charged with o-aminophenol (1, 10.9 g, 0.10 mol) and chlo-
roacetic acid (47.2 g, 0.50 mol), was added an aqueous solution of
Synthesis 2005, No. 11, 1838–1844 © Thieme Stuttgart · New York

PAPER
APTRA Derivatives for Fluorescent Ca2+ Indicators
1841
Table 1 Synthesis and Use of Key Intermediate 4-Amino APTRA Triester 12 and Its Model Compound 4-Aminoveratrol (14)
Product
15
R1
R2
R3
Cl
R4
Yield (%)
OMe
OMe
93
16
N(CH2COOEt)2
OMe
OCH2COOEt
OMe
83
17
48
18
OMe
OMe
72a
55
19a
19b
20
N(CH2COOMe)2
N(CH2COOEt)2
OMe
OCH2COOMe
OCH2COOEt
OMe
35
Br
Cl
Br
68
21
N(CH2COOMe)2
N(CH2COOMe)2
OMe
OCH2COOMe
OCH2COOMe
OMe
28
22
66
23
47
24
N(CH2COOMe)2
OMe
OCH2COOMe
OMe
n.d.
56
25
H
26
N(CH2COOMe)2
N(CH2COOMe)2
OMe
OCH2COOMe
OCH2COOMe
OMe
H
15,b 33c
22,b tracec
52
27
CHO
29
30
N(CH2COOMe)2
OCH2COOMe
20
a In a one-pot reaction: 14, KSCN, Br2 in MeOH.
b Using 8 equiv DMF.
c Using 2 equiv DMF.
HR–MS (EI): m/z calcd for C18H25NO7: 367.1631; found: 367.1632.
4-Bromo-2-methoxycarbonylmethoxy-N,N-bis(methoxycarbo-
nylmethyl)aniline (4a)
Trimethyl o-Aminophenol-N,N,O-triacetate (3a)
Compound 4a was prepared by the same procedure as 4b giving 4a
Compound 3a was prepared in the same way as 3b. Yield: 15.8 g
(48.4% starting from o-aminophenol); white crystals after crystalli-
sation from diisopropyl ether; mp 65 °C (Lit.7 75 °C).
(3.92 g, 97%) as white crystals; mp 72–73 °C (Lit.7 58–60 °C).
1H NMR and 13C NMR spectra were identical to those previously
reported.7
1H NMR and 13C NMR spectra were identical to the previously re-
ported ones.7
2-Ethoxycarbonylmethoxy-N,N-bis(ethoxycarbonylmethyl)-4-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (5)
A solution of 4b (530 mg, 1.20 mmol), bis(pinacolato)diboron (329
mg, 1.30 mmol), KOAc (294 mg, 3.00 mmol) and Pd[1,1¢-
bis(diphenylphosphino)ferrocene]Cl2 (30 mg, 3 mol%) in ethylene
glycol dimethyl ether (DME, 2 mL) was heated in a microwave
oven to 150 °C at a power of 200 W. After 15 min the solution was
cooled and filtered through a layer of Celite. The Celite was washed
with Et2O. After removing of the solvent, the residue was purified
by column chromatography on silica gel (eluent: CH2Cl2–EtOAc,
20:1). Compound 5 (370 mg, 63%) was obtained as a pale brown
viscous oil.
1H NMR (300 MHz, CDCl3): d = 1.22–1.27 (m, 9 H, OCH3), 1.30
[s, 12 H, C(CH3)3], 4.14–4.26 (m, 10 H, OCH2, NCH2), 4.66 (s, 2 H,
OCH2), 6.82 (d, J = 8.1 Hz, 1 H, C6H3), 7.20 (d, J = 1.5 Hz, 1 H,
C6H3), 7.38 (d, J = 8.1, 1.5 Hz, 1 H, C6H3).
13C NMR (75 MHz, CDCl3): d = 171.5, 169.2, 148.9, 142.6, 130.0,
120.5, 118.7, 83.9, 66.0, 61.4, 61.0, 54.1, 25.2, 14.5.
4-Bromo-2-ethoxycarbonylmethoxy-N,N-bis(ethoxycarbonyl-
methyl)aniline (4b)
A solution of 3b (1.84 g, 5.00 mmol) in CH2Cl2 (80 mL) was cooled
to 0 °C in an ice bath. Br2 (880 mg, 282 mL 5.50 mmol) dissolved in
CH2Cl2 (10 mL) was added in a dropwise manner to the mixture.
