Synthesis of 18F-labeled phenolphthalein and naphtholphthalein

Article abstract of DOI:10.1016/j.jfluchem.2013.03.021

The fluorination of phenolphthalein and naphtholphthalein was performed with diluted fluorine gas under acidic conditions. For both compounds we observed an electrophilic fluorination into ortho position to the hydroxyl group. Through the use of this reaction we synthesized and characterized mono- and difluorinated derivatives of phenolphthalein and naphtholphthalein. The compounds were also prepared in the ¹⁸F labeled form, which are usable as a new type of probe for in vivo pH measurement in biological objects using Cerenkov imaging or combination of light absorption and PET.

Full text of DOI:10.1016/j.jfluchem.2013.03.021

Journal of Fluorine Chemistry 151 (2013) 1–6
Contents lists available at SciVerse ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
18
Synthesis of F-labeled phenolphthalein and naphtholphthalein
Alexander V. Kachur a, , Andrey A. Popov a,b, E. James Delikatny a, Joel S. Karp a,
*
Anatoliy V. Popov a
a Department of Radiology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
b Rensselaer Polytechnic Institute, Troy, NY 12180, USA
A R T I C L E I N F O
A B S T R A C T
Article history:
The fluorination of phenolphthalein and naphtholphthalein was performed with diluted fluorine gas
under acidic conditions. For both compounds we observed an electrophilic fluorination into ortho
position to the hydroxyl group. Through the use of this reaction we synthesized and characterized mono-
and difluorinated derivatives of phenolphthalein and naphtholphthalein. The compounds were also
prepared in the 18F labeled form, which are usable as a new type of probe for in vivo pH measurement in
biological objects using Cerenkov imaging or combination of light absorption and PET.
ß 2013 Elsevier B.V. All rights reserved.
Received 13 February 2013
Received in revised form 25 March 2013
Accepted 26 March 2013
Available online 6 April 2013
Keywords:
F-18
Electrophilic fluorination
Phenolphthalein
Naphtholphthalein
1. Introduction
University of Pennsylvania using a custom-made fluorination
a)
unit. The [18F]-F2 gas was prepared by the 20Ne(d, 18F reaction
Direct electrophilic fluorination is an infrequently used
synthetic method for the labeling of complex organic molecules
with the positron emitting isotope 18F. The electrophilic fluorina-
tion reaction produces compounds with relatively low specific
activity and in most cases results in non-specific decomposition of
the reagent. Despite this, we have recently achieved the specific
electrophilic fluorination of phenolsulfonphthalein [1] and its
derivatives [2], and used this reaction for the synthesis of 18F-
labeled pH indicators [3].
In the current paper we report the synthesis of 18F-labeled
derivatives of two other types of triarylmethane indicators,
phenolphthalein and naphtholphthalein. These compounds repre-
sent a new class of dual-mode indicators with high potential as in
vivo pH probes in biological objects.
using an IBA 18/9 Cyclone accelerator. The labeling reaction was
performed by bubbling of 0.1% F2 or [18F]-F2 in Ne (total 100–
200 mCi of activity in 35 micromoles of fluorine carrier) for 5 min
through a solution of the correspondent indicator in trifluor-
oacetic acid (2 mg/mL). The reaction mixture was evaporated in a
vacuum at 100 8C, redissolved in 2 mL of water/acetonitrile
mixture, injected into a semi-preparative HPLC column (Phenom-
˚
enex, Synergi 4
m
m Hydro-RP 80 A 10 mm ꢀ 250 mm) and eluted
with 40% acetonitrile–water for phenolphthalein and 55%
acetonitrile–water buffer for naphtholphthalein at a rate of
3 mL/min with simultaneous detection of radioactivity and
absorption at 276 nm. Under these conditions the elution times
for the products of interest were in the range of 20–35 min with
good separation of initial precursor and fluorination products on
both UV and radioactivity detectors.
In order to analyze the reaction products, corresponding
fractions were collected and analyzed by analytical HPLC, mass-
spectroscopy, UV–vis spectroscopy, and 1H and 19F NMR. The
presence of individual compounds in the fractions was
confirmed by analytical HPLC performed with a 4.6 mm ꢀ
250 mm column of the same type and using a gradient of
acetonitrile–ammonium acetate buffer (0.1 M, pH 4.7). The
incorporation of fluorine atoms into the indicator molecule was
proven by mass-spectroscopy on a Thermo Fisher Scientific
(Bremen, Germany) Orbitrap Exactive with Xcalibur software.
