Anilinomethylrhodamines: PH sensitive probes with tunable photophysical properties by substituent effect

Article abstract of DOI:10.1021/jo401323g

A series of pH dependent rhodamine analogues possessing an anilino-methyl moiety was developed and shown to exhibit a unique photophysical response to pH. These anilinomethylrhodamines (AnMR) maintain a colorless, nonfluorescent spirocyclic structure at high pH. The spirocyclic structures open in mildly acidic conditions and are weakly fluorescent; however, at very low pH, the fluorescence is greatly enhanced. The equilibrium constants of these processes show a linear response to substituent effects, which was demonstrated by the Hammett equation.

Full text of DOI:10.1021/jo401323g

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Article
AnilinoMethylRhodamines: pH Sensitive Probe with
Tunable Photophysical Properties by Substituent Effects
Quinn A. Best, Chuangjun Liu, Paul D van Hoveln, Matthew E Mccarroll, and Colleen Nicola Scott
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo401323g • Publication Date (Web): 19 Sep 2013
Downloaded from http://pubs.acs.org on September 20, 2013
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AnilinoMethylRhodamines: pH Sensitive Probes
with Tunable Photophysical Properties by
Substituent Effect
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Quinn A. Best, Chuangjun Liu, Paul D. van Hoveln, Matthew E. McCarroll, Colleen N. Scott*
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Department of Chemistry & Biochemistry, Southern Illinois University, Carbondale, IL 62901-4409,
USA
cscott@chem.siu.edu
Received Date (will be automatically inserted after manuscript is accepted)
A series of pH dependent rhodamine analogs possessing an anilino-methyl moiety was
developed and shown to exhibit a unique photophysical response to pH. These
Anilinomethylrhodamines (AnMR) maintain a colorless, non-fluorescent spiro-cyclic structure at
high pH. The spiro-cyclic structures open in mildly acidic conditions and are weakly fluorescent;
however at very low pH, the fluorescence is greatly enhanced. The equilibrium constants of
these processes show a linear response to substituent effects, which was demonstrated by the
Hammett equation.
.
INTRODUCTION
The pH of biologica l s ys te ms ca n be diverse, ranging from slightly ba s ic to s trongly a cidic.
Mos t of these systems are known to operate under neutral or near neutral conditions; however
certain a nima l's and cell's organelles function under very acidic conditions. For example,
lysosomes are acidic organelles where proteins and de bris pa rticle s are digested under
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relatively acidic conditions (pH 5.5-4.5) by a variety of enzymes. . Abnormal deviations from
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these pH ranges can perturb cellular functions, which may lead to diseases. For example,
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abnormalities in lysosomal pH values have been linked to human breast cancer and
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neurodegenerative disorders. Another strongly a cidic orga n is the stomach, which contains
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ga s tric juice s ranging in acidity pH from 1.5 – 3.0. This very acidic environment activates
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digestive enzymes and also serves to protect the body from ingested microorganisms by acting
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as a barrier for bacteria. Stomach diseases can occur if the pH of the stomach in not operating
at optimal conditions.6
Given the fundamental importance of pH in biology and the recognition of its relationship to
diseases, it has become increasingly important to develop techniques to accurately measure pH
with high spatiotemporal resolution for the various cellular and physiological compartments.
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Some of the techniques available to measure pH are positron emission topography and
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magnetic resonance imaging (MRI); however, fluorescence based techniques are one
alternative to PET and MRI. Fluorescence based techniques are advantageous because of the
relatively low technical costs, high sensitivity, and high spatiotemporal resolution. A variety of
fluorescence-based sensors for pH have been designed over the years. 9 The main design
criteria for a fluorescent pH sensor are the fluorophore's optical qualities, na me ly, mola r
absorptivity, quantum yield, and the emission and excitation wavelengths. Another important
+
crite rion is tuning the pKa of the H recognition element to the organelle or cellular pH values of
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interest. The phenolic proton in fluorescein and fluorescein analogs drastically influences the
fluorescent properties of these molecules, which can be tuned by introducing halogen atoms to
the xanthene core.10 Carbocyanines covalently linked with amino or anilino substituents have
been used to introduce a pH dependent property in the development of fluorescent pH probes.11
However, a major drawback in the use of these fluorophores is their inherent tendency to
undergo photobleaching, which is problematic for applications involving biological imaging. On
the contrary, rhodamine-based fluorophores have advantages in biological imaging because of
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their superior photophysical properties such as, photostability and their relative ease of
de riva tiza tion.
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A multitude of fluorescent probes have been designed around the rhodamine spiro-cyclic
amide scaffold,12 which allows for a distinct "off-on" colorimetric and fluorescent response
toward an intended analyte, e.g. H+.13 Recently, we reported an analogous rhodamine spiro-
cyclic amine scaffold used in the optical detection of pH.14 Since then, these
AminoMethylRhodamines (AMR), aka rhodamine deoxy-lactams, have also been utilized by
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Han et. al. in the detection of nerve agents, phosgene, and biologically relevant aldehydes.
Furthermore, our work on the AMR scaffold, and more recently the work of Peterson et al.,18
has shown that the pH range in which these probes respond can be tuned by introducing
different functional groups to change the basicity of the amine moiety.
We are interested in exploring functionalities that can be used to tune the pH dependent
properties of the AMR scaffold. We have chosen to use aniline in place of the amino group, with
the goal of altering the basicity by attaching various electron withdrawing groups (EWG) or
electron donating groups (EDG) substituents on the benzene ring. Herein, we report our findings
on a new class of rhodamine based pH probes, which we've termed AnilinoMethylRhodamines
(AnMR).
.
RESULTS AND DISCUSSION
We investigated a series of pH dependent rhodamines derived from both rhodamine B and
rhodamine 6G (Figure 1). This work is a continuation of our previous work expanding on the
development of optical pH probes based on rhodamine B aminomethylrhodamine (AMR). We
included the rhodamine 6G fluorophore in the study because they are known to have better
19
photophysical properties compared to rhodamine B. It was also believed that the presence of
the methyl groups on rhodamine 6G could influence the spiro-cyclic ring opening through steric
effects. Acid/base titrations were carried out and monitored by both fluorescence and UV-visible
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absorption techniques. The AMR a nd AMR6G were found to
be colorless and non-fluorescent under strongly basic
conditions, i.e. pH 9-12, which is attributed to the spiro-cyclic
form (SF). However, as the solutions approach acidic pH, the
characteristic colors of the AMR (λabs = 564nm, pH = 4.6) and
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Figure 1. Structures of
aminomethylrhodamines (AMR)
and anilinomethylrhodamines
AMR6G (λabs = 535 nm, pH = 3.3) fluorophores become
(
AnMR).
apparent and a constant absorption maximum is maintained
from pH 7-3 (Figure 2A-B). Similarly, a fluorescence enhancement is observed as the solutions
of AMR (λFl = 584 nm) and AMR6G (λFl = 550 nm) become increasingly acidic, which correlates
well with the rise in absorbance (Figure 2C-D).
Figure 2. Spectroscopic properties of a 5µM solution of AMR (A and C) and AMR6G (B and D) in 0.1 M
sodium phosphate buffer and 0.5% DMSO at the various pH values shown. (A and B) absorbance and
fluorescence with excitation at 520 nm and 500 nm for the AMR and AMR6G respectively; (C and D)
mapping of fluorescence and absorbance.
Next, we set out to investigate the effects of pH on AnMR and AnMR6G. We hypothesized
that AnMR and AnMR6G would respond similarly to AMR and AMR6G, albiet at a lower pH.
We synthesized AnMR and AnMR6G from the corresponding rhodamine precursors and
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analyzed their structures by NMR and mass spectrometry. In the C- NMR spectrum, both
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compounds display a quaternary carbon at a chemical shift, indicative of the s piro-cyclic form
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(see supporting information).
Fluorescence and UV-visible spectroscopy were also consistent with the spiro-cyclic form as
both AnMR and AnMR6G appear colorless and non-fluorescent, which is similar to the
analogous AMR based system. As expected, the anilino-analogs respond colorimetrically under
more acidic conditions with respect to the corresponding AMR compounds. Figure 3A and 3B
displays the absorption spectra for AnMR and AnMR6G respectively. The color from AnMR
becomes noticeable around pH 5 (λabs = 550 nm), whereas AnMR6G begins to respond around
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pH 7 (λabs = 528 nm). Like the AMR system, the absorbance of AnMR and AnMR6G increases
steadily as the pH decreases; however, an unexpected red-shift in the absorption ma xima
occurred under very acidic conditions with little change in the actual absorption va lue .
Interestingly, the fluorescence response of AnMR (λFl = 591 nm, Figure 3C) and AnMR6G (λFl
=
559 nm, Figure 3D) is greatly enhanced and mirrors the red-s hift observed in the absorption
spectra (AnMR Figure 3E and AnMR6G 3F). The maximum absorbance shifts to 568 nm around
pH = 3.5, and 536 nm around pH = 4 for AnMR and AnMR6G, respectively (Figure 3G and 3H).
