Synthesis and pharmacological evaluation of pomalidomide derivatives useful for sickle cell disease treatment

Article abstract of DOI:10.1016/j.bioorg.2021.105077

Fetal hemoglobin (HbF) induction constitutes a valuable and validated approach to treat the symptoms of sickle cell disease (SCD). Here, we synthesized pomalidomide–nitric oxide (NO) donor derivatives (3a–f) and evaluated their suitability as novel HbF in

Full text of DOI:10.1016/j.bioorg.2021.105077

Bioorganic Chemistry 114 (2021) 105077
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and pharmacological evaluation of pomalidomide derivatives
useful for sickle cell disease treatment
a
b,c
Thais Regina Ferreira de Melo , Brian M. Dulmovits
,
a
d
d
Guilherme Felipe dos Santos Fernandes , Cristiane M. de Souza , Carolina Lanaro ,
b,
c
b
,
c
a
b
d
Minghzu He , Yousef Al Abed , Man Chin Chung , Lionel Blanc , Fernando Ferreira Costa ,
a,*
Jean Leandro dos Santos
a
S a˜ o Paulo State University (UNESP), School of Pharmaceutical Sciences, Araraquara 14800-903, Brazil
b
c
Department of Molecular Medicine and Pediatrics, Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY 11549, USA
Laboratory of Developmental Erythropoiesis, Les Nelkin Memorial Pediatric Oncology Laboratory, The Feinstein Institutes for Medical Research, Manhasset, NY 11030,
USA
d
Faculty of Medical Sciences, State University of Campinas - UNICAMP, Campinas 13083-970, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Fetal hemoglobin (HbF) induction constitutes a valuable and validated approach to treat the symptoms of sickle
Fetal hemoglobin inducers
Nitric oxide
cell disease (SCD). Here, we synthesized pomalidomide–nitric oxide (NO) donor derivatives (3a–f) and evaluated
their suitability as novel HbF inducers. All compounds demonstrated different capacities of releasing NO, ranging
NO-donors
+
0
.3–30.3%. Compound 3d was the most effective HbF inducer for CD34 cells, exhibiting an effect similar to that
Epigenetics
of hydroxyurea. We investigated the mode of action of compound 3d for HbF induction by studying the in vitro
alterations in the levels of transcription factors (BCL11A, IKAROS, and LRF), inhibition of histone deacetylase
enzymes (HDAC-1 and HDAC-2), and measurement of cGMP levels. Additionally, compound 3d exhibited a
Pomalidomide
Sickle Cell Disease
potent anti-inflammatory effect similar to that of pomalidomide by reducing the TNF-
α
levels in human
mononuclear cells treated with lipopolysaccharides up to 58.6%. Chemical hydrolysis studies revealed that
compound 3d was stable at pH 7.4 up to 24 h. These results suggest that compound 3d is a novel HbF inducer
prototype with the potential to treat SCD symptoms.
1
. Introduction
Sickle Cell Disease (SCD) is a monogenic hemoglobinopathy that
recommended only for selective patients. Currently, four drugs are
approved for SCD: hydroxyurea (HU), L-glutamine, voxelotor, and cri-
zanlizumab [2]. Among them, HU is the oldest drug being used for
almost two decades, including in pediatric patients. The drug acts by
increasing the fetal hemoglobin (HbF) levels—an established and vali-
dated approach for the treatment.
affects millions of people worldwide, affecting 300,000–400,000 new-
borns annually, mainly in developing countries such as those in sub-
Saharan Africa [1]. Despite the wide phenotypic variability of SCD,
the vast majority of patients face common hallmarks of reduced life
expectancy and quality of life, high medical cost, chronic pain, and lack
of multiple therapeutic options.
Several small molecules have been identified as HbF inducers—na-
tural products (i.e., resveratrol), metformin, methyltransferase in-
hibitors (i.e., azacitidine and decitabine), histone deacetylase inhibitors
(i.e., butyrate and panobinostat), and immunomodulatory imide drugs
(IMiDs) (i.e., thalidomide and pomalidomide) [3]. Among these, IMiDs
are interesting HbF inducers prototypes and are the only ones that have
showed a therapeutic effect in vivo [4]. Pomalidomide, an approved
treatment for relapse and refractory multiple myeloma, regulates
erythropoiesis, slows down the erythroid maturation, and upregulates
Owing to the advances in technology in recent years, gene therapy
and hematopoietic stem cell transplantation have been considered as
potential cures; however, we are yet to overcome the challenges of
finding compatible donors, allogeneic graft rejection, and high cost.
Therefore, small molecule drugs are still essential to mitigate the di-
versity of symptoms since gene therapy and transplantation are
*
Corresponding author.
E-mail address: jean.santos@unesp.br (J.L. dos Santos).
https://doi.org/10.1016/j.bioorg.2021.105077
Received 17 February 2021; Received in revised form 3 June 2021; Accepted 7 June 2021
Available online 10 June 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

T.R.F. de Melo et al.
Bioorganic Chemistry 114 (2021) 105077
γ-globin gene expression by transcriptional mechanisms. Thus, it in-
duces potent fetal hemoglobin production without causing cytotoxic
side effects common to other HbF inducers, like HU [4,5].
dimethylaminopyridine (DMAP) as coupling reagents to obtain the
pomalidomide derivatives (3a–f) with yields ranging 13–43%. We
evaluated the purity of all compounds (3a–f) using high performance
liquid chromatographic (HPLC) analysis and confirmed them to be su-
Pomalidomide-dependent induction of HbF is due to reversion of
γ-globin silencing in erythroblasts by targeting the transcription factors,
BCL11A and SOX6, independently of IKZF1 degradation. Several
γ-globin repressors have been implicated in the globin switch, such as
BCL11A, SOX6, GATA-1, KLF1, and LSD1, with BCL11A being one of the
most known modulators of the fetal-to-adult globin switch. Pomalido-
mide downregulated all these repressors, albeit to different degrees, in
1
perior to 98.5%. Proton nuclear magnetic resonance ( H NMR) spectra
of all of the intermediates (8a, b; 10a, b; and 12a, b) and final com-
pounds (3a–f) revealed one signal that corresponded to the ylidene
hydrogen of the E-diastereomers [14,15].
2
.2. Nitrite quantification
+
CD34 cells [6].
Previously, we have demonstrated that another IMIDs (i.e. thalido-
mide) analogue containing a nitric oxide (NO) release subunit could
Nitrite quantification is an indirect method to measure the ability of
compounds to release NO. This assay is based on the reaction between
the released NO from compounds, oxygen, and water, providing nitrite
in the medium; this nitrite is further derivatized using the Griess reagent,
consisting of 0.2% N-(1-naphthyl)ethylenediamine dihydrochloride,
and 2% sulfanilamide in 5% phosphoric acid [16–18]. We studied the
release of NO after incubating 100 µM of each of the pomalidomide
+
induce gamma-globin expression in K562 and CD34 cells, although at
moderate levels [7–11]. In vivo studies using the transgenic sickle mice
revealed that these analogs increased HbF levels, reduced the amount of
proinflammatory cytokines, and surprisingly, reversed priapism
[
12,13].
Therefore, in a continuing effort to discover new HbF inducer agents
ꢀ 4
derivatives (3a–f) with a large excess of L-cysteine (50 × 10 M) for 1 h
for treating SCD and as an alternative to HU, we aimed to synthesize and
evaluate the pharmacological properties of new pomalidomide ana-
logues containing an NO donor subunit. As a potent HbF inducer, we
hypothesized that conjugation of pomalidomide derivatives with NO-
donor subunit could improve the HbF-inducer effect observed for its
parental drug (2) and thalidomide analogues (1) previously described
[
19]. Table 1 summarizes the main results, expressed as a percentage of
ꢀ
nitrite (NO
2
; mol/mol).
Compounds 3a–f induced nitrite formation at levels ranging
.1–30.3%, after the 1-h incubation (Table 1). The control, isosorbide
1
dinitrate (DNS), induced 12.0% nitrite formation. We observed a direct
relationship between the pattern of substitution in the furoxan subunits
and the ability to release NO. Furoxan subunits containing an electron-
withdrawing substituent at position 3 released more NO than those
containing electron-withdrawing groups at other positions. Phenyl-
sulfonyl derivatives (3c–d) released high levels of NO (28.1–30.3%),
followed by phenyl-furoxan derivatives (3a–b; 5.0–6.3%). Methyl-
furoxan derivatives (3e–f) released low levels of NO (0.3–1.1%) under
the same experimental condition. In the absence of L-cysteine, there was
no detectable production of nitrite in the medium (not shown).
(
Fig. 1). These new derivatives could also release NO, acting through
pleotropic effects useful to treat some of the SCD symptoms. The drug
design explored the bioisosteric replacement of phthalimide subunit (1)
to 3-amino-phthalimide present in the pomalidomide (2) (Fig. 1). In
addition, the molecular optimization evaluated the contribution of
furoxan with different NO-release profiles. Then, we studied the mech-
anisms of action of the new compounds involved in the activation of
γ-globin gene expression.
2
. Results
+
2
.3. Evaluation of γ-globin and HbF expression in CD34 cells
2
.1. Chemistry
Based on the different NO-release profiles, we evaluated compounds
3
b, 3d, and 3f for inducing γ-globin gene expression and HbF production
Scheme 1 summarizes the synthesized pomalidomide derivatives
+
+
using CD34 cells. Adult peripheral blood CD34 hematopoietic stem
and progenitor cells were isolated, expanded, and differentiated using a
(
3a–f). The first step of synthesis involved the coupling of furoxan de-
rivatives (6, 9, and 11) with hydrazides, 7a and 7b, in a solution con-
taining ethanol and acetic acid with variable yields ranging 34–75%.
The nitro group present in (4) was reduced to amino group (5) using
hydrogenation reaction catalyzed by palladium on carbon (Pd/C) in
acetone, at yields of 98%. In the last step, 3-aminophthalic anhydride (5)
and amino derivatives (8a, b; 10a, b; and 12a, b) were condensed using
3
-phase culture system that recapitulates human erythropoiesis,
including enucleation, in the presence of pomalidomide and compounds
b, 3d, and 3f at different concentrations of 0.1 µM, 1 µM, and 10 µM
20]. After 14 days of culture, only compound 3d at 1 µM induced sig-
3
[
nificant γ-globin gene expression (Fig. 2A). For this compound, the
concentration of 10 µM reduced substantially the number of cells, sug-
gesting toxicity. At 2.5 µM compound 3d induced potent γ-globin gene
1
-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 4-
Fig. 1. Drug design of pomalidomide–NO donor compounds (3a–f).
2

