Synthesis of phenanthridine spiropyrans and studies of their effects on G-quadruplex DNA

Article abstract of DOI:10.1039/C7OB00300E

G-quadruplex (G4) DNA structures are involved in many important biological processes and can be linked to several human diseases. Drug-like low molecular weight compounds that target G4 structures are therefore interesting not only for their potential therapeutic properties but also for their potential use as chemical research tools. We report here on the development of methods to synthesize spiropyrans using a condensation-cyclisation reaction of quaternary salts of α-methyl quinoline or phenanthridine with salicylaldehydes. Evaluation of the synthesized phenanthridine spiropyrans' interactions with G4 DNA was performed with a Thioflavin T displacement assay, circular dichroism, Taq DNA polymerase stop assay, and NMR. This revealed that the substitution pattern on the phenanthridine spiropyrans was very important for their ability to bind and stabilize G4 structures. Some of the synthesized low molecular weight spirocyclic compounds efficiently stabilized G4 structures without inducing structural changes by binding the first G-tetrad in the G4 structure.

Full text of DOI:10.1039/C7OB00300E

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
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PAPER
Synthesis of phenanthiridine spiropyrans and studies of their
effects on G-quadruplex DNA
M. Livendahl,a J. Jamroskovic,b M. Hedenström,a T. Görlich,a N. Sabourib* and E. Chorella*
Received 00th January 20xx,
Accepted 00th January 20xx
G-quadruplex (G4) DNA structures are involved in many important biological processses and can be linked to several
human diseases. Drug-like low molecular weight compounds that target G4 structures are therefore interesting for their
potential therapeutic properties but also for their potential use as chemical research tools. We report here on the
development of methods to synthesize spiropyrans using a condensation-cyclisation reaction of quaternary salts of α-
methyl quinoline or phenanthridine with salicylaldehydes. Evaluation of the synthesized phenanthridine spiropyrans
interactions with G4 DNA was performed with a ThT displacement assay, circular dichroism (CD), Taq DNA polymerase
stop assay, and NMR. This revealed that the substitution pattern on the phenanthridine spiropyrans was very important
for their ability to bind and stabilize G4 structures. Some of the synthesized low molecular weight spirocyclic compounds
efficiently stabilized G4 structures without inducing structural changes by binding the first G-tetrad in the G4 structure.
DOI: 10.1039/x0xx00000x
www.rsc.org/
structures have great potential as drug candidates and as
6
chemical research tools.4c, We recently performed a high-
Introduction
throughput screen (HTS) with over 28,000 compounds, which
Guanine-rich sequences of single stranded DNA or RNA can
fold into secondary DNA structures called G-quadruplex (G4)
structures. The G4 structures are composed of two or more
stacks of guanines (G-tetrads) that align on top of each other.
Each G-tetrad is composed of four guanines that interact
through Hoogsteen hydrogen bonds,1 and are further
stabilized by a central monovalent cation, such as potassium or
sodium.2 Although this central arrangement is similar for all G4
structures, their overall structure can differ significantly, as the
structures can form inter- or intra-molecularly, and comprise
of different number of guanine stacks. Also the nucleotides
bridging the central guanines (known as loops) can differ, and
the sequences that build up the G4 structure can be in parallel,
antiparallel, or hybrid orientations.3
The evolutionary conserved genomic locations of many G4
sequences suggest that they are involved in biological
functions, such as gene regulation, DNA replication, and
telomere maintenance.4 G4 sequences are also associated to
cancer and a variety of other human diseases.5 The number of
sequences with potential to form a G4 structure in the human
genome is immense, but which sequences that form a G4
structure in vivo and their biological functions are still not fully
elucidated.
identified new G4-binding small molecules.7 Among these
structures was a phenanthridine based spiropyran compound
(7, Table 1). Spirocycles are a common moiety in many natural
products and frequently appear in drug development.8
Spirocyclic compounds are structurally interesting as they have
an intrinsic three-dimensional feature, which is beneficial
compared to the many flat aromatic compounds used as lead
compounds in medicinal chemistry in general,9 and as G4
stabilizing compounds in particular.4c, 10 Here, we report the
development of synthetic methods to phenanthridine
spiropyrans that were used to synthesize over 20 analogues.
The abilities of these low molecular weight spirocyclic
compounds to interact with G4 structures were evaluated in
orthogonal assays. This not only revealed compounds with
high binding and stabilization efficiencies, but also information
about how the compounds bind to the G4 structure.
Results and discussion
Synthetic procedures
A previous report on the synthesis of these types of central
fragments inspired us to begin our investigation by developing
a general reaction between quaternary salts of α-methyl
quinoline and phenanthridine in a condensation, cyclisation
reaction with different salicylaldehydes.11
Low molecular weight compounds that target G4
To probe the viability of the synthetic steps we initially
made the triflate salt of 1,2-dimethylisoquinolin-2-ium (1) by
methylation of commercially available 1-methylisoquinoline
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Table 1. Synthesis of spiropyransa
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DOI: 10.1039/C7OB00300E
OH
O
OH
O
OTf
N
OTf
X
N
N
R
N
N
, piperidine
O
MeOTf
, piperidine
O
X
R
DCE, rt, 89%
MeOH, 80 °C 2h,
77%
MeOH, 80 °C, 2h
1
2
5
6-28
O
OTf
N
O
poly-phosphoric acid
140 °C, 2.5 h, 85%
MeOTf
Br
, LiOH
N
NH2
N
H
DCE, rt, 89%
DCM, 0 °C-rt,
2h, 93%
OMe
N
N
N
N
O
O
O
O
3
4
5
OMe
Me
Scheme 1. Synthesis of spiropyran 2 and phenanthridinium salt 5
Br
6 (74%)
7, 8-OMe (69%)
8, 7-OMe (71%)
9, 6-OMe (52%)
10, 5-OMe (52%)
11, 8-Me (70%)
12, 6-Me (77%)
13 (74%)
and reacted it with salicylaldehyde. This led to the isolation of
spiropyran 2 in 77% yield (Scheme 1). Inspired by this, we
started to develop the synthesis of the triflate salt of 5,6-
dimethylphenanthridin-5-ium (5), as this was structurally more
closely related to our hit compound. The synthesis was
accomplished in three steps starting with acetylation of
commercially available 2-aminobiphenyl to give 3 followed by
a Bischler-Napieralski reaction generating the phenanthridine
core in 4. The nitrogen was then easily methylated using
methyl triflate at room temperature resulting in the desired
quarternary salt 5 (Scheme 1).
R
R1
N
N
N
O
N
O
O
O
N
R2
R
16, R1, R2 = Br (61%)
17, R1, R2 = Cl (85%)
14, R = OH (66%)
15, R = Et (87%)
18, R = F (76%)
19, R = Br (71%)
20, R = tBu (72%)
21, R = Ph (78%)
26 (52%)
22, R = NEt2 (11%)
N
O
N
O
23, R = COOMe (77%)
24, R = I (70%)
25, R = COOH (30%)
O
NH
28 (56%)
27 (81%)
O
The final spiropyran products were obtained through a
one-pot condensation, cyclisation reaction between triflate
salt 5 and different salicylaldehydes (Table 1, top). Key to the
success of the reaction proved to be the portion-wise addition
of the triflate salt to drive the complete consumption of the
salicylaldehyde. In almost all cases the product precipitated
out of the reaction, driving the equilibrium of the reaction
towards product. The products were isolated in high yields
from the reaction mixtures through filtration. By using this
method, over 20 phenanthiridine spiropyrans were
synthesized with both mono- and di-substitutions in different
positions on the benzopyran ring system. A wide range of
substituents proved to be tolerated in the reaction such as
halides, tert-butyl, phenyl, methoxy, amide, methyl ester, and
even a carboxylic acid substituent, albeit the latter resulted in
a lower yield (Table 1).
In the cases where no precipitation of the product
occurred, for example in the synthesis of phenanthridine
spiropyrans 14, 22, and 26, the isolation was more
complicated because of the formation of ring-opened
spiropyran merocyanine forms, which also resulted in lower
yields. The ring-opening to the merocyanine form was not
induced by near UV light but seem to be substrate dependent.
These synthetic methods, both for the triflate salt 5 as well
as the following spiropyran formation, present efficient routes
towards these types of scaffolds. The routes are amenable to a
high degree of variation both in the salicylaldehyde part as
well as in the quaternary salts.
O
a Reaction conditions: Phenanthridinium salt 5 (1 equiv.) was added portion-wise
to salicylaldehydes (1 equiv.) and piperidine (1 equiv.) in refluxing methanol (0.1
M) over 2h. Trace amounts of ring-opened merocyanine forms are present in 14
and 22.
