Design and synthesis of diazine-based panobinostat analogues for HDAC8 inhibition

Article abstract of DOI:10.3762/bjoc.16.59

Guided by computational analysis, herein we report the design, synthesis and evaluation of four novel diazine-based histone deacetylase inhibitors (HDACis). The targets of interest (TOI) are analogues of panobinostat, one of the most potent and versatile HDACi reported. By simply replacing the phenyl core of panobinostat with that of a diazine derivative, docking studies against HDAC2 and HDAC8 revealed that the four analogues exhibit inhibition activities comparable to that of panobinostat. Multistep syntheses afforded the visualized targets TOI1, TOI2, TOI3-rev and TOI4 whose biological evaluation confirmed the strength of HDAC8 inhibition with TOI4 displaying the greatest efficacy at varying concentrations. The results of this study lay the foundation for future design strategies toward more potent HDACis for HDAC8 isozymes and further therapeutic applications for neuroblastoma.

Full text of DOI:10.3762/bjoc.16.59

Design and synthesis of diazine-based panobinostat
analogues for HDAC8 inhibition
Sivaraman Balasubramaniam1, Sajith Vijayan1, Liam V. Goldman2, Xavier A. May2,
Kyra Dodson1, Sweta Adhikari1, Fatima Rivas3, Davita L. Watkins1
and Shana V. Stoddard*2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2020, 16, 628–637.
1Department of Chemistry and Biochemistry, University of Mississippi,
University, MS 38677-1848, USA, 2Department of Chemistry, Rhodes
College, Memphis, TN 38112, USA and 3Department of Chemical
Biology and Therapeutics, St. Jude Children’s Research Hospital,
Memphis, TN 38105-3678, USA
doi:10.3762/bjoc.16.59
Received: 04 December 2019
Accepted: 24 March 2020
Published: 07 April 2020
Email:
Associate Editor: J. Aubé
Shana V. Stoddard* - stoddards@rhodes.edu
© 2020 Balasubramaniam et al.; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
diazine; histone deacetylase; inhibitors; isozymes; panobinostat
Abstract
Guided by computational analysis, herein we report the design, synthesis and evaluation of four novel diazine-based histone
deacetylase inhibitors (HDACis). The targets of interest (TOI) are analogues of panobinostat, one of the most potent and versatile
HDACi reported. By simply replacing the phenyl core of panobinostat with that of a diazine derivative, docking studies against
HDAC2 and HDAC8 revealed that the four analogues exhibit inhibition activities comparable to that of panobinostat. Multistep
syntheses afforded the visualized targets TOI1, TOI2, TOI3-rev and TOI4 whose biological evaluation confirmed the strength of
HDAC8 inhibition with TOI4 displaying the greatest efficacy at varying concentrations. The results of this study lay the founda-
tion for future design strategies toward more potent HDACis for HDAC8 isozymes and further therapeutic applications for
neuroblastoma.
