Comparative Evaluation of Substituent Effect on the Photochromic Properties of Spiropyrans and Spirooxazines

Article abstract of DOI:10.1021/acs.joc.6b01193

Spiropyrans and spirooxazines represent an important class of photochromic compounds with a wide variety of applications. In order to effectively utilize and design these photoswitches it is desirable to understand how the substituents affect photochromic properties, and how the different structural motifs compare under identical conditions. In this work a small library of photoswitches was synthesized in order to comparatively evaluate the effect of substituent modifications and structure on photochromism. The library was designed to modify positions that were believed to have the greatest effect on C-O bond lability and therefore the photochromic properties. Herein we report a comparative analysis of the UV and visible light responses of 30 spiropyrans, spiroindolinonaphthopyrans, and spirooxazines. The influence of gadolinium(III) binding was also investigated on the library of compounds to determine its effect on photoswitching. Both assays demonstrated different trends in substituent and structural requirements for optimal photochromism.

Full text of DOI:10.1021/acs.joc.6b01193

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Article
Comparative Evaluation of Substituent Effect on the
Photochromic Properties of Spiropyrans and Spirooxazines
Edward I. Balmond, Brandon K. Tautges, Andrea L. Faulkner, Victor
W. Or, Blanka M. Hodur, Jared T. Shaw, and Angelique Y. Louie
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01193 • Publication Date (Web): 16 Aug 2016
Downloaded from http://pubs.acs.org on August 20, 2016
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Comparative Evaluation of Substituent Effect on the Photochromic Properties of
Spiropyrans and Spirooxazines
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Edward I. Balmond[a][§], Brandon K. Tautges[a][§], Andrea L. Faulkner , Victor W. Or ,
[a]
[a]
[
a]
[a]
[b]
Blanka M. Hodur , Jared T. Shaw* , Angelique Y. Louie*
[
a] Department of Chemistry, University of California at Davis, One Shields Avenue,
Davis, CA, 95616, United States; [b] Department of Biomedical Engineering and
Chemistry Graduate Group, University of California at Davis, One Shields Avenue,
Davis, CA, 95616, United States; [§] These authors contributed equally to this work.
jtshaw@ucdavis.edu, aylouie@ucdavis.edu
EDG
EWG
H3C CH3
H3C CH3
H3C CH3
R
UV
EDG
Gd3+
N
O
R'
N
O
N
O
CH3
CH3
CH3
Abstract
Spiropyrans and spirooxazines represent an important class of photochromic compounds
with a wide variety of applications. In order to effectively utilize and design these
photoswitches it is desirable to understand how the substituents affect photochromic
properties, and how the different structural motifs compare under identical conditions. In
this work a small library of photoswitches was synthesized in order to comparatively
evaluate the effect of substituent modifications and structure on photochromism. The
library was designed to modify positions that were believed to have the greatest effect on
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C-O bond lability and therefore the photochromic properties. Herein we report a
comparative analysis of the UV and visible light responses of 30 spiropyrans,
spiroindolinonaphthopyrans, and spirooxazines. The influence of gadolinium(III) binding
was also investigated on the library of compounds to determine its effect on
photoswitching. Both assays demonstrated different trends in substituent and structural
requirements for optimal photochromism.
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Introduction
Photochromic compounds represent a growing area of research as the utility of these
compounds begins to be realized. Numerous photochromic compounds have recently
been explored for their utility, such as azobenzenes,1 stilbenes,2 fulgides,3
dithienylethenes,4 and spiropyrans/spirooxazines (Scheme 1),5 among others.6
Photochromic compounds have been employed for a myriad of applications including
microfluidics,7
biochemical assays,8
modulating
electric
potential,9
and
photopharmacology.10 Spiropyrans and spirooxazines are two of the most widely studied
photochromic compounds due to their reversible activation by a wide range of external
stimuli. Examples of light-,11 thiol-,12 redox-,13 metal ion-,14 and pH-15 sensitive
spiropyrans and spirooxazines have previously been demonstrated. An advantage of
spiropyrans and spirooxazines over other photochromic compounds is the dramatic
difference in the properties of the two isomers (Scheme 1). In the closed spiro (SP) form,
spiropyrans and spirooxazines are pale yellow or colorless in solution, uncharged,
hydrophobic, and occupy less volume than the merocyanine (MC) form.16 The MC-form
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is intensely colored in solution, zwitterionic, hydrophilic, and has a much higher dipole
moment (14-18 D) than the SP-form (~4 D).5, 17
In many applications, for spiropyran- or spirooxazine-based dynamic materials to be
robust and behave reliably they are covalently attached to a surface to prevent leaching,18
reduce photodegradation,19 enhance biocompatibility,20 and otherwise improve
performance.21 Tethered spiropyrans have been used in a wide range of applications
including “smart electrodes,”22 photo-controlled enzymatic activity,23 photo-controlled
release of therapeutics,24 photo-control of nanoparticle solubility,25 and modulation of
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magnetic resonance imaging (MRI) contrast agent efficiency. To aid the development of
future spiropyran- and spirooxazine-based systems, knowledge of how these different
structural motifs compare under identical experimental conditions is required.
Our goal in this work is twofold; first to better understand how substituents affect
photoswitching and to identify strong switches under mild irradiation and temperature
conditions more amenable to biological applications.27 We systematically vary
substituents with different electron withdrawing or donating power and study them under
identical experimental conditions to allow direct comparison for substituent effects on
photochromic properties. The results of these studies will help develop a platform for
future MRI contrast agent design and development.
Despite much work in this field, a comparative study between spiropyrans,
spiroindolinonaphthopyrans, and spirooxazines with the same substituent pattern has not
been conducted (Figure 1). Previous literature reports have demonstrated that electron-
withdrawing groups on the chromene ring enhance the UV response of photoswitches.28
However, little work has been done to understand how these groups affect the visible
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light response of photochromic compounds. Even less is known how indoline substituents
affect photoswitching, and among compounds similar to those reported here, very few
have been subjected to identical experimental conditions for direct comparison between
substitution patterns.
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Scheme 1: A General isomerization of commonly employed photoswitching compounds,
B General isomerization of spiropyrans (X = CH) and spirooxazines (X = N).