Compound evaluation
To investigate the compounds abilities to bind G4
structures, we used a Thioflavin T (ThT) displacement assay
(see supplemental information).7, 12 Using this assay we tested
binding of the different analogues to a known G4 structure
forming oligonucleotide, i.e., the ribosomal DNA (rDNA)
sequence from Schizosaccharomyces pombe (Figure 1A), which
forms a parallel-stranded G4 structure.13 To calculate the half
maximum displacement concentration (DC50), the compounds
were tested at 16 different concentrations ranging from 0.19
nM to 50 μM (Figure 1B, top).
The structural variations in the synthesized analogues
resulted in large variations in their activity in this assay and
several compounds demonstrated similar or even improved
activities compared to the original hit structure (7). The most
active compound in the series was the 7-methoxy substituted
phenanthiridine spiropyran 8 with a DC50 value of 0.2 μM. The
following regioisomer, the 6-methoxy analogue 9, had a
slightly higher DC50 value, about two-fold, but was still
submicromolar. Interestingly the following regioisomer, the 5-
methoxy analogue 10, showed almost 11-fold reduced binding
compared to the hit structure with a DC50 value of 3.3 μM. The
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Figure 1. ThT displacement assay. (A) Oligonucleotide templates used in the ThT
displacement assay. (B) DC50 values of ThT displacement (μM) with DNA
a
concentration of 1 μM. *Estimated DC50 values as the ThT signal was not completely
reduced at the highest compound concentrations.
positioning of the substituents on the benzopyran ring system
is thus of key importance for the activity. Furthermore, the
methoxy substituent is preferred over the structurally related
8-methyl substituted analogue 11, 8-hydroxy analogue 14, and
8-ethyl substituted analogue 15, which have about 2-, 4-, and
5-fold less efficient binding than 7, respectively.
Figure 2. The melting temperature is increased in the presence of phenathridine
spiropyran compounds. (A) CD spectra of 5 µM rDNA supplemented with 1.25% (v/v)
DMSO and 80 µM compounds at 25 °C. (B and C) Melting curves of the same samples
were recorded at 264 nm at temperature range 25-90 °C. Tm corresponds to the
estimated value when 50% of the DNA is melted. Tm of the rDNA treated with 1.25%
(v/v) DMSO is 68 °C. ∆Tm describes Tm difference between Tm of the DNA treated with
compounds compared to DMSO. All the data are shown in °C. The reason to the initial
increase in ellipticity is examined in supporting figure S2.
Generally, the most efficient binders in this assay have
small substituents, such as methyl, methoxy or fluorine in
position 6, 7, or 8 on the benzopyran ring system. Larger
substituents such as the 6-tert-butyl in analogue 20, or the 6-
phenyl in analogue 21 showed significantly reduced activity,
with DC50 values over 10 μM. Methyl ester 23, benzoic acid 25
and amide 28 all had fairly high DC50 values ranging from
around 5 to over 10 μM whereas the 6-diethylamino analogue
22 had a DC50 value of around 1 μM. The pyridine analogue 26
was not very active in this assay, with a DC50 value of around 7
μM, potentially because of the difference in electron density
and basicity induced by the heteroaromatic ring. The addition
of a second 5-bromo-substituent to the benzopyran ring
system of phenathridine spiropyran 7, as in the 8-methoxy-5-
bromo substituted analogue 13, reduced the activity and
resulted in a DC50 value of 1.1 μM. The 8,6-dibromo and 8,6-
dichloro substitutions in analogues 16 and 17 resulted in DC50
values of 3.1 μM and 1.2 μM, respectively.
Additionally, we also performed the ThT displacement
assay with the Pu24T c-MYC promoter sequence (Figure 1A),14
which also forms a parallel-stranded G4 structure, with our
seven best binders 6, 7, 8, 9, 11, 12 and 18. Also with this G4
structure, the methoxy substituent (as in 7, 8, and 9) proved to
be highly important for a low DC50 value as well as the
positioning of the substituents in position 7 or 8 on the
benzopyran ring system (compare phenanthridine spiropyran 7
versus 9 and 11 versus 12 in Figure 1B, bottom). Overall, the
compounds’ binding efficiency for this G4 structure were
slightly lower than with the rDNA G4 structure, but showed
the same relative trend.
ThT displacement assay 6, 7, 8, 9, 11, 12, and 18 were
evaluated with the rDNA G4 structure at 8:1 (compound:DNA)
molar ratios (Figure 2A). None of the tested compounds
induced any significant structural change in the G4 structure,
which is beneficial from a research tool perspective and is also
in contrast to the large structural changes induced by many of
the most frequently used G-quadruplex stabilizing compounds
today.15 The absorbance spectra for compounds 6, 7, 8, 9, 11,
12, and 18 are displayed in supporting information figure S1.
Further, the ability of these compounds to stabilize the
rDNA G4 structure was also studied by using CD spectroscopy
and stepwise increasing the temperature (Figure 2B and C). All
of the tested phenanthridine spiropyrans stabilized the rDNA
G4 structure. Analogues 7 and 8 resulted in the strongest
stabilization with an increased estimated G4 structure melting
temperature (Tm) of +17 °C. In accordance with the
observations in the ThT displacement assay, moving or
replacing the methoxy substituent in 7 and 8 substantially
reduced the G4 stabilization capacity of the compounds. The 6-
methoxy analogue 9 increased the Tm with +11 °C compared to
+17 °C for both the 7- and 8-methoxy analogues 7 and 8.
Replacing the methoxy substituent with a methyl group as in
11 and 12 or with a fluorine as in 18 increased the G4
stabilization with +8 °C, +13 °C, and +7 °C, respectively.
Removing the methoxy substituent as in analogue 6 reduced
the G4 stabilization from +17 °C to +9 °C. Some of the
compounds resulted in an initial increase in ellipticity. This
maybe due to the existence of both a parallel and antiparallel
structural motifs in the rDNA G4 structure, which unfolds and
folds differently in the presence of the compounds (supporting
information figure S2).
To investigate if the compounds induce a structural change
of the G4 structure upon binding, circular dichroism (CD)
spectroscopy was used. The most active compounds from the
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F
The effect of the changes in the benzopyran ring system of rDNA G4 template or a non-G4 template from S. pombe ade6+
the compounds was also examined by the Taq DNA gene as a control (Figure 3A).
polymerase stop assay (Figure 3). In this assay, stabilization of
First, we examined the effect of the compounds on the
the DNA results in an enhanced pausing of the DNA primer extension of non-G4 DNA. We observed similar
polymerase, which result in a reduced amount of full-length increase of the full product in all the samples, treated either
product (measured after 0.5, 1, 5, and 10 minutes reactions). with compounds 7, 8, 20, 27, and 28 or DMSO (Figure 3B and
We used a 5’ fluorescently labeled primer annealed to the D). An increased termination probability in the early time
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points (0.5 and 1 min) was observed with compound 8 but We have developed synthetic methods to phenanthridine
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these changes were not present in the later time points (5 and spiropyrans that are applicable to a wideDrOaIn: g10e.1o0f39s/uCb7sOtBit0u0e3n0t0sE.
10 min).
The compounds ability to bind and stabilize G4 structures
Next, we examined if the compounds stabilized the rDNA proved to be highly dependent on the substitution pattern on
G4 structure and thus blocked DNA synthesis. If so, the the spiropyrans. Methoxy substituents in position 7 or 8 in the
amount of full-length product should be significantly benzopyran ring system resulted in efficient G4 structure
decreased and most of the signal should be detected at the stabilization without induction of structural changes, by
pausing sites before the G-tracts. Phenanthridine spiropyrans 7 binding to the first G-tetrad in the G4 structure. The acquired
and 8 significantly stabilized the rDNA G4 structure and binding information and structure activity relationships is of
reduced the amount of full-length product almost three-fold importance for future optimizations of this low molecular
after 10 min incubation (Figure 3C and E). Pausing by the DNA weight spirocyclic class of compounds and also for their use
polymerase caused by 7 and 8 was observed two nucleotides and further development as chemical research tools.
upstream of the first and second G-tract (Figure 2, arrow a and
b). In addition, the small structural difference between 7 and 8
resulted in the enrichment of another pausing site for 8, two
Experimental
nucleotides upstream of another G-tract, containing only two
guanines, instead of three guanines (Figure 3C, arrow c).
Synthesis
General Synthesis. Unless stated otherwise, all reagents and
solvents were used as received from commercial suppliers. 3-
Ethyl-2-hydroxybenzaldehyde was prepared according to a
previously published procedure.17 TLC was performed on
aluminum backed silica gel plates (median pore size 60 Å,
fluorescent indicator 254 nm) and detected with UV light.