Introduction
One of the most important posttranslational modifications and non-histone proteins. The balance between acetylation and
involve acetylation/deacetylation of histone proteins by histone deacetylation is pivotal for typical cell function. Abnormal or
deacetylases (HDACs) [1]. HDACs belong to an important increased HDAC expression has been reported in several
family of enzymes consisting of 18 isozymes. They control pro- human tumors and cancer cell lines [2]. As such, the develop-
tein acetylation, which is a change that occurs after translation. ment of novel HDAC inhibitors (HDACis) has become a
In addition, they regulate gene transcription, cell differentiation, rapidly evolving area where targeted inhibition has emerged in
cell cycle progression and apoptosis by targeting both histone clinical research as a potential therapeutic approach for the
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treatment of various cancers as well as neurodegenerative disor- TOI3-rev in this article is different from TOI3 in the previous
ders and immune related diseases [3-5]. Of specific interests are reporting [12]. Here, the 1,2-diazole ring has been replaced with
Class I HDAC isozymes, HDAC2 and HDAC8, which are im- that of a pyrimidine core. Given the abundance of literature
portant targets in cancer models as both are associated with regarding analogues having modifications of the indole amine
high risk diseases such as prostate cancer and neuroblastoma unit and vinylogous hydroxamic moieties [13-15], the non-
[6-8]. Compounds such as vorinostat, givinostat and panobino- availability of the central core modification stimulated our
stat have been successfully applied as HDAC inhibitors [3]. interest toward altering the central core to evaluate efficacy. We
Among these drugs, panobinostat (Farydak, Novartis) an FDA found particular interest in the replacement of the phenyl ring
approved drug, has been recognized as a pan-deacetylase inhibi- with diazine cores (pyridazines, pyrimidines and pyrazines) as
tor [9,10]. As a hydroxamic acid pan-HDACi, it is zinc-depend- their docking values were on par with that of the parent mole-
ent, capable of binding in a bidentate fashion to the zinc-con- cule, panobinostat (Figure 1). Given that the diazine-containing
taining catalytic domain of the HDACs, and classified as highly compounds are considered to be one of the most important
potent amongst traditional HDACis [11]. According to previous classes of heterocycles, their presence in a plethora of pharma-
reports, panobinostat not only induces apoptosis in cells, but cology and drug molecules motivated us to synthesize these an-
also stimulates cell growth inhibition, and cell-cycle arrest in a alogues and subject them towards biological evaluation [16-19].
time- and dose-dependent manner. Thus, panobinostat has In addition, our envisioned dinitrogen heterocycle cores may
demonstrated high therapeutic potential in anticancer efforts. have increased interactions in the binding pockets, and thus
Although panobinostat offers a versatile approach for the inhi- leading to a better therapeutic activity.
bition of cancer cell growth and survival, a lack of selectivity
and bioavailability can cause negative molecular and clinical Aiming to provide a strategy to address our global objective of
effects, specifically in combination therapies.
developing single agent therapeutic hydroxamate derivatives,
we began the synthesis of four leading HDAC8 diazine-based
Despite advances in Class I HDAC inhibition, there remains an HDACis: TOI1, TOI2, TOI3-rev and TOI4 (Figure 1).
obvious need to develop compounds having better therapeutic Herein, we provide a summary of the design process followed
properties as a single-agent therapeutic drug. Our recent by an outline of the multistep synthesis and preliminary biologi-
research based on computational studies indicated heterocyclic cal evaluation of each target. HDAC8 was selected for testing
cores as suitable surrogates for the central core of the hydroxa- due to its unique structure and multifaceted functional activities
mate derivative, panobinostat [12]. It should be noted that [4,5,20,21]. HDAC8 is also upregulated in neuroblastoma, a
Figure 1: Chemical structures of the target diazine-based surrogates for the central core of panobinostat.
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childhood pediatric cancer, hence considered a drug target for respectively, with panobinostat possessing a docking score of
this cancer subtype [22,23]. Despite a multifaceted array of al- 8.47. Considering the effects of structural diversity, TOI3-rev
ternative treatment options for neuroblastoma, some patient was included in the library and computationally evaluated to de-
cohorts who are considered high risk at the time of diagnosis termine its potential as an HDAC8 inhibitor. TOI3-rev pos-
face poor prognosis [24,25]. Recent studies indicated that sesses a −log(Kd) value of 8.36 suggesting that it too would be
HDAC8 inhibition induces differentiated phenotypes and on a par with panobinostat (8.47).
reduces neuroblastoma growth in vitro and in vivo with few
adverse effects [22]. However, there are very few effective ther- Our previous study showed that the hydroxamate tail of TOI1,
apeutic options in neuroblastoma that inhibit HDAC8 [26]. TOI2, and TOI4 formed a bidentate interaction with the Zn2+
Thus, the design of novel HDAC8 inhibitors as potential ion at the base of the HDAC8 receptor, which is consistent with
neuroblastoma therapeutics could be valuable to expand the the crystal structure of this class of inhibitors in HDACs [31-
treatment options for this patient population [27]. Therefore, we 34]. Again, our work here extends the investigation focusing on
sought to focus on the biological evaluation of our proposed in- HDAC8 due to the need for novel neuroblastoma therapeutics.