T.R.F. de Melo et al.
Bioorganic Chemistry 114 (2021) 105077
Scheme 1. Synthesis of pomalidomide derivatives with NO-donor properties (3a–f). Reagents and conditions: a) acetone, Pd/C, H
2
; b) aminobenzohydrazides,
+
ethanol, H , room temperature, 4–24 h; c) 3-aminophthalic anhydride (5), EDC, DMAP, DMF anhydrous, N
2
atmosphere, room temperature, 24–30 h.
We monitored the terminal erythroblast differentiation at days 7, 11,
and 14 of culture using flow cytometry. Erythroblasts were defined as
Table 1
NO-release Data.
pos
GPA cells, and maturation level was determined by the presence of
–
% NO
2
(mol/mol) 50 £ 10–4 M of L-Cysa
Compounds
α
4-integrin and band 3 proteins [20]. Flow cytometry revealed erythroid
+
DNS
12.0 ± 1
precursors and terminal erythroblast differentiation of CD34 cells
treated with all compounds, highlighting the differentiation that
occurred at day 14 (Fig. 5). Moreover, we verified the cellular
morphology using the May–Grunwald–Giemsa method, in which we
observed the predominance of control and control processes of diseases
with embryonic stem cells with proerythroblasts and basophilic eryth-
roblasts (supplementary material).
3
3
3
3
3
3
a
b
c
d
e
f
5.0 ± 1.0
6.3 ± 0.1
28.1 ± 1.0
30.3 ± 3.0
1.1 ± 0.1
0.3 ± 0.2
a
All values are the mean ± SEM. Determined by Griess reaction, after incu-
◦
bation for 1 h at 37 C in pH 7.4 buffered water, in the presence of 1:50 M excess
of L-cysteine. DNS, isosorbide dinitrate.
2
2
.4. Investigation of mechanisms for HbF induction
.4.1. Transcriptional/repressor factors
expression, without interfere with β-globin expression (Fig. 2B). At this
concentration, the 3d-treated cells exhibited comparable proliferation
to that of HU (10 µM)-treated cells; however, it was less potent than that
of pomalidomide (1 µM)-treated cells (Fig. 2B).
We evaluated the comprehensive network of transcription factors
+
involved in the regulation of γ-globin gene expression in CD34 cells
using western blot. Among its several gene repressors, BCL11A, IKAROS,
and LRF play key roles in the regulation of γ-globin gene expression
+
We measured HbF levels after the treatment of CD34 cells with
[
21,22]. After four days of differentiation, we observed that compound
compound 3d, HU, and pomalidomide on day 14 using HPLC. Repre-
sentative chromatograms for DMSO, pomalidomide, HU and 3d-treated
cultures can be found in the supplementary material (S25-S28). The HbF
level induced by 3d (2.5 µM) was significant in comparison to control
3
d (2.5 µM) and HU (10 µM) did not affect the expression of BCL11A and
LRF, however an increased levels in the expression of IKAROS was
observed (Fig. 6). On the contrary, pomalidomide (1 µM) reduced the
levels of BCL11A and IKAROS; however, the LRF levels were unaltered.
(
DMSO) (Fig. 3). HU did not interfere in hemoglobin A (HbA) levels,
whereas 3d caused a marginal reduction. The growth curves of treated
2
.4.2. Inhibition of histone deacetylase (HDAC 1 and 2)
+
CD34 cells exhibited similar profiles for 3d (2.5 µM) and HU (10 µM)
Epigenetic enzymes, such as histone deacetylase (HDAC), regulate
(
Fig. 4).
the γ-globin gene expression [23]. Previous studies have demonstrated
3

T.R.F. de Melo et al.
Bioorganic Chemistry 114 (2021) 105077
+
+
Fig. 2. Expression of γ-globin in CD34 cells. (A) CD34 cells were treated for 14 days with different concentrations of pomalidomide and compounds 3b, 3d, and 3f.
+
(
B) CD34 cells were treated for 14 days with 1 µM pomalidomide (POM), 10 µM hydroxyurea (HU), and 2.5 µM compound 3d. DMSO (0.1%) was used as a negative
control. Cell lysates were analyzed by western blot using γ-, β-, and α-globin antibodies. The mean relative γ-globin and β-globin expression levels were normalized to
that of GAPDH by analyzing the band density using ImageJ software. The error bars represent the SD from three individual experiments. *p < 0.05 compared with
DMSO (ANOVA followed by Dunnet test).
+
Fig. 3. Quantification of hemoglobin in CD34 cells. Hemoglobin production was assessed in supernatants from H
2
O-lysed cells, using high-performance liquid
chromatography (HPLC). Blotplots from DMSO, pomalidomide, HU, and 3d-treated cultures. The area under the curve (AUC) was calculated for each sample, and
relative hemoglobin composition was expressed as the mean percent (A) HbF/(HbF + HbA) ± SEM (N = 3), *p < 0.05 and (B) the mean percent HbA/(HbA + HbF) ±
SEM, *p < 0.05 compared with DMSO (ANOVA followed by Dunnet test).
that class I HDAC inhibitors, mainly those acting through HDAC-1 and
HDAC-2 inhibition, can induce γ-globin gene expression and increase
HbF levels in a dose-dependent manner using cultured primary cells
derived from sickle cell patients [23,24]. Thus, to evaluate its ability as
an HDAC inhibitor, we performed an enzymatic assay using 2.5 µM of
compound 3d. The percentage of inhibition for HDAC-1 and –2 induced
by 3d was 6% and 2%, respectively (Table 2). In this experiment, we
used 30 nM of vorinostat (also called suberanilohydroxamic acid, SAHA)
as a drug reference. At 30 nM vorinostat inhibited HDAC-1 and –2 by
87% and 72%, respectively. These data suggested that the HbF induction
mechanism is unrelated to HDAC inhibition.
4