Initia lly, the low fluorescence response indicates the presence of a non-fluorescent spiro-cyclic
form (SF) that is observed under basic conditions. Under weakly acidic conditions, solutions
containing the probe become colored; albeit only a weakly fluorescent. It is not until s trongly
acidic conditions are introduced that the probes display strong fluorescence intensity. Based on
these results, we propose the following mechanism to describe the behavior of the AnMR system
and its ‘unique’ behavior with respect to the AMR system. Both systems maintain the spiro-
cyclic form (SF) under basic conditions. Mono-protonation of either the amine or the aniline
+
nitroge n fa cilita te s ring-opening, resulting in the rhodamine cationic open form (OF ), which can
++
be further protonated to the dicationic open form (OF ) under strong acidic conditions.
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+
The equilibrium between the OF /SF can be observed as the increase in the absorption,
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whereas the equilibrium between the OF /OF can be observed by the increase in
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fluorescence. In this mechanism, the weak fluorescence of the OF state may be a result of
partial quenching by the lone pair electrons on the anilino or amino groups via photo- induced
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electron transfer (PeT). In the OF state, PeT is disrupted when the lone pair electrons are now
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involved in bonding to the hydrogen, and therefore can no longer quench the fluorophore.
Figure 3. Spectroscopic properties of a 5µM solution of AnMR and AnMR6G in 0.1 M sodium phosphate
buffer and 0.5% DMSO at the various pH values shown. (A and B) absorbance; (C and D) fluorescence,
with excitation at 520 nm and 500 nm for AnMR and AnMR6G respectively; (E and F) mapping of
fluorescence and absorbance; (G and H) demonstration of the shifting in the absorbance at low pH.
+
++
Equilibrium constants for these two states (OF and OF ) can be approximated from the
absorption and fluorescence-based titration curves. Here we define the equilibrium constant of
+
++
+
the OF /SF (pKOF+) process at half the change in absorbance, and for the OF /OF (pKOF++
)
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process at half the change in fluorescence. From this analysis, we have estimated that the
pKOF+ (AnMR) is 4.3, (AnMR6G) is 5.6, and the pKOF++ (AnMR) is 2.6 and (AnMR6G) is 3.8
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(Table 1). The red-shift in the absorption spectrum highlights the change in the AnMR ground
state. The AMR system does not display a red-shift, nor is there a distinct difference between
the absorption or fluorescence pH titration curves, which seems to indicate that the pKOF+ and
the pKOF++ are too close to be observed separetely by these techniques.
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Table 1. Spectroscopic properties and equilibrium constants of the AnMR and AnMR6G
series.
a
b
Conditions: Measured in pH 1 buffer solution. Measured in pH 1 buffer solution (Fl) and pH 10
buffer solution (Fl ).
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We then examined a variety of AnMR and AnMR6G compounds with variation of the
subsitution on the anilino phenyl ring. These compounds were synthesized in a method similar
to their AnMR and AnMR6G analogs (Figures 4 and 5) and were characterized by NMR, mass
spectrometry, UV-vis, and fluorescence. All of the AnMR compounds dis pla y a s imila r type of
behavior as their AnMR parent compound, i.e. a separate and distinct colorimetric and
fluorescence response (Table 1). The low pKa value observed for the response of these
compounds (pKOF++ ~ 4 – 1.5) suggest possible applications in very acidic regions of biological
systems such as the stomach.
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In general, the absorption and fluorescence for the AnMR series show a 30-40 nm red-s hift
compared to the AnMR6G series. There was no trend observed in the molar absorptivity data
-1
-1
for these compounds and the average molar absorptivity is 56,000 cm M . There were a few
compounds that deviated from the average; noteworthy was pF-AnMR with a very high molar
-
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-AnMR with low values of 38,000 cm-
M and 39,000 cm M respectively (Table 1). The limited number of compounds in the
AnMR6G series hinders the formation of a definite conclusion; however, one compound, pCF
absorptivity of 100,000 cm M and mMe-AnMR and pCF
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AnMR6G (64,000 cm M ), stands out as having a higher molar absorptivity than the others.
There was no noticeable trend for the fluorescence enhancement (Fl/Fl ) for either series.
However, some compounds show very low fluorescence enhancement among the AnMR and
AnMR6G series. Among the AnMR series, pCF -AnMR showed a remarkably low fluorescence
o
3
enhancement of 6, while mMeO-AnMR and mMe-AnMR also showed a small fluorescence
enhancement of 14 and 16 respectively. In the AnMR6G series, one low fluorescence
enhancement was observed in pCl-AnMR6G with a fluorescence enhancement of 14. The
precise nature of the lower fluorescence enhancement observed by these species remains
unknown, but presumably some quenching by the substituents themselves may be occurring.
The quantum yield was measured for two representative compounds from each series to
compare the two probe systems. Acidic conditions (pH 1.5), where a maximum amount of
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fluorescence was observed, were invoked when measuring pMeO-AnMR and pMeO-AnMR6G,
which had quantum yields of 14% and 65%, respectively, using rhodamine B in ethanol
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(
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Figure 4. Synthesis of the anilinomethylrhodamine (AnMR) and their substituted analogs
Figure 5. Synthesis of the AnMR6G series
Although the fluorescence and UV-vis data provides strong evidence for the proposed
mechanism, we carried out NMR experiments on an AnMR derivative to elucidate the structural
changes occuring under similar conditions. A solution of pMeO-AnMR was prepared in d
DMSO and titrated with aliquots of D SO while changes in the structure were monitored by
NMR (Figure 6). New signals in the NMR spectrum corresponding to a drastic change in the
pMeO-AnMR structure were observed upon adding D SO . The benzylic signal gave the
6
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clearest signal and was ultimately used in determing the stuctural changes that were occuring.
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In the absence of D
The addition of 1 equivalent of D
ppm and a reduction in the 4.98 ppm signal intensity. By 2 equivalents of D
2
SO
4
, pMeO-AnMR maintains the SF with a benzylic signal at 4.98 ppm.
SO creates a new set of signals, notably a new signal at 4.05
SO , there is no
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longer a signal at 4.98 ppm, nor is there anymore increase in the signal intensity at 4.05, which
is believed to be a new strucuture. The generation of the distinctly new and unique proton signals
in the NMR structure is indicative of the molecule reacting and undergoing a major change in
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0
1
2
3
4
5
6
7
8
9
0
the structure when D
2
SO is added. Interestingly, further changes in the NMR spectrum occur
4
1
Figure 6. H-NMR titration of a 0.5 mM solution of pMeO-AnMR in d
6
-DMSO with D
2
SO
4
as additions of D
2
SO leading to 10 equivalents causes downfield shift in the benzylic signal
4
from 4.05 ppm to 4.35 ppm and is thought to occur from the formation of the anilinium cation or
++
the OF . A comparison to the AMR series was made (see Supporting Information), where
+
similar changes between the SF and OF were observed; however, there was no further shifting
of the benzylic signal with increasing equivalents of D
2
SO indicating that the ammonium cation
4
+
++
is quickly formed and there is no distinction between the OF and OF . From the NMR, the
+
+
++
distinct structural changes between the SF to OF and the OF to OF can be distinguished by
1
1
either the formation of new H-NMR signals or from the shift in the benzylic H-NMR signal.
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The Journal of Organic Chemistry
These results corroborate the differences in the AnMR and AMR mechanism observed by
fluorescence and UV-vis .
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
As predicted, the acid/base properties of the AnMR system were affected by the presence of
EDG or EWG. From the UV-vis and fluorescence titrations curves, the pKOF+ and the pKOF++
were estimated for each compound (see Supporting Information). The effects of the substituents
on the equilibrium constants of the reaction can be predicted by the Hammett equation. The
pKOF+ and pKOF++ for each AnMR and AnMR6G compound were plotted against their respective
Hammett σ constants and compared with published experimental data from similar aniline
derivatives.21 We were pleased to find that the overall trend of the pKOF+ and the pKOF++ for
AnMR analogs follows typical Hammet-like behavior, i.e. a negative slope in the pKa with respect
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
++
to the corresponding σ constants (Figure 7). The R-squared values for the AnMR OF and the
AnMR6G OF++ both had comaparable fit to that of the literature aniline series, although there
++
+
+
are some anomal values for the AnMR6G OF series. The AnMR OF and AnMR6G OF gave
poor R-squared values, perhaps due to deviation of a few data points from the mean. It is evident
from these plots however, that the equlibrium between the spiro-cyclic ring (SF) and the open
+
+
++
form (OF ), and the (OF ) and (OF )
structures were being influenced
substantially by the substituents on the
aniline ring and thus produces
difference in the pKa for the protonated
(pKOF+) and di-protonated (pKOF++
)
anilines.
Figure 7. A comparison of the Hammett plots for the pKOF+
and pKOF++ of the AnMR and AnMR6G series and aniline.