hibitors in HDAC8. This study aimed at offering additional Each compound was also shown to have two parallel-displaced
therapeutic options to be used in conjunction with or in place of π–π interactions, one with Phe-152 and the other with Phe-208.
panobinostat while providing a rationale design guideline The mode of binding for each compound was similar to panobi-
towards HDACis.
nostat in HDAC8 (Figure 2). Two differences were however
observed in the mode of binding of TOI4. It was shown that the
pyridazine ring did not lay as planar in the gorge as the pyrazine
and pyrimidine rings of TOI1 and TOI2, respectively. The
Results and Discussion
Molecular design
Docking analysis is a well-established technique that is utilized indole ring of TOI4 was also contorted upward, forming a
to evaluate interactions in important biological receptors such T-shaped π–π interaction with Phe-207 while both the indole
as that of HDACs [28-30]. Previously, we implored docking to rings of TOI1 and TOI2 were shown to be flipped downward
predict the interactions between the active sites of HDAC2 and forming a T-shaped π–π interaction with Phe-208.
HDAC8 molecular frameworks with similar structures to that of
panobinostat [12]. In developing such analogues, the hydroxa- TOI4 is the only diazine compound having two nitrogens
mate tails and indole capping moiety were maintained as both directly next to each other in the core ring structure. The
are essential to binding at the active site of HDAC2 and nitrogen atoms in the pyridazine ring are positioned so that they
HDAC8. Results suggested that TOI1, TOI2, and TOI4 [12] do not sit in the center of the phenyl rings of Phe-152 or Phe-
would be inhibitors exhibiting similar potency as that of panobi- 208. This results in the pyridazine ring of TOI4 shifted closer
nostat. The reported −log(Kd) values were 8.93, 8.64 and 8.25, to the side of the gorge having Phe-152 compared to the other
Figure 2: Docking pose for panobinostat and panobinostat derivatives in the HDAC8 receptor. (a) Overlay of all compounds investigated in this study
in the HDAC8 active site: panobinostat (green), TOI1 (purple), TOI2 (yellow) and TOI4 (grey); (b) TOI3-rev (pink) docking pose in active site; (c) TOI4
(grey) docking pose in the active site.
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derivative (Figure 2). This shift allows for the indole ring of panobinostat. Thus, aiming to develop improved compounds to
TOI4 to fit in a small hydrophobic pocket at the surface, which effectively target HDAC8, the synthesis of TOI inhibitors and
is created by the phenyl ring of Phe-207. Like the previously their evaluation was undertaken.
studied compounds (TOI1, TOI2, and TOI4), TOI3-rev is also
shown to bind to the HDAC8 receptor by forming a bidentate Synthesis
interaction with the Zn2+ ion. TOI3-rev, like that of TOI1, Equipped with molecular targets achieved via theory, the syn-
TOI2, and TOI4 produces two parallel-displaced π–π interac- thesis of the hypothesized compounds commenced with com-
tions with Phe-152 and Phe-208 (Figure 2). The pyrimidine ring mercially available starting materials (Figure 3). The central
of TOI3-rev also lays planar in the gorge similar to the core building blocks for TOI1, TOI2, TOI3-rev and TOI4
pyrazine and pyrimidine rings of TOI1 and TOI2. The indole were selected based on their ability to tether the indole amine
ring of TOI3-rev is tilted downward forming a T-shaped π–π and the zinc binding group in a para-relationship to each other.
interaction with Phe-208.
Initial efforts focused on synthesizing analogue TOI1
In review of the computational results, the inclusion of the (Scheme 1). Acid 1 was converted to the corresponding methyl
nitrogen atoms in the core ring structure of panobinostat pro- ester 5 via esterification reaction using methanol mediated by
duced compounds with predicted binding affinities similar to sulfuric acid under heating conditions to provide the compound
Figure 3: General building blocks for the visualized targets.