After 2 h stirring at r.t., the reaction mixture was quenched with an
aq NaOH solution (10%). The organic layer was washed with water
and then dried over MgSO4. After evaporation of the solvent, 4b
(1.72 g, 77%) was obtained as a pale brown oil.
1H NMR (300 MHz, CDCl3): d = 1.16–1.26 (m, 9 H, OCH3), 4.10–
4.19 (m, 8 H, NCH2, OCH2), 4.26 (q, J = 7.1 Hz, 2 H, OCH2), 4.60
(s, 2 H, OCH2), 6.73 (d, J = 8.8 Hz, 1 H, C6H3), 6.87 (d, J = 2.2 Hz,
1 H, C6H3), 7.05 (dd, J = 8.8, 2.2 Hz, 1 H, C6H3).
13C NMR (75 MHz, CDCl3): d = 171.4, 168.9, 150.6, 140.0, 131.9,
124.5, 121.9, 84.2, 66.6, 61.6, 61.1, 53.9, 43.2, 14.6, 14.4.
LR–MS (CI): m/z = 446–448 [MH+].
LR–MS (CI): m/z = 494 [MH+].
HR–MS (EI): m/z calcd for C18H24BrNO7: 445.0736; found:
445.0726.
Synthesis 2005, No. 11, 1838–1844 © Thieme Stuttgart · New York

1842
B. Metten et al.
PAPER
HR–MS (EI): m/z calcd for C24H36BNO9: 493.2483; found:
493.2488.
2-Methoxycarbonylmethoxy-N,N-bis(methoxycarbonylmeth-
yl)-4-(1H-phenathro[9,10-d]-imidazol-2yl)aniline (11)
A solution of 6a (177 mg, 0.50 mmol), 9,10-phenanthrenequinone
(10, 104 mg, 0.50 mmol) and ammoniumacetate (385 mg, 5.00
mmol) in glacial AcOH was charged in a 10 mL glass tube which
was then tightly sealed with an aluminum/Teflon crimp and heated
at 180 °C by irradiation at 250 W for 2 min in a CEM-Discover
mono-mode microwave apparatus. The cooled solid mixture was
poured in crushed ice and then neutralized with an aq NH3 solution
(10%). The precipitate was filtered and purified by column chroma-
tography on silica gel (eluent: CH2Cl2 to CH2Cl2–EtOAc, 90:10).
Compound 11 (95 mg, 35%) was obtained as a white-yellow pow-
der; mp 199–200 °C.
1H NMR (300 MHz, CDCl3): d = 3.66 (s, 6 H, OCH3), 3.77 (s, 3 H,
OCH3), 4.29 (s, 4 H, NCH2), 4.93 (s, 2 H, OCH2), 6.95 (s, J = 8.1
Hz, 1 H, C6H3), 7.63 (t, J = 7.3 Hz, 2 H), 7.75 (t, J = 7.3 Hz, 2 H),
7.77 (s, 1 H, C6H3), 7.83 (d, J = 8.8 Hz, 1 H, C6H3), 8.54 (d, J = 8.1
Hz, 2 H), 8.85 (d, J = 8.1 Hz, 2 H), 13.38 (br s, 1 H, NH).
4-Formyl-2-ethoxycarbonylmethoxy-N,N-bis(ethoxycarbonyl-
methyl)aniline (6b)
To a solution of 3b (1.47 g, 4.00 mmol), pyridine (388 mg, 0.40 mL,
5.00 mmol) in DMF (3.76 g, 4,0 mL, 50.0 mmol) was added POCl3
(4.94 g, 3.00 mL, 32.0 mmol) in a dropwise manner at 0 °C over 5–
10 min. The reaction was heated for 1–2 h at 65 °C until completion.
The reaction was monitored by the disappearance of the starting
material on silica TLC plate. After completion of the reaction, the
reaction mixture was poured into ice-water . After extraction with
EtOAc the organic layer was washed with brine and dried over
MgSO4. After removal of the solvent under reduced pressure, the
residue was purified by column chromatography on silica gel (elu-
ent: CH2Cl2–EtOAc, 9:1). This afforded 6b (1.33 g, 61%) as white
crystals; mp 59–60 °C.
