Mixed alkoxy/hydroxy 1,8-naphthalimides: expanded fluorescence colour palette and: In vitro bioactivity

Article abstract of DOI:10.1039/d0cc01251c

An efficient and functional group tolerant route to access hydroxy 1,8-naphthalimides has been used to synthesise a range of mono- and disubstituted hydroxy-1,8-naphthalimides with fluorescence emissions covering the visible spectrum. The dialkoxy substit

Full text of DOI:10.1039/d0cc01251c

View Article Online
View Journal
ChemComm
Chemical Communications
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: E. E. Rudebeck, R.
P. Cox, T. D. M. Bell, R. Acharya , Z. Feng, N. Guven, T. D. Ashton and F. M. Pfeffer, Chem. Commun.,
2020, DOI: 10.1039/D0CC01251C.
Volume 54
Number
January 2018
Pages 1-112
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
1
4
ChemComm
Chemical Communications
rsc.li/chemcomm
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
shall the Royal Society of Chemistry be held responsible for any errors
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
ISSN 1359-7345
COMMUNICATION
S. J. Connon, M. O. Senge et al.
Conformational control of nonplanar free base porphyrins:
towards bifunctional catalysts of tunable basicity
rsc.li/chemcomm

Page 1 of 4
Please d oC hn eo mt Ca do mj u ms t margins
View Article Online
DOI: 10.1039/D0CC01251C
COMMUNICATION
Mixed alkoxy/hydroxy 1,8-naphthalimides: expanded
fluorescence colour palette and in vitro bioactivity
a
d
d
e
e
Elley E. Rudebeck, Rosalind. P. Cox, Toby D. M. Bell, Rameshwor Acharya, Zikai Feng, Nuri
Received 00th January 20xx,
Accepted 00th January 20xx
e
a,b,c
a
Gueven, Trent D. Ashton * and Frederick M. Pfeffer *
DOI: 10.1039/x0xx00000x
An efficient and functional group tolerant route to access hydroxy
Nevertheless, the majority of examples reported to date
8
1
,8-naphthalimides has been used to synthesise a range of mono- possess relatively simple, single, substituents. Multi-
and disubstituted hydroxy-1,8-naphthalimides with fluorescence substituted 1,8-naphthalimides can offer interesting
9
,10
emssions covering the visible spectrum. The dialkoxy substituted photophysical properties
and recently, Jolliffe and New
compounds prepared possess high quantum yields (up to 0.95) and demonstrated that, for 4-amino-1,8-naphthalimides, additional
1
0
long fluorescent lifetimes (up to 14 ns). The method has been used substituents significantly impact emission properties.
to generate scriptaid analogues that successfully inhibit HDAC6 in Nevertheless, examples in which disubstituted hydroxy/alkoxy
9
,11
vitro with tubulin acetylation assays confirming that these 1,8-naphthalimides have been studied are rare.
compounds are more effective than tubastatin. The most common approach to access 4-alkoxy (and 4-acyloxy)
The 4-hydroxy-1,8-naphthalimide scaffold is, like its 4-amino derivatives involves reacting the 4-hydroxy-1,8-naphthalimide
relative, a photostable fluorophore that possesses a large with an alkylating (or acylating) agent. Critical to this approach,
Stokes shift and two-photon absorption cross-section, as well as is the hydroxylation of the 1,8-naphthalimide core and this has
1
a high quantum yield suitable for imaging applications. With a conventionally been accomplished using a two-step sequence
2
pK
a
~5.5 the 4-hydroxy-1,8-naphthalimdes are, essentially, involving (i) conversion to the methoxy then (ii) cleaving the
1
2
13
fully deprotonated to the corresponding “phenoxide” (1, Figure ether with strong acid (generally HI or Al/I
2
). These
1
) at physiological pH. Under the same conditions the 4-alkoxy conditions are clearly incompatible with acid sensitive
derivatives are blue emissive species (λem ~440 nm) and the functional groups. Other methods include the multi-step
7
controllable interconversion of these spectrally distinct species Staudinger/Sandmeyer approach of Tsukamoto and the one-
1
4
has driven their increasing popularity as ratiometric fluorescent step Pd-catalysed hydroxylation developed by Fleming. Acid-
3
-7
sensors in physiologically relevant conditions (Figure 1).
labile groups were tolerated by the latter method, although
extended reaction times (20–48 h) and high loading (8 mol%) of
the t-BuXPhos or bippyphos ligand were required. A boronate
1
5
ester/H
2
O
2
approach has also been successfully used.