Scheme 1: Reaction conditions: a) MeOH, H2SO4 (5 drops), MS 4 Å (2 pieces), 68 °C, 8 h, 81%; b) DIBAL-H (1.2 equiv), 6 h, −78 °C, THF, 78%;
c) phosphorane 8 (2.0 equiv), THF, 8 h, 60 °C, 72%; d) SeO2, dioxane, 110 °C, 8 h, 61%; e) indolamine 10 (1.1 equiv) DCE, sodium triacetoxyboro-
hydride (STAB, 1.1 equiv), TEA (2 equiv), rt, 63%; f) NaOH at −10 °C, NH2OH·H2O at −10 °C, MeOH , rt, 12 h, 55%.
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in 81% yield. The methyl ester 5 was reduced to aldehyde 6 solvent systems, we identified an optimized water/methanol
using DIBAL-H at −78 °C. While TLC analysis revealed com- mixture to provide the pure product TOI1 in 55% yield. The
plete conversion of the ester to aldehyde, the isolated yield was isolated compound was thoroughly characterized by spectros-
poor (20%). The obstacle was overcome by using a modified copic techniques.
Fieser work-up procedure to yield the aldehyde 6 in high yield,
78%. Then, aldehyde 6 was converted to the α,β-unsaturated Having successfully establish reaction conditions for the syn-
trans-ester 7 through a Wittig reaction with the phosphorane thesis of TOI1, we then focused our efforts on the generation of
synthon 8, which was derived from ethyl bromoacetate at 60 °C regioisomers TOI2 and TOI3-rev, respectively. Initial attempts
for 8 h in THF in 72% yield. The exclusive formation of the to oxidize the methyl group at the benzylic position in starting
trans-isomer was confirmed by 1H NMR studies, namely the materials 2 and 3 to provide the corresponding aldehyde com-
presence of the olefin at δ 7.00 and 7.71 with a J value of 15 Hz pounds 13 and 14 failed, despite using rigorous reaction condi-
(Supporting Information File 1, Figure S3).
tions of SeO2 or alternative strong oxidizing agents (e.g., MnO2
and oxone). Thus, we considered the critical role of the elec-
Next, oxidation of the methyl group of 7 under SeO2 condi- tronic effects of the nitrogen atoms on this cyclic substrate, and
tions at 110 °C provided the ethyl acrylate aldehyde 9 in 61% then we revised our synthetic strategy by a) tethering an alkene
yield. The next step involved the crucial reductive amination functional group on the aromatic ring and b) then conducting
reaction between aldehyde 9 with indolamine 10, which had the oxidation of the benzylic group to afford the aldehyde prod-
been obtained via Fischer indole synthesis – the reaction of uct. Towards this end, we performed a Suzuki coupling reac-
phenylhydrazine with 5-chloro-2-pentanone [35]. Initial reduc- tion between boronic acid 15 with chloro compound 2
tion attempts using sodium triacetoxyborohydride (STAB) as (Scheme 2). To the best of our knowledge, there is no report of
the reducing agent provided predominantly starting material and a Suzuki coupling reaction using boronic acid 15 in the litera-
negligible potential product as monitored by TLC. However, ture. However, we generated this required boronic acid from the
addition of 2 equivalents of triethylamine to the reaction mix- corresponding methyl propiolate [38].
ture facilitated the formation of the product, compound 11 in
63% yield. The product was confirmed by 1H NMR and
13C NMR as shown in Supporting Information File 1.
Finally, the ethyl ester 11 was converted to the hydroxamic acid
derivative, TOI1 using the bidentate nucleophile hydroxyl-
amine either under neutral or basic conditions [36,37]. We first
explored neutral conditions where aqueous hydroxylamine was
added to compound 11 in methanol, and a predominant polar
spot was observed by TLC. However, the isolated product was
not the expected TOI1, as 1H NMR revealed two new peaks at
δ 4.53 and 2.66 ppm (Supporting Information File 1, Figure
S36) presumably indicating that a favorable Michael addition
followed by intramolecular cyclization or vice versa provided
compound 12, which was validated by 13C and DEPT NMR
studies (Supporting Information File 1 Figures S37and S38).
