Modular synthesis of 4-aminocarbonyl substituted 1,8-naphthalimides and application in single molecule fluorescence detection

Article abstract of DOI:10.1039/c7cc07922b

Robust methodology to install amide, carbamate, urea and sulfonamide functionality to the 1,8-naphthalimide scaffold has been developed and exemplified. New benzamidonaphthalimide 6, synthesised using this approach, was found to be sensitive to base whereupon fluorescence emission strongly increases (>10-fold) and red-shifts (>4000 cm^-1). The optical properties of deprotonated 6 allow for single molecule fluorescence detection, the first example of such behaviour from this class of fluorophore.

Full text of DOI:10.1039/c7cc07922b

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DOI: 10.1039/C7CC07922B
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Modular synthesis of 4-aminocarbonyl substituted 1,8-
naphthalimides and application in single molecule fluorescence
detection
Received 00th January 20xx,
Accepted 00th January 20xx
K. N. Hearn,a T. D. Nalder,a, b R. P. Cox,c H. D. Maynard,c T. D. M. Bell,c F. M. Pfeffera and T. D.
Ashton,a*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Robust methodology to install amide, carbamate, urea and approaches. For example, the typical synthesis of an amide
sulfonamide functionality to the 1,8-naphthalimide scaffold has begins with 4-nitro- or 4-azido-1,8-naphthalic anhydride (Figure
been developed and exemplified. New benzamidonaphthalimide 1), the latter requiring an SNAr step from 4-bromo-1,8-
6, synthesised using this approach, was found to be sensitive to naphthalic anhydride. In each case formation of the imide and
base whereupon fluorescence emission strongly increases (>10- a reduction step (usually involving stoichiometric SnCl2, PPh3 or
fold) and red-shifts (>4000 cm-1). The optical properties of NaSH) are required to give the 4-amino analogue. This amine is
deprotonated 6 allow for single molecule fluorescence detection, a poor nucleophile and can only be modified to accommodate
the first example of such behaviour from this class of fluorophore. one extra substituent, a fact highlighted by a lack of lactam
derivatives in the literature. In addition, converting the 4-amino
In light of their photophysical properties, including high
fluorescence quantum yields and inherent photostability,
amine substituted 1,8-naphthalimides are extensively used as
colourimetric and fluorescent probes.1 While less common,
amide and carbamate substituted 1,8-naphthalimides have also
been employed for sensing and as intracellular probes.2
Hydrolysis of amide or carbamate containing 1,8-
moiety to a carbamate relies on the use of toxic triphosgene to
generate the intermediate carbamoyl chloride.2
naphthalimides to the corresponding amine elicits
a
bathochromic shift of emission maxima (from λem ~470 to ~550
nm), a property exploited in the design of ratiometric probes.
In addition, Tian et al. showed that a 4-benzamide substituted
1,8-naphthalimide can be deprotonated to effect red-shifted
emission (λem = 583 nm, MeCN) that enabled the detection of
fluoride.3 This sensing mechanism also functions in carbamates
and has been applied to the detection of carbon dioxide.4
Importantly, the 1,8-naphthalimide is also a common scaffold in
medicinal chemistry with scriptaid, amonafide, and their
derivatives possessing potent anti-cancer activity.5
Figure 1. Summary of traditional approaches and current work.
Despite the demonstrated utility of 1,8-naphthalimide-
based fluorescent probes, the synthesis of functionalised 1,8-
naphthalimides remains remarkably underdeveloped with a
dependence on highly functional group specific, linear,
While amine substituted 1,8-naphthalimide derivatives can be
directly accessed using copper and palladium-mediated cross-
coupling reactions,6 for carboxamide substituents, examples
are limited to the introduction of phthalimide using
stoichiometric copper(I)7 and the single example of palladium-
catalysed bisarylation of urea reported by Fabrizzi et al.8 With
the diverse utility of 1,8-naphthalimides in medicinal chemistry
and fluorescence spectroscopy there remains a need for
methodology that can efficiently attach functional groups to the
1,8-naphthalimide core.
a.Centre for Chemistry and Biotechnology, School of Life and Environmental
Sciences, Deakin University, Waurn Ponds, 3216, Australia. E-mail;
t.ashton@deakin.edu.au.
b.Marine Products Team, The New Zealand Institute for Plant & Food Research
Limited, 293-297 Akersten Street, Nelson 7010, New Zealand.
c. School of Chemistry, Monash University, Clayton, 3800, Australia.