Herein we report a comparative study on the photochromic properties of different
spiropyrans, spiroindolinonaphthopyrans, and spirooxazines, with similar substitution
patterns under identical experimental conditions. Also examined was the effect of
gadolinium(III) ions on photoswitching in this array of compounds, which is important
for applications in contrast agent development and analytical chemistry.26, 29 Based on
previous literature, we believed that varying the lability of the C-O bond would have the
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greatest effect on the photochromic properties of the compounds.5, 30 Therefore, two
positions for substitution were selected on either side of the switches that would likely
have the most significant effect on the C-O bond lability and consequently their
photochromic properties. Initially 36 compounds were chosen as targets for this study
with varying substituents (NO2, H, and OCH3) on either side of the switches (Figure 1).
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Figure 1: Target compounds for this study
Results and Discussion
Synthesis
The commercially available starting materials indoline 1, salicylaldehydes 2-4, 2-
hydroxy-1-naphthaldehyde 5, and 1-nitroso-2-naphthol 6 were employed. The synthesis
of additional starting materials was required in order to generate all of the target
compounds (Scheme 2), for full experimental details and compound characterization see
experimental section. Indolium Iodide Synthesis: The 4-methoxy- and 4-nitro- indolium
iodides, 7 and 8 respectively, were synthesized in two steps from their respective p-
hydrazine hydrochlorides. Initially, an interrupted Fischer indole reaction was conducted
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between the hydrazines and 3-methyl-2-butanone, followed by methylation with
iodomethane.31 Naphthaldehyde Synthesis: 2-Hydroxy-6-methoxynaphthalene-1-
carbaldehyde 9 was obtained in three steps from 2,6-dihydroxynaphthalene. 2,6-
Dihydroxynaphthalene was initially methylated using NaH and iodomethane to afford
2,6-dimethoxynaphthalene,32 which was subsequently formylated with N-
methylformanilide and POCl3.33 Selective deprotection of the o-methoxy group of 2,6-
dimethoxy-1-naphthalenecarbaldehyde was achieved by treating it with MgBr2 and NaI
in CH3CN at 150 °C. Nitration of 2-hydroxy-1-naphthaldehyde with concentrated nitric
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acid
afforded
the
required
2-hydroxy-6-nitro-1-naphthaldehyde
Nitrosonaphthalenol Synthesis: 6-Methoxy-1-nitrosonaphthalen-2-ol 11 was synthesized
from 6-methoxynaphthalen-2-ol by nitrosation using NaNO2 in acetic acid and water.35
The synthesis of 24 of the initial 36 target compounds was achieved in 13-97% yield,
through the condensation reaction between the required indoline or indolium salt, and the
corresponding salicylaldehydes, 2-hydroxy-1-naphthaldehydes or 1-nitroso-2-naphthols.
A
OH
O
H3C CH3
H3C CH3
R
H
N
N
O
R'
CH3
CH3
R'
1
2, R' = OCH3
3, R' = NO2
SP1-SP9
(
Et3N or
4, R' = H
piperidine)
or
+
or
or
EtOH or
i-PrOH
reflux
H3C CH3
O
H3C CH3
R
X
R
X
CH3
CH3
HO
R'
N
N
O
CH3
I
R'
7
8
, R = OCH3
, R = NO2
5, X = CH, R' = H
6, X = N, R' = H
X = CH, NP1-NP9
X = N, SO1-SO3 and SO7-SO9
9
, X = CH, R' = OCH3
10, X = CH, R' = NO2
1, X = N, R' = OCH3
1
B
H3C CH3
H3C CH3
R
N
R
N
HNO3
NO2
N
O
H2SO4, 0 °C
N
O
CH3
CH3
SO1, R = H
SO4, R = H
SO2, R = NO2
SO3, R = OCH3
SO5, R = NO2
SO6, R = OCH3
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R’
H
NO2
OCH3
SP1, 97%
NP1, 67%
SO1, 47%
SP2, 63%
NP2, 64%
SO2, 20%
SP3, 44%
NP3, 73%
SO3, 45%
SP4, 73%
NP4, 26%
SO4, 15%
SP5, 77%
NP5, 67%
SO5, N/A*
SP6, 69%
NP6, 15%
SO6, N/A*
SP7, 86%
NP7, 40%
SO7, 38%
SP8, 56%
NP8, 67%
SO8, 13%
SP9, 93%
NP9, 50%
SO9, 15%
H
R
NO2
OCH3
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Scheme 2: Synthesis of target spiropyrans SP1-SP9, spiroindolinonaphthopyrans NP1-
NP9, and spirooxazines SO1-SO9. *SO5 could not be reproducibly synthesized and SO6
could not be synthesized.
The required starting material, 6-nitro-nitroso-2-naphthol, for the synthesis of
spirooxazines SO4, SO5 and SO6, was neither commercially or synthetically obtainable.
In an attempt to install the nitro group on the naphthalene ring system the
unfunctionalized spirooxazines SO1, SO2, and SO3 were subjected to nitration
conditions, employing HNO3 and H2SO4 (Scheme 2, B).36 Spirooxazine SO4 was
obtained in 15% yield, however, attempts to synthesize spirooxazines SO527c and SO6
were irreproducible and unsuccessful, respectively. We found that for SO6 there was
competing nitration of the indoline ring, resulting in an inseparable mixture of products.
In addition, despite spirooxazines originating from o-nitrosophenols being known,37 the
required spirooxazines for this study have never been synthesized. o-Nitrosophenols are
relatively difficult to synthesize, isolate, and handle;38 and while o-nitrosophenols can be
accessed through the Baudisch reaction,39 it was not possible to isolate the required
compounds using this methodology. As a result the corresponding nine spirooxazines
based on the o-nitrosophenols could not be synthesized. Thus, in total 25 of the initial 36
target compounds were obtained and evaluated for their photochromic properties. Of the
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SP1-SP8,27a,
31b,
40
synthesized
compounds
(Scheme
2)
spiropyrans
spiroindolinonaphthopyrans NP1-NP5,27a, 41 and spirooxazines SO1-SO4,27c, 41-42 and
SO743 are known in the literature. Spiropyran SP9, spiroindolinonaphthopyrans NP6-
NP9, and spirooxazines SO8 and SO9 have not previously been reported in the literature.