Marred with these observations, compound 11 was treated with
aqueous hydroxylamine in the presence of strong base (i.e.,
10 equivalents of methanolic sodium hydroxide or aqueous so-
dium hydroxide) at 0 °C. The reaction was monitored by TLC
and it revealed that methanolic sodium hydroxide provided a
cleaner reaction than aqueous sodium hydroxide conditions.
The reaction mixture was quenched with a saturated ammoni-
Scheme 2: Reaction conditions: a) boronic acid 15 (1.3 equiv),
um chloride solution at 0 °C after 12 h, the solvent was evapo-
PdCl2(PPh3)2 (0.1 equiv), dioxane/H2O (3:1), Na2HPO4 (2.0 equiv),
rated, and the compound was subjected to reversed-phase
column chromatography using C-18 silica gel. After initial un-
successful purification protocols with water/ACN or water/THF
TEA (4.0 equiv), 90 °C, 15 h, 55% for 16 and 71% for 18; b) SeO2 ,
different conditions, 0%; c) SeO2 (2.0 equiv), dioxane, 110 °C, 12 h,
54%.
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Next we investigated reaction conditions for the reaction of Confounded by this observation, extensive experimentation
compound 2 with boronic acid 15 using different variables varying solvent and temperature were evaluated, but none were
(Supporting Information File 1, Table S1). Gratifyingly, after fruitful. Alternatively to synthesize compound 17, we adapted
surveying several reaction conditions, we successfully obtained the strategy as depicted in Scheme 3. Compound 20 was con-
the desired product 16 in 35% yield using PdCl2(PPh3)2 and verted into compound 17 via intermediate 21 using a previ-
Na2HPO4 as the base in a dioxane/water system under heating ously reported literature procedure [39]. The intermediate 21
conditions of 90 °C.
was then subjected to the Suzuki reaction using conditions
already developed to provide the ester aldehyde 17 in overall
To improve the overall chemical yields, we evaluated several yield of 16% for two steps. Having identified synthetically suit-
organic bases and found that TEA (4.0 equiv) improved the able conditions for compound 17, we scaled up the reaction to
yield to 55%. This success was attributed to the improved in- complete the final two steps, the reductive amination reaction
creased solubility of TEA in the reaction mixture under heating and the hydroxamic acid preparation. Using the same reaction
conditions. In parallel, similar reaction conditions were used for conditions developed for TOI1, we proceeded with precursors
compound 3 and the final coupled product 18 was obtained in 22 and 23, which were obtained in 61% and 68% yield, respec-
71% yield. The product was confirmed by the expected chemi- tively. The desired hydroxamic acid TOI2 and TOI3-rev were
cal shift at δ 6.50 and 7.54 ppm with a J value of 15 Hz for obtained in 49% and 51% yield, respectively, after C18 silica
compound 16 (Supporting Information File 1, Figure S7) and δ gel purification procedure using a methanol/water (1:1) solvent
6.57 and 7.63 ppm with a J value of 15 Hz for compound 18 system.
(Supporting Information File 1, Figure S9) as inferred by 1H
NMR analysis.
Finally, we focused our efforts towards the synthesis of the last
analogue TOI4. From the above observations, we hypothesize
The resulting Suzuki-coupled products 16 and 18, were subject- that successful synthesis of TOI4 would rely on generating the
ed to benzylic oxidation expecting the olefin functionality key intermediate aldehyde 25 (Scheme 4).
would facilitate the corresponding aldehydes 17 and 19, respec-
tively. Surprisingly, the methyl group in compound 16 did not Initially, we explored the Suzuki coupling reaction for sub-
undergo oxidation under SeO2 conditions as observed for TOI1 strate 4. Gratifyingly, the reaction product 24 was produced in
whereas under the same reaction conditions, compound 18 41% yield from our developed synthetic strategy. Then, com-
readily underwent oxidation to yield compound 19 in 54% pound 24 was subjected to an SeO2 oxidation reaction. The oxi-
yields.