Since the relative amount of fluorine-18 in radioactive [18F]-F2
gas was very small, and as thus would not give any distinct
2. Materials and methods
All reagents were purchased from Sigma–Aldrich, with the
exception of the fluorine-neon mixture, which was obtained from
Matheson TriGas Inc. (Twinsburg, OH). Labeling of the molecules
with F2 and [18F]-F2 was performed at the Cyclotron facility of the
* Corresponding author at: 420 Curie Blvd., Cyclotron Facility, University of
Pennsylvania, School of Medicine, Philadelphia, PA 19104, USA.
Tel.: +1 215 662 7550; fax: +1 215 662 7550.
E-mail address: kachur@mail.med.upenn.edu (A.V. Kachur).
0022-1139/$ – see front matter ß 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jfluchem.2013.03.021

2
A.V. Kachur et al. / Journal of Fluorine Chemistry 151 (2013) 1–6
signal on mass-spectra, the molecular mass of the products was
determined after complete decay of the radioactive label.
UV–vis spectroscopic investigation of the absorption and
indicator properties of the products was performed on a Beck-
man-Coulter DU 530 spectrophotometer using acetate, phos-
phate, TRIZMA, and borate buffers for different pH values. A
combination of radioactivity and optical properties was used as a
supplementary method for confirming the incorporation of the
fluorine atoms into the indicator molecule. The position of the
fluorination in the molecules was determined from 1H and 19F
NMR spectra obtained for neutral (colorless) forms of the
indicators on a Bruker DMX-360 spectrometer using deuterated
methanol solvent. Decoupling experiments were carried out with
set parameters O1 = 4.5 ppm, O2 = ꢁ138 ppm for 1H {19F} and
O1 = ꢁ100 ppm, O2 = 6.95 ppm for 19F {1H}, respectively.
3.5. (RS)-3-(3-fluoro-4-hydroxynaphthalen-1-yl)-3-(4-
hydroxynaphthalen-1-yl)isobenzofuran-1(3H)-one
(monofluoronaphtholphthalein, MFNP)
m/z 435.100 amu (negative), 437.119 amu (positive); pKa =
7.66,
1H (360 MHz, CD3OD)
J = 7.9 Hz, 1H); 7.165 (td, J = 7.9, 1.4 Hz, 1H);
1.1 Hz, 1H); 7.330 (t, J = 7.6 Hz, 1H); 7.617 (d, J = 6.5 Hz, 1H);
7.624 (td, J = 7.6, 1.4 Hz, 1H); 7.683 (td, J = 7.6, 1.4 Hz, 1H);
(d, J = 8.6 Hz, 1H); 7.873 (d, J = 8.6 Hz, 1H);
1.1 Hz 1H); 8.220 (d, J = 7.9 Hz 1H); 8.229 (d, J = 7.9 Hz, 1H).
19F (338 MHz, CD3OD):
-143.965 (bs, 1F).
lbasic = 659 nm.
d
6.725 (d, J = 8.3 Hz, 1H);
d 7.104 (t,
d
d
7.306 (td, J = 7.6,
d
d
d
d
d
7.861
d
d 7.997 (dd, J = 7.6,
d
d
d
3.6. 3,3-Bis(3-fluoro-4-hydroxynaphthalen-1-yl)isobenzofuran-
1(3H)-one (difluoronaphtholphthalein, DFNP)
3. Experimental
m/z 453.090 amu (negative), 455.110 amu (positive); pKa =
The reactants and synthesized products were characterized by
mass spectra, visible spectrophotometry, and proton and fluorine
NMR. The results are given below.
7.24,
1H (360 MHz, CD3OD)
J = 7.6 Hz, 2H); 7.667 (d, J = 6.8 Hz, 1H);
1H); 7.799 (td, J = 7.6, 1.4 Hz, 1H); 7.858 (d, J = 8.6 Hz, 2H);
8.043 (dd, J = 7.2, 1.4 Hz 1H); 8.247 (d, J = 7.9 Hz 2H).
19F (338 MHz, CD3OD):
-143.955 (bs, 2F).
lbasic = 665 nm.
d
7.161 (td, J = 7.9, 1.4 Hz, 2H);
d 7.367 (t,
d
d
7.693 (td, J = 7.6, 1.4 Hz,
d
d
d
3.1. Phenolphthalein
d
d
m/z 317.079 amu (negative), 319.097 amu (positive); pKa =
9.75,
1H (360 MHz, CD3OD)
7.089 ppm (dd, J = 6.5, 2.1 Hz 4H);
l
basic = 553 nm.
