Direct amidation to access 3-amido-1,8-naphthalimides including fluorescent scriptaid analogues as hdac inhibitors

Article abstract of DOI:10.3390/cells10061505

Methodology to access fluorescent 3-amido-1,8-naphthalimides using direct Buchwald– Hartwig amidation is described. The protocol was successfully used to couple a number of substrates (including an alkylamide, an arylamide, a lactam and a carbamate) to 3-bromo-1,8-naphthalimide in good yield. To further exemplify the approach, a set of scriptaid analogues with amide substituents at the 3-position were prepared. The new compounds were more potent than scriptaid at a number of histone deacetylase (HDAC) isoforms including HDAC6. Activity was further confirmed in a whole cell tubulin deacetylation assay where the inhibitors were more active than the established HDAC6 selective inhibitor Tubastatin. The optical properties of these new, highly active, compounds make them amenable to cellular imaging studies and theranostic applications.

Full text of DOI:10.3390/cells10061505

cells
Article
Direct Amidation to Access 3-Amido-1,8-Naphthalimides
Including Fluorescent Scriptaid Analogues as HDAC Inhibitors
Kyle N. Hearn 1,2, Trent D. Ashton
and Frederick M. Pfeffer *
1,3,4
, Rameshwor Acharya , Zikai Feng 5 , Nuri Gueven 5
5
1
,
1
2
3
4
5
School of Life and Environmental Sciences, Deakin University, Waurn Ponds, VIC 3216, Australia
STEM College, RMIT University, Melbourne, VIC 3000, Australia; kyle.hearn@rmit.edu.au
Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; ashton.t@wehi.edu.au
Department of Medical Biology, The University of Melbourne, Parkville, VIC 3010, Australia
School of Pharmacy and Pharmacology, College of Health and Medicine, University of Tasmania,
Hobart, TAS 7001, Australia; ra26@utas.edu.au (R.A.); zikai.feng@utas.edu.au (Z.F.);
nuri.guven@utas.edu.au (N.G.)
*
Correspondence: fred.pfeffer@deakin.edu.au
Abstract: Methodology to access fluorescent 3-amido-1,8-naphthalimides using direct Buchwald–
Hartwig amidation is described. The protocol was successfully used to couple a number of substrates
(including an alkylamide, an arylamide, a lactam and a carbamate) to 3-bromo-1,8-naphthalimide in
good yield. To further exemplify the approach, a set of scriptaid analogues with amide substituents
at the 3-position were prepared. The new compounds were more potent than scriptaid at a number
of histone deacetylase (HDAC) isoforms including HDAC6. Activity was further confirmed in a
whole cell tubulin deacetylation assay where the inhibitors were more active than the established
HDAC6 selective inhibitor Tubastatin. The optical properties of these new, highly active, compounds
make them amenable to cellular imaging studies and theranostic applications.
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ꢁꢂꢃꢄꢅꢆ
Citation: Hearn, K.N.; Ashton, T.D.;
Acharya, R.; Feng, Z.; Gueven, N.;
Pfeffer, F.M. Direct Amidation to
Access 3-Amido-1,8-Naphthalimides
Including Fluorescent Scriptaid
Keywords: Buchwald–Hartwig; scriptaid; histone deacetylase; HDAC; 1,8-naphthalimide; fluores-
cence; imaging; tubulin deacetylase
Analogues as HDAC Inhibitors. Cells
2
021, 10, 1505. https://doi.org/
1
0.3390/cells10061505
1
. Introduction
Interest in functionalised 1,8-naphthalimides has primarily focussed on substitution at
the 4-position to generate fluorophores suitable for sensing and imaging applications [ ].
Academic Editors: Doug Brooks,
Alexandra Sorvina and Shane Hickey
1–4
Received: 11 May 2021
Accepted: 7 June 2021
Published: 15 June 2021
Examples where sensors have been modified in the 3-position are less common, with
notable examples including those reported by Zhang et al., Guo et al. and Elmes et al.
for the detection of CO , ClO and reductive stress, respectively [
−
5–
7]. Examples that
2
incorporate a 3-amido substituent are particularly rare [
5
,
8
–
10], likely due to the multistep
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
nature of the synthetic protocols required to access them. We have recently demonstrated
that in the synthesis of 4-amido-1,8-naphthalimides, the usual three-step approach can be
avoided using a Buchwald–Hartwig cross-coupling protocol in which a range of amides as
well as lactams, carbamates and urea can be introduced in a single step [11]. Nicotinamides
were also successfully coupled and the resultant probes shown to act as reversible indicators
of the cellular redox state [12]. This direct coupling approach has not yet been evaluated
for introducing substituents at the 3-position.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
The 1,8-naphthalimide core has also been employed in medicinal chemistry [13
Relevant examples (Figure 1) include (i) scriptaid [15] (an inhibitor of histone deacetylases
HDAC)) and amonafide [16 19] (a DNA intercalator and topoisomerase II inhibitor).
,14].
(
–
Amonafide is converted in vivo into the bioactive N-acetyl-amonafide.
The HDAC family has become well-studied due to their roles in a number of disease
states [20
–22]. Indeed, a number of HDAC inhibitors are FDA-approved for clinical use as
treatments for T-cell lymphoma or myeloma [23
–
27]. In an effort to mitigate side effects, the
4
.0/).
Cells 2021, 10, 1505. https://doi.org/10.3390/cells10061505
https://www.mdpi.com/journal/cells