dation reaction was performed at 110 °C for 16 h to furnish the
Scheme 3: Reaction conditions: a) 5-bromo-2-chloropyrimidine (1 equiv), ethyl formate (1.5 equiv), THF (20 mL), n-butyllithium (0.6 equiv, 2.5 M) in
hexane, −100 °C, 2 h, 42%; b) boronic acid (1.3 equiv), PdCl2(PPh3)2 (0.1 equiv), dioxane/water (8:2), Na2HPO4 (2.0 equiv), TEA (4.0 equiv), 95 °C,
15 h, 40%; c) indolamine 10 (1.1 equiv) DCE, STAB (1.0 equiv), TEA (2 equiv), rt, 61% for 22, 68% for 23; d) NaOH at −10 °C, NH2OH·H2O at
−10 °C, MeOH, rt, 12 h, 49% for TIO2 and 51% for TIO3-rev.
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Scheme 4: Reaction conditions: a) boronic acid 15 (1.3 equiv), PdCl2(PPh3)2 (0.1 equiv), dioxane/H2O (8:2, Na2HPO4 (2.0 equiv), TEA (4.0 equiv),
95 °C, 15 h, 41%; b) SeO2 (2.0 equiv), dioxane , 110 °C, 16 h, 52%; c) indolamine 10 (1.1 equiv), DCE, STAB (1.0 equiv), TEA (2 equiv), rt, 38%; d)
NaOH at −10 °C, NH2OH·H2O at −10 °C, MeOH, rt, 12 h, 44%.
aldehyde 25 in 52% yield. Next, successful coupling of the 1 µM concentration of the designed inhibitors and panobinostat.
aldehyde 25 with indolamine 10 yielded the expected product All compounds displayed HDAC8 inhibition as predicted by
26 in 38% yield. Compound 26 was converted to hydroxamic our computational studies [12]. Complete inhibition of HDAC8
acid TIO4 in 44% yield under NaOH conditions in methanol was observed for panobinostat at 100 µM or 10 µM concentra-
and purified using C18 column chromatography. All the final tion, while only 89% inhibition was recorded at 1 µM concen-
compounds were thoroughly characterized by NMR and mass tration. The in vitro IC50 values for panobinostat against
spectrometry (see Supporting Information File 1).
HDAC8 have been shown to be 277 nM [11]. In vivo studies
measuring the IC50 values of panobinostat have also been
Having successfully synthesized the targets TOI1 to TOI4, our performed, however, since panobinostat is a pan-DAC inhibitor
next aim was to evaluate them biologically.
it has been difficult for researchers to specifically correlate
its IC50 value for HDAC8 in a physiological system [40]. Theo-
retical studies predicted TOI1 as the most potent inhibitor
Inhibition assay
The biochemical evaluation of the proposed inhibitors TOI1, over TOI2, TOI3-rev, and TOI4. TOI1 was shown to be
TOI2, TOI3-rev, and TOI4 was performed to experimentally the most potent inhibitor designed in this study producing
determine the potency of HDAC8 inhibition. The obtained 100% inhibition at a 100 µM, 90% inhibition at a 10 µM,
results, as shown in Table 1, aligned with the predicted compu- and 44% at a 1 µM concentration. At a 100 µM concentration,
tational studies. In a homogeneous fluorogenic assay, the TOI2, TOI3-rev and TOI4 inhibited 79%, 89%, and 93%
HDAC activity is quenched with a fluorescent dye that is teth- of HDAC8 activity, respectively. Strong inhibition was
ered to an acetyllysine-containing peptide. If the acetyl moiety seen at a 10 µM concentration for all inhibitors. At a concentra-
of the fluorophore is enzymatically hydrolyzed by HDAC8, it tion of 1 µM all inhibitors showed less inhibition of HDAC8
will produce a strongly fluorescent signal at 360 nm. Table 1 than panobinostat. It was noted that TOI2 and TOI3-rev
shows the percentage of HDAC8 inhibition at 100, 10, and produced similar inhibition results against HDAC8. Both of
Table 1: Percent inhibition of HDAC8 by hydroxamate inhibitors.
concentration
compound
100 µM
10 µM
1 µM
panobinostat (n = 4)a
TOI1 (n = 4)
TOI2 (n = 4)
TOI3-rev (n = 4)
TOI4 (n = 4 @ 100 and 10 µM,
n = 3 @ 1 µM)
100 0.0
100 0.0
79 8.1
89 7.0
93 8.1
100 0.0
90 21.1
75 13.4
75 26.9
89 13.4
89 13.4
44 11.5
23 26.9
37 18.1
65 21.4
TCAb
93 14.0
an = number of replicates for the assay; btrichostatin A (TCA) as an inhibitor control.