Electronic Supplementary Information (ESI) available: [ESI containing full
characterisation details for all new compounds]. See DOI: 10.1039/x0xx00000x
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a high-yielding, modular an equimolar ratio of reagents are particularly advantageous if
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method that directly attaches a variety of functionalities to the two synthetically valuable substrates areDtOoI:b1e0.c10o3u9p/Cle7dC.C0W79h2e2nB
1,8-naphthalimide scaffold. This method allows ready access to performing the reaction at 80 °C (Entry 6) longer reaction times
underrepresented derivatives including benzamides, lactams were required (23 h) however a high yield (82%) was again
and sulphonamides, as well as commonly encountered analyte obtained. Increasing the amount of coupling partner (1.5 equiv.
responsive triggers and cellular localisation moieties. Of of benzamide, Entry 7) failed to improve the reaction rate
considerable interest the new benzamide analogues have been (22 h).
shown to be readily detectable at the level of single molecule
(SM). This opens the way for 1,8-naphthalimides to be used in investigated. A small series of aryl carboxamides were
applications such as SM-FRET and SM-sensing. synthesised from 4-bromo imide (Scheme 1). In general,
Xantphos is compatible with 1,8-naphthalimide amination6a yields were greater than 90% when 1.2 equivalents of neutral
With methodology in hand, substrate scope could be
3
and fluorophore synthesis,9 and as such we sought inspiration
(4–5) and electron deficient benzamides (6–9) were used as
from the seminal work by Yin and Buchwald.10 In preliminary coupling partners. The 4-methoxybenzamide was incorporated
experiments, the treatment of 4-bromo-N-propyl-1,8- in 83% but two equivalents were required to achieve full
naphthalimide (1) (Entry 1, Table 1) with benzamide in the conversion within a suitable timeframe.
presence of Pd(OAc)2 (1.0 mol%) and Xantphos (1.5 mol%) led
to the consumption of starting material after 20 h at 100 °C.
Complete reaction occurred despite conducting the reactions
10-fold more dilute than previously reported,10 a requirement
for the poorly soluble starting material. After completion of the
reaction, dilution using H2O led to precipitation and easy
isolation of the secondary benzamide 2 in good yield (71%).
Table 1. Conditions for amidation.
Entry
1
BzNH2
(equiv.)
1.2
Pd-L
Pd
(mol%)
1.0a
Temp Time
Yield
(%)
71
Scheme 1. Conditions: Bromide (0.25 or 0.5 mmol, 0.1 M) Amide (1.2 equiv)., G3-
Xantphos (1 mol%), Cs2CO3 (1.4 equiv.), 100 °C; a) Amide (2.0 equiv.); b) G3-Xantphos
(2 mol%).
(°C)
100
(h)
20
Pd(OAc)2/
Xantphos
2
3
4
5
6
7
1.2
2.0
1.2
1.0
1.2
1.5
G3-Xantphos
G3-Xantphos
G3-Xantphos
G3-Xantphos
G3-Xantphos
G3-Xantphos
1.0
1.0
0.5
1.0
1.0
1.0
100
100
100
100
80
2.5
0.25
6.5
7
23
22
93
90
76
82
82
78
Nicotinamide was also coupled to the 1,8-naphthalimide
scaffold to give 11 in very good yield (83%). In contrast, the best
yield achieved using picolinamide was 54%, a reaction that
required 2 mol% of G3-Xantphos. Increasing the equivalents of
amide was detrimental in this instance. The long reaction time
(24 h) also suggested that the reaction may be hindered by
potential chelation of the substrate to palladium.
80
a) Pd(OAc)2 (1.0 mol%), Xantphos (1.5 mol%).
In an effort to enhance the reaction rate the palladacycle
precatalyst G3-Xantphos was investigated. The G3-precatalyst
offers a number of advantages including ease of preparation
(and also commercial availability) and insensitivity to both air
and moisture.11 The Pd to Xantphos ratio is controlled and H2O
is not required for reduction of Pd(II), which can negate the
potential for competitive hydroxylation.12 Repeating the trial
reaction using 1 mol% G3-Xantphos led to the consumption of
Simple aliphatic primary amides were coupled smoothly to
a number of 4-bromo-1,8-naphthalimides (Scheme 2). Linear
aliphatic amides (13
87%. Cyclohexanecarboxamide
adamantanecarboxamide 21 were also incorporated in
–19) were installed in yields greater than
(
20 and 1-
)
(
)
excellent yields (96 and 90%, respectively). Lactams, 1-
pyrrolidinone and isoindolin-1-one were coupled to give tertiary
amides (23–24), again, in near quantitative yields. To date there
starting material in 2.5 h (Entry 2) providing 2 in an isolated yield
are only two reported structures of 1,8-naphthalimides bearing
a lactam at the 4-position; neither of which contain any
preparative information.13 Secondary amides (e.g. N-methyl
benzamide) failed to give any product.