Flash column chromatography was performed using silica gel
with an average particle diameter 50 μm (range 40−65 μm,
pore diameter 53 Å), and eluents are given in brackets. DMF,
THF and DCM were dried in a solvent drying system (THF and
DCM drying agent: neutral alumina ; DMF drying agent:
activated molecular sieves, also equipped with an isocyanate
scrubber) and were collected fresh prior to every reaction. 1H
and 13C NMR spectra were recorded on a Bruker 400 MHz
spectrometer at 298 K or on a Bruker 600 MHz spectrometer
at 298 K, and calibrated by using the residual peak of the
solvents as the internal standard (CDCl3: δ H= 7.26 ppm; δ C =
77.16 ppm. DMSO-d6: δ H = 2.50 ppm; δ C = 39.50 ppm). LC-
MS was conducted on an Agilent 6100 Series Quadropole
LC/MSD system . HRMS was performed by using a Agilent 1290
binary LC System connected to a Agilent 6230 Accurate-Mass
TOF LC/MS (ESI+); calibrated with Agilent G1969-85001 ES-TOF
Reference Mix containing ammonium trifluoroacetate, purine
and hexakis(1H, 1H, 3H-tetrafluoropropoxy)phosphazine in
90:10 acetonitrile:water. Preparatory HPLC was performed on
a C18 reversed-phase column (25 cm x 21.2 mm, 5 mm) with
H2O/MeCN mixtures as the eluent.
Pausing at this site was also detected in an earlier study and
may suggest that this region also is part of the G4 structure.7
As expected, the phenanthridine spiropyrans 20, 27, and 28
did not display any large stabilization of the G4 structure,
which is in alignment with the DC50 values (Figure 1B).
NMR analysis
To understand how the compunds interact with G4 DNA
1
we evaluated compounds 7 and 8 by using H-NMR. Spectra
were recorded on 100 µM folded Pu24T c-MYC G4 structure
(that also was used in the ThT assay, Figure 1B, bottom) in
absence and presence of equimolar amounts of either 7 or 8.
Analysis of the imino-region of the spectra showed line-
broadening of most guanine imino peaks in the presence of
both 7 and 8 (Figure 4). In particular, the signals from the
guanine bases in positions 4, 8, 13, and 17 were broadened to
such a degree that they disappeared in the baseline. These
guanine bases form the first G-tetrad in this particular G4
structure, which indicate that the top tetrad is involved
directly in the binding of 7 and 8. Similar effects have been
reported in previous studies of small molecules binding to
Pu24T c-MYC G4 DNA.14, 16
Abbreviations. DMF – dimethylformamide, MeOH – methanol,
THF - tetrahydrofuran, EtOAc – ethyl acetate, TFA – trifluoro
acetic acid, DCE – dichloroethane, DCM – dichloromethane,
MeCN – acetonitrile, DMAP – dimethylaminopyridine, TLC –
thin layer chromatography, HPLC – high pressure liquid
Figure 4. The imino-region of 1H spectra recorded for Pu24T c-MYC G4 DNA in absence
(bottom) and presence of compound 7 (middle) and 8 (top). Imino protons from
guanine bases in the top tetrad are marked with numbers and dashed lines.
chromatography, LCMS
– liquid chromatography mass
spectrometry, rt – room temperature
1,2-dimethylisoquinolin-2-ium trifluoromethanesulfonate (1):
Methyltrifluoromethansulfonate (307 µl, 2.71 mmol) was
added to
a solution of 1-methylisoquinoline (360 µl,
Conclusions
2.71 mmol) in 1,2-dichloroethene (5 ml). The reaction mixture
was stirred at rt for 5 h. A white solid precipitated from the
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solution. After filtering off the solid, it was washed with cold solution was then extracted with DCM and the organic phase
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diethyl ether (0 °C) and dried under reduced pressure. The was dried with sodium sulfate. EvapoDraOtIi:o1n0.10o3f9/tCh7eOBs0o0lv30e0nEt
product was isolated as a white solid in 87 % yield (722 mg, under reduced pressure gave the product as a white solid in
2.35 mmol) 1H-NMR (400 MHz, DMSO-d6): δ = 8.77 (d, J = 87 % yield (165 mg, 854 mmol). 1H-NMR (400 MHz, CDCl3):
8.7 Hz, 1H), 8.68 (d, J = 6.9 Hz, 1H), 8.38 (d, J = 6.9 Hz, 1H), 8.29 δ = 8.64 (d, J = 8.3 Hz, 1H), 8.55 (d, J = 8.1 Hz, 1H), 8.23 (d, J =
(d, J = 8.1 Hz, 1H), 8.21 (t, J = 7.1 Hz, 1H), 8.04 (ddd, J = 8.4, 6.9, 8.2 Hz, 1H), 8.10 (d, J = 8.1 Hz, 1H), 7.85 (ddd, J = 8.3 Hz, 7.1,
1.3 Hz, 1H), 4.38 (s, 3H), 3.21 (s, 3H). 13C-NMR (100 MHz, 1.3 Hz, 1H), 7.71 (m, 2H), 7.63 (ddd, J = 8.3, 7.1, 1.3 Hz, 1H),
DMSO-d6): δ = 160.6, 136.6, 136.5, 135.8, 130.9, 128.4, 127.9, 3.06 (s, 3H). 13C-NMR (100 MHz, CDCl3): δ = 159.0, 143.8,
127.1, 123.4, 121.1 (JC-F = 321 Hz), 46.4, 16.8. LCMS: Calculated 132.7, 130.6, 129.5, 128.8, 127.5, 126.7, 126.5, 126.1, 123.9,
M+ (cationic part only) (158); Found (M+) 158 m/z, Rt = 1.29 122.5, 122.1, 23.6. LCMS: Calculated MH+ (194); Found (MH+)
min.
194 m/z, Rt = 3.91 min.
2'-Methyl-3,8a-dihydro-2'H-spiro[chromene-2,1'-
5,6-dimethylphenanthridin-5-ium trifluoromethanesulfonate
isoquinoline] (2) (ECH23): Made using the general procedure (5): 4 (719 mg, 3.72 mmol) was dissolved in DCM (40 mL) at rt.
(vide infra) from 1 (100 mg, 0.33 mmol) and salicylaldehyde Methyl trifluoromethanesulfonate (505 μL, 4.47 mmol) was
(34 µl, 0.33 mmol).The crude mixture was concentrated and then added dropwise and the reaction was left stirring at rt
purified by preparative HPLC using MeCN:water with 0.75% over night. The formed precipitate was filtered off, washed
formic acid as eluent, giving the open zwitterionic with Et2O and dried under vacuum. The product was isolated
1
intermediate after condensation with the aldehyde. The in 89% yield (1.18 g, 3.30 mmol) as white needles. H NMR
isolated intermediate was then dissolved in DMSO and sodium (400 MHz, DMSO-d6) δ 9.14 (d, J = 8.0 Hz, 2H), 8.91 (d, J = 8.5
carbonate (35 mg, 0.33 mmol) was added to the solution. The Hz, 1H), 8.63 (d, J = 8.3 Hz, 1H), 8.35 (t, J = 7.6 Hz, 1H), 8.16 –
suspension was stirred at rt for 12 h. The reaction mixture was 8.01 (m, 3H), 4.57 (s, 3H), 3.43 (s, 3H). 13C NMR (100 MHz,
diluted with dichloromethane and washed with water three DMSO) δ 165.9, 136.8, 135.0, 133.1, 131.7, 130.6, 130.2,
times. The organic layer was dried with sodium sulfate and the 129.6, 124.5, 124.4, 124.4, 123.3, 120.7 (JC-F = 321 Hz), 120.0,
solvent was removed under reduced pressure. The product 41.3, 19.6. LCMS: Calculated M+ (cationic part only) (208);
was a red solid. Yield: 77 % (65 mg, 0.25 mmol).1H-NMR (400 Found (M+) 208 m/z, Rt = 0.95 min.
MHz, DMSO-d6): δ = 7.27 (t, J = 7.2 Hz, 1H), 7.24 (d, J = 7.0 Hz, General procedure for preparation of spiropyrans. 2-
1H), 7.23 (t, J = 7.3 Hz, 1H), 7.16 (d, J = 7.0 Hz, 1H), 7.11 (t, J = hydroxybenzaldehyde (salicyl aldehyde) (1 equiv.) was
6.8 Hz, 1H), 7.11 (t, J = 6.8 Hz, 1H), 7.06 (d, J = 10.1 Hz, 1H), dissolved in methanol (0.2 M) in a capped microwave vial at rt
6.84 (t, J = 7.4 Hz, 1H), 6.54 (t, J = 8.2 Hz, 2H), 5.93 (d, J = and piperidine (1 equiv.) was added. The mixture was then
10.0 Hz, 1H), 5.70 (d, J = 7.5 Hz 1H), 3.01 (s, 3H).13C-NMR (151 heated to reflux before it was removed from the oilbath and
MHz, DMSO-d6): δ = 152.2, 134.4, 130.4, 130.1, 128.5, 127.4, 0.25 equiv. of triflate salt of 5,6-dimethylphenanthridin-5-ium
127.1, 127.1, 126.7, 124.8, 123.5, 121.6, 120.1, 118.4, 113.6, (5) in methanol (0.2 M) was added. The reaction was then
40.4. HRMS ESI-TOF+: m/z calcd for C18H16NO+ [M+H+]: refluxed for 20 min and then again removed from the oilbath
262.1226; found 262.1235.
N-([1,1'-biphenyl]-2-yl)acetamide (3): Lithium hydroxide was repeated until a total of 1 equiv. of 5 had been added. The
(727 mg, 17.3 mmol) was added to solution of 2- reaction was then left to reflux for an additional hour and then
and an additional 0.25 equiv. of 5 was added. This sequence
a
aminobiphenyl (1.00 g, 5.91 mmol) in DCM (88 mL). The allowed to cool down under stirring over night. Unless
suspension was cooled to 0 °C before a solution of acetyl otherwise noted, the formed precipitate was filtered off over a
bromide (1.31 ml, 17.7 mmol) in DCM (8 mL) was added small glass-frit funnel and then dried under vacuum to give the
dropwise. The reaction mixture was stirred for 4 h at rt, phenanthridine spiropyrans as racemates. No additional
filtered and concentrated under reduced pressure. The residue purification was needed.
was dissolved in DCM and washed with water. The organic 5'-Methyl-5'H-spiro[chromene-2,6'-phenanthridine]
(6)
layer was dried over sodium sulfate and concentrated under (ECH24): Made using the general procedure from 5 (53 mg,
reduced pressure. The crude product was purified by 0.15 mmol) and salicyl aldehyde (15 μL, 0.15 mmol), giving 6 as
1
crystallization at 0 °C from a DCM:n-heptane solution giving a white solid in 74% yield (34 mg, 0.11 mmol). H NMR (600
the product as colourless needles in 93 % yield (1.16 g, MHz, DMSO-d6) δ 8.14 (d, J = 8.0 Hz, 1H), 8.10 (d, J = 8.0 Hz,
1
5.48 mmol). H-NMR (400 MHz, CDCl3): δ = 8.27 (d, J = 8.1 Hz, 1H), 7.49 (t, J = 7.4 Hz, 1H), 7.41 – 7.34 (m, 3H), 7.27 (d, J = 7.5,
1H), 7.35 – 7.51 (m, 6H), 7.25 (d, J = 7.1 Hz, 1H), 7.18 (t, J = 1H), 7.17 (d, J = 10.0 Hz, 1H), 7.10 – 7.01 (m, 3H), 6.86 (t, J =
7.3 Hz, 1H), 7.11 (s, 1H), 2.02 (s, 3H). 13C-NMR (100 MHz, 7.4, 1H), 6.46 (d, J = 8.0 Hz, 1H), 6.02 (d, J = 10.0 Hz, 1H), 3.10
CDCl3): δ = 168.4, 138.3, 134.8, 132.3, 130.2, 129.4, 129.2, (s, 3H). 13C NMR (151 MHz, DMSO) δ 151.9, 140.8, 131.4,
128.6, 128.1, 124.5, 121.8, 24.8. LCMS: Calculated MH+ (212); 130.1, 129.6, 129.0, 128.2, 127.8, 127.5, 127.2, 127.0, 123.1,
Found (MH+) 212 m/z, Rt = 4.68 min.
123.0, 122.1, 120.4, 119.6, 119. 2, 118.4, 114.9, 114.0, 91.2,
6-methylphenanthridine (4): 3 (208 mg, 985 µmol) and 33.0. HRMS ESI-TOF+: m/z calcd for C22H18NO+ [M+H+]:
polyphosphoric acid (3 ml, 115 % H3PO4 equiv.) were mixed 312.1383; found 312.1393.
and stirred for 2.5 h at 140 °C. The reaction was then cooled 8-Methoxy-5'-methyl-5'H-spiro[chromene-2,6'-
down to 0 °C and saturated aqueous sodium hydroxide phenanthridine] (7) (ECH25): Made using the general
solution was added until pH 14 was reached. The aqueous procedure from 5 (50 mg, 0.14 mmol) and 2-hydroxy-3-
6 | J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
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methoxybenzaldehyde (18 μL, 0.14 mmol), giving 7 as a white mmol). 1H NMR (600 MHz, DMSO-d6) δ 8.13 (dd, JV=iew8.A0rt,ic1le.2OnHlinze,
1
solid in 69% yield (33 mg, 0.10 mmol). H NMR (600 MHz, 1H), 8.09 (dd, J = 7.8, 1.6 Hz, 1H), 7.49 (dDdOdI,: J10=.170.399,/C77.1O,B10.0530H0zE,
DMSO-d6) δ 8.15 (d, J = 7.9, 1H), 8.11 (d, J = 7.9, 1H), 7.49 (td, J 1H), 7.41 (dd, J = 7.9, 1.5 Hz, 1H), 7.36 (dddd, J = 8.0, 7.0, 4.2,
= 7.3, 1.9 Hz, 1H), 7.41 – 7.34 (m, 3H), 7.13 (d, J = 10.0 Hz, 1H), 1.4 Hz, 2H), 7.14 (d, J = 10.1 Hz, 1H), 7.10 (dd, J = 7.5, 1.7 Hz,
7.06 (d, J = 8.4 Hz, 1H), 7.02 (t, J = 7.3 Hz, 2H), 6.88 (dd, J = 7.0, 1H), 7.06 – 7.00 (m, 2H), 6.96 (ddd, J = 7.5, 1.8, 0.9 Hz, 1H),
2.2 Hz, 1H), 6.84 – 6.78 (m, 2H), 5.98 (d, J = 10.0 Hz, 1H), 3.46 6.77 (t, J = 7.5 Hz, 1H), 6.04 (d, J = 10.0 Hz, 1H), 3.03 (s, 3H),
(s, 3H), 3.09 (s, 3H). 13C NMR (151 MHz, DMSO) δ 146.6, 141.0, 1.66 (s, 3H). 13C NMR (151 MHz, DMSO) δ 149.8, 140.9, 132.1,
140.6, 131.2, 129.6, 129.0, 128.1, 127.5, 126.8, 123.1, 123.0, 131.2, 129.5, 128.9, 128.5, 127.6, 127.3, 127.3, 124.8, 123.5,
122.0, 119.9, 119.4, 119.3, 119.1, 118.7, 113.8, 113.7, 91.2, 123.0, 122.7, 122.1, 119.9, 119.2, 118.2, 113.9, 90.6, 32.7,
55.4, 32.9. HRMS ESI-TOF+: m/z calcd for C23H20NO2+ [M+H+]: 14.7. HRMS ESI-TOF+: m/z calcd for C23H20NO+ [M+H+]:
342.1489; found 342.1498.
326.1539; found 326.1548.
7-Methoxy-5'-methyl-5'H-spiro[chromene-2,6'-
5',6-Dimethyl-5'H-spiro[chromene-2,6'-phenanthridine] (12)
phenanthridine] (8) (ECH26): Made using the general (ECH30): Made using the general procedure from 5 (50 mg,
procedure from 5 (50 mg, 0.14 mmol) and 2-hydroxy-4- 0.14 mmol) and 2-hydroxy-5-methylbenzaldehyde (19 mg, 0.14
methoxybenzaldehyde (14 μL, 0.14 mmol), giving 8 as a white mmol), giving 12 as a white solid in 77% yield (35 mg, 0.11
1
solid in 71% yield (34 mg, 0.10 mmol). H NMR (600 MHz, mmol). 1H NMR (600 MHz, DMSO-d6) δ 8.13 (d, J = 7.9 Hz, 1H),
DMSO-d6) δ 8.17 – 8.13 (d, J = 8.1 Hz, 1H), 8.10 (d, J = 8.1 Hz, 8.09 (d, J = 7.9 Hz, 1H), 7.49 (t, J = 7.4 Hz, 1H), 7.41 – 7.34 (m,
1H), 7.50 (t, J = 7.3 Hz, 1H), 7.44 – 7.34 (m, 3H), 7.17 (d, J = 8.4 3H), 7.12 (d, J = 10.0 Hz, 1H), 7.08 – 7.02 (m, 3H), 6.89 – 6.86
Hz, 1H), 7.11 (d, J = 10.0 Hz, 1H), 7.08 (d, J = 8.1 Hz, 1H), 7.06 – (d, J = 8.4 Hz, 1H), 6.36 (d, J = 8.2 Hz, 1H), 6.00 (d, J = 10.0 Hz,
7.02 (t, J = 7.4 Hz, 1H), 6.43 (dd, J = 8.4, 2.4 Hz, 1H), 6.08 (d, J = 1H), 3.09 (s, 3H), 2.21 (s, 3H). 13C NMR (151 MHz, DMSO) δ
2.4 Hz, 1H), 5.84 (d, J = 10.0 Hz, 1H), 3.61 (s, 3H), 3.11 (s, 3H). 149.8, 140.8, 131.5, 130.5, 129.6, 129.1, 129.0, 128.2, 127.8,
13C NMR (151 MHz, DMSO) δ 161.1, 153.3, 140.8, 131.4, 129.5, 127.5, 127.1, 123.2, 123.0, 122.1, 119.6, 119.2, 118.2, 114.7,
129.0, 128.2, 128.0, 127.9, 127.5, 126.7, 123.0, 122.0, 120.1, 113.9, 91.0, 33.0, 20.1. HRMS ESI-TOF+: m/z calcd for
119.6, 119.2, 114.0, 111.5, 107.0, 100.1, 91.5, 55.1, 33.0. C23H20NO+ [M+H+]: 326.1539; found 326.1548.
HRMS ESI-TOF+: m/z calcd for C23H20NO2+ [M+H+]: 342.1489; 5-Bromo-8-methoxy-5'-methyl-5'H-spiro[chromene-2,6'-
found 342.1499.
6-Methoxy-5'-methyl-5'H-spiro[chromene-2,6'-
phenanthridine] (13) (ECH31): Made using the general
procedure from 5 (30 mg, 0.08 mmol) and o (19 mg, 0.08
phenanthridine] (9) (ECH27): Made using the general mmol), giving 13 as a white solid in 74% yield (26 mg, 0.06
procedure from 5 (20 mg, 0.06 mmol) and 2-hydroxy-5- mmol). 1H NMR (600 MHz, DMSO-d6) δ 8.17 (d, J = 8.1 Hz, 1H),
methoxybenzaldehyde (7 μL, 0.06 mmol), giving 9 as a white 8.12 (d, J = 7.9 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.43 – 7.36 (m,
1
solid in 52% yield (10 mg, 0.03 mmol). H NMR (600 MHz, 2H), 7.35 (d, J = 8.0 Hz, 1H), 7.29 (d, J = 10.2 Hz, 1H), 7.11 –
DMSO-d6) δ 8.13 (d, J = 7.9 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.03 (m, 3H), 6.80 (d, J = 8.8 Hz, 1H), 6.17 (d, J = 10.2 Hz, 1H),
7.48 (t, J = 6.7 Hz, 1H), 7.38 – 7.34 (m, J = 2.0 Hz, 2H), 7.14 (d, J 3.47 (s, 3H), 3.08 (s, 3H). 13C NMR (151 MHz, DMSO) δ 146.6,
= 10.0 Hz, 1H), 7.07 – 7.00 (m, 2H), 6.89 (d, J = 3.0 Hz, 1H), 6.65 142.4, 140.3, 130.5, 129.7, 129.3, 128.1, 128.1, 127.7, 125.2,
(dd, J = 8.7, 3.0 Hz, 1H), 6.40 (d, J = 8.7 Hz, 1H), 6.04 (d, J = 10.0 125.0, 123.2, 123.1, 122.1, 119.4, 119.2, 117.6, 114.6, 113.9,
Hz, 1H), 3.70 (s, 3H), 3.08 (s, 3H). 13C NMR (151 MHz, DMSO) δ 111.7, 91.8, 55.6, 33.0. HRMS ESI-TOF+: m/z calcd for
153.0, 145.8, 140.9, 131.4, 129.5, 129.0, 128.2, 127.7, 127.4, C23H19BrNO2+ [M+H+]: 420.0594; found 420.0598.
127.1, 123.9, 123.0, 122.1, 119.6, 119.1, 118.9, 115.7, 115.6, 5'-Methyl-5'H-spiro[chromene-2,6'-phenanthridin]-8-ol (14)
113.9, 111.7, 90.8, 55.4, 33.1. HRMS ESI-TOF+: m/z calcd for (ECH32): Made using the general procedure from 5 (20 mg,
C23H20NO2+ [M+H+]: 342.1489; found 342.1496.
5-Methoxy-5'-methyl-5'H-spiro[chromene-2,6'-
0.06 mmol) and 2,3-dihydroxybenzaldehyde (8.0 mg, 0.06
mmol). The crude mixture was concentrated and purified by
phenanthridine] (10) (ECH28): Made using the general preparative HPLC using MeCN:water with 0.75% formic acid as
procedure from 5 (30 mg, 0.08 mmol) and methyl 2-hydroxy-6- eluent, giving the open zwitterionic intermediate after
methoxybenzaldehyde (13 mg, 0.08 mmol), giving 10 as a condensation with the aldehyde. The isolated intermediate
1
white solid in 52% yield (15 mg, 0.04 mmol). H NMR (400 was then dissolved in a small amount of methanol and poured
MHz, DMSO-d6) δ 8.09 (dd, J = 8.0, 7.7 Hz, 2H), 7.48 (t, J = 7.6 into 5% KOH in water. The closed spirocyclic compound was
Hz, 1H), 7.44 – 7.29 (m, 4H), 7.06 – 7.01 (m, 3H), 6.52 (d, J = 8.3 then extracted by EtOAc and dried under vacuum giving 14 in
Hz, 1H), 6.09 (d, J = 8.2 Hz, 1H), 5.93 (d, J = 10.2 Hz, 1H), 3.86 66% yield (12 mg, 0.04 mmol). Trace amounts of ring-opened
(s, 3H), 3.09 (s, 3H). 13C NMR (100 MHz, DMSO) δ 155.2, 152.5, merocyanine form still present. 1H NMR (400 MHz, DMSO-d6) δ
140.7, 131.4, 130.3, 129.6, 129.0, 128.2, 127.9, 127.5, 123.0, 8.14 (d, J = 7.9 Hz, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.48 (t, J = 7.4
122.1, 121.4, 121.0, 119.6, 119.2, 114.0, 107.9, 107.2, 102.9, Hz, 1H), 7.41 – 7.32 (m, 3H), 7.10 – 6.98 (m, 3H), 6.74 – 6.61
90.9, 55.7, 32.9. HRMS ESI-TOF+: m/z calcd for C23H20NO2+ (m, 3H), 5.92 (d, J = 10.0 Hz, 1H), 3.07 (s, 3H). 13C NMR (151
[M+H+]: 342.1489; found 342.1497.
MHz, DMSO) δ 144.3, 140.8, 139.9, 131.5, 129.5, 128.9, 128.2,
5',8-Dimethyl-5'H-spiro[chromene-2,6'-phenanthridine] (11) 127.4, 126.9, 123.0, 122.9, 121.8, 119.9, 119.2, 119.0, 118.9,
(ECH29): Made using the general procedure from 5 (50 mg, 117.7, 117.1, 113.7, 90.7, 32.9. HRMS ESI-TOF+: m/z calcd for
0.14 mmol) and 2-hydroxy-3-methylbenzaldehyde (17 μL, 0.14 C22H18NO2+ [M+H+]: 328.1332; found 328.1338.
mmol), giving 11 as a white solid in 70% yield (32 mg, 0.10
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Organic & Biomolecular Chemistry
Page 8 of 12
ARTICLE
8-Ethyl-5'-methyl-5'H-spiro[chromene-2,6'-phenanthridine]
Journal Name
mg, 0.14 mmol) and 5-bromosalicylaldehyde (V2ie8w Amrtigcl,e O0n.l1in4e
DOI: 10.1039/C7OB00300E
(15) (ECH33): Made using the general procedure from 5 (20 mmol), giving 19 as a white solid in 71% yield (39 mg, 0.10
mg, 0.06 mmol) and 3-ethyl-2-hydroxybenzaldehyde (8.0 mg, mmol). 1H NMR (400 MHz, DMSO-d6) δ 8.15 (d, J = 8.0 Hz, 1H),
0.06 mmol), giving 15 as a white solid in 87% yield (17 mg, 0.05 8.10 (dd, J = 7.9, 1.5 Hz, 1H), 7.55 – 7.47 (m, 2H), 7.41 – 7.34
mmol). 1H NMR (600 MHz, DMSO-d6) δ 8.13 (d, J = 8.0 Hz, 1H), (m, 3H), 7.23 – 7.15 (m, 2H), 7.11 – 7.02 (m, 2H), 6.45 (d, J =
8.09 (d, J = 7.8 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 7.43 – 7.33 (m, 8.6 Hz, 1H), 6.11 (d, J = 10.1 Hz, 1H), 3.09 (s, 3H). 13C NMR (151
3H), 7.15 (d, J = 10.0 Hz, 1H), 7.10 (dd, J = 7.6, 1.7 Hz, 1H), 7.06 MHz, DMSO) δ 151.1, 140.5, 132.3, 130.9, 129.7, 129.4, 129.2,
– 7.00 (m, 2H), 6.95 (dd, J = 7.5 Hz, 1H), 6.78 (t, J = 7.5 Hz, 1H), 128.2, 127.8, 127.6, 126.0, 124.3, 123.1, 122.2, 120.7, 119.5,
6.04 (d, J = 10.0 Hz, 1H), 3.06 (s, 3H), 2.10 – 2.02 (m, 2H), 0.65 119.4, 117.2, 114.0, 111.4, 91.8, 33.1. HRMS ESI-TOF+: m/z
(t, J = 7.5 Hz, 3H). 13C NMR (151 MHz, DMSO) δ 149.5, 140.9, calcd for C22H17BrNO+ [M+H+]: 390.0488; found 390.0497.
131.9, 129.9, 129.7, 129.4, 128.9, 128.5, 127.6, 127.4, 127.3, 6-(tert-Butyl)-5'-methyl-5'H-spiro[chromene-2,6'-
124.9, 122.9, 122.7, 122.1, 120.1, 119.9, 119.2, 118.3, 113.9, phenanthridine] (20) (ECH38): Made using the general
90.6, 32.9, 22.3, 13.7. HRMS ESI-TOF+: m/z calcd for procedure from 5 (50 mg, 0.14 mmol) and 5-tert-butyl-2-
C24H22NO+ [M+H+]: 340.1696; found 340.1703.
6,8-Dibromo-5'-methyl-5'H-spiro[chromene-2,6'-
hydroxybenzaldehyde (24 μL, 0.14 mmol), giving 20 as a white
solid in 72% yield (37 mg, 0.10 mmol). H NMR (400 MHz,
1
phenanthridine] (16) (ECH34): Made using the general DMSO-d6) δ 8.13 (d, J = 7.9 Hz, 1H), 8.09 (dd, J = 7.9, 1.5 Hz,
procedure from (30 mg, 0.08 mmol) and 3,5- 1H), 7.48 (dt, J = 7.4, 2.3 Hz, 1H), 7.41 – 7.33 (m, 3H), 7.28 (d, J
5
dibromosalicylaldehyde (23 mg, 0.08 mmol), giving 16 as a = 2.4 Hz, 1H), 7.17 (d, J = 10.0 Hz, 1H), 7.12 – 6.99 (m, 3H), 6.39
1
white solid in 61% yield (24 mg, 0.05 mmol). H NMR (600 (d, J = 8.5 Hz, 1H), 5.98 (d, J = 10.0 Hz, 1H), 3.08 (s, 3H), 1.25 (s,
MHz, DMSO-d6) δ 8.19 (d, J = 7.9 Hz, 1H), 8.13 (d, J = 7.9 Hz, 9H). 13C NMR (151 MHz, DMSO) δ 149.7, 142.5, 140.8, 131.6,
1H), 7.57 (dd, J = 8.5, 2.3 Hz, 2H), 7.53 (t, J = 7.7 Hz, 1H), 7.43 – 129.6, 129.0, 128.2, 127.8, 127.5, 127.4, 127.0, 123.9, 123.0,
7.34 (m, 3H), 7.19 (d, J = 10.1 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 122.8, 122.1, 119.6, 119.2, 117.5, 114.2, 113.9, 91.0, 33.8,
7.06 (t, J = 7.5 Hz, 1H), 6.21 (d, J = 10.1 Hz, 1H), 3.07 (s, 3H). 13
C
33.0, 31.3. HRMS ESI-TOF+: m/z calcd for C26H26NO+ [M+H+]:
NMR (151 MHz, DMSO) δ 148.0, 140.2, 134.4, 130.5, 129.6, 368.2009; found 368.2018.
129.4, 129.0, 128.4, 127.8, 127.6, 125.8, 124.9, 123.1, 122.2, 5'-Methyl-6-phenyl-5'H-spiro[chromene-2,6'-phenanthridine]
121.8, 119.6, 119.5, 114.0, 111.4, 109.6, 93.3, 32.9. HRMS ESI- (21) (ECH39): Made using the general procedure from 5 (20
TOF+: m/z calcd for C22H16Br2NO+ [M+H+]: 467.9593; found mg, 0.06 mmol) and 29 (11 mg, 0.06 mmol), giving 21 as a
1
467.9597.
white solid in 78% yield (17 mg, 0.04 mmol). H NMR (600
6,8-Dichloro-5'-methyl-5'H-spiro[chromene-2,6'-
MHz, DMSO-d6) δ 8.17 (d, J = 8.0 Hz, 1H), 8.12 (d, J = 7.8 Hz,
phenanthridine] (17) (ECH35): Made using the general 1H), 7.66 – 7.59 (m, 4H), 7.53 (t, J = 7.5 Hz, 1H), 7.47 – 7.35 (m,
procedure from 5 (30 mg, 0.08 mmol) and 3,5-dichloro-2- 5H), 7.32 (t, J = 7.3 Hz, 1H), 7.27 (d, J = 10.1 Hz, 1H), 7.10 (d, J =
hydroxybenzaldehyde (16 mg, 0.08 mmol), giving 17 as a white 8.2 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 6.57 (d, J = 8.2 Hz, 1H), 6.09
1
solid in 85% yield (27 mg, 0.07 mmol). H NMR (600 MHz, (d, J = 10.1 Hz, 1H), 3.14 (s, 3H). 13C NMR (151 MHz, DMSO) δ
DMSO-d6) δ 8.20 (d, J = 8.0 Hz, 1H), 8.14 (d, J = 7.8 Hz, 1H), 151.6, 140.7, 139.6, 132.4, 131.3, 129.6, 129.1, 128.9, 128.3,
7.54 (t, J = 7.3 Hz, 1H), 7.44 (d, J = 2.5 Hz, 1H), 7.43 – 7.34 (m, 128.2, 127.8, 127.6, 127.1, 126.8, 126.1, 125.5, 123.5, 123.1,
4H), 7.22 (d, J = 10.1 Hz, 1H), 7.11 (d, J = 8.3 Hz, 1H), 7.07 (t, J = 122.1, 119.6, 119.3, 118.7, 115.4, 114.0, 91.6, 33.0. HRMS ESI-
7.5 Hz, 1H), 6.24 (d, J = 10.1 Hz, 1H), 3.09 (s, 3H). 13C NMR (151 TOF+: m/z calcd for C28H22NO+ [M+H+]: 388.1696; found
MHz, DMSO) δ 148.0, 140.2, 134.4, 130.5, 129.7, 129.4, 129.0, 388.1708.
128.4, 127.8, 127.6, 125.8, 124.9, 123.1, 122.2, 121.8, 119.6, N,N-diethyl-5'-methyl-5'H-spiro[chromene-2,6'-
119.5, 114.0, 111.4, 109.6, 93.3, 32.9. HRMS ESI-TOF+: m/z phenanthridin]-6-amine (22) (ECH40): Prepared using the
calcd for C22H16Cl2NO+ [M+H+]: 380.0603; found 380.0611.
general procedure from 5 (30 mg, 0.08 mmol) and 4-
6-Fluoro-5'-methyl-5'H-spiro[chromene-2,6'-phenanthridine]
diethylaminosalicylaldehyde (16 mg, 0.08 mmol). The crude
(18) (ECH36): Made using the general procedure from 5 (50 mixture was concentrated and purified by preparative HPLC
mg, 0.14 mmol) and 5-fluorosalicylaldehyde (20 mg, 0.14 using MeCN:water with 0.75% formic acid as eluent, giving the
mmol), giving 18 as a white solid in 76% yield (35 mg, 0.11 open zwitterionic intermediate after condensation with the
mmol).1H NMR (600 MHz, DMSO-d6) δ 8.14 (d, J = 8.0 Hz, 1H), aldehyde, as a deep purple solid. The isolated solid was then
8.10 (d, J = 7.9 Hz, 1H), 7.50 (m, 1H), 7.41 – 7.34 (m, 2H), 7.21 – dissolved in a small amount of methanol and poured into 5%
7.14 (m, 2H), 7.07 (d, J = 8.4 Hz 1H), 7.04 (t, J = 7.5 Hz, 1H), KOH in water. The closed spirocyclic compound was then
6.89 (td, J = 8.4, 3.0 Hz, 1H), 6.48 (dd, J = 8.7, 4.5 Hz, 1H), 6.12 extracted by EtOAc and dried under vacuum giving 22 in 11%
(d, J = 10.0 Hz, 1H), 3.09 (s, 3H). 13C NMR (151 MHz, DMSO) δ yield (3.5 mg, 0.0092 mmol). Trace amounts of ring-opened
156.8, 155.3, 148.1, 140.7, 131.1, 129.6, 129.1, 128.2, 127.7, merocyanine form still present. 1H NMR (400 MHz, DMSO-d6) δ
127.6, 126.4, 124.5, 123.1, 122.2, 119.6, 119.5, 119.4, 119.3, 8.14 – 8.06 (dd, J = 8.2, 7.9 Hz, 2H), 7.50 – 7.40 (m, 2H), 7.38 –
116.3, 116.1, 116.1, 114.0, 113.2, 113.0, 91.4, 33.1. HRMS ESI- 7.33 (m, 2H), 7.08 – 6.96 (m, 4H), 6.16 (dd, J = 8.5, 2.5 Hz, 1H),
TOF+: m/z calcd for C22H17FNO+ [M+H+]: 330.1289; found 5.68 (d, J = 2.5 Hz, 1H), 5.62 (d, J = 9.9 Hz, 1H), 3.19 (q, J = 7.0
330.1294.
Hz, 4H), 3.10 (s, 3H), 0.96 (t, J = 7.0 Hz, 6H). 13C NMR (151 MHz,
6-Bromo-5'-methyl-5'H-spiro[chromene-2,6'-phenanthridine]
DMSO) δ 153.5, 149.4, 141.0, 131.9, 129.5, 128.8, 128.2,
(19) (ECH37): Made using the general procedure from 5 (50 128.1, 128.0, 127.4, 126.8, 123.0, 121.9, 119.5, 118.9, 117.5,
8 | J. Name., 2012, 00, 1-3
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1
113.9, 106.6, 103.9, 96.8, 91.1, 43.5, 32.9, 12.5. HRMS ESI- under vacuum giving 26 in 52% yield (16 mg, 0.05 mmol). H
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TOF+: m/z calcd for C26H27N2O+ [M+H+]: 383.2118; found NMR (400 MHz, DMSO-d6) δ 8.18 (d, J = 7.9 Hz, 1H), 8.13 (d, J =
DOI: 10.1039/C7OB00300E
383.2128.
7.7 Hz, 1H), 7.91 (dd, J = 5.0, 2.0 Hz, 1H), 7.71 (dd, J = 7.3, 2.0
Methyl 5'-methyl-5'H-spiro[chromene-2,6'-phenanthridine]- Hz, 1H), 7.55 – 7.51 (m, 1H), 7.44 – 7.34 (m, 3H), 7.22 (d, J =
6-carboxylate (23) (ECH41): Made using the general procedure 10.0 Hz, 1H), 7.14 – 7.07 (m, 2H), 6.93 (dd, J = 7.3, 5.0 Hz, 1H),
from
5 (50 mg, 0.14 mmol) and methyl 3-formyl-4- 6.16 (d, J = 10.0 Hz, 1H), 3.11 (s, 3H). HRMS ESI-TOF+: m/z
hydroxybenzoate (21 μL, 0.14 mmol), giving 23 as a white solid calcd for C21H17N2O [M+H+]: 313.1335; found 313.1339.
in 77% yield (40 mg, 0.11 mmol). 1H NMR (400 MHz, DMSO-d6) 5'-Methyl-5'H-spiro[benzo[f]chromene-3,6'-phenanthridine]
δ 8.17 (d, J = 8.0 Hz, 1H), 8.12 (d, J = 7.6 Hz, 1H), 7.95 (d, J = 2.2 (27) (ECH45): Made using the general procedure from 5 (50
Hz, 1H), 7.67 (dd, J = 8.5, 2.2 Hz, 1H), 7.54 – 7.50 (m, 1H), 7.41 mg, 0.14 mmol) and 2-hydroxy-1-naphtaldehyde (24 mg, 0.14
– 7.36 (m, 3H), 7.32 (d, J = 10.2 Hz, 1H), 7.12 – 7.03 (m, 2H), mmol), giving 27 as a gray solid in 81% yield (41 mg, 0.11
6.57 (d, J = 8.5 Hz, 1H), 6.14 (d, J = 10.2 Hz, 1H), 3.81 (s, 3H), mmol). 1H NMR (400 MHz, DMSO-d6) δ 8.31 (d, J = 8.5 Hz, 1H),
3.11 (s, 3H). 13C NMR (100 MHz, DMSO) δ 165.7, 155.9, 140.4, 8.17 (d, J = 8.1 Hz, 1H), 8.13 (d, J = 7.5 Hz, 1H), 7.98 (d, J =
131.5, 130.8, 129.7, 129.3, 128.8, 128.2, 127.8, 127.7, 126.6, 10.4 Hz, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.69 (d, J = 8.9 Hz, 1H),
123.8, 123.1, 122.2, 121.6, 119.6, 119.5, 118.4, 115.3, 114.1, 7.58 (t, J = 7.6 Hz, 1H), 7.54 – 7.43 (m, 2H), 7.42 – 7.33 (m, 3H),
92.7, 51.9, 33.0. HRMS ESI-TOF+: m/z calcd for C24H20NO3+ 7.11 – 7.03 (m, 2H), 6.77 (d, J = 8.9 Hz, 1H), 6.13 (d, J = 10.4 Hz,
[M+H+]: 370.1438; found 370.1447.
6-Iodo-5'-methyl-5'H-spiro[chromene-2,6'-phenanthridine]
1H), 3.13 (s, 3H). 13C NMR (151 MHz, DMSO) δ 150.0, 140.6,
131.4, 130.4, 129.6, 129.6, 129.1, 128.4, 128.4, 128.2, 127.6,
(24) (ECH42): Made using the general procedure from 5 (20 127.0, 123.5, 123.1, 122.7, 122.0, 121.6, 121.2, 119.6, 119.3,
mg, 0.06 mmol) and 5-iodosalicylaldehyde (14 mg, 0.06 mmol), 117.3, 114.0, 110.1, 91.3, 32.9. HRMS ESI-TOF+: m/z calcd for
1
giving 24 as a white solid in 70% yield (17 mg, 0.04 mmol). H C26H20NO+ [M+H+]: 362.1539; found 362.1550.
NMR (400 MHz, DMSO-d6) δ 8.15 (d, J = 8.0 Hz, 1H), 8.10 (d, J = N-(3,4-dimethoxyphenyl)-5'-methyl-5'H-spiro[chromene-2,6'-
7.9, 1H), 7.65 (d, J = 2.2 Hz, 1H), 7.52 – 7.48 (m, 1H), 7.43 – phenanthridine]-6-carboxamide (28) (ECH46): Made using the
7.31 (m, 4H), 7.16 (d, J = 10.1 Hz, 1H), 7.10 – 7.01 (m, 2H), 6.32 general procedure from 5 (35 mg, 0.10 mmol) and 31 (42 mg,
(d, J = 8.5 Hz, 1H), 6.08 (d, J = 10.1 Hz, 1H), 3.09 (s, 3H). 13C 0.10 mmol), giving 28 as a gray solid in 56% yield (27 mg, 0.06
NMR (151 MHz, DMSO) δ 151.8, 140.5, 138.2, 135.2, 131.0, mmol). 1H NMR (600 MHz, DMSO-d6) δ 9.92 (s, 1H), 8.18 (d, J =
129.6, 129.2, 128.2, 127.8, 127.6, 125.9, 124.0, 123.1, 122.1, 8.0 Hz, 1H), 8.13 (d, J = 7.9 Hz, 1H), 7.92 (d, J = 2.3 Hz, 1H), 7.71
121.2, 119.5, 119.4, 117.6, 114.0, 91.7, 82.5, 33.0. HRMS ESI- (dd, J = 8.5, 2.3 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 7.46 – 7.37 (m,
TOF+: m/z calcd for C22H17INO+ [M+H+]: 438.0349; found 4H), 7.34 – 7.26 (m, 2H), 7.11 (d, J = 8.3 Hz, 1H), 7.09 – 7.05 (t,
438.0350.