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CONCLUSION
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
In conclusion, a series of novel fluorescent probes for the optical measurement of a cidic pH
has been developed based on the AnMR system. These probes display distinct and separate
colorimetric and fluorescent responses at different pH values. We propose that the differences
in the colorimetric and fluorescence response can be explained by an initial opening of the spiro-
cycle form (SF) upon protonation in weakly acidic conditions, resulting in the weakly fluorescent
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
monocationic open form (OF ). Further protonation under increasingly acidic conditions results
+
+
in the highly fluorescent dication open form (OF ). The e quilibrium constants for these
+
++
+
reactions, OF /SF and OF /OF can be measured by UV-vis absorption and fluorescence
respectively. The results of NMR based studies corroborate the proposed mechanism. The
equilibrium constants, display a linear relationship when plotted against the Hammett
σ constants for the AnMR OF++ and AnMR6G OF++ series based on the R-squared values;
however, the linear plot for the AnMR OF+ and AnMR6G OF series did not fit as well. More
data points will be developed to e s ta blis h a more accurate relationship for all the series. We will
probe the nature of the low fluorescence enhancement in some of the substituted AnMR and
AnMR6G series. Biologica l s tudie s utilizing AnMR a s a probe for me a s uring intra ce llula r pH a re
ongoing.
+
EXPERIMENTAL
General chemicals were purchased from commercial sources and used without further
purification unless otherwise stated. DMF, acetone, methylene chloride, chloroform and ethyl
acetate were all reagent grade and used without further purification unless otherwise mentioned.
THF was obtained from solvent purification system. Analytical thin layer chromatography (TLC)
was conducted on pre-coated TLC plates, silica gel 60 F254, layer thickness 0.25 mm. Column
chromatography was performed on an is ole ra 4 with an ultra violet detector.
1
13
H NMR, C NMR, DEPT, and NOESY spectra were recorded on a 400 MHz or 300 MHz
instrument using deuterated chloroform (CDCl ), methanol (CD OD) and dimethyl sulfoxide
3
3
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The Journal of Organic Chemistry
[
(CD
3
)
2
SO]. Chemical shifts are reported in delta (δ) pa rts pe r million (ppm). S plitting pa tte rns
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
are abbreviated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad.
Mass spectra (MS) were measured on a GC-MS, a Quattro II, or a Q-Tof Ultima .
Absorbance data were obtained using a UV-Vis spectrometer. Fluorescence spectra were
recorded on a modular spectrofluorometer.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
pH Titrations. The following procedure was used for the spectroscopic analysis of the AnMR
pH probes. In a 200 mL beaker, 50 mL of either a 2.5 µM (fluorescence) or a 5 µM (UV-vis)
solution of the corresponding sensor in 0.1 M sodium phosphate buffer and 0.5 % DMSO was
allowed to stir in open air for approximately 0.5 hours allowing the solution to become saturated
with oxygen. AnMR6G solutions were similarly prepared, with the exception that a 2.5 µM
concentration was used for both fluorescence and UV-vis. Using a digital pH meter equipped
with a glass electrode (pH/ATC calomel) the pH was monitored and adjusted to acidic or basic
conditions using small aliquots of conc. HCl or a 4.0 M NaOH solution, respectively. The pH was
allowed to stabilize for ~1 minute and then 3.0 mL of the solution was added to a quartz cuvette
for analysis. The spectra are found in the supporting information.
Synthesis of compounds 1-11
A general procedure for preparing the rhodamine B amides (1-11) was used. To a solution
of rhodamine B (400 mg, 0.84 mmol) in 1,2-dichloroethane (10 mL) was added phosphorus
oxychloride (0.3 mL, 3 mmol) dropwise. The solution was refluxed for 4 hours. The reaction
mixture was cooled and concentrated under reduced pressure to give the rhodamine B acid
chloride, which was used in the next step without further purification. The crude acid chloride
was dissolved in 1,2-dichloroethane (10 mL) and to the solution was added 0.15 mL of the
corresponding aniline. This solution was allowed to stir for 24 hours, which was then transferred
to a separatory funnel with an additional 20 mL of dichloromethane (DCM). The organic layer
was washed with 2.0 M H
2
SO solution (3 x 20 mL), then with water (2 x 20 mL), and finally 0.10
4
M solution (2 x 20 mL). The organic layer was then dried over anhydrous MgSO and evaporated
4
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Page 14 of 28
in vacuo. The products were purified by column chromatography on alumina (neutral) with 70:30
hexanes:EtOAc
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
',6'-bis(diethylamino)-2-phenyl-2,3-dihydrospiro[isoindole-1,9'-xanthene]-3-one
2
2
1
(
1). Previously synthesized. Yield 368 mg (85%); R
MHz, CDCl ) δ 8.00 (dd, J = 4.6, 2.4 Hz, 1H), 7.59 – 7.42 (m, 2H), 7.21 – 6.94 (m, 4H), 6.79 (dd,
J = 8.0, 1.8 Hz, 2H), 6.63 (d, J = 8.8 Hz, 2H), 6.30 (dd, J = 8.8, 2.6 Hz, 2H), 6.25 (s, 2H), 3.31
F
= 0.4 in 70:30 Hex:EtOAc. H NMR (400
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
1
3
(
q, J = 7.2 Hz, 8H), 1.14 (t, J = 7.0 Hz, 12H). C NMR (101 MHz, CDCl
3
) δ 167.7, 153.3, 153.1,
1
1
52.6, 148.7, 136.7, 132.8, 131.0, 128.8, 128.5, 128.1, 127.3, 126.7, 124.0, 123.3, 108.2, 108.1,
06.4, 106.0, 105.0, 97.8, 91.6, 67.5, 44.5, 44.3, 12.6.
3',6'-bis(diethylamino)-2-(4-methylphenyl)-2,3-dihydrospiro[isoindole-1,9'-
xanthene]-3-one (2). Pink amorphous solid; Yield 360 mg (81%); R
1H NMR (400 MHz, CDCl
) δ 8.02 (dd, J = 7.0, 2.2 Hz, 1H), 7.59 – 7.40 (m, 2H), 7.15 (dd, J =
.0, 2.2 Hz, 1H), 6.90 (d, J = 7.9 Hz, 2 H), 6.64 (m, 4H), 6.31 (dd, J = 8.9, 2.6 Hz, 2H), 6.25 (d,
F
= 0.38 in 70:30 Hex:EtOAc.
3
7
1
3
J = 2.6 Hz, 2H), 3.31 (q, J = 7.2 Hz, 8H), 2.20 (s, 3H), 1.15 (t, J = 7.1 Hz, 12H). C NMR (101
MHz, CDCl ) δ 168.2, 167.7, 153.1, 153.18, 153.17, 151.9, 149.4, 148.7, 136.3, 133.8, 132.7,
31.2, 129.27, 129.25, 129.24, 128.9, 128.12, 128.05, 127.32, 127.27, 124.0, 123.3, 110.0,
3
1
1
08.2, 108.1, 106.5, 97.8, 67.3, 44.5, 44.3, 27.0, 21.08, 21.06, 14.7, 12.6. HRMS (ESI-TOF)
+
m/z: [M + H] Calcd for C35
H
37
N
3
2
O 532.2964; Found 532.2955
3
',6'-bis(diethylamino)-2-(3-methylphenyl)-2,3-dihydrospiro[isoindole-1,9'-
xanthene]-3-one (3). Pink amorphous solid; Yield 338 mg (76%); R = 0.38 in 70:30 Hex:EtOAc.
1H NMR (400 MHz, CDCl
) δ 8.05 – 7.90 (m, 1H), 7.54 – 7.43 (m, 2H), 7.21 – 7.14 (m, 1H),
.98 (t, J = 7.7 Hz, 1H), 6.90 (d, J = 7.6 Hz, 1H), 6.63 (d, J = 8.8 Hz, 2H), 6.57 (d, J = 2.0 Hz,
H), 6.49 (d, J = 7.8 Hz 1H), 6.32 (dd, J = 8.8, 2.6 Hz, 2H), 6.24 (d, J = 2.6 Hz, 2H), 3.32 (q, J
F
3
6
1
=
1
3
7.2 Hz, 8H), 2.11 (s, 3H), 1.14 (t, J = 7.0 Hz, 12H). C NMR (101 MHz, CDCl ) δ 167.5, 153.2,
3
153.1, 151.0, 149.4, 148.7, 146.0, 138.0, 136.4, 132.7, 131.4, 128.9, 128.5, 128.2, 128.1,
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The Journal of Organic Chemistry
1
3
5
27.64, 127.60, 124.32, 124.26, 124.0, 123.3, 108.2, 108.1, 106.6, 97.8, 91.4, 69.2, 67.5, 44.4,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
8.3, 21.3, 14.7, 12.6. HRMS (ESI-TOF) m/z: [M + H] Calcd for C35
32.2964
H
37
N
3
2
O 532.2964; Found
3
',6'-bis(diethylamino)-2-(4-methoxyphenyl)-2,3-dihydrospiro[isoindole-1,9'-
xanthene]-3-one (4). Pink amorphous solid; Yield 289 mg (63%); R = 0.28 in 70:30 Hex:EtOAc.