Ar/Lossen-
Hydroxylation of halobenzenes by a tandem S
N
1
6
rearrangement was reported by Maloney using AcNHOH as
the hydroxyl source. Similarly, Du used N-hydroxyphthalimide
and K
naphthalimide to the corresponding 4-hydroxy derivative.
While encouraging, this methodology, with regards to 1,8-
2 3
CO to effect conversion of N-butyl-4-chloro-1,8-
1
7
Figure 1: The physiologically relevant form of 4-hydroxy-1,8-naphthalimide (1) and naphthalimides with multiple hydroxy/alkoxy substituents has
5
,6,9
examples of substituted derivatives.
not been explored.
Herein, using N-hydroxysuccinimide (NHS) as the key
reagent, both 4-hydroxy and 3,4-disubstituted-1,8-
naphthalimides have been accessed and the photophysical
properties of the resultant fluorophores measured. The
method has been exemplified in the development of a small
family of highly potent inhibitors of tubulin deacetylation.
Beginning with the conditions reported by Du N-propyl-4-
chloro-1,8-naphthalimide was heated (80°C) in the presence of
a. School of Life and Environmental Sciences, Deakin University, Waurn Ponds, 3216,
Australia. E-mail: fred.pfeffer@deakin.edu.au
Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3052,
b.
Australia. E-mail: ashton.t@wehi.edu.au
Department of Medical Biology, The University of Melbourne, Parkville, 3010,
Australia.
School of Chemistry, Monash University, Clayton, 3800, Australia
School of Pharmacy and Pharmacology, College of Health and Medicine,
University of Tasmania, Hobart, 7001, Australia.
c.
d.
e.
Electronic Supplementary Information (ESI) available: Full details of synthesis,
optical measurements and in-vitro studies. See DOI: 10.1039/x0xx00000x
Please do not adjust margins

Please d oC hn eo mt Ca do mj u ms t margins
Page 2 of 4
COMMUNICATION
Journal Name
2 3
N-hydroxyphthalimide (1.1 equiv.) and K CO
(3.3 equiv.) in using these conditions. Similarly, Spring and Cui's probe 111
8
View Article Online
1
7
DOI: 10.1039/D0CC01251C
DMSO for 6 hours. However, following the published work-up that has demonstrated lysosomal accumulation was also
2
(dilution in H O and acidification to pH 3), the product collected prepared in 69% yield, although isolation was achieved by
from the reaction mixture, contained an aromatic impurity extraction with 10% MeOH in CH
2
Cl
2
after the product failed to
1
(apparent in the H NMR spectrum). In order to remove this precipitate after acidification to pH 3. For the synthesis of the
impurity (likely derived from N-hydroxyphthalimide) column acid-labile tetrahydropyranyl (THP) protected hydroxamic acid
chromatography was required. 7, DMF was used as the reaction solvent in place of DMSO to
We reasoned that the related N-hydroxysuccinimide (NHS) facilitate solvent removal prior to extractive workup. Following
should partake in the same mechanistic sequence proposed by trituration with Et O, compound 7 was isolated in 69% yield.