J = 7.4 Hz, 1H), 6.92 (d, J = 8.7 Hz, 1H), 6.59 (d, J = 8.4 Hz, 1H),
6.15 (d, J = 10.1 Hz, 1H), 3.75 (s, 3H), 3.73 (s, 3H), 3.13 (s, 3H).
5'-Methyl-5'H-spiro[chromene-2,6'-phenanthridine]-6-
carboxylic acid (25) (ECH43): Made using the general 13C NMR (151 MHz, DMSO) δ 164.3, 154.5, 148.4, 145.0, 140.5,
procedure from 5 (50 mg, 0.14 mmol) and 30 (23 mg, 0.14 132.9, 131.0, 129.7, 129.3, 128.2, 127.8, 127.6, 127.1, 127.1,
mmol). Removal of unknown byproduct was accomplished by 126.8, 123.7, 123.1, 122.2, 119.6, 119.5, 118.0, 114.7, 114.1,
several recrystalisations using DCM and MeOH, finally giving 112.1, 111.9, 105.4, 92.3, 55.7, 55.4, 33.1. HRMS ESI-TOF+: m/z
25 as a beige solid in 30% yield (15 mg, 0.04 mmol). H NMR calcd for C31H26N2O4+ [M+H+]: 491.1965; found 491.1968.
1
(400 MHz, DMSO-d6) δ 8.17 (d, J = 7.9 Hz, 1H), 8.13 (d, J = 7.8
Hz, 1H), 7.91 (d, J = 2.2 Hz, 1H), 7.65 (dd, J = 8.4, 2.2 Hz, 1H),
Folding of the G4 structures
7.54 – 7.50 (m, 1H), 7.42 – 7.38 (m, 3H), 7.30 (d, J = 10.1 Hz, The oligonucleotides used in this study were purchased from
1H), 7.11 – 7.05 (m, 2H), 6.54 (d, J = 8.5 Hz, 1H), 6.12 (d, J = Eurofins MWG Operon (Germany). To fold the
10.1 Hz, 1H), 3.12 (s, 3H). 13C NMR (151 MHz, DMSO) δ 166.8, oligonucleotides, 50 µM oligonucleotide solution was prepared
155.6, 140.5, 131.6, 130.9, 129.7, 129.3, 129.0, 128.2, 127.9, in 10 mM Tris(hydroxymethyl)aminomethane hydrochloride
127.7, 126.7, 123.6, 123.1, 122.2, 119.5, 118.1, 115.0, 114.1, (Tris–HCl; pH 7.5) and 100 mM KCl, heated to 95 °C for 5 min,
92.5, 33.0. HRMS ESI-TOF+: m/z calcd for C23H18NO3+ [M+H+]: and slowly cooled to room temperature overnight.
356.1281; found 356.1294.
Thioflavin T (ThT) displacement assay
5-Methyl-5H-spiro[phenanthridine-6,2'-pyrano[2,3-
b]pyridine] (26) (ECH44): Made using the general procedure 1 µM folded oligonucleotides was incubated in 0.5 µM ThT, 10
from
5 (35 mg, 0.10 mmol) and 2-oxo-1,2-dihydro-3- mM Tris pH=7.5, 100 mM KCl treated with 0.0002, 0.0008,
pyridinecarbaldehyde (12 mg, 0.10 mmol), giving the open 0.003, 0.01, 0.25, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.13, 6.25, 12.5,
cationic intermediate after condensation with the aldehyde, as 25 or 50 µM concentration of the compounds in a final volume
1
a beige solid. H NMR (400 MHz, DMSO-d6) δ 12.30 (bs, 1H), of 40 µl. 2.5% v/v DMSO was used as a reference sample. All
9.17 (d, J = 7.8 Hz, 2H), 8.72 – 8.51 (m, 3H), 8.36 (t, J = 7.7 Hz, compounds were dissolved in 100% v/v DMSO. The final
1H), 8.23 – 7.90 (m, 3H), 7.66 (d, J = 5.6 Hz, 1H), 7.26 (d, J = concentration of DMSO in each sample was 2.5%. 10 µL from
16.2 Hz, 1H), 6.45 (t, J = 6.5 Hz, 1H), 4.57 (s, 3H).
the mixture was transferred into three wells in 384-well
The isolated solid was then dissolved in a small amount of Corning black flat-bottom microplates to obtain technical
methanol and poured into 5% KOH in water. The closed triplicates. BioTek Synergy H4 Microtiter plate reader was used
spirocyclic compound was then extracted by EtOAc and dried for readout at λ = 435/480 nm (ex/em). Background
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fluorescence of the buffer containing appropriate compound All spectra were acquired on a Bruker 850MHz Avance III HD
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was subtracted from all the measurements. The relative spectrometer equipped with a 5mm TCIDcOryI:o1-0p.1r0o3b9e/C. 72O5B600sc3a00nEs
fluorescence was calculated as a fold change (increase or were added with a relaxation delay of 1s and with excitation
decrease) to reference sample treated with DMSO. DC50 values sculpting for water suppression. 1Hz line-broadening was
of ThT displacement were calculated using Origin 8.5 software applied before fourier transform. All processing was done in
by fitting a dose response function with fixed bottom Topspin 3.2 (www.bruker.com).
asymptote to 0.
CD measurements
Acknowledgements
For CD measurements, 50 µM folded oligonucleotide were
diluted to 5 µM concentration in a buffer containing 10 mm
Tris–HCl (pH 7.5) and 100 mm KCl and in the presence or
absence of 80 µM of the tested compound. The blank sample
contained the same buffer without the compound but with
1.25% (v/v) DMSO. A JASCO-720 spectropolarimeter with
Peltier temperature control was used for the measurements.
Four accumulations of CD spectra of 5 µM oligonucleotide
were recorded at 25 °C over λ = 230–350 nm. The
measurements were performed in a quartz cuvette with a path
length of 0.1 cm. For melting curves, the wavelength was held
constant at λ =264 nm and melting curves were recorded in a
range of 25-90 °C. The Tm value is defined as the temperature
at which 50% of the G4 structures are unfolded. Tm values
were estimated by fitting the melting curves into a dose
response function in Origin 8.5 Software.
This work was supported by the Kempe foundations
(SMK1449). In addition, NS was also supported by the Swedish
Society for Medical Research and Knut and Alice Wallenberg
foundation, EC was also supported by Lion's Cancer Research
Foundation, Umeå University.
Notes and references
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Williamson, T. R. Cech and D. M. Prescott, Nature, 1991, 350,
718.
Taq-polymerase stop assay
1 µM TET-labeled primer was annealed to 1.25 µM template
DNA in 100 mM KCl by heating at 95 °C for 5 min and was
slowly cooled to room temperature, overnight. Annealed DNA
(40 nm) was incubated with 25 µM compound or 5% (v/v)
DMSO for 30 min in 50 mM KCl, 10 mM Tris-HCl (pH 7.5), 1.5
mM MgCl2, and 200 mM dNTPs (dATP, dTTP, dCTP, dGTP) at
room temperature. Primer extension assay was accomplished
by the addition of 1 U of Taq DNA polymerase (Thermo
Scientific) diluted in 10 mM Tris-HCl (pH 7.5) and 50 mM KCl
into the reaction mixture at 50 °C. The final volume of the
reaction was 45 µL. Reaction was stopped after 0.5, 1, 5 or 10
minutes by transferring 10 uL of the reaction mixture into a
stop solution (containing 95% formamide, 20 mM
5 (a) S. Balasubramanian, L. H. Hurley and S. Neidle, Nat. Rev. Drug
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ethylenediaminetetraacetic
acid
(EDTA),
and
0.1%
bromophenol blue). 5 uL of the final mixture was loaded onto
a 10% polyacrylamide gel containing 8 M urea, 25% formamide
and 1×Tris/borate/ethylenediaminetetraacetic acid (TBE). The
gel was visualized with a Typhoon Scanner 9400 (GE
Healthcare) and quantified with the Image Quant 5.2 software
(GE Healthcare).
7 J. Jamroskovic, M. Livendahl, J. Eriksson, E. Chorell and N. Sabouri,
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NMR Analysis
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The reference sample of Pu24T was prepared by adding 20mL
D2O to 200mL stock solution of Pu24T (100 mM Pu24T in PBS
buffer 10mM phosphate, 25mM KCl, pH 7) and transferring
the solution to a 3mm NMR tube.
Samples containing compound 7 or 8 were prepared in the
same way as the reference sample but with addition of 2mL of
stock solution of 7 or 8 (10mM in DMSO) to get a 1:1 ratio
between Pu24T and the added compound.
10 (a) D. Monchaud and M. P. Teulade-Fichou, Org. Biomol. Chem.,
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10 | J. Name., 2012, 00, 1-3
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View Article Online
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