1H NMR (300 MHz, CDCl3): d = 1.25–1.31 (m, 9 H, OCH3), 4.21–
4.27 (m, 10 H, NCH2, OCH2), 4.65 (s, 2 H, OCH2), 6.80 (d, J = 8.1
Hz, 1 H, C6H3), 7.27 (d, J = 1.8 Hz, 1 H, C6H3), 7.44 (dd, J = 8.1,
1.8 Hz, 1 H, C6H3,), 9.76 (s, 1 H, CHO).
13C NMR (75 MHz, CDCl3): d = 190.3, 170.7, 168.0, 148.7, 145.3,
129.8, 127.3, 117.2, 111.6, 65.6, 61.4, 61.0, 54.0, 14.2, 14.1.
13C NMR (75 MHz, CDCl3): d = 171.9, 168.7, 154.6, 150.4, 146.1,
143.4, 140.4, 138.8, 129.4, 129.3, 128.0, 125.1, 120.8, 120.4, 120.3,
119.1, 117.6, 115.8, 114.0, 108.2, 65.5, 60.7.
LR–MS (CI): m/z = 542 [MH+].
HR–MS (EI): m/z calcd for C30H27N3O7: 541.1849; found:
541.1838.
LR–MS (CI): m/z = 396 [MH+].
HR–MS (EI): m/z calcd for C19H25NO8: 395.1580; found: 395.1574.
2-Ethoxycarbonylmethoxy-N,N-bis(ethoxycarbonylmeth-
yl)benzene-1,4-diamine (12b)
4-Formyl-2-methoxycarbonylmethoxy-N,N-bis(methoxycarbo-
nylmethyl)aniline (6a)
Compound 6a was prepared in the same way as 6b giving 6a (4.24
To a solution of 3 (6.85 g, 18.6 mmol) in AcOH (60 mL) was added
fuming HNO3 (1.40 g, 1.00 mL, 22.3 mmol) dissolved in AcOH (10
mL) in a dropwise manner at 0 °C over 5–10 min. The reaction was
monitored by the disappearance of the starting material on a silica
TLC plate. After completion, the reaction mixture was poured on to
ice-water. After extraction with CH2Cl2, the organic layer was dried
over MgSO4 and filtered. After removing of the solvent under re-
duced pressure, the residue was purified by column chromatogra-
phy on silica gel (eluent: CH2Cl2). Compound 13b (3.94 g, 51%)
was obtained as yellow crystals; mp 76–78 °C.
1H NMR (300 MHz, CDCl3): d = 1.25–1.30 (m, 9 H, OCH3), 4.21–
4.27 (m, 10 H, OCH2, NCH2), 4.66 (s, 2 H, OCH2), 6.71 (d, J = 8.8
Hz, 1 H, C6H3), 7.61 (d, J = 2.6 Hz, 1 H, C6H3), 7.83 (dd, J = 8.8 Hz,
J = 2.6 Hz, 1 H, C6H3).
g, 60%) as white crystals; mp 94 °C.
1H NMR (300 MHz, CDCl3): d = 3.78 (s, 6 H, OCH3), 3.80 (s, 3 H,
OCH3), 4.29 (s, 4 H, NCH2), 4.67 (s, 2 H, OCH2), 6.80 (d, J = 8.8
Hz, 1 H, C6H3), 7.27 (d, J = 1.5 Hz, 1 H, C6H3), 7.43 (dd, J = 8.8,
1.5 Hz, 1 H, C6H3).
13C NMR (75 MHz, CDCl3): d = 190.7, 171.5, 168.5, 148.9, 145.5,
130.3, 127.8 (CH), 117.5 (CH), 111.9, 65.8, 54.2, 52.6, 52.4.
LR–MS (CI): m/z = 354 [MH+].
HR–MS (EI): m/z calcd for C16H19NO8: 353.1111; found: 353.1111.
4-(3,5-Dimethyl-1,7-diphenyl-1,7-dihydrodipyrazolo[3,4-b:4,3-
e]-2-ethoxycarbonylmethoxy-N,N-bis(ethoxycarbonylmeth-
yl)aniline (9)
13C NMR (75 MHz, CDCl3): d = 170.9, 168.0, 147.8, 145.8, 141.1,
119.4, 116.8, 109.1, 66.2, 62.0, 61.9, 61.6, 60.9, 54.5, 14.5, 14.4.