Maloney16 to form the more soluble β-alanine which would be
The use of NHS permits access to 3- and 4-substituted 1,8-
2
easily removed during aqueous workup. When NHS (1.1 equiv.) napthalimides with different oxygen containing functional
was used in place of N-hydroxyphthalimide (Scheme 1) groups, a feat not possible using traditional preparations. To
consumption of starting material and formation of the trial the synthesis of disubstituted 1,8-naphthalimides, a series
fluorescent product (λex = 254 nm) was observed after 85 min of 3-alkoxy-4-chloro-1,8-naphthalimides was prepared by
2
using TLC analysis. Dilution of the reaction mixture with H O, treating the corresponding 3-alkoxy-1,8-naphthalimide with N-
then adjustment of the pH to 1 resulted in the precipitation of chlorosuccinimide (NCS) (see ESI for full details). In contrast to
1
9
the desired product 2 which was isolated by vacuum filtration many reports describing NCS mediated chlorination, acid was
1
in 97% yield. There was no evidence by H NMR that the β- not required to effect the transformation.
Using the
3-methoxy-4-chloro-1,8-
3
naphthalimde was reacted with NHS in the presence of K CO .
alanine by-product persisted through the work-up.
hydroxylation
methodology,
2
In comparison with the single substituted 1,8-naphthalimides a
higher temperature (100 °C vs 80 °C) was required to effect the
reaction in a comparable timeframe, nevertheless, the desired
hydroxylated product 12 was isolated as a precipitate in 92%
yield. Similarly, 3-benzyloxy-4-hydroxy-1,8-naphthalimide 13
was obtained in 77% yield, while a 3-(4-bromobenzyloxy)
analogue 14 was isolated in 79% yield. The aryl bromide of 14
provides a synthetic handle which may permit access to
2
0
numerous self-immolative linkers.
Scheme 1: Substrate scope for N-hydroxysuccinimide mediated hydroxylation.
Unless otherwise stated X = Cl: a) X = Br; b) DMF was used in place of DMSO; c)
Scheme 2: Preparation of 3,4-dialkoxy and 3-hydroxy-4-methoxy-1,8-napthalimides.
1
00 °C.
Further functionalisation to afford differentially substituted
A range of N-substituted 4-halo-1,8-naphthalimides were 3,4-dialkoxy 1,8-naphthalimides was readily accomplished
then evaluated as substrates using the modified conditions (Scheme 2). For example, treatment of 3-benzyloxy-4-hydroxy-
Scheme 1). The desired hydroxy product 3 was prepared from 1,8-naphthalimide 13 with benzyl bromide and K CO gave the
both the 4-chloro and 4-bromo-1,8-naphthalimide in high yields corresponding bis-benzyloxy compound 16 in 62% yield. A
87 and 88%, respectively). Various functional groups on the N- similar result was obtained using iodomethane to give 15 in 63%
(
2
3
(
imide were well-tolerated, including carboxylic acids 4 and 5 (98 yield. The 4-chloro-3-alkoxy-1,8-naphthalimides could also be
and 81% yield, respectively), methyl ester 6 (94%), a propargyl directly converted to the corresponding 4-methoxy analogues
handle 8 (88%) and an aryl bromide 9 (98%).
using K
2 3
CO in MeOH at elevated temperatures (≥100 °C) using
The endoplasmic reticulum targeting fluorescent probe 10
,
microwave irradiation. This procedure gives 15, 18 and 19 in
which was reported by Xu et al., was prepared in 92% yield yields of 80–93% while avoiding the use of iodomethane.
1
3
2
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 4
Please d oC hn eo mt Ca do mj u ms t margins
Journal Name
COMMUNICATION
Finally, the 3-hydroxy-4-methoxy derivative 22 was accessed in greener emission is seen (λem = 508 nm). For the 4-hydroxy
View Article Online
DOI: 10.1039/D0CC01251C
analogue 3, in the presence of Et N, a single green-yellow
3
6
2
7% yield by hydrogenolysis (Pd/C, H , MeOH) of 20.