1H NMR (400 MHz, CDCl
) δ 8.06 – 7.95 (m, 1H), 7.56 – 7.44 (m, 2H), 7.17 (dd, J = 5.3, 3.3
Hz, 1H), 6.71 – 6.53 (m, 6H), 6.42 – 6.17 (m, 4H), 3.68 (s, 3H), 3.32 (dd, J = 15.1, 8.0 Hz, 8H),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
3
1
3
1
1
.15 (t, J = 7.0 Hz, 12H). C NMR (101 MHz, CDCl ) δ 167.6, 158.2, 153.2, 148.7, 132.6, 131.4,
3
29.01, 128.95, 128.1, 124.0, 123.3, 113.9, 108.1, 97.8, 67.3, 55.2, 44.4, 12.5. HRMS (ESI-
+
TOF) m/z: [M + H] Calcd for C35
H
38
N
3
3
O 548.2913; Found 548.2915
3
',6'-bis(diethylamino)-2-(3-methoxyphenyl)-2,3-dihydrospiro[isoindole-1,9'-
xanthene]-3-one (5). Pink amorphous solid; Yield 446 mg (97%); R = 0.31 in 70:30 Hex:EtOAc.
1H NMR (400 MHz, CDCl
) δ 8.08 – 7.96 (m, 1H), 7.57 – 7.43 (m, 2H), 7.22 – 7.12 (m, 1H),
.01 (t, J = 8.1 Hz, 1H), 6.64 (d, J = 8.7 Hz, 3H), 6.46 (d, J = 7.9 Hz, 1H), 6.39 – 6.21 (m, 5H),
F
3
7
1
3
3.48 (s, 3H), 3.31 (q, J = 6.8 Hz, 8H), 1.14 (t, J = 7.0 Hz, 12H). C NMR (101 MHz, CDCl
3
) δ
1
1
67.4, 159.3, 153.2, 153.0, 148.7, 137.6, 132.8, 131.2, 129.0, 128.9, 128.1, 124.0, 123.7, 119.8,
+
14.0, 111.4, 108.0, 106.4, 97.7, 67.5, 54.8, 44.3, 12.5. HRMS (ES I-TOF) m/z: [M + H] Ca lcd
548.2913; Found 548.2905
',6'-bis(diethylamino)-2-(4-fluorophenyl)-2,3-dihydrospiro[isoindole-1,9'-
xanthene]-3-one (6). Pink amorphous solid; Yie ld 265 mg (59%); R = 0.41 in 70:30 Hex:EtOAc.
1H NMR (400 MHz, CDCl
) δ 8.01 (dd, J = 7.1, 1.6 Hz, 1H), 7.51 (dd, J = 3.8, 3.2 Hz, 2H), 7.18
dd, J = 7.1, 1.4 Hz, 1H), 6.81 (t, J = 8.5 Hz, 2H), 6.69 (dd, J = 8.5, 5.1 Hz, 2H), 6.65 – 6.57 (m,
for C35
H
38
N
3
O
3
3
F
3
(
2
H), 6.32 (dd, J = 8.8, 2.1 Hz, 2H), 6.25 (d, J = 2.2 Hz, 2H), 3.42 – 3.20 (m, 8H), 1.15 (t, J = 7.0
1
3
Hz, 12H). C NMR (101 MHz, CDCl ) δ 167.6, 162.5, 160.1, 153.2, 152.9, 148.8, 132.8, 132.41,
3
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1
4
32.38, 131.1, 129.4, 129.3, 128.8, 128.2, 124.1, 123.4, 115.6, 115.3, 108.1, 106.1, 97.7, 67.5,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
4.3, 12.6. HRMS (ES I-TOF) m/z: [M + H] Calcd for C34
H
35
N
3
2
O F 536.2713; Found 536.2718
3
',6'-bis(diethylamino)-2-(3-fluorophenyl)-2,3-dihydrospiro[isoindole-1,9'-
xanthene]-3-one (7). Pink amorphous solid; Yield 234 mg (52%); R = 0.45 in 70:30 Hex:EtOAc.
1H NMR (400 MHz, CDCl
) δ 8.04 – 7.97 (m, 1H), 7.56 – 7.45 (m, 2H), 7.17 – 7.12 (m, 1H),
.12 – 7.03 (m, 1H), 6.83 – 6.73 (m, 1H), 6.73 – 6.65 (m, 2H), 6.64 – 6.58 (m, 2H), 6.31 (d, J =
.6 Hz, 1H), 6.29 (d, J = 1.5 Hz, 3H), 3.32 (qd, J = 7.1, 1.4 Hz, 8H), 1.15 (t, J = 7.1 Hz, 12H).
) δ 167.7, 163.6, 161.1, 153.4, 153.0, 148.8, 138.5, 138.4, 133.1,
30.3, 129.4, 129.3, 128.6, 128.2, 123.9, 123.4, 122.2, 122.1, 114.0, 113.8, 113.4, 113.2, 108.2,
F
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
7
2
13C NMR (101 MHz, CDCl
3
1
+
106.1, 97.8, 67.5, 44.3, 12.6. HRMS (ESI-TOF) m/z: [M + H] Calcd for C34
H
35
N
3
O F 536.2713;
2
Found 536.2707
3
',6'-bis(diethylamino)-2-(4-chlorophenyl)-2,3-dihydrospiro[isoindole-1,9'-
xanthene]-3-one (8). Pink amorphous solid; Yield 459 mg (99%); R = 0.48 in 70:30 Hex:EtOAc.
F
1H NMR (400 MHz, CDCl
) δ 8.01 (d, J = 6.2 Hz, 1H), 7.58 – 7.41 (m, 2H), 7.20 – 7.13 (m, 1H),
3
7.09 (d, J = 8.8 Hz, 2H), 6.78 (d, J = 8.8 Hz, 2H), 6.61 (d, J = 8.8 Hz, 2H), 6.36 – 6.24 (m, 4H),
13
3
1
1
.33 (q, J = 7.3 Hz, 8H), 1.16 (t, J = 7.0 Hz, 12H). C NMR (101 MHz, CDCl ) δ 167.7, 153.2,
3
53.1, 148.8, 135.3, 133.0, 132.1, 130.6, 128.74, 128.68, 128.3, 128.2, 124.0, 123.4, 108.2,
+
06.0, 97.8, 67.5, 44.3, 12.6. HRMS (ESI-TOF) m/z: [M + H] Calcd for C34
35
H N
3
O Cl 552.2418;
2
Found 552.2418
3',6'-bis(diethylamino)-2-(3-chlorophenyl)-2,3-dihydrospiro[isoindole-1,9'-
xanthene]-3-one (9). Pink amorphous solid; Yield 375 mg (81%); R = 0.48 in 70:30 Hex:EtOAc.
1H NMR (400 MHz, CDCl
) δ 8.00 (d, J = 6.4 Hz, 1H), 7.59 – 7.43 (m, 2H), 7.16 (d, J = 6.3 Hz,
H), 7.09 – 6.97 (m, 2H), 6.90 (s, 1H), 6.68 (d, J = 7.5 Hz, 1H), 6.60 (d, J = 8.7 Hz, 2H), 6.38 –
F
3
1
6
13
.25 (m, 4H), 3.32 (q, J = 7.4 Hz, 8H), 1.15 (t, J = 7.0 Hz, 12H). C NMR (101 MHz, CDCl ) δ
3
167.5, 153.1, 148.9, 138.0, 133.8, 133.1, 130.6, 129.4, 128.4, 128.2, 127.3, 126.6, 124.8, 124.0,
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The Journal of Organic Chemistry
+
1
23.4, 108.3, 106.1, 97.9, 67.6, 44.4, 12.6. HRMS (ESI-TOF) m/z: [M + H] Calcd for
Cl 552.2418; Found 552.2415
',6'-bis(diethylamino)-2-[4-(trifluoromethyl)phenyl]-2,3-dihydrospiro[isoindole-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
C
34
H
35
N
3
O
2
3
1
,9'-xanthene]-3-one (10). Pink amorphous solid; Yield 357 mg (73%); R = 0.65 in 80:20
F
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 7.99 (d, J = 1.9 Hz, 1H), 7.60 – 7.44 (m, 2H), 7.39 (d,
J = 8.5 Hz, 2H), 7.16 – 7.04 (m, 3H), 6.61 (d, J = 9.0 Hz, 2H), 6.29 (m, 4H), 3.32 (q, J = 7.2 Hz,
1
3
8
1
6
H), 1.15 (t, J = 7.0 Hz, 12H). C NMR (101 MHz, CDCl ) δ 168.3, 153.4, 152.9, 148.8, 140.0,
3
33.4, 129.8, 128.40, 128.35, 126.4, 125.63, 125.59, 125.55, 124.0, 123.3, 108.2, 105.5, 97.8,
+
7.9, 44.2, 12.4. HRMS (ESI-TOF) m/z: [M + H] Calcd for C35
H
35
N
3
O
2
3
F 586.2681; Found
586.2675
',6'-bis(diethylamino)-2-[3-(trifluoromethyl)phenyl]-2,3-dihydrospiro[isoindole-
3
1
,9'-xanthene]-3-one (11). P ink amorphous solid; Yie ld 313 mg (64%); R = 0.56 in 70:30
F
1
Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 8.02 (dd, J = 6.0, 2.1 Hz, 1H), 7.57 – 7.48 (m, 2H),
7
.34 (d, J = 7.9 Hz, 1H), 7.25 – 7.18 (m, 2H), 7.03 – 6.93 (m, 2H), 6.61 (d, J = 8.7 Hz, 2H), 6.32
(dd, J = 8.9, 2.6 Hz, 2H), 6.24 (d, J = 2.6 Hz, 2H), 3.31 (q, J = 7.1 Hz, 8H), 1.14 (t, J = 7.0 Hz,
1
1
1
2H). 13C NMR (101 MHz, CDCl
) δ 168.3, 153.4, 152.9, 148.9, 140.0, 133.4, 129.8, 128.4,
3
2
3
1
28.0 (q, J = 32.5 Hz) 126.4, 125.6 (q, J = 3.7 Hz), 124.0, 123.9 (q, J = 272 Hz) 123.3, 108.2,
+
05.5, 97.8, 67.8, 44.4, 12.4. HRMS (ESI-TOF) m/z: [M + H] Ca lcd for C35
H
35
N
3
O
2
3
F 586.2681;
Found 586.2697
Synthesis of the anilinomethylrhodamine (AnMR) and their substituted analogs
A general procedure for AnMR, pMe-AnMR, mMe-AnMR, pMeO-AnMR, mMeO-AnMR,
pF-AnMR, mF-AnMR, pCl-AnMR, mCl-AnMR, pCF -AnMR, and mCF -AnMR was used. In a
0 mL round bottom flask, 100 mg of the rhodamine amide was dissolved in 30 mL of dry THF.