Compound 6b (99 mg, 0.25 mmol) and 5-amino-1-phenyl-3-meth-
ylpyrazole (8, 108 mg, 0.75 mmol) were heated in a long tube under
Ar to 120–160 °C for 1 h and 2 min at 200 °C. After cooling, EtOH
(8 mL) was added and the mixture was heated at reflux temperature
for 20 min. The solvent was evaporated and the obtained residue
was purified by column chromatography on silica gel (eluent:
CH2Cl2 to CH2Cl2–EtOAc, 80:20) to give 9 (30 mg, 17%) as yellow
glass film; mp 141 °C.
1H NMR (300 MHz, CDCl3): d = 1.20–1.30 (m, 9 H, OCH3), 2.10
(s, 6 H, pyrazole-CH3), 4.21–4.42 (m, 10 H, OCH2, NCH2), 4.67 (s,
2 H, OCH2), 6.87 (s, 1 H, C6H3), 6.99–7.05 (m, 2 H, C6H3), 7.31 (t,
J = 8.1 Hz, 2 H, C6H5), 7.52 (t, J = 8.1 Hz, 4 H, C6H5), 7.52 (d, J =
8.1 Hz, 4 H, C6H5).
LR–MS (CI): m/z = 413 [MH+].
HR–MS (EI): m/z calcd for C18H24N2O9: 412.1482; found:
412.1493.
To a solution of the nitrated APTRA derivative 13b (1.65 g, 4.00
mmol) in Et2O (100 mL) was added Pd/C (5%, 200 mg) and the sus-
pension was left under H2 at atmospheric pressure. After stirring
overnight the mixture was filtered through a layer Celite. The resi-
due, after evaporation of the solvent, was purified by flash column
chromatography on silica gel (eluent: EtOAc), giving 12b (968 mg,
63%) as an oil that turned brown after long-time exposure to air.
1H NMR (300 MHz, CDCl3): d = 1.21–1.29 (m, 9 H, OCH3), 3.49
(br s, 2 H, NH2), 4.10–4.17 (m, 8 H, NCH2, OCH2), 4.25 (q, 2 H,
OCH2), 4.67 (s, 2 H, OCH2), 6.22 (d, J = 2.6 Hz, 1 H, C6H3), 6.28
(dd, J = 8.4, 2.6 Hz, 1 H, C6H3), 6.88 (d, J = 8.4, 2.6 Hz, 1 H, C6H3).
13C NMR (75 MHz, CDCl3): d = 171.6, 168.7, 150.9, 149.2, 144.9,
141.4, 140.8, 129.3, 127.9, 125.5, 123.4, 120.6, 119.2, 115.1, 114.0,
66.3, 61.7, 61.3, 54.2, 15.3, 14.6, 14.5.
13C NMR (75 MHz, CDCl3): d = 171.3, 169.2, 151.6, 142.6, 131.4,
122.8, 109.1, 103.4, 66.4, 61.4, 60.4, 54.1, 14.2, 14.1.
LR–MS (CI): m/z = 705 [MH+].
HR–MS (EI): m/z calcd for C39H40N6O7: 704.2958; found:
LR–MS (CI): m/z = 383 [MH+].
704.2971.
Compound 12a was prepared in the same way as 12b giving 13a
(6.20 g, 84%) after nitration (pure on TLC, no additional purifica-
Synthesis 2005, No. 11, 1838–1844 © Thieme Stuttgart · New York

PAPER
APTRA Derivatives for Fluorescent Ca2+ Indicators
1843
tion) and 12a (5.71 g, 96%) after reduction (no purification by chro-
matography necessary).
13C NMR (75 MHz, CDCl3): d = 172.0, 170.2, 167.0, 149.8, 149.1,
134.7, 124.4, 113.2, 105.3, 66.4, 54.1, 52.6, 52.1.
LR–MS (CI): m/z = 398 [MH+].
4-Nitro-2-methoxycarbonylmethoxy-N,N-bis(methoxycarbon-
ylmethyl)aniline (13a)
Mp 78–80 °C.
HR-MS (EI): m/z calcd for C16H19N3O7S: 397.0944; found:
397.0946.
1H NMR (300 MHz, CDCl3): d = 3.77 (s, 6 H, OCH3), 3.81 (s, 3 H,
OCH3), 4.22 (s, 4 H, NCH2), 4.68 (s, 2 H, OCH2), 6.72 (d, J = 8.8
Hz, 1 H, C6H3), 7.62 (d, J = 2.2 Hz, 1 H, C6H3), 7.86 (dd, J = 8.8,
2.2 Hz, 1 H, C6H3).
13C NMR (75 MHz, CDCl3): d = 170.8, 168.0, 147.4, 145.3, 140.8,
119.1, 116.4, 108.7, 65.7, 53.9, 52.2, 51.9.
7-Ethoxycarbonylmethoxy-N6,N6-bis(ethoxycarbonylmethyl)-
2,6-diaminobenzothiazole (19b)
To a solution of 3b (855 mg, 2.24 mmol) and KSCN (325 mg, 3.36
mmol) in MeOH (20 mL) was added bromine (223 mg, 72 mL, 0.63
mmol) in MeOH (5.0 mL) in a dropwise manner at 0 °C. The mix-
ture was stirred for 30 min at r.t and then the solvent was evaporated
under reduced pressure. The residue was dissolved in CH2Cl2 (20
mL) and then washed with a 10% aq HCl solution, a concentrated
NaHCO3 solution, brine and distilled water. The organic layer was
dried over MgSO4, filtered and the solvent was partially evaporated.
The residue was purified by column chromatography on silica gel
(eluent: EtOAc). Compound 19b was obtained (340 mg, 35%) as a
black oil.
1H NMR (300 MHz, CDCl3): d = 1.21–1.27 (m, 9 H, OCH3), 4.12–
4.35 (m, 10 H, OCH2, NCH2), 4.70 (s, 2 H, OCH2), 5.38 (br s, 2 H,
NH2), 6.99–7.03 (m, 1 H, C6H2), 7.23 (s, 1 H, C6H2).
13C NMR (75 MHz, CDCl3): d = 171.6, 171.5, 169.2, 166.4, 150.5,
148.4, 135.9, 125.1, 113.6, 105.8, 66.7, 61.6, 61.0, 60.8, 54.5, 21.4,
14.5.
LR–MS (CI): m/z = 371 [MH+].
HR–MS (EI): m/z calcd for C15H18N2O9: 370.1012; found:
370.1012.
2-Methoxycarbonylmethoxy-N,N-bis(methoxycarbonylmeth-
yl)benzene-1,4-diamine (12a)
Mp 85–88 °C.
1H NMR (300 MHz, CDCl3): d = 3.52 (br s, 2 H, NH2), 3.68 (s, 6 H,
OCH3), 3.72 (s, 3 H, OCH3), 4.10 (s, 4 H, NCH2), 4.67 (s, 2 H,
OCH2), 6.20 (d, J = 8.8 Hz, 1 H, C6H3), 6.28 (dd, J = 8.8, 2.2 Hz, 1
H, C6H3), 6.87 (d, J = 2.9 Hz, 1 H).
13C NMR (75 MHz, CDCl3): d = 172.2, 170.0, 158.9, 143.3, 131.6,
123.1 (CH), 109.4 (CH), 103.5 (CH), 66.6, 54.3, 52.5, 52.0.
LR–MS (CI): m/z = 341 [MH+].
LR–MS (CI): m/z = 440 [MH+].
HR–MS (EI): m/z calcd for C19H25N3O7S: 439.1413; found:
439.1402
HR–MS (EI): m/z calcd for C15H20N2O7: 340.1271; found:
340.1271.
2-Chloro-7-methoxycarbonylmethoxy-N6,N6-bis(methoxycar-
bonylmethyl)-6-aminobenzothiazole (21)
4-Isothiocyanato-2-ethoxycarbonylmethoxy-N,N-bis(ethoxy-
carbonylmethyl)aniline (16)
To a solution of 19a (397 mg, 1.00 mmol) and CuCl2 (203 mg, 1.50
mmol) in MeCN (20 mL) was added a solution of tert-butyl nitrite
(90%, 172 mg, 200 mL, 1.50 mmol) in MeCN (10 mL) in a dropwise
manner at 0 °C. The mixture was stirred for 1 h at r.t. and then 1 h
at 65 °C. The mixture was taken up in EtOAc and washed with an
aq HCl solution (6 M) and then with a concd NaHCO3 solution,
brine and water. The organic layer was dried over MgSO4, filtered
and concentrated. The residue was purified by column chromatog-
raphy on silica gel (eluent: CH2Cl2–EtOAc, 9:1). Compound 21
(113 mg, 28%) was obtained as a grey powder; mp 114–115 °C.