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next generation of HDAC inhibitors have been developed to be selective for specific HDAC
classes or isoforms [28–30]. In this context, HDAC6 (Class IIb) has emerged as a valuable
target as it has a clear role in the progression of a number of cancer types, and, unlike other
isoforms, mouse models in which this isoform has been deleted are viable [31,32].
Figure 1. Examples of 1,8-naphthalimides in medicinal chemistry.
The entrance to the HDAC6 active site is slightly larger [33,34] than that of the other
isoforms, and therefore a successful strategy for enhancing selectivity for this isoform is
the modification of the pharmacophoric capping group. Amides offer not just a means to
introduce additional size; they present both an H-bond donor and acceptor to maximise
potential interactions with residues at the active site periphery [35–37].
There are currently only three published examples of scriptaid analogues with substi-
tution at the 3-position, and only compound
3 has full HDAC IC50 activity data recorded
(
Figure 2) [38 40]. There are no scriptaid analogues with 3-amido substituents described
–
in the literature. As such, the generation of a small set of 3-amido-substituted scriptaid
analogues presents the opportunity to (i) test the Buchwald–Hartwig amidation method-
ology beyond the 4-position and to (ii) further explore the structure-activity relationship
of functionalised scriptaid analogues. Our own recent work (CF010 and CF011, Figure 2)
has identified that, for the 4-position, relatively small substituents can dramatically influ-
ence isoform selectivity and fluorescence properties [41]. While our efforts have focussed
on modifying scriptaid to develop highly fluorescent anticancer agents, related recent
examples exist for identifying biofilms and detecting influenza [42,43].
Figure 2. Top: All published examples of 3-substituted scriptaid analogues [38
selected examples of 4-substituted scriptaid analogues from our recent work [41]. No activity data
for 1 and 2 has been published. Selectivity factor (SF) = HDAC1 IC /HDAC6 IC .
–40], and Bottom:
5
0
50