3
3
5
To this solution, 80 mg of LiAlH was slowly added and the mixture was stirred at room
4
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temperature for 12 hours. The reaction was quenched by slowly adding 0.5 mL of water, followed
by 1 mL of 15% NaOH. The mixture was filtered and the filtrate dried using MgSO . The THF
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
was removed under reduced pressure. The products were purified by column chromatography
on alumina (neutral) with 90:10 hexanes:EtOAc.
3
'-N,3'-N,6'-N,6'-N-tetraethyl-2-phenyl-2,3-dihydrospiro[isoindole-1,9'-xanthene]-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
',6'-diamine (AnMR). Pink viscous oil; Yield 70 mg (72%); R
F
= 0.5 in 90:10 Hex:EtOAc. H
NMR (400 MHz, CDCl
3
) δ 7.39 (d, J = 7.6 Hz, 1H), 7.20 (t, J = 7.4 Hz, 1H), 7.12 (t, J = 7.5 Hz,
1H), 7.05 (t, J = 7.7 Hz, 2H), 6.88 (d, J = 7.7 Hz, 1H), 6.65 (m, 4H), 6.57 (t, J = 7.2 Hz, 1H), 6.42
d, J = 2.5 Hz, 2H), 6.19 (dd, J = 8.8, 2.5 Hz, 2H), 5.06 (s, 2H), 3.29 (q, J = 7.1 Hz, 8H), 1.13 (t,
(
1
3
J = 7.0 Hz, 12H). C NMR (101 MHz, CDCl
3
) δ 188.2, 174.7, 167.4, 151.9, 151.1, 148.1, 144.4,
1
9
43.2, 132.6, 128.54, 128.48, 128.0, 126.7, 124.0, 121.8, 115.8, 113.5, 113.2, 108.1, 105.0,
+
7.9, 67.4, 55.0, 44.2, 29.4, 12.7. HRMS (ESI-TOF) m/z: [M + H] Calcd for C34
H
38
3
N O
504.3015; Found 504.3019
3
'-N,3'-N,6'-N,6'-N-tetraethyl-2-(4-methylphenyl)-2,3-dihydrospiro[isoindole-1,9'-
xanthene]-3',6'-diamine (pMe-AnMR). P ink vis cous oil; Yie ld 87 mg (89%) R = 0.55 in 90:10
F
1
Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 7.37 (d, J = 7.5 Hz, 1H), 7.19 (t, J = 7.4 Hz, 1H), 7.11
(t, J = 7.5 Hz, 1H), 6.86 (m, 3H), 6.65 (d, J = 8.6 Hz, 2H), 6.55 (d, J = 8.6 Hz, 2H), 6.41 (d, J =
2.5 Hz, 2H), 6.18 (dd, J = 8.8, 2.6 Hz, 2H), 5.04 (s, 2H), 3.29 (q, J = 7.0 Hz, 8H), 2.13 (s, 3H),
1
3
1
.13 (t, J = 7.0 Hz, 12H). C NMR (101 MHz, CDCl ) δ 151.9, 151.2, 148.1, 142.2, 132.9, 129.2,
3
128.5, 128.0, 126.7, 124.6, 124.0, 121.9, 113.5, 113.4, 108.1, 97.9, 67.3, 55.1, 44.3, 20.5, 12.8,
+
1
2.7. HRMS (ESI-TOF) m/z: [M + H] Calcd for C35
H
40
3
N O 518.3171; Found 518.3170
3
'-N,3'-N,6'-N,6'-N-tetraethyl-2-(3-methylphenyl)-2,3-dihydrospiro[isoindole-1,9'-
xanthene]-3',6'-diamine (mMe-AnMR). P ink vis cous oil; Yie ld 92 mg (95%) R = 0.55 in 90:10
F
1
Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 7.38 (d, J = 7.5 Hz, 1H), 7.20 (t, J = 7.3 Hz, 1H), 7.12
(t, J = 7.4 Hz, 1H), 6.93 – 6.81 (m, 2H), 6.65 (d, J = 8.7 Hz, 2H), 6.53 (t, J = 1.9 Hz, 1H), 6.46 –
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The Journal of Organic Chemistry
6
1
1
4
.32 (m, 4H), 6.19 (dd, J = 8.8, 2.6 Hz, 2H), 5.05 (s, 2H), 3.29 (q, J = 7.1 Hz, 8H), 2.22 (s, 3H),
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
.13 (t, J = 7.0 Hz, 12H). C NMR (101 MHz, CDCl ) δ 151.9, 151.2, 148.1, 144.4, 138.1, 132.8,
3
28.6, 128.3, 128.0, 126.7, 124.1, 121.8, 116.8, 114.0, 113.4, 111.1, 108.2, 97.9, 67.4, 55.1,
+
4.3, 22.0, 12.7. HRMS (ESI-TOF) m/z: [M + H] Calcd for C35
H
40
3
N O 518.3171; Found
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
518.3173
'-N,3'-N,6'-N,6'-N-tetraethyl-2-(4-methoxyphenyl)-2,3-dihydrospiro[isoindole-1,9'-
3
xanthene]-3',6'-diamine (pMeO-AnMR). Pink viscous oil; Yield 55 mg (56%) R = 0.42 in 90:10
F
1
Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 7.39 (d, J = 7.5 Hz, 1H), 7.20 (t, J = 7.4 Hz, 1H), 7.12
(t, J = 7.5 Hz, 1H), 6.88 (d, J = 7.6 Hz, 1H), 6.67 (d, J = 9.2 Hz, 4H), 6.58 (d, J = 9.1 Hz, 2H),
6.43 (s, 2H), 6.21 (d, J = 8.8 Hz, 2H), 5.04 (s, 2H), 3.66 (s, 3H), 3.30 (q, J = 7.0 Hz, 8H), 1.14
t, J = 7.5Hz, 12H). 13C NMR (101 MHz, CDCl
) δ 152.0, 151.3, 150.7, 148.1, 139.0, 133.1,
(
3
128.6, 128.0, 126.7, 124.1, 121.9, 114.4, 114.1, 113.6, 108.1, 98.0, 67.4, 55.6, 55.3, 44.3, 12.8.
+
HRMS (ESI-TOF) m/z: [M + H] Calcd for C35
H
40
N
3
O 534.3121; Found 534.3116.
2
3
'-N,3'-N,6'-N,6'-N-tetraethyl-2-(3-methoxyphenyl)-2,3-dihydrospiro[isoindole-1,9'-
xanthene]-3',6'-diamine (mMeO-AnMR). Pink viscous oil; Yield 28 mg (29%). R = 0.42 in
F
1
9
7
2
8
0:10 Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 7.39 (d, J = 7.6 Hz, 1H), 7.21 (t, J = 7.0 Hz, 1H),
.13 (t, J = 7.4 Hz, 1H), 6.95 (t, J = 8.2 Hz, 1H), 6.86 (d, J = 7.7 Hz, 1H), 6.64 (d, J = 8.8 Hz,
H), 6.38 (d, J = 2.6 Hz, 2H), 6.27 – 6.11 (m, 5H), 5.02 (s, 2H), 3.57 (s, 3H), 3.29 (q, J = 7.1 Hz,
13
H), 1.13 (t, J = 7.0 Hz, 12H). C NMR (101 MHz, CDCl ) δ 159.9, 152.0, 150.9, 148.2, 145.9,
3
133.1, 129.2, 128.9, 128.1, 126.9, 124.3, 121.9, 113.1, 108.3, 106.3, 102.3, 98.9, 97.6, 67.5,
+
5
5
5.1, 54.7, 44.3, 12.7. HRMS (ESI-TOF) m/z: [M + H] Calcd for C35
H
40
N
3
O 534.3121; Found
2
34.3115.
3
'-N,3'-N,6'-N,6'-N-tetraethyl-2-(4-fluorophenyl)-2,3-dihydrospiro[isoindole-1,9'-
xanthene]-3',6'-diamine (pF-AnMR). Pink viscous oil; Yield 79 mg (81%) R = 0.53 in 90:10
F
1
Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 7.38 (d, J = 7.5 Hz, 1H), 7.20 (t, J = 7.4 Hz, 1H), 7.13
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(
t, J = 7.5 Hz, 1H), 6.87 (d, J = 7.6 Hz, 1H), 6.76 (dd, J = 12.9, 4.7 Hz, 2H), 6.64 (dd, J = 8.8,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
1
.2 Hz, 2H), 6.60 – 6.48 (m, 2H), 6.47 – 6.36 (m, 2H), 6.20 (dd, J = 8.8, 1.4 Hz, 2H), 5.02 (s,
13
H), 3.30 (q, J = 7.0 Hz, 8H), 1.14 (t, J = 7.5, 6.5 Hz, 12H). C NMR (101 MHz, CDCl ) δ 156.2,
3
53.8, 152.0, 151.1, 148.2, 141.0, 140.9, 132.7, 128.5, 128.1, 126.8, 124.1, 121.9, 115.2, 115.0,
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
113.83, 113.76, 113.0, 108.0, 97.8, 67.6, 55.4, 44.3, 12.7. HRMS (ES I-TOF) m/z: [M + H] Ca lcd
for C34
H
37
N
3
OF 522.2921; Found 522.2914
3
'-N,3'-N,6'-N,6'-N-tetraethyl-2-(3-fluorophenyl)-2,3-dihydrospiro[isoindole-1,9'-
xanthene]-3',6'-diamine (mF-AnMR). Pink viscous oil. Yield 39 mg (41%) R = 0.53 in 90:10
F
1
Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 7.39 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 7.4 Hz, 1H), 7.13
(t, J = 7.5 Hz, 1H), 6.95 (dd, J = 15.7, 7.8 Hz, 1H), 6.87 (d, J = 7.7 Hz, 1H), 6.64 (d, J = 8.7 Hz,
2
8
H), 6.42 (d, J = 2.5 Hz, 2H), 6.41 – 6.32 (m, 2H), 6.26 (td, J = 8.4, 2.4 Hz, 1H), 6.21 (dd, J =
13
.8, 2.5 Hz, 2H), 5.02 (s, 2H), 3.30 (q, J = 7.0 Hz, 8H), 1.14 (t, J = 7.0 Hz, 12H). C NMR (101
) δ 164.7, 162.3, 151.9, 150.9, 148.3, 146.3, 146.2, 132.3, 129.5, 129.4, 128.4,
28.2, 126.9, 124.1, 121.9, 112.7, 109.4, 109.3, 108.1, 102.5, 102.3, 100.7, 100.4, 97.9, 67.8,
MHz, CDCl
3
1
+
55.3, 44.3, 12.7. HRMS (ESI-TOF) m/z: [M + H] Calcd for C34
22.2926
'-N,3'-N,6'-N,6'-N-tetraethyl-2-(4-chlorophenyl)-2,3-dihydrospiro[isoindole-1,9'-
H
37
N OF 522.2921; Found
3
5
3
xanthene]-3',6'-diamine (pCl-AnMR). Pink viscous oil; Yield 54 mg (55%) R = 0.53 in 90:10
F
1
Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 7.38 (d, J = 7.3 Hz, 1H), 7.20 (t, J = 7.4 Hz, 1H), 7.13
(t, J = 7.5 Hz, 1H), 6.98 (d, J = 9.0 Hz, 2H), 6.86 (d, J = 7.6 Hz, 1H), 6.61 (d, J = 8.8 Hz, 2H),
6
3
1
9
.54 (d, J = 9.0 Hz, 2H), 6.40 (d, J = 2.2 Hz, 2H), 6.19 (dd, J = 8.8, 2.3 Hz, 2H), 5.01 (s, 2H),
13
.30 (q, J = 7.0 Hz, 8H), 1.14 (t, J = 7.0 Hz, 12H). C NMR (101 MHz, CDCl ) δ 151.9, 150.9,
3
48.2, 143.0, 132.4, 128.43, 128.41, 128.2, 126.9, 124.1, 121.9, 120.9, 114.4, 112.7, 108.1,
+
7.8, 67.6, 55.1, 44.3, 12.7. HRMS (ESI-TOF) m/z: [M + H] Calcd for C34
H
37
N OCl 538.2625;
3
Found 538.2618
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The Journal of Organic Chemistry
3
'-N,3'-N,6'-N,6'-N-tetraethyl-2-(3-chlorophenyl)-2,3-dihydrospiro[isoindole-1,9'-
1
2
xanthene]-3',6'-diamine (mCl-AnMR). Pink viscous oil; Yield 49 mg (51%) R
F
= 0.53 in 90:10
) δ 7.39 (d, J = 7.5 Hz, 1H), 7.22 (t, J = 7.4 Hz, 1H), 7.14
t, J = 7.5 Hz, 1H), 6.86 (dd, J = 9.6, 8.5 Hz, 2H), 6.70 (d, J = 2.0 Hz, 1H), 6.63 (d, J = 8.8 Hz,
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
Hex:EtOAc. H NMR (400 MHz, CDCl
3
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2H), 6.58 – 6.49 (m, 1H), 6.48 – 6.38 (m, 3H), 6.21 (dd, J = 8.8, 2.5 Hz, 2H), 5.02 (s, 2H), 3.31
13
(
q, J = 7.0 Hz, 8H), 1.14 (t, J = 7.0 Hz, 12H). C NMR (101 MHz, CDCl ) δ 152.0, 150.8, 148.3,
3
145.7, 134.3, 132.3, 129.4, 128.5, 128.2, 127.0, 124.2, 121.9, 115.8, 113.1, 112.6, 112.0, 108.2,
+
9
7.9, 67.8, 55.1, 44.3, 12.7. HRMS (ESI-TOF) m/z: [M + H] Calcd for C34
H
37
N OCl 538.2625;
3
Found 538.2618.
3'-N,3'-N,6'-N,6'-N-tetraethyl-2-[4-(trifluoromethyl)phenyl]-2,3-dihydrospiro-
[
isoindole-1,9'-xanthene]-3',6'-diamine (pCF
3
-AnMR). Pink viscous oil; Yield 89 mg (91%) R
F
1
=
9
8
0.51 in 90:10 Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 7.40 (d, J = 7.5 Hz, 1H), 7.27 (d, J =
.4 Hz, 3H), 7.24 – 7.19 (m, 1H), 7.14 (t, J = 7.4 Hz, 1H), 6.87 (d, J = 7.6 Hz, 1H), 6.66 (d, J =
.7 Hz, 2H), 6.61 (d, J = 8.8 Hz, 2H), 6.41 (d, J = 2.5 Hz, 2H), 6.20 (dd, J = 8.8, 2.6 Hz, 2H),
13
5.07 (s, 2H), 3.30 (q, J = 7.1 Hz, 8H), 1.14 (t, J = 7.0 Hz, 12H). C NMR (101 MHz, CDCl ) δ
3
3
1
1
1
51.9, 150.6, 148.3, 146.8, 132.1, 128.4, 127.1, 125.9 (q, J = 3.8 Hz), 125.2 (q, J = 269 Hz),
2
24.2, 121.9, 117.3 (q, J = 32.4 Hz), 112.8, 112.3, 108.1, 97.8, 67.9, 55.1, 44.3, 12.7. HRMS
+
(
ESI-TOF) m/z: [M + H] Calcd for C35
H
37
N
3
OF 572.2889; Found 572.2882
3
3
'-N,3'-N,6'-N,6'-N-tetraethyl-2-[3-(trifluoromethyl)phenyl]-2,3-
dihydrospiro[isoindole-1,9'-xanthene]-3',6'-diamine (mCF
3
-AnMR). Pink viscous oil; Yield
1
9
1
2 mg (94%); R
F
= 0.54 in 90:10 Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 7.41 (d, J = 7.5 Hz,
H), 7.23 (td, J = 7.4, 1.2 Hz, 1H), 7.18 – 7.11 (m, 1H), 7.01 (t, J = 8.0 Hz, 1H), 6.91 (t, J = 2.1
Hz, 1H), 6.87 (d, J = 7.6 Hz, 1H), 6.79 (d, J = 7.3 Hz, 1H), 6.67 (dd, J = 8.5, 2.6 Hz, 1H), 6.61
d, J = 8.8 Hz, 2H), 6.41 (d, J = 2.5 Hz, 2H), 6.20 (dd, J = 8.8, 2.6 Hz, 2H), 5.05 (s, 2H), 3.29 (q,
(
1
3
J = 7.1 Hz, 8H), 1.13 (t, J = 7.0 Hz, 12H). C NMR (101 MHz, CDCl ) δ 152.0, 150.6, 148.3,
3
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1
1
5
44.6, 132.4, 130.6, 128.7, 128.5, 128.2, 127.0, 124.2, 121.9, 116.5, 112.3, 112.3, 109.3, 109.3,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
08.2, 97.8, 67.7 55.0, 44.3, 12.6. HRMS (ESI-TOF) m/z: [M + H] Calcd for C35
72.2889; Found 572.2896.
H
37
N
3
OF
3
Synthesis of the AnMR6G series
Compound 13 was synthesized according to the literature.22
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A general procedure for preparing the rhodamine 6G amides (14 - 17) was used. To a
solution of 13 (200 mg, 0.44 mmol) in 1,2-dichloroethane (10 mL) was added phosphorus
oxychloride (0.3 mL, 3 mmol) dropwise. The solution was refluxed for 4 hours. The reaction
mixture was cooled and evaporated under reduced pressure to give the rhodamine 6G acid
chloride, which was used in the next step without further purification. To the crude acid chloride
dissolved in 1,2-dichloroethane (10 mL) was added 0.1 mL of the corresponding aniline. The
mixture was added to a separatory funnel with an additional 20 mL of DCM. The organic layer
was washed with 2.0 M H
2
SO
4
(3 x 20 mL), then with water (2 x 20 mL), and finally 0.10 M NaOH
and the volatiles were
(2 x 20 mL). The organic layer was then dried over anhydrous MgSO
4
removed under reduced pressure. The products were purified by column chromatography on
alumina (neutral) with 70:30 hexanes:EtOAc.
3
',6'-bis(ethylamino)-2',7'-dimethyl-2-phenyl-2,3-dihydrospiro[isoindole-1,9'-
xanthene]-3-one (14). Pink amorphous solid; Yield 180 mg (83%); R = 0.23 in 70:30
F
1
Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 8.15 – 7.94 (m, 1H), 7.50 (dd, J = 5.7, 3.1 Hz, 2H),
7
.18 – 6.98 (m, 4H), 6.76 (dd, J = 7.9, 1.9 Hz, 2H), 6.44 (s, 2H), 6.23 (s, 2H), 3.48 (s, 4H), 1.94
1
3
(s, 6H), 1.30 (t, J = 7.1 Hz, 6H). C NMR (101 MHz, CDCl
3
) δ 167.7 , 153.3 , 151.5 , 147.2 ,
1
3
36.6 , 132.9 , 131.0, 128.5, 128.1, 127.1, 126.6, 124.0, 123.4, 117.9, 106.9, 105.0, 96.6, 67.5,
+
8.4, 29.7, 28.0, 16.8, 14.7. HRMS (ESI-TOF) m/z: [M + H] Ca lcd for C32
H
32
N
3
2
O 490.2495;
Found 490.2498.
',6'-bis(ethylamino)-2-(4-methoxyphenyl)-2',7'-dimethyl-2,3-dihydrospiro
3
[
isoindole-1,9'-xanthene]-3-one (15). Pink amorphous solid; Yie ld 150 mg (65%); R = 0.13 in
F
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7
0:30 Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 8.18 – 7.95 (dd, J = 5.9, 2.8 Hz, 1H), 7.60 –
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7.40 (m, 2H), 7.17 – 7.06 (dd, J = 5.8, 2.8 Hz, 1H), 6.67 – 6.51 (m, 4H), 6.43 (s, 2H), 6.22 (s,
2
6
1
1
H), 3.67 (s, 3H), 3.56 – 3.28 (S, 2H), 3.16 (q, J = 7.1 Hz, 4H), 1.95 (s, 6H), 1.30 (t, J = 7.1 Hz,
H). 13C NMR (101 MHz, CDCl ) δ 151.4, 150.6, 150.3, 146.5, 139.1, 133.1, 128.8, 128.0,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
26.7, 124.1, 121.8, 117.5, 114.3, 114.3, 114.1, 114.0, 113.9, 96.6, 67.4, 55.6, 55.3, 38.5, 17.0,
+
4.9. HRMS (ESI-TOF) m/z: [M + H] Calcd for C33
H
34
N
3
3
O 520.2600; Found 520.2600
2-(4-chlorophenyl)-3',6'-bis(ethylamino)-2',7'-dimethyl-2,3-dihydrospiro[isoindole-
1
,9'-xanthene]-3-one (16). Pink amorphous solid; Yield 161 mg (70%); R = 0.39 in 70:30
F
1
Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 8.01 (d, J = 3.4 Hz, 1H), 7.66 – 7.37 (m, 2H), 7.22 –
6
.94 (m, 3H), 6.73 (d, J = 8.7 Hz, 2H), 6.39 (s, 2H), 6.24 (s, 2H), 3.49 (s, 2H), 3.17 (q, J = 7.0
1
3
Hz, 1H), 1.93 (s, 6H), 1.31 (t, J = 7.1 Hz, 6H). C NMR (101 MHz, CDCl ) δ 167.7, 153.3, 151.5,
3
147.4, 135.3, 133.1, 132.1, 130.6, 128.7, 128.3, 128.3, 128.1, 124.0, 123.3, 118.0, 106.4, 105.0,
+
9
6.6, 67.6, 38.3, 38.2, 16.8, 14.7, 14.7, 14.6. HRMS (ESI-TOF) m/z: [M + H] Calcd for
32 31 3
C H N O
2
Cl 524.2105; Found 524.2098
3
',6'-bis(ethylamino)-2',7'-dimethyl-2-[4-(trifluoromethyl)phenyl]-2,3-
dihydrospiro[isoindole-1,9'-xanthene]-3-one (17). Pink amorphous solid; Yield 180 mg
1
(73%); R
F
= 0.48 in 70:30 Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 8.07 – 7.94 (m, 1H), 7.59 –
7
.44 (m, 2H), 7.36 (d, J = 8.5 Hz, 2H), 7.10 (d, J = 8.3 Hz, 3H), 6.41 (s, 2H), 6.27 (s, 2H), 3.50
13
(
s, 2H), 3.25 – 3.01 (m, 4H), 1.92 (s, 6H), 1.31 (t, J = 7.1 Hz, 6H). C NMR (101 MHz, CDCl
3
)
δ 168.1, 153.7, 151.3, 147.5, 140.4, 133.6, 133.4, 129.8, 128.6, 128.3, 128.2, 128.1, 128.0,
1
27.8, 127.7, 127.4, 125.7, 125.7, 125.6, 125.6, 125.6, 125.5, 125.4, 124.0, 123.9, 123.9, 123.6,
123.5, 123.4, 122.7, 118.2, 106.4, 96.7, 67.6, 38.3, 16.8, 14.6. HRMS (ESI-TOF) m/z: [M + H]+
31 3
Calcd for C33H N O
F
2 3
558.2368; Found 558.2366
A general procedure for AnMR6G, pMeO-AnMR6G, pCl-AnMR6G, and pCF
3
-AnMR6G
was used. In a 50 mL round bottom flask, 100 mg of the rhodamine amide was dissolved in 30
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mL of dry THF. To this solution, 80 mg of LiAlH was slowly added and the mixture was stirred
4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
at room temperature for 12 hours. Then the reaction was quenched by slowly adding 0.5 mL of
water, followed by 1 mL of 15% NaOH. The mixture was filtered and the filtrate dried using
MgS O
column chromatography on alumina (neutral) with 90:10 hexanes:EtOAc.
'-N,6'-N-diethyl-2',7'-dimethyl-2-phenyl-2,3-dihydrospiro[isoindole-1,9'-xanthene]-
4
. The volatiles were removed under reduced pressure. The products were purified by
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
',6'-diamine (AnMR6G). Pink amorphous solid; Yield 54 mg (56%); R = 0.32 in 90:10
F
1
Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 7.43 (d, J = 7.5 Hz, 1H), 7.23 (t, J = 7.4 Hz, 1H), 7.13
(t, J = 7.5 Hz, 1H), 7.05 (t, J = 7.8 Hz, 2H), 6.84 (d, J = 7.7 Hz, 1H), 6.66 (d, J = 8.1 Hz, 2H),
6
4
1
.57 (t, J = 7.0 Hz, 1H), 6.46 (s, 2H), 6.40 (s, 2H), 5.08 (s, 2H), 3.34 (s, 2H), 3.21 (q, J = 7.2 Hz,
1
3
H), 1.84 (s, 6H), 1.31 (t, J = 7.1 Hz, 6H). C NMR (101 MHz, CDCl ) δ 151.2, 150.2, 146.5,
3
44.5, 132.7, 128.7, 128.6, 128.5, 128.1, 126.7, 124.2, 121.8, 117.5, 115.7, 113.6, 113.5, 113.4,
+
96.6, 67.5, 55.0, 38.5, 17.0, 14.7. HRMS (ESI-TOF) m/z: [M + H] Calcd for C32
H
34
N
3
O
4
76.2702; Found 476.2702
'-N,6'-N-diethyl-2-(4-methoxyphenyl)-2',7'-dimethyl-2,3-dihydrospiro[isoindole-
3
1,9'-xanthene]-3',6'-diamin (pMeO-AnMR6G). Pink amorphous solid; Yie ld 88 mg (90%); R
F
=
1
0
.19 in 90:10 Hex:EtOAc. H NMR (400 MHz, CDCl
3
) δ 7.40 (d, J = 7.5 Hz, 1H), 7.21 (t, J = 6.9
Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 6.85 – 6.76 (m, 1H), 6.64 (d, J = 9.2 Hz, 2H), 6.56 (d, J = 9.3
Hz, 2H), 6.44 (s, 2H), 6.37 (s, 2H), 5.02 (s, 2H), 3.64 (s, 3H), 3.33 (s, 2H), 3.19 (q, J = 7.1 Hz,
1
3
4
1
6
H), 1.84 (s, 6H), 1.30 (t, J = 7.1 Hz, 6H). C NMR (101 MHz, CDCl ) δ 167.7, 158.2, 153.1,
3
51.7, 147.2, 132.7, 131.4, 129.0, 128.9, 128.6, 128.1, 124.1, 123.3, 117.8, 113.9, 106.8, 96.6,
+
7.5, 55.11, 38.4, 16.8, 14.7. HRMS (ESI-TOF) m/z: [M + H] Calcd for C33
H
36
N
3
2
O 506.2808;
Found 506.2805
-(4-chlorophenyl)-3'-N,6'-N-diethyl-2',7'-dimethyl-2,3-dihydrospiro[isoindole-1,9'-
2
xanthene]-3',6'-diamine (pCl-AnMR6G). Pink amorphous solid; Yie ld 75 mg (77%); R = 0.32
F
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in 90:10 Hex:EtOAc. H NMR (400 MHz, CDCl ) δ 7.40 (d, J = 7.5 Hz, 1H), 7.22 (t, J = 7.4 Hz,
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1H), 7.12 (t, J = 7.5 Hz, 1H), 6.95 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 7.7 Hz, 1H), 6.53 (d, J = 8.9
Hz, 2H), 6.39 (s, 2H), 6.36 (s, 2H), 5.01 (s, 2H), 3.35 (s, 2H), 3.19 (q, J = 7.2 Hz, 4H), 1.84 (s,
1
3
6
1
1
H), 1.30 (t, J = 7.1 Hz, 6H). C NMR (101 MHz, CDCl ) δ 150.9, 150.2, 146.7, 143.1, 132.5,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
28.5, 128.4, 128.2, 126.8, 124.2, 121.8, 120.8, 117.6, 114.3, 113.1, 96.6, 67.7, 55.1, 38.4,
+
7.0, 14.8. HRMS (ESI-TOF) m/z: [M + H] Calcd for C32
H
33
3
N OCl 510.2312; Found 510.2319.
3'-N,6'-N-diethyl-2',7'-dimethyl-2-[4-(trifluoromethyl)phenyl]-2,3-dihydrospiro
isoindole-1,9'-xanthene]-3',6'-diamine (pCF -AnMR6G). Pink amorphous solid; Yield 71 mg
= 0.30 in 90:10 Hex:EtOAc. H NMR (400 MHz, CDCl ) δ 7.42 (d, J = 7.5 Hz, 1H),
.25 (m, 3H), 7.14 (t, J = 7.5 Hz, 1H), 6.82 (d, J = 7.7 Hz, 1H), 6.65 (d, J = 8.5 Hz, 2H), 6.37 (s,
H), 5.07 (s, 2H), 3.37 (s, 2H), 3.20 (q, J = 7.2 Hz, 4H), 1.84 (s, 6H), 1.31 (t, J = 7.1 Hz, 6H).
) δ 150.7, 150.2, 146.9, 146.8, 132.1, 128.5, 128.4, 127.0, 125.8,
25.8, 125.7, 124.3, 121.9, 117.7, 112.8, 112.7, 112.6, 96.6, 68.0, 55.1, 38.4, 17.0, 14.8. HRMS
[
3
1
(
73%); R
F
3
7
4
13C NMR (101 MHz, CDCl
3
1
+
(
ESI-TOF) m/z: [M + H] Calcd for C33
H
33
N
3
OF 544.2576; Found 544.2582
3
Synthesis of AMR and AMR6G
AMR was synthesized according to procedures in the literature.14
3',6'-bis(ethylamino)-2',7'-dimethyl-2,3-dihydrospiro[isoindole-1,9'-xanthene]-3-
one (18). In a 250 mL Erlenmeyer flask, 10 g of NH Cl was added to a mixture of DMF (25 mL)
4
and TEA (25 mL). The flask was capped and the mixture was allowed to stir for two hours before
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adding 1g (2.1 mmol) of Rhodamine 6G. This mixture was then stirred for an additional 48 hours.
The product was isolated by pouring the reaction mixture in 125 mL of ice/cold water and then
transferring to a separatory funnel. The product was extracted from the aqueous mixture using
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
DCM (3 x 25 mL). The combined organic layers were dried using Na
2
SO
4
and the solvent was
removed in vacuo. The crude oil was purified by column chromatography using 70/30
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Hex/EtOAc) yielding 310 mg (37%) of 2-9 as a white solid that turned pink soon after.
1H NMR (400 MHz, CDCl
3
) δ 7.89 (dd, J = 5.6, 3.2 Hz, 1H), 7.42 (dd, J = 5.7, 3.1 Hz, 2H), 7.00
(dd, J = 5.7, 3.0 Hz, 1H), 6.54 (s, 2H), 6.42 (s, 1H), 6.35 (s, 2H), 3.49 (s, 2H), 3.21 (q, J = 7.1
1
3
Hz, 4H), 1.94 (s, 6H), 1.32 (t, J = 7.1 Hz, 6H). C NMR (101 MHz, CDCl ) δ 169.9, 155.8, 150.7,
3
147.2, 132.9, 129.4, 128.0 (d, J = 15.1 Hz), 123.6, 123.2, 117.8, 107.7, 96.7, 76.7, 60.0, 38.4,
+
1
6.7, 14.7. HRMS (ESI-TOF) m/z: [M + H] Calcd for C26
H
28
N
3
2
O 414.2182; Found 414.2178
3
'-N,6'-N-diethyl-2',7'-dimethyl-2,3-dihydrospiro[isoindole-1,9'-xanthene]-3',6'-
diamine (AMR6G). In a 100 mL round bottom flask, 200 mg of 2-9 was dissolved in 50 mL of
dry THF. To this solution, 180 mg of LAH was added slowly. The solution was then refluxed
under argon for 48 hours. The reaction mixture was cooled using an ice bath and then quenched
using 0.5 mL water and 1.5 mL 10% NaOH solution. The mixture was filtered and the filtrate
dried with MgSO followed by removal of the volatiles under reduced pressure. The crude was
4
purified by column chromatography to give 116 mg (60%) of AMR6G as a pink amorphous solid.
1H NMR (400 MHz, CDCl
3
) δ 7.42 – 7.05 (m, 3H), 6.90 (d, J = 7.6 Hz, 1H), 6.57 (s, 2H), 6.49 (s,
1
7
1
H), 6.33 (s, 2H), 4.50 (s, 2H), 3.39 (s, 2H), 3.20 (q, J = 6.7 Hz, 4H), 1.96 (s, 6H), 1.30 (t, J =
.1 Hz, 6H). 13C NMR (101 MHz, CDCl
3
) δ 149.9, 148.6, 146.4, 146.0, 140.3, 131.3, 130.5,
30.3, 129.2, 127.7, 127.1 – 126.8 (m), 124.9, 122.0, 117.4, 116.8, 115.8, 111.9, 97.4, 96.6,
77.4, 65.7, 51.3, 43.6, 38.5 (d, J = 4.3 Hz), 17.0, 16.8, 14.9 (d, J = 3.5 Hz). HRMS (ESI-TOF)
+
m/z: [M + H] Calcd for C26
H
28
N
3
2
O 414.2182; Found 414.2178
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The Journal of Organic Chemistry
ACKNOWLEDGEMENTS
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The authors gratefully acknowledge support fully or in part from the National Institutes of Health
(NIH-1R15GM080721-01A1), the National Science Foundation (NSF-CHEM-0719185). The
authors also thank the Department of Chemistry and Biochemistry, Southern Illinois University
for partial support of this research.
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SUPPORTING INFORMATION AVAILABLE
UV/Vis and Fluorescence pH titration curves, quantum yield studies, analog structures, NMR
experiments and NMR spectra. These materials are available free of charge via the Internet at
http://pubs.acs.org.
REFERENCES
(
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(9) Han, J.; Burgess, K. Chem. Rev., 2010, 110, 2709.
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Desai, S.; Pulsinelli, W. Anal. Biochem. 1990, 187,109.
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