The optical (absorption and emission) properties of all emission band (λem = 558 nm) is recorded which provides a red-
2
1
compounds were determined in DMSO, with hydroxy shifted alternative to 4-amino-1,8-napthalimides. Inclusion of
substituted examples evaluated both in the presence of acid a 3-alkoxy group (e.g. 13 with Et N) results in a bathochromic
TFA) and base (Et N) (See ESI for full details, selected examples shift emission maxima (λem = 590 nm) versus 3. Base induced
are highlighted in Table 1 and Figures 2 and 3). The red-shifts have also been recorded for amide containing 1,8-
3
(
3
2
2
disubstituted 3,4-dialkoxy series (e.g. 15, 16 and 20, Table 1) naphthalimides. The 3-hydroxy-1,8-naphthalimide S11 has a
were efficient cyan fluorophores (λem ~490 nm, λex ~390 nm, Φ red emission (λem = 621 nm). Despite the quantum yields of
F
up to 0.95) with absorption and emission maxima that were red- hydroxy derivatives in DMSO being heavily reduced (<0.10),
shifted approximately 20 and 40 nm, respectively, compared to these compounds have considerable potential to be developed
the blue emissive 4-methoxy derivative 21. Time-resolved as more sophisticated cellular probes and sensing systems
fluorescence studies of the 3,4-dialkoxy examples identified covering a wide range of spectral and intensity readouts.
long fluorescence lifetimes (>12 ns) comprising a single
exponential component (Table 1 and Figure 2). The substitution
pattern, or the effect of a methyl versus benzyl substituent, had
little influence on emission wavelength.
Table 1. Photophysical data for selected alkoxy substituted compoundsa
Compd.

abs (nm)
391

em (nm)
493

F
τ
F
(ns)
χ2
1
1
2
2
5
6
0
1
0.76
0.86
0.95
0.47
14.4
12.3
14.1
5.6
1.05
1.13
1.08
1.07
387
487
386
490
367
449
aAll experiments performed in DMSO. For full details see ESI.
Figure 3: Emission spectra in DMSO of S13 (purple), 21 (blue), 15 (cyan), 22
green), 3 (Et N, yellow), 13 (Et N, orange), S11 (red).
(
3
3
To further illustrate the utility of the method we sought an
additional application. The 1,8-naphthalimide scaffold is
present in the histone deacetylase (HDAC) inhibitor scriptaid,
which has been the subject of a number of recent reports to
2
3,24
develop isoform selective HDAC inhibitors (namely HDAC6).
Primarily, 4-amino-scriptaid analogues have emerged as potent
2
3
and selective inhibitors of HDAC6, however the development
of alkoxy variants has, only recently, received attention when
2
5
Ho et al. reported JW-1 (Figure 4) as a HDAC selective inhibitor
with equivalent activity to the benchmark HDAC6 inhibitor,
2
6
tubastatin (1000-fold selective for HDAC6 over HDAC1), in the
in vitro inhibition of tubulin acetylation.
Using the 4-amino-scriptaid analogues as templates23
a
small library, consisting of seven alkoxy scriptaid analogues, was
synthesised (23–29, Figure 4, see ESI for full details) and
evaluated in the human lung cancer cell line A549 using
immunostaining against acetylated tubulin combined with high
content imaging. While in this assay, increased tubulin
acetylation by tubastatin did not reach statistical significance (p
Figure 2: For compounds 21 (blue), 16 (red), 20 (purple) and 15 (cyan) in DMSO.
Top: Absorption (solid) and emission (dashed) spectra. Bottom: TCSPC decay = 0.06) compared to non-treated and negative (nullscript
histograms and fitted mono-exponential decay functions (λex = 375 nm). The
instrument response function (IRF) is shown in light-green.
treated) controls (Figure 4), all alkoxy analogues that were
prepared as part of this series displayed significantly higher
Depending on the position and combination of 3- and 4- levels (up to ~1.5-fold higher) of tubulin acetylation compared
alkoxy/hydroxy substituents, emission maxima which span a with the non-treated and negative controls. This outcome
significant portion of the visible spectrum can be accessed suggests that the new analogues retain comparable HDAC6
(Figure 3). A single 3-benzyloxy substituent (e.g. S13) results in inhibitory activity to either the 4-amino scriptaid analogues or
a hypsochromic shift of emission maxima (λem = 421 nm) relative JW-1 and further investigation into the influence of the oxy
to 4-alkoxy substitution (e.g. 21). As shown in Table 1, 3,4- substituents on HDAC selectivity is warranted.
dialkoxy substitution results in a cyan emission (λem ~490 nm)
while the 3-hydroxy-4-methoxy substituent pattern 22 a
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please d oC hn eo mt Ca do mj u ms t margins
Page 4 of 4
COMMUNICATION
Journal Name
Notes and references
View Article Online
DOI: 10.1039/D0CC01251C
1
(a) R. M. Duke, E. B. Veale, F. M. Pfeffer, P. E. Kruger and T.