Based on the results obtained from our assays of the initial targets in the absence and
presence of Gd(NO3)3 (vide infra) five additional novel spiropyrans SP10-SP14 were
synthesized to further explore the observed results. The synthesis of spiropyrans SP10-
SP13, containing different electron-withdrawing groups (EWGs) on the chromene
portion and the electron-donating methoxy substituent on the indoline portion, required
the synthesis of three salicylaldehyde derivatives 12-14. Both the 4-(trifluoromethyl)- and
4-cyano- phenols were formylated with hexamethylenetetramine in TFA, to afford the
desired 5-substituted salicylaldehydes, 12 and 13.44 4-Hydroxynicotinaldehyde 14 was
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synthesized through the hydrolysis of 4-chloronicotinaldehyde with HCl and H2O2. The
three spiropyrans SP10-SP12 were then synthesized in an analogous fashion to the initial
2
4 compounds in 16-67% yield, with spiropyran SP12 methylated with iodomethane to
afford the final spiropyran SP13 in 60% yield (Scheme 3). Spiropyran SP14 was
synthesized, in which an alternate electron-donating group (EDG) was incorporated on
the chromene ring. The required 5-dimethylamino-2-hydroxy-benzaldehyde 15 was
obtained by the hydrogenation of 4-nitrophenol in the presence of aqueous
46
formaldehyde. Benzaldehyde 15 was then reacted with 4-methoxyindolium iodide 7 and
Et3N to afford the desired spiropyran SP14 in 47% yield (Scheme 3).
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OH
R
O
7
H3C CH3
Et3N or piperidine
H3CO
H
EtOH, reflux
N
O
R
CH3
12, R = CF3
3, R = CN
5, R = N(CH3)2
SP10, R = CF3, 67%
SP11, R = CN, 60%
SP14, R = N(CH3)2, 47%
1
1
H3C CH3
10
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OH
O
7
H3CO
piperidine
H
i-PrOH, reflux
16%
N
O
N
N
CH3
1
4
SP12
H3C CH3
CH3I
acetone
0%
H3CO
I
6
N
O
N
CH3
CH3
SP13
Scheme 3: Synthesis of spiropyrans SP10-SP14
Photochromic Analysis
Evaluation of response to UV and visible light irradiation was conducted in
absolute ethanol at 100 μM for all photoswitches. Samples were irradiated with UV for
1
5 minutes using an 8-Watt UV source; both 365 nm and 302 nm were tested, with 302
nm light produced the greatest changes in absorbance among spiropyrans and
spirooxazines (SI, Figure 103 and Figure 104). For the Job’s plot, different volumes of
1
00 μM solutions of photoswitch and Gd(NO3)3 in ethanol were mixed together to a
constant volume keeping the sum total of photoswitch and Gd(NO3)3 concentrations equal
to 100 μM. Visible light was supplied with one-minute irradiations by a 150-Watt
tungsten halogen lamp. The power output was stable from 500-800 nm (SI, Figure 99).
All photoswitching solutions were prepared under ambient light conditions and displayed
strong absorbance bands between 200 and 400 nm corresponding to the SP-form of the
photoswitches. Of the 30 compounds tested, only 10 photoswitches demonstrated an
observable shift to the open form upon UV light irradiation (Figure 2A). Five
photochromic compounds showed minor photoswitching (<0.1 A.U. SI, Figure 100 and
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Figure 101). The presence of the electron-withdrawing nitro substituent on the
chromene/naphthoxazine rings imparted the greatest UV response among photoswitches
with final absorbances of 1.88, and 1.76 A.U. for spiropyran SP4 (Figure 2A, blue) and
spirooxazine SO4 (Figure 2A, red) respectively. Spiropyrans SP13 (Figure 2A, light
blue) and SP6 (Figure 2A, green) were the next best at 1.12 and 0.70 A.U. respectively.
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Figure 2: A Photochemical properties of photoswitches SP4 (blue), SP6 (green), SP11
(purple), SP13 (light blue) and SO4 (red). B Kinetics of spiropyran SP4 at 100 μM in
ethanol. C Kinetics of spiropyran SP6 at 100 μM in ethanol. D Kinetics of spiropyran
SP13 at 100 μM in ethanol.
Under these assay conditions, none of the spiroindolinonaphthopyrans displayed a
UV response, however, previous literature suggests stronger UV sources could induce
spiroindolinonaphthopyrans isomerization.47 In the case of both the spirooxazines and
spiropyrans, the presence of the methoxy substituent on the indoline ring reduced the UV
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response of photoswitches. This was particularly noticeable between spiropyran SP4,
which had a maximal absorbance of 1.88 A.U. (Figure 2A, blue), and spiropyran SP6,
which achieved only 0.70 A.U. after 15 minutes of UV irradiation (Figure 2A, green).
Utilizing a stronger electron withdrawing “chromene ring” was capable of restoring some
of this UV responsiveness with spiropyrans SP13 giving a final absorbance of 1.12 A.U.,
an increase of 0.52 A.U. from spiropyrans SP6 after 15 minutes of UV irradiation (Figure
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A, light blue).
All UV-responsive photoswitches shown in Figure 2 could be reverted back to the
MC-form, to varying degrees, with visible light irradiation. The application of
photoswitches to in vivo light sensing requires sensitivity at potentially very low levels of
light. Three of the four best photoswitches from Figure 2A were further evaluated for
their low light sensitivity. Spiropyrans SP4, SP6 were exposed to decreasing levels of
light until no ring closing was observed (SI, Figure 97 and 98). Although spirooxazine
SO4 also had good UV response, spirooxazine photoswitches were not included in this
evaluation due to the inherent difficulty in their synthesis. The lower limit of sensitivity
for all three switches was 2.29 ± 0.03 mW. The kinetics of these photoswitches was
tested at this power level to determine the relative response of photoswitches upon low
light irradiation.
Absorbances of photoswitch solutions were tracked every minute for both UV and
low power visible light irradiation. Kinetic response times for spiropyrans SP4, SP6, and
SP13 were determined by fitting the decay curve of their visible light response with an
exponential function under the assumption of a first order decay process (Equation 1) to
find the rate of ring closing. Kinetics assays illustrated that the solutions of spiropyrans
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SP4, SP6, and SP13 were saturated after 10 minutes of UV irradiation, with a half-
maximal time of approximately 2.16, 2.33, and 0.85 minutes respectively (Figure 2B-D).