4. Results
d
6.735 ppm (dd, J = 6.5, 2.1 Hz 4H);
7.593 (t, J = 7.6 Hz, 1H); 7.603
7.754 (td, J = 7.5, 1.3 Hz, 1H); 7.882 (dt, J = 7.2,
d
d
d
The reaction between phenolphthalein and [18F]-F2 gas in
trifluoroacetic acid demonstrates a certain degree of similarity
with the previously observed fluorination of Phenol Red and its
derivatives [1–3]. In both cases significant amounts of the product
of fluorine-hydrogen substitution were obtained via specific
fluorination of the precursor molecules. Separation of the reaction
mixture by HPLC indicated the presence of the non-reacted
precursor and the synthesis of two major radioactive components.
These products had higher retention time on the reverse-phase
column, which is typical for more hydrophobic fluorinated
derivatives [1–3]. The ratio of the radioactivity to UV absorption
was almost 1–2 for the obtained products, suggesting successive
incorporation of two fluorine atoms into the phenolphthalein
molecule.
(d, J = 7.2 Hz, 1H);
d
d
1.3 Hz, 1H).
3.2. (RS)-3-(3-fluoro-4-hydroxyphenyl)-3-(4-
hydroxyphenyl)isobenzofuran-1(3H)-one
(monofluorophenolphthalein, MFPP)
m/z 335.069 amu (negative), 337.087 amu, (positive);
pKa = 9.33, basic = 558 nm.
1H (360 MHz, CD3OD)
6.891 (dd, JHF = 8.0 Hz, JHH = 8.3 Hz 1H);
2.1 Hz, 1H); 6.948 (dd, JHF = 12.1 Hz, JHH = 2.1 Hz 1H);
(ddd, JHH = 8.9, 3.0, 2.2 Hz 4H); 7.619 (td, JHH = 7.5, 0.9 Hz, 1H);
l
d
6.750 (ddd, JHH = 8.9, 3.0, 2.2 Hz 2H);
6.914 (dd, JHH = 8.5,
7.089
d
d
d
d
d
d
7.640 (d, JHH = 7.8 Hz, 1H);
(d, JHH = 7.6 Hz, 1H).
d
7.779 (td, JHH = 7.6, 1.1 Hz, 1H);
d
7.897
Mass-spectroscopic investigation of the three HPLC fractions
reveal main peaks at m/z 317.079, 335.069, and 353.060 amu for
negative mode and 319.097, 337.087, and 355.079 amu for positive
mode. These data indicate the successive substitution of one or two
hydrogen atoms by fluorine in the phenolphthalein molecule.
Based on the mass-spectroscopic data and radioactivity, two
reaction products were identified as mono- (MFPP) and difluor-
ophenolphthalein (DFPP). The corrected radiochemical yield of
these products varied for the two products and was in the ranges of
10–15% and 3–4%, respectively. The chemical yield of the products
was calculated from the absorption of their basic forms using the
extinction coefficient of phenolphthalein, 3 ꢀ 104 Mꢁ1 cmꢁ1 [4].
The value of the chemical yield was the same as the radiochemical
for MFPP and halved for DFPP.
Both compounds acted as pH indicators with properties similar
to phenolphthalein. Colorless neutral forms of these molecules
have weak absorption maxima in the UV range (270–280 nm),
while the purple basic forms of indicators absorbed at 558 nm for
the monofluorinated and 564 nm for the difluorinated derivative.
The pKa values for the transfer between these two forms were
found to be 9.33 for MFPP and 8.70 for DFPP. Thus the successive
incorporation of fluorine atoms into the phenolphthalein molecule
19F (338 MHz, CD3OD):
d
ꢁ138.185 (dd, JFH = 8.1, 11.9 Hz, 1F).
3.3. 3,3-Bis(3-fluoro-4-hydroxyphenyl)isobenzofuran-1(3H)-one
(difluorophenolphthalein, DFPP)
m/z 353.060 amu (negative), 355.079 amu (positive); pKa =
8.70,
1H (360 MHz, CD3OD):
JHF = 8.3 Hz, 2H); 6.921 (dd, JHH = 9.7, 1.8 Hz, 2H);
JHF = 11.7 Hz, JHH = 1.8 Hz 2H); 7.638 (td, JHH = 7.6, 0.7 Hz, 1H);
lbasic = 564 nm.
d
6.897 (ddd, JHH = 8.3, 0.7 Hz,
6.951 (dd,
d
d
d
d
7.675 (d, JHH = 7.8 Hz, 1H);
(d, JHH = 7.6 Hz, 1H).
d
7.799 (td, JHH = 7.6, 1.1 Hz, 1H);
d
7.909
19F (338.8 MHz, CD3OD):
d
ꢁ137.954 (dd, JFH = 11.8, 7.5 Hz, 2F).