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Herein we outline the methodology for the direct synthesis of 3-amido-1,8-naphthalimides
and the use of this approach to construct a set of scriptaid analogues. The results of the photo-
physical and biological assessment (IC50 and in cell tubulin acetylation) are also presented.
2. Results and Discussion
2.1. Methodology
To trial the Buchwald–Hartwig amidation methodology, 3-bromo-1,8-naphthalimide 4 was
first required. The treatment of 1,8-naphthalic anhydride with 1.1 equiv. N-bromosuccinimide in
H SO provided the desired brominated compound in >90% purity after trituration with
EtOH [44]. The bromoanhydride was then converted to the corresponding methoxyethyl
imide using microwave irradiation (Scheme 1, see supplementary material for full details).
4
2
4
5
Scheme 1. Direct synthesis of 3-amido and 3-carbamato-1,8-naphthalimides.
Using the reported conditions to introduce amides to the 4-position as a guide [11],
a test reaction was performed in which 3-bromo-1,8-naphthalimide
5 was reacted with
◦
benzamide, Cs CO and G3-xantphos at 100 C (Scheme 1). Monitoring the reaction
2
3
progress using thin layer chromatography (TLC) indicated the consumption of the starting
material and the appearance of a new blue spot (using UV visualisation) within 2.5 h,
whereupon the reaction mixture was poured over H O to provide a yellow solid. The
2
1
analysis of the solid using H NMR spectroscopy revealed new aryl resonances centred at
8
.08 and 7.60 ppm and a new broad singlet at 10.81 ppm that was assigned to the amide
N-H proton. Evaluation using HRMS further confirmed that the desired 3-benzamido-
,8-naphthalimide had been synthesised. The product was obtained in good yield (84%)
1
6
and as such several other substrates—an aliphatic amide (propionamide), a lactam (pyrro-
lidinone) and a carbamate (tert-butyl carbamate)—were also trialled as reaction partners.
These additional reactions also provided the desired 3-substituted 1,8-naphthalimide in
under 3 h and in good yields (61–81%, Scheme 1). The substrates were well tolerated with
yields and reaction times comparable to those observed for the coupling reactions that
used 4-bromo-1,8-naphthalimide [11]. In terms of reactivity, 3-bromo-1,8-naphthalimides
are an excellent, readily accessible substrate for palladium-mediated amidation reactions.
The new compounds showed an absorption maximum and a secondary maximum
(~340 and ~380 nm, respectively, Table 1 and Figure 3) and a single emission (~440 nm,
Table 1 and Figure 3). Consistent with the decreased extent of intramolecular charge
transfer (as compared to the amino substituted examples), the quantum yields for the new
compounds ranged from 0.02 to 0.06 (Table 1). For comparison, the 4-benzamido isomer of
6
has Φf = 0.33 and for the 4-fluorobenzamido Φf = 0.05 [11]. It is our experience that even
with low values the compounds are amenable to imaging applications [45].

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Table 1. Photophysical properties of 3-amido-1,8-naphthalimides in DMSO.
†
Φ
f
Compound
λ
(nm)
λem (nm)
Stokes Shift (nm)
abs
‡
6
7
8
9
344, 385
341, 383
343, 383
345, 367
435
439
441
442
91, 50
98, 56
98, 58
97, 75
0.02
0.04
0.02
0.06
‡
‡
‡
†
Average of two independent samples. ‡ Secondary absorption maxima.
Figure 3. Normalised absorption and emission spectra of 6 in DMSO.
.2. Scriptaid Analogues
The most potent of the previously synthesised 4-amino series were CF010 and CF011
2
(Figure 2) possessing propyl and benzyl substituents, respectively [41]. As such, two
aromatic and one aliphatic amide were chosen as suitable substituents for the new 3-amido
analogues (KNH019, 020 and 021, respectively, Figure 4).
Figure 4. Target 3-amido scriptaid analogues.
The strategy reported by Fleming et al. for the construction of 4-substituted scriptaid
analogues was followed here for the synthesis of the 3-substituted relatives [41]. First,
3
(
-bromo-1,8-naphthalic anhydride
96% over two steps, see supplementary material for details). Next, the key Buchwald–
Hartwig reaction was used to introduce the amido-substituent (Scheme 2). For example, in
4 was transformed into imide 11 in an excellent yield
the synthesis of KNH019 a solution of 3-bromo-1,8-naphthalimide 11, benzamide, Cs CO
2
3
◦
and G3-xantphos in 1,4-dioxane were heated at 100 C with the reaction progress being
monitored using TLC. Following reaction completion (3.5 h), the desired product 12 was
isolated as a light brown solid (63% yield). The methyl ester was carefully removed