T.R.F. de Melo et al.
Bioorganic Chemistry 114 (2021) 105077
Fig. 4. Growth curves (number of cells) for DMSO-, HU-, pomalidomide (POM)-, and 3d-treated cultures at days 4, 7, 11, and 14. The data are shown as mean cells/
mL ± SEM (N = 3). *p < 0.05 compared with DMSO (ANOVA followed by Tukey test).
2
.4.3. Quantification of cGMP levels
3. Discussion
The activation of the soluble guanylyl cyclase (sGC) pathway is
another mechanism that induces HbF production [25]. Since 3d acts as
NO donor, soluble cyclic guanosine monophosphate (cGMP) pathway
could be activated after treatment with this compound. Therefore, in
this experiment, we measured the levels of cGMP in human umbilical
vein endothelial cells (HUVEC) after incubation with 3d (2.5 µM), HU
In recent years, novel drugs have been approved for SCD treatment;
however, HU remains the only approved drug capable of increasing HbF
levels. Long-term treatment using HU leads to a significant reduction of
morbidity and mortality [26,27]. High levels of HbF decrease hemo-
globin S polymerization inside erythrocytes; thus, preventing the sick-
ling process. In addition, HU reduces the adhesiveness of the red blood
cells, platelets, and leukocytes, preventing the vaso-occlusive process
[28,29]. Despite all those beneficial effects, severe adverse events, such
as myelosuppression and lack of effectiveness in a significant patient
population, limit its use, justifying the need for novel HbF inducers [30].
The complex heterogeneity of SCD pathophysiology demands efforts
for identifying novel compounds that act through multiple mechanisms.
Previously, we demonstrated that thalidomide derivatives designed to
release NO could contribute through multiple effects in SCD treatment,
including HbF induction, anti-inflammatory, and antiplatelet effects
(
10 µM), and pomalidomide (1 µM) or DMSO (vehicle control; 0.1%) for
4 h (Fig. 7). Both HU and compound 3d elevated cGMP levels in
2
HUVEC cells to 9.6 and 24.4 pmol cGMP/µg protein, respectively. In
contrast, pomalidomide did not increase the cGMP level in HUVEC cells.
Similar observation was found after measurement of cGMP levels using
CD34 + cells, however, we found lesser amount of cGMP compared to
HUVEC cells (supplementary material).
2
.5. Measurement of TNF- level in human mononuclear cells culture
α
[
8–10,12]. Although the mechanisms of these effects of the thalidomide
We employed enzyme-linked immunosorbent assay (ELISA) to
determine the level of the proinflammatory cytokine, tumor necrosis
factor (TNF- ), in the supernatant of mononuclear cell cultures treated
derivatives were not completely established, they had distinct effects
compared with thalidomide [10].
α
α
Despite these effects, thalidomide is less potent than pomalidomide
in inducing HbF production [4,5]. In addition, the anti-inflammatory
activity of pomalidomide is greater than that of thalidomide, partially
with lipopolysaccharide (LPS) and co-incubated with either DMSO,
pomalidomide, HU, or 3d (Fig. 8). LPS (50 mg/mL) was used as an in-
flammatory stimulus (data not shown). We performed a dose–response
assay to characterize the cellular viability. Only those concentrations
exhibiting viability greater than 90% were considered in this study.
explained by its ability to inhibit TNF- [31]. Pomalidomide also in-
α
creases erythropoiesis and preserves bone marrow function [5]. Some
studies suggested that the HbF-inducing mechanism of pomalidomide is
selectively related to BCL11A and SOX-6 repression factors [6]. Based on
all promising effects of pomalidomide, a phase I trial was conducted to
assess its effectiveness, safety, and maximum tolerated dose in SCD
patients treated with pomalidomide (US ClinicalTrials.gov identifier:
NCT01522547). Therefore, considering the potential of pomalidomide
as a novel prototype HbF-inducer, we evaluated the effectiveness of
hybrid pomalidomide–NO donor subunit to optimize the beneficial ef-
fects previously identified in the thalidomide–NO donors.
Synthetic procedures allowed the preparation of compounds 3a–f
through coupling reactions with yields ranging 13–43%. We chose
furoxan (1,2,5-oxadiazole 2-N-oxide) as the NO-donor subunit as it is
advantageous over other NO-donors (i.e., organic nitrate esters) in terms
of the possibility to modulate the levels of NO released in the medium.
The insertion of electron-withdrawing substituents at position 3 of
furoxan enhances the NO-release compared with that of electron-
enriching substituents. Therefore, for this study, we selected furoxan
Previously, we demonstrated that HU did not reduce TNF-
this experiment, cultured cells were treated with pomalidomide (5 µM)
levels
α
levels [8]. In
and 3d (1 µM and 2.5 µM). All tested compounds reduced TNF-
α
compared with LPS (425 pg/mL). Compound 3d and pomalidomide
exhibited comparable effects in reducing the production of this proin-
flammatory cytokine with TNF-
α
levels of 180 pg/mL (57.6%) and 176
pg/mL (58.6%), respectively (Fig. 8).
2
.6. In vitro stability study
To determine the in vitro stability of compound 3d at pH 1.0 and 7.4,
we performed a chemical hydrolysis study using the HPLC method. After
h, we observed about 96% degradation of the compound at pH 1.0,
1
suggesting that compound 3d is unstable under highly acid conditions.
On the contrary, at pH 7.4 that mimics the plasma pH, the compound
was stable up to 12 h, after which there was marginal degradation of
4
.86% and 8.06% at 20 h and 24 h, respectively (Fig. 9).
5

T.R.F. de Melo et al.
Bioorganic Chemistry 114 (2021) 105077
Fig. 5. Flow cytometric characterization of erythroid precursors and terminal erythroblast differentiation. Terminal differentiation was monitored via 4-integrin
α
pos
hi
lo
lo
hi
and band 3 levels of GPA cells on indicated days. Cells with
α
4-integrin /band 3 represent less mature erythroblasts, whereas those with 4-integrin /band3
α
are further differentiated (N = 3).
containing phenylsulfonyl (strong), phenyl (moderate), and methyl
weak) substituents with the aim to characterize a possible relationship
endothelium and inducing HbF production [34]. The NO-induced
augmentation of HbF was a result of the activation of the sGC
pathway in both erythroleukemic and primary erythroblast cells
[25,35]. cFOX is a transcription factor activated by the NO/cGMP
pathway; it binds to the hypersensitive site 2 in the β-globin locus
control region and the Sp1 in the CACCC box [36–38].
(
between the NO release and HbF induction. We determined that the
levels of nitrite ranged from 28.1 to 30.3% for phenylsulfonyl de-
rivatives (3c and 3d), 5.0–6.3% for phenylfuroxan (3a and 3b), and
0
.3–1.1% for methylfuroxan (3e and 3f).
In sickled cells, vascular hemolysis releases the heme group arising
We selected three compounds, 3b, 3d, and 3f, with different NO-
release profiles to study their ability to induce γ-globin gene expres-
from hemoglobin into circulation, which reduces the endothelial NO
bioavailability and favors a vasoconstrictor response. In the vascular
endothelium, chronic injury associated with low levels of NO contrib-
utes to inflammation and hypercoagulability states, leading to vaso-
occlusion and prothrombotic events [32]. Abnormal adhesion of
erythrocytes to vascular endothelium is caused due to augmented
adhesion molecules. Additional interactions with leukocytes and acti-
vated platelets induce cellular heteroaggregation that is responsible for
vaso-occlusion [2,26,33].
+
sion in CD34 cells. Of these, only compound 3d induced the gene
expression. It was observed an appropriate balance between optimum
effect and cellular toxicity at 2.5 µM. Interestingly, this phenylfuroxan
derivative (3d) was extremely potent at releasing NO; it generated
around 30.3% of nitrite, suggesting that high levels of NO-release may
be necessary to induce γ-globin gene expression. Both compound 3d and
HU exhibited similar levels of γ-globin gene expression; however, this
effect was lesser than that induced by pomalidomide. It has been re-
ported that after metabolism, HU gets bioconverted to NO. HU-treated
patients exhibited augmentation in their plasma levels of nitrite,
NO has a pleiotropic effect in SCD, contributing as vasodilator; thus,
decreasing platelet aggregation and cellular adhesion in the
6