3.4. Naphtholphthalein
m/z 417.110 amu (negative), 419.128 amu (positive); pKa =
7.83, basic = 653 nm.
1H (360 MHz, CD3OD)
J = 7.9, 1.4 Hz, 2H); 7.290 (td, J = 7.6, 1.1 Hz, 2H);
J = 7.6 Hz, 1H); 7.600 (td, J = 7.6, 1.4 Hz, 1H);
1.4 Hz, 1H); 7.885 (d, J = 9.0 Hz, 2H); 7.976 (dd, J = 7.6, 1.4 Hz
1H); 8.215 (d, J = 8.3 Hz 2H).
l
d
6.712 (d, J = 7.9 Hz, 2H);
d
7.129 (td,
d
d 7.590 (d,
d
d
7.657 (td, J = 7.6,
(pKa = 9.75,
and a bathochromic shift of
behavior of fluorinated derivatives of Phenol Red [1–3].
l
basic = 553 nm) causes a decrease in the pKa values
d
d
l
basic, which is consistent with the
d

A.V. Kachur et al. / Journal of Fluorine Chemistry 151 (2013) 1–6
3
Fig. 1. 1H NMR spectra of phenolphthalein and its fluorinated derivatives. Successive introduction of fluorine atoms into the phenolphthalein molecule (a) causes only a minor
shift of the signals from the carboxylic ring ( 7.6–7.9 ppm) and gradual replacement of the signals from phenolic rings ( 6.7 and 7.1 ppm) by a new group of peaks ( 6.8–
d
d
d
7.0 ppm) for mono- (b) and difluorophenolphthalein (d). Decoupling of the fluorine-hydrogen coupling (spectra (c) and (e) for mono- and difluorophenolphthalein,
respectively) shows, that these new peaks are coupled with fluorine and allows an assignment of the proton signals.
The position of fluorine incorporation into the phenolphthalein
molecule was determined by comparison of the 1H and 19F NMR
spectra of the compounds. The 1H NMR spectrum of phenolphtha-
The 1H NMR data indicate that the first fluorine atom
substitutes a hydrogen in the phenol ring, and the second fluorine
atom is located in the identical positions on the second phenol ring.
The latter conclusion is also supported by 19F NMR data (Fig. 2),
showing only one signal from two identical nuclei in the
difluorophenolphthalein spectrum. The possibility of substitutions
in the carboxyl ring, or introduction of both fluorine atoms into the
same phenol ring, are thus both ruled out as well. The exact
position of the substitution can be deduced from the chemical shift
in 19F NMR spectra (Fig. 2). Location of the signals at ꢁ138 ppm is
characteristic of fluorine atoms in ortho positions in fluorophenols
and excludes the possibility of meta location (which has much
lower value chemical shift of ꢁ113 ppm) [5]. From these data we
can identify the monofluorophenolphthalein (MFPP) as (RS)-3-(3-
fluoro-4-hydroxyphenyl)-3-(4-hydroxyphenyl)isobenzofuran-
1(3H)-one and difluorophenolphthalein (DFPP) as 3,3-bis(3-
fluoro-4-hydroxyphenyl)isobenzofuran-1(3H)-one and propose a
mechanism of the reaction for fluorination of phenolphthalein,
shown in Fig. 3.
lein (Fig. 1a) has four signals in the range of d 7.6–7.9 ppm from the
protons of the aromatic ring containing the carboxyl group.
Incorporation of fluorine atoms into the molecule causes only
minor (<0.1 ppm) shifts of these signals without any alteration of
their shape. From these results we gather that substitution of
hydrogen by fluorine does not occur in the carboxyl ring. Two other
signals in the 1H NMR spectrum of phenolphthalein have
d
6.735 ppm (4H) and 7.089 ppm (4H) and correspond to protons
located in the phenolic rings in ortho and meta positions to the
hydroxyl groups, respectively. Introduction of one fluorine atom
into the molecule (Fig. 1b) decreases their integral intensity by a
half, while a new group of signals appears in the range of
d 6.8–
7.0 ppm (3H). The second fluorine atom (Fig. 1d) causes complete
disappearance of the signals from the non-substituted phenol rings
of phenolphthalein, while the new group of peaks is doubled in
integral intensity without significant change in their shape.
Spectra with fluorine decoupling (Fig. 1c and e) shows that these
new signals represent protons adjacent to 19F.