1H NMR (300 MHz, CDCl3): d = 3.71 (s, 6 H, OCH3), 3.79 (s, 3 H,
OCH3), 4.27 (s, 4 H, NCH2), 4.72 (s, 2 H, OCH2), 7.27 (s, 1 H,
C6H2), 7.31 (s, 1 H, C6H2).
13C NMR (75 MHz, CDCl3): d = 171.8, 178.6, 169.0, 150.5, 146.9,
139.4, 130.2, 111.6, 107.4, 66.2, 54.1, 52.68, 52.3.
To a solution of 12b (444 mg, 1.16 mmol), thiophosgene (159 mg,
106 mL, 1.39 mmol) in CH2Cl2 (20 mL) at 0 °C was added Et3N
(4 × 100 mL, 288 mg, 2.80 mmol). After stirring for 1.5 h at r.t., H2O
(10 mL) was added. The organic layer was washed with brine and
water and dried over MgSO4. After evaporation of the solvent under
reduced pressure, 16 (410 mg, 83%) was obtained as a brown oil.
1H NMR (300 MHz, CDCl3): d = 1.22–1.30 (m, 9 H, CH3), 4.17–
4.21 (m, 8 H, OCH2, NCH2), 4.28 (q, J = 7.1 Hz, 2 H, OCH2), 4.62
(s, 2 H, OCH2), 6.64–6.65 (m, 1 H, C6H3), 6.81–6.82 (m, 2 H,
C6H3).
13C NMR (75 MHz, CDCl3): d = 207.0, 170.9, 168.2, 149.6, 139.1,
134.2, 124.5, 120.0, 119.9, 111.8, 66.0, 61.4, 60.9, 53.7, 30.94,
14.2, 14.1.
LR–MS (CI): m/z = 425 [MH+].
LR–MS (CI): m/z = 417–419 [MH+].
HR–MS (EI): m/z calcd for C19H24N2O7S: 424.1304; found:
424.1291.
HR–MS (EI): m/z calcd for C16H17ClN2O7S: 416.0445; found:
416.0443.
7-Methoxycarbonylmethoxy-N6,N6-bis(methoxycarbonylmeth-
yl)benzothiazolo-2,6-diamine (19a)
2-Bromo-7-methoxycarbonylmethoxy-N6,N6-bis(methoxycar-
bonylmethyl)-6-aminobenzothiazole (22)
Compound 22 was prepared by the same procedure as for 21 except
that Cu(I)Br was used instead of Cu(II)Cl2. Compound 22 (304 mg,
66%) was obtained as a grey powder; mp 104–105 °C.
1H NMR (300 MHz, CDCl3): d = 3.72 (s, 6 H, OCH3), 3.79 (s, 3 H,
OCH3), 4.26 (s, 4 H, NCH2), 4.79 (s, 2 H, OCH2), 7.28 (s, 1 H,
C6H2), 7.32 (s, 1 H, C6H2).
13C NMR (75 MHz, CDCl3): d = 171.7, 169.0, 150.3, 148.2, 139.4,
137.1, 131.5, 111.2, 107.1, 66.2, 54.1, 52.4, 52.3.
LR–MS (CI): m/z = 461–463 [MH+].
A solution of 12a (1.46 g, 4.30 mmol) and dithiocyanogen (6.00
mmol) in MeOH–Et2O [prepared from Pb(SCN)2 (1.95 g, 6.00
mmol) and bromine (770 mg, 250 mL, 4.80 mmol) in Et2O (50 mL)]
was stirred for 30 min at r.t. The solvent was evaporated under re-
duced pressure. The residue was dissolved in EtOAc, heated for a
few minutes at reflux temperature and then cooled. The precipitate
was filtered affording 19a (930 mg, 55%) as a grey powder; mp 145
°C.
1H NMR (300 MHz, CDCl3): d = 3.60 (s, 6 H, OCH3), 3.70 (s, 3 H,
OCH3), 4.14 (s, 4 H, NCH2), 4.83 (s, 2 H, OCH2), 6.91 (s, 1 H,
C6H2), 7.44 (s, 1 H, C6H2), 9.35 (br s, 2 H, NH2).
Synthesis 2005, No. 11, 1838–1844 © Thieme Stuttgart · New York

1844
B. Metten et al.
PAPER
HR–MS (EI): m/z calcd for C16H17BrN2O7S: 459.9940; found:
459.9927.
combined organic layers were dried over MgSO4, filtered and the
solvent was evaporated. The residue was purified by column chro-
matography on silica gel (eluent: CH2Cl2 to CH2Cl2–EtOAc, 90:10).
Compound 30 (75 mg, 20%) was obtained as a yellow powder; mp
89–92 °C.
2-Methoxycarbonylmethoxy-N4-acetyl-N,N-bis(methoxycarbo-
nylmethyl)benzene-1,4-diamine (24)
To a solution of 3a (2.20 g, 6.47 mmol) and Et3N (712 mg, 0.99 mL,
7.11 mmol) in CH2Cl2 (50 mL) was added acetyl chloride (420 mg,
462 mL, 6.47 mmol) in CH2Cl2 (15 mL) in a dropwise manner at 0
°C. The mixture was stirred for 1 h at r.t. The solvent was evaporat-
ed and the residue was used without purification in the next step.
1H NMR (300 MHz, CDCl3): d = 2.68 (s, 3 H, pyrazole-CH3), 3.78
(s, 6 H, OCH3), 3.83 (s, 3 H, OCH3), 4.36 (s, 4 H, NCH2), 4.84 (s, 2
H, OCH2), 7.25 (s, 1 H, Ar), 7.29 (s, 1 H, Ar), 7.46–7.53 (m, 3 H,
C6H5), 8.29 (s, 1 H, Ar), 8.42 (d, J = 8.8 Hz, 2 H, C6H5).
13C NMR (75 MHz, CDCl3): d = 171.9, 168.7, 154.6, 150.4, 146.1,
143.4, 140.4, 138.8, 129.4, 129.3, 128.0, 125.1, 120.8, 120.4, 120.3,
119.1, 117.6, 115.8, 114.0, 108.2, 65.5, 60.7.
2-Chloro-3-formyl-8-methoxycarbonylmethoxy-7-bis(meth-
oxycarbonylmethyl)aminoquinoline (26) and 2-Chloro-3,6-di-
formyl-8-methoxycarbonylmethoxy-7-bis(methoxycarbonyl-
methyl)aminoquinoline (27)
LR–MS (CI): 506–508 [MH+].
To a mixture of POCl3 (13.8 g, 8.40 mL, 16 equiv) and the residue
of the previous step (ca 6.5 mmol) was added DMF (3.76 g, 4.00 Acknowledgment
mL, 8 equiv) in a dropwise manner at 0 °C. After heating the reac-
The authors are grateful to the Research Council of the University
tion mixture at 80 °C for 3 h, the mixture was poured in crushed ice
and stirred for 3 h. The precipitate was filtered and the strongly col-
ored filtrate was extracted with EtOAc. The combined organic lay-
ers were dried over MgSO4, filtered and the solvent was evaporated.
The residue from the filtrate was purified by column chromatogra-
phy on silica gel (eluent: EtOAc). The combined yield of the unsep-
arated compounds 26 (15%) and 27 (21%) was 1.03 g (36%) as a
grey-yellow powder.
of Leuven (IDO-grant IDO/00/001) for a doctoral fellowship to B.
M. The Fonds voor Wetenschappelijk Onderzoek Vlaanderen
(FWO grant G.0172.02) and the Ministerie voor Wetenschapsbeleid
(Belgium, DWTC-IUAP-V-03) are thanked for continuing support.
M. S. thanks the FWO for a postdoctoral fellowship.
References
Compound 26
(1) (a) Otten, P. A.; London, R. E.; Levy, L. A. Bioconjugate
Chem. 2001, 12, 76. (b) Otten, P. A.; London, R. E.; Levy,
L. A. Bioconjugate Chem. 2001, 12, 203.
(2) Tsien, R. Y. Biochemistry 1980, 19, 2396.
(3) (a) Tsien, R. Y. Annu. Rev. Neurosci. 1989, 12, 227.
(b) Scheenen, W. J. J. M.; Hofer, A. M.; Pozzan, T. In Cell
Biology: A Laboratory Handbook, Vol. 3, 2nd ed.; Celis, J.
E., Ed.; Academic Press: New York, 1998, 363.
(c) Takahashi, A.; Camacho, P.; Lechleiter, J. D.; Herman,
B. Physiol. Rev. 1999, 79, 1089.
1H NMR (300 MHz, CDCl3): d = 3.74 (s, 6 H, OCH3), 3.78 (s, 3 H,
OCH3), 4.28 (s, 4 H, NCH2), 4.77 (s, 2 H, OCH2), 7.08 (s, 1 H,
C6H2), 7.13 (s, 1 H, C6H2), 8.43 (s, 1 H, C5NH), 10.39 (s, 1 H,
CHO).
13C NMR (75 MHz, CDCl3): d = 189.0, 171.4, 168.0, 155.9, 148.8,
147.5, 141.6, 138.2, 125.2, 123.1, 115.6, 108.4, 65.8, 57.0, 52.8,
52.47.
LR–MS (CI): m/z = 439–441 [MH+].
(4) (a) Berridge, M. J.; Bootman, M. D.; Lipp, P. Nature
(London) 1998, 395, 645. (b) Berridge, M. J.; Lipp, P.;
Bootman, M. D. Nature Rev. Mol. Cell Biol. 2000, 1, 11.
(c) Berridge, M. J. Cell Calcium 2002, 32, 235.
(5) Haugland, R. P. In Handbook of Fluorescent Probes and
Research Products, 9th ed.; Molecular Probes: Eugene, OR,
USA, 2002.
(6) Freedman, H. H.; Frost, A. E. J. Org. Chem. 1958, 23, 1292.
(7) (a) Cielen, E.; Stobiecka, A.; Tahri, A.; Hoornaert, G. J.; De
Schryver, F. C.; Gallay, J.; Vincent, M.; Boens, N. J. Chem.
Soc. Perkin Trans. 2 2002, 6, 1197. (b) Cielen, E. PhD
Dissertation; University of Leuven: Belgium, 1999.
(8) Appukkuttan, P.; Van der Eycken, E.; Dehaen, W. Synlett
2003, 1204.
HR–MS (EI): m/z calcd for C19H19ClN2O8: 438.0829; found:
438.0828.
Compound 27
1H NMR (300 MHz, CDCl3): d = 3.65 (s, 6 H, OCH3), 3.81 (s, 3 H,
OCH3), 4.19 (s, 4 H, NCH2), 4.89 (s, 2 H, OCH2), 7.37 (s, 1 H,
C6H2), 9.68 (s, 1 H, C5NH), 10.39 (s, 1 H, CHO), 10.88 (s, 1 H,
CHO).
13C NMR (75 MHz, CDCl3): d = 194.4, 189.0, 170.9, 167.8, 159.3,
151.1, 149.2, 147.1, 138.7, 130.1, 126.6, 120.5, 113.3, 65.6, 57.1,
53.0, 52.3.
LR–MS (CI): m/z = 467–469 [MH+].
HR–MS (EI): m/z calcd for C20H19ClN2O9: 466.0780; found:
(9) Abramov, M. A.; Ceulemans, E.; Jackers, C.; Van der
Auweraer, M.; Dehaen, W. Tetrahedron 2001, 9123.
(10) Hiskey, R. G.; Carroll, F. I.; Babb, R. M.; Bledsoe, J. O.;
Pucket, R. T.; Roberts, B. W. J. Org. Chem. 1961, 26, 1152.
(11) Hunter, R. F.; Jones, J. W. T. J. Chem. Soc., Abstr. 1930,
941.
(12) Meth-Cohn, O.; Narine, B.; Tarnowski, B.; Hayes, R.;
Keyzad, A.; Rhouati, S.; Robinson, A. J. Chem. Soc., Perkin
Trans. 1 1981, 9, 2509.
466.0793.
7-Methoxycarbonylmethoxy-N6,N6-bis(methoxycarbonylmeth-
yl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b]quinolin-6-amine
(30)
Compound 12a (340 mg, 1.00 mmol) and 5-chloro-4-formyl-3-
methyl-1-phenylpyrazole (28, 220 mg, 1.00 mmol) were charged in
a 10 mL glass tube which was then tightly sealed with an aluminum/
Teflon crimp and heated at 180 °C by irradiation at 250 W for 2 min
in a CEM-Discover mono-mode microwave apparatus. The cooled
solid mixture was dissolved in EtOAc. The organic phase was
washed with a concd aq NaHCO3 solution, brine and water. The
(13) Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
(14) Stille, J. K. Angew. Chem., Int. Ed. Engl. 1986, 25, 508;
Angew. Chem. 1986, 98, 504.
Synthesis 2005, No. 11, 1838–1844 © Thieme Stuttgart · New York