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these inhibitors have pyrimidine rings in the core; thus, the data (DCM), and dimethylformamide (DMF) were degassed in 20 L
suggests that while this ring structure did produce inhibition, it drums and passed through two sequential purification columns
was not as effective as the inclusion of a pyrazine ring. (activated alumina; molecular sieves for DMF) under a positive
This finding is consistent with our computational data which argon atmosphere. Thin-layer chromatography (TLC) was per-
demonstrated that TOI1 would slightly outperform TOI2 and formed on SiO2-60 F254 aluminum plates with visualization by
TOI3-rev.
ultraviolet (UV) detection at 254 nm or staining. Flash column
chromatography was performed using Purasil SiO2-60,
It is interesting to note that TOI1, which was shown to be better 230–400 mesh from Whatman. NMR spectra were recorded on
than TOI4 at the 100 µM level, was less potent than TOI4 at a BRUKER AV500 spectrometer (operating at 500 MHz for 1H
low concentration (1 µM). TOI4, which was predicted to be and 125 MHz for 13C acquisitions). Chemical shifts were re-
slightly less potent than TOI1, TOI2, and TO3-rev computa- ported as ppm relative to the solvent residual peak (CHCl3:
tionally was revealed via experiment to be a better inhibitor 7.26 ppm for 1H, 77.2 ppm for 13C). Data are reported as
than TOI2 and TOI3-rev. The result was observed presumably follows: chemical shifts, multiplicity (s = singlet, d = doublet,
due to a variation in the binding mode for TOI4 that differs t = triplet, q = quartet, quint = quintet, m = multiplet,
from that of TOI1, TOI2, and TOI3-rev. TOI4 was shown to br = broad), coupling constant J (Hz), and integration. High-
observe a slightly different orientation of the core heterocyclic resolution mass spectra were recorded using an ESI–TOF mass
ring as well as a change in the orientation of the terminal indole spectrometer (Agilent 6220 Time-of-Flight), gas temperature –
ring. It is not clear if this difference in binding mode affected 350 °C, drying gas (N2) – 8.0 L/min, mobile phase(s): metha-
the ability of TOI4 to maintain a higher potency at lower con- nol with 0.1% formic acid, flow rate: 0.2 mL/min, sample prep-
centrations. However, this observation does raise an interesting aration: The sample was dissolved in 1 drop of chloroform and
question: “Are certain modes of binding more effective at dif- diluted with 1 mL methanol.
ferent concentrations and if so how can this be accurately
modelled computationally?” Taken together, this data indicates Additional chemical synthesis details can be found in Support-
that while TOI1 is the best inhibitor of the four compounds ing Information File 1.
presented herein, TOI4 remains effective against HDAC8 even
at lower concentrations.