T.R.F. de Melo et al.
Bioorganic Chemistry 114 (2021) 105077
+
Fig. 6. (A) Western blot for BCL11A and IKAROS in CD34 cells treated with 0.1% DMSO (negative control), 1 µM POM, 10 µM HU, and 2.5 µM compound 3d. Cell
lysates were analyzed on day 4 of the treatment. The mean relative BCL11A and IKAROS expression levels were normalized to that of GAPDH by analyzing the band
density using the ImageJ software. The error bars represent the SD from three individual experiments, *p < 0.05 compared with DMSO (ANOVA followed by Dunnet
+
test). (B) Western blot for LRF in CD34 cells treated with 0.1% DMSO, 1 µM POM, 10 µM HU, and 2.51 µM 3d at day 4 of culture. The mean relative LRF expression
level was normalized to that of GAPDH by analyzing the band density using the ImageJ software. The error bars represent the SD from three individual experiments,
*p < 0.05 compared with DMSO (ANOVA followed by Dunnet test).
Table 2
Percentage of HDAC-1 and –2 inhibition.
%
inhibition
Treatment
HDAC1
HDAC2
3
d *
6
2
vorinostat, 30 nM
87
72
The enzymatic assays were performed in triplicate at each concentration (360
nm). The percent activity in the presence of each compound was calculated
according to the following equation: % activity = (F ꢀ Fb)/(Ft ꢀ Fb), where F =
fluorescent intensity in the presence of vorinostat (SAHA).
*
2
.5 µM of compound 3d was used.
nitrate, and iron nitrosyl hemoglobin, 2 h after the oral intake of HU
39]. Therefore, there exists a relationship between NO levels and
[
γ-globin gene expression [10].
Distinct stresses can activate γ-globin gene expression in erythroid
cells [40]. Thus, in addition to gene expression, we must measure HbF
levels during phenotypic assays to avoid false-positive results. On day
Fig. 7. Effects 0.1% DMSO, 1 µM POM, 10 µM HU, and 2.5 µM compound 3d
on intracellular cGMP levels in HUVEC cells. HUVEC cells were used at the
confluence, and the results were measured after 48 h of treatment. Values
represent means ± SEM (n = 3). *p < 0.05.
1
4, we measured levels of HbF after treatment with compound 3d, HU,
+
and pomalidomide in CD34 cells. The HbF level induced by 3d (2.5 µM)
was significant in comparison to control (DMSO) . These results
confirmed the findings observed in the gene expression assay.
We also studied the mechanism of action of 3d-induced γ-globin gene
expression and HbF production. Among the several transcription factors
that silence the globin genes, the effect of BCL11A is notorious; it binds
within the β-globin locus and silences
41]. As pomalidomide downregulates several transcription factors,
including BCL11A and IKAROS involved in γ-globin gene expression, we
ε- and γ-globin gene expressions
[
7

T.R.F. de Melo et al.
Bioorganic Chemistry 114 (2021) 105077
has been associated with the teratogenic effects of thalidomide [42,44].
Molecular studies revealed that the binding of IMiDs to cereblon re-
quires the glutarimide subunit as pharmacophore [45]. In our drug
design, we speculated the suppression of glutarimide subunit to elimi-
nate the asymmetric center. The absence of glutarimide subunit for 3d
could explain the lack of effect on transcription factors, such as IKAROS.
Epigenetic sites act as recognition sites that facilitate interactions
with proteins or complexes and then regulate the pattern of gene
expression. Similarly, chromatin remodeling is a regulated phenomenon
that allows the switch between
ε-, γ-, and β-globin expression during the
different development stages from embryo to adulthood. The regulation
of γ-globin gene expression involves several epigenetic mechanisms,
including histone acetylation and methylation [36]. Since the discovery
of the effect of butyrate on rising HbF levels, HDAC inhibitors have
received attention as promising inducers of HbF [2]. It was demon-
strated that selective inhibition of HDAC class I, specifically HDAC-1 and
–
2, increases HbF levels without interfering with cell cycle proliferation
[
23]. To assess the inhibitory effect of 3d on HDAC-1 and –2, we per-
Fig. 8. Level of the proinflammatory cytokine, TNF- , determined using ELISA
α
formed an enzymatic assay that revealed extremely weak inhibition of
these enzymes by 3d. At 2.5 µM, compound 3d inhibited only 6% and
2% of HDAC-1 and –2, respectively, suggesting that it did not induce
HbF via this mechanism.
in the supernatant of human mononuclear culture treated with LPS and co-
incubated with test drugs. Pomalidomide (POM) was used as positive anti-
inflammatory controls. LPS at 50 mg/mL was used as an inflammatory stim-
ulus. Data are represented as the mean ± SEM; p < 0.05 compared with lipo-
polysaccharide (LPS) (ANOVA followed by Tukey’s test); N = 3 experiments; C
Based on the NO-release effect of 3d, we investigated the activation
of the sGC pathway. We measured the levels of cGMP in HUVEC cells
after incubation with 3d (2.5 µM), HU (10 µM), and pomalidomide (1
µM) or DMSO (vehicle control). Pomalidomide did not affect cGMP
levels. Conversely, compound 3d was 2.5-fold more potent than HU in
increasing the cGMP level. The activation of the NO–cGMP pathway
increases Jun mRNA levels [46,47], which modulate γ-globin gene
expression through a cAMP response element (CRE). The way CRE acts is
comparable with that of transcription factor CRE binding protein 1
(CREB1) [48]. Increased levels of NO and cGMP were also related to the
increase in phosphorylation of p38 MAPK [49]. Additionally, CREB1
activated γ-globin gene expression through the p38 MAPK pathway in
erythroid cells [50]. Thus, the NO–cGMP pathway can stimulate the
expression of the γ-globin gene through the pathways involving both
p38 MAPK and JUN.
(
ꢀ ) : DMSO.
investigated similar effects for 3d [6]. Although our study confirmed
that pomalidomide downregulated BCL11A and IKAROS, we did not
observe such effects for both HU and 3d. On the contrary, the levels of
IKAROS were slightly increased for the group treated with 3d, sug-
gesting a different mode of action for HbF induction compared to
pomalidomide. In addition, another transcription factor, leukemia/
lymphoma-related factor (LRF), was not altered by pomalidomide,
2 2
HU, or 3d. LRF binds DNA through its C-terminal C H -type zinc finger
domain and recruits a transcriptional repressor complex through its N-
terminal domain. LRF also represses the expression of the γ-globin gene
[
22].
Pomalidomide binds to cereblon, an E3 ligase protein and a substrate
receptor for the CRL4 (CUL4 ꢀ RBX1 ꢀ DDB1) ubiquitin ligase complex,
and induces the degradation of IKAROS, involved in the silencing of
γ-globin genes [42]. This occurs through the combined effect of IKAROS
and GATA-1, suppressing γ-globin genes [43]. Additionally, cereblon
Despite its effectiveness as an HbF inducer, in vitro and in vivo studies
demonstrated that HU did not reduce the levels of TNF-
this pro-inflammatory cytokine is commonly found in SCD patients
[8,12,51,52]. The high levels of TNF- contribute to an increase in
α. High levels of
α
Fig. 9. In vitro determination of chemical stability for compound 3d in buffers at pH 1.0 and 7.4. Data are represented as means ± SEMs and expressed as %.
8

T.R.F. de Melo et al.
Bioorganic Chemistry 114 (2021) 105077
adhesion molecules (i.e., ICAM and VCAM-1), worsening vaso-
occlusion, and maintaining active chronic inflammation with the
multiplet (m). Chemical shifts were expressed in parts per million (ppm)
relative to tetramethylsilane. Elemental analyses (C, H, and N) were
performed on a Perkin-Elmer model 240C analyzer, and the data were
within ± 0.4% of the theoretical values.
recruitment of inflammatory cells [53]. Moreover, TNF- acts directly
α
on the nociceptor, contributing to the primary complaint of chronic pain
in sickle cell patients [54,55].
Our results demonstrated that both 3d and pomalidomide reduce the
5.2. General procedures for the synthesis of N-oxide derivatives (8a-b;
10a-b and 12a-b)
levels of TNF-
α
in the supernatants of human mononuclear cultures
treated with LPS. We did not observe differences between 3d and
pomalidomide; but 3d was more potent than the thalidomide de-
rivatives previously described [10]. Based on the structure activity
Compounds 5, 6, 9 and 11 were synthesized according to previously
described methodologies [38,56,57]. In a nitrogen atmosphere, an
equivalent amount of furoxan derivatives (6, 9 or 11) (1.5 mmol) and 2-
(7a) or 3-aminobenzhydrazides (7b) (1.5 mmol) was stirred under room
temperature at different times (4–24 h) in a medium containing ethanol
and 0.1 mL of anhydrous acetic acid. The reaction was monitored by TLC
using as a mobile phase a mixture of hexane (50%): ethyl acetate (50%).
After, it was carried out a filtration under reduced pressure to collect N-
oxide intermediates (8a-b; 10a-b and 12a-b), which were washed with
cold ethanol to remove residual reagents. If necessary, an additional
purification was performed by column chromatography using Isolera
Biotage equipment, using a 20 µm (25 g) Sphere silica column, with a
flow rate of 25 mL / min and phase mobile 50% ethyl acetate / 50%
hexane. The N-oxide derivatives (8a-b; 10a-b and 12a-b) were obtained
at yields ranging from 34 to 75%.
relationship for phthalimide derivatives and its effect of reducing TNF-
α
levels, it is possible to comprehend that the 3-amino-phthalimide sub-
unit, found for 3d, is the pharmacophore responsible for the anti-
inflammatory effect [8–11]. In addition, it is notorious that this com-
pound was stable at pH 7.4 up to 12 h; however, it was unstable in acidic
conditions; thus, demanding appropriate formulation for further studies.
4
. Conclusions
In this study, we successfully synthesized a novel series of pomali-
domide derivatives with NO-donor properties (3a–f) and evaluated their
suitability as HbF inducers for SCD treatment. The new pomalidomide
derivatives were optimized aiming to not only induce HbF production,
but also contribute for pleiotropic effects by releasing NO. The de-
rivatives (3a–f) released 0.3–30.3% of nitrite in the medium. Strong NO-
donor, such as phenyl-sulfonyl 3d, was able to induce gamma-globin
gene expression at profile similar to that of HU. Compound 3d also
induced HbF production and did not alter the expression of transcription
factors involved in the γ-globin gene regulation, such as BCL11A and
LRF. Although, a slightly increase in the levels of IKAROS was found.
Compound 3d did not inhibit the enzymes, HDAC-1 and –2, involved in
the regulation of gamma-globin gene expression. Interestingly, 3d
significantly increased the levels of cGMP in HUVEC cells compared
with HU, while pomalidomide did not exhibit this effect. Moreover, it
(E)-4-(4-((2-(2-aminobenzoyl)hydrazono)methyl)phenoxy)-3-
phenyl-1,2,5-oxadiazole 2-N-oxide (8a)
◦
ꢀ 1
White powder; yield: 55%; mp: 201–203 C. IR
max
–
–
(cm ; KBr pel-
lets): 3113 (C
–
H
aromatic
), 2928 (C
–
H
alkyl
), 1636 (C
C aromatic), 1479
–
–
–
1
(C
C aromatic), 1382 (N O oxide). H NMR (300 MHz, DMSO‑d , δ ppm)
6
δ: 11.69 (s, 1H), 8.41 (s, 1H,), 8.07 (dd, 2H), 7.8 (dd, 2H, J = 7.8 Hz),
7.55–7.65 (m, 6H), 7.18 (t, 1H, J = 8.0 Hz e J = 1.7 Hz), 6.77 (dd, 2H, J
1
3
= 7.0 Hz), 6.55 (ddd, 1H, J = 7.0 Hz), 6.39 (s, 2H, NH ). C NMR (75
2
MHz, DMSO‑d , δ ppm) δ: 161.92, 153.64, 150.13, 132.95, 132.85,
6
131.01, 129.17, 128.67, 126.73, 121.71, 120.60, 116.44, 114.69,
108.41. Anal. Calcd (%) for C22H17N O : C: 63.61; H: 4.13; N: 16.86.
5 4
reduced TNF-
α
levels in mononuclear cells treated with LPS, similar to
Found: C: 63.64; H: 4.14; N: 16.87. (MW: 415.14, Rf: 0.27, eluent hex-
ane: ethyl acetate 1: 1 (v/v)).
the effect of pomalidomide. Although unstable in acid conditions, 3d
exhibited good chemical stability at pH 7.4 up to 24 h. In summary,
compound 3d emerged as a novel HbF inducer with anti-inflammatory
properties and may prove useful as an alternative to treat the symp-
toms of SCD.
(E)-4-(4-((2-(4-aminobenzoyl)hydrazono)methyl)phenoxy)-3-
phenyl-1,2,5-oxadiazole 2-N-oxide (8b)
◦
ꢀ 1
White powder; yield: 63%; mp: 217–219 C. IR
max
–
–
(cm ; KBr pel-
lets): 3111 (C
–
H
aromatic
), 2926 (C
–
H
alkyl
), 1638 (C
C aromatic), 1475
–
–
aromatic), 1382 (N–O oxide
1
(
C
C
). H NMR (300 MHz, DMSO‑d
6
, δ ppm)
5
. Experimental section
δ: 11.54 (s, 1H); 8.42 (s, 1H), 8.08 (dd, J = 8.2 Hz, 2H), 7.82 (d, J = 7.6
Hz, 2H), 7.58 – 7.63 (m, 3H), 7.60 (m, 4H), 6.60 (dd, J = 7.6 Hz, 2H),
1
3
5
.1. Chemistry. General information
5.81 (s, 2H, NH
2
). C NMR (75 MHz, DMSO‑d
6
, δ ppm) δ: 161.94,
1
53.51, 152.42, 133.03, 131.02, 129.17, 128.55, 126.73, 121.71,
Reagents and solvents were purchased from commercial suppliers at
120.58, 119.43, 112.69, 108.40. Anal. Calcd (%) for C22 : C:
17 5 4
H N O
reagent purity grade and were used without any further purification.
Dry solvents used in the reactions were obtained by distillation of
technical grade materials over appropriate dehydrating agents. Thin-
layer chromatography (TLC), precoated with silica gel 60 (HF-254;
Merck) to a thickness of 0.25 mm, was used for monitoring all reactions.
The plates were revealed under UV light (254 nm). All compounds were
purified on a chromatography column with silica gel (60 Å pore size,
63.61; H: 4.13; N: 16.86. Found: C: 63.63; H: 4.15; N: 16.86. (MW:
415.14, Rf: 0.30, eluent hexane: ethyl acetate 1: 1 (v/v)).
(E)-4-(4-((2-(2-aminobenzoyl)hydrazono)methyl)phenoxy)-3-(phe-
nylsulfonyl)-1,2,5-oxadiazole 2-N-oxide (10a)
◦
ꢀ 1
White powder; yield: 59%; mp: 175–178 C. IR max (cm ; KBr pel-
–
H
aromatic), 2927 (C
–
H
alkyl), 1636 (C
–
C aromatic), 1480
lets): 3108 (C
–
–O oxide
1
(C
–
C
aromatic), 1381 (N
). H NMR (300 MHz, DMSO‑d
6
, δ ppm)
3
5–75-µm particle size) and the following solvents were used as mobile
δ: 11.70 (s, 1H), 8.42 (s, 1H), 8.06 (d, J = 7.4 Hz, 2H), 7.93 (t, J = 7.5 Hz
e J = 1.2, 1H), 7.87 – 7.73 (m, 4H), 7.55 (m, 3H), 7.21 (t, J = 8.3 Hz,
1H), 6.76 (d, J = 7.9 Hz, 1H), 6.58 (t, J = 7.5 Hz and J = 1.5, 1H), 6.40
phase: dichloromethane, hexane, ethyl acetate and petroleum ether. In
addition, the purity analyzed by HPLC for all tested compounds was
superior to 98%. Melting points (mp) were determined in open capillary
tubes using an electrothermal melting point apparatus (SMP3; Bibby
Stuart Scientific). Infrared (IR) spectroscopy (KBr disc) was performed
on an FTIR-8300 Shimadzu spectrometer, and the frequencies are
1
3
(s, 2H, NH
2
). C NMR (75 MHz, DMSO‑d
6
, δ ppm) δ: 158.23, 153.59,
150.13, 136.92, 136.35, 132.94, 132.40, 130.10, 128.69, 120.15,
116.43, 114.68, 111.37. Anal. Calcd (%) for C22 S: C: 55.11; H:
17 5 6
H N O
3.57; N: 14.61. Found: C: C: 55.10; H: 3.57; N: 14.63. (MW: 479.47, Rf:
0.15, eluent hexane: ethyl acetate 1: 1 (v/v)).
ꢀ
1
1
expressed per cm . The nuclear magnetic resonance (NMR) for H and
1
3
C of all compounds were scanned on a Bruker Fourier with Dual probe
(E)-4-(4-((2-(4-aminobenzoyl)hydrazono)methyl)phenoxy)-3-(phe-
nylsulfonyl)-1,2,5-oxadiazole 2-N-oxide (10b)
1
3
1
C/ H (300-MHz) NMR spectrometer using dimethyl sulfoxide
◦
ꢀ 1
(
DMSO‑d
6
), as solvent. Chemical shifts were expressed in parts per
White powder; yield: 75%; mp: 173–175 C. IR max (cm ; KBr pel-
–
H
aromatic), 2925 (C
–
H
alkyl), 1638 (C
–
C aromatic), 1477
). H NMR (300 MHz, DMSO‑d
million (ppm) relative to tetramethylsilane. The signal multiplicities are
reported as singlet (s), doublet (d), doublet of doublet (dd), and
lets): 3110 (C
–
–
aromatic), 1382 (N–O oxide
1
(C
C
6
, δ ppm)
9

T.R.F. de Melo et al.
Bioorganic Chemistry 114 (2021) 105077
δ: 11.52 (s, 1H), 8.42 (s, 1H), 8.06 (dd, 2H, J = 8 Hz), 7.77–7.94 (m, 5H),
NMR (300 MHz, DMSO‑d
6
, δ ppm) δ: 12.04 (s, 1H), 8.48 (s, 1H),
7
.67 (dd, 2H, J = 7.5 Hz), 7.51 (dd, 2H, J = 7.5 Hz), 6.6 (dd, 2H, J = 8.0
8.09–8.02 (m, 4H), 7.90 (dd, 2H), 7.61–7.51 (m, 8H), 7.09–7.05 (m,
1
3
13
Hz), 5.8 (2H, s, NH
2
). C NMR (75 MHz, DMSO‑d
6
, δ ppm) δ: 158.65,
2H), 6.58 (s, 2H). C NMR (75 MHz, DMSO‑d
6
, δ ppm) δ: 168.54,
1
1
5
4
53.83, 152.82, 144.96, 136.75, 130.51, 129.98, 129.91, 128.57,
167.33, 163.29, 162.30, 154.27, 147.46, 147.37, 136.14, 132.45,
131.45, 129.59, 129.37, 128.65, 127.33, 127.15, 122.16, 122.03,
121.05, 111.73, 109.06, 108.84, 40.16, 40.02, 39.88, 39.74, 39.60,
20.54, 119.65, 113.07, 111.75. Anal. Calcd (%) for C22 S: C:
17 5 6
H N O
5.11; H: 3.57; N: 14.61. Found: C: C: 55.15; H: 3.58; N: 14.62. (MW:
79.47, Rf: 0.22, eluent hexane: ethyl acetate 1: 1 (v/v)).
20 6 6
39.46, 39.32, 30.85. Anal. Calcd (%) for C30H N O : C, 64.28; H, 3.60;
(
E)-4-(4-((2-(2-aminobenzoyl)hydrazono)methyl)phenoxy)-3-
methyl-1,2,5-oxadiazole 2-N-oxide (12a)
White powder; yield: 65%; mp: 165–168 C. IR max (cm ; KBr pel-
N, 14.99. Found: C, 64.27; H, 3.61; N, 14.98. (MW: 545.50, Rf: 0.32,
eluent ether petroleum (55%): ethyl acetate (45%)).
(E)-4-(4-((2-(2-(4-amino-1,3-dioxoisoindolin-2-yl)benzoyl)hydra-
zono)methyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-N-oxide
(3c)
◦
ꢀ 1
–
C aromatic), 1475
lets): 3103 (C
–
H
aromatic), 2924 (C
–
H
alkyl), 1635 (C
–
–
–O oxide). H NMR (300 MHz, DMSO‑d
1
(
C
–
C
aromatic), 1381 (N
6
, δ ppm)
◦
ꢀ 1
δ: 11.67 (1H, s), 8.41 (1H, s), 7.80 (2H, dd, J = 8 Hz), 7.48–7.57 (3H, m),
Yellowish powder; yield: 13%; mp: 216–218 C. IR max (cm ; KBr
7
6
1
1
.2 (1H, ddd, J = 7 Hz), 6.75 (1H, dd, J = 7 Hz), 6.57 (1H, J = 8 Hz),
.39 (s, 2H), 2.13 (s, 3H). 13C NMR (75 MHz, DMSO‑d
, δ ppm) δ:
65.73, 163.30, 154.02, 150.40, 145.70, 132.64, 129.05, 120.48,
16.68, 114.91, 113.64, 107.94, 7.29. Anal. Calcd (%) for C17
pellets): 3058 (C
–
H
aromatic), 2931 (C
–
H
alkyl
), 1780 and 1720 (C–
O
–
–
–
–
–
1
6
imide), 1610 (C
–
C
aromatic), 1465 (C
C aromatic), 1360 (N O oxide). H
NMR (300 MHz, DMSO‑d
6
, δ ppm) δ: 12.06 (s, 1H), 8.48 (s, 1H), 8.06 (d,
H
15
5
N O
4
:
2H, J = 7.1 Hz, 2H), 8.02 (dd, 1H, J = 8.0), 7.89 (dd, 2H), 7.63–7.59 (m,
7H), 7.52 (ddd, 1H, J = 7,0), 7.09 (dd, 1H, J = 7.08), 7.05 (dd, 1H, J =
C: 57.79; H: 4.28; N: 19.82. Found: C: 57.81; H: 4.29; N: 19.82. (MW:
53.33, Rf: 0.22, eluent dichloromethane (95%): methanol (5%)).
E)-4-(4-((2-(4-aminobenzoyl)hydrazono)methyl)phenoxy)-3-
methyl-1,2,5-oxadiazole 2-N-oxide (12b)
White powder; yield: 66%; mp: 160–162 C. IR max (cm ; KBr pel-
3
7,07), 6.58 (s, 2H). 13C NMR (75 MHz, DMSO‑d
6
, δ ppm) δ: 168.54,
(
167.35, 163.36, 162.30, 147.44, 136.17, 135.52, 132.58, 132.43,
131.47, 129.60, 129.40, 128.66, 127.36, 127.16, 122.24, 122.00,
◦
ꢀ 1
aromatic), 1475
20 6 8
121.05, 111.78, 109.05, 108.85. Anal. Calcd (%) for C30H N O S: C,
–
H
aromatic), 2925 (C
–
H
alkyl), 1634 (C
–
C
57.69; H, 3.23; N, 13.46. Found: C, 57.70; H, 3.24; N, 13.46. (MW:
624.58, Rf: 0.20, eluent ether petroleum (55%): ethyl acetate (45%)).
(E)-4-(4-((2-(4-(4-amino-1,3-dioxoisoindolin-2-yl)benzoyl)hydra-
zono)methyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-N-oxide
(3d)
lets): 3101 (C
–
–O oxide). H NMR (300 MHz, DMSO‑d
1
(
C
–
C
aromatic), 1382 (N
6
, δ ppm)
δ: 11.51 (s, 1H), 8.41 (s, 1H), 7.80 (dd, 2H, J = 8 Hz), 7.67 (dd, 2H, J =
7
2
1
1
.5 Hz), 7.50 (dd, 2H, J = 7.5 Hz), 6.60 (dd, 2H, J = 7.5 Hz), 5.80 (s,
H), 2.13 (s, 3H). 13C NMR (75 MHz, DMSO‑d
, δ ppm) δ: 162.98,
53.58, 152.47, 144.75, 132.79, 129.08, 128.64, 120.08, 119.40,
12.71, 107.74, 7.14. Anal. Calcd (%) for C17 : C: 57.79; H: 4.28;
6
◦
ꢀ 1
Yellowish powder; yield: 20%; mp: 221–223 C. IR max (cm ; KBr
H
15
N
5
O
4
pellets): 3057 (C
–
H
aromatic), 2927 (C
–
H
alkyl
), 1775 and 1718 (C–
O
–
–
–
–
), 1361 (N–O oxide). H
1
N: 19.82. Found: C: 57.83; H: 4.29; N: 19.85. (MW: 353.33, Rf: 0.15,
eluent dichloromethane (95%): methanol (5%)).
imide), 1610 (C
NMR (300 MHz, DMSO‑d
.02–8.07 (m, 4H), 7.74–7.94 (m, 5H), 7.49–7.62 (m, 5H), 7.03–7.09
C
aromatic), 1461 (C
–
C
aromatic
6
, δ ppm) δ: 12.02 (s, 1H), 8.49 (s, 1H),
8
1
3
5
.3. General procedures for the pomalidomide-NO donor derivatives (3a-
(m, 2H), 6.61 (s, 2H). C NMR (75 MHz, DMSO‑d
166.58, 162.42, 157.96, 153.46, 146.80, 146.39, 136.63, 136.06,
35.36, 134.75, 132.39, 131.91, 129.81, 128.63, 128.36, 127.93,
126.59, 121.68, 119.89, 111.00, 110.90, 108.39. Anal. Calcd (%) for
S: C, 57.69; H, 3.23; N, 13.46. Found: C, 57.68; H, 3.24; N,
6
, δ ppm) δ: 167.82,
f)
1
In a nitrogen atmosphere, a mixture containing 3-amino-phthalic
′
anhydride (5) (1 mmol), N-ethyl-N -(3-dimethylaminopropyl)carbodii-
mide hydrochloride (EDC) (1.5 mmol), N-oxide derivatives (8a-b; 10a-b
and 12a-b) (2 mmol) and 4-dimethylaminopyridine (0.2 mmol) in 20
mL of dry dimethylformamide (DMF) was stirred and maintained at
room temperature for 24–30 h. After, it was added around 10 mL of ice-
water to precipitate the compounds (3a-f), which were collected by
filtration under reduced pressure and washed with cold water to remove
residual reagents. If necessary, an additional purification was performed
by column chromatography using Isolera Biotage equipment, using a 20
µm (25 g) Sphere silica column, with a flow rate of 25 mL / min and
phase mobile 50% ethyl acetate / 50% hexane. The pomalidomide NO-
donor derivatives (3a-f) were obtained at yields ranging from 13 to 43%.
30 20 6 8
C H N O
13.45. (MW: 624.58, Rf: 0.30, eluent ether petroleum (55%): ethyl ac-
etate (45%)).
(E)-4-(4-((2-(2-(4-amino-1,3-dioxoisoindolin-2-yl)benzoyl)hydra-
zono)methyl)phenoxy)-3-methyl-1,2,5-oxadiazole 2-N-oxide (3e)
◦
ꢀ 1
White powder; yield: 15%; mp: 217–220 C. IR max (cm ; KBr pel-
), 2933 (C ), 1778 and 1720 (C O imide),
lets): 3060 (C
–
H
aromatic
aromatic), 1475 (C
(300 MHz, DMSO‑d
–
H
alkyl
–
1
–
1600 (C
–
–
), 1358 (N–O oxide). H NMR
–
C
C
aromatic
6
, δ ppm) δ: 12.1 (s, 1H), 8.35 (s, 1H), 7.77–7.81 (m,
2H), 7.71–7.62 (dd, 2H, J = 7 Hz), 7.54–7.52 (m, 4H), 7.02–7.06 (m,
2H), 6.87 (dd, 1H), 6.57 (s, 2H), 2.11 (s, 3H). 13C NMR (75 MHz,
DMSO‑d
6
, δ ppm) δ: 168.4, 167.18, 162.87, 153.90, 151.51, 146.90,
(
E)-4-(4-((2-(2-(4-amino-1,3-dioxoisoindolin-2-yl)benzoyl)hydra-
146.40, 139.30, 135.64, 132.60, 132.17, 130.44, 128.16, 125.01,
128.16, 125.01, 121.68, 120.01, 111.21, 108.67, 107.75, 7.02. Anal.
zono)methyl)phenoxy)-3-phenyl-1,2,5-oxadiazole 2-N-oxide (3a)
◦
ꢀ 1
Yellowish powder; yield: 32%; mp: 250–252 C. IR max (cm ; KBr
pellets): 3061 (C
imide), 1608 (C–
18 6 6
Calcd (%) for C25H N O : C, 60.24; H, 3.64; N, 16.86. Found: C, 60.26;
–
H
aromatic), 2929 (C
–
aromatic
H
alkyl), 1776 and 1721 (C
–
O
H, 3.65; N, 16.85. (MW: 498.45, Rf: 0.22, eluent ether petroleum (55%):
ethyl acetate (45%)).
–
–
), 1362 (N–O oxide). H
1
–
C
aromatic), 1472 (C
C
NMR (300 MHz, DMSO‑d
6
, δ ppm) δ: 12.11 (s, 1H), 8.36 (s, 1H), 8.07
(E)-4-(4-((2-(4-(4-amino-1,3-dioxoisoindolin-2-yl)benzoyl)hydra-
zono)methyl)phenoxy)-3-methyl-1,2,5-oxadiazole 2-N-oxide (3f)
(
(
dd, J = 8,0 Hz, 2H), 7.78–7.83 (m, 3H), 7.58–7.56 (m, 6H), 7.54–7.47
13
◦
ꢀ 1
m, 3H), 7.02–7.06 (m, 2H), 6.53 (s, 2H). C NMR (75 MHz, DMSO‑d
6
, δ
White powder; yield: 19%; mp: 218–221 C. IR max (cm ; KBr pel-
), 2930 (C ), 1777 and 1719 (C O imide),
lets): 3059 (C
–
H
aromatic
aromatic), 1470 (C
(300 MHz, DMSO‑d
–
H
alkyl
–
1
ppm) δ: 168.34, 167.12, 162.62, 161.85, 153.82, 146.91, 146.34,
–
–
–
), 1360 (N–O oxide). H NMR
1
1
6
5
35.43, 132.67, 132.36, 130.97, 129.15, 128.88, 126.71, 121.68,
20.54, 111.06, 109.11, 108.40. Anal. Calcd (%) for C30 : C,
1608 (C
–
C
C
aromatic
H
20
N
6
O
6
6
, δ ppm) δ: 12.02 (s, 1H), 8.49 (s, 1H), 8.02 (dd, J =
4.28; H, 3.60; N, 14.99. Found: C, 64.29; H, 3.59; N, 14.99. (MW:
45.50, Rf: 0.10, eluent ether petroleum (55%): ethyl acetate (45%)).
8.0 Hz, 2H), 7.88 (dd, J = 8 Hz, 2H), 7.63 (d, 2H, J = 7.5 Hz), 7.5 (dd,
2H, J = 7.5 Hz), 7.06–7.10 (m, 2H), 6.87(m, 1H), 6.61 (s, 2H), 2.13 (s,
3H). 13C NMR (75 MHz, DMSO‑d
, δ ppm) δ: 168.10, 166.86, 162.77,
(
E)-4-(4-((2-(4-(4-amino-1,3-dioxoisoindolin-2-yl)benzoyl)hydra-
6
zono)methyl)phenoxy)-3-phenyl-1,2,5-oxadiazole 2-N-oxide (3b)
153.90, 151.51, 147.08, 146.75, 139.24, 135.64, 135.10, 132.19,
130.88, 128.92, 128.14, 126.86, 124.96, 121.75, 119.97, 111.18,
◦
ꢀ 1
Yellowish powder; yield: 43%; mp: 205–208 C. IR max (cm ; KBr
pellets): 3060 (C
–
H
aromatic), 2930 (C
–
aromatic
H
alkyl), 1779 and 1720 (C
–
O
18 6 6
108.67, 107.68, 7.02. Anal. Calcd (%) for C25H N O : C, 60.24; H, 3.64;
–
–
–
), 1361 (N–O oxide). H
1
imide), 1607 (C
C
aromatic), 1470 (C
–
C
N, 16.86. Found: C, 60.25; H, 3.63; N, 16.84. (MW: 498.45, Rf: 0.4,
1
0