Cells 2021, 10, 1505
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(
1.7 equiv. of LiOH in THF/H O), and the resultant carboxylic acid 13 was converted to
2
the desired hydroxamic acid via the mixed anhydride (formed using ethyl chloroformate
and an in-situ treatment with a solution of freshly prepared NH OH in MeOH). After
2
2
4 h, the solvent was removed and the residue was triturated with H O to provide the
2
1
desired hydroxamic acid KNH019 in a good yield (76% over two steps). In the H NMR
spectrum, two broad singlets at 10.34 and 8.68 ppm (each integrating for one proton) were
characteristic of the newly installed hydroxamic acid N-H and O-H protons, respectively.
The singlet corresponding to the benzamide N-H proton at 10.85 ppm was also present,
indicating the successful formation of the 3-amido scriptaid analogue.
Scheme 2. Synthesis of 3-benzamido scriptaid analogue KNH019.
The remaining scriptaid analogues KNH020 and KNH021 were accessed in a good
yield using the same reaction sequence (see supplementary material for full details). As
with the 3-amido-1,8-naphthalimides synthesised at the outset (6–9), the quantum yield of
the new inhibitors ranged from 0.03 to 0.05 (Table 2) with emission at λem ~437 nm.
Table 2. Photophysical properties of 3-amido scriptaid analogues in DMSO.
†
Φ
f
Compound
λ
(nm)
λem (nm)
Stokes Shift (nm)
abs
‡
KNH019
KNH020
KNH021
343, 386
343, 374
341, 383
437
437
435
94, 51
94, 63
94, 54
0.03
0.05
0.04
‡
‡
†
Average of two independent samples. ‡ Secondary absorption maxima.
2.3. HDAC Inhibition
The new scriptaid analogues were initially assessed using a single point assay where
enzyme activity is reported as a percentage of full enzyme activity (Table 3). For HDAC
isoforms 1, 3, 8 and 11, a concentration of 10 M of the inhibitor was used. Against
HDAC6, inhibition was determined using 0.01 M of the inhibitor. All 3-amido-1,8-
µ
µ
naphthalimide analogues were significantly more effective inhibitors of HDAC6 than the
control compound scriptaid. However, no substantial difference in HDAC6 enzyme activity
was observed between the benzamide, p-methoxybenzamide and propionamide analogues.
Furthermore, when the 3-amido scriptaid analogues were evaluated at the “off-target”
HDAC isoforms (Table 3), considerable activity was noted; again, they were more effective
than scriptaid.

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Table 3. HDAC activity (percentage of full enzyme activity) for 3-amido scriptaid analogues.
Average Enzyme Isoform Activity (%) †
HDAC6: 0.01 µM; HDAC1 3, 8, 11: 1.0 µM
Compound
HDAC1
HDAC3
HDAC6
HDAC8
HDAC11
scriptaid
KNH019
KNH020
KNH021
43.2%
36.3%
38.7%
29.8%
43.8%
24.1%
17.2%
20.1%
39.8%
20.4%
19.7%
20.3%
60.3%
69.3%
93.4%
50.4%
78.6%
67.5%
58.7%
90.7%
†
Average of two independent measurements, margin of error ±2% (see supplementary material for details).
The IC50 for the three compounds against the same isoform panel was then measured
Table 4). In agreement with the results of the single point assay, all compounds were
(
potent inhibitors of HDAC6, with similar IC50 values (ranging from 0.58 nM to 1.0 nM).
Compared to our previously reported 4-amino analogues CF010 and CF011 [41], the new
compounds were more effective inhibitors of HDAC6 (by an order of magnitude) and
compared favourably with the 3-methoxy analogue
3 reported by Ho [39]. Based on
the inhibition of other isoforms, the selectivity of the new compounds was considered
fair to good (ranging from 38 to 150 for HDAC6 over HDAC1); however, when the 3-
substituted series were compared to the previous 4-amino analogues CF010 and CF011 (up
to 566-fold selectivity for HDAC6, Table 4), they were universally less selective. While the
set of compounds produced for this study was not extensive, it would appear that amido
substitution at the 3-position of the 1,8-naphthalimide core leads to a slightly enhanced
activity at all HDAC isoforms to ultimately provide highly potent but only moderately
selective HDAC6 inhibitors.
Table 4. HDAC activity (IC ) and isoform selectivity factor for 3-amido scriptaid analogues.
5
0
Isoform IC50 (µM) † and Selectivity Factor (SF) against HDAC6
Compound
scriptaid
CF010
HDAC1
HDAC3
HDAC6
HDAC8
HDAC11
1
0
1
.74 ± 0.04
0.37 ± 0.04
1.52 ± 0.007
0.36 ± 0.02
0.012 ± 0.002
(145)
(31)
(127)
(30)
.59 ± 0.02
123)
.98 ± 0.04
566)
.091
0.11 ± 0.004
(23)
1.52 ± 0.08
(317)
0.08 ± 0.03
(16)
0.0048 ± 0.0002
0.0035 ± 0.0002
0.0010 ± 0.000005
0.00058 ± 0.000002
0.00097 ± 0.00005
(
0.36 ± 0.0007
2.46 ± 0.11
0.15 ± 0.02
CF011
(
(103)
(703)
(43)
0
0
0
±
0.006 0.064 ± 0.0002
2.95 ± 0.46
(2950)
0.29 ± 0.035
(290)
KNH019
KNH020
KNH021
(
91)
0.007
150)
.037 0.002
38)
(64)
0.027 ± 0.003
(47)
.087
±
4.33 ± 0.39
0.24 ± 0.011
(
(7466)
(413)
±
0.032 ± 0.001
0.95 ± 0.05
(981)
2.16 ± 0.14
(2234)
(
(332)
†
Average of two independent measurements (see supplementary material for details).
To confirm the observed activities of the selected test compounds against HDAC6,
the acetylation status of tubulin in the human lung cancer cell line A549 was investigated
by immunostaining combined with automated high content image analysis (see supple-
mentary material for full details). In addition to the previously published 4-benzylamino
scriptaid analogue CF011 (HDAC6 IC50 = 3.5 nM, SF = 566, Table 4), the structurally related
3-benzamido scriptaid KNH019 (HDAC 6 IC50 = 1.0 nM, SF = 91) was tested with regards
to dose response and time dependency, where both acetylated tubulin and acetylated his-
tone were detected (Figure 5A–E). Compared to untreated cells, both CF011 and KNH019
rapidly increased tubulin acetylation in a dose-dependent manner by up to
∼
1.5-fold from
−
9
−6
1
0
mM and 10 mM, respectively. In contrast, the reference compound tubastatin only
−
3
demonstrated a significantly increased tubulin acetylation from 10 mM in the same
test system (Figure 5A). When acetylated histone was detected for both test compounds,
−
6
CF011 showed a significant increase from 10 mM, while KNH019 only demonstrated