HDAC8 enzymatic activity assay
A fluorogenic assay (BPS Bioscience, catalog # 50008) was
performed to evaluate the inhibition potential of the designed
Conclusion
Our investigation provides a successful synthetic strategy inhibitors. The assay was carried out using the supplier’s
towards four new analogues of panobinostat having diazines as instructions. TOI1, TOI2, TOI3-rev, TOI4, and panobinostat
the central core and details the results of their biochemical eval- were evaluated at concentrations of 100 µM, 10 µM, and 1 µM
uation. Computational data corroborated that the substitution of in a 96 well plate. Trichostatin A (TCA), which was used as an
benzene in the molecular framework of panobinostat for a inhibitor control, was also evaluated at 100 µM. The blank con-
nitrogen-containing heterocycle in the core ring structure would sisted of HDAC assay buffer without the addition of inhibitor or
enhance the pharmacological properties while maintaining the HDAC8, while the positive control consisted of HDAC8 with-
level of HDAC8 inhibition. The targets TOI1–4 were synthe- out inhibitor added. The substrate concentration was 5 µM, and
sized from commercially available starting materials in moder- HDAC8 concentration per well was 4 ng/µL. The reaction was
ate yields. The synthesized compounds displayed potent activi- initiated by addition of enzyme. The fluorogenic substrate was
ty against HDAC8; thus, emphasizing the advantages of drug excited at 360 nm and the emission signal was detected at
design on a theoretical basis. These efforts led to the design of 460 nm using a Biotek Synergy HTX multimodal plate reader
potential analogues that warrant further studies to develop ther- after incubation for 30 minutes.
apeutic agents for neuroblastoma. Future research will be aimed
at investigating the HDAC class specificity of these designed
Preparation of the HDAC8 receptor for
analogues and evaluating their overall potential to inhibit docking
neuroblastoma cell growth.
The HDAC8 crystal structure (protein database pdb: 1W22)
[32] was utilized as the docking receptor for all compounds.
This receptor is crystalized as a dimer thus only the A chain
was prepared for docking using Sylbyl-X 2.1. A protocol
Experimental
General
Reagents and solvents were purchased from commercial defining the regions of hydrogen donor, acceptor and hydro-
sources and used without further purification unless otherwise phobic character was created using the SFXC protocol [41-43].
specified. Tetrahydrofuran (THF), ether, dichloromethane The Zn2+ ion which is known to be essential in the hydroxa-
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4. Chakrabarti, A.; Oehme, I.; Witt, O.; Oliveira, G.; Sippl, W.; Romier, C.;
Pierce, R. J.; Jung, M. Trends Pharmacol. Sci. 2015, 36, 481–492.
doi:10.1016/j.tips.2015.04.013
mate class of HDAC inhibitors was included in the preparation
of the receptor.
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Compounds were drawn in ChemDraw and the converted for
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Supporting Information
Supporting Information File 1
Experimental and analytical data.
[https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-16-59-S1.pdf]
17.Flefel, E.; Tantawy, W.; El-Sofany, W.; El-Shahat, M.; El-Sayed, A.;
Abd-Elshafy, D. Molecules 2017, 22, 148.
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19.Robarge, K. D.; Lee, W.; Eigenbrot, C.; Ultsch, M.; Wiesmann, C.;
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Funding
For the synthesis of the nitrogen containing heterocyclic cores,
S.B., S.V., K.D. S.A. and D. L. W. appreciate financial support
from the National Science Foundation under Grant Numbers
CHE-1652094. X. A. M., L. V. G. and S. V. S. would like to
thank the William Randolph Hearst Fellowship for the funding.
doi:10.1016/j.bmcl.2014.08.008
20.Banerjee, S.; Adhikari, N.; Amin, S. A.; Jha, T. Eur. J. Med. Chem.
2019, 164, 214–240. doi:10.1016/j.ejmech.2018.12.039
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112–126. doi:10.1002/bip.22135
ORCID® iDs
Davita L. Watkins - https://orcid.org/0000-0002-0943-7220
Shana V. Stoddard - https://orcid.org/0000-0002-0721-084X
22.Rettig, I.; Koeneke, E.; Trippel, F.; Mueller, W. C.; Burhenne, J.;
Kopp-Schneider, A.; Fabian, J.; Schober, A.; Fernekorn, U.;
von Deimling, A.; Deubzer, H. E.; Milde, T.; Witt, O.; Oehme, I.
Cell Death Dis. 2015, 6, e1657. doi:10.1038/cddis.2015.24
23.Oehme, I.; Deubzer, H. E.; Wegener, D.; Pickert, D.; Linke, J.-P.;
Hero, B.; Kopp-Schneider, A.; Westermann, F.; Ulrich, S. M.;
von Deimling, A.; Fischer, M.; Witt, O. Clin. Cancer Res. 2009, 15,
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