T.R.F. de Melo et al.
Bioorganic Chemistry 114 (2021) 105077
eluent ether petroleum (55%): ethyl acetate (45%)).
calculated by integrating the area under the curve of chromatograms,
and expressed as a percent of total hemoglobin (HbA + HbF, normal
controls).
5
5
.4. Pharmacology
5
.4.2.3. Flow cytometric analysis of erythropoiesis. Terminal erythroblast
.4.1. Quantification of nitrite
differentiation was monitored at D7 and 14 of culture. Cells were pel-
leted and stained with a cocktail of antibodies consisting of an anti-band
The quantification of nitrite in the medium was characterized using
Griess reaction after incubation of all compounds (3a-f) according
procedures previously described [8,9,38]. All experiments were carried
out in triplicate and repeated three independent times. In the conditions
3
FITC-conjugated (produced by Dr. Mohandas Narla), anti-GPA PE-
conjugated, and anti- 4-integrin APC-conjugated (MACS) antibodies for
5 min at RT. Erythroblasts were defined as GPApos cells, and the level
of maturation was determined by 4 and band 3 expression. Immature
erythroblasts (i.e. proerythroblasts) were 4-integrinhi/band 3lo more
differentiated erythroblasts such as orthochromatic and reticulocytes
represent 4-integrinlo/band 3hi populations. Dead cells were excluded
α
1
without L-cysteine was nor observed nitrite formation. The results were
α
ꢀ
shown as a percentage of nitrite (% NO
2
; mol/mol) and for statistical
α
analysis was used ANOVA followed by Tukey’s Multiple Comparison
Test at a significance level of p < 0.05.
α
from analysis through 7-AAD staining. Flow cytometric analyses were
performed on a BD Fortessacytometer and FlowJo software. Unless
otherwise indicated, all antibodies used were from BD Biosciences.
5
.4.2. Evaluation of gamma-globin expression using CD34 + cells
CD34 + cells were isolated from de-identified control patient pe-
ripheral blood leukoreduction filters. To limit variability from one in-
dividual to another, CD34 + isolated from 20 leukoreduction filters were
pooled for each experiment. Blood components were separated using
Ficoll-Opaque, and CD34 + cells were purified using anti-CD34 manual
cell separation columns and conjugated microbeads according to man-
ufacturer’s instructions (Miltenyi). Isolated peripheral blood CD34 +
cells were expanded in H3000 media supplemented with CC100 (Stem
5
.4.3. Histone deacetylase inhibition
Compound 3d was evaluated through an in vitro enzymatic assay
using recombinant HDAC-1 and HDAC-2. This compound was dissolved
in DMSO at initial concentration of 1 mM. After, it will then get directly
diluted 10x fold into assay buffer for an intermediate dilution of 10%
DMSO in HDAC assay buffer and 5 µL of the dilution was added to a 50
µL reaction so that the final concentration of DMSO is 1% in all of re-
actions. The enzymatic reactions for the HDAC enzymes were carried
5
Cell Technologies) for 4 days at a density of 10 cells/mL. CD34 + cells
were differentiated toward erythrocytes using an adapted version of a
previously described in vitro 3-phase culture system [20].Briefly, CD34
◦
out in triplicate at 37 C for 30 min in a 50 µL mixture containing HDAC
assay buffer, 5 µg BSA, an HDAC substrate (10 µM HDAC Substrate 3;
BPS Bioscience, #50037), a HDAC enzyme (HDAC-1, 7.2 ng/reaction,
BPS Bioscience, catalog #50051; and HDAC-2, 7.2 ng/reaction, BPS
+
cells were cultured in 3% (vol/vol) AB serum, 2% (vol/vol) human
plasma, 10 g/mL insulin, 3U/mL heparin, 200 g/mL transferrin sup-
μ
μ
plemented with either 10 ng/mL stem cell factor (SCF), 1 ng/mL
interleukin-3 (IL-3) and 3 IU/mL erythropoietin (EPO) (Phase I; D0-7) or
Bioscience, catalog #50052) and compound 3d (0.3
nM and 30 nM. After enzymatic reactions, 50 L of 2x HDAC Developer
(BPS Bioscience, catalog #50030) was added to each well for the HDAC
μ
M) or vorinostat at
1
0 ng/mL SCF and 3 IU/mL EPO (Phase II; D7-11) or 1 mg/Ml trans-
ferrin and 3 IU/mL EPO (Phase III; D11-16). The treatments (0.1–10
pomalidomide, 10 M compounds 3b, 3d, 3f and DMSO)
3
μ
μ
M
(
μ
M HU, 0.1–10 μ
enzymes and the plate was incubated at room temperature for an
additional 15 min. Fluorescence intensity was measured at an excitation
of 360 nm and an emission of 460 nm using a Tecan Infinite M1000
microplate reader. HDAC activity assays were performed in triplicate, in
three different experiments. The fluorescent intensity data were
analyzed using the computer software, Graphpad Prism. In the absence
were added to erythroid differentiation cultures at every culture me-
dium change.
5
.4.2.1. Western Blot. Cells were lysed in 1X RIPA buffer (Thermo Sci-
entific) supplemented with 1:100 protease inhibitor cocktail (Sigma
Aldrich) on ice, and then centrifuged at maximum speed. Supernatants
were mixed 1:1 with 2X Laemmli sample buffer (BioRad) under reducing
conditions and boiled for 5 min. Following this, samples were separated
via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) for 1.5 h at 110 V, and transferred to nitrocellulose membranes
for 1 hr at 90 V. Membranes were blocked with 4% (wt/vol) milk 1%
of the compound, the fluorescent intensity (F ) in each data set was
t
defined as 100% activity. In the absence of HDAC, the fluorescent in-
tensity (F ) in each data set was defined as 0% activity. The percent
b
activity in the presence of each compound was calculated according to
the following equation: % activity = (F-F )/(F -F ), where F = the
b
t
b
fluorescent intensity in the presence of the compound.
(
wt/vol) BSA 1X Phosphate Buffer Saline 0.1% (vol/vol) Tween20 (PBS-
T), and incubated with the following primary antibodies overnight at 4
5.4.4. Quantification of cGMPc levels
◦
C: BCL11-A (Abcam, MA), IKAROS (Santa Cruz Biotechnologies, CA),
The levels of intracellular accumulation of cGMP were quantified
using HUVEC cells [58]. HUVEC cells were grown in 60 mm plates in
medium M199 supplemented with 15% fetal calf serum, 200 Ag/ml
endothelial cell growth supplement, 4 U/ml heparin sodium, 100 U/ml
penicillin–streptomycin and 50 Ag/ml gentamycin. Cultures were
(
(
Abcam, MA), and
α-globin (Santa Cruz Biotechnologies, CA), β- globin
Santa Cruz Biotechnologies, CA), γ-globin (Santa Cruz Biotechnologies,
CA), and GAPDH (Millipore, Germany). Membranes were washed 3
times with PBST and incubated with horseradish peroxidase (HRP)-
conjugated secondary antibodies for 1 h at room temperature (RT).
Bands were detected using enhanced chemiluminescence (ECL) (Thermo
Scientific). All western blots presented in the manuscript are represen-
tative of multiple experiments, as detailed in the figure legend section.
Quantifications were obtained with ImageJ software.
◦
maintained at 37 C, 5% CO and 100% humidity and used when in
2
confluence. The cells were treated and incubated for a period of 4 h. The
incubation with the tested compounds was interrupted by removing the
supernatant from the culture and the cells were then immediately
covered with lysis buffer and scraped off. The extract was collected,
◦
centrifuged for 5 min at 5000x g and stored at ꢀ 20 C until analyzed. To
5
.4.2.2. High Performance Liquid Chromatography (HPLC). At D14 of
normalize the cGMP values, the protein content in each well was
measured by Bradford trial assay and normalized to have the same
concentration in a final volume of 250uL. Results were presented in
order of fmol cGMP / µg of protein. Intracellular cGMP levels were
measured using the kit GMPc EIA (R & D Systems), according to the
manufacturer’s instructions.
5
differentiation, 5x10 cells were pelleted and lysed with 100
ice for 30 min. Alternatively, 1 L of packed RBCs derived from cord or
peripheral blood was pelletized and lysed with 500 L H O on ice for 30
μ
2
L H O on
μ
μ
2
min. Following centrifugation at max speed, hemolysates were
collected, and hemoglobin molecules were measured using a polyCAT
HPLC column (100x4.6-mm, 3
μ
m, 1000 Å) (PolyLC inc., MD) and dual
wavelength absorbance at 415 and 690 nm. Hemoglobin content was
1
1

T.R.F. de Melo et al.
Bioorganic Chemistry 114 (2021) 105077
5
.4.5. Cellular viability and measurement of TNF-
α
level in macrophage
Faculdade de Ci ˆe ncias Farmac ˆe uticas da UNESP – PADC and Conselho
cells
Nacional de Desenvolvimento Científico
e
Tecnol o´ gico (CNPq
This study was approved by the Research Ethics Committee of the
Faculty of Medical Sciences of UNICAMP, under No. 1,169,997 and
CAAE no.: 45878215.8.0000.5404. Human mononuclear cells were
separated from peripheral blood by Ficoll gradient (GE Healthcare,
Pittsburgh, PA, USA) and subsequently Percoll 45% (GE Healthcare,
Ref. Process: 479468/2013-3 and 310026/2011-3), the National In-
stitutes of Health HL144436 (to LB).
Appendix A. Supplementary data
Pittsburgh, PA, USA) to obtain a more purified cell population. After, a
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105077.
6
volume of 100
μ
L containing 5 × 10 cells suspended in RPMI medium
and 10% of calf bovine serum was added to the 24-well plates and
◦
incubated for 1 h (37 C, atmosphere 5% of CO
2
). Those non-adherent
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