Gunnlaugsson, Chem. Soc. Rev., 2010, 39, 3936; (b) Z. Luo, B.
Yang, C. Zhong, F. Tang, M. Yuan, Y. Xue, G. Yao, J. Zhang and
Y. Zhang, Dyes Pigm., 2013, 97, 52; (c) X. Wu, L. Li, W. Shi, Q.
Gong, X. Li and H. Ma, Anal. Chem., 2016, 88, 1440.
2
3
(a) X.-X. Zhang, H. Wu, P. Li, Z-J. Qu. M.-Q. Tan and K.-L. Han,
Chem. Commun., 2016, 53, 8283; (b) Z. Qu, P. Li, X. Zhang, E.
Wang, Y. Wang and P. Zhou, J. Lumin., 2016, 177, 197.
(a) T. D. Ashton, K. A. Jolliffe and F. M. Pfeffer, Chem. Soc.
Rev., 2015, 44, 4547; (b) J. Tu, M. Xu, S. Parvez, R. T.
Peterson and R. M. Franzini, J. Am. Chem. Soc., 2018, 140,
8
410.
4
Z-R. Dai, G-B. Ge, L. Feng, J. Ning, L-H. Hu, Q. Jin, D-D. Wang,
X. Lv, T-Y. Dou, J-N. Cui and L. Yang, J. Am. Chem. Soc., 2015,
1
37, 14488.
5
6
T. D. Nalder, T. D. Ashton, F. M. Pfeffer, S. N. Marshall and C.
J. Barrow, Biochimie, 2016, 128-129, 127.
L. Feng, Q. Yan, B. Zhang, X. Tian, C. Wang, Z. Yu, J. Cui, D.
Guo, X. Ma and T. D. James, Chem. Commun., 2019, 55,
3
548.
7
8
K. Tsukamoto, S. Shimabukuro, M. Mabuchi and H. Maeda,
Chem. Eur. J., 2016, 22, 8579.
(a) N. I. Georgiev and V. B. Bojinov, J. Lumin., 2012, 132,
2
235; (b) N. V. Marinova, N. I. Georgiev and V. B. Bojinov, J.
Photoch. Photobio. A, 2013, 254, 54.
9
1
R. W. Middleton, J. Parrick, E. D. Clarke and P. Wardman, J.
Heterocycl. Chem., 1986, 23, 849.
0 K. G. Leslie, D. Jacquemin, E. J. New and K. A. Jolliffe, Chem.
Eur. J., 2018, 24, 5569.
1
1
1 T. Kasai, 1969, Kogyo Kagaku Zasshi, 72, 1128.
2 W. Adam, X. Qian and C. R. Saha-Möller, Tetrahedron, 1993,
4
9, 417.
1
3 S. Xu, H.-W. L, X.-X. Hu, S.-Y. Huan, J. Zhang, Y.-C. Liu, L. Yuan,
F.-L. Qu, X.-B. Zhang and W. Tan, Anal. Chem., 2017, 89, 7641
Figure 4. Top: Structures of tubastatin, nullscript, JW-1 and the compounds
prepared in this study. Bottom: Effect of scriptaid analogues on tubulin
acetylation compared against non-treated cells, tubastatin as +ve control and 14 C. L. Fleming, T. D. Nalder, E. H. Doeven, C. J. Barrow, F. M.
nullscript as -ve control. Cells were treated with 1 µM of each of the test
compounds for 24 hours and acetylated tubulin levels were quantified by
automated high content imaging from at least 1000 cells per condition. Data
represent the average of 4 different experiments with 4 replicate wells for each
experiment. Statistical significance was determined using Welsh t-test (Graph pad
Prism). Significance was set as p ≥ 0.05 = non-significant, 0.05 > p ≥ 0.01 = *, 0.01
Pfeffer and T. D. Ashton, Dyes Pigm., 2016, 126, 118.
5 (a) W. Sun, W. Li, J. Li, J. Zhang, L. Du and M. Li, Tetrahedron,
2012, 68, 5363; (b) B. Zhu, H. Jiang, B. Guo, C. Shao, H. Wu,
B. Du and Q. Wei, Sensor. Actuators. B Chem., 2013, 186,
1
>
p ≥ 0.002 = **, and p < 0.002 = *** Error bars represent SD.
6
81.
1
1
6 P. S. Fier and K. M. Maloney, Org. Lett., 2016, 18, 2244.
7 W. Shu, L. Yan, J. Liu, Z. Wang, S. Zhang, C. Tang, C. Liu, B.
Zhu and B. Du, Ind. Eng. Chem. Res., 2015, 54, 8056.
In conclusion a facile, functional group tolerant, method for
the hydroxylation of 4-halo-1,8-naphthalimides has been
developed. The method uses inexpensive reagents, and the 18 T. Liu, Z. Xu, D. R. Spring and J. Cui, Org. Lett., 2013, 15, 2310.
desired products are isolated in excellent yield (typically >80%) 19 (a) T. Mahajan, L. Kumar, K. Dwivedi and D. D. Agarwal, Ind.
Eng. Chem. Res., 2012, 51, 3881; (b) W. M. Gołębiewski and
M. Gucma, Synthesis, 2007, 23, 3599.
0 A. Alouane, R. Labruére, T. Le Saux, F. Schmidt and L. Jullien,
Angew. Chem. Int. Ed., 2015, 54, 7492.
with no requirement for chromatography. Based on the readily
accessed 4-hydroxynaphthalimide scaffold a highly active series
of tubulin acetylation inhibitors were synthesised. The method
2
is equally effective using either 4-bromo- and 4-chloro-1,8- 21 C. L. Fleming, T. D. Ashton and F. M. Pfeffer, Dyes Pigm.,
naphthalimides and can be used to produce previously
inaccessible 3-alkoxy-4-hydroxy-1,8-naphthalimides. The newly
described hydroxylation conditions ultimately allow for the
simple, functional-group tolerant synthesis of mixed 3,4-
alkoxy/hydroxy 1,8-naphthalimides, a feat that was impossible
to achieve using other literature conditions. These new
fluorophores possess emissions covering the visible spectrum
with the 3,4-dialkoxy analogues displaying high quantum yields
and long fluorescent lifetimes.
2014, 109, 135.
2
2
2
2 K. N. Hearn, T. D. Nalder, R. P. Cox, H. D. Maynard, T. D. M.
Bell, F. M. Pfeffer and T. D. Ashton, Chem. Commun., 2017,
5
3, 12298.
3 C. L. Fleming, A. Natoli, J. Schreuders, M. Devlin, P.
Yoganantharajah, Y. Gibert, K. G. Leslie, E. J. New, T. D.
Ashton and F. M. Pfeffer, Eur. J. Med. Chem., 2019, 162, 321.
4 (a) C. L. Fleming, T. D. Ashton, C. Nowell, M. Devlin, A. Natoli,
J. Schreuders and F. M. Pfeffer, Chem. Commun., 2015, 51,
7
2
827; (b) Y. Zhang, J. Yan and T-P. Yao, Eur. J. Med. Chem.,
017, 141, 596.
2
2
5 Y.-H. Ho, K.-J. Wang, P.-Y. Hung, Y.-S. Cheng, J.-R. Liu, S.-T.
Fung, P.-H. Liang, J.-W. Chern and C.-W. Yu, Org. Biomol.
Chem., 2018, 16, 7820.
6 K. V. Butler, J. Kalin, C. Brochier, G. Vistoli, B. Langley and A. P.
Kozikowski, J. Am. Chem. Soc., 2010, 132, 10842.
Conflicts of interest
There are no conflicts to declare
4
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins