Highly fluorescent and HDAC6 selective scriptaid analogues

Article abstract of DOI:10.1016/j.ejmech.2018.11.020

Fluorescent scriptaid analogues with excellent HDAC6 selectivity (HDAC1/6 > 500) and potency (HDAC6 IC₅₀ < 5 nM) have been synthesised and evaluated. The highly fluorescent nature of the compounds (up to Φ_F = 0.83 in DMSO and 0.38 in aqueous buffer) makes them ideally suited for cellular imaging and visualisation of their cytoplasmic localisation was readily accomplished. Whole organism imaging in zebrafish confirmed both the vascular localisation of the new inhibitors and the impact of HDAC6 inhibition on in vivo development.

Full text of DOI:10.1016/j.ejmech.2018.11.020

Accepted Manuscript
Highly fluorescent and HDAC6 selective scriptaid analogues
Cassandra L. Fleming, Anthony Natoli, Jeannette Schreuders, Mark Devlin,
Prusothman Yoganantharajah, Yann Gibert, Kathryn G. Leslie, Elizabeth J. New,
Trent D. Ashton, Frederick M. Pfeffer
PII:
S0223-5234(18)30978-4
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.020
EJMECH 10877
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 August 2018
Revised Date: 28 October 2018
Accepted Date: 8 November 2018
Please cite this article as: C.L. Fleming, A. Natoli, J. Schreuders, M. Devlin, P. Yoganantharajah,
Y. Gibert, K.G. Leslie, E.J. New, T.D. Ashton, F.M. Pfeffer, Highly fluorescent and HDAC6 selective
scriptaid analogues, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.11.020.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Highly Fluorescent and HDAC6 Selective Scriptaid Analogues
Cassandra L. Fleming,a Anthony Natoli,b Jeannette Schreuders,b Mark Devlin,b Prusothman
Yoganantharajah,c Yann Gibert,c Kathryn G. Leslie,d Elizabeth J. New,d Trent D. Ashton,a,e,f and
Frederick M. Pfeffera*
a School of Life & Environmental Sciences, Deakin University, Waurn Ponds, Victoria, 3216,
Australia.
b Peter MacCallum Cancer Centre, Parkville, Melbourne, Victoria, 3000, Australia.
c School of Medicine, Deakin University, Waurn Ponds, Victoria, 3216, Australia.
d School of Chemistry, The University of Sydney, Sydney, New South Wales, 2006, Australia.
e Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3052, Australia.
f Department of Medical Biology, The University of Melbourne, Parkville, Victoria, 3010, Australia.
Keywords
Naphthalimide; Fluorescence; Imaging; Zebrafish; Scriptaid; 4MS, HDAC
Abstract
Fluorescent scriptaid analogues with excellent HDAC6 selectivity (HDAC1/6 > 500) and potency
(HDAC6 IC50 < 5 nM) have been synthesised and evaluated. The highly fluorescent nature of the
compounds (up to ΦF = 0.83 in DMSO and 0.38 in aqueous buffer) makes them ideally suited for
cellular imaging and visualisation of their cytoplasmic localisation was readily accomplished. Whole
organism imaging in zebrafish confirmed both the vascular localisation of the new inhibitors and the
impact of HDAC6 inhibition on in vivo development.
1 INTRODUCTION
Given their important physiological roles, members of the histone deacetylase (HDAC) family are
attractive targets for therapeutic intervention,1-6 with four HDAC inhibitors (HDACi)—vorinostat
(2006, Zolinza®),7 romidepsin (2009, Istodax®),8 belinostat (2014, Belodaq®)9 and panobinostat (2015,
Farydak®)10— FDA-approved to date for the treatment of peripheral T-cell lymphoma (PTCL) and
multiple myeloma. In addition, chidamide (2015, Epidaza®)11 has been approved for use in China.
Recent concerns regarding the off-target effects associated with non-isoform-selective (pan) inhibitors
have spurred efforts towards agents that are selective amongst the 18 HDAC isoforms, in particular
those that target HDAC6.2, 12-14 This class IIb isoform is located primarily in the cytoplasm and has
demonstrated roles including α-tubulin deacetylation and the regulation of cytoskeletal dynamics.1, 13,
15-19
Like other isoforms HDAC6 is clearly linked to cancer progression, however, its phenotype is
unique in that mice with HDAC6 deletion are viable.20 As such targeting HDAC6 represents a
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potentially less cytotoxic avenue to cancer treatment,21 a strategy that has been shown to have merit in
a melanoma xenograft mouse model.22 As such, the design and synthesis of HDAC6 selective
inhibitors has become an important pursuit (examples shown in Figure 1).16, 21, 23-30
Figure 1. Selected examples of HDAC6 inhibitors: Ricolinostat (ACY-1215),23 Tubacin,24 isoxazole (1),25
Tubastatin A,26 Nexturastat A,27 oxazole (2)28 and HPOB.29 Ricolinostat, tubacin and isoxazole (1) have been
highlighted to show the typical HDACi pharmacophore of capping group-spacer-Zn binding group. a SF;
selectivity for HDAC6 vs HDAC1.
Inhibitors of HDAC, regardless of whether they are pan-inhibitors or isoform specific, typically
consist of a zinc binding group (ZBG) linked through a spacer to a capping group (highlighted as red,
blue and black respectively in Figure 1).31 The similarity between the active sites of the different
isoforms makes achieving selectivity a non-trivial task and HDACi designed using the typical
template are in many instances active against a number of isoforms.32-34 Nevertheless, selectivity for
HDAC6 has been successfully achieved through modifications of the capping group of the HDACi
(see Figure 1) as this section of the inhibitor interacts with a region known to be less conserved
amongst the HDAC family and hence is a valuable option for discrimination.13, 26, 32 Similarly, it has
been noted that the entrance to the active site of HDAC6 is slightly larger than that of the other
isoforms and as a consequence HDAC6 selective inhibitors often feature a larger capping group (for
example Tubacin and Tubastatin A, Figure 1).24, 26, 35 The introduction of H-bond donor/acceptor
groups to the capping group has also been shown to be an effective strategy for targeting HDAC6.25,
29, 36, 37
Many of these HDAC6 selective inhibitors have been investigated as anticancer agents in their
own right,38 and considerable evidence is now available to support their use in combination therapy
with other anticancer agents.23, 39-42
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Many HDACi have therapeutic potential beyond cancer, for example, the well-known scriptaid
44
(Figure 2) identified in 2000 by Su et al,43,
has been investigated for the treatment of HIV,
neurodegenerative disorders and to enhance muscle metabolism.5, 45-47 Scriptaid is a hydroxamic acid-
based inhibitor and its structure follows that of the archetypal HDAC pharmacophore.13 Scriptaid has
a modest preference for HDAC6,‡ 48-50 yet, despite its well established anticancer activity,51, 52 SAR
studies regarding the capping group are limited, but do include a 3-nitro derivative53 and a
naphthalenediimide.54 Other examples include 4-bromo, 4-alkyl and 3-hydroxy substituents on the
naphthalimide ring, but these analogues employ the aminobenzamide moiety in place of the
hydroxamic acid as the zinc-binding unit,55 a modification known to favour class 1 HDAC
inhibition.56
Figure 2. Structure of scriptaid and 4MS (ΦF reported for DMSO).50 a SF; selectivity for HDAC6 vs HDAC1.
In a recent report, a morpholine substituted scriptaid derivative (4MS, Figure 2) was identified as a
fluorescent analogue that closely matched scriptaid in terms of inhibitory profile and in vitro
activity.50 A subsequent report by Zhang described a successful modification of 4MS in which an
arylhydroxamic acid (similar to tubastatin A) was used as the zinc binding unit.57 Fluorescent
molecular probes are of remarkable utility in elucidating and quantifying fundamental biological
processes.58-64 In addition, biologically active fluorescent compounds have great potential as
theranostic agents.65-69 Examples such as 4MS are particularly valuable as, in contrast to the ‘tagging’
approach, only minor structural modifications are required to generate the fluorescent analogue and
essential parameters such as molecular weight and polar surface area are not greatly impacted.
Although 4MS is only weakly fluorescent (ΦF = 0.03 in DMSO) rapid cellular uptake and subsequent
localisation in the cytoplasm of human breast cancer (MDA-MB-231) cells was readily visualised.50
Cytoplasmic localisation of HDACi has in itself been proposed as a means to elicit selectivity as
HDAC6 is primarily found outside the nucleus.66 Reports of fluorescent HDACi are not yet
widespread in the literature,57, 66, 70, 71 however, they have already been employed as displacement
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indicators72 and theranostics.66 Nevertheless, inhibitors that combine HDAC6 selectivity with strong
fluorescence are rare.
1.1 Design
With the dual aims of (i) creating analogues with increased fluorescence quantum yields and (ii)
investigating modifications to the capping group to enhance HDAC6 selectivity, we designed 4-amino
analogues 3–8 for synthesis and evaluation in the current study (Figure 2, 4-aminoaryl 3 and 4, 4-
aminoalkyl 5–7, and 4-nitro 8). The aminobenzamide analogue of 4MS was also of interest (9, Figure
2) for comparison against the aminobenzamide previously prepared by Delorme (10) that possessed
no morpholine substituent at the 4-position of the naphthalimide ring.55
Figure 3. Design of scriptaid analogues 3–10 investigated herein.
The synthesis, together with the photophysical and biological evaluation, of this collection is
presented herein. The utility of these compounds is demonstrated by cellular imaging and whole
organism in vivo imaging, in which the distribution and the developmental impact of the inhibitors
can be clearly visualised.
2 RESULTS AND DISCUSSION
2.1 Synthesis
In the 4-aminoscriptaid series, compound 7, despite being disubstituted, was readily constructed; both
the methylbenzyl and N-alkyl carboxylic acid moieties could be introduced in one-pot (Scheme 2).73
Commercially available 4-chloronaphthalic anhydride 11 was heated with 6-aminohexanoic acid 13
and N-methylbenzylamine 12 using microwave irradiation at 140 °C for 30 mins to afford carboxylic
acid 14 (70%, see ESI for full details). Conversion of the carboxylic acid 14 to the corresponding
hydroxamic acid 7 was achieved using a two-step protocol involving (i) carbodiimide mediated
coupling of commercially available O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (NH2OTHP) to the
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carboxylic acid, followed by (ii) deprotection of the O-protected hydroxamic acid under mild
conditions (p-TsOH in i-PrOH).
Scheme 1. Reagents and conditions: (a) 12, 13, DMSO, µw, 140 °C, 30 min; (b) NH2OTHP, EDCI·HCl, HOBt,
MeCN, 21 °C, 24 h (35%); (c) p-TsOH·H2O, i-PrOH, 21 °C, 24 h (42%).
For the remainder of the 4-amino substituted analogues 3–6 the first step in the synthesis was the
reaction of commercially available 4-bromo-1,8-naphthalic anhydride with methyl 6-aminohexanoate
hydrochloride to give the corresponding imide 15 (Scheme 1). Heating imide 15 with the requisite
amine in the presence of Pd2(dba)3·CHCl3 (4 mol%), xantphos (4 mol%) and Cs2CO3 (3 equiv.) at 40
or 80 °C for 24 h afforded the desired aminonaphthalimides 16a–e in high yields (81–98%).74, 75
Hydrolysis gave the corresponding carboxylic acids 17a–d, also in excellent yields (92–98%), and the
hydroxamic acid was again installed using EDCI mediated coupling of NH2OTHP and subsequent
deprotection to give analogues 3–6.
Scheme 2. Reagents and conditions: (a) Pd2(dba)3·CHCl3 (4 mol%), xantphos (4 mol%), amine, Cs2CO3,
PhCH3, 40 or 80 °C, 24 h; (b) LiOH·H2O, 1:1 THF/H2O, 21 °C, 24 h; (c) NH2OTHP, EDCI·HCl, HOBt, MeCN,
21 °C; (d) p-TsOH·H2O, i-PrOH, 21 °C.
The 4-nitro derivative 8 was synthesised in a similar fashion using a three-step sequence starting with
commercially available 4-nitronaphthalic anhydride 18 (Scheme 3).
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Scheme 3: Reagents and conditions: (a) 13, EtOH, MW, 100 °C, 60 min (76%); (b) NH2OTHP, EDCI·HCl,
HOBt, MeCN, 21 °C, 24 h (50%); (c) p-TsOH·H2O, i-PrOH, 80 °C, 16 h (33%).
The aminobenzamide analogues of 4MS and scriptaid were accessed from the carboxylic acid
precursors 2050 and 21, the latter prepared from naphthalic anhydride (See ESI for full details).
Treatment of the acids with 1,2-diaminobenzene, EDCI·HCl and HOBt55 gave the target compounds,
9 and 10 in yields of 67% and 43%, respectively (Scheme 4).
Scheme 4. Reagents and conditions: (a) o-phenylenediamine, EDCI·HCl, HOBt, MeCN, 21 °C, 24 h.
2.2 HDAC Isoform Inhibition.
The scriptaid analogues were assessed for inhibitory activity against HDAC isoforms 1, 3 and 8 (class
I), HDAC6 (class IIb) and HDAC11 (class IV). Hydroxamic acids bearing nitrogen containing
substituents in the 4-position (3–7) typically inhibited HDAC6 at least as effectively as scriptaid (IC50
= 12 nM). Of these examples, the propylamine and benzylamine substituted derivatives 5 and 6
possessed low nanomolar activity (IC50 = 4.8 and 3.5 nM respectively); better than scriptaid and
comparable to that of HDAC6 selective HDACi rocilinostat23 and nexturastat27 (HDAC6 IC50 of 4.7
and 5.0 nM respectively). The 4-anilino 3 and 4 mirrored scriptaid in terms of potency towards
HDAC6 (IC50 = 13 and 14 nM respectively).
It is noteworthy that the N-alkyl compounds (5–7) showed significantly enhanced selectivity for
HDAC6 over HDAC1 (Table 1), which compares well against scriptaid and known HDAC6 specific
agents. The most potent of these (5 and 6) were subsequently evaluated against the remaining HDAC
isoforms (HDAC2, 4, 5, 7 9 and 10, see ESI S2.1) and compound 6 was identified as more than 500-
fold selective for HDAC6 compared with HDAC1 (IC50 = 1.98 µM), 100-fold more selective against
HDAC3 (IC50 = 0.36 µM) and 190-fold more selective against the other class IIb isoform HDAC10
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(IC50 = 0.66 µM). The enhanced selectivity of compound 6 for HDAC6 over HDAC1 exceeds that of
the reported HPOB29 and HPB37 and also Tubacin24 and Tubastatin A.26
The presence of the aminobenzamide ZBG typically favours inhibition at class I HDACs, and this
preference was observed for 9 and 10, with no activity at both HDAC6 and HDAC8 below the
starting concentration (10 µM) of the assay. Both of these derivatives exhibited sub-micromolar
inhibition of HDAC3 (IC50 = 0.29 and 0.20 µM for 9 and 10, respectively) and HDAC1 (IC50 = 0.43
and 0.74 µM) with reasonable selectivity against HDAC11.
Table 1: Inhibition of individual HDAC isoforms, IC50 (µM)a and the selectivity factorb (SF) vs HDAC6.
HDAC Isoform (SF)b
Compound
1
0.032 ± 0.009
(5)
3
0.013 ± 0.003
(2)
6
8
0.99 ± 0.11
(167)
1.52 ± 0.007
(127)
11
0.015 ± 0.006
(2)
0.36 ± 0.02
(30)
TSA
scriptaid
4MS
0.0059 ± 0.001
1.74 ± 0.04
(145)
1.43 ± 0.09
(119)
0.37 ± 0.04
(31)
0.32 ± 0.03
(27)
0.012 ± 0.002
0.012 ± 0.001
1.81 ± 0.12
(151)
0.29 ± 0.01
(24)
2.38 ± 0.007
(183)
3.06 ± 0.16
(219)
0.47 ± 0.003
(36)
0.55 ± 0.01
(39)
4.09 ± 0.02
(315)
4.07 ± 0.05
(291)
0.19 ± 0.01
(14)
0.21 ± 0.001
(15)
0.013 ± 0.0006
0.014 ± 0.002
3
4
0.59 ± 0.02
(123)
1.98 ± 0.04
(566)
2.38 ± 0.06
(245)
0.63 ± 0.09
(111)
0.11 ± 0.004
(23)
0.36 ± 0.0007
(103)
0.70 ± 0.003
(72)
1.62 ± 0.06
(284)
1.52 ± 0.08
(317)
2.46 ± 0.11
(703)
1.40 ± 0.03
(144)
0.62 ± 0.02
(109)
0.08 ± 0.03
(16)
0.15 ± 0.02
(43)
1.22 ± 0.13
(126)
1.02 ± 0.11
(179)
0.0048 ± 0.0002
0.0035 ± 0.0002
0.0097 ± 0.0007
0.0057 ± 0.0007
5
6
7
8
0.43 ± 0.03
0.29 ± 0.01
(-)
>10
(-)
>10
(-)
8.12 ± 0.91
(-)
9
(-)c
0.74 ± 0.06
(-)
0.20 ± 0.004
(-)
>10
(-)
>10
(-)
9.35 ± 0.96
(-)
10
a IC50 performed in duplicate using 10-dose IC50 mode with 3-fold serial dilution starting from 10 µM solutions;
b
Selectivity for HDAC6 over other isoforms calculated by dividing the IC50 value for the relevant isoform
against that for HDAC6. c Not determined due to lack of HDAC6 activity.
Given that the naphthalimide core, the linker and ZBG are identical for scriptaid, 4MS and
compounds 3–8 the strong and selective inhibition of HDAC6 observed for compounds 5–7 could
reasonably be attributed to additional interactions of the position 4 substituents of the naphthalimide
with the residues surrounding the rim of the active site; a region less conserved across the various
HDAC isoforms.13, 26, 32
2.3 Molecular Modelling
To shed more light on the observed results a number of compounds (scriptaid, 4MS and compounds
3–8) were docked into the active site of HDAC6 (co-crystallised with TSA; PDB accession code:
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5EDU).76 As expected the N-alkyl hydroxamic acid projects away from the heterocyclic core through
the tunnel towards the zinc ion of the active site. This perpendicular geometry is common for 1,8-
naphthlaimides as it relieves steric strain between the carbonyl groups of the imide substituent.77 In
each case the 1,8-napthalimide core sits close to the solvent exposed surface of the enzyme and is
largely solvent exposed itself. The proximity of this ring system to the protein surface appears to be
governed by the minimised conformation of the 4-substituent, which is in turn accommodated by
flexibility in the linker portion of the molecule.
Figure 4: Docking of (a) Scriptaid, (b) 4MS and compounds (c) 5 and (d) 6 with the crystal structure of
HDAC6 obtained by removing bound trichostatin.76 The potential for additional hydrophobic interactions of the
N-substituent of 5 (b) and 6 (c) with the surface of HDAC6 can be clearly seen.
For 4MS and 3 the inhibition at HDAC6 is equivalent to that of scriptaid (0.012, 0.013 and 0.012 µM
respectively). These results are in accordance with the absence of any additional interactions (e.g.
electrostatic). For compounds 5 and 6 which displayed ~2-fold better inhibition (4.8 and 3.7 nM,
respectively) it could be rationalised that the heterocyclic ring could adopt two possible conformations
which are “flipped” with relation to each other (Figure 4b and c). Additional flexibility of the
aliphatic substituent may serve to increase van der Waals interactions with the protein surface.
Whereas analogue 3 (4-NHPh) does not have the flexibility to adopt such a pose (Figure S3.1).
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2.4 Activity against KASUMI-1 cancer cells
Cancer is an established target for many HDACi including scriptaid51, 52 and as such the in-vitro
antiproliferative activity of the new scriptaid analogues to inhibit cell growth was evaluated in
KASUMI-1 cell lines (Table 2, see ESI section S4 for details). Compared to scriptaid, slightly
enhanced activity was noted for compounds 3–6. The 4-propylamino derivative 5 was of interest,
inhibiting the growth of the KASUMI-1 cell line with an IC50 of 0.096 µM, a five-fold improvement
over scriptaid (0.49 µM, Table 2). Of interest, the aminobenzamide analogues targeting HDAC1 (9
and 10) were less effective than scriptaid in this assay. Given HDAC1 is primarily localised in the
nucleus the lesser activity is potentially a result of poor nuclear penetration.
Table 2. Anticancer activity in the KASUMI-1 cell line.a
Compound
IC50 (µM)
0.49b
0.29
0.28
0.32
0.096
0.36
0.81
0.57
0.64
1.09
scriptaid
4MS
3
4
5
6
7
8
9
10
a Compounds were incubated with KASUMI-1 cells for 72 hr then viability determined using resazurin reagent.
IC50 values were determined from the mean of three experiments conducted in duplicate. b Error < 1% (n = 3).
See ESI section S4 for full details.
2.5 Photophysical Evaluation
The absorption and emission spectra obtained for compounds 3–7 (Table 3) are typical for 4-
aminonaphthalimides with λabs ≈ 440 nm, λem ≈ 530 nm in DMSO and λabs ≈ 440 nm, λem ≈ 540 nm in
aqueous buffer.78, 79 Of particular relevance to the aim of developing potent and highly fluorescent
scriptaid analogues, several of the most biologically active compounds were also strongly fluorescent,
for example, the quantum yield for propyl analogue 5 (ΦF = 0.81 in DMSO and ΦF = 0.28 in buffer)
made this compound ideally suited for further studies. In contrast, the 4-N-phenyl analogues (3 and 4)
were weakly fluorescent (ΦF < 0.01), as was the disubstituted 4-NBn(Me)N 7 (ΦF = 0.07 in DMSO).
For 3 and 4 the electron withdrawing aryl group restricts electron transfer to the ICT system and for
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disubstituted 7 the steric clash between the 4-amino-substituents and the hydrogen at the 5-position
(peri position) disfavours the planar structure required for ICT.80
Table 3. Photophysical properties of selected compounds in DMSO and aqueous buffer.
c
Compound
scriptaid
Solventa
λabs (nm)b
λem (nm)b
Stokes Shift
ΦF
DMSO
PB
336
-
386
-
50
-
<0.01
-
4MS50
DMSO
PB
399
396
448
-
534
460
544
-
135
64
99
-
0.03
<0.01
<0.01
-
DMSO
PB
3
4
5
6
7
9
DMSO
PB
443
-
536
-
93
-
<0.01
-
DMSO
PB
446
456
439
446
422
431
402
-
526
548
523
543
528
544
535
80
92
84
97
106
113
133
-
0.81
0.28
0.83
0.38
0.07
0.15
0.04
-
DMSO
PB
DMSO
PB
DMSO
PB
-
a PB = 1% DMSO in 10 mM phosphate buffer (pH 7.40); Wavelengths of maximum absorbance (λabs) and
b
emission intensity (λem); c Values represent the average of three measurements.
2.6 Cellular Imaging
To examine cellular uptake and localisation, A549 cells were incubated with the most promising
candidates 5, and 6 (1 µM) for 20 mins and 2 hours (Figure 5). As a representative of the
aminobenzamide class of inhibitors compound 9 was also included in the cellular imaging studies.
Cellular fluorescence was visualised by confocal microscopy following excitation with a 405 nm
laser. All three dyes showed good intracellular fluorescence, with clear localisation in the cytoplasm
and similar intracellular fluorescence intensities for all three compounds. Localisation did not change
markedly over time. There was a distinct lack of fluorescence within nuclear regions and we have
previously shown that 4MS fluorescence is not quenched in the presence of DNA,50 indicating that the
lack of fluorescence in the nucleus is due to a lack of nuclear accumulation rather than due to
quenching of fluorescence by DNA
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Figure 5: Confocal microscopy images of A549 cells incubated for 2 hours with (a) 5, (b) 6, (c) 9, (1 µM, λex =
405 nm, λem = 460–560 nm) highlighting uptake and cytoplasmic distribution.
The aminobenzamide 9 showed some degree of lysosomal, as well as cytoplasmic, localisation.
(Figure 6). An established method of lysosomal targeting uses tertiary amines such as the morpholine
group, which can be protonated in the acidic environment of the lysosome, preventing diffusion back
into the cytoplasm.81 However, for compound 9, the morpholine nitrogen is conjugated with the
aromatic naphthalimide system, reducing basicity and explaining why only partial accumulation in the
lysosomes was observed. This partial localisation was confirmed with co-localisation studies with
Lysotracker Red, with a Pearson’s correlation coefficient of 0.58 (Figure S6.3). The localisation
study supports the postulation above that the inability of the compound to access HDAC1 in the
nucleus leads to decreased activity. For compound 6 at 10 µM incubation, protrusions of the
membrane were observed (Figure 6). Such blebbing is the initial phase of cell disassembly in
apoptosis, consistent with the cytotoxicity of these compounds.
Figure 6: Confocal microscopy image (λex = 405 nm, λem = 460–560 nm) of A549 cells incubated with (a) 9 at 1
µM for 2 hours with punctate staining consistent with lysosomal localisation and (b) 6 at 10 µM for 20 minutes
showing blebbing consistent with toxicity.
2.7 Zebrafish imaging
Of the analogues prepared, the 4-aminopropyl and the benzyl analogues 5 and 6 exhibited favourable
properties in terms of HDAC6 activity, selectivity, cellular potency and high fluorescence quantum
yields, and were therefore selected for further in vivo studies using developing zebrafish embryos.
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Class IIa HDACs are known to be distributed in the heart, brain and skeletal muscle (for HDAC4, 5,
9) and in the heart, skeletal muscle, pancreas and placenta for HDAC7, while Class IIb HDAC have
been detected in the heart, liver, kidney, placenta and vascular system (HDAC6) and in the liver
spleen and kidney (HDAC10).20
In order to characterise whole organism localisation a time course study was performed in which
zebrafish embryos were treated with 1, 10 and 20 µM of 5 or 6 from 24–50 hours post fertilization
(hpf) for 24 hours. Following the incubation of the fluorescent scriptaid derivatives, imaging
experiments were performed 72 hpf, (Figure 7, See ESI section S7 for fluorescent images for
analogue 5 and superimposed fluorescence & brightfield images for 5 and 6). Due to strong
autofluorescence from the yolk sac (correlating with the known high lipid content), fluorescence from
derivatives 5 and 6 was only clearly visualised posterior to the yolk sac extension, specifically in the
trunk and tail. Again autofluorescence was noted in this section (due to melanocytes in the dorsal
section), however, for the zebrafish treated at 10 and 20 µM the distribution of the fluorescent
analogues 5 and 6 could be clearly visualised—confirming ready uptake of the compounds and
localisation in the vasculature; a site rich in HDAC6. Compounds 5 and 6 have high affinity for
HDAC6 in-vitro, however, it is impossible to state unequivocally that this determines their in vivo
behaviour as distribution is influenced by a number of factors.82 A similar scenario, involving the
distribution of fluorescent naphthalimide based Zn binders in zebrafish, was encountered by Goldup
and Watkinson.83, 84
Figure 7: Fluorescent images of posterior trunk and tail section of zebrafish embryo after exposure to 6.
Zebrafish were exposed to (a) 0, (b) 1, (c) 10 or (d) 20 µM of 6 during the time period 24–50 hpf and visualised
72 hpf using fluorescence microscopy (λex = 480 nm, λem = 517 nm). Arrows highlight auto-fluorescence due to
lipids in the yolk sac and also from melanocytes in the dorsal section.
The pan-inhibitor Trichostatin A (TSA) has been shown to have a strong effect on blood vessel
formation in developing zebrafish embryos.85 An additional HDAC6 specific transient knock-down
study in zebrafish has clearly demonstrated that the absence of this isoform results in malformations
of the vascular system, including vessel maturation, an outcome that matches those identified in a
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mouse model.17 To investigate the impact of 5 and 6 on organism development, zebrafish embryos
were exposed from 24–50 hours post fertilisation (hpf) to 10 µM of 5 and 6 with scriptaid and
Trichostatin A (TSA) employed as controls. The vascular system was visualised at 72 hours by
alkaline phosphatase staining (Figure 8).86
All compounds had a negative impact on blood vessel development especially the formation of the
subintestinal vein, the blood vessel that lies on the top of the yolk sac. These results confirm that, like
the known HDAC inhibitors, analogues 5 and 6 affect blood vessel formation. However, compared to
the damage resulting from exposure to TSA and even scriptaid, the subintestinal vein defects caused
by compounds 5 and 6 were not as pronounced. Given that the new analogues would be expected to
have similar pharmacokinetic behaviour to scriptaid, and at 10 µM they are clearly present in the
vasculature (indicated by the fluorescence studies outlined above), it is postulated that the improved
selectivity of both 5 and 6 for HDAC6 leads to the milder phenotype that was observed, corroborating
the idea that selectively targeting HDAC6 leads to ‘viable’ organisms.20, 21, 86
Figure 8. Images of Zebrafish embryos 72 hpf after treatment with HDACi. All compounds were used at a
concentration of 10 µM. Embryos were exposed from 24-50 hpf with (a) vehicle only, (b) scriptaid (10 µM),
(c) TSA (10 µM), (d) 5 (10 µM) and (e) 6 (10 µM) and stained at 72 hpf with alkaline phosphatase to visualise
blood vessels.
3 CONCLUSION
This study has identified a number of scriptaid analogues that inhibit HDAC6 as effectively as known
inhibitors rocilinostat and nexturastat and are also highly selective (compound 6 is more than 500 fold
selective for HDAC6 over HDAC1). Furthermore, the compounds are highly fluorescent and both
their distribution and activity confirmed in a zebrafish model organism. It is expected that these
compounds will be of considerable use to researchers examining the biological effects of scriptaid and
the selective inhibition of HDAC6.
13

ACCEPTED MANUSCRIPT
4 EXPERIMENTAL SECTION
General reagents and solvents for the synthesis of compounds were purchased from commercial
sources and used as supplied. Column chromatography was performed using 230–400 Mesh silica gel.
Melting points are uncorrected. All NMR spectra (1H and 13C) were collected on a 270, 400 or 500
MHz spectrometer as specified. Samples were dissolved (0.5 mL) in either deuterated chloroform
(CDCl3) or deuterated dimethyl sulfoxide (DMSO-d6). NMR chemical shifts were assigned using 2D
1
NMR experiments (HSQC and HMBC). In instances when either the H or 13C resonance was
coincidental with the deuterated solvent or residual H2O, the resonance was confirmed by HSQC
experiments. High Resolution Mass Spectra (HRMS) analysis were conducted and recorded on an
HRMS-ESI-TOF. Those reactions that employed microwave irradiation were conducted using a CEM
Discover S-Class Microwave reactor, operating at a frequency of 50/60 Hz and continuous irradiation
power from 0 to 200 W. Reaction mixture temperatures were monitored by an external infrared
sensor. All reactions were conducted in either 10 mL or 35 mL microwave vials sealed with a Teflon®
crimp cap.
6-(6-(Benzyl(methyl)amino)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)hexanoic acid (14)
In a 5 mL microwave vial, a mixture of 4-chloro-1,8-naphthalic anhydride 11 (235 mg, 1.01 mmol),
6-aminohexanoic acid 13 (131 mg, 0.999 mmol) and N-benzylmethylamine 12 (390 µL, 3.02 mmol)
in DMSO (2 mL) was heated using microwave irradiation at 140 °C for 30 min. The reaction mixture
was diluted with H2O (15 mL) and acidified with 2 M HCl (5 mL) and extracted using EtOAc (2 × 30
mL). The combined organic phase was washed with brine (30 mL), dried (MgSO4), filtered and
concentrated under reduced pressure. The crude solid was then triturated in 2 M HCl (20 mL) to
afforded the title compound 14 (305 mg, 70%), as an orange solid, which was then used in the
1
following step without further purification; H NMR (500 MHz, DMSO-d6): δ 11.98 (s, 1H, COOH),
8.52 (d, J = 8.5 Hz, 1H, H-9), 8.45 (d, J = 7.6 Hz, 1H, H-7), 8.34 (d, J = 8.1 Hz, 1H, H-4), 7.75 (dd, J
= 8.4, 7.3 Hz, 1H, H-8), 7.40–7.33 (m, 4H, Ar, H-5), 7.32–7.27 (m, 2H, Ar), 4.58 (s, 2H, CH2Ar),
4.01 (t, J = 7.5 Hz, H-6'), 2.95 (s, 3H, NCH3), 2.21 (t, J = 7.3 Hz, 2H, H-2'), 1.64–1.58 (m, 2H, H-
13
3'/H-5'), 1.57–1.51 (m, 2H, H-3'/H-5'), 1.37–1.29 (m, 2H, H-4'); C NMR (125 MHz, DMSO-d6): δ
174.5, 163.6, 163.0, 155.8, 137.3, 132.1, 130.7 (C × 2), 129.5, 128.6 (C × 2), 127.7 (C × 2), 127.4,
126.0, 125.0, 122.5, 115.0, 114.4, 60.1, 41.5, 39.7, 33.5, 27.4, 26.1, 24.2; HRMS (m/z, ESI+):
Calculated for C26H26N2O4 m/z = 413.1971 [M+H]+. Found m/z = 431.1978.
6-(6-(Benzyl(methyl)amino)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-N-
hydroxyhexanamide (7)
14

ACCEPTED MANUSCRIPT
A mixture of 6-(6-(benzyl(methyl)amino)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)hexanoic
acid 14 (110 mg, 0.256 mmol), EDCI·HCl (83 mg, 0.433 mmol), anhydrous HOBt (17 mg, 0.126
mmol) and O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (62 mg, 0.529 mmol) in MeCN (5 mL) was
stirred at 21 °C for 24 h. The precipitate that had formed in solution was collected by vacuum
filtration to afford the THP-protected hydroxamic acid (47 mg, 35%) as a yellow solid; 1H NMR (270
MHz, DMSO-d6): δ 10.88 (s, 1H), 8.54 (d, J = 8.4 Hz, 1H), 8.47 (d, J = 7.2 Hz, 1H), 8.35 (d, J = 7.9
Hz, 1H), 7.77 (app. t, Japp. = 7.9 Hz, 1H), 7.46–7.24 (m, 6H), 4.74 (s, 1H), 4.59 (s, 2H), 4.01 (t, J = 7.3
Hz, 2H), 3.92–3.83 (m, 1H), 3.45–3.37 (m, 1H), 2.96 (s, 3H), 1.98 (t, J = 7.1 Hz, 2H), 1.71–1.38 (m,
10H), 1.35–1.27 (m, 2H); 13C NMR (125 MHz, DMSO-d6): δ 169.0, 163.6, 163.0, 155.8, 137.3,
132.1, 130.7 (C × 2), 129.5, 128.6 (C × 2), 128.0 (C × 2), 127.4, 125.6, 125.0, 122.6, 115.0, 114.4,
100.8, 61.2, 60.0, 41.5, 39.5, 32.1, 27.8, 27.4, 26.0, 24.7, 24.6, 18.3. Next, a solution of the THP-
protected hydroxamic acid (28 mg, 0.053 mmol) in 2-propanol (1 mL) was treated with TsOH·H2O (1
mg, 0.005 mmol) at 50 °C for 16 h. During this time a yellow precipitate formed in solution which
was isolated by vacuum filtration to afford the title compound 7 (10 mg, 42%) as a bright yellow
solid; 1H NMR (500 MHz, DMSO-d6): δ 10.31 (s, 1H, NHOH), 8.65 (s, 1H, NHOH), 8.54 (d, J = 8.5
Hz, 1H, H-9), 8.47 (d, J = 6.6 Hz, 1H, H-7), 8.36 (d, J = 8.2 Hz, 1H, H-4), 7.77 (app. t, Japp. = 7.5 Hz,
1H, H-8), 7.42–7.33 (m, 4H, Ar), 7.32–7.28 (m, 2H, H-5, Ar), 4.59 (s, 2H, CH2Ar), 4.01 (t, J = 7.4
Hz, 2H, H-6'), 2.96 (s, 3H, CH3), 1.95 (t, J = 7.3 Hz, 2H, H-2'), 1.64–1.58 (m, 2H, H-3'/H-5'), 1.57–
1.48 (m, 2H, H-3'/H-5'), 1.33–1.27 (m, 2H, H-4'); 13C NMR (125 MHz, DMSO-d6): δ 169.0, 163.6,
163.0, 155.8, 137.3, 132.1, 130.70, 130.67, 129.5, 128.6 (C × 2), 127.7 (C × 2), 127.4, 125.6, 125.0,
122.6, 115.0, 114.4, 60.0, 41.5, 39.8, 32.1, 27.4, 26.1, 24.9; m.p. 136–138 °C; HRMS (m/z, ESI+):
Calculated for C26H28N3O4 m/z = 446.2074 [M+H]+. Found m/z = 446.2076.
6-(1,3-Dioxo-6-(phenylamino)-1H-benzo[de]isoquinolin-2(3H)-yl)hexanoic acid (17a)
A solution of methyl 6-(1,3-dioxo-6-(phenylamino)-1H-benzo[de]isoquinolin-2(3H)-yl)hexanoate 16a
(160 mg, 0.38 mmol) and LiOH·H2O (161 mg, 3.84 mmol) in THF/H2O (1:1, 3 mL) was stirred at 21
°C for 24 h. The solvent volume was halved in vacuo and the remaining mixture was diluted with H2O
and treated with 2 M HCl until pH 3 was obtained. The resulting precipitate was collected by vacuum
1
filtration to afford the title compound 17a (148 mg, 97%) as a yellow solid; H NMR (270 MHz,
DMSO-d6): δ 9.40 (s, 1H, NH), 8.82 (d, J = 8.5 Hz, 1H, H-9), 8.50 (d, J = 7.2 Hz, 1H, H-7), 8.27 (d, J
= 8.5 Hz, 1H, H-4), 7.79 (app. t, Japp. = 8.3 Hz, 1H, H-8), 7.49–7.38 (m, 4H, H-2'', H-3'', H-5'', H-6''),
7.26–7.16 (m, 2H, H-5, H-4''), 4.01 (t, J = 7.2 Hz, 2H, H-6'), 2.21 (t, J = 7.2 Hz, 2H, H-2'), 1.67–1.49
(m, 4H, H-3', H-5'), 1.38–1.27 (m, 2H, H-4'); 13C NMR (125 MHz, DMSO-d6): δ 174.5, 163.7, 162.8,
147.9, 140.4, 133.5, 131.1, 129.6 (C × 2), 129.5, 129.0, 125.1, 124.2, 122.7 (C × 2), 122.1, 121.6,
111.1, 107.8, 39.0, 33.7, 27.4, 26.1, 24.3; m.p. 119–122 °C; HRMS (m/z, ESI+): Calculated for
C24H23N2O4 m/z = 403.1652[M+H]+. Found m/z = 403.1659.
15

ACCEPTED MANUSCRIPT
6-(6-((3-Fluorophenyl)amino)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)hexanoic acid (17b)
A
solution of methyl 6-(6-((3-fluorophenyl)amino)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-
yl)hexanoate 16b (174 mg, 0.40 mmol) and LiOH·H2O (167 mg, 4.00 mmol) in THF/H2O (1:1, 3 mL)
was stirred at 21 °C for 24 h. The solvent volume was halved in vacuo and the remaining mixture was
diluted with H2O and treated with 2 M HCl until pH 3 was obtained. The resulting precipitate was
collected by vacuum filtration to afford the title compound 17b (164 mg, 98%) as an orange solid; 1H
NMR (500 MHz, DMSO-d6): δ 11.98 (br s, 1H, COOH), 9.42 (s, 1H, NH), 8.73 (dd, J = 8.5, 0.8 Hz,
1H, H-9), 8.47 (dd, J = 7.4, 0.8 Hz, 1H, H-7), 8.29 (d, J = 8.4 Hz, 1H, H-4), 7.79 (dd, J = 8.5, 7.4 Hz,
1H, H-8), 7.46–7.42 (m, 1H, H-5''), 7.40 (d, J = 8.4 Hz, 1H, H-5), 7.22 (d, J = 8.1, Hz, 1H, H-6''),
7.18 (dt, J = 11.0, 2.1 Hz, 1H, H-2''), 6.94 (td, J = 8.4, 2.1 Hz, 1H, H-4''), 3.99 (t, J = 7.5 Hz, 2H, H-
6'), 2.21 (t, J = 7.4 Hz, 2H, H-2'), 1.64–1.58 (m, 2H, H-5'), 1.57–1.51 (m, 2H, H-3'), 1.36–1.30 (m,
1
2H, H-4'); 13C NMR (125 MHz, DMSO-d6): δ 174.4, 163.6, 162.80 (d, JC-F = 241.5 Hz), 162.78,
146.6, 142.8 (d, 3JC-F = 10.0 Hz), 133.1, 131.06 (d, 3JC-F = 8.8 Hz), 131.05, 129.3, 129.0, 125.4, 122.2,
4
2
2
122.1, 117.3 (d, JC-F = 1.3 Hz), 112.3, 109.9 (d, JC-F = 21.3 Hz), 109.3, 108.1 (d, JC-F = 23.8 Hz),
39.6, 33.5, 27.3, 26.1, 24.2; m.p. 88–90 °C; HRMS (m/z, ESI+): Calculated for C24H22FN2O4 m/z =
421.1558 [M+H]+. Found m/z = 421.1558.
6-(1,3-Dioxo-6-(propylamino)-1H-benzo[de]isoquinolin-2(3H)-yl)hexanoic acid (17c)
A solution of methyl 6-(1,3-dioxo-(6-propylamino)-1H-benzo[de]isoquinolin-2(3H)-yl)hexanoate 16c
(54 mg, 0.14 mmol) and LiOH·H2O (60 mg, 1.44 mmol) in THF/H2O (1:1, 2 mL) was stirred at 21 °C
for 24 h. The solvent volume was halved in vacuo and the remaining mixture was diluted with H2O
and treated with 2 M HCl until pH 3 was obtained. The resulting precipitate was collected by vacuum
1
filtration to afford the title compound 17c (47 mg, 92%) as an orange solid; H NMR (270 MHz,
DMSO-d6): δ 11.98 (br s, 1H, COOH), 8.72 (dd, J = 8.6, 1.2 Hz, 1H, H-9), 8.43 (dd, J = 7.3, 1.0 Hz,
1H, H-7), 8.26 (d, J = 8.7 Hz, 1H, H-4), 7.80 (t, J = 5.6 Hz, 1H, NH), 7.68 (dd, J = 8.4, 7.3 Hz, 1H,
H-8), 6.78 (d, J = 8.7 Hz, 1H, H-5), 3.99 (t, J = 7.4 Hz, 2H, H-6'), 3.35 (2H, CH2CH2CH3)α, 2.21 (t, J
= 7.3 Hz, 2H, H-2'), 1.80–1.66 (m, 2H, CH2CH2CH3), 1.65–1.43 (m, 4H, H-3', H-5'), 1.39–1.26 (m,
2H, H-4'), 0.99 (t, J = 7.4 Hz, 3H, CH2CH2CH3); 13C NMR (125 MHz, DMSO-d6): δ 174.4, 163.8,
162.9, 150.7, 134.3, 130.7, 129.5, 128.6, 124.2, 121.9, 120.1, 107.5, 103.8, 44.6, 39.2, 33.5, 27.4,
26.1, 24.2, 21.2, 11.6; m.p. 211–213 °C; HRMS (m/z, ESI+): Calculated for C21H25N2O4 m/z =
369.1809 [M+H]+. Found m/z = 369.1823. α Confirmed by HSQC.
6-(6-(Benzylamino)-1,3-dioxo-6-1H-benzo[de]isoquinolin-2(3H)-yl)hexanoic acid (17d)
A solution of methyl 6-(6-(benzylamino)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)hexanoate 16d
(55 mg, 0.13 mmol) and LiOH·H2O (54 mg, 1.28 mmol) in THF/H2O (1:1, 2 mL) was stirred at 21 °C
for 24 h. The solvent volume was halved in vacuo and the remaining mixture was diluted with H2O
and treated with 2 M HCl until pH 3 was obtained. The resulting precipitate was collected by vacuum
16

ACCEPTED MANUSCRIPT
filtration to afford the title compound 17d (50 mg, 94%) as a bright yellow solid; 1H NMR (270 MHz,
DMSO-d6): δ 8.78 (d, J = 8.7 Hz, 1H, H-9), 8.50 (t, J = 6.1 Hz, 1H, NH), 8.45 (d, J = 7.5 Hz, 1H, H-
7), 8.17 (d, J = 8.6 Hz, 1H, H-4), 7.73 (app. t, Japp. = 7.4 Hz, 1H, H-8), 7.42–7.22 (m, 5H, Ar), 6.67 (d,
J = 8.6 Hz, 1H, H-5), 4.67 (d, J = 6.1 Hz, 2H, CH2Ar), 3.98 (t, J = 7.4 Hz, 2H, H-6'), 2.20 (t, J = 7.4
Hz, 2H, H-2'), 1.64–1.47 (m, 4H, H-3', H-5'), 1.36–1.23 (m, 2H, H-4'); 13C NMR (125 MHz, DMSO-
d6): δ 174.5, 163.7, 162.9, 150.4, 138.5, 134.0, 130.8, 129.4, 128.6 (C × 3), 127.1, 127.0 (C × 2),
124.6, 122.0, 120.3, 108.1, 104.6, 45.9, 39.1, 33.5, 27.4, 26.1, 24.3; m.p. 191–192 °C; HRMS (m/z,
ESI+): Calculated for C25H25N2O4 m/z = 417.1809 [M+H]+. Found m/z = 417.1829.
6-(1,3-Dioxo-6-(phenylamino)-1H-benzo[de]isoquinolin-2(3H)-yl-N-hydroxyhexanamide (3)
A mixture of 6-(1,3-dioxo-6-(phenylamino)-1H-benzo[de]isoquinolin-2(3H)-yl)hexanoic acid 17a
(156 mg, 0.388 mmol), EDCI·HCl (90 mg, 0.582 mmol), anhydrous HOBt (26 mg, 0.194 mmol) and
O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (95 mg, 0.815 mmol) in MeCN (8 mL) was stirred at 21
°C for 24 h. After solvent was removed, the crude material was then suspended in H2O (10 mL) and
extracted with EtOAc (3 × 30 mL). The combined organic phase was washed with KH2PO4 (10 mL)
and brine (10 mL), dried (MgSO4), filtered and concentrated under reduced pressure. Purification by
flash column chromatography (9:1 CH2Cl2/MeOH with 1% NH4OH) afforded the THP-protected
1
hydroxamic acid (144 mg, 74%, Rf = 0.50) as an orange solid; H NMR (500 MHz, DMSO-d6): δ
10.89 (s, 1H), 9.41 (s, 1H), 8.83 (d, J = 8.4 Hz, 1H), 8.50 (d, J = 7.2 Hz, 1H), 8.27 (d, J = 8.5 Hz, 1H),
7.80 (app. t, Japp. = 7.9 Hz, 1H), 7.46 (app. t, Japp. = 7.7 Hz, 2H), 7.40 (d, J = 7.8 Hz, 2H), 7.25 (d, J =
8.5 Hz, 1H), 7.19 (t, J = 7.3 Hz, 1H), 4.76–4.72 (m, 1H), 4.01 (t, J = 7.4 Hz, 2H), 3.92–3.84 (m, 1H),
13
3.45–3.39 (m, 1H), 1.98 (t, J = 7.3 Hz, 2H), 1.65–1.38 (m, 10H), 1.35–1.25 (m, 2H); C NMR (125
MHz, DMSO-d6): δ 169.0, 163.7, 162.9, 147.9, 140.4, 133.5, 131.1, 129.6 (C × 2), 129.5, 129.0,
125.2, 124.2, 122.7 (C × 2), 122.1, 121.6, 111.1, 107.8, 100.8, 61.3, 39.8, 32.0, 27.8, 27.4, 26.0, 24.8,
24.7, 18.3. Next, a solution of THP-protected hydroxamic acid (78 mg, 0.156 mmol) in 2-propanol (3
mL) was treated with TsOH·H2O (9 mg, 0.047 mmol). The reaction mixture was allowed to stir at 40
°C for 16 h, during which, a red precipitate formed in solution. The precipitate was isolated by
1
vacuum filtration to afford the title compound 3 (39 mg, 60%) as a red solid; H NMR (270 MHz,
DMSO-d6): δ 10.35 (br s, 1H, NHOH), 9.40 (s, 1H, NHOH), 8.83 (d, J = 8.4 Hz, 1H, H-9), 8.65 (s,
1H, NH), 8.50 (d, J = 7.4 Hz, 1H, H-7), 8.28 (d, J = 8.5 Hz, 1H, H-4), 7.80 (app. t, Japp. = 7.6 Hz, 1H,
H-8), 7.49–7.38 (m, 4H, H-2'', H-3'', H-5'', H-6''), 7.25 (d, J = 8.5 Hz, 1H, H-5), 7.19 (t, J = 7.1 Hz,
1H, H-4''), 4.01 (t, J = 7.1 Hz, 2H, H-6'), 1.95 (t, J = 7.1 Hz, 2H, H-2'), 1.66–1.48 (m, 4H, H-3', H-5'),
1.35–1.23 (m, 2H, H-4'); 13C NMR (125 MHz, DMSO-d6): δ 169.5, 164.1, 163.3, 148.4, 140.8, 133.9,
131.5, 130.02 (C × 2), 130.00, 129.6, 125.6, 124.7, 123.2 (C × 2), 122.5, 122.1, 111.5, 108.2, 39.5,
32.6, 27.9, 26.6, 25.4; m.p. 188–190 °C; HRMS (m/z, ESI+): Calculated for C24H24N3O4 m/z =
418.1761 [M+H]+. Found m/z = 418.1774.
17

ACCEPTED MANUSCRIPT
6-(6-((3-Fluorophenyl)amino)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-N-
hydroxyhexanamide (4)
A mixture of 6-(6-((3-fluorophenyl)amino)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)hexanoic
acid 17b (167 mg, 0.397 mmol), EDCI·HCl (92 mg, 0.596 mmol), anhydrous HOBt (27 mg, 0.199
mmol) and O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (98 mg, 0.834 mmol) in MeCN (9 mL) was
stirred at 21 °C for 48 h. The reaction mixture was cooled (0 °C) and the precipitate isolated by
vacuum filtration to afford the THP-protected hydroxamic acid (120 mg, 58%) as a bright yellow
1
solid; H NMR (270 MHz, DMSO-d6): δ 10.89 (s, 1H), 9.46 (s, 1H), 8.77 (dd, J = 8.5, 1.0 Hz, 1H),
8.51 (dd, J = 7.3, 1.0 Hz, 1H), 8.32 (d, J = 8.4 Hz, 1H), 7.83 (dd, J = 8.5, 7.4 Hz, 1H), 7.53–7.35 (m,
2H), 7.29–7.10 (m, 3H), 7.03–6.84 (m, 1H), 4.74 (s, 1H), 4.02 (t, J = 7.2 Hz, 2H), 3.94–3.75 (m, 1H),
13
3.51–3.37 (m, 1H), 1.99 (t, J = 7.3 Hz, 2H), 1.70–1.39 (m, 10H), 1.38–1.22 (m, 2H); C NMR (125
MHz, DMSO-d6): δ 169.0, 163.6, 162.9, 162.8 (d, 1JC-F = 241.5 Hz), 146.6, 142.9 (d, 3JC-F = 10.6 Hz),
3
133.2, 131.13, 131.11 (d, JC-F = 9.5 Hz), 129.4, 129.1, 125.5, 122.3, 122.2, 117.3, 112.4, 110.2 (d,
2JC-F = 29.9 Hz), 109.4, 108.1 (d, 2JC-F = 23.7 Hz), 100.8, 61.2, 39.7, 32.0, 27.8, 27.4, 26.0, 24.8, 24.7,
18.3. Next, a solution of THP-protected hydroxamic acid (63 mg, 0.121 mmol) in 2-propanol (4 mL)
was treated with TsOH·H2O (7 mg, 0.036 mmol). The reaction mixture was allowed to stir at 40 °C
for 16 h, during which, an orange precipitate formed in solution. The precipitate was isolated by
1
vacuum filtration to afford the title compound 4 (40 mg, 77%) as an orange solid; H NMR (270
MHz, DMSO-d6): δ 10.33 (s, 1H, NHOH), 9.46 (s, 1H, NHOH), 8.77 (d, J = 8.5 Hz, 1H, H-9), 8.66
(br s, 1H, NH), 8.51 (d, J = 7.1 Hz, 1H, H-7), 8.33 (d, J = 8.4 Hz, 1H, H-4), 7.83 (app. t, Japp. = 7.9
Hz, 1H, H-8), 7.50–7.41 (m, 2H, H-5, H-5''), 7.24–7.17 (m, 2H, H-2'', H-6''), 6.95 (td, J = 8.7, 2.3 Hz,
1H, H-4''), 4.01 (t, J = 7.2 Hz, 2H, H-6'), 1.95 (t, J = 7.2 Hz, 2H, H-2'), 1.69–1.46 (m, 4H, H-3', H-5'),
1
1.35–1.26 (m, 2H, H-4'); 13C NMR (125 MHz, DMSO-d6): δ 169.5, 164.1, 163.28 (d, JC-F = 241.3
3
3
Hz), 163.26, 147.1, 143.3 (d, JC-F = 10.4 Hz), 133.7, 131.5 (d, JC-F = 9.8 Hz), 129.8, 129.6, 129.4,
125.9, 122.7, 122.6, 117.7 (d, 4JC-F = 1.9 Hz), 112.8, 110.4 (d, 2JC-F = 20.9 Hz), 109.8, 108.6 (d, 2JC-F
=
23.9 Hz), 39.5, 32.6, 27.9, 26.6, 25.4; m.p. 193–195 °C; HRMS (m/z, ESI+): Calculated for
C24H23FN3O4 m/z = 436.1667 [M+H]+. Found m/z = 436.1665.
6-(1,3-Dioxo-6-(propylamino)-1H-benzo[de]isoquinolin-2(3H)-yl)-N-hydroxyhexanamide (5)
A mixture containing 6-(1,3-dioxo-6-(propylamino)-1H-benzo[de]isoquinolin-2(3H)-yl)hexanoic acid
17c (244 mg, 0.662 mmol), EDCI·HCl (226 mg, 1.46 mmol), anhydrous HOBt (45 mg, 0.331 mmol)
and O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (163 mg, 1.39 mmol) in CH2Cl2 (20 mL) was stirred
at 21 °C for 48 h. The reaction mixture was then transferred into a separatory funnel and washed with
KH2PO4 (10 mL) and brine (10 mL), dried (MgSO4), filtered and concentrated under reduced
pressure. Purification by flash column chromatography (9:1 CH2Cl2/MeOH with 1% NH4OH)
1
afforded the THP-protected hydroxamic acid (262 mg, 85%, Rf = 0.58) as a yellow solid; H NMR
18

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(500 MHz, DMSO-d6): δ 10.88 (s, 1H), 8.71 (dd, J = 8.6, 1.2 Hz, 1H), 8.42 (dd, J = 7.3, 1.0 Hz, 1H),
8.25 (d, J = 8.5 Hz, 1H), 7.79 (t, J = 5.5 Hz, 1H), 7.67 (dd, J = 8.5, 7.3 Hz, 1H), 6.78 (d, J = 8.7 Hz,
1H), 4.76–4.73 (m, 1H), 3.99 (t, J = 7.4 Hz, 2H), 3.90–3.83 (m, 1H), 3.47–3.40 (m, 1H), 3.34 (2H)α,
1.98 (t, J = 7.3 Hz, 2H), 1.74–1.68 (m, 2H), 1.62–1.43 (m, 10H), 1.28–1.25 (m, 2H), 0.99 (t, J = 7.4
Hz); 13C NMR (125 MHz, DMSO-d6): δ 169.0, 163.8, 162.9, 150.7, 134.3, 130.7, 129.5, 128.6, 124.2,
121.9, 120.1, 107.5, 103.8, 100.8, 61.3, 44.6, 39.3, 32.1, 28.8, 27.5, 26.0, 24.7, 21.2, 19.6, 18.3, 11.6.
Next, a solution of THP-protected hydroxamic acid (239 mg, 0.511 mmol) in 2-propanol (3 mL) was
added TsOH·H2O (30 mg, 0.153 mmol). The reaction mixture was allowed to stir at 21 °C for 16 h,
during which, a fine yellow precipitate formed in solution. The solvent was removed under reduced
pressure and crude material was triturated in MeCN (1 mL) at 21 °C for 12 h to afford the title
compound 5 (52 mg, 27%) as yellow solid; 1H NMR (400 MHz, DMSO-d6): δ 10.32 (s, 1H, NHOH),
8.72 (d, J = 8.8 Hz, 1H, H-9), 8.65 (s, 1H, NHOH), 8.44 (d, J = 7.3 Hz, 1H, H-7), 8.27 (d, J = 8.8 Hz,
1H, H-4), 7.79 (t, J = 5.4 Hz, 1H, NH), 7.68 (app. t, Japp. = 8.3 Hz, 1H, H-8), 6.79 (d, J = 8.8 Hz, 1H,
H-5), 3.99 (t, J = 7.3 Hz, 2H, H-6'), 3.37 (2H, CH2CH2CH3)α, 1.95 (t, J = 7.4 Hz, 2H, H-2'), 1.77–1.68
(m, 2H, CH2CH2CH3), 1.62–1.49 (m, 4H, H-3', H-5'), 1.32–1.23 (m, 2H, H-4'), 0.99 (t, J = 7.3 Hz,
3H, CH2CH2CH3); 13C NMR (125 MHz, DMSO-d6): δ 169.5, 164.2, 163.4, 151.2, 134.8, 131.1,
129.9, 129.0, 124.7, 122.3, 120.5, 107.9, 104.3, 45.0, 39.6, 32.6, 28.0, 26.6, 25.4, 21.6, 12.0; m.p.
194–196 °C; HRMS (m/z, ESI+): Calculated for C21H26N3O4 m/z = 384.1918 [M+H]+. Found m/z =
384.1933. α Confirmed by HSQC.
6-(6-(Benzylamino)-1,3-dioxo-6-1H-benzo[de]isoquinolin-2(3H)-yl)-N-hydroxyhexanamide (6)
A mixture of 6-(6-benzylamino)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)hexanoic acid 17d
(338 mg, 0.812 mmol), EDCI·HCl (277 mg, 1.79 mmol), anhydrous HOBt (55 mg, 0.406 mmol) and
O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (200 mg, 1.71 mmol) in CH2Cl2 (20 mL) was stirred at
21 °C for 48 h. The reaction mixture was then transferred into a separatory funnel and washed with
KH2PO4 (10 mL) and brine (10 mL), dried (MgSO4), filtered and concentrated under reduced
pressure. Purification by flash column chromatography (9:1 CH2Cl2/MeOH with 1% NH4OH)
1
afforded the THP-protected hydroxamic acid (358 mg, 86%, Rf = 0.55) as a yellow solid; H NMR
(500 MHz, DMSO-d6): δ 10.88 (s, 1H), 8.77 (dd, J = 8.5, 1.1 Hz, 1H), 8.49 (t, J = 6.1 Hz, 1H), 8.45
(dd, J = 7.4, 1.0 Hz, 1H), 8.16 (d, J = 8.6 Hz, 1H), 7.73 (dd, J = 8.4, 7.3 Hz, 1H), 7.40 (d, J = 6.9 Hz,
2H), 7.33 (t app., Japp. = 7.6 Hz, 2H), 7.24 (t, J = 7.3 Hz, 1H), 6.67 (d, J = 8.6 Hz, 1H), 4.75–4.72 (m,
1H), 4.66 (d, J = 5.9 Hz, 2H), 3.97 (t, J = 7.4 Hz, 2H), 3.89–3.82 (m, 1H), 3.44–3.38 (m, 1H), 1.97 (t,
13
J = 7.3 Hz, 2H), 1.60–1.41 (m, 10H), 1.30–1.22 (m, 2H); C NMR (125 MHz, DMSO-d6): δ 169.0,
163.7, 162.9, 150.4, 138.5, 134.0, 130.7, 129.4, 128.5 (C × 3), 127.02, 126.92 (C × 2), 124.6, 122.0,
120.3, 108.2, 104.6, 100.8, 61.3, 45.9, 39.5, 32.0, 27.8, 27.5, 26.0, 24.8, 24.7, 18.3. Next, a solution of
THP-protected hydroxamic acid (93 mg, 0.180 mmol) in 2-propanol (2 mL) was treated with
TsOH·H2O (10 mg, 0.054 mmol). The reaction mixture was allowed to stir at 21 °C for 16 h, during
19

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which, a fine yellow precipitate formed in solution. The solvent was removed under reduced pressure
and the crude material was triturated in MeCN (1 mL) at 21 °C for 12 h to afford the title compound 6
1
(43 mg, 56%) as yellow solid; H NMR (500 MHz, DMSO-d6): δ 10.31 (s, 1H, NHOH), 8.78 (d, J =
8.4 Hz, 1H, H-9), 8.65 (s, 1H, NHOH), 8.50 (t, J = 6.0 Hz, 1H, NH), 8.46 (d, J = 7.2 Hz, 1H, H-7),
8.17 (d, J = 8.5 Hz, 1H, H-4), 7.73 (app. t, Japp. = 7.9 Hz, 1H, H-8), 7.40 (d, J = 7.6 Hz, 2H, H-2'', H-
6''), 7.34 (app. t, Japp. = 7.6 Hz, 2H, H-3'', H-5''), 7.25 (app. t, Japp. = 7.3 Hz, 1H, H-4''), 6.68 (d, J = 8.6
Hz, 1H, H-5), 4.67 (d, J = 5.9 Hz, 2H, CH2Ar), 3.97 (t, J = 7.5 Hz, 2H, H-6'), 1.93 (t, J = 7.3 Hz, 2H,
H-2'), 1.61–1.47 (m, 4H, H-3', H-5'), 1.31–1.23 (m, 2H, H-4'); 13C NMR (125 MHz, DMSO-d6): δ
169.1, 163.8, 162.9, 150.4, 138.5, 134.1, 130.8, 129.4, 128.6 (C × 3), 127.1, 127.0 (C × 2), 124.6,
122.0, 120.3, 108.1, 104.6, 45.9, 39.1, 32.2, 27.5, 26.2, 25.0; m.p. 179–182 °C; HRMS (m/z, ESI+):
Calculated for C25H26N3O4 m/z = 432.1917 [M+H]+. Found m/z = 432.1930.
N-Hydroxy-6-(6-nitro-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)hexanamide (8)
A mixture of 6-(6-nitro-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)hexanoic acid 19 (315 mg,
0.884 mmol), EDCI·HCl (206 mg, 1.33 mmol), anhydrous HOBt (60 mg, 0.442 mmol) and O-
(tetrahydro-2H-pyran-2-yl)hydroxylamine (217 mg, 1.86 mmol) in MeCN (20 mL) was stirred at 21
°C for 24 h. The solvent was removed under reduced pressure and the resulting crude residue was
triturated in MeCN (2 mL) to afford the THP-protected hydroxamic acid (247 mg, 50%) as a tan
1
solid; H NMR (270 MHz, DMSO-d6): δ 10.88 (s, 1H), 8.71 (dd, J = 8.7, 1.0 Hz, 1H), 8.63 (dd, J =
7.3, 1.1 Hz, 1H), 8.61 (d, J = 8.1 Hz, 1H), 8.55 (d, J = 8.1 Hz, 1H), 8.10 (dd, J = 8.7, 7.3 Hz, 1H),
4.71 (s, 1H), 4.03 (t, J = 7.3 Hz, 2H), 3.93–3.76 (m, 1H), 3.41–3.37 (m, 1H), 1.98 (t, J = 7.3 Hz, 2H),
1.70–1.38 (m, 10H), 1.38–1.26 (m, 2H); 13C NMR (125 MHz, DMSO-d6): δ 168.9, 163.0, 162.2,
149.2, 131.7, 130.1, 129.6, 128.8, 128.4, 126.7, 124.3, 122.83, 122.78, 100.7, 61.2, 39.6, 32.0, 27.7,
27.2, 25.9, 24.7, 24.6, 18.2. Next, a solution of the THP-protected hydroxamic acid (112 mg, 0.246
mmol) in 2-propanol (3 mL) was added TsOH·H2O (13 mg, 0.074 mmol). The reaction mixture was
allowed to stir at 80 °C for 16 h, during which, a tan precipitate formed in solution. The precipitate
was isolated by vacuum filtration and triturated in MeCN (1 mL) to afford the title compound 8 (30
mg, 33%) as a tan solid; 1H NMR (270 MHz, DMSO-d6): δ 10.33 (s, 1H, NHOH), 8.72 (d, J = 8.7 Hz,
1H, H-9), 8.68–8.59 (m, 2H, H-4, H-7), 8.56 (d, J = 8.0 Hz, 1H, H-5), 8.10 (dd, J = 8.7, 7.4 Hz, 1H,
H-8), 4.03 (t, J = 7.4 Hz, 2H, H-6'), 1.95 (t, J = 7.3 Hz, 2H, H-2'), 1.74–1.45 (m, 4H, H-3', H-5'),
1.42–1.23 (m, 2H, H-4'); 13C NMR (125 MHz, DMSO-d6): δ 169.1, 163.0, 162.2, 149.2, 131.8, 130.2,
129.7, 129.0, 128.8, 128.5, 126.7, 124.3, 122.8, 39.6, 32.1, 27.2, 26.1, 24.9; m.p. 175–177 °C; HRMS
(m/z, ESI+): Calculated for C18H17N3O6 m/z = 372.1196 [M+H]+. Found m/z = 372.1201.
N-(2-Aminophenyl)-6-(6-morpholino-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)hexanamide
(9)
20

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To a mixture of 6-(6-morpholino-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)hexanoic acid 20 (266
mg, 0.671 mmol) in MeCN (15 mL), was added EDCI·HCl (229 mg, 1.48 mmol), anhydrous HOBt
(45 mg, 0.336 mmol) and O-phenylenediamine (152 mg, 1.48 mmol). After stirring at 21 °C for 24 h,
the solvent was removed under reduced pressure. Purification by flash column chromatography (9:1
CH2Cl2/MeOH with 1% NH4OH) afforded the title compound 9 (219 mg, 67%, Rf = 0.50) as a yellow
solid; 1H NMR (500 MHz, DMSO-d6): δ 9.06 (s, 1H, NH), 8.51–8.49 (m, 2H, H-7, H-9), 8.43 (d, J =
8.1 Hz, 1H, H-4), 7.82 (dd, J = 8.5, 7.4 Hz, 1H, H-8), 7.37 (d, J = 8.2 Hz, 1H, H-5), 7.10 (dd, J = 7.8,
1.2 Hz, H-6'''), 6.87 (td, J, = 7.3, 1.4 Hz, 1H, H-4'''), 6.69 (dd, J = 8.0, 1.3 Hz, H-3'''), 6.49 (td, J, =
7.7, 1.4 Hz, 1H, H-5'''), 4.79 (s, 2H, NH2), 4.05 (t, J = 7.4 Hz, 2H, H-6'), 3.91 (app. t, Japp. = 4.7 Hz,
4H, H-2'', H-6''), 3.23 (app. t, Japp. = 4.5 Hz, 4H, H-3'', H-5''), 2.31 (t, J = 7.4 Hz, 2H, H-2'), 1.69–1.61
(m, 4H, H-3', H-5'), 1.41–1.35 (m, 2H, H-4'); 13C NMR (125 MHz, DMSO-d6): δ 171.1, 163.6, 163.1,
155.5, 141.9, 132.2, 130.7, 130.6, 129.2, 126.2, 125.7, 125.32, 125.29, 123.5, 122.6, 116.1, 115.9,
115.8, 115.2, 66.2 (C × 2), 53.1 (C × 2), 39.8, 35.6, 27.5, 26.2, 25.1; m.p. 66–67 °C; HRMS (m/z,
ESI+): Calculated for C28H31N4O4 m/z = 487.2340 [M+H]+. Found m/z = 487.2341.
5 ASSOCIATED CONTENT
1
13
Supporting Information. Full H NMR and C NMR spectra of novel compounds. This material is
available free of charge via the Internet at…….
6 AUTHOR INFORMATION
Corresponding Author
*Phone: +613 5227 1439. Email: fred.pfeffer@deakin.edu.au
Author Contributions
All authors contributed equally and have given approval of the final version of the manuscript.
Funding Sources
Notes
The authors declare no competing financial interests.
7 ACKNOWLEDGEMENT
CLF and TDA thank the Centre for Chemistry and Biotechnology at Deakin University for a top up
scholarship and a salary contribution respectively. The work performed by YG was supported by
21

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grants from the Centre for Molecular and Medicinal research at Deakin University. YG and PY thank
the Deakin University zebrafish facility. We thank Westpac Bicentennial Foundation for a Research
Fellowship (EJN) and the Australian government for a Research Training Program Scholarship
(KGL). The authors acknowledge Alexander P. Ray for helping with the in vivo experiments in
zebrafish and the scientific and technical assistance of the Australian Microscopy and Microanalysis
Research Facility at the Australian Centre for Microscopy and Microanalysis (ACMM) at the
University of Sydney.
8 ABBREVIATIONS
HDAC:
HDACi:
hpf:
histone deacetylase
histone deacetylase inhibitor
hours post fertilisation
9 FOOTNOTES
‡ The degree of HDAC6 selectivity (vs HDAC1) reported for scriptaid varies from simply suggested
(Langley et al.),48 to 6-fold (Yanik et al. and Bradner et al.),49, 87 to >100 (Fleming et al.).50 There are
reports (Cremer et al.) indicating scriptaid favours HDAC1 over HDAC6.88
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Highly Fluorescent and HDAC6 Selective Scriptaid Analogues
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Highlights:
•
•
•
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New highly fluorescent HDAC inhibitors based on scriptaid.
Low-nanomolar activity against HDAC6 and more than 500-fold selectivity for HDAC6 over HDAC1.
Imaging studies show uptake and localisation in cellular cytoplasm and zebrafish vasculature.
The selective HDAC6 inhibition leads to a mild phenotypical outcome in zebrafish