By decoupling fluorine from the proton spectrum, we were also
able to identify all the signals in the fluorophenol ring and assign

4
A.V. Kachur et al. / Journal of Fluorine Chemistry 151 (2013) 1–6
The direct fluorination of naphtholphthalein occurs in a similar
manner. We were also able to detect only two major radioactive
products, which were separable by HPLC due to their higher
retention time on the reverse-phase column. The ratio of
radioactivity to absorption was doubled for the second product,
suggesting the presence of one and two fluorine atoms in the
molecules of the fluorinated derivatives, respectively. The mass-
spectroscopy of the initial compound and the fluorinated products
showed m/z values of 417.110, 435.100, and 453.090 amu for
negative mode, and 419.128, 437.119, and 455.110 amu for
positive mode. Collectively these data indicate the synthesis of
mono- (MFNP) and difluoronaphtholphthalein (DFNP). The cor-
rected radiochemical yields of these products varied for different
experiments in the range of 3–5% and 6–8%, respectively, while the
chemical yields were virtually the same.
Both compounds act as pH indicators, having a pKa value of 7.66
and absorption maximum for the basic form at 659 nm for MFNP,
and pKa = 7.24 and
fluorine atom into the molecule of naphtholphthalein (pKa = 7.83,
basic = 653 nm) also causes a decrease of the pKa values and a
lbasic = 665 nm for DFNP. Introduction of the
l
bathochromic shift of the absorption maximum of the basic form,
which seems to be a common behavior for all the triarylmethane
indicators.
Analysis of 1H and 19F NMR spectra of naphtholphthalein and its
fluorinated derivatives reveals a mechanism for its fluorination
reaction. Comparison of the 1H NMR spectra of naphtholphthalein
and its derivatives (Fig. 4) shows that only one proton signal is
drastically affected by successive introduction of the fluorine
atoms. The signal from two protons at d 6.7 ppm in the spectrum of
naphtholphthalein loses half of its intensity for MFNP and
completely disappears for DFNP, indicating that these two protons
represent the position of the fluorine-hydrogen substitution.
Disappearance of the signal in the spectrum of DFNP also confirms
that the two fluorine atoms are introduced into identical positions,
which suggests a hydrogen–fluorine exchange in the naphthol
rather than benzoic rings. This signal is located in the spectrum of
naphtholphthalein in the most shielded field and therefore is the
most prone to electrophilic attack. By modeling the NMR spectra in
various online programs, we determined that this signal belongs to
the proton located in the ortho position to the hydroxyl group and
marked as 3 and 30 on Fig. 5.
The effect of the introduction of fluorine atoms into the
molecule of naphtholphthalein on other proton NMR signals
supports the presumption that fluorine substitution occurs in the
naphthol systems in the ortho position. Introduction of the
electron-withdrawing fluorine atom causes a significant shift of
the signals from the naphthol aromatic system without alteration
of their shape. As both naphthol systems in DFNP are substituted
and therefore identical, for these naphthol protons we should
observe only shifted peaks with the same intensity as compared
with naphtholphthalein. In contrast, both substituted and non-
substituted naphthol systems are present in the molecule of MFNP.
As a result, the signals from the naphthol protons should be
doubled and halved in the spectrum of MFNP, and its spectrum
Fig. 2. 19F NMR spectra of mono- (a) and di- (c) fluorophenolphthalein show that all
the signals have a chemical shift close to
d
ꢁ138 ppm, which is specific for fluorine
atoms in ortho position to hydroxyl group of the phenolic rings. Decoupling of the
hydrogen-fluorine coupling (b and d, respectively) indicates that for both
compounds no isomers are present and both fluorine atoms in
difluorophenolphthalein molecule are identical.
them to three different protons, as presented in Section 3. The
signal at
numbering is shown in Fig. 3 on the MFPP structure);
from proton 6, and 6.95 ppm from 2. The close values of chemical
d
6.90 ppm corresponds to the proton at carbon 5 (the
d
6.92 ppm is
d
shift for these protons can be explained if the opposite effects of the
hydroxyl group and fluorine atom are considered. In the spectrum
of phenolphthalein, the hydroxyl group activates protons in the
ortho position, shifting signals from protons 3 and 5 upfield to
6.74 ppm, and deactivates protons in the meta position, shifting
signals from protons 2 and 6 downfield to 7.09. The introduction
d
d
of the fluorine atom into position 3 has just the opposite effect:
shifting protons at 2 and 6 upfield, and 5 downfield. The fluorine-
hydrogen coupling on both protons 2 and 5 creates a very
complicated multiplet structure, which we were able to resolve
only after fluorine-hydrogen decoupling.
HO
F
HO
HO
HO
OH
F
OH
OH
F
OH
5
2
6
4
3
+
+
+
C
C
C
F
2
+
F
2
O
+
COOH
CO
COOH
COOH
CF3COOH
DFPP
MFPP
Fig. 3. Mechanism of fluorination of phenolphthalein in trifluoroacetic acid. In trifluoroacetic acid solution the compound is turned into the cation form, which is fluorinated in
the ortho-positions to the hydroxyl group.

A.V. Kachur et al. / Journal of Fluorine Chemistry 151 (2013) 1–6
5
7.1–7.4, which might be attributed to these protons. 19F NMR
spectra of MFNP and DFNP also differ from the data we observed
for phenolphthalein derivatives. They contain only one broad
fluorine signal, which does not demonstrate any splitting on the
adjacent proton. Both these observations can be attributed to steric
hinderance in the molecule of naphtholphthalein. Bulk naphthol
substitute groups are not able to face each other, placing 2 and 20
protons in very close position, subsequently increasing their
relaxation time and potentially causing a coupling with variable
distance, causing broadening of the signal.
5. Discussion
We were recently able to perform specific electrophilic
fluorination of phenolsulfonphthalein [1] and cresolsulfonphtha-
lein [2] with dilute fluorine gas in acetic acid solution. This reaction
was used to synthesize new types of pH indicators, 18F-labeled
derivatives of phenolsulfonphthalein, which have a potential for in
vivo pH measurement in biological objects using Cerenkov imaging
or combination of light absorption and PET [3].
Fig. 4. 1H NMR spectra of naphtholphthalein (top) and its mono- (middle) and di-
(bottom) fluorinated derivatives. Disappearance of the signal at 6.7 indicates the
Our initial attempts to accomplish the same fluorination for
phenolphthalein were unsuccessful and resulted in non-specific
destruction of the reagent [1]. The failure of the reaction was
attributed to different structures of the molecules of these
indicators in acetic acid solution. The acidic form of phenolsul-
fonphthalein exists as a zwitterion [6] with a positive charge
distributed through the conjugated system of two phenol rings and
a central carbon atom [1]. This structural feature decreases the
overall energy of the fluorination reaction, promoting a specific
substitution in the phenol rings rather than destruction of the
molecule [2]. In contrast, at neutral and mild acidic conditions
phenolphthalein exists as an uncharged cyclic lactone structure
and does not survive an attack by the aggressive fluorine molecule.
Analyzing the literature we found [7] that under strong acidic
conditions, such as in sulfuric acid, phenolphthalein forms a yellow
solution, which contains its molecule in the form of positively
charged ion. This charge is distributed through the conjugated
phenol rings of phenolphthalein, forming a structure similar to the
zwitterion of the uncharged form of phenolsulfonphthalein. This
similarity led us to the conclusion that in very strong acidic
conditions we might be able to avoid the non-specific destruction
of the molecule and perform a specific electrophilic fluorination of
phenolphthalein.
d
position of the fluorine-hydrogen substitution; assignment of other signals is
discussed in the text.
should look like overlapped spectra of naphtholphthalein and
DFNP. We observe this effect for four signals: doublets at
(from protons at 5 and 50 in Fig. 5) and 7.9 ppm (8 and 80) and
triplets at
7.1 ppm (6 and 60) and 7.3 ppm (7 and 70), thus also
d 8.2 ppm
d
d
d
supporting the successive introduction of fluorine into the
naphthol rings.
Signals from the benzoic ring do not demonstrate any change in
intensity in the spectra of MFNP and DFNP, showing only a slight
shift of the peaks to lower field due to successive addition of
fluorine atoms into the molecule. This is observed for the doublet
at
d d 7.6–7.7 ppm
8.0 ppm (proton at 300) and a group of peaks at
(overlapping doublet from 600 and two triplets from 400 and 500), and
provides further evidence for the absence of substitution in the
benzoic ring.
19F NMR spectra of MFNP and DFNP have only one signal at
d
ꢁ144 ppm, which is also attributable to the fluorine atom located
in the ortho- position to the hydroxyl group in the aromatic system.
Based on these data, we can identify monofluoronaphtholphtha-
lein (MFNP) as (RS)-3-(3-fluoro-4-hydroxynaphthalen-1-yl)-3-(4-
hydroxynaphthalen-1-yl)isobenzofuran-1(3H)-one and difluoro-
naphtholphthalein (DFNP) as 3,3-bis(3-fluoro-4-hydroxynaphtha-
Trifluoroacetic acid is a much stronger acid in comparison with
the acetic acid (with pKa of 0.5 vs. 4.76, respectively [8]), which we
initially used as a solvent for the fluorination reaction. We found
that trifluoroacetic acid could act similarly to sulfuric acid,
len-1-yl)isobenzofuran-1(3H)-one and as such propose
a
mechanism of fluorination of naphtholphthalein (Fig. 5) identical
to phenolphthalein and phenolsulfonphthaleins [1–3].
dissolving phenolphthalein and forming a yellow solution.