This methodology was compatible with a number of
functional groups important in both medicinal chemistry and
fluorescence sensing and labelling applications. Methyl ester
of 93% after precipitation. When two equivalents of benzamide
were employed (Entry 3) a yield of 90% was obtained after
0.25 h. Lower G3-Xantphos loading (0.5 mol%, Entry 4) or use
of equimolar amounts of benzamide (Entry 5) also gave
secondary amides in very good yields (76 and 82%, respectively)
but with longer reaction times of (>6.5 h). Conditions involving
2 | J. Name., 2012, 00, 1-3
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(
14) and terminal alkyne (15) were well tolerated, as were access to probes that contain an established trigger for the
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tertiary amines, which facilitates access to amonafide cellular monitoring of nitroreductase5b aDnOdI:h1y0d.1r0o3g9e/Cn7sCuCl0fi7d9e2.2B
derivatives (such as 24) and lysosome targeting groups (for Ethyl and tert-butyl carbamates also underwent smooth
example 25). Similarly, the secondary sulphonamide recently coupling giving 32 and 33 in 94 and 55% yield, respectively.
reported by Tang14 as an endoplasmic reticulum targeting group
16
was compatible with these conditions giving 17 in 90% yield.
dos Santos’ fluoride trigger15 could also be incorporated using
these reaction conditions highlighting that acid sensitive
protecting groups are also well tolerated. Here the D-serine
amide bearing tert-butyldimethylsilyl ether and secondary tert-
butoxycarbonyl groups was incorporated to give 22 in 82%
yield.
A
THP-protected hydroxamic acid was also
accommodated by these conditions giving rise to a protected
scriptaid analogue5b 27 in moderate yield (67%).
Scheme 3. Conditions: Bromide (0.25 or 0.5 mmol, 0.1 M) R2-NH2 (1.2 equiv)., G3-
Xantphos (1 mol%), Cs2CO3 (1.4 equiv.), 100 °C; a) G3-Xantphos (2 mol%); b) N-
Phenylurea (2.0 equiv.); c) Sulfonamide (2.0 equiv.).
The coupling of 1,1-dimethylurea afforded 34 in reasonable
yield (71%) after purification by column chromatography. N-
Phenylurea could also be incorporated to give the 1,8-
naphthalimide 35, albeit in moderate yield (43%). We also
trialled phenylthiourea as a coupling partner to access
analogues described by Gale et al. as transmembrane chloride
transporters,17 however no appreciable conversion was
detected. Incorporation of 4-chlorobenzenesulfonamide was
successful with 36 isolated in excellent yield (90%). Examples of
sulfonamides appended to 1,8-naphthalimide scaffold are
rare18 presumably owing to the difficulty of the transformation.
Optical characterisation of a number of the compounds
reported herein was undertaken with photophysical data
compiled (see ESI, Table S1). Of particular interest is benzamide
6
which showed a dramatic >10-fold increase in fluorescence
emission on addition of NaOH to a solution in DMSO (Figure 2a).
Neutral shows an absorption maximum at 357 nm and weak
6
emission at 461 nm (ΦF = 0.05). On addition of NaOH (~5–20
equivalents), a new absorption grows at 486 nm with emission
shifting to 575 nm, a bathochromic shift of >4000 cm-1, and the
quantum yield increases to 0.69 upon full deprotonation with
excess NaOH (>100 equivalents). Time-resolved fluorescence
Scheme 2. Conditions: Bromide (0.25 or 0.5 mmol, 0.1 M) Amide (1.2 equiv.), G3-
Xantphos (1 mol%), Cs2CO3 (1.4 equiv.), 100 °C; a) Bromide (1.5 mmol).
To circumvent the use of triphosgene in the synthesis of
carbamate derivatives, benzyl carbamate was directly attached
to the 1,8-naphthalimide scaffold (28–30, Scheme 3) in very
good (73% for 30) to excellent yields (94% for 28). The 4-
nitrobenzyl carbamate group (synthesised using CDI, see ESI),
was also coupled to give 31 in 91% yield. This approach gave
measurements show that emission from benzamide 6 in DMSO
(monitored at 470 nm) is short-lived and dominated by a decay
component with a lifetime of 0.56 ns (Figure 2b). Addition of
excess NaOH leads to emission (monitored at 570 nm) with a
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significantly increased lifetime of 6.91 ns, a value similar to
other reported 1,8-naphthalimides.19
Notes and references
View Article Online
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6 often stayed visible for over
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from a 1,8-naphthalimide has been observed. It is envisaged
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Conflicts of interest
The Authors declare no conflict of interest.
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