Spiropyran SP4 was the least stable undergoing thermal relaxation of 21% from the
maximum absorbance after a 10 minute dark incubation, and 30% decrease after 15
minute dark incubation (Two Tailed students t-test, p = 0.0086) (Figure 2C, green line).
In contrast, both spiropyrans SP6 and SP13 showed no significant decrease from
maximal absorbance after 10 minutes incubation in the dark (Figure 2B and 2D, green
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line). Of the three photoswitches, spiropyran SP6 had the fastest rate at 1.43 × 10 s (R
0.9788) with spiropyrans SP4 and SP13 an order of magnitude lower at 3.45 × 10-3 s-1
=
2
-3 -1
2
(
R = 0.9887) and 2.5 × 10 s (R = 0.9939) respectively (SI, Figures 31-33).
퐴(푡) = 퐴(0)푒−푘ꢀ
(1)
These three compounds highlight the effect that minor variations in substituents can
have on photoswitch properties. These results demonstrate again that the absolute
absorbance changes are greatest for the 6-Nitro-BIPS (SP4), but that addition of an
electron donating group speeds up the ring closing of SP6 by an order of magnitude
under low visible light irradiation. These assays also illustrate the importance of a
comparative study under identical conditions. While SP4 has long been considered the
best for UV light response, these kinetics assays demonstrate that SP13 reaches
saturation much faster and possesses greater thermal stability.
The kinetics results validate previous literature reports that nitro groups on the
chromene ring significantly enhance the UV response of photoswitches. These results
corroborate previous literature reports that increasing the C-O bond lability enhances
photoswitching. We also demonstrated that the UV response could be recovered to a
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certain degree by increasing the electron-withdrawing nature of the “chromene ring” as
shown with spiropyrans SP6 and SP13. The most important aspect of these visible light
assays was determining which substituents provided the best visible light response. From
these results we found that among compounds with nitro groups on the chromene ring,
having an electron donating substituent provided the best light response. In contrast, the
additional presence of a nitro group on the indoline ring shut down photoswitching
completely as was seen with spiropyrans SP2, SP5, and SP8.
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Table 1: Comparative analysis of different substituent combinations on the absolute
change in absorbance upon 15 minutes of UV irradiation.
Table 1 presents photoresponse as a function of the electron donating or withdrawing
capacity of the chromene substituent when the indoline methoxy is fixed. With the
methoxy group at the 5’ on the indoline side, there is a trend toward improved UV
response with strong electron withdrawing groups in the 6-position on the chromene side.
As seen in Table 1 the UV response seems to improve with the electron-withdrawing
capacity on the chromene side, and progressively increases with trifluoromethyl (8.9 ×
-
2
10 A.U.) , cyano (0.25 A.U.), and nitro groups (0.70 A.U.), respectively. There is very
little photoswitching in the absence of strong electron withdrawing groups on the
chromene side, and methoxy, hydrogen and dimethyl amino groups afford little to no
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improvement in photoresponse. This trend also holds for the switching response in the
reverse direction to visible light with trifluoromethyl, cyano, and nitro groups affording
increasing visible light response (SI, Figure 105). In contrast, if we fix the nitro group as
a strong electron withdrawing group on the chromene side there is no clear trend for
photoresponse, UV or visible, against increasing electron-donating ability for 5’
substituents on the indoline side (SI, Figure 106).
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Gadolinium(III) Responses
We are interested in the photoswitches as vehicles for modulating the behavior of
gadolinium-based contrast agents. In the MC-form, spiropyrans and spirooxazines have
been previously observed to coordinate to gadolinium(III) to alter the magnetic properties
of a gadolinium-chelate.26 It is currently hypothesized that in the MC-form, the
phenoxide of spiropyrans binds gadolinium(III) blocking a coordination site for water,
keeping the contrast agent in a low relaxivity state. Upon visible light irradiation the
spiropyran closes, removing this coordinated phenoxide and opening a coordination site
for water. Before the lengthy synthesis to couple the prepared photoswitches to
gadolinium-chelates, we wished to evaluate the capacity for these photoswitches to
interact with gadolinium(III). Ideal photoswitches would naturally open in the presence
of gadolinium(III) corresponding to a natural tendency to equilibrate to an “off” position
when conjugated to a gadolinium-chelate. Thus, all photoswitches were tested for
photoresponse in the presence of one equivalent of gadolinium(III) nitrate (Gd(NO3)3). A
solution of Gd(NO3)3 was added to photoswitch solutions prior to assays with a final
concentration of 1:1 photoswitch to Gd(NO3)3 (addition volumes were limited to 5 μL so
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concentration effects could be neglected). These assays were conducted in absolute
ethanol with the final concentration of both photoswitch and Gd(NO3)3 being 100 μM.
After addition of Gd(NO3)3, the solutions were allowed to equilibrate in the dark for 15
minutes to determine their gadolinium(III) ion response. The solutions were then
subjected to UV irradiation for 15 minutes, dark incubation for one minute, and visible
light irradiation for one minute with absorbance taken at each time point (Figure 3).
Several of the photoswitches showed sensitivity to the Gd(NO3)3 alone, isomerizing
to the MC-form upon incubation in the dark. The photoswitches with the greatest
absorbance after 15 minute incubations with Gd(NO3)3 were photoswitches SP3 (0.39
A.U., Fig 3A red), SP9 (0.51 A.U. Fig 3A orange), NP3 (0.29 A.U. Fig 3B red) NP9
(0.21 A.U.) (Fig 3B light blue) and, to a lesser degree, SP1 (0.15 A.U. Fig 3A blue). In
addition, the presence of Gd(NO3)3 induced a UV response for several photoswitches that
previously had shown no photoswitching including SP7 (Figure 3A, light blue), SP9
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(Figure 3A, orange), SP14 (Figure 3A, navy blue), NP3 and NP9 (Figure 3B).
Photoswitches that are not shown on Figure 3 had absolute responses of <0.1 A.U. (SI,
Figure 101).
The presence of Gd(NO3)3 diminished the UV response for the photoswitches SP4
(Figure 3A, light green), and SP6 (Figure 3A, purple) with final absorbances of 0.26 A.U.
for both compounds corresponding to decreases of 1.62 A.U. , and 0.44 A.U. from
previous assays in the absence of Gd(NO3)3. To verify that this was not a result of the
nitro groups coordinating the gadolinium(III) ion, reducing the free gadolinium(III) ion
concentration, spiropyran SP9 was incubated with p-methoxynitrobenzene and Gd(NO3)3
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(
1:1:1 mole ratio). The presence of nitrobenzene, or p-methoxynitrobenzene had no effect
on the Gd(NO3)3, UV, or visible light response of spiropyran SP9 (SI, Figure 95).
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Figure 3: A Photophysical properties of spiropyrans with one equivalent of Gd(NO3)3
added. B Photophysical properties of spiroindolinonaphthopyrans with one equivalent of
Gd(NO3)3 added.
Spiroindolinonaphthopyrans NP3, and NP9 rapidly isomerized to the open form
after one minute of Gd(NO3)3 incubation (Figure 3B). There was no significant change
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from one to 15 min of incubation with gadolinium(III) ions for
spiroindolinonaphthopyrans NP3 and NP9. Irradiation with UV light showed some
capacity for additional conversion to the MC-form for spiroindolinonaphthopyrans NP3,
and NP9. These results suggest a lack of visible light response for any of these
spiroindolinonaphthopyrans in the presence of gadolinium(III) ions. However, utilizing
shorter scan times (one second) we were able to demonstrate that
spiroindolinonaphthopyrans NP3 and NP9 were light-responsive, and after removing the
visible light source they rapidly equilibrated back to the MC-form (Figure 4B). For
gadolinium(III) ion sensitivity alone, methoxy substitutions on the indoline ring and
chromene ring were most effective for spiroindolinonaphthopyrans. However, none of the
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substitutions
tested
were
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produce
photoswitching
for
the
spiroindolinonaphthopyrans core in the absence of gadolinium(III) ions.
The photoswitches that were least responsive to Gd(NO3)3 were the spirooxazines
SO1-SO9, which showed no switching response to the presence of Gd(NO3)3. The
presence of the gadolinium(III) ions did, however, negatively impact UV response of
spirooxazine SO4 and final absorbance going from 1.76 A.U. in the absence of Gd(NO3)3
to 1.23 A.U. in its presence (SI, Figure 101). The combined results of these Gd(NO3)3
assays suggest that in the presence of gadolinium(III) ions the electron density of the
phenoxide ion is a more important factor for photoswitching than the lability of the spiro
C-O bond. The presence of a nitro group on the indoline ring eliminated photoswitching,
with spiropyrans SP2, SP5, and SP8 showing no UV response in the presence and
absence of gadolinium(III) ions. This implies that stabilizing the iminium ion is important
for photoswitching both in the presence and absence of gadolinium(III) ions.
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Figure 4: A UV response of spiropyran SP3 in the absence (blue) and presence (red) of 1
equivalent of Gd(NO3)3. B Visible light response of spiroindolinonaphthopyrans NP3 and
3
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One of the more surprising results for the spiropyrans was the inversion in UV
sensitivity of spiropyran SP3 in the presence of Gd(NO3)3 (Figures 4A, red). Without
Gd(NO3)3, spiropyran SP3 followed showed no significant decrease in absorbance after
UV irradiation (P > 0.05). However, addition of Gd(NO3)3 to spiropyran SP3 induces a
significant shift to the MC-form (0.40-0.48 A.U.) and upon UV irradiation instead of an
increase in the MC-form, as expected, the compound reverted back to the SP-form (0.15
A.U., Figure 4A, red) (p = 0.0001). While we currently do not have an explanation for
this change in photochromic properties, this will be explored further with additional
metal ion salts and computational studies. Spiropyran SP3 however, still follows full
conversion to the SP-form under visible light (0.07 A.U.) (Figure 4A) as normally
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expected and was slightly more responsive to visible light irradiation (k = 0.6909 M s )
-
1 -1
than SP9 (k = 0.6909 M s ) (SI, Figures 109 and 110). Both SP3 and SP9 based on their
low light responses and gadolinium(III) interactions are ideal candidates for gadolinium-
chelate conjugation and contrast evaluation.
The nature of the complex between spiropyran SP9 and Gd(NO3)3 was investigated
by the continuous variations method, Job’s plot analysis (Figure 4C). The absorption
values were taken at the absorbance maximum (481 nm) and plotted against Gd(NO3)3
concentration under the condition of an invariant total concentration. From this plot the
maximum molar fraction of 0.1 indicates a 1:9 stoichiometry of Gd(NO3)3 to SP9 in the
complex. This observation is quite unique to the stoichiometric ratio for the metal ion/SP
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derivative (2:1 and 1:1) reported previously, but this stoichiometric ratio does match the
known coordination chemistry of gadolinium(III), which is nine coordinate.49
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In contrast to our initial assays in the absence of the lanthanide, the Gd(NO3)3 assays
suggest EDGs on both ring systems would provide better photochromic properties when
conjugated to a gadolinium-based MRI contrast agent. The Gd(NO3)3 assays also suggest
that analyzing the photochromic behavior of switches alone in solution are not indicative
of the best possible photochromic properties when coordinated to a contrast agent.
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Conclusion
In summary, we have synthesized 30 spiropyrans, spiroindolinonaphthopyrans, and
spirooxazines and identified 10 compounds that demonstrate reversible response to
UV/visible irradiation under our experimental setup. As expected, the magnitude of
response to UV irradiation was found to be greater for spiropyrans and spirooxazines
containing nitro groups on the chromene and naphthoxazine rings respectively. However,
we were intrigued to find that increasing the electron donating character at the 6-position
of the indoline ring improved the rate of UV and Visible light responses. The addition of
an electron donating methoxy to the indoline side significantly improved rate of visible
light response, but there was no clear trend for degree of electron donating capacity on
the indoline side and the amplitude of the photoresponse. However, in the presence of
Gd(NO3)3 the behavior of photoswitches was significantly altered, where compounds
with EDGs on both ring systems showed greatest photochemical responses. Interestingly,
the direction of switching was reversed for spiropyran SP3 with a methoxy substituent on
the indoline ring. These results suggest that the behavior of photoswitches in the absence
of gadolinium(III) ions are not absolutely predictive for their performance in the presence
of gadolinium(III) ions, such as might be found when coupled to a gadolinium-chelate.
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Overall, EDGs tend to improve the thermal conversion to MC-form in the presence of
gadolinium(III) ions while maintaining the ability to isomerize to the SP-form under
visible light irradiation. From these gadolinium(III) assays two lead spiropyrans, SP3 and
SP9, were identified as ideal candidates for light activated contrast agent development.
This study highlights the importance of taking a comparative approach to evaluating
photoswitches. While several compounds in our studies exhibited no photochromic
activity, these compounds can be found in the literature demonstrating photochromism.
However, these results were obtained using extreme conditions of high temperature27a or
exceptionally high intensity light sources27b-d to observe photochromism. Unlike these
previous literature reports, we utilized irradiation sources and temperatures closer to what
one might use for biological systems.27 These results can be used to intelligently design
photoswitches for various applications through the optimization of UV and visible light
characteristics in the absence and/or presence of a metal. Future work will focus on
exploring the effect of various metal salts on the photochromic properties of this library
of spiropyrans, spiroindolinonaphthopyrans and spirooxazines in order to utilize the
results to develop novel, light sensitive contrast agents.
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Experimental Section
General experimental. All reagents were purchased from commercial sources and used
without further purification unless stated otherwise. Solvents were dried over an activated
alumina solvent system or purchased anhydrous where required. Reactions requiring
anhydrous conditions were performed under argon; glassware was flame dried under
vacuum immediately prior to use and allowed to cool under reduced pressure; liquid
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reagents, solutions or solvents were added via syringe through rubber septa; solid
reagents were added under a flow of argon. Reactions were monitored by TLC on Silica
Gel 60 F254, and detected by examination under UV light (254 nm and 365 nm). Flash
column chromatography was performed using silica gel [230−400 mesh (40−63 μm)],
unless otherwise stated. Microwave reactions were conducted in a Biotage® Initiator
Classic microwave reactor, using 2.45 GHz microwaves with temperatures measured
with an integrated IR sensor. Accurate mass measurements were recorded on positive ESI
mode in CH3OH or CH3CN. Extracts were concentrated in vacuo using both a rotary
evaporator at a pressure of 15 mmHg (diaphragm pump), and a high vacuum line at a
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pressure of 0.1 mmHg (oil pump) at room temperature. H and C spectra were measured
in the solvent stated at 400 or 600 MHz, and 101 or 151 MHz respectively. H and 13
1
C
NMR chemical shifts are quoted in parts per million (ppm) and referenced to the residual
1
13
1
solvent peak (CDCl3: H = 7.26 ppm and C = 77.2 ppm, DMSO-d6: H = 2.50 ppm and
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1
13
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C = 39.5 ppm, CD3CN: H = 1.94 ppm and C = 1.3 ppm, CD3OD: H = 3.31 ppm and
13C = 49.0 ppm), coupling constants (J) are given in Hertz (Hz). Multiplicities are
abbreviated as: br (broad), s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet)
or combinations thereof. UV irradiation (302 nm) was achieved with a UV viewing
cabinet with 8-Watt UV Lamp. Visible light irradiation was provided by a 150W tungsten
halogen lamp. Absorption spectra were recorded in 1.0 cm path length 0.7-mL quartz
cuvettes on a UV/Vis spectrophotometer. Light power was determined using a hand held
power meter with photodiode sensor. All lamps and spectrometers were allowed to warm
for 15 minutes prior to use to ensure consistent spectral output.
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-Methoxy-1,2,3,3-tetramethyl-3H-indol-1-ium iodide (7).31b Following the literature
procedure,50 to a solution containing 3-methylbutan-2-one (5.90 mL, 55.2 mmol) in
glacial acetic acid (88 mL) was added 4-methoxyphenylhydrazine hydrochloride (4.815
g, 27.57 mmol). The solution was stirred at reflux for 5.5 h. The solution was allowed to
cool to room temperature and neutralized with KOH pellets. The crude material was
extracted with Et2O (3 x 100 mL), dried over MgSO4, filtered, and concentrated in vacuo.
The crude product was purified by flash column chromatography (70:30,
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hexanes:EtOAc) to afford 2,3,3-trimethyl-5-methoxy-3H-indole as a red amorphous solid
1
(5.149 g, 99%). H NMR (600 MHz, CDCl3) δ 7.43 (d, J = 8.3 Hz, 1H), 6.84-6.80 (m,
1
2
H), 3.82 (s, 3H), 2.24 (s, 3H), 1.28 (s, 6H). H NMR spectrum is consistent with
published data.51 Following the literature procedure,31b to a solution of 2,3,3-trimethyl-5-
methoxy-3H-indole (0.133 g, 0.716 mmol) in anhydrous CH3CN (14.3 mL) was added
iodomethane (0.048 mL, 0.76 mmol). The solution was stirred at reflux for 21 h. The
solution was allowed to cool to room temperature, concentrated in vacuo, and suspended
in CHCl3 (2.5 mL) and hexanes (20 mL). The suspension was sonicated for 30 min and
1
filtered to afford indolium 7 as a pink amorphous solid (0.117 g, 49%). H NMR (600
MHz, CDCl3) δ 7.56 (d, J = 8.7 Hz, 1H), 7.07-7.01 (m, 2H), 4.23 (s, 3H), 3.90 (s, 3H),
.04 (s, 3H), 1.65 (s, 6H). H NMR spectrum is consistent with published data.31b
1
3
1,2,3,3-Tetramethyl-5-nitro-3H-indolium iodide (8).52 Following a modified literature
procedure,31a to a solution containing 3-methylbutan-2-one (10.16 mL, 94.91 mmol) in
glacial acetic acid (68 mL) were added 4-nitrophenylhydrazine (9.002 g, 47.50 mmol)
and concentrated H2SO4 (6.9 mL). The solution was stirred at reflux for 23 h, allowed to
cool to room temperature and then concentrated in vacuo. The crude material was
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purified by flash column chromatography (60:40, hexanes:EtOAc) to afford 2,3,3-
1
trimethyl-5-nitro-3H-indole as a light brown amorphous solid (4.515 g, 47%). H NMR
(600 MHz, CDCl3) δ 8.26 (dd, J = 8.5, 2.3 Hz 1H), 8.17 (d, J = 2.2 Hz, 1H), 7.62 (d, J =
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.5 Hz, 1H), 2.36 (s, 3H), 1.38 (s, 6H). H NMR spectrum is consistent with published
data.31a Following a modified literature procedure,31b to a solution of 2,3,3-trimethyl-5-
nitro-3H-indole (2.04 g, 10.0 mmol) in anhydrous CH3CN (50 mL) was added
iodomethane (1.25 mL, 20.1 mmol). The reaction mixture was stirred at reflux for 24 h.
The solution was concentrated in vacuo, dissolved in CHCl3 (15 mL) and hexanes (30
mL), and sonicated for 30 min. After filtration, indolium 8 was afforded as a light brown
1
amorphous solid (3.27 g, 95%). H NMR (400 MHz, CD3CN) δ 8.59 (d, J = 2.3 Hz, 1H),
8.50 (dd, J = 8.8, 2.2 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 3.98 (s, 3H), 2.78 (s, 3H), 1.62 (s,
H). H NMR was consistent with that reported in the literature.52b
1
6
2-Hydroxy-6-methoxynaphthalene-1-carbaldehyde (9).53 Following
a
literature
procedure,32 a solution of 2,6-dihydroxynapthalene (4.20 g, 26.2 mmol) in DMF (200
mL) was cooled to 0 °C and NaH (60% in mineral oil, 2.63 g, 65.8 mmol) was added in 5
portions. The solution was allowed to warm to room temperature and stirred for 30 min,
then cooled back to 0 °C and iodomethane (4.1 mL, 66 mmol) was added dropwise. The
reaction mixture was allowed to slowly warm to room temperature and stirred for 16 h.
The reaction mixture was quenched with CH3OH (1.0 mL), concentrated in vacuo, and
purified by flash column chromatography (95:5, hexanes:EtOAc) to afford 2,6-
1
dimethoxynapthalene as a white amorphous solid (3.91 g, 80%). H NMR (600 MHz,
1
CDCl3) δ 7.65 (d, J = 8.9 Hz, 2H), 7.17-7.08 (m, 4H), 3.91 (s, 6H). H NMR was
consistent with that reported in the literature.54 Following a literature procedure,33
a
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solution of N-methylformanilide (3.2 mL, 26 mmol), 2,6-dimethoxynapthalene (4.40 g,
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3.4 mmol) and POCl3 (2.4 mL, 26 mmol) was stirred at 100 °C for 18 h. The solution
was added to DMF (50 mL) and the resulting solution was added to cold 1 M HCl (200
mL), with vigorous stirring. The resulting precipitate was filtered and purified by flash
column chromatography (50:50 to 30:70, hexanes:CH2Cl2) afforded 2,6-dimethoxy-1-
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naphthalenecarbaldehyde as a yellow amorphous solid (3.25 g, 64%). H NMR (600
MHz, CDCl3) δ 10.88 (s, 1H), 9.22 (d, J = 9.4 Hz, 1H), 7.95 (d, J = 9.1 Hz, 1H), 7.31
(dd, J = 9.4, 2.8 Hz, 1H), 7.27 (d, J = 9.1 Hz, 1H), 7.09 (d, J = 2.8 Hz, 1H), 4.03 (s, 3H),
1
.92 (s, 3H). H NMR was consistent with that reported in the literature.33 2,6-
3
Dimethoxy-1-naphthalenecarbaldehyde (3.24 g, 15.0 mmol), MgBr2 (5.52 g, 30.0 mmol),
NaI (4.49 g, 30.0 mmol), and CH3CN (100 mL) were added to a reaction vessel that was
sealed and the stirred at 150 °C for 2 h. The resulting solution was added to CH2Cl2 (60
mL), washed with 1 M HCl (20 mL), H2O (60 mL), dried over Na2SO4, and filtered. The
filtrate was concentrated in vacuo and the residue was purified by flash column
chromatography (60:40, hexanes:CH2Cl2) to afford the desired carbaldehyde 9 as a
1
yellow amorphous solid (2.91 g, 96%). H NMR (400 MHz, CDCl3) δ 12.79 (s, 1H),
1
1
0.78 (s, 1H), 8.27 (d, J = 9.2 Hz, 1H), 7.90 (d, J = 9.0 Hz, 1H), 7.29 (dd, J = 9.2, 2.4 Hz,
1
H), 7.16-7.12 (m, 2H), 3.92 (s, 3H). H NMR was consistent with that reported in the
literature.53
-Hydroxy-6-nitro-1-naphthaldehyde (10).34 Following a modified literature procedure,34
2
to an aqueous solution of 85% nitric acid (10 mL) at -5 °C, 2-hydroxy-1-naphthaldehyde
0.994 g, 5.77 mmol) was added in small portions over 10 min. The mixture was stirred
for another 10 min at -5 °C before being poured over ice (20 g). The precipitate was
(
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filtered, washed with water and dried, followed by recrystallization from hexanes:EtOAc
(10:1) to afford the desired naphthaldehyde 10 as a pale yellow powder (0.459 g, 37%).
1H NMR (600 MHz, DMSO-d6) δ 12.30 (s, 1H), 10.74 (s, 1H), 9.14 (d, J = 9.7 Hz, 1H),
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.88 (d, J = 2.0 Hz, 1H), 8.40 (d, J = 9.3 Hz, 1H), 8.30 (dd, J = 9.5, 2.4 Hz, 1H), 7.41 (d,
J = 9.0 Hz, 1H); 13C NMR (151 MHz, DMSO-d6) δ 191.8, 166.5, 143.2, 139.5, 135.0,
1
1
26.4, 125.0, 124.4, 122.3, 121.1, 113.0. H NMR was consistent with that reported in the
literature.34
6-Methoxy-1-nitrosonaphthalen-2-ol (11).35 Following the reported literature,35 6-
methoxy-2-naphthol (1.125 g, 6.458 mmol) was dissolved in a mixture of AcOH (5.0
mL) and water (10 mL) before being cooled to 0 °C. NaNO2 (0.453 g, 6.57 mmol) was
added as a amorphous solid and stirred for 15 min at 0 °C before the solution was filtered
and washed with cold water. The product was dried in vacuo to afford the desired
nitrosonaphthalenol 11 as a dark brown amorphous solid (0.664 g, 51%, ~80% purity).
1H NMR (600 MHz, CDCl3) δ 8.24 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 9.8 Hz, 1H), 7.09 (dd,
J = 8.8, 2.6 Hz, 1H), 6.93 (d, J = 2.6 Hz, 1H), 6.57 (d, J = 9.8 Hz, 1H), 3.89 (s, 3H)
(hydroxyl proton not observed); 13C NMR (151 MHz, CDCl3) δ 183.2, 160.8, 147.7,
13
1
44.7, 130.0, 126.5, 125.1, 123.7, 117.9, 113.4, 55.7 ( C NMR has been tentatively
assigned due to a purity of ~80%. The compound could not be purified further as it is
known to readily degrade under recrystallization conditions.);35 HRMS (ESI) m/z calcd
+
+
for C11H10NO3 (M + H) 204.0655, found 204.0662.
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-Hydroxy-5-(trifluoromethyl)benzaldehyde (12).44b Following a modified literature
procedure,55 to a solution of p-trifluoromethylphenol (0.069 g, 5.4 mmol) in TFA (10.7
mL), was added hexamethylenetetramine (0.751 g, 5.36 mmol) under argon. The reaction
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mixture was stirred at reflux under argon for 15 h. The solution was allowed to cool to
room temperature and the solvent was removed in vacuo. The crude mixture was
dissolved in CH2Cl2 (5.0 mL) and was diluted with water (5.0 mL). The aqueous phase
was acidified with concentrated HCl (1.2 mL) and extracted with CH2Cl2 (2 × 25 mL).
The combined organic layers were washed with 3.5 M HCl (10.0 mL), saturated aqueous
NaHCO3 (10.0 mL) and water (10.0 mL). The organic layer was dried over MgSO4,
filtered, and concentrated in vacuo. The crude amorphous solid was purified by flash
column chromatography (98:2, hexanes:EtOAc) to afford the desired benzaldehyde 12 as
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a white amorphous solid (0.311 g, 31%). H NMR (600 MHz, CDCl3) δ 11.31 (s, 1H),
9
.96 (s, 1H), 7.87 (d, J = 1.6 Hz, 1H), 7.76 (d, J = 8.8, 1.6 Hz, 1H), 7.11 (d, J = 8.7 Hz,
1H). H NMR spectrum is consistent with published data.44b
1
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-Formyl-4-hydroxybenzonitrile (13).44a Following a modified literature procedure,55 to a
solution of 4-cyanophenol (0.200 g, 1.68 mmol) in TFA (1.7 mL), was added
hexamethylenetetramine (0.471 g, 3.36 mmol) under argon. The reaction mixture was
stirred at reflux for 5 h. The solution was cooled to 0 °C, followed by the addition of 1 M
HCl (11 mL) and stirred for 30 min. The aqueous solution was extracted with of CH2Cl2
(3 × 25 mL) and the organic layers were combined, dried over MgSO4, filtered, and
concentrated in vacuo. The crude amorphous solid was purified by flash column
chromatography (85:15, hexanes:EtOAc) to afford the desired benzonitrile 13 as a white
1
amorphous solid (0.016 g, 17%). H NMR (600 MHz, CDCl3) δ 11.45 (s, 1H), 9.93 (s,
1
1
H), 7.94 (d, J = 1.8 Hz, 1H), 7.78 (dd, J = 8.7, 1.9 Hz, 1H), 7.11 (d, J = 8.7 Hz, 1H). H
NMR spectrum is consistent with published data.44a
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-Hydroxynicotinaldehyde (14). Following the literature procedure, to a round bottom
flask containing 4-chloronicotinaldehyde (0.092 g, 0.70 mmol) was added 3 M HCl (1.0
mL) and two drops of 30% H2O2. The reaction mixture was stirred at reflux for 17.5 h
before being allowed to cool to room temperature, neutralized with Na2CO3, and
concentrated in vacuo. The crude product mixture was suspended in EtOH (10.0 mL) and
filtered. The filtrate was concentrated in vacuo and purified by flash column
chromatography (9:1, CH2Cl2:CH3OH) to afford the desired nicotinaldehyde 14 as a
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white amorphous solid (0.083 g, 96%). H NMR (600 MHz, DMSO-d6) δ 11.91 (br s,
1
7
H), 10.09 (s, 1H), 8.15 (d, J = 1.1 Hz, 1H), 7.70 (dd, J = 7.6, 1.1 Hz, 1H), 6.36 (d, J =
.5 Hz, 1H). H NMR spectrum is consistent with published data.45
1
5-Dimethylamino-2-hydroxy-benzaldehyde (15).46 Following the reported literature
procedure,46 EtOH (30 mL) and aqueous formaldehyde solution (37%, 3.8 mL) were
added to a flask containing 2-hydroxy-5-nitrobenzaldehyde (0.160 g, 0.957 mmol) and
Pd/C (20 wt%, 0.098 g). The solution was purged with argon and then H2. The mixture
was then stirred under a balloon of H2 for 18 h at room temperature. Additional aqueous
formaldehyde solution (37%, 2.0 mL) was added, and the mixture was again purged with
argon and H2, and then stirred for an additional 24 h under a balloon of H2. The mixture
was filtered through Celite and the filtrate was acidified with 1 M HCl (15 mL) and
concentrated in vacuo. The residue was neutralized with saturated aqueous NaHCO3 and
extracted with CH2Cl2 (3 x 35 mL). The combined organic extracts were dried over
Na2SO4 concentrated in vacuo. Purification by flash chromatography (8:2,
1
hexanes:EtOAc) afforded aldehyde 15 as a red oil (0.117 g, 74%). H NMR (600 MHz,
CDCl3) δ 10.45 (s, 1H), 9.86 (s, 1H), 7.08 (dd, J = 9.0, 3.1 Hz, 1H), 6.91 (d, J = 9.0 Hz,
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