Addition of dilute fluorine gas to this solution validated our initial
assumptions about the reactivity of the positively charged form of
phenolphthalein. Instead of the destruction of the target molecule,
which we observed in acetic acid, the reaction between dilute
fluorine gas and phenolphthalein in trifluoroacetic acid resulted in
The 1H NMR spectra of naphtholphthalein and its derivatives
have one quite unusual feature: in all the cases we were not able to
detect any distinct signal from the protons at carbon atoms 2 and
20. On the other hand, we can see a broad signal in the range of
d
F
F
F
3
HO
OH
HO
OH
HO
OH
HO
5
3'
OH
2
2'
+
+
+
C
C
C
5'
6'
O
CO
6
8
8'
COOH
F2
+
F
2
6"
5"
7
7'
COOH
+
COOH
CF3COOH
3"
4"
MFNP
DFNP
Fig. 5. A proposed mechanism for the reaction of naphtholphthalein fluorination. Trifluoroacetic acid converts the reagent into the cation form, which can survive direct
fluorination producing mono- and difluorinated products. The formula of monofluorinated derivative has a numbering scheme for the carbon atoms, which is used in the text.

6
A.V. Kachur et al. / Journal of Fluorine Chemistry 151 (2013) 1–6
formation of two major fluorination products (Fig. 3). We
characterized these as mono- and difluorinated derivatives of
phenolphthalein, which were produced with quite significant
chemical yields, 10–15% and 1.5–2%, respectively. We can thus
expand the list of novel 18F-labeled indicators with the addition of
various derivatives of phenolphthalein and other similar phtha-
leins. These results also support our initial presumption [1,2] that
the presence of the positive charge in phenolsulfonphthalein
derivatives is a necessary condition for their successful specific
fluorination, making strong acidic conditions suitable for reactions
of electrophilic fluorination [9].
In general, phenolphthalein demonstrates lower stability
toward fluorination reaction in comparison with phenolsul-
fonphthalein derivatives. Although it has four available ortho-
positions, it forms only two fluorination products (vs. up to four for
phenolsulfonphthaleins [2,3]) with relatively lower yields. Never-
theless, the reaction leads to the synthesis of fluorinated products
in amounts sufficient for their practical application.
the presence of porphyrin-based molecules in biological objects.
For example, hemoglobin has strong absorption peaks for both Hb
(555 nm) and HbO2 (542 nm and 577 nm) [12] and will interfere
with the color change of indicator in the range of 500–600 nm. The
parental compound
a-naphtholphthalein has pKa = 7.95 and
absorption maximum of the basic form at 653 nm, which makes
it more suitable for biological objects in comparison with
phenolphthalein and phenolsulfonphthalein derivatives. Introduc-
tion of fluorine atoms into the molecule of
a-naphtholphthalein
leads to additional improvement of its properties, since the
absorption maxima of the basic form undergoing a bathochromic
shift to 659 nm for MFNP and 665 nm DFNP, moving them even
further away from the porphyrins. Moreover, the values of pKa are
shifted to 7.66 for MFNP and 7.24 for DFNP, meaning that the
strongest color change occurs in the range of physiological pH. This
combination of the properties makes both compounds particularly
useful for in vivo pH measurement in biological objects.
Incorporation of the fluorine atoms into molecule of phenol-
phthalein alters the properties as indicators in a similar fashion to
that observed for phenolsulfonphthalein derivatives. The value of
pKa for phenolphthalein is equal to 9.75 [10] and the maximum
wavelength of absorption of its basic form is 553 nm. Successive
incorporation of fluorine atoms into molecule of phenolphthalein
decreases its pKa value to 9.33 and 8.70 and causes a bathochromic
shift of the basic form maximum absorption to 558 nm and
564 nm, respectively. Although these compounds are not ideal for
pH measurements in biological objects, our ability to perform the
reaction itself is quite a fascinating result. Through the use of the
same reaction we can almost certainly synthesize other indicators
of this class, namely fluorinated derivatives of cresolphthalein and
thymolphthalein. This, however, is of only theoretical interest as
the effects of methyl and fluorine substituting groups on pH and
absorption are additive [2], and as such these indicators should
have even higher values of pKa.
6. Conclusions
Direct fluorination of phenolphthalein and naphtholphthalein
with [18F]-F2 in acidic conditions leads to synthesis of 18F-labeled
mono- and difluorinated derivatives. The fluorinated products act
like pH indicators with decreased values of pKa and a bathochromic
shift of absorption maxima for their basic forms. Derivatives of
phenolphthalein demonstrate a shift of pKa to 9.33 and 8.70 and
lbasic to 558 nm and 564 nm for mono- and difluorinated
compounds, respectively. Fluorination of
results in the synthesis of two compounds with optimal values of
pKa (7.66 and 7.24) and high wavelengths of absorption (659 nm
and 665 nm). Both mono- and difluorinated derivatives of
a-naphtholphthalein
a-
naphtholphthalein have very high potential for in vivo pH
measurement in biological objects.
Acknowledgements
The reaction of
a-naphtholphthalein fluorination is similar to
that of phenolphthalein. Naptholphthalein was also destroyed by
electrophilic fluorination in acetic acid, but in trifluoroacetic acid
naphtholphthalein forms a blue-green solution, which produced
only two fluorinated products in the reaction with dilute fluorine
gas. Given the various different ways in which the fluorination could
occur in the naphthol rings, this is quite an unexpected result.
Nevertheless, our results indicate an incorporation of only one
fluorine atom per naphthol group into the ortho position to the
hydroxyl group. A lack of products with higher number of fluorine
atoms is most likely associated with the lower stability to oxidation
of trialylphthaleins as compared to triarylsulfonphthaleins.
Supported by the Institute for Translational Medicine and
Therapeutics (ITMAT) Transdisciplinary Awards Program in
Translational Medicine and Therapeutics Translational Biomedical
Imaging Core Grant (TAPITMAT-TBIC) UL1RR024134 from the
National Center For Research Resources and by Grant # IRG -78-
002-31 from the American Cancer Society. The authors would like
to thank Diana Sardelis and Charles N. McEwen at the University of
the Sciences for help with mass-spectroscopy.
References
Fluorinated derivatives of
a-naphtholphthalein demonstrate a
[1] A.V. Kachur, A.V. Popov, J.S. Karp, E.J. Delikatny, Cell Biochem. Biophys. (2012),
DOI: 10.1007/s12013-12012-19390-x..
[2] A.V. Kachur, D. Sardelis, C. Bentzley, A.V. Popov, E.J. Delikatny, J. Karp, J. Fluorine
Chem. 145 (2013) 112–117.
[3] A.V. Kachur, E.J. Delikatny, J.S. Karp, J. Nucl. Med. 53 (S1) (2012) 187.
[4] M.D. Barnes, V.K. La-Mer, J. Am. Chem. Soc. 64 (1942) 2312–2316.
[5] W.R. Dolbier, Guide to Fluorine NMR for Organic Chemists, John Wiley & Sons,
Inc., Hoboken, NJ, 2009.
very high potential for their practical applications. As discussed
previously [3], 18F-labeled pH indicators represent a new type of
probe for in vivo investigation of hydrogen ion concentration.
These compounds have three potential modes of detection: pH-
dependent optical absorption, pH dependent Cerenkov emission
[11], and concentration-dependent g-radiation by PET technique.
[6] Z. Tamura, M. Maeda, Yakugaku Zasshi 117 (1997) 764–770.
[7] A.C. Hopkinson, P.A.H. Wyatt, J. Chem. Soc. B (1970) 530–535.
[8] J.A. Dean (Ed.), Lange’s Handbook of Chemistry, 13th ed., McGraw-Hill Book
Company, New York, 1985.
[9] W.R. Dolbier, A.R. Li, C.J. Koch, C.Y. Shiue, A.V. Kachur, Appl. Radiat. Isot. 54 (2001)
73–80.
[10] J.R. Lalanne, J. Chem. Educ. 48 (1971) 266–268.
[11] J. Czupryna, A. Kachur, E. Blankemeyer, A. Popov, J. Karp, E. Delikatny, J. Nucl. Med.
53 (S1) (2012) 73.
The combination of the 3-dimensional distribution of the basic
form of the indicator with the PET image of its concentration will
lead to in vivo 3D mapping of pH in biological objects. A functional
probe for this kind of investigation should contain a radioactive
PET label (18F) within the indicator molecule with an optimal value
of pKa (between 6 and 8), and an appropriate absorption maximum
of the basic form (above 600 nm.) The latter limitation considers
[12] B.L. Horecker, J. Biol. Chem. 148 (1943) 173–183.