Cells 2021, 10, 1505
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a significant activity at 10 2 mM. Surprisingly, the reference compound tubastatin in-
−
−
3
creased histone acetylation from 10 mM, similar to its activity against tubulin acetylation
Figure 5A,B). The apparent drop in histone acetylation by both compounds above 10
(
µM
is indicative of a potential cytotoxicity at higher concentrations (data not shown). Why this
effect did not also manifest as reduced tubulin acetylation is unclear at this point.
Figure 5. Cell-based assessment of HDAC activity and selectivity. Dose and time-dependency of
tubulin and histone acetylation by test compounds. A549 cells were treated with (
concentrations (0–50 M) of CF011 KNH019 and tubastatin for 24 h or with ( ) 1 M over different
time intervals before ( ) tubulin acetylation or ( ) histone acetylation were automatically quan-
A,B) different
µ
,
C
,D
µ
A,
C
B,D
tified using high content imaging. Data represent the average of four independent experiments with
four replicate wells/experiments. The statistical significances of effects compared to the untreated
control were analysed using Brown–Forsythe and Welch one-way ANOVA analyses using Graph
Pad Prism. The significance was set as * 0.05 > p
the Standard Error of Mean (SEM). ( ) Representative fluorescence images show tubulin acetylation
yellow), histone acetylation (red) and nuclear counterstain (DAPI, blue) after 24 h of exposure to test
compounds. Secondary antibodies were detected using excitation/emission filters for CY3 (excitation:
42/27 nm, emission: 597/45 nm), CY5 (excitation: 632/22 nm, emission: 684/25 nm) and DAPI
excitation: 390/18 nm, emission: 435/48 nm), respectively.
≥
0.01, ** 0.01 > p
≥
0.002. Error bars represent
E
(
5
(

Cells 2021, 10, 1505
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In A549 cells, all test and reference compounds rapidly increased tubulin and histone
acetylation within only a few hours (Figure 5C,D). After 4 h, the increases in tubulin acety-
lation reached a plateau that was sustained for up to 24 h (data not shown) (Figure 5C,D)
Representative immunostaining images also demonstrate that both CF011 and KNH019
at 10 M strongly induce tubulin and histone acetylation. This illustrates that the anti-
.
µ
bodies used can differentiate between nuclear and cytoplasmic targets and that at this
concentration both compounds therefore do not seem to be HDAC6 selective, which pre-
dominantly resides in the cytoplasm (Figure 5E). Similar results have been generated in
the hepatocarcinoma cell line HepG2 (See supplementary material Figure S24 for details),
which highlights that the observed effects are not cell line-specific but are likely relevant
for other cells and tissues.
Overall, the data clearly indicate that the new compounds retain HDAC6 activity in
cells with a significantly higher potency compared to the reference compound tubastatin.
A broader HDAC activity was confirmed for the test and reference compounds when
measuring both tubulin and histone acetylation, which reflected the residual activity of
isoforms other than HDAC6. The discrepancy between the cell-free and cellular activity
observed in this study was reported for tubastatin before and should be considered when
using tubastatin as a reference compound [46].
3. Conclusions
In summary, the direct amidation of 3-bromo-1,8-naphthalimides was readily achieved
using the Buchwald–Hartwig methodology to give amido, lactam and carbamato products.
The method was used to produce a set of novel fluorescent scriptaid analogues, and, as
identified using direct IC50 measurements and whole cell tubulin deacetylation assays,
the analogues were potent (but less selective) inhibitors of histone deacetylase enzymes.
The fluorescent nature of these compounds makes them well-suited as tools for additional
cell-based studies.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/
0.3390/cells10061505/s1, Pages S2–S11: Experimental procedures, Figures S1–S16: 1H and
13
C
1
NMR of synthesised compounds, Table S1: Photophysical properties, Figures S17–S24: UV/Vis and
fluorescence data, Figure S24: Cell-based assessment of HDAC activity and selectivity in HepG2 cells.
Author Contributions: Conceptualisation, T.D.A. and F.M.P.; methodology, K.N.H., T.D.A., N.G. and
F.M.P.; investigation, K.N.H., R.A., Z.F. and N.G.; writing—original draft preparation, K.N.H., N.G.
and F.M.P.; writing—review and editing, K.N.H., T.D.A., N.G. and F.M.P. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data has been presented in this article or associated supplemen-
tary material.
Acknowledgments: The authors would like to acknowledge the assistance of Shane Hickey (University
of South Australia) in the characterisation of new compounds using high-resolution mass spectrometry.
Conflicts of Interest: The authors declare no